is there a relationship between disease duration and p-wave dispersion in patients with psoriasis? full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 is there a relationship between disease duration and p-wave dispersion in patients with psoriasis? bilge bulbul sen, emine nur rifaioglu, ozlem ekiz, eyup buyukkaya & nihat sen to cite this article: bilge bulbul sen, emine nur rifaioglu, ozlem ekiz, eyup buyukkaya & nihat sen (2013) is there a relationship between disease duration and p-wave dispersion in patients with psoriasis?, upsala journal of medical sciences, 118:3, 204-205, doi: 10.3109/03009734.2013.785614 to link to this article: https://doi.org/10.3109/03009734.2013.785614 © informa healthcare published online: 27 mar 2013. submit your article to this journal article views: 378 view related articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.785614 https://doi.org/10.3109/03009734.2013.785614 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.785614 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.785614 upsala journal of medical sciences. 2013; 118: 204–205 letter to the editor is there a relationship between disease duration and p-wave dispersion in patients with psoriasis? bilge bulbul sen1, emine nur rifaioglu1, ozlem ekiz1, eyup buyukkaya2 & nihat sen2 1department of dermatology, mustafa kemal university school of medicine, hatay, turkey, and 2department of cardiology, mustafa kemal university school of medicine, hatay, turkey dear editor we have read with great enthusiasm the recently published article entitled ‘electrocardiographic pwave characteristics in patients with psoriasis vulgaris’ by bacaksız and co-workers (1). they concluded that pmax and p-wave dispersion (pwd) were significantly higher in psoriasis patients than in controls (108.8 ± 21.3 ms versus 93.3 ± 13.0 ms, p < 0.01; 67.4 ± 22.9 ms versus 45.0 ± 19.6 ms, p < 0.01, respectively). furthermore, pmin was significantly lower in the psoriasis group (41.3 ± 12.3 ms versus 48.3 ± 14.3 ms, p = 0.04) (1). recently, ahlehoff et al. showed that psoriasis is associated with an increased risk of atrial fibrillation (af) (2). prolongation of pwd has been demonstrated to be an independent risk factor for the development of af. therefore, the subject is important in daily practice, and the study deserves further attention, keeping in mind its successful design and results. bacaksız et al. showed (1) that the psoriasis area and severity index (pasi) score was correlated with pmax and pwd (p = 0.002 and p = 0.005, respectively). in addition, there was a significant positive correlation between hscrp and pwd (r = 0.229, p = 0.001). it was speculated that although the exact mechanism still remains unclear, chronic inflammation may be responsible for the correlation between pasi score and pwd in these patients. psoriasis is a chronic inflammatory disease, and the inflammation continues for the duration of the disease. hence, a significant positive correlation between duration of the disease and pwd would be expected. we believe that an evaluation of the relationship between duration of the disease and pwd will help us to understand the increased frequency of af in patients with psoriasis. in addition, patients with coronary artery disease (cad) were excluded in the study of bacaksiz et al (1). epidemiological studies in patients with severe psoriasis have revealed that there is an increased risk of cardiovascular mortality (3,4), and the cardiovascular disease risk has been found to be particularly high in young patients with a severe form of the disease (5,6). it has been postulated that premature occurrence of atherosclerosis is related to why psoriasis patients have an increased risk for developing cardiovascular disease. as we know, there is an important relationship between psoriasis and subclinical coronary atherosclerosis, independent of conventional cardiovascular disease risk factors (7-9). bacaksız et al. (1) did not specify how cad was excluded, but there might be some subclinical atherosclerosis in their patients. in a previous study, pwd was found to be more significant in patients with stable cad than in patients with normal coronary angiograms and to be associated with severity of the disease as well (10). therefore, it would have been helpful if bacaksiz and co-workers had clearly identified these factors. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. correspondence: bilge bulbul sen, md, department of dermatology, mustafa kemal university, tayfur ata sokmen medical school, serinyol, hatay 31005, turkey. fax: +90 326 245 5305. e-mail: bilgebulbul@yahoo.com (received 5 february 2013; accepted 10 march 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.785614 references 1. bacaksiz a, erdogan e, tasal a, vatankulu ma, kul s, sevgili e, et al. electrocardiographic p-wave characteristics in patients with psoriasis vulgaris. ups j med sci. 2013;118:35–41. 2. ahlehoff o, gislason gh, jørgensen ch, lindhardsen j, charlot m, olesen jb, et al. psoriasis and risk of atrial fibrillation and ischaemic stroke: a danish nationwide cohort study. eur heart j. 2012;33:2054–64. 3. mehta nn, azfar rs, shin db, neimann al, troxel ab, gelfand jm. patients with severe psoriasis are at increased risk of cardiovascular mortality: cohort study using the general practice research database. eur heart j. 2010;31:1000–6. 4. prodanovich s, kirsner rs, kravetz jd, ma f, martinez l, federman dg. association of psoriasis with coronary artery, cerebrovascular, and peripheral vascular diseases and mortality. arch dermatol. 2009;145:700–3. 5. brauchli yb, jick ss, miret m, meier cr. psoriasis and risk of incident myocardial infarction, stroke or transient ischaemic attack: an inception cohort study with a nested case–control analysis. br j dermatol. 2009;160:1048–56. 6. daudén e, castañeda s, suárez c, garcía-campayo j, blasco aj, aguilar md, et al. [integrated approach to comorbidity in patients with psoriasis]. actas dermosifiliogr. 2012; 103:1–64. 7. yiu kh, yeung ck, zhao ct, chan jc, siu cw, tam s, et al. prevalence and extent of subclinical atherosclerosis in patients with psoriasis. j intern med. 2013;273:273–82. 8. gelfand jm, neimann al, shin db, wang x, margolis dj, troxel ab. risk of myocardial infarction in patients with psoriasis. jama. 2006;296:1735–41. 9. gisondi p, fantin f, del giglio m, valbusa f, marino f, zamboni m, et al. chronic plaque psoriasis is associated with increased arterial stiffness. dermatology. 2009;218: 110–13. 10. yilmaz r, demirbag r. p-wave dispersion in patients with stable coronary artery disease and its relationship with severity of the disease. j electrocardiol. 2005;38:279–84. letter to the editor 205 mandibular bone exposure and osteonecrosis as a complication of general anaesthesia full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 mandibular bone exposure and osteonecrosis as a complication of general anaesthesia mohammad kharazmi, lillemor björnstad, pär hallberg, jonas wanbro, anders-petter carlsson, samandar habib & gunnar warfvinge to cite this article: mohammad kharazmi, lillemor björnstad, pär hallberg, jonas wanbro, anders-petter carlsson, samandar habib & gunnar warfvinge (2015) mandibular bone exposure and osteonecrosis as a complication of general anaesthesia, upsala journal of medical sciences, 120:3, 215-216, doi: 10.3109/03009734.2015.1010667 to link to this article: https://doi.org/10.3109/03009734.2015.1010667 © informa healthcare published online: 17 mar 2015. submit your article to this journal article views: 334 view related articles view crossmark data citing articles: 5 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2015.1010667 https://doi.org/10.3109/03009734.2015.1010667 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1010667 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1010667 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1010667&domain=pdf&date_stamp=2015-03-17 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1010667&domain=pdf&date_stamp=2015-03-17 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1010667#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1010667#tabmodule upsala journal of medical sciences. 2015; 120: 215–216 letter to the editor mandibular bone exposure and osteonecrosis as a complication of general anaesthesia mohammad kharazmi1,2, lillemor björnstad1, pär hallberg3, jonas wanbro4, anders-petter carlsson1,5, samandar habib6 & gunnar warfvinge7 1department of oral and maxillofacial surgery, central hospital, västerås, sweden, 2section of orthopaedics, department of surgical sciences, uppsala university, uppsala, sweden, 3department of medical sciences, uppsala university, uppsala, sweden, 4department of anaesthesia and intensive care, central hospital, västerås, sweden, 5section of oral and maxillofacial surgery, department of surgical sciences, uppsala, sweden, 6school of medicine, uppsala university, uppsala, sweden, and 7department of oral pathology, malmö university, malmö, sweden trauma to the oral structures sometimes occurs as a complication to general anaesthesia. we here report two cases of an unusual type, mandibular osteonecrosis. to our knowledge, there has been only one previous publication of this condition (1). the first case was a 69-year-old man who underwent oral examination 3 months prior to planned cardiac surgery. his medical history included hypertension, diabetes mellitus, and mitral insufficiency, and he was on treatment only with enalapril. three molars in his lower jaw were extracted due to periodontitis, and healing was uneventful at follow-up. at cardiac surgery, he was intubated with a standard endotracheal tube (number 8) and was placed on a cardio-pulmonary bypass (cpp) machine. two weeks later, he complained of pain in the right side of his mandible, which had started directly after recovery from surgery and successively increased. examination revealed a 4 � 8 mm area of exposed non-vital bone at the right mylohyoid ridge. there was no sign of infection, and radiography was unremarkable. he was treated with 2 mg/ml chlorhexidine mouth rinse twice daily and amoxicillin 500 mg three times daily. three weeks later there was spontaneous exfoliation of a sequestrum, and there was complete healing 2 months after his cardiac surgery. the other case was an 86-year-old man with progressive aorta stenosis and a history of myocardial infarction. medication included enalapril, isosorbide mononitrate, simvastatin, acetylsalicylic acid, and glyceryl trinitrate. he was planned for cardiac surgery and underwent oral examination 2 months before. two upper and three lower molars were extracted due to apical periodontitis, and healing was uneventful at follow-up. at cardiac surgery, the patient was intubated with a standard endotracheal tube (number 8) and placed on a cpp machine. two weeks later, he complained about right-sided mandibular pain, which had started directly after recovery from surgery. examination revealed an 8 � 5 mm area of non-vital exposed bone at the right mylohyoid ridge. there was no sign of infection, and radiography was unremarkable. he was treated with 2 mg/ml chlorhexidine mouth rinse twice daily and phenoxymethylpenicillin 1 g three times daily, and there was complete healing 2 months after surgery preceded by exfoliation of a small sequestrum. the incidence of osteonecrosis as here described is unknown. it is probably low, but the location at the medial aspect of the mandible could make it easily mistaken for discomfort after intubation (2). the pathogenesis is unclear, but the prominence of correspondence: mohammad kharazmi dds, department of oral and maxillofacial surgery, central hospital, västerås, se-721 89 västerås, sweden. fax: +46 21 17 54 30. e-mail: kharazmi.mohammad@gmail.com (received 22 december 2014; accepted 8 january 2015) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1010667 http://informahealthcare.com/journal/ups mailto:kharazmi.mohammad@gmail.com the mandibular shelf, covered by only a thin layer of oral mucosa, is probably vulnerable to trauma, which may affect the blood supply to the periosteum leading to local ischemia and osteonecrosis. another possibility could be soft tissue necrosis caused by several hours of pressure from the endotracheal tube or the transesophageal echocardiograph with its bite blocks. although the anaesthesiologists reported no procedural complications, reduced blood pressure could have aggravated the condition through diminished blood flow in the soft tissue. in a previously published case series (table i), it appears that the affected area was at the right side of the posterior part of the mandible in all cases, indicating that trauma from the laryngoscope blade was a possible cause (1). although the exact mechanism involved in this type of osteonecrosis remains unknown (2), we believe that an association with general anaesthesia in our cases is very likely. there was no history of bisphosphonate use or radiation treatment, and follow-up oral examination prior to surgery showed no sign of osteonecrosis. also, the onset of mandibular pain was right after recovery from anaesthesia in both cases. factors that may predispose to this type of osteonecrosis include prominent mandibular shelves, limited mouth opening, and extensive oral manipulation while managing the airway during anaesthesia. previous or ongoing treatment with bisphosphonates or radiation might also lead to more severe complications in affected patients. we hope that our reported cases will increase the awareness of this type of complication to general anaesthesia. acknowledgements all authors contributed to this study and in the writing of the manuscript. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. almazrooa sa, chen k, nascimben l, woo sb, treister n. case report: osteonecrosis of the mandible after laryngoscopy and endotracheal tube placement. anesth analg. 2010;2: 437–41. 2. fisher qa. true, true–but how related? bony necrosis and sequestration in the mandible after endotracheal intubation. anesth analg. 2010;2:272–3. table 1. summary of cases in the literature and the present report. age/ gender location of osteonecrosis duration of intubation (hours) osteonecrosis visualized by radiography symptoms onset of symptoms after general anaesthesia treatment time until healing case 1a 49f posterior, right side of mandible 2.5 yes (ct) oral pain after 1 week non-surgical removal of the sequestrum. chlorhexidine rinse. within 1 week case 2a 60m posterior, right side of mandible 3 yes (plain x-ray) oral pain immediately after recovery non-surgical removal of the sequestrum. chlorhexidine and saline rinse. 2 weeks case 3a 47f posterior, right side of mandible 1.5 na oral pain immediately after recovery non-surgical removal of the sequestrum. amoxicillin. na case 4a 67f posterior, right side of mandible 1.75 na soreness and oral pain after 1 day non-surgical removal of the sequestrum. chlorhexidine rinse. within 1 week case 1b 69m posterior, right side of mandible 4.5 no (plain x-ray) oral pain immediately after recovery amoxicillin. chlorhexidine rinse. 6 weeks case 2b 86m posterior, right side of mandible na no (plain x-ray) oral pain immediately after recovery phenoxymethylpenicillin. chlorhexidine rinse. 7 weeks aalmazrooa sa et al, 2010 (1). bpresent report. na = not available. 216 m. kharazmi et al. http://www.ncbi.nlm.nih.gov/pubmed/20495140?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20495140?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20664092?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20664092?dopt=abstract ss1 acknowledgements declaration of interest references further improvement of our metrics—will plan s affect them? editorial further improvement of our metrics—will plan s affect them? traditionally, the last issue—no. 4—of each volume of ujms informs our readers about the performance of our journal in terms of metrics. it is a much-debated phenomenon (1), questioned by many researchers but loved by research administrators (2). as long as figures are pointing in the right direction, we tend to belong to the lovers. thus, this time we can report new record figures for all three parameters of special interest—the 2and 5-year impact figures (clarivate) and the newly launched citescore (elsevier). the 5-year score closed just below 3.0, and the most frequently used score— the 2-year value—a couple of tenths lower (figure 1). we prefer to be judged by the former, not just because it gave us the highest score ever this year, but rather because small journals with few issues and articles are very sensitive to the fate of individual papers. a blockbuster can be very valuable, but its disappearance from the denominator after 2 years is equally detrimental. for ujms the citescore figure, 2.34, is very close to these traditional impact factor (if) scores. this is noteworthy, since most of the prestigious journals have a five-fold lower citescore value when compared with their if figures (3). this is because of the fact that they all—and especially new england journal of medicine—have to include all published articles in the denominator of the citescore calculation. when estimating different metric values, the number of citations in recognised journals forms the basis for such calculations. taking into account the electronic supervision of all activities at the journal’s website nowadays, it is possible to monitor all downloads of published articles and also see who the viewer is (4). one pertinent question, then, is to what extent there is a correlation between the viewings of an article and the number of citations of the same paper. now and then you can hear the argument that articles should be judged by the numbers of readers—approximately the number of downloads on the journal’s website—rather than by the actual citation figures in different data bases. we therefore looked at these figures in one volume—volume 119, published in 2014—at a time point when both new citations and viewings had started to level off. perhaps not surprisingly, there was quite a strong correlation between the numbers of citations and viewings (r ¼ 0.88; p < 0.0001) (figure 2). there are, however, quite a few extreme cases where many viewings do not necessarily predict a future as a top-score citation candidate. in the present volume of our journal, we have an example of one such article that was downloaded many times immediately upon its release in mid-april. thus, we want to highlight the paper by milutinovic et al. in issue 2, 2019, on surface glycans in prostasomes (5). now, half a year later, no less than 1700 viewings have been registered. will that paper become a citation classic? we would like to interpret this to indicate that this field of research, pioneered in a masterly way by our former editor gunnar ronquist, is becoming hotter and hotter (6). we encourage people active in this field to submit their papers to our journal. as always, we guarantee quick and reliable handling of manuscripts, as well as the open access strategy of our journal without apcs (article publication charges; see below). the issues dwelt upon above are trivial when considering the big and most controversial subject in scholarly publishing these days—coalition s, or more frequently presented as ‘plan s’. when visiting their website, it says that plan s is ‘making full and immediate open access a reality’ (7). the plan is supported by an international consortium of funders, and the task of coalition s is to take action towards the implementation of plan s. there are many well-known supporters of this initiative and prestigious funders like the eu, wellcome, the research council of norway, the academy of finland, and some swedish funders such as vinnova, formas, and forte. interestingly, the swedish research council has postponed their signing of the contract. when it was launched in september 2018, the start was planned for 2020, but this has now been postponed until 2021, because of lack of time for the implementation and some firm resistance amongst researchers. what impact could these changes have for our journal? quite likely, forcing researchers funded by public grants provided by national and european research councils to publish under open access policies would benefit our journal. upsala journal of medical sciences has applied gold open access for the last 10 years, i.e. all published articles have been fully accessible immediately upon release without a publication paywall. it is most likely that this feature of our journal has enhanced our performance substantially. when investigating the influence of the open access publishing format on the if values of open access and closed access journals over a 5year period, we found no obvious effect on the journals chosen for the review (4). in order to have a longer time perspective, we looked at the same journals 5 years later when the if values for 2018 were released (tables 1 and 2). interestingly, there was no obvious increase of the values during this 10-year period. this was despite a remarkable increase of both the number of journals and articles. but, again, no differences between the two groups of journals were discernible. it is worthy of note that two uppsala-based journals, one in the closed category (amyloid; more than two-fold) and one in the open access (ujms; almost fourfold) display quite obvious improvements. so, in essence we have nothing to fear. open access publishing will most probably slowly increase, and fees will have to be paid in some upsala journal of medical sciences 2019, vol. 124, no. 4, 215–217 https://doi.org/10.1080/03009734.2019.1691688 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1691688&domain=pdf&date_stamp=2019-12-09 https://doi.org/10.1080/03009734.2019.1691688 http://www.tandfonline.com way for the service publishers provide. apcs will become a more common phenomenon, and the level will depend on the quality and reputation characterising the journal. at present the board of our society has no plans for a change of our no-apc policy. of course, such an offer attracts many contributions from research environments with meagre economic resources. it also constitutes a substantial work load for the editorial board. there is only room for some 40–50 published papers a year, and, with submission figures close to 300, there is a fairly high rejection rate. from the editors’ point of view, we have to attract more papers good enough for acceptance. and we take this opportunity to repeat that we offer a fast track for high-quality papers. let us finish this annual report with an apology. perhaps some of you have noticed that the journal launched a new submission portal in may 2019. as editors of scholarly journals you would not notice this if it wasn’t for the sudden increase in your inbox of e-mails from prospective authors complaining about difficulties in crossing a veritable wall created by this new electronic manuscript central. it also quite soon became clear that resubmitting a revised manuscript involved many problems. the reasons for these shortcomings have not been resolved in detail as yet. still, almost half a year later, there are complaints of the same nature, and the problems mainly involve the log-in process. we are struggling to return to the use of the old portal. meanwhile, our advice is to either report problems to the technical support of taylor & francis or, alternatively, to contact the editor of the journal. it is impossible to assess how many authors have given up and sent their papers to other journals. luckily, some manuscripts have nevertheless arrived at the editorial office, and submission figures are close to those of last year. therefore, besides begging for your patience, we would like to hear from you if, or when, you face problems with your submissions. press stop as of december 2, 2019; the old submission portal has now been reinstalled and, not surprisingly, the inconveniencies with the new version disappeared. disclosure statement no potential conflict of interest was reported by the authors. references 1. ioannidis jpa, thombs bd. a user’s guide to inflated and manipulated impact factors. eur j clin invest. 2019;49:e13151. 2. metze k. bureaucrats, researchers, editors, and the impact factor: a vicious circle that is detrimental to science. clinics (sao paulo). 2010;65:937–40. 3. andersson a, lau borjesson j. a new contribution to research metrics. ups j med sci. 2018;123:191–3. figure 1. impact factor or citescore of upsala journal of medical sciences. figure 2. viewings on taylor & francis correlate to total citations. table 1. non-open access journals: impact factors. non-open access journals if 2009 if 2018 factor change new england journal of medicine 47.050 70.670 1.50 the lancet 30.758 59.102 1.92 journal of clinical investigation 15.387 12.282 0.80 diabetologia 6.551 7.113 1.09 diabetes 8.585 7.199 0.84 endocrinology 4.752 3.800 0.80 embo journal 8.993 11.227 1.25 pnas 9.432 9.580 1.02 amyloid 2.115 4.919 2.33 acta oncologica 2.265 3.298 1.46 mean factor change: 1.30 ± 0.16. table 2. open access journals: impact factors. open access journals if 2009 if 2018 factor change plos one 4.351 2.776 0.64 plos medicine 13.050 11.048 0.85 plos biology 12.916 8.386 0.65 bmc biology 5.636 6.723 1.19 bmc medicine 3.985 8.285 2.08 virology journal 2.435 2.464 1.01 chinese medical journal 0.952 1.555 1.63 swiss medical weekly 1.681 1.821 1.08 journal of translational medicine 3.407 4.098 1.20 upsala journal of medical sciences 0.733 2.747 3.75 mean factor change: 1.41 ± 0.30. 216 a. andersson et al. 4. andersson a, borjesson jl. operating in an era of impact factor mania. ups j med sci. 2015;120:124–31. 5. milutinovic b, goc s, mitic n, kosanovic m, jankovic m. surface glycans contribute to differences between seminal prostasomes from normozoospermic and oligozoospermic men. ups j med sci 2019;124:111–8. 6. ronquist g. prostasomes are mediators of intercellular communication: from basic research to clinical implications. j intern med. 2012;271:400–13. 7. coalition s. available at: https://www.coalition-s.org/ arne andersson (editor), daniel espes (statistical consultant), and joey lau b€orjesson (editorial assistant) joey.lau@mcb.uu.se received 7 november 2019; accepted 7 november 2019 � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ 4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 217 https://www.coalition-s.org/ outline placeholder disclosure statement references strong development of research based on national quality registries in sweden article strong development of research based on national quality registries in sweden jack lysholma and bertil lindahlb adepartment of public health and clinical medicine, umeå university, umeå, sweden; bdepartment of medical sciences and uppsala clinical research center, uppsala university, uppsala, sweden abstract the aim of the present paper is to describe how the use of national quality registries (nqrs) for research has evolved over the past decade in sweden. all swedish nqrs have reported their scientific activity (publications per year in peer-reviewed scientific journals) to the swedish association of local authorities and regions since 2009, and the present report is based on available data from 2009–2016. the yearly number of publications of the 69 registries active in 2009 has increased from 121 to 496 in 2016. seventeen of these registries published more than 10 papers in 2016; however, 12 nqrs did not publish any papers in 2016. an additional 77 papers were published in 2016 by the 34 nqrs started after 2009. in summary, there has been a strong development of quality registry-based research in sweden over the last decade. however, there is still room for further increase of the use of research based on nqrs in sweden. article history received 23 august 2018 revised 4 september 2018 accepted 4 september 2018 keywords quality registries; research; peer-reviewed journals according to swedish law a quality registry is a structured collection of information with the primary aim to develop and safeguard the quality of care. however, once the information has been gathered, data in the registry can also be used for statistics and research. quality registries that have applied for and been granted national funding are referred to as national quality registries (nqrs). since the first nqr in sweden, the knee arthroplasty registry, was started in 1975 many nqrs have been initiated, covering many different areas of the swedish health care sector. currently (2018), there are 108 ongoing nqrs in sweden. the participation in a nqr is voluntary both for health care providers and patients, which differentiates them from health and population registries managed by government agencies like the national board of health and welfare (e.g. cause of death registry and national patient registry) and statistics sweden (e.g. on socioeconomic condition). participation in the last-mentioned registries is mandatory by law both for the health care providers and for patients. in contrast, for nqr an “opt-out” principle is used; it is assumed that the patient gives consent unless he/she does not actively say no to participation after being informed of registration in a nqr. a high-quality nqr contains detailed data on the individual patient that can be very useful for observational studies, especially if combined with data from the mandatory health registries in sweden. recently, a few nqrs have also been used for performing pragmatic randomized clinical trials incorporated in the registry (1). for a long time nqrs were underutilized for research. however, in the last decade there have been several efforts to increase the use of nqrs for research. therefore, the aim of the present paper was to describe how the use of nqrs for research has evolved over the past decade. method swedish nqrs have reported their scientific activity (number of publications per year) to the swedish association of local authorities and regions (salar) since 2009. as the use of a nqr for research has an effect on both the level of certification (a criterion-based classification system, which certifies the register as level 1–3, where 1 is the highest level) of the register and the annual grant from salar and the swedish government, it is important for the register to compile and report their list of scientific publications for the previous year. there were 69 registers in 2009 that still reported in 2016. these lists of publications, available via salar, were scrutinized in order to exclude all publications that did not qualify by being included in peer-reviewed scientific journals. titles and if necessary abstracts were checked to ensure that the paper to a significant degree was based on data from a nqr. moreover, the list was cleared from double registrations (e.g. e-publication ahead of print). to obtain the total contact bertil lindahl bertil.lindahl@ucr.uu.se department of medical sciences and uppsala clinical research center, uppsala university, uppsala, sweden. � 2018 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 1, 9–11 https://doi.org/10.1080/03009734.2018.1520761 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1520761&domain=pdf http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1520761 http://www.tandfonline.com number of publications from swedish nqrs in 2016, peerreviewed scientific publications reported by the 34 nqrs started since 2009 were added. results the yearly number of publications in peer-reviewed journals based on results from nqrs increased from 121 in 2009 to 496 in 2016 (figure 1). there was a special national campaign in order to strengthen nqrs in 2012–2016. during this campaign the number of publications increased almost three-fold (by a factor of 2.93). data from many nqrs were extensively used for research. the number of nqrs reporting 10 or more articles increased from 3 in 2009 to 17 in 2016. moreover, during the same time period the number of nqrs reporting no publications at all decreased from 37 to 12 (figure 2). at the end of 2016 another 34 nqrs had been included during the campaign period. some of them had been started recently, and still had a lot of development to do in terms of participation, coverage, and data quality. in 2016 data from these nqrs were reported in 77 articles. twenty-two of these 34 registers reported no publications at all, but a few (n ¼ 3) reported 10 or more publications. the total number of peerreviewed papers based on data from all ongoing nqrs in 2016 was 573. discussion there has been an impressive increase in the number of peer-reviewed publications from the swedish nqrs during the study period. the increase is due to the fact that the proportion of registers reporting publications, as well as the number of publications per register, has increased. unfortunately, the increased publication activity does not guarantee a corresponding increase in the quality of research. however, many papers from the nqrs were published in high-ranked journals like new england journal of medicine, jama, and the lancet (2–6). furthermore, a review of the swedish nqrs performed by the swedish agency for health and care services analysis stated that the papers based on nqrs were published in journals with higher impact factors than the average clinical research paper in sweden (7). despite this strong development, there is still room for a further increase in the use of nqr for research, since many nqrs still publish few or even no papers. there are several possible explanations for this favorable development of the quality registry-based research. firstly, during this time period a special national campaign in order to strengthen nqrs (2012–2016) took place, in which the funding of the nqrs was substantially increased. although this campaign was primarily focused on strengthening the nqr for their use in improving the quality of health care, the resulting increase in e.g. data quality and coverage has also been beneficial for research. in addition, several efforts were also aimed directly at strengthening research, e.g. an annual research meeting and increased support to the regional registry competence centers (e.g. uppsala clinical research center) for statistical competence and capacity. secondly, simultaneously, an increased interest among funding agencies to fund registry-based research has been noticed. thirdly, and most importantly, more nqrs have achieved the maturity required to enable high-quality research to be conducted. there is a substantial lag between the start of a registry and the point when it has reached high coverage and enough high-quality data have been gathered. in summary, during the last decade there has been a strong development of the quality registry-based research in sweden. however, there is still room for further increase of the use of research based on nqrs in sweden. 0 100 200 300 400 500 600 2009 2010 2011 2012 2013 2014 2015 2016 year n um be ro f p ub lis he d ar �c le s figure 1. yearly numbers of peer-reviewed publications based on swedish national quality registers (n ¼ 69) 2009–2016. 10 j. lysholm and b. lindahl disclosure statement the authors report no conflicts of interest. notes on contributors jack lysholm, md, phd, professor emeritus in orthopedics, umeå university and scientific advisor for swedish association of local authorities and regions regarding national quality registries. bertil lindahl, md, phd, professor in cardiology uppsala university and former scientific advisor for swedish association of local authorities and regions regarding national quality registries. references 1. james s, rao sv, granger cb. registry-based randomized clinical trials–a new clinical trial paradigm. nat rev cardiol 2015;12:312–6. 2. rawshani a, rawshani a, franz�en s, eliasson b, svensson am, miftaraj m, et al. mortality and cardiovascular disease in type 1 and type 2 diabetes. n engl j med 2017;376:1407–18. 3. jernberg t, johanson p, held c, svennblad b, lindb€ack j, wallentin l, et al. swedeheart/riks-hia. association between adoption of evidence-based treatment and survival for patients with st-elevation myocardial infarction. jama 2011;305: 1677–84. 4. chung sc, gedeborg r, nicholas o, james s, jeppsson a, wolfe c, et al. acute myocardial infarction: a comparison of short-term survival in national outcome registries in sweden and the uk. lancet. 2014;383:1305–12. 5. stenberg e, szabo e, ågren g, ottosson j, marsk r, l€onroth h, et al. closure of mesenteric defects in laparoscopic gastric bypass: a multicentre, randomised, parallel, open-label trial. lancet. 2016; 387:1397–404. 6. hofmann r, james sk, jernberg t, lindahl b, erlinge d, witt n, et al. oxygen therapy in suspected acute myocardial infarction. n engl j med 2017;377:1240–9. 7. myndigheten f€or vårdoch omsorgsanalys [swedish agency for health and care services analysis (vårdanalys)]. registrera flera eller analysera mera? rapport 2014:9 [register more or analyse more? report 2014:9] (in swedish). available at: www.vardanalys. se/rapporter/registrera-flera-eller-analysera-mera. 0 5 10 15 20 25 30 35 40 2009 2010 2011 2012 2013 2014 2015 2016 0 1 − 4 5 − 9 ≥10 year n um be ro f p ub lis he d ar �c le s figure 2. the swedish national quality registers were divided into four subgroups characterized by the yearly number of published peer-reviewed articles. blue bars denote no articles, brown bars 1–4 articles, grey bars 5–9 articles, and yellow bars 10 or more articles. upsala journal of medical sciences 11 http://www.vardanalys.se/rapporter/registrera-flera-eller-analysera-mera http://www.vardanalys.se/rapporter/registrera-flera-eller-analysera-mera abstract method results discussion disclosure statement notes on contributors references cancer treatment of today in view of the nobel prize commentary cancer treatment of today in view of the nobel prize the idea that we can activate our immune system to attack cancer is old. already in 1808, personal physician to louis xviii, dr alibert, tried to vaccinate himself against cancer by injecting tumour tissue. at the end of the nineteenth century, the surgeon william coley drew the conclusion from a sarcoma case that infections can counteract cancer by eliciting an immune response and started to treat cancer patients with streptococci (1). later observations—such as the notion that tumours in rare instances may undergo spontaneous regression, that immunosuppressant drugs increase the risk for cancer, and that the infiltration of t cells in the tumour goes with a better prognosis—have also indicated the important role of the immune system in cancer. however, it is not until recently that immunotherapy was established as a main treatment for cancer. indeed, research has increased our knowledge of the immune system and led to better techniques, not least for antibody production. the nobel prize in medicine 2018 is shared between james allison and tasuku honjo. checkpoint antibodies, which bind to and block inhibitory proteins on the t cells, have in a few years become an established treatment for several cancer types. allison’s research has paved the way in making the first checkpoint antibody reaching the clinic, and honjo’s research has been decisive in the development of the most commonly used checkpoint antibodies. also this year’s nobel prize in chemistry rewards discoveries that have been of great importance for the development of antibodies. george p. smith and greg winter share the nobel prize in chemistry for improving the manufacturing of antibodies through ‘phage display’. this technique means that virus infecting bacteria, bacteriophages, can be utilized to develop new proteins such as antibodies (2). winter has also developed a technique to manufacture antibodies with less mouse protein, yielding humanized antibodies. the advantages of humanized and human (no mouse protein) antibodies are that the risk for acute reaction decreases and, since they are not degraded as quickly as chimeric antibodies, their half-lives are longer. since a couple of decades antibodies are routinely used in cancer care, and new antibodies have steadily been introduced. most of them are antagonistic (blocking), non-conjugated (not coupled, naked), but a few are conjugated to a toxic substance or a radioactive compound. the majority of them are also monospecific, i.e. binding with their two arms to the same structure and predominantly to a tumour-associated antigen on the tumour cell. unconjugated antibodies can kill cancer cells in several ways. they are, after binding to the tumour cell, able to affect intracellular signal pathways and thereby induce the cell to undergo apoptosis. additionally, after binding to the tumour cell, they can by their constant parts activate receptors on immune cells, receptors on immune cells, mostly nk cells which kill the cancer cell. this mechanism is called antibody-dependent cellular toxicity (adcc). furthermore, antibodies having bound to cancer cells can activate the complement system via the classical pathway. a membrane attack complex is formed, making a hole in the cancer cell, and death is followed by complement-dependent cytotoxicity (cdc) through osmosis. yet another mechanism enhancing the anti-tumour effect is that the number of antibodies can increase by the idiotypic network. bevacizumab, which before the introduction of the checkpoint inhibitors has been the antibody used in most cancer types, does not bind to cancer cells but to vegf-a. thereby, the binding of vegf-a to vegfr1 and vegfr2 is inhibited, reducing angiogenesis in the tumour and consequently tumour growth. examples of bispecific antibodies are antibodies binding with one arm to cd3 and with the other to a tumour-associated antigen. since cd3 is found on all t cells, these antibodies can join the cancer cells with t cells, resulting in tumour death. treatment with checkpoint antibodies is recognized as specific immunotherapy even though they activate the immune system in an unspecific way. the general activation means a risk for autoimmune reactions, and the most common side effects are fatigue, colitis (diarrhoea as cardinal symptom), and skin reaction (dryness, pruritus, and exanthema). in some circumstances it is preferable to give an extra powerful treatment with two types of antibodies. it has been shown that the treatment benefit with this combination immunotherapy is greater for patients who need to stop the treatment due to side effects. if the side effects become severe, treatment with steroids is needed. it is, however, important to avoid steroid treatment as much as possible, since the anti-tumour effect otherwise may be hampered (3). the blocking of inhibitory signals in the immune system by the checkpoint antibodies leads to an activation of cytotoxic t cells. these t cells release perforin, which makes holes in the cancer cell, and granzymes entering the cell through these holes activate the caspase chain leading to cell death. the t cells themselves do not die by the attack but can carry on as serial killers of cancer cells. tumour types with many mutations, such as malignant melanoma, lung cancer, and urinary bladder cancer, respond better to treatment with checkpoint antibodies. mutations make the cell express more foreign structures, facilitating the break of tolerance to mount an effective immune response. when treating with chemotherapy, the cancer sooner or later always becomes resistant, but with the introduction of checkpoint inhibitors an increasing number of patients respond completely, i.e. complete remission is achieved. several of these patients are most likely cured, which is fantastic news in each single case. � 2018 informa uk limited, trading as taylor & francis group upsala journal of medical sciences 2018, vol. 123, no. 4, 205–206 https://doi.org/10.1080/03009734.2018.1548528 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1548528&domain=pdf https://doi.org./10.1080/03009734.2018.1548528 http://www.tandfonline.com as for other treatments within the field of oncology, a lot of effort has been made to find predictive markers for selecting the right patients for the treatment. in some cancer types the checkpoint inhibitors are, according to study results, used only in patients exhibiting a certain amount of pdl1 in their tumours. since the most commonly used checkpoint inhibitors block the binding to pdl1, it is logical that a high expression of this protein yields a better response. interestingly, there are reports indicating that intestinal bacteria are of importance for how well patients respond to treatment with checkpoint inhibitors, and studies are underway to change the microbiome of the patients to increase the response rate (4). despite the above-described advances, so far most cancer patients do not benefit from immunotherapy. however, the development in the field continues rapidly, and the treatment will soon be used routinely also in the adjuvant situation in malignant melanoma patients (5). other important lines of research are combinations of different immunotherapies (for example vaccine þ checkpoint antibody) and immunotherapy preceded by chemotherapy, which is called conditioning. to succeed with conditioning, it is important to choose the right chemotherapy in the correct dosage, and the timing is also essential. conditioning is a way to make ‘cold’ tumours ‘hot’, i.e. to make them immunogenic and thereby responsive to immunotherapy. our most common cancer type, prostate cancer, is one example of a type we hitherto have not succeeded in treating with immunotherapy, and we need to try to make this cancer ‘hot’. antibodies are usually administered intravenously, but in recent years some antibodies have been registered for subcutaneous delivery, which facilitates delivery for both patients and health care. of note, it is not easy to launch a competitor when the patent for an established antibody expires. contrary to pills which can be synthesized, antibodies are manufactured in biological systems and are categorized as biosimilars. hence, new studies are needed in man to prove acceptable toxicity and effect before a competitor is allowed for usage. we must not forget that there are other important immunotherapies that are used routinely for cancer. the most powerful immunotherapy is to change immune system, and allogeneic stem cell transplantation is a main treatment in the field of leukaemia. another type of successful immunotherapy used frequently for decades is that of bcg vaccination of superficial bladder cancer patients. another important immunotherapy is treatment with autologous t cells for malignant melanoma, although it has not as yet become part of routine practice. moreover, car t cells have been registered for treatment of lymphoma patients (6). car t cells are t cells genetically changed to produce a chimeric antigen receptor (car) partly consisting of an antibody and in part of the enhanced signal domains of the t cell receptor. thus, car t cells can, quite in contrast to normal t cells without antigen presentation, become activated to kill tumour cells. a hot research field is immunostimulating gene therapy (7). for example, a study with a virus acting as a vector for immune-stimulating genes is ongoing in uppsala. the virus is injected repeatedly intratumourally and cancer cells are destroyed (oncolysis), hopefully stimulating an immune response against the cancer that also is effective in noninjected metastases. the best is of course not having to get cancer treatment at all, and the cancer type with the fastest-growing incidence in recent decades is malignant melanoma. the advice is therefore not to go to thailand. in addition to a decrease in the risk for developing malignant melanoma, the likelihood for future patients to receive the new immunotherapy increases. the reason is that there is a risk that carbon dioxide emissions might disable modern health care, which is a prerequisite for advanced cancer treatment. in conclusion, in recent years immunotherapy has—in addition to surgery, radiotherapy, and chemotherapy— emerged as the fourth cornerstone in cancer treatment. in view of the fact that almost 100,000 swedes will be diagnosed with cancer in 2040 according to the swedish cancer society, the discoveries behind the nobel prize in medicine 2018 are particularly appreciated. at the same time, it is of utmost importance to continue our efforts in the field of research with the vision that all who are struck with this widespread disease will be cured. references 1. mccarthy ef. the toxins of william b. coley and the treatment of bone and soft-tissue sarcomas. iowa orthop j. 2006;26:154–8. 2. winter g, griffiths ad, hawkins re, hoogenboom hr. making antibodies by phage display technology. annu rev immunol. 1994;12: 433–55. 3. brahmer jr, lacchetti c, schneider bj, atkins mb, brassil kj, caterino jm, et al. management of immune-related adverse events in patients treated with immune checkpoint inhibitor therapy: american society of clinical oncology clinical practice guideline. j clin oncol. 2018;36:1714–68. 4. routy b, le chatelier e, derosa l, duong cpm, alou mt, daillere r, et al. gut microbiome influences efficacy of pd-1-based immunotherapy against epithelial tumors. science. 2018;359:91–7. 5. weber j, mandala m, del vecchio m, gogas hj, arance am, cowey cl, et al. adjuvant nivolumab versus ipilimumab in resected stage iii or iv melanoma. n engl j med. 2017;377:1824–35. 6. june ch, o’connor rs, kawalekar ou, ghassemi s, milone mc. car t cell immunotherapy for human cancer. science. 2018;359:1361–5. 7. loskog a, maleka a, mangsbo s, svensson e, lundberg c, nilsson a, et al. immunostimulatory adcd40l gene therapy combined with low-dose cyclophosphamide in metastatic melanoma patients. br j cancer. 2016;114:872–80. gustav ullenhag scientific secretary, swedish society of oncology associate professor and consultant in oncology, uppsala university hospital 206 editorial outline placeholder references vol_117_004_sups_a_686125 411..414 full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 routine intubation in the prone position klaus baer & bo nyström to cite this article: klaus baer & bo nyström (2012) routine intubation in the prone position, upsala journal of medical sciences, 117:4, 411-414, doi: 10.3109/03009734.2012.686125 to link to this article: https://doi.org/10.3109/03009734.2012.686125 © informa healthcare published online: 10 may 2012. submit your article to this journal article views: 627 view related articles citing articles: 6 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2012.686125 https://doi.org/10.3109/03009734.2012.686125 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2012.686125 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2012.686125 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2012.686125#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2012.686125#tabmodule upsala journal of medical sciences. 2012; 117: 411–414 original article routine intubation in the prone position klaus baer & bo nyström clinic of spinal surgery, löt, 64594 strängnäs, sweden abstract background. tracheal intubation in the prone position has previously been reported only as a necessity in a very few emergency situations. it emerged at our clinic as a routine after invention of a test aimed at pinpointing a painful motion segment in patients with chronic low back pain who were candidates for lumbar fusion operation. material and methods. during a 6-year period 247 consecutive patients were treated at our clinic, 91 men and 156 women, mean age 42.8 years, range 25.3–62.8. classification of the pharyngeal structures according to mallampati et al. was done the day before surgery, and grading of visualization of the glottis as described by cormack and lehane was done during intubation, with the aim of revealing factors of importance for the possibility of performing tracheal intubation in the prone position. results. the large majority of patients classified preoperatively as mallampati class 1 had cormack and lehane grade 1 at laryngoscopy, although some patients had grades 2, 3, and 4. most problems with intubation in the prone position were anticipated among those classified preoperatively as mallampati class 3, but tracheal intubation in the prone position was still possible in 21 of the 23 patients in this group. in all, tracheal intubation in the prone position was successful in 244 of the 247 patients (98.8%). conclusion. routine tracheal intubation in the prone position can be performed effectively by experienced anaesthesiologists, but this requires continuous training and good support from the anaesthesiology staff. key words: anaesthesia, endotracheal intubation, laryngoscopy, pharyngeal anatomy, prone position introduction spine surgery is almost invariably performed with the patient in the prone position. development of this position and its physiological effects and risks were recently reviewed by edgcombe et al. (1). in this planned type of surgery tracheal intubation has been performed in the supine position and the patient then turned to the prone position for the surgical procedure. the need for tracheal intubation in the prone position has been reported in only a few emergency situations (2,3), and this need has been met (2) or resolved by using a laryngeal mask (4,5). in some types of surgery in the prone position with short operation times, induction of anaesthesia and use of a laryngeal mask with the patient in the prone position facilitate the procedure, since the patient can position himself/herself comfortably (6–8). spine surgery with long operation times such as in fusion surgery is a good example in which induction of anaesthesia and tracheal intubation are normally performed in the supine position, after which the patient is turned to the prone position and positioned on a framework to minimize intra-abdominal pressure and thereby bleeding, and also possible pressure on the knees, hips, male genitals, face, and eyes. at our clinic a test aimed at pinpointing a painful motion segment in patients with chronic low back pain was invented in order to select as accurately as possible the segment that might be responsible for the patient’s back pain. after performing this test under local anaesthesia with the patient awake in the prone position and an open surgical wound in the lumbar back, we found that turning the patient to the supine position for induction of anaesthesia and intubation was time-consuming and somewhat risky regarding correspondence: bo nyström, clinic of spinal surgery, löt, 64594 strängnäs, sweden. fax: +46 152 26370. e-mail: bo.nystrom@ryggkirurgiska.se (received 8 march 2012; accepted 13 april 2012) issn 0300-9734 print/issn 2000-1967 online � 2012 informa healthcare doi: 10.3109/03009734.2012.686125 infection. the present article describes our experience using routine intubation in the prone position under these circumstances. material and methods the study represents the experience of a single centre over a 6-year period in treating a group of 247 consecutive patients (91 men and 156 women, mean age 42.8 years, range 25.3–62.8), all subjected to prone intubation after giving their informed consent. all had asa status i or ii. preoperatively the range of neck movement was tested, and the visibility of the pharyngeal structures according to mallampati et al. (9) was documented. during intubation the following day the extent of exposure of the glottis was graded according to cormack and lehane (10) in order to examine and determine what observations were important for the final goal, i.e. the possibility of performing prone tracheal intubation. open mechanical provocation test with the patient awake and in the prone position, mepivacaine + adrenaline, 5 mg/ml + 5 mg/ml, usually 15–20 ml, are administered subcutaneously over the spinal processes in the lower lumbar region. the processes are exposed and gently tapped, permitting patients to report about recognition and localization of their ordinary pain. if there seems to be a definitive localization of the patient’s pain to a specific segment, fusion of that segment is decided upon. since the patients had to be focused and as mentally clear as possible concerning the localization of their back pain, no premedication was given. anaesthesia and laryngeal intubation procedure prior to anaesthesia induction, laryngoscopes and tubes of different sizes, and stylets for use inside the tube if necessary, are at hand. the patient lies prone on the operating table with the head turned to the right. a nurse anaesthetist stands on the left side of the table to assist and support the patient’s head during induction. the anaesthesiologist stands at the head of the table and informs the patient regarding the procedure. fentanyl 0.2 mg i.v. is given as premedication. the anaesthesiologist places his/her right hand underneath the patient’s head and elevates it slightly, and the mask is applied and oxygen 50% is administered. preoxygenation continues into assisted spontaneous ventilation. blood pressure (nibp), ventilatory frequency, and oxygenation are checked continuously by pulse oxymetry. during assisted ventilation midazolam 0.2 mg/kg is given i.v. oxygen saturation as determined by pulse oxymetry should be at least 98%. when the patient is asleep and well ventilated, rocuronium bromide 0.6 mg/kg is given i. v. for relaxation. when the patient is relaxed, verified by muscle stimulation, the head is elevated by the anaesthesiologist using the right index finger around the upper molars while at the same time using the left hand to elevate and slightly extend the head dorsally. the macintosh laryngoscope with an adult blade is cautiously applied with the left hand until the right part of the tongue is pressed down and no longer visible. the epiglottis is localized, the laryngoscope adjusted, and the glottis inspected. laryngeal intubation is performed with a smaller tube at hand as well as a stylet for insertion into the tube in case of problems in getting the tube in place. if intubation in the prone position turned out to be impossible the decision to discontinue the attempt was taken at 2 minutes. the patient was manually ventilated, a sterile dressing was applied to the open surgical wound in the patient’s back, and the patient turned to the supine position for intubation. staff training in this procedure is done regularly, and an extra operating table is always on standby in case this happens. the practical performance of tracheal intubation in the prone position has been described previously in more detail (11,12). when considered necessary to facilitate laryngeal intubation, external laryngeal manipulation was applied (burp manoeuvre = backward, upward, and rightward pressure on the larynx) (13). results of the 247 patients investigated and treated during the study period, 163 were classified preoperatively as mallampati class 1. among them there was one patient in whom not even the epiglottis could be seen at laryngoscopy, cormack and lehane grade 4; in this patient intubation in the prone position was not possible (table i). sixty-one patients were preoperatively classified as mallampati class 2, and all were possible to intubate in the prone position. in 8 of the 23 patients classified preoperatively as mallampati class 3 only the epiglottis was seen, but intubation in the prone position was still possible. in another four not even the epiglottis could be seen at laryngoscopy. it was nevertheless possible to perform intubation in the prone position in two of them by using the burp manoeuvre (table i). thus in 3 out of the 247 patients intubation in the prone position was not possible (1.2%). intubation was always completed within a maximum of 2 minutes following rocuronium injection. extubation after surgery was usually performed in the prone position when the patient was awake and 412 k. baer & b. nyström breathing spontaneously. no systematic difference was noted between men and women concerning difficulties during this intubation procedure. there were no complications from the procedure, although one patient sustained a tooth injury. discussion in ordinary clinical work there is seldom a need for tracheal intubation in the prone position, but in extreme emergency situations such a need may arise (2,3). in these situations fibre-optic intubation may be considered, but this technique requires the patient’s co-operation, special equipment, and extensive training as noted by van zundert et al. (2). these authors tried fibre-optic intubation without success in a patient with a traumatic thoracic injury, but handled the situation using direct laryngoscopy in the prone position. although it was found during this study that preoperative inspection of the patient’s pharyngeal structures was of value in preparing for the intubation, there were exceptional patients with mallampati class 1 who were classified at laryngoscopy as grades 3 and 4 according to cormack and lehane (10). such a discrepancy between visibility of the pharyngeal structures and the findings at laryngoscopy was also noted by charters et al. (14), who abandoned oral inspection as a useful tool for predicting difficult tracheal intubation. in previous studies the sensitivity of the mallampati test has been found to vary from 33 to 84, and the specificity from 65 to 89 (15–19). in the original study by mallampati et al. (9) the figures were 50 and 99.5, respectively, and in our study they are 42.8 and 94.9, respectively. the reason for these differences is probably differences between samples of patients as well as differences in evaluations of the mallampati and cormack and lehane scales. differences are also reported concerning whether or not mallampati class 2 should predict a difficult intubation (15). although the test has a low sensitivity and specificity, there is no doubt that most problems with tracheal intubation occur in patients with less visualization of the pharyngeal structures, as seen in our study and also previously in larger studies with patients in the supine position (20,21). among the 247 patients in our study, failure to perform intubation in the prone position occurred in only 3 (1.2%), which is markedly higher than the incidence of failed tracheal intubations in the supine position (22) but nevertheless very low. the present study shows that laryngeal intubation in the prone position can be performed almost as well as in the supine position in young and middle-aged patients. however, continuous training and good support from the anaesthesiology staff are necessary. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. edgcombe h, carter k, yarrow s. anaesthesia in the prone position. br j anaesth. 2008;100:165–83. 2. van zundert a, kuczkowski km, tijssen f, weber e. direct laryngoscopy and endotracheal intubation in the prone position following traumatic thoracic spine injury. j anesth. 2008; 22:170–2. 3. santos ia, oliveira ca, ferreira l. life-threatening ventilatory obstruction due to a defective tracheal tube during spinal surgery in the prone position. anesthesiology. 2005;103: 214–15. 4. valero r, serrano s, adalia r, tercero j, blasi a, sanchezetayo g, et al. anesthetic management of a patient in prone position with a drill bit penetrating the spinal canal at c1-c2, using a laryngeal mask. anesth analg. 2004;98:1447–50. 5. raphael j, rosenthal-ganon t, gozal y. emergency airway management with a laryngeal mask airway in a patient placed in the prone position. j clin anesth. 2004;16:560–1. 6. ng a, raitt dg, smith g. induction of anesthesia and insertion of a laryngeal mask airway in the prone position for minor surgery. anesth analg. 2002;94:1194–8. 7. brimacombe jr, wenzel v, keller c. the proseal laryngeal mask airway in prone patients: a retrospective audit of 245 patients. anaesth intensive care. 2007;35:222–5. 8. weksler n, klein m, rozentsveig v, weksler d, sidelnik c, lottan m, et al. laryngeal mask in prone position: pure exhibitionism or a valid technique. minerva anestesiol. 2007;73:33–7. 9. mallampati sr, gatt sp, gugino ld, desai sp, waraksa b, freiberger d, et al. a clinical sign to predict difficult tracheal intubation: a prospective study. can anaesth soc j. 1985;32: 429–34. 10. cormack rs, lehane j. difficult tracheal intubation in obstetrics. anaesthesia. 1984;39:1105–11. 11. baer k. är det mycket svårare att intubera i bukläge? läkartidningen. 1992;89:3657–60. 12. baer k. endotracheale intubation in bauchlage. fortschr anästh. 1992;6:30–2. table i. correlation between mallampati grading of pharyngeal structures (see text) seen before laryngoscopy and visualization of the glottis according to cormack and lehane at intubation in 247 consecutive patients undergoing spinal fusion surgery. cormack and lehane grade mallampati class g 1 g 2 g 3 g 4 total c 1 137 23 2 1a 163 c 2 22 26 13 0 61 c 3 3 8 8 2 + 2a 23 total 162 57 23 5 247 apatients in whom intubation in the prone position was not possible. routine intubation in the prone position 413 13. ulrich b, listyo r, gerig hj, gabi k, kreienbuhl g. die schwierige intubation. der nutzen von burp und die aussagekraft von prädiktoren. anaesthesist. 1998;47: 45–50. 14. charters p, perera s, horton wa. visibility of pharyngeal structures as a predictor of difficult intubation. anaesthesia. 1987;42:1115. 15. pottecher t, velten m, galani m, forrler m. valeur comparée des signes cliniques d’intubation difficile chez la femme. ann fr anesth réanim. 1991;10:430–5. 16. savva d. prediction of difficult tracheal intubation. br j anaesth. 1994;73:149–53. 17. laplace e, bénéfice s, marti flich j, patrigeon rg, combourieu e. intubation difficile: évaluation prospective des tests de mallampati et de wilson. cahiers d’anesth. 1995;43:205–8. 18. tse jc, rimm eb, hussain a. predicting difficult endotracheal intubation in surgical patients scheduled for general anesthesia: a prospective blind study. anesth analg. 1995; 81:254–8. 19. raouf el-ganzouri a, mccarthy rj, tuman kj, tanck en, ivankovich ad. preoperative airway assessment: predictive value of a multivariate risk index. anesth analg. 1996;82: 1197–204. 20. cohen sm, laurito ce, segil lj. oral exam to predict difficult intubations: a large prospective study. anesthesiology. 1989;71:a937. 21. samsoon glt, young jrb. difficult tracheal intubation: a retrospective study. anaesthesia. 1987;42:487–90. 22. benumof jl. management of the difficult adult airway. with special emphasis on awake tracheal intubation. anesthesiology. 1991;75:1087–110. 414 k. baer & b. nyström swedish guidelines for registry-based randomized clinical trials article swedish guidelines for registry-based randomized clinical trials karl nyberg and peter hedman uppsala clinical research center, uppsala university, uppsala, sweden abstract during the last decade sweden has invested in a national infrastructure for collection of structured clinical data in the form of healthcare registries (in sweden known as kvalitetsregister). these data can be combined with other public data using the national personal identifiers that are issued to swedish citizens. the healthcare registries have an almost complete coverage of swedish healthcare, and a large network of clinicians is involved in the quality assurance and continuous improvement of healthcare using these registries. uppsala clinical research center (ucr) has been a technology provider of large-scale national registries and has a strong background in clinical trial management. this effort combines the areas of healthcare registries and clinical trials into a novel way of performing clinical trials to be able to: 1) run clinical trials as an integrated part of normal clinic workflow; and 2) leverage the nationwide network of outcome reporting. this strategy was shown to be successful in the taste (thrombus aspiration in myocardial infarction) study. when taste had been published, the new england journal of medicine wrote a perspective on the study calling it ‘the randomized registry trial—the next disruptive technology in clinical research?’ since then several studies have been conducted in this way with great success. ucr has been appointed, by clinical studies sweden and the swedish research council, to develop the swedish national guidelines for registry-based randomized clinical trials in order to ensure the possibility for more organizations to run this kind of study. this paper describes key concepts of register-based randomized clinical trials and the development of swedish national guidelines. article history received 17 june 2018 revised 16 november 2018 accepted 16 november 2018 keywords cost efficiency; healthcare registries; national guidelines; register-based randomized clinical trial; taste study registry-based randomized clinical trials— key benefits introduction a prospective trial contains several important elements that are necessary for its successful performance and completion: identification of eligible patients, obtaining consent, randomization, collection of baseline variables, endpoint detection, and endpoint adjudication. a clinical registry can be used for most of these aspects of a trial. by including the possibility of randomization in a clinical quality registry, it is possible to combine some of the critical attributes of a prospective randomized trial with the practical features of a large-scale clinical registry including the key strength of minimally selected consecutive enrollment and automated patient identification and follow-up. we describe a prospective randomized trial linked to a registry as a prospective registry-based randomized clinical trial (rrct) by way of analogy with a prospective randomized clinical (or controlled) trial (rct). the rrct is an efficient and effective mechanism to assess hard clinical endpoints in large patient cohorts. hitherto, registry-based randomized trials have evaluated treatment strategies and devices or simple pharmaceutical agents, but there is no strict limit to what therapy can be evaluated with the registry link, as long as patient safety is assured and there is adherence to existing regulations. registry-based rcts with more efficient and streamlined trial conduct may also be possible for evaluation of approved pharmaceutical agents for new indications and labels. the benefit of the rrct is related to the ability to identify patients, enroll larger numbers of patients in relatively short time, and a possibility of indefinite follow-up. this together with the fact that an rrct often is much more inexpensive than an ordinary rct has made the rrct renowned on a global scale. evidence-based selection of inclusion criteria the registry data collections provide a unique reference set when deciding on suitable inclusion criteria for the clinical trial. statisticians work closely with investigators to infer the optimal balance between population size and event rates based on the large body of historical data in the registries. in-registry enrollment of patients enrollment of patients to the study can be done in the registry application. this inclusion can be based on reported patient demographics and medical background available in contact peter hedman peter.hedman@ucr.uu.se uppsala clinical research center, uppsala university, uppsala, sweden. � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 1, 33–36 https://doi.org/10.1080/03009734.2018.1550453 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1550453&domain=pdf http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1550453 http://www.tandfonline.com the registry, like age, sex, comorbidity, previous trial participation, etc. the inclusion can also be extended with trial-specific data that form a prerequisite for the enrollment of e.g. informed consent and specific laboratory analyses. in-registry randomization to strategy randomization to a treatment strategy can be done in the registry for rrcts. this is typically done either prior to or during an intervention. in-registry randomization can be achieved when the registry is used for reporting prior to the treatment strategy selection, for instance during an intervention in the operating room. the randomization can be stratified (e.g. by site) and be tailored to support any number of strategies. distributed clinical outcome reporting endpoints and specified events can be designed to be automatically collected from the normal reporting workflow in the registry. given the nationwide adoption of the registries, this means that any site can provide outcome reporting for a trial participant as a part of routine follow-ups following the procedure. a common endpoint like death is required to be reported to the population registry within 48 h, and this can be automatically added to the trial data set to have a current status on safety and lost-to-follow-up. in the case where the data normally captured during follow-ups are not enough for the trial objective, it is possible to add clinical trial-specific question panels to the registry follow-up forms. long-term follow-up sometimes the trial needs to be continued outside the temporal scope of workflow in the registry. this can be achieved by feeding a separate ecrf with the clinical trial data model data and use this system for the long-term follow-up. given that the participant is properly identified in the study data model, it is possible to interlink the study database with the registry or other sources at a later stage to add data to the study. continuous adjudication of clinical events as clinical outcome reporting is distributed and happens in conjunction with the event, adjudication of the event can be done continuously during the trial. this means that the trial duration can be shortened, as the adjudication is performed while the trial progresses. it also means that a data and safety monitoring board report can be kept current and validated during the clinical trial. any specified event that has to be adjudicated can be automatically sent to a clinical events committee (cec) group along with the personal identifier, site identifier, date, and other required information. the cec group can then handle the collection of data and adjudication in their normal workflow. registry-based randomized clinical trial framework uppsala clinical research center (ucr) has developed a framework for registry clinical trials as an initiative to standardize and facilitate implementation of clinical trials in registries. the framework specifies the domain of clinical trials and defines business rules for legal compliance. the building blocks of the framework are combined to support the clinical trial at hand and coupled to the registry after being validated against the study protocol. the clinical trial implementation is treated as a stand-alone application which shares life-cycle with the registry. this architecture handles the incompatible legal requirements between clinical trials and registries with regard to patient/participant opt-out. it is important to note that the framework is not the same as a product and that there is a need for systems developers to implement the framework with the registry. clinical trial data model at the heart of the rct is the clinical trial data model. this is an extension of the traditional clinical report form. the clinical trial data model is a specification of the variables that are required for the inclusion and exclusion of patients as well as the ones needed for analysis of the primary and secondary endpoints. the clinical trial data model is: � defined in the study protocol � a basis for the data management plan � a part of the ethical approval application this means that the data model should contain all data that are required to analyze the outcome of the trial. this in turn means that the study data set can be handled separately from the registry data to provide support for the different legal requirements on registries and clinical trials. any change to the study database that handles the clinical data set defined by the clinical data model is fully audited. electronic data capture as an implementation of the data model the study can be performed using an electronic data capture system (edc) as the backend for the rrct framework. the clinical trial model is then implemented as ecrfs in the edc. the framework provides a seamless data transfer from the registry to the edc, which has built-in functions for data quality monitoring and on-line reporting to continuously monitor the integrity of the data. 34 k. nyberg & p. hedman this architecture also allows for hybrid studies where some of the sites provide data through the registry and some through the ecrfs in the edc. clinical trial criteria a number of criteria can be configured for key features in the study. inclusion of patients. the framework provides the formal requirements for defining inclusion criteria for patients. opening and closing a trial. the framework provides support for defining criteria for the conditions where the trial accepts inclusion of participants. this can for instance be a time interval, a maximum number of participants, or based on available stratification by site. patient opt-out. the framework supports participant withdrawal from the trial. this means that the record for the participant cannot accept new data once the opt-out has been reported. this requirement is built into the data model handler in the framework. lost-to-follow-up. in the event that a participant cannot be contacted for follow-up, the participant might have to be marked for exclusion from the trial. this requirement is built into the data model handler in the framework. randomization service the framework provides support for a randomization service that randomizes participants to a treatment strategy. this includes configurations like stratification by site and sex. the stratification and randomization can be pre-compiled for the study. data capture two different strategies are currently employed for data capture. event listeners. the framework defines event listeners that can be configured to eavesdrop on the information flow in the registry. this generates events that are sent to the clinical trial data model. this can be used for endpoints that are designed to use existing variables in the registry. in-registry trial panels. in-registry trial panels can be used to add trial-specific data capture to existing registry forms. this can also be used to add branding to the clinical trial, enroll a participant, and provide the possibility for the user to randomize a participant to a treatment strategy. reporting the framework has an export function to export the study data at the end of the study. it is also possible to review the screening log during the study. swedish national guidelines in 2017 uppsala clinical research center was appointed by swedish clinical studies and the swedish research council to develop sweden’s national guidelines for rrcts. the purpose was to use existing knowledge and technology in order to spread the use of rrcts and to give everyone a head start instead of having to invent processes and technology themselves. the objective is to have the guidelines publicly available for everyone who wants to run a rrct. the guidelines consist of documentation on how the process works and how it differs from a rct. it also contains the different roles involved and when one should, and should not, use rrct. the guidelines also contain the actual technical framework together with documentation on how it works and how to implement it. the guidelines are available to use and adapt to a specific study and technical environment. the guidelines can be used freely, but appropriate credit must always be given. (the national guidelines for randomized registry-based trials are available from the regional node for clinical studies or the regional office of the national registry center.) case study: thrombus aspiration in myocardial infarction (taste) taste study details are given in table 1. objective treatment of myocardial infarction (blood clot in the arteries of the heart) has improved after introduction of 24/7 balloon angioplasty to open the blocked artery. the clot itself is not routinely removed, but recent data in smaller trials indicate that this might improve recovery and prognosis. in this multicenter study of 7000 patients referred to scandinavian hospitals for myocardial infarction, the investigators test the hypothesis that patients randomized to treatment with thrombus aspiration (removing the blood clot by manual suction) before conventional angioplasty will have a reduced risk of death as the primary outcome, and fewer rehospitalizations, fewer new myocardial infarctions, reduced risk of heart failure, better coronary artery flow after angioplasty, and greater reduction of infarct size compared to patients randomized to conventional angioplasty alone. the study was run using the swedeheart registry from 27 sites in sweden, iceland, and denmark (1,2). the registry helped identifying patients suitable for inclusion by selecting those with a swedish personal identification number above the age of 18 with stemi, registered with an indication for pci, and no previous enrollment. when the system proposed table 1. thrombus aspiration in myocardial infarction (taste). taste study details enrollment 7243 study start date july 2010 study completion date august 2013 primary completion date march 2013 clinicaltrials.gov nct01093404 upsala journal of medical sciences 35 enrollment, the treating physician only had to check a few exclusion criteria and get verbal consent from the patient. the ethics committee requested a written confirmation of the verbal consent as soon as possible after the procedure. randomization was performed directly in the registry clinical report form. no additional study variables were required for follow-up of study endpoints. all endpoints were automatically collected from national registries. technical implementation to support the taste study swedeheart was complemented with the possibility to randomize the treatment strategy and to enroll participants. randomization was stratified by site. addition of study sites was performed by making the randomizations available to the sites. endpoints were inferred from the variables in the registry as a part of the study analysis. declaration of interest the authors report no conflicts of interest. note on contributors karl nyberg is an information technology consultant and systems architect with a msc in biotechnology engineering. peter hedman is a director of digital r&d with a ma in mediaand communications. references 1. fr€obert o, lagerqvist b, olivecrona gk, omerovic e, gudnason t, maeng m, et al. thrombus aspiration during st-segment elevation myocardial infarction. n engl j med. 2013;369:1587–97. 2. lagerqvist b, fr€obert o, olivecrona gk, gudnason t, maeng m, alstr€om p, et al. outcomes 1 year after thrombus aspiration for myocardial infarction. n engl j med. 2014;371:1111–20. 36 k. nyberg & p. hedman abstract registry-based randomized clinical trialskey benefits introduction evidence-based selection of inclusion criteria in-registry enrollment of patients in-registry randomization to strategy distributed clinical outcome reporting long-term follow-up continuous adjudication of clinical events registry-based randomized clinical trial framework clinical trial data model electronic data capture as an implementation of the data model clinical trial criteria randomization service data capture reporting swedish national guidelines case study: thrombus aspiration in myocardial infarction taste objective technical implementation declaration of interest note on contributors references lars grimelius and his silver impregnation method—commentaries on the paper in upsala journal of med full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 lars grimelius and his silver impregnation method—commentaries on the paper in upsala journal of medical sciences with the highest number of citations per westermark to cite this article: per westermark (2015) lars grimelius and his silver impregnation method—commentaries on the paper in upsala�journal�of�medical�sciences with the highest number of citations, upsala journal of medical sciences, 120:2, 113-116 to link to this article: https://doi.org/10.3109/03009734.2015.1023910 © informa healthcare published online: 17 mar 2015. submit your article to this journal article views: 457 view related articles view crossmark data https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://doi.org/10.3109/03009734.2015.1023910 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1023910 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1023910 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1023910&domain=pdf&date_stamp=2015-03-17 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1023910&domain=pdf&date_stamp=2015-03-17 upsala journal of medical sciences. 2015; 120: 113–116 lars grimelius and his silver impregnation method—commentaries on the paper in upsala journal of medical sciences with the highest number of citations per westermark department of immunology, genetics and pathology, uppsala university, uppsala, sweden key words: amyloidosis, diabetes, hormones, pathology citation counting is a comparably new procedure that has become widely used to evaluate single papers but also to rank journals by the so-called impact factor (if), often determined to the third decimal. this is a phenomenon of the information society and therefore only reliable for later years. however, we can go back to the 1960s. when doing so for upsala journal of medical sciences, there is one paper which has been cited much more than any other, and that is lars grimelius’s ‘a silver nitrate stain for a2 cells in human pancreatic islets’, published in the journal in 1968 (1). according to the thomson reuters database it has been cited 897 times to date. none of the journal’s other papers is even close to that record. the value would have been even higher if the method had not been incorporated in many laboratories as a routine staining for which a citation was regarded as unnecessary. silver impregnation (‘staining’) has been used in histology and histopathology for a long time and was initially particularly developed for studies of nervous structures. like in photography, the methods utilize reduction of a silver salt to inorganic silver or silver oxide which is deposited on specific structures. subsequently, it was found that silver techniques were able to visualize certain specific cells outside the nervous system. several methods had been developed that turned out to be useful for the studies of the endocrine pancreas. one often-used technique was the gross–schultze method, which was extensively studied by grimelius’s and my teacher professor gösta t. hultquist (2). the gross–schultze technique was later severely questioned, however, since it was found not to be completely specific and small changes in the staining protocol resulted in other impregnation patterns (3). when this story started, the most frequent pancreatic islet cells were basically defined and included b-cells, a1 (d), and a2 (a) cells. b-cells were known to produce insulin, and a2 cells were supposed to produce glucagon, while the function of a1 was unknown. in addition to the gross–schultze technique there were a couple of other silver methods which were often employed for pancreatic studies. one was the silver proteinate method, originally developed by bodian (4) and further modified by several investigators including lars grimelius (5). another silver impregnation method was that of davenport, modified by hellerström and hellman in uppsala (6). this last method was found to discriminate between two populations of non-b-cells shown to include a1 and a2 cells. when lars grimelius (figure 1) entered the scene the importance of islet composition in the pathogenesis of diabetes (type 1 and type 2 were not clearly defined at that time) was still somewhat unclear. a number of trials to determine the total mass of the different islet cells had produced controversial results (for review, see warren et al. (7)). the professor who recruited lars grimelius, the above-mentioned gösta hultquist, who preferred to be called hqt, had a long history of studies of islets of langerhans. hqt was a brilliant pathologist, particularly in anatomical pathology, but his heart beat more strongly for experimental pathology. he used a technique to ligate correspondence: per westermark, rudbeck laboratory, se 751 85 uppsala, sweden. e-mail: per.westermark@igp.uu.se (received 11 february 2015; accepted 20 february 2015) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1023910 http://informahealthcare.com/journal/ups mailto:per.westermark@igp.uu.se the rat pancreatic duct in order to induce atrophy of the exocrine but not endocrine tissue. such tissue was then transplanted into the anterior chamber of diabetic or control rats (8). it was possible by such means to study effects of diabetes and of antidiabetic drugs (9). we should remember that when lars grimelius started his career at the department of pathology at uppsala university, in the 1950s, there were no graduate student programmes, no graduate courses, and not necessarily any projects to start with. rather, there was a demand for the students themselves to find a suitable problem to solve and also find useful methods for the task. at best there could be a suggestion of a broad problem to study— not so easy for an inexperienced student. luckily, for a student in pathology there were a limited set of methods to use, and most of them were based on light microscopy. the time for writing a doctoral thesis was not restricted, and it could sometimes take a decade or more. it was suggested to grimelius that he should study the effects of a new antidiabetic drug on the rat pancreas. i can already here unveil that this study was never accomplished. instead of taking up the suggested problem, grimelius (a methodical person) started to evaluate different staining methods for islet cells. the mentioned silver proteinate method gave very nice results. however, the supply of the chemical at the department was limited, and therefore grimelius ordered the substance from 10 different suppliers. only one of the received substances gave satisfactory results. grimelius therefore ordered 1 kg of this specific preparation but got the response from the company that they had tried to produce more but results were unreliable. this sad fact led grimelius to develop his so successful silver impregnation method. like many advances in science, this one sprang from a failure. against this background lars grimelius decided at least to try to develop a method based only on simple, wellcontrolled chemicals. since there was no firm biochemical knowledge on how silver impregnation methods worked or what exactly they demonstrated, development of a procedure was basically empiric. that meant a lot of experimental work. lars grimelius identified the following variables: concentration of silver salt, concentration of reduction agent, ph, temperature, duration and fixation of tissue, and, with human material, autolysis. it is easy to understand that combinations of all these variables can create an almost endless number of possibilities. grimelius, of course, was assisted in performing sections of all tissue materials needed, but he carried out the mixing of solutions, the incubations, mountings, and evaluations himself, without assistance. after years of work, a method finally saw the light of day. this was about the time when i came to the department as a young and enthusiastic medical student with an intention to start with a research project. grimelius was about to finish the laboratory work, and it was time to write the papers. part of that time i came to share an office with him. the room was a somewhat historical one since it had been the office of probably the most famous pathologist in uppsala, robin fåhraeus (1888–1968), and it had still some of his furniture and fixtures, including a large hibiscus and a green sofa on which grimelius lay down and slowly wrote his papers with tiny handwriting with a short oldfashioned pencil. i also remember the frequent use of an eraser. now and then he went out to the laboratory to do some late checking experiments. above us both, a copy of a greek (or roman?) sculpture was looking down. i am looking back at this time through a nostalgic mist, but the situation in the laboratory was certainly very relaxed. all this writing took its time for grimelius. there was no real feeling of hurry since there was no fixed time for a thesis work, and today’s severe competition was unknown. funding was safe through money directly allocated to the department. we should also remember that there were no computers, so all text and the large number of figure 1. lars grimelius on a tour in swedish lapland together with the author in august 1969. 114 p. westermark tables had to be written and re-written on a typewriter. finally, two meticulous papers describing and evaluating the grimelius silver impregnation method were finished, and it was time for submission. the method manuscript was sent to the journal stain technology, which was the journal that most often published new methods. the journal, however, unexpectedly rejected the paper. this was, as it turned out, a big mistake by the editor. what to do then? luckily, there was acta societatis medicorum upsaliensis, later renamed to upsala journal of medical sciences, a journal very often used for doctoral thesis papers. lars grimelius has told me how he submitted the papers there. he went to the editor, gunnar ågren, professor of medical biochemistry, who inspected them and asked, ‘has professor hultquist looked at them?’ the answer was yes, and then the papers were accepted. the first paper, ‘a silver nitrate stain for a2 cells in human pancreatic islets’ (1), which is the description of the method, rapidly became used and has been frequently cited, until now 897 times according to the thomson reuters database. it became a ‘citation classic’ in 1984. it is obvious that the rejection by stain technology (present impact factor 1.0) was a big mistake, as it became the most cited of any single papers in upsala journal of medical sciences (present impact factor 1.71). grimelius later got his revenge when he was invited to contribute with a review article on silver staining methods for the journal stain technology, now renamed to biotechnology and histochemistry (10). lars grimelius later got a second ‘citation classic’ with the paper that shows that pancreatic a1 cells (d cells) store somatostatin (11). the second paper in grimelius’s thesis (12) contains all control experiments with a great number of variables. included in the doctoral thesis, defended in the spring of 1969, was also an electron microscopic study of the distribution of silver particles in labelled cells. the electron microscope, a zeiss em9, had been bought a few years earlier, partially with money raised by the thoracic surgeon viking olov björk, who wanted hqt to study heart tissue electron microscopically, which he also did (13). grimelius showed that silver particles were localized to secretory granules and more specifically in the electron-lucent peripheral area (14). this was an interesting observation, since while the electron-dense area contains glucagon, other granule components are present in the translucent periphery. among these is chromogranin a, which is one of the substances that have been found to be responsible for the argyrophilic properties of endocrine cells (15,16). other components are certainly of importance as well (15). i had much benefit both from the use of the silver method and from grimelius himself for one of the papers in my own thesis (17). examples of the grimelius silver impregnation are shown in figure 2. lars grimelius widened the use of his own as well as other silver staining methods to other areas in endocrine anatomy and pathology. grimelius’s stain turned out to visualize a number of cells, e.g. in gastrointestinal tract and parathyroid and thyroid glands, and it played an important role in the mapping of such cells (figure 2) a b c d figure 2. examples of grimelius stain, all performed in 1972. a: normal human islet of langerhans. b: human islet of langerhans with large deposits of amyloid. most remaining cells are a2 (a) cells. (patient with type 2 diabetes.) c: part of adrenal gland. cells in medulla are argyrophilic, while cortical cells are unlabelled. (same case as a.) d: section from anterior pituitary. one population of cells is argyrophilic. (same case as a). grimelius’s silver staining method 115 (18,19). the grimelius silver impregnation method turned out to be very useful for discrimination of neuroendocrine tumours from other neoplasms. grimelius was wise to understand that he should widen his methodological arsenal, and in 1974–1975 he was guest scientist in the laboratory of professor a. g. everson pearse at hammersmith hospital, london. pearse was a pioneer in histochemistry, well-known to everyone working in histopathology. this started a long period of collaboration and friendship with pearse and with julia m. polak, who succeeded as professor at the department. during his stay in london, grimelius learnt immunohistochemistry, which was under development, and brought the technique back to sweden. he then turned more and more towards endocrine tumour pathology, employing immunohistochemistry, and started a new career in clinical pathology. but that is another story. declaration of interest: the author reports no conflicts of interest. the author alone is responsible for the content and writing of the paper. references 1. grimelius l. a silver nitrate stain for alpha2 cells in human pancreatic islets. acta soc med upsal. 1968;73:243–70. 2. hultquist gt, dahlen m, helander cg. über die technik darstellung und zählung der sogen. silberzellen in den langerhansschen inseln. schweiz zeitschr path bakt. 1948;11:570–89. 3. creutzfeldt w, theodossiou a. die relation der aund b-zellen in den pankreasinseln bei nichtdiabetikern und diabetikern. beitr pathol anat. 1957;117:235–52. 4. bodian d. a new method for staining nerve fibres and nerve endings in mounted paraffin sections. anat rec. 1936;65:89–97. 5. grimelius l. a modified silver protein method for studying the argyrophil cells of the islets of langerhans. in brolin se, hellman b, knutsson h, editors. the structure and metabolism of pancreatic islets. oxford: pergamon press: 1964. p. 99–104. 6. hellerström c, hellman b. some aspects of silver impregnation of the islets of langerhans in the rat. acta endocrinol (copenh). 1960; 35:518–32. 7. warren s, lecompte pm, legg ma. the pathology of diabetes mellitus. 4th ed. philadelphia: lea & febiger; 1966. 8. hultquist gt. the ultrastructure of pancreatic tissue from duct-ligated rats implanted into anterior chamber of rat eyes. ups j med sci. 1972;77:8–18. 9. grimelius l, hultquist gt, thorell j, winbladh l. studies on islet tissue transplants in the anterior chamber of the eye in rats. in brolin se, hellman b, knutsson h, editors. the structure and metabolism of the pancreatic islets. oxford: pergamon press; 1964. p 173– 8. 10. grimelius l. silver stains demonstrating neuroendocrine cells. biotech histochem. 2004;79:37–44. 11. polak jm, pearse ag, grimelius l, bloom sr. growth-hormone release-inhibiting hormone in gastrointestinal and pancreatic d cells. lancet. 1975;1:1220–2. 12. grimelius l. the argyrophil reaction in islet cells of adult human pancreas studies with a new silver nitrate procedure. acta soc med ups. 1968;73:271–94. 13. björk vo, hultquist g. ultrastructural, enzyme histochemical and light microscopic investigation of the myocardium in cases undergoing open-heart surgery. scand j thorac cardiovasc surg. 1967;1:27–41. 14. grimelius l. an electron microscopic study of silver stained adult human pancreatic islet cells, with reference to a new silver nitrate procedure. ups j med sci. 1969;74:28–48. 15. lundqvist m, arnberg h, candell j, malmgren m, wilander e, grimelius l, et al. silver stains for identification of neuroendocrine cells. a study of the chemical background. histochem j. 1990;22:615–23. 16. cetin y. chromogranin a immunoreactivity and grimelius’ argyrophilia. a correlative study in mammalian endocrine cells. anat embryol (berl). 1992;185:207–15. 17. westermark p, grimelius l. the pancreatic islet cells in insular amyloidosis in human diabetic and non-diabetic adults. acta pathol microbiol scand a. 1973;81:291–300. 18. grimelius l, wilander e. silver stains in the study of endocrine cells of the gut and pancreas. invest cell pathol. 1980;3:3–12. 19. delellis ra, balogh k. histochemical characteristics of parafollicular cells and medullary thyroid carcinoma. am j pathol. 1973;72:119– 28. 116 p. westermark http://www.ncbi.nlm.nih.gov/pubmed/4185638?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/18121801?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/18121801?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/13471461?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/4559613?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15223752?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/48838?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/4185639?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/6078790?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/6078790?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/4187673?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/4187673?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/1705926?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/1705926?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/1349463?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/1349463?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/4129056?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/6156147?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/4124194?dopt=abstract ss1 declaration of interest references complete response with combined braf and mek inhibition in braf mutated advanced low-grade serous ovarian carcinoma case report complete response with combined braf and mek inhibition in braf mutated advanced low-grade serous ovarian carcinoma bengt tholandera,b, anthoula koliadia,b, johan botlingb, hanna dahlstranda,b, anne von heidemana,b, håkan ahlstr€omc, kjell €obergd,e and gustav j. ullenhaga,b adepartment of oncology, uppsala university hospital, uppsala, sweden; bdepartment of immunology, genetics and pathology, science for life laboratory, uppsala university, uppsala, sweden; cdepartment of surgical sciences, uppsala sweden, division of radiology, uppsala university hospital, uppsala, sweden; ddepartment of oncologic endocrinology, uppsala university hospital, uppsala, sweden; edepartment of medical sciences, uppsala university, uppsala, sweden abstract more effective treatments are needed for low-grade serous ovarian carcinoma (lgsoc). our patient, who suffers from metastatic lgsoc, had received all established treatments. sequencing analysis revealed an activating braf mutation. therefore, combined treatment with braf and mek inhibitors, which is the gold standard in malignant melanoma, was initiated. after eight months of therapy, the response was assessed as complete and the treatment is still, 3.5 years after initiation, of benefit. to our knowledge, no complete response on combined braf and mek inhibitor treatment of low-grade serous ovarian cancer has previously been reported. article history received 10 august 2020 revised 14 september 2020 accepted 16 september 2020 keywords braf inhibitor; chemotherapy; low-grade serous ovarian cancer; mek inhibitor; next-generation sequencing; surgery; targeted therapy; v600e mutation introduction low-grade serous ovarian carcinoma (lgsoc) is a less common subtype, affecting around 5% of all patients with epithelial ovarian cancer (1). however, in contrast to high-grade serous ovarian carcinoma (hgsoc), lgsoc more often affects young, fertile women. low-grade serous ovarian carcinoma has also different clinical and molecular biologic characteristics and course of disease, compared to hgsoc. primary surgery is standard of care, and is often curative in lgsoc, but both early and late relapses are common. in stages ic–iv, postoperative platinum-based chemotherapy is recommended, but the response is often poor, especially at relapse (2). repeated surgeries with curative or palliative intent are regularly needed. however, the course of disease may be indolent, with slow progression over many years, and with periods with stable disease even without treatment. hormonal therapy may be effective (3). nevertheless, therapy resistance, disease generalization, and progression often finally lead to death. there is need for more effective treatments for patients with lgsoc, possibly individually tailored, molecular biology-driven targeted therapies (4). mutations in braf, kras, and nras genes have been detected in lgsoc (4). in serous borderline tumours, the premalignant form preceding lgsoc, braf mutations are present in almost half of the cases (5). in hgsoc, braf mutations are rare (6), while the reported frequency of braf mutations is 5–14% in lgsoc (5,7). treatment with a mek inhibitor alone has been compared to chemotherapy in a clinical randomized trial in lgsoc patients regardless of braf status. a median progression-free survival (pfs) of 13 months, compared to 7 months for the group receiving physician’s choice, was achieved (8). in a phase 3 study (mek inhibitor in low-grade serous ovarian cancer, milo), patients with lgsoc were randomized to treatment with the mek inhibitor binimetinib or chemotherapy. there were no significant differences for the primary endpoint pfs (9). a few cases with responses on braf inhibitor treatment have been reported in ovarian cancer (7). furthermore, long-term survival has been reported in a patient with lgsoc treated with the braf inhibitor vemurafenib (10). in addition, there are a few recent reports on braf and mek inhibitor combination therapy in patients with lgsoc (6,11). combination treatment with braf and mek inhibitors is recommended in patients with braf mutated malignant melanoma. combining dabrafenib and the mek inhibitor trametinib or the braf inhibitor encorafinib with the mek inhibitor binimetinib results in impressive and durable responses and also prolonged survival in patients with metastatic melanoma (12–14). one might assume that all patients with braf mutant cancer would benefit from treatment with braf inhibitors. however, colon cancer patients harbouring the braf oncogenic lesion have a poor prognosis and do not respond to contact bengt tholander bengt.tholander@telia.com department of oncology, uppsala university hospital, uppsala, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 4, 325–329 https://doi.org/10.1080/03009734.2020.1826612 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1826612&domain=pdf&date_stamp=2020-10-19 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1826612 http://www.tandfonline.com vemurafenib therapy. it was shown that this resistance is mediated through feedback activation of egfr (15). case presentation patient characteristics, course of disease, and conventional treatments received our patient was diagnosed in 1986, at the age of 20, with serous borderline tumour, figo (international federation of gynaecology and obstetrics) stage ic at uppsala university hospital. she had bilateral ovarian masses, both 8 cm in diameter with no capsules, but rough, rugged surface. furthermore, loose tissue in the pouch of douglas and 5 litres of ascites were found. macroscopically radical primary surgery was performed, with bilateral salpingo-oophorectomy, hysterectomy, and omentectomy. no microscopic invasion in the ovaries, neither in the pelvic peritoneum nor the omentum, and no macroor microscopic carcinomatosis were found. postoperative chemotherapy with seven cycles of doxorubicin and cisplatin was delivered. second-look surgery revealed no residual disease, and no further therapy was given. the patient was prescribed oral oestrogen and had regular follow-ups during 11 years with clinical examinations and serum levels of the tumour marker ca-125, without signs of recurrence. however, in 1997, first relapse was evident in lymph nodes in the abdomen and the left groin, with node calcifications on ct scan. the nodes in the groin were removed. relapse was verified, but now with invasive lgsoc. the patient was treated with seven cycles of paclitaxel and carboplatin. the disease was stable at evaluation. new nodal progression was evident in 1999, which prompted hormonal therapy with tamoxifen and medroxyprogesterone, and this treatment turned out effective. in 2003, lymph node progression was again verified, now also in the left axillary and supraclavicular nodes. treatment with uracil and tegafur resulted in a durable partial response, lasting to 2010. thereafter, a slow continuous and symptomatic nodal progression was evident, prompting repeated palliative abdominal surgeries. neither weekly paclitaxel plus bevacizumab treatment nor mtor inhibition with everolimus was effective. initiation, conduct, and modulation of combined braf and mek inhibitor therapy in the autumn of 2016, therapy-resistant rapid progression and spread of the disease were evident. miliary small calcified lung metastases were present with obstructive breathing. node metastases in the abdomen and the left groin caused pain and lymph-oedema in the left leg. performance status was fair, ecog 1–2. however, there was a rapid exponential increase of the serum tumour marker ca-125. targeted next-generation sequencing (a custom haloplex/ agilent capture and illumina miseq sequencing) was performed on a lymph node metastasis, and an activating p.v600e braf mutation was detected (16). combination treatment with the braf-inhibitor dabrafenib and the mekinhibitor trametinib at full doses was initiated in december 2016, after informed consent from the patient had been obtained. initially, during the first month, three short treatment interruptions, for 2–3 days each, were necessary to handle repeated fever reactions up to 40 �c (common side effect of dabrafenib), which prompted dose reduction. after one month of treatment, a more than 50% decrease of ca-125 from 3900 to 1668 u/ml was noted. after 35 days, oral prednisolone 10–20 mg daily was introduced to control the fever episodes, as described by lee at al. (17). this measure was effective and enabled reintroduction of dabrafenib at full figure 1. serum levels of ca-125 during course of treatment with combination of braf and mek inhibitors in a patient with low-grade serous ovarian cancer. 326 b. tholander et al. dose, while the prednisolone dose could be reduced and kept fairly low. prompt symptom relief and rapid further decrease of ca-125 levels followed, with normal serum level achieved after 8 months of braf–mek inhibitor combination therapy (figure 1). a complete response was also verified radiologically after 8 months (figure 2). after a total treatment period of one year, therapy was stopped in december 2017, as was the prednisolone medication. six months later, ca-125 started to rise again. after 12 months, progressive disease was evident radiologically in lymph nodes in the left groin and on the left pelvic wall, with escalating pain. radiotherapy was given to these lymph nodes, which resulted in a partial response and pain relief. half a year later, in july 2019, after confirming that the tumour still figure 2. baseline and eight months ct scans demonstrating complete response for a patient with low-grade serous ovarian cancer treated with combination of braf and mek inhibitors. (a) lung metastases. (b, c) lymph node metastases at left pelvic wall. (d) lymph node metastases in left groin. upsala journal of medical sciences 327 harboured a braf p.v600e mutation, combined treatment with braf and mek inhibitors was reintroduced, due to progressive disease with multiple lung metastases. this time, the braf inhibitor encorafenib and mek inhibitor binimetinib were given due to the side effects experienced with dabrafenib and trametinib. after treatment at full doses for five months, the encorafenib dose was reduced, due to side effects, including abdominal pain and bowel discomfort. since the side effects were successfully treated and a rise in ca-125 was evident along with radiological progression, full doses of encorafenib and binimetinib were reintroduced, and treatment is today ongoing. at present, 3.5 years after initiation of treatment, the response is assessed as stable disease. the patient is in fairly good shape, fully ambulatory with performance status ecog 1. consent for publication in print has been obtained from the patient. discussion our patient with metastatic lgsoc had symptomatic and widespread disease, resistant to conventional therapy, rapid progression, and no obvious treatment alternative. however, with next-generation sequencing, a braf p.v600e mutation was detected. this finding, the good results of braf–mek combination treatment in malignant melanoma (12), and a recently published case reporting a response on single treatment with a braf inhibitor in a patient with lgsoc (10) formed the rationale to initiate braf–mek inhibitor combination therapy for our patient. furthermore, experiences in melanoma show that with single-drug dabrafenib therapy, the risk for some adverse reactions is higher than with combination treatment with dabrafenib and trametinib (18). thus, we decided to start combination therapy. the grading system for malignant serous ovarian carcinoma has changed and is today binary (i.e. low grade or high grade), which has performed better for outcome prediction than the old three-tier grading (1,19). in low-grade serous ovarian carcinoma the mitogen-activated protein kinase (mapk) pathway is activated, a kinase cascade that mediates the transmission of growth signals into the nucleus, via mutations in kras and braf, the upstream regulators of the mapk pathway. expression of active mapk has been detected in up to 80% of lgsoc and 78% in serous borderline tumours (4). mutations in braf, kras, and nras genes have been reported in lgsoc (3,4,20). in a review of earlier studies the frequency of braf mutations ranged from 23% to 48% in serous borderline tumours, and from 0% to 33% in low-grade serous cancers (average 5%) (3). we report a long-lasting complete response with combined braf and mek inhibition in a patient with advanced lgsoc having received established treatments. the patient is still benefiting from treatment 3.5 years after start of therapy. in comparison, in malignant melanoma patients the median pfs is 11–15 months, and complete response is observed in 8–13% of the cases (14,18). there are a few recent reports on braf and mek inhibitor combination therapy in patients with lgsoc (6,11). however, to our knowledge, there is no previously described case where complete response has been achieved in lgsoc with this treatment and no prior report on a patient benefiting for years. in conclusion, we demonstrate that complete response and long-term pfs can be achieved in advanced lgsoc with combined braf and mek inhibitor treatment. hence, this treatment may be an option when established medical treatments, i.e. hormonal and chemotherapy, are no longer effective. acknowledgements the authors are grateful to pierre fabre for supplying encorafenib and binimetinib without costs. disclosure statement the authors declare that there are no conflicts of interest. references 1. pauly n, ehmann s, ricciardi e, ataseven b, bommert m, heitz f, et al. low-grade serous tumors: are we making progress? curr oncol rep. 2020;22:8. 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conventional treatments received initiation, conduct, and modulation of combined braf and mek inhibitor therapy discussion acknowledgements disclosure statement references twelve-month follow-up of advance provision of emergency contraception among teenage girls in sweden full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 twelve-month follow-up of advance provision of emergency contraception among teenage girls in sweden—a randomized controlled trial maria ekstrand, tanja tydén, elisabeth darj & margareta larsson to cite this article: maria ekstrand, tanja tydén, elisabeth darj & margareta larsson (2013) twelve-month follow-up of advance provision of emergency contraception among teenage girls in sweden—a randomized controlled trial, upsala journal of medical sciences, 118:4, 271-275, doi: 10.3109/03009734.2013.841308 to link to this article: https://doi.org/10.3109/03009734.2013.841308 © informa healthcare published online: 09 oct 2013. submit your article to this journal article views: 632 view related articles citing articles: 5 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.841308 https://doi.org/10.3109/03009734.2013.841308 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.841308 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.841308 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.841308#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.841308#tabmodule upsala journal of medical sciences. 2013; 118: 271–275 original article twelve-month follow-up of advance provision of emergency contraception among teenage girls in sweden—a randomized controlled trial maria ekstrand1,2,3, tanja tydén2, elisabeth darj1 & margareta larsson1 1department of women’s and children’s health, uppsala university, uppsala, sweden, 2department of public health and caring sciences, uppsala university, uppsala, sweden, and 3department of care science, malmö university, malmö, sweden abstract objective. the objective of this study was to evaluate the effect of an intervention with advance provision of emergency contraceptive pills (ecp), condoms, and extended information to a targeted group of teenage girls, compared with a control group, 12 months after intervention. material and methods. a randomized controlled trial among 420 girls, 15–19 years old, requesting emergency contraception at a youth clinic in sweden was carried out. data were collected by a questionnaire at the initial visit and structured telephone interviews 12 months after enrolment. differences between the intervention group and the control group regarding ecp use, time interval from unprotected intercourse to ecp intake, contraceptive use, and sexual risk-taking were analysed. results. one year after the intervention 62% of the girls could be reached for follow-up. the girls in the intervention group reported a shorter time interval (mean 15.3 hours) from unprotected intercourse to ecp intake compared to the control group (mean 25.8 hours) (p = 0.019), without any evidence of decreased use of contraceptives or increased sexual risk-taking. conclusion. even up to 12 months following the intervention, advance provision of ecp at one single occasion, to a specific target group of adolescent girls, shortens the time interval from unprotected intercourse to pill intake, without jeopardizing contraceptive use or increasing sexual risk-taking. considering the clinical relevance of these results, we suggest that advance provision of ecp could be implemented as a routine preventive measure for this target group. key words: adolescents, advance provision, emergency contraception, sexual risk-taking introduction a single-dose administration of levonorgestrel 1.5 mg is the most widely used emergency contraceptive pill (ecp) worldwide. despite few side effects and increased sale statistics, the method has not yet met the expectations regarding reduced abortion rates, especially among teenagers, that many had hoped for (1). ecp is recommended to be taken as soon as possible after unprotected intercourse; however, obstacles for prompt use are many, including: underestimation of pregnancy risk, limited knowledge, worries about side effects, high cost, cultural barriers, and restricted availability. different strategies and interventions such as information campaigns, over-the-counter sales, subsidies, and advance provision have, therefore, been adopted in order to increase availability and thereby decrease the underutilization (2,3). theoretically, advance provision of ecp is a costeffective method that has the potential to prevent unintended pregnancies (4). most international studies are concordant; women provided with an advance provision of ecp use them to a greater extent and more rapidly after unprotected intercourse without engaging in increased sexual risk-taking behaviour (5,6). correspondence: maria ekstrand, faculty of health and society, department of care science, malmö university, malmö, sweden. e-mail: maria.ekstrand@mah.se (received 8 july 2013; accepted 2 september 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.841308 http://informahealthcare.com/journal/ups mailto:maria.ekstrand@mah.se since most studies evaluating advance provision of emergency contraception have only conducted a 6-month follow-up post intervention, the aim of this study was to evaluate an intervention of advance provision of ecp to teenage girls, 12 months after enrolment. main outcome measures were time frames for ecp use, contraceptive use, and sexual risk-taking. material and methods we conducted a randomized controlled trial among teenage girls requesting ecp at a local youth clinic in a medium-sized university town in sweden. the hypothesis was that advance provision of ecp, condoms, and extended information would reduce the time interval for ecp use and improve contraceptive use among participants in the intervention group. a power calculation based on a previous study by gold et al. (3) estimated that 150 women in an intervention group (ig) and 150 in a control group (cg) would be needed to detect a difference between the groups of 11 hours’ interval before use of ecp after unprotected sex with 80% power, at 5% significance level. we assumed a 30% drop-out rate, which made us include 210 women in each group. in total 667 girls between 15–19 years who visited the youth clinic in order to get ecp were consecutively invited to participate, and 420 out of those who met the inclusion criteria and accepted participation were randomly assigned to either ig or to cg. participation rates are shown in figure 1. girls who met the inclusion criteria and accepted participation received verbal and written information aboutthestudy.participantsprovidedcontactinformation and signed an informed consent. the randomization procedure was organized in blocks of four. pre-coded envelopes were sequentially labelled with a study identification number that had been generated by atableofrandomdigitsandhandedoutconsecutivelyto the participants, allocating them into ig or cg. the envelopescontainedabaselinequestionnaire,whichthe participants completed in a private area at the clinic, before receiving the requested ecp. the cg received standard care, including ecp (1.5 mg levonorgestrel in a single dose). additionally to the standard care, participants in the ig were provided with an intervention kit containing one extra package of ecp, ten condoms, and a specially designed leaflet with information of protection against unwanted pregnancy and sexually transmitted infections (sti). the girls were followed up after 3, 6, and 12 months by a structured telephone interview with 12 questions regarding contraceptive use and sexual risk-taking behaviour. data fromthe3-and6-monthfollow-uphavebeenpublished previously (7). the study was approved by the local medical research committee in uppsala. n = 476 invited to participate intervention group n = 214 control group n = 206 n = 420 enrolled and randomized n = 667 registered visits 3-month follow-up n = 154 3-month follow-up n = 150 n = 56 re-visits n = 106 refused n = 54 did not meet inclusion criteria n = 31 12-month follow-up n = 119 12-month follow-up n = 142 not invited due to lack of time (patient or staff) figure 1. flow chart of participants. 272 m. ekstrand et al. the baseline questionnaire, before the intervention, consisted of 29 questions covering the following areas: demographic data, reproductive and sexual health history, contraceptive use, and sexual risktaking behaviour. there were no differences between the groups concerning socio-demographic background at baseline, or at follow up 1 year later. at the 12-month follow-up, the mean age was 18 years in both groups, 95.1% in the ig and 94.1% in the cg were of nordic origin, 20.4% in the ig and 21.0% in the cg had immigrant parents, 88.6% in the ig and 83.9% in the cg were students, and 20.4% in the ig and 18.5% in the cg were daily smokers. their previous sexual experiences were also similar; first intercourse was reported at a mean age of 15.2 years in the ig and 15.4 in the cg, condom use at first intercourse was reported by 76.1% in the ig and 65.5% in the cg, the mean number of sexual partners was 4.3 in the ig and 4.6 in the cg, 8.5% in the ig and 6.9% in the cg had a history of sti, 6.3% in the ig and 7.6% in the cg had experienced an abortion, and 48.6% in the ig and 49.6% in the cg had used ecp before inclusion into the trial. girls who were lost after 1 year differed from those who completed the study only with respect to their own and their parents’ origin. furthermore, girls who completed the study were more likely to have a nordic origin, 94.6% compared with 88.1% (p = 0.02) of those lost to follow-up. of the girls who completed the study 20.7% had parents with an immigrant background compared with 34.0% (p = 0.01) of the girls who dropped out. there were no differences in sexual experiences between those who completed the study and those who were lost to follow-up. data analysis the statistical package for social sciences (spss) for windows (14.0) was used when entering and analysing data. differences between groups were tested using student’s t test, fischer’s exact test, and pearson’s chisquare test for two independent samples. differences were considered significant if p < 0.05. results during the study period 667 adolescent girls requested ecp at the selected youth clinic. a total of 54 girls did not meet the inclusion criteria (50 were not within the age span, 2 did not speak swedish, and 2 were excluded for other unspecified reasons); 31 were not invited due to staff’s lack of time. of the remaining girls, 106 refused to participate, and 56 were revisits who had already been invited. after 12 months 72 girls in the intervention group and 87 girls in the control group were lost to follow-up, leaving a 62% follow-up rate (n = 142 versus n = 119) (figure 1). the main outcome measure, the time interval for intake of ecp after an unprotected intercourse at the follow-up after 12 months, was markedly reduced in the ig when compared with that in the cg (table i). almost half, 45% (n = 64) of the girls in the ig had not used their extra ecp package, 40% (n = 57) had used it, 8% (n = 11) had given it to a friend, and 7% (n = 10) did not remember or were unsure if they had used it. after 12 months, there were no differences in contraceptive use and sexual risk-taking between the two groups. in total 83.1% in the ig and 89.1% in the cg had used oral contraception and/or condom at their latest intercourse, but 18.3% in the ig and 23.7% in the cg had at some time during the last 3 months been engaged in unprotected sex (table ii). discussion by directing the intervention to a special group, at high risk for unintended pregnancies, our aim was to evaluate if this one-dose advance supply of ecp would make any difference in time frames for ecp intake, contraceptive use, and sexual risk-taking behaviour. our study sample did not have the power to show any differences in pregnancy or abortion rates between groups; however, this was not the aim of the study. the most important finding was that even with this limited intervention, a difference between the groups in the time interval from unprotected intercourse to ecp intake was still notable after 1 year. concerns table i. time interval in hours (mean, sd) from unprotected intercourse to ecp intake in the intervention group versus the control group. n time interval (h) mean (sd) at 3-month follow-up intervention group (n = 154) 31 13.6 (10.4) control group (n = 150) 17 25.5 (18.7) p = 0.007 a at 12-month follow-up intervention group (n = 142) 57 15.3 (12.1) control group (n = 119) 26 25.8 (19.8) p = 0.019 a astudent’s t test. advance provision of emergency contraception 273 about increased sexual risk-taking in the ig were not confirmed, which is congruent with our previous results from the 3-month and the 6-month followup. however, there was still considerable risk-taking among all girls regardless of group allocation, indicating that girls requesting ecp are well-suited candidates for advance provision of ecp. some girls had not used the extra dose themselves, but had given it to a friend, which was a positive finding, since it could have helped another girl to prevent an unwanted pregnancy. advance provision of ecp as a routine preventive measure could therefore be beneficial for this target group, especially since youth clinics in sweden, where ecp is distributed to young people free of charge, are only open during week days and the need for ecp may arise at all times of the week. the benefits of increased availability was also discussed by polis et al. (5), who concluded that women should have easy access to emergency contraception because it can decrease unwanted pregnancies. how this could be best implemented remains to be demonstrated. in a widespread community-based study by glasier et al. (8) five courses of ecp were distributed to 17,800 women aged 16–29 years. despite the large sample size and the generous intervention, no effect on the abortion rates could be demonstrated; consequently, the authors raised the question whether they targeted the right women. it is most likely not cost-effective to provide ecp in advance to every woman. instead, choosing certain groups at high risk for unintended pregnancies could be a better strategy. the potential clinical feasibility of this patient-oriented trial was therefore considered important. in order to enhance a clinical implementation the advance ecp supply was limited to one dose only together with condoms and some extra information. we did several follow-ups in order to evaluate both shortand long-term effects. our conclusion that teenage girls requesting ecp could benefit from an advance supply of ecp is supported by other researchers, who have also suggested other potential target groups such as women who undergo a legal abortion, condom users, and women who have recently given birth (9-12). girls were scheduled for a structured telephone interview at 3, 6, and 12 months post enrolment. the follow-up procedure of this quite mobile target group of teenage girls required therefore thorough logistics and a flexible and creative set-up. in order to minimize drop-outs each girl received a notifying text message before the scheduled telephone call. several attempts to call back were made in case the girls did not answer the first time or if they wished to reschedule the call to a different day or time. having in mind the mobility of this age group, the drop-out rate of 38% 1 year after enrolment must be deemed as reasonable. research has shown that telephone interviews are equally reliable as face to face interviews (13). it is our impression that most girls responded openly and honestly to our questions and were eager to provide information for the study, also 12 months post enrolment. it cannot be ruled out that the three followup calls had an effect on the girls, regardless of group belonging, and that awareness of contraception and emergency contraception may have improved during the study. our previous study on advance provision of ecp to teenage girls showed significantly quicker use of ecp at follow-up 3 and 6 months post intervention. from the results in this paper, we conclude that the results were maintained even after 12 months; onedose advance supply of ecp to teenage girls requesting ecp shortens the time interval from unprotected intercourse to pill intake, without jeopardizing contraceptive use and/or increasing sexual risk-taking. considering the clinical relevance of these results, we suggest that advance provision of ecp could be implemented as a routine preventive measure for this target group. acknowledgements funding from the swedish research council and contribution from bayer-schering made this study possible. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. table ii. contraceptive use and sexual risk-taking at 12-month follow-up in the intervention group versus the control group. intervention group n = 142 control group n = 119 p-value intercourse without any contraception during the last 3 months 26 (18.3%) 28 (23.7%) ns condom use at latest intercourse 39 (27.5%) 37 (31.1%) ns oral contraceptive use at latest intercourse 79 (55.6%) 69 (58.0%) ns unprotected latest intercourse 13 (9.2%) 13 (10.9%) ns 274 m. ekstrand et al. references 1. baecher l, weaver ma, raymond eg. increased access to emergency contraception: why it may fail. hum reprod. 2009; 24:815–19. 2. raine tr, harper cc, rocca ch, fischer r, padian n, klausner jd, et al. direct access to emergency contraception through pharmacies and effect on unintended pregnancy and stis: a randomized controlled trial. jama. 2005;293: 54–62. 3. gold ma, wolford je, smith ka, parker am. the 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hanrahan kj. comparing telephone and faceto-face qualitative interviewing: a research note. qual res. 2004;4:107–18. advance provision of emergency contraception 275 www.ncbi.nlm.nih.gov/pubmed/19095666?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19095666?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15632336?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15632336?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15632336?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15050984?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15050984?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15050984?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15050984?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20620913?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20620913?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18055735?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18055735?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20869281?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20869281?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20869281?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18307077?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18307077?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18307077?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15105057?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15105057?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11535209?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11535209?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11535209?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16643822?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16643822?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17484192?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17484192?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17484192?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20399951?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20399951?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20399951?dopt=abstract abstract introduction material and methods data analysis results discussion acknowledgements declaration of interest references predictors of liver failure in primary biliary cirrhosis full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 predictors of liver failure in primary biliary cirrhosis pan zhao, wei-wei liu, jin-feng li, chun-ya wang, hao wang, jun xu, ruifang wang, hao-zhen yang, cheng jin & zhen-man wei to cite this article: pan zhao, wei-wei liu, jin-feng li, chun-ya wang, hao wang, jun xu, rui-fang wang, hao-zhen yang, cheng jin & zhen-man wei (2015) predictors of liver failure in primary biliary cirrhosis, upsala journal of medical sciences, 120:1, 47-51, doi: 10.3109/03009734.2014.985763 to link to this article: https://doi.org/10.3109/03009734.2014.985763 © informa healthcare published online: 28 nov 2014. submit your article to this journal article views: 553 view related articles view crossmark data citing articles: 3 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2014.985763 https://doi.org/10.3109/03009734.2014.985763 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2014.985763 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2014.985763 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2014.985763&domain=pdf&date_stamp=2014-11-28 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2014.985763&domain=pdf&date_stamp=2014-11-28 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2014.985763#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2014.985763#tabmodule upsala journal of medical sciences. 2015; 120: 47–51 original article predictors of liver failure in primary biliary cirrhosis pan zhao1,8, wei-wei liu2, jin-feng li3, chun-ya wang4, hao wang5, jun xu6, rui-fang wang7, hao-zhen yang8, cheng jin1 & zhen-man wei1 1clinical trial center, beijing 302 hospital (pla 302 hospital), beijing 100039, china, 2postgraduate division, academy of military medical science, beijing 100850, china, 3radiology department, pla general hospital, beijing 100853, china, 4emergency & critical care center, beijing anzhen hospital, capital medical university, beijing 100029, china, 5medical information center, beijing 302 hospital (pla 302 hospital), beijing 100039, china, 6clinical laboratory, beijing 302 hospital (pla 302 hospital), beijing 100039, china, 7ultrasonography department, beijing 302 hospital (pla 302 hospital), beijing 100039, china, and 8liver failure therapy and research center, beijing 302 hospital (pla 302 hospital), beijing 100039, china abstract background. the disease progression of patients with primary biliary cirrhosis (pbc) varies significantly, and the prognostic markers that identify those patients who will develop liver failure have been scarcely studied from a chinese cohort. aims. we aimed to determine the predictive factors of liver failure in patients with pbc. methods. patients who were first diagnosed as pbc with hepatic compensation between january 2007 and december 2009 were enrolled in this cohort study. results. altogether 398 patients were finally included. of these patients, 80% were women, 98% had positive antimitochondrial antibodies, and 45% had positive antinuclear antibodies (ana). to december 2012, a total of 38 patients developed liver failure. according to the outcome, patients who developed liver failure had had higher serum concentration of baseline total bilirubin (tbil) (p = 0.013) and total bile acid (tba) (p < 0.001), and lower concentrations of baseline total cholesterol (tch) (p = 0.008), than patients who did not develop liver failure. additionally, the proportion of ana positivity was statistically different between the two groups (p = 0.009). in the established model for predicting liver failure in pbc, three variables were finally selected out, including tch (odds ratio (or) 0.552, 95% confidence interval (ci) 0.394–0.774, p < 0.001), tba (or 1.006, 95% ci 1.002–1.010, p = 0.002), and ana (+ versus –, or 5.518, 95% ci 1.155–26.376, p = 0.032). conclusions. ana, tch, and tba are predictors of liver failure in pbc. key words: primary biliary cirrhosis, liver failure, predictor introduction primary biliary cirrhosis (pbc) is an autoimmune liver disease characterized by the destruction of intrahepatic bile ducts, which can lead to hepatic cirrhosis and eventually liver failure and death (1,2). ursodeoxycholic acid (udca) is the only drug accepted internationally for the treatment of pbc (3-5). however, a recent meta-analysis of 16 randomized clinical trials demonstrated no significant benefits of udca on all-cause mortality or liver transplantation in patients with pbc (6). in fact, the disease progression varies markedly among patients with pbc (7); moreover, substantial divergences of clinical characteristics exist because of variations in the populations under different studies (8,9). thus, it is necessary in the early stage to determine the prognostic variables associated with the development of end-stage liver disease, so that physicians can closely monitor the disease progress and adjust treatment measures in a timely manner before fatal events occur. in the present study, we aim to study the clinical characteristics correspondence: zhen-man wei, no. 100 of west fourth ring middle road, beijing, china. e-mail: weizhenman@sina.com (received 21 may 2014; accepted 4 november 2014) issn 0300-9734 print/issn 2000-1967 online � 2014 informa healthcare doi: 10.3109/03009734.2014.985763 http://informahealthcare.com/journal/ups mailto:weizhenman@sina.com and risk factors associated with the development of liver failure in a prospective cohort with pbc. patients and methods patients patients who were first diagnosed as pbc with hepatic compensation between january 2007 and december 2009 in beijing 302 hospital were enrolled in this cohort study. all of these included patients had received udca therapy at the initial diagnosis of pbc. exclusion criteria included the concurrence of autoimmune hepatitis or extra-hepatic autoimmune diseases; infection with hepatitis a, b, c, d, e, epstein–barr virus, cytomegalovirus, or human immunodeficiency virus; the presence of other forms of liver diseases such as alcoholic liver disease, druginduced hepatitis, or wilson’s disease; the use of corticosteroids or immunosuppressive drugs for a period of more than 2 weeks; and udca non-responders. liver failure in this study was defined as coagulopathy (prothrombin activity (pta) £40% or international normalized ratio (inr) ‡1.5) and jaundice (serum total bilirubin (tbil) ‡171 mmol/l or a daily increase ‡17.1 mmol/l). the study was performed in accordance with the ethical guidelines of the 1975 declaration of helsinki and was approved by the ethics committee of beijing 302 hospital. laboratory tests biochemical profiles, including alanine aminotransferase (alt), aspartate aminotransferase (ast), total bilirubin (tbil), gamma glutamyl transferase (ggt), alkaline phosphatase (alp), albumin, total cholesterol (tch), and total bile acid (tba) were measured using standard laboratory procedures. normalized serum concentrations of alt, ast, tbil, ggt, alp, albumin, tch, and tba were, respectively, <40 u/l, <40 u/l, <17.1 mmol/l, 7–32 u/l, 40–150 u/l, 35–55 g/l, 2.8–5.2 mmol/l, and 0–10 mmol/l. serum autoantibodies, including antimitochondrial antibodies (ama) and antinuclear antibodies (ana), were tested using indirect immunofluorescence with standardmethods(euroimmunmedizinnischelabordiagnostika ag, lubeck, germany), and sera were consideredtobepositivewhentheyproducedareaction at a dilution of ‡1:100. immunoglobulin (ig) was assayed by means of immunological turbidometry (diasys diagnostic systems, shanghai, china). normal serum concentrations of iga, igg, and igm were 0.69–3.28 g/l, 7.23–16.6 g/l, and 0.63–2.77 g/l, respectively. statistical analysis data analyses were performed using sas 9.2 software (sas institute inc., cary, nc, usa). continuous data were expressed as medians (interquartile range). categorical data were expressed as the number of subjects. group comparisons were performed using the wilcoxon rank sum test for continuous variables, and chi-square test or fisher exact test for categorical variables. logistic regression was used for evaluating prognostic predictors of liver failure. a probability (p) value of less than 0.05 was considered statistically significant. results demographic and clinical characteristics finally, 398 patients were included. in these patients, 317 (80%) were female and 81 (20%) were male. the average age was 57 (12) years. a total of 389 patients had positive ama, and 9 had negative ama; 182 patients had positive ana, and 216 had negative ana. in the years 2007, 2008, and 2009, 80, 147, and 171 patients were enrolled (figure 1). to december 2012, a total of 38 patients had developed liver failure (figure 2). univariate analysis for baseline variables in pbc according to the outcome, patients who developed liver failure had had higher serum concentrations of baseline tbil (43.7 mmol/l versus 23.2 mmol/l, p = 0.013) and tba (92.5 mmol/l versus 46.0 mmol/l, p < 0.001) and lower serum concentrations of baseline tch (3.09 mmol/l versus 4.52 mmol/l, p = 0.008) than patients who did not develop liver failure (table i). in addition, the proportion of ana positivity differed between the two groups. thus, a majority of the patients who developed liver failure had positive ana (65.79% versus 43.61%, p = 0.009). predictors of liver failure in pbc three variables were eventually selected out to predict the development of liver failure in pbc using logistic regression, including tch (odds ratio (or) 0.552, 95% confidence interval (ci) 0.394–0.774, p < 0.001), tba (or 1.006, 95% ci 1.002–1.010, p = 0.002), and ana (+ versus –, or 5.518, 95% ci 1.155–26.376, p = 0.032) (table ii). 48 p. zhao et al. discussion pbc is a chronic and progressive cholestatic disease, and its pathogenesis remains unclear (10-12). due to lack of curative therapeutics, liver failure is an evitable severe outcome in the majority of such patients. so, studying factors associated with the development of liver failure has vital and practical value. beijing 302 hospital is the largest hospital specializing in hepatology in china. therefore, such an investigation on the risk of incipient liver failure in a large number of pbc patients possesses certain representativeness. previous studies have shown that patients with pbc often have higher serum concentrations of cholesterol (13-15). because the synthetic and metabolic process of cholesterol is closely associated with the function of the liver, the development and progression of liver diseases can influence the serum concentrations of cholesterol. in our study, patients with lower total serum cholesterol concentrations were more likely to develop liver failure than patients with higher concentrations. tba has been less studied than other serum markers in the evaluation of the prognosis of pbc. based on our study, it was an independent risk factor for liver failure in pbc. as previously shown, cholestasis is a main physiopathological characteristic of pbc. it can cause the accumulation of hydrophobic bile acids in the liver, which are toxic to cellular membranes (16,17). so, an increase of serum tba may mirror the disease severity in pbc. it has been reported that ana can be found in 30%–50% of all pbc patients, and disease-specific ana are associated with a more severe and rapidly progressing disease (18-20). in the present study, ana were detected in 46% of the patients and proved to be related to the occurrence of liver failure. regarding other early variables, such as alp, ggt, and igm, no correlations with the development of liver year c u m u la ti v e i n c id e n c e o f li v e r fa il u re ( % ) 2007 2008 2009 2010 2011 2012 10 8 6 4 2 0 figure 2. cumulative incidence of liver failure in patients with primary biliary cirrhosis during follow-up. evaluation of clinical information at entry exclusion criteria: (i) concurrence of autoimmune hepatitis or extra-hepatic autoimmune diseases; (ii) infection with hepatitis a, b, c, d, e, epstein-bar virus, cytomegalovirus or human immunodeficiency virus; (iii) presence of other forms of liver diseases such as alcoholic liver disease, drug-induced hepatitis or wilson’s disease; (iv) use of corticosteroids or immunosuppressive drugs (≥ 2weeks); (v) non-responders to ursodeoxycholic acid 398 patients with hepatic compensation were enrolled, including 80 in the year of 2007, 147 in 2008 and 171 in 2009 follow-up: check-up in hospital pr telephone interview a total of 38 patients developed liver failure to december 2012 figure 1. patient enrollment. predictors of liver failure in primary biliary cirrhosis 49 failure were observed, though some of them had definitely diagnostic value. in conclusion, ana positivity, a lower serum concentration of tch, and higher serum concentration of tba are all associated with the development of liver failure in pbc. acknowledgements we are grateful to professor yu-kun han and dr xin-ying liu for their great help in the patient enrollment. zhen-man wei was the guarantor; pan zhao designed the study; wei-wei liu, jin-feng li, and pan zhao analyzed the data; hao-zhen yang and pan zhao enrolled the patients; pan zhao, hao wang, jun xu, rui-fang wang, and cheng jin collected the data; pan zhao and chun-ya wang wrote the manuscript. all authors read and approved the final manuscript. pan zhao, wei-wei liu, jin-feng li and chun-ya wang contributed equally to this work. declaration of interest: this work was partly supported by the 302 hospital research project (ynkt2013009). the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. czul f, peyton a, levy c. primary biliary cirrhosis: therapeutic advances. clin liver dis. 2013;17:229–42. 2. balmer ml, dufour jf. treatment of hypercholesterolemia in patients with primary biliary cirrhosis might be more beneficial than indicated. swiss med wkly. 2008;138: 415–19. 3. silveira mg, brunt em, heathcote j, gores gj, lindor kd, mayo mj. american association for the study of liver diseases endpoints conference: design and endpoints for clinical trials in primary biliary cirrhosis. hepatology. 2010; 52:349–59. 4. zhao p, han y. low incidence of positive smooth muscle antibody and high incidence of isolated igm elevation in table ii. predictors of liver failure in primary biliary cirrhosis. parameters odds ratio (95% ci) 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http://www.ncbi.nlm.nih.gov/pubmed/23009110?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23009110?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21212566?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21212566?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/6240147?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/6240147?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/6240147?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23184345?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23184345?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23876351?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23876351?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21134832?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21134832?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/12117892?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/12117892?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22531844?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22531844?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23672463?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23672463?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22414767?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22414767?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22414767?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22414767?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23354620?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23354620?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23354620?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23467321?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23467321?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23777462?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23777462?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23777462?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21185272?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21185272?dopt=abstract abstract introduction patients and methods patients laboratory tests statistical analysis results demographic and clinical characteristics univariate analysis for baseline variables in pbc predictors of liver failure in pbc discussion acknowledgements declaration of interest references the uppsala anatomist ivar sandström and the parathyroid gland full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 the uppsala anatomist ivar sandström and the parathyroid gland henry johansson to cite this article: henry johansson (2015) the uppsala anatomist ivar sandström and the parathyroid gland, upsala journal of medical sciences, 120:2, 72-77 to link to this article: https://doi.org/10.3109/03009734.2015.1027426 © informa healthcare published online: 27 apr 2015. submit your article to this journal article views: 377 view related articles view crossmark 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henry johansson department of surgical sciences, uppsala university, uppsala, sweden key words: anatomy, endocrinology, parathyroid gland the last anatomical discovery the discovery of the parathyroid gland by the uppsala anatomist ivar sandström (figure 1) is often called the last anatomical discovery. that remarkable discovery was made in 1877 at the anatomical department in uppsala. at that time sandström was a young medical student, who had worked as an assistant at the department since 1873. his discovery was published in upsala läkareförenings förhandlingar, in 1880 (1). his publication was in swedish, and the paper was entitled ‘on a new gland in man and several animals’. the article comprised 30 pages, and in the introduction sandström writes: almost three years ago i found on the thyroid gland of a dog a small organ, hardly as big as a hemp seed, which was enclosed in the same connective tissue as the thyroid, but could be distinguished therefrom by the light colour. microscopically the examination revealed glandular tissue completely different from that of the thyroid (1). afterwards he continued his dissections and found similar glands in cats and rabbits. stimulated by these findings he started examinations in human autopsy subjects, and in his article he writes: the existence of a hitherto unknown gland in what has so often been a subject of anatomical examination called for a thorough approach to the region around the thyroid gland even in man. although the probability of finding something hitherto unrecognised seemed so small that it was exclusively with the purpose of completing the investigation, rather than with the hope of finding something new, that i began a careful examination of this region. so much the greater was my astonishment therefore when in the first individual examined i found on both sides at the inferior border of the thyroid gland an organ of the size of a small pea, which, judging from its exterior did not appear to be a lymph gland, nor an accessory thyroid gland, and upon histological examination showed a rather peculiar structure (2). the strength of his work lay in his careful dissections and examinations. the dissections included 50 autopsy cases, and in 43 of them he found four glands, two on each side of the thyroid. he presented a detailed gross description of the position, size, colour, and the various forms of the gland (figure 2). he mentioned that the glands should be looked upon in the vicinity of the thyroid. sometimes the glands were only 3 mm in diameter, in other cases up to 15 mm. histologically he noted that there was variable distribution between the parenchymal and fat cells. sandström thought that the glands were embryonal remnants of the thyroid and suggested that the new structures should be named ‘glandulae parathyroideae’. he was not able to allow even himself to make a guess of the possible physiological role of the glands but suggested that they could be of pathological importance and clinical relevance. many of sandström’s epoch-making anatomical and histological observations are of interest also for modern pathology and surgery. correspondence: henry johansson, department of surgical sciences, uppsala university, uppsala, sweden. e-mail: henry.johansson29@gmail.com (received 25 february 2015; accepted 26 february 2015) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1027426 http://informahealthcare.com/journal/ups mailto:henry.johansson29@gmail.com figure 1. ivar sandström, 1852–1889. figure 2. drawing by sandström of human parathyroid glandular anatomy. the uppsala anatomist ivar sandström and the parathyroid gland 73 no international recognition to obtain international recognition, sandström translated his manuscript into german with the hope of getting the article published in rudolf virchow’s journal, one of the most famous at that time. for sandström it was a big disappointment when virchow declined its publication in his journal, because of the length of the paper (30 pages). many believe, however, that the truth was different. virchow had himself noted some structures in the region of the thyroid and may have realized when he read sandström’s manuscript that it was the parathyroid gland he had observed (3,4). he was probably not willing to give the young swede full credit for the discovery of a new organ (5). therefore sandström’s paper was published only in swedish (1), and if two abstracts had not been published in a german yearbook of 1880 his work would probably have remained unrecognized for many years. the discovery of the parathyroid on a rhinoceros like virchow, prior researchers had observed structures near the thyroid in humans. they did not pay much attention to these findings; the structures were usually considered accessory glands of the thyroid. however, sandström was not first to detect the parathyroid gland—it was the english professor of comparative anatomy sir richard owen. he made the discovery in 1850—the year of sandström’s birth—during a dissection of an indian rhinoceros at the london zoo. his observation was reported to the zoological society of london, but the paper was not published until 1862, when it appeared in the transactions of the society (6). in his article owen describes the parathyroid gland as ‘a small compact yellow glandular body attached to the thyroid gland at the point where the veins emerge’. owen had probably no prior knowledge of the presence of this organ, no microscopic examination was performed, and he never mentioned such glands in other animals. owen’s anatomical finding passed unnoticed until 1905, when it was mentioned in a paper in the british medical journal. therefore, the credit for the discovery of the parathyroid gland has justly been given to ivar sandström, who was the first to observe the gland in human and to make a thorough description microscopically. in 1906, the american r. l. thompson praised sandström’s discovery with the following words: this paper of sandström is so thorough, that little has been added to our knowledge of the anatomy or histology of these glands since their discovery and it is so complete as to leave no doubt, that he deserved all credit for the discovery of these organs (4). the man behind the discovery ivar sandström was born in stockholm in 1852 as the fifth child in a family of seven brothers and sisters. his father was a surveyor who died of cholera when ivar was only six years old. his mother became guardian of all the children. facing financial difficulties, she gave up ivar and his younger sister anna (who became a famous teacher and pedagogue) to foster parents. after seven years in foster care ivar returned to his family. with the economic support of his brother nils he was able to rejoin the family and return to his studies. in 1872 he began his medical studies at the university of uppsala, where he became a medicine candidate in 1878. he worked as an assistant at the department of anatomy (figure 3), and during 1879–80 was appointed ‘prosector’ at the department. in 1881, he became an extra teacher in histology and remained in this position until 1886, when he became a licensed physician at uppsala university. he married in 1885 and had two children. the family was poor, and sandström had to split his time between his own medical studies, his research, and jobs that would earn money for his family. his wife became more and more distressedbyhislonghoursandfinancialstruggles.afterafewyears,shelefthim,takingthetwochildrenwithher(7). sandström suffered from a hereditary mental disease and was plagued by psychiatric, alcoholic, and drug problems from a young age. sandström was disappointed with the reception of his scientific work. his impression of the scandinavian meeting of natural sciences in stockholm in 1880 was of ‘a big scandinavian humbug’. his disillusionment is reflected in a letter to his sister, dated 8 august 1880, where he writes: . . . i accepted an invitation from stockholm to attend an international meeting of the natural scientists in order that i might do my part so that we swedes should not succumb to our guests—which later on proved not to be the case at all. . . . one should, of course, at a personal meeting of so many men who devote themselves to science, be inclined to expect less of a hurried reading of, more or less unimportant products of genius whose creators in any case would not forget to publish them if they are of any value, than rather a friendly exchange of 74 h. johansson thoughts and of trustful communication of personal experiences and impressions. nothing of the kind was seen . . . everyone seemed to be there with the intention of showing what ‘discoveries’ he had made and at the same time give the astonished world the opportunity to have a look at the fortunate discoverer. but for the discovery itself, for the revealed truth, the interest was little or none. in time, sandström became more depressed. in 1886 he was treated for an acute psychotic episode at ulleråker’s hospital in uppsala. his psychiatric problems worsened, and in early 1889 his older brother nils took care of ivar at his home in askesta, where he was manager for a sawmill industry. the intention was to give ivar a quiet and calm existence, but these hopes were dashed. in june, 1889, at the age of 37 years, ivar sandström committed suicide. the day before, he was sitting together with nils and his family saying ‘it would have been nice to become a professor and get a name’ (7). sandström never became a professor, but his discovery has given him a name to be remembered in the medical history, and his scientific work has often been praised for its accuracy. his discovery is forever associated with his name and the department of anatomy in uppsala. the parathyroid gland and calcium at the time of sandström’s discovery, the physiological function of the parathyroid gland was unknown. a decade later (in 1891) eugéne gley, professor of physiology in paris, published the first report on a relationship between the parathyroid glands and tetany. in canine experiments he found that thyroidectomy was followed by tetany and death onlyiftheparathyroidswerealsoremoved(8).someyearslater(in1896)twoitalians,giorliovassala andfrancesco generali, observed that tetany could be caused by parathyroidectomy alone. they hypothesized that tetany was caused by an intoxication and that the parathyroids were the organs that removed the toxins—later on believed to be guanidine—from the body. regardless, the parathyroid glands were now recognized as vital organs to be managed with great care in thyroid surgery. tetany was at that time a common and harmful complication to thyroid surgery. the theory that tetany was caused by an intoxication persisted for many years. however, in 1924 it became clear through the work of william maccollum, pathologist at johns hopkins hospital, that tetany was the result of calcium deficiency. about the same time the norwegian physiologist harald salvesen proved that the hypocalcemic tetany could be treated with injections of crude parathyroid extract. further research led to the isolation of the parathyroid hormone by the canadian professor james collip in 1925, and it became clear that the hormone was calcium-regulating (9). the parathyroid gland and bone disease in 1891 the german pathologist friedrich van rechlinghausen described the parathyroid cystic bone disease, which was given the name ‘osteitis fibrosa cystica’ (10). the association between this typical bone disorder and figure 3. the building where sandström discovered the parathyroid gland. the uppsala anatomist ivar sandström and the parathyroid gland 75 parathyroid disease was established in 1904, when max askanazy of tubingen, germany, described a patient with bone changes and a parathyroid tumour (11). in 1907 jakob erdheim, a viennese pathologist, reported that patients dying of advanced skeletal disease frequently exhibited enlarged parathyroid glands (12). he suggested that the enlargement of the parathyroids accompanying the bone changes was a compensatory phenomenon and that the skeletal disease should be treated with parathyroid extract. in 1915 friedrich schlagenhaufer questioned the validity of this interpretation. he had observed that mostly only one of the four glands was enlarged, and proposed that the changes in the parathyroid glands were the primary events leading to the bone disease (13). on these grounds it seemed logical to treat these patients by removal of the diseased gland. the first parathyroidectomy the first parathyroidectomy was carried out by felix mandl in vienna in 1925 (14). mandl’s patient albert jahne was a 38-year-old trolley conductor suffering from a severe, crippling bone disease. he had fractured his femur, and his blood calcium level was very high. his disorder was recognized as a typical parathyroid bone disease. mandl thought that the disease was due to a parathyroid deficiency, and the patient was first treated by parathyroid extract and grafted fresh parathyroid tissue. this, however, made his condition worse, and mandl decided to explore albert jahne’s neck surgically. the operation was performed under local anaesthesia and a parathyroid tumour measuring 21 � 12 � 12 mm was removed. the three remaining glands were normal. the result of this operation was dramatic reversal of the bone changes. mandl had for the first time shown that schlagenhaufer’s hypothesis was correct: the bone disease was secondary to a lesion of the parathyroid gland. news of mandl’s operation spread, and in 1931 about 20 more operations had been performed in europe and the united states. around 1950 several surgeons had experience with parathyroid surgery, and a decade later the number of patients receiving parathyroidectomy for hyperparathyroidism had grown. by the 1960s routine biochemical screening, including serum calcium determinations, was introduced. since then hyperparathyroidism has been more frequently diagnosed leading to a rapid development of parathyroid surgery. in sweden, john hellström, professor of surgery at the karolinska hospital in stockholm, was the great pioneer of parathyroid surgery. his first operation was performed in 1930, but few patients were treated prior to 1950. in 1962 he presented his series of 138 patients. in his studies he confirmed the association between renal stones and hyperparathyroidism (15). twenty per cent of hellström’s patients were reoperative cases, who had been operated on at other hospitals. the failed primary operation was mostly the result of an incomplete neck operation. hellström strongly emphasized that parathyroid surgery should be carried out ‘in hospitals where the services of experienced surgeons and pathologists were available’ (15). in uppsala, the first parathyroid neck exploration was performed in 1941 on a woman with the diagnosis of ‘rachitis tarda’. only a single normal-sized gland was found, and the patient was unimproved following the operation. the first operation where an abnormal gland was identified and removed was, in fact, a reoperation undertaken in 1946 on a woman with multiple spontaneous fractures. the operation was performed by olle hultén, professor of surgery in uppsala 1942–1964. the patient had previously undergone a parathyroid operation, which had failed to cure her. at the reoperation, a kidney-shaped, bean-sized adenoma was removed from the mediastinum by sternotomy. the serum calcium decreased dramatically after the operation, and the patient developed severe tetany. despite intravenous infusion of both calcium and parathyroid hormone the patient died postoperatively. the parathyroid—an ‘uppsala’ organ in uppsala parathyroid surgery has held a prominent position. a hundred years after sandström’s discovery göran åkerström presented another unique study from the surgical department on the anatomy and histology of the parathyroid glands. his study became valuable for surgeons involved in parathyroid surgery. along with studies of the anatomy and history of the glands, hyperparathyroidism itself has been extensively studied in uppsala. the natural course of the disease has been examined, and it was found that an increased mortality was associated with hypercalcemia and successful surgery resulted in a better survival. in one study it was shown that as many as 65%–80% of patients with hyperparathyroidism had psychiatric symptoms that could be eliminated with parathyroidectomy. in some studies it has been demonstrated that even mild forms of hyperparathyroidism are associated with renal stone disease as well as disturbances of lipid and skeletal metabolism; successful surgery provides an important benefit for such patients. 76 h. johansson the pathogenesis of hyperparathyroidism has also been the subject of several studies in uppsala. it has been demonstrated, for example, that defective calcium regulation can result from reduced expression of calcium receptors on the parathyroid cells. the research has also shown that the disease can be caused by a gene variant that lowers the expression of vitamin d receptors, reducing the regulatory effect of the vitamin on the secretion of parathyroid hormone. an interesting observation is that the parathyroid glands possess an enzyme that can convert vitamin d to its active metabolite and hence have influence on growth and hormone production. benign parathyroid tumours—adenomas—show increased expression of this enzyme and thus inhibited growth, whereas cancers have reduced expression and thereby a more aggressive growth. in a recent study, it was found that genetic aberrations may be a common pathway to development of parathyroid tumours. modern parathyroid surgery primary hyperparathyroidism is caused by a single adenoma in 90% of cases. today, the enlarged gland can often be localized preoperatively by using imaging techniques. this has led to a wide use of minimally invasive surgical approaches for removal of the adenomas. conventional neck explorations are becoming rarer. of course, such approaches are still indicated in cases when hyperplastic glands are expected, in reoperations, or when removal of a goitre is needed. modern literature indicates that results and complication rates after a minimally invasive surgery approach is comparable to those obtained by neck exploration via standard open surgery. declaration of interest: the author reports no conflicts of interest. the author alone is responsible for the content and writing of the paper. references 1. sandström iv. on a new gland in man and several mammals – glandulae parathyroideae. upsala läk förenings förh. 1879–1880;15: 441–71. 2. seipel cm. on a new gland in man and several mammals (glandulae parathyroideae) by ivar sandström (english translation). baltimore: johns hopkins press; 1938. 3. kock w. medicinska klassiker. v. ivar viktor sandström. svenska läkartidningen. 1944;34:2073–82. 4. kock w. ivar viktor sandström och bisköldkörtlarna. en svensk insats i anatomiens historia. nordisk medicinhistorisk årsbok. 1969; suppl ii:8–15. 5. johansson h. ivar sandström, uppsala-anatomen som upptäckte bisköldkörteln. medicinhistoriska museet i uppsala 2013. isbn 97891-981137-1-6. 6. owen r. on the anatomy of the indian rhinoceros (rh. unicornis, l). trans zool soc lond. 1862;4:31–58. 7. ask-upmark e, rexed b, sandström b. ivar sandstöm and the parathyroid glands. a 90-years-anniversary. acta univ ups. 1967;13: 4–14. 8. gley e. sur les functions du corps thyroid. c r séances soc biol. 1891;43:841–3. 9. collip jp. extraction of parathyroid hormone which will prevent or control parathyroid tetany and regulates level of blood calcium. j biol chem. 1925;63:395–438. 10. von recklinghausen fd. die fibröse oder deformierende ostitis, die osteomalacie und die osteoplastische karzinose in ihren gegenseitigen beziehungen. in festschrift fur rudolf virchow reimer. berlin: 1891. p 1–89. 11. askanazy m. uber ostitis deformans ohne ostoides gewebe. arb geb path anat inst tubingen. 1903;4:398–422. 12. erdheim j. uber die dentinverkalkung in nagezahn bei der epithelkörerchen transplantation. frankfurt z path. 1911;7:295–342. 13. schlagenhaufer f. zwei fälle von parathyroideatumoren. wien klin wschr. 1915;28:1362. 14. mandl f. therapeutischer versuch bei ostitis fibrosa generalisata mittels extirpation eines epithelkörperchentumors. wien klin wschr. 1925;50:1343–4. 15. hellström j. primary hyperparathyroidism. triangle. 1962;5:171–8. the uppsala anatomist ivar sandström and the parathyroid gland 77 http://www.ncbi.nlm.nih.gov/pubmed/13953630?dopt=abstract the last anatomical discovery no international recognition the discovery of the parathyroid on a rhinoceros the man behind the discovery the parathyroid gland and calcium the parathyroid gland and bone disease the first parathyroidectomy the parathyroid&mdash;an &lsquo;uppsala&rsquo; organ modern parathyroid surgery declaration of interest references correlation analysis of coagulation dysfunction and liver damage in patients with novel coronavirus pneumonia: a single-center, retrospective, observational study original article correlation analysis of coagulation dysfunction and liver damage in patients with novel coronavirus pneumonia: a single-center, retrospective, observational study sai chena , hanting liua, tie lib, rong huanga, rong guia and junhua zhanga adepartment of blood transfusion, the third xiangya hospital of central south university, changsha, china; bdepartment of clinical laboratory, the first people’s hospital of yueyang, yueyang, china abstract background: the novel coronavirus disease 2019 (covid-19) is currently breaking out worldwide. covid-19 patients may have different degrees of coagulopathy, but the mechanism is not yet clear. we aimed to analyse the relationship between coagulation dysfunction and liver damage in patients with covid-19. methods: a retrospective analysis of 74 patients with covid-19 admitted to the first people’s hospital of yueyang from 1 january to 30 march 2020 was carried out. according to the coagulation function, 27 cases entered the coagulopathy group and 47 cases entered the control group. a case control study was conducted to analyse the correlation between the occurrence of coagulation dysfunction and liver damage in covid-19 patients. results: alanine aminotransferase (alt) and aspartate aminotransferase (ast), markers of liver damage, were positively correlated with coagulopathy (p ¼ 0.039, or 2.960, 95% ci 1.055–8.304; and p ¼ 0.028, or 3.352, 95% ci 1.137–9.187). alkaline phosphatase (alp), c-glutamyl transpeptidase (c-gt), and total bilirubin (tbil) were not statistically correlated with coagulopathy. according to the diagnosis and treatment plan, the included cases were classified into mild, moderate, severe, and critical. the results showed that the occurrence of coagulation dysfunction had no statistical correlation with the severity of covid-19. conclusion: coagulation dysfunction in patients with covid-19 is closely related to liver damage. a longer course of the disease may cause a vicious circle of coagulopathy and liver damage. clinicians need to closely monitor coagulation and liver function tests and to give prophylactic or supportive therapy when needed. article history received 6 july 2020 revised 4 september 2020 accepted 8 september 2020 keywords blood coagulation dysfunction; covid-19; liver damage; pneumonia; sarscov-2 introduction the novel coronavirus disease (covid-19) is currently breaking out worldwide, threatening human health and quality of life seriously. its main clinical manifestations are fever, dry cough, and fatigue. in some severe cases, acute respiratory distress, multiple organ failure, and even death may occur (1). covid-19 is caused by severe acute respiratory syndrome coronavirus-2 (sars-cov-2), belonging to the group of b-coronaviruses. it has the characteristics of a long incubation period (1–14 days, average 6.4 days), a long onset period, and strong infectivity. the s protein on the surface of sarscov-2 binds to angiotensin-converting enzyme ii (ace2), which leads sars-cov-2 to enter the host cell (2,3). some researchers (4–6) have reported that patients with covid-19 have varying degrees of coagulopathy and liver damage as the disease progresses. the liver is closely related to the synthesis of coagulation factors, which means that when the liver is damaged, it will directly affect the coagulation function. the relationship between coagulation dysfunction induced by covid-19 and liver damage is unclear. this study retrospectively analysed the clinical data of 74 confirmed cases of covid-19 to explore the correlation between covid-19 patients’ coagulopathy and liver damage. methods a total of 74 patients with covid-19 admitted to the first people’s hospital of yueyang, hunan province from 1 january to 30 march 2020 were enrolled. the hospital is the designated hospital for the treatment of covid-19 patients. the ethics committee of the third xiangya hospital of central south university approved this study. inclusion criteria were in line with the ‘novel coronavirus infection pneumonia diagnosis and treatment program (trial version 7)’ (7) diagnosis requirements: (1) have fever and/or respiratory symptoms; (2) have lung-imaging features of new contact rong gui aguirong@163.com; junhua zhang xy3zhangjunhua@csu.edu.cn department of blood transfusion, the third xiangya hospital of central south university, changsha, china � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution-noncommercial license (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 4, 293–296 https://doi.org/10.1080/03009734.2020.1822960 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1822960&domain=pdf&date_stamp=2020-10-19 http://orcid.org/0000-0003-4677-0169 http://orcid.org/0000-0001-7790-0291 http://orcid.org/0000-0001-7028-0632 http://creativecommons.org/licenses/by-nc/4.0/ https://doi.org/10.1080/03009734.2020.1822960 http://www.tandfonline.com coronavirus pneumonia; (3) real-time fluorescent rt-pcr detection of sars-cov-2 nucleic acid is positive. on this basis, according to coagulation function, patients with coagulation dysfunction were included in the coagulation dysfunction group, and those without coagulation dysfunction were included in the control group. exclusion criteria: (1) history of coagulation disease or taking coagulationrelated drugs; (2) liver and/or kidney dysfunction; (3) hepatobiliary diseases; (4) incomplete clinical data. we collected clinical data from electronic medical records of all confirmed patients, including past history, gender, age, length of hospital stay, clinical symptoms and laboratory findings, mainly liver and coagulation function test results. patients’ first test results were selected for inclusion in the analysis. diagnostic criteria for coagulopathy a fully automatic blood coagulation analyser (cs5100, sysmex corp., japan) was used to detect the blood coagulation function. prior to testing, all projects were subject to strict quality control testing. when one or more of the indicators listed in table 1 were abnormal, it was defined as coagulation dysfunction. diagnostic criteria for liver damage a fully automatic biochemical analyser (bs800, mindray corp., china) was used to detect liver function. prior to testing, all projects were subject to strict quality control testing. when one or more of the following indicators exceeded the upper limit of normal value, it was defined as liver damage: serum alanine aminotransferase (alt), serum aspartate aminotransferase (ast), total bilirubin (tbil), c-glutamyl transpeptidase (c-gt), and alkaline phosphatase (alp). the reference values were: alt, 7–40 u/l; ast, 13–35 u/l; tbil, 1.7–25 lmol/l; c-gt, 7–45 u/l; and alp, 50–135 u/l. statistical analysis this study was conducted during the outbreak of covid-19. therefore, we did not estimate the sample size by formal hypotheses, and we have included the maximum number of patients who met the inclusion criteria. qualitative data were expressed in number of cases, percentage, or composition ratio. we used chi-square test to compare the qualitative data between the two groups. the quantitative data were tested for normality and homogeneity of variance. normally distributed quantitative data were expressed as mean ± standard, and we used student’s t test for two independent samples to compare the qualitative data of the two groups. tests were two-sided with significance set at a < 0.05. spss 23.0 for windows (spss inc.) was applied for all analysis. results following the inclusion and exclusion criteria strictly, 74 patients (36 female and 38 male) with covid-19 were included; 27 cases entered the coagulopathy group, and 47 cases entered the control group. general information between the two groups was compared (table 2). there was no statistically significant difference. according to the ‘new coronavirus infected pneumonia diagnosis and treatment program (trial version 7)’ (7), patients with different severity of pneumonia were classified into mild, moderate, severe, and critical. in the coagulation group, 4 cases were mild (14.8%), 12 were moderate (44.4%), and 11 were severe/critical (40.8%). in the control group, 14 cases (29.8%) were mild, 19 cases (40.4%) were moderate, and 14 cases (29.8%) were severe/critical. there was no significant correlation between coagulation dysfunction and covid-19 severity (chi-square ¼ 3.012, p > 0.05). this study analysed the relationship between changes in liver damage markers and covid-19 coagulation dysfunction. alt and ast are markers that can reflect liver damage. covid-19 coagulation dysfunction was associated with alt and ast (pearson chi-square test, both p < 0.05) (table 3). however, alp, c-gt, and tbil, reflecting the function of the bile duct system, showed no significant correlation with coagulopathy in the covid-19 patients (pearson chi-square test, all p > 0.05). the above single factor analysis showed a statistically significant difference of alt and ast levels between the coagulation dysfunction group and the control group. therefore, a logistic regression analysis model of the influencing factors of coagulation dysfunction was constructed. taking whether the patient has coagulation dysfunction as the dependent variable (normal ¼ 0, abnormal ¼ 1), and ast and alt as the independent variable (normal ¼ 0, abnormal ¼ 1), a binary logistic regression analysis was performed. it was found that ast and alt levels were the main risk factors affecting covid-19 patients’ coagulopathy (p ¼ 0.039, or 2.960, 95% ci 1.055–8.304; and p ¼ 0.028, or 3.352, 95% ci 1.137–9.187). table 1. diagnostic criteria for coagulopathy. indicator reference values diagnostic criteria pt 9.8–14.8 s extension > 3 s tt 14–21 s extension > 3 s inr 0.86–1.27 >1.3 aptt 23.3–32.5 s extension > 10 s fibrinogen content 2–4 g/l <2 g/l d-dimer <500 lg/l >500 lg / l platelet count 125–350 � 109/l <125 � 109/l aptt: activated partial thromboplastin time; inr: international normalised ratio; pt: prothrombin time; tt: thrombin time. table 2. general information on the two evenly matched groups. parameters coagulopathy group (n ¼ 27) control group (n ¼ 47) p age (y) 52.5 ± 12.1 47.8 ± 17.1 >0.05 gender male 14 (51.9%) 24 (51.1%) >0.05 female 13 (48.1%) 23 (48.9%) basic diseases hypertension 5 (18.5%) 4 (8.5%) >0.05 diabetes 1 (3.7%) 1 (2.1%) >0.05 copd 1 (3.7%) 2 (4.3%) >0.05 other 7 (25.9%) 10 (21.3%) >0.05 copd: chronic obstructive pulmonary disease. 294 s. chen et al. discussion at present, it has been observed that covid-19 can lead to different degrees of coagulation dysfunction. endothelial cells play a key role in the regulation of blood coagulation and fibrinolysis. the immune response in vivo of patients with covid-19 implies the release of a variety of inflammatory mediators such as interleukin 6 (il-6). there is damage of vascular endothelial cells, initiating endogenous coagulation pathways. the damaged endothelial cells increase the release of von willebrand factor (vwf) and tissue factor (tf), and then subendothelial tissues become exposed. in the presence of calcium ions, fvii is activated to start the exogenous coagulation system and accelerates the production of thrombin, resulting in activation of the coagulation or fibrinolysis system (8,9). in addition, systemic inflammation may activate nox2, and reactive oxygen species (ros) derived from nox2 will adversely affect blood coagulation. several studies have shown that, in addition to coagulation dysfunction, covid-19 patients will have different degrees of liver damage as well. the pathogenesis of liver damage complicated by covid-19 is not yet clear; however, current mainstream views on the mechanism of liver injury include: (1) angiotensin-converting enzyme 2 (ace2) mediates sars-cov-2 invasion of target cells. bile duct epithelial cells highly express ace2, and its expression level is similar to alveolar type ii cells, which is the main target cell type of sars-cov-2 in the lung. but according to current clinical data, the levels of bile duct injury markers such as alp and c-gt in covid-19 patients have not increased, so this mechanism needs further studies (10–12). (2) after infection with sars-cov-2, the immune cells are activated, and excessive accumulation of immune cells occurs. the excessively activated inflammatory cells infiltrating the tissue release many proinflammatory cytokines, oxygen free radicals, etc., which can cause damage. the cytokine storm triggered may be one of the mechanisms of liver injury (13,14). (3) hypoxaemia and respiratory distress syndrome may occur in severe and critical pneumonia. when the tissue is hypoxic, an oxidative stress response may be triggered, resulting in liver function damage. at the same time, it promotes reactive oxygen species increasing continuously, which further initiates the release of various proinflammatory factors to induce liver damage. (4) patients with covid-19, especially those with severe and critical disease, usually receive different kinds of pharmacological therapy. a drug in itself or the interaction between different drugs may cause liver damage (15–18). recent research has shown that the hypercoagulable state of covid-19 patients alters intrahepatic vascular structures and causes a variable degree of luminal thrombosis. this may add to other mechanisms of liver damage in covid-19 patients (19). the synthesis of coagulation factors is closely related to the liver. liver, as a production site for multiple clotting factors, might play a huge role in covid-19 coagulopathy. liver damage leads to reduced coagulation factors, plasma plasminogen activator inhibitors, and tissue-type plasminogen activator secretion, causing bleeding tendency. fibrinogen (fbg), one of the important proteins of the blood coagulation system, is synthesised by liver parenchymal cells. it is the final substrate for the successive activation of coagulation factors during coagulation. because of its functions of hemostasis and mediating platelet aggregation, liver damage will cause a decrease in fbg synthesis, which will affect blood coagulation. it is worth mentioning that some studies have pointed out that the incidence of liver damage is related to the severity of covid-19, and that patients with severe disease are more likely to have liver damage. in this study, however, it was found that there was no correlation between coagulopathy and the severity of covid-19, which is inconsistent with a liver–covid-19 relationship. rather, it may suggest that the occurrence of coagulation dysfunction is not related to liver abnormalities. moreover, we found that alt and ast were positively correlated with covid-19 coagulopathy, suggesting that after covid-19 infection liver damage might be detrimental. it might also be envisaged that the coagulation dysfunction in covid-19 patients may cause liver damage due to thrombosis. a longer course of covid-19 may cause a vicious circle of coagulation dysfunction and liver damage, which does not promote patient survival. although there is yet no highly efficient specific therapy for sars-cov-2, it is important to prevent thrombosis and to identify coagulation and liver dysfunction by laboratory monitoring to enable supportive therapy. in this study we found a correlation between coagulopathy and elevated liver transferases in covid-19 patients. the coagulopathy was, in contrast to other studies, not related to the severity of the disease. the mechanism behind the association between coagulopathy and liver damage is still unclear and needs further investigation. acknowledgements we thank all patients and their families involved in the study. disclosure statement the authors declare that they have no conflicts of interest. table 3. the correlation between biochemical test index with coagulopathy in covid-19. group abnormala alt abnormala ast abnormala tbil abnormala alp abnormala c-gt coagulopathy group 12 11 7 3 6 control group 10 8 5 2 14 chi-square 4.406 5.056 1.932 0.423 0.498 p 0.036 0.025 0.165 0.516 0.481 aabnormal means that the indicator exceeds the upper limit of normal value. upsala journal of medical sciences 295 notes on contributors sai chen, mm, department of blood transfusion, the third xiangya hospital of central south university, changsha, china. hanting liu, mm, department of blood transfusion, the third xiangya hospital of central south university, changsha, china. tie li, mb, department of clinical laboratory, the first people’s hospital of yueyang, yueyang, china. rong huang, md, department of blood transfusion, the third xiangya hospital of central south university, changsha, china. rong gui, md, is a professor of clinical transfusion immunology, department of blood transfusion, the third xiangya hospital of central south university, changsha, china junhua zhang, md, department of blood transfusion, the third 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https://doi.org/10.1016/j.blre.2014.09.003 https://doi.org/10.1055/s-0035-1556586 https://doi.org/10.1101/2020.02.03.931766 https://doi.org/10.1016/j.cell.2020.03.045 https://doi.org/10.1016/j.cell.2020.02.058 https://doi.org/10.1016/j.ebiom.2020.102763 https://doi.org/10.1001/jama.2020.1585 https://doi.org/10.1016/s2213-2600(20)30076-x https://doi.org/10.5582/bst.2020.01020 https://doi.org/10.5582/bst.2020.01020 https://doi.org/10.1016/j.toxlet.2020.01.026 https://doi.org/10.1007/s00204-020-02734-1 https://doi.org/10.1007/s00204-020-02734-1 https://doi.org/10.1111/liv.14601 abstract introduction methods diagnostic criteria for coagulopathy diagnostic criteria for liver damage statistical analysis results discussion acknowledgements disclosure statement orcid references effect of dexmedetomidine on plasma brain-derived neurotrophic factor: a double-blind, randomized an full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 effect of dexmedetomidine on plasma brainderived neurotrophic factor: a double-blind, randomized and placebo-controlled study lin yang, jun-mei xu, xinghua jiang, wei ruan, yulong cui & liang he to cite this article: lin yang, jun-mei xu, xinghua jiang, wei ruan, yulong cui & liang he (2013) effect of dexmedetomidine on plasma brain-derived neurotrophic factor: a double-blind, randomized and placebo-controlled study, upsala journal of medical sciences, 118:4, 235-239, doi: 10.3109/03009734.2013.808295 to link to this article: https://doi.org/10.3109/03009734.2013.808295 © informa healthcare published online: 20 jun 2013. submit your article to this journal article views: 661 view related articles citing articles: 10 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.808295 https://doi.org/10.3109/03009734.2013.808295 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.808295 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.808295 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.808295#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.808295#tabmodule upsala journal of medical sciences. 2013; 118: 235–239 original article effect of dexmedetomidine on plasma brain-derived neurotrophic factor: a double-blind, randomized and placebo-controlled study lin yang, jun-mei xu, xinghua jiang, wei ruan, yulong cui & liang he department of anesthesiology, the second xiangya hospital, central south university and anesthesiology research institute, central south university, changsha, hunan, people’s republic of china abstract background. dexmedetomidine (dex) has neuro-protective effects, but the clinical mechanism remains unclear. method. forty patients were randomly divided into two groups: group a (control) and group b (treated with dex). plasma concentrations of brain-derived neurotrophic factor (bdnf) were determined in blood samples using enzymelinked immunosorbent assays at five time points: t1 (baseline), t2 (15 minutes after intubation and before the surgery was started), t3 (the end of surgery), t4 (10 minutes after extubation in the post-anesthesia care unit), and t5 (24 hours after the surgery). changes in bispect (bis) index, heart rates, and doses of anesthetics used for induction were also recorded. results. baseline plasma concentrations of bdnf did not differ between group a and group b; 15 minutes after induction, concentrations of plasma bdnf were significantly reduced in group a. twenty-four hours after surgery, the concentration was still higher in group b than in group a. in contrast, plasma concentrations of bdnf at other time points tested did not differ between the two groups. conclusion. it appears that dex could reverse the reduced plasma concentrations of bdnf caused by anesthetics, and this effect lasted for 24 hours after surgery. key words: anesthetic drugs, brain-derived neurotrophic factor (bdnf), dexmedetomidine introduction as a member of the neurotrophin family of growthpromoting proteins, brain-derived neurotrophic factor (bdnf) plays an important role in neuronal survival, axon growth, and synaptic plasticity (1). in rodents, bdnf is highly correlated with learning, memory, and other advanced neuronal functions (2). elevation of bdnf in the central nervous system (cns) may significantly attenuate neuronal injuries caused by ischemia and also be beneficial for the treatment of degenerative diseases (3). importantly, bdnf is detectable in blood, and a positive correlation has been found between serum and cortical bdnf concentrations (4,5). thus, determinations of serum bdnf concentrations could help to investigate changes of bdnf concentrations in the cns. anesthetics are known to inhibit neuronal activity in the cns (6) and cause sedation, amnesia, and post-operative cognitive dysfunction. further studies have shown that such drugs could inhibit the release of bdnf from cortical neurons (7). it has been proven clinically that perioperative use of anesthetics could lead to decreased plasma concentrations of bdnf in patients (8). the highly selective a2-adrenoceptor agonist, dexmedetomidine (dex) has marked effects on hypnosis, anti-anxiety, and analgesia (9). recent animal studies have revealed that dex also has neuro-protective effects (10-12), due in part to the elevation of bdnf concentrations in the cortex and hippocampus (13). however, in humans, the impact of dex on bdnf concentrations remains elusive. therefore, we investigated the effect of dex on plasma bdnf correspondence: jun-mei xu, md, department of anesthesiology and anesthesiology research institute second xiangya hospital, central south university, central south university, changsha, hunan, china. fax: +86 073185295970. e-mail: 13975148864@139.com (received 22 january 2013; accepted 21 may 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.808295 http://informahealthcare.com/journal/ups mailto:13975148864@139.com concentrations in patients who underwent general anesthesia and were classified as physical status i and ii patients (american society of anesthesiologists (asa)). subjects and methods our trial was registered in the chinese clinical trial registry with the registration number chictrtrc-12002714 and approved by the ethics committee of the second xiangya hospital, central south university. all patients agreed to join our research project and signed an informed consent statement. from 1 august 2012 to 31 december 2012 in the second xiangya hospital, central south university, asa physical status i and ii patients, who were scheduled for lumbar discectomy, were enrolled. remembering that adult plasma bdnf concentrations are correlated with age, gender, and body mass index (bmi) (14), we planned to choose only male patients, from 40 to 60 years old, and with a bmi of 20 to 30. patients with a history of alcohol abuse or use of antipsychotic drugs, as well as those with underlying organ disease or psychiatric illness, were not considered. patients received 5 mg diazepam orally, 30 minutes before entering the operating room. in the waiting room, they were randomly allocated to one of the two groups by drawing lots; lidocaine cream was applied to the skin of each patient’s arms in order to attenuate the pain of puncture. two intravenous catheters were inserted, one in each of the patient’s arms; one was used exclusively for the measurement of bdnf plasma concentrations and the other for anesthesia and volume management. after entering the operating room, 250 ml of ringer’s lactate was administered to ensure rapid volume expansion. routinely, we monitored (dash 3000, ge company, milwaukee, wi, us) a fivelead ecg, non-invasive blood pressure, heart rate and peripheral capillary oxygen saturation. bis index (bistm complete 2-channel monitor and 4 electrode sensor, both from covidien, mansfield, ma, us) was used to measure the depth of anesthesia. in order to maintain the double-blind nature of the study, an anesthetist who did not participate in our research program prepared the ‘study drug solutions’ containing either dex 2 mg/ml or 0.9% saline, while a member of our research team supervised the whole anesthesia management. in group b, 0.7 mg/kg dex was administered by intravenous infusion for 15 minutes, and propofol (diprivan, astrazeneca, paddington, london, uk) was administered until the patient’s eyelid reflex was lost. then, sulfentanil (eurocept, ankeveen, the netherlands) 0.15 mg/kg and cis-atracurium 0.1 mg/kg were infused intravenously until neuromuscular relaxation was achieved. the same anesthetist carried out the intubation. in group a, however, an equal amount of 0.9% saline instead of dex was administered, while the rest of the steps were the same as for group b. another member of the research team was in charge of recording the doses of propofol and sulfentanil used for induction, changes in bis values, and heart rates during induction. anesthesia was maintained by the continuous infusion of propofol; sulfentanil and cis-atracurium were administered intermittently for analgesia and muscular relaxation, respectively. in group b, 0.6 mg/kg/h dex was additionally administered by continuous infusion. controlled mechanical ventilation was adjusted to maintain the petco2 between 4.7 and 6.0 kpa, and bis index was monitored between 35 and 45. the infusion of all anesthetics and dex was halted at skin closure, when 6–8 mg sulfentanil was given to each patient in order to reduce post-operative pain. five venous blood samples (5 ml each) were drawn from each patient: at t1 (baseline), t2 (15 minutes after intubation before the operation was started), t3 (after skin closure), t4 (10 minutes after extubation in the pacu), and t5 (24 hours after surgery). blood samples were placed in ethylene diamine tetraacetie acid (edta) tubes and centrifuged for 15 minutes at 3,000 rpm. the plasma obtained was stored at –70�c until required for subsequent analysis of its contents. bdnf plasma concentrations were measured by enzyme-linked immunosorbent assays (elisa) (human bdnf immunoassay, r&d system, minneapolis, us). using this assay, the minimum detectable concentration of bdnf is less than 20 pg ml-1. statistical analysis for the design of this study, an estimation of the required minimum sample size was determined based on results of our pre-experimental investigations (not included in the real study) and on a previous study (8). using type i error (0.05) and type ii error (0.1), a power of test of 90%, the number of cases calculated per group would be 17. thus, we recruited 20 patients in each group in order to prevent unforeseen difficulties. data were expressed as mean ± standard deviation, maximum or minimum. plasma bdnf concentrations were compared between the two groups and within each group, by using the non-parametric mann–whitney u test. a t test was used for comparison of doses of induction anesthetics and changes of the heart rates and bis index at each time point 236 l. yang et al. between the two groups. p < 0.05 was considered to be statistically significant. all data were analyzed with spss 19.0 software (ibm company, armonk, new york city, us). results forty patients were enrolled. data from three patients were excluded: one from group a due to a change of the surgical procedure, and the other two from group b due to technical problems of blood processing. in group a, bdnf plasma concentrations were 196.1 ± 36.7 pg/ml at baseline and decreased to 120.9 ± 17.6 pg/ml 15 minutes after anesthesia induction (table i). at the time point of skin closure, the bdnf plasma concentration increased to 167.3 ± 19.9 pg/ml and remained high (154.9 ± 28.6 pg/ml) 5 minutes after extubation. twenty-four hours after the operation, the bdnf plasma concentrations fell below the baseline (126.8 ± 28.2 pg/ml). in group b, the baseline bdnf plasma concentration was 173.8 ± 40.0 pg/ml, which was comparable with that in group a. fifteen minutes after anesthesia induction, the bdnf concentration tended to decrease (167.3 ± 36.8 pg/ml). however, it was still higher than in group a. twenty-four hours after the operation, bdnf plasma concentrations were maintained at 176.0 ± 26.9 pg/ml, which was higher than in group a. both at skin closure and 10 minutes after the extubation, bdnf plasma concentrations increased to 165.3 ± 34.9 pg/ml and 164.8 ± 29.4 pg/ml, but there were no differences between the two groups. in general, heart rates were lower in group b than in group a at the time point of injection of dex, anesthesia induction, and intubation (table ii). the lowest heart rate in group b was 49 bpm after anesthesia induction, and it returned to 70 bpm after intra-tracheal intubation. there was no serious hypotension recorded. bis values were recorded during the operation (table iii). compared with group a, the bis values in group b were lower at the procedures of intubation and skin cut. at the other time points checked there were no differences between the two groups. basic characteristics, duration of the surgery, and the amount of bleeding and volume therapy did not differ between the two groups. however, doses of propofol and sulfentanil for induction were lower in group b than in group a (table iv). discussion this study demonstrated that dex did reverse anesthetic-induced reductions of bdnf plasma concentrations. bdnf in blood could readily be detected, and according to previous studies it is highly correlated with bdnf concentrations in the brain (3,4). it is mainly stored in platelets (15) and may also be synthesized and secreted by visceral epithelia (16), vascular endothelia (17), and inflammation cells (18) (activated t-helper th1 and th2 cd4+ cell lines, especially). there is evidence to suggest that intravenous table i. bdnf plasma concentrations. group a (n = 19) group b (n = 18) t1 196.1 ± 36.7 (141.4–268.6) 173.8 ± 40.0 (113.7–274.6) t2 120.9 ± 17.6 (92.5–159.5) 167.3 ± 36.8a (109.4–264.9) t3 167.3 ± 19.9 (113.6–192.5) 165.3 ± 34.9 (119.2–255.6) t4 154.9 ± 28.6 (107.8–194.5) 164.8 ± 29.4 (114.2–254.4) t5 126.8 ± 28.2 (90.4–190.4) 176.0 ± 26.9a (149.4–260.4) t1 = baseline; t2 = 15 minutes after intubation before the operation started; t3 = skin closure; t4 = 10 minutes after extubation in the pacu; t5 = 24 hours after the operation. ap < 0.05 versus control. table ii. changes in patients’ heart rates. group a group b baseline 78 ± 6.6 78 ± 7.0 study drug injection 78 ± 6.8 63 ± 4.2a anesthesia induction 65 ± 4.4 53 ± 2.7a intubation 93 ± 7.0 86 ± 9.1a ap < 0.05 versus control. table iii. bis value variation during surgery. group a group b baseline 98.6 ± 0.5 98.2 ± 0.6 anesthesia induction 40.3 ± 2.3 39.1 ± 3.5 intubation 64.5 ± 4.3 51.4 ± 2.8a skin cut 54.3 ± 4.9 46.8 ± 2.5a skin closure 59.6 ± 4.4 59.5 ± 3.2 ap < 0.05 versus control. effect of dexmedetomidine on plasma bdnf 237 administration of bdnf labeled with 125i can result in entry into the cns to promote neuron protection and regeneration, and after intracerebroventricular injection of exogenous bdnf an efflux of bdnf from brain to blood was also detected (4). clinically depressed patients were found to have lower levels of bdnf, both in the cortex (19) and in plasma (20). in healthy individuals, bernward winter (21) demonstrated that the improvement of short-term learning was highly correlated with an elevation in the serum concentration of bdnf. it appears that elevation of blood concentrations of bdnf could contribute to a better neural function. therefore, one can suppose that if dex could increase bdnf concentrations in plasma, it might produce neurological benefits. anesthetics could inhibit the release of bdnf from cortical neurons (7). this reduction of plasma bdnf concentrations was proven to cause bdnf-dependent neuroapoptosis in the neonatal rat brain (22), which indicated that the bdnf signal was crucial to the cns and could partly explain the mechanisms of neurotoxicity induced by anesthetics (23). without the interference of noxious stimuli and with strict management to maintain the bis index within a narrow range, 15 minutes after induction may be the best time to assess the effects of the dex and anesthetics on plasma bdnf. our present study, together with other studies (8), showed that the administration of anesthetics could reduce the bdnf concentrations in plasma in both groups. we believe that measurements of bdnf concentrations in plasma may reflect, to some extent, the effect of anesthetics on bdnf expression in cns. in contrast, dex was proven to have neuro-protective effects, and it could attenuate the cognitive impairment induced by anesthetics (24) and elevate the bdnf levels in the hippocampus (12). in the present study, we found that when combined with dex, anesthetics-induced reduction of bdnf appeared to be reversible. this could be explained by two mechanisms: firstly, dex administration obviously permitted a reduction in the doses of propofol and sulfentanil used for induction, as was also demonstrated in other studies (25). thus, dex may weaken the attenuated effect of the anesthetics on the release of bdnf. secondly, dex itself might increase the bdnf levels both in cns (proven in animal research (12)) and in plasma. however, further studies are still needed to figure out the actual peripherally or centrally acting site of dex and anesthetics. at the time point of skin closure and 10 minutes after extubation in the pacu, the bdnf plasma concentrations in both groups increased markedly compared to the concentrations 15 minutes after induction. we supposed that these changes resulted from the gradual reduction in the depth of anesthesia and analgesia, as the infusion of all the drugs was stopped at the beginning of the skin closure. neuronal excitability recovered gradually, which might have caused a rapid release of bdnf in the cns. the change of bdnf plasma concentrations 24 hours after surgery may well reflect the longlasting effects of anesthetics on patients. in our present study, it was found that after anesthesia the bdnf plasma concentrations were decreased 24 hours after surgery, which is consistent with results from previous research (8). importantly, we showed that dex combined with general anesthesia reversed the decrease of the bdnf plasma concentration induced by anesthetics. clinical trials (26) with large sample sizes have already reported that postoperative sedation with dex instead of propofol or midazolam is associated with significantly lower rates of postoperative delirium (which is a form of neurological impairment) and care costs. we believe that this elevation of plasma bdnf concentrations might serve as a potential mechanism to explain the neuro-protective effect of dex in humans. perioperative noxious stimuli are known to be important factors affecting the post-operative neurologic recovery. in the present study, we found that compared with group a, the dex-treated patients in group b had more stable heart rates and bis values in the face of intubation and skin cut, most probably because of the anti-sympathetic effect of dex. whether it is correlated with the elevation of bdnf concentrations needs to be clarified. a limitation of our research was that we did not carry out stratified research of the population (only well-selected male patients were enrolled), and the number of patients used was limited and thus might not be representative of the general population. we did not extend our investigation to include studies of our patients’ cognition functions, because patients (aged 40–60 years old) rarely suffer from postoperative cognition dysfunction after the surgery of lumbar discectomy. table iv. duration of surgery, amount of bleeding and volume therapy, and the dose of induction anesthetics. group a group b duration of surgery (min) 109.3 ± 7.1 104.6 ± 10.6 bleeding (ml) 102.1 ± 10.3 103.9 ± 9.2 volume therapy (ml) 1442.1 ± 161.0 1436.1 ± 173.9 propofol for induction (mg) 92.1 ± 15.1 36.9 ± 6.0a sulfentanil for induction (mg) 36.8 ± 5.06 20.4 ± 5.1a ap < 0.05 versus control. 238 l. yang et al. overall, our experiments showed that dex combined with general anesthesia could reverse the reduced bdnf plasma concentrations caused by anesthetics during and 24 hours after surgery. whether the elevation of plasma bdnf is beneficial to the patient remains to be unequivocally established. acknowledgements here we want to thank the technician chun-lin wu, who was in charge of measuring the plasma concentrations of bdnf by elisa. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. sossin ws, barker pa. something old, something new: bdnf-induced neuron survival requires trpc channel function. nat neurosci. 2007;10:537–8. 2. chao mv, rajagopal r, lee fs. neurotrophin signalling in health and disease. clin sci. 2006;110:167–73. 3. nagahara ah, merrill da, coppola g, tsukada s, schroeder be, shaked gm, et al. neuroprotective effects of brain-derived neurotrophic factor in rodent and primate models of alzheimer’s disease. nat med. 2009;15:331–7. 4. pan w, banks wa, fasold mb, bluth j, kastin aj. transport of brain-derived neurotrophic factor across 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www.ncbi.nlm.nih.gov/pubmed/17453053?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17453053?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16411893?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16411893?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19198615?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19198615?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19198615?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19198615?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9886678?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9886678?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9886678?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12147321?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12147321?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12147321?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12650981?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12650981?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18270817?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18270817?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18270817?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10730549?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21241763?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21241763?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21241763?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18514174?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18514174?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15585351?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15585351?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/8869564?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/8869564?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/8869564?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/8869564?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10514401?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10514401?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10514401?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10514401?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10734218?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10734218?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16402109?dopt=abstract 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methods statistical analysis results discussion acknowledgements declaration of interest references experiences of sexual violence among women seeking services at a family planning unit in sweden article experiences of sexual violence among women seeking services at a family planning unit in sweden mariella €oberga , alkistis skalkidoua and gun heimerb adepartment for women’s and children’s health, uppsala university, uppsala, sweden; bnational centre for knowledge on men’s violence against women, uppsala university, uppsala, sweden abstract background: experiences of sexual violence among women can lead to ill health and increase the risk of lifetime co-occurrence of violence. identifying risk factors and victims facilitates development of effective programmes for treatment and prevention of additional violence. the primary aim of this study was to assess the prevalence and correlates of sexual violence experiences among women seeking care at a family planning unit in sweden. a secondary aim was to examine associations between sexual violence and other types of violence. methods: women (n ¼ 1226) seeking services at a family planning unit, uppsala university hospital, sweden, answered a questionnaire and were interviewed about experiences of sexual violence. bivariate associations were examined using the chi-square test. results: experiences of sexual violence were reported by 27% of the participants, of which 57% were exposed when they were younger than 18 years old. women with experiences of sexual violence were more likely to have lower education (p ¼ 0.024), were students or without occupation (p ¼ 0.037), and were not in a current relationship (p < 0.001). women with experiences of non-partner sexual violence were more likely to have experiences of intimate partner violence (p < 0.001). conclusion: prevalence of sexual violence was high among the respondents. many women were young when they were exposed to violence, and lifetime co-occurrence of violence was common among women with experiences of non-partner sexual violence. article history received 7 november 2018 revised 25 february 2019 accepted 4 april 2019 keywords intimate partner violence; polyvictimization; psychological violence; physical violence; revictimization; sexual violence introduction sexual violence against women can be a major threat to the health of millions of girls and women all over the world. prevalence studies have indicated that 35.6% of women worldwide have experiences of either physical and/or sexual violence (1), and the estimated prevalence of physical and/or sexual violence was 33% among women in european union countries (2). in a national prevalence study of lifetime exposure to sexual violence in sweden, 38% of women reported a history of sexual victimization (3). sexual violence has been defined by the world health organization (who) as: ‘any sexual act, attempt to obtain a sexual act, unwanted sexual comments or advances, or acts to traffic or otherwise directed against a person’s sexuality using coercion, by any person regardless of their relationship to the victim, in any setting, but not limited to home and work’ (4). it can take place in a relationship where a partner perpetrates physical, sexual, and/or psychological violence. the offenders of sexual violence can also be non-partners (e.g. stranger, acquaintance, friend, family member, colleague, military, etc.). the health consequences of sexual violence against women are well known. negative effects on health can be shortand/or long-term and include a variety of symptoms and complications such as gynaecological trauma, unintended pregnancy, sexually transmitted infections, sexual dysfunction, and chronic pelvic pain (5–10). experiences of sexual violence can also have a major impact on mental health, with women reporting depression, posttraumatic stress syndrome, sleeping disorders, and suicidal behaviour (11,12). according to the ecological model for understanding violence, a number of risk factors have been identified to be associated with experiences of sexual violence on a societal, community, relationship, and individual level such as young age, low education, low socioeconomic status, and exposure to prior abuse (4,13). research has shown that lifetime co-occurrence of violence, revictimization, or polyvictimization, among survivors of any type of violence (e.g. physical, sexual, or psychological) is common and can be perpetrated by more than one person during the lifetime of the women exposed (14,15). the patterns of polyvictimization are though not yet fully understood. contact mariella €oberg mariella.oberg@kbh.uu.se department for women’s and children’s health, uppsala university, uppsala, sweden � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 2, 135–139 https://doi.org/10.1080/03009734.2019.1604587 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1604587&domain=pdf&date_stamp=2019-05-31 http://orcid.org/0000-0002-1244-7278 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2019.1604587 http://www.tandfonline.com many women with experiences of sexual violence may seek medical help, due to the adverse health consequences described above. the health care system constitutes therefore a significant setting to identify exposed women, and in turn can help to develop effective intervention programmes to prevent further ill health and polyvictimization. a research project aiming to elucidate different aspects of experiences of violence among women seeking family planning services in uppsala was undertaken during 2005–2006 (16). the primary aim of the current study was to assess the prevalence and correlates of sexual violence experiences among women seeking care at a family planning unit in sweden. a secondary aim was to examine associations between sexual violence and other types of violence. materials and methods subjects the details of the recruiting procedure of the study have been described previously (16). eligible to participate were swedish-speaking girls and women from 15 years of age seeking services at the family planning unit in uppsala’s university hospital during the period of october 2005 to october 2006. uppsala is the fourth largest city of sweden with a substantial student population, and the family planning unit services are both for girls and women seeking termination of pregnancy and contraceptive counselling. information on the study and a self-administered questionnaire were provided to the women at registration. by answering the questionnaire and allowing trained staff to interview them, women gave their consent to participate. instructions were given to the women to place the questionnaire in a locked box regardless of whether they answered it or not. confidentiality was assured, and the interviews took place in absence of their partners. if needed, the participants were offered counselling in accordance with current clinical practice. variables the questions for the interviews consisted of a modified, translated version of the abuse assessment screen (aas), a validated tool for detection of intimate partner violence and sexual violence (17). a short version of the norvold abuse questionnaire (noraq), a tool validated in a swedish population with questions about psychological, physical, and sexual violence, was used in the self-administered questionnaire (18,19). a number of background questions were also included, for example, age, educational level, occupation, birthplace, and current relationship status. questions about experiences of sexual violence in the questionnaire were divided into three categories ranging from mild, to moderate and severe: 1. mild sexual violence. ‘has anybody against your will touched parts of your body other than genitals in a sexual way or forced you to touch parts of his or her body in a sexual way?’ ’have you in any other way been sexually humiliated; e.g. by being forced to watch a porno movie or similar against your will, forced to show your body naked, or forced to watch when somebody else showed his/her body?’ 2. moderate sexual violence. ‘has anybody against your will touched your genitals, used your body to satisfy him/ herself, or forced you to touch anybody else’s genitals?’ 3. severe sexual violence. ‘has anybody against your will put or tried to put his penis into your vagina, mouth, or rectum; put or tried to put an object or other parts of the body into your vagina, mouth, or rectum?’ for each of these questions the women could answer who the perpetrator was and if the violence was experienced during the past year. women answering ‘yes’ to any of the questions regarding sexual violence were categorized as women with experiences of sexual violence. intimate partner violence. furthermore, the participants answered questions about experiences of psychological violence (have you experienced anybody systematically and for any longer period trying to repress, degrade, or humiliate you? have you experienced anybody systematically and by threat or force trying to limit your contacts with others or totally control what you may or may not do? have you experienced living in fear because somebody systematically and for a longer period has threatened you or somebody close to you?) or physical violence (have you experienced anybody hitting you, smacking your face, or holding you firmly against your will? have you experienced anybody hitting you with his/her fist(s) or with a hard object, kicking you, pushing you violently, giving you a beating, thrashing you, or doing anything similar to you? have you experienced anybody threatening your life by, for instance, trying to strangle you, showing a weapon or a knife, or by any other similar act?). if the participants answered ‘yes’ to any of those questions and had a present or former partner as perpetrator they were categorized as women with experiences of intimate partner violence. non-partner sexual violence. women who answered ‘yes’ to any of the questions regarding sexual violence and the perpetrator was other than a partner or former partner were categorized as women with experience of non-partner sexual violence. the women who answered ‘yes’ to any of the questions regarding sexual violence and the perpetrator was a partner or former partner were excluded from the ‘nonpartner sexual violence’ group. data analysed in this study came from the questionnaire answers and the interviews. no additional information was collected from the women’s medical journals. statistical analysis spss version 24.0 was used for the statistical analyses. the prevalence of sexual violence among the participants was assessed. comparisons were made between the women who confirmed experiences of sexual violence and those who did not, regarding known risk factors (i.e. age, educational level, 136 m. €oberg et al. occupation, and place of birth). furthermore, lifetime prevalence of intimate partner violence was compared between women with and without experiences of non-partner sexual violence to examine associations between sexual violence from a non-partner and other forms of violence. bivariate associations were examined using the chi-square test. statistical significance was set at a value of p < 0.05. the study was approved by the regional ethical review board in uppsala university (dnr 2005:219). results prevalence of sexual violence a total of 1517 questionnaires were distributed to women seeking care at the family planning unit. of those, 1286 (85%) women agreed to participate. sixty women did not state their background information and were therefore excluded from the analyses. the age of the women ranged from 15 to 55 years. among the participants (n¼ 1226), 326 (27%) answered ‘yes’ to any of the questions regarding experiences of sexual violence during the interview or in the questionnaire. during the interviews, 192 (16%) of the participants reported that they had been sexually abused, 19.2% (37/192) before they were 13 years of age, 40.1% (77/192) when they were 13–18 years of age, and 41.1% (79/192) when they were over 18 years of age. in the questionnaire, 310 (25%) of the women answered ‘yes’ to some or all four questions regarding experiences of mild, moderate, or severe sexual violence. of those women, 157 (51%) reported experiences of severe sexual violence. sexual violence during the past year was reported by 38 (3%) of the participants. of all the women who reported exposure to sexual violence in the questionnaire, 220 (71%) stated that the perpetrator was a non-partner. experiences of sexual violence and background characteristics it was more common that women with experiences of sexual violence had 9 or less years of education (p¼ 0.024), were students or without occupation (p¼ 0.037), and were not in a current relationship (p< 0.001) (table 1). women with no experiences of sexual violence were more likely to be in longer relationships. there was also a difference between the two groups, with the women with experiences of sexual violence having a higher total lifetime experience of psychological and/ or physical violence from a present or former partner (p< 0.001). of the women who reported experiences of sexual violence during the interviews or in the questionnaires, 186 (57%) were abused when they were younger than 18 years of age. in the questionnaire, 71 (23%) respondents stated that they had been subjected to severe penetrating sexual violence when they were younger than 18 years of age. in 55 of those cases, the violence was committed by a nonpartner (data not presented). experience of non-partner sexual violence and intimate partner violence the women with experience of non-partner sexual violence were more likely to report experiences of psychological and physical violence from a present or former partner compared to women with no experiences of non-partner sexual violence table 1. distribution of study participants by lifetime sexual violence exposure, a series of background characteristics, and experience of emotional and/or physical violence from a partner or former partner. characteristics experience of sexual violence n (%) no experience of sexual violence n (%) missing values p valuea current age (years) 0.156 15–19 42 (34) 83 (66) 20–30 171 (26) 479 (74) >31 113 (25) 338 (75) relationship 27 <0.001 not in relationship 85 (35) 156 (65) less than 1 year 69 (31) 159 (69) 1–5 years 106 (26) 295 (74) over 6 years 57 (18) 262 (82) place of birth 22 0.131 scandinavia 279 (26) 813 (74) other than scandinavia 36 (32) 76 (68) education 0.024 �9 years of school 64 (33) 129 (67) >9 years of school 262 (25) 771 (75) occupation 0.037 gainfully employed 159 (24) 512 (76) student 113 (31) 255 (69) without occupationb 54 (29) 133 (71) experience of psychological and/or physical violence from a partner or former partner 10 <0.001 yes 151 (47) 170 (53) no 179 (20) 716 (80) apearson’s chi-square test. bunemployed, parental leave, sick leave. upsala journal of medical sciences 137 (p< 0.001). there were no significant differences between the groups regarding experiences of intimate partner violence during the past year (table 2). discussion results of the present study indicate that the prevalence of different types of sexual violence is high within the context of a family planning unit in sweden. experiences of sexual violence were reported by 27% of the participants in this study. this rate can be compared to other prevalence studies in sweden, the nordic countries, and europe. in a national survey from 2014 in sweden with a representative sample of 5681 women, 42% of the women reported experiences of sexual violence (3). a study among swedish adolescents aged 17–23 years old showed that the prevalence of sexual violence was 32% (20). a nordic crosscountry study revealed a prevalence of sexual violence of 24.1% of the 3641 respondents (19). in europe, the prevalence of sexual violence has been estimated to an average of 11% among european union countries (2). sexual violence during the past year was reported by 3% of the women in this study. this is consistent with other studies where the 12-month prevalence of sexual violence was 2%–5.8% (2,3). among the participants who experienced sexual violence, 57% (15% of all the respondents in the study) specified that they were subjected to violence when they were younger than 18 years of age. the european survey showed an occurrence of sexual violence before 15 years of age of 12% of the respondents (2). this is in accordance to who global prevalence studies, including 133 countries, reporting 20% of women having been sexually abused as children (21). the prevalence of non-partner sexual violence was 71% in this study. the respective prevalence has been estimated globally to 7.2% (1) and 6% in the european union countries (2). in addition to acquaintance, friend, or unknown persons, etc., family members that commit sexual violence are also included as non-partner perpetrators. the higher prevalence of nonpartner sexual violence in our study is in line with the large number of participants who reported exposure of sexual violence when they were younger than 18 years of age. women with experience of sexual violence were not as often in a relationship in comparison to women without sexual violence experiences, and the latter group was more likely to be in longer relationships. considering known risk factors such as educational level and occupation, women with experiences of sexual violence reported lower educational level, were students or without occupation compared to women without experiences of sexual violence. socioeconomic status was thus strongly associated with experiences of sexual violence in this setting in sweden, in line with studies from other countries (22). our study results of a high prevalence of sexual violence among teenagers raise the question on the need for preventive efforts in this group. of the women who confirmed experience of non-partner sexual violence in particular, 31% stated that they also had lifetime experience of psychological and/or physical violence from an intimate partner. this could be explained by the fact that the majority of the women in our study reported experience of sexual violence when they were younger than 18 years of age, and that one of the consequences of child sexual abuse is the risk of lifetime co-occurrence of violence (23,24). our results are in line with findings from the swedish national survey which showed a distinct association between experience of sexual violence before 18 years of age and exposure to violence as an adult (3). among the strengths of this study are the large number of participants and available information on the individual level of many related parameters, as well as the specification of perpetrator of sexual violence (partner versus non-partner). a limitation of the study could be the participants’ self-report on experience of different types of violence. nevertheless, by asking specifically structured questions about violence, we minimized the risk of misinterpretations regarding the definition of violence. the study data were collected some years ago, but there have been no big legislative or policy changes during this period. family planning units are structured in similar ways throughout table 2. distribution of participants by sexual violence perpetrator and type of intimate partner violence. type of intimate partner violence n experience of non-partner sexual violence n (%) no experience of non-partner sexual violence n (%) p valuea interviews have you ever experienced physical or psychological violence from a present or former partner? npsv: 220 no npsv: 909 58 (26) 138 (15) <0.001 have you during the past year been slapped, kicked, shoved, or in other ways harmed by a present or former partner? npsv: 219 no npsv: 908 10 (5) 21 (2) 0.067 questionnaire experience of psychological violence from a partner or former partner npsv: 218 no npsv: 909 67 (31) 121 (13) <0.001 experience of physical violence from a partner or former partner npsv: 217 no npsv: 908 75 (24) 177 (13) 0.003 total experience of intimate partner violence (interviews þ questionnaire) npsv: 220 no npsv: 911 69 (31) 281 (20) <0.001 apearson’s chi-square test. npsv ¼ experiences of non-partner sexual violence. 138 m. €oberg et al. sweden, but using solely a family planning unit from one region as a recruitment area may have affected the degree of generalizability of the findings. although all women wanting termination of pregnancy need to visit the family planning unit, the questionnaire being available only in swedish probably resulted in failure to include a number of immigrants who did not speak swedish. the impact of the exclusion of this group of women on the results is hard to hypothesize about, but based on the literature, associations present in this study have been reported in other cultures and thus are not expected to greatly differ in immigrant populations in sweden (1). experiences of sexual violence were common among women participating in this study. many of the respondents were young when they were exposed to violence, and lifetime co-occurrence of violence was common. thus, the results of this study have important clinical implications. identification of victims within the health care system would not only enable the development of effective interventions for treatment but also a possibility to prevent future violence. disclosure statement no potential conflict of interest was reported by the authors. notes on contributors mariella €oberg is a ph.d-student and specialist of obstetrics and gynecologi at the university hospital. alkistis skalkidou is currently a professor of obstetrics and gynecology in uppsala university and works as a senior consultant in the university hospital. research activities include peripartum depression and its effects on the offspring, and ultrasound pregnancy dating. gun heimer is currently a professor and director of national centre for knowledge on men’s violence against women and senior consultant at the university hospital. research activities includes prevalence of men’s physical, psychological and sexual violence against women, health effect of violence, and violence during pregnancy. orcid mariella €oberg http://orcid.org/0000-0002-1244-7278 references 1. world health organization. global and regional estimates of violence against women: prevalence and health effects of intimate partner violence and non-partner 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treatments. mcgill j med. 2006;9:111–18. 13. world health organization/london school of hygiene and tropical medicine. preventing intimate partner violence and sexual violence against women: taking action and generating evidence. geneva: world health organization; 2010. 14. simmons j, wijma b, swahnberg k. lifetime co-occurrence of violence victimization and symptoms of psychological ill health: a cross-sectional study of swedish male and female clinical and population samples. bmc public health. 2015;15:1979. 15. hamby s, grych j. the web of violence. exploring connections among different forms of interpersonal violence and abuse. springer briefs in sociology. new york: springer; 2013. 16. €oberg m, stenson k, skalkidou a, heimer g. prevalence of intimate partner violence among women seeking termination of pregnancy compared to women seeking contraceptive counseling. acta obstet gynecol scand. 2014;93:45–51. 17. soeken kl. the abuse assessment screen: a clinical instrument to measure frequency, severity and perpetrator of abuse against women. in: campbell j, editor. empowering survivors of abuse: health care for battered women and their children. newbury park, ca: sage; 1998. p. 195–203. 18. swahnberg k, wijma b. the norvold abuse questionnaire (noraq): validation of new measures of emotional, physical and sexual abuse, and abuse in the health care system among women. eur j public health. 2003;361:2107–13. 19. wijma b, schei b, swahnberg k, hilden m, offerdal k, pikarinen u, et al. emotional, physical and sexual abuse among patients visiting gynaecology clinics: a nordic cross-sectional study. lancet. 2003;361:2107–13. 20. danielsson i, blom h, nilses c, heimer g, h€ogberg u. gendered patterns of high violence exposure among swedish youth. acta obstet gynecol scand. 2009;88:528–35. 21. world health organization. global status report on violence prevention 2014. geneva: world health organization; 2014. 22. garc�ıa-moreno c, st€ockl h. protection of sexual and reproductive rights: addressing violence against women. int j gynaecol obstet. 2009;106:144–7. 23. simma c, postmus lj, lee i. sexual revictimization in adult women: examining factors associated with their childhood and adulthood experiences. j child sex abus. 2012;21:593–611. 24. de haas s, van berlo w, bakker f, vanwesenbeeck i. prevalence and characteristics of sexual violence in the netherlands, the risk for revictimization and pregnancy: results from a national population’s survey. violence vict. 2012;27:592–608. upsala journal of medical sciences 139 abstract introduction materials and methods subjects variables statistical analysis results prevalence of sexual violence experiences of sexual violence and background characteristics experience of non-partner sexual violence and intimate partner violence discussion disclosure statement notes on contributors references authors' reply: ‘mean platelet volume could be a promising biomarker to monitor dietary compliance i full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 authors' reply: ‘mean platelet volume could be a promising biomarker to monitor dietary compliance in celiac disease' tugrul purnak, yavuz beyazit, cumali efe, ersan ozaslan, osman yuksel & emin altiparmak to cite this article: tugrul purnak, yavuz beyazit, cumali efe, ersan ozaslan, osman yuksel & emin altiparmak (2013) authors' reply: ‘mean platelet volume could be a promising biomarker to monitor dietary compliance in celiac disease', upsala journal of medical sciences, 118:3, 208-208, doi: 10.3109/03009734.2013.806617 to link to this article: https://doi.org/10.3109/03009734.2013.806617 © informa healthcare published online: 10 jul 2013. submit your article to this journal article views: 311 view related articles citing articles: 1 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.806617 https://doi.org/10.3109/03009734.2013.806617 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.806617 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.806617 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.806617#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.806617#tabmodule upsala journal of medical sciences. 2013; 118: 208–208 letter to the editor authors’ reply: ‘mean platelet volume could be a promising biomarker to monitor dietary compliance in celiac disease’ tugrul purnak1, yavuz beyazit1, cumali efe3, ersan ozaslan1, osman yuksel4 & emin altiparmak4 1ankara numune education and research hospital, department of gastroenterology, ankara, turkey, 2ankara yuksek ihtisas education and training hospital, department of gastroenterology, ankara, turkey, 3hacettepe university medical school, department of gastroenterology, ankara, turkey, and 4ankara diskapi education and research hospital, department of gastroenterology, ankara, turkey we thank dr varol for his interest in our article (1). first of all, we agree with dr varol regarding the methodological concern of mean platelet volume (mpv) assessments (2). blood samples must be studied within 2 hours, as in our study, to avoid falsely elevated results. also, when commenting mpv values in different studies, one must be cautious about the analyzer device. different analyzer devices might produce different mpv results, and this fact should be taken into account before generalization of the results. therefore, we tried to use the same analyzer device (beckman coulter analyzer) as in our previous studies (3,4). secondly, dr varol mentions in great detail variables which affect mpv. currently, almost all diseases may influence mpv. excluding mpv-related diseases is practically impossible, but to some extent, however, researchers might take appropriate measures to avoid fluctuations of mpv. moreover, our main driving force behind this study was to find a useful marker for dietary compliance of celiac patients other than the change in mpv. finally, we find dr varol’s hypothesis very intriguing regarding a relationship between celiac disease and cardiovascular disease. hitherto, with respect to cardiovascular mortality among celiac patients, studies have yielded conflicting results. however, in a recent study performed in a large group of patients, it was concluded that celiac patients have more favorable cardiac profiles compared with controls (5). as a result of that, our current knowledge about celiac disease is evolving, and all clues including dr varol’s hypothesis will broaden our horizon. references 1. purnak t, efe c, yuksel o, beyazit y, ozaslan e, altiparmak e. mean platelet volume could be a promising biomarker to monitor dietary compliance in celiac disease. ups j med sci. 2011;116:208–11. 2. varol e. comment on ‘mean platelet volume could be a promising biomarker to monitor dietary compliance in celiac disease’. ups j med sci. 2013;118:206–7. 3. purnak t, olmez s, torun s, efe c, sayilir a, ozaslan e, et al. mean platelet volume is increased in chronic hepatitis c patients with advanced fibrosis. clin res hepatol gastroenterol. 2013;37:41–6. 4. beyazit y, sayilir a, torun s, suvak b, yesil y, purnak t, et al. mean platelet volume as an indicator of disease severity in patients with acute pancreatitis. clin res hepatol gastroenterol. 2012;36:162–8. 5. emilsson l, carlsson r, holmqvist m, james s, ludvigsson jf. the characterisation and risk factors of ischaemic heart disease in patients with coeliac disease. aliment pharmacol ther. 2013; 37:905–14. correspondence: tugrul purnak, md, ankara numune education and research hospital, department of gastroenterology, talatpasa bulvari, samanpazari 06100 sihhiye, ankara, turkey. fax: +90 312 3125026. e-mail: purnakt@yahoo.com (received 13 may 2013; accepted 14 may 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.806617 http://informahealthcare.com/journal/ups www.ncbi.nlm.nih.gov/pubmed/21679011?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21679011?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22572524?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22572524?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22572524?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22572524?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22572524?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22572524?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22088974?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22088974?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/23451861?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/23451861?dopt=abstract mailto:purnakt@yahoo.com ss1 references upsala j med sci 88: 25-31, 1983 composition of surface layers in unfixed autologous fascia lata heart valve grafts a transmission electron microscopical study roger willcn’, helena willcn’, wieslaw t. dubie12 and lennart johansson’ from the department of histopathology’. university hospital, lund, and department of thoracic and cardiovascular surgery’, university hospital, uppsala, sweden abstract sixty-nine p a t i e n t s underwent a o r t i c valve replacement w i t h frame mounted non-fixated f a s c i a l a t a g r a f t s . nine p a t i e n t s s t i l l have t h e i r f a s c i a l a t a valve i n p l a c e a f t e r a period of between 9 t o 1 1 y e a r s . fifty-two g r a f t s were a v a i l a b l e f o r h i s t o p a t h o l o g i c examination. the mechanism of valve t h i c k e n i n g was s t u d i e d by t r a n s m i s s i o n e l e c t r o n microscopy ( t e m ) . the major f a c t o r c o n t r i b u t i o n t o t h e t h i c k e n i n g seems t o be a s u r f a c e accumulation of complex p r o t e i n s , f i b r i n and p l a t e l e t s . w i t h i n t h e v a l v u l a r t i s s u e i t s e l f , s u b c e l l u l a r d e g e n e r a t i v e products i n c e l l cytoplasm, t h e i n t e r s t i t i u m and i n t h e sub-surface l a y e r a l s o c o n t r i b u t e d . the t h i c k e n i n g never exceeded one f i f t h of t h e o r i g i n a l t h i c k n e s s of t h e valve. introduction unfixed autologous f a s c i a l a t a h e a r t valve g r a f t m a t e r i a l i s f a i r l y rough on one s u r f a c e , with s l i g h t l y p r o t r u d i n g smooth c o l l a g e n bundles a t t h e o t h e r ( 1 4 ) . the rough s i d e i s i n i t i a l l y covered by f a t , c o n n e c t i v e t i s s u e and v e s s e l s . a f t e r s u r g i c a l t a i l o r i n g , some remnants of small damaged f i b e r s of f a s c i a l t i s s u e remain. implanted i n t h e blood s t r e a m , t h e f a s c i a l tissue i s r a p i d l y covered by a f i n e , g r a n u l a r m a t e r i a l , probably p r o t e i n ( 2 , 1 4 ) . small fragments of f i b r i n and p l a t e l e t s can be v i s u a l i z e d (14). t h i s covering makes t h e s u r f a c e smoother and may e x p l a i n t h e f r e q u e n t l a c k of c l i n i c a l thrombus formation w i t h t h e s e p r o s t h e s e s ( 6 , 10, 1 1 ) . the blood born elements which adhere t o t h e s u r f a c e l a t e r become organized mainly on t h e rough s u r f a c e . eventually t h e v a l v u l a r t i s s u e i t s e l f undergoes f o c a l d e g e n e r a t i v e and hyper p l a s t i c changes ( 3 , 4 , 151, probably a s a r e s u l t of mechanical s t r e s s ( 5 ) . d i f f e r e n t s t a g e s o f t i s s u e breakdown can be i l l u s t r a t e d ( 1 4 , 1 5 ) . f i n a l l y , shrinkage c a l c i f i c a t i o n and v a l v e t h i c k e n i n g appear i n some of t h e valves w i t h subsequent f a i l u r e due t o r u p t u r e o r dysfunction (4). the aim of t h i s paper i s t o d i s c u s s t h e p o s s i b l e c o n t r i b u t i o n t o valve t h i c k e n i n g by the endogenous breakdown of f a s c i a l tissue i t s e l f which becomes enveloped by the 25 s u r f a c e pseudomembrane. patients between november 13, 1969, and june 7 , 1972, 69 p a t i e n t s underwent i s o l a t e d a o r t i c valve replacement w i t h a frame-mounted f a s c i a l a t a g r a f t . of the 55 p a t i e n t s r e l e a s e d from t h e h o s p i t a l , 30 d i e d a f t e r between 5 months and 83 y e a r s . in 3 o f t h e s e c a s e s t h e cause of d e a t h was g r a f t f a i l u r e and, i n 2 7 , myocardial f a i l u r e sometimes i n combination w i t h s e p t i c i n f e c t i o n ( 5 p a t i e n t s ) . twenty-two p a t i e n t s were r e o p e r a t e d due t o g r a f t f a i l u r e w i t h a o r t i c r e g u r g i t a t i o n and 6 of t h e s e died during o r soon a f t e r t h e r e o p e r a t i o n . nine p a t i e n t s s t i l l have t h e i r o r i g i n a l f a s c i a l a t a valve g r a f t i n p l a c e a f t e r a period of between 9 and 1 1 y e a r s and most of them a r e doing well ( f i g . 1 ) . died q x o r postop died in cardiae failure, 5 with septic infection. ,6 op. or postop. dead \16 well with other 22 reop. prosthesis. 9 well with fascialata graft. f i g . 1 . c l i n i c a l m a t e r i a l . ultimate f a t e of 69 f a s c i a l a t a h e a r t a o r t i c valve o p e r a t e d p a t i e n t s . methods the t i s s u e was inbedded f o r scanning e l e c t r o n microscopy (sem) a s p r e v i o u s l y d e s c r i b e d ( 1 4 , 1 5 ) . in o r d e r t o c a r r y o u t t r a n s m i s s i o n e l e c t r o n microscopy (tem) t h e scanning e l e c t r o n microscopy t i s s u e was trimmed i n t o small blocks 3 x 3 mm and p u t i n t o a mixture of propylenoxide/epon 1 : l over n i g h t . the following morning t h e t i s s u e was l e f t i n epon f o r 4 t o 6 hours. i t was then p u t i n beemr-capsules f i l l e d w i t h f r e s h l y made r e s i n mixture and hardened a t 6ooc f o r 3 t o 4 days. the blocks were c u t i n t h e usual way on a 26 l k b u l t r a t o m e r . the s e c t i o n s were mounted on formvar c o a t e d grids, s t a i n e d w i t h l e a d c i t r a t e f o r 4 minutes, rinsed and d r i e d , c o u n t e r s t a i n e d w i t h uranyl a c e t a t e f o r 30 minutes a t 37oc, and f i n a l l y r i n s e d and d r i e d . the gold l a y e r p u t on t h e v a l v u l a r s u r f a c e i n the p r e p a r a t i o n f o r t h e sem i n v e s t i g a t i o n served a s a marker f o r i d e n t i f i c a t i o n of f a s c i a 1 s u r f a c e . results central p a r t s of t h e normal v a l v u l a r t i s s u e showed a r e a s w i t h well pre served f i b r o c y t e s and f i brobl a s t s surrounded by numerous col 1 agen f i b e r s ( 1 4 , 1 5 ) . sometimes markedly degenerated a r e a s were observed, showing f i b r o b l a s t s with e n d o c y t i c a c t i v i t y , and i n t e r s t i t i a l t i s s u e which c o n t a i n e d amorphous m a t e r i a l and only a very few c o l l a g e n f i b r i l s ( f i g . 2 ) . some of t h e c e l l s contained p a r t l y degenerated rough and smooth endopl asmic r e t i c u l u m , primary and secondary lysosomes and o t h e r c e l l d e b r i s ( f i g s . 3 , 4 ) . some f i b r o b l a s t s fig. 2 . e l e c t r o n micrograph of f a i r l y well preserved f i b r o b l a s t with endo c y t i c a c t i v i t y (arrow) and s l e n d e r p r o t r u s i o n s . degeneration i s e v i d e n t i n t h e i n t e r c e l l u l a r space where mostly amorphous material and only s c a n t c o l l a g e n bundles a r e seen ( o r i g . mag. x 5 0 0 0 ) . showed s l i g h t bulging of t h e plasma membrane i n a s s o c i a t i o n w i t h lysosomes and o u t s i d e t h e c e l l e x p e l l e d r e s i d u a l bodies were v i s u a l i z e d ( f i g . 4 ) . sometimes t h e i n t e r s t i t i u m was f i l l e d w i t h well preserved c o l l a g e n bundles showing c r o s s s t r i a t i o n ( f i g . 5 ) . s t u d i e s of a r e a s c l o s e r t o t h e s u r f a c e showed abundant c o l l a g e n f i b r i l s t o g e t h e r w i t h l i g h t ground s u b s t a n c e , c e l l d e b r i s and degenerated p l a t e l e t s ( f i g . 6 ) . i t was thus p o s s i b l e t o f i n d s u b c e l l u l a r breakdown products w i t h i n a l l p a r t s of t h e v a l v u l a r t i s s u e ; i n c e l l cytoplasm a s well a s i n t h e i n t e r s t i t i u m . an accumulation o f t h e s e products was seen a t 27 fig. 3 . electron micrograph o f f i b r o b l a s t . p a r t of nucleus (bottom) and c y t o plasm f i l l e d with rough endoplasmic r e t i c u l u m autophagocytic vacuoles (arrow h e a d ) , primary lysosomes (arrow) and c e l l d e b r i s ( o r i g . mag. x 16 0 0 0 ) . fig. 4. electron micrograph of p a r t i a l l y degenerated f i b r o b l a s t c o n t a i n i n g primary and secondary lysosomes ( l a r g e arrow-head), s l i g h t bulging of plasma membrane i n a s s o c i a t i o n with a lysosome (small a r r o w ) , and e x p e l l e d lysosomal m a t e r i a l (small arrow-head). in t h e i n t e r f a c e abundant c o l l a g e n f i b e r s , showing c r o s s s t r i a t i o n ( l a r g e a r r o w ) , ( o r i g . mag. x 5 0 0 0 ) . t h e s u r f a c e , b u t t h e t h i c k n e s s of t h i s accumulation never exceeded one f i f t h o f t h e o r i g i n a l v a l v u l a r t h i c k n e s s . 28 f i g . 5 . e l e c t r o n micrograph of c o l l a g e n bundles, p a r t l y degenerated ( a r r o w ) , p a r t l y showing e v i d e n t c r o s s s t r i a t i o n . in t h e i n t e r f a c e secondary lysosomes of d i f f e r e n t s i z e s and shapes ( o r i g . mag. x 20 0 0 0 ) . fig. 6 . s u r f a c e a r e a c o n t a i n i n g degenerated p l a t e l e t s ( a r r o w ) , amorphous m a t e r i a l a n d c e l l d e b r i s . outer s u r f a c e i s covered by e l e c t r o n opaque m a t e r i a l . discussion from t h i s and e a r l i e r s t u d i e s (2, 4 , 6 , 7, 9, 1 0 , 1 4 , 15) i t i s e v i d e n t t h a t the events t a k i n g p l a c e a t the s u r f a c e o f and c e n t r a l l y i n b i o p r o s t h e t i c v a l v u l a r tissue a r e very complex and t h a t t h e c a u s e s of t h i c k e n i n g , s h r i n k a g e a n d d y s f u n c t i o n i n t h e s e p r o s t h e s e s a r e not f u l l y understood. in t h i s s t u d y i t has been shown t h a t t r a n s i t o r y adhesion of complex p r o t e i n s a n d p l a t e l e t s 29 t a k e s p l a c e on t h e s u r f a c e of t h e t i s s u e v a l v e , b u i l d i n g u p a s u r f a c e membrane. within t h i s membrane and i n t h e c e n t e r of t h e c u s p , breakdown products from v i a b l e f a s c i a l c e l l s could be v i s u a l i z e d both w i t h i n t h e c e l l cytoplasm a s well a s w i t h i n t h e i n t e r s t i t i a l t i s s u e . i t was thought t h a t these two p r o c e s s e s i n combination c o n t r i b u t e d t o t h e t h i c k e n i n g of t h e valve a s adhesion and d e g r a d a t i o n products occupied so much space a s v i s u a l i z e d i n t h e tem i n v e s t i g a t i o n . t h e formation of a complex p r o t e i n l a y e r a t the s u r f a c e was thought t o prevent f u r t h e r p l a t e l e t adhesion and t h a t a t i s s u e antithrombogenic inducing f a c t o r e x i s t s i n t h e proteoglycan f r a c t i o n ( 2 ) . however, continuous adhesions of f i b r i n fragments o r thrombous m a t e r i a l seem t o t a k e p l a c e on t h e cusp s u r f a c e . these o r g a n i z e t o form a c o n n e c t i v e t i s s u e pseudointima ( 3 , 10, 1 1 , 1 2 , 1 3 ) . in o r d e r t o prevent t h i s p r o c e s s , t h e use of a n t i c o a g u l a n t s i n t h e i n i t i a l phase a f t e r valve implantation e . g . 6 weeks t o 1 0 months, has been advocated ( 1 , 5 , 9 ) . when using non-fixed b i o p r o s t h e t i c v a l v u l a r t i s s u e i t was e v i d e n t from t h i s s t u d y t h a t breakdown products from v i a b l e c e l l s w i t h i n t h e f a s c i a l t i s s u e i t s e l f , c o n t r i b u t e t o t h e t h i c k e n i n g o f t h e cusp. this f a c t o r can only be reduced by f i x a t i o n of the v a l v u l a r t i s s u e b e f o r e i n s e r t i o n i n t o t h e h o s t by e . g . formaldehyd o r g l u t a r a l d e h y d . the l a t t e r i s now in common p r a c t i c e . and 1. 2. 3. 4. 5. 6. 7. 30 acknowledgement this work was supported by t h e swedish national a s s o c i a t i o n a g a i n s t heart chest diseases. references bernhard, a . , thiede, a . , muller-hermelink, h k . , krug, a , , f i s c h e r , k. & yankah, a . c . : mitral v a l v e replacement w i t h autologous f a s c i a l a t a . result of morphologic examinations. j . thorac. cardiovasc. surg. 64:94, 1973. c a r p e n t i e r , a , , relland, j . , c a r p e n t i e r , s . , lessana, a. & gory, g . : t i s s u e antithrombogenic inducing f a c t o r : experimental evidence and p r a c t i c a l a p p l i c a t i o n s i n t h e c o n s t r u c t i o n of c a r c i a c v a l v e s and the a r t i f i c i a l h e a r t . thromb. diates. haemorrag. 34:557, 1975. dubiel, w.t., johansson, l . & willen, r . : p o s t o p e r a t i v e changes i n autologous f a s c i a l a t a h e a r t valve g r a f t s . a n n . thorac. surg. 15:140, 1973. dubiel, w.t. & willen, r . : v i a b i l i t y p r o p e r t i e s i n autologous fascia lata heart valve g r a f t s , l a t e o b s e r v a t i o n s i n : s h i l e y european c a r c i o v a s c u l a r conference, chamonix, france, 1979, p . 161. , f . : uberlagungen z u r f u n k t i o n e l l e n s t r u k t u r d e r f a s c i e . zbl vete? 9 reie a ) 13:85, 1966. jonescu, m.i., p a k r a s h i , b . c . , mary, d.a.s., bartek, i.t. & wooler, g . h . : long term e v a l u a t i o n of t i s s u e v a l v e s . 3 . thorac. cardiovasc. surg. 68: 361, 1974. senning, 8 . : f a s c i a l a t a replacement of a o r t i c v a l v e s . j . thorac. cardio vasc. surg. 54:465, 1967. surg. 68:379, 1974. 9. s h i l e y european c a r d i o v a s c u l a r c o n f e r e n c e , chamonix, f r a n c e , 1979, p. 108. 1 0 . s i l v e r , m . d . , hudson, r . e . b . & t r i m b l e , a.s.: morphologic o b s e r v a t i o n s on h e a r t v a l v e p r o s t h e s e s made o f f a s c i a l a t a . j . thorac. c a r d i o v a s c . surg. 70:360, 1975. 1 1 . s i l v e r , m . d . & t r i m b l e , a.s.: s t r u c t u r e o f a u t o l o g o u s f a s c i a l a t a h e a r t v a l v e p r o s t h e s e s . arch. p a t h . 93:109, 1972. 1 2 . t r i m b l e , a.s., gunstensen, j . , s i l v e r , m . d . , a l d r i d g e , h . e . , s c h w a r t z , l . & morch, j.e.: a o r t i c v a l v e r e p l a c e m e n t w i t h f a s c i a l a t a : an e n c o u r a g i n g l a t e s t u d y . j . thorac. c a r d i o v a s c . surg. 68:219, 1974. 13. welch, w . , p o v l i e g e , p. & promo, g . : autologous f a s c i a l a t a c a r d i a c v a l v e r e p l a c e m e n t . thorax 26:271 , 1971. 14. willgn, r . , d u b i e l , w.t. & j o h a n s s o n , l . : v i a b i l i t y and s u r f a c e t w o p e r t i e s o f a u t o l o g o u s f a s c i a l a t a h e a r t v a l v e g r a f t s . ann.-thorac. s u r g . ' 18':597, 1974. 15. willgn, r . m d u b i e l , w.t. & j o h a n s s o n , l . : v i a b i l i t y p r o p e r t i e s i n a u t o l o g o u s f a s c i a l a t a h e a r t v a l v e g r a f t s . a n n , thorac. surg. 19:494, 1975. address f o r r e p r i n t s : roger willgn, m . d . department o f p a t h o l o g y u n i v e r s i t y h o s p i t a l 5-221 8 5 l u n d , sweden 31 placental glucocorticoid receptors are not affected by maternal depression or ssri treatment article placental glucocorticoid receptors are not affected by maternal depression or ssri treatment åsa edvinssona,b , angela hoyera, malin hanssona, theodora kunovac kallaka , inger sundstr€om-poromaaa , alkistis skalkidoua and susanne lagera adepartment of women’s and children’s health, uppsala university, uppsala, sweden; bdepartment of immunology, genetics and pathology, uppsala university, uppsala, sweden abstract background: prenatal depression is common, with an estimate that up to one in five pregnant women suffers from depressive symptoms. maternal depression is associated with poor pregnancy outcomes such as preterm birth and low birth-weight. such outcomes possibly affect offspring development. previous studies suggest placental rna levels of the glucocorticoid receptor are altered by maternal depression or anxiety; this stress may affect the placenta of male and female foetuses differently. however, it is unknown if the protein levels and activity of this receptor are additionally affected in women with depressive symptoms or being pharmacologically treated for depression. methods: in this study, we investigated whether the glucocorticoid receptor (nr3c1) in the placenta is affected by maternal depression and/or selective serotonin reuptake inhibitor (ssris) treatment. placentas from 45 women with singleton, term pregnancies were analysed by western blot to determine glucocorticoid receptor levels, and by dna-binding capacity to measure glucocorticoid receptor activation. results: there were no differences in levels of the glucocorticoid receptor or activity between groups (control, depressive symptoms, and ssri treatment; n ¼ 45). similarly, there was no difference in placental glucocorticoid receptor levels or activity dependent upon foetal sex. conclusion: maternal depression and ssri treatment do not affect the glucocorticoid receptors in the placenta. article history received 21 august 2019 revised 23 november 2019 accepted 4 december 2019 keywords nr3c1; pregnancy; prenatal depression; ssri; western blot introduction during pregnancy, women experience major physical and psychological changes which increase their risk for development of mental illness. it is estimated that approximately 10–20% of pregnant women experience depressive symptoms (1). the proportion of women suffering from major depressive disorder in pregnancy is lower, but it still affects many women, with a prevalence of about 3–5% (2,3). to manage depression, selective serotonin reuptake inhibitors (ssris) are commonly prescribed medications during pregnancy. in europe, up to 4.5% of pregnant women are prescribed ssris (4). in the usa, it is twice as common that women use antidepressant drugs during pregnancy (5). both maternal depression and ssri treatment are associated with poor pregnancy outcomes (6–9). it is common that pregnant women experience anxiety, depression, and psychosocial distress. such distress often stimulates the hypothalamic–pituitary–adrenal (hpa) axis, resulting in release of stress hormones (for instance cortisol) (10). it has been shown that direct transfer of cortisol from the maternal circulation to the foetus across the placenta is limited (11). but cortisol can also exert its effects through the glucocorticoid receptor (nr3c1) (12), which is present in the placenta (13). maternal stress has been shown to directly affect the placenta. for instance, increased placental rna levels of the glucocorticoid receptor have been observed in association with maternal perceived stress or depressive symptoms (14–16). interestingly, the effects of maternal stress may affect the placenta from female and male infants differently (17,18). previous studies have focussed on glucocorticoid receptor mrna levels, which may not reflect the protein amount of receptor present in the tissue. it is currently unknown if protein levels or activation of the placental glucocorticoid receptor are affected as well. therefore, the aim of this study was to determine if maternal depression or ssri treatment affects placental protein levels of glucocorticoid receptor. material and methods study population samples utilized in this study are from the biology, affect, stress, imaging, and cognition (basic) cohort conducted at uppsala university hospital, department of women’s and children’s health (19). the study was approved by the contact susanne lager susanne.lager@kbh.uu.se department of women’s and children’s health, uppsala university, uppsala, 751 85, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 1, 30–36 https://doi.org/10.1080/03009734.2019.1702126 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1702126&domain=pdf&date_stamp=2020-02-19 http://orcid.org/0000-0002-6246-7218 http://orcid.org/0000-0002-2112-8674 http://orcid.org/0000-0002-2491-2042 http://orcid.org/0000-0002-4935-7532 http://orcid.org/0000-0003-3556-065x http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2019.1702126 http://www.tandfonline.com regional ethical review board in uppsala (dnr 2009/171 with amendments). all participants gave informed written consent. briefly, women who were registered for a routine ultrasound scan at akademiska university hospital in uppsala around 17 weeks gestation were asked about participation in the basic study. from participating women, data were collected through web-based surveys, including the edinburgh postnatal depression scale (epds) (20), at 17 and 32 weeks gestation. a sub-set of women was invited to a visit in late pregnancy (around 38 weeks gestation) for a structured psychiatric interview (mini international neuropsychiatric interview, mini). from the basic cohort, 45 women with singleton pregnancies were selected for the present study. the women were divided into three groups: control, depressive symptoms, and ssri treatment. women in the control group had a maximum epds score of 9 during pregnancy and no history of psychiatric illness according to medical records (n ¼ 17). the women in the depressive symptoms group had an epds score of 12 or greater in gestational weeks 17 and/or 32 (n ¼ 14). six of these women were depressed according to mini conducted in late pregnancy. the women in the ssri treatment group used ssri medication during at least half of the pregnancy (n ¼ 14). all the women in the ssri treatment group had measurable blood concentrations of their prescribed ssri at delivery (except one woman prescribed sertraline where no blood sample was available for testing). clinical characteristics of the selected pregnancies are presented in table 1. tissue collection placental tissue collection was carried out as soon as possible after delivery. a full-thickness biopsy was sampled from the central part of the placenta, selecting areas devoid of calcifications and infarcts. the samples were rinsed in sterile phosphate-buffered saline, frozen on dry ice within 30 min after delivery, and then stored at �70 �c until further processing. from the frozen tissue biopsies, small samples were carefully cut from the villous tissue to exclude tissue from the basal and chorionic plates. western blot placental villous tissue samples were homogenized in ripa buffer (25 mm tris-hcl ph 7.6, 150 mm nacl, 1% np-40, 1% sodium deoxycholate, 0.1% sds; cat# 89900, thermo fisher scientific) containing halt protease inhibitor cocktail (cat# 87785, thermo fisher scientific). protein concentrations of homogenates were determined using the bradford assay (b6919, sigma-aldrich, st. louis, mo). western blot was performed on 4–12% bis-tris pre-cast gels (np0323, thermo fisher scientific, waltham, ma) and transferred onto pvdf membranes (ipfl00010, merck millipore, burlington, ma). after transfer, membranes were stained for total protein with amido black staining solution (a8181, sigma-aldrich) and/or ponceau s staining solution (p7170, sigma-aldrich). total protein stains have been previously proposed as a good reference for western blotting of placental proteins (21). thereafter, membranes were blocked for 1 h at room temperature in odyssey blocking buffer (927–40000, li-cor biosciences, lincoln, ne). after blocking, the membranes were probed with beta-actin (final concentration 0.2 mg/ml; sc-47778, santa cruz biotechnology, dallas, tx; 1 h at room temperature) or glucocorticoid receptor (nr3c1; final concentration 81 ng/ml; ab109022, abcam, cambridge, uk; table 1. clinical characteristics. healthy control depressive symptoms ssri treatment mother n 17 14 14 age at delivery (years) 30 ± 3 31 ± 5 31 ± 5 bmi (kg/m2) 24.9 (21.5–32.2) 23.7 (21.1–26.4) 24.9 (22.5–29.3) primiparous women 8 (47%) 6 (43%) 4 (29%) preeclampsia 0 (0%) 0 (0%) 0 (0%) hypertension 0 (0%) 0 (0%) 0 (0%) diabetes 0 (0%) 0 (0%) 0 (0%) ethnicity (born in scandinavia) 17 (100%) 14 (100%) 14 (100%) college/university education 15 (88%) 10 (71%) 10 (77%) missing 0 0 1 mode of delivery vaginal 13 (76%) 11 (79%) 11 (79%) elective cs 1 (6%) 2 (14%) 2 (14%) intrapartum cs 3 (18%) 1 (7%) 1 (7%) epds, gestational week 17 2 (1–7) 13 (12–16) 9 (5–12) missing 0 0 1 epds, gestational week 32 3 (1–6) 15 (14–16) 7 (5–9) missing 1 0 2 ssri 0 (0%) 0 (0%) fluoxetine, 5 (36%); sertraline, 5 (36%); citalopram, 4 (29%) newborn gestational length (days) 281 (276–288) 286 (280–290) 277 (272–280) birth-weight (kg) 3.76 ± 0.68 3.90 ± 0.42 3.66 ± 0.38 sex, proportion female 7 (41%) 7 (50%) 6 (35%) nicu care after delivery 0 (0%) 0 (0%) 1 (7%) data are presented as mean ± sd, median (iqr), or number (%). bmi: body mass index; cs: caesarean section; epds: edinburgh postnatal depression scale; iqr: interquartile range; nicu: neonatal intensity care unit; sd: standard deviation; ssri: selective serotonin reuptake inhibitor. upsala journal of medical sciences 31 overnight at þ4 �c). immunolabeling was visualized with fluorescently labelled secondary antibodies (926–68070 or 926–32211, li-cor biosciences) in an odyssey sa scanner (licor biosciences). images were analysed with imagej (version 1.52a). placental levels of the glucocorticoid receptor (gr) were adjusted for total protein staining intensity (ponceau s staining). transcription factor activity nuclei were isolated from 40 mg of placental villous tissue using the nuclear extraction kit (ab113474; abcam). protein concentrations of isolated nuclei were measured with the bradford assay (sigma-aldrich). the dna binding capacity of nr3c1 was determined with the glucocorticoid receptor transcription factor assay kit (ab207207; abcam) using 3.9 mg of nuclear extract per well. nuclei extraction and gr transcription factor assay were performed according to the manufacturer’s instructions. the gr transcription factor activity was tested in duplicate for each placenta. statistics statistical analysis was carried out using ibm spss statistics, version 25. placental glucocorticoid receptor levels and activity were tested for normality using the shapiro–wilk test; differences between the groups were evaluated by one-way anova test, t test, kruskal–wallis, or mann–whitney u test as appropriate. results western blotting loading control prior to examining placental levels of the glucocorticoid receptor, three different approaches were evaluated as potential loading controls for the western blot analysis: total protein staining by amido black and ponceau s, as well as probing the membrane for beta-actin (figure 1). ponceau s was selected as the appropriate loading control and used in subsequent experiments. placental nr3c1 protein the glucocorticoid receptor was detected as a main band at approximately 100 kda (figure 2(a)). in 45 placentas, levels of the glucocorticoid receptor were measured and adjusted for total protein (ponceau s staining; figure 2(b)). detectable level of the glucocorticoid receptor was present in all but two samples. the results were similar when including these two samples (the value of glucocorticoid receptor level as zero) or excluding the samples completely from the analysis. when comparing placentas from healthy controls, women with depressive symptoms, and women using ssris during pregnancy, no difference in glucocorticoid receptor levels between the groups was observed (figure 2(c)). response to these stressors did not differ depending on foetal sex (figure 2(d)). similarly, there was no difference in glucocorticoid receptor levels when comparing placentas from female and figure 1. western blot loading control. for evaluation of loading controls, one membrane was first stained with: (a) ponceau s; followed by (b) amido black; and lastly (c) the membrane was probed for beta-actin. (d) quantification of total protein stains and beta-actin. far left lane contained a molecular weight marker. ponceau s displayed values closest to the expected doubling and was used as loading control for subsequent experiments. the mean values of the 10 mg protein loading signals were assigned a value of 1. data are presented as mean ± sem. 32 å. edvinsson et al. male foetuses (mean nr3c1/total protein levels 0.042 ± 0.004 versus 0.038 ± 0.004; n ¼ 20–25/group, p > 0.05, t test). placental glucocorticoid receptor dna binding activity dna binding activity of the glucocorticoid receptor was detected in all 45 placentas investigated. there was no difference in glucocorticoid receptor activity between the three groups of women (healthy controls, depressive symptoms, and ssri treatment; figure 3(a)). further, there was no difference in activation of the glucocorticoid receptor relating to foetal sex, neither when comparing placentas from healthy controls, depressive symptoms, and ssri treatment (figure 3(b)), nor when comparing placentas from female versus male foetuses (median gr activation 0.057 [95% ci 0.050–0.083] versus 0.074 [95% ci 0.067–0.107]; n ¼ 20–25/ group, p > 0.05, mann–whitney u test). discussion in this study, we show that maternal depressive symptoms or use of ssris during pregnancy do not affect placental glucocorticoid receptor protein levels. our finding contrasts with some previous reports describing an association between maternal prenatal stress and higher placental glucocorticoid receptor mrna levels. maternal depressive symptoms or ssri treatment affects placental rna levels of multiple genes (22,23). ssris can also influence the function of placental cells in vitro (24–26). these studies clearly demonstrate that such circumstances (maternal depression and ssri treatment) affect the placenta. what factors are causative of such alterations need to be determined. however, several differences in circulating factors have been reported with maternal depression or ssri treatment (27–30). it is possible that one or more of these factors could alter placental rna and protein levels, including the glucocorticoid receptor. the human placenta expresses several isoforms of the glucocorticoid receptor (13). for this study, we used an antibody which detects both nr3c1a and nr3c1b isoforms of the glucocorticoid receptor. in our western blot analysis, one main band for the glucocorticoid receptor isoform was observed. this band likely consists of full-length glucocorticoid receptor a and b. detecting both isoforms together might be considered a limitation in the current study. however, it is in accordance with many previous studies focussing on placental rna levels of nr3c1 (14–16,23,31–34). furthermore, a strong positive correlation between total nr3c1 and nr3c1a mrna levels has been reported, a relationship also observed to a lesser extent for nr3c1b levels (17). this suggests that total glucocorticoid receptor levels are informative. we did not observe an effect of maternal ssri treatment upon placental glucocorticoid receptor levels. this finding is figure 2. glucocorticoid receptor protein in the placenta. (a, b) representative western blot of nr3c1 in placenta and corresponding ponceau s stain; 20 mg of placental samples from healthy controls (healthy), women with depressive symptoms (depres.), and women with ssri treatment (ssri) were loaded on each western blot gel together with two control samples (cnt.). the two control samples were loaded on all gels. in the far left lane, a molecular weight marker was loaded. nr3c1 was detected as a main band at �100 kda; a weaker band was detected between 55 and 75 kda. (c) quantification of placental nr3c1 (�100 kda band) separated into groups (healthy control, depressive symptoms, and ssri treatment). amount of nr3c1 was adjusted for total protein. n ¼ 14–17/group; p > 0.05, one-way anova. (d) foetal sex and placental nr3c1 protein levels. n ¼ 6–10/group; p > 0.05, one-way anova. data in graphs are presented as mean ± sem. upsala journal of medical sciences 33 in accordance with a previous study investigating rna levels by microarray (23). olivier and co-workers did, however, show that maternal ssri treatment can affect the placenta, as genes relating to for instance cellular function and maintenance were differentially expressed (23). this suggests that, although ssris may have an impact on the placenta, its capacity to respond to glucocorticoids remains. in this study, we found no effect of maternal depressive symptoms upon placental glucocorticoid receptor levels. our finding is in accordance with a study by st-pierre and coworkers, which did not observe an association between maternal depressive symptoms and placental nr3c1 mrna levels (17). however, several other studies have suggested that maternal depressive symptoms (15,16) or high perceived stress (14) affect placental glucocorticoid receptor mrna levels. there may be several possible causes for these divergent results. first, the timing of experienced stress may influence effects on the glucocorticoid receptor. that is, if stress occurs in early, middle, or late pregnancy. capron (15) estimated depressive symptoms the day before delivery, whereas in our study depressive symptoms were measured at 17 and 32 weeks gestation. second, degree of stress may also affect the placental glucocorticoid receptor. a continuous low-grade stress may have different effects than a sudden, very stressful event. finally, the type of stress experienced may also have an impact. maternal stress can be estimated with a range of different tools, such as epds [current study and (15,17)], centre for epidemiologic studies–depression scale (16), perceived high stress (14), and experience of a natural disaster (17). therefore, maternal stress can be viewed as an umbrella term capturing a range of states that may or may not affect cellular signalling in the placenta. multiple types of stress experienced by the pregnant mother and the effects upon placental glucocorticoid receptor have been investigated. some stresses appear to affect placenta from female and male foetuses differently. the stress of experiencing a natural disaster during pregnancy reduces glucocorticoid receptor rna levels in placentas from male foetuses but not female foetuses (17), whereas high perceived emotional distress in the mother increases nr3c1a rna levels in placentas from female but not male foetuses (18). furthermore, mina and co-workers reported that the levels of nr3c1a mrna differ between placentas from female and male foetuses (18). we could not confirm such findings in our study. neither the response to maternal depressive symptoms and ssri treatment, nor overall glucocorticoid receptor levels differed depending on foetal sex in our cohort. another factor suggested as influencing how the placenta responds to stress is ethnicity. in a study by capron and coworkers, maternal depressive symptoms were associated with increased placental nr3c1 rna levels but only in caucasian mothers (15). in our study, we did not observe such an association in this cohort of scandinavian mothers. it is well established that glucocorticoids are very important for adequate foetal development, but also that excess exposure can have potentially harmful effects, such as growth restriction (35). glucocorticoids exert numerous effects on placenta, including influence of placental development, regulating placental nutrient transport and release of hormones (36). we report that protein levels of glucocorticoid receptor are unchanged for maternal depressive symptoms or ssri treatment. therefore, the placentas retain their capacity to respond to glucocorticoids. this observation is further supported by a similar level of glucocorticoid receptor activation, independent of maternal depressive symptoms or ssri treatment. an altered sensitivity to glucocorticoids could have major effects on placental function, and subsequently the intrauterine environment, as glucocorticoids can regulate a substantial number of genes in our genome (35). in conclusion, maternal depression and ssri treatment do not affect glucocorticoid receptor protein levels or activity in placenta. further research is needed for confirmation of the effects of prenatal stress upon placental biological mechanisms, as well as additional effects upon offspring. acknowledgements the authors are grateful to all basic study participants and staff at akademiska university hospital delivery unit for sample collection. figure 3. placental glucocorticoid receptor activity. activity of the glucocorticoid receptor measured in placental nuclear isolates by dna binding capacity. (a) glucocorticoid receptor activity in the three groups of women (healthy control, depressive symptoms, and ssri treatment). n ¼ 14–17/group; p > 0.05, kruskal–wallis. (b) foetal sex and placental glucocorticoid receptor activation. n ¼ 6–10/group, p > 0.05, kruskal–wallis. data in graphs are presented as median, interquartile range, and 95% confidence interval. 34 å. edvinsson et al. disclosure statement a.s. occasionally serves as an invited speaker at scientific meetings for ferring pharmaceuticals. i.s.p. occasionally serves on advisory boards or act as invited speaker at scientific meetings for msd, bayer health care, peptonics, shire/takeda, and lundbeck a/s. å.e., a.h., m.h., t.k.k., and s.l. have no conflicts of interests to declare. funding this study was supported by grants from the swedish research council [vr:521–2013-2339 and vr:523–2014-2342]; f€odelsefonden; and lions. notes on contributors åsa edvinsson, phd, is a postdoc at uppsala university. she received her master of medical science, biomedicine and her phd from uppsala university. her doctoral thesis focused on inflammation, placenta, and peripartum depression. angela hoyer, msc, is a research assistant at karolinska institutet in the field of asthma research in children. she received her master degree in molecular medicine from uppsala university, and her bachelor degree from erlangen university, germany. malin hansson, md, works as a doctor at hudiksvall hospital, sweden. she received her medical degree from uppsala university. theodora kunovac kallak, phd, is a researcher at uppsala university. her research activities cover genitourinary symptoms of menopause, infertility, gynecological cancers, maternal health during pregnancy and the effect of the future health of the offspring. inger sundstr€om-poromaa, md, phd, is a professor of obstetrics and gynecology at uppsala university. her research interests include adverse mood effects of hormonal contraception, premenstrual dysphoric disorder, peripartum depression, and brain imaging. alkistis skalkidou, md, phd, is a professor of obstetrics and gynecology at uppsala university, senior consultant at akademiska university hospital, and serves as president of the gynecologic endocrinology group of the swedish society of obstetricians and gynecologists. her research activities include peripartum depression and its effects on the offspring, ultrasound pregnancy dating, and studies on vulvodynia. susanne lager, phd, is a researcher and a docent in experimental obstetrics and gynecology at uppsala university. her research interests include pregnancy complications, infections, maternal substance abuse, and placenta. orcid åsa edvinsson http://orcid.org/0000-0002-6246-7218 theodora kunovac kallak http://orcid.org/0000-0002-2112-8674 inger sundstr€om-poromaa http://orcid.org/0000-0002-2491-2042 alkistis skalkidou http://orcid.org/0000-0002-4935-7532 susanne lager http://orcid.org/0000-0003-3556-065x references 1. woody ca, ferrari aj, siskind dj, whiteford ha, harris mg. a systematic review and meta-regression of the prevalence and incidence of perinatal depression. j affect disord. 2017;219: 86–92. 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and infant temperament: the moderating role of placental gene expression. infancy. 2018;23:211–31. 35. clifton vl, cuffe j, moritz km, cole tj, fuller pj, lu nz, et al. review: the role of multiple placental glucocorticoid receptor isoforms in adapting to the maternal environment and regulating fetal growth. placenta. 2017;54:24–9. 36. fowden al, forhead aj, sferruzzi-perri an, burton gj, vaughan or. review: endocrine regulation of placental phenotype. placenta. 2015;36:s50–s9. 36 å. edvinsson et al. abstract introduction material and methods study population tissue collection western blot transcription factor activity statistics results western blotting loading control placental nr3c1 protein placental glucocorticoid receptor dna binding activity discussion acknowledgements disclosure statement funding references tf-iups190012 193..198 article clusterin as a potential marker of brain ischemia-reperfusion injury in patients undergoing carotid endarterectomy joanna ił_zeckaa, marek ił_zeckib, aneta grabarskac, shawn daved, marcin feldob and tomasz zubilewiczb aindependent neurological rehabilitation unit, medical university of lublin, lublin, poland; bdepartment of vascular surgery and angiology, medical university of lublin, lublin, poland; cdepartment of biochemistry and molecular biology, medical university of lublin, lublin, poland; duniversity of oklahoma health sciences center in oklahoma city, oklahoma, usa abstract introduction: carotid endarterectomy (cea) is a surgical procedure used in the prevention of ischemic stroke. however, this procedure can cause complications of ischemia-reperfusion injury to the brain. clusterin (clu) is a cytoprotective chaperone protein that is released from neurons in response to various neurological injuries. the objective of the study was to report the changes in serum clu concentrations of patients undergoing cea. materials and methods: the study involved 25 patients with severe internal carotid artery stenosis. serum samples were taken from patients at three different times: within 24 hours preoperatively to cea, 12 hours postoperatively, and 48 hours postoperatively. serum clu concentrations were measured using a commercially available enzyme-linked immunosorbent assay. results: when compared to concentrations preoperatively, the serum clu concentration initially decreased during the 12 hours following cea. however, 48 hours following the procedure there was an increase in the clu concentration. after statistical analysis, differences were detected in serum clu concentration between all three recorded measurements (p < 0.05). conclusion: data from our study indicate that serum clu concentrations are affected after cea. we hypothesize that serum clu concentrations may depend on brain ischemia-reperfusion injury following this surgical procedure. article history received 22 october 2018 revised 16 july 2019 accepted 17 july 2019 key words brain injury; carotid endarterectomy; clusterin; potential markers introduction carotid endarterectomy (cea) is a minimally invasive surgical procedure used in the prevention of ischemic stroke. however, this treatment can cause various complications, including embolism resulting in brain ischemia. it was also observed that clamping and declamping of the internal carotid artery during cea may lead to ischemia-reperfusion injury and brain edema (1,2). clusterin (clu) is also called apolipoprotein j (apoj), sulfated glycoprotein-2, secreted glycoprotein gp80, complement lysis inhibitor, testosterone-repressed prostate message 2 (trpm-2), or complement-associated protein sp40-40. it is found at high concentrations in physiological fluids and is expressed constitutively at variable levels in a wide variety of tissues (3). expression of the clu gene (located on chromosome 8p21-p12) leads to the production of the secretory form of this protein found in plasma, cerebrospinal fluid (csf), and other body fluids (4). clu is expressed in the human brain by subpopulations of neurons in the neocortex, glial cells in the hippocampus/neocortex, and ependymal cells (5–7). data from previous literature showed that clu is a cytoprotective chaperone protein whose concentration is increased in response to a diverse range of stresses including heat, pro-apoptotic insults, oxidative stress, ionizing radiation, and proteotoxicity (8–10). it has been demonstrated that this protein is involved in diverse cellular processes, including apoptosis, cell cycle regulation, dna repair, and the acquisition of cell resistance against multiple conventional therapies (11). in animal models of ischemic stroke, both neuronal and astroglial cells express high levels of clu early following ischemic damage (12). clu binds to a wide range of misfolded client proteins and either sequesters them into stable, soluble complexes or inhibits the formation and accumulation of toxic amyloid assemblies (13,14). this glycoprotein can also inhibit protein aggregation (15). according to gregory et al. (16), clu protects neurons against intracellular proteotoxicity. clu is also a chaperone protein that has the ability to bind to a wide array of physiological ligands involved in the pathogenesis of alzheimer’s disease. one of these ligands is b-amyloid, for which clusterin mediates clearance from the brain through the blood–brain barrier to the peripheral circulation via megalin (5,17). moreover, in the peripheral circulation, clu may prevent low-density lipoprotein (ldl) oxidation by the arterial wall endothelium (18). it was revealed that clu also exists as a nuclear, unglycosylated contact joanna ił_zecka, md, phd joanna.ilzecka@umlub.pl independent neurological rehabilitation unit, s. staszica 4/6, 20-081 lublin, poland. � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 3, 193–198 https://doi.org/10.1080/03009734.2019.1646359 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1646359&domain=pdf&date_stamp=2019-09-10 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2019.1646359 http://www.tandfonline.com 60-kda protein (19). according to leskov et al. (20), nuclear clu can promote cell death. on the other hand, accumulation of clu in the cytosol promotes cell survival. nizard et al. (19) showed stressinduced translocation of clu from the endoplasmic reticulum to the cytosol. additionally, this glycoprotein can prevent apoptosis by antagonizing bax, therefore preventing mitochondrial release of cytochrome c and consequent caspase activation (21). pereira et al. (22) described protective molecular mechanisms of clu against apoptosis; according to the authors, clu can act directly or indirectly on apoptosis by regulating several intracellular pathways. these pathways include: the oxidant and inflammatory program, insulin growth factor 1 (igf-1) pathway, ku70/bcl-2-associated x protein (bax) pathway, tumor necrosis factor alpha (tnf-a) pathway, bcl-2 antagonist of cell death (bad) pathway, and mitogen-activated protein kinase (mapk) pathway. according to zinkie et al. (23), the regulation and biological effects of clu are complex and circumstance-dependent. experimental investigation conducted on mice showed that intraventricular apolipoprotein apoj infusion acts protectively in traumatic brain injury by suppressing the inflammatory response (glial activation, cytokine expression), blood–brain barrier disruption, and cerebral edema (24). taking into account the fact that the clu concentrations are affected in various neurological diseases, we have hypothesized that brain ischemia-reperfusion injury after cea can also change the concentration of this molecule. this change may be due to the previously described neuroprotective function of clu against intracellular toxicity within the central nervous system. the objective of our study was to report the changes in serum clu concentrations of patients undergoing cea. materials and methods the patients were admitted in the department of vascular surgery and angiology, medical university of lublin, poland, and were scheduled to undergo cea due to internal carotid artery stenosis. based on doppler studies, candidates were qualified for the cea procedure as determined by the guidelines set forth by the european society of vascular surgery. patients with severe carotid artery stenosis were identified using criteria established by nascet (north american symptomatic carotid endarterectomy trial) (25,26). the study involved 25 patients aged from 55 to 83 years with a mean age of 69 years. the degree of the internal carotid artery stenosis ranged from 60% to 92%. neurological examination was performed by a neurologist prior to and after cea. in this neurological study there were no deviations from the normal. the demographic information and pertinent past medical histories of patients are summarized in table i. serum samples were taken from the antecubital vein of patients at three different times: within 24 hours preoperatively to cea, 12 hours postoperatively, and 48 hours postoperatively. serum clu concentrations were measured using a commercially available enzyme-linked immunosorbent assay (circulex human clusterin/apo-j elisa kit, cyclex co., ltd, nagano, japan). statistical analysis was performed using statistica, version 12 software (statsoft, inc., poland). the mann–whitney and friedman tests were used to measure the differences between groups of patients. non-parametric methods were used because the data were not normally distributed. the clu concentration was measured in ng/ml. the level of statistical significance was p < 0.05. the study was approved by the ethics committee of medical university in lublin (ke-0254/218/2014). results serum clu concentrations in patients and a comparative analysis are presented in figure 1 and table ii. data from the study revealed that the serum clu concentration initially decreased 12 hours after cea when compared to concentration preoperatively, and then increased 48 hours postoperatively. the friedman test indicated there were differences in serum clu concentrations between all three recorded measurements (p < 0.05). there was no difference in serum clu concentrations in three measurements between males and females (p > 0.05). the difference in serum clu concentrations between younger (�69 years) and older (>69 years) patients in three measurements was not significant (p > 0.05). there was no difference in serum clu concentrations between preoperative symptomatic and asymptomatic patients (p > 0.05). the difference in serum clu concentrations between the left and right carotid artery stenosis in three measurements was not significant (p > 0.05). there was no difference between the groups of patients divided according to degree of carotid artery stenosis (p > 0.05). there was no significant difference between the degree of carotid artery stenosis in patients dependent on their age (p ¼ 0.78). discussion our study revealed that serum clu concentrations are affected following cea. clu concentrations were decreased in the earliest period postoperatively. it has been previously hypothesized that ischemia-reperfusion injury may play a crucial role in brain damage following cea. therefore, alterations in clu concentrations in the serum of patients may be reflective of the important function of this glycoprotein during central nervous system injury. studies conducted on animal models support the neuroprotective role of clu in early ischemic events, and have demonstrated that this glycoprotein plays a central role in the remodeling of ischemic damage (27). this study further suggests that clu may also participate in ischemia-reperfusion injury after cea. data from current literature indicate that clu concentrations may be affected in the csf and plasma of patients during the pathogenesis of different neurological diseases and may also play a role as a neurological biomarker. changes in the serum and csf concentration of clu are also present in patients with cerebrovascular diseases. 194 j. ił _zecka et al. according to wąsik et al. (28), inflammation following subarachnoid hemorrhage (sah) involves numerous mediators with biomarker properties. these authors aimed to clarify the status of clu in sah. they observed that, following sah, mean csf clu concentration decreased after 5–7 days, and then increased 10–14 days later. however, the clu concentration failed to differentiate between good and poor prognosis at 0–3 days and 10–14 days after sah, but significantly higher levels of csf clu were found 5–7 days after sah in patients with good outcome. thus, sah was associated with a significant decrease in csf clu in their patients. however, there were no significant differences between clu table i. characteristics of patients. patient id sex age carotid artery stenosis past medical history: stroke and tia symptoms other diseaseslocation % 1 f 72 l 90 tia transient hemiparesthesia right arterial hypertension 2 m 74 l 92 none none none 3 m 76 l 90 none none none 4 m 67 r 80 tia transient hemiparesis left chronic obstructive pulmonary disease 5 f 70 l 60 tia vertigo arterial hypertension, diabetes, ischemic heart disease 6 m 67 r 87 hemorrhagic stroke none none 7 m 70 l 75 none none none 8 f 74 r 85 none none arterial hypertension 9 f 65 r 82 2 ischemic strokes hemiparesis right arterial hypertension 10 m 69 l 80 none none none 11 f 66 r 79 tia transient hemiparesthesia left arterial hypertension 12 m 77 r 88 none none none 13 m 67 r 80 tia, ischemic stroke hemiparesis left arterial hypertension 14 f 67 l 85 tia transient hemiparesis right diabetes 15 f 78 r 80 tia transient hemiparesthesia left none 16 f 70 l 90 tia, ischemic stroke transient hemiparesis right none 17 m 56 r 84 none none metabolic syndrome, ischemic heart disease, hypothyreosis 18 f 68 l 90 none none none 19 f 62 l 90 tia transient amaurosis in left eye none 20 m 73 r 90 none none none 21 m 62 r 89 none none arterial hypertension 22 m 83 l 86 ischemic stroke aphasia, hemiparesis right arterial hypertension, atrial fibrillation 23 f 55 r 79 none none none 24 f 78 r 70 tia transient amaurosis in right eye arterial hypertension, diabetes 25 m 66 l 85 none none arterial hypertension, diabetes, parkinson’s syndrome f ¼ female; l ¼ left; m ¼ male; r ¼ right; tia ¼ transient ischemic attack. figure 1. serum clu concentrations in patients. upsala journal of medical sciences 195 concentration in csf at three time points (0–3 days after sah versus 5–7 days after sah versus 10–14 days after sah). according to the authors, decreased clu concentrations might be explained by the immediate binding of clu to proteins and lipids from the extravasated blood. moreover, the fact that the significant difference in clu levels between patients with good and poor outcomes was observed only on days 5–7 after sah might indicate that patients with good outcome more rapidly induce synthesis and secretion of clu into the csf. similarly, the initial decrease in the serum clu concentrations seen in our study after cea may be due to the inclusion of this molecule in the mechanisms of nervous system protection after ischemia-reperfusion injury. furthermore, song et al. (29) measured serum clu in acute ischemic stroke patients within 24 hours of stroke onset and observed significantly higher serum clu concentrations in stroke patients than in healthy controls. the serum values of clu were also positively correlated with the nih stroke scale (nihss) scores, the time interval after stroke onset, as well as major stroke risk factors associated with lipid profile. the authors concluded that elevated serum clu concentration is independently associated with ischemic stroke and may serve as peripheral biomarker to aid in clinical assessment of ischemic stroke and its severity. according to the authors, the results from their study are consistent with previous findings suggesting that ischemic insult is associated with an overall downregulation of beneficial neurotrophic molecules and an upregulation of inflammatory signaling proteins and cytoskeletal components. additionally, according to yu et al. (30), the clu concentration was decreased in the csf of human models diagnosed with epilepsy. therefore, measurement of clu concentrations in the csf may be helpful in generating differential diagnosis of neurodegenerative disorders (31). it has been observed that clu concentration was also decreased in the plasma of patients diagnosed with alzheimer’s disease when compared to control populations (32). the aim of the study conducted by maskanakis et al. (33) was to clarify the predictive value of apoj in internal carotid artery restenosis following cea. the serum apoj levels of 100 patients were examined; 56 patients who underwent cea constituted the vascular group (vg), and 44 patients constituted the control group (cg). apoj samples were obtained preoperatively, 24 h after the surgical procedure, and at 1, 6, and 12 months thereafter during the follow-up period. the preoperative differences in apoj levels between the cg and vg were statistically significant; the mean values were higher in the vg. in the vg, the serum apoj concentrations were higher at postoperative day 1 compared to preoperative levels, and were decreased at 1, 6, and 12 months postoperatively. meanwhile, the apoj levels of patients in the cg remained unchanged. further subdivision of the vg into patients with or without restenosis revealed that restenotic patients presented with significantly higher mean apoj values than those that were non-restenotic in the vg patients. according to the authors, apoj seems to be an important predictor for carotid restenosis at 6 and 12 months postoperatively. in our study there was no difference in serum clu concentrations depending on the degree of artery stenosis. previously we assessed different markers of brain damage after cea, and we obtained data similar to our present study. our earlier study showed that microtubule-associated protein tau (mapt) and myelin basic protein (mbp) concentrations were also significantly decreased 12 hours after cea when compared to the level before the surgery. furthermore, levels of these parameters were also normalized 48 hours after cea. mapt and mbp levels showed a characteristic time curve in patients who underwent cea and did not experience any neurological deficit in the perioperative period. we concluded that possible alterations of this time curve may potentially be an index of neurological event occurrence (34). in our next study we observed that serum carnosine dipeptidase 1 (cndp1) and ubiquitin c-terminal hydrolase l1 (uchl1) concentrations were also significantly decreased 12 hours after cea when compared to these concentrations before the surgery. just as with the previous studies, these table ii. serum clu concentrations in patients. group of patients n median (iqr) [ng/ml] comparisonbefore surgery [a] 12 h after cea [b] 48 h after cea [c] total 25 13.43 (8.65, 23.32) 9.30 (0, 18.96) 16.31 (4.41, 21.42) [a-b-c] p ¼ 0.001a males 13 13.61 (9.39, 25.05) 8.73 (0, 10.85) 14.84 (7.25, 21.59) [a] p ¼ 0.58 [b] p ¼ 0.53 [c] p ¼ 0.60 females 12 15.61 (1.63, 23.43) 11.24 (1.21, 22.14) 17.53 (4.41, 20.38) younger (�69 years) 13 13.00 (9.39, 26.81) 10.85 (3.66, 26.02) 18.18 (9.64, 25.72) [a] p ¼ 0.56 [b] p ¼ 0.14 [c] p ¼ 0.20 older (>69 years) 12 13.52 (5.95, 18.36) 6.52 (0, 11.24) 11.29 (3.80, 20.38) symptomatic 12 14.47 (8.02, 21.02) 10.41 (1.21, 20.03) 17.57 (8.59, 21.42) [a] p ¼ 0.93 [b] p ¼ 0.76 [c] p ¼ 0.70 asymptomatic 13 13.43 (8.65, 23.32) 8.73 (0, 18.96) 13.36 (3.80, 20.41) left artery stenosis 12 13.49 (7.44, 20.91) 8.45 (0, 14.40) 13.36 (0, 20.38) [a] p ¼ 0.64 [b] p ¼ 0.26 [c] p ¼ 0.32 right artery stenosis 13 13.43 (8.65, 26.81) 10.85 (2.42, 26.02) 16.92 (6.90, 25.72) artery stenosis �85% 14 13.84 (3.26, 26.81) 6.19 (0, 26.02) 12.03 (2.20, 25.58) [a] p ¼ 0.68 [b] p ¼ 0.93 [c] p ¼ 0.75 artery stenosis >85% 11 13.43 (12.92, 23.32) 10.34 (4.32, 18.31) 17.60 (8.59, 20.41) astatistically significant. iqr ¼ interquartile range. 196 j. ił _zecka et al. enzyme concentrations also normalized 48 h after cea. thus, we concluded that cea significantly affects serum cndp1 and uchl1 concentrations, and therefore these enzyme concentrations seem to reflect brain ischemia resulting from severe internal carotid artery stenosis in patients undergoing cea. however, these observed changes in serum cndp1 and uchl1 concentrations do not necessarily warrant a change in recommendations concerning the use of cea in patients with high-grade internal carotid artery stenosis (35). as previously mentioned, ischemia-reperfusion injury can play a critical role following cea. liu et al. (36) revealed that clu reduces cold ischemia-reperfusion injury in heart transplantation through regulation of nf-kb signaling and bax/ bcl-xl expression. according to the authors, clu has a protective effect caused by inhibition of cell apoptosis/death and reducing the pre-inflammatory response. li et al. (37) also observed reduction of cold ischemia-reperfusion injury by using graft samples expressing clu during heart transplantation. additionally, other authors demonstrated promotion of cell proliferation by clu in the renal tissue repair phase after ischemia-reperfusion injury (38). moreover, it was revealed that loss of clu expression worsened renal ischemia-reperfusion injury (39). these studies clearly provide evidence of the protective effects of clu during the ischemia-reperfusion injury. in summary, data from our present study indicate that serum clu concentrations are affected following cea. clu produced in the brain can be measured in the serum secondary to blood–brain barrier rupture possibly following ischemia-reperfusion injury. as previously mentioned, the initial reduction of serum clu concentrations in our study may indicate the involvement of this molecule in the central nervous system protectant mechanisms against ischemia-reperfusion injury. however, it should not be excluded that a significantly decreased serum clu concentration during the earliest period (12 hours after cea) of ischemiareperfusion injury may disrupt the cytoprotective effect of this protein. when compared to the preoperative serum clu concentrations, we hypothesize that the increase in serum clu levels 48 hours postoperatively may indicate clu’s neuroprotective function on the brain after cea, and thus might explain the absence of neurological complications in our examined patients. therefore, it is possible that serum clu concentrations can be dependent upon brain ischemia-reperfusion injury following cea. as in our previous studies, the clu concentration also showed a characteristic time curve in our patients without neurological deficit in the perioperative period. thus, any deviant alteration of this time curve might be a reflection of potential neurological complication. our study presents new data on the clu after cea. however, the study has some limitations, and therefore it cannot be excluded that additional factors, e.g. anesthesia or/and physical injury caused by surgery, may influence clu concentrations. another such limitation is also the low power of the study to reveal group differences. disclosure statement the authors declare no conflict of interest. funding the study was supported by the foundation for the development of vascular surgery at the department of vascular surgery and angiology, medical university of lublin, lublin, poland. notes on contributors joanna ił_zecka, md, phd, is a practicing neurologist, professor, and department head in the independent neurological rehabilitation unit, medical university of lublin, poland. marek ił _zecki, md, phd, is a vascular surgeon and associate professor in the department of vascular surgery and angiology, medical university of lublin, poland. aneta grabarska, phd, is a research coordinator in the department of biochemistry and molecular biology, medical university of lublin, poland. shawn dave, md, is a graduate of the medical university of lublin, poland, and a resident physician at the university of oklahoma health sciences center in oklahoma city, oklahoma, usa. marcin feldo, md, phd, is a vascular surgeon and research coordinator at the department of vascular surgery and angiology, medical university of lublin, poland. tomasz zubilewicz, md, phd, is a vascular surgeon, professor and department head at the department of vascular surgery and angiology, medical university of lublin, poland. references 1. gupta n, corriere ma, dodson tf, chaikof el, beaulieu rj, reeves jg, et al. the incidence of microemboli to the brain is less with endarterectomy than with percutaneous revascularization with distal filters or flow reversal. j vasc surg. 2011;53:316–22. 2. ballesteros-pomar m, alonso-arg€ueso g, tejada-garc�ıa j, vaqueromorillo f. cerebral hyperperfusion syndrome in carotid revascularisation surgery. rev neurol. 2012;55:490–8. 3. rizzi f, coletta m, bettuzzi s. chapter 2: clusterin (clu): from one gene and two transcripts to many proteins. adv cancer res. 2009; 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renal physiol. 2014;306:724–33. 39. zhou w, guan q, kwan cc, chen h, gleave me, nguan cy, et al. loss of clusterin expression worsens renal ischemia-reperfusion injury. am j physiol renal physiol. 2010;298:568–78. 198 j. ił _zecka et al. abstract introduction materials and methods results discussion disclosure statement references a novel vars2 gene variant in a patient with epileptic encephalopathy case report a novel vars2 gene variant in a patient with epileptic encephalopathy lucija ruzmana�, ivana kolica�, jelena radic nisevica,b, antonija ruzic barsicc, ingrid skarpa prpicd and igor prpica,b achild neurology and child psychiatry department, pediatric clinic, clinical hospital center rijeka, rijeka, croatia; buniversity of rijeka, school of medicine rijeka, rijeka, croatia; cradiology department, thalassoterapia opatija, opatija, croatia; dneurology clinic, clinical hospital center rijeka, rijeka, croatia abstract background: mitochondrial disorders are heterogeneous clinical syndromes caused by defective activity in the mitochondrial respiratory chain, resulting in a faulty oxidative phosphorylation system. these inherited disorders are individually rare, and furthermore they are phenotypic variables. the genetically characterized mitochondrial disorders are rarely associated with epileptic encephalopathies. case presentation: we present the clinical phenotype, biochemical analysis, and electrographic and neuro-radiological features of a 5-month-old girl with epileptic encephalopathy, microcephaly, severe psychomotor delay, hypertrophic cardiomyopathy, and abnormal mri scan. using whole-genome sequencing technique, compound heterozygous mutations of the vars2 gene were revealed, with one previously unreported frameshift mutation. conclusion: our report extends the phenotypic spectrum of vars2-related disorders with an initial presentation of epileptic encephalopathy and early death due to malignant arrhythmia. article history received 1 july 2019 revised 6 september 2019 accepted 17 september 2019 keywords encephalocardiomyopathy; epileptic encephalopathy; mitochondrial disease; vars2 introduction mitochondrial disorders are heterogeneous clinical syndromes caused by defective activity in the mitochondrial respiratory chain (mrc), resulting in a faulty oxidative phosphorylation system (oxphos) (1), associated with a broad range of nuclear and mitochondrial gene mutations. these inherited disorders are rare and phenotypically variable, and thus generally unfamiliar to clinicians. epileptic encephalopathies (ee) constitute a heterogeneous group of disorders characterized by intractable seizures with specific abnormalities on electroencephalography (eeg) and severe developmental delay or regression (2); however, a clearly defined aetiology and a diagnostic algorithm for these clinical entities have not been fully established. in our case study we present a clinical phenotype, a biochemical analysis, as well as electrographic and neuroradiological features of a patient with ee. a genetic analysis revealed an mrc defect. methods acquisition of a clinical case we made a comprehensive evaluation of our patient, including molecular genetics investigations. the patient’s parents signed an informed consent in agreement with the declaration of helsinki. molecular genetics genomic dna from the proband, father, and mother was isolated from peripheral blood samples, and whole-exome sequencing (wes) was completed. wes, bioinformatic analysis, variant confirmation, and segregation analysis was performed in the cegat gmbh laboratory (t€ubingen, germany). coding and flanking intronic regions were enriched using the agilent in solution technology and were sequenced using the illumina hiseq/novaseq system (illumina inc., san diego, california, usa). variants (snvs/small indels) with a minor allele frequency (maf) <1.5% were evaluated. known disease-causing variants (according to hgmd) were evaluated up to 5% maf. the maf were taken from the 1000 genomes, dssnp, gnomad, and in-house database. for the index case, 95.61% of the targeted regions were covered by a minimum of 30 high-quality sequencing reads per base. all filtered variants were further analysed using mutation taster, fathmm, mutation assessor, sift, fathmm-mkl coding, lrt, and provean for pathogenicity prediction. two algorithms were applied (3) for assessments of the consequences of variants on splicing. wes identified compound heterozygous mutations in the vars2 gene (nm_001167734.1) in our contact igor prpic igor.prpic@medri.uniri.hr child neurology and child psychiatry department, pediatric clinic, clinical hospital center rijeka, istarska 43, 51000 rijeka, croatia �lucija ruzman and ivana kolic contributed equally to this study. � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 4, 273–277 https://doi.org/10.1080/03009734.2019.1670297 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1670297&domain=pdf&date_stamp=2019-12-09 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2019.1670297 http://www.tandfonline.com patient. both parents are heterozygous carriers of variants in vars2. results a female child was born at term after an uneventful pregnancy and delivery. she was the first child from the first pregnancy of healthy non-consanguineous parents. the family history was unremarkable. the birth weight, length, and head circumference were in accordance with the gestational age (4030 g, 51 cm, and 34 cm, respectively). computed tomography (ct) was performed at one month of age because of failure of head growth (50th percentile at birth and 10th after neonatal period). craniosynostosis was excluded, with no other pathological findings. neurohabilitation was started due to delayed acquisition of milestones. at 5 months of age she developed infantile spasms consistent with hypsarrhythmia on eeg studies. clinical examinations showed microcephaly with ‘pear-shaped face’, hypertelorism, and a wide nasal bridge. global motor development was delayed by 2–3 months. language and social skills were in accordance with the patient’s age. laboratory findings revealed an increased concentration of alanine in plasma (537 lmol/l, normal range 100–439 lmol/l) and cerebrospinal fluid (36.1 lmol/l, normal range 19.9–31.3 lmol/l), as well as mildly elevated concentrations of lactate in plasma (2.0–5.4 mmol/l, normal range 0.33–2.0 mmol/l) and cerebrospinal fluid (3.0 mmol/l, normal range 1.1–2.8 mmol/) indicative of mitochondrial disorder, while others were normal. magnetic resonance imaging (mri) performed at that time revealed diffuse cerebral atrophy with more prominent hypoplasia of the cerebellum (especially vermis), brainstem, and corpus callosum (figure 1(a)). at the age of 6 months multiple seizure types (mastication, eyelid and lip twitching, myoclonic seizures of the trunk and extremities, tonic and clonic seizures) were noticed. treatment attempts with several antiepileptic drugs including folic acid, cholecalciferol, pyridoxine and modified atkins diet were not successful. eeg studies showed continuous burst– suppression pattern. all in all, the clinical status of the child developed into ee. microcephaly, severe global hypotonia, failure to thrive, and feeding and breathing difficulties were more and more pronounced. pendular nystagmus and oedema of hands and feet were periodically noticed. echocardiography showed hypertrophic obstructive cardiomyopathy with fast progression, and atenolol treatment was initiated. figure 1. (a) first mri scan, age 5 months. sagittal t1-weighted and coronal t2-weighted imaging showing diffuse white matter reduction, marked cerebral and cerebellar atrophy, corpus callosum thinning, and brainstem hypoplasia. (b) mri scan, age 9 months. coronal t2-weighted imaging showing marked progression of cerebral and cerebellar atrophy and ventriculomegaly (dominantly left occipital ventricle) due to diffuse white matter necrosis, while mri spectroscopy showed markedly increased myoinositol peak, increased lactate peak, and reduced n-acetyl aspartate peak. 274 l. ruzman et al. ta b le 1. c lin ic al , b io ch em ic al an d m ol ec ul ar fin d in g s in p re vi ou sl y re p or te d p at ie n ts w it h v a rs 2 m ut at io n . o ur ca se d io d at o et al . (6 ); pe re ir a et al . (7 ); br un i et al . (1 0) pr on ic ka et al . (9 ) br un i et al . (1 0) ta yl or et al . (5 ) ba er tl in g et al . (8 ) br un i et al . (1 0) br un i et al . (1 0) br un i et al . (1 0) br un i et al . (1 0) v ar ia n t in v a rs 2 g en e c. 11 00 c > t; p .t h r3 67 ile c. 60 3_ 60 6d up g a tg ; p .a rg 20 3a sp fs � 3 7 c. 11 00 c > t; p .t h r3 67 ile c. 11 00 c > t; p .t h r3 67 ile c. 14 90 g > a ; a rg 49 7h is c. 12 58 g > a ; p .a la 42 0t h r c. 11 35 g > a ; p .a la 37 9t h r c. 18 77 c > a ; p .a la 62 6a sp c. 60 1c > t; p .a rg 20 1t rp c. 11 00 c > t; p .t h r3 67 ile c. 25 57 -2 a > g ; ab er ra n t sp lic in g c. 11 00 c > t; p .t h r3 67 ile c. 15 46 g > t; p .g lu 51 6� c. 22 39 g > a ; p .a la 74 7t h r c. 11 00 c > t; p .t h r3 67 ile c. 11 50 g > a ; p .a sp 38 4a sn c. 11 00 c > t; p .t h r3 67 ile c. 14 90 g > a ; p a rg 49 7h ys se x fe m al e fe m al e (n ¼ 4) , m al e (n ¼ 2) m al e fe m al e (n ¼ 1) , m al e (n ¼ 2) m al e m al e fe m al e m al e fe m al e m al e a g e of on se t 5 m on th s n eo n at al p er io d n eo n at al p er io d n eo n at al p er io d < 1 ye ar n eo n at al p er io d n eo n at al p er io d n eo n at al p er io d 3 m on th s n eo n at al p er io d fi rs t sy m p to m se iz ur es (in fa n ti le sp as m s) n eu ro lo g ic al sy m p to m s (s ei zu re s, h yp ot on ia , an d /o r d d ) st ri d or an d re sp ir at or y fa ilu re h yp ot on ia , se iz ur es , an d fe ed in g d iff ic ul ti es h yp ot on ia la ct ac id os is an d re sp ir at or y fa ilu re st ri d or an d ap n oe a st ri d or an d p oo r fe ed in g se iz ur es st ri d or , h yp ot on ia , an d re sp ir at or y fa ilu re n eu ro lo g ic al sy m p to m s se iz ur es , h yp ot on ia , d d , p en d ul ar n ys ta g m us se iz ur es , d d , sp as ti c te tr ap ar es is , an d /o r p en d ul ar n ys ta g m us h yp ot on ia , lim b sp as ti ci ty , se iz ur es d d , se ve re h yp ot on ia , se iz ur es h yp ot on ia , at ax ia , se iz ur es sp as ti c m ov em en t d is or d er , se iz ur es h yp ot on ia , h yp or ef le xi a, h yp er ek p le xi a, se iz ur es h yp ot on ia , d d h yp ot on ia , d d h yp ot on ia , ab se n ce of sw al lo w in g re fle x fa ci al d ys m or p h is m pr es en t pr es en t or ab se n t n /p n /p n /p n /p pr es en t pr es en t n /p n /p c ar d ia c si g n s h c m h c m or ab se n t ca rd ia c si g n s h c m h c m h c m h c m h c m , p er ic ar d ia l ef fu si on h c m h c m h c m a d d it io n al cl in ic al si g n s m ic , oe d em a of h an d s an d fe et , fa ilu re to th ri ve , fe ed in g an d b re at h in g d iff ic ul ti es m ic , fe ed in g s d iff ic ul ti es , fa ilu re to th ri ve c ry p to rc h id is m , ch ro n ic p an cr ea ti ti s h ep at os p le n om eg al y pr og re ss iv e ex te rn al op h th al m op le g ia an d p to si s m ic c on g en it al h ip d is lo ca ti on , m ic m ic , la ry n g om al at ia fe ed in g d iff ic ul ti es , m ic n /p la b or at or y te st s " la c an d a la in p la sm a an d c sf " a la in p la sm a " la c in p la sm a n /p n /p " la c in p la sm a " la c an d a la in c sf an d p la sm a " la c in p la sm a " la c an d a la in p la sm a " la c in p la sm a ee g ep ile p ti fo rm ab n or m al it ie s ep ile p ti fo rm ab n or m al it ie s n /p n /p n /p ep ile p ti fo rm ab n or m al it ie s n /p n /p ep ile p ti fo rm ab n or m al it ie s n /p m ri fe at ur es c er eb ra l at ro p h y w it h h yp op la si a of ce re b el lu m , b ra in st em , an d co rp us ca llo su m c er eb ra l or ce re b el la r at ro p h y an d /o r co rp us ca llo su m h yp op la si a c er eb ra l at ro p h y an d ve rm is h yp op la si a n /p sy m m et ri ca l b as al g an g lia ca lc ifi ca ti on s c or p us ca llo su m an d ce re b el la r h yp op la si a c er eb ra l an d ce re b el la r at ro p h y n /p c or p us ca llo su m an d ce re b el la r h yp op la si a n /p a g e of d ea th 10 m on th s n ¼ 4 d ie d at ag e fr om 28 m on th s to 8 ye ar s; n ¼ 2 st ill al iv e 9 ye ar s n ¼ 2 d ie d b ef or e 3 m on th s of ag e; n ¼ 1 st ill al iv e a liv e at ag e of 18 ye ar s n /p 3. 5 m on th s 19 m on th s 5 m on th s 3 m on th s a la : al an in e; c sf : ce re b ro sp in al flu id ; d d : d ev el op m en ta l d el ay ; h c m : h yp er tr op h ic ca rd io m yo p at h y; la c: la ct at e; m ic : m ic ro ce p h al y; n /p : in fo rm at io n n ot p ro vi d ed b y th e au th or . upsala journal of medical sciences 275 genetic analysis was performed at the age of 9 months, and it revealed compound heterozygous mutations of the vars2 gene: previously reported pathogenic variant (c.1100c>t; p.thr367ile) transmitted from the father and one unreported frameshift mutation (c.603_606dupgatg; p.arg203aspfs�37) transmitted from the mother. an additional mri with spectroscopy showed a further significant loss of the brain parenchyma and a high lactate peak (figure 1(b)). a multidisciplinary treatment approach was applied to treat the neurologic, cardiac, respiratory, and gastrointestinal complications. in accordance with the established diagnosis, treatment with l-carnitine and coenzyme q10 was initiated. despite multiple supportive and symptomatic treatment regimens, the child died of malignant arrhythmia at the age of 10 months. discussion combined oxidative phosphorylation deficiency 20 (coxpd20) (omim #615917) is an autosomal recessive mitochondrial encephalocardiomyopathy caused by variants in the vars2 gene (omim #612802) located on chromosome 6p21 (https://www.omim.org/entry/615917). the vars2 gene contains 30 exons and encodes mitochondrial valyl trnasynthetase, which participates in mitochondrial protein synthesis (4). despite the estimated prevalence of mrc defects being 1 in 5000 live births, it is often difficult to find the molecular basis. the underlying genetic basis is not possible to identify in 40% to 70% of the patients, even though there is biochemical evidence of mrc defects (5). presently, there are 10 reported pathogenic variants in the vars2 gene (https://www.ncbi.nlm.nih.gov/clinvar/?term=vars2[all]). when considering our patient in comparison with previously reported patients with vars2 mutations (5–10), it appears that the main phenotypic features are seizures, various degrees of developmental delay, facial dysmorphism, brain atrophy, and cardiomyopathy. the main clinical features, biochemical studies, and molecular findings are shown in table 1. bruni et al. (10) elaborated clinical, biochemical, and molecular features in 13 patients from 9 families diagnosed by wes. two previously reported cases by taylor et al. (5) and by pronicka et al. (9) were also included. bruni et al. (10) presented a new homozygous (c.1258g>a; p.ala420thr) variant and four new compound heterozygous variants, along with already known pathogenic variants (table 1). the study found the mutant vars2 allele c.1100c>t; p.thr367ile the most common—in six patients in homozygous and in five patients in heterozygous states. laboratory findings implied a mitochondrial disease, while the eeg and brain mri of these patients did not show any disease-characteristic pattern. the common problem in these patients (n ¼ 11/17) is hypertrophic cardiomyopathy, with only a few patients not being affected. the c.1100c>t; p.thr367ile variant is considered to have less of an effect on the heart (10). alsemari et al. (11) published a study describing four patients with angelman-like syndrome and mutation in the vars2 gene, providing new insight into genotype–phenotype correlation. to date, our case is one of the 17 described cases of severe infantile mitochondrial disorder associated with vars2 pathogenic variants. the phenotypic spectrum of vars2-related gene variants is complex, and no clear genotype–phenotype correlations have been established. therefore, a detailed description of phenotype related to specific mutations is of great importance. our report extends the phenotypic spectrum of vars2-related disorders in the context of ee, severe neurodevelopmental delay, abnormal brain mri scan, and one novel frameshift mutation (c.603_606dupgatg; p.arg203aspfs�37) in vars2 (nm_001167734.1). with the exception of supportive care, currently no disease-modifying treatment exists for the disease. the genetically characterized mitochondrial disorders are rarely associated with ee, with very few clinical reports available (12). a timely evaluation of patients with ee resulting in a specific diagnosis is of great value. we want to emphasize the importance of genetic analysis in the early diagnostic efforts of these patients. wes is a very powerful, noninvasive, accessible, and not overly expensive diagnostic tool. the finding of a specific gene mutation is of exceptional importance to clinicians for further management, and for the family to cope with the diagnosis and prognosis. genetic counselling about familial risk, as well as for potential prenatal diagnostics, is of great value to the family. although the field of mitochondrial encephalomyopathies is highly dynamic, the genetic diagnosis of these disorders, which may offer the possibility to design targeted therapy, remains challenging. in the near future, the wes approach will provide valuable information regarding the genes implicated in mrc defects, which are important in the differential diagnosis of ee. acknowledgements the authors would like to thank our patient’s family for participating in the study. they thank dr saskia biskup and cegat gmbh, who performed whole-exome analysis of the patient and parents. also, they thank their colleagues from the clinical hospital centre rijeka for ensuring multidisciplinary treatment for their patient. disclosure statement no potential conflict of interest was reported by the authors. notes on contributors lucija ruzman is a ph.d. student at the university of rijeka, school of medicine. she works as a pediatric resident at the department of pediatrics at the clinical hospital center rijeka. her main topic of interests are genetic disorders. ivana koli�c is a pediatrician and pediatric neurologist. she works at child neurology and child psychiatry department at the clinical hospital center rijeka and is a ph.d. student at the university of rijeka, school of medicine. her main topic of interests interest are neurodevelopment disorders. 276 l. ruzman et al. https://www.omim.org/entry/615917 https://www.ncbi.nlm.nih.gov/clinvar/?term=vars2 jelena radic nisevic, ph.d., is a pediatrician and pediatric neurologist at child neurology and child psychiatry department at the clinical hospital center rijeka and university of rijeka, school of medicine. antonija ruzic barsic is a radiologist, ph.d. student of the university of osijek. she is the head of radiology department at the thalassotherapia opatija hospital. her main topic of interest is pediatric neuroradiology. ingrid skarpa prpic, ph.d., is an assistant professor of neurology. she works at clinical hospital center rijeka and university of rijeka, school of medicine. her main topics of interest are demyelinating disease and paroxysmal disorders. igor prpic, ph.d., is a full time professor of pediatrics-permanent election, and head of child neurology and child psychiatry department at the clinical hospital center rijeka and university of rijeka, school of medicine. he is subspecialist in pediatric neurology and in neonatology. since 2009 he runs croatian referral centre of ministry of health for childhood epilepsy and convulsive disorders. references 1. ghezzi d, zeviani m. assembly factors of human mitochondrial respiratory chain complexes: physiology and pathophysiology. adv exp med biol. 2012;748:65–106. 2. scheffer ie, berkovic s, capovilla g, connolly mb, french j, guilhoto l, et al. ilae classification of the epilepsies: position paper of the ilae commission for classification and terminology. epilepsia. 2017;58:512–21. 3. jian x, boerwinkle e, liu x. in silico prediction of splice-altering single nucleotide variants in the human genome. nucleic acids res. 2014;42:13534–44. 4. bonnefond l, fender a, rudinger-thirion j, giege r, florentz c, sissler m. toward the full set of human mitochondrial aminoacyltrna synthetases: characterization of asprs and tyrrs. biochemistry. 2005;44:4805–16. 5. taylor rw, pyle a, griffin h, blakely el, duff j, he l, et al. use of whole-exome sequencing to determine the genetic basis of multiple mitochondrial respiratory chain complex deficiencies. jama. 2014;312:68–77. 6. diodato d, melchionda l, haack tb, dallabona c, baruffini e, donnini c, et al. vars2 and tars2 mutations in patients with mitochondrial encephalomyopathies. hum mutat. 2014;35:983–9. 7. pereira s, adriao m, sampaio m, basto ma, rodrigues e, vilarinho l, et al. mitochondrial encephalopathy: first portuguese report of a vars2 causative variant. jimd rep. 2018;42:113–9. 8. baertling f, alhaddad b, seibt a, budaeus s, meitinger t, strom tm, et al. neonatal encephalocardiomyopathy caused by mutations in vars2. metab brain dis. 2017;32:267–70. 9. pronicka e, piekutowska-abramczuk d, ciara e, trubicka j, rokicki d, karkuci�nska-wierckowska a, et al. new perspective in diagnostics of mitochondrial disorders: two years’ experience with wholeexome sequencing at a national paediatric centre. j transl med. 2016;14:174. 10. bruni f, di meo i, bellacchio e, webb bd, mcfarland r, chrzanowska-lightowlers zma, et al. clinical, biochemical, and genetic features associated with vars2-related mitochondrial disease. hum mutat. 2018;39:563–78. 11. alsemari a, al-younes b, goljan e, jaroudi d, binhumaid f, meyer bf, et al. recessive vars2 mutation underlies a novel syndrome with epilepsy, mental retardation, short stature, growth hormone deficiency, and hypogonadism. hum genomics. 2017;11:33. 12. paciorkowski ar, thio ll, dobyns wb. genetic and biologic classification of infantile spasms. pediatr neurol. 2011;45:355–67. upsala journal of medical sciences 277 abstract introduction methods acquisition of a clinical case molecular genetics results discussion acknowledgements disclosure statement references effects of dapagliflozin on cardiac substrate uptake, myocardial efficiency, and myocardial contractile work in type 2 diabetes patients—a description of the dapacard study article effects of dapagliflozin on cardiac substrate uptake, myocardial efficiency, and myocardial contractile work in type 2 diabetes patients—a description of the dapacard study axel åkerbloma, jonas oldgrena, aino latva-raskub,c, lars johanssond, vera lisovskajae, cecilia karlssone, jan oscarssone and pirjo nuutilab,c auppsala clinical research center and department of medical sciences, uppsala university, uppsala, sweden; bturku pet centre, university of turku, turku, finland; cdepartment of endocrinology, turku university hospital, turku, finland; dantaros medical ab, gothenburg, sweden; eastrazeneca, gothenburg, sweden abstract background: diabetes increases the risk for cardiovascular (cv) events. it has recently been shown that the use of sodium-glucose cotransporter 2 (sglt2) inhibitors leads to a reduction in cv outcomes in patients with type 2 diabetes mellitus (t2dm), including mortality and heart failure hospitalization. the exact mechanisms of how sglt2 inhibitors lead to this cv risk reduction remain incompletely understood. the study of dapagliflozin on cardiac substrate uptake, myocardial efficiency and myocardial contractile work in type 2 diabetes patients (dapacard) (nct03387683) explores the possible effects of dapagliflozin, an sglt2 inhibitor, on cardiac work, metabolism, and biomarker levels. methods: dapacard is an international, randomized, double-blind trial that aims to examine the effects of dapagliflozin versus matching placebo in 52 patients with t2dm that are on stable metformin therapy prior to and during the 6 weeks of treatment. the primary efficacy endpoint is change in global longitudinal strain of the left ventricle (glslv) measured with magnetic resonance imaging (mri) between baseline (pre-treatment) and end of study (on-treatment). the secondary endpoint is the corresponding change in myocardial efficiency measured as external left ventricular work divided by total left ventricular work, which is estimated using [11c]-acetate clearance using positron emission tomography (pet). conclusion: the dapacard study is an extensive investigation of cardiac function and metabolism, by advanced imaging with pet and mri, as well as biomarkers, performed in order to further explore how the sglt2 inhibitor dapagliflozin could influence cardiovascular outcomes in patients with t2dm. article history received 21 june 2018 revised 6 august 2018 accepted 13 august 2018 keywords diabetes; experimental diabetes; magnetic resonance; molecular biology; nuclear medicine background type 2 diabetes mellitus (t2dm) is associated with a 2–4-fold increased risk for cardiovascular (cv) events as compared to patients without t2dm (1). although antidiabetic drugs reduce hyperglycemic complications and morbidity, to date few antidiabetic drugs have been proven to reduce cv mortality (2–4). the sodium-glucose cotransporter 2 (sglt2) inhibitor empagliflozin showed a reduction in cv outcomes, including cv mortality and heart failure admissions, in patients with t2dm (4). another sglt2 inhibitor, canagliflozin, recently showed similar results including a decrease in heart failure admissions, but not in all-cause mortality, in patients with t2dm (5,6). for both of the sglt2 inhibitors, the positive cardiovascular effects became apparent within months from treatment start, suggesting that mechanisms beyond improved glucose control and reduced atherosclerosis are involved in the cv risk reduction (5). the effect of dapagliflozin on cv outcomes in a broad t2dm population is currently investigated in a large phase iii study (multicenter trial to evaluate the effect of dapagliflozin on the incidence of cardiovascular events—declare-timi 58 [nct01730534]), with results expected in the second half of 2018 (7). a number of potential mechanisms have been suggested to explain the cv benefits observed in studies with sglt2 inhibitors. the primary effect of these drugs is a reduction in reabsorption of glucose in proximal tubuli, which results in increased urinary excretion of glucose and sodium and thus increased diuresis. consequently, sglt2 inhibition not only leads to a decrease in hemoglobin a1c (hba1c) levels, but also a lower body weight and lower blood pressure, as well as an increase in hematocrit (hct) levels (4,5,8). however, none of these effects is believed to fully explain the reported cv benefits. the need for mechanistic studies is therefore warranted to elucidate the mechanisms behind the beneficial effects of sglt2 inhibitors on cv outcomes (8). global longitudinal strain of the left ventricle of the heart (glslv) is a marker of cardiomyocyte contractile work of the contact axel åkerblom axel.akerblom@ucr.uu.se uppsala clinical research center and department of medical sciences, uppsala university, uppsala, sweden � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 1, 59–64 https://doi.org/10.1080/03009734.2018.1515281 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1515281&domain=pdf http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1515281 http://www.tandfonline.com heart. a decrease in glslv has been associated with cv events, including heart failure and a composite of cv death, myocardial infarction, or stroke (9). changes in myocardial efficiency and contractile work can be measured with positron emission tomography (pet) and magnetic resonance imaging (mri), respectively (10–12). it has previously been shown that patients with t2dm have a reduced myocardial efficiency both in the fasting state and during insulin infusion (10,13). in the current ‘effects of dapagliflozin on cardiac substrate uptake, myocardial efficiency and myocardial contractile work in type 2 diabetes patients—a description of the dapacard study’ (nct03387683) we aim to further explore the effects of dapagliflozin on myocardial metabolism and contractility with advanced pet and mri scans, as well as with an extensive array of biomarkers. the dapacard study study objectives the objective of the study is to explore the potential effects of dapagliflozin on myocardial perfusion, substrate uptake, and measures of contractile performance of the myocardium including left ventricle (lv) strain, as a surrogate for myofibril contractile work. study design the dapacard is an international, randomized, doubleblind, parallel-group, phase iv trial that aims to include 52 patients with t2dm on a stable dose of metformin, randomized to 6 weeks of treatment with either dapagliflozin 10 mg once daily (od) or matching placebo. the study design is depicted in figure 1. for each patient, five visits are planned. the screening visit (visit 1) takes place 0–21 days prior to randomization and start of treatment (visit 2), and is primarily aimed at determining the suitability of an inclusion of a patient in the study and collection of background information. the efficacy data will be collected at the randomization (visit 2; baseline) and end of treatment visit (visit 4, which takes place after 6 weeks on study drug). during these two visits blood and urine samples for biomarkers are collected, and pet and mri scans are performed. it is required that patients should be fasting prior to collection of these measurements. pet and mri scans can, if preferred, be performed on two consecutive days. a crossover study design was discussed; however, this would have prolonged the study period for participants with a risk for an increased number of drop-outs and would have exposed the participants to additional pet scans (higher radiation exposure), and was consequently discarded. finally, there are two safety check-up visits planned: visit 3 (28 days after the start of the randomized treatment) and visit 5 (1 week after the cessation of the study drug). both of these visits are scheduled telephone calls. a steering committee with academic experts and representatives from the sponsor (astrazeneca) and from antaros medical (gothenburg, sweden) designed the dapacard study in collaboration. the steering committee, together with project managers at uppsala clinical research center (uppsala, sweden), oversees the medical, scientific, and operational conduct of the study. the steering committee, together with statisticians, has planned the analyses and will perform the final statistical analyses. the study adheres to the ethical principles of the declaration of helsinki, and to good clinical practice. the study protocol has been approved by an independent ethics committee and/or institutional review board at each site. the protocol requires investigators to obtain each subject’s signed informed consent before initiating study procedures or recording any information into the study database. blood samples a predefined set of safety blood samples will be collected at screening, randomization, and at the 6 weeks visit. furthermore, blood samples for biomarker collection will be obtained at randomization and 6 weeks visits. the biomarkers (such as glucose, hba1c, non-esterified fatty acids, betahydroxybutyrate, hct, nt-probnp, and fgf21) could help to understand changes in metabolism and/or function of the heart. study population, study conduct, and follow-up the study subjects are recruited either from outpatient visits to the participating centers or via registries of patients previously admitted to the participating centers. the patients have to meet all the inclusion criteria (table 1) before randomization, including: age of �40 years but <75 years, t2dm on a stable dose of metformin for at least 6 weeks, and willing to undergo both pet and mri scans. patients are allowed to have had coronary artery disease, but no ongoing cardiac signs or symptoms (e.g. angina pectoris, dyspnea or fatigue on exertion). no patients with decreased lvef or overt heart failure are allowed. a complete list of the predefined exclusion criteria can be found in table 2. all patients eligible for participation in the trial must receive metformin, and be on a steady dose for at least figure 1. overview of the dapacard trial. screening visit (visit 1) is 0 to 21 days prior to randomization. the randomization (visit 2) is day 1, and the patients perform both pet and mri scans prior to starting the randomized treatment. visit 3 is a telephone call. visit 4 is with both pet and mri scans. a final follow-up call (visit 5) is also planned. 60 a. åkerblom et al. 6 weeks before enrollment. other antidiabetic treatments, including other sglt2 inhibitors, insulin, glitazones, pramlinitides, dpp-iv inhibitors, glp-1 analogues, or sulfonylurea, are not allowed. likewise, loop diuretics are not allowed in the trial. overall, all pre-existing concomitant medications are asked to be kept on stable doses throughout the study from 6 weeks before screening and throughout the study, including, for example, thyroid hormones, thiazides, beta-blockers and arbs, and ace inhibitors (any drug affecting plasma volume and/or heart function). study participants are asked to not intentionally try to change body weight or to attend weight loss programs, start other medications, or change diets or current rate of physical practice during the 6 weeks of study duration. the investigational product (ip) is dapagliflozin 10 mg once daily (od) versus placebo (matched to ip). the dapagliflozin 10 mg once daily was chosen because it is well tolerated and is approved for long-term t2dm treatment. study treatment compliance will be assessed by the return of all unused investigational products and empty packages. table 1. inclusion criteria. provision of signed and dated, written informed consent prior to any study-specific procedures, and: 1. females or males �40 years up to 75 years of age. 2. patients with type 2 diabetes on stable dose of metformin for at least 6 weeks prior to screening and hba1c at screening visit of �42 mmol/mol and �75 mmol/mol. 3. no significant signs or symptoms of coronary artery disease or, if known coronary artery disease, currently free of symptomsa and (i) all major epicardial vessels with <50% stenosis within 12 months prior to screening, or (ii) if revascularized, with all major epicardial vessels with <50% remaining stenosis after stenting or bypass surgery procedure between 3 and 12 months prior to screening. 4. normal left ventricular ejection fraction (�50%) assessed within 1 year prior to informed consent, and, if applicable, after most recent acute episode of coronary artery syndrome, or at screening visit. 5. subjects with bmi �25 kg/m2. 6. female subjects must be 1 year post-menopausal, surgically sterile, or using an acceptable method of contraception (an acceptable method of contraception is defined as a barrier method in conjunction with a spermicide) for the duration of the study (from the time they sign consent) to prevent pregnancy.b acardiac symptoms include, but are not limited to, angina pectoris, dyspnea, and fatigue on exertion judged by a physician to be of cardiac origin. bin addition, oral contraceptives, approved contraceptive implant, long-term injectable contraception, intrauterine device, or tubal ligation are allowed. oral contraception alone is not acceptable; additional barrier methods in conjunction with spermicide must be used. table 2. exclusion criteria. medical conditions: 1. blood pressure at screening that would require a change in blood pressure treatment over the study period or any of the following: systolic blood pressure >160 mmhg or diastolic blood pressure >100 mmhg. 2. history of stroke or other clinically significant cerebrovascular disease. 3. any of the following cardiovascular diseases known within 3 months prior to signing the consent at enrollment: a. atrial fibrillation, or other unstable or severe arrhythmia affecting heart function; b. unstable heart failure or any heart failure with nyha class iii and iv; c. significant valvular disease; d. significant peripheral artery disease. 4. planned cardiac surgery or angioplasty within 3 months from enrollment. 5. clinical diagnosis of type 1 diabetes, maturity onset diabetes of the young (mody), secondary diabetes, or diabetes insipidus. 6. verified body weight variability of >3 kg during the 3 months before screening (by interviews). 7. active malignancy requiring treatment at the time of visit 1 (with the exception of successfully treated basal cell or treated squamous cell carcinoma). 8. patients with severe hepatic impairment (child–pugh class c). 9. unstable or rapidly progressing renal disease. 10. clinically significant disease or disorder which, in the opinion of the investigator, may either put the subject at risk because of participation in the study, or influence the results or the subject’s ability to participate in the study. prior/concomitant therapy: 11. ongoing treatment with other antidiabetic drugs than metformin. 12. ongoing treatment with loop diuretics. 13. ongoing weight loss diet (hypocaloric diet) or use of weight loss agents. 14. contraindications to dapagliflozin therapy. 15. ongoing treatment with systemic steroids at time of informed consent or change in dosage of thyroid hormones within 6 weeks prior to informed consent or any other uncontrolled endocrine disorder except for t2dm. prior/concurrent clinical study experience: 16. previous enrollment in the present study or participation in another clinical study with an investigational product during the last 1 month prior to screening. diagnostic assessments: 17. estimated glomerular filtration rate (egfr) < 45 ml/min/1.73 m2, based on the mdrd study equation (www.kidney.org/content/mdrd-study-equation) (egfr ¼175 � (scr) � 1.154 � (age) � 0.203 � 0.742 [if female] � 1.212 [if black]). 18. alcohol or drug abuse within the 3 months prior to informed consent that would interfere with trial participation or any ongoing condition leading to a decreased compliance to study procedures or study drug intake. 19. any condition in which mri and pet are contraindicated such as, but not limited to, having a metallic implant (such as pacemaker or cochlear implant), permanent make up, claustrophobia, or bmi �40 kg/m2. other exclusions: 20. involvement in the planning and/or conduct of the study (applies to astrazeneca, ucr, and/or antaros staff and/or staff at the study site). 21. plasma donation within one month of screening or any blood donation/blood loss >450 ml during the 3 months prior to screening. 22. women who has a positive pregnancy test at enrollment or randomization, or are breastfeeding. upsala journal of medical sciences 61 http://www.kidney.org/content/mdrd-study-equation randomization between placebo and ip will be 1:1 (figure 1). study endpoints and statistics the primary endpoint is the change in glslv between the baseline and the end of treatment visits, measured with mri. myocardial strain is a surrogate for myocardial contractile work that is dependent on atp production capacity, which in turn is dependent on myocardial perfusion and mitochondrial function in the myocardium (14). the secondary endpoint is change in myocardial efficiency between the baseline and the end of treatment visits. the myocardial efficiency is calculated as the mechanical work divided by the total work, with mechanical work defined as mean arterial pressure (map) � stroke volume (sv) � heart rate (hr)/myocardial mass. total work is defined as the total myocardial oxygen consumption per myocardial mass measured by [11c]-acetate pet (15). the myocardial oxygen consumption is directly proportional to the clearance of [11c]-acetate. [11c]-acetate is immediately converted into [11c]-acetyl coa in the cell, and after entering the citric acid cycle the label 11 c is released as [11c]-co2. also, it is assumed that the myocardial washout of [11c]-acetate is a marker of the citric cycle activity and hence mitochondrial function. [11c]-acetate pet will also be used to estimate myocardial perfusion. a key exploratory objective in the study is to investigate the change in uptake of free fatty acids in the myocardium by measuring [18f]-ftha uptake with pet. [18f]-ftha pet will also be applied to investigate the effect of dapagliflozin as compared to placebo on fatty acid uptake in other tissues, e.g. the brain and the liver. [18f]-ftha uptake will be measured in a subset of the patients as described below. all personnel involved with the analysis of the study will remain blinded until the study is finalized and the database is locked. a comprehensive statistical analysis plan (sap) has been prepared. any subsequent amendments to the analysis will be documented, with final amendments completed prior to the unblinding of the data. statistical analyses—sample size estimation the common standard deviation in estimated change from baseline of glslv measured using cardiac mri is assumed to be 2.0%, which is a conservative approximation of the results found in the literature (1.73%) (16). the effect size after 6 weeks of treatment is difficult to predict, as there are no prior short interventional studies. however, based on the meta-analysis performed (9), a change in gls of the magnitude of 1 sd has a significant association with mortality. baseline sd of glslv using cardiac mri has been shown to be 2.25 (16) and 2.5 (17). therefore, we judge the effect of the same magnitude as the baseline sds, namely 2% improvement in glslv, to be clinically significant, and used it to power the study. based on these assumptions, it is estimated that 17 evaluable patients/arm provides 80% power at a two-sided alpha level of 0.05 to detect a treatment difference of 2% in the change from baseline in glslv between dapagliflozin and placebo, assuming a common standard deviation in estimated change from baseline in glslv of 2.0%. change in myocardial efficiency from baseline to the end of treatment (%, measured as external lv work per gram [defined as map � sv � hr/lv mass] divided by total lv work per gram [linearly proportional to mvo2, in turn linearly proportional to [11c]-acetate clearance/lv mass]), is the secondary endpoint in the dapacard study. healthy subjects had in two different studies baseline myocardial efficiency of 54.3 (n ¼ 36) and 49.3 (n ¼ 8) (pooled mean of 50.21) mmhg � l � g�1, with a standard deviation (sd) of 8.9 and 14.2 mmhg � l � g�1, respectively (15,18). a 25% increase in myocardial efficiency is regarded as clinically significant and translates into 12.55 mmhg � l � g�1 with a sd of 14.2 mmhg � l � g�1 between dapagliflozin and placebo treatment group. altogether 22 evaluable subjects per group are needed to detect a treatment difference of 12.55, at a twosided significance level of 0.05 and 80% power. assuming a 15% non-evaluable rate, a total of 52 (26 per group) subjects will be randomized. the [18f]-ftha examinations are exploratory and will not be performed in all the randomized patients. instead, a minimum 40 and a maximum of 44 of the randomized patients will be scheduled for examination for [18f]-ftha uptake in heart, liver, kidney, and the brain. this is due to ethical reasons and aims to limit unnecessary exposure to radioactivity. viljanen et al. (19) reported sd of myocardial fatty acid uptake to be 0.4 (before diet) and 0.2 (after diet) in a healthy population, leading to a pooled sd of 0.32. no information is available regarding the magnitude of the treatment effect in the planned study, but an effect of approximately 0.3–0.4 (approximately 10% relative change) is judged to be clinically relevant. using this assumption, and the pooled estimate of the sd as a conservative approximation of the variability of measurement differences between baseline and end of treatment, leads to approximately 80–90% power with 40 randomized patients (at a two-sided alpha level of 0.05 and assuming 15% non-evaluable rate). analysis of the efficacy endpoints all the analyses will be based using measurements that were taken in accordance to the planned procedures. since the main objective of the study is to search for mechanisms, the per-protocol population will be used for the analyses (e.g. if a patient uses prohibited medication during the study, this patient may be removed from the analysis). as the primary, secondary, and exploratory objectives of this study are of the same nature, namely to detect a systematic difference between the dapagliflozin and the placebo groups in measurements that can be regarded as continuous, all the endpoints will be analyzed in the same manner. an ancova model will be used which, besides the treatment group indicator, includes the baseline value of the respective endpoint as a co-variate. the results will be summarized through least 62 a. åkerblom et al. square means (lsm) for each of the treatment groups, obtained from the model, as well as the difference thereof, with the corresponding standard deviations, confidence intervals, and, in the case of the difference in lsm, p values. evaluation of safety throughout the study, safety findings, such as serious adverse events and adverse events leading to discontinuation of treatment, will be collected. these will later be summarized, with the frequencies of different types of adverse events tabulated. the details of the events will be provided in listings. safety will also be evaluated through safety labs, and the results will be summarized in terms of the values of the labs before and after the treatment period, as well as the differences thereof. study sites and collaborators sites are selected on the basis of availability of pet and mri facilities with a prerequisite of the possibility to produce the radioactive pet tracers, namely [11c]-acetate and [18f]-ftha. the facilities must be adjacent to a clinic with the capacity of including subjects for the study, based on feasibility data. only two sites have been identified that fulfill these requirements: uppsala university hospital, uppsala, sweden and turku university hospital, turku, finland. the uppsala clinical research center (ucr) and antaros medical are responsible for site and data management, as well as imaging management (including analyses), respectively. covance laboratories is the central lab involved in the study. astrazeneca is the sponsor of the study. present status the first patient was enrolled on 28 february 2018, at the turku university hospital, turku, finland. the first patient in sweden to be recruited is scheduled for august 2018. it is expected that all the patients planned to participate in the study will be enrolled within a year of study start. discussion the beneficial effects of the sglt2 inhibitors on cv outcomes, including cv mortality and decreased rate of heart failure admissions, is a major progress in the treatment of patients with diabetes (4,5). a number of potential mechanisms have been suggested to explain the beneficial effects on cv outcomes (8,20–23). one of these is the reduction of plasma volume due to osmotic diuresis, followed by reduced preload, decreased arterial stiffness and blood pressure, which, in turn, would reduce afterload, leading to an improved coronary circulation with increased subendocardial blood flow (20). increased hematocrit and an increase in plasma levels of ketones have been suggested to improve oxygenation and mitochondrial energy metabolism of the myocardium, respectively (8,14,21,24). a recent meta-analysis has demonstrated a possible renoprotective effect (6,23). however, the cv and renoprotective mechanisms are still unclear and go beyond effects on glucose control, and, based on the fast onset, primary effects on atherosclerosis are unlikely. therefore, this study focuses on the early effects, within weeks, of sglt2 inhibition on cardiac function and metabolism. an additional hypothesis is based on the increased urinary glucose excretion caused by sglt2 inhibition, which leads to enhanced night-time catabolism, followed by increased glycogenolysis, gluconeogenesis, and production of ketone bodies (24). the increased gluconeogenesis during night-time would inhibit mammalian target of rapamycin (mtor), which would increase autophagy/mitophagy of damaged mitochondria and biogenesis, as well as fusion of mitochondria, maximizing bioenergetic efficiency (22,25), and, possibly, improved myocardial efficiency and contractile work, which will be investigated in this study. in the current dapacard study, we explore in depth the effect of the sglt2 inhibitor dapagliflozin on myocardial contractile work, myocardial efficiency, substrate uptake, and cardiac metabolism with mri, pet, and biomarkers and may help unravel these, and other, effects of dapagliflozin. in conclusion, the overall aim with the dapacard study is to gain further knowledge about beneficial effects of dapagliflozin on cv outcomes by advanced imaging with mri and pet as well as biomarkers. this will potentially help laying the foundation to tailor treatment strategies with sglt2 inhibitors in patients with t2dm in the future. disclosure statement axel åkerblom received speaking fees from astrazeneca. jonas oldgren received fees to his institution from astrazeneca, boehringer ingelheim, bayer, bristol-myers squibb, daichii sankyo, pfizer and sanofi. aino latva-rasku and pirjo nuutila have nothing to declare. lars johansson is employed by antaros medical ab, gothenburg, sweden. vera lisovskaja, cecilia karlsson, jan oscarsson are employed by astrazeneca, gothenburg, sweden. funding the dapacard trial is funded by astrazeneca. notes on contributors axel åkerblom is a senior consultant in cardiology at the uppsala university hospital, and a researcher at the department of medical sciences and uppsala clinical research center, uppsala university, uppsala, sweden. cecilia karlsson, md, phd, holds a position as senior medical lead at cardiovascular, renal and metabolism global medicines development (cvrm gmd), astrazeneca gothenburg, sweden and has an affiliation to department of molecular and clinical medicine, institute of medicine, sahlgrenska academy, university of gothenburg, sweden. vera lisovskaja is a senior statistician in astrazeneca, sweden, primarily working with analysis of clinical trials in later phases of development. jonas oldgren is a cardiologist and professor of coagulation research at department of medical sciences, uppsala university, uppsala, sweden. upsala journal of medical sciences 63 pirjo nuutila, professor of metabolic research, turku pet centre, university of turku; head of the department of endocrinology, turku univ hospital. aino latva-rasku, md, doctoral candidate, university of turku. lars johansson, a phd, is the chief scientific officer of antaros medical with 30 years background in clinical imaging with pet and mri. jan oscarsson is director physician at astrazeneca gothenburg, sweden and professor in physiology, especially endocrinology and integrative metabolism at university of gothenburg. references 1. haffner sm, lehto s, r€onnemaa t, py€or€al€a k, laakso m. mortality from coronary heart disease in subjects with type 2 diabetes and in nondiabetic subjects with and without prior myocardial infarction. n engl j med. 1998;339:229–34. 2. effect of intensive blood-glucose control with metformin on complications in overweight patients with type 2 diabetes (ukpds 34). lancet. 1998;352:854–65. 3. marso sp, daniels gh, brown-frandsen k, kristensen p, mann jfe, nauck ma, et al. liraglutide and cardiovascular outcomes in type 2 diabetes. n engl j med. 2016;375:311–22. 4. zinman b, wanner c, lachin jm, fitchett d, bluhmki e, hantel s, et al. empagliflozin, cardiovascular outcomes, and mortality in type 2 diabetes. n engl j med. 2015;373:2117–28. 5. neal b, perkovic v, mahaffey kw, de zeeuw d, fulcher g, erondu n, et al. canagliflozin and cardiovascular and renal events in type 2 diabetes. n engl j med. 2017;377:644–57. 6. rådholm k, figtree g, perkovic v, solomon sd, mahaffey kw, de zeeuw d, et al. canagliflozin and heart failure in type 2 diabetes mellitus: results from the canvas program (canagliflozin cardiovascular assessment study). 2018; circulation. mar 11 [epub ahead of print]. 7. raz i, mosenzon o, bonaca mp, cahn a, kato et, silverman mg, et al. declare-timi 58: participants’ baseline characteristics. diabetes obes metab. 2018;20:1102–10. 8. inzucchi se, zinman b, fitchett d, wanner c, ferrannini e, schumacher m, et al. how does empagliflozin reduce cardiovascular mortality? insights from a mediation analysis of the empa-reg outcome trial. dia care. 2018;41:356–63. 9. kalam k, otahal p, marwick th. prognostic implications of global lv dysfunction: a systematic review and meta-analysis of global longitudinal strain and ejection fraction. heart. 2014;100:1673–80. 10. mather kj, hutchins gd, perry k, territo w, chisholm r, acton a, et al. assessment of myocardial metabolic flexibility and work efficiency in human type 2 diabetes using 16-[18 f]fluoro-4-thiapalmitate, a novel pet fatty acid tracer. am j physiol metab. 2016;310: e452–60. 11. hansson nh, tolbod l, harms j, wiggers h, kim wy, hansen e, et al. evaluation of ecg-gated [11c]acetate pet for measuring left ventricular volumes, mass, and myocardial external efficiency. j nucl cardiol. 2016;23:670–9. 12. levelt e, rodgers ct, clarke wt, mahmod m, ariga r, francis jm, et al. cardiac energetics, oxygenation, and perfusion during increased workload in patients with type 2 diabetes mellitus. eur heart j. 2016;37:3461–9. 13. leung m, wong vw, hudson m, leung dy. impact of improved glycemic control on cardiac function in type 2 diabetes mellitus. circ cardiovasc imaging. 2016;9:e003643. 14. neubauer s. the failing heart-an engine out of fuel. n engl j med. 2007;356:1140–51. 15. tuunanen h, engblom e, naum a, någren k, hesse b, airaksinen kej, et al. free fatty acid depletion acutely decreases cardiac work and efficiency in cardiomyopathic heart failure. circulation. 2006; 114:2130–7. 16. singh a, steadman cd, khan jn, horsfield ma, bekele s, nazir sa, et al. intertechnique agreement and interstudy reproducibility of strain and diastolic strain rate at 1.5 and 3 tesla: a comparison of feature-tracking and tagging in patients with aortic stenosis. j magn reson imaging. 2015;41:1129–37. 17. gjesdal o, yoneyama k, mewton n, wu c, gomes as, hundley g, et al. reduced long axis strain is associated with heart failure and cardiovascular events in the multi-ethnic study of atherosclerosis. j magn reson imaging. 2016;44:178–85. 18. tuunanen h, kuusisto j, toikka j, j€a€askel€ainen p, marjam€aki p, peuhkurinen k, et al. myocardial perfusion, oxidative metabolism, and free fatty acid uptake in patients with hypertrophic cardiomyopathy attributable to the asp175asn mutation in the alphatropomyosin gene: a positron emission tomography study. j nucl cardiol. 2007;14:354–65. 19. viljanen apm, karmi a, borra r, p€arkk€a jp, lepom€aki v, parkkola r, et al. effect of caloric restriction on myocardial fatty acid uptake, left ventricular mass, and cardiac work in obese adults. am j cardiol. 2009;103:1721–6. 20. lytvyn y, bjornstad p, udell ja, lovshin ja, cherney dzi. sodium glucose cotransporter-2 inhibition in heart failure: potential mechanisms, clinical applications, and summary of clinical trials. circulation. 2017;136:1643–58. 21. ferrannini e. sodium-glucose co-transporters and their inhibition: clinical physiology. cell metab. 2017;26:27–38. 22. esterline rl, vaag a, oscarsson j, vora j. mechanisms in endocrinology: sglt2 inhibitors: clinical benefits by restoration of normal diurnal metabolism? eur j endocrinol. 2018;178:r113–25. 23. jardine mj, mahaffey kw, neal b, agarwal r, bakris gl, brenner bm, et al. the canagliflozin and renal endpoints in diabetes with established nephropathy clinical evaluation (credence) study rationale, design, and baseline characteristics. am j nephrol. 2017; 46:462–72. 24. ferrannini e, muscelli e, frascerra s, baldi s, mari a, heise t, et al. metabolic response to sodium-glucose cotransporter 2 inhibition in type 2 diabetic patients. j clin invest. 2014;124:499–508. 25. rambold as, cohen s, lippincott-schwartz j. fatty acid trafficking in starved cells: regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics. dev cell. 2015;32:678–92. 64 a. åkerblom et al. abstract background the dapacard study study objectives study design blood samples study population, study conduct, and follow-up study endpoints and statistics statistical analysessample size estimation analysis of the efficacy endpoints evaluation of safety study sites and collaborators present status discussion disclosure statement notes on contributors references reduced 10-year risk of developing cardiovascular disease after participating in a lifestyle programme in primary care original article reduced 10-year risk of developing cardiovascular disease after participating in a lifestyle programme in primary care lena l€onnberga,b , elin ekblom-bakc and mattias damberga,b acenter for clinical research, county of v€astmanland, uppsala university, v€asterås, sweden; bdepartment of public health and caring sciences, family medicine and preventive medicine, uppsala university, uppsala, sweden; cthe swedish school of sports and health sciences, stockholm, sweden abstract background: despite well-known preventive effects for future cardiovascular disease (cvd) risk through lifestyle changes, scientific evaluations of lifestyle programmes in primary care are scarce. moreover, structured lifestyle counselling is still not integrated in everyday clinical practice. we aimed to evaluate change in cardiovascular risk factors and framingham 10-year risk score of developing cvd in men and women at high cardiovascular risk after participation in a structured lifestyle programme over 1 year. a single-group study was carried out with a 1-year follow-up including before and after measurements. methods: the lifestyle programme comprised five appointments to a district nurse over 1 year, focussing on lifestyle habits based on motivational interviewing. fasting blood samples and anthropometric measurements were obtained at baseline and 1-year follow-up. the 10-year risk of cvd was calculated according to framingham general cvd risk score. results: a total of 404 patients were included in the study. there was a positive change over 1 year in the total study population for all risk factors evaluated. this included improvements in weight, waist circumference, blood pressure, blood lipids, and fasting glucose. the 10-year risk of developing cvd decreased for the total population from 24.8% to 21.4% at 1 year, equivalent to a 14% decrease. conclusions: a structured lifestyle programme in primary care contributes to significant improvements of cardiovascular risk factors and the reduction of 10-year risk for cvd for both men and women at high cardiovascular risk. article history received 23 august 2019 revised 31 january 2020 accepted 3 february 2020 keywords cardiovascular prevention; cardiovascular risk factors; framingham heart study; hypertension; lifestyle counselling; type 2 diabetes mellitus introduction although there has been substantial improvement in cardiovascular disease (cvd) outcomes, non-communicable diseases including ischaemic heart disease, type 2 diabetes mellitus (t2dm), stroke, and chronic obstructive pulmonary disease account for the majority of deaths and disabilityadjusted life-years (dalys) world-wide (1). the leading underlying risk factors in 2017 for death or dalys were: high systolic blood pressure, causing 10.4 million deaths in 1 year, followed by smoking, high fasting plasma glucose, high body-mass index (bmi), and high concentrations of low-density lipoprotein cholesterol (ldl). guidelines from the american heart association, european heart association as well as the swedish national board of health and welfare emphasise that the highest clinical priority for cvd prevention should be directed towards patients with prevalent cvd or those at high risk of developing cvd (2–4). despite the guidelines and the knowledge of the preventive effect for future cvd through lifestyle changes such as improved diet, increased physical activity, and smoking cessation, many patients with high cvd risk do not comply with this and have uncontrolled blood pressure and high concentrations of lipids and blood glucose (3,5–7). a teambased, comprehensive, patient-centred approach that addresses all aspects of a patient’s lifestyle habits has previously been proposed as an effective strategy for cvd prevention in clinical practice (4,8,9). however, scientific evaluations of lifestyle programmes in primary care are scarce (10–12), and structured lifestyle counselling is still not integrated in everyday clinical practice. to enhance the care of patients recently diagnosed with hypertension, t2dm, or impaired glucose tolerance (igt) and address their cardiovascular risk profile, we started a structured lifestyle programme at a primary care unit in v€asterås, sweden. in a previous publication, we observed favourable changes of lifestyle habits including physical activity, diet, smoking, and stress over 1 year (13). the results support the utility of a multifactorial, structured approach in clinical practice to change unhealthy lifestyle habits. the aim of the present study was to evaluate changes in cardiovascular risk factors and 10-year risk for cvd according contact lena l€onnberg lena.lonnberg@regionvastmanland.se centrum f€or klinisk forskning, v€asterås hospital, 721 89, v€asterås, sweden supplemental data for this article can be accessed here. � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution-noncommercial license (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 3, 250–256 https://doi.org/10.1080/03009734.2020.1726533 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1726533&domain=pdf&date_stamp=2020-06-29 http://orcid.org/0000-0003-4706-6915 http://orcid.org/0000-0002-3901-7833 http://orcid.org/0000-0001-7654-7553 https://doi.org/10.1080/03009734.2020.1726533 http://creativecommons.org/licenses/by-nc/4.0/ https://doi.org/10.1080/03009734.2020.1726533 http://www.tandfonline.com to the framingham general cvd risk score in men and women at high cardiovascular risk after participation in a structured lifestyle programme in primary care for 1 year. we hypothesised that a structured lifestyle programme would reduce cardiovascular risk factors and 10-year risk of cvd. methods study design and population we conducted a single-group study with a 1-year follow-up including before and after measurements. all data were collected consecutively and registered in a database. patients registered at citypraktiken, a primary care unit in v€asterås, sweden, were enrolled during a 5-year period between october 2009 and september 2014. inclusion criteria were men and women aged 30–74 years, newly diagnosed with either hypertension (blood pressure >140/90 mmhg), t2dm (fasting plasma glucose concentration >7 mmol/l), or igt (2h glucose concentration of 7.8–11.0 mmol/l on the 75-g oral glucose tolerance test). antihypertensive or cholesterol-lowering medication was prescribed, when needed, according to hypertension and diabetes guidelines (14–16). exclusion criteria were diagnosed dementia or severe psychiatric disease. a total of 448 patients were referred by their physician to join the lifestyle programme. eleven patients did not meet the inclusion criteria, three patients died, and 30 patients did not provide consent. thus, 404 patients were included in the analyses. sixty-three patients (16%) were lost to follow-up. the study was conducted according to the ethical guidelines of the declaration of helsinki and the good clinical practice guidelines. the swedish ethical review authority approved the study (reference number: 2014/497). all patients provided written informed consent. the study was registered at www.clinical-trials.gov (clinicaltrial.gov id: dnr 2014/497). the structured lifestyle programme the structured lifestyle programme has been described in detail previously (13). in short, the structured lifestyle programme comprised five appointments to a district nurse with post-graduate credits in diabetes care and the metabolic syndrome, at baseline and after 3, 6, 9, and 12 months. every appointment focussed primarily on lifestyle habits. fasting blood samples were obtained one week before the baseline and at the 1-year follow-up. anthropometric variables were measured at baseline and at the 1-year follow-up. blood pressure and waist circumference were measured at every appointment. clinical examinations at baseline and 1-year follow-up, all patients were weighed to the nearest 0.1 kg using an electronic scale. height was measured to the nearest 0.5 cm. body mass index was calculated from the measured weight and height as kg/m2. waist circumference was measured with the participant in a standing position, midway between the lower rib margin and the iliac crest to the nearest 0.5 cm. blood pressure was measured using a standard auscultatory method with the participant in a seated position after a 10-min rest. laboratory measurements a blood sample was drawn from the antecubital vein after overnight fasting. concentrations of total cholesterol (mmol/ l), low-density lipoprotein (ldl) (mmol/l), high-density lipoprotein (hdl) (mmol/l), and triglycerides (mmol/l) were estimated by standard methods at aleris medilab (stockholm, sweden, iso/iec 151 89, certified). fasting plasma glucose concentrations were analysed at the primary care unit from venous blood sample, using the hemocuevr glucose 201 rt system. the framingham 10-year risk of cvd framingham general cvd risk score (2008) calculates the risk of developing cardiovascular events in 10 years (17). the prediction variables used are age, sex, total cholesterol, hdl cholesterol, systolic blood pressure, blood pressure treatment or not, diabetes mellitus or not, and current smoking. endpoints assessed in the framingham general cvd risk score are a composite of coronary heart disease (coronary death, myocardial infarction, coronary insufficiency, and angina), cerebrovascular events (including ischaemic stroke, haemorrhagic stroke, transient ischaemic attack), peripheral artery disease (intermittent claudication), and heart failure. a calculated 10-year risk of <5% is considered as low, �5 to <7.5% as borderline, �7.5% to <20% as intermediate, and �20% as high risk (18). the metabolic syndrome the metabolic syndrome was classified according to the national cholesterol education programme (ncep) adult treatment panel iii (atpiii) (19) as �3 of the following risk determinants; waist circumference >102 cm (men) and >88 cm (women); systolic blood pressure �130/85 mmhg; triglycerides �1.7 mmol/l; hdl <1.0 (men) and <1.3 (women) mmol/l; or fasting plasma glucose >6.1 mmol/l. patients with anti-hypertensive or statin medication have been included in the high blood pressure or triglyceride groups. the care need index the care need index is used to evaluate a population’s need for primary care, with a high index indicating an increased need for health care (20). the care need index measures socio-economic factors and comprises seven variables: age >65 years; born in eastern europe, asia, africa, or south america; unemployed; single parent with child younger than 17 years; children under 5 years; poor education level; and highly mobile people. upsala journal of medical sciences 251 http://www.clinical-trials.gov statistics results have been presented in the total population, by sex or by diagnosis (hypertension or t2dm þ igt). an intentionto-treat approach was used, and last observation was carried forward for missing data for all variables. data were checked for normality by visual inspection of histograms for each variable. parametric statistical methods should be used for all cardiovascular risk factors and framingham risk score. a twotailed paired t test was used to study within-group changes over 1 year, and an unpaired t test to study subgroup differences. the rejection criteria for each of the individual nullhypotheses were adjusted according to the bonferroni–holm method to counteract the problem of multiple comparisons (i.e., type i error). alpha-levels after correction with bonferroni–holm ranged from p < 0.005 for the highest ranked result to p < 0.05 for the lowest ranked result of the paired t test. chi-square test was used to study differences at baseline for medication and metabolic syndrome prevalence between study population and patients lost to follow-up. mcnemar’s test was used to detect changes over 1 year of proportions of patients at increased risk for each cvd risk factor and 10-year risk of cvd. analyses were performed using statistical package for social science (ibm spss statistics for windows, version 24.0; ibm corp. armonk, ny, usa). results baseline characteristics the population comprised 404 participants (52% women), median age 59 years (range 43–74 years), recently diagnosed with either hypertension (73%), t2dm (25%), or igt (2%) (table 1). the metabolic syndrome was present in 50% of the study population, and 6% had a previous history of cvd. subjects lost to follow-up during the study were slightly younger, more often daily smokers, and were more often treated with statins than patients in the study population (p < 0.05). the care need index was 0.86 for the present primary care unit in 2014 as compared with 1.08 (±0.20) in the county of v€astmanland. cardiovascular risk factors there was a positive change over 1 year in the total study population for all risk factors evaluated (table 2). changes in systolic and diastolic blood pressure (bp) were observed in table 1. baseline characteristics of the study population and patients lost to follow-up. total sample (n ¼ 404) men (n ¼ 195) women (n ¼ 209) lost to follow-up (n ¼ 63) age, years 59 (±8.8) 59 (±9.0) 60 (±8.6) 55 (±10.1)a sex, women 52% (209) 52% (33) hypertension 73% (294) 65% (127) 78% (162) 79% (50) daily smoking 10% (41) 9% (18) 11% (23) 19% (12)a anti-hypertensive medication 56% (226) 59% (114) 54% (112) 57% (36) statin medication 13% (52) 17% (33) 9% (19) 5% (3)a glucose-lowering medicationb 38% (42) 41% (28) 34% (14) 46% (6) previous cardiovascular disease 6% (25) 8% (15) 5% (10) 6% (4) metabolic syndrome 50% (201) 59% (114) 42% (87) 46% (29) data are presented as mean (sd) for age and % (number of patients) for all other variables. asignificantly different from study population (p < 0.05). bonly individuals with type 2 diabetes mellitus or impaired glucose tolerance (n ¼ 109) were included for this variable. table 2. cardiovascular risk factors at baseline, 1 year, and change over 1 year, total sample. n ¼ 404 baseline mean (sd) 1 year mean (sd) change over 1 year mean (95% ci) p value weight (kg) 86.7 (18.7) 84.5 (18.6) �2.1 (�1.6 to �2.6)c <0.001a bmi (kg/m2) 29.3 (5.5) 28.6 (5.5) �0.7 (�0.5 to �0.9)c <0.001a waist circumference (cm) 101.2 (13.7) 98.2 (13.5) �2.9 (�2.5 to �3.4) <0.001a systolic bp (mmhg) 149.2 (17.1) 140.5 (15.4) �8.7 (�7.1 to �10.4)c <0.001a anti-hypertensive medication, n ¼ 268 150.3 (18.1) 139.3 (15.3) �11.0 (�9.0 to �13.1)c <0.001a no medication, n ¼ 136 147.0 (14.8) 142.8 (15.5) �4.2 (�1.7 to �6.8) 0.001a diastolic bp (mmhg) 88.0 (10.5) 84.1 (8.8) �4.0 (�3.0 to �4.9)c <0.001a anti-hypertensive medication, n ¼ 268 88.4 (11.1) 83.3 (8.6) �5.1 (�3.9 to �6.3) <0.001a no medication, n ¼ 136 87.4 (9.3) 85.5 (9.0) �1.8 (�0.3 to �3.3)c 0.017a total cholesterol (mmol/l) 5.94 (1.18) 5.72 (1.10) �0.22 (�0.13 to �0.31) <0.001a with statin medication, n ¼ 65 6.19 (1.63) 5.22 (1.36) �0.97 (�0.58 to �1.37) <0.001a no statin medication, n ¼ 339 5.89 (1.06) 5.81 (1.02) �0.08 (0.00 to �0.15) 0.051 ldl (mmol/l) 3.86 (0.99) 3.72 (0.99) �0.14 (�0.06 to �0.21) <0.001a with statin medication, n ¼ 65 3.89 (1.32) 3.21 (1.19) �0.68 (�0.38 to �0.98) <0.001a no statin medication, n ¼ 339 3.86 (0.92) 3.82 (0.91) �0.04 (�0.03 to 0.10) 0.249 hdl (mmol/l) 1.37 (0.37) 1.40 (0.38) 0.04 (0.02 to 0.05)c <0.001a triglycerides (mmol/l) 1.66 (1.06) 1.51 (0.82) �0.15 (�0.08 to �0.23)b,c 0.001a fasting plasma glucose (mmol), n ¼ 109 8.51 (2.99) 7.15 (1.70) �1.36 (�0.85 to �1.88) <0.001a glucose-lowering medication, n ¼ 42 10.17 (3.75) 7.85 (1.92) �2.32 (�1.19 to �3.46) <0.001a no medication, n ¼ 67 7.48 (1.75) 6.72 (1.39) �0.76 (�0.35 to �1.17) <0.001a values are presented as mean (sd) for baseline and 1-year measurements, and as mean (95% ci) for change over 1 year. asignificantly different after bonferroni–holm correction. bsignificantly different between sex. csignificantly different between diagnosis (p < 0.05). 252 l. lönnberg et al. patients with or without anti-hypertensive medication, with the greatest decrease in patients with medication. total cholesterol and ldl decreased mainly in patients treated with statin medication. fasting plasma glucose decreased regardless of glucose-lowering medication for patients with t2dm or igt. longitudinal data for blood pressure and waist circumference (five measurements over 1 year) exposed a continuous decrease over 1 year (supplementary figures 1 and 2, available online). sex-specific subgroup analyses exposed positive changes for all risk factors, similar to the analysis of the total study group (supplementary table 1, available online). diagnosis-specific subgroup analyses exposed that all risk factors decreased for the two diagnosis subgroups, except for diastolic blood pressure and ldl in patients with t2dm or igt. reduction of systolic blood pressure was more prominent for patients with hypertension than with t2dm or igt. patients with t2dm or igt had a larger reduction of weight and body mass index compared to patients with hypertension. increases in hdl concentrations were more prominent for patients with t2dm or igt (supplementary table 2, available on line). moreover, all analyses of cardiovascular risk factors have additionally been performed excluding participants lost to follow-up. the results are similar to the results from the study population where intention-to-treat was applied (supplementary tables 3–5, available online, for analyses of patients with data available at baseline and at 1 year). the framingham 10-year risk of cvd the 10-year risk of developing cvd decreased for the total population over 1 year, equivalent to a relative decrease of 14% compared with baseline values (table 3; figure 1). men had an almost doubled 10-year risk for cvd compared with women at both baseline and 1-year follow-up. patients with t2dm or igt had a higher 10-year risk than patients with hypertension. there was a decreased 10-year risk of developing cvd in both men and women, and for both diagnosis subgroups (table 3; supplementary figure 3, available online). change in proportions of patients at increased risk for different risk factors the proportions of patients at increased risk over 1 year decreased regarding blood pressure, triglycerides, body mass index, waist circumference, and framingham risk score (figure 2). discussion we observed significant improvements for all risk factors and in 10-year cvd risk after participation in a 1-year structured lifestyle programme in primary care, in both men and women with high cardiovascular risk and irrespective of whether they were diagnosed with hypertension, t2dm, or igt. this included improvements in weight, waist circumference, blood pressure, blood lipids, and fasting glucose. the 10-year cvd risk decreased from 24.8% to 21.4% in the total study population equivalent to an absolute mean decrease of 3.4% in the total study population (3.0% in women and 3.9% in men). it is worthy of note that the total study population except for women were all at high risk for cvd, exceeding 20% risk for future cvd. after participation in the lifestyle programme, patients with hypertension instead had an intermediate 10-year risk of cvd. patients with t2dm þ igt had the highest 10-year risk at both the baseline and 1-year follow up, indicating that this subgroup is in crucial need of both drug therapy and lifestyle counselling. the reduction in systolic blood pressure in our study is in line with previous studies. for example, a swedish randomised study of 151 middle-aged men and women assigned to either lifestyle intervention or control group presented a 4.9 mmhg reduction of systolic blood pressure and a 2.2 cm reduction of waist circumference in the intervention group compared with the control group (11). similar results were reported in a review article by dickinson et al. in which a mean reduction in systolic blood pressure of 5.0 mmhg after participation in different lifestyle programmes was found (21). the us preventive services task force presented comparable results in their updated version of a systematic table 3. estimated 10-year risk of developing cvd according to framingham risk score at baseline, 1 year, and change over 1 year. baseline, % mean (sd) 1 year, % mean (sd) change over 1 year, % mean (95% ci) p value total (n ¼ 402) 24.8 (15.6) 21.4 (13.5) �3.4 (�2.7 to �4.1) <0.001a men (n ¼ 193) 31.8 (16.8) 27.9 (14.2) �3.9 (�2.7 to �5.0) <0.001a women (n ¼ 209) 18.3 (11.2) 15.4 (9.6) �3.0 (�2.1 to �3.8) <0.001a hypertension (n ¼ 293) 21.1 (12.5) 18.3 (11.2) �2.8 (�2.1 to �3.5) <0.001a t2dm þ igt (n ¼ 109) 34.8 (18.7) 29.8 (15.7) �5.0 (�3.1 to �6.9)b <0.001a values are presented as mean (sd) for baseline and 1-year measurements, and as mean (95% ci) for change over 1 year. asignificantly different after bonferroni–holm correction. bsignificantly different between diagnosis (p < 0.05). -50 -40 -30 -20 -10 0 10 20 30 % d el ta v al ue o ve r 1 y ea r 10-year risk of developing cvd, individual δvalues mean= -3.4% figure 1. ten-year risk of developing cvd, individual d-values, total study population (n ¼ 404). upsala journal of medical sciences 253 https://doi.org/10.1080/03009734.2020.1726533 https://doi.org/10.1080/03009734.2020.1726533 https://doi.org/10.1080/03009734.2020.1726533 https://doi.org/10.1080/03009734.2020.1726533 https://doi.org/10.1080/03009734.2020.1726533 https://doi.org/10.1080/03009734.2020.1726533 review regarding ‘behavioral counselling to promote a healthful diet and physical activity for cardiovascular disease prevention’ (22). they concluded that even a small reduction in blood pressure (2 mmhg systolic blood pressure and 1 mmhg diastolic blood pressure) may result in long-term decrease in cvd morbidity and mortality. furthermore, epidemiological data suggest that this small reduction is associated with a decreased risk for coronary artery disease by 6% and for cerebrovascular event by 16% (23). in contrast, according to a report from the swedish council on health and technology assessment, 20%�30% of those who have been prescribed blood pressure-lowering drugs do not reach the treatment goal of blood pressure <140/90 mmhg (24). the results of our study indicate that the number of patients that reach the blood pressure goal of <140/90 can be increased with a structured lifestyle programme and counselling, regardless of anti-hypertensive medication or not. patients receiving statin medication showed reduced serum concentrations of cholesterol and ldl, which is in line with previous observations suggesting fairly low effects of reduced or modified dietary fat on serum (25). according to the aha/acc guidelines to prevent cvd, all patients at intermediate and high risk of cvd should be given statin treatment aiming to reduce serum ldl by 30%�40% and 50% respectively (2). similar recommendations are also stated in the national guidelines for diabetes care from the swedish national board of health and welfare (26). it is also stressed that enhancement of lifestyle habits should always accompany medical treatment, for all patients at high cardiovascular risk (2), which is in agreement with the intention of the structured lifestyle programme in our study. an increase of hdl was detected in the total study population as well as in sex and diagnosis subgroups. this was most prominent for patients with t2dm. it is well known that the serum concentration of hdl depends on to which extent a person is physically active, and that increased physical activity increases serum hdl (27). in our previous study on how lifestyle habits changed after participating in the structured lifestyle programme we observed an increase in physical activity, which could be an explanation of the present increase of hdl (13). the relative decline by 14% of the 10-year risk of cvd in the total study population is similar to another swedish study of 100 patients at high cardiovascular risk who participated in a 1-year lifestyle programme presenting a 15% decrease over 1 year (28). on the other hand, there are diverse results of the effects on 10-year risk of cvd after participating in lifestyle counselling. in a canadian randomised controlled treatment study consisting of 315 patients, the authors reported a 24% decreased 10-year risk after participating in a telehealth counselling for patients at high cardiovascular risk (29). in contrast, a study from the finnish diabetes prevention programme with 2250 patients presented a 2.5% decreased 10-year risk after lifestyle counselling offered in primary health care (30). the intensity of the lifestyle programme and groups targeted are partly different from the population in our study, which may explain the various results, although we find it promising that the 10year risk of cvd can be reduced. despite the modest reduction in the finnish study, the authors calculated that 25 men or 59 women would need to change their lifestyle to prevent one cvd in 1 year (30). 0 10 20 30 40 50 60 70 80 90 100 % change in propor�ons (%) of individuals at increased risk for cvd risk factors and framingham risk score baseline 1-year * * * * * * * figure 2. change in proportions of patients at increased risk for cvd risk factors and framingham risk score, total sample (n ¼ 404). � significantly different from baseline after bonferroni–holm correction. 254 l. lönnberg et al. this study was carried out in an ordinary primary care setting, performed in everyday practice, using only the limited resources available at the clinic. these ‘real world data’ strengthen the external validity even though the lack of randomisation limits the inference of the results. as the lack of control group limits the analyses of casual relationship between participation in the programme and change in cardiovascular risk, regression towards the mean must be considered. although the results of repeated measurements of blood pressure and waist circumference may indicate a possible relationship to the reduced cvd risk seen in our material (as the regression towards the mean effect gets smaller with repeated measurements), this limitation has to be taken into account when interpreting our results. this is an intention-to-treat study, where 16% of the patients were lost to follow-up. as this may influence the interpretation of the results, we completed an analysis of the data excluding patients lost to follow-up. the results regarding change in cardiovascular risk factors and cvd risk over 1 year for the study population excluding participants lost to follow-up were well in line with the results where intention-to-treat analysis was applied. we therefore find that the results of the intention-to-treat analysis are valid, despite the fact that there were participants lost to follow-up. as lifestyle counselling is a heterogeneous phenomenon, an evaluation of a real-life setting is of importance. the high socio-economic index for the family care unit in this study might impair the generalisation to other family care units in less prosperous communities. we find that the study design and the rather large number of patients included in our study give an indication of what effect a structured lifestyle programme carried out in primary care might have on cardiovascular risk factors and 10-year risk of cvd. as all patients with a recent diagnosis with hypertension, t2dm, or igt were invited to participate in the structured programme, the population studied is a typical representation of patients receiving care at a primary care unit. it also indicates that the intervention is feasible to carry out in a primary care setting and might be implemented at other primary care units. we conclude that a structured lifestyle programme in primary care contributes to significant improvements of cardiovascular risk factors and the reduction of 10-year risk for cvd for both men and women at high cardiovascular risk. since this is a single-group study there is a need for future randomised controlled trials to confirm our findings. acknowledgements we thank all staff at citypraktiken, in particular the nurses who performed all counselling sessions. we also thank professor mai-lis hellenius for the inspiration to start the structured lifestyle programme. disclosure statement the authors report no conflict of interest. funding funding was received from praktikertj€anst ab, 103 55 stockholm. notes on contributors lena l€onnberg physiotherapist, doctoral student at centre for clinical research, v€asterås, sweden; main research focus on lifestyle counselling for patients at high cardiovascular risk. elin ekblom-bak, associate professor at the swedish school of sport and health sciences in stockholm, sweden; main research focus on the association between sedentary behaviour, physical activity, and cardiorespiratory fitness on health and disease risk in the adult population. mattias damberg, associate professor at 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doi:10.1186/s12872-018-0792-6 29. wister a, loewen n, kennedy-symonds h, mcgowan b, mccoy b, singer j. one-year follow-up of a therapeutic lifestyle intervention targeting cardiovascular disease risk. cmaj 2007;177:859–65. doi: 10.1503/cmaj.061059 30. rautio n, jokelainen j, p€ol€onen a, oksa h, peltonen m, vanhala m, et al. changes in lifestyle modestly reduce the estimated cardiovascular disease risk in one-year follow-up of the finnish diabetes prevention programme (fin-d2d). eur j cardiovasc nurs. 2015;14: 145–52. doi:10.1177/1474515114521713 256 l. lönnberg et al. https://doi.org/10.1371/journal.pone.0005195 https://doi.org/10.1080/03009734.2019.1602088 https://doi.org/10.1093/eurheartj/ehm316 https://doi.org/10.1161/circulationaha.107.699579 https://doi.org/10.1161/cir.0000000000000638 https://doi.org/10.1161/cir.0000000000000638 https://doi.org/10.1177/14034948980260021301 https://doi.org/10.1097/01.hjh.0000199800.72563.26 https://doi.org/10.1001/jama.2017.7171 https://doi.org/10.1001/jama.2017.3303 https://doi.org/10.1371/journal.pone.0176436 https://doi.org/10.1186/s12872-018-0792-6 https://doi.org/10.1503/cmaj.061059 https://doi.org/10.1177/1474515114521713 abstract introduction methods study design and population the structured lifestyle programme clinical examinations laboratory measurements the framingham 10-year risk of cvd the metabolic syndrome the care need index statistics results baseline characteristics cardiovascular risk factors the framingham 10-year risk of cvd change in proportions of patients at increased risk for different risk factors discussion acknowledgements disclosure statement references in vitro biomechanical evaluation of four fixation techniques for distractive–flexion injury stage 3 full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 in vitro biomechanical evaluation of four fixation techniques for distractive–flexion injury stage 3 of the cervical spine thomas henriques, bryan w. cunningham, paul c. mcafee & claes olerud to cite this article: thomas henriques, bryan w. cunningham, paul c. mcafee & claes olerud (2015) in�vitro biomechanical evaluation of four fixation techniques for distractive–flexion injury stage 3 of the cervical spine, upsala journal of medical sciences, 120:3, 198-206, doi: 10.3109/03009734.2015.1019684 to link to this article: https://doi.org/10.3109/03009734.2015.1019684 © informa healthcare published online: 06 mar 2015. submit your article to this journal article views: 768 view related articles view crossmark data citing articles: 1 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2015.1019684 https://doi.org/10.3109/03009734.2015.1019684 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1019684 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1019684 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1019684&domain=pdf&date_stamp=2015-03-06 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1019684&domain=pdf&date_stamp=2015-03-06 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1019684#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1019684#tabmodule upsala journal of medical sciences. 2015; 120: 198–206 original article in vitro biomechanical evaluation of four fixation techniques for distractive–flexion injury stage 3 of the cervical spine thomas henriques1, bryan w. cunningham2, paul c. mcafee3 & claes olerud4 1stockholm spine center, löwenströmska hospital, upplands väsby, sweden, 2orthopaedic spinal research institute, the university of maryland st. joseph medical center, baltimore, maryland, usa, 3scoliosis and spine center, the university of maryland st. joseph medical center, baltimore, maryland, usa, and 4department of orthopaedics, uppsala university hospital, uppsala, sweden abstract purpose. anterior plate fixation has been reported to provide satisfactory results in cervical spine distractive flexion (df) injuries stages 1 and 2, but will result in a substantial failure rate in more unstable stage 3 and above. the aim of this investigation was to determine the biomechanical properties of different fixation techniques in a df-3 injury model where all structures responsible for the posterior tension band mechanism are torn. methods. the multidirectional three-dimensional stiffness of the subaxial cervical spine was measured in eight cadaveric specimens with a simulated df-3 injury at c5–c6, stabilized with four different fixation techniques: anterior plate alone, anterior plate combined with posterior wire, transarticular facet screws, and a pedicle screw–rod construct, respectively. results. the anterior plate alone did not improve stability compared to the intact spine condition, thus allowing considerable range of motion around all three cardinal axes (p > 0.05). the anterior plate combined with posterior wire technique improved flexion–extension stiffness (p = 0.023), but not in axial rotation and lateral bending. when the anterior plate was combined with transarticular facet screws or with a pedicle screws–rod instrumentation, the stability improved in flexion–extension, lateral bending, and in axial rotation (p < 0.05). conclusions. these findings imply that the use of anterior fixation alone is insufficient for fixation of the highly unstable df-3 injury. in these situations, the use of anterior fixation combined with a competent posterior tension band reconstruction (e.g. transarticular screws or a posterior pedicle screws–rod device) improves segmental stability. key words: biomechanical analysis, cervical spine, distractive–flexion injury, internal fixation, transarticular screws, pedicle screws introduction distractive flexion stage 3 injury (df-3) of the cervical spine is characterized by rupture of the posterior soft tissue elements causing instability in flexion (1). both facet joints are dislocated, and there is a translational deformity not exceeding 50% (figure 1). in order to dislocate both facet joints all posterior structures including the posterior annulus fibrosus and the posterior longitudinal ligament (pll) must be ruptured (2). there still is a clinical debate on how best to manage these injuries surgically. an anterior approach has obvious advantages. however, when the posterior annulus is ruptured, disk fragments may shift into the spinal canal when the facet joints are reduced (3-9). an anterior plate fixation is technically simple, and the fusion is under compression, thus optimizing bone healing. the anterior approach leaves the patient with less pain and stiffness (10), and provides acceptable clinical outcomes (11). also, the patient does not correspondence: professor claes olerud, md phd, department of orthopaedics, uppsala university hospital, se751 85 uppsala, sweden. fax: +46 18 50 94 27. e-mail: claes.olerud@surgsci.uu.se (received 11 december 2014; accepted 10 february 2015) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1019684 http://informahealthcare.com/journal/ups mailto:claes.olerud@surgsci.uu.se have to be turned to the prone position with a highly unstable injury, when placed on the operation table. however, koller et al. (12) reported a 31% incidence of construct failure in patients treated with an anterior plate alone, and henriques et al. (13) also reported high failure rates in patients with df-3 injuries and concomitant severe neurological injury when managed with anterior reconstruction alone. from a biomechanical standpoint, it is hypothesized that anterior fixation alone is less than ideal for a df-3 stage injury—characterized by significant posterior column disruption. when the posterior tension band fails, the only remaining stabilizing structures are located anteriorly. a stand-alone anterior plate reconstruction technique will be positioned close to these structures—providing suboptimal stability for a posterior column injury. angle stable screws that lock to the plate will improve stabilization of the construct (14); however, the addition of posterior fixation will create a more ideal biomechanical situation (15-23). using an in vitro human cadaveric cervical spine model with distractive–flexion stage 3 injury at c5–c6, the present study aims to quantify the multidirectional stability provided by four different reconstruction techniques: anterior plate fixation alone (a), and combined with one of three posterior fixation techniques—triple wire technique (24) (aw), transarticular facet joint screws (25) (at), or a posterior pedicle screw–rod device (ap). materials and methods preparation and experimental groups eight human cadaveric spines from c1 to t2 were harvested from fresh cadavers (5 males and 3 females; age range 49–82 years, mean 67 years), frozen immediately in double-wrapped plastic bags and stored at �20�c until testing. pre-experimentation radiographs were obtained to identify and exclude any specimen that demonstrated spinal pathology. prior to biomechanical testing, the specimens were thawed to room temperature and the surrounding soft tissue and muscles were removed, with care being taken to preserve osseous and pertinent ligamentous structures. prior to biomechanical testing, all specimens were sectioned at c2–c3 proximally and t1– t2 interbody levels. the c3–c4 (proximal) and c7–t1 (distal) motion segments were rigidly fixed with bone screws, with care taken not to disrupt the operative c5–c6 site and adjacent proximal (c4–c5) and distal (c6–c7) intervertebral levels. the specimens were mounted to the six-degree-of-freedom spine simulator with transfixation pins and polyester adhesive resin (bondo�, 3m corporation, st. paul, mn, usa) at c3–4 and c7–t1, respectively, leaving the motion segments c4–c5, c5–c6, and c6– c7 unconstrained (figure 2). the specimens were kept moistened with saline during the mechanical testing, which never exceeded 8 hours (26). to enable each specimen to serve as its own control, the specimens were first tested intact and then figure 1. schematicdrawingofthedistractive–flexionstage3injury, df-3. the upper vertebra is dislocated in flexion in relation to the lower one. both facet joints are dislocated, but the overall anterior displacement is less the 50%. an observation from the creation of the injury in the specimen was that the posterior longitudinal ligament had to be torn in order to allow dislocation of the joints. figure 2. the test setup in the six-degree-of-freedom spinal simulator. evaluation of fixation techniques in cervical spine injury 199 tested after destabilization and reconstruction at the c5–c6 level. the destabilization consisted of simulating a distractive flexion injury stage 3 (df-3) by transecting the c5–c6 supraspinous ligament, interspinous ligament, facet joint capsules, ligamentum flavum, posterior longitudinal ligament, and the posterior half of annulus fibrosus. the facet joints were then manually dislocated and reduced. spinal constructs the spinal specimens were reconstructed and tested in the following order. anterior plate (a): anterior plate reconstruction with the cervical spine locking plate (cslp, depuysynthes, inc. raynham, ma, usa) device and tricortical interbody bone graft between c5 and c6. the cslp plate has monocortical angle stiff locking screws. angle stable screws improve stability compared to plates with non-locking screws (20). this necessitated transecting of the remaining annulus fibrosus and the anterior longitudinal ligament. the bone graft was harvested from the parietal bone of the cranium of the same cadaver that was tested. anterior plate–wire (aw): the anterior plate was left in place. a bohlman triple wire reconstruction using 1-mm stainless steel wire and bone graft from the parietal bone of the cranium of the same specimen was added (24). the wire was tightened manually with pliers until just before the wire started to cut into the bone substance. to standardize this procedure, the same surgeon performed the wire application in all specimens, trying to use the same force each time. anterior plate–transarticular facet screws (at): the posterior wire reconstruction was removed, and 3.5-mm standard bone screws were applied as transarticular facet joint screws bilaterally over the c5–c6 facet joints. the screw holes were drilled with a 2.5-mm drill bit. the screw hole was tapped with an appropriate tap both in the proximal and distal facets. anterior plate–pedicle screw instrumentation (ap): the oc fixation system was utilized (anatomica ab, göteborg, sweden). the transarticular screws were removed, and 4.0-mm pedicle screws were implanted into c5 and c6 bilaterally. the screw holes were prepared according to the recommendation by the manufacturer by first probing the pedicles with a blunt probe, then tapping the hole with the appropriate tap. longitudinal 3.5-mm rods were then applied to complete the fixation device (figure 3). multidirectional flexibility analysis multidirectional flexibility testing was performed utilizing a custom-designed six-degree-of-freedom spine simulator interfaced with an optotrak 3020 motion analysis system (optotrak 3020, northern digital inc., waterloo, ontario, canada) and labview software (national instruments corporation, austin, tx, usa). the six-degree-of-freedom gimbal apparatus contains three independent stepper motors, harmonic drives, and electromagnetic clutches, which apply pure, unconstrained rotational moments (±) about three axes—x, y, and z. unconstrained translations (±) are permitted using linear bearing guide rails (x and z) and 858 bionix materials testing system (mts) servo-controlled linear actuator (y axis) (mts systems corporation, eden prairie, mn, usa) (figure 2). the intact and reconstructed cervical motion segments were evaluated under axial rotation (y axis, ±1.5 nm), flexion/extension (x axis, ±1.5 nm), and lateral bending (z axis, ±1.5 nm) testing modes using a pure moment loading condition. intersegmental motion was quantified using specialized rig markers containing three non-co-linear infrared light-emitting diodes (leds) rigidly attached figure 3. various fixation methods as they were applied on the specimen mounted on plastic models. a = anterior plate alone; aw = anterior plate combined with posterior wire; at = anterior plate combined with transarticular screws; ap = anterior plate combined with a pedicle screw–rod construct. 200 t. henriques et al. to the vertebral elements at c5 and c6 and oriented to permit detection by an optoelectronic motion analysis system. each test was repeated for three loading and unloading cycles at a rate of three degrees/second, with data from the third cycle used for computational analysis. statistical analysis for non-destructive multidirectional flexibility analysis, the peak intervertebral range of motion (euler angles, degrees) for each loading mode was calculated as the sum of motions [maximum ± rotation for torsion, flexion–extension, and left + right bending (degrees)] observed at the third loading cycle rangeof-motion (rom). the raw data set consisted of angle measurements around three axes (fe: flexion–extension, lb: lateral bending, and ar: axial rotation) for the intact condition and following reconstruction using four different preparations: a, aw, at, and ap. the analysis data set was created from the raw data set by normalizing the angles dividing them by the corresponding value for the intact specimen, e.g. (fe, angular plate angle)/(fe, intact angle). the resulting value was expressed as a percentage. thus, the value 100 indicates that the angle was identical to the corresponding angle for the intact condition. the three rotational axes (fe, lb, and ar) were analyzed separately. for each axis, the four stabilization methods were compared pairwise. as the data failed to show normality when examined with the shapiro–wilk w test, the wilcoxon signed ranks test for paired data was utilized. the target parameter is the pseudomedian for the difference in percentage discussed above. the pseudomedian of a variable x is the median of (x–y)/2, where y is an independent copy of x. for symmetric distributions, it coincides with the ordinary median. results are presented as 95% confidence intervals. no correction has been made for multiple testing. the missing value for specimen 1, fe, ap is disregarded in these analyses. thus, comparisons to this group have been made on seven pairs only, as opposed to the other ones, performed on eight pairs. one should note that the statistical power of these analyses is limited due to the small sample size. hence, moderately large p values should not be interpreted as strong evidence against any difference between the groups. the data analysis was performed using r version 3.0.2. results during the multidirectional testing of the reconstruction with anterior plate alone, the 95% ci of range-ofmotion (rom) was 19–116 percentage points (pp) of the intact spine in flexion–extension which was close to but not significant (p = 0.15) (table i; figure 4). a closer analysis of the data revealed that the anterior plate alone provided stability mainly in extension whereas the stability in flexion was poorer. the 95% ci for the rom for lateral bending was 67– table i. the data set. values are percentages (angle/ intact angle). flexion–extension lateral bending axial rotation a aw at ap a aw at ap a aw at ap spec 1 3.4 2.9 0.6 – 36.7 33.8 0.3 1.1 4.3 41.4 5.6 34.5 spec 2 113.3 1.0 97.6 5.5 194.4 237.4 8.3 14.7 275.9 253.9 42.9 17.4 spec 3 115.5 2.2 28.3 10.0 422.2 492.3 1.5 29.7 64.1 108.9 14.6 9.5 spec 4 78.5 13.2 53.1 4.2 555.9 515.7 21.1 10.2 123.8 127.4 9.9 13.4 spec 5 131.9 6.7 38.8 7.1 101.7 104.6 11.7 3.0 132.6 85.0 15.9 15.5 spec 6 37.9 3.7 2.9 10.7 97.0 78.7 100.8 15.5 55.2 78.1 11.5 4.5 spec 7 7.1 9.3 0.5 10.6 36.7 206.0 68.5 26.1 43.0 142.8 21.8 67.6 spec 8 35.6 7.2 10.8 11.8 103.2 107.8 28.8 18.2 84.9 98.7 11.0 20.2 median 58 5 20 10 102 157 16 15 75 104 13 16 range 3–132 1–13 0–98 4–12 37–556 34–516 0–101 1–30 4–276 41–254 6–43 4–68 95% ci 19, 116 2, 10 2, 63 6, 11 67, 375 71, 377 4, 65 6, 24 34, 180 70, 181 9, 29 9, 43 p 0.15 0.008 0.008 0.016 0.38 0.15 0.016 0.008 0.55 0.74 0.008 0.008 for the test sequence flexion–extension—ap in specimen 1, a mechanical failure of the construct occurred, thus no data could be retrieved. a = anterior plate alone; ap = anterior plate combined with pedicle-screw construct; at = anterior plate combined with posterior transarticular facet screws; aw = anterior plate combined with posterior wire. evaluation of fixation techniques in cervical spine injury 201 375 pp of intact (p = 0.38), and for axial rotation it was 34–180 pp of intact (p = 0.55). thus, in none of the tested modes did the anterior plate alone provide improved stability compared to the normal mobility of the intact spine. flexion–extension when posterior instrumentation was added to the anterior plate alone construct (a), the stability improved for all the applied techniques: with bohlman wire construct (aw) the segmental range of motion decreased by 59 pp compared to a (p = 0.023), with the transarticular screws (at) it decreased by 28 pp compared to a (p = 0.008), and with the pedicle screw construct (ap) it decreased by 66 pp compared to a (p = 0.031). there were no significant differences between the different posterior techniques in flexion–extension (p = 0.22) (table ii). lateral bending when the anterior plate stabilization was supplemented with the bohlman wire construct (aw) there was no improvement in stability in lateral bending compared to the stability provided by the anterior plate alone (p = 0.38). when the anterior plate was combined with transarticular facet screws (at) or a pedicle screw construct (ap), the segmental range of motion decreased by 121 pp compared to a (p = 0.039) and 131 pp compared to a (p = 0.008), respectively. the at (–158 pp; p = 0.016) and ap constructs (–171 pp; p = 0.008) were significantly more stable when compared to the the data set r a n g e o f m o ti o n ( % o f in ta c t) 600 500 400 300 200 intact 0 a aw at ap flexion-extension lateral bending axial rotaton median 25%–75% non-outlier range outliers a aw at ap a aw at ap figure 4. boxplots of the data set for the different testing moments. data have been normalized and expressed as the percentage of range-ofmotion for the intact specimen for each test sequence. table ii. analysis of flexion–extension. reconstruction methods in the left column have been compared to methods in the top row. thus, values above zero mean that the method to the left gives larger values than the method above. entries are: estimate (ci) p. a aw at aw –59 (–113, –13) 0.023 – – at –28 (–64, –9) 0.008 17 (–3, 61) 0.15 – ap –66 (–115, –12) 0.031 3 (–4, 7) 0.30 –20 (–62, 8) 0.22 a = anterior plate alone; ap = anterior plate combined with pedicle-screw construct; at = anterior plate combined with posterior transarticular facet screws; aw = anterior plate combined with posterior wire. 202 t. henriques et al. aw construct. there was no difference between the at and ap constructs in lateral bending (p = 0.25) (table iii). axial rotation when the anterior plate stabilization was supplemented with the bohlman wire construct (aw) there was no improvement in stability in axial rotation compared to the stability provided by the anterior plate alone (p = 0.31). when the anterior plate was combined with transarticular facet screws (at) or a pedicle screw construct (ap) the segmental range of motion decreased by 71 pp compared to a (p = 0.016) and 62 pp compared to a (p = 0.039), respectively. the at (–94 pp; p = 0.008) and ap constructs (–86 pp; p = 0.008) were significantly more stable when compared to the aw construct. there was no difference between the at and ap constructs in axial rotation (p = 0.64) (table iv). discussion methodology—df-3 injury procedure in patients traumatized with a df-3 injury, the extent of insufficiency of the posterior tension band mechanism probably varies, resulting in a pronounced instability. in order to dislocate both facets on the specimen, all posterior ligaments including the pll and the posterior part of the intervertebral disc were transected. in further preparation, the anterior longitudinal ligament was sectioned and anterior disc removed in order to place the bone graft, leaving only the anterolateral remnants of annulus fibrosus to stabilize the operative motion segment. hence, the tested specimens were more unstable than most clinical cases where the muscles and fasciae may contribute to stability (27). also, the tested specimens were most likely older and with a decreased bone quality compared to the average patient, which correlates with inferior purchase of screws (28). due to the scarcity of human cadaver specimens, we chose to perform non-destructive testing and omitted cyclic loading in spite of the known importance of this as shown by weis et al. in a bovine df-3 model. they described fatigue failure in extension–flexion in two out of six specimens with posterior wire fixation, and in one specimen with anterior plate alone (19). thus, our study probably overestimates the stabilization provided by anterior plate alone and anterior plate combined with posterior wiring. the purpose of this study was to evaluate how the stability was affected by adding different posterior fixations to an anterior fixation. in the clinical situation anterior exploration has certain advantages and may therefore be some surgeons’ first choice, although many would consider a ‘posterior alone’ fixation for these injuries, especially when a previous mri has shown that no disk fragment has been dislodged to the spinal canal. we are also fully aware that posterior wire techniques probably have very little table iv. analysis of axial rotation. reconstruction methods in the left column have been compared to methods in the top row. thus, values above zero mean that the method to the left gives larger values than the method above. entries are: estimate (ci) p. a aw at aw 19 (–22, 61) 0.31 – – at –71 (–153, –21) 0.016 –94 (–153, –62) 0.008 – ap –62 (–162, –10) 0.039 –86 (–157, –41) 0.008 3 (–13, 27) 0.64 a = anterior plate alone; ap = anterior plate combined with pedicle-screw construct; at = anterior plate combined with posterior transarticular facet screws; aw = anterior plate combined with posterior wire. table iii. analysis of lateral bending. reconstruction methods in the left column have been compared to methods in the top row. thus, values above zero mean that the method to the left gives larger values than the method above. entries are: estimate (ci) p. a aw at aw 17 (–19, 87) 0.38 – – at –121 (–360, –2) 0.039 –158 (–362, –34) 0.016 – ap –131 (–363, –46) 0.008 –171 (–364, –63) 0.008 –10 (–48, 10) 0.25 a = anterior plate alone; ap = anterior plate combined with pedicle-screw construct; at = anterior plate combined with posterior transarticular facet screws; aw = anterior plate combined with posterior wire. evaluation of fixation techniques in cervical spine injury 203 clinical use nowadays, but we wanted to see the effect of this technique mainly as a historical reflection. stability of the cervical reconstructions anterior plate alone. when reconstructed with a, the range of motion equaled the intact spine in axial rotation and lateral bending, whereas the stability in flexion–extension was only marginally improved. a detailed analysis of the flexion–extension data revealed that the anterior plate stabilized foremost in extension, and was poorer in flexion. in a df-3 injury, the posterior tension band mechanism is absent. therefore, a flexural moment will rotate the motion segment anteriorly, separating its posterior structures. ananteriorplate will resistthemoment closetotheaxis of segmental rotation. as a result, the fixation device by itself has to withstand the flexion moment. in extension, on the other hand, the posterior structures are compressing the bone graft, and the anterior plate will serve to improve stability of the anterior tension band mechanism. the fact that an anterior plate only stabilizes themotionsegmentatonepoint probablyexplains why the device performs comparably poorly in axial rotation and lateral bending. in agreement with the present results and clinical experience (12,13), several studies have indicated poor properties of an anterior plate alone for reconstruction of a three-column ligamentous injury, in contrast to some reports on good clinical results (11,29-32). one explanation may be that in a series of patients the severity of the injuries varies also within the same fracture class. incomplete disruption of the pll may provide enough posterior tension band to allow a successful result with an anterior plate alone. another explanation may be that the stability provided by an anterior plate, although weaker than the combined reconstructions, may be sufficient for patients where the bone stock is of good quality. some support for this latter hypothesis is provided by a biomechanical report in a df-3 injury model (33). however, their specimens were all male and considerably younger (mean age 44 years, range 21–65) compared to those utilized in the current study 67 years (range 49–82), which could indicate better bone quality. anterior plate and bohlman triple wire technique. this two-point fixation (one anteriorly, one posteriorly) is effective in restricting motions in extension–flexion compared to when the spine is fixed with an anterior plate alone, as the posterior tension band mechanism is reconstructed. the wires connect to the spinous processes providing a longer lever arm for the posterior component compared to the other posterior fixation techniques in the study, which may explain the slightly higher stiffness in extension–flexion compared to these techniques. however, the reduced stabilization demonstrated in axial rotation loading may be secondary to the inability to tighten a stainless steel wire, creating enough compression, and thus friction, between the bone surfaces. the wire resists tension loads, whereas axial and lateral bending moments are poorly controlled. in axial rotation, the geometry of the triple wire construct is almost 90 degrees with respect to therotationalmoment,thusexplainingthepoorresults. anterior plate and transarticular facet screws or pedicle screw–rod fixation. the combination of an anterior plate with transarticular facet screws or pedicle screw–rod device rigidly stabilizes at three circumferential points around the operative motion segment. this three-point fixation effectively controls the applied rotational moments as no significant difference could be detected between these two fixation techniques. hence, from a biomechanical point of view they are similar, but they differ significantly from a clinical point of view. the transarticular facet screw technique is difficult to implant superior to c4 due to the prominence of the occiput. it is often difficult to locate the correct screw trajectory, without coming in conflict with the cranium. also, the facets have to be perfectly reduced in order for this screw technique to be applicable. if the facets are subluxed, too little bone may be available for the screw canal, posing a risk for fracture or nerve root compromise when the screw is inserted. the cervical pedicle screw technique described by abumi and kaneda (34), on the other hand, can be applied superior to c4. it permits for segmental compression over the rod, thus aiding in the facet reduction. this technique has been demonstrated to improve segmental fixation and stability (35) and probably offers the best purchase in the cervical spine (36). there are obvious neurovascular risks involved in placing cervical pedicle screws, and from a clinical point of view safer techniques such as lateral mass screws have been shown to be sufficient (37). however, for the present study, pedicle screws were included as they probably offer the best stabilization that can be achieved in the cervical spine. it is possible that other anterior constructs, e.g. utilizing plates with bicortical screws and/or other designs of the bone graft may have yielded better results for the ‘anterior fixation alone’, but as we do not have any data on such constructs we cannot comment on this any further. other fixation devices primarily designed for anterior cervical fusion in degenerative conditions have also been tested biomechanically in fracture models. two low-profile devices—intracorporal cages with locked and variable 204 t. henriques et al. angle screw anchorage, respectively—were compared in a recent study (38). both devices had very similar features. the authors concluded that the stability provided by the locked screw device probably was sufficient for distractive-flexion stage 3 injury if combined with an external immobilization, while the device with variable angle screws was not suited for patients with distractive-flexion injuries. conclusions in this in vitro study using human spine specimens we evaluated four fixation methods in a df-3 injury of the cervical spine. we found that anterior plate fixation alone is not sufficient to stabilize a df-3, but by combining it with a competent posterior tension band reconstruction, i.e. transarticular facet screws or pedicle screw–rod instrumentation, segmental stability is improved. acknowledgements funding was provided by orthopaedic associates research foundation, inc., towson, maryland, usa. cervical spinal implants were provided by anatomica ab, göteborg, sweden. the statistical analysis was performed by lars lindhagen, phd, biostatistician at uppsala clinical research center, uppsala, sweden. declaration of interest: none of the authors has any conflict of interest with regard to the content of this article. references 1. allen bl, ferguson rl, lehmann tr, o’brien rp. a mechanistic classification of closed indirect fractures and dislocations of the lower cervical spine. spine. 1982;7:1–27. 2. vaccaro ar, madigan l, schweitzer me, flanders ae, hilibrand as, albert tj. magnetic resonance imaging analysis of soft tissue disruption after flexion-distraction injuries of the 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http://www.ncbi.nlm.nih.gov/pubmed/2797387?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/2028339?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/2028339?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/2028339?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/2028341?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/2028341?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/2028341?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/8171985?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/8171985?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/8171985?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/19127153?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/19127153?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/19127153?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/19127153?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/9280021?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/9280021?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7855677?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7855677?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7855677?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7855677?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/9152447?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/9152447?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/9152447?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/12859066?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/12859066?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22850940?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22850940?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22850940?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22850940?dopt=abstract abstract introduction materials and methods preparation and experimental groups spinal constructs multidirectional flexibility analysis statistical analysis results flexion&ndash;extension lateral bending axial rotation discussion methodology&mdash;df-3 injury procedure stability of the cervical reconstructions anterior plate alone anterior plate and bohlman triple wire technique anterior plate and transarticular facet screws or pedicle screw&ndash;rod fixation conclusions acknowledgements declaration of interest references comment on: ‘mean platelet volume could be a promising biomarker to monitor dietary compliance in ce full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 comment on: ‘mean platelet volume could be a promising biomarker to monitor dietary compliance in celiac disease’ ercan varol to cite this article: ercan varol (2013) comment on: ‘mean platelet volume could be a promising biomarker to monitor dietary compliance in celiac disease’, upsala journal of medical sciences, 118:3, 206-207, doi: 10.3109/03009734.2013.803503 to link to this article: https://doi.org/10.3109/03009734.2013.803503 © informa healthcare published online: 19 jun 2013. submit your article to this journal article views: 358 view related articles citing articles: 1 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.803503 https://doi.org/10.3109/03009734.2013.803503 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.803503 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.803503 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.803503#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.803503#tabmodule upsala journal of medical sciences. 2013; 118: 206–207 letter comment on: ‘mean platelet volume could be a promising biomarker to monitor dietary compliance in celiac disease’ ercan varol department of cardiology, faculty of medicine, suleyman demirel university, isparta, turkey i have read the article published by purnak et al. with great interest (1). they examined the mean platelet volume (mpv), an indicator of platelet reactivity, in patients with celiac disease. they showed that mpv values in patients with celiac disease were significantly higher than in controls. they concluded that mpv could be a useful clinical marker for monitoring of dietary compliance in celiac disease patients. this is a very interesting study. however, i would like to put forward some minor criticism with regard to some methodological and pathophysiological aspects. basically, the method used for mpv assessments is correct. blood samples were studied within 2 hours to prevent edta-induced swelling. it has to be kept in mind, however, that there are significant associations between mpv and type 2 diabetes mellitus, prediabetes, acute coronary syndromes, smoking, hypertension, hypercholesterolemia, obesity, and the metabolic syndrome (2,3). although co-morbidities like heart failure, peripheral vascular disease, acute or chronic infection, cancer, and hematologic and hepatic disorders were excluded, the authors do not bring up body mass index, blood pressure values, and serum glucose and cholesterol concentrations in their patients with celiac disease and the control subjects. these factors can greatly influence the mpv values. platelet size is regulated at the level of the megakaryocyte. it has been reported that cytokines such as interleukin-3 and interleukin-6 (il-6) influence megakaryocyte ploidy and can lead to the production of more reactive and larger platelets (4). on the other hand, serum il-6 concentrations have been shown to be elevated in patients with celiac disease (5). so, il-6, a major inflammatory cytokine, which is increased in patients with celiac disease, may cause an increase in mpv values by stimulating the megakaryocyte ploidy. platelet activation plays a major role in the pathophysiology of diseases prone to thrombosis and inflammation, and in line with this it has been argued that mpv might be a link between thrombosis and inflammation (6). it might be speculated that low-grade chronic inflammation exists in patients with celiac disease, and this in turn causes increased platelet reactivity as measured by mpv in these patients. this is an important issue because this can increase the cardiovascular long-term risks in patients with celiac disease. declaration of interest: the author reports no conflicts of interest. the author alone is responsible for the content and writing of the paper. references 1. purnak t, efe c, yuksel o, beyazit y, ozaslan e, altiparmak e. mean platelet volume could be a promising biomarker to monitor dietary compliance in celiac disease. ups j med sci. 2011;116:208–11. 2. vizioli l, muscari s, muscari a. the relationship of mean platelet volume with the risk and prognosis of cardiovascular diseases. int j clin pract. 2009;63:1509–15. 3. varol e, akcay s, icli a, yucel h, ozkan e, erdogan d, et al. mean platelet volume in patients with prehypertension and hypertension. clin hemorheol microcirc. 2010;45: 67–72. 4. debili n, masse jm, katz a, guichard j, breton-gorius j, vainchenker w. effects of the recombinant hematopoietic correspondence: ercan varol, md, suleyman demirel univesitesi tip fakultesi, isparta, turkey. fax: +90 2462324510. e-mail: drercanvarol@yahoo.com (received 18 april 2013; accepted 5 may 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.803503 http://informahealthcare.com/journal/ups www.ncbi.nlm.nih.gov/pubmed/21679011?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21679011?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19769707?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19769707?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19769707?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20571231?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20571231?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/7686791?dopt=abstract mailto:drercanvarol@yahoo.com growth factors interleukin-3, interleukin-6, stem cell factor, and leukemia inhibitory factor on the megakaryocytic differentiation of cd34s cells. blood. 1993;82:84–95. 5. kapoor a, patwari ak, kumar p, jain a, narayan s. serum soluble interleukin-2 receptor, interleukin-6 and tumor necrosis factor alpha as markers of celiac disease activity. indian j pediatr. 2013;80:108–13. 6. gasparyan ay, ayvazyan l, mikhailidis dp, kitas gd. mean platelet volume: a link between thrombosis and inflammation? curr pharm des. 2011;17:47–58. comment 207 www.ncbi.nlm.nih.gov/pubmed/7686791?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/7686791?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/7686791?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22766904?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22766904?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22766904?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21247392?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21247392?dopt=abstract doi4 references psychosocial aspects of identity-release gamete donation – perspectives of donors, recipients, and offspring review article psychosocial aspects of identity-release gamete donation – perspectives of donors, recipients, and offspring agneta skoog svanberga, gunilla sydsj€ob and claudia lampicc adepartment of women’s and children’s health, uppsala university, uppsala, sweden; bdepartment of obstetrics and gynaecology and department of clinical and experimental medicine, link€oping university, link€oping, sweden; cdepartment of women’s and children’s health, karolinska institutet, stockholm, sweden abstract donor conception creates families with varying genetic linkage between family members. this may have short-term as well as lifelong psychosocial consequences for all involved. gamete donors have traditionally been anonymous to recipients and offspring, but there is a growing trend towards identity-release donor programmes that give offspring the right to obtain the donor’s identity. this review aims to provide an overview of the perspectives of donors and recipients and offspring involved in identity-release donation. the results show that both oocyte and sperm donors have primarily altruistic motives, and recipients, in particular lesbian and single women, are generally open about the donation to their offspring. the few existing studies on offspring perspectives indicate that those who are aware of their donor conception appear to be interested in contact with the donor, and most donors are open to such contact. investigations of donors and recipients indicate a need for more counselling and support to manage family life with varying genetic linkage within and outside the family unit. this includes preparing for and managing future contact between the donor and his/her family and donor offspring and their family, as well as between donor siblings and their respective families. article history received 7 october 2019 revised 19 november 2019 accepted 19 november 2019 keywords disclosure; donor offspring; gamete donation; openidentity donors introduction the desire to have a child is based both on psychological and social reasons. assisted reproduction treatments have made it possible for many individuals/couples to have a family even when sperm and oocytes are reduced or lacking. large numbers of children are born following donor conception, and there is an increasing demand for donor conception worldwide. depending on legislation and regulations, treatment with donor sperm, oocytes, and embryos is available to various groups of recipients, including heterosexual couples, lesbian couples, and single women. donor conception has traditionally been performed with anonymous donors, while it has been less common to use a donor who is ‘known’ to the recipient(s), most often a female relative who donated oocytes. there is a global trend towards programmes using donors that are identifiable to the resulting offspring at maturity, commonly labelled ‘identity-release’ or ‘open-identity’ donors. the use of identity-release donors implies that the donor is anonymous to the recipients, although they may receive some non-identifying information about the donor. upon request from a donor-conceived child that has reached mature age, the donor’s identity is released to the child. legislation on identity-release gamete donation was first introduced in 1985 in sweden (1) and later in other jurisdictions (e.g. austria, switzerland, new zealand, and the uk). donor conception creates families where the genetic linkage between family members is varying and where there are genetic links to individuals outside the family unit. the presence and/or absence of genetic linkage may have psychosocial consequences for all involved parties, i.e. for the donor and his/her family, as well as for the recipient(s), the donorconceived child, and their larger family. there is a relatively large body of research on the psychosocial aspects of anonymous donor conception, and to a lesser extent on ‘known’ donation. systematic reviews in the field show that both oocyte and sperm donors’ psychosocial wellbeing was good throughout all donor groups (anonymous, known, eggsharers, and open-identity) (2,3), and families created through the use of donor gametes appeared to be welladjusted (4). during recent years, the attitude towards disclosure in gamete donation has shifted from secrecy to openness, and disclosure is now strongly encouraged by the ethics committee of the american society of reproductive medicine (5). a recent systematic review focussing on donorconceived offspring showed that genetic ties are perceived as important, especially during adolescence and adulthood, and that many were interested in receiving more information about the donor and for potential contact (6). in contrast to contact agneta skoog svanberg agneta.skoog_svanberg@kbh.uu.se department of women’s and children’s health, uppsala university, uppsala, 751 05, sweden � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 2, 175–182 https://doi.org/10.1080/03009734.2019.1696431 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1696431&domain=pdf&date_stamp=2020-05-21 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2019.1696431 http://www.tandfonline.com the relatively large body of research on anonymous and known gamete donation, research on the psychosocial aspects of conception with gametes from identity-release donors is more limited, mostly due to the fact that this type of donor conception has been less common. the purpose of the present review is to summarize the available research on psychosocial aspects of identity-release gamete donation. furthermore, we want to present an overview of the specific perspectives of donors, recipients, and offspring, including motivations for participating in this type of donor conception, as well as perceptions of disclosure issues and potential contact between donor and offspring/ family. pubmed and psycinfo databases were used in order to search for relevant empirical studies. due to the diverse use of terminology for this type of donation (e.g. openidentity, identity-release, open donation) it is possible that some studies including relevant groups were missed. this overview covers only studies that specifically noted the inclusion of donors, recipients, or offspring involved in identityrelease donation, but does not include studies on surrogacy, although such arrangements frequently include donor gametes. in several studies, a small subset of participants concerned identity-release donation. such studies were included provided that they presented results from these groups separately, and were otherwise excluded [e.g. (7,8)]. not surprisingly the largest number of identified studies was performed in sweden, where identity-release gamete donation has been mandatory since 1985, and the remaining studies were conducted in other european countries and in the usa. perspectives of identity-release donors motives and characteristics by definition, the term donation implies altruism, and both male and female donors who agree that their identity may be released to offspring at maturity have been found to donate primarily or solely based on altruistic reasons, i.e. they want to help involuntarily childless people (9–13). however, also other motives such as receiving confirmation of one’s own fertility potential, spreading one’s good genes, and donating as a way to have a child/children in the future have been reported (11,14,15), with sperm donors more often reporting spreading their genes as a salient motive compared to oocyte donors (11). although the dominant motive for donating was altruistic, two small survey studies found that a subgroup of identity-release sperm donors also reported financial motives (9,10). a danish interview study with sperm donors, five of whom had opted for identityrelease status, found that financial compensation was a factor for the decision to allow more information about them to be made available to recipients, but the child’s wellbeing was also considered when providing extended information about oneself (15). also, in a large finnish study of oocyte donors, one in four reported that the financial compensation had at least some influence on their decision to donate (13). in contrast, in a recent interview study including 24 oocyte and sperm donors in the uk, all rejected the idea that they had been financially motivated, and it was particularly important for sperm donors to frame their donation as a purely altruistic ‘gift’, as a financial motive was perceived to be incompatible with a beneficial potential relationship with offspring from their donation (16). oocyte donors, on the other hand, were more comfortable to incorporate the fact that they received financial compensation in the narrative of their donation as a ‘gift’ to recipients longing for a child. the personalities and characters of gamete donors are of interest for the recipients but also for the resulting offspring. in a swedish national study, the temperament and character inventory (17) was used to assess both oocyte and sperm donors. the sperm donors were found to all be in the normal range of character, which means that the donors perceived themselves as autonomous individuals with capacity to take responsibility and with the ability to behave in a goal-oriented manner (18). the sperm donors described themselves as persons well integrated in society and having a capacity for relatively high identification with and acceptance of other people. concerning the personality and character of the oocyte donors, it was evident that they described themselves as less worried, shy, and fatigued, and as more persistent compared to a comparison group of women in fertile age (19). these findings are reassuring for all involved, both in the donation and treatment process, and for the future families, and suggest well-functioning screening procedures in a non-commercial donor programme. openness about donating little is known about donors’ view of informing others, such as partners and biological children, about their donation. in two swedish survey studies, each including 30 sperm donors, almost all had shared information with their partner about their intention to donate (10) or of being a donor (20). the involvement and support from the partner seemed to be important factors for the decision to donate sperm, particularly among younger men (9). many donors in the ekerhovd et al. (10) study planned to inform their own children of the donation, particularly if the donation did result in a child. similarly, in a well-designed finnish study, almost all oocyte donors who were mothers either had or planned to share this information with their children (21). thoughts about offspring and potential contact for identity-release gamete donors, the number of children conceived with their gametes is of particular interest, since the offspring will be able to obtain the donor’s identity and may attempt to contact the donor. however, only one study was found investigating donors’ views regarding the number of children a donor may conceive (22). about half of 235 oocyte and sperm donors 5–8 years after their donation regarded 1–10 children to be an acceptable number of offspring from one donor, with oocyte donors more often supporting an upper limit than sperm donors. following identity-release donation, a majority of both oocyte and sperm donors would like to be informed if their donation results in pregnancy and birth (10,23). in a swedish 176 a. skoog svanberg et al. follow-up study of gamete donors, sperm donors reported a higher level of emotional involvement with offspring from their donation compared to oocyte donors (23). this included wanting to know how the child fares in life and feeling responsibility for the child if anything happened to his/her parents. in two swedish qualitative studies, each including 30 sperm donors, most of them were positive or neutral towards contact with adult offspring from their donation (10,20). this finding was confirmed in a large follow-up study of sperm donors and oocyte donors 5–8 years post-donation (23,24). ten percent of donors were negative towards being contacted by an offspring, and some comments indicate that this was based on a desire that the child would feel no need for contact and be ‘happy in their real family’ (24). among oocyte donors in finland, a majority stated that they were positive or neutral towards future contact with an offspring, but they were more uncertain regarding potential contact between their own children and a donor offspring (21). furthermore, in a qualitative study of 11 oocyte donors from the uk, women were happy to be contacted by offspring, but some expressed concerns regarding potential negative impact of such contact on the offspring’s parents and on the donor’s own family (25). so far, only one study has reported on identity-release donors’ position when adult offspring from their donation seek information about them (26). in that study, from one sperm bank in the usa, clinic staff contacted sperm donors when information about them was being requested, and 39 out of 43 men responded that they were open for contact with their offspring. satisfaction with the donation and need for counselling the experiences, satisfaction, and consequences of being an identity-release gamete donor, i.e. with respect to the medical care and treatment, have been investigated in a few structured follow-up studies. in a finnish follow-up study of 428 former oocyte donors, 67 of which were identity-release, almost all were satisfied with their donation (13). similarly, in a swedish study of 300 oocyte and sperm donors, most of them were satisfied with the donation (11). those who expressed ambivalence before the donation (but after being accepted in the programme) reported lower satisfaction 2 months after their donation (27). in a qualitative study from the netherlands, male donors expressed a need for counselling in order to discuss the emotional consequences of their donation, disclosure to their own children, family, and friends, and potential future contact with an offspring (28). in a swedish follow-up study of 210 oocyte and sperm donors several years following donating, one in four donors reported a need for counselling about how to manage potential future contact with offspring from their donation (24). more than half of these donors wanted to be notified when an offspring requested information about them in order to prepare for potential contact, while one-third were negative to receiving such information, partly to avoid potential disappointment if no contact attempts would follow. in the above-mentioned finnish study, the oocyte donors indicated a high level of satisfaction with the support offered during the process and a relatively low need for additional support (13). perspectives of recipients motivation for choosing identity-release donation in general, reasons for choosing treatment with donor gametes include having a biological tie to the child, desiring to experience a pregnancy and to have a child who has a genetic link to at least one parent. the most common reasons for choosing identity-release sperm donation stated by heterosexual-couple, lesbian-couple, and single-woman parents in a us study were that this gave the child the option of getting more information about the donor, including his identity, and the option to be able to meet him (29). in a us study of 129 lesbian mothers, most of them were satisfied with their choice of an anonymous, known, or identifiable donor (30). those who had selected an open-identity donor were most satisfied with their choice, both because they avoided potential custody conflicts and/or involvement from a third person, and because offspring would have access to information about the donor. disclosure identity-release donation gives the offspring an option to obtain identifying information about the donor. however, offspring can only make use of this option if he/she has been made aware of the donor conception, most often by his/her parents. in a swedish study of 148 heterosexual couples with children conceived through donor insemination in the years directly after the law was introduced, only 11% had informed the child about the donor conception, 46% planned to disclose later, and one-third were unsure or planned not to disclose (31). those who had disclosed did not regret their decision to disclose and thought that being open about the donor conception had been beneficial to the child. in a follow-up interview study including 19 couples from the above study, participants said that healthcare staff had influenced their thinking, and a majority of those who had been encouraged to tell their children about the donation had done so (32). another interview study of 31 heterosexual-couple parents with 1to 7-year-old children conceived with donor sperm during 1997–2003 found that 75% already had or planned to talk with the child about the donation (33). in a later swedish study of 111 heterosexual couples with 1to 4-year-old children following oocyte or sperm donation (34), 78% planned to disclose to the child about the donation, and 16% had already started the disclosure process. a subset of 30 heterosexual sperm recipients from that study also participated in an interview study when the child had reached 7 years of age (35). the authors concluded that sharing information about donor conception with offspring was a complex process that involves different levels, and in which parents’ beliefs and the child’s responses serve as driving or impeding forces. upsala journal of medical sciences 177 a uk study investigated disclosure to offspring among 31 heterosexual solo mothers and 47 heterosexual-partnered mothers with donor-conceived children aged 4–8 (36). about half of the solo mothers and one-third of the partnered mothers had already told the child about their conception with donor sperm. among those who had not yet disclosed, partnered mothers were significantly more reluctant or negative to disclose than solo mothers. in line with this finding, a us study showed that all single women and lesbian couples with adolescent offspring had disclosed their use of donor insemination to their children, while this was the case for 70% of heterosexual couples (29). disclosure did not seem to have any negative impact on the families, regardless of the parents’ sexual orientation or relationship status. several studies have found that large groups of recipients had told other persons about their use of gamete conception (32–34,37–39), with no differences in disclosure behaviour with regard to sex or type of donation (oocyte/sperm) (34). one stated reason to refrain from sharing this information with people outside a close circle of friends and family was that the child should learn about the donation before other people did (34). among parents who plan not to share information about the donor conception with their child, disclosure to others increases the risk of accidental disclosure. managing family life when a couple conceives with donor oocytes or sperm, this creates a family where the child has a genetic link to only one of the parents. the presence and/or absence of genetic linkage, as well as the existence of an identifiable donor, may have psychosocial consequences for the couple and the family. two swedish interview studies of heterosexual couples following sperm donation concerned family life. leeblundberg et al. (33) reported that some parents had worried that the lack of a genetic link to the father would create an unequal relationship with the child, but these concerns had disappeared over time. isaksson et al. (40) found that resemblance between child and parent was an important theme, and parents were reported to navigate between the importance of genetic connectedness and of ‘doing parenthood’ through social interactions. non-resemblance between parent and child was described to bring the donor to the front and the donor constituted an ‘absent presence’, as also described in a qualitative study of single mothers in the uk (41). in the longitudinal ‘swedish study of gamete donation’, recipient couples of donor oocytes and sperm were followed 2–5 years after treatment. heterosexual couples that had been treated with sperm donation expressed satisfaction with their relationship (42), and couples using oocyte donation treatment had a balanced and solid view of their relationship (43), where having children or not after treatment had no effect on the nature of the relationship. lesbian couples following sperm donation reported stable relationships and a high satisfaction with their relationship, also after unsuccessful treatment (44). they also reported less parenting stress compared to heterosexual-couple parents following ivf with their own gametes and parents following a spontaneous pregnancy (45). while a previous study in the usa indicates that, following donor insemination, heterosexual couples, lesbian couples, and single women were positive about their decision and were quite open about the donor conception (29), lesbian-couple families may face specific challenges related to their non-traditional family formation. results from interviews with 20 female-partnered mothers of young children indicate that participants had lacked psychological support in the process of planning and becoming a parent (46). they expressed a desire to be treated as equally valid mothers and as a proper family by professionals at child healthcare services (47). also, the results from a web survey with 145 belgian and swedish participants, 36 of whom had used identifiable donors, showed that donor-conceived families were challenged by cultural norms and values and responses from friends, healthcare professionals, and teachers (39). when children conceived with gametes from identityreleased donors grow up, parents need to deal with the question of disclosure to the child. in a study of 111 heterosexual couples following oocyte and sperm donation, onethird were not in agreement about what to disclose to their child about his/her conception, and these couples reported a lower relationship quality than couples who agreed about disclosure (34). however, incomplete couple agreement about disclosure did not appear to have a negative impact on parental stress (48). contact with donor and donor-siblings in a swedish study including 279 heterosexual recipient couples of donor oocytes or donor sperm, about half believed that it was in the best interest of the child to be able to obtain identifying information about the donor, while the remaining were unsure, neutral, or negative (37). while few believed that contact between the child and the donor could be harmful for the offspring or family, about one-third could not form an opinion about this. in a subsequent qualitative study of 30 heterosexual parents with a 7-year-old child, some parents were curious about the sperm donor and hoped that the child would make contact in the future, while other parents expressed concerns about potential contact between the child and the donor (35). with openness about donor treatment, both parents and offspring may want to get information and contact others who share the same donor. the sperm bank of california has established a service that connects families who share the same donor, and it has been used predominantly by families headed by lesbian couples and single women, most of whom had used identityrelease donation (49). these groups’ motivations for contact with other families who share the same sperm donor included to create a family for the child, obtain support for their children and/or themselves, and to get information about shared traits and medical problems (49,50). femalepartnered women most often described their own and their children’s relationship with ‘linked’ families as a unique type 178 a. skoog svanberg et al. of relationship, a ‘special bond’, an ‘extended family’, or merely ‘acquaintances’ (51). perspectives of offspring there is a dearth of research including the perspectives of persons conceived with gametes from donors who originally chose to donate within an identity-release programme, and all concern persons conceived with donor sperm. scheib and co-workers have presented several studies based on donors, recipients, and their offspring from one sperm bank that has offered identity-release donors since 1983. one interview study included 29 adolescents conceived with sperm from open-identity donors, from households led by lesbian couples, single mothers, and heterosexual couples (52). a majority (76%) reported always knowing about their donor conception and were comfortable with their conception origins. most also planned to request the donor’s identity and pursue contact in order to learn more about themselves. these results are in line with two longitudinal studies of families headed by lesbian couples in the netherlands and the usa, where half of adolescents conceived with sperm from an open-identity donor reported a desire to meet their donor (53) and 67% of adolescents planned to contact the donor at the allowed age of 18 (8). only one study was found that reported on the final step of an identity-release donor programme, i.e. requests and provision of identifying donor information to adult offspring. in a follow-up study, scheib et al. (26) reported on the first 10 years of this practice at one sperm bank. during this period, adult offspring from 256 families were eligible to receive such information, and a total of 85 offspring (35%) contacted the clinic for this purpose. being a female offspring and belonging to a single-parent household increased the probability of requesting donor information, while having heterosexual-couple parents decreased the likelihood of a request. a large majority of offspring contacted the clinic for information within the first three years after turning 18. the most common motivations for requesting the donor’s identity were to gain information about who the donor is as a person, his motives for donating, and medical or health information. many believed that information about the donor would help them learn something about themselves and help to ‘fill in the missing links’. while a majority (75%) expressed an interest in contacting the donor, most had low or no specific expectations of a potential contact, and very few expressed a desire for a close relationship. four offspring were informed by the clinic that their donor was not open to contact and were reported to be very disappointed and upset. discussion the aim of this review was to provide an overview of the perspectives of donors, recipients, and offspring involved in identity-release donation. identifiable donors of oocytes and sperm predominantly reported altruistic motives, in line with motives reported for anonymous and known donors (2,3). still, the prospect of a potential future meeting with donor offspring may influence how donors reflect about and frame their motives for donating. identity-release oocyte and sperm donors were found to be mature and well-adjusted individuals (18,19), which is reassuring and indicates that the screening procedures are well-functioning. the studies that have investigated long-term consequences of donating in an identity-release donor programme indicate that most donors were satisfied with their decision (11,13) and had positive or neutral attitudes towards being contacted by offspring from their donation (21,23,24). however, subgroups of donors expressed a need for support and counselling, both to handle their own situation and to prepare for a potential situation when an offspring seeks contact (or not) (24,28). while a recent review concluded that families following gamete donation in general are well-functioning (6), conception with oocytes or sperm from a donor who will be identifiable to the child at maturity may have specific psychosocial consequences. the present results indicate that recipients of gametes from identity-release donors had stable relationships (42–45) and were increasingly open about having used donor conception (33,34), with female-partnered and single women being most positive towards disclosure to the offspring (29,36). parents were generally satisfied with their choice of an identity-release donor as this gives their child the option to obtain more information about their genetic origin (29,30). some parents had own interest in contact with the donor and/or with families who had used the same donor (49,50). concerning the perspectives of offspring conceived with gametes from identity-release donors, the present review highlighted the very limited knowledge base for this specific group. only four studies were found (8,26,52,53), all concerning adolescent and young adult offspring conceived with donor sperm. the results indicate that about half of the offspring who are aware of their donor conception, and have the possibility to obtain the identity of their donor, plan to do so, and many also intend to contact the donor. their motivations for learning the donor’s identity and meeting the donor are in line with those reported by offspring conceived with gametes from anonymous donors (6). the study by scheib et al. (26) is the first to report on the percentage of offspring eligible to receive identifying donor information who actually made such a request, which was about onethird of the total sample. also, it was recently reported that only 5% of eligible adult offspring from heterosexual-couple families in sweden had requested donor information (54). at the moment, there is no information available about the large groups that have not (yet) requested information about their donor. are they aware of their donor conception and the possibility to obtain the donor’s identity? what is their level of interest in this information? are there any practical issues, concerns, or considerations that impact their decision not to seek donor information? in view of the relatively low disclosure rates reported by heterosexual couples who conceived with donor gametes in the 1980s and 1990s (i.e. whose offspring have reached adulthood by now) (55), is it upsala journal of medical sciences 179 reasonable to assume that at least some of those offspring are unaware of their origin with donor gametes. the studies covered in the present overview were performed in europe and the usa and included quantitative and qualitative studies with cross-sectional, retrospective, and longitudinal designs. studies of individuals involved in gamete donation are frequently based on single clinics and suffer from relatively low response rates, which limits the generality of the findings. research on donor-conceived offspring is hampered by specific difficulties identifying this population, as it is ethically only possible to approach individuals who are aware of their donor conception. recruitment of participants through self-selection, e.g. membership in networks for donor conception, is feasible but introduces a selection bias. thus, there is a lack of knowledge about the perspectives of donors, recipients, and offspring who are not interested in seeking information about genetically related persons. also, the number of long-term follow-up studies in this field is limited, and attrition is a matter of concern, as in all longitudinal designs. changes in legislation, dna-based voluntary contact registers, and direct-to-consumer genetic tests constitute new challenges and may have great influence for the future for assisted reproduction with donor gametes. the possibility of identification of genetically related individuals may have an impact not only on donors, recipients, and offspring, but also on their extended families (56). the need to ensure that all this information is handled with the best safety and privacy rights has been stressed (57). however, reflections on the future of genetic testing and/or screening must be distinguished from the long-standing debate about disclosure of donor conception to children (58). in conclusion, donor conception creates families with varying genetic linkage between family members and where there are genetic links to individuals outside the family unit. in the case of identity-release donation, the offspring has the opportunity to obtain identifying information about his/her donor. existing research about the perspectives of donors and recipients involved in identity-release donation indicates that both oocyte and sperm donors primarily have altruistic motivations and that recipients are increasingly open about having used donor conception. furthermore, several studies indicate that the offspring are interested in contact with the donor, and most donors are open to such contacts. keeping in mind the lifelong consequences of identity-release donor conception, recipient families and donors could benefit from support and counselling to increase their confidence in managing family life. in view of the present findings, as well as the rapid development and increasing use of resources to identify genetic relatives, more high-quality research is warranted on the long-term psychosocial consequences of gamete donation. disclosure statement no potential conflict of interest was reported by the authors. notes on contributors agneta skoog svanberg, registered nurse/midwife. phd and professor in reproductive health, department of women’s and children’s health, uppsala university, uppsala, sweden. gunilla sydsj€o, certified psychotherapist/ma behavioural scientist. phd and professor in psychosocial obstetrics and gynaecology, department of obstetrics and gynaecology, department of clinical and experimental medicine, link€oping university, link€oping, sweden. claudia lampic, lic psychologist. phd and associate professor in caring sciences, department of women’s and children’s health, karolinska institutet, stockholm, sweden. references 1. stoll j. swedish donor offspring and their legal right to information. uppsala: uppsala university; 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gender differences and similarities among oocyte and sperm donors in a national sample. acta obstet gynecol scand. 2013;92: 1049–56. 28. visser m, mochtar mh, de melker aa, van der veen f, repping s, gerrits t. psychosocial counselling of identifiable sperm donors. hum reprod. 2016;31:1066–74. 29. scheib je, riordan m, rubin s. choosing identity-release sperm donors: the parents’ perspective 13-18 years later. hum reprod. 2003;18:1115–27. 30. gartrell nk, bos h, goldberg ng, deck a, van rijn-van gelderen l. satisfaction with known, open-identity, or unknown sperm donors: reports from lesbian mothers of 17-year-old adolescents. fertil steril. 2015;103:242–8. 31. lindblad f, gottlieb c, lalos o. to tell or not to tell–what parents think about telling their children that they were born following donor insemination. j psychosom obstet gynaecol. 2000;21: 193–203. 32. lalos a, gottlieb c, lalos o. legislated right for donorinsemination children to know their genetic origin: a study of parental thinking. hum reprod. 2007;22:1759–68. 33. leeb-lundberg s, kjellberg s, sydsj€o g. helping parents to tell their children about the use of donor insemination (di) and determining their opinions about open-identity sperm donors. acta obstet gynecol scand. 2006;85:78–81. 34. isaksson s, sydsjo g, skoog svanberg a, lampic c. disclosure behaviour and intentions among 111 couples following treatment with oocytes or sperm from identity-release donors: follow-up at offspring age 1-4 years. hum reprod. 2012;27:2998–3007. 35. isaksson s, skoog-svanberg a, sydsjo g, linell l, lampic c. it takes two to tango: information-sharing with offspring among heterosexual parents following identity-release sperm donation. hum reprod. 2016;31:125–32. 36. freeman t, zadeh s, smith v, golombok s. disclosure of sperm donation: a comparison between solo mother and two-parent families with identifiable donors. reprod biomed online. 2016;33: 592–600. 37. isaksson s, skoog svanberg a, sydsjo g, thurin-kjellberg a, karlstrom po, solensten ng, et al. two decades after legislation on identifiable donors in sweden: are recipient couples ready to be open about using gamete donation? hum reprod. 2011;26: 853–60. 38. gottlieb c, lalos o, lindblad f. disclosure of donor insemination to the child: the impact of swedish legislation on couples’ attitudes. hum reprod. 2000;15:2052–6. 39. indekeu a, lampic c. the interaction between donor-conceived families and their environment: parents’ perceptions of societal understanding and attitudes regarding their family-building. hum fertil (camb). forthcoming. [2018 nov 2]:[1–10]. 40. isaksson s, sydsj€o g, skoog svanberg a, lampic c. managing absence and presence of child-parent resemblance: a challenge for heterosexual couples following sperm donation. reprod biomed soc online. 2019;8:38–46. 41. zadeh s, freeman t, golombok s. absence or presence? complexities in the donor narratives of single mothers using sperm donation. hum reprod. 2016;31:117–24. 42. sydsjo g, svanberg as, bladh m, lampic c. relationships in couples treated with sperm donation a national prospective followup study. reprod health. 2014;11:62. 43. sydsj€o g, lampic c, bladh m, skoog svanberg a. relationships in oocyte recipient couples a swedish national prospective followup study. reprod health. 2014;11:38. 44. borneskog c, lampic c, sydsjo g, bladh m, svanberg as. relationship satisfaction in lesbian and heterosexual couples before and after assisted reproduction: a longitudinal follow-up study. bmc womens health. 2014;14:154. 45. borneskog c, lampic c, sydsj€o g, bladh m, skoog svanberg a. how do lesbian couples compare with heterosexual in vitro fertilization and spontaneously pregnant couples when it comes to parenting stress? acta paediatr. 2014;103:537–45. 46. appelgren engstrom h, haggstrom-nordin e, borneskog c, almqvist al. mothers in same-sex relationships describe the process of forming a family as a stressful journey in a heteronormative world: a swedish grounded theory study. matern child health j. 2018;22:1444–50. 47. appelgren engstr€om h, h€aggstr€om-nordin e, borneskog c, almqvist al. mothers in same-sex relationships-striving for equal parenthood: a grounded theory study. j clin nurs. 2019;28: 3700–9. 48. gebhardt aj, sydsj€o g, skoog svanberg a, indekeu a, lampic c. parenting stress and its association with perceived agreement about the disclosure decision in parents following donor conception. acta obstet gynecol scand. 2017;96:968–75. 49. scheib je, ruby a. contact among families who share the same sperm donor. fertil steril. 2008;90:33–43. 50. goldberg ae, scheib je. female-partnered and single women’s contact motivations and experiences with donor-linked families. hum reprod. 2015;30:1375–85. 51. goldberg ae, scheib je. female-partnered women conceiving kinship: does sharing a sperm donor mean we are family? j lesbian stud. 2016;20:427–41. 52. scheib je, riordan m, rubin s. adolescents with open-identity sperm donors: reports from 12-17 year olds. hum reprod. 2005; 20:239–52. upsala journal of medical sciences 181 53. bos h, van rijn-van gelderen l, gartrell n. self-esteem and problem behavior in dutch adolescents conceived through sperm donation in planned lesbian parent families. j lesbian stud. forthcoming. [2019 jun 20]:[1–15]. 54. lampic c, lessons from three decades of non-anonymity. proceedings of the 35th annual meeting of the european society of human reproduction and embryology; 2019 jun 23–26; vienna, austria. 2019. 55. van den akker o. a review of family donor constructs: current research and future directions. hum reprod update. 2006;12:91–101. 56. beeson d, jennings p, kramer w. a new path to grandparenthood: parents of sperm and egg donors. j fam stud. 2013;34:1295–316. 57. harper jc, kennett d, reisel d. the end of donor anonymity: how genetic testing is likely to drive anonymous gamete donation out of business. hum reprod. 2016;31:1135–40. 58. zadeh s. disclosure of donor conception in the era of nonanonymity: safeguarding and promoting the interests of donorconceived individuals? hum reprod. 2016;31:2416–20. 182 a. skoog svanberg et al. abstract introduction perspectives of identity-release donors motives and characteristics openness about donating thoughts about offspring and potential contact satisfaction with the donation and need for counselling perspectives of recipients motivation for choosing identity-release donation disclosure managing family life contact with donor and donor-siblings perspectives of offspring discussion disclosure statement references tf-iups200022 240..249 original article dietary advice and oral nutritional supplements do not increase survival in older malnourished adults: a multicentre randomised controlled trial lisa s€oderstr€oma,b , andreas rosenbladc,d , leif bergkvista, hanna fride and eva thors adolfssona acentre for clinical research, region v€astmanland, uppsala university, v€asterås, sweden; bdepartment of food studies, nutrition and dietetics, uppsala university, uppsala, sweden; cdepartment of statistics, stockholm university, stockholm, sweden; ddepartment of medical sciences, clinical diabetology and metabolism, uppsala university, uppsala, sweden; edepartment of child and adolescence psychiatry, v€astmanland hospital, v€asterås, sweden abstract objectives: the study aimed to investigate the effect on survival after 6 months of treatment involving individual dietary advice and oral nutritional supplements in older malnourished adults after discharge from hospital. methods: this multicentre randomised controlled trial included 671 patients aged 65 years who were malnourished or at risk of malnutrition when admitted to hospital between 2010 and 2014, and followed up after 8.2 years (median 4.1 years). patients were randomised to receive dietary advice or oral nutritional supplements, separate or in combination, or routine care. the intervention started at discharge from the hospital and continued for 6 months, with survival being the main outcome measure. results: during the follow-up period 398 (59.3%) participants died. at follow-up, the survival rates were 36.9% for dietary advice, 42.4% for oral nutritional supplements, 40.2% for dietary advice combined with oral nutritional supplements, and 43.3% for the control group (log-rank test p ¼ 0.762). after stratifying the participants according to nutritional status, survival still did not differ significantly between the treatment arms (log-rank test p ¼ 0.480 and p ¼ 0.298 for the 506 participants at risk of malnutrition and the 165 malnourished participants, respectively). conclusions: oral nutritional supplements with or without dietary advice, or dietary advice alone, do not improve the survival of malnourished older adults. these results do not support the routine use of supplements in older malnourished adults, provided that survival is the aim of the treatment. trial registration: clinicaltrials.gov with id: nct01057914 article history received 3 february 2020 revised 27 march 2020 accepted 29 march 2020 keywords dietary advice; malnutrition; older adults; oral nutritional supplementation; randomised controlled trial; survival analysis introduction malnutrition is still a common problem in older adults in any setting (1), and the condition is associated with many negative health outcomes (2–5), including mortality (6–8). nutritional treatment strategies aiming at increasing survival among older adults is highly relevant, since the life expectancy at 65 years is around 20 years in sweden and many other countries. however, the effectiveness of nutritional interventions is still uncertain (9). evidence suggests that dietary advice and oral nutritional supplements may help maintain body weight (10) or cause a modest weight gain (9,11,12), and improve body composition (11–13) and grip strength (11,12), but the effect on survival is unclear (9,11,12,14). mortality is considered a critical outcome to analyse when evaluating nutritional interventions aiming at preventing or treating malnutrition in older adults (15). all-cause mortality is preferred over cause-specific mortality as an outcome measure, since it avoids such problems as misclassification bias related to the true cause of death (16). moreover, it balances out the harmful and beneficial impacts an exposure may have on health, thus giving the net effect of an exposure on mortality. the results from a cochrane review (14) including data from 42 randomised controlled trials (rcts) in older adults (aged 65 years) with different nutritional statuses reported no reduced mortality in groups that received oral nutritional supplements compared with a control group. subgroup analyses indicated an effect in older adults who were already malnourished (21% reduction in mortality rate) and older adults who received oral nutritional supplements of 400 kcal a day (11% reduction in mortality rate). however, no single trial has had sufficient statistical power or length of followup to investigate mortality as a primary outcome. additional data from large-scale multicentre trials are required to strengthen the evidence base. the present multicentre rct included older adults with malnutrition, or at risk of malnutrition, with the aim of investigating the effect on survival after 6 months of intervention consisting of individual dietary advice, oral nutritional contact lisa s€oderstr€om lisa.soderstrom@regionvastmanland.se centre for clinical research, region v€astmanland, uppsala university, v€astmanland hospital, v€asterås s-721 89, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 3, 240–249 https://doi.org/10.1080/03009734.2020.1751752 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1751752&domain=pdf&date_stamp=2020-07-06 http://orcid.org/0000-0002-8367-1189 http://orcid.org/0000-0003-3691-8326 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1751752 http://www.tandfonline.com supplements, separately or in combination, or routine care. the hypothesis was that the survival differed between the intervention groups and the control group. materials and methods study design and setting this was a multicentre rct involving three intervention groups that received individual dietary advice, oral nutritional supplements, or a combination thereof. a fourth group served as the control group. the patients were recruited to the study when admitted to an internal medicine, surgical, or orthopaedic ward, for miscellaneous reasons, at five small-to-large-scale hospitals in central sweden between february 2010 and december 2014. the trial was conducted by registered dietitians. the dietitians engaged in the study were employed by the participating hospitals. some were recruited to work with the study directly after their dietetic studies, while others had been working for several years. before they started to recruit patients, the dietitians received 2days of instruction about the study protocol, including how to use the mini nutritional assessment (mna) instrument, by the project leaders. thereafter, the dietitians had weekly telephone conferences where they could discuss any uncertainties or other issues that had arisen. twice a year during the study period the dietitians met in person and practised the implementation of the study protocol, to decrease interrater variability. participants patients aged 65 years were included (figure 1). the patients were informed both verbally and in writing about the study by the dietitians and were then asked to participate. they were screened with the mna instrument (17–19) and the short portable mental status questionnaire (spmsq) (20–22). the mna has been validated in previous studies and been shown to predict mortality (8,23,24). the primary inclusion criterion was malnutrition or risk of malnutrition as indicated by a full 18-item mna score of 23.5. participants also had to have five incorrect answers on the spmsq, indicating no or at most moderate cognitive impairment. the exclusion criteria were: inability to communicate, does not speak swedish, decreased cognitive ability, having a body mass index (bmi) �35 kg/m2, receiving a dietary intervention, living in a nursing home, or having an expected survival of <1 year. since the expected survival was hard to estimate, in practice only those who were given palliative support were excluded according to the last-mentioned criterion. randomisation procedure a computerised block randomisation procedure with random block sizes varying between 8 and 32, stratified on nutritional status (malnourished or at risk of malnutrition), was performed by the responsible statistician (a.r.). the randomisation sequences were placed in sequentially numbered and sealed opaque envelopes by the project leader (h.f.), and the envelopes were distributed to and kept at the participating hospitals. the dietitian asked a research assistant who was not otherwise involved in this study to open the envelope and inform the dietitian which intervention group the participant was allocated to. the intervention was not blinded since this was not possible for practical reasons. figure 1. flow chart describing the participant recruitment and randomisation process in five hospitals in central sweden. da: dietary advice; mna: mini nutritional assessment; mna sf: mna short form; ons: oral nutritional supplements; spmsq: short portable mental status questionnaire. upsala journal of medical sciences 241 intervention and control groups information about the intervention was given during the hospitalisation, and the participants were told to start the intervention at discharge (intervention groups) or continue their usual habits (control group). dietary advice group patients randomised to receive dietary advice were counselled by a registered dietitian before they were discharged from hospital, and no further appointments were given once they had returned home. the patients were asked to describe their dietary habits and intake, and to discuss possible improvements with the dietitian to optimise the diet according to national dietary recommendations (25). the advice was semi-standardized and was based on the answers given in the mna. table 1 displays the dietary advice given to participants in the intervention groups that received dietary advice or dietary advice þ oral nutritional supplements (n ¼ 337). the semi-standardized approach was used to minimise interpersonal differences between the dietitians. it was developed by the project-leading dietitians and the dietitians working with the recruitment in accordance with national guidelines. the patient received a written copy of the advice. to increase the consensus between the dietitians’ dietary advice, a telephone conference was held each week during the recruitment period. oral nutritional supplements group all patients randomised to oral nutritional supplements were asked to drink 1–2 bottles per day, depending on the energy content of the supplement, to provide 400 kcal/day and 12–20 g protein. the participants were allowed to choose between different flavours and brands to increase their compliance. protein-dense supplements with a complete vitamin and mineral content were offered first. only if these were not tolerated were the participants offered supplements with lower protein content. the supplements had a volume of 125–200 ml/bottle, energy density 1.25–2.4 kcal/ml, and protein content 4–9.4 g/100 ml. the oral nutritional supplements were paid for by grants unrelated to the manufacturers. combined group the patients received dietary advice as described above for the dietary advice group. in addition, they were encouraged to drink the oral nutritional supplements in the same way as the oral nutritional supplements group. control group the patients were informed about the screening result and that the dietitian in the study would not give any further instructions about their nutrition. however, they were free to contact a health-care professional if they were concerned about their nutritional status. the control group was contacted by a dietitian by telephone at 1, 3, and 6 months after discharge to answer questions about health-care consumption and side effects to be able to compare possible side effects with the intervention groups. follow-up all four groups were asked questions according to a question guide, specifically developed for the present study, with both closed and open-ended questions about their visits to a general practitioner, district nurse, or dietitian, whether the patient had home care, and, if so, to what extent. the participants also answered questions about side effects such as nausea, vomiting, diarrhoea, constipation, and other problems in the gastrointestinal tract. the three intervention groups were contacted by the dietitian by telephone at 1, 3, and 6 months after discharge to check their compliance with table 1. dietary advice given to participants based on the answers from the mini nutritional assessment instrument. advice aiming at improving … other advice regarding … mna question with low scores energy densitya nutrient densityb texture of food number of meals per dayc advice to relatives how to complete meals protein-dense foods increased vegetable or fruit intake increased fluid intake declined food intake � � � � recent weight loss � � � mobility � neuropsychological problems � body mass index � � living independently � number of full meals � protein intake � vegetable intake � fluid intake � mode of feeding � mid arm circumference � � calf circumference � � � aenergy density: dietary advice regarding energy supplementation with energy-dense food, e.g., butter, margarine, full-fat dairy products (milk/yoghurt/cream), oil, and sugar. beverages should be energy-dense. bnutrient density: improved content of protein, vitamins, and minerals in meals. cnumber of meals per day: the energy and nutrient content should be distributed into 3 main meals and 1–3 in-between meals. the overnight fast should not exceed 11 h. 242 l. söderström et al. the treatment. to assess the compliance to the oral nutritional supplements, the dietitian asked the participants if they had consumed the prescribed number of supplements at 1, 3, and 6 months. following the same question guide, participants had the opportunity to ask questions, receive new dietary advice, or change the flavour or type of oral nutritional supplementation, if needed. controls were contacted by the dietitian at the same intervals as the intervention groups to minimise the risk of bias caused by increased attention given to the intervention groups. outcome survival of the intervention groups and the control group was followed up through the swedish population register until 16 april 2018, i.e., between 3.4 years and 8.2 years after starting the nutritional treatment. ethical considerations the study was approved by the uppsala ethical review board (approval number: 2009/203). before the patients entered the study, all provided written informed consent. all patients received at least routine treatment at the hospital. however, some participants received information about their risk of malnutrition, which gave them the opportunity to consider whether they needed or wanted to take further actions, such as consulting a dietitian. participation in the rct was registered in each patient’s medical record along with information on the purpose of the study and to which intervention group the patient was randomised. the trial is registered at clinicaltrials.gov with id: nct01057914. statistical analyses descriptive statistics are given as frequencies and percentages, n (%), for categorical data and as means and standard deviations (sds) for discrete and continuous data. an intention-to-treat (itt) approach was used for the analyses. six participants were misclassified in terms of their nutritional status group; four were categorised as malnourished but had only a risk of malnutrition, and two were classified as being at risk of malnutrition but were malnourished. these people were analysed according to their allocated group. differences in survival between the four randomisation groups were tested using log-rank tests, stratified according to nutritional status group, and illustrated using kaplan–meier plots. to examine the effect of the dietary advice and oral nutritional supplements on all-cause mortality, cox proportional hazards (ph) regression models stratified according to nutritional status group were used with time to death as outcome and dietary advice and oral nutritional supplements as predictors. this allowed the whole sample to be included since the combined (dietary adviceþ oral nutritional supplements) group contributed to the effects of both the dietary advice and the oral nutritional supplements predictors, thus giving a better power. the results are presented as hazard ratios (hrs) with accompanying 95% confidence intervals (cis). as potential confounders, the following baseline variables were considered: age (years), sex (men/women), bmi, smoking (never/former/current), living alone (yes/no), length of overnight fast (hours), cooks independently (always/sometimes/never), receiving home care service (yes/no), charlson comorbidity index, and number of medications. length of overnight fast was defined as the time between the last eating episode in the evening and the first eating episode the morning after. these variables were chosen because of their potential association with mortality (7,26). the proportional hazards (ph) assumption of the cox regression model was tested separately for each included explanatory variable using the grambsch–therneau test (27). variables that failed this test were included as piecewise variables with change point at 2 years (731 days) of follow-up, in which case they did not fail the test any more. the statistical analyses were performed using ibm spss statistics 24 and r 3.5.0, with p values <0.05 considered statistically significant. sample size calculation the sample size calculation was based on two studies with similar design, in which the mortality was approximately 20% in untreated patients and 10% in patients who had received nutritional treatment (10,28). to detect a 10-percentage point difference in mortality while obtaining a power (1–) of 80% at a two-sided significance level of 0.05 with a z test for difference in percentages, we calculated that each group should include at least 199 patients. with mortality as end-point, the drop-out is negligible since mortality is followed up in registers. accordingly, we calculated that 800 patients were needed for the study. a tentative interim analysis was performed after 560 participants had been included. this was not planned at the start of the study but decided upon when recruitment was slow and we needed to know if it was meaningful to continue. the analysis showed that there were no significant differences in mortality between the intervention and control groups. to obtain a statistically significant difference, an extreme difference in mortality in the remaining group of 240 patients would have been needed. since this seemed unlikely, we decided to terminate the study early. for logistical and personnel reasons, the termination was set to december 2014. at that time, a total of 671 individuals had been included in the study, which formed the study population of the present study. results participant characteristics in total, 671 patients were included in the present study. the median age was 79.0 (mean 78.7, sd 7.7) years with a range of 65–96years, and 61% (n¼ 410) were women. the mean bmi was 24 (sd 4) kg/m2, and 10% (n¼ 67) were current smokers. most of them (81%, n¼ 529) had normal cognitive functioning according to the spmsq, and only 16% (n¼ 107) and 3% (n¼ 20) had mild or moderate cognitive impairment, respectively. table 2 shows the baseline characteristics of the upsala journal of medical sciences 243 participants grouped according to the four treatment arms. the groups differed slightly in terms of age and the variable ‘receiving home care service’. of the excluded patients 56% were women, while the mean age was 80.6years and the mean bmi 25.6kg/m2. overall survival the median follow-up time was 4.1 years (3.4–8.2) generating 2588 person-years. at 6 months, i.e., when the intervention was completed, a total of 94 (14%) patients had died, an equal proportion in each arm. at the end of follow-up 273 (40.7%) were still alive. the survival rate was 36.9% in the dietary advice group, 42.4% in the supplement-only group, 40.2% in the combined group, and 43.3% in the control group. the differences, however, did not attain statistical significance (log-rank test p ¼ 0.762). kaplan–meier curves illustrate survival in the whole group (figure 2), the malnourished group (figure 3), and the group at risk of malnutrition (figure 4). there were, however, no statistically table 2. characteristics of the 671 participants at baseline. da ons da and ons control (n ¼ 168) (n ¼ 170) (n ¼ 169) (n ¼ 164) variables malnourished, % 24.4 24.7 24.9 24.4 age, y – mean (sd) 79.9 (7.9) 77.6 (7.5) 79.0 (7.6) 78.2 (7.7) women, % 58.3 65.9 58.6 61.6 body mass index, kg/m2 – mean (sd)a 23.7 (3.8) 24.2 (4.2) 23.8 (4.1) 23.7 (4.2) smoking, % never smoker 47.3 44.1 42.6 43.9 former smoker 43.7 47.1 45.0 46.3 current smoker 9.0 8.8 12.4 9.8 living alone, % 55.1 44.7 49.1 49.4 length of overnight fast, h – mean (sd) 12.4 (1.9) 12.5 (1.9) 12.3 (1.8) 12.3 (2.0) cooks independently, % always 42.5 39.4 43.8 45.7 sometimes 44.3 46.5 45.6 40.2 never 13.2 14.1 10.7 14.0 receiving home care service, % 31.3 22.6 27.2 17.8 charlson comorbidity index – mean (sd) 1.2 (1.4) 1.4 (1.8) 1.3 (1.6) 1.5 (1.8) number of medications – mean (sd) 6.9 (3.7) 6.9 (3.7) 6.9 (3.8) 6.7 (3.7) all characteristics except survival rate are as per baseline in 2010–2014. atwo participants had a bmi �35 kg/m2. da: dietary advice; ons: oral nutritional supplements; sd: standard deviation. p = 0.762 0.00 0.25 0.50 0.75 1.00 0 1 2 3 4 5 6 7 8 time (years) s u rv iv a l p ro b a b ili ty 168 138 115 103 85 55 32 26 5 170 131 118 103 85 61 42 22 8 169 133 114 95 88 72 44 25 5 164 133 122 103 82 60 41 27 6control da + ons ons da 0 1 2 3 4 5 6 7 8 time (years) tr e a tm e n t number at risk treatment da ons da + ons control figure 2. kaplan–meier survival curves for participants in the four intervention groups (n ¼ 671). log-rank test for any difference between groups (p ¼ 0.762). da: dietary advice; ons: oral nutritional supplements. 244 l. söderström et al. significant differences. unadjusted and adjusted hazard ratios for the respective groups are displayed in table 3. compliance the three intervention groups were contacted by the dietitian by telephone at 1, 3, and 6 months after discharge to check their compliance with the treatment. the self-reported compliance rates for the two groups who received oral nutritional supplements (including both malnourished patients and patients at risk of malnutrition) were 79% (n ¼ 215) at 1 month, 79% (n ¼ 194) at 3 months, and 74% (n ¼ 157) at 6 months. in total, 70% (n ¼ 104) in the group at risk of malnutrition and 63% (n ¼ 34) in the malnourished group reported taking the oral nutritional supplements according to the prescribed amount at each of the three follow-up times. the highest compliance rate (74%) to the prescribed supplements was observed among malnourished individuals in the intervention group who received both dietary advice and oral nutritional supplements (n ¼ 20). there were no significant differences in side effects such as nausea, vomiting, diarrhoea, constipation, and other problems in the gastrointestinal tract between the intervention groups and the control group, and there were no significant associations between compliance and reported side-effects at telephone interview 1, 3, and 6 months after discharge. discussion the findings from this multicentre rct imply that use of oral nutritional supplements and dietary advice does not improve survival in older adults with malnutrition or at risk of malnutrition. this confirms and adds to previous research pointing in the same direction (9,11,12,14,29). no single trial has had sufficient statistical power or length of follow-up to investigate mortality as a primary outcome (14). two cochrane reviews that examined the effect of oral nutritional supplements on mortality reported conflicting results. in a 2005 review that included data from 32 trials, the mortality was reduced by 26% in the supplemented group compared with the control groups (30). almost half of the trials did not report an itt analysis. the authors concluded that there may be beneficial effects of supplementation on mortality, although doubt remains due to the poor quality of most of the included trials. in fact, excluding the largest study (31), which also had the lowestquality rating, made the overall results non-significant (32). in an updated review from 2009 including data from 42 rcts in older adults with varying nutritional statuses, no reduced mortality was detected in the group that received oral nutritional supplements compared with a control group (14). most of these trials (60%) did not report an itt analysis (14,30). the conflicting results regarding mortality may be explained partly by inclusion of the multicentre food trial in the 2009 meta-analysis. the food trial provided oral p = 0.480 0.00 0.25 0.50 0.75 1.00 0 1 2 3 4 5 6 7 8 time (years) s u rv iv a l p ro b a b ili ty 127 108 90 80 64 43 25 21 4 128 102 90 77 63 47 33 19 8 127 104 91 79 75 61 39 24 5 124 105 98 83 65 48 33 24 6control da + ons ons da 0 1 2 3 4 5 6 7 8 time (years) tr e a tm e n t number at risk treatment da ons da + ons control figure 3. kaplan–meier survival curves for participants at risk of malnutrition in the four intervention groups (n ¼ 506). log-rank test for any difference between groups (p ¼ 0.480). da: dietary advice; ons: oral nutritional supplements. upsala journal of medical sciences 245 nutritional supplements or the ordinary hospital diet to stroke patients who were mainly well-nourished. no improved survival was detected at the 6-month follow-up (33). since the food trial included 4023 patients and thus constituted 50% of the study population in the 2009 metaanalysis, it may have diluted any potential treatment effects (14). subgroup analyses in the 2009 cochrane review indicated that a reduction in mortality applied only to subgroups of older adults who were already malnourished and older adults who received 400 kcal/day of oral nutritional supplements (14). a systematic review from 2019 including one study with 100 elderly subjects found no effect on mortality table 3. all-cause mortality for the three interventions and the control group. nutritional status intervention unadjusted hr (95% ci)a p value adjusted hr (95% ci)b p value all participants da 1.16 (0.88–1.53) 0.296 1.21 (0.83–1.75) 0.317 ons 1.05 (0.79–1.39) 0.749 1.08 (0.73–1.60) 0.691 da þ ons 1.08 (0.81–1.43) 0.603 1.08 (0.74–1.56) 0.693 control 1.00c 1.00c at risk of malnutrition da 1.24 (0.89–1.72) 0.201 1.20 (0.83–1.74) 0.342 ons 1.11 (0.79–1.55) 0.553 1.08 (0.73–1.60) 0.688 da þ ons 0.98 (0.70–1.39) 0.929 1.08 (0.74–1.56) 0.691 control 1.00c 1.00c malnourished da 0.97 (0.58–1.64) 0.918 1.01 (0.53–1.92) 0.984 ons 0.90 (0.53–1.52) 0.686 1.22 (0.64–2.33) 0.538 da þ ons 1.39 (0.85–2.29) 0.193 1.03 (0.53–2.01) 0.931 control 1.00c 1.00c results of separate cox regression analyses for the participants at risk of malnutrition and the malnourished participants, respectively. aresults based on 506 (100.0%) participants at risk of malnutrition and 165 (100.0%) malnourished participants. badjusted for age, sex, body mass index, smoking, living alone, length of overnight fast, cooks independently, receiving home care service, charlson comorbidity index, and number of medications. age is included as piecewise with change point at 731 days of follow-up for both nutritional status groups, as are smoking, cooks independently, and number of medications for the malnourished group. for the total group, age is included as piecewise with change points at 731 and 1461 days of followup. the results are based on 423 (83.6%) participants at risk of malnutrition and 125 (75.8%) malnourished participants with complete values for all variables. creference category. da: dietary advice; hr: hazard ratio; ons: oral nutritional supplements. p = 0.298 0.00 0.25 0.50 0.75 1.00 0 1 2 3 4 5 6 7 8 time (years) s u rv iv a l p ro b a b ili ty 41 30 25 23 21 12 7 5 1 42 29 28 26 22 14 9 3 0 42 29 23 16 13 11 5 1 0 40 28 24 20 17 12 8 3 0control da + ons ons da 0 1 2 3 4 5 6 7 8 time (years) tr e a tm e n t number at risk treatment da ons da + ons control figure 4. kaplan–meier survival curves for malnourished participants in the four intervention groups (n ¼ 165). log-rank test for any difference between groups (p ¼ 0.298). da: dietary advice; ons: oral nutritional supplements. 246 l. söderström et al. of oral nutritional supplements, or dietary advice in combination with oral nutritional supplements in older people (29). strengths and limitations of this study this study had several methodological strengths. the patients were recruited from a wide range of hospitals in central sweden, came from the general population, and had a variety of geriatric conditions, all of which help to increase the generalizability. however, the study population in the present study is less frail than the average population of older adults admitted to hospital, as the study only included patients who were living at home (not in a nursing home), had no cognitive impairment, and were not terminally ill. randomisation was performed centrally, was secure, and involved concealment of the allocations. the follow-up of deaths was almost 100% complete (one participant emigrated). this is so far one of the largest multicentre rcts examining the effect of oral nutritional supplements and dietary advice on mortality. in a cochrane review including data about mortality from 42 rcts of older adults receiving nutritional treatment, the study populations in most studies numbered fewer than 100 participants (14). moreover, most previous studies were conducted during a hospital stay or in nursing homes (10,11,14). in the cochrane review, 85% of the patients were admitted to hospital or lived in a nursing home during the treatment. however, today most older adults in sweden live at home. therefore, another strength was that the effect of nutritional treatment was studied in older people living at home. moreover, in the aforementioned cochrane review the duration of the intervention varied from 2 weeks to 18 months, and the follow-up time was in general the same as the duration of the treatment (14). in the present study, the duration of nutritional treatment was 6 months, with follow-up of survival for up to 8 years. registered dietitians at each centre performed the nutritional screening and the interventions. compliance with oral nutritional supplements was followed up by telephone and recorded by the dietitians at three time points during the treatment period. the compliance with dietary supplements in the present study was high and is consistent with an overall mean compliance of 78% (37–100%) reported in previous studies (34). in our study, malnourished individuals in the intervention group who received both dietary advice and oral nutritional supplements had the highest compliance with prescribed supplements (74%). the present study could be criticised for not obtaining information on nutritional outcomes (e.g., weight change) during treatment and follow-up. however, given the absence of evidence for effects on our primary outcome (survival), the relevance of any effect on such surrogate outcomes is questionable. to use weight change as a measure of compliance to supplements or as an outcome is problematic, since it is only possible to measure weight on those participants who have survived until the day of follow-up, thus introducing severe survivorship bias. the relevance of mortality as a primary outcome is further stressed in a systematic review by a panel of geriatricians and experts in nutrition. besides mortality, morbidity, and functional status, nutritional status and quality of life were considered a critical outcome for research in nutrition interventions for the prevention and treatment of malnutrition in older people (15). a further limitation was that the adherence to oral nutritional supplements and dietary advice was only followed up during the 6month treatment period, which could weaken the reliability of the results during the long-term follow-up. a further limitation of our study was that we did not reach our primary goal for inclusion. because the recruitment rate was slower than expected, a tentative interim analysis was performed after about 70% of the planned number of participants had been included. the interim analysis showed no difference in survival between the exposure groups, despite a higher than expected mortality in all groups. an extreme difference in outcome among the remaining 30% of planned inclusions would have been needed to change the overall results. this was considered unlikely, and we decided to end recruitment at the end of 2014. however, the results are still of interest, pointing to the large number-needed-totreat for the interventions considered in the present study. a possible explanation of why survival did not differ significantly between the intervention groups and the control group could be that the standard care of malnourished patients at the hospitals fully met the nutritional needs of the patients. in the dietary advice group (n ¼ 168) and control group (n ¼ 164), a total of 38 individuals had visited a dietitian other than those in the current study. this was significantly more visits to the dietitians compared to the two groups that received oral nutritional supplements. another concern may be that although the mna has been shown to predict a high risk of early death among individuals that have been classified as malnourished or at risk of malnutrition according to the mna criteria, no one has shown that nutritional support to these groups may alter the cause of these conditions. conclusions we could not confirm the anticipated benefit on survival from oral nutritional supplements or dietary advice. contrary to our hypothesis, oral nutritional supplements with or without dietary advice, or dietary advice alone, did not improve the survival in older adults who were malnourished or at risk of malnutrition. these results do not support the unselective, routine use of oral supplementation in older malnourished adults in the general population, if survival is the aim of the treatment. however, this does not mean that oral nutritional supplements should not be used at all, since the supplements may have other beneficial effects such as increased quality of life or increased performance in activities of daily living. however, the indication for providing the oral nutritional supplements must be clearly understood, both by the patient and the health-care professionals. it should also be noted that several factors, such as environmental factors and social isolation, influence nutritional intake. these factors might thus be important to include in future studies. upsala journal of medical sciences 247 acknowledgements the authors thank the dietitians who recruited the patients for the study and performed the nutritional treatments. disclosure statement no potential conflict of interest was reported by the author(s). funding this research was supported by grants from region v€astmanland, uppsala-€orebro regional research foundation (rfr), and the swedish national board of health and welfare. the funders had no role in the design or conduct of the study; collection, management, analysis, or interpretation of the data; preparation, review, or approval of the manuscript; or the decision to submit the manuscript for publication. notes on contributors lisa s€oderstr€om is a dietitian and obtained her phd in medical sciences in 2016 at uppsala university. previously a lecturer in nutrition at uppsala university, she is currently working at the centre for clinical research in v€asterås, sweden. andreas rosenblad, previously karlsson, born 1973, obtained his phd in statistics from uppsala university in 2006, and has been associate professor in applied medical statistics and epidemiology at uppsala university since 2014. previously a senior lecturer in statistics at stockholm university, he is currently an affiliated researcher at the department of medical sciences, uppsala university, and employed as a statistician at the regional cancer centre stockholm-gotland. leif bergkvist, md, phd, received his phd in 1987 at uppsala university, associate professor 1996, and professor of breast surgery 2007. he was head of the breast unit in v€asterås over 20 years, and working part time at the centre for clinical research from 1999 to retirement, but is still affiliated. main interest is clinical research in breast cancer, epidemiology, and nutrition. hanna frid is a dietitian at the department of child and adolescence psychiatry, v€astmanland county hospital v€asterås, sweden. eva thors adolfsson is a dietitian and obtained her phd in medical sciences in 2008 at uppsala university. she became associate professor in care science at uppsala university 2017. she has worked as a developer in region v€astmanland from 2009 to 2019 and since 2019 as a part-time researcher at the centre for clinical research in v€asterås, sweden. orcid lisa s€oderstr€om http://orcid.org/0000-0002-8367-1189 andreas rosenblad http://orcid.org/0000-0003-3691-8326 references 1. rojer ag, kruizenga hm, trappenburg mc, reijnierse em, sipila s, narici mv, et al. the prevalence of malnutrition according to the new espen definition in four diverse populations. clin nutr. 2016; 35:758–62. 2. abizanda p, sinclair a, barcons n, lizan l, rodriguez-manas l. costs of malnutrition in institutionalized and community-dwelling older adults: a systematic review. j am med dir assoc. 2016;17: 17–23. 3. lim sl, ong kc, chan yh, loke wc, ferguson m, daniels l. malnutrition and its impact on cost of hospitalization, length of stay, readmission and 3-year mortality. clin nutr. 2012;31:345–50. 4. luger e, haider s, kapan a, schindler k, lackinger c, dorner te. association between nutritional status and quality of life in (pre) frail community-dwelling older persons. j frailty aging 2016;5: 141–8. 5. marshall s, 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hospital (food): a multicentre randomised controlled trial. lancet. 2005;365: 755–63. 34. hubbard gp, elia m, holdoway a, stratton rj. a systematic review of compliance to oral nutritional supplements. clin nutr. 2012;31: 293–312. upsala journal of medical sciences 249 abstract introduction materials and methods study design and setting participants randomisation procedure intervention and control groups dietary advice group oral nutritional supplements group combined group control group follow-up outcome ethical considerations statistical analyses sample size calculation results participant characteristics overall survival compliance discussion strengths and limitations of this study conclusions acknowledgements disclosure statement references brain death due to fat embolism — could moderate hypercapnia and prone position be blamed for the to full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 brain death due to fat embolism — could moderate hypercapnia and prone position be blamed for the tonsillar herniation? rafael kawati & anders larsson to cite this article: rafael kawati & anders larsson (2013) brain death due to fat embolism — could moderate hypercapnia and prone position be blamed for the tonsillar herniation?, upsala journal of medical sciences, 118:4, 276-278, doi: 10.3109/03009734.2013.818600 to link to this article: https://doi.org/10.3109/03009734.2013.818600 © informa healthcare published online: 27 aug 2013. submit your article to this journal article views: 524 view related articles citing articles: 2 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.818600 https://doi.org/10.3109/03009734.2013.818600 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.818600 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.818600 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.818600#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.818600#tabmodule upsala journal of medical sciences. 2013; 118: 276–278 case report from uppsala university hospital brain death due to fat embolism — could moderate hypercapnia and prone position be blamed for the tonsillar herniation? rafael kawati & anders larsson central intensive care unit, department of anesthesia and intensive care, uppsala university hospital, uppsala, sweden abstract fat embolism to the systemic circulation in polytrauma patients is very common. the fat embolism syndrome (fes), however, is a rare condition. we describe a case of traumatic femur fracture with fes that was presented as acute tonsillar herniation (coning) and brain death postoperatively. we believe that in this case the prone position and moderate hypercapnia contributed to the acute coning. key words: fat embolism, hypercapnia, prone position, trauma introduction fat embolism to the systemic circulation in polytrauma patients is very common and up to 80% of trauma patients have had fat embolization at autopsy (1,2). the fat embolism syndrome (fes), however, is a rarer condition consisting of a constellation of neurological, pulmonary, cutaneous, and hemodynamic changes. an incidence of 1% following long-bone fractures has been reported (3,4). there are a vast number of publications describing different presentations of fat embolism. however, cerebral edema and fatal tonsillar herniation is an unusual presentation of fes described in the literature only a few times (5-7). in one case, cerebral fat embolism syndrome caused brain death after long-bone fractures and acetazolamide therapy (8). we describe a case of traumatic femur fracture with fes that was presented as acute tonsillar herniation (coning) and brain death postoperatively. case report a 20-year-old man was involved in a motor vehicle accident resulting in an isolated fracture of the right mid femur. the patient was awake and had a glasgow coma scale (gcs) of 15 at admission to the emergency department. the patient was examined and stabilized according to the advanced trauma life support (atls) principles. whole-body computerized tomography did not show any head injury. there were multiple displaced costal fractures on the right side with lung contusion and minimal pneumothorax that did not need drainage. the patient was admitted to the intensive care unit (icu) while waiting for surgical stabilization of the fracture. during the 6-hour preoperative stay in the icu, the patient was stable and awake. the patient was operated upon in general anesthesia. intramedullary insertion of a femur nail was attempted but was quite difficult to achieve. a great deal of manipulation and drilling was required in order to insert a 400 mm nail. the patient was stable peroperatively. three hours later he was transported back to the icu, still intubated and on 0.5 fraction inspired oxygen (fio2). a wake-up test was performed at arrival to the icu, and the patient seemed to be awake but suffered from pain. the patient deteriorated during the next 6 hours. he developed a high correspondence: rafael kawati, phd, central intensive care unit, department of anesthesia and intensive care, uppsala university hospital, uppsala, se 75185, sweden. e-mail: rafael.kawati@gmail.com (received 15 may 2013; accepted 17 june 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.818600 http://informahealthcare.com/journal/ups mailto:rafael.kawati@gmail.com fever, and his oxygen saturation decreased. a new chest x-ray showed 2.5 cm pneumothorax that was drained successfully, but saturation continued to decrease during the next 6 hours. his oxygen demand increased ending up at 0.9 fio2. a decision was made to increase sedation and apply a higher positive end expiratory pressure. in addition the patient was turned into a prone position. the patient was kept sedated during the next 24 hours, and the fio2 was decreased to 0.4. moderate hypercapnia was tolerated (7.5–8.5 kpa). a wake-up test was not performed while the patient was in the prone position but the patient’s pupils were examined regularly. upon turning the patient to a supine position, his pupils dilated and they were not responsive to light. the patient was hyperventilated and mannitol was given. in addition a computerized tomography scan (ct) of the head was performed (figures 1 and 2) revealing cerebral edema and evidence of coning. ct with angiography was also performed to exclude dissection of the great vessels in the neck. no dissection was found. examination of the patient revealed petechial hemorrhages mainly on the torso, extremities, and conjunctivas. transesophageal echocardiography revealed no intracardiac shunting. twenty-four hours later the patient was declared brain dead according to the brain death clinical criteria and four-vessel angiography. autopsy showed signs of massive fat embolism in the lungs and brain. discussion we present a case of fes with all criteria of that syndrome including neurological, pulmonary, cutaneous, and hemodynamic changes. neurological manifestations of fes can vary from mild cognitive changes to coma, but severe cerebral edema including coning is a quite unusual presentation. therefore one might speculate on why our patient developed such a severe cerebral edema. the patient did not have a head injury that might have been worsened postoperatively. he was awake before turning him into a prone position. pneumothorax and lung contusion contributed to the deterioration of his pulmonary status and also delayed the diagnosis of fes. moreover, he was deeply sedated and therefore no wake-up test was performed during the period when he was in the prone position. prone position and deep sedation were clinically motivated because of his high fio2. moderate hypercapnia was also tolerated in line with the latest recommendation in managing patients with acute respiratory distress syndrome (ards) (9). the effect of prone position on intra-cranial pressure (icp) has not been studied extensively. however, in one study it was found that the prone position did not influence icp and the cerebral perfusion pressure, but significantly improved the patient’s pao2 (10). in another study oxygenation was also improved but icp increased significantly (11). theoretically speaking, turning the patient into a prone position and tilting the head to one side (at 90 degrees angle) could affect the venous return from the brain, resulting in cerebral edema. hypercapnia induces cerebral vasodilation and increases cerebral blood flow, which might worsen the cerebral edema (12). the combination of prone position and moderate hypercapnia might have contributed to the severe cerebral edema and eventually coning and brain death. figure 1. head ct (sagittal view) showing cerebral edema and tonsillar herniation. figure 2. head ct (axial view) showing cerebral edema and tonsillar herniation. brain death due to fat embolism 277 to our knowledge this is the first reported case with this combination and leading to such a tragic outcome. deep sedation and permissive hypercapnia are established recommendations in managing severe ards. in addition, prone position is an effective method to improve saturation in patients needing high fio2 (13). in patients with head injuries, icp monitoring is a routine procedure to monitor cerebral edema. in awake multitrauma patients without head injuries one would not expect a need for icp monitoring. with the risk of fes in mind we advocate performing wake-up tests regularly. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. hulman g. the pathogenesis of fat embolism. j pathol. 1995; 176:3–9. 2. eriksson ea, pellegrini dc, vanderkolk we, minshall ct, fakhry sm, cohle sd. incidence of pulmonary fat embolism at autopsy: an undiagnosed epidemic. j trauma. 2011;71: 312–15. 3. bulger em, smith dg, maier r, jurkovich gj. fat embolism syndrome. a 10-year review. arch surg. 1997; 132:435–9. 4. muller c, rahn b, pfister u, meinig rp. the incidence, pathogenesis, diagnosis and treatment of fat embolism. orthop rev. 1994;23:107–17. 5. bracco d, favre jb, joris r, ravussin a. fatal fat embolism syndrome: a case report. j neurosurg anesthesiol. 2000;12:221–4. 6. beretta l, calvi mr, frascoli c, anzalone n. cerebral fat embolism, brain swelling, and severe intracranial hypertension. j trauma. 2008;65:e46–8. 7. etchells ee, wong dt, davidson g, houston pl. fatal cerebral fat embolism associated with a patent foramen ovale. chest. 1993;104:962–3. 8. walshe cm, cooper jd, kossmann t, hayes i, iles l. cerebral fat embolism syndrome causing brain death after long-bone fractures and acetazolamide therapy. crit care resusc. 2007;9:184–6. 9. the acute respiratory distress syndrome network. ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. n engl j med. 2000;342:1301–8. 10. thelandersson a, cider a, nellgård b. prone position in mechanically ventilated patients with reduced intracranial compliance. acta anaesthesiol scand. 2006;50:937–41. 11. nekludov m, bellander bm, mure m. oxygenation and cerebral perfusion pressure improved in the prone position. acta anaesthesiol scand. 2006;50:932–6. 12. kety ss, schmidt cf. the effects of altered arterial tensions of carbon dioxide and oxygen on cerebral blood flow and cerebral oxygen consumption of normal young men. j clin invest. 1948;27:484–92. 13. guérin c, reignier j, richard jc, beuret p, gacouin a, boulain t, et al. proseva study group. prone positioning in severe acute respiratory distress syndrome. n engl j med. 2013;368:2159–68. 278 r. kawati & a. larsson www.ncbi.nlm.nih.gov/pubmed/7616354?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21825932?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21825932?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9108767?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9108767?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/8196970?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/8196970?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10905570?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10905570?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18349713?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18349713?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18349713?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/8365324?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/8365324?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17536990?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17536990?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10793162?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10793162?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10793162?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10793162?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16923087?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16923087?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16923087?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16923086?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16923086?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/23688302?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/23688302?dopt=abstract abstract introduction case report discussion declaration of interest references biobank linked to swedeheart quality registry—routine blood sample collection opens new opportunities for cardiovascular research article biobank linked to swedeheart quality registry—routine blood sample collection opens new opportunities for cardiovascular research tomasz barona,b, anna beskowb,c, stefan jamesa,b and bertil lindahla,b adepartment of medical sciences, cardiology, uppsala university, uppsala, sweden; buppsala clinical research center, uppsala, sweden; cuppsala biobank, uppsala, sweden abstract high-quality biobanking within routine health services, through the use of existing health-care practices and infrastructure, with respect to safety and integrity of patients in line with the swedish biobank act, enables large-scale collection of biological material at reasonable costs. complementing the extensive information on myocardial infarction patients from a national registry gives unique opportunities for research focusing on better understanding of cardiovascular disease occurrence and prognosis, developing of new diagnostic methods, and personalized treatments with greater efficacy and fewer side effects. article history received 28 march 2018 revised 1 july 2018 accepted 5 july 2018 key words biobank; cardiovascular research; quality registry; swedeheart biobanks the term ‘biobank’ is primarily used to describe collections of biological material of different size—from large-scale population collections to human biological specimens collected for health-care purposes. today, the material is stored in many different ways across sweden, ranging from large specially designed freezing rooms to single freezers spread in hospitals and research departments. currently a lot of work is being done and has been done to transform many small biobanks into sample collections in a larger biobank organization, for example, uppsala biobank in sweden. the concept of a biobank is relatively new. the first article using this term was published by loft and poulsen in 1996 (1). collection and storing of tissue samples began, however, far earlier, and already in 1858 virchow published a work on biological samples (2). according to a survey conducted recently by the swedish national biobank council, there are currently approximately 150 million blood, cell, or tissue samples collected in biobanks for care and treatment and approximately 7.5 million samples collected in biobanks for purposes of research or clinical trials (3); every year about 2–3 million new samples are stored in the swedish biobanks (4). however, most are collected as part of a clinical trial or other temporary studies from highly selected patient groups. biobank research biological material, such as tissue or blood samples, can be used to study genetic factors or protein expression in various diseases. a detailed phenotype description required for most research projects is commonly retrieved from patient records, registers, or questionnaires. samples collected primarily for health-care purposes can be used in research if the patient/donor has given consent. when the primary purpose of the sample collection is research, both an approved ethical application and an informed consent from the donors are required. quality registry for coronary heart disease the swedish web-system for enhancement and development of evidence-based care in heart disease evaluated according to recommended therapies (swedeheart) is a national quality registry for cardiovascular care, coronary angiography, percutaneous coronary intervention (pci), cardiac surgery, secondary prevention, and cardio-genetic diseases, which was formed in 2009 by the merging of four existing registries (5). the purpose of the registry is to develop and improve cardiovascular care by continuously providing information on care needs and treatments on local, regional, and national levels. the swedeheart registry and its earlier four separate registries have contributed to the enormous improvements of cardiac care and dramatic increase of survival after myocardial infarction in the last decade (6,7). however, despite the fact that diagnosis, treatment, and secondary prevention of coronary heart disease have developed favorably and that cardiac care has been improved in accordance with national guidelines, there are still shortcomings, which are reflected in, among other things, significant variations between different hospitals and regions regarding contact tomasz baron tomasz.baron@ucr.uu.se uppsala clinical research center, dag hammarskj€olds v€ag 38, se-751 85 uppsala, sweden. � 2018 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 1, 12–15 https://doi.org/10.1080/03009734.2018.1498957 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1498957&domain=pdf http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1498957 http://www.tandfonline.com treatment regimens, with consequences for public health and health economics. quality registry research in order to achieve even better treatment results in the future, new and better knowledge about the genetic background to cardiovascular disease, new diagnostic and predictive markers, and new effective and individualized treatments are needed. already extensive research is being carried out using data from swedeheart (5), which can be escalated by linking biobank data and samples to the registry. concept of a biobank linked to the swedeheart registry there are currently several regional initiatives where blood samples are collected as part of individual research projects in which phenotypic information is obtained from the swedeheart registry. cooperation between the various regional initiatives enables samples from the different biobanks to be available nationally. starting the collection of samples linked to the swedeheart registry in the uppsala–€orebro region aimed to create a whole workflow entirely within routine clinical care. high-quality blood samples collected from consecutive patients with myocardial infarction could be then used in future research. after approval by the regional ethical review board in uppsala, the blood sample collection was started on 12 september 2011 at the department of cardiology, uppsala university hospital, in close collaboration with the clinical laboratory, uppsala clinical research center, and uppsala biobank. the inclusion of patients is performed by routine healthcare professionals, and the samples are collected on two occasions: first, during the hospitalization at the cardiac intensive care unit (icu) due to the acute myocardial infarction (acute phase); and second, before or in connection with the regular follow-up visit after 6–10 weeks (stable phase). on these occasions, the information that the samples have been taken is recorded in the swedeheart registry. the collected specimens are: whole blood for later dna extraction, as well as edta-plasma and citrate-plasma for biomarkers specific for e.g. cardiac function, atherosclerosis, inflammation, coagulation, and renal function. inclusion and exclusion criteria are presented in table i. practical setup patients hospitalized at the cardiac icu, uppsala university hospital, who meet criteria for inclusion in the biobank are informed about the sample collection by a routine nurse and asked for participation. written consent is collected and scanned to be stored in the electronic patient record. samples for biobanking are ordered electronically via the electronic patient record in the same way as, and usually with, regular samples ordered for laboratory analysis. the samples are sent to the hospital clinical laboratory, department of clinical chemistry and pharmacology (kkf). at first sampling, usually the day after admission, edta whole blood, edta plasma, and citrate plasma are taken; in the second sampling after 6–10 weeks, only edta plasma and citrate plasma are taken. at kkf, the samples are handled and sorted by a pre-analytic robot according to the sample type specified by a four-digit extension on the tube id. tubes with ‘whole blood’ are registered and sorted out and frozen directly. the edta plasma tubes are registered, centrifuged (2400g for 7min), and de-capped before being transferred to another rack for aliquoting. citrate plasma tubes are registered and sorted into a rack for manual centrifugation, 2000g for 20minutes, to ensure that it is free from platelets. plasma from each primary tube is then transferred to eight microtubes with 225ll each in a 96-format using a fluidhandling robot. the volume of 225 ll per microtube is more than sufficient for most modern analyses. the samples are then frozen to –80 �c and stored in uppsala ice hotel (biobank premises). all information about the handling and sampling is automatically transferred from the laboratory information management system (lims) flexlab together with the information from the fluid management robot to the biobank lims, where complete traceability is available and information about the sample’s quality based on pre-analytical treatment is recorded. in the swedeheart registry, information about consent and date of sampling is entered. the concept’s layout is presented graphically in figures 1 and 2. primary results, experiences, and strength of the concept during the period 12 september 2011 to 31 december 2017, biobank samples were collected from a total of 534 patients of which 181 (34%) have also left samples in the second round. a detailed report one year after the start of sample collection showed that more than two-thirds of all patients with myocardial infarction who met the inclusion criteria and lacked exclusion criteria have their specimens stored in the biobank. patients with non-st-elevation myocardial infarction (nstemi) constitute 65% of the collected material, reflecting the distribution in the total infarct population. the most common reason for non-inclusion of patients appeared to be a concurrent participation in other parallel research studies, unclear initial diagnosis, lack of significant coronary artery stenosis at coronary angiography, in-hospital death before scheduled sampling, logistic reasons (transfer between departments), language deficiencies, or cognitive disorders. table i. the inclusion and exclusion criteria for participation in the swedeheart biobank. inclusion criteria (all fulfilled) exclusion criteria (at least one of the following) � myocardial infarction diagnosis � age �75 � patient included in swedeheart signed informed consent � patient transferred from another hospital � >72 hours from myocardial infarction diagnosis to planned biobank sampling � procedure-related myocardial infarction (type 4a or 5) upsala journal of medical sciences 13 out of all respondents, during the first year of sample collecting only one patient declined participation (6). the strength of the concept is that all parts of the workflow, including sampling and specimen management, are routinely done, using existing care routines and infrastructures, and do not require additional resources in the form of special staff, which also means that the study experience is perceived as part of routine care. this means that time and resources are saved, while automated handling of samples ensures consistency and high quality. the department of cardiology is paid for the nurse time spent on the biobank project. the simple method that works in routine health care allows for the collection of samples of large, non-selected patient materials and where the phenotype can be described figure 1. practical setup of biobank sampling in routine care within the framework of the swedeheart registry. figure 2. workflow in the clinical chemistry and pharmacology laboratory, academic hospital. (cosmic ¼ patient data journal; flexlab and evoware ¼ laboratory data systems; lims ¼ laboratory information management system). 14 t. baron et al. in detail at the individual level using registry data. biobank sampling does not exclude patients from parallel participation in other research projects. future development there are ongoing efforts to enable similar biobanking in the entire health-care region. the university hospital in €orebro is next to start the corresponding biobanking linked to the swedeheart register. at the same time, work is under way with swedeheart’s steering group and with researchers in other regions who are planning or have already started biobanking linked to the registry, as in the region of skåne and stockholm, to standardize sampling and testing, as well as to create a common set of rules for sampling. the biobank material, together with the registry’s possibilities for careful phenotyping and follow-up, will provide a unique national resource for future research. disclosure statement no potential conflict of interest was reported by the authors. notes on contributors dr tomasz baron, md, phd, senior consultant cardiology and clinical physiology, department of medical sciences, uppsala university and uppsala clinical research centre, uppsala, sweden. dr anna beskow, phd, director, uppsala biobank and uppsala clinical research centre, uppsala, sweden. professor stefan james, senior consultant cardiology, department of medical sciences, uppsala university and uppsala clinical research centre, uppsala, sweden. professor bertil lindahl, senior consultant cardiology, department of medical sciences, uppsala university and uppsala clinical research centre, uppsala, sweden. references 1. loft s, poulsen he. cancer risk and oxidative dna damage in man. j mol med (berl). 1996;74:297–312. 2. wagner rp. rudolph virchow and the genetic basis of somatic ecology. genetics. 1999;151:917–20. 3. biobanker och biobankstj€anster 2011. inventering utf€ord av nationellt biobanksråd (nbr). 2012. [cited 2012 december 11]. 4. biobanken resurs f€or livet. 2009. [cited 2012 december 11]. 5. jernberg t, attebring mf, hambraeus k, ivert t, james s, jeppsson a, et al., the swedish web-system for enhancement and development of evidence-based care in heart disease evaluated according to recommended therapies (swedeheart). heart. 2010;96:1617–21. 6. baron t, beskow a, james s, lindahl b. [biobank linked to swedeheart, a resource for future research]. lakartidningen. 2013;110:1669–71. 7. larsson s, lawyer p, garellick g, lindahl b, lundstr€om m. use of 13 disease registries in 5 countries demonstrates the potential to use outcome data to improve health care’s value. health aff (millwood). 2012;31:220–7. 8. puymirat e, simon t, steg pg, schiele f, gu�eret p, blanchard d, et al. association of changes in clinical characteristics and management with improvement in survival among patients with stelevation myocardial infarction. jama. 2012;308:998–1006. 9. http://www.ucr.uu.se/swedeheart/component/edocman/swedeheartarsrapport-2017-swedish-svensk/viewdocument?itemid¼ upsala journal of medical sciences 15 http://www.ucr.uu.se/swedeheart/component/edocman/swedeheart-arsrapport-2017-swedish-svensk/viewdocument?itemid&hx003d; http://www.ucr.uu.se/swedeheart/component/edocman/swedeheart-arsrapport-2017-swedish-svensk/viewdocument?itemid&hx003d; abstract biobanks biobank research quality registry for coronary heart disease quality registry research concept of a biobank linked to the swedeheart registry practical setup primary results, experiences, and strength of the concept future development disclosure statement notes on contributors references dexmedetomidine pretreatment alleviates propofol injection pain upsala journal of medical sciences. 2014; 119: 338–342 original article dexmedetomidine pretreatment alleviates propofol injection pain liang he1, jun-mei xu2, tao he1, lei liu2 & rong zhu2 1department of anesthesiology, loudi central hospital of university of south china, loudi 417000, hunan province, china, and 2department of anesthesiology, the second xiangya hospital of central south university, changsha 410011, hunan province, china abstract objective. the incidence of propofol injection pain during induction of general anesthesia varies from 28% to 90%. this prospective, randomized, double-blind, placebo-controlled study evaluated the effect of dexmedetomidine (dex) for reducing the incidence and severity of propofol injection pain. methods. patients undergoing elective surgical procedures were randomly allocated into seven groups of 30 patients each. experimental treatments were intravenously administered over 10 min (total volume 10 ml) prior to intravenous propofol injection, as follows: group i, the control group, was given isotonic saline. patients in groups ii, iii, and iv received dex 0.25 mg/kg, 0.5 mg/kg, or 1.0 mg/kg, respectively, mixed with isotonic saline immediately before propofol injection. patients in groups v, vi, and vii received dex as above, but 5 minutes before propofol injection. propofol consisted of 1% long-chain triglyceride propofol (2.5 mg/kg) injected at 1 ml/s. results. median propofol injection pain score was 0.00 (iqr 0.00–3.00) in patients who received 1.0 mg/kg dex 5 min before the propofol injection (group vii), and only 1 patient (of 30) in this group received a pain score >2. the median pain score and number of patients with pain scores >2 in group vii were both significantly less than in the control (group i; p = 0.000, both). there were no differences in either mean arterial pressure or heart rate at any time point after dex injection among the groups. conclusions. pretreatment with intravenous dex 1 mg/kg 5 min prior to injection of long-chain triglyceride propofol is effective and safe in reducing the incidence and severity of pain due to propofol injection. key words: dexmedetomidine, injection pain, propofol introduction propofol is widely administered during anesthetic induction. however, the pain of injection is undesirable, and may cause hand withdrawal and dislodging of the venous cannula (1,2). the incidence of propofol injection pain varies from 28% to 90% (3). many methods have been used to relieve the pain of propofol injection, such as pretreatment with lidocaine, ondansetron, and methylene blue, but the effectiveness of these methods remains uncertain (4–8). the alpha-2 adrenoceptor agonist clonidine was foundtoalleviatethepainofinjectedpropofoleffectively (9). dexmedetomidine (dex; jiangsu singch pharmaceutical, lianyungang, jiangsu province, china) is also an alpha-2 adrenoceptor agonist, but is more selective than clonidine and has analgesic and sedative properties (2). dex has been evaluated for reducing the incidence and intensity of propofol-induced pain, but reported results are inconsistent (1,2). we hypothesized that dex injection before propofol would reduce propofol injection pain. we also studied the effectiveness of different doses of dex and the time interval between dex and propofol injection. methods after the approval of the hospital ethics committee of second xiangya hospital of central south correspondence: rong zhu, md, phd, department of anesthesiology, the second xiangya hospital of central south university, changsha 410011, hunan province, china. e-mail: zhurong0807@hotmail.com (received 29 april 2014; accepted 29 june 2014) issn 0300-9734 print/issn 2000-1967 online � 2014 informa healthcare doi: 10.3109/03009734.2014.941049 http://informahealthcare.com/journal/ups mailto:zhurong0807@hotmail.com university, 210 patients, aged 18 to 60 years, asa (american society of anesthesiologists) physical classification i to ii, and scheduled for minor elective surgery, were included in the study. all patients signed a written informed consent form. patients were excluded if they had a history of drug abuse, chronic use of any medication, presence of neurological or psychiatric diseases, uncontrolled hypertension, or renal or hepatic insufficiency. patients were also excluded if they had a known history of hypersensitivity to the study drugs. before surgery (24 h) the patients did not receive analgesics or sedatives. upon arrival to the operating room, a 20-gauge cannula was inserted into the dorsum of the patient’s hand and connected to a t-connector for drug administration. standard asa monitors were attached, including non-invasive arterial pressure, electrocardiography, and pulse oximetry. patients were randomly allocated to seven groups (i–vii; n = 30 each) using a computer-generated table with random numbers (table i). patients in group i, the control group, were given 10 ml of isotonic saline intravenously over 10 min via a micro-infusion pump. patients in groups ii, iii, and iv received dex (200 mg/2 ml; singch pharm, lianyungang, china) 0.25 mg/kg, 0.5 mg/kg, or 1.0 mg/kg, respectively, mixed with isotonic saline over 10 min (final volume, 10 ml), and then 1% long-chain triglyceride (lct) propofol (propofol 1%; fresenius kabi, beijing, beijing municipality, china) was immediately injected intravenously. patients in groups v, vi, and vii were given dex 0.25 mg/kg, 0.5 mg/kg, or 1.0 mg/kg, respectively, mixed with isotonic saline, over 10 min (final volume, 10 ml). five minutes later, 1% lct propofol was injected. all groups received 2.5 mg/kg of 1% lct propofol, injected at a rate of 1 ml/s. all study medications were prepared in a 10-ml syringe that was covered with black tape by an anesthesiologist who was not involved in the study. all study drugs were maintained at room temperature and were used within 30 min after preparation. another anesthesiologist, who was unaware of the group assignment, assessed the intensity of pain after propofol injections. the assessing anesthesiologist used a specially designed composite pain scale described by rochette and colleagues (7) to evaluate the level of propofol injection pain. the pain score is based on assessments of patients’ motor and verbal reactions, from the time of propofol injection to loss of consciousness (table ii). pain is graded on a 0– 6 scale, with a score >2 considered unacceptable (8). mean arterial pressure (map) and heart rate (hr) were recorded immediately before injection of the study drug, and then every 5 min until propofol injection. all patients received 1% lct propofol 2.5 mg/kg (fresenius kabi, beijing, beijing municipality, france) at a rate of 1 ml/s with different pretreatments. for the design of this study, an estimation of the required minimum sample size was determined based on a previous report that pain caused by propofol injection was experienced by 70% of adults (8), and an assumption that a pretreatment with dex would cause a 50% reduction in the injection pain. with a probability of less than 5% of making a type i error, (i.e. significance level a = 0.05) and the probability of less than 10% of making a type ii error (accepting a null hypothesis that is false, b = 0.10), we were required to enroll at least 24 patients in each group; we recruited 30 patients in each group. statistical analyses were performed using statistical product for social sciences (spss) software v. 18.0. the continuous normally distributed data (age and weight) are described as mean ± standard deviation and compared using one-way anova. changes in hr and bp over time were tested for normality using the mean ± standard deviation and then compared table i. experimental treatment groups. group drug and dose time to propofol injection control i isotonic saline immediately before treatment ii dex 0.25 mg/kg immediately before treatment iii dex 0.50 mg/kg immediately before treatment iv dex 1.00 mg/kg immediately before treatment v dex 0.25 mg/kg 5 minutes treatment vi dex 0.50 mg/kg 5 minutes treatment vii dex 1.00 mg/kg 5 minutes table ii. scoring system for propofol injection pain (8). score motor events no movement 0 slight hand withdrawal 1 marked withdrawal, rubbing, trying to tear off the line 2 general restlessness 3 verbalization scale no vocalization 0 purposeless moaning 1 explicit protest 2 screams, cries 3 total 0–6 dexmedetomidine and propofol injection pain 339 using two-way repeated measures anova followed by post hoc bonferroni correction. pain scores are expressed as median (interquartile range (iqr)) and compared using the kruskal–wallis test. categorical data such as gender, asa status, and the number of patients having pain scores >2 were expressed as number, percent, or both, and were compared using the chi-square test or fischer’s exact test as appropriate. whenever statistically significant discrepancies appeared, group i was compared separately with others to analyze the differences with the bonferroni correction when appropriate. a corrected p value of 0.05/6 was considered significant. results all patients completed the study. there were no statistically significant differences among the seven groups with regard to age, weight, gender, or asa class (p > 0.05) (table iii). there was no difference in median propofol injection pain scores among groups i (3.00 (iqr 2.00– 4.00)), ii, iii, v, and vi. however, the median pain score of group iv (2.00 (iqr 0.00–3.00)) was significantly lower than that of group i (p = 0.003), and the median propofol injection pain score of group vii was 0.00 (iqr 0.00–3.00), which was significantly lower than that of group i (p = 0.000). the incidence of pain scored >2 was 17/30 in the control group i (figure 1). there were no differences in this incidence rate among groups i, ii, iii, iv, v, and vi. however, the incidence of pain scored >2 in group vii was 1/30, which was the lowest among all the groups and was significantly lower than that of group i (p = 0.000). there were no differences between the groups as regards map and hr at any time point after dex injection (p > 0.05, detailed data not shown). discussion this prospective, randomized, double-blind, placebo-controlled study was carried out in order to evaluate the effect of dex for reducing the incidence and severity of propofol injection pain. besides the control group (given isotonic saline vehicle), group i group ii group iii group iv group v group vi group vii 0 20 40 60 80 100 a p a in fu l in je c ti o n ( % ) figure 1. percentages of patients experiencing pain scored >2. acompared with group i, p = 0.000. table iii. demographics of each group (n = 30, each)a. control treatment groups i ii iii iv v vi vii age, y 37.5 ± 8.5 37.3 ± 9.8 36.7 ± 10.6 39.0 ± 10.9 42.6 ± 9.9 37.5 ± 8.5 37.5 ± 8.5 male/female, n 17/13 15/15 14/16 16/14 17/13 18/12 17/13 weight, kg 53.3 ± 5.1 52.6 ± 6.0 55.1 ± 6.1 56.2 ± 5.9 54.7 ± 5.5 55.3 ± 7.0 53.6 ± 6.4 asa i/ii, n 19/11 20/10 20/10 18/12 17/13 19/11 18/12 aall patients completed the present study. there were no statistically significant differences among the seven groups with regard to age, weight, gender, or asa class (p > 0.05). 340 l. he et al. treatments consisted of 0.25, 0.5, or 1.0 mg/kg dex, each delivered either immediately before or 5 minutes prior to lct propofol injection. we found that pretreatment with 1 mg/kg dex 5 min before propofol reduced the incidence and severity of pain due to propofol injection. various other pretreatments have also been evaluated, such as parecoxib with venous occlusion (10), tourniquet-controlled lidocaine (4), ondansetron (5), intravenous methylene blue (6), alfentanil and lidocaine (11), and a small dose of ketamine (12). a recent systematic review and meta-analysis showed that propofol infusion via the antecubital vein and pretreatment with lidocaine in conjunction with venous occlusion were the two most efficient interventions to reduce pain on injection of propofol (13). however, some unexpected adverse side effects have been associated with the two methods. for some patients undergoing short-time surgery with general anesthesia, propofol infusion via a hand vein is more convenient than via an antecubital vein. tourniquets are the most common compressive devices for venous occlusion, but can cause tourniquet-induced hypertension or even ischemia-reperfusion injury (14–17). therefore, venous occlusion before propofol injection may be contraindicated in patients with moderate to severe hypertension. dex has been demonstrated to have significant analgesic effects (18–23). although the mechanisms of the analgesic effect of dex have not been fully elucidated, many studies have shown that dex acted by inhibiting the release of substance p from the dorsal horn of the spinal cord (24,25). a recent study reported that dex effected strong analgesia through inhibition of the spinal erk1/2 signaling pathway (26). these studies suggest that dex has an important role in nociceptive transmission at the spinal level. boehm et al. (27) demonstrated in rats that when dex was administered intraperitoneally, the onset of profound analgesia was not reached until 5– 10 min after the injection. so, when an infusion of dex was administered, a time interval was allowed for equilibrium of dex concentrations between the plasma and effect sites. so far, there have been only a few studies investigating the inhibiting effect of dex on the pain of propofol injection, and the question of its efficacy remains controversial. ayo�glu et al. (1) demonstrated that pretreatment with 0.25 mg/kg dex was not effective in reducing propofol injection pain (1). yet the research done by turan and his colleagues (2) contradicted this, showing that pretreatment with 0.25 mg/kg dex decreased propofol injection pain as effectively as pretreatment with lidocaine 0.50 mg/kg (2). our study demonstrated that the reduction of propofol injection pain through pretreatment with dex depended on the dex dose, and 0.25 or 0.5 mg/kg dex could not reduce the intensity and incidence of propofol injection pain. however, when the pretreatment dose of dex was increased to 1.0 mg/kg, the incidence rate of pain scores >2 decreased from 17/30 to 1/30. results of other studies demonstrating analgesic effects of dex are in accord with ours. for example, park et al. (28) demonstrated that dex had a dose-dependent analgesic effect in rat models, and ebert and colleagues (29) showed that increasing concentrations of dex in humans resulted in a progressive increase in analgesic effect. another finding in our study was that the interval between dex and propofol infusion influenced the analgesic effect of dex on propofol injection pain. dex was most effective when 1 mg/kg was injected 5 min before propofol injection. it is possible that, given this time interval, dex concentrations at the spinal level increased enough to result in an analgesic effect. pretreatment with dex has been reported to cause significant hemodynamic adverse side effects (30). however, a recent study showed that dex at doses of 0.5 mg/kg or 1 mg/kg can be safely used preoperatively, with stable hemodynamics (31). koroglu et al. (32)foundthatadministrationwithahighdoseofdex (a bolus of 2–3 mg/kg over 10 min, or infusion of 1.5– 3.0 mg�kg-1�h-1) provided adequate sedation in most of the children aged 1–7 years, without hemodynamic changes or adverse sequelae, and no specific treatment required. in our study, none of the patients who received dex 0.25, 0.5, or 1 mg/kg infusion developed bradycardia or hypotension. therefore, we believe 1 mg/kg dex is safe for the general population. in conclusion, 1 mg/kg dex given intravenously 5 min before administration of intravenous lct propofol (2.5 mg/kg) is an effective and safe way to reduce the intensity and incidence of propofol injection pain. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. ayo�glu h, altunkaya h, ozer y, yapakçi o, cukdar g, ozkoçak i. does dexmedetomidine reduce the injection pain due to propofol and rocuronium? eur j anaesthesiol. 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http://www.ncbi.nlm.nih.gov/pubmed/10320175?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/10320175?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/10320175?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/11573599?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/11573599?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/11573599?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20304348?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20304348?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20304348?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20304348?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/19542104?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/19542104?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/19542104?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22608582?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22608582?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22608582?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21406529?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21406529?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20676375?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20676375?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20676375?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17294989?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17294989?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17294989?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23347808?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23347808?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23347808?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15505442?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15505442?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15505442?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21685111?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21685111?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17646493?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17646493?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17646493?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23362890?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23362890?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23362890?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21953329?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21953329?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21953329?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23166426?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23166426?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21040379?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21040379?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21040379?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23198038?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23198038?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/10910487?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/10910487?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/10910487?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/19702517?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/19702517?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22335390?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22335390?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22335390?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15764627?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15764627?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15764627?dopt=abstract abstract introduction methods results discussion declaration of interest references upsala j med sci 88: 141-149, 1983 what do patients know about their digitalis? a comparison between two different areas in sweden kurt boman,' hans mollerberg' and jan-erik ogren3 from the 'department of internal medicine, 'central laboratory, and 'the pharmacy of skellefieii hospital, skellefteii, sweden abstract o u t of 1183 u n s e l e c t e d o u t p a t i e n t s i n s k e l l e f t e a and 620 i n u p s a l a , 200 p a t i e n t s from each p l a c e were s e l e c t e d a t random t o b e s e n t a q u e s t i o n n a i r e on t h e i r m e d i c a t i o n w i t h d i g i t a l i s . answers t o t h e q u e s t i o n n a i r e s were ob t a i n e d from 196 p a t i e n t s (98 p e r c e n t ) i n s k e l l e f t e d and from 163 p a t i e n t s i n upsala ( 8 2 p e r c e n t ) . about 8 5 p e r c e n t s t a t e d t h a t t h e y took t h e i r d i g o x i n a s p r e s c r i b e d o n c e a day. about 60 p e r c e n t knew c o r r e c t l y why d i g o x i n t r e a t ment was g i v e n and 2 0 p e r c e n t were u n c e r t a i n a s t o why t h e y t o o k d i g o x i n . about 4 5 p e r c e n t s t a t e d t h a t t h e y f e l t improved t h a n k s t o t h e d i g o x i n t h e r a p y . 55 p e r c e n t d i d n o t know a b o u t d i g i t a l i s s i d e e f f e c t s . about 50 p e r c e n t de n i e d h a v i n g r e c e i v e d any i n f o r m a t i o n a b o u t d i g i t a l i s and 50 p e r c e n t were un s a t i s f i e d w i t h t h e i n f o r m a t i o n t h e y had been g i v e n . only 1 5 p e r c e n t were con t e n t w i t h t h e i n f o r m a t i o n . methods f o r improving t h e i n f o r m a t i o n t o p a t i e n t s a r e p r o p o s e d . introduction s e v e r a l s t u d i e s h a v e d i s c l o s e d t h a t o u t p a t i e n t s know v e r y l i t t l e a b o u t t h e i r d r u g s and f i n d i t d i f f i c u l t t o comply w i t h t h e c o n d i t i o n s o f t h e p r e s c r i p t i o n ( 1 , 6 , 8 ) . due t o t h e n a r r o w t h e r a p e u t i c r a n g e , i t i s p a r t i c u l a r l y i m p o r t a n t t h a t t h e p r e s c r i b i n g o f d i g i t a l i s , o n e of t h e d r u g s most commonly p r e s c r i b e d f o r e l d e r l y p e o p l e , is a d h e r e d t o c l o s e l y . two b r i t i s h s t u d i e s ( 9 , l o ) showed t h a t a b o u t h a l f o f t h e o u t p a t i e n t s d i d n o t t a k e t h e i r doses of d i g i t a l i s e x a c t l y a s p r e s c r i b e d . the p u r p o s e o f t h i s s t u d y was t o f i n d o u t , what p a t i e n t s i n a s m a l l town ( s k e l l e f t e d ) knew a b o u t t h e i r d i g i t a l i s m e d i c a t i o n a s compared w i t h p a t i e n t s i n a u n i v e r s i t y c i t y ( u p s a l a ) . 141 patients during t h e t h r e e months, j u l y t o september 1978, 1345 p r e s c r i p t i o n s o f d i g o x i n ( l a n a c r i s t ( r ) , draco) were made up a t t h e pharmacy o f s k e l l e f t e . 5 h o s p i t a l and t h e pharmacy ( n o r d s t j a r n a n ) i n s k e l l e f t e . 5 . t h e s e p h a r m a c i e s s e r v e an a r e a w i t h 40 0 0 0 i n h a b i t a n t s . out o f t h e s e 1345 p r e s c r i p t i o n s 1183 c o u l d b e u s e d t o i d e n t i f y p a t i e n t s e l i g i b l e for t h e s t u d y . t h e r e were 678 f e m a l e s and 505 males. a random sample o f 250 p a t i e n t s was t a k e n from t h i s p o p u l a t i o n , and 200 o f t h e s e p a r t i c i p a t e d i n t h e s t u d y . the 50 e x t r a p a t i e n t s were u s e d i f some of t h e f i r s t 2 0 0 p a t i e n t s had moved, d i e d or c o u l d n o t be found. t h i s was t r u e f o r 1 0 p a t i e n t s . the 2 0 0 p a t i e n t s p a r t i c i p a t i n g i n t h e s t u d y were randomized i n t o t w o g r o u p s : t h e a g r o u p w i t h 56 f e m a l e s and 44 males and t h e b g r o u p w i t h 71 fe males and 29 males. i n t h e u p s a l a pharmacy (kronan) 671 p r e s c r i p t i o n s were h a n d l e d d u r i n g t h e same p e r i o d as i n s k e l l e f t e h . 6 2 0 o f t h e p r e s c r i p t i o n s were e l i g i b l e f o r t h e s t u d y . a random sample o f 3 0 0 p a t i e n t s was c h o s e n , and o u t o f t h e s e 200 were s e l e c t e d f o r t h e s t u d y . t h e r e were 1 0 0 p a t i e n t s k e p t i n r e s e r v e , 50 f o r e a c h a and b g r o u p . of t h e f i r s t 200 p a t i e n t s i n u p s a l a 76 had moved, d i e d or c o u l d n o t b e f o u n d , so 76 p a t i e n t s from t h e r e s e r v e s were added t o t h e material. the a g r o u p c o m p r i s e d 48 f e m a l e s and 52 males and t h e b g r o u p 57 f e m a l e s and 43 males. the a g e d i s t r i b u t i o n s o f t h e random s a m p l e s i n s k e l l e f t e : and u p s a l a are shown i n f i g u r e 1 . percent 0 n i m n ,n 0 j f i g 1 a. the a g e d i s t r i b u t i o n o f 2 0 0 p a t i e n t s (random s a m p l e ) on d i g o x i n i n s k e l l e f t e : . r e e i n yebr5 f i g 1 b. the a g e d i s t r i b u t i o n o f 200 p a t i e n t s (random s a m p l e ) on d i g o x i n i n u p s a l a . 142 the h e a l t h d i s t r i c t o f s k e l l e f t e d had 80 0 0 0 i n h a b i t a n t s i n 1981. t h e r e were 66 m e d i c a l p o s t s , 36 o f which were a t t a c h e d t o t h e h o s p i t a l , and 30 t o d i s t r i c t m e d i c a l o f f i c e r s . during t h e main p a r t o f 1978 o n l y s e v e n o u t o f 2 5 posts f o r d i s t r i c t m e d i c a l o f f i c e r s were p e r m a n e n t l y o c c u p i e d . the r e s t were a t t e n d e d t o by locums for s h o r t p e r i o d s . t h i s is a common s i t u a t i o n i n t h e n o r t h e r n p a r t o f sweden b u t n o t i n c e n t r a l sweden. so a comparison w a s made w i t h u p s a l a , where t h e r e were 594 p h y s i c i a n s , 459 o f whom worked a t t h e u n i v e r s i t y hospital, and 135 b e i n g d i s t r i c t m e d i c a l o f f i c e r s . the u p s a l a area had 1.3 m i l l i o n i n h a b i t a n t s . methods the p a r t i c i p a n t s were s e n t a q u e s t i o n n a i r e w i t h n i n e q u e s t i o n s a b o u t e.g. t h e d u r a t i o n o f d i g i t a l i s t r e a t m e n t , i t s d o s a g e and how t h e p a t i e n t s c o m p l i e d w i t h it. the p a t i e n t s were a l s o a s k e d i f t h e y knew why t h e y were t a k i n g d i g i t a l i s , i f t h e i r symptoms had improved f o l l o w i n g t r e a t m e n t w i t h d i g i t a l i s , and i f t h e y knew a n y t h i n g a b o u t p o s s i b l e s i d e e f f e c t s . t h e r e were q u e s t i o n s a b o u t t h e s o u r c e s o f t h e i r i n f o r m a t i o n on t h e d r u g and whether t h e y c o n s i d e r e d t h e i n f o r m a t i o n t o be a d e q u a t e . the n i n e q u e s t i o n s o f t h e q u e s t i o n n a i r e were i d e n t i c a l f o r a l l t h e p a t i e n t s b u t t h e a and b g r o u p were g i v e n d i f f e r e n t a d d i t i o n a l i n f o r m a t i o n t o f i n d o u t i f t h e l a n g u a g e u s e d i n t h e q u e s t i o n n a i r e c o u l d b i a s t h e a n s w e r s . the a g r o u p w a s t o l d : "it i s i m p o r t a n t n o t t o t a k e more o f t h e d r u g t h a n needed. t h i s i s e s p e c i a l l y t r u e for l a n a c r i s t ( r ) . i f you t a k e too much o f t h e d r u g , c e r t a i n u n p l e a s a n t s i d e e f f e c t s may a r i s e " . the b g r o u p was warned: " i t is i m p o r t a n t n o t t o t a k e too l i t t l e of t h e d r u g which i s needed f o r your t r e a t m e n t . t h i s i s e s p e c i a l l y t r u e f o r l a n a c r i s t ( r ) . you s h o u l d t a k e e x a c t l y t h e d o s e p r e s c r i b e d and n o t be careless". the p r e s c r i b e d d i g o x i n d o s a g e i n r e l a t i o n t o t h e a g e o f t h e p a t i e n t s was r e g i s t e r e d . p a t i e n t s i n b o t h u p s a l a and s k e l l e f t e d who f a i l e d t o r e p l y were r e q u e s t e d o n c e a g a i n t o answer t h e q u e s t i o n s . t h e u s e of p r e s c r i p t i o n s f o r i d e n t i f y i n g t h e p a r t i c i p a n t s w a s approved by s o c i a l s t y r e l s e n s l a k e m e d e l s a v d e l n i n g ( n a t i o n a l board o f h e a l t h and w e l f a r e , department o f d r u g s ) . results reply r a t e answers t o t h e q u e s t i o n n a i r e s were r e c e i v e d from 196 p a t i e n t s i n s k e l l e f t e d (98 p e r c e n t ) and from 163 p a t i e n t s (82 p e r c e n t ) i n u p s a l a . the d i f f e r e n c e i n t h e r e p l y r a t e s i s s t a t i s t i c a l l y s i g n i f i c a n t ( p < o . o o l ) . i n s k e l l e f t e 6 and u p s a l a a n s w e r s were r e c e i v e d from 97 and 7 7 p a t i e n t s r e s p e c t i v e l y i n g r o u p a , compared w i t h 9 9 and 86 p a t i e n t s r e s p e c t i v e l y i n g r o u p b. t h e r e were n o s i g n i f i c a n t d i f f e r e n c e s i n t h e a n s w e r i n g r a t e s between t h e a and b g r o u p n e i t h e r i n 143 s k e l l e f t e d (97, 9 9 ) n o r i n upsala ( 7 7 , 8 6 ) . i n s k e l l e f t e d t h e e x t r a i n f o r m a t i o n g i v e n seems t o h a v e b i a s e d t h e a n s w e r s , as t h e p a t i e n t s o f t h e b g r o u p who were reminded n o t t o be c a r e l e s s i n t a k i n g t h e i r m e d i c i n e , r e p o r t e d a s i g n i f i c a n t l y (p. 0.05) h i g h e r c o m p l i a n c e t h a n p a t i e n t s of t h e a group. a s i m i l a r t e n d e n c y was found i n u p s a l a , where none i n t h e b g r o u p b u t f i v e i n t h e a g r o u p a d m i t t e d t h a t t h e y had been careless. digoxin d o s a g e and a g e the mean d a i l y d o s a g e o f d i g o x i n c a l c u l a t e d from t h e r e s p e c t i v e 1152 and 619 p r e s c r i p t i o n s was 0.193 mg p e r d a y i n s k e l l e f t e d and 0.212 mg p e r d a y i n u p s a l a . t h i s i n d i c a t e s t h a t more p a t i e n t s t o o k 0.25 mg p e r d a y (57 p e r c e n t v e r s u s 50 p e r c e n t ) i n u p s a l a t h a n i n s k e l l e f t e i . the d o s e f e l l g r a d u a l l y w i t h i n c r e a s i n g a g e i n b o t h s k e l l e f t e i and u p s a l a ( f i g 2 ) s k e l l e f t e d 0.291 and u p s a l a 0.243 and a r e ( p c 0.001). * d i eux i n d k drily the c o r r e l a t i o n c o e f f i c i e n t were i n s i g n i f i c a n t l y d i f f e r e n t from z e r o gux i n rg drily . 3 . 2 f i g 2 a. the r e l a t i o n between t h e f i g 2 b. the r e l a t i o n between t h e a g e and d i g o x i n d o s a g e o f 1152 a g e and d i g o x i n d o s a g e o f 619 p a t i e n t s i n s k e l l e f t e 5 . the correp a t i e n t s i n upsala. the corre l a t i o n c o e f f i c i e n t r=0.291. l a t i o n c o e f f i c i e n t r=0.243. (*= the mean d i g o x i n d o s a g e f o r ( * = the mean d i g o x i n d o s a g e f o r e a c h f i v e y e a r a g e i n t e r v a l ) . e a c h f i v e y e a r a g e i n t e r v a l ) . r e s u l t s o f t h e q u e s t i o n n a i r e s ( p e r c e n t a g e o f 196 p a t i e n t s i n s k e l l e f t e d and 163 i n u p s a l a ) skelleftea q u e s t i o n 1 . n o answer n o t q u i t e s u r e more t h a n 5 y e a r s 2-5 y e a r s 1-2 y e a r s l e s s t h a n 1 y e a r upsala "how l o n g have you been t a k i n g l a n a c r i s t ? " 2 3 3 4 39 49 27 21 16 6 ( p 4 0 . 0 1 ) 1 1 1 have d i s c o n t i n u e d 2 100 10 100 144 skelleetea upsala q u e s t i o n 2. n o answer d o n o t know 0.13 mg 0.25 mg q u e s t i o n 3. n o answer 4 t a b l e t 1 t a b l e t 1 % t a b l e t 2 t a b l e t s another d o s a g e one p a t i e n t who had t a k e 1 2 t a b l e t s p e r q u e s t i o n 4. n o answer every d a y s k i p i t sometimes s k i p it a l w a y s only on demand have d i s c o n t i n u e d q u e s t i o n 5. n o answer n o t q u i t e s u r e headache i r r e g u l a r p u l s e insomnia ankle s w e l l i n g high b l o o d l i p i d s body p a i n "what i s t h e d o s e p e r t a b l e t ? " (it i s p r i n t e d o n t h e b o t t l e ) 0 4 4 6 50 100 7 2 34 5 1 100 " h o w many t a b l e t s do you t a k e e a c h t i m e ? " 1 1 91 4 1 2 100 8 2 83 4 3 0 1 0 0 m i s t a k e n n i t r o g l y c e r i n f o r l a n a c r i s t s t a t e d t h a t h e c o u l d day. " h o w o f t e n d o you r e a l l y t a k e l a n a c r i s t ? " 1 9 1 5 6 85 2 1 1 2 100 2 1 0 0 "for what c o m p l a i n t s do you t a k e lanacr i s t ? " 4 18 3 1 5 5 7 2 2 r e s p i r a t o r y d i s t r e s s 14 c h e s t p a i n 18 hyper t e n s i o n 3 h e a r t f a i l u r e 53 o t h e r s s u c h as h e a r t e n l a r g e m e n t , a f t e r m y o c a r d i a l i n f a r c t i o n 8 some p a t i e n t s g a v e s e v e r a l a n s w e r s . 9 1 7 1 9 1 9 2 1 1 6 14 44 12 ( p ( o . 0 1 ) 6 i r r e g u l a r p u l s e , a n k l e s w e l l i n g , r e s p i r a t o r y d i s t r e s s , and h e a r t f a i l u r e were r e g a r d e d a s t h e r i g h t i n d i c a t i o n s f o r d i g o x i n t r e a t m e n t . q u e s t i o n 6. "did you improve on l a n a c r i s t ? " n o answer 3 n o 4 y e s 49 d i f f i c u l t t o s a y 44 1 0 0 1 0 3 38 49 1 0 0 145 skelleetea upsala q u e s t i o n 7 . "what a r e t h e u n p l e a s a n t s i d e e f f e c t s o f l a n a c r i s t ? " n o t q u i t e s u r e headache nausea, v o m i t i n g d i a r r h o e a a n o r e x i a e v e r y t h i n g is l o o k i n g y e l l o w t i r e d n e s s r e s p i r a t o r y d i s t r e s s i r r e g u l a r p u l s e v e r t i g o i n s o m n i a ankle s w e l l i n g h e a r t b u r n o t h e r 5 7 3 6 2 5 5 3 4 1 2 3 6 1 1 0 4 4 8 4 3 1 5 some p a t i e n t s g a v e s e v e r a l answers. q u e s t i o n 8. "who or what h a s t o l d you what you know a b o u t l a n a c r i s t ? " n o answer nobody h a s t o l d m e r e l a t i v e s , f r i e n d s newspaper r a d i o or t e l e v i s i o n n u r s e doctor pharmacy or o t h e r s 4 5 4 4 5 2 2 31 1 1 2 (p(o.01) 50 2 5 0 2 3 3 3 some p a t i e n t s g a v e s e v e r a l answers. only 37 p e r c e n t of t h e p a t i e n t s i n s k e l l e f t e d who had been i n f o r m e d by t h e i r d o c t o r s , were s a t i s f i e d w i t h t h e i n f o r m a t i o n t h e y r e c e i v e d . q u e s t i o n 9. "is t h e i n f o r m a t i o n s u f f i c i e n t ? " n o answer 9 y e s 1 4 n o 51 26 100 d o n o t know 1 5 1 8 48 1 9 1 0 0 discussion the u n u s u a l l y h i g h r e p l y r a t e i n s k e l l e f t e a i n d i c a t e d t h a t t h e s e p a t i e n t s were i n t e r e s t e d i n t h e s t u d y . the s i g n i f i c a n t l y lower r e p l y r a t e i n u p s a l a may be b e c a u s e t h e s t u d y was r e m o t e l y c o n t r o l l e d from t h e d i s t r i c t o f s k e l l e f t e ; . moreover it was easier f o r t h e a u t h o r s t o r e a c h t h e p a t i e n t s i n s k e l l e f t e d , where many were known p e r s o n a l l y . s i g n i f i c a n t l y (p<o.ool) more p a t i e n t s i n u p s a l a were a l s o t a k e n from t h e r e s e r v e s . another d i s c r e p a n c y was t h a t s i g n i f i c a n t l y (p<o.o5) more women i n s k e l l e f t e 6 were randomly c h o s e n f o r t h e s t u d y . t h i s c o u l d p e r h a p s i n f l u e n c e t h e a n s w e r s as t h e r e m i g h t b e sex-dependent d i f f e r e n c e s i n m o r b i d i t y and m e d i c a t i o n . maybe t h e r e were o t h e r r e a s o n s f o r t h i s d i s c r e p a n c y i n r e p l y r a t e , s u g g e s t i n g t h a t c o n c l u s i o n s b a s e d o n t h e d i f f e r e n c e s between u p s a l a and s k e l l e f t e a s h o u l d be made c a u t i o u s l y . the r e s u l t s of t h e q u e s t i o n n a i r e a s a whole s h o u l d a l s o be i n t e r p r e t e d w i t h c a u t i o n , as t h e a n s w e r s c o u l d have been i n f l u e n c e d by t h e l a n g u a g e u s e d . however, t h e d o s e s and d o s a g e s o f d i g o x i n i s s u e d a g r e e d w e l l w i t h t h e r e s u l t s o f a n o t h e r s t u d y i n t h e d i s t r i c t of s k e l l e f t e d ( 5 ) . i n t e r e s t i n g l y , a b o u t 1 5 p e r c e n t o f p a t i e n t s i n s k e l l e f t e a answered " i r r e g u l a r p u l s e " , which p r o b a b l y meant a t r i a l f i b r i l l a t i o n , and c o r r e s p o n d e d f a i r l y w e l l t o t h e 2 0 p e r c e n t ecg-verified o c c u r r e n c e o f a t r i a l f i b r i l l a t i o n i n d i g o x i n t r e a t e d p a t i e n t s ( 5 ) . the lower d i g o x i n d o s a g e i n t h e e l d e r l y when compared w i t h t h e younger p a t i e n t s i n b o t h s k e l l e f t e d and u p s a l a p r o b a b l y r e f l e c t s t h e p h y s i c i a n s ' f e a r of c a u s i n g d i g i t a l i s i n t o x i c a t i o n i n t h e e l d e r l y . the a g e o f t h e p a t i e n t i s s a i d t o b e o n e o f t h e f a c t o r s i n f l u e n c i n g s e n s i t i v i t y t o d i g o x i n ( 7 ) , d u e t o d i m i n i s h e d r e n a l f u n c t i o n and l o w body weight.moreover, a m a j o r i t y o f d i g i t a l i s i n t o x i c a t e d g e r i a t r i c p a t i e n t s i n s k e l l e f t e a ( 4 ) had serum d i g o x i n l e v e l s w i t h i n or below t h e t h e r a p e u t i c r a n g e , s u g g e s t i n g an a g e d e p e n d e n t s e n s i t i v i t y t o d i g i t a l i s . one o f t h e few s i g n i f i c a n t d i f f e r e n c e s i n t h e a n s w e r s between s k e l l e f t e d and u p s a l a was t h e l a r g e number i n u p s a l a who t h o u g h t t h a t l a n a c r i s t w a s g i v e n f o r h y p e r t e n s i o n ( p < o . o l ) . the d i f f e r e n c e i s d i f f i c u l t t o e x p l a i n . i t c o u l d be a t t r i b u t e d t o c h a n c e or maybe t h e f a c t t h a t t h e i n d i c a t i o n f o r t r e a t m e n t was more o f t e n p r i n t e d on t h e p r e s c r i p t i o n s i n s k e l l e f t e a t h a n i n u p s a l a ( 7 0 p e r c e n t v e r s u s 4 0 p e r c e n t ) . o t h e r w i s e t h e s i m i l a r i t i e s i n t h e a n s w e r s t o t h e q u e s t i o n n a i r e between up s a l a and s k e l l e f t e d a r e o b v i o u s . l a c k o f knowledge or m i s u n d e r s t a n d i n g a s t o why t h e p a t i e n t s were t a k i n g d i g i t a l i s may have s e v e r a l c a u s e s . the i n f o r m a t i o n was n o t p r o v i d e d , it was n o t u n d e r s t o o d , it had been f o r g o t t e n , t h e d o c t o r p a t i e n t c o n t a c t was t o o p o o r , t h e d o c t o r s were changed too o f t e n , t h e p a t i e n t was h a r d o f h e a r i n g and s o on. i t was q u i t e o b v i o u s from q u e s t i o n s 9 and 1 0 t h a t many p a t i e n t s knew l i t t l e or n o t h i n g a b o u t t h e i r d i s e a s e or i t s t r e a t m e n t . they also knew l i t t l e a b o u t t h e p o s s i b l e d i g i t a l i s s i d e e f f e c t s . i n t o x i c a t i o n by d i g i t a l i s r e m a i n s a major c l i n i c a l problem. b e l l e r e t a 1 ( 2 ) found t h a t as many as 30 p e r c e n t o f t h e i n p a t i e n t s were s u f f e r i n g from d i g i t a l i s i n t o x i c a t i o n . of 9 1 o u t p a t i e n t s i n s k e l l e f t e a , 5 p e r c e n t were found t o be c e r t a i n l y i n t o x i c a t e d and 2 p e r c e n t p o s s i b l y so ( 5 ) . i n s k e l l e f t e d a b o u t 5 p e r c e n t knew a b o u t t h e a d v e r s e e f f e c t s o f d i g i t a l i s , b u t on t h e o t h e r hand j u s t as many l i s t e d symptoms t h a t c o u l d n o t be a s c r i b e d t o it. if p a t i e n t s e r r o n e o u s l y a t t r i b u t e a d v e r s e r e a c t i o n s t o t r e a t m e n t by d i g i t a l i s t h e y m i g h t d i s c o n t i n u e t h e d i g i t a l i s t h e r a p y . the o c c u r r e n c e o f i n t o x i c a t i o n by d i g i t a l i s c o u l d be r e d u c e d i f p a t i e n t s knew more a b o u t i t s s i d e e f f e c t s . they m i g h t t h e n e a r l i e r r e c o g n i s e t h e s i g n s o f i n t o x i c a t i o n and so r e p o r t e a r l i e r to t h e i r d o c t o r s . many p a t i e n t s found it d i f f i c u l t t o s t a t e i f t h e y had been improved by d i g i 147 t a l i s t r e a t m e n t . t h i s m i g h t have been b e c a u s e t h e t r e a t m e n t and/or t h e d i a g n o s is were wrong or d i u r e t i c t r e a t m e n t had made it d i f f i c u l t t o a s s e s s t h e e f f i c a c y o f d i g i t a l i s . t h i s a g r e e s w i t h t h e r e s u l t s o f o t h e r s t u d i e s showing t h a t many g e r i a t r i c p a t i e n t s a r e d i g i t a l i z e d u n n e c e s s a r i l y ( 3 ) . from t h i s s t u d y it i s o b v i o u s t h a t t h e i n f o r m a t i o n g i v e n t o or u n d e r s t o o d by p a t i e n t s i s i n s u f f i c i e n t . i t was n o t known how many p a t i e n t s were c o r r e c t l y i n f o r m e d , b u t f o r g o t or m i s u n d e r s t o o d t h e i n f o r m a t i o n g i v e n . i t i s i m p o r t a n t t o m a k e s u r e t h a t t h e p a t i e n t h a s u n d e r s t o o d t h e i n f o r m a t i o n and t o r e p e a t it, i n o r d e r t o a c h i e v e e f f e c t i v e m e d i c a t i o n w i t h d i g i t a l i s . one o f t h e most common r e a s o n s f o r a breakdown i n p a t i e n t d o c t o r communication i s p r o b a b l y f r e q u e n t c h a n g e s o f d o c t o r s . the p a t i e n t s were g e n e r a l l y informed by t h e i r d o c t o r s which i s b o t h n a t u r a l and d e s i r a b l e . a d d i t i o n a l i n f o r m a t i o n and e x p l a n a t i o n c o u l d b e g i v e n by n u r s e s t o b o t h o u t p a t i e n t s and i n p a t i e n t s . p h a r m a c i s t s may s u p p l e ment t h i s i n f o r m a t i o n t h r o u g h p r a c t i c a l a d v i c e a b o u t t h e s t o r a g e o f t h e d r u g , i t s s t a b i l i t y , t h e i m p o r t a n c e o f t a k i n g t h e correct d o s e s a t t h e correct t i m e and how t o t a k e it i n r e l a t i o n t o food. a n e c e s s a r y c o n d i t i o n f o r a w e l l f u n c t i o n i n g p a t i e n t i n f o r m a t i o n s y s t e m is t h a t t h e p a t i e n t a l w a y s meets t h e same d o c t o r f o r c o n s u l t a t i o n . another p r e r e q u i s i t e f o r good communication i s t h a t t h e d o c t o r u s e s a l a n g u a g e t h a t t h e p a t i e n t c a n u n d e r s t a n d . maybe o t h e r c h a n g e s i n t h e m e d i c a l c a r e a r e n e c e s s a r y . good c o n t i n u i t y i s also i m p o r t a n t when j u d g i n g t h e e f f i c a c y o f t h e d i g i t a l i s t r e a t m e n t , when a d e c i s i o n i s made t o d i s c o n t i n u e or c o n t i n u e a g i v e n t r e a t ment. i n t h i s way u n n e c e s s a r y d i g i t a l i s m e d i c a t i o n may b e a v o i d e d . guided by t h e r e s u l t s o f t h e p r e s e n t s t u d i e s w e hope t h a t t h e f o l l o w i n g s t e p s w i l l assist i n improving t h e p a t i e n t s knowledge o f t h e i r m e d i c a t i o n . when d i g o x i n t r e a t m e n t is s t a r t e d : t e l l t h e p a t i e n t t h e name o f t h e c a r d i a c g l u c o s i d e b e i n g u s e d and why t r e a t ment h a s t o be s t a r t e d . t e l l t h e p a t i e n t what symptoms w i l l p r o b a b l y b e r e l i e v e d by t h e t r e a t m e n t . s t a t e t h e d o s a g e , t h e l e n g t h o f t r e a t m e n t and t h e i m p o r t a n c e of t a k i n g t h e d o s e s e x a c t l y a s p r e s c r i b e d . make t h e p a t i e n t aware o f t h e a d v e r s e e f f e c t s which n e c e s s i t a t e s a new c o n s u l t a t i o n w i t h t h e d o c t o r . give a l l t h i s i n f o r m a t i o n t o t h e p a t i e n t i n w r i t i n g as w e l l a s v e r b a l l y . write on t h e p r e s c r i p t i o n t h e symptom(s) which t h e d r u g w i l l r e l i e v e or c u r e . record t h e i n d i c a t i o n f o r t h e t r e a t m e n t . a t t h e n e x t v i s i t : a s k f o r d e t a i l s o f any improvement i n h e a r t symptoms and a s k f o r any a d v e r s e e f f e c t s . a t e a c h v i s i t r e c o n s i d e r i f t h e d o s a g e s h o u l d be changed or d i g i t a l i s s h o u l d b e withdrawn. a s k t h e p a t i e n t i f h e / s h e u n d e r s t o o d t h e i n f o r m a t i o n and s u p p l e m e n t it i f n e c e s s a r y . 148 emphasize the importance of taking the medicine in the doses and at the intervals exactly as prescribed. many of these suggestions are familiar and obvious and are already followed widely. however, it appears to be very difficult to get information across to patients. if these proposals are followed, we hope that digitalis intoxications and unnecessary digitalis prescribing will decrease and that improved coope ration by patients will be achieved, particularly among those who need digi talis therapy. acknowledgements we would like to thank the national corporation of swedish pharmacies, pharmacist gunnar eriksson at the pharmacy (kronan) in upsala, and ass. prof. goran sundlof , department of internal medicine, akademiska sjuk huset, upsala, for valuable cooperations. r e f e r e n c e s 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. ander, s . & tibblin, s.: manniskors medicinska kunskaper. soc med tidskr hafte 1 9 7 4 ; 1 0 : 6 5 4 660. beller, g.a., smith, t.w., abelman, w.h., haker, e. & hood, w.b.: digitalis intoxication. a prospective clinical study with serum level correlations. n engl j med. 1 9 7 1 ; 284: 9 8 9 997. boman, k., allgulander, s. & skoglund, m.: is maintenance digoxin necessary in geriatric patients? acta med scand 1 9 8 1 ; 210: 4 9 3 495. boman, k.: digitalis intoxication in geriatric in-patients. acta med scand. accepted for publication. boman, k. & mollerberg, h.: digoxinintoxikation i ett oselekterat patientmaterial. lakartidningen 1 9 7 9 ; 76: 4 1 0 8 4 1 1 0 . bottiger, l.e.: kan patienten ratt skota sin medicinering? nord med 1969; 82: 1 6 0 5 1606. ewy, g.a., kapadia, g.g., yao, l., lullin, m. & marcus, f.i.: digoxin metabolism in the elderly. circulation 1 9 6 9 ; 3 9 : 4 4 9 453. hellstrom, k. & leijd, b.: vad vet patienter om sina sjukdomar och deras medikamentella behandling? lakartidningen 1 9 7 6 ; 7 3 : 9 6 8 9 7 0 . johnston, g.d. & mc devitt, d.g.: digoxin compliance in patients from general practice. brit j clin pharmacol 1 9 7 8 ; 3 3 9 343. johnston, g.d., kelly, j.g. & mc devitt, d.g.: do patients take digoxin? br heart j 1 9 7 8 ; 40: 1 7. adress for reprints: kurt boman, m.d. department of internal medicine skelleftez hospital 931 86 skelleftew sweden 149 perinatal outcome in children born after assisted reproductive technologies review article perinatal outcome in children born after assisted reproductive technologies ulla-britt wennerholma and christina berghb adepartment of obstetrics and gynaecology, institute of clinical sciences, sahlgrenska academy, gothenburg university, sahlgrenska university hospital east, gothenburg, sweden; bdepartment of obstetrics and gynaecology, institute of clinical sciences, sahlgrenska academy, gothenburg university, reproductive medicine, sahlgrenska university hospital, gothenburg, sweden abstract over the past 40 years access and effectiveness of assisted reproductive technologies (art) have increased, and to date more than 8 million children have been conceived after art globally. most pregnancies resulting from art are uncomplicated and result in the birth of healthy children. yet, it is well known that pregnancies following art are more likely to be affected by obstetric complications such as hypertensive disorders in pregnancy, preterm birth, and low birth weight compared with spontaneously conceived pregnancies. art children are also at increased risk of birth defects. the majority of the problems arise as a result of multiple pregnancies and can be reduced by transferring a single embryo, thereby avoiding multiple pregnancies. new art technologies are constantly introduced, and monitoring of the health of art children is crucial. article history received 25 january 2020 revised 3 february 2020 accepted 3 february 2020 keywords assisted reproductive technology; art; icsi; ivf; perinatal; short-term follow-up introduction infertility affects one in seven couples, and many of these need assisted reproductive technology (art). to date, more than 8 million children have been conceived after art globally (1), and up to 6% (range between 0.2% and 6.4%) of the european birth cohorts is conceived by art (2). art involves standard in vitro fertilisation (ivf) and intracytoplasmic sperm injection (icsi). icsi is the more advanced method, where a single sperm is injected into the cytoplasm of the oocyte. icsi was originally used for severe male-factor infertility, but nowadays it is also used to treat mild male-factor infertility, mixed infertility, unexplained infertility, and fertilisation failures. there has been an increasing global use of icsi, with 71.3% of fresh ivf/icsi cycles performed with icsi in europe in 2014, as shown in the latest reports from european society of human reproduction and embryology (eshre) (2). among fresh ivf cycles in the united states, icsi use increased from 36.4% in 1996 to 76.2% in 2012, with the largest relative increase among cycles without male-factor infertility (3). further, cryopreservation (freezing and thawing of embryos) has gained popularity. in europe, cryopreservation constituted 27.4% of all cycles in 2014, with the highest rate in switzerland, 41.1% (2). elective freezing of all good-quality embryos and transfer in subsequent cycles, i.e. elective frozen embryo transfer (efet), has recently been introduced as a way to reduce ovarian hyperstimulation syndrome and improve reproductive outcome (4). while most births after art are uncomplicated, art is associated with potential adverse obstetric outcomes for both mothers and infants, including hypertensive disorders of pregnancy, preterm delivery, and low birth weight (5,6). art has also been associated with an increased risk of birth defects (7,8). many of these adverse outcomes can be attributed to a higher rate of multiple pregnancies after art (2,9). with the increasing use of single embryo transfer, the multiple pregnancy rate has been significantly reduced but is still unacceptably high in many countries. in 2014, eshre reported a multiple birth rate of 17.5% in art in europe (range 4.3% to 30.6%) (2). in 2016, in the us, 31.5% of artconceived infants were born after multiple birth pregnancies compared with 3.4% of all infants in the general population (9). however, most data also show that art singletons have a more compromised perinatal outcome compared to singletons born after spontaneous conception, e.g. higher rates of preterm birth and low birth weight (10). new art technologies are continuously being introduced, and it is important to monitor the safety of art and the health of art offspring. the aim of this narrative review is to give a summary of the current literature on perinatal outcomes in singletons born after art, including ivf, icsi, freezing/thawing, oocyte donation (od), and more recent methods such as blastocyst culture and vitrification. the review is based on recent systematic reviews (srs) and large cohort studies. in case of rare outcomes, cohort studies with limited numbers of children have been included. studies on perinatal outcomes after medically assisted reproduction including intrauterine insemination, preimplantation diagnostics, preimplantation genetic testing for aneuploidy, and oocyte vitrification have been excluded. contact ulla-britt wennerholm ulla-britt.wennerholm@vgregion.se department of obstetrics and gynaecology, institute of clinical sciences, sahlgrenska academy, gothenburg university, sahlgrenska university hospital east, gothenburg se 416 85, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 2, 158–166 https://doi.org/10.1080/03009734.2020.1726534 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1726534&domain=pdf&date_stamp=2020-05-21 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1726534 http://www.tandfonline.com perinatal outcome ivf/icsi versus general population several srs and large cohort studies have analysed perinatal outcomes in art singletons versus singletons from the general population (6,11–17). most studies have included children born after ivf and icsi as well as fresh and frozen-thawed embryo transfer (fet). the latest sr and meta-analysis from 2017 included more than 180,000 art singleton pregnancies from 52 cohort studies from all over the world (6). results from the srs consistently show that singletons born after art have more adverse perinatal outcomes compared with singletons born after spontaneous conception, even after adjustment for relevant confounders (table 1). art singletons have significantly higher rates of preterm birth, with adjusted risks in the range of 1.4–2.0 for preterm birth and 1.7–3.1 for very preterm birth. preterm birth is a syndrome with multifactorial aetiology. art pregnancies have more placental complications (i.e. hypertensive disorders of pregnancy, placenta previa, and placental abruption) which entail an increased risk for indicated preterm birth (18–20). an increased risk of spontaneous preterm birth (<37 weeks [10.1% versus 5.5%] and <34 weeks [3.6% versus 2.2%]) in art versus spontaneous conception has recently been reported in a sr and metaanalysis (21). analogously, art singletons have higher rates of low birth weight (adjusted risks 1.6–1.7) and very low birth weight (adjusted risks 1.8–3.0) compared with spontaneous conception (6,11–15,17) (table 1). most studies also show an increased risk for art singletons being born small for gestational age (sga), with adjusted risks around 1.5, and increased risks of perinatal mortality, with adjusted risks between 1.7 and 2.0. an australian study found that art singletons had a 2-fold increased risk of stillbirth (17). a large nordic collaborative study from the committee of nordic art and safety (conartas) including 62,485 art singletons found an increased risk for stillbirth only before 28 weeks (adjusted risk 2.0) (16). sibling studies where the same mother has given birth to both an art and a spontaneously conceived singleton is a way to examine art methods per se under the assumption that maternal factors (except maternal age) remain constant across pregnancies. in a meta-analysis by pinborg et al., the risk of preterm birth in a singleton sibling born after art was higher than in a singleton sibling born after spontaneous conception (aor 1.27, 95% ci 1.08–1.49) (22). in summary, it is well documented that art singletons have an increased risk for preterm birth and low birth weight compared with singletons born after spontaneous conception. both parental characteristics, such as age and underlying subfertility, and the art technique per se may contribute to the more adverse outcome in art children. ivf versus icsi when comparing icsi with standard ivf, most large studies have found similar or lower risks of preterm birth, low birth weight, and peri/neonatal mortality in singletons born after icsi. pinborg et al. analysed singletons born after icsi (fresh or frozen/thawed cycles) versus singletons born after ivf (fresh or frozen/thawed cycles) (22). five studies were included in a meta-analysis on preterm birth. the pooled estimate for icsi singletons versus ivf singletons showed a lower risk of preterm birth in icsi singletons (aor 0.80, 95% ci 0.69–0.93). a possible explanation for the better outcome in icsi singletons may be that in icsi the majority of the women are reproductively healthy, which could give a more favourable perinatal outcome. in summary, children born after icsi have a better perinatal outcome compared with standard ivf. transfer of blastocysts versus transfer of cleavage stage embryos blastocyst culture (day 5–6) compared with cleavage stage culture (day 2–3) is considered to improve the selection of the most viable embryo and to increase pregnancy and live birth rates per transfer and potentially result in more healthy infants (23). yet, srs and meta-analyses show that the cumulative live birth rate, including a fresh transfer and all subsequent frozen embryo transfers from one oocyte retrieval, is similar for blastocyst transfer and cleavage stage transfer (23,24). however, blastocyst culture, by improving embryo selection, may encourage elective single embryo transfer and thus reduce multiple birth rates. two recent systematic reviews and meta-analyses on perinatal outcomes after blastocyst transfer versus cleavage table 1. perinatal outcome in singletons born after art versus spontaneous conception. results from systematic reviews and meta-analyses. author, year of publication (ref.) helmerhorst et al., 2004 (11) jackson et al., 2004 (12) mcgovern et al., 2004 (13) mcdonald et al., 2009 (14) pandey et al., 2012 (15) qin et al., 2017 (6) rr (95% ci) or (95% ci) rr (95% ci) rr (95% ci) rr (95% ci) art vs sc, % no. of studies (no. of art children) 14 (5361) 14 (12,283) 27 (14,748) 15 (31,032) 22 (27,819) 52 (181,741) preterm birth <37 weeks 2.04 (1.80 � 2.32) 1.95 (1.73 � 2.20) 1.98 (1.77 � 2.22) 1.84 (1.54 � 2.21) 1.54 (1.47 � 1.62) 10.9 vs 6.4� <32 weeks 3.27 (2.03 � 5.28) 3.10 (2.00 � 4.80) 2.49 (0.86 � 7.21) 2.27 (1.73 � 2.97) 1.68 (1.48 � 1.91) 2.4 vs 1.2� low birth weight <2500 g 1.70 (1.50 � 1.92) 1.77 (1.40 � 2.22) – 1.60 (1.29 � 1.98) 1.65 (1.56 � 1.75) 8.7 vs 5.8� <1500 g 3.00 (2.07 � 4.36) 2.70 (2.31 � 3.14) – 2.65 (1.83 � 3.84) 1.93 (1.72 � 2.17) 2.0 vs 1.0� small for gestational age 1.40 (1.15 � 1.71) 1.60 (1.25 � 2.04) – 1.45 (1.04 � 2.00) 1.39 (1.27 � 1.53) 7.1 vs 5.7� perinatal mortality 1.68 (1.11 � 2.55) 2.19 (1.61 � 2.98) – – 1.87 (1.48 � 2.37) 1.1 vs 0.6� �p ¼ 0.000. art: assisted reproductive medicine; ci: confidence interval; or: odds ratio; rr: relative risk. upsala journal of medical sciences 159 transfer included more than 100,000 singletons born after fresh cycles (25,26). both found a higher rate of preterm birth (<37 weeks; relative risks [rrs] 1.15 and 1.16, respectively) and very preterm birth (<32 weeks; rrs 1.16 and 1.16, respectively) and a lower rate of being sga (rrs 0.83 and 0.84, respectively) after blastocyst transfer compared with cleavage stage transfer. there was no difference with regard to low birth weight. abnormal placentation and implantation may cause the increased risk of preterm birth after blastocyst transfer. a population-based registry study found increased rates of placenta previa and placental abruption after blastocyst transfer (27). blastocyst transfer is associated with a higher risk for monozygotic twinning (mzt) (28–30). in a sr and meta-analysis of 38 studies, rates of mzt after blastocyst transfer varied between 0% and 13.3%, and there was a 2-fold increased risk compared with cleavage transfer (or 2.18, 95% ci 1.93–2.48) (29). the authors suggest—besides extended culture time—culture media and the age of the mother as the underlying mechanisms (29). further, blastocyst transfer is associated with a higher male-to-female ratio (28,31–33). in summary, blastocyst transfer compared with cleavage transfer is associated with a small increased risk of adverse perinatal outcomes, particularly preterm birth. further, a higher rate of mzt and an altered sex-ratio have been observed after blastocyst transfer. fresh versus frozen/thawed embryo transfer several srs and meta-analyses have observed that perinatal outcomes are better in children conceived following frozen/ thawed embryo transfer (fet) compared with fresh embryo transfers, with reduced risks of preterm birth and low birth weight (22,34–36). maheshwari et al. performed an updated sr and metaanalysis (26 studies and almost 300,000 deliveries) and confirmed that singletons conceived from fet were at lower risk of preterm birth (rr 0.90, 95% ci 0.84–0.97), low birth weight (rr 0.72, 95% ci 0.67–0.77), and sga (rr 0.61, 95% ci 0.56–0.67) compared with those conceived from fresh embryo transfers (37). yet, they also found that singletons born after fet had an increased risk of being born large for gestational age (lga) (rr 1.54, 95% ci 1.48–1.61) and being macrosomic (birth weight more than 4000 g) (rr 1.85, 95% ci 1.46–2.33). there was no difference in the risk of perinatal mortality in children born after fet versus children born after fresh embryo transfer, but the risk of hypertensive disorders of pregnancy was increased (rr 1.29, 95% ci 1.07–1.56) in pregnancies after fet. theories of the underlying mechanisms behind this overgrowth include a selection of better embryos surviving the freezing and thawing procedure. further, epigenetic modifications may occur at the early embryonic stages and affect the growth potential of the foetus. a third suggested theory is that the uterine environment in a fet cycle is more natural than in fresh ivf, as most fet cycles do not use the ovarian stimulation which is used in fresh ivf cycles. recent studies have shown a link between the absence of corpus luteum and a higher risk of pre-eclampsia (38,39). the obstetric outcome after fet depending on protocol used has recently been investigated (40). programmed cycles (no corpus luteum, n ¼ 1446) were associated with higher rates of hypertensive disorders in pregnancy, postpartum haemorrhage, postterm birth, and macrosomia compared with natural cycles (n ¼ 6297). there were no differences regarding preterm birth and low birth weight. the results support the hypothesis of a link between absence of corpus luteum in programmed cycles and adverse perinatal outcomes. with the increasing number of art cycles worldwide performed as fet, this finding is important and may support the use of natural cycles in fet. a recent sr (11 randomised controlled studies) evaluated perinatal outcome after efet versus fresh embryo transfers (5379 women) (4). there were higher risks of pre-eclampsia after efet (rr 1.79, 95% ci 1.03–3.09), whereas mean birth weight, preterm birth, or birth defects were the same. concerning safety, efet significantly decreased the risk of moderate and severe ovarian hyperstimulation syndrome. vitrification versus slow freezing vitrification is an ultrarapid cryopreservation method, which instead of slow freezing has become the dominant method for cryopreservation in recent years. it has been associated with higher post-thaw survival rates and higher clinical pregnancy rates when compared with slow freezing (41,42). however, the high concentrations of cryoprotectants used for vitrification have raised concerns about possible negative health effects for the children. when comparing vitrification and slow freezing of day-3 embryos (43) or blastocysts (44), similar outcomes were found. a nordic study compared vitrified blastocyst transfers with slow freezing day-2–3 embryo transfer (45). except for a higher risk of preterm birth in the vitrified blastocyst group (aor 1.33, 95% ci 1.09–1.62), there were no other differences. the higher risk of preterm birth was considered to be related to extended culture. in summary, the change of strategy from slow freezing to vitrification seems to be reassuring. oocyte donation (od) the number of od treatments has increased during recent years, and a total of 17,259 deliveries after od was reported in 2014 from eshre (2), which considerably exceeds the number of deliveries reported in 2013 (þ45.5%); 65% of the recipients were 40 years or older. in a recent sr including 23 studies, rates of hypertensive disorders in pregnancy (including pregnancy-induced hypertension, pre-eclampsia, severe pre-eclampsia), preterm birth, very preterm birth, low birth weight, and very low birthweight were increased after od compared with ivf/icsi with autologous oocytes (pooled odds ratio 2.64, 1.57, 1.80, 1.25, and 1.37, respectively) (46). the high occurrence of obstetric complications after od has been associated with advanced maternal age. yet, also in young women, aged <35 years, the use of donated oocytes 160 u.-b. wennerholm and c. bergh compared with autologous oocytes was associated with a higher rate of preterm birth and low birth weight (47). in the sr by moreno-sepulveda et al., there was no difference in the rate of preterm birth and low birth weight when adjusted for pre-eclampsia (46). the fact that the foetus is allogenic to the mother’s immunological mechanisms may explain the higher risk of pre-eclampsia in od pregnancies. there was a lower prevalence of pre-eclampsia in od pregnancies when the donor was related to the recipient (48). since od pregnancies have nearly three times the risk of pre-eclampsia in comparison to spontaneous pregnancies, od pregnancies should be considered as high-risk pregnancies, and single embryo transfer is highly recommended, as multiple pregnancies further add to the perinatal risks (49). in summary, od may constitute an independent risk factor for a more adverse perinatal and maternal outcome than pregnancies after art with autologous oocytes. birth defects art versus general population large registry-based cohort studies and srs with meta-analyses have assessed birth defects in art singletons compared with spontaneously conceived singletons. most studies have found an increased rate of birth defects in art children, ranging between 30% and 70% (table 2) (6,7,14,15,50–52). the latest review included 22 studies (40,746 art singletons), and the point estimate for any birth defect was rr 1.41 (95% ci 1.38–1.52) (52). the risk of any birth defects (rr 1.36) and major birth defects (rr 1.41) for art versus spontaneous conception was similar in the sr by hansen et al. (7). a nordic cohort study from the conartas comparing art singletons (n ¼ 62,379) with singletons born after spontaneous conception (n ¼ 362,215) observed an increased risk for major birth defects (3.4% versus 2.9%; aor 1.14, 95% ci 1.08–1.20) (53). increased rates of birth defects occurred in different organ systems: central nervous system; eye; ear, face, and neck; heart; gastrointestinal system; urinary system; and the musculo-skeletal system, with congenital heart defects being the most common defects. a sr of congenital heart defects in art versus spontaneous conceptions (5 studies, 13,396 art singletons) showed an increased risk of congenital heart defects in art singletons: 1.0% versus 0.7% (or 1.55, 95% ci 1.21–1.99) (54). ivf versus icsi neither lie et al. (4 studies) nor wen et al. (24 studies) in their meta-analyses including both singletons and multiples found any increase in the risk of birth defects in icsi compared with standard ivf (rr 1.12, 95% ci 0.97–1.28; and rr 1.05, 95% ci 0.91–1.20, respectively) (55,56). in contrast, an australian study of 6163 art children (singletons and multiples) found that ivf was associated with a reduced risk of any birth defect as compared with icsi (aor 0.68, 95% ci 0.53–0.87) (57). the risk was reduced for fresh cycles but not ta b le 2. bi rt h d ef ec ts in si n g le to n s b or n af te r a rt ve rs us sp on ta n eo us co n ce p ti on . re su lt s fr om sy st em at ic re vi ew s an d m et aan al ys es . a ut h or , ye ar of p ub lic at io n (r ef .) ri m m et al ., 20 04 (5 1) h an se n et al ., 20 05 (5 0) m cd on al d et al ., 20 05 (1 4) pa n d ey et al ., 20 12 (1 5) h an se n et al ., 20 13 (7 ) q in et al ., 20 17 (6 ) a rt vs sc , % zh ao et al ., 20 20 (5 2) n o. of st ud ie s (n o. a rt ch ild re n ) iv f: 8 (2 06 4) ic si : 6 (3 94 8) 15 (1 3, 05 9) 7 (4 03 1) 7 (4 38 2) 23 (4 8, 94 4) 29 (7 7, 63 0) 22 (4 0, 74 6) bi rt h d ef ec ts : p oo le d es ti m at es (9 5% c i) iv f: 1. 51 (0 .8 5 � 2. 70 ) ic si : 1. 33 (0 .9 0 � 1. 95 ) 1. 31 (1 .1 7 � 1. 46 ) 1. 41 (1 .0 5 � 1. 88 ) 1. 67 (1 .3 3 � 2. 09 ) a n y: 1. 36 (1 .3 0 � 1. 43 ) m aj or : 1. 41 (1 .3 3 � 1. 50 ) 5. 7 vs 3. 9a 1. 41 (1 .3 0 � 1. 52 ) a p ¼ 0. 00 0. a rt : as si st ed re p ro d uc ti ve te ch n ol og y; c i: co n fid en ce in te rv al ; ic si : in tr ac yt op la sm ic sp er m in je ct io n ; iv f: in vi tr o fe rt ili za ti on ; sc : sp on ta n eo us co n ce p ti on . upsala journal of medical sciences 161 for frozen cycles. an overall higher rate of urogenital defects in icsi versus ivf was found in a sr by massaro et al. (58). icsi with ejaculated versus non-ejaculated sperm there seems to be no difference in the rate of birth defects in children conceived by icsi using non-ejaculated sperm compared with icsi using ejaculated sperm (59–61). the rate of major birth defects in children conceived where the fathers had non-obstructive azoospermia, obstructive azoospermia, and aspermia (n ¼ 359 children) was assessed in a nationwide norwegian study (62). birth defects were not significantly associated with sperm origin or the cause of malefactor infertility. fresh versus frozen/thawed embryo transfer the rate of birth defects in children born after fet from slow freezing procedures seems comparable to that of art children born after fresh cycles (34,63,64). two recent srs including singletons born after fet from vitrification of slow freezing found no difference in the risk of birth defects between fet and fresh cycles. (26,37). a single-centre belgian study reported that the rate of any or major birth defects in singletons born after vitrification (n ¼ 827) was similar to that after fresh cycles (n ¼ 1374) (65). transfer of blastocysts versus transfer of cleavage stage embryos earlier studies have claimed that blastocyst transfer is associated with an increased risk of birth defects compared with cleavage stage (32,66). two recent reviews and one cohort study assessed birth defects after blastocyst transfer and found no increased risk compared with cleavage stage transfer, irrespective of whether any cryopreservation procedure had been used (25,27,67). in summary, art is associated with a modestly increased risk of birth defects, when compared with spontaneous conception. there seems to be no increased risk after cryopreservation, while the risk after icsi is still unresolved. on an individual level the increase in birth defects is small. chromosomal anomalies early studies form the belgian group have shown a higher rate of de novo, non-inherited chromosomal abnormalities in icsi children (n ¼ 1586) compared with the rate in the general population (1.6% versus 0.5%) (68). this was related mainly to a higher number of sex chromosomal anomalies and partly to a higher number of autosomal structural anomalies. the finding was associated with sperm concentration and motility. in children born to fathers with reduced sperm concentration the incidence of de novo abnormalities was higher compared with children born to fathers with a normal sperm concentration (2.1% versus 0.24%) (68). the incidence of de novo chromosomal anomalies was comparable in children conceived from non-ejaculated sperm (testicular n ¼ 530, epididymal n ¼ 194) versus ejaculated sperm (n ¼ 2516) (69). in summary, based on few studies, icsi may be related to a modestly increased risk of chromosomal abnormalities associated with sperm parameters. trends over time a nordic study from the conartas group analysed trends in perinatal health in art singletons (n ¼ 62,379) versus a control group of spontaneously conceived singletons (n ¼ 362,215) (70). the rates of perinatal outcomes were stratified into four time periods: 1988–1992, 1993–1997, 1998–2002, and 2003–2007. there was a substantial decline in the risk of being born preterm and very preterm for singletons conceived after art but not for singletons born after spontaneous conception. rates of low birth weight, stillbirth, and infant deaths also declined among art singletons. a possible explanation for the positive development may be a change in the art population, with healthier women with shorter time of infertility undergoing art treatment. other factors are increased use of icsi for male-factor infertility, cryopreservation, and single embryo transfer. single embryo transfer reduces the risk of the vanishing twin phenomenon, a risk factor for preterm birth (71). another study from the conartas group assessed the risk of major birth defects and the risk over time between 1988 and 2007 in art singletons compared with spontaneously conceived singletons (53). the rate of children born with a major birth defect increased in both groups over time, but the difference in risk of a major birth defect between art children and spontaneously conceived children remained unchanged. in summary, singletons born after art have a higher risk for adverse perinatal outcomes compared with singletons born after spontaneous conception. there is a positive trend with improved outcomes, mainly for rates of preterm birth during recent years. comments the conclusion from numerous studies is that art is a safe and successful treatment for infertility. further, perinatal outcomes have improved over time. the increased use of single embryo transfer (set), thus avoiding multiple pregnancies, is the main contributor to the better outcome seen during recent years. nordic countries, sweden in particular, have been the leading countries in reduction of multiple pregnancies by implementing set as the main strategy (72). several studies have shown that perinatal outcome is better in art singletons compared with art multiples including twins (73–75). yet, there is a modestly increased risk of adverse perinatal outcomes including birth defects in art singletons compared with the general population. whether this is attributable to patient characteristics related to infertility or the art technique is uncertain. art children have mostly been compared with children in the general population born after spontaneous conception. some studies, however, 162 u.-b. wennerholm and c. bergh included only spontaneous conceptions in a subfertile population as controls, while others have excluded the subfertile population. subfertility with or without assisted conception is found to be significantly associated with a higher rate of adverse perinatal outcomes and increased risk of birth defects (22,57,76). patients with infertility may be older and more likely to have pre-existing comorbidity, which may predispose to adverse perinatal outcomes. most outcome data come from retrospective observational studies with a wide heterogeneity and differences in control groups, and, as in all observational studies, there may be unknown and unmeasured confounders. there are several pitfalls and methodological limitations when birth defects are studied. the definition of birth defects, mode and frequency of preand postnatal surveillance, length of follow-up, inclusion or exclusion of pregnancy terminations for birth defects, source of data, etc. may differ between art pregnancies and spontaneous conception. most early studies included small sample sizes with inadequate power to assess differences in rates of birth defects. the increasing and often unnecessary use of icsi worldwide is a matter of concern, as there are still conflicting results concerning the risk of birth defects. therefore, until further research can demonstrate safety, icsi should mainly be reserved for its original intended use, male-factor infertility. caution should be applied about embarking on a policy of electively freezing all embryos in art as there seems to be increased risks for hypertensive disorders of pregnancy, macrosomia, and lga babies after fet. the implications of the findings of macrosomia and lga after fet, and the consequences for future health and risk of obesity in offspring, are unclear. therefore, efet should be used in specific cases such as high risk of ovarian hyperstimulation syndrome, fertility preservation, or in the context of randomised trials. concerning management during pregnancy, closer surveillance during pregnancy and prophylactic treatment for preeclampsia with low-dose aspirin may be indicated in highrisk pregnancies such as pregnancies after od (77). the higher risk of spontaneous preterm birth may indicate a benefit of screening with transvaginal ultrasound measurements of cervical length in the second trimester and subsequent treatment with progesterone if the cervix is short (78). continuous supervision after art is needed to ensure safety and quality, especially when new techniques are introduced. national art registries such as those existing in the nordic countries enable follow-up studies of art children and should be encouraged. furthermore, research collaborations between countries should be supported as well (79). disclosure statement no potential conflict of interest was reported by the author(s). funding the study was financed by grants from the swedish state under the agreement between the swedish government and the county councils, the alf-agreement [lua/alf 70940], and the hjalmar svensson research foundation. notes on contributors ulla-britt wennerholm is associated professor in obstetrics and gynaecology at sahlgrenska university hospital. christina bergh is professor in reproductive medicine at sahlgrenska university hospital. references 1. adamsson g, dyer s, 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birth and adverse perinatal outcomes in singleton gestations with a short cervix: a meta-analysis of individual patient data. am j obstet gynecol. 2018;218:161–80. doi:10.1016/j.ajog.2017.11.576 79. opdahl s, henningsen aa, bergh c, gissler m, romundstad lb, petzold m, et al. data resource profile: the committee of nordic assisted reproductive technology and safety (conartas) cohort. int j epidemiol. 2019; doi:10.1093/ije/dyz228 166 u.-b. wennerholm and c. bergh https://doi.org/10.1136/bmj.38919.495718.ae https://doi.org/10.1016/j.ajog.2017.11.576 https://doi.org/10.1093/ije/dyz228 abstract introduction perinatal outcome ivf/icsi versus general population ivf versus icsi transfer of blastocysts versus transfer of cleavage stage embryos fresh versus frozen/thawed embryo transfer vitrification versus slow freezing oocyte donation (od) birth defects art versus general population ivf versus icsi icsi with ejaculated versus non-ejaculated sperm fresh versus frozen/thawed embryo transfer transfer of blastocysts versus transfer of cleavage stage embryos chromosomal anomalies trends over time comments disclosure statement references introduction of the uppsala traumatic brain injury register for regular surveillance of patient char full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 introduction of the uppsala traumatic brain injury register for regular surveillance of patient characteristics and neurointensive care management including secondary insult quantification and clinical outcome lena nyholm, tim howells, per enblad & anders lewén to cite this article: lena nyholm, tim howells, per enblad & anders lewén (2013) introduction of the uppsala traumatic brain injury register for regular surveillance of patient characteristics and neurointensive care management including secondary insult quantification and clinical outcome, upsala journal of medical sciences, 118:3, 169-180, doi: 10.3109/03009734.2013.806616 to link to this article: https://doi.org/10.3109/03009734.2013.806616 © informa healthcare view supplementary material published online: 10 jul 2013. submit your article to this journal article views: 432 view related articles citing articles: 15 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.806616 https://doi.org/10.3109/03009734.2013.806616 https://www.tandfonline.com/doi/suppl/10.3109/03009734.2013.806616 https://www.tandfonline.com/doi/suppl/10.3109/03009734.2013.806616 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.806616 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.806616 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.806616#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.806616#tabmodule upsala journal of medical sciences. 2013; 118: 169–180 original article introduction of the uppsala traumatic brain injury register for regular surveillance of patient characteristics and neurointensive care management including secondary insult quantification and clinical outcome lena nyholm, tim howells, per enblad & anders lewén department of neuroscience, neurosurgery, uppsala university, uppsala, sweden abstract background. to improve neurointensive care (nic) and outcome for traumatic brain injury (tbi) patients it is crucial to define and monitor indexes of the quality of patient care. with this purpose we established the web-based uppsala tbi register in 2008. in this study we will describe and analyze the data collected during the first three years of this project. methods. data from the medical charts were organized in three columns containing: 1) admission data; 2) data from the nic period including neurosurgery, type of monitoring, treatment, complications, neurological condition at discharge, and the amount of secondary insults; 3) outcome six months after injury. indexes of the quality of care implemented include: 1) index of improvement; 2) index of change; 3) the percentages of ‘talk and die’ and ‘talk and deteriorate’ patients. results. altogether 314 patients were included 2008–2010: 66 women and 248 men aged 0–86 years. automatic reports showed that the proportion of patients improving during nic varied between 80% and 60%. the percentage of deteriorated patients was less than 10%. the percentage of talk and die/talk and deteriorate cases was <1%. the mean glasgow coma score (motor) improved from 5.04 to 5.68 during the nic unit stay. the occurrences of secondary insults were less than 5% of good monitoring time for intracranial pressure (icp) >25 mmhg, cerebral perfusion pressure (cpp) <50 mmhg, and systolic blood pressure <100 mmhg. favorable outcome was achieved by 64% of adults. conclusion. the uppsala tbi register enables the routine monitoring of nic quality indexes. key words: database, neurointensive care, outcome, quality register, secondary insults, traumatic brain injury introduction quality of treatment and care of patients with traumatic brain injury (tbi) is traditionally measured in clinical outcome and related to patient characteristics. there are many other factors that affect clinical outcome, e.g. the amount of secondary insults (high intracranial pressure (icp), hypotension, fever, etc.), types of treatment, and the occurrence of complications during neurointensive care (nic). these factors, which are related to nic, have so far not been followed regularly for quality assurance of the management of tbi. during the last decades of the twentieth century, the development of nic contributed to substantially improved results (1).to improve further the care and outcome for these patients it is crucial to monitor quality parameters routinely in the nic unit. such parameters include monitoring time spent over/ under predefined secondary insult levels (e.g. proportion of time with icp >25 mmhg) and many other measures. this was the purpose for us establishing the uppsala tbi register, an internet (world wide web)based quality register for traumatic brain-injured patients treated at the nic unit in uppsala, sweden. the quality register was established in 2008 in collaboration with uppsala clinical research center (ucr, www.ucr.uu.se, uppsala university). the objectives behind the uppsala tbi register were to be able correspondence: anders lewén, md, phd, department of neuroscience, neurosurgery, uppsala university, uppsala university hospital, se-751 85 uppsala, sweden. fax: +46 18 558617. e-mail: anders.lewen@neuro.uu.se (received 22 december 2012; accepted 15 may 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.806616 http://informahealthcare.com/journal/ups http://www.ucr.uu.se mailto:anders.lewen@neuro.uu.se regularly to: 1) obtain information about demographic data, clinical outcome, and how treatments and care affected outcome; 2) identify patients who did not have the expected result or the right treatment and why that happened; 3) provide data and select patients for research studies. the specific purpose of this paper is to present the design of the quality register including definitions and to demonstrate the functionality by reporting the first results from the register. material and methods standardized management protocol system and treatment goals routines and treatment goals at the nic unit in uppsala are described in a standardized management protocol system which is based on good laboratory practice (glp) principles (2). in the system it is, for example, declared that patients with tbi who are not responding to commands should be intubated and icp should be monitored. these rules represent two management principles relevant for quality assurance of the nic. the treatment and care of these patients are focused on avoiding secondary insults. there are, for example, treatment goals of keeping icp <20 mmhg and cerebral perfusion pressure (cpp) >60 mmhg written in the standardized management protocol system (1). these rules represent other important management goals important to follow in a quality assurance program. the blood pressure is measured at heart level, and icp is measured 2 cm below the head’s highest point. the head was elevated 30�. the uppsala tbi register the department of neurosurgery in uppsala receives patients from a region with a population of 1.9 million. most patients are secondary admissions from local hospitals in the region. all patients with tbi admitted to the nic unit at uppsala university hospital are entered in the register. the register was developed in collaboration with ucr. ucr provides comprehensive solutions for web-based quality registers including e.g. design and maintenance of the database, electronic reports, and statistical analysis. the web-based register allows easy ‘push-button’ statistics of predefined parameters (standardized summary reports) with data up to the previous day included as well as spreadsheet downloading options of the entire register for detailed analysis of specific questions. data elements data are extracted from the medical charts by a small group of persons. predefined criteria are set up for the data set (see supplementary tables 1–3, only available in the online version of the journal; please find this material with the following direct link to the article: http://www. informahealthcare.com/doi/ abs/10.3109/03009734.2013.806616). there are three columns for each patient where data are inserted (table i). the first column includes admission data (table i; supplementary table 1). the second column includes data from the nic period concerning surgery, types of monitoring, if and how long the patient was intubated, complications, and neurological condition at discharge. in this column it is also possible to table i. outline of the uppsala tbi register.a first column: admission to nic unit second column: nic period third column: six-month follow-up date of accident deceased or not evaluation of outcome using gose for adults and gos for children £15 years. name, age, and address number of days spent at the nic unit registration of vp shunt operation after discharge medical history rls and gcs assessment at discharge from the nic unit cause of accident treatments accident circumstances artificial ventilation events pre nic period neuromonitoring rls and gcs (supplementary table 4) secondary insult occurrence dominant finding on first ct scan complications other injuries afor details and definitions see supplementary material. ct = computerized tomography; gcs = glasgow coma scale (11); gos = glasgow outcome scale (8); gose = glasgow outcome scale extended (8); nic = neurointensive care; rls = reaction level scale (10); vp = ventriculoperitoneal. 170 l. nyholm et al. http://informahealthcare.com/doi/suppl/10.3109/03009734.2013.806616/suppl_file/10.3109/03009734.2013.806616_suppl.doc http://www. informahealthcare.com/[doi number] http://www. informahealthcare.com/[doi number] http://informahealthcare.com/doi/suppl/10.3109/03009734.2013.806616/suppl_file/10.3109/03009734.2013.806616_suppl.doc register the amount of secondary insults as assessed from the monitoring data (see below) (table i; supplementary table 2). the third column is used for sixmonth outcome follow-up (table i; supplementary table 3). secondary insult quantification the secondary insults are organized in different categories: icp, cpp, and blood pressure (bp). the amount of secondary insults is presented as the proportion (%) of good monitoring time (gmt) and hours spent at predefined insult levels: icp >25 and >35 mmhg; cpp <60, <50, <40 mmhg, and >70, >80 mmhg; systolic blood pressure (sbp) <100, <90 mmhg, and >160, >180 mmhg; and mean arterial pressure (map) <80, <70 mmhg, and >110, >120 mmhg. the threshold levels for secondary insults were chosen according to existing guidelines and based on the occurrence of secondary insults in a detailed secondary insult quantification study performed earlier at our nic unit (3-5). gmt is the time left when all gaps in monitoring data associated to, for example, radiology examinations or surgical procedures are removed together with clear artifacts. the odin monitoring system developed by tim howells and colleagues in edinburgh and uppsala was used for artifact screening and for the calculations of percent of gmt that the patients spent at insult level (6). outcome the clinical outcome was assessed using the extended glasgow outcome scale (gose) (7) after 6 months (mean 8.2 months). in practice, specially trained nurses interviewed the patients by phone using the standard questionnaire (8). children (£15 years) were followed up by interviewing their parents or guardian after 6 months (mean 7.2 months) using the original glasgow outcome scale (gos) (8,9). quality assurance components automatic daily standardized summary reports on demand. the uppsala tbi register provides standardized summary reports on the web page (table ii). the data are updated every night. the mean values of the reaction level scale (rls) (see supplementary table 4 for criteria, only available in the online version of the journal; please find this material with the following direct link to the article: http://www.informahealthcare.com/doi/abs/10.3109/ 03009734.2013.806616) (10) at admission and discharge are automatically calculated for the last 20 patients, patients treated during the last 12 months, and for all patients treated since 2008. from these values the index of improvement and the index of change are calculated (for these special calculations untreatable patients in rls 7–8 with bilateral unreactive pupils were excluded; for all other calculations all patients in the register are included). index of improvement is calculated as the difference between mean rls at arrival and mean rls at discharge. index of change shows the difference between rls at arrival and rls at discharge and divides the patients into three groups: improved, unchanged, and deteriorated. in the talk and deteriorate report and talk and die report, all patients who have talked (rls 1–2 on admission) and then deteriorated (rls 3–8 at discharge) or died during nic were registered. detailed analysis of database. it is possible to export all data from all patients into a spreadsheet for detailed analysis and research. every case consists of 1 row and 114 columns. from this excel file it is possible to study, for example, the amount of secondary insults for every single patient. all data from the start of the registry 2008 to the end of 2010 is summarized and presented in the results section of this study. means are presented ± standard deviations. table ii. overview of automatic daily standardized summary reports. figures denote series of patients reported (1 = all patients last 12 months; 2 = all patients since 2008; 3 = patients by year; 4 = last 20 patients). age (1+2) sex (1+2) referring hospitals (1+3) cause of accident (1+3) rls and gcs-m assessment on admission to the nic unit (1+3) monitoring (1+3) index of improvement (1+2+3+4)a index of change (improved, unchanged, and deteriorated) (1+2+3+4)a talk and deteriorate (1+2+3+4)a talk and die (1+2+3+4)a clinical outcome for all patients ‡16 years (gose) (1+2+3) clinical outcome for all patients ‡16 years (gose) by age (1+2) clinical outcome for all patients ‡16 years (gose) by neurological status on admission (1+2) clinical outcome for children £15 years (gos) (1+2+3) a for definitions see text. gcs-m = glasgow coma scale–motor (11); gos = glasgow outcome scale (9); gose = glasgow outcome scale extended (8); nic = neurointensive care; rls = reaction level scale (10). the uppsala tbi register and nic management 171 http://informahealthcare.com/doi/suppl/10.3109/03009734.2013.806616/suppl_file/10.3109/03009734.2013.806616_suppl.doc http://informahealthcare.com/doi/suppl/10.3109/03009734.2013.806616/suppl_file/10.3109/03009734.2013.806616_suppl.doc http://informahealthcare.com/doi/suppl/10.3109/03009734.2013.806616/suppl_file/10.3109/03009734.2013.806616_suppl.doc http://www. informahealthcare.com/[doi number http://www. informahealthcare.com/[doi number specific reviews of compliance with standardized management protocols. the occurrence of patients not responding to command who did not receive artificial ventilation and icp monitoring, respectively, as prescribed in the standardized management protocol system, is investigated. a specific medical chart review is done for these cases to find explanations. specific reviews of deteriorating cases. a specific medical chart review was performed in cases with rls 1– 5 at arrival who deteriorated, to find reasons for their deterioration and to identify possible poor management. ethics the study was approved by the local ethics committee. results between 1 january 2008 and 31 december 2010 the uppsala tbi register contained 314 patients. automatic daily standardized summary reports every day, on command, the system displays the mean rls at admission and discharge for different time periods. the mean rls at admission were slightly different during 2008–2010 (3.43 in 2008; 2.95 in 2009; and 3.37 in 2010). index of improvement and index of change are presented by year in figure 1. the patients improved on average during the nic stay (1.2 rls levels in 2008; 0.7 rls levels in 2009; and 0.9 rls levels in 2010). the proportion of patients improved in rls scale during nic varied between 80% in 2008 and 60% in 2009. the proportion of deteriorated patients was stable and less than 10% during the period. figure 1 shows the occurrence of talk and die and talk and deteriorate by year. in 2008 there were no patients who talked and died and none who talked and deteriorated. detailed analysis of database the 314 patients studied included 66 women and 248 men with an age of 0–86 years (mean 42.9 years, ±22.2). out of these 314 cases, 33 were children aged £ 15 years (mean 8.9 years, ±5.4) (figure 2). admission to the nic unit.. the mean gcs-m was 5.04 ± 1.23 (rls 3.4 ± 1.6) (table iii) (11). the gcs classification was mild (gcs 13–15) (22%), moderate (gcs 9–12) (27%), and severe (gcs 3–8) (51%) (figure 3). the co-occurrence of some specific diseases that may influence the outcome after tbi is presented in table iv. the most frequent causes of injury were fall accidents (44%) and vehicle accidents (30%) (table iv). in 24% of the cases the injury occurred under the influence of alcohol or other drugs (anamnestic or positive serum levels) (table iv). the patients were transferred with specialized intensive care helicopter in 33% of the cases (table iv). acute evacuation of an extracerebral hematoma was done at the referral hospital in 8% of the patients before admission to the nic unit in uppsala (table iv). the primary findings on the initial brain ct scan were contusions (33%) and 0 0.2 0.4 0.6 0.8 1 1.2 1.4 2008 n =101 2009 n = 87 2010 n = 117 in d e x o f im p ro v e m e n t 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 2008 n = 101 2009 n = 87 2010 n = 117 in d e x o f c h a n g e ( % ) deteriorated unchanged improved 0 1 2 2008 n = 101 2009 n = 87 2010 n = 117 n u m b e r o f p a ti e n ts talk and die talk and deteriorate figure 1. the automatic standardized summary report on demand in treatable patients (n = 305) included in the uppsala tbi register 2008–2010. the upper panel shows ‘index of improvement’ calculated by the difference between mean rls at arrival and mean rls at discharge. the middle panel shows ‘index of change’ calculated by the difference between rls on arrival and rls at discharge. the lower panel shows the number of patients talk (rls 1–2 on admission) and die (dead during nic), and talk and deteriorate (rls 3–8 at discharge). 172 l. nyholm et al. acute subdural hemorrhage (23%) (table iv). the most common injuries beside the brain injury were thoracic injuries (23%) followed by extremity injuries (15%), facial injuries (15%), and spinal column injuries (11%) (table iv). neurointensive care. the patients stayed at the nic unit for 0–86 days (mean 11 days, ± 10). craniotomy for evacuation of intracranial hematomas and/or contusions was the most common treatment registered (performed in 30% of all cases), followed by barbiturate coma treatment (8%) and decompressive craniectomy (6%) (table v). nine percent of the patients went through multiple neurosurgical operations (insertion of monitoring devices excluded). icp was monitored in approximately half of the cases, and, of these, 16% had ventricular drainage, 64% had parenchymal probe, and 20% had both (table v). icp was monitored between 1 and 50 days (mean 11.1 days, ±7.3) (table v). artificial ventilation was used in 75% of all cases between 0 and 46 days (mean 8.6 days, ±7.6) (table v). other types of neuromonitoring applied was microdialysis (11% of the cases), brain tissue oxygenation (3%), and jugular bulb (2%) (table v). occurrence of secondary insults. analysis of the occurrence of secondary insults for all patients according to predefined insult thresholds showed that icp >25 and >35 mmhg, cpp <50 and <40 mmhg, sbp <100 and <90 mmhg, and map <70 and >120 mmhg occurred in less than 5% of gmt (figure 4). cpp <60, >70, and >80 mmhg, sbp >160 and >180 mmhg, and map <80 and >110 mmhg were present in a larger proportion of gmt (figure 4). eleven cases were excluded from the analysis of secondary insults since no monitoring data were stored for these patients because they had initially been treated in the general intensive care unit. neurological status at discharge. at discharge from the nic unit, the mean gcs-m was 5.68 ± 0.8 (rls 2.5 ± 2.0) compared to 5.04 ± 1.23 (rls 3.4 ± 1.6) on admission (table iii). the pupil reaction and size became more normal during the stay at the nic unit, but the amount of paresis was almost the same at discharge (34%) as at arrival (36%) (table iii). complications during the stay at nic unit. severe pulmonary problems occurred in 3% of the cases, and 2% of the patients had meningitis with positive bacterial cultures. outcome forty-one percent of the adult patients (‡16 years) had good recovery (gr), 23% moderate disability (md), 19% severe disability (sd), and two (1%) patients remained in a vegetative state (vs). five percent died at the nic unit, and 8% died within six months after discharge (table vi). among the children (£15 years), 61% showed gr, 15% md, 9% 0 2 4 6 8 10 12 14 0 8 13 18 23 28 33 39 44 49 54 59 64 70 75 81 n u m b e r o f p a ti e n ts age female male figure 2. age and sex in all 314 patients included in the uppsala tbi register 2008–2010. the uppsala tbi register and nic management 173 sd; no children persisted in vs, and 9% died at the nic unit (table vii). figure 5 shows the clinical outcome for children and adults. clinical outcome for all adult patients (‡16 years) is presented by age in table viii and by the severity of the injury in figure 3. specific medical chart review compliance with standardized management protocols. the compliance with standardized management protocols was spot-checked by analyzing two management principles selected from the standardized table iii. neurological status on arrival at the nic unit and at discharge from the nic unit in all 314 patients included in the uppsala tbi register 2008–2010. admission n = 314 (%) discharge n = 297 (%) gcs-m 6 obeys commands 141 (45) 244 (82) 5 localizing pain 110 (35) 26 (9) 4 withdrawal from pain 32 (10) 17 (6) 3 abnormal flexion 7 (2) 5 (2) 2 extending 15 (5) 5 (2) 1 no response 9 (3) 0 (0) rls 1 alert response 34 (11) 104 (33) 2 delayed response 64 (20) 118 (38) 3a very delayed response 43 (15) 31 (10) 3b wards off pain 81 (25) 7 (2) 4 localizes pain 32 (10) 10 (3) 5 withdrawing movements 28 (9) 21 (7) 6 stereotype flexion 8 (2) 3 (1) 7 stereotype extension 15 (5) 3 (1) 8 no response 9 (3) 0 (0) dead 0 (0) 17 (5) pupil reaction right–left right–left normal 224 (71)–218 (69) 272 (92)–266 (90) sluggish 47 (15)–51(16) 13 (4)–21 (7) unreactive 32 (10)–33(10) 9 (3)–8 (3) unknown 11 (4)–12 (5) 20 (1)–19 (1) pupil size right–left right–left small 50 (16)–52 (17) 7 (2)–7 (2) normal 233 (74)–221 (70) 273 (93)–273 (92) dilated 21 (7)–29 (9) 13 (4)–13 (4) unknown 10 (3)–12 (4) 4 (1)–4 (1) paresis yes 113 (36) 106 (34) arm or leg paresis 45 (40) 46 (43) hemiparesis 58 (51) 50 (47) paraparesis 2 (2) 7 (7) tetraparesis 8 (7) 3 (3) no 181 (57) 188 (63) unknown 20 (7) 3 (1) gcs-m = glasgow coma scale–motor (11); rls = reaction level scale (10). 174 l. nyholm et al. management protocols, i.e. if patients not responding to commands received icp monitoring and were artificially ventilated as prescribed. among 173 cases who did not respond to commands (rls 3b-8, gcsm 1–5) on arrival at the nic unit, icp was not monitored in 36 (21%) cases. explanations for not monitoring icp were found in the medical records and are presented in table ix. three cases who did not respond to commands (rls 3b-8) on arrival at the nic unit were not intubated and artificially ventilated. according to the medical records, the reason for not intubating those three cases was that all of them made a very quick clinical improvement. deterioration in neurological status. out of all patients who arrived in rls 1–5 (n = 282) at the nic unit 20 patients deteriorated. likely patient-related explanations could be found in 19 cases (table x). 0% 10% 20% 30% 40% 50% 60% gr md sd vs d dm % o f p a ti e n ts mild (gcs-m 6) moderate (gcs-m 4–5) severe (gcs-m 1–3) mild (gcs 13–15) moderate (gcs 9–12) severe (gcs 3–8) 0% 10% 20% 30% 40% 50% 60% 70% gr md sd vs d dm % o f p a ti e n ts figure 3. six-month outcome (gos) divided by severity of injury at admission to the nic unit in adult patients ‡16 (n = 181) years included in the uppsala tbi register 2008–2010. the severity of injuries was classified as mild, moderate, and severe using the gcs sum score. untestable reactions were scored as 1 (no reaction) according to common practice. to avoid the problem with untestable reactions and over-classification of severity, a modified classification of the severity of the injury based on the gcs motor score was also used. (gr = good recovery; md = moderate disability; sd = severe disability; vs = vegetative state; d = dead within six months; dm = data missing). table iv. admission data in all 314 patients included in the uppsala tbi register 2008–2010a. n (%) unknown n (%) medical history prior brain diseases 40 (13) 17 (6) diabetes mellitus 18 (6) 19 (6) cardiovascular diseases 49 (16) 21 (7) alcohol addiction 49 (16) 31 (10) anticoagulation treatment 37 (12) 20 (7) cause of accident fall accident 138 (44) vehicle 94 (30) sports 17 (6) assault 16 (5) walker 14 (4) cyclist hit by other vehicle 12 (4) remaining 23 (7) accident circumstances influence of alcohol/drugs 74 (24) 62 (20) work-place accident 17 (6) 1 (~0) events pre nic severe global ischemia 8 (3) 5 (2) hypothermia 1 (1) 3 (1) urgent surgery at referral hospital 23 (8) 0 (0) transportation helicopter 105 (33) 8 (3) dominant finding on first ct scan acute subdural hematoma 74 (23) contusions 104 (33) epidural hematoma 23 (7) diffuse axonal injury 32 (10) traumatic subarachnoid hemorrhage 24 (8) impression fracture 9 (3) mixed injuries 33 (11) other 10 (3) normal examination 5 (2) other injuries spinal column injury 34 (11) spinal cord injury 4 (1) facial injury 48 (15) thoracic injury 71 (23) abdominal injury 20 (6) pelvic injury 15 (5) extremities 47 (15) large bleeding 14 (4) asee supplementary material for definitions. the uppsala tbi register and nic management 175 out of these 20 patients, two talked (rls 1–2) and died: in one this was due to cardiac arrest, and the other one died at the nic unit due to direct consequences of the tbi. both were older than 70 years and were on anticoagulation treatment. one patient talked (i.e. rls 1–2 on admission) and deteriorated; this patient was older than 70 years and had anticoagulation treatment. no treatable children deteriorated. the three children who died arrived in rls 8. deteriorating patients had a similar amount of secondary insults compared to all patients (figure 4). discussion the main goal of establishing the uppsala tbi register was to obtain an instrument for regular quality assurance of the management of tbi with particular focus on nic. therefore, a battery of quality assurance components suitable for nic was introduced to reflect the quality of nic in different aspects. the introduced quality assurance components and the results in general will be discussed in the following sections. automatic daily standardized summary reports on demand the idea with the standardized summary reports was to be able to get updated reports on demand, with 0 10 20 30 40 50 60 70 % o f g m t ic p >2 5 ic p >3 5 cp p <6 0 cp p <5 0 cp p <4 0 cp p >7 0 cp p >8 0 sb p <1 00 sb p <9 0 sb p >1 60 sb p >1 80 m ap < 80 m ap < 70 m ap > 11 0 m ap > 12 0 all patients deteriorated patients figure 4. mean occurrence of secondary insults (% of gmt) according to predefined threshold levels (mmhg) in tbi patients with icp monitoring ‡16 years (n = 146) and in deteriorated patients ‡16 years (n = 20), included in the uppsala tbi register 2008–2010. (icp = intracranial pressure; cpp = cerebral perfusion pressure; sbp = systolic blood pressure; map = mean arterial pressure). table v. treatment and neuromonitoring in all 314 patients included in the uppsala tbi register 2008–2010. time (days) n (%) mean (±) min max treatments craniotomy 94 (30) decompressive craniectomy 19 (6) barbiturate coma 24 (8) 5.9 (3.4) 1 13 artificial ventilation 237 (75) 8.6 (7.6) 0 46 neuromonitoring icp monitoring 170 (54) 11.1 (7.3) 1 50 ventricular drainage 28 (16) parenchymal probe 109 (64) both 33 (20) jugular bulb oxygenation 6 (2) microdialysis 36 (11) brain tissue oxygenation 10 (3) table vi. six-month outcome after traumatic brain injury in all tbi patients ‡16 years included in the uppsala tbi register 2008– 2010. gose n (%) good recovery—higher 72 (26) good recovery—lower 43 (15) moderately disabled—higher 37 (13) moderately disabled—lower 27 (10) severely disabled—higher 20 (7) severely disabled—lower 34 (12) vegetative state 2 (1) dead at the nic unit 14 (5) dead within 1 week after discharge from the nic unit 9 (3) dead 1–24 weeks after discharge from the nic unit 15 (5) missing data 8 (3) total 281 (100) table vii. six-month outcome after traumatic brain injury in all children £15 years included in the uppsala tbi register 2008–2010. gos n (%) good recovery 20 (61) moderately disabled 5 (15) severely disabled 3 (9) vegetative state 0 (0) dead at the nic unit 3 (9) missing data 2 (6) total 33 (100) 176 l. nyholm et al. predefined selections of patients for overview and comparison (all patients last year, all patients since the start of the register 2008, patients by year, and last 20 patients). the reports would include traditional demographic data, crude outcome data, and outcome in relation to established prognostic admission factors. furthermore, new quality measures were included in the reports with inspiration from the description of patients with head injuries who talked and died due to secondary brain injury in the 1970s in glasgow (12), i.e. occurrence of talk and die cases, and occurrence of talk and deteriorate cases. concerning using index of improvement, index of change, talk and die, and talk and deteriorate, the idea was to introduce new quality measures for the intensive care period specifically. if, for example, the number of talk and die cases suddenly increased, this would be a severe warning, indicating an audit of care. in this material,<1% of the tbi patients talked and died, while around 6% have been reported by others (13,14). talk and deteriorate cases, which means patients who are awake on admission and then deteriorate (15), were also rare and occurred in less than 1% of the patients (figure 1). our impression is that the standardized summary reports on demand provide a valuable tool to monitor demographic changes over time and the quality of nic in tbi patients. the possibility to get updated reports on demand every day is a great advantage. inclusion of the different quality measures developed from the talk and die concept adds valuable information to ordinary long-term outcome analysis by reflecting the nic period specifically. these measures need to be evaluated further. review of deteriorating cases the researchers from glasgow, who described the talk and die cases, found a number of secondary 0% 10% 20% 30% 40% 50% 60% 70% gr md sd vs d* d° d' md % o f p a ti e n ts adults ≥ 16 years children ≤ 15 years figure 5. clinical six-month gose and gos outcome for adults ‡16 years (n = 181) and children £15 years (n = 33) in all 314 patients included in the uppsala tbi register 2008–2010. (gr = good recovery; md = moderate disability; sd = severe disability; vs = vegetative state; d* = dead at the nic unit; dp = dead within 1 week after discharge from the nic unit; d’ = dead 1–24 weeks after discharge from the nic unit; dm = data missing). table viii. age-stratified six-month outcome in all adult tbi patients ‡16 years included in the uppsala tbi register 2008–2010 (% of patients in each age group). age (years) clinical outcome 16–39 (n = 110) n (%) 40–59 (n = 79) n (%) ‡60 (n = 92) n (%) good recovery—higher 39 (35) 17 (22) 16 (17) good recovery—lower 14 (13) 12 (15) 17 (18) moderate disability—higher 18 (16) 12 (15) 7 (8) moderate disability—lower 16 (15) 6 (8) 5 (5) severe disability—higher 4 (3) 6 (7) 10 (11) severe disability—lower 8 (7) 16 (20) 10 (11) vegetative state 2 (2) 0 (0) 0 (0) dead at the nic unit 4 (4) 4 (5) 6 (7) dead within 1 week after discharge from the nic unit 0 (0) 0 (0) 9 (10) dead 1–24 weeks after discharge from the nic unit 1 (1) 4 (5) 10 (11) missing data 4 (4) 2 (3) 2 (2) table ix. medical chart review—explanations why and the occurrence of patients not responding to command who did not receive icp monitoring according to standardized management protocol among all 314 patients included in the uppsala tbi register 2008–2010. possible explanation number of patients quick improvement 16 coagulopathy and quick improvement 5 other severe diseases 2 fast deterioration after arrival 1 fatal brain herniation during evacuation of acute subdural hematoma 2 minor mass effect on ct scan and improvement 1 coagulopathy 3 poor neurological status and coagulopathy 1 poor neurological status 5 total 36 the uppsala tbi register and nic management 177 insults which could explain the fatal clinical courses and judged that those insults to some extent could have been avoided (12). in order to understand better why some patients (both initially conscious and unconscious) deteriorated during their stay at the nic unit and to identify suboptimal care, we did a specific medical chart review in all patients who were rls 1–5 on admission and then deteriorated or died. in this series of patients, the specific medical chart review revealed that 17 out of 20 deteriorating patients had patient-related factors (i.e. high age, on-going anticoagulation treatment or coagulopathy, severe neurological status on arrival) contributing to the deterioration. two patients had complications (basilar dissection, n = 1; sinus thrombosis, n = 1) which were judged not to have been preventable. only one patient had no obvious reason for deterioration, and he deteriorated from rls 3b to rls 4. this structured way to investigate patients who deteriorate during the stay at nic unit illustrates a way to survey the occurrence of avoidable factors contributing to poor outcome, e.g. misjudgments, incorrect treatment, and complications. reviews of compliance with standardized management protocols it is well established that application of management protocols improves care (1,16). however, the compliance with management protocols has been found to be as low as around 50% (17,18). at the nic unit in uppsala, a standardized management protocol system has been developed and maintained by the nursing staff in collaboration with the doctors for many years (1). by involving doctors and nurses we hoped that the guidelines would be followed in daily care. in this study, we checked for the compliance with two crucial standardized management principles, i.e. icp monitoring and artificial ventilation. the compliance with the indication for icp monitoring was 79% and for artificial ventilation 98%. when the reasons were investigated for not monitoring icp when indicated according to the management protocol, reasonable explanations were found, e.g. coagulopathy. there were somewhat fewer patients monitored with icp than treated with artificial ventilation. the reason is that some patients arrived at the nic unit intubated in order to make the transport secure, and after arrival patients who obeyed commands were extubated and not in need of icp monitoring. it is important in our opinion that quality assurance programs include evaluation of compliance with applied management protocols and reasons for exclusions. detailed analysis of database—occurrence of secondary insults to our knowledge, quantification of secondary insults during nic has never been mandatory in any quality assurance program for tbi management, although specific studies of secondary insults have been performed (3,19-21). no ideal quantitative measure of secondary insult burden exists. mean icp per day or for the whole nic period are obviously too crude as summary measures. we believe that proportion of gmt above/below a defined threshold level for certain types of insults provides better information (3,15). quantification of secondary insults within this quality assurance program revealed that all investigated parameters except four had less than 10% of gmt out of the threshold, i.e. high levels of cpp >70 mmhg and >80 mmhg, sbp >160 mmhg, and map <80 mmhg. icp >25 mmhg occurred in 4.5% of gmt and sbp <100 mmhg in 1.7% of gmt. thus, the occurrence of secondary insults appears to be low. quantification of secondary insults during nic is in our opinion of utmost importance in any quality assurance program concerning tbi management. detailed analysis of database—general results admission to the nic unit. patients of all ages were admitted to the nic unit, and all ages are represented in the material with two peaks around 20 and 60 years. several patients obeyed command (44%) and only a few (10%) were gcs-m 1–3 at arrival, i.e. there were only a few severely injured patients. falls (44%) and vehicle (30%) accidents were the most common causes of tbi, which is rather similar to studies from finland (22) but different from some other countries (23,24). the elderly patients had more table x. medical chart review—possible patient-related explanations and occurrence of patients in rls 1–5 on arrival at the nic unit who deteriorated among all 314 patients included in the uppsala tbi register 2008–2010. possible explanations number of patients old patient (>69 years) 1 anticoagulantia or coagulopathy 1 rls 4–5 on arrival 4 two or more of the above-mentioned explanations 11 severe complicationa 2 no explanations 1 total 20 abasilaris dissection (n = 1) and sinus thrombosis (n = 1). 178 l. nyholm et al. fall accidents, and they were often afflicted with an acute subdural hematoma. the younger patients had more vehicle accidents. contusions (33%) and acute subdural hematoma (23%) were the most common findings on the first ct scan. regular evaluation of the automatic summary reports on demand in combination with the detailed analysis of the database can be used to follow any changes in demographic patterns over time for tbi. nic period. it is difficult to compare the length of stay at the nic unit between different centers due to different structure and organization of health care. analysis of the tbi cases managed in uppsala revealed that length of stay varied considerably (0– 86 days) but was around 11 days on average. another study from austria reported a mean of 10 days at the intensive care unit for tbi patients (25). it was also interesting to see that intracranial hematomas/ contusions were evacuated in 30% of all cases and that barbiturate coma treatment and decompressive craniectomy were required in 8% and 6% of the patients, respectively, which underlines the need for highly specialized care of tbi. the number of complications during nic appeared to be small with pulmonary complications in 3% of patients and meningitis in 2%. clinical outcome. the uppsala tbi register includes all patients managed at the nic unit without selections, which is preferable when the overall results are reported and for comparisons. it is also important that the clinical outcome is assessed in an established and validated way. the glasgow outcome scale, both the original form and the extended version, is assessed reliably by a structured interview, and the result describes an overall social outcome (9). however, there are many sources of bias which need to be considered when making interviews with tbi patients. patients in poor pre-traumatic state only need a small deterioration to become dependent. patients may lack insight and be unconcerned about his/her deficits. patients may return early to home or work because of a caring family or employer (9). these confounding factors may have influenced the results in this material as well. looking at the follow-up results of this patient material, 64% of the adult patients had a favorable outcome (good recovery or moderately disabled). this result can be compared with other studies which reported favorable outcome in 50%–70% of cases (26–30) and the earlier results from uppsala presented by elf et al. who reported favorable outcome in 78% of the cases treated 1996–1997 (1). it should be emphasized that it is difficult to compare overall results between different studies because of differences in e.g. the selection of patients and demographics. the tbi patients treated in uppsala 1996–1997 were younger but in poorer gcs-m grade at admission compared to the present series, and patients potentially not possible to treat were excluded (n = 18) (1). the results from the present study also showed that younger patients and patients with better neurological status at arrival had better outcomes overall which is in accordance with other studies (31,32). the children in our material made good recovery in 61% of the cases, and 9% died at the nic unit, which is comparable with another swedish study (33). concluding remarks the specific objectives of establishing the uppsala tbi register were to be able regularly to: 1) obtain information about demographic data, clinical outcome, and how the treatments and care affected outcome; 2) identify patients who did not have the expected result or the right treatment and to determinate why that happened; 3) provide data and select patients for research studies. the conclusion of this study, presenting the design of the register and the first results, is that the uppsala tbi register is functional in these objectives. if quality is measured routinely, problem areas can be identified and corrected continuously, which should produce improvements in neurointensive care and outcome for tbi patients. the uppsala tbi register is internet-based which makes it possible for other centers to enter their data. acknowledgements agneta ohlén is acknowledged for entering parts of the data into the uppsala tbi register. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. the study was supported by grants from the swedish research council. references 1. elf k, nilsson p, enblad p. outcome after 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clin nurs. 2011;20:1518–32. 33. emanuelson i, wendt l. epidemiology of traumatic brain injury in children and adolescents in south-western sweden. acta paediatr. 1997;86:730–5. supplementary material available online supplementary tables 1–4. 180 l. nyholm et al. http://www.epa.gov/quality/qs-docs/g6-final.pdf http://www.epa.gov/quality/qs-docs/g6-final.pdf www.ncbi.nlm.nih.gov/pubmed/15854244?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15854244?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15854244?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17511557?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17511557?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9202767?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9202767?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9726258?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9726258?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9726258?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9726257?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9726257?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9726257?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/6453957?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/6453957?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/6453957?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/3394542?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/3394542?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/4136544?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/4136544?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/51187?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/51187?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17297312?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17297312?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17297312?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17297312?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/6864295?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/6864295?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17986931?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17986931?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17686165?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17686165?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17686165?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19077619?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19077619?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19077619?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19077619?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19077619?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22488001?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22488001?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22488001?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17522844?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17522844?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17522844?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11198763?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11198763?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11198763?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20589400?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20589400?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20589400?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18297592?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18297592?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18297592?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10955671?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10955671?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17318747?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17318747?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10214478?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10214478?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9824083?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9824083?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9824083?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11300376?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11300376?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11300376?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11300376?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19226191?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19226191?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22149445?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22149445?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22149445?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12561359?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12561359?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21453293?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21453293?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9240881?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9240881?dopt=abstract abstract introduction material and methods standardized management protocol system and treatment goals the uppsala tbi register data elements secondary insult quantification outcome quality assurance components automatic daily standardized summary reports on demand detailed analysis of database specific reviews of compliance with standardized management protocols specific reviews of deteriorating cases ethics results automatic daily standardized summary reports detailed analysis of database admission to the nic unit. neurointensive care occurrence of secondary insults neurological status at discharge complications during the stay at nic unit outcome specific medical chart review compliance with standardized management protocols deterioration in neurological status discussion automatic daily standardized summary reports on demand review of deteriorating cases reviews of compliance with standardized management protocols detailed analysis of database&mdash;occurrence of secondary insults detailed analysis of database&mdash;general results admission to the nic unit nic period clinical outcome concluding remarks acknowledgements doi43 references targeted deletion of vglut2 expression in the embryonal telencephalon promotes an anxiolytic phenoty full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 targeted deletion of vglut2 expression in the embryonal telencephalon promotes an anxiolytic phenotype of the adult mouse karin nordenankar, assar bergfors & åsa wallén-mackenzie to cite this article: karin nordenankar, assar bergfors & åsa wallén-mackenzie (2015) targeted deletion of vglut2 expression in the embryonal telencephalon promotes an anxiolytic phenotype of the adult mouse, upsala journal of medical sciences, 120:3, 144-156, doi: 10.3109/03009734.2015.1032454 to link to this article: https://doi.org/10.3109/03009734.2015.1032454 © informa healthcare view supplementary material published online: 09 apr 2015. submit your article to this journal article views: 585 view related articles view crossmark data citing articles: 1 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2015.1032454 https://doi.org/10.3109/03009734.2015.1032454 https://www.tandfonline.com/doi/suppl/10.3109/03009734.2015.1032454 https://www.tandfonline.com/doi/suppl/10.3109/03009734.2015.1032454 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1032454 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1032454 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1032454&domain=pdf&date_stamp=2015-04-09 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1032454&domain=pdf&date_stamp=2015-04-09 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1032454#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1032454#tabmodule upsala journal of medical sciences. 2015; 120: 144–156 original article targeted deletion of vglut2 expression in the embryonal telencephalon promotes an anxiolytic phenotype of the adult mouse karin nordenankar, assar bergfors & åsa wallén-mackenzie department of neuroscience, unit of functional neurobiology and unit of developmental genetics, uppsala university, box 593, s-75214 uppsala, sweden abstract background. anxiety is a natural emotion experienced by all individuals. however, when anxiety becomes excessive, it contributes to the substantial group of anxiety disorders that affect one in three people and thus are among the most common psychiatric disorders. anxiolysis, the reduction of anxiety, is mediated via several large groups of therapeutical compounds, but the relief is often only temporary, and increased knowledge of the neurobiology underlying anxiety is needed in order to improve future therapies. aim. we previously demonstrated that mice lacking forebrain expression of the vesicular glutamate transporter 2 (vglut2) from adolescence showed a strong anxiolytic behaviour as adults. in the current study, we wished to analyse if removal of vglut2 expression already from mid-gestation of the mouse embryo would give rise to similar anxiolysis in the adult mouse. methods. we produced transgenic mice lacking vglut2 from mid-gestation and analysed their affective behaviour, including anxiety, when they had reached adulthood. results. the transgenic mice lacking vglut2 expression from mid-gestation showed certain signs of anxiolytic behaviour, but this phenotype was not as prominent as when vglut2 was removed during adolescence. conclusion. our results suggest that both embryonal and adolescent forebrain expression of vglut2 normally contributes to balancing the level of anxiety. as the neurobiological basis for anxiety is similar across species, our results in mice may help improve the current understanding of the neurocircuitry of anxiety, and hence anxiolysis, also in humans. key words: affective, anxiolysis, behaviour, development, psychostimulant introduction glutamate is the main excitatory neurotransmitter in the mammalian brain, and glutamatergic transmission is solidly regulated both pre-and post-synaptically during healthy conditions. dysregulated glutamatergic transmission is implicated in several neuropsychiatric disorders, including schizophrenia, addiction and anxiety disorders (for examples of recent reviews see (1-4)). better understanding of the neurobiology and neurocircuitry of glutamatergic neurons is therefore needed to identify future potential therapeutic targets. the presence of vesicular glutamate transporters (vgluts) 1 and/or 2 (encoded by the vglut1 and vglut2 genes, aka slc17a7 and slc17a6, respectively) defines the presynaptic glutamate site (5-10). expression analyses of the adult telencephalic area in the mouse and rat have shown a strong predominance for vglut1 mrna across the cerebral cortex and hippocampus, areas in which vglut2 is only found regionally distributed. more specifically, vglut2 mrna is correspondence: åsa wallén-mackenzie, department of neuroscience, unit of functional neurobiology and unit of developmental genetics, uppsala university, box 593, 751 24 uppsala, sweden. fax: +46 18 511 540. e-mail: asa.mackenzie@neuro.uu.se (received 15 january 2015; accepted 13 march 2015) this is an open-access article distributed under the terms of the cc-by-nc-nd 3.0 license which permits users to download and share the article for noncommercial purposes, so long as the article is reproduced in the whole without changes, and provided the original source is credited. issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1032454 http://informahealthcare.com/journal/ups mailto:asa.mackenzie@neuro.uu.se localized to the retrosplenial group (rsg), layers iii and v/vi of the neocortex, and to the endo-piriform cortex. within the hippocampal formation, the expression is confined to the subiculum (7,9,11-13). both vglut1 and vglut2 mrnas are found in the olfactory bulb (14), and both are localized to the amygdaloid complex, albeit to different subpopulations. vglut2 mrna is detected only in the medial (me), anterior cortical (aco), and anterior basomedial (bm) nuclei, while vglut1 mrna is found in all other amygdaloid populations (13,15). vglut2, the predominant vglut in deep structures of the adult brain, is broadly distributed already at midgestation of the developing mouse embryo (16,17). this expression is subsequently down-regulated in most areas postnatally populated by vglut1 mrna (7-9,18-20). behavioural phenotyping of mice gene-targeted for either vglut1 or vglut2 has implied a role for the presynaptic glutamate site in behaviour of relevance for psychiatric conditions. mice heterozygous for vglut1, the predominant vglut in the telencephalon, showed an anxiogenic phenotype as well as depressionand schizophrenia-related behaviours (21,22). demonstrating the importance of vglut2 in the brainstem, a full knock-out of vglut2 led to immediate neonatal lethality due to respiratory failure (23,24). by using the cre/loxp conditional gene targeting system (reviewed in (25)), the functional role of vglut2 in neuropsychiatric-like conditions has been further addressed. for example, several studies of mice gene-targeted specifically for vglut2 within dopamine neurons (26) have demonstrated alterations in the response to psychostimulants, leading to a proposed role of vglut2 in mechanisms of importance for addiction (16,17,27-29). the regional distribution of vglut2 in the forebrain was previously targeted in our laboratory at the adolescent stage by use of the camkiia-cre transgenic mouse line, which can be detected from postnatal week 3 (30). by behavioural and biochemical analysis of adult vglut2f/f;camkii-cre conditional knock-out (cko), we identified an anxiolytic phenotype alongside several behaviours relevant for animal models of schizophrenia (13). in the current study, we aimed to approach whether vglut2 gene expression during embryo development is of any relevance for affective behaviour at adulthood. to analyse this issue, we used the previously described emx1ires-cre knock-in mice (31) to drive dorsal telencephalic deletion of vglut2 expression from mid-gestation onwards. we analysed adult vglut2f/f;emx1-cre cko and control mice for functions of relevance to psychiatric conditions, including measures of psychostimulant-induced behavioural activation, sociability, and various aspects of anxiety. material and methods animals all mice used in the study were housed in the animal facility at the bmc, uppsala university, in accordance with the swedish regulation guidelines (animal welfare act sfs 1998:56) and european union legislation (convention ets123 and directive 2010/63/eu). ethical approval was obtained from the uppsala animal ethical committee. mice were housed at constant temperature (21 ± 1�c) and humidity (50%–60%) with 2–8 mice per cage unless otherwise stated. all behavioural experiments took place during the light phase, between 06.00 and 18.00. food (r3, lactamin/lantmännen, sweden) and water were provided ad libitum unless otherwise stated. all behaviour tests were performed on adult (>10 weeks) mice. generation of vglut2f/f;emx1-cre mice the vglut2f/f;emx1-cre mouse line was produced by using the breeding procedure established for conditional knock-out mice to ensure identical genetic background (32). by crossing mice homozygous for the floxed allele of vglut2 (vglut2f/f) (24) with emx1ires-cre knock-in mice (31), hereafter referred to as emx1-cre mice, both vglut2f/f;emx1-cre(tg/wt) conditional knock-out mice (cko) and vglut2f/f;emx1-cre(wt/wt) control mice (which do not express the emx1-cre transgene and therefore have normal vglut2 expression) were produced as littermates which allows for behavioural phenotyping and comparison between genotype groups (33). the vglut2f/f;emx1-cre cko and ctrl mice were thus of the same genetic background, a mixture of c57/bl6 and sv129. genotyping was performed as previously described (24). tissue sectioning and rna probes mice were intraepidermally injected with a 1:1 mixture of dormitor (medetomidine hydrochloride, 70 mg/g body weight; orion pharma, espoo, finland) and ketalar (ketamine hydrochloride, 7 mg/g body weight, pfizer, groton, ct, usa). transcardial perfusion was performed with phosphate-buffered saline (pbs) followed by 4% formaldehyde (histolab, västra frölunda, sweden). the brain was excised and stored in 4% formaldehyde overnight, after which it was washed in pbs and embedded in 4% agarose and sectioned (70 mm) on a leica vt1000s vibratome (leica biosystems, nussloch, germany). sections were dehydrated through a series of methanol washes (25%, 50%, and 75% methanol) and deletion of vglut2 expression in the embryonal telencephalon 145 lastly stored in 100% methanol at –20�c until further processing. templates for in situ probes were derived from commercialcdnaclonesbyusinggene-specificpromotors (asdescribedatwww.genepaint.org).thevglut2probe covers nucleotides (nts) 1616-2203 (nm_080853.2), the vglut1 probe nts 462-1067 (nm_182993), and the vesicular inhibitory amino acid transporter (viaat) probe nts 1578-1889 (nm_009508.1). the probes were synthesized by using t7, t3, or sp6 rna polymerase in the presence of digoxigenin-11-utp (roche diagnostics scandinavia, stockholm, sweden). probes were controlled and quantified by using the nanodrop nd-1000 spectrophotometer (nanodrop technologies, wilmington, de, usa). in situ hybridization on free-floating sections mouse brain sections were step-wise rehydrated from 100% methanol to pbt (pbs with 0.1% tween-20 (sigma-aldrich sweden ab, stockholm, sweden)) and bleached in 6% hydrogen peroxidase in pbt. thereafter, the sections were permeabilized with 0.5% tritonx-100 (sigma-aldrich sweden ab, stockholm, sweden), digested with 20 mg/ml proteinase k (invitrogen, nordic biolabs, täby, sweden) and post-fixed in 4% formaldehyde (histolab) with pbt washes between all steps. sections were then pre-hybridized at 55�c in hybridization buffer (50% formamide, 5 � ssc, ph 4.5, 1% sds, 50 mg/ml of trna (sigma), 50 mg/ml of heparin (sigma), and pbt). the digoxigenin-labelled probe (1 mg/ml) diluted in hybridization buffer was heat-denatured at 80�c, cooled on ice, and added to the sections for hybridization overnight at 55�c (14– 16 h). unbound probe was removed by sequential washes of buffer 1 (50% formamide, 5 � ssc, ph 4.5, and 1% sds in pbt) followed by buffer 2 (50% formamide, 2 � ssc, ph 4.5, and 0.1% tween-20 in pbt) at 55�c. the sections were further washed in 0.1% tween-20 tris-buffered saline (tbst) before incubation in blocking solution (1% blocking reagent (roche diagnostics scandinavia, stockholm, sweden)) together with 1:5,000 diluted anti-digoxigenin alkaline phosphates conjugated antibody (roche diagnostics scandinavia) overnight at 4�c. unbound antibodywasremovedby sequentialwashes with2mm levamisole (gtf fisher, stockholm, sweden) in tbst followed by washes with 2 mm levamisole in ntmt (100 mm nacl, 10 mm tris-hcl, ph 9.5, 50 mg mgcl2, and 0.1% tween-20). sections were developed in bm purple ap substrate (roche diagnostics scandinavia) at 37�c. after mounting in glycerol,thesectionswerephotographedinaleicamz16f dissection microscope using adfc300fx cameraand firecam software (leica microsystem). images were adjusted in adobe photoshop cs3 (san jose, ca, usa) by adjusting the levels and brightness/contrast. figures were assembled in adobe illustrator cs3. ethical considerations the number of mice used in the study was reduced by allowing one batch of mice (n = 17 ctrl; n = 12 cko) for analysis in the elevated plus maze (epm), the multivariate concentric square field� (mcsf), the social interaction, and the dominance tube test in this order, before being processed for dissection and monoamine analysis by high-pressure liquid chromatography (hplc). two separate batches of mice were analysed in the porsolt forced swim test (n = 19 ctrl; n = 13 cko) and in an amphetamine provocation set-up (n = 9 ctrl; n = 9 cko), respectively. amphetamine challenge amphetamine-induced activity was analysed in automated activity boxes, so-called locoboxes (locobox, kungsbacka reglerteknik ab, kungsbacka, sweden) that each consists of a plastic cage (55 � 55 � 22 cm) placed inside a ventilated and illuminated (10 lux) cabinet. inside the locobox, a grid of photo beams (beams spaced by 5 cm) records the movement of the mouse. on the first experimental day, each mouse was allowed to explore the cage for 30 min in order to measure baseline activity, and was thereafter injected with 10 ml/kg saline (i.p.) and allowed back to the locobox for an additional 90 min of monitoring. twenty-four hours later, the same protocol was used, but instead of saline the mice were injected i.p. with 1.5 mg/kg of amphetamine. the locoboxparameters scored were locomotion, peripheral activity, and corner activity as previously described (16). locomotion is defined by two beam breaks anywhere in the box; peripheral activity is defined by two beam breaks in the periphery of the box; corner activity is defined by two beam breaks in any of the four corners of the box. data were analysed by two-way anova by prism software (graphpad) (variables: genotype, cko versus ctrl; time, 30 min for pre-injection and 90 min for post-injection). epm, mcsf, and social interaction analyses followed by monoamine analysis the behavioural set-ups for the epm, mcsf, social interaction, and social dominance tests, performed in this order, have been described previously (13). after cervical dislocation, brains were rapidly dissected and mounted into a coronal mouse brain matrix (ted pella, 146 k. nordenankar et al. http://www.genepaint.org inc., redding, ca, usa)kept on icefrom which 1-mm slices were prepared. the nucleus accumbens, dorsal striatum, hippocampus, and prefrontal cortex (pfc) from cko mice and littermate controls were microdissected from these slices. samples were instantly frozen on dryice and kept frozen at �80�c until analysis. after ultrasound homogenization (sonifier cell disruptor b30, branson ultrasonics, danbury, ct, usa) in 0.1 m perchloricacid containing 2.5mm of na2edta and subsequent centrifugation (10,000 rpm, 10 min), the supernatant was collected for analysis. the supernatantwasremoved,andthepelletwashedwithdoubledistilledwaterthreetimes.amixtureof0.2maceticacid and0.2mphosphoricacid(8:2,vol/vol)wasadded,and samples were shaken again for 15 minutes. the eluate was analysed for dopamine (da), 3,4-dihydroxyphenylacetic (dopac) and noradrenaline (na), 3-methoxytyramine (3-mt) and serotonin (5-ht) using hplc with electrochemical detection as previously described (13,34,35). the porsolt forced swim test the porsolt swim test represents a model for interpretation of animal depression-like behaviour (36,37). each mouse was placed in a plexiglas cylinder (ø20 cm) filled with 25 ± 2�c water. the mice were videotaped during two consecutive 12-minute trials with 24 hours in between and scored (anitracker software) for total time spent swimming (defined as at least three paws paddling). statistical analysis a non-parametric mann–whitney u test was used for statistical analyses of differences between genotype groups (behaviour and monoamine content) except in the case of behaviour analyses over time when twoway anova or repeated measures two-way anova were implemented. multiple testing included bonferroni’s post-hoc comparisons unless otherwise stated. the chi-square test was used for statistical comparison in the elevated plus maze to evaluate the difference in the number of mice entering and not entering the outer open arm. values in graphs are expressed as mean ± sem. results conditional deletion of vglut2 expression in the olfactory bulb, cortex, hippocampus, and part of the amygdala complex the emx1-driven cre activity in the emx1ires-cre knock-in mouse line was investigated previously by reporter-gene analysis of the r26r strain and shown to be dorsally located in cortical subdivisions of the telencephalon from embryonic day (e) 10.5 and to stay regionally distributed also in the adult telencephalic area (31). areas characterized by emx1-driven cre activity in the adult included the whole neocortex and the entire hippocampal formation as defined by the subiculum, hippocampus proper, and the dentate gyrus. the olfactory bulb and several, but not all, subpopulations of the amygdala were also characterized by emx1-driven cre activity. cre-active areas included the lateral (l), centrolateral (cl), basolateral (bl), and basomedial (bm) amygdala, while leaving untouched the adjacently located medial (me) and central (c) amygdaloid nuclei as well as the bed nucleus of stria terminalis (bst) (the two last-mentioned a part of the so-called extended amygdala). somewhat weaker activity was detected in the anterior cortical (aco) amygdala (31). guided by this previous report, brain sections from vglut2f/f;emx1-cre(tg/wt) cko mice and littermate vglut2f/f;emx1-cre(wt/wt) control mice, produced as described in ‘materials and methods’, were analysed by in situ hybridization (ish) at the adult stage in order to ascertain the targeted deletion of vglut2. as we previously reported, expression of vglut2 in the adult telencephalon was found in the retrosplenial group (rsg) and layers iii and v/vi (figure 1e, q) (13). expression was also found in the mitral and deep periglomerular cells of the olfactory bulb (figure 1a, i), much resembling the expression previously reported in the rat olfactory system during embryonal development (14). in accordance with the reported cre-activity of the emx1ires-cre mouse line (31), vglut2 mrna expression was found deleted in all of these telencephalic areas in the cko brains (figure 1r, e, b and j). further, as described before, vglut2 expression in controls was evident in the subiculum, but not in the rest of the hippocampal formation (figure 1q), and in several subnuclei of the amygdala complex, including the aco, the bm, and anterior and posteroventral me nuclei (figure 1m, o). in the cko mice, the subicular and bm vglut2 expression was found absent, again in accordance with the reported cre activity (figure 1r, n) (31). however, vglut2 expression in the aco appeared reduced only (figure 1n), in line with the limited cre activity described in this area (31), while the expression in the me appeared normal, fitting the apparent lack of cre activity in these areas (figure 1n, p) (31). in addition, we addressed vglut2 expression in all of the remaining brain and found it normal compared to control mice (figure 1g, h; table i) (supplementary table s1, available online). thus, in the adult vglut2f/f;emx1-cre(tg/wt) cko mice, deletion of vglut2 expression in the embryonal telencephalon 147 http://informahealthcare.com/doi/suppl/10.3109/03009734.2015.1032454/suppl_file/10.3109/03009734.2015.1032454_suppl.doc the targeted deletion of vglut2 is specific to the olfactory bulb, cortical subregions, the subiculum, and the bm and aco areas of the amygdala, regions that all express the emx1ires-cre bicistronic construct. normal overall histology and distribution of cellular marker genes to verify that the overall brain anatomy was normal in the vglut2f/f;emx1-cre(tg/wt) cko mice, ish analysis of one additional glutamatergic marker, vglut1, and of one marker for inhibitory neurons, the vesicular amino acid transporter (viaat), was performed. in contrast to the restricted expression of vglut2 in the telencephalon, vglut1 is prominently expressed in this area (7,9,11-13,38). strong vglut1 expression was found throughout the neocortex and hippocampal formation and also in several subnuclei of the amygdala complex (figure 2a, c), which is in accordance with previous studies. vglut1 was most prominently expressed in the bl, bm, and me nuclei of the amygdala. no altered distribution was seen in the cko brains (figure 2b, d; and data not shown). further, expression of vglut1 in the mid-brain, a b i j k l m n o p q r c d e f g h mi control vglut2f/f;emx1-cre vglut2 mrna vglut2 mrna control vglut2f/f;emx1-cre mi mi mi 3.92 3.92 3.92 3.92 rsg rsg rsg bl bl bm bm aco mepd mepd -1.94 -1.94 rsgrsg sub -2.80 sub -2.80 mepv mepv aco me me -1.70 -1.70 -1.22 -1.22 rsg rsg rsg -2.2-1.22 -2.2-1.22 -1.34-1.80 -1.34-1.80 -3.80-3.40 gl gl gl gl figure 1. specific deletion of vglut2 in selected forebrain target areas. floating in situ hybridization on coronal brain (70 mm) sections from control mice and vglut2f/f;emx1-cre cko mice (a–h) using a dig-labelled vglut2 probe. close-ups as indicated in i–r, which demonstrate that cells expressing vglut2 mrna was absent in the mitral cell (mi) layer and gi layer in the olfactory bulb (i–j). vglut2 mrna was also absent in the rsg of the medial cortex in the vglut2f/f;emx1-cre mice (k, l). there is a loss of vglut2 mrna in the bma, in the aco the mrna is partially deleted, and the me amygdala and mepv are unaltered in the vglut2f/f;emx1-cre mice (m–p). vglut2 mrna positive cells were present in the sub in control mice but not in vglut2f/f;emx1-cre cko mice (q, r). aco = anterior cortical amygdaloid area; bl = basolateral amygdaloid nucleus; bm = basomedial amygdaloid nucleus anterior part; cko = conditional knock-out; dig = digoxigenin; gi = periglomerular layer; me = medial amygdaloid nucleus; mepv = posteriorventral medial amygdaloid nucleus; mi = mitral cell layer; rsg = retrosplenial group; sub = subiculum; bregma interval (dorsal, ventral) is shown in lower right corner. 148 k. nordenankar et al. cerebellum, and pons appeared normal in the cko brains compared to controls (figure 2e, f). viaat expression was also detected, as expected, in inhibitory populations and appeared normal in the cko brain compared to controls (figure 2g, h). together, these results showed normal cellular distribution of excitatory and inhibitory populations in the vglut2f/f;emx1-cre(tg/wt) cko mice, and although this was not quantitative, by ish analysis, we did not detect differences in expression levels in these areas. normal basal and amphetamine-induced activity in agreement with unaltered da levels the behavioural analyses of the previously reported vglut2f/f;camkii-cre cko mice revealed a spontaneous hyperactivity which was further accentuated above the levels of the controls when mice were challenged with an acute dose of amphetamine (13). this increased behavioural activation was correlated with increased basal levels of dopamine in the striatum, which we suggested as the underlying cause of the hyperactivity (13), in accordance with the role of dopamine in table i. summarized results from in situ hybridization evaluating the presence or not of vglut2 mrna in brain structures from three adult vglut2f/f;emx1-cre cko mice and three control mice. structure ctrl cko pallium neocortex + – rsg + – subiculum + – hippocampus ca1, ca3, dg – – septohippocampus – – ventral endopiriform nucleus + –* piriform cortex + –* claustrum + –* border regions lateral septum – – medial septum – – rostroventral septum – – endopiriform nucleus – – lateral amygdala – – bm + –* me + + aco + –* subpallium olfactory tuberculum + + striatum – – ventral pallidum – – nucleus accumbens nd nd bed nucleus stria terminalis nd nd lateral olfactory tract nd nd olfactory bulb mitral cell layer + – external plexiform – – internal plexiform – – periglomerular layer + –* granule cell – – ependymal layer – – the expression for the emx1-cre transgene was described previously (gorski et al. 2002 (31)), and those structures in which emx1-cre was there shown to be expressed were analysed for vglut2 mrna and listed here. + = expression of vglut2 mrna emx1-cre transgene; – = no expression; –* = low amounts of expression; cko = conditional knock-out; dg = dentate gyrus; nd = not determined. a b c d e f g h me mebm bm aco aco -1.22 control vglut2f/f;emx1-cre v ia a t m r n a v g lu t1 m r n a -1.22 -1.22 -1.22 -5.80-6.64-5.80 -1.22-1.58 -1.22-1.82 bl bl figure 2. verification that the overall gross anatomy is normal in the vglut2f/f;emx1-cre mice compared to control mice. floating in situ hybridization on coronal brain (70 mm) sections from control and vglut2f/f;emx1-cre cko mice using a dig-labelled viaat (a, b) or vglut1(c–h) probe. close-ups show that aco and bm do not express vglut1 mrna, while bl and part of me express vglut1 mrna. aco = anterior cortical amygdaloid area; bl = basolateral amygdaloid nucleus; bm = basomedial amygdaloid nucleus anterior part; me = medial amygdaloid nucleus. bregma interval (dorsal, ventral) is shown in lower right corner. deletion of vglut2 expression in the embryonal telencephalon 149 general activational response (39). caretaker handling of the vglut2f/f;emx1-cre(tg/wt) cko mice now revealed that these mice were calmer than the vglut2f/f;camkii-cre cko mice, which we previously had experienced as difficult to handle due to the strong hyperactivity. this observation tentatively suggested that the telencephalic deletion of vglut2 from embryo development (vglut2f/f;emx1-cre cko) had a different effect on behaviour than had the postnatal deletion (vglut2f/f;camkii-cre cko). by comparing spontaneous and amphetamine-induced activity between the vglut2f/f;emx1-cre cko mice and their baseline activity locomotion saline 10 mg/kga. l o c o m o ti o n ( b e a m b re a k s ) injection 1000 750 500 250 0 time (min) -30 -10 0 10 30 50 70 90 baseline activity locomotion amphetamine 1.5 mg/kgb. l o c o m o ti o n ( b e a m b re a k s ) injection 1000 750 500 250 0 time (min) -30 -10 0 10 30 50 70 90 control vgt2f/f;emx1-cre periferal activity periferal activity c. l o c o m o ti o n ( b e a m b re a k s ) 1000 750 500 250 0 time (min) -30 -10 0 10 30 50 70 90 d. l o c o m o ti o n ( b e a m b re a k s ) 1000 750 500 250 0 time (min) -30 -10 0 10 30 50 70 90 corner activity corner activitye. l o c o m o ti o n ( b e a m b re a k s ) 0 100 200 300 l o c o m o ti o n ( b e a m b re a k s ) 0 100 200 300 time (min) -30 -10 0 10 30 50 70 90 f. time (min) -30 -10 0 10 30 50 70 90 figure 3. the vglut2f/f;emx1-cre mice do not show any altered response to amphetamine. the initial response to novel environment (30 min) and overall response after i.p. injection of saline (90 min), 10 mg/kg, in locomotion, periferal activity, and corner activity are unaltered in the vglut2f/f;emx1-cre cko mice compared to control mice (b, d, f). on day 2, the mice were subjected to the activity boxes for 30 min and were thereafter administered 1.5 mg/kg amphetamine i.p. and were recorded for 90 min (a, c, e). the arrows in the graphs depict the time of saline and amphetamine injection. data were analysed with two-way anova and showed no significant interactions (effect of genotype). data are represented as mean ± sem. cko = conditional knock-out. 150 k. nordenankar et al. littermate control mice, no statistically relevant difference in either locomotion, corner activity, or peripheral activity was detected between genotypes either preor post-injection by saline or amphetamine (figure 3a–f) (all statistical data are shown in supplementary table s2, available online). further, we did not find any weight differences between either male (ctrl n = 11, 30.4 ± 0.7 g; cko n = 6, 28.7 ± 0.4 g, p = 0.13) or female mice (n = 10, 22.1 ± 0.8 g; cko n = 8, 22.5 ± 0.9 g, p = 0.75) of the different genotype groups, indicating normal food intake and activational levels in the cko mice. biochemical detection of da and its metabolite dopac as well as noradrenalin, 3-mt, and 5-ht in tissue dissected from the dorsal (caudate putamen) and ventral (nucleus accumbens) striatum did not reveal any differences between the vglut2f/f;emx1-cre(tg/wt) cko and control mice (table ii). taken together, these results show that adult mice lacking vglut2 in selected telencephalic areas from early developmental stages show normal spontaneous and amphetamine-induced activity as well as normal levels of monoamines da, na, 5-ht, and their metabolites in the striatum. decreased avoidance of open areas and time spent in shelter suggest an anxiolytic phenotype altered social skills, as measured by the social interaction test and the dominance tube test, were part of the behavioural phenotypes described for the vglut2f/f;camkii-cre cko (13). by using the same paradigms here, no differences between the vglut2f/f;emx1-cre(tg/wt) cko and control mice were detected either in the social interaction test (p = 0.85) or in the dominance tube test (p = 0.58) (figure 4a, b). also, no difference between genotype groups could be discerned in the porsolt forced swim test, a model for despair-like behaviour (trial 1, p = 0.86; trial 2 p = 0.54) (figure 4c), which previously revealed an altered response in the vglut2f/f; camkii-cre cko mice (13). behavioural analysis of relevance to anxiety in humans include an apparatus which takes into advantage the rodent’s preference for familiar, dark, and/or enclosed areas (40). the epm, which allows exploration of open versus enclosed areas is perhaps the most commonly used such method. the mcsf, which contains multiple challenges including both an open field and a dark, sheltered space, as well as challenges related to risk-assessment and risk-taking, is another valuable paradigm which we have used before (24,41,42). during a 10-minute trial, vglut2f/f;emx1-cre cko and control mice were analysed in the epm. the number of entries (frequency) into each area; i.e. the centre, the closed, and the inner and outer segments of the open arm were scored (figure 4d), as was the time spent (duration) in each of these compartments (figure 4d). no differences in either total activity (the sum of all frequencies) in the maze (p = 0.84) or in any other parameter were identified (centre frequency, p = 0.83; closed arm frequency, p = 0.60; inner arm frequency, p = 0.57; outer arm frequency, table ii. levels of monoamines and dopamine metabolites (ng/g of wet tissue) of vglut2f/f;emx1-cre cko (n = 12) and control (n = 18) mice. the data show no statistically significant alterations between control and cko mice. data are presented as mean ± sem. brain region metabolite ctrl vg2f/f;emx1-cre p value nucleus accumbens dopamine 0.0129 ± 9.1e-04 0.0122 ± 9.5e-04 0.21 dopac 0.0053 ± 3.9e-04 0.0049 ± 5.7e-04 0.48 dopac/da 0.4784 ± 3.6e-02 0.4708 ± 4.6e-02 0.99 na 0.0015 ± 2.1e-04 0.0015 ± 3.1e-04 0.82 3-mt 0.0032 ± 2.7e-04 0.0025 ± 2.9e-04 0.26 5-ht 0.0040 ± 3.1e-04 0.0030 ± 3.8e-04 0.15 caudatus putamen dopamine 0.0197 ± 1.4e-03 0.0200 ± 2.2e-03 0.08 dopac 0.0059 ± 5.2e-04 0.0066 ± 7.0e-04 0.51 dopac/da 0.3386 ± 4.7e-02 0.2958 ± 2.1e-02 0.47 na 0.0004 ± 8.0e-05 0.0003 ± 4.7e-05 0.51 3-mt 0.0038 ± 5.0e-04 0.0045 ± 4.8e-04 0.36 5-ht 0.0012 ± 2.2e-04 0.0015 ± 2.1e-04 0.24 p < 0.05 (mann–whitney u test). nucleus accumbens corresponds to the ventral striatum; caudatus putamen to the dorsal striatum. 3-mt = 3-methoxytyramine; 5-ht = 5-hydroxytryptamine; cko, conditional knock-out; dopac = 3,4-dihydroxypheneylacetic acid; na = noradrenalin. deletion of vglut2 expression in the embryonal telencephalon 151 http://informahealthcare.com/doi/suppl/10.3109/03009734.2015.1032454/suppl_file/10.3109/03009734.2015.1032454_suppl.doc http://informahealthcare.com/doi/suppl/10.3109/03009734.2015.1032454/suppl_file/10.3109/03009734.2015.1032454_suppl.doc social interaction total activity closed arm total activity central circle central circle central field dark corner outer inner arm outer open arm not entering entering dominance tube test porsolt swin test elevated plus maze * * * * * multivarite concentric square field a. b. c. d u ra ti o n ( s ) 100 80 40 60 20 0 d. e. f re q u e n c y ( n u m b e r o f e n tr ie s ) 80 40 60 20 0 d u ra ti o n ( s ) d u ra ti o n ( s ) 500 400 300 200 100 0 d u ra ti o n ( s ) 50 40 30 20 10 0 n u m b e r o f e n tr ie s i n o u te r o p e n a rm 0 5 10 15 20 0 5 10 15 20 d u ra ti o n ( s ) 0 50 100 150 200 n u m b e r o f w in s 0 0.5 1.0 1.5 0 5 10 15 im m o b il e ( s ) 150 100 50 0 f re q u e n c y ( n u m b e r o f e n tr ie s ) f re q u e n c y ( n u m b e r o f e n tr ie s ) 150 100 50 0 trial 1 trial 2 control vgt2f/f;emx1-cre figure 4. no social deficits or altered despair-like behaviour, but decreased avoidance of open areas and time spent sheltered. social behaviour analysis on adult vglut2f/f;emx1-cre cko mice and control littermates (a, b). the total time spent interacting during a 10-min social interaction session shows no alteration in the vglut2f/f;emx1-cre cko mice compared to control mice (a). the number of wins in the social dominance tube test shows that the vglut2f/f;emx1-cre cko mice do not display any altered dominance compared to control (b). the vglut2f/f;emx1-cre mice spent equal time swimming during two 12 min swimming sessions separated 24 h apart in the porsolt swim test (c). the total activity and the duration in the threedifferentareasintheelevatedplusmazeshownosignificantdifferencesbetweengenotypes.thenumberoftotalentriesintheouteropenarm is statistically different between the vglut2f/f;emx1-cre cko mice compared to control mice (*p < 0.05, chi-square test) (d). the total activity as measured in the multivariate concentric square field (mcsf) shows no altered behaviour for the vglut2f/f;emx1-cre cko mice compared to control mice.thefrequencyinthecentralcircleanddurationinthecentralcircleaswellasincentralfieldaresignificantlyincreasedforthevglut2f/f;emx1-cre mice compared to control mice; the duration in the dark corner is significantly decreased in the vglut2f/f;emx1-cre cko mice compared to control mice (e). values represent mean ± sem. * p < 0.05 compared to control littermates. cko = conditional knock-out. p = 0.39; centre time, p = 0.20; closed arm time, p = 0.77; inner open arm time, p = 0.28; and outer open arm time; p = 0.65). however, when analysing the number of mice that entered the outer open arm, we found that significantly more cko than control mice actually visited this exposed area (chi-square, p = 0.010) (figure 4d). to analyse this putatively anxiolytic phenotype further, we turned to the mcsf paradigm (all statistical data are shown in supplementary table s3, available online). while displaying the same overall activational level as the controls (figure 4e), the vglut2f/f;emx1-cre(tg/wt) cko mice showed a significantly decreased avoidance of the open areas as displayed by higher frequency of visits inthecentralcircleandthetimespentthere(figure4e). theckomicealsodisplayeddecreasedshelter-seeking in the dark room and increased time in the central field. decreasedavoidanceofopenareasandofthedarkroom were previously identified in the vglut2f/f;camkii-cre cko mice which also showed a general hyperactivity and increased risk-assessment and exploratory behaviour. compared to the vglut2f/f;camkii-cre cko mice, the vglut2f/f;emx1-cre cko mice thus show a milder behavioural phenotype, but one which is more specifically centred around anxiolysis instead of strong hyperactivity in several different aspects. taken together, the behavioural alterations identified in the epm and the mcsf show that the vglut2f/f;emx1-cre cko mice have an anxiolytic phenotype. discussion contrary to the strong behavioural phenotype observed when vglut2 was gene-targeted in the forebrain from postnatal week 3 and onwards (vglut2f/f;camkii-cre mouse line) (13), the embryonic onset of deletion used in the current study (the vglut2f/f;emx1-cre mouse line) produced a milder behavioural phenotype focused around anxiolysis. as vglut2 is broadly expressed in the embryonic mouse brain, including within the dorsal telencephalon (16,17), the embryonal gene targeting in the current study is likely to affect brain development and adult function profoundly differently than a targeting event occurring from 3 weeks of age. further, the earlier temporal onset of vglut2 deletion in the vglut2f/f;emx1-cre mouse line (e10.5 onwards) compared to the vglut2f/f;camkii-cre mouse line (p20 onwards) might lead to the milder behavioural phenotype due to compensations made possible during embryogenesis, but which fail to rescue functions in the postnatal life. in addition to developmental differences between the vglut2f/f;camkii-cre and the vglut2f/f;emx1-cre transgenic mouse lines, an important factor that may contribute to the identified permutations is the genetic background of the mice. although both kept on a hybrid of c57/bl6j and sv129, the exact quantity of each contributor is difficult to ascertain. however, as the cko mice of each line are compared to littermate control mice of the identical genetic background, the impact of genetic background for the overall interpretation of the result is limited. importantly, although the spatial extent of gene targeting appeared the same in the neocortex, subiculum, and olfactory bulb in the cko mice of both the vglut2f/f;emx1-cre and vglut2f/f;camkii-cre mouse lines, vglut2 expression was somewhat differentially targeted within the amygdaloid subnuclei. in the vglut2f/f;camkii-cre cko mice, all amygdaloid expression of vglut2 was lacking as identified by our previous ish analysis (13). in the vglut2f/f;emx1-cre cko mice, on the other hand, only the vglut2 expression in the bm amygdala was completely lacking, while expression in the aco nucleus was merely blunted and the me amygdala showed normal vglut2 expression. this observation shows that the spatial activity within the amygdala differs between the emx1-cre and the camkii-cre transgenes. the amygdaloid complex, consisting of a series of heterogeneous subnuclei, is known to be important for aspects of both fear and anxious behaviour. anxiety, experienced as unease, dread, apprehension, and similar, is a negativevalenced emotion characterized by sustained hyperarousal in response to uncertainty (42-44). experienced by all individuals, anxiety serves to guide decision-making by contributing to evaluation of risk and need for defence or avoidance. when anxiety becomes excessive or pathological, however, it may lead up to the substantial group of anxiety disorders that include generalized anxiety disorder and obsessive-compulsive disorder (42,44). a vast number of studies have shown that patients with various kinds of anxiety disorder show amygdala hyperactivity in response to anxiogenic stimuli, leading to the view of amygdala hyperfunction as a key component of human anxiety disorder (45-49). the contribution of specific amygdaloid subnuclei is not firmly established yet, but both inhibitory and excitatory amygdaloid nuclei are implicated in the neurocircuitry of anxiety. in the mouse, the excitatory subnuclei express either vglut1 or vglut2, or both, as shown above. the interconnectivity between the amygdaloid nuclei has been investigated thoroughly (described in detail in (50)). the lateral (l; vglut1-expressing) and the me (vglut1/vglut2-expressing) nuclei send reciprocal projections to the basal (b) and accessory basal (ba) amygdaloid nuclei, which also receive an excitatory drive from the subiculum (vglut1/vglut2expressing). excitatory bl amygdala (vglut1) and deletion of vglut2 expression in the embryonal telencephalon 153 http://informahealthcare.com/doi/suppl/10.3109/03009734.2015.1032454/suppl_file/10.3109/03009734.2015.1032454_suppl.doc http://informahealthcare.com/doi/suppl/10.3109/03009734.2015.1032454/suppl_file/10.3109/03009734.2015.1032454_suppl.doc local inhibitory projections gate the activity of the inhibitory nucleus central amygdala which in turn projects out from the amygdala to brain-stem, basal forebrain, and hypothalamus regions that control autonomic, hormonal, and behavioural responses to emotional situations. additional connectivity between the above-mentioned amygdaloid nuclei is also substantially involved in the regulation of emotional responses, which were only briefly summarized here for an overview of vglut1 and vglut2 expression. although a vglut2-expressing nucleus, the me amygdala was not targeted in the vglut2f/f;emx1-cre cko mice due to the lack of emx-cre-activity in this particular nucleus; thus the anxiolytic phenotype of these cko mice cannot be attributed to a loss of excitatory drive from this specific subnucleus. on the other hand, the me nucleus was vglut2-targeted in the vglut2f/f;camkii-cre cko mice, possibly explaining the more profound anxiolytic phenotype in these mice, which included increased risk-taking. in the vglut2f/f;emx1-cre cko mice, vglut2 was deleted in the bm amygdala and partly in the aco. further studies would be required to reveal if the loss of vglut2 in these particular areas contributes to the observed behavioural phenotypes. importantly, based on the rather broad deletion of vglut2 in the entire telencephalon during embryonal development, it is conceivable that the cause of the anxiolytic phenotype is independent on the restricted targeting of vglut2 in the bma and aco. for example, as mentioned above, the importance of the subiculum, which expresses both vglut1 and vglut2 and which we show is lacking vglut2 in the cko mice (both in the vglut2f/f;camkii-cre and vglut2f/f;emx1-cre mouse lines), should not be neglected. the subiculum innervates the b/ba amygdaloid nuclei, and loss of vglut2 in this hippocampal area may substantially decrease the glutamatergic drive into the amygdala, altering the final balance of output from the cea so that the level of anxiety is measurably decreased. hippocampus function in the cko mice of a similar vglut2f/f;emx1-cre mouse line was in fact previously shown to be characterized by reduced evoked glutamatergic transmission and release probability in the ca3-ca1 region (51), a feature that might also contribute to the anxiolytic phenotype. further addressing the functional roles ascribed to the amygdaloid nuclei expressing vglut2, the bm, m, and aco amygdala nuclei have all been shown to mediate feeding behaviour via extensive connectivity directly with e.g. the hypothalamus (52,53). no alteration in body weights were observed in our cko mice, an observation which, taken together with the normal extent of spontaneous activity, indicates a normal amount of food intake. in summary, the main findings of the current study indicate that expression of vglut2 in the dorsal telencephalic area from mid-gestation is not of major importance for establishing the general activational level, responsiveness to the psychostimulant amphetamine, or for sociability, behaviours that all are affected when vglut2 is removed in the same areas during adolescence. however, expression of vglut2 in the dorsal telencephalic area from mid-gestation is shown to be involved in the regulation of anxiety, possibly by providing excitatory input into the amygdala or by ascertaining a balanced intraamygdaloid connectivity. given this restricted behavioural phenotype, the vglut2f/f;emx1-cre mouse line might be considered a useful tool for further analysis of the neurocircuitry and neurobiology of anxiety. acknowledgements the authors thank professor klas kullander, uppsala university, for constructive input during the initiation of this study; professor kevin jones, university of colorado-boulder, and professor rudiger klein, max-planck institute for neurobiology, for providing the emx-cre mouse line. we thank dr carolina birgner, uppsala university, for performing the hplc analysis, and drs daniel andersson and erik stuber, both at gothenburg university, for providing technical expertise in hplc analysis and behaviour analysis, respectively. funding: this work was supported by grants from the swedish medical research council (2007-5742, 20114747), uppsala university, the swedish brain foundation, the hållsten research foundation, and the foundations of åke wiberg and åhlén. declaration of interest: the authors 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medial nucleus of the amygdala: a phal study in the rat. j comp neurol. 1995;360:213–45. supplementary material available online supplementary table s1. supplementary table s2. supplementary table s3. 156 k. nordenankar et al. http://www.ncbi.nlm.nih.gov/pubmed/14743184?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/14743184?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/14743184?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/11840475?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/11840475?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/11840475?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/1986388?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/1986388?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/18495312?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/18495312?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/18495312?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/16356558?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/16356558?dopt=abstract 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http://www.ncbi.nlm.nih.gov/pubmed/18483136?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21215728?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21215728?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23136427?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23136427?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/12724147?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/8522644?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/8522644?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/8522644?dopt=abstract http://informahealthcare.com/doi/suppl/10.3109/03009734.2015.1032454/suppl_file/10.3109/03009734.2015.1032454_suppl.doc http://informahealthcare.com/doi/suppl/10.3109/03009734.2015.1032454/suppl_file/10.3109/03009734.2015.1032454_suppl.doc http://informahealthcare.com/doi/suppl/10.3109/03009734.2015.1032454/suppl_file/10.3109/03009734.2015.1032454_suppl.doc abstract introduction material and methods animals generation of vglut2f/f;emx1-cre mice tissue sectioning and rna probes in situ hybridization on free-floating sections ethical considerations amphetamine challenge epm, mcsf, and social interaction analyses followed by monoamine analysis the porsolt forced swim test statistical analysis results conditional deletion of vglut2 expression in the olfactory bulb, cortex, hippocampus, and part of the amygdala complex normal overall histology and distribution of cellular marker genes normal basal and amphetamine-induced activity in agreement with unaltered da levels decreased avoidance of open areas and time spent in shelter suggest an anxiolytic phenotype discussion acknowledgements declaration of interest references eighty years of research on islet amyloidosis in uppsala full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 eighty years of research on islet amyloidosis in uppsala gunilla t. westermark, marie oskarsson, arne andersson & per westermark to cite this article: gunilla t. westermark, marie oskarsson, arne andersson & per westermark (2015) eighty years of research on islet amyloidosis in uppsala, upsala journal of medical sciences, 120:2, 117-123 to link to this article: https://doi.org/10.3109/03009734.2015.1037032 © informa healthcare published online: 22 apr 2015. submit your article to this journal article views: 461 view related articles view crossmark data https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://doi.org/10.3109/03009734.2015.1037032 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1037032 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1037032 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1037032&domain=pdf&date_stamp=2015-04-22 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1037032&domain=pdf&date_stamp=2015-04-22 upsala journal of medical sciences. 2015; 120: 117–123 eighty years of research on islet amyloidosis in uppsala gunilla t. westermark1, marie oskarsson1, arne andersson1 & per westermark2 1department of medical cell biology, uppsala university, uppsala, sweden, and 2department of immunology, genetics and pathology, uppsala university, uppsala, sweden key words: alzheimer’s disease, amyloid, iapp, senile amyloidosis, type 2 diabetes introduction scienceissaidtobefreeinswedeninthesensethatscientistsareallowedtodoresearchonanysubject,butthereisone veryseriouslimitation:theworkhastobefunded.thelatteraspecthasbecomeincreasinglyevidentduringlateryears. in many ways, research was more free decades ago when more money was directly allocated to universities and their departments.inthisway,islandsofresearchcouldmoreeasilydeveloparoundasuccessfulscientist,andifhe(itusedto bemen,atleastinmedicalresearch)wasluckyhemightrecruitanumberofpupilswhocouldtaketheresearchfurther. themostwell-knownexampleofthisscenarioinuppsalaofcourseiscarlvonlinné(1707–1778)withhislargegroup of famous disciples. on the other hand, it is a risk that a scientific line fades out rapidly when a leading and charismatic person dies or moves. there is a multitude of examples of this phenomenon. the first demonstration of islet amyloid in uppsala during last decades amyloidosis has become a hot subject in medical research. quite evidently, the number of scientists working on different aspects of amyloid has increased, also in uppsala. but how was the situation earlier at uppsala university? the first scientist interested in amyloid seems to have been a professor of pathology, nils gellerstedt (figure 1). gellerstedt was known as an excellent clinical pathologist and seems to have had quite wide interests in a number of different conditions. he and some of his pupils published several papers on age-related (‘senile’) amyloid forms. most interesting is perhaps an article published in 1938 dealing with amyloid in the islets of langerhans (1). that article by gellerstedt was initiated by his observation of the presence of islet amyloid in two patients with diabetes mellitus. the scientific activity in upsala medical society (upsala läkareförening) must have been very high at that time with many oral presentations each month. in the record of a meeting on 13 december 1935 it appears that gellerstedt gave a talk entitled ‘elective amyloidosis of islet of langerhans as a cause of diabetes’ (figure 2). unfortunately there is no written report from that meeting, but gellerstedt refers to these two cases as a background to his following, extended study. according to the title of his presentation in uppsala gellerstedt regarded the found islet amyloid as the cause of diabetes in these two patients. in his more elaborated investigation from 1938 he studied sections from the pancreatic tail in 181 consecutive autopsies among which 110 were from subjectsaged50yearsormore.gellerstedtmostcertainlywasdisappointedbythenewfindingsofhislargerstudy.as expected, he found islet amyloid in subjects with diabetes but perhaps more surprisingly he found the alteration to be quite common among non-diabetic individuals as well, some of them with quite pronounced depositions. gellerstedt finally concluded that it was not possible to see a direct causal relationship between islet amyloid correspondence: per westermark, rudbeck laboratory, se 751 85 uppsala, sweden. e-mail: per.westermark@igp.uu.se (received 26 march 2015; accepted 27 march 2015) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1037032 http://informahealthcare.com/journal/ups mailto:per.westermark@igp.uu.se and diabetes. this view has since then been the predominating one in the field of diabetes research until the last 10 years or so. remarks by gellerstedt on the histological appearance of islet amyloid for a very long time there was a discussion on whether hyaline in the islets of langerhans was to be regarded as amyloid or not. amyloid was at that time only defined by its appearance in the light microscope, i.e. a hyaline substance with some tinctorial properties, particularly metachromasy with certain aniline dyes and affinity for congo red. the latter is still very much used in research and clinical practice although our present definition of amyloid is more sophisticated and based on protein molecular arrangements. gellerstedt entered that discussion figure 1. nils gellerstedt. professor of pathology, uppsala university 1948–1954. figure 2. extract from upsala läkareförenings förhandlingar, 1935. 118 g. t. westermark et al. and proposed that islet hyaline actually should be regarded as amyloid as judged from his experience with staining with methyl violet and congo red, beautifully illustrated by hand-coloured pictures in his article. however, he must have felt some uncertainty, since he put in a ‘para’ in the title and gave the hyaline deposits the designation (para)amyloid. the amyloid nature discussion was not finished with this paper but principally went on (2,3) for decades until amyloid in general was defined by its cross-b-sheet fibrillar structure (4-6), shown to be valid also for the islet form. the study by gellerstedt contains several other interesting points. he noted that the islet amyloid shows ‘classical colour reactions’ and that the metachromasy with methyl violet is very clear but the colour slightly different from general amyloid (‘allgemeine amyloid’, most probably aa amyloidosis), also shown in his illustration (figure 3). this observation shows that with an open mind it is possible to find interesting facts without very sophisticated methods. interestingly, the same colour difference can be seen with congo red as we use this stain today (figure 4). it is now understood that amyloid is not a single substance but a conformational figure 3. figure form gellerstedt’s paper from 1938 showing islet amyloid in a patient with systemic amyloidosis. although we do not know what type of systemic amyloidosis it is, aa amyloidosis was by far the most common form in sweden at that time. note the colour differences between the amyloids. the depicted section was stained with methyl violet. figure 4. a modern pancreatic section from a patient with aa amyloidosis due to rheumatoid arthritis and with islet amyloidosis of iapp nature. a similar colour difference as in figure 3 is seen in the two amyloid forms. congo red. islet amyloid in uppsala 119 state that at least 30 human proteins can obtain in vivo, resulting in deposition of protein fibrils (7). although these fibrils look similar by means of electron microscopy and have the same general protein conformation and some properties in common, it is clear that there are differences (8). however, gellerstedt did not speculate much about the nature of the amyloid material although he mentioned the possibility of an antigen–antibody reaction. this was a common theory concerning the nature of amyloid in general at that time. the article ends with the prophetic statement that ‘the problem “senile amyloidosis” contains many interesting questions, studies of which may open new ways in research’ (figure 5). this was certainly true as we will see in the following. reappearance of islet amyloid research in uppsala gellerstedt published some additional articles on senile amyloidosis but nothing more about islet amyloid. after his decease in 1954 gellerstedt’s knowledge capital on amyloid seems to have faded, and when one of us (p.w.) started his career in pathology in the late 1960s no one at the department knew anything about amyloid or even knew that someone had been interested in that subject. it was instead quite by accident that studies on islet amyloid started from a project on mast cells in diabetes (9). in an early quantitative study published in upsala journal of medical sciences it was shown that islet amyloid is initiated in a few islets but as the degree of amyloid infiltration augments the number of affected islets increases as well (10), as confirmed in a later study (11). another important finding, obtained by serial sectioning, was that deposits usually are spread throughout islets, particularly along capillaries, even in cases with comparably little amyloid (10). these novel and quite intriguing findings fit very well with the present conception of formation of amyloid as a nucleation-dependent process (12) and that amyloid actually can spread between islets in this way (13). iapp and the precipitation of islet amyloid gellerstedt, like most other researchers at that time, thought that amyloid hardly could be a cause of diabetes since it occurs in non-diabetic subjects as well and there are always a number of unaffected or less affected islets left in diabetic patients (1). at that time the nature of amyloid was only a matter of speculation, and its origin from blood vessels or fibroblasts was much discussed. later electron microscopic studies showed a close relationship between islet amyloid and b-cells (14). this finding was important in two respects. first, it indicated that islet amyloid is a figure 5. gellerstedt’s prophetic statements on p. 11 of his paper (1). in translation: ‘the problem “senile amyloidosis” contains many interesting questions, studies of which perhaps may open new ways in research. the most important is again serology. it is important to put more efforts than earlier into studies of the serological changes in the senium as well as immunobiological and allergic reactions in the healthy as well as sick organism, also in animal experiments. the studies must also widen into the psychiatric field; there are interesting problems regarding the development of senile and presenile psychoses due to antigen–antibody reactions and so on. — however, the pathologicalanatomical research is not to be disregarded. the author tends to believe that a systematic and careful study of the different body tissues for presence of atypical amyloid deposits in old humans, perhaps also in animals, could discover additional, non-rare findings of similar nature’. 120 g. t. westermark et al. product of b-cells. second, it showed that even if deposits were slight, they were widely spread within the islet and resulting in virtual holes in b-cell membranes, something that should result in functional loss. however, not much happened on islet amyloid until its composition of a previously unknown regulatory peptide was untangled (15). this peptide, named islet amyloid polypeptide (iapp, or amylin) was shown to be a 37-amino-acid residue peptide, most closely related to calcitonin-gene related peptide (cgrp). it was demonstrated to be expressed by b-cells where it is stored and released together with insulin (reviewed in (16)). initially, iapp was believed to cause insulin resistance in type 2 diabetes, but when it turned out that a superphysiological plasma concentration is needed to initiate such an effect the interest in function of iapp declined somewhat. however, the interest in iapp has increased considerably again, but now as an amyloid-forming peptide rather than for its effects via receptors. deposition of iapp amyloid is associated with a decline in the number of b-cells in human beings (17,18), even in the absence of diabetes (17). presence of islet amyloid is associated with b-cell apoptosis, and no apoptotic cells are detected in non-amyloid containing islets (11). why islet amyloid cytotoxicity is limited to b-cells and a-cells are spared (19) may suggest differences in cell surface-associated molecules. the mechanism is unclear, however. like the situation with alzheimer’s disease and the ab peptide, the interest has moved from the mature amyloid fibrils to pre-fibrillar aggregation states, often named oligomers or protofibrils. such aggregates of misfolded peptides, including ab and iapp, have been shown to exert toxic effects on cells in many systems in vitro. therefore, there is strong evidence that a major cause of the reduction of and impaired function of b-cells in type 2 diabetes is aggregation of iapp in the islets of langerhans. amyloidosis in transplanted islets studies based on transplantation of pancreatic endocrine tissue into nude mice—diabetic or non-diabetic—were performed in one of our laboratories in the 1970s. thus, for instance isolated human islets were injected intraperitoneally (20), and porcine islet-like cell clusters (21) or human fetal pancreas (22) were grafted under the renal capsule. when isolated human islets became more regularly available, thanks to the collaboration with the laboratory in brussels, we carried out extensive studies on the long-term in vivo effects of high glucose concentrations (23) and alloxan (24) on human islet tissue. it was then logical to look for the possible development of amyloid deposits in the same type of grafted tissue. to some surprise such deposits were demonstrated even in islet grafts from young, non-diabetic donors (25). these first studies were carried out on renal, subcapsular islet grafts, but extended studies revealed that islet amyloid developed in intrasplenic (figure 6) and liver grafts as well (26). we were also able to demonstrate the deposition of amyloid in transplanted islets prepared from transgenic mice expressing human iapp (27). against this strong evidence of an exaggerated development of amyloid in transplanted human islets it was with tense expectations that we undertook the first investigations of liver sections from a deceased diabetic patient grafted intraportally with isolated islets about 5 years prior to his death (28). quite a few islets were identified, and interestingly enough many of them (40%) contained iapp amyloid deposits as assessed by means of congo red figure 6. human islet from a non-diabetic subject transplanted into the spleen of a nude mouse. pronounced amyloid deposits had developed within the islet after 1 month. congo red. islet amyloid in uppsala 121 staining and immunolabelling with specific antibodies. in a follow-up study there was further evidence for amyloid deposition in clinical pancreatic islet grafts in that one patient from edmonton and one patient from milan also had such deposits in their islet grafts (29). since there is a decline in function in such islets (30) it is tempting to believe that islet amyloid formation is an important cause (16,29,31). our findings of amyloid in human islets, both grafted and cultured specimens, have been repeatedly reproduced. perhaps the most informative one of these reports is that of potter et al. (32) demonstrating that while human islet grafts suffer from the deposition of amyloid after transplantation into nude mice, porcine islets do not. the explanation for this is that porcine iapp lacks the ability to form amyloid due to its amino-acid sequence. therefore porcine islets might be useful for clinical islet transplantation. islet amyloidosis and alzheimer’s disease—is there a link? in this context it may be relevant with a reflection. alzheimer’s disease is an age-related condition which is associated with deposition of amyloid as plaques in the cerebral cortex and in cerebral blood vessels. these pathological traits have been known for a long time, but the importance of the alterations has not been well understood. alzheimer research was for a long time very much focused on disturbances in cerebral transmitters, and not much happened in the field. thus, when glenner (33,34) and beyreuther and masters (35) discovered the ab peptide it meant a sudden, unexpected, and completely new direction in alzheimer research. this field completely exploded and almost everyone moved to ab research. why did the same not happen with islet amyloid and iapp? one may suspect that researchers in the diabetes field have had difficulties to liberate themselves from old ideas and to accept new ones, and this is probably true generally in science. another likely possibility is that for a long time type 2 diabetes was regarded as a disease dependent on peripheral insulin insensitivity with no direct bcell lesion other than that these cells ‘burnt out’ due to constant stress exerted by high plasma glucose concentrations. in a way this may not be completely wrong since hyperglycemia is believed to drive iapp overexpression, impaired processing of proiapp, and dissociation from parallel expression with insulin (reviewed in (16)). however, slowly, iapp amyloid or pre-amyloid assemblies are increasingly accepted as important players in the pathogenesis of type 2 diabetes. interestingly, several epidemiological studies support a link between type 2 diabetes and alzheimer’s disease, two conditions that have local amyloid formation in common as a component in their respective pathogenesis. comparison of ab and iapp amino-acid sequences point to a 50% identity which is slightly higher in regions identified as important for amyloid formation (36). in in vitro studies on amyloid seeding, iapp and ab can crossseed (36) and injections of preformed ab-fibrils to human iapp transgenic mice trigger islet amyloid formation (13). further studies on brain tissue from patients with alzheimer’s disease with the antibody-based proximity ligation assay revealed co-localization of iapp and ab in both diffuse and dense plaques and in amyloid deposits present in cerebral vessel walls. therefore, it is possible that cross-seeding could be a mechanism that links the two diseases. our increasing understanding of the importance of iapp aggregation in the pathogenesis of type 2 diabetes as well as the development in the alzheimer field underlines that gellerstedt was right when he in 1938 wrote ‘the problem “senile amyloidosis” contains many interesting questions, studies of which perhaps may open new ways in research’ (1). acknowledgement the authors thank nils gellerstedt, kungsbacka, for providing a photo of his father. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. gellerstedt n. die elektive, insuläre (para-)amyloidose der bauchspeicheldrüse. zugleich ein beitrag zur kenntnis der “senilen amyloidose”. beitr path anat. 1938;101:1–13. 2. ehrlich jc, ratner im. amyloidosis of the islets of langerhans. a restudy of islet hyalin in diabetic and nondiabetic individuals. am j path. 1961;38:49–59. 3. warren s, lecompte pm, legg ma. the pathology of diabetes mellitus. 4th ed. philadelphia: lea & febiger; 1966. 122 g. t. westermark et al. http://www.ncbi.nlm.nih.gov/pubmed/13726023?dopt=abstract 4. cohen as, calkins e. electron microscopic observations on a fibrous component in amyloid of diverse origins. nature. 1959;183:1202–3. 5. eanes ed, glenner gg. x-ray diffraction studies on amyloid filaments. j histochem cytochem. 1968;16:673–7. 6. glenner gg, eanes ed, bladen ha, linke rp, termine jd. b-pleated sheet fibrils. a comparison of native amyloid with synthetic protein fibrils. j histochem cytochem. 1974;22:1141–58. 7. sipe jd, benson md, ikeda s, merlini g, saraiva mj, westermark p. updated nomenclature 2014: amyloid fibril proteins and clinical classification of the amyloidoses. amyloid. 2014;21:221–4. 8. eisenberg d, jucker m. the amyloid state of protein in human diseases. cell. 2012;148:1188–203. 9. westermark p. mast cells in the islets of langerhans in insular amyloidosis. virchows arch path anat. 1971;354:17–23. 10. westermark p. quantitative studies of amyloid in the islets of langerhans. upsala j med sci. 1972;77:91–4. 11. jurgens ca, toukatly mn, fligner cl, udayasankar j, subramanian sl, zraika s, et al. b-cell loss and b-cell apoptosis in human type 2 diabetes are related to islet amyloid deposition. am j pathol. 2011;178:2632–40. 12. rochet j-c, lansbury ptj. amyloid fibrillogenesis: themes and variations. curr opin struct biol. 2000;10:60–8. 13. oskarsson me, paulsson jf, schultz sw, ingelsson m, westermark p, westermark gt. in vivo seeding and cross-seeding of localized amyloidosis: a molecular link between type 2 diabetes and alzheimer’s disease. am j pathol. 2015;185:834–46. 14. westermark p. fine structure of islets of langerhans in insular amyloidosis. virchows arch a pathol pathol anat. 1973;359:1–18. 15. westermark p, wernstedt c, wilander e, sletten k. a novel peptide in the calcitonin gene related peptide family as an amyloid fibril protein in the endocrine pancreas. biochem biophys res commun. 1986;140:827–31. 16. westermark p, andersson a, westermark gt. islet amyloid polypeptide, islet amyloid and diabetes mellitus. physiol rev. 2011;91: 795–826. 17. westermark p, grimelius l. the pancreatic islet cells in insular amyloidosis in human diabetic and non-diabetic adults. acta path microbiol scand a. 1973;81:291–300. 18. clark a, wells ca, buley id, cruickshank jk, vanhegan ri, matthews dr, et al. islet amyloid, increased a-cells, reduced b-cells and exocrine fibrosis: quantitative changes in the pancreas in type 2 diabetes. diabetes res. 1988;9:151–9. 19. law e, lu s, kieffer tj, warnock gl, ao z, woo m, et al. differences between amyloid toxicity in alpha and beta cells in human and mouse islets and the role of caspase-3. diabetologia. 2010;53:1415–27. 20. lundgren g, andersson a, borg h, buschard k, groth cg, gunnarsson r, et al. structural and functional integrity of isolated human islets of langerhans maintained in tissue culture for 1-3 weeks. transplant proc. 1977;9:237–40. 21. korsgren o, jansson l, eizirik d, andersson a. functional and morphological differentiation of fetal porcine islet-like cell clusters after transplantation into nude mice. diabetologia. 1991;34:379–86. 22. sandler s, andersson a, schnell a, mellgren a, tollemar j, borg h, et al. tissue culture of human fetal pancreas. development and function of b-cells in vitro and transplantation of explants to nude mice. diabetes. 1985;34:1113–19. 23. jansson l, eizirik dl, pipeleers dg, borg la, hellerström c, andersson a. impairment of glucose-induced insulin secretion in human pancreatic islets transplanted into diabetic nude mice. j clin invest. 1995;96:721–6. 24. eizirik dl, pipeleers dg, ling z, welsh n, hellerström c, andersson a. major species differences between humans and rodents in the susceptibility to pancreatic beta-cell injury. proc natl acad sci usa. 1994;91:9253–6. 25. westermark p, eizirik dl, pipeleers dg, hellerström c, andersson a. rapid deposition of amyloid in human islets transplanted into nude mice. diabetologia. 1995;38:543–9. 26. 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gt. is aggregated iapp a cause of beta-cell failure in transplanted human pancreatic islets? curr diab rep. 2005;5:184–8. 32. potter kj, abedini a, marek p, klimek am, butterworth s, driscoll m, et al. islet amyloid deposition limits the viability of human islet grafts but not porcine islet grafts. proc natl acad sci usa. 2010;107:4305–10. 33. glenner gg, wong cw. alzheimer’s disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. biochem biophys res commun. 1984;120:885–90. 34. glenner gg, wong cw. alzheimer’s disease and down’s syndrome: sharing of a unique cerebrovascular amyloid fibril protein. biochem biophys res commun. 1984;122:1131–5. 35. masters c, simms g, weinman na, multhaup g, mcdonald bl, beyreuther k. amyloid plaque core protein in alzheimer disease and down syndrome. proc natl acad sci usa. 1985;82:4245–9. 36. o’nuallian b, williams ad, westermark p, wetzel r. seeding specificity in amyloid growth induced by heterologous fibrils. j biol chem. 2004;279:17490–9. islet amyloid in uppsala 123 http://www.ncbi.nlm.nih.gov/pubmed/13657054?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/5723775?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/4443557?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/4443557?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/4443557?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/25263598?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/25263598?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22424229?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/4940454?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/4116019?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21641386?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21641386?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21641386?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/10679462?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/25700985?dopt=abstract 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http://www.ncbi.nlm.nih.gov/pubmed/7635965?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7635965?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7937750?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7937750?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7489836?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7489836?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15000457?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15000457?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/18753660?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22193043?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22193043?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15983207?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15929864?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20160085?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20160085?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/6375662?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/6375662?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/6375662?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/6236805?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/6236805?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/6236805?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/3159021?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/3159021?dopt=abstract introduction the first demonstration of islet amyloid in uppsala remarks by gellerstedt on the histological appearance of islet amyloid reappearance of islet amyloid research in uppsala iapp and the precipitation of islet amyloid amyloidosis in transplanted islets islet amyloidosis and alzheimer&rsquo;s disease&mdash;is there a link? acknowledgement declaration of interest references further improvements of our journal performance figures upsala journal of medical sciences. 2014; 119: 295–297 editorial further improvements of our journal performance figures when we approached our readers two years ago to comment upon the newly released impact factor scores from thomson reuters (tr) we were happy to announce that for the first time we had passed the critical 1.0 level (1). this time we are glad to let you know about a further increase of this long-questioned instrument for measuring the quality of scientific journals (figure 1). after a 33% increase last year and 21% this year we have now established ourselves in the 1.0– 2.0 interval with the new figure of 1.708 as a new top score for the journal. besides the actual increase of the impact figure we have also advanced in the ranking of 150 journals in our category ‘medicine, general and internal’ from 83rd place two years ago up to 55th this year. last time we promised/aimed at belonging to the better half of the ranking list in the list to come. that goal has been fulfilled in full measure. perhaps belonging to the best third, next time? one more figure that has increased over the last few years is that of ‘total cites’. that is not only citations during one particular year from the ‘impact factor years’ but all other years as well. by necessity that type of figures should constantly increase, keeping in mind the increasing numbers of journals and articles. well above 500 for our journal this year mainly reflects increases of citations to latest years’ papers. it is also worthy of note that the figure is that high despite a fairly low self-citation figure for our journal—3%. for many journals that figure approaches 10%. this is not to say that we intend to become more active in that respect. thus, so-called coercive citation (forced citations to own journal) is not an instrument that we will use or have used. a prerequisite for increasing the impact factor figure is of course that the quality of our published papers is high enough to attract the interest of our readers. for that purpose a high submission rate is crucial, and we are glad to see that preliminary figures for this year tell that we will have handled more than 300 manuscripts. our four issues will accommodate some 50–60 contributions, which means that no less than some 80% of thesubmittedmanuscriptshave tobe rejected. perhaps one reason for the high submission rate is our policy of not charging authors any handling fees. processing that many manuscripts is costly and even with the introductionofasystemwithmanyassociateeditorstoshare the burden (2) it might be necessary to change our policy with regard to processing charges. one obvious reason for our increase of the impact factor figure this year is the inclusion in the 2012 volume of a special issue on tumour biology edited by bengt westermark and carl-henrik heldin (3). all of the published articles have been cited, and the most highly cited one, that by nazarenko and co-authors (4), has got 20 citations to date (august 2014). that means that it will belong to the group of the 1% most-cited (22 citations required) articles published in 2012 (5). fiveotherarticleshavepassedthe10%limit(8citations required). of course this stimulates us to pursue our plan with publication of one such issue with a specified topic every second year. antibiotic resistance was the subject of this year’s issue (6), and in the year 2016 we intend to publish one on diabetes research. and even before that, that is next year, we are going to celebrate our 150th anniversary with a special jubilee issue. as pointed out at the very beginning of this editorial, the use of impact factor figures for measuring different quality aspects of scientific journals has been much debated. however, there are not very many useful alternatives. the citation database scopus of elsevier publishes a journal analyser that amongst many things contains the sjr (scimago journal rank) metrics. although it is hard to understand how the actual figures have been calculated, the ranking is said to be ‘weighted by the prestige of a journal, etc.’ an impressive three-fold increase of the value for our journal is, nevertheless, obvious from 2010 to 2013 (figure 2). more clear-cut are the figures in that database concerning ‘percent not cited’. interestingly, the highest ranked journal of our category in thomson reuters (new england journal of medicine) has got much higher figures (mainly around 30%) than our journal (between 10% and 20%) (figure 3). most certainly, it reflects how much the citation rates of individual articles vary in each separate journal. it also demonstrates that the issn 0300-9734 print/issn 2000-1967 online � 2014 informa healthcare doi: 10.3109/03009734.2014.960308 http://informahealthcare.com/journal/ups 0.733 0.636 upsala journal of medical sciences im p a c t fa c to rs 1.083 1.420 1.708 2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 jcr years 2009 2010 2011 2012 2013 figure 1. a graph published on isi web of knowledge (thomson reuters) (2013 jcr science edition). s j r 0.7 0.6 0.4 0.5 0.2 0.3 0.1 0 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 figure 2. scimago journal rank for upsala journal of medical sciences as of 13 june 2014 in scopus (elsevier). % a rt ic le s n o t c it e d 0 10 20 30 40 50 60 70 80 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013 2014 figure 3. percentage of papers not cited; upsala journal of medical sciences (diamonds—green symbols) and new england journal of medicine (circles—black symbols) as of 29 june 2014 in scopus (elsevier). 296 editorial acceptance of a manuscript for publication in a highly ranked journal does not by necessity guarantee that it will be much cited. although we have dwelt upon this before, we would like to stress that the number of local, uppsala-based contributions—less than 40% last year—is fairly low, which means that there is room for more of them. it can alsobearguedthatthispointstothefactthatourjournal isatrulyinternationalone.indeed,lastyearwereceived submissions from more than 30 countries. finally, we would like to announce that our editorial assistant rikard fred has moved to lund for a post-doc position down there. we thank him very much for many valuable contributions over the last two years. he will be replaced by joey lau, and we warmly welcome her to this task. arne andersson editor declaration of interest: the author reports no conflicts of interest. the author alone is responsible for the content and writing of the paper. references 1. andersson a, ronquist g. a substantial increase of the impact factor. ups j med sci. 2012;117:353–4. 2. andersson a, karlsson t. editorial. a change of the editorial machinery. ups j med sci. 2013;118:1–2. 3. weinberg r. bengt westermark and our current understanding of tumor pathogenesis. ups j med sci. 2012;117:81–2. 4. nazarenko i, hede s-m, he x, hedren a, thompson j, lindström ms, et al. pdgf and pdgf receptors in glioma. ups j med sci. 2012;117:99–112. 5. karlsson s. den svenska produktionen av högt citerade vetenskapliga publikationer. vetenskapsrådets lilla rapportserie. 2010;1:1–21. 6. davies j. antibiotic resistance and the golden age of microbiology. ups j med sci. 2014;119:65–7. editorial 297 http://www.ncbi.nlm.nih.gov/pubmed/23110366?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23110366?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22512243?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22512243?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22509804?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24836049?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24836049?dopt=abstract ss1 references pragmatic clinical trials in the context of regulation of medicines article pragmatic clinical trials in the context of regulation of medicines rolf gedeborg, charles cline, bj€orn zethelius and tomas salmonson swedish medical products agency, uppsala, sweden abstract the pragmatic clinical trial addresses scientific questions in a setting close to routine clinical practice and sometimes using routinely collected data. from a regulatory perspective, when evaluating a new medicine before approving marketing authorization, there will never be enough patients studied in all subgroups that may potentially be at higher risk for adverse outcomes, or sufficient patients to detect rare adverse events, or sufficient follow-up time to detect late adverse events that require long exposure times to develop. it may therefore be relevant that post-marketing trials sometimes have more pragmatic characteristics, if there is a need for further efficacy and safety information. a pragmatic study design may reflect a situation close to clinical practice, but may also have greater potential methodological concerns, e.g. regarding the validity and completeness of data when using routinely collected information from registries and health records, the handling of intercurrent events, and misclassification of outcomes. in a regulatory evaluation it is important to be able to isolate the effect of a specific product or substance, and to have a defined population that the results can be referred to. a study feature such as having a wide and permissive inclusion of patients might therefore actually hamper the utility of the results for regulatory purposes. randomization in a registry-based setting addresses confounding that could otherwise complicate a corresponding non-interventional design, but not any other methodological issues. attention to methodological basics can help generate reliable study results, and is more important than labelling studies as ‘pragmatic’. article history received 17 june 2018 revised 1 august 2018 accepted 13 august 2018 keywords drug approval; government regulation; methods; pharmaceutical preparations; pragmatic clinical trial what is a pragmatic study? a distinction is sometimes made between explanatory and pragmatic clinical trials. while explanatory trials aim to estimate the efficacy of an intervention under optimized conditions, the term pragmatic trials usually refers to what an intervention accomplishes when applied in wider clinical practice (1). the traditional phase iii randomized clinical trial in a drug development programme would typically be an explanatory trial, focusing on establishing an ideal situation to estimate the efficacy of the drug in relation to a sufficiently well characterized safety profile. the pragmatic clinical trial concept addresses questions with a focus on estimating effects in a setting closer to routine clinical practice and sometimes using routinely collected data. the pragmatic trial is therefore expected to more adequately inform a clinical or policy decision by providing evidence more relevant for use of a medicine in daily clinical practice. this is sometimes also referred to as ‘real-world evidence’ (2). an evaluation tool (precis-2) has been proposed to allow a structured grading of specific domains that defines explanatory versus pragmatic aspects of a trial (3). it tries to quantify aspects such as how similar the participants in the trial are to the target population in routine clinical practice, and how much extra effort is made to recruit patients compared to the usual care setting. the differences in setting where the trial is conducted, differences in the resources needed to deliver the intervention, and measures applied to promote adherence to treatment are also compared to expectations from routine clinical care. questions on how closely participants are followed up, how relevant the outcome measure is to participants, and completeness of data are also assessed in the precis-2 tool. a pragmatic trial might therefore use less strict control over which patients to include, the exposure, and standard of care. study procedures may not actively promote adherence to treatment, and may accept that follow-up of patients and measurement of outcomes are not fully standardized and optimal (4). the design and conduct of pragmatic trials offer distinct challenges, and the appropriate framework for this type of studies is under development (5). for the purpose of the present discussion we will not consider non-interventional studies or single-arm studies that may also be considered pragmatic designs, but instead focus on randomized trials. some aspects of pragmatic trials are of particular relevance or concern from a regulatory perspective. protection of human subjects is of key importance, e.g. concerning informed consent and compliance with good clinical practice (gcp) (6). the concept of ‘low-intervention’ clinical trials has therefore been introduced in the legislation, contact rolf gedeborg rolf.gedeborg@mpa.se swedish medical products agency, po box 26, se-751 03 uppsala, sweden � 2018 medical products agency. published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 1, 37–41 https://doi.org/10.1080/03009734.2018.1515280 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1515280&domain=pdf http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1515280 http://www.tandfonline.com to allow a risk-based approach to using less rigid rules, e.g. as regards monitoring and traceability of investigational medicinal products (7). these aspects are of major importance but not further discussed in this article. it is apparent that there is no distinct boundary between explanatory and pragmatic trials (4). both types of study can answer relevant questions, and the most appropriate study design should therefore be determined based on the specific scientific question to be answered. are pragmatic studies of value for regulators? one important requirement for the approval to market a medicinal product is having sufficient evidence that the expected beneficial effects outweigh potential risks in a defined population. this conclusion on the benefit–risk balance is always expected to be reconsidered whenever new relevant evidence becomes available during the life-cycle of a medicinal product (8). even if there are large studies underlying a marketing authorization, there will never be enough patients studied in all subgroups that may potentially be at higher risk for adverse outcomes, or sufficient patients to detect rare adverse events, or sufficient follow-up time to detect late adverse events that require long exposure times to develop. there is therefore always a need for continued safety surveillance and often also a need for systematic postmarketing studies of specific safety concerns. sometimes there is also uncertainty regarding the efficacy of the new drug that warrants further studies post approval. it may not be feasible, or even necessary, to require large randomized clinical trials before making a decision to approve a new drug or a new indication. the degree of certainty in the characterization of effects that is considered necessary to obtain before approval must be weighed against the urgency of the unfilled medical need for the product, in order to make new treatments available to patients as soon as possible. some drugs are therefore approved without having large randomized trials documenting efficacy and safety. over the period 1 january 1999 to 8 may 2014 the european medicines agency (ema) approved 44 new indications for 35 drugs without a randomized controlled study, among 415 approvals of new indications reviewed (9). generic drugs, biosimilars, diagnostics, medical devices, vaccines, antimicrobials, blood products, and fixeddose combinations of existing products were excluded from this review. the majority of the new indications approved without a randomized controlled study were for haematological malignancies, oncology, and metabolic conditions. such approvals may be possible if the natural course of disease allows for the isolation of drug effects without a comparator. though the effect size in the target population may remain uncertain due to the lack of reliable calibration against a standard of care, such limitations may be acceptable in often fatal orphan diseases, for which there are no other therapeutic options, or situations when an ethically acceptable control group cannot be specified. specific legal frameworks have been created for conditional approval and approval under exceptional circumstances, to be used in situations when data are limited but medical need is great. obligations to perform further clinical studies post approval are then imposed on the drug company. these studies may e.g. focus on endpoints different from the primary endpoints of the studies available at the time of initial authorization, evaluate longer treatment duration, and/or the number of subjects receiving the product as part of imposed studies may be higher compared to studies available before approval (10). in some cases data in closely related patient populations also contribute to the generation of comprehensive data in support of the granted indication, and it is not uncommon that the data from post-marketing trials after conditional approval have led to a change in the definition of target population in the therapeutic indication (10). it may therefore be relevant that post-marketing trials have more pragmatic characteristics, if this is needed to provide the information on efficacy and safety that is missing. from a regulatory perspective, both explanatory and pragmatic trials are therefore relevant. potential concerns with pragmatic trials from a regulatory perspective—the completeness and validity of the data from registries and health records one key feature of a pragmatic trial may be that data collection is based largely on routinely available data in electronic health records or disease registries (11). there are some distinct advantages in terms of reduced effort for both investigators and study subjects, and consequently reduced cost. this may facilitate recruitment of patients to the trial and enable a larger sample size. the potential concerns lie in the validity and completeness of data. a pragmatic non-interventional study, e.g. conducted using prescription and/or disease registries, may seem appropriate in that it is representative of use in clinical practice. a prescription registry, however, often does not include medications administered in-hospital. this may be a major limitation since many drug treatments are initiated in-hospital, and hospitalized patients may be particularly frail and prone to adverse events. another example is the use of hospital discharge registries where primary care encounters are not registered (12), or registers based on general practitioner data, where data from patients cared for in hospital-based specialist clinics are not available unless linked with hospital data (13,14). the potential problem that the definition of the study population can introduce selection bias therefore needs to be considered irrespective of the type of study, also for non-interventional studies. selection bias can present major limitations also for studies labelled as pragmatic with a very permissive inclusion of patients. the completeness of data collection is a key issue for any type of trial and in focus for gcp (6). an example is when data on outcomes routinely collected in the electronic patient records are not accessible during the study and are found to be frequently missing during analyses (15). a study design and study conduct that prevents missing information is always preferable to statistical handling of missing 38 r. gedeborg et al. information at the analysis stage (16). such methods often have unverifiable assumptions. sensitivity analyses to evaluate the impact of the handling of missing data and associated assumptions are essential for the interpretation of study results. the importance of choosing an appropriate estimand during the planning stage of a study, so that attempts to prevent missing data can be tailored to that choice, and appropriate estimation methods can be specified, must be stressed (17). it is essential to detect and adequately handle intercurrent events, such as use of alternative treatment, discontinuation of treatment, and treatment switches, in the analyses. otherwise, these types of events may lead to invalid conclusions regarding treatment effects (17). these two fundamental considerations in the study design, how to prevent missing information and defining the appropriate estimand, may be a greater concern for pragmatic study designs. another risk with the use of routinely collected healthcare data is that the measurement of outcomes may be a less precise. for an outcome such as long-term all-cause mortality this is usually not a major problem. mortality is a well-defined outcome that can be reliably captured by administrative data. for outcomes with insidious onset, or outcomes requiring accurate measurements and strict definitions, this can on the other hand be a substantial problem. an example is the difficulty to identify severe infections based on hospital discharge diagnoses in health-care databases (18). patients with a well-defined diagnosis such as meningitis, often also being the primary reason for hospitalization, can be accurately identified using such a data source. this is in contrast to a common and important diagnosis such as sepsis, which is a less well-defined condition often complicating a hospital stay rather than being the primary cause for admission. the sensitivity with which patients with sepsis can be identified from hospital discharge diagnoses is expected to be very low (18). the use of randomization integrated in a registry-based setting is an important step towards increased reliability of patient-relevant research aimed to reflect clinical practice (19). randomization offers the unique property of addressing not only known and measured confounding, but also taking care of unmeasured and unknown confounding. it should, however, be recognized that randomization does not address any other methodological problem discussed in this article, such as selection bias or misclassification. adding randomization in a registry-based setting should consequently be seen as a means to address confounding that could complicate the corresponding non-interventional study design. other methodological issues remain, however. to what extent does the study population in randomized clinical trials need to be representative of the target population? an argument in favour of a pragmatic approach for clinical studies is that this can make the results generalizable to a wider target population, i.e. the population where the results of the study are expected to be applicable. it is, however, not an imperative feature for a comparative study that the study population accurately mirrors the target population in terms of the distribution of relevant patient characteristics (20). this thinking most likely stems from the process of survey-sampling. in a purely descriptive study, such as a survey, it is essential to have a representative sample of the population of interest. in an inferential comparative study, this may instead hamper the ability to make internally valid inferences (20). concerns regarding generalizability are only meaningful once the results are deemed internally valid, i.e. that they are sufficiently unbiased to warrant causal inference, for the restricted study population. in a comparative study the assumption of homogeneity of the effect in the study population is central to a data analysis that ultimately presents an overall effect estimate for the study population. in this situation the result is assumed to be the same in all parts of the distributions of all relevant patient characteristics that are well represented in the study population. this means that e.g. the age distribution need not be exactly the same as in the target population but all parts of the age distribution in the target population must be reasonably well represented in the study population. this could justify over-sampling, e.g. of some parts of the age distribution, in order to have sufficient representation of all parts of the relevant age range for the target population in the source population for the study (20). it is also important to remember that this assumes that there is no effect modification, meaning that the conclusions of therapeutic efficacy (and safety) apply consistently across relevant subgroups of the clinical trial population. an obvious example is that all relevant age categories must be adequately represented in the study population. if one states that results from an explanatory randomized trial are unreliable regarding their prediction of expected effect in the overall target population, this is the same as stating that there is important effect modification by some patient characteristic. consequently, further efforts should aim to characterize this effect modification, and not simply estimate another overall effect estimate. if clinical data fail to establish statistically persuasive evidence of effect, there may be an interest in exploratory analyses to further characterize the effect in relevant subgroups (21). it is otherwise difficult to understand how to interpret the totality of evidence. from a regulatory perspective there is a focus on identifying the patient population where the benefits outweigh the risks. the problem with exploring subgroups is closely related to the problem of multiple testing, and carries an increased probability of false-positive findings. it is therefore important to conduct reliable analyses of potential effect modification from e.g. age, renal, or hepatic function, whether through direct such analyses of interaction or through pre-planned subgroup analyses (21). how can the results be generalized outside the study population in an explanatory phase iii trial? a study aiming for high internal validity through an explanatory type of trial design may for this purpose select a upsala journal of medical sciences 39 homogeneous population that can differ in important ways from the intended broad target population in clinical practice. these studies have therefore sometimes been questioned for uncertainty about generalizability of the results. extrapolation of the study results is always needed to some extent, otherwise they are not meaningful. there is a need to generalize the results at least in time, so that we consider them valid not only for the patients studied but also for future patients. although this may be seen as selfevident, there may be an evolution of the treatment effect over time (22). this is something that may need to be considered in cases where data have evolved over a period of time in parallel to changes in the population and in health care, i.e. new diagnostic criteria and treatment guidelines, and in an evaluation that should be based on the totality of data. apart from generalizability over time there is often a need for extrapolation also in relation to other characteristics of the target population. a common example is age, and to what extent results can be generalized to the extremes of age—children and the elderly. the basis for extrapolation to a paediatric target population rests on the relevance of results obtained in an adult study population. this can be quantified in terms of drug exposure being dependent on body size and organ maturation (pharmacokinetic data), differences in pharmacodynamic response, and age-dependent differences in disease characteristics (23). simply generating an overall effect estimate including all age groups, children as well as adults, is clearly not appropriate. extrapolation is in some instances not justifiable, while in other cases extrapolation is uncomplicated, based on wellestablished understanding of the drug pharmacology and the disease (23). it becomes evident that a targeted approach to data collection is needed, so that children participate only in trials with specific objectives to obtain the information needed for a specific intervention and patient group. the aim should be to characterize patient factors that influence the efficacy and safety of the drug, and not only to estimate an overall effect size. a relevant aim for a post-marketing trial could also be to further elucidate the effects in an elderly population, if they were not adequately represented in the populations already studied. if this is the concern, then it may be more effective to target a study specifically at this subpopulation, rather than performing a wide pragmatic clinical trial covering the entire target population and all age groups (24). in this scenario an overall effect estimate for the entire target population may be difficult to interpret. to clarify if the effect is age-dependent the study must be dimensioned specifically with the question of potential effect modification by the age in focus (21). the overall effect estimate cannot be the only aim of the study. it becomes more important to understand the impact of patient characteristics, such as age, body size, and renal and hepatic function, rather than to calculate an overall effect estimate in a broad population representative of the entire target population. pragmatism may make a randomized trial more relevant for questions relating to using the medicine in clinical practice and also less expensive, but comes at a cost from other aspects an example of how pragmatic trials can be questioned based on their pragmatic design is provided by the regulatory procedures concerning potential adverse effects of hydroxyethyl starch (hes) products finalized in 2013 (25). key evidence in these procedures were some academic clinical trials that could be considered pragmatic (26,27). criticism against these studies focused on difficulties with defining and controlling the exposure in these studies, and the study population being poorly defined due to the broader pragmatic inclusion of patients (28). in a regulatory evaluation it is important to be able to isolate the effect of the product or substance in focus of the investigation, and to have a defined population that the results can be referred to. a feature such as having a wide and permissive inclusion of patients might therefore actually hamper the utility of the results for regulatory purposes. simplifying the data collection process in a clinical study is expected to require trade-offs, but the actual consequences must be evaluated for each specific outcome in relation to the research question at hand. is the accuracy of data sufficient to support reliable conclusions from the study? it may e.g. be problematic to claim effectiveness of an intervention without reliable concomitant estimation of relevant safety endpoints. safety reporting in published randomized trials has been found to be variable but largely inadequate (29). this is an area where simplification of data collection may be a concern, particularly for new medicines. for a study investigating a well-characterized medicine, e.g. where the investigational medicinal product is covered by a marketing authorization, and the quality, safety, and efficacy have already been assessed in the course of the marketing authorization procedure, the intervention poses only very limited additional risk to the subject compared to normal clinical practice. such ‘low-intervention’ clinical trials may be of crucial importance for assessing standard treatments and diagnoses, thereby optimizing the use of medicinal products. those clinical trials could be subject to modified requirements, e.g. as regards monitoring and traceability of investigational medicinal products (19). in other situations reliable collection of all relevant data according to gcp remains essential. conclusion regulatory decision-making should always be based on the totality of data and not on whether a specific study is labelled as explanatory or pragmatic. the regulators have to be convinced that the expected benefits of the new treatment outweigh the potential risks. the need for further understanding of efficacy and safety should be addressed by specifying the appropriate research questions, and tailoring the study design and analyses to those particular questions. this process must be informed by the challenges arising 40 r. gedeborg et al. from the specific clinical context of the new therapy, current available evidence, and patients’ need for timely access to new treatment options. this article has attempted to highlight some general sources of bias related to study design. there may be a risk that access to large amounts of clinical data and sophisticated analytical tools is used to generate results without paying sufficient attention to fundamental methodological concepts that limit what conclusions can be drawn from a specific clinical trial. attention to methodological basics can help generate reliable study results, and is more important than labelling studies as ‘pragmatic’ or ‘realworld evidence’. disclosure statement no potential conflict of interest was reported by the authors. notes on contributors associate professor rolf gedeborg, md, phd, specialist in anaesthesiology and intensive care, desa, edicm. clinical assessor, efficacy and safety 1, licensing, swedish medical products agency. charles cline, md, phd, specialist in cardiology & internal medicine. clinical assessor, dept. clinical trials and special permissions, swedish medical products agency. associate professor bj€orn zethelius, md, phd, specialist in internal medicine, endocrinology and diabetology. scientific director pharmacotherapy, department of scientific expertise, swedish medical products agency, uppsala, sweden. tomas salmonson, msc (pharm), phd in pharmacokinetics and pharmacodynamics, senior scientific advisor, scientific support, swedish medical products agency. references 1. friedman lm, furberg cd, demets dl. fundamentals of clinical trials. heidelberg: springer international publishing; 2015. 2. sherman re, anderson sa, dal pan gj, gray gw, gross t, hunter nl, et al. real-world evidence what is it and what can it tell us? n engl j med. 2016;375:2293–7. 3. loudon k, treweek s, sullivan f, donnan p, thorpe ke, zwarenstein m. the precis-2 tool: designing trials that are fit for purpose. bmj. 2015;350:h2147. 4. ford i, norrie j. pragmatic trials. n engl j med. 2016;375:454–63. 5. weinfurt kp, hernandez af, coronado gd, debar ll, dember lm, green bb, et al. pragmatic clinical trials embedded in healthcare systems: generalizable lessons from the nih collaboratory. bmc med res methodol. 2017;17:144. 6. international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use. guideline for good clinical practice e6 (r2). london: european medicines agency; 2016. 7. the european parliament and the council of the european union. regulation (eu) no 536/2014 of the european parliament and of the council of 16 april 2014 on clinical trials on medicinal products for human use, and repealing directive 2001/20/ec 2014. 8. evans sjw, leufkens hgm. regulatory decision-making: are we getting it right? pharmacoepidemiol drug saf. 2014;23:1012–16. 9. hatswell aj, baio g, berlin ja, irs a, freemantle n. regulatory approval of pharmaceuticals without a randomised controlled study: analysis of ema and fda approvals 1999-2014. bmj open. 2016;6:e011666. 10. european medicines agency. conditional marketing authorisation. london, uk: european medicines agency; 2017. 11. feltelius n, gedeborg r, holm l, zethelius b. utility of registries for post-marketing evaluation of medicines. a survey of swedish health care quality registries from a regulatory perspective. ups j med sci. 2017;122:136–47. 12. ludvigsson jf, andersson e, ekbom a, feychting m, kim jl, reuterwall c, et al. external review and validation of the swedish national inpatient register. bmc public health. 2011;11:450. 13. herrett e, thomas sl, schoonen wm, smeeth l, hall aj. validation and validity of diagnoses in the general practice research database: a systematic review. br j clin pharmacol. 2010;69:4–14. 14. ludvigsson jf, otterblad-olausson p, pettersson bu, ekbom a. the swedish personal identity number: possibilities and pitfalls in healthcare and medical research. eur j epidemiol. 2009;24:659–67. 15. heim n, van stel hf, ettema rg, van der mast rc, inouye sk, schuurmans mj. help! problems in executing a pragmatic, randomized, stepped wedge trial on the hospital elder life program to prevent delirium in older patients. trials. 2017;18:220. 16. lavange lm, permutt t. a regulatory perspective on missing data in the aftermath of the nrc report. stat med. 2016;35:2853–64. 17. committee for human medicinal products. ich e9 (r1) addendum on estimands and sensitivity analysis in clinical trials to the guideline on statistical principles for clinical trials. london, uk: european medicines agency; 2017. 18. gedeborg r, furebring m, michaelsson k. diagnosis-dependent misclassification of infections using administrative data variably affected incidence and mortality estimates in icu patients. j clin epidemiol. 2007;60:155–62. 19. james s, rao sv, granger cb. registry-based randomized clinical trials–a new clinical trial paradigm. nat rev cardiol. 2015;12:312–16. 20. rothman k, greenland s, lash t. validity in epidemiological studies. modern epidemiology. 3rd ed. philadelphia: lippincott williams & wilkins; 2008. p. 128–47. 21. committee for medicinal products for human use (chmp) european medicines agency. guideline on the investigation of subgroups in confirmatory clinical trials (draft). london, uk: european medicines agency; 2014. ema/chmp/539146/2013. 22. ioannidis j, lau j. evolution of treatment effects over time: empirical insight from recursive cumulative metaanalyses. proc natl acad sci u s a. 2001;98:831–6. 23. european medicines agency. reflection paper on the use of extrapolation in the development of medicines for paediatrics (draft). london, uk: european medicines agency, 2017. ema/ 199678/2016. 24. bourgeois ft, olson kl, tse t, ioannidis jp, mandl kd. prevalence and characteristics of interventional trials conducted exclusively in elderly persons: a cross-sectional analysis of registered clinical trials. plos one. 2016;11:e0155948. 25. european medicines agency. assessment report for solutions for infusion containing hydroxyethyl starch (ema/667674/2013). london, uk: european medicines agency; 2013. contract no.: ema/667674/2013. 26. perner a, haase n, guttormsen ab, tenhunen j, klemenzson g, aneman a, et al. hydroxyethyl starch 130/0.42 versus ringer’s acetate in severe sepsis. n engl j med. 2012;367:124–34. 27. myburgh ja, finfer s, bellomo r, billot l, cass a, gattas d, et al. hydroxyethyl starch or saline for fluid resuscitation in intensive care. n engl j med. 2012;367:1901–11. 28. chappell d, jacob m. hydroxyethyl starch the importance of being earnest. scand j trauma resusc emerg med. 2013;21:61. 29. ioannidis jp, lau j. completeness of safety reporting in randomized trials: an evaluation of 7 medical areas. jama. 2001;285:437–43. upsala journal of medical sciences 41 abstract what is a pragmatic study? are pragmatic studies of value for regulators? potential concerns with pragmatic trials from a regulatory perspectivethe completeness and validity of the data from registries and health records to what extent does the study population in randomized clinical trials need to be representative of the target population? how can the results be generalized outside the study population in an explanatory phase iii trial? pragmatism may make a randomized trial more relevant for questions relating to using the medicine in clinical practice and also less expensive, but comes at a cost from other aspects conclusion disclosure statement notes on contributors references a national survey on routines regarding sedation in swedish intensive care units article a national survey on routines regarding sedation in swedish intensive care units oskar talsia, ritva kiiski berggrena,b, g€oran johanssona and ola wins€oa adepartment of surgical and perioperative sciences, anaesthesiology and intensive care medicine, umeå university, umeå, sweden; bswedish national quality registry for intensive care (sir), karlstad, sweden abstract background: previous studies concerning sedation in swedish intensive care units (icu) have shown variability in drug choices and strategies. currently, there are no national guidelines on this topic. as an update to a nordic survey from 2004, and as a follow-up to a recently introduced quality indicator from the swedish intensive care registry, we performed a national survey. methods: a digital survey was sent to the icus in sweden, asking for sedation routines regarding hypnosedatives, analgosedatives, protocols, sedation scales, etc. results: fifty out of 80 icus responded to the survey. all units used sedation scales, and 88% used the rass scale; 80% used written guidelines for sedation. propofol and dexmedetomidine were the preferred short-term hypnosedatives. propofol, dexmedetomidine, and midazolam were preferred for long-term hypnosedation. remifentanil, morphine, and fentanyl were the most frequently used agents for analgosedation. conclusions: all icus used a sedation scale, an increase compared with previous studies. concerning the choice of hypnoand analgosedatives, the use of dexmedetomidine, clonidine, and remifentanil has increased, and the use of benzodiazepines has decreased since the nordic survey in 2004. article history received 19 february 2019 revised 15 april 2019 accepted 3 may 2019 keywords analgesics; intensive care; mechanical ventilation; sedation scale; sedatives introduction sedation is crucial for treatment in the intensive care unit (icu). the concept is wide and includes everything from light sedation with patients being awake and able to communicate, to heavy sedation where patients cannot manage their own respiration. the purpose of sedation from the patient perspective is relief of pain and anxiety, which means that nursing care, communication, and drugs interact to achieve these goals. nowadays, the general opinion on sedation among icu staffs and recent studies is pointing towards less sedation. today more focus is directed on analgesia, since heavy sedation has been reported to prolong the length of stay (los) at the icu and to increase the rate of complications (1–5). lower levels of sedation also permit easier evaluation of disease progress, easier follow-up of analgesic therapy, and they simplify clinical examinations. previous studies have shown that the level of sedation in the icu has a tendency to be too deep and that the analgesic therapy is insufficient (6). nowadays it is well known that monitoring of sedation levels and utilization of validated sedation scales, with or without a sedation protocol, give a more accurate sedation level. more accurate and lighter sedation gives shorter los, shorter time on mechanical ventilation, and fewer complications in the icu (7,8). too heavy sedation can also contribute to an increase of post-traumatic stress disorder (ptsd) symptoms in icu survivors, though many aspects are involved (9). earlier swedish and nordic observational studies from 2003 and 2004 have shown varying results in usage of sedation scales and goals, ranging from 16% to about 50% (10,11). similar studies in europe and north america show data comparable to the use of sedation scales in the nordic countries (12–14). subjective scales evaluating the level of sedation are considered more accurate than objective measures of brain activity, e.g. bispectral index (bis) (1). there are multiple sedation scales in clinical use, though a ‘gold standard’ has not been identified. the recommendations today promote the use of a validated scale, e.g. the richmond agitation sedation scale, which is one of the most frequently used sedation scales in europe (1,15–18). icu care is expensive, and the number of beds is often very limited. it is therefore relevant, both for patients and hospitals, that knowledge on strategies which can reduce los and complications is used. utilization of sedation scales and definition of sedation goals are some of the swedish intensive care registry’s eight quality indicators, but there are currently no national guidelines on which sedation scale to use. moreover, there are no recent swedish studies on the compliance to this new quality indicator. contact ola wins€o ola.winso@umu.se department of surgical and perioperative sciences, anesthesiology and intensive care medicine, umeå university, umeå, sweden � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 3, 199–202 https://doi.org/10.1080/03009734.2019.1616339 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1616339&domain=pdf&date_stamp=2019-09-09 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2019.1616339 http://www.tandfonline.com since there are no written national guidelines on this subject in sweden, the type of treatment varies between hospitals. previous studies have shown that propofol and midazolam were the most frequently used hypnosedatives in sweden (10). recently, the use of dexmedetomidine was promoted due to the shorter time on mechanical ventilation and shorter los at the icu (19). the aim of this study was to investigate the current sedation practice in sweden based on a national survey. focus in the survey was on the use of sedation scales, drug administration methods, and choice of pharmacological agents for sedation and analgesia. material and methods a national survey was created in cooperation with the swedish national quality registry for intensive care (sir). the survey was sent out on 12 april 2017 to 80 icus in sweden, through sir’s member register. the survey was an electronic questionnaire consisting of multiple-choice questions, with a possibility to add written answers at the end. in the survey, we asked for routines regarding the use of sedation scales, sedation goals, and preferred analgosedatives and hypnosedatives (see appendix). the respondent was asked to rank the three most frequently used hypnosedatives and analgosedatives. additional data were assembled through email. ethics it was voluntary to participate in the study, and a written reply was considered as informed consent. the principal study concept was approved by the local ethical committee on 10 april 2018 (epn 2018/13-31). statistical analysis ibm spss statistics (version 24.0) was used. the mann–whitney test and the kruskall–wallis test were used for statistical calculations; 95% confidence interval was used, and significance level was set at p < 0.05. results the total number of responders was 50 out of 80 (63%) surveys sent out. the responding units were categorized according to the swedish intensive care society (sis) classification from 1 to 3, where 3 is the most advanced level of icu. eight of the responders were from category 1; 29 from category 2; and 13 from category 3. median number of ventilators possible to use simultaneously per icu was 8 (min. 2, max. 18). all the responding units used a sedation scale. forty-four (88%) of the units used the rass scale, making it the predominantly used sedation scale. the motor activity assessment scale (maas) was used by four units; one paediatric icu used the comfort b scale; and one unit used an unspecified scale. local written guidelines were used by 80% of the responding icus, and 10% were working on such guidelines. daily ‘wake-up calls’ were used in 58% of the units. the two most frequently used hypnosedatives for shortterm sedation (<24 h) were propofol (78%) and dexmedetomidine (20%). the most popular combination was to use propofol as first choice and dexmedetomidine as second choice. for long-term hypnosedation (>24 h) propofol (52%) was the most frequently used agent. the most popular combination was to use propofol as first choice and dexmedetomidine as second choice. there is one more first choice than responders in the category long-term sedation, because one respondent gave two drugs as first choice (table 1). for analgosedation remifentanil (36%), morphine (30%), and fentanyl (26%) were the preferred agents. the most popular combination of analgosedatives was to use remifentanil or morphine as first choice and ketobemidon or fentanyl as second choice (table 2). in one part of the survey we asked for the use of alpha-2-agonists. all the responding units use dexmedetomidine. the most common indications for dexmedetomidine were non-invasive ventilation (88%), shallow sedation (88%), and nightly sedation (80%). the most common indications for clonidine were shallow sedation (rass > -2) (64%), noninvasive ventilation (46%), nightly sedation, and deep sedation (rass < -2) (32%) (table 3). continuous infusion was the preferred administration method for all the responding units (50/50) in the category hypnosedation. thirty-one out of 50 used intermittent table 1. hypnosedation, shortand long-term use. choice first second third short term use, <24 hours propofol 39 10 0 dexmedetomidine 10 31 6 clonidine 1 5 10 diazepam 0 1 0 esketamine 0 0 6 lorazepam 0 0 0 midazolam 0 1 18 other 0 2 4 long term use, >24 hours propofol 26 15 6 dexmedetomidine 16 16 5 midazolam 7 8 12 clonidine 2 7 14 diazepam 0 0 0 esketamine 0 1 2 lorazepam 0 1 2 thiopental 0 0 2 other 0 0 2 data presented as preferred choices from the participating national swedish icus. table 2. analgosedation. choice first second third remifentanil 18 6 9 morphine 15 6 11 fentanyl 13 12 3 ketobemidone 3 9 5 alfentanil 0 4 6 esketamine 0 1 5 clonidine 0 6 5 sufentanil 0 0 0 other 1 4 1 data presented as preferred choices from the participating national swedish icus. 200 o. talsi et al. intravenous injections as second choice. in the category analgosedation, 45 out of 50 (90%) used continuous infusions as first choice, and four out of 50 units (8%) used intermittent injections as first choice. the most frequently used method for follow-up on sedation level was that nurses and assistant nurses examined the patient (40/50, 80%). in this group of 40 answers, 31 (78%) made a note in the patient chart and reported at rounds; five units only made a note in the patient chart; and four units (10%) only reported at rounds. discussion of the responding units 80% used written guidelines for sedation, an increase from 44% in 2004 (10). all the responding icus used a sedation scale, which also is an increase from 2004 when 54% of swedish icus responded that they used a sedation scale; 100% is a high number and might not fully represent the situation nationwide, since there were 30 units missing in the survey. but comparable numbers have been observed in a recent similar study from great britain (20). eighty-eight per cent of the icus used the rass scale, making it the predominantly used sedation scale. this is a change from the 2004 survey, where maas was the most frequently used scale (58%) and no use of the rass scale was registered. most probably, the increase in usage of sedation scales reflects the current state of knowledge on sedation in icu settings. furthermore, the increase in utilization of the rass scale might be due to ease of use and that it perhaps is more detailed in the span of light sedation. the rass scale is recommended in both american and german guidelines and was the most frequently used sedation scale in a recent british icu survey (1,18,20). in our survey, it was possible to identify two favourite drugs in the category hypnosedation, propofol and dexmedetomidine. these drugs were preferred in both the category shortand long-term sedation. however, in the category longterm sedation, midazolam was preferred as first choice by 14% of the responders. the change of use in both shortand long-term hypnosedation reflects recent studies, promoting a lighter level of sedation and supporting a non-benzodiazepine sedation strategy (18,19). this pattern has also been observed in recent studies and guidelines, where deep sedation is recommended only in specific patient groups (1,18). it was difficult to identify a clear favourite in the category analgosedation, but remifentanil was preferred by most responders. this is a change from 2004 when no use of remifentanil was registered (10), partially because it was a relatively new drug at that time. due to its pharmacokinetic properties, remifentanil is mostly administered as an infusion. because of its short-acting nature, it needs to be withdrawn carefully. the short-acting nature makes it practical for patients with renal or hepatic failure, which is often the case in the icu (21). remifentanil is also well tolerated by icu patients, facilitates a fast and predictable extubation, and may shorten icu los and time on mechanical ventilation (22). continuous infusions were preferred by all icus in the category hypnosedation and by most responders in the category analgosedation, even though continuous infusions have certain drawbacks. thus, continuous infusions of benzodiazepines have been associated with higher risk of delirium (1,23). continuous intravenous sedation has also been linked to prolonged icu los and increased duration of mechanical ventilation and hospitalization (24). one explanation to the high reported use of continuous infusions might be the increased use of remifentanil and dexmedetomidine, drugs only administered as intravenous infusions. the responses in the survey reflect the current state in swedish icus. the response rate (63%) is comparable to similar studies elsewhere in europe (10,13,17,25). however, this survey does not say anything about quantities of the drugs used and actual compliance to sedation scales and local guidelines. our survey was filled out by one physician for each icu, and there might be a discrepancy from the actual clinical practice. a similar survey carried out in 2013 (25), sent out to icu nurses in europe, recorded no answers with dexmedetomidine as the preferred first choice sedative. this raises questions on whether there has been a shift in drug choice over a short period of time or if the answers in this or previous studies overor underestimate the use of dexmedetomidine. to get a more representative picture of the current practice in swedish icus, prospective multicentre studies or point prevalence studies on site need to be performed. to conclude, we observed a few changes from the study published in 2004 (10). all the responding units use sedation scales, and the preferred scale is rass. similar changes are reported from other countries, e.g. the uk (17,20), and are supported by international guidelines (1,18). dexmedetomidine and clonidine are used regularly. remifentanil was reported as the first-choice analgesic agent, even though a clear favourite in this category was not possible to identify. disclosure statement the authors report no conflicts of interest. acknowledgements the authors would like to acknowledge gunnar malmqvist for valuable input. table 3. indications for the use of dexmedetomidine and clonidine. n dexmedetomidine non-invasive ventilation 44 sedation (rass > -2) 44 nightly sedation 40 sedation (rass < –1) 7 short procedures 6 other 5 clonidine sedation (rass > –2) 32 non-invasive ventilation 23 nightly sedation 18 sedation (rass < –1) 16 abstinence 9 weaning 6 short procedures 5 do not use 4 hypertension 3 other 2 data presented as preferred choices from the participating national swedish icus. upsala journal of medical sciences 201 funding this work was supported by the department of surgical and perioperative sciences, anaesthesiology, and intensive care medicine, umeå university, umeå, sweden. notes on contributors oskar talsi conducted this study as a mandatory thesis project during his medical studies in umeå. he has now finished his studies and is currently working as a physician. the work was performed in collaboration with the swedish intensive care registry, represented by ritva kiiski berggren. ritva kiiski berggren is a senior consultant, working as anaesthesiologist at umeå university hospital, umeå. g€oran johansson is a research engineer. he has for several years helped medical students and others with data analyses and staristical calculations. ola wins€o is currently professor emeritus, since he has retired from the work as professor in anaesthesiology and intensive care medicine at umeå university. references 1. barr j, fraser gl, puntillo k, ely ew, gelinas c, dasta jf, et al. clinical practice guidelines for the management of pain, agitation, and delirium in adult patients in the intensive care unit. crit care med. 2013;41:263–306. 2. bassett r, adams km, danesh v, groat pm, haugen a, kiewel a, et al. rethinking critical care: decreasing sedation, increasing delirium monitoring, and increasing patient mobility. jt comm j qual patient saf. 2015;41:62–74. 3. brook ad, ahrens ts, schaiff r, prentice d, sherman g, shannon w, et al. effect of a nursing-implemented sedation protocol on the duration of mechanical ventilation. crit care med. 1999;27: 2609–15. 4. sessler cn, varney k. patient-focused sedation and analgesia in the icu. chest. 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fraser gl, coursin db, riker rr, fontaine d, wittbrodt et, et al. clinical practice guidelines for the sustained use of sedatives and analgesics in the critically ill adult. crit care med. 2002;30: 119–41. 16. schweickert wd, kress jp. strategies to optimize analgesia and sedation. crit care. 2008;12(suppl 3):s6. 17. reschreiter h, maiden m, kapila a. sedation practice in the intensive care unit: a uk national survey. crit care. 2008;12:r152. 18. martin j, heymann a, basell k, baron r, biniek r, burkle h, et al. evidence and consensus-based german guidelines for the management of analgesia, sedation and delirium in intensive care – short version. ger med sci. 2010;8:doc02. 19. chen k, lu z, xin yc, cai y, chen y, pan sm. alpha-2 agonists for long-term sedation during mechanical ventilation in critically ill patients. cochrane database syst rev. 2015;1:cd010269. 20. richards-belle a, canter rr, power gs, robinson ej, reschreiter h, wunsch h, et al. national survey and point prevalence study of sedation practice in uk critical care. crit care. 2016;20:355. 21. westmoreland cl, hoke jf, sebel ps, hug cc jr, muir kt. pharmacokinetics of remifentanil (gi87084b) and its major metabolite (gi90291) in patients undergoing elective inpatient surgery. anesthesiology. 1993;79:893–903. 22. battershill aj, keating gm. remifentanil: a review of its analgesic and sedative use in the intensive care unit. drugs. 2006;66: 365–85. 23. zaal ij, devlin jw, hazelbag m, klein klouwenberg pm, van der kooi aw, ong ds, et al. benzodiazepine-associated delirium in critically ill adults. intensive care med. 2015;41:2130–7. 24. kollef mh, levy nt, ahrens ts, schaiff r, prentice d, sherman g. the use of continuous i.v. sedation is associated with prolongation of mechanical ventilation. chest. 1998;114:541–8. 25. egerod i, albarran jw, ring m, blackwood b. sedation practice in nordic and non-nordic icus: a european survey. nurs crit care. 2013;18:166–75. appendix the questionnaire sent to the icus was designed as a multiple-choice form with the possibility to add written answers. background data were also asked for. questions: 1. do you use written guidelines for sedation? 2. which sedation scale do you use? 3. do you use daily interruption of sedation, so-called ‘wake up-calls’? 4. how are drugs for hypnosedation administered? 5. how are drugs for analgosedation administered? 6. which are the main drugs used for short-term hypnosedation (<24 h)? 7. which are the main drugs used for long-term hypnosedation (>24 h)? 8. which are the main drugs used for analgosedation? 9. to which patient groups/situations do you use dexmedetomidine? 10. to which patient groups/situations do you use clonidine? 11. are you reporting data to sir, according to sir’s quality indicator for sedation scales and sedation goals? 202 o. talsi et al. abstract introduction material and methods ethics statistical analysis results discussion disclosure statement acknowledgements notes on contributors references effect of physical training on fat-free mass in patients with chronic obstructive pulmonary disease full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 effect of physical training on fat-free mass in patients with chronic obstructive pulmonary disease (copd) margareta emtner, runa hallin, ragnheiður harpa arnardottir & christer janson to cite this article: margareta emtner, runa hallin, ragnheiður harpa arnardottir & christer janson (2015) effect of physical training on fat-free mass in patients with chronic obstructive pulmonary disease (copd), upsala journal of medical sciences, 120:1, 52-58, doi: 10.3109/03009734.2014.990124 to link to this article: https://doi.org/10.3109/03009734.2014.990124 © informa healthcare published online: 28 nov 2014. submit your 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https://www.tandfonline.com/doi/citedby/10.3109/03009734.2014.990124#tabmodule upsala journal of medical sciences. 2015; 120: 52–58 original article effect of physical training on fat-free mass in patients with chronic obstructive pulmonary disease (copd) margareta emtner1,2, runa hallin1, ragnheiður harpa arnardottir1,3,4 & christer janson1 1department of medical sciences, respiratory, allergy and sleep research, uppsala university, uppsala, sweden, 2department of neuroscience, physiotherapy, uppsala university, uppsala, sweden, 3school of health sciences, university of akureyri, akureyri, iceland, and 4department of rehabilitation, akureyri hospital, akureyri, iceland abstract background. weight loss and depletion of fat-free mass are common problems in patients with chronic obstructive pulmonary disease (copd) and are related to muscular weakness and exercise intolerance. physical training of copd patients has good effect on exercise tolerance and quality of life. the aim of this study was to examine factors that affect change in fat-free mass after physical training, in patients with copd. patients. patients were examined before and after a 4-month exercise period. weight and height were measured, and bioelectrical impedance was performed. fat-free mass (ffm) was calculated, by a three-compartment model, and fat-free mass index (ffmi) was calculated as ffm kg/m2 and body mass index (bmi) as kg/m2. a symptom-limited ramp ergometer test and 12-minute walk test (12mwt) were performed. dyspnoea score of daily activities was determined by chronic respiratory disease questionnaire (crdq). blood was taken for analyses of c-reactive protein (crp) and fibrinogen. patients with a bmi <21 kg/m2 were given nutritional support during the training period. results. a total of 27 patients completed the training (64 years, fev1 31% of predicted). patients with low ffmi gained 1.2 kg, whereas those with normal ffmi lost 0.7 kg (p = 0.04). in multivariate analyses high age (p = 0.03), low fev1 (p = 0.02), and a high level of dyspnoea (p = 0.01) at baseline were found to be negative predictors for increase in ffm. conclusions. difficulties in increasing the fat-free mass in copd patients by physical training seem to be associated with dyspnoea in daily life and impaired lung function (fev1). key words: chronic obstructive pulmonary disease, dyspnoea, exercise training, fat-free mass, nutrition introduction there is growing evidence that chronic obstructive pulmonary disease (copd) is a multi-organ systemic disease. skeletal muscle weakness, muscle wasting, and impaired exercise performance, which are poorly related to air flow limitation, have commonly been reported in copd patients (1). exercise-limiting factors include muscular weakness (2), dynamic hyperinflation resulting in increased work of breathing (3), increased load on the respiratory muscles (4), and an intensified perception of respiratory discomfort. in addition, symptoms perceived by the patients with anxiety and depression have impact on training tolerance and physical activity (5). loss of body weight is a common and serious finding in patients with copd (6) and is associated with increased mortality and morbidity independent of gender, smoking, and lung function (7). patients with low fat-free mass (ffm) often have muscular weakness, especially in the lower limbs (8). low weight is also related to low health-related quality of life and worsening dyspnoea (9). correspondence: margareta emtner, department of medical sciences, respiratory, allergy and sleep research, uppsala university, uppsala, sweden. e-mail: margareta.emtner@neuro.uu.se (received 13 october 2014; accepted 17 november 2014) issn 0300-9734 print/issn 2000-1967 online � 2014 informa healthcare doi: 10.3109/03009734.2014.990124 http://informahealthcare.com/journal/ups mailto:margareta.emtner@neuro.uu.se pulmonary rehabilitation is an important component in the management of subjects with copd (10) and is effective in improving exercise performance and health status (10). exercise training is a core component of pulmonary rehabilitation and has been shown to be the best available means of improving muscle function, exercise capacity, and cardiovascular function (10). exercise training has also been shown to result in less dyspnoea in daily life (11) and to less mood disturbance. before training start, an adequate assessment including evaluation of body composition, i.e. ffm, needs to be performed in order to optimize intervention strategies (10). body mass index (bmi) is a simple way to assess body composition. however, it is possible to have normal bmi and still be nutritionally depleted (12). ffm reduction is a better predictor of peak exercise performance than bmi (13), and it has been suggested that ffm is the best parameter to assess body composition. a few studies have investigated the efficacy of multimodal intervention, including nutrition and anabolic steroids integrated into pulmonary rehabilitation for advanced copd patients (14-16). although the intervention was successful in improving body weight, ffm, and exercise tolerance, the influence of the various components could not be determined. the underlying mechanisms contributing to depletionofskeletalmusclestillremainunclear.associations between various circulating markers of inflammation and the loss of muscle mass have been made in chronic inflammatory disease conditions, including copd. the aim of this study was to examine factors that affect change in fat-free mass after a period of physical training, in patients with copd. material and methods the study population was recruited from patients included in an exercise study comparing different training methods (17). patients with moderate to severe copd, according to the global initiative for obstructive lung disease (gold) criteria, were consecutively invited to participate in the exercise study (1). inclusion criteria were copd with a post-bronchodilator fev1/fvc ratio <0.7 and a fev1 <60% of the predicted value. all patients (n = 51) included in the training study were asked if they wanted to participate in the current study. forty-nine patients (96%) agreed to participate, of whom 27 patients completed the 4-month training period. patients were considered drop-outs if they attended less than 75% of the training sessions. reasons for drop-out were exacerbations (n = 16), other diseases (n = 2), and lack of motivation or transportation problems (n = 4). measurements were made before and after the 4-month training period. information on smoking historywasobtainedusingastructuredinterview.informed consent was obtained from all the participants. exercise training exercise sessions were performed twice a week. each session included cycle ergometer training at an intensity ‡65% of baseline peak exercise capacity (wpeak) for 27 min. for warming up (6 min) and cooling down (6 min) the patients cycled at 30%–40% of baseline wpeak. after the cycling, once a week the session proceeded with flexibility and relaxation training, while the other session of the week proceeded with resistance training for upper and lower limbs as well as abdominal muscles (10 repetitions, two sets at about 70% of 1 repetition maximum). exercise load was kept as high as tolerated at all times, above the target value when possible. patients who desaturated on exercise (spo2 <90%) were given supplemental oxygen during training to keep spo2 ‡90%. all patients with bmi <21 kg/m2 were given nutritional support ad libitum. the threshold of 21 kg/m2 was chosen because mortality has been shown to be increased below this value in copd populations (18). body composition body height was measured to the nearest 0.5 cm, and weight was measured on a balance scale to the nearest 0.1 kg. bioelectrical impedance analysis was performed in the morning with patients in a fasting state using a hydra efc/icf, model 4200, xitron tech (san diego, ca, usa). ffm was calculated by a three-compartment model, and the fat-free mass index (ffmi) was calculated as ffm kg/body height m2 and bmi as kg/m2. limits for depleted ffm were employed: ffmi £16 kg/m2 was used for men and £15 kg/m2 for women. the two outcome variables used were change in ffm, and ffm in per cent of baseline values (change in ffm of baseline). physical capacity peak exercise capacity in watt (wpeak) was determined by a progressive symptom-limited cycle ergometer test (case 8000 exercise testing system, ge medical systems, milwaukee, wi, usa). functional exercise capacity was measured by the self-paced 12-min walk test (12mwt) (19). the walking tests were performed in a level corridor, and the total distance walked (in meters) was measured. two tests were performed both before and after the training period. the second test was repeated factors associated with change in fat-free mass in copd 53 on a different day within one week. the better of the two tests was used. dyspnoea for evaluation of dyspnoea during activities in daily life the dyspnoea scale from the chronic respiratory disease questionnaire (crdq) was used (20). patients scored dyspnoea experienced during five self-chosen activities of daily living. the scale for each of the five activities is 7-graded, and a higher score indicates less dyspnoea. the score from the five activities were combined, and the combined dyspnoea score thereby ranged from 5 to 35. systemic inflammation blood samples were taken for analyses of c-reactive protein (crp) and fibrinogen. high-sensitivity crp was measured with a two-point nephelometry method with a prospec instrument (dade behring, marburg, germany) using monoclonal mouse antibodies (dade behring, marburg, germany). the total analytical imprecision was 1.4% at 1.23 and 5.49 mg/l, with a lowest detectable level of 0.17 mg/l. measurement of fibrinogen was performed using a prospec instrument (dade behring, marburg, germany) with a rabbit antibody against fibrinogen (dade behring, marburg, germany). the total analyticalimprecisionwas5.7%at2.2g/land4.4%at4.7g/l. analyses were carried out at the department of clinical chemistry at uppsala university hospital, sweden. lung function forced expiratory volume in one second (fev1) was measured with a jaeger master scope spirometer (jaeger, höchberg, germany). the best of three acceptable manoeuvres was used in accordance with the american thoracic society guidelines for standardization of spirometry (21). fev1 was expressed as a percentage of the predicted value using swedish reference values (22,23). the ethics committee at the medical faculty at uppsala university approved the study (2002-07-02, dnr 02-307). statistics results are expressed as mean ± sd. spearman rank correlation and multiple linear regression analysis were used when analysing the relation between change in ffm expressed in absolute values and per cent of the baseline values and baseline characteristics. spearman rank correlation was also used when analysing association between change in ffm and change in 12mwt and wpeak. variables associated with change in ffm at p < 0.10 in bivariate analyses were included in the multiple linear regression model. crp was log-transformed before being entered into the analyses. statistically significant difference was assumed when p < 0.05. results the study included 27 patients who completed the 4-month physical training period. the attendance rate was 29 ± 3 of 32 possible sessions. patients with low ffmi at training start were more likely to gain weight than patients with normal ffmi (table i). nutritional supplementation during the exercise training period was given to all patients with bmi below 21 kg/m2 at baseline; this meant that most patients with low ffmi received supplementation, whereas none of the patients with normal ffmi received supplementation (figure 1). the median change in ffm was 0.6 kg, while the median change in 12mwt and wpeak was 54 m and 10 w, respectively (figure 2). statistically significant correlations were found between changes in ffm and fev1, ffmi, dyspnoea score, and fibrinogen at baseline (table ii). there were no significant associations between change in ffm and change in 12mwt or change in wpeak. inthe multivariate analyses approximately 70% ofthe variation in ffm change was accounted for when age, table i. baseline characteristics and changes in body composition and physical capacity, for patients with low or normal fat-free mass index (ffmi). low was defined as ffmi £16 kg/m2 for men and £15 kg/m2 for women. low ffmi (n = 15) normal ffmi (n = 12) p female (%) 48 26 0.09 age (years) 64 ± 7 65 ± 7 0.73 fev1 % predicted 32 ± 10 31 ± 11 0.68 fibrinogen (mg/l) 4.1 ± 1 3.9 ± 1 0.71 crp (mg/l) 6.5 ± 10.1 8.2 ± 11.5 0.70 pack-years 40 ± 9 40 ± 9 0.94 dyspnoea score 17 ± 4 17 ± 3 0.90 d weight (kg) 1.2 ± 2.4 –0.67 ± 2.1 0.04 d fibrinogen (mg/l) –0.3 ± 0.9 –0.1 ± 0.8 0.53 d crp (mg/l) –4.4 ± 10.9 –1.4 ± 12.9 0.58 d ffm (kg/m2) –0.2 ± 2.4 1.3 ± 1.6 0.07 d wpeak (watt) 20 ± 28 11 ± 10 0.40 d 12mwt (m) 99 ± 90 35 ± 66 0.06 54 m. emtner et al. sex,fev1,dyspnoea,andfibrinogenwerecombinedina multiple regression model (table iii). change in ffm was negatively correlated with age and positively with fev1 and a higher dyspnoea score indicating less dyspnoea at the start of the training period. discussion our main finding is that patients with lower age, higher fev1, and a lower level of dyspnoea were more likely to increase in muscle mass during a 4-month physical training period. moreover, patients with a low fev1 were less likely to increase in ffm. it has been shown that muscle strength and fev1 are factors limiting exercise capacity (2), and that expiratory airflow obstruction is a limiting factor to maximal ventilation and thereby limits exercise tolerance (24). there are multiple possibilities for the association between airflow obstruction and exercise limitation, for example dynamic hyperinflation (3) resulting in increased work of breathing, increased load on the respiratory muscles (4), and the intensified perception of respiratory discomfort. another possible reason is that a low fev1 is related to hypoxic muscles due to insufficient oxygen caused by insufficient blood supply or hypoxemia (25). we found that dyspnoea was a risk factor for not increasing ffm during an exercise programme. one recent study reported that dyspnoea on exertion is correlated with general anxiety, indicating that exercise training may be influenced negatively by anxiety–10 –7.5 –5 –2.5 0 2.5 5 7.5 10 12.5 15 δ ffm % of baseline low ffmi kg/m2 at baseline normal ffmi kg/m2 at baseline figure 1. changesinfat-freemass(ffm)inpatientswithlowfat-free mass index (ffmi) and normal ffmi between baseline and four months of training. low ffmi is defined as ffmi £16 kg/m2 for men and £15 kg/m2 for women. filled-symbols = patients given no supplementation; open symbols = patients given supplementation. table ii. associations between changes in fat-free mass (ffm), and variables showing lung function, body composition, systemic inflammation, physical capacity, and dyspnoea at baseline. d ffm d ffm % baseline rho p rho p age (years) 0.06 0.76 0.09 0.66 sex female –0.003 0.99 –0.03 0.89 fev1 % of predicted 0.48 0.01 0.48 0.02 pack years (years) 0.09 0.65 0.09 0.64 bmi (kg/m2) –0.18 0.37 –0.26 0.19 ffmi (kg/m2) –0.35 0.07 –0.44 0.03 crp (mg/l) 0.18 0.39 0.19 0.38 fibrinogen (mg/l) 0.47 0.03 0.49 0.02 12mwt (m) 0.08 0.68 0.07 0.75 wpeak (watt) 0.20 0.30 0.16 0.42 dyspnoea 0.48 0.02 0.44 0.03 0 1 2 3 4 5 c o u n t –6 –5 –4 –3 –2 –1 0 1 2 3 4 5 d ffm (kg) median change 0.6 kg figure 2. distribution of changes in fat-free mass (ffm) between baseline and four months of training. table iii. associations between changes in fat-free mass (ffm), and age, sex, lung function, dyspnoea, and fibrinogen. estimates (beta coefficients) are adjusted for all the variables in the table. d ffm d ffm of baseline r2 = 0.697 r2 = 0.682 coeff. p coeff. p age (years) –0.120 0.03 –0.24 0.07 female sex –0.519 0.58 –0.658 0.77 fev1 % of predicted 0.104 0.02 0.255 0.03 dyspnoea crdq 0.336 0.01 0.661 0.03 fibrinogen (mg/l) 0.588 0.16 1.63 0.11 factors associated with change in fat-free mass in copd 55 worsened dyspnoea (26). as dyspnoea has been shown to be related to all subscores of st georges respiratory questionnaire, a low health-related quality of life is obvious in subjects with dyspnoea (27). dyspnoea can of course have many different explanations, and some of them can probably be treated. in malnourished patients with copd nutritional interventions can improve quality of life (28). strength training results in less dyspnoea during exercise, thereby making this strategy easier to tolerate than aerobic training (29). our finding that age is correlated with difficulties to increase ffm is consistent with the fact that cachexia is more common in older age (30). crp in our patients with low and normal ffmi was within the normal range, and there was no significant correlation in the bivariate analysis between crp at baseline and change in ffmi after training. this is in accordance with results of eagan et al. who found, in a large copd population, that crp was positively associated with ffmi (31), thus crp was not elevated in patients having lower ffmi. they concluded that crp is not elevated in cachectic copd patients. our results and the results from eagan et al. are in contrast to a prominent theory suggesting that pathological weight loss is at least partly explained by an increase in systemic inflammation reflected in enhanced plasma or serum concentrations of inflammatory markers (32). a probable explanation for increased crp in copd patients might instead be that active fat mass tissue in obese patients is associated to higher systemic levels of crp (33). there was a training response in our patients, which was seen in both increased maximal capacity (wpeak) and 12-min walk distance (34-36), and there was no significant difference in this response between the low ffmi group and the normal ffmi group. we found that ffm decreased slightly in the low ffmi group and increased in the normal ffmi group, but the difference between the two groups did not attain statistical significance. in accordance with our results, berton et al. reported that both their depleted patients (who were given nutritional supplementation and endurance and strength training for 12 weeks) and their non-depleted patients (who just got the 12 weeks of training) slightly increased ffm and that there was no statistically significant difference between the groups (34). two recent studies have shown improvements in ffm in depleted patients as a result of exercise training and nutritional supplementation, whereas depleted control patients who just received exercise decreased in ffm (36,37). our depleted patients received one oral nutritional supplementation after training, i.e. twice a week, whereas in the study by berton et al. patients received a daily polysaccharide supplementation if caloric ingestion was judged inadequate (34). in the two studies that improved ffm, one study gave essential amino acid supplementation twice a day for 12 weeks (37), and in the other study the patients were given three oral liquid supplements per 24 hours for four months (36). thus, the amount and type of supplement may be an important factor to consider. as exercise capacity increases as a result of exercise training independently of bmi (38), we suggest that to improve ffm in depleted patients nutritional supplementation has to be extensive. while muscle mass can be readily increased by progressive strength training, this would not be expected from an aerobic training programme such as ours. strength training has been shown to have greater potential to improve muscle mass and strength than endurance training (39). in addition, it was recently shown that cachectic patients can retain the potential for skeletal muscle remodelling as a result of strength training (40). it was not possible to study the effectiveness of supplementation during exercise training in patients with low ffmi because of our small number of patients. another limitation is that potential confounding factors including physical activity and metabolic syndrome features were not measured. our training programme was not primarily aimed at improving muscle mass but rather endurance capacity. another limitation is that we did not control for daily caloric intake, i.e. patients might have dropped in the normal dietary intake. our conclusion is that in copd old age, low fev1, and high level of dyspnoea in daily life were, independently of each other, related to less chance of increasing fat-free mass by physical training. funding this paper was supported by grants from the swedish heart and lung association e011-02, the swedish heart-lung foundation 2004-10-33, and 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http://www.ncbi.nlm.nih.gov/pubmed/19734323?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20110400?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20110400?dopt=abstract abstract introduction material and methods exercise training body composition physical capacity dyspnoea systemic inflammation lung function statistics results discussion funding declaration of interest references u22 protocol as measure of symptomatic improvement after catheter ablation of atrial fibrillation full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 u22 protocol as measure of symptomatic improvement after catheter ablation of atrial fibrillation niklas höglund, folke rönn, titti tollefsen, steen m. jensen & milos kesek to cite this article: niklas höglund, folke rönn, titti tollefsen, steen m. jensen & milos kesek (2013) u22 protocol as measure of symptomatic improvement after catheter ablation of atrial fibrillation, upsala journal of medical sciences, 118:4, 240-246, doi: 10.3109/03009734.2013.821190 to link to this article: https://doi.org/10.3109/03009734.2013.821190 © informa healthcare published online: 09 oct 2013. submit your article to this journal article views: 352 view related articles citing articles: 2 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.821190 https://doi.org/10.3109/03009734.2013.821190 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.821190 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.821190 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.821190#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.821190#tabmodule upsala journal of medical sciences. 2013; 118: 240–246 original article u22 protocol as measure of symptomatic improvement after catheter ablation of atrial fibrillation niklas höglund, folke rönn, titti tollefsen, steen m. jensen & milos kesek department of cardiology, heart centre, university hospital, s-901 85 umeå, sweden abstract introduction. left atrial catheter ablation is useful as symptomatic treatment in selected patients with atrial fibrillation (af). evaluation requires measurement of arrhythmia-related symptoms. many of the published protocols have drawbacks and have been used in af only, with no possible comparison to other ablations that compete for the same resources. u22 is a published protocol that quantifies paroxysmal tachycardia symptoms through scales with 11 answer alternatives, translated into discrete numerical scales 0–10. it has been shown to reflect the clinical improvement after ablation of supraventricular tachycardia. here we report the use of u22 in measuring improvement after catheter ablation for af. material and methods. a total of 105 patients underwent first-time ablation for af and answered u22 and sf-36 forms at baseline and follow-up 304 (sd 121) days after ablation. independently, the patients underwent a clinical follow-up. all decisions regarding medication and reablation were taken without knowledge of the symptom scores. results. the u22 scores for well-being, arrhythmia as cause for impaired well-being, derived time-aspect score for arrhythmia, and discomfort during attack detected relevant improvements of symptoms after the ablation. u22 showed larger improvement in patients undergoing only one procedure than in patients who later underwent repeated interventions, thus reflecting the independent clinical decision for reablation. conclusion.u22 quantifies the symptomatic improvement after af ablation with adequate internal consistency and construct validity. u22 mirrors aspects of the arrhythmia symptomatology other than sf-36. keywords: arrhythmia symptoms, atrial fibrillation, catheter ablation, quality of life, symptom-specific protocol introduction left atrial catheter ablation is a suitable treatment in selected patients with atrial fibrillation. the patients experience improvement due both to decreased incidence of arrhythmia and decreased symptoms from remaining arrhythmias (1). the procedure is demanding and competes for limited resources with other electrophysiological interventions. no prognostic gain has yet been shown, and the indication for the procedure presently is only symptomatic. tools for measurement of arrhythmia-related symptoms are therefore much needed for evaluation and comparison between different groups. several questionnaires that quantify symptoms associated with tachyarrhythmias have been published. the symptom checklistfrequency and severity scale exists in multiple versions that are not well-described in the literature (2-6). the asta-questionnaire (7) is a three-part protocol that measures arrhythmia-specific symptoms and health-related quality of life in connection with arrhythmias. most of the other protocols have been developed and used only in atrial fibrillation (8-16), thus offering no comparison with the large groups of other arrhythmias that also undergo ablation on symptomatic indication and compete for the same resources. u22 is a patient-administered protocol that quantifies paroxysmal symptoms associated with tachyarrhythmias. the protocol has been published and shown to reflect the clinical improvement after correspondence: milos kesek, department of cardiology, heart centre, university hospital, s-901 85 umeå, sweden. fax: +46 90 127630. e-mail: milos.kesek@medicin.umu.se (received 2 april 2013; accepted 26 june 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.821190 http://informahealthcare.com/journal/ups mailto:milos.kesek@medicin.umu.se ablation of paroxysmal supraventricular tachycardia, a procedure considered as curative with a distinct endpoint and a high success rate (17,18). our present aim is to quantify the symptomatic effect of left atrial catheter ablation for atrial fibrillation with u22 and relate the results to the incidence of reablation based on the clinical evaluation that serves as reference. material and methods patients the study group consists of patients who underwent left atrial catheter ablation for paroxysmal and persistent atrial fibrillation at the heart centre, university hospital, umeå, sweden, between 2006 and 2011. on admission, the patients were invited to answer the baseline u22 and sf-36 forms. radiofrequency ablation was subsequently performed as segmental pulmonary vein isolation, wide antral circumferential isolation with an irrigated tip catheter, and the carto� mapping system (biosense webster, inc., diamond bar, ca, usa) or isolation with a multi-polar circular ablation catheter (pvac�, medtronic, inc., minneapolis, mn, usa). a questionnaire follow-up with u22 and sf-36 was performed 6–9 months after the ablation. the answers were prospectively entered into a database by a co-author (t.t.), blinded with respect to the clinical picture. independently from the u22 protocol, the patients underwent a clinical follow-up (co-authors n.h., f.r., s.m.j., m.k.) and a 7-day holtermonitor recording. all decisions regarding medication and possible repeated ablation were taken without knowledge of the symptom scores. the protocol data were subsequently retrieved for the analysis. at review time the clinical data and the catheterization reports were analysed and coded according to a pre-specified scheme by co-authors blinded with respect to the u22 results (n.h., f.r., s.m.j., m.k.). questionnaires the arrhythmia-related symptoms were measured with the u22 protocol. detailed description and the definitions of the individual u22 scores have been published previously (17,18). symptoms are quantified by positioning the answers on verbally described scales with 11 horizontally aligned answer alternatives, an approach similar to that by härden et al. (10). the answers are translated into discrete numerical scales with a range of 0–10 (nrs-10, a likert scale that is a discrete alternative of the common visual analogue scale). in the current analysis the following u22 questions were used: q01: ‘on the whole, how have you felt over the past month?’ [miserable–very well], [0–10] q02 (follow-up only): ‘compared to the time before the treatment, do you now feel:’ [very much worse– very much better], [0–10] q03: ‘do you take any prescribed medications for your heart rhythm problems?’ [no, yes] q06 (follow-up only): ‘have you experienced any problems with the heart rhythm after the treatment? – please disregard the first 3 months after the treatment –’ [no; yes, of the same type as before the treatment; yes, of different type] q08: ‘how often do you experience problems with heart rhythm? (despite taking any medication)’ [never, rarely, a few times a month, a few times a week, daily, all the time] q10: ‘how long does a spell usually last?’ [seconds, more than a minute but less than 15 min, 15 min to 1 hour, 1 to 4 hours, more than 4 hours, all the time] q11: ‘how much do the spells affect your wellbeing?’ [not at all–very much], [0–10] q12. ‘how bothered are you while you are experiencing a spell?’ [not at all–very much], [0–10] a time-aspect with range 0–10 was computed by summarizing the scores from q08 and q10 (18). sf-36 (19) was used as a generic measure of quality of life. it quantifies the mental and physical wellbeing in eight scales with range 0–100, together with two summary scores, physical component summary (pcs) score and mental component summary (mcs) score. answers to u22 and sf-36 were compared between baseline and follow-up, group-wise and in individual patients. statistics data are presented as mean (sd), unless otherwise stated. differences between groups in continuous variables were examined by paired and unpaired two-tailed t test and global f test. pearson’s r was used for correlation between continuous variables. cronbach’s alpha was used for estimating internal consistency of the scores. for analysis of freedom from subsequent reablation, kaplan–meier curves were constructed, and differences between dichotomized groups were evaluated by log-rank test. twotailed fisher exact text and chi-square test were used for testing differences in proportions. a p-level of 0.05 was considered as significant. the data were analysed in r 2.15.2 (r foundation for statistical computing, 2012, http://www.r-project.org). the study was approved by the ethics committee at the umeå university faculty of medicine. measuring symptoms in atrial fibrillation with u22 241 http://www.r-project.org results a total of 105 patients undergoing a first-time left atrial ablation on clinical indication answered all four required forms (u22 and sf-36 at baseline and follow-up). the group consisted of 78 men and 27 women aged 58 (sd 9) years. clinical description the atrial fibrillation was paroxysmal in 50%, persistent in 48%, and long-standing persistent in 2% of the patients. at baseline, the chads2 scores were 0, 1, 2, 3, and 4 in 49%, 35%, 11%, 4%, and 1% of patients, respectively. the mean left atrial diameter was 44 (sd 7) mm. segmental pulmonary vein isolation was performed in 36 cases (34%), wide antral circumferential isolation in 43 cases (41%), and isolation with the multi-polar catheter in 26 cases (25%). the procedure and fluoroscopy times were 208 (sd 50) and 36 (sd 13) minutes. at the clinical follow-up 136 (sd 58) days after the ablation, 62% of the patients had no atrial fibrillation in the 7-day holter-monitor recording. in 61% of the patients, the symptomatic result was considered satisfactory, and antiarrhythmic drugs were discontinued. in 20% the result was considered as satisfactory, but the antiarrhythmic therapy was continued. in 14% the result was not satisfactory, and a second catheter ablation was intended. in 5% the result was not satisfactory, but no catheter intervention was planned at the clinical follow-up (due to patient’s wish, permanent atrial fibrillation, or planned surgical ablation). the patient records were analysed at review time 873 (sd 490) days after the first-time ablation. at that time the incidence of reablation was 34%, while 66% had only undergone the initial ablation. u22 in all first-time ablations at the questionnaire follow-up 304 (sd 121) days after the ablation, 29 patients (28%) reported freedom from arrhythmia symptoms, 48 (46%) had the same type of symptoms as before the ablation, and 27 (26%) had symptoms of different type (q06). eighty (78%) patients reported taking some medication (including beta-blockers) against the arrhythmia, compared to 96 (91%) prior to the ablation (q03). the main results are summarized in tables i and ii. compared to the state before ablation, significant improvements were recorded at follow-up in the u22 scores for well-being (q01), arrhythmia as cause for impaired well-being (q11), the time-aspect score for arrhythmia derived from q08 and q10 (17,18), discomfort during an attack (q12), and the sf-36 summary scores pcs and mcs. no significant differences between the three ablation techniques were seen in improvement of well-being (expressed as difference q01follow-up – q01baseline, f = 1.26, p = 0.3), arrhythmia as cause for impaired well-being (difference q11follow-up – q11baseline, f = 0.17, p > 0.5), or discomfort during an attack (difference q12follow-up – q12baseline, f = 0.12, p > 0.5). no significant differences in these parameters were detected between the groups with chads2 = 0, 1, and >1. the score for patients’ retrospective estimation of improvement in well-being measured at followup (q02) was 7.1 (sd 2.5). this retrospective estimate of improvement correlated moderately to the improvement computed as difference between the score for well-being at follow-up and before ablation (q01follow-up – q01baseline) (r = 0.55, p < 0.0001). at baseline, u22 score for well-being (q01) correlated moderately to the sf-36 summary variables pcs and mcs (r = 0.65 and 0.49). the correlations between q11, q12, and time-aspect on one side and pcs and mcs on the other side were weak (r £ 0.4) or non-significant. at follow-up, q01 correlated strongly to pcs and moderately to mcs (r = 0.75 and 0.54); and q11, q12, and time-aspect correlated moderately to pcs (r = 0.59, 0.57, and 0.61) and weakly to mcs (r £ 0.4). the strong and moderate correlations were significant (p £ 0.0004, corrected for multiple comparisons by holm’s method). patients had a low incidence of reablation at review time if their first-time ablation was associated with a high improvement of u22 scores (figure 1). u22 in singular procedure, compared to the first of repeated procedures of the above patients 69 underwent only one procedure. they were compared to the 36 patients who later underwent repeated procedures (table ii). at followup, u22 detected a significantly larger symptomatic improvement in the scores for well-being (q01), arrhythmia as cause for impaired well-being (q11), discomfort during an attack (q12), and derived timeaspect score for arrhythmia in the patients who underwent a singular ablation than in the group with the first of multiple ablations (figure 2). most recent of repeated procedures nineteen patients who had undergone repeated procedures (mean 2.11/patient) answered all four forms in connection with their most recent procedure. after 242 n. höglund et al. this last ablation, the u22 scores and the sf-36 summary scores had improved to a similar degree as after a singular ablation (table ii). their improvement in the u22 scores and the sf-36 summary scores did not significantly differ from that of the 75 patients undergoing a singular procedure. freedom from atrial fibrillation in the follow-up holter recording the patients with freedom from atrial fibrillation in the 7-day holter-monitor recording at clinical followup had significantly better mean u22 scores for well-being (q01), arrhythmia as cause for impaired well-being (q11), the time-aspect score for arrhythmia, and discomfort during an attack (q12) compared to those with some episode of atrial fibrillation (6.9 versus 4.4; 3.9 versus 6.7; 3.3 versus 5.7; and 4.1 versus 6.8, respectively, p < 0.0005 for all comparisons). at review time 27% of the former had undergone reablation, compared to 67% of the latter (log-rank test, p < 0.0001). validity of u22 cronbach’s alpha for the set of u22 scores at baseline, follow-up, and individual patients’ score differences were 0.79, 0.94, and 0.91, indicating a satisfactory to excellent internal consistency (table iii). the construct validity of u22 for measurement of symptoms in atrial fibrillation is supported by the relation of the u22 scores to the symptomatic effect of the initial table i. patient profile, u22, and sf-36 in 105 ablated patients. all first-time ablations paroxysmal non-paroxysmal p n 105 52 52 men (%) 74 67 81 0.2 age (years) 58 (9) 56 (10) 60 (7) 0.03 chads2 score 0 (%) 49 48 50 > 0.5 left atrial diameter (mm) 44 (7) 44 (6) 45 (8) 0.3 structurally normal echo (%) 75 77 69 0.5 procedure time (min) 208 (50) 214 (50) 202 (51) 0.2 fluoroscopy time (min) 36 (13) 38 (13) 35 (13) 0.2 no af in post-ablation holter (%) a 62 44 78 0.001 days to questionnaire follow-up b 304 (121) 272 (119) 335 (117) 0.007 u22 score q01baseline c 4.1 (2.6) } p < 0.0001 3.8 (2.4) 4.5 (2.8) 0.2 u22 score q01follow-up 6.1 (3.0) 5.7 (3.0) 6.7 (2.8) 0.07 u22 score q11baseline d 8.1 (1.9) } p < 0.0001 8.4 (1.7) 7.9 (2.1) 0.2 u22 score q11follow-up 4.9 (3.8) 5.3 (3.7) 4.3 (3.9) 0.2 u22 score time-aspectbaseline e 6.6 (1.9) } p < 0.0001 6.6 (1.6) 6.5 (2.1) > 0.5 u22 score time-aspectfollow-up 4.0 (3.1) 4.5 (2.9) 3.4 (3.2) 0.08 u22 score q12baseline f 8.0 (1.8) } p < 0.0001 8.5 (1.4) 7.7 (2.1) 0.03 u22 score q12follow-up 5.0 (3.8) 5.5 (3.8) 4.4 (3.9) 0.2 sf-36 score pcsbaseline 39.8 (9.9) } p = 0.003 39.4 (10.1) 40.5 (9.6) > 0.5 sf-36 score pcsfollow-up 43.9 (11.5) 43.1 (12.1) 45.1 (10.8) 0.4 sf-36 score mcsbaseline 41.7 (12.5) } p < 0.0001 40.8 (12.0) 42.4 (13.2) 0.5 sf-36 score mcsfollow-up 47.6 (10.9) 47.5 (10.5) 47.6 (11.5) > 0.5 days to review g 873 (490) 959 (570) 801 (378) 0.1 reablated at review time (%) 43 50 35 0.16 afreedom from atrial fibrillation in a 7-day holter recording at time of a clinical follow-up. btime between ablation and answering the follow-up forms. cu22 score for well-being, 0–10 (miserable–very well). du22 arrhythmia cause for impaired well-being, 0–10 (not at all–very much). ecomputed from u22 q08 and q10, 0–10 (10 is most severe). fu22 discomfort during attack, 0–10 (not at all–very much). gtime between ablation and review of the patient record with respect to subsequent reablation. measuring symptoms in atrial fibrillation with u22 243 intervention: improvement in the scores after a clinically inefficient first ablation was significantly smaller than after a singular ablation (figure 2). similarly, a larger improvement in the scores was associated with a significant increase in time to reablation (figure 1). discussion ablation of atrial fibrillation resulted in symptomatic improvement that was recorded by the u22 scores. in the group undergoing repeated procedures, the table ii. singular and repeated ablations. singular procedure first of repeated procedures last of repeated procedures n 69 36 19 men 48 (70%) 30 (83%) 14 (74%) age (years) 59 (9) 57 (8) 58 (9) days to questionnaire follow-up a 331 (121) 250 (103) 299 (91) baseline at follow-up p baseline at follow-up p baseline at follow-up p u22 q01 b 4.3 (2.8) 7.0 (2.8) < 0.0001 3.8 (2.1) 4.4 (2.5) 0.1 4.1 (2.2) 6.5 (2.7) 0.0001 u22 q11 c 8.0 (2.1) 3.5 (3.8) < 0.0001 8.4 (1.4) 7.4 (2.4) 0.02 8.0 (1.7) 3.1 (3.8) 0.0002 u22 time-aspect d 6.3 (2.1) 2.7 (3.0) < 0.0001 7.1 (1.4) 6.4 (1.6) 0.03 7.1 (1.1) 2.7 (2.8) 0.0001 u22 q12 e 7.8 (2.1) 3.6 (3.9) < 0.0001 8.4 (1.2) 7.5 (2.1) 0.03 8.4 (1.2) 2.6 (3.1) <0.0001 sf-36 pcs 39.9 (10.2) 45.6 (11.6) < 0.000139.8 (9.4) 40.7 (10.8) > 0.5 39.6 (11.8) 42.8 (13.7) 0.05 sf-36 mcs 41.9 (12.5) 48.6 (11.0) < 0.000141.3 (12.7) 45.6 (10.6) 0.1 40.7 (13.7) 49.4 (10.2) 0.03 atime between ablation and answering the follow-up form. bu22 score for well-being, 0–10 (miserable–very well). cu22 arrhythmia cause for impaired well-being, 0–10 (not at all–very much). dcomputed from u22 q08 and q10, 0–10 (10 is most severe). eu22 discomfort during attack, 0–10 (not at all–very much). 0 500 1000 1500 2000 0.0 0.2 0.4 0.6 0.8 1.0 lower median upper median figure 1. symptomatic improvement in 105 patients and freedom from subsequent reablation. survival curves for freedom from reablation as a function of days after first-time ablation. the population was dichotomized into two subsets along the median of individual patients’ differences in u22 scores, computed as (scorefollow-up – scorebaseline). the plot is shown for the u22 score q11 (effect of arrhythmia on the well-being), p < 0.0001 for the difference between the survival curves. a similar pattern was seen in the u22 scores q01, q12, and time-aspect of arrhythmia (p = 0.0006, p < 0.0001, and p < 0.0001, respectively). −10 −5 0 5 10 q01 diff q11 diff q12 diff time aspect diff p = 0.0002 p < 0.0001 p < 0.0001 p < 0.0001 figure 2. differences in u22 scores in singular ablations compared to the first of multiple ablations. the differences for q01, q11, q12, and time-aspect in individual patients were computed as (scorefollow-up – scorebaseline). singular ablations are represented by white boxes, the first of multiple ablations by grey boxes. the boxes are delimited by mean ± 1 sd. the central line depicts the mean, and the whiskers are placed at the extreme values. for all scores the singular ablations resulted in significantly larger improvements than the first of multiple ablations. 244 n. höglund et al. clinical indication for the reablation is clearly reflected in the diminutive improvement in u22 scores after the first ablation. the latter (£1 points difference in the u22 scores; figure 2) may be explained by a positive but clinically insufficient effect of the initial ablation or by a placebo effect. the symptomatic improvement in u22 after the last of repeated ablations is comparable to that after a singular ablation. this is in line with the established clinical experience regarding symptomatic gain from repeated procedures. the patients’ retrospective estimate of improvement at follow-up correlated modestly to the improvement expressed by the difference in the individual scores for well-being between the follow-up form and the baseline form. a retrospective estimation of improvement is therefore at best a rough measure for the change in well-being after ablation of atrial fibrillation. cronbach’s alpha indicated an excellent internal consistency for the set of u22 scores at follow-up and for the individual patients’ score differences between baseline and follow-up. also the consistency of the u22 scores in the baseline measurement was satisfactory (table iii). the correlation between u22 scores and sf-36 scores was weak to moderate. u22 is directed towards measurement of arrhythmia symptoms and their effect on well-being. sf-36, on the other hand, is a generic measurement of quality of life and mirrors aspects of atrial fibrillation other than u22. the present patients with atrial fibrillation have a greater impairment of well-being and higher level of symptoms both before and after an ablation than patients undergoing first-time ablation of av-nodal re-entry tachycardia or accessory pathway and evaluated by the u22 protocol (18). study limitations we use incidence of reablation at review time as a clinically relevant hard end-point. due to the nature of atrial fibrillation, relapses occur even after successful ablations. it is therefore not entirely correct to interpret reablation as a sign of an unsuccessful firsttime procedure. added co-morbidity and progress of the arrhythmia might shift a decision towards conservative treatment, in spite of significant symptomatology. some patients may have developed an asymptomatic permanent atrial fibrillation after the initial ablation. they would not be reablated, and their first-time ablation will thus be considered as successful. formally this may be correct, since ablation of atrial fibrillation is performed on symptomatic indication. most cardiologists would nevertheless hesitate to consider such an ablation as successful. these factors may decrease the specificity of reablation as a marker for continued symptoms. they should, however, not affect the conclusion that the symptom quantification by u22 is related to the independent decision regarding a reablation. in conclusion, u22 measures the symptomatic effect of left atrial ablation for atrial fibrillation. u22 has several practical advantages. it consists of a limited number of questions. the scales have a high resolution (scored 0–10), while many of the other symptom protocols only use four levels. the u22 scores are well suited for a statistical comparison between groups of arrhythmia patients and easily clinically interpreted in individual patients. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. hindricks g, piorkowski c, tanner h, kobza r, gerds-li jh, carbucicchio c, et al. perception of atrial fibrillation before and after radiofrequency catheter ablation: relevance of asymptomatic arrhythmia recurrence. circulation. 2005; 112:307–13. 2. bubien rs, knotts-dolson sm, plumb vj, kay gn. effect of radiofrequency catheter ablation on health-related quality of life and activities of daily living in patients 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www.ncbi.nlm.nih.gov/pubmed/9817107?dopt=abstract abstract introduction material and methods patients questionnaires statistics results clinical description u22 in all first-time ablations u22 in singular procedure, compared to the first of repeated procedures most recent of repeated procedures freedom from atrial fibrillation in the follow-up holter recording validity of u22 discussion study limitations declaration of interest references gunnar blix and his discovery of sialic acids. fascinating molecules in glycobiology full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 gunnar blix and his discovery of sialic acids. fascinating molecules in glycobiology arne lundblad to cite this article: arne lundblad (2015) gunnar blix and his discovery of sialic acids. fascinating molecules in glycobiology, upsala journal of medical sciences, 120:2, 104-112 to link to this article: https://doi.org/10.3109/03009734.2015.1027429 © informa healthcare published online: 28 apr 2015. submit your article to this journal article views: 644 view related articles view crossmark data citing articles: 2 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://doi.org/10.3109/03009734.2015.1027429 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1027429 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1027429 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1027429&domain=pdf&date_stamp=2015-04-28 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1027429&domain=pdf&date_stamp=2015-04-28 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1027429#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1027429#tabmodule upsala journal of medical sciences. 2015; 120: 104–112 gunnar blix and his discovery of sialic acids. fascinating molecules in glycobiology arne lundblad g.kjellbergs väg 5, 75643, uppsala, sweden key words: glycoprotein, glycolipid, sialic acid gunnar blix (figure 1) was born in lund in 1894. he was the son of a famous physiologist, magnus blix. after medical school, he started in research and defended his thesis entitled studies on diabetic lipaemia in 1925 at the university of lund. previously he had been employed by the university as a research fellow in anatomy and medical chemistry, and for shorter periods of time he also practised as a physician. directly after his dissertation he moved to uppsala, where he obtained a position in 1926 as associate professor (laborator) and four years later succeeded carl thore mörner as professor in medical and physiological chemistry. this professorship had been created in 1853. gunnar blix held the professorship for 31 years (1930–1961). during the years 1956–1961 he also served as deputy vice chancellor in the university administration. he was editor-in-chief for acta societatis medicorum upsaliensis (from 1972 upsala journal of medical sciences) between 1943 and 1969. after his retirement he continued to work until 1970 as executive member of the swedish foundation for nutritional research (stiftelsen svensk näringsforskning). gunnar blix died on 10 june 1981 at the age of 87 years. those who met and got to know gunnar blix remember him as an honest, dutiful, and humble person who generously gave of his ideas and devoted much of his time to teaching students in the medical school and graduate students in his research group. he became a highly worthy successor to his predecessors of the professorship (table i, figure 2). his time as professor coincided with an important period in the development of the scientific field of medical and physiological chemistry. he became internationally well known and a leading scientist within his own research field. gunnar blix enjoyed public confidence, and it was by no means accidental that he was assigned to numerous commissions of trust within his own faculty, within the university, and also nationally. he was a member of the organizing committees for the formation of both gothenburg and umeå medical schools in 1947 and 1958, respectively. in the case of umeå, all existing universities in sweden voted against the establishment of a medical school there. blix, however, made a reservation against the negative attitude from uppsala and thereby he succeeded in convincing the swedish government to proceed with the plans, which later on led to the formation of umeå university. the faculty of medicine in uppsala initially became ‘mother faculty’ to the new medical faculties in both gothenburg and umeå and later on also in linköping. when blix became a member of the faculty of medicine in uppsala, his department of medical chemistry was housed in an old building called ‘gamla kemikum’ (old chemistry institute) located in engelska parken (the english park). this building (figure 3) was erected in 1853–1859 and in time became more and more worn out. the sanitary conditions, for instance, made it gradually impossible to continue to work there. the government therefore correspondence: arne lundblad, g.kjellbergs väg 5, 75643, uppsala, sweden. e-mail: arne.lundblad@comhem.se (received 26 february 2015; accepted 28 february 2015) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1027429 http://informahealthcare.com/journal/ups mailto:arne.lundblad@comhem.se decided (in 1941) to construct a new building, to be shared by the departments of medical chemistry and pharmacology. blix took a very active role in the planning of the various facilities and in the moving to the new building (figure 4). due to the second world war the construction materials were not of the best quality, and the whole building project was also delayed. not until 1946 did the transfer actually take place. gunnar blix was also engaged as an inspector for two ‘student nations’ in uppsala: 1938–1943 for ‘gotlands nation’ and 1943–1961 for ‘norrlands nation’. when he resigned from his inspectorship he was honoured with a festschrift in which blix’s instrumental role for the establishment of umeå university is emphasized. in addition, the student dormitories ‘norrlands-gårdarna’ were built during blix’s time as inspector. blix covered a broad area in his research. his interest in lipid chemistry, which was documented already in his thesis, continued through the years. in this context two important discoveries should be mentioned. the first came already in 1933, in his studies of lipids in human brain tissue, when he isolated and characterized the first sulfatide known at this time. he showed that it was composed of sulfuric acid, cerebronic acid, sphingosine, and galactose (figure 5) (1). the other important discovery was made together with arne tiselius and harry svensson in 1941 when blix demonstrated the existence of lipoproteins and that the blood lipids preferentially were transported in the bglobulin fraction obtained on serum electrophoresis (2). the most important discovery, however, was made in 1936. this year gunnar blix isolated a crystalline compound from bovine submaxillary mucin (3). this compound was later designated ‘sialic acid’ by blix. much of the further work on sialic acid(s) was carried out during the 1950s and in competition with several groups worldwide. figure 1. gunnar blix in 1970. table i. predecessors to gunnar blix for the professorship in medical and physiological chemistry at uppsala university. elof wallquist 1853–1857 sten stenberg 1858–1859 august almén 1861–1883 olof hammarsten 1883–1906 sven gustaf hedin 1908–1924 carl thore mörner 1925–1929 gunnar blix and his discovery of sialic acids 105 two of these important discoveries were published in hoppe-seyler’s zeitschrift für physiologische chemie, published by the springer verlag in germany. this journal had high impact, as we say today, and german was an important scientific language in those days. mainly during the 1960s, but also after his retirement, blix was very interested in research related to nutrition, food intake, and public health. the importance of iron in food and the problem of sufficient iron intake in connection with a low total calorie intake was discussed in some of his publications. in his role as executive member in the swedish foundation for nutritional research and also chief editor for the swedish journal näringsforskning (nutritional research) he was able to influence the quality of ongoing nutritional research in sweden at the time. blix became a member of the royal swedish academy of science in 1956 and received ‘björkén’s award’ (björkénska priset) in 1960 from uppsala university for his studies on sialic acid. in 1965 he was awarded an honorary doctoral degree in medicine at the university of oslo. the early years and the discovery the discovery of sialic acid was, as mentioned above, blix’s most important contribution to science. this made him known internationally. in a lecture that blix presented at the ivth international congress of biochemistry in vienna 1958 (4) he looks back, and in a fascinating resumé he describes his view on how the discovery was made in 1936 and what actually happened during the following 22 years. there were many initial difficulties and mistakes, partly due to the peculiar distribution and properties of the molecules, association to both proteins and lipids, ability to form mixed crystals, lability, and tendency to decompose. on the other hand one remarkable circumstance greatly favoured the study: the molecules occur mainly in terminal positions of the parent molecules and are easy to split off. furthermore materials could be found that figure 2. four generations of professors in medical and physiological chemistry. sitting from left: carl thore mörner, sven gustaf hedin, olof hammarsten. standing: gunnar blix (1930). figure 3. ‘gamla kemikum’, later called philologicum. 106 a. lundblad contained up to 20% or more of sialic acid. however, not until an isolation procedure giving a sufficient yield had been developed would it become possible to perform a more detailed structural analysis. in the mid-1930s blix got interested in the chemistry of salivary mucin. at that time the knowledge was restricted to the fact that the mucin contained hexosamine. blix prepared the mucin using a method that his predecessor olof hammarsten had developed as early as 1881. when blix arrived in uppsala as associate professor in 1926, professor hammarsten was around 85 years old. it is tempting to guess that he visited his old laboratory in the english park and inspired the young blix to look a little closer into the chemistry of mucin. hammarsten died in 1932 and never witnessed the international breakthrough for sialic acid. blix continues his story in the lecture and tells us that ordinary acid hydrolysis resulted in the formation of black humins and, as known before, free hexosamine. different enzymes were tried with no results. however, when water-insoluble submaxillary mucin was heated at 100�c and at its isoelectric point (ph 2.5–3.5) some peptide material together with the bulk of the carbohydrate was dissolved. after fractionation of the methanol-soluble part of this material with less polar solvents, one day suddenly there was, unexpectedly, growth of nice crystal colonies on the walls of the vessel (figure 6, left). blix does not tell us how his discovery was celebrated and if anyone other than himself was present. we can only guess that his loyal assistant ewert lindberg was there and that probably no celebration took place; but a toast in champagne would certainly have been appropriate. this was how it started. it might be added that blix in the course of his intensive studies of saliva personally developed a technique to contract the muscles around one of his submaxillary glands. by doing so he was able to eject a jet of saliva straight into a test tube. his method might have simplified the purification and certified the origin of the saliva. figure 4. the departments of medical chemistry and pharmacology, located at stockholmsvägen 19 (later named dag hammarskjölds väg). ceramide sphingosine oh chchch ho h o o h h 3 hor h oh ch2oh ch c ch(oh) (ch2)21 ch3(ch2)12ch3 galactose fatty acid (eg. cerebronic acid) ch2 o h n figure 5. structure of a galactocerebroside (today usually called galactosphingolipid). in sulfatides the r at carbon 3 in galactose is a sulfate group. a glycosphingolipid containing sialic acid in the carbohydrate unit of the cerebroside is named ganglioside. gunnar blix and his discovery of sialic acids 107 a positive thing with the original experiment when crystals of sialic acid were discovered was that an optimal ph (2.5–3.5) had been chosen. the sialic acid was released but not decomposed. on the other hand it was most unfortunate that bovine submaxillary mucin was chosen as it would turn out later. if instead sheep or pig mucin had been used, a relatively stable form of sialic acid would have been found directly, and crystals like those in figure 6 (right) might have been seen. bovine mucin, however, contains at least two extremely labile forms of sialic acid. the crystals obtained were recrystallized and elementary analyses were performed. the results showed a composition of c14h23cno11. acetyl determination showed two groups, one probably bound to nitrogen. the molecule gave a pink colour with ehrlich’s reagent (p-dimethyl-amino-benzaldehyde). colorimetric analytical methods in studies of carbohydrates (hexoses, fucose, hexosamines, etc.) were standard procedures at a time long before nuclear magnetic resonance (nmr) and mass spectrometry (ms) became available. the analytical results pointed towards a disaccharide composed of n-acetylhexosamine and a poly-hydroxy-acid with six carbon atoms. mucin research temporarily put aside. international competition evolves since no methods were available for a large-scale preparation of sialic acid that would enable a detailed characterization, further research on mucin was put aside in blix’s laboratory for about 10 years. instead blix resumed his lipid research, and in that context he found in 1938 (6) a product with striking similarities to sialic acid. in his studies of normal brain lipids he found a subfraction that gave a beautiful violet colour with bial’s reagent (contains orcinol that forms coloured complexes with different carbohydrates when shaken in isoamylalcohol). the material was purified and hydrolysed with acid. fatty acids, sphingosine, and carbohydrate were liberated, but in contrast to the behaviour of cerebrosides this material became black like in humin formation. it turned out that klenk a few years earlier (in 1935) (7) had found a lipid in brain tissue from a patient with niemann–pick’s disease. klenk’s lipid fraction also gave black humin formation in acid hydrolysis, which led blix to test his purified brain lipid with ehrlich’s and bial’s reagents: pink and violet colours, respectively, were obtained with both the brain lipid and the previously isolated sialic acid from mucin. klenk later gave his new glycolipid the name ganglioside (figure 5). a few years later (in 1941) (8) klenk degraded his ganglioside in methanolic (5%) hydrochloric acid and succeeded in isolating a crystalline methoxy derivative of an acid that he named neuraminic acid. methoxyneuraminic acid was more stable than sialic acid and had in contrast to the latter a free amino group. it reacted in the same way with ehrlich’s and bial’s reagents as sialic acid and displayed the same tendency to humin formation. obviously it was a matter of closely related or possibly identical compounds. only many years later was the relationship between the two compounds clarified. because of the introduction of freeze-drying techniques blix returned to the sialic acid problem. it became possible to obtain fairly good yields of the substance from bovine submaxillary mucin. it could be demonstrated that the mucin contained sialic acid and galactosamine in approximately equimolar amounts. it was at this time (in 1952) (9) that blix introduced the name sialic acid internationally and also suggested that neuraminic acid simply was identical to deacetylated sialic acid. this result was later confirmed by klenk’s group. influenza virus releases a carbohydrate with properties similar to those of sialic acid the development of the field after 1950 was very much influenced by the studies on influenza virus haemagglutination carried out in frank m. burnet’s laboratory in melbourne. from their studies it was known that the influenza virus attaches itself to erythrocytes which thereby agglutinate. on incubating the agglutinated cells at figure 6. mixed crystals of sialic acid isolated from bovine submaxillary mucin by blix in 1936 (4) (left) and crystals of n-acetylneuraminic acid isolated by svennerholm in 1956 (5) (right). 108 a. lundblad 37�c, the virus is released in intact form and the cells disaggregate. at the same time they lose their ability to bind the virus (10). burnet et al. also coined the term rde (receptor-destroying enzyme) for the activity (later identified as neruraminidase) of the virus (11). of special interest in this context was a work by gottschalk and lind (of 1949) (12) where they described that the influenza virus was able to release a carbohydrate from ovomucin with properties similar to those of sialic acid. blix describes in a letter (in 1975) to a friend (klaus störiko, behringwerke ag, marburg, germany) how he first came in contact with gottschalk: ... i recall how i at the first international biochemist congress in cambridge 1949 talked with stacey and gottschalk, when the latter described the general properties of the carbohydrate component in mucins that inhibited virus haemagglutination and asked for advice from stacey. it struck me immediately that some of the properties gottschalk mentioned strongly reminded me about those of sialic acid. when i got home i mailed gottschalk what we had written about sialic acid and told him that we intended to test the matter closer. gottschalk obviously did not, at this time, know about sialic acid, which outside cologne and uppsala still was of low interest. i had on my part limited knowledge about burnet’s investigations. we agreed later on to study in each of our laboratories the carbohydrate component in the well defined tamm–horsfall urinary protein and to publish our results in 1952 simultaneously in nature... in 1952 lars odin in blix’s group reported in addition to the tamm–horsfall urinary protein a long series of biological materials (blood serum, plasma glycoproteins, human tears, cow’s milk, various epithelial mucins, human saliva, and meconium) inhibiting virus haemagglutination and all containing sialic acid (as judged from colorimetric measurements) (13). odin also showed that bovine submaxillary mucin had a lower inhibitory ability than corresponding mucin from sheep, although the content of sialic acid was the same. the explanation to this phenomenon was that the submaxillary mucin from sheep, pig, horse, and cow all contained different forms of sialic acid, as was also demonstrated by different x-ray diffraction patterns. in sheep, n-acetylneuraminic acid was found, whereas in pig, n-glycolylneuraminic acid dominated. in horse and cow different forms of nand o-diacetylated forms were isolated. preparative chromatography carried out on special cellulose columns turned out to be an effective way to separate different forms of sialic acid. a review on all the achievements from blix’s group was published in acta societatis medicorum upsaliensis in 1956 (14). blix received many letters from research groups asking for samples of the new crystalline compounds. in general, blix was very generous and sent out hundreds of vials with sialic acid to scientists around the world. this contributed to the intensive international race that took place in the mid-1950s to find out the correct structure. beside blix’s group in uppsala there were the groups of ernst klenk in cologne, richard kuhn in heidelberg, tamio yamakawa in tokyo, alfred gottschalk in melbourne, and noboro hiyama in sendai. several suggestions were published, but finally gottschalk reported a structure that would turn out to be correct (15). a short note was published in nature in 1957 (16) informing readers that blix, gottschalk, and klenk had reached an agreement on the nomenclature of the substance, i.e.: in order to avoid further confusion we propose to call the basic, unsubstituted compound neuraminic acid. sialic acid is suggested as a group name for the acylated neuraminic acids (for example n-acetylneuraminic acid, n-glycolylneuraminic acid, diacetylneuraminic acids). for the enzyme which splits the glycosidic linkage joining the terminal sialic acid to the residual oligoor polysaccharide the names neuraminidase and sialidase may be used synonymously (16). international breakthrough for sialic acid today it is nearly 80 years since blix made his discovery, and about 60 years since its international breakthrough started. around 1980, more than 30 types of sialic acid were known, and after the finding of 2-keto-3-deoxynononic acid (abbreviated kdn) (figure 7) the family has expanded to more than 50 members. structures, biosynthesis, distribution, and biological roles have been studied by several groups. there are a number of recent and excellent review articles covering these fields (17-22). a few aspects of general interest have been selected and are emphasized below. n-acetylneuraminic acid (neu5ac), n-glycolylneuraminic acid (neu5gc), and n-acetyl-9-o-acetylneuraminic acid (neu5,9ac2) are the three most frequently occurring forms. only neu5ac is ubiquitous, while the others are not found in all species. the animal with the largest number of different sialic acids known so far is the cow. in its submandibular (previously: submaxillar) gland gunnar blix and his discovery of sialic acids 109 15 types were detected. in man the number of different forms is much smaller, with neu5ac as the most abundant. sialic acid has not been found in plants, despite intensive search. neu5ac and neu5gc (figure 7) differ by a single oxygen atom, which is added to cmp-neu5ac via a reaction catalysed by the enzyme cytidine monophosphate n-acetylneuraminic acid hydroxylase (cmah). an interesting chemical difference exists between man and the great apes. in contrast to the latter, man has lost the expression of neu5gc due to a mutation in the gene encoding the hydroxylase (cmah). the genes of these apes exhibit over 98% sequence homology with man. the event when the hydroxylase activity was lost is assumed to have occurred 2.5 million years ago. one can only speculate to what extent the lack of neu5gc has influenced the development of man and especially his brain. neu5gc was first considered to be an onco-fetal antigen in humans since it was absent in normal adult tissues but found in fetal tissues and in some human tumours. furthermore, antibodies directed against neu5gc were found in patients with cancer and certain infectious diseases. with sensitive techniques traces of neu5gc have now been found also in normal human tissues. it is generally believed, however, that this represents incorporation from dietary sources such as red meat and milk products. neu5gc is today considered to be a xeno-antigen in man, since most healthy humans have some circulating anti-neu5gc antibodies. the question has also been raised whether this might account for the higher frequency of atherosclerosis and epithelial cancers in humans, diseases that are considered to be correlated with red meat consumption. general functions the external position of sialic acid on glycoproteins and gangliosides and the high expression on outer cell membranes (e.g. more than 10 million molecules per human erythrocyte) imply a strong influence in cell biology. some functions are certainly related to their strong electronegative charge, for instance stabilizing the conformation of proteins and enhancing the viscosity of mucins. sialic acids are also involved in the binding and transport of positively charged molecules including pharmaceuticals as well as in the attraction and repulsion of cells and molecules. another important role is their ability to mask antigenic sites, receptors, and penultimate galactose residues. after desialylation, molecules and cells can be bound, for instance by macrophages and hepatocytes via galactoserecognizing receptors, and also be taken up and degraded. this has been extensively studied with serum glycoproteins and blood cells. industrial production of glycoprotein pharmaceuticals using recombinant techniques often results in incomplete glycosylation. the carbohydrate chains become truncated and are lacking the terminal sialic acid. the halflife of such molecules in circulation is short because of rapid uptake in the liver but can be extended again by correct sialylation, which can be carried out on an industrial scale. the role of sialic acid in receptors for microbial lectins sialic acids themselves can also serve as receptors for a variety of microbial and animal lectins. some of these lectins were first discovered in viruses (see above) because of their ability to agglutinate red cells in vitro, and by the loss of this ability after sialidase treatment of the target cells. ho ho ho ho ho ho ho ho ho ho o oh nh n ho oh coo– coo– coo– o oh oh oh o oh oh o neu5ac kdn neu5gc figure 7. structures of n-acetylneuraminic acid (neu5ac), n-glycolylneuraminic acid (neu5gc), and 2-keto-3-deoxynononic acid (kdn). 110 a. lundblad most sialic acid-containing virus receptors contain terminal sialic acid attached to a penultimate galactose in either an a(2–6)or an a(2–3)-linkage. the different tissue distribution of a(2–6)-and a(2–3)-linked sialic acid influences the tissue tropism of many viruses. for instance certain adenoviruses have a tropism for the eye and bind preferentially to a(2–3)-linked sialic acid which in fact is the most frequent linkage type in the corneal and conjunctival cells. human influenza virus is another example where its haemagglutin (ha) preferentially binds to a(2–6)-linked sialic acid. this type of linkage is most abundant in the upper respiratory tract. avian influenza ha on the other hand binds best to the a(2–3)-linked sialic acid, more frequent in the lower respiratory tract. following virus replication in the infected cell, the neuraminidase also present in the influenza virus membrane removes its substrate (sialic acid) from infected cell surfaces so that newly made viruses are released to infect more cells. many other viruses also attach to cells via sialic acid, the most prominent being corona, polyoma, paramyxo, and rota viruses. bacteria produce carbohydrate-specific adhesins located on their fimbriae or pili. examples of colonization via sialic acid-containing receptors are certain strains of escherichia coli, streptococci, and helicobacter pylori. bacterial toxins, such as cholera, tetanus, and diphtheria toxins, bind firmly to sialic acid-containing receptors, mostly those located on gangliosides, as basis for their pathophysiological activity. the receptor–ligand interactions may be inhibited with soluble ligands such as sialic acid-containing oligosaccharides or glycoproteins. in human milk and especially in the colostrum there is a high content of sialic acidrich oligosaccharides. the milk from each mammalian species contains different and more or less sialylated glycans. these carbohydrates are considered to regulate the species-specific colonization of the intestine by microorganisms and to prevent the attachment of pathogenic bacteria, such as e. coli and helicobacter strains, or pathogenic viruses, e.g. rota viruses, which is especially important in newborns. essential nutrients for brain development in this context it should be noted that the high content of sialic acid in breast milk, especially during the first days after birth, was shown to increase the brain ganglioside content in rats, pigs, and man. it is considered important to have a sufficient amount of biochemical precursors available during the rapid neural growth that takes place postnatally. polysialylated neural cell adhesion molecules (ncam) and neural gangliosides both play critical roles in mediating cell–cell interactions which are important for neuronal outgrowth, synaptic connectivity, and memory formation. it has even been suggested that the sialic acid-rich glycans given to the child via breast milk permanently promote higher intelligence. infant formulae, which are based on bovine milk, have in contrast to the human milk a much lower content of sialic acid. furthermore human milk contains mainly neu5ac, whereas bovine milk has got both neu5ac and neu5gc. the role of sialic acid in receptors for mammalian internal lectins one example of sialic acid-binding molecules is the selectins, which belong to the group of internal lectins. they are located on endothelial cells and participate in the initial stage of adhesion of white blood cells to endothelia. in the course of this process the leukocytes roll along the vascular walls, adhere, and penetrate into the tissues below which have been damaged by the lack of oxygen (after transplants or infarcts for example) or in connection with an inflammation. cytokines play an essential role in this context. selectins specifically recognize sialylated lewis structures such as sialyl-lex, which are bound to glycoproteins or glycolipids (figure 8). since the sialyl-lex structure is also present on the surface of some tumour cells, selectins can be involved in the formation of metastases. in order to prevent further tissue damage by evading leukocytes and to prevent metastases of tumour cells, great efforts have been made to prevent cell adhesion to endothelial cells by the application of either selectin antibodies or sialyl-lex analogues. using ultrasensitive analysis by mass spectrometry of glycans linked to the human zona pellucida (zp), it has been shown that this matrix is coated with a high density of complex type n-glycans terminated with the sialyl-lex sequence, the universal selectin ligand. subsequent inhibition studies showed that the free sialyl-lex tetrasaccharide or neoglycoproteins terminated with this sequence inhibited sperm–zp binding by 70% (23). the results support the hypothesis that both lectin-like and protein–protein interactions play an essential role in human gamete interactions. another group of mammalian internal lectins are the siglecs (sialic acid-binding immunoglobulin-like lectins). many members of this family have been discovered during recent years, mainly due to the increased availability of expressed sequence tags (est) and genomic dna sequences. the first well-characterized type was macrophage gunnar blix and his discovery of sialic acids 111 sialoadhesin, involved in binding and nursing of maturing blood cells. most other documented siglecs participate in the interactions between band t-lymphocytes and are thereby regulators of the immune system. these lectins vary in the recognition of different sialic acids and can also distinguish between various glycosidic linkages. concluding remarks in recent years carbohydrate chains, or glycans, have emerged from historical obscurity to the formation of the specialized field of glycobiology, which refers to the molecular and cellular biology of glycans. the reason for the relatively late development of glycobiology is mainly technical, as glycans were more complex and difficult to study compared with nucleic acids, proteins, and lipids. the story around blix’s discovery of sialic acid is a good illustration of this. this brief review provides only some examples of the role of sialic acid in glycobiology. maybe we are only seeing ‘the tip of the iceberg’ and many more functions of sialic acids remain to be uncovered. future research is much needed and will most likely increase our possibilities to fight against the many infectious, immunological, malignant, psychiatric, and degenerative diseases in which sialic acids are involved. declaration of interest: the author reports no conflicts of interest. the author alone is responsible for the content and writing of the paper. references 1. blix g. zur kenntnis der schwefelhaltigen lipoidstoffe des gehirns. über cerebronschwefelsäure. z physiol chem. 1933;219:82–98. 2. blix g, tiselius a, svensson h. lipids and polysaccharides in electrophoretically separated blood serum proteins. j biol chem. 1941;137: 485–94. 3. blix g. über die kohlenhydratgruppen des submaxillaris mucins. z physiol chem. 1936;240:43–54. 4. blix g. sialic acids. proc intern congr biochem 4th vienna. 1958;1:94–106. 5. svennerholm l. on the isolation and characterization of n-acetyl-sialic acid. acta soc med upsalien. 1956;61:75–85. 6. blix g. einige beobachtungen über eine hexosaminhaltige substanz in der protagonfraktion des gehirns. scand arch physiol. 1938;80: 46–51. 7. klenk e. über die natur der phosphatide und anderer lipoide des gehirns und der leber bei der niemann-pickschen krankheit. z physiol chem. 1935;235:24–36. 8. klenk e. neuraminsäure, das spaltprodukt eines neuen gehirnlipoide. z physiol chem. 1941;268:50–8. 9. blix g, svennerholm l, werner i. the isolation of chondrosamine from gangliosides and from submaxillary mucin. acta chem scand. 1952;6:358–62. 10. hirst g. the agglutination of red cells by allantoic fluid of chick embryos infected with influenza virus. science. 1941;94:22–3. 11. burnet fm, stone jd. the receptor-destroying enzyme of v. cholerae. aust j biol med sci. 1947;3:227–33. 12. gottschalk a, lind pe. product of interaction between influenza virus enzyme and ovomucin. nature. 1949;164:232–3. 13. odin l. carbohydrate residue of a urine mucoprotein inhibiting influenza virus haemagglutination. nature. 1952;170:662–3. 14. blix g, lindberg e, odin l, werner i. studies on sialic acids. acta soc med upsalien. 1956;61:1–25. 15. gottschalk a. structural relationship between sialic acid, neuraminic acid and 2-carboxypyrrole. nature. 1955;176:881–2. 16. blix g, gottschalk a, klenk e. proposed nomenclature in the field of neuraminic and sialic acids. nature. 1957;179:1088. 17. schauer r. sialic acids: fascinating sugars in higher animals and man. zoology. 2004;107:49–64. 18. schauer r. sialic acids as regulators of molecular and cellular interactions. curr opin struct biol. 2009;19:507–14. 19. varki a, schauer s. sialic acids. in varki a, cummings rd, esko jd, et al. editors. essentials of glycobiology. 2nd ed. 2009. chapter 14 available at http://www.ncbi.nlm.nih.gov/books/nbk1920/#ch14.s1. 20. varki a. sialic acids in human health and disease. trends mol med. 2008;14:351–60. 21. gamblin sj, skehel jj. influenza hemagglutinin and neuraminidase glycoproteins. j biol chem. 2010;285:28403–9. 22. wang b. molecular mechanism underlying sialic acid as an essential nutrient for brain development and cognition. 2012;3:465s–72s. 23. clark gf. the role of carbohydrate recognition during human sperm-egg binding. hum reprod. 2013;28:566–77. ho ho h3c ho o o o o o o o oh ohoh oh oh oh oh co2h oh ohachn achn figure 8. structure of sialylated lewis x. a tetrasaccharide linked to a galactose unit in either glycoproteins or glycolipids. (the formula is: neu5ac-a(2,3)galb(1,4)(fuc-a1,3)-glcnac). 112 a. lundblad http://www.ncbi.nlm.nih.gov/pubmed/13339505?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17777315?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20270643?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/18135944?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/13002403?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/13339502?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/16351927?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/19699080?dopt=abstract http://www.ncbi.nlm.nih.gov/books/nbk1920/&num;ch14.s1 http://www.ncbi.nlm.nih.gov/books/nbk1920/&num;ch14.s1 http://www.ncbi.nlm.nih.gov/pubmed/18606570?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20538598?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22585926?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23315069?dopt=abstract ss1 the early years and the discovery mucin research temporarily put aside. international competition evolves influenza virus releases a carbohydrate with properties similar to those of sialic acid international breakthrough for sialic acid general functions the role of sialic acid in receptors for microbial lectins essential nutrients for brain development the role of sialic acid in receptors for mammalian internal lectins concluding remarks declaration of interest references does the antisecretory peptide af-16 reduce lung oedema in experimental ards? article does the antisecretory peptide af-16 reduce lung oedema in experimental ards? annelie barrueta tenhunena� , fabrizia massaroa,b�, hans arne hanssonc, ricardo feinsteind, anders larssone , anders larssona and gaetano perchiazzia ahedenstierna laboratory, department of surgical sciences, uppsala university, uppsala, sweden; bcardiac anesthesia and intensive care, anthea hospital, gvm care & research, bari, italy; cinstitute of biomedicine, university of gothenburg, g€oteborg, sweden; ddepartment of pathology and wildlife diseases, national veterinary institute, uppsala, sweden; edepartment of medical sciences, uppsala university, uppsala, sweden abstract background: acute respiratory distress syndrome (ards) is an acute inflammatory condition with pulmonary capillary leakage and lung oedema formation. there is currently no pharmacologic treatment for the condition. the antisecretory peptide af-16 reduces oedema in experimental traumatic brain injury. in this study, we tested af-16 in an experimental porcine model of ards. methods: under surgical anaesthesia 12 piglets were subjected to lung lavage followed by 2 hours of injurious ventilation. every hour for 4 hours, measurements of extravascular lung water (evlw), mechanics of the respiratory system, and hemodynamics were obtained. results: there was a statistically significant (p ¼ 0.006, two-way anova) reduction of evlw in the af16 group compared with controls. however, this was not mirrored in any improvement in the wet-todry ratio of lung tissue samples, histology, inflammatory markers, lung mechanics, or gas exchange. conclusions: this pilot study suggests that af-16 might improve oedema resolution as indicated by a reduction in evlw in experimental ards. article history received 1 july 2019 revised 28 september 2019 accepted 21 october 2019 keywords af-16 antisecretory factor; ards; extravascular lung water; pulmonary oedema introduction acute respiratory distress syndrome (ards) is an inflammatory lung injury with acute onset, characterized by increased pulmonary vascular permeability, pulmonary oedema, increased lung weight, and loss of aerated lung tissue (1). ashbaugh and colleagues described this syndrome in 1967 (2), and, despite reductions in both incidence and mortality (3), ards is still a significant health problem with high mortality (4). survivors often have a reduced quality of life (5). other than treatment of the underlying condition leading to ards, the therapy is mainly focussed on limiting ventilatorinduced lung injury (vili) and on adequate fluid management (3,6,7). currently, there are no effective pharmacologic interventions specific for ards (6). antisecretory factor (af) is an endogenous 41 kda protein (8,9), detectable in most tissues and plasma (10,11). the protein has antisecretory and anti-inflammatory properties (11–13). the biologically active site of the protein resides in a 16-peptide fragment, af-16, with the sequence (i)vchsktr (8,13). in experimental models, af-16 reduces brain oedema (14–17) and interstitial fluid pressure in solid tumours, but does not affect healthy tissue (18). in clinical trials increased concentrations of af in plasma is associated with a decrease of symptoms in different conditions such as inflammatory bowel disease, gastroenteritis, and m�eni�ere’s disease (19–21). it is unknown whether af or af-16 has any effect on oedema resolution in the lungs. we hypothesised that the peptide af-16 could reduce pulmonary oedema formation in a porcine model of ards by altering the quantity of extravascular lung water (evlw), the inflammatory response, and the pressure–volume (pv) relation of the lung. materials and methods the study was approved by the animal ethics committee in uppsala (decision 5.8.18–01054/2017), and the care of the animals followed the national institute of health guide for the care and use of laboratory animals (nih publications no 8023, revised 1978). the study was performed at the hedenstierna laboratory, uppsala university, sweden anaesthesia and instrumentation twelve piglets (25–30 kg), of mixed swedish, hampshire, and yorkshire breeds, were sedated with an intramuscular contact annelie barrueta tenhunen annelie.barrueta@surgsci.uu.se hedenstierna laboratory, department of surgical sciences, uppsala university, 75185 uppsala, sweden supplemental data for this article can be accessed here. �these authors contributed equally to this work. � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 4, 246–253 https://doi.org/10.1080/03009734.2019.1685029 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1685029&domain=pdf&date_stamp=2019-12-09 http://orcid.org/0000-0003-0815-375x http://orcid.org/0000-0002-0702-8343 http://orcid.org/0000-0001-6834-6399 https://doi.org/10.1080/03009734.2019.1685029 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2019.1685029 http://www.tandfonline.com injection of zoletil forte (tiletamine and zolazepam) 6 mg/kg and rompun (xylazine) 2.2 mg/kg. a peripheral intravenous catheter was inserted in an ear vein. after 5–10 min the animals were placed supine on a table and anaesthesia was induced with fentanyl 5–10 mg/kg i.v. and maintained with a continuous i.v. infusion of ketamine 30 mg/kg/h, midazolam 0.1–0.4 mg/kg/h, and fentanyl 4 mg/kg/h. after established anaesthesia, controlled by absence of reaction to painful stimulation between the front hooves, esmeron (rocuronium) 25mg/kg/h was added as muscle relaxant. during the first hour, 30ml/kg/h of ringer’s acetate was infused i.v. during the second hour, until established lung injury, ringer’s acetate was infused at a rate of 20ml/kg/h, followed by a maintenance infusion of 10ml/kg/h during the rest of the protocol. after induction of anaesthesia, the animals were tracheostomized, and an 8 mm internal diameter tube (mallinckrodt medical, athlone, ireland) was inserted in the trachea and connected to a ventilator (servo i, maquet, solna, sweden). until the initiation of lung injury the lungs were ventilated with tidal volume (vt) 8 ml/kg, respiratory rate (rr) 30/min, inspiratory/expiratory time (i:e) 1:2, inspired oxygen concentration (fio2) 0.7, and positive end-expiratory pressure (peep) 5 cmh2o. an oesophageal catheter (oesophageal catheter, erich jaeger gmbh, h€ochberg, germany) was positioned in the distal third of the oesophagus, and the correct position was assessed by a modified baydur procedure by finding less than 10% difference between simultaneous measurement of the oesophageal and the occluded airway pressures during compression of the chest wall (22), in order to ascertain that the oesophageal measurements reflected the pressure changes at the pleural surface. the procedure consisted in temporarily occluding the airways by a clamp and externally compressing the chest wall while recording both airway and oesophageal pressure. the catheter provided continuous measurements of oesophageal pressure (peso). airway pressure (paw) and airway flow (v’aw) were measured at the airway opening during the entire protocol. three different pressure transducers (digimaclic pressure transducers, special instruments gmbh, n€ordlingen, germany) were used to measure paw, peso, and gastric pressure (pga), while v’aw was acquired by a fleisch pneumotachograph (laminar flow element type pt, special instruments gmbh, n€ordlingen, germany) positioned between the endotracheal tube and the ventilator, connected to a differential pressure transducer (diff-cap pressure transducer, special instruments gmbh, n€ordlingen, germany). a triple-lumen central venous catheter for fluid infusions and a pulmonary artery catheter (edwards life-science, irvine ca, usa) for measurement of cardiac output (co) and pulmonary artery pressures were inserted via the right jugular vein. an arterial catheter was inserted in the right carotid artery for blood sampling and blood pressure measurement, and a pulse contour cardiac output (picco) catheter (pv2015l20, pulsion, munich, germany) was placed in the right femoral artery for estimation of evlw evolution. blood gases were analyzed on an abl 3 analyzer (radiometer, copenhagen, denmark) immediately after sampling, and venous admixture was calculated according to the shunt equation (23). a midline mini-laparotomy was performed for catheterization of the urinary bladder for urine drainage. study protocol preparation was followed by at least 30 min of stabilization, after which baseline measurements were performed (figure 1). lung injury was then induced using a two-hit injury model according to an established protocol (24). lung lavages with 30 ml/kg of warmed isotonic saline were repeated until the arterial oxygen tension/inspired oxygen tension (pao2/fio2) ratio was less than 27 kpa. injurious ventilation consisting of plateau pressure of 36 cmh2o, rr 20/ min, and i:e 1:2, with zero end-expiratory pressures, was then initiated and maintained for 2 h. after the induction of lung injury, the animals were randomized to the intervention with af-16 (n ¼ 6) or to the control group (n ¼ 6). directly after established lung injury the intervention group received af-16 (sample no. 05501, kj ross petersen figure 1. experimental timeline. lung lavages were repeated until the arterial oxygen tension/inspired oxygen tension (pao2/fio2) ratio was less than 27 kpa. injurious ventilation consisting of plateau pressure of 36 cmh2o, fr 20/min, and inspiratory/expiratory time (i:e) 1:2, with zero end-expiratory pressures, was then initiated and maintained for 2 h. h1, h2, h3, h4 ¼ measurements 1, 2, 3, and 4 h after intervention, respectively. upsala journal of medical sciences 247 aps, copenhagen, denmark) 20 mg/kg in a solution of 50 mg/ml, administered as an infusion over 10 min in a central vein, while the control group received an equal amount of the vehicle (0.9% nacl) at the same time point. a lung recruitment manoeuvre was performed for 2 min, by applying pressure-controlled ventilation; rr 6/min, peep 10 cmh2o, peak pressure of 40 cmh2o, and i:e 1:1. thereafter, the following settings for mechanical ventilation were applied and maintained until the end of the protocol: volume-controlled ventilation, vt 6 ml/kg, peep 14 cmh2o, fio2 0.7, and rr 40/min. this rate was chosen since it has previously been associated with formation of pulmonary oedema in this model (24). at baseline, after establishment of lung injury and every hour during the following 4 hours’ duration of the protocol, evlw, pao2, paco2, ph, lactate, and base excess were measured. at the same time points, the main hemodynamic parameters (systemic and pulmonary pressures, co, heart rate) were recorded. pressure–volume (pv) curves of the respiratory system were obtained at baseline, directly after the induction of lung injury, and at 4 h after the intervention/vehicle, in steadystate conditions, just prior to euthanasia. all respiratory signals were acquired by an analog-todigital converter card (daq-card ai-16xe50, national instruments corp., austin, tx, usa) controlled by the biobench software (ver. 1.0, national instruments corp., austin, tx, usa), at a sampling frequency of 200 hz. inspiratory and expiratory airway volumes were obtained by integration of the airway flow (v’aw). the pv relation was measured by delivering eight monotonically decreasing lung volumes, from paw 25 cmh2o to 0 cmh2o over peep. each volume was delivered during steadystate ventilation, in volume control mode, and followed by an inspiratory hold manoeuvre (ihm) and an expiratory hold manoeuvre. before the beginning of the decreasing ramp, in order to standardise the history of volume, we performed a recruitment manoeuvre applying a paw of 40 cmh2o for 40 s (25). having the oesophageal catheter in place, we could measure the variation of transpulmonary pressure (ptp) as: dptp ¼ ðpaw, plat � paw, eeþ�ðpeso, plat � peso, eeþ (1) where paw,plat is airway pressure during ihm, peso,plat is the oesophageal pressure at the same time, and paw,ee and peso,ee are the corresponding airway and oesophageal pressures at the end of expiration (26). this way we could draw the pv curve of the lung in the different mentioned conditions. the animals were euthanized with 100 mmol kcl i.v. at the end of the experiment under deep anaesthesia. the chest wall was then opened. ventilation was maintained identical to the protocol in order to keep the pressure gradient between airway and vascular pressures during the sampling. the heart and the lungs were excised en bloc. lung tissue samples were collected from both lungs from the following regions: apical-medial, medial-medial, caudal-dorsal, caudal-medial, and caudal-ventral. the samples were immediately immersed in 10% buffered formalin. a veterinary pathologist who was blinded to the experimental groups evaluated the samples histologically. in addition, samples from both lungs were analyzed for cytokines (tnf-a, il-6) using an elisa method. wet-to-dry ratio was measured in the same lung regions from the right lung, and an average for each location was calculated. samples were weighed, and dried in an oven, at 50 �c, until the weight did not differ between two measurements as described by matute-bello et al. (27). statistics and data analysis the number of animals to be included in this pilot study was determined according to the mead resource equation (28) that allows for determination of the sample size when the effect of a treatment is difficult to estimate a priori. we expressed values as means ± standard deviation (sd), or median and range where appropriate. the overall differences were analyzed by a two-way anova using time and groups, and then the one-way anova for repeated measurements (after established lung injury and every hour until the end of the protocol) to evaluate the within-group differences after starting treatment. the null hypothesis was that neither time nor treatment had an effect on the sampled data. the course of evlw over time, in the treated versus non-treated group, was studied by applying a robust polynomial fitting (matlab r2018, curve fitting toolbox, mathworks, natick, usa) of the second degree, having a model of: y ¼ p1 � x2 þ p2 � x þ p3 (2) where y is the evlw in ml and x is the time [hours]. the effect of ards induction was assessed by a paired t test. a difference of p < 0.05 was considered as statistically significant. sigmaplot 12.5 (systat inc. software, usa) was used for this statistical analysis. in order to analyze and compare the pv curves from the different animals, the following procedure was applied. each pressure value of the pv curves was divided by the maximum pressure that the same animal presented in healthy conditions at the highest volume. this indexing allowed for standardization of the comparison among different individuals and experimental phases while maintaining the morphology of the curve, as already published by perchiazzi at al. for an analogous issue (29). the deflation limbs of all the pv curves were subjected to polynomial regressions of the second degree (matlab r2018, curve fitting toolbox, mathworks, natick, ma, usa), in order to verify whether they obeyed this model and in order to allow a further comparison between different individuals and phases of the experiments. the pv models were then compared using the f test (matlab r2018, statistics toolbox, mathworks, natick, ma, usa), separately for treated and non-treated individuals in order to assess whether there was any statistically significant difference, firstly, between baseline conditions and postlavage pv curves (hence attesting the validity of the lung injury model), and, secondly, between post-lavage pv curves and measurements taken after 4 h of treatment (hence attesting the presence of an effect after drug or vehicle administration). 248 a. barrueta tenhunen et al. results no differences were found between the groups at baseline regarding hemodynamics, respiratory parameters, or body weight (table 1). all animals survived the experiment until euthanasia. extravascular lung water after the induction of lung injury, the amount of evlw increased (p < 0.001) in comparison to baseline. evlw decreased over time in the intervention group as compared to the control group (p ¼ 0.0057, difference of the means -51 ml, 95% confidence interval 86–15 ml) (figure 2). in contrast, there were no statistically significant changes in the quantity of evlw (p ¼ 0.87) or by time (p ¼ 0.12) in the control group after administration of the vehicle. the robust polynomial fitting yielded: for the intervention group: y ¼ 0:7 � x2�8:5 � x þ7:8 ðr2 ¼ 0:81þ (3) for the controls: y ¼ �0:3 � x2 þ 1:2 � x�0:8 ðr2 ¼ 0:04þ (4) wet-to-dry ratio there were no statistically significant differences in wet-todry ratio when pooling the regional samples from each animal (figure 3) or when the different lung regions were analyzed separately. lung mechanics the regression of the pv relations yielded statistically significant regression curves, including pooled data and data derived from treated and non-treated animals. the regressions separately subtending the three different phases of the experiment—healthy lungs, after induction of lung injury, and after administration of drug/vehicle—were statistically significant as well. using the regression curves for estimating compliance at the same applied pressure in a standardized way, it is possible to infer that at 20 cmh2o compliance passes from 36.6 to 20 and then to 23.1 ml/cmh2o in the treated group, and from 39.9 to 22.6 and to 22.3 ml/cmh2o in the control group when considering the values sampled in the three different phases of the experiment. using the f test to ascertain whether the curves displayed changes of their course, it is possible to note that in the case of the passage from healthy conditions to lung injury the pv curves were statistically different. the intervention with af-16 did not change this course in a statistically significant way; the same happened in controls after the administration of vehicle (figure 4). gas exchange after established lung injury the pao2/fio2 decreased from 70.8 (sd 4.9) to 12.6 (sd 4.4) kpa (p < 0.0001). no statistically significant difference in gas exchange could be distinguished between the intervention and control groups after start of administration (table 2). likewise, there was no difference in venous admixture or in ph between the two groups throughout the study. cytokines lung homogenates from the two groups did not differ in il6 or tnf-a concentrations in a statistically significant way. the il-6 concentration was 511 (sd 377) pg/ml and 551 (sd 472) pg/ml in the af-16 group and the control group, table 1. measurements at baseline and at the end of the experiment. values expressed as mean (sd). no statistically significant difference was found between the groups at baseline. parameters af-16 control baseline final data baseline final data body weight (kg) 27.8 (1) – 27.4 (1.7) – arterial ph 7.50 (0.05) 7.32 (0.08) 7.51 (0.06) 7.33 (0.1) paco2 (kpa) 5 (0.6) 7.5 (1.4) 5.2 (0.7) 7.9 (2.8) pao2 (kpa) 49.8 (4.6) 33.7 (10.7) 49.3 (2.1) 29.9 (12.9) peak pressure (cmh2o) 15 (1.8) 28.7 (2.9) 15.7 (1) 28 (2.3) plateau pressure (cmh2o) 11.2 (1.7) 19.5 (3) 11.5 (1.4) 19.8 (3.9) dynamic compliance (ml/cmh2o) 25.6 (4.2) 11.4 (2.4) 23.2 (3.6) 11.1 (2.1) mean arterial pressure (mmhg) 91 (5.6) 74 (17) 82 (10.7) 74 (13) cardiac output (l/min) 4 (0.6) 3 (0.7) 3.8 (0.5) 3.5 (1) extravascular lung water (ml) 324 (26) 425 (54) 331 (43) 462 (96) paco2: arterial carbon dioxide tension; pao2: arterial oxygen tension. figure 2. extravascular lung water (evlw) in ml at baseline, immediately after ventilator-induced lung injury and at 1, 2, 3, and 4 h post-damage. black dots represent intervention group (af-16), white dots represent control group. upsala journal of medical sciences 249 respectively. the values for tnf-a were 132 (sd 72) pg/ml and 135 (sd 88) pg/ml. histology the histological analysis identified interstitial oedema, leucocyte infiltration, emphysema, and atelectasis to a varying extent (figure 5). there was no significant difference between the two groups with respect to oedema or inflammatory activity (supplementary table 1, available online). hemodynamics mean arterial pressure, co, and heart rate decreased after established vili with no significant difference between figure 3. wet-to-dry ratio, comparison between intervention (af-16) and control group at the end of the experiment. regional samples pooled from each animal. no statistically significant difference between groups. figure 4. pressure volume curves at airway opening; x-axes depict transpulmonary pressure (ptp), y-axes show inspired volume (v). both axes are expressed in arbitrary units obtained by scaling both co-ordinates by the maximum value of pressure that the single animals have in healthy conditions. using the same scaling factor, the morphology of the single curves remains unaffected. 250 a. barrueta tenhunen et al. https://doi.org/10.1080/03009734.2019.1685029 groups. circulatory parameters were stable throughout the experiment in both groups (supplementary table 2, available online). fluid administration was standardized according to the protocol, and both groups maintained an adequate diuresis throughout the experiment. discussion this pilot study in experimental ards suggests that the antisecretory peptide af-16 might improve oedema resolution, indicated by a decrease in evlw. at the same time, there were no effects on gas exchange, respiratory mechanics, inflammatory response, or alveolar damage. we used a two-hit model for ards, consisting of lung lavage followed by injurious ventilation. the surfactant depletion by lung lavage primes the lung for injurious ventilation, and we established a ventilator-induced lung injury with a resultant mean pao2/fio2 of 12.6 (sd 4.4), which equals severe ards in humans, according to the berlin definition (1). the model was stable as indicated by no change in evlw, gas exchange, and lung mechanics over the time following the induction of injury, in the control group. the ards model used in this protocol has several features similar to human ards, such as rapid onset, histological findings of tissue injury with evidence of an inflammatory response, increased pulmonary vascular permeability, and severe hypoxaemia, all being important features of acute lung injury in an animal model (27,30). thus, the inflammatory, histological, and lung mechanical characteristics can be assumed to mirror ards in patients. table 2. gas-exchange data. after established lung injury the pao2/fio2 decreased from 70.8 kpa (sd 4.9) to 12.6 kpa (sd 4.4) (p < 0.0001), pooled data. no statistically significant difference could be distinguished in gas exchange between treated and non-treated groups after intervention. values shown as mean (sd). group baseline vili vili þ 1h vili þ 2h vili þ 3h vili þ 4h po2 (kpa) af-16 49.8 (4.6) 14.4 (4.4) 32.2 (15.6) 32.3 (10.1) 33.4 (10.2) 33.7 (10.7) control 49.3 (2.1) 10.8 (3.8) 27 (14) 30.6 (12.7) 32 (11.1) 29.9 (12.9) pao2/fio21 (l/kpa) af-16 71.1 (6.6) 14.4 (4.4) 46.1 (22.4) 46.1 (14.4) 47.7 (14.5) 48.1 (15.2) control 70.4 (3) 10.8 (3.8) 38.6 (20) 43.7 (18.1) 45.7 (15.9) 42.7 (18.5) pco2 (kpa) af-16 5 (0.6) 4.3 (2.5) 7.6 (1.5) 7.6 (1.5) 7.5 (1.3) 7.5 (1.4) control 5.2 (0.7) 4 (1) 7.7 (2.1) 7.7 (2.6) 7.7 (2.7) 7.9 (2.8) ph af-16 7.5 (0.05) 7.55 (0.22) 7.29 (0.09) 7.3 (0.09) 7.31 (0.08) 7.32 (0.08) control 7.51 (0.06) 7.58 (0.09) 7.31 (0.09) 7.32 (0.1) 7.33 (0.1) 7.33 (0.1) lactate (mmol/l) af-16 1.8 (0.48) 3.6 (0.9) 2.4 (1) 1.7 (0.5) 1.4 (0.3) 1.1 (0.2) control 1.5 (0.64) 2.8 (0.7) 1.5 (0.6) 1.2 (0.6) 1.1 (0.4) 1.1 (0.5) peak pressure (cmh2o) af-16 15 (1.8) 42.2 (4.4) 28.8 (2.5) 29 (2.4) 28.3 (2.9) 28.7 (2.9) control 15.7 (1) 44 (4.2) 27.8 (1.6) 28.3 (2.1) 28.5 (2.6) 28 (2.3) plateau pressure (cmh2o) af-16 11.2 (1.7) 31.2 (3.1) 18.7 (4) 19.5 (3.4) 17.7 (4.5) 19.5 (3) control 11.5 (1.4) 34 (2.3) 20 (3.5) 20 (3.7) 19.7 (4.1) 19.8 (3.9) driving pressure (cmh2o) af-16 6.2 (1.7) 31.2 (3.1) 4.7 (4) 5.5 (3.4) 5.3 (3.3) 5.5 (3) control 6.5 (1.4) 34 (2.3) 7.7 (5) 6 (3.7) 5.7 (4.1) 5.8 (3.9) dynamic compliance (ml/cmh2o) af-16 25.6 (4.2) 19.5 (5.6) 11.1 (2) 11 (2.3) 11.3 (2.3) 11.4 (2.4) control 23.2 (3.6) 20.5 (3.9) 11.6 (1.8) 11.4 (1.8) 11.1 (2) 11.1 (2.1) paco2: arterial carbon dioxide tension; pao2: arterial oxygen tension; pao2/fio2: arterial oxygen tension inspired oxygen tension; vili: ventilator-induced lung injury. figure 5. representative lung tissue samples from an animal treated with af-16 (a) and a control animal (b). both images show prominent oedema in the interlobular septa with leukocyte infiltration and dilated lymphatic capillaries. upsala journal of medical sciences 251 https://doi.org/10.1080/03009734.2019.1685029 af-16 has been found to reduce oedema formation in different conditions, e.g. brain oedema related to mild-tomoderate traumatic brain injury (14), and to reduce interstitial pressure in solid tumours (18). in the airways endogenous af is localized to the epithelium of the trachea and the bronchial tree, as well as in mononuclear cells in the lamina propria (8). in the pulmonary acini af is localized to type ii cells and lung macrophages, the former localization indicating a possible regulatory role of af in the secretion of pulmonary surfactant (8). af also has neuromodulatory effects and inhibits chloride permeation over neuronal membranes (8,11,17,31–33). our hypothesis was that administration of af-16 would improve oedema resolution in this porcine ards model. indeed, we found that evlw decreased in the af-16-treated group, and although there was a tendency to a reduced wet-to-dry ratio of the lung samples and a positive effect on lung mechanics, neither reached statistical significance. we hypothesized that the significant reduction of evlw of approximately 50 ml was too small to be detected in a statistically significant way in the small samples that were subjected to the drying process. on the other hand, it is worth mentioning that the assessment by the wet-to-dry ratio includes a blood component (which inevitably remains inside the sample) while evlw does not. this last is ‘the amount of water that is contained in the lungs outside the pulmonary vasculature, that is, the sum of interstitial, alveolar, intracellular, and lymphatic fluids’ (34). furthermore, we could not discern any positive effect on gas exchange. this might be explained by the longer time it takes for inflammatory compared with non-inflammatory oedema to be resorbed and thus to any improvement in respiratory compliance and oxygenation. af-16 downregulates the inflammatory response in several models (8,9,11,12). however, in this study there was no difference in cytokine levels or in histology between the controls and the af-16 group. these results were indeed expected, since the observation period was short, and any immunological effect would first appear after a longer time. on the other hand, this study was not designed to primarily assess the immune response by af-16, but mainly to assess the possible oedema resolution properties. the study of pv curves was performed in order to observe any possible lung mechanical effect of the intervention. notably, the induction of lung injury reduced the lung compliance, confirming the validity of the applied model. dealing with different individuals and different experimental phases, we decided to standardize the curves dividing the pressure component by its maximum value measured in healthy condition. lung compliance is also a function of body weight, and this procedure allowed this potential problem to be eliminated. a careful examination of the graph of the treated animals shows that the curves before and after the administration of af-16 seem to follow different courses, rendering a better compliance after the treatment. although this difference cannot be considered statistically different, the observation opens the question whether we could have had a better signal-to-noise ratio using other doses/timings of drug administration or simply by increasing the sample size. this study has many limitations; first, it is an animal study with an artificial lung condition, where the surfactant depletion by lung lavage primes the lung for injurious ventilation. human ards, in contrast, is often the result of complex interactions of disease (directly or indirectly affecting the lung), co-morbidities, and genetic predisposition (3,30). no animal model reproduces the full characteristics of human ards, and interspecies variability in lung injury mechanisms and response to intervention can by no means be ruled out (30). furthermore, although af-16 was given i.v. in a dose that was higher than in the experimental models of brain injury, the insult was also more severe, and we cannot disprove that the intervention with af-16 would be more effective at an even higher dose. moreover, the number of studied animals was limited, and this affected the magnitude of the treatment effects that could have been considered statistically significant: small variation between the groups might not have been detected. finally, the intervention was not blinded to the investigators, since this could not be achieved for practical reasons. the analyzing pathologist, however, was blinded. in summary, we found that evlw was reduced by the antisecretory peptide af-16, but we could not find any major effect on inflammation, gas exchange, or lung mechanics. thus, further long-term experimental studies are necessary to assess whether af-16 has any important effect on oedema resolution in ards and to pinpoint underlying mechanisms. acknowledgements the authors express their sincere gratitude to kerstin ahlgren, agneta roneus, liselotte pihl, mariette andersson, and maria sw€alas for their assistance and support during the experiments at the hedenstierna laboratory of uppsala university, sweden. the peptide af-16 was provided by lantm€annen as faktor ab, stockholm, sweden. disclosure statement the authors abt, fm, rf, al1, al5, and gp declare the absence of conflicts of interests. hah has patents and patent applications related to af peptides; he has not been involved in the practical execution of the experiments and has been blinded to the results. funding this study was supported by the swedish heart and lung foundation (grant no. 20170531), the swedish research council (grant no. x201599x-22731–01-4), and alf grants of uppsala university hospital. notes on contributors annelie barrueta tenhunen is a phd student at the department of surgical sciences, university of uppsala, uppsala, sweden. fabrizia massaro is a specialist in cardiac anaesthesia and intensive care at the anthea hospital, gvm care & research, bari, italy. hans arne hansson is professor emeritus at the institute of biomedicine, university of gothenburg, g€oteborg, sweden. 252 a. barrueta tenhunen et al. ricardo feinstein is associate professor at the department of pathology and wildlife diseases, national veterinary institute, uppsala, sweden. anders larsson is professor at the department of medical sciences, uppsala university, uppsala, sweden. anders larsson is professor at the department of surgical sciences, uppsala university, uppsala, sweden. gaetano perchiazzi is researcher at the department of surgical sciences, uppsala university, uppsala, sweden. orcid annelie barrueta tenhunen http://orcid.org/0000-0003-0815-375x anders larsson http://orcid.org/0000-0002-0702-8343 gaetano perchiazzi http://orcid.org/0000-0001-6834-6399 references 1. ranieri vm, rubenfeld gd, thompson bt, ferguson nd, caldwell e, fan e. acute respiratory distress syndrome: the berlin definition. jama. 2012;307:2526–33. 2. ashbaugh dg, bigelow db, petty tl, levine be. acute respiratory distress in adults. lancet. 1967;290:319–23. 3. thompson bt, chambers rc, liu kd. acute respiratory distress syndrome. n engl j med. 2017;377:562–72. 4. bellani g, laffey jg, pham t, fan e, brochard l, esteban a, et al. epidemiology, patterns of care, and mortality for patients with 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antisecretory factor modulates gabaergic transmission in the rat hippocampus. regul pept. 2005;129:109–18. 32. lange s, l�onnroth i, palm a, hyd�en h. the effect of antisecretory factor on the permeability of nerve cell membrane to chloride ion. pflugers arch. 1987;410:648–51. 33. rapallino mv, cupello a, lange s, l€onnroth i, hyd�en h. further studies on the effect of asf factor on cl-permeability across the deiters’ neurone plasma membrane. int j neurosci. 1989;46:93–5. 34. jozwiak m, silva s, persichini r, anguel n, osman d, richard c, et al. extravascular lung water is an independent prognostic factor in patients with acute respiratory distress syndrome. crit care med. 2013;41:472–80. upsala journal of medical sciences 253 abstract introduction materials and methods anaesthesia and instrumentation study protocol statistics and data analysis results extravascular lung water wet-to-dry ratio lung mechanics gas exchange cytokines histology hemodynamics discussion acknowledgements disclosure statement references vol_117_004_sups_a_724732 355..369 full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 cyclic amp dynamics in the pancreatic β-cell anders tengholm to cite this article: anders tengholm (2012) cyclic amp dynamics in the pancreatic β-cell, upsala journal of medical sciences, 117:4, 355-369, doi: 10.3109/03009734.2012.724732 to link to this article: https://doi.org/10.3109/03009734.2012.724732 © informa healthcare published online: 13 sep 2012. submit your article to this journal article views: 1426 view related articles citing articles: 59 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2012.724732 https://doi.org/10.3109/03009734.2012.724732 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2012.724732 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2012.724732 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2012.724732#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2012.724732#tabmodule upsala journal of medical sciences. 2012; 117: 355–369 review article cyclic amp dynamics in the pancreatic b-cell anders tengholm department of medical cell biology, uppsala university, biomedical centre, box 571, se-751 23 uppsala, sweden abstract insulin secretion from pancreatic b-cells is tightly regulated by glucose and other nutrients, hormones, and neural factors. the exocytosis of insulin granules is triggered by an elevation of the cytoplasmic ca2+ concentration ([ca2+]i) and is further amplified by cyclic amp (camp). cyclic amp is formed primarily in response to glucoincretin hormones and other gs-coupled receptor agonists, but generation of the nucleotide is critical also for an optimal insulin secretory response to glucose. nutrient and receptor stimuli trigger oscillations of the camp concentration in b-cells. the oscillations arise from variations in adenylyl cyclase-mediated camp production and phosphodiesterase-mediated degradation, processes controlled by factors like cell metabolism and [ca2+]i. protein kinase a and the guanine nucleotide exchange factor epac2 mediate the actions of camp in b-cells and operate at multiple levels to promote exocytosis and pulsatile insulin secretion. the camp signaling system contains important targets for pharmacological improvement of insulin secretion in type 2 diabetes. key words: epac2, insulin secretion, oscillations, protein kinase a winner of the eric k. fernström award for young investigators 2011 at the medical faculty of uppsala university. correspondence: anders tengholm, department of medical cell biology, uppsala university, biomedical centre, box 571, se-751 23 uppsala, sweden. e-mail: anders.tengholm@mcb.uu.se (received 20 august 2012; accepted 23 august 2012) issn 0300-9734 print/issn 2000-1967 online � 2012 informa healthcare doi: 10.3109/03009734.2012.724732 introduction the pancreatic b-cells are adapted to respond to changes in the extracellular concentrations of glucose and other nutrients as well as hormones and neurotransmitters by releasing appropriate amounts of insulin to promote the uptake and storage of glucose in liver, muscle, and fat. functional defects in the b-cells may lead to glucose intolerance and eventually clinically manifest diabetes mellitus. insulin, like many other hormones, is released in pulses with a period of approximately 3–6 minutes (1-3). the pulses are important for the action of insulin on the targets, in particular the liver, probably by preventing down-regulation of the insulin receptors. the pulsatile pattern of insulin secretion arises from an endogenous rhythmicity of the individual pancreatic b-cell that involves several intracellular messengers, including atp, ca2+, phospholipid-derived messengers, and cyclic amp (camp) (2,4,5). there is consensus that glucose stimulates insulin secretion via its metabolism and atp/adp-dependent closure of atp-sensitive k+ channels (katp channels), which leads to membrane depolarization and opening of voltage-gated ca2+ channels (6,7). the resulting increase of the cytoplasmic ca2+ concentration ([ca2+]i) triggers exocytosis of insulin secretory granules. cyclic variations in cell metabolism and atp/adp ratio are thought to underlie the oscillations of [ca2+]i and pulsatile insulin secretion (2,4,8,9). additional signals generated by glucose metabolism are important for a proper insulin secretory response by amplifying exocytosis at steps distal to the elevation of [ca2+]i, but their identities have not been elucidated (7). in addition to ca2+, which is the primary triggering signal, camp is the most important regulator of exocytosis in b-cells. cyclic amp is a ubiquitous intracellular messenger involved in the regulation of a wide range of processes in many types of cells. the first indication that the nucleotide is involved in the b-cell secretory response came from the observation that camp-generating glucagon promotes insulin secretion (10), and other studies soon confirmed a link between camp and insulin release (11–14). it is now well established that camp promotes secretion at multiple levels, such as by increasing electrical activity and [ca2+]i signaling, by recruiting granules and by acting directly on the exocytosis machinery (previously reviewed in (15)). cyclic amp is also important for b-cell function by stimulating e.g. insulin synthesis, cell differentiation and proliferation, and by protecting the cells from apoptosis (reviewed in (16,17)). the action of camp in b-cells is primarily linked to effects of certain hormones, in particular the glucoincretin hormones glucagon-like peptide-1 (glp-1) and glucose-dependent insulinotropic polypeptide (gip), which are released from intestinal land kcells, respectively, to reduce postprandial glucose levels by enhancing insulin secretion. cyclic amp generation by receptor agonists has been reported to be required for normal glucose-responsiveness (18– 20). however, as will be discussed in detail below, glucose also stimulates camp production in the absence of neuro-hormonal inputs. the effects of the nucleotide are mediated by protein kinase a (pka) and exchange protein directly activated by camp (epac), also known as camp-dependent guanine nucleotide exchange factor (figure 1) (21). despite the importance of camp in b-cell function, it is only recently that it has become possible to investigate the intracellular dynamics of the messenger. this review summarizes recent advances in our understanding of camp signaling dynamics in the context of insulin secretion. cyclic amp generation by adenylyl cyclases cyclic amp is formed exclusively from atp via adenylyl cyclases (acs). the classical pathway for camp generation involves activation of transmembrane acs by gs-coupled receptors. there are nine isoforms of transmembrane acs with different regulatory properties (22). most, if not all, of these are expressed in pancreatic islets and insulinoma cells (23–25). early studies identified a close link between camp and ca2+ (26–28), and particular attention has therefore been paid to ac isoforms regulated by this ion. the activities of ac1 and ac8 are stimulated by ca2+ or ca2+/calmodulin, and, despite relatively low expression, ac8 is functionally important by integrating g-protein and ca2+ signals in b-cells (25). recent studies also provided evidence that ac8 is required for glp-1 generation of [ca2+]i signals (29). ac8 is preferentially found in raft-like domains of the plasma membrane where it interacts with the a-kinase anchoring protein akap79/150 and is regulated by store-operated ca2+ entry (22,30–32). although b-cells indeed exhibit store-operated ca2+ influx, it is quantitatively minor compared to voltage-dependent ca2+ entry (33), and its importance for ac regulation in b-cells is uncertain. the more abundantly expressed ac5 and ac6 isoforms are inhibited by ca2+ and by pka-mediated phosphorylation. little is known about their functional importance, but the regulatory properties indicate involvement in feedback inhibition of camp 356 a. tengholm formation. several acs isoforms are regulated by protein kinase c, and there is evidence for a stimulatory effect of the kinase on ac activity in mouse islets (34–36). recent data indicate that b-cell ac activity can be directly regulated by cell metabolism, probably via atp (37). apart from the nine transmembrane acs there is a structurally distinct soluble ac (sac) which at least is expressed in ins-1 cells (29,38) and which has been implicated in glucose-induced camp generation (38). in being regulated by ca2+, hco3 -, and physiological concentrations of atp (39–41), sac seems well suited as a metabolic sensor, but functional evaluation of sac is complicated by side-effects of the commonly used inhibitor kh7 (38,42). the importance of sac for b-cell function therefore remains to be clarified. cyclic amp degradation by phosphodiesterases the intracellular camp level is determined by a balance between camp production by acs and degradation by cyclic nucleotide phosphodiesterases (pdes). the pdes constitute a large family of enzymes which catalyze the hydrolysis of camp and/or cgmp to 5¢-amp and 5¢-gmp. there are 11 sub-families with >50 isoforms differing in structure, regulation, and substrate preferences (43). the role of pdes in islets has previously been reviewed (44,45). the pde1, pde3, and pde4 families are generally regarded as most important for camp regulation in islet cells. pancreatic islets were early found to have ca2+/calmodulin-sensitive pde activity (46–48), and later studies have identified pde1c as a prominent isoform (49–51). pharmacological inhibition or genetic down-regulation of pde1 thus enhances glucose-stimulated insulin secretion both from insulinoma cells and pancreatic islets (49,51). pde3 is a membrane-associated dual-specificity isoform degrading both camp and cgmp with kinetic properties that result in cgmp-inhibition of camp degradation. pde3b is expressed in bcells and is probably quantitatively the most important pde in islets, constituting up to 70% of the total pde activity in some studies (45,52). the enzyme is activated by glucose, insulin, and camp via changes in protein kinase aand b-mediated phosphorylation (53). pde3b is a major regulator of camp at sites important for insulin secretion. overexpression of pde3b in b-cells or insulinoma cells consequently reduces insulin secretion (54,55), whereas genetic down-regulation or pharmacological inhibition of the enzyme amplifies secretion (51,52,56,57), probably by regulating the most distal steps of granule fusion (58). moreover, igf-1-induced attenuation of insulin secretion is mediated by activation of pde3b (56). pde4 is present in islets and insulin-secreting cells (51,52,59), but studies with inhibitors have yielded conflicting results. while the pde4 family-selective inhibitor rolipram lacked effect on glucose-induced insulin secretion from islets (52,59), secretion was enhanced in both ins-1 cells and rat islets after selective pharmacological inhibition of the enzyme with roflumilast or l-826,141 and by sirna-mediated knock-down of pde4c (51). recent studies have also identified members of the pde7, pde8, pde10, and pde11 families in rodent and human islets and insulin-secreting cell lines (50,51,53). these pde isoforms probably constitute a relatively small fraction of the total pde activity in b-cells but may nevertheless play important functional roles. for example, pharmacological inhibition of pde10a (60) and knock-down of pde8b (50) potentiate insulin secretion from rat islets, and the latter isoform was recently implicated in camp oscillations and pulsatile insulin secretion from min6 cells (61). glucose metabolism atp camp k+ ca2+ pka epac2 depolarization pulsatile insulin secretion glucagon glp-1 gip _ rap1 figure 1. cyclic amp signaling in insulin secretion. schematic drawing of a b-cell and the involvement of camp in insulin secretion stimulated by glucose and amplified by hormones. glucose metabolism generates atp, which inhibits atp-sensitive k+ channels and causes voltage-dependent ca2+ influx. elevation of [ca2+]i triggers exocytotic release of insulin granules. atp also promotes formation of camp, which amplifies secretion via epac2 and protein kinase a (pka). activation of gs-coupled receptors by e.g. glucagon, glp-1, or gip leads to camp formation and enhancement of insulin release. cyclic variations in metabolism, [ca2+]i and camp concentration caused by incompletely understood feedback circuits result in pulsatile insulin secretion. cyclic amp dynamics in the pancreatic b-cell 357 cyclic amp signaling triggered by neuro-hormonal stimuli several gs-coupled receptor agonists, including glucagon, glp-1, gip, pituitary adenylyl cyclaseactivating polypeptide (pacap), and acth, are known to enhance glucose-stimulated insulin secretion, effects which correlate with their ability to increase camp in b-cells (62–64). on the contrary, gi-coupled agonists like adrenaline, noradrenaline, somatostatin,galanin, ghrelin, and melatonin suppress insulin secretion, in part by reducing camp (65–68). measurements of single-cell camp dynamics beneath the plasma membrane have revealed that the b-cell camp response to glucagon and glp-1 is oscillatory in both rat insulinoma cells (69) and primary mouse b-cells within intact islets (figure 2) (42). higher glp-1 concentrations increase the timeaverage camp by prolonging the periods of camp elevation until the oscillations are replaced by stable elevation. the glp-1-induced camp oscillations in insulinoma cells are synchronized with oscillations of [ca2+]i and abolished upon removal of the ion from the extracellular medium, consistent with a close connection between the two messengers (69). such co-ordination of the triggering [ca2+]i and amplifying camp signals, which provides distinct stimulation of exocytosis, has been reproduced in modeling studies (70,71). however, elevated [ca2+]i is not necessary for the camp response to gs-coupled receptor agonists, since both glucagon and glp-1 can trigger camp oscillations in mouse islets at sub-stimulatory glucose concentrations (42). the camp oscillations are synchronized among different b-cells within the islet, reinforcing the idea that b-cells are functionally coupled (72,73). glucose-induced camp signaling glucose has long been recognized to increase the camp content of pancreatic b-cells (26,74–76), an effect regarded to be secondary to elevation of [ca2+]i (26–28). since the magnitude was modest and camp alone was unable to stimulate secretion, the interest for camp as a messenger in glucose-stimulated insulin secretion declined. from experiments demonstrating that purified b-cells have lower camp content, glucose-induced camp formation, and insulin secretion than intact islets and that the camp content and insulin secretion are restored by addition of glucagon or glucagon-releasing a-cells, it was suggested that camp has a permissive role in insulin secretion and that the main effect of glucose is to amplify camp formation by glucagon (77,78). a 1.2 1.0 c a m p ( a .u .) glucagon 10 nm 10 min b 1.4 0.8 glp-1 1 nm 10 min 1 2 2 1 1.3 1.0 0 ca2+/ 2 mm egta glucose 11 mm 5 min c 1.0 1.6 c a 2 + ( a .u .) 1.0 1.3 glucose 20 mm 5 min ca2+ camp c a m p ( a .u .) d c a m p ( a .u .) c a m p ( a .u .) figure 2. cyclic amp oscillations in hormoneand glucose-stimulated b-cells. total internal reflection fluorescence (tirf) microscopy recordings of the sub-membrane camp concentration in mouse b-cells within intact pancreatic islets. a, b: cyclic amp oscillations evoked by 10 nm glucagon and 1 nm glp-1 in b-cells exposed to 3 mm glucose. oscillations are synchronized among different b-cells within the islet as illustrated by graphs from the numbered cells in the tirf image (b). c, d: elevation of the glucose concentration from 3 to 11 or 20 mm evokes co-ordinated oscillations of camp and ca2+ beneath the plasma membrane. the camp oscillations are amplified by ca2+ but are maintained also when ca2+ entry is prevented (d). 358 a. tengholm glucose has indeed been found to amplify hormone-induced elevations of camp, an effect attributed to the elevation of [ca2+]i (25). when it became possible to measure camp dynamics at the single-cell level it was shown that glucose also induces pronounced increases of camp in both clonal bcells (37,79) and isolated primary mouse b-cells (37) devoid of paracrine influences. landa et al. (79) observed that the glucose effect is strictly ca2+-dependent and mimicked by depolarizing agents. however, when [ca2+]i oscillations are evoked by a combination of high glucose and tetraethylammonium the very pronounced peaks of [ca2+]i coincide with nadirs of camp, probably reflecting activation of ca2+-sensitive pdes (79,80). measurements of camp in the sub-plasmamembrane space showed that glucose not only increases the camp levels, but that the camp concentration often oscillates in synchrony with [ca2+]i with a periodicity of 2–10 minutes (figure 2) (37). in intact islets of langerhans these oscillatory responses become synchronized among neighboring b-cells (42), and the co-ordinated camp and ca2+ signals are critical for generating pulsatile insulin secretion. the glucose-induced camp elevation is amplified by ca2+, but lowamplitude oscillations remain also after removal of extracellular ca2+ or inhibition of voltage-dependent ca2+ influx (37,42). glucose also triggers camp elevation and often with oscillations under conditions when [ca2+]i is clamped by high k + in the presence of the katp channel-opener diazoxide, and a similar effect is observed with the mitochondrial substrate a-ketoisokaproic acid (37). glucose-induced elevation of camp independent of ca2+ has also been reported in b-cells from mice transgenically expressing a fret-based camp indicator (81). together, these data provide strong evidence that cell metabolism is a potent stimulator of camp production. the mechanisms by which metabolism stimulates camp formation are unknown. since camp is formed from atp it seems likely that its concentration directly regulates ac activity. in support for this idea, lowering of sub-membrane atp consumption by na+/k+-atpase inhibition was found to trigger camp elevation, and atp stimulates camp formation in permeabilized min6 b-cells (37). a problem with the hypothesis is that the in vitro-km for atp of the islet acs is ~0.3 mm (82), which is an order of magnitude below the atp concentration believed to prevail in the cytoplasm. on the other hand, affinities in vitro may not properly reflect the atp dependence in living cells. the soluble ac has a higher km for atp (40), and experiments in ins-1 cells have indicated that glucose-induced camp production might be mediated by sac (38). however, in both min6 and mouse b-cells the glucose-induced rise of camp is completely suppressed by a selective inhibitor of transmembrane acs. the sac inhibitor kh7 abolished both camp and [ca2+]i elevations, but this effect could be ascribed to an inhibitory effect on glucose oxidation unrelated to camp (42). further work is required to clarify the mechanisms underlying the stimulation of camp production by cell metabolism. available data obviously cannot exclude that atp also may have indirect effects. the camp oscillations are driven by variations in ac rather than pde activity. partial inhibition of pdes with an intermediate concentration of ibmx thus induces camp oscillations in the presence of a sub-stimulatory glucose concentration, indicating that variations in the rate of camp production under basal conditions are balanced by degradation of pdes (61). variations in the rate of camp degradation do not seem to drive camp oscillations since they are prevented by an ac inhibitor. pdes are obviously crucial for lowering camp levels during each oscillation cycle, but no isoform alone is responsible for this effect. use of pde-selective pharmacological inhibitors identified pde3 and pde1 as most important for shaping glucose-induced camp oscillations in clonal min6 and primary mouse b-cells. in addition, sirna-mediated knock-down of the ibmxinsensitive pde8b in min6 cells was found to perturb both camp oscillations and pulsatile insulin secretion (61). does camp account for the metabolic amplification of glucose-induced insulin secretion? the observations that glucose metabolism promotes camp accumulation (37,81) and that atp can stimulate exocytosis at distal steps in a pka-dependent fashion (83) are consistent with such an action of camp. on the other hand, with the observations that pka is not involved in the amplifying pathway, that the correlation between camp and insulin secretion is sometimes poor, and that camp is ineffective in enhancing ca2+-dependent secretion in the absence of glucose, it has been concluded that camp is not the main metabolic amplification signal (84–86). however, the studies have not taken into account that conventional measurements of average camp will underestimate the levels reached during the peaks of oscillations, in particular if the changes primarily occur in a specific sub-compartment. moreover, these studies are typically based on insulin secretion evoked by high concentrations of k+, which may involve a different pool of granules than that induced by glucose (87). further studies seem required to clarify if camp is or contributes to the metabolic amplifying signal or whether the two pathways are distinct and operate in parallel. cyclic amp dynamics in the pancreatic b-cell 359 role of pka in insulin secretion pka is a major effector of camp in b-cells, and the kinase is involved in mediating the stimulatory effects of the incretin hormones and other camp-elevating agents on insulin secretion. many proteins have been identified as targets for pka phosphorylation (reviewed in (15,88)). anchoring of the kinase to specific sub-cellular localizations via a-kinase anchoring proteins is important for its actions on insulin secretion (89–93). pka is highly dynamic, and camp oscillations have been found to be directly translated into oscillations of enzyme activity (80). the oscillations may contribute to keep signaling locally restricted. this idea is supported by the observation that brief elevations of camp do not provide sufficient time for the pka catalytic subunits to diffuse through the nuclear pores and enter the nucleus, which requires prolonged camp elevations (69,80,94). cyclic amp has long been known to promote b-cell electrical activity and ca2+ signaling (95–97). the enhancement of [ca2+]i signals involves both voltage-dependent entry and intracellular mobilization (98–101) and can largely be explained by pka phosphorylation of voltage-gated channels (102,103), katp channels (18,104), and ip3 receptors (101,105). effects of glp-1 on intracellular ca2+ stores have also been suggested to involve the ca2+-mobilizing messengers cyclic adp ribose and nicotinic acid adenine dinucleotide phosphate (106) and ca2+-induced ca2+ release via ryanodine receptors (107). these mechanisms were reported to involve both pka and epac. cyclic amp also stimulates exocytosis by actions distal to the elevation of ca2+ (102,108–110). pka is involved in sensitizing the secretory machinery to ca2+ (111). pka also increases secretory vesicle mobility and accounts for replenishment of the readily releasable granule pool (112–114), in particular by increasing the number of granules which are highly sensitive to ca2+ (115,116). despite the undisputed importance of pka in mediating camp signals on exocytosis, inhibitors of pka have surprisingly small effects on glucosestimulated insulin secretion from rat islets (117,118). the explanation may be that pka is primarily important during initiation of insulin secretion, as shown by time-resolved measurements of insulin release from single b-cells using two-photon excitation imaging with polar tracers (119). a detailed analysis of camp action in glucose-stimulated min6 cells demonstrated that pka controls the magnitude of the secretory response by affecting the co-ordination of ca2+ and camp signals (figure 3) (120). although pka promotes ca2+ entry via voltage-dependent channels (102,121), inhibition of the kinase neither suppressed the glucose-induced [ca2+]i response nor the glucose-induced camp elevation. instead, inhibition of pka accelerated glucose-induced membrane depolarization, such that the resulting [ca2+]i elevation triggered exocytosis before the amplifying camp signal was manifested (120). one potential explanation for this effect is that both the channel-forming kir6.2 and sulphonylurea receptor-1 (sur1) subunits of the katp channel under basal conditions are phosphorylated by pka at sites that increase channel activity (122,123). the regulation of katp channels by pka is complex, and whether phosphorylation is activating or inactivating depends e.g. on the levels of intracellular adp (124). this intricate regulation may perhaps explain the apparent paradox that glp-1 stimulates b-cell depolarization by closing katp channels via a pka-dependent mechanism (18,125). pka is thus required for establishing an initial insulin response to glucose stimulation, but pka inhibitors lack effects on already established pulsatile insulin secretion (figure 4) or on secretion triggered by ca2+. the latter observations indicate that the camp-dependence of glucose-induced insulin secretion is mediated mainly by effectors other than pka. role of epac in insulin secretion while pka was long regarded as the only camp effector in b-cells, it was evident that some camp effects on exocytosis are independent of the kinase (113). it was soon discovered that the pkaindependent effects of camp on exocytosis are mediated by epac (126), a guanine nucleotide exchange factor for the rap family of small gtpases (21). the role of epac in insulin secretion has previously been reviewed (15,127–129). there are two epac isoforms, epac1 and epac2, which are expressed in pancreatic islets (130–132), but it is mainly epac2 that has been implicated in exocytosis. of the three splice variants of epac2, b-cells only express the full-length version (133). epac-specific cyclic nucleotide analogues have been found to amplify glucose-induced insulin secretion from ins-1 cells and from mouse and human islets (131,132,134). although the specific activator does not activate pka, its effect on human islets is prevented by inhibitors of pka, indicating that pka has a permissive role for insulin secretion in human islets. capacitance measurements have demonstrated that epac2 accounts for the rapid camp-dependent potentiation of exocytosis and that pka has slower effects (113,114). epac has also been reported to recruit granules to the plasma membrane (87,135) 360 a. tengholm and together with pka to stimulate granule-granule fusion events (135). the small gtpase rap1 has been found to link activation of epac2 to stimulation of insulin secretion, probably by stimulating the recruitment of secretory granules to the membrane, but the detailed mechanism of action has not been clarified (87). one possibility is that rap1 activates vav2 and tiam (87), guanine nucleotide exchange factors for the small gtpases cdc42 and rac, which regulate insulin secretion via modulation of the actin cytoskeleton (136,137). another alternative is that rap1 stimulates mobilization of intracellular ca2+ via activation of phospholipase c-e (138). in support of the latter idea knock-out of phospholipase c-e has been found to disrupt epac-selective potentiation of insulin secretion (139). it has been suggested that camp stimulates intracellular ca2+ mobilization primarily via epac activation of ryanodine receptors (140,141), but this conclusion has been questioned (105). the study by dyachok et al. (105) is instead consistent with the idea that camp-stimulated ca2+ mobilization mainly occurs via a phospholipase c–ip3-mediated mechanism. epac was originally found to interact with the sur1 subunit of the katp channel (126). this interaction may result in modification of the atp-sensitivity of the channel (142). interestingly, the pka-independent component of camp-stimulated secretion is absent in sur1-/mice (114), suggesting that interaction between epac and sur1 is important for granule priming. epac2 has also been found to bind to the rab3-binding protein rim2 (126,143,144), and this interaction is important for the stimulatory effect of incretin hormones on insulin secretion. also the ca2+-binding protein piccolo, a neural active zone protein, is expressed in b-cells and interacts with epac2 (145,146). in addition, interaction between epac2 and the t-snare protein snap25 may be a prerequisite for the fast pka-independent effects of camp on exocytosis (147). whether these actions of epac are mediated by rap1 or not has not been determined. recent observations indicate that epac2, in addition to mediating the amplification of insulin release by incretins and other camp-elevating agents, is involved in glucose generation of pulsatile insulin secretion (120). thus, studies in min6 cells demonstrated that an epac-selective camp analogue restored not only the initial glucose-induced insulin secretion suppressed by pka inhibition, but also subsequent pulsatile secretion perturbed by adenylyl cyclase inhibition (figure 5). conversely, when the expression of epac2 was knocked down by sirna there was a marked reduction of both the initial and a b 2.2 1.0 1 min 1.0 1.3 control pka inhibited glucose 11 mm insulin ca2+ in s u li n s e c re ti o n (p ip 3 a .u .) in s u li n s e c re ti o n (p ip 3 a .u .) 1.0 2.2 1.0 1.3 c a 2 + ( a .u .) c a 2 + ( a .u .) δt = 164 ± 5 s δt = 134 ± 7 s control camp 1 min in s u li n s e c re ti o n (p ip 3 a .u .) in s u li n s e c re ti o n (p ip 3 a .u .) insulin 1.0 2.2 1.0 2.2 glucose 11 mm δt = 14 ± 6 s δt = – 27 ± 9 s c a m p ( a .u .) c a m p ( a .u .)1.6 1.0 1.0 1.6 pka inhibited figure 3. temporal relationship between glucose-induced ca2+ and camp signals and insulin secretion. a: tirf microscopy recordings of sub-membrane ca2+ concentration (dotted curves) and the insulin secretory response (solid curves) in min6 b-cells show that inhibition of pka markedly shortens the delay between glucose stimulation and the initial ca2+ elevation triggering secretion. b: simultaneous recordings of camp (dotted curve) and insulin secretion (solid curve) showing that pka inhibition shifts the timing such that secretion, which normally follows the amplifying camp signal, instead precedes the camp elevation. cyclic amp dynamics in the pancreatic b-cell 361 subsequent pulsatile insulin secretion. these findings contrast with results from epac2 knock-out mouse islets where the glucose response is not significantly decreased despite markedly reduced camp amplification of glucose-induced insulin exocytosis (87,148). this discrepancy may reflect an inherent difference between min6 cells and mouse islets or that compensatory mechanisms are differently activated by the knock-down and knock-out strategies. however, there is no information on secretion dynamics from the knock-out islets, and secretion was either measured in static incubation experiments (148) or estimated by imaging of single exocytosis events (87). it has been reported that epac regulates exocytosis of small synaptic-like vesicles rather than that of insulin-containing dense-core granules (149). however, this conclusion is supported neither by granule-imaging of knock-out mouse b-cells (87) nor by studies of the autocrine effects of insulin in min6 cells (120). the sulphonylurea class of anti-diabetic drugs, which depolarize the b-cell by inhibiting katp channel conductance after binding to the sur1 subunit of the channel, was recently found to directly bind and activate epac2 (148). this observation has gained support from another study, which even identified an epac2 mutation that abolished the sulphonylurea interaction (150), whereas other studies have failed to demonstrate a direct interaction between epac2 and sulphonylurea (151,152). a link between sulphonylureas and epac2 activation is supported by suppression of the insulin secretory response to sulphonylureas in epac2-knock-out mice (148). from the available data it is not clear whether the activation of epac2 by sulphonylureas is direct or indirect, mediated for example by an increase of camp. even if no overall elevation of camp was detected in the study by zhang et al. (148), sulphonylureas may interact with pdes (153,154) and thereby increase camp in local sub-compartments, which might be sufficient for epac2 activation. future studies will establish the nature of the link between sulphonylureas and epac2. cyclic amp signaling in type 2 diabetes type 2 diabetes is characterized by loss of first phase and impaired second phase insulin release (155) with disappearance of the regular pulsatile secretory pattern (156). it is now broadly accepted that the rp-camps 11 mm glucose 10 min 2.5 1.0 10 min glucose 11 mm rp-camps in s u li n s e c re ti o n (p ip 3 a .u .) 10 min glucose 11 mm dda a b c in s u li n s e c re ti o n (p ip 3 a .u .) 1.0 2.5 in s u li n s e c re ti o n (p ip 3 a .u .) 1.0 2.5 in s u li n s e c re ti o n (p ip 3 a .u .) glucose 20 mm dda 10 min 1.0 1.2 d figure 4. cyclic amp dependence of glucose-induced pulsatile insulin secretion. tirf microscopy recordings of the insulin secretory response from individual min6 b-cells. a: the pka inhibitor rp-8-cpt-camps (100 mm) barely affects pulsatile insulin secretion triggered by glucose. b: in contrast, if added prior to glucose stimulation, the inhibitor markedly suppresses the subsequent secretory response. c, d: glucose-induced pulsatile insulin secretion critically depends on camp generation as 50 mm of the ac inhibitor dideoxyadenosine (dda) inhibits secretion in both min6 (c), and primary mouse pancreatic b-cells (d). 362 a. tengholm disease develops as a result of b-cell dysfunction (157,158). it is not known whether camp generation is impaired in b-cells from patients with type 2 diabetes. however, several aberrations in diabetic subjects may be envisaged to affect b-cell camp handling, such as the reduced incretin effect and alterations of glucagon secretion (159,160). single nucleotide polymorphisms that correlate with fasting blood glucose and type 2 diabetes have been identified in genes linked to camp signaling, including the gip receptor, the a2 adrenergic receptor and ac5 (161–163). alterations in b-cell camp signaling have been reported from several animal models of diabetes. decreased glucose-induced camp generation and insulin secretion were thus found in diabetic chinese hamsters (164), neonatal streptozotocin diabetic rats (165), and gk rats (166), and b-cell function was regained by treatment with camp-elevating agents (165–167). in some animal models of type 2 diabetes there are increased basal camp levels and exaggerated responses to ac activators, which may be linked to an increased expression of several ac isoforms (24,166,168–170) and decreased expression of pdes (166). altered camp handling has also been found after prolonged culture of b-cells in high glucose (29,171). ins-1e cells exposed to 20 mm glucose for 3 days showed reduced camp accumulation in response to forskolin and ibmx. microarray analysis of gene expression showed several changes in the camp-signaling pathways, including a reduction of ac8, a finding confirmed also in rat and human islets (29). recently developed treatment strategies for type 2 diabetes are based on mechanisms that increase b-cell camp levels (reviewed in (172–176). the most successful approaches are based on activation of b-cell acs via incretin hormones, but the rapid degradation of the hormones via dipeptidylpeptidase4 (dpp4) is a problem. however, inhibitors of dpp4 increase the availability of endogenous circulating glp-1 and gip, and stable incretin hormone analogues as well as the glp-1 mimetic exendin4 have successfully been employed in diabetes treatment. the fatty acid receptor gpr119, a gs-coupled receptor predominantly expressed in islets, has also been identified as a promising drug target (174,177). strategies based on inhibition of pdes seem less promising due to low tissue specificity. glucose 11 mm dda 007-am 10 min 5 min glucose 11 mm control glucose 11 mm 10 min control 007-am a b epac2 knockdown c in s u li n s e c re ti o n (p ip 3 a .u .) 1.0 2.5 in s u li n s e c re ti o n (p ip 3 a .u .) 1.0 2.5 + rp-camps 1.0 4.0 in s u li n s e c re ti o n (p ip 3 a .u .) figure 5. involvement of epac in glucose-induced pulsatile insulin secretion. tirf microscopy recordings of the insulin secretory response from individual min6 b-cells. a, b: the epac-selective camp analogue 8-pcpt-2’-o-me-camp-am (007-am, 1 mm) restores the magnitude of insulin secretion initiated by glucose in the presence of the pka inhibitor rp-8-cpt-camps (a), as well as that of established glucose-induced insulin pulses suppressed by ac inhibition with dideoxyadenosine (dda) (b). c: knock-down of epac2 with sirna reduces the magnitude of both initial and subsequent pulsatile insulin secretion in response to glucose. cyclic amp dynamics in the pancreatic b-cell 363 conclusions and future perspectives nearly half a century after the discovery of the link between camp and insulin secretion, camp signaling in b-cells is still a topic for intense research. methodological advances in the past few years have provided novel insights into the spatio-temporal dynamics of the messenger and the regulation of its downstream effectors. it has become increasingly clear that the camp concentration often show complex spatio-temporal patterns that contribute to the versatility and specificity of the signaling. there are yet many unresolved questions. future studies will clarify the detailed molecular organization of the local camp signaling circuits and the precise mechanisms by which pka and epac potentiate insulin secretion, particularly in human b-cells. moreover, potential defects in camp handling of b-cells from diabetic human islet donors need to be explored. cyclic amp is important also for the release of other pancreatic islet hormones. clarification of the intricate interplay between the different endocrine cell types in the islet is a prerequisite for fully understanding normal b-cell function and the pathophysiology of impaired hormone secretion in diabetes as well as for improving treatment strategies. acknowledgements i wish to express my gratitude to all present and past members of my laboratory as well as to colleagues within and outside the department for valuable discussions. the work in my laboratory is supported by grants from the european foundation for the study of diabetes/msd, family ernfors foundation, novo nordisk foundation, swedish diabetes association, and the swedish research council. declaration of interest: the author reports no conflicts of interest. the author alone is responsible for the content and writing of the paper. references 1. pørksen n. the in vivo regulation of pulsatile insulin secretion. diabetologia. 2002;45:3–20. 2. hellman b. pulsatility of insulin release—a clinically important phenomenon. ups j med sci. 2009;114:193–205. 3. schmitz o, rungby j, edge l, juhl cb. on high-frequency insulin oscillations. ageing res rev. 2008;7:301–5. 4. tengholm a, gylfe e. oscillatory control of 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specific antibodies were detected with a protein a-gold method, modified for transmission electron microscopy of air-dried blastocysts. the results showed that c b a i h blastocyst incubated in d b a 2 immune serum were positive for protein a-gold labelling, while control blastocysts only possessed a few irregularily scattered gold particles. t h u s , it seems a s a deposition of antigens in the spleen tissue with persistence of the antigens at this site will result in detectable afitibodies in the peripheral blood. introduction raising antiserum hgsinst blastocyst antigens is laborious, since a con ventional immunization requires thousands of blastocysts ( 1 , 4 , 6 ) . a more rational way could be the use of a spleen cell culture, that i s , a few blasto cysts ( t h e antigens) are co-cultured with spleen cells which then are used to produce monoclonal antibodies ( 2 ) . in the present report we describe experi ments with a technique where the blastocysts were deposited directly into the spleen tissue, where they seemed capable to trigger an antibody response. material anc methgds blastocysts were obtained from c b a / h mice in delayed implantation. the blastocysts were recovered 18 h a f t e r an injection of oestrogen, that i s , when they were about to attach onto the uterine surface. the flushings were made with pbs containing 1 p e r cent serum from the future host o r 5 0 0 ieiml hepa rin. since gup prime goal was to obtain antibodies against surface constituents 151 of attaching trophoblast cells, the blastocysts were irradiated, before t h e transfer into the spleen, with 5000 r from a cesium source to block their ability to grow. recipients were 4 male mice, aged two months, of the dea2 strain. the spleen was reached through a dorso-lateral incision, and the caudal end of the spleen was carefully taker. out of the abdominal cavity. a micropipette containing 2-3 donor blastocysts in a small amount of pbs with recipient serum o r heparin was inserted underneath the splenic capsule, and the blastocysts were extruded from the pipette. the fluid remaining in the pipette was examined in o r d e r to determine whether all of t h e blastocysts had been extruded. a f t e r a successful transfer the spleen was replaced in position, arid the incision was closed. after four weeks, a booster inimunization was performed by transfer of sin;ilsrily treated blastocysts. between one and three weeks later, blood samples were taken frcm each recipient animal by retro -orbital puncture, and serum was prepared for testing for the presence of immunoglobulins directed against trophcblast determinants. the presence of specific antibodies was detected with a protein a-gold method (3,5) modified for transmission electron microscopy of blastocysts (svalander, ljung arid nilsson, in preparation). blastocysts in the same functional state as those used for immunization were flushed from the uterine horns of c b a i h mice with a fixative of 0 . 5 % glutaraldehyde in pds into a watchglass. the blastocysts were left in the fixative for a maximum of 20 rnin, washed four times in p s s and incubated in the dba2 immune serum diluted 1:20 for 45 min at room temperature. controls were incubated in pbs or in dea2 non-immune serum. after three subsequent washings in p b s , the blastocysts were incubated in rabbit anti-mouse-ig antiserum diluted 1: 5g for 30 min at room temperature, and washed three times in pbs. the blastocysts were then transferred to the protein a-colloidal gold solution, in which they were left for 30 min at r o o m temperature. a f t e r this labelling, the blastocysts were washed three times in pbs and twice in double-distilled, ultrafiltered water. finally, they were placed on a hexagonal grid ( h e x 700 t b , polaron eq. ltd., england) without membrane in a microdroplet of water, and were left to d r y in the air. the air-dried blastocysts were examined in a philips 400 stem electron microscope with field emission gun operated at 100 kv to evaluate the number of gold particles. results a n d comkents blastocysts flushed from cba/h mice 18 h after an injection of estrogen and incubated in d b a 2 immune sera were positive for protein a-gold labelling. the particles observed in the micrographs imaged gold colloid from both the upper and lower surface of the flat blastocyst. the particles were 152 irregularily scattered in the field of view. the control blastocysts, which were incubated in pbs o r in d b a 2 non-immune serum instead of the immune serum at the first incubation s t e p , possessed only a few irregularily scattered gold particles. t h u s , it seems a s b deposition of antigens in the spleen tissue with persistence of the antigens at this site will result in a detectable amount of antibody in the peripheral blood. this preliminary finding is now f u r t h e r tested using, among other things, gel plugs with bovine serum albumin a s antigen. r e feren ce s 1. johnson, l. v . & calarco, p. g . : stage-specific embryonic antigens detected by an antiserum against nouse blastocysts. dev eiol 7 9 : 224-231, 1980. antibodies to hypothalamic growth hormone-releasing factor with picornoles of antigen. science 218 : 887-889, 1 9 8 % . quantitative approach for antigen localizatior, on thin sections. in: techniques in immuriocytochemistry (eds. g . r . bullock & p. petrusz), pp. 107-133. academic press, london, 1982. 4 . shevinsky, l . h . , knowles, b.b., damjanov i . & solter, d . : monoclonal antibody to murine embryos defines a stage-specific embryonic antigen expressed on mouse embryos and human teratocarcinoma cells. cell 2 . luben, r.a., brazeau, p . , bohlen, p. & guillemin, r . : monoclonal 3 . roth, j.: the protein a-gold (pag) technique. qualitative and 3 0 : 6 9 7 7 0 5 , 1982. 5 . slot, j . w . & geuze, h.j.: sizing of protein a-colloidal gold probes for 6 . wiley, l . m . & calarca, p . g . : the effects of anti-embryo sera and their immunoelectron microscopy. j cell biol 90: 533-536, 1 9 8 1 . localization on the cell surface during mouse preiniplaritation development. dev biol 4 7 : 4 0 7 4 1 8 , 1 9 7 5 . address for reprints: €3. ove nilssori department of human anatomy eiomedical centre box 5 7 1 s-751 23 uppsala sweden 153 pulmonary artery coil embolisation prevented tumour progression in a patient with advanced squamous cell lung carcinoma case report pulmonary artery coil embolisation prevented tumour progression in a patient with advanced squamous cell lung carcinoma virginija �sileikien_ea, viktorija gurskyt_eb, ingrida zeleckien_ec, elena bernotien_ea and sigitas �cibirasd aclinic of chest diseases, immunology and allergology, institute of clinical medicine, faculty of medicine, vilnius university, vilnius, lithuania; bfaculty of medicine, vilnius university, vilnius, lithuania; ccentre of radiology and nuclear medicine, vilnius university hospital santaros klinikos, vilnius, lithuania; dcentre of cardiology and angiology, vilnius university hospital santaros klinikos, vilnius, lithuania abstract background: squamous cell lung carcinoma (sqclc) is a type of non-small-cell lung cancer, accounting for 25–30% of all lung cancer cases with a median advanced stage survival of 8–11 months. here we present a rare case of long-term survival with metastatic sqclc following coil embolisation of the right pulmonary artery. case presentation: the 49-year-old patient was diagnosed with stage iv (ct4n3m1) sqclc in 2007 due to a biopsy-proven central malignant tumour in the right lung and bilateral mediastinal lymphadenopathy. a magnetic resonance imaging scan also revealed a metastatic lesion in the liver. soon after the diagnosis, the patient experienced pulmonary haemorrhage, which was managed by obturating the intermediate bronchus and performing coil embolisation of the right pulmonary artery. the patient also received chemotherapy in 2007 and 2009 without radiological changes. at three different time points in years 2010–2019, biopsies of the primary tumour were taken. all showed dense connective tissue with no indication of cancer growth. in 2020, a positron emission tomography scan showed no pathological metabolic activity in the lungs and liver. currently, the patient remains in a stable clinical condition with a good performance status. conclusion: the long-term clinical benefit indicates a direct effect of coil embolisation on tumour progression. we suggest that coil embolisation of tumour-feeding arteries could be considered as a potential treatment method for patients with sqclc. article history received 13 march 2020 revised 3 april 2020 accepted 3 april 2020 keywords alternative therapy; coil embolisation; non-small cell lung cancer; spontaneous regression; squamous cell carcinoma introduction lung cancer remains the leading cause of malignancy-related mortality worldwide, accounting for approximately 25% of all cancer deaths (1,2). one of the most prevalent histological types of lung cancer is squamous cell lung carcinoma (sqclc), which constitutes 25–30% of all lung cancer cases and is strongly associated with cigarette smoking (3,4). the survival rates of lung cancer are largely dependent on the cancer stage at the time of diagnosing and the histological type of the tumour. on average, 50.3% of patients with localised stage lung cancer survive for 5 years (2). unfortunately, 48.7–57% of non-small cell lung cancer (nsclc) patients are diagnosed in late stages of the disease, when the options for treatment are limited and the chances of surviving are poor (2,5,6). for instance, the median survival of patients with advanced sqclc receiving first-line platinum-based chemotherapy is only 8–11 months (7). nevertheless, there have been nearly 40 cases of spontaneous regression and longer survival in patients with advanced nsclc documented in literature from 1950 to 2020, most of whom were diagnosed with sqclc (8,9). in this paper, we present a rare case of long-term survival with metastatic sqclc following chemotherapy and endovascular embolisation of the right pulmonary artery with 33 coils due to massive haemoptysis. case presentation in june 2007, a 49-year-old male suffering from chronic nonproductive cough, episodic fever, and night sweats was referred to a pulmonologist. the symptoms lasted for approximately 6 months. the patient’s medical history revealed a myocardial infarction at the age of 47 years, new york heart association (nyha) functional class iii chronic heart failure, and smoking. upon chest auscultation, diminished vesicular breathing sounds with crackles during forced expiration were heard in the right lung. laboratory findings showed a slight leukocytosis (10,110/ll), elevated c-reactive protein level (40.2 mg/l) and high erythrocyte sedimentation rate (86 mm/h). a computed tomography (ct) scan was performed, which revealed a centrally located mass in the right lung, invading the mediastinum and occluding the right lower lobe bronchus. multiple bilaterally enlarged paratracheal and subcarinal lymph nodes were also present (figure 1). the patient underwent an endobronchial biopsy contact viktorija gurskyt_e gurskyte.viktorija@gmail.com faculty of medicine, vilnius university, m. k. �ciurlionio str. 21/27, lt-03101, vilnius, lithuania � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 3, 257–261 https://doi.org/10.1080/03009734.2020.1753863 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1753863&domain=pdf&date_stamp=2020-06-25 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1753863 http://www.tandfonline.com of the mass, which led to the diagnosis of sqclc. furthermore, a 2.6 � 2.3 � 2.0 cm hypovascular lesion in segment vi of the liver was detected in an abdominal ct scan. although no biopsy of the lesion was performed, it was considered as a possible metastasis based on gadoliniumenhanced magnetic resonance imaging (mri) scan results (figure 2). therefore, the sqclc was clinically staged as ct4n3m1 (stage iv). since the disease was diagnosed at an advanced stage, the patient was not a candidate for pneumonectomy and was scheduled for chemotherapy treatment instead. however, the initiation of chemotherapy was delayed as the patient experienced massive haemoptysis soon after being diagnosed with sqclc. the pulmonary haemorrhage was managed by obturating the intermediate bronchus and performing ct pulmonary angiography and coil embolisation of the right pulmonary artery in june 2007 (figure 3). a total of 33 gianturco 3–8 mm diameter coils were used for the embolisation, allowing only minimal residual blood flow to the right lung. two weeks later, the bronchial obturator was removed and the haemorrhage did not recur after the procedure. in july 2007, first-line chemotherapy with cisplatin and gemcitabine was initiated for six cycles of treatment, which resulted in primary tumour size reduction from 6.4 � 3.0 � 6.5 cm to 1.5 � 1.2 � 1.2 cm in axial, coronal, and sagittal planes, with no effect on the liver metastasis. the patient remained stable until august 2009, when increased lung tumour size (2.8 � 2.2 cm) was observed in a follow-up ct scan and the disease was termed as progressive according to the response evaluation criteria in solid tumours (recist). at this point, no rebiopsy was performed. the patient was then included in a clinical trial and received six cycles of second-line chemotherapy with docetaxel and either a tyrosine kinase inhibitor nintedanib or a placebo. it is unknown whether the patient was in the treatment or in the control group. despite the chemotherapy, the disease appeared to progress further as the primary tumour continued to grow in size based on evaluation of the ct scans (figure 4). in contrast, the metastatic lesion in the liver has no longer been visible on ultrasound or ct imaging since 2011, which could be regarded as a mixed response to the treatment. in 2010, 2011, and 2019, three biopsies of the lung tumour were performed due to radiological changes suggestive of sqclc progression, i.e., occurrence of several new pulmonary lesions and increased primary tumour volume. however, only excess connective tissue formation with no sqclc cells was observed in each of the tissue samples. since the biopsies have been non-informative, a whole-body 18f-fluorodeoxyglucose (18fdg) positron emission tomography-ct (pet-ct) scan was performed in january 2020 in order to exclude disease progression. the only notable finding in the 18fdg pet-ct scan was an area of low metabolic activity among the coils, which can be considered physiological. no pathological metabolic activity in the liver or figure 1. thoracic ct scan image at the time of diagnosing sqclc. contrastenhanced ct image with soft-tissue window showing a centrally located mass in the right lung with mediastinal invasion (arrow), and mediastinal lymphadenopathy. figure 2. a hypovascular metastatic lesion in the liver. liver mri ti w image on post-contrast arterial phase showing a hypo-intense lesion with subtle concentric perilesional enhancement in segment vi of the liver (arrow). figure 3. thoracic ct scan image after the pulmonary artery embolisation procedure. contrast-enhanced ct image with soft-tissue window showing multiple coils embolised to the right main pulmonary artery, as well as a partially decreased tumour and reduced occlusion of the right lower lobe bronchus. 258 v. šileikienė et al. elsewhere in the body was detected. due to the lack of evidence indicating cancer progression, the patient has not received treatment for sqclc since 2009. currently, the patient remains in a stable clinical condition with a good performance status—0 on the eastern cooperative oncology group (ecog) scale. discussion we have demonstrated a rare case of spontaneous regression of advanced stage sqclc in a patient who has already survived for nearly 13 years since receiving the diagnosis. spontaneous regression is frequently defined as the ‘partial or complete disappearance of a malignant tumour in the absence of treatment or in the presence of therapy considered inadequate to exert a significant influence on the disease’ in cases when the diagnosis of cancer is proven by histopathological examination in the first place (10,11). according to the widely accepted criteria formulated by everson and cole, the term can be applied to both the regression of the primary tumour as well as its metastases, the presence of which can be either confirmed histopathologically or suspected using radiological imaging (8,10). in our patient’s case, it appears that the phenomenon of spontaneous regression occurred both in the primary tumour and the metastatic lesion of the liver. it is important to note that the diagnosis of metastatic disease was established solely on the basis of hepatic lesion appearance in the mri scan. at the time, a pet scan was not available in our country for detection of metastatic lesions, and no biopsy of the suspected liver metastasis was taken. nevertheless, according to a meta-analysis of 10 studies, gadolinium-enhanced mri scan is an excellent diagnostic tool for metastatic lesions in the liver. the pooled specificity of gadolinium-enhanced mri and 18fdg pet-ct is 0.99 and 1.00, respectively (12). hence, only a minuscule possibility remains that the hepatic lesion in our patient was an unrelated finding, such as a haemangioma or an area of liver steatosis. the most prevalent explanations for spontaneous lung cancer regression involve activation of the immune system. for instance, it has been observed that the phenomenon often occurs soon after acute infections, which led some authors to believe that the activation of innate immunity is responsible for the disease regression (11). although the exact biological mechanism of this effect is unknown, it is thought that immunological response results in either t-cell-mediated apoptosis or inflammatory necrosis of the tissues (13,14). however, infection-related immune response in our patient is unlikely as there was no evidence of significant infections during the observation period. tumour regression has also been observed after performing invasive procedures, such as a biopsy or surgery of the neoplasm, possibly due to the release of antigens into the bloodstream and subsequent activation of the immune response (15,16). another theory involving the immune system is based on the psychoneuroimmunological mechanisms, suggesting that positive psychological changes can stimulate the cellular immune response and may thus induce cancer regression in some patients (13,17,18). naturally, it is extremely difficult to prove or disprove this mechanism being responsible for disease regression in any given specific case, including the one we describe. we believe that in our case cancer regression was induced by performing endovascular embolisation of the right pulmonary artery with 33 coils due to pulmonary haemorrhage. we speculate that coil embolisation resulted in locally impaired angiogenesis, which led to primary tumour cell death and prevented further progression. when the tumour cells become deprived of oxygen and nutrients, they produce angiogenic growth factors which bind to the receptors located in the endothelium of pre-existing blood vessels. this results in morphological changes of the original vessels and formation of new ones during a multi-step process (19,20). however, in our patient’s case, the vascular supply of the neoplasm became inadequate after coil embolisation as the blood flow via the main tumour-feeding artery was drastically reduced. therefore, both the blood supply and angiogenic potential of the tumour could have been impaired by this procedure. upon review of the literature, we were unable to find any similar cases attributing primary lung cancer regression to endovascular embolisation of tumour-feeding arteries. to our knowledge, only metastatic pulmonary lesions from primary renal cell carcinoma as well as some benign thoracic tumours have been reported to regress following transarterial embolisation (21). furthermore, the disappearance of our patient’s metastatic lesion in the liver is also obscure. it has been previously suggested that the removal of the primary tumour may provoke an immune response which can eliminate metastatic lesions (22). it is possible that a similar mechanism may have been triggered by the coil embolisation procedure and subsequent regression of the primary tumour. nevertheless, similarly to the other suggested possible underlying mechanisms of cancer regression, these theories are only hypothetical and require extensive further research to be confirmed. it should also be taken into account that our patient has received two courses of chemotherapy treatment. the firstline treatment with cisplatin and gemcitabine appeared to be rather effective in reducing the primary tumour size. however, the initiation of chemotherapy coincided with the figure 4. thoracic ct scan image in 2009. a follow-up contrast-enhanced ct image with soft-tissue window after 2 months of treatment with second-line chemotherapy showing an increased tumour in the right lung. upsala journal of medical sciences 259 endovascular embolisation procedure, which could have also led to tumour volume reduction (21). even if the initial radiological improvement can be attributed to systemic treatment, according to the literature, the chemotherapy regimens used for treating our patient are highly unlikely to induce a longterm remission. for instance, the median survival of advancedstage sqclc patients treated with cisplatin and gemcitabine is only 10.8months. moreover, the median progression-free survival is 5.1 months, and the survival rate at 24months posttreatment is 14% (23). regarding second-line chemotherapy, the median survival of patients treated with docetaxel is 6 months, while the median progression-free survival is only 2.8 months (24). it is unknown whether a tyrosine kinase inhibitor was added to our patient’s treatment regimen along with docetaxel as he received the treatment in a clinical trial setting. either way, the radiological appearance of the primary tumour did not improve after six cycles of second-line chemotherapy. thus, it seems improbable that the systemic treatment led to cancer remission in our patient’s case. conclusions in this paper, we present a case of long-term sqclc remission following the procedure of pulmonary artery coil embolisation. we suggest that coil embolisation of tumour-feeding arteries should be considered as a potential treatment method in patients with sqclc and other histological types of nsclc. however, further studies are needed to support our proposition and elucidate the molecular mechanisms behind cases of spontaneous cancer regression. consent for publication written consent was obtained from the patient for publication of this case report and for the use of accompanying images. the authors have fully anonymized the patient. disclosure statement no potential conflict of interest was reported by the author(s). notes on contributors virginija �sileikien_e, phd, is an assistant professor in pulmonology at the faculty of medicine, vilnius university, and a consulting pulmonologist at the centre of pulmonology and allergology, vilnius university hospital santaros klinikos, vilnius, lithuania. viktorija gurskyt_e is a medical student at the faculty of medicine, vilnius university, and a laboratory assistant at the department of anatomy, histology and anthropology, faculty of medicine, vilnius university, vilnius, lithuania. ingrida zeleckien_e is a doctor radiologist at the centre of radiology and nuclear medicine, vilnius university hospital santaros klinikos, vilnius, lithuania. the doctor specializes in cardiovascular and thoracic radiology. she is a member of the lithuanian association of radiology (lra), the european society of radiology (esr). elena bernotien_e is a pulmonologist specialising in lung cancer at the centre of pulmonology and allergology, vilnius university hospital santaros klinikos, and the supervisor of resident doctors in pulmonology at the faculty of medicine, vilnius university, vilnius, lithuania. sigitas �cibiras, phd, is an interventional cardiologist at the centre of cardiology and angiology, vilnius university hospital santaros klinikos, vilnius, lithuania. references 1. herbst rs, morgensztern d, boshoff c. the biology and management of non-small cell lung cancer. nature 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poddubskaya e, et al. nivolumab versus docetaxel in advanced squamous-cell non–small-cell lung cancer. n engl j med. 2015; 373:123–35. doi:10.1056/nejmoa1504627 upsala journal of medical sciences 261 https://doi.org/10.3390/cells8091102 https://doi.org/10.1016/j.cpet.2009.04.011 https://doi.org/10.1055/s-0039-1692658 https://doi.org/10.1016/j.rmed.2010.04.026 https://doi.org/10.1200/jco.2007.15.0375 https://doi.org/10.1056/nejmoa1504627 abstract introduction case presentation discussion conclusions consent for publication disclosure statement references changes of arterial pressure following relief of obstruction in adults with hydronephrosis article changes of arterial pressure following relief of obstruction in adults with hydronephrosis ammar al-mashhadia , michael h€aggmanb, g€oran l€ackgrena, sam ladjevardib, tryggve nev�eusc , arne stenberga, a. erik g. perssond and mattias carlstr€ome apediatric surgery section, department of women’s and children’s health, uppsala university, uppsala, sweden; bdepartment of surgical sciences, uppsala university, uppsala, sweden; cpediatric nephrology unit, department of women’s and children’s health, uppsala university, uppsala, sweden; ddepartment medical cell biology, uppsala university, uppsala, sweden; edepartment of physiology and pharmacology, karolinska institutet, stockholm, sweden abstract background: as much as 20% of all cases of hypertension are associated with kidney malfunctions. we have previously demonstrated in animals and in pediatric patients that hydronephrosis causes hypertension, which was attenuated by surgical relief of the ureteropelvic junction (upj) obstruction. this retrospective cohort study aimed to investigate: (1) the proposed link between hydronephrosis, due to upj obstruction, and elevated arterial pressure in adults; and (2) if elevated blood pressure in patients with hydronephrosis might be another indication for surgery. materials and methods: medical records of 212 patients undergoing surgical management of hydronephrosis, due to upj obstruction, between 2000 and 2016 were assessed. after excluding patients with confounding conditions and treatments, paired arterial pressures (i.e. before/after surgery) were compared in 49 patients (35 years old; 95% ci 29–39). split renal function was evaluated by using mercaptoacetyltriglycine (mag3) renography before surgical management of the hydronephrotic kidney. results: systolic (�11 mmhg; 95% ci 6–15 mmhg), diastolic (�8 mmhg; 95% ci 4–11 mmhg), and mean arterial (-9mmhg; 95% ci 6–12) pressures were significantly reduced after relief of the obstruction (p < 0.001). split renal function of the hydronephrotic kidney was 39% (95% ci 37–41). no correlations were found between mag3 and blood pressure level before surgery or between mag3 and the reduction of blood pressure after surgical management of the upj obstruction. conclusions: in adults with hydronephrosis, blood pressure was reduced following relief of the obstruction. our findings suggest that elevated arterial pressure should be taken into account as an indication to surgically correct hydronephrosis. article history received 9 june 2018 revised 31 august 2018 accepted 4 september 2018 key words blood pressure; hydronephrosis; hypertension; kidney; renal function; ureteral obstruction introduction cardiovascular disease, including hypertension (�140/90mmhg) is a major health problem, associated with increased morbidity and mortality. secondary forms of hypertension are found in 5–20% of the hypertensive population, and most of these cases can be linked to renal abnormalities (1,2), but the role of hydronephrosis has so far received only scant attention. published reports of hypertension obviously caused by hydronephrosis are few, and the numbers of patients included in these reports are low (3,4). the treatment of symptomatic ureteropelvic junction (upj) obstruction is surgical. it has been shown that the function of the hydronephrotic kidney is rather well preserved in young children (5–7). based on this finding, the management policy concerning asymptomatic hydronephrosis has consequently become much more conservative. besides the need for lifelong follow-up with repeated investigations, the potential cardiovascular risk of this new treatment strategy is still unknown. previous experimental studies in animals have demonstrated that hydronephrosis, due to chronic partial unilateral ureteral obstruction, is linked to the development of renal injuries and hypertension (8–14). moreover, the causal relationship is demonstrated by the fact that arterial pressure is significantly lowered by surgical relief of the obstruction (15). in children, there are currently only a few reports investigating the association between hydronephrosis and abnormal arterial pressure (16,17). in a recent prospective study, we demonstrated that arterial pressure and markers of oxidative stress were elevated in pediatric patients with hydronephrosis compared with healthy controls, and that these disturbances were normalized following surgical management of the obstruction. the aim of this study was to investigate the contact mattias carlstr€om mattias.carlstrom@ki.se department of physiology and pharmacology, karolinska institutet, biomedicum q5b, solnav€agen 9, s-17177, stockholm, sweden supplemental data for this article can be accessed here. � 2018 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2018, vol. 123, no. 4, 216–224 https://doi.org/10.1080/03009734.2018.1521890 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1521890&domain=pdf http://orcid.org/0000-0001-9578-1783 http://orcid.org/0000-0002-4590-4957 http://orcid.org/0000-0001-9923-8729 https://doi.org/10.1080/03009734.2018.1521890 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1521890 http://www.tandfonline.com hypothesis that arterial pressure preoperatively is higher than arterial pressure postoperatively in patients aged more than 15 years old. if correct, elevated blood pressure in patients with hydronephrosis should be considered as another indication for surgery. material and methods we studied the medical records and hospital charts of 212 patients with hydronephrosis, due to congenital upj, who were operated between 2000 and 2016 at the urology department of uppsala university hospital (table 1). all patients were more than 15 years old. from the total population with operated upj, 49 patients fulfilled our inclusion criteria, i.e. patients with unilateral hydronephrosis not associated with any other chronic diseases that may be predisposing for hypertension (e.g. cardiovascular disease, diabetes, obesity), who were not treated with antihypertensive drugs, and with available arterial pressure values before and after surgical management of the hydronephrosis (figure 1, table 2). according to medical records the majority of upj was associated with aberrant vessel (n ¼ 37; 76%), and to a lesser extent it was due to inflammation and fibrosis (n ¼ 8; 16%) and pelviureteral junction stricture (n ¼ 4; 8%). we included those blood pressure measurements when the patients were not in an acute situation nor in pain for any reason, even before temporary treatment with nephropyelostomy catheter or double j-stent. the same method of blood pressure monitoring (i.e. office) was used for all patients, and it was consistent in terms of location and time of the day (i.e. daytime). the procedure for all included patients was office blood pressure measurements during non-stressed situations and in the absence of any acute pain. we performed previously a similar study in which we included two control groups, i.e. one healthy not operated group and one healthy operated group and found no effects of preoperative stress on the blood pressure in patients with hydronephrosis (18). moreover, due to the retrospective nature of this study, and that blood pressure results were acquired from the patient’s digital record, it is difficult to know if the measurements were made in supine or prone position, but the procedure should be the same for each patient. twenty-five (52%) of these patients had right side hydronephrosis. thirty-eight patients were operated by laparoscopic pyeloplasty, while nine patients were operated by robot-assisted pyeloplasty, and one patient underwent open surgery. the patients’ age at the time of surgery ranged from 16 to 71 years, and 24 (49%) of them were males. renal obstruction was relieved temporarily before pyeloplasty with a double j-stent and/or percutaneous nephropyelostomy in 40 patients. the duration of this preoperative relief of obstruction ranged between two weeks and one year. nephrostomy was performed preoperatively due to acute pain during the period the patient was waiting for the pyeloplastic surgery. pre-relief arterial pressure was measured one day to one year before temporary relief of obstruction in these 40 patients, while pre-relief arterial pressure for the table 1. characteristics of the population with upj obstruction. total population n ¼ 212 age (y) 37 (95% ci 35–40) female/male (%) 53/47 unilateral/bilateral (%) 93/7 chronic disorders (%) 20 cvd, including hypertension (%) 15 others (%) 12 antihypertensive drugs (%) 10 pre relief of upj obstruction (n) n ¼ 203 systolic pressure (mmhg) 133 (95% ci 130–136) diastolic pressure (mmhg) 81 (95% ci 79–83) mean arterial pressure (mmhg) 99 (95% ci 97–100) post relief of upj obstruction (n) n ¼ 60 systolic pressure (mmhg) 122 (95% ci 119–127)� diastolic pressure (mmhg) 76 (95% ci 72–78)� mean arterial pressure (mmhg) 91 (95% ci 88–94)� �p < 0.005 compared with pre relief of upj obstruction. cvd: cardiovascular disease; upj: ureteropelvic junction. (n=164) (n=49) figure 1. schematic illustration of the study diagram. medical records and hospital charts of 218 patients who were operated (2000–2016 at the urology department of uppsala university hospital) due to hydronephrosis were studied. a total of 212 patients displayed hydronephrosis due to ureteropelvic junction (upj) obstruction. patients with other chronic disorders, bilateral hydronephrosis, antihypertensive treatment, or with missing data on blood pressure (i.e. before or after surgical management) were not included in the analysis. from the total population with operated upj obstruction 49 patients fulfilled the inclusion criteria, and their systolic, diastolic, and mean arterial pressure were analyzed before and after surgery, and linear regression analysis was carried out comparing changes in arterial pressure with renal function by mercaptoacetyltriglycine (mag3) renography. upsala journal of medical sciences 217 rest of patients was measured one day before pyeloplastic surgery. post-relief arterial pressures were measured between two weeks to two years after relief of obstruction. seven (14.3%) of the investigated 49 patients had recurrent upj obstruction after pyeloplasty, but in the total population the recurrence frequency was 6.6% (14 of 212 patients). three of these had undergone robotic pyeloplasty, while four relapses occurred after laparoscopic pyeloplasty (table 3). four of these seven patients were operated again with open pyeloplasty, whereas two were nephrectomized and one underwent ureterotomy. the arterial pressure of these seven patients was measured again before and after relief of the obstruction relapse. as a part of the routine management, preoperative evaluations of bilateral renal function were carried out using mercaptoacetyltriglycine (mag3) renography with forced diuresis. this examination was performed in a standardized fashion. in nine patients the preoperative mag3 renography was substituted with computed tomography (ct) urography, which has been proven an accurate and useful non-invasive imaging technique for the surgical planning (treatment) of upj obstruction (19). statistical analysis non-parametric kruskal–wallis test, followed by dunn’s test, was used for multiple comparisons among groups. matched blood pressure values (before and after relief) were analyzed by non-parametric wilcoxon test (i.e. matched-pairs signed rank test). linear regression analysis (least squares ordinary fit) was used to compare age-grouped populations (i.e. total population, �30 and >30 years of age). computed p values are indicated, and p< 0.05 denotes statistical significance. results arterial pressure in the total population of hydronephrotic patients, unpaired analysis suggested that systolic, diastolic, and mean arterial pressures (map) were higher before surgical management of upj (table 1, figure 2). after excluding patients with chronic disease, bilateral hydronephrosis, patients already on antihypertensive treatment, and those with missing blood pressure data, paired analysis of blood pressure values (i.e. pre and table 2. patient characteristics preand postoperatively. age sex operation side mag3 ap (pre) ap (post) 16 f lap pyeloplasty rt 34% 138/78 120/80 16 m lap pyeloplasty lt 55% 115/75 120/70 17 f lap pyeloplasty rt 50% 105/65 115/65 17 m robotic pyeloplasty lt 46% 130/80 120/80 17 m lap pyeloplasty lt not done 130/80 130/70 19 f lap pyeloplasty lt 20% 120/90 120/81 19 m lap pyeloplasty lt 47% 130/70 130/65 19 m lap pyeloplasty rt 30% 120/90 120/60 20a f robotic pyeloplasty lt 54% 100/60 95/55 20 m lap pyeloplasty lt 41% 110/70 110/70 20 m lap pyeloplasty lt 41% 110/70 110/70 21 f lap pyeloplasty lt 37% 135/80 110/60 21 f lap pyeloplasty rt 41% 110/70 120/70 22 f lap pyeloplasty rt 30% 120/80 110/60 22 m lap pyeloplasty rt 23% 123/81 120/85 22 m lap pyeloplasty lt 37% 160/85 120/85 23a m robotic pyeloplasty rt 40% 140/80 105/63 24 m lap pyeloplasty rt 47% 145/90 120/80 25 f lap pyeloplasty lt 41% 120/80 100/80 25 m lap pyeloplasty lt 48% 130/75 120/80 26 f robotic pyeloplasty lt 38% 120/75 120/70 26 m lap pyeloplasty lt 19% 170/105 130/90 26 m lap pyeloplasty lt not done 140/90 120/70 27 m lap pyeloplasty rt not done 110/80 108/73 32 f lap pyeloplasty rt not done 105/60 110/63 32 f robotic pyeloplasty rt 28% 165/110 138/90 33 f lap pyeloplasty rt 25% 110/65 104/70 35 f lap pyeloplasty rt 39% 120/80 100/70 35 m lap pyeloplasty lt not done 154/109 140/95 36 f lap pyeloplasty lt 39% 120/80 110/80 37 f lap pyeloplasty lt 60% 120/75 120/75 37 m robotic pyeloplasty rt 15% 130/90 120/70 41 f open pyeloplasty rt 31% 150/90 120/80 41 f lap pyeloplasty lt 62% 125/100 125/90 41 m lap pyeloplasty lt 48% 160/80 136/84 43a m lap pyeloplasty rt not done 145/80 150/95 44 f lap pyeloplasty rt 46% 130/80 140/90 46a m lap pyeloplasty rt not done 145/90 120/70 49 m lap pyeloplasty rt 38% 130/82 130/85 50 f robotic pyeloplasty rt 29% 130/80 120/85 50a f robotic pyeloplasty rt 42% 150/100 120/70 50 m robotic pyeloplasty rt 40% 130/95 130/65 55 m lap pyeloplasty rt not done 160/110 140/95 56 f lap pyeloplasty rt 39% 140/90 140/90 67 f lap pyeloplasty rt 57% 169/117 140/90 68 f lap pyeloplasty lt 31% 111/82 110/60 68 f lap pyeloplasty rt 58% 195/90 150/90 71a f lap pyeloplasty lt 39% 190/70 150/80 71 m lap pyeloplasty lt not done 160/90 150/70 apatients who had recurrent ureteropelvic junction obstruction after pyeloplasty. ap: arterial pressure (mmhg); f: female; lap: laparoscopic; lt: left kidney; m: male; mag3: mercaptoacetyltriglycine; rt: right kidney. table 3. recurrent cases of upj obstruction with pre-relief (before second operation) and post-relief (after second operation) arterial pressure, and methods of relief of the obstruction for the second time. age sex operation side ap (pre) ap (post) delta map 20 f pyeloplasty lt 110/60 95/50 12 23 m pyeloplasty rt 118/67 105/63 7 41 m nephrectomy lt 160/80 136/84 6 43 m ureterotomy rt 150/95 140/90 4 46 m pyeloplasty rt 140/80 120/75 10 50 f pyeloplasty rt 175/90 missed – 71 f nephrectomy lt 140/80 missed – ap: arterial pressure (mmhg); f: female; lt: left kidney; m: male; map: mean arterial pressure (mmhg); rt: right kidney. 0 50 100 150 200 250 a rt e ri a l p re s s u re (m m h g ) pre post pre post pre post systolic diastolic map p < 0.001 p < 0.001 p < 0.001 figure 2. systolic, diastolic, and mean arterial pressure (map) in hydronephrotic patients (n ¼ 203; male 47%; female 53%) before (n ¼ 203) and after (n ¼ 60) surgical management of ureteropelvic junction obstruction. 218 a. al-mashhadi et al. post relief) was performed on the remaining 49 patients. in agreement with the total population, systolic (d: �14mmhg), diastolic (d: �10mmhg), and map (d: �11mmhg) were all significantly reduced after relief of the obstruction (p values <0.001 for all variables) (table 2, figure 3). as mentioned above, seven patients had recurrence of upj stenosis 2–4 months after surgical management of the hydronephrosis and were thus re-operated (table 3). analysis of paired blood pressure values (n ¼ 5), pre and post relief, showed significant reduction of systolic (d: �16 mmhg; p ¼ 0.002) and map (d: �8 mmhg; p ¼ 0.002), whereas diastolic pressure was not significantly reduced (d: �4 mmhg; p ¼ 0.15). in order to investigate if the arterial pressure elevation and the reduction of blood pressure following surgery were influenced by age, a subanalysis was performed. the study population was divided into patients aged �30 years and patients aged >30 years at the time of surgery. systolic, diastolic, and map were all significantly higher in the older patient group (figure 4(a–c)). the blood pressure was significantly reduced following surgery in both age groups, and the magnitude of blood pressure was not significantly different, although there was a clear trend towards greater reduction in systolic arterial pressure in older patients (p ¼ 0.08) (figure 4(d)). finally, a long follow-up period before reassessing blood pressure after surgery may influence the interpretation of the surgical intervention. in our study the shortest duration before obtaining post-relief values was 0.5 month, and the longest duration was 30 months. we stratified the blood pressure data based on the duration of the follow-up period. linear regression analysis did not show any significant correlation between the length of the postoperative period and the change in arterial pressure (figure 5(a)). when separating data into four groups with different time spans for the post-relief follow-up (i.e. 0 to <3, 3 to <6, 6 to <9, and 9–30 months), pre post 0 80 100 120 140 160 180 200 (a) (b) (c) (d) (e) (f) s y s to lic p re s s u re (m m h g) p < 0.0001 pre post 0 40 60 80 100 120 d ia s to lic p re s s u re (m m h g) p < 0.0001 pre post 0 60 80 100 120 140 m e a n a rt e ri a l p re s s u re (m m h g) p < 0.0001 pre post 0 110 120 130 140 s y s to lic p re s s u re (m m h g) p < 0.0001 pre post 0 60 70 80 90 d ia s to lic p re s s u re (m m h g) p < 0.0001 pre post 0 85 90 95 100 105 m e a n a rt e ri a l p re s s u re (m m h g) p < 0.0001 figure 3. matched systolic (a, b), diastolic (c, d), and mean arterial pressure (e, f) in hydronephrotic patients (n ¼ 49; male 49%; female 51%) before and after surgical management of ureteropelvic junction obstruction. upsala journal of medical sciences 219 a significant reduction of blood pressure was observed in all groups compared with pre-relief values. however, there were no differences among the groups (figure 5(b)). renal function linear regression analysis did not reveal any correlation between systolic, diastolic, or map before surgery and the split renal function of the hydronephrotic kidney (mag3) (supplementary figure s1(a–c), available online). in addition, there was no correlation between the changes in systolic, diastolic, or map following surgery and mag3 (supplementary figure s2(a–c), available online). further analysis, following separation by age groups (�30 and >30 years of age) at the time of surgical management did also not reveal any significant correlations between reduction of 0 80 100 120 140 160 180 200 s y s to lic p re s s u re (m m h g) pre post pre post < 30 years > 30 years p < 0.001 p < 0.001 p < 0.001 p < 0.001 0 60 70 80 90 100 110 120 130 140 m e a n a rt e ri a l p re s s u re (m m h g) pre post pre post < 30 years > 30 years p < 0.001 p < 0.001 p < 0.001 p < 0.001 0 50 60 70 80 90 100 110 120 130 d ia s to lic p re s s u re (m m h g) pre post pre post < 30 years > 30 years p < 0.001 p < 0.001 p < 0.001 p < 0.001 sys dia map sys dia map 0 5 10 15 20 d e lt a a rt e ri a l p re s s u re (m m h g) < 30 years > 30 years p = 0.08 (a) (b) (c) (d) figure 4. systolic (a), diastolic (b), and mean arterial pressure (c) and changes in arterial pressure (d) in hydronephrotic patients separated in age groups (i.e. aged �30 years, or aged >30 years) before and after surgical management of ureteropelvic junction obstruction. 0 3 6 9 12 15 18 21 24 27 30 −30 −20 −10 0 10 20 30 (a) (b) duration post-relief (months) d e lt a m e a n a rt e ri a l p re s s u re (m m h g) r2 = 0.004 0 < 3 3 < 6 6 < 9 9 30 −20 −15 −10 −5 0 duration post-relief (months) d e lt a m e a n a rt e ri a l p re s s u re (m m h g) * * * * n=13 n=13n=11 n=12 figure 5. impact of the length of the follow-up period before reassessing blood pressure after surgery. linear regression analysis did not show any significant correlation between the length of the postoperative period and the change in arterial pressure (a). significant reduction of blood pressure was observed in all groups compared with pre-relief values, independent of the length of the duration of the post-relief period, i.e. 0 to <3, 3 to <6, 6 to <9, and 9–30 months (b). no differences were observed among the different groups. �p < 0.05 compared with pre-relief; n ¼ number of patients in each group. 220 a. al-mashhadi et al. https://doi.org/10.1080/03009734.2018.1521890 https://doi.org/10.1080/03009734.2018.1521890 blood pressure and mag3 (supplementary figure s2(d–i), available online). discussion unilateral hydronephrosis appears to be sufficient to cause elevation of blood pressure, regardless of the presence of a normal contralateral kidney (3). some authors describe resolution of hypertension following removal of the affected kidney, while others—in agreement with our study—show that relief of obstruction may also lead to normalization of blood pressure. it therefore appears that the intrarenal mechanism leading to hypertension is reversible (3). there are only few previously published case reports on the reduction or normalization of blood pressure following relief of renal obstruction in adults with hydronephrosis (3,20). greminger et al. demonstrated that hypertension, associated with hydronephrosis, could be considered as a curable form of hypertension (21). in a case report by chalisey et al., a patient was described presenting with acute kidney injury and bilateral hydronephrosis secondary to pelvic malignancy. peripheral venous renin and aldosterone levels were elevated, and the patient was hypertensive. the high blood pressure reduced rapidly and was normalized 10 days following insertion of bilateral nephrostomies (22). so far, only the study by wanner et al. (4) addressed the question using a larger population. the authors found hypertension in 20% of 101 consecutive patients with unilateral hydronephrosis. these patients were followed for 35 months after surgery. hypertension was cured in 62% of all cases, improved in 19%, and left unchanged in only 19% of the subjects. the authors concluded that, in unilateral hydronephrosis, high blood pressure is reversible by surgery (4). in children with hydronephrosis with the same underlying pathology (i.e. upj obstruction), there are currently only three clinical studies that have investigated the effects of surgical relief of the obstruction on arterial pressure. the first study by de waard et al. was a retrospective investigation that suggested reduction of arterial pressure after surgical management of dilated or obstructed upper urinary tracts (17). the second and third studies (16), conducted by our research group, were prospective and demonstrated that elevated systolic and diastolic pressures before surgery were significantly reduced following surgical correction of hydronephrosis in children with upj obstruction (16). in the current study, we scrutinized all hydronephrotic patients (n ¼ 212) that were operated in uppsala university hospital between 2000 and 2016, but in the final analysis only those patients with unilateral hydronephrosis due to upj obstruction that were not previously on antihypertensive therapy and had no underlying chronic disease were included. the exclusion of patients already on medication was made since the relationship (if any) between hydronephrosis and hypertension in such patients is difficult to evaluate. moreover, it is very difficult to evaluate the short-term effect of obstruction relief on blood pressure if the patients are already on continuous antihypertensive treatment. we find it unlikely that this exclusion biases the results in a way that invalidates the conclusions made in this study. of note, none of the patients included in this study initiated antihypertensive treatment after surgery or developed any other diseases before post-relief values were obtained. life style questionnaires do not exist so we cannot exclude changes in their dietary habits or physical activity. however, no changes in bmi were observed between the preand post-relief examination. moreover, the retrospective nature of this study is associated with several limitations, e.g. our hospital is a tertiary center and patients come from other governorates’ hospitals. the evaluation and operation were performed at our clinical site, but the long-term follow-up of the patients was carried out in the governorates’ hospitals. our hospital digital patient record system is limited, and we cannot access all patients’ follow-up visits in the governorates’ hospital patient record system. this limitation explains why repeated blood pressure and mag3 data were not routinely obtained preand postoperatively for all the patients. the post-relief period in our study was variable, ranging from 0.5 to 30 months. long duration before reassessment of blood pressure may certainly impact on the interpretation of the surgical intervention, since changes in many other parameters may occur. however, only few patients in this study had a post-relief period of more than 12 months. we found a similar degree of blood pressure reduction in all groups when data had been divided into different follow-up duration periods, thus supporting the conclusion that our results can be linked with the surgical intervention performed. the current results are in agreement with previously mentioned clinical studies and case reports and also with our experimental research on hydronephrosis. we confirmed previously experimental (8,9,15) and clinical studies (16) that hydronephrosis is associated with elevated arterial pressure, which can be reduced by surgical relief. the current study also indicates that recurrence of obstruction, following surgical management of the hydronephrosis, was associated with elevated arterial pressure, and that the pressure was again reduced after relief of the obstruction. this finding gives further support to our hypothesis about the causal link between upj obstruction and elevation of the arterial pressure. findings demonstrating that the function of the hydronephrotic kidney is rather well preserved in young children (5–7) have led in recent decades to a shift towards non-surgical management of unilateral upj obstruction. a recent systematic review by weitz et al. of non-surgical management of hydronephrosis could not resolve the ongoing controversy, and the authors concluded that recommendations cannot be made in favor of or against the non-surgical treatment of upj obstruction in children (23). as pointed out by the authors, in the short term approximately 80% of children with upj obstruction may be treated safely (at least regarding split renal function) with non-surgical management, but the follow-up periods available were too short to evaluate the long-term consequences. against this background, the evidence for effective non-surgical management in children with unilateral upj obstruction needs to be upsala journal of medical sciences 221 https://doi.org/10.1080/03009734.2018.1521890 critically assessed, accompanied by serial functional imaging with radiation exposure, anesthesia and/or analgosedation, which could potentially lead to serious adverse effects. in a study by menon et al. (24) in children with hydronephrosis and less than 20% renal function in the affected unit only three out of 62 patients developed hypertension, which in two cases resolved after surgery. furthermore, they showed that no case of hypertension occurred in this group of patients without signs of obstruction after surgery, during the long-term follow-up (up to 8 years). in our current study, we tried to investigate if there is a correlation between renal function results according to mag3 and blood pressure changes after relief of the obstruction. based on findings in our current study, it was clear that in spite of preserved split renal function of the hydronephrotic kidney, the arterial pressure was elevated. moreover, there was no correlation between mag3 split renal function and the reduction in arterial pressure after relief of the obstruction. it has, however, previously been established that children with reflux nephropathy, i.e. renal parenchymal defects associated with vesicoureteral reflux, have an increased risk for hypertension (25). what is not known is whether this risk pertains to all scintigraphic uptake defects or only those that are due to acquired renal damage (‘scarring’) as opposed to congenital hypoplasias—since these subgroups are difficult or impossible to differentiate clinically. the same ambiguities pertain to children with hydronephrosis. the current retrospective study does not provide evidence regarding the mechanism, but in several previous experimental studies we have investigated underlying mechanisms contributing to hydronephrosis-induced hypertension. these include increased adenosine receptor-mediated contraction in the kidney, activation of renin-angiotensin-aldosterone system, enhanced renal sympathetic nerve activity (8,11,13), together with altered prostaglandin and thromboxane signaling (26,27), oxidative stress, and impaired nitric oxide signaling in the affected kidney (10–13). in support of these findings, our recent clinical study also demonstrated that children with hydronephrosis have abnormal prostaglandin and thromboxane signaling, oxidative stress, nitric oxide homeostasis (18), and increased plasmin levels in the urine (28). in conclusion, this study demonstrates that hydronephrosis in adult patients is associated with arterial pressure that is higher before relief of the renal obstruction if compared with values after relief of the obstruction. importantly, higher arterial pressure, or the reduction of arterial pressure following surgery, did not significantly correlate with split renal function of the hydronephrotic kidney. although future prospective studies using gold standard 24-h ambulatory blood pressure measurements with validated devices are warranted (29,30), we propose that arterial pressure measurements should be done routinely and frequently in patients with dilated or obstructed upper urinary tracts. any sign of high arterial pressure (i.e. equal to or above 140/90 mmhg) in the absence of other chronic disorders should indicate the need for surgery. there are health benefits even with reduction of blood pressure from high normal to population median values. our study suggests, but does not prove, that the urologist and nephrologist may have to pay more attention to the risk of development of high blood pressure in patients with hydronephrosis. the clinical importance of the current finding is that surgical management may prevent the development of chronic hypertension and associated comorbidities in patients with hydronephrosis. ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee (epn; protocol number 2017/017, uppsala, sweden), and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. disclosure statement the authors report no conflicts of interest. funding this work was supported by grants from the swedish research council [2016-01381 mc, 65x-03522-43-3 aegp], the swedish heart and lung foundation [20140448 mc, 20170124 mc], by research funds from the karolinska institutet [2-560/2015 mc], and her highness the crown princess lovisa’s fund for scientific research. notes on contributors ammar al-mashhadi is consultant at the pediatric surgery section, department of women’s and children’s health, uppsala university, uppsala, sweden. his research is focused on the link between hydronephrosis and development of hypertension, and was published in j pediatr urol, am j physiol renal physiol and pediatr nephrol. michael h€aggman is consultant at the department of surgical sciences, urology, uppsala university, uppsala, sweden. his research was published in various journals, including j urology, scand j urology, british j urology, eur urology and n engl j med. g€oran l€ackgren is professor at uppsala university, department of women’s and children’s health, and consultant at the pediatric surgery section, akademiska hospital, uppsala, sweden. his research is focused on urogenital disorders including vesicoureteral reflux and unilateral congenital hydronephrosis, and was published in various journals, including pediatric radiology, journal of pediatric urology, j urology, journal of urology, urology; he is a contributor and reviewer for journals, including curr urol rep, indian j urol and urology. sam ladjevardi is consultant at the department of surgical sciences, urology, uppsala university, uppsala, sweden. his research was published in various journals, including acta radiol, j urology, scand j urol, eur urol. 222 a. al-mashhadi et al. tryggve nev�eus is associate professor at uppsala university, department of women’s and children’s health, and consultant at the pediatric nephrology unit, akademiska hospital, uppsala, sweden. his research involves studies of nocturnal enuresis and is published in various journals, including journal of pediatric urology, pediatric nephrology, pediatric radiology, acta paediatrica, british j. urology, j urology, pediatrics, scand j urol nephrol; he is a contributor and reviewer for journals including nat clin pract urol and curr opin pediatr. arne stenberg is associate professor at uppsala university, department of women’s and children’s health, and consultant at the pediatric surgery section, akademiska hospital, uppsala, sweden. his research is focused on urogenital disorders including unilateral congenital hydronephrosis, and is published in various journals, including journal of pediatric urology, journal of urology, pediatric nephrology, therapeutic advances in urology. a. erik g. persson is emeritus professor at uppsala university, department of medical cell biology, uppsala, sweden. his research is focused on renal mechanisms contributing to the development and progression of arterial hypertension, including abnormal tubuloglomerular feedback response. his findings are published in various journals, including kidney international, hypertension, am j physiol renal physiol, cardiovascular research. he has contributed with review articles for acta physiol (oxf), curr opin nephrol hypertens and cardiovascular research. mattias carlstr€om is associate professor of physiology at the karolinska institutet, department of physiology and pharmacology. his research is focused on reno-cardio-metabolic interaction in health and disease, and his findings are published in various journals, including pnas, antioxidant redox signaling, free radical biology medicine, hypertension, cardiovascular research, circulation and diabetologia. he has contributed with review articles for cell metabolism, physiological reviews, journal of internal medicine, cardiovascular research and molecular aspects of medicine. orcid ammar al-mashhadi http://orcid.org/0000-0001-9578-1783 tryggve nev�eus http://orcid.org/0000-0002-4590-4957 mattias carlstr€om http://orcid.org/0000-0001-9923-8729 references 1. 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arterial pressure renal function discussion ethical approval disclosure statement funding notes on contributors references equal palliative care for patients with copd? a nationwide register study article equal palliative care for patients with copd? a nationwide register study ingela henocha,b , peter strangc,d, claes-g€oran l€ofdahle,f and ann ekberg-janssong,h adepartment of research and development, angered local hospital, gothenburg, sweden; bthe sahlgrenska academy, institute of health and care sciences, university of gothenburg, sweden; cdepartment of oncology-pathology, karolinska institutet, stockholm, sweden; dstockholms sjukhem foundation�s research and development unit, stockholm, sweden; euniversity of lund, sweden; fcopd center, institute of medicine, sahlgrenska university hospital, university of gothenburg, sweden; gdepartment of research and development, region halland, sweden; hthe sahlgrenska academy, institute of medicine, university of gothenburg, sweden abstract background: although chronic obstructive pulmonary disease (copd) is a life-limiting disease with a significant symptom burden, the patients are more often referred to nursing homes (nh), than to specialist palliative care (spc) at the end of life (eol). this study aimed to compare patients with copd in spc with those in nh and to compare the care provided. methods: a national register study was carried out where the swedish national airway register and the swedish register of palliative care were merged. copd patients who died in nhs or short-term facilities were included in the nh group (n ¼ 415) and those who died in spc were included in the spc group (n ¼ 355). demographic and clinical variables were included from the swedish national airway register and variables concerning eol care from the swedish register of palliative care. results: symptom prevalence was similar in nhs and spc, but symptom assessment (32% vs 20%), symptom relief medication (93-98% in spc vs 74-90% in nh), eol discussions (88% vs 66%), and bereavement support (94% vs 67%) were more likely in spc (in all comparisons p < 0.001). younger age and co-habiting increased the probability of dying in spc (p < 0.001). conclusion: despite similar symptom prevalence, older persons are more likely to be referred to nhs. if applying a palliative care philosophy in nhs, routine symptom assessment and prescription of rescue medication for frequent symptoms, would be more likely. promoting advance care planning and eol discussions at an earlier stage would result in more prepared patients and families. article history received 12 december 2018 revised 22 january 2019 accepted 19 february 2019 keywords palliative care; chronic obstructive pulmonary disease; symptoms; advance care planning; register study introduction chronic obstructive pulmonary disease (copd), a progressive and life-limiting disease, is predicted to be the third-leading cause of death by the year of 2020, globally (1). despite troublesome symptoms in advanced stages, copd still receives considerably less attention in specialized palliative care (spc) services, than does cancer (2,3). prominent symptoms in copd include an increasing number of exacerbations with pneumonia (4), anxiety (5), and depression (4,6), which is associated with impaired prognosis (7). the patients also exhibit increasing dyspnea, partly due to respiratory failure (4,8–10), decreased exercise capacity (4), low bmi (8), dry mouth (10), cough (10), sleep problems (10), and pain (10). moreover, copd patients are often affected by comorbidities (11), such as heart failure, thrombo-embolic episodes, osteoporosis and renal failure. in copd, physical symptoms and psychological wellbeing interact, as breathlessness and anxiety often appear in a cycle; breathlessness triggers anxiety, and anxiety triggers breathlessness (12). pharmacologic respiratory treatment of the disease can positively influence severe dyspnea, and, in the most severe cases, when combined with opioids, can decrease breathlessness (13,14). severe copd also has psychological, social and existential consequences, such as social isolation, loss of hope, and a struggle to maintain meaning in life (12). in a review, hrqol in palliative care patients was related to symptom burden and health care issues, but also to cognitive, emotional, social, and spiritual aspects, and to issues about personal autonomy, and preparedness (15). most of these aspects are highly relevant to patients with copd and their families. the latter aspects, i.e., awareness and preparedness, are central to palliative and end of life (eol) care. however, there are several barriers to promoting these. in a survey of respiratory physicians, only one-third regularly discussed palliative care issues with their patients (16). given the gradual progression and the prognostic uncertainty of these individuals (17), health care professionals might be unaware of the patient with copd being in the palliative phase, which may result in limited planning and provision of palliative care (18). the consequences of this non-awareness contact ingela henoch ingela.henoch@gu.se sahlgrenska academy at the university of gothenburg, institute of health and care sciences, box 457, se405 30 gothenburg, sweden � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 2, 140–147 https://doi.org/10.1080/03009734.2019.1586803 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1586803&domain=pdf&date_stamp=2019-05-31 http://orcid.org/0000-0002-1987-5419 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2019.1586803 http://www.tandfonline.com and organizational barriers, may result in a reluctance to initiate eol discussions or advance care planning (18). as copd is a life-limiting disease, associated with a significant symptom burden and comorbidities, these patients should be considered as candidates for specialist palliative care (spc) at the eol, as spc home care and in-patient care units have special attention on symptom control, as well as on psychosocial and existential support to patients and their families. in sweden, spc is provided by palliative home care teams or by palliative in-patient care units. palliative home care teams often co-operate with hospitals which provide disease-modifying treatments whereas the home care team is responsible for symptom control, support, management of nutrition, follow-up visits and so on. typically, the length of care is for some months. the palliative in-patient care facilities are mainly used in the dying phase for the last 2–3 weeks of life. obviously, spc units are experts on symptom control and support. still, we know that a considerable number of copd patients are referred to nursing homes. for the above reasons, the aim of this research was to compare patients diagnosed with copd treated in specialized palliative care with those in nursing homes, regarding the following research questions: � what are the characteristics of patients diagnosed with copd who received specialized palliative care compared to patients cared for in nursing homes? � what are the characteristics of the care provided to patients diagnosed with copd in specialized palliative care and in nursing homes? � is the provided care equal in quality and equity regardless of place of care? � what predicts health care admission to specialized palliative care or nursing home care in the eol care of patients with copd? methods this is a register study including two national quality registers, the swedish national airway register and swedish register of palliative care (srpc). the swedish national airway register is a register of patients diagnosed with either copd or asthma, where registrations of each patient visit were made by healthcare professionals in out-patient units, mostly primary healthcare and only a minority, 14%, in specialized pulmonary clinics. registrations concerned demographic, clinical, and patient-reported variables. in the present study, the last registrations were identified. in the srpc, healthcare professionals at the unit where the patients had died made registrations of demographic and clinical characteristics of the patients, place of death, and some characteristics of the eol care, and symptoms in the last week of life. sample an extract from the swedish national airway register was obtained in 2016 and a total of 20 668 patients diagnosed with copd were identified, with each attending at least one visit. of these, deceased patients were identified through the swedish tax agency, and we found 3113 patients who had died. the identities of the deceased patients were crossreferenced with the identities of patients in the srpc and 1842 of the deceased patients were also found there. the two databases were then merged. the patients whose place of death was a nursing home (n ¼ 279) or short-term service for older people (n ¼ 136) were identified and merged to one group called the “nursing home group” (n ¼ 415). the patients who died in specialized in-patient palliative care (n ¼ 252) or in their homes with support from specialized palliative home health care (n ¼ 103) were merged to one group called the “specialized palliative care group” (n ¼ 355). variables the variables from the swedish national airway register related to demographic characteristics, i.e., age, gender, and social situation. clinical characteristics related to stage of copd according to the gold stages (1), number of exacerbations the last 12 months, number of hospitalizations due to copd in the last 12 months, fev1% of predicted value, comorbidities, and exercise capacity, measured by the number of days per week that the patient had been physically active. patient-reported variables comprised smoking habits, divided into non-smokers, ex-smokers, and still smokers. dyspnea was measured by the modified medical research council (mmrc) dyspnea scale (19), which is a patient-rated, single-item scale where severity of the dyspnea experience is reported, ranging from 0, corresponding to “not troubled by breathlessness except on strenuous exercise”, to 4, corresponding to “breathless when dressing or undressing”. hrqol was measured by the clinical copd questionnaire (ccq) (20), a patient-rated questionnaire with ten items, measuring dyspnea at rest, dyspnea during physical activities, cough, phlegm, how concerned the patient is about the dyspnea; and how the dyspnea had limited the patient’s activities, i.e., strenuous physical activities, moderate physical activities, daily activities, and social activities. all items are scored by the patient from 0, “never”, to 6, which corresponds with “almost all the time”. a mean score of 4.0–6.0 is interpreted as a large or very large impact on hrqol; 2.0–3.9 corresponds with moderate impact on hrqol, and 0.0–1.9, no or small impact on hrqol. minimal clinical important difference in the ccq was found to be 0.4 (21). in later registrations, health status was measured by the copd assessment test (cat) (22). the cat consists of eight items relating to cough, phlegm, pressure on chest, dyspnea on exertion, limitations in performing activities, risks due to lung condition, sleep quality, and level of energy. all items are scored by the patients from 0, “without problems”, to 5, which corresponds with “severe problems”. all scores of the items are added together to obtain a single score ranging from 0 to 40. from the srpc, the following variables were retrieved: whether death was expected; whether the patient was able upsala journal of medical sciences 141 to express his/her wishes the last part of life; whether the place of death was preferred by the patient; whether anyone was present at death; and whether there had been any eol discussion about the impending death with either the patient or the family; whether the family was invited to participate in the after-death dialogue; and whether members of other professions were consulted. data about symptoms during the latter part of life were also retrieved, such as presence of pressure ulcers, symptom assessments, symptoms prevalence during the last week of life, assessments made, prescribed medications, and data on whether the symptom was alleviated. the following symptoms: pain, rattle, nausea, anxiety, dyspnea, and delirium, were registered. data analysis descriptive statistics were used for describing the sample and their registered treatments, with mean values and standard deviations (sd) calculated for continuous variables and numbers and percentages of the total sample for categorical variables. t-test was used to make comparisons between continuous variables. relationships between dichotomous categorical experience and treatment variables and age, gender, and social situation were analyzed with the mantelhaenszel chi-square test. in order to explore predictors of place of death, we performed bivariate logistic regression analyses with a dependent variable with nursing homes as place of death scored as 0 and specialized palliative care as place of death scored as 1. independent variables were: age; gender; social situation; fev1% of predicted value; number of exacerbations in the last 12 months; number of hospital admissions due to copd in the last 12 months; bmi; exercise capacity; smoking; hrqol measured by ccq; dyspnea measured by mmrc; and comorbidities. secondly, independent variables that significantly predicted the dependent variable with p < 0.20 in the bivariate analyses were entered in the multivariate stepwise logistic regression analyses with the same dependent variable. table 1. differences in patients who died in either specialized palliative care or in nursing homes (n ¼ 770). patients receiving specialized palliative care, in-patient or in their own home (n ¼ 355) nursing home and short-term facilities (n ¼ 415) p-value, diff between sp pall care and nursing homemean (sd) mean (sd) time between last visit and death, days 662.6 (518.5) 618.9 (521.4) 0.24 demographic variables age 73.9 (8.0) 79.3 (7.7) <0.001 n (%) n (%) gender men 168 (47.3%) 204 (49.2%) 0.61 women 187 (52.7%) 211 (50.8%) social situation: living alone 48 (32.9%) 68 (56.7%) <0.001 co-habiting 98 (67.1%) 52 (43.3) clinical variables n (%) n (%) stage of copd 0.28 i: (80-100% of predicted fev1) 11 (4.3%) 6 (2.7%) ii: (50-79% of predicted fev1) 86 (33.6%) 73 (32.7%) iii: (30-49% of predicted fev1) 103 (40.2%) 98 (43.9%) iv: (0-29% of predicted fev1) 56 (21.9%) 43 (19.3%) mean (sd) mean (sd) number of exacerbations in the last 12 months 1.37 (2.06) 1.24 (2.04) 0.48 number of hospitalizations in the last 12 months 0.62 (1.30) 0.76 (1.82) 0.32 fev% of predicted value 43.50 (18.42) 43.52 (16.65) 0.99 exercise capacity 2.42 (2.79) 1.79 (2.58) 0.012 patient -reported variables n (%) n (%) smoking: non-smoker 15 (4.7%) 40 (13.5%) <0.001 have quit smoking 205 (64.9%) 188 (63.5%) still smoking 96 (30.4%) 68 (23%) mean (sd) mean (sd) dyspnea 2.61 (1.30) 2.80 (1.23) 0.113 hrqol (ccq) 2.29 (1.34) (n ¼ 150) 2.41 (1.22) (n ¼ 124) 0.42 hrqol (cat) 17.95 (7.61) (n ¼ 59) 17.78 (6.90) (n ¼ 45) 0.91 comorbidity n (%) n (%) heart failure 45 (18.4%) 92 (36.7%) <0.001 ischemic heart disease 60 (24.4%) 102 (39.8%) <0.001 stroke 18 (8.9%) 21 (11.5%) 0.39 hypertension 118 (45.0%) 173 (60.3%) <0.001 diabetes 35 (13.3%) 62 (24.7%) 0.001 osteoporosis 39 (17.9%) 57 (26.9%) 0.025 depression/anxiety 65 (25.0%) 88 (33.3%) 0.036 lung cancer 19 (9.5%) 17 (8.9%) 0.84 alpha-1-antitrypsin deficiency 3 (1.7%) 0 0.094 t-test was used for comparisons between continuous variables, chi 2 for comparisons between categorical, variables. 142 i. henoch et al. results in total, 355 and 415 patients admitted to specialized palliative care (spc) and nursing homes (nh), respectively, were included in the study. differences between patient groups patients receiving spc were significantly younger (mean 73.9 vs 79.3 years, respectively) and more often co-habiting than nh patients (table 1). patients in specialized palliative care were also to a larger extent still smokers (30.4% vs 23.0%, p < 0.001). nh patients had significantly more comorbidities than patients in spc, i.e., a significantly higher proportion were affected by heart failure, ischemic heart disease, hypertension, diabetes, osteoporosis, and depression (p ¼ 0.036 – 0.001, see table 1). characteristics and content of care: symptom management there were no significant differences in symptom prevalence between spc and nhs for any of the symptoms of pain, death rattle, nausea, anxiety, dyspnea, or delirium (table 2). neither was there a difference as regards the prevalence of pressure ulcers at admission or at death. there were, however, significant differences for other parameters and activities. compared to nh patients, spc table 2. characteristics of symptom experience, assessment, and management registered in specialized palliative care and in nursing homes. palliative care characteristics patients receiving specialized palliative care (n ¼ 355) nursing home and short-term facilities (n ¼ 415) p-value, diff between sp pall care and nursing home pressure ulcer on admission 0.53 no pressure ulcer 285 (85.3%) 292 (85.1%) grade 1 21 (6.3%) 19 (5.5%) grade 2 17 (5.1%) 14 (4.1%) grade 3 7 (2.1%) 11 (3.2%) grade 4 4 (1.2%) 7 (2.0%) pressure ulcer at death 0.88 no pressure ulcer 242 (71.6%) 255 (72.9%) grade 1 43 (12.7%) 41 (1.7%) grade 2 37 (10.9%) 25 (7.1%) grade 3 11 (3.3%) 17 (4.9%) grade 4 5 (1.5%) 12 (3.4%) symptoms or assessments during the last week of life mouth health assessment 253 (78.6%) 193 (62.9%) <0.001 symptom assessment other than pain 101 (32.0%) 67 (20.5%) 0.001 pain, prevalence 254 (73.0%) 240 (69.8%) 0.36 pain assessment 184 (54.1%) 116 (34.3%) <0.001 prescribed rescue medication for pain 344 (98.3%) 317 (90.3%) <0.001 pain alleviated 0.74 totally 213 (80.4%) 199 (81.6%) partly 52 (19.6%) 45 (18.4%) not at all 0 0 rattle, prevalence 204 (58.3%) 183 (53.5%) 0.206 prescribed rescue medication for rattle 336 (96.3%) 316 (89.8%) 0.001 rattle alleviated 0.88 totally 113 (52.8%) 98 (52.4%) partly 90 (42.1%) 84 (44.9%) not at all 11 (5.1%) 5 (2.7%) nausea, prevalence 45 (13.0%) 47 (14.2%) 0.21 prescribed rescue medication for nausea 324 (92.6%) 259 (74.0%) <0.001 nausea alleviated 0.117 totally 41 (69.5%) 27 (52.9%) partly 15 (25.4%) 23 (45.1%) not at all 3 (5.1%) 1 (2.0%) anxiety, prevalence 194 (57.2%) 201 (62.2%) 0.19 prescribed rescue medication for anxiety 344 (98.3%) 311 (88.4%) <0.001 anxiety alleviated 0.49 totally 142 (68.6%) 134 (65.4%) partly 65 (31.4%) 71 (34.6%) not at all 0 0 dyspnea, prevalence 179 (52.2%) 150 (44.9%) 0.058 dyspnea alleviated 0.060 totally 83 (43.7%) 48 (31.4%) partly 104 (54.7%) 101 (66.0%) not at all 3 (1.6%) 4 (2.6%) delirium, prevalence 77 (22.7%) 85 (26.5%) 0.26 delirium alleviated 0.59 totally 30 (33.0%) 23 (26.1%) partly 49 (53.8%) 51 (58.0%) not at all 12 (13.2%) 14 (15.9%) upsala journal of medical sciences 143 patients were more likely to receive symptom assessments during their last week of life as regards mouth health (p < 0.001), pain (p < 0.001), and assessment of symptoms other than pain (p < 0.001). they were also more likely to have prescriptions of rescue medications for pain (opioids), nausea (mainly metoclopramide or haloperidol), anxiety (benzodiazepines), and death rattle (anti-cholinergic medication, e.g., glycopyrronium bromide) (p < 0.001). characteristics and content of care: psychosocial and existential aspects according to the health care staff�s own registrations, death was, to a larger extent, expected more in spc than in nh (98.3% vs 84.5%, p < 0.001) and spc was more often the preferred place of death according to the patients (77.2% vs 62.0%, respectively, p < 0.001). eol discussions were, to larger extent, held in spc (88.1 vs 66.2%, p < 0.001), and dialogues and eol discussions with relatives were more often held in spc, both about changes in care and transition to eol palliative care (p < 0.001, table 3). relatives in spc settings were more often present at the time of death (65.3% vs 41%, respectively). they were also offered bereavement follow-up more often. health care staff at spc units were more satisfied with the care they had been able to provide. predictors of place of care in the bivariate logistic regression analyses the following variables became predictors with p < 0.20: age (p < 0.001), social situation (p < 0.001), bmi (p ¼ 0.015), exercise capacity (p ¼ 0.013), smoking (p ¼ 0.001 and p < 0.001), heart failure (p < 0.001), ischemic heart disease (p < 0.001), hypertension (p < 0.001), diabetes (p ¼ 0.001), osteoporosis (p ¼ 0.026), and depression/anxiety (p ¼ 0.036) (table 4). in the multivariate stepwise logistic regression analysis, younger age and cohabiting increased the probability of dying in spc. table 3. characteristics of palliative care registered in specialized palliative care and in nursing homes. (chi-square). palliative care characteristics patients receiving specialized palliative care (n ¼ 355) nursing home and short-term facilities (n ¼ 415) p-value, diff between sp pall care and nursing home death was expected 349 (98.3%) 337 (84.5%) <0.001 could the patient express his/ her wishes? <0.001 yes, all the time 44 (12.7%) 61 (18.2%) lost ability express hours before death 146 (42.2%) 102 (30.4%) lost ability express days before death 143 (41.3%) 119 (35.5%) lost ability to express weeks before death 12 (3.5%) 27 (8.1%) lost ability to express months before death 1 (0.3%) 26 (7.8%) (eol) communication 280 (88.1%) 210 (66.2%) <0.001 patients’ preferred place of death? <0.001 yes 234 (77.7%) 176 (62.0%) do not know 50 (16.6%) 93 (32.7%) no 17 (5.6%) 15 (5.3%) anyone present at time of death <0.001 none 68 (19.2%) 88 (21.2%) relatives 169 (47.6%) 109 (26.3%) relatives and hcp 63 (17.7%) 61 (14.7%) hcp 52 (14.6%) 153 (36.9%) do not know 3 (0.8%) 4 (1.0%) (eol) communication with relatives/family <0.001 have no relatives yes 3 (0.9%) 8 (2.3%) no 297 (88.4%) 242 (69.3%) do not know 27 (8.0%) 73 (20.9%) family invited to bereavement dialogue 9 (2.7%) 26 (7.4%) <0.001 yes 329 (93.5%) 271 (66.6%) no 10 (2.8%) 74 (18.2%) do not know 13 (3.7%) 62 (15.2%) parenteral or enteral infusion of fluids last 24 hours 0.65 no 314 (93.5%) 336 (96.3%) yes 21 (6.3%) 9 (2.6%) do not know 1 (0.3%) 4 (1.1%) consultation with pain unit 2 (0.6%) 4 (1.0%) 0.53 consultation with palliative care team 17 (4.8%) 37 (8.9%) 0.025 consultation with other hospital unit 12 (3.4%) 22 (5.3%) 0.20 contact with physiotherapists, occupational therapists and social workers 4 (1.1%) 10 (2.4%) 0.18 spiritual counselor 5 (1.4%) 2 (0.5%) 0.18 144 i. henoch et al. discussion despite similar prevalence of symptoms, some patients were referred to spc, whereas others were admitted to nhs. a higher mean age for persons residing in nhs, when compared to patients cared for at spc, is a common finding, as there is a tendency to refer frail older patients with multiple illnesses, to geriatric services or to nhs. however, if aiming at equal and equitable care, the main focus should be on actual care needs. in our register study, the symptom prevalence was similar in spc and nhs. moreover, nh patients had a higher prevalence of comorbidities, including cardio-vascular diseases. regardless, they were admitted to nhs, which constitute a lower level of care in sweden. most of the staff at spc units in sweden are registered nurses or physicians, whereas a majority of the staff at nhs are assistant nurses. our results support the results from cohen et al., who analyzed place of death for patients diagnosed with lung cancer and patients diagnosed with copd in fourteen countries (23). compared to patients diagnosed with lung cancer, patients affected by copd were less likely to die at home or in a specialist palliative care facility, but were more likely to die in hospital or to be referred to nhs (23). the reasons for the possible inequality as regards referral to spc cannot be revealed from our register study. although we have measurements of symptom prevalence, we do lack data on symptom severity and complexity, which are important factors. one possible reason for the differences that we found might be that patients affected by more intense symptoms, or symptoms that were more difficult to alleviate, were admitted to spc. as the proportion of relieved symptoms was similar in spc and nh despite a great difference in the formal level of competence, this might be an indication of a difference in the initial symptom intensity and complexity. if so, the patients were referred to an adequate level of care. however, another explanation might be that older, frail people with multiple illnesses are routinely referred to nhs, without sufficient consideration of the actual symptom burden (24). in a qualitative study, barriers to admitting copd patients to palliative care were, among others, found to be that exacerbations and death in copd are unpredictable, that professional caregivers lack a coherent and proactive plan and have insufficient experience, and that they hold a negative view of palliative care for end-stage copd (25). moreover, there may be a vague or insufficient communication between patients and professional caregivers about care possibilities for end-stage copd (25). this, together with our findings, indicate that there is a need to discuss palliative care with patients and relatives before routinely admitting patients to nh. although symptom prevalence and the proportion of alleviated symptoms were similar, there was a great difference in awareness and in use of a holistic palliative care approach as recommended in the who definition of palliative care (26). the staff�s awareness of the impending death was higher in the spc units, which translated into pre-emptive table 4. logistic regression with nursing home or specialized palliative care as place of death as dependent variable. nursing home as reference variable, i.e. 0 and specialized palliative care as 1. specialized palliative care as place of death compared to nursing home bivariate multivariate independent variables or (95% ci) p or (95% ci) p constant 1171.14 p < 0.001 time between last visit and death 1.00 (1.00, 1.00) 0.24 age, divided by 10 years 0.42 (0.34, 0.51) <0.001 0.36 (0.22, 0.61) <0.001 gender (1¼ man, 2 ¼ woman) 0.93 (0.70, 1.23) 0.61 social situation (0¼ living alone; 1¼ co-habiting) 2.67 (1.62, 4.40) <0.001 4.57 (2.17, 9.62) <0.001 fev1% of predicted value 1.00 (0.99, 1.01) 0.99 number of exacerbations in the last 12 months 1.03 (0.95, 1.12) 0.47 number of hospitalizations in the last 12 months 0.94 (0.84, 1.06) 0.32 bmi 1.02 (0.99, 1.06) 0.15 exercise capacity 1.09 (1.02, 1.17) 0.013 smoking: non-smoker 1 have quit smoking 2.91 (1.56, 5.44) 0.001 still smoking 3.77 (1.93, 7.34) <0.001 quality of life (ccq) (0-6) 1.00 (0.95, 1.06) 0.90 dyspnea (mmrc) (0-4) 1 0.98 (0.38, 2.51) 0.97 0.96 (0.37, 2.49) 0.94 0.58 (0.23, 1.44) 0.24 0.72 (0.30, 1.76) 0.48 comorbidity heart failure 0.39 (0.26, 0.59) <0.001 ischemic heart disease 0.49, (0.33, 0.72) <0.001 stroke 0.75 (0.38, 1.45) 0.39 hypertension 0.54 (0.38, 0.76) <0.001 diabetes 0.47 (0.30, 0.74) 0.001 osteoporosis 0.59 (0.37, 0.94) 0.026 depression/anxiety 0.67 (0.46, 0.98) 0.036 lung cancer 1.08 (0.54, 2.12) 0.84 upsala journal of medical sciences 145 measures, not only in symptom management, but also as regards psychosocial and existential domains. prn rescue medications for pain, nausea, anxiety, and death rattles were generously prescribed for patients at spc units. moreover, eol discussions with patients and relatives were performed to a higher extent. for this reason, it is likely that the relatives were more prepared of the impending death, were present when their loved ones died, and were offered bereavement follow-up meetings more frequently. this is perhaps the greatest difference in the care delivered: whereas traditional care is focused on day-to-day care, the palliative care approach and the palliative care philosophy underline the importance of preparing for a good death. not only in physical terms, but also as regards the psychological, social, and existential/spiritual domains, both for the patient and his or her family (27). the results of our study indicate that health care staff should make informed decisions when referring patients with diagnosis other than cancer to eol care. despite having a similar prevalence of symptoms but a higher proportion of co-morbidities, older patients were still referred to nhs. judging from the frequency of successful symptom relief, nhs probably constitute an adequate level of care for a proportion of elderly copd patients. however, the palliative care approach, including a higher awareness, would probably further increase their outcomes. if a conscious palliative care philosophy is also applied at nhs for patients who have prognostic uncertainty, symptom assessment of common symptoms, as well as prescription of rescue medication for frequent symptoms would be more likely, as a result. moreover, it would be possible to initiate advance care planning and eol discussions at an earlier stage, which would likely result in well-informed and more prepared patients. this would be an example of effective resource utilization. limitations to our study include its retrospective approach. the strengths are that the swedish national airway register includes a representative number of patients diagnosed with copd, with different severities and about two-thirds of deaths in sweden are registered in the srpc. however, merging two registers resulted in a substantial number of missing participants. increasing the number of patients registered in both registers will improve the data quality in subsequent studies. we conclude that despite similar symptom prevalence, older persons are more likely to be referred to nhs. if applying a palliative care philosophy in nhs, routine symptom assessment and prescription of rescue medication for frequent symptoms, would be more likely. promoting advance care planning and eol discussions at an earlier stage would result in more prepared patients and families. disclosure statement no potential conflict of interest was reported by the authors. notes on contributors ingela henoch, rn, phd, associate professor, researcher at angered hospital and senior lecturer at sahlgrenska academy, institute of health and care sciences, university of gothenburg, sweden. peter strang, md, phd, karolinska institutet, professor at department of oncology-pathology, stockholm, and researcher at stockholms sjukhem foundation research and development unit, stockholm, sweden. claes-g€oran l€ofdahl, md, phd, senior professor at university of lund and at copd center, institute of medicine, sahlgrenska university hospital, university of gothenburg, sweden. ann ekberg-jansson, md, phd, research manager at department of research and development, region halland and associate professor at the sahlgrenska academy, institute of medicine, university of gothenburg, sweden. orcid ingela henoch http://orcid.org/0000-0002-1987-5419 references 1. global strategy for the diagnosis mapoc. global initiative for chronic obstructive lung disease (gold). volume 2017, 2017. 2. faes k, de frene v, cohen j, annemans l. resource use and health care costs of copd patients at the end of life: a systematic review. j pain symptom manage. 2016;52:588–99. 3. gore jm, brophy cj, greenstone ma. how well do we care for patients with end stage chronic obstructive pulmonary disease (copd)? a comparison of palliative care and quality of life in copd and lung cancer. thorax 2000;55:1000–6. 4. henoch i, strang s, lofdahl cg, ekberg-jansson a. health-related quality of life in a nationwide cohort of patients with copd related to other characteristics. eur clin respir j. 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duostim – a reproducible strategy to obtain more oocytes and competent embryos in a short time-frame aimed at fertility preservation and ivf purposes. a systematic review review article duostim – a reproducible strategy to obtain more oocytes and competent embryos in a short time-frame aimed at fertility preservation and ivf purposes. a systematic review alberto vaiarellia, danilo cimadomoa, cecilia petrigliab, alessandro confortic, carlo alviggic, nicol�o ubaldid, sergio leddae, susanna ferreroa, laura rienzia and filippo maria ubaldia aclinica valle giulia, g.en.e.r.a. centers for reproductive medicine, rome, italy; bdepartment of obstetrics and gynecology, university of cagliari, policlinico universitario duilio casula, monserrato, cagliari, italy; cdepartment of neuroscience, reproductive science and odontostomatology, university of naples federico ii, naples, italy; dcatholic university of the sacred heart, rome, italy; edepartment of veterinary medicine, university of sassari, sassari, italy abstract recent evidence suggests that follicular development occurs in a wave-like model during the ovarian cycle, where up to three cohorts of follicles are recruited to complete folliculogenesis. this understanding overtakes the previous dogma stating that follicles grow only during the follicular phase of the menstrual cycle. therefore, in in vitro fertilization (ivf), novel protocols regarding ovarian stimulation have been theorized based on the use of gonadotrophins to prompt the growth of antral follicles at any stage of the menstrual cycle. these unconventional protocols for ovarian stimulation aim at a more efficient management of poor-prognosis patients, otherwise exposed to conflicting outcomes after conventional approaches. duostim appears among these unconventional stimulation protocols as one of the most promising. it combines two consecutive stimulations in the follicular and luteal phases of the same ovarian cycle, aimed at increasing the number of oocytes retrieved and embryos produced in the short time-frame. this protocol has been suggested for the treatment of all conditions requiring a maximal and urgent exploitation of the ovarian reserve, such as oncological patients and poor responders at an advanced maternal age. at present, data from independent studies have outlined the consistency and reproducibility of this approach, which might also reduce the drop-out between consecutive failed ivf cycles in poor-prognosis patients. however, the protocol must be standardized, and more robust studies and cost-benefit analyses are needed to highlight the true clinical pros and cons deriving from duostim implementation in ivf. article history received 8 november 2019 accepted 21 february 2020 keywords advanced maternal age; bologna criteria; double ovarian stimulation; fertility preservation; duostim; pgt; poor-prognosis patients; poor-responder patients; reduced ovarian reserve introduction innovations implemented in in vitro fertilization (ivf) such as blastocyst culture, single embryo transfer (set), cryopreservation, and preimplantation genetic testing for aneuploidies (pgt-a) represent important advances in our clinical practice for the management of infertile couples (1). the cryopreservation of gametes and embryos in particular has become fundamental in each treatment, the influence of which has been further boosted by the safety and efficiency of vitrification protocols. in fact, this method ensures higher cryo-survival rates compared with slow-freezing at any stage of preimplantation development (2). all efforts invested in refining ivf during the last decades have aimed at improving its efficacy (number of children born per intention-to-treat) and efficiency (time, drop-out, and risks related to each treatment). with regard to this, an individualized approach (according to each couple’s specific characteristics) has become pivotal for many ivf specialists. if, on the one hand, patients with expected high or normal response to the ovarian stimulation might benefit from validated and reproducible strategies, on the other, the management of poor-prognosis patients is still challenging (3,4). this latter category embraces both advanced maternal age and poorresponder patients. the assessment of the predicted response to controlled ovarian stimulation (cos) is therefore crucial for the personalization of the treatment and to accurately estimate chances of success and inherent risks in addition to complications. currently, the tailoring of cos is based on: (i) different daily doses and type of gonadotrophins; (ii) the use of gnrh antagonists or agonists to inhibit the luteinizing hormone (lh) peak; (iii) the kind of medications chosen to trigger final oocyte maturation (hgc or gnrh agonist); (iv) the application of fresh or cryopreserved embryo transfer (et) policy; and (v) whether embryo selection is conducted through pgt-a or solely morphological/ morphokinetic criteria. nevertheless, although several strategies have been proposed, aiming at a maximization of ovarian response and success in poor-prognosis patients (in contact alberto vaiarelli alberto.vaiarelli@gmail.com g.en.e.r.a. centers for reproductive medicine, via g. de notaris 2b, rome, 00197, italy � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 2, 121–130 https://doi.org/10.1080/03009734.2020.1734694 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1734694&domain=pdf&date_stamp=2020-05-21 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1734694 http://www.tandfonline.com particular poor responders), no standard management has yet been outlined for them. the evidence that multiple follicular waves can arise during a single ovarian cycle in humans (5) represented a novel model to describe human folliculogenesis and paved the way to the introduction of unconventional stimulation protocols to manage specific groups of infertile patients (6). the extreme dynamism of folliculogenesis overtakes the classic theory in which a single cohort of follicles starts growing after luteal regression. today, two more theories have been proposed: the first states that the follicles start growing and regress continuously during the ovarian cycle, and the second states that 2–3 cohorts of antral follicles are recruited in a single ovarian cycle according to the duration of the ovarian cycle. these theories supported the definition of four unconventional protocols for ovarian stimulation: (a) random start, in which cos is started at any phase of the ovarian cycle, a regimen common for fertility preservation purposes to minimize the time invested before starting oncological treatment; (b) late follicular phase stimulation (fps) in which the stimulation starts after the selection of the dominant follicle or immediately before ovulation in case of fertility preservation; (c) luteal phase stimulation (lps) in which cos begins with gonadotrophin administration between the 17th and the 21st day of the cycle, a strategy which has been proposed to patients with reduced ovarian reserve or previous cancellation due to no response; (d) double stimulation in the same ovarian cycle (duostim) which complements fps with lps in the same ovarian cycle, a strategy which has been proposed to poor-prognosis patients, especially due to reduced ovarian reserve and advanced maternal age, but also for fertility preservation purposes (7,8). duostim is useful to all patients who might benefit from increasing the number of oocytes retrieved to maximize the cumulative live birth rate (clbr) per intention-to-treat (itt) (9), that is, the current measure of success in ivf (10). likewise, duostim seems to reduce the time to obtain euploid blastocysts and, as bosch and colleagues suggested, avoid treatment discontinuation (11). the aim of this systematic review is to summarize the evidence already published on putative advantages and disadvantages of the duostim protocol for fertility preservation and ivf purposes. a glimpse of double stimulation in animal models ovarian follicular dynamics have been described in different large-animal models by daily transrectal ultrasound (12–14). along the inter-ovulatory interval, which varies according to the species (ranging from 17 to 28 days), 2–4 waves of follicular growth might emerge during both the follicular and luteal phase throughout the ovarian cycle. for instance, folliculogenesis in bovine has been extensively studied. a wave of follicular recruitment in cattle is characterized by the synchronous growth of several follicles followed by the selection of dominant ones and the consequent regression of the subordinates. during the oestrus cycle, usually two waves start on day 0 and day 10, but also three waves might be detected on day 0, day 9, and day 16. in the last decades, many authors started to routinely collect oocytes in vivo from bovine (once or twice a week). these oocyte retrievals can occur in the presence of the corpus luteum (cl) that in this species covers a large part of the oestrus cycle (16–17 days) (15–17). even in the luteal phase, the follicles are sensitive to exogenous gonadotrophins that determine an increase in their size and, if the cl is lysed through prostaglandin administration, ovulation can occur as well, and the oocytes might even undergo regular fertilization. many studies were therefore successfully carried out also in vitro and highlighted the fact that oocytes retrieved after lps might result in good-quality blastocysts and viable offspring after assisted reproductive technology (art) (18). also, in the horse, it is well known that follicles respond to gonadotrophins and are selected in the presence of cl. specifically, during the early gestation in mares (35–45 days post-fertilization), a 4–8-follicle wave starts to grow, stimulated by the secretion of equine chorionic gonadotrophin (ecg) produced by the endometrial cups (an early formation of the chorion placenta). as a consequence of the double activity of folliclestimulating hormone (fsh) and lh, the cg not only induces the follicular growth but also determines their luteinization, resulting in the formation of accessory cl. it is therefore clear that, in both cattle and horses, the follicles remain sensitive to gonadotrophins even though they are growing under high and/or long-lasting progesterone influence. moreover, no alteration of the follicular dynamics (e.g. no extension or alteration of the oestrus cycle and no ovarian pathologies) has been observed when the animals were left following their regular reproductive activity after several consecutive ovarian pick-ups (opu), including those resulting from lps (19). of note, in cattle, numerous consecutive opus are possible only thanks to the ablation of dominant follicles that would otherwise have negatively affected the recruitment of the new cohorts of antral follicles. interestingly, the presence of cl and high levels of progesterone seems to modulate the effect of gonadotropins. for instance, in sheep, progesterone has been proposed to be a key endocrine signal governing periodic increases in both serum fsh concentrations and number of follicular waves per cycle. whether these effects of luteal progesterone on antral follicle lifespan are local, systemic (i.e. mediated by changes in fsh/lh secretion), or both remains to be elucidated. hence, under the influence of luteal progesterone, the sensitivity of fsh-producing gonadotrophins to gnrh may increase, resulting in a higher secretion of fsh from the pituitary gland. moreover, circulating progesterone concentrations may dictate the clearance rate of circulating fsh (14). when is duostim indicated? oocyte cryopreservation is a great challenge for oncological patients urgently needing fertility preservation prior to undergoing chemotherapy and/or radiotherapy (20). in these patients, it is crucial to maximize the number of cryopreserved oocytes after cos in the short time-frame, to increase the chance of future conception(s). in this regard, an ideal number of oocytes to cryopreserve can be considered to be 122 a. vaiarelli et al. at least 10–15, mainly depending on maternal age (20–22). for this reason, a random start protocol is used to speed up fertility preservation and therefore reduce the delay to cancer treatment. such a protocol is possible since there is no need for ovarian–endometrial synchrony. however, in many patients, there are not enough oocytes collected to ensure reasonably good chances of pregnancy. based on this, when the time is limited and the oocytes collected from one stimulation are insufficient, duostim protocols might be discussed with the oncologist together with the patients as a valuable option for fertility preservation (23–26). duostim, combining two consecutive stimulations spanning a 5-day interval, has been put forward as a valuable opportunity also for the management of poor-prognosis patients such as women with reduced ovarian reserve and/or advanced maternal age. the aim then is to maximize the number of oocytes retrieved in a single ovarian cycle, or to rescue patients in whom no oocytes were retrieved or competent embryos were not produced after conventional fps (27–30). for these thorny populations of patients, there is insufficient evidence to outline an ideal management since, regardless of the cos protocol adopted, consistently low live birth rates have been reported. in fact, oocyte quantity and quality, which are both critical to increase clbr per itt, could have suffered a dramatic physiological decline in these women. of note, if ageing impairs oocyte competence due to insults such as mitochondria and cohesion dysfunction, shortening of the telomeres, and spindle instability (31), also ‘young’ oocytes suffer from impairments that shape the window of the woman’s fertility. specifically, the oocyte aneuploidy rate follows a u-shaped curve with its highest prevalence before menarche and just before menopause, and its lowest prevalence at the age of 25 years (32). conversely, the oocyte competence to develop as a blastocyst seems constant across the age range of the woman until the age of 40 years (33), when it abruptly decreases. both these curves outline a sharp increase in the aneuploid blastocyst rate, which from a rate of 25–30% in women younger than 35 years might reach rates higher than 90% in patients older than 42 years of age (34,35). these data affect also the choice of an effective cos strategy depending on the age range of the patient (<35 y, 35–40 y, or >40 y) (36). moreover, beyond being responsible for a decreased fertility, aneuploidies cause an increased prevalence of vital chromosomal abnormalities, increased miscarriage rates, as well as an increased prevalence of numerical chromosomal abnormalities in new-borns (31). nevertheless, no therapy is available at present to minimize the ageing-related damage listed above. the only available strategy is to compensate the physiological decline in oocyte and embryonic competence by collecting the highest possible number of mature oocytes (36,37). in our setting, the duostim protocol is always combined with pgt-a and single vitrified–warmed euploid blastocyst transfer, independently of the number and morphological quality of the embryos obtained after the two stimulations (38). the aim of this approach is to try to reduce both the frustrating reiterated implantation failures and the miscarriage rate after ivf (39,40). these aspects are especially crucial to reduce the drop-out in poor-prognosis patients (as for instance the patient fulfilling the bologna criteria) (41) without compromising the overall efficacy of treatment (42). performance of the duostim protocol the duostim protocol entails two consecutive stimulations in a single ovarian cycle with the intent to increase the number of oocytes retrieved and the blastocysts available for transfer or pgt. the protocol involves a pre-treatment with luteal oestradiol priming (4 mg/day of oestradiol valerate) on day 21 of the previous menstrual cycle to promote the synchronization and coordination of the follicular growth (43). transvaginal ultrasound and basal assessment of the ovaries are performed on day 2 to day 3 of the menstrual cycle, then luteal oestradiol priming is stopped, and fps is started with a fixed dose of recombinant fsh (r-fsh) 300 iu/day plus r-lh 150 iu/day for 4 days. follicular growth is monitored on day 5 and then every 2–3 days depending on the progress of the ovarian response. flexible gnrh antagonist is administered daily after the identification of a leading follicle with a diameter �13–14 mm in fps and lps until the day of ovulation trigger. the final maturation of oocytes is triggered with a subcutaneous bolus of buserelin (dose 0.5 ml) to reduce the time of luteolysis (44). egg retrieval is performed 35 h after the trigger. five days after the first retrieval, lps is started with the same protocol and daily dose regardless of the number of visible antral follicles. in our group we propose duostim protocol combined with pgt-a and single euploid vitrified–warmed blastocyst transfer. search procedure this systematic review was conducted by searching the medline (pubmed), scopus, and embase databases up to october 2019. combinations of the following keywords and search terms were used: ‘duostim’, ‘luteal phase stimulation’, ‘luteal phase ovarian stimulation’, ‘dual stimulation’, ‘double stimulation’, ‘ovarian stimulation’, ‘assisted reproductive technique’, ‘in vitro fertilization’. no time or language restriction was adopted, and queries were limited to human studies. the reference lists of relevant reviews and articles in press were also hand-searched. three reviewers (av, dc, ac) evaluated titles and abstracts. duplicates were removed using endnote online software and manually. disagreements were discussed and ultimately resolved by consensus between all authors with the involvement of the most experienced ones (ca, lr, fmu). we included all studies published that comprised case series and case reports in which two consecutive stimulations were performed in the same menstrual cycle in infertile women undergoing ivf or fertility preservation programmes. a total of 264 search items were identified. after removal of duplicates a total of 175 papers were scrutinised. fifty-one papers were assessed for eligibility. in the following review, we included 21 papers, while 30 were excluded because they were reviews, abstracts, or studies comparing fps and upsala journal of medical sciences 123 lps not in the same patient or ovarian cycle (figure 1). table 1 represents a summary of all the studies included in this systematic review and is presented as an electronical supplement. updated body of evidence regarding the clinical implementation of duostim the first case report of lps subsequent to fps was published in 2013 by xu and li. they conducted lps in a 41-year-old woman in whom no eggs were retrieved from a first oocyte pick-up (45). since then, several studies have been published of different design and number of patients undergoing double stimulation for either fertility preservation or ivf purposes. in 2014, kuang et al. (8) reported more opportunities for retrieving oocytes in a single month thanks to what is known as the ‘shanghai protocol’. according to this protocol, the first stimulation was a conventional fps, whereas lps started on the subsequent day of the first oocyte retrieval, when two or more antral follicles were identified. regarding these two stimulations, two different regimens were adopted: for fps, a combination of clomiphene citrate 25mg/day starting on day 3 of the cycle and until the triggering of ovulation, letrozole 2.5 mg/day starting on day 3 for a total of 4 days, and human menopausal gonadotrophin (hmg) 150 iu/day starting on day 6 and until the triggering of ovulation; for lps, letrozole 2.5 mg/day and hmg 225 iu/day, both starting from the day of first oocyte retrieval and until the second triggering of ovulation. medrossiprogesterone acetate (mpa) was also administered at the end. for both stimulations, ovulation was triggered with triptorelin 0.1 ml when follicular maturation was finally achieved (8). in 2016, wei et al. (46) confirmed the same results as those of kuang et al., with the same protocol adopted in patients aged >40y, with a prior history of poor response defined as �3 oocytes retrieved and an antral follicle count (afc) <6. in the same year, zhang et al. (47) showed, in a retrospective study based on 153 patients fulfilling the bologna criteria, that lps results in more cumulus–oocyte complexes, metaphase ii (mii) oocytes, and zygotes and that embryos obtained after lps are characterised by higher implantation rates. moreover, ubaldi et al. (38) published a proof of concept that defines the first application of duostim (gnrh antagonist protocol with a fixed r-fsh 300 iu/day dose combined with r-lh 75 iu/day in both fps and lps) in 51 poorprognosis patients (anti-m€ullerian hormone [amh] < 1.5 ng/ ml, afc <6 follicles, and/or <5 oocytes retrieved in previous ivf cycles) undergoing icsi and pgt-a. gnrh agonist trigger was then adopted with the aim of reducing the half-life of the cl after oocyte collection and facilitate the recruitment of follicles from the luteal wave. here, no statistically significant difference was found in terms of number of mii oocytes retrieved, fertilisation, blastocyst, and euploid blastocyst rates between fps and lps. as a consequence, duostim increased the final transferable blastocyst yield per ovarian cycle with respect to fps-only (38). these results were confirmed in a larger number of patients one year later (7). in 2017, two retrospective studies reported that double stimulation increases the number of oocytes retrieved in a short period of time: cardoso et al. (48) compared the conventional records iden�fied through database searching (n = 264 ) sc re en in g in cl ud ed el ig ib ili ty id en �fi ca �o n addi�onal records iden�fied through other sources (n = 2) records a�er duplicates removed (n = 89) records screened (n = 175) records excluded (n = 124) full-text ar�cles assessed for eligibility (n = 51) full-text ar�cles excluded (n = 30) studies included in qualita�ve synthesis (n = 21) figure 1. prisma 2009 flow diagram. 124 a. vaiarelli et al. ta b le 1. su m m ar y of th e cl in ic al ev id en ce p ro d uc ed vi a d ou b le st im ul at io n in th e sa m e ov ar ia n cy cl e to d at e. st ud y d es ig n in cl us io n cr it er ia n um b er of p at ie n ts fp s p ro to co l tr ig g er lp s p ro to co l to av oi d th e lh su rg e c on cl us io n s x u an d li (4 5) c as e re p or t po or -r es p on d er p at ie n t 41 y ol d 1 c c (5 0– 10 0 m g /d ay ) þ fs h 15 0 iu /d ay g n rh -a (t ri p to re lin 0. 2 m g ) an d h c g 10 ,0 00 iu c c (5 0– 10 0 m g /d ay ) þ fs h 15 0 iu /d ay ib up ro fe n lp s m ig h t re sc ue un su cc es sf ul oo cy te re tr ie va ls af te r fp s ku an g et al . (8 ) pi lo t a fc �6 ; �5 oo cy te s re tr ie ve d fr om a p re vi ou s cy cl e; h is to ry of ov ar ia n su rg er y; fs h le ve l: 10 – 19 iu /l ; m at er n al ag e �4 0 y 38 c c 25 m g /d ay ; le 2. 5 m g /d ay (4 d ay s) ; h m g 15 0 iu /d ay g n rh -a 0. 5 m g le 2. 5 m g /d ay þ h m g 22 5 iu /d ay ib up ro fe n 0. 6 g þ m pa h ig h er ch an ce to re tr ie ve oo cy te s in a si n g le ov ar ia n cy cl e m of fa t et al . (2 4) c om m en ta ry fe rt ili ty p re se rv at io n an d co n d it io n s re q ui ri n g la rg er co h or ts of oo cy te s n r rfs h 30 0 iu /d ay g n rh -a (t ri p to re lin 0. 2 m g ) rfs h 30 0 iu /d ay g n rh -a n t in cr ea se d n um b er of oo cy te s co lle ct ed in le ss th an 30 d ay s w it h a p at ie n tfr ie n d ly ap p ro ac h u b al d i et al . (3 8) pr oo f of co n ce p t a m h �1 .5 n g /m l; a fc �6 ; �5 oo cy te s re tr ie ve d fr om a p re vi ou s cy cl e; m at er n al ag e �3 5 y 51 rfs h 30 0 iu /d ay þ rlh 75 iu /d ay g n rh -a (b us er el in 50 iu ) rfs h 30 0 iu /d ay þ rlh 75 iu /d ay g n rh -a n t lp s in cr ea se d th e n um b er of p at ie n ts p ro d uc in g at le as t 1 eu p lo id b la st oc ys t an d un d er g oi n g a tr an sf er in a si n g le ov ar ia n cy cl e w ei et al . (4 6) re tr os p ec ti ve m at er n al ag e > 40 y; p ri or h is to ry of p oo r re sp on se ; �3 oo cy te s re tr ie ve d ; a fc < 6. 23 c c 50 m g /d ay þ le 2. 5 m g /d ay (5 d ay s) þ h m g 15 0 iu /d ay g n rh -a 0. 1 m g (f ps ) an d h c g 10 ,0 00 iu (l ps ) c c 50 m g /d ay þ le 2. 5 m g /d ay (5 d ay s) þ h m g 15 0 iu /d ay m pa m or e oo cy te s co lle ct ed af te r lp s w it h re sp ec t to fp s zh an g et al . (4 7) re tr os p ec ti ve po r d ef in ed ac co rd in g to th e bo lo g n a cr it er ia 15 3 c c 50 m g /d ay þ h pfs h 15 0 iu /d ay g n rh -a (t ri p to re lin 0. 2 m g ) c c 50 m g /d ay þ h pfs h 15 0– 22 5 iu /d ay ib up ro fe n 30 0 m g ev er y 6 h fr om g n rh -a in je ct io n to th e d ay of fo lli cl e as p ir at io n lp s re su lt s in m or e c o c , m ii, an d zy g ot es ; lp sd er iv ed em b ry os re su lt ed in h ig h er ir ts am p ra s et al . (2 3) pi lo t fe rt ili ty p re se rv at io n in on co lo g ic al p at ie n ts 10 on co lo g ic al p at ie n ts h m g 15 0– 45 0 iu /d ay h c g 50 00 iu h m g 15 0– 45 0 iu /d ay g n rh -a n t in cr ea se d n um b er of oo cy te s vi tr ifi ed af te r c o s w it h n o d el ay in st ar ti n g ch em ot h er ap y v ai ar el li et al . (7 ) o b se rv at io n al a m h �1 .5 n g /m l; a fc �6 ; �5 oo cy te s re tr ie ve d fr om a p re vi ou s cy cl e; m at er n al ag e �3 5 y 12 8 rfs h 30 0 iu /d ay þ rlh 75 – 15 0 iu /d ay g n rh -a (b us er el in 50 iu ) rfs h 30 0 iu /d ay þ rlh 75 – 15 0 iu /d ay g n rh -a n t n o d iff er en ce in th e em b ry ol og ic al ou tc om es b et w ee n fp s an d lp s (f er ti liz at io n , b la st ul at io n , an d eu p lo id y ra te s) c ar d os o et al . (4 8) re tr os p ec ti ve pr ev io us ly fa ile d iv f tr ea tm en t( s) 13 rfs h 22 5 iu / d ay þ h m g 75 iu /d ay g n rh -a (t ri p to re lin 0. 2 m g ) rfs h 22 5 iu /d ay þ h m g 75 iu /d ay g n rh -a n t h ig h er n um b er of oo cy te s in a si n g le ov ar ia n cy cl e li u et al . (4 9) c as eco n tr ol w om en �3 8 y 11 6 rfs h 15 0– 30 0 iu / d ay þ rlh 75 – 15 0 iu /d ay rh c g 25 0 m g h m g 22 5 iu /d ay lo n g ag on is t (1 3p z) ; sh or t ag on is t (2 7p z) ; g n rh -a n t (5 3p z) ; m pa (2 3p z) a h ig h er n um b er of oo cy te s in a si n g le ov ar ia n cy cl e c im ad om o et al . (5 2) pa ir ed ca se -c on tr ol st ud y a m h �1 .5 n g /m l; a fc �6 ; �5 oo cy te s re tr ie ve d fr om a p re vi ou s cy cl e; m at er n al ag e �3 5 y 18 8 rfs h 30 0 iu /d ay þ rlh 75 – 15 0 iu /d ay g n rh -a (b us er el in 50 iu ) rfs h 30 0 iu /d ay rlh 75 – 15 0 iu /d ay g n rh -a n t lp s g en er at es la rg er co h or ts of oo cy te s w it h co m p ar ab le d ev el op m en ta l an d ch ro m os om al co m p et en ce th an p ai re d -f ps d er iv ed on es zh an g et al . (7 4) re tr os p ec ti ve po or re sp on d er s fu lfi lli n g th e bo lo g n a cr it er ia b 61 c c 50 – 10 0 iu /d ay (5 d ay s) þ h m g 75 – 15 0 iu /d ay rh c g 25 0 m g c c 50 – 10 0 iu /d ay (5 d ay s) þ h m g 75 – 15 0 iu /d ay d uf as to n m or e oo cy te s re tr ie ve d b ut lo w er m ii ra te af te r lp s th an af te r fp s; si m ila r lb r an d c pr ra sh ti an an d zh an g (5 1) re tr os p ec ti ve fs h le ve l > 15 iu /m l; a fc 1– 8; 1 p re vi ou s fa ile d co n ve n ti on al iv f cy cl e 69 c c 50 m g /d ay þ le 2. 5 m g /d ay (5 d ay s) þ fs h 75 iu /d ay g n rh -a (f ps )/ rh c g (l ps ) c c 50 m g /d ay þ le 2. 5 m g /d ay (5 d ay s) þ fs h 75 iu /d ay g n rh -a n t n o d iff er en ce in th e n um b er of oo cy te s re tr ie ve d b et w ee n fp s an d lp s (c on ti nu ed ) upsala journal of medical sciences 125 ta b le 1. c on ti n ue d . st ud y d es ig n in cl us io n cr it er ia n um b er of p at ie n ts fp s p ro to co l tr ig g er lp s p ro to co l to av oi d th e lh su rg e c on cl us io n s m ad an i et al . (5 3) pr os p ec ti ve po or re sp on d er s fu lfi lli n g th e bo lo g n a cr it er ia b 10 4 c c 25 m g /d ay þ le 2. 5 m g /d ay (4 d ay s) þ h m g 15 0 iu /d ay g n rh -a le 2. 5 m g /d ay þ h m g 22 5 iu /d ay ib up ro fe n 0. 6 g þ m pa fe rt ili za ti on ra te an d n um b er of fr oz en em b ry os h ig h er af te r fp s th an lp s; d ou b le st im ul at io n is a ti m esa vi n g an d p at ie n tfr ie n d ly re g im en ji n et al . (5 0) re tr os p ec ti ve po or re sp on d er s ac co rd in g to bo lo g n a cr it er ia b 76 c c 50 m g /d ay þ le 10 0 m g /d ay or 5 m g /d ay (5 d ay s) þ h m g 15 0– 30 0 iu /d ay g n rh -a (t ri p to re lin 0. 1 m g ) or h c g 50 00 – 10 ,0 00 iu c c 50 – 10 0 m g / d ay þ h m g 15 0– 30 0 iu /d ay g n rh -a n t m or e oo cy te s an d em b ry os ob ta in ed , as w el l as lo w er ca n ce lla ti on ra te w it h in an ov ar ia n cy cl e w it h d ou b le st im ul at io n v ai ar el li et al . (2 7) m ul ti ce n te r ob se rv at io n al a m h �1 .5 n g /m l; a fc �6 ; �5 oo cy te s re tr ie ve d fr om a p re vi ou s cy cl e; m at er n al ag e �3 5 y 31 0 rfs h 30 0 iu /d ay þ rlh 15 0 iu /d ay g n rh -a (b us er el in 50 iu ) rfs h 30 0 iu /d ay þ rlh 15 0 iu /d ay g n rh -a n t h ig h er ra te of p at ie n ts ob ta in in g at le as t 1 eu p lo id b la st oc ys t in a si n g le ov ar ia n cy cl e th an ks to th e co n tr ib ut io n of lp s si g h in ol fi et al . (2 5) c om m en ta ry fe rt ili ty p re se rv at io n in on co lo g ic al p at ie n ts n r rfs h 20 0 iu /d ay or h m g 20 0 iu /d ay g n rh -a rfs h 30 0 iu /d ay þ rlh 75 iu /d ay g n rh -a n t in cr ea se d n um b er of oo cy te s co lle ct ed an d vi tr ifi ed in a sh or t ti m efr am e li n et al . (7 5) pi lo t po or re sp on d er s fu lfi lli n g th e bo lo g n a cr it er ia b 60 h m g 22 5 iu /d ay þ c c 10 0 iu /d ay rh c g þ g n rh -a n t rfs h 30 0 iu /d ay þ rlh 15 0 iu /d ay m pa 10 m g m or e oo cy te s an d d ay -3 em b ry os af te r lp s th an af te r fp s h at ir n az et al . (5 5) re tr os p ec ti ve st ud y po ic 51 si n g le d os e of rfs h 22 5 iu th e d ay of tr ig g er h c g 10 ,0 00 iu le 5 m g /d ay þ si n g le d os e of rfs h 22 5 iu th e d ay of tr ig g er h c g 10 ,0 00 iu d ua l st im ul at io n co m p ar ed to su b se q ue n t co n ve n ti on al st im ul at io n s re d uc ed th e n um b er of oo cy te re tr ie va ls p er fo rm ed to ob ta in at le as t 2 cl ea va g e st ag e em b ry os a ls b je rg et al . (5 4) c as e se ri es po or re sp on d er s fu lfi lli n g th e bo lo g n a cr it er ia (< 42 y) b 54 c or ifo lli tr op in al fa þ rfs h 30 0– 37 5 iu /d ay o r rfs h 30 0 iu / d ay þ rlh 15 0 iu /d ay g n rh -a (f ps ) or h c g (l ps ) c or ifo lli tr op in -a lfa þ rfs h 30 0– 37 5 iu /d ay o r rfs h 30 0 iu /d ay þ rlh 15 0 iu /d ay g n rh -a n t m or e oo cy te s re tr ie ve d af te r lp s; lo w er ca n ce lla ti on ra te af te r d ou b le st im ul at io n v ai ar el li et al . (4 2) c as e se ri es po or re sp on d er s fu lfi lli n g th e bo lo g n a cr it er ia b 10 0 p at ie n ts ch oo si n g d uo st im ve rs us 19 4 ch oo si n g co n ve n ti on al c o s rfs h 30 0 iu /d ay þ rlh 15 0 iu /d ay g n rh -a (b us er el in 50 iu ) rfs h 30 0 iu /d ay þ rlh 15 0 iu /d ay g n rh -a n t d uo st im p re ve n ts d ro p -o ut af te r a fir st fa ile d at te m p t, th er eb y in cr ea si n g th e c lb r p er it t a lo n g ag on is t: co n tr ol le d ov ar ia n st im ul at io n p ro to co l w it h lo n g g n rh ag on is t; sh or t ag on is t: co n tr ol le d ov ar ia n st im ul at io n p ro to co l w it h sh or t g n rh ag on is t. b po or re sp on d er s (f ul fil lin g bo lo g n a cr it er ia ): ac co rd in g to th e bo lo g n a cr it er ia th e p at ie n ts sh ou ld h av e at le as t tw o of th es e ch ar ac te ri st ic s: (i) m at er n al ag e (� 40 ye ar s) ; (ii ) a p re vi ou s ov ar ia n re sp on se �3 oo cy te s w it h a co n ve n ti on al st im ul at io n p ro to co l; (ii i) an ab n or m al ov ar ia n re se rv e te st (i. e. , a fc 5– 7 fo lli cl es or a m h 0. 5– 1. 1 n g /m l) . c p re m at ur e ov ar ia n in su ff ic ie n cy (p o i) d ef in ed as fs h le ve ls h ig h er th an 40 iu /l , up to tw o ov ar ia n fo lli cl es (2 – 9 m m ) at th e b as el in e p el vi c sc an , p re se n ce of ol ig om en or rh ea /a m en or rh ea , an d lo w le ve ls of a m h < 0. 30 p g /m l. a fc : an tr al fo lli cl e co un t; a m h : an ti -m € ul le ri an h or m on e; c c : cl om ip h en e ci tr at e; c lb r: cu m ul at iv e lb r; c o c : cu m ul us oo cy te co m p le x; c o s: co n tr ol le d ov ar ia n st im ul at io n ; c pr : cu m ul at iv e p re g n an cy ; fp s: fo lli cu la r p h as e st im ul at io n ; fs h : fo lli cl est im ul at in g h or m on e; g n : g on ad ot ro p h in ; g n rh -a : g n rh ag on is t tr ig g er ; g n rh -a n t: co n tr ol le d ov ar ia n st im ul at io n p ro to co l w it h an ta g on is t p ro to co l; h c g : h um an ch or io n ic g on ad ot ro p h in ; h m g : h um an m en op au sa l g on ad ot ro p h in ; h pfs h : h ig h ly p ur ifi ed fs h ; ir : im p la n ta ti on ra te ; it t: in te n ti on -t otr ea t; iv f: in vi tr o fe rt ili sa ti on ; lb r: liv e b ir th ra te ; le : le tr oz ol e; lh : lu te in iz in g h or m on e; lp s: lu te al p h as e st im ul ati on ; m ii: m et ap h as e ii; m pa : m ed ro xy p ro g es te ro n e ac et at e; n r: n ot re p or te d ; po i: p re m at ur e ov ar ia n in su ff ic ie n cy ; rfs h : re co m b in an t fs h ; rh c g : re co m b in an t h c g ; rlh : re co m b in an t lh . 126 a. vaiarelli et al. antagonist protocol to duostim in 13 patients with a previous history of failed ivf treatments, while liu et al. (49) investigated the efficacy of duostim compared with fps-only in advanced maternal age patients (mean age: 42 ± 3 years). in both cases, twice as many embryos were obtained with duostim. in liu’s study also, the cancellation rate decreased from 37% to 18% with duostim. a further pilot study published in 2017 by tsampras et al. (23) tested the efficacy of double stimulation for fertility preservation in oncological patients. ten patients underwent double stimulation with gnrh antagonist and hmg protocol. this protocol increased the number of oocytes retrieved and consequently vitrified, without delaying cancer treatment. in 2018, another two studies (50,51) demonstrated how double stimulation could be effective in patients with poor ovarian reserve. jin et al. (50) in a retrospective study compared double stimulation (group a, n ¼ 76 poor responders) to lpsonly (group b) and to mild ovarian stimulation (group c). although after fps fewer oocytes were collected and fewer embryos produced in group a than in groups b and c, their overall numbers in a single ovarian cycle were significantly higher with the contribution of lps (50). rashtian and zhang assessed whether duostim in advanced maternal age patients (mean age: 42 years) might produce a higher number of oocytes compared with fps-only. in their study, 69 women with diminished ovarian response underwent a gnrh antagonist protocol with r-fsh, letrozole, and clomiphene citrate for both stimulations. the ovulation was triggered with gnrh agonist in fps and with hcg in lps. there was no statistically significant difference between the number of oocytes retrieved. therefore, the addition of lps to fps doubled the number of inseminated oocytes in a single ovarian cycle (51). two more studies were published in 2018: cimadomo et al. (52) reported that the cohorts of oocytes obtained after lps from 188 patients are larger than their paired-fpsderived cohorts and showed a comparable competence in terms of blastulation rate and euploidy rate. vaiarelli et al. (27), on the other hand, conducted a multicentre study which confirmed the reproducibility of the results with consistently superior outcomes utilising the duostim application in 310 poor-prognosis patients from four ivf centres. in particular, 65.5% of the patients obtained at least one euploid blastocyst after duostim rather than 42% if only fps had been carried out. in 2019, madani et al. (53) published a prospective clinical study based on the adoption of duostim to treat 121 patients fulfilling the bologna criteria. double stimulation was performed by letrozole, clomid, hmg, and gnrh agonist and was found to be a time-saving and patient-friendly regimen. alsbjerg et al. (54) reported a case series of 54 poor responders classified according to the bologna criteria (mean age: 37 years), who were treated with duostim performed with corifollitropin-alfa. also in this study, duostim was confirmed as a valuable alternative to conventional fps to increase the overall number of oocytes retrieved and decrease the risk for cycle cancellation. hatirnaz et al. (55) demonstrated in a retrospective study including 51 women that double stimulation is convenient in the management of patients with premature ovarian insufficiency (poi). they reported that double stimulation halves the number of oocyte retrievals required to obtain at least two transferable good-quality cleavage stage embryos compared with several consecutive conventional stimulations. in a recent observational study of patients fulfilling the bologna criteria, 100 out of 297 patients had agreed to undergo duostim after extensive counselling (42). in these couples, the clbr per itt was 15% in a single ovarian cycle, whereas the corresponding figure was 8% among the 197 patients choosing a conventional cos strategy and undergoing up to two oocyte retrievals in a 2-year period. in fact, only 17 patients not conceiving after a first failed attempt returned for a second one in the latter study arm. therefore, the authors underlined that duostim application in patients fulfilling the bologna criteria prevents cycle discontinuation, thereby conferring a higher chance to conceive in a shorter time-frame (42). furthermore, cimadomo et al. (56) identified the mirnomic signatures of the follicular fluids collected from 15 poor-prognosis patients after fps and paired-lps. no difference was reported, thereby further suggesting the safety of lps. lastly, an ongoing non-selection study, the interim analysis of which has been presented at the eshre annual meeting held in barcelona in 2018 (57), outlined the absence of differences in terms of obstetrical and neonatal outcomes between fps-derived and lps-derived live births. weaknesses, risks, and concerns related to duostim the personalisation of cos has represented a game-changer for the management of poor-prognosis patients undergoing ivf (29). clearly, duostim fits into this scenario, and its strengths and weaknesses must therefore be outlined. duostim still needs a cost-benefit analysis and a randomised controlled trial (rct) that compares it with consecutive conventional fps. more data regarding its safety from a biological, clinical, and neonatal perspective are still required. the mandatory need for a freeze-all strategy represents an inevitable limitation (27). then, a consensus regarding duostim protocol should be built in terms of timing, kind, and dosage of the medications adopted in the lps. the luteal phase is characterised by the presence of the cl, higher progesterone and oestrogen levels, and evidence demonstrating that, after gnrh agonist trigger in antagonist protocols, luteolysis differs greatly among patients and depends on: (i) levels of progesterone on the day of final oocyte maturation and oocyte retrieval; (ii) duration of ovarian stimulation; (iii) number of days of suppression; (iv) total dosage of the medications used for ovarian suppression; and (v) number of oocytes retrieved (44). therefore, the beginning of the menstruation following agonist triggering is to be considered patient-specific. the use of gonadotropins a few days after agonist trigger in the luteal phase of the ovarian cycle allows the rescue of small antral follicles that otherwise would have been lost because they are recruited from a physiologically anovulatory luteal wave (6,7). yet, the choice of the medications in a duostim protocol should be based on their effectiveness. although the shanghai protocol upsala journal of medical sciences 127 suggested the use of clomiphene citrate and letrozole in the fps, a cochrane review published in 2017 did not support their use, also due to an increased risk for cycle cancellation as well as for a lower number of oocytes retrieved in poor responders (58). similarly, several rcts (59–62) and metaanalyses (63–65) reported better efficacy in terms of oocyte retrieval after cos with r-fsh compared with hmg. moreover, the total amount of gonadotrophins used is lower when cos is performed with r-fsh compared with hmg (66,67). all these aspects must be considered when we treat poor-prognosis patients where even a single oocyte can make a huge difference in terms of cumulative results and cost-benefit (28). finally, the choice of a r-lh supplementation during duostim is based on its role in promoting steroidogenesis and folliculogenesis (68). indeed, lh increases androgen production, stimulates early stages of follicular growth, increases the recruitment of pre-antral and antral follicles, and increases the expression of fsh receptors in the granulosa cells (69). all these aspects are especially important in patients of advanced maternal age or poor/sub-optimal responders subject to ageing-derived reduction in androgen production and to inadequate levels of endogenous androgens, in turn associated with a decreased ovarian sensitivity and responsiveness to exogenous fsh (29,70,71). although there is no consensus about lh measurement and a correct therapeutic window, its supplementation in cos seems helpful for the treatment of advanced maternal age patients undergoing a gnrh antagonist protocol in terms of lower dose of r-fsh and higher ivf outcomes with no increase of the overall costs (72,73). all these issues indirectly related with duostim are still controversial and debated in the scientific community. conclusions the wave theory of follicle recruitment was developed in several animal models before it was confirmed in women. that evidence introduced important clinical implications for the personalized approach of cos in specific patients undergoing ivf. the exploitation of both the ovulatory (major) and the anovulatory (minor) waves has allowed the implementation of new unconventional cos protocols, among which duostim is one of the most promising, especially for the treatment of poor-prognosis patients (advanced maternal age and/or reduced ovarian response) and patients requiring fertility preservation for medical reasons. although the quality of the clinical studies focussed on the implementation of duostim is moderate–low, all of them highlighted that a double stimulation is a valid option to increase the number of oocytes/embryos in a single ovarian cycle. the lps-derived cohorts of oocytes were also larger than their paired-fpsderived ones while showing a comparable competence in terms of blastulation and euploidy rate. these results certainly support the important contribution lps has in poorprognosis and oncological patients, with a limited amount of time and insufficient numbers of oocytes to grant a reasonable chance of ivf success. nevertheless, additional clinical and basic research studies on this topic are needed to further encourage the personalisation of cos in specific populations of patients. disclosure statement av, fmu, lr, and ca report personal fees and honoraria outside the submitted work. dc, sf, ac, sl, nu, and cp have nothing to disclose. notes on contributors filippo maria ubaldi, md, phd, is the clinical director of the g.en.e.r.a. centres for reproductive medicine. laura rienzi, msc, is the laboratory director of the g.en.e.r.a. centres for reproductive medicine. danilo cimadomo, phd, is the scientific coordinator of the g.en.e.r.a. centres for reproductive medicine. alberto vaiarelli, md, phd, is a specialist in obstetrics and gynaecology and has a master degree in reproductive medicine, andrology, and ivf. he works at the g.en.e.r.a. centre for reproductive medicine of rome. carlo alviggi, md, phd, is associate professor in obstetrics and gynaecology at the department of neuroscience, reproductive science and odontostomatology, university federico ii of naples. alessandro conforti, md, phd, is a specialist in obstetrics and gynaecology at the department of neuroscience, reproductive science and odontostomatology, university federico ii of naples. sergio ledda, phd, is professor in veterinary medicine at the university of sassari. cecilia petriglia, md, is a resident in obstetrics and gynaecology at the university of cagliari, policlinico universitario duilio casula of monserrato. susanna ferrero, md, is a specialist in obstetrics and gynaecology. she works at the g.en.e.r.a. centre 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a, revelli a, et al. probabilistic cost-effectiveness analysis of controlled ovarian stimulation with recombinant fsh plus recombinant lh vs. human menopausal gonadotropin for women undergoing ivf. reprod biol endocrinol. 2018;16:68. 74. zhang w, wang m, wang s, bao h, qu e, zhang n, et al. luteal phase ovarian stimulation for poor ovarian responders. jbra assist reprod. 2018;22:193–98. 75. lin lt, vitale sg, chen sn, wen zh, tsai hw, chern cu, et al. luteal phase ovarian stimulation may improve oocyte retrieval and oocyte quality in poor ovarian responders undergoing in vitro fertilization: preliminary results from a single-center prospective pilot study. adv ther. 2018;35:847–56. 130 a. vaiarelli et al. abstract introduction a glimpse of double stimulation in animal models when is duostim indicated? performance of the duostim protocol search procedure updated body of evidence regarding the clinical implementation of duostim weaknesses, risks, and concerns related to duostim conclusions disclosure statement references tf-iups200019 211..216 original article using total plasma triacylglycerol to assess hepatic de novo lipogenesis as an alternative to vldl triacylglycerol leanne hodsona,b, sion a. parrya, thomas cornfielda, catriona charltona, wee suan lowa, charlotte j. greena and fredrik rosqvista,c aoxford centre for diabetes, endocrinology and metabolism, churchill hospital, university of oxford, oxford, uk; bnational institute for health research oxford biomedical research centre, oxford university hospitals foundation trust, oxford, uk; cdepartment of public health and caring sciences, clinical nutrition and metabolism, uppsala university, uppsala, sweden abstract background: hepatic de novo lipogenesis (dnl) is ideally measured in very low-density lipoprotein (vldl)-triacylglycerol (tag). in the fasting state, the majority of plasma tag typically represents vldltag; however, the merits of measuring dnl in total plasma tag have not been assessed. this study aimed to assess the performance of dnl measured in vldl-tag (dnlvldl-tag) compared to that measured in total plasma tag (dnlplasma-tag). methods: using deuterated water, newly synthesised palmitate was determined in fasting plasma vldl-tag and total tag in 63 subjects taking part in multiple studies resulting in n ¼ 123 assessments of dnl (%new palmitate of total palmitate). subjects were split into tertiles to investigate if dnlplasma-tag could correctly classify subjects having ‘high’ (top tertile) and ‘low’ (bottom tertile) dnl. repeatability was assessed in a subgroup (n ¼ 16) with repeat visits. results: dnlvldl-tag was 6.8% (iqr 3.6–10.7%) and dnlplasma-tag was 7.5% (iqr 4.0%�11.0%), and the correlation between the methods was rs ¼ 0.62 (p < 0.0001). bland–altman plots demonstrated similar performance (mean difference 0.81%, p ¼ 0.09); however, the agreement interval was wide (�9.6% to 11.2%). compared to dnlvldl-tag, 54% of subjects with low dnl were correctly classified, whilst 66% of subjects with high dnl were correctly classified using dnlplasma-tag. repeatability was acceptable (i.e. not different) at the group level, but the majority of subjects had an intra-individual variability over 25%. conclusion: dnl in total plasma tag performed similarly to dnl in vldl-tag at the group level, but there was large variability at the individual level. we suggest that plasma tag could be useful for comparing dnl between groups. article history received 20 february 2020 revised 2 march 2020 accepted 4 march 2020 keywords de novo lipogenesis; dnl; hepatic; human; triacylglycerol; vldl introduction de novo lipogenesis (dnl), the process whereby non-lipid precursors (e.g. sugar/carbohydrate) are synthesised to fat, primarily occurs in the liver in humans (1). it is often suggested that enhanced dnl is related to increased risk of cardiometabolic diseases including type 2 diabetes, nonalcoholic fatty liver disease (nafld), and insulin resistance (2–6). as the major end product of dnl is the saturated fatty acid palmitate, hepatic dnl is commonly inferred from fatty acid composition in various circulating lipid fractions (e.g. phospholipids and erythrocytes) (1–5). we recently demonstrated, in a large group of healthy men and women, that fatty acid markers were poor proxies for isotopically assessed fasting hepatic dnl during habitual dietary conditions (7). the gold standard method for measuring hepatic dnl in humans involves stable-isotope tracers, where the appearance of the stable-isotope label (from deuterated water [2h2o] or [ 13c]acetate) is measured in palmitate in very low-density lipoprotein (vldl) triacylglycerol (tag; equivalent to the commonly used term ‘triglycerides’) (8). however, the process of isolating vldl using ultracentrifugation is costly, time-consuming, has a low throughput, and can require up to 3 ml of plasma, depending on the ultracentrifugation method used. thus, it is not feasible to assess hepatic dnl in larger-scale studies using the gold standard method. a method whereby vldl isolation is not required could make the measurement of dnl more accessible. total plasma tag (instead of vldl-tag) is seldom used to assess dnl, but in the fasting state a large proportion (�60–75%) of plasma tag represents vldl-tag (9,10). thus, fasting dnl assessed in total plasma tag may be reflective of fasting dnl assessed in vldl-tag. using total plasma tag would offer several advantages (in terms of cost, throughput, plasma volume, and time), but a direct comparison between dnl assessed in total plasma tag and vldl-tag has not previously been performed. contact fredrik rosqvist fredrik.rosqvist@pubcare.uu.se department of public health and caring sciences, clinical nutrition and metabolism, uppsala university, uppsala 752 36, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 3, 211–216 https://doi.org/10.1080/03009734.2020.1739789 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1739789&domain=pdf&date_stamp=2020-07-08 http://orcid.org/0000-0002-8982-6129 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1739789 http://www.tandfonline.com here, we utilised stable-isotope methodology (deuterated water) to measure newly synthesised palmitate in vldl-tag (‘gold standard hepatic dnl’) and compared this to newly synthesised palmitate in total plasma tag (‘poor man’s dnl’). subjects and methods subjects subjects were recruited from the oxford biobank (www. oxfordbiobank.org.uk) or were from the oxfordshire area. all volunteers were non-diabetic and free from any known metabolic disease, were not taking medication known to affect lipid or glucose metabolism, and did not consume alcohol above recommended uk limits (�14 units per week; 1 unit equals 8 g of pure alcohol). the data and plasma used for the present analysis were from subjects taking part in published or ongoing trials with different primary aims (clinicaltrials.gov identifiers: nct03090347, nct02478541, nct01936779, and nct03145350). data and plasma from n ¼ 63 individuals (46 males, 17 females) were used. because most individuals (n ¼ 60; 44 males, 16 females) took part in two separate study visits (pre–post intervention) and data and plasma from both study visits were used when available, a total of n ¼ 123 data points were used for the current analysis. prior to study days subjects were encouraged to refrain from strenuous exercise and alcohol intake and to consume a low-fat meal the evening before measurement in order to standardise the last meal. subjects had fasted �10 h prior to collection of blood samples. a subgroup of n ¼ 16 subjects who were not involved in any intervention were asked to maintain their habitual lifestyle for 4 weeks before repeated measurements were performed. all studies were approved by the respective research ethics committees (reference numbers 12/sc/0267, 15/ns/0117, 17/ne/0031, and 16/nw/ 0751), and all subjects provided written informed consent. measurements of dnl deuterated water (3 g/kg body water) was administered the evening prior to measurement. a fasting whole-blood sample was collected the following morning, and plasma was immediately separated by centrifugation at 4 �c. separation of the vldl-rich fraction (sf20–400) was made by sequential flotation using density gradient ultracentrifugation (11,12). lipids in the vldl-rich fraction and total plasma were extracted using chloroform-methanol, and tag was separated by solidphase extraction (12). fatty acid methyl esters were prepared using methanolic sulphuric acid, and fatty acid relative abundance (mol%) was determined by gas chromatography (12). dnl was assessed based on the incorporation of deuterium in plasma water into vldl-tag palmitate and plasma tag palmitate using gc-ms, monitoring ions with mass-to-charge ratios of 270 (m þ 0) and 271 (m þ 1) (13). background isotopic enrichment in plasma water and vldl-tag was measured in a fasting blood sample taken before subjects consumed deuterated water. clinical chemistry blood was collected into heparinised tubes, and plasma was separated by centrifugation at 4 �c. plasma glucose (werfen, warrington, uk, 18250840; cv 1.3%), triacylglycerol (werfen, 18255640; cv 1.7%), total cholesterol (werfen, 18250540; cv 1.7%), and hdl cholesterol (randox, ch2652; cv 2.5%) were analysed enzymatically (ilab 600/650 clinical chemistry; werfen), and insulin was analysed by radioimmunoassay (millipore, uk). statistics data were analysed using jmp 13.1.0 (sas institute inc.). the correlation between the two assessments was analysed using spearman’s rank correlation. a bland–altman plot (difference plot) was used to analyse agreement between the two assessments and for analysing repeatability. subjects were split into tertiles (based on dnl) in order to investigate if dnl assessed in total plasma tag could correctly classify/distinguish subjects having ‘high’ (top tertile) and ‘low’ (bottom tertile) dnl. using tertiles was an arbitrary choice, but has the benefits of creating distinct groups without compromising group sizes too much. an alluvial plot was used to visualise misclassification when using total plasma tag. results characteristics of the 63 individuals are given in table 1. association and agreement between dnlvldl-tag and dnlplasma-tag the distribution of dnlvldl-tag and dnlplasma-tag was similar, with the medians being 6.8% (iqr 3.6 � 10.7%) and 7.5% (iqr 4.0�11.0%), respectively (figure 1). there was a strong positive correlation between the two methods (figure 2). using a bland–altman plot to compare the two methods, it was found that although the two methods performed similarly (mean difference 0.81%, p ¼ 0.09), the agreement interval was wide (�9.6 to 11.2%) (figure 3). we classified subjects into tertiles based on dnlvldl-tag and dnlplasma-tag and found the medians for the respective tertiles to be 3.0% (2.1�3.6%), 6.8% (6.0�8.2%), and 12.8% (10.6�16.5%) for dnlvldl-tag and 4.2% (2.3�6.8%), 6.0% (3.5�9.7%), and 11.4% (8.0�15.9%) for dnlplasma-tag (figure 4). although there was a strong positive correlation between table 1. subject characteristics. characteristics mean ± sd age, years 46 ± 6 sex, m/f 46/17 bmi 29.3 ± 3.9 glucose, mmol/l 5.5 ± 0.9 insulin, mu/l 13.5 ± 8.5 triglycerides, mmol/l 1.5 ± 0.9 total cholesterol, mmol/l 5.1 ± 1.0 hdl cholesterol, mmol/l 1.2 ± 0.4 data (mean ± sd) are based on baseline/first visit data (n ¼ 63); follow-up/second visit data were similar (data not shown). 212 l. hodson et al. http://www.oxfordbiobank.org.uk http://www.oxfordbiobank.org.uk methods, 50% of individuals were misclassified from their dnlvldl-tag tertile when classified using dnlplasma-tag. for the top tertile of dnlvldl-tag 66% (27/41) were correctly classified, whereas this number was 32% (13/41) for the middle tertile and 54% (22/41) for the bottom tertile (coloured points in figure 4). repeatability of dnlvldl-tag and dnlplasma-tag repeatability of dnlvldl-tag and dnlplasma-tag was assessed in 16 subjects (8 males, 8 females) not taking part in any intervention but with repeated measurements separated by 4 weeks. for dnlvldl-tag, the first and second measurements were similar (p ¼ 0.53) at the group level with a mean difference of 0.94%; however, 12/16 subjects had an intra-individual difference of more than 25% (figure 5(a,b)). for dnlplasma-tag, the first and second measurements were similar (p ¼ 0.44) at the group level with a mean difference of 1.8%; however, 13/16 subjects had an intra-individual difference of more than 25% (figure 5(c,d)). discussion we assessed the use of plasma tag to measure hepatic dnl and compared this to vldl-tag in a large sample of metabolically healthy adult humans. we found that plasma tag performed similarly in terms of mean and distribution compared to vldl-tag. although misclassification was evident at the individual level, plasma tag worked well for determining dnl at the group level and performed best in individuals having higher dnl. using a less intensive method of measuring hepatic dnl (based on plasma and not requiring ultracentrifugation for isolation of vldl) could aid our understanding of the role hepatic dnl has in health and disease. we have recently demonstrated that fatty acid composition of circulating tag was not a good reflection of isotopically measured dnl in healthy humans (7). therefore, in the present work, we used deuterated water to assess dnl in circulating total plasma tag and compared this with dnl in isolated vldl-tag. as the majority (�60–75%) of plasma tag in the fasting state typically is from vldl-tag (9,10), it would be reasonable to assume that dnl in plasma tag may be reflective of dnl in vldl-tag. using plasma tag instead of vldl-tag potentially increases the accessibility for mean±sd 8.1±6.0 median (iqr) 6.8 (3.6-10.7) mean±sd 8.9±7.0 median (iqr) 7.5 (4.0-11.0) dnl (%) vldl-tag dnl (%) plasma-tag figure 1. histograms showing the distributions, means, and medians of dnlvldl-tag and dnlplasma-tag. n ¼ 123. figure 2. correlation between dnlvldl-tag and dnlplasma-tag. n ¼ 123. figure 3. bland–altman plot showing the agreement between dnlvldl-tag and dnlplasma-tag. n ¼ 123. upsala journal of medical sciences 213 assessing hepatic dnl as well as making it cheaper, as vldltag isolation would not be required and plasma could be retrospectively analysed in batches instead of continuously during an ongoing study. compared to simple fatty acid markers of hepatic dnl (as reported in our previous paper [7]), the assessment reported here (dnlplasma-tag) showed dnl vldl-tag 3.0% (2.1-3.6%) dnl vldl-tag 6.8% (6.0-8.2%) dnl vldl-tag 12.8% (10.6-16.5%) dnl vldl-tag 4.2% (2.3-6.8%) dnl vldl-tag 6.0% (3.5-9.7%) dnl vldl-tag 11.4% (8.0-15.9%) t1 t2 t3 t1 t2 t3 ter�les dnl vldl-tag ter�les dnl plasma-tag figure 4. alluvial plot with subjects split into tertiles based on dnlvldl-tag and dnlplasma-tag, showing misclassification of subjects when using dnlplasma-tag. median (iqr) dnlvldl-tag is shown beside respective tertile. coloured dots represent composition of the tertile; e.g. red dots (subjects) in the top tertile of dnlplasma-tag actually belong to the middle tertile when using dnlvldl-tag. figure 5. repeatability of dnl measured in vldl-tag and plasma-tag. (a) bland-altman plot showing mean and difference of the two measurements of dnl in vldl-tag; (b) bland-altman plot showing mean and difference of the two measurements of dnl in plasma-tag; (c) matched-pair plot showing the individual differences between first and second measurement of dnl in vldl-tag; (d) matched-pair plot showing the individual differences between first and second measurement of dnl in plasma-tag. n ¼ 16. 214 l. hodson et al. 2–3-fold stronger associations with dnlvldl-tag. furthermore, the dnlplasma-tag and dnlvldl-tag assessments were shown to perform similarly in terms of mean (differing only 0.81%) and distribution of dnl. however, the agreement interval between the two assessments was wide (�21 percentage points), and overall 50% of subjects were misclassified using plasma tag, suggesting that plasma tag is not an optimal substitute and likely to be more useful for separating individuals with high and low dnl, rather than investigating subtle individual changes, such as those that may be observed in response to an intervention. when analysing repeatability in a subgroup, both dnlvldl-tag and dnlplasma-tag were found to be repeatable at the group level; however, the majority of subjects had a large or very large intra-individual variability. variability of dnl at the individual level has not previously been reported and may be due to both methodological and biological reasons, for example: (i) subjects did not receive a standardised diet the days preceding measurement, hence both macronutrient composition and energy balance were uncontrolled for; (ii) deuterium labelling may have been too low (increasing the noise) or too short (susceptible to intrahepatic fatty acid turnover) (6); and (iii) factors regulating intrahepatic fatty acid partitioning may have changed between measurements, potentially affecting the amount of secreted vldl-tag. it would be valuable to investigate intraindividual variability during more tightly controlled standardisation settings in order to disentangle methodological and biological reasons. in line with the above, another potential reason for the discrepancy between dnlvldl-tag and dnlplasma-tag is that the proportion of vldl-tag in total tag may differ between individuals; i.e. dnlplasma-tag should be more reflective of dnlvldl-tag in subjects where a larger proportion of plasma tag is vldl-tag. the proportion of vldl-tag in plasma tag would be an unknown factor in most cases and thus constitutes a potential limitation of using plasma tag. limitations of the current study include the relatively low number of subjects used for repeatability analyses as well as not controlling for background diet in this analysis. furthermore, we compared the assessments in the fasting state only, and further studies are needed to investigate if plasma tag could be used in the postprandial state. it could be speculated, however, that plasma tag may potentially underestimate dnl postprandially due to the presence of chylomicrons and chylomicron remnants in circulation. a strength of the study is the large sample size with isotopically measured hepatic dnl. furthermore, subjects were metabolically healthy and included both males and females. however, further work would need to be performed in patient populations (e.g. nafld) and across a wider phenotype. in conclusion, dnl measured in total plasma tag is more reliable than simple fatty acid markers and performed similarly to dnl measured in vldl-tag at the group level, but demonstrated large inconsistencies at the individual level. this simplified method could be useful for distinguishing between groups having higher and lower dnl (e.g. based on tertiles) but not used as a continuous variable. acknowledgements authors thank louise dennis, rachel craven-todd, and cru staff for excellent nursing provision, and niall dempster and jonathan hazlehurst for medical cover. this work was supported by the nihr biomedical research centre, oxford, and authors thank the volunteers from the oxford biobank, nihr oxford biomedical research centre, for their participation. the oxford biobank (www.oxfordbiobank.org.uk) is also part of the nihr national bioresource which supported the recalling process of the volunteers. a small number of samples analysed in this paper were from cjg’s novo nordisk postdoctoral fellowship run in partnership with the university of oxford. disclosure statement the authors report no conflicts of interest. funding fr is supported by henning and johan throne-holsts foundation, swedish society for medical research, swedish society of medicine, and the foundation blanceflor. lh is a british heart foundation senior research fellow in basic science (fs/15/56/31645). notes on contributors leanne hodson, phd is a professor of metabolic physiology at the oxford centre for diabetes, endocrinology and metabolism (ocdem), university of oxford, united kingdom. sion a. parry, phd is a researcher at the oxford centre for diabetes, endocrinology and metabolism (ocdem), university of oxford, united kingdom. thomas cornfield, bsc is a research technician at the oxford centre for diabetes, endocrinology and metabolism (ocdem), university of oxford, united kingdom. catriona charlton, bsc was a research technician at the oxford centre for diabetes, endocrinology and metabolism (ocdem), university of oxford, united kingdom at the time of the work, now a laboratory analyst at essentra laboratories. wee suan low, phd was a doctoral student at the oxford centre for diabetes, endocrinology and metabolism (ocdem), university of oxford, united kingdom at the time of the work, now a research fellow at a*star – agency for science, technology and research, singapore. charlotte j. green, phd was a researcher at the oxford centre for diabetes, endocrinology and metabolism (ocdem), university of oxford, united kingdom at the time of the work, now a scientific liaison manager at the university of dundee, united kingdom. fredrik rosqvist, phd was a postdoctoral researcher at the oxford centre for diabetes, endocrinology and metabolism (ocdem), university of oxford, united kingdom at the time of the work, now a researcher at the department of public health and caring sciences, clinical nutrition and metabolism, uppsala university, sweden. orcid fredrik rosqvist http://orcid.org/0000-0002-8982-6129 references 1. hellerstein mk, schwarz jm, neese ra. regulation of hepatic de novo lipogenesis in humans. annu rev nutr. 1996;16:523–57. 2. jacobs s, jager s, jansen e, peter a, stefan n, boeing h, et al. associations of erythrocyte fatty acids in the de novo lipogenesis upsala journal of medical sciences 215 http://www.oxfordbiobank.org.uk pathway with proxies of liver fat accumulation in the epicpotsdam study. plos one. 2015;10:e0127368. 3. ma w, wu jh, wang q, lemaitre rn, mukamal kj, djousse l, et al. prospective association of fatty acids in the de novo lipogenesis pathway with risk of type 2 diabetes: the cardiovascular health study. am j clin nutr. 2015;101:153–63. 4. wu jh, lemaitre rn, imamura f, king ib, song x, spiegelman d, et al. fatty acids in the de novo lipogenesis pathway and risk of coronary heart disease: the cardiovascular health study. am j clin nutr. 2011;94:431–8. 5. zong g, zhu j, sun l, ye x, lu l, jin q, et al. associations of erythrocyte fatty acids in the de novo lipogenesis pathway with risk of metabolic syndrome in a cohort study of middle-aged and older chinese. am j clin nutr. 2013;98:319–26. 6. smith gi, shankaran m, yoshino m, schweitzer gg, chondronikola m, beals jw, et al. insulin resistance drives hepatic de novo lipogenesis in nonalcoholic fatty liver disease. j clin invest. 2020;130: 1453–60. 7. rosqvist f, mcneil ca, pramfalk c, parry sa, low ws, cornfield t, et al. fasting hepatic de novo lipogenesis is not reliably assessed using circulating fatty acid markers. am j clin nutr. 2019;109: 260–8. 8. pinnick ke, gunn pj, hodson l. measuring human lipid metabolism using deuterium labeling: in vivo and in vitro protocols. methods mol biol. 2019;1862:83–96. 9. pramfalk c, pavlides m, banerjee r, mcneil ca, neubauer s, karpe f, et al. fasting plasma insulin concentrations are associated with changes in hepatic fatty acid synthesis and partitioning prior to changes in liver fat content in healthy adults. diabetes 2016;65: 1858–67. 10. pramfalk c, pavlides m, banerjee r, mcneil ca, neubauer s, karpe f, et al. sex-specific differences in hepatic fat oxidation and synthesis may explain the higher propensity for nafld in men. j clin endocrinol metab. 2015;100:4425–33. 11. hodson l, bickerton ast, mcquaid se, roberts r, karpe f, frayn kn, et al. the contribution of splanchnic fat to vldl triglyceride is greater in insulin-resistant than insulin-sensitive men and women: studies in the postprandial state. diabetes 2007;56:2433–41. 12. heath rb, karpe f, milne rw, burdge gc, wootton sa, frayn kn. selective partitioning of dietary fatty acids into the vldl tg pool in the early postprandial period. j lipid res. 2003;44: 2065–72. 13. semple rk, sleigh a, murgatroyd pr, adams ca, bluck l, jackson s, et al. postreceptor insulin resistance contributes to human dyslipidemia and hepatic steatosis. j clin invest. 2009; 119:315–22. 216 l. hodson et al. abstract introduction subjects and methods subjects measurements of dnl clinical chemistry statistics results association and agreement between dnlvldl-tag and dnlplasma-tag repeatability of dnlvldl-tag and dnlplasma-tag discussion acknowledgements disclosure statement references excess deaths from covid-19 correlate with age and socio-economic status. a database study in the stockholm region original article excess deaths from covid-19 correlate with age and socio-economic status. a database study in the stockholm region peter stranga,b, per f€ursta,c and torbj€orn schultzb adepartment of oncology-pathology, karolinska institutet, stockholm, sweden; br&d department, stockholms sjukhem foundation, stockholm, sweden; cpalliative care unit, stockholms sjukhem foundation, stockholm, sweden abstract background: the covid-19 pandemic has affected the entire health care system, internationally as well as in sweden. we aimed to study excess deaths (all death causes, but also covid-19-related deaths) during the covid-19 pandemic regarding age, socio-economic status, the situation in nursing homes, and place of death for nursing home residents. design: we performed a descriptive regional registry data study using val, the stockholm regional council’s central data warehouse, which covers almost all health care use in the county of stockholm. t tests and chi-square tests were used for comparisons. results: compared with 2016–2019, there were excess deaths in march–may 2020 (p < 0.0001), mainly explained by covid-19, but in april there were also unexplained excess deaths. individuals dying from covid-19 were older than patients dying from other causes (p < 0.0001). there were more patient deaths among people residing in less advantaged socio-economic areas (p < 0.0001). nursing home residents dying from covid-19 were more often admitted to acute hospitals than residents dying from other causes (p < 0.0001). also, the proportion of admissions of nursing home residents dying from other causes increased from april to may 2020 (p < 0.0001). conclusions: dying from covid-19 mainly affects the elderly, nursing home residents, and persons from less advantaged socio-economic groups. the pandemic has resulted in an increase in acute admissions of dying nursing home residents to acute hospitals. article history received 30 july 2020 revised 21 september 2020 accepted 22 september 2020 keywords covid-19; excess deaths; hospital care; nursing homes introduction although covid-19 probably already existed during the autumn of 2019, the outbreak in wuhan, china was formally announced by the who in december 2019. initially, it was uncertain whether the virus would reach countries like sweden, but in march 2020 it was obvious that it had done so, with the first confirmed death from covid-19 occurring in the middle of the month. soon, it was evident that sweden had a rapid dissemination of the disease, especially in the stockholm region that covers about 2.3 million inhabitants. initially, the major concerns were regarding the capacity and the limits of intensive care units (icus) in the region, as well as worries about limited access to personal protective equipment (ppe). however, already from the beginning, it was also clear that frail elderly people with comorbidities had the highest risk of dying from covid-19 and that age in itself was an established important risk factor (1,2). in a swedish context, the impact of socio-economic factors was also apparent in march. regarding the stockholm region, patients from less affluent areas seemed to already be overrepresented among deaths at the beginning of the pandemic, and there were alarming early reports stating that individuals from certain non-european backgrounds were overrepresented among the deceased. this was confirmed in a study where deaths in 2020 were compared with deaths in 2016–2019, month by month (3). immigrants from somalia, syria, and iraq who typically live in less affluent areas had a much higher rate of death than other groups. for obvious reasons, nursing homes also received a great deal of attention, as a substantial proportion of the deaths occurred in these services among the elderly, frail residents. due to the organization of swedish elderly care, with the principle of ‘aging at home’ (kvarboendeprincipen), the goal is to stay in one’s own home with the aid of home-help services for as long as possible. a person is accepted for nursing home care after a municipal needs-assessor’s decision, and only when 6–8 scheduled visits per day by home-help services, including nightly visits, are insufficient to support a person with high needs of assistance with activities of daily living (adl). for this reason, the mean age of the residents is over 85 years. more than 60% suffer from cognitive failure or diagnosed dementia, and most have several comorbidities (4). moreover, expected survival is limited in nursing homes. in a survey in the stockholm region, one-tenth of the contact peter strang peter.strang@ki.se department of oncology-pathology, karolinska institutet, stockholm, sweden; r&d department, stockholms sjukhem foundation, po box 12230, stockholm, 102 26, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 4, 297–304 https://doi.org/10.1080/03009734.2020.1828513 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1828513&domain=pdf&date_stamp=2020-10-22 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1828513 http://www.tandfonline.com residents died within a week of admission, one-third within six months, and almost 50% died within the first year (5). in the county of stockholm, the municipalities are responsible for nursing homes and staffing, except for physicians who are provided by the region stockholm (formerly ‘stockholm county council’). nursing homes provide help with adl and offer basic health care, when needed. about 36% of all deaths in sweden take place in nursing homes (6). if the care provided by a nursing home is not sufficient in an individual case, admission to an acute hospital (including a geriatric clinic) is possible. when the pandemic was established, region stockholm strengthened the existing services and decided on five levels of care for elderly patients infected with covid-19: 1) nursing homes for people already residing in such homes; 2) nursing home care with the aid of specialized palliative home care units (with a higher degree of medical staffing); 3) geriatric clinics with designated covid-19 wards; 4) acute hospitals; and 5) icus. the staffing in nursing homes is basic: a great majority of the staff are assistant nurses, and only about 5% are registered nurses. for this reason, nursing homes provide a basic level of care and nursing, but most cannot offer oxygen therapy or intravenous antibiotics which would require more staffing by registered nurses. if oxygen is needed to support a resident affected by covid-19 this can, in a limited number of cases, be offered in the nursing home, with or without the help of palliative home care teams. in other cases, acute admission to a geriatric clinic with special covid-19 wards or to an acute hospital is needed. with this strategy in place and these levels of care, almost 65% of nursing home residents with covid-19 in the stockholm region recovered from the disease during the first months, whereas about 35% died from covid-19, some of whom died after referral to hospitals (7). the pandemic has led to an urgent need for research data concerning those at risk of dying from covid-19. however, studying deaths that are attributed to covid-19 is not simple. first, there was a worldwide delay in testing during february and march 2020, meaning that laboratory-confirmed cases only constituted as little as 10–15% of all cases in some countries (8). moreover, due to a shortage of tests, only severe hospital cases were initially tested, implying that patients with less obvious symptoms in nursing homes might have been overlooked. a general way to partially overcome this problem is to study excess deaths (all causes of death), which is what has been done to track influenza mortality for more than a century (9,10). we aimed to study excess deaths (all death causes, but also covid-19-related deaths) during the covid-19 pandemic, with special reference to age, socio-economic status, and the situation in nursing homes. a further aim was to study place of death for nursing home residents. patients and methods the methods and the results sections are, when possible, reported based on the strengthening the reporting of observational studies in epidemiology (strobe) criteria (11). study design the study is based on information retrieved from administrative databases. in sweden, administrative databases can be used to study health care consumption, place of care, and place of death. this also applies now to covid-19. in the stockholm region, all appointments, hospital visits, diagnoses, and major costs are registered and stored in val, the stockholm regional council’s central data warehouse. socioeconomic status can also be studied, as region stockholm subscribes to mosaictm, a commercial, internationally used database developed by the company experian. the mosaic database can be used for several purposes, but, in the form that it is applied within the stockholm region, three socioeconomic groups are defined, based on neighbourhood characteristics. thus, the mosaic groups characterize areas, rather than individuals. these groups or areas are based on several variables, of which education, income, family situation, and living arrangements, and also phase of life, origin, and ethnicity are the most important (12–14). we conducted a descriptive regional registry data study using val, the stockholm region’s central data warehouse. within val, there are separate registers for outpatient visits to hospitals (ovr) and hospital stays (slv). in swedish health care a person’s health care consumption can be followed between different administrative systems as sweden uses unique personal numbers for each individual. data in val registers are based on these personal numbers but encrypted, meaning that a person’s health care consumption can be followed, without revealing personal identity. the monthly deaths for january to may 2020 were identified and compared with the corresponding months over four consecutive years, 2016–2019. the data were further analysed in relation to age, sex, living arrangements (residents in nursing homes versus all others), and socio-economic status by means of mosaic. populations study population all monthly deaths registered in val databases during january to may 2020 (data retrieved 29 june 2020) were included. in accordance with the guidelines by the public health agency of sweden (folkh€alsomyndigheten), any death with a covid-19 diagnosis according to icd-10 should be considered as a death from covid-19 (15). reference population all monthly deaths registered in val databases during january to may 2016–2019 (four year-cohorts) were included. based on these data, mean values with 95% confidence intervals (ci) were calculated. the reason for choosing a mean from the four previous years (instead of just data from the previous year) was to smooth out any short-term spikes, e.g. due to an influenza outbreak. 298 p. strang et al. variables deaths (all causes) as well as deaths with a covid-19 diagnosis were used as outcome measures. age, sex, living arrangements (nursing homes versus all others), and mosaic groups were used as explanatory variables. the mosaic methodology is based on the assumption that people tend to settle in neighbourhoods where others are quite similar to themselves, and the final mosaic groups are based on iterative cluster analyses, based on more than 40 socio-economic variables. thus, mosaic provides socio-economic information and allows the council to define and allocate different areas of residence to three different socio-economic classes (mosaic 1–3), mainly based on income and education, but also, for example, on family situation (single/cohabiting/children, etc.) and living arrangements (owned or rented housing, etc.), phase of life, origin and ethnicity, and degree of urbanization. the county of stockholm is divided into 1300 small areas, and each area is classified as mosaic 1, 2, or 3. the three groups are approximately equal in size. mosaic group 1 refers to persons living in the most affluent areas. as people residing in nursing homes have moved in from their ordinary homes, this may affect their belonging to a certain mosaic group, although we know that people prefer a nursing home in their local area. with this in mind, we performed an extra pair-wise comparison of mosaic groups for individuals during their last year of life, compared to their allocated mosaic group four years previously when they resided in their ordinary home. the differences on a mosaic group level differed only with 1–2% (e.g. the proportion of nursing home residents allocated to mosaic group 2 were 37.3% during their last year of life and 37.1% four years previously). thus, mosaic groups were found to be rather stable and reliable enough to be used also when studying nursing home residents. selection bias drop-outs as reporting data to val constitutes the basis for the respective clinic’s/care unit’s remuneration, data are complete with very few missing values. this means that any individual who has used public health care during the actual year is included in the val databases, which is also the case for most forms of private care, as private health care suppliers have economic agreements with the regional council. immediacy the data in val are updated every month, thus, it is possible to retrieve even very recent data. nursing home residents nursing home residents were identified through registrations of medical interventions by physicians, as such care use is exclusive to nursing home residents and has a unique, identifiable code. it is most unlikely that a nursing home resident does not have a single registration; nursing home residents without registrations were not included in the analysis. study size the study covers total cohorts, i.e. all deaths (all causes) as well as all reported covid-19-related deaths during january–may 2020, and data have been compared with data for four similar year cohorts (2016–2019). therefore, no power calculations were made. statistical methods, missing data the 95% confidence intervals (95% ci) were calculated. t tests and chi-square tests were used to compare proportions. the few missing data were not substituted. the sas version 9.4 and spss version 25 software programs were used for statistics. ethics the study was approved by the national ethics authority (etikpr€ovningsmyndigheten, dnr 2020–02186). results excess deaths (all causes) january–may 2020 compared with 2016–2019 all deaths the mean age of all the deceased from january to may 2020 was 79.5 years (median 83 years), which was higher than for the deaths during the corresponding months in 2016–2019, 78.8 years (median 82 years), p < 0.0001. in 2020, 49.6% were female, compared with 52.1% for 2016–2019 (chi-square ¼ 18, 1 df), p < 0.0001. in a first comparison, death rates for january to may 2020 were compared with corresponding calculated means and 95% ci for the corresponding months in 2016–2019. whereas january and february were similar to the calculated means, the proportions of deaths were significantly higher for march, april, and may (23%, 113%, and 44%, respectively), p < 0.0001, for each comparison (table 1). regarding age groups, only patients over 80 years of age had excess deaths in march, whereas all the studied age groups were affected in april. in may, excess deaths were mainly attributed to patients aged 70–79 years and to those aged 80 years or more (table 1). nursing homes versus other places of death the proportion of patients dying in nursing homes as a fraction of all deaths in 2020 was 32% in march, 43% in april, and 34% in may. when specifically studying the percentage of excess deaths in nursing homes, compared with deaths in 2016–2019, the proportions were found to be significantly higher: 11% in march, 167% in april, and 46% in may (table 2). there were also excess deaths (all causes) for march–may, upsala journal of medical sciences 299 for those dying in other places than nursing homes. in march, the excess deaths (%) were higher for other places of deaths than nursing homes, 30% versus 11%, whereas the situation was reversed for april. for details, see table 2. dying from covid-19 the mean age of patients who died with a covid-19 diagnosis was 81 years (median 83 years), which was higher than for other causes (table 3); 45% were female, compared with 51% in 2016–2019 (chi-square ¼ 23, 1 df), p < 0.0001. the proportions of deaths with a registered covid-19 diagnosis compared with all deaths were 10% for march, 37% for april, and 32% for may. covid-19 and socio-economic status per month when stratifying for socio-economic mosaic groups, where mosaic group 1 represents individuals from the most affluent and mosaic 3 the least affluent socio-economic areas, there were significant differences as regards covid-19-related deaths for each month during march to may 2020, with more deaths in mosaic group 3 (table 4). per age group covid-19-related deaths were also calculated in relation to every 1000 inhabitants, stratified both for mosaic groups and for age groups. data for the most affected month, april 2020, are presented in table 5. deaths were consistently higher in mosaic group 3 compared with mosaic group 1. excess deaths not explained by covid-19 april was the most affected month, with 1096 confirmed covid-19 deaths and a total of 2934 deaths. when removing the number of those who died from covid-19, the remaining number is 1838 deaths in april, which is significantly higher than for the reference years (95% ci 1300–1452). nursing home residents: changes in place of care during the last two weeks of life changes in place of care for nursing home residents were mapped for the last two weeks of life, for the months march–may. in a first comparison, we studied trends for 2016–2019 compared with 2020. for the period 2016–2019, 28.3% (95% ci 26.7%–30.0%) had at least one change as regards place of care, compared with 15.2% for the corresponding period in 2020 (chi-square ¼ 162, 1 df; p < 0.0001) (table 6). in a second comparison, we did a more detailed comparison for 2020, where we compared those who had died with a covid-19 diagnosis with all others. for the whole period (march–may), 24% and 12% of those who died with versus without a covid-19 diagnosis had at least one change ofta b le 1. m or ta lit y an d ag e ca te g or ie s. m ar ch a p ri l m ay 20 16 – 20 19 n, m ea n (9 5% c i) 20 20 ex ce ss m or ta lit y si g n ifi ca n ce a (c h isq ua re )b 20 16 – 20 19 n, m ea n (9 5% c i) 20 20 ex ce ss m or ta lit y si g n ifi ca n ce a (c h isq ua re )b 20 16 – 20 19 n, m ea n (9 5% c i) 20 20 ex ce ss m or ta lit y si g n ifi ca n ce a (c h isq ua re ) b m on th ly m or ta lit y 14 74 (1 32 6– 16 22 ) 18 19 23 % �� � (3 8. 9) 13 76 (1 30 0– 14 52 ) 29 34 11 3% �� � (1 02 0. 5) 12 87 (1 24 4– 13 30 ) 18 60 44 % �� � (1 47 .4 ) a g e ca te g or ie s 60 – 69 ye ar s 15 4 (1 34 – 17 4) 15 3 �1 % 14 5 (1 26 – 16 5) 24 6 69 % �� (4 4. 8) 15 1 (1 25 – 17 7) 16 2 7% 70 – 79 ye ar s 34 7 (3 02 – 39 1) 37 8 9% 32 3 (2 61 – 38 4) 63 2 96 % �� � (1 56 .7 ) 30 6 (2 79 – 33 3) 40 6 33 % �� � (1 3. 7) �8 0 ye ar s 84 0 (7 68 – 91 1) 11 41 36 % �� � (4 6. 6) 77 9 (7 12 – 84 6) 18 86 14 2% �� � (8 21 .1 ) 69 4 (6 45 – 74 3) 11 56 67 % �� � (1 68 .1 ) th e p ro p or ti on s of d ea th s w er e si g n ifi ca n tl y h ig h er fo r m ar ch , ap ri l, an d m ay (2 3% , 11 3% , an d 44 % , re sp ec ti ve ly ). th e ch isq ua re va lu es w er e w el l b el ow th e lim it fo r p < 0. 00 1 (c h isq ua re ¼ 10 .8 3) in al l m on th ly co m p ar is on s. a le ve ls of si g n ifi ca n ce : � p < 0. 05 ; �� p < 0. 01 ; �� � p < 0. 00 1. b in al l ch isq ua re co m p ar is on s: 1 d f (d eg re e of fr ee d om ), m ea n in g th at th e ch isq ua re va lu e fo r p < 0. 05 is 3. 84 ; p < 0. 01 is 6. 64 ; an d fo r p < 0. 00 1 is 10 .8 3. 300 p. strang et al. care (chi-square ¼ 59.8, 1 df; p < 0.0001). for details, see table 7. residents dying from causes other than covid-19 had more changes of place in may than in april (table 7). whereas 5% were referred acutely during the last two weeks of life in april, the corresponding figure had increased to 13% in may (chi-square ¼ 24.6, 1 df; p < 0.0001). place of death for nursing home residents when specifically studying the proportion of nursing home residents who eventually died either in an acute hospital or in a geriatric hospital ward, the proportion for residents dying from covid-19 during march–may was 19%, and for residents dying from other causes 5% (chi-square ¼ 109, 1 df; p < 0.0001) (table 8). table 2. proportion of all deaths (all causes), and excess deaths (all causes) in march–may 2020, in comparison with data for respective month in 2016–2019, in nursing homes versus in all other places of death. nursing homes all others month 2016–2019 (95% ci) 2020 excess mortality 2016–2019 (95% ci) 2020 excess mortality march 531 (482–580) 590 11%a 944 (828–1059) 1229 30%a april 475 (437–511) 1269 167%a 898 (852–951) 1665 85%a may 428 (368–488) 625 46%a 855 (795–924) 1235 44%a ap < 0.05 in all comparisons between 2016–2019 and 2020. table 3. age at death. cause of death p valuecovid-19, mean (median) other causes, mean (median) all patients (years) 81.0 (83) 79.2 (82) <0.0001 female patients (years) 84.2 (86) 82.0 (86) <0.0001 male patients (years) 78.3 (80) 76.3 (79) <0.001 patients dying from covid-19 were significantly older than patients dying from other causes. table 4. covid-19-related deaths per month during march–may 2020, in relation to socio-economic mosaic groups 1 and 3. month mosaic group 1 mosaic group 3 chi-square (1 df) p value march 36/703478 91/715494 22.9 <0.0001 april 230/703576 451/715666 68.0 <0.0001 may 163/703508 226/715875 9.2 0.003 there were more covid-19-related deaths in mosaic group 3. table 5. covid-19-related deaths/1000 inhabitants in april 2020. age group (years) mosaic group deaths n deaths/1000 inhabitants chi-square (mosaic 1 versus 3) p value (mosaic 1 versus 3) 40–59 1 11 0.05 2 14 0.06 3 26 0.15 8.6 0.003 60–69 1 17 0.25 2 28 0.31 3 54 0.86 22.4 <0.00001 70–79 1 58 1 2 95 1.19 3 110 2.12 22.5 <0.00001 �80 1 143 5 2 274 6.98 3 265 7.88 19.8 <0.00001 for each age group, the proportion of deaths was significantly higher in mosaic group 3, compared to mosaic group 1. table 6. proportion of nursing home residents with at least one change of place of care during their last 2 weeks of life. 2016–2019, march–may, % (95% ci) with changes in place of care 2020, march–may, % with changes in place of care chi-square (1 df) p value 28.3% (26.7%–30.0%) 15.2% 162.0 <0.0001 march–may 2016–2019 is compared with march–may 2020. table 7. proportion of nursing home residents with at least one change of place of care during their last 2 weeks of life, march–may 2020. month covid-19 deaths, n (%) with changes in place of care other causes, n (%) with changes in place of care chi-square (1 df) p value march 22/39 (56%) 111/551 (20%) 27.9 <0.0001 april 99/449 (22%) 43/820 (5%) 80.8 <0.0001 may 48/213 (22%) 55/412 (13%) 8.6 0.003 march–may 169/701 (24%) 209/1783 (12%) 59.8 <0.0001 residents dying from covid-19 are compared with residents dying from other causes. upsala journal of medical sciences 301 discussion our register data showed significant excess deaths (all causes) for each of the months of march to may, with peak values for april. the excess deaths correlated with more advanced age and consequently also with being resident in a nursing home, as the mean age of swedish nursing home residents is about 86 years. there was also a correlation with excess covid-19-related deaths and lower socio-economic status as measured by the mosaic groups, in good agreement with a detailed report from region stockholm (16). an additional finding was that nursing home residents dying from covid-19 were more often admitted to hospitals than residents dying from other causes. our data are in good agreement with similar data from other countries who also report excess deaths, not only related to a covid-19 diagnosis but also to other causes (9,10,17–19). such unexplained deaths were also seen in the stockholm region in april, the most affected month. excess deaths related to other causes are believed to occur indirectly through delayed care for acute emergencies, exacerbations of chronic diseases, and psychological distress (e.g. drug overdoses) (17). age has been a known risk factor already from the start of the epidemic (1,2). for this reason, the swedish strategy attempted to protect citizens above the age of 70 years from contact with others through a number of recommendations aimed at social distancing. still, our data show that the covid-19-related excess mortality in the peak month (april) was most pronounced in these groups, with 86% of the covid-19-related deaths in persons over 70 years of age, in good agreement with other data from region stockholm (16). a disproportionately large proportion of these individuals were residents in nursing homes compared with other living arrangements. however, the proportion dying in nursing homes with a covid-19 diagnosis (37% in april), is similar to the data from other countries, with figures ranging from 19% in hungary to 62% in canada (20). moreover, people dying from covid-19 were older than people dying from other causes. socio-economic status in itself, as well as belonging to a minority, has been associated with a higher risk of contracting covid-19 and, also, with a higher risk of death (16,19,21–23). in many studies, socio-economic status and belonging to a minority are strongly intercorrelated, but the risk of contracting covid-19 and dying from the disease is not explained merely by socio-economic status or comorbidities. as shown by lassale et al. in their study, black individuals had an increased risk of covid-19 infection compared with white individuals, even when adjusting for age, sex, and other potential explanatory factors which included neighbourhood deprivation, household crowding, smoking, body size, inflammation, glycated haemoglobin, and mental illness (22). similar data have been published by williamson et al. (23). sweden has a relatively large proportion of immigrants, with approximately 20% of the population born abroad (24). in a recent study, the number of deaths was 220% higher for immigrants from somalia, syria, and iraq in 2020, compared with deaths in previous years, and much higher than for individuals born in sweden (3). in our current study, we used mosaic groups instead of comparing immigrants with persons born in sweden. mosaic includes immigration status as one of the defining variables, but also several other unrelated variables, of which education, income, family situation, and living arrangements are the most salient factors. for each age group, covid-19-related deaths were significantly more frequent for persons living in mosaic group 3 areas than in mosaic group 1 areas. in sweden, there has been an animated debate concerning the optimal place of care for nursing home residents who are infected with covid-19, especially considering the high number of deaths in nursing homes. critics have argued that ‘no one is being admitted to hospitals’, despite inadequate access to oxygen treatment in nursing homes (25). care of residents with covid-19 in nursing homes has even been compared with euthanasia by public persons, in an infected debate (26). therefore, according to the critics, more admissions would mean more saved lives. others have argued that most of these frail, dying, nursing home residents would not benefit from an acute admission, as an acute transfer from a well-known environment often triggers acute delirium in this patient group and they would not benefit from hospital care. for this reason, it was of interest to study whether, and to what degree, hospital admissions were made for dying nursing home residents. when comparing the proportion of all changes in place of care in nursing homes in 2020 with figures from previous years (2016–2019), it is obvious that the total number of changes was lower in 2020, implying that there was a general reluctance to admit dying residents to acute hospitals. however, data show that, in 2020, a substantially larger proportion of residents dying from covid-19, compared with residents dying from other causes, were referred to acute hospitals or geriatric hospital wards. an interesting finding is that although 24% of those dying from covid-19 and 12% of the others were acutely admitted to hospitals during their last two weeks of life, only 19% and 5% of these patients actually died in hospitals, implying that some of them were sent back to the nursing homes. therefore, it is not possible to draw any conclusions about the net benefits, i.e. how many lives were saved by acute admissions and how many died in the unfamiliar hospital environment. estimates by individual providers of nursing table 8. proportion of nursing home residents who eventually died in acute hospitals or geriatric wards, march–may 2020. months covid-19 deaths, n (%) hospital deaths other causes, n (%) hospital deaths chi-square (1 df) p value march–may 132/701 (19%) 96/1687 (5%) 109.1 <0.0001 residents dying from covid-19 are compared with residents dying from other causes. 302 p. strang et al. home care say that relatively few of those admitted to acute hospitals survived (7). because the debate in the media included a great deal of criticism of nursing home care, we also decided to study to what degree nursing home residents dying from other causes were acutely admitted to other services during the last two weeks of life, i.e. in the dying phase. we found that whereas only 5% of these residents were admitted to hospitals in april, the figure was significantly higher in may, 13%. a possible explanation is that the general discussion, where the medical competence of nursing homes was questioned in the media, affected the public’s view on nursing home care in general, with subsequent requirements for referrals to hospitals. our analyses were based on administrative data from the regional val databases. as all health care, with very few exceptions, is financed by taxes and reporting to the val databases is a mandatory basis for remuneration, the data have extremely few missing values. this is also the case for private health care, as most of the private care providers have agreements with region stockholm. a possible limitation is that only persons with health care utilization provided by the regional council are registered. this means that basic care in nursing homes, care that is provided by the municipalities, is not registered. however, as all consultations and medical interventions by physicians are provided by the regional council, these interventions are registered with an identifiable coding. in this way, nursing home residents were indirectly identified, but we might have missed some cases. another possible limitation is that people in nursing homes that are located in certain socio-economic mosaic areas (e.g. mosaic group 2 area) might have moved there from another area belonging to a different mosaic group. however, specifically for this study, we have compared the actual mosaic allocation for nursing home residents with their previous mosaic allocation four years earlier (see methods section) and found only minor differences, in the range of a few percent. thus, mosaic groups are rather stable and reliable enough also when studying nursing home residents. finally, we used the definition proposed by the public health agency of sweden, that any death with an icd-10 code of covid-19 should be counted as a death from covid-19. future studies will show to what extent people actually died from covid-19 or died from other causes with covid-19 as a secondary diagnosis. conclusions dying from covid-19 mainly affects the elderly, and those dying from covid-19 are, in fact, older than those dying from other causes. nursing home residents as well as elderly individuals from less advantaged socio-economic groups are at a higher risk. the pandemic has changed the patterns of care: a higher proportion of nursing home residents with severe forms of covid-19 are referred to hospitals, and this has, in turn, also affected decisions for residents dying from other causes, with more acutely dying persons admitted to acute hospitals. acknowledgements the authors thank region stockholm for generously providing us with the data for the study. the stockholm sjukhem foundation is acknowledged for providing excellent facilities in their research & development unit. david boniface is acknowledged for linguistic revision. disclosure statement the authors have nothing to disclose. funding dr strang reports grants from the stockholm sjukhem foundation’s jubilee fund [519102] during the conduct of the study. notes on contributors peter strang is an oncologist and professor in palliative medicine, karolinska institutet in stockholm. per f€urst is a geriatrician and consultant in palliative medicine at the palliative care unit, stockholms sjukhem, 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community-based cohort study. brain behav immun. 2020;88:44–9. 23. williamson ej, walker aj, bhaskaran k, bacon s, bates c, morton ce, et al. factors associated with covid-19-related death using opensafely. nature 2020;584:430–6. 24. statistiska centralbyrån (scb). utrikes f€odda i sverige [people born abroad in sweden]. 2020. available at: https://www.scb.se/hittastatistik/sverige-i-siffror/manniskorna-i-sverige/utrikes-fodda/ (last accessed 21 july 2020). 25. r€ostlund l, gustafsson a. de valdes bort av vården – fast vårdplatser stod tomma [they have been prioritized away – despite available hospital beds]. dagens nyheter; 6 june 2020. 26. radio s. hård kritik mot €aldreboenden: snarare d€odshj€alp €an vård 22 maj 2020 [harsh criticism of nursing homes: “it is euthanasia rather than care”]. 2020. available at: https://sverigesradio.se/artikel/7479262 (last accessed 22 may 2020). 304 p. strang et al. https://www.folkhalsomyndigheten.se/smittskydd-beredskap/utbrott/aktuella-utbrott/covid-19/statistik-och-analyser/bekraftade-fall-i-sverige/ https://www.folkhalsomyndigheten.se/smittskydd-beredskap/utbrott/aktuella-utbrott/covid-19/statistik-och-analyser/bekraftade-fall-i-sverige/ https://www.folkhalsomyndigheten.se/smittskydd-beredskap/utbrott/aktuella-utbrott/covid-19/statistik-och-analyser/bekraftade-fall-i-sverige/ https://ces.sll.se/globalassets/verksamheter/forskning-och-utveckling/centrum-for-epidemiologi-och-samhallsmedicin/folkhalsoguiden/rapporter-och-faktablad/rapport-2020.6-covid-19-i-stockholms-lan-till-och-med-mitten-av-juni-2020_uppdaterad-2020-07-13.pdf https://ces.sll.se/globalassets/verksamheter/forskning-och-utveckling/centrum-for-epidemiologi-och-samhallsmedicin/folkhalsoguiden/rapporter-och-faktablad/rapport-2020.6-covid-19-i-stockholms-lan-till-och-med-mitten-av-juni-2020_uppdaterad-2020-07-13.pdf https://ces.sll.se/globalassets/verksamheter/forskning-och-utveckling/centrum-for-epidemiologi-och-samhallsmedicin/folkhalsoguiden/rapporter-och-faktablad/rapport-2020.6-covid-19-i-stockholms-lan-till-och-med-mitten-av-juni-2020_uppdaterad-2020-07-13.pdf https://ces.sll.se/globalassets/verksamheter/forskning-och-utveckling/centrum-for-epidemiologi-och-samhallsmedicin/folkhalsoguiden/rapporter-och-faktablad/rapport-2020.6-covid-19-i-stockholms-lan-till-och-med-mitten-av-juni-2020_uppdaterad-2020-07-13.pdf https://ces.sll.se/globalassets/verksamheter/forskning-och-utveckling/centrum-for-epidemiologi-och-samhallsmedicin/folkhalsoguiden/rapporter-och-faktablad/rapport-2020.6-covid-19-i-stockholms-lan-till-och-med-mitten-av-juni-2020_uppdaterad-2020-07-13.pdf https://ltccovid.org/wp-content/uploads/2020/05/mortality-associated-with-covid-3-may-final-6.pdf https://ltccovid.org/wp-content/uploads/2020/05/mortality-associated-with-covid-3-may-final-6.pdf https://ltccovid.org/wp-content/uploads/2020/05/mortality-associated-with-covid-3-may-final-6.pdf https://www.scb.se/hitta-statistik/sverige-i-siffror/manniskorna-i-sverige/utrikes-fodda/ https://www.scb.se/hitta-statistik/sverige-i-siffror/manniskorna-i-sverige/utrikes-fodda/ https://sverigesradio.se/artikel/7479262 https://sverigesradio.se/artikel/7479262 abstract introduction patients and methods study design populations study population reference population variables selection bias drop-outs immediacy nursing home residents study size statistical methods, missing data ethics results excess deaths (all causes) january–may 2020 compared with 2016–2019 all deaths nursing homes versus other places of death dying from covid-19 covid-19 and socio-economic status per month per age group excess deaths not explained by covid-19 nursing home residents: changes in place of care during the last two weeks of life place of death for nursing home residents discussion conclusions acknowledgements disclosure statement funding references growth differentiation factor 15 (gdf-15) is a potential biomarker of both diabetic kidney disease and future cardiovascular events in cohorts of individuals with type 2 diabetes: a proteomics approach article growth differentiation factor 15 (gdf-15) is a potential biomarker of both diabetic kidney disease and future cardiovascular events in cohorts of individuals with type 2 diabetes: a proteomics approach axel c. carlssona, christoph nowaka, lars lindb, carl johan €ostgrenc, fredrik h. nystr€omc, johan sundstr€omb, juan jesus carrerod, ulf riseruse , erik ingelssonf,g,h,i, tove falli and johan €arnl€ova,j adepartment of neurobiology, care sciences and society (nvs), karolinska institutet, huddinge, sweden; bdepartment of medical sciences, uppsala university, uppsala, sweden; cdepartment of medical and health sciences, link€oping university, link€oping, sweden; ddepartment of medical epidemiology and biostatistics, karolinska institutet, stockholm, sweden; edepartment of public health and caring sciences, clinical nutrition and metabolism, uppsala university, uppsala, sweden; fdepartment of medicine, division of cardiovascular medicine, stanford university school of medicine, stanford, ca, usa; gstanford cardiovascular institute, stanford university, stanford, ca, usa; hstanford diabetes research center, stanford university, stanford, ca, usa; imolecular epidemiology and science for life laboratory, uppsala university, uppsala, sweden; jschool of health and social studies, dalarna university, falun, sweden abstract background: diabetic kidney disease (dkd) is a leading risk factor for end-stage renal disease and is one of the most important risk factors for cardiovascular disease in patients with diabetes. it is possible that novel markers portraying the pathophysiological underpinning processes may be useful. aim: to investigate the associations between 80 circulating proteins, measured by a proximity extension assay, and prevalent dkd and major adverse cardiovascular events (mace) in type 2 diabetes. methods: we randomly divided individuals with type 2 diabetes from three cohorts into a two-thirds discovery and one-third replication set (total n ¼ 813, of whom 231 had dkd defined by estimated glomerular filtration rate <60 mg/ml/1.73 m2 and/or urinary albumin-creatinine ratio �3 g/mol). proteins associated with dkd were also assessed as predictors for incident major adverse cardiovascular events (mace) in persons with dkd at baseline. results: four proteins were positively associated with dkd in models adjusted for age, sex, cardiovascular risk factors, glucose control, and diabetes medication: kidney injury molecule-1 (kim-1, odds ratio [or] per standard deviation increment, 1.65, 95% confidence interval [ci] 1.27–2.14); growth differentiation factor 15 (gdf-15, or 1.40, 95% ci 1.16–1.69); myoglobin (or 1.57, 95% ci 1.30–1.91), and matrix metalloproteinase 10 (mmp-10, or 1.43, 95% ci 1.17–1.74). in patients with dkd, gdf-15 was significantly associated with increased risk of mace after adjustments for baseline age, sex, microalbuminuria, and kidney function and (59 mace events during 7 years follow-up, hazard ratio per standard deviation increase 1.43 [95% ci 1.03–1.98]) but not after further adjustments for cardiovascular risk factors. conclusion: our proteomics approach confirms and extends previous associations of higher circulating levels of gdf-15 with both microand macrovascular disease in patients with type 2 diabetes. our data encourage additional studies evaluating the clinical utility of our findings. article history received 29 august 2019 revised 17 november 2019 accepted 19 november 2019 keywords albumin-creatinine ratio; biomarker; diabetic kidney disease; glomerular filtration rate; proteomics; risk factor; type 2 diabetes mellitus introduction diabetic kidney disease (dkd) contributes to up to half of all cases of end-stage renal disease in the world and is one of the most important risk factors for cardiovascular disease in patients with diabetes (1). the definition of dkd relies on assessment of both kidney function and kidney damage and is defined as an estimated glomerular filtration rate (egfr) of less than 60 mg/ml/1.73 m2 and/or microor macroalbuminuria in patients with diabetes (1). recent technological advances have made it possible to simultaneously measure a large number of proteins in biological samples (2,3). these ‘proteomics’ assays could offer new ways to discover pathophysiologic pathways and identification of novel disease biomarkers in dkd. yet, despite the substantial clinical relevance of dkd as one of the most common complications of both types of diabetes, only a few prior proteomics studies have focussed on dkd (4,5), and in most of these prior studies, the definition of dkd did not include albuminuria assessments (4). we believe that proteomics analyses can provide novel insights into underlying mechanisms leading to dkd but also to mechanisms that mediate the risk of future cardiovascular disease. estimated gfr and albuminuria are well-established biomarkers of kidney disease progression. however, much is still unknown contact axel c. carlsson axelcefam@hotmail.com department of neurobiology care sciences and society, division for family medicine and primary care, 141 83 huddinge, sweden � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 1, 37–43 https://doi.org/10.1080/03009734.2019.1696430 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1696430&domain=pdf&date_stamp=2020-02-19 http://orcid.org/0000-0002-8620-4586 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2019.1696430 http://www.tandfonline.com about the pathophysiology of cardiovascular disease-specific to dkd. we reasoned that associations between circulating proteins previously linked to cardiovascular disease (cvd) and inflammation could provide new insights into cardiovascular disease pathways involved in dkd. the kidney is one of the best-perfused organs in the body and shares many biomarkers that are also of interest for cardiovascular pathology. therefore, we aimed to explore and validate the associations between 80 circulating proteins involved in cardiovascular pathology or inflammation with dkd in persons with type 2 diabetes enrolled in three separate cohort studies. we also aimed to study if the identified proteins were associated with the incidence of major adverse cardiovascular events beyond established risk factors in those with prevalent dkd. methods study cohorts data were used from individuals with type 2 diabetes enrolled in three cohorts where egfr and microalbuminuria were measured on at least one occasion, and a biobank with samples available for proteomic analysis: the cardiovascular risk factors in patients with diabetes: a prospective study in primary care (cardipp) (6), the prospective investigation of the vasculature in uppsala seniors (pivus) (7), and uppsala longitudinal study of adult men (ulsam) (8). the cardipp study was launched in 2005, and the baseline data collection was completed in november 2008. patients with type 2 diabetes aged 55–65 were consecutively recruited during their usual annual follow-up assessments at 22 primary healthcare diabetes clinics in the swedish counties of €osterg€otland and j€onk€oping (9). the centres varied in size and were located in different sociodemographic areas, but all followed the national guidelines for diabetes care. out of 761 consecutively enrolled patients, 621 with available data on proteomics, cardiovascular risk factors, dkd status, and outcome data on cardiovascular events were included in the present analyses. all 70-year-old men and women living in uppsala, sweden, between 2001 and 2004 were invited to participate in the pivus study (http://www.medsci.uu.se/pivus/pivus. htm) (6) and were re-investigated with blood samples and urine biochemistry at the age of 75 years. at the re-investigation, 77 participants had diabetes and were thus included in this study. the ulsam study was initiated in 1970 (7). all 50-year-old male residents of uppsala, sweden, who had been born in 1920–24 were invited to participate in a health survey of cardiovascular risk factors (described in detail here: http://www. pubcare.uu.se/ulsam) (8). at the fourth examination cycle, when participants were approximately 77 years old, 1398 were invited and 838 (60%) participated, of which 115 persons had diabetes and could be included in the present study. outcome definitions, inclusion criteria, and number of eligible participants we defined type 2 diabetes as fulfilling at least one of the following criteria: (i) self-reported type 2 diabetes; (ii) physician-diagnosed type 2 diabetes according to hospital records; (iii) fasting glucose �7.0 mmol/l (126 mg/dl); or iv) hba1c >6.5% (48 mmol/mol). participants without available frozen plasma or serum samples or with missing data on proteomics, egfr, or microalbuminuria were excluded. dkd was defined as an egfr below 60 mg/ml/1.73 m2 and/or urinary albumin-creatinine ratio (acr) �3 g/mol. single measurements of egfr and acr were assessed. major adverse cardiovascular events (mace) were defined as fatal or non-fatal myocardial infarction (international classification of diseases, 10th ed., i21) or stroke (i60–i63), whichever occurred first after baseline assessment. these were obtained from follow-up in national swedish registers that started after the baseline investigation in each individual. by combining data from the three cohorts, the total study population was 813, of whom 231 had prevalent dkd. there were 59 mace recorded after baseline in those with dkd. ethical permission participants provided written informed consent, and the study was conducted according to the declaration of helsinki. ethical permission was granted by the ethics committees of link€oping university and uppsala university. multiplex protein assay the olink proseek multiplex cardiovascular i 96 x 96 kit was used to measure proteins in plasma (cardipp, pivus) and serum (ulsam) by real-time polymerase chain reaction (pcr) using the fluidigm biomark hd real-time pcr platform. the assay attempts to quantify the abundance of 92 proteins and uses the standard 96-wells plate format. of the 96-wells, one serves as negative control, whilst three wells contain positive controls. the resulting relative values obtained were log2transformed for subsequent analysis. twelve proteins with <85% valid measurements were removed, leaving 80 proteins for the present analysis. if values were below the lower limit of detection (lod), they were imputed by lod/2. each protein was normalized by plate (by setting the mean ¼ 0, and standard deviation ¼ 1 within each plate) and by storage time (correction based on the observed values and predicted values from a spline model). in a previous validation study of the proteomics assay, the mean intra-assay coefficient of variation was found to be 8%, and the mean inter-assay coefficient of variation was 12% (10). detailed information about the methods used in the assay and on the coefficients of variation of specific proteins can be found on the olink website (www.olink.com). 38 a. c. carlsson et al. http://www.medsci.uu.se/pivus/pivus.htm http://www.medsci.uu.se/pivus/pivus.htm http://www.pubcare.uu.se/ulsam http://www.pubcare.uu.se/ulsam http://www.olink.com statistical analysis we used mixed-effects logistic regression to assess associations between protein abundance (standardized to a mean of 0 and a standard deviation of 1) and dkd, with adjustments for age, sex (fixed effects), and cohort (random effect). data were divided into a discovery data set and a replication data set. samples were combined at the individual person-level and randomly split into a two-thirds training and one-third hold-out test set using the ‘createdatapartition’ function in the ‘caret’ package in r. the function balances the dkd case proportion across both samples. proteins associated at a 5% false discovery rate (fdr) in the discovery sample were tested in the replication sample and were considered successfully replicated at the nominal significance level of 0.05 (11). missing covariate values were imputed by multivariate imputation by chained equations (mice) by predictive mean matching based on all other covariates and averaged across five iterations (12). imputed values were compared against complete values to assess accuracy. as a second step, we used the whole cohort to perform additional multivariable modelling adjusted for cohort (random effects), age, sex, glucose control/diabetes factors (hba1c, oral antidiabetic drug use, and insulin treatment), and cardiovascular risk factors (cardiovascular disease at baseline, low-and high-density lipoprotein cholesterol, triglycerides, bmi, cardiovascular disease at baseline, systolic and diastolic blood pressure, antihypertensive therapy, statins, and smoking status). finally, we used cox regression with frailty effect for cohort adjusted for age, sex, gfr, and microalbuminuria to study if any of the proteins associated with dkd were associated with risk of mace beyond baseline acr and egfr in individuals with dkd (13). we also adjusted a model additionally for cardiovascular risk factors (prevalent cardiovascular disease at baseline, systolic blood pressure, lowdensity lipoprotein cholesterol, and smoking). all statistical analyses were performed with r version 3.3.2, 2016–1031 (14). results baseline characteristics a total of 813 subjects were included in the present study, of whom 231 had prevalent dkd. the sample was divided into discovery 542 (two-thirds) and replication 271 (onethird). baseline characteristics are shown in table 1 for the whole cohort and also stratified by dkd status. microalbuminuria was more common than an egfr <60 mg/ ml/1.73 m2 among those with dkd, 71% versus 39%. systolic blood pressure was higher in those with dkd (147 mmhg) versus those without dkd (139 mmhg), and previous cardiovascular disease was also more common, 44% versus 25%. associations between proteins and prevalent dkd a total of 14 proteins were positively associated with dkd in the discovery sample at <5% fdr in age-, sex-, and cohortadjusted models. four of these 14 proteins were associated with dkd in the replication sample: kidney injury molecule-1 (kim-1), growth differentiation factor 15 (gdf-15), myoglobin, and matrix metalloproteinase 10 (mmp-10) (figure 1). higher levels of all four proteins remained significantly associated with prevalent dkd in additional multivariable models that were adjusted for cardiovascular risk factors, glucose control, and treatment for type 2 diabetes (table 2). associations of proteins and mace incidence in individuals with prevalent dkd over a median of 7.9 ± 1.5 years of follow-up, 59 persons out of the 231 with dkd at baseline experienced a mace event. in these, higher levels of gdf-15 at baseline were associated with a higher risk of incident mace after additional adjustment for egfr and acr, while neither kim-1, myoglobin, nor mmp-10 was associated with future mace (table 3). when we adjusted for cardiovascular risk factors the results were attenuated and not statistically significant. table 1. baseline characteristics. variables all (n ¼ 813) dkd (n ¼ 231) no dkd (n ¼ 582) age, y 64 ± 7 68 ± 8 63 ± 6 women 234 (29%) 62 (27%) 174 (30%) glomerular filtration rate (egfr, ml/min) 77 ± 14 70 ± 17 80 ± 12 glomerular filtration rate (egfr, ml/min) <60 90 (11%) 90 (39%) 0 microalbuminuria (albumin-creatinine ratio �3 g/mol) 164 (20%) 164 (71%) 0 body mass index (bmi, kg/m2) 30 ± 4.6 30 ± 4.5 30 ± 4.6 systolic blood pressure (mmhg) 142 ± 20 147 ± 19 139 ± 20 diastolic blood pressure (mmhg) 81 ± 11 82 ± 10 81 ± 11 fasting glucose (mmol/l) 8.7 ± 2.6 8.9 ± 3.0 8.6 ± 2.4 triglycerides (mmol/l) 1.8 ± 1.1 1.9 ± 1.1 1.8 ± 1.1 low-density lipoprotein cholesterol (mmol/l) 2.7 ± 0.8 2.7 ± 0.8 2.8 ± 0.8 high-density lipoprotein cholesterol (mmol/l) 1.3 ± 0.3 1.2 ± 0.3 1.3 ± 0.3 glycated haemoglobin (hba1c, mmol/l) 51 ± 12 53 ± 13 51 ± 12 treatment with insulin 209 (26%) 66 (29%) 143 (25%) oral antidiabetic drug treatment 480 (59%) 152 (66%) 328 (56%) previous cardiovascular disease 247 (30%) 101 (44%) 146 (25%) statin treatment 415 (51%) 125 (54%) 290 (50%) smoking 123 (15%) 28 (12%) 95 (16%) data are shown as mean ± sd, or as n (%). upsala journal of medical sciences 39 discussion main findings in our cross-sectional analysis of individuals with type 2 diabetes from three different cohorts, a multiplex proteomics assay identified four circulating proteins associated with dkd: kim-1, gdf-15, myoglobin, and mmp-10. the essentially unchanged associations of these biomarkers with dkd after adjustment for glycemic control, diabetes medication, and cardiovascular risk factors point to a possible independent prediction of these biomarkers beyond factors that are generally assessed in clinical practice. moreover, in prospective analyses in participants with prevalent dkd in the present study, higher levels of gdf-15 were associated with a higher risk of incident mace after adjustments for baseline egfr and acr. additional adjustments for established cardiovascular risk factors attenuated this association slightly so that it was no longer statistically significant. comparison with previous studies although the prevalence of dkd parallels the type 2 diabetes and obesity epidemic (15), there has been little advancement in the discovery of clinically relevant biomarkers for dkd. there are several examples of previous studies investigating individual proteins as biomarkers of dkd (4,5,16–18). however, we are aware of only few previous studies that have simultaneously evaluated multiple proteins as biomarkers of dkd progression in serum or plasma. in a recent report in patients with type 2 diabetes in scotland, 205 circulating proteins were evaluated, of which 30 proteins (including gdf-15) were associated with rapid progression of egfr decline (19). in another study in 82 patients with type 2 diabetes, a panel of 13 biomarkers representing fibrosis, angiogenesis, inflammation, mineral metabolism, and endothelial function was found to improve the prediction of egfr decline (20). few of the proteins evaluated in these two prior studies overlapped with the proteins evaluated in the present study. it should also be noted that, in these studies, albuminuria was not included as a kidney disease outcome. moreover, none of these studies evaluated whether dkdassociated biomarkers predicted incident cardiovascular disease in those with prevalent dkd. gdf-15 gdf-15 is a cytokine-induced as a stress response in inflammatory states, after tissue injury and as a response to oxidative stress (21). a comprehensive research effort into gdf-15 (22), and its cardiometabolic associations, is currently ongoing by several research groups worldwide. gdf-15 is of interest in individuals with diabetes and has been shown to be a marker of elevated glucose during an oral glucose tolerance test and to be a marker of impaired fasting glucose, as well as a marker of metformin treatment (23–25). however, metformin treatment did not seem to affect the associations of gdf-15 with dkd in the present study, since diabetic treatment was adjusted for in our full model and as figure 1. discovery and replication of the association between 80 circulating proteins measured by a proximity extension assay, and prevalent diabetic kidney disease. proteins associated at a 5% false discovery rate in the discovery sample were tested in the replication sample, and considered successfully replicated at the nominal significance level of 0.05. two-thirds of the subjects (n ¼ 542) were analyzed in the discovery sample, and one-third of the subjects (n ¼ 271) were analyzed in the replication sample. table 2. multivariable logistic regression models for the association between discovered and replicated proteins and diabetic kidney disease in the whole sample. protein model a model b model c kim-1 or (95% ci) 1.67 (1.31–2.13) 1.59 (1.25–2.03) 1.61 (1.24–2.09) p value 3.23 � 10�5 1.6 � 10�4 3.18 � 10�4 gdf-15 or (95% ci) 1.47 (1.22–1.77) 1.43 (1.20–1.70) 1.38 (1.14–1.67) p value 3.66 � 10�5 5.6 � 10�5 7.34 � 10�4 mb or (95% ci) 1.51 (1.25–1.83) 1.56 (1.30–1.88) 1.55 (1.28–1.89) p value 1.83 � 10�5 2.9 � 10�6 8.29 � 10�6 mmp-10 or (95% ci) 1.46 (1.20–1.78) 1.42 (1.19–1.70) 1.42 (1.67–1.73) p value 1.32 � 10�4 1.2 � 10�4 4.82 � 10�4 the following was adjusted for in the logistic regression models: model a: age, sex, cardiovascular risk factors and cohort; model b: age, sex, glucose control/diabetes factors, and cohort; model c: all relevant factors in model c ([a and b combined], hba1c, oral antidiabetic drug use and insulin treatment, lowand high-density lipoprotein, triglycerides, body mass index, cardiovascular disease at baseline, systolic and diastolic blood pressure, antihypertensive therapy, statins, and smoking status). gdf-15: growth differentiation factor 15; kim-1: kidney injury molecule 1; mb: myoglobin; mmp-10: matrix metalloproteinase 10. table 3. cox regression models for the association between discovered and replicated circulating proteins and time to major adverse cardiovascular events. protein model a: hazard ratio (95% ci) p value model b: hazard ratio (95% ci) p value kim-1 1.15 (0.88–1.50) 0.31 1.07 (0.79–1.45) 0.66 gdf-15 1.43 (1.03–1.98) 0.03 1.34 (0.96–1.88) 0.09 mb 1.17 (0.88–1.55) 0.30 1.12 (0.83–1.51) 0.45 mmp-10 1.21 (0.89–1.65) 0.23 1.27 (0.93–1.75) 0.13 model a was adjusted for age, sex, frailty effect for cohort, microalbuminuria, and kidney function; model b for all variables in model a and cardiovascular disease at baseline, smoking, low-density lipoprotein, and systolic blood pressure. ci: confidence interval; kim-1: kidney injury molecule 1; gdf-15: growth differentiation factor 15; mb: myoglobin; mmp-10: matrix metalloproteinase 10. 40 a. c. carlsson et al. metformin is the most common oral antidiabetic drug in sweden. furthermore, reference intervals for gdf-15 have been suggested in dkd (26). higher levels of circulating gdf-15 have been linked to an increased risk for several adverse outcomes, including a recent study showing an association with incident type 2 diabetes (27), deteriorating microalbuminuria (28), progression of albuminuria in persons with type 2 diabetes (28), kidney function decline and cardiovascular risk in persons with type 1 diabetes (29), early death in patients undergoing haemodialysis (30), as well as incident heart failure and cardiovascular events in the general population (31). the fact that gdf-15 was the only biomarker that was associated with incident cardiovascular events is interesting and also supported by several studies showing associations between gdf-15 levels and both cardiovascular morbidity and mortality (32–35). kim-1 kim-1 is expressed in the proximal tubule and excreted in the urine, and urinary kim-1 is used as a clinical marker of acute kidney damage (36–38). less is known about plasma levels of kim-1, but, out of 80 tested proteins, circulating kim-1 was the biomarker that had the strongest association with acr in the general population (3). apart from the mechanisms of kim-1 in acute kidney damage, experimental studies have shown that kim-1 is active in the regulation of immune responses activated by the t helper cell (39). circulating kim-1 has been associated with the number of carotid arteries affected by atherosclerotic plaques in the general population (40), as well as coronary artery atherosclerosis, and the risk of cardiovascular death in dialysis patients (41). whether circulating levels of kim-1 reflect atherosclerosis in the kidney in these diabetes patients remains to be established. myoglobin rhabdomyolysis is well known to be associated with acute kidney injury, and one of the proteins that are used as markers of rhabdomyolysis and its associated acute kidney injury is myoglobin (42). although myoglobin has not been put forward as a dkd biomarker, plasma levels of myoglobin have been associated with chronic kidney disease, and higher levels of myoglobin with higher stages of chronic kidney disease (43). our findings of an association between myoglobin and dkd suggest that myoglobin, in addition to its use in acute kidney injury, maybe a marker of slowly deteriorating kidney function in diabetes patients. mmp-10 matrix metalloproteinases have been suggested to be causally involved in many processes leading to kidney disease progression and cardiovascular disease (44). elevated levels of mmp-10 were independently associated with the severity of atherosclerosis in patients with chronic kidney disease (ckd) (45), and also associated with nephropathy in patients with type 1 diabetes (46). interestingly, mmp-10 was not associated with egfr-decline or acr in previous communitybased studies using the same assay (2,3) and may thus be dkd-specific. matrix remodelling properties of mmp-10 and its degradation products favour expansion of a thin membrane supporting the capillary loops in renal glomeruli called the mesangium, which may explain some of its effects in the development of dkd (47). in fact, glucose-induced mesangial matrix remodelling has been suggested as a mechanism leading to nephropathy, and thus mmp-10 has been suggested as a potential drug target to slow down diabetic nephropathy and retinopathy (46). strengths and limitations strengths of our investigation include the discovery/replication approach in multiple study samples, which add to the validity and generalizability of our findings. we cannot infer causality in the present study as it is of observational design. limitations include a possible selection bias for persons participating in cohort studies that in general often are healthier than the average patient population. another limitation is the fact that our study was based on single assessments of the proteins and kidney phenotypes. limitations of the proteomics assay include that only relative levels of the proteins are obtained, which makes defining relevant cut-off limits impossible. furthermore, the selection of the specific proteins on the olink cvd-i assay was not based on potential relevance for dkd. neither can we determine if it is the protein that has an effect on the kidney nor if it is the reduced clearance as an effect of reduced kidney function that explains our findings. since we did not perform kidney biopsies in our study participants, we were not able to rule out the misclassification of dkd due to other causes. finally, the limited sample size in our longitudinal analyses precluded stratified analyses in participants with versus without prevalent cardiovascular disease at baseline. conclusions we discovered and replicated four blood proteins associated with prevalent dkd. circulating levels of gdf-15 were associated with incident cardiovascular events in models adjusted for age, sex, kidney function, and microalbuminuria; however, the association was attenuated when adjusted for established cardiovascular risk factors. our study encourages more studies evaluating large-scale proteomics in order to discover new pathways leading to dkd and pinpoint prognostic markers of cardiovascular risk. author contributions acc conceived the study, drafted the manuscript, and interpreted data. cn analysed the data and revised/edited the manuscript. js, jjc, ei, and tf contributed to methodology and reviewed/edited the manuscript. ur contributed with data from ulsam and read and reviewed/edited manuscript. cj€o and fhn reviewed/edited the manuscript and collected the cardipp data. data from the pivus study were collected by ll, and he upsala journal of medical sciences 41 also reviewed/edited the manuscript. j€a interpreted data, reviewed/ edited manuscript, contributed to discussion, and provided funding. disclosure statement the study was investigator-initiated and -driven. erik ingelsson is a scientific advisor for precision wellness, and has received consulting fees from olink proteomics for work unrelated to the present project. the company had no influence over design, analysis, or interpretation of data in the present study, and did not provide any funding for the study. the other authors report no conflicts of interests. notes on contributors axel c. carlsson, msc, phd is an associate professor at the department of neurobiology, care sciences and society (nvs), karolinska institutet, huddinge, sweden. christoph nowak, phd, bm bch, dipl-psych, is a post-doctoral researcher at the division of family medicine and primary care, department of neurobiology, care sciences and society (nvs), karolinska institutet, huddinge, sweden. lars lind, md, phd is a professor at the department of medical sciences, uppsala university, uppsala, sweden. carl johan €ostgren, md, phd is a professor at the department of medical and health sciences, link€oping university, link€oping, sweden. fredrik h. nystr€om, md, phd is a professor at the department of medical and health sciences, link€oping university, link€oping, sweden. johan sundstr€om, md, phd is a professor at the department of medical sciences, uppsala university, uppsala, sweden. juan jesus carrero, msc pharm, phd is a professor at the department of medical epidemiology and biostatistics, karolinska institutet, stockholm, sweden ulf riserus, phd is a professor at the department of public health and caring sciences, clinical nutrition and metabolism, uppsala university, uppsala, sweden. erik ingelsson, md, phd is a professor at the stanford cardiovascular institute, and the stanford diabetes research center, stanford university, stanford, ca 94305, and the molecular epidemiology and science for life laboratory, uppsala university tove fall, phd, is a associate professor at the department of medical sciences, molecular epidemiology and scilife laboratory, uppsala university, uppsala, sweden. johan €arnl€ov, md, phd, is a professor of family medicine at the division of family medicine and primary care, department of neurobiology, care sciences and society (nvs), karolinska institutet, huddinge, sweden, and the school of health and social studies, dalarna university, falun, sweden. orcid ulf riserus http://orcid.org/0000-0002-8620-4586 references 1. tuttle kr, bakris gl, bilous rw, chiang jl, de boer ih, goldsteinfuchs j, et al. diabetic kidney disease: a report from an 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2010;78:1275–80. 46. toni m, hermida j, goni mj, fernandez p, parks wc, toledo e, et al. matrix metalloproteinase-10 plays an active role in microvascular complications in type 1 diabetic patients. diabetologia. 2013;56:2743–52. 47. vestra saller dm, mauer a, fioretto m. p. role of mesangial expansion in the pathogenesis of diabetic nephropathy. j nephrol. 2001;14:s51–s7. upsala journal of medical sciences 43 abstract introduction methods study cohorts outcome definitions, inclusion criteria, and number of eligible participants ethical permission multiplex protein assay statistical analysis results baseline characteristics associations between proteins and prevalent dkd associations of proteins and mace incidence in individuals with prevalent dkd discussion main findings comparison with previous studies gdf-15 kim-1 myoglobin mmp-10 strengths and limitations conclusions author contributions disclosure statement references alternative diagnosis to heparin-induced thrombocytopenia in two critically ill patients despite a p full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 alternative diagnosis to heparin-induced thrombocytopenia in two critically ill patients despite a positive pf4/heparin-antibody test gregor hron, folke knutson, thomas thiele, karina althaus, christoph busemann, sigrun friesecke, andreas greinacher & norbert lubenow to cite this article: gregor hron, folke knutson, thomas thiele, karina althaus, christoph busemann, sigrun friesecke, andreas greinacher & norbert lubenow (2013) alternative diagnosis to heparin-induced thrombocytopenia in two critically ill patients despite a positive pf4/heparin-antibody test, upsala journal of medical sciences, 118:4, 279-284, doi: 10.3109/03009734.2013.838811 to link to this article: https://doi.org/10.3109/03009734.2013.838811 © informa healthcare published online: 09 oct 2013. submit your article to this journal article views: 697 view related articles citing articles: 5 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.838811 https://doi.org/10.3109/03009734.2013.838811 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.838811 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.838811 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.838811#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.838811#tabmodule upsala journal of medical sciences. 2013; 118: 279–284 case report from uppsala university hospital alternative diagnosis to heparin-induced thrombocytopenia in two critically ill patients despite a positive pf4/heparin-antibody test gregor hron1, folke knutson2, thomas thiele1, karina althaus1, christoph busemann3, sigrun friesecke4, andreas greinacher1 & norbert lubenow2 1abteilung für transfusionsmedizin, institut für immunologie und transfusionsemedizin, ernst-moritz-arndt university greifswald, germany, 2department of clinical immunology and transfusion medicine, uppsala university hospital, uppsala, sweden, 3klinik für innere medizin c, ernst-moritz-arndt university greifswald, germany, and 4klinik für innere medizin b, ernst-moritz-arndt university greifswald, germany abstract thrombocytopenia can cause diagnostic challenges in patients who have received heparin. heparin-induced thrombocytopenia (hit) is often considered in the differential diagnosis, and a positive screening can be mistaken as confirmation of the disorder. we present two patients who both received low-molecular-weight heparin for several days. in the first patient, clinical judgment rejected the suspicion of hit despite a positive screening assay, and treatment for the alternative diagnosis of posttransfusion purpura was correctly initiated. in the second patient, the inaccurate diagnosis hit was pursued due to a positive screening assay, while the alternative diagnosis of drug-dependent thrombocytopenia caused by piperacillin/tazobactam was rejected. this resulted in re-exposure to piperacillin/tazobactam which caused a second episode of severe thrombocytopenia. a positive screening assay for platelet factor 4/heparin-antibody should be verified by a functional assay, especially in patients with low pretest probability for hit. key words: drug-induced thrombocytopenia, heparin-induced thrombocytopenia, hit, piperacillin-induced thrombocytopenia, post-transfusions purpura, ptp introduction patients developing thrombocytopenia during treatment with heparin or low-molecular-weight heparin for more than four days can pose a diagnostic challenge. if these patients have no other obvious reason for a rapid decrease in platelet counts, such as platelet consumption in severe bleeding or sepsis, an immunological cause is the most likely reason for thrombocytopenia (1). the accurate differential diagnosis between the antibody-mediated procoagulant syndrome of heparin-induced thrombocytopenia (hit) and other antibody-mediated thrombocytopenic bleeding disorders is important, as management strategies are diametrically opposed (2). the situation is especially difficult as commercially available antigen assays for detection of antibodies against platelet factor 4 (pf4)/heparin complexes— the cause of hit—show a poor specificity for clinically relevant pf4/heparin antibodies (3,4). hit is usually caused by high-titer igg antibodies directed against a complex of pf4 and heparin. iga, igm, and low-titer igg antibodies directed against the pf4/ heparin complexes usually do not cause hit, but can occur in up to 75% of patients in some patient cohorts (5). this can result in a positive pf4/heparin antigen assay caused by non-pathogenic, non-plateletactivating antibodies. these test results are often misleading and can even distract the clinician from the correct alternative diagnosis. as only igg antibodies to correspondence: norbert lubenow, md, department of clinical immunology and transfusion medicine, uppsala university hospital, 75185 uppsala, sweden. fax: +46 18 538280. e-mail: norbert.lubenow@akademiska.se (received 8 july 2013; accepted 25 august 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.838811 http://informahealthcare.com/journal/ups mailto:norbert.lubenow@akademiska.se pf4/heparin activate platelets (3), the risk for an erroneous interpretation of results is especially high with antigen assays recognizing igg, iga, and igm antibodies combined. clinical judgment, preferably with the use of clinical pretest scores such as the ‘4 t’ score (assessing degree of thrombocytopenia, timing of platelet count fall, thrombosis or other sequelae, and other causes of thrombocytopenia (6)) or the recently proposed hit expert probability score (hep score) (7), and the hit simple scoring system (8) should be applied before screening tests for antibodies directed against pf4/heparin complexes are performed. we demonstrate two patients who presented with thrombocytopenia and a positive pf4/heparin antigen test, but a low clinical likelihood for hit. in the first patient application of systematic scoring systems for clinical hit almost immediately led to the right non-hit diagnosis. in contrast, clinical judgment in the second patient resulted in a misdiagnosis of hit, although use of a systematic score could have guided the clinician towards an alternative diagnosis. case presentations all unnecessary patient details such as sex and exact age were omitted to prevent identification. approval for publication has been obtained from the ethics committees responsible for greifswald university hospital (patient 1) and uppsala university hospital (patient 2), respectively. case 1 clinical presentation. an elderly patient presented to the emergency department with fever and pancytopenia (platelets 23 g/l, hemoglobin 4.9 mmol/l, white blood cells 1.8 g/l) caused by oral methotrexate. methotrexate was administered for rheumatoid arthritis and was immediately withdrawn. imipenem was started to treat febrile neutropenia, and the patient received two red blood cell concentrates and one platelet concentrate. subsequently the platelet count rose from 23 to 69 g/l, and on day 2 lowmolecular-weight heparin for thrombosis prophylaxis, fluconazole for fungus-induced mucositis, and amiodarone for supraventricular tachycardia were started. on day 9 the patient developed fever, tachycardia, and hypotension, and the platelet count fell from 234 to 2 g/l (figure 1). for possible sepsis-associated thrombocytopenia antibiotic treatment with ciprofloxacin was initiated. on the same day, the patient developed diffuse, transfusion-dependent bleeding from peripheral and central line punctures. due to the low platelet count one platelet concentrate was given. this caused a febrile transfusion reaction but no platelet count increment after 1 hour. differential diagnosis and treatment. the treating physician suspected hit due to the fall in the platelet count. however, the 4 t score was only 3 (platelet count nadir <10 g/l, onset between days 5 and 10, no new thrombosis, and other causes for thrombocytopenia (such as drug-dependent or sepsis) were possible). the hep score was 1 (magnitude of fall >50%, days after admission p la te le t c o u n t (g /l ) h e m o g lo b in le v e l (m m o l/l ) platelet count hb red blood cell transfusion platelet transfusion ig g i.v. ig g i.v. dalteparin 5,000 u/d 2 4 6 8 10 12 14 0 100 200 300 400 2 4 6 8 10 12 14 4 5 6 7 8 9 figure 1. platelet count profile and relevant medication of patient 1. 280 g. hron et al. fall began 5–10 days after heparin exposure, nadir £20 g/l, presence of bleeding, and newly initiated non-heparin medication known to cause thrombocytopenia), and the simple scoring system was 0 (antibody-mediated thrombocytopenia as a significant competing cause for thrombocytopenia). therefore, the pretest probability for hit was low, and other causes for thrombocytopenia had to be considered. thrombotic thrombocytopenic purpura and disseminated intravascular coagulation can also cause low platelet counts but were promptly excluded due to the absence of fragmented red cells, elevated fibrinogen, and only slightly elevated d-dimer levels, respectively. the abrupt fall in the platelet count and the absence of a platelet count increase 1 hour after transfusion of a platelet concentrate suggested an antibody-mediated disorder. post-transfusion purpura, drug-dependent thrombocytopenia, and autoimmune thrombocytopenia were considered. antibiotics are relatively frequent causes of drug-dependent thrombocytopenia. however, as thrombocytopenia occurred rapidly after ciprofloxacin was given, drug-dependent thrombocytopenia to this drug was regarded as very unlikely, and ciprofloxacin was continued. since a febrile reaction after transfusion of platelet concentrates is typical for post-transfusion purpura, this disorder was considered most likely. however, in the acute situation no clear diagnosis could be made on clinical grounds. as the patient showed severe bleeding, intravenous igg (1 g/kg body weight) was administered to treat post-transfusion purpura and/or autoimmune thrombocytopenia. furthermore, all drugs started since admission of the patient to the hospital were withdrawn to interrupt the immune response in potential drugdependent thrombocytopenia. this resulted in cessation of bleeding within 1 day and recovery of the platelet count within 3 days. subsequent laboratory testing. laboratory testing by an antigen test revealed pf4/heparin igm antibodies at a lowopticaldensity(0.56),whilenoiggorigaantibodies were found. a glycoprotein-specific assay confirmed the presence of strongly reacting alloantibodies against the human platelet antigen-1a, establishing the diagnosis of post-transfusion purpura (9). furthermore, pseudomonas aeruginosa sensitive to ciprofloxacin was found in the blood cultures from day 9. septic shock caused by pseudomonas aeruginosa may have contributed to the clinical presentation, especially the high temperature, which is typical for neither post-transfusion purpura nor drug-dependent thrombocytopenias. case report 2 clinical presentation. an elderly patient without relevant medical history presented with a traumatic fractured neck of femur (day 1, figure 2). on day 3 a hip endoprosthesis was inserted. during surgery the patient developed hypoxemia and hypotension, which 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 0 100 200 300 400 500 600 700 800 days after admission p la te le t c o u n t (g /l ) platelet count dalteparin 5,000 u/d piperacillin/ tazobactam danaparoid platelet transfusion s u rg e ry figure 2. platelet count profile and relevant medication of patient 2. thrombocytopenia misdiagnosed as hit 281 eventually necessitated admission to the critical care unit. cement or fat embolism was suspected, and the patient received one dose of enoxaparin 40 mg postsurgery (no heparin was given before surgery). thrombosis prophylaxis was changed to dalteparin 5000 units once daily from day 5 onwards. on day 10 piperacillin/tazobactam was commenced for pneumonia. from day 18 to day 20, the platelet count fell from 208 g/l to 5 g/l. the patient developed petechiae on the trunk, while no clinical signs of thromboembolism were present. differential diagnosis and treatment. hit or piperacillin/ tazobactam-induced thrombocytopenia was suspected, and both piperacillin/tazobactam and dalteparin were stopped. three platelet concentrates were transfused at a platelet count of 5 g/l without an increment. a particle-gel immunoassay (pagia, diamed gmbh, cressier, switzerland) for anti-pf4/heparin antibodies was positive, which seemed to be compatible with the diagnosis of hit, and danaparoid 2 � 750 units s.c. was started for thrombosis prophylaxis. subsequently the platelet count rose to a stable plateau until day 30, which seemed to confirm hit. at that time the patient again developed fever, and piperacillin/tazobactam was restarted at a platelet count of 446 g/l. the next day the platelet count fell to 8 g/l although the patient still received danaparoid, and the alternative diagnosis of piperacillin/ tazobactam-induced thrombocytopenia was reconsidered. both piperacillin/tazobactam and danaparoid were withdrawn and the platelet count recovered over the following 5 days. hit was initially suspected by the treating physician due to the fall in the platelet count. as in case 1 the 4 t score was low at only 2 points (platelet count nadir <10 g/l, onset after day 10, no new thrombosis, and other causes for thrombocytopenia (such as drug-dependent) were possible). the hep score was 0 (magnitude of fall >50%, fall 11–14 days after heparin exposure, nadir £20 g/l, presence of bleeding, and newly initiated non-heparin medication known to cause thrombocytopenia) and the simple scoring system was also 0 (antibody-mediated thrombocytopenia as a significant competing cause for thrombocytopenia). therefore, the pretest probability for hit was low in three scoring systems, and other causes for thrombocytopenia should have been considered. subsequent laboratory testing. further laboratory testing revealed strongly reactive piperacillin-dependent platelet antibodies, whereas the anti-pf4/heparinantibody elisa was positive for anti-pf4/heparin iga antibodies only, but negative for anti-pf4/heparin igg or igm antibodies. the functional assay for platelet-activating clinically relevant hit antibodies (heparin-induced platelet activation test) (10) was negative. the assay indicating drug-dependent antibodies (2), the anti-pf4/heparin-antibody elisa (3), and the heparin-induced platelet activation (hipa) (10) test were performed as described elsewhere. discussion patients who develop sudden and/or severe thrombocytopenia without an obvious reason during their hospital stay can cause a diagnostic and management dilemma. these patients have often received unfractionated heparin or low-molecular-weight heparin for several days, but also several other new drugs and/ or transfusion of blood products. whereas in hit a high risk of thrombosis caused by increased thrombin generation mandates alternative anticoagulation despite low platelet counts (11), other thrombocytopenias are associated with an increased risk of severe bleeding (2). the timing of platelet count decrease is a very important parameter to differentiate a normal early platelet count decrease after major surgery from immune-mediated thrombocytopenias (1). while a platelet count nadir until day 3 after major orthopedic or cardiac surgery is normal and should be expected, a rapid decrease in platelet counts that begins after day 4, and after the platelet count has already started to increase again, is typical for immune-mediated causes in the absence of other causes such as sepsis (1). in both patients, the drop in platelet counts began 8 and 13 days, respectively, after the start of lowmolecular-weight heparin. in patient 1, the platelet count started falling 8–9 days after the first transfusions and after exposure to novel drugs. in patient 2, the platelet count fall started 6 days after the start of antibiotic treatment and almost 2 weeks after surgery. in both patients the platelet count fall was within the typical time frame for immune-mediated thrombocytopenias (drug-dependent thrombocytopenia, hit, and post-transfusion purpura). hit is the most frequent of these three causes. it occurs in 0.2% of patients treated with low-molecular-weight heparin after hip replacement surgery, according to a metaanalysis of orthopedic and surgical patients (12). piperacillin-induced immune thrombocytopenia on the other hand is rare, and only a few cases (13-17) and a recent series of 13 patients have been reported (18). interestingly, rousan et al. reported a patient with sepsis and disseminated intravascular coagulation (dic) in whom hit or piperacillin-induced immune thrombocytopenia were also considered (and the 282 g. hron et al. patient was shown to have the latter). in contrast to our patient (case 2) they were able to rule out hit by a negative pf4/heparin elisa (18). post-transfusion purpura is even rarer than drug-dependent thrombocytopenias, and fewer than 300 cases have been reported (19). even if thrombocytopenia is a common feature of hit, platelet count values <20 g/l do not usually occur in hit, but are typical for drug-dependent thrombocytopenia and post-transfusion purpura (20). the bleeding signs observed in both patients (diffuse bleeding and petechiae, respectively) also favored the diagnosis of non-hit-mediated thrombocytopenia. in clinical practice it is often difficult to weigh all signs and symptoms in a given thrombocytopenic patient. in this regard assessment of the patient by a systematic score for hit, e.g. the 4 t score (6), can be helpful to guide further assessment and diagnosis. in fact, the 4 t score in both patients was only 2 and 3, respectively, out of 8, ranking them in the low pretest probability group where laboratory testing might not even be necessary (21). in addition, both patients had a low probability for hit in the hep and the simple scoring system. the particle-gel immunoassay for anti-pf4/heparin-antibodies (patient 2) as well as the anti-heparin/pf4 elisa (both patients) has a high sensitivity but low specificity for clinical hit (22). their strength is to rule out hit when negative. however, as demonstrated by the present cases, reliance on a positive antigen assay alone can lead to a wrong suspicion or even a misdiagnosis of hit. in patient 2 the erroneous diagnosis of hit even caused a second period of severe thrombocytopenia due to reexposure to the causing antibiotic, luckily without major bleeding complications. these cases underscore that a positive antigen assay in patients with suspicion of hit needs further clinical assessment, especially if there is a low pretest probability of hit, which is particularly important if the test is not igg-specific (23). suitable methods for verification are functional assays, which are the only assays that can demonstrate the platelet-activating properties of hit-antibodies (non-activating low-titer igg-antibodies seem to lack clinical significance). conclusion in patients with pronounced thrombocytopenia and clinical features favoring other diagnoses than hit, management can be misguided by a positive hitantibody assay. we recommend the use of a systematic approach for the diagnosis of hit, i.e. using a clinical score such as the 4 t score for hit as well as further laboratory evaluation of a positive antigen test by a functional assay, especially in patients with low pretest probability of hit. acknowledgements we are grateful to dr daniel lahner, medical university of vienna, austria, for his thoughtful comments and critical revision of the manuscript. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. greinacher a, selleng k. thrombocytopenia in the intensive care unit patient. hematology am soc hematol educ program. 2010;2010:135–43. 2. greinacher a, eichler p, lubenow n, kiefel v. drug-induced and drug-dependent immune thrombocytopenias. rev clin exp hematol. 2001;5:166–200. 3. greinacher a, juhl d, strobel u, wessel a, lubenow n, selleng k, et al. 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www.ncbi.nlm.nih.gov/pubmed/15985543?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15985543?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15985543?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19761734?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19761734?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12791166?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12791166?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/23036037?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/23036037?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9770174?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/9770174?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/1287819?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/1287819?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19802882?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19802882?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19802882?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19224780?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19224780?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11108897?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11108897?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11108897?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11108897?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19422442?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19422442?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11442479?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11442479?dopt=abstract abstract introduction case presentations case 1 clinical presentation differential diagnosis and treatment subsequent laboratory testing case report 2 clinical presentation differential diagnosis and treatment subsequent laboratory testing discussion conclusion acknowledgements declaration of interest references atrial fibrillation in patients undergoing coronary artery surgery is associated with adverse outcome article atrial fibrillation in patients undergoing coronary artery surgery is associated with adverse outcome gorav batraa,b, anders ahlssonc, bertil lindahla,b, lars lindhagena, anders wickbomc and jonas oldgrena,b auppsala clinical research center, uppsala, sweden; bdepartment of medical sciences, cardiology, uppsala university, uppsala, sweden; cdepartment of cardiothoracic and vascular surgery, school of medicine and health, €orebro university, €orebro, sweden abstract background: the aim was to determine the association between atrial fibrillation (af) and outcome in patients undergoing coronary artery bypass grafting (cabg). methods: all patients undergoing cabg between january 2010 and june 2013 were identified in the swedish heart surgery registry. outcomes studied were all-cause mortality, cardiovascular mortality, myocardial infarction, congestive heart failure, ischemic stroke, and recurrent af. patients with history of af prior to surgery (preoperative af) and patients without history of af but with af episodes post-surgery (postoperative af) were compared to patients with no af using adjusted cox regression models. results: among 9,107 identified patients, 8.1% (n ¼ 737) had preoperative af, and 25.1% (n ¼ 2,290) had postoperative af. median follow-up was 2.2 years. compared to no af, preoperative af was associated with higher risk of all-cause mortality, adjusted hazard ratio with 95% confidence interval (hr) 1.76 (1.33–2.33); cardiovascular mortality, hr 2.43 (1.68–3.50); and congestive heart failure, hr 2.21 (1.72–2.84). postoperative af was associated with risk of all-cause mortality, hr 1.27 (1.01–1.60); cardiovascular mortality, hr 1.52 (1.10–2.11); congestive heart failure, hr 1.47 (1.18–1.83); and recurrent af, hr 4.38 (2.46–7.78). no significant association was observed between preor postoperative af and risk for myocardial infarction and ischemic stroke. conclusions: approximately 1 in 3 patients undergoing cabg had preor postoperative af. patients with preor postoperative af were at higher risk of all-cause mortality, cardiovascular mortality, and congestive heart failure, but not of myocardial infarction or ischemic stroke. postoperative af was associated with higher risk of recurrent af. article history received 12 march 2018 revised 10 july 2018 accepted 15 july 2018 keywords atrial fibrillation; cardiovascular disease; coronary artery bypass grafting introduction atrial fibrillation (af) is a common arrhythmia with an increasing incidence due to aging population (1). patients with af have an increased risk of mortality and morbidity, including ischemic stroke (2,3). history of preoperative af is a common finding among patients undergoing coronary artery bypass grafting (cabg), with prevalence varying between 7% and 9% (4,5). to our knowledge, only two single-center studies have reported outcome in patients with preoperative af undergoing cabg, both reporting an increased risk of long-term mortality (4,5). moreover, previous studies have reported that postoperative af may occur in approximately one-third of patients undergoing cabg (6,7). to date, most studies on patients undergoing cabg have studied the effects of postoperative af on mortality and stroke (6–11). previous studies have found postoperative af to be associated with increased risk of short-term mortality and stroke (6,8,9). also, some data suggest that postoperative af might be associated with longterm mortality (7, 9–11). however, only one small single-center study has simultaneously evaluated both preand postoperative af in relation to outcome in patients undergoing cabg (4). in addition, only two single-center studies have assessed the risk of recurrent af in patients with new-onset postoperative af, with data suggesting an increased risk (7,12). in this study on patients undergoing cabg, based on data from swedish registers with complete national coverage, we sought to answer the following question: in patients undergoing isolated cabg, what are the impacts of preoperative af and new-onset postoperative af on all-cause and cardiovascular mortality, myocardial infarction, congestive heart failure, ischemic stroke, and recurrent af? materials and methods study population and data collection clinical and procedural data were obtained from the swedish web-system for enhancement and development of evidencecontact gorav batra, md, phd, e-mail: gorav.batra@ucr.uu.se uppsala clinical research center, uppsala science park, hubben, dag hammarskj€olds v€ag 38, s-751 85 uppsala, sweden supplemental data for this article can be accessed here. � 2018 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 1, 70–77 https://doi.org/10.1080/03009734.2018.1504148 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1504148&domain=pdf https://doi.org/10.1080/03009734.2018.1504148 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1504148 http://www.tandfonline.com based care in heart disease evaluated according to recommended therapies (swedeheart) registry and was enriched with data from the national patient registry and the national dispensed drug registry. swedeheart is a national quality registry that contains several sub-registries, including the swedish heart surgery registry which captures data about all patients undergoing thoracic surgery in sweden. the swedeheart registry also includes data about all patients admitted to coronary care units due to symptoms indicative of acute coronary syndrome and data on patients undergoing coronary angiography (13). patients included in swedeheart are informed about their participation in the registry but do not provide written consent; however, they have the right to withdraw their participation. mortality data were obtained from the swedish cause of death registry, which is a mandatory nationwide registry that has collected vital status of all swedish citizens since 1961. the national patient registry is a mandatory nationwide registry that includes discharge diagnosis for all patients admitted to swedish hospitals since 1987. previous studies have shown that the registry has high validity for several diagnoses, including myocardial infarction, congestive heart failure, and stroke (14). the national dispensed drug registry has captured data about all prescribed drugs dispensed at swedish pharmacies since 2005 (15). in our study, baseline pharmacologic treatment was determined based on drugs dispensed 6 months prior to admission and within 30days from surgery. data linkage between registries was approved and performed by the national board of health and welfare in sweden using the unique 10-digit identifier relating to all swedish citizens. see supplementary table 1 (available online) for detailed description of data collection, linkage, and variable definition. the ethics committee at karolinska institute, stockholm approved the study protocol. in the present study, inclusion period was defined between january 2010 and june 2013. during this period, 10,008 consecutive patients underwent isolated cabg at eight sites and were identified in swedeheart. among identified patients, 9,107 individuals were eligible for inclusion. patients were excluded if they died within 30 days from surgery, had missing discharge date, were discharged over 30 days after surgery, or had a previous registration in swedeheart due to cabg (figure 1). definition of exposure the main exposure was af. history of af, i.e. preoperative af, was considered to be present if indicated as occurring before surgery in swedeheart, or if the diagnosis of af appeared in the national patient registry prior to admission. new-onset postoperative af was considered in patients with no history of af according to swedeheart and the national patient registry, but with an episode of af post-surgery according to swedeheart. as controls, we used patients with no diagnosis of af preor post-cabg. definition of outcome outcomes under investigation were all-cause mortality, cardiovascular mortality, myocardial infarction, hospitalization due to congestive heart failure, ischemic stroke, and recurrent af. outcomes were identified in the national patient registry and in the national cause of death registry using international classification of diseases 10th revision codes; see supplementary table 2 (available online) for details. for recurrent af, direct-current (dc) cardioversion was used as a surrogate marker of symptomatic relapse in af as previously reported (16). as a sensitivity analysis, diagnosis of recurrent af in the national patient registry was accounted for, however, with the risk of af diagnosis codes being used for both follow-up visits and for true relapses. time at risk was counted from 30 days after cabg surgery. this blanking period was applied to avoid registration of in-hospital complications and double-counting of events when patients moved between hospital wards. when analyzing outcome, complete follow-up was available for all cases. patients were censored at the end of follow-up, which lasted up to 31 december 2013, giving a minimum follow-up of 6 months. figure 1. study population. upsala journal of medical sciences 71 https://doi.org/10.1080/03009734.2018.1504148 https://doi.org/10.1080/03009734.2018.1504148 for all-cause mortality, no other censoring scheme was applied. for cardiovascular mortality, myocardial infarction, congestive heart failure, ischemic stroke, and recurrent diagnosis of af, patients were also censored for allcause mortality. statistical analyses demographics, baseline characteristics, euroscore i scoring system for prediction of mortality in patients undergoing cardiac surgery (17), hospital course, perioperative data, and discharge medication among patients with no af, preoperative af, and postoperative af were presented descriptively using percentages for categorical variables and with median and interquartile range for continuous variables. to test for differences, pearson’s chi-square test was used for categorical variables and the kruskal–wallis test for continuous variables. the risk of all-cause mortality and recurrent diagnosis of af in relation to preand postoperative af was illustrated using kaplan–meier survival plots. event rates according to the number of events per 100 person-years were calculated. unadjusted and adjusted cox proportional-hazards regression models were estimated for each outcome and presented using hazard ratios (hr) with 95% confidence interval (ci). in the unadjusted models, af status was entered as the sole variable. in the adjusted models, factors in the cha2ds2vasc scoring system (congestive heart failure, hypertension, age [3 knot restricted cubic spline], diabetes mellitus, stroke, transient ischemic attack or thromboembolism, vascular disease, and sex) (18), hospital (c distributed random frailty effect), and year of inclusion were accounted for. no data were missing in regard to the variables included in the adjusted cox proportional-hazards regression models. as sensitivity analysis, a propensity-score matched analysis was performed in which propensity scores for the likelihood of no af or postoperative af were obtained using random effects logistic regression models with all variables in the adjusted cox proportional-hazards regression models as explanatory variables. matching was done in 1:1 based on estimated propensity scores with a caliper of 0.001, resulting in 2,211 patients in the no af cohort and 2,211 patients in the postoperative af cohort (19). all statistical analyses were conducted at uppsala clinical research center using r version 3.1.0. all statistical tests were two-sided using a p values of <0.05 as significant. results study population and patient characteristics the study population had a median age of 68 years, and 18.9% were women. a history of af prior to cabg was documented in 737 (8.1%) patients, and 2,290 (25.1%) patients had new-onset postoperative af. table 1 summarizes patient characteristics stratified by af status. patients with preoperative af were older than patients with postoperative af, and accordingly more likely to have concomitant diseases. compared to patients with no af, patients with postoperative af were older and more likely to have comorbidities such as hypertension, history of myocardial infarction, congestive heart failure, and chronic obstructive pulmonary disease. compared to patients with no af, a higher proportion of patients with preand postoperative af had reduced left ventricular ejection fraction and higher creatinine values. likewise, the euroscore 1 was numerically higher among patients with preand postoperative af. additional perioperative and postoperative data are presented in table 1. at discharge, patients with preoperative af versus postoperative af were more likely to receive oral anticoagulants, while patients with postoperative af were more likely to receive aspirin and/or p2y12 inhibitors. however, compared to patients with no af, patients with postoperative af were more likely to receive oral anticoagulants, diuretics, digoxin, sotalol, and amiodarone at discharge. outcome analyses clinical outcomes post-cabg were evaluated according to preoperative af versus no af and postoperative af versus no af. median follow-up was 2.2 years. figures 2 and 3 present unadjusted kaplan–meier plots illustrating cumulative event rates for all-cause mortality and recurrent af within 4 years of post-surgery, with exclusion of a 30-day blanking period, stratified by af status at baseline. unadjusted cumulative event rates per 100 person-years for all-cause mortality and cardiovascular mortality were numerically higher in patients with preand postoperative af compared to no af. using cox proportional-hazards regression models with no af as reference, and with adjustment for clinically relevant variables, preoperative af and postoperative af were associated with higher risk of allcause mortality, adjusted hazard ratio (hr) with 95% confidence interval 1.76 (1.33–2.33) and hr 1.27 (1.01–1.60), respectively (figures 2 and 4). for cardiovascular mortality, a similar risk was observed for patients with preand postoperative af, hr 2.43 (1.68–3.50) and hr 1.52 (1.10–2.11), respectively. as compared to no af, patients with preand postoperative af had a higher number of hospitalizations due to congestive heart failure during follow-up, with similar findings found in adjusted analyses, hr 2.21 (1.72–2.84) and hr 1.47 (1.18–1.83), respectively. in patients with preoperative af versus no af, a higher non-adjusted hr was recorded in regard to myocardial infarction, but with a non-significant adjusted hr of 1.37 (0.97–1.92). for postoperative af versus no af, no significant observation was made in regard to myocardial infarction. preoperative af and postoperative af were not associated with ischemic stroke (figure 4). the cumulative incidence rate per 100 person-years for recurrent symptomatic af after discharge was numerically higher for patients with new-onset postoperative af compared with no af (0.7 versus 0.2). this association persisted after adjustments; hr 4.38 (2.46–7.78) (figures 3 and 4). 72 g. batra et al. table 1. baseline table: patient characteristics, admission year, and clinical and in-hospital characteristics of coronary artery bypass graft surgery patients in relation to atrial fibrillation status. variable no af (n ¼ 6,080) preoperative af (n ¼ 737) postoperative af (n ¼ 2,290) p value demographics age, median (iqr), years 66 (60–72) 72 (67–77) 70 (64–75) <0.001 sex, women 1,171 (19.3) 141 (19.1) 406 (17.7) 0.27 smoking, n ¼ 8,493 1,031 (18.3) 84 (12.2) 302 (14.0) <0.001 bmi, median (iqr), n ¼ 8,990 27.1 (24.7–29.7) 27.2 (24.6–30.1) 27.0 (24.7–29.7) 0.55 admission year 0.009 2010 1,901 (31.3) 202 (27.4) 643 (28.1) 2011 1,762 (29.0) 206 (28.0) 660 (28.8) 2012 1,670 (27.5) 223 (30.3) 657 (28.7) 2013 747 (12.3) 106 (14.4) 330 (14.4) comorbidities and presentation at admission diabetes mellitus 1,851 (30.4) 285 (38.7) 675 (29.5) <0.001 hypertension 4,128 (67.9) 594 (80.6) 1,697 (74.1) <0.001 myocardial infarction 3,595 (59.1) 560 (76.0) 1,421 (62.1) <0.001 congestive heart failure 974 (16.0) 280 (38.0) 406 (17.7) <0.001 peripheral vascular disease 264 (4.3) 56 (7.6) 110 (4.8) <0.001 thromboembolism 14 (0.2) 3 (0.4) 10 (0.4) 0.26 ischemic / unknown stroke 287 (4.7) 60 (8.1) 114 (5.0) <0.001 transient ischemic attack 115 (1.9) 37 (5.0) 69 (3.0) <0.001 hemorrhagic stroke 44 (0.7) 4 (0.5) 15 (0.7) 0.83 any bleeding 291 (4.8) 63 (8.5) 98 (4.3) <0.001 renal failure 125 (2.1) 50 (6.8) 56 (2.4) <0.001 chronic obstructive pulmonary disease 319 (5.2) 62 (8.4) 138 (6.0) 0.002 cancer diagnosis within 3 years 146 (2.4) 33 (4.5) 61 (2.7) 0.004 previous pci 1,365 (22.5) 184 (25.0) 490 (21.4) 0.13 previous cabg 99 (1.6) 21 (2.8) 35 (1.5) 0.04 hospital course and perioperative data indication for cabg, n ¼ 8,886 <0.001 stable coronary artery disease 2,533 (42.8) 232 (32.4) 921 (41.0) unstable angina / acute mi 3,389 (57.2) 485 (67.6) 1,326 (59.0) euroscore i, median (iqr), n ¼ 9,062 4 (2–6) 6 (4–8) 5 (3–7) <0.001 extracorporeal circulation 6,014 (98.9) 724 (98.2) 2,256 (98.5) 0.14 number of central anastomoses, n ¼ 7,434 <0.001 0 413 (8.5) 48 (8.4) 96 (4.8) 1 2,153 (44.1) 254 (44.4) 876 (44.2) 2 2,163 (44.3) 250 (43.7) 938 (47.3) �3 149 (3.1) 20 (3.5) 74 (3.7) number of peripheral anastomoses, n ¼ 7,434 <0.001 0 250 (5.1) 17 (3.0) 33 (1.7) 1 149 (3.1) 24 (4.2) 59 (3.0) 2 919 (18.8) 106 (18.5) 372 (18.8) �3 3,560 (73.0) 425 (74.3) 1,520 (76.6) internal mammary artery, left, n ¼ 7,434 4,352 (89.2) 497 (86.9) 1,834 (92.4) <0.001 internal mammary artery, right, n ¼ 7,434 92 (1.9) 6 (1.0) 30 (1.5) 0.24 vein graft, n ¼ 7,434 4,465 (91.5) 525 (91.8) 1,882 (94.9) <0.001 radial artery graft, n ¼ 7,434 59 (1.2) 15 (2.6) 49 (2.5) <0.001 postoperative bleeds, n ¼ 9,080 207 (3.4) 31 (4.2) 113 (4.9) 0.005 postoperative stroke, n ¼ 9,093 48 (0.8) 12 (1.6) 35 (1.5) 0.003 left ventricular ejection fraction <50%, n ¼ 8,160 1,466 (27.0) 307 (44.7) 609 (29.7) <0.001 creatinine, median (iqr), mmol/l, n ¼ 9,100 87 (75–105) 100 (83–129) 93 (79–120) <0.001 cha2ds2-vasc score at discharge <0.001 cha2ds2-vasc score ¼0 0 (0.0) 0 (0.0) 0 (0.0) cha2ds2-vasc score ¼1 638 (10.5) 13 (1.8) 113 (4.9) cha2ds2-vasc score �2 5,442 (89.5) 724 (98.2) 2,177 (95.1) discharge medication aspirin 5,539 (91.1) 512 (69.5) 1,958 (85.5) <0.001 p2y12 inhibitors 955 (15.7) 61 (8.3) 311 (13.6) 0.52 oral anticoagulants 278 (4.6) 338 (45.9) 417 (18.2) <0.001 acei/arb 4,158 (68.4) 522 (70.8) 1,558 (68.0) <0.001 calcium channel blockers 1,146 (18.8) 158 (21.4) 473 (20.7) <0.001 diuretics 2,155 (35.4) 402 (54.5) 1,075 (46.9) <0.001 statins 5,492 (90.3) 617 (83.7) 2,034 (88.8) 0.54 digoxin 37 (0.6) 89 (12.1) 44 (1.9) <0.001 b-blockers 5,433 (89.4) 629 (85.3) 1,973 (86.2) <0.001 sotalol 39 (0.6) 27 (3.7) 229 (10.0) <0.001 amiodarone 127 (2.1) 111 (15.1) 417 (18.2) <0.001 verapamil / diltiazem 59 (1.0) 16 (2.2) 30 (1.3) <0.001 characteristics were reported using percentages for categorical variables, or with median and iqr for continuous variables (as noted). acei ¼ angiotensin-converting enzyme inhibitors; af ¼ atrial fibrillation; arb ¼ angiotensin ii receptor blockers; bmi ¼ body mass index; cabg ¼ coronary artery bypass grafting; cha2ds2-vasc ¼ congestive heart failure, hypertension, age �75 years, diabetes mellitus, prior stroke, transient ischemic attack or thromboembolism, vascular disease, age 65–74 years, female sex; iqr ¼ interquartile range; pci ¼ percutaneous coronary intervention. upsala journal of medical sciences 73 sensitivity analyses two sensitivity analyses were conducted. first, risk of relapse in af was analyzed in a model in which all recurrent diagnoses of af in the national patient registry, not only dc cardioversion, were accounted for. in the analysis, similar increased risk for recurrent af as in the main analysis was observed among patients with postoperative af, hr 3.60 (2.99–4.35). second, propensity score analyses were performed for all outcomes. in such analyses, similar results as in the main analyses were observed between postoperative af versus no af in regard to all outcomes; see supplementary table 3 (available online). discussion in this nationwide cohort of 9,107 consecutive patients undergoing isolated cabg, 8.1% of the patients had a preoperative history of af prior to surgery, and new-onset postoperative af complicated 25.1% of the patients. preand postoperative af were both significantly associated with figure 3. event rate of recurrent symptomatic atrial fibrillation in relation to atrial fibrillation status. figure 2. event rate of all-cause mortality in relation to atrial fibrillation status. 74 g. batra et al. https://doi.org/10.1080/03009734.2018.1504148 higher risk of long-term all-cause mortality, cardiovascular mortality, and hospitalization due to congestive heart failure. however, no significant association was observed between af status and risk for ischemic stroke and myocardial infarction. compared with no af, postoperative af was associated with a higher risk of recurrent symptomatic af after discharge. to our knowledge, this study is the first and only large multicenter study with complete coverage to compare several long-term outcomes, including all-cause mortality, cardiovascular mortality, myocardial infarction, congestive heart failure, and ischemic stroke, in patients with preand postoperative af after cabg. moreover, this is the only nationwide study to assess the long-term risk of recurrent af after cabg in patients with postoperative af. further, we believe this is the only multicenter study to compare different subtypes of af (preand postoperative af) versus no af, which has previously only been done in one small single-center study (4). several studies, preceding ours, have shown an association between postoperative af and long-term mortality (7,9–11), e.g. in an australian multicenter study based on 19,497 patients undergoing isolated cabg where postoperative af was associated with a higher long-term risk of all-cause mortality (odds ratio [or] 1.19 [1.06–1.32]) (10), as well as a us single-center study based on 16,169 patients undergoing isolated cabg (hr 1.21 [1.12–1.32]) (11), but a slightly higher mortality rate was reported in a prior small swedish singlecenter study based on 571 patients undergoing cabg (hr 1.57 [1.05–2.34]) (7). in our study we also found a strong association between postoperative af and cardiovascular mortality. in addition, we found that 8.1% of our cabg population had a history of af preceding surgery which was significantly associated with an increased long-term risk of all-cause mortality and cardiovascular mortality. despite an increased risk of cardiovascular mortality, no association was seen in regard to myocardial infarction for patients with postoperative af, while patients with preoperative af had a non-significant 37% higher risk of myocardial infarction. this leads to the notion that other causes of cardiovascular mortality might have an influence, such as the substantially higher risk of congestive heart failure among patients with preand postoperative af as observed in our study. several other speculative mechanisms for the association of af and mortality have been proposed in the literature. these include af recurrence leading to hemodynamic instability (20), and negative proarrhythmic effects due to higher prescription of antiarrhythmic medication among patients with af (21,22). however, these theories are hypothetical, and to our knowledge no biological and pathophysiological studies are available clarifying casual pathways. another important finding in our study was that postoperative af predicts recurrent af. during a median follow-up of 2.2 years, patients with postoperative af had a more than 4-times higher risk of relapse in symptomatic af as compared to patients with no af at baseline. these results are similar to the above-mentioned swedish single-center study, in which the authors reported an increased risk of development of recurrent af in patients with postoperative af (or 8.31 [4.20–16.43]) (7). also, similar findings with increased risk of recurrent af have been reported in a korean singlecenter study based on 1,171 patients undergoing cabg (hr 5.25 [1.75–15.77]) (12). despite the high recurrence rate of af in our study, we were not able to show a significant increased risk of ischemic stroke during follow-up. to our figure 4. event rates according to the number of events per 100 person-years. unadjusted and adjusted hazard ratios with a 95% confidence interval in relation to atrial fibrillation status, with no atrial fibrillation as reference. upsala journal of medical sciences 75 knowledge, only two large studies have explored the relationship between postoperative af and long-term risk of ischemic stroke. in a us multicenter study based on 73,543 patients undergoing any cardiac surgery, postoperative af was associated with higher risk of ischemic stroke during long-term follow-up (hr 1.3 [1.1–1.6]) (23). similar estimates were shown in a canadian single-center study based on 8,058 patients undergoing isolated cabg (hr 1.26 [1.08–1.47]) (24). the differences in results between our study and the two above-mentioned studies might be explained by the number of patients receiving oral anticoagulants at discharge. in our study, 18.2% of the patients with postoperative af received oral anticoagulants at discharge compared to 11.9% in the canadian study (data about discharge medication were only available in 14.8% of the population in the canadian study, and no data about discharge medication were available in the us study). also, in comparison, the two previous studies had even longer follow-up than ours and encountered a higher number of ischemic stroke events post-surgery. moreover, it has previously been shown that patients post-cabg, irrespectively of presence of af, have an increased risk for ischemic stroke (25), which to some extent might make it more difficult to show an isolated effect of postoperative af on the risk of ischemic stroke. given the current lack of clinical evidence, af guidelines recommend oral anticoagulants as reasonable for patients with postoperative af and with risk for ischemic stroke (class iia, level b recommendation) (26,27). however, this strategy is currently not proven, with only one observational single-center study suggesting that warfarin at discharge might be associated with a lower risk of all-cause mortality (11). nevertheless, the risk associated with postoperative af and treatment with oral anticoagulants is an area in need of further research. limitations this retrospective observational cohort study has some limitations that have to be taken into consideration when interpreting the results. first, patients were not and cannot be randomized, thereby residual confounding may occur. however, we present multicenter data based on all patients undergoing cabg in sweden with a large sample size and are able to adjust for relevant clinical variables. furthermore, our findings were confirmed in a sensitivity analysis with propensity-score matching. still, unknown and unmeasured confounders may exist, and the results should be interpreted with caution. second, the definition of postoperative af was based on data entered by physicians on the swedeheart data collection form. however, undetected episodes of af might be misclassified into the no af group. likewise, patients with silent and undiagnosed af before admission might be misclassified. third, the definition of af during follow-up had some inherent limitations. the outcome was based on dc cardioversion as a surrogate marker of symptomatic relapse in af, probably resulting in us underestimating the relapse risk. however, a similar finding was noted in a sensitivity analysis in which all diagnosis codes of af during follow-up were accounted for. unfortunately, this method has its own drawbacks as patients with follow-up visits due to earlier episodes of af might be wrongly classified as having a relapse in af, resulting in us overestimating the risk. despite these limitations, this multicenter cohort of patients after isolated cabg surgery with complete long-term followup in routine health care seems to provide valuable information about risks associated with preand postoperative af. conclusions among patients undergoing isolated cabg, preand postoperative af was associated with an increased long-term risk of all-cause mortality, cardiovascular mortality, and hospitalization due to congestive heart failure. moreover, postoperative af was associated with higher risk of relapse in symptomatic af. no significant association was observed between af status and risk for ischemic stroke and myocardial infarction. disclosure statement the authors report no conflicts of interest. acknowledgements we thank all staff members at all cardiothoracic surgery departments and coronary care units in sweden for their help and cooperation in contributing data to the swedeheart register. all participating centers, medical doctors, nurses, and the details of the swedeheart register are presented on the registry’s website (www.ucr.uu.se/swedeheart). funding this work was supported by the swedish foundation for strategic research, sweden [grant number kf10–0024] and the uppsala–€orebro regional research council, sweden [grant number rfr-561761]. notes on contributors gorav batra is a cardiology resident at the department of medical sciences, uppsala university, uppsala, sweden. anders ahlsson is a cardiac surgeon and associate professor and managing director at karolinska university hospital, stockholm, sweden. bertil lindahl is a cardiologist and professor of cardiology at the department of medical sciences, uppsala university, uppsala, sweden. lars lindhagen is a biostatistician and phd at uppsala clinical research center, uppsala, sweden. anders wickbom is a cardiothoracic surgery resident at the department of cardiothoracic and vascular surgery, €orebro university hospital, and phd candidate at the institution of medical sciences at €orebro university, örebro sweden. jonas oldgren is a cardiologist and professor of coagulation research at the department of medical sciences, uppsala university, uppsala, sweden. references 1. krijthe bp, kunst a, benjamin ej, lip gyh, franco oh, hofman a. projections on the number of individuals with atrial fibrillation in the european union, from 2000 to 2060. eur heart j. 2013;34:2746–51. 76 g. batra et al. http://www.ucr.uu.se/swedeheart 2. 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after coronary artery bypass grafting. jama. 2011;305:381–90. 26. january ct, wann ls, alpert js, calkins h, cigarroa je, cleveland jc, et al. aha/acc/hrs guideline for the management of patients with atrial fibrillation: executive summary a report of the american college of cardiology/american heart association task force on practice guidelines and the heart rhythm society. j am coll cardiol. 2014;2014:64e1–76. 27. kirchhof p, benussi s, kotecha d, ahlsson a, atar d, casadei b, et al. 2016 esc guidelines for the management of atrial fibrillation developed in collaboration with eacts. eur heart j. 2016;37:2893–962. upsala journal of medical sciences 77 abstract introduction materials and methods study population and data collection definition of exposure definition of outcome statistical analyses results study population and patient characteristics outcome analyses sensitivity analyses discussion limitations conclusions disclosure statement acknowledgements notes on contributors references improved unhealthy lifestyle habits in patients with high cardiovascular risk: results from a structured lifestyle programme in primary care article improved unhealthy lifestyle habits in patients with high cardiovascular risk: results from a structured lifestyle programme in primary care lena l€onnberga,b , elin ekblom-bakc and mattias damberga,b acenter for clinical research, county of v€astmanland, uppsala university, v€asterås, sweden; bdepartment of public health and caring sciences, family medicine and preventive medicine, uppsala university, uppsala, sweden; cthe swedish school of sports and health sciences, stockholm, sweden abstract background. physical activity, healthful dietary habits, and not smoking are associated with reduced cardiovascular morbidity and mortality. however, few studies have examined how counselling to improve poor lifestyle habits might be carried out in clinical practice. in swedish primary care, structured lifestyle counselling is still not integrated into everyday clinical practice. the aim of the present study was two-fold: (1) to describe a novel lifestyle intervention programme in primary care; and (2) to evaluate change in unhealthy lifestyle habits over 1 year in men and women with high cardiovascular risk who participated in the lifestyle intervention programme. method. a single-group study with a 1-year follow-up was carried out. a total of 417 people was enrolled, median age 62 years (54% women), with either hypertension (69%), type 2 diabetes mellitus, or impaired glucose tolerance. the 1-year intervention included five counselling sessions that focused on lifestyle habits, delivered by a district nurse with postgraduate credits in diabetes care and the metabolic syndrome. all patients were offered in-depth counselling for one or more lifestyle habits when needed. lifestyle habits were assessed by a questionnaire at baseline and 1-year follow-up. total change was assessed using a nine-factor unhealthy lifestyle habit index. results. favourable, significant changes were observed for physical activity, dietary habits, smoking, and stress over 1 year. similar improvements were seen for both sexes and type of diagnosis. conclusions. the results support the utility of a multifactorial, structured approach to change unhealthy lifestyle habits for cardiovascular risk prevention in a primary care setting. article history received 8 january 2019 revised 22 march 2019 accepted 28 march 2019 keywords cardiovascular prevention; general practice; hypertension; lifestyle habits; structured lifestyle programme; type 2 diabetes mellitus introduction heart attack and stroke are major killers in all parts of the world. about 80% of premature deaths from these causes could be avoided by controlling for the main risk factors such as physical inactivity, unhealthy diet, and tobacco use (1). international guidelines on cardiovascular disease (cvd) prevention include lifestyle counselling to improve unhealthy lifestyle habits with the aim of reducing cardiovascular risk (2). these guidelines emphasize that the highest clinical priority for prevention should be directed towards patients at high cardiovascular risk, such as those with type 2 diabetes mellitus (t2dm), impaired glucose tolerance (igt), and hypertension (3). the prevalence of t2dm is 4%–5% in sweden for people of all ages and increases with age up to 20% for people older than 70 years (4,5). there is a strong relationship between t2dm and overweight, especially abdominal obesity (4–6). the prevalence of hypertension is about 25% in sweden and increases with advancing age; e.g. it is >60% in people aged 60 years and over (4,7). for prevention of future cvd, behavioural interventions, such as increased physical activity, weight reduction, and smoking cessation, are essential for treating both t2dm and hypertension (5,7). unhealthy lifestyle habits are common in the population. according to a national survey by the public health agency of sweden about lifestyle habits and living conditions in 2016, half of all women and two-thirds of all men have at least one unhealthy lifestyle habit (8). smoking, alcohol overconsumption, physical inactivity, and poor dietary habits account for 20% of total health-care costs in sweden (9). the recently published guidelines—national guidelines for prevention and treatment of unhealthy lifestyle from the swedish national board of health and welfare—highlight the importance of health-care professionals in providing patients with the knowledge, tools, and support needed to improve their unhealthy lifestyle habits (10). however, scientific evaluations of programmes to improve lifestyle habits are scarce, despite the knowledge that healthy lifestyle habits are important for reducing cardiovascular risk (3,11–14). structured lifestyle counselling is still not integrated into everyday clinical practice in primary care (15–17). contact lena l€onnberg lena.lonnberg@regionvastmanland.se centrum f€or klinisk forskning, v€asterås hospital, 721 89, v€asterås, sweden supplemental data for this article can be accessed here. � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 2, 94–104 https://doi.org/10.1080/03009734.2019.1602088 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1602088&domain=pdf&date_stamp=2019-06-04 http://orcid.org/0000-0003-4706-6915 http://orcid.org/0000-0002-3901-7833 http://orcid.org/0000-0001-7654-7553 https://doi.org/10.1080/03009734.2019.1602088 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2019.1602088 http://www.tandfonline.com it is important to evaluate these programmes because of the lack of evaluation of structured lifestyle counselling programmes which use only the limited resources available at the primary care level. thus, the aim of the present study is two-fold: (1) to describe a novel lifestyle intervention programme in primary care; and (2) to evaluate change in unhealthy lifestyle habits over 1 year in men and women with high cardiovascular risk that participated in the lifestyle intervention programme. we hypothesized that the structured lifestyle counselling programme would improve lifestyle habits in these patients with high cardiovascular risk. methods study design and population we conducted a single-group study with a 1-year follow-up with before and after measurements. all data were collected consecutively and were registered in a database by one of five district nurses. people registered at citypraktiken, a primary care unit in v€asterås, sweden, were enrolled during a 5-year period between october 2009 and september 2014. the inclusion criteria were age 18–75 years and for the first time meeting the diagnosis criteria of either hypertension (blood pressure >140/90mm hg), t2dm (fasting blood glucose level >7 mmol/ l), or igt (two-hour glucose levels of 7.8–11.0mmol/l on the 75-g oral glucose tolerance test). antihypertensive or cholesterol-lowering medication was prescribed when needed according to hypertension and diabetes guidelines. people with dementia or severe psychiatric disease were excluded (table 1). a total of 448 people was referred by their physician to join the lifestyle programme. one did not meet the inclusion criteria, and 30 did not provide written consent. thus, 417 participants were included in the present study. however, 101 were lost to follow-up: 69 did not complete the questionnaire at the baseline and 1-year follow-up, 30 did not complete the programme, and two died (figure 1). the study was conducted according to the ethical guidelines of the declaration of helsinki and the good clinical practice guidelines. the swedish ethical review authority approved the study (reference number: 2014/497). all participants provided written informed consent. the study was registered at www.clinical-trials.gov (clinicaltrial.gov id: dnr 2014/497). the structured lifestyle programme the structured lifestyle programme comprised five appointments with the same district nurse, with postgraduate credits in diabetes care and the metabolic syndrome, at the baseline and after 3, 6, 9, and 12 months. fasting blood samples were obtained 1 week before, and a submaximal cycle ergometer test was performed the same day as the baseline and 1-year follow-up appointments. anthropometric variables were measured, and the questionnaire was completed at baseline and the 1-year follow-up appointment. blood pressure and waist circumference were measured at every appointment (figure 2). at both the baseline and 1-year follow-up appointment, the results of the clinical examination, anthropometric measurements, answers from the questionnaire, and laboratory data were discussed between the nurse and participant. every appointment focused primarily on lifestyle habits and involved motivational interviewing to strengthen the participant’s ability to modify one or more lifestyle habits. each participant received a prescription for physical activity in accordance with professional associations for physical activity, physical activity in the prevention and treatment of disease, fyss 2008 (17,18). dietary counselling was performed in accordance with the nordic nutrition recommendations (19). if the participant table 1. baseline characteristics of the study population (n ¼ 316). total, n ¼ 316 men, n ¼ 146 women, n ¼ 170 t2dm þ igt, n ¼ 99 hypertension, n ¼ 217 sex (% women) 54% 40% 60% diagnosis 31% 69% age, years 62.0 (54.0–66.0) 62.0 (54.8–66.0) 62.0 (53.8–67.0) 63.0 (58.0–67.0) 61.0 (53.0–66.0) height, cm 170.0 (164.0–179.0) 180.0 (175.0–183.2) 165.0 (161.0–168.5) 172.5 (165.0–180.0) 170.0 (164.0–177.5) weight, kg 83.8 (73.0–96.0) 92.0 (82.8–104.0) 76.4 (68.0–87.0) 93.0 (80.8–108.2) 80.0 (71.8–92.3) body mass index, kg/m2 28.0 (25.6–32.0) 28.2 (26.0–31.9) 28.0 (25.0–32.7) 31.0 (27.2–35.8) 27.5 (25.0–31.0) waist circumference, cm 100.9 (92.0–109.0) 103.0 (98.0–112.0) 94.0 (87.4–105) 106.8 (97.6–115.5) 97.0 (89.5–103.5) blood pressure, mmhg 150/90 (140–160/80–95) 150/90 (140–160/80–95) 150/90 (140–160/80–95) 140/80 (130–150/75–90) 155/90 (140–165/85–100) predicted maximal oxygen uptake, ml o2/kg/min a 22.0 (18.0–25.5) 22.0 (17.0–26.0) 22.0 (18.0–25.0) 20.0 (15.3–23.0) 22.9 (18.4–26.0) total cholesterol, mmol 5.9 (5.2–6.7) 5.6 (4.8–6.6) 6.1 (5.5–6.9) 5.4 (4.6–6.5) 6.1 (5.5–6.8) low-density lipoprotein, mmol 3.8 (3.2–4.6) 3.8 (3.0–4.5) 3.9 (3.3–4.7) 3.4 (2.7–4.4) 4.0 (3.4–4.7) high-density lipoprotein, mmol 1.3 (1.1–1.6) 1.2 (1.0–1.4) 1.5 (1.3–1.8) 1.2 (1.0–1.4) 1.4 (1.2–1.7) triglycerides, mmol 1.4 (1.0–2.0) 1.5 (1.1–2.2) 1.3 (1.0–1.7) 1.7 (1.3–2.2) 1.2 (0.9–1.7) fasting blood glucose, mmol 5.5 (5.0–6.6) 5.9 (5.1–7.2) 5.3 (4.9–6.1) 7.7 (6.5–9.1) 5.2 (4.7–5.7) metabolic syndrome 51% 60% 43% 77% 39% previous cardiovascular disease 6% 8% 4% 15% 2% antihypertensive medication 56% 60% 54% 66% 52% cholesterol-lowering medication 15% 21% 10% 36% 6% continuous data are presented as median (q1–q3). a269 individuals performed a bicycle ergometer test. upsala journal of medical sciences 95 http://www.clinical-trials.gov needed extended counselling about one or more lifestyle habits, he or she was referred within the primary care unit, e.g. to a physiotherapist regarding physical activity or to a nurse trained in smoking cessation counselling. at the 1-year follow-up appointment, the nurse summarized the past 12-month period and new goals were set for the future. an oral referral response was given to the referring physician. during the 12-month intervention, all participants were allowed to participate in three evening group sessions, alone or together with a spouse or a friend. the group session focused on: (1) cardiovascular risk factors and physical activity; (2) healthy food and alcohol and tobacco use; and (3) stress, sleeping habits, and behavioural change. the participation rate varied over time, and between 15% and 25% of all participants attended one or more of the group sessions. clinical examination at the baseline and 1-year follow-up, all participants were weighed in light indoor clothing without shoes to the nearest 0.1 kg using an electronic balance. height was measured without shoes to the nearest 0.5 cm using a scale fixed to the wall. body mass index was calculated from the measured weight and height as kg/m2. waist circumference was measured with the participant in a standing position midway between the lower rib margin and the iliac crest with a tape measure to the nearest 0.5 cm. blood pressure was measured using the standard auscultatory method with an appropriatesized cuff on the right arm with the participant in a seated position after a 10-min rest. maximal oxygen uptake was estimated using the åstrand submaximal cycle ergometer test (20,21) on a monark e 818 or monark e 928 cycle ergometer. laboratory measurements the concentrations of total cholesterol (mmol/l), low-density lipoprotein (ldl, mmol/l), high-density lipoprotein (hdl, mmol/l), and triglycerides (mmol/l) were analysed by standard methods at aleris medilab (stockholm, sweden). the laboratory was quality-certified according to iso/iec 151 89. fasting blood glucose concentration was measured by the laboratory at the primary care unit. all equipment was calibrated regularly. questionnaire information about lifestyle habits was obtained using a self-administered questionnaire comprising 25 questions covering physical activity, dietary habits, alcohol consumption, tobacco use, stress, and sleeping habits. the questionnaire combined validated and reliability tested questions from other questionnaires regarding lifestyle habits. however, the complete questionnaire is not validated and tested for reliability in its present form. questions about physical activity covered daily activity, exercise, and time being sedentary using a 1–4 scale and enrolment in the lifestyle programme at cityprak�ken healthcare centre 2009– 2014 assessed for eligibility n=448 excluded (n=31) did not fulfil inclusion criteria (n= 1) did not consent (n=30) included in the study n=417 1-year follow-up n=316 lost to follow-up (n=101) did not complete the ques�onnaire at baseline and 1 year (n=69). did not complete the ques�onnaire at baseline and declined to par�cipate during the 12 months (n=30) deceased (n=2) figure 1. flow chart. baseline •individual counselling •fas�ng blood sample •anthropometric measurements •submax vo2 test •ques�onnaire 3 months •individual counselling •blood pressure and waist circumference 6 months • individual counselling • blood pressure and waist circumference 9 months • individual counselling • blood pressure and waist circumference 1-year follow-up • individual counselling • fas�ng blood sample • anthropometric measurements • submax vo2 test • ques�onnaire figure 2. the structured lifestyle programme. 96 l. l€onnberg et al. overall physical activity using a 0–10 visual analogue scale. dietary habits were assessed in eight questions about the intake of fish, fruit and vegetables, fast food, extra calories, and soft drinks. alcohol consumption was assessed in two questions about the frequency and quantity. smoking and snuff use were assessed in questions covering daily consumption (yes/no) and number of cigarettes/portions of snuff. stress and sleep habits were evaluated in two questions using a 1–4 scale. the questions have been listed in tables 2–4. for the full questionnaire, see appendix. unhealthy lifestyle habits and the unhealthy lifestyle habit index each continuous lifestyle habit variable was dichotomized further into unhealthy or healthy, considering the lowest rank on each question as unhealthy except for fruit and vegetable consumption where the two lowest ranks were considered as unhealthy (see tables 2–4). to study the clustering of unhealthy lifestyle habits, a nine-factor unhealthy lifestyle habit index was constructed. this index included daily smoking, high alcohol intake, low daily physical activity, low exercise level, high sedentary time, low intake of fruit and vegetables, high fast-food consumption, sleeping difficulties, and high level of stress. the care need index the care need index (cni) is used to evaluate a population’s need for primary care. the cni measures socio-economic factors and comprises seven variables; age >65 years; born in eastern europe, asia, africa, or south america; unemployed; single parent with child younger than 17 years; children under 5 years; low educational level; and highly mobile people. a high index indicates an increased need for health care (22). the metabolic syndrome and previous cardiovascular disease the metabolic syndrome was classified according to the national cholesterol education program (ncep) adult treatment panel iii (atpiii) (23). the ncep/atpiii has defined the metabolic syndrome as three or more of the following: waist circumference >102 cm in men and >88 cm in women; blood pressure �130/85 mmhg; triglyceride concentration �1.7 mmol/l; hdl concentration <1.0 mmol/l in men and <1.3 mmol/l in women; and fasting plasma glucose concentration >6.1 mmol/l. individuals with antihypertensive or cholesterol-lowering medication were included in the high blood pressure or triglyceride groups. previous cardiovascular disease was defined as myocardial infarction, coronary insufficiency, angina, ischemic stroke, haemorrhagic stroke, transient ischaemic attack, peripheral artery disease, or heart failure. statistics subgroup analyses were based on diagnosis set by the referring physician—individuals with hypertension, or individuals with either t2dm or igt (t2dm þ igt)—and on sex. continuous characteristic data were checked for a normal distribution. for questionnaire data, values from baseline were carried forward for missing data for all variables. although the questionnaire responses were ordinal data, the mean and standard deviation values are presented to facilitate the interpretation of the results. however, to detect significant changes within groups over the year, the wilcoxon signed-rank test was used. the bonferroni–holm correction was applied to reduce the possibility of getting a statistically significant result (i.e. type i error) when multiple hypothesis tests were performed. mcnemar’s test was used to identify changes in percentages of each unhealthy lifestyle habits over the year. a paired-sample t test was used to detect changes in the unhealthy lifestyle index over the year. the analyses were performed using statistical package for social science (ibm spss statistics for windows, version 24.0, armonk, ny, usa). results baseline characteristics the population comprised 54% women, with a median age of 62 years (range 54–66 years), recently diagnosed with either hypertension (69%), type 2 diabetes mellitus (29%), or impaired glucose tolerance (2%) (table 1). patients lost to follow-up were significantly younger (p < 0.001), median age of 56 years (range 50–63.5 years), and had cholesterol-lowering medication to a lesser extent than the study population (5% for lost to follow-up compared to 15% in the study population, p ¼ 0.006) (table 1). regarding weight, body mass index, and prevalence of metabolic syndrome, there were no differences between patients lost to follow-up and the study population. the care need index (cni) was 0.86 for the present primary care unit in 2014 compared with 1.08 (±0.20) in the county. changes in physical activity and sedentary time in the total sample, continuous levels of daily activity, exercise, and overall physical activity increased, and sedentary time decreased over the year (all p < 0.05) (table 2). the percentages of participants with unhealthy (low) levels of daily activity, exercise, and overall physical activity decreased over the year. sex-specific analyses revealed that all activity-related habits improved in men, except that there was no significant improvement of sedentary time. by contrast, women showed a slight decrease in sedentary time and improved exercise and overall physical activity habits. similar significant trends were seen for changes in the percentages of participants with the dichotomized unhealthy physical activity habits. in participants with hypertension, daily activity, exercise, and overall physical activity increased over the year. there upsala journal of medical sciences 97 ta b le 2. se lfre p or te d p h ys ic al ac ti vi ty -r el at ed lif es ty le h ab it s. to ta l se xsp ec ifi c d ia g n os is -s p ec ifi c c on ti n uo us lif es ty le h ab it q ue st io n n ai re sc or in g ba se lin e m ea n (s d ) 1 ye ar m ea n (s d ) p va lu e ov er 1 ye ar ba se lin e m ea n (s d ) 1 ye ar m ea n (s d ) p va lu e ov er 1 ye ar ba se lin e m ea n (s d ) 1 ye ar m ea n (s d ) p va lu e ov er 1 ye ar d ai ly p h ys ic al ac ti vi ty (n ¼ 31 5) 3. 13 (0 .8 0) 3. 25 (0 .7 2) 0. 00 1a m en (n ¼ 14 6) 2. 95 (0 .8 2) 3. 14 (0 .7 0) < 0. 00 1a t2 d m þ ig t (n ¼ 99 ) 3. 14 (0 .7 1) 3. 22 (0 .6 8) n .s . w om en (n ¼ 16 9) 3. 29 (0 .7 4) 3. 34 (0 .7 3) n .s . h t (n ¼ 21 6) 3. 13 (0 .8 3) 3. 26 (0 .7 4) 0. 00 2a ex er ci se (n ¼ 31 3) 2. 23 (1 .1 0) 2. 71 (1 .0 3) < 0. 00 1a m en (n ¼ 14 6) 2. 27 (1 .1 2) 2. 71 (1 .0 4) < 0. 00 1a t2 d m þ ig t (n ¼ 98 ) 2. 18 (1 .1 2) 2. 73 (1 .0 1) < 0. 00 1a w om en (n ¼ 16 7) 2. 20 (1 .0 8) 2. 71 (1 .0 2) < 0. 00 1 h t (n ¼ 21 5) 2. 26 (1 .1 0) 2. 70 (1 .0 4) < 0. 00 1a o ve ra ll p h ys ic al 4. 45 (1 .7 9) 5. 92 (1 .7 0) < 0. 00 1a m en (n ¼ 14 4) 4. 54 (1 .9 0) 5. 92 (1 .6 2) < 0. 00 1a t2 d m þ ig t (n ¼ 96 ) 4. 35 (1 .7 8) 5. 99 (1 .6 7) < 0. 00 1a ac ti vi ty (n ¼ 31 2) w om en (n ¼ 16 8) 4. 37 (1 .7 0) 5. 93 (1 .7 7) < 0. 00 1a h t (n ¼ 21 6) 4. 50 (1 .8 0) 5. 90 (1 .7 1) < 0. 00 1a ti m e se d en ta ry (n ¼ 31 2) 1. 86 (0 .7 2) 1. 79 (0 .6 7) 0. 01 5a m en (n ¼ 14 3) 1. 93 (0 .7 3) 1. 87 (0 .6 8) n .s . t2 d m þ ig t (n ¼ 95 ) 1. 95 (0 .7 2) 1. 86 (0 .6 9) n .s . w om en (n ¼ 16 9) 1. 79 (0 .7 1) 1. 72 (0 .6 6) n .s . h t (n ¼ 21 7) 1. 82 (0 .7 2) 1. 76 (0 .6 6) n .s . % w it h un h ea lt h y lif es ty le h ab it s ba se lin e 1 ye ar d iff (9 5% c i) ba se lin e 1 ye ar d iff (9 5% c i) ba se lin e 1 ye ar d iff (9 5% c i) lo w d ai ly p h ys ic al ac ti vi ty le ve l 19 .4 % 13 .7 % �5 .7 % (� 9. 6 to �2 .0 ) m en 26 .7 % 15 .8 % �1 0. 9% (� 17 .6 to �4 .5 ) t2 d m þ ig t 19 .2 % 12 .1 % �7 .1 % (� 14 .8 to 0. 3) (< 30 m in /d ay ) w om en 13 .0 % 11 .8 % �1 .2 % (� 5. 7 to 3. 2) h t 19 .4 % 14 .4 % �5 .0 % (� 9. 7 to �0 .7 ) lo w ex er ci se le ve l 56 .5 % 36 .4 % �2 0. 1% (� 25 .3 to �1 4. 7) m en 57 .5 % 41 .1 % �1 6. 4% (� 24 .1 to �8 .4 ) t2 d m þ ig t 58 .2 % 34 .7 % �2 3. 5% (� 33 .1 to �1 2. 9) (< 1 h ou r/ w ee k) w om en 55 .7 % 32 .3 % �2 3. 4% (� 30 .3 to �1 5. 9) h t 55 .8 % 37 .2 % �1 8. 6% (� 24 .7 to �1 2. 2) lo w ov er al l p h ys ic al ac ti vi ty 33 .3 % 9. 0% �2 4. 3% (� 29 .6 to �1 9. 2) m en 36 .8 % 6. 9% �2 9. 9% (� 37 .9 to �2 1. 7) t2 d m þ ig t 32 .3 % 7. 3% �2 5. 0% (� 34 .9 to �1 5. 1) (� 3 on a 10 -p oi n t sc al e) w om en 30 .4 % 10 .7 % �1 9. 6% (� 26 .5 to �1 2. 9) h t 33 .8 % 9. 7% �2 4. 1% (� 30 .3 to �1 7. 9) h ig h am ou n t of se d en ta ry ti m e 16 .0 % 12 .8 % �3 .2 % (� 6. 8 to 0. 3) m en 18 .9 % 17 .5 % �1 .4 % (� 7. 2 to 4. 4) t2 d m þ ig t 18 .9 % 15 .8 % �3 .1 % (� 10 .1 to 3. 6) (� 9 h ou rs /d ay ) w om en 13 .6 % 8. 9% �4 .7 % (� 9. 6 to �0 .2 ) h t 14 .7 % 11 .5 % �3 .2 % (� 7. 7 to 1. 1) d ai ly p h ys ic al ac ti vi ty w as as se ss ed w it h th e q ue st io n , ‘h ow p h ys ic al ly ac ti ve ar e yo u d ur in g a d ay ?’ th e re sp on se op ti on s w er e: 1, n ot at al l p h ys ic al ly ac ti ve ; 2, < 30 m in /d ay ; 3, 30 – 60 m in /d ay ; 4, > 60 m in /d ay . ex er ci se w as as se ss ed us in g th e q ue st io n , ‘h ow m uc h ex er ci se d o yo u p er fo rm in a w ee k’ . th e re sp on se op ti on s w er e: 1, n o ac ti vi ty at al l; 2, < 1 h ou r w ee kl y; 3, 1– 2 h ou rs w ee kl y; 4, > 2 h ou rs w ee kl y. o ve ra ll p h ys ic al ac ti vi ty w as as se ss ed b y as ki n g p ar ti ci p an ts , ‘p le as e ra te yo ur ov er al l p h ys ic al ac ti vi ty fr om 0 to 10 , w it h 0 in d ic at in g n ot at al l p h ys ic al ly ac ti ve an d 10 in d ic at in g ve ry p h ys ic al ly ac ti ve ’. se d en ta ry ti m e w as as se ss ed b y as ki n g p ar ti ci p an ts , ‘p le as e es ti m at e th e am ou n t of ti m e yo u si t ea ch d ay ’. th e re sp on se op ti on s w er e: 1, 0– 4 h ou rs /d ay ; 2, 5– 8 h ou rs /d ay ; 3, 9– 12 h ou rs /d ay ; 4, 13 h ou rs a d ay or m or e. a si g n ifi ca n t ch an g e ov er 1 ye ar , p va lu e < 0. 05 w it h bo n fe rr on i– h ol m co rr ec ti on . h t: h yp er te n si on ; ig t: im p ai re d g lu co se to le ra n ce ; n .s .: n on -s ig n ifi ca n t ch an g e; t2 d m : ty p e 2 d ia b et es m el lit us . 98 l. l€onnberg et al. ta b le 3. se lfre p or te d d ie ta ry -r el at ed lif es ty le h ab it s. t ot al se xsp ec ifi c d ia g n os is -s p ec ifi c c on ti n uo us lif es ty le h ab it q ue st io n n ai re sc or in g ba se lin e m ea n (s d ) 1 ye ar m ea n (s d ) p va lu e ov er 1 ye ar ba se lin e m ea n (s d ) 1 ye ar m ea n (s d ) p va lu e ov er 1 ye ar ba se lin e m ea n (s d ) 1 ye ar m ea n (s d ) p va lu e ov er 1 ye ar fi sh (n ¼ 31 6) 3. 00 (0 .7 8) 3. 12 (0 .7 6) < 0. 00 1a m en (n ¼ 14 6) 2. 94 (0 .7 8) 3. 07 (0 .7 3) 0. 00 9a t2 d m þ ig t (n ¼ 99 ) 3. 11 (0 .7 1) 3. 14 (0 .7 4) n .s . w om en (n ¼ 17 0) 3. 06 (0 .7 7) 3. 16 (0 .7 8) 0. 01 7a h t (n ¼ 21 7) 2. 95 (0 .8 0) 3. 11 (0 .7 6) < 0. 00 1a fa st fo od (n ¼ 31 6) 2. 27 (0 .8 7) 2. 19 (0 .8 7) 0. 03 6a m en (n ¼ 14 6) 2. 41 (0 .8 8) 2. 34 (0 .8 3) n .s . t2 d m þ ig t (n ¼ 99 ) 2. 40 (0 .8 7) 2. 36 (0 .9 0) n .s . w om en (n ¼ 17 0) 2. 15 (0 .8 5) 2. 06 (0 .8 9) n .s . h t (n ¼ 21 7) 2. 21 (0 .8 7) 2. 11 (0 .8 5) 0. 02 7a fr ui t an d ve g et ab le s (n ¼ 31 6) 3. 63 (0 .8 8) 3. 80 (0 .8 2) < 0. 00 1a m en (n ¼ 14 6) 3. 40 (0 .8 8) 3. 66 (0 .6 6) < 0. 00 1a t2 d m þ ig t (n ¼ 99 ) 3. 61 (0 .7 4) 3. 80 (0 .5 2) 0. 00 1a w om en (n ¼ 17 0) 3. 84 (0 .4 8) 3. 91 (0 .3 2) n .s . h t (n ¼ 21 7) 3. 65 (0 .7 2) 3. 80 (0 .5 2) < 0. 00 1a ex tr a ca lo ri es (n ¼ 31 6) 2. 14 (0 .8 8) 2. 03 (0 .8 2) 0. 00 3a m en (n ¼ 14 6) 2. 16 (0 .9 0) 2. 10 (0 .8 8) n .s . t2 d m þ ig t (n ¼ 99 ) 1. 43 (0 .6 4) 1. 34 (0 .5 4) n .s . w om en (n ¼ 17 0) 2. 12 (0 .8 6) 1. 96 (0 .7 5) 0. 00 3a h t (n ¼ 21 7) 1. 43 (0 .5 8) 1. 39 (0 .5 7) 0. 02 3a so ft d ri n ks /j ui ce (n ¼ 31 6) 1. 43 (0 .6 0) 1. 38 (0 .5 6) 0. 04 1a m en (n ¼ 14 6) 1. 59 (0 .6 5) 1. 51 (0 .6 2) n .s . t2 d m þ ig t (n ¼ 99 ) 1. 43 (0 .6 4) 1. 34 (0 .5 4) n .s . w om en (n ¼ 17 0) 1. 29 (0 .5 2) 1. 26 (0 .4 6) n .s . h t (n ¼ 21 7) 1. 43 (0 .5 8) 1. 39 (0 .5 7) n .s . % of p ar ti ci p an ts w it h un h ea lt h y lif es ty le h ab it s ba se lin e 1 ye ar d iff (9 5% c i) ba se lin e 1 ye ar d iff (9 5% c i) ba se lin e 1 ye ar d iff (9 5% c i) lo w in ta ke of fis h 4. 7% 2. 5% �2 .2 % (� 4. 7 to �0 .2 ) m en 5. 5% 2. 1% �3 .4 % (� 7. 9 to 0. 0) t2 d m þ ig t 3. 0% 2. 0% �1 .0 % (� 6. 4 to 4. 1) (r ar el y or n ev er ) w om en 4. 1% 2. 9% �1 .2 % (� 4. 6 to 1. 9) h t 5. 5% 2. 8% �2 .7 % (� 5. 9 to �0 .3 ) h ig h in ta ke of fa st fo od (� a 8. 5% 5. 7% �2 .8 % (� 6. 2 to 0. 4) m en 11 .0 % 6. 2% �4 .8 % (� 10 .6 to 0. 7) t2 d m þ ig t 12 .1 % 8. 1% �4 .0 % (� 11 .8 to 3. 4) co up le of ti m es a m on th ) w om en 6. 5% 5. 3% �1 .2 % (� 5. 5 to 3. 0) h t 6. 9% 4. 6% �2 .3 % j( �6 .1 to 1. 2) lo w in ta ke of fr ui t an d 9. 8% 4. 1% �5 .7 % (� 8. 9 to �3 .0 ) m en 17 .1 % 7. 5% �9 .6 % (� 15 .2 to �4 .8 ) t2 d m þ ig t 11 .1 % 5. 1% �6 .0 % (� 12 .5 to �0 .8 ) ve g et ab le (� 3 ti m es a w ee k) w om en 3. 5% 1. 2% �2 .3 % (� 6. 3 to 1. 0) h t 9. 2% 3. 7% �5 .5 % (� 9. 6 to �2 .2 ) h ig h in ta ke of ex tr a 8. 2% 6. 0% �2 .2 % (� 5. 1 to 0. 5) m en 10 .3 % 8. 9% �1 .4 % (� 5. 9 to 3. 0) t2 d m þ ig t 6. 1% 3. 0% �3 .1 % (� 8. 7 to 1. 5) ca lo ri es (d ai ly ) w om en 6. 5% 3. 5% �3 .0 % (� 7. 3 to 0. 9) h t 9. 2% 7. 4% �1 .8 % (� 5. 7 to 1. 8) h ig h in ta ke of so ft 3. 8% 2. 5% �1 .3 % (� 3. 6 to 0. 8) m en 6. 2% 4. 1% �2 .1 % (� 6. 3 to 1. 6) t2 d m þ ig t 4. 0% 1. 0% �3 .0 % (� 8. 8 to 1. 6) d ri n ks /j ui ce (d ai ly ) w om en 1. 8% 1. 2% �0 .6 % (� 3. 8 to 2. 4) h t 3. 7% 3. 2% �0 .5 % (� 3. 3 to 2. 2) th e am ou n t of fis h co n su m ed w as as se ss ed w it h th e q ue st io n , ‘h ow of te n d o yo u ea t fis h ?’ th e re sp on se op ti on s w er e: 1, ra re ly /n ev er ; 2, a co up le of ti m es a m on th ; 3, on ce a w ee k; 4, a co up le of ti m es a w ee k or m or e. th e am ou n t of fa st fo od w as as se ss ed w it h th e q ue st io n , ‘h ow of te n d o yo u ea t sa us ag es , h am b ur g er , or p iz za ?’ th e re sp on se op ti on s w er e: 1, ra re ly /n ev er ; 2, a co up le of ti m es a m on th ; 3, on ce a w ee k; 4, a co up le of ti m es a w ee k or m or e. th e am ou n t of fr ui t an d ve g et ab le s w as as se ss ed w it h th e q ue st io n , ‘h ow of te n d o yo u ea t fr ui t an d ve g et ab le s? ’ th e re sp on se op ti on s w er e: 1, a fe w ti m es a m on th ; 2, 1– 2 ti m es a w ee k; 3, 3– 5 ti m es a w ee k; 4, d ai ly . c on su m p ti on of ex tr a ca lo ri es w as as se ss ed w it h th e q ue st io n , ‘h ow of te n d o yo u ea t “e xt ra ” ca lo ri es ?’ th e re sp on se op ti on s w er e: 1, a fe w ti m es a m on th ; 2, 1– 2 ti m es a w ee k; 3, 3– 5 ti m es a w ee k; 4, d ai ly . c on su m p ti on of so ft d ri n ks /j ui ce w as as se ss ed w it h th e q ue st io n , ‘h ow of te n d o yo u d ri n k sw ee te n ed so ft d ri n ks or ju ic e? ’ th e re sp on se op ti on s w er e: 1, a fe w ti m es a m on th ; 2, 1– 2 ti m es a w ee k; 3, 3– 5 ti m es a w ee k; 4, d ai ly . a si g n ifi ca n t ch an g e ov er 1 ye ar , p va lu e < 0. 05 w it h bo n fe rr on i– h ol m co rr ec ti on . h t: h yp er te n si on ; ig t: im p ai re d g lu co se to le ra n ce ; n .s . ¼ n on -s ig n ifi ca n t ch an g e ov er 1 ye ar ; t2 d m : ty p e 2 d ia b et es m el lit us . upsala journal of medical sciences 99 ta b le 4. se lfre p or te d le ve ls of al co h ol co n su m p ti on , to b ac co us e, st re ss , an d sl ee p in g d iff ic ul ti es . to ta l se xsp ec ifi c d ia g n os is -s p ec ifi c c on ti n uo us lif es ty le h ab it q ue st io n n ai re sc or in g ba se lin e m ea n (s d ) 1 ye ar m ea n (s d ) p va lu e ov er 1 ye ar ba se lin e m ea n (s d ) 1 ye ar m ea n (s d ) p va lu e ov er 1 ye ar ba se lin e m ea n (s d ) 1 ye ar m ea n (s d ) p va lu e ov er 1 ye ar a lc oh ol , fr eq ue n cy (n ¼ 31 5) 2. 52 (1 .0 5) 2. 48 (1 .0 3) n .s . m en (n ¼ 14 5) 2. 74 (1 .0 3) 2. 69 (1 .0 3) n .s . t2 d m þ ig t (n ¼ 99 ) 2. 46 (0 .9 8) 2. 38 (0 .9 6) n .s . w om en (n ¼ 17 0) 2. 33 (1 .0 2) 2. 30 (1 .0 0) n .s . h t (n ¼ 21 6) 2. 54 (1 .0 8) 2. 52 (1 .0 6) n .s . a lc oh ol , in ta ke /o cc as io n 0. 99 (0 .4 6) 0. 97 (0 .4 4) n .s . m en (n ¼ 14 5) 1. 09 (0 .5 5) 1. 06 (0 .5 2) n .s . t2 d m þ ig t (n ¼ 99 ) 0. 99 (0 .4 8) 0. 97 (0 .4 6) n .s . (n ¼ 31 5) w om en (n ¼ 17 0) 0. 91 (0 .3 4) 0. 89 (0 .3 3) n .s . h t (n ¼ 21 6) 1. 00 (0 .4 5) 0. 97 (0 .4 2) n .s . st re ss (n ¼ 31 5) 2. 82 (0 .7 9) 2. 73 (0 .8 ) 0. 00 9a m en (n ¼ 14 5) 2. 59 (0 .8 0) 2. 58 (0 .8 2) n .s . t2 d m þ ig t (n ¼ 98 ) 2. 58 (0 .8 0) 2. 58 (0 .8 4) n .s . w om en (n ¼ 17 0) 3. 02 (0 .7 3) 2. 86 (0 .7 8) 0. 00 1a h t (n ¼ 21 7) 2. 93 (0 .7 7) 2. 80 (0 .8 0) 0. 00 1a sl ee p in g d iff ic ul ti es (n ¼ 31 5) 2. 59 (0 .9 7) 2. 54 (0 .8 9) n .s . m en (n ¼ 14 5) 2. 32 (0 .9 0) 2. 33 (0 .8 6) n .s . t2 d m þ ig t (n ¼ 98 ) 2. 55 (0 .9 0) 2. 57 (0 .8 1) n .s . w om en (n ¼ 17 0) 2. 82 (0 .9 6) 2. 71 (0 .8 7) n .s . h t (n ¼ 21 7) 2. 61 (1 .0 1) 2. 52 (0 .9 2) n .s . % w it h un h ea lt h y lif es ty le h ab it s ba se lin e 1 ye ar d iff (9 5% c i) ba se lin e 1 ye ar d iff (9 5% c i) ba se lin e 1 ye ar d iff (9 5% c i) d ai ly sm ok er 8. 6% 6. 0% �2 .6 % (� 5. 1 to �0 .3 ) m en 9. 0% 6. 2% �2 .8 % (� 6. 7 to 0. 6) t2 d m þ ig t 7. 1% 7. 1% 0. 0% (� 4. 7 to 4. 7) w om en 8. 2% 5. 9% �2 .3 % (� 6. 4 to 1. 3) h t 9. 2% 5. 5% �3 .7 % (� 7. 2 to �0 .7 ) sn uf f us er 8. 3% 8. 3% 0. 0% (� 1. 7 to 1. 7) m en 15 .4 % 16 .1 % 0. 7% (� 2. 5 to 4. 0) t2 d m þ ig t 10 .3 % 10 .3 % 0. 0% (� 4. 6 to 4. 6) w om en 2. 4% 1. 8% �0 .6 % (� 3. 4 to 1. 9) h t 7. 4% 7. 4% 0. 0% (� 2. 2 to 2. 2) a lc oh ol , h ig h fr eq ue n cy (� 4 1. 3% 1. 6% 0. 3% (� 1. 0 to 1. 9) m en 2. 1% 2. 8% 0. 7% (� 2. 2 to 4. 0) t2 d m þ ig t 0. 0% 0. 0% 0. 0% (� 3. 4 to 3. 4) ti m es a w ee k) w om en 0. 6% 0. 6% 0. 0% (� 2. 5 to 2. 5) h t 1. 9% 2. 3% 0. 4% (� 1. 5 to 2. 7) a lc oh ol , h ig h in ta ke p er 9. 5% 7. 3% �2 .2 % (� 4. 7 to 0. 0) m en 18 .6 % 15 .2 % �3 .4 % (� 8. 1 to 0. 9) t2 d m þ ig t 11 .1 % 9. 1% �2 .0 % (� 7. 5 to 3. 0) oc ca si on (� 5 g la ss es ) w om en 1. 8% 0. 6% �1 .2 % (� 4. 4 to 1. 4) h t 8. 8% 6. 5% �2 .3 % (� 5. 4 to 0. 4) h ig h le ve l of st re ss (o ft en ) 19 .0 % 17 .1 % �1 .9 % (� 5. 9 to 2. 1) m en 11 .0 % 13 .1 % 2. 1% (� 2. 8 to 7. 2) t2 d m þ ig t 11 .2 % 12 .2 % 1. 0% (� 6. 3 to 8. 4) w om en 25 .9 % 20 .6 % �5 .3 % (� 11 .6 to 1. 0) h t 22 .6 % 19 .4 % �3 .2 % (� 8. 2 to 1. 7) sl ee p in g d iff ic ul ti es (o ft en ) 19 .7 % 13 .0 % �6 .7 % (� 10 .5 to �3 .0 ) m en 9. 0% 8. 3% �0 .7 % (� 5. 1 to 3. 6) t2 d m þ ig t 16 .3 % 12 .2 % �4 .1 % (� 11 .7 to 3. 3) w om en 28 .8 % 17 .1 % �1 1. 7% (� 17 .8 to �5 .8 ) h t 21 .2 % 13 .4 % �7 .8 % (� 12 .4 to �3 .6 ) a lc oh ol fr eq ue n cy w as as se ss ed w it h th e q ue st io n , ‘h ow of te n d o yo u d ri n k al co h ol ic b ev er ag es ?’ th e re sp on se op ti on s w er e: 1, n ev er ; 2, le ss th an on ce a m on th ; 3, 2– 4 ti m es a m on th ; 4, 1– 3 ti m es a w ee k; 5, �4 ti m es /w ee k. a lc oh ol co n su m p ti on w as as se ss ed w it h th e q ue st io n , ‘h ow m an y g la ss es d o yo u ty p ic al ly d ri n k w h en yo u d ri n k al co h ol ?’ th e re sp on se op ti on s w er e: 0, n on e; 1, 1– 4 g la ss es ; 2, 5– 9 g la ss es ; 3, �1 0 g la ss es . d ai ly sm ok in g w as as se ss ed w it h th e q ue st io n , ‘d o yo u sm ok e? ye s/ n o’ . th e us e of sn uf f w as as se ss ed w it h th e q ue st io n , ‘d o yo u us e sn uf f? ye s/ n o’ . th e am ou n t of st re ss w as as se ss ed w it h th e q ue st io n , ‘d o yo u fe el st re ss ed ?’ th e re sp on se op ti on s w er e: 1, n ev er ; 2, ra re ly ; 3, so m et im es ; 4, of te n . sl ee p in g d iff ic ul ti es w er e as se ss ed w it h th e q ue st io n , ‘h av e yo u ex p er ie n ce d d iff ic ul ti es w it h yo ur sl ee p ?’ th e re sp on se op ti on s w er e: 1, n ev er ; 2, ra re ly ; 3, so m et im es ; 4, of te n . a si g n ifi ca n t ch an g e ov er 1 ye ar , p va lu e < 0. 05 w it h bo n fe rr on i– h ol m co rr ec ti on . h t: h yp er te n si on ; ig t: im p ai re d g lu co se to le ra n ce ; n .s . ¼ n on -s ig n ifi ca n t ch an g e; t2 d m : ty p e 2 d ia b et es m el lit us . 100 l. l€onnberg et al. were similar significant improvements in the percentages of participants with low levels of these activity variables. in participants with t2dm, continuous scoring and the percentages of participants with low levels of exercise and overall activity improved over the year. change in dietary habits continuous scoring of the intakes of fish, fast food, fruit and vegetables, extra calories, and soft drinks improved significantly over the year in the total sample (all p < 0.05) (table 3). the percentages of participants with unhealthy low intakes of fish and fruit and vegetables decreased. sex-specific analyses revealed that both men and women increased their intake of fish. men increased intake of fruit and vegetables, and women reduced their intake of extra calories (all p < 0.05). men had a higher percentage of participants with unhealthy dietary habits than women at both the baseline and 1-year follow-up. participants with hypertension increased their intakes of fish and fruit and vegetables, and reduced their intakes of fast food and extra calories (all p < 0.05). in participants with t2dm, continuous intakes of fruit and vegetables increased (p < 0.001). the percentages of participants with unhealthy intake of fruit and vegetables decreased for both the t2dm and hypertension groups. changes in alcohol consumption, tobacco use, stress, and sleeping habits the number of daily smokers decreased in the total sample and for individuals with hypertension. levels of stress decreased in the total sample, for women and individuals with hypertension respectively (all p < 0.01). the percentages of participants with unhealthy levels of stress and sleeping difficulties were nearly twice as high for women compared with men at both the baseline and 1-year follow-up. the percentages reporting sleeping difficulties decreased over the year in participants with hypertension (p < 0.05). a higher percentage of participants with hypertension had unhealthy levels of stress at the baseline. changes in the unhealthy lifestyle habit index the mean value of the unhealthy lifestyle habit index decreased over the year from 1.67 (±1.40) at the baseline to 1.16 (±1.22) at the 1-year follow-up (p < 0.001). at the end of the year, the percentage of the total sample with one or no unhealthy lifestyle habits had increased, and the percentages of those with two to eight risk factors had decreased (p < 0.001) (figure 3). men had a higher mean index at both the baseline (1.80 [±1.46]) and 1-year follow-up (1.30 [±1.32]) compared with women (1.56 [±1.34] and 1.03 [±1.11], respectively). the mean index decreased for both men and women over the year (p < 0.001). the mean index also decreased in both the hypertension and t2dm groups over the year, i.e. from 1.69 (±1.46) at the baseline to 1.15 (±1.20) at the 1-year follow-up (p < 0.001) in those with hypertension and from 1.65 (±1.25) to 1.16 (±1.26), respectively (p < 0.001), for those with t2dm (figure 4). discussion the main findings of this study are the significant, favourable changes in physical activity levels, dietary habits, smoking, and feelings of stress and sleeping difficulties after participation in a 1-year structured lifestyle programme in people at high cardiovascular risk provided at a primary care unit. this was seen in both men and women, and in participants with hypertension or t2dm. to our knowledge, this is the largest figure 3. unhealthy lifestyle habit index using nine factors, total sample. data are expressed as the percentages of participants in the total sample. mean value (sd) for number of unhealthy risk factors at baseline and 1-year follow-up (�p � 0.001). upsala journal of medical sciences 101 swedish study to evaluate a structured lifestyle programme performed in an ordinary clinical setting using only the limited resources available at a primary care centre. in people at high cardiovascular risk, changing lifestyle habits after participating in lifestyle counselling has been reported by eriksson et al., whose study included 151 participants (16), and by lidin et al., whose study included 100 participants (13). in this study, the percentages of participants with one or no unhealthy lifestyle habits increased over the year from 51.5% to 69.4% (p < 0.001). this finding is important because having few unhealthy lifestyle habits is associated with reduced risk of incident ischemic cvd and all-cause mortality. clustering of unhealthy lifestyle habits is, in our study, evaluated by a nine-factor unhealthy lifestyle habits index. it combines information about physical activity, diet, alcohol, smoking, stress, and sleeping difficulties. in 2013, carlsson et al. could detect a 70% reduction for all-cause mortality in individuals with the healthiest lifestyle, using a seven-factor lifestyle habit index, in a representative population-based study of 4232 sixty-year-old men and women (24). since the questions in our study’s questionnaire is not identical to the index used by carlsson et al., we are not able to compare our results; however, a positive trend for all-cause mortality could be found in our study population as well. there are different views on whether it is effective to address more than one lifestyle habit at the same time (4, 10). however, a structured lifestyle programme addressing one or more lifestyle habits has been shown to be successful in cvd prevention in other parts of europe and in sweden (12,13,16). a study by gibson et al. that included 521 people with increased cvd risk reported significant favourable improvements in physical activity, dietary habits, and smoking cessation after a 16-week programme to improve unhealthy lifestyle habits (25). the structured lifestyle programme in our study provided people at high cardiovascular risk with the knowledge and tools to improve their unhealthy lifestyle habits, as stated in several guidelines to decrease the risk for future cvd (5,7,10). our results are consistent with previous research evidence that individual counselling and support from a specialized nurse lead to improved lifestyle habits (13,16,25). the technique of motivational interviewing was chosen to provide person-centred care and makes it possible for individual participants to change one or more lifestyle habits. we regard this method as being both well-suited for clinical practice and feasible for implementing in primary care. the increased physical activity level is important because even small increases in moderate-intensity physical activity provide health benefits (11,26). in our study, men reported a slightly higher level of both exercise and sedentary time than women, but a reverse relationship was observed for daily activity. this movement pattern is consistent with a swedish cohort study of 948 swedish men and women aged 50–64 years by ekblom-bak et al. (27). 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 0 1 2 3 4 5 6 7 8 unhealthy lifestyle habit index: hypertension baseline 1-year follow up unhealthy lifestyle factors baseline 1.69 (±1.46) 1 year: 1.15* (±1.20) % 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 0 1 2 3 4 5 6 7 8 % unhealthy lifestyle factors unhealthy lifestyle habit index: men baseline 1-year follow up baseline 1.80 (±1.46) 1 year: 1.30* (±1.32) 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 0 1 2 3 4 5 6 7 8 unhealthy lifestyle habit index: t2dm+igt baseline 1-year follow up baseline: 1.65 (±1.25) 1 year: 1.16*(±1.26) % unheathy lifestyle factors 0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 0 1 2 3 4 5 6 7 8 unhealthy lifestyle habit index: women baseline 1-year follow up unheathy lifestyle factors baseline: 1.56 (±1.34) 1 year: 1.03* (±1.11) % figure 4. unhealthy lifestyle habit index using nine factors, sexand diagnosis-specific. data are expressed as the percentages of participants in the total sample. mean value (sd) for number of unhealthy risk factors at baseline and 1-year follow-up (�p � 0.001). (igt: impaired glucose tolerance; t2dm: type 2 diabetes mellitus). 102 l. l€onnberg et al. the total sample showed changes in dietary habits to a healthier pattern. similar results have been reported in both national and international studies (13,16,25). this is encouraging because following a healthy diet is associated with a reduced risk of cvd (28,29). in our study, men had a less healthy dietary pattern, which is consistent with other swedish reports on dietary habits (30,31). sex, low income, and low educational level are factors that should be considered when providing dietary counselling (10). one-third of daily smokers stopped smoking after participating in the programme. this result is consistent with other reports of a 30%–40% success rate when participants are offered qualified counselling for smoking cessation, compared with 2%–3% when not given any support (31). a concern that needs to be addressed is what the natural course of lifestyle changes would be for individuals after being diagnosed with either hypertension or t2dm. a study from canada comprising 1281 persons newly diagnosed with hypertension showed that one of five persons quit smoking but that it did not lead to lasting lifestyle changes for e.g. physical activity, weight control, and alcohol use over a period of 2 years (33). lifestyle change after a diagnosis of t2dm shows a similar pattern, with minimal changes in lifestyle factors after receiving a diagnosis, except for smoking cessation which was more common among persons with t2dm compared with those with no t2dm diagnosis according to an australian study of persons newly diagnosed with t2dm by chong et al. (34). this supports the possibility that the structured lifestyle programme might have contributed to the lifestyle changes seen in our study rather than the natural cause of lifestyle changes after being diagnosed. the strength of this relatively large study is that the number of included participants has made subgroup analyses possible. it also provides a rather high external validity since the structured lifestyle programme was performed at an ordinary primary care unit. although the results achieved in this study are promising, there are several limitations. the study was not designed as a randomized controlled trial, and hence we cannot compare the changes in lifestyle habits in responses to this programme with those in standard care. this also means that regression towards the mean should be considered when interpreting the results. the results must be seen in the context of a primary care unit with low cni, indicating a population with a low proportion of individuals who are manual workers, unemployed, or foreign-born from non-westerns countries (22). this can possibly limit the transference of the results of the structured lifestyle programme to primary care units with higher cni. the study involves the testing of many separate null hypotheses, which may entail problems with inflated type i error rates. to address this potential misinterpretation, we have undertaken a more stringent criterion for statistical significance level by using the bonferroni–holm method which reduces the possibility of getting a statistically significant result when performing multiple tests (35). all lifestyle habits were self-reported, and there may have been problems with misreporting or answering in a perceived socially acceptable manner (recall bias). however, the use of questionnaires is common and has relevance to the clinical setting. the questionnaire in our study had dual purposes, both to evaluate change before and after participating in the structured lifestyle programme, and to enhance awareness of current lifestyle behaviour for the individual. another aspect is the ‘hawthorne effect’, i.e. a change in behaviour of the research participants and/or the health-care provider due to the attention they receive regardless of the intervention (36). this might influence the results of our study, and both this and the aspect of recall bias should be considered interpreting the results. our results support the utility of a multifactorial, structured approach in cardiovascular risk prevention for change in unhealthy lifestyle habits in a primary care setting. however, since this study is a single-group study there is a need for future randomized controlled studies to confirm our findings. acknowledgements we thank all personnel at citypraktiken, especially the nurses who performed all counselling sessions. we also thank professor mai-lis hellenius for the inspiration to start the structured lifestyle programme. disclosure statement no potential conflict of interest was reported by the authors. author contributions the study was designed by md and ll together with personnel from citypraktiken. data were analysed by ll, md, and ebb. ll wrote the first draft of the manuscript. all authors critically assessed the manuscript and approved the final manuscript. funding funding was received from praktikertj€anst ab, 103 55 stockholm, sweden. notes on contributors lena l€onnberg, physiotherapist, doctoral student at center for clinical research, v€asterås sweden; main research focus on lifestyle counselling for patients at high cardiovascular risk. elin ekblom-bak, associate professor at the swedish school of sport and health sciences in stockholm, sweden; main research focus on the association between sedentary behavior, physical activity and cardiorespiratory fitness on health and disease risk in the adult population. mattias damberg, associate professor at uppsala university, uppsala, sweden; main research focus on the cardiovascular prevention. orcid lena l€onnberg http://orcid.org/0000-0003-4706-6915 elin ekblom-bak http://orcid.org/0000-0002-3901-7833 mattias damberg http://orcid.org/0000-0001-7654-7553 references 1. naghavi m, abajobir aa, abbafati c, abbas km, abd-allah f, abera sf, et al. global, regional, and national age-sex specific mortality for 264 causes of death, 1980-2016: a systematic analysis for the global burden of disease study 2016. lancet. 2017;390:1151–210. upsala journal of medical sciences 103 2. kotseva k, wood d, de bacquer d, backer g, ryd�en l, jennings c, et al. euroaspire iv: a european society of cardiology survey on the lifestyle, risk factor and therapeutic management of coronary patients from 24 european countries. eur j prev cardiol. 2016;23: 636–48. 3. avanzini f, marzona i, baviera m, barlera s, milani v, caimi v, et al. improving cardiovascular prevention in general practice: results of a comprehensive personalized strategy in subjects at high risk. eur j prev cardiol. 2016;23:947–55. 4. ståhle a, hagstr€omer, janson e, editors. professional associations for physical activity, physical activity in the prevention and treatment of disease, fyss 2017. 3rd ed. stockholm: l€akartidningens f€orlag ab; 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118: 217–221 original article inhibition of hyaluronan synthesis in rats reduces renal ability to excrete fluid and electrolytes during acute hydration sara stridh1, fredrik palm1,2 & peter hansell1 1department of medical cell biology, division of integrative physiology, uppsala university, uppsala, sweden, and 2department of medical and health sciences, linköping university, linköping, sweden abstract background. hyaluronan (ha) is the dominant glycosaminoglycan in the renomedullary interstitium. renomedullary ha has been implicated in tubular fluid handling due to its water-attracting properties and the changes occurring in parallel to acute variations in the body hydration status. methods. ha production was inhibited by 4-methylumbelliferone (4-mu in drinking water for 5 days, 1.45 ± 0.07 g/day/ kg body weight) in rats prior to hydration. results. following hypotonic hydration for 135 min in control animals, diuresis and osmotic excretion increased while sodium excretion and glomerular filtration rate (gfr) remained unchanged. the medullary and cortical ha contents were 7.85 ± 1.29 ng/mg protein and 0.08 ± 0.01 ng/mg protein, respectively. medullary ha content after 4-mu was 38% of that in controls (2.98 ± 0.95 ng/g protein, p < 0.05), while the low cortical levels were unaffected. baseline urine flow was not different from that in controls. the diuretic response to hydration was, however, only 51% of that in controls (157 ± 36 versus 306 ± 54 ml/g kidney weight/135 min, p < 0.05) and the osmolar excretion only 47% of that in controls (174 ± 47 versus 374 ± 41 mosm/g kidney weight/135 min, p < 0.05). sodium excretion, gfr, and arterial blood pressure were similar to that in control rats and unaltered during hydration. conclusions. reduction of renomedullary interstitial ha using 4-mu reduces the ability of the kidney to respond appropriately upon acute hydration. the results strengthen the concept of renomedullary ha as a modulator of tubular fluid handling by changing the physicochemical properties of the interstitial space. key words: diuresis, fluid balance, hyaluronan, hydration, interstitium, kidney, matrix, medulla introduction the glycosaminoglycan hyaluronan (ha) dominates in the extracellular matrix of the renal medulla. the specific physicochemical properties of ha enable a unique hydration capacity (1). in the last decade it was revealed that in the interstitium of the renal medulla the ha levels change depending on the body hydration status while that of the cortex remains unchanged at very low levels (2-5). the rapid changes in ha occurring in response to acute hydration require intact nitric oxide and prostaglandins, while dehydration for 24 h primarily seems to affect the mrna expression of hyaluronan synthase 2 (4). the renomedullary interstitial cell (rmic) is a major producer of ha, and the turnover depends on the osmolality of the growth media and classical hormones involved in renal fluid handling, such as vasopressin and angiotensin ii (6-8). the involvement of ha in pathological kidney function has also been well described where severely elevated levels have been found during for example ischemia-reperfusion injury, transplant rejection, experimentally induced diabetes, and tubulointerstitial inflammation (9-14). the proinflammatory and waterattracting properties of ha have been implicated in the organ dysfunction associated with kidney disease (5). correspondence: peter hansell, department of medical cell biology, biomedical centre, box 571, se-751 23 uppsala, sweden. e-mail: peter.hansell@mcb.uu.se (received 18 june 2013; accepted 6 august 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.834013 http://informahealthcare.com/journal/ups mailto:peter.hansell@mcb.uu.se since the renomedullary ha content varies with hydration status and the rmic turnover of ha depends on osmotic conditions and fluid regulatory hormones, it has been suggested that the physicochemical properties of ha will affect tubular fluid handling by regulating the properties of the interstitial space of the renal medulla where fine-tuning of fluid volume occurs. this system would thus function in parallel with vasopressin-regulated aquaporins. to test the concept of an involvement of renomedullary interstitial ha in renal fluid handling, we reduced the synthesis of ha by administering 4-methylumbelliferone (4-mu) to rats in the drinking water during five days and then challenging them with an acute hypotonic hydration. materials and methods all chemicals were from sigma-aldrich (saint louis, mi, usa) and at the highest grade available unless otherwise stated. all experiments were performed in accordance with the nih guidelines for use and care of laboratory animals and approved by the animal care and use committee of uppsala university. all animals were kept in a room with controlled temperature of 24�c and a 12 h dark–light cycle and had free access to standard rat chow and water. male sprague-dawley rats (charles river, sulzfeld, germany; n = 15, body weight 274 ± 32 g) received the ha synthesis inhibitor 4-mu (1.45 ± 0.07 g/kg body weight/24 h) in the drinking water or the corresponding vehicle for five consecutive days. the drinking water was made fresh every day with 4-mu. the dose was calculated after the experiments from the concentration in the drinking water (15 mg/ml) and by measuring daily water consumption. after five days the rats were anaesthetized with an intraperitoneal injection of thiobutabarbital (inactin, 5-ethyl-5-(1-methyl-propyl)-2-thio-barbiturate sodium, 120 mg/kg body weight) and were placed on a servo-controlled heating pad to maintain the core temperature at 37.5�c. surgery after tracheotomy, polyethylene catheters were inserted into the right femoral vein and artery, the former for infusion of isotonic saline (0.9% nacl) and hypotonic glucose-saline (0.25% nacl, 0.5% glucose) containing 3h-inulin, and the latter for measurement of mean arterial blood pressure (map). the urinary bladder was catheterized through a suprapubic incision for urine sampling. protocol after a post-surgery equilibration period of 45 minutes, another 45-minute period followed for determination of the baseline glomerular filtration rate (gfr) estimated from inulin clearance. therefore, 3h-inulin (185 kbq/ml; bionuclear scandinavian ab, bromma, sweden) dissolved in isotonic saline was infused (5 ml/h/kg body weight) intravenously from the start of the equilibration period. urine and arterial blood samples were taken for subsequent analyses. after this control period the rats were hydrated by changing the infusion to a hypotonic glucose-saline solution and increasing the infusion rate (15 ml/h/kg body weight). then three consecutive periods followed lasting 45 minutes each, resulting in 135 minutes of hypotonic infusion corresponding to hydration of the animal. after completion of the experimental procedure, the kidneys were excised and weighed, and samples from cortex and inner medulla (papilla) were taken and frozen for subsequent analysis of ha content. measurement of blood and urine parameters for gfr gfr was estimated from the clearance of 3h-inulin. the radioactivity of 3h-inulin in plasma (10 ml) and urine (1 ml) was measured by liquid scintillation counting. urine volumes were measured gravimetrically, osmolality by use of a freezing point technique (model 210, the fiske micro-sample osmometer advanced instruments, boston ma, usa), and urinary sodium concentrations by use of flame photometry (il943, instrumentation lab, milan, italy). measurement of ha content excised and frozen samples of kidney tissue were dried at 68�c over night, then disrupted in 0.5 m nacl (fp120, thermo electron corporation, marietta, ohio, usa). protein content was measured using a commercial assay (dc protein assay, biorad laboratories, hercules, ca, usa), and samples were analysed for ha content using a commercial elisa kit (echelon biosciences, inc., salt lake city, ut, usa). statistical analysis all values are expressed as mean ± sem. one-way analysis of variance (anova) and dunnett’s multiple comparison tests were used for comparing effects over time within each group. bonferroni’s multiple comparison tests were used for comparisons of treatment effects against controls at each time point. 218 s. stridh et al. a p-value of less than 0.05 was considered statistically significant. results during the five days of treatment prior to acute experiments the animals ingested similar amounts of fluid suggesting similar hydration (control group 21.8 ± 1.4 ml/day; 4-mu group 25.7 ± 1.2 ml/day). in the control group (n = 7; body weight 283 ± 11 g; kidney weight 1.10 ± 0.03 g) the medullary ha content was 7.85 ± 1.29 ng/mg protein while that of the cortex was 0.09 ± 0.01 ng/mg protein (figure 1). during hydration, gfr, sodium excretion, and arterial blood pressure remained unchanged, while urine flow increased (table i). the excreted urine volume during hydration was 306 ± 54 ml/g kidney weight/135 min (figure 2), and the excretion of osmotically active particles during hydration was 374 ± 41 mosm/g kidney weight/135 min (figure 3). in the group treated with the ha synthesis inhibitor 4-mu (n = 8; body weight 266 ± 4 g; kidney weight 1.10 ± 0.02 g) the renomedullary ha content was 2.98 ± 0.95 ng/g protein, while that of the cortex was 0.08 ± 0.01 ng/mg protein (figure 1). the renomedullary ha content was thus only 38% of that in the control animals (p < 0.05), whereas it was similarly low in the cortex. baseline gfr, urine flow, and arterial blood pressure were similar to that in the control group (table i). the excreted urine volume during hydration was 157 ± 36 ml/g kidney weight/ 135 min (figure 2), which was only 51% of that in the control animals (p < 0.05). the excretion of osmotically active particles during hydration was 174 ± 47 mosm/g kidney weight/135 min, which was only 47% of that in the control animals (figure 3, p < 0.05). discussion the present study demonstrates that if the renomedullary ha level is reduced with the ha synthesis inhibitor 4-mu the ability to respond to a hydration challenge is suppressed. this suggests that the interstitial matrix component ha changes the physicochemical characteristics of the medullary interstitial table i. glomerular filtration rate (gfr), mean arterial blood pressure (map), urine flow rate, and sodium excretion in control rats (c) and in rats treated with the hyaluronan synthesis inhibitor 4-mu. each period corresponds to 45 min in consecutive order, p < 0.05 versus control period of same group. values are means ± sem. control period hydration 1 hydration 2 hydration 3 gfr (ml/min/g kw) c 0.90 ± 0.27 0.78 ± 0.22 0.93 ± 0.16 0.90 ± 0.10 4-mu 0.84 ± 0.12 0.65 ± 0.12 0.80 ± 0.15 0.82 ± 0.15 map (mmhg) c 109 ± 11 104 ± 11 108 ± 9 105 ± 8 4-mu 97 ± 4 96 ± 4 96 ± 3 92 ± 3 urine flow rate (ml/min/g kw) c 1.31 ± 0.45 1.59 ± 0.32 2.61 ± 0.89 2.54 ± 0.49 (p < 0.05) 4-mu 0.82 ± 0.13 1.16 ± 0.34 1.48 ± 0.41 1.52 ± 0.30 (p < 0.05) sodium excretion (ml/min/g kw) c 0.05 ± 0.01 0.07 ± 0.02 0.08 ± 0.02 0.07 ± 0.05 4-mu 0.07 ± 0.02 0.06 ± 0.02 0.06 ± 0.02 0.07 ± 0.02 kw = kidney weight. control h a ( n g /m g p ro te in ) cortex medulla * 0 2 4 6 8 10 4-mu figure 1. hyaluronan (ha) content in cortex and inner medulla (papilla) in control rats and in rats treated with the hyaluronan synthesis inhibitor 4-mu. *p < 0.05 versus control. renomedullary hyaluronan and fluid handling 219 space, which directly affects fluid transport from the tubular lumen through the interstitial space. such a suggestion is corroborated by earlier findings on changes in medullary ha in relation to hydration status (2,3,5). interstitial ha is elevated during hydration which will reduce reabsorption in conjunction with reduced vasopressin-regulated aquaporins. in contrast, during dehydration lower interstitial ha and up-regulated aquaporins will increase fluid transport across the interstitial space of the medulla, leading to increased reabsorption. the concept of an involvement of ha in renal water handling was first proposed by ginetzinsky in 1958 (15). he suggested that the action of the antidiuretic hormone arginine vasopressin (adh) in the renal medulla is exerted through activation of hyaluronidases, which changes the interstitial properties for fluid transport by reducing the amounts of glucosaminoglycans. these findings have been forgotten, and the discovery of aquaporins (16) has further shifted the interest from a possible involvement of the interstitial matrix component ha in renal fluid regulation. we have previously demonstrated that adh infusion not only reduces renomedullary ha levels in vivo but also reduces the ha content in the supernatant of cultured rmic (8). it should be noted that these cells are the major producers of ha in the renal medulla. the effect primarily involves the v1-receptor, since the v2-receptor agonist desmopressin did not alter the ha levels (2). it would thus seem that adh exerts its antidiuretic effect via two separate pathways: first, by changing the permeability of the apical membranes of the medullary tubular system through regulation of the number of aquaporins over the v2-receptor; and second, by changing the physicochemical characteristics of the interstitial space which will reduce or increase the permeability for fluid by changing the ha content over the v1-receptor. the present study suggests that ha directly participates in renal water handling by changing the physicochemical characteristics of the interstitial matrix of the medulla and possibly by affecting the interstitial hydrostatic pressure (2,17,18). it could thereby function as an important regulator of water diffusion in the interstitium regardless of organ origin. a possible mechanistic view of the effects is the following: 1) the repulsion between the negatively charged carboxylate groups of the sugar moieties in ha (glucuronic acid) protrude outward at regular intervals and contribute to the structure and size of the ha molecule; 2) the negatively charged gel attracts positive ions and increases osmosis, which will attract water into the gel matrix. when water is immobilized within the gel, further water reabsorption is antagonized. furthermore, the medullary thick ascending limb of the loop of henle (mtal), which generates the medullary osmotic gradient, may be functionally compromised when interstitial diffusion characteristics are changed due to elevated ha. also, the efficiency of the vasa recta (‘counter-current exchanger’), which help to maintain the osmotic gradient by recirculating fluid and electrolytes in the medulla, may also be affected when diffusion characteristics are changed. this would also result in altered urine concentration capacity. finally, the interstitial swelling (‘functional oedema’), which occurs in response to elevations in ha, increases the diffusion distances between the tubules and blood vessels, which also will affect the reabsorption rate. in the present study, 4-mu reduced the medullary ha content which corresponded to increased ability to reabsorb water demonstrated by a reduced diuretic response upon hydration. the mechanisms underlying the reduced ha production by 4-mu are not completely understood. however, it is clear that 4-mu has no direct influence control u ri n e e x c re ti o n ( ml /g k w /1 3 5 m in ) * 0 100 200 300 400 4-mu figure 2. accumulated urine excretion during hydration in control rats and in rats treated with the hyaluronan synthesis inhibitor 4-mu. *p < 0.05 versus control. (kw = kidney weight). control o s m o la r e x c re ti o n ( mo s m /g k w /1 3 5 m in ) * 0 100 200 300 500 400 4-mu figure 3. accumulated osmolar excretion during hydration in control rats and in rats treated with the hyaluronan synthesis inhibitor 4-mu. *p < 0.05 versus control. (kw = kidney weight). 220 s. stridh et al. on the ha synthases as studied in vitro (19). quite in contrast, the substance has been suggested to deplete the cellular stores of the substrate udp-glucuronic acid which will reduce ha synthesis (20). in summary, when renomedullary ha is reduced using the ha synthesis inhibitor 4-mu, the ability to respond appropriately to hydration is suppressed. the study strengthens the concept that the properties of the medullary interstitium are modulated by the glycosaminoglycan ha, which directly affects the transport of fluid. acknowledgements the technical assistance of angelica fasching is gratefully acknowledged. declaration of interest: support for this study was provided by the swedish research council. the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. fraser jr, laurent tc, laurent ub. hyaluronan: its nature, distribution, functions and turnover. j intern med. 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www.ncbi.nlm.nih.gov/pubmed/11736696?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15031336?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15031336?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15031336?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18441392?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18441392?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18441392?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20933085?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20933085?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20933085?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/13590279?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/13590279?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/13590279?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19096770?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19096770?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/1864772?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/1864772?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/1310233?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/1310233?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12383266?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12383266?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15190064?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15190064?dopt=abstract abstract introduction materials and methods surgery protocol measurement of blood and urine parameters for gfr measurement of ha content statistical analysis results discussion acknowledgements declaration of interest references effects of exercise with or without blueberries in the diet on cardio-metabolic risk factors: an exp full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 effects of exercise with or without blueberries in the diet on cardio-metabolic risk factors: an exploratory pilot study in healthy subjects sofia nyberg, edvard gerring, solveig gjellan, marta vergara, torbjörn lindström & fredrik h nystrom to cite this article: sofia nyberg, edvard gerring, solveig gjellan, marta vergara, torbjörn lindström & fredrik h nystrom (2013) effects of exercise with or without blueberries in the diet on cardio-metabolic risk factors: an exploratory pilot study in healthy subjects, upsala journal of medical sciences, 118:4, 247-255, doi: 10.3109/03009734.2013.825348 to link to this article: https://doi.org/10.3109/03009734.2013.825348 © informa healthcare published online: 27 aug 2013. submit your article to this journal article views: 869 view related articles citing articles: 12 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.825348 https://doi.org/10.3109/03009734.2013.825348 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.825348 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.825348 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.825348#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.825348#tabmodule upsala journal of medical sciences. 2013; 118: 247–255 original article effects of exercise with or without blueberries in the diet on cardio-metabolic risk factors: an exploratory pilot study in healthy subjects sofia nyberg, edvard gerring, solveig gjellan, marta vergara, torbjörn lindström & fredrik h nystrom department of medical and health sciences, faculty of health sciences, linköping university, linköping, sweden abstract background. the improvement of insulin sensitivity by exercise has been shown to be inhibited by supplementation of vitamins acting as antioxidants. objective. to examine effects of exercise with or without blueberries, containing natural antioxidants, on cardio-metabolic risk factors. methods. fifteen healthy men and 17 women, 27.6 ± 6.5 years old, were recruited, and 26 completed a randomized cross-over trial with 4 weeks of exercise by running/jogging 5 km five times/week and 4 weeks of minimal physical activity. participants were also randomized to consume 150 g of blueberries, or not, on exercise days. laboratory variables were measured before and after a 5 km running-race at maximal speed at the beginning and end of each period, i.e. there were four maximal running-races and eight samplings in total for each participant. results. insulin and triglyceride levels were reduced while hdl-cholesterol increased by exercise compared with minimal physical activity. participants randomized to consume blueberries showed an increase in fasting glucose levels compared with controls, during the exercise period (blueberries: from 5.12 ± 0.49 mmol/l to 5.32 ± 0.29 mmol/l; controls: from 5.24 ± 0.27 mmol/l to 5.17 ± 0.23 mmol/l, p = 0.04 for difference in change). triglyceride levels fell in the control group (from 1.1 ± 0.49 mmol/l to 0.93 ± 0.31 mmol/l, p = 0.02), while hdl-cholesterol increased in the blueberry group (from 1.51 ± 0.29 mmol/l to 1.64 ± 0.33 mmol/l, p = 0.006). conclusions. ingestion of blueberries induced differential effects on cardio-metabolic risk factors, including increased levels of both fasting glucose and hdl-cholesterol. however, since it is possible that indirect effects on food intake were induced, other than consumption of blueberries, further studies are needed to confirm the findings. key words: blueberries, cholesterol, exercise, glucose, running, troponin introduction common strategies in the primary prevention of cardiovascular disease include recommendations to perform physical exercise several times a week in order to counteract components of the metabolic syndrome and to increase physical fitness (1). although exercise can lead to improved blood lipid levels, reduced blood pressures, and lower glucose levels, several studies have shown that strenuous long-distance races, such as marathons and triathlons, increase markers of inflammation (2-6). it has been proposed that this pro-inflammatory effect was caused by inclusion of subjects that were less physically fit than the actual exercise required, as inflammation seems to occur less frequently following a period of regular exercise (7), although this has not been specifically investigated in a randomized manner. interestingly, several earlier trials have shown that highly strenuous exercise, such as running a correspondence: professor fredrik h. nystrom, md, phd, department of medical and health sciences, linköping university, se 581 85 linköping, sweden. fax: +46 13 145949. e-mail: fredrik.nystrom@lio.se (received 6 april 2013; accepted 10 july 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.825348 http://informahealthcare.com/journal/ups mailto:fredrik.nystrom@lio.se marathon, can induce release of troponin-t (2,8,9), which is a protein that is used clinically to diagnose myocardial infarction since it is a specific marker for cardiac myocyte damage (10). oxidative stress is implicated in human disease, and in experimental studies antioxidants decrease oxidative damage (11). many subjects are using antioxidant supplements, which are often marketed as to improve health and to prevent disease (12-14). whether antioxidant supplements are indeed beneficial or harmful is uncertain, but a meta-analysis of randomized trials suggested that treatment with beta carotene, vitamin a, and vitamin e may increase mortality, while the effect of vitamin c was less clear (15). ingestion of berries, with natural antioxidants, have, on the other hand, been demonstrated to improve many cardiovascular risk factors (16). however, antioxidants in the form of pills containing vitamin c and e specifically inhibited the increased insulin sensitivity following 4 weeks of exercise, and this effect was present in moderately as well as in highly fit participants (17). we know of no study specifically designed to compare the effects of running a distance common in recreational exercise on components of the metabolic syndrome, in which the effects of natural antioxidants and vitamins in blueberries were analyzed. the aim of this randomized cross-over trial was to compare the effects of a 4-week exercise protocol with or without blueberries to inactivity on markers of cardio-metabolic disease in healthy subjects. the participants were randomized to starting with the exercise period or with the period of minimal physical activity, and also to consuming 150 g of blueberries on the day they exercised, or to keeping eating habits unchanged. material and methods by local advertising at linköping university we recruited 32 participants as volunteers for the study. the participants had to be free from major disease as judged by medical check-up and history, and they had to have some earlier experience of exercise in the form of running. the trial consisted of two main periods: 4 weeks of running (or jogging), and 4 weeks during which the participant should strive for minimal levels of physical activity, without any exercise whatsoever. the study period was from beginning of september 2010 to the end of december 2010, and the study organizers aimed for one month of return to individual regular exercise habits (wash-out) in between the running and minimal-physical-activity periods. however, if anything arose that affected the participants’ ability to exercise or the evaluation in these two main periods, such as musculoskeletal problems or upper respiratory infections, the priority was set to achieve evaluation of the two main periods, and hence it was possible to prolong either period by up to a week, and thus the wash-out period could be shortened correspondingly. participants were randomized to start with either the exercise period or with the 4 weeks of minimal physical activity. at the beginning and end of each trial period the participants ran 5 km at the fastest possible time in the evening (at 6 p.m.), here denoted ‘running-race’. venous blood was drawn for analysis of cardiometabolic risk markers after an overnight fast (10 hours between 6 p.m. and 9 a.m.) on the morning of the day of the evening run, and the corresponding morning after, except for troponin-t which was analyzed only from samples after the races. figure 1 shows a schematic presentation of the study design for a participant randomized to start with the exercise period when reading the figure from left to right. each participant who completed the whole trial thus ran a total of 4 running-races at maximal speed, and venous blood was correspondingly drawn 8 times in the fasting state; thus he or she had one period of exercise, a washout period, and also a period of minimal physical activity in this cross-over trial. the participants were instructed to exercise in the form of running or jogging 5 km five times a week during the exercise period. the randomized design to test effects of antioxidants on the presumed insulinsensitizing effect of exercise was based on a study by ristow et al. in which it was found that vitamin c and e in combination hinders benefits on insulin sensitivity of regular exercise in humans (17). however, in our study the participants were randomized to consume 150 g of frozen blueberries (berries that have a high natural content of several antioxidants (18-20)) on each day of running, or to keep eating habits unchanged, during the training period. no particular instructions were given on how or when to consume the blueberries on the exercise days, in order to make potential findings easily incorporated in regular eating habits. the blueberries had a content of 40 kcal/100 g and also had 0.5 g protein, 8 g carbohydrates, and 0.5 g fat per 100 g of the product. all blueberries were bought on the same occasion from a regular local grocery store, in order to assure that there were no changes in nutrient composition during the study period. the participants were subjected to determination of body fat content with bodpod (life measurement, inc., concord, ca, usa) at the end of both main periods. at these time points resting blood pressures were measured in the seated position with manual technique. all analyses of laboratory variables including high-sensitivity c-reactive protein (hs-crp) were done at the department of clinical chemistry, 248 s. nyberg et al. linköping university hospital as part of their regular clinical routine analyses. troponin-t was analyzed with an electrochemical luminescence method (troponin-t stat cardiac t, cobas e411 equipment, roche, basel, switzerland). serum-insulin was determined by an immunoassay (autodelfia, perkin elmer, linköping, sweden). total-cholesterol, hdl-cholesterol, and triglycerides were determined by colorimetric analyses (siemens, liederbach, germany), and ldl-cholesterol was calculated according to friedewald (total-cholesterol – hdl-cholesterol – 0.456 � total triglyceride concentration). the methods for analyses of the other routine samples have been published (21,22). ethics the study was approved by the regional ethics committee of linköping and performed in accordance with the declaration of helsinki. written informed consent was obtained from all participating subjects. the study was registered at clinicaltrials.gov (nct01274078). statistics statistical estimates were calculated using spss 19.0 software (ibm corporation, somers, new york, usa). comparisons within and between groups were done with student’s paired and unpaired 2-tailed t test or as stated in the results section. mean values and standard deviations are given. statistical significance refers to 2-sided p £ 0.05. since the lower limit of detection of troponin-t was 5 ng/l, the results were set at 2.5 ng/l in calculations of non-detectable levels. correspondingly, serum hs-crp that was undetectable was set as 0.15 mg/l (limit of detection =0.3 mg/l) in calculations. the sample size was based on the trial by ristow et al. in which 20 untrained subjects performed regular exercise for 4 weeks and were randomized to consume antioxidants or not (17). the sample size in our study was increased 60% to allow for dropouts during the longer total study period of three months. analyses were performed per protocol. results we recruited 32 subjects with a mean age of 27.6 ± 6.5 years (15 men and 17 women, age range 21–48 years) for the trial. six subjects could not complete the whole trial (two because of upper airway infections and four because they found the trial to be too time-consuming). three subjects were unable to run all the laps during the complete exercise period due to musculoskeletal problems and performed strenuous bicycling on training bikes instead. thirteen subjects did the last runningrace indoors (n = 5) or on a treadmill (n = 8), due to cold weather and snow which made the race-track difficult to run at a high speed in a secure way. the participants who started with the exercise period shortened the times to finish the race from 1772 ± 371 s to 1621 ± 288 s (p = 0.002), and those who began with minimal physical activity required 1681 ± 341 s to finish the race after the four weeks of little physical activity compared with 1611 ± 348 s at the beginning of the study (p = 0.032). the racetimes were not statistically different at baseline between the groups (p = 0.21). since the particular track that was used for the maximal runningraces became difficult to run on at high speed due bloodsampling: maximal speed running-race exercise-period running/jogging 5 times/week with or without blueberries minimal physical activity washout time (week): alt.1 0 4 8 12 alt.2 12 8 4 0 figure 1. study design description. the figure shows the design for a participant who started with the exercise period (when read from left to right). the subjects were randomized to whether they started with exercise or with the period of little physical activity. during the exercise period participants were randomized to consume blueberries on exercise days, or to be asked not to make any changes in regular eating habits. blood sampling was performed in the fasting state in the morning; running-races at maximal speed were performed in the evening at 6 p.m. exercise, troponin-t and blueberries 249 to large amounts of snow and slippery conditions, the corresponding race-times achieved at the end of the study period (late autumn 2010) were not used in these particular analyses of race-times. results in the mixed population as seen in table i, exercise compared with a minimal physical activity induced lower insulin and triglyceride levels, and higher hdl cholesterol levels, when data from all participants, with or without blueberry supplementation, were analyzed as one group. levels of creatine kinase (ck) almost doubled on average after the running-races (levels before race 2.45 ± 3.2 mkat/l; levels after race 4.44 ± 4.7 mkat/l, p < 0.0001, n = 112). the levels of ck were elevated similarly after the running-race when levels at the end of the exercise period were compared with those after minimal physical activity, while body mass but not fat mass was lower at the end of the period with low physical activity (table i). serum troponin-t was found to be present at a detectable level after 51 out of the total 112 runningraces (45.5%) in the study, 7 of which (6.2%) were above the reference limit of 14 ng/l. among participants who ran at least two running-races 75% showed detectable troponin-t levels in plasma after at least one race. there was no detectable change in postrace troponin-t when the individual levels after the races at the end of the exercise period and after that of minimal physical activity were compared in the mixed population. when analyzed according to gender, however, a reduction in post-race troponin-t after exercise was found in women (table i). the levels of hs-crp increased after the runningraces in general (data from all races accumulated, hscrp before race 0.49 ± 0.7 mg/l; after 0.58 ± 0.7 mg/l, p = 0.017, after exclusion of two subjects with hscrp > 5 mg/l, considered to be outliers, n = 108). levels of hs-crp tended to be lower after the race when results after the exercise period were compared with those at the end of the minimal physical activity period (table i). acute effects of the running-races on metabolic markers the design of the study allowed us to analyze effects of a single running-race on levels of cardiometabolic risk markers. when fasting levels on the morning of the running-race were compared with the corresponding levels the morning after the runningrace, glucose levels were found to be lowered by the races (data from all running-races accumulated, levels before 5.28 ± 0.44 mmol/l; after 5.11 ± 0.39 mmol/l, p < 0.0001), while corresponding insulin levels did not change statistically significantly (levels before 56 ± 23 pmol/l; after 53 ± 25 pmol/l, p = 0.13). analyzed in the same manner, triglyceride levels also decreased the morning after a running-race (levels before 0.99 ± 0.52 mmol/l; after 0.88 ± 0.45 mmol/l, p = 0.008), while levels of hdl-cholesterol or ldl-cholesterol did not change significantly (not shown). effects of blueberries during the exercise period table ii shows changes in fasting levels of markers of insulin sensitivity and lipids (mean values of the levels before and after the race) in the groups randomized to consume blueberries or to keep eating habits unchanged. interestingly, fasting glucose levels tended to increase despite exercise in the blueberry group, and the difference in changes between the two groups was statistically significant (p = 0.044). correspondingly, triglyceride levels decreased only in the control group. these changes were in contrast to effects on hdland ldl-cholesterol which improved in the blueberry-group, although these changes only bordered on statistical significance between groups. effects of blueberries on the corresponding levels of apolipoprotein concentrations showed similar tendencies as on cholesterol levels (table ii). ingestion of blueberries had no discernible effect on levels of hs-crp (table ii). however, there was a diminution of troponin-t levels after the races at the end of the exercise period compared with baseline levels of the same period within the group randomized to blueberries (table ii). discussion as expected, based on the study by ristow et al. (17), the participants in our study improved cardio-metabolic risk markers by exercise, hdl-cholesterol increased, and triglyceride levels fell after exercise, when compared with a period when participants were asked to perform minimal physical activity. fasting insulin levels also differed when comparing changes between these two main periods, suggesting that there indeed was a favorable effect of the exercise and/or an unfavorable effect of a sedentary life-style. a bit more concerning was that in participants who ran at least two races 75% displayed detectable troponint levels after at least one race. however, in women there was a diminution of the troponin-t levels when participants were relatively fit, at the end of the exercise period, as compared with less pronounced fitness at the end of the period with minimal exercise. it has earlier been reported that also a short bout of spinning exercise can induce detectable troponint in serum in healthy individuals (23), which 250 s. nyberg et al. table i. anthropometrics, laboratory variables, and body fat content during the trial in the mixed population, i.e. without separate analysis depending on whether blueberries were consumed or not, n = 26. all laboratory variables, except s-ck and hs-crp, were the mean of the analyses before and after the 5 km race that was performed at the beginning and at the end of both trial periods, i.e. the period of exercise or the period of minimal physical activity. there were no differences between the groups at baseline, i.e. baseline of exercise compared with baseline of exercise periods. p values correspond to student’s paired 2-tailed t test. the delta value is the level of a measurement at the end of the period minus the corresponding level at baseline for the same period. variable baseline before exercise period after 4 weeks of exercise baseline before minimal physical activity period after 4 weeks of minimal physical activity p for difference between the end of the two periods p for difference of delta values between the two periods weight (kg) all – 70.3 ± 9.7 – 69.8 ± 9.7 0.010 – women – 64.9 ± 11 – 64.6 ± 10 0.26 – men – 73.7 ± 7.6 – 73.1 ± 7.9 0.022 – body fat (kg) all – 14.4 ± 8.3 – 14.4 ± 8.4 0.8 – women – 19.1 ± 7.7 – 19.3 ± 7.8 0.43 – men – 11.5 ± 7.5 – 11.3 ± 7.4 0.26 – blood pressure (mmhg) all – 116 ± 10/73 ± 7.2 – 116 ± 11/72 ± 6.5 0.7/0.9 – women – 112 ± 11/72 ± 8.7 – 107 ± 11/68 ± 5.1 0.067/0.13 – men – 119 ± 8.5/73 ± 6.6 – 121 ± 6.6/75 ± 5.8 0.25/0.30 – s-ldl-chol (mmol/l) all 2.68 ± 0.73 2.62 ± 0.75 2.76 ± 0.69 2.77 ± 0.80 0.091 0.18 women 2.46 ± 0.39 2.32 ± 0.33 2.77 ± 0.46 2.66 ± 0.40 0.042 0.063 men 2.85 ± 0.89 2.80 ± 0.88 2.75 ± 0.85 2.85 ± 0.99 0.62 0.65 s-hdl-chol (mmol/l) all 1.50 ± 0.34 1.55 ± 0.38 1.50 ± 0.36 1.50 ± 0.35 0.003 0.039 women 1.70 ± 030 1.76 ± 0.33 1.70 ± 0.34 1.73 ± 0.32 0.13 0.34 men 1.34 ± 0.28 1.43 ± 0.36 1.35 ± 0.30 1.34 ± 0.29 0.017 0.071 s-triglycerides (mmol/l) all 0.94 ± 0.41 0.88 ± 0.31 0.91 ± 0.56 1.0 ± 0.45 0.046 0.036 women 0.71 ± 0.29 0.70 ± 0.29 0.72 ± 0.23 0.85 ± 0.35 0.056 0.27 men 1.1 ± 0.40 0.99 ± 0.27 1.1 ± 0.69 1.1 ± 0.48 0.20 0.087 s-apob (g/l) all 0.875 ± 0.19 0.854 ± 0.19 0.891 ± 0.20 0.891 ± 0.21 0.082 0.21 women 0.814 ± 0.08 0.784 ± 0.12 0.872 ± 0.12 0.855 ± 0.13 0.11 0.73 men 0.925 ± 0.24 0.898 ± 0.22 0.905 ± 0.24 0.915 ± 0.25 0.39 0.23 s-apoa1 (g/l) all 1.38 ± 0.22 1.41 ± 0.23 1.35 ± 0.23 1.38 ± 0.23 0.058 0.71 women 1.45 ± 0.25 1.50 ± 0.26 1.45 ± 0.25 1.49 ± 0.27 0.59 0.86 men 1.32 ± 0.19 1.35 ± 0.20 1.28 ± 0.18 1.30 ± 0.16 0.063 0.76 fs-glucose (mmol/l) all 5.15 ± 0.40 5.25 ± 0.27 5.17 ± 0.37 5.24 ± 0.36 0.9 0.85 women 4.91 ± 0.38 5.14 ± 0.22 5.02 ± 0.27 5.18 ± 0.36 0.95 0.38 men 5.35 ± 0.30 5.31 ± 0.28 5.28 ± 0.41 5.29 ± 0.37 0.92 0.77 exercise, troponin-t and blueberries 251 suggests that it might be quite normal to detect troponin-t in serum after a relatively short period of exercise in people who are generally healthy. but we know of no earlier study that specifically was designed to study effects of increased physical fitness. more bothersome, detectable troponint levels, when measured in subjects without specific symptoms of ischemia, have been shown to be related to increased incidence of mortality (10), thus suggesting that a detectable troponin-t is useful marker of cardiovascular risk. it has been suggested that it is the strain per se, and the ensuing increase in oxidation, that induces the increase in insulin sensitivity following exercise, as recently tested by ristow et al. (17), although more recent studies in rats have not confirmed this (24). our trial design was based on the study by ristow et al., but we aimed to investigate the effects of blueberries rather than supplementation of vitamin c and e in combination. blueberries contain antioxidants such as resveratrol and vitamins (18-20) and are naturally growing berries in europe in late summer and autumn. interestingly we extended the findings in the study by ristow et al. and found that also blueberries seem to affect unfavorably the insulinsensitizing effects of exercise as shown by the difference in delta-values of fasting glucose in the two groups. however, we are not able to pinpoint what particular component in the berries that affected fasting glucose levels, and we are also not able to discern whether these effects were indirect, i.e. that other changes in food intake or behavior were induced by the intake of blueberries per se, as no data on dietary changes were available. the reason for the lack of such data from dietary records was that we did not anticipate that it would give useful reproducible information. although food records might seem to give exact and detailed information, we have earlier table i. (continued). variable baseline before exercise period after 4 weeks of exercise baseline before minimal physical activity period after 4 weeks of minimal physical activity p for difference between the end of the two periods p for difference of delta values between the two periods fs-insulin (pmol/l) all 57 ± 25 49 ± 17 54 ± 17 58 ± 17 0.015 0.009 women 54 ± 25 49 ± 20 58 ± 19 62 ± 17 0.16 0.14 men 59 ± 26 49 ± 16 51 ± 16 55 ± 17 0.044 0.038 s-ck before race (mkat/l) all 2.50 ± 1.6 1.85 ± 0.71 3.00 ± 5.1 1.6 ± 0.62 0.024 0.48 women 1.6 ± 0.98 1.8 ± 0.96 4.2 ± 7.7 1.9 ± 1.6 0.16 0.29 men 4.2 ± 4.3 2.0 ± 0.55 2.4 ± 1.3 1.8 ± 0.49 0.094 0.26 s-ck after race (mkat/l) all 5.3 ± 5.0 3.1 ± 1.9 3.6 ± 4.2 3.6 ± 4.2 0.6 0.28 women 3.0 ± 2.3 2.4 ± 1.4 4.4 ± 6.7 1.9 ± 1.6 0.014 0.66 men 7.8 ± 6.5 3.5 ± 2.0 5.8 ± 5.0 4.7 ± 4.9 0.36 0.31 hs-crp before race (mg/l) all 0.49 ± 0.59 0.42 ± 0.51 0.46 ± 0.52 0.58 ± 0.94 0.28 0.48 women 0.63 ± 0.56 0.56 ± 0.70 0.68 ± 0.72 0.74 ± 1.1 0.41 0.91 men 0.38 ± 0.61 0.35 ± 0.39 0.32 ± 0.31 0.52 ± 0.88 0.46 0.45 hs-crp after race (mg/l) all 0.63 ± 0.58 0.45 ± 0.54 0.57 ± 0.62 0.72 ± 0.86 0.054 0.19 women 0.73 ± 0.59 0.59 ± 0.79 0.82 ± 0.82 1.2 ± 1.2 0.058 0.48 men 0.56 ± 0.59 0.37 ± 0.35 0.41 ± 0.42 0.48 ± 0.47 0.47 0.27 troponin-t after race (ng/l) all 7.1 ± 5.7 5.3 ± 3.3 6.4 ± 6.7 5.2 ± 4.8 0.96 0.92 women 6.8 ± 5.4 5.0 ± 2.5 3.6 ± 2.1 3.4 ± 1.8 0.038 0.68 men 7.2 ± 6.1 5.4 ± 3.7 8.3 ± 8.1 6.3 ± 5.6 0.53 0.86 chol = cholesterol; ck = creatine kinase; fs = fasting serum; hs-crp = high-sensitivity c-reactive protein; s = serum. 252 s. nyberg et al. reported that caloric intake based on food records does not match basal metabolic rate even when physical activity was similar in lean and obese subjects (25), and lof et al. have confirmed bias of caloric intake from food records with regard to bmi (26). finally, the need to weigh and make notes of all food consumed for several days could also by itself affect dietary habits as suggested by under-reporting of vegetable and fruit intake by food records (27). data from 24-h recalls of food intake would also not have been sufficient in this study, in which blueberries were not consumed on every day. insulin levels, known to have large intraindividual variation, showed no statistically significant changes in relation to blueberry intake, but there was a trend towards lowered levels by blueberries. triglycerides, which are related to many components of the metabolic syndrome, decreased only in the control group. in contrast, levels of ldl-cholesterol decreased and hdl-cholesterol increased in participants who had been randomized to blueberries. indeed, as pointed out earlier, an increase in hdl cholesterol by intake of berries has previously been shown by erlund et al. (16). interestingly, we found that ingestion of blueberries was linked with lower troponin-t levels after exercise when levels after the races at baseline of the exercise period were compared with those at the end of the same study period. this would be in accordance with cardio-protective effects of blueberries. however, due to the small number of subjects of each gender, these data should be interpreted with some caution, also since the difference in changes between the groups (blueberries versus controls) regarding troponin-t was not statistically significant. it is also important to point out that, in a recent study of 12 weeks of exercise and the same dose of vitamin e but lower vitamin c dose (500 mg instead of 1000 mg/day), the inhibition of improved insulin sensitivity reported earlier by ristow et al. (17) was not confirmed (28). this suggests that the specific doses of vitamins and/or the duration of the exercise are of importance for affecting the insulin-sensitizing consequences of exercise in humans. the particular trial design, incorporating a total of four running-races in which fasting blood samples were drawn before and after each such race, allowed for a study of the effects of exercise in the evening on fasting levels of insulin and glucose the following morning with high statistical power. when all 112 running-races from the participants were pooled together (irrespective of the period of the study they were taken) it became apparent that fasting glucose is reduced 12 hours after a running-race in healthy subjects, while the insulin levels were unaffected. this finding is in line with known effects of muscular activity to increase levels of adenosine monophosphate (amp) which is formed from adenosine diphosphate that follows from hydrolysis of adenosine triphosphate. amp can induce insulin-independent glucose uptake in fat and muscle cells through activation of amp-kinase (29), and that this indeed was the explanation of our findings was in line with the fact that insulin levels tended to be lower the day after the running-races. hence, when screening for diabetes, the sensitivity to detect the disease is probably diminished if sampling is performed in the fasting state the day after strenuous physical exercise. it is of importance for the interpretation of the data reported in this paper to keep these semi-acute effects (after 12 hours) of exercise in mind, since most analyses were based on average values before and after the race at maximal speed. the increase in inflammation following the maximal races in our study tended to be reduced by the end of the exercise period. this inflammatory activity, that was measured as hs-crp, could emanate from several bodily tissues and is thus unspecific. we would assume that at least some part was derived from large muscle groups such as leg muscles, and thus it seems reasonable that the levels tended to be reduced when physical fitness was achieved at the end of the exercise period when the running technique probably improved in participants, leading to a shortening of the time required to finish the race. these findings are in line with an earlier observation in subjects training in preparation for a marathon run (7). a limitation of our study was that we did not have control of the period of minimal physical activity. but, despite the need to rely on compliance with this presumed period of sedentary life-style, we did indeed find changes in race-times at the beginning of the study, and also changes in markers of the metabolic syndrome, such as hdl-cholesterol and insulin levels, when comparing exercise with the presumed sedentary life-style, which was indicative of compliance with the protocol. we also acknowledge the limitation that not all participants were able to follow the running protocol, either due to side-effects of the exercise or due to the cold weather at the end of the trial period. however, these are common culprits in recreational running in a country such as sweden, and the sample size of the study had been adjusted to allow for several such non-completers. the choice for each participant to consume the blueberries in any way preferred is a limitation from a strict scientific viewpoint. the reason for this design, however, was to make the findings potentially applicable to ordinary life and to differing habits. it is important to note that the design of our study did permit analysis of longexercise, troponin-t and blueberries 253 term effects of exercise in combination with blueberries. indeed, in contrast to lack of positive effects in most studies of high doses of vitamins with presumed antioxidant effects (15), several observations support beneficial health effects of blueberries in humans. these potential effects include reduction of neuronal and cardiovascular diseases (30,31). blueberry extract has also been shown to possess direct antioxidant effects that can be detected in human serum (32). we acknowledge that the small number of participants that completed the study of the potential effects of blueberries on cardiometabolic risk factors, 13 in each group, is a limitation of the trial. in conclusion, we found beneficial effects of blueberries on hdland ldl-cholesterol levels, and also that the troponin-t release after exercise was blunted in subjects who had been randomized to blueberry ingestion. these findings point to the complexity when analyzing effects in humans of a naturally occurring supplement containing several antioxidants and vitamins and were in contrast to unfavorable effects of blueberry ingestion on fasting levels of glucose, as was part of the main hypothesis tested in the study. it is important to note that we cannot discern whether the effects of ingestion of blueberries were direct, or indirect, due to other concomitant dietary changes, and thus we call for further studies on the subject. acknowledgements the study was supported by research funds from the university hospital of linköping, linköping university, and the county council of östergötland. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. orchard tj, temprosa m, goldberg r, haffner s, ratner r, marcovina s, et al. the effect of metformin and intensive lifestyle intervention on the metabolic syndrome: the diabetes prevention program randomized trial. ann intern med. 2005; 142:611–19. table ii. change in fasting levels of laboratory variables in the groups when comparing the 4 week exercise period with (n = 13), or without (n = 13), intake of blueberries. delta values denote difference from baseline to the end of the period. variable supplement baseline before exercise period after 4 weeks of exercise p for difference 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www.ncbi.nlm.nih.gov/pubmed/20384847?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20384847?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22907211?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12323088?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12323088?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12323088?dopt=abstract abstract introduction material and methods ethics statistics results results in the mixed population acute effects of the running-races on metabolic markers effects of blueberries during the exercise period discussion acknowledgements declaration of interest references pharmacological limitations of phage therapy review article pharmacological limitations of phage therapy anders s. nilsson department of molecular biosciences, the wenner-gren institute, stockholm university, stockholm, sweden abstract clinical trial results of phage treatment of bacterial infections show a low to moderate efficacy, and the variation in infection clearance between subjects within studies is often large. phage therapy is complicated and introduces many additional components of variance as compared to antibiotic treatment. a large part of the variation is due to in vivo pharmacokinetics and pharmacodynamics being virtually unknown, but also to a lack of standardisation. this is a consequence of the great variation of phages, bacteria, and infections, which results in different experiments or trials being impossible to compare, and difficulties in estimating important parameter values in a quantitative and reproducible way. the limitations of phage therapy will have to be recognised and future research focussed on optimising infection clearance rates by e.g. selecting phages, bacteria, and target bacterial infections where the prospects of high efficacy can be anticipated, and by combining information from new mathematical modelling of in vivo pharmacokinetic and pharmacodynamic processes and quantitatively assessed experiments. article history received 27 august 2019 revised 27 october 2019 accepted 30 october 2019 keywords bacteriophage; phage therapy; pharmacodynamics; pharmacokinetics; pharmacology introduction the ability of bacteria to develop resistance against antibiotics is probably as old as the bacteria themselves and has been a concern ever since the introduction of the antibiotics we use today (1). however, the current overuse of antibiotics has led to an accelerating spread of antibiotic resistance, and there is no development of new antibiotics taking place (2). the use of bacteriophages, i.e. phage therapy, for the treatment of bacterial infections is not a new idea but has gained attention over the past 20 years as a possible alternative treatment method due to the emergence of antibiotic-resistant bacterial strains (3). the gained interest has mainly been fuelled by the fact that phages can be shown to specifically kill almost any bacteria, are easy and cheap to isolate, and do not interfere with normal human bacterial flora nor the environment (4,5). however, in spite of the need for new ways to cure bacterial infections, and a long history of trials, clinically applied phage therapy is not routinely carried out. there are probably two main reasons for this. firstly, phages are very different from conventional antibiotics. they can obviously kill bacteria, but that does not necessarily imply that they can be used therapeutically; phages are host strain-specific and have special pharmacokinetics (pk) and pharmacodynamics (pd) which demands that methods, from isolation to clinical use, will have to be developed and tailored to each individual phage–bacteria combination (see (6,7) for a definition of phage therapy pk and pd). the pk and pd of phages applied in vivo are, however, poorly understood and not part of the current research agenda. secondly, the long history of using conventional antibiotics has led to the establishment of socioeconomic structures and drug regulation policies which taken together makes it virtually impossible to establish phage therapy. in other words, the push from the scientific community is too weak, with no comprehensive studies demonstrating a sufficiently strong and clinically relevant result of phage therapy that could motivate continued development. likewise, the pull from society and pharma industry is equally weak to make huge investments in a completely new way of treating bacterial infections without relevant proof of concept, and with major regulatory problems. the deadlock is presumably not going to be broken by more in vitro studies of particular phages being effective in killing a certain strain of a pathogen, or by the results from well-designed murine infection models (even though these have contributed substantially to the understanding of the complexity of phage therapy pharmacology). moreover, there are far more economically interesting pharmacological research and development projects for the pharma industry to get involved in. what is needed is a number of clinical trials showing a generally high level of infection clearance comparable to antibiotics and significantly higher than in the trials conducted so far. clinical trials of phage therapy the outcome of clinical trials with different phage–bacteria combinations is heterogeneous, as the variation among trial contact anders s. nilsson anders.s.nilsson@su.se department of molecular biosciences, the wenner-gren institute, stockholm university, se-10691, stockholm, sweden � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 4, 218–227 https://doi.org/10.1080/03009734.2019.1688433 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1688433&domain=pdf&date_stamp=2019-12-09 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2019.1688433 http://www.tandfonline.com participants ranges from complete clearance of bacteria to no effect at all (8–10). reports of randomised and doubleblind clinical trials that have been carried out discuss that the reason for phage treatment failure could be the complexity of intestinal bacterial infections due to other coinfecting bacteria (11), or, in the case of burn wounds, technical difficulties including interfering treatments with antibiotics or too low titres of phages or target bacteria (12,13). another careful trial reports a reduction of the mean of pseudomonas aeruginosa counts in the outer ear of chronic otitis patients after 1, 3, and 6 weeks, as compared to placebo treatment (14). the infection was cleared in 3 out of 12 cases but remained in the other cases with only a minor reduction, or even increase, of bacterial counts (figure 1). apparently, phage therapy worked in some but not all cases, and the average reduction mainly depended on the cases where the therapy worked. possible explanations to the varying results in this case may be the very low dose of phages applied, 2 � 104, of each phage in a cocktail, development of phage resistance, or variation in obstructive biofilm formation, which is a known problem with p. aeruginosa infections (14). the largest and possibly most comprehensive report of clinical phage therapy in general, from the phage therapy unit in wrocław, poland, shows inconclusive results. the patient infection status after treatment was assessed and classified into seven categories. patients in categories a, ‘pathogen eradication’, b, ‘good clinical result’, and c, ‘clinical improvement’, were considered to be good results of the treatment and constituted 40%, whereas categories d, ‘questionable clinical improvement’, e, ‘transient clinical improvement’, f, ‘no response to treatment’, and g, ‘clinical deterioration’, constituted 60% of the patients (10). however, the trials were not randomised, double-blind, clinical trials, and the report involves many different types of infections, bacteria, phage preparations, and treatment procedures. the overall infection clearance rate was, however, significantly lower than previously reported by the polish researchers (15,16). this difference is explained by adopting more stringent assessment criteria of treatment results (10). taken together, the results of clinical trials of phage therapy clearly show a broad variation in efficacy, which makes it very difficult to predict the outcome of a treatment in individual cases. however, many smaller trials and single compassionate treatments have been carried out lately (17,18). phage therapy of single patients is occasionally effective, but due to the severity of their infections, simultaneous antibiotic therapy is common, and negative controls are naturally missing. there are also many preclinical and a few clinical phase 1–2 trials being planned to start soon (19). phage therapy research phage therapy as a field of research has been dominated by experimentalists, and the majority of the experiments referred to above were carried out without much phage therapy-specific theoretical consideration, applying a phage or phage cocktail that had been proven to be highly effective in vitro. such in vitro efficacy can be delusive (e.g. if based on qualitative spot test screening only (20)), and planning of the experiments and assessment of results were subsequently focussed on the overall efficacy of the treatments and not on how, or why, it worked or not. as a consequence, pharmacological data from experiments are more often than not missing, and values of the many parameters that affect the outcome of phage therapy still remain unknown. c fu /g (l og 10 6 7 8 9 10 11 0 ) 7 21 42 days 0 figure 1. a randomised double-blind clinical trial of phage therapy against pseudomonas aeruginosa otitis media. patients were divided into two groups, one treated with phages (solid lines, �) and the other with placebo (dashed lines, �). there is a positive effect of the treatment in three patients as shown by non-detectable counts of the bacterium (cfu/g) after 7 days, but the infection remains in the majority of patients as in most of the patients treated with placebo. symbols under the x-axis indicate the number of patients in the two groups with non-detectable counts of p. aeruginosa at the different time points. data from (14). upsala journal of medical sciences 219 phage therapy theory, on the other hand, is mostly based on results from mathematical models of phages infecting bacteria in bioreactors or chemostats (21–24). these are mostly mass-action-based models that consequently reduce the probability of infection to be dependent on the titre of free phages, the uninfected bacteria, and the adsorption rate only; spatial distribution of phages and bacteria is considered to be uniform, and the diffusion rate infinite. phage biologists have, however, studied phages–bacteria infection dynamics for a long time, and the values of many in vitro infection parameters—e.g. adsorption rate, latency times, and burst sizes—of many phage–bacteria combinations are known. efforts have been made to modify mathematical models, and encompass more and other infection parameters to more closely fit actual conditions, including e.g. better simulation of adsorption dynamics, biofilm formation, and distribution of latency time and burst size (24–26, and references therein). as such, the models do not completely reflect the pharmacology of phage therapy, but they can nevertheless contribute with data on some parameters, and their relative importance, albeit under idealised conditions. current pd models can become the foundation for new refined models with the addition of in vivo data, but models of pk based on in vivo data must also be developed and incorporated into new mathematical models. phage therapy pharmacological complications the aim of phage therapy is to maximise the number of phages that reach and infect as many bacteria as possible, and that these phage infections eventually result in clinically insignificant levels of bacteria without causing unwanted side effects. in order to accomplish this, not only the titre of phages but also the titre of the bacteria must be sufficiently high at the site of infection, and the phage titre must pass the ‘inundation threshold’ where the phage replication outruns the bacterial replication (27,28). this can be achieved either through a single phage infection cycle or by the following cycles of phage infection and reproduction (i.e. active or productive phage infection), but also by repeated administration of phages. it may seem counter-intuitive, but the effectiveness of phage therapy increases as the concentration of bacteria increases. the likelihood that a phage hits a bacterium increases as does the production of more phages. looking back at the results from phage therapy experiments and clinical trials, it seems that the unpredictable or poor results in many cases are due to the combined effect of the pk properties of phages and the in situ pd of phages–bacteria. phages have been applied clinically without recognising the full complex dynamics arising from interactions between the human body, bacteria, and phages, and the quantitative data that would allow reproducing the trials are often missing (29,30). phage pharmacokinetics the unusual pk is a consequence of phage particles being a million times larger than any antibiotics molecule, and consisting of several different proteins. their size limits the dose that can be given as well as lowers the uptake and transportation rates, and their protein nature causes them to be eliminated by the mononuclear phagocytic system (31). in comparison, low-molecular-weight antibiotics have far better properties. ofloxacin, a common broad-spectrum fluoroquinolone antibiotic, reaches a concentration of around 2 mg/ ml in serum after a standard oral dose of 200 mg, equivalent to about 3 � 1015 molecules/ml, with a half-life in plasma of around 30 min, and 12 h in tissue (32,33). in comparison, phages are distributed and taken up by most organs regardless of route of administration (34, and references therein), but the uptake is lower and hence also the titres in different tissues. as a consequence of the size of phages, a phage suspension cannot contain more than approximately 1013–1014 plaque-forming units/ml (pfu/ml). it is, however, technically difficult to achieve more than 1011 pfu/ml crude lysate, and the titre is further reduced after purification (5). there are no comparable human studies on the phage titre in plasma following oral administration of phages, but a human phage therapy safety test reports a peak average of 3 � 104 pfu/ml in stool samples after drinking mineral water supplied with 105 or 107 t4 phages for 2 days, according to different schedules (35). it can, however, be assumed that phages are taken up and transported similarly in other vertebrates (36), and pk of phages have been studied in many animal models (37,38). the results from a rat model showed an increase in pfu in six organs after intraperitoneal or subcutaneous injection of 108 pfu in rat pups. the phage titre was about 107 pfu/ml in blood 2 h after intraperitoneal injection and dropped to below 104 pfu/ml after 24 h. the concentration of phages in spleen and kidney was, after the same time, above 106 pfu/g (37). a study of phage pk in rat reports an overall pfu/ml of a phage cocktail over 108 in serum after a 1 ml intravenously administered bolus of 1010 pfu/ml, and a decline down to 105 pfu/ml after 24 h, indicating an elimination half-life of about 2.3 h (39). continuous infusion of 0.1 ml/h for 24 h of the same phage cocktail and titre resulted, however, in a serum concentration of 107 pfu/ ml. the rate of clearance of phages caused by neutralisation by antibodies, and subsequent phagocytosis, lowers the titre (40–42) and affects the efficacy of a treatment when phages are administered during a longer period of time, but the loss differs between different phages. another reason for inefficient outcome of phage therapy might be a tendency for phages to bind to bacterial debris resulting from already lysed bacteria, blocking their tail fibre receptor-binding protein, and preventing them from adsorbing to live bacteria (43,44). this is not studied empirically in detail in the context of phage therapy, but there is an indication that phage titres from the second and following cycles of phage infection become unexpectedly low in in vitro experiments (45,46). however, there are many different structures that phages potentially can bind to, and the interference with other structures, e.g. exopolysaccharides, may play a more significant role for inactivation of phages. the influence on the infection kinetics from inactivation factors, as well as the importance of uneven distribution of cells and phages, e.g. as would be the case with biofilm formation, 220 a. s. nilsson has been investigated in models by bull et al. (47). phages may also exhibit differences in their propensity to bind to cells of different tissues, which in some cases may lead to an even greater loss of phage titre, but it has been hypothesised that this can be turned into an advantage as it might be utilised for homing of phages to particular tissues (48). the phage dose reaching the site of infection can thus be assumed to be substantially lower than the given dose in the majority of phage treatments, and rapidly drop to even lower levels if phages are not added continuously. this does not necessarily mean that a treatment will fail, since the success of phage therapy is a matter of probability that relies on many factors, e.g. the density of both phages and bacteria and the phage adsorption rate, but the probability that it does fail increases. a low dose of phages will require that a sufficient number of bacteria get infected (which can happen if the bacterial density is high enough), that the phages produced can spread to all infected sites, and that the rate of phage amplification is higher than the bacterial growth rate at particular sites of infection (28,49). the last-mentioned leads to the importance of also understanding phage infection pd in vivo. phage pharmacodynamics recognising the shortcomings of phage therapy due to their special pk properties mentioned above, it can be assumed that the explanation for phage therapy being occasionally successful is phages’ ability to replicate and compensate for a low dose. different mathematical models and experiments with phages–bacteria, in batch or continuous cultures, have contributed to the understanding of their kinetics during in vitro infection (21–23,45,50–53). with basic mathematical models, it is easy to show that any virulent phage with ordinary infection characteristics will completely eliminate all bacteria if the titres of phages and bacteria are sufficiently high and if the bacteria are not allowed to become resistant (50). on the other hand, infection experiments e.g. in chemostats in most cases show something different; after an initial dramatic decline, bacteria grow back to high titres, and the phages are often maintained at high titres as well. the bacteria appear not to become uniformly resistant, and there must be enough susceptible bacteria to be able to sustain a large phage population. mathematical models and other experiments, including experiments with periodic nutrient supply, seem under many conditions also to result in stable coexistence of bacteria and phages (21,54). there could be many reasons for this, one being the obvious development of bacterial phage resistance through acquired mutations of receptor genes or by regular genetic systems (55,56), followed by counter-mutations by the phages and a continued and endless arms race. however, a high rate (10�5 per cell per hour) of genetic transition from resistant to susceptible cells has also been observed, without this being explained by host range mutations in the phage population (57). a bacterial population is not a homogeneous collection of uniform and equally susceptible cells. apart from cells being genetically different, i.e. carrying different mutations of which some may confer complete or partial resistance, the susceptibility to infection and ability to produce phages may also show spatial and phenotypic variation upon infection. spatial heterogeneity in a growing culture may arise from bacteria protecting themselves in surface biofilm or hiding in crevices in the vessel wall from where they can regrow once the phage titre is low enough to permit it (24,58). in vivo dynamics, i.e. titres of phage and bacteria during phage therapy, is probably even more complicated as bacteria will have better chances of producing biofilm in a more diverse environment, or hide deep in tissue or intracellularly, and avoid getting infected. susceptible bacteria will not be evenly distributed, their growth will be affected by nutrient availability, and phages will be more concentrated around lysed bacteria. as a consequence, there will be local differences in growth of bacteria and phages and accumulation of biofilm, released debris, and metabolites that may further impede phage replication, and the pd is also affected by the bacteria’s ability to form micro-colonies or other arrangements (7,47,59). phenotypic variation between individual cells of bacteria, and the physiological state of the bacteria, may also affect the probability of infection and the number of released phages from lysed bacteria (59,60). phenotypic variation in susceptibility between cells may arise due to phase shifting between different states, most often resulting in changes in cell surface structures (61). while the frequency of such random shifts may be small, selection by phages would rapidly increase the number of non-susceptible cells and the population would eventually recover. if the shift varies at random between cells, and is genetically inheritable, an equilibrium between phages and bacteria would eventually occur. other phenotypic changes affecting cell surface structures may be explained by epigenetic gene regulation events. dna methylation is likely to be involved in the regulation of expression of certain pili and outer membrane proteins which potentially can act as phage receptors (62, and references therein). it is also possible that some bacterial receptors vary naturally in such a way that the rate of adsorption will vary in the population and allow selection of bacteria with a low adsorption rate, leading to the coexistence of bacteria and phages (59). after successful adsorption by a phage, and when its nucleic acid has been introduced into the cell, bacteria may also show phenotypic variation in the capacity of replicating the phage. individual bacteria can be dormant depending either on limited access to nutrition or random fluctuations between cells in the expression of key genes. depending on the availability of nutrition, bacteria may lie dormant as spores or have their metabolism running low or completely turned off. it has been shown in vitro that, when infecting bacteria with phages under low-nutrition conditions, some cells will produce just a few phages and that phage production increases significantly after the addition of nutrients. a fraction of phage-infected cells can either be hibernating or be in a pseudolysogenic state when starved, but this is also dependent on the infection biology of the phage. bryan et al. (60) showed that phage t4 can ‘scavenge’ on available resources and produce a small number of phages, but, in upsala journal of medical sciences 221 most infected cells, t4 hibernates and does not carry out the final steps of dna degradation and subsequent reproduction until nutrients are added. phage t7, in contrast, is less dependent on the nutritional status of the host bacteria. it has a smaller genome which requires fewer resources for reproduction and is adapted to take advantage of the resources readily available for its dna replication and production of structural components (60,63). pseudolysogeny of virulent phages, i.e. a stalled development which does not rule out a later full lytic cycle, may happen under nutrientlimited conditions but also when bacteria, having special growth requirements affecting their metabolism (e.g. ph, temperature, or salinity), grow under non-optimal conditions (64,65). superinfection, the infection of an already phageinfected cell by the same or in some cases unrelated phages, can also fail as a result of the inability of the second phage to inject its dna, i.e. superinfection exclusion, or it may lead to an extension of the length of the infection period, i.e. lysis inhibition (66–68). phage therapy using phage cocktails have been reported in a number of studies (13,69). there are two main reasons for simultaneous use of more than one phage against a bacterial infection. firstly, when the bacterial strain and its susceptibility for phages are known, the phages in a cocktail can be chosen to infect the target bacteria by different receptors which likely will reduce the probability of development of resistance. secondly, when the infecting bacterial strain and sensitivity to different phages is unknown, at least one phage in the cocktail may be able to infect. however, the composition of cocktails into liquid formulations limits the individual titre of each phage due to physical reasons, with the result that an already low dose of the phage or phages being potentially effective becomes even lower. phage cocktails also bring additional complexity to the pd. co-infection with two or more phages can in some combinations result in synergism, e.g. when one phage releases an enzyme that depolymerases biofilm, facilitating the infection by another phage (70), but phages competing for resources, as would be the case when more than one phage is able to infect the host, will in most cases interfere with one another. during co-infection, the phage with the highest adsorption rate, shortest latency time, and largest burst size will inevitably have a selective advantage, and slower phages will be eliminated; the result would be equal to an infection with a single phage at a lower titre. furthermore, co-infection can also result in superinfection exclusion or lysis inhibition already mentioned, but also in cross-resistance where the resistance developed under infection of one phage results in resistance against other phages as well. hence, the bacteria can become resistant to all phages infecting by the same receptor through a single mutation altering the structure of that receptor molecule, or against phages utilising different receptors by mutations affecting a common global regulator of multiple receptors (71). bacteria appear to have both genetically controlled resistance systems as well as regular phenotypic ‘contingency plans’ to quickly cope with phage infections. the extent of the various types of resistance in different bacteria and the reason for low efficiency, especially of the mechanisms described above, must be investigated more closely in order to understand the pd of phage therapy. most mathematical models and in vitro experiments do not reflect all aspects of the interactions between phages and bacteria and are not particularly true to a real phage therapy treatment, as the majority of models are presuming phages and bacteria to be planktonic and the phage infection to follow mass-action kinetics. more recently, however, models have been developed which take into account some of the pharmacodynamical problems associated with the low efficacy of phage therapy. recent theoretical models have for instance been developed that include the complexity arising from biofilm or spatial heterogeneity (47), as well as bacteria expressing ‘leaky resistance’ where a fraction of the resistant bacteria reverts to susceptibility at a high rate in spite of being surrounded by virulent phages (57), but every phage–bacteria combination is indeed different and general models may prove to be very challenging to construct. future research and development the in vivo pk and pd of phage therapy, and hence the outcome, cannot be anticipated from phage–bacteria in vitro infection experiments. there is a need for new models, animal experiments, and clinical trials reflecting genetic and phenotypic changes of bacteria and phages during in vivo phage therapy. the outcome of phage therapy trials at present varies from one individual to another and between different studies in such a way that the results are not reproducible. the aim of optimising is to eventually make the result of a phage treatment predictable for a large part of treated infections. in the first place, it will become necessary to focus the efforts and reduce the variation by limiting the number of phages, target bacterial strains, and infections to a few type cases. secondly, the important parameters during in vivo experiments or clinical trials must be quantitatively assessed under different conditions to make it possible to get an idea of the importance and contribution of different components of variance and allow statistical analysis. phage therapy experiments are generally evaluated at different endpoints and by qualitative, or semi-quantitative, measurements, but the optimisation of the efficacy of phage therapy is completely dependent on a systematic generation of quantitative in vivo data (29). selection and basic characterisation of phages in general, reducing the components of variance of phage therapy is theoretically easy but in practice more difficult to do because of the huge variation of phages and bacteria. while there are no virulent phages found for some pathogenic bacteria, e.g. clostridium difficile and helicobacter pylori, virulent phages can be isolated for most pathogens, but the number of phages needed to cover the variation among strains within a particular bacterium varies. it is essential that the phage not only possess good antibacterial properties against the bacterium, but equally important is that no gene products are expressed that add to unexplained variation 222 a. s. nilsson and unwanted side effects. firstly, there are genes with unknown function in virtually every phage genome. genome sequencing data could reveal not only if a phage is truly virulent, which is desirable, but could possibly also rule out phage gene products that may be harmful to man or compromise a treatment (72–74). secondly, phages may exhibit surface proteins that evoke the innate immune system as well as cause elevated levels of antibodies (40,41). phages are, however, part of the microflora that we are exposed to in everyday life, the immune response varies from phage to phage depending on tissue, and it should be possible to find phages which do not give rise to, or only cause insignificant, immunological responses (42,75). however, it is difficult to compare the outcome of studies of immunogenicity originating from the phages themselves as there are many methods for purification, each leaving trace amounts of different fractions of bacterial debris from shattered bacteria which may confound the assessment. reducing the variation coming from differences in immune response would require that phages that are going to be used in clinical trials be produced meeting the requirements set by international regulatory bodies for medicines. there is, however, currently a lack of a common best practice and standardised purification methods, and the degree of purity varies between methods (76,77). the best purifying method would need to be scaled up for fast production of larger volumes, and the phages would in most cases need to be formulated into stable medical preparations without reducing phage titres or phage function. phages can also be selected in vivo for improved stability and extended circulation time by e.g. mutation selection, or formulated by encapsulation, lyophilisation, or pegylation (78–82). many other formulation strategies, e.g. nebulisation or freeze-drying, have also been elaborated that have resulted in greater stability and higher titres at the infection site (9,83). phages’ suitability for phage therapy should be quantitatively shown in efficiency of plating (eop) analyses and not merely in spot tests, since the latter may result in false positives and overestimation of the host range (20,84). the high efficacy of the phage on the bacterial strain should also be reproducible in vitro, with insignificant variance between experiments. that would in turn indicate absence of defence mechanisms against the actual phage, bacterial phase or antigen shifting, masking or sporulation, as well as a low rate of development of any type of resistance against the phage. induction of prophages, or genes within prophage genomes, may also contribute to unwanted variation. this may seem like a benefit, as the bacterial host cell lyses and the released temperate phages can continue to infect other bacteria, but overall this can have the opposite effect; as the number of hosts decreases, the efficacy of the virulent phage goes down and bacteria containing the prophage would be immune to superinfections by the same phage. interference with an induced prophage would not matter if the rate of induction is completely predictable, but it may be triggered by unknown and varying environmental factors and cause random variation. it is well known that prophage genes that get induced interfere with infecting virulent phages; expression of non-essential genes in prophages have been shown to block other phages from replicating, and are often constitutively transcribed from prophages in the bacterial genome (85). prophages are also frequently encoding bacterial virulence factors that can be induced by unknown factors, possibly including infection by virulent phages (86,87). in addition, the bacterial strain should preferably not produce exotoxins and only produce small amounts of endotoxins released upon lysis, and bacteria causing intracellular infections must of course also be ruled out. optimising the pharmacokinetics and pharmacodynamics the pharmacokinetic parameters that primarily influence the outcome of in vivo phage therapy are the phage titre (dose and dosage administered), delivery routes, pharmaceutical formulation, phage adsorption rate, phage decay or elimination rate, and the rate of diffusion or transportation of phages. most of these parameters have not been subjected to optimisation, and it must be pointed out that the efficacy of phage therapy is highly dependent on the pharmacodynamics too. phage therapy may fail even if the pharmacokinetics is optimised, e.g. a very high single dose of phages will be in vain if the bacterial concentration, movement of phages and bacteria, or the adsorption rate is too low. although this may lead to a reduction of bacterial titres, it will not lead to productive phage infection (only to a local increase in phage titre) and subsequent spread of phages, and the bacterial population would consequently continue to grow after the phages have been degraded or eliminated. a phage dose as high as it can possibly be, without inducing the immune system, would be a good start when optimising a phage treatment, but the phage adsorption rate has a large influence on the efficacy too and has to be considered. under ideal conditions, assuming constant mixing of planktonic cells and phages, the number of phages that actually adsorb and infect a bacterium follows the poisson distribution. as the mean probability of infection goes down, the number of uninfected bacteria increases. this has led to the concept of actual multiplicity of infection (moiactual) in contrast to the added moiinput (moiinput ¼ concentration of phages/concentration of bacteria): moiactual ¼ ð1 � e�kctþmoiinput where k is the adsorption rate constant, c is the concentration of bacteria/ml (cfu/ml) of bacteria and moiactual the number of phages bound to bacteria at time k (figure 2). at low titres of bacteria and a low adsorption rate, it may take a long time for the maximum number of bacteria to become infected even though a high titre of phage is added (49,88). maintaining a high titre is especially important if the adsorption rate is low. phages might otherwise become degraded before they can adsorb to the bacteria. to optimise phage therapy, knowing the adsorption rate under the conditions that apply during treatment is essential, and the best alternative would be to choose phages with a very high adsorption rate (for instance t1-like phages: t1 itself has an upsala journal of medical sciences 223 adsorption rate of 3 � 10�9 ml � min�1 (89)). in vivo phage adsorption rates are, however, affected by the movements of both phages and bacteria, and a high in vitro adsorption rate alone does not guarantee phage therapy success. however, small phages might be better than large phages in terms of diffusion and mobility in vivo. in addition to the fact that small phages should have a higher diffusion and transportation rate, some large phages can be disadvantageous as they may have protrusions that can bind to mucus layers, which reduces their mobility (30). it has been suggested that binding to certain cell receptors could be turned into an advantage by adding surface epitopes to the phage, causing improved affinity for a certain tissue and a homing of phages to infected tissues (48). another way to solve the phage-dosing problem is to supply phages in repeated high doses and sustain a high titre over a long period of time (39). this, of course, requires that the phages do not give rise to any serious immunological reaction (which is the main explanation to why the efficiency of phage therapy need to be optimised, not maximized). from a pharmacodynamic perspective, the latency time should be short and the burst size large, but it is of equal importance that the phage infection is independent of the nutritional status of the bacteria and that the phage does not enter pseudolysogeny or causes superinfection exclusion, lysis inhibition, or other obstacles for replicating. all of these parameters can be assessed in vitro as well as the propensity for development of phage resistance or induction of inhibiting genetic systems, taking into account that these properties may very well be observed only under ideal conditions and not reflect what is going to happen in vivo. are cocktails better than a repeated dose of the same or alternatively different phages? cocktails may have advantages when the infecting bacteria are unknown or to delay the development of resistance of the infecting bacterial strain, but this must be balanced against the risk of interference between the phages, that cross-resistance arises, or that the individual dose of each phage becomes lower. however, it has been shown that some phages produce depolymerases with the ability to degrade biofilm and that this can potentially lead to synergies between phages (90–92). such synergies can compensate for the disadvantages and motivate the use of cocktails, especially as biofilm is a major problem in many infections. development of evaluation methods optimisation would not be possible without the development of new quantitative methods that allow for precise monitoring of pharmacological parameters of importance for phage therapy. these methods must be applicable to quantify cfus and pfus in diverse tissues after delivering different formulations of phages by different routes (93). in animal experiments, it is fairly easy to determine phage and bacteria titres at endpoints, and sometimes quantitatively during treatment. alternative techniques such as bioluminescent imaging of phages and bacteria in live animals might supply more detailed information of importance for optimisation of later clinical trials in humans. imaging of phage infection in vivo, e.g. during phage therapy of mice, has not been carried out, although the technique has been established (94). monitoring of infection parameters is much easier with skin infections, where direct counts of titres can be performed (13). it can, however, be demanding since sampling must be carried out at short intervals and in a quantitative way. thus, there is an urgent need for faster and easier methods for phage quantification. if the pk and pd of such treatments become reproducible, and if the bacterial titres 6 7 8 9 10 p fu /m l (l og 10 11 k = 10 -8 k = 10 -9 k = 10 -10 k = 10 -11 k = 10 -12 3 4 5 6 7 8 9 10 cfu/ml (log10) ) figure 2. phage titre needed to reach an actual multiplicity of infection (moiactual) of five after 1 h of infection at different adsorption rate constants (k ¼ adsorbed phages/ml min), and as a function of the bacterial titre, in an ideal pelagic system. at moiactual ¼ 5, the probability that a bacterium gets infected is 0.99. the functions converge to the diagonal line when moiactual ¼ moiinput. for example, at a cfu/ml of 108, a moiinput of 5 equals moiactual if the adsorption rate is higher than 10�9, but if it is lower than 10�12 a moiinput of about 1000 (10 11 pfu/ml) is needed. function values are constant at lower cfu/ml where moiinput has to be substantially higher especially if the adsorption rate is low. 224 a. s. nilsson after treatment are negligible, phage therapy could become an accepted treatment option. however, if a successful result of phage therapy of a topical infection cannot be predicted, it is hard to understand how treatment of other more complicated infections should be planned. conclusion when comparing outcomes of antibacterial treatment, administration of antibiotics often results in complete infection clearance, whereas the results of phage treatment show variation from no effect at all to significant elimination of the infecting bacterium. the goal of phage therapy research should be to reduce that variation in efficiency, i.e. increase the number of cases where the bacterial infection completely clears to substantially higher levels. treatments that could show the same good and reproducible results as treatments with antibiotics have done would most likely lead to increased interest and enable the development of alternatives. however, the list of factors limiting phage therapy is quite long. much of the phage therapy research reported so far has been descriptive, typically showing the ability of a phage to infect a particular bacterial strain, and often not in quantitative terms, advocating the particular phage for therapeutic purposes. such reports will still have some value in the future, but it is desirable that phage therapy research moves on to study how phage therapy works under different conditions rather than just if it works or not, e.g. the cases where phage therapy fails are far more interesting than reports of the sheer percentage of successful cases. it is apparently possible to get phage therapy to work, but single successful cases have shown that it takes extraordinary efforts (95,96), and it is currently difficult to amass such efforts on a broader scale. the special pharmacokinetics and pharmacodynamics of phage therapy need to be studied in depth and phage properties as well as methods outlined that maximise the number of successful treatments. data on a relevant number of bacteria causing serious illness, which have been proven in vitro to be successfully and effectively eliminated by phages, are highly warranted. some strains will be resistant to particular phages, and many phages would be needed to cover all bacterial strains. the number of relevant bacterial species and strains to be selected as targets for phage therapy depends, however, heavily on the financial resources allocated. the primary focus should undoubtedly be on multi-resistant bacteria, severe infections, and those causing a high societal burden. phages should be optimally selected by showing high adsorption rate, large burst size, and, less important, short latency time on at least one bacterial strain. in addition, it might be possible to find phages having a broad host range with comparable eops on many strains for some bacterial species. the establishment and development of collections of phages for therapy may be faster than the development of a new antibiotic, but too slow within a local context (country). although data on the efficacy and host range of phages in many cases are published, available data are varying, as are the phages themselves. there is a need for a centralised initiative on the collection, standardisation of methods (e.g. purification methods), and quantitative evaluation of phages suggested for therapy. initiatives have, however, been taken recently to coordinate resources and share phages and information from research institutions around the world (97–99). disclosure statement the author reports no conflicts of interest. funding this work was supported by the swedish research council for environment, agricultural sciences and spatial planning (formas) coordinated by the animal health and welfare (anihwa) project within the european research area (era-net) under grant number 221–2015-1894 and the olle engkvist byggm€astare foundation under grant number 2015/419. notes on contributor anders s. nilsson is a senior lecturer at the department of molecular biosciences, the 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collection of microorganisms and cell cultures gmbh [internet]. leibniz, germany: leibniz institute. available at: https://www.dsmz.de/ (accessed august 22, 2019). 98. phage directory [internet]. atlanta, ga, usa. available at: https:// phage.directory/ (accessed august 22, 2019). 99. sacher jc, zheng j, mccallin s. sourcing phages for compassionate use. microbiol aust 2019;40:24–7. upsala journal of medical sciences 227 https://www.dsmz.de/ https://phage.directory/ https://phage.directory/ abstract introduction clinical trials of phage therapy phage therapy research phage therapy pharmacological complications phage pharmacokinetics phage pharmacodynamics future research and development selection and basic characterisation of phages optimising the pharmacokinetics and pharmacodynamics development of evaluation methods conclusion disclosure statement references persistent environmental endocrine-disrupting chemicals in ovarian follicular fluid and in vitro fertilization treatment outcome in women review article persistent environmental endocrine-disrupting chemicals in ovarian follicular fluid and in vitro fertilization treatment outcome in women richelle d. bj€orvang and pauliina damdimopoulou division of obstetrics and gynecology, department of clinical science, intervention and technology, karolinska institutet and karolinska university hospital, stockholm, sweden abstract several international organizations have recently highlighted endocrine-disrupting chemicals (edcs) as factors of concern in human reproduction. since successful reproduction is dependent on timely and appropriate action of hormones, disruption of the endocrine system could lead to difficulties in conceiving or carrying a pregnancy to term. edcs are chemicals that disrupt the endocrine system by activating or inhibiting receptors of the endocrine system, and/or altering hormone receptor expression; signal transduction; epigenetic marks; hormone synthesis, transport, distribution, and metabolism; and the fate of hormone-producing cells. due to the increasing production of industrial chemicals over the past century and their lenient control, edcs are now common contaminants in the environment. consequently, everyone faces a life-long exposure to mixtures of chemicals, some of which have been identified as edcs. as birth rates in humans are declining and the use of assisted reproductive technologies increasing, it is timely to consider possible effects of edcs on human reproduction and fertility. in this review, we focus on persistent edcs, their occurrence in ovarian follicular fluid, and associations to treatment outcomes in assisted reproduction. our summary shows that despite being banned decades ago, mixtures of persistent edcs are still detected in the ovarian follicular fluid, demonstrating direct exposure of oocytes to these chemicals. in addition, there are several reported associations between exposure and worse outcome in in vitro fertilization. further research is therefore warranted to prove causality, which will lead towards better regulation and exposure reduction. article history received 21 november 2019 revised 27 january 2020 accepted 4 february 2020 keywords assisted reproduction; endocrine disrupting chemical; follicular fluid; persistent organic pollutant 1. introduction the phenomenon of endocrine disruption started gaining attention in the 1990s after a group of experts concluded that many compounds introduced into the environment by human activity are capable of disrupting the endocrine system of animals and humans with possibly profound consequences (1). the concept of endocrine disruption was popularized by theo colborn’s book, our stolen future, that proposed that chemical pollution is threatening the intelligence, fertility, and survival of the human race (2). today, 25 years after the term endocrine-disrupting chemical (edc) was coined, endocrine disruption remains a highly relevant area of research and debate in society, and the methods to identify and regulate these chemicals are still under development. there are no international registries of numbers of chemicals in the market. the us toxic substance control act (tsca) inventory contains over 86,000 existing chemicals. in the european union, over 22,000 unique substances are registered under the registration, evaluation, authorisation and control (reach) regulation. these databases only have chemicals produced or imported over 10,000 kg/year (tsca) or 1000 kg/year (reach), so it is safe to assume the actual number of different chemicals that are or have been in the market is higher1. the chemical industry is one of the most profitable businesses in the world with a revenue of us$5.7 trillion in 2019. the biggest chemical producers being china, europe, and the united states (4,5). unfortunately, the speed of production of new chemicals has far exceeded the speed of development of chemical health risk assessment. the side effects of uncontrolled chemical use were first discovered in 1950s when wildlife populations of birds, reptiles and mammals started drastically declining due to uncontrolled use of organochlorine pesticides like ddt (dichlorodiphenyltrichloroethane), lindane (gamma-hexachlorocyclohexane), and chlordane (octachloro4,7-methanohydroindane). in the baltic sea region, organochlorine chemicals nearly caused the extinction of the baltic grey seal and the white-tailed sea eagle (6,7). in the united states, populations of bald eagles and alligators declined in polluted areas (8,9). these alarming occurrences among others led little by little to the establishment of international agreements for the restriction of chemicals, such as � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. contact pauliina damdimopoulou pauliina.damdimopoulou@ki.se division of obstetrics and gynecology, department of clinical science, intervention and technology, karolinska institutet and karolinska university hospital, stockholm, 141 86, sweden 1a new estimate of chemicals in global commerce was published after this review was accepted, suggesting that there are 330,000 chemicals that are or have been in the market (3). upsala journal of medical sciences 2020, vol. 125, no. 2, 85–94 https://doi.org/10.1080/03009734.2020.1727073 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1727073&domain=pdf&date_stamp=2020-05-21 http://orcid.org/0000-0002-3619-2257 http://orcid.org/0000-0001-8458-0855 https://doi.org/10.1080/03009734.2020.1727073 http://www.tandfonline.com the stockholm convention (ratified in 2004) as well as to the development of tests for chemical risk assessment. by the time the first validated oecd guidelines for the testing of chemicals were in place in the 1980s, thousands of chemicals were already in the market. although organochlorine chemicals were regulated starting from the 1970s, and later internationally restricted by the stockholm convention, they still persist in the environment due to their extremely long half-lives. sadly, they also still threaten the reproductive success and survival of long-lived species like killer whales (10). in addition, they are now accompanied by a plethora of newer chemicals. current requirements for chemical safety testing in the european union (and elsewhere) are imperfect, in particular for endocrine-disruptive activity (11). the required regulatory structure having a clear definition of edcs, guidance documents, suitable tests, test requirements, and risk management is not in place for any sector of chemical legislation (11). in practice, this means that no regulatory risk assessment concerning endocrine-disruptive activity has been carried out for the chemicals currently in the market. according to estimates by the united nations environment programme and world health organization (unep/who), there are at least 800 chemicals with known endocrine-disruptive activity (12). the european union has formally recognised 13 chemicals as edcs (11). several international organisations in the field of public and reproductive health have recently expressed their concerns about edcs and human reproduction. unep/who prepared an extensive summary of edcs in 2012 and concluded that there are many gaps in our knowledge of endocrine disruption of the female reproductive system and that test methods for screening of chemicals for endocrine disruption on female reproduction are missing (12). a few years later, the endocrine society released their second scientific summary on edcs stating that several classes of chemicals ranging from pesticides to plasticisers can impair ovarian development and function, suggesting that exposure to edcs may be associated, for example, with reduced fertility, infertility, polycystic ovarian syndrome, endometriosis, and fibroids (13). following this, trasande and colleagues estimated that the uncontrolled use of edcs in europe is associated with increased incidence of uterine fibroids and endometriosis, with an estimated annual cost of 1.4 billion euros to the taxpayers (14). in 2013, the american college of obstetricians and gynaecologists (acog) published a committee opinion on exposure to toxic environmental agents stating that ‘the evidence that links exposure to toxic environmental agents and adverse reproductive and developmental health outcomes is sufficiently robust’ to call for timely action to identify and reduce exposure while addressing the consequences (15). the international federation of gynaecology and obstetrics (figo) joined this view in their 2015 opinion (16). both acog and figo also acknowledge that while the exposure to chemicals is ubiquitous, it disproportionally affects people with low income. hence, actions taken to prevent harm of edc exposure in women is not only a question of gender equality, but also a matter of equality in society at large. with this review, we wish to bring the attention of the clinicians working with reproductive-age patients to environmental chemicals as factors affecting fertility and reproductive health in women. we will first briefly outline some central concepts of endocrine disruption, and then focus on three topics: the extensive mixture exposure of all populations to industrial chemicals; the occurrence of persistent environmental chemicals with endocrine-disruptive activities in patients seeking assisted reproduction; and the potential implications of this exposure. 2. central concepts of endocrine disruption 2.1. definitions and mechanism of action an edc is defined as an ‘exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations’ (17). this definition is complex as it needs both an endocrine activity and a demonstration of adverse effects as its consequence in living organisms. adversity in the context of endocrine disruption is defined as ‘a change in morphology, physiology, growth, reproduction, development or lifespan of an organism which results in impairment of functional capacity or impairment of capacity to compensate for additional stress or increased susceptibility to the harmful effects of other environmental influences’ (18). there is a wide range of mechanisms by which edcs can interfere with the endocrine system and cause adverse effects. classically, edcs are thought to act via receptormediated disruption where they mimic actions of endogenous hormones such as oestrogen and androgen (agonists) or blocking interaction of the ligand with the receptor (antagonists) (19). recently, ten key characteristics for the identification of edcs have been proposed (19). in addition to activation or inhibition of receptors of the endocrine system, alteration of hormone receptor expression, signal transduction, epigenetic marks, hormone synthesis, transport, distribution and metabolism, and/or the fate of hormoneproducing cells are listed as mechanisms of action of edcs (19). in the classical mechanism of action, edcs bind to nuclear hormone receptors, which then bind to specific response elements and influence transcription of their target genes (18). by contrast, they can also act as antagonists by binding to the receptor but not triggering the normal response (18). for example, oestrogen-disruptive activity could result from the edc binding to the oestrogen receptor and subsequently activating (agonist) or repressing (antagonist) its downstream activity in the cell. in addition, many edcs also bind to the aryl hydrocarbon receptor (ahr), which like the hormone receptors is a ligand-activated transcription factor. ahr is evolutionary conserved, widely expressed, and activated by a variety of xenobiotics. in response, it triggers the expression of genes involved in xenobiotic and hormone metabolism, such as cyp enzymes (e.g. cyp1a1) and udpgt1a, by binding 86 r. d. björvang and p. damdimopoulou to specific response elements on dna in the promoters of these genes (18). moreover, ahr can also cross-talk with other nuclear receptors, implying that it can indirectly interfere with hormonal signalling pathways at large (18). 2.2. features of endocrine disrupting chemicals edcs have diversified the field of toxicology by challenging traditional toxicological dogmas. it was originally thought that substances cause toxicity in a monotonic dose–response with consequences seen at high doses, that is, dose makes the poison. however, this is not the case with edcs. similar to natural hormones, edcs can produce non-monotonic dose–response curves, where the slope of the curve changes from positive to negative or vice versa, thereby having a uor inverted u-shape. some of the mechanisms behind this response are receptor selectivity, receptor competition, feedback loops, and receptor number (20). this can lead to significant effects even at low doses, implying that biological effects can be observed at exposure levels typical to human exposure or lower. the endocrine system responds to very low concentrations of endogenous hormones due to high affinity of hormones to their receptors, among others. similarly, as edcs mimic natural hormones, they can also trigger a response at low levels (21). for example, the plastic additive bisphenol a leaching from plastic mouse cages caused disruption of meiotic spindles in mouse oocytes at exposure levels corresponding to 1 lg/day per mouse (22). for comparison, the estimated human intake of bisphenol a varies between 10–60 ng/kg per day, suggesting exposure of 0.6–3.6 lg/day for a person weighing 60 kg (23). it has also been shown that there can be a long lag time from exposure until the adverse effect is seen. for example, exposure to edcs during organogenesis is associated with increased risk of development of diseases later in life (24). moreover, this also suggests that chemicals can cause more damage when exposure takes place during certain windows of susceptibility such as the prenatal and early postnatal period because they disrupt essential organ development (25,26). an example of this is diethylstilbestrol (des), a synthetic non-steroidal oestrogen prescribed from 1930s to 1970s to prevent miscarriages as well as decrease risk of pregnancy complications and premature delivery. as des interfered with the reproductive tract development in utero, des-exposed daughters had higher primary infertility, were less likely to have full-term births, and had higher likelihood of premature births, spontaneous miscarriages, and ectopic pregnancies compared with unexposed women (27–29). the des incidence has also illustrated the multigenerational effects of edcs as the grandchildren of des-exposed women have increased risk of irregular menstrual cycles, amenorrhoea, ectopic pregnancy, and preterm delivery (30). since edcs are ubiquitous and can be found in various consumer products, we are not exposed to a single chemical but to multiple chemicals at the same time. common routes of exposure to edcs are oral, respiratory, and dermal. they can also enter the body through intravenous, intramuscular, or subcutaneous routes for example during medical treatments such as ivf procedures. developing foetuses can be exposed through placental transfer of chemicals from the mother, and neonates via breastmilk (12,31). the extensive exposure to edcs can be seen in various biomonitoring programmes in different countries where pesticides, phthalates, bisphenols, aromatic hydrocarbons, benzophenones, perfluoroalkyl substances (pfas), chlorinated chemicals, and metals are commonly detected in the general population (32,33). this mixture exposure can lead to combinatory effects of chemicals called cocktail effects. as chemicals are usually assessed individually, the hazards and risks could be underestimated because possible additive (1 þ 1 ¼ 2), synergistic (1 þ 1 > 2), or antagonistic (1 þ 1 < 2) properties are not accounted for. there is continuous effort on designing and optimising statistical approaches to quantifying the effect of mixtures. various statistical approaches have been proposed from machine learning to classical linear regression, but there is no single best approach that outperforms the others (34). 3. exposures and outcomes in ivf patients 3.1. persistent organic pollutants there are various different groups of edcs, but for the purpose of this review we focus on persistent organic pollutants (pops). pops are halogenated organic chemical substances that are toxic to both human and wildlife, bioaccumulative, and resistant to environmental degradation because of their stability. while most pops are lipophilic in nature and accumulate to fatty tissues, pfas are amphiphilic and bind to proteins. in general, pops are also volatile at certain temperatures and may travel long distances in the atmosphere. hence, they can be found even in areas where they were never used (12,35). for humans, the largest source of pop exposure is diet. contaminated baltic sea fish remains a significant source of pops in scandinavian countries. a list of the pops in focus of this review, their uses, and regulations are given in table 1. we choose to focus on pops since organochlorine chemicals, which form a large part of the group, are historically the chemicals that are associated with disruption of reproductive activities in wildlife. they accumulate in humans with increasing age due to their long half-lives, and the levels therefore reflect the life-history of exposure. currently, women postpone childbearing. the average age of first-time mothers in sweden is 27.3 years for the whole country, and 30.3 years for its capital stockholm (51). delaying starting of a family means longer cumulative exposure to environmental factors, including pops. particularly for older women, whose oocyte quality is already declining (52), the increasing cumulative exposure to chemicals could further worsen the chances of pregnancy. in contrast to other edcs that are easily metabolised such as phthalates, it is difficult to reduce the body burden of pops only through lifestyle modification. for women, the body burden of lipophilic pops is reduced when bearing a child because these chemicals cross the placenta and deposit to the foetus (53). in addition, they are also transferred to the neonate via breast milk (54). upsala journal of medical sciences 87 3.2. pops in follicular fluid and associations to outcomes with the advent of assisted reproductive technologies, follicular fluid has become accessible for evaluating the direct exposure of oocytes to edcs. several studies have measured concentrations of pops in follicular fluid, and some also analysed the associations to treatment outcomes. we summarise the literature on pops in follicular fluid in table 2. the lipophilic pops have been adjusted for sample lipid content in some studies. although the exposure levels were reported in different units (e.g. ng/ml or ng/g wet weight or ng/g lipids), which made direct comparisons between studies challenging, the summary shows direct exposure of oocytes to mixtures of pops, which could lead to cocktail effects. only three studies (57,61,65) gave account of this mixture exposure with the use of principal component analysis. as these cohorts were composed of women undergoing ivf treatment, information on ovarian reserve, endometrial thickness, oocyte quality, fertilization rate, embryo quality, and live birth were readily available to further investigate the impact of pops on human reproduction, specifically on ivf endpoints. approximately half of the studies analysed association between exposure and outcome. common outcomes evaluated were oocyte quality, implantation rate, and live birth as well as endometrial thickness. while some studies did not find any association between chemicals and ivf outcomes, others found that in particular the lipophilic pops were associated with lower fertilisation rates and poorer embryo quality after adjusting for covariates such as age, body mass index, and oestradiol. for example, dichlorodiphenyldichloroethylene (dde), a metabolite of ddt, was found to be associated with lower oocyte quality in three studies (43,57,60), while two failed to find associations (56,59). the indicator pcbs (pcbs 28, 52, 101, 138, 153, and 180) were associated with lower oestradiol, thinner endometrium, and lower fertilization rates in most studies (57,60,62). the pfas compounds were evaluated in two studies and found to be associated with higher androgen levels and higher embryo quality (65,66). it is clear that more studies are warranted, both experimental and epidemiological, to interpret these associations. it should also be noted that the reported cohort studies are relatively small, most having fewer than 100 participants, which clearly limits the statistical power. 3.3. a way forward studying the effects of pops on fertility in women is challenging, as fertility and fecundability depend on multiple factors. in addition, women (and couples) are exposed to multiple pops, which makes statistical analyses challenging. ideally, similar chemicals are grouped together, allowing comparison of toxic equivalency values which is currently done for dioxin and dioxin-like compounds. alternatively, statistical methods that can handle highly correlated exposures and non-linear relationships that are typical for these chemicals should be further developed. effects seen only in some quantiles of exposure should not be disregarded but rather explored further. lastly, human folliculogenesis lasts for months, and during this time cytoplasmic and nuclear maturation take place including epigenetic changes and germline imprinting. exposure assessment during preconception could help identify chemicals with adverse effects on oocyte quality. population studies give a good starting point for gauging associations between exposures and reproductive outcomes. however, for proving causality, experimental models will be needed. better understanding of mechanisms underlying table 1. use, source, and regulation of pops and their suggested reproductive health effects in women. chemical use/sources regulationa associated health effects in women references pecb and hcb fungicide; unintentional production during industrial processes annex a and c failed implantation, increased spontaneous abortion mahalingaiah et al. (36); younglai et al. (37) hch (lindane) agricultural insecticide and treatment for lice and scabies annex a increased spontaneous abortion, premature delivery, endometriosis upson et al. (38); us department of health and human services (39) chlordane termite treatment in food crops (e.g. corn and citrus) annex a altered cycle length chen et al. (40) ddt and dde disease vector control (e.g. malaria) annex b impaired fertilization, impaired lactation, infertility, reduced parity, longer time-to-pregnancy, uterine fibroids gesink law et al. (41); trabert et al. (42); younglai et al. (37) (43) pcbs electrical insulation, heat transfers, hydraulic systems and capacitors, paints, plasticizers, dyes for carbonless duplicating paper annex a and c impaired response to ovulation induction, impaired lactation, reduced parity and fecundability, longer time to pregnancy, uterine fibroids gennings et al. (44); gesink law et al. (41); trabert et al. (42); younglai et al. (37) pbdes flame retardants added to fabrics, textiles, plastics, carpets, and electronical appliances annex a failed implantation, decreased fecundability, endometriosis johnson et al. (45); harley et al. (46); ploteau et al. (47) pfass consumer products that are water-, oil-, and stain-resistant (e.g. scotchgard, teflon) annex b longer time-to-pregnancy, infertility, endometriosis buck louis et al. (48); campbell et al. (49); fei et al. (50) aregulation under the stockholm convention: annex a, elimination of production and use; annex b, restrict production and use; annex c, reduce unintentional releases. dde: dichlorodiphenyldichloroethylene; ddt: dichlorodiphenyltrichloroethane; hcb:hexachlorobenzene; hch: hexachlorocyclohexane; pbde: polybrominated diphenyl ether; pcb: polychlorinated biphenyl; pecb: pentachlorobenzene; pfas: perfluoroalkyl substance. 88 r. d. björvang and p. damdimopoulou ta b le 2. le ve ls of po ps in fo lli cu la r flu id an d th e as so ci at io n s to iv f ou tc om e. c h em ic al sa m p lin g p er io d n a g e (y ea rs ) lo ca ti on le ve l in fo lli cu la r flu id : m ea n {g eo m et ri c m ea n } [m ed ia n ] (s d or ra n g e) a a ss oc ia ti on to iv f ou tc om es b re fe re n ce s h c b 19 94 – 20 03 72 25 – 44 u sa [0 .0 35 ] n g /g w et w ei g h t – m ee ke r et al . (5 5) 20 00 12 28 – 32 it al y 73 n g /g lip id s – d e fe lip et al . (5 6) 20 08 – 20 09 40 25 – 43 be lg iu m 32 (1 9) p g /m l lo w er fe rt ili za ti on ra te ; fe w er h ig h -q ua lit y em b ry os pe tr o et al . (5 7) d d e 19 94 – 20 03 72 25 – 44 u sa [0 .3 63 ] n g /g w et w ei g h t – m ee ke r et al . (5 5) 20 00 12 28 – 32 it al y 63 0 n g /g lip id s – d e fe lip et al . (5 6) 20 02 – 20 03 61 9 19 – 50 sa ud i a ra b ia 0. 40 7 l g /l n o as so ci at io n w it h iv f ou tc om es a lsa le h et al . (5 8) 20 03 – 20 04 99 25 – 41 c ze ch re p ub lic 33 03 .3 (4 20 5. 2) n g /g lip id s n o as so ci at io n w it h iv f ou tc om es ji rs ov � a et al . (5 9) 20 07 – 20 08 32 28 – 42 u sa 0. 68 (0 .9 2) n g /m l h ig h er p ea k oe st ra d io l le ve l; lo w er lik el ih oo d fo r re tr ie va l of m at ur e oo cy te bl oo m et al . (6 0) 20 08 – 20 09 40 25 – 43 be lg iu m 39 2 (3 48 ) p g /m l lo w er fe rt ili za ti on ra te ; fe w er h ig h -q ua lit y em b ry os pe tr o et al . (5 7) 20 13 12 7 20 – 35 c h in a 84 .3 (3 4. 2) n g /g lip id s – zh u et al . (6 1) n r 21 28 – 38 c an ad a 26 77 (1 58 4) p g /m l n eg at iv el y co rr el at ed w it h fe rt ili za ti on yo un g la i et al . (4 3) d d d 20 02 – 20 03 61 9 19 – 50 sa ud i a ra b ia 0. 00 04 l g /l n o as so ci at io n w it h iv f ou tc om es a lsa le h et al . (5 8) 20 03 – 20 04 99 25 – 41 c ze ch re p ub lic 13 .1 (1 5. 6) n g /g lip id s n o as so ci at io n w it h iv f ou tc om es ji rs ov � a et al . (5 9) 20 13 12 7 20 – 35 c h in a 5. 8 (1 6. 8) n g /g lip id s – zh u et al . (6 1) d d t 19 94 – 20 03 72 25 – 44 u sa [0 .0 14 ] n g /g w et w ei g h t – m ee ke r et al . (5 5) 20 02 – 20 03 61 9 19 – 50 sa ud i a ra b ia 0. 00 44 l g /l n o as so ci at io n w it h iv f ou tc om es a lsa le h et al . (5 8) 20 03 – 20 04 99 25 – 41 c ze ch re p ub lic 98 .1 (7 5. 9) n g /g lip id s n o as so ci at io n w it h iv f ou tc om es ji rs ov � a et al . (5 9) 20 07 – 20 08 32 28 – 42 u sa 0. 01 (0 .0 3) n g /m l n o as so ci at io n w it h iv f ou tc om es bl oo m et al . (6 0) 20 08 – 20 09 40 25 – 43 be lg iu m 35 (5 ) p g /m l n o as so ci at io n w it h iv f ou tc om es pe tr o et al . (5 7) 20 10 – 20 13 94 20 – 38 eg yp t 21 .1 (3 .8 ) l g /l th in n er en d om et ri um , lo w er n um b er of sa cs a lh us sa in i et al . (6 3) 20 13 12 7 20 – 35 c h in a 8. 39 (4 .1 6) n g /g lip id s – zh u et al . (6 1) ah c h 20 13 12 7 20 – 35 c h in a 29 .3 (1 5. 1) n g /g lip id s – zh u et al . (6 1) b -h c h 20 08 – 20 09 40 25 – 43 be lg iu m 34 (3 5) p g /m l lo w er fe rt ili za ti on ra te ; fe w er h ig h -q ua lit y em b ry os pe tr o et al . (5 7) 20 13 12 7 20 – 35 c h in a 27 .3 (2 6. 1) n g /g lip id s – zh u et al . (6 1) g -h c h 20 08 – 20 09 40 25 – 43 be lg iu m 34 (1 ) p g /m l n o as so ci at io n w it h iv f ou tc om es pe tr o et al . (5 7) 20 13 12 7 20 – 35 c h in a 18 .5 (6 .7 4) n g /g lip id s – zh u et al . (6 1) d -h c h 20 13 12 7 20 – 35 c h in a 72 .7 (4 9. 7) n g /g lip id s – zh u et al . (6 1) o xy ch lo rd an e 19 94 – 20 03 72 25 – 44 u sa [0 .0 12 ] n g /g w et w ei g h t – m ee ke r et al . (5 5) 20 08 – 20 09 20 25 – 43 be lg iu m n d n o as so ci at io n w it h iv f ou tc om es pe tr o et al . (5 7) tr an sn on ac h lo r 19 94 – 20 03 72 25 – 44 u sa [0 .0 20 ] n g /g w et w ei g h t – m ee ke r et al . (5 5) 20 08 – 20 09 20 25 – 43 be lg iu m n d n o as so ci at io n w it h iv f ou tc om es pe tr o et al . (5 7) pc b 28 20 07 – 20 08 32 28 – 42 u sa 0. 13 (0 .0 3) n g /m l lo w er lik el ih oo d fo r im p la n ta ti on bl oo m et al . (6 0) 20 10 – 20 13 94 20 – 38 eg yp t 45 .5 (9 .4 ) l g /l th ic ke r en d om et ri um , h ig h er n um b er of eg g s re tr ie ve d a lh us sa in i et al . (6 2) 20 13 12 7 20 – 35 c h in a 25 (3 9) n g /g lip id s – h ua n g et al . (6 3) pc b 44 20 03 – 20 04 99 25 – 41 c ze ch re p ub lic 1. 8 (2 .5 ) n g /g lip id s n o as so ci at io n w it h iv f ou tc om es ji rs ov � a et al . (5 9) 20 07 – 20 08 32 28 – 42 u sa 0. 09 (0 .0 3) n g /m l n o as so ci at io n w it h iv f ou tc om es bl oo m et al . (6 0) pc b 47 20 03 – 20 04 99 25 – 41 c ze ch re p ub lic 13 .1 (3 4. 0) n g /g lip id s n o as so ci at io n w it h iv f ou tc om es ji rs ov � a et al . (5 9) pc b 49 20 07 – 20 08 32 28 – 42 u sa 0. 07 (0 .0 3) n g /m l n o as so ci at io n w it h iv f ou tc om es bl oo m et al . (6 0) n r 21 28 – 38 c an ad a 62 .4 (6 .8 ) p g /m l o ut co m e re su lt n ot sh ow n yo un g la i et al . (4 3) pc b 52 20 07 – 20 08 32 28 – 42 u sa 0. 16 (0 .0 4) n g /m l n o as so ci at io n w it h iv f ou tc om es bl oo m et al . (6 0) 20 10 – 20 13 94 20 – 38 eg yp t 37 0. 6 (5 4. 1) l g /l th in n er en d om et ri um , h ig h er n um b er of sa cs a lh us sa in i et al . (6 2) 20 13 12 7 20 – 35 c h in a 43 (3 7) n g /g lip id s – h ua n g et al . (6 3) pc b 66 20 07 – 20 08 32 28 – 42 u sa 0. 09 (0 .0 4) n g /m l lo w er lik el ih oo d fo r im p la n ta ti on an d liv e b ir th bl oo m et al . (6 0) pc b 74 20 07 – 20 08 32 28 – 42 u sa 0. 06 (0 .0 2) n g /m l lo w er lik el ih oo d fo r liv e b ir th bl oo m et al . (6 0) pc b 87 20 07 – 20 08 32 28 – 42 u sa 0. 05 (0 .0 2) n g /m l th in n er en d om et ri um ; lo w er em b ry o q ua lit y bl oo m et al . (6 0) pc b 99 20 00 12 28 – 32 it al y 20 n g /g lip id s – d e fe lip et al . (5 6) 20 07 – 20 08 32 28 – 42 u sa 0. 04 (0 .0 2) n g /m l th in n er en d om et ri um bl oo m et al . (6 0) pc b 10 1 20 00 12 28 – 32 it al y 40 n g /g lip id s – d e fe lip et al . (5 6) 20 03 – 20 04 99 25 – 41 c ze ch re p ub lic 8. 1 (9 .1 ) n g /g lip id s n o as so ci at io n w it h iv f ou tc om es ji rs ov � a et al . (5 9) 20 07 – 20 08 32 28 – 42 u sa 0. 11 (0 .0 4) n g /m l lo w er lik el ih oo d fo r liv e b ir th bl oo m et al . (6 0) (c on ti nu ed ) upsala journal of medical sciences 89 ta b le 2. c on ti n ue d . c h em ic al sa m p lin g p er io d n a g e (y ea rs ) lo ca ti on le ve l in fo lli cu la r flu id : m ea n {g eo m et ri c m ea n } [m ed ia n ] (s d or ra n g e) a a ss oc ia ti on to iv f ou tc om es b re fe re n ce s 20 13 12 7 20 – 35 c h in a n d – h ua n g et al . (6 3) pc b 10 5 20 07 – 20 08 32 28 – 42 u sa 0. 02 (0 .0 2) n g /m l lo w er lik el ih oo d fo r re tr ie va l of m at ur e oo cy te ; h ig h er lik el ih oo d fo r oo cy te fe rt ili sa ti on bl oo m et al . (6 0) 20 08 – 20 09 40 25 – 43 be lg iu m n d n o as so ci at io n w it h iv f ou tc om es pe tr o et al . (5 7) pc b 11 0 20 07 – 20 08 32 28 – 42 u sa 0. 09 (0 .0 4) n g /m l n o as so ci at io n w it h iv f ou tc om es bl oo m et al . (6 0) pc b 11 8 19 94 – 20 03 72 25 – 44 u sa [0 .0 32 ] n g /g w et w ei g h t – m ee ke r et al . (5 5) 20 00 12 28 – 32 it al y 77 n g /g lip id s – d e fe lip et al . (5 6) 20 07 – 20 08 32 28 – 42 u sa 0. 06 (0 .0 4) n g /m l n o as so ci at io n w it h iv f ou tc om es bl oo m et al . (6 0) 20 08 – 20 09 40 25 – 43 be lg iu m 15 (8 ) p g /m l lo w er fe rt ili za ti on ra te ; fe w er h ig h -q ua lit y em b ry os pe tr o et al . (5 7) 20 13 12 7 20 – 35 c h in a 9 (1 9) n g /g lip id s – h ua n g et al . (6 3) n r 8 29 – 44 be lg iu m 60 (2 8� 87 ) p g /g w et w ei g h t – pa uw el s et al . (6 4) pc b 13 8 19 94 – 20 03 72 25 – 44 u sa [0 .0 47 ] n g /g w et w ei g h t – m ee ke r et al . (5 5) 20 00 12 28 – 32 it al y 33 0 n g /g lip id s – d e fe lip et al . (5 6) 20 07 – 20 08 32 28 – 42 u sa 0. 05 (0 .0 3) n g /m l lo w er p ea k oe st ra d io l le ve l bl oo m et al . (6 0) 20 08 – 20 09 40 25 – 43 be lg iu m 49 (3 2) p g /m l lo w er fe rt ili za ti on ra te ; fe w er h ig h -q ua lit y em b ry os pe tr o et al . (5 7) 20 10 – 20 13 94 20 – 38 eg yp t 14 6. 2 (2 1. 3) l g /l th ic ke r en d om et ri um a lh us sa in i et al . (6 2) 20 13 12 7 20 – 35 c h in a n d – h ua n g et al . (6 3) n r 8 29 – 44 be lg iu m 16 1 (6 3� 39 6) p g /g w et w ei g h t – pa uw el s et al . (6 4) pc b 14 6 20 07 – 20 08 32 28 – 42 u sa 0 (0 .0 1) n g /m l h ig h er lik el ih oo d fo r im p la n ta ti on bl oo m et al . (6 0) pc b 14 9 20 07 – 20 08 32 28 – 42 u sa 0. 05 (0 .0 3) n g /m l th in n er en d om et ri um ; lo w er em b ry o q ua lit y bl oo m et al . (6 0) pc b 15 1 20 07 – 20 08 32 28 – 42 u sa 0. 01 (0 .0 1) n g /m l fe w er b as el in e an tr al fo lli cl es , fe w er oo cy te s re tr ie ve d , an d th in n er en d om et ri um bl oo m et al . (6 0) pc b 15 3 20 00 12 28 – 32 it al y 50 0 n g /g lip id s – d e fe lip et al . (5 6) 19 94 – 20 03 72 25 – 44 u s [0 .0 72 ] n g /g w et w ei g h t – m ee ke r et al . (5 5) 20 07 – 20 08 32 28 – 42 u sa 0. 07 (0 .0 4) n g /m l lo w er p ea k oe st ra d io l le ve l bl oo m et al . (6 0) 20 08 – 20 09 40 25 – 43 be lg iu m 72 (4 4) p g /m l lo w er fe rt ili za ti on ra te ; fe w er h ig h -q ua lit y em b ry os pe tr o et al . (5 7) 20 13 12 7 20 – 35 c h in a 0. 04 (0 .2 8) n g /g lip id s – h ua n g et al . (6 3) n r 8 29 – 44 be lg iu m 17 1 (9 3� 41 1) p g /g w et w ei g h t – pa uw el s et al . (6 4) n r 21 28 �3 8 ye ar s c an ad a 73 .3 (7 .0 ) p g /m l o ut co m e re su lt n ot sh ow n yo un g la i et al . (4 3) pc b 15 6 20 00 12 28 – 32 it al y 30 n g /g lip id s – d e fe lip et al . (5 6) pc b 15 7 20 00 12 28 – 32 it al y 6. 7 n g /g lip id s – d e fe lip et al . (5 6) pc b 15 8 20 03 – 20 04 99 25 – 41 c ze ch re p ub lic 10 .2 (1 0. 9) n g /g lip id s n o as so ci at io n w it h iv f ou tc om es ji rs ov � a et al . (5 9) pc b 16 7 20 00 12 28 – 32 it al y 25 n g /g lip id s – d e fe lip et al . (5 6) pc b 17 0 20 00 12 28 – 32 it al y 10 0 n g /g lip id s – d e fe lip et al . (5 6) 20 07 – 20 08 32 28 – 42 u sa 0. 01 (0 .0 1) n g /m l fe w er b as el in e an tr al fo lli cl es bl oo m et al . (6 0) 20 08 – 20 09 40 25 – 43 be lg iu m 21 (1 3) p g /m l lo w er fe rt ili za ti on ra te ; fe w er h ig h -q ua lit y em b ry os pe tr o et al . (5 7) pc b 18 0 20 00 12 28 – 32 it al y 40 0 n g /g lip id s – d e fe lip et al . (5 6) 19 94 – 20 03 72 25 – 44 u s [0 .0 45 ] n g /g w et w ei g h t – m ee ke r et al . (5 5) 20 07 – 20 08 32 28 – 42 u sa 0. 04 (0 .0 2) n g /m l fe w er b as el in e an tr al fo lli cl es bl oo m et al . (6 0) 20 08 – 20 09 40 25 – 43 be lg iu m 51 (3 3) p g /m l lo w er fe rt ili za ti on ra te ; fe w er h ig h -q ua lit y em b ry os pe tr o et al . (5 7) 20 10 – 20 13 94 20 – 38 eg yp t 10 1. 5 (1 9. 2) l g /l th in n er en d om et ri um , lo w er n um b er of fe rt ili se d oo cy te s, lo w er n um b er of cl ea ve d em b ry os a lh us sa in i et al . (6 2) 20 13 12 7 20 – 35 c h in a 0. 03 (0 .3 1) n g /g lip id s – h ua n g et al . (6 3) n r 21 28 �3 8 ye ar s c an ad a 62 .2 (5 .2 ) p g /m l o ut co m e re su lt n ot sh ow n yo un g la i et al . (4 3) n r 8 29 – 44 be lg iu m 16 1 (6 4� 37 2) p g /g w et w ei g h t – pa uw el s et al . (6 4) pc b 18 3 20 00 12 28 – 32 it al y 50 n g /g lip id s – d e fe lip et al . (5 6) 20 07 – 20 08 32 28 – 42 u sa 0 (0 .0 1) n g /m l n o as so ci at io n w it h iv f ou tc om es bl oo m et al . (6 0) 20 08 – 20 09 40 25 – 43 be lg iu m 13 (3 ) p g /m l n o as so ci at io n w it h iv f ou tc om es pe tr o et al . (5 7) pc b 18 7 20 00 12 28 – 32 it al y 63 n g /g lip id s – d e fe lip et al . (5 6) 20 07 – 20 08 32 28 – 42 u sa 0. 02 (0 .0 2) n g /m l n o as so ci at io n w it h iv f ou tc om es bl oo m et al . (6 0) 20 08 – 20 09 40 25 – 43 be lg iu m 18 (1 0) p g /m l n o as so ci at io n w it h iv f ou tc om es pe tr o et al . (5 7) (c on ti nu ed ) 90 r. d. björvang and p. damdimopoulou ta b le 2. c on ti n ue d . c h em ic al sa m p lin g p er io d n a g e (y ea rs ) lo ca ti on le ve l in fo lli cu la r flu id : m ea n {g eo m et ri c m ea n } [m ed ia n ] (s d or ra n g e) a a ss oc ia ti on to iv f ou tc om es b re fe re n ce s pc b 18 9 20 00 12 28 – 32 it al y 90 n g /g lip id s – d e fe lip et al . (5 6) pc b 19 4 20 00 12 28 – 32 it al y 23 n g /g lip id s – d e fe lip et al . (5 6) pb d e 28 19 94 – 20 03 65 27 – 44 u s 0. 00 1 n g /g w et w ei g h t n o as so ci at io n w it h fa ile d im p la n ta ti on jo h n so n et al . (4 5) 20 13 12 7 20 – 35 c h in a n d – h ua n g et al . (6 3) pb d e 47 19 94 – 20 03 65 27 – 44 u s 0. 02 6 n g /g w et w ei g h t n o as so ci at io n w it h fa ile d im p la n ta ti on jo h n so n et al . (4 5) 20 08 – 20 09 40 25 – 43 be lg iu m 12 p g /m l n o as so ci at io n w it h iv f ou tc om es pe tr o et al . (5 7) 20 13 12 7 20 – 35 c h in a 0. 32 (0 .9 1) n g /g lip id s – h ua n g et al . (6 3) pb d e 99 19 94 – 20 03 65 27 – 44 u s 0. 01 4 n g /g w et w ei g h t n o as so ci at io n w it h fa ile d im p la n ta ti on jo h n so n et al . (4 5) 20 08 – 20 09 40 25 – 43 be lg iu m 14 p g /m l n o as so ci at io n w it h iv f ou tc om es pe tr o et al . (5 7) 20 13 12 7 20 – 35 c h in a 13 (1 3) n g /g lip id s – h ua n g et al . (6 3) pb d e 10 0 19 94 – 20 03 65 27 – 44 u s 0. 00 6 n g /g w et w ei g h t n o as so ci at io n w it h fa ile d im p la n ta ti on jo h n so n et al . (4 5) 20 13 12 7 20 – 35 c h in a 35 (2 1) n g /g lip id s – h ua n g et al . (6 3) pb d e 15 3 19 94 – 20 03 65 27 – 44 u s 0. 00 7 n g /g w et w ei g h t in cr ea se d od d s fo r fa ile d im p la n ta ti on jo h n so n et al . (4 5) 20 13 12 7 20 – 35 c h in a 1. 1 (3 .3 ) n g /g lip id s – h ua n g et al . (6 3) pb d e 15 4 19 94 – 20 03 65 27 – 44 u s 0. 00 3 n g /g w et w ei g h t n o as so ci at io n w it h fa ile d im p la n ta ti on jo h n so n et al . (4 5) 20 13 12 7 20 – 35 c h in a 0. 43 (2 .1 ) n g /g lip id s – h ua n g et al . (6 3) pb d e 18 3 19 94 – 20 03 65 27 – 44 u s 0. 00 0 n g /g w et w ei g h t n o as so ci at io n w it h fa ile d im p la n ta ti on jo h n so n et al . (4 5) 20 13 12 7 20 – 35 c h in a 0. 24 (1 .1 ) n g /g lip id s – h ua n g et al . (6 3) pf o s 20 08 – 20 09 38 25 – 43 be lg iu m [7 .5 ] (3 0. 3) n g /m l h ig h er fe rt ili za ti on ra te ; h ig h er p ro p or ti on of h ig h q ua lit y em b ry o pe tr o et al . (6 5) 20 15 59 20 – 45 u k {2 } (3 .6 9) n g /m l ir re g ul ar m en st ru al cy cl es ; lo w er fr ee an d ro st en ed io n e in d ex h ef fe rn an et al . (6 6) pf o a 20 08 – 20 09 38 25 – 43 be lg iu m [1 .8 ] (3 ) n g /m l h ig h er fe rt ili za ti on ra te ; h ig h er p ro p or ti on of h ig h q ua lit y em b ry o pe tr o et al . (6 6) 20 15 59 20 – 45 u k {1 .8 2} (6 .2 1) n g /m l h ig h er te st os te ro n e le ve ls h ef fe rn an et al . (6 6) pf n a 20 08 – 20 09 38 25 – 43 be lg iu m [0 .4 ] (1 .9 ) n g /m l h ig h er fe rt ili za ti on ra te ; h ig h er p ro p or ti on of h ig h q ua lit y em b ry o pe tr o et al . (6 5) 20 15 59 20 – 45 u k {0 .4 1} (1 .4 2) n g /m l h ig h er te st os te ro n e an d an d ro st en ed io n e le ve ls h ef fe rn an et al . (6 6) pf h xs 20 08 – 20 09 38 25 – 43 be lg iu m [0 .3 ] (1 .3 ) n g /m l h ig h er fe rt ili za ti on ra te ; h ig h er p ro p or ti on of h ig h q ua lit y em b ry os pe tr o et al . (6 5) 20 15 59 20 – 45 u k {0 .8 8} (1 0) n g /m l h ig h er te st os te ro n e le ve ls h ef fe rn an et al . (6 6) a u n it s us ed as re p or te d in th e st ud y. b a n ex p os ur e st ud y is in d ic at ed b y – . d d e: d ic h lo ro d ip h en yl d ic h lo ro et h yl en e; d d t: d ic h lo ro d ip h en yl tr ic h lo ro et h an e; h c b: h ex ac h lo ro b en ze n e; h c h : h ex ac h lo ro cy cl oh ex an e; n r: n ot re p or te d ; pb d e: p ol yb ro m in at ed d ip h en yl et h er ; pc b: p ol yc h lo ri n at ed b ip h en yl ; po ps : p er si st en t or g an ic p ol lu ta n ts . upsala journal of medical sciences 91 folliculogenesis, oocyte quality, ovarian aging, and endometrial receptivity will be needed in order to tailor better assays for chemical safety testing. 4. conclusions multiple studies have identified cocktails of pops in ovarian follicular fluid of reproductive-aged women across the world, although the use of most of the chemicals in focus in this review was restricted decades ago (tables 1 and 2). specifically, lipophilic organochlorine chemicals such as dde and pcbs were associated with worse outcomes in ivf treatments (table 2). in addition, these compounds have also been linked to other adverse reproductive outcomes in women (table 1). originally, organochlorine chemicals were found to be toxic for reproduction in wildlife animal populations. our review suggests that they may also worsen the chances of successful fertility treatments in humans. although cohort studies give information about significant associations between exposures and outcomes, they cannot demonstrate causality or inform about associated mechanisms. therefore, experimental models will be needed for proving endocrine mechanisms of action as well as causality. because edcs affect not only reproductive function of women but also the health of the offspring, it is of utmost importance that all action should be taken to reduce exposure. advising women and families on the use of consumer products, healthy diets, and home supplies represents a good start and raises awareness. however, populations can only truly be protected with better chemical regulations that prevent harmful chemicals from entering the market. therefore, research proving causal effects between edc exposures and adverse effects in humans is urgently needed. disclosure statement no potential conflict of interest was reported by the author(s). funding this work was supported by jane and aatos erkko foundation, swedish research council formas, and european union’s horizon 2020 research and innovation programme under grant agreement [no 825100; freia]. notes on contributors richelle d. bj€orvang, md, is a phd student at karolinska institutet, stockholm, sweden. pauliina damdimopoulou, phd, is an associate professor and a senior researcher at karolinska institutet. her research group focuses on chemicals and fertility in women. orcid richelle d. bj€orvang http://orcid.org/0000-0002-3619-2257 pauliina damdimopoulou http://orcid.org/0000-0001-8458-0855 references 1. colborn t, clement c. chemically-induced alterations in sexual and functional development: the wildlife/human connection. in: advances in modern environmental toxicology. princeton, nj: princeton scientific publishing co.; 1992. 2. colborn t, dumanoski d, peterson myers j. our stolen future. new york: dutton; 1996. 3. wang z, walker gw, muir dcg, nagatani-yoshida k. toward a global understanding of chemical pollution: a first comprehensive analysis of national and regional chemical inventories. environ sci technol. 2020. doi: 10.1021/acs.est.9b06379 4. cefic. facts and figures of the european chemical industry [internet]. 2018. 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disruption definitions and mechanism of action features of endocrine disrupting chemicals exposures and outcomes in ivf patients persistent organic pollutants pops in follicular fluid and associations to outcomes a way forward conclusions disclosure statement references functional stimulation of graft nerves has minor effects on insulin release from transplanted rat pa full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 functional stimulation of graft nerves has minor effects on insulin release from transplanted rat pancreatic islets leif jansson, caroline kampf & örjan källskog to cite this article: leif jansson, caroline kampf & örjan källskog (2013) functional stimulation of graft nerves has minor effects on insulin release from transplanted rat pancreatic islets, upsala journal of medical sciences, 118:4, 209-216, doi: 10.3109/03009734.2013.818601 to link to this article: https://doi.org/10.3109/03009734.2013.818601 © informa healthcare published online: 27 aug 2013. submit your article to this journal article views: 380 view related articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.818601 https://doi.org/10.3109/03009734.2013.818601 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.818601 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.818601 upsala journal of medical sciences. 2013; 118: 209–216 original article functional stimulation of graft nerves has minor effects on insulin release from transplanted rat pancreatic islets leif jansson1, caroline kampf2 & örjan källskog1 1department of medical cell biology, uppsala university, uppsala, sweden, and 2department of immunology, genetics and pathology, uppsala university, uppsala, sweden abstract introduction. morphological evidence for reinnervation of pancreatic islet grafts is plentiful. however, to what extent intragraft nerves influence the endocrine functions of the islet transplant is largely unknown. we therefore aimed to directly stimulate nerves leading to islet grafts with electrodes and measure insulin secretion in response to this. methods. we implanted syngeneic islets under the renal capsule of rats, and examined them 1 or 7–9 months later. in anesthetized rats blood samples were collected from the renal vein and femoral artery, respectively, during electrode stimulation of the nerves leading to the islet grafts. results. as expected, nerve stimulation decreased renal blood flow. however, serum insulin concentrations in samples derived from the renal vein or femoral artery changed in concert with one another, both during normoglycemia and acute hyperglycemia. conclusion. reinnervation which occurs after islet transplantation under the renal capsule has minor effects on graft endocrine function. key words: insulin release, islet transplantation, pancreatic islets, reinnervation introduction the endocrine pancreas contains a rich and complex innervation, encompassing sympathetic, parasympathetic, sensory, peptidergic, entero-pancreatic, and nitric oxide synthase-containing neurons (1-3), all of which have important modulating effects on the secretion of islet hormones. in general, insulin secretion is stimulated by parasympathetic and inhibited by sympathetic nerves or their neurotransmitters (1,2,4,5). in addition to nerve fibers, some islets also contain nerve cell bodies, so called neuro-insular complexes (6), and most islets seem to be surrounded by schwann cells (7,8). to what extent reinnervation occurs after transplantation of pancreatic islets has been studied extensively from a morphological point of view (9). during the first week after implantation some nerve cells survive, but they disappear soon thereafter (10). reinnervation predominantly from sympathetic nerves occurs during the next 3–4 months, mainly by axonal ingrowths along the blood vessels (11-13). nervemediated effects on graft blood perfusion appear with time, as demonstrated after implantation of fetal islets (14). effects of intra-graft nerves on endocrine transplant function have, however, not been demonstrated other than by indirect means (9). however, in a recent series of experiments it has been demonstrated that islets implanted into the anterior chamber of the eye become reinnervated and functionally affected by mainly parasympathetic nerves, even though mouse strain differences were noted (15). the aim of the present study was to evaluate functionally if intra-graft nerves affect insulin secretion from the grafts by directly stimulating these nerves with electrodes. to achieve this we implanted correspondence: leif jansson, department of medical cell biology, biomedical centre, box 571, husargatan 3, se-751 23 uppsala, sweden. fax: +46 18 4714059. e-mail: leif.jansson@mcb.uu.se (received 13 june 2013; accepted 19 june 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.818601 http://informahealthcare.com/journal/ups mailto:leif.jansson@mcb.uu.se syngeneic islets under the renal capsule of rats, i.e. at a site where mainly sympathetic reinnervation occurs, and examined them 1 or 7–9 months later. materials and methods animals male, inbred wistar-furth rats weighing 300–350g were purchased from scanbur (sollentuna, sweden). all animals had free access to tap water and pelleted rat food throughout the experiments. all experiments were approved by the local animal ethics committee at uppsala university. islet isolation and transplantation pancreatic islets were isolated by a collagenase digestion method, as previously described (16). the islets were cultured free-floating, with 150 islets in each culture dish, in 5 ml culture medium, rpmi 1640 (sigma-aldrich; stockholm, sweden) supplemented with l-glutamine (sigma-aldrich), 11.1 mmol/l d-glucose, benzyl penicillin (100 u/ml; roche diagnostics scandinavia, bromma, sweden), streptomycin (0.1 mg/ml; sigma-aldrich), and 10% (vol/vol) fetal calf serum (sigma-aldrich). the medium was changed every second day. a total of 17 syngeneic male, wistar-furth rats were used as recipients. these animals were anesthetized with an intraperitoneal injection of ekvicitine (a mixture of pentobarbital and chloral hydrate). the left kidney was exposed through a flank incision, and 250 islets were implanted under the renal capsule. an additional 12 rats served as untreated, age-matched control animals. surgical preparation of the animals for nerve stimulation the transplanted or age-matched control rats were anesthetized with an intraperitoneal injection of thiobutabarbital (inactin�; research biochemicals, natick, ma, usa; 120 mg/kg) 1 or 7–9 months after transplantation. the animals were placed on a servocontrolled heated operating table to maintain body temperature and breathed spontaneously through a tracheostomy. polyethylene catheters were inserted into the femoral artery and vein. the arterial catheter was connected to a pressure transducer (pdcr 75/1; druck ltd, groby, uk), thereby allowing constant monitoring of the mean arterial blood pressure, whereas the venous catheter was used to infuse ringer solution continuously (6 ml/kg body weight/h). a subcostal left flank incision was made to visualize the graft-bearing left kidney. the kidney and its vascular stalk was gently dissected free from surrounding tissues and immobilized in a lucite cup. through the suprarenal vein a catheter was inserted with its tip in the lumen of the renal vein to allow for blood sampling. the nerve plexus of the renal artery was then located. between this plexus and the kidney one or several fairly large (diameter approximately 0.2–0.3 mm) discrete nerves, usually accompanied by a small blood vessel, could be seen with the aid of the operating microscope (17), and this was used for the subsequent stimulations. some of the nerve fibers were embedded in the adventitia of the renal artery and vein. the nerves were carefully dissected free from surrounding tissues as atraumatically as possible. after this, the nerves were hooked on to a bipolar 100 mm thick platinum-iridium electrode. stimulation was made with a custom-made pulse generator and isolated stimulator with square-wave signals. the width of these signals was 1 ms and the intensity 5 v, with a frequency of 5–10 hz. to control that the nerve stimulation induced a physiological response we continuously recorded renal blood perfusion. to achieve this we used an ultrasound flow probe (irb/206; transonic systems inc., ithaca, ny, usa). the probe was, after careful dissection, placed around the central parts of the renal artery. an ultrasound gel was applied around the probe, and the kidney was then embedded in pieces of cotton wool soaked in ringer solution and covered with mineral oil (apoteksbolaget; uppsala, sweden) heated to body temperature to prevent evaporation and thereby keeping the kidney moist and at body temperature. experimental protocol after an initial equilibration period of approximately 30 min, blood samples (50 ml each) were obtained from the femoral artery and the renal vein and stored in heparinized microcapillary tubes (110.690; kebo grave, stockholm, sweden). nerve stimulation was then initiated (1 ms, 5 v, 10 hz) and maintained for 60–90 s. during this time period blood samples were again secured. a 10-min resting period followed, and at the end of this period new blood samples were obtained. a second nerve stimulation, similar to the first one, with simultaneous blood sampling was then performed, once again followed by a 10-min long resting period. after this 1 ml of a 30% (w/v) d-glucose solution was given intravenously. three minutes later blood samples were secured. an additional 2 min later a third nerve stimulation with simultaneous blood sampling was performed. this means that a total of 14 blood samples, amounting to a volume of 210 l. jansson et al. approximately 700 ml, were taken from each animal. the graft-bearing kidney was removed and fixed in 4% formaldehyde, after which the animal was killed by an intravenous injection of saturated kcl. throughout the experiment renal blood flow was monitored, and the effectiveness of the nerve stimulation was assessed by the induced decrease in renal blood perfusion, which amounted to a 30–70% decrease from the basal value. whenever blood samples from the renal vein and femoral artery were obtained, blood glucose concentrations were measured with test reagent strips (medisense svenska ab, stockholm, sweden) on blood secured from the femoral artery. analysis of blood samples the microcapillary tubes were centrifuged and the hematocrit was recorded. serum was then removed and stored at –20�c. these samples were later analyzed for insulin content with an elisa technique (supersensitive rat elisa�; mercodia ab, uppsala, sweden). morphological examination the formaldehyde-fixed kidneys were dehydrated, embedded in paraffin, and sectioned to a thickness of 4 mm. the sections were placed onto superfrost/ plus� slides (braunschweig, mentzel, germany), deparaffinized in xylene, and rehydrated in graded alcohols. incubation with peroxidase blocking reagent� (dako, stockholm, sweden) for 10 min, to block endogenous peroxidase, was followed by washing in wash buffer� (dako). the pgp 9.5 antibody (dakopatts, glostrup, denmark) diluted 1:100 in antibody diluent with background reducing components� (dako) was incubated for 30 min at room temperature followed by washing in wash buffer�. the chemmate envision detection kit� (dako, stockholm, sweden) was used according to the manufacturer’s instructions. the envision kit� was developed with diaminobenzidine before being counterstained with hematoxylin. the sections were dehydrated and mounted in pertex� mounting medium (histolab, göteborg, sweden). negative control slides were processed identically except that the primary antibody was omitted. the immunohistochemical staining was done at room temperature using an automated immunostaining instrument (autostainer plus�; dako). statistical method all values are given as means ± sem. probabilities (p) of chance differences between the groups were calculated with student’s paired or unpaired t test or anova. all calculations were made with sigmastat� (sssp; erfart, germany). results all rats tolerated the transplantation without any complications, and no signs of infirmity were noted. a total of two transplanted animals were excluded from the study since the surgical preparation and identification of nerves failed. the body weight of the animals was similar in transplanted and non-transplanted rats of the same age (data not shown). blood glucose concentrations were similar at the beginning of the experiment in all animals, and they all responded with a prompt increase after glucose administration (figure 1). the glucose response 3 min after injection of exogenous glucose in 9-month-old control rats was slightly lower than in the other groups (figure 1). mean arterial blood pressure varied between 100 and 120 mmhg in all animals, and did not differ between the groups (detailed data not shown). likewise hematocrit, which remained at 44%–46%, was similar in all animals. occasional nerve cells (figure 2a) as well as nerve fibers within the grafts, and especially so around blood vessels, were seen in all studied specimens (figure 2b), and more extensively in the older age 0 ctr 1 mo b lo o d g lu c o s e ( m m o l/ l) ctr 9 mo tx 1 mo tx 9 mo 5 10 15 20 25 30 * * * *§ figure 1. blood glucose concentrations in anesthetized, islettransplanted (tx) wistar-furth rats, 1 or 9 months after transplantation. ctr represents age-matched, non-transplanted rats. samples were taken after surgical preparation (black bars) or 3 min after an intravenous injection of 1 ml 30% d-glucose (grey bars). values are means ± sem for 5–9 experiments. *p < 0.001 when compared to the corresponding basal value (student’s unpaired t test); §p < 0.05 versus other glucose-stimulated groups as calculated with anova. nerve stimulation of transplanted islets 211 group. due to the scarcity of the nerves no attempts at quantification were made. renal blood flow was higher in transplanted rats implanted 9 months earlier than in the other groups (figure 3). when renal nerves were stimulated with an electrode, renal blood flow decreased to the same extent in transplanted and non-transplanted animals in both age groups. however, the third stimulation was less pronounced in all groups (figure 4; p < 0.01 when compared to the basal values for all comparisons with anova on ranks). in all test groups the ratio between insulin concentrations in samples from the renal vein and femoral artery varied between 0.6 and 0.8 during basal conditions (figure 5). after glucose administration the ratio increased in both control and transplanted rats except in the 9-month-old control rats (figure 5). the most pronounced increase (p < 0.05 compared to the other groups; anova on ranks) was seen in the 1-month control rats. there were no differences in the ratio between serum insulin concentrations in the renal vein and femoral artery during any of the nerve stimulations (figure 6). renal flow values and insulin concentrations in the renal vein were used to calculate the amount of insulin released into the renal vein during basal conditions (figure 7a) and during glucose stimulation (figure 7b). as expected, the amount of insulin markedly increased after glucose administration (p < 0.001 for all comparisons against normoglycemic control groups with anova). however, there were no differences when values during basal conditions and hyperglycemia, respectively, were compared between transplanted and non-transplanted rats (figure 7a and 7b). during nerve stimulation the amount of insulin released into the renal vein decreased to the same extent in transplanted and non-transplanted control rats 1 month (figure 8a) or 7–9 months (figure 8b) after transplantation. discussion previous experimental studies have demonstrated that reinnervation occurs in transplanted pancreatic islets, even though it usually takes several months until any significant number of nerves can be recognized (9,11,15). the types of nerves differ from those seen in endogenous islets (9,11,18), mainly encompassing sympathetic nerves in association with blood vessels, a finding which was confirmed in the present study. this is likely to reflect the choice of implantation organ, since parasympathetic nerves have been demonstrated in islets grafted in the anterior eye chamber (15). we identified nerves adjacent to the renal artery and vein under a stereo-microscope during surgery, 0 1 2 3 4 5 6 7 ctr 1 mo r e n a l b lo o d f lo w ( m l/ m in ) ctr 9 mo tx 1 mo tx 9 mo * figure 3. left kidney blood flow measured, after surgical preparation, with an ultrasound flow probe in anesthetized, islettransplanted (tx) wistar-furth rats, 1 or 9 months after transplantation. ctr represents age-matched, non-transplanted rats. values are means ± sem for 5–9 experiments. *p < 0.05 when compared to the other groups as calculated with anova. a b figure 2. light micrographs of islet transplants under the renal capsule 1 month (a) and 8 months (b) after transplantation. the sections were immunohistochemically stained for the presence of pgp 9.5 and counterstained with hematoxylin. a neuron (arrow) can be seen in a, and a blood vessel with positive nerves in the adventitia in b. scale bar 100 mm in a and 200 mm in b. 212 l. jansson et al. and after careful dissection it was possible visually to ascertain that some branches from the nerves that we stimulated entered the islet graft. the nerves, ramifying into both the renal and islet parenchyma, were then stimulated with electrodes in a way similar to that used in our previous studies on whole kidneys (17). this was the reason for us to choose this implantation site, since it allowed us to confirm in vivo that the nerves we stimulated really entered the graft. it should be noted that we cannot evaluate to what extent we stimulate both efferent and afferent nerves, or if they belonged to the sympathetic or parasympathetic nervous system. there are suggestions that neurotransmitters characteristic of afferent nerves, e.g. calcitonin gene-related peptide (cgrp) and substance p, can be found in islet grafts implanted under the renal capsule (9,18), but their importance for graft function is unknown. there is a marked increase in cgrpcontaining nerves after experimental whole-pancreas transplantation (19), but, despite this, graft insulin secretion is unaffected. this argues against an important contribution of these intrapancreatic nerves for metabolic regulation. in the endogenous pancreas cgrp reduces islet blood perfusion (20), but if this is the case also in transplanted islets is at present unknown. in view of previous studies in islets, suggesting that mainly sympathetic reinnervation occurs, even though this depends somewhat on the implantation site (10,13,21), we believe that stimulation of intragraft nerves would mainly release sympathetic neurotransmitters such as noradrenaline and neuropeptide y. these substances preferentially inhibit insulin secretion (1,2). however, there were only minor effects on the release of insulin into the renal vein, which drains also the islet grafts. this would argue against that any functionally meaningful reinnervation occurs, at least at this implantation site. this is in contrast to some previous experiments where physical exercise induced a decrease in serum insulin concentration from islets implanted into the liver in rats, also after adrenalectomy (22,23). also direct stimulation of hepatic nerves in the perfused liver of rats transplanted intraportally with neonatal islets demonstrated an inhibition of insulin release 9 months post-transplantation (24), presumably through a2adrenoceptors (25). islets implanted into the portal vein also exhibit pulsatile insulin secretion, which is likely to be due to reinnervation (26-28). it is also worthy of note that exercise in islet-transplanted rats leads to hypoglycemia, irrespective of whether the grafts are located in the liver, kidney, or the peritoneal cavity (29). the last-mentioned finding was interpreted to reflect an islet dysfunction per 40 0 1 2 control 1 month t× 1 month 3 4 5 6 7 8 50 60 70 80 90 100 110 r e n a l b lo o d f lo w ( % o f b a s a l) a b 40 0 1 2 3 4 5 6 7 8 50 60 70 80 90 100 110 r e n a l b lo o d f lo w ( % o f b a s a l) control 1 month t× 1 month figure 4. renal blood flow as a fraction of renal basal blood flow (given as 100% at 1, 3, 5, and 7; for absolute values, see figure 3) during the three nerve stimulations (at 2, 4, and 6) in islet-transplanted rats (grey dots) and non-transplanted control rats (black dots), 1 month (a) or 9 months (b) after transplantation. values are means ± sem for 5–9 experiments. see ‘materials and methods’ for further details. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 ctr 1 mo r a ti o i n s u li n r e n a l/ fe m o ra l b lo o d v e s s e ls ctr 9 mo tx 1 mo tx 9 mo ** * * figure 5. ratio between serum insulin concentrations in samples from the left renal vein and femoral artery during basal conditions (black bars) and 3 min after an intravenous injection of 1 ml 30% d-glucose (grey bars) in transplanted (tx) and non-transplanted control (ctr) rats, 1 or 9 months after transplantation. values are means ± sem for 5–9 experiments. *p < 0.05; **p < 0.01 when compared to the corresponding basal value (student’s unpaired t test). nerve stimulation of transplanted islets 213 se, and since it was seen also in animals carrying microencapsulated islets it is difficult to reconcile the findings with the presence of reinnervation. thus, there are several studies suggesting that reinnervation affecting islet graft function may occur. the question is why we did not see this in our study. one possibility is that it varies depending on the implantation organ or animal strain used. most of the studies referred to above were performed in intraportally transplanted rats, whereas we used intrarenal, subcapsularly grafted islets. studies on nerve stimulation of islets implanted into the liver showed that such islets stimulate hepatocyte glycogenolysis (30,31). thus, the local release of glucose and other substances within the liver during nerve stimulation and perfusion may affect the local milieu of the implanted islets. it may also be that there are differences in functional importance of reinnervation between these sites (13). as a control that the nerve stimulation was sufficient to induce a physiologic response we continuously measured renal blood flow with an ultrasound-doppler device and found a pronounced decrease in blood perfusion whenever we stimulated the renal nerves. it can of course be argued that possible changes induced by nerve stimulation were masked by the simultaneous reduction in blood flow. however, in view of the results in studies suggesting a role for intra-graft nerves, i.e. a reduction in insulin secretion, such an effect would be even more pronounced if studied during vasoconstriction. in the present study we also mainly observed sympathetic nerves, which are more prone to inhibit insulin secretion (1). thus, in our opinion the reduced renal blood flow is unlikely to explain our findings. we chose to collect blood for insulin measurements both from the renal vein, representing insulin preferentially released from the transplanted islets but 0.0 0 1 2 3 4 5 6 7 8 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 control t× 1 month r a ti o i n s u li n a b 0.0 0 1 2 3 4 5 6 7 8 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 r a ti o i n s u li n control t× 9 month figure 6. ratio of serum insulin concentrations in samples from the renal vein and femoral artery during the three nerve stimulations (at 2, 4, and 6) in transplanted (grey dots) and non-transplanted control (black dots) rats, 1 (a) or 9 months (b) after transplantation. the value at basal conditions (1, 3, 5, and 7) is normalized to 1 (for absolute values see figure 5). values are means ± sem for 5–9 experiments. see ‘materials and methods’ for further details. 0 2 4 6 8 10 12 14 16 ctr 1 mo in s u li n r e le a s e i n to re n a l v e in ( n g /m in ) ctr 9 mo tx 1 mo tx 9 mo 0 10 20 30 40 50 60 70 ctr 1 mo in s u li n r e le a s e i n to re n a l v e in ( n g /m in ) a b ctr 9 mo tx 1 mo tx 9 mo figure 7. total amount of insulin released into the left renal vein in transplanted (grey bars) and non-transplanted control (black bars) rats, 1 month or 9 months after transplantation during basal conditions (a) and 3 min after an intravenous injection of 1 ml 30% d-glucose (b). values are means ± sem for 5–9 experiments. 214 l. jansson et al. also from the endogenous islets, as well as from the femoral artery. to what extent the latter contains insulin primarily due to graft or endogenous insulin production is difficult to ascertain. to interpret this, the vascular anatomy of the graft must be taken into consideration. the afferent, arterial blood vessels to grafts under the renal capsule are derived from the interlobular arteries and thereby penetrate the graft from below, i.e. the renal side (32,33). the venous outflow is derived mainly from large vessels on the surface of the graft, which empties into intralobular veins some distance from the grafts (34,35). thus, the venous outflow contains a mixture of blood from the islet graft and the renal parenchyma. during normal blood glucose concentrations the ratio between insulin in the renal vein and femoral artery was approximately 0.6–0.8 in both transplanted and control rats, that is, the systemic insulin concentrations were higher than those in the renal vein. besides the liver, the kidney is normally the major site of insulin removal from the systemic circulation with approximately 50% of peripheral insulin removed by this organ (36). the major route is by glomerular filtration, followed by proximal tubular reabsorption and intracellular degradation or transcellular transport (37,38). insulin is cleared also from the post-glomerular, peritubular circulation by receptor-mediated processes (39). thus, the ratio we have measured can be accounted for by renal degradation of insulin, especially that from the systemic circulation, namely the endogenous pancreas. as outlined above, the insulin released from the graft is unlikely to be affected by renal degradation for anatomical reasons and can explain that our measured femoral vein insulin constitutes 60%–80% of that of the systemic circulation. during hyperglycemia, i.e. stimulated insulin secretion, the ratio between insulin concentrations in the renal and femoral vein was approximately 1.2–1.4 in both control and transplanted rats. this is likely to reflect the higher systemic insulin concentrations, allowing more insulin to escape destruction in the kidney. interestingly, the finding also suggests that the contribution from the graft is minor during hyperglycemia, since the values are similar in transplanted and control rats. the major finding in the present study was that the reinnervation occurring after islet transplantation seems to be of minor importance for the insulin release from the grafted islets. another issue of interest with regard to nerves is the finding that the autoimmune assault in type 1 diabetes is not restricted to the islet b-cells, but also encompasses intra-islet nervous structures (40). it has even been suggested that autoimmunity against such nervous structures might be involved in triggering the attack also on b-cells (41). if, and to what extent, nervous structures in islet grafts can be subjected to or help to trigger an autoimmune assault post-transplantation remains to be established. acknowledgements financial support was received from the swedish medical research council (55x-00109), the swedish diabetes association, the juvenile diabetes research foundation, and the family ernfors fund. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. ahrén b. autonomic regulation of islet hormone secretion– implications for health and disease. diabetologia. 2000;43: 393–410. 30 40 50 60 70 80 90 100 110 0 1 2 3 4 5 6 7 8 control t× 1 month in s u li n r e le a s e o n to r e n 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methods animals islet isolation and transplantation surgical preparation of the animals for nerve stimulation experimental protocol analysis of blood samples morphological examination statistical method results discussion acknowledgements declaration of interest references when do we need clinical endpoint adjudication in clinical trials? article when do we need clinical endpoint adjudication in clinical trials? claes held uppsala clinical research center, department of medical sciences, cardiology, uppsala, sweden abstract clinical endpoint adjudication (cea) is a standardized process for assessment of safety and efficacy of pharmacologic or device therapies in clinical trials. cea plays a key role in many large clinical trials with the aim of achieving consistency and accuracy of the study results, by applying independent and blinded evaluation of suspected clinical events reported by investigators. however, due to high costs there are different opinions regarding the use of central adjudication versus more simplified strategies or site-based assessments and whether the final results differ significantly. there is a lack of scientific evaluation of different adjudication strategies, and more knowledge is needed on the optimal adjudication process and how to achieve the best cost-effectiveness. new methodologies using national registry data and artificial intelligence may challenge the traditional adjudication strategy and could potentially reduce cost considerably with a similar result. further research and evidence in this field of clinical trials methodology are essential. article history received 9 july 2018 revised 22 august 2018 accepted 23 august 2018 keywords adjudication; clinical trials; endpoints introduction clinical endpoint adjudication (cea) is a standardized process for the assessment of safety and efficacy of pharmacologic or device therapies in clinical trials. central adjudication plays a key role in many large clinical trials with the aim of achieving consistent, accurate, independent, unbiased, and blinded evaluation of suspected clinical events reported by investigators. however, there is a controversy regarding the use of central adjudication committees versus simplified strategies or site-based assessments, and, thus, the value has been debated (1,2). arguments have been made that the final results do not differ from the investigator judgement and that it is a costly process. despite a long history of centralized adjudication of both cardiovascular (cv) and non-cv endpoint events, there is a need for the clinical trials community to establish a set of best practices to inform how cea committees should be structured and operated. for a long time there has not been a standardized and commonly accepted cea process in the scientific community, and there is a need for standardization of the cea methodology. in addition, historically the endpoint definitions have been inconsistent between studies, which has been a challenge when comparing results between studies. one example is the definition of bleeding, where there are several sets of definitions available. often, a single study includes several bleeding definitions to adjust for this challenge. however, recently an international expert group, the bleeding academic research consortium (barc), developed the barc standardized bleeding criteria which are now widely accepted (3). another important endpoint in cv outcome trials is the definition of myocardial infarction (mi), which has been updated several times by the universal definitions expert group (4), and a new updated definition is awaited shortly. recently, an expert committee, led by the food and drug administration (fda), published a joint suggestion on endpoint definitions of the most important cardiovascular events (5), which is now becoming a standard. for device trials the academic research consortium (arc) just published suggestions for revised endpoint definitions, such as periprocedural mis and stent thrombosis in coronary device trials (6). previous studies have shown that the rate of mi assessed by standardized adjudication or by investigator-reported results differed significantly (7). differential ways of identifying endpoints, such as using triggered events or screening of laboratory data or ecg for mi, could possibly increase the detection of mi (8) or bleedings (9). at uppsala clinical research center (ucr), we have offered cea services for clinical trials for 10 years and have now substantial experience of adjudication of outcomes, from small academic studies to registry-based randomized clinical trials and large big pharma phase iii trials for registration purposes. this review aims to provide an overview and highlight the advantages but also the challenges of performing centralized cea and to provide some future perspectives for the role of central adjudication in future clinical trials. selection of endpoints when a prospective clinical trial is planned, the primary and secondary outcomes are usually defined as both a composite contact claes held claes.held@ucr.uu.se uppsala clinical research center, department of medical sciences, uppsala, 75185 sweden � 2018 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 1, 42–45 https://doi.org/10.1080/03009734.2018.1516706 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1516706&domain=pdf http://orcid.org/0000-0001-9402-7404 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1516706 http://www.tandfonline.com of clinically relevant outcomes, such as cv death, myocardial infarction (mi), and stroke, and its separate components. in studies on antithrombotic or anticoagulant therapy, safety endpoints are mandatory, of which bleeding complication is an important and common endpoint. when total mortality is included, subcategories such as cv death and non-cv death (infection, cancer, or other organ-specific endpoints) are often being evaluated. subcategories of cv endpoints, such as non-st elevation mi versus st-elevation mi (nstemi versus stemi) or type 1–5 mi, or ischemic versus hemorrhagic stroke, are used for subgroup analyses. despite the fact that clinical endpoint adjudication has been applied for several decades, the evidence is surprisingly scarce on what endpoint definitions should be used and how to capture the information. updates and revisions are necessary since the diagnostic tools such as biomarkers and new technology with highly sensitive methods develop with time. the cea process the cea process is believed to enhance the validity of clinical trial cv outcome measures through independent, systematic, and standardized identification, processing, and review of cv events. however, little is published that critically and consistently reports on the specifics of cea organization and the process methodology, and there is no gold standard. the cea charter is the document that regulates all aspects of the study specifics regarding cea and to which there should be a reference in the study protocol. here, the cea committee is specified, usually composed of clinical specialists in the respective fields, such as cardiology, stroke medicine/neurology, or nephrology. the endpoint office (epo) collects clinically relevant documents, such as hospital notes, relevant ecgs, lab reports, ct reports, and angiography documentation. the epo is usually composed of cea coordinators, monitors, and assistants. the coordinator leads the daily work together with the cea chair or co-chair. collection of predefined source documents has always been challenging in large studies with a high number of endpoints. it used to be paper or fax documents, but with modern technology commercial software is now available that enables the cea to be carried out completely electronically. the epo creates pdf files with the relevant endpoint information and submits the package to the reviewers after careful review for completeness (figure 1). there is no evidence-based scientific evidence on how the flow of events and review process should be handled. however, the most common strategy used in the large global cea centers is that two independent reviewers evaluate each potential event. if there is agreement on all aspects, the event is complete. in case of disagreement, a process of resolving this is initiated. depending on the level of disagreement, it may be a committee meeting with several senior specialists coming to a consensus decision or that the two initial reviewers come to a mutual agreement. collection of the required source documents should be specified for each type of endpoint. this should be handled by a query process at each site in the study. when this is completed a package of all documents is created as a pdf file. this also often includes some pre-selected relevant data from the ecrf (often called patient profile). registry-based studies during the last few years, registry-based randomized controlled studies (rrct) in cardiology have become a new exciting concept in clinical research, using quality improvement registries for prospective randomized trials (10). such studies are characterized by pragmatism, simple design, and non-complex outcome measures, such as total mortality. endpoint detection through the use of public registries (as used in rrcts) can be employed in randomized clinical trials instead of the more traditional approach with active screening and central adjudication (10–12). there are several advantages with endpoint detection through public registries, including a rapid inclusion rate, a more representative all-comer population, as well as substantially lower costs compared to a traditional randomized clinical trial (rct) (13). furthermore, official healthcare registries collect a much larger array of possible outcomes. however, registry follow-up is largely based on icd codes that are dependent on the judgement of the local hospital physician and not on a systematic review of study-specific event definitions. icd codes are often fairly crude and do not catch granularities around an event, such as sub-categories of a death or an mi. thus, they may sometimes be inaccurate, and there is also a risk of underreporting of events in registries, such as type 2 mis or bleedings. a standard rct may thus offer a higher degree of accuracy regarding a limited number of pre-specified clinical endpoints compared to an rrct. during the last year two rrcts were completed in which endpoint adjudication was applied in the ascertainment of endpoints (14,15). the reason was that primary and secondary endpoints, such as bleedings and unplanned revascularization, were slightly more complex and difficult to accurately collect in standard registries. the accuracy of clinical endpoint detection using registries only, as compared to active screening, follow-up, and clinical adjudication (as employed in rcts), is currently unknown and under investigation (15). published data from danish registries comparing hospital medical records with and without central adjudication showed modest agreement on an individual patient basis, but the estimated intervention effects were similar. however, the study addressed the issue of central adjudication or its absence and did not compare active patient follow-up (as in rcts) compared to using registries only (16). future developments there is a great need for research in the field of cea, from evaluation of concordance between investigators and adjudication, endpoint definitions, and the optimal cea process, to whether other ways of endpoint ascertainment can be applied, such as using icd codes from registries. we have to compare the quality of data and concordance with the newer technologies. technical innovations are also needed with better adjudication software, user-friendly both from the perspective of a reviewer and from the administrative side. better and more detailed reports that can be used for upsala journal of medical sciences 43 monitoring of the study progress are another important aspect of improvement. finally, electronic health records and registries now offer the potential ability to accelerate evidence development, and applying artificial intelligence methodology in the adjudication of clinical endpoints and is a scenario that might become reality in the near future. hopefully, that possibly could increase the efficacy and considerably reduce the costs. conclusions central adjudication of endpoints, with the aim of achieving consistent and accurate evaluation of suspected clinical events reported by investigators, is currently the gold standard in clinical trials. more knowledge is needed on the optimal adjudication process and how to achieve the best cost-effectiveness. new methods using national registry data and artificial intelligence may challenge the traditional adjudication strategy and could potentially reduce cost considerably, with similar results. about the contributor claes held is professor of cardiology at clinical research center, uppsala and is serving as a faculty member. he is the chair of the clinical endpoint adjudication committee. disclosure statement the author reports no conflicts of interest. orcid claes held http://orcid.org/0000-0001-9402-7404 references 1. granger cb, vogel v, cummings sr, held p, fiedorek f, lawrence m, et al. do we need to adjudicate major clinical events? clin trials. 2008;5:56–60. 2. sepehrvand n, zheng y, armstrong pw, welsh r, goodman sg, tymchak w, et al. alignment of site versus adjudication committee-based diagnosis with patient outcomes: insights from the providing rapid out of hospital acute cardiovascular treatment 3 trial. clin trials. 2016;13:140–8. 3. mehran r, rao sv, bhatt dl, gibson cm, caixeta a, eikelboom j, et al. standardized bleeding definitions for cardiovascular clinical trials: a consensus report from the bleeding academic research consortium. circulation 2011;123:2736–47. 4. thygesen k, alpert js, jaffe as, simoons ml, chaitman br, white hd, et al. third universal definition of myocardial infarction. eur heart j. 2012;33:2551–67. 5. hicks ka, mahaffey kw, mehran r, nissen se, wiviott sd, dunn b, et al. 2017 cardiovascular and stroke endpoint definitions for clinical trials. j am coll cardiol. 2018;71:1021–34. 6. garcia-garcia hm, mcfadden ep, farb a, mehran r, stone gw, spertus j, et al. standardized end point definitions for coronary intervention trials: the academic research consortium-2 consensus document. eur heart j. 2018;39:2192–207. 7. mahaffey kw, harrington ra, akkerhuis m, kleiman ns, berdan lg, crenshaw bs, et al. disagreements between central clinical events committee and site investigator assessments of myocardial infarction endpoints in an international clinical trial: review of the pursuit study. curr control trials cardiovasc med 2001;2:187–94. 8. mahaffey kw, held c, wojdyla dm, james sk, katus ha, husted s, et al. ticagrelor effects on myocardial infarction and the impact of event adjudication in the plato (platelet inhibition and patient outcomes) trial. j am coll cardiol. 2014;63:1493–9. 9. kosmidou i, mintz gs, jatene t, embacher m, redfors b, wong sc, et al. impact of bleeding assessment and adjudication methodology on event rates and clinical trial outcomes: insights from the horizons-ami trial. eurointervention 2018;14:e580–7. 10. frobert o, lagerqvist b, olivecrona gk, omerovic e, gudnason t, maeng m, et al. thrombus aspiration during st-segment elevation myocardial infarction. n engl j med. 2013;369:1587–97. 11. frobert o, lagerqvist b, gudnason t, thuesen l, svensson r, olivecrona gk, et al. thrombus aspiration in st-elevation myocardial infarction in scandinavia (taste trial). a multicenter, prospective, randomized, controlled clinical registry trial based on the swedish angiography and angioplasty registry (scaar) platform. study design and rationale. am heart j. 2010;160:1042–8. 12. erlinge d, koul s, eriksson p, scherst�en f, omerovic e, linder r, et al. bivalirudin versus heparin in non-st and st-segment elevation myocardial infarction–a registry-based randomized clinical trial in the swedeheart registry (the validate-swedeheart trial). am heart j. 2016;175:36–46. site records new clinical event collection of source documents cec office data check event package final review by cea coordinator event package submitted to two physicians for review incomplete source data adjudication reviewer 1 adjudication reviewer 2 disagreements resolved at phase ii meeting or other solution database completed events figure 1. flow chart of the cea process. 44 c. held 13. james s, frobert o, lagerqvist b. cardiovascular registries: a novel platform for randomised clinical trials. heart. 2012;98: 1329–31. 14. gotberg m, christiansen eh, gudmundsdottir ij, sandhall l, danielewicz m, jakobsen l, et al. instantaneous wave-free ratio versus fractional flow reserve to guide pci. n engl j med. 2017; 376:1813–23. 15. erlinge d, omerovic e, frobert o, linder r, danielewicz m, hamid m, et al. bivalirudin versus heparin monotherapy in myocardial infarction. n engl j med. 2017;377:1132–42. 16. kjoller e, hilden j, winkel p, galatius s, frandsen nj, jensen gb, et al. agreement between public register and adjudication committee outcome in a cardiovascular randomized clinical trial. am heart j. 2014;168:197–204. e1–4. upsala journal of medical sciences 45 abstract introduction selection of endpoints the cea process registry-based studies future developments conclusions about the contributor disclosure statement references new insights into the roles of the foxo3 and p27kip1 genes in signaling pathways article new insights into the roles of the foxo3 and p27kip1 genes in signaling pathways sabah mayahia,b,c, masood golalipourc, ahad yamchid, gagan deep jhingane and majid shahbazic,f adepartment of molecular medicine, school of advanced technologies in medicine, golestan university of medical sciences, gorgan, iran; bdepartment of medical mycology and parasitology, school of medicine, mazandaran university of medical sciences, sari, iran; cmedical cellular and molecular research center, golestan university of medical sciences, gorgan, iran; ddepartment of biotechnology, gorgan university of agricultural sciences and natural resources, gorgan, iran; evproteomics, ground floor green park main, new delhi, india; faryatinagene biopharmaceutical company, gorgan, iran abstract background: the forkhead box o3 (foxo3) and p27kip1 are two important genes in breast cancer progression. in the present study we analyzed the effect of simultaneous foxo3 silencing and p27kip1 activation on breast cancer cell survival and the potential targets of these changes in cancer molecular pathways. materials and methods: the present study involved the cloning of foxo3a shrna and p27kip1 genes under the control of the bidirectional survivin promoter to downand up-regulate foxo3 and p27kip1 genes, respectively. after transfection of the recombinant expression vector into the breast cancer cell line, the inhibition of cell growth was assessed by mts and flow cytometry assays. following the extraction of total mrna and protein, the expression of target genes was evaluated by qpcr and western blotting in both treated and untreated cell lines. then, the downstream protein responses were examined by 2 d electrophoresis. the differentially expressed proteins were also identified by mass spectrometry. results: rates of cell proliferation were significantly inhibited in the transfected cell line 72 h posttransfection. proteomic profiling of the cell line resulted in the identification of seven novel protein markers in breast cancer responsive to these changes in expression of foxo3 and p27kip1. the changes in expression of these markers suggested that certain signaling pathways contribute to the development of breast cancer. conclusion: simultaneous silencing of foxo3 and activation of p27kip1 in mda-mb-231 cells caused alterations in the expression level of several genes involved in apoptosis, cell proliferation, cell cycle control, tissue invasion, drug resistance, and metastasis. it seems that the identified genes might serve as useful biomarkers for breast cancer. article history received 31 december 2018 revised 17 may 2019 accepted 21 may 2019 keywords breast cancer; cell signaling; foxo3a; gene therapy; p27kip1; proteomics introduction breast cancer is an important global health concern and the second leading cause of cancer-related mortality among women (1). omics studies have revealed several pathways and genes related to breast cancer (2), such as p27kip1 and forkhead box o3 (foxo3). the p27kip1 protein interacts with cyclin e and cyclin-dependent kinase 2 (cdk2). in addition, the p27kip1 gene arrests the progression of the cell cycle and leads to apoptosis, tumor suppression, and cell adhesion (3). poor prognosis in patients with colorectal, gastric, pulmonary, and breast cancers is associated with a low expression level of the p27kip1 gene (4). the foxo proteins, a subgroup of forkhead transcription factors, are involved in apoptosis, differentiation, stress response, cell cycle control, and cell metabolism (5). overall, the foxo3 gene is involved in many signaling pathways and makes major contributions to several processes, including cancer development (6). therefore, the identification of foxo3 inhibitors presents a promising strategy for future anticancer drugs (7). proteomic analysis is generally applied in breast cancer serving two ends, namely the identification of novel molecular markers for breast tumor profiling and determination of the intracellular signaling pathways contributing to the growth of breast cancer cells (8). to our knowledge, there is no information on the effect of simultaneously decreasing foxo3 gene and increasing p27kip1 gene expression, respectively, to inhibit breast cancer. with this background in mind, the present study aimed to analyze the effect of the foxo3 and p27kip1 genes on the control of the breast cancer mda-mb-231 cell line, using a bidirectional survivin promoter. moreover, the changes in the protein profile contact majid shahbazi shahbazimajid@yahoo.co.uk medical cellular and molecular research center, golestan university of medical sciences, 4916668197, iran supplemental data for this article can be accessed here � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 3, 149–157 https://doi.org/10.1080/03009734.2019.1623351 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1623351&domain=pdf&date_stamp=2019-09-09 https://doi.org/10.1080/03009734.2019.1623351 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2019.1623351 http://www.tandfonline.com of the transfected cell line were investigated in order to find cancer molecular markers. materials and methods cell line, plasmid constructs, and transfection the mda-mb-231 cell line adopted in this study was supplied by the pasteur institute (tehran, iran). cells were cultured in a high-glucose dulbecco’s modified eagle’s medium (thermofisher scientific, waltham, ma, usa), supplemented with penicillin-streptomycin (100 units/ml) and fetal bovine serum (10% fbs; gibco bi102-100) in a damp atmosphere (5% co2) at 37 �c. the cells were transfected with a plasmid construct designed in a bidirectional promoter (dual targeting of foxo3a shrna and simultaneous induction of p27kip1 gene). in addition, an empty plasmid of pcdna3.1þ expression vector with egfp was used as the control (figure 1). the vectors were purchased from biomatic, toronto, ontario, canada. to verify the identity of the inserted sequences, all plasmids were examined through direct sequence analysis. twenty-four hours before transfecting dnas into the cells, some of the cells were applied in the growth medium without antibiotics. transfection was performed as recommended by the manufacturer using lipofectamine 2000 (invitrogen, waltham, ma, usa). cell viability assay the viability of the cells (2–5 � 103/well) was tested in a 96well plate in a final volume of 200 ll/well, incubated in a co2 incubator (5% co2) at 37 �c overnight. then, 20 ll/well of mts reagent (promega, fitchburg, wi, usa) were added to each well and incubated for 4 h at 37 �c. the plate was shaken briefly, and then the absorbance of the treated and untreated cells was measured at 490 nm 24 h, 48 h, and 72 h after transfection, using a plate reader (biotek, winooski, vt, usa). quantitative real-time polymerase chain reaction assay the extraction of total rna was accomplished using trizol solution (invitrogen). total rna (2 mg) was used for complementary dna synthesis as recommended by the manufacturer (thermo scientific, vilnius, lithuania). quantitative realtime reverse transcriptase polymerase chain reaction (qpcr) assay was performed using abi 7300 rt-pcr systems (applied biosystems, foster city, ca, usa) to determine the relative expression of the p27kip1 and foxo3a genes (table 1). the threshold cycle (ct) value was estimated, and the rest 2009 software was used to assess the amplification plots. in addition, the comparative ct (2�ddct) method was applied to analyze the data (8). apoptosis assay to identify apoptotic cells, the fitc annexin v detection kit with propidium iodide (pi) (biolegend, san diego, ca, usa) was exclusively developed. after seeding the cells (2 � 105 cells/ml) in tissue culture plates, they were incubated overnight, and then treated after 24 h, 48 h, and 72 h. in the next stage, the cells were washed with a cell-staining buffer, and then re-suspended in the annexin v binding buffer at a concentration of 0.25–1.0 � 107 cells/ml. subsequently, the pi solution (10 ll) and fitc annexin v (5 ll) were added. the cells were vortexed and incubated at 25 �c for 15 min in darkness. then, the annexin v binding buffer (400 ll) was figure 1. (a) map of pcdna3.1(þ) egfp; (b) psur (shfoxo3-p27kip1) vector with a survivin promoter. 150 s. mayahi et al. added to the tubes. finally, a bd accuritm c6 cytometer (seattle, wa, usa) was used to perform flow cytometry. protein extraction and immunoblotting a protein lysis buffer (100 mm tris-hcl, 150 mm nacl, 5% sds, 1 mm dithiothreitol, 5 mm edta, ph of 7.4, 5% sodium deoxycholate, and 10% glycerol) was used for lysing the harvested cells collected via trypsinization. a protease inhibitor cocktail (sigma-aldrich, st. louis, mo,usa) mixture was added to 1 mm of pmsf. the total protein in the resultant supernatant was used for western blotting analysis. after separating the total protein using polyacrylamide gel electrophoresis, it was transferred to a nitrocellulose membrane, which was blocked with bovine serum albumin (5% w/v bovine serum albumin; sigma-aldrich) in tbst. furthermore, the primary antibody was used against foxo3a and p27kip1 antibodies (santa cruz, santa cruz, ca, usa). visualization of the bands was accomplished by means of the enhanced chemiluminescence detection kit (parstous, tehran, tehran, iran) following the manufacturer’s protocol. in addition, the national institutes of health imagej program (bethesda, md, usa) was used to determine the band density. two-dimensional gel electrophoresis cell pellets were resuspended by adding an adequate amount of 2 d lysis buffer, containing 2 m thiourea, 1% dithiothreitol, 4% chaps, 7 m urea, 0.001% bromophenol blue, and 0.5% immobilized ph gradient (ipg) buffer (ph of 3–10; ge healthcare, usa), followed by extracting the total protein. in addition, a 2 d quant kit (ge healthcare) was used to measure the protein level with bovine serum albumin as the standard. to this end, after rehydrating the ipg strips (7 cm 3–10 nl, immobilone dry strip; ge healthcare) at a ph gradient of 3–10, 300 mg of each protein sample was loaded on the ipg strip. the 2 d electrophoresis was carried out based on our previously published method (9). for staining the gels, a colloidal coomassie brilliant blue dye was used as described by candiano et al. (10). a scanner (ge healthcare life sciences, chicago, il, usa) was also employed to scan the stained gels. the analysis of the protein spots in the gel images was performed by the imagemaster 2 d platinum 6.0 (ge healthcare life sciences). finally, the intensities of the treated and untreated gels were compared, and their ratios were determined. protein identification via liquid chromatography-mass spectrometry after cutting the protein spots into 1-mm3 pieces, they were removed from the stained gel manually and added to tubes. the pieces were destained with an equal solution of ammonium bicarbonate and acetonitrile. then, they were digested with trypsin (promega, usa) at 37 �c for a minimum of 16 h. trifluoroacetic acid (tfa) 10% was also added to prevent digestion. extraction of the gel pieces was accomplished using an equal solution of 0.1% tfa and acetonitrile (50 ll). supernatants from the pooled samples were dried, using speed-vac centrifugation (thermo scientific, waltham, ma, usa). then, 2% acetonitrile (10 ll) and 0.1% tfa were used for dissolving the samples. the q-trap nlc-ms/ms system (applied biosystems) was used for all analyses, as described (11). mass data were collected and analyzed using the analyst software (applied biosystems). for finding the swissprot-trembl subdatabase, the ms/ms lists were searched, and mascot 3 (version 2.2., matrix science, boston, ma, usa) was used to blast the data lists against databases. data analysis data were analyzed in prism, version 6.0 (graphpad software inc, san diego, ca, usa) using two-way anova and student’s t test. the data were also presented as mean± standard error of mean of at least three independent analyses. a p values less than 0.05 was considered statistically significant. results combinatorial effects of induced p27kip1 and shfoxo3a on cell viability the p27kip1 mrna expression level was significantly higher, and foxo3a mrna expression significantly lower, in treated cells 72 h post-transfection in comparison with that found in controls (p < 0.05). analysis of the qpcr results showed a 0.41-fold change in foxo3a gene expression (down-regulation) and more than a three-fold change in p27kip1 gene expression (up-regulation) 72 h after transfection (figure 2). foxo3 expression decreased (60%), while p27kip1 expression increased 72 h after transfection, compared to the control expression (figure 3). the expression of construct in mda-mb-231 significantly reduced the growth of the cell line (p < 0.05) (figure 4). the effect of the construct on the rate of cell proliferation was not statistically significant 24 h post-transfection. however, it was significantly inhibited at 72 h after treatment (p < 0.05; figure 5). flow cytometry apoptotic cells included both early and late apoptosis (annexin vþ/piand annexin vþ/piþ, respectively), while annexin v and pi were negative for viable cells. in the controls, the majority of the cells (99.9%) were viable and nonapoptotic at baseline. however, early apoptotic cells showed a significant increase after treatment, compared to the untreated cells after 72 h (from 5.0% to 36.4%; annexin vþ/ pi–; figures 6(a, b)). effect of overexpression p27kip1 and foxo3a shrna on downstream proteins to identify the downstream responsive proteins associated with the changes in p27kip1 and shfoxo3a expression at upsala journal of medical sciences 151 72 h after transfection, the treated and untreated samples were compared by 2 d gel electrophoresis. there was differential expression of 10 protein spots in the breast cancer cell line (figure 7). out of the 10 proteins, 3 and 7 proteins showed up-regulated and down-regulated expression, respectively (figure 7). table 2 presents the mass spectrometry (ms) information of the appeared spots. succinate dehydrogenase (sdh; t1/c1), albumin (alb; t3/c3), and serine/threonine-protein kinase nlk (nlk; t4/c4) were significantly up-regulated, while tubulin alpha (tuba1c; t2/c2), figure 2. relative expression of foxo3 and p27kip1 genes in the mda-mb-231 cell line after transfection was significantly down-regulated and up-regulated, respectively, 72 h after transfection. expression of the genes was analyzed by the rest program (p < 0.05). figure 3. relative band densitometry of western blotting data, showing: (a) the incidence of the highest down-regulation of foxo3a gene 72 h after transfection; (b) reduction of foxo3a gene expression in the cells treated with the construct after 72 h, compared to the control; (c) the highest up-regulation of p27kip1 gene in mda-mb-231 cells occurring 72 h post-transfection; and (d) an increase in p27kip1 expression in the cells treated with the construct 72 h after transfection, compared to the control. implementation of quantification in three repeats (gapdh was used as the control). figure 4. microscopic morphology of mda-mb-231 cells under a fluorescent microscope after transfection for 24 h, 48 h, and 72 h with: (a) an empty vector; (b) a vector containing the genes after 24 h, (c) 48 h, (d) 72 h; and (e) negative signal of control cells without transfection. 152 s. mayahi et al. tubulin beta chain (tubb; t10/c10), b-actin (actb) with two isoforms (t8/c8 and t9/c9), and vimentin (vim) with three isoforms (t5/c5, t6/c6, and t7/c7) were significantly downregulated (figure 7). the theoretical and experimental masses and the isoelectric points of these differentially expressed proteins were also nearly the same, verifying the identified ms results (table 2). gene ontology (go) analysis of 10 differentially expressed proteins consisted of protein– protein interactions using the genemania (https://genemania.org) and kegg (https://www.genome.jp/kegg/kegg2.html) databases (table 3). based on the result of two-way anova, after the transfection of genes in mda-mb-231 cells for 72 h, foxo3 gene expression showed the greatest reduction (p < 0.05), while p27kip1 gene showed the highest increase, compared to the control (figure 7). the 2 d gel figure 5. inhibition of mda-mb-231 cell proliferation after 24 h, 48 h, and 72 h of treatment in wells (in triplicate) using mts assay. data are expressed as the mean cell viability percentage versus that of the control. figure 6. apoptosis detection via flow cytometry. a: use of annexin v-fitc staining in the treated and untreated mda-mb-231 cells after 24 h, 48 h, and 72 h. the upper and lower right quadrants (annexin vþ/pi þ and annexin vþ/pi�, respectively) indicate late and early apoptosis, respectively (10.4% of cells died by lipofectamine, as shown in supplementary figure 1, available online). b: apoptosis in the treated and control mda-mb-231 cells 24 h, 48 h, and 72 h post-transfection. data were presented as mean ± sd (p < 0.05). upsala journal of medical sciences 153 https://genemania.org https://genemania.org https://www.genome.jp/kegg/kegg2.html https://doi.org/10.1080/03009734.2019.1623351 electrophoresis of the plasmid construct and empty vectortransfected mda-mb-231 cells indicated changes in the expression level. out of the collected 25 spots, 10 spots (indicated by arrows, figure 7) that had the highest scores were selected and confirmed to be involved in breast cancer. discussion there are studies regarding the independent effect of p27kip1 and foxo3a genes on the inhibition of breast cancer; however, they have reported a poor prognosis (12,13). the present study involved the design of a bidirectional expression vector suppressing foxo3a and inducing p27kip1 expression. our results showed that the synergistic effects of p27kip1 and shfoxo3a induced 36.4% apoptosis in cells. however, the overexpression of p27kip1 alone has been reported to induce 12.73% apoptosis (12). recently, proteomic methods have been used as a strategy for the detection of protein biomarkers (14). expression of seven proteins changed after foxo3a suppression and p27kip1 induction (figure 8). these proteins are all involved in apoptosis, metastasis, drug resistance, proliferation, and cell cycle control. three up-regulated proteins included sdha, albumin (with cell source), and nlk, while four down-regulated proteins included tuba1c, tubb, actb with two isoforms, and vim with three isoforms. the sdh is identified as a mitochondrial enzyme, involved in the krebs cycle and electron transfer chain, and contributes to tumor suppression. this enzyme has four subunits, namely sdha, sdhb, sdhc, and sdhd (15). research over the past decades has shown an inverse relationship between albumin level and severity/risk of the progression of different cancers, including pulmonary, gastric, ovarian, and breast cancers. there are various mechanisms for the anticancer effects of albumin, such as dna stabilization and duplication, calcium preservation and regulation, and antioxidant properties. in a study performed by xiao-jing du et al., the growth of human cancer cells, such as breast cancer cells, was inhibited by high concentrations of albumin (16). the main cause of the reduction of the albumin level, especially in cancer patients, is the inhibition of albumin-regulating gene by tumor necrosis factor which results in mrna expression reduction of approximately 90% in the liver (17). nlk, which belongs to the mitogen-activated protein kinase family, is a vital regulator of various cancer types. in a study conducted by lv et al., nlk inhibited non-small-cell lung cancer through the wnt signaling pathway (18). in addition, huang et al. showed that nlk inhibits cellular proliferation in breast cancer and induces apoptosis. similar studies confirm that nlk is a tumor suppressor gene in ovarian cancer (19). the theoretical molecular weight of nlk is about 58 kda, while its experimental molecular weight is about 35 kda, which can be attributed to the degradation of this protein. actin and its proteins have a major functional and structural role in cell motility, endocytosis, cell adhesion and division, and maintenance of cell morphology. the expression of actb increases in a metastatic state (20). accordingly, chunmei guo et al. reported that actb figure 7. two-dimensional gel electrophoresis of mda-mb-231 cell line in treated and untreated cells, showing the increased expression of tubulin beta chain, vimentin (three isoforms), and b-actin (two isoforms) and decreased expression of succinate dehydrogenase complex, albumin, and nlk. protein spots 6 and 7 were absent in the treated cells. the experiment was performed in two biological repeats. a ¼ absence. table 1. primers used in this study. primer sequence (5’!3’) foxo3a (r) gactatgcagtgacaggttgtg foxo3a (f) tctacgagtggatggtgcgtt p27 (r) tccattccatgaagtcagcg p27 (f) gagcagacgcccaagaagc gapdh (r) aggcagggatgatgtggagag gapdh (f) atcgtggaactcagtaccaca 154 s. mayahi et al. ta b le 2. pr ot ei n sp ot s co lle ct ed fr om tw od im en si on al g el s in m d a -m b23 1 ce ll lin e tr an sf ec te d w it h th e p la sm id co n st ru ct in co m p ar is on w it h th e co n tr ol (e m p ty ve ct or ) af te r 72 h . sp ot id a cc es si on n am e of p ro te in g en e th eo re ti ca l m w (k d a) /p i ex p er im en ta l m w (k d a) /p i ra ti o pe p ti d e sc or e 1 p3 10 40 su cc in at e d eh yd ro g en as e sd h a 72 .6 /7 .3 9 72 .2 /7 .8 2. 1 (u p -r eg ul at ed ) v g sv lq eg g k 2. 65 2 q 9b q e3 a -t ub ul in tu ba 1c 51 /6 .0 49 .9 /7 .1 0. 2 (d ow n re g ul at ed ) a v fv d le pt v id ev r; ei id lv ld r; ed a a n n ya r; d v n a a ia ti k; ti g g g d d sf n tf fs et g a g k; ls v d yg kk ; ls v d yg k 12 .4 9 3 p0 27 68 a lb um in a lb 69 .3 /6 .2 8 65 .1 /6 .5 1. 2 (u p -r eg ul at ed ) kv pq v st pt lv ev sr ; a ef a ev sk ; lv td lt k 2. 49 4 q 9u be 8 se ri n e/ th re on in ep ro te in ki n as e n lk n lk 8. 13 /5 8. 28 34 ..6 /8 .7 4. 5 (u p -r eg ul at ed ) tq ev v tq yy ra pe il m g sr h ys n a id iw sv g if a el lg r; pn v fq n lv sc k; ic d fg la rv ee ld es r 2. 6 5 p0 86 70 v im en ti n v im 53 .6 5/ 5. 12 49 .8 /5 .5 0. 3 (d ow n -r eg ul at ed ) q d v d n a sl a r; q v q sl te v d a lk ; v el q el n d r; q q ye sv a a k; ee a en tl q sf r; et n ld sl pl v d th sk ; ey q d ll n v k; lq ee m lq r; 19 .8 2 6 p0 86 70 v im en ti n v im 53 .6 5/ 5. 12 52 .5 /5 .7 a/ p (d ow n -r eg ul at ed ) ee en fa v ea a n yq d ti g r; a ld ie ia ty r; ek lq ee lq r; q v q sl te v d a lk ; q d v d n a sl a r; v es lq ee ia fl k; v el q el n d r; ee a en tl q sf r; et n ld sl pl v d th sk ; q q ye sv a a k; lq ee m lq r; ll eg ee sr ; d n la ed ir ; q v d q lt n d k 35 .7 1 7 p0 86 70 v im en ti n v im 53 .6 5/ 5. 12 50 .4 /5 .6 a/ p (d ow n -r eg ul at ed ) v el q el n d r; fa d ls ea a n r; q ye sv a a kl g d ly ee em r; ea en tl q sf r; a ld ie ia ty r; lq ee m lq r; q es te yr ; et n ld sl pl v d th sk ; q v q sl te v d a lk ; lg d ly ee er ; d n la ed ir ; ll eg ee sr ; lq d ei q n ke ea r; fa n yi d k 27 .5 8 8 p6 07 09 b -a ct in a c tb 41 .7 /5 .4 8 40 .5 /6 .5 0. 2 (d ow n -r eg ul at ed ) q ey d es g ps iv h r; g ys ft tt a er ; d lt d yl m k; a pe eh pv ll te a pl n pk ; d sy v g d ea q sk r; d sy v g d ea q sk ; ei ta la ps tk ; d lt d yl k; d ly a n tv ls g g tt yp g ia d r 17 .0 2 9 p6 07 09 b -a ct in a c tb 41 .7 /5 .4 8 40 .8 9/ 6. 7 0. 1 (d ow n -r eg ul at ed ) q ey d es g ps iv h r; g ys ft tt a er ; d lt d yl m k; a pe eh pv ll te a pl n pk ; d sy v g d ea q sk r; d sy v g d ea q sk ; ei ta la ps tk ; d lt d yl k; d ly a n tv ls g g tt yp g ia d r 10 .7 2 10 p0 74 37 tu b ul in b et a ch ai n tu bb 51 .2 /5 49 .6 /5 .8 0. 3 (d ow n -r eg ul at ed ) in tf sv v ps pk ; ta v d ip pr ; is v yy n ea tg g k; n a a d pr ; la v n v pf pr 46 .3 8 a/ p : ab se n ce /p re se n ce ; m w : m ol ec ul ar w ei g h t. ta b le 3. pr ot ei n – p ro te in in te ra ct io n s of al l p ro te in s fo r co lle ct in g th e d at a. g en e v im a c tb tu ba 1c tu bb sd h a n lk a lb ex p an d ph ys ic al in te ra ct io n se rp in h , m a g o h , ei fa 3, sp rm 4, kr t1 8, u pp 1, it g b4 , ba sp 1, pp l, u rg d p, x pn pe p3 c fl 1, c fl 2, pf n 1, c c t8 , tc p1 , c c t3 , c c t4 , ru v bl 1, m yh 11 tu ba 4b , tu bb 6, tu bb , tu ba 4a , tu bb 4a , tu bb 4b , tu bb 3, tu bb 2a , tu bb 6, tu bb 2b , g a pd h , th bs 1, c li p1 , tc p1 1l 1, tu bb 2a , yb x 1, tu ba 1a , tu ba 1b , tu ba 1c , tu ba 1a , tu ba 4b , pn kd , tn po 2, d n a ja 4 sl c 2a 14 , c pn e1 , ra b1 4, lr rc 8a , h ig d 1a , sd h a f2 , sd h a f2 , sd h c , sd h b, u bc m yb , ra n g a p1 , zh x 3, m a p3 k7 , ta b1 , h ip k2 , le f1 , tp 53 , tc f7 l2 f2 , h p, it ih 1, a m bp , a h sg , a po a 2, rb p4 , fc g rt c oex p re ss io n se rp in h , tt n c fl 1, pf n 1 tu bb 6, tu bb , tu ba 4a , tu bb 4b , tu bb 3, tu bb 2a , tu bb 6, tu bb 2b , g a pd h , tu ba 1a , tu ba 1b , tb c b, pf d n 1, st m n 1 tu ba b, tu bb 2a , tu bg 2, yb x 1, tu ba 1b , tu ba 1c , tu ba 4a , tu bb 3, tu bb 4b , tu bg 1 ra b1 4, h ig d 1a , sd h c , sd h b, n d u fv 1, u bc , a la d , u q c rc 1 — f2 , h p, it h 1, g c , a m bp , a fm , a h sg , a fp , a po 2, rb p4 , sl c o 1b 1, sl c o 1b 3 c olo ca liz at io n se rp in h i — st m n 1, tb c b, tu ba 1b , tu ba 1a , tu bb 2a — n d u fv 1, u q c rc 1, n d u fs 1 ra n g a p1 , m a p3 k7 , ta b1 f2 , h p, it h 1, g c , a m bp , a h sg , a po 2, rb p4 , sl c o 1b 1, sl c o 1b 3 g en e in te ra ct io n — — — — — tp 53 — ro le pr ol ife ra ti on , ti ss ue in va si on , an d m et as ta si s c el l m ob ili ty , en d oc yt os is , ce ll ad h es io n , d iv is io n , d ru g re si st an ce , an d m et as ta si s c el lu la r m ob ili ty c el lu la r m ob ili ty tu m or su p p re ss or in h ib it ce llu la r p ro lif er at io n , in d uc e ap op to si s st ab ili zi n g d n a , re g ul at io n ca lc iu m , an ti ox id an t p ro p er ti es upsala journal of medical sciences 155 increased in many cancers of the liver, kidneys, colon, stomach, pancreas, esophagus, lung, breast, prostate, and ovaries (21). these tumors are associated with increased drug resistance and metastasis, especially in mda-mb-231 cells (21). tubulins consist of microtubules, with two alpha and beta subunits (approximate weight of 50 kda). they play a large role in cellular activities, such as cellular mobility. dysfunction of the microtubule network causes disturbance in apoptosis and differentiation (20). in addition, vim is one of the most filamentous proteins, expressed in various types of cancers, such as prostate, gastrointestinal, central nervous system, pulmonary, and breast cancers. in a study carried out by satelli, it was shown that the increased invasive/migratory potential of cancer cells is related to vim overexpression (22). the vim is generally considered as an indicator of poor prognosis (22). in conclusion, the simultaneous silencing of foxo3 and activation of p27kip1 in mda-mb-231 cells caused alterations in the expression level of several genes which are involved in cell apoptosis, cell proliferation, cell cycle control, tissue invasion, drug resistance, and metastasis. therefore, it seems that these genes may serve as useful biomarkers for breast cancer. acknowledgements we thank the golestan university of medical sciences and arya tina gene (atg) biopharmaceutical company, gorgan, iran. disclosure statement no potential conflict of interest was reported by the authors. funding this work was supported by the golestan university of medical sciences and health services (grant number: 940714170 ) and aryatinagene biopharmaceutical company. notes on contributors sabah mayahi is a ph.d. student of molecular medicine at golestan university of medical sciences, gorgan, iran. she works in the department of parasitology and mycology, faculty of medicine, mazandaran university of medical sciences. dr. masood golalipour is an associate professor of molecular genetics at the department of genetic and member of the cellular and molecular research center of golestan university of medical sciences. dr. ahad yamch is an assistant professor of genetic engineering at the department of biotechnology, gorgan university of agricultural sciences and natural resources. dr. gagan deep jhingan completed his masters and ph.d. in biotechnology from jawaharlal nehru university. he has worked as wellcome/dbt early career fellow at national institute of immunology, new delhi. he has specialization in the field of proteomics and is responsible for the development and management of vproteomics platform technology. majid shahbazi is a professor of genetics working on target therapy and director of cellular and molecular research center; head of the department of molecular medicine, school of advanced technologies in medicine of golestan university of medical sciences; also ceo of aryatinagene biopharmaceutical company. references 1. abdulkareem ih. aetio-pathogenesis of breast cancer. niger med j. 2013;54:371–5. 2. sadeghi h, golalipour m, yamchi a, farazmandfar t, shahbazi m. cdc25a pathway toward tumorigenesis: molecular targets of cdc25a in cell-cycle regulation. j cell biochem. 2019;120:2919–28. 3. sgambato a, cittadini a, faraglia b, weinstein ib. multiple functions of p27(kip1) and its alterations in tumor cells: a review. j cell physiol. 2000;183:18–27. 4. kudoh s, kumaravel ts, kuramavel b, eguchi m, asaoku h, dohy h, et al. protein expression of cell cycle regulator, p27kip1, correlates with histopathological grade of non-hodgkin’s lymphoma. jpn j cancer res. 1999;90:1262–9. 5. obsil t, obsilova v. structure/function relationships underlying regulation of foxo transcription factors. oncogene 2008;27:2263–75. 6. fu z, tindall dj. foxos, cancer and regulation of apoptosis. oncogene 2008;27:2312–9. 7. taylor s, lam m, pararasa c, brown je, carmichael ar, griffiths hr. evaluating the evidence for targeting foxo3a in breast cancer: a systematic review. cancer cell int. 2015;15:1–9. figure 8. effect of simultaneous foxo3a suppression and p27kip1 induction on protein expression in treated cells. 156 s. mayahi et al. 8. golalipour m, mahjoubi f, sanati mh, alimoghaddam k, kamran a. gene dosage is not responsible for the upregulation of mrp1 gene expression in adult leukemia patients. arch med res. 2007; 38:297–304. 9. yamchi a, ben c, rossignol m, zareie sr, mirlohi a, sayedtabatabaei be, et al. proteomics analysis of medicago truncatula response to infection by the phytopathogenic bacterium ralstonia solanacearum points to jasmonate and salicylate defence pathways. cell microbiol. 2018;20:e12796. 10. candiano g, bruschi m, musante l, santucci l, ghiggeri gm, carnemolla b, et al. blue silver: a very sensitive colloidal coomassie g-250 staining for proteome analysis. electrophoresis. 2004;25:1327–33. 11. boudart g, jamet e, rossignol m, lafitte c, borderies g, jauneau a, et al. cell wall proteins in apoplastic fluids of arabidopsis thaliana rosettes: identification by mass spectrometry and bioinformatics. proteomics. 2005;5:212–21. 12. jiang d, wang x, liu x, li f. gene delivery of cyclin-dependent kinase inhibitors p21waf1 and p27kip1 suppresses proliferation of mcf-7 breast cancer cells in vitro. breast cancer. 2014;21:614–23. 13. storz p, doppler h, copland ja, simpson kj, toker a. foxo3a promotes tumor cell invasion through the induction of matrix metalloproteinases. mol cell biol. 2009;29:4906–17. 14. gamez-pozo a, trilla-fuertes l, berges-soria j, selevsek n, lopezvacas r, diaz-almiron m, et al. functional proteomics outlines the complexity of breast cancer molecular subtypes. sci rep 2017;7: 1–13. 15. bardella c, pollard pj, tomlinson i. sdh mutations in cancer. biochim biophys acta. 2011;1807:1432–43. 16. du xj, tang ll, mao yp, sun y, zeng ms, kang tb, et al. the pretreatment albumin to globulin ratio has predictive value for longterm mortality in nasopharyngeal carcinoma. plos one 2014;9: 1–8. 17. seaton ka. concentration controls cancer. j natl med assoc 2001; 93:490–3. 18. lv l, wan c, chen b, li m, liu y, ni t, et al. nemo-like kinase (nlk) inhibits the progression of nsclc via negatively modulating wnt signaling pathway. j cell biochem. 2014;115:81–92. 19. huang y, jiang y, lu w, zhang y. nemo-like kinase associated with proliferation and apoptosis by c-myb degradation in breast cancer. plos one 2013;8:1–10. 20. lee s, terry d, hurst dr, welch dr, sang qx. protein signatures in human mda-mb-231 breast cancer cells indicating a more invasive phenotype following knockdown of human endometase/matrilysin-2 by sirna. j cancer. 2011;2:165–76. 21. guo c, liu s, wang j, sun mz, greenaway ft. actb in cancer. clin chim acta. 2013;417:39–44. 22. satelli a, li s. vimentin in cancer and its potential as a molecular target for cancer therapy. cell mol life sci. 2011;68:3033–46. upsala journal of medical sciences 157 abstract introduction materials and methods cell line, plasmid constructs, and transfection cell viability assay quantitative real-time polymerase chain reaction assay apoptosis assay protein extraction and immunoblotting two-dimensional gel electrophoresis protein identification via liquid chromatography-mass spectrometry data analysis results combinatorial effects of induced p27kip1 and shfoxo3a on cell viability flow cytometry effect of overexpression p27kip1 and foxo3a shrna on downstream proteins discussion acknowledgements disclosure statement funding notes on contributors references patients’ experience of outsourcing and care related to magnetic resonance examinations upsala journal of medical sciences. 2014; 119: 343–349 original article patients’ experience of outsourcing and care related to magnetic resonance examinations parvin tavakol olofsson1, peter aspelin1, lott bergstrand2 & lennart blomqvist3 1department of clinical science, intervention and technology at karolinska institutet, division of medical imaging and technology, stockholm, sweden, 2department of radiology, ersta hospital, stockholm, sweden, and 3department of radiology, karolinska university hospital in solna, stockholm, sweden abstract background. outsourcing radiological examinations from public university hospitals affects the patient, who has to attend a different clinic or hospital for the radiological examination. we currently have a limited understanding of how patients view outsourcing and their care related to mr examinations. aim. to examine the experiences of patients who are sent to private radiology units when their referrals for mr examinations are outsourced from a university hospital, as well as to explore factors which influence patient satisfaction regarding the quality of care related to the mr examination. methods. a group of patients (n = 160) referred for mr examinations and either examined at a university hospital or at an external private unit were interviewed. the interview was designed as a verbal questionnaire. data were analyzed using student’s t test, analysis of variance (anova), and pearson’s correlation. results. sixty-nine percent of the patients could neither choose nor influence the location at which they were examined. for those who could, aspects that influenced the patient’s choice of radiology department were: short waiting time 79% (127/160), ease of traveling to the radiology department 68% (110/160), and short distance to their home or work 58% (93/160). for 40% (60/160) of the patients, a short time in the waiting room was related to a positive experience of the mr examination. conclusion. if patients were informed about outsourcing and could also choose where to have their examination, key factors contributing to patient satisfaction could be met even when mr examinations are outsourced. key words: care quality, decision-making, health policy, outsourcing radiology introduction about 60 million magnetic resonance (mr) examinations are performed annually worldwide (1), and numbers have increased over recent years. the ability of magnetic resonance imaging (mri) to provide high soft-tissue contrast resolution images without ionizing radiation is important for many diagnoses, and this contributes to the modality being highly coveted (2). according to a report on the census of the radiology workforce in the uk in 2008, 18% of radiology clinics outsourced a number of radiological imaging procedures, the median of the requested mr examinations being 49% (3). in sweden, increased demand for mr examinations has resulted in longer waiting times for these examinations. in this setting, many public hospitals in sweden outsource a proportion of their referrals for radiological examinations, including mr examinations, to external hospitals or units, usually private radiology departments. outsourcing radiological examinations from a university hospital to external private units affects the patient, who has to attend a different clinic or hospital for the radiological examination. we currently have a limited correspondence: parvin tavakol olofsson, department of clinical science, intervention and technology at karolinska institutet, division of medical imaging and technology, stockholm, sweden. e-mail: parvin.tavakol@ki.se (received 22 april 2014; accepted 29 july 2014) issn 0300-9734 print/issn 2000-1967 online � 2014 informa healthcare doi: 10.3109/03009734.2014.951133 http://informahealthcare.com/journal/ups mailto:parvin.tavakol@ki.se understanding of how patients feel when their mr examination is outsourced and how they view the quality of their care related to these examinations. the aim of this study was to examine the patients’ reactions about being sent to private radiology units when their referrals for mr examinations are outsourced from a university hospital, as well as to explore factors which influence patient satisfaction regarding the quality of care related to the mr examination. material and methods this study was conducted at the two private radiological units that received most outsourced referrals and at karolinska university hospital in stockholm, sweden. a total of 160 patients who were referred for mr examinations, either to the karolinska university hospital or to external private units, were interviewed over a two-month period. the interviews took place in the radiology departments just after the patients had completed their mr examination. for practical reasons, the interviews were scheduled on three days per week over a two-week period in each radiology department: monday, tuesday, and wednesday during the first week, and wednesday, thursday, and friday during the second week. sixty patients at each private unit and 40 patients at the university hospital were interviewed. the average duration of each interview was 20 min. participation was voluntary, and all informants were free to make an independent decision about taking part in the study. each patient gave consent verbally before the interview. all participants were verbally informed about the overall purpose of the research and its main features. all participants were ensured confidentiality about their identity and were also informed that the data from the interviews would only be used in this study for the purpose of providing knowledge through understanding their experience. data were collected by one of the researchers (p.t.o.). the project in its entirety was approved as quality assurance by the regional ethical review board at the karolinska institute. the interview had a structured design which consisted of alternative questions and a few open-ended questions (4). the interview questions also included background questions, which sought to obtain patient socio-demographic data, such as age, education level, occupation, and physical and psychological wellbeing. the remaining questions were divided into a number of domains concerning patients’ expectations of care such as: the caring attitude of the staff, waiting time, availability, and the patient’s freedom of choice regarding selection of radiology clinic. these questions were based on two criteria: comprehensiveness and importance, e.g. each question regarding health care experiences and related to the mr examination was followed by a question about the importance of the question (5). the interview consisted of 34 questions of which 10 questions were follow-up questions. in these follow-up questions patients were asked to indicate on a five-point scale (a = 5, of greatest importance; b = 4, of great importance; c = 3, of little importance; d = 2, of no importance; e = 1, cannot take a position on the issue) how important they considered the subject. degree of perceived satisfaction was measured on a five-point scale: e = 1, very bad; d = 2, bad; c = 3, neither good nor bad; b = 4, good; and a = 5, very good. degree of information quality was also measured on a similar five-point scale. degree of access, including waiting times for radiological examinations, was measured on a fivepoint scale: e = 1, do not know; d = 2, three months or more; c = 3, one to two months; b = 4, one to four weeks; and a = 5, less than a week. degree of satisfaction regarding the staff at the radiology department where the patients had their mr examinations was assessed on a three-point scale: 1 = do not know; 2 = no; and 3 = yes. in total, 20 of the items about patient experiences had a five-point response scale, three had a four-point scale, and seven questions hade a three-point scale (see appendix). analysis statistical analyses were carried out using spss software, version 20. the patients’ satisfaction with their care was calculated by the mean satisfaction scores in each dimension, compared with patient characteristics using student’s t test and anova to compare differences between independent and dependent variables, as appropriate. correlations were analyzed by pearson’s test, where satisfaction was defined for p < 0.05. analysis of the text from open-ended questions proceeded as follows. in order to pick up relevant information, only those sentences in the responses that were clear and created context were transcribed. the data were organized in a commonly used spreadsheet format with microsoft office excel 2010 11.6560.6568 sp3 software by microsoft� and microsoft word 2010 (6,7). two questions guided analysis of these responses. first, what specific aspects of care or outsourcing mr examinations are the interview texts discussing? second, what are the issues regarding care or outsourcing mr examinations that are of concerntothepatients interviewed?inorder toidentify common themes each response was read carefully. the second step was to develop coding categories for each response. data were organized through coding 344 p. t. olofsson et al. http://informahealthcare.com/doi/suppl/10.3109/03009734.2014.951133/suppl_file/10.3109/03009734.2014.951133_suppl.doc categories so that text providing similar context on a given theme could be separated from the other data. the third step was labeling each response with one or several coding categories. the final step was to find out what categories were related to each other and to identify the common theme. the most common types of coding categories that emerged in this study were: codes of situation (which define a setting: for example, patients’ views on the staff’s work) and codes of activity (related to commonly occurring varieties of behavior: for example, patients’ visits to the radiology department). a total of four themes emerged: quality ofpatient care,office waiting time, choice ofradiology department, and improvement of the patient’s satisfaction. results of the 160 patients who participated in this study, 67 were men and 93 were women, between 18 and 81 years old (median age for men was 43, and for women 61). table i also shows the other characteristics of the socio-demographic background of the patients interviewed, such as education and occupational status. thirty-three patients (20.6%) considered their physical health to be very good; 71 (44.4%) good; 28 (17.5%) neither good nor bad; 27 (17%) bad; and 1 (0.6%) very bad. sixty-four patients (40%) judged their psychological health to be very good; 72 (45%) good; 10 (6.3%) neither good nor bad; 9 (5.6%) bad; and 5 (3.1%) very bad. the most common mr examinations the patients had undergone were those of the knee or spine, which together constituted 58% of the examinations (figure 1). two different groups of patients were separated in the analysis: those who had previously had an mr examination (group a, n = 105; 66%) and those who had not (group b, n = 55; 34%). group a consisted of two smaller subgroups, namely patients who had both their current and previous mr examinations in the same radiology department (subgroup a1, n = 23; 14%) and patients who had their previous and current mr examinations in different radiology departments (subgroup a2, n = 82; 51%) (figure 2). patients’ satisfaction and the caring attitude of the staff based on the result of this study, patients’ satisfaction levels with the four dimensions of care quality (information given by radiology staff, communication between the staff and patients, level of expertise, and caring attitude of the staff) are presented in table ii. the majority of the patients were very satisfied with the amount of information given by the radiology staff. this included both written and oral information in 64/160 (40%), radiographers’ communication skills in 94/160 (58.8%), perceived level of expertise in 142/160 (88.8%), and the staff’s caring attitude 145/160 (90.6%). fifty-nine percent (94/160) of the patients answered that the attitude of the staff was of major importance, 40% (64/160) of great importance, and 1% (2/160) of no importance. in general, the table i. socio-demographic characteristic background of the patients interviewed. background n % age groups 18–39 y 44 27.5 40–65 y 74 46.25 66–81 y 42 26.25 gender female 93 58.1 male 67 41.9 education primary school 15 9.4 polytechnic school/high school 64 40 college education 77 48.1 other degree 4 2.5 occupational status student 6 3.8 employed 78 48.8 employer 25 15.6 unemployed 1 0.6 retired 50 31.2 0 5 10 15 20 25 30 35 40 45 50 number and types of radiological examinations ab do m en c he st he ad & n ec k kn ee lo we r e xt re m itie s up pe r e xt re m itie s w ho le s pi ne & s pi ne figure 1. mr examinations: number of examinations of different anatomic regions. outsourcing and care related to magnetic resonance examinations 345 majority of patients, 146/160 (91%), were very satisfied with their care during their visit to a radiology department, while others, 14/160 (9%), were quite satisfied. fifty-three percent (84/160) believed this point was of major importance, 44% (70/160) of great importance, 2% (4/160) of no importance, and 1% (2/160) could not take a position. sixty-six patients (41.2%) who were dissatisfied with radiographers’ communication skills were of the opinion that radiographers’ communication during the performance of mr examinations is very important and necessary in order to increase the patient’s sense of security. patient satisfaction regarding the staff’s ability to provide care, the quality and amount of information, and patient age were inversely related. radiographers’ communication skills were significantly lower according to reports from patients between 18 and 58 years old (mean age = 34.8) than from patients between 45 and 81 years old (mean age = 68) (figure 3). patients in both groups (a and b) reported high satisfaction relative to their mr examination. no significant correlations were found between satisfaction and patient age or gender. waiting time including office waiting time twenty-nine percent (46/160) of the patients had waited less than one week between their referral and the mr examination; 60% (96/160) between one and four weeks; 6% (10/160) between one and two months; 2% (3/160) had waited three months or more; and 3% (5/160) did not know their waiting time. when patients were asked what an acceptable waiting time for an mr examination would be, 23.1% (37/160) answered less than one week; 64.4% (103/ 160) one to four weeks; 9.4% (15/160) one to two months; and 3.1% (5/160) did not have an opinion. the reported acceptable waiting time was significantly (p < 0.001) lower among the patients between 18 and 38 years old than among the patients between 48 and 81 years old who specified that an acceptable waiting time was between one and four weeks. the age of the 15 patients who reported that it was group b, n = 55 patients with no previous experience group a, n = 105 patients with previous experience patients who had their current and previous examinations in the same radiology department subgroup ±1, n = 23 subgroup ±2, n = 82 patients who had their current and previous examinations in different radiology departments the patients interviewed, n = 160 figure 2. number of patients interviewed, stratified according to previous experience and radiology department. table ii. patients’ level of satisfaction within four dimensions of assessment of care quality. very good good neither good nor bad bad cannot judge level of satisfaction freq. % freq. % freq. % freq. % freq. % information given by radiology staff 64 40 54 33.7 4 2.5 19 11.9 19 11.9 radiographers’ communication skills 94 58.8 66 41.2 level of expertise 142 88.8 6 3.8 1 0.63 2 1.3 9 5.6 caring attitude of the staff 145 90.6 15 9.4 346 p. t. olofsson et al. acceptable to wait one to two months for an mr examination was between 69 and 81 years old. for 40% (64/160) of the patients, a short time in the waiting room was related to a positive response regarding returning for a further mr examination. choice of radiology department and patients’ freedom of choice when patients were asked whether they could choose or influence where their mr examination would be performed, 27.5% (44/160) of the patients answered yes; 69.4% (111/160) answered no; and 3.1% (5/160) replied partly. when patients were asked how important this freedom of choice was, 23.1% (37/160) replied that it was of major importance; 30% (48/160) of great importance; 24.3% (39/ 160) of little importance; 11.3% (18/160) of no importance; and 11.3% (18/160) had no opinion on the issue. ninety of the patients in this study (56.3%) believed that it was their physician who decided where their mr examination should be performed. aspects that influenced the patient’s choice of radiology department were: short waiting time 79.4% (127/160); ease of traveling to the radiology department 68.8% (110/160); and short distance to their home or work 58% (93/160). improvement of the patients’ satisfaction forty-eight percent (77/160) of the patients suggested that better information about the examination would increase their satisfaction related to the mr examination, and 52% (83/160) desired more instructions during the procedure. discussion the main finding of this study is that the majority of patients referred for an mr examination (140/160; 91%) were satisfied with the level of care that they received in the radiology department, whether it was the one in the university hospital or the external caregiver, indicating that the patients’ perception was that they received the same level of care at either public or private radiology departments. fifty-six percent of the patients (90/160) believed that it was their referring physician who decided where the mr examination should be performed. it is natural that the patients, being unaware of the hospital’s policies, generally expect the referring physician to take on the role of decision-maker (8). in reality, this decision is made in the radiology department and depends mostly on the inflow of referrals. the physician is the closest link between the patient and radiology. although radiologists play a crucial role in patients’ health, they are invisible to patients (9) and are not perceived as decision-makers in the health care process. sixty percent of the patients in this study waited between one and four weeks from when their referrals were written until the mr examinations were performed, and 65% of the patients regarded that as acceptable. this indicates that radiology departments within university hospitals and private radiology units are, together, meeting the patients’ expectations fairly well. results showed that older patients were generally more satisfied with the staff’s ability to communicate, including the quality and amount of information they received. it is important to note that the nature of expectations could be different between older and younger patients, as well as between those patients who had previously had an mr examination versus r2 = 0.5721 age satisfaction level regarding radiographers¢ communication skills l e v e l o f s a ti s fa c ti o n 4 3.5 3 2.5 2 1.5 1 0.5 0 1 21 41 61 81 figure 3. satisfaction with the radiographers’ communication skills and patient age was correlated at the level of about r = 0.76 and r2 = 0.57. outsourcing and care related to magnetic resonance examinations 347 those undergoing one for the first time (10). satisfaction with care usually arises when there is no discrepancy between patients’ expectations and the care received (11). according to the results of this study, more instructions during mr examinations and better information about the examination itself would increase patients’ satisfaction relative to the mr examination. one definite way to increase patient satisfaction is to focus on the patients’ views about the care they receive and their expectations of it (12). the advantage of using structured interviews, i.e. the verbal questionnaire, was that the questions could be clarified for the informants if necessary. this diminished the risk of collecting an incorrect response or partial non-response. we believe that a written questionnaire would have been of limited use in this study, because of the chance that patients would fail to recall situations. both alternative questions and open-ended questions were used in the interview. the goal was to attract the strengths and minimize the limitations of the quantitative and qualitative analyses (13). we believe that the combination of quantitative and analytical open-ended questions followed by text analyses provides a better understanding of patients’ expectations, satisfaction, and communication about outsourcing their mr examination, because the experience of satisfaction cannot be measured fully by statistical methods alone. but this study also has several limitations to consider. the interviews may result in a biased sample by attracting respondents who could or were willing to participate. the result showed high satisfaction with the patient care, which could be caused by the fact that displeased patients did not participate (14). for this reason, we may not be able to generalize the results. despite this, we believe that this study could be of interest to other public hospitals which choose outsourcing as one solution for making their radiology departments more efficient. another limitation in this study is that we studied those dimensions of care quality during mr examinations that were most related to patient nursing. however, the quality of care related to mr examinations involves other factors, such as the radiologist’s level of expertise, work experience, knowledge, work-load pressure, as well as work satisfaction, all of which may have a major impact on the quality of the interpretation. indeed these factors should be studied further. patients undergoing mr examinations usually come into contact with radiographers but they seldom have direct contact with radiologists (15). in conclusion, the patients interviewed in this study were unaware of the university hospital’s policy regarding the outsourcing of mr referrals. when considering outsourcing, the patients’ desires and requirements regarding information must be considered. if this information is adequate, patients are likely to feel that they have experienced a high level of care irrespective of whether their mr examination is outsourced or not. ways to improve communication between patients and the referring physicians regarding their radiological examinations should be studied further. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. sutton r, kanal e, wilkoff bl, bello d, luechinger r, jenniskens i, et al. safety of magnetic resonance imaging of patients with a new medtronic enrhythm mri surescan pacing system: clinical study design. trials. 2008;9:68. 2. götte mj, rüssel ik, de roest gj, germans t, veldkamp rf, knaapen p, et al. magnetic resonance imaging, pacemakers and implantable cardioverter-defibrillators: current situation and clinical perspective. neth heart j. 2010;18: 31–7. 3. the royal college of radiologists. teleradiology and outsourcing census. london: the royal college of radiologists; 2010. available 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satisfaction with medication: an overview of conceptual, methodological, and regulatory issues. value health. 2004;7:204–15. 11. auquier p, pernoud n, bruder n, simeoni mc, auffray jp, colavolpe c, et al. development and validation of a perioperative satisfaction questionnaire. anesthesiology. 2005;102: 1116–23. 12. scardina sa. servqual: a tool for evaluating patient satisfaction with nursing care. j nurs care qual. 1994;8:38–46. 13. johnson rb, onwuegbuzie aj. mixed methods research: a research paradigm whose time has come. educ res. 2004;33:14–26. 348 p. t. olofsson et al. http://www.ncbi.nlm.nih.gov/pubmed/19055703?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/19055703?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/19055703?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20111641?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20111641?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20111641?dopt=abstract 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http://www.ncbi.nlm.nih.gov/pubmed/15915023?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15915023?dopt=abstract 14. kinnersley p, egbunike jn, kelly m, hood k, owen-jones e, button la, et al. the need to improve the interface between in-hours and out-of-hours gp care, and between out-of-hours care and self-care. fam pract. 2010;27:664–72. 15. camponovo ej. radiologist-patient contact: a different perspective. j am coll radiol. 2004;12:998–9.author reply 1000 comment on radiologist-patient contact during the performance of cross-sectional examinations. j am coll radiol. 2004. supplementary materials available online appendix. outsourcing and care related to magnetic resonance examinations 349 http://www.ncbi.nlm.nih.gov/pubmed/20671001?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20671001?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20671001?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17411745?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17411745?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17411745?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17411745?dopt=abstract http://informahealthcare.com/doi/suppl/10.3109/03009734.2014.951133/suppl_file/10.3109/03009734.2014.951133_suppl.doc abstract introduction material and methods analysis results patients&rsquo; satisfaction and the caring attitude of the staff waiting time including office waiting time choice of radiology department and patients&rsquo; freedom of choice improvement of the patients&rsquo; satisfaction discussion declaration of interest references corrigendum corrigendum the editors are sorry for giving the incorrect name for the third guest editor of our special issue on uppsala clinical research center. his name is johan sundstr€om. � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 2, 148 https://doi.org/10.1080/03009734.2019.1626656 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1626656&domain=pdf&date_stamp=2019-06-06 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2019.1626656 http://www.tandfonline.com palliative care in copd—web survey in sweden highlights the current situation for a vulnerable group full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 palliative care in copd—web survey in sweden highlights the current situation for a vulnerable group of patients susann strang, ann ekberg-jansson, peter strang & lars-olof larsson to cite this article: susann strang, ann ekberg-jansson, peter strang & lars-olof larsson (2013) palliative care in copd—web survey in sweden highlights the current situation for a vulnerable group of patients, upsala journal of medical sciences, 118:3, 181-186, doi: 10.3109/03009734.2013.801059 to link to this article: https://doi.org/10.3109/03009734.2013.801059 © informa healthcare published online: 28 may 2013. submit your article to this journal article views: 675 view related articles citing articles: 12 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.801059 https://doi.org/10.3109/03009734.2013.801059 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.801059 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.801059 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.801059#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.801059#tabmodule upsala journal of medical sciences. 2013; 118: 181–186 original article palliative care in copd—web survey in sweden highlights the current situation for a vulnerable group of patients susann strang1,2, ann ekberg-jansson1,2, peter strang3 & lars-olof larsson1,4 1angered local hospital, angered, sweden, 2the sahlgrenska academy, university of gothenburg, sweden, 3department of oncology-pathology, karolinska institutet, stockholm and stockholms sjukhem, stockholm, sweden, and 4department of medicine, division of respiratory medicine, karolinska institutet, stockholm, sweden abstract background. chronic obstructive pulmonary disease (copd) is a common cause of death. despite the heavy symptom burden in late stages, these patients are relatively seldom referred to specialist palliative care. methods. a web-based survey concerning medical and organizational aspects of palliative care in copd was distributed to respiratory physicians in sweden. there were 93 respondents included in the study. results. palliative care issues were regularly discussed with the patients according to a third of the respondents. about half of the respondents worked in settings where established routines for co-operation with palliative units were available at least to some extent. less than half of the respondents (39%) were aware of current plans to develop palliative care, either as a cooperative effort or within the facility. palliative care is focused on physical, psychological, social, and existential dimensions, and the proportions of respondents providing support within these dimensions, ‘always’ or ‘often’, were 83%, 36%, 32%, and 11%, respectively. thus, to treat the physical dimensions was perceived as much more obvious than to address the other dimensions. conclusions. the survey indicates that the priorities and resources for palliative care in copd are insufficient in sweden. the data, despite limitations, reveal a lack of established team-work with specialized palliative care units and actual plans for such co-operation. key words: care management, copd, holistic care, palliative care, survey introduction chronic obstructive pulmonary disease (copd) is a progressive, disabling, and life-limiting disease. in sweden, 5%–15% of the population over 45 years of age is believed to suffer from copd; the number of hospitalizations due to copd has increased by 40%, and the number of deaths has doubled during the last decades (1,2). copd is the only endemic modern western illness where the number of deaths is still increasing (3,4). shortness of breath, fatigue, and cough are the most troublesome and well-known symptoms, and living with copd imposes limitations on daily living (5-7). the available treatment regimens are mainly focused on physical symptoms and give only limited relief from these symptoms. the comprehensive palliative care philosophy goes beyond the focus on physical symptoms, and, according to the world health organization (who), palliative care should comprise physical, psychological, social, and existential/spiritual dimensions throughout the continuum of the disease (8). the physical dimensions of copd include severe breathlessness, acute exacerbations, cough, fatigue, anorexia and muscle wasting, osteoporosis, pain, increased risk for thrombosis, and heart failure (6,9-11). the correspondence: susann strang, phd, sahlgrenska academy, göteborg university, institute of health and caring sciences, box 457, se-405 30 göteborg, sweden. fax: +46 31 786 6050. e-mail: susann.strang@gu.se (received 13 february 2013; accepted 28 april 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.801059 http://informahealthcare.com/journal/ups mailto:susann.strang@gu.se psychological burden is characterized by depression, anxiety, risk for cognitive impairment, and sleep disorders (10,12). the social consequences are mainly related to limitations in everyday life, affecting both the patient and the family (13,5), whereas the existential burden is a suffering connected to feelings of meaninglessness, guilt, and impending death (14-16). thus, copd in its advanced stages is definitely a disease requiring palliative care; still it is seldom discussed in a palliative context. the comprehensive palliative care philosophy and the need for palliative care services are self-evident in advanced stages in cancer care, whereas this approach is not generally offered to copd patients, despite the heavy symptom burden in the late stages of the disease. questionnaires have earlier been distributed in england, wales, and northern ireland since it was unclear to what extent specialist palliative care was available for those with non-malignant lung diseases. the study revealed that patients with advanced copd had less universal access to specialist palliative care services than patients with malignant lung diseases (17). further, a self-assessment survey in the uk showed that 42% of the hospitals had formal palliative care arrangements for patients with copd (18). the response rates for these studies were only 51% and 38%, respectively, which may reflect a less pronounced interest in these questions. the aim of the study was to survey respiratory physicians’ views of copd as a potential palliative care diagnosis, where physical, psychological, social, and existential domains are addressed. a further aim was to survey the perceived availability of resources for palliative care and the trajectory through the health care system at the end of life in our setting, i.e. in sweden. materials and methods a web-based survey concerning palliative care in copd was distributed by e-mail through the swedish society of pulmonary medicine (slmf), initially to all members (n = 259) in sweden in august 2011. the survey was distributed twice to the members with an interval of four weeks. the slmf includes not only clinically active respiratory physicians but also retired members, other professionals within respiratory medicine, and supportive members. after exclusion of physicians who were retired or not in clinical work (but, e.g., within administrative positions, pharmaceutical companies) and exclusion of members who were not physicians, the response rate was calculated to be 41% (93 respondents). the address register for health and medical care personnel (hsra) records in 2010 was used for comparison, and 222 clinically active respiratory physicians were identified, 126 men and 96 women. the web survey responses were returned anonymously, and neither respondents nor hospitals could be identified. the survey consisted of five questions. the possible alternatives for the four first questions were: ‘yes’, ‘partly’, ‘seldom’, ‘not at all’, and ‘do not know’. for the fifth question the alternatives were: ‘always’, ‘sometimes’, ‘often’, ‘seldom’, and ‘not at all’. the questions were: (1) do you consider any of your copd patients as needing palliative care? (2) when a patient is considered as such, do you usually discuss palliative care with the patient and the family members? (3) are there any established routines at your unit for patients with copd to receive palliative care, if needed? (4) are there any plans at your unit to develop, or further develop, palliative care services for patients with copd? (5) according to who, palliative care is focused on physical, psychological, social, and existential dimensions. to what extent do you provide support in each of these dimensions? ethical considerations the study was carried out in accordance with the ethical standards of the helsinki declaration (world medical association declaration, 2000). ethical approval of the study was obtained from the regional ethical review board in gothenburg (dnr: 209-11). results the 93 respondents provided some demographic data such as gender, working place, working years within respiratory medicine, and whether they provided care for copd patients or not. there was a predominance of male respondents and fewer female respondents compared to the general respiratory specialist gender ratio. there were, however, no significant gender differences in the patterns of the answers (table i). an absolute majority of the respondents provided care for patients whom they considered in need of palliative care (table ii). about one-third of the respondents discussed palliative care issues with their patients only partly or not at all. one of those who did not discuss palliative care commented: ‘the patient is expected to realize that all medical efforts in copd are palliative and thus palliation is not discussed as for cancer patients’. the optimal timing for palliative care discussions was commented on in 182 s. strang et al. the web survey. there were different suggestions about when such discussions should take place, e. g. when the patient was hospitalized for intensive care, when the patient was listed for home care, after joint therapy conferences, or at the regular visits. one respondent stressed the need to discuss palliative care while the patient was still in a stable condition and not when the patient was admitted to hospital for an exacerbation. about 53% of the respondents worked in settings where routines for co-operation with palliative units were available, although they were only partly established according to 24% of the respondents. as a spontaneous comment, several respondents (10%) emphasized the need for the availability of palliative at-home or in-patient services for copd patients while still delivering profiled medicine respiratory care. almost 39% of the respondents were aware of current plans to develop palliative care, either as a co-operative effort or within the facility. there were more definitive plans according to 20% of the respondents and partly decided plans according to 19%. there were, however, no known plans for further development of palliative care services according to one-third of the respondents. those respondents who had been working for 10 years or more as respiratory physicians were more likely to report plans to develop palliative care compared to those who had been working for 5 years or less (47% versus 13%) (p < 0.05, fisher’s exact test). since who’s definition of palliative care focuses on physical, psychological, social, and existential/ spiritual dimensions the final question addressed these issues. the fact that a doctor should treat the physical dimensions was perceived as obvious in contrast to the psychological and social dimensions. few of the respondents expressed the view that the existential/ spiritual issues should be always or often addressed, and half of the respondents said that they seldom or never provided support in these dimensions, despite the poor prognosis in the late stages of the disease, as shown in table iii. the open-ended comments showed that this last question is a broad issue and difficult to answer only by ticking a box in a questionnaire. furthermore, it was perceived as a difficult task to separate these dimensions, and there is also a stated lack of time to address these issues in clinical practice. the crosstabulation revealed that those who usually do not discuss palliative care with their patients and their families addressed neither the existential (0%) nor the psychological dimension (0%). those who had worked less than 5 years in pulmonary medicine said that they never (9%) or seldom respond to an existential/spiritual dimension, and none of them felt that they always do. discussion almost all of the physicians had patients in a palliative phase, and over two-thirds also did discuss palliative care issues with patients and their relatives. however, one-third of the physicians only partially or never discussed palliative care. this may be due to several reasons, e.g. the difficulty of prognosis in advanced copd as well as predicting whether the patient will recover or not in an exacerbated phase of copd (19). in studies from the uk there were even more notable figures, as 87% had no routines for initiating end-oflife discussions (17,20). further, studies have shown that patients themselves are reluctant to discuss palliative care issues with their doctor (12,21). this may be due to the patients’ own perception of copd as a non-fatal disease, because most have suffered from copd for a long time and have somewhat learned to live with the disease (19). only half of the physicians stated that they had established routines for working with palliative care units. even fewer knew about any plans to develop further the palliative care for patients with copd. there might be several reasons, e.g. lack of health staff members designated and responsible for palliative care of non-malignant respiratory diseases within the facility and collaborating with specialized palliative care medicine. further, respiratory specialists might question if the palliative care services have sufficient competence in specific copd issues. however, some health authorities in sweden are now requiring excellence in palliative care of malignant as well as of non-malignant diseases, such as copd, for those who are providing advanced care of patients in their homes (22). in an early stage of copd, it is reasonable for respiratory specialists or general practitioners within primary care to have sole responsibility for the patients, while at a later stage of the disease cooperation with palliative care services would most likely benefit the patient. studies have identified the obvious thresholds during the copd disease course, i.e. when there is a risk of death within one year of complications to copd, e.g. respiratory failure (23-25). despite this knowledge, there seems to be a reluctance to initiate palliative care in time. still, who emphasizes that palliative intervention preferably should be incorporated early in the course of the disease, along with disease-specific treatment. palliative care services should be offered at least as a complement to specialist copd care, if the symptom burden and palliative care in copd 183 the emotional suffering are obvious, regardless of prognosis (26). as a comparison, early palliative care interventions have proven to improve quality of life and prolong survival for newly detected lung cancer patients (27). moreover, in a recent study where the symptom burden was compared for hospitalized cancer and copd patients, the frequency of distressing symptoms was comparable, but the symptoms were more difficult to relieve in copd patients (28). who strongly recommends team-based palliative care, ‘the third corner-stone’ (29). team-based care seems to provide synergistic outcomes whether if the focus is on palliative care (3). the team members, with different professional profiles, create the possibility for improved management of complex symptoms and fulfilment of needs beyond medical ones. effective and well-functioning team-work further creates trust as well as support for the team members themselves. professionals within respiratory medicine who have the excellence and interest in palliative care could be part of teams collaborating with palliative medicine and could support more integrated models of care for all patients in an end-of-life situation (2). only a minority of copd patients are treated by respiratory specialists; instead the majority of these patients receive medical help from primary care services. although the survey was addressed to respiratory specialists, it still highlights considerable shortcomings as regards a palliative care approach for the copd population. the cause has not been studied but may be related to insufficient chains of care for co-operative measures in advanced copd and palliative care, the large number of copd patients, and difficulties to communicate palliative care with the patient and the family, as well as a feeling of uncertainty and powerlessness for this vulnerable group. copd as a major cause of death is of course a wellknown fact for copd specialists, but as the medical trajectory is difficult to predict, co-operation with a palliative care team is rarely established. further, there is still a widespread misconception: palliative care is believed to be limited to cancer care (30,31). the psychological dimensions were only regarded as important by 36%. this is in good agreement with a comparative study of patients with lung cancer and patients with copd, where the authors were able to demonstrate that copd patients had more anxiety and depression, but were more seldom for treated (32). this is a great concern, as studies show that depression in copd patients is highly related to mortality, hospital readmissions, and longer lengths of stay (33,34). barely one-third of the physicians considered the social dimensions important. this shortcoming may be related to a perceived lack of time, but may also be attributed to a lack of priority. still, recent research highlights the need of chains of care and formalized co-operation between copd and palliative care (31). among all aspects, the existential dimension of copd care was the most neglected one: only 11% table i. the study population (n = 93). gendera femaleb 34% maleb 66% health care providers university hospital 38% other health care providers 62% working years within pulmonary medicinea £10 31% >10 69% providing care for copd patients yes 96% no 4% a data incomplete for two study respondents. b among all clinically active physicians 43% are female and 57% are male. table ii. distribution of the responses to questions 1–4 in the web survey. questions yes n (%) partly n (%) no n (%) do not know n (%) data incomplete n 1. do you consider any of your copd patients as being a palliative care patient? 85 (93%) n/a 1 (1%) 5 (6%) 2 2. when a patient is considered as palliative, do you usually discuss palliative care with the patient and the family members? 63 (69%) 26 (28%) 5 (5%) 2 (2%) 1 3. are there any established routines in your unit for patients with copd to receive palliative care, if needed? 29 (32%) 24 (26%) 38 (41%) 2 (2%) 1 4. are there any plans to develop, or further develop, palliative care services for patients with copd? 18 (20%) 17 (19%) 27 (30%) 27 (30%) 4 184 s. strang et al. of the physicians considered support in existential issues to be a task for them, despite the high mortality rate of copd patients, which activates thoughts of life and death issues and triggers existential anxiety (35). study data show that these issues are of great importance to patients (13), but they are seldom addressed, possibly due to the lack of competence, lack of awareness, and a feeling of powerlessness. still, existential discussions are probably addressed by the physicians, at least to some degree, even if they do not label them as being existential or spiritual (36). the core question remains to be answered in a swedish setting: should access to specialist palliative care be restricted to cancer care or should it be expanded also to include copd? end-stage copd is associated with a heavy symptom burden, not only from the respiratory system, but also in the form of pain, fatigue, weight loss, death anxiety, and depression, just like cancer diagnoses. data indicate that an interactive and integrated process between copd care and palliative care would benefit the patient and also optimize the resource utilization (37). this web survey has its limitations since it is difficult to achieve adequate answers on such complex questions with only fixed-format questions and the possibility of adding open-ended comments. the interpretation of the survey was also impacted by the relatively low response rate (41%). a pessimistic view is that the low response rate at least partly reflects the limited interest in palliative care for copd patients and the respondents might be those with an interest of palliative care. despite the limitations of the study, it points to deficiencies in the palliative care of copd patients. patients with copd are a vulnerable, non-prioritized group as seen from the inadequate actions from the local authorities as well as from the health care services. further initiatives must be taken to provide these patients with equal and decent care in the palliative phase of the disease. one way to establish these measures might be to increase education and information for health care providers as well as politicians about copd. patient organizations are also important to accomplish a sustainable change. acknowledgements we would like to thank all the physicians who contributed to the survey, the swedish society of pulmonary medicine who helped with the distribution of the survey, and the swedish address register for health and medical care personnel (hsra) for assistance with demographic data. declaration of interest: this study received generous support from the swedish national association for heart and lung patients.the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. all authors, (susann strang, lars-olof larsson, ann ekbergjansson, and peter strang) have made a substantial contribution throughout the process and take public response for the whole content. references 1. jansson c, 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www.ncbi.nlm.nih.gov/pubmed/17989116?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16334970?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16334970?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16334970?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18454613?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18454613?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18454613?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18454613?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18373613?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18373613?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18373613?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15540669?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15540669?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20348366?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20348366?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20348366?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20348366?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19176708?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18838492?dopt=abstract 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www.ncbi.nlm.nih.gov/pubmed/20818875?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22371388?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22371388?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22371388?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22351651?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22351651?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22116516?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22116516?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11083884?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11083884?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11083884?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11083884?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12006426?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12006426?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17210879?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17210879?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17210879?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17210879?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19825897?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19825897?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19825897?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19648222?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19648222?dopt=abstract abstract introduction materials and methods ethical considerations results discussion acknowledgements declaration of interest references upsala j med sci 88: 3 3 4 1 , 1983 intraarterial and intraportal in vivo catheterization of the regenerating rat liver effects upon body and liver weights and dna synthesis lillernor lewan', herman arnneus', ola forsberg3 and kerstin lundberg4 department of zoophysiology', university of lund, lund, and department of physical biolo y 2 , central hospital, vaster&, and department of experimental medicine4, pharmacia a b , uppsala, sweden the gustaf werner institute, university of uppsala, uppsala, and department of surgery b , abstract a method f o r c o n c o m i t a n t p a r t i a l hepatectomy and c a t h e t e r i z a t i o n of t h e a r t e r i a l and p o r t a l systems of t h e l i v e r i n t h e r a t i s d e s c r i b e d . c a t h e t e r s were i n s e r t e d i n t o t h e g a s t r o d u o d e n a l a r t e r y and t h e i l e o c o l i c v e i n . continuous s a l i n e p e r f u s i o n was performed d u r i n g 36 h o u r s . i n c a t h e t e r i z e d r a t s r e c o v e r y of l i v e r and body w e i g h t l a g g e d b e h i n d t h a t of n o n c a t h e t e r i z e d r a t s . the more e x t e n s i v e s u r g e r y and t h e p r e s e n c e of c a t h e t e r s a l s o c a u s e d d e c r e a s e d i n c o r p o r a t i o n of 3h-thymidine i n t o l i v e r dna 24 h o u r s p o s t o p e r a t i v e l y . the v a r i a t i o n i n t h y m i d i n e i n c o r p o r a t i o n between a n i m a l s was l a r g e . it was shown t h a t by pre l a b e l l i n g l i v e r dna w i t h "c-thymidine t h e r a t s c a n s e r v e a s t h e i r own c o n t r o l s d u r i n g a c u t e e x p e r i m e n t s i n v o l v i n g 'h-thymidine, t h u s r e d u c i n g t h e i n c o n s i s t a n c y of i n d i v i d u a l v a r i a t i o n . introduction p a r t i a l r e s e c t i o n of 213 of t h e l i v e r i n r a t s i n d u c e s h y p e r t r o p h y and h y p e r p l a s i a of t h e r e m a i n i n g l i v e r t i s s u e . t h i s i s c h a r a c t e r i z e d by changes i n enzyme a c t i v i t i e s , i n rnaand dna-synthesis, and i n m i t o t i c a c t i v i t y . almost a l l c e l l s a r e s t i m u l a t e d t o s y n t h e s i z e dna and p a s s t h r o u g h t h e c e l l c y c l e 1-2 t i m e s . r e s t o r a t i o n of o r g a n w e i g h t is u s u a l l y s e e n w i t h i n a week. the c o u r s e of t i m e f o r t h e s e e v e n t s a r e w e l l known ( 2 , 5 , 8 , 1 3 , 1 4 ) . i n t h e p r e s e n t s t u d y w e d e s c r i b e a model d e s i g n e d t o a n a l y z e t h e e f f e c t s of blood f l o w m o d i f i c a t i o n s on l i v e r c e l l k i n e t i c s i n u n a e s t h e t i z e d r a t s . we e v a l u a t e d t h e e f f e c t s of c o n c o m i t a n t c a t h e t e r i z a t i o n of t h e h e p a t i c a r t e r y a n d p o r t a l v e i n i n c o n n e c t i o n w i t h p a r t i a l hepatectomy on body and l i v e r w e i g h t s , a s w e l l as t h e e f f e c t of c a t h e t e r i z a t i o n upon i n c o r p o r a t i o n of 3h-thymidine and 14c-thymidine i n t o l i v e r dna, u s i n g e a c h animal a s i t s own c o n t r o l . material and methods animals young, a d u l t male sprague dawley r a t s weighing 170-210 g (spf q u a l i t y , 3-832859 33 l i v e r of fig. 1. schematic view of c a t h e t e r s i n t h e i l e o c o l i c v e i n , i n t h e gastroduodenal a r t e r y and of t h e f r e e c a t h e t e r emptying i n t o t h e p e r i t o n e a l c a v i t y . for d e t a i l s of t h e a r t e r i a l and p o r t a l systems r e f e r t o green ( 7 ) . from pump t o a r t e r i a l \ and p o r t a l c a t h e t e r p e r i t : t e r :oneal f i g . 2 . c a t h e t e r i z e d r a t i n cage. 34 anticimex, sweden) were u s e d f o r t h e e x p e r i m e n t . the a n i m a l s were k e p t u n d e r c o n s t a n t c o n d i t i o n s and had f r e e access t o s t a n d a r d l a b o r a t o r y d i e t b e f o r e and a f t e r s u r g e r y . s u r g i c a 1 p r o c e d u r e s the a n i m a l s were a n a e s t h e t i z e d w i t h a n i p i n j e c t i o n of mebumal, 30 mg/kg body w e i g h t . a f t e r m i d l i n e i n c i s i o n and r e t r a c t i o n of t h e l i v e r , t h e o p e r a t i o n w a s performed under a microscope ( 4 x). a p o l y e t h y l e n e c a n n u l a ( i n t r a m e d i c pe 50) w i t h a narrow t i p was i n s e r t e d i n t o t h e g a s t r o d u o d e n a l a r t e r y i n t h e r e t r o g r a d e d i r e c t i o n , a c c o r d i n g t o l e i v e s t a d and malt ( 1 2 ) . the i l e o c o l i c v e i n was c a t h e t e r i z e d i n t h e a n t e r o g r a d e d i r e c t i o n . to a l l o w i n t r a p e r i t o n e a l i n f u s i o n , t h e t i p of a t h i r d c a t h e t e r w a s p o s i t i o n e d i n t h e p e r i t o n e a l c a v i t y , f i g . 1. a f t e r c a t h e t e r i z a t i o n t h e median and l e f t l a t e r a l l o b e s of t h e l i v e r were r e s e c t e d a c c o r d i n g t o h i g g i n s and anderson ( 9 ) . i n s e p a r a t e e x p e r i m e n t s p a r t i a l hepatectomy w a s accomplished b e f o r e c a t h e t e r i z a t i o n . the c a t h e t e r s were p u l l e d s u b c u t a n e o u s l y t o t h e neck and t h e abdominal wound w a s s t i t c h e d . a small p l a s t i c v i a l a t t a c h e d t o t h e neck s e c u r e d t h e c a t h e t e r s from damage. from t h e v i a l t o t h e t o p of t h e c a g e , t h e c a t h e t e r s were p r o t e c t e d by a m e t a l h e l i x , f i g . 2 . the i n t r a v a s c u l a r c a t h e t e r s were c o n n e c t e d t o pumps (brown-melsungen, t y p e 71100) which s u p p l i e d 1 m1/24 h r of f y s k o s a l / h e p a r i n (500 i e hep/100 c c f y s k ) . the p e r i t o n e a l c a t h e t e r was f i l l e d w i t h t h e same s o l u t i o n and c l o s e d a t i t s f r e e end. f i v e r a t s s u b j e c t e d t o p a r t i a l hepatectomy o n l y s e r v e d as c o n t r o l s . p a r t i a l hepatectomy a l o n e was u s u a l l y accomplished w i t h i n 5 m i n u t e s . cathe t e r i z a t i o n r e q u i r e d a n o t h e r 15-25 m i n u t e s . to minimize e f f e c t s of d i u r n a l v a r i a t i o n s , o p e r a t i v e work was r e s t r i c t e d t o a 1-2 hour p e r i o d i n t h e morning. one group of r a t s w e r e d e t a c h e d from t h e pumps 36 h o u r s a f t e r t h e o p e r a t i o n . the metal h e l i x was removed, t h e c a t h e t e r s were f l u s h e d , m e l t e d s h u t , c o i l e d and p u t i n t o p l a s t i c v i a l s . t h i s group a n d t h e c o n t r o l g r o u p of non c a t h e t e r i z e d r a t s were weighed d a i l y . seven days a f t e r o p e r a t i o n t h e r a t s were k i l l e d and t h e l i v e r s removed and weighed. animals aimed f o r r a d i o n u c l i d e s t u d i e s were g i v e n r a d i o a c t i v e thymidine 24 h u r s a f t e r t h e o p e r a t i o n v i a t h e p e r i t o n e a l c a t h e t e r o r i n t h e c a t h e t e r i n t h e i l e o c o l i c v e i n . slow f l u s h i n g w i t h 5 i.ll a t a t i m e was accomplished w i t h a r e p e a t i n g d i s p e n s e r (hamilton comp) u n t i l 100 i.ll c o n t a i n i n g 185 kbq ( 5 pci) of (methyl-i4c) thymidine (185 gbq, amersham) a n d / o r 100 i.l1 c o n t a i n i n g 3 . 7 mbq (100 pci) ( m e t h ~ l ~ h ) thymidine (555-1110 gbq, amersham) w a s a d m i n i s t e r e d . for d o u b l e l a b e l l i n g , where e a c h a n i m a l s e r v e d a s i t s own c o n t r o l , t4c-thymidine was i n j e c t e d f i r s t and 3h-thymidine one o r two h o u r s l a t e r . one hour a f t e r t h e l a s t i n j e c t i o n t h e r a t s were k i l l e d and t h e l i v e r s were removed,and t h e d i f f e r e n t p a r t s of t h e r e g e n e r a t i n g l o b e were i d e n t i f i e d , f r o z e n 35 on s o l i d c o z , and s t o r e d a t 7 o o c u n t i l a n a l y s i s . o t h e r , one group of a n i m a l s r e c e i v e d 3h-thymidine o n l y and a n o t h e r group 3h thymidine and 14c-thymidine c o n c o m i t a n t l y . the same d o s e s as d e s c r i b e d above were used. to e n s u r e t h a t t h e two i n j e c t i o n s of t h y m i d i n e d i d n o t i n t e r f e r e w i t h e a c h to show m e t a b o l i c e f f e c t s on thymidine i n c o r p o r a t i o n i n l i v e r dna one group of a n i m a l s was g i v e n 3h-thymidine 24 h o u r s a f t e r p a r t i a l hepatectomy and one hour l a t e r 14c-thymidine mixed w i t h 0 . 0 0 1 o r 0 . 2 mmol "cold" t h y m i d i n e . number body w e i g h t of r a t s m sd 0 1 2 3 4 5 6 7 day 5 w i t h 202g 100% 90% 92% 95% 97% 100% 1 0 7 % l ) no c a t h . + + + + + + + 4 w i t h 206g 100% 87% 87% 89% 90% 91% 98% c a t h . -5 -2 -2 -3 -4 -4 -4 24 -6 -3 -4 4 -3 -3 -2 23 + + + + + + + i a n a l y s i s of dna specimens t a k e n from t h r e e d i f f e r e n t p a r t s of t h e r e g e n e r a t i n g l o b e were a n a l y z e d by two methods. dna was e x t r a c t e d a c c o r d i n g t o schmidt-tannhauser as m o d i f i e d by munro & f l e c k ( 1 6 ) and q u a n t i f i e d a c c o r d i n g t o burton ( 4 ) . dna w a s a l s o i s o l a t e d by p r o t e i n a s e k a c c o r d i n g t o kasche-amneus (10) and q u a n t i f i e d by w s p e c t r o p h o t o m e t r y . r a d i o a c t i v i t y was a n a l y s e d i n a s c i n t i l l a t i o n c o u n t e r (mark 11) and dna s p e c i f i c r a d i o a c t i v i t y d e t e r m i n e d . the two methods f o r dna a n a l y s e s were i n good agreement. only r e s u l t s from a n a l y s e s performed a c c o r d i n g t o t h e schmidt tannhauser method are r e p o r t e d h e r e . f o r c a l c u l a t i o n of t h e r a t i o between t h e s p e c i f i c 3hand 1 4 c a c t i v i t y i n dna, t h e measured cpm/mg dna w a s d i v i d e d by t h e cpm of i n j e c t e d 3h-thymidine a n d 14c-thymidine, r e s p e c t i v e l y . l i v e r w e i g h t m sd day 7 7 . 4gl) ?0.3 6.4g 2) 20.9 results the e f f e c t s of s u r g e r y and c a t h e t e r i z a t i o n upon t o t a l body and l i v e r w e i g h t s a r e g i v e n i n t a b l e 1. d i f f e r e n c e s between c a t h e t e r i z e d and non-cathe t e r i z e d r a t s were small, b u t r e c o v e r y i n r a t s w i t h c a t h e t e r s l a g g e d b e h i n d of t h e c o n t r o l group. t a b l e 1. e f f e c t of c a t h e t e r i z a t i o n on body a n d l i v e r w e i g h t 1-7 d a y s a f t e r p a r t i a l hepatectomy. c a t h e t e r s were c o n n e c t e d t o t h e pumps f o r 36 h o u r s . mean f s t a n d a r d d e v i a t i o n . t h a t 36 n e i t h e r p r e n o r c o a d m i n i s t r a t i o n o f 14c-thymidine i n t e r f e r r e d s i g n i f i c a n t l y w i t h t h e i n c o r p o r a t i o n of 3h-thymidine i n t o l i v e r dna ( t a b l e 2 ) . t a b l e ' 2 . e f f e c t of preo r c o a d m i n i s t r a t i o n of 14c-thymidine ( 1 0 4 mmol) on 3h thymidine ( 5 x mmol) i n c o r p o r a t i o n i n t o l i v e r dna 24 h o u r s a f t e r p a r t i a l hepatectomy. r a d i o n u c l i d e s were a d m i n i s t e r e d t h r o u g h a per manent c a t h e t e r emptying i n t o t h e p e r i t o n e a l c a v i t y . mean * s t a n d a r d d e v i a t i o n , r e l a t i v e v a l u e s . number of a n i m a l s 4 3 5 ~~ hours a f t e r p a r t i a l hepatectomy cpm p e r mg dna p e r i n j e c t e d cpm i n j e c t i o n of 14c 3h k i l l e d 'i 4c-thymidine 3h-thymidine m sd m s d 24 25 2.43?0.61 24 24 25 3.55?0.55 2 . 9020. 72 23 24 25 4.17?0.72 3.08:o. 70 i n t a b l e 3 , two g r o u p s of r a t s , h a v i n g e i t h e r a n i n t r a p e r i t o n e a l c a t h e t e r o n l y (a1) o r h a v i n g an i n t r a p e r i t o n e a l c a t h e t e r as w e l l as c a t h e t e r s i n t h e g a s t r o d u o d e n a l a r t e r y and i l e o c o l i c v e i n (b ) are compared. both g r o u p s were t a b l e 3. e f f e c t of c a t h e t e r s i n t h e g a s t r o d u o d e n a l a r t e r y and t h e i l e o c o l i c v e i n on t h y m i d i n e i n c o r p o r a t i o n i n t o l i v e r dna 24 h o u r s a f t e r p a r t i a l hepatectomy. i4c-thymidine o r 3h-thymidine was a d m i n i s t e r e d t h r o u g h a permanent c a t h e t e r emptying i n t o t h e p e r i t o n e a l c a v i t y . mean s t a n d a r d d e v i a t i o n . 1 i n j e c t i o n of i k i l l e d i m ? sd r a d i o n u c l i d e c a t h e t e r s i n sd % of 1 a 1 c a v i t y b 1 a2 b2 p e r i t o n e a l c a v i t y p e r i t o n e a 1 g a s t roduodena 1 a. i l e o c o l i c v . per i t one: 1 c a v i t y p e r i t o n e a l c a v i t y g a s t r o d u o d e n a l a. i l e o c o l i c v . 23 23 24 24 radio n u c l i d e (numb er of r a t s ) ( 5 r a t s ) 4c-thymidine 'c-thymidine ( 3 r a t s ) 3h-thymid i n e (12 r a t s ) 3ht hymid i n e (7 r a t s ) 25 4.17 * 0.72* 17 25 2 . 3 8 0.49* 2 1 25 2.82 * 0.68** 24 25 1.75 0.86** 49 hours a f t e r p a r t i a l cpm p e r mg dna p e r hepatectomy i n j e c t e d cpm * the d i f f e r e n c e i s s i g n i f i c a n t 0 . 0 1 > p > 0.002 i 1 i 1 1 1 ** 1 1 0.02 > p > 0 . 0 1 g i v e n 14c-thymidine t h r o u g h t h e i n t r a p e r i t o n e a l c a t h e t e r . the e x p e r i m e n t was r e p e a t e d i n two similar g r o u p s , a2 a n d b2, g i v e n 3h-thymidine ( t a b l e 3 ) . rats w i t h i n t r a v a s c u l a r c a t h e t e r s had a l a r g e r v a r i a t i o n i n t h y m i d i n e i n c o r p o r a t i o n i n t o dna of t h e r e g e n e r a t i n g l i v e r , and l e s s i n c o r p o r a t i o n compared t o a n i m a l s 37 t a b l e 4. v a r i a t i o n i n dna l a b e l l i n g i n g r o u p s of r a t s and i n i n d i v i d u a l r a t s . 14c-thymidine w a s a d m i n i s t e r e d 23 h o u r s and 3h-thymidine 24 h o u r s p o s t o p e r a t i v e l y a n d t h e r a t s were k i l l e d one hour l a t e r . cpm p e r mg dna p e r i n j e c t e d cpm ' 4 c 3h mean sd mean sd + 2.19 0.66 2.10 -+ 0.90 p e r i t o n e a l p e r i t o n e a l ly.;s) /cavity 3 h / ' 4 c i n dna mean sd 1 . 0 3 0.16 p e r i t o n e a l p e r i t o n e a l c a v i t y c a v i t y g a s t roduodenal a r t e r y i l e o c o l i c ( 3 r a t s ) i l e o c o l i c i l e o c o l i c i e i n e i n ( 8 r a t s ) v e i n 2 . 1 3 1 . 7 9 1 . 4 6 0.56 cpm p e r mg dna p e r i n j e c t e d cpm 2) 4 ) 0 . 4 3 0.13 + 1) 0.79 0.49 0.29 0.04 0.36 _t 0.01 3) m ~ s d 4.17?0.72 2.38+0.49 2.19?0.66 sd : of m 1 7 2 1 30 3h m%d 3.08:o. 7c l . 8 s 2 0 . 5 4 ? . l o + o . 90 sd : of m 23 29 4 3 t a b l e 5. e f f e c t of a d d i t i o n of "coldll t h y m i d i n e on i n c o r p o r a t i o n 3 h / ' 4 c i n dna i n e a c h r a t of i 1 1 6 1 6 of r a d i o a c t i v e thymidine i n r e g e n e r a t i n g rat l i v e r . a l l i n j e c t i o n s were made t h r o u g h a c a t h e t e r i n t h e i l e o c o l i c v e i n . 0.001 o r 0.02 mnol l ' c o l d l l thymidine w a s mixed w i t h t h e i n j e c t e d 3h-thymidine. i4c-thymidine was adminis t e r e d 23 h o u r s and 3h-thymidine w i t h c o l d thymidine 24 h o u r s p o s t o p e r a t i v e l y and t h e r a t s were k i l l e d one hour l a t e r . "cold" thymidine mn01/100 1.11 a (8 r a t s ) b 0.001 (2 r a t s ) c 0.2 ( 2 r a t s ) . ~~ .~ ~~~~ ~ 1) d i f f e r e n c e a s compared t o 3 h a c t i v i t y i n group a i s n o t s i g n i f i c a n t . 0 . 1 > p > 0.05 ( s t u d e n t ' s t t e s t ) 0.05 > p > 0.02 1 1 i 1 11 i ' 3h/14c i n group a i s s i g n i f i c a n t . 1 1 i 1 i 1 3 h a c t i v i t y i n g r o u p a i s s i g n i f i c a n t . 1 1 1 1 1 1 3h/14c i n group a i s s i g n i f i c a n t . 2) 3) 4) 0.002 > p > 0.001 0.001 > p. 38 w i t h o u t i n t r a v a s c u l a r c a t h e t e r s . the s p e c i f i c a c t i v i t y of dna w a s lower a f t e r i n t r a v a s c u l a r a d m i n i s t r a t i o n of l a b e l l e d thymidine t h a n a f t e r i n t r a p e r i t o n e a l a d m i n i s t r a t i o n i n a n i m a l s w i t h o u t i n t r a v a s c u l a r c a t h e t e r s ( t a b l e 4 ) . the s t a n d a r d d e v i a t i o n of t h e dna s p e c i f i c a c t i v i t y i n t h e d i f f e r e n t groups of a n i m a l s w a s 17-43% of t h e mean, whereas t h e s t a n d a r d d e v i a t i o n of t h e h/ c of dna i n e a c h a n i m a l was 1-16% of t h e mean. thus, w i t h a p r e t r e a t m e n t i n j e c t i o n of l a b e l l e d thymidine t h e l e v e l of i n c o r p o r a t i o n i n t o dna f o r e a c h a n i m a l c o u l d be d e t e r m i n e d . 3 1 4 the e f f e c t of a n i n t e r f e r i n g s u b s t a n c e c o u l d b e d e m o n s t r a t e d a t t h e t i m e of t h e second i n j e c t i o n of l a b e l l e d thymidine ( t a b l e 5 ) . the a d d i t i o n of " c o l d " thymidine t o t h e i n j e c t e d 3h-thymidine d e c r e a s e d t h e i n c o r p o r a t i o n of l a b e l i n t o dna i n agreement w i t h known m e t a b o l i c e f f e c t s ( 6 , l l ) . discuss i o n the e x p e r i m e n t a l u s e of t h e c o n t i n u o u s l y p e r f u s e d r a t l i v e r i n v i v o h a s been p r e v i o u s l y d e s c r i b e d ( 1 2 ) . i n t h i s s t u d y w e have shown t h a t t h e p o r t a l and t h e a r t e r i a l systems c a n be c o n c o m i t a n t l y p e r f u s e d i n r a t s i n c o m b i n a t i o n w i t h p a r t i a l hepatectomy. p a r t i a l hepatectomy s t i m u l a t e s t h y m i d i n e p h o s p h o r y l a t i o n and dna s y n t h e s i s i n l i v e r c e l l s . thus, t h e e x p e r i m e n t a l s y s t e m d e s c r i b e d i s of i n t e r e s t f o r i n v e s t i g a t i o n s of c e l l k i n e t i c s . because of t h e d u a l blood f l o w t h e l i v e r i s more c o m p l i c a t e d t h a n o t h e r models. it o f f e r s , however, i n t e r e s t i n g p o s s i b i l i t i e s i n t h e s t u d y of e f f e c t s of blood f l o w r e s t r i c t i o n and compensa t o r y mechanisms. i n t h e p r e s e n t s t u d y , i n f u s i o n was i n t e r r u p t e d 36 h o u r s p o s t o p e r a t i v e l y b u t i t c o u l d have b e e n c o n t i n u e d w i t h o u t d i f f i c u l t y . a t l a t e r times f o l l o w i n g p a r t i a l hepatectomy, however, o p e r a t e d r a t s seem t o b e no more s e n s i t i v e t o t h e e x p e r i m e n t a l s i t u a t i o n t h a n are o r d i n a r y r a t s , which t o l e r a t e i n f u s i o n q u i t e w e l l ( 1 2 ) . a p r o l o n g e d a n d c o m p l i c a t e d o p e r a t i v e p r o c e d u r e a n d / o r t h e p r e s e n c e of c a t h e t e r s a f f e c t e d body and l i v e r weight a s w e l l a s l i v e r c e l l meta b o l i s m . l i v e r dna s y n t h e s i s was d e c r e a s e d o r d e l a y e d and t h e v a r i a t i o n b e t ween a n i m a l s i n c r e a s e d . these d i f f e r e n c e s show t h a t i t i s e s s e n t i a l t o u s e c a t h e t e r i z e d r a t s g i v e n sham i n j e c t i o n s a s c o n t r o l s . i n t h i s s t u d y t h e incon s i s t a n c y of i n d i v i d u a l v a r i a t i o n w a s d e c r e a s e d when a n i m a l s were used as t h e i r own c o n t r o l s u s i n g t h e d o u b l e l a b e l l i n g t e c h n i q u e . the c h o i c e of l a b e l l e d t h y m i d i n e i n t h i s s e r i e s of e x p e r i m e n t s was r e l a t e d t o t h e v a s t documentation of t h e f a t e of t h i s p r e c u r s o r i n t h e r e g e n e r a t i n g l i v e r ( 1 3 ) . it i s w e l l known t h a t t h e d i u r n a l rhythm of t h e r a t i s r e f l e c t e d i n l i v e r m e t a b o l i s m d u r i n g t h e r e g e n e r a t i v e p r o c e s s (1) and t h a t a g e and stress f a c t o r s i n f l u e n c e dna s y n t h e s i s ( 3 ) . we c o u l d p o s s i b l y have improved t h e synchrony o f l i v e r r e g e n e r a t i o n by u s i n g a r e g u l a r f e e d i n g s c h e d u l e (17) t h u s d e c r e a s i n g v a r i a t i o n i n t h y m i d i n e i n c o r p o r a t i o n between a n i m a l s . we do n o t 39 t h i n k , however, t h a t t h e v a r i a t i o n between t h e r a t s due t o c a t h e t e r i z a t i o n would b e d e c r e a s e d s i g n i f i c a n t l y . t h e r e f o r e , t h e d o u b l e l a b e l l i n g method s h o u l d b e a v a l u a b l e t o o l i n improving t h e s i g n i f i c a n c e of t e s t r e s u l t s . it i s p o s s i b l e t h a t t h e i n t e r v a l between i n j e c t i o n s c a n be p r o l o n g e d , b u t b e c a u s e of d i u r n a l v a r i a t i o n t h i s must b e i n v e s t i g a t e d f u r t h e r . high c o n c e n t r a t i o n s of "cold" t h y m i d i n e i n j e c t e d w i t h 3h-thymidine d i l u t e s t h e r a d i o a c t i v e m o l e c u l e s r e s u l t i n g i n d e c r e a s e d l a b e l l i n g o f dna ( 1 1 ) . thymidine a l s o d i s t u r b s d e o x y n u c l e o t i d e metabolism and n u c l e i c a c i d s y n t h e s i s r e s u l t i n g i n growth i n h i b i t i o n (6,111. the n e g a t i v e e f f e c t of added t h y m i d i n e on t h e i n c o r p o r a t i o n of 3h-thymidine i n t o dna was obvious i n t h i s s t u d y . we c o n s i d e r dna e s p e c i a l l y w e l l s i u t e d f o r t h e d e t e r m i n a t i o n of f i n a l r e s u l t s of m e t a b o l i c a l t e r a t i o n s b e c a u s e of i t s v e r y low t u r n o v e r . other compounds w i t h a low t u r n o v e r s u c h a s c e r t a i n rnas and p r o t e i n s s h o u l d a l s o b e u s e f u l . acknowledgements we t h a n k mrs i n g e g e r d k a r l s s o n , dept of s u r g e r y , u n i v e r s i t y h o s p i t a l a t uppsala f o r v a l u a b l e a s s i s t a n c e a t o p e r a t i v e work and mrs l i l l i a n n l s l u n d , department of p h y s i c a l b i o l o g y , the gustaf werner i n s t i t u t e , f o r c a r e f u l b i o c h e m i c a l a n a l y s e s . references 1. 2 . 3. 4 . 5 . 6. 7. 8. 9. 1 0 . 11. 12. 40 barbason, h. & c a n t f o r t , j . : n y c t o h e m a l v a r i a t i o n s o f t i s s u l a r and divi s i o n f u n c t i o n 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symptomatic colloid cysts in the third ventricle of monozygotic twins elisabeth ronne-engström & edith popek to cite this article: elisabeth ronne-engström & edith popek (2015) symptomatic colloid cysts in the third ventricle of monozygotic twins, upsala journal of medical sciences, 120:1, 59-62, doi: 10.3109/03009734.2014.988309 to link to this article: https://doi.org/10.3109/03009734.2014.988309 © informa healthcare published online: 10 dec 2014. submit your article to this journal article views: 436 view related articles view crossmark data citing articles: 2 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2014.988309 https://doi.org/10.3109/03009734.2014.988309 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2014.988309 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2014.988309 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2014.988309&domain=pdf&date_stamp=2014-12-10 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2014.988309&domain=pdf&date_stamp=2014-12-10 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2014.988309#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2014.988309#tabmodule upsala journal of medical sciences. 2015; 120: 59–62 case report from uppsala university hospital symptomatic colloid cysts in the third ventricle of monozygotic twins elisabeth ronne-engström1 & edith popek2 1department of neuroscience, section of neurosurgery, uppsala university, uppsala, sweden, and 2department of neurology, örebro university hospital, örebro, sweden abstract in this case report we describe colloid cysts in the third ventricles of monozygotic twin sisters. they were 26 years old when their condition was discovered. one woman was admitted to us on an emergency basis, with signs of high intracranial pressure such as unconsciousness and extension posturing. her sister was also brought to the hospital since she had a history of attacks of headache. they were both operated with removal of the colloid cysts, and the clinical courses are described in the case report. in reviewing the literature another 30 familial cases were found. of these were two pairs of monozygotic and one pair of dizygotic twins. key words: intraventricular colloid cyst, twins introduction colloid cysts in the third ventricle are of uncertain origin and occur in 1% of intracranial tumours. there have been previous reports on the familial occurrence of this condition. we were able to identify 30 published familial cases. of these there were two pairs of monozygotic and one pair of dizygotic twins, the remaining being either parent–child or non-twin sibling pairs. the first case of monozygotic twin boys (1) had parents who were cousins. the boys had other malformations as well, including cataracts, retinal detachment, squint, and amblyopia. it was suggested that the cysts were a part of a diencephalic congenital malformation. one boy was symptomatic and was operated, while the other was followed. the second case of monozygotic twins was again two boys, one with symptoms and the other without. in the case of the dizygotic twin boys, both eventually developed symptoms, but more than a decade apart. we can now report a third case involving monozygotic twins, in this case two sisters, admitted to our clinic when 26 years old. both were operated for symptomatic colloid cysts in the third ventricle within the same week. they also have a younger sister, and her mri was found to be normal. the twin sisters gave their permission for publication of this case report. case report the sisters were born full-term after a normal pregnancy and without previous medical problems. sister a sister a was admitted to us on an emergency basis. she had displayed some attacks that were initially interpreted as panic disorders. in the weeks before admission she had increasing problems with headache and neck pain, but she did not seek medical attention. on the day before admission she had a severe headache. her boyfriend reported that she had attacks approximately every hour, during which she became unconscious and had spasms of the arms and legs. after these spasms she woke up and vomited. the following day they went to the emergency room. a ct correspondence: elisabeth ronne-engström, department of neurosurgery, uppsala university hospital, 751 85 uppsala, sweden. fax: +46 18 55 86 17. e-mail: elisabeth.ronne.engstrom@akademiska.se (received 24 october 2014; accepted 11 november 2014) issn 0300-9734 print/issn 2000-1967 online � 2014 informa healthcare doi: 10.3109/03009734.2014.988309 http://informahealthcare.com/journal/ups mailto:elisabeth.ronne.engstrom@akademiska.se scan was done and a contrast-enhancing cyst in the third ventricle was found (figure 1a). there was also a pronounced obstructive hydrocephalus. it was decided to transfer the patient immediately to the department of neurosurgery, uppsala university hospital. during the preparations for this she deteriorated again, became unconscious and displayed extension posturing. she also had bradycardia and arrhythmia. her pupils dilated but still showed some slow reaction to light. she was intubated and transferred with intensive care helicopter to us, a 45-min flight. on arrival to uppsala she was withdrawing from painful stimuli. she went directly from the helicopter to the neurosurgical operating room and received a ventricular drain. she was treated with csf drainage. further investigations were done with mri, supporting the diagnosis of an intraventricular colloid cyst. she was operated one week later, when we felt she had recovered sufficiently from the emergency event. surgery was carried out through a right-sided frontal craniotomy with a transcortical/transventricular approach to the cyst. histopathology showed a single-layered epithelial cyst. surgery was uneventful from a neurosurgical perspective, and she woke up soon afterwards. however, she contracted pneumonia and had to be reintubated. she also developed suspected meningitis, but bacterial growth could not be verified. the treatment of pneumonia and suspected meningitis was complicated by a severe adverse drug reaction to antibiotics. she developed a skin rash followed by a hepatic reaction and finally a transient kidney failure. after the pneumonia had been treated and the meningitis found to be aseptic, she was treated with steroids, which made the symptoms of meningitis disappear. a ct scan before discharge showed bilateral infarctions in the territories of the posterior cerebral arteries. this was interpreted as caused by the impending herniation at the emergency event. on discharge to the home-town hospital one month later she was awake, but had a tracheostomy and was still on haemodialysis. since then she has gradually improved, and her kidney function has improved. sister b the day after sister a had been admitted we got to know that she had a twin sister under investigation for severe headache. contact was made with her hometown hospital. they brought her in the same day for a ct scan. this showed a large contrast-enhancing cyst in the third ventricle with a pronounced obstructive hydrocephalus (figure 1b). she was in good clinical condition otherwise, but was transferred to us on an emergency basis with intensive care helicopter. she told us that she had suffered from headache attacks for a couple of years. these attacks were associated with blurred vision that lasted for only a short time. the headache was interpreted as migraine. the last year she had sometimes fainted in association with these attacks, and other persons had reported that she then had spasms in her arms. after arrival to us she was further investigated with mri and was then operated, three days before her sister. surgery was carried out through a right-sided frontal craniotomy and a transcortical/transventricular approach to the cyst. histopathology showed a benign cyst with a single-layered epithelium, who grade i. the postoperative course was uneventful, and she soon recovered fully. discussion including these monozygotic twin sisters, 32 cases of familial colloid cysts in the third ventricle have been reported (1-14): two previous reports of monozygotic twin brothers (1,9) and one pair of dizygotic twin brothers (14), and the remaining cases are siblings or parent–child. one-third of all twins are monozygotic, according to the swedish twin register (15). if we include the now published cases, 3 out of 4 twin couples with colloid cysts were monozygotic, which is more than would be expected based on the natural prevalence (p = 0.11). this suggests that there may be a genetic component in the development of the cysts. a b figure 1. a: ct scan of sister a, carried out when she went to the emergency room. there is a contrast-enhancing lesion in the third ventricle in the region of foramen monroi. also, there is a pronounced obstructive hydrocephalus with dilatation of the lateral ventricles. 201 � 201 mm (72 � 72 dpi). b: ct scan of sister b, carried out when we got to know that she was under investigation for severe headache attacks. as in her twin sister, there is a contrastenhancing lesion in the third ventricle in the region of foramen monroi. there is a pronounced obstructive hydrocephalus with dilatation of the lateral ventricles as well. 201 � 201 mm (72 � 72 dpi). 60 e. ronne-engström & e. popek however, the genetic background for the familial colloid cysts is far from clear, and autosomal dominant as well as recessive genes have been suggested (9,11). we observed that 18 of 32 (56%) of the known familial cases were women. this is in contrast with many published series that show a male dominance (16). it is possible that the familial colloid cysts are of another nature than the single-appearing cysts. also, the mean age for the cases becoming symptomatic in the familial cases seems to differ between men (mean 41 ± 21 years) and women (mean 35 ± 15 years). the natural course of the cysts has been studied, and it seems that patients with asymptomatic colloid cysts can safely be followed with serial neuroimaging (17). neurosurgical intervention is necessary, however, if a patient shows symptoms, the cyst enlarges, or hydrocephalus develops (18). in that study the overall mortality of patients with symptoms was 12% during the 5-year follow up. it is surprising that our patients had severe and life-threatening symptoms for quite a long time before they had a ct scan done. ct scans on all patients with headache give a low yield, with only 22 significant findings and 1 colloid cyst in 1876 patients (19). still, there must be a point when a physician has to rethink the postulated diagnosis. the combination of headache attacks with unconsciousness and blurred vision should lead to an immediate ct scan, and only when intracranial conditions have been proved normal can the headache investigation continue as before. the complication with aseptic meningitis after cyst extirpation is a recognized clinical problem but only occasionally described. wang and colleagues (20) reported a case with aseptic meningitis after extirpation of a colloid cyst in the fourth ventricle. their cyst was adherent to the brain stem and could not be completely removed. the patient developed fever and an elevated csf leukocyte count, but the microbiological examination was negative. the symptoms disappeared after steroid treatment had been initiated. we had a similar experience. our patient developed pneumonia together with signs of meningitis. once the pneumonia was treated and it was established that the meningitis was aseptic, treatment with steroids was started and the fever quickly disappeared. the pathophysiology of the aseptic reaction is unknown, but it is assumed that an inflammatory reaction to the cyst content is involved. surgical treatment of these cysts is a matter frequently discussed. transcortical (21) or transcallosal (22,23) extirpation of the cyst are probably the mostused methods. they carry different risks, with epilepsy and memory deficits being the most severe, respectively. in recent years, less invasive techniques have been introduced, including endoscopic removal (24) as well as stereotactic aspiration (25) and cyst wall rupture (26). it still has to be proven though whether or not these innovations are preferable to micro-neurosurgical removal. endoscopic removal has been demonstrated to have fewer complications, but on the other hand more recurrence of the cysts (27). declaration of interest: the authors report no conflicts of 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http://www.ncbi.nlm.nih.gov/pubmed/12066904?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/12066904?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/12066904?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15606567?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15606567?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22405542?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22405542?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22405542?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/14530924?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/14530924?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/18295836?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/18295836?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/18295836?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7099406?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7099406?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7099406?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21150763?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21150763?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21150763?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22236766?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22236766?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/10420147?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/10420147?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17415197?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17415197?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17415197?dopt=abstract abstract introduction case report sister a sister b discussion declaration of interest references operating in an era of impact factor mania full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 operating in an era of impact factor mania arne andersson & joey lau börjesson to cite this article: arne andersson & joey lau börjesson (2015) operating in an era of impact factor mania, upsala journal of medical sciences, 120:2, 124-131 to link to this article: https://doi.org/10.3109/03009734.2015.1034899 © informa healthcare published online: 15 apr 2015. submit your article to this journal article views: 441 view related articles view crossmark data citing articles: 6 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://doi.org/10.3109/03009734.2015.1034899 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1034899 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1034899 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1034899&domain=pdf&date_stamp=2015-04-15 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1034899&domain=pdf&date_stamp=2015-04-15 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1034899#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1034899#tabmodule upsala journal of medical sciences. 2015; 120: 124–131 editorial operating in an era of impact factor mania arne andersson & joey lau börjesson department of medical cell biology and department of medical sciences, uppsala university, uppsala, sweden introduction when one of us (a.a.) served as chairman of our society (upsala läkareförening) for a 3-year period some 15 years ago, one of the most challenging tasks was to find a reasonable and fruitful handling strategy for our journal, which at that time was 135 years old. actually the question, expressed by a task force (gunnar ronquist, editor; bengt westermark, vice rector; arne andersson, chairman) in an editorial on the subject (1), was: should the journal be kept alive or should we let it die? actually, we asked our colleagues to go on ‘investigating, writing, and publishing’ and preferably in the upsala journal of medical sciences because ‘we don’t intend to perish’. tools in that survival strategy included first of all starting electronic publishing. we also tried to stimulate different award winners to write reviews on their research for our journal. one more important move was to introduce special issues on comprehensive research with extramural classes at our faculty. the first one of that sort was that on ‘diabetes research in uppsala’ with claes hellerström serving as guest editor. after some years we realized that electronic publishing also had to include the initial manuscript handling. that led us to sign up with a highly experienced publisher, informa healthcare, to run a scholarone-based manuscript centre with standardized digital procedures for manuscript submission, peer review, editorial decisions, manuscript production, and the use of a very handy and informative web page. and all this at a cost that was in fact lower than we had paid previously. with this strategy shift, we faced a fascinating change 5 years later. the submission rate of new manuscripts increased by a factor of 10, and we immediately decided to go from a release of three issues per year to one issue per quarter. very soon we also noticed that numbers of all sorts of citations increased. in order to enhance the attraction of the journal—by that we meant attracting manuscripts from teams with highly visible research—we had to enter the field of ‘impact factor mania’ (2), or ‘impactitis’ (3) as some people prefer to call it. thus, when discussing these issues with potential submitters of manuscripts to our journal the most common comment we heard was that ‘your impact factor (if) value is too low’. and basically they were right. at that time our if values were well below 0.50. this article will present figures for various aspects of the daily running of a journal of this kind and size. comparisons have also been made with other, mainly uppsala-based, journals to see how different handling strategies affect the performance figures of those particular journals. by using such information to make improvements to our journal management, we hope to increase further the impact of our esteemed publication, which is 150 years old this year. journal report one very useful piece of information in scholarly publishing is the publisher’s annual report with statistics on more or less every aspect of journal management. we have extracted some basic data (figure 1) from the latest report of november 2014 and added some internal data. in times when almost every experienced scientist receives a couple correspondence: arne andersson, department of medical cell biology, uppsala university, po box 571, se-75123, uppsala, sweden. e-mail: arne.andersson@mcb.uu.se (received 22 march 2015; accepted 24 march 2015) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1034899 http://informahealthcare.com/journal/ups mailto:arne.andersson@mcb.uu.se of invitations each week to publish their latest articles in different new, obscure journals, we are glad to report a fairly constant and high inflow of new submissions—some 250–300 on an annual basis. authors from a total of 41 different countries submitted their papers last year, documenting that our journal indeed is a true international publication. besides contributions from sweden, there are very many from turkey and china as well. one understands previous predictions that by the year 2020 every fifth biomedical publication will be written by chinese authors. the high figure for manuscripts from turkey most certainly reflects the fact that there are some 60 medical faculties in that country. the backbone of scholarly publishing is the use of classical peer review for selection of papers for publication. the system, though, has limitations, one of which is that of convincing suitable people to contribute. the editor’s responsibility is then to avoid the use of reviewers for decisions that appear obvious at a quick glance. in our hands, that would mean that about half of the submitted papers would be rejected without a traditional review using two referees (figure 1). to that should be added a handful of papers that never manage to pass the ‘scholarone’ filter checking that authors have followed the instructions for authors reasonably well. about 20% of the submitted papers have been sent out for classical review. some papers—last year about 10%—were accepted as submitted, figure 1. data from journal report presented by the publisher concerning submitting countries, rejection rate, handling strategies, and lead times for upsala journal of medical sciences. operating in an era of impact factor mania 125 but it has to be kept in mind that a great majority of them were invited papers. all in all, it means that the official rejection rate of our journal is fairly high—about 80%—but the figure is, for different reasons, a bit shaky. one reason for the high number of rejected papers, of course, is that many of them have faults of different kinds. perhaps less experienced and trained scientists have chosen our journal since we do not charge authors any submission fees despite the fact that we publish under the premises of open access. in order to keep authors happy we strive for quick decisions and information. therefore, it is with considerable satisfaction that we can report on fairly short lead times—2 weeks to the first decision and 3 weeks to the final decision (figure 1). one more advantage with a well-designed web page for a scholarly journal, and especially an open access journal, is that numbers of downloads of separate articles can be followed in great detail. it is not surprising to see that the most downloaded papers are quite newly released. thus, when viewing download figures for our journal during 2014 we found that 5 out of 10 most downloaded papers had been published the same year (figure 2a). one of them (4) was published as late as in the third issue of that 2014 volume. one more token of the immediate a b figure 2. a: most downloaded articles 2014 from the web page of upsala journal of medical sciences. b: downloading countries the same year. 126 andersson & lau börjesson interest for this article among the general public was that it was the subject of one of the ‘sunday scientific reports’ of our local newspaper authored by åke spross. there is, however, one remarkable exception from the assumption that much-downloaded papers also become the most cited ones, and that is a more than 30-year-old italian study (5). for several years this almost non-cited publication (according to thomson reuters, there are a total of two citations of this paper) has been number one in our top download tables. it is tempting to speculate that reasons other than pure interest in the scientific content of the report have brought about the downloads. such events have been designated ‘crawlers’, and it has been claimed that it should be possible to correct for this type of contamination of these attention-based figures, making them more useful for qualitative assessments of scientific publications. it is also interesting and stimulating to see that those who most frequently download our articles are us citizens (figure 2b). citations when eugene garfield some 60 years ago first launched his idea of a citation index for scientific publications he probably could not have dreamt what an enormous impact such instruments would have on scholarly publishing. garfield himself has summarized experiences and feelings on the use of the journal impact factor (6), and that article was concluded with a citation from a paper by hoeffel (7). the last sentence of that citation states that ‘the use of impact factor as a measure of quality is widespread because it fits well with the opinion we have in each field of the best journals in our speciality’. memories from the early days of research education for one of us (a.a.) reveal that there was an informal journal ranking system ‘built in the walls’ of the laboratory. some journals were regarded as esteemed, and others were extremely exclusive and in practice impossible even to think of as possible candidates for submission. problems come when using the journal impact factor for evaluations of individual scientists and in particular their specific publications. so, regardless of all conflicting opinions, we have to take notice of our own impact figures and perhaps also adapt the journal operation to be compatible with the maintenance of honourable measures. we have had the pleasure of reporting increasing if figures more or less every year since 2003 (figure 3). the first critical level to pass was that of 1.0—a value of great importance in our own internal faculty budget formula. in 2011 that event could be celebrated, and as a token of appreciation we served champagne at our annual editorial board meeting that year. looking back we can see a 10-fold increase of the impact factor over the last 10 years. we are at present approaching the 2.0 level, which was previously regarded as the lowest figure for ‘esteemed journals’ as defined by the swedish funding agencies. one objection that should be kept in mind when using the impact factor instrument is that the whole business is run by a commercial, private company. the influence of different scientific organizations on how to run it is probably very small. our own experience is, however, that thomson reuters are keen and willing to help when you suggest a correction due to inaccuracies in their databases (missing issues, invalid citations, etc.). it is anyhow of interest to direct some attention to other databases as well. among the few that exist, the scopus database run figure 3. impact factor development during a 10-year period for upsala journal of medical sciences. operating in an era of impact factor mania 127 by elsevier has been available for some years. as we pointed out in a recent editorial on newly released impact figures (8), a major problem with them is that knowledge on how these figures have been calculated is meagre. nevertheless, we found that there was a parallelism in the increase of our impact factor and the scimago journal rank figure. another parameter reported in scopus is the so-called snip factor. snip stands for source normalized impact per paper. this indicator measures the average citation impact of the articles of a journal. it has been calculated by leiden university’s centre for science and technology studies (cwts) based on the scopus bibliographic database produced by elsevier. unlike the well-known journal impact factor, snip corrects for differences in citation practices between scientific fields, thereby allowing for more accurate between-field comparisons of citation impact. as with the sci figure there is a brisk increase of this factor for our journal as well—a 2–3-fold increase over the last 5 years (figure 4). three other uppsala-based journals—acta oncologica, acta dermatovenereologica, and amyloid—display more constant figures. one more piece of information that can be extracted from the scopus database is the percentage of not-cited papers for different journals. we have recently pointed out (8) that substantial numbers (about one-third) of the total number of publications in one of the most esteemed journals (new england journal of medicine) were not cited at all. according to eugene garfield, out of 38 million items cited from 1900 to 2005 half were not cited at all and only 0.5% more than 200 times (6). we are very pleased to find that our publications are cited to a very high extent—only between 10% and 20% have not been cited 3–10 years after publication (8). one more issue of interest in this context is to what extent self-citations have been included in figures of this type. in the traditional measures released in late july from thomson reuters of journal impact factor figures selfcitations are included, but for each journal there is information on their self-citation rates. of our total cites 3% were self-cites, a figure that is fairly common amongst journals in our subject category—medicine, general and internal. whether the editor’s possible use of so-called coercive citations (forced citations of articles in their own journals) influences these figures to any major extent is difficult to judge. there is, however, evidence to suggest that journals have been excluded from these ranking lists due to proven use of this behaviour. we are happy to say that, to the best of our knowledge, we do not apply this practice. impact of publication characteristics on citation figures when editors talk to scientists about problems with low impact figures the answer has always been—go for more reviews. when the diabetologia editorial office moved to claes hellerström and his group in uppsala in the mid1980s, we were told by the publisher that the number of original articles should be reduced as much as possible and be replaced by reviews. the idea was to increase the impact factor to the level of its american counterpart. however, it is not that simple, so we decided to look at citation figures for different types of articles in upsala journal of medical sciences. we then categorized all articles published in our journal for a 5-year period with regard to type of article, research subject, and also number of authors. out of 240 published items, 26 were denominated reviews, and the average citation frequency of these articles was more than 11 over this 5-year period (figure 5). figure 4. source normalized impact per paper (snip) for four different uppsala-based biomedical journals: red circles = amyloid; green triangles = acta oncologica; yellow triangles = acta dermato-venereologica; black circles = upsala journal of medical sciences. 128 andersson & lau börjesson this is well above the corresponding figure for original articles, i.e. 3.8 citations, and it supports previous assumptions on this matter. to become a highly cited review it is, however, quite evident that a review has to be written by a very distinguished person in the field on a topic of great relevance for the moment. quite evidently, also in conformity with usual statements, case reports are very seldom cited. when remembering that letters are as much cited without burdening the denominator of the impact factor, it is not surprising to see this category is increasing at the expense of classical case reports. in our journal we, however, intend to keep the case report window open for cases from our own hospital. we also tried to figure out as to whether the number of authors on the papers to any extent influenced the citation figures. there were no obvious differences with regard to this article characteristic (figure 6), but it is worthy of note that the highest figure was found for single-author papers. most probably that reflects the fact that many of these articles were reviews. we also looked for differences with regard to the subject of the article. four categories of research were delineated. some articles covered more than one subject area, and therefore not all articles published during this timespan have been included. no great differences were found between the different subject areas (figure 7). somewhat surprisingly, articles dealing with social medicine aspects had the highest citation figures. perhaps one explanation for this is the fact that our most cited papers had to be excluded because they were reviews with a broad scope of topics. impact of open access when we decided to go for a professional publisher and to use an electronic manuscript centre we were quite clear about using open access for our performance. in combination with the scanning of our volume archive in order to give everyone the opportunity to read all our recent articles for free on the web page of the journal, the open access feature of our journal most certainly worked as very efficient advertising for the existence of our journal. when we also refrained from charging authors any kind of publication fee it was quite logical that papers started to flood our inbox. but basically we were an open access journal even before being published by informa. it was therefore of figure 5. impact of article type on number of citations. figure 6. impact of number of authors on number of citations. figure 7. impact of research subject on number of citations. operating in an era of impact factor mania 129 interest to see whether citation figures for other journals had been influenced over the years by the use of open access. in a very ‘non-scientific’ way we looked at the development of the impact figure over a 5-year period (2009–2013) of 10 journals of interest that have been open access all the time and 10 other journals being nonopen access over the same time period (figure 8). there was a small difference between the two types of journals. open access journals tended to increase their impact factors more than non-open access ones. besides the selection bias in our study groups, open access journals are most often ‘younger’ and thus still in a phase of natural growth. we are quite convinced that we have benefited from the openness of our journal. however, it then has to be kept in mind that we are privileged in the sense that we are owned and run by a non-profit society. how to proceed from these observations? as can be seen from this presentation, editors live in busy times at present. new journals pop up like mushrooms, and the existence of predatory journals makes it necessary to explain to everyone that our task is not to create wealth or make shareholders happy. on top of that, there seems to be a kind of a war out there where an endless number of editors/journals are offering you space for your scientific articles. remember the times when you spent hours and days trying to figure out where to submit your paper that had been rejected on the grounds of being too conventional but otherwise sound. figure 8. impact factors from journal citation reports at two different occasions (2009 and 2013) have been given and differences between the two were determined. means for the two categories, open access or non-open access journals, were calculated. 130 andersson & lau börjesson our journal operates from a very solid platform. one hundred and fifty years of scholarly publishing means that we are not ephemeral and planning should be long-term. we will go on printing the quarterly issues of the journal for the sake of the members of the society. in order to keep up the good reputation of the journal and perhaps also the curiosity of potential submitters, we have to be aware of the importance of the impact factor. there are no selfplaying pianos! inclusion of contributions as before from prize winners in the faculty and also an annual special issue will meet the need for reviews. it would also be nice to publish more editorials and commentaries. however, the major bulk of publications still has to be original articles. finding out what is new knowledge which can be of great benefit to the scientific community is the task of the whole editorial staff. seeing the number of citations for such papers escalating is probably the best reward for the editor. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. andersson a, ronquist g, westermark b. on the revival of an old and venerable journal. ups j med sci. 2000;105:3–4. 2. casadevall a, fang fc. causes for the persistence of impact factor mania. mbio. 2014;5:e00064–14. 3. van dienst pj, holzel h, burnett d, crockner j. impactitis: new cures for an old disease. j clin pathol. 2001;54:817–19. 4. westermark p, westermark g, suhr ob, berg s. transthyretin-derived amyloidosis: probably a common cause of lumbar spinal stenosis. ups j med sci. 2014;119:223–8. 5. baroldi g. pathophysiology of acute myocardial infarction. ups j med sci. 1983;88:159–68. 6. garfield e. the history and meaning of the journal impact factor. jama. 2006;295:90–3. 7. hoeffel c. journal impact factors. allergy. 1998;53:1225. 8. andersson a. further improvements of our journal performance figures. ups j med sci. 2014;119:295–7. operating in an era of impact factor mania 131 http://www.ncbi.nlm.nih.gov/pubmed/24643863?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24620715?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/6675233?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/16391221?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/9930604?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/25231005?dopt=abstract introduction journal report citations impact of publication characteristics on citation figures impact of open access how to proceed from these observations? doi18 references is progress in clinical reproductive medicine happening fast enough? editorial is progress in clinical reproductive medicine happening fast enough? more than 8 million babies have been born from in vitro fertilization (ivf) around the world since louise brown’s birth in 1978. the number of ivf treatment cycles has globally increased to more than 2 million per year (1). in europe, treatment numbers lately grew by an estimated 7% per year, with denmark and belgium at the top of the league table, each offering more than 2500 ivf treatment cycles per million people (2). assisted reproduction has arrived to take its place within mainstream medicine. infertility has also lost its social stigma: a quick google search on the keywords infertility & support and infertility & stigma yields 119 million and 2.3 million results, respectively, a 51:1 ratio in favour of support. important milestones in assisted human reproduction were the cryopreservation of embryos (1984) and oocytes (2003), the introduction of icsi (1992), the use of testicular sperm (1993), preimplantation genetic testing (1989), ovarian tissue transplantation (2000), uterus transplantation (2015) and ‘three-parent babies’ after mitochondrial transfer (2016). these techniques, together with the availability, on a global scale, of gamete donation and gestational surrogacy, provide a wide range of treatment options for people faced with subfertility or infertility. the chance of having a full-term, normal birth weight and singleton live birth per ivf cycle using fresh embryos from non-donor eggs for women younger than 35, according to us figures from 2015, is 21.3% (3). in women 38–40 years of age, this number drops to 11.1% (3). for australia, a live birth rate of 23.3% for fresh autologous embryo transfers has recently been reported (4). of note, it is estimated that, for example, in denmark, only approximately 50% of infertile couples initiating ivf treatment will eventually have a baby within a 5-year span (5). ivf: a success story with some obvious limitations while the successes of modern assisted reproductive technology (art) are obvious, so are the limitations: a high treatment failure rate per cycle, widespread use of ivf on a purely empirical basis, a lack of management options in case of age-related ovarian depletion, medical risks associated with ovarian stimulation and oocyte retrieval, a high psychological burden associated with infertility itself but also with the treatment, high financial costs especially for couples not on reimbursement schemes, a high multiple pregnancy rate, insufficient knowledge on the long-term risks of some aspects of ivf treatment. new technologies are therefore constantly proposed to improve ivf outcomes (6). the list of currently offered, presumably beneficial adjuncts is seemingly endless: from pharmacological add-ons (dehydro-epiandrostenedione, growth hormone, testosterone, coenzyme q10, heparin, lowdose aspirin, vasodilators, myo-inositol, etc.) to laboratory technology (sperm dna fragmentation testing, sperm selection procedures, time-lapse embryo monitoring, preimplantation genetic screening, assisted hatching, endometrial injury, embryo adherence compounds, etc.) to the different approaches to regenerate, rejuvenate or reactivate germ cells in the human ovary (intra-ovarian injection of calcium gluconate-activated autologous platelet-rich plasma, the use of autologous mitochondrial transfer into oocytes, the generation of artificial gametes from putative ovarian stem cells or the in vitro activation of dormant follicles by hippo-signalling disruption, etc.). of note, all innovations to ivf treatment have to be judged from one perspective: will a new technology help more couples have a baby with less effort, less burden, less financial costs, and less parental and foetal risk? the recent history of reproductive medicine, however, indicates that very few, if any, new developments have been introduced into clinical practice after thorough and rigorous clinical validation. that is why, more than 20 years after the introduction of some of the technologies named above, there is still no clear picture of the clinical utility. rcts: how much have they really helped? it is commonly accepted that randomized controlled trials (rcts) are best suited to evaluate the effectiveness of interventions. when the outcome of interest is binary (in reproductive medicine it is, broadly speaking, ‘baby or no baby’), large sample sizes are necessary to detect clinically relevant effects with sufficient confidence. in a recent systematic review on rcts and meta-analyses published within the field of reproductive medicine (7), it was found that the vast majority of rcts are underpowered for this purpose. even meta-analyses, synthesizing multiple rcts, could not make up for this. within the cochrane library, not a single sufficiently powered rct could be identified, and only 2% of the meta-analyses were large enough to detect a difference of 5% in live birth rate (e.g. an increase from 25–30%) with a given intervention (7). moreover, researchers have so far been using differently assessed and defined outcomes, which contributes to the inability to compare and combine individual rcts (8). despite the widespread call for rcts in our field (9), the progress achieved through performing rcts as we did so far has been, taking a sober view at it, rather limited. upsala journal of medical sciences 2020, vol. 125, no. 2, 65–67 https://doi.org/10.1080/03009734.2020.1734991 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1734991&domain=pdf&date_stamp=2020-05-21 https://doi.org/10.1080/03009734.2020.1734991 http://www.tandfonline.com why is progress in ivf so slow? rcts are a powerful instrument, but collaborative efforts are needed for sufficiently large trials. while both collaboration and competition are essential for scientific progress, the ivf world appears to be occupied with competition on the economic side of art provision rather than the science of human reproduction. ivf is seeing industrialization of medical services at rapid speed and large scale and conglomeration of individual treatment centres into large networks, often under one brand name and often funded by private equity. the necessity of constant economic growth under such circumstances necessitates competition for patients in need of services to solve their infertility problems or expanding existing treatments to newly identified groups of patients (‘social freezing’ being a good example of the latter). patients use social media and the internet as a knowledge resource, but the information available freely on the net is often of unclear validity or biased outright by commercial interest. doctors try to advertise their services on the internet either openly (where legally permissible) or hidden within press releases about putative novel techniques and treatment breakthroughs. all these driving forces tempt researchers and practicing clinicians to jump on the moving bandwagon of the new and superficially promising treatment option often too early. the moment that money is made from a new intervention, the incentive for further research with the risk of disproving initial expectations is often gone. what next as a science policy in human reproductive medicine? certification of ivf centres may therefore in the future take different levels of scientific engagement within the field as a prerequisite. borrowing from oncology, a distinction could be made between simple care providers, specialized centres (e.g. pgd-m centres or fertility preservation centres) and comprehensive centres with major research aims. certification schemes of centres on a national or european level could consider, within the catalogue of requirements, different levels of research activity as a requirement for admittance to general care as well as admittance to more advanced or experimental treatments. for example, in a first step, it could be a requirement for all ivf centres to include a given fraction of the overall treated population in registered trials. of note, the implementation of quality control systems in our field has not been based on voluntary action alone, given the ramifications and costs for implementing and auditing these systems. why would that not also work for research? reproductive medicine and the research priorities identified within the field need to be put back into the focus of public interest, and public funding is of essence for research networks to be built and large collaborative trials to be conducted. for achieving that, we will not only need to beat the drum for our field, we will also need to demonstrate that we are structurally capable and inherently professionally motivated to run these trials. finally, we must look beyond the rct. as an educated guess, nearly all ivf centres in the industrialized world are using electronic patient files. we have large national and international ivf data registries by now already. in other words, we are already sitting on a mountain of data, but have not been making intelligent use of these data. observational data can be an easily accessible and cheap method to look at the safety and effectiveness of different treatment strategies. moreover, large-scale observational data stem from real-world circumstances and therefore include those patients that rcts typically exclude. sophisticated statistical methods including multivariable logistic regression analysis and propensity-matched analysis will be needed, but the establishment of collaborations with biostatisticians and epidemiologists may be, in many instances, an easier, faster and much cheaper option as compared to the design, conduct and analysis of large-sized rcts. new technologies, new drugs and new treatments will continue to enter our field. these novelties will improve the efficiency, effectiveness, quality, sustainability, safety and/or affordability of reproductive medicine, but only if we as a medical community manage, in a timely manner, to sort out the useless, and then focus our attention and power on the promising. orcid georg griesinger http://orcid.org/0000-0002-0606-5804 references 1. adamson gd, de mouzon j, chambers gm, zegers-hochschild f, mansour r, ishihara o, et al. international committee for monitoring assisted reproductive technology: world report on assisted reproductive technology, 2011. fertil steril. 2018;110: 1067–80. doi:10.1016/j.fertnstert.2018.06.039 2. de geyter c, calhaz-jorge c, kupka ms, wyns c, mocanu e, motrenko t, et al. european ivf-monitoring consortium (eim) for the european society of human reproduction and embryology (eshre). art in europe, 2014: results generated from european registries by eshre: the european ivf-monitoring consortium (eim) for the european society of human reproduction and embryology (eshre). hum reprod. 2018;33:1586–601. doi:10.1093/ humrep/dey242 3. sart. society for assisted reproductive technology in the united states. assisted reproductive technology national summary report 2015. 2015 [retrieved 2019 oct 31]. available from: https:// www.cdc.gov/art/artdata/index.html 4. chambers gm, wand h, macaldowie a, chapman mg, farquhar cm, bowman m, et al. population trends and live birth rates associated with common art treatment strategies. hum reprod. 2016; 31:2632–41. doi:10.1093/humrep/dew232 5. malchau ss, henningsen aa, loft a, rasmussen s, forman j, nyboe andersen a, et al. the long-term prognosis for live birth in couples initiating fertility treatments. hum reprod. 2017;32: 1439–49. doi:10.1093/humrep/dex096 6. harper j, jackson e, sermon k, aitken rj, harbottle s, mocanu e, et al. adjuncts in the ivf laboratory: where is the evidence for ‘add-on’ interventions? hum reprod. 2017;32:485–91. doi:10.1093/ humrep/dex004 7. stocking k, wilkinson j, lensen s, brison dr, roberts sa, vail a. are interventions in reproductive medicine assessed for plausible and clinically relevant effects? a systematic review of power and 66 g. griesinger https://doi.org/10.1016/j.fertnstert.2018.06.039 https://doi.org/10.1093/humrep/dey242 https://doi.org/10.1093/humrep/dey242 https://www.cdc.gov/art/artdata/index.html https://www.cdc.gov/art/artdata/index.html https://doi.org/10.1093/humrep/dew232 https://doi.org/10.1093/humrep/dex096 https://doi.org/10.1093/humrep/dex004 https://doi.org/10.1093/humrep/dex004 precision in trials and meta-analyses. hum reprod. 2019;34: 659–65. doi:10.1093/humrep/dez017 8. duffy jmn, bhattacharya s, curtis c, evers jlh, farquharson rg, franik s, et al. commit: core outcomes measures for infertility trials. a protocol developing, disseminating and implementing a core outcome set for infertility. hum reprod open. 2018;2018: hoy007. doi:10.1093/hropen/hoy007 9. van steirteghem a. what next for assisted reproductive technology? a plea for an evidence-based approach. hum reprod. 2008; 23:2615–6. doi:10.1093/humrep/den422 georg griesinger department of gynecological endocrinology and reproductive medicine, university hospital of schleswig-holstein, l€ubeck, germany georg.griesinger@uni-luebeck.de received 5 november 2019; revised 16 february 2020; accepted 21 february 2020 � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/ 4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 67 https://doi.org/10.1093/humrep/dez017 https://doi.org/10.1093/hropen/hoy007 https://doi.org/10.1093/humrep/den422 outline placeholder ivf: a success story with some obvious limitations rcts: how much have they really helped? why is progress in ivf so slow? what next as a science policy in human reproductive medicine? references upsala journal of medical sciences 2021, 126, e5653 http://dx.doi.org/10.48101/ujms.v126.5653 original article contact jonas selmeryd jonas.selmeryd@regionvastmanland.se. supplemental data for this article can be accessed here. © 2021 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. staphylococcus aureus bacteremia and cardiac implantable electronic devices in a county hospital setting: a population-based retrospective cohort study sara pichtchoulina, ingrid selmerydb, elisabeth freyhultc, pär hedberga,d and jonas selmeryda,d adepartment of clinical physiology, västmanland county hospital, västerås, sweden; bdepartment of infectious diseases, västmanland county hospital, västerås, sweden; cdepartment of microbiology, västmanland county hospital, västerås, sweden; dcentre for clinical research, uppsala university, västmanland county hospital, västerås, sweden abstract background: due to a high incidence of cardiac implantable electronic device-associated infective endocarditis (cied-ie) in cases of staphylococcus aureus bacteremia (sab) and high mortality with conservative management, guidelines advocate device removal in all subjects with sab. we aimed to investigate the clinical course of sab in patients with a cied (sab+cied) in a swedish county hospital setting and relate it to guideline recommendations. methods: all cied carriers with sab, excluding clinical pocket infections, in the county of västmanland during 2010–2017 were reviewed retrospectively. results: there were 61 cases of sab+cied during the study period, and cied-ie was diagnosed in 13/61 (21%) cases. in-hospital death occurred in 19/61 (31%) cases, 34/61 (56%) cases were discharged with cied device retained, and 8/61 (13%) cases were discharged after device removal. subjects dying during hospitalization were elderly and diseased. no events was seen if the cied was removed. among four discharged cases with conservatively managed cied-ie one relapse occured. among 30 cases discharged with retained cied and no evidence of ie, 22/30 (73%) cases had an uneventful follow-up, whereas adverse events secondary to overlooked cied-ie were likely in 1/30 (3%) cases and could not be definitely excluded in additionally 4/30 (13%) cases. conclusions: during the study period, management became more active and prognosis improved. the heterogeneity within the population of sab+cied suggests that a management strategy based on an individual risk/benefit analysis could be an alternative to mandatory device removal. article history received 28 september 2020 revised 13 january 2021 accepted 9 february 2021 published 5 march 2021 keywords staphylococcus aureus; cardiac implantable electronic device; endocarditis; pacemaker introduction many elderly in the western world are equipped with cardiac implantable electronic devices (cieds), probably >2–3% among subjects aged >75 years, and the number of implanted devices is increasing (1). the incidence of cied-associated infective endocarditis (cied-ie) among cied carriers with staphylococcus aureus bacteremia (sab) is high and has been estimated to be 27–45% in different studies (2–5). device removal is strongly recommended for patients with cied-ie (6–9). diagnosis of cied-ie in sab cases is challenging, and multiple clinical parameters must be weighed together to reach one (9, 10). also, multiple comorbidities, old age, and short life expectancy complicate the management of such patients (11). because of the high incidence of cied-ie in patients with sab, diagnostic difficulties, and poor prognosis with conservative management, swedish national guidelines from 2016 have advocated device removal in all subjects with sab, including cases where cied-ie cannot be demonstrated (8). the 2020 european heart rhythm  association (ehra) and the 2017 heart rhythm society (hrs) consensus papers have similar recommendations (6, 7). with an evident pocket infection, the diagnostic challenges are minor and management is well established (6). in contrast, the management of sab in cied carriers (sab+cied), when cied-ie is not evident upon presentation, is more complex. the objective of this study was to describe the background characteristics, management, and outcome of sab+cied, excluding pocket infections, within the county of västmanland, sweden, and relate them to current guidelines. materials and methods all sab+cied in the county of västmanland, sweden (population of 275,000), from 2010 to 2017 were reviewed retrospectively. cases of sab+cied were identified by cross-linking all hospitalacquired blood cultures positive for s. aureus in the database of the microbiology laboratory during 2010–2017 with 1) the swedish pacemaker registry (12) containing data on all pacemakers implanted since 1989 and onward; and 2) the local http://dx.doi.org/10.48101/ujms.v126.5653� mailto:jonas.selmeryd@regionvastmanland.se http://dx.doi.org/10.48101/ujms.v126.5653 http://creativecommons.org/licenses/by/4.0/� https://orcid.org/0000-0001-5731-966x https://orcid.org/0000-0002-1355-0250 2 s. pichtchoulin et al. hospital’s registries of diagnoses according to the international statistical classification of diseases and related health problems (icd-10) for cied-related diagnostic codes (t8xx, z9xx, z4xx, fpxxx, tfp00, df016, and df031) in 2008–2017. for all matches, the presence of a cied at the time of sab was verified in the patient’s medical journal. cases with clinical pocket infections were excluded. data were collected by reviewing digital hospital medical records from all departments in the county. background data were collected on age, gender, comorbidities according to the charlson comorbidity score (ccs) (13), residential care status (home dwelling or nursing home residency), general medical conditions according to the highest national early warning score version 2 (news2) (14) during the week following the taking of blood culture, sab community acquisition (15), type of device (pacemaker, implantable cardioverter-defibrillator [icd], or cardiac resynchronization therapy [crt]), and time of implantation and last revision. information was also collected on diagnostic procedures and treatments: if the diagnosis of cied-ie was considered, diagnostic imaging, antibiotic treatment, clinical diagnosis, and whether device removal was performed. each case  was classified by the modified duke criteria (10) based on available clinical information, autopsy results, and lead cultures, into cied-ie or not. cied-ie was defined as either lead endocarditis or valvular ie in a cied carrier. analogously to previous studies, we defined cied-ie as the fulfillment of the criteria for definite ie (using the criteria for possible ie would have classified all cases as cied-ie since sab, as a major criterion, and presence of an intracardiac device, as a minor criterion, are sufficient for possible ie) (3, 4). similar to previous studies (2–4), the outcome was measured as relapse of sab or death from any cause during hospitalization or within 90 days of discharge. the rationale for using this timeframe is that sab episodes occurring >70 days after initial blood culture are much more likely to be reinfection than a relapse (16, 17). the regional ethics board of uppsala, sweden, approved the study protocol 2018-11-29 (dnr 2017/513/1). statistical analyses continuous variables are reported as medians and interquartile ranges (iqrs), and categorical variables are reported as counts and percentages. to account for clustering when comparing groups (a few subjects had more than one sab episode during the study period), confidence intervals (cis) calculated by generalized linear models with the cluster bootstrap were calculated and compared (18). the clustering effect was small, with negligible differences in results compared to those obtained by more conventional methods. therefore, approximate independence between observations was assumed, and differences between groups were finally evaluated with the wilcoxon rank-sum for continuous variables and fisher’s exact test for categorical variables. to illustrate trends of dichotomous variables graphically over time, predictions based on univariate logistic regression models were plotted with 95% cis. p-values < 0.05 were considered statistically significant. r version 3.6.2 was used for all analyses (https://www.r-project.org). the r packages eulerr and clusterbootstrap were used in the analysis. results from the microbiology database, 1,035 unique patients were identified with sab during 2010–2017. when cross-linked against the national pacemaker registry, there were 72 unique patients with 98 sab episodes. of these, 66 episodes occurred in subjects where a cied was in place when afflicted by sab. after excluding four episodes with clinical pacemaker pocket infections and one episode where the subject was transferred to another hospital early after admission, the study sample consisted of 61 sab cases in 55 unique patients. we also crosslinked subjects with sab with pacemaker-related icd-10 codes from the hospital registry to validate our search strategy. this gave an identical subset of patients except for one, who was present in the national pacemaker registry but had no pacemaker-related icd-10 code registered. the incidence of sab+cied increased with time (figure 1). basic characteristics, outcome, and management stratified by discharge and cied removal status are illustrated in tables 1 and 2, figures 2 and 3, and supplementary figure 1. in the study sample, 37/61 (61%) cases were male, and the median age was 80 years (iqr 73–85). one case had methicillin-resistant s. aureus (mrsa), whereas the majority of cases had methicillin-susceptible s. aureus (mssa). a clinical diagnosis of cied-ie was established in 13/61 (21%) cases. one of these cases did not fulfill the duke definite criteria but was clinically diagnosed as cied-ie, despite negative transthoracic echocardiography (tte), in the context of recurring sab after a recent cied-ie diagnosis. death during hospital care in-hospital death occurred in 19/61 (31%) of cases. these cases were older (86 vs. 77 years; p < 0.001), were more frequently nursing home residents (53% vs. 7%; p < 0.001), and were in a worse general condition, as reflected by a higher news2 score (10 vs. 6; p < 0.001), compared with cases discharged from figure 1. the yearly incidence of hospitalization due to staphylococcus aureus bacteremia in cardiac implantable electronic device carriers (sab+cied) in the county of västmanland, sweden. https://www.r-project.org http://dx.doi.org/10.48101/ujms.v126.5653 a population-based retrospective cohort study 3 the hospital. of the cases dying during hospital care, 8/19 (42%) died within 3 days of care, tte was performed in 6/19 (32%) cases, and transesophageal electrocardiography (tee) was performed in none. three cases were diagnosed with cied-ie with vegetations on a native valve, a prosthetic valve, and a cied lead, respectively. none of these cases were in a clinical state where endocarditis surgery or cied extraction was deemed feasible. only two of the cases without cied-ie diagnosis were autopsied (without evidence of cied-ie). discharged after cied removal of the 42/61 (69%) cases discharged from hospital, the cied was explanted in 8/42 (19%) cases. these cases were younger (73 vs. 79 years; p = 0.061) and had lower ccs (1.5 vs. 4; p = 0.002) compared with those discharged with a retained cied. there were no relapses or deaths in the explanted cases. in 5/8 cases, clinical duke definite criteria were fulfilled and lead cultures were positive in two and negative in three cases, but this was after >4 days of intravenous (i.v.) antibiotics. in 3/8 cases, clinical duke definite criteria were not fulfilled: one subject with fever 3 h postimplantation of an icd device (lead culture not taken); one 58-year-old patient with severe heart failure, crt, and persistent bacteremia on day 3 (negative lead culture after >4 days of i.v. cloxacillin); and finally, one 63-year-old patient with persistent bacteremia on day 3 and a recently implanted pacemaker where an indication for pacemaker treatment no longer existed (positive lead culture). discharged with cied retained among the 34/61 cases (56%) discharged with a retained cied, 4/34 (12%) had a diagnosis of cied-ie. these conservatively treated cied-ie patients received 6 weeks of i.v. and/or peroral (p.o.) antibiotic suppression treatment. there were no deaths among cases of conservatively treated cied-ie. however, one relapse was detected in an 80-year-old woman with lymphoma where a cied lead infection had been proven, but device removal was deemed unfeasible. she received 45 days of i.v. antibiotics and relapsed 42 days postdischarge, after which she was put on chronic suppressive antibiotics. among the 30 cases without a cied-ie diagnosis, there were two sab relapses: there was one early relapse in an 86-year-old man with chronic obstructive pulmonary disease (copd) and heart failure, 9 days after the termination of a 12-day treatment with i.v. cloxacillin in the context of a negative tte. this patient did not survive his relapsing sab. another patient, an 83-yearold woman with metastatic cancer under palliation, had 14 days of i.v. cloxacillin and a negative tte and relapsed 80 days postdischarge. the relapse was treated with a short course of i.v. antibiotics, and the patient lived another 4 months without apparent signs of infection. there were six deaths without table 1. characteristics of cases with sab+cied (staphylococcus aureus bacteremia+cardiac implantable electronic device) stratified by in-hospital death and discharge status. variable all cases (n = 61) in-hospital death (n = 19) discharged (n = 42) pa male 37 (61) 15 (79) 22 (52) 0.088 age 80 (73–85) 86 (79–90) 77 (73–83) <0.001 age > 80 years 29 (48) 13 (68) 16 (38) 0.051 charlson comorbidity score (ccs) 4 (3–5) 5 (3–5.5) 4 (2–5) 0.153 ccs > 4 22 (36) 10 (53) 12 (29) 0.089 national early warning score 2 (news2) 7 (6–10) 10 (7.5–13) 6 (5–8) <0.001 symtomatic days before blood culture (bc) 1 (0–3) 2 (1–4.5) 1 (0–2.8) 0.148 nursing home resident 13 (21) 10 (53) 3 (7.1) <0.001 methicillin-resistant s. aureus (mrsa) 1 (1.6) 0 (0) 1 (2.4) 1.000 type of cied       0.703   cardiac resynchronization therapy (crt) or crt-d 7 (11) 1 (5.3) 6 (14)     implantable cardioverter-defibrillator (icd) 6 (9.8) 2 (11) 4 (9.5)     pacemaker 48 (79) 16 (84) 32 (76)   acquisition       0.394   community aquired 14 (23) 3 (16) 11 (26)     healthcare associated 30 (49) 12 (63) 18 (43)     nosocomial 17 (28) 4 (21) 13 (31)   known non-infective endocarditis (ie) focus 22 (36) 4 (21) 18 (43) 0.151 implantation (years) 6.8 (3.8–11) 7.2 (3.9–13) 6.7 (3.9–9.6) 0.503 last revision or implantation (years) 4.6 (2.7–7.8) 4.6 (2.7–7.6) 4.6 (2.8–7.5) 0.950 possibility of cied-ie discussed 26 (43) 6 (32) 20 (48) 0.276 transthoracic echocardiography (tte) or transesophageal echocardiography (tee) performed 42 (69) 6 (32) 36 (86) <0.001 tee performed 10 (16) 0 (0) 10 (24) 0.023 cied-ie diagnosis 13 (21) 3 (16) 10 (24) 0.737 values are counts and (percentages) for categorical variables and medians (with interquartile range) for continuous variables. awilcoxon rank-sum test or fisher’s exact test. 4 s. pichtchoulin et al. microbiologically documented sab relapse. in three cases, blood cultures were negative for s. aureus upon clinical deterioration, and plausible alternative death causes were documented: gastrointestinal bleeding, gram-negative sepsis, and heart failure secondary to terminal cardiac amyloidosis. in three cases, blood cultures were not taken, and undiagnosed sab relapse could not be ruled out: one 92-year-old female patient on chronic suppressive p.o. antibiotics for an s. aureus hip prosthesis infection died in her dementia nursing home 8 days after discharge; one 87-year-old male patient with copd died 10 days after discharge in respiratory and cardiac failure without signs of  infection; one 72-year-old male patient with dilated cardiomyopathy died 27 days after discharge in his home. trends during the inclusion period as illustrated in figure 4 and supplementary figures 2 and 3, management, basic characteristics, and outcomes were not constant over the inclusion period 2010–2017. when comparing the period 2010–2013 with 2014–2017, the risk of having an adverse event decreased from 92 to 35% (p < 0.001) and inhospital mortality from 67 to 22 % (p = 0.005). there was no table 2. characteristics of sab+cied (staphylococcus aureus bacteremia+cardiac implantable electronic device) cases discharged from hospital stratified by cied removal status. variable all discharged cases (n = 42) cied removed (n = 8) cied retained (n = 34) pa male 22 (52) 5 (62) 17 (50) 0.700 age 77 (73–83) 73 (66–74) 79 (73–83) 0.061 age > 80 years 16 (38) 1 (12) 15 (44) 0.127 charlson comorbidity score (ccs) 4 (2–5) 1.5 (1–2.2) 4 (3–5.8) 0.002 ccs > 4 12 (29) 0 (0) 12 (35) 0.080 national early warning score 2 (news2) 6 (5–8) 6.5 (4.8–8.2) 6 (5–7.8) 0.783 symtomatic days before blood culture (bc) 1 (0–2.8) 2.5 (0–4.5) 1 (0–2) 0.379 nursing home resident 3 (7.1) 0 (0) 3 (8.8) 1.000 methicillin-resistant s. aureus (mrsa) 1 (2.4) 1 (12) 0 (0) 0.190 type of cied       0.031   cardiac resynchronization therapy (crt) or crt-d 6 (14) 1 (12) 5 (15)     implantable cardioverter-defibrillator (icd) 4 (9.5) 3 (38) 1 (2.9)     pacemaker 32 (76) 4 (50) 28 (82)   acquisition       0.005   community aquired 11 (26) 5 (62) 6 (18)     healthcare associated 18 (43) 0 (0) 18 (53)     nosocomial 13 (31) 3 (38) 10 (29)   known non-infective endocarditis (ie) focus 18 (43) 1 (12) 17 (50) 0.109 implantation (years) 6.7 (3.9–9.6) 4.3 (2–8.1) 7 (4.3–11) 0.210 last revision or implantation (years) 4.6 (2.8–7.5) 3.7 (2–5.1) 5 (3.3–7.5) 0.222 possibility of cied-ie discussed 20 (48) 8 (100) 12 (35) 0.001 transthoracic echocardiography (tte) or transesophageal echocardiography (tee) performed 36 (86) 8 (100) 28 (82) 0.576 tee performed 10 (24) 4 (50) 6 (18) 0.075 cied-ie diagnosis 10 (24) 6 (75) 4 (12) 0.001 outcome       0.473   no event 33 (79) 8 (100) 25 (74)     death from any cause 6 (14) 0 (0) 6 (18)     relapse 3 (7.1) 0 (0) 3 (8.8)   values are counts and (percentages) for categorical variables and medians (with interquartile range) for continuous variables. awilcoxon rank-sum test or fisher’s exact test. figure 2. euler diagrams illustrating the distribution and overlapping of some background characteristics stratified by discharge and device removal status. medians were used for dichotomization. the sizes of the outer black circles are proportional to the sizes of the subgroups. cied: cardiac implantable electronic device; ccs: charlson comorbidity score. http://dx.doi.org/10.48101/ujms.v126.5653 http://dx.doi.org/10.48101/ujms.v126.5653 a population-based retrospective cohort study 5 figure 3. outcomes stratified by discharge and device removal status. cied: cardiac implantable electronic device; ie: infective endocarditis; sab: staphylococcus aureus bacteremia. 1) echocardiography was not performed in 13 cases. 2) echocardiography was not performed in six cases. figure 4. trends in outcome and management during the study period. the probabilities of having an adverse event (a), having transesophageal echocardiography (tee) performed (b), cardiac implantable electronic device (cied) removal considered (c), and cied removal performed (d), expressed as functions of the year of inclusion are illustrated by unadjusted logistic regression probability estimates. individual measurements are indicated as circles. p-values were calculated by logistic regression for association with the year of inclusion. 6 s. pichtchoulin et al. significant association between inclusion time and age, ccs,  news2 score, or nursing home residency. with regard to management, device removals were more frequently considered, 48–96 h blood cultures were more frequent, and there were trends toward referring for more echocardiography and device removals. discussion here, we have described the background characteristics, management, and outcome of all cases of sab+cied without signs of pocket infections between 2010 and 2017 in a county hospital. the incidence of hospitalizations due to sab+cied increased steeply during the study period. in the most recent 2016 national swedish guidelines on ie  management, the indication for device removal was broadened by recommending it for all subjects with sab, regardless of diagnostic findings (8). the profound effect these recommendations had on management can be seen in figure 4c, where device removal from 2016 and later, was considered in a majority of patients. however, the recommendations seem to have been challenging to implement in real life as only a fraction of patients were finally explanted. among the 34 cases discharged with a retained cied, 30 had no cied-ie diagnosis. most of these (22/30; 73%) had an uneventful follow-up, but among the six deaths and two relapses, there might have been cases of missed cied-ie. based on available clinical information, an overlooked cied-ie was probable in one case with early relapse, possible in four cases (late relapse or early death with very limited clinical data), and unlikely in three (negative blood cultures and/or no clinical signs of infection at the time of death). if all cases of nonproven cied-ie had been explanted, in line with current guideline recommendations, 25–29 out of 30 (83–97%) device removals would, retrospectively, have been carried out in vain, whereas it would potentially have been advantageous in 1–5 out of 30 cases (3–17%). this potential benefit needs to be compared with the potential harm associated with device removal. lead extraction is a safe procedure in general, but for specific subgroups, the risk of major complications and death is increased to >5% (19–21). the risk of adverse outcome increases, for instance, with active sepsis, multiple leads, female gender, long lead dwell time, implantation at young age, multiple previous cied procedures, and anemia (19–21). as the potential benefits and risks might be of similar gross magnitude for specific subgroups, a strategy based on an individual risk/ benefit analysis might be preferable over mandatory device removal: if the probability of cied-ie is high (i.e. persistent bacteremia or positive imaging) and the risk of complications is low (i.e. low procedural risk), cied removal would be warranted, whereas a low cied-ie probability and/or a high risk of complications might favor a more conservative approach. several risk scoring systems have been put forward that potentially could be used to identify subjects with an elevated risk of adverse outcome with extraction (19–21). unfortunately, scoring systems for predicting cied-ie in sab+cied are scarce. to our knowledge, only one exists, the predict-sab, which, however, does not take imaging findings, or lack thereof, into account (4). with predict-sab, subjects without any risk factors, including persistent bacteremia, had a predicted probability for having cied-ie of 7% before imaging. similarly, in a recent study, subjects with negative imaging findings and without persistent bacteremia were shown to have a relapse rate of 5% with cied retention, and it was suggested that cied retention might be a viable option in such subjects if the risk with extraction was increased (22). the cied-ie prevalence in the present study was 21% compared with 27–34% in previous studies performed in tertiary referral centers (device pocket infections excluded) (2–4). a referral bias, where subjects with a high cied-ie probability are transferred and concentrated to tertiary referral centers, could in part explain this. however, another likely explanation was probably underdiagnosis because of the infrequent utilization of diagnostic imaging. for instance, the subjects dying during hospital care were rarely evaluated for the possibility of cied-ie, as illustrated by a tte frequency of 33% and tee frequency of 0%. while this might be perceived as negligent, it is likely the result of rapid clinical deterioration in the patients (42% of them died within 3 days) and/or a short expected survival not perceived to be modifiable by diagnostic or therapeutic approaches because of advanced age (median age 86 years), frailty (53% were nursing home residents), multiple comorbidities (median ccs 5), and a poor general condition (median news2 score 10). also, many of the cases were managed prior to contemporary awareness of cied-ie. in this study, 46% of the cases had an adverse event (relapse or death from any cause within 90 days). the overall mortality was 41%, in-hospital mortality was 31%, and relapse rate was 5%. these values are similar to the outcomes in previous studies on similar populations; for example, uslan et al. reported inhospital mortality of 32% (3), chamis et al. reported an all-cause mortality of 36% and a relapse rate of 6% (2), and sohail et al. described an all-cause mortality of approximately 40% (4). nevertheless, the similarities in mortality and relapse rates with the current study are surprising given that our population was considerably older (80 years vs. 70–73) (2–4), there was a lower utilization of diagnostic modalities (tee 16% vs. 64–67%) (2–4) and a lower frequency of device removal (13% vs. 36%) (2). one reason for this pattern might be a bias toward the referral of complicated cases with a high suspicion of cied-ie to tertiary referral centers. also, mrsa was very uncommon in the present population compared with the abovementioned studies (2% vs. 39–55%) (2–4), which might explain a better than expected prognosis in our population as mssa is associated with better outcomes (23). recently, in a similarly designed swedish study on sab in 33 cied carriers, snygg-martin et al. demonstrated frequencies of device removal (12%), echocardiography (63%), tee (33%), and mortality (30-day: 36%; 1-year: 65%) very similar to those of the present study (5). during the study, the prognosis for patients improved substantially (figure 4a), consistent with previous findings for sab (24), s. aureus ie (25), and sepsis cohorts (26–28). there are probably several explanations for this. we observed signs of a population-based retrospective cohort study 7 more active management strategies over time, as exemplified by more use of echocardiography and more device removals. the large impact national ie guidelines published in 2016 (8) had on management can be appreciated in an increased awareness of cied-ie and utilization of 48–96 h blood cultures (supplemental figure 3d). however, the onset of the improvement in prognosis seemed to precede the shifts in management, indicating other factors at play. changes in population characteristics might have contributed, even though the relative importance of such factors seems to be less pronounced as no significant linear association between age, news2 score, ccs, nursing home residential status, community acquisition, lead dwelling time and type of cied with time could be detected (supplementary figures 2 and 3). improvements in different sab care processes, not analyzed specifically here – for instance, increased sepsis awareness, standardization of management (29), a shortened time to antistaphylococcal therapy (24), and improvements in intensive care management (28) were likely to be essential factors in explaining the observed improvements seen in prognosis. limitations there were several important limitations to our study. first, the restricted sample size and number of events made it impossible to analyze the effect of multiple background factors and management on the outcomes while adjusting for confounding effects. second, as observations were cases and not subjects, the assumption of independence among observations was violated to a degree. however, the violation was deemed to be of minor importance as only a few within-subject repeated observations were present, and analysis accounting for clustering yielded similar results (data not shown). third, the long inclusion time of 8 years “diluted” the results over the years, which hampered our ability to focus on and describe current practices accurately. fourth, any generalizability of results must be done with caution because of the study’s single-center county hospital setting. fifth, since most cases were included before the contemporary cied-ie criteria of the european society of cardiology (esc) and ehra, we chose to use the older modified duke criteria frequently employed in previous sab+cied studies. the main difference between esc/ ehra criteria and the modified duke criteria is additional diagnostic modalities (intra-cardiac ultrasound, leukocyte scintigrahy, gated computer tomography, and positron emission tomography) within the imaging major criteria. however, as these novel imaging modalities were not implemented in clinical practice during the study period and as the weighting of criteria has not changed, the classification would have been similar to the esc/ehra criteria. sixth, the reported prevalence of cied-ie in this study was likely underestimated because of the infrequent utilization of diagnostic imaging. conclusions during the study period, management of sab+cied became more active, paralleling new guideline recommendations, and the patients’ prognosis improved, illustrating the importance of the active management advocated by current guidelines. however, the heterogeneity of the population concerning age, comorbidities, and outcomes and a low frequency of sab relapses among subjects discharged with cied retained suggest that, instead of device removal in all sab+cied, a strategy based on an individual risk/benefit analysis of device retention vis-àvis removal might be an option. however, further research on the development and validation of clinical prediction tools to aid in such risk/benefit analysis is needed. disclosure statement the authors have nothing to disclose. funding this work was supported by the county of västmanland, sweden. notes on contributors sara pichtchoulin. medical technologist and sonographer at the department of clinical physiology, västerås, sweden. ingrid selmeryd, md. infectious disease specialist at the department of infectious diseases, västerås, sweden. elisabeth freyhult, md. medical microbiologist at the department of clinical microbiology, västerås, sweden. pär hedberg, md. clinical physiologist at the department of clinical physiology, västerås, sweden. associate professor at the centre for clinical research, uppsala university, västerås, sweden. jonas selmeryd, md. clinical physiologist at the department of clinical physiology, västmanland county hospital, västerås, sweden. phd student at the centre for clinical research, uppsala university, västerås, sweden. references 1. bradshaw pj, stobie p, knuiman mw, briffa tg, hobbs mst. trends in the incidence and prevalence of cardiac pacemaker insertions in an ageing population. open hear. 2014;1 :e000177. doi: 10.1136/ openhrt-2014-000177 2. chamis al, peterson ge, cabell ch, corey gr, sorrentino ra, greenfield ra, et al. staphylococcus aureus bacteremia in patients with permanent pacemakers or implantable cardioverter-defibrillators. circulation 2001;104:1029–33. doi: 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http://dx.doi.org/10.1007/s00134-009-1738-3 palpable maculopapular rash with elevated esr: what is your diagnosis? full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 palpable maculopapular rash with elevated esr: what is your diagnosis? subhankar chakraborty, dominick dimaio & trevor vanschooneveld to cite this article: subhankar chakraborty, dominick dimaio & trevor vanschooneveld (2013) palpable maculopapular rash with elevated esr: what is your diagnosis?, upsala journal of medical sciences, 118:4, 285-286, doi: 10.3109/03009734.2013.831504 to link to this article: https://doi.org/10.3109/03009734.2013.831504 © informa healthcare published online: 27 aug 2013. submit your article to this journal article views: 513 view related articles citing articles: 1 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.831504 https://doi.org/10.3109/03009734.2013.831504 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.831504 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.831504 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.831504#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.831504#tabmodule upsala journal of medical sciences. 2013; 118: 285–286 letter to the editor palpable maculopapular rash with elevated esr: what is your diagnosis? subhankar chakraborty1, dominick dimaio2 & trevor vanschooneveld3 1department of internal medicine, university of nebraska medical center, omaha, ne, usa, 2departments of pathology and microbiology, university of nebraska medical center, omaha, ne, usa, and 3infectious disease, antimicrobial stewardship program, university of nebraska medical center, omaha, ne, usa dear editor leucocytoclastic vasculitis (lcv) is a necrotizing vasculitis that complicates infections, malignancies, and collagen vascular diseases and can also occur as a reaction to medications (1). it is difficult to distinguish clinically from a drug eruption. we present a case of drug rash resembling lcv. diagnosis was established by skin biopsy and responded to prompt discontinuation of the medication. the case emphasizes the importance of considering skin biopsy in distinguishing a suspected drug reaction from vasculitis. a 65-year-old man was admitted to the hospital with cellulitis of the left leg. he was started empirically on intravenous vancomycin and piperacillin-tazobactam. based on results of blood cultures, he was treated with oxacillin and then cefazolin for methicillinsensitive staphylococcus aureus. after three doses of cefazolin, however, the patient developed a rash on both legs and back (figure 1). the rash was palpable but non-blanching (figure 2a and b), non-tender, and there were no blisters. laboratory investigation revealed an elevated erythrocyte sedimentation rate of 70 mm/h (reference range 0–20 mm/h), rheumatoid factor of 20 iu/ml (reference range < 15 iu/ml), and negative antinuclear antibodies. other significant findings included microscopic hematuria (> 50 rbcs/hpf) and microscopic proteinuria (protein/ creatinine ratio 0.3; reference range 0–0.2 mg/mg of creatinine). histopathology of skin biopsy revealed mild lymphocytic infiltration into the dermis without any evidence of fibrinoid necrosis (figure 2c and d). a diagnosis of drug rash was made, presumably due to oxacillin. cefazolin was continued and the patient discharged home 2 weeks later with instructions to complete a 6-week course. on follow-up his rash had completely resolved. cutaneous lcv is characterized by a palpable purpuric rash that usually begins 7–21 days after exposure to the offending antigen, involves the lower extremities, and is accompanied by dependent lower extremity edema, abdominal and joint pains, and renal involvement. involvement of the forearms, hands, back, sacral, and gluteal regions is less common. when fully evolved, the skin lesions form vesicles, bullae nodules, or ulcers (2). histologically, it is characterized by acute necrotizing inflammation involving small blood vessels in the upper dermis (3). drug-induced rash, on the other hand, is characterized by a superficial, mostly perivascular mononuclear cell infiltrate and some eosinophilia. interface dermatitis, characterized by epidermal basal cell damage, may also be seen with a drug rash (4). the two conditions can only be distinguished histologically. the management of cutaneous lcv depends on the severity. in mild cases, support stockings and elevation of the edematous legs can be tried. antihistamines, non-steroidal anti-inflammatory drugs, corticosteroids, and cytotoxics have been employed with variable success in moderate to severe cases. small-vessel vasculitis with internal organ involvement requires aggressive immunosuppressive therapy. drug rash, on the other correspondence: subhankar chakraborty, md, phd, department of internal medicine, university of nebraska medical center, omaha, ne-68198-2055, usa. e-mail: schakra@unmc.edu (received 4 april 2013; accepted 19 july 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.831504 http://informahealthcare.com/journal/ups mailto:schakra@unmc.edu hand, responds promptly to discontinuation of the offending medication as in this case. in summary, we present a case of drug rash that mimicked leucocytoclastic vasculitis based on elevated acute-phase reactants and negative antinuclear antibodies. diagnosis was established by histopathology and responded to avoidance of the drug. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. koutkia p, mylonakis e, rounds s, erickson a. cutaneous leucocytoclastic vasculitis associated with oxacillin. diagn microbiol infect dis. 2001;39:191–4. 2. bhattacharyya a, yeddula k, nwankwo n, senussi mh. leucocytoclastic vasculitis. bmj case rep. 2013;2013. 3. koutkia p, mylonakis e, rounds s, erickson a. leucocytoclastic vasculitis: an update for the clinician. scand j rheumatol. 2001;30:315–22. 4. khan da. cutaneous drug reactions. j allergy clin immunol. 2012;130:1225. a b c d figure 1. palpable purpuric rash on the left lower extremity (a and b) and back (c and d) following antibiotic therapy. a b c d figure 2. demonstration of non-blanchability of the purpuric lesions (a and b). histopathology of skin lesions from the purpuric lesions (c). the epidermis demonstrates no inflammatory changes. within the papillary dermis there is a mild inflammatory infiltrate associated with extravasated red blood cells (� 100, h&e). the inflammatory infiltrate is composed predominately of lymphocytes with rare eosinophils (d). there are scattered extravasated red blood cells. no neutrophils are present. the vessels show no fibrinoid necrosis or fibrin thrombi (� 200, h&e). 286 s. chakraborty et al. www.ncbi.nlm.nih.gov/pubmed/11337188?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11337188?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11846048?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11846048?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/23116633?dopt=abstract references population-based cardiovascular cohort studies in uppsala article population-based cardiovascular cohort studies in uppsala lars lind department of medical sciences, uppsala university, sweden abstract the first population-based cohort study in uppsala with the aim to study cardiovascular disease was initiated in 1970 (ulsam). this cohort of 2300 middle-aged men has since then been followed in a longitudinal fashion for almost 50 years. this study has been followed by the pivus study, investigating 1000 men and women at ages 70, 75, and 80. a very detailed examination has also been performed in 500 subjects aged 50 years, the poem study. in recent years, a high-throughput study conducted in 13000 subjects has also been performed, named epihealth. uppsala also collects data in 5,000 subjects in the nationwide scapis study. taken together, these cardiovascular-oriented studies constitute a very rich source for cardiovascular epidemiological research in uppsala. this review summarizes the design of these studies and highlights some of the important results published based on data from these studies. article history received 26 june 2018 revised 13 august 2018 accepted 20 august 2018 keywords cardiovascular; cohort study; heart; longitudinal; vessels introduction framingham heart study (fhs) was initiated in 1948 to study the natural course of cardiovascular disease (cvd) and its risk factors. since then this cohort study has been the leading epidemiological cvd study. inspired by fhs, g€osta tibblin and co-workers started the study of ‘men born 1913’ in 1963 in gothenburg. at that time, it was recognized that coronary heart disease mainly was a disease of men, and therefore only men were included. the ulsam study inspired by these two milestone studies in the usa and in sweden, hans hedstrand and co-workers at the department of medicine at uppsala university hospital initiated in 1970 a cohort study of 2,322 men all aged 50 years that was later named ‘the uppsala longitudinal study of adult men’ (www. pubcare.uu.se/ulsam) (1). the aim from the beginning was to investigate risk factors for cvd, and then to randomize the individuals to a more intense intervention or not, in order to evaluate if future cvd could be prevented. this aim of the study was not really fulfilled, but this cohort study has served as the basis for more than 40 phd theses and 350 publications during the years. unique features of the initial investigation at age 50 years were an intravenous glucose tolerance test with frequently sampled insulin determinations, to determine glucose-stimulated insulin secretion, and a detailed characterization of the fatty acid profile in cholesterol esters, a marker of dietary fat quality intake. following the first examination cycle, hans lithell at the department of geriatrics took over the responsibility of the study. although an examination was performed at age 60, it was the examination at age 70 that put ulsam on the international scene. apart from traditional cvd risk factors, an oral glucose tolerance test (ogtt) with insulin determinations was performed together with the hyperinsulinemic euglycemic clamp that allowed an evaluation of both insulin secretion and sensitivity. this is still the largest effort in a single study to characterize these two major determinants of diabetes development. other unique features of the 70-year examination cycle were an echocardiographic examination including determinations of left ventricular geometry and function, as well as skeletal muscle biopsies to assess the fiber composition in half of the sample. based on the ‘development origin of adult disease (doad)’ hypothesis also the birth weight was included, and a 24-h ambulatory blood pressure measurement was performed. at the 70-year examination, and even more in the following years, also diseases other than cvd have been studied, and data from dietary records, cognitive function tests, and measurements of bone mineral content by dual x-ray absorptiometry (dxa) and bone fractures have been studied (2). major findings published over the years regarding cvd in ulsam include the following: proinsulin is an important risk factor for cvd (3); left ventricular mass determined both at echocardiography and at ecg are additive risk factors for cvd (4); insulin sensitivity is a risk factor also for stroke (5) and heart failure (6); metabolic syndrome is a risk factor for cvd (7); atrial fibrillation is a risk factor for poor cognitive contact lars lind e-mail: lars.lind@medsci.uu.se department of medical sciences, uppsala university, uppsala, sweden � 2018 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 1, 16–20 https://doi.org/10.1080/03009734.2018.1515282 http://www.pubcare.uu.se/ulsam http://www.pubcare.uu.se/ulsam http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1515282&domain=pdf http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1515282 http://www.tandfonline.com function (8); isolated ambulatory hypertension is a risk factor for cvd (9); increase in fasting glucose during treatment with beta-blockers increased the risk of myocardial infarction (10); a set of biomarkers predicted cardiovascular (cv) mortality (11); obesity without metabolic risk factors, so-called metabolically healthy obesity, was related to premature cvd (12); and a certain fatty acid profile, including high proportions of saturated fat and a low proportion of linoleic acid, was related to incident myocardial infarction (13). the major strength of the ulsam study is the many reexaminations performed, which allows for collection of longitudinal trends of measured variables as well as for collection of incident cases of a number of diseases, not only cvd, over more than 40 years. figure 1 shows an overview of the number of participants at each examination cycle. the pivus study with the major aim to study if measurements of endothelial function would add predictive power on top of traditional risk factors regarding cvd, the prospective study of the vasculature in uppsala seniors (pivus) study was initiated in 2001 (www.medsci.uu.se/pivus) (14). in this study, all 1,016 participants were 70 years old, and 50% of the sample were women. apart from endothelial function the vasculature was characterized by arterial compliance and carotid artery ultrasound measurements to evaluate atherosclerosis, and total atherosclerotic burden by magnetic resonance angiography. the heart was examined by echocardiography and magnetic resonance imaging (mri) with late enhancement. fat distribution was characterized by dxa and abdominal mri. dietary data were recorded, and fa composition in cholesterol esters were determined. at age 75, the sample was reinvestigated, and at this time mri of the brain and three cognitive function tests were performed. these investigations were also repeated at age 80 (see figure 1 for numbers of participants). incident cases of cvd have been recorded over 10 years, and will be updated after 15 years. more than 250 papers have been published, and more than 20 phd students have had pivus data in their thesis. major results being published showed that endothelial function in the forearm resistance vessels, but not in the brachial conduit artery, was related to future cvd (15); silent myocardial scars were more common than previously thought (16); silent myocardial scars were related to cerebral infarcts in women only (17); and troponin i levels were related to both cv and non-cv mortality (18). since the results in many studies probably are false positive findings and cannot be reproduced by others, we have in a number of studies used data from both ulsam and pivus to evaluate the use of biomarkers. by this approach, it has been shown that plasma levels of endostatin (19) and cathepsin s (20) are related to mortality in these two independent samples. since the first genome-wide association studies (gwas) were published some 10 years ago, ulsam and pivus have participated in a number of the big consortia trying to find the genetic architecture of obesity, diabetes, coronary heart disease, and stroke. there are two major conclusions from these studies. first, the top gene findings, the fto gene, the tf gene, and the 9.21 loci, were largely unknown gene regions before the gwas era and could therefore not be found by a candidate gene approach. second, in none of these outcomes was a single or a handful of genes of major importance; rather, hundreds of genes have a small impact each. during the recent years, ulsam and pivus have put a large effort into proteomics and metabolomics and published that low levels of lyophosphatidylcholine (18:2) are associated with a high risk of myocardial infarction (21), that certain bile acids are related to diabetes risk (22), and have identified a number of proteins being associated with carotid atherosclerosis (23), incident stroke (24), and atrial fibrillation (25). the pivus study has become famous in the field of environmental research in that we have measured >50 different environmental contaminants in plasma and found, amongst other things, that persistent organic pollutants, such as pcbs, perfluorinated compounds, and pesticides, are related to atherosclerosis (26,27) and incident stroke (28). the major strength of the pivus study is the detailed characterization of the cardiovascular system at repeated examinations. the poem study the population-based prospective investigation of obesity, energy and metabolism (poem) study was conducted in inhabitants of uppsala, sweden, all aged 50 years. between october 2010 and october 2016, 502 individuals were investigated (50% women). the primary aim was to explore the links between obesity and cvd. a great number of markers of subclinical cv disease (three tests of endothelial function, three tests of arterial compliance, carotid atherosclerosis, left ventricular mass, systolic and diastolic function, heart rate variability, a maximal figure 1. number of participants at each examination cycle in the ulsam and pivus studies. upsala journal of medical sciences 17 http://www.medsci.uu.se/pivus bicycle test with vo2 determinations) were recoded. an ogtt with insulin determinations has been made. wholebody mri with determinations of visceral/subcutaneous adipose tissue and liver and pancreatic fat has been performed. fat mass has been recorded by dxa. also genomic, proteomic, and metabolomics measurements have been analyzed. based on data from mri in poem, a novel concept to analyze relationships between a phenotype and whole-body images has been developed at the department of radiology, called ‘imiomics’ (29). by this approach, each of the >2 million image elements in the 3 d magnetic resonance image is quantified in terms of volume and lipid content, and each image element could be related to any phenotype, and later correlation maps of the body could be constructed in 3 d. figure 2 shows such an analysis when fat mass measured by bioimpedance is related to the relative volume of each image element. the major strength of the poem study is the extremely detailed characterization of metabolism and the cardiovascular system. the epihealth study from april 2011, men and women in the age range 45–75 years in two swedish towns, uppsala and malm€o, have been invited in a random fashion to an on-going health screening survey, epihealth (epidemiology for health) (www. epihealth.se) (30). the study was started as a part of the government-supported strategic research areas, and the major aim of the study is to investigate interactions between genes and lifestyle factors regarding common diseases in the elderly. by 31 december 2017, data on approximately 24,000 individuals have been collected (13,000 individuals in uppsala). traditional cv risk factors and fat mass (bioimpedance) have been recorded. genomic, metabolomic, and proteomic data have been collected in a subsample (n ¼ 2500). in spring 2017, incident cv diseases were updated by swedish in-hospital care and mortality registers. in a substudy, atherosclerosis has been measured, and a whole-body pet/mri scan with fdg-tracer has been performed with special reference to inflammation of atherosclerosis in the aorta and carotid arteries (n ¼ 100). the major strength of epihealth is the rather large size of the cohort, which will enable a fairly rapid collection of incident cvd endpoint, and provide a good power for ‘omics’ studies. the scapis study as a collaboration project between six swedish universities, 30,000 subjects aged 50–65 years will be investigated in the swedish cardiopulmonary imaging study (scapis) with a planned ‘last subject in’ in q4 2018 (5,000 subjects in uppsala) (www.scapis.se) (31). the major aims are to discover novel mechanisms and biomarkers for cvd and pulmonary diseases to improve risk stratification and discover new drug targets and preventive strategies. in addition to the traditional cv risk factors, an extensive imaging program is performed, including ct coronary angiography, ct scanning of lungs and abdomen for visceral/subcutaneous adipose tissue and liver fat (including ‘inflamed adipose tissue’ determinations), as well as ultrasound for carotid artery atherosclerosis (and mri if carotid plaques are found). a detailed pulmonary function testing battery has been performed together with measurements of the ankle–brachial index to assess leg atherosclerosis. genomic, proteomic, and metabolomics measurements are planned in a subsample of 5,000 during 2017 and 2018. figure 2. ‘imiomics analysis’ of fat mass measured by bioimpedance in females (left) and males (right). a correlation analysis was performed between fat mass and the relative volume of each image element in the 3d magnetic resonance image. each image element is then color-coded according to the correlation coefficient, where dark red represents a high positive correlation coefficient and blue a high negative correlation coefficient. unpublished data from robin strand, joel kullberg, and håkan ahlstr€om at the department of radiology, uppsala university. 18 l. lind http://www.epihealth.se http://www.epihealth.se http://www.scapis.se the scapis study is the largest academic collaborative project in sweden in medicine with a budget of >40 million efor collection of data. table i summarizes the major phenotypes collected in the cvd studies in uppsala, and figure 3 shows the when the studies were performed. discussion two extremes of epidemiological studies exist. first, studies with a limited number of subjects (some hundreds to a couple of thousands) that have been extensively phenotyped; ulsam, pivus, and poem are examples of such studies. second, large high-throughput studies with less deep phenotyping; epihealth is one such example. the development of ‘omics’ technologies, especially gwas, demands large-scale studies to deal with the multiple testing issue. on the other hand, to evaluate mechanisms involved in the development of cvd, the small-scale studies are needed with deep phenotyping of markers of subclinical cvd, such as endothelial function, atherosclerosis, and myocardial function. thus, both largeand small-scale epidemiological studies are needed in order to move the knowledge of cvd forward. (table i) figure 4 illustrates the costs and output in terms of measured phenotypes and outcomes in two examples of largescale and small-scale uppsala epidemiological studies. in the scapis study, the features of both the largeand small-scale studies are merged into a large study with deep phenotyping. however, to accomplish that goal in a reasonable time-frame, this study has to be performed as a multicenter study and at a high cost. in conclusion, several cvd cohort studies with different characteristics exist in uppsala which makes it possible to explore hypotheses demanding both a large number of participants, as well as a deep phenotyping of markers of subclinical cvd and long follow-up periods. in addition, having access to several cohorts makes it possible to replicate findings in one sample in another study, an important task to ascertain the validity of novel research findings. disclosure statement no potential conflict of interest was reported by the authors. notes on contributor lars lind is professor of medicine at uppsala university with an expertise in cardiovascular epidemiology. references 1. hedstrand h. a study of middle-aged men with particular reference to risk factors for cardiovascular disease. ups j med sci suppl. 1975;19:1–61. table 1. table showing which kinds of investigations were performed in the different studies. ulsam pivus poem epihealth scapis n at baseline 2322 1016 503 13000 5000 traditional risk factors � � � � � fatty acid composition � � � dxa � � � dietary data � � � � � echocardiography � � � carotid atherosclerosis � � � � coronary atherosclerosis � endothelial function/arterial compliance � � ogtt � � hyperinsulinemic clamp � 24-h blood pressure � � � abdominal mri/ct � � � myocardial mri � ct: computer tomography; dxa: dual x-ray absorptiometry; mri: magnetic resonance imaging; ogtt: oral glucose tolerance test. figure 3. this calendar shows when the different studies and their examination cycles were performed. figure 4. costs and outcome during three years of data collection in two extremes of epidemiological studies. upsala journal of medical sciences 19 2. michaelsson k, lithell h, vessby b, melhus h. serum retinol levels and the risk of fracture. n engl j med. 2003;348:287–94. 3. zethelius b, byberg l, hales cn, lithell h, berne c. proinsulin is an independent predictor of coronary heart disease: report from a 27-year follow-up study. circulation. 2002;105:2153–8. 4. sundstrom j, lind l, arnlov j, zethelius b, andren b, lithell ho. echocardiographic and electrocardiographic diagnoses of left ventricular hypertrophy predict mortality independently of each other in a population of elderly men. circulation. 2001;103:2346–51. 5. wiberg b, sundstrom j, zethelius b, lind l. insulin sensitivity measured by the euglycaemic insulin clamp and proinsulin levels as predictors of stroke in elderly men. diabetologia. 2009;52:90–6. 6. ingelsson e, sundstrom j, arnlov j, zethelius b, lind l. insulin resistance and risk of congestive heart failure. jama. 2005;294: 334–41. 7. sundstrom j, riserus u, byberg l, zethelius b, lithell h, lind l. clinical value of the metabolic syndrome for long term prediction of total and cardiovascular mortality: prospective, population based cohort study. bmj. 2006;332:878–82. 8. kilander l, andren b, nyman h, lind l, boberg m, lithell h. atrial fibrillation is an independent determinant of low cognitive function: a cross-sectional study in elderly men. stroke. 1998;29: 1816–20. 9. bjorklund k, lind l, zethelius b, andren b, lithell h. isolated ambulatory hypertension predicts cardiovascular morbidity in elderly men. circulation. 2003;107:1297–302. 10. dunder k, lind l, zethelius b, berglund l, lithell h. increase in blood glucose concentration during antihypertensive treatment as a predictor of myocardial infarction: population based cohort study. bmj. 2003;326:681. 11. zethelius b, berglund l, sundstrom j, ingelsson e, basu s, larsson a, et al. use of multiple biomarkers to improve the prediction of death from cardiovascular causes. n engl j med. 2008;358: 2107–16. 12. arnlov j, ingelsson e, sundstrom j, lind l. impact of body mass index and the metabolic syndrome on the risk of cardiovascular disease and death in middle-aged men. circulation. 2010;121: 230–6. 13. ohrvall m, berglund l, salminen i, lithell h, aro a, vessby b. the serum cholesterol ester fatty acid composition but not the serum concentration of alpha tocopherol predicts the development of myocardial infarction in 50-year-old men: 19 years follow-up. atherosclerosis. 1996;127:65–71. 14. lind l, fors n, hall j, marttala k, stenborg a. a comparison of three different methods to evaluate endothelium-dependent vasodilation in the elderly: the prospective investigation of the vasculature in uppsala seniors (pivus) study. arterioscler thromb vasc biol. 2005;25:2368–75. 15. lind l, berglund l, larsson a, sundstrom j. endothelial function in resistance and conduit arteries and 5-year risk of cardiovascular disease. circulation. 2011;123:1545–51. 16. barbier ce, bjerner t, johansson l, lind l, ahlstrom h. myocardial scars more frequent than expected: magnetic resonance imaging detects potential risk group. j am coll cardiol. 2006;48:765–71. 17. barbier ce, nylander r, themudo r, ahlstrom h, lind l, larsson em, et al. prevalence of unrecognized myocardial infarction detected with magnetic resonance imaging and its relationship to cerebral ischemic lesions in both sexes. j am coll cardiol. 2011;58: 1372–7. 18. eggers km, venge p, lindahl b, lind l. cardiac troponin i levels measured with a high-sensitive assay increase over time and are strong predictors of mortality in an elderly population. j am coll cardiol. 2013;61:1906–13. 19. arnlov j, ruge t, ingelsson e, larsson a, sundstrom j, lind l. serum endostatin and risk of mortality in the elderly: findings from 2 community-based cohorts. arterioscler thromb vasc biol. 2013;33:2689–95. 20. jobs e, ingelsson e, riserus u, nerpin e, jobs m, sundstrom j, et al. association between serum cathepsin s and mortality in older adults. jama. 2011;306:1113–21. 21. ganna a, salihovic s, sundstrom j, broeckling cd, hedman ak, magnusson pk, et al. large-scale metabolomic profiling identifies novel biomarkers for incident coronary heart disease. plos genet. 2014;10:e1004801. 22. fall t, salihovic s, brandmaier s, nowak c, ganna a, gustafsson s, et al. non-targeted metabolomics combined with genetic analyses identifies bile acid synthesis and phospholipid metabolism as being associated with incident type 2 diabetes. diabetologia. 2016;59:2114–24. 23. lind l, arnlov j, lindahl b, siegbahn a, sundstrom j, ingelsson e. use of a proximity extension assay proteomics chip to discover new biomarkers for human atherosclerosis. atherosclerosis. 2015; 242:205–10. 24. lind l, siegbahn a, lindahl b, stenemo m, sundstrom j, arnlov j. discovery of new risk markers for ischemic stroke using a novel targeted proteomics chip. stroke. 2015;46:3340–7. 25. lind l, sundstrom j, stenemo m, hagstrom e, arnlov j. discovery of new biomarkers for atrial fibrillation using a custom-made proteomics chip. heart. 2017;103:377–82. 26. lind pm, salihovic s, van bavel b, lind l. circulating levels of perfluoroalkyl substances (pfass) and carotid artery atherosclerosis. environ res. 2017;152:157–64. 27. lind pm, van bavel b, salihovic s, lind l. circulating levels of persistent organic pollutants (pops) and carotid atherosclerosis in the elderly. environ health perspect. 2012;120:38–43. 28. lee dh, lind pm, jacobs dr jr, salihovic s, van bavel b, lind l. background exposure to persistent organic pollutants predicts stroke in the elderly. environ int. 2012;47:115–20. 29. strand r, malmberg f, johansson l, lind l, sundbom m, ahlstrom h, et al. a concept for holistic whole body mri data analysis, imiomics. plos one. 2017;12:e0169966 30. lind l, elmstahl s, bergman e, englund m, lindberg e, michaelsson k, et al. epihealth: a large population-based cohort study for investigation of gene-lifestyle interactions in the pathogenesis of common diseases. eur j epidemiol. 2013;28:189–97. 31. bergstrom g, berglund g, blomberg a, brandberg j, engstrom g, engvall j, et al. the swedish cardiopulmonary bioimage study: objectives and design. j intern med. 2015;278:645–59. 20 l. lind abstract introduction the ulsam study the pivus study the poem study the epihealth study the scapis study discussion disclosure statement notes on contributor references surrogacy relationships: a critical interpretative review review article surrogacy relationships: a critical interpretative review jenny gunnarsson paynea , elzbieta korolczuka and signe mezinskaa,b adepartment of historical and contemporary studies, sodertorns hogskola, huddinge, sweden; bfaculty of medicine, university of latvia, riga, latvia abstract based on a critical interpretative review of existing qualitative research investigating accounts of ‘lived experience’ of surrogates and intended parents from a relational perspective, this article proposes a typology of surrogacy arrangements. the review is based on the analysis of 39 articles, which belong to a range of different disciplines (mostly sociology, social psychology, anthropology, ethnology, and gender studies). the number of interviews in each study range from as few as seven to over one hundred. countries covered include australia, canada, greece, india, iran, israel, italy, mexico, norway, russia, sweden, uk, ukraine, and the usa. most studies focus only on surrogacy practices in one country (although often with intended parents from other countries), and some include several countries (e.g. interviewees from several countries or fieldwork in different field-sites). the proposed typology goes beyond the division between altruistic versus commercial, and traditional versus gestational surrogacy, in order to inform further research and to contribute to bioethical and policy debates on surrogacy in a transnational context. four types of relations are identifiable: open, restricted, structured, and enmeshed. the criteria which influence these relationships are: the frequency and character of contact preand post-birth; expectations of both parties; the type of exchange involved in surrogacy arrangements; and cultural, legal, and economic contexts. the theoretical contribution of the article is to further the development of a relational justice approach to surrogacy. article history received 3 november 2019 revised 27 january 2020 accepted 2 february 2020 keywords assisted reproduction; critical interpretative review; ethnography; qualitative interviews; qualitative methods; relational ethics; reproductive justice; surrogacy; surrogate motherhood introduction and background surrogacy, the situation where a person intentionally gets pregnant and carries a child for someone else, began flourishing in the usa in the 1980s, and today it has grown into a global trend. sometimes arrangements are non-paid (altruistic), but often they are contracted and paid (commercial). in traditional or partial surrogacy, the surrogate’s own eggs are used, hence making the surrogate and the surrogacyconceived child genetically related. in gestational or full surrogacy, the egg of either the intended mother or an egg-donor is used. partial surrogacy was more common in the earlier phase of contemporary surrogacy (1), but in contemporary surrogacy practices it is far more common to use the eggs from one of the intended parents or from a donor, often anonymous. like other forms of collaborative reproduction, surrogacy involves new parties in the process of reproduction and creates new relationships, leading to questions about emotions, rights, and responsibilities of all people involved. in current debates, there are strong disputes whether surrogacy strengthens reproductive rights (e.g. women’s right to choose, involuntarily childless people’s reproductive rights, lgbtq rights) or impedes them (e.g. surrogates’ right to abortion or to keep the child). as such, the ethical and political debates on surrogacy are intrinsically intertwined not only with medical and psychological risks, but also with issues concerning autonomy, agency, and justice, as well as risks of coercion and exploitation. moreover, the laws regulating surrogacy differ widely between countries. research question and aim through critical review of existing qualitative research, this article seeks to explore the possibilities of developing a relational justice approach to surrogacy. specifically, we seek to investigate existing accounts of the ‘lived experience’ of surrogates and intended parents from a relational perspective, focussing in particular on the way surrogates and intended parents narrate the relationship to each other and the child. the goal is to propose a typology of surrogacy arrangements going beyond the division between altruistic versus commercial and traditional/partial versus gestational surrogacy, in order to inform further research and to contribute to bioethical and policy debates on surrogacy in a transnational context. thereby, we seek to identify under what circumstances the reproductive parties themselves describe the relationship as satisfactory, fair, and just, and by extension what experts and lawmakers need to take into account when debating contact jenny gunnarsson payne jenny.gunnarsson.payne@sh.se department of historical and contemporary studies, sodertorns hogskola, huddinge 141 89 sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 2, 183–191 https://doi.org/10.1080/03009734.2020.1725935 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1725935&domain=pdf&date_stamp=2020-05-21 http://orcid.org/0000-0001-7764-6326 http://orcid.org/0000-0002-8263-5530 http://orcid.org/0000-0002-3190-100x http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1725935 http://www.tandfonline.com not only whether to permit surrogacy or not, but also how a framework of relational justice might be formulated in a way that takes the rights, well-being, and emotions of all reproductive parties equally into account throughout the surrogacy process and after the birth. previous research while there are no official global statistics, it is well established that surrogacy has recently increased exponentially (2), as has research in this field. even though there are a few notable exceptions, review articles analysing the effects of surrogacy on the surrogate, intended parents, and children are still remarkably few, and the relational aspect of surrogacy remains surprisingly understudied. s€oderstr€om-anttila et al. (3) have conducted a systematic review focussing on obstetric, medical, and psychological outcomes for surrogates, surrogacy-conceived children, and their parents. only 55 out of 1795 studies were deemed of sufficient quality for review. their review concludes that obstetric, medical, and psychological outcomes in existing studies show no significant difference between surrogacy pregnancies and other pregnancies, but argues that due to lacking numbers and quality of studies results should be interpreted with caution. van den akker (4) points out strongly selective samples in existing studies but shows that research indicates that openness and disclosure to surrogacy-conceived children about their conception are common, and that most intended parents intend to keep in touch with surrogates after birth. the study also discusses how in the uk, where much of the research is carried out, it is common for intended parents and surrogates to develop a close relationship during the surrogacy process. edelman (5) points out the lack of research but concludes that surrogacy generally seems to be a positive experience for everyone involved, and that surrogates are often motivated by altruism. likewise, ciccarelli and beckman (6) argue that although most studies are methodologically inadequate, existing evidence points in the same direction: surrogates are motivated mainly by altruism, relationships between surrogates and intended parents are experienced as satisfying, and surrogates rarely have problems relinquishing the child. teman’s (7) anthropological critical review of psychosocial research on surrogacy argues that the reviewed studies approach the issue from normative ideas about motherhood, family, and women. teman calls for research to take surrogates’ own narrated experience at face value and suggests that this may lead to new approaches in research and important implications for policy. busby and vun (8) have conducted a qualitative review of 40 empirical studies on surrogacy in order to compare it with feminist critique of surrogacy and assess to what extent their ethical concerns match empirically documented outcomes. also this review article suggests that common anxieties about exploitation and surrogate’s bonding with the baby are countered by existing evidence. it should be noted that existing review articles disproportionally deal with research conducted in more affluent countries, where financial inequalities between surrogates and intended parents are generally less stark than in transnational surrogacy arrangements in poorer countries. it is reasonable to assume that this may have significant consequences for the results. in most reviews ethnographic research is excluded altogether. with its specific focus on how the insights from existing qualitative ethnographicand interview-based research on surrogacy can contribute to debates on surrogacy, this review article addresses this lack. beyond medical risks, psychological assessments, and individual choice: a relational approach to reproductive justice the theoretical paradigm of reproductive justice seeks to go beyond both simplistic liberal notions of choice and allencompassing structural ideas of exploitation, and can therefore offer a framework for more complex and nuanced analyses of surrogacy, taking into account the agency of surrogates, potential vulnerability of intended parents, and the moral issues that arise when surrogacy services are ‘outsourced’ to less affluent countries in the world (9,10). the concept of reproductive justice has been defined as ‘the normative concept that all women, regardless of their ethnic, racial, national, social, or economic backgrounds, should be able to make healthy decisions about their bodies and their families’ (11). other researchers have argued for the need to extend the concept to include other groups that might be negatively affected by policies on reproductive rights, including hbtq þ persons (12,13), and to emphasise ‘intersectional social identities’ and the need for ‘community-developed solutions to structural inequalities’ (14). drawing on a reproductive rights paradigm we argue that surrogacy is intrinsically neither ethical or unethical, but rather that its ethical status must be assessed contextually, depending on how well it is possible in a given context to: (1) negotiate potentially conflicting interests; (2) to secure reproductive rights and needs of all the involved parties in a satisfactory way; and (3) to build a relationship that is sustainable for surrogates, intended parents, and surrogacy-conceived children during and after conception, pregnancy, and birth. for this to be conceptualized and possibly put into practice in legislation and policy, however, it is necessary to combine the more structural perspectives on reproductive justice with a relational approach to ethics. relational ethics views persons not only as autonomous agents, but also as human beings involved in relationships. each person is the centre of their own decision-making, but is at the same time related to others on different levels (family, social groups, society, etc.). this involvement in relationships influences individual decisions and embeds an understanding of justice in the context of human interaction. relational ethics is closely connected to feminist ethics and ethics of care (15). some scholars make a distinction between a ‘causal view’ and a ‘constitutive view’ in relational ethics. the ‘causal view’ emphasizes the relational context of autonomous choices influencing individual decisions. the ‘constitutive view’ looks at persons as directly involved in relations, concerns, and care for others (16). our approach is 184 j. gunnarsson payne et al. built on a constitutive view on relational ethics, and asks for justice in the context of the relationship between intended parents, the surrogate, and the surrogacy-conceived child. method we performed a critical interpretive literature review (17) specifically tailored to respond to bioethical inquiry, thereby emphasising relevance and quality of insights rather than ‘quantity for its own sake’. the review has been conducted in an iterative rather than a linear or stepwise process, using our initial research question flexibly, and continuously refining it in accordance with the results of the searches. importantly, the quality and relevance of each article have been assessed in relation to how it can help responding to the qualitative and normative research question. this means that also articles that are not easily adaptable to simple assessments of methods have been included, such as ethnographic or mixed-methods studies, which allows for the inclusion of any potentially useful insights the study can bring. only seriously flawed studies have been excluded (18). with the research question in mind we included articles which: (1) focus solely or predominantly on surrogacy; (2) are based on ethnographic data and/or in-depth interviews with surrogate mothers and intended parents, preferably including substantial quotes from respondents’ own narratives (including some relevant studies of online forums or email interviews); and (3) include analysis of relations between surrogates and intended parents, and surrogacy-conceived children and other parties involved, such as clinic staff, doctors, and agents. to ensure that all studies included have undergone assessment of quality based on their own (rather than external) disciplinary norms, the sample has been limited to articles published in peer-reviewed international journals published in english. initial searches were broad, and included hundreds of articles, but with the use of keywords such as ‘surrogates’, ‘surrogate mothers’, ‘surrogacy’, ‘intended parents’, ‘interviews’, ‘ethnography’, ‘qualitative method’, and ‘relation(s)’ it was narrowed down to more than a hundred texts. complementary searches were made to increase the geographical distribution of studies and to ensure a range of studies from different decades, spanning from early surrogacy practices in the usa in the 1980s and 1990s to recently published research. we ended up with a total of 39 articles that met all of our criteria. articles were continuously read through carefully and were manually recorded in a table, including themes, quotes, and findings relevant for this review. results selected articles belonged to a range of different disciplines, the majority of which from the interpretative human and social sciences (sociology, social psychology, anthropology, ethnology, and gender studies) and a smaller number from psychology, social work, or nursing. the number of interviews in each study ranges from as few as seven to over one hundred. countries covered include australia, canada, greece, india, iran, israel, italy, mexico, norway, russia, sweden, uk, ukraine, and the usa. most studies focus only on surrogacy practices in one country (although often with intended parents from other countries), and some include several countries (e.g. interviewees from several countries or fieldwork in different field-sites). many studies (some with overlapping samples) focus solely or mainly on surrogacy practices in a single country, e.g. thirteen on indian (19–31), nine on the us (32–40), and five articles on the israeli context (36,41–44). the least studied country contexts in this sample include greece (45), iran (46), italy (47), mexico (48,49), norway (50), sweden (51,52), and russia/ukraine (53,54). in countries where surrogacy is not legal, only intended parents through transnational surrogacy have been interviewed (e.g. italy, norway, sweden), and some studies have interviewed intended parents or surrogates in several countries (55,56). the vast majority of studies investigate commercial surrogacy arrangements, but some investigate altruistic surrogacy. these have either been conducted in countries such as the uk and greece where only non-paid surrogacy is permitted, or have been specifically designed to investigate altruistic surrogacy drawing on interviews with surrogates in several countries (56). in addition, there are some examples of this in berend’s online ethnography (32–35). most studies concern gestational surrogacy and focus exclusively or mainly on heterosexual intended parents, with only few including same-sex couples and non-heterosexual individuals (40,44). characteristics of the relation between surrogates and intended parents: closeness, expectations, and satisfaction the following analysis has been conducted in three steps. first, we identified four frequently occurring themes or ‘areas of negotiation’ in the literature, that is, areas of real or imagined potential tension or difficulty that is resolved differently in the different samples and contexts. second, we have constructed a table of ‘ideal types’ of surrogacy relationships, based on the empirical findings. third, we analysed the implications of our findings for future research, ethical considerations, and policy debate. to bond or not to bond: varieties and strategies of proximity and distance the first theme concerns the existence (or non-existence), creation, or dissolution of bonds between surrogate and intended parents, between surrogate and child, and between intended parents and child. this was significantly affected not only by geographic proximity or distance but also through the creation of emotional closeness or personal boundaries. in some cases, relationships between surrogate and intended parents were very close; especially intended mothers were very engaged throughout the pregnancy—coming to pregnancy appointments, birth classes, and delivery, often upsala journal of medical sciences 185 ‘managing the pregnancy’ by being in charge of medical aspects. in these relationships, both surrogates and intended mothers would engage in emotional work to define the pregnant belly as an extension of the intended mother. surrogates would often ‘transfer’ pregnancy symptoms to intended mothers by reporting on them (e.g. phoning when feeling nausea). some intended mothers reported a ‘pseudopregnancy’ or (psycho)somatic symptoms such as gaining weight or the secretion of colostrum or feeling contractions when the surrogate went into labour (1,42). while surrogates are continuously reported to use strategies or mental frames to separate the pregnancy and foetus from themselves (e.g. to prevent bonding or to communicate never having felt a bond), there are examples of intended parents (mainly mothers) who wear a ‘dummy belly’ or record their voice for the surrogate to play to the foetus in order to aid the bonding process (23). in some cases, physical proximity and frequent contact were used as a means to control the surrogate (diet, exercise, etc.), especially in cases where she was living together with the intended parents or in-laws (25,42). in studies where geographical distance was larger, bonds could be created and upheld over the phone or through online communication (27,33,39,47,57). choosing a distant country for surrogacy is sometimes deliberate: it may be a way to distance oneself from the surrogate and strengthen the borders of the newly established nuclear family (47). in stark contrast, israeli gay couples interviewed by ziv and freund-eschar (44) experienced significant frustration and anxiety over lack of contact with surrogates, describing an ‘abstract’, ‘theoretical’, and ‘disembodied’ pregnancy, and expressed feelings that the pregnancy consisted of documents, was lacking in emotions, and that they missed being closer to the surrogate so as to bond with the child (e.g. stroke belly). related or not related: the use of relationship and kinship vocabularies the second theme concerns the kind of relationship between surrogate and intended parents as well as the one between the surrogate and the child. we found that these are often negotiated through identification or disidentification using a family, kinship, or kinship-like vocabulary. although surrogates, most notably in the us and israeli studies, express clearly that they are not related to the baby, some differences in emphasis were detected. in the us studies, parentage was more often described in terms of intentions, while in israel the genetic link to intended parents was foregrounded (32–35). although these surrogates would generally denounce any kinship to the child, for example by likening themselves to an ‘oven’, ‘kindergarten teacher’, ‘nanny’, or ‘babysitter’ (35,53), in other studies kinship metaphors were used to describe the surrogate’s relationship to the child, like ‘auntie’ (35,47) or ‘tummy mommy’ (55), or exceptionally even ‘mom’ (23). in the indian context there are several examples of surrogates expressing a non-genetic kinship relationship to the children they carried referring to ‘blood, sweat, and milk’ (standing for both work and substances) to claim kinship or kinship-like relationships or expressing how they would miss ‘their babies’ (21,22,27). in contexts where a close relationship between surrogates and intended parents is the norm (uk, usa), kinship and family vocabulary is used to describe the relationship, e.g. that they are ‘like family’ (e.g. 35,39). in other contexts, the family bond is resisted and kinship metaphors are rarely used (35). in the indian context, the metaphor of surrogates and intended mothers being ‘sisters’ frequently recurs (20–23,25,27). context: culturally available narratives, norms, expectations, and disappointments the third theme concerns how norms and expectations of the surrogacy relationship are differently constructed and interpreted in different contexts through culturally available narratives. significantly, the correspondence or discrepancy between these narratives and the experienced reality seems to impact satisfaction and disappointment. more or less idealist narratives of surrogacy as a mutual ‘journey’ undertaken by surrogates and intended parents are widespread in some contexts where relationships are more equal (32–35,43,56). in berend’s us study, romantic metaphors were used, including language from dating and falling in love (e.g. about finding the ‘perfect couple’, ‘the ones’, ‘we clicked’) (33). even though strong similarities in narration have been identified in earlier studies (1), in contexts where surrogates and/or intended parents form online communities, narratives and norms have been shown to become more standardized, sometimes almost identical. a romanticized narrative may also work as an incentive for women to become surrogates, constructing a norm against which their own experience is measured, which can lead to disappointment (43). the reviewed studies include evidence of how surrogates consistently feel disappointment or even grief when the relationship with the intended parents does not live up the expectations. surrogates express feeling ‘ditched’, ‘tossed aside’, and betrayed when a close relationship does not materialize, fades, or deteriorates during or after pregnancy (36), or if the intended parents turn their backs in case of failed treatments or pregnancy loss (32). conversely, more precautionary cultural narratives of the surrogacy relationship can lead to distrust and are sometimes used to legitimate monitoring by agencies. these include media narratives that warn about surrogates who do not want to relinquish the baby and intended parents who do not want to claim the child, especially in case the child is ill or disabled. in other cases, precautionary narratives are cultivated by agencies and intermediaries who warn intended parents of ‘greedy surrogates’ (25,48), thereby strengthening their own position and ability to fully control the relations between them and the surrogates. in some other countries, such as russia, surrogates prefer to frame their relationship as business-like and based on legal regulations and monetary exchange, rather than trust and emotional closeness. such a narrative, as siegl (53) has observed, 186 j. gunnarsson payne et al. leaves little room for expressing feelings, which in turn may cause distress when emotions might be felt anyway. ‘the gift of life’ or monetary transaction: conceptualising the exchange the fourth theme concerns the coexistence and sometimes tension between the idea of surrogacy as a form of ‘gift-giving’ or as a business transaction. the relationship between altruism and monetary exchange is complex and is negotiated in a variety of ways across different contexts. among swedish intended parents the surrogacy relationship is described as a ‘win–win’ situation (e.g. surrogate gets money, intended parents receive their long-awaited child) (51). money might be described as a motive simultaneous to altruism, and/or affective narratives are used to downplay the economic exchange (34,40). in some contexts, such as india, expectations of extra-contractual gifts were reported (25), along with intended parents’ discomfort by being reminded of the surrogate’s poverty or requests for additional remuneration (48,49). money, however, tends to be symbolically charged in different ways, and there are many clear examples where paid surrogates do not want the relationship to be reduced to a business transaction, and cases where they retroactively regret not charging the intended parents what they felt they were entitled to, when their emotional needs were not fulfilled (32,33). narratives of altruism are frequently used by agencies in advertising, and in some countries such as india and ukraine the agency controls communication between surrogates and intended parents to avoid surrogates requesting more money (54). the russian case, where ideals of the surrogate as an entrepreneur or business person are conveyed in the interviews with surrogates (53), appears to differ significantly from other contexts. this tendency can, however, be interpreted as yet another strategy to avoid stigma and manage conflicting emotions on the part of surrogates. analysis based on the outcomes of the studies included in this review, we propose a typology of relations between surrogates and intended parents. four types are identifiable: open, restricted, structured, and enmeshed (table 1). the types take into account criteria which influence these relationships, such as frequency and character of contact pre-and postbirth; expectations of both parties; the type of exchange involved in surrogacy arrangements; as well as the cultural, legal, and economic context. open relationships open relationships are characterized by regular, caring, and intense contact between the intended parents and the surrogate. the frequency depends mostly on conditions, e.g. geographical proximity, but is often spontaneous, initiated ad-hoc. face-to-face contact often occurs at least a couple of times during pregnancy, which may be initiated by both surrogate and table 1. tentative typology of relations between surrogates, intended parents, and child. type of relation characteristics of contact expectations towards surrogacy exchange / what is being exchanged context open regular, caring, intense, also spontaneous. face-to-face at least a couple of times, with few or no intermediaries. fulfilled, high level of satisfaction on both parties. emotions: love, joy; solidarity, friendship, sense of shared experiences, as well as money and material gifts. mostly (but not exclusively) emerging in contexts where power disparities are relatively low. restricted no contact or sporadic contact, although in some cases frequent contact if surrogate lives with intended parents during pregnancy. low expectations, some surrogates are not interested in relationship, but others report that they would need more contact and support during pregnancy, while intended parents are withdrawn or absent. focus on monetary compensation and low expectations as to other aspects of exchange; no space for re-negotiating the terms. defined mostly by power inequalities, structural conditions of surrogacy industry in countries of the global south or the post-communist region, but also reinforced by diverging cultural narratives. structured planned at regular intervals: more often pre-birth, occasional post-birth (postcard or a phone call from time to time). fulfilled to a large extent, based on contractual frames, relatively low expectations as to emotional and relational aspects of surrogacy. surrogacy is structured as labour, which needs to be compensated; limited possibilities for renegotiations. dependent on the cultural scripts, a big role of legal contract for this type of relation. enmeshed characterized by tendency to expand the boundaries established in contract or expected by one party, contact is often intense in frequency or emotional content. unfulfilled or failed, due to divergent expectations and/or different cultural scripts regarding parenthood and surrogacy. diverging visions of what is being exchanged: surrogate focuses on ‘gift of life’ and assumes that obligations of intended parents go beyond contract, whereas intended parents either do not understand or do not accept any emotional or other extra-contractual obligations. mostly contexts characterized by power inequalities, where low compensation for surrogate accompanies cultural narratives around surrogacy as a precious gift, but it can also occur in other contexts. upsala journal of medical sciences 187 intended parents. this type of relationship is marked by emotional openness and direct contact with few or no intermediaries between the reproductive parties. the intended parents are usually present at birth, and the surrogate sometimes maintains regular contact with the child/parents after birth: we wanted one [surrogate] who was very open … this summer [we] spent one week with them in california. and the kids skype with her frequently. and they call her tummy mummy. (55) in open relations there is a matching level of expectations of both parties as to the development of the relationship, thus the expectations of both parties are usually fulfilled and reported levels of satisfaction high. close contact with intended parents may help the surrogate to focus her emotional bond on the intended parents rather than the baby: it is an amazing feeling to find the perfect couple … ahhh, the romance, i miss it! (33) in some situations, it is expected that the surrogate will develop a relationship with intended parents and the child as a friend or even relative. we bond more with the couples than the babies [ … ] our friendship doesn’t end at birth; hopefully it grows into more of a family bond than what’s already there! (35) [t]hey are like family to us … (39) what is being exchanged between the reproductive parties is described as emotions such as love, joy, solidarity, friendship, and sense of shared experiences, as well as material gifts: [it’s] a journey of shared love … (33) [t]hey’d be there for me anytime i need them and i’m there for them and yeah i can confide in them … they’re good friends … (57) they visit me weekly and every time they bring biscuits, fruits, and dry fruits. … they really love me a lot. (27) open relations emerge mostly (but not exclusively) in contexts where power disparities are relatively low, and where cultural narratives of surrogacy tend to match the expectations and socio-economic status of the surrogate. in case of altruistic surrogacy it is measured mostly in a close, nurturing relationship with the intended parents, and in the case of commercial surrogacy it is a combination of security, financial gratification, and emotional support according to individual needs (see also 58). restricted relationships in contrast, in restricted relationships there is normally no contact or sporadic contact between the surrogate and the intended parents, although in some cases (e.g. in india) frequent contact occurs if the surrogate lives with intended parents during pregnancy. often, there is very limited or no face-to-face contact pre-birth, and just one meeting during delivery. such relationships are mostly or fully controlled by the intermediaries (agency, doctors) and/or intended parents: [m]y first contact was through video call when i was two months pregnant, and i only met them personally when the baby was born … (49) restricted relationships are characterized by low expectations as to the quality of contact, especially on the part of the surrogates. some surrogates report not being interested in a continuous relationship as ‘their work ended with the delivery’ (35), but others claimed that they would need more contact and support during pregnancy, while intended parents were withdrawn or absent: being in contact with the parents would help her emotionally, she says, because then it would feel much clearer that the child is not hers—now, without the contact, it instead feels like she is carrying her own child. (53) lack of contact was also problematic for some intended parents, who spoke of how such an ‘abstract pregnancy’ made it difficult for them to prepare mentally for parenthood (44). for surrogates, restricted relationships might be especially traumatic in case of failed pregnancy: when intended parents walk away as if from a failed transaction, they deny the value of the surrogate’s gift and the magnitude of her sacrifice. (32) restricted relationships are framed by a focus on monetary compensation and low expectations as to other aspects of surrogacy. surrogates often reported that their needs and expectations remained unfulfilled, because intended parents often saw the surrogate just as a ‘vessel’ or a hired labourer, as expressed in the following quote: … i did not find that lady [previous surrogate] right. her words and demeanor was not very convincing after the unsuccessful conception. (26) this type of relationship increases the probability that the surrogate would regret her decision to become one: most of the surrogates in the study spoke of their distaste in doing what they had done or where undertaking, saying that they would not do it again and had been forced to do it by the circumstances. (25) the development of restricted relationships is defined mostly by already-existing power inequalities and structural conditions of the surrogacy industry, for example in countries of the global south or the post-communist region. they can also be reinforced by diverging cultural narratives, when the elevated status of the surrogate as the giver of the precious ‘gift of life’ is not matched by good conditions, monetary compensation, and/or appreciation and support. this type of relationship reflects highly unequal power relations and status between the reproductive parties and was not reported in studies on altruistic surrogacy. structured relationships structured relationships are highly contingent on pre-existing conditions, often described in the contract. such contracts usually include contact planned at regular intervals: more often pre-birth, ocasionally post-birth, a postcard or phone call from time to time. in practice, these may mean a couple of meetings, either face-to-face or internet-mediated, initiated by the surrogate or the intended parents. often the meetings are mediated through an agency, but not strictly controlled. 188 j. gunnarsson payne et al. contact before and after the birth is regulated by the contract, including: (1) lifestyle rules and behavioural restrictions; (2) rules governing breastfeeding, and ‘intimate’ form of contact with the baby; and (3) rules governing viewing, handling, and future relationships with the newborn (37). the expectations of both parties are often fulfilled to a large extent, based on contractual regulations: i’m satisfied, it’s what we wanted. if she felt she wanted more contact we’d definitely do it more [ … ] i think we’re on the same page. (47) what do i need this personal contact [with intended parents] for? i do my job and get money for it; that’s all i need to know. (53) dependent on the cultural scripts, e.g. preference for a business-like relation by russian and ukrainian surrogates (53,54), it also reflects a strategy on part of the surrogate to mediate social stigma attached to surrogacy in most cultural contexts, and facilitate emotional detachment in relation to the baby. within the framework of structured relations, surrogacy is seen as a form of labour which needs to be compensated, and there is a big role of the legal contract for the ways in which it is organized. the more inclusive it is of unexpected and changing needs, the more possibility there is for the surrogate to get the support she needs. both parties tend to report that their formal expectations were fulfilled, but these are often relatively low with regard to emotional and relational aspects of surrogacy. sometimes emotional expectations change over the course of or after pregnancy, but there is very little or no room for negotiations: she is my first baby girl. i have two sons and i always craved for a girl. i know she looks japanese but i think of her as my own daughter. [intended mother agreed that she was] on my breast for two whole months. … i miss my daughter, you don’t know how much. (23) enmeshed relationships the last type of relation is the enmeshed one characterized by a tendency to expand the boundaries established in the contract or expected by one party, as well as intensive contact both in terms of frequency and accompanied emotions. the surrogate and the intended parents may get in touch face-to-face and through social media. sometimes contact continues with increasing frequency and/or intensity postbirth, generally initiated by the surrogate rather than intended parents. relationships between the parties are often controlled via intermediaries but only to a certain point, as there is usually no mediation post-birth: clinics and agencies encourage [surrogates to demand] informal payments [post-birth] to offset the low compensation they offer to the surrogate. (25) the expectations of both intended parents and surrogates are often unfulfilled or failed, due to divergent expectations and/or different cultural scripts: i used to think they would invite us to america. i used to think of her as a sister—all of it went to waste. forget an invitation, they did not even call to see if we are dead or alive. (23) the practice of gift-giving [in india] also follows a particular form of obligatory logic, wherein the surrogate seeks compensation through gift and extra contractual money from the intended parents. (25) this type of relationships occurs mostly in contexts characterized by power inequalities, where low compensation for surrogates accompanies cultural narratives around surrogacy as a precious gift, e.g. india or mexico. such outcomes, however, can happen also in other contexts, e.g. the usa, when expectations prior to the surrogacy arrangement do not meet the quality of the relationship with intended parents during pregnancy or after birth. the surrogate focuses on ‘gift of life’ and assumes that obligations of the intended parents go beyond contract, whereas the intended parents either do not understand or do not accept any emotional or other extra-contractual obligations, even though they may eventually give in to requests: i have paid the surrogate a lot of money. i want her to understand that we are done now. she cannot keep on asking for more. (49) [h]ere i am doing something for you and you treat me like you are scared of me … (39) i went through so much physically and emotionally for them to have a family and … they vanish into the great unknown like a stranger in the grocery store. (32) conclusion our analysis highlights the ways in which the framework of relational justice can inform bioethical and policy debates. we point to the importance of a longitudinal perspective to surrogacy, and the need for recognizing changing expectations and needs of the people involved. whereas new conceptualizations of parenthood increasingly recognize that people having no genetic or gestational connection to children are part of an extended kinship network (e.g. ‘bonus parents’ in patchwork families), in the case of surrogacy there is a tendency to perceive the surrogate as either ‘the mother’ (or someone who wants to keep the child) or just a ‘vessel’ or ‘gestational carrier’. existing research shows that while most surrogates do not identify as mothers, they often wish to sustain contact with intended parents and children, and such contact is key to emotional stability and satisfaction. thus, there is a need for an informed debate and policies taking into consideration the possibility for promoting sustainable relations between the parties involved in surrogacy arrangements over a longer period of time. this requires a certain amount of flexibility and open-endedness inscribed in both legal ramifications and everyday practices, which should be adjusted for the evolving needs and emotional perspectives preand postbirth, also in case of failure at any given stage of the procedure. the possibility for including a relational justice perspective, however, depends largely on the intermediaries, especially the agencies managing commercial surrogacy, as well as non-governmental organisations helping in altruistic arrangements and state institutions. the key challenge for upsala journal of medical sciences 189 these actors is to recognize the temporal changes and openendedness of the relationship between the surrogate and intended parents, taking more responsibility not only for financial and medical risk management but also for the quality of relational aspects of surrogacy arrangements over longer periods of time. adequate medical and psychological evidence is imperative for ethical debates and policy discussions on the issue, but we need to recognize that controversies are intimately tied to issues of values that go far beyond scientific facts. surrogacy cannot be detached from cultural meanings and ideological ideals concerning motherhood/parenthood, kinship, rights and justice, fairness and equality, and ideas of agency and ‘free choice’. there is simply no ‘neutral’ way of approaching the issue. considering this complexity, we conclude that it is necessary to develop a more nuanced approach to surrogacy, one that includes a relational justice approach and merges deep knowledge of cultural norms and socio-economic inequalities in the local context with recognition of both the agency and potential vulnerabilities of surrogates and intended parents alike. acknowledgements we would like to thank the reviewer and editor for useful feedback on our article. disclosure statement the authors declare that there is no conflict of interest. funding the work on this article was funded by the foundation for baltic and east european studies [grant no. 3152–3 1.1.2017]. notes on contributors jenny gunnarsson payne, phd, professor of ethnology, department of historical and contemporary studies, s€odert€orn university, huddinge, sweden. elzbieta korolczuk, phd, researcher, school of historical and contemporary studies, s€odert€orn university, sweden. signe mezinska, phd, associate professor in bioethics at the university of latvia, faculty of medicine and researcher at s€odert€orn university, sweden. orcid jenny gunnarsson payne http://orcid.org/0000-0001-7764-6326 elzbieta korolczuk http://orcid.org/0000-0002-8263-5530 signe mezinska http://orcid.org/0000-0002-3190-100x references 1. ragon�e h. chasing the blood tie: surrogate mothers, adoptive mothers and fathers. american ethnolo. 1996;23:352–65. doi:10. 1525/ae.1996.23.2.02a00090 2. 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parents of children conceived by donor insemination, egg donation and surrogacy. reprod biomed online. 2011;22:485–95. doi:10.1016/j. rbmo.2011.01.014 upsala journal of medical sciences 191 https://doi.org/10.1177/0973174118778481 https://doi.org/10.1186/s13010-016-0040-6 https://doi.org/10.1186/s13010-016-0040-6 https://doi.org/10.1177/0069966719836883 https://doi.org/10.1177/0069966719836883 https://doi.org/10.1177/0891243213486832 https://doi.org/10.1215/10679847-3320149 https://doi.org/10.1186/1747-5341-8-10 https://doi.org/10.1086/671018 https://doi.org/10.1086/671018 https://doi.org/10.1111/j.1548-1387.2010.01099.x https://doi.org/10.1111/j.1573-7861.2012.01362.x https://doi.org/10.1111/aman.12444 https://doi.org/10.1080/13648470.2017.1401825 https://doi.org/10.1111/lasr.12125 https://doi.org/10.1016/j.fertnstert.2016.08.013 https://doi.org/10.1300/j137v04n01_05 https://doi.org/10.5153/sro.4312 https://doi.org/10.1525/maq.2003.17.1.78 https://doi.org/10.1177/1357034x09337780 https://doi.org/10.1177/1357034x09337780 https://doi.org/10.1080/01459740.2018.1532423 https://doi.org/10.1080/01459740.2018.1532423 https://doi.org/10.1177/1066480714565107 https://doi.org/10.1016/j.wombi.2015.07.005 https://doi.org/10.1111/jjns.12158 https://doi.org/10.1093/humrep/dex245 https://doi.org/10.1177/0891243218823344 https://doi.org/10.1177/0891243218823344 https://doi.org/10.1177/0308518x18769652 https://doi.org/10.1177/0308518x18769652 https://doi.org/10.1080/13648470.2017.1392100 https://doi.org/10.1080/13648470.2017.1392100 https://doi.org/10.1371/journal.pone.0126518 https://doi.org/10.1371/journal.pone.0126518 https://doi.org/10.1016/j.rbms.2018.08.001 https://doi.org/10.3167/ajec.2018.270202 https://doi.org/10.1093/hsw/hly018 https://doi.org/10.1177/1464700117700048 https://doi.org/10.1016/j.rbmo.2014.06.004 https://doi.org/10.1016/j.rbmo.2011.01.014 https://doi.org/10.1016/j.rbmo.2011.01.014 abstract introduction and background research question and aim previous research beyond medical risks, psychological assessments, and individual choice: a relational approach to reproductive justice method results characteristics of the relation between surrogates and intended parents: closeness, expectations, and satisfaction to bond or not to bond: varieties and strategies of proximity and distance related or not related: the use of relationship and kinship vocabularies context: culturally available narratives, norms, expectations, and disappointments ‘the gift of life’ or monetary transaction: conceptualising the exchange analysis open relationships restricted relationships structured relationships enmeshed relationships conclusion acknowledgements disclosure statement references daily systemic energy expenditure in the acute phase of aneurysmal subarachnoid hemorrhage article daily systemic energy expenditure in the acute phase of aneurysmal subarachnoid hemorrhage christoffer nyberga, elisabeth ronne engstr€oma, lars hillereda and torbj€orn karlssonb adepartment of neuroscience, section of neurosurgery, uppsala university, uppsala, sweden; bdepartment of surgical sciences, section of anesthesiology and intensive care, uppsala university, uppsala, sweden abstract background: patients with subarachnoid hemorrhage often have impaired consciousness and cannot regulate nutritional intakes themselves. previous studies have demonstrated elevated energy expenditure in the acute phase, but it is not known whether the energy demand is constant during the first week after onset of the disease. in this study, we performed daily measurements of energy expenditure with indirect calorimetry during the first 7 days after aneurysmal subarachnoid hemorrhage in mechanically ventilated patients. methods: metabolic measurements were performed daily with indirect calorimetry in 26 patients with aneurysmal subarachnoid hemorrhage. all patients were intubated and mechanically ventilated. the measured value was compared to the predicted values from the harris–benedict equation and the penn state university 1998 equation. urinary nitrogen excretion was measured daily. results: there was a significant increase in energy expenditure during days 2–3 compared to days 5–6. the harris–benedict equation underestimated metabolic demand. the penn state 1998 equation was closer to the measured values, but still underestimated caloric need. urinary nitrogen excretion increased throughout the first week from initially low values. conclusions: there is a dynamic course in energy expenditure in patients with aneurysmal subarachnoid hemorrhage, with increasing metabolic demand during the first week of the disease. indirect calorimetry could be used more often to help provide an adequate amount of energy. article history received 11 january 2019 revised 14 august 2019 accepted 20 august 2019 keywords energy expenditure; indirect calorimetry; subarachnoid hemorrhage introduction spontaneous subarachnoid hemorrhage (sah) is a potentially life-threatening condition that activates a stress response. this in turn triggers numerous events that prepares the body to meet the challenges of severe illness, including activation of the hypothalamic-pituitary-adrenal axis and of the sympathetic nervous system (1,2). the stress response also may influence the patient’s metabolic demand. apart from the initial impact at the time of onset of disease, there are several severe complications (e.g. vasospasm, rebleeding, hydrocephalus, and infections) that may affect energy expenditure (ee) in the acute phase. in critically ill patients, who cannot regulate nutritional intake themselves, nutritional treatment is especially important. it has previously been shown that the nutritional balance may have impact on the outcome after stroke (3,4). underfeeding as well as overfeeding may affect outcome. predictive equations to estimate the nutritional need have been developed and refined since the early 1900s. the original equation by harris and benedict (h-b) from 1919 is still being used to calculate the basal metabolic rate (5). several attempts have been made to make more accurate predictive equations, taking into account different factors that may influence energy balance. a patient with severe illness, sedation, and a need for mechanical ventilation presents an even more complex challenge. numerous ways to predict ee in this heterogeneous group have been suggested. the equation from penn state 1998 (psu-98) (6) has previously been shown to correlate with measurements by indirect calorimetry (ic) for patients with severe traumatic brain injury (7). in this equation, h-b is combined with body temperature and respiratory minute volume to compensate for increased metabolism caused by the systemic inflammatory response. ic is currently considered to be the most precise and widely available method for measuring ee in critically ill patients (8). however, the equipment is expensive, and in most centers ic is not routinely used. measurements with ic on patients with hemorrhagic stroke have been reported. in most of these studies, single short measurements have been performed once or twice during the acute stage of the disease (9–14). in patients with sah, measured ee has generally then been elevated compared to the energy demand predicted by equations. the aim of this study was to investigate ee during the first week in patients with aneurysmal sah. we carried out daily measurements with indirect calorimetry during the first contact christoffer nyberg christoffer.nyberg@neuro.uu.se department of neurosurgery, uppsala university hospital, 751 85 uppsala, sweden � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 4, 254–259 https://doi.org/10.1080/03009734.2019.1659888 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1659888&domain=pdf&date_stamp=2019-12-09 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2019.1659888 http://www.tandfonline.com week after onset of sah to investigate if ee had a dynamic course. daily urinary nitrogen excretion was measured to estimate protein catabolism. we also performed comparisons between measured and calculated ee using predictive equations. materials and methods patients and sah management patients were included from october 2010 until july 2014. mechanically ventilated patients with spontaneous sah were considered for inclusion. twenty-six patients were included with at least two measurements using indirect calorimetry during the first week after sah. the patients were managed according to the department of neurosurgery’s protocols for sah (15). briefly, this includes early diagnosis and treatment of aneurysm. patients with impaired consciousness were intubated and normo-ventilated. a ventricular catheter was placed for drainage of cerebrospinal fluid and monitoring of intracranial pressure. sedation in mechanically ventilated patients was obtained, mainly with propofol and bolus doses of morphine. enteral nutrition was administered through a gastro-enteral tube. intravenous nutrition was only used in selected cases. nimodipine was administered for three weeks. symptoms of delayed cerebral ischemia were considered to be due to vasospasm when other causes of deterioration were ruled out. vasospasm treatment was then given with increase in blood volume and blood pressure and in selected cases intra-arterial administration of nimodipine. secondary insults putting further strain on the brain’s supply and use of energy and oxygen were systematically detected and treated if present (16). the uppsala university regional ethical review board for clinical research granted ethical permission. predictive equations calculations of the metabolic rate (r; kcal/day) were done for each patient with the harris–benedict equation, the penn state 1998 equation, and by using 25 kcal per kilogram body weight. h-b men: r ¼ 66:473 þ ð13:7516 � wþ þ ð5:0033 � lþ � ð6:755 � aþ (1) h-b women: r ¼ 655:0955 þ ð9:5634 � wþ þ ð1:8496 � lþ�ð4:6756 � aþ (2) penn state 1998: r ¼ ð1:1 � harris–benedictþ þ ð140 � tmaxþ þ ð32 � _veþ–5:340 (3) where w is weight in kg, l is height in cm, a is age in years, tmax is maximum temperature in �c in the past 24 hours, and v_e is minute volume in l/min. metabolic measurements the energy expenditure was determined by measurements with the indirect calorimeter quark rmr (cosmed, rome, italy). before each measurement, the equipment was calibrated according to instructions from the manufacturer. the measurements lasted for approximately 4.5 hours. records without v_o2 and v_co2 values were removed. in order to exclude invalid measurements, values were removed that deviated more than two standard deviations from the mean. from the resulting data, daily energy expenditure was calculated by taking the median from all collected data points. measurements were started as soon as possible after admission, typically on the day after admission, and continued daily throughout the first week after sah, or until the patient was no longer mechanically ventilated or had respiratory problems requiring >50% fio2. on some occasions, patients were unavailable for measurements because of time-consuming examinations or surgical procedures. in order to compare metabolic measurements between patients, the ratio of the measured value to h-b was calculated. ratios of the measured values were also calculated to the psu-98 estimates of ee, and to body weight. to be able to compare early and late measurements despite the fact that some patients were missing measurements, the mean of days 2–3 and the mean of days 5–6 were calculated for all patients. urine was collected for 24 hours repeatedly and analyzed for urea by the routine method used in the hospital chemistry department. from this value, daily urinary nitrogen excretion was calculated. statistics statistical analyses were performed with r (r foundation for statistical computing, vienna, austria). changes in ee and nitrogen excretion over the entire time period were evaluated with the skillings–mack test. wilcoxon matched pairs test was used to compare early and late measurements in days 2–3 and days 5–6. values of p < 0.05 were considered statistically significant. results the mean age of the included patients was 59 years (range 41–81). eighteen patients were of female gender. world federation of neurosurgical societies sah grade (17) at admission was i in 1 patient, ii in 5 patients, iii in 1 patient, iv in 15 patients, and v in 4 patients. all patients were diagnosed as having a ruptured aneurysm. the aneurysms were located in the anterior circulation in 21 patients, and in the posterior circulation in 5 patients. the aneurysms were treated with endovascular methods in 22 patients and with open surgery in 4 patients. fourteen patients were conscious at discharge from the neurosurgical unit, 9 patients were unconscious, and 3 patients died before discharge. in total, 118 ic measurements were performed during the first week after onset of sah. the measured values increased upsala journal of medical sciences 255 in the later part of the first week (figure 1). when comparing days 2–3 with days 5–6, the difference was statistically significant (p ¼ 0.017). measured values versus predictive equations for each patient and ic measurement, the ratio of ic to the expected value was calculated using the h-b equation (figure 2) and the psu-98 equation (figure 3). the ratio of ic to the h-b equation increased during the first week after sah, peaking at day 6 when the measured value was about 60% higher than expected. there was a difference between days 2–3 and days 5–6, p ¼ 0.020. the ratio of measured ic to the psu-98 equation also increased in a similar manner. the variations were, however, smaller than with the h-b equation. on the day with the peak value, day 6, the measured value was less than 20% higher than expected. figure 1. daily ee measured with indirect calorimetry. significance testing over time with the skillings–mack test, p ¼ 0.030. median with 25%–75% interquartile range and minimum–maximum values. figure 2. ee measured with indirect calorimetry related to the harris–benedict equation. significance testing over time with the skillings–mack test, p ¼ 0.030. median with 25%–75% interquartile range. 256 c. nyberg et al. the ee as measured with ic was also calculated to kg of body weight (figure 4). ee was close to 25 kcal/kg in the first measurements and increased to more than 30 kcal/kg on day 6. urinary nitrogen excretion from initially low values on the second and third day after sah, there was a distinct increase throughout the first week (figure 5). discussion many patients with acute aneurysmal hemorrhage have impaired consciousness and cannot regulate their nutritional intake. ensuring these patients an adequate caloric intake may be one factor affecting the outcome. to better understand their caloric requirements, we performed a study with repeated measurements using ic during the first week after sah. to our knowledge, this is the first study with this high number of patients and daily repeated measurements with ic during the first 7 days after sah. figure 3. ee measured with indirect calorimetry related to the penn state 1998 equation. significance testing over time with the skillings–mack test, p ¼ 0.12. median with 25%–75% interquartile range. figure 4. ee measured with indirect calorimetry related to body weight. significance testing over time with the skillings–mack test, p ¼ 0.041. median with 25%–75% interquartile range. upsala journal of medical sciences 257 one difficulty when comparing ee between and within patients is the fact that the basal metabolism varies with multiple factors such as age, body weight, height, gender, and systemic inflammatory state. in order to be able to compare measured ee in this group of patients, we used the ratio of measured ee to the h-b equation. the predicted ee with the h-b equation is constant during the first week, since it is based on constant or slowly changing factors. this means that the calculated ratio can be used not only to compare ee within patients during the first week after sah, but also between patients. this calculation can also be used to compare ee measured with ic to the expected value from the h-b equation. from our results, it seems clear that the hb equation underestimates the metabolic demand in this patient group. since the h-b equation was developed for prediction of the basal metabolic rate in healthy individuals, it is not surprising that the equation does not apply without modification on patients with severe illness. we performed the same type of calculation with another predictive method, the psu-98 equation. this is one of the equations developed to predict caloric need in an intensive care unit (icu) setting. it is based on the h-b equation, but also includes information about respiration and body temperature to compensate for the increased metabolism caused by the systemic inflammatory response. our results indicate that the psu-98 equation may underestimate ee in patients with sah, although the difference between predicted and calculated ee is smaller than when the h-b equation is used. also, the increase in ee that we found during the course of the disease was accounted for by the psu-98 equation since no significant difference was found. this suggests that the psu-98 equation is a better alternative than the h-b equation for this patient group. the variation between patients is still substantial, and the perfect predictive equation for sah patients is yet to be found. one way of deciding how much energy to provide to a patient in intensive care is to estimate 25 kcal per kg body weight (18). this calculation is most likely widely used because of its simplicity. with the patients in this study, using the 25 kcal/kg equation underestimates ee, especially in the later part of the first week. on day 6, measured ee was above 30 kcal/kg. urinary nitrogen excretion can be used as an indicator of protein turnover. our measurements showed low nitrogen excretion the first days after onset of disease. thereafter, there seemed to be a linear increase in nitrogen excretion. a similar pattern has been demonstrated in plasma measurements in previous studies (19). one possible interpretation is a shift in protein metabolism towards catabolism. whether the possible catabolic state is only due to the disease itself is unclear. the increase in ee during the acute phase after sah is almost parallel to nitrogen excretion, raising the suspicion that the proteolytic state could be enhanced by underfeeding. in this study, we did not collect detailed information about nitrogen intake. this means we cannot rule out high protein intake as a factor influencing nitrogen excretion, but the measurements of ee or nitrogen excretion did not influence the type of nutrition or energy provided to the patients. one factor possibly contributing to the low values the first day is the fact that enteral nutrition in our unit is not provided the first day after onset of disease, thereby causing low protein metabolism. measured ee increased gradually during the first 6 days, and there was a significant difference in ee between days 2–3 compared to days 5–6, in line with recent investigations (14). an increase in metabolic demand could be expected figure 5. daily nitrogen excretion in urine. significance testing over time with the skillings–mack test, p < 0.001. median with 25%–75% interquartile range. 258 c. nyberg et al. after an intracranial aneurysm rupture and the subsequent triggering of a stress response. the findings in this study suggest that ee has a dynamic course after sah, and it is difficult to rely on a predictive equation to determine nutritional requirements. in order to provide an adequate amount of energy to sah patients, it would be beneficial to routinely use ic. our results illustrate that a single measurement with ic is only valid on the day of the measurement and cannot be used universally during the first week after sah. there are some limitations of our study. the metabolic measurements with ic was only performed on mechanically ventilated patients, meaning that mostly poor-grade patients were included. furthermore, the limited number of patients in the study made it impossible to compare between different groups of patients based on treatment modality, complications, and outcome. we conclude that energy expenditure in mechanically ventilated patients with aneurysmal sah increases gradually during the first week after sah. prediction of metabolic demand with equations is often unreliable. in this study, the psu-98 equation performed best of those tested but, on the whole, underestimated ee. these results suggest that ic could be beneficial in patients with sah and mechanical ventilation. acknowledgements we would like to thank the staff in the neurointensive care unit for helping with the measurements. disclosure statement no potential conflict of interest was reported by the authors. notes on contributors christoffer nyberg is a neurosurgeon and neurointerventionist at uppsala university hospital. elisabeth ronne-engstr€om is an adjunct professor of neurosurgery at uppsala university hospital. lars hillered is a professor of neurosurgery at uppsala university hospital. torbj€orn karlsson is an associate professor of anesthestesiology and intensive care at uppsala university hospital. references 1. espiner ea, leikis r, ferch rd, macfarlane mr, bonkowski ja, frampton cm, et al. the neuro-cardio-endocrine response to acute subarachnoid haemorrhage. clin endocrinol (oxf). 2002;56:629–35. 2. zetterling m, engstrom be, hallberg l, hillered l, enblad p, karlsson t, et al. cortisol and adrenocorticotropic hormone dynamics in the acute phase of subarachnoid haemorrhage. br j neurosurg. 2011;25:684–92. 3. davalos a, ricart w, gonzalez-huix f, soler s, marrugat j, molins a, et al. effect of malnutrition after acute stroke on clinical outcome. stroke. 1996;27:1028–32. 4. gariballa se, parker sg, taub n, castleden cm. influence of nutritional status on clinical outcome after acute stroke. am j clin nutr. 1998;68:275–81. 5. harris ja, benedict fg. a biometric study of basal metabolism in man. washington: carnegie institution of washington; 1919. 6. frankenfield d, smith js, cooney rn. validation of 2 approaches to predicting resting metabolic rate in critically ill patients. jpen j parenter enteral nutr. 2004;28:259–64. 7. krakau k. energy balance out of balance after severe traumatic brain injury. uppsala, sweden: acta universitatis upsaliensis; 2010. 8. oshima t, berger mm, de waele e, guttormsen ab, heidegger cp, hiesmayr m, et al. indirect calorimetry in nutritional therapy. a position paper by the icalic study group. clin nutr. 2017;36: 651–62. 9. esper dh, coplin wm, carhuapoma jr. energy expenditure in patients with nontraumatic intracranial hemorrhage. jpen j parenter enteral nutr. 2006;30:71–5. 10. kasuya h, kawashima a, namiki k, shimizu t, takakura k. metabolic profiles of patients with subarachnoid hemorrhage treated by early surgery. neurosurgery. 1998;42:1268–74; discussion 1274–5. 11. nagano a, yamada y, miyake h, domen k, koyama t. increased resting energy expenditure after endovascular coiling for subarachnoid hemorrhage. j stroke cerebrovasc dis. 2016;25:813–18. 12. piek j, zanke t, sprick c, bock wj. resting energy expenditure in patients with isolated head injuries and spontaneous intracranial haemorrhages. clin nutr. 1989;8:347–51. 13. touho h, karasawa j, shishido h, morisako t, numazawa s, yamada k, et al. measurement of energy expenditure in acute stage of cerebrovascular diseases. neurol med chir (tokyo). 1990; 30:451–5. 14. shimauchi-ohtaki h, tosaka m, ohtani t, iijima k, sasaguchi n, kurihara h, et al. systemic metabolism and energy consumption after microsurgical clipping and endovascular coiling for aneurysmal subarachnoid hemorrhage. acta neurochir (wien). 2018;160: 261–8. 15. persson l, enblad p. neurointensive care of aneurysmal sah. acta neurochir suppl. 1999;72:73–80. 16. enblad p, persson l. impact on clinical outcome of secondary brain insults during the neurointensive care of patients with subarachnoid haemorrhage: a pilot study. j neurol neurosurg psychiatry. 1997;62:512–16. 17. teasdale gm, drake cg, hunt w, kassell n, sano k, pertuiset b, et al. a universal subarachnoid hemorrhage scale: report of a committee of the world federation of neurosurgical societies. j neurol neurosurg psychiatry. 1988;51:1457. 18. cerra fb, benitez mr, blackburn gl, irwin rs, jeejeebhoy k, katz dp, et al. applied nutrition in icu patients. a consensus statement of the american college of chest physicians. chest 1997;111: 769–78. 19. ronne-engstrom e, cesarini kg, enblad p, hesselager g, marklund n, nilsson p, et al. intracerebral microdialysis in neurointensive care: the use of urea as an endogenous reference compound. j neurosurg. 2001;94:397–402. upsala journal of medical sciences 259 abstract introduction materials and methods patients and sah management predictive equations metabolic measurements statistics results measured values versus predictive equations urinary nitrogen excretion discussion acknowledgements disclosure statement notes on contributors references upsala j med sci 88: 1-8, 1983 bile salt sulphation in man liver bile salt sulphotransferase activity in patients with primary biliary cirrhosis lars loof and anders nyberg department of internal medicine, university hospital, uppsala, sweden abstract bile salt sulphation in primary biliary cirrhosis was studied by measure ments of the liver bile salt sulphotransferase levels in 16 patients. although the enzyme activity varied among the patients it did not correlate with the severity of cholestasis. furthermore, the mean bile salt sulphotransferase magnitude in patients with primary biliary cirrhosis did not differ signifi cantly from corresponding enzyme activity in patients with non-cholestatic, alcohol induced liver disease. the present data indicates that chronic chole stasis, as evidenced in patients with primary biliary cirrhosis, does not lead to increased levels of liver bile salt sulphotransferase. it is suggested that mechanisms other than enzymic induction are responsible for the increased bile salt sulphate synthesis as observed in primary biliary cirrhosis. introduction sulphation, identified by palmer (14) as one pathway of the human bile salt metabolism, occurs to only a minor extent i n the healthy man (1, 12, 21, 26). patients with cholestasis eliminate large amounts o f bile salt sulphates in the urine (1, 1 2 , 21, 23, 26). although the pathophysiological significance of bile salt sulphation has not been determined it is regarded to be important for the elimination o f potentially toxic bile salts (22). however, few data concerning the enzymatic mechanisms for bile salt sulphate synthesis in clini cal conditions with cholestasis are available. the enzyme, bile salt sulpho transferase, which transfers sulphate groups form 3'-phosphoadenosine-5'-phos phosulphate (paps) to bile salts, has been identified in the human liver, (5, 8), small intestine ( 9 ) and adrenals (11). in the present investigation bile salt sulphation in primary biliary cirrhosis (pbc), a liver disease characte rised by a chronic intrahepatic cholestasis ( 1 6 ) , has been studied by measu rements of the bile salt sulphotransferase levels in percutaneous liver biopsy specimens. 1-832859 1 material and methods patients the study was carried out o n patients admitted for clinical investigation of liver disease to the department of internal medicine, university hospital, uppsala, sweden. sixteen patients with primary biliary cirrhosis were examined (table i). drug treatment at the time of the study included prednisolone (pat. n o . 2, 5 and l o ) , spironolactone (pat. n o . 1 and 2), cholestyramine (pat. n o . 4 , 7, 11 and 1 4 ) , cimetidine i levothyroxin (pat. n o . 14). the criteria f o r the diagnosis were: a) blood chemistry analyses of liver function indicative of cholestasis; b) liver histology diagnostic o r highly suggestive of pbc. c) normal extrahepatic bile ducts on cholangiography. d) positive serum mitocond rial antibody. routine percutaneous liver biopsies were performed ad modum menghini (13). liver histology was analysed and classified in four stages by one pathologist. (stage 1 florid duct lesion, stage 2 = ductular proliferation, lymphoid aggregates with preserved lobular architecture, stage 3 = fibrosis with septum formation and disturbed lobular architecture, stage 4 = cirrhosis (16). liver function was also evaluated by the intravenous galactose tolerance test ( 2 4 ) . eight patients with alcoholic liver disease were also included in the study (table 11). the patients had no drug therapy. the diagnosis was based on a history of alcohol abuse together with abnormal liver function tests. the clinical investigation did not reveal any alternative etiology to the liver disease. i n these patients blood chemistry analyses of liver function were not indicative of cholestasis. chemicals chemicals used were of analytical grade and obtained from kebo grave, stockholm, sweden, unless otherwise specified. the sodium salt of glycolitho cholate (calbiochem) was stored dessicated at +4oc. for preparation of buffers and other solutions deionized water was used. carrier-free (35s)-labelled sodium sulphate was purchased from the radio chemical centre ltd, amersham, england. radioactive 3' -phosphoadenosine-5 ' -phosphosulphate ( (35s)paps) was biosyn thesised and purified as earlier described (10). radioactive samples were counted in 5 ml lumagelb in a packard tricarb 3000 liquid scintillation coun ter. total serum bile salts were determined by a ready made enzymatic kit (sterognost-3 ci flu, nygaard & co, o s l o , norway). 2 tissue p repa rat ion the percutaneous liver biopsy specimens were divided into two parts, one for histological examination and the other for analysis of sulphotransferase activity. the latter specimen was immedeatly transported to the laboratory in ice cold 0.15m kc1 containing 0.02 m tris/hcl (ph 7.5), 0.001 m, edta and 0 . 0 0 1 m 2-mercaptoethanol. unless otherwise specified all further procedures were carried out at + 4 o c . the tissue was homogenised in 0 . 5 ml of the aforementioned buffer and after addition of another 0.5 ml of the buffer the homogenate was centrifuged for 60 min. at 105000 x g in a beckman l2-65b ultracentrifuge. the clear supernatant was pipetted off and stored at -7ooc in 0.2-0.3 ml portions until used. sulphotransferase assay a modification (10) of the sulphotransferase assay described previously ( 8 ) was used. the incubation mixture contained: a) 2.5 m o l glycolithocholate added as an ethanolic solution and evaporated to dryness in vacuo; b) 7.5 nmol (35s)paps, specific activity 50 cpm/pmol, in 100 pl 0.15 m k c 1 containing 0 . 0 2 m tris/hcl (ph 7.5), 0 . 0 0 4 m edta and 0.001 m 2-mercaptoethanol; c) 50 1-11 of liver cytosol containing 10-40 1-18 of protein. incubations were carried out in duplicate for 30 min. at 37oc followed by butanol extraction and quantification of radioactive sulphate esters by liquid scintillation (8). one enzyme unit was defined as the amount of enzyme neces sary to catalyze the formation of one pmol glycolithocholate sulphate per 3 minute. specific enzyme activity is expressed as units/mg cytosol protein. the protein content of liver cytosol was determined by the method of lowry (7). statistics students t-test was used for testing the statistical significance between two means. means (x) and standard deviation (s.d.) were calculated by conven tional methods (2). results liver bile salt sulphotransferase activity towards glycolithocholate in patients with pbc, table i, varied between 42 and 193 units/mg cytosol protein. no obvious difference with respect to the sulphotransferase activity was found between drug treated patients and patients without drugs. the lowest enzyme activities belonged to the two men included in the pbc group. no correlation was found between bile salt sulphotransferase activity and serum concentration o f bilirubin, total bile salt and alkaline phosphatases or to the intravenous 3 p t a b le 1 . p ri m ar y b il ia ry c ir rh o s is . p a ti e n t d a ta . p a ti e n t a ge s ex l iv e r s -b il ir u b in s -a lk . s -b il e am a i .v . g a la k to se l iv e r b il e s a lt n o. y e a rs h is to lo g y p m o l/ l p h o sp h . s a lt ti tr e to le ra n c e su lp h o tr a n sf e ra se st a g e p k a t/ l p rn o ll l (n e g ) te s t u n it sl m g c y to so l iiv (4 2 1 )+ < (0 .8 -4 .8 ) (2 .7 -7 .3 ) t + m in p ro te in (< 1 7 m in ) 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 15 1 6 74 0 i1 5 2 0 i1 1 43 0 i1 1 48 0 i1 1 6 4 6 11 1 47 6 i1 1 5 7 0 iv 6 6 0 iv 5 6 0 iv 6 2 0 iv 5 6 0 iv 39 0 iv 42 0 iv 36 0 iv 5 9 0 iv 59 0 iv 7 19 9 82 50 27 7 8 1 7 4 1 4 20 19 29 15 1 3 4 3 1 2 3 4 1 1 .5 1 4 .0 1 6 .7 4 2 .0 6 .0 1 7 .5 1 1 .7 9 .3 1 1 .6 1 0 .4 2 9 .0 2 5 .0 3 5 .0 1 9 .2 3 7 .0 1 2 .8 2 .0 2 2 .0 7 .0 1 7 .0 1 7 .0 2 2 .0 3 9 .3 5 4 .0 4 .6 3 1 .6 1 7 .0 1 2 .0 5 9 .0 2 7 0 .0 2 5 .0 1 0 .0 m ea nk sd 5 4 2 1 1 ~ _ _ _ _ ~ 66 ?8 9 1 9 .3 2 1 1 .o 3 8 2 6 4 .0 1 1 4 00 11 40 0 11 40 0 1 /1 0 0 11 40 0 1 /1 0 0 1 / 4 00 11 10 0 11 40 0 1 /4 0 0 11 40 0 1 /2 5 11 10 0 11 40 0 1 /4 0 0 11 40 0 2 1 1 3 18 1 8 19 1 5 2 3 44 2 3 1 3 17 11 1 6 28 2 1 9 4 11 7 10 0 12 6 42 76 18 8 79 14 6 1 2 1 13 3 1 2 5 72 12 2 1 2 4 1 9 3 20 28 11 6+ 40 ;‘i n or m al ra n g e o f se ru m v a lu e s i s g iv e n i n p a ra n th e si s. galactose tolerance test. moreover, no correlation was found between the bile salt sulfotransferase level and the known duration of the disease. the bile salt sulphotransferase activity in patients with alcoholic liver disease, table 11, varied between 70 and 136 units/mg cytosol protein. also in this group the lowest values were found in the male patients. when the mean liver bile salt sulphotransferase activity in patients with pbc (116 2 40 units/mg cytosol protein) and patients with alcoholic liver disease (106 % 26 units/mg cytosol protein) were compared no significant diffe rence was found. d i s c u s s i o n in the present work liver bile salt sulphotransferase activity was measured in primary biliary cirrhosis. the patients had varying degree of cholestasis as indicated by the serum concentrations of bilirubin and bile salts. the enzyme levels differed by 500 % when the highest and lowest values were compared. however, the variations in bile salt sulphotransferase activity were not correlated to the degree of cholestasis. furthermore no correlation was found between the bile salt sulphotransferase activity and liver function as evidenced by the intravenous galactose tolerance test. we have not measured the amounts of bile salt sulphates excreted in the urine from these patients. however, other investigations have shown that patients with p b c constantly have an increased urinary elimination of bile salts which mainly appear in the sulphated form ( 1 5 ) . moreover, the magnitude of the bile salt sulphate eli mination in the urine seem to correlate well with the serum bile salt concen tration (15, 2 6 ) . thus, the present data suggest that patients with pbc, a clinical condition characterized mainly by the symptoms of a chronic chole stasis, do not increase their sulphation capacity by induction of liver bile salt sulphotransferase when the cholestasis progresses. this assumption is further supported by the observation that the mean bile salt sulphotransferase activities in p b c and in alcoholic liver disease with no clinical signs of cholestasis, respectively, do not differentiate significantly. these results also agree with the reported absence of bile salt sulphotransferase induction in the livers from bile duct ligated hamsters ( 4 ) . the individual data of the male patients in the p b c group and in the alcohol liver disease group with respect to bile salt sulphotransferase activity had a tendency to be lower than the corresponding enzyme activity in females. we have not observed a sex diffe rence with respect to human bile salt sulphotransferase activity in the liver in previous studies (9). however, steroid sulphotransferase activity, including bile salt sulphotransferase, in the rat and hamster seem to be significantly influenced by sex hormones ( 3 , 17, 18, 1 9 , 2 0 ) . female rat and hamster livers 5 ta bl e 2. a lc oh ol ic l iv er d is ea se . pa ti en t da ta . pa ti en t ag e se x li ve r sbi li ru bi n sal k. i . v . li ve r bi le sa lt sbi le n o . ye ar s hi s t o l og y pm ol /l ph os ph . sa lt ga la kt os e su lp ho tr an sf er as e pk at /l p m o l/ l to le ra nc e un it s/ mg cy to so l (4 -2 1) (0 .8 -4 .8 ) (2 .7 -7 .3 ) te st t k m in pr ot ei n (< 17 m in ) 6 7 8 54 41 38 45 73 63 22 44 d 0 0 _ _ n or ma l st ea to si s st ea to si s ci rr ho si s in fl am ma ti on , si de ro si s no rm a 1 no rm al s t ea to si s 8 4. 8 1. 5 15 6 5. 3 4. 5 12 20 2. 1 6. 5 14 11 2. 0 3. 5 23 4 3. 3 4. 5 31 9 2. 8 2. 5 20 7 2. 2 2. 5 11 19 3. 5 10 .0 16 70 78 12 7 10 6 12 9 80 11 8 13 6 me an ts d 48 t 16 11 t 6 3. 3t l. 2 4 . 4 f 2. 7 18 f 7 10 6 t 26 contain two to six times the activity in males (17, 20). even if such a sex difference might be present in man and is taken into consideration because of the uneven sex distribution between the pbc and alcoholic liver group it would rather support the observed absence of bile salt sulphotransferase induction in primary biliary cirrhosis. the increased biosynthesis of bile salt sulphates as observed in pbc (15, 26) may instead be caused by a better substrate availability for bile salt sulphotransferase when the bile salt concentration increases in the cholestatic liver as suggested by barnes (4). in addition extrahepatic sulphation, e.g. in the small instestine or adrenals (9) may contribute to the enhanced sulphation of bile salts in primary biliary cirrhosis. acknowledgements this work was supported by grants from the swedish medical research council, project n o . b 81-03p-5727-02 and b81-03x-5449-03. references 1 2. 3. 4. 5 6 7. 8. 9. 10. 11. 12. almc, b., bremmelgaard, a., sjovall, j. & thomassen, p.j.: analysis of metabolic profiles of bile acids in urine using a lipophilic anion exchanger and computerized gas-liquid chromatography-mass spectrometery. lipid res 18:339-362, 1977. armitage, p.: statistical methods in medical research. blackwell scien tific publications, oxford, 1973. barnes, s., burhol, p . , zander, r., haggstrom, g., settine, r. & hirscho witz, b.: enzymatic sulphation o f glycochenodeoxycholic acid by tissue fractions from adult hamsters. j lipid res 20:952-959, 1979. barnes, s., burhol, p., zander, r. & hirschowitz, b.: the effect of bile duct ligation on hepatic bile acid sulphotransferase activity in the hamster. biochem med 22:165-174, 1979. chen, l.-j., bolt, r.j. 6: admirand, w.: cholyl sulphokinase. enzymatic sulphation of bile salts. gastroenterology 67;782, 1974. chen, l.-j.: development and regulation of bile salt sulphotransferase in rat liver. int. workshop on sulphate conjugation and sulphate metabolism. noordwijkerhout, the netherlands, 20-23 sept, 1981. to be published. lwory, o.h., rosenbrough, n., farr, a.l. & randall, r.j. protein measure ment with the folin phenol reagent. j biol chem 193:265-275, 1951. m6f, l. & wengle, b.: enzymatic sulphation of bile salts in human liver. biochim biophys acta 530:451-460, 1978. loof, l. & wengle, b.: enzymatic sulphation of bile salts in man. scand j gastroenterol 14:513-519, 1979. loof, l. & hjertcn, s.: partial purification of a human liver sulphotrans ferase active towards bile salts. biochim biophys acta 617:192-204, 1980. loof, l.: enzymatic sulphation of bile salts in man: bile salt sulpho transferase activity in human adrenal. digestion 21:297-303, 1981. makino, i., hashimoto, h., shinosaki, k., yoshino, k. & nakagava, s.: sulphated and non-sulphated bile acids in urine, serum and bile of patients with hepatobiliary diseases. gastroenterology 68:545-553, 1975. 7 13. 14. 15. 16. 17, 18. 19. 20. 21. 22. 23. 24. 25. 26. menghini, g.n.: one-second biopsy of the liver-problems of its clinical application. n engl j med 283:582-585, 1970. palmer, r. : the formation of bile acid sulphates: a new pathway of bile acid metabolism in humans. proc natl acad sci 58:1047-1050, 1967. samuelsson, k., aly, a . , johansson, c. & norman, a . : serum an urinary bile acids in patients with primary biliary cirrhosis. scand j gastroenterol 1981. (to be published). sherlock, s.: primary biliary cirrhosis. progress in liver diseases 5:559-574, 1976. singer, s . , giera, d., j o h n s o n , j. & sylvester, s.: enzymatic sulphation of steroids. i. the enzymatic basis for the sex difference in cortisol sulphation by rat liver preparations. endocrinology 98;963-974, 1976. singer, s . , gebhard, j. & hess, e.: enzymatic sulphation of steroids. v. partial purification and some properties of sulphotransferase 111, the major glycocorticoid sulphotransferase of liver cytosols from male rats. can j biochem 56:1028-1035, 1978. singer, s . : enzymatic sulphation of steroids. i v . control of the hepatic glycocorticoid sulphotransferase activity and the individual glucocor ticoid sulphotransferases from male and female rats by adrenal glands and corticosteroids. endocrinology 103:66-73, 1978. singer, s . , kutzer, t. & lee, a.: enzymatic sulphation of steroids. viii. control of hepatic cortisol sulphation and glucocorticoid sulphotrans ferases of rats by the pituitary gland. endocrinology 104:571-575, 1979. stiehl, a . : bile salt sulphates in cholestasis. eur j clin invest 4:59-63, 1974. stiehl, a . : disturbances of bile acid metabolism in cholestasis. clinics in gastroenterology 6:45-67, 1977. stiehl, a . , becker, m., czygan, p., frohling, w., kommerell, b., rott hauwe, h. & senn, m.: bile acids and their sulphated and glucuronidated derivatives in bile, plasma and urine of children with intrahepatic cholestasis: effects of phenobarbital treatment. eur j clin invest 10: 307-316, 1980. tengstrgm, b., hjelm, m., deverdier, c.-h. & werner, i.: intravenous galactose tolerance test with the use of an enzymatic method for the determination of galactose. am j digest dis 12:853-861, 1967. thomassen, p.: urinary bile acids in late pregnancy and recurrent chole stasis of pregnancy. eur j clin invest 9:425-432, 1979. vanberge henegouwen, g . , brandt, k.-h., eyssen, h. & parmentier, g.: sulphated and unsulphated bile acids in serum, bile and urine of patients with cholestasis. gut 17:861-869, 1976. address for reprints: lars liiiif, m.d. department of internal medicine university hospital sweden s-750 14 uppsala 8 remarkable developments in our performance editorial remarkable developments in our performance most certainly, this is the last time you will hold a newly printed issue of upsala journal of medical sciences (ujms) in your hands. for quite a long time we have discussed whether we should join the present trend in scholarly publishing, i.e. terminating the printing of scientific journals. in our case, it will mean an interruption of a 155-year-old tradition (1). it should, however, save us money since both printing and distribution costs have increased lately. an introduction of article processing charges (apcs) paid by the authors might have solved these financial problems. we are, however, not particularly fond of that alternative and wanted to avoid that type of burden on scholarly publishing. you might remember last year’s editorial dwelling on coalition s and how that could affect our journal (2,3). that combination of gold open access publication at no cost for the authors is unique and should attract manuscripts from all over the world with, we hope, interesting research news. on top of that, one should not underestimate the impact of a farewell to the printing era on our aim to produce a sustainable journal. all in all, this has made us look for an alternative publishing process. last february, the editors of our journal and the scandinavian journal of urology gathered editors from medical journals edited in sweden for a 1-day conference at svenska l€akares€allskapet (i.e. the swedish medical society) in stockholm. amongst the speakers a small company had been invited – open academia – run by two people with great experience in scholar publishing who at present serve nine other journals. they offer the exact key services we need and, what’s more, at a modest price. most importantly, we can retain control over the design and maintenance of our journal website and layout. moreover, finances are based figure 1. metrics of ujms over the latest 10 years. upsala journal of medical sciences 2020, vol. 125, no. 4, 263–264 https://doi.org/10.1080/03009734.2020.1830579 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1830579&domain=pdf&date_stamp=2020-10-19 https://doi.org/10.1080/03009734.2020.1830579 http://www.tandfonline.com on a cost-per-published-page model. we are very much looking forward to this collaboration. there will be a lot of work but also opportunities to give both our journal and our website a fresh look. at this point in time, it might be a good idea to look back to see what we have achieved together with taylor & francis (t&f) over the last 10 years. the number of submitted manuscripts has doubled (figure 1(a)), the number of total cites in the clarivate statistics has increased by a factor of four (figure 1(b)), and, finally, the 5-year impact factor has now passed the 3.0 level (figure 1(c)). a couple of comments on these figures might be of interest. first of all, the lack of an increase in the submission figure last year was due to a maintenance failure of the submission portal during a 5month period. this year the figure will be close to 400 submitted manuscripts. combining this figure with the number of published papers per year, 40–50 per volume, gives a rejection rate of about 80–90%. this might be the back of the coin. naturally, the lack of apcs is of great interest to researchers in financially disadvantaged research environments. numbers of total cites – perhaps the most relevant citation figure published – have also increased over the years from one per day to more than three at present. finally, this year’s impact factor value – we prefer to show the 5-year value since it is the most reliable figure for small journals – has passed another whole number level for the first time. in our editorial 2 years ago we elaborated on the introduction of a new bibliometric scoring system introduced by elsevier – the so-called citescore (4). since then, they have changed the calculation procedure. rather than using citation figures for the last 3 years, they now use the last 4 years. for ujms it has meant an obvious change for the better. we are still placed just above the top 90th percentile amongst journals in ‘general medicine’, but our score value has more than doubled over the last 10 years (figure 1(d)). we are grateful for this opportunity to thank some of our collaborators at t&f – håkan pårup, therese granlund, and sarah hands – all of whom have contributed extensively over this 10-year period. one major problem for all journals of our size and reputation is finding referees for the submitted papers. we truly pay tribute to all of you who have graciously offered your time to read our manuscripts. without better support for this system – yes, it might be regarded as old-fashioned, but still there is no better alternative – it might all collapse and be handed over to strictly commercial interests. this is an issue, very much emphasized in the summing-up document of the editor conference mentioned above. so, although this is the last issue of the journal produced on paper in the classical way, we plan to continue to publish four issues a year as before. when each issue has been finalized it will be sent as a pdf file to all members of the upsala medical society by e-mail. you will be able to glance through it as you can do with many of our daily journals nowadays or indeed even print it out. in the latter case, you will be able to save it on your bookshelf in the time-honoured way for simple and reliable consultation whenever you like. references 1. karlsson t. progress and milestones in scientific communication-a 150 years perspective. ups j med sci. 2015;120:63–4. 2. andersson a, espes d, lau b€orjesson j. further improvement of our metrics-will plan s affect them? ups j med sci. 2019;124: 215–7. 3. coalition s, plan s: making full and immediate open access a reality. available at: https://www.coalition-s.org/ 4. andersson a, lau b€orjesson j. a new contribution to research metrics. ups j med sci. 2018;123:191–3. arne andersson editor of upsala journal of medical sciences arne.andersson@mcb.uu.se joey lau b€orjesson editorial assistant kerstin westermark chair of upsala medical society � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 264 a. andersson et al. https://www.coalition-s.org/ outline placeholder references somatostatin receptor 1–5; expression profiles during rat development full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 somatostatin receptor 1–5; expression profiles during rat development eva ludvigsen, carina carlsson, eva tiensuu janson, stellan sandler & mats stridsberg to cite this article: eva ludvigsen, carina carlsson, eva tiensuu janson, stellan sandler & mats stridsberg (2015) somatostatin receptor 1–5; expression profiles during rat development, upsala journal of medical sciences, 120:3, 157-168, doi: 10.3109/03009734.2015.1035413 to link to this article: https://doi.org/10.3109/03009734.2015.1035413 © informa healthcare published online: 30 apr 2015. submit your article to this journal article views: 661 view related articles view crossmark data citing articles: 6 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 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ludvigsen1, carina carlsson1, eva tiensuu janson2, stellan sandler1 & mats stridsberg2 1department of medical cell biology, uppsala university, uppsala, sweden, and 2department of medical sciences, uppsala university, uppsala, sweden abstract background. somatostatin acts through five receptor subtypes (sstrs 1–5). we aimed to investigate sstrs mrna expression and protein distribution in whole rat embryos, with special emphasis on the pancreas. material and methods. rat embryos were collected on embryonal days 10, 11, 12, 14, 15, 17, 19, 21, and at birth. presence of sstrs was investigated with rt-pcr techniques and immunohistochemistry. results. there was no sstr5 mrna expression in the whole rat embryos. all sstr1–5 proteins were observed at embryonal day 10, but the localization varied between the different subtypes. from day 11 to birth sstrs protein presence increased with time in major structures such as skin and cartilage. it remained similar over time in the heart and liver. in the fetal pancreas mrna expression of sstr2 and 4 was detected at day 14, and there was an increase up to birth. only sstr1 protein colocalized to a higher extent with the islet hormones studied. sstr2 was present in all islet endocrine cells except for b-cells. in contrast, the immunostaining for sstr3–4 was co-localized with insulin and pp, and, finally, sstr5 with glucagon and pancreatic polypeptide. in mrna isolated from whole rat embryos sstr1–2 and sstr4 expression showed a peak at day 14, while sstr3 mrna was not present until day 15. conclusion. the present data suggest a role for sstrs during the development of the rat embryo. subsequent functional studies may elucidate regulatory roles of specific sstrs for the growth and differentiation of the pancreas as well as other organs. key words: development, mrna expression, pancreas, rat embryo, somatostatin receptors introduction somatostatin is a cyclic polypeptide hormone with two biologically active forms, somatostatin-14 and the nterminal extended version, somatostatin-28. the hormone was initially described in the brain, but is today known to exist in a variety of tissues and organs (1-3). the peptide is known to inhibit secretion of hormones from endocrine cells associated with growth, development, and metabolism (2,3). the inhibitory effects of somatostatin are mediated by specific g-protein coupled membrane receptors (sstrs). these sstrs are able to activate a variety of intracellular signalling mechanisms, leading todifferentcellular responses. to datefivemammaliansstrshavebeencloned,studied, and named according to the order of identification (4-7). they are distinguished by their pharmacological specificity for different somatostatin analogues, tissue distribution, regulation, and intracellular signalling pathways (2,3,8). inhibition of hormone secretion and growth is mainly considered to be mediated by sstr1, sstr2, and sstr5 (9,10). in the pancreas, decreasedinsulinsecretionisthoughttoberegulatedvia sstr5,whiledecreasedglucagonsecretionismediated via sstr2 (11,12). inhibition of growth hormone secretion is more complex and is considered to be a combined effect of signalling through both sstr2 and sstr5 (13). moreover, signalling via sstr2 and sstr3 is believed to be involved in apoptosis (14,15).so far, not muchis known about thefunctional correspondence: dr mats stridsberg, department of medical sciences, akademiska sjukhuset, ing 40, 5 tr, 751 85 uppsala, sweden. fax: +46 18 552562. e-mail: mats.stridsberg@medsci.uu.se (received 19 february 2015; accepted 11 march 2015) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1035413 http://informahealthcare.com/journal/ups mailto:mats.stridsberg@medsci.uu.se effects mediated via sstr4. there are reasons to believe that two or more sstrs may operate together toelicitspecificfunctions,whichissupportedbythefact that several sstrs are expressed in the same tissues or organs (2,16,17), and recent findings also support the idea of cross-talk between different sstrs (18,19). previous studies on sstrs distribution have mainly focused on tissues or organs from adult animals. however, some studies have implied a critical role for somatostatin and its receptors during embryonic development in different species (20-23). regional distribution of sstr mrnas in the rat brain at embryonic day 17–18 has been investigated using in situ hybridization (23). high expression of sstr3 mrna and low expression of sstr1 and sstr2 mrnas were demonstrated, whereas sstr4 and sstr5 mrnas were absent in distinct areas of the brain. in the adult rat brain the sstr mrnas were highly expressed, and it was suggested that some sstrs might play an important role during neurogenesis (23). during early neurogenesis in the rat sstrs were present in a transient manner, with a peak around embryonal day 14, and it was suggested that the transient expression of sstrs was associated with critical episodes in the development of a cohort of neurons and that somatostatin may play a fundamental role during neurogenesis (22). moreover, in a recent study in orange-spotted grouper, the presence of sstr1–3 and sstr5 mrna expression during development was demonstrated (24). however, teleost fish lack sstr4, but they have sstr6 instead which is believed to be lost in mammals and birds (25). the pancreas is a foregut derivative and develops from a dorsal and a ventral outgrowth of the primitive duodenum. in rodents early pancreatic buds become visible around embryonal day 9. subsequently, the ventral pancreatic bud rotates around the stomach and duodenum and fuses with the dorsal pancreatic bud. during organogenesis, the pancreatic epithelium proliferates and invadesthesurroundingmesenchyme. epithelial cells then differentiate into ductal, endocrine, and acinar cells. cells containing glucagon have been detected as early as embryonic day 9.5, and one day later insulin-containing cells have been demonstrated. cells secreting somatostatin appeared atday15, andpp-producing cellswere identifiedatday 18.5 (26,27). in the latter contextit is interestingthat in previous studies of fetal murine pancreases coexpression of more than one of the four major islet hormones during development was observed, but also co-localization with other polypeptides not demonstrated in the adult pancreas (28). it could be envisaged that embryonic islet cell expression of sstrs may play a role in the further differentiation of islet cells. the aim of this study was to follow the mrna and protein expression profiles of the sstrs during normal development in rat embryos, with special emphasis on the pancreas, from embryonic day 10. materials and methods animals embryos were obtained from pregnant sprague dawley rats (bomholtgaardresearch and breeding center, ry, denmark) and from a local outbred sprague dawley rat strain (29). the animals were housed in a room with a 12-h light/dark cycle and had free access to water and pelleted food. the regional laboratory animal ethics committee in tierp, sweden, approved all experiments. rats were killed by cervical dislocation at embryonic day 10, 11, 12, 14, 15, 17, 19, 21, and at postnatal day 1 (newborn). embryos were dissected out, and deciduas and membranes were carefully removed. further dissection of the pancreas was performed on some of the embryos. all whole embryos (n = 1–8) or pancreases isolated from embryos (n = 2– 10) of one pregnant rat were pooled and considered as one observation (n = 1–3). after birth, rna from one pancreas of one fetus was treated as one observation. pancreases from adult animals were used as controls. pcr measurements of sstr mrnas, preparation of total rna expression profiles of the sstr mrna in both total embryos and embryonic pancreas were studied. total rna from embryonic pancreases from pregnant rats (n = 15) was collected from embryonic day 14 up to birth. total rna from either pooled whole embryos or pooled fetal pancreases was isolated using rneasy mini kits (qiagen ag, basel, switzerland) according to the manufacturer’s description. briefly, embryos or pancreases from embryos of one pregnant rat were homogenized in buffer rlt. next, 70% ethanol was added to the homogenates, and the samples were mixed and applied to rneasy mini columns. homogenates were centrifuged, the flow-through was discarded, and the columns were washed. membrane-bound rna was treated with dnase i to exclude chromosomal dna. columns were subsequently washed and rna eluted using rnase-free water. rna was precipitated with 0.3 m naac and ice-cold 99% ethanol, washed and dissolved in water at a final volume of 10 ml. 158 e. ludvigsen et al. preparation of cdna synthesis of cdna was performed using a reversed transcription system (promega, madison, wi, usa). foreachreactionof20ml,9mloftotalrna,5mmol/l mgcl2, 1 � reverse transcription buffer, 1 mmol/l of each dntp, 1 u/ml recombinant rnasin� ribonuclease inhibitor, 15 u/mg amv reverse transcriptase, and 0.5 mg (dt)15 primer were used. the rna and primers were preincubated for 5 min at 60�c, then incubated for 60 min at 42�c, followed by 5 min at 99�c, and subsequently stored at –20�c. analysis of sstrs mrna expression the cdna synthesized was amplified using a lightcycler (roche diagnostics gmbh, mannheim, germany). specific primers for the sstrs were designed by tib molbiol (berlin, germany) (table i). duplicates of the cdna synthesized were amplified according to the lightcycler protocol in a final volume of 10 ml containing 5 ml faststart dna master sybr green (roche molecular biochemicals, mannheim, germany) and 0.5 mm of the sense and anti-sense primers. we assessed the stability of expression of various housekeeping genes, such as tata-binding protein (tbp) (cybergene ab, stockholm, sweden), glucose-6-phosphate dehydrogenase (g6pdh), and porphobilinogen deaminase haem biosynthetic enzyme (pbg) (tib molbiol syntheselabor, berlin, germany). the tbp mrna was found to be most stable in accordance with the total rna concentration, in embryos at day 10–21 (data not shown) and was therefore used as housekeeping gene. the lightcycler run version 5.32 was used with the following parameters: 1) denaturation: 95�c 10 min; and 2) cycling: 95�c 15 s, 60�c 10 s, and 72�c 15 s. data were analysed using meltingpoint curves, temperature-dependent dissociation of the products, verifying that the correct product was amplified. furthermore, the purity of the products was checked using agarose gel separation. all samples had a crossing point between 19 and 39 cycles. melting curve analyses confirmed no primer-dimer products. results were recalculated comparing the difference between crossing point (cp) values of the amplified sample and the housekeeping mrna using the formula 2-d (cpssts-cptbp). immunohistochemistry the entire embryo or its pancreas was immediately fixed in 4% formalin (merck, darmstadt, germany) for 24 h at room temperature, and then moved into 70% ethanol and embedded in paraffin. sections of 5 mm were attached to polysine� glass slides (menzel-gläser, braunschweig, germany) for further treatment as below. sstr1–5 antibodies. all pancreases were immunostained for the five sstrs as previously described (29). the production and specificity of subtype-specific somatostatin receptor antibodies have been described and discussed earlier (30,31). briefly, the immune reaction was amplified by an avidin-biotin complex coupled to alkaline phosphates (vectastain abc-ap; vector laboratories inc., burlingame, ca, usa) and visualized with vector red (vector laboratories) as substrate (30). when sstr-specific antibodies were preincubated with the peptides used for immunization, the immunoreactivity for each receptor was blocked (data not shown) (30). morphological evaluation. the expression and distribution of sstrs were evaluated in a light microscope. the anatomical distribution of sstrs in rat fetuses during development was noted, and the intensities of sstr immunoreactivities were scored semi-quantitatively: ++ = marked positive immunoreactivity; + = weak positive immunoreactivity; – = not detected. theratembryoorpancreaticspecimenswereevaluated with the examiner being unaware of the origin of the sections and the identity of the antibody used. double immunofluorescence of sstr1–5 and islet hormones in the fetal pancreas.. to investigate the cotable i. pcr primers. primer sequences tbp forward acccttcaccaatgactcctatg reverse atgatgactgcagcaaatcgc pbg forward cctggcatacagtttgaaatcat reverse tttccctaaaaacaacagcat g6pdh forward attgaccactacctgggcaa reverse gagatacacttcaacactttgacct sstr1 forward caagatgaagaccgcaacc reverse cacgatgggcaagataacc sstr2 forward gctcgtggaaaagcaagatgt reverse ccgttgaggtcaaagggag sstr3 forward tggctgtgctctggtggta reverse ggattcagtcagcagccaactag sstr4 forward ggaagcaggtggtggtcag reverse atttgcattcgcaggaagtct sstr5 forward ctgtcctgcacagagacacg reverse cattggcgatgctgagc somatostatin receptors in development 159 expression of sstr1–5 in pancreatic islet cells of the rat fetus we used a previously described immunofluorescence method (30). fetal pancreatic specimens were collected, paraffin-embedded, and stained for sstr1–5 in a cocktail with chicken anti-insulin (1:750, immunsystem, uppsala, sweden), chicken anti-glucagon (raised against human glucagon, 1:400, a kind gift from associate professor anders larsson; novo nordisk, bagsvaerd, denmark), sheep anti-somatostatin (1:25; guildhay, guildford, uk), or sheep anti-pp (1:25; serotech, oxford, uk) as previously described (30). the immune reaction was visualized with a cocktail consisting of secondary antibodies, cy3-conjugated donkey anti-rabbit igg (1:100; jackson immunoresearch, west grove, pa, usa), and cy2-conjugated donkey anti-sheep igg (1:100; jackson immunoresearch) or cy2-conjugated donkey anti-chicken igg (1:100; jackson immunoresearch). using different dilutions of the antibodies as well as omitting the primary antibodies, we tested the specificity of the commercial antihormone antibodies. evaluation of immunofluorescence. pancreatic sections were examined in a leica leitz dmr fluorescence microscope (leica microsystems, wetzlar, germany) equipped with filters of 492 nm to 510 nm for cy2 (green) and 550 nm to 570 nm for cy3 (red). pictures from a zeiss axiocam camera (carl zeiss, oberkochen, germany) of pancreatic islets, using both filters, were merged together with adobe photoshop 7.0 software (adobe, san jose, ca, usa), in which a yellow colour indicated co-expression of sstr subtypes with one of the four islet hormones tested in this study. results were expressed as a percentage of sstr-positive cells in relation to the total number of the respective islet cell type in a specific pancreatic islet. statistical analysis data are presented as means ± sem, and groups of data were compared using student’s t test, where p < 0.05 was considered as statistically significant. the computer program used was sigmastat 2.0 (spss science, chicago, il, usa). results immunocytochemical detection of sstrs during rat development to investigate the protein expression of sstrs in different embryonic tissues, whole rat embryos from embryonic day 10 to birth were collected and immunostained for sstr1–5. tissues were identified using light microscopy, and sstr expression was graded (for details see table ii). in figure 1 representative sections from rat embryos immunostained for sstrs at different stages of development are demonstrated. sstr1 protein expression was identified in the neural tube, yolk sac, and aorta on day 10 and 11, and in aorta from day 11 (figure 1a), while expression in other tissues was detected from day 14–15 and onwards. retina was an exception, with sstr1 expression present from day 19 (figure 1f). the expression was usually weak, but in skin and cartilage it was more intense for 2 and 3 consecutive days. expression of sstr2 was similar to sstr1 with a few exceptions: expression in the heart was not detected until day 14, while sstr2 was detected in the retina from day 15 (figure 1b). sstr3 was expressed during early development in all organs except for the heart, where it was detected from day 11 and skin from day 14. the expression gradually decreased in some organs beginning in the cartilage and retina from day 17, later in the liver, and lens from day 19. expression was usually weak, but the lens and muscle showed a more intense staining (figure 1d). sstr4 expression was found early in the neural tube, yolk sac, skin, heart, liver, and brain, while cartilage, retina, and lens showed expression on day 15 or later (figure 1e, g). sstr4 was not expressed in muscle. sstr5 was the most universally expressed receptor, being present in all tissues investigated and with an intense staining in the skin, lens, muscle, and brain (figure 1c). sstrs mrna expression during development of the embryonic rat pancreas sstr1: the expression of sstr1 was faint at day 14. at day 15 the levels were higher and peaked at day 17. subsequently, at day 19 it disappeared, but returned with high expression in the newborn. in the adult pancreas, expression of sstr1 was very weak (figure 2a). sstr2: expression of sstr2 was present at day 14–15 with a peak at day 17, but then there was a decline in the mrna levels. in the pancreas of adult rats sstr2 expression was absent (figure 2b). sstr3: levels of sstr3 were under the detection level at day 14–19. in the newborn the levels increased, and there was high expression of sstr3 in the adult rat pancreas (figure 2c). sstr4: low sstr4 levels were detected at days 14–19. in the newborn the levels were much higher 160 e. ludvigsen et al. table ii. summarized protein expression of sstr1–5 in major structures identified in rat embryos from day 10 to birth. structure embryonal day sstr1 sstr2 sstr3 sstr4 sstr5 neural tube 10 + + ++ + ++ 11 ++ ++ ++ + + 12 + – + ++ ++ yolk sac 10 ++ ++ + ++ + 11 + + + + + 12 n.i. n.i. n.i. n.i. n.i. aorta 10 + + + ++ + 11 + ++ + + + 12 – – + + + skin 10 n.i. n.i. n.i. n.i. n.i. 11 n.i. n.i. n.i. n.i. n.i. 12 – – – + + 14 + ++ + ++ ++ 15 ++ ++ + ++ ++ 17 ++ ++ + ++ ++ 19 + + – – ++ newborn + ++ ++ ++ ++ heart 10 – – – – + 11 ++ – ++ + + 12 + – + ++ + 14 + + + + + 15 + + + + + 17 + + – + + liver 14 + + + + + 15 + + + + + 17 – + + + + 19 + + – – + newborn n.i. n.i. n.i. n.i. n.i. cartilage 14 – + + – + 15 + + + – + 17 ++ ++ – + + 19 ++ ++ – + + newborn ++ + – + + retina 14 – – + – + 15 – + + – + 17 – ++ – + + 19 ++ ++ – – + newborn n.i. n.i. n.i. n.i. n.i. lens 14 – – ++ – + 15 + – ++ + ++ 17 + + + ++ ++ 19 – + – – + newborn n.i. n.i. n.i. n.i. n.i. somatostatin receptors in development 161 table ii. (continued). structure embryonal day sstr1 sstr2 sstr3 sstr4 sstr5 muscle 14 n.i. n.i. n.i. n.i. n.i. 15 + + ++ – ++ 17 + + ++ – ++ diencephalon 14 + + + ++ ++ 15 + + + + ++ 17 ++ + + – + mesencephalon 14 + + + ++ ++ 15 + + + + ++ 17 n.i. n.i. n.i. n.i. n.i. rhomencephalon 14 + + + ++ ++ 15 + + + + ++ 17 n.i. n.i. n.i. n.i. n.i. the distribution and intensity of sstr staining were scored semi-quantitatively. the immunostainings are reported for sstr1, sstr2, sstr3, sstr4, and sstr5 for all major structures in rat embryos. + weak positive staining; ++ clear positive staining; – not detected; n.i. tissue not investigated. a: day 11 sst1 d: day 15 sst3 eye e: day 15 sst4 skin f: day 19 sst4 eye g: day 22 sst4 skin b: day 15 sst1 c: day 17 sst5 d d figure 1. the immunohistochemical staining for sstr subtypes. a: at embryonal day 11 (sstr1, magnification 100�); b: day 15 (sstr2, magnification 16�); and c: day 17 (sstr5, magnification 16�) in rat embryos. positive staining by sstr antibodies is highlighted by red colour. d–g: structures in rat embryos from day 15 to birth (magnification 200�). d: positive staining of sstr3 in the lens at day 15. e: the outer part of the skin expresses sstr4 at day 15; f: part of the eye is positive for sstr1 at day 19; and g: the outer part of the skin is immunostained for sstr5 in the newborn. 162 e. ludvigsen et al. and comparable to the high expression levels of sstr1. in adult pancreatic tissues there was no sstr4 expression (figure 2d). sstr5: expression of sstr5 was low in all fetal pancreatic specimens. this was in contrast to the adult pancreas where mrna levels were high (figure 2e). sstrs mrna expression in entire rat embryos total rna of rat embryos (n = 11) was collected from embryonal day 10 up to birth. embryos from one pregnant rat were considered as one observation. sstr1: there was no expression of sstr1 in rat embryos 10–11 days old. it increased markedly from figure 2. expression of sstr mrnas in the embryonic rat pancreas amplified using real-time pcr. a: sstr1; b: sstr2; c: sstr3; d: sstr4; e: sstr5. data are given as mean relative expression (2-d(cpssts-cptbp)) of embryonal pancreases from the same day. somatostatin receptors in development 163 day 12 with a peak at day 14. at day 15–19 the sstr1 expression decreased, but increased slightly again in the newborn (figure 3a). sstr2: the expression of sstr2 was barely detectable at day 10 in the embryos, but increased markedly up to day 14 followed by a decline during development (figure 3b). sstr3: we were unable to detect sstr3 mrna before embryonic day 15. sstr3 expression increased 5-fold at day 19 but disappeared in the newborn (figure 3c). sstr4: there was some expression of sstr4 at day 11, and it then increased with a peak at day 14 (figure 3d). figure 3. expression of sstr mrnas of entire rat embryos amplified using real-time pcr. a: sstr1; b: sstr2; c: sstr3; d: sstr4; e): sstr5. embryos pooled from one pregnant rat (n = 11) were considered as one observation. 164 e. ludvigsen et al. sstr5: at all time points investigated, there was no expression of sstr5 mrna (figure 3e). presence of sstrs in endocrine cells of the embryonal pancreas pancreatic specimens from embryonal days 19, 21, in the newborn, and from adult rats were used to investigatethedistribution ofsstrsinpancreaticisletcells. in all investigated pancreatic sections at least 10 islets (n=10–20) were identifiedandtheirislet cellscounted. the results are summarized in table iii. it should be noted that sstr immunofluorescence was not analysed in the exocrine part of the pancreas. sstr1 was highly expressed in band pp-cells and had a moderate to low expression in aand d-cells. expression was especially low in a-cells on day 21 and in the newborn. sstr2 displayed a very weak expression in embryonal b-cells, while the expression was moderate in adult rats. in the other pancreatic islet cell types sstr2 showed a high to moderate expression. sstr3 showed a low to moderate expression in band -cells, while the expression was high in the other two cell types, although declining with time in pp-cells. sstr4 showed a high expression in embryonal b-cells at day 21 and in the newborn, while the expression in the other islet cells was low or absent. in the adult pancreatic islets expression of sstr4 was low to moderate except for the d-cells. sstr5 was not expressed at all in embryonal b-cells, while adult rat pancreas had a high expression. the highest expression was found in -cells, while dand pp-cells had a moderate expression. discussion mrna expression in whole rat embryos in whole rat embryos the sstr mrna expression was very weak at day 10 but showed a peak already at day 14, followed by a decrease at day 15 in the fetus. a similar expression profile was demonstrated in the optic pathways and cerebellum during rat table iii. quantitative analysis of the expression of sstrs in b-cells, a-cells, d-cells, or pp-cells in the fetal rat pancreas from day 19, 21 and in the newborn. percentages are proportion of cells in which immunofluorescence was detected. embryonal day a sstr1 (%) sstr2 (%) sstr3 (%) sstr4 (%) sstr5 (%) b-cells e19 80 2 15 0 0 e21 5 2 30 67 0 newborn 85 2 2 65 0 adult b 50 40 40 35 60 a-cells e19 50 75 2 5 80 e21 5 65 15 10 15 newborn 5 60 5 30 40 adult b 40 80 20 20 85 d-cells e19 35 55 5 0 20 e21 15 18 45 0 5 newborn 10 60 75 10 15 adult b 60 70 70 80 40 pp-cells e19 60 85 100 20 30 e21 20 20 40 15 35 newborn 80 30 35 20 15 adult b 30 15 15 15 20 a developmental stage of pancreatic specimens investigated. b for comparative purposes observations from a previous study (30) on adult rat pancreases have been included. somatostatin receptors in development 165 development (22). the most highly expressed mrna was sstr1, followed by sstr4 and sstr2 mrnas (figure 3). interestingly, according to a phylogenetic analysis, the sstr1 and sstr4 subtypes are more closely related to each other, forming a subfamily, than to the other three sstrs which form a separate subfamily (25). thus, our finding of similar gene regulation for sstr1 and sstr4 is consistent with their closer evolutionary relationship. the expression of sstr5 was weak, while sstr3 expression was absent during day 10–14 in the fetus. recent studies demonstrated that the sstrs genes might be agedependent. this may suggest that different sstrs are expressed not only in a particular tissue, but also in a critical time frame during development of rodents (25,32,33). this is in line with our data in that the sstrs mrna expression varied over time during development of the pancreas as well as the whole fetus. organ-specific protein expression to investigate the organ-specific sstr protein expression, subtype-specific sstr antibodies were used on paraffin-embedded embryos collected at embryonal days 10 to birth. because of their size, newly born fetuses could not be cut, therefore only the neck and head were cut and stained. due to this, the comparison of protein expression data obtained from these tissues with sstr mrna expressions from pooled whole embryos must be interpreted with caution. this circumstance may explain the discrepancy with, for example, the rapid drop for sstr1 at e19 to e21 (80% to 5%). this pattern was observed in several of the animals included in this study, but further studies are needed to explore this issue. in the skin, sstr4 and sstr5 were the subtypes first detected, and then the other sstrs followed at day 14 (table ii). in a previous study proliferating epithelial cells were demonstrated to express mainly sstr2 and sstr5, which may suggest a developmental role for sstrs in angiogenesis (34). another study reported that all five sstrs mrnas were expressed in adult retina, with sstr2 and sstr4 as the most abundant, suggesting an important role for the sstrs in the eye (35). in our study the retina expressed only sstr3 and sstr5 at embryonic day 14, and sstr2 appeared later. sstr4 expression was first observed at day 17 in the retina, which was at the same time point as the sstr3 expression disappeared. the immunostaining of sstr1 was not detectable until day 19 in the retina. we showed that all five sstrs were present in the brain at day 14 and that sstr4–5 were strongly expressed. in diencephalon the intensity of sstr4 seemed to decline with time. the function and role of sstr4 is as yet mainly unknown. in our study we demonstrate that during development the sstr4 expression is detected almost in all tissues studied, with time suggesting an important developmental role for sstr4, but further studies will be needed to confirm this. furthermore, a counterregulatory pattern of the sstrs expression was demonstrated in various tissues in the present study. for example, in diencephalon the expression of sstr1 increased with time, while sstr4–5 decreased. interestingly, the protein expression of sstr3–4 seemed to vary reciprocally in the neural tube as well as in cartilage, lens, and retina. moreover, sstr2–3 had a counter-regulatory pattern in lens, retina, and cartilage tissue. both sstr2–3 are known to be involved in the induction of apoptosis (14,15). this may suggest that the protein expression of sstrs during rat development is influenced differently by developmental signals, and may thereby have opposing effects during differentiation of specific tissues. alternatively, it cannot be excluded that such changes suggest that different sstrs may substitute for each other in order to maintain biological actions of somatostatin in developing tissues. the occasionally observed discrepancies between levels of mrna transcripts detected by pcr and protein expressions as detected by immunocytochemical methods may reflect methodological limitations concerning antibody specificity and cross-reactivity and very low abundant expressions of mrna transcripts. on the other hand, it could also reflect dynamic and rapid changes during embryonic development. for individual findings in our investigation it is thus difficult to be certain to what extent such a result represented a true biological discrepancy. sstr mrna expression in endocrine cells of the pancreas pancreatic specimens were collected from rat fetuses at embryonic days 19, 21, and after birth. sections were double-stained with sstr subtype-specific antibodies and antibodies directed towards the four major islet hormones (table iii). in a previous study in adult rat pancreas we suggested that there exists a possible cross-talk between sstr2 and sstr5 on b-cells (18,19), as also supported by others (10,12,25,36). in the present study it was found that the protein expression of sstr5 in embryonic pancreas was not detectable by the experimental settings chosen for this study. this was also the case for the sstr5 mrna levels in embryonic rat pancreas (figure 2e) as well as for the whole rat embryo (figure 3e). however, both the protein expression of sstr5 and the mrna levels were very high in 166 e. ludvigsen et al. adult pancreatic tissue (table iii, figure 2e). the receptor has been shown to play a role during insulin secretion; and the low prenatal pancreatic expression indicates that at this time point the physiological need for glucose sensitization is not yet prioritized in the rat fetus, while this is required in the rat after birth. glucagon secretion is thought to be inhibited by sstr2 (12), and the high co-expression of sstr2 observed in rat pancreatic a-cells all through rat pancreatic development and adult pancreatic tissue may suggest that glucagon secretion has an important function already in the rat embryo. moreover, a peak at embryonic day 14 was observed for sstr2 during pancreas development (also seen for sstr1) (figure 2a, b), suggesting that this may be an important time point in glucagon secretion development. in line with this the majority of sstr1 was co-expressed on a-cells early in development. this finding is in line with previous observations showing that the cross-talk between sstr1 and sstr2 is important for a maximal inhibitory effect on glucagon secretion (18,19,30). in addition, the majority of a-cells also express sstr5, making it possible that the cross-talk also includes this receptor for maximum regulatory effects, but further studies are needed to support this. there is little information about d-cells in the pancreas, but our findings indicate that the co-expression of sstr2–3 is similar in the newborn and in the adult pancreatic tissue, suggesting a possible basic role in somatostatin secretion. moreover, the high co-expression of sstr3 at embryonic day 21, in the newborn, and in adult tissue is in line with the increasing mrna levels of the same receptor (figure 2c). pp-cells are also poorly investigated, and not much is known about their function and role in the developing rat pancreas. therefore we can only put forward the idea that the high co-expression of sstr1–3 in these cells during development compared with that in the adult pancreas may indicate a possible important role for these sstrs in developmental pp regulation. the co-localization of sstr4–5 remained more or less the same during the development as well as in adult pancreas (about 20%). the reason for this is not clear, and more studies are needed to explore this issue further. in summary, we have described the expression profiles of sstrs both as total mrna and as protein in the developing rat embryo and especially so in the pancreas. expression profiles revealed mostly increased expression of the sstrs up to embryonic day 14 and thereafter a decline. these results suggest an important role for sstrs during the developmental process, and subsequent functional studies may elucidate the role of specific sstrs for the growth, differentiation, and metabolism of different organs. acknowledgements the technical expertise by lisbeth sagulin and handling of the animals by dr mattias gäreskog and dr parri wentzel are gratefully acknowledged. funding: this work was supported by grants from the swedish research council [72x-8273], the swedish diabetes association, the swedish cancer foundation, the novo nordisk foundation, the swedish childhood diabetes foundation, the family ernfors fund, the european foundation for the study of diabetes, magnus bergvalls foundation, and the lions foundation for cancer research at the university hospital, uppsala, sweden. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. brazeau p, vale w, burgus r, ling n, butcher m, rivier j, et al. hypothalamic polypeptide that inhibits the secretion of immunoreactive pituitary growth hormone. science. 1973; 179:77–9. 2. moller ln, stidsen ce, hartmann b, holst jj. somatostatin receptors. biochim biophys acta. 2003;1616:1–84. 3. patel yc. somatostatin and its receptor family. front neuroendocrinol. 1999;20:157–98. 4. 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et al. the effect of global sstr5 gene ablation on the endocrine pancreas and glucose regulation in aging mice. j surg res. 2005;129:64–72. 34. adams rl, adams ip, lindow sw, zhong w, atkin sl. somatostatin receptors 2 and 5 are preferentially expressed in proliferating endothelium. br j cancer. 2005;92:1493–8. 35. cristiani r, petrucci c, dal monte m, bagnoli p. somatostatin (srif) and srif receptors in the mouse retina. brain res. 2002;936:1–14. 36. sprecher u, mohr p, martin re, maerki hp, sanchez ra, binggeli a, et al. novel, non-peptidic somatostatin receptor subtype 5 antagonists improve glucose tolerance in rodents. regul pept. 2010;159:19–27. 168 e. ludvigsen et al. abstract introduction materials and methods animals pcr measurements of sstr mrnas, preparation of total rna preparation of cdna analysis of sstrs mrna expression immunohistochemistry sstr1&ndash;5 antibodies morphological evaluation double immunofluorescence of sstr1&ndash;5 and islet hormones in the fetal pancreas. evaluation of immunofluorescence statistical analysis results immunocytochemical detection of sstrs during rat development sstrs mrna expression during development of the embryonic rat pancreas sstrs mrna expression in entire rat embryos presence of sstrs in endocrine cells of the embryonal pancreas discussion mrna expression in whole rat embryos organ-specific protein expression sstr mrna expression in endocrine cells of the pancreas acknowledgements declaration of interest references martin h:son holmdahl full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 martin h:son holmdahl to cite this article: (2015) martin h:son holmdahl, upsala journal of medical sciences, 120:2, 132-133 to link to this article: https://doi.org/10.3109/03009734.2015.1044055 © informa healthcare published online: 05 may 2015. submit your article to this journal article views: 156 view related articles view crossmark data https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://doi.org/10.3109/03009734.2015.1044055 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1044055 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1044055 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1044055&domain=pdf&date_stamp=2015-05-05 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1044055&domain=pdf&date_stamp=2015-05-05 upsala journal of medical sciences. 2015; 120: 132–133 in memoriam martin h:son holmdahl uppsala medical society has the sad duty to inform about the death of martin h:son holmdahl, who died in his 92nd year on 11 march 2015. he is remembered with gratitude for his important work for science, education, humanity, and medicine in the academic community in uppsala. he was born in gothenburg in 1923, the son of henrik holmdahl, md, into a family of academics, priests, and medical doctors. he began his medical studies in uppsala in 1942 and finished them in 1950. he completed his thesis at the department of physiology in 1956 on ‘apnoic oxygenation’ under the mentorship of professor henrik enghoff. already in the late 1940s martin h:son holmdahl was contacted by the professor of surgery, olle hultén, to give guidance on anaesthesia. his leadership for anaesthesia in uppsala was formalized in 1953. he did part of his training at the royal postgraduate medical school in london. when uppsala, in 1956, got an independent department of anaesthesia, martin holmdahl became its first head. he developed it into a strong and modern university clinic. using his experience from the polio epidemic in 1953, he was one of the first to introduce modern intensive care in sweden. his knowledge in the field of respiratory physiology was of course of utmost importance. in 1965 martin h:son holmdahl became the first professor of anaesthesia at uppsala university. his scientific work focused on mentorship for young anaesthetists, and he was anxious to support his students in their scientific projects. respiratory physiology, local anaesthesia, resuscitation, and patient-controlled analgesia are some of the fields in which about 35 doctoral theses were published under his stewardship. over the years he became more engaged in the leadership of the faculty and later the university. after a period as dean of the medical faculty between 1969 and 1970 he was elected rector magnificus of uppsala university in 1978, a position he held until his retirement in 1989. even after this he remained very active and took an important role as an adviser and discussion partner for his younger colleagues in the department. we saw him almost daily at work just a few months ago. for uppsala medical society he was the person who provided insights in discussions. he encouraged contacts between generations and often championed the viewpoints of the young medical students. whenever martin h:son holmdahl took part in the society meetings he added quality and depth to the discussions. he was a strong spokesman for this journal, and he supported the initiative, together with the medical faculty, to arrange the annual rudbeck day. his autobiography portrays his life and his contribution to national and international academic study of anaesthesia well. from his youngest days martin was a spokesman for humanity and everyone’s right to freedom. on his 80th birthday uppsala university honoured him by instituting a prize for human rights martin h:son holmdahl issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1044055 http://informahealthcare.com/journal/ups in his name. in 2010 the selander foundation initiated a lecture to be held every year in martin h:son holmdahl’s name, and uppsala medical society is one of the organizers. martin h:son holmdahl left a lasting imprint in many of us who met him. characterized by his scientific interest, never-ending curiosity, enthusiasm, and humanism, his memory will persist in the minds and hearts of very many friends, students, colleagues, and trainees. for uppsala medical society lars wiklund, henry johansson, and torbjörn karlsson in memoriam: martin henriksson holmdahl 133 ss1 platelet-derived growth factor in glioblastoma—driver or biomarker? upsala journal of medical sciences. 2014; 119: 298–305 review article platelet-derived growth factor in glioblastoma—driver or biomarker? bengt westermark department of immunology, genetics and pathology, uppsala university, uppsala, sweden abstract the platelet-derived growth factor (pdgf) family of mitogens exerts vital functions during embryonal development, e.g. in the central nervous system, where pdgf drives the proliferation of oligodendrocyte precursors. pdgf and pdgf receptors are co-expressed in human glioblastoma (gbm). whether an aberrant activation of the pdgf receptor pathway is a driving force in glioma development has remained an open question. in experimental animals, overexpression of pdgf has convincingly been shown to induce tumors, both in wild-type animals (marmoset, rat, mouse) and in mice with targeted deletions of suppressor genes, e.g. tp53 or ink4a. targeting the pdgf receptor in tumor-bearing mice leads to growth inhibition and reversion of the transformed phenotype. findings of pdgf receptor amplification or mutations in human gbm are strong indicators of a causative role of the pdgf receptor pathway. however, clinical trials using pdgf receptor antagonists have been disappointing. in conclusion, a pdgf receptor profile may be a biomarker for a subgroup of gbm originating from a pdgf receptor-responsive cell. although compelling experimental and clinical evidence supports the notion that the pdgf receptor pathway is a driver in gbm, formal proof is still missing. key words: brain tumors, molecular biology, oncogenes, tumor biology introduction the widely cited articles by hanahan and weinberg on the hallmarks of cancer (1) have highlighted the importance of self-sufficiency in growth factor receptor signaling, mediated by aberrant ligandinduced activation, constitutively activated and mutated receptors, or aberrant activation of downstream signals. the idea that cancer cells are growthstimulated by autocrine growth factors has been around for quite a while, even before the term ‘autocrine’ was used to describe the phenomenon (2). for instance, howard temin referred to it in his pioneering work on the mechanism of rna tumor virus-induced cell transformation (3,4). the field was tremendously boosted by the discovery that the transforming v-sis gene of simian sarcoma virus (ssv) is a retroviral version of the cellular platelet-derived winner of the rudbeck award, 2012 at the medical faculty of uppsala university, for his outstanding research on growth regulation of normal and neoplastic cells. correspondence: bengt westermark, department of immunology, genetics and pathology, uppsala university, se-751 85 uppsala, sweden. e-mail: bengt.westermark@igp.uu.se (received 22 september 2014; accepted 24 september 2014) issn 0300-9734 print/issn 2000-1967 online � 2014 informa healthcare doi: 10.3109/03009734.2014.970304 http://informahealthcare.com/journal/ups mailto:bengt.westermark@igp.uu.se growth factor (pdgf) b-chain gene (5,6) and that the transforming activity of the gene is mediated by autocrine pdgf-bb (7). in my own search for suitable sources for novel growth factors, i was encouraged by the early studies by temin and others to use serum-free conditioned culture media from human established cancer cell lines. the human osteosarcoma cell line u-2 os, established by jan pontén (8), turned out positive as it was found to produce growth-promoting factor (s) which could be partially purified (9). further purification and structural and functional analyses revealed a striking resemblance to pdgf (10). subsequently, the osteosarcoma cell-derived growth factor was shown to be identical to pdgf-aa (11). using the recently developed pdgf receptorbinding assay, i found that one of our clonal human glioblastoma (gbm) cell lines, u-343 mga cl2, produced large amounts of pdgf receptorcompeting activity. through subsequent studies it became evident that the cells produce significant amounts of pdgf (12,13), mainly pdgf-aa (14). this finding was the starting-point for a new research avenue, pdgf in human and experimental brain tumors (reviewed in (15,16)). pdgf and pdgf receptors the platelet-derived growth factor (pdgf) family consists of covalently linked heteroor homodimers of a-, b-, c-, and d-chains (pdgf-aa, -ab, -bb, -cc, and -dd) (for comprehensive reviews on pdgf and pdgf receptors, see (17,18)). the ligands bind to and activate heterodimeric a and b tyrosine kinase receptors with ligand specificities outlined in figure 1. several intracellular signaling pathways are engaged by pdgf receptor activation among which the rasmapk, pi3k, and plcg pathways are most studied. receptor activation culminates in cell cycle initiation, dna synthesis, and mitosis. pdgf is not only involved in cell cycle regulation but also cell migration and chemotaxis (19-21). pdgf and pdgf receptors fulfill important functions in development, e.g. in kidney, lung, intestine, and brain tissues, mainly through paracrine receptor activation (17). pdgf in glial development a ground-breaking study by richardson et al. (22) showed that cultured oligodendrocyte precursor cells (opcs) from the developing rat optic nerve are growth-stimulated by pdgf, present in astrocyteconditioned medium. a subsequent study showed that the opcs express pdgfra (23). these findings have been confirmed by in vivo experiments in mice, where pdgf-aa has been shown to drive the proliferation of opcs (24-26). opcs are likely to be stimulated in vivo by paracrine pdgf released from astrocytes and neurons (27). no such autocrine stimulation by normal opcs during development has been demonstrated. interestingly, pdgf-aa seems to be a rate-determining factor for opc proliferation (28), but not for myelination; it is only in the total absence of pdgf that myelination becomes negatively affected (26). continuous infusion of pdgf into the subventricular zone of the mouse brain induces precursor cell proliferation and hyperplastic, glioma-like lesions. these lesions are reversible and cannot be defined as full-blown malignancies (29). in conclusion, in normal development pdgf drives the expansion of undifferentiated opcs, and an excess of pdgf leads to an increase in opc number beyond the physiological need. apparently, there is no negative feedback mechanism operating in opcs to control their pdgf-induced proliferation. rather opc proliferation seems to be controlled at the level of pdgf available for receptor activation. pdgf-induced gliomas in animal models experiments performed by friedrich deinhardt and collaborators showed that ssv is tumorigenic in marmoset monkeys (30,31). a complete record of deinhardt’s experiments was unfortunately never published, but we were lucky to obtain the original paraffin-embedded material from deinhardt’s experiments and could publish the histology of brain tumors induced by intracerebral ssv injections into newborn pdgf-aa pdgf-cc pdgf-ab pdgf-bb pdgf-dd pdgfr-ββpdgfr-αβpdgfr-αα figure 1. binding specificities of pdgf ligands and receptors. the pdgf isoforms are made up as homoor heterodimers of antiparallel subunits covalently linked by two s-s bonds. the ligands bind to and dimerize pdgf a and b receptors with specificities depicted in the figure. the receptors have extracellular domains with five immunoglobulin domains. each receptor has an intracellular, split tyrosine kinase domain. platelet-derived growth factor in glioblastoma 299 animals (32). interestingly, the virus caused lesions indistinguishable from human glioblastoma with all its hallmarks: cellular pleomorphism, necroses, and microvascular proliferations. the morphology was mixed, with oligodendroglioma-like, astrocytic, and anaplastic areas present. we now know that the transforming gene v-sis of ssv is a retroviral version of the pdgfb gene and that the transforming activity of v-sis is exerted by an autocrine pdgf receptor activation. thus, an autocrine and uncontrolled pdgf receptor activation of target cells in primate brain in a retroviral context leads to the development of brain tumors displaying all the hallmarks of gbm. in this respect, ssv infection in marmosets has a more dramatic effect than continuous infusion in mouse brain (29). a number of studies have shown that intracerebral delivery of pdgf-encoding retroviral vectors in rodents leads to the development of brain tumors at high frequency. a recombinant moloney murine leukemia virus encoding pdgfb (momulv/ pdgfb) injected together with replicationcompetent helper virus (momulv) was found to induce glioma-like lesions in wild-type mice (33). the tumors mostly resembled human glioblastoma with extensive necrotic areas. some tumors displayed characteristics of primitive neuroectodermal tumors, and some were phenocopies of human low-grade oligodendrogliomas. only a minority of the cells, identified as infiltrating astrocytes, were glial fibrillary acidic protein (gfap)-positive, whereas a high proportion were nestin and pdgfra-positive. these characteristics suggested that the tumors were derived from a pdgfra-positive neural progenitor transformed by a persistent autocrine activation of the pdgf receptor signaling pathway. eric holland and collaborators have made important discoveries and significantly contributed to our understanding of pdgf-induced brain tumors in the mouse. a useful mouse model was developed, where pdgf is encoded by a recombinant avian rcas virus and delivered intracerebrally in transgenic mice with a targeted expression of the virus receptor tv-a under the control of gfap (gtv-a mice) or nestin (ntv-a mice) promoters (34). rcas-pdgfb generated brain tumors in both types of transgenic mice: oligodendrogliomas in ntv-a mice, and oligodendrogliomas or oligoastrocytomas in gtv-a mice. tumor latency was shortened, and malignancy was enhanced in mice with homozygous deletion of the ink4a-arf locus, which is a common aberration in human gbm. using the 2’,3’-cyclic nucleotide 3’-phosphodiesterase (cnpase) promoter to drive the expression of tv-a in transgenic mice (ctv-a mice), lene uhrbom’s research group was able to target pdgf expression to cells of the oligodendrocyte lineage. in the absence of other genetic aberrations, rcaspdgfb generated low-grade oligodendrogliomas (35), whereas high-grade oligodendrogliomas as well as astrocytomas were induced in cdkn2a null (p16ink4a-/-, p19arf-/-) mice (36). collectively, these data show that a single oncogene, i.e. pdgfb, can cause tumors with different cellular origin (gfap-positive astrocytes, nestin-positive neural stem cells, and cnpase-positive oligodendrocyte progenitors). the impact of these findings on the identification of the cell of origin of human glioblastoma has been discussed (37). pdgfb has also been shown to induce gbm-like tumors when expressed as a transgene under the control of the gfap promoter in tp53 null, but not wild-type, mice (38). high expression of pdgfb, generated by deleting the translation-inhibiting sequence in the 5� end of pdgfb cdna inserted into the rcas vector, was shown to give rise to high-grade tumors in wild-type mice, whereas the full-length sequence mostly induced low-grade oligodendrogliomas (39). tumor grade is thus dose-dependent. low expression of pdgf causes low-grade tumors, and high expression yields high-grade tumors. in this context it is interesting to note that the v-sis gene lacks this particular sequence (40), which may explain why ssv causes high-grade tumors in marmosets. the finding that an oncogene coding for a structurally and functionally normal growth factor can cause high-grade tumors is difficult to understand given the widely accepted concept that malignant tumors are caused by multiple genetic events and display a diversity of phenotypic aberrations (1). this question was addressed decades ago (41) but has not yet been fully answered. there are three main alternatives. (1) mitogenic stimulation and unrestricted cell proliferation induced by excessive amounts of pdgf is accompanied by secondary genetic aberrations causing tumor progression. this idea was adopted in a recent review (42), where replicative stress was supposed to be the driving force behind the mutagenic events. direct evidence for this hypothesis, e.g. whole-genome sequence data for pdgf-induced tumors, is, however, lacking. (2) pdgf synthesis driven by retroviral vectors synergizes with insertional mutagenesis of complementary oncogenes or suppressor genes. retroviral long terminal repeats are strong promoters and enhancers, which could activate cellular genes at a distance from the insertion sites. further, integrated proviral dna could theoretically interrupt genes and give rise to 300 b. westermark truncation or inactivation of the gene product. in order to probe this idea and search for genes that synergize with pdgf in gliomagenesis, uhrbom et al. (33) constructed a recombinant momulv coding for a full-length pdgfb cdna. the recombinant virus was combined with a replication-competent helper virus to increase the frequency of proviral insertions in order to render the genetic screen more effective. the experiment generated 108 brain tumors from which 647 insertion sites were obtained with as many as 66 common insertion sites (43), reviewed in (44). a number of the tagged genes are known to be involved in oncogenesis, e.g. trp53, eef1a1, gli, fos, and ccnd, providing proof of concept for the genetic screen. the vast majority of tagged genes, however, had not previously been implicated in oncogenesis or pdgf signaling. one of these, sox5, was subsequently found to be a suppressor of pdgf-induced brain tumors and cause cellular senescence (45). five insertions in the nfix locus were found, the functional implication of which is still unknown and worth pursuing. nfix has been shown to be a critical factor in development and to induce quiescence in neural stem cells (46,47). insertion into the nfix locus may interfere with the differentiation program and thereby synergize with pdgf in tumor development. whether insertional mutagenesis also operates in the rcas-pdgf-induced brain tumors is an open question. the oncogenic potential of retroviruses was dramatically illustrated by the clinical trials on gene therapy in x-linked severe combined immunodeficiency (xscid) patients a few years ago. three out of 11 patients developed t cell tumors, all of which caused by proviral insertions in the lmo2 locus (48,49). (3) the generation of high-grade gliomas by pdgf overexpression is caused by an autocrine activation of transformed cells as well as recruitment of non-transformed cells, which are attracted to the tumor site by paracrine activation. in fact, pdgf virus-induced gliomas in the rat contain a large number of untransformed cells, which apparently have migrated and populated the tumor (50). according to this model, pdgf is sufficient to drive tumor formation in the absence of other genetic aberrations. this model may seem unlikely and less attractive than those described above. the model presumes that an excessive, pdgf-induced, cell proliferation is not controlled by any feedback regulation. interestingly, this may indeed be the case. in pdgftransgenic mice, the number of pdgfresponsive oligodendrocyte precursors was proportional to the supply of pdgf and seemingly unsaturable (51). in the case of a local production of autocrine/paracrine pdgf, the pool of stimulated cells may increase progressively to the point where hypoxia drives angiogenesis and where necroses may occur in severely hypoxic areas. such a process may then yield tumors, which closely resemble glioblastoma multiforme (see figure 2 for a schematic representation of this model). pdgf and pdgf receptors in human glioma our observation that human glioma cells in culture produce pdgf became an incentive for studies of the expression of pdgf and pdgf receptors in human glioma. a rather coherent picture has emerged from these studies, pointing to a causative role for pdgf in gliomagenesis. pdgfa and pdgfa receptors are co-expressed in glioblastoma (52) and oligodendroglioma (53). the pdgfra gene is amplified, mutated, or rearranged in a fraction of glioblastoma tumors (54-60). these findings strongly suggest that genetic aberrations and overactivity of the pdgfa receptor signaling pathway are important events in the development of a subset of glioblastomas. this view is strengthened by transcriptome analysis of glioblastoma, which has indicated that the proneural subgroup of gbm is characterized by aberrations in the a b c d figure 2. schematic model for the development of high-grade gliomas in experimental animals following infection with pdgfencoding retroviruses. a. cells are infected, produce pdgf, and start to proliferate in an uncontrolled fashion. b. the pdgftransformed population expands, and neighboring cells are attracted by paracrine stimulation. c. cell density has increased, and local hypoxia elicits an angiogenic response. d. the expanding population has grown beyond the supply of nutrients, and necroses appear. pink areas denote pdgf released by the tumor cells. platelet-derived growth factor in glioblastoma 301 pdgf/pdgfr pathway (60). indeed, overactivity of the pdgfa receptor pathway may be an initiating event in human glioblastoma as a result of an increased pdgfa gene dosage generated by chromosomal non-disjunction by the occurrence of multiple copies of chromosome 7, which harbors the pdgf a-chain gene (61). driver or biomarker? in the discussion on the causative role of pdgf and pdgfr in the development of human brain tumors, we may be guided by an analogous discussion over a century ago regarding micro-organisms as etiologic agents in human disease. this subject was brought up by henle and koch and resulted in what became known as koch’s postulates (reviewed in (62)). although koch’s postulates cannot be directly translated into the etiology or pathogenesis of a non-viral cancer, such as glioblastoma multiforme, they can be adjusted as follows: (1) pdgf receptor signaling must be aberrantly activated in brain tumor cells. as has been summarized above, pdgf and pdgfr are expressed at high levels in most gbm. since the cell of origin of human gbm is yet unknown, we cannot make any direct comparison between the levels of activation in the normal cell(s) of origin versus gbm cells. however, the pdgfra gene is amplified in a subset of gbm, which stronglyindicatesthatitconfers aselectivegrowth advantage and thus has a mechanistic role in the pathogenesis of the tumor. the recurrent findings of receptor mutations and rearrangements, albeit rare, are strong indicators of a mechanistic role, although we cannot formally rule out the possibility that these changes are passenger mutations and not drivers. (2) aberrant activation of pdgf receptors in the cell of origin must cause gbm in vivo. as described above, studies in marmosets, rats, and mice have provided convincing evidence that overexpression of pdgf, mainly pdgfb but also pdgfa (61), causes gbm in experimental animals. it needs to be said, however, that these findings in no way prove that similar mechanisms operate in human gbm. (3) blocking the pdgfr signal transduction pathway in gbm should retard the growth of glioma cells in vivo. there are a few examples where pdgf antagonists have been shown to retard cell growth in gbm cultures (63,64). however, clinical trials on the use of pdgf receptor kinase inhibitors have been disappointing (65,66). these results are in striking contrast to studies on pdgfb-induced gliomas in mice, where lowmolecular-weight pdgf receptor kinase inhibitors are efficacious in reverting the transformed phenotype and cause tumor growth retardation in vivo (67). it is also notable that imatinib, a lowmolecular-weight pdgfr inhibitor, is an effective drug in the treatment of other pdgf-driven tumors such as gastrointestinal stroma tumors with pdgfra mutations and dermatofibrosarcoma tuberans with an activating pdgfb translocation (reviewed in (68,69)). in the absence of in-depth pharmacokinetic and pharmacodynamic studies in gbm, we cannot draw firm conclusions regarding the potential of pdgf receptor antagonists in the treatment of gbm. however, the possibility remains that aberrant activation of the pdgfr signaling pathway is an initial event in the proneural and mesenchymal subgroups of gbm (61) but is rendered redundant during tumor progression. this would point to a hit-and-run mechanism in contrast to the clinically more favorable situation of oncogene addiction (70,71) where oncogene inactivation leads to the death of tumorcells but spares normal cells. a third possibility could be that pdgfr activation is a driver in a subset gbm in all stages but intervention of the pathway needs to be combined with an interference with other aberrations, e.g. in the pten, tp53, ink4a/arf pathways. in conclusion, a pdgf receptor profile, such as has been demonstrated in the proneural subgroup of gbm, is likely to be a marker of the cell of origin. such tumors are probably derived from pdgfrapositive cells. there is also considerable evidence that a constitutively active pdgfr pathway is a driver in a subset of gbm, although formal proof is still lacking. targeting the pdgfr, or other signaling pathways, in gbm has so far not been clinically successful. maybe an unprejudiced search for gbm antagonists by high-throughput screening will be more fruitful than the ‘intelligent design’ of targeted drugs. a promising substance has recently been identified (72). there is currently a vibrant activity in basic, translational, and clinical research on gbm as well as in cancer research in general. it is my personal belief that these research activities will bear fruit and translate into a better treatment of gbm, which until now has remained a tumor with a dismal prognosis. acknowledgements my own work cited in the text has been generously supported by grants from the swedish cancer 302 b. westermark society, the swedish research council, and the swedish childhood cancer foundation. declaration of interest: the author reports no conflicts of interest. the author 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biomarker? acknowledgements declaration of interest references gynecologists are afraid of prescribing hormone replacement to endometrial/ovarian cancer survivors despite national guidelines—a survey in sweden article gynecologists are afraid of prescribing hormone replacement to endometrial/ ovarian cancer survivors despite national guidelines—a survey in sweden sandra halldorsdottira, hanna dahlstrandb and karin stålberga adepartment of women’s and children’s health, uppsala university, uppsala, sweden; bdepartment of immunology, genetics and pathology, uppsala university, uppsala, sweden abstract background: prolonged survival in ovarian and endometrial cancer patients increases the importance of paying attention to quality of life. hormone replacement therapy (hrt) after gynecologic cancer has been controversial. with this survey, we sought to describe swedish gynecologists’ and gynecologic oncologists’ attitudes towards prescribing hrt to these cancer survivors and see if prescribing practice is consistent with the available evidence and national guidelines. material and methods: a web-based survey containing three hypothetical cases with a total of 15 questions was distributed to gynecologists and gynecologic oncologists in sweden. respondents were asked about their hrt prescription practices in endometrial/ovarian cancer patients with moderate to severe menopausal symptoms. results: in total 262 gynecologists and 24 gynecologic oncologists answered the survey. in the lowrisk endometrial cancer case a majority of the gynecologists (55%) and gynecologic oncologists (66.7%) would prescribe local estrogen. a total of 30% of the gynecologists would prescribe estrogen replacement therapy (ert) in the high-risk endometrial cancer case compared to 58.3% of the gynecologic oncologists. the gynecologic oncologists felt more comfortable treating patients with endometrial cancer than did gynecologists, and the gynecologists were more likely to read the national guidelines. in the ovarian cancer case, 63.7% of the gynecologists would prescribe hrt compared to 92% of the gynecologic oncologists. conclusion: swedish gynecologic oncologists have a more favorable attitude towards hrt for endometrial/ovarian cancer patients and feel more comfortable treating their patients than do gynecologists. this study illustrates a need for education in these matters in order not to withhold hrt from women due to doctors’ sometimes unjustified anxiety. article history received 10 october 2018 revised 1 november 2018 accepted 1 november 2018 keywords endometrial cancer; hormone replacement therapy; ovarian cancer; survey introduction symptoms of iatrogenic menopause are usually considerably more severe in comparison to those following a naturally occurring menopause and might adversely affect the quality of life in young female cancer survivors (1). progress in the treatment of gynecological cancer has given many women prolonged survival, and therefore it is increasingly important to pay attention to long-term consequences of estrogen deficiency. the most effective treatment for menopausal symptoms is hormone replacement therapy (hrt). hrt is effective for the treatment of vasomotor symptoms, preventing osteoporosis, reducing the risk of cardiovascular disease (in women <60 years old), perimenopausal low mood, sexual dysfunction, and urogenital symptoms (2–4). endometrial cancer is the most common gynecological malignancy in sweden, and its incidence has increased over the last decades from 18.5/100,000 persons/year in 1970 to 28.9/100,000 persons/year in 2014 (5). twenty percent of women with endometrial cancer are premenopausal, and the standard treatment of endometrial cancer includes bilateral salpingo-oophorectomy. the use of estrogen replacement therapy (ert) in women with a history of endometrial cancer has been controversial. the fear is that estrogen up-regulates estrogen receptor expression and stimulates growth in endometrial cancer cells (6), but the theory has not been consolidated in clinical studies. a few retrospective studies and case-control studies have been published and have not been able to show increased risk of recurrence after ert treatment of patients surgically treated for endometrial adenocarcinoma (7–9). the swedish national guidelines on endometrial cancer state that the risk of endometrial cancer recurrence after ert treatment is low or possibly none (10). ovarian cancer is the second most common gynecological cancer in sweden. the incidence has decreased over the last decades from 23.8/100,000 persons/year in 1970 to 14.4/ 100,000 persons/year in 2014 (5). even though the incidence increases with age, a significant proportion of cases are contact karin stålberg, md, phd karin.stalberg@kbh.uu.se department of women’s and children’s health, uppsala university, 751 85 uppsala, sweden. supplemental data for this article can be accessed here. � 2018 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2018, vol. 123, no. 4, 225–229 https://doi.org/10.1080/03009734.2018.1544597 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1544597&domain=pdf https://doi.org/10.1080/03009734.2018.1544597 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1544597 http://www.tandfonline.com diagnosed in premenopausal and perimenopausal women. as for endometrial cancer, physicians have been reluctant to give ovarian cancer survivors ert because of fear of relapse and decreased survival. studies have demonstrated a favorable outcome in women treated with hrt following ovarian cancer in comparison to non-users (11–14). the swedish national guidelines for ovarian cancer recommend that women with iatrogenic menopause symptoms after primary treatment for epithelial ovarian cancer can be treated with hrt without any known risk of recurrence of disease or decreased survival. therefore hrt can be offered to women with a history of endometrial or ovarian cancer with moderate to severe menopausal symptoms (10). after hysterectomy, estrogen only should be offered after the assessment of risk factors (15). hancke et al. published a survey in 2010 where german gynecologists/gynecologic oncologists were asked about prescribing practice in women with lowor high-risk endometrial cancer and menopausal symptoms (16). in a followup study by yokoyama et al. in 2015, the same survey was used on japanese gynecologists (17). the aim with the present study was to illustrate swedish gynecologists’ and gynecologic oncologists’ attitudes concerning prescription of ert to women treated for endometrial/ovarian cancer using the same endometrial cancer cases as in the german and japanese survey studies and to see if prescribing practice is consistent with the available evidence and the swedish national guidelines. material and methods a link to a web-based survey (surveymonkey) was distributed to the e-mail membership list of the swedish society of gynecologic oncology (66 members) in january 2017 and to members of the swedish society of obstetrics and gynecology (2100 members) in march 2017. a follow-up e-mail was sent 2 weeks later. the survey contained questions regarding gender, age, workplace, and sub-specialization. respondents were presented with three hypothetical cases (appendix i) and asked to answer multiple-choice questions regarding ert. the first case was a 41-year-old woman with figo (international federation of gynecology and obstetrics) stage ib grade 2 endometrial cancer and moderate menopausal symptoms. a second case regarded a 38-year-old woman with stage iiic1 grade 3 endometrial cancer with severe menopausal symptoms. these cases have been featured in previous surveys in germany and japan (16,17). in addition, we created one hypothetical case with a stage iia ovarian cancer patient with moderate menopausal symptoms. the answers were gathered in an anonymous database. the results of the endometrial cancer cases were compared with the results from the german and japanese studies. statistics a p value was calculated with fisher’s exact test, and a value of <0.05 was considered to be statistically significant. ethical approval an application to the ethical review board in uppsala, sweden was submitted, but the board found that a permit for this study was not applicable. results the aim was to distribute the survey to the 2100 swedish gynecologists registered as members of the swedish society of obstetrics and gynecology. there was an uncertainty about how many actually received the survey due to doubt regarding the status of the membership e-mail list. additionally, an e-mail filter in the respondents’ mail system might have sifted out the survey. therefore, we are unfortunately unable to calculate the actual response rate for the gynecologists. in total, we received answers from 362 gynecologists, out of whom 100 did not complete the survey (reasons being because they never handled these kinds of patients, were retired, etc.). the survey was also distributed to 66 gynecologic oncologists: 28 started the survey, and 4 terminated (residents), yielding a response rate of 38.7% (24/62). there was no difference regarding distribution of age, gender, and specialization, but a higher proportion of gynecologic oncologists worked at a university hospital (62.5%) compared to gynecologists (33%) (table 1). low-risk endometrial cancer in the first case, illustrating a patient with low-risk endometrial cancer and decreased libido, dyspareunia due to vaginal atrophy, and intermittent moderate hot flushes, a total of 53.1% (139/262) of the gynecologists and 41.7% (10/24) of the gynecologic oncologists answered that ert was contraindicated (figure 1). a majority of the gynecologists (55%) and gynecologic oncologists (66.7%) would prefer to prescribe local estrogen, whereas 14.9% (39/262) of the gynecologists and 4.2% (1/24) of the gynecologic oncologists would not prescribe estrogen at all (p ¼ 0.22). the gynecologic oncologists felt more comfortable treating patients with low-risk endometrial cancer than did the gynecologists (75% versus table 1. characteristics of the respondents. gynecologists gynecologic oncologists p valuean ¼ 262 % n ¼ 24 % age <50 years 106 40.1% 12 50% 0.39 female 191 72.9% 16 66.7% 0.48 specialization, completed survey (started survey) 262 (362) 24 (28) gynecologic oncologist 30 (30) 11.5% 19 (19) 79.2% tumor surgeon 20 (20) 7.6% 3 (3) 12.5% general gynecology 136 (148) 52% 0 0% other (obs/repro) 42 (75) 16% 1 (1) 4.2% resident 20 (74) 7.6% 0 (4) 0% no answer of title 14 (15) 5.3% 1 (1) 4.2% work location 0.0062 university hospital 86 32.8% 15 62.5% non-university hospital 130 49.6% 7 29.2% private practice 37 14.1% 1 4.2% no answer 9 3.4% 0 0% afisher’s exact test. 226 s. halldorsdottir et al. https://doi.org/10.1080/03009734.2018.1544597 42.7%) (p ¼ 0.003). of the gynecologists, 18.7% (49/262) would seek opinion from a more experienced colleague, and 37.0% (97/262) would read the swedish national guidelines; the corresponding rates for the gynecologic oncologists were 8.3% (ns) and 16.7% (p ¼ 0.047) respectively. high-risk endometrial cancer the second case was a patient with high-risk endometrial cancer with persistent severe hot flushes and additionally a family history of osteoporosis. a higher proportion thought that ert was contraindicated in this case, 70.9% (186/262) of the gynecologists and 50% (12/24) of the gynecologic oncologists (p ¼ 0.039) (figure 2). thirty percent (79/262) of the gynecologists stated that they would prescribe ert in this case compared to 58.3% (14/24) of the gynecologic oncologists (p ¼ 0.011). as non-hormonal alternative 43.1% of the gynecologists would prescribe ssri as first choice, compared to 45.8% of the gynecologic oncologists. five percent of the gynecologists would recommend naturopathy instead of ert, and 14% would choose other drugs. these numbers among the gynecologic oncologists were 4.2% (1/24) for naturopathy and 4.2% for other drugs (ns) (figure 2). the gynecologic oncologists felt more comfortable (54.2%) treating patients with high-risk endometrial cancer compared to gynecologists (31.3%) (p ¼ 0.039). there was no statistically significant difference between gynecologists and gynecologic oncologists in how many would read the national guidelines (39% versus 25%) and seek second opinion (26% versus 20.8%). epithelial ovarian cancer the third case was a patient with stage iia high-grade serous ovarian cancer suffering from sleeping problems and flushing. she had no known brca (breast cancer gene) mutation. all of the gynecologic oncologists and 66.4% of the gynecologists stated that ert was not contraindicated in this case. sixty-four percent (167/262) of the gynecologists and 91.7% (22/24) of gynecologic oncologists would prescribe ert (p ¼ 0.006). thirty-nine percent (103/262) of the gynecologists felt comfortable treating the patient compared to 79.2% of the gynecologic oncologists (p ¼ 0.0002). in total 22.9% (60/262) of the gynecologists would seek second opinion about treatment compared to 4.2% (1/24) of gynecologic oncologists (p ¼ 0.035). of the gynecologists, 34.7% (91/ 262) would read the national guidelines compared to 16.7% (4/24) of the gynecologic oncologists (ns). in the three hypothetical cases where respondents would not prescribe ert the most common reason among both gynecologists (62.5%) and gynecologic oncologists (50%) was fear of increased risk of cancer recurrence (ns). of the gynecologists, 16% said it was because of fear of both cancer recurrence and breast cancer, compared to 8.3% of the gynecologic oncologists. comparison to japanese and german studies hancke et al. published in 2010 a survey study regarding german physicians’ attitude towards prescribing ert after endometrial cancer. physicians were asked about their prescribing practice concerning moderate to severe menopausal symptoms for patients having undergone hysterectomy and salpingo-oophorectomy for low-risk and high-risk endometrial cancer. the response rate was 39.8% (165/420) (16). yokoyama et al. published in 2015 a survey study on japanese gynecologists’ view on ert using the same hypothetical cases. in total 880 members of the japanese gynecology oncology group were asked, with a response rate of 40.9% (17). we used the same type of endometrial cancer cases as the two studies, enabling comparisons of results. the ratio of respondents that would prescribe systemic ert in the low-risk endometrial cancer case was 43% in japan, 28.3% in sweden, and 13% in germany (table 2). a majority of respondents in germany (67%) and sweden (55.9%) would prefer local estrogen. in japan, only 15% would prescribe local estrogen, with a higher proportion (43%) giving systemic ert. in the high-risk endometrial cancer case 33% in the swedish study would prescribe ert in comparison to 37% in japan (ns) and 18% in germany. in japan 50% of respondents would choose traditional japanese drugs, and none ns ns ns 0% 10% 20% 30% 40% 50% 60% 70% 80% no ert systemic ert local estrogen figure 1. percentage of gynecologic oncologists (blue) and gynecologists (red) who agree/strongly agree that estrogen replacement therapy (ert) is contraindicated (no ert), prescribe systemic ert or local estrogen therapy. non-significant differences between gynecologic oncologists and gynecologists (fisher’s exact test). * ** ns ns ns 0% 10% 20% 30% 40% 50% 60% 70% 80% no ert systemic ert ssri naturopathy other figure 2. percentage of gynecologic oncologists (blue) and gynecologists (red) who agree/strongly agree that estrogen replacement therapy (ert) is contraindicated (no ert) and drug of choice in the high-risk endometrial case. �p ¼ 0.039; ��p ¼ 0.011 (fisher’s exact test). upsala journal of medical sciences 227 would prescribe ssri. responders in sweden and germany seek second opinion more often than their colleagues in japan. discussion prescribing ert to women with a history of ovarian/endometrial cancer is a difficult decision. based on this survey many of the respondents had a favorable attitude towards ert after treatment of epithelial ovarian or endometrial cancer. the gynecologic oncologists had a more favorable attitude than the gynecologists in the high-risk endometrial cancer case and the ovarian cancer case. the gynecologic oncologists were members of the swedish society of gynecologic oncology, and according to the national board of health and welfare a total of 62 gynecologic oncologists were professionally active in 2015 (18). consequently, the questionnaire reached almost all gynecologic oncologists in sweden. the response rate was fairly low (38.7%) but similar to those in the japanese (40.9%) and german (39.8%) survey studies. a problem appeared when sending the survey to the gynecologists because of a possible mass e-mail filter in the responders’ mail system. in fact, it might have sifted out the survey, leading to an uncertainty in the number of members who actually received the mail, and this made it impossible to calculate a true response rate (262/ 2100). a possible bias may appear if those who choose to respond to the survey have more knowledge or experience of the subject, resulting in a false high frequency of adherence to national guidelines. another potential problem might be that respondents may not give accurate answers or feel comfortable providing answers that present themselves in an unfavorable manner even though the study is anonymous. in the low-risk endometrial cancer case, the japanese respondents were the most likely to prescribe ert more often than the swedish and german participants. in the high-risk case swedish and japanese respondents were more likely to prescribe hrt than germans. the japanese study was published in 2013 whereas the german one was published in 2006, which might influence the respondents’ attitude towards hrt. concerns regarding hrt in postmenopausal women with no history of gynecologic cancer are that it may increase the risk of venous thromboembolic events (19), cerebrovascular accidents, breast cancer, and coronary heart disease (chd). the prescription of hrt increased from the beginning of the 1980s until the late 1990s but decreased dramatically after publication of e.g. the women’s health initiative study (whi) in 2002, where results showed that hrt increased the risk of chd and breast cancer in postmenopausal women (20). subsequent re-analyses of data from the whi study now consistently show reductions in chd and mortality when hrt is initiated close to menopause (2). hrt with estrogen and progesterone can be associated with an increase in the risk of breast cancer, with an additional 8 cases diagnosed per 10,000 women over 5 years. the risk is related to treatment duration and reduced after stopping hrt (21). however, data from the whi study showed that treatment with estrogen alone among women with prior hysterectomy did not increase the risk of breast cancer, chd, or all-cause mortality (22). hrt use in sweden in the age group 50–59 years decreased from a peak of 36% in 1999 to 9% in 2007 (information from national pharmacy data) (23), and according to the swedish national board of health and welfare it was almost unchanged in 2015 (8.8%) (24). retrospective studies of postoperative hrt for endometrial cancer (mostly stage i–ii) show no significant differences in cancer recurrence or survival of women who take hrt (7–9). barakat et al. published in 2006 the only randomized study on ert after early-stage endometrial cancer treatment. the study included 1236 patients randomized to receive either ert or placebo after cancer treatment during 1997–2003. the study was stopped early because of difficulties recruiting patients after the results of the whi study 2002. because of lack of study power they could not conclusively support or disprove the safety of ert with regard to risk of endometrial cancer recurrence, but the absolute recurrence rate (2.1%) was low when 55% of the patients had been taking estrogen for more than 2 years (8). guidozzi and daponte published in 1999 a randomized controlled study about ert in epithelial ovarian carcinoma survivors: 130 patients <59 years old treated for invasive epithelial ovarian cancer were randomized for ert versus placebo and followed for 48 months. the study could not show any difference in disease-free and overall survival between groups (12). a swedish prospective cohort study of 649 patients with ovarian cancer where 150 patients received hrt after primary treatment showed a better survival among women who used hrt after diagnosis (11). in 2015 eeles et al. published an article on hrt effect on survival and disease outcome in women with epithelial ovarian cancer. in total 150 ovarian cancer patients were randomized to hrt versus placebo in 1990–1995. overall survival was improved in the hrt group after follow-up for 19 years (14). a meta-analysis published in 2015 suggests that hrt use by women with a history of epithelial ovarian cancer did not lead to a significantly increased risk of death or recurrence of disease (25). non-hormonal management of postmenopausal symptoms includes lifestyle modification, diet, and application of behavioral and alternative medicine therapies, though the results of the impact of these treatments are conflicting. the swedish national guidelines on epithelial ovarian and endometrial cancer state that ert can be used in survivors in selected cases. as mentioned, hrt is the most effective table 2. comparison of prescription intentions between swedish, japanese, and german doctors. sweden japan germany lowrisk highrisk lowrisk highrisk lowrisk highrisk number (n) 286 286 363 363 165 165 ert not contraindicated 47% 28% 65% 49% 46% 25% systemic ert 28%a,b 33%c 43%a 37% 13%b 18%c local ert 56% 15% 67% ssri 43%d 0% 29%d phytoestrogen/naturopathy 5% 2% 3% 45% second opinion 18% 26% 2% 3% 36% 50% comparison of prescription intention between swedish, japanese, and german respondents for ert in low-risk versus high-risk endometrial cancer cases. ap < 0.00001; bp ¼ 0.00016; cp ¼ 0.0007; dp ¼ 0.004 (fisher’s exact test). 228 s. halldorsdottir et al. treatment for vasomotor symptoms associated with menopause. benefits such as positive effects on menopausal symptoms as well as the protective effect on the cardiovascular disease and osteoporosis have to be considered and are likely to outweigh risks for symptomatic women before the age of 60 years or within 10 years after menopause. the risk and benefit of hrt should thus be individualized for every woman in premature menopause. the results from the present study demonstrate a sometimes unjustified fear of using ert in endometrial/ovarian cancer survivors and that the swedish national guidelines are not properly followed. those more experienced with gynecologic cancer patients (gynecologic oncologists) have a more favorable attitude towards ert for endometrial/ovarian cancer patients than do general gynecologists, and they feel more comfortable treating their patients. this study illustrates a need for more education on these matters. quality of life as well as other benefits with hrt should continuously be addressed in the follow-up visits. more studies regarding the benefits and risks of hrt in gynecologic cancer survivors are needed and might be possible with improved pharmaceutical registers. acknowledgements we are grateful to professor alkistis skalkidou, department of women’s and children’s health, uppsala university and the office of swedish society of obstetrics and gynecology and the secretary of swedish society of gynecologic oncology for helping us with distribution of this survey. declaration of interest the authors whose names are listed immediately below certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript: sandra halldorsdottir, hanna dahlstrand, karin stålberg. funding no specific funding was obtained for this study. notes on contributors sandra halldorsdottir, md, is a consultant in obstetrics and gynecology, uppsala university hospital. hanna dahlstrand, md, phd, is an associate professor (docent), senior consultant in gynecologic oncology, uppsala university hospital. karin stålberg, md, phd, is an associate professor (docent), senior consultant in obstetrics and gynecology, uppsala university hospital. references 1. rodriguez m, shoupe d. surgical menopause. endocrinol metab clin north am. 2015;44:531–42. 2. rossouw je, prentice rl, manson je, ko m, lacroix az, margolis kl, et al. postmenopausal hormone therapy and risk of cardiovascular disease by age and years since menopause. jama. 2007;297:1465–78. 3. bagger yz, tank�o lb, alexandersen p, hansen hb, møllgaard a, ravn p, et al. two to three years of hormone replacement treatment in healthy women have long-term preventive effects on bone mass and osteoporotic fractures: the perf study. bone. 2004;34:728–35. 4. the north american menopause society (nams). the 2012 hormone therapy position statement of the north american menopause society. menopause. 2012;19:257–71. 5. socialstyrelsen cancer incidence in sweden 1970–2014. 6. holinka ce, hata h, kuramoto h, gurpide e. responses to estradiol in a human endometrial adenocarcinoma cell line. j steroid biochem. 1986;24:85–9. 7. chapman ja, disaia pj, osann k, roth pd, gillotte dl, berman ml. estrogen replacement in surgical stage i and ii endometrial cancer survivors. am j obstet gynecol. 1996;175:1195–200. 8. barakat rr. randomized double-blind trial of estrogen replacement therapy versus placebo in stage i or ii endometrial cancer: a gynecologic oncology group study. j clin oncol. 2006;24:587–92. 9. suriano ka, mchale m, mclaren ce, li kt, re a, disaia pj. estrogen replacement therapy in endometrial cancer patients: a matched control study. obstet gynecol. 2001;97:555–60. 10. swedish national guidelines for endometrial cancer. 2011. available from: https://www.cancercentrum.se/samverkan/ cancerdiagnoser/ gynekologi/livmoderkropp/vardprogram/gallande-vardprogram/ 11. mascarenhas c, lambe m, bellocco r, bergfeldt k, riman t, persson i, et al. use of hormone replacement therapy before and after ovarian cancer diagnosis and ovarian cancer survival. int j cancer. 2006;119:2907–15. 12. guidozzi f, daponte a. estrogen replacement therapy for ovarian carcinoma survivors: a randomized controlled trial. cancer. 1999; 86:1013–18. 13. eeles ra, tan s, wiltshaw e, fryatt i, a’hern rp, shepherd jh, et al. hormone replacement therapy and survival after surgery for ovarian cancer. bmj. 1991;302:259–62. 14. eeles ra, morden jp, gore m, mansi j, glees j, wenczl m, et al. adjuvant hormone therapy may improve survival in epithelial ovarian cancer: results of the aht randomized trial. j clin oncol. 2015;33:4138–44. 15. swedish national guidelines for ovarian cancer. 2015. available from: https://www.cancercentrum.se/samverkan/cancerdiagnoser/gynekologi/ aggstock/vardprogram/gallande-vardprogram-aggstock cancer/ 16. hancke k, foeldi m, zahradnik hp, gitsch g, gilbert l, denschlag d. estrogen replacement therapy after endometrial cancer: a survey of physicians’ prescribing practice. climacteric. 2010;13:271–7. 17. yokoyama y, ito k, takamatsu k, takehara k, nakanishi t, harano k, et al. how do japanese gynecologists view hormone replacement therapy for survivors of endometrial cancer? japanese gynecologic oncology group (jgog) survey. int j clin oncol. 2015;20:997–1004. 18. socialstyrelsen: statistik €over h€also-och sjukvårdspersonal. 2015. available from: shttps://www.socialstyrelsen.se/publikationer2016/ 2016-10-15 19. daly e, vessey mp, hawkins mm, carson jl, gough p, marsh s. risk of venous thromboembolism in users of hormone replacement therapy. lancet. 1996;348:977–80. 20. rossouw j, andersson g. risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results from the women’s health initiative randomized controlled trial. jama. 2002;288:321–33. 21. marsden j. long-term benefits and risks of hrt: breast cancer. post reprod health. 2016;22:85–91. 22. lacroix az, chlebowski rt, manson je, aragaki ak, johnson kc, martin l, et al. health outcomes after stopping conjugated equine estrogens among postmenopausal women with prior hysterectomy: a randomized controlled trial. jama. 2011;305:1305–14. 23. lambe m, wigertz a, holmqvist m. reduction in use of hormone replacement therapy: effects on swedish breast cancer incidence trends only seen after several years. breast cancer res treat. 2010; 121:679–83. 24. socialstyrelsen statistic database for drugs. 2010 and 2015. available from: https://www.socialstyrelsen.se/statistik/statistikefteramne/lakemedel 25. zhang y-l, chen j-h, lu w, li b-l, zhu q-y, wan x-p. efficacy of postoperative hormone replacement therapy on prognosis of patients with serous ovarian carcinoma. chin med j (engl). 2016; 129:1316. upsala journal of medical sciences 229 https://www.cancercentrum.se/samverkan/cancerdiagnoser/gynekologi/livmoderkropp/vardprogram/gallande-vardprogram/ https://www.cancercentrum.se/samverkan/cancerdiagnoser/gynekologi/livmoderkropp/vardprogram/gallande-vardprogram/ https://www.cancercentrum.se/samverkan/cancerdiagnoser/gynekologi/aggstock/vardprogram/gallande-vardprogram-aggstockscancer/ https://www.cancercentrum.se/samverkan/cancerdiagnoser/gynekologi/aggstock/vardprogram/gallande-vardprogram-aggstockscancer/ https://www.socialstyrelsen.se/publikationer2016/2016-10-15 https://www.socialstyrelsen.se/publikationer2016/2016-10-15 https://www.socialstyrelsen.se/statistik/statistikefteramne/lakemedel https://www.socialstyrelsen.se/statistik/statistikefteramne/lakemedel abstract introduction material and methods statistics ethical approval results low-risk endometrial cancer high-risk endometrial cancer epithelial ovarian cancer comparison to japanese and german studies discussion acknowledgements declaration of interest funding notes on contributors references work at inpatient care units is associated with an increased risk of sars-cov-2 infection; a cross-sectional study of 8679 healthcare workers in sweden original article work at inpatient care units is associated with an increased risk of sars-cov-2 infection; a cross-sectional study of 8679 healthcare workers in sweden anna-karin lidstr€oma, fredrik sunda, bo albinssonb , johan lindb€ackc and gabriel westmana adepartment of medical sciences, section of infectious diseases, uppsala university, uppsala, sweden; blaboratory of clinical microbiology, uppsala university hospital, uppsala, sweden; cuppsala clinical research center, uppsala university, uppsala, sweden abstract background: during the covid-19 pandemic, the protection of healthcare workers has been in focus throughout the world, but the availability and quality of personal protective equipment has at times and in some settings been suboptimal. materials and methods: a total of 8679 healthcare workers and healthcare support staff in the county of uppsala, north of stockholm, were included in this cross-sectional study. all subjects were analysed for igg anti-sars-cov-2, and predictors for positive serostatus were analysed in a logistic regression model including demographic parameters and self-reported employment characteristics. results: overall, 577 (6.6%) were classified as seropositive, with no statistically significant differences between healthcare workers and support staff. among healthcare workers, age (or 0.987 per year, 95% ci 0.980–0.995), time to sampling (or 1.019 per day, 95% ci 1.004–1.035), and employment at an outpatient care unit (or 0.620, 95% ci 0.487–0.788) were statistically significantly associated with risk of infection. covid-19 specific units were not at particular risk, compared to other units with comparable characteristics and staff demography. conclusion: our findings indicate that sars-cov-2 transmission is related to inpatient healthcare work, and illustrate the need for a high standard of basic hygiene routines in all inpatient care settings. article history received 23 june 2020 revised 3 july 2020 accepted 3 july 2020 keywords covid-19; healthcare workers; igg; sars-cov-2; transmission introduction the sars-cov-2 pandemic began in late 2019, originating from the hubei province in china (1). compared to seasonal influenza, the pattern of human-to-human transmission appears more clustered around super-spreaders of the virus, causing national and regional differences that are yet to be completely understood (2). similar to the sars-cov-1 outbreak in 2003–2004, there were early reports of frequent transmission to healthcare professionals, including several with fatal outcome (3,4). even though most countries put extensive effort into acquiring personal protective equipment in order to provide adequate protection for caregivers, numerous reports describe shortages in quantity or quality of the protective equipment provided (5). in swedish hospital units dedicated to care for covid-19 patients, single-use protective masks were by necessity to a large extent replaced by multi-use filter masks intended for military or civilian purposes. although the technical filter performance is superior to ffp3-class devices, the total protective effect of these devices in a healthcare setting is not known (6). the adaptive immune response to sars-cov-2 infection is multi-faceted, including both cellular and humoral components, but for diagnostic purposes the development of virus specific igg has been a gold standard for convalescent patients where rna no longer can be amplified from upper airway specimens (7). early in the pandemic point-of-care antibody tests dominated the market but have now to a variable extent been replaced with antibody detection kits on high throughput platforms. the sensitivity and specificity of the abbott architect sars-cov-2 igg assay has previously been evaluated, but it is still not clear to what extent the severity of covid-19 disease affects the level of seroconversion. this could result in lower sensitivity in populations with mild or asymptomatic disease (8). region uppsala is a swedish public health region located just north of stockholm, delivering health care to the population in the county of uppsala as well as specialized care in the university hospital to inhabitants referred from surrounding counties. from 27 may 2020, all healthcare and healthcare administrative support staff in region uppsala were offered testing for igg anti-sars-cov-2. here, we present the rate of infection and investigate professional and demographic factors associated with transmission. contact gabriel westman gabriel.westman@medsci.uu.se department of medical sciences, uppsala university, uppsala, 751 85, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 4, 305–310 https://doi.org/10.1080/03009734.2020.1793039 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1793039&domain=pdf&date_stamp=2020-10-22 http://orcid.org/0000-0002-1184-9267 http://orcid.org/0000-0002-6473-8798 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1793039 http://www.tandfonline.com materials and methods study subjects between 27 may and 25 june 2020, all healthcare staff, including support staff, in region uppsala were offered free testing for igg anti-sars-cov-2 within the study. the largest employers include uppsala university hospital (approximately 8000 employees), primary health care (approximately 1400 employees), and enk€oping hospital (approximately 550 employees). however, as also part-time workers and private healthcare providers were allowed to participate, the exact number of potential participants is unknown. study subjects were to be above the age of 18 and without symptoms of airway infection for at least seven days. after informed consent was obtained, sampling was performed in line with clinical routines at their unit of employment or at general sampling units, at the discretion of the study subject. all subjects’ self-reported place of work was coded in the analysis into three independent variables: primary versus hospital care, outpatient versus inpatient care, and covid-19 (specific and possible) units versus other units. coding was done up to the level of detail provided by the study subject, with missing data leading to differences in the denominators presented in the results section. in covid-19 possible units such patients could have been cared for, whereas in covid19 specific units most patients were diagnosed with covid19. personal protective measures in covid-19 possible and specific units (including intensive care units) were standardized and included both multi-use filter face masks and long-sleeved gowns at all times. the study was approved by the swedish ethical review authority (no. 2020–02688). igg anti-sars-cov-2 immunoassay the analysis of sars-cov-2 igg antibodies was carried out using the ce-labelled sars-cov-2 igg kit with nucleoproteinbased antigen on the architect i2000sr analyser (abbott, abbott park, il, usa). the assay is an automated, two-step immunoassay for detection of igg antibodies to sars-cov-2 in human serum and plasma using chemiluminescent microparticle immunoassay (cmia) technology. sample, sars-cov-2 antigen-coated paramagnetic microparticles, and assay diluent are combined. the igg antibodies to sars-cov-2 present in the sample bind to sars-cov-2 antigen-coated microparticles. after washing, an anti-human igg acridinium-labelled conjugate is added to create a reaction mixture. following a wash cycle, pre-trigger and trigger solutions are added. the resulting chemiluminescent reaction is measured as a relative light unit (rlu). the presence or absence of igg antibodies to sars-cov-2 in the sample is determined by comparing the chemiluminescent rlu in the reaction to the calibrator rlu. there is a direct relationship between the amount of igg antibodies to sars-cov-2 in the sample and the rlu detected by the system optics. this relationship is reflected in the calculated index (s/c). a positive/ negative cut-off of 1.4 s/c was used in line with the manufacturer’s instructions. 0.0 0.5 1.0 1.5 0.01 0.10 1.00 10.00 cmia igg (log10) d e n si ty figure 1. density plot illustrating the distribution of igg anti-sars-cov-2. all values shifted 0.01 to allow logarithmic transformation. all subjects included in the analysis. 306 a.-k. lidström et al. statistical analyses statistical analyses were performed with r version 3.4.1, with packages plyr version 1.8.4 and ggplot2 version 3.0 (9). proportions of seropositive subjects were estimated and compared using uniand multivariable logistic regression models. the association with age and calendar time was assumed to be linear on the log-odds scale. unadjusted results are presented as the estimated prevalence with corresponding 95% confidence intervals. associations between the prevalence and the studied factors in the multivariable models are presented as odds ratios with corresponding 95% confidence intervals and p value less than 0.05. for all tests, a statement of statistical significance implies a p values less than 0.05. results igg anti-sars-cov-2 immunoassay during the four-week testing period a total of 8679 individuals (77% women and 23% men) participated in the study. all blood samples were analysed for igg anti-sars-cov-2, with 577 (6.6%) positive results using the manufacturer’s cut-off at 1.4 s/c. antibody levels were distributed into two populations with a geometrically neutral cut-off at approximately 1.0 s/c (figure 1). this is in line with a borderline result category recently introduced in clinical use between 0.9 and 1.39 s/c, which would expand the seropositive population to 635 (7.3%) subjects. baseline characteristics and subgroup prevalence igg positive study subjects were slightly younger and more often working at an inpatient care unit, compared to igg negative subjects. evaluation of the unadjusted subgroup prevalence of igg anti-sars-cov-2 seropositive subjects shows higher proportions of igg positive subjects among male participants and those working in inpatient care units and covid-19 specific units (table 1; figure 2). predictors of igg anti-sars-cov-2 serostatus to analyse the overall effect of healthcare employment versus administrative support employment on the risk of sarscov-2 infection, a logistic regression model was created using age, gender, and sampling time as covariates (table 2). working with direct patient contact versus healthcare support did not appear to have a statistically significant effect, but lower age and male sex were both associated with an increased risk of infection. table 1. characteristics of igg anti-sars-cov-2 positive and negative study subjects. igg positive igg negative age, years 42 (18–78) 45 (18–85) sampling time from project start, days 12 (0–28) 12 (0–29) male sex 164/577 (28.4%) 1855/8102 (22.9%) working in health care 495/577 (85.8%) 6799/8102 (83.9%) working in primary health care 72/577 (12.5%) 1351/8060 (16.8%) working with outpatient care 186/465 (40.0%) 3301/6311 (52.3%) working in covid-19 specific unit 39/577 (6.8%) 387/8060 (4.8%) working in covid-19 possible unit 111/577 (19.2%) 1682/8060 (20.9%) data presented as medians (range) or proportions. subgroup sex female male working in health care no yes working in primary health care no yes working with outpatient care no yes working in a covid−19 unit no covid−19 specific covid−19 possible igg pos/n 413/6659 164/2020 82/1385 495/7294 505/7214 72/1423 279/3289 186/3487 427/6418 39/426 111/1793 prevalence [95% ci] 0.062 [0.056, 0.068] 0.081 [0.070, 0.094] 0.059 [0.047, 0.073] 0.068 [0.062, 0.074] 0.070 [0.064, 0.076] 0.051 [0.040, 0.063] 0.085 [0.076, 0.095] 0.053 [0.046, 0.061] 0.067 [0.061, 0.073] 0.092 [0.066, 0.123] 0.062 [0.051, 0.074] 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 igg anti−sars−cov−2 seroprevalence ● ● ● ● ● ● ● ● ● ● ● figure 2. forest plot of igg anti-sars-cov-2 prevalence. subgroup prevalence and confidence intervals of igg anti-sars-cov-2 positivity. n is the total number of subjects in each category. all subjects included in the analysis. table 2. predictors of igg anti-sars-cov-2 serostatus. odds ratio 95% confidence interval p value age, years 0.984 0.978–0.991 <0.001 time from study start, days 1.005 0.992–1.019 0.41 male sex 1.334 1.104–1.612 0.003 working in health care 1.175 0.918–1.505 0.2 multivariable logistic regression model. all study subjects included in the analysis. upsala journal of medical sciences 307 fi g u re 3. u n iv ar ia b le lo g is ti c m od el lin g of re la ti on b et w ee n ag e (p an el a ,l ef t) an d ti m e to sa m p lin g (p an el b, ri g h t) an d ri sk of ig g an ti -s a rs -c ov -2 p os it iv it y. a ll su b je ct s in cl ud ed in an al ys is . 308 a.-k. lidström et al. subsequently, the risk of sars-cov-2 infection in relation to type of workplace was analysed in a logistic regression model using the same covariates as described above but including only staff with direct healthcare tasks. in addition to lower age and later sampling time (figure 3), also work at an inpatient care unit was statistically significantly associated with an increased risk of infection (table 3). there were no statistically significant risk differences related to working at covid-19 specific care units or at primary healthcare centres. discussion the covid-19 pandemic has put healthcare systems across the world at stress, to an extent that has rarely been seen in developed countries. in sweden, the just-in-time logistics system for delivering personal protective equipment failed within days to weeks when the demand quickly increased to a level that was beyond the imagination of the regional pandemic planning in early 2020 (10). given that staff availability and experience are key to healthcare capacity and quality, protecting healthcare professionals from hospital-acquired infections is pivotal. in contrast to early reports of numerous cases of severe infections and deaths among chinese and italian healthcare professionals (3,4), the overall rate of igg anti-sars-cov-2 in our study is relatively low. however, our analyses show that healthcare workers at inpatient care units have a statistically significantly increased risk of infection that cannot be explained by demographic differences or transmission at covid-19 specific care units only. the age-dependent association likely represents two factors, both a higher level of community transmission among the young, and a correlation between age and level of physical contact with patients during inpatient care, which relates to differences in length of training and the pyramid-shaped age-related hierarchy among different categories of clinical staff. our findings emphasize the need for a high standard in basic hygiene routines in all settings, especially in inpatient care where physical contact with patients is more extensive, as covid-19 patients are not always easily identifiable through symptom-based triage. in contrast, covid-19 specific units with routines of high hygiene standard and protective measures do not appear to be at an additional risk compared to other comparable inpatient and outpatient care settings. the performance of the igg anti-sars-cov-2 immunoassay used in this study is well characterized in patients with symptomatic disease, but less is known about pcr-positive patients with mild or asymptomatic disease where the rate of igg seroconversion has been shown to be lower (11). as this affects the interpretation of seroepidemiological data, studies are needed to further characterize the cellular immune response and cross-reactivity with other circulating corona viruses (12). however, this should not affect our within-study comparisons of healthcare worker sub-groups. we believe our results are relevant and generalizable to other swedish healthcare settings and to some extent also to international inpatient care settings. also, our findings are comparable with previously published studies of seroprevalence in healthcare workers in contact with covid-19 patients (13). the total rate of igg positivity and the temporal trend indicate ongoing community transmission of sars-cov-2 in the uppsala county. however, the vast majority of the population has not been infected. this is in line with the current knowledge of the cluster-like spread of the virus and has implications for public health strategies. unless there is a high proportion of occult infection not resulting in igg seroconversion detected by the assay used in this study, the remaining spread of sars-cov-2 needed to achieve herd immunity appears to be a goal too far away to be feasible in terms of the associated disease burden. acknowledgements the authors are very thankful to all staff at uppsala university hospital involved in sampling, analysis of samples, and data management. disclosure statement none of the authors have any disclosures to report in relation to this study. funding this study was funded by uppsala county council (alf grant). notes on contributors anna-karin lidstr€om, md. infectious disease specialist at the department of infectious diseases, uppsala university hospital, uppsala, sweden. fredrik sund, md and phd. head of department of infectious diseases, uppsala university hospital, uppsala, sweden. bo albinsson, md, phd stud. clinical virology and bacteriology specialist at the laboratory of clinical microbiology, uppsala university hospital, uppsala, sweden. johan lindb€ack, phlic/msc mathematical statistics. biostatistician at uppsala clinical research center, uppsala university, uppsala, sweden. gabriel westman, md, phd, and msceng. infectious disease specialist at the department of infectious diseases, uppsala university hospital, uppsala, sweden and at the swedish medical products agency, uppsala, sweden. orcid bo albinsson http://orcid.org/0000-0002-1184-9267 johan lindb€ack http://orcid.org/0000-0002-6473-8798 table 3. predictors of igg anti-sars-cov-2 serostatus. odds ratio 95% confidence interval p value age at sampling, years 0.988 0.980–0.995 0.001 time from study start, days 1.019 1.004–1.035 0.014 male sex 1.107 0.879–1.394 0.387 working in primary health care 0.711 0.493–1.026 0.068 working with outpatient care 0.631 0.497–0.801 <0.001 working in covid-19 specific unit 1.114 0.766–1.619 0.572 working in covid-19 possible unit 1.275 0.945–1.721 0.112 multivariable logistic regression model. only healthcare staff included in the analysis. upsala journal of medical sciences 309 references 1. huang c, wang y, li x, ren l, zhao j, hu y, et al. clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet. 2020;395:497–506. 2. on kwok k, hin chan hh, huang y, cheong hui ds, anantharajah tambyah p, in wei w, et al. inferring super-spreading from transmission clusters of covid-19 in hong kong, japan and singapore. j hosp infect 2020. doi:10.1016/j.jhin.2020.05.027 3. lapolla p, mingoli a, lee r. deaths from covid-19 in healthcare workers in italy – what can we learn? infect control hosp epidemiol. 2020. doi:10.1017/ice.2020.241 4. zhan m, qin y, xue x, zhu s. death from covid-19 of 23 health care workers in china. n engl j med. 2020;382:2267–8. 5. kamerow d. covid-19: the crisis of personal protective equipment in the us. bmj. 2020;369:m1367. 6. f€orsvarsmaktens information om skyddsmask 90. available at: https://www.forsvarsmakten.se/sv/information-och-fakta/materieloch-teknik/cbrn-materiel/skyddsmask-90/ 7. long qx, liu bz, deng hj, wu gc, deng k, chen yk, et al. antibody responses to sars-cov-2 in patients with covid-19. nat med. 2020;26:845–8. 8. bryan a, pepper g, wener mh, fink sl, morishima c, chaudhary a, et al. performance characteristics of the abbott architect sarscov-2 igg assay and seroprevalence in boise, idaho. j clin microbiol 2020. doi:10.1128/jcm.00941-20 9. r core team. r: a language and environment for statistical computing. vienna (austria): r foundation for statistical computing; 2017. 10. sveriges radio [radio sweden]. lagren av skyddsutrustning tog slut på fyra dagar [stocks of protective equipment ran out in four days]. 2020 jun 16. available at: https://sverigesradio.se/sida/artikel.aspx?programid=83&artikel=7496947 11. long qx, tang xj, shi ql, li q, deng hj, yuan j, et al. clinical and immunological assessment of asymptomatic sars-cov-2 infections. nat med. 2020. doi:10.1038/s41591-020-0965-6 12. grifoni a, weiskopf d, ramirez si, mateus j, dan jm, moderbacher cr, et al. targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals. cell 2020;181:1489–501.e15. 13. korth j, wilde b, dolff s, anastasiou oe, krawczyk a, jahn m, et al. sars-cov-2-specific antibody detection in healthcare workers in germany with direct contact to covid-19 patients. j clin virol. 2020;128:104437. 310 a.-k. lidström et al. https://doi.org/10.1016/j.jhin.2020.05.027 https://doi.org/10.1017/ice.2020.241 https://www.forsvarsmakten.se/sv/information-och-fakta/materiel-och-teknik/cbrn-materiel/skyddsmask-90/ https://www.forsvarsmakten.se/sv/information-och-fakta/materiel-och-teknik/cbrn-materiel/skyddsmask-90/ https://doi.org/10.1128/jcm.00941-20 https://sverigesradio.se/sida/artikel.aspx?programid=83&artikel=7496947 https://sverigesradio.se/sida/artikel.aspx?programid=83&artikel=7496947 https://doi.org/10.1038/s41591-020-0965-6 abstract introduction materials and methods study subjects igg anti-sars-cov-2 immunoassay statistical analyses results igg anti-sars-cov-2 immunoassay baseline characteristics and subgroup prevalence predictors of igg anti-sars-cov-2 serostatus discussion acknowledgements disclosure statement references progress and milestones in scientific communication – a 150 years perspective full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 progress and milestones in scientific communication – a 150 years perspective torbjörn karlsson to cite this article: torbjörn karlsson (2015) progress and milestones in scientific communication – a 150 years perspective, upsala journal of medical sciences, 120:2, 63-64 to link to this article: https://doi.org/10.3109/03009734.2015.1022669 © informa healthcare published online: 14 apr 2015. submit your article to this journal article views: 293 view related articles view crossmark data citing articles: 3 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://doi.org/10.3109/03009734.2015.1022669 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1022669 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1022669 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1022669&domain=pdf&date_stamp=2015-04-14 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1022669&domain=pdf&date_stamp=2015-04-14 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1022669#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1022669#tabmodule upsala journal of medical sciences. 2015; 120: 63–64 foreword progress and milestones in scientific communication – a 150 years perspective torbjörn karlsson chairman of the upsala medical society, uppsala, sweden science expands knowledge not only through understanding but also through communication. words form ideas, and words are tools for thoughts. the world looked very different 150 years ago. scientific communication had a completely different language than now, and we were more strictly dependent on the spoken and printed word. the dissemination of new ideas was much slower than today, leading to a lower tempo in the questioning of new as well as older thoughts. a scientific journal published in uppsala, sweden, and most often in swedish, had difficulties in the nineteenth century in reaching the wider scientific community. the relatively new digitalized and immediate scientific communication by means of the internet and database searches has given us a completely new landscape. proudly we can say that our journal has evolved much since the early days and is today globally accessible. this is thanks to scientists presenting their work, and to editors and referees for reading and criticizing it. true open access is today an important cornerstone in the idea of scientific publication, and we want the journal to remain accessible for everyone. publications were initially predominately from authors at uppsala university and other swedes, and still we welcome good manuscripts from locally active scientists. however, the journal is open for everyone, and we welcome important science reports from wherever they are produced. we have had the opportunity to read many papers in this journal through the years (1-4). some of the publications are still much cited and are still of high scientific impact. others reflect their time and ideas and thoughts which are difficult for us to understand and accept today. finding a completely new gland in the human body (5) is not possible today. seeking the human mind and awareness in a decapitated head is hopefully the scientific scope of a bygone age (holmgren, 1876, ‘on decapitation, from a physiological viewpoint’) (6), but today we can deepen our understanding of human physiology, e.g. by means of the genetic knowledge given us in more recent decades. our increased computer power means that we can create better images than ever before and with a resolution down to the molecular level. today we can easily make complex calculations and models of dynamic procedures with powerful tools not foreseen by our predecessors. the very first papers of our journal were published in swedish (e.g. by frithiof holmgren) and had to be translated in order to be accessible for a greater scientific community. important work was later published in english, but because of the turbulent war times and the lack of scientific databases and congresses it took a long time before they reached the international community (e.g. lundsgaard, 1939, ‘on the mode of action of insulin’) (7). now we live in a world where information is at our fingertips, a world where science and education have come closer to everybody. the thoughts of dr marshall mcluhan in the classical ‘the gutenberg galaxy’ 1962 have evolved another step. the upsala journal of the medical sciences has been published for 150 years. what is the future of the journal? will we still read journals printed on paper in the years to come? we have to stay open-minded and ready for the communication tools of the future, not pointing in any specific direction but ready to adapt. reading the journal through the years means finding many interesting thoughts but also some that we do not agree with correspondence: torbjörn karlsson, upsala medical society, uppsala, sweden. e-mail: torbjorn.karlsson@surgsci.uu.se (received 3 february 2015; accepted 20 february 2015) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1022669 http://informahealthcare.com/journal/ups mailto:torbjorn.karlsson@surgsci.uu.se today. perspective changes, and all subjects and findings presented in the journal should be open for debate. the first title of the journal was upsala läkareförenings förhandlingar (proceedings of the upsala medical society), the name in itself indicating that the findings published were to be discussed. views changed over the years, and thus it is necessary to apply a historical perspective even when we look at some items in this specific issue. the primary goal of the upsala medical society is to promote medical science and education. it stands, as ever, close to the faculty of medicine of uppsala university. in 1865 the publication of a scientific and educational journal was a way of fulfilling the purpose of the society; as it is today. the journal has an important place in the history of medical science, and the upsala medical society is proud to own and run a modern scientific journal (8). references 1. sandström i. on a new gland in humans and several vertebrates. upsala läkareförenings förhandlingar. 1880;15:441–71. 2. holmgren f. about the retinal stream. upsala läkareförenings förhandlingar. 1870–1871;6:419–55. 3. grimelius l. a silver nitrate stain for alfa2 cells in human pancreatic islets. acta soc med ups. 1968;73:243–70. 4. westermark p. quantitative studies of amyloid in the islets of langerhans. ups j med sci. 1972;77:91–4. 5. johansson h. the uppsala anatomist ivar sandström and the parathyroid gland. ups j med sci. 2015;120:72–7. 6. holmgren f. on decapitation, from a physiological viewpoint. upsala läkareförenings förhandlingar. 1876;7–8:588–648. 7. lundsgaard e. on the mode of action of insulin. upsala läkareförenings förhandlingar. 1939;45:143–51. 8. andersson a. further improvements of our journal performance figures. ups j med sci. 2014;119:295–7. 64 foreword ss1 references vol_117_004_sups_a_725511 353..354 full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 a substantial increase of the impact factor arne andersson & gunnar ronquist to cite this article: arne andersson & gunnar ronquist (2012) a substantial increase of the impact factor, upsala journal of medical sciences, 117:4, 353-354, doi: 10.3109/03009734.2012.725511 to link to this article: https://doi.org/10.3109/03009734.2012.725511 © informa healthcare published online: 30 oct 2012. submit your article to this journal article views: 296 view related articles citing articles: 2 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2012.725511 https://doi.org/10.3109/03009734.2012.725511 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2012.725511 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2012.725511 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2012.725511#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2012.725511#tabmodule upsala journal of medical sciences. 2012; 117: 353–354 editorial a substantial increase of the impact factor arne andersson1 & gunnar ronquist2 1editor, basic sciences, and 2editor, clinical sciences when we contracted, some 4 years ago, an established publisher (informa healthcare) for the publication of our journal, we had to set a reasonable goal for our achievements. perhaps, not surprisingly, we looked at the so much debated impact factor institute and found that by reaching the critical 1.0 level—used by our local faculty in their budgetary system—a realistic and meaningful aim had been set. as a physical token of this planning we promised our editorial board members a champagne party the day we had brought about this considerable effort. so, it is our great pleasure to announce that we have succeeded. as published late june by thomson reuters (tr) in their annual report, our 2-year impact factor has risen to 1.063, a more than 70% increase as compared with last year’s figure (table i). the increase of the 5-year impact factor figure was less impressive, indicating that the citations of the last 2 years’ articles have contributed to a higher extent. we can also see a sharp increase of the immediacy index, telling us that many of our articles are cited very soon after they have been released on the ‘early online’ site at the website of the journal. it is also interesting to see that the numbers of total cites in both databases (t&r and scopus) have increased substantially fulfilling our objective to be cited once every day all days of the year. as we pointed out in our latest editorial on the performance of ujms (1), we have managed to keep up the percentage of our papers that indeed become cited—75%–80% after 3–5 years—which is very close to figures for other more well-known and wellreputed scientific journals. concerning the rank figures, it can be seen that we are close to entering the best half of all journals belonging to the category ‘medicine, general and internal’. a reasonable next goal of our journal will be to establish ourselves in the better half. of course, these increases in different parameters all depend on the fact that we have successfully selected good papers for publication. in that process our editorial board members and reviewers have all done a tremendous job. we take this opportunity to thank them all! the champagne expert has been called upon. in order to make this selection successful a good submission rate is a prerequisite. we have had some 150–200 original papers submitted each year, and we accept about 50 manuscripts for publication. so almost three out of four papers have to be rejected, and that is a cumbersome process. in this context it is interesting to see how one single article can be the difference between failure and success. amongst the citations for the 2011 impact factor figure there is one paper that stands out. the review by niklas nordquist and lars oreland on ‘serotonin, genetic variability, behaviour, and psychiatric disorders’ (2) was cited 16 times last year and then was responsible for more than 20% of the citations to the 2010 and 2011 volumes last year. at present (august 2012) that particular article has been cited 24 times. an article cited that much belongs to the 1% most-cited publications (3). for that type of calculations citations during the publication year and the two following years are counted. the number of cites for the 1% limit is 22, and for the corresponding 10% limit 8 citations. we can foresee that a couple of our articles will reach that lower level as well since almost half a year of citation collection still remains. apart from the fact that the quality of our published papers undoubtedly has increased, there might be at least one more issue that speaks in favour of our issn 0300-9734 print/issn 2000-1967 online � 2012 informa healthcare doi: 10.3109/03009734.2012.725511 journal when authors look for an attractive publication source. also the ‘open access’ entrance on the publication arena has become more and more important. ever since the start of the electronic publication of our journal, all papers—old (since a few years back) as well as brand-new—have been freely accessible at our website. this in combination with the fact that we have not charged our authors any type of publication fees has made our journal quite unique. most probably, however, we will have to change this somewhat in the sense that we will introduce some sort of handling fee for our published articles. at least for non-faculty members. this then should be interpreted as one more invitation to our researchers at the faculty to submit their reports to our journal. last year some 40% of our published papers came from uppsala-based teams. we would very much welcome more of them. references 1. editorial. lessons from the recent journal citation report figures. ups j med sci. 2010;115:161–2. 2. nordquist n, oreland l. serotonin, genetic variability, behaviour, and psychiatric disorders—a review. ups j med sci. 2010;115:2–10. 3. karlsson s. den svenska produktionen av högt citerade vetenskapliga publikationer. vetenskapsrådets lilla rapportserie. 2010;1:1–21. table i. 5-year trends in the performance of the journal. year impact factor 2-year tr impact factor 5-year tr immediacy index tr rank tr total cites tr total cites scopus % not-cited articles scopus 2007 0.500 0.570 0.033 79/100 229 247 23 2008 0.677 0.683 0.067 82/107 262 274 20 2009 0.733 0.745 0.083 90/103 259 281 26 2010 0.636 0.623 0.209 99/153 271 310 45 2011 1.063 0.770 0.395 80/153 347 378 63 354 editorial a framework for monitoring of new drugs in sweden article a framework for monitoring of new drugs in sweden thomas carsa, lars lindhagenb and johan sundstr€oma,b adepartment of medical sciences, uppsala university, uppsala, sweden; buppsala clinical research center uppsala university, uppsala, sweden abstract in order to monitor the net public health benefit of new drugs, especially in the light of recent stepwise approval approaches, there is a need to optimize real-time post-marketing evaluation of new drugs using data collected in routine care. sweden, with its unique possibilities for observational research, can provide these data. we herein propose a framework for continuous monitoring of the effectiveness, safety, and cost-effectiveness of new drugs, using prospectively determined protocols designed in collaboration between all relevant stakeholders. we believe that this framework can be a useful tool for healthcare authorities and reimbursement agencies in the introduction of new drugs. article history received 29 june 2018 revised 16 november 2018 accepted 16 november 2018 keywords comparative effectiveness research; pharmacoepidemiology; propensity score; real-world evidence; sequential monitoring introduction before the marketing of a new drug, its efficacy and safety are evaluated using randomized controlled trials (rcts). rcts are invaluable for establishing the relative treatment benefit of a new drug. however, the population eventually treated with a new drug often differs in many respects from the sample studied in the rcts. in routine care, drugs are often used by elderly patients with more comorbidities and with multiple concomitant drug use. rcts are also typically powered to detect only the most common adverse events. consequently, rare or late adverse effects are not expected to be identified in rcts. hence, understanding of the realizable net public health benefit from a new drug requires other study designs than the rct. some of the data needed for such estimations may be available before the new drug is marketed, but other data are not available until the drug is actually used. society needs these estimations as soon as possible, for determination of long-term safety, net public health benefit, and regulatory decisions. we herein propose a framework for such studies, using data collected in routine care. observational studies of comparative effectiveness and safety comparative effectiveness research (cer) involves the comparison of healthcare interventions, aiming to produce evidence regarding the effectiveness and safety of medical products (1). a drug’s efficacy is defined as ‘the extent to which a specific health intervention produces a beneficial result under ideal conditions’ (i.e. a response to the question: ‘can it work?’), whereas effectiveness can be defined as ‘the extent to which a specific health intervention produced a beneficial result when deployed in the field under routine conditions’ (i.e. a response to the question: ‘does it work in practice?’) (2). the ultimate goal of comparative effectiveness research is to improve health by developing and disseminating evidence to patients, healthcare professionals, and policy-makers regarding the effectiveness of specific interventions. the exponential developments in the quantity, quality, and availability of digital healthcare data generated in routine care hold great promise for development of observational cer in digitally mature countries. this development parallels a trend towards more use of adaptive licensing of new drugs (3), with healthcare authorities and regulators relying increasingly on observational data for evaluation of drug effectiveness and safety (4–6). unique possibilities in sweden sweden provides unique opportunities for observational cer. the advantages lie in the country’s civic registration system involving a 12-digit personal identity number, unique to all swedish citizens (7), and the fact that all residents have universal access to healthcare with a negligible co-payment for healthcare visits, hospitalizations, and drugs (8). using the personal identity number to link healthcare data to a variety of nationwide health registers (classifying diagnoses using the international classification of diseases [icd] system (9), surgical procedures using the nordic medico-statistical committee classification of surgical procedures [ncsp] system (10), and filled drug prescriptions using the anatomical therapeutic chemical classification [atc] system (11)), quality contact thomas cars thomas.cars@medsci.uu.se department of medical sciences, uppsala university, uppsala, sweden. � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 1, 46–50 https://doi.org/10.1080/03009734.2018.1550454 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1550454&domain=pdf http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1550454 http://www.tandfonline.com registers, and registers on sociodemographics and socioeconomics allows for research on large populations with nearzero loss to follow-up. although many countries have registry data on prescription drugs at the individual level in ambulatory care, such data are often missing for drugs administered in hospitals (12, 13). lack of individual-level data for hospital-based drugs is a large and increasing problem, since we are currently observing a trend towards more hospital-administered drugs. electronic health records (ehrs) provide an opportunity to include hospital drugs in observational cer. sweden started to implement ehrs in the 1990s, and all regions had implemented ehrs in all healthcare areas by 2012 (14). today, eight different ehr systems account for 97% of all ehr usage in sweden, and most regions in sweden have chosen to implement one single ehr system in their region (15). ehrs also contain more detailed clinical information than data from national health registries. since the medical records are recorded as part of patient care, ehrs are instantly updated. this opens up for new and better opportunities to monitor treatments continuously with regard to its utility, safety, and cost-effectiveness. in a phd thesis, ‘real-time monitoring of healthcare interventions in routine care. effectiveness and safety of newly introduced medicines’ (16), we developed and validated a ‘sequentially evaluated non-randomized comparative effectiveness (sence)’ framework for continuous follow-up of new treatments using data from routine care in sweden. this model is based on ehr data but can also utilize data from other sources. the model is built to collect and analyze data continuously as soon as new information is generated in the clinical data sources, making it possible to evaluate drugs sequentially and provide observational effectiveness evidence in as timely a manner as possible. the following is a summary of the proposed framework. proposal for a framework for continuous postmarketing monitoring of new drugs in sweden we have developed a generic sequential cohort model for real-time head-to-head (drug a versus drug b) comparisons of new drugs when used in routine care. we propose that this model is set up prospectively before a new drug enters the market, and sequential monitoring is launched when the drug is marketed. other interventions than drugs may also be analysed. to maximize the credibility and utility of the results produced by the model, we propose that this framework is carried out in collaboration between all relevant stakeholders (regulators, payers, and manufacturers). process before the launch of a new therapy, regulators and authorities set the requirements for the post-marketing monitoring of the new drug. a project steering group and a scientific project group are formed. the steering group includes representatives from regulators, payers, and the drug manufacturer. the scientific project group includes experts in the actual therapeutical area, epidemiologists, and statisticians. initiation phase. in the initiation phase, a study protocol, statistical analysis plan (sap), and an ethics committee application are developed by the project group. permission to extract and include data is also obtained from data holders. a significant amount of work is dedicated to meticulously defining samples, data sources, exposures, best practice comparator drugs, covariates, and outcomes. a central task in this phase is the development of the causal models (17) and mimicking a target clinical trial (18). for development of causal models, we propose using the directed acyclic graphs (dags) approach (www.dagitty.net) in order to minimize potential bias (19). statistical models used in sequential cohort design are defined (20–22). before entering the data extraction and data management phase, all assumptions and the protocol are agreed upon between all stakeholders in the steering group, documented, and made available in the public domain (23). the monitoring should begin when the drug is marketed. the data extraction and data management phase and the data analysis phase (see below) are repeated until stable results have been obtained. data extraction and data management phase. in this phase, data are extracted from ehrs and pseudonymized. data from ehrs may also be linked to other data sources if necessary and defined in the initiation phase. all data should figure 1. in each recruitment cycle, new users of drug a and new users of drug b are included and added to the cohort to continuously increase the study sample size. upsala journal of medical sciences 47 http://www.dagitty.net undergo quality checks including but not limited to logical checks, outlier detection, and investigation of missingness patterns. this step results in an analysis database and a data management report. data analysis phase. in the analysis phase, data are analysed according to the sap. the model is sequentially updated in order to evaluate data as they are collected, at a frequency determined by the projected uptake of the drug figure 2. effect estimates are published after each recruitment cycle. figure 3. standardized differences in baseline covariates between new users of drug a and new users of drug b before and after adjusting on the propensity score. a standardized difference <0.1 indicates negligible imbalance. 48 t. cars et al. on the market. in a proof-of-concept study, we updated the model every six months, but the model can be updated at any desired frequency. in each time period (recruitment cycle), new users of the new treatment (drug a) and new users of a comparator treatment (drug b) are included and added to the cohort to continuously increase the study sample size (figure 1). at the end of each recruitment cycle, a propensity score (the probability of treatment assignment conditional on observed baseline covariates) (24) is estimated for all patients, and all patients are assessed for study outcomes. at the end of each recruitment cycle, a comparative effectiveness and/or comparative safety analysis is carried out for the entire sample (all new users of drugs a and b over the whole time at risk) and plotted sequentially for each recruitment cycle (figure 2). results are continuously made available in a timely fashion in the public domain. in this observational monitoring framework, we have chosen not to account for the sequential nature of the analysis. the rationale for this is that the intention of the sequential analysis was never to point out a single significant estimate or to terminate the study when a satisfactory result was observed. as a result of this strategy, each confidence interval presented in figure 2 is only valid one at a time. the goal of using a propensity score is to achieve covariate balance between groups of treated and controls, which is a fundamental step in the proposed process. several methods to assess balance have been proposed. one frequently used method is to estimate standardized differences and represent the number of standard deviations by which the two groups differ (25). we propose to visualize the standardized differences using the plot presented in figure 3. the legitimacy of causal inference in observational studies is based on the assumption that no unmeasured confounding exists. this is a very strong assumption, and analyses should therefore be accompanied by sensitivity analyses investigating how the study findings may be affected by the presence of unmeasured confounding. we have in this framework included one commonly used approach proposed by lin et al. (26) that involves evaluating how powerful an unmeasured confounder would have to be to change the observed results (figure 4). conclusion in order to ensure the effectiveness, safety, and cost-effectiveness of new drugs, especially in the light of recent stepwise approval approaches, there is a need for regulators, payers in healthcare, and the pharmaceutical industry to optimize real-time post-marketing evaluation of new drugs using data collected in routine care. sweden, with its unique possibilities for observational research, can have a strong position in the post-marketing monitoring of new drugs. we suggest that the herein proposed framework can provide timely and comprehensive observational evidence of effectiveness, safety, and costs of new drugs, using a prospectively determined protocol designed in collaboration between all relevant stakeholders. we further suggest that analyses are repeated regularly as long as they provide value, and that all results are published without delay in the public domain. we believe that this framework can be a useful tool for healthcare authorities and reimbursement agencies in the introduction of new drugs. declaration of interest the authors report no conflicts of interest. figure 4. evaluation of how powerful an unmeasured confounder would have to be to change the observed results. for example, if the prevalence of a potential unmeasured confounder is 40% in the drug a group (x-axis) and 10% in the drug b group, then the unmeasured confounder must have a risk estimate (hazard ratio) of the outcome close to 3 to fully explain the advantage of drug a over drug b. upsala journal of medical sciences 49 notes on contributors thomas cars, msc pharm, phd, is a postdoctoral research fellow at the department of medical sciences, uppsala university. lars lindhagen, msc, phd, is a biostatistician at uppsala clinical research center, uppsala university. johan sundstr€om is professor of epidemiology at uppsala university, and a cardiologist at uppsala university hospital. references 1. conway ph, clancy c. comparative-effectiveness research—implications of the federal coordinating council’s report. n engl j med. 2009;361:328–30. 2. haynes b. can it work? does it work? is it worth it? the testing of healthcareinterventions is evolving. bmj. 1999;319:652 3. eichler hg, baird lg, barker r, bloechl-daum b, børlum-kristensen f, brown j, et al. from adaptive licensing to adaptive pathways: delivering a flexible life-span approach to bring new drugs to patients. clin pharmacol ther. 2015;97:234–46. 4. sherman re, anderson sa, dal pan gj, gray gw, gross t, hunter nl, et al. real-world evidence what is it and what can it tell us? n engl j med. 2016;375:2293–7. 5. european medicines agency (ema). annual report 2017 [internet]. 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https://www.inera.se/globalassets/om-inera/styrdokument-och-rapporter/ehalsa-i-landstingen/ehalsa-it-ilandstingen-2017.pdf http://uu.diva-portal.org/smash/get/diva2:1015130/fulltext01.pdf http://uu.diva-portal.org/smash/get/diva2:1015130/fulltext01.pdf https://www.hsph.harvard.edu/miguel-hernan/causal-inference-book/ https://www.hsph.harvard.edu/miguel-hernan/causal-inference-book/ http://www.encepp.eu/encepp_studies/e_register.html http://www.encepp.eu/encepp_studies/e_register.html abstract introduction observational studies of comparative effectiveness and safety unique possibilities in sweden proposal for a framework for continuous post-marketing monitoring of new drugs in sweden process conclusion declaration of interest notes on contributors references repeated measures of body mass index and waist circumference in the assessment of mortality risk in patients with myocardial infarction article repeated measures of body mass index and waist circumference in the assessment of mortality risk in patients with myocardial infarction lars berglunda,b� , ulf ris�erusc and kristina hambraeusa,d,e auppsala clinical research center, uppsala university, uppsala, sweden; bdepartment of public health and caring sciences/geriatrics, uppsala university, uppsala, sweden; cdepartment of public health and caring sciences, uppsala university, clinical nutrition and metabolism, uppsala, sweden; ddepartment of cardiology, falun hospital, falun, sweden; edepartment of medical sciences, uppsala university, uppsala, sweden abstract aims: weight loss is recommended for myocardial infarction (mi) patients with overweight or obesity. it has, however, been suggested that obese patients have better prognosis than normal-weight patients have, but also that central obesity is harmful. the aim of this study was to examine associations between repeated measures of body mass index (bmi) and waist circumference (wc), and all-cause mortality. methods and results: a total of 14,224mi patients aged <75 years in sweden between the years 2004 and 2013 had measurements of risk factors at hospital discharge. the patients’ bmi and wc were recorded in secondary prevention clinics two months and one year after hospital discharge. we collected mortality data up to 8.3 years after the last visit. there were 721 deaths. we used anthropometric measures at the two-month visit and the change from the two-month to the one-year visit. with adjustments for risk factors and the other anthropometric measure the hazard ratio (hr) per standard deviation in a cox proportional hazard regression model for mortality was 0.64 (95% confidence interval [ci] 0.56–0.74) for bmi and 1.55 (95% ci 1.34–1.79) for wc, and 1.43 (95% ci 1.17–1.74) for a bmi decrease from month two to one year of more than 0.6 kg/m2. low bmi and high wc were associated with the highest mortality. conclusion: high wc is harmful regardless of bmi in mi patients. reduced bmi during the first year after mi is, however, associated with higher mortality, potentially being an indicator of deteriorated health. article history received 22 may 2018 accepted 22 june 2018 key words body mass index; myocardial infarction; obesity paradox; repeated measurements; waist circumference introduction in the year 2016 a total of 25,700 individuals in sweden were affected by a myocardial infarction (mi). the proportion that died within 28 days after the event was 25%. cardiovascular diseases continue to be the most common cause of death among both men and women (1). for mi patients several pharmacological and lifestyle interventions are recommended at hospital discharge in order to reduce the risk of early mortality. overweight and obesity are conditions associated with increased cardiovascular and non-cardiovascular mortality risks (2). overweight and obese mi patients are generally recommended to decrease their body weight (3). this recommendation is, however, supported by very weak evidence, and the most recent guidelines on cardiovascular disease prevention from the european society of cardiology recognizes that the optimal level of body mass index (bmi) after a cardiovascular event still is one of the important remaining gaps in evidence (4). several observational studies show an inverse association between bmi and mortality in subjects with coronary artery disease (cad). this phenomenon has been called the ‘obesity paradox’ (5–7). waist circumference (wc), a measure of central obesity, may be a more valid measure of fat distribution than bmi in cad patients, although wc carries larger measurement errors than bmi (8). there are only few studies examining the effect of central obesity on mortality in patients with cad (9,10). coutinho et al. conclude from a meta-analysis of four studies with 16,000 patients that central obesity is directly associated with higher mortality in individuals with cad, whereas the opposite is valid for bmi (10). the direct effect of central obesity on mortality was observed even in subjects with normal bmi. these studies use a single measurement of anthropometry (at hospital discharge) for each patient and are thus not able to consider fluctuations of bmi and wc. a single measurement of a predictor may not be sufficient to reveal its impact on an outcome, as post-mi changes in bmi and wc may also influence the risk, and it provides no basis for contact lars berglund lars.berglund@pubcare.uu.se department of public health and caring sciences/geriatrics, box 564, 751 22 uppsala, sweden. � 2018 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 1, 78–82 https://doi.org/10.1080/03009734.2018.1494644 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1494644&domain=pdf http://orcid.org/0000-0002-7437-9047 http://orcid.org/0000-0002-8620-4586 http://orcid.org/0000-0002-0768-2484 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1494644 http://www.tandfonline.com the recommendation for overweight and obese people to aim for a weight change after a myocardial infarction. we examined how mortality, up to 8.3 years after a secondary prevention visit at one year after the infarction, was related to levels and changes of bmi and wc during the first year after hospital discharge. material and methods patient population in this prospective cohort study, patients in sweden with mi between the years 2004 and 2013 were treated at hospital, and their data, including bmi, were collected in the swedish web-system for enhancement and development of evidencebased care in heart disease evaluated according to recommended therapies (swedeheart) registry (11). after the hospital stay the patients returned at two months and one year after hospital discharge to secondary prevention clinics, and bmi and wc data from these visits were entered into the swedeheart sub-register for secondary prevention after ihcu care (sephia) (1). dates for death, up to 8.3 years after the one-year visit, were collected from the swedish national population registry. inclusion criteria to the analysis sample were that the patient was younger than 75 years at hospital discharge (since the sephia registry had an upper age limit of 75), and was alive one year after hospital discharge. a further criterion was that the patient had complete data on variables from the hospital stay and had available anthropometric data from the two-month and one-year visits. the study was approved by the local ethics committee (dnr epn 2014/152). anthropometric measures well-trained nurses or physicians measured anthropometric indices, with patients wearing only minimal clothing with no footwear during measurements. weight and height were measured, with the patient standing, to the nearest 0.1 kg and 1 cm, respectively. if measurements were not possible, self-reported data were registered if the patient was able to communicate those. if not, data were registered as missing. bmi was calculated in the standard way: weight (kg) divided by square of height (m). wc was measured to the nearest cm with the patient standing, at the level midway between the lower rib margin and the iliac crest. the patient was asked to breathe out gently and relax during the measurement. statistics continuous variables were described by means and standard deviations, and categorical variables were described by numbers and percentages. in order to examine linearity of the associations between anthropometric variables and mortality we fitted and visualized a generalized additive model (gam) (12) with a binomial link function. in the gam model the anthropometric variables were adjusted for each other. in cox proportional hazards regression models we examined how all-cause mortality after the one-year visit was related to levels and changes of bmi and wc. the models were estimated with adjustments for the other anthropometric variable and covariates age and gender and further with adjustments for the other anthropometric variable and covariates age, gender, smoking, serum creatinine, diabetes, hypertension, hyperlipidaemia, previous myocardial infarction, previous stroke, previous congestive heart failure, diagnosis (stemi/nstemi), percutaneous coronary intervention, coronary artery bypass grafting, and year of hospital discharge (2004–2013). covariates were registered at the hospital stay at the time of the index mi. estimated models were presented with hazard ratios (hr) with 95% confidence intervals per standard deviation of bmi and wc levels, and p values. the changes of bmi and wc between the two-month and one-year visits were categorized into three groups based on estimates of normal biological variation from a reliability study by nordhamn et al. (8) the cutoffs used were two standard deviations of intra-individual variation from their study. for bmi and wc the cutoffs were 0.6 kg/m2 and 5.8 cm, respectively. in the reference group the patient’s changes between the twomonth and one-year visits were less than or equal to the cutoff. patients in the other two groups had decreased and increased, respectively, their bmi and wc values more than the cutoff. in a sensitivity analysis we examined if the analysis sample would indicate biased estimates. a cox regression model with all covariates and bmi and wc levels at the two-month visit was estimated and compared between all available data and the analysis sample. we confirmed the proportional-hazards assumptions of the cox models with schoenfeld’s test. in kaplan–meier curves survival was displayed for four groups: bmi <25 kg/m2 and wc <102 (male)/88 (female) cm (13); bmi <25 kg/m2 and wc �102/88 cm; bmi �25 kg/m2 and wc <102/88 cm; and bmi �25 kg/m2 and wc �102/88 cm in which the mean of anthropometric measures from two months and one year after hospital discharge was used. all statistical tests and confidence intervals were twosided. results with p values <0.05 were considered statistically significant without adjustments for multiplicity. the statistical analyses were performed with the statistical program package sas version 9.4 (sas institute inc., cary, nc, usa). results mean age of the 14,224 patients was 62 years (table 1). seventy-six per cent were men. there were 721 deaths up to 8.3 years after the one-year visit. median follow-up time was 3.2 years. estimated gam models displaying associations between anthropometric variables and mortality risk showed that the associations were linear except for small deviations for high bmi values and for low wc values (figure 1). upsala journal of medical sciences 79 with adjustments for covariates and the other anthropometric measure the hazard ratio (hr) per standard deviation (sd) in a cox proportional hazard regression model for mortality was 0.64 (95% confidence interval [ci] 0.56–0.74) for bmi and 1.55 (95% ci 1.34–1.79) for wc, and 1.43 (95% ci 1.17–1.74) for a bmi decrease from month two to one year of more than 0.6 kg/m2 (table 2). no interaction effects were found between anthropometric variables and age and gender. kaplan–meier curves showed that the group with low bmi and high wc had the highest mortality, and the group with high bmi and low wc had the lowest mortality (figure 2). the other two groups had similar mortality. in the sensitivity analysis with a cox regression model with all covariates and bmi and wc levels at the two-month visit was estimated with all available data (n ¼ 21,214) and with the analysis sample (n ¼ 14,224). hrs for bmi were 0.66 (95% ci 0.59–0.74) and 0.68 (95% ci 0.60–0.79), respectively, and hrs for wc were 1.48 (95% ci 1.32–1.66) and 1.46 (95% ci 1.27–1.67), respectively. discussion we examined a sample of 14,224 mi patients whose bmi and wc were measured two months and one year after hospital discharge. levels of bmi and wc at two months were inversely and directly associated, respectively, with risk of all-cause mortality. low bmi combined with high wc was associated with the highest mortality. further, we showed that a decreased bmi, but not wc, from two months to one year was significantly associated with higher mortality risk. to our knowledge, this study was the largest that examined the combined effects of bmi and wc on all-cause mortality for mi patients. the study was the only one that also considered fluctuations over time for these measures. in line with our results, coutinho et al. (9,10) showed that cad patients with normal weight and central obesity had the highest risk of mortality. kragelund et al. (14) showed that in patients with acute mi bmi was inversely related to mortality and that abdominal obesity appeared to be an independent predictor of mortality in men. angerås et al. (7) examined associations between bmi and mortality in patients with acute coronary syndromes. they found that the risk for 20 25 30 bmi (kg/m2) wc (cm) 35 40 −1 0 1 2 80 90 100 110 120 130 −1 0 1 2 figure 1. logit of mortality risk with 95% confidence intervals versus bmi and wc, adjusted for each other. table 1. characteristics of the analysis sample, mean (±sd) or number (%). variable n ¼ 14,224 age hia (years) 62.0 (±8.3) female gender 3433 (24.1%) current smoker 4240 (29.8%) diabetes 2208 (15.5%) hypertension 5815 (40.9%) hyperlipidaemia 13,736 (96.6%) creatinine (mmol/l) 84.2 (±34.1) previous pci 1601 (11.3%) previous mi 2104 (14.8%) previous stroke 537 (3.8%) previous chf 443 (3.1%) nstemi 8496 (59.7%) pci hia 11,149 (78.4%) cabg hia 518 (3.6%) bmi hia (kg/m2) 27.5 (±4.1) bmi 2 months (kg/m2)a 27.5 (±4.2) bmi 12 months (kg/m2)a 27.8 (±4.3) wc 2 months (cm)a 99.7 (±11.5) wc 12 months (cm)a 100.4 (±11.8) amonths after hospital discharge. bmi ¼ body mass index; cabg ¼ coronary artery bypass grafting; chf ¼ congestive heart failure; hia ¼ hospital stay; mi ¼ myocardial infarction; nstemi ¼ non-st-segment elevation myocardial infarction; pci ¼ percutaneous coronary intervention; stemi ¼ st-segment elevation myocardial infarction; wc ¼ waist circumference. 80 l. berglund et al. mortality decreased with increasing bmi up to 35 kg/m2 and then increased. khalid et al. (15) demonstrated that patients who were overweight or obese before heart failure development had lower mortality compared with normal bmi patients. the above-mentioned studies used bmi and wc as categorical variables. in our study we show that to a high degree of approximation the relations between bmi and wc, and mortality are linear. the implication is that decreased bmi at a given wc, or increased wc at a given bmi, is hazardous over the entire range of bmi and wc. central obesity, partly reflecting increased visceral fat, is coupled with the metabolic syndrome, blood lipid disorders, inflammation, insulin resistance or full-blown diabetes, and increased risk of developing cardiovascular disease (16). high bmi without central obesity may be due to muscle mass instead of increased adiposity, but could also reflect a metabolically healthy obesity, representing a phenotype with peripheral fat distribution but lower visceral fat accumulation (17). in addition, gluteofemoral body fat (subcutaneous fat in hips and legs), which is manifested in bmi, is associated with healthy metabolic profiles (18) and may thus imply lower mortality risk. the high-risk group with central obesity and normal bmi may have received less dietary advice than overweight and obese patients. sui et al. (19) showed that fitness is a significant mortality predictor in older adults, independent of overall or abdominal adiposity. hence, patients with normal bmi but central obesity may have low fitness, which partly can explain the increased mortality risk for this group in our study. furthermore, it would be interesting and clinically relevant to examine if lifestyle or drug strategies that prevent abdominal fat accumulation per se may increase survival in mi patients. limitations this prospective observational cohort study has some limitations. residual confounding may occur. however, we were able to adjust for relevant clinical variables. our design implies that we have not considered mortality during the first year after hospital discharge. also, patients in the analysis sample must have performed visits at secondary prevention clinics both at two months and one year after hospital discharge. however, these restrictions entail a healthier sample than the general mi population and our risk assessments are probably underestimated. furthermore, our findings were confirmed in a sensitivity analysis with a simple model. the age limit of 75 years of age in the registry does not allow for conclusions to be made for patients older than this at the time of the mi. since comorbidities potentially affecting weight may be more prevalent among the very elderly, more detailed information on existing and added comorbidities will be needed to study this group of patients. a limitation is that no wc measurements were available from the hospital stay. thus, we used levels from the two-month visit. this may though imply that temporal bmi and wc fluctuations in the acute disease state, due to the infarction or interventions, were avoided in the analyses. figure 2. kaplan–meier survival estimates (from one year after hospital discharge) by anthropometric groups: low-bmi, low-wc ¼ bmi<25 kg/m2, wc<102 (male)/88 (female) cm; low-bmi, high-wc ¼ bmi<25 kg/m2, wc�102/88 cm; high-bmi, low-wc ¼ bmi�25 kg/m2, wc<102/88 cm; high-bmi, high-wc ¼ bmi�25 kg/m2, wc�102/88 cm. table 2. associations between bmi and wc, and all-cause mortality (from one year after hospital discharge), n ¼ 14,224 (721 deaths). variable effect basic modelb extended modelc hr (95% ci) p value hr (95% ci) p value bmi 2 monthsa one sd (4.1 kg/m2) increase 0.62 (0.54–0.72) <0.0001 0.64 (0.56–0.74) <0.0001 bmi change 2 months to 1 yeara decrease >0.6 kg/m2 1.45 (1.19–1.76) 0.0002 1.43 (1.17–1.74) 0.0004 change �0.6 kg/m2 1.00 1.00 increase >0.6 kg/m2 0.96 (0.80–1.15) 0.66 0.92 (0.77–1.10) 0.36 wc 2 monthsa one sd (11.5 cm) increase 1.84 (1.59–2.13) <0.0001 1.55 (1.34–1.79) <0.0001 wc change 2 months to 1 yeara decrease >5.6 cm 0.80 (0.61–1.04) 0.09 0.82 (0.63–1.07) 0.15 change �5.6 cm 1.00 1.00 increase >5.6 cm 1.45 (1.18–1.78) 0.0005 1.22 (0.98–1.50) 0.07 amonths after hospital discharge. badjusted for the other anthropometric variable, age, and gender. cadjusted for the other anthropometric variable, age, gender, smoking, creatinine, diabetes, hypertension, hyperlipidaemia, previous myocardial infarction, previous stroke, previous congestive heart failure, diagnosis (stemi/nstemi), percutaneous coronary intervention, coronary artery bypass grafting, and year of hospital discharge (2004–2013). bmi ¼ body mass index; hr ¼ hazard ratio from cox regression model; sd ¼ standard deviation; wc ¼ waist circumference. upsala journal of medical sciences 81 conclusion patients with mi with normal bmi and central obesity have the highest all-cause mortality compared with other anthropometric groups. also, a reduction of bmi implies an even higher mortality risk. bmi only is thus an inaccurate anthropometric measurement, and our results indicate that combining bmi and wc in the assessment of mortality risk in mi patients is preferable to the use of bmi alone. acknowledgements we would like to thank felicia franz�en, pharma consulting group, uppsala, sweden for skilful assistance with statistical programming. disclosure statement the authors report no conflicts of interest. funding the research for this paper was financially supported by svenska f€ors€akringsf€oreningen [grant number 2014/152]. notes on contributors lars berglund, ph.d., is associate professor of medical epidemiology at department of public health and caring sciences/geriatrics, uppsala university, uppsala, sweden. ulf ris�erus, ph.d., is associate professor in clinical nutrition and metabolism at department of public health and caring sciences/geriatrics, uppsala university, uppsala, sweden, with research focus on obesity, nutrition and cardiometabolic disease. kristina hambraeus, ph.d., is a cardiologist at the department of cardiology, falun hospital, falun, sweden, co-founder of the sephia-registry. orcid lars berglund http://orcid.org/0000-0002-7437-9047 ulf ris�erus http://orcid.org/0000-0002-8620-4586 kristina hambraeus http://orcid.org/0000-0002-0768-2484 references 1. swedeheart 2011 annual report 2012. 2. prospective studies collaboration; 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2002. 13. expert panel on detection, evaluation, and treatment of high blood cholesterol in adults. executive summary of the third report of the national cholesterol education program (ncep) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel iii). jama. 2001;285: 2486–97. 14. kragelund c, hassager c, hildebrandt p, torp-pedersen c, kober l; trace study group. impact of obesity on long-term prognosis following acute myocardial infarction. int j cardiol. 2005;98: 123–31. 15. khalid u, ather s, bavishi c, chan w, loehr lr, wruck lm, et al. pre-morbid body mass index and mortality after incident heart failure: the aric study. j am coll cardiol. 2014;64:2743–9. 16. despres jp, lemieux i. abdominal obesity and metabolic syndrome. nature. 2006;444:881–7. 17. bluher m. the distinction of metabolically ‘healthy’ from ‘unhealthy’ obese individuals. curr opin lipidol. 2010;21:38–43. 18. manolopoulos kn, karpe f, frayn kn. gluteofemoral body fat as a determinant of metabolic health. int j obes (lond). 2010;34: 949–59. 19. sui x, lamonte mj, laditka jn, hardin jw, chase n, hooker sp, et al. cardiorespiratory fitness and adiposity as mortality predictors in older adults. jama. 2007;298:2507–16. 82 l. berglund et al. abstract introduction material and methods patient population anthropometric measures statistics results discussion limitations conclusion acknowledgements disclosure statement funding notes on contributors references intratumoral expression of foxp3-positive regulatory t-cells in t-cell lymphoma: no correlation with survival article intratumoral expression of foxp3-positive regulatory t-cells in t-cell lymphoma: no correlation with survival josefine lundberga,c, david berglunda, daniel molinb and amelie kinchc adepartment of immunology, genetics and pathology, section of clinical immunology, uppsala university, uppsala, sweden; bdepartment of immunology, genetics and pathology, section of experimental and clinical oncology, uppsala university, uppsala, sweden; cdepartment of medical sciences, section of infectious diseases, uppsala university, uppsala, sweden abstract background. in cancer, regulatory t-cells (tregs) were previously believed to inhibit tumor immunity, leading to reduced survival. however, in hematologic malignancies, including t-cell lymphoma (tcl), a correlation between increased numbers of tumor-infiltrating tregs and a favorable prognosis has been reported. we aimed to investigate the expression of the treg biomarker forkhead box protein 3 (foxp3) in tcl in immunocompetent individuals and explore a possible correlation to overall survival. methods. in total, 35 diagnostic biopsies of tcl were stained using a foxp3-specific monoclonal antibody (clone 236a/e7). visual scoring was performed by counting positive cells in 15 high-power fields. clinical data were collected retrospectively from medical records. results. all the tcls contained foxp3þ cells, median 342 foxp3þ cells/mm2 (range 1–3047). the degree of intratumoral expression of foxp3 varied between the different subtypes of tcl, with the highest frequency found in angioimmunoblastic tcl. the frequency of intratumoral foxp3þ cells had no impact on overall survival; neither when using a cutoff value of 200 foxp3þ cells/mm2 (p ¼ 0.84) nor with foxp3 as a continuous variable (p ¼ 0.63). conclusions. intratumoral tregs are frequently found in tcl in immunocompetent individuals. in this heterogeneous group of tcl, there was no correlation between the density of intratumoral foxp3þ cells and overall survival. article history received 25 october 2018 revised 27 november 2018 accepted 29 november 2018 keywords foxp3; outcome; t-cell lymphoma; tregs; tumor microenvironment introduction regulatory t-cells (tregs) are a specialized subpopulation of t-cells with the capacity to suppress anti-self immune responses, thus maintaining peripheral tolerance and preventing autoimmune diseases (1). tregs are also believed to play a vital part in inducing tumor-specific immune tolerance (2). natural tregs are produced in the thymus (3) and express cd4, cd25, and the transcription factor forkhead box protein 3 (foxp3) (4). peripheral cd4þ foxp3� cells may also upregulate foxp3 in the presence of cytokines such as tgf-b and il-2; this population of extrathymically generated foxp3þ tcells are known as peripherally derived tregs (ptregs) (5). the transcription factor helios (6) and the cell surface molecule neuropilin-1 (7) have been suggested as markers to distinguish ntregs from ptregs, but the usefulness of these markers in humans has been subject to debate (8,9), and consensus has not yet been reached. despite the fact that cd4þ cd25� t-cells have been shown to express foxp3 upon activation, and that the levels of expression do not necessarily correlate to suppressive function in these cells, foxp3 is still considered the most reliable molecular marker of tregs (4, 10). regarding the role of tregs in cancer, several studies have shown a correlation between increased numbers of intratumoral tregs and decreased survival (e.g. ovarian carcinoma (11), prostate cancer (12), and non-small cell lung cancer (13)). in contrast, a higher frequency of intratumoral tregs has been associated with improved survival in colorectal carcinoma (14) as well as in several types of lymphoma—for example, follicular lymphoma, hodgkin lymphoma, and germinal center-like diffuse large b-cell lymphoma (15). in previous studies of tregs in t-cell lymphoma (tcl), a positive impact on survival with increased numbers of intratumoral tregs has been reported in mycosis fungoides (mf), unspecified cutaneous tcl (ctcl) (16), and extranodal nk/t-cell lymphoma (enktl) (17). one study compared lymph node biopsies from patients with angioimmunoblastic tcl (aitl) or follicular lymphoma with reactive lymph nodes and found significantly lower numbers of tregs in the aitl biopsies, which, according to the authors, might at least in part explain the autoimmune symptoms typical for aitl and thus the poor prognosis (18). another study on adult t-cell leukemia/lymphoma (atll) focused on the expression of foxp3 in the malignant cell population and reported that patients with foxp3-negative tumors showed a tendency toward contact amelie kinch amelie.kinch@medsci.uu.se department of medical sciences, section of infectious diseases, uppsala university, s-751 85 uppsala, sweden � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 2, 105–110 https://doi.org/10.1080/03009734.2018.1555195 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1555195&domain=pdf http://orcid.org/0000-0002-9646-7283 http://orcid.org/0000-0003-3420-8590 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1555195 http://www.tandfonline.com better survival in comparison with those with strong or intermediate intratumoral expression of foxp3 (19). in a previous study we found that none of the t-cell posttransplant lymphoproliferative disorders were foxp3þ (20). we speculated that this finding in part might be due to the immunosuppressive treatment that the patients typically receive after solid organ transplantation, and we questioned if tcl from patients without any previous immunosuppression would differ in their expression of foxp3, which led us to initiate this present study. we hypothesized that increased numbers of intratumoral tregs may have an impact on survival in different types of tcl, and in this study we have correlated the number of foxp3þ cells in biopsies of different subtypes of tcl to outcome. materials and methods we included 35 tcl patients diagnosed 1999–2002 with sufficient tissue available for further analysis from a swedish–danish case-control study (scale; scandinavian lymphoma etiology) (21). the 35 tissue samples were reevaluated in the scale study by experienced hematopathologists according to the world health organization (who) classification of tumors in hematopoietic and lymphatic tissue. after re-examination of the pathological reports in combination with the clinical data, we reclassified one of the biopsies from primary cutaneous cd30þ tcl to tcl not otherwise specified. another biopsy was reclassified as highly malignant tcl not otherwise specified, after consulting an experienced hematopathologist, since the original diagnosis of ‘peripheral large cell tcl’ was not consistent with any of the diagnostic entities of the who classification. clinical data for each patient were collected retrospectively from the different swedish hospitals where the patients had been primarily treated for their tcl. immunohistochemistry for foxp3 from formalin-fixed, paraffin-embedded diagnostic biopsies of tcl, 4 lm thick tissue sections were sliced. pt-link was used for performing deparaffinization and antigen retrieval. immunohistochemical (ihc) staining was performed with dako autostainer plus with clone 236a/e7 (ebioscience) used as primary antibody to detect foxp3, diluted 1:100 in antibody-solution (dako). in order to visualize the primary antibody, envision dab-kit (dako) was used. the samples were scanned and examined using aperio imagescope v12.1.0.5029 (leica biosystems). in the majority of the samples the positive cells were distributed in a heterogeneous manner, therefore the stained cells were counted manually in 15 arbitrarily chosen fields in each biopsy, at 40� magnification. the area of each field was measured. the number of positive cells per mm2 in the total analyzed area was calculated. statistics comparison of frequencies of foxp3þ cells in different subtypes of tcl was made using the mann–whitney u-test. overall survival (os) was defined as the time from initial diagnosis of tcl until death or last date of follow-up, which was 19 february 2016. survival curves were generated using the kaplan–meier method. difference in survival was calculated using cox proportional hazards regression model with foxp3 as a continuous variable or log-rank test with a cutoff value of 200 foxp3þ cells/mm2. statistical significance was defined as p < 0.05. all statistical analyses were performed using statistica software (version 13, stat soft inc., tulsa, ok, usa). table 1. clinical and biological characteristics of the patient group. characteristics number (%) (available data) sex (35/35) male 21 (60%) female 14 (40%) age at tcl diagnosis (35/35) 18–20 2 (6%) 41–50 8 (23%) 51–60 13 (37%) 61–70 11 (31%) >70 1 (3%) tcl localization (35/35) nodala 15 (43%) extranodal 20 (57%) bone marrow 6 (17%) skin 7 (20%) small intestine 5 (14%) otherb 6 (17%) stage (ann arbor) (31/35) i 9 (29%) ii 4 (13%) iii 11 (35%) iv 7 (23%) performance status (who/ecog) (35/35) 0–1 33 (94%) 2–3 2 (6%) ipi (26/35) low risk 11 (42%) low/high intermediate risk 14 (54%) high risk 1 (4%) serum ldh concentration (28/35) elevated 15 (54%) within normal range 13 (46%) recurrence (35/35) yes 19 (54%) no 16 (46%) subtypes of tcl (35/35) anaplastic large cell lymphoma 12 (34%) peripheral tcl, not otherwise specified 6 (17%) unspecified tcl 5 (14%) angioimmunoblastic tcl 3 (9%) enteropathy-associated tcl 3 (9%) cutaneous tcl 2 (6%) mycosis fungoides 2 (6%) t-cell prolymphocytic leukemia 2 (6%) cytotoxic chemotherapy treatment (first-line) (35/35) chop/choep 22 (63%) vacop-b 4 (11%) abvd 1 (3%) other 1 (3%) none 7 (20%) anodal localization: malignancy in lymph nodes only. bextranodal localization, other: pleura, lung, meninges, bone marrow, testis. abvd ¼ adriamycin (doxorubicin), bleomycin, vinblastine, dacarbazine; choep ¼ addition of etoposide to the standard chop-regimen; chop ¼ cyclophosphamide, hydroxydaunorubicin, oncovin (vincristine), prednisone or prednisolone; ecog ¼ eastern cooperative oncology group; ipi ¼ international prognostic index; ldh ¼ lactate dehydrogenase; tcl ¼ t-cell lymphoma; vacop-b ¼ vp-16 (etoposide), adriamycin (doxorubicin), cyclophosphamide, oncovin (vincristine), prednisone, bleomycin; who ¼ world health organization. 106 j. lundberg et al. ethics approval the study was approved by the regional ethical review board in stockholm, sweden, and was conducted in accordance with the declaration of helsinki. results clinical characteristics and subtypes of tcl the median age at the time of diagnosis was 57 years (range 18–73) (table 1). there were 21 men and 14 women. the majority of the patients (57%) had extranodal localization of tcl, most commonly the skin (20%). the most common subtype of tcl was anaplastic large cell lymphoma (alcl; n ¼ 12, 34%). other subtypes included peripheral tcl, not otherwise specified (ptcl-nos; n ¼ 6, 17%), unspecified tcl (n ¼ 5, 14%), aitl (n ¼ 3, 9%), enteropathy-associated tcl (eatl; n ¼ 3, 9%), ctcl (n ¼ 2, 6%), mf (n ¼ 2, 6%), and t-cell prolymphocytic leukemia (t-pll, n ¼ 2, 6%). expression of foxp3 in tcl all 35 tissue samples expressed foxp3 to some degree. overall, the median expression of foxp3 was 342 positive cells/mm2 (mean 610 positive cells/mm2), and the range was large (1–3047 positive cells/mm2). the degree of intratumoral expression of foxp3 tended to vary between the different subtypes of tcl (figure 1). the highest frequency of foxp3þ cells was detected in aitl (n ¼ 3, median 1057 and range 849–1642 positive cells/mm2) compared with all other subtypes, although this difference did not reach statistical significance (p ¼ 0.053). t-pll (n ¼ 2) tended to have the lowest frequency of foxp3þ cells/mm2 (p ¼ 0.14). survival at the end of follow-up 21 patients had died, 18 of whom due to lymphoma. the median follow-up time for the 14 surviving patients was 11.8 years (range 5.7–14.4 years). oneand five-year overall survival were 89% and 46%, and oneand five-year relapse-free survival were 72% and 44% for all patients. there was no difference in os between cases with high or low expression of foxp3, neither when analyzed as a dichotomous variable (> or < 200 foxp3þ cells per mm2, p ¼ 0.84, figure 2), nor as a continuous variable (p ¼ 0.63). this result did not change when the cases of mf, which figure 1. degree of intratumoral expression of foxp3þ cells/mm2 in the eight different subtypes of t-cell lymphoma. aitl: angioimmunoblastic t-cell lymphoma; alcl: anaplastic large cell lymphoma; ctcl: unspecified cutaneous t-cell lymphoma; eatl: enteropathy-associated t-cell lymphoma; mf: mycosis fungoides; ptcl-nos: peripheral t-cell lymphoma, not otherwise specified; tpll: t-cell prolymphocytic leukemia; tcl-nos: unspecified t-cell lymphoma. figure 2. the frequency of intratumoral foxp3þ cells (more or less than 200 foxp3þ cells/mm2) in t-cell lymphomas had no impact on overall survival. figure 3. degree of intratumoral expression of foxp3 in the 35 biopsies, with cutoff level set arbitrarily at 200 foxp3þ cells/mm2. upsala journal of medical sciences 107 have a better prognosis than the rest of the tcls, were omitted from the survival analysis (log-rank test, p ¼ 0.55; cox proportional hazards, p ¼ 0.76). considering that no difference in os was detected when analyzing foxp3 as a continuous variable, it is of minor consequence that the cutoff was set arbitrarily based on visual assessment of a scatter chart (figure 3) when foxp3 was used as a dichotomous variable. discussion in this study we found that all tcls expressed foxp3 to some degree but that the frequency varied considerably between different subtypes. there was no association between the number of intratumoral tregs and os. many studies have shown a correlation between a high density of tregs in tumor tissue and a worse prognosis in different malignancies (11–13), but at the same time several studies have shown the opposite (14–17). the negative impact of tregs on outcome in cancers may be explained as one of several immune evasion strategies by tumors, where the tumors escape the immune system by suppressing antitumoral effector cells (11,22–24). in the malignancies where tregs instead are associated with a favorable outcome, a possible explanation could be that tregs, due to their anti-inflammatory capacity, reduce tissue damage and restrict inflammation-related carcinogenesis (1,25,26). in tcl, the function of tregs seems particularly complex, partly because the malignant cells in these tumors are lymphoid and thus potentially could be suppressed by tregs, and partly because some studies have suggested that the malignant cells could in fact be treg-derived (19,27–29). berger et al. (29) suggested that ctcls are tumors of malignant tregs, based on findings that ctcl-cells could adopt treg phenotype and function after stimulation by apoptotic material in vitro. gjerdrum et al. (16) showed that only a small minority of ctcl cases showed expression of foxp3 in the malignant cells while all contained foxp3þ cells in the tumor microenvironment to some degree. of note, higher numbers of intratumoral tregs were associated with improved survival in these tcls. there were only two mf and two ctcl unspecified in our case series, and we have not used the same method for evaluation; this complicates comparisons between the two studies, but the frequencies of tregs seem similar. regarding alcl, ptcl-nos, and aitl, we found higher frequencies of foxp3þ cells than previous studies (15,18,27). the largest subgroup in our case series was alcl (n ¼ 12), where we found a mean of 621 foxp3þ cells/mm2 as compared with 15 foxp3þ cells/mm2 in the four cases of alcl in the study by tzankov et al. (15). bonzheim et al. (27) detected foxp3þ cells in four of six alkþ and in one of 14 alkalcl, but the specific number of positive cells was not reported. this is in sharp contrast to our case series where the majority of alcl were alk(seven of nine with known alk status) and despite this were foxp3þ. however, the limited number of cases prevents a meaningful comparison between alkþ and alkalcl. in ptcl-nos we found a mean of 965 foxp3þ cells/ mm2 compared with 34 foxp3þ cells/mm2 in the 27 cases in the study by tzankov et al. (15), whereas bonzheim et al. (27) only detected expression of foxp3 in one of 14 cases of ptcl-nos, and that was in the malignant population. regarding aitl, we detected a mean of 1183 foxp3þ cells/mm2 in three cases as compared with a mean of 61 foxp3þ cells/mm2 in 23 cases by tzankov et al. (15) and mean of 90 foxp3þ cells/high-power field (hpf; 400�) in 30 cases by bruneau et al. (18). bonzheim et al. (27) found no foxp3þ tumor cells and only a few foxp3þ cells in the reactive infiltrate in 23 cases of aitl. the differences in expression of foxp3 can in part be explained by the use of different antibodies for the detection of foxp3. tzankov et al. (15) used the mouse monoclonal antibody 22510 (abcam), bonzheim et al. (27) used a rabbit polyclonal antibody from abcam (clone number not specified), whereas bruneau et al. (18) and gjerdrum et al. (16) used the same clone as we did, namely the mouse monoclonal 236a/e7 (abcam). clone 236a/e7 has been reported to be predictive of improved outcome in follicular lymphoma (30), hodgkin lymphoma (31), and ctcl (16). in our case series there was no correlation between expression of foxp3þ and outcome. an association between high numbers of intratumoral tregs and superior survival has been reported for some types of tcls, e.g. ctcl (16) and enktl (17), but not for the largest subgroups in our case series, alcl and ptclnos, to the best of our knowledge. in a previous study with the use of the same antibody as in the present study, we found that 13 cases of t-cell posttransplant lymphoproliferative disorder occurring after solid organ transplantation were completely negative for expression of foxp3 and in one case foxp3þ cells were detected at a low density (10 positive cells/mm2) (20). this finding is in contrast with the observation in the present study where all tcl expressed foxp3 to some degree. a plausible explanation for this striking difference is the influence of the immunosuppressive drugs in solid organ transplant recipients. the limitations of this study include the small subgroups of tcl subtypes, which hinders comparisons between groups. further, the foxp3þ cells in the biopsies have not been defined as belonging to either the neoplastic or reactive cell population within the tumors. the frequency of intratumoral tregs is based on the expression of foxp3, which has limitations but still is considered the most reliable biomarker for tregs (10). strengths include that the biopsies were collected prospectively on a national level and re-evaluated by experienced hematopathologists. furthermore, 15 arbitrarily chosen hpfs were manually counted which is more than the standard five hpfs. in conclusion, in this study we found distinctly higher levels of expression of foxp3 in aitl, ptcl-nos, and alcl when compared with previous studies of these lymphomas in immunocompetent individuals. the finding that all of the tcls showed expression of foxp3 to some degree is interesting when compared with the results of our previous study of tregs in posttransplant lymphoproliferative 108 j. lundberg et al. disorder, where all but one of the t-cell posttransplant lymphoproliferative disorders were entirely lacking expression of foxp3. thus, the role of tregs seems to differ between tcls in immunocompetent and immunosuppressed hosts. in this heterogeneous group of tcl, there was no correlation between the density of intratumoral foxp3þ cells in biopsies of tcl and os. declaration of interest d.m. has received honoraria from roche, merck, bristol-meyers squibb, and takeda. the other authors report no conflicts of interest. funding the work was supported by the lion's cancer research foundation. notes on contributors josefine lundberg, master of science in medicine, is at the beginning of her clinical internship at nyk€opings hospital, sweden. david berglund, md, phd, trained in immunology and transplant/reconstructive surgery and is an associate professor at the department of immunology, genetics and pathology, section of immunology, uppsala university, uppsala, sweden. daniel molin, md, phd, is a senior consultant at the department of oncology, uppsala university hospital and associate professor at the department of immunology, genetics and pathology, section of experimental and clinical oncology, uppsala university, uppsala, sweden. amelie kinch, md, phd, is a senior consultant at the department of infectious diseases, uppsala university hospital and researcher at the department of medical sciences, section of infectious diseases, uppsala university, uppsala, sweden. orcid daniel molin http://orcid.org/0000-0002-9646-7283 amelie kinch http://orcid.org/0000-0003-3420-8590 references 1. mougiakakos d, choudhury a, lladser a, kiessling r, johansson cc, regulatory t cells in cancer. advances in cancer research [internet]. elsevier; 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tumor-infiltrating foxp3positive regulatory t cells are associated with improved overall survival in follicular lymphoma. blood 2006;108:2957–64. 31. greaves p, clear a, coutinho r, wilson a, matthews j, owen a, et al. expression of foxp3, cd68, and cd20 at diagnosis in the microenvironment of classical hodgkin lymphoma is predictive of outcome. j clin oncol. 2013;31:256–62. 110 j. lundberg et al. abstract introduction materials and methods immunohistochemistry for foxp3 statistics ethics approval results clinical characteristics and subtypes of tcl expression of foxp3 in tcl survival discussion declaration of interest notes on contributors references increased anxiety-like behavior but no cognitive impairments in adult rats exposed to constant light full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 increased anxiety-like behavior but no cognitive impairments in adult rats exposed to constant light conditions during perinatal development erika roman & oskar karlsson to cite this article: erika roman & oskar karlsson (2013) increased anxiety-like behavior but no cognitive impairments in adult rats exposed to constant light conditions during perinatal development, upsala journal of medical sciences, 118:4, 222-227, doi: 10.3109/03009734.2013.821191 to link to this article: https://doi.org/10.3109/03009734.2013.821191 © informa healthcare published online: 01 aug 2013. submit your article to this journal article views: 663 view related articles citing articles: 9 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.821191 https://doi.org/10.3109/03009734.2013.821191 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.821191 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.821191 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.821191#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.821191#tabmodule upsala journal of medical sciences. 2013; 118: 222–227 original article increased anxiety-like behavior but no cognitive impairments in adult rats exposed to constant light conditions during perinatal development erika roman & oskar karlsson department of pharmaceutical biosciences, uppsala university, box 591, se-751 24 uppsala, sweden abstract background. shift-work is suggested to affect fetal development negatively. in particular, maternal hormonal disturbance arising from sleep deprivation or circadian rhythm changes may disturb fetal growth or lead to complications during pregnancy. exposure to constant light is an environmental stressor that can affect the circadian system and has been shown to induce neurochemical and behavioral changes when used during the prenatal and/or postnatal period in experimental animals. however, studies investigating long-term effects of constant light in the offspring are sparse. methods. an accidental power outage resulted in pregnant females being housed under constant light (ll) conditions for seven days of the offspring perinatal development (embryonic day 20 to postnatal day 4). the long-term effects of constant light on the behavior in the adult offspring were assessed by means of open field, object recognition, and water maze tests. results. in adulthood, ll-animals displayed an intact recognition memory and no deficits in spatial learning or memory. in the open field test, ll-animals exhibited higher anxiety-like behavior, observed as significantly more thigmotaxis and less ambulation. these results were confirmed in the other behavioral tests as the ll-animals spent less time exploring the objects in the object recognition test, and showed thigmotactic behavior also in the water maze test. conclusion. the results confirm that early life experience can cause changes in brain development that shape brain function and add to the sparse literature on long-term effects of constant light conditions during perinatal development on specific behaviors in adulthood. key words: anxiety, biological rhythms, circadian rhythm, cognitive function, development, open field, shift-work, stress, water maze introduction early life experiences can shape brain function and behavior in adulthood. developmental processes throughout the perinatal period and the final postnatal maturation and reorganization include a variety of adaptation processes that affect brain function. during these developmental time windows the brain is highly sensitive to toxic or physical agents and environmental input (1-4). the belief that a mother’s emotional or psychological state during pregnancy may influence the development of her fetus has existed since ancient times. for example, shift-work during pregnancy is a suspected risk factor for fetal development. in particular, maternal hormonal disturbance arising from sleep deprivation or circadian rhythm changes may disturb fetal growth or lead to complications of pregnancy (5). constant light conditions is an environmental stressor that can induce neurochemical changes during preand/or postnatal development as well as modify the rat circadian system, resulting in physiological and behavioral alterations (6,7). however, knowledge about the long-term consequences of exposure to constant light during the perinatal period is sparse. in the present study an accidental power outage resulted in pregnant females being housed under constant light (ll) conditions for 7 days of correspondence: dr oskar karlsson, uppsala university, department of pharmaceutical biosciences, box 591, se-751 24 uppsala, sweden. fax: +46 18 4714253. e-mail: oskar.karlsson@farmbio.uu.se (received 22 may 2013; accepted 26 june 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.821191 http://informahealthcare.com/journal/ups mailto:oskar.karlsson@farmbio.uu.se the offspring perinatal development (embryonic day 20 to postnatal day 4). the aim of the present study was therefore to examine the long-term behavioral effects of constant light for a total of 7 days during fetal and postnatal development in the adult offspring. material and methods time-mated, outbred wistar rats were obtained from taconic (ejby, denmark) and arrived at gestational day 15. each dam was singly housed in polysulfone cages (59 � 38 � 20 cm) containing wood-chip bedding and nesting material. on postnatal day (pnd) 1, all litters were arranged to contain an equal distribution of males and females. the animals were maintained on standard lab chow and water ad libitum, and were housed in temperature-controlled (21 ± 1�c) and humidity-controlled (50% ± 10%) cabinets in an animal room with a 12-h light/ dark cycle (lights on at 6 a.m.). as a consequence of a power outage that interfered with the computer system that automatically controls the light/dark cycle in the animal facility 12 dams were exposed to 7 days of constant light (ll) from embryonic day (e) 20 and onwards after the birth of pups: e 20 until pnd 4. four of these dams were kept for investigation of effects of ll in the adult offspring. litters from 12 dams housed under normal 12-h light/dark cycle served as controls. pups from each housing condition were given one daily subcutaneous injection (20 ml/g) of hanks’ balanced salt solution on pnds 9 and 10. the injections were given to all pups since the control group served as controls in a parallel study in which the ll-animals also were meant to be included (8). after weaning on pnd 22 and onwards, the offspring were housed three per cage in polysulfone cages (59 � 38 � 20 cm). on pnd 31 and onwards, the rats were housed under a reversed 12-h light/dark cycle (lights off at 7 a.m.) to allow for behavioral testing during the active period. the animals were allowed to adapt to the reversed light/dark cycle for over 2 months before the behavioral tests started at 13 weeks of age. the experimental procedures followed a protocol approved by the university veterinarian and the local uppsala animal ethical committee and were in accordance with the guidelines of the swedish legislation on animal experimentation (animal welfare act sfs1998:56) and the european communities council directive (86/609/eec). the object recognition test the object recognition (or) test is based on the natural behavior of animals to spend more time exploring a novel, rather than a familiar, object. the test consists of three different phases: habituation, sample, and discrimination (9) and was conducted in an open field (of) arena. the arena used in this study (circular, 90 cm in diameter) was as described elsewhere (10,11). the of was divided into zones: that is, a central circle 30 cm in diameter (the center) surrounded by a middle circle (width 15 cm), which was surrounded by an outer circle (width 15 cm). the test was conducted at 13 weeks of age. the habituation phase consisted of two consecutive days with 10-min trials under dimmed light. exploration of a novel of arena, as in the habituation phase, is a commonly used test for studies of general exploration, ambulation, and emotional reactivity, and the 2-day trial protocol enables studies of habituation. for the two habituation trials, the time in seconds until the first visit to the defined zones (latency; lat), number of visits (frequency; frq), duration (dur) of visits in seconds, number of animals visiting the defined zones or performing the scored behavior (occurrence; occ), total number of visits to the defined zones (totact), the distance moved (totdistance; centimeters), and rearing were registered. in the sample phase, conducted 24 h after the last habituation trial, the samples (s) consisting of two identical objects (s1: cube 1 and cube 2; or s2: glass 1 and glass 2) were presented to the animals. the samples were placed in fixed positions in the middle circle for 5 min of exploration. the animals were then returned to their home cages for 1 h before the discrimination phase started. during the discrimination phase, one of the familiar objects was replaced by a novel object (n) for 5 min of exploration. the use of the two objects as sample or novel object was shifted equally between the animals, and the object locations were counterbalanced (8). exploration of an object was defined as directing the nose at the object at a distance of less than 2 cm and/or touching it with the nose (9). the total exploration time (s1 + s2) or (s + n) and the discrimination ratio, n/(n + s) were calculated. between each animal the of arena was wiped clean with 10% ethanol and allowed to dry. the water maze test the water maze (wm) test is commonly used in studies of spatial learning and memory. the arena (160 cm in diameter) and the experimental conditions used in this study were as described elsewhere (8). the tank was divided into four equal quadrant zones, and the escape platform was placed in the south-west (sw) quadrant (target quadrant) during all acquisition trials. the acquisition trials were long-term effects of constant light conditions 223 conducted for five consecutive days with four trials per day. the rats were started facing the wall using randomized starting positions, and allowed to search for the platform for 90 seconds. the animals were tested at 15–16 weeks of age. parameters such as escape latency, latency to target quadrant, and swim distance were scored. swimming distance to the wall was measured for evaluation of thigmotactic behavior. the retention test was conducted 72 h after the last acquisition session, as a 90-second single trial without the platform (probe trial). the animals were started in the north-east (ne) quadrant. in addition to the parameters analyzed during the acquisition trials, quadrant analyses were performed in the probe trial. latency in the first crossing of the former platform location (target zone), number of crossings of the target zone, and number and duration of visits to the different quadrants were scored. activity, defined as percentage of total time swimming faster than 2 cm/s, was scored for each quadrant. behavioral recordings the animals were monitored on a tv-video set-up in all tests. behavioral parameters in the or tests were scored manually using score 3.3 software (pär nyström; soldis, uppsala, sweden) by an observer blinded as to the treatment. a visit to one of the defined zones was scored when both hind legs had crossed over into that section. distance moved during the habituation phase of the or test was automatically registered using the computerized tracking systems ethovision version 2.3 (noldus information technology, wageningen, the netherlands). performance in the wm test was registered automatically using viewer2 (biobserve gmbh, bonn, germany). statistical analysis twelve animals per group were studied in the behavioral tests. one male from 12 different dams was tested in the control group (n = 12), and three males per litter from four different dams were tested in the ll-group (n = 12). the statistical analysis was based on number of litters (control group n = 12; llgroup n = 4). the non-parametric mann–whitney u test was used for intergroup comparisons of behavioral parameters. measurements over time in the behavioral tests were analyzed using the nonparametric friedman two-way anova test and the wilcoxon matched pairs test. differences were considered statistically significant at p < 0.05. values are expressed as mean ± sem. results short-term findings the exposure of the dams to constant light did not induce preterm birth or malformation in the offspring. no differences between the groups were detected in body weight gain during the postnatal period or in body weight in adulthood (data not shown). adult behavioral tests the or test consisted of three different phases, habituation, sample, and discrimination, and was conducted at 13 weeks of age, in a circular of arena. the two consecutive days of habituation were used for assessment of explorative strategies in a novel environment. the ll-animals had a significantly reduced locomotor activity (i.e. total activity and total distance moved) compared to the control group (figure 1a and table ia). the ll-animals moved significantly less in the center compared to controls in both trials, indicating higher levels of thigmotaxis (figure 1b). the data analysis of trial 1 (table ia) revealed that the ll-animals spent significantly more time in the outer circle and had fewer visits to the middle circle and center compared to controls. the time per visit in the outer circle was also significantly longer in the llgroup compared with the controls. a similar pattern was seen in the second habituation trial (table ib). in the or sample phase, the ll-animals spent less time exploring the objects compared to controls, 33 ± 4 s and 44 ± 3 s, respectively (u = 8, p < 0.06). no difference was observed between ll-animals and controls in the cognitive part of the test. the llgroup had significantly (u = 6, p < 0.05) longer time to first exploration of the novel object compared to controls, 19 ± 5 s and 6 ± 1 s, respectively. however, both groups explored the novel object more than the familiar sample in the discrimination phase. the control group had a discrimination ratio of 0.70 and the ll-group had a ratio of 0.65, which both were significantly different from the probability discrimination ratio 0.5 (t test of single means). the wm test was conducted at 15–16 weeks of age. there was no significant difference between llanimals and controls in escape latency, and all groups learned the task in a similar manner, as indicated by reduced escape latencies over the 5 days of acquisition (figure 1c). on the first acquisition day, the llanimals swam closer to the wall when compared to the controls, 10 ± 1 cm and 13 ± 1 cm, respectively, indicating increased thigmotactic behavior, but the difference did not reach statistical significance (u = 9, p < 0.08). both ll-animals and controls had reduced 224 e. roman & o. karlsson thigmotactic behavior over time (figure 1d). in the probe trial, without the platform present, the llgroup swam longer and was more active (higher percentage of time with swim speed > 2 cm/s) compared to the controls, but the differences did not reach statistical significance (u = 12, p < 0.2 and u = 9, p < 0.08, respectively) (table ii). discussion shift-work is suggested to affect fetal development negatively. in particular, maternal hormonal disturbance arising from sleep deprivation or circadian rhythm changes may disturb fetal growth or lead to complications during pregnancy (5). in the present study, rats were exposed to constant light conditions for 7 days during preand postnatal development (e 20–pnd 4), which corresponds to the last trimester of pregnancy in humans (12). to our knowledge this is the first study examining cognitive behaviors in adult rats after perinatal exposure to constant light. the ll-animals had increased anxiety-like behavior but no impairments in recognition memory or spatial learning and memory. these findings suggest that the brain systems important for these cognitive functions are not the main target for developmental changes due to constant light during this period of perinatal development. the reason for this is unknown, but granule cells of the dentate gyrus are one of the cell types in the brain with the last developmental trajectory, and cognitive behavior that is dependent on hippocampal integrity develops ontogenetically at about 1 month of age in rats (12,13). it is therefore possible that this environmental stressor only affects more mature neuronal systems directly or that the on-going development of hippocampus may reverse potential negative effects induced by constant light conditions on this brain region. in the of test, ll-rats displayed significantly more thigmotaxis and less ambulation compared to controls, commonly interpreted as anxiety-like behavior. the results were further strengthened by the findings in the or test and the first wm trial. interestingly, the increased anxiety-like behavior in the ll-animals after mainly postnatal light exposure is in accordance with a previous study, which showed that constant light during prenatal development (e 10–e 21) resulted in offspring with anxiety-like behavior in adulthood (7). the hypoactivity in the of and the reduced exploration of the objects in the or test, respectively, are most likely due to the anxietylike characteristics in the ll-animals, but may also 4000 60 25 ## ## $$20 15 10 5 50 40 30 20 10 0 0 1 2 3 acquisition probe trial acquisition probe trial 4 5 6 0 1 2 3 4 5 6 to ta l d is ta n c e ( c m ) e s c a p e l a te n c y ( s ) d is ta n c e f ro m t h e w a ll ( c m ) open field 1 open field 2 open field 1 open field 2 3000 2000 1000 0 300 * * ** * 200 100 d is ta n c e c e n te r (c m ) 0 control control controlll ll control ll ll a c b d figure 1. a-b: spontaneous activity of rats exposed to constant light (ll) during the perinatal development or a 12-h light/dark cycle (control) examined in the open field (of) test. a: the ll-animals had a significantly reduced locomotor activity compared to the control group. neither of the groups had significantly reduced total distance moved in the arena between the two days. b: the ll-animals traveled a significantly shorter distance in the center compared to controls in both trials, interpreted as higher anxiety-like behavior. c-d: the performance in the water maze (wm) test. c: the escape latency (s), i.e. climbing up on the platform during the acquisition trials, and latency in first visiting the target zone during the probe trial. d: the average wall distance (cm) of the animals, used for interpretation of thigmotactic behavior. values represent mean ± sem. * p < 0.05, ** p < 0.01, compared to controls (mann–whitney u test); ## p < 0.01 compared to the first acquisition trial; $$ p<0.01 compared to the last acquisition trial (wilcoxon matched pairs test). twelve animals per group were tested, and the statistical analysis was based on number of litters (control group n = 12; ll-group n = 4). long-term effects of constant light conditions 225 result from a direct effect on neuronal systems that control locomotion and exploration (14). on the other hand, the ll-animals swam longer and were more active compared to the controls in the probe trial of the wm test, which indicates that the effects on activity and exploration are somewhat context-dependent. table i. behavioral parameters recorded during 10 min in the first (a) and second (b) open field (of) tests in rats exposed to a 12-h light/dark cycle (control) or constant light (ll) during the perinatal development. values represent mean ± sem. *p < 0.05, ** p < 0.01 compared to controls (mann–whitney u test). twelve animals per group were tested, and the statistical analysis was based on number of litters (control group n = 12; ll-group n = 4). parameters control ll a: open field test 1 total activity 56 ± 4 36 ± 3** rearing 24 ± 1 28 ± 2 outer circle frq 20 ± 1 14 ± 1** dur 482 ± 9 522 ± 12* dur/frq 25 ± 2 39 ± 2** middle circle lat 9 ± 2 32 ± 5** frq 28 ± 2 17 ± 1** dur 97 ± 7 67 ± 11* dur/frq 3.6 ± 0.2 3.8 ± 0.6 center lat 72 ± 20 135 ± 24 frq 8 ± 1 4 ± 1* dur 23 ± 3 12 ± 1 dur/frq 2.8 ± 0.3 3.2 ± 0.2 b: open field test 2 total activity 52 ± 4 35 ± 3* rearing 25 ± 1 27 ± 2 outer circle frq 18 ± 1 13 ± 1* dur 489 ± 8 525 ± 7* dur/frq 29 ± 3 46 ± 6* middle circle lat 40 ± 16 70 ± 22 frq 26 ± 2 17 ± 2* dur 84 ± 8 57 ± 5* dur/frq 3.3 ± 0.2 3.4 ± 0.3 center lat 95 ± 21 151 ± 13 frq 8 ± 1 5 ± 1* dur 29 ± 3 19 ± 3 dur/frq 3.7 ± 0.5 3.7 ± 0.2 dur = duration (s), total time spent in zone; dur/ frq = duration per visit (s); frq = frequency, number of visits; lat = latency (s), time to first visiting a zone; rearing = total number of rearings; total activity = total number of visits to the defined zones. table ii. behavioral parameters recorded during the 90-second probe trial of the water maze (wm) test. values represent mean ± sem. * p < 0.05 compared to controls (mann–whitney u test). twelve animals per group were tested, and the statistical analysis was based on number of litters (control group n = 12; ll-group n = 4). parameters control ll target zone crossings 5.1 ± 0.6 4.5 ± 0.5 swim speed 25 ± 0.8 27 ± 0.7 distance 2228 ± 72 2434 ± 59 activity 78 ± 2 86 ± 1 nw quadrant lat 9 ± 3 6 ± 1 frq 7 ± 0.5 7 ± 0.4 dur 22 ± 2 19 ± 0.3 distance 481 ± 42 490 ± 23 activity 75 ± 3 87 ± 3* target (sw) quadrant lat 3 ± 0.4 5 ± 0.6 frq 9 ± 1 9 ± 0.6 dur 36 ± 2 36 ± 1 distance 742 ± 45 834 ± 37 activity 78 ± 3 85 ± 1 ne quadrant frq 6 ± 1 7 ± 0.3 dur 14 ± 1 16 ± 1 distance 367 ± 44 425 ± 20 activity 79 ± 3 84 ± 1 se quadrant lat 7 ± 2 5 ± 1 frq 7 ± 1 8 ± 0.4 dur 18 ± 1 19 ± 1 distance 421 ± 38 516 ± 27 activity 82 ± 2 89 ± 3 activity = percentage of time in the whole pool or a defined quadrant with swim speed of more than 2 cm/s; distance = swim distance (cm); dur = duration (s), total time spent in zone; frq = frequency, number of visits; lat = latency (s), time to first visiting a zone; swim speed = average swim speed (cm/s) in the pool; target zone crossing = target zone (former location of the platform) crossings. 226 e. roman & o. karlsson in mammals, the suprachiasmatic nucleus (scn) of the hypothalamus contains a biological clock that controls the rhythmic expression of a number of physiological processes and behaviors including locomotor activity, sleep, and wakefulness. disturbance in circadian rhythm has been associated with, and proposed to be a contributing factor to, mood disorders (15,16). the effects of constant light and constant darkness, respectively, during adulthood on physiological and behavioral processes have previously been assessed (14). for example, the circadian rhythm of the hypothalamus–pituitary–adrenal (hpa) axis, which is central for control of emotional processes (17), is synchronized via the scn and has been shown to be dysregulated by constant light (18). here, the animals were exposed to constant light during preand postnatal development, both of which may play a crucial role in the final organization of the circadian system and could be of importance for the observed behavioral effects (6,19,20). in conclusion, the exposure of the dams to constant light from e 20 to pnd 4 did not induce preterm birth or any observed short-term effects in the offspring. however, the 7 days of perinatal exposure to constant light induced an increased anxiety-like behavior in the offspring in adulthood. these findings add to the sparse knowledge of long-term consequences of constant light during early development and confirm that early life experiences can cause changes in brain development that shape brain function and affect specific behaviors in adulthood. acknowledgements ms marita berg, professor eva brittebo, and dr samuel rowley uppsala university are gratefully acknowledged for technical assistance, for valuable comments during the preparation of the manuscript, and for language editing, respectively. declaration of interest: funding from the swedish society for medical research (e.r.), the facias foundation (e.r. and o.k.), the helge ax:son johnson foundation (o.k.), and nn jubileumsfond b (o.k.) supported this study. the authors report no conflicts of interest. the authors alone are responsible for the content and 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www.ncbi.nlm.nih.gov/pubmed/18417120?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/23178164?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/23178164?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/3746430?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/3746430?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/3746430?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10586927?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10586927?dopt=abstract abstract introduction material and methods the object recognition test the water maze test behavioral recordings statistical analysis results short-term findings adult behavioral tests discussion acknowledgements declaration of interest references social freezing of oocytes: a means to take control of your fertility review article social freezing of oocytes: a means to take control of your fertility anna-lena wennberg nordic ivf, gothenburg, sweden abstract in the last few decades, there have been tremendous developments of assisted reproductive technologies, but the outcome of in vitro fertilization is highly dependent on the age of the oocyte. the introduction of vitrification offers a possibility to freeze eggs proactively in younger years and use them at later ages, also called social egg freezing. knowledge about age-related fertility decline is insufficient among many women, and there is an overoptimistic trust that in vitro fertilization can overcome this. the awareness of proactive egg freezing is limited, both among doctors and in the general population. this review aims at increasing the knowledge about proactive egg freezing and offers a means to advise women correctly. it deals with national guidelines, the best age and optimal number of eggs to freeze, and the chance to succeed. creating more public awareness about age-linked fertility decline and elective egg freezing may help women reproduce at their own pace – to take control of their fertility. article history received 1 october 2019 revised 23 november 2019 accepted 15 december 2019 keywords age; elective; fertility; oocyte; pregnancy; results; social egg freezing; vitrification introduction ever since the birth of the first child from in vitro fertilization (ivf) in 1978 (1) there have been great developments in the area of assisted reproductive technology (art). many different ways to achieve parenthood exist, and there are now available treatments for most types of male and female infertility. however, a growing problem in the developed world is that we start families at a continuously later stage in life. the mean age for giving birth to the first child has increased all over the world. in sweden it now reaches 29.4 years (2), with similar figures in all the nordic countries. parallel to this, female fertility drops with age. a recent study from denmark showed that fertility decreases approximately 15% between 35 and 40 years and 35% annually above 40 years (3). studies have shown that there is insufficient knowledge about agerelated fertility decline (4–6). nonetheless, many women are aware of their declining fertility potential, and it causes stress to women who have not yet found a partner to form a family with. on the other hand, the awareness of proactive egg freezing is limited, and there is often an overoptimistic belief that ivf will help to overcome age-related infertility (4,7). creating more public awareness about age-linked fertility decline and elective egg freezing, also called social freezing, is therefore a key factor to help women reproduce at their own pace. elective egg freezing can also serve as a means not only to have one child, but to increase the possibility of having a second and third child, for couples who have their first child fairly late. egg freezing cryopreservation of spermatozoa, to protect male fertility, was established already in the 1950s (8), and techniques to cryopreserve embryos were developed during the 1980s (9). because of their size, unfertilized eggs are more sensitive to cryopreservation than sperm and embryos. the first birth after oocyte freezing, followed by warming and ivf, was described already in 1986 by chen in south korea (10), but the results were difficult to reproduce. cryopreservation techniques at that time were limited, with low oocyte survival after thawing, low fertilization rates, and poor pregnancy results. in later years a novel technique – vitrification – has been introduced, showing good warming results and with good fertilization and pregnancy rates (11). the technique means that the oocytes are cooled ultra-rapidly in liquid nitrogen to �196 �c, in a minimal volume of cryoprotectant. in addition, a hands-on tool has made the method easier to introduce into clinical practice than previously. spanish and italian groups have, in large randomized and cohort studies, demonstrated that live birth rates after ivf with vitrified oocytes are comparable to ivf using fresh oocytes (12,13). importantly, international follow-up of children born after oocyte vitrification have not revealed any increased risk of adverse obstetric outcome or increased risk of birth defects (14,15). becoming pregnant with cryopreserved oocytes requires ivf. the woman’s ovaries need to be stimulated before the eggs can be collected and cryopreserved. after warming, the contact anna-lena wennberg annalena@nordicivf.se nordic ivf, odinsgatan 10, gothenburg, se-411 03, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 2, 95–98 https://doi.org/10.1080/03009734.2019.1707332 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1707332&domain=pdf&date_stamp=2020-05-21 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2019.1707332 http://www.tandfonline.com eggs are fertilized, with the partner’s or donated sperm, and an embryo is transferred to the uterus. oocyte freezing is valuable for a variety of medical reasons such as egg donation, genetic predisposition of premature ovarian failure, severe endometriosis, or any gonadotoxic treatment that may result in premature ovarian failure, e.g. cancer treatment. there may also be ethical or religious reasons to freeze unfertilized eggs instead of embryos. elective egg freezing without medical reasons is somewhat more controversial, and there was a big debate when the method was introduced. however, most people today have a positive attitude towards the procedure. a swedish questionnaire study among 2000 women of reproductive age showed that a majority were positive to elective egg freezing, both for medical and for non-medical purposes, and almost half of the women would consider the procedure themselves (16). the swedish national council of medical ethics (smer) has also made a statement in favour of nonmedical egg freezing (17), as have the european society of human reproduction and endocrinology (eshre) (18) and the nordic fertility society (nfs). elective egg freezing for non-medical reasons was introduced in sweden by nordic ivf gothenburg in 2011 and is now offered by most private clinics in sweden. there is no specific law regulating oocyte cryopreservation in sweden. national guidelines have therefore been created by the swedish society of obstetrics and gynaecology (sfog) (19) based on guidelines from smer (17) and eshre (18). the guidelines state that elective egg freezing for non-medical reasons is ethically acceptable provided that the woman receives thorough information about the procedure, the risks, and the chance of having a child by use of the cryopreserved eggs. the importance of information is also stressed in the recommendations by eshre, but the organization at the same time points out that it should be each woman’s free choice weather to freeze eggs or not. according to smer the cost for non-medical egg freezing should be covered by the individual. from 1 october 2013 drugs are not subsidized by the swedish state for women who freeze eggs just by choice. one argument is that only a small percentage of the banked oocytes will probably ever be used (20–22), and the procedure is thus not cost-effective (23). it is, however, not obvious that this approach is equal and just. one can argue for various systems of public funding, part-funding, or pay-back systems for women who proactively freeze their eggs in younger years, similarly to funding ivf with public means (24). younger women have better egg quality, better chance of becoming pregnant, and require a lower hormonal dose at stimulation than older women. it is also likely that is more cost-effective to try and get pregnant with own frozen young eggs than by egg donation at a more advanced age. moreover, there are conditions that are neither purely medical nor non-medical, such as congenital low ovarian reserve or reduced ovarian reserve due to endometriosis, benign ovarian cysts, or unilateral adnexal operations. egg freezing in this intermediate group is today not subsidized by the state. when to freeze? the debate is ongoing regarding the suitable age for elective oocyte freezing. the biologically optimal time would be in the mid-20s, when fertility is at its peak, but it may not be cost-effective in relation to the low usage. in one study from the united states, a decision-tree model was constructed to determine the cost-effectiveness of oocyte preservation in comparison to no action at ages 25–40 years (25). in this model, a mathematical calculation was made of the probability of a live birth seven years after the decision of whether to freeze eggs or to take no action. the highest probability of live birth was seen when oocyte cryopreservation was performed at ages >34 years. elective oocyte freezing compared to no action gave the greatest improvement in likelihood of live births at the age of 37 years (52% after oocyte freezing and 22% at no action), but there was little benefit over no action at ages 25–30 years. furthermore, in this us setting, the cost-effectiveness for elective oocyte freezing was highest at the age of 37 years. there is a distinction between effectiveness and costeffectiveness, and it may be misleading to suggest an optimal age of 37 years for freezing in general. advice regarding elective egg freezing should always be based on individual assessments. also, based on data that follow, there may be reasons for women to consider egg freezing if they have not yet found a partner or are not planning a pregnancy when approaching the age of 35 years. how many eggs to freeze? another important issue is how many eggs to preserve in order to have at least one child. it is challenging to determine the ideal number of oocytes since it depends on many factors, including maternal age and health, ovarian reserve, reproductive goals, and also paternal health. no number of oocytes can offer a guarantee. many clinics recommend banking around 20 eggs, which for most women means going through the procedure more than once. two american prediction models, based on ivf/icsi-treatments, estimate that approximately 20 oocytes are needed, to have around 75% likelihood of achieving at least one child provided that the woman is younger than 38 years (table 1) (26,27). the models also show that the chance of success is highly age-dependent and confirm that attempting fertility preservation in women at an age of over 40 years is unlikely to succeed. appropriate information about the potential to bank a sufficient number of oocytes is vital for women’s decisionmaking. cryopreservation of too few oocytes would limit the possibility of success, while an excessive number would increase the physical discomfort and the expense. most table 1. live birth predictions by age, with 20 mature oocytes banked (24). age (y) probability of one live birth (%) probability of two live births (%) probability of three live births (%) 34 90 66 38 37 75 39 15 42 37 7 1 96 a.-l. wennberg clinics use a combination of age, antral follicle count, and measurement of anti-m€ullerian hormone (amh) to advise women properly. observing a low ovarian reserve at a young age would cause a stronger incitement to preserve oocytes, but it would also increase the cost because of the necessity to repeat the freezing procedure. what is the chance of having a child? an appropriate question is evidently what the chance to have a child is. previous studies have shown that frozen eggs are as good as fresh ones (12,13), but direct pregnancy results from non-medical elective egg freezing are scarce. to a great extent we have had to rely on outcomes from conventional ivf, but data from egg freezing are now coming up. cobo et al. (20) presented a series of 137 out of 1468 women who returned for autologous ivf. the women preserved eggs for both medical and non-medical reasons and constituted 9% of the total number of women freezing eggs electively. the cumulative ongoing pregnancy rate/live birth rate (opr/lbr) per patient was almost 60%. in an australian study, 95 women who had stored oocytes for non-medical reasons answered a non-anonymous questionnaire (21). six of these women had returned for autologous ivf (in total 6% of the responders), and three of them had given birth as a result. at nordic ivf gothenburg we recently published our own data from 1 august 2011 to 31 october 2018 (22). during this period of time 254 women had frozen their eggs for non-medical reasons, and 38 of them (15%) had returned to use their banked oocytes. the cumulative lbr/opr was 63% for women 36–37 years of age at freezing and 26% for women 38–39 years of age at freezing. there were unfortunately no births or ongoing pregnancies for women who were 40 years of age or older at the time of the freezing. although our study includes only a limited population, it clearly demonstrates that the chance of having a child drops with the age at freezing. age-limits in the work with national guidelines age-limits have been discussed. there may be reasons to consider age-limits both for freezing eggs and for using them. since the probability of a pregnancy is highly related to the age of the woman at freezing, elective egg freezing is usually discouraged after the age of 38 years (18). it is, however, agreed that setting an upper age-limit for freezing is up to the individual clinic. it is more difficult to define an upper age-limit for attempting pregnancy with frozen eggs. pregnancies at advanced maternal age are generally considered more hazardous than at younger age. the risks of pregnancy diabetes, hypertensive disorders of pregnancy, and preeclampsia are increased, as is the risk of perinatal complications. the number of caesarean sections is also very high (28–31). based on this knowledge and on international practice, the general recommendation in sweden is not to thaw eggs at a higher age than 46 years. on the other hand, the surveys suggest that the risk of maternal and neonatal complications depend on the mother’s preconceptional health (28–31). smer states that it is improper to define a certain age-limit as ageing is individual. according to smer, the basis should be a woman’s medical capacity of coping with a pregnancy and the best interest of the future child, rather than her actual age (32). social egg freezing the expression ‘social egg freezing’ can be questioned since it implies that women deliberately postpone childbearing for various social reasons, e.g. career, studies, or simply because a child does not fit in to their present situation of life. several studies have shown that this assumption is wrong. most women who freeze eggs have a strong wish for a child, but wish to share their parenthood with a partner rather than being a single parent (33,34). elective egg freezing is often a last resort. at nordic ivf, where almost 300 women have banked their eggs, the mean age for freezing eggs is around 37 years, which is in line with others (20–22). it reflects the fact that women hope to find a partner and wait to the last second to freeze their eggs. even after thorough information, however, many women state that freezing is better that doing nothing, and that they would regret it if not (35,36). the women in the study by gold et al. (35) also stated that they would have frozen earlier if they had been aware of the possibility. our mission as gynaecologists must be to inform that the possibility of having a child is greatest at younger ages, but also to inform about elective egg freezing alongside other reproductive alternatives. egg freezing by choice means no guarantee to have a child but offers women a means to take control of their fertility. many women are insufficiently aware that fertility drops with age, and, by adding information of the possibility to freeze eggs to the message of fertility decline, women may feel helped rather than lectured. disclosure statement no potential conflict of interest was reported by the authors. notes on contributor anna lena wennberg, md, phd, nordic ivf g€oteborg-eugin, gothenburg, sweden. references 1. steptoe pc, edwards rg. birth after the reimplantation of a human embryo. lancet. 1978;2:366. 2. 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jones c, copperman ab. a motivational assessment of women undergoing elective egg freezing for fertility preservation. fertil steril. 2006;86:s201. 36. greenwood ea, pasch la, hastie j, cedars mi, huddleston hg. to freeze or not to freeze: decision regret and satisfaction following elective oocyte cryopreservation. fertil steril. 2018;109: 1097–104.e1. 98 a.-l. wennberg https://www.sfog.se/start/raadriktlinjer/sfog-raad-gynekologi/reproduktion/ https://www.sfog.se/start/raadriktlinjer/sfog-raad-gynekologi/reproduktion/ abstract introduction egg freezing when to freeze? how many eggs to freeze? what is the chance of having a child? age-limits social egg freezing disclosure statement references can costs of screening for hypertension and diabetes in dental care and follow-up in primary health full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 can costs of screening for hypertension and diabetes in dental care and follow-up in primary health care be predicted? sevek engström, lars borgquist, christian berne, lars gahnberg & kurt svärdsudd to cite this article: sevek engström, lars borgquist, christian berne, lars gahnberg & kurt svärdsudd (2013) can costs of screening for hypertension and diabetes in dental care and followup in primary health care be predicted?, upsala journal of medical sciences, 118:4, 256-262, doi: 10.3109/03009734.2013.818599 to link to this article: https://doi.org/10.3109/03009734.2013.818599 © informa healthcare published online: 19 aug 2013. submit your article to this journal article views: 495 view related articles citing articles: 2 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.818599 https://doi.org/10.3109/03009734.2013.818599 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.818599 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.818599 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.818599#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.818599#tabmodule upsala journal of medical sciences. 2013; 118: 256–262 original article can costs of screening for hypertension and diabetes in dental care and follow-up in primary health care be predicted? sevek engström1, lars borgquist2, christian berne3, lars gahnberg4 & kurt svärdsudd1 1department of public health and caring sciences, family medicine and preventive medicine section, uppsala university, uppsala sweden, 2department of medical and health sciences, family medicine, linköping university, linköping, sweden, 3department of medical sciences, university hospital, uppsala, sweden, and 4department of behavioural and community dentistry, institute of odontology, university of gothenburg, göteborg, sweden abstract aim. the purpose was to assess the direct costs of screening for high blood pressure and blood glucose in dental care and of follow-up in primary health care and, based on these data, arrive at a prediction function. study population. all subjects coming for routine check-ups at three dental health clinics were invited to have blood pressure or blood glucose measurements; 1,623 agreed to participate. subjects screening positive were referred to their primary health care centres for follow-up. methods. information on individual screening time was registered during the screening process, and information on accountable time, costs for the screening staff, overhead costs, and analysis costs for the screening was obtained from the participating dental clinics. the corresponding items in primary care, i.e. consultation time, number of follow-up appointments, accountable time, costs for the follow-up staff, overhead costs, and analysis costs during follow-up were obtained from the primary health care centres. results. the total screening costs per screened subject ranged from e7.4 to e9.2 depending on subgroups, corresponding to 16.7–42.7 staff minutes. the corresponding follow-up costs were e57–e91. the total resource used for screening and followup per diagnosis was 563–3,137 staff minutes. there was a strong relationship between resource use and numbers needed to screen (nns) to find one diagnosis (p < 0.0001, degree of explanation 99%). conclusions. screening and follow-up costs were moderate and appear to be lower for combined screening of blood pressure and blood glucose than for separate screening. there was a strong relationship between resource use and nns. key words: costs, dental care, diabetes mellitus type 2, early diagnosis, high blood pressure, primary health care introduction because of the asymptomatic nature, most subjects are unaware of hypertension and diabetes in the early course but have nevertheless an increased risk of developing cardiovascular and other complications (1,2). it is also well known that treatment reduces the risk of complications (3). it has been assumed that early detection and treatment reduce or postpone the risk of complications, even though no firm evidence has been forwarded so far. prevention of complications not only benefits patients, but also potentially reduces overall health care expenditure (4,5). blood pressure and blood glucose screening is one possible means of finding subjects unaware of their disease. the question is how best to screen subjects who consider themselves healthy and therefore have no reason to see a physician. in industrialized countries, the dental care (dc) service is usually the only health care organization to which healthy people systematically come for regular correspondence: sevek engström, department of public health and caring sciences, family medicine and preventive medicine section, po box 564, se-751 22 uppsala, sweden. e-mail: sevek.engstrom@pubcare.uu.se (received 24 january 2013; accepted 18 june 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.818599 http://informahealthcare.com/journal/ups mailto:sevek.engstrom@pubcare.uu.se check-ups (6). it has been estimated that during a 2-year period approximately 80% of the adult population in sweden see a dentist for routine dental check-up, whereas regular check-ups in primary health care of healthy people hardly exist (6). dc may therefore be a suitable venue for screening large segments of the general population. in previous publications from this project the possibilities of using dc for screening of blood pressure and blood glucose, with follow-up at primary health care (phc) centres participating in the project, were tested (7-9). the results showed that this way of combining the work done by dc and phc was highly feasible. in blood pressure screening a new incident hypertension diagnosis was found on average for every 18 subjects screened (number needed to screen (nns) = 18), and a new incident diabetes diagnosis for every 196 subjects screened (nns = 196). the aim of this study was to analyse the direct costs of screening for hypertension and diabetes in dc, and for follow-up in phc, and to arrive at a resource (staff time needed) prediction instrument by which screening and follow-up costs may be predicted. methods setting in sweden, the vast majority of medical care is operated by the county councils, which are responsible for health care in their geographical areas, either as county council-operated health care units or as private subcontractors, at the time of the study a minority. for dc the situation is similar, except that approximately half of the units are county council-operated and the remaining ones are private subcontractors. all units, whether operated by county councils or by private subcontractors, follow the same regulations. this study was performed at two county counciloperated dc clinics located in the municipality of ovanåker (population 12,000) and one country council operated dc in the strömsbro area of the city of gävle (population 92,000), located in gävleborg county, central sweden. the three phc centres in the municipalities, all county council-operated, were partners in the project and were responsible for calibration and quality of the blood pressure and blood glucose meters and for the blood pressure and blood glucose follow-up in subjects screening positive. study population a detailed description of the study population has been given elsewhere (8,9). briefly, all consecutive patients scheduled for an annual examination at any of the clinics in ovanåker and strömsbro 2003–2005, and living in the area, were invited by letter to participate in the study. the screening measurements were performed before the scheduled dental examination. participants were asked for their height, weight, and whether they had a hypertension or a diabetes diagnosis. those who did not know their height or weight had it measured, without shoes, on a fixed wall measure to the nearest centimetre or on a lever balance, wearing indoor clothing, to the nearest tenth of a kilogramme, respectively. body mass index (bmi) was calculated as weight in kilogrammes/(height in metres)2, used to delineate the screening population and motivate screening in the younger ages. subjects with no known hypertension or diabetes and who were in the age range 20–39 with a bmi >25 kg/m2, or in the age range 40–65 (ovanåker) or 40–75 (strömsbro) regardless of bmi, were eligible for screening (figure 1). the study design was influenced by the fact that the phc centre in strömsbro agreed screening only for blood glucose and that blood pressure measurements could not be performed. of the 1,623 subjects who participated, 55 were screened for blood pressure only, 475 for blood glucose only, and 1,093 for blood pressure and blood glucose. altogether, 1,149 were screened for blood pressure regardless of blood glucose screening, and 1,568 were screened for blood glucose regardless of blood pressure screening. moreover, as shown previously, more restricted screening criteria (age ‡30 and body mass index (bmi) ‡30 or age ‡40 and bmi ‡25) resulted in lower nns, and therefore this subpopulation was analysed as well. thus six possible subgroups were analysed. data collection blood pressure was measured in a sitting position, in the left arm, after resting for 5 minutes, with an automatic blood pressure reading device omron m4� (omron co, kyoto, japan). if systolic blood pressure was >160 mmhg or diastolic blood pressure was >90 mmhg, a second reading was taken after the dental examination and the lowest recorded value was used as the screening blood pressure. samples for non-fasting blood glucose analysis were obtained as capillary blood from the patient’s third fingertip and analysed immediately with an accuchek compact device (roche diagnostics scandinavia ab,stockholm,sweden)orwithahemocueb-glucose analyser (hemocue ab, ängelholm, sweden). data measured at the dc clinics were registered in pre-prepared protocols and entered into the study costs of hypertension and diabetes screening 257 database. subjects with screening systolic blood pressure >160 mmhg, diastolic blood pressure >90 mmhg, or a screening blood glucose concentration of ‡6.7 mmol/l, were asked for permission to be referred to their phc centre (all accepted). a copy of the dental service protocol served as referral document for follow-up at the subject’s phc centre, where an appointment was arranged. phc follow-ups were standardized to three nurse appointments and one general practitioner (gp) appointment for blood pressure followup and two nurse appointments and one gp appointment for blood glucose follow-up. information on whether the referral resulted in a hypertension or diabetes diagnosis was obtained by scrutiny of patient records from all appointments at the three phc centres for the complete study population, regardless of screening result, for the 3 years following the screening appointment. a 3-year follow-up time was estimated to be long enough to catch late-comers, and short enough to minimize the risk of hypertension or diabetes development among those not referred. to check for completeness, appointment log-books were also scrutinized. data included date of appointment and gp and hypertension or diabetes nurse appointments diagnoses. moreover, discharge diagnoses after hospital admissions within the 3 years following screening, obtained from the national hospital discharge register covering all hospital admissions in sweden, attending phc n = 5 d c p h c attending phc n = 37 high blood pressure only n = 204 high blood pressure diagnoses n = 1 diabetes diagnoses n = 4 high blood pressure diagnoses n = 75 diabetes diagnoses n = 5 high blood pressure or diabetes diagnoses n = 80 high blood glucose only n = 81 high blood pressure and high blood glucose n = 28 attending phc n = 313 blood pressure screening n = 55 blood glucose screening n = 475 blood pressure and glucose screening n = 1,093 1,623 subjects screened figure 1. flow chart displaying the number of participants in the screening process from dental care (dc) to the follow-up in primary health care (phc). 258 s. engström et al. were scrutinized for hypertension and diabetes diagnoses. mortality data for those who died (date of death and underlying diagnoses) were obtained from the national cause of death register. phc diagnoses, hospital discharge diagnoses, and mortality diagnoses were coded according to the international classification of diseases (icd) (10). the main outcome used was whether a hypertension or diabetes diagnosis was established during the first 3 years of follow-up from the date of screening. the time per subject spent on the screening procedure (screening time) was measured in minutes and entered into the study database. it included history-taking regarding inclusion criteria (known hypertension or diabetes, age, height, weight, and in some instances measurements of height and weight), blood pressure or blood glucose measurements, and data registration, but not time for writing and sending invitation letters or other administrative procedures. cost calculations only direct costs were measured. information on wages, employer’s fees, institutional overheads, accountable time, and measurements costs were obtained from the swedish association of local authorities and regions (11), and from the gävleborg county council central administration. all swedish employers pay employer’s fees to the central government, calculated as a percentage of the gross wages of each employee and used to cover national health insurance fees, pensions, etc. institutional overheads are charged on all financial transactions in health care units, to cover administration costs. accountable time is the fraction of working time that can be charged for, in this case direct health care activities, and was estimated to be 65%. the remaining working time, used for administration, preparations for new work tasks, etc., is the unaccountable time which, although not chargeable, has to be covered. the following cost analysis models were used: . dc costs: number of screened subjects, multiplied by screening time adjusted for unaccountable time, multiplied by wages adjusted for employer’s fee and overhead costs, plus analysis costs. . phc costs: number of follow-up appointments, multiplied by consultation time adjusted for unaccountable time, multiplied by wages adjusted for employer’s fees and overhead costs, plus analysis costs. all costs are given in euros (e), e1 approximately corresponding to 9 sek and us$1.4. in dc dental hygienists performed the screening. the crude price per screening minute was e0.29 (table i). at the phc centres, hypertension, diabetes, or district nurses performed the initial follow-up (including history-taking and repeated blood pressure and blood glucose measurements), and a gp made the final decision as to whether the subject had a hypertension or a diabetes diagnosis. the crude minute costs for a nurse was e0.29, and for a gp e0.67. the employer’s fee was 43% for all three staff types, and the overhead costs were 36% in phc and 50% in dc. the total minute price was then e0.62 in dc, e0.56 for nurses, and e1.30 for gps. the cost per blood glucose analysis was e0.64 at all units. for blood pressure measurements there were no specific costs other than time for measurements. blood pressure screening only, blood glucose screening only, or both types of screening on the same occasion formed the three screening groups. cost per diagnosis in dc was obtained by multiplying price per screened subject by the numbers needed to screen (nns) to identify a person with undiagnosed diabetes. nns was computed as the reciprocal of the proportion of new cases found by screening minus the number of subjects who would have been detected anyway (1,12). in phc, the standard follow-up was three appointments with blood pressure measurements performed by a nurse or two appointments with fasting blood glucose measurements performed by a nurse, in both cases followed by a single appointment with the gp for diagnosis assessment. the total time used for follow-up was the number of subjects multiplied by number of appointments per person, and the total wages cost was total time used multiplied by price per minute. total price per subject for follow-up was total wages cost for nurses and gps plus analysis costs divided by number of subjects followed up, and total price per diagnosis was total price divided by number of diagnoses. data were analysed with the sas software, version 9.3 (13). the relationship between resource use and nns was analysed with linear regression, using crude table i. financial characteristics. all costs are given in euros. dental care primary health care dental hygienist nurse gp costs per minute 0.29 0.29 0.67 employer tax, % 43 43 43 overhead costs, % 50 36 36 total minute cost 0.62 0.56 1.30 costs per blood glucose analysis 0.64 0.64 – costs of hypertension and diabetes screening 259 data as well as data weighted by subpopulation size. both types of analyses gave similar results. therefore, only crude data analysis results are shown. r2 was used as measure of degree of explanation. p < 0.05 indicated statistical significance. ethics all participants gave their written informed consent. the study was performed in accordance with the helsinki declaration and was approved several times during the data collection process by the research ethics committee at uppsala university and later by the regional research ethics board. results characteristics of the study population approximately half (50.5%) were women. mean age was 48.0 years (standard deviation (sd) 10.5), and mean bmi was 26.4 (sd 4.0). mean screening systolic blood pressure was 135.1 (sd 20.0) mmhg, mean diastolic blood pressure 82.1 (sd 11.2) mmhg, and mean blood glucose 5.4 (sd 1.1) mmol/l. after adjustment for unaccountable time in dc, the time used for screening of high blood pressure or high blood glucose was 12.0 minutes, and time used for screening of both conditions was 13.8 minutes. after adjustment for unaccountable time in phc, the time for a follow-up consultation was 30.8 minutes for subjects screening positive for high blood pressure only, 15.4 for a follow-up consultation for high blood glucose only, and 30.8 minutes for a follow-up appointment for both conditions. the gp consultation time was estimated to be 30 minutes. screening and follow-up costs the cost calculations were based on the six subgroups mentioned previously (table ii). the nns levels found covered a broad range, 15–196. the analysis costs were marginal as compared with salary costs. total screening costs per screened subject were similar in all subgroups (range e7.44–9.20). the costs for the phc follow-up were higher, but fairly similar in table ii. resource use in terms of time (minutes) and costs (e) in blood pressure and blood glucose screening. bps regardless of bgs bgs regardless of bps bgs (restricted screening pop) bps only bgs only bps + bgs combined screening number screened 1,149 1,568 766 55 475 1,093 adjusted screening time per screened 12 12 12 12 12 13.8 total time for screening 13,788 18,816 9,192 660 5,700 15,083 total analysis costs 0 1,004 490 0 304 700 total screening costs 8,549 12,669 6,189 409 3,838 10,052 total screening cost per screened 7.44 8.08 8.08 7.44 8.08 9.20 follow-up number followed up 237 155 97 5 37 291 adjusted follow-up time per subject 30.8 15.4 15.4 30.8 15.4 30.8 total nurse time 21,899 4,774 2,988 462 1,140 22,823 total gp time 7,110 4,650 2,910 150 1,110 8,730 total analysis costs 0 198.40 124.16 0 47,4 140 total follow-up costs per subject 91 58 58 91 57 83 number of subjects with diagnosis found 76 9 9 1 4 80 numbers needed to screen (nns) 18 196 96 55 119 15 total resource use per screened total time used 25.2 18.0 19.7 23.1 16.7 42.7 analysis costs 0 0.77 0.80 0 0.74 0.77 total resource use per diagnosed subject total time used 563 3,137 1,677 1,272 1,988 583 bps = blood pressure screening; bgs = blood glucose screening. 260 s. engström et al. the subgroups (range e57–91). the total resources used for screening and follow-up per screened subject were 16.7–42.7 staff minutes, and the analysis cost range was e0–0.80 depending on subgroup. the corresponding resource use per diagnosed subject was 563–3,137 staff minutes. the resource use range was thus rather narrow per screened subject but considerably wider per diagnosed subject. to analyse whether the latter condition was due to the variable nns levels the resource use measured as staff minutes was plotted against nns (figure 2). there was an obvious linear relationship, irrespective of subgroup, between nurse/dental hygienist time per diagnosed subject and nns (p < 0.0001), gp time and nns (p < 0.001), and total time used per diagnosed subject and nns (p < 0.0001). the functional form for the lastmentioned relationship was time used = 371 + 14.0 � nns. the degree of explanation was 99%. discussion the screening cost per screened subject was thus moderate, as compared with the follow-up costs for those screening positive. the costs for high blood pressure screening were lower than the corresponding costs for blood glucose screening and the combined screening because of lower analysis costs. however, the cost for follow-up was lower for those screening positive for blood glucose than for those screening positive for blood pressure, owing to the need of more follow-up visits among the latter. the total cost per diagnosis found was lowest for combined blood pressure and blood glucose screening, since two possible diagnoses were being sought. the strengths of this study include that the screened cohort was fairly large, the measurement procedures were standardized, and the equipment was provided and calibrated by the participating phc. the followup procedures were standardized, and follow-up was complete in the sense that the screened subjects who did or did not appear for follow-up were included and very few were lost to follow-up. the limitations include the scanty clinical information from phc. however, we have reason to believe that the phc centres were following the national guidelines for hypertension and diabetes diagnosis assessment, or local practice recommendations based on established guidelines. furthermore, the time required for the diagnosis assessment by the gp had to be estimated (14), and the time spent for checking inclusion criteria for those not fulfilling the criteria had to be neglected. the assumption that all referred subjects had a final follow-up consultation with the gp may be an over-estimate. the nurse may have forwarded the gp’s decision to the subjects. however, even in this situation the gp had to spend time making the assessment, and in any case this circumstance probably affected all groups equally. the stakeholders potentially considering the results of this study include dc or phc centres planning screening activities, since either the patient or his or her insurance company, or as in the swedish setting the national social insurance, a government agency, pays for the screening activity. a third party, at least in countries with a national social insurance system, may be the body responsible for primary prevention, in sweden the county councils, for which the costs per diagnosis found are important, since this cost is a measure of screening effectiveness. the time spent by the dental staff per subject screened, 8–9 minutes, is similar to time reported from other studies (15). the total screening cost per screened subject in dc was e7.4 for blood pressure screening only, e8.1 for blood glucose only, and e9.2 for combined blood pressure and blood glucose screening. it is obvious that history-taking, information, and creating a calm screening atmosphere all influence the total screening time more than the time for the actual screening procedure at the dc. this appears to be the reason why screening for blood pressure and blood glucose took only marginally more time than screening for a single abnormality. the screening costs were low compared to the corresponding follow-up costs in phc. 0 0 500 1,000 1,500 2,000 2,500 3,000 3,500 20 40 60 80 100 numbers needed to screen r e s o u rc e n e e d e d , m in u te s 120 140 160 180 200 total time needed nurse/dental hygienist time gp time figure 2. relationship between numbers needed to screen (nns) to find one case ending up with a diagnosis, and resources needed in the form of nurse/dental hygienist time, gp time, and total nursedental hygienist-gp time. solid lines indicate results based on screening for blood pressure only, blood glucose only, or combined screening. symbols with no line indicate results based on other subgroups (cf. table ii). costs of hypertension and diabetes screening 261 the nns for blood glucose screening only is high as compared with those reported from other studies (16,17). in the previous blood glucose screening study from this project it was shown that by narrowing the screening population a nns level of 96 could be achieved (9). a more targeted study population of this kind might have decreased dc costs by about 50%, and total costs, including all follow-up costs, might have been substantially lower. the cost for finding undiagnosed subjects with diabetes varies with study population, cut-off level, and general health care cost level. a study performed in the usa in subjects aged 45–74 years showed that the most efficient cut-off level was approximately 6.7 mmol/l, the same as in this study, resulting in a total screening and follow-up cost ranging from $392 to $671 (e280 to e479) per case. if indirect costs, such as patient travel costs and patient wage loss, were included, the costs ranged from $504 to $990 (e360 to e707) (18). opportunistic screening, as used in this study, carries no extra costs for patient time spent on travelling for the screening procedure at the dental care appointment. the relationship between resource use, measured as staff time used, and nns proved to be very strong and almost linear. based on this finding it may be possible to calculate resource need for screening in advance, based on measured or assumed nns. this is a novel finding, not presented earlier. conclusions using dc for opportunistic screening is a new avenue for early detection of diabetes and high blood pressure. the total resource need for screening and follow-up per diagnosis seems to be lower when blood pressure screening is combined with blood glucose screening as compared to separate screening for the two disease entities. resource need may be calculated based on known or assumed nns. acknowledgements this study was supported by grants from the centre for clinical research at uppsala university/ gävleborg county council, the public dental service, gävleborg county council, and uppsala university. special thanks go to the staff at the dental and primary health care centres where the study was performed. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. waugh n, scotland g, mcnamee p, gillett m, brennan a, goyder e, et al. screening for type 2 diabetes: literature review and economic modelling. health technol assess. 2007;11: iii–v; ix–xi, 1–125. 2. sbu. moderately elevated blood pressure. a report from sbu, the swedish 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www.ncbi.nlm.nih.gov/pubmed/11769300?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18808662?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18808662?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18808662?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15920046?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15920046?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15920046?dopt=abstract abstract introduction methods setting study population data collection cost calculations ethics results characteristics of the study population screening and follow-up costs discussion conclusions acknowledgements declaration of interest references an intervention in contraceptive counseling increased the knowledge about fertility and awareness of preconception health—a randomized controlled trial article an intervention in contraceptive counseling increased the knowledge about fertility and awareness of preconception health—a randomized controlled trial yvonne skogsdala , helena fadlb , yang caoc , jan karlssond and tanja tyd�ene amaternal health care unit, faculty of medicine and health, €orebro university, €orebro, sweden; bdepartment of obstetrics and gynecology, faculty of medicine and health, €orebro university, €orebro, sweden; cclinical epidemiology and biostatistics, school of medical sciences, €orebro university, €orebro, sweden; duniversity health care research center, faculty of medicine and health, €orebro university, €orebro, sweden; edepartment of women’s and children’s health, akademiska sjukhuset, uppsala, sweden abstract background: reproductive life plan counseling (rlpc) is a tool to encourage women and men to reflect upon their reproduction, to avoid unintended pregnancies and negative health behavior that can threaten reproduction. the aim was to evaluate the effect of rlpc among women attending contraceptive counseling. outcomes were knowledge about fertility and awareness of preconception health, use of contraception, and women’s experience of rlpc. material and methods: swedish-speaking women, aged 20–40 years, were randomized to intervention group (ig) or control group (cg). participants (n ¼ 1,946) answered a questionnaire before and two months after (n ¼ 1,198, 62%) the consultation. all women received standard contraceptive counseling, and the ig also received the rlpc, i.e. questions on reproductive intentions, information about fertility, and preconception health. results: women in the ig increased their knowledge about fertility: age and fertility, chances of getting pregnant, fecundity of an ovum, and chances of having a child with help of ivf. they also increased their awareness of factors affecting preconception health, such as to stop using tobacco, to refrain from alcohol, to be of normal weight, and to start with folic acid before a pregnancy. the most commonly used contraceptive method was combined oral contraceptives, followed by long-acting reversible contraception. three out of four women (76%) in the ig stated that the rlpc should be part of the routine in contraceptive counseling. conclusions: knowledge about fertility and awareness of preconception health increased after the intervention. the rlpc can be recommended as a tool in contraceptive counseling. article history received 1 july 2019 accepted 28 july 2019 keywords contraceptive counseling; fertility; lifestyle factors; preconception care; preconception health; pregnancy; reproductive life plan introduction preconception health and care are of growing interest as they can affect fertility, pregnancy outcomes, and the health of the offspring both in the short and in the long term (1–4). lifestyle factors such as smoking, obesity, and alcohol consumption have a negative impact on fertility and pregnancy outcomes. when planning a pregnancy, it would be optimal to change an unhealthy lifestyle to a healthier one in order to improve one’s outcomes. there are preconception guidelines for women with chronic diseases, but only fragmentary guidelines exist for healthy women (5). an increased intake of folate through food or vitamin supplementation is recommended in many countries for all women who might become pregnant, in order to prevent neural tube defects (6). however, in a swedish study, only 4 out of 10 women took folic acid prior to pregnancy, despite the fact that the pregnancy was very planned (7). for promotion of preconception health and care, moos et al. developed a tool called reproductive life plan (rlp). it is intended for both women and men to reflect upon their reproductive intentions, to find strategies for successful family planning, and to avoid unwanted pregnancies and adverse health outcomes that may adversely affect reproduction (8). the rlp focuses on the individual’s goal of having children or not, as well as a plan for how to achieve this goal. jack et al. recommend that women of fertile age should be screened for their intentions to become or not become pregnant, both from a shortand a long-term perspective, and also screened for their risk of an unwanted pregnancy (9). an rlp can help women to choose a suitable contraceptive method according to their reproductive goals. however, a us study did not find support for the increased use of effective contraceptives following reproductive life plan counseling (rlpc) among women (10). a swedish study on female university students found that after rlpc the contact yvonne skogsdal yvonne.skogsdal@regionorebrolan.se maternal health care unit, region €orebro county, box 1613, se-701 16, €orebro, sweden supplemental data for this article is available online at here. � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 3, 203–212 https://doi.org/10.1080/03009734.2019.1653407 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1653407&domain=pdf&date_stamp=2019-09-09 http://orcid.org/0000-0003-2412-4676 http://orcid.org/0000-0002-2691-7525 http://orcid.org/0000-0002-3552-9153 http://orcid.org/0000-0002-2559-5456 http://orcid.org/0000-0002-2172-6527 https://doi.org/10.1080/03009734.2019.1653407 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2019.1653407 http://www.tandfonline.com knowledge of preconception health increased and the women planned to have their last child earlier in life (11). the rlpc has also been used among men with positive results (12). midwives who used the rlpc considered it as a health-promoting concept, but they emphasized the importance of tactfulness and were aware that social norms influenced decisions regarding childbearing (13). since screening tools on pregnancy intentions have gained attention, it has been suggested that the use of these tools in clinical settings needs to be further examined in a general population (14–16). the specific aim of this study, which is part of a planned longitudinal study, was to evaluate the effect of using the rlpc among a representative sample of swedish-speaking women visiting registered nurse-midwives (rnm) for contraceptive counseling. the main outcomes were knowledge of fertility and awareness of preconception health. secondary outcomes were use of contraception and women’s experiences of the rlp. material and methods a randomized controlled trial was conducted between february 2015 and march 2016. all women (n ¼ 6,354) attending contraceptive counseling during the study period were assessed for eligibility. inclusion criteria were: aged between 20 and 40 years and able to read and understand swedish. a flow diagram of the study is presented in figure 1. sample size in addition to the evaluation of primary and secondary outcomes in the present study, the plan is to follow the participants with their future pregnancies. accordingly, the sample size calculation is based on both immediate outcomes, as well as on the assumption that the intervention might have a possible effect on future preconception health. assessed for eligibility (n=6,354) excluded (n=4,357) • not meeting inclusion criteria (n=2,374) • declined to participate (n=1,059) • other reasons (n=924) baseline questionnaire (n=1,997) allocated to intervention (n=1,004) • received intervention and standard care (n=976) • did not receive allocated intervention: age under 20 and over 40 (n=13) lack of completed written consent (n=15) allocated to control group (n=993) • received standard care (n=970) • did not receive allocated control group: age under 20 and over 40 (n=8) lack of completed written consent (n=15) lost to follow-up: • wrong or no address (n=16) • declined to continue to participate (n=2) • did not send back follow-up questionnaire (n=346) analyzed (n=592) analyzed (n=606) randomized (n=1,997) lost to follow-up: • wrong or no address (n=19) • declined to continue to participate (n=1) • did not send back follow-up questionnaire (n=364) figure 1. flow diagram. 204 y. skogsdal et al. for the present study, assuming that knowledge about folic acid would increase from 20% to 40% after the intervention (11), with a power of 0.8 and a significance level of p < 0.05, 82 women would be needed in each group. for the planned future longitudinal study, assuming that the intervention would affect women to make lifestyle changes before a future pregnancy, the sample size was calculated accordingly: a) to reduce hazardous use of alcohol by 50% (from 6% to 3%); b) to reduce smoking 3 months before the pregnancy by 50% (from 13.5% to 6.75%); and c) to reduce the proportion of women with bmi �25 in early pregnancy from 40% to 32%. with a power of 0.8 and a significance level of p < 0.05, a) 749, b) 312, and c) 564 would be needed in each group. it was not possible to accurately estimate how many of the women who participated in the study would become pregnant in the future. however, assuming that approximately 50% would become pregnant, it was decided that 1,000 individuals would be included in each group. setting in sweden, midwives are licensed to insert contraceptive devices and prescribe hormonal contraceptives to healthy women. a total of 86 midwives at 28 outpatient clinics in central sweden participated in the data collection. before the commencement of the study, the midwives were informed about the study rationale and the intervention to ensure equivalent implementation. they were instructed how to counsel the intervention and control group as well. as described by skogsdal et al. (17), women were informed about the study when they called the clinic to make their appointment. later, when they attended the clinic, they received written and oral information. they were also informed that participation was voluntary and that they could interrupt their participation at any time without explanation. those who agreed to participate signed an informed consent and were randomly assigned to a control group (cg) or an intervention group (ig). they were informed that the signed consent with their personal identification number should be kept separate from the questionnaire. all data were coded and stored in secure servers in order to minimize risk of unauthorized access. randomization procedure randomization was done within each maternal healthcare unit in clusters. sealed envelopes were prepared with notes for the ig or cg, together with a baseline questionnaire and a prepaid envelope. the midwife opened the envelope containing instructions for either the cg or the ig. before the counseling, participants completed the baseline questionnaire in the waiting room. the questionnaire was then put into a sealed envelope and handed over to the midwife. part of the results from the baseline questionnaire has been presented previously (17). intervention both the ig and the cg received routine contraceptive counseling according to the swedish medical product agency (18), as described in supplement 1 (available online). in addition to the routine counseling, women in the ig also received the rlpc, which was conducted in the similar way as in a study on female university students (11). the midwives had access to a template with discussion points to choose from, depending on the woman’s answer to the first question, ‘do you wish to have children/more children in the future?’ for those who did not want children/more children, the discussion focused on the importance of choosing effective contraceptives and general information about preconception health. for women who wished to have children, information was focused on preconception health, including folic acid supplementation and use of contraception until a pregnancy was planned. for women who wanted to postpone childbearing for years, the focus was on contraception for a long time, preconception health, and information about fertility and age. for those who were unsure about their reproductive intention, the discussion was about contraception and a healthy lifestyle. the midwife gave the women standardized information based on a checklist (11). they also gave the women in the ig a specially designed 18-page booklet to read. the booklet contained information/facts about fertility and preconception health. questionnaires the outcome was evaluated using two questionnaires: at baseline (q1) and at follow-up (q2) (questionnaires 1 and 2, available online). the questions were developed on the basis of the previous study of university students (19). some questions were modified, and some new questions were added. before the present study, the questionnaire was piloted on 28 women attending contraceptive counseling, whereupon a few questions were modified. q1 consisted of 41 multiple-choice questions on background and demographics (table 1), knowledge about fertility (table 3), preconception health (table 4), the use of contraception at latest intercourse (table 6), including the question ‘how satisfied are you with your current contraceptive?’ in a five-grade response scale, and reproductive intention: ‘do you want children/more children in the future?’ and ‘how many children do you want?’ we also asked about when they wanted to have their first and last child, as well as the probability of having an unplanned pregnancy in the future. q2 was sent by post, two months after the counseling, with two reminders. most questions in q2 were repeated from q1 (table 2). we also asked them: ‘in the last 2 months, have you at any time talked to someone close to you, for example, your partner or a friend, about fertility (ability to become pregnant)?’ and ‘what did you talk about?’ the ig also received additional questions about the experiences of the rlpc: ‘did you read the brochure you received from the midwife?’ ‘what did you think when the midwife asked you upsala journal of medical sciences 205 https://doi.org/10.1080/03009734.2019.1653407 about your reproductive life plan?’ and ‘do you think that the midwife or other categories of healthcare professionals, e.g. doctors, should have as a routine to discuss the reproductive life plan with their patients?’ the cg received additional questions about if they had talked with the midwife about ‘having children in the future’, ‘importance of age for fertility’, ‘probability of becoming pregnant after an unprotected sexual intercourse’, ‘factors that can affect the fertility’, ‘factors that can increase the probability of having a healthy pregnancy’, ‘chances of having a baby via ivf’, and the ‘lifespan of ovum and sperm’. statistical analysis participants’ demographic characteristics and responses are presented in percentages, proportions, or median with a corresponding interquartile range wherever suitable. chi-square or fisher’s exact tests were used for categorical variables. mann–whitney u test was used for ordinal table 1. background and demographics among those who answered the baseline questionnaire and comparison between those who answered and did not answer the follow-up questionnaire. answer baseline n ¼ 1946 answer baseline and follow-up n ¼ 1204 answer baseline and not follow-up n ¼ 742 p age, median year (interquartile range) 25 (22, 31) 26 (22, 31) 24 (3.4) 0.003 bmi, n (%) underweight (<18.5) 14 (0.7) 26 (2.2) 25 (2.3) 0.263 normal weight (18.5–24.99) 1198 (64.3) 763 (65.5) 435 (61.1) overweight (25–29.99) 430 (22.0) 259 (22.3) 171 (24.0) obese class i (30–34.99) 138 (7.1) 80 (6.9) 58 (8.1) obese class ii (>35) 47 (2.4) 36 (3.1) 24 (3.4) education, n (%) non-completed education (<9 years) 14 (0.7) 4 (0.3) 10 (1.3) <0.001 elementary school (9 years) 82 (4.2) 33 (2.8) 49 (6.6) high school (12 years) 1145 (58.9) 678 (56.6) 467 (62.7) professional education (non-academic) 159 (21.3) 99 (8.3) 60 (8.1) college/university 543 (27.8) 384 (32.1) 159 (8.1) main occupation, n (%) working 1105 (56.9) 644 (53.8) 461 (61.8) <0.001 student 535 (27.5) 361 (30.2) 174 (23.3) parental leave 171 (8.8) 123 (10.3) 48 (6.4) unemployed 66 (3.4) 32 (2.7) 34 (4.6) sick leave 52 (2.7) 28 (2.3) 24 (3.2) other 14 (0.7) 9 (0.8) 5 (0.7) country of birth, n (%) sweden 1798 (92.9) 1129 (94.7) 669 (90.0) 0.002 other nordic country 14 (0.7) 6 (0.5) 8 (1.1) other european country 50 (2.6) 25 (2.1) 25 (3.4) outside europe 74 (3.8) 33 (2.8) 41 (5.5) sexual orientation, n (%) heterosexual 1823 (94.4) 1135 (95.1) 688 (93.2) 0.109 bisexual 80 (4.1) 47 (3.9) 33 (4.5) homosexual 1 (0.1) 1 (0.1) 0 (0.0) do not know/unsure 21 (1.1) 9 (0.8) 12 (1.6) other 7 (0.4) 2 (0.2) 5 (0.7)) stable relationship, n (%) 1476 (76.3) 927 (77.7) 549 (74.0) 0.062 reproduction, n (%) had tried to get pregnant 652 (33.7) 412 (34.5) 240 (32.4) 0.348 had been pregnant 881 (45.4) 512 (42.8) 369 (49.6) 0.003 had given birth 656 (75.5) 413 (81.5) 243 (67.1) <0.001 experience of abortion 280 (32.3) 135 (26.7) 145 (40.1) <0.001 experience of miscarriage 112 (12.9) 78 (15.4) 34 (9.4) 0.009 smoking, n (%) smoking daily 199 (10.2) 102 (8.6) 97 (13.1) <0.001 smoking, but not daily 221 (11.5) 108 (9.1) 113 (15.3) former smoker 429 (22.0) 276 (23.2) 153 (20.7) never smoked 1077 (55.9) 702 (59.1) 375 (50.8) swedish snuff, n (%) snuff daily 126 (6.4) 65 (5.5) 61 (8.3) 0.013 use snuff, but not daily 81 (4.2) 47 (4.0) 34 (4.6) former user of snuff 157 (8.1) 93 (7.8) 64 (8.7) never used snuff 1563 (81.1) 984 (82.8) 579 (78.5) drinking alcohol, 4 standard glasses or more at the same time, n (%)a daily 1 (0.1) 1 (0.1) 0 (0) 0.073 once/week 94 (4.8) 54 (4.5) 40 (5.5) once/month 495 (25.8) 290 (24.3) 205 (28.0) less than once/month 972 (49.7) 618 (51.9) 354 (48.4) never 360 (18.7) 228 (19.1) 132 (18.1) aone standard glass is: beer (<3.5%) 50 cl; beer (>3.5%) 33 cl; wine (8%–15%) 12–15 cl; wine (15%–22%) 8 cl; or liquor 4 cl. 206 y. skogsdal et al. variables and asymmetrically distributed continuous variables. the difference in the response rate between the groups was tested using mantel–haenszel chi-square test, adjusting for the same question in the first questionnaire. the association between the intervention and the participants’ responses was also presented using relative risk (rr), with a corresponding 95% confidence interval (ci). spss statistics version 22 (ibm corp., armonk, ny, usa) was used in all statistical analyses. in all of the analyses, twosided p values <0.05 were considered statistically significant. ethical approval the regional ethical review board in uppsala, sweden approved the study (dnr 2012/101). the study was registered in isrctn 32759. results in total, after exclusions, 1,946 women answered q1 and 1,198 q2 (cg, n ¼ 606; and ig, n ¼ 592), which is a response rate of 62%. the internal response rate was high for most questions in both questionnaires (95.5%–100%). non-responders to q2 had lower education, and a greater proportion were smokers, users of snuff, workers, and born abroad. they also had different experiences of pregnancies (table 1). there were more women with overweight and more smokers in the ig. there were no other differences between the ig and the cg regarding the background characteristics (table 2). fertility knowledge at baseline, knowledge about fertility was low, and 64% (756/1,174) of the participants thought that the probability of a 25-year-old woman becoming pregnant if she had unprotected intercourse with a man of the same age was 60% or more (the correct answer is 30%–35%). a total of 16.7% (195/1,165) answered correctly that the fecundity of an ovum is around 1 day, and 27% (314/1,165) thought it was 5 days or more. the responses to when a woman’s fertility markedly begins to decline varied between 1 and 65 years, with a mean of 35.5 years (the correct answer was around 35 years with individual fluctuations). the chance of giving birth after one attempt of in vitro fertilization is 25%–30%, and 22.3% (256/1,144) answered correctly, while 51.9% (594/1,144) thought the chance was 50% or more. after the intervention, women in the ig increased their knowledge on all questions about fertility (p < 0.05; table 3). awareness of preconception factors at baseline, to quit smoking before a pregnancy was stated as being the most important. importance of taking folic acid was unknown for 28%, and nearly 30% thought it was neither important nor unimportant or rather/very unimportant. more parous (69%) than nulliparous (43%) stated that taking folic acid was very or fairly important at baseline (table 4). after the intervention a larger proportion of women in the ig thought that it was more important to make lifestyle changes before a pregnancy (table 4). adjustment for bmi or smoking did not change the results. more women in the ig planned to make lifestyle changes after the intervention, especially those with lower education (table 5). when the question was adjusted for bmi or smoking, no noticeable changes were found in the rrs and corresponding cis. table 2. demographic background of study population answering q2. q2 intervention q2 control p age, median year (interquartile range) 25 (22, 31) 26 (23, 31) 0.518 bmi, n (%) underweight (<18.5) 12 (2.1) 14 (2.4) 0.037 normal weight (18.5–24.99) 359 (62.7) 404 (68.4) overweight (25–29.99) 140 (24.4) 119 (20.1) obese class i (30–34.99) 44 (7.7) 36 (6.1) obese class ii (>35) 18 (3.1) 18 (3.0) education, n (%) non-completed education (<9 years) 3 (0.5) 1 (0.2) 0.088 elementary school (9 years) 16 (2.7) 17 (2.8) high school (12 years) 349 (59.0) 329 (54.3) professional education (non-academic) 47 (7.9) 52 (8.6) college/university 177 (29.9) 207 (34.2) main occupation, n (%) working 315 (53.2) 329 (54.4) 0.882 student 184 (31.1) 177 (29.3) parental leave 60 (10.1) 63 (10.4) unemployed 13 (2.2) 19 (3.1) sick leave 15 (2.5) 13 (2.1) other 5 (0.8) 4 (0.7) country of birth, n (%) sweden 557 (94.7) 572 (94.5) 0.291 other nordic country 1 (0.2) 5 (0.8) other european country 11 (1.9) 14 (2.3) outside europe 19 (3.2) 14 (2.8) sexual orientation, n (%) heterosexual 656 (95.6) 570 (94.5) 0.719 bisexual 22 (3.7) 25 (4.1) homosexual 0 (0.0) 1 (0.2) do not know/unsure 3 (0.5) 6 (1.0) other 1 (0.2) 1 (0.2) stable relationship, n (%) 453 (76.8) 474 (78.6) 0.448 reproduction, n (%) had tried to get pregnant 209 (35.3) 203 (33.7) 0.551 had been pregnant 255 (43.1) 257 (42.4) 0.796 had given birth 208 (81.9) 205 (81.0) 0.803 experience of abortion 65 (25.7) 70 (27.7) 0.615 experience of miscarriage 43 (17) 35 (13.8) 0.325 smoking, n (%) smoking daily 54 (9.2) 48 (8.0) 0.034 smoking, but not daily 65 (11.0) 43 (7.2) former smoker 138 (23.4) 138 (23.0) never smoked 332 (56.4) 370 (61.8) swedish snuff, n (%) snuff daily 30 (5.1) 35 (5.8) 0.881 use snuff, but not daily 20 (3.4) 27 (4.5) former user of snuff 53 (9.0) 40 (6.6) never used snuff 115 (82.4) 113 (83.1) drinking alcohol, 4 standard glasses or more at the same time, n (%)a daily 1 (0.2) 0 (0) 0.416 once/week 28 (4.8) 26 (4.3) once/month 133 (22.7) 157 (26.0) less than once/month 310 (52.8) 308 (51.0) never 115 (19.6) 113 (18.7) aone standard glass is: beer (<3.5%) 50 cl; beer (>3.5%) 33 cl; wine (8%–15%) 12–15 cl; wine (15%–22%) 8 cl; or liquor 4 cl. upsala journal of medical sciences 207 contraception there were no differences between the ig and the cg on the use of contraception after the intervention (table 6). in total, 79% (n ¼ 920) were very or fairly satisfied with the current contraceptive method, and those who used the combined hormonal contraceptive pill were the most satisfied (87%, n ¼ 217, p > 0.001). in total, 101 women did not use any contraception when they answered q2, and 57% (n ¼ 41 out of 72 who answered the question) were very or fairly satisfied with no contraceptives, 32% (n ¼ 23) were neither satisfied nor dissatisfied, and 11.1% (n ¼ 8) were fairly or very dissatisfied. among these 101 women, 69 women had talked about fertility with a relative, 38 had talked about possibilities of becoming pregnant when it was time, 15 planned their pregnancies or were already pregnant, and 15 were worried about not getting pregnant. seven had discussed age and fertility, five preconception health, five how to avoid pregnancy, and for two participants it was unclear what they had discussed. age desired for the first and last child there was no difference between the ig and the cg regarding the age at which the women wanted their first (28 years) and last (33.5 years) child, nor regarding how many children they wanted; on average, the ig and the cg wanted 2.4. pregnancy plan/unplanned pregnancy of those who answered q2, 82% (980/1,195) stated that they had great influence on if and when they will become pregnant. of those who wished to have children/more children, 65% (516/794) thought it was important to become pregnant table 3. percentage of women in the intervention group and the control group who responded correctly or almost correctly to questions about fertility. before after intervention control intervention control n (%) n (%) n (%) n (%) adjusted rr 95% ci how likely is it for a 25-year-old woman to become pregnant if she has unprotected intercourse with a man at the same age at the time of ovulation? (correct or almost correct 25%–40%) 69 (11.9) 66 (11.1) 273 (46.5) 121 (20.1) 2.297 1.915–2.755 for how long is it possible for an ovum to be fertilized? (correct or almost correct 1–2 days) 182 (33.7) 199 (36.0) 306 (54.3) 244 (42.4) 1.245 1.098–1.412 at what age does a woman’s ability to become pregnant begin to decline? (correct or almost correct 33–37 years) 203 (34.6) 181 (30.2) 300 (50.8) 199 (33.2) 1.541 1.341–1.771 what are the chances of giving birth to a child after in vitro fertilization per attempt? (correct or almost correct 20%–35%) 177 (31.5) 182 (31.3) 306 (52.9) 228 (39.0) 1.354 1.190–1.540 rr was adjusted for the same question before the intervention. table 4. women’s awareness of preconception health before and after the intervention. before after intervention control intervention control n (%) n (%) n (%) n (%) p how important is it to stop smoking before pregnancy? very and fairly important 557 (94.2) 575 (95.4) 573 (97.6) 574 (94.9) 0.019 very and fairly unimportant, neither important/unimportant 26 (4.4) 24 (4.0) 14 (2.4) 27 (4.5) do not know 8 (1.4) 4 (0.7) 0 (0) 4 (0.7) how important is it to stop using snuff before pregnancy? very and fairly important 525 (88.8) 552 (91.5) 548 (93.4) 530 (88.0) 0.004 very and fairly unimportant, neither important/unimportant 50 (8.5) 46 (7.6) 33 (5.6) 55 (9.1) do not know 16 (2.7) 5 (0.8) 6 (1.0) 17 (2.8) how important is it to start taking folic acid before pregnancy? very and fairly important 261 (44.2) 255 (42.4) 458 (78) 330 (54.5) <0.001 very and fairly unimportant, neither important/unimportant 168 (28.4) 180 (29.9) 73 (12.4) 165 (27.3) do not know 162 (27.4) 167 (27.7) 56 (9.5) 110 (18.2) how important is it to be of normal weight before pregnancy? very and fairly important 468 (79.2) 460 (76.2) 533 (90.8) 510 (84.7) 0.003 very and fairly unimportant, neither important/unimportant 10 (17.4) 114 (18.9) 50.(8.5) 80 (13.3) do not know 20 (3.4) 30 (5.0) 4 (0.7) 12 (2.0) how important is it to refrain from alcohol before pregnancy? very and fairly important 468 (79.2) 460 (76.2) 507 (86.6) 441 (73.0) <0.001 very and fairly unimportant, neither important/unimportant 103 (17.4 114 (18.9) 69 (11.8) 147 (24.3) do not know 20 (3.4) 20 (3.4) 11 (1.9) 16 (2.6) how important is it to start taking vitamin c? very and fairly important 133 (22.5) 127 (21.1) 261 (44.8) 164 (27.1) <0.001 very and fairly unimportant, neither important/unimportant 311 (54.6) 319 (53.1) 258 (44.3) 328 (54.2) do not know 135 (22.9) 155 (25.8) 64 (11.0) 113 (18.7) the answer options in each question was ‘very important’, ‘fairly important’, ‘neither important/unimportant’, ‘fairly unimportant’, ‘very unimportant’, and ‘do not know’. in this table, the response options are congregated. p value was adjusted for the same question before the intervention. 208 y. skogsdal et al. according to their own time plans. in response to questions regarding the hypothetical case, ‘if they were to become pregnant today’, 46.5% (557/1,198) responded that they would continue the pregnancy, 26% (311/1,198) would have an induced abortion, and 27.5% (329/1,198) were unsure. among those who did not want children/more children (229/ 1,198), 32.3% (n ¼ 74) would continue an unplanned pregnancy today, 34.5% (n ¼ 79) would have an induced abortion, and 33.2% (n ¼ 76) were unsure. women who were considering induced abortion if they became pregnant did not have a different pattern of contraceptive use compared with others. experience of the intervention the ig answered three additional questions to evaluate the women’s experiences of the intervention. three out of four (437/585) had read the brochure. also, 59.2% (342/577) considered the rlpc as being very or fairly positive, 37.6% (217/ 577) thought it was neither positive nor negative, 3.1% (18/ 577) thought it was fairly negative, and no one thought it was very negative. three out of four women (76%, 443/58) stated that the rlpc should be part of the routine during visits to midwives or other healthcare providers, while 18.2% (106/583) were unsure, and 5.7% (33/583) were negative. spillover effect to evaluate a possible spillover effect of answering the questionnaires, the women in the cg were asked questions in the follow-up questionnaire about whether they had discussed topics related to our questions with the midwife during the counseling. half of the women, 50% (307/607), had discussed questions regarding having children in the future, 16% (98/ 607) had discussed age and fertility. a further 17% (102/607) had discussed the probability of becoming pregnant as a result of unprotected intercourse. there were also 10% who had discussed lifestyle factors that can affect fertility and the chance of a healthy pregnancy. another 10% had discussed the lifespan of the ovum and the sperm, and 3% had discussed ivf. discussion we have shown that women’s knowledge about fertility and preconception health was low at baseline; however, the intervention increased their knowledge about fertility and the awareness of factors that affect preconception health, i.e. to stop using tobacco, to refrain from drinking alcohol, to be of normal weight, and to supplement with folic acid before a pregnancy. we found no differences between the ig and the cg with regard to their future reproductive planning and no differences in their choice of contraceptives. the majority of women appreciated the intervention. we did not expect the ig to achieve 100% knowledge because some women were young and did not plan to have children for many years, while others did not want more children, which may have made them less interested in issues on fertility and preconception health. we also found a ‘spillover effect’ in the cg, suggesting that the study table 5. imagine that you want to get pregnant. would you take any action before trying to conceive to increase the chances of having a healthy pregnancy and healthy child? women who answered ‘yes’ before and after the intervention. before after intervention control intervention control n (%) n (%) n (%) n (%) adjusted rr 95% ci all women 335 (57.4) 346 (58.6) 450 (77.6) 407 (67.9) 1.140 1.063–1.223 overweight/obesity 116 (58.3) 101 (59.8) 155 (78.7) 111 (64.5) 1.227 1.073–1.403 smokers 38 (70.4) 34 (70.8) 44 (84.6) 34 (72.3) 1.170 0.947–1.445 using snuff 27 (90.0) 29 (82.9) 28 (96.6) 25 (71.4) 1.352 1.084–1.685 binge drinkinga 99 (62.7) 123 (68.0) 136 (86.6) 136 (75.6) 1.147 1.037–1.269 college/university 98 (55.4) 122 (60.4) 141 (80.1) 136 (67.3) 1.187 1.051–1.340 elementary school (9 years) 11 (68.8) 9 (52.9) 14 (93.3) 10 (58.8) 1.587 1.042–2.415 experience of abortion 44 (67.7) 49 (71.0) 51 (81.0) 46 (65.7) 1.214 0.989–1.491 had given birth 100 (48.8) 103 (52.0) 141 (69.5) 126 (61.8) 1.121 0.973–1.291 rr was adjusted for the same question before the intervention. table 6. use of contraceptives at the women’s latest intercourse before and after the intervention. before after ig cg ig cg n (%) n (%) n (%) n (%) p combined hormonal contraceptive pill 240 (40.7) 234 (38.9) 241 (40.7) 239 (39.4) 0.654 condom 113 (19.2) 100 (16.6) 68 (11.5) 59 (9.7) 0.325 long-acting reversible contraception 128 (21.6) 122 (20.1) 167 (28.2) 159 (26.2) 0.443 hormonal intrauterine device 39 (6.6) 35 (5.8) 88 (14.9) 78 (12.9) 0.318 copper intrauterine device 40 (6.8) 40 (6.7) 37 (6.3) 39 (6.4) 0.895 progestin implant 49 (8.3) 47 (7.8) 42 (7.1) 42 (6.9) 0.912 combined hormonal contraceptive ring 34 (5.8) 41 (6.8) 41 (6.9) 53 (8.7) 0.241 progestin-only pill 26 (4.4) 39 (6.5) 33 (5.6) 40 (6.6) 0.458 progestin-only injection 7 (1.2) 3 (0.5) 13 (2.2) 6 (1.0) 0.095 other 5 (0.8) 11 (1.8) 11 (1.9) 17 (2.8) 0.278 no method 67 (11.4) 69 (11.4) 45 (7.6) 56 (9.2) 0.307 p values are analyzed after the intervention. upsala journal of medical sciences 209 questions about fertility and preconception health may have stimulated women to reflect on these topics during and after the counseling. based on the questions posed to the women in the cg, we know that some of them also discussed these topics with their midwives. all midwives working in the outpatient clinics in the study region participated in the data collection, which is a strength because they could recruit participants from different social groups, different education levels, and different occupations. we believe that our results can be generalized to other regions in sweden, with a similar socioeconomic profile and mean childbirth age. the response rate for q2 was 62%, which is satisfactory, as it has gradually become more difficult to recruit people for studies. in public health surveys, the response rate has decreased from 60.8% in 2004 to 47% in 2016, and the response rate was particularly low among young adults (20). in a nationwide study about the use of contraception in 2015, the response rate was 25.3% (21). when comparing attendance rates, we are satisfied with our participation rate. one limitation is that only swedish-speaking women were included. the intervention was partly based on direct communication between the midwife and the client, and we did not have financial resources to include interpreters or to print the booklet in other languages. in a previous survey at antenatal clinics, great efforts were made to also include immigrant women, but this was very difficult in spite of having translated questionnaires and interpreters (19). another limitation was that we included women aged 20–40 years, since we believe that reproductive life planning is of more interest in this specific age range. the non-responders to the follow-up had lower education, a greater proportion were smokers, users of snuff, workers, and born abroad. they had more experience of pregnancies and abortion, but less experience of giving birth. there were more smokers and women with overweight/obesity in the ig; however, we have adjusted for these factors, and the results did not change. there was notably a low awareness about the impact of lifestyle factors on reproductive health. the women were largely aware that smoking before a pregnancy is unhealthy, but they did not have the same awareness about swedish snuff, which has similar risks as smoking (22–24). the awareness of the importance of being of normal weight was low among women in our study. in sweden, 40% are overweight or obese in early pregnancy, and as obesity is increasing in women of fertile ages, prevention of obesity must start years before a pregnancy in order to optimize future pregnancy outcomes (1,25–27). it was surprising that one-quarter of the women were drinking four glasses or more either once a month or more often, a drinking pattern that women need to recognize as unfavorable and not recommended if planning pregnancy. the knowledge to refrain from drinking alcohol before a pregnancy was also low. the facts that alcohol can cause a miscarriage (28,29) and that fetal alcohol spectrum disorders (fasds) can occur even in moderate alcohol consumption or binge drinking are important information for women (30–32). knowledge about taking folic acid before a pregnancy was lower than expected among the women in our study. only 30% were aware of the importance of folic acid supplementation when planning a pregnancy, and our results are similar to findings in the study on female university students (11). however, parous women were more aware of this factor than nulliparous, albeit a lower awareness than expected. parous women have most likely been informed in their earlier pregnancy about the importance of folic acid, but perhaps they did not fully understand that they should start before the pregnancy. our results show that the swedish national recommendations about taking folic acid have not reached women of fertile age, and this finding is similar to results in other studies (33,34). many countries, for example canada and the usa, have fortified flour with folic acid. the swedish agency for health technology assessment and assessment of social services (sbu) carried out a systematic review in 2007 (6) of the scientific evidence on the benefits and risks of fortifying the flour, and they concluded that there was not enough evidence to fortify flour. their recommendation was that women should be informed about the importance of using folic acid before and during early pregnancy—a strategy that clearly has not been successful, based on the results in this study. healthcare providers have an important role in informing about folic acid supplementation for women of fertile age. to our knowledge, there have not been any public campaigns in sweden to increase knowledge about folic acid, as they have had in the netherlands, but despite their national campaigns the knowledge still remains low (35,36). what kind of strategy or intervention is reasonable to implement in healthcare for improving knowledge is unclear; clearly, improved efforts need to be made both in schools and healthcare in the future. the first step in making lifestyle changes is to understand what changes are needed and why. our results show a clear need for lifestyle changes prior to pregnancy in a large proportion of women. the challenge is how these changes in lifestyle could be implemented. a control question was used in the questionnaire: ‘is it important to supplement with vitamin c before pregnancy?’ even though there is no recommendation for taking vitamin c. however, a large proportion of women in the ig stated at follow-up that it was important to add vitamin c; perhaps, they mixed this up with folic acid. it could also be that they wanted to answer correctly and, therefore, it might be a weakness of the study. there was no difference in the choices of contraceptives between the ig and the cg, similar with previous studies (10,11,37). interestingly, 7.6%–9.2% (n ¼ 101) in our study did not use any contraceptive at their latest intercourse, despite having received contraceptive counseling two months earlier. among these women, 11.1% answered that they were dissatisfied with their contraceptives and could therefore have stopped. another reason was that some of them had changed their minds and wanted to become pregnant or were already pregnant. low compliance can also be related to no sexual activity or fear of hormones, side effects, or not finding any suitable method (21,38). surprisingly many 210 y. skogsdal et al. women would continue an unplanned pregnancy, compared to the reported unintended pregnancies worldwide (39). because eight out of ten women thought that they can influence when they will become pregnant, and two-thirds answered that it was important to become pregnant at the time they had planned, we assume that many women want to plan their pregnancies. it has been suggested that healthcare providers should find ways of identifying women who are planning a pregnancy and make use of existing platforms for delivery of preconception care (3). we suggest that contraceptive counseling is an opportunity to talk about preconception health, since three out of four women stated that the rlpc should be part of the routine during visits to midwives. in our study, the midwives gave the women in the ig a specially designed brochure with information about fertility and preconception health, and three out of four women had read this brochure. this evidence-based information has been translated into english and some other languages and is now available on a mobile-friendly website (www.reproduktivlivsplan.se) (40). the follow-up in our study was conducted after two months, and we have no information on whether the increased knowledge in the ig will lead to a behavioral change when women are planning a pregnancy in the future. the challenge in healthcare is to find prevention strategies that will have long-lasting effects (41). to study the long-term effects of the rlpc intervention, we plan to conduct a follow-up study and evaluate future pregnancy outcomes by linking the study cohort with existing national pregnancy and health registers in sweden, when the number of future pregnancies in the cohort is sufficiently large. conclusion the knowledge of fertility and awareness of reproductive health were low at baseline but increased among women in the ig. most women appreciated the rlpc that can be recommended in contraceptive counseling. however, long-term evaluation of the effects of the intervention on future pregnancies is needed. acknowledgements thanks to all the midwives at the participating clinics for their help with recruitment and with the intervention. also, thanks to wilma rutfj€all, jennifer drevin, jenny stern, marie lid�en, and elina backman for help with data entry. declaration of interest professor tanja tyd�en participated to one meeting of bayer’s advisory board in sweden in 2017. yvonne skogsdal was an unpaid speaker for nordic employees at the bayer winter conference in 2018. there are no other conflicts of interest. funding this work was supported by research, development and education (alf) from uppsala county council [grant no. as 2014-0831], region €orebro county [grant no. oll-640211, oll-734371], and bayer ab. none of these parties had any influence on the questions asked, analysis, or interpretation of results. notes on contributors yvonne skogsdal is a coordinating midwife in antenatal care at region €orebro l€an, and phd student at €orebro university. helena fadl, phd, senior consultant obstetrics and gynecology, department of obstetrics and gynecology, faculty of medicine and health, €orebro university. yang cao, phd, is associate professor of biostatistics at clinical epidemiology and biostatistics, school of medical sciences, €orebro university. jan karlsson, phd, is associate professor at university health care research center, faculty of medicine and health, €orebro university. tanja tyd�en, phd, is a registered nurse midwife, and senior professor at uppsala university. orcid yvonne skogsdal http://orcid.org/0000-0003-2412-4676 helena fadl http://orcid.org/0000-0002-2691-7525 yang cao http://orcid.org/0000-0002-3552-9153 jan karlsson http://orcid.org/0000-0002-2559-5456 tanja tyd�en http://orcid.org/0000-0002-2172-6527 references 1. stephenson j, heslehurst n, hall j, schoenaker d, hutchinson j, cade je, et al. before the beginning: nutrition and lifestyle in the preconception period and its importance for future health. lancet. 2018;391:1830–41. 2. fleming tp, watkins aj, velazquez ma, 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petersen kb, andersen an, pinborg a, et al. status one year after fertility assessment and counselling in women of reproductive age – a qualitative study. ups j med sci. 2018;123:264–70. 212 y. skogsdal et al. https://www.folkhalsomyndigheten.se/folkhalsorapportering-statistik/statistikdatabaser-och-visualisering/nationella-folkhalsoenkaten/ https://www.folkhalsomyndigheten.se/folkhalsorapportering-statistik/statistikdatabaser-och-visualisering/nationella-folkhalsoenkaten/ https://www.folkhalsomyndigheten.se/folkhalsorapportering-statistik/statistikdatabaser-och-visualisering/nationella-folkhalsoenkaten/ abstract introduction material and methods sample size setting randomization procedure intervention questionnaires statistical analysis ethical approval results fertility knowledge awareness of preconception factors contraception age desired for the first and last child pregnancy plan/unplanned pregnancy experience of the intervention spillover effect discussion conclusion acknowledgements declaration of interest references gonadotropin-releasing hormone agonist for ovulation trigger – ohss prevention and use of modified luteal phase support for fresh embryo transfer review article gonadotropin-releasing hormone agonist for ovulation trigger – ohss prevention and use of modified luteal phase support for fresh embryo transfer juan carlos castilloa, thor haahrb,c, mar�ıa mart�ınez-moyaa and peter humaidanb,c ainstituto bernabeu, alicante, spain; bdepartment of clinical medicine, aarhus university, aarhus, denmark; cthe fertility clinic skive, skive regional hospital, skive, denmark abstract the introduction of gonadotrophin-releasing hormone agonist (gnrha) trigger greatly impacted modern ivf treatment. patients at low risk of ovarian hyperstimulation syndrome (ohss) development, undergoing fresh embryo transfer and gnrha trigger can be offered a virtually ohss-free treatment with non-inferior reproductive outcomes by using a modified luteal phase support in terms of small boluses of human chorionic gonadotrophin (hcg), daily recombinant luteinizing hormone lh (rlh) or gnrha. in the ohss risk patient, gnrha trigger can safely be performed, followed by a ‘freeze-all’ policy with a minimal risk of ohss development and high live birth rates in the subsequent frozen embryo transfer cycle. importantly, gnrha trigger opened the ‘black box’ of the luteal phase, promoting research in the most optimal steroid levels during the luteal phase. gnrha trigger allows highdose gonadotropin stimulation to achieve the optimal number of oocytes and embryos needed to ensure the highest chance of live birth. this review thoroughly discusses how the gnrha trigger concept adds safety and efficacy to modern ivf in terms of ohss prevention. furthermore, the optimal luteal phase management after gnrha trigger in fresh embryo transfer cycles is discussed. article history received 30 october 2019 revised 11 february 2020 accepted 26 february 2020 keywords gnrha trigger; hcg; ivf; ovarian hyperstimulation syndrome; ovulation trigger introduction traditionally a bolus of human chorionic gonadotrophin (hcg) has been the gold standard for ovulation induction and final oocyte maturation in assisted reproductive technology (art) cycles as a surrogate for the natural mid-cycle luteinizing hormone (lh) surge. however, the increased glycosylation of hcg results in a prolonged bioactivity which in combination with the sustained luteotropic activity may induce ovarian hyperstimulation syndrome (ohss), one of the most serious complications of controlled ovarian stimulation (cos) (1). additionally, studies have reported adverse effects of hcg in terms of reduced endometrial receptivity and a negative impact on oocyte as well as embryo quality (2–4). when gonadotrophin-releasing hormone (gnrh) antagonist protocols were introduced for the prevention of a premature lh surge (5) it became possible to trigger final oocyte maturation and ovulation with a single bolus of a gnrh agonist (gnrha) as an alternative to hcg. induction of final oocyte maturation with a bolus of gnrha in patients undergoing ovarian stimulation for ivf could be considered to be more physiologic because the elicited surge mimics the natural cycle surge of gonadotropins (figure 1). however, when compared to the natural cycle, the total amount of gonadotrophins released during the gnrha-triggered surge is significantly reduced, but a possible advantage of gnrha trigger over hcg trigger is the simultaneous induction of an fsh (follicle-stimulating hormone) surge (8). the exact role of the mid-cycle fsh surge in the natural cycle is not fully understood, but available evidence suggests that fsh promotes oocyte nuclear maturation, i.e. resumption of meiosis (9,10), cumulus expansion, and the induction of lh receptors on granulosa cells to promote corpus luteum (cl) formation (11,12). with this in mind, a bolus of gnrha induces a more physiologic final oocyte maturation; however, this comes at the expense of a deficient luteal phase due to the short duration of the induced lh/fsh peak (figure 1). it also prevents the secretion of vasoactive substances, mainly vegf (vascular endothelial growth factor), from the corpora lutea (13–15), thereby acting as a luteolytic agent. luteolysis induced by gnrha is the key to prevention of ohss (16). avoiding ohss in risk patients with the use of gnrha trigger the syndrome ohss was already described in 1941 (17). in spontaneous pregnancy, ohss is an extremely rare event (18). hence, ohss is an iatrogenic complication in almost all cases and occurs almost exclusively in the presence of sustained lh activity as seen after hcg trigger in art cycles. furthermore, the severity of ohss is proportional to the dose of hcg administered and the number of corpora lutea obtained after trigger. ohss occurs less frequently after mild contact juan carlos castillo jcastillo@institutobernabeu.com instituto bernabeu, av. albufereta 31, alicante, 03016, spain � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 2, 131–137 https://doi.org/10.1080/03009734.2020.1736696 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1736696&domain=pdf&date_stamp=2020-05-21 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1736696 http://www.tandfonline.com ovarian stimulation using clomiphene citrate or low-dose gonadotrophins for the development of a few follicles in intrauterine insemination (iui) cycles (18,19). it has, however, previously been reported to be as high as 30% in polycystic ovarian syndrome (pcos) patients submitted to ivf (13). the most significant benefit of gnrha trigger is its ability to induce a quick and reversible luteolysis and, thus, reduce the risk of ohss development. in gnrh antagonist cycles, gnrha trigger has been utilized to induce the final stage of follicular maturation while reducing the risk of ohss. this protocol works because gnrh agonists have a greater affinity for the gnrh receptor than gnrh antagonists have. thus, a bolus of gnrha can displace the gnrh antagonist from the receptor, resulting in a surge of lh as well as fsh. the lower mean lh concentrations and decreased lh pulse amplitude and activity over the luteinized granulosa/theca cells in addition to an upregulation of the apoptosis of granulosa cells induce the process of luteolysis early in the luteal phase, which seems to be key factors for ohss prevention after gnrha trigger (20,21). interestingly, in a small pilot study, it was reported that converting ohss risk patients during stimulation from a long gnrha protocol to a gnrh antagonist protocol and subsequent gnrha trigger is a plausible rescue option in patients at high risk of ohss development (22). currently, however, as the benefits and risks of this rescue strategy are not quite clear, most clinicians probably opt for cycle segmentation and frozen–thawed embryo transfer in a subsequent cycle. from a clinical point of view, the most optimal strategy to prevent ohss starts with the identification of ohss risk patients in order to adapt the ovarian stimulation regimen accordingly and subsequently plan the trigger agent. in patients at risk of ohss, some clinicians might opt for elective frozen embryo transfer (efet). it has, however, been proven, during a series of randomized controlled trials (rcts), that gnrha trigger enables a fresh transfer if modifications of the luteal phase support (lps) are performed, and, importantly, the reproductive outcome is non-inferior to hcg trigger (4,23–26). however, only gnrha trigger and efet (segmentation) will result in a virtually ‘ohss-free clinic’ (27). nevertheless, on rare occasions, ohss can be encountered even when using gnrha trigger and efet (28). gnrha trigger and segmentation (freeze-all) oocyte/embryo freezing, so-called ‘freeze-all’, segmentation or efet, was recently proposed for all gnrha-triggered ivf cycles (27,29) and in particular so for women at risk of ohss (30), avoiding early as well as late-onset ohss. the segmented treatment approach involves: (1) controlled ovarian hyperstimulation (coh) using a gnrha trigger; (2) vitrification of oocytes/embryos; and (3) embryo transfer in a subsequent, natural or artificial cycle. by segmenting the treatment into these steps, the concern about specific and adequate luteal support after gnrha triggering disappears. an additional benefit of postponing embryo transfer is avoiding embryo exposure to non-physiologic, elevated circulating steroid levels observed in fresh transfer ivf cycles. in recent years, the pregnancy and live birth rates after the transfer of frozen embryos have improved, most certainly as a result of the use of vitrification for embryo cryopreservation (31). a recent meta-analysis comparing fresh transfer to fet supports the use of efet in hyper-responder patients and patients undergoing pgt-a (preimplantation genetic testing for aneuploidy) (32); however, from a clinical point of view, it is warranted to make a study comparing reproductive outcomes in fresh and efet cycles triggered with gnrha in polycystic ovary (pco) patients, who are at high risk of ohss. unfortunately, such a study is not available, and results of a multicenter, randomized clinical trial comparing fresh transfer with efet in hcg-triggered cycles among infertile women with pcos showed that efet was associated with a higher live birth rate than fresh embryo transfer (49.3% versus 42.0%). this difference was explained by a lower early pregnancy loss rate in the efet group (22.0% versus 32.7%). however, it is also worth highlighting that a higher incidence of preeclampsia was seen in the efet group compared with the fresh transfer group (4.4% versus 1.4%) (33). thus, some concerns and drawbacks regarding the freeze-all approach need to be weighed against the potential benefits. a frozen cycle involves higher economical costs and additional emotional/physical burden associated with deferring the programmed fet, and the additional endometrial preparation protocol (ultrasound controls, medication, office visits). importantly, additional manipulations (freezing/ figure 1. comparison of ovulation triggers. schematic graphic showing lh activity of different types of trigger agents when compared with natural mid-cycle surge. gnrha: gonadotrophin-releasing hormone agonist; hcg: human chorionic gonadotropin. adapted from hoff et al. (6) and casper (7). 132 j. c. castillo et al. thawing) at early embryonic stages might induce epigenetic changes only visible during adulthood (34), and the oestrogen contents of the artificial cycle might have an impact on placentation. finally, despite the freeze-all policy clearly preventing the development of late-onset ohss, it does not completely prevent severe early ohss, as recently reported (35). although there is a lack of randomized clinical trials comparing gnrha-triggered fresh and efet cycles, evidence from observational studies suggests that gnrha triggering combined with oocyte/embryo vitrification and embryo transfer in a subsequent cycle provides patients at high risk of developing ohss a safe and efficient alternative. in this high-risk population, a freeze-only approach to minimize the risk of ohss may outweigh the known and unknown risks and the financial costs involved with the efet strategy. nonetheless, results from large long-term follow-up trials in children conceived as a result of frozen–thawed embryo transfer and the exploration of possible obstetric complications are mandatory before the widespread adoption of a ‘freeze-all ‘policy can be recommended. gnrh trigger and the optimal modified luteal phase support (mlps) during fresh embryo transfer luteal phase insufficiency, resulting in an unacceptably high early pregnancy loss rate, despite the use of a standard lps was seen during the early studies of gnrha trigger (36,37). following these first disappointing reports, several studies explored how to most optimally rescue the luteal phase after gnrha trigger (8). current evidence supports that gnrha trigger and fresh transfer followed by a modified lps (mlps) policy results in reproductive outcomes comparable to those of hcg trigger – and notably with a reduction in ohss (4). this introduced the idea of individually tailoring the luteal phase after gnrha trigger according to the response to ovarian stimulation and luteal steroid levels (25,38–40). mlps: a single low-dose bolus of hcg as noted previously, while the lower lh activity commencing early in the luteal phase was a beneficial key factor for ohss prevention, it was also responsible for the disappointing lower reproductive outcomes in gnrha-triggered compared with hcg-triggered cycles. thus, the question was: how can lh activity be introduced after gnrha triggering to most optimally restore the luteal phase? based on two small pilot studies in iui patients (41,42), the concept of an early lps with a single low-dose hcg (1500 iu) on the day of ovum pick-up (opu) combined with a standard lps (vaginal progesterone and oral oestradiol) was investigated (38). the small bolus of hcg clearly rescued the luteal phase after gnrha triggering, resulting in a satisfying reproductive outcome. the protocol was subsequently investigated in a large rct showing a non-significant difference in delivery rates between gnrha-triggered cycles supplemented with a single bolus of hcg versus 10,000 iu hcg for trigger (24). importantly, no ohss was found in the gnrha group versus 2% in the 10,000 iu hcg group, even though more than one-third of the patients in each group had �14 follicles �11 mm on the day of triggering. to further explore whether this procedure was safe for women at risk of ohss, a total of 118 patients at risk of ohss were randomized to either hcg trigger or the modified gnrha trigger protocol (25). of note, women with more than 25 follicles were excluded from the trial. no ohss was reported after gnrha trigger using this upper follicular limit compared with 3% of women experiencing ohss after hcg trigger. additional retrospective studies also explored the use of the same protocol in at-risk patients, resulting in similar outcomes: high pregnancy rates and a very low ohss incidence (0.72%�1.4%). it is worthy of note, however, that these retrospective analyses included cycles with no upper limit in terms of number of follicles present on the trigger day (43,44). in contrast, one retrospective cohort study included 23 women at high risk of ohss development who underwent gnrha trigger and also received a bolus of 1500 hcg on the day of oocyte retrieval as previously reported (24). in this ohss high-risk group, the severe ohss risk was reported to be high, 26% (6/23), which seems conceivable as no upper limit of follicles for fresh transfer at trigger was applied and as many as 50–65 oocytes were retrieved in some patients (45). a recent trial investigated the advantage of adding gnrha (triptorelin 0.1 mg s.c.) on day 6 after opu (opu þ 6) in an rct compared with gnrha trigger and modified lps with 1500 iu hcg at opu (46). the intervention with gnrha at opu þ 6 showed a small but non-significant increase in live birth rates (or 1.3; 95% ci 0.8–2.0). based on available evidence, gnrha trigger followed by a single low-dose bolus of hcg and fresh transfer prevents or significantly reduces the risk of ohss while providing high pregnancy rates. however, it is crucial to have an upper cutoff limit of follicles before embarking on this strategy. based on our previous studies and daily clinical experience we suggest a cut-off of >25 follicles �11 mm (47). mlps: recombinant lh (rlh) theoretically, the addition of recombinant lh (rlh) will overcome the defective luteal endocrine/endometrial environment and restore luteal function after gnrha trigger. due to the shorter half-life of rlh compared with hcg, the risk of ohss may be further reduced. papanikolaou et al. explored this interesting concept in a pilot study, applying repeated doses of rlh as a luteal supplementation (six alternate doses of 300 iu rlh, starting on the day of oocyte retrieval in addition to vaginally administered micronized progesterone) post gnrha trigger and compared this concept to a standard hcg trigger and lps (48). similar implantation rates were achieved with the rlh luteal supplementation scheme compared with the standard luteal progesterone protocol (25.0% versus 26.7%, respectively). no cases of ohss were noticed in this group of normal-responder patients. thus, studies exploring rlh supplementation in patients at risk of ohss are lacking. at present, more extensive studies are warranted to draw robust conclusions regarding the cost/efficacy and upsala journal of medical sciences 133 dose of rlh for the most optimal luteal support after gnrha trigger, specifically in the at-risk population. it is noteworthy that the cost of rlh and patient convenience might hinder the use of rlh in a standard luteal support practice. mlps: exogenous intensive progesterone and oestradiol supplementation an early small study in ohss risk patients by babayof et al. (13) explored the concept of steroids-only luteal phase supplementation after gnrha trigger and also investigated markers of luteal phase function in order to identify patients in need of intense support after gnrha trigger. more recently, the approach was investigated in a rct, comparing gnrha trigger to hcg trigger, and using intensive lps and monitoring of serum steroid levels in the gnrha-triggered group (49). all patients received 50 mg intramuscular progesterone daily starting the evening after oocyte retrieval and continued until 10 weeks of gestation. in addition, patients received three 0.1-mg oestradiol (e2) transdermal patches every other day starting on the day after oocyte retrieval. serum e2 and progesterone were measured on the day of embryo transfer, 1 week after oocyte retrieval, and weekly thereafter. the dose of transdermal e2 patches was increased, if necessary, to a maximum of four 0.1-mg patches every other day, and/or the addition of oral micronized e2 to maintain a serum e2 level >200 pg/ml. the intramuscular progesterone dose was also increased, if necessary, to a maximum of 75 mg daily and/or the addition of micronized vaginal progesterone to maintain a serum progesterone level >20 ng/ml. an ongoing pregnancy rate of 53% and no ohss was reported in the gnrha trigger group compared with 34% (10/29) in the hcg trigger group. some groups, applying a similar exogenous intensive luteal support regimen, reported favourable results after fresh transfer in retrospective studies (44,50). however, others were not able to corroborate the initial findings (51). finally, the use of daily intramuscular injections of progesterone for up to 10 weeks and the perceived reduced patient-friendliness of this lps policy is a potential drawback for the widespread use of this approach in clinical practice. despite the potential limitations, but considering the encouraging results, the engmann group evaluated the factors predicting the probability of successful outcomes after gnrha trigger, reporting that a peak serum e2 � 4000 pg/ml and serum lh levels on the day of trigger are the most important predictive factors for success (52). the implantation (34.4% versus 25.3%; p ¼ 0.02) and clinical pregnancy rates (53.6% versus 38.1%; p ¼ 0.02) were significantly higher in patients with peak e2 levels �4000 pg/ml compared with those with peak e2 levels <4000 pg/ml. patients within these limits might also have higher lh levels during the luteal phase, leading to a hypothetically higher degree of cl rescue, higher progesterone and e levels, and subsequently higher implantation rates. the authors hypothesized that some form of low-dose lh-like activity might rescue cl function in patients with low peak serum e2 levels without significantly increasing the risk of ohss. following this, the authors suggested administering low-dose adjuvant hcg in women with peak serum e2 < 4000 pg/ml, in line with the proposed protocol by humaidan et al. (23–25,38). mlps: the dual trigger concept – concomitant use of gnrha and low-dose hcg shapiro et al. first reported the use of the so-called dual trigger (gnrha plus low-dose hcg) (53). in their retrospective analysis, patients with significant risk factors for ohss received a combination of 4 mg leuprolide acetate and hcg in a dose ranging from 1000 iu to 2500 iu for trigger, depending on body weight (mean dose 26.2 iu hcg/kg) and follicle number >25. the authors reported good pregnancy rates and no ohss cases, using this protocol. of note, this trial did not include a control group for comparison. in a subsequent larger study in a total of 182 women at risk of ohss who had individual dosing of the hcg bolus used for dual trigger (54), the same group reported that concomitant low-dose hcg with a gnrha trigger was associated with higher ongoing pregnancy and implantation rates and reduced pregnancy loss rates when compared with gnrha trigger only. moreover, a low ohss rate of 0.5% was reported. another retrospective analysis including a total of 102 ivf/icsi (intracytoplasmic sperm injection) cycles in ohss high-risk patients having peak serum e2 levels <4000 pg/ml reported that the use of dual trigger (gnrha and a fixed dose of 1000 iu hcg) resulted in a significantly higher live birth rate (52.9% versus 30.9%), implantation rate (41.9% versus 22.1%), and clinical pregnancy rate (58.8% versus 36.8%) compared with gnrha trigger only (55). in both groups, an intensive lps was used, consisting of 50 mg intramuscular progesterone daily and 0.3 mg transdermal e patches every other day, starting from the day after opu. additional oral e and intramuscular progesterone were used up to 10 weeks of gestation, maintaining levels above 200 pg/ml and 20 ng/ml, respectively. one mild ohss case was reported in the dual trigger group, whereas there was no ohss in the gnrha trigger group. the authors concluded that the dual trigger concept using a combination of gnrha and low-dose hcg in high responders with peak e2 < 4000 pg/ml improved live birth rates without increasing the risk of clinically significant ohss. taken together, data from observational analyses support the idea, that, taking into account oestradiol levels on the trigger day and luteal function markers, monitoring with appropriate dose adjustment seems important when deciding whether dual trigger or steroids only should be used to support the luteal phase of patients at risk of ohss development. this is in the absence of rcts evaluating the optimal timing and dosage of hcg to be given alongside gnrha trigger. 134 j. c. castillo et al. mlps: intranasal gonadotropin-releasing hormone agonist the idea of only supporting the luteal phase after gnrha with daily doses of intranasal gnrha was initially explored in 17 and 40 patients, respectively (56,57). more recently, the concept was tested in a retrospective study, including 46 ohss high-risk patients, using a daily intranasal dose of gnrha for lps in gnrha-triggered cycles. importantly, no exogenous steroids were used for lps (58). of the 46 patients analysed, 52.1% achieved an ongoing pregnancy. the treatment was well tolerated by all patients, and none of the patients developed clinical signs of early or late ohss. the same group subsequently retrospectively reported the outcomes of 1436 cycles triggered with hcg, using repeated daily nasal gnrha administration as the sole lps. higher live birth rates were reported with this protocol when compared with a retrospective cohort of 1093 patients having daily vaginal lps after hcg trigger (59). the potential advantages of this approach over current strategies are: (i) convenient nasal self-administration compared with hcg injections or multiple daily vaginal administrations of progesterone; (ii) use of a purely synthetic polypeptide with no biological fluid residues; (iii) the opportunity for early pregnancy diagnosing (56). larger prospective trials are clearly needed to draw firm conclusions regarding the true benefits of the use of intranasal gnrha for lps. conclusions the introduction of the gnrha trigger in modern ivf revolutionized ovarian stimulation and ovarian trigger concepts. moreover, it opened the ‘black box’ of the luteal phase. currently, patients undergoing fresh embryo transfer who are not at risk of ohss development can be offered a virtually ohss-free treatment in addition to good reproductive outcomes with modifications of the lps after gnrha trigger in terms of small boluses of hcg, daily rlh, or gnrha. although recent rcts investigate further fine-tuning of the modified lps, more studies are needed to reach a consensus on the optimal strategy. in the ohss risk patient gnrha trigger can be safely performed, followed by a ‘freeze-all’ policy with a minimal risk of ohss development and high live birth rates in the subsequent fet cycle. taken together, the gnrha trigger concept adds safety, efficacy, and patientfriendliness to modern ivf. disclosure statement not related to this manuscript, t.h. received honoraria for lectures from ferring, ibsa, besins, and merck. p.h. received unrestricted research grants from msd, merck, and ferring as well as honoraria for lectures from msd, merck, gedeon-richter, theramex, and ibsa. p.h. and t.h. are listed as inventors in an international patent application (pct/uk2018/ 040882). the remaining authors have no disclosures. notes on contributors juan carlos castillo is a senior consultant (md phd) at instituto bernabeu, alicante, spain. thor haahr is a medical doctor and a phd student at the fertility clinic skive, and the department of clinical medicine, aarhus university, denmark. mar�ıa mart�ınez-moya is a consultant (md) at instituto bernabeu, alicante, spain. peter humaidan is a senior consultant (md, dmsc) and professor at the fertility clinic skive, and the department of clinical medicine, aarhus university, denmark. references 1. delvigne a, rozenberg s. epidemiology and prevention of ovarian hyperstimulation syndrome (ohss): a review. hum reprod update. 2002;8:559–77. doi:10.1093/humupd/8.6.559 2. forman r, fries n, testart j, belaisch-allart j, hazout a, frydman r. evidence for an adverse effect of elevated serum estradiol concentrations on embryo implantation. fertil steril. 1988;49:118–22. doi:10.1016/s0015-0282(16)59661-7 3. valbuena d, martin j, de pablo jl, remoh�ı j, pellicer a, sim�on c. increasing levels of estradiol are deleterious to embryonic implantation because they directly affect the embryo. fertil steril. 2001;76:962–8. doi:10.1016/s0015-0282(01)02018-0 4. haahr t, roque m, esteves sc, humaidan p. gnrh agonist trigger and lh activity luteal phase support versus hcg trigger and conventional luteal phase support in fresh embryo transfer ivf/icsi cycles—a systematic prisma review and meta-analysis. front endocrinol. 2017;8:116. 5. albano c, smitz j, camus m, riethm€uller-winzen h, van steirteghem a, devroey p. comparison of different doses of gonadotropin-releasing hormone antagonist cetrorelix during controlled ovarian hyperstimulation. fertil steril. 1997;67:917–22. doi: 10.1016/s0015-0282(97)81407-0 6. hoff jd, quigley me, yen ss. hormonal dynamics at midcycle: a reevaluation. j clin endocrinol metab. 1983;57:792–6. doi:10.1210/ jcem-57-4-792 7. casper rf. reducing the risk of ohss by gnrh agonist triggering. j clin endocrinol metab. 2015;100:4396–8. doi:10.1210/jc.20153676 8. humaidan p, kol s, papanikolaou eg; 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analogue as sole luteal support in antagonist-based assisted reproductive technology cycles. fertil steril. 2017;107:130–5.e1. doi:10.1016/j.fertnstert.2016.10.011 upsala journal of medical sciences 137 https://doi.org/10.1093/humrep/det304 https://doi.org/10.1093/humrep/det304 https://doi.org/10.1093/humrep/det124 https://doi.org/10.1093/humrep/det287 https://doi.org/10.1016/j.fertnstert.2010.09.023 https://doi.org/10.1016/j.fertnstert.2010.09.023 https://doi.org/10.1016/j.fertnstert.2007.02.002 https://doi.org/10.1016/j.fertnstert.2007.02.002 https://doi.org/10.1093/humrep/der463 https://doi.org/10.1093/humrep/der463 https://doi.org/10.1016/j.rbmo.2012.03.005 https://doi.org/10.1016/j.fertnstert.2011.04.050 https://doi.org/10.1016/j.fertnstert.2011.04.050 https://doi.org/10.1016/j.fertnstert.2007.06.030 https://doi.org/10.1016/j.fertnstert.2011.03.109 https://doi.org/10.1016/j.fertnstert.2012.03.015 https://doi.org/10.1093/humrep/deh830 https://doi.org/10.1155/2015/727569 https://doi.org/10.1016/j.fertnstert.2016.04.004 https://doi.org/10.1016/j.fertnstert.2016.04.004 https://doi.org/10.1016/j.fertnstert.2016.10.011 abstract introduction avoiding ohss in risk patients with the use of gnrha trigger gnrha trigger and segmentation (freeze-all) gnrh trigger and the optimal modified luteal phase support (mlps) during fresh embryo transfer mlps: a single low-dose bolus of hcg mlps: recombinant lh (rlh) mlps: exogenous intensive progesterone and oestradiol supplementation mlps: the dual trigger concept – concomitant use of gnrha and low-dose hcg mlps: intranasal gonadotropin-releasing hormone agonist conclusions disclosure statement references u-shaped association between serum uric acid concentration and mortality in hypertrophic cardiomyopathy patients article u-shaped association between serum uric acid concentration and mortality in hypertrophic cardiomyopathy patients ziqiong wang, ying xu, hang liao, xiaoping chen and sen he department of cardiology, west china hospital, sichuan university, chengdu, china abstract background. no study has examined the effect of low serum uric acid (sua) concentrations on mortality in hypertrophic cardiomyopathy (hcm) patients. the aim of the present study was to assess the relations between both low and high sua concentrations and the risk of mortality across the full range of sua concentrations in a retrospective cohort of hcm patients. methods. a total of 454 hcm patients were enrolled in the study, and sua concentrations were measured at baseline. the primary and secondary endpoints were all-cause mortality and hcm-related mortality, respectively. the associations between sua concentrations and endpoints were analysed. results. during a median follow-up of 3.8 years, there were 80 (17.6%) all-cause mortality events, and 52 of them (11.5%) were ascribed to hcm-related mortality. patients with sua concentrations of 250–350mmol/l had the lowest all-cause mortality rate (11.8%) and hcm-related mortality rate (5.0%). both low and high sua concentrations were associated with increased all-cause and hcm-related mortality. adjusted hrs were 2.52 (95% ci 1.13–5.61, p ¼ 0.024) and 4.86 (95% ci 1.74–13.58, p ¼ 0.003) for all-cause mortality and hcm-related mortality in the lowest sua group (<250 mmol/l) when compared with the reference group (250–350mmol/l), respectively. the corresponding hrs in the highest sua group (�450mmol/l) were 2.73 (95% ci 1.42–5.23, p ¼ 0.003) and 4.14 (95% ci 1.70–10.13, p ¼ 0.002), respectively. conclusions. both low and high sua concentrations were significantly associated with increased risk of all-cause mortality and hcm-related mortality, which supported a u-shaped association between sua concentrations and mortality in hcm patients. article history received 13 january 2020 accepted 16 january 2020 keywords hypertrophic cardiomyopathy; mortality; serum uric acid; u-shaped association introduction uric acid is the end product of purine metabolism (1). it has been reported that high serum uric acid (sua) concentrations are associated with increased risk of cardiovascular diseases and death (2,3). however, the role of sua in this constellation still remains controversial (4,5). recently, several studies have also indicated that low sua concentrations could predict cardiovascular death and all-cause mortality, which supports a jor u-shaped association between sua concentrations and mortality (6–9). hypertrophic cardiomyopathy (hcm) is a genetic cardiac disease with marked heterogeneity in clinical expression, natural history, and prognosis (10). some factors have been identified for risk stratification in hcm patients, such as new york heart association (nyha) class, atrial fibrillation (af), maximal wall thickness (mwt), and left ventricular outflow tract obstruction (lvoto), etc. (11). in a retrospective cohort study, zhu et al. illustrated that high sua concentrations were associated with adverse outcomes in hcm patients (12). however, no longitudinal studies have evaluated the risk of all-cause and hcm-related mortality across the full range of sua concentrations in hcm patients while considering both low and high sua concentrations. therefore, the purpose of the present study was to evaluate the association between both low and high sua concentrations with all-cause and hcm-related mortality in a cohort of hcm patients. methods study population this was a retrospective and longitudinal study. a total of 508 patients with a diagnosis of hcm were consecutively enrolled in the study from december 2008 to may 2016 at west china hospital of sichuan university (a tertiary referral centre). the diagnosis of hcm was based on the echocardiographic demonstration of an increase in wall thickness of �15 mm in any left ventricular myocardial segment, which was not solely explained by abnormal load conditions (13). nine patients with inherited metabolic disease or syndromic causes of hcm were excluded from the study (cardiac contact sen he hesensubmit@163.com department of cardiology, west china hospital, sichuan university, 37 guo xue xiang, chengdu, 610041 sichuan province, china ziqiong wang and ying xu contributed equally to this work. � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 1, 44–51 https://doi.org/10.1080/03009734.2020.1719245 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1719245&domain=pdf&date_stamp=2020-02-19 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1719245 http://www.tandfonline.com amyloidosis n ¼ 5, restrictive cardiomyopathy n ¼ 2, dilated cardiomyopathy n ¼ 1, myocarditis n ¼ 1). we also excluded patients who were lost to follow-up after the first evaluation (n ¼ 41), as well as patients with incomplete biochemistry data (n ¼ 4). the final sample size consisted of 454 hcm patients. detailed information about those patients has been reported elsewhere (14,15). the study was approved by the ethics committee on medical research of west china hospital of sichuan university, and performed according to the principles of the declaration of helsinki. due to the retrospective nature of the study, informed consent was waived. data collection blood samples were obtained at admission for all patients. sua concentrations were measured by the uricase method. estimated glomerular filtration rate (egfr) was calculated using the four-variable modification of diet in renal disease study equation: egfr (ml/min/1.73 m2) ¼ 186.3 � (serum creatinine)�1.154� age�0.203 (� 0.742 if female) (16). normal kidney function was defined as egfr � 60 ml/min/1.73 m2 (6). all patients underwent standard two-dimensional transthoracic echocardiography examinations by standard techniques (17). the presence of lvoto was defined as a gradient >30 mmhg at rest. other data of baseline characteristics were collected from medical records. follow-up and outcomes follow-ups were carried out by clinical consultations, medical records, or telephone interviews. the primary endpoint was all-cause mortality, and the secondary endpoint was hcmrelated mortality, which included: (1) sudden cardiac death, (2) heart failure-related death, (3) stroke-related death, and (4) perioperative death due to septal myectomy. statistical analysis patients were divided into four groups according to concentrations of sua: <250 mmol/l (n ¼ 43), �250 mmol/l and <350 mmol/l (n ¼ 161), �350 mmol/l and <450 mmol/l (n ¼ 148), and �450 mmol/l (n ¼ 102). descriptive statistics were used to summarise baseline characteristics. baseline characteristics among the four groups were analysed by anova for parametric variables, kruskal–wallis test for non-parametric variables, and chi-square or fisher exact tests for categorical variables. a kaplan–meier method was used to estimate the survival in each group, and a log-rank test was used for comparisons. to assess the role of sua as an independent predictor of mortality, cox proportional hazard regression analysis was used. the lowest mortality incidence group was defined as the reference group. age and gender were forced into five multivariable models. other variables entered a model on the basis of clinical relevance and a univariate relation with mortality (p < 0.05). for the final model, the predictors were sought using a stepwise backward modelling approach (p ¼ 0.05 for inclusion, p ¼ 0.10 for exclusion) including all variables from models 1 to 4. the proportional hazard assumption was verified by means of multivariate cox regressions. restricted cubic splines were used to further explore the shape of the dose–response relation between sua concentrations and risk of all-cause mortality and hcm-related mortality. finally, we assessed the relation between sua concentrations and mortality in the patients who did not take hydrochlorothiazide and patients with normal kidney function as sensitivity analyses. all analyses were performed by empowerstats software (www.empowerstats.com, x&y solutions, inc., boston, ma, usa) and spss version 25.0 (spss inc., chicago, il, usa). all statistical testing was two-sided. results baseline characteristics the median age was 57.5 (interquartile range: 46.0–67.0) years (table 1). male patients accounted for 55.7%. sua concentrations ranged from 42.0 to 913.0 mmol/l. patients with higher sua concentrations had higher male percentage (p < 0.001), higher serum creatinine (p < 0.001), larger left atrium (la) and left ventricle sizes (p ¼ 0.006 and p ¼ 0.001, respectively), but lower egfr (p ¼ 0.046), high-density lipoprotein–cholesterol (p < 0.001), and left ventricular ejection fraction (lvef) (p < 0.001). other variables did not differ between the four groups. clinical outcomes during a median follow-up of 3.8 years (range: 0.1–9.4), there were 80 (17.6%) all-cause mortality events, and 52 (11.5%) were ascribed to hcm-related mortality. patients with sua concentrations in the interval 250–350 mmol/l had the lowest mortality rate (table 2). likewise, a kaplan–meier analysis showed that the survival freedom of all-cause mortality and hcm-related mortality was highest in this group of patients (p ¼ 0.014 and 0.002, respectively, figure 1). a further decrease or increase of sua was associated with higher risk of mortality. relation of sua to all-cause mortality and hcmrelated mortality table 3 presents the results of univariate cox proportional hazard analysis. the lowest sua group (<250 mmol/l) was found to have an increased risk of all-cause and hcm-related mortality. the corresponding hrs for all-cause and hcmrelated mortality, comparing <250 mmol/l sua with 250–350 mmol/l sua, were 2.11 (95% confidence interval [ci]: 0.98–4.55, p ¼ 0.056) and 3.98 (95% ci: 1.49–10.62, p ¼ 0.006), respectively. the highest sua group (�450 mmol/l) was significantly associated with increased risk of all-cause and hcm-related mortality. among the remaining variables, nyha iii/iv, af, warfarin, serum glucose, and la were also identified as significant risk factors for both all-cause mortality and hcm-related mortality, while triglycerides, low-density upsala journal of medical sciences 45 http://www.empowerstats.com lipoprotein–cholesterol (ldl-c), and lvef were protective factors. after adjusting for potential confounding factors, the association between sua concentrations and endpoints remained consistent. in the final model—after adjusting for age, sex, nyha iii/iv, chronic obstructive pulmonary disease, af, triglycerides, ldl-c, and la—hrs for all-cause mortality and hcm-related mortality, comparing �450 mmol/l sua with 250–350 mmol/l sua, were 2.73 (95% ci: 1.42–5.23, p ¼ 0.003) and 4.14 (95% ci: 1.70–10.13, p ¼ 0.002), respectively. the corresponding adjusted hrs in sua <250 mmol/l for all-cause mortality and hcm-related mortality were 2.52 (95% ci: 1.13–5.61, p ¼ 0.024) and 4.86 (95% ci: 1.74–13.58, p ¼ 0.003) (table 4). restricted cubic spline in the multivariable-adjusted spline (adjusted for the same variables as in model 5), there was a u-shaped association between sua concentrations and all-cause mortality and hcm-related mortality, with a nadir of risk at sua around 300 mmol/l. deviation of sua from 300 mmol/l was significantly associated with higher mortality risk (figure 2(a,b)). for sua concentrations higher than 300 mmol/l, a 10 mmol/l increase of sua showed a 4.6% (p < 0.001) increase of allcause mortality and 5.2% (p ¼ 0.001) increase of hcm-related mortality. for sua concentrations less than 300 mmol/l, a 10 mmol/l decrease of sua showed a 9.4% (p ¼ 0.03) increase of all-cause mortality and 15.6% (p ¼ 0.004) increase of hcmrelated mortality. sensitivity analyses when including patients with normal kidney function (n ¼ 384), there were 56 (14.6%) all-cause mortality and 38 (9.9%) hcm-related mortality events. the adjusted hrs maintained a u-shaped relation, with a nadir of risk at sua around 300 mmol/l (figure 3(a,b)). for sua concentrations higher than 300 mmol/l, a 10 mmol/l increase of sua was associated with a 2.6% (p ¼ 0.213) increase of all-cause table 2. primary and secondary endpoint of the present study. endpoints whole cohort serum uric acid concentration (mmol/l) <250 �250, <350 �350, <450 �450 no. of patients 454 43 161 148 102 all-cause mortality no. of deaths 80 10 19 25 26 mortality rate (%)a 17.6 23.3 11.8 16.9 25.5 hcm-related mortality no. of deaths 52 8 8 17 19 mortality rate (%)a 11.5 18.6 5.0 11.5 18.6 abinary event rate. table 1. baseline characteristics of the study cohort. variables whole cohort (n ¼ 454) serum uric acid concentration (mmol/l) p value<250 (n ¼ 43) �250, <350 (n ¼ 161) �350, <450 (n ¼ 148) �450 (n ¼ 102) basic information age (y) 57.5 (46.0–67.0) 59.0 ± 15.1 59.0 (46.0–68.0) 56.5±±4.8 52.1 ± 16.6 0.067 gender (male) 253 (55.7%) 10 (23.3%) 72 (44.7%) 95 (64.2%) 76 (74.5%) <0.001 fhhcm 42 (9.3%) 2 (4.7%) 15 (9.3%) 15 (10.1%) 10 (9.8%) 0.739 fhscd 18 (4.0%) 2 (4.7%) 4 (2.5%) 6 (4.1%) 6 (5.9%) 0.579 nyha iii/iv 156 (34.4%) 20 (46.5%) 49 (30.4%) 47 (31.8%) 40 (39.2%) 0.121 medical history hypertension 141 (31.1%) 9 (20.9%) 46 (28.6%) 51 (34.5%) 35 (34.3%) 0.280 diabetes 37 (8.1%) 5 (11.6%) 14 (8.7%) 13 (8.8%) 5 (4.9%) 0.517 copd 29 (6.4%) 6 (14.0%) 11 (6.8%) 5 (3.4%) 7 (6.9%) 0.092 af 77 (17.0%) 5 (11.6%) 23 (14.3%) 23 (15.5%) 26 (25.5%) 0.067 medications/devices/procedures aspirin/clopidogrel 98 (21.6%) 10 (23.3%) 29 (18.0%) 37 (25.0%) 22 (21.6%) 0.512 warfarin 41 (9.0%) 2 (4.7%) 8 (5.0%) 13 (8.8%) 18 (17.6%) 0.004 statins 123 (27.1%) 10 (23.3%) 37 (23.0%) 48 (32.4%) 28 (27.5%) 0.279 beta-blockers 325 (71.6%) 29 (67.4%) 109 (67.7%) 117 (79.1%) 70 (68.6%) 0.109 acei/arb 88 (19.4%) 8 (18.6%) 30 (18.6%) 27 (18.2%) 23 (22.5%) 0.837 hctz 27 (5.9%) 3 (7.0%) 10 (6.2%) 9 (6.1%) 5 (4.9%) 0.959 icd/pacemaker 59 (13.0%) 5 (11.6%) 23 (14.3%) 17 (11.5%) 14 (13.7%) 0.393 obstruction intervention 41 (9.0%) 4 (9.3%) 13 (8.1%) 19 (12.8%) 5 (4.9%) 0.091 laboratory test egfr (ml/min/1.73 m2) 82.8 (68.6–100.7) 94.2 (79.3–110.3) 88.5 (74.5–107.0) 80.0 ± 24.3 74.4 (56.0–91.2) 0.046 creatinine (mmol/l) 80.6 (67.0–94.7) 63.1 (55.0–75.0) 74.0 ± 16.7 84.6 (74.0–99.2) 94.7 (80.3–115.3) <0.001 glucose (mmol/l) 5.4 (4.9–6.5) 5.4 (4.6–6.7) 5.4 (4.7–6.2) 5.4 (5.0–6.3) 5.5 (5.0–6.8) 0.437 triglycerides (mmol/l) 1.2 (0.9–1.9) 1.2 (0.9–1.4) 1.2 (0.9–1.6) 1.4 (1.0–2.0) 1.2 (0.9–2.1) 0.054 hdl-c (mmol/l) 1.3 (1.0–1.6) 1.3 (1.1–1.5) 1.4 (1.1–1.7) 1.2 (1.0–1.5) 1.1 (1.0–1.5) <0.001 ldl-c (mmol/l) 2.4 ± 0.8 2.6 ± 0.9 2.4 ± 0.8 2.4 ± 0.7 2.4 ± 0.8 0.597 echocardiographic data la (mm) 40.0 (35.0–46.0) 39.2 ± 6.6 38.0 (34.0–45.0) 40.4 ± 6.7 42.6 ± 8.0 0.006 lv (mm) 43.0 (40.0–46.0) 40.0 (36.3–43.0) 43.0 (39.3–46.0) 43.5 (40.0–47.0) 44.0 (40.0–49.0) 0.001 mwt (mm) 19.0 (16.0–22.0) 19.1 ± 5.0 19.0 (17.0–22.0) 20.0 (16.3–22.0) 19 (16.0–21.0) 0.309 lvef (%) 68.0 (63.0–72.0) 69.0 (65.0–73.0) 70.0 (65.0–73.0) 68.0 (63.0–71.0) 65.0 (59.0–71.0) <0.001 lvoto 181 (39.9%) 20 (46.5%) 72 (44.7%) 57 (38.5%) 32 (31.4%) 0.165 acei: angiotensin-converting-enzyme inhibitor; af: atrial fibrillation; arb: angiotensin receptor blocker; copd: chronic obstructive pulmonary disease; egfr: estimated glomerular filtration rate; fhhcm: family history of hypertrophic cardiomyopathy; fhscd: family history of sudden cardiac death; hcm: hypertrophic cardiomyopathy; hctz: hydrochlorothiazide; hdl-c: high-density lipoprotein–cholesterol; icd: implantable cardioverter defibrillator; la: left atria; ldl-c: low-density lipoprotein–cholesterol; lv: left ventricle; lvef: left ventricular ejection fraction; lvoto: left ventricular outflow tract obstruction; mwt: maximal wall thickness; nyha: new york heart association; tg: triglycerides. 46 z. wang et al. mortality and 6.3% (p ¼ 0.008) increase of hcm-related mortality. for sua concentrations less than 300 mmol/l, a 10 mmol/l decrease of sua was associated with a 11.1% (p ¼ 0.026) increase of all-cause mortality and 19.3% (p ¼ 0.002) increase of hcm-related mortality. upon excluding patients who were taking hydrochlorothiazide, 427 patients remained. there were 75 (17.5%) allcause mortality and 49 (11.4%) hcm-related mortality events. the association between sua concentrations and mortality did not change materially (figure 3(c,d)). for sua concentrations higher than 300 mmol/l, the risk of all-cause mortality and hcm-related mortality increased 4.5% (p ¼ 0.001) and 5.0% (p ¼ 0.003) with each 10 mmol/l increase in sua concentrations, respectively. for sua less than 300 mmol/l, the risk of all-cause mortality and hcm-related mortality increased 9.9% (p ¼ 0.029) and 15.6% (p ¼ 0.006) with each 10 mmol/l decrease in sua concentration, respectively. discussion in the present study, patients with either low or high sua concentrations were found to have a higher risk of all-cause mortality and hcm-related mortality. our study is the first study to reveal a u-shaped association between sua concentrations and all-cause mortality and hcm-related mortality in hcm patients. the inflection point is approximately 300 mmol/l sua. our findings have some similarities with previous studies but also presented with certain differences (6–9). in a general us population, it was reported that there was a u-shaped association between sua concentrations and cardiovascular mortality. however, this relation was no longer statistically significant after adjusting for egfr and albumin–creatinine ratio (acr) (6). in our study, the association remained stable after adjusting for egfr. we were unable to examine the effect of acr due to data unavailability. in another study comprising korean adults with normal kidney function, kang et al. found that the overall mortality rate had a u-shaped association with sua concentrations in males but not in females (7). furthermore, in a large cohort study of korean general populations, the authors demonstrated that low sua concentrations were independently associated with increased risk of all-cause mortality in both genders and increased risk of cardiovascular disease in females only (9). there was no gender-specific relation in our study. to our knowledge, only one study has been carried out to illustrate the relation between sua concentrations and allcause mortality and cardiovascular death in hcm patients (12). in that study, sua was categorised into tertiles. the adjusted hrs for all-cause mortality and cardiovascular death of subjects in the highest tertile of sua were 2.33 (95% ci: 1.11–4.89, p ¼ 0.025) and 3.10 (95% ci: 1.37–7.04, p ¼ 0.007) when compared to that of subjects in the lowest tertile. there was no statistical difference between the second tertile versus the first tertile with regard to the aforementioned outcomes. the cut-off points overlapped but not the same when categorising patients into different groups between that study and our study. by grouping sua into tertiles, intracategory variations in mortality risk could not be detected, probably leading to a failure of examining the influence of very low and very high sua concentrations on mortality. in vitro and animal studies have revealed that high sua concentrations might produce an inflammatory reaction, as evidenced by increased expression of inflammation cytokines, such as interleukin (il)-6, il-8, and tumour necrosis factor-a (tnf-a) in endothelial cells. this process was associated with activation of transcription factor nf-jb (18). high sua concentrations could also stimulate monocyte chemoattractant protein-1 in vascular smooth muscle cells through mitogen-activated protein kinase and cyclooxygenase-2 (19). a clinical study based on community-dwelling older persons revealed a positive and significant association between sua concentrations and several inflammatory markers, including neutrophil count, c-reactive protein (crp), il-6, il18, and tnf-a (20). the above findings supported a role of high sua concentrations in the process of inflammation. recently, wang et al. reported that elevated high-sensitivity crp was associated with increased risk of adverse outcomes in patients with hcm, suggesting a possible association of an inflammatory state and the clinical progression of hcm (21). therefore, these inflammatory events induced by sua might figure 1. freedom from all-cause mortality (a) and hcm-related mortality (b) according to different serum uric acid (sua) concentrations during follow-up period in hcm patients. upsala journal of medical sciences 47 partially explain the poor prognosis of hcm patients with high sua concentrations in the present study. in addition, the impaired nitro-oxide bioavailability and oxidative stress produced by xanthine oxidase may also be mechanisms behind high sua-related all-cause and hcm-related mortality (22,23). the mechanism underlying the increased risk of mortality related to low sua is not fully understood. sua acting as an antioxidant may be one of the possible explanations. it has been reported that sua may exert antioxidant protection against the damage of free radical and reactive oxygen species in ischaemic brain tissue, illustrating the protective effect of sua for the central nervous system (24). there is also one previous study showing that extremely low sua concentrations were associated with endothelial dysfunction and vascular damage (25). therefore, when sua decreased even more, antioxidant defense might contribute to the relatively high risk of all-cause mortality and hcm-related mortality in hcm patients with low sua in our study. this study has several limitations. first, we did not adjust for urate-lowering medications. there were 39.4% of patients diagnosed as having hyperuricaemia (>420 mmol/l for male table 3. univariate cox proportional hazard analysis for all-cause mortality and hcm-related mortality in hcm patients. variables change all-cause mortality hr (95% ci), p hcm-related mortality hr (95% ci), p age per 1-year increase 1.02 (1.01–1.04), 0.006 1.01 (0.99–1.03), 0.219 gender female vs male 1.12 (0.72–1.73), 0.625 1.33 (0.77–2.30), 0.302 fhhcm yes vs no 0.71 (0.31–1.63), 0.419 0.94 (0.37–2.36), 0.893 fhscd yes vs no 1.51 (0.61–3.73), 0.376 1.38 (0.43–4.46), 0.582 nyha iii/iv yes vs no 2.91 (1.87–4.53), <0.001 2.44 (1.41–4.20), 0.001 hypertension yes vs no 0.81 (0.49–1.33), 0.405 0.76 (0.40–1.42), 0.387 diabetes yes vs no 1.04 (0.48–2.27), 0.913 0.91 (0.33–2.53), 0.861 copd yes vs no 3.18 (1.75–5.76), <0.001 2.13 (0.91–4.99), 0.083 af yes vs no 2.23 (1.39–3.58), 0.001 3.59 (2.07–6.23), <0.001 warfarin yes vs no 2.38 (1.33–4.24), 0.003 3.77 (2.01–7.07), <0.001 hctz yes vs no 1.09 (0.44–2.71), 0.846 1.01 (0.31–3.23), 0.993 devices none 1 1 pacemaker 1.55 (0.67–3.57), 0.304 2.49 (1.06–5.86), 0.036 icd 0.56 (0.20–1.53), 0.257 0.69 (0.21–2.22), 0.529 procedures none 1 1 alcohol septal ablation 0.48 (0.15–1.53), 0.216 0.25 (0.03–1.77), 0.163 septal myectomy 1.06 (0.15–7.60), 0.957 1.64 (0.23–11.87), 0.627 egfr per 1 unit increase 0.99 (0.98–0.99), 0.004 0.99 (0.98–1.00), 0.128 glucose per 1 mmol/l increase 1.12 (1.03–1.22), 0.008 1.11 (0.99–1.24), 0.070 triglycerides per 1 mmol/l increase 0.67 (0.49–0.93), 0.015 0.55 (0.35–0.87), 0.010 ldl-c per 1 mmol/l increase 0.64 (0.48–0.85), 0.002 0.67 (0.46–0.96), 0.029 la per 1 mm increase 1.04 (1.01–1.07), 0.016 1.06 (1.03–1.10), <0.001 mwt per 1 mm increase 1.01 (0.96–1.05), 0.826 0.98 (0.92–1.04), 0.439 ef per 1 percent increase 0.97 (0.95–0.99), 0.004 0.96 (0.94–0.98), 0.002 lvoto yes vs no 1.07 (0.68–1.69), 0.757 1.10 (0.63–1.92), 0.749 serum uric acid (mmol/l) <250 2.11 (0.98–4.55), 0.056 3.98 (1.49–10.62), 0.006 �250, <350 1 1 �350, <450 1.65 (0.91–3.00), 0.098 2.65 (1.14–6.14), 0.023 �450 　 2.56 (1.42–4.64), 0.002 4.43 (1.94–10.15), <0.001 abbreviations as in table 1. table 4. multivariate cox proportional hazard models for all-cause mortality and hcm-related mortality in hcm patients. models serum uric acid concentration (mmol/l) <250 �250, <350 �350, <450 �450 all-cause mortality, hr (95% ci), p model 1 1.64 (0.75–3.58), 0.215 1 1.86 (1.01–3.43), 0.047 2.73 (1.47–5.07), 0.001 model 2 1.58 (0.71–3.50), 0.261 1 1.91 (1.04–3.54), 0.038 2.75 (1.47–5.15), 0.002 model 3 2.02 (0.93–4.41), 0.076 1 1.73 (0.94–3.20), 0.080 2.59 (1.39–4.84), 0.003 model 4 2.72 (1.21–6.10), 0.016 1 1.72 (0.92–3.24), 0.091 2.53 (1.26–5.06), 0.009 model 5 2.52 (1.13–5.61), 0.024 1 1.99 (1.06–3.72), 0.031 2.73 (1.42–5.23), 0.003 hcm-related mortality, hr (95% ci), p model 1 3.10 (1.14–8.40), 0.026 1 3.13 (1.33–7.36), 0.009 5.16 (2.19–12.18), <0.001 model 2 3.16 (1.17–9.06), 0.024 1 3.27 (1.38–7.76), 0.007 4.63 (1.94–11.09), 0.001 model 3 4.13 (1.52–11.25), 0.005 1 3.03 (1.28–7.18), 0.012 4.20 (1.77–9.96), 0.001 model 4 4.68 (1.71–12.81), 0.003 1 2.97 (1.24–7.09), 0.014 4.38 (1.77–10.85), 0.001 model 5 4.86 (1.74–13.58), 0.003 1 3.18 (1.33–7.61), 0.010 4.14 (1.70–10.13), 0.002 model 1: adjusted for age, sex, fhhcm, fhscd, nyha. model 2: adjusted for age, sex, hypertension, diabetes, copd, af. model 3: adjusted for age, sex, warfarin, hctz, obstruction intervention and devices. model 4: adjusted for age, sex, egfr, glucose, triglycerides, ldl-c, la, ef. model 5: adjusted for age, sex, nyha, copd, af, tg, ldl-c, la. abbreviations as in table 1. 48 z. wang et al. figure 2. u-shaped association between serum uric acid concentration and all-cause mortality (a) and hcm-related mortality (b). figure 3. sensitivity analyses including patients with normal kidney function (a,b) or excluding patients taking hydrochlorothiazide (c,d). u-shaped association between serum uric acid concentration and all-cause mortality (a,c) and hcm-related mortality (b,d). upsala journal of medical sciences 49 and >360 mmol/l for female (26)). some of them might take urate-lowering agents, which in turn would reduce the effect of high sua on endpoints in our study. however, patients with sua concentrations less than 250 mmol/l might not be affected by the treatment. and thus the u-shaped association between sua concentrations and all-cause mortality and hcm-related mortality in hcm patients is credible to some extent. second, the mortality rate in the present study is higher than in previous studies (11), which might be partially explained by collection bias of patients. all patients were enrolled at the inpatient department of a tertiary referral hospital, and their diseases might be more severe than general hcm populations. patients with nyha class �3 accounted for 34.1% in the present study. third, there is a relatively small number of patients (n ¼ 43) with sua level <250 mmol/l in our study. fourth, some unknown covariates could not be excluded, although extensive adjustment was performed for many important covariates. fifth, this is a retrospective study from a single centre. multicentre-based prospective studies are needed to confirm and extend the present findings. disclosure statement no potential conflict of interest was reported by the author(s). funding this study was supported by the national natural science foundation of china [grant number: 81600299]. notes on contributors ziqiong wang is a resident from the cardiology department in sichuan university at west china hospital. ying xu is a chief nurse from the cardiology department in sichuan university at west china hospital. hang liao is an attending doctor from the cardiology department in sichuan university at west china hospital. xiaoping chen is a cardiologist. she is the head of cardiology department in sichuan university at west china hospital. sen he is an associate professor from the cardiology department in sichuan 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https://doi.org/10.1016/j.cjca.2019.05.035 https://doi.org/10.1097/00041552-200111000-00009 https://doi.org/10.1097/00041552-200111000-00009 https://doi.org/10.1016/j.echo.2014.10.003 https://doi.org/10.1016/j.echo.2014.10.003 https://doi.org/10.3892/br.2017.966 https://doi.org/10.1161/01.hyp.0000072820.07472.3b https://doi.org/10.1093/eurheartj/ehi879 22. shah a, keenan rt. gout, hyperuricemia, and the risk of cardiovascular disease: cause and effect?. curr rheumatol rep. 2010;12: 118–24. doi:10.1007/s11926-010-0084-3 23. puddu p, puddu gm, cravero e, vizioli l, muscari a. the relationships among hyperuricemia, endothelial dysfunction, and cardiovascular diseases: molecular mechanisms and clinical implications. j cardiol. 2012;59:235–42. doi:10.1016/j.jjcc.2012. 01.013 24. �alvarez-lario b, macarr�on-vicente j. is there anything good in uric acid? qjm. 2011;104:1015–24. doi:10.1093/qjmed/hcr159 25. iso t, kurabayashi m. extremely low levels of serum uric acid are associated with endothelial dysfunction in humans. circ j. 2015; 79:978–80. doi:10.1253/circj.cj-15-0232 26. fang j, alderman mh. serum uric acid and cardiovascular mortality: the nhanes i epidemiologic follow-up study, 1971. jama. 2000;283:2404–10. doi:10.1001/jama.283.18.2404 upsala journal of medical sciences 51 https://doi.org/10.1007/s11926-010-0084-3 https://doi.org/10.1016/j.jjcc.2012.01.013 https://doi.org/10.1016/j.jjcc.2012.01.013 https://doi.org/10.1093/qjmed/hcr159 https://doi.org/10.1253/circj.cj-15-0232 https://doi.org/10.1001/jama.283.18.2404 abstract introduction methods study population data collection follow-up and outcomes statistical analysis results baseline characteristics clinical outcomes relation of sua to all-cause mortality and hcm-related mortality restricted cubic spline sensitivity analyses discussion disclosure statement references re: neural injury after use of vasopressin and adrenaline during porcine cardiopulmonary resuscitati full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 re: neural injury after use of vasopressin and adrenaline during porcine cardiopulmonary resuscitation eric m rottenberg to cite this article: eric m rottenberg (2015) re: neural injury after use of vasopressin and adrenaline during porcine cardiopulmonary resuscitation, upsala journal of medical sciences, 120:3, 213-214, doi: 10.3109/03009734.2015.1021936 to link to this article: https://doi.org/10.3109/03009734.2015.1021936 © informa healthcare published online: 15 mar 2015. submit your article to this journal article views: 316 view related articles view crossmark data https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2015.1021936 https://doi.org/10.3109/03009734.2015.1021936 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1021936 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1021936 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1021936&domain=pdf&date_stamp=2015-03-15 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1021936&domain=pdf&date_stamp=2015-03-15 upsala journal of medical sciences. 2015; 120: 213–214 letter to the editor re: neural injury after use of vasopressin and adrenaline during porcine cardiopulmonary resuscitation eric m rottenberg columbus, ohio, usa dear editor, halvorsen and colleagues (1) investigated cerebral and cardiac tissue injury subsequent to use of vasopressin and adrenaline in combination compared with vasopressin alone during cardiopulmonary resuscitation (cpr). they concluded that combined use of vasopressin and adrenaline caused greater signs of cerebral and cardiac injury than use of vasopressin alone during experimental cpr. however, given that epinephrine was administered after 12 min of untreated ventricular fibrillation (vf), these results are not surprising. first, neurologically intact survival to hospital discharge after in-hospital cardiac arrest has been found to be significantly more likely after earlier epinephrine administration (2). this recent retrospective evaluation of more than 25,000 patients (at 570 us hospitals), who were not in intensive care units or emergency departments and who exhibited initial rhythms of asystole or pulseless electrical activity (pea), found that delayed administration of epinephrine (>3 min) was associated significantly with lower chance for survival to hospital discharge, in stepwise fashion (12%, 10%, 8%, and 7% survival, respectively, for patients receiving their first epinephrine dose £3, 4–6, 7–9, and >9 min after arrest (p < 0.001). second, as the authors already understand from their previous work in a swine model of hemorrhagic circulatory arrest that applied 15 min of open-chest cpr after 8 min of untreated vf, intracranial pressure (icp) in the post-resuscitation phase is greaterwith use of adrenalin versus vasopressin, but with no significant difference in neuronal injury (3). however, one possible explanation for the lack of difference in neuronal injury could be that open-chest cpr does not raise intrathoracic pressure; therefore, it does not raise icp. it has been postulated that without effective-depth chest compressions with complete recoil and/or gasping to maintain lower icp, adrenaline administration has a strong potential to result in poor survival and neurological outcome (4). the elevation of intrathoracic pressure during chest compression generates carotid pressure and flow but also increases icp, which may be what limits cerebral blood flow (5-7). the lower the icp is, the lower is the resistance to forward blood flow to the brain. allowing complete sternal recoil after effective-depth compressions results in less intrathoracic pressure; this in turn produces greater coronary and cerebral perfusion (because of the greater perfusion pressures produced by the effective-depth chest compressions) and lower increases in icp (because of the complete sternal recoil). while lund university cardiopulmonary assist system (lucas) cpr ensures effective-depth compression with complete recoil, it may not mitigate enough the increased icp generated during chest compressions without the aid of gasping, unless perhaps an inspiratory impedance valve is used (8). gasping alone during vf arrest improves cerebral perfusion and decreases icp (9). therefore, without a means to mitigate intracranial pressure adequately during cpr, late adrenaline administration resulted in greater neural injury and, as evidenced, has a strong potential to result in poor survival and neurological outcome. declaration of interest: the author reports no conflicts of interest. correspondence: eric m. rottenberg, aas, 301b fenway road, columbus, ohio 43214, usa. e-mail: rottenberg.1@osu.edu (received 8 february 2015; accepted 17 february 2015) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1021936 http://informahealthcare.com/journal/ups mailto:rottenberg.1@osu.edu references 1. halvorsen p, sharma hs, basu s, wiklund l. neural injury after use of vasopressin and adrenaline during porcine cardiopulmonary resuscitation. ups j med sci. 2015;120:11–19. 2. donnino mw, salciccioli jd, howell md, cocchi mn, giberson b, berg k, et al. time to administration of epinephrine and outcome after in-hospital cardiac arrest with nonshockable rhythms: retrospective analysis of large in-hospital data registry. bmj. 2014;348:g3028. 3. semenas e, sharma hs, wiklund l. adrenaline increases blood-brain-barrier permeability after haemorrhagic cardiac arrest in immature pigs. acta anaesthesiol scand. 2014;58: 620–9. 4. rottenberg em. should unobstructed gasping be facilitated and confirmed before administering adrenaline, otherwise, give titrated vasopressin? am j emerg med 2015;33:286–9. 5. yannopoulos d, mcknite s, aufderheide tp, sigurdsson g, pirrallo rg, benditt d, et al. effects of incomplete chest wall decompression during cardiopulmonary resuscitation on coronary and cerebral perfusion pressures in a porcine model of cardiac arrest. resuscitation. 2005;64:363–72. 6. rubertsson s, karlsten r. increased cortical cerebral blood flow with lucas; a new device for mechanical chest compressions compared to standard external compressions during experimental cardiopulmonary resuscitation. resuscitation. 2005;65:357–63. 7. guerci ad, shi ay, levin h, tsitlik j, weisfeldt ml, chandra n. transmission of intrathoracic pressure to the intracranial space during cardiopulmonary resuscitation in dogs. circ res. 1985;56:20–30. 8. pantazopoulos in, xanthos tt, vlachos i, troupis g, kotsiomitis e, johnson e, et al. use of the impedance threshold device improves survival rate and neurological outcome in a swine model of asphyxial cardiac arrest. crit care med. 2012; 40:861–8. 9. srinivasan v, nadkarni vm, yannopoulos d, marino bs, sigurdsson g, mcknite sh, et al. spontaneous gasping decreases intracranial pressure and improves cerebral perfusion in a pig model of ventricular fibrillation. resuscitation. 2006; 69:329–34. 214 e. m. rottenberg http://www.ncbi.nlm.nih.gov/pubmed/24846323?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24846323?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24846323?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24846323?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24580085?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24580085?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24580085?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15733767?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15733767?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15733767?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15733767?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15919574?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15919574?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15919574?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15919574?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/3967345?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/3967345?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/3967345?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21983368?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21983368?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21983368?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/16494991?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/16494991?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/16494991?dopt=abstract ss1 declaration of interest references physiology of the senses—a prominent area of science in uppsala at the end of the nineteenth century full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 physiology of the senses—a prominent area of science in uppsala at the end of the nineteenth century bo s. lindberg to cite this article: bo s. lindberg (2015) physiology of the senses—a prominent area of science in uppsala at the end of the nineteenth century, upsala journal of medical sciences, 120:2, 78-89 to link to this article: https://doi.org/10.3109/03009734.2015.1029653 © informa healthcare published online: 20 apr 2015. submit your article to this journal article views: 380 view related articles view crossmark data citing articles: 1 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://doi.org/10.3109/03009734.2015.1029653 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1029653 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1029653 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1029653&domain=pdf&date_stamp=2015-04-20 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1029653&domain=pdf&date_stamp=2015-04-20 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1029653#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1029653#tabmodule upsala journal of medical sciences. 2015; 120: 78–89 physiology of the senses—a prominent area of science in uppsala at the end of the nineteenth century bo s. lindberg malma ringväg 41a, 75645 uppsala, sweden key words: anatomy, physiology, senses the first volume of upsala läkareförenings förhandlingar (proceedings of the upsala medical society) in 1865– 1866 contains a report from a lecture given by the newly appointed professor of physiology, frithiof holmgren, with the title ‘a method to make objective the effect of light on the retina’ (1). neither holmgren nor his listeners at that time understood that he had made a discovery of great scientific value. he had shown for the first time that impressions from a sense organ are transmitted to the brain through electrical impulses. instead of the subjective perception, an objective registration of impulses between the sensitive organ and the brain was possible. its importance for the understanding of the function of all sense organs cannot be overestimated. fifteen years earlier, holmgren’s great teacher, emil du bois-reymond, had shown that when a nerve is stimulated electrically, the electric impulse is transmitted along the nerve. the same phenomenon appeared after a physiological stimulation of the nerve as an index of the activity, and it was called the action potential current. another of holmgren’s teachers, herman von helmholtz, was the first to measure the velocity of the current. holmgren had worked in du bois-reymond’s laboratory in berlin in 1863–1864. when he returned to uppsala, he thought it would be possible to register impulses between the cornea and the optical nerve when the retina was exposed to light. for that purpose, he used eyes from frogs. in order to be able to register the weak currents he needed a very sensitive galvanometer. the best he could find at that time was wiedemann’s mirror bussol (bussola is italian for ‘compass’). he registered the current between an electrode on the cornea and another on the optical nerve and found that the eye responded with a current both when the light was shut on and off. holmgren thought that he had registered what he had intended to do, but the nerve had very little to do with the phenomenon. the apparatus (figure 1) was too insensitive to register the rapid, weak currents on the nerve sheath. it took him several years to understand what he had found. when holmgren continued his studies, he moved the electrodes against each other in different diameters of the eye bulb and understood that the potential had nothing to do with the optical nerve, but could emanate only from the retina. he published his new findings in swedish five years later (2), but they became internationally known only when they were printed in a german paper edited by his friend willy kühne, who had succeeded herman von helmholtz as professor of physiology in heidelberg (3). already in his first publication holmgren had discussed the possibility of studying the effect of coloured light on the retinal current. he thought, in analogy with the auditory organ, that oscillations with different wave-lengths should give different deviations on the galvanometer, but he did not proceed on these projects. the reason for that may have been that the galvanometers of that time were too insensitive and laborious. with the development of correspondence: bo s. lindberg, uppsala university, uppsala, sweden. e-mail: bo.sven.lindberg@gmail.com (received 25 february 2015; accepted 11 march 2015) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1029653 http://informahealthcare.com/journal/ups mailto:bo.sven.lindberg@gmail.com modern electronic equipment, electroretinography has today become a valuable tool in the diagnosis of eye diseases in humans and animals. problems with colour vision were to occupy most of holmgren’s thoughts during the rest of his life and made him much more known among the general public than the scientifically more important work on the retinal current. the problems with perception of colours could not be solved with the electro-physiological methods he had used earlier, but required experiments on human beings. only once did he return to problems related to the current. the anatomist heinrich müller had found orange and red pigments (rhodopsin or visual purple) in the retina, and it had been proposed that these were important for the formation of pictures on the retina. kühne had shown that the pigments were rapidly bleached by light and regenerated rapidly in darkness. holmgren’s aim was to elucidate the relationship between the pigments and the electrical current. after experiments with eyes from frogs and rabbits, he found that the visual purple and the retinal current are independent of each other. he even drew the conclusion that the visual purple is of no importance for the vision at all, a too rash deduction (4). studies with modern electro-physiological techniques have made it evident that reactions when light is shut on and off are indispensable for visual discrimination. the outlines of the visual objects are brought into relief, i.e. enhanced, when the eye is moved with small oscillations and when rhodopsin rapidly is bleached and reactivated. rhodopsin is also necessary for adaptation to darkness and bright light. colour-blindness in 1867, a new physiological laboratory was built after a donation from anders fredrik regnell (5). for the first time, physiology had got a place of its own. six rooms and sufficient equipment were now available, which proved to be necessary. in the next decades holmgren had to examine several hundreds of individuals for colourblindness for his studies of its occurrence in the population. during a year from august 1869 to august 1870, holmgren worked in heidelberg under the guidance of one of the greatest physiologists ever, herman von helmholtz. among his many interests, helmholtz studied colourblindness. in 1802, thomas young had postulated the existence of three types of photoreceptors (now known as cone cells) in the eye, each of which was sensitive to a particular range of visible light. helmholtz developed the theory further in the 1850s. according to the young–helmholtz theory of colour vision, there are three receptors in the retina that are responsible for the perception of colour. one receptor is sensitive to the colour red, another to green, and a third to blue (or violet). these three colours can then be combined to form any visible colour of the spectrum. light stimulating all the types of cones is perceived as white. helmholtz was conscious that the threecolour theory not could be proven anatomically. he therefore tried to bridge the gap between the microscopic picture of the retina and the physiological analyses of colour vision. helmholtz divided the retina in three chromatic zones, which had different distributions of cones and rods. during his stay in heidelberg, holmgren took part in the practical experiments on these zones. he had his vision field examined with a perimeter by the figure 1. the equipment holmgren used when he detected the retina current. the apparatus belongs to the museum of medical history in uppsala. physiology of the senses 79 russian ophthalmologist mikhail mikhailowitsch woinow. it turned out that he had a small defect in the redblind middle zone. the central zone merged over to the peripheral zone directly at this spot. the interpretation was that such variations of the colour vision may occur normally (6). when holmgren had returned to uppsala, he published a series of studies on the subject (7). he also made an appeal to the medical profession in sweden to report to him all cases of colour-blindness they had detected. in that way he got access to the greatest statistical material of colour-blindness at the time (32,165 men and 7,119 women) and found that 3.23% of males and 0.26% of females in sweden were colour-blind. he also noted that colour-blindness was hereditary and was inherited from the mother’s side (8). once, holmgren had the opportunity to examine two colour-blind sisters, but he did not mention if their parents had impaired colour vision (9). the chromosomes and the connection with the x chromosome were unknown at that time. red–green colourblindness is passed from mother to son on the x chromosome, which is known as the sex chromosome because it also determines sex. females have two x chromosomes and males one x and one y chromosome. the faulty gene for colour-blindness is found only on the x chromosome. so, for a male to be colour-blind the faulty gene only has to appear on his single x chromosome. for a female to be colour-blind it must be present on both of her x chromosomes. if a woman has only one colour-blind gene she is known as a carrier, but she will not be colourblind herself. when she has a child she will give one of her x chromosomes to the child. if she gives the x chromosome with the faulty gene to her son, he will be colour-blind, but if he receives the ‘good’ chromosome he will not be colour-blind. the lack of knowledge about the causes of colour-blindness explains why holmgren devoted himself to two laborious studies we now know to have been futile. in one, he tested a statement in a german book grundzüge der physiologischen optik by herman aubert that colour-blind persons had a shorter distance between the pupils of their eyes. holmgren constructed a special apparatus for the study and then tested 100 colour-blind individuals and 100 without any colour vision deficiency. he concluded that the distance between the pupils had no correlation to colour-blindness (10). in may 1880 frithiof holmgren proclaimed a reward of 2,000 swedish crowns to those who put at his disposal for investigation a person with a complete, inborn colour-blindness on one eye and a normal colour vision on the other (11). the prize had been put at his disposal by a person whose name not was given. it corresponds to over 100,000 crowns today. in several papers he later reported a few cases of alleged one-sided colour-blindness, but in no case had those individuals been examined earlier in life. thus, the deficient colour vision may have been due to a retinal disease in one eye. there were several different methods for testing colour-blindness before holmgren’s time. most often colour vision deficiency was reported simply by subjective descriptions of what the test person saw. in 1837 august seebeck used a more advanced technique. he used a set of more than 300 coloured papers and let people match or find a colour closely related to a sample colour. this type of colour vision test abandoned the naming of colours, which differs a lot between persons. holmgren adapted this kind of test by using skeins of wool instead of coloured papers. the examined person had to match one piece of wool to the samples in a box. there were light and dark shades to confuse the person. the advantage to all other methods was that it was simple, rapid, and inexpensive. an examination took less than a minute, compared with about an hour with the old methods. the holmgren wool test was widely used and even commercially available more than 100 years later (12). in the night of 15 november 1875 a railway accident occurred in lagerlunda near the bankeberg station in ostrogothia. nine people were killed, among them the engineer, the oiler, and the firemen. if only death and injury were considered, the lagerlunda incident was not remarkable. every year more than 1,000 people were killed in england in railway accidents during the 1870s. one reason that contributed most to the national and international horror of the disaster was a sketch by the later famous artist carl larsson, then part time illustrator for ny illustrerad tidning. the sketch was reproduced by a woodcut procedure and published in many newspapers around the world (figure 2). during the ensuing trial, only the stationmaster at bankeberg was found guilty and sentenced to jail. the court’s decision was met with criticism, and it was commonly thought that he had been made a scapegoat by the management. according to a recent review of the event, there were a lot of circumstances pointing at several broken regulations by higher-placed persons in the railway company (13). holmgren suspected that the engineer had been colour-blind, but he had lost his life. on 15 july 1876, holmgren gave an account of his method of screening with the wool test at a nordic medical congress in gothenburg. the assembled physicians resolved that it was necessary to investigate the occurrence of colour80 b. s. lindberg blindness among the staff of the railways. holmgren also wrote a letter to the management of statens järnvägar (swedish state railways); they were, however, sceptical: if colour-blindness really existed, it could not, at any rate, be amongst their employees, or it would undoubtedly have been noticed. they had had a lot of possibilities to prove their ability to distinguish signals. but holmgren got permission to test all employed by the railroad between uppsala and gävle. among the 266 tested, he found 13 that were colour-blind. he also got permission to perform a demonstration before the board of statens järnvägar, which took place on 13 october 1876 in uppsala. in a dark room, the members of the board were sitting on one side of the room. at each end of the room, holmgren had stationed one red-blind and one green-blind conductor, each with a signal lantern. they were instructed that when one signalled to the other, the second should respond with the same signal. the general director of statens järnvägar was to order what signal to show. he chose ready, i.e. white. the first conductor showed red to which the second responded with green. in the silence that followed, holmgren said: ‘gentlemen, now that we have seen this, it is clear that if any accident occurs as a result of colour-blindness, there is no doubt about where the responsibility lies’. three days later, statens järnvägar issued an order requiring all railroad physicians to screen for colour deficiency with holmgren’s method. his article ‘colour-blindness in its relation to accidents by rail and sea’ was translated to french, english, german, russian, and italian and was referred to in danish, polish, and spanish publications (14). as a consequence of this, the laboratory in regnellianum was visited by numerous scientists from near and far. what neither the colour-blind conductors nor the members of the board knew was that holmgren had manipulated the lanterns. he knew that colour-blind persons were accustomed to judge colours by their luminosities and had placed coloured glass of different thickness in the lanterns. in a paper two years later, he reported on a method to make a correct diagnosis on a person who simulates colour-blindness or is trying to conceal his handicap, but he did not mention that he had used this trick in front of the railway board (15).the secret was revealed only after his death in a footnote in the obituary written by his successor as professor, hjalmar öhrvall (16). politically, holmgren was liberal, and his wife ann-margret tersmeden was a member of the radical student association verdandi and a member of its editorial board. thus, it is worth noting that holmgren, although he stressed that a colour-blind person could not get a position in a railway company, he cared about those already working as engineers or in other positions where perfect colour vision was needed. in his paper, he elaborated at length about the problems of discharging people with long, immaculate service on the grounds that they had an inborn error. he suggested some sort of compensation or that they should be transferred to new posts, where colour-blindness was no hindrance. gustaf göthlin, professor of physiology in uppsala 1918–1939, found that some persons had a weak colour sense in spite of normal results with holmgren’s wool test. a person having normal vision will need a particular intensity ratio of red, green, and blue primary colours to see white; he is a normal trichromate. some individuals, however, need more of one of the primary colours than the other two; these are anomalous trichromates. with a double spectroscopic method devised by göthlin, these persons could be found (17). his method, however, was too complicated, and in 1934 pseudo-isochromatic pictures were introduced for the testing in sweden and most other countries. holmgren’s wool method had then been used for 58 years. in the 1880s, holmgren became more interested in the theoretical aspects of colour vision. he was an ardent believer in the young–helmholtz theory, but there were other competing theories. the best known was proposed figure 2. medal engraved in 1964 in commemoration of the 100th anniversary of holmgren’s detection of the retina current by the royal swedish academy of sciences. sculptor leo holmgren. on the medal is engraved ‘luci retinam electrice respondere primus demonstravit’, i.e. ‘he was the first who demonstrated that retina responded on light with an electric current’. uppsala university coin cabinet. physiology of the senses 81 by the german physiologist ewald hering. he disagreed with young and helmholtz that colours are based on three primary colours: red, green, and purple (or blue). he instead believed that colour vision is based on a system of colour opponency. in this model colours are perceived through receptors sensitive to three couples of opposing colours: red–green, yellow–blue and white–black. his colour concept gave rise to mostly fruitless controversies, because it appeared to be incompatible with the theory put forward by young and helmholtz. holmgren defended the young–helmholtz theory in a sometimes very excited debate (18). it must be said, however, that helmholtz himself took a much more active part in the dispute. in a lecture in honour of gustaf göthlin when he retired from the chair of physiology in 1939, ragnar granit (1900–1991), professor of neurophysiology at the karolinska institute and nobel prize laureate in 1967, presented a method to register impulses from single nerve fibres of the retina with microelectrodes. he used glass tubes with silver cores pulled out to fine tips and applied them directly onto the retina of frogs. the experiments were carried out under micro-illumination. the spectral lights were reflected internally from the silver-coated glass rods. when the light spot and the microelectrode were adjusted relative to one another in order to obtain a precise response, the distance between the two tips was such that only the optic nerve fibres themselves could be considered as sources of the spikes obtained (19). later, granit found further experimental support of the trichromate theory of young and helmholtz (20). his pupil, the finnish-swedish-venezuelan neurophysicist gunnar svaetichin found experimental support for the opponent theory of hering (21). it is now clear that the two colour concepts can be reconciled with one another. nowadays, we know that the human being possesses three types of receptors, which are combined in three opponent channels as proposed by hering. thus, both hering and helmholtz were right. frithiof holmgren, always very keen on applied physiology, would have been pleased if he had lived to see this development. as a young medical student the future professor of physiology at the karolinska institute, jöns johansson, was holmgren’s amanuensis in physiology. holmgren inspired him to study the colour sense in the area just around the blind spot. in an investigation on his own right eye, johansson found that the perception of red and green ceases gradually towards the macula until a complete colour-blindness occurs, just as in the periphery of the retina (22). in a later report, holmgren stated that johansson’s colour vision was not quite normal, which is why he repeated his investigation of the conditions around the blind spot in his own eyes. he confirmed johansson’s finding but extended it to a scotoma he had had in the central part of his right eye since 1880. he found that colour-blindness appeared around the scotoma, just as around the blind spot. he stated that in any part of the vision field near a blind area, normal colour vision does not cease instantly, but there is a gradual transition through a red-blind zone before total colour-blindness develops (23). the scotoma gave him a partial macropsy, a defect of vision in which objects appear to be larger than their actual size. towards the end of his life, he tried to determine the nature of this scotoma. in order to do so, he measured the enlargement by comparing the size of a sheet of paper, as seen by the right eye with the size seen by his healthy left eye. he calculated the enlargement to be 2.5 times vertically and 1.14 times horizontally. he also tried to determine the size of the scotoma through an examination with a perimeter, but was not satisfied with the results. he wrote: ‘a more accurate information about the scotoma can only be accomplished through a direct microscopic examination of the eye. i have thus donated the eye to my colleague professor j. a. hammar’. on 14 august 1897, when holmgren died, it became evident that it was not legal to take out the eye immediately, but only after 24 hours. his widow ann-margret then said: ‘had frithiof known this, he would certainly have tried to get exemption from the law’; now it was too late. after 24 hours a post-mortem examination was performed and the right eye removed and examined. the postmortem decomposition prevented a detailed microscopic examination, but some old haemorrhages close to the central fovea were found. the results from the examination were printed in proceedings of the upsala medical society, the same journal where his greatest scientific discovery had been published 32 years before (24). holmgren was not the only person at this time to donate an organ to science. many neuroscientists thought that it was possible to find a correlation between the size and configuration of the brain and the person’s intelligence and character. the famous scientists gustaf retzius, sonja kovalevsky, and salomon eberhard henschen had their brains examined. holmgren’s own pupil, magnus blix, donated his brain to his friend magnus fürst, professor of anatomy in lund. the result of the investigation was published 14 years later in a publication in honour of the 250th anniversary of the foundation of the university of lund (25). holmgren was the first professor of physiology in uppsala (figure 3). he put his stamp on the department and his pupils, many of whom were to continue his research in various fields of physiology of the senses. 82 b. s. lindberg the specific nerve energies of the skin magnus blix magnus blix was the first of holmgren’s pupils to make his own scientific career. he had been an assistant in physiology since 1872 and demonstrator since 1882 until 1885, when he was appointed professor of physiology in lund. ever since antiquity, sense organs had been considered as a kind of entrances, into which the impressions from objects in the surrounding world penetrated along the nerves and were integrated into what was called sensorium. the law of specific nerve energies, first proposed by johannes müller in 1826, rather states that the nature of perception is defined by the pathway over which the sensory information is carried. therefore, differences in perception of seeing, hearing, and touch are not caused by differences in the stimuli themselves but by the different nervous structures that these stimuli excite. for example, pressing on the eye elicits sensations of flashes of light because the neurons in the retina send a signal to the occipital lobe. despite the sensory input being mechanical, the experience is visual. the previous conception was that all perceptions could be provoked from every spot on the skin, which was not in accordance with müller’s law. blix thought that, by applying a localized excitation, it should be possible to register different local responses from the nervous end organs in the skin. he thus applied a weak faradic current with a thin metal point. the current evoked distinct sensations of warm or cold on the skin surface. in his most famous work blix demonstrated the existence of specific end organs in the skin for the different perceptions of touch, cold, and warmth (26). he showed that there were different end organs for different sensations and called them cold, warmth, and pressure points. in order to study them, he constructed a simple but ingenious piece of equipment (figure 4). by putting the fine cone on the skin and marking with different colours the sensations he or his assistant andersson perceived when the cone was cold, warm, or just pressed on the skin, blix could map the areas for the different sensations.hefoundthat theyveryrarelycoincide.hewrote,however,thatsensationsofwarmthandcoldsometimes could be evoked from the same spot, a view torsten thunberg later confirmed. blix’s research was not well known internationallyuntil itwaspublishedingerman in1884.independentlyofeachother,alfred goldscheider,professor at theuniversity of berlin in 1884, and henry donaldson,in 1885 at johnshopkins university, reported that the skin was not continuously sensitive, but instead, had specific loci for specific tactual senses. blix also tried to demonstrate separate trigger points for pain, but writes: ‘if there are such points, other methods are needed in order to prove them’. this was done in 1894 when max von frey, professor of physiology in würtsburg and zürich, proposed that pain is an independent tactile quality, together with touch, heat, and cold. hjalmar öhrvall, holmgren’s successor as professor of physiology in uppsala, characterized blix’s findings in the following exuberant words: ‘since olof rudbeck in 1650 detected the lymphatic vessels, the swedish physiology has not made such an important scientific discovery as the discovery of the cold-, warm-, and pressure points, one of the most important within the physiology of the senses. it has already given inspiration to several other scientists’ (27). figure 3. the railway collision in lagerlunda. a sketch by carl larsson in ny illustrerad tidning, 20 november 1875. physiology of the senses 83 blix proved the universality of müller’s law of specific nerve energies, and the idea that a nerve fibre could propagate impulses of different kinds was forever abandoned. when blix became professor of lund, he did not pursue his investigations of the punctuate sensitivity of the skin, but devoted the rest of his life to the study of muscular physiology. torsten thunberg one of the scientists inspired by holmgren and blix was torsten thunberg, successor to blix as professor of physiology in lund in 1905. he continued the studies of the senses of the skin. in order to determine the threshold of the sensitivity for warmth and cold, he constructed a series of silver plates with an area of 2 cm2 and a weight between 2 and 500 mg. the plates were glued to a cylinder of cork in order to make them easier to handle. silver was chosen because of its high warm conduction. the warm capacity of the silver plates correlated with their thickness. when a plate of a certain temperature and mass was applied to the skin, it deprived the skin of warmth. thunberg could by such means estimate the minimum perceptible stimulus (28). with the equipment that thunberg had constructed (figure 5) he also investigated the simultaneous occurrence of the sensation of warmth and cold. he set up two separate spiral brass tubes that were connected by rubber tubes to retorts with warm and cold water. the coils of the tubes were interlocked as shown to the left in the figure; a modern thunberg grill is shown to the right. when thunberg applied his coil to the skin, he made an unexpected observation. innocuous warm and cool coils that were spatially interlaced produced a painful burning sensation. he could not wholly explain his finding. his first hypothesis was that the warm and cold nerve endings were situated at different depths from the skin surface, but he dismissed this idea. instead he speculated on some sort of direct and indirect nerve stimulation. the nerve endings were thought to be extra sensitive to weak thermal stimulation, but were reacting with a long latency. with direct nerve stimulation, the latency time disappeared. simultaneous stimulation on both these manners entailed the painful sensation (29,30). figure 4. equipment constructed by magnus blix to define the three different sense points. a: 1, a bottle with cold water; 2, a retort with hot water; 3, a hollow electroplate cone. b: cold and warmth points on 1, the dorsal side of blix’s hand; 2, his wrist; 3, his elbow; and 4, dorsal side of j. h. andersson’s left hand. 84 b. s. lindberg thunberg’s discovery was not fully understood during his lifetime and was forgotten for many years, but it has now been re-evaluated. it provides an interesting model of integrative mechanisms in the nervous system, supposed to be relevant in explaining the hypersensitivity found in chronic pain of unclear etiology. it is now called the thunberg (or thermal) grill illusion, or just the thunberg illusion. it has been suggested that the thermal grill illusion could be of clinical relevance, casting light on mechanisms involved in neuropathic pain including cold-allodynia. the occurrence of cold-allodynia may also be related to the pathological processes involved in chronic wide-spread pain and is, for instance, common in fibromyalgia (31). the illusion is created by an interlaced grill of warm (e.g. 40�c) and cool (20�c) bars. when someone’s hand presses against the grill, he or she experiences the illusion of burning heat. but if the person presses against only a cool bar, only coolness is experienced; if the person presses against only a warm bar, only warmth is experienced. hjalmar öhrvall hjalmar öhrvall had been an amanuensis at the physiological laboratory, and when he had defended his doctoral thesis in 1889 he became a demonstrator. in 1899, he succeeded holmgren as professor. in his thesis (32), öhrvall first discussed how taste should be defined. according to older opinions, there were about 10 different tastes. linné, for example, mentioned 11 in sapor medicamentorum. his basis of division was the use of medicaments (33). at the end of the nineteenth century, however, physiologists had agreed on just four tastes: salt, sweet, sour, and bitter. öhrvall translated some of helmholtz’s books into swedish and was an ardent supporter of his theory of modality, which states that there are various types of sensation, such as vision, hearing, smell, temperature, pressure, and taste. within each sense, there are differences in quality, such as red, blue, yellow, etc., for vision, and low and high pitch for sound. when two colours are put together, for example blue and green, they create a new, yellow, and when two simple tones are put together they create a complex tone. taste was different. a mixture of salt and sweet did not create a new taste, but still tasted both salt and sweet. accordingly, taste should not be considered as a single modality with four qualities, but as four different senses without any merging between them. öhrvall was inspired by blix’s method of stimulating separate end-points in the skin when he proved that there were separate points for the different qualities of sense. the existence of the taste buds had independently been discovered by gustav schwalbe and christian lovén in 1867. öhrvall decided to use blix’s method in order to investigate if there were different taste buds for the taste of salt, sweet, sour, and bitter. he used a concave mirror, which gave a sufficient magnification so that he could distinguish the different buds on his own tongue (figure 6). solutions of the tested substances were applied to the different buds with a fine brush. öhrvall found that in the papillae fungiformis at the tip of the tongue, of the 91 sensitive to acid, only 12 papillae were sensitive exclusively to acid. and whereas 79 were sensitive to sweet, only three papillae were sensitive to just sweet. no papillae responded only to bitter, although 71 responded to bitter and another taste solution. he excluded the results obtained with salt, because the sensations elicited were not distinct enough. his conclusion was that there were different end organs for the different taste perceptions. these end organs are present in different combinations on the tongue (32). öhrvall did not pursue his studies of the sense of taste, but almost 50 years later, his doctoral thesis was reported in detail in emil von skramlik’s comprehensive textbook psychophysiologie der tastsinne. figure 5. thunberg’s brass coils from 1894 (left) and a modern thunberg’s grill (right), in which fifteen 1-cm-wide silver bars are set about 3 mm apart. alternate (evenand odd-numbered) bars can be heated or chilled independently. physiology of the senses 85 sydney alrutz and the perception of heat sydney alrutz had quite another background than the other scientists working at the department of physiology in regnellianum. his father was the owner of an iron export company in stockholm, and when sydney had passed his high school examination in 1886 it was taken for granted that he would join the family business. during the following years he studied iron melting and passed falu bergsskola in 1888. but he became more interested in philosophy and theosophy. in july 1891 he was the swedish delegate at the first annual convention in london of the theosophical society of europe. in 1892, alrutz attended lectures in philosophy in uppsala and at the same time followed courses in physiology, anatomy, and histology at the faculty of medicine. both frithiof holmgren and hjalmar öhrvall welcomed him, and he started to participate in the physiological experiments of the senses of the skin. in alrutz’s first publication, he did not merely repeat blix’s studies from 1882 of the law of the specific energies of the nerves, but could also convincingly show that the criticism against blix’s findings was inaccurate and based on faulty techniques (34). in a following paper ‘on the sensation “hot”’ (35) he used a series of thin silver plates of different thickness designed by thunberg to test the thermal sensations released by cold and hot stimulus. he described a perception called heat, a quality to be distinguished from warm. at that time, alrutz was just a student at the faculty of science, but he wanted to pass the examination for filosofie licentiat. according to the rules, the examination must comprise three subjects; two of them were theoretical and practical philosophy. as a third subject, alrutz wanted to include physiology, but that belonged to the faculty of medicine. he had to apply to his majesty the king for an exemption from the rules. his application was supported both by the two professors of philosophy and by the faculty of medicine. he argued for a combination of philosophy and physiology because he thought psychologists needed insight in the functions of the human body and the physiologists needed knowledge about the mind. his application got a forceful support from the faculty of medicine and was approved by the king (36). in 1900 alrutz passed his degree as a licentiate, and in 1901 he defended his thesis ‘investigations into the sense of pain’ (37) and became a docent the same year. in 1902, he established a psychological laboratory in two rooms at the bottom floor of regnellianum. it was used for courses in which 530 participants took part during the years 1902–1908. later, regular laboratory courses started, and in all of them he was supported by öhrvall and the other physiologists. after some years, however, alrutz’s theosophical interests took over, and he started studies of occult phenomena such as telepathy and clairvoyance. through what he called nerve radiation, he argued that he could affect a person at a distance by moving his hand upwards or downwards over the body without touching him. he called these movements passing. upward passes increased the sensitivity and downward passes decreased it (figure 7). alrutz performed numerous experiments � mainly with a single participant and himself as the experimenter � in order to eliminate alternative explanations such as telepathic phenomena or suggestion. his assumption was that information could be transferred over distance, and that this transference could not be explained within the framework of ordinary physical laws. his main study person was karl wennersten, a bicycle repairer, who had consulted him because of nervousness. all these activities must have appeared strange to the rational scientist öhrvall, but it is difficult to find official statements from him on what he thought about the line of research going on in the two rooms at regnellianum. öhrvall seems to have reacted only once. in april 1905, alrutz gave a lecture at the association minerva, whose figure 6. hjalmar öhrvall and the tip of his tongue magnified twice. the picture is drawn according to a photograph and shows the position and relative size of the papillae fungiformis referred to in his thesis. (uub, kartoch bildenheten, id 7255). 86 b. s. lindberg goal was to promote co-operation between practitioners from different disciplines. he talked about ‘the phenomenon of table-turning and its explanation’. the newspaper dagens nyheter then received an anonymous letter under the heading ‘the occult science in uppsala’. according to the letter, öhrvall had been present, and he stated that he was not familiar with the literature within the field, but if the occult science was true, many of the findings of modern natural sciences were wrong. he also pointed out that if the forces alrutz talked about were true, it would be difficult to do chemical analyses and would even destroy the confidence in provision and poultry dealers, when they, in spite of legally hallmarked weights, could make unlawful profit because of the peculiar psychical force. the letter pointed out that öhrvall’s opinion was that alrutz’s lecture was ridiculous and puerile. in a reply alrutz protested vehemently against the content of the letter and that his 11/2 hour long lecture only was related in a few lines. professor öhrvall’s short contribution to the discussion, on the other hand, was related almost completely, but he did not write that öhrvall was quoted incorrectly. this statement of alrutz indicates that öhrvall in fact was very critical (38). during the following years, alrutz studied the influence of hypnosis on the senses. the results have been summarized in his opus magnum, ‘the dynamics of the nervous system’ (39). the book comprises 517 pages and has seven appendices. according to the book, hjalmar öhrvall and gustaf göthlin, professors of physiology, both took part in the experiments at the physiological laboratory and were sometimes performing the passes themselves. professor swedberg, the nobel prize winner in chemistry 1926, participated in many séances both at the department of chemistry and in alrutz’s dwelling in kåbo in the years around 1911–1914. he was even performing passes himself and is quoted as being positive to the séances. among other prominent professors from the faculty of medicine in uppsala taking part in alrutz’s séances were gunnar forssner, ragnar friberger, johan wilhelm hultcrantz, ivar thorling, gustaf bergmark, august hammar, and frey svensson. professors johan edgren and frithjof lennmalm from the karolinska institute participated in experiments in stockholm. alrutz also demonstrated his passing method before the upsala medical society and the minerva society. from the faculty of theology bror jonzon, later bishop, and emmanuel linderholm, professor of church history, and from the faculty of science the professor of philosophy, erik olof burman, were all present at many occasions and even made passes themselves. thus, a lot of the leading professors at the university accepted alrutz’s methods and showed a positive interest in his performances; what they really thought is difficult to know as the account is based solely on alrutz’s own statement. as a medical student, the future chairman of the swedish medical association and professor of paediatrics at the karolinska institute, curt gyllenswärd, gave an account of an experiment he had carried out on a hysterical patient of the effect of hypnotic passes on the sensibility of the skin and the colour vision. gyllenswärd concludes: ‘as regards the changes of the sensibility brought about by the passes, i must regard these to be caused by some sort of transferred nervous energy’. as a medical student, bernhard jacobowsky, professor of psychiatry in uppsala 1932–1960, investigated the influence of hypnosis on the perception of red and the stimulation threshold (minimum perceptible) for the sense of light on the same patient. upwards passes decreased the threshold for the sense of light and the figure 7. sydney alrutz and karl wennersten during an experiment on 27 april 1913. according to the figure text in ‘till nervsystemets dynamic’, alrutz is doing downward passes with both hands at the same time. photographer: östling, uppsala. physiology of the senses 87 threshold for red, and the downward passes acted in the opposite direction. his conclusion was that nothing contradicts the hypothesis of alrutz that the treatments imply the transference of nervous energy from one person to another through some sort of hitherto unknown form of radiation. both these experiments were performed in the dark-room of the physiological laboratory in 1917. alrutz believed very strongly in the effect of passes on nerve radiation and wanted to convince others about its effect. this, perhaps, is why he invited so many prominent men in the scientific community to attend his séances. he tried to convince them empirically through experiments rather than by presenting ideas that could make the phenomenon understandable and lead to testable hypotheses. alrutz’s contributions in the physiology of the senses are not comparable to the previously mentioned scientists, but he started the psychological laboratory at the department of physiology, which was the first in sweden. alrutz’s educational programme was also a pioneering achievement in swedish psychology. in spite of his deviations from established science, alrutz was an important pioneer for experimental psychology in sweden. his book ‘on the study of mental life on the basis of physiology’ from 1899 was a powerful plea for psychology as an independent academic discipline, but a department of psychology was not established until 1948, when the department of education was divided into psychology and education. e. louis backman and the sense of smell the last contribution from uppsala to the physiology of the senses at the turn of the previous century stems from eugène louis backman. already in 1891, öhrvall had written about some physical peculiarities of organic compounds with strong scent, but he had not continued this research until late in his time as professor (40). in 1917, the year öhrvall retired from his chair as professor, backman published an experimental study where he found that the intensity of a smell is increased when the water solubility of the compound is decreased and is increased with increased lipid solubility. backman’s conclusion was that the ability of the compounds to stimulate the smell sensation must be due to these changes and to the concentrations of them in the peripheral nerve endings for smell (41). backman’s conclusion is consistent with ernest overton’s findings some 20 years earlier. overton investigated the osmotic properties of cells and noticed that the permeability of molecules through membranes is related to the lipid solubility of the compound. this is of fundamental importance for the action of anaesthetic agents (42). backman also tested alrutz’s allegation that the patients had a heightened sense of smell during light hypnosis. the experiment subject was first tested for acetic acid and benzaldehyde. alrutz then made the patient’s left side insensible through a downward pass on this side and hypersensible on the right side through an upward pass and applied a light hypnosis. when backman then tested the patient’s ability to smell, he found that the ability to recognize acetic acid had increased twofold and benzaldehyde fivefold as compared with the awake state. it must be stressed, however, that just one, unidentified, test person was examined and that only three different concentrations of the test substances were used, each concentration just once. declaration of interest: the author reports no conflicts of interest. the author alone is responsible for the content and writing of the paper. references 1. holmgren f. metod att objektivera effecten af ljusintryck på retina [a method to make objective the effect of light on the retina]. upsala läkareförenings förhandlingar. 1865–1866;1:184–98. 2. holmgren f. om retinaströmmen. upsala läkareförenings förhandlingar. 1870–1871;6:419–55. 3. holmgren f. über die retina-ströme. 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j. sapor medicamentorum; thesis defended 23 february 1751 under the presidium of linné. uppsala university, uppsala. 34. alrutz s. bidrag till kännedomen om hudens kalloch varmpunkter. upsala läkareförenings förhandlingar. 1896;2:246–63. 35. alrutz s. om förnimmelsen hett [on the sensation ‘hot’]. upsala läkareförenings förhandlingar. 1896;2:340–59. 36. alrutz s. om själslifvets studium på fysiologisk grundval [on the study of mental life on the basis of physiology]. stockholm: skoglund; 1899. 37. alrutz s. undersökningar öfver smärtsinnet [investigations into the sense of pain]. uppsala universitets årsskrift. 1901. 38. debate article: den ockulta vetenskapen i uppsala. dagens nyheter, 23 may 1905. 39. alrutz s. till nervsystemets dynamik [the dynamics of the nervous system]. almqvist & wiksell, uppsala; 1917. 40. öhrvall h. om några fysikaliska egendomligheter hos luktande ämnen af organiskt ursprung. upsala läkareförenings förhandlingar. 1890–1891;26:249–58. 41. backman el. experimentella undersökningar öfver luktsinnets fysiologi. upsala läkareförenings förhandlingar. 1917;22:319–470. 42. overton e. studien über die narkose zugleich ein beitrag zur allgemeinen pharmakologie. jena: gustav fischer verlag; 1901. physiology of the senses 89 http://www.ncbi.nlm.nih.gov/pubmed/22301271?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/13444020?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21423614?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21423614?dopt=abstract ss1 colour-blindness the specific nerve energies of the skin magnus blix torsten thunberg hjalmar &ouml;hrvall sydney alrutz and the perception of heat e. louis backman and the sense of smell declaration of interest references uppsala clinical research center—development of a platform to promote national and international clinical science article uppsala clinical research center—development of a platform to promote national and international clinical science lars wallentin and bertil lindahl uppsala clinical research center, uppsala university, uppsala, sweden abstract uppsala clinical research center (ucr) is a non-profit organization that provides service for clinical research aiming for development and improvement of health care in sweden and worldwide. ucr was started in 2001 with the ambition to shift the focus of clinical research from new medications or devices launched by the industry to problem-based research on issues identified in clinical reality, for example through the national quality registries. in order to accomplish these goals, ucr has established services in: 1) clinical trials of new and old methods in health care; 2) quality development of the health care system supported by internet-based national quality registries; 3) biostatistics, epidemiology, and data management; 4) biobanking of biological materials (uppsala biobank); 5) high-throughput biochemical analyses (ucr laboratory); and 6) academic leadership by the members of the ucr research faculty. the ucr clinical trials group provides services for investigator-driven projects in all areas of health care, for global mega-trials on new pharmaceutical treatments and devices, for biobanking including biomarker and genetics analyses, and for clinical events adjudication in national as well as global mega-trials. during the last few years, ucr has been a pioneer in establishing the registry-based randomized clinical trial (r-rct), which today is an international model on how to perform cost-effective pragmatic randomized trials in the real-world environment. in 2002, ucr started the first national competence center for national quality registries, which pioneered the development of the current internet-based technologies for registering, reporting, and supporting continuous systematic improvement of health care. ucr is currently harboring around 20 national quality registries in all areas of health care. today, ucr is the leading european center for registry-based quality development and evaluation of new medical treatments in cardiovascular care and has started to support other european countries in implementing the ucr registry platform in order to improve quality of care in the european union. article history received 2 october 2018 revised 22 october 2018 accepted 22 october 2018 key words biobank; biomarkers; clinical research; clinical trials; genetics; health economy; quality development; registries introduction uppsala clinical research center (ucr) is a non-profit organization that supports research and quality development in health care in sweden and internationally. the center was founded on 1 july 2001, based on a proposal from the authors of this manuscript. the reasons for the creation of ucr were the needs to support the nationally and internationally successful clinical research groups on cardiovascular, metabolic, cancer, and infectious diseases, and the close collaboration of these research groups with the advanced clinical and basic science laboratories in uppsala (1–4). there was also an urgent need to support the national quality registries in cardiovascular care, which at this time pioneered the utilization of internet-based technologies as well as quality development and research in this area (5–7). at this time, clinical research in sweden was in a period of crisis because of low status for medical science and medical faculties, few opportunities for systematic training and education, few career positions, and decreasing grant funding (den kliniska forskningens kris och pris, mfr-rapport 5; 1998 (isbn:91-85547-12-3)). clinical research projects were often limited to either being a minor contributor in industry-related international mega-trials or performing local, underpowered, underfunded research projects with small chances to produce results with international impact. ucr was started with the ambition to shift the focus of clinical research from new medications or devices launched by the industry to problem-based research on issues identified in clinical reality, for example through the national quality registries. the objectives of clinical research needed to be renewed by updating the knowledge on occurrence and natural courses of disease, utilization of old and new treatments, and their effects in real-life health care. both old and new methods for diagnosis, prognostication, decision support, and treatment should be continuously evaluated by scientific methods. a similar systematic scrutiny should be applied for interventional treatments with surgery, endoscopic, and catheter-based treatments as for new medications. the ambition of ucr was to support the development of a systematic learning process, which should also facilitate a better controlled introduction of new methods and elimination of old contact lars wallentin lars.wallentin@ucr.uu.se department of medical sciences and uppsala clinical research center (ucr), uppsala university, uppsala science park, hubben, dag hammarskj€olds v€ag 38, se-751 85 uppsala, sweden � 2018 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 1, 1–5 https://doi.org/10.1080/03009734.2018.1540506 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1540506&domain=pdf http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1540506 http://www.tandfonline.com ones in the health care system (figure 1). in addition, a modern scientific approach was also needed to support the inclusion of issues on quality of life, health economy, and prioritization in all clinical research projects (table i). from the start, ucr has had the same overriding main objective, which is to provide service for clinical research aiming to develop and improve health care (figure 1). ucr has had its main activities in six areas (figure 2). the main reason for the development of ucr was the universally recognized need for improved services in (i) clinical trials of new and old methods in health care. however, the first leaders of ucr also took the, at that time rather unique, initiative to include also (ii) quality development of the health care system supported by internet-based national quality registries as an equally important part of the services. as for all other research services (iii) biostatistics, epidemiology, and data management were also important components of the initial organization. over the years, the ucr organization has also integrated services for (iv) biobanking of biological materials (uppsala biobank) and (v) high-throughput biochemical analyses in plasma samples from large international clinical trials and observational materials (ucr laboratory). since the start of ucr both the whole organization and most individual projects have been initiated and led by members of the (vi) ucr research faculty. as cardiologists started ucr, the three ucr directors as well as most faculty members have been senior researchers in the cardiovascular area. the faculty has gradually been growing to include researchers at all levels from senior professors to research students whose presence at ucr enriches the discussions and acts both as a solid foundation for ongoing projects and as a spearhead for future innovative activities. the ucr faculty group is also the hub for the extensive networking with other national and international researchers and research centers who are often the key contacts for new projects. over the years, the activities at ucr have gradually expanded and included researchers also in other areas, for example cancer, neurology and care of the elderly, diabetes, nutrition, psychiatry, infection, radiology, and pulmonary, gastrointestinal, and orthopedic diseases. clinical trials in order to perform academic clinical trials, it is today almost a necessity to have support from a clinical research center. very few clinical trialists can keep the competence needed to perform larger clinical trials within their own organization. without this support, today’s complicated regulation around clinical trials will form an almost insurmountable obstacle even for the startup of any investigator-initiated trial. in order for a clinical trials center to manage planning, performance, analysis, and reporting of larger multi-center clinical trials, there is a need for a critical mass of personnel and technical resources within several different areas. therefore, the support for clinical trials concerning new or old methods of diagnosis and treatment is a key part of the development of ucr. the ucr clinical trials group started with two project managers and three research nurses and has over the years grown to a considerably larger group of around 50 persons. today, the group provides complete services including study design, protocols, patient information, negotiations, agreements, contracts, applications to authorities, recruitment of countries, centers and investigators, case report form (crf) and internet-based remote data entry, monitoring and quality assurance, planning and management of biological samples including transportation and storing in the ucr biobank, biochemical and genetic analyses, project coordination, logistics, administration and archiving, consultation, and education. in addition, during the first 10 years ucr was running the ucr clinic, which performed and supported clinical trials in healthy volunteers and outpatients. the primary purpose for the clinical trials group is to support physicians and other investigators, especially in investigator-initiated and registry-based clinical trials. the same services are also provided for clinical trials in collaboration with external sponsors. the group has been working on investigator-initiated projects within many different areas: cardiovascular disease, internal medicine, obstetrics, rheumatology, renal disease, physiology, odontology, nutrition, cancer, ear–nose–throat diseases, infectious diseases, registers – self evaluation scientific evidence – guidelines quality development clinical trials of new treatments figure 1. evidence-based health care development. ucr’s main objective is to provide services for clinical research aiming for development and improvement of health care based on ucr’s combination of systematic monitoring and continuous evaluation of health care performances and outcomes in registers together with performance of prospective randomized clinical trials of new treatments and finally support of implementation by quality development programs; this has led to the new concept ‘evidence-based health care development’. table i. planned services at the foundation of ucr. computerand internet-based information and communication data management for registries and clinical studies statistical advice, consultation, and computation services epidemiological analyses behavioral science service project management quality control biobank service biochemical laboratory service presentation and publication service economic assistance and legal support methodological research development courses and tutoring in research and clinical trial methodology coordination of regional, national, and international research projects new focus areas of clinical research: � registry-based research � registry-based clinical trials � translational research � implementation research � disease-oriented research � organizational research � care-oriented research � research in primary care � patient-related outcomes 2 l. wallentin and b. lindahl psychology, and quality development. the projects concern research on pharmaceutical agents, functional foods, medical implants and devices, quality development methods, and national quality registries. they have been, and are, performed in collaboration with governmental departments, swedish board of health and welfare, swedish federation of county councils, swedish heart–lung foundations, swedish cancer foundation, center for clinical trials of foods, and pharmaceutical and medical device companies. the international breakthrough for the clinical trials group was being one of the leading centers in the development and performance of four global phase iii mega-trials in the cardiovascular area, each including 15,000 to 19,000 patients: the rely (8), plato (9), aristotle (10), and stability (11) trials which were started between 2005 and 2007. through these trials, ucr has become an appreciated member of a global network of academic research organizations, vigour, which is continuously taking on new assignments in international clinical trials and has its center at duke clinical research institute (dcri) in durham, nc, usa (12). a second breakthrough by these trials was the establishment of the ucr laboratory in 2008, which was a necessity as ucr researchers took the initiative and the responsibility for an extensive biomarker and genetic substudy program with analyses of blood samples from all patients in these megatrials (13–18). in addition, the large repository of samples from these trials substantially contributed to the third breakthrough, the establishment of uppsala biobank in 2008, which was later integrated with the ucr organization. a fourth breakthrough by participation in these mega-trials was the need to set up a center for clinical events adjudication (cec) (19). this cec organization has thereafter rapidly expanded and become one of the world-leading centers for cec, with assignments for these services in many international trials. finally, it should be emphasized that a less recognized fifth breakthrough occurred during the first years of ucr clinical trials services, which was the initiation and performance of the very first registry-based randomized clinical trials (r-rct). these first trials were based on cluster randomization of hospitals to registry-based quality development or usual care and nicely showed the superior results with registry-based care (20, 21). during the last few years the concept of r-rct has been the largest international breakthrough of ucr services which is further discussed in other papers (22, 23). quality registries since its start in 2002, the first national competence center for national quality registries—the swedish cardiovascular registries—constitutes a large part of uppsala clinical research center. the basis for this activity is the development and maintenance of an advanced internet-based technology for registration, reporting, and support for systematic improvement of health care processes. the focus of the work in the registry group is to maintain and further develop the concept of internet-based interactive and intuitive data entry systems, preferably integrated with electronic patient records, and to further improve the online information with interactive as well as imperative illustrative analyses. the purpose is also to promote all centers to be involved in a continuous quality improvement process supported by internetbased tools for ‘evidenced-based health care development’. the specific tasks are accordingly to develop, maintain, and improve the national quality registries within cardiovascular diseases; to expand the analysis and reporting from these registries; and to provide support to other registries (6). the competence center today has the responsibility for around 20 national quality registries, 10 of which are in the cardiovascular area and around 10 in other areas. today, the ucr registry activities seem to cover all health care areas in life from before birth (in-vitro fertilization registry) to very old age (senior alert). the internet-based registries usually include mainly hospital-based clinics in the around 70 hospitals in sweden. however, the senior alert registry accepts data from as many as 40,000 different care units all over the country. the ucr quality registry group started with as few as three software developers but rapidly grew to 10 system developers in 2006 and further increased to around 40 people in 2018. in addition, the group also contains several data managers and statisticians for the integrated statistical and scientific analysis. the registry group collaborates closely with a large number of physicians and research nurses, project leaders, and registry coordinators over the whole country and even internationally. the group provides the following services: national and international internet-based databases and registries; electronic computer forms and remote online data entry; imports from and transfer to external data sources; uppsala biobank head ceo senior researcher (administrative assistent) administrative officer (personnel assistent economy assistent) senior researcher (research coordinator publications assistent) biostatistics and epidemiology clinical trials cec ucr laboratory it-services it-security quality manager quality registers software services research faculty rearchers fellows. students figure 2. ucr organization 2013. upsala journal of medical sciences 3 interactive, simple, and advanced statistical analysis; online reports with tabular and graphical presentations; education, support, and monitoring; data security; maintenance and support; quality development and projects supported by consultant and internet-based education; and finally internet portals for information, communication, collaboration, and education. ucr’s it-based registration technology is also used in international projects and in other countries for evaluation of the effects of new treatments in health care. therefore, ucr is today established as the leading european center for registry-based quality development and evaluation of new medical treatments in cardiovascular care. in addition, the registry technology has over the last years been developed to integrate tools for registry-based randomized clinical trials (r-rct). the international interest for taking part in this development has currently placed ucr as one of the world leaders in technologies for cost-effective pragmatic randomized clinical trials within the usual health care system (6, 24). biostatistics biostatistics and epidemiology, supported by data management, are key resources in all medical research. at the beginning, ucr biostatistics group consisted of three statisticians and two data managers. after five years, the group had grown to contain eight biostatisticians, two epidemiologists, and three data managers. today the group has once more doubled in size and contains several statisticians at the phd level and specialists in bioinformatics, genetics, and big data analysis. currently, the group is one of the largest and most experienced in sweden for management and analysis of quantitative biological data. the group provides services in planning, recording, management, analysis, and interpretation of data in all areas of clinical research and also develops new statistical models and innovative online tools for interactive statistical analyses. the activities started mainly with services for cardiovascular research but the group now has experience from collaboration in many different areas. the group is now responsible for basic courses of biostatistics in a biomedical program at uppsala university. several of the statisticians and epidemiologists are running their own research programs and are acting as tutors for research students. the epidemiology group has recently grown to establish its own organization as an epidemiological center within uppsala university. administration during the early years, ucr had a very limited single-person administration, handling personnel, finance, localities, it services, and general services. however, over the years also this group has had to grow in order to handle the increasing demands from an expanding organization, including it solutions and it security, quality assurance, budget planning, accounting, contracts, etc. in addition, new services have been added including a communications officer and publication services. finally, after a few years it became apparent that an internal quality system was a necessity to provide a solid framework for the continuous work with standardized operational procedures (sops) and all other quality routines. although starting on a small and improvised scale in the hands of a few dedicated people, all main activities at ucr became over the years well-established and were shown to work with high quality, able to cope with the increasing service demand. based on a combination of assignments from research groups, research foundations, hospitals, county councils, national board of health and welfare, and pharmaceutical, device, and diagnostic companies, the ucr finances have gradually been growing and during most years kept in a good balance. organization ucr was thus founded as, and still is, a collaborative project between the faculty of medicine at uppsala university and uppsala university hospital within uppsala county council. from 2007, ucr has been organized as an independent unit reporting directly to the dean of the faculty of medicine and the director of uppsala university hospital. the dean of the faculty of medicine and the director of uppsala university hospital appoint the director of ucr for three years. the board of ucr consists of five voting members (three from uppsala university, two from uppsala county council/uppsala university hospital) and the ucr director. a scientific advisory committee has for many years supported the ucr director concerning the strategic planning. the ucr director is further supported by an internal steering group, which has regular meetings concerning planning and performance of the daily operations. the number of employees at ucr has gradually increased in association with an increasing demand for services. ucr started its activities in 2001 with only around 10–15 employees, in 2006 reaching 40–45 fullor part-time employees, and in 2012 around 100. the ucr finances are managed as a combination of department-based finances within the uppsala university administration and project-based finances within the uppsala university hospital administration. the personnel can thereby be employed either by the university or by the university hospital. the director of ucr is responsible for the finances of all projects within both administrations. all research groups collaborating with ucr have full responsibility for their own finances. before the start of collaborative projects, the sharing of costs between the respective group and ucr is established in separate contracts. this organization is ideally suited for its purpose to perform costeffective clinical research and development by integrating the available resources both in the laboratory-based medical research in the university and in the clinical medicine departments in the hospital environment, with direct access to patients, patient records, registries, and hospital-based resources for clinical investigations and care. future after almost 20 years of development, ucr has now established a stable infrastructure for support of continuous quality development, clinical trials, and translational research in 4 l. wallentin and b. lindahl all areas of health care. currently, ucr is world-leading in the areas of internet-based interactive registry-supported quality development and registry-based randomized clinical trials (r-rct). ucr has recently been assigned the task to try to implement the swedeheart registry and clinical trials platform in as many european countries as possible. in addition, ucr is one of the world-leading centers for all types of services in conventional randomized clinical trials. in this area, ucr has pioneered international collaboration on biobanking and omics research in order to make personalized and precision medicine an integral part of global collaborative trials. at present, ucr is therefore well positioned to play a gradually expanding role in the national and international development and improvement of health care. declaration of interest the authors report no conflicts of interest. notes on contributors lars wallentin, senior professor of cardiology, founder and former director of uppsala clinical research center and the swedish cardiovascular quality registries; currently senior researcher with focus on prospective clinical trials, registrybased outcome research and biomarker research in international biobanks. bertil lindahl, professor of cardiology, former director of uppsala clinical research center; currently leading registrybased randomized clinical trials, performing experimental 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upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 tumor length in elderly patients with esophageal squamous cell carcinoma: is it a prognostic factor? ji-feng feng, ying huang & qiang zhao to cite this article: ji-feng feng, ying huang & qiang zhao (2013) tumor length in elderly patients with esophageal squamous cell carcinoma: is it a prognostic factor?, upsala journal of medical sciences, 118:3, 145-152, doi: 10.3109/03009734.2013.792887 to link to this article: https://doi.org/10.3109/03009734.2013.792887 © informa healthcare published online: 26 apr 2013. submit your article to this journal article views: 584 view related articles citing articles: 15 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.792887 https://doi.org/10.3109/03009734.2013.792887 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.792887 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.792887 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.792887#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.792887#tabmodule upsala journal of medical sciences. 2013; 118: 145–152 original article tumor length in elderly patients with esophageal squamous cell carcinoma: is it a prognostic factor? ji-feng feng1,2, ying huang3 & qiang zhao1 1department of thoracic surgery, zhejiang cancer hospital, no. 38 guangji road, banshan bridge, hangzhou 310022, china, 2key laboratory diagnosis and treatment technology on thoracic oncology, zhejiang province, hangzhou 310022, china, and 3department of nursing, zhejiang cancer hospital, hangzhou 310022, china abstract background. several researchers have determined the tumor length to be an important prognostic indictor of esophageal cancer (ec). however, controversy exists concerning the optimal cut-off points for tumor length to predict overall survival. the aim of this study was to determine the prognostic value of tumor length and propose the optimum cut-off point for tumor length in predicting survival difference in elderly patients with esophageal squamous cell carcinoma (escc). methods. from january 2001 to december 2009, a retrospective analysis of 132 consecutive patients older than 70 years with escc was conducted. a receiver-operating characteristic (roc) curve for survival prediction was plotted to verify the optimum cut-off point for tumor length. univariate and multivariate analyses were performed to evaluate prognostic parameters for survival. results. a roc curve for survival prediction was plotted to verify the optimum cut-off point for tumor length, which was 4.0 cm. patients with tumor length £4.0 cm had significantly better 5-year survival rate than patients with a tumor length >4.0 cm (60.7% versus 31.6%, p = 0.007). multivariate analyses showed that tumor length (>4.0 cm versus £4.0 cm, p = 0.036), differentiation (poor versus well/moderate, p = 0.032), n staging (n1-3 versus n0, p = 0.018), and t grade (t3-4 versus t1-2, p = 0.002) were independent prognostic factors. conclusion. tumor length is a predictive factor for long-term survival in elderly patients with escc, especially in t3-4 grade or nodal-negative patients. we conclude that 4.0 cm may be the optimum cut-off point for tumor length in predicting survival in elderly patients with escc. key words: esophageal cancer, esophagectomy, prognostic factor, squamous cell carcinoma, survival, tumor length introduction esophageal cancer (ec) is the eighth most common type of cancer worldwide. it is endemic in many parts of the world, particularly in developing nations, and accounts for more than 200,000 deaths every year in china (1). as a result of worldwide increases in the elderly population, there has been a concomitant increase in the number of ec patients (2). therefore, assessing the prognostic factors in elderly patients with ec will become increasingly important. tumor length is still a controversial prognostic factor in ec patients. several researchers have determined tumor length to be an important prognostic indictor of ec after surgery (3-5). however, there have been few studies regarding tumor length in elderly ec patients (6,7). furthermore, controversy exists concerning the optimal cut-off points for tumor length to predict overall survival (4,5,8). different study sizes, different histological types, variable inclusion criteria, and, most importantly, unreliable statistical methods used to determine the cut-off points have contributed to this controversy (4,5,8-10). correspondence: qiang zhao, md, associate professor, department of thoracic surgery, zhejiang cancer hospital, no. 38 guangji road, banshan bridge, hangzhou 310022, china. e-mail: cdbzjzl@163.com (received 6 january 2013; accepted 22 march 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.792887 we have chosen to study esophageal squamous cell carcinoma (escc) as it is the most common type of ec in china, in contrast to the predominance of adenocarcinoma in the western world. thus, the aim of this study was to determine the prognostic value of tumor length and propose the optimum cutoff point for tumor length in predicting survival difference in elderly patients with escc. patients and methods patients from january 2001 to december 2009, a retrospective analysis was conducted of 132 consecutive patients older than 70 years with escc who underwent curative esophagectomy in the department of thoracic surgery, zhejiang cancer hospital, hangzhou, china. all of the patients included in the analysis fit the following criteria: 1) escc confirmed by histopathology; 2) older than 70 years; 3) curative esophagectomy with r0 resection (en bloc resection with margins histologically free of disease); 4) at least six lymph nodes were examined for pathological diagnosis; and 5) surgery was neither preceded nor followed by adjuvant chemotherapy and/or radiotherapy. all of the above patients were followed up by posting letters or by telephone interviews. the last follow-up was 30 november 2011. all subjects gave written informed consent to the study protocol, which was approved by the ethical committees of zhejiang cancer hospital, hangzhou, china. surgery all patients were treated with radical resection. the standard surgical approach consisted of a limited thoracotomy on the right side and intrathoracic gastric reconstruction (the ivor lewis procedure) for lesions at the middle/lower third of the esophagus. upper third lesions were treated by cervical anastomosis (the mckeown procedure). in our institute, two types of lymphadenectomy were carried out as a standard procedure for escc. the majority of patients underwent two-field (thoracoabdominal) lymphadenectomy. in this cohort of patients, thoracoabdominal lymphadenectomy was performed, including the subcarinal, paraesophageal, pulmonary ligament, diaphragmatic and paracardial lymph nodes, as well as those located along the lesser gastric curvature, the origin of the left gastric artery, the celiac trunk, the common hepatic artery, and the splenic artery. three-field (cervicalthoracoabdominal) lymphadenectomy was performed only if the cervical lymph nodes were thought to be abnormal upon preoperative evaluation. pathological analysis the fresh specimen was routinely dissected and measured by surgeons immediately after resection of the tumor. the length of each tumor was measured with a hand-held ruler. then the specimens were sent for pathology examination after preservation in 10% formalin. differentiation, depth of tumor invasion (t grades), and nodal involvement (n stagings) were recorded according to the results of pathology reports. in addition, pathologists also described the tumor length (measured on the 10% formalin fixed specimens). in this study, the surgeons’ measure was used. all patients were restaged according to the seventh edition of the american joint committee on cancer (ajcc) cancer staging manual (11). statistical analysis statistical evaluation was conducted with spss 17.0 (spss, inc., chicago, il, usa). the mean values are presented as the means ± standard deviations (sd). independent t test was used to compare groups of continuous, normally distributed variables. the pearson chi-square test was used to determine the significance of differences for dichotomous variables. a receiver-operating characteristic (roc) curve for survival prediction (survival versus death) was plotted to verify the optimum cut-off point for tumor length. the area under curve (auc) was used as an estimation of diagnostic accuracy. the youden index (sensitivity + specificity – 1) was used to identify the tumor length threshold values corresponding to the value of the roc curve farthest from the identity line. this index corresponds to the optimal cut-off, defined as the value with the highest average of sensitivity and specificity. the overall cumulative probability of survival was calculated by the kaplan–meier method, and the difference was assessed by the log-rank test. univariate and multivariate analyses of cox regression proportional hazard model were performed to evaluate the prognostic parameters for survival. hazard ratios (hrs) with 95% confidence intervals (cis) were used to quantify the strength of the association between predictors and survival. a p value less than 0.05 was considered to be statistically significant. results patient characteristics among the 132 patients, 11 (8.3%) were women, and 121 (91.7%) were men (table i). the mean age was 146 j.-f. feng et al. 73.7 ± 2.6 years, with an age range from 70 to 85 years. analysis of tumor length the tumor length in these 132 patients ranged from 0.8 to 9.2 cm, mean 4.59 ± 1.74 cm. a roc curve for survival prediction was plotted to verify the optimum cut-off point for tumor length, which was 4.0 cm (figure 1). then, patients were divided into two groups for survival analysis (patients with tumors £4.0 cm in length and patients with tumors >4.0 cm in length). tumors >4.0 cm in length had a 66.0% chance of being t3-4, whereas tumors £4.0 cm in length had a 65.7% chance of being t1-2 (p = 0.001) (table ii). the 5-year overall survival was 43.9% in our study. patients with tumor length £4.0 cm had significantly better 5-year survival rate than patients with a tumor length >4.0 cm (60.7% versus 31.6%, p = 0.007) (figure 2). in the group of t1-2 disease, the 5-year survival of patients with tumor length £4.0 cm was similar in patients with tumor length >4.0 cm (78.3% versus 75.0%, p = 0.771) (figure 3a). in the t3-4 group, however, the 5-year survival of patients with tumor length £4.0 cm was better than that of patients with tumor length >4.0 cm (48.5% versus 23.4%, p = 0.035) (figure 3b). the 5-year survival in patients with tumor length £4.0 cm was better than patients with a tumor length >4.0 cm in n0 staging (79.3% versus 48.3%, p = 0.046) (figure 3c). however, no significant differences in 5-year survival were found between the patients with tumor length £4.0 cm and >4.0 cm in n1-3 staging (40.7% versus 21.3%, p = 0.168) (figure 3d). analyses of prognostic factors univariate analyses showed that vessel involvement (p = 0.037), differentiation (p = 0.027), perineural invasion (p = 0.009), tumor length (p = 0.007), table i. baseline characteristics of 132 elderly patients with escc. cases (n, %) age (mean ± sd, years) 73.6 ± 2.6 gender female 11 (8.3) male 121 (91.7) tumor length (mean ± sd, cm) 4.59 ± 1.74 tumor location upper 6 (4.5) middle 55 (41.7) lower 71 (53.8) differentiation well 17 (12.9) moderate 81 (61.3) poor 34 (25.8) t grade t1 19 (14.4) t2 16 (12.1) t3 89 (67.4) t4 8 (6.1) n staging n0 58 (43.9) n1 42 (31.8) n2 18 (13.7) n3 14 (10.6) tln (mean ± sd, nodes) 22.7 ± 9.7 mln (mean ± sd, nodes) 2.2 ± 3.7 escc = esophageal squamous cell carcinoma; mln = metastatic lymph nodes; tln = total lymph nodes. 0.0 0.0 0.2 0.4 1 specificity 0.6 0.8 1.0 0.2 0.4s e n s it iv it y 0.6 0.8 1.0 figure 1. a roc curve plots the sensitivity on the y-axis against 1 minus the specificity on the x-axis. a diagonal line at 45 degrees, known as the line of chance, would result from a test which allocated subjects randomly. each point on the roc curve corresponds to a value of tumor length. in general, a good cut-off point is one which produces both a large sensitivity and a large specificity. this can be interpreted as choosing the point on the roc curve with the largest vertical distance from the line of chance (two-way arrow). the auc for tumor length was 67.1% with a sensitivity of 79.7% and a specificity of 53.4% (1 – 46.6%) by youden index (dotted lines). the threshold value corresponding to the tumor length was 4.0 cm. tumor length in elderly patients with escc 147 n staging (p = 0.000), and t grade (p = 0.000) were predictive of survival. then multivariate analyses were performed with the cox proportional hazards model. in that model, we demonstrated that tumor length (p = 0.036), differentiation (p = 0.032), n staging (p = 0.018), and t grade (p = 0.002) were independent prognostic factors (table iii). discussion the aging of the population and a longer life expectancy have led to more elderly patients with cancers being referred for treatment. for many of them, in particular for ec, surgery remains the mainstay of treatment. there is no established cut-off to define a patient as ‘elderly’ in relation to surgery, but most studies available so far set the age limit at 70 (12,13). in our study, we determined the prognostic value of tumor length in escc patients older than 70 years. it was found that tumor length is a predictive factor for long-term survival in elderly patients with escc, especially in t3-4 grade or nodal-negative patients. we conclude that 4.0 cm may be the optimum cut-off point for tumor length in predicting survival in elderly patients with escc. before 1987, the ajcc staging system used tumor length (t1, <5 cm; t2, >5 cm; and t3, evidence of extraesophageal spread) to predict patient prognosis (14). however, at the 1987 ajcc annual meeting, the current tnm staging system was adopted, in which tumor length is not a staging criterion (15,16). the recent edition of the ajcc tnm staging system was published in 2009. in this newly published staging system for ec, adenocarcinoma and escc are regarded as two different entities and should be staged separately. it also proposes depth of tumor invasion, number of metastatic lymph nodes, histologic type, and tumor location as independent staging factors for ec. however, it does not emphasize the prognostic role of tumor length in ec (11). tumor length is still a controversial prognostic factor in ec patients. tachibana et al. (9) evaluated 129 patients with escc and indicated that tumor length was related to survival but was not an independent prognostic factor on multivariate analysis. bollschweiler et al. (10) and igaki et al. (17) demonstrated similar results. however, several researchers have determined tumor length to be an important prognostic indictor of ec after surgery (3-5). eloubeidi et al. (8) analyzed the outcomes of 10,441 ec patients from the national cancer institute surveillance, epidemiology, and end results database. their results demonstrated that tumor length was an important prognostic factor of overall survival for patients with ec. however, there have been few studies regarding tumor length in elderly patients. in our study, we determined that the tumor length (£4.0 cm versus >4.0 cm) is a predictive factor for long-term survival in elderly patients with escc. many studies have shown that tumor length of 3.0 cm was the optimum cut-off point for survival prediction in ec (4,5,9,10). eloubeidi et al. (8) table ii. characteristics of patients with tumor length more or less than 4.0 cm. tumor length (n, %) p value£4.0 cm >4.0 cm age (years) 0.020 £75 47 (48.5) 50 (51.5) >75 9 (25.7) 26 (74.3) gender 0.832 female 5 (45.5) 6 (54.5) male 51 (42.1) 70 (57.9) differentiation 0.864 well/moderate 42 (42.9) 56 (57.1) poor 14 (41.2) 20 (58.8) tumor location 0.454 upper/middle 28 (45.9) 33 (55.1) lower 28 (39.4) 43 (60.6) n staging 0.119 n0 29 (50.0) 29 (50.0) n1-3 27 (36.5) 47 (63.5) t grade 0.001 t1-2 23 (65.7) 12 (34.3) t3-4 33 (34.0) 64 (66.0) 0.0 0 1 2 3 4 time (y) 5 6 tumor length ≤ 4.0 cm > 4.0 cm ≤ 4.0 cm-censored > 4.0 cm-censored p = 0.007 7 8 0.2 0.4 0.6 c u m s u rv iv a l 0.8 1.0 figure 2. patients with tumor length £4.0 cm had a significantly better 5-year survival rate than patients with a tumor length >4.0 cm (60.7% versus 31.6%, p = 0.007). 148 j.-f. feng et al. suggested a cut-off point of tumor length in ec between 3.0 cm and 4.0 cm. in our study, however, a roc curve for survival prediction was plotted to verify the optimum cut-off point for tumor length, which was 4.0 cm (figure 1). our results showed that tumors >4.0 cm in length had a 66.0% chance of being t3-4, whereas tumors £4.0 cm in length had a 65.7% chance of being t1-2 (p = 0.001). bhutani et al. (18) showed that tumors ‡5 cm in length had an 89% chance of being t3 or greater, whereas tumors <5 cm in length had a 92% chance of being t1 or t2. our study also showed that patients with tumor length £4.0 cm had a significantly better 5-year survival than patients with tumor length >4.0 cm (60.7% versus 31.6%, p = 0.007). it has been widely agreed upon that lymph node status, depth of tumor invasion, and overall tnm stage are strong, independent prognostic factors in ec (19,20). it may well be that the influence of tumor length on the subgroup with different t grades and n stagings is important for the understanding of its role in overall survival in elderly patients with ec. in our study, the esophageal tumor length had a significant impact on survival of n0 patients (p = 0.046). our finding is quite consistent with the previous studies showing that tumor length had a greater prognostic value for localized ec than for cancer with metastases (5,8), but is contrary to the result of khan et al. (21), who suggested that tumor length is not a prognostic factor for ec patients with n0 staging (p = 0.861). in our study, tumor length was also a prognostic factor 0.0 0 1 tumor length p = 0.771 ≤ 4.0 cm 2 3 4 time (y) 5 6 7 8 0.2 0.4 c u m s u rv iv a l 0.6 0.8 1.0 > 4.0 cm ≤ 4.0 cm-censored > 4.0 cm-censored 0.0 0 1 tumor length p = 0.035 ≤ 4.0 cm 2 3 4 time (y) 5 6 7 8 0.2 0.4 c u m s u rv iv a l 0.6 0.8 1.0 > 4.0 cm ≤ 4.0 cm-censored > 4.0 cm-censored 0.0 0 1 tumor length p = 0.046 ≤ 4.0 cm 2 3 4 time (y) 5 6 7 8 0.2 0.4 c u m s u rv iv a l 0.6 0.8 1.0 > 4.0 cm ≤ 4.0 cm-censored > 4.0 cm-censored 0.0 0 1 tumor length p = 0.168 ≤ 4.0 cm 2 3 4 time (y) 5 6 7 0.2 0.4 c u m s u rv iv a l 0.6 0.8 1.0 > 4.0 cm ≤ 4.0 cm-censored > 4.0 cm-censored a b c d figure 3. kaplan–meier survival curves stratified by tumor length in (a) t1-2 patients, (b) t3-4 patients, (c) n0 patients, and (d) n1-3 patients. tumor length in elderly patients with escc 149 after controlling the factor of t grades. its predictive value was significant for t3-4 lesions between patients with tumor length £4.0 cm and >4.0 cm (p = 0.035). from the database of 132 elderly patients with escc who underwent surgery, our results clearly demonstrate that tumor length can serve as an independent predictor of long-term survival for elderly patients with escc, especially in t3-4 grade or nodalnegative patients. the question of how many lymph nodes should be dissected has been a point of debate in previous studies. rizk et al. (22) reported that the prognosis of patients after esophagectomy worsens significantly when four or more lymph nodes have metastases, irrespective of t stage. a consensus conference of experts meeting in 1995 suggested that accurate pathological staging of ec required resection of at least 15 lymph nodes (23). greenstein et al. (24) and yang et al. (25) recommended 18 nodes as the minimum number of resectable lymph nodes. however, it is proposed by international union against cancer (uicc) and ajcc that at least six lymph nodes should be removed during resection of ec (26,27). accordingly, we excluded patients who had fewer than six lymph nodes dissected (range: 6–61 nodes). table iii. univariate and multivariate analyses in elderly patients with escc. cases (n, %) survival(%) univariate analysis hr (95% ci) p value multivariate analyses hr (95% ci) p value age (years) 0.780 0.322 £75 97 (73.5) 45.4 1.000 1.000 >75 35 (26.5) 40.0 1.075 (0.647–1.785) 0.743 (0.413–1.337) gender 0.420 0.175 female 11 (8.3) 54.5 1.000 1.000 male 121 (91.7) 43.0 1.453 (0.586–3.606) 1.989 (0.737–5.372) tumor location 0.642 0.422 upper/middle 61 (46.2) 42.6 1.000 1.000 lower 71 (53.8) 45.1 1.115 (0.706–1.761) 1.231 (0.741–2.045) tln (nodes) 0.288 0.176 £15 32 (24.2) 37.5 1.000 1.000 >15 100 (75.8) 46.0 0.756 (0.452–1.266) 0.672 (0.378–1.194) vessel involvement 0.037 0.194 no 100 (75.8) 46.0 1.000 1.000 yes 32 (24.2) 37.5 1.733 (1.032–2.911) 1.456 (0.825–2.569) perineural invasion 0.009 0.281 no 113 (85.6) 47.8 1.000 1.000 yes 19 (14.4) 21.1 2.130 (1.205–3.766) 1.432 (0.745–2.752) differentiation 0.027 0.032 well/moderate 98 (74.2) 49.0 1.000 1.000 poor 34 (25.8) 29.4 1.715 (1.053–2.794) 1.774 (1.049–3.000) tumor length (cm) 0.007 0.036 £4 56 (42.4) 60.7 1.000 1.000 >4 76 (57.6) 31.6 1.963 (1.191–3.237) 1.769 (1.038–3.016) n staging 0.000 0.018 n0 58 (43.9) 63.8 1.000 1.000 n1-3 74 (56.1) 28.4 3.059 (1.838–5.091) 1.949 (1.119–3.395) t grade 0.000 0.002 t1-2 35 (26.5) 77.1 1.000 1.000 t3-4 97 (73.5) 32.0 4.450 (2.129–9.298) 3.342 (1.538–7.261) ci = confidence interval; escc = esophageal squamous cell carcinoma; hr = hazard ratio; tln = total lymph nodes. 150 j.-f. feng et al. potential limitations of the present study include the relatively small number of patients, the use of a retrospective analysis, and the short duration of the mean follow-up duration. in addition, because the study used data from a single institution but with different pathologists and different surgeons, there may have been a lack of uniformity in measurement methods. in our study, tumor length was measured immediately after resection by surgeons. however, pathologists also described the tumor length (measured on the 10% formalin-fixed specimens) in their pathology reports. thus, there were some differences between these two measures. furthermore, due to the limited number of elderly patients with escc, our analysis may suffer from type i or type ii errors. the results of the study should therefore be regarded with caution. larger prospective studies will need to be performed to confirm these preliminary results and determine the optimum cut-off point. conclusion in summary, our study showed that tumor length is a predictive factor for long-term survival in elderly patients with escc, especially in t3-4 grade or nodal-negative patients. we 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6th ed. new york: wiley & sons; 2002. 152 j.-f. feng et al. the impact of standardized care pathway on reporting time for invasive melanoma – results from one pathology department in sweden article the impact of standardized care pathway on reporting time for invasive melanoma – results from one pathology department in sweden margr�et agnarsd�ottira,b , helen p€a€arena,b and ismini vassilakic adepartment of clinical pathology, akademiska university hospital, uppsala, sweden; bdepartment of immunology, genetics and pathology, rudbeck laboratory, uppsala university, uppsala, sweden; cdermipath, consulting dermatopathology service, stockholm, sweden abstract background: standardized care pathway (scp) was introduced by the swedish health authorities to eliminate unwanted delay in the diagnostics of cancer patients; for melanoma, scp started in 2016. the aim of this study was to investigate the impact of scp on reporting time for invasive melanomas. materials and methods: information on reporting time was collected on all samples handled according to the scp and on all invasive melanomas diagnosed in 2016–2018 at the department of clinical pathology, akademiska university hospital, uppsala, sweden. results: during the study period, 205 samples were handled according to the scp, resulting in 53 cases (26%) diagnosed with invasive melanomas. a total of 301 invasive melanomas from 286 patients were diagnosed during the study period; 67 (22%) were submitted as scp, 36 (12%) as a general priority case, and 198 (66%) as non-priority. the reporting time for the scp cases was 8 days, for general priority cases 6 days, and for non-priority cases it was 24 days. the reporting time increased from 18 to 31 days for the non-priority cases and from 15 to 25 days for all cases with invasive melanomas during the study period. conclusion: this study demonstrates prolonged reporting times for invasive melanomas since the implementation of scp. this is probably caused by the crowd-out effect of the scp samples, limited personnel resources, and inaccuracy of the clinical diagnosis. scp might therefore be a suboptimal method to shorten reporting times for invasive melanomas. article history received 23 january 2019 revised 13 september 2019 accepted 27 september 2019 keywords diagnosis; melanoma; pathology; quality; reporting time; standardized care pathway introduction invasive melanoma is the leading cause of skin-related deaths in the western world (1,2). in sweden, melanoma is the fifth most common cancer diagnosis in women and the sixth for men (3). it is important to diagnose these tumours at an early stage to secure good prognosis of the patients and to reduce waiting times – a significant cause of patients’ anxiety. in many swedish pathology departments, the reporting time for non-priority cases is long due to high workload, as there is a shortage of both pathologists and laboratory technicians. traditionally, physicians are able to mark a case as priority on the pathology requisition form, resulting in a shorter reporting time, but at an additional cost. standardized care pathway (scp) was introduced by the swedish health authorities to eliminate unwanted delay in the diagnostics of cancer patients; for melanoma, scp started in may 2016. similar programmes have been established in denmark and norway (4–6). according to the scp clinical melanoma guidelines, it is important that the physician clearly writes on the pathology requisition form that the clinical diagnosis is melanoma, or high suspicion thereof, as scp for skin lesions is only intended for melanomas. scp is not to be used for ruling out melanomas or diagnosing other skin malignancies. for patients to be managed according to scp, the pathology requisition is marked in a specific way. therefore, these cases are treated as priority cases resulting in shorter reporting time but without any additional cost for the healthcare provider. the aim of this study was to investigate the impact of scp on reporting time for invasive melanomas. materials and methods all the cases were diagnosed at the department of clinical pathology, akademiska university hospital, uppsala, sweden. the department provides clinical diagnostic services to other departments located at the university hospital (i.e. dermatology, plastic surgery, head and neck surgery, among others) and many general practitioner practices located outside of the hospital. in addition, it serves a small municipal hospital and a few private surgeons. the population of the area that the department serves was 360,124 in 2016 (7). during that year, the department received a total of 7701 contact margr�et agnarsd�ottir margret.agnarsdottir@igp.uu.se department of immunology, genetics and pathology, rudbeck laboratory, uppsala university, uppsala, se-75185, sweden � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 4, 260–264 https://doi.org/10.1080/03009734.2019.1675102 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1675102&domain=pdf&date_stamp=2019-12-09 http://orcid.org/0000-0003-3197-0053 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2019.1675102 http://www.tandfonline.com dermatopathology cases, and all cases were taken care of by dermatopathologists. patients with invasive melanoma the patients included in the study were diagnosed with invasive melanoma during the years 2016 and 2017, in addition to the first half of the year 2018 (january until june). the patients were identified by a search through the laboratory information system (lis) by using specific diagnostic snomed (systematized nomenclature of medicine) codes. the following codes were used for the search: superficial spreading melanoma (ssm), m87433; nodular melanoma (nm), m87213; lentigo malignant melanoma (lmm), m87423; acral lentiginous melanoma (alm), m87443; desmoplastic melanoma, m87453; and malignant melanoma unspecified, m87203. the information registered for each patient was collected from the pathology requisition form, the pathology report, and from the lis (table 1). the glass slides were not reviewed. if the tumour thickness was reported as an interval, the higher number was registered as the thickness. if the tumour was biopsied and the remaining tumour later removed with an excision, information on the pathology requisition form coupled to the biopsy was registered. during the years 2016 and 2017, the t-stage was registered according to the american joint committee of cancer (ajcc), 7th edition 2009 (8). in 2018, the t-stage was registered according to the updated 8th edition 2017 (9). the reporting time was defined as the time in calendar days between the date of arrival of the specimen to the pathology department and the date of signature of the electronic report by the pathologist. patients with pathology requisition forms marked as scp but with a form not filled out correctly according to guidelines and therefore not treated as scp were included among the non-priority cases (n ¼ 12). forms where the case was marked with both scp and as a general priority case were included among the scp cases (n ¼ 12). cases where scp was missed – at the department of clinical pathology, or the clinicians failed to mark the pathology form (information found in the lis) – were included among the non-priority cases (n ¼ 6). for the clinical diagnosis found on the pathology requisition form, the diagnoses were divided into the following groups: (a) melanoma or high suspicion thereof; (b) different melanocytic lesions (including benign naevus, dysplastic naevus, in situ and invasive melanomas); (c) various different tumour types (e.g. melanoma/ seborrhoeic keratosis, basal cell carcinoma/melanoma); (d) basal cell carcinoma; (e) benign lesions (e.g. seborrhoeic keratosis, pyogenic granuloma, hemangioma); and (f) other, i.e. no specific diagnosis revealed on the pathology requisition form. patients diagnosed with invasive melanomas in the year 2015 (a comparison group) this group was identified with a search in the lis employing the same snomed codes as for the patients diagnosed in the years 2016–2018 (see above). for the patients diagnosed in the year 2015, the only information collected was the reporting time and whether or not the pathology requisition form was marked with priority. in the year 2015 scp had not been introduced. patients handled according to the scp in the lis all patients handled according to the scp are marked in a specific way that is independent of the diagnostic snomed code. to identify the patients a search was carried out in the lis during the included time period (2.5 years), and the diagnosis for each patient was registered. statistics the results are presented as descriptive statistics in the form of absolute numbers and percentage; median and average calculations were performed in microsoft excel. ethical statement the uppsala regional ethical review board was consulted, and they had no ethical objections for the implementation of the study (ref. 2017/438). results patients handled according to the scp during the study period, a total of 205 cases were handled according to the scp at the department of clinical pathology. fifty-three cases (25.9%) were diagnosed as invasive melanomas and 40 as in situ melanomas (19.5%). the remaining cases represented various other melanocytic lesions (n ¼ 63), benign non-melanocytic lesions (n ¼ 29), basal cell carcinoma (n ¼ 10), and in situ squamous cell carcinoma (n ¼ 2). the majority of the scp cases with the histopathological diagnosis of invasive melanoma (n ¼ 53) were submitted from hospital departments (department of dermatology, n ¼ 17; other departments, n ¼ 27). table 1. information collected for each patient/tumour. patient/tumour details collected gender age at diagnosis tumour localization case marked with priority (scp or a general priority case) healthcare provider clinical diagnosis/question on pathology requisition form surgical excision or biopsy material reporting time in calendar days additional glass slides generated at the histopathology laboratory immunohistochemistry performed subtype (ssm, nm, lmm, other) t-stage found in pathology report tumour thickness in mm pre-existing melanocytic naevus discussed at a multidisciplinary meeting the pathology form for the melanoma quality register completed lmm: lentigo malignant melanoma; nm: nodular melanoma; scp: standardized care pathway; ssm: superficial spreading melanoma. upsala journal of medical sciences 261 patients diagnosed with invasive melanomas during the study period, a total of 301 invasive melanomas from 286 patients were diagnosed at the department of clinical pathology. the median age was 67 years (sd 15). the cases were evenly distributed between men and women, the most common location was the trunk, and the dominant morphological subtype was ssm when stated (table 2). the clinical diagnosis of melanoma or the differential diagnosis of a melanocytic lesion was only found on half of the pathology forms (table 2). these clinical diagnoses were more often noted on pathology requisition forms in the year 2018 (n ¼ 37; 63.8%) compared with the year 2016 (n ¼ 60; 51.8%). the clinical diagnosis of melanoma was more often noted on pathology requisition forms submitted from departments located at the hospital (n ¼ 82/114; 72%; department of dermatology n ¼ 35; other departments n ¼ 47) compared with healthcare providers outside of the hospital (n ¼ 32/114; 28.0%; general practitioners n ¼ 17; others n ¼ 15). for the cases where the clinical diagnosis was melanoma or high suspicion thereof (n ¼ 114), only nine were biopsied (table 2). among these 114 cases, 76 were marked as priority cases, either according to scp or as general priority cases. during the study period, there was an increase in the number of pathology reports where the subtype was stated by the dermatopathologist (2016: n ¼ 40, 34.5%; 2017: n ¼ 75, 59.1%; 2018: n ¼ 50, 86.2%). the median thickness of the tumours was 0.7 mm, and the majority were thin melanomas, pt1a and pt1b (table 2). the majority of the pt1 tumours were from women (n ¼ 103; 34.2%) compared with men (n ¼ 91; 30.2%). as for the pt2–pt4 tumours, the majority were from men (n ¼ 57; 18.9%) compared with women (n ¼ 45; 15.0%). information on t-stage was found in 91.4% (n ¼ 275) of the pathology reports. according to the pathology reports, 109 tumours (36.2%) originated from a pre-existing naevus. the majority of the patients were discussed at a multidisciplinary meeting (n ¼ 265; 88.7%). the pathology form for the melanoma quality register in sweden was completed for the vast majority of the cases (n ¼ 295; 98.0%). table 2. demographic and clinical data for the invasive melanomas diagnosed in 2016–2018 (n ¼ 301). demographic and clinical data number (%) healthcare provider department of dermatology 79 (26.2) other hospital departments 67 (22.3) general practitioners 105 (34.9) others outside of the hospital 50 (16.6) clinical diagnosis on pathology form melanoma 114 (37.9) ddx melanocytic lesion 55 (18.3) various tumour types 51 (16.9) basal cell carcinoma 10 (3.3) benign lesion 7 (2.3) no specific diagnosis 64 (21.3) gender men 151 (50.2) women 150 (49.8) treatment local excision 239 (79.4) biopsy 62 (20.6) location trunk 170 (56.5) lower extremity 48 (16.0) upper extremity 47 (15.6) head and neck 16 (5.3) face 18 (6.0) ear 1 (0.3) missing information 1 (0.3) morphological subtype ssm 126 (41.9) lmm 16 (5.3) nm 14 (4.7) other types 9 (3.0) not stated 136 (45.1) t-stage pt1 194 (64.5) pt2 48 (15.9) pt3 25 (8.3) pt4 29 (9.6) ptx 5 (1.7) ddx: differential diagnosis; lmm: lentigo malignant melanoma; nm: nodular melanoma; ssm: superficial spreading melanoma. table 3. the reporting time for the invasive melanoma cases in the different priority groups including the standardized care pathway (scp). the number of cases in each group and the healthcare provider submitting the scp cases (percentage in parentheses). 2015a 2016 2017 2018 2016–2018 reporting time all cases 21 15 20 25 18 scp 7 10 6 8 general priority 6 5 7 6 6 scp and priority 5 9 6 7 non-priority 26 18 25 31 24 number scp 14 (12.0) 34 (26.8) 19 (32.8) 67 (22.2) general priority 28 (24.8) 22 (19.0) 11 (8.7) 3 (5.2) 36 (12.0) non-priority 85 (75.2) 77 (66.4) 75 (59.0) 28 (48.3) 180 (59.8) othersb 3 (2.6) 7 (5.5) 8 (13.7) 18 (6.0) total 113 116 127 58 301 healthcare provider scp department of dermatology 4 (6.0) 13 (19.4) 5 (7.4) 22 (32.8) other hospital departments 8 (11.9) 14 (21.0) 8 (11.9) 30 (44.8) general practitioners 2 (3.0) 6 (9.0) 4 (6.0) 12 (18.0) others outside of the hospital 0 1 (1.5) 2 (3.0) 3 (4.5) total 14 34 19 67 ascp not implemented. bcases where scp or priority was missed or the pathology form not correctly filled out and therefore not handled as scp. 262 m. agnarsd�ottir et al. reporting time of the patients with invasive melanoma and the application of scp the median reporting time during the study period was 8 days for the scp cases, 6 days for the general priority cases, and 24days for the non-priority cases. the reporting time increased from 18 to 31days for the non-priority cases and from 15 to 25days for all cases with invasive melanomas during the study period (table 3). for comparison, in the year 2015, before scp was introduced, the reporting time for priority cases was 6 days, for non-priority cases 26days, and for all cases 21days. there was a gradual increase in the number of scp cases during the study period, with a corresponding decrease in the number of general priority cases. hospital departments were more prone to use scp than other healthcare providers (table 3). the majority of the scp cases were pt1 tumours (table 4); among the thick melanomas pt4, only 12 out of 29 cases (41.4%) were marked with priority (scp n ¼ 9; general priority n ¼ 3). additional laboratory procedures and the effect on reporting time for 107 cases (35.5%), more slides were generated at the histopathology laboratory before signing out the case, mostly deeper sections. for 123 cases (40.9%), immunohistochemistry was added. for 62 cases (20.6%), both applied. if additional laboratory procedures were added the reporting time during the study period increased by 4 days for the scp cases and by 3 days for the general priority cases. for the non-priority cases the reporting time increased by 4 days. discussion this study demonstrates prolonged reporting times for invasive melanomas since the implementation of scp at a pathology department located at a university hospital in sweden. during the study period, the median reporting time for all cases was long, 18 calendar days. for cases handled with priority (general priority case and scp), the reporting time was in the range of 5–9 days, which is within the scp’s recommended time period. according to scp, the recommended time period from excision to information to the patient is 14 days. for non-priority cases, the reporting time gradually became longer during the study period, from 18 days in 2016 to 31 days in 2018. the variation in the reporting time observed for the non-priority cases is related to high workload, shortage of both dermatopathologists and laboratory technicians, and the possible crowd-out effect of the scp samples. in addition, inaccuracy in the clinical diagnosis resulted in more than half of the melanomas were underdiagnosed clinically and were found among the nonpriority cases. the scp for melanoma started formally in may 2016, but the first case was received at the department of clinical pathology in the end of march 2016. however, problems were observed with implementation of this new routine at the department, e.g. other tumour types being marked as scp, pathology forms not filled out correctly, cases marked both with scp and also as a general priority case. this resulted in problems with implementation and handling at the department. however, during the study period there was a gradual increase in the number of cases handled according to the scp. still, it is of concern that during the first half of 2018, when scp had been implemented for 2 years, half of the invasive melanomas were submitted with no priority at all, including both general priority and scp. it is also of concern that among the cases that were handled according to the scp during the study period, less than half were actually melanomas, either in situ or invasive. the drawback of this study is that we have not found similar studies to compare our results with, and the study includes only the first years after the implementation of scp. the strength of the study is the systematic review of the cases found at one pathology department where all the cases were taken care of by dermatopathologists, not general pathologists. the pathway for patients undergoing diagnosis and treatment for melanoma is complex and involves many factors. this study reveals that despite efforts to shorten the reporting time for melanomas, the introduction of the scp has not been successful, as the majority were not submitted as a scp case, and the reporting time for the non-priority cases – the group where the vast majority of the melanomas was found – increased. we can only speculate why the scp is not more successful in lowering the reporting time during the included study period. the number of physicians seeing this patient group is vast; therefore, adequate and repeated information about this new routine and its consequences is very important. the clinical experience varies among treating physicians, as does the clinical appearance of this tumour type, making it difficult to diagnose, even for experienced dermatologists. this study also demonstrates the possible crowd-out effect of the scp cases on the reporting time of non-priority cases at a pathology department with limited personnel resources, when a subgroup of cases is handled with priority. perhaps the optimal solution would have been to invest funding, time, and effort to support the pathology departments in order to facilitate decreased lead times during the complex laboratory and diagnostic processes. in conclusion, this study demonstrates prolonged reporting times for invasive melanomas since the implementation of scp. this is probably caused by the crowd-out effect of the scp samples, limited personnel resources, and inaccuracy of the clinical diagnosis. scp might therefore be a suboptimal method to shorten reporting times for invasive melanomas, but reports and studies from other departments are also necessary to evaluate the long-term effect. table 4. t-stage of the standardized care pathway (scp) cases and the healthcare providers submitting the cases. pt1 pt2 pt3 pt4 total department of dermatology 17 2 2 1 22 other hospital departments 17 4 2 7 30 general practitioners 10 1 0 1 12 others outside of the hospital 2 1 0 0 3 total 46 8 4 9 67 upsala journal of medical sciences 263 disclosure statement no potential conflict of interest was reported by the authors. funding this work was supported by the local uppsala university hospital ‘alf’ grants. notes on contributors margr�et agnarsd�ottir, md, phd, specialist in pathology, senior consultant at the department of clinical pathology, akademiska university hospital, uppsala sweden. researcher at the department of immunology, genetics and pathology, rudbeck laboratory, uppsala university, uppsala, sweden. helen p€a€aren, md, specialist in dermatovenerology and pathology, consultant at the department of clinical pathology, akademiska university hospital, uppsala sweden. researcher at the department of immunology, genetics and pathology, rudbeck laboratory, uppsala university, uppsala, sweden. ismini vassilaki, md, specialist in pathology, senior consultant at dermipath, consulting dermatopathology service, stockholm, sweden. orcid margr�et agnarsd�ottir http://orcid.org/0000-0003-3197-0053 references 1. tryggvadottir l, gislum m, hakulinen t, klint a, engholm g, storm hh, et al. trends in the survival of patients diagnosed with malignant melanoma of the skin in the nordic countries 1964-2003 followed up to the end of 2006. acta oncol. 2010;49:665–72. 2. sacchetto l, zanetti r, comber h, bouchardy c, brewster dh, broganelli p, et al. trends in incidence of thick, thin and in situ melanoma in europe. eur j cancer. 2018;92:108–18. 3. official statistics of sweden. health and medical care. 2018. available at: https://www.socialstyrelsen.se/globalassets/sharepointdokument/artikelkatalog/statistik/2018-12-51.pdf 4. regional cancer centres (rcc). the national board of health and welfare. available at: https://www.cancercentrum.se/samverkan/ vara-uppdrag/kunskapsstyrning/vardforlopp/ 5. the danish health authority. 2018. available at: https://www.sst.dk/-/ media/udgivelser/2019/pakkeforloeb-kraeft-2015-2019/pakkeforloebog-opfoelgningsprogrammer-begreber-forloebstider-og-monitorering.ashx 6. the norwegian directorate of health. available at: https://www. helsedirektoratet.no/pakkeforlop/generell-informasjon-for-alle-pakkeforlopene-for-kreft/pakkeforlop-pa-kreftomradet 7. statistics sweden, the statistical database. available at: http:// www.statistikdatabasen.scb.se/pxweb/en/ssd/ 8. balch ce, gershenwald je, atkins mb, buzaid ac, cascinelly n, cochran aj, et al. melanoma of the skin. in: edge se, byrd dr, compton cc, fritz ag, greene fl, trotty a, editors. ajcc cancer staging manual. 7th ed. new york: springer; 2009. 9. gershenwald je, scolyer ra, hess kr, thompson jf, long gv, ross ml, et al. melanoma of the skin. in: amin mb, edge s, greene f, byrd dr, brookland rk, washington mk, et al., editors. ajcc cancer staging manual. 8th ed. new york: springer; 2017. 264 m. agnarsd�ottir et al. https://www.socialstyrelsen.se/globalassets/sharepoint-dokument/artikelkatalog/statistik/2018-12-51.pdf https://www.socialstyrelsen.se/globalassets/sharepoint-dokument/artikelkatalog/statistik/2018-12-51.pdf https://www.cancercentrum.se/samverkan/vara-uppdrag/kunskapsstyrning/vardforlopp/ https://www.cancercentrum.se/samverkan/vara-uppdrag/kunskapsstyrning/vardforlopp/ https://www.sst.dk/-/media/udgivelser/2019/pakkeforloeb-kraeft-2015-2019/pakkeforloeb-og-opfoelgningsprogrammer-begreber-forloebstider-og-monitorering.ashx https://www.sst.dk/-/media/udgivelser/2019/pakkeforloeb-kraeft-2015-2019/pakkeforloeb-og-opfoelgningsprogrammer-begreber-forloebstider-og-monitorering.ashx https://www.sst.dk/-/media/udgivelser/2019/pakkeforloeb-kraeft-2015-2019/pakkeforloeb-og-opfoelgningsprogrammer-begreber-forloebstider-og-monitorering.ashx https://www.sst.dk/-/media/udgivelser/2019/pakkeforloeb-kraeft-2015-2019/pakkeforloeb-og-opfoelgningsprogrammer-begreber-forloebstider-og-monitorering.ashx https://www.helsedirektoratet.no/pakkeforlop/generell-informasjon-for-alle-pakkeforlopene-for-kreft/pakkeforlop-pa-kreftomradet https://www.helsedirektoratet.no/pakkeforlop/generell-informasjon-for-alle-pakkeforlopene-for-kreft/pakkeforlop-pa-kreftomradet https://www.helsedirektoratet.no/pakkeforlop/generell-informasjon-for-alle-pakkeforlopene-for-kreft/pakkeforlop-pa-kreftomradet http://www.statistikdatabasen.scb.se/pxweb/en/ssd/ http://www.statistikdatabasen.scb.se/pxweb/en/ssd/ abstract introduction materials and methods patients with invasive melanoma patients diagnosed with invasive melanomas in the year 2015 (a comparison group) patients handled according to the scp statistics ethical statement results patients handled according to the scp patients diagnosed with invasive melanomas reporting time of the patients with invasive melanoma and the application of scp additional laboratory procedures and the effect on reporting time discussion disclosure statement funding references tf-iups190017 213..213 membership announcement meeting with a lecture by professor carl-henrik heldin followed by dinner time: september 18th, 2019 5.30 pm place: gunne hall, university hospital entrance 10, uppsala hour: 17:30 professor heldin is a molecular biologist and medical researcher. he was appointed director of the uppsala branch of ludwig cancer research 1986 and professor in molecular cell biology at the medical faculty of uppsala university 1992. he is the chairman of the nobel foundation sicne 2013. his research has focused on the mechanisms of signal transduction by growth regulatory factors like platelet-derived growth factor (pdgf) and transforming growth factor (tgf). the best dissertation during the academic year 20182019 will be awarded. after the meeting the members are invited to dinner at 19:00 in the orangerie in the linnean garden, uppsala. please use the e-mail anna.rask-andersen@medsci.uu.se for registration for the dinner or call þ46 70 425 06 54. please indicate any food allergies. the olof rudbeck day time: october 18th, 2019 place: gr€onwall hall, university hospital entrance 70, uppsala. hour: 9:00 15.45 this year’s theme will be autoimmunity. you will find the program here: https://www.medfarm.uu.se/rudbeckdagen/ a warm welcome to all our members of upsala society of medicine! � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 3, 213 https://doi.org/10.1080/03009734.2019.1653571 https://www.medfarm.uu.se/rudbeckdagen/ http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1653571&domain=pdf&date_stamp=2019-09-13 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2019.1653571 http://www.tandfonline.com outline placeholder meeting with a lecture by professor carl-henrik heldin followed by dinner the olof rudbeck day bioactive glass s53p4 eradicates staphylococcus aureus in biofilm/planktonic states in vitro original article bioactive glass s53p4 eradicates staphylococcus aureus in biofilm/planktonic states in vitro torstein grønsetha,b, lene k. vestbyc, live l. nessed, magnus von ungee,f and juha t. silvolaa,b,e ainstitute for clinical medicine, university of oslo, oslo, norway; bdepartment of otolaryngology, head and neck surgery, oslo university hospital, oslo, norway; cdepartment of analysis and diagnostic, norwegian veterinary institute, oslo, norway; ddepartment of animal health and food safety, norwegian veterinary institute, oslo, norway; edepartment of otolaryngology, head and neck surgery, akershus university hospital, akershus and oslo, norway; fcenter for clinical research, uppsala university, v€asterås, sweden abstract background: increasing antimicrobial resistance to antibiotics is a substantial health threat. bioactive glass s53p4 (bag) has an antimicrobial effect that can reduce the use of antibiotics. the aim of this study was to evaluate the antimicrobial efficacy of bag in vitro on staphylococci in biofilm and in planktonic form. secondary aims were to investigate whether supernatant fluid primed from bag retains the antibacterial capacity and if ciprofloxacin enhances the effect. methods: bag-s53p4 granules, <45 mm, primed in tryptic soy broth (tsb) were investigated with granules present in tsb (100 mg/ml) and after removal of granules (100, 200, and 400 mg/ml). the efficacy of bag to eradicate staphylococcus aureus biofilm in vitro was tested using 10 different clinical strains and 1 reference strain in three test systems: the biofilm-oriented antiseptic test based on metabolic activity, the biofilm bactericidal test based on culturing surviving bacteria, and confocal laser scanning microscopy (clsm) combined with live/dead staining. results: exposure to 48 h primed bag granules (100 mg/ml) produced bactericidal effects in 11/11 strains (p ¼ 0.001), and clsm showed reduction of viable bacteria in biofilm (p ¼ 0.001). supernatant primed 14 days, 400mg/ml, reduced metabolic activity (p < 0.001), showed bactericidal effects for 11/ 11 strains (p ¼ 0.001), and clsm showed fewer viable bacteria (p ¼ 0.001). the supernatant primed for 48 h, or in concentrations lower than 400mg/ml at 14 days, did not completely eradicate biofilm. conclusion: direct exposure to bag granules, or primed supernatant fluid, effectively eradicated s. aureus in biofilm. the anti-biofilm effect is timeand concentration-dependent. when bag had reached its full antimicrobial effect, ciprofloxacin had no additional effect. article history received 15 september 2019 revised 20 april 2020 accepted 4 may 2020 keywords bag; biofilm; confocal laser scanning microscopy; staphylococcus aureus introduction bacteria can alternate between a planktonic and a biofilm state. as biofilm, bacteria are attached to a substratum, or to each other, and are embedded in a self-produced extracellular polymeric substance. since the 1970s, when nils høiby observed a link between the aetiology of a persistent infection and aggregates of bacteria in cystic fibrosis patients (1), biofilm infections have become increasingly recognised in chronic infections. a common pathogen is staphylococcus aureus, a potent biofilm producer (2–6). bacteria living in biofilm display an altered antimicrobial resistance phenotype through a lower metabolic rate and a reduced rate of cell division (7,8). in addition, the biofilm can act as a diffusion barrier, and also cause deactivation of antimicrobial substances (9–11). as a result, the minimum inhibitory concentration of antimicrobial compounds on bacteria in biofilm can reach 500–1000 times that of their planktonic counterparts (9,12). mature biofilm can further shed off planktonic bacteria or micro-colonies that may travel to other parts of the body, causing relapsing infections (10). because of these biofilm defense mechanisms and growing antimicrobial resistance (13,14), we need innovative new treatments in addition to conventional antimicrobial treatment. one such option is bioactive glass (bag), a synthetic silica-based material. bag gradually releases ions from the granules’ surfaces when in contact with biological fluids. this leads to an increase in osmotic pressure and ph, thus making the surrounding environment unsuitable for microbial growth (15–17). there are several types of bag with differing antibacterial activity, one of which, bag-s53p4, has been well studied (15,18). in clinical practice, bag has been used to fill bone cavities (19) and treat osteomyelitis in orthopaedic surgery (20), chronic frontal sinusitis during frontal sinus obliteration (21), and chronic otitis media during mastoid obliteration (22,23). the diameter of the granules is usually between 0.5 mm and 3.15 mm. contact torstein grønseth t.gronseth@gmail.com ørenese og halsavdelingene, oslo universitetssykehus hf, postboks 4950 nydalen, oslo, 0424, norway � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 3, 217–225 https://doi.org/10.1080/03009734.2020.1765908 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1765908&domain=pdf&date_stamp=2020-06-29 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1765908 http://www.tandfonline.com the main aim of the study was to evaluate the antimicrobial efficacy of bag-s53p4 in vitro on clinical strains of s. aureus in biofilm and planktonic form. the sub-aims were to investigate whether the supernatant fluid primed from bag retained anti-biofilm properties after removal of the granules, and to evaluate whether addition of ciprofloxacin causes an increased anti-biofilm effect. methods study design ten clinical strains of s. aureus and one reference collection strain were first studied to verify their biofilm-producing capacity. the 11 bacterial strains were then tested in a planktonic state for sensitivity to bag (broth dilution test) and ciprofloxacin (disc diffusion test). if a strain was not sensitive in the planktonic state, it was excluded from further tests in the biofilm form. the antimicrobial effect on bacteria living in biofilm in vitro was then tested using three different methods: biofilm-oriented antiseptic test (boat); biofilm bactericidal test; and confocal laser scanning microscopy (clsm). bacterial strain collection and verification ten clinical strains of s. aureus were collected from 10 randomly selected unique individuals with draining ears at oslo university hospital, in a period from april to october 2014. s. aureus atcc 29213, a previously described strain known for its biofilm-producing capabilities, was used as a positive control (24,25). the strains from the draining ears were obtained by using an otomicroscope and a sterile swab (vwr transport swabs, copan, breschia, italy). the identification and antibiotic susceptibility testing did not reveal any mrsa strains (maldi-tof-ms, bruker daltonik gmbh, bremen, germany; vitekv r 2, biom�erieux s.a. france). the bacteria were stored in a freezing broth at �70 �c (frysebuljong, oslo university hospital, oslo, norway). before the tests, each strain was plated onto blood agar plates (blod agar plater, oslo university hospital, oslo, norway) for amplification and verification of purity. the blood agar plates were incubated for 24 h at 37 ± 1 �c. test substance our study evaluated the antimicrobial efficacy of smaller granules of bag-s53p4, <45 mm, (donated by bonalive biomaterials, turku, finland), which have previously been shown to have greater antimicrobial activity compared with larger-sized granules (16). the concentrations and exposure times used are shown in table 1. the concentrations were selected based on our broth dilution test and on previous studies (26). the test substance was obtained by adding bags53p4 < 45 mm to tryptic soy broth (tsb) with 1% glucose/1% nacl, ph 7.0, and vortexed (60 s) to ensure that all granules were in contact with the broth. the test substances were placed at room temperature prior to the experiment. one test substance was prepared for 48 h, and one 14 days prior to the test day. the time that the tsb was exposed to the bag granules prior to test day is called priming time. on the test day, the 48-h solutions were further divided into one group with granules (granule contact test) and one group with only the supernatant (supernatant test). the 14day solutions were only tested in the supernatant form (supernatant test) (table 1). granule contact test only 100 mg/ml bag primed for 48 h was tested because of its complete eradication of biofilm at this concentration. supernatant test bag granules were removed from the test solutions, and only the supernatant was used for further testing. it was divided into two sub-tests: i) addition of ciprofloxacin; and ii) without added ciprofloxacin. the concentration of ciprofloxacin was 0.5 mg/ml, based on a previously published study on the expected concentration of ciprofloxacin in the middle ear mucosa (27). no comparisons were made between the efficacy of bag þ ciprofloxacin and bag alone for the concentrations of 14-day primed 400 mg/ml and 200 mg/ml. the reason is that the bactericidal test for bag alone showed 0 strains (400 mg/ml) and 3 strains (200 mg/ml) surviving of the total 11 strains. tsb (with 1% glucose/1% nacl) broth was ph-adjusted with naoh, to ph 9.7, 10.7, and 11.7 and used as controls to similar ph-level bag. the ph was chosen as a result of the highest measured ph from the bag in tsb for 14 days. all ph measurements used a ph metre (inolabv r ph 7310 p, wtw gmbh, weilheim, germany). table 1. test substance and exposure time. test substance priming time bag-s53p4 < 45 mm, tsb with 1% glucose/1% nacl granule test bag tsb, 100 mg/ml 2 days supernatant test bag tsb, 100 mg/ml 2 days bag tsb, 200 mg/ml 2 days bag tsb, 400 mg/ml 2 days bag tsb, 100 mg/ml 14 days bag tsb, 200 mg/ml 14 days bag tsb, 400 mg/ml 14 days bag tsb, 100 mg/ml with ciprofloxacin 5 mg/ml 2 days bag tsb, 200 mg/ml with ciprofloxacin 5 mg/ml 2 days bag tsb, 400 mg/ml with ciprofloxacin 5 mg/ml 2 days bag tsb, 100 mg/ml with ciprofloxacin 5 mg/ml 14 days bag tsb, 200 mg/ml with ciprofloxacin 5 mg/ml 14 days bag tsb, 400 mg/ml with ciprofloxacin 5 mg/ml 14 days ph-adjusted (naoh) tsb with 1% glucose/1% nacl ph-adjusted tsb, ph 7.0 ph-adjusted tsb, ph 9.7 ph-adjusted tsb, ph 10.7 ph-adjusted tsb, ph 11.7 all tsb with 1% glucose/1% nacl solutions were from the norwegian veterinary institute, oslo, norway. bag: bioactive glass; tsb: tryptic soy broth. 218 t. grønseth et al. disc diffusion susceptibility testing of ciprofloxacin each of the strains of the s. aureus was tested in its planktonic state to evaluate the efficacy of ciprofloxacin by a disc diffusion test according to the eucast disc diffusion method, version 5. single colonies from a fresh overnight bacterial culture on blood agar were collected and transferred into sterile saline. the suspension was measured to mcfarland 0.5 and the spread on m€uller hinton agar plates using an automated plate spreader. diffusion discs (cip 5, sensi-disctm, bbltm, discs, becton, dickinson and company, sparks, md, usa) with ciprofloxacin 5 mg were applied to the agar plates. inhibition zones were evaluated with callipers after 18 h of incubation at 36 ± 1 �c. broth dilution test for planktonic bacteria a modified broth dilution test was employed to evaluate the efficacy of bag on planktonic-state bacterial strains (28). first, 48-h suspensions of tsb with bag were made at concentrations of 50, 100, 200, and 400 mg/ml, separately. two test sets were prepared for the granule contact and supernatant test. in the granule test, further tests were carried out with the bag granules in the test solution. in the supernatant test, the supernatant of the primed fluid was pipetted into new sterile tubes without the granules for further testing. bacterial preparation mcfarland standard 0.5 turbidity suspensions of 1 � 108 colony-forming units (cfu)/ml in sterile saline were prepared from 24-h agar incubations of pure cultures from each bacterial strain. the mcfarland 0.5 solutions were then diluted in sterile 48-h primed test solution as described above to reach the standard inoculum of 5 � 105 cfu/ml. in the granule test, the supernatant fluid was first moved into new test tubes. in the new test tubes the desired inoculum concentration was reached. thereafter the supernatant with the inoculum was transferred back to the original test tubes with the bag. in the supernatant test, the supernatant of the primed fluid was pipetted into new sterile tubes without granules. from the supernatant the described inoculum concentration was prepared, and 200 ml of bacterial inoculum with different primed supernatant concentrations was transferred in triplets to a sterile 96-well microtiter plate (nunclon delta surface, thermo fisher scientific, roskilde, denmark). prepared samples were used within 30 min of preparation. sterility control with broth only and each concentration of tsb/bag were included in all experiments. each bacterial strain was grown in tsb to provide a positive growth control. the test tubes for the granule test and the microplates for the supernatant test were kept at 35 ± 1 �c for about 20 ± 0.5 h before being visually evaluated for growth or no growth (28). biofilm assay the ability of the s. aureus strains to form biofilm was tested in a 96-well microtiter plate (nunclon delta surface) according to a previously published method (29). one colony of each bacterial strain was inoculated in 5 ml of tsb, which was cultured overnight at 37 ± 1 �c. the next day, 180 ml of tsb with 1% glucose/1% nacl was transferred to each of the wells on the microtiter plate, except for the first three blank control wells, where 200 ml was transferred. the overnight cultures were then vortexed for 40 s, and 20 ml was transferred to all the wells, except for the blank control. each strain of the s. aureus was tested in three parallel wells. the microtiter plate was incubated at 37 ± 1 �c for 48 h. the wells were then washed three times with 220 ml of tap water and left to dry at room temperature for 30 min. after drying, 220 ml of crystal violet (1% solution, sigma aldrich, st louis, mo, usa) was added and incubated for 30 min. the wells were washed five times with 220 ml of tap water. to extract the crystal violet from the biofilm, 220 ml of ethanol:acetone (70:30 w:w) was added. the results were calculated by measuring the optical density at 570 nm (siemens bep 2000 advance, germany). the experiment was repeated three times. biofilm-oriented antiseptics test (boat) in the presence of viable metabolically active bacteria, tetrazolium chloride (ttc) is reduced from a colourless compound to red formazan, which correlates to the number of viable cells (30–32), utilised in the boat method (33). ten clinical strains of s. aureus and the reference collection strain s. aureus atcc 29213 were tested. the same 96well microtiter plate was used as in the biofilm assay and the biofilm was produced as described above with 12 parallel wells for each strain. after 48 h of incubation, the wells were washed with 220 ml sterile 0.85% nacl, before adding the active or control substances. the microtiter plate was incubated at 37 ± 1 �c for 24 h. for each strain, three parallel wells were exposed to bag at concentrations of 400 mg/ml, 200 mg/ml, and 100 mg/ml. three wells were used as controls. the content in all wells was then removed and dey engley neutralising broth was added for 5 min. the wells were filled with 200 ml of tsb:ttc in the ratio of 20:1. the microtiter plate was incubated at 37 ± 1 �c for 7.5 h. the results were evaluated visually by colour change and measured calorimetrically. the amount of formazan produced was calculated calorimetrically by measuring the optical density at 492 nm (siemens bep 2000 advance, germany). the experiment was repeated three times (34). biofilm bactericidal test to confirm the eradication effects of bag solutions on staphylococcus biofilm, a model described by t. mah was used, modified for staphylococci (33). all 11 strains were tested. the first steps of establishing a biofilm, and applying the test solution, sterile 0.85% nacl, and neutralising broth upsala journal of medical sciences 219 were identical to those of the boat method described above and as described in our previous study (34). however, instead of then adding tsb:ttc, 200 ml of tsb was added to each well and incubated at 37 ± 1 �c for 24 h. of the overnight culture 5 ml was transferred from each well onto a blood agar plate and incubated at 37 ± 1 �c for 24 h before the results were evaluated visually. if there was no growth, the tested substance was considered bactericidal. the experiment was repeated three times. confocal laser scanning microscopy (clsm) the first steps of making biofilm, applying test solutions, and adding neutralising broth were identical to those of the boat method described above, except that a lab-tek ii chambered coverglass with cover 8-well (thermo fisher scientific, inc., waltham, ma, usa) was used instead of a microtiter plate. the slides were stained with filmtracertm live/deadv r biofilm viability kit, (molecular probes, thermo fisher scientific, inc., waltham, ma, usa) according to the manufacturer’s specifications. images of the stained biofilm were generated on a clsm (zeiss lsm710, germany). a 488 nm argon laser line was used for the sytov r 9 and a 561 nm dpss laser line for the propidium iodide. the ratio of dead or dying cells to the total number of cells in the biofilm was determined by imagej software (open source, public domain). the experiment was repeated twice. statistical analysis statistical analyses were performed using spss statistical software (release 25.0 spss inc., chicago, il, usa, and stata mp/ 15.1, statacorp, college station, tx, usa). p < 0.05 was considered to be statistically significant. in the boat, paired t test was performed to identify any significant differences between the test substance groups compared to the controls. in the bactericidal test, a mcnemar asymptotic test (using stata mp/15.1) was performed to identify any significant differences between the test substance groups compared to the controls. in clsm, significant difference between the treated groups and control was calculated by paired t test. approval the collection of specimens from human subjects was approved by the regional ethical committee (2014/1956, rek sør-øst c). results disc diffusion susceptibility testing of ciprofloxacin ciprofloxacin showed clear inhibition zones �21 mm in the disc diffusion tests, indicating sensitive bacteria for all strains, except strain 3, which was not used for further testing with ciprofloxacin. broth dilution test for planktonic bacterial strains a bag concentration of 50 mg/ml showed growth for all strains (11/11) in both the granule and the supernatant tests. in the test with bag granules, 100 mg/ml, there was no growth for any strains (p ¼ 0.001), but the supernatant test, 100 mg/ml, showed growth in 9/11 strains (p ¼ 0.160). the concentrations of 200 and 400 mg/ml of bag showed no growth for any of strains (p ¼ 0.001) in either test. biofilm assay all strains of staphylococci grew biofilm within 48 h in 96well microtiter plates. mean optical density ±1 sd of biofilm produced by 11 s. aureus stains was 1.864 ± 0.945. the minimum optical density was 0.851, and the maximum optical density was 3.00. biofilm-oriented antiseptics test (boat) boat was only performed in supernatants, since it was impossible to remove the bag granules before measuring optical density, thus giving a misleading optical density. the reduction in optical density as an indirect measure of metabolic activity was significant for all concentrations of the test solutions primed for 14 days. after 48 h of priming only the 400 mg/ml concentration showed a significant reduction (table 2). the optical densities in the 14-day primed test at 400 mg/ml and 200 mg/ml concentrations were similar to the blank control, while the other concentrations and durations showed some metabolically active bacteria in biofilm (table 2). this was also observed visually, where all wells treated with the 400 mg/ml concentration for 14 days appeared blank (figure 1). the wells exposed to other test concentrations produced different shades of red, which indicated surviving metabolically active bacteria for at least some of the strains. a ph-adjusted tsb solution was used as a control and shown to have a significant effect on the reduction of staphylococcal metabolic activity at ph 11.7 and 10.7, but not ph 9.7. all wells were completely blank with no visible shades of red colour only for the highest ph of 11.7. the efficacy of bag in the supernatant test was performed with and without ciprofloxacin. the tests showed an additional reduction of viable bacteria with ciprofloxacin only in concentrations of 100 mg/ml (p ¼ 0.005) and 200 mg/ml primed for 48 h (p ¼ 0.001). there was no additional effect of ciprofloxacin at any concentration of the supernatants primed for 14 days. biofilm bactericidal test in the granule test, all 11 strains of staphylococci biofilm were eradicated. in the supernatant tests, no bactericidal effects were recorded for the 48-h primed 100 and 200 mg/ ml or the 14-day primed 100 mg/ml. in the supernatant test, only the 14-day primed 400 mg/ml concentration eradicated all strains (table 2). for the remaining concentrations 220 t. grønseth et al. ta b le 2. a c om p ar is on of ba g ef fe ct s ve rs us co n tr ol . bo a t ba ct er ic id al te st c on fo ca l la se r sc an n in g m ic ro sc op y w it h li v e/ d ea d st ai n in g te st so lu ti on n m ea n ± 1 sd p v al ue su rv iv in g st ra in s/ to ta l n o. st ra in s p v al ue ra ti o d ea d / to ta l ce ll n o. ± 1 sd p v al ue u n tr ea te d b ac te ri al st ra in s (c on tr ol ) 11 2. 88 4 0. 11 – 11 /1 1 – 0. 19 0 0. 01 – su p er n at an t te st ba g -s 53 p4 < 45 m m , 10 0 m g /m l, 2 d ay s 11 2. 70 4 0. 49 0. 30 11 /1 1 – 0. 17 6 0. 12 0. 85 ba g -s 53 p4 < 45 m m , 20 0 m g /m l, 2 d ay s 11 2. 40 2 0. 96 0. 15 11 /1 1 – 0. 74 6 0. 35 0. 07 ba g -s 53 p4 < 45 m m , 40 0 m g /m l, 2 d ay s 11 0. 78 9 0. 72 < 0. 00 1 8/ 11 0. 08 0. 93 1 0. 21 0. 01 ba g -s 53 p4 < 45 m m , 10 0 m g /m l, w it h ci p ro flo xa ci n , 2 d ay s 10 1. 63 5 0. 86 0. 00 1 10 /1 0 – 0. 81 1 0. 32 0. 04 7 ba g -s 53 p4 < 45 m m , 20 0 m g /m l, w it h ci p ro flo xa ci n , 2 d ay s 10 0. 75 4 0. 80 < 0. 00 1 10 /1 0 – 0. 83 6 0. 11 0. 00 2 ba g -s 53 p4 < 45 m m , 40 0 m g /m l, w it h ci p ro flo xa ci n , 2 d ay s 10 0. 20 8 0. 09 < 0. 00 1 4/ 10 0. 01 1. 09 9 0. 07 < 0. 00 1 ba g -s 53 p4 < 45 m m , 10 0 m g /m l, 14 d ay s 11 1. 20 8 1. 07 0. 00 1 11 /1 1 – 0. 68 6 0. 13 0. 00 8 ba g -s 53 p4 < 45 m m , 20 0 m g /m l, 14 d ay s 11 0. 17 3 0. 05 < 0. 00 1 3/ 11 0. 00 5 0. 95 8 0. 26 0. 01 5 ba g -s 53 p4 < 45 m m , 40 0 m g /m l, 14 d ay s 11 0. 15 2 0. 03 < 0. 00 1 0/ 11 0. 00 1 1. 08 6 0. 05 < 0. 00 1 ba g -s 53 p4 < 45 m m , 10 0 m g /m l, w it h ci p ro flo xa ci n , 14 d ay s 10 1. 16 3 0. 44 0. 00 2 10 /1 0 – 0. 87 1 0. 13 0. 00 3 ba g -s 53 p4 < 45 m m , 20 0 m g /m l, w it h ci p ro flo xa ci n , 14 d ay s 10 0. 25 7 0. 10 < 0. 00 1 2/ 10 0. 00 8 1. 09 9 0. 12 0. 00 1 ba g -s 53 p4 < 45 m m , 40 0 m g /m l, w it h ci p ro flo xa ci n , 14 d ay s 10 0. 20 7 0. 11 < 0. 00 1 0/ 10 0. 00 2 1. 12 9 0. 12 0. 00 1 g ra n ul e te st ba g -s 53 p4 < 45 m m , 10 0 m g /m l 11 n /a n /a n /a 0/ 11 0. 00 1 1. 04 6 0. 11 0. 00 1 p h -a d ju st ed ts b ts b w it h 1% g lu co se /1 % n ac l, p h 9. 7 11 2. 91 5 0. 11 0. 56 7 11 /1 1 – 0. 23 6 0. 05 0. 17 ts b w it h 1% g lu co se /1 % n ac l, p h 10 .7 11 0. 74 9 0. 80 < 0. 00 1 6/ 11 0. 02 5 0. 73 1 0. 20 0. 02 ts b w it h 1% g lu co se /1 % n ac l, p h 11 .7 11 0. 12 9 0. 02 < 0. 00 1 0/ 11 0. 00 1 1. 17 0 0. 04 < 0. 00 1 si g n ifi ca n ce ca lc ul at ed b y p ai re d t te st or m cn em ar as ym p to ti c te st . ba g : b io ac ti ve g la ss ; bo a t: b io fil m -o ri en te d an ti se p ti cs te st ; n /a : n ot ap p lic ab le ; sd : st an d ar d d ev ia ti on ; ts b: tr yp ti c so y b ro th . upsala journal of medical sciences 221 figure 1. biofilm-oriented antiseptics test (boat). supernatant test: bag 14-day priming time (a); bag 2-day priming time (b). bag concentration from left to right is 400 mg/ml, 200 mg/ml, 100 mg/ml, and 0 mg/ml; 3 wells per concentration. rows 1–7: strains 1–7; row 8: negative control. red formazan is a sign of viable cells. (96-well microtiter plate; nunclon delta surface, thermo fischer scientific). figure 2. confocal laser scanning microscopy (clsm) stacks of staphylococcus aureus biofilm. 1) control; ph-adjusted tsb: 2) ph 9.7; 3) ph 10.7; 4) ph 11.7; 5) granule test; supernatant test, 2-day exposure: 6) 100 mg/ml; 7) 100 mg/ml with ciprofloxacin; 8) 200 mg/ml; 9) 200 mg/ml with ciprofloxacin; 10) 400 mg/ml; 11) 400 mg/ml with ciprofloxacin; supernatant test, 14-day exposure: 12) 100 mg/ml; 13) 100 mg/ml with ciprofloxacin; 14) 200 mg/ml; 15) 200 mg/ml with ciprofloxacin; 16) 400 mg/ml; 17) 400 mg/ml with ciprofloxacin. the units are in mm. 222 t. grønseth et al. and durations, there was a significant bactericidal effect, but not complete eradication for all strains (table 2). there was an additional effect of ciprofloxacin in the bactericidal test only with 400 mg/ml, 48-h primed (p ¼ 0.046). ciprofloxacin showed no additional bactericidal effect for the other concentrations. confocal laser scanning microscopy (clsm) in the granule test, the ratio of compromised cells to the total number of cells was close to 1, indicating that all bacteria were dead or dying (table 2; figure 2). in the supernatant test, there was a reduction in the 48-h primed solutions only for the concentration of 400 mg/ml (table 2; figure 2). some viable biofilm bacteria were still observed. there was a significant reduction in viable cells for all concentrations primed for 14 days. the ratio of dead to total number of cells was close to 1 only for the concentrations of 200 and 400 mg/ml (table 2). clsm showed an additional effect of ciprofloxacin only in the group with 100 mg/ml (48 h primed). summary of results � bag granules (100 mg/ml) eradicated staphylococci aureus in planktonic form. � bag supernatant (200 mg/ml) eradicated staphylococci aureus in planktonic form. � bag granules (100 mg/ml) eradicated staphylococci aureus in biofilm in the biofilm bactericidal test. � bag supernatant (400 mg/ml) primed for 14 days eradicated staphylococci aureus in biofilm in the biofilm bactericidal test. � boat tests showed that there was a reduction in metabolic activity for all concentrations after 14-day priming, but only for the 400 mg/ml concentration after 48 h priming. � bag granules (100 mg/ml) showed reduction in viable bacteria in biofilm on clsm. � bag supernatant showed reduction in viable bacteria in biofilm for all concentrations primed for 14 days, and for 400 mg/ml primed for 48 h on clsm. � ciprofloxacin had an additional effect only with a 48 h priming time. discussion several studies have indicated a difference in the antimicrobial sensitivity of laboratory and clinical strains (35–37). the strength of the present study is the relatively large number of clinically relevant strains compared to previous studies, and the use of three separate test systems. all three tests showed significant antibacterial properties exhibited by bag against staphylococci in biofilm and in planktonic states. our results show that primed bag granules in direct contact with biofilm eradicate all viable bacteria even at the lowest concentration and shortest priming time. this indicates that bag is a potent anti-biofilm substance. previous studies have indicated that small granules have a significantly stronger antimicrobial effect, and reach a higher ph in solution, compared with larger granules, supposedly because of a greater surface area (16). therefore we used the smallest available granules (bag-s53p4, <45 mm). our results support the use of small granule size to rapidly gain full antimicrobial effect. it is of great interest to know if the supernatant fluid primed with granules retains its antimicrobial and anti-biofilm properties after physical removal of the granules. to our knowledge, this has not been previously investigated for staphylococcal biofilms. the results show that the surrounding fluid itself assumes anti-biofilm properties after long enough priming, even when isolated from the physical granules. complex bone cavities, such as the mastoid cell system, are often difficult to surgically pack, and may have pockets that remain incompletely filled with granules despite efforts to pack them completely. therefore it is important to know whether the supernatant has the same effect as granules. when the supernatant from the lowest concentration and 48-h priming time was tested, there was no reduction in viable biofilm (table 2). the same concentration with 14-day priming resulted in reduction in viable biofilm bacteria when using clsm and boat. however, only the supernatant from the concentration of 14-day primed 400 mg/ml bag eradicated all the biofilm bacteria strains in all three test systems. this shows that the anti-biofilm effect on biofilm-associated staphylococci is dependent on concentration and priming time and that it could be beneficial to use pre-primed saline solution. this can be of importance when packing chronically infected bone cavities with biofilm-associated staphylococci. ciprofloxacin, which has good penetration into biofilms (38), was evaluated regarding possible additional antimicrobial effect when combined with bag. clsm and boat showed a significant reduction in viable bacteria in biofilm by combining bag and ciprofloxacin for the two lowest concentrations primed for 48 h, compared with bag only (table 2). however, the combination did not show complete bactericidal effects. this indicates that the addition of ciprofloxacin to a bag treatment could render some additional antimicrobial effect, which could be clinically beneficial during the first days of treatment before bag reaches its maximal effect on biofilm-associated bacteria. the antibacterial properties of bag have been attributed to its high ph and osmotic effects caused by the nonphysiological concentration of dissolved ions (39). pre-adjusted ph-buffered (naoh) tsb broth was used as a control to compare with bag at similar ph. the most alkaline control was set to ph 11.7, the same as achieved by bag primed for 14 days at 400 mg/ml. at this ph level, there was a significant reduction in amount of viable bacteria in biofilm in all three test systems. it was also the only ph that eradicated all bacteria in the bactericidal biofilm test. previous studies have shown that s. aureus can survive up to a ph of 9.83 (40,41). this explains why the tsb control broth (ph 9.7) and bag supernatant (100 mg/ml, primed for 48 h) (ph 9.9) upsala journal of medical sciences 223 showed no reduction in the amount of viable biofilmassociated bacteria. when the ph of the tsb control broth was raised to 10.7, five out of eleven strains were eradicated. supernatant from bag 400 mg/ml 48-h priming resulted in a ph of 10.6, and three out of eleven strains were eradicated. this finding is in line with previous studies (42) and indicates that high ph is an important factor against biofilm-associated bacteria. these in vitro results are promising, but several factors in vivo can affect the clinical response. if the environment around the biofilm does not achieve bactericidal ph, s. aureus can upregulate a naþ/hþ antiporter system in response to a sudden alkaline shock environment (43). although further clinical studies are required to explore the antimicrobial effects on biofilm in vivo, this in vitro study indicates that both supernatant and granules of bag can give bactericidal anti-planktonic and anti-biofilm properties. this reduces the likelihood of recurrent infections after incomplete filling of the bone cavity with bag (20,44). the tested granules are smaller than the granules typically used in bone packing. our study suggests that the risk of recurrent infections could possibly be reduced by using small granules, <45 mm, which proved to give a more rapid and higher rise in ph. we also showed that the bactericidal effect of bag is dependent on exposure time and concentration. when bag has reached its full antimicrobial effect it is as efficient as ciprofloxacin in vitro and could reduce the use of conventional antibiotics where s. aureus biofilm is suspected. our results also suggest that short-term use of antibiotics at the beginning of treatment may be beneficial. we conclude that bag-s53p4, <45 mm, has powerful antimicrobial effects on s. aureus in both planktonic and biofilm states. duration of exposure and concentration are important factors for achieving maximal effect on biofilm-associated bacteria. ciprofloxacin may have an additional effect during the first days of treatment. acknowledgements the authors thank the staff at the department of microbiology at the oslo university hospital and the norwegian veterinary institute for their excellent support that allowed us to perform the sample analysis in this paper. disclosure statement the authors have no conflict of interest in the subject matter or materials discussed in this manuscript. author contributions tg and jts conceived the study; tg, lkv, lln, mvu, and jts conducted research and analysed data; tg wrote the first draft and reviewed it with jts. all authors contributed to, read, and approved the manuscript. funding the project was funded by the university of oslo. notes on contributors torstein grønseth is a consultant at the department of otolaryngology, head and neck surgery, oslo university hospital, oslo, norway and a research fellow at the institute for clinical medicine, university of oslo, oslo, norway. lene k. vestby is head of section for immunology and virology, department of analysis and diagnostics at the norwegian veterinary institute. she is also a senior researcher and hold a phd in bacteriology. live l. nesse, dvm, phd, dr.med.vet, is a senior research scientist at the norwegian veterinary institute. magnus von unge, md, phd, is a ear surgeon and a professor emeritus at the department of clinical medicine, university of oslo, norway. juha t. silvola is a senior consultant in otology-otoneurology and 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https://doi.org/10.1097/mao.0b013e31825f249e https://doi.org/10.1097/mlg.0b013e3181826e24 https://doi.org/10.1097/mlg.0b013e3181826e24 https://doi.org/10.1080/08927014.2014.904294 https://doi.org/10.1080/08927014.2014.904294 https://doi.org/10.1080/000163598422901 https://doi.org/10.1034/j.1600-0501.2002.130106.x https://doi.org/10.1034/j.1600-0501.2002.130106.x https://doi.org/10.1111/j.1574-695x.2010.00736.x https://doi.org/10.1302/0301-620x.96b6.33014 https://doi.org/10.1302/0301-620x.96b6.33014 abstract introduction methods study design bacterial strain collection and verification test substance granule contact test supernatant test disc diffusion susceptibility testing of ciprofloxacin broth dilution test for planktonic bacteria bacterial preparation biofilm assay biofilm-oriented antiseptics test (boat) biofilm bactericidal test confocal laser scanning microscopy (clsm) statistical analysis approval results disc diffusion susceptibility testing of ciprofloxacin broth dilution test for planktonic bacterial strains biofilm assay biofilm-oriented antiseptics test (boat) biofilm bactericidal test confocal laser scanning microscopy (clsm) summary of results discussion acknowledgements disclosure statement author contributions references attitudes towards embryo donation in swedish women and men of reproductive age full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 attitudes towards embryo donation in swedish women and men of reproductive age kjell wånggren, frida prag & agneta skoog svanberg to cite this article: kjell wånggren, frida prag & agneta skoog svanberg (2013) attitudes towards embryo donation in swedish women and men of reproductive age, upsala journal of medical sciences, 118:3, 187-195, doi: 10.3109/03009734.2013.808294 to link to this article: https://doi.org/10.3109/03009734.2013.808294 © informa healthcare view supplementary material published online: 20 jun 2013. submit your article to this journal article views: 550 view related articles citing articles: 12 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.808294 https://doi.org/10.3109/03009734.2013.808294 https://www.tandfonline.com/doi/suppl/10.3109/03009734.2013.808294 https://www.tandfonline.com/doi/suppl/10.3109/03009734.2013.808294 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.808294 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.808294 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.808294#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.808294#tabmodule upsala journal of medical sciences. 2013; 118: 187–195 original article attitudes towards embryo donation in swedish women and men of reproductive age kjell wånggren, frida prag & agneta skoog svanberg department of women’s and children’s health, uppsala university, 751 85 uppsala, sweden abstract background. when performing in-vitro fertilization (ivf), more embryos than needed are often derived. these embryos are usually frozen and stored, but as ruled by swedish law they have to be discarded after 5 years. in other countries it is legal to donate the excess embryos to other infertile couples who for different reasons cannot undergo the procedure of ivf. the aim of the present study was to investigate public opinion in sweden regarding different aspects of embryo donation. methods. a questionnaire regarding attitudes towards aspects of embryo donation was sent to a randomized sample of 1,000 swedish women and men of reproductive age. results. a total of 34% responded to the questionnaires. a majority of the respondents (73%) were positive towards embryo donation. seventy-five per cent agreed that it should be possible to donate embryos to infertile couples. approximately half of the participants (49%) supported embryo donation to single women. a majority of the participants emphasized that demands should be imposed on the recipient’s age (63%), alcohol addiction (79%), drug addiction (85%), and criminal record (67%). forty-seven per cent of the respondents agreed that the recipient should be anonymous to the donor, and 38% thought that the donor should remain anonymous to the child. conclusions. the results of the present study indicate support for embryo donation among a subset of the swedish population of reproductive age. if embryo donation were to be allowed in sweden, strategies for treatment and counselling need to be developed. key words: attitudes, disclosure, embryo donation, gender, swedish population introduction infertility is a common problem worldwide (1). the most common and effective treatment for infertility today is in-vitro fertilization (ivf) (2). in sweden more than 3,500 out of approximately 120,000 (3.1%) of children are born annually after ivf treatment (3). during ivf treatment, quite often more good-quality embryos will be created than the couples will use (4). these supernumerary embryos are usually frozen and stored for later use. embryo donation is legal in certain countries, such as australia, canada, england, finland, france, new zealand, and usa (5,6). for couples who carry a genetic disease, or where neither the male nor the female partner has viable gametes, embryo donation could be an alternative. in most countries, adoption is the only option for these couples. embryo donation could also be an option for couples who have undergone several unsuccessful ivf treatments. embryo donation is cost-effective and involves lower costs than ivf treatment, or egg and sperm donation (7). however, embryo donation requires many ethical considerations and difficult decisions (8,9). on the one hand, embryo donation makes it possible for the couple to experience pregnancy, childbirth, and early childhood (10). on the other hand, the consequences for the child of not being genetically related to any of the parents or of having genetic siblings in other families may be psychologically difficult to handle. correspondence: kjell wånggren, department of women’s and children’s health, uppsala university, 751 85 uppsala, sweden. e-mail: kjell.wanggren@kbh.uu.se (received 21 march 2013; accepted 16 may 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.808294 http://informahealthcare.com/journal/ups mailto:kjell.wanggren@kbh.uu.se there are a lot of embryos which continuously have to be discarded due to time limits for cryopreservation which could be used for treatment in selected cases of infertile couples (11). in sweden embryos have to be discarded after five years’ storage (12). if embryo donation is allowed, couples who undergo ivf treatment may have to make a decision whether they want to donate their surplus frozen embryos or have them discarded. most couples undergoing ivf treatment have a hard time deciding what to do with their surplus embryos, and the decision process is often stressful for parents (13,14). swedish figures have shown that 92% of infertile couples would choose to donate their superfluous embryos for stem cell research, rather than having them discarded (15). infertile couples who have had embryos frozen are generally in favour of donating to research but are less likely to donate to other infertile couples (16-18). however, in practice it is most common that the couples, either by an active decision or by not responding to requests from ivf clinics, choose to have their stored embryos discarded, even in countries where donation is a legal option (19). the majority of the swedish population, over 70%, are positive to oocyte donation to infertile couples (20). oocyte donation and sperm donation in conjunction with ivf treatment has been permitted since 2003 in sweden. according to swedish law, the donor’s identity should be traceable for the donorconceived person when they reach adulthood (12). however, embryo donation for reproduction, surrogacy, and assisted reproduction of single women is not allowed in sweden. some patients therefore may have to go abroad for infertility treatment. attitudes among embryo donors and recipients have earlier been studied (18,21). popular attitudes towards embryo donation to other couples have, however, to our knowledge not previously been reported. the aim of this study was to evaluate attitudes towards donation of embryos in a random sample of the swedish population of reproductive age. materials and methods potential participants received by post a letter with a questionnaire and a stamped, addressed envelope. the accompanying letter described the purpose of the study, provided brief information about embryo donation and current legislation, and asked participants for voluntary anonymous participation in the study (supplement a, only available in the online version of the journal; please find this material with the following direct link to the article: http://www. informahealthcare.com/doi/abs/10.3109/03009734. 2013.808294). a reminder was sent four weeks later to those who had not responded to the first questionnaire. this reminder letter stressed the importance of participation for the study’s outcome and also clarified the selection criteria. a third letter was sent two weeks later to non-responders, where again the importance of participation and the assurance of anonymity was emphasized. approval of ethics committee the study was approved by the regional ethics committee of uppsala university, d.nr. 2010/455. study population participants in the study were randomly selected from a data registry covering the whole swedish population. a total of 500 women in the age group 18–45 years and 500 men in the age group 18–55 years were selected. the age groups were chosen to reflect the reproductive part of the population and thus those who might be concerned about donors and recipients of embryos. socio-demographic data of the study participants are presented in table i. the responses were registered and handled in confidence, and anonymously coded to be processed in a database. no fee has been paid for the voluntary participation in the study. altogether 200 women out of 500 (40%) responded to the questionnaire and 138 of the 500 men (28%) responded to the questionnaire. twenty-three (0.02%) questionnaires were returned because they did not reach the respondent. the questionnaire the survey consisted of two parts. part one covered the personal data and contained 16 questions, of which 14 were composed of two sub-questions. part two contained 22 statements regarding embryo donation, donors, and recipients, on age, criminal background, tobacco use, etc. for the answers, we used a likert scale where one agrees with the statements in varying degrees (22). the scale was a 5-point scale and contained the options ‘strongly agree’, ‘agree to a large extent’, ‘neither’, ‘agree just a bit’, and ‘completely disagree’. furthermore, the option ‘do not know/cannot take a position’ was included in all claims. at the presentation of material in the tables, the two positive categories ‘strongly agree’ and ‘agree to a large extent’ were conjoined, as were the more negative categories ‘agree just a bit’ and ‘completely disagree’. the new variables are named ‘agree’ and ‘disagree’. the option ‘neither’ remains the sole 188 k. wånggren et al. http://www. informahealthcare.com http://www. informahealthcare.com alternative in the form of a ‘neutral’ opinion (tables ii–iv). two versions of the questionnaire were sent out, one aimed at women (supplement b, only available in the online version of the journal;please find this material with the following direct link to the article: http://www.informahealthcare.com/doi/abs/10.3109/ 03009734.2013.808294)and the other one at men (supplement c, only available in the online version of the journal;please find this material with the following direct link to the article: http://www. informahealthcare.com/doi/abs/10.3109/03009734. 2013.808294). the two questionnaires had different wordings in the section concerning personal information. the woman was asked if she had donated eggs, and the man if he had donated sperm. the wording of question 4 differed regarding whether the woman had been pregnant/if the man has given rise to pregnancies. apart from these two questions the survey designs were identical for men and women. statistics the program statistical package for the social sciences (spss) was used for recording responses and for statistical processing. to compare the differences between men and women in individual items, mann–whitney u test was used on the 3-degree scale. p < 0.05 was set as the threshold for significance. median values were calculated on the original data (5-point scale) for each item. results attitudes towards embryo donation (table ii) the statement ‘embryo donation should be allowed in sweden’ was supported by over 73% of respondents, which suggests a positive attitude towards embryo donation in sweden. an even a larger part, 75%, of respondents was in favour of allowing embryo donation to infertile couples, while fewer of the respondents (49%) were positive to embryo donation to single women. just over half (51%) of the respondents were in favour of embryos being donated for research (table ii). men were more willing than women to donate embryos to research. special requirements for the recipient of donated embryos (table iii) a majority of respondents believed that one should be able to make demands on the recipient’s age. the calculated mean of the appropriate minimum age of the recipient women was around 25 years and maximum age about 43 years (table iii). a majority of respondents thought that requirements should be imposed on the recipient’s alcohol dependency, and here more women than men agreed with the statement. an even larger number of the respondents thought that requirements should be imposed on the table i. socio-demographic data regarding female and male participants. the figures are given in frequency % (n) of the total number of respondents to each question/statement. the mean age is presented with standard deviations (sd) and the number of years living in sweden as means (m). women (n = 200) men (n = 138) % (n) % (n) mean age (sd) 32.5 (8.1) 38.8 (11.3) level of education university 46.2 (92) 33.3 (46) high school at least three years 36.7 (73) 36.2 (50) upper secondary school 9.0 (18) 18.8 (26) elementary school 8.0 (16) 11.6 (16) marital status single 17.5 (35) 25.4 (35) lives with wife/husband/partner 72.0 (144) 71.7 (99) lives alone but has a steady partner 10.5 (21) 2.9 (4) caused/experienced pregnancy 61.5 (123) 65.0 (89) difficulties in becoming pregnant/ causing pregnancy 37.0 (74) 4.3 (6) own children 55.0 (110) 65.9 (91) by natural means 95.4 (103) 94.4 (85) through assisted reproductive technologies (art) 3.7 (4) 3.3 (3) by art + naturally 0.9 (1) 2.2 (2) cryo-preserved fertilized eggs 0.5 (1) 2.2 (3) donated sperm/eggs 0.0 (0) 0.0 (0) born abroad 12.5 (25) 12.5 (17) living in sweden one’s entire life 83.4 (166) 85.3 (116) number of years (m) (distribution women 1–38, men 1–50) 13.4 20.3 religious community/church none 22.8 (45) 29.4 (40) swedish church 70.1 (138) 63.2 (86) other 7.1 (14) 7.4 (10) active in help organization 16.5 (33) 17.6 (24) is/has been blood donor 14.6 (29) 22.1 (30) familiar with infertility in the surroundings 77.0 (154) 64.7 (88) attitudes towards embryo donation in sweden 189 http://www. informahealthcare.com http://www. informahealthcare.com http://www. informahealthcare.com recipient’s drug addiction. this issue was found to be more important for women than for men. with regard to the recipient’s criminal background a majority of respondents agreed that one should be able to set specific requirements, and more women than men considered this as an important issue (table iii). attitudes to embryo donation and anonymity (table iv) more than one-third of the respondents thought that the embryo donor should remain anonymous to the child. almost half of the respondents stated that the embryo donor should remain anonymous to the recipient. about half of the respondents agreed that the recipient should be anonymous to the embryo donor. discussion the presented results indicate that there is a positive attitude in sweden towards allowing embryo donation. a majority of the participants in this swedish population study supports embryo donation, especially to other infertile couples but also for research. almost half of the respondents supported embryo donation to single women. the positive attitudes towards embryo donation are consistent with the previously demonstrated positive attitudes towards egg donation in sweden (20). even though a majority had a positive attitude towards oocyte donation, only 16% actually wanted to donate. in the present study, men were more positive than women towards donation of embryos for research purposes. this might be explained by the view that many women to a greater extent than men regard embryos as potential human beings (23). in a previous australian study, women who had embryos frozen regarded their embryos as human beings to a larger extent than men did, and were therefore less willing to donate them to research (24). it has also been shown that people who want children define embryos as people in earlier stages of the embryo’s development than do people without such a desire (23). women having frozen embryos are more willing to donate their embryos to scientific research than to infertile couples (25). the decision to donate embryos for fertility treatment is regarded as the most difficult decision (26). in a previous study, patients who were positive to donating embryos before treatment changed their minds after table ii. women’s and men’s attitudes towards embryo donation in sweden. all (n = 338) women (n = 200) men (n = 138) statement answer % (n) % (n) mda % (n) mda p embryo donation should bepermitted in sweden agree 72.8 (246) 74.5 (149) 4 70.3 (97) 4 ns neutral 5.9 (20) 3.0 (6) 10.1 (14) i disagree 13.0 (44) 14.0 (28) 11.2 (16) cannot decide 8.2 (28) 8.5 (17) 8.0 (11) embryos should be allowed to be donated to research agree 51.2 (173) 45.0 (90) 3 60.1 (83) 4 0.005 neutral 13.6 (46) 17.5 (35) 8.0 (11) i disagree 14.8 (50) 10.0 (20) 21.7 (30) cannot decide 20.4 (69) 27.5 (55) 10.1 (14) embryos should be allowed to be donated to infertile couples agree 75.4 (255) 77.0 (154) 4 73.2 (101) 4.5 ns neutral 5.0 (17) 3.5 (7) 7.2 (10) i disagree 11.5 (39) 12.0 (24) 10.9 (15) cannot decide 8.0 (27) 7.5 (15) 8.7 (12) embryos should be allowed to be donated to single women agree 49.1 (166) 54.0 (108) 4 42.0 (58) 3 ns neutral 6.8 (23) 5.0 (10) 9.4 (13) i disagree 32.8 (111) 31.0 (62) 35.5 (49) cannot decide 11.2 (38) 10.0 (20) 13.0 (18) the figures are given in frequency (%) and number (n) of the total number of respondents to each question/statement, unless otherwise indicated.differences between women and men are statistically evaluated according to mann–whitney u test. p < 0.05 is set as the threshold for significance. amedian is calculated on original data (5-point scale) for each statement. ns = not significant. 190 k. wånggren et al. table iii. attitudes by two cohorts of swedish women and men of reproductive age on embryo donation. statement all (n = 338) women (n = 200) men (n = 138) requirements should be imposed on answer % (n) % (n) mda/mb % (n) mda/mb p recipient woman’s age agree 67.1 (221) 68.8 (137) 4 63.6 (84) 4 ns neutral 12.1 (40) 7.0 (14) 9.8 (13) i disagree 8.2 (27) 13.1 (26) 10.6 (14) cannot decide 12.5 (41) 11.1 (22) 15.9 (21) appropriate minimum age for recipient woman, m <25 years 30.1 (82) 25.0 (49) 24.8 26.0 (33) 24.5 ns 25–35 years 34.6 (94) 48.0 (94) 40.2 (51) 36–45 years 2.2 (6) 1.0 (2) 3.1 (4) >45 years 0.0 (0) 0.0 (0) 0.0 (0) cannot decide 33.0 (90) 26.0 (51) 30.7 (39) appropriate maximum age for recipient woman, m <25 years 0.0 (0) 0.0 (0) 42.8 0.0 (0) 42.6 ns 25–35 years 5.9 (19) 5.1 (10) 7.1 (9) 36–45 years 54.5 (176) 56.2 (110) 52.0 (66) >45 years 10.2 (33) 9.7 (19) 11.0 (14) cannot decide 29.4 (95) 29.1 (57) 29.9 (38) recipient male partner’s age agree 62.4 (204) 64.6 (128) 4 58.9 (76) 4 ns neutral 8.6 (28) 7.6 (15) 10.1 (13) i disagree 14.0 (46) 15.7 (31) 11.6 (15) cannot decide 15.0 (49) 12.1 (24) 19.4 (25) appropriate minimum age for recipient male partner, m <25 years 23.7 (75) 22.9 (44) 25.1 25.0 (31) 24.5 ns 25–35 years 44.0 (139) 46.4 (89) 40.3 (50) 36–45 years 0.9 (3) 1.0 (2) 0.8 (1) >45 years 0.3 (1) 0.0 (0) 0.8 (1) cannot decide 31.0 (98) 29.7 (57) 33.1 (41) appropriate maximum age for recipient male partner, m <25 years 0.0 (0) 0.0 (0) 44.4 0.0 (0) 45.3 ns 25–35 years 3.4 (11) 3.6 (7) 3.1 (4) 36–45 years 39.6 (127) 41.2 (80) 37.0 (47) >45 years 20.9 (67) 18.6 (36) 24.4 (31) cannot decide 36.1 (116) 36.6 (71) 35.4 (45) recipient’s level of education agree 12.0 (40) 11.1 (22) 1 13.6 (18) 1 ns neutral 12.0 (40) 14.1 (28) 9.1 (12) i disagree 63.4 (210) 65.8 (131) 59.8 (79) cannot decide 12.4 (41) 9.0 (18) 17.4 (23) recipient’s economic situation agree 36.9 (122) 38.2 (76) 2 34.8 (46) 2 ns neutral 10.0 (33) 8.5 (17) 12.1 (16) i disagree 42.0 (139) 44.7 (89) 37.9 (50) cannot decide 11.2 (37) 8.5 (17) 15.2 (20) recipient’s sexual orientation agree 17.0 (56) 14.6 (29) 1 20.5 (27) 1 ns neutral 11.8 (39) 10.1 (20) 14.4 (19) i disagree 57.0 (188) 62.1 (123) 49.2 (65) cannot decide 14.2 (47) 13.1 (26) 15.9 (21) attitudes towards embryo donation in sweden 191 they became parents, and had their embryos discarded (19). some of the female participants (18%) in the present study have chosen not to state a position, indicating that it is a difficult question. interesting to note is that embryo donation to infertile couples, in the present study, was supported by a large majority of both women and men, while donation to research did not have the same support among women. this is despite the fact that embryo donation for research is permitted in sweden but not donation of embryos for reproduction to infertile couples. it might be the case that more participants would support donation if the type of research that the embryos would be used for was more clearly defined (27). the participants had, to a large degree, requirements regarding the age of the recipient of the embryo donation. the participants believed that the appropriate maximum age for the female recipient would be around 43 years. at such an advanced age it can be difficult for the woman to conceive, and thus the need for help with donated eggs or embryos is great. approximately 10% of the respondents stated that an upper age limit should be above 45 years, an age when it is very difficult for women to become pregnant and an age at which assisted reproduction under current rules is not allowed in sweden (12). the recommended mean minimum and maximum age for the male recipient was 25 and 45 years, respectively. table iii. (continued). statement all (n = 338) women (n = 200) men (n = 138) requirements should be imposed on answer % (n) % (n) mda/mb % (n) mda/mb p recipient woman’smedical history agree 38.7 (128) 37.7 (75) 2 40.2 (53) 3 ns neutral 11.5 (38) 9.0 (18) 15.2 (20) i disagree 33.2 (110) 37.7 (75) 26.5 (35) cannot decide 16.6 (55) 15.6 (31) 18.2 (24) recipient’s male partner’s medical history agree 38.0 (126) 36.7 (73) 2 40.2 (53) 3 ns neutral 11.2 (37) 9.5 (19) 13.6 (18) i disagree 34.1 (113) 38.2 (76) 28.0 (37) cannot decide 16.6 (55) 15.6 (31) 18.2 (24) recipient’s tobacco use agree 39.3 (130) 42.7 (85) 3 34.1 (45) 3 ns neutral 11.8 (39) 8.5 (17) 16.7 (22) i disagree 37.8 (125) 39.2 (78) 35.6 (47) cannot decide 11.2 (37) 9.5 (19) 13.6 (18) recipient’s alcohol abuse agree 79.2 (262) 84.4 (168) 5 71.2 (94) 5 0.006 neutral 2.7 (9) 0.5 (1) 6.1 (8) i disagree 9.7 (32) 8.5 (17) 11.4 (15) cannot decide 8.5 (28) 6.5 (13) 11.4 (15) recipient’s drug abuse agree 84.6 (280) 89.9 (179) 5 76.5 (101) 5 0.002 neutral 1.8 (6) 0.0 (0) 4.5 (6) i disagree 5.7 (19) 4.0 (8) 8.3 (11) cannot decide 7.9 (26) 6.0 1 (2) 10.6 (14) recipient’s criminal background agree 65.9 (218) 71.4 (142) 4 57.6 (76) 4 0.008 neutral 6.0 (20) 5.5 (11) 6.8 (9) i disagree 16.0 (53) 13.6 (27) 19.7 (26) cannot decide 12.1 (40) 9.5 (19) 15.9 (21) the figures are given in frequency (%) and number (n) of the total number of respondents to each question/statement, unless otherwise indicated. differences between women and men are statistically evaluated according to mann–whitney u test. p < 0.05 is set as the threshold for significance. amedian is calculated on original data (5-point scale) for each statement. bmean age calculated from original data for statements related to appropriate minimum/maximum age for recipient woman/man. ns = not significant. 192 k. wånggren et al. one-quarter of men responded that the male recipient should have an upper age limit of 45 years. a common maximum age for men to receive statefunded ivf treatment in sweden is, however, 55 years. the recipient’s educational level was generally not considered so important. it is interesting to note that a majority of the participants (56%) disagreed on the statement that you should be able to have requirements on the recipient’s sexual orientation. this is in agreement with the swedish regulations regarding sperm donation, allowing treatment also to lesbian couples. as expected, the vast majority of participants thought that requirements should be imposed on the recipient’s alcohol and drug addiction. women disagreed, to a higher degree than men, with alcohol abuse among couples receiving embryo donation. a majority of respondents believed that demands should be imposed on the recipient’s criminal background, and more women than men thought that this was important. a limited proportion (38%) of participants in the study was of the opinion that the embryo donor should remain anonymous to the children. a larger proportion (45%) thought that the donor should be anonymous to the recipient, and 47% thought that the recipient should be anonymous to the donor. a british study showed that nearly half of the mothers of children born after embryo donation were not going to tell their children about this (28). many studies suggest that parents of children being conceived through various types of donations would rather not want that the child knew about the donation. despite this, sweden has chosen to protect the rights of the children and their statutory right to know who their genetic parents are. in a previous study of swedish public attitudes to egg donation it was shown that a large majority felt that children should know their genetic origin (20). the view that the embryo donor should remain anonymous to the child is in conflict with the swedish legislation regarding gamete donation, where children born after gamete donation are entitled to receive identifying information about the donor, when the child has reached mature age (12). in sweden, ivf staff, psychologists, and family therapists recommend that children should know their origin (20). the question regarding children’s knowledge of the donor has been studied more in terms of gamete donation than embryo donation (29). however, studies of potential recipients of donated embryos emphasize the importance of openness and of the possibility to be able to access information regarding the child’s genetic heritage (5). embryo donation is more complicated than gamete donation, since it involves the full genetic material of the donating couple and might lead to two genetic full siblings of the same age living in the same city without knowing each other. there is a risk that the physical table iv. swedish women’s and men’s attitudes towards embryo donation and anonymity. all (n = 338) women (n = 200) men (n = 138) statement answer % (n) % (n) mda % (n) mda p the embryo donor should be anonymous to the child agree 39.0 (130) 36.5 (72) 2 42.6 (58) 2 ns neutral 11.1 (37) 9.6 (19) 13.2 (18) i disagree 27.3 (91) 23.9 (47) 20.6 (28) cannot decide 22.5 (75) 29.9 (59) 23.5 (32) the embryo donor should be anonymous to the recipients agree 46.1 (154) 44.4 (88) 3 48.5 (66) 2 ns neutral 9.6 (32) 8.1 (16) 11.8 (16) i disagree 18.3 (61) 21.7 (43) 13.2 (18) cannot decide 26.0 (87) 25.8 (51) 26.5 (36) the recipients should be anonymous to the embryo donor agree 47.9 (160) 49.0 (97) 3 46.3 (63) 2 ns neutral 11.7 (39) 9.1 (18) 15.4 (21) i disagree 15.0 (50) 16.2 (32) 13.2 (18) cannot decide 25.4 (85) 25.8 (51) 25.0 (34) the figures are given in frequency (%) and number (n) of the total number of respondents to each question/statement, unless otherwise indicated. differences between women and men are statistically evaluated according to mann–whitney u test. p < 0.05 is set as the threshold for significance. amedian is calculated on original data (5-point scale) for each statement. ns = not significant. attitudes towards embryo donation in sweden 193 similarities are commented on, and it would be traumatic for the children if they formed a relationship. the fact that the child is conceived by embryo donation can be revealed by chance, which could also be a traumatic experience for the child. openness regarding the donation can prevent this. embryo donation from one couple needs to be limited maybe to one recipient couple (30). potential embryo donors must be offered counselling regarding the future use of their embryos. they need to be certain that they do not want to use the embryos for their own reproduction, and agree with the fact that other couples might conceive and have children as a result of their embryo donation (9). the well-being of the existing and coming children is of utmost importance and needs to be guaranteed (30). counselling for donors and recipients can assist them in making the right decisions about donating or receiving an embryo and will also make them more positive towards disclosure regarding the donation (31). if embryo donation for reproduction were to be allowed in sweden, rules and regulations need to be developed for treatment strategies and for counselling of the donor and recipient couples. permitting embryo donation in sweden would be beneficial for some infertile patients who under the current legislation have to go abroad for treatment. the relatively low percentage of responses is a limitation to the study that may make it difficult to draw definitive conclusions from the material. there is, however, often a low response rate reported in studies that involve attitudes to embryo donation. previously conducted studies, that were completely anonymous, reported response rates ranging between 29% and 45% (13). to analyse the effect of the dropouts on the validity of the study the respondents were compared to the studied population. for the 40% of women who responded to the questionnaire the mean age was 32.5 years, which was equal to the mean age of the women who were sent a questionnaire, indicating that the women responding could accurately represent the attitudes of the population. the fact that only 26% of men returned the questionnaire makes the attitudes of men uncertain. thirty-seven per cent of the responding women had experienced difficulties in becoming pregnant, and 55% had children of their own. these numbers differ from the 10%–15% prevalence of infertility in the swedish population (32). there might be a selection of respondents, interested in the issues affecting assisted reproduction and embryo donation, who were more likely to answer the questionnaire. altogether 4.7% of the women and 3.3% of the men had had children through assisted reproduction, which is close to the 3.1% of children born after ivf treatment in sweden (3). a higher proportion of the participating women had university education compared to the male respondents, which is in agreement with the respective levels of education seen in the swedish population (33). in conclusion, the results from the study indicate that a majority of the swedish population in reproductive age are in favour of donation of embryos, both for reproduction to infertile couples and for research. if embryo donation is to be allowed in sweden, strategies for treatment and counselling need to be developed. acknowledgements the authors want to thank dr torbjörn bergh, carl von linné kliniken, uppsala, sweden, for help with design of the study and advice regarding the manuscript, and dr lena wånggren, department of english literature, university of edinburgh, united kingdom, for linguistic review. declaration of interest: the study was supported by uppsala university. the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. all authors have participated in study design, analysis, manuscript drafting and critical discussion. the collection of data was mainly performed by f. prag. references 1. boivin j, bunting l, 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16–74 years of age by highest level of education, age and sex. year 1985–2011. in statistics sweden, editor. statistics sweden. 2012. accessed from: http://www. scb.se/default____2154.aspx supplementary material available online supplement a (letter) supplement b (questionnaire) supplement c (questionnaire) attitudes towards embryo donation in sweden 195 www.ncbi.nlm.nih.gov/pubmed/15823217?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15823217?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16716313?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16716313?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16716313?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16716313?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19136479?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19136479?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19136479?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12773471?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12773471?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21247922?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21247922?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21247922?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21247922?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15310730?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15310730?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15310730?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18053994?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18053994?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15760957?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15760957?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15760957?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15760957?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12721192?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12721192?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11387280?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11387280?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/16448734?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12660288?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12660288?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21411080?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21411080?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15196710?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15196710?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20413349?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20413349?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20413349?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19102601?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19102601?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10655327?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10655327?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/10655327?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20817740?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20817740?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20817740?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20817740?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19133052?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19133052?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19133052?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19133052?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19133052?dopt=abstract abstract introduction materials and methods approval of ethics committee study population the questionnaire statistics results attitudes towards embryo donation (table ii) special requirements for the recipient of donated embryos (table iii) attitudes to embryo donation and anonymity (table iv) discussion acknowledgements declaration of interest references a new contribution to research metrics editorial a new contribution to research metrics the last issues of our two latest volumes have both contained editorials dealing with the performance of upsala journal of medical sciences (ujms) in terms of numbers of submissions and citations of articles. thus, we have reported on the decline of the number of submitted manuscripts, most probably reflecting the extensive appearance of socalled predatory journals (1–3), but also on the joy of announcing the passage for the first time of the critical 2.0 level of the much-discussed impact factor (4, 5). in that last report we dwelt on how to maintain or even increase these impact figures. this is an attempt to brief you on the progress of that proposal. perhaps it would be most suitable at this time point to inform you on the latest impact factor figures from clarivate analytics in june, last summer, before discussing the proposed moves. we have to announce a slight drop—from 2.389 to 1.971—of the 2-year impact factor figure (figure 1). that most likely illustrates the fate of many smaller and not frequently published journals in that single articles have a very strong impact on the value, resulting in a fairly bumpy ride. quite in contrast, the 5-year factor for ujms has been constantly growing for more than 10 years. for the sake of clarity, this factor is calculated after introducing in the denominator the number of documents the last five years before the calculation year. this year, we reached a record high 2.355 (figure 1). and more will come. that is what can be seen from the preliminary figures of this year. in the year 2016 editorial (1) we discussed the fate of individual articles in terms of citation figures. we then determined the number of articles belonging to the 99th percentile most-cited papers during a specific year (year three after the publication). for the period 2010–2014 with 227 publications, three papers qualified for the 1% (more than 21 cites) and 23 for the 10% category (more than 7 cites). during the two following years one was identified as a ‘1% cited paper’ and six more as belonging to 10% category. thus, highly cited papers show up as frequently these days as before. the most cited paper by anders larsson et al. (6) on ‘the state of point-of-care testing’ is another piece of evidence that review articles tend to become most cited. one of the 10% articles, the rudbeck award review by lena claesson-welsh (7), illustrates the fact that papers published late in the year are very much hampered by the shorter exposure time in year 1. in her case the citation figure, 15 for years 2015–2017, has more than doubled at this time point in the year 2018. interestingly, scopus has recently launched an alternative research metric besides their sjr and snif scores, the socalled citescore (8). it measures the average citations per document published within three years ahead of the year for the calculation of the pertinent citescore. in other words, one more impact factor estimate, but now on a three-year basis. it, however, differs from the traditional clarivate metric in that it includes all available document types. thus, case reports, letters, editorials, commentaries, etc. will all be added in the denominator. to that should be added the fact that the scopus database contains about 50% more journals. as a consequence of that, citescores for most biomedical journals will be considerably lower than the clarivate impact factor value. this difference is perhaps most evidently displayed for the new england journal of medicine: 79.830 is their 2017 clarivate score and 14.75 the citescore. a more than five-fold decrease when all categories of published documents have to be taken into account. likewise, the lancet had a six-fold drop from 53.254 (clarivate) to 8.60 (citescore). it is worthy of note that the corresponding difference for ujms is close to zero, since our citescore is as high as 1.94 for the year 2017 (figure 2). this new research metric also offers a so-called citescore tracker for the year under evaluation. this gives everyone an opportunity to continuously monitor the increase of the citescore value, since it is updated monthly. at present, in mid-october, the value for ujms is above 1.60, suggesting a considerable increase compared with last year. there is also a rank system for journals belonging to different research fields. ujms has become sorted into the general medicine category that consists of 841 journals. our rank position is 61 corresponding to the 92th percentile (figure 2). the corresponding figure for ujms in clarivate rank for that group of journals is 54 out of 155. the most obvious explanation for this discrepancy most probably is the inclusion of more nonprestigious journals in the scopus database. one more citation figure worthwhile to mention is that for the number of total cites (figure 3). this is perhaps the most important value to follow. the figure shows one more year with a substantial increase—about 10%—for ujms that cannot simply be explained by the fact that one more volume is out there to be cited and also many more journals active in scholarly publishing. i can remember when reporting to our board of the upsala medical society some years ago when our cooperation with taylor & francis had just commenced that we then proudly could claim that each day � 2018 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2018, vol. 123, no. 4, 191–193 https://doi.org/10.1080/03009734.2018.1542644 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1542644&domain=pdf http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1542644 http://www.tandfonline.com there was a new citation of a ujms article. now, this figure is rapidly approaching three new citations a day. over the last few years we have faced decreasing numbers of submissions. actually, last year we had to report an ‘all-time low’ with only 175 submitted papers. when this is being written (mid-october) that number has already been passed. preferably, one would like to interpret that as if our measures presented a year ago have been effective—free color-prints, fast-track opportunities (admittedly, we have not had many), free printed issues distributed to all society members, and maintenance of the ‘free-of-charge’ system despite our open-access publishing. perhaps we could have exaggerated our presence on social media. for an old-fashioned editor the efficiency of the work load is difficult to estimate, but many high altmetric scores (9) are noteworthy and rewarding. therefore, in this context we have to rely very much on you, readers and authors. one more measure to facilitate and convince potential authors to choose ujms for their next submission is that of ‘format-free submission’. that is supposed to make the process of preparing research papers for submission simpler and less time-consuming. the post-acceptance reformatting will be, as much as possible, managed by our publisher. hopefully, this will ensure that the research of the authors can be disseminated quicker through a speedier publication process. by such means we should be able to attract highquality papers, a prerequisite for remaining successful in times of impactitis (10, 11). finally, we mentioned last time that special issues have been very valuable when discussing how to improve the impact of our journal. the next to follow—that from uppsala clinical research centre—will appear as the first issue of next year. the two most recent ones, denominated ‘remembering claes hellerstr€om’ (12) and ‘preconceptional health and care’ (13) have been very successful in terms of citations, and to a great extent they can explain our fairly high citation figures. i will take this opportunity to invite researchers, who want to present their research as well as that of excellent colleagues both from their own research environments and abroad, to edit such a special issue. we can offer quick and efficient editorial assistance. we are looking forward to a most pleasant collaborative effort for the sake of scholarly publishing in general and our old and venerable journal in particular. references 1. andersson a, lau b€orjesson l. scholarly publishing threatened?. ups j med sci. 2016;121:205–6. 2. available at: https://www.thinkchecksubmit.org 3. beall j. what i learned from predatory publishers. biochem med (zagreb). 2017;27:273–8. 4. garfield e. the history and meaning of the journal impact factor. jama 2006;295:90–3. 5. andersson a. a milestone reached?. ups j med sci. 2017;122: 205–6. 6. larsson a, greig-pylypczuk r, huisman a. the state of point-ofcare testing: a european perspective. ups j med sci. 2015;120: 1–10. figure 1. figure 2. figure 3. 192 editorial https://www.thinkchecksubmit.org 7. claesson-welsh l. vascular permeability-the essentials. ups j med sci. 2015;120:135–43. 8. ziljstra h, mccullough r, citescore: a new metric to help you choose the right journal. available at: https://www.elsevier.com/ editors-update/story/journal-metrics/. 9. available at: https://www.altmetricexplorer.com 10. andersson a, lau b€orjesson l. operating in an era of impact factor mania. ups j med sci. 2015;120:124–31. 11. van diest pj, holzel h, burnett d, crocker j. impactitis: new cures for an old disease. j clin pathol. 2001;54:817–9. 12. andersson a. remembering claes hellerstr€om and those around him. ups j med sci 2016;121:71–2. 13. tyd�en t. why is preconception health and care important?. ups j med sci. 2016;121:207. arne andersson editor joey lau b€orjesson editorial assistant upsala journal of medical sciences 193 https://www.elsevier.com/editors-update/story/journal-metrics/ https://www.elsevier.com/editors-update/story/journal-metrics/ https://www.altmetricexplorer.com outline placeholder references upsala journal of medical sciences 2021, 126, e5660 http://dx.doi.org/10.48101/ujms.v126.5660 original article contact michael eklund michael.eklund@regionorebrolan.se supplemental data for this article can be accessed here. © 2021 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. effects of spironolactone on extrasystoles and heart rate variability in haemodialysis patients: a randomised crossover trial michael eklunda, olof hellberga, hans furulandb, yang caoc,d and erik nilssone,f adepartment of internal medicine, school of medical sciences, örebro university, örebro, sweden; bdepartment of medical sciences, uppsala university hospital, uppsala, sweden; cclinical epidemiology and biostatistics, school of medical sciences, örebro university, örebro, sweden; dunit of integrative epidemiology, institute of environmental medicine, karolinska institutet, stockholm, sweden; edepartment of medical epidemiology and biostatistics, karolinska institutet, stockholm, sweden; fschool of medical sciences, örebro university, örebro, sweden abstract background: spironolactone treatment reduces mortality in haemodialysis (hd) patients. the objective of this study was to evaluate if spironolactone affects cardiac electric activity in this population. methods: participants were randomised to start with spironolactone 50 mg daily or observation (12 weeks) with subsequent washout (6 weeks) and crossover to the other intervention (12 weeks). long-term electrocardiograms were recorded and assessed with blinding to treatment. the primary outcome was premature ventricular complexes (pvc), and secondary outcomes were atrial premature contractions (apc) and heart rate variability (hrv). results: thirty participants were recruited, and data for 16 participants were included in the analysis. treatment was associated with an increase in pvcs by 9.7 [95% confidence interval (ci): 1.5 to 18] h−1. hrv time-domain variables increased during treatment, the standard deviation of all beat-to-beat intervals by 18 (95% ci: 3.3 to 32) milliseconds (ms) and the standard deviation of the averages of beat-to-beat intervals in all 5-min segments of the entire recording by 16 (95% ci: 1.5 to 30) ms. there were no significant differences in other variables. conclusion: spironolactone treatment increases pvcs in hd, indicating a possible proarrhythmic effect. however, improved cardiac autonomic function, as indicated by an increased hrv, may contribute to the survival benefit from spironolactone treatment in hd patients. article history received 11 september 2020 revised 16 october 2020 accepted 10 november 2020 published 25 january 2021 keywords haemodialysis spironolactone; clinical trial; long-term ecg; arrhythmias; heart rate variability introduction cardiovascular disease is a leading cause of morbidity and mortality in end-stage renal disease (esrd) (1). in two recent trials, treatment with the mineralocorticoid receptor antagonist (mra) spironolactone improved survival and reduced the risk of  cardiovascular events in haemodialysis (hd) patients (2,3). however, the mechanisms by which mras may improve survival in hd patients are not clear. multiple studies suggest a link between the mineralocorticoid aldosterone and cardiac electric activity (4–6). animal studies show that overexpression of the mineralocorticoid receptor in cardiac myocytes increases the occurrence of ventricular arrhythmias (6). it also reduces heart rate variability (hrv) (6), which is a marker for autonomic function (7). in line with this, mras decrease atrial arrhythmias in rodents (5). a reduction in hrv, as well as the presence of premature ventricular complexes (pvcs) and atrial premature contractions (apcs), has been associated with an increased risk of death due to cardiovascular events (8–11). these electrocardiogram (ecg) markers are also associated with the risk of arrhythmias (10,12–14). this study aimed to investigate the effects of mra on cardiac electrical activity in hd patients. we hypothesised that spironolactone treatment would be associated with a change in the number of pvcs on long-term ecg (ltecg). materials and methods this was an open-label, randomised crossover trial controlled using untreated observation with blinded assessment of outcomes. the primary outcome was the frequency of pvcs, and secondary outcomes were the frequency of apcs and change in hrv indices. patients were recruited in sweden, from the dialysis units at örebro university hospital and uppsala university hospital, http://dx.doi.org/10.48101/ujms.v126.5660 mailto:michael.eklund@regionorebrolan.se http://dx.doi.org/10.48101/ujms.v126.5660 http://creativecommons.org/licenses/by/4.0/ 2 m. eklund et al. from february 2013 until december 2014. inclusion criteria were age 18 years or older, three or more weekly hd-sessions, compliance during the previous three months and adequate cognitive function. exclusion criteria were cardiac pacemaker, persistent or permanent atrial fibrillation, plasma potassium >  6.5 mmol/l at any time during the two months preceding inclusion, life-expectancy of less than 12 months, and pregnancy or breastfeeding. power calculation was based on pvcs and yielded a minimum of 14 subjects completing the crossover study. the analysis was based on results from trials in congestive heart failure (15–17) and used a two-sided significance level of 5% and 80% power. the sample size was set to 30 as a dropout rate of 50% was expected, mainly due to renal transplantation, mortality and side effects of spironolactone. randomisation and allocation were conducted by a person not otherwise involved in the study. sequence generation in blocks of six with a ratio of 1:1 was performed using clinstat (martin bland, united kingdom). sequentially numbered, sealed and opaque envelopes were used for allocation. subjects were assigned to a sequence of 12 weeks 50 mg oral spironolactone daily, followed by 6 weeks washout phase and finishing with 12 weeks of no treatment, or the reverse order. spironolactone and its active metabolites have half-lives of 1.4–16.5 h in healthy individuals, but the effects of chronic kidney disease and hd on the elimination of spironolactone are unknown (18). the lack of knowledge on the elimination of spironolactone in the study population motivated the use of a long washout phase. the four study visits took place on midweek dialysis sessions and were conducted before and after both of the 12week intervention periods. blood draw and measurements were performed before dialysis treatment. blood pressure was measured using an automated oscillometric blood pressure monitor (omron m6 ac, omron healthcare, japan). the accredited local hospital laboratories analysed routine biochemical parameters. ltecg recordings were started at the onset of the dialysis session and continued for a minimum of 24 h, using a medilog ar12 plus ecg recorder (schiller ag, switzerland). the swedish renal registry was accessed to obtain information on the primary renal disease of the participants. a clinical laboratory scientist and a physician, blinded to treatment allocation, curated the ltecg recordings, using medilog darwin 2 enterprise v 2.1.0 software (schiller ag, switzerland). hrv analysis was conducted using r-statistics v 3.4.1 (r development core team, austria) and the package rhrv v 3.0.7 (19). hrv variables were defined in accordance with malik et al. (7) as follows: time-domain variables included the standard deviation of all beat-to-beat intervals (sdnn), the standard deviation of the averages of beat-tobeat intervals in all 5-min segments of the entire recording (sdann) and the square root of the mean of the sum of the squares of differences between adjacent beat-to-beat intervals (rmssd). frequency-domain variables were based on power in different frequency ranges, 0.15–0.4 hz – high frequency (hf), 0.04–0.15 hz – low frequency (lf), 0.003– 0.04  – very low frequency ( vlf) and ≤ 0.003 hz – ultra-lowfrequency (ulf). after treatment, remaining spironolactone tablets were collected. compliance was calculated as the ratio of tablets consumed, based on the amount returned, to expected consumption. baseline data were expressed as median and interquartile range, or as percentage. a generalised linear mixed model with random patient effects was used to assess the treatment effect in all statistical analyses, as described by senn (20). period effects and baseline values were considered fixed effects. log transformation to normality was used when mandated by the assessment of model residuals. all analyses were conducted using r-statistics v 3.4.1 (r development core team, austria). per-protocol analysis with listwise deletion of incomplete cases was pre-specified in the study analysis plan, motivated by the study aim of elucidating mras mechanisms of action rather than to assess clinical efficacy. the study received ethical approval by the regional ethical review board in uppsala, sweden (reference number 2011/316). the board decision covers both study centres of this trial. all participants gave written informed consent before enrolment. the declarations of helsinki was adhered to, and good clinical practice was applied. the trial was approved by the swedish medical products agency (eudractnr 2011-002773-39) and was registered on 28 september 2011 at http://www.clinicaltrialsregister.eu/ (eudractnr 2011-00277339). monitoring was performed to ensure adherence to the study protocol, complete documentation and complete registration of adverse events. results baseline characteristics a total of 30 participants were recruited, of which 18 (60%) were enrolled in örebro and 12 (40%) in uppsala. of these, 18 persons (60%) completed follow-up (figure 1). there was a larger proportion of participants completing follow-up in örebro [13, (72%)] than in uppsala [5, (42%)]. two participants had corrupt ltecg data files from single recordings and were excluded, resulting in a total of 16 participants (53%) included in analysis. of the analysed population, the median age was 66 (61.5–67) years, and 12 (75%) were male. only two (13%) of the included participants were current tobacco users, and three (19%) participants were previous tobacco users. eight participants (50%) were treated with angiotensin-converting enzyme inhibitors or angiotensin receptor blockers (table 1). further, eight persons (50%) were treated with a betaadrenoceptor antagonist. no other class of anti-arrhythmic medication was used. the potassium binder polystyrene sulphonate was used at inclusion in two cases (12%), and one participant (6.2%) was treated with potassium chloride supplementation. concomitant medications have been listed in supplementary table 1. http://www.clinicaltrialsregister.eu/ http://dx.doi.org/10.48101/ujms.v126.5660 effects of spironolactone on extrasystoles in hd patients 3 outcomes the 16 participants included in the analysis had a mean compliance of 97%. spironolactone treatment was associated with an increase in pvcs compared to observation [mean treatment effect 9.7, 95% confidence interval (ci): 1.5 to 18 h-1]. there was no significant difference in apcs during treatment compared to observation (ratio 2.5, 95% ci: 0.58 to 11). all hrv time-domain variables (sdnn, sdann, rmssd) increased during treatment, although statistical significance was reached only for sdnn, which increased by 18 (95% ci: 3.3 to 32) milliseconds (ms), and sdann, which increased by 16 (95% ci: 1.5 to 30) ms (table 2). the effects of spironolactone on these variables on the individual level are presented in figure 2. hrv frequency-domain variables (hf, lf, vlf, ulf) did not change significantly during treatment compared to observation (table 3). note that in tables 2 and 3, the ltecg variables measured at the end of the spironolactone treatment period were compared to the measurement after the observation period, and since the estimate was adjusted for both baselines, it corresponds to the change in each variable that is due to spironolactone treatment. during the study period, the following changes in antiarrhythmic treatment were made: three participants initiated beta-blocker treatment (bisoprolol in all cases), three discontinued treatment (two cases of atenolol, and one case of metoprolol) and three had a reduction in dose (bisoprolol in one case, and metoprolol in two cases). all changes, except a dose reduction of metoprolol for one participant, occurred during the non-intervention period of follow-up. there was no apparent effect of spironolactone on plasma potassium concentrations, with an estimated mean difference in plasma potassium between the end of treatment and the end of observation of −0.046 mmol/l (95% ci: −0.57 to 0.47). potassium binder polystyrene sulphonate was initiated for one participant during spironolactone treatment, and no other changes in potassium binder treatment occurred during followup. serum bicarbonate concentration decreased by −2.0 (95% ci: −3.5 to −0.52) mmol/l during treatment. effects on electrolytes have been presented in supplementary table 2. there were no changes in systolic blood pressure (13 mmhg, 95% ci: −1.8 to 28) or diastolic blood pressure (5.6 mmhg, 95% ci: −1.1 to 12) during treatment compared to observation (supplementary table 2). during the study, 62 adverse events were reported for the study population that underwent at least one study visit (n = 29). one serious adverse event was reported, with a participant passing away during the wash-out period of follow-up. the case review did not indicate any association between death and exposure to the study drug. one case of  muscular cramps was considered associated with spironolactone treatment. all other events were judged as not being associated with the intervention. the most common adverse events were infections (12 occurrences), chronic obstructive pulmonary disease exacerbations (10 occurrences) and hypotension (four occurrences, of which two were during spironolactone treatment). discussion the major novel findings of this study were that spironolactone treatment is associated with an increased occurrence of pvcs and that it increases hrv time-domain indices in persons undergoing hd. although an increase in pvcs during treatment was unexpected, effects on ecg variables are in line with mineralocorticoid receptor action on cardiac electrical conductivity (4–6). spironolactone has been shown to act as a partial mineralocorticoid receptor agonist during certain intracellular conditions in vitro (21, 22), and we speculate that figure 1. flow chart of the study procedure. study design, recruitment of participants and follow-up for long-term electrocardiogram data. http://dx.doi.org/10.48101/ujms.v126.5660 http://dx.doi.org/10.48101/ujms.v126.5660 4 m. eklund et al. the relatively high dose of spironolactone used in this study may have contributed to an increased occurrence of pvcs, thus having a proarrhythmic effect. hrv reflects autonomic function (7), and the observed increase in hrv may be due to inhibition of aldosterone action in the central nervous system, leading to a reduction in cardiac sympathetic stimulation, as has been shown in animal studies (23). in individuals with congestive heart failure, sympathoexcitation has been associated with a reduction in hrv (24) and increased risk of death (25). increased hrv, as demonstrated here, could, therefore, be a mechanism underlying the beneficial effect of mra in hd patients (2, 3). we also found an association between spironolactone treatment and a decrease in plasma bicarbonate concentration, which may be due to aldosterone action on na+/h+ exchange in the intestine (26) or remaining nephrons, with secondary effects on bicarbonate reabsorption. anti-arrhythmic effects of spironolactone have been suggested as an important mechanism of action leading to improved survival in congestive heart failure and ischemic heart disease, reducing the occurrence of pvcs (16, 27, 28) and apcs (16). to our knowledge, no prior studies have investigated the effect of spironolactone on these markers in esrd. in contrast to studies on heart disease patients (16, 27, 28), we found that mra treatment increased the frequency of pvcs and did not demonstrate an effect on apcs. differences in serum potassium concentrations (29), cardiovascular pathology and concomitant medications between these populations are possible explanations for these discrepancies. for example, in this study, only 25% of the participants had congestive heart failure, and 25% had a history of myocardial infarction, compared to 50–52% with previous myocardial ischemia in the congestive heart failure population (16). furthermore, previous studies in chronic heart disease patients reported the use of angiotensinconverting enzyme inhibitors in more than 90% of cases (16, 27), compared to 50% using angiotensin-converting enzyme inhibitors or angiotensin receptor blockers in our study. these differences could indicate that a beneficial effect of spironolactone on apcs and pvcs is dependent on pathological processes that are less prevalent in hd patients than in congestive heart failure and ischemic heart disease, or that the effect requires a more extensive renin–angiotensin–aldosterone table 1. baseline characteristics. variable comorbidities number % dialysis data median interquartile range (iqr) cardiovascular 8 50 time in dialysis, days 480 1,000 myocardial infarction 4 25 angina pectoris 6 38 physiological data median iqr congestive heart failure 4 25 heart rate, min−1 76 11 arrhythmia 2 12 systolic blood pressure, mmhg 140 30 peripheral vascular disease 3 19 diastolic blood pressure, mmhg 72 18 stroke 3 19 extra systoles diabetes mellitus 7 44 premature ventricular complex, h−1 2.7 7.2 diabetes mellitus, type 1 1 6.2 atrial premature contraction, h−1 4 21 diabetes mellitus, type 2 6 38 heart rate variability (hrv) time-domain other comorbidities standard deviation of all beat-to-beat intervals (sdnn), milliseconds (ms) 110 43 dyslipidaemia 11 69 standard deviation of the averages of beat-to-beat intervals in all 5 min segments of the entire recording (sdann), ms 92 42 obstructive sleep apnoea syndrome 5 31 square root of the mean of the sum of the squares of differences between adjacent beat-to-beat intervals (rmssd), ms 22 33 hrv frequency-domain* biochemical data median iqr high frequency (hf), ms2 28 100 potassium, mmol/l 4.8 0.76 low frequency (lf), ms2 100 120 sodium, mmol/l 140 2.2 very low frequency (vlf), ms2 220 250 magnesium, mmol/l 0.9 0.13 ultra-low-frequency (ulf), ms2 50 26 calcium, mmol/l 2.3 0.18 bicarbonate, mmol/l 23 2.8 phosphate, mmol/l 1.6 0.63 albumin, g/l 38 3 haemoglobin, g/l 120 18 c-reactive protein, mg/l 3.5 12 parathyroid hormone, pmol/l 140 210 baseline characteristics of the 16 persons included in long term electrocardiogram analysis, reported as number and percentage or as median and interquartile range. *power in different ranges, hf 0.15–0.4 hz, lf 0.04–0.15 hz, vlf 0.003–0.04 hz, ulf ≤0.003 hz. effects of spironolactone on extrasystoles in hd patients 5 system inhibition, achieved through concomitant angiotensinconverting enzyme inhibitors treatment. a few trials have investigated the effect of spironolactone on hrv in esrd. in a study by flevari et al. (30), 14 hd patients without congestive heart failure were treated with spironolactone 25 mg three times weekly for 4 months. in line with our results, the authors found that hrv increased in the time-domain variable rmssd during treatment but also that hrv frequency-domain indices (ulf and vlf) decreased. effects on sdnn, the most commonly used marker for cardiac autonomic function, were not reported. lin et al. (2) conducted a trial with 253 esrd patients, who received spironolactone 25  mg daily, or placebo, for two years. contrary to our results, spironolactone treatment did not affect hrv variables, and although lin et al. had a larger sample size than our study, differences in dosing and treatment duration should be noted. in congestive heart failure, and consistent with our results, spironolactone 50–100 mg daily increased sdnn, sdann (31) as well as hf and lf/hf (32) after 4–8 weeks of treatment. although results from esrd patients are not consistent, an increase in hrv has been found in multiple studies in different populations, indicating a beneficial effect of  spironolactone on cardiac autonomic function as measured by hrv. contrary to our expectations and previous findings by flevari et al. (30), we did not observe blood pressure reduction with spironolactone, but rather a slight but not statistically significant increase. it should be noted that the participants in the study by flevari et al. had more extensive use of antihypertensive agents at baseline, and a slightly longer intervention period (4 months), which may explain the differences. the use of mras in individuals with reduced renal function is limited by the risk of hyperkalemia (33). in this study, we observed no cases of severe hyperkalemia, and the treatment did not affect mean plasma potassium concentration. multiple studies of spironolactone in esrd have been conducted, each one individually not showing an increased risk of severe hyperkalemia in spironolactone treatment, although a recent meta-analysis weighted estimate indicates that the risk is increased (34). in consequence, and despite the lack of effect on potassium in this study, the risk of hyperkalemia during spironolactone treatment in esrd should be recognised and appropriate monitoring conducted. some limitations apply to this study. although the number of patients analysed was sufficient based on the power analysis, our sample size was limited. however, the crossover design increases statistical power, and we emphasise that according to table 2. effect of spironolactone on premature ventricular complexes and heart rate variability time-domain variables. variable estimate 95% confidence interval p premature ventricular complex, h-1 9.7 1.5 to 18 0.024 standard deviation of all beat-to-beat intervals (sdnn), milliseconds (ms) 18 3.3 to 32 0.02 standard deviation of the averages of beat-to-beat intervals in all 5-min segments of the entire recording (sdann), ms 16 1.5 to 30 0.033 square root of the mean of the sum of the squares of differences between adjacent beat-to-beat intervals (rmssd), ms 8 −5.7 to 22 0.23 estimates of the treatment effect of spironolactone, corresponding to absolute change in the respective variable. estimates, confidence intervals and p-values were calculated from a generalised linear mixed model with random effects, using fixed effects for treatment, intervention order and baseline values as well as random effects for patients. figure 2. effect of spironolactone on premature ventricular complexes and heart rate variability time-domain. treatment centred residuals were calculated from the generalised linear mixed model with random effects residuals, which was added to treatment estimates, representing both treatment effect and random influences not eliminated by study design or adjustment. diamonds signify means and horizontal bars 95% confidence intervals. individual values are plotted as circles. abbreviations: ms, milliseconds; pvc, premature ventricular complexes; rmssd, the square root of the mean of the sum of the squares of differences between adjacent beat-to-beat intervals; sdann, the standard deviation of the averages of beat-to-beat intervals in all 5-min segments of the entire recording; sdnn, the standard deviation of all beat-to-beat intervals. table 3. effect of spironolactone on heart rate variability frequency-domain variables. variable* estimate 95% confidence interval (ci) p high frequency (hf) 1.8 0.81 to 4.1 0.13 low frequency (lf) 1.3 0.77 to 2.3 0.29 very low frequency (vlf) 1.3 0.94 to 1.7 0.11 ultra-low frequency (ulf) 1.2 0.83 to 1.6 0.35 estimates of the treatment effect of spironolactone, corresponding to the ratio between treatment and observation. values were log-transformed, and estimates, confidence intervals and p-values were calculated with a generalised linear mixed model with random effects. treatment, intervention order and baseline values were used as covariates, and patient effects were treated as random. estimates and confidence intervals were antilogged to get ratios. *power in different ranges, hf 0.15–0.4 hz, lf 0.04–0.15 hz, vlf 0.003–0.04 hz, ulf ≤0.003 hz. 6 m. eklund et al. the power calculation performed before study start, the analysis has sufficient power for detecting relevant changes in the primary outcome of pvcs. for secondary outcomes, the sample size may have been too small to show relevant effects, although it should be noted that all hrv variables measured showed a trend towards increasing values after treatment, which is concordant with the finding of statistically significant increases in sdnn and sdann. further, we did not use placebo during the control period, which allows for the theoretical possibility of an anticipation effect among participants. however, it is unlikely that such an effect would affect ecg variables and we employed blinded assessment of ecg recordings to avoid assessment bias. an additional limitation is that only four participants were female, and results could therefore not be analysed separately by sex. we also acknowledge the multiplicity of analyses and emphasise that change in pvcs was postulated as the primary endpoint in this study and that all analyses were specified a priori. strengths of the study include treatment randomisation, blinded assessment of outcomes and the crossover design, which limits confounding and increases statistical power. also, this study included hd patients with and without congestive heart failure and may, therefore, be valid for a relatively unselected hd population. we conclude that improved cardiac autonomic function, as indicated by an increased hrv, may contribute to the survival benefit from spironolactone treatment seen in previous studies in hd patients (2, 3). however, an increase in pvcs was observed and could indicate a possibly harmful effect with an increased risk of arrhythmias. these findings mandate cautious dosing of spironolactone or ltecg monitoring in subgroups at especially high risk of arrhythmia or sudden cardiac death. acknowledgements johan sundholm assisted with planning, project administration and investigation. disclosure statement the authors report no conflict of interest. funding this work was supported by the uppsala-örebro regional research council under grants rfr-309221, rfr-480961 and rfr-562621; region örebro county research committee under grants oll-213701, oll-261511 and oll-329591; the swedish kidney foundation; and the cuwx counties renal patients’ association. notes on contributors michael eklund, md, phd student in medical sciences at the school of medical sciences, örebro university, örebro, sweden. olof hellberg, md, phd in medical sciences. retired researcher at the school of medical sciences, örebro university, örebro, sweden. hans furuland, md, phd in medical sciences, department of medical sciences, uppsala university hospital, uppsala, sweden. yang cao, phd in biostatistics. associate professor in biostatistics and epidemiology at the unit of clinical epidemiology and biostatistics, school of medical sciences, örebro university, örebro, sweden. researcher at the unit of integrative epidemiology, institute of environmental medicine, karolinska institutet, stockholm, sweden. erik nilsson, md, phd in medical sciences. researcher at the school of medical sciences, örebro university, örebro, sweden, and researcher at the department of medical epidemiology and biostatistics, karolinska institutet, stockholm, sweden. orcid michael eklund https://orcid.org/0000-0001-7706-1758 olof hellberg hans furuland http://orcid.org/0000-0002-9001-614x yang cao http://orcid.org/0000-0002-3552-9153 erik nilsson http://orcid.org/0000-0001-6968-6934 references 1. sarnak mj, levey as, schoolwerth ac, coresh j, culleton b, hamm ll, et al. kidney disease as a risk factor for development of cardiovascular disease: a statement from the american heart association councils on kidney in cardiovascular disease, high blood pressure research, clinical cardiology, and epidemiology and prevention. hypertension. 2003;42:1050–65. doi: 10.1161/01.hyp.0000102971.85504.7c 2. lin c, zhang q, zhang h, lin a. long-term effects of low-dose 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http://dx.doi.org/10.1016/s0735-1097(01)01243-8 http://dx.doi.org/10.1016/s0735-1097(01)01243-8 http://dx.doi.org/10.1016/j.jacc.2012.07.048 http://dx.doi.org/10.1053/j.ajkd.2016.04.011 upsala j med sci 88: 4 3 4 9 , 1983 irradiation effects upon ischemic regenerating rat liver cells lillemor lewan' , ola forsberg', herman amneus3, kerstin lundberg4 and borje larsson3 department of zoophysiology', university of lund, lund, and department of surgery2, central hospital, vasterds, and department of physical biology3, the gustaf werner institute, university of uppsala, uppsala, and department of experimental medicine4, pharmacia a b , uppsala, sweden abstract s t a r c h p a r t i c l e s i n j e c t e d i n t o t h e a r t e r i a l and p o r t a l s y s t e m s of t h e l i v e r of t h e r a t c a u s e d a temporary b l o c k a g e of t h e l i v e r c i r c u l a t i o n and consequent hypoxia i n t h e l i v e r c e l l s . i n t h e r e g e n e r a t i n g l i v e r t h i s r e s u l t e d i n a 30-402 d e c r e a s e of thymidine i n c o r p o r a t i o n i n t o d n a , when a n a l y s e d 1.5 h o u r s a f t e r i n j e c t i o n . i r r a d i a t i o n i n d u c e d c e l l damage, e v a l u a t e d by thymidine i n c o r p o r a t i o n 1.5 h o u r s a f t e r i r r a d i a t i o n w i t h a s i n g l e d o s e of x-rays, was n o t a m e l i o r a t e d by t h e i s c h e m i c c o n d i t i o n . it i s s u g g e s t e d t h a t t h i s depends on a n i n h i b i t e d n u c l e o t i d e metabolism and dna s y n t h e s i s l e a d i n g t o a n a d d i t i v e meta b o l i c hypoxic e f f e c t of t h e s t a r c h p a r t i c l e s on r a d i a t i o n damage. an e q u a l l e v e l of thymidine i n c o r p o r a t i o n , however, w a s found i n an i s c h e m i c and a non i s c h e m i c group of a n i m a l s 1 6 h o u r s a f t e r i r r a d i a t i o n . i n t h i s c a s e t h e l i v e r c e l l s i n t h e i s c h e m i c group had overcome t h e a d d i t i o n a l i n h i b i t i o n of dna s y n t h e s i s c a u s e d by temporary h y p o x i a . introduction low let-radiation i n d u c e s dna damage by f r e e r a d i c a l s . a n e x t r e m e l y low t i s s u e c o n c e n t r a t i o n of m o l e c u l a r oxygen r e d u c e s damage. the d e g r e e of damage can be s t u d i e d h i s t o l o g i c a l l y (5,6) o r , a t t h e m o l e c u l a r l e v e l , e f f e c t s on dna s y n t h e s i s c a n b e s t u d i e d by t h e i n c o r p o r a t i o n of r a d i o a c t i v e l y l a b e l l e d p r e c u r s o r s e . g . t h y m i d i n e ( 1 , 1 4 ) . r a p i d l y growing l i v e r c e l l s o f f e r a good o p p o r t u n i t y f o r such s t u d i e s . a f t e r p a r t i a l hepatectomy l i v e r c e l l s a r e f a i r l y w e l l s y n c h r o n i z e d when e n t e r i n g t h e s-phase of t h e c e l l c y c l e w i t h i n 12-30 h o u r s of r e g e n e r a t i o n . the v a r i o u s s t a g e s o f t h e c e l l c y c l e have b e e n t h o r o u g h l y i n v e s t i g a t e d and t h e w e l l known t i m e c o u r s e f o r t h e growth of t h e r e g e n e r a t i n g l i v e r ( 9 , l o ) a l l o w s s t u d i e s of r a d i a t i o n e f f e c t s a t d i f f e r e n t s t a g e s ( 1 , 2 , 8 ) . i n t h e p r e s e n t s t u d y we a n a l y s e d t h e e f f e c t s of i r r a d i a t i o n on dna-synthesis i n r e g e n e r a t i n g i s c h e m i c l i v e r t i s s u e . ischemia was t e m p o r a r i l y induced by means of i n t r a v a s c u l a r l y i n j e c t e d , d e g r a d a b l e s t a r c h m i c r o s p h e r e s ( 4 ) . the a n i m a l s 43 were w h o l e b o d y i r r a d i a t e d d u r i n g t h e p r e r e p l i c a t i v e o r t h e r e p l i c a t i v e phase of l i v e r growth. dna-synthesis w a s e v a l u a t e d by thymidine i n c o r p o r a t i o n i n t o dna d u r i n g t h e r e p l i c a t i v e p h a s e of growth. material and methods animals young a d u l t male sprague-dawley r a t s weighing 160-180 g ( s p f q u a l i t y , anti cimex, sweden) were u s e d . the a n i m a l s were k e p t under c o n s t a n t c o n d i t i o n s , and had f r e e a c c e s s t o s t a n d a r d l a b o r a t o r y d i e t b e f o r e and a f t e r t h e e x p e r i m e n t a l p r o c e d u r e s . s u r g i c a l p r o c e d u r e s the s u r g i c a l p r o c e d u r e s have been t h o r o u g h l y d e s c r i b e d e a r l i e r (11). the e x p e r i m e n t s were accomplished i n f o u r d i f f e r e n t s e r i e s . s e r i e s i and i11 com p r i s e d sham-operated and p a r t i a l l y h e p a t e c t o m i z e d a n i m a l s . series i1 and i v comprised p a r t i a l l y h e p a t e c t o m i z e d a n i m a l s w i t h one c a t h e t e r i n t h e g a s t r o d u o d e n a l a r t e r y , one i n t h e i l e o c o l i c v e i n and one c a t h e t e r emptying i n t o t h e p e r i t o n e a l c a v i t y . the c a t h e t e r s were i n s e r t e d i n t o t h e v e s s e l s o r f i x e d t o t h e p e r i t o n e a l w a l l i n c o n n e c t i o n w i t h p a r t i a l hepatectomy as d e s c r i b e d p r e v i o u s l y ( 1 1 ) . each c a t h e t e r w a s f l u s h e d , f i l l e d w i t h f y s k o s a l (pharmacia) + heparin (vitrum) 500 ie/100 m l and plugged. the c a t h e t e r s were k e p t open by i n t e r m i t t e n t f l u s h i n g . i n j e c t i o n of s t a r c h p a r t i c l e s i n t o t h e v a s c u l a r c a t h e t e r s was p e r f o r med 8 o r 25 h o u r s a f t e r s u r g e r y . to a v o i d d i u r n a l v a r i a t i o n a l l o p e r a t i o n s were performed between 8 11 a . m . i r r a d i a t i o n the r a t s were exposed t o t o t a l d o s e s of 4 16 gy whole body i r r a d i a t i o n . s e r i e s i, i1 and i v w e r e i r r a d i a t e d w i t h 8 mv x-rays from a l i n e a r a c c e l e r a t o r a t a dose r a t e of 4 gy/min. s e r i e s 111 was i r r a d i a t e d i n a n i r r a d i a t i o n chamber w i t h y r a y s from a 1 3 7 c e s o u r c e ( s c a n d i t r o n i x , b 349, s c a n d i t r o n i x i n s t r u m e n t s a b , uppsala, sweden) a t a d o s e r a t e o f 0 . 8 4 gy/min. n o n i r r a d i a t e d c o n t r o l g r o u p s were i n c l u d e d i n e a c h series. s t a r c h p a r t i c l e s d i g e s t i b l e s t a r c h p a r t i c l e s (spherex 60 mg/ml, b a t c h 78 08 2 4 , pharmacia) were a d m i n i s t e r e d i n t o t h e p o r t a l and a r t e r i a l c a t h e t e r s 3.5 m i n u t e s b e f o r e i r r a d i a t i o n ( s e r i e s i1 and iv). mean d i a m e t e r of t h e s p h e r e s was 30 p. 200 p1 of spherex s u s p e n s i o n was i n j e c t e d i n t o e a c h c a t h e t e r and t h e c a t h e t e r s were f l u s h e d w i t h 200 p 1 s a l i n e ( f y s k o s a l , pharmacia) . 44 r a d i o n u c l i d e s thymidine i n c o r p o r a t i o n i n t o dna was a n a l y s e d on t h e second day a f t e r p a r t i a l hepatectomy and 16 o r 1 . 5 h o u r s a f t e r i r r a d i a t i o n . 100 ~1 c o n t a i n i n g 185 kbq ( 5 pci) of ( m e t ~ l ' ~ c ) thymidine (185 gbq, amersham) o r 100 1.11 c o n t a i n i n g 3.7 mbq (100 vci) of (metylh) thymidine (555-1110 g b q , amersham) was adminis t e r e d i . p . i n a n i m a l s w i t h p e r i t o n e a l c a t h e t e r s i n j e c t i o n w a s made by way of t h i s c a t h e t e r . animals i r r a d i a t e d 1 . 5 h o u r s b e f o r e a n a l y s i s of dna s y n t h e s i s were p r e l a b e l l e d w i t h 3h-thymidine one hour b e f o r e i r r a d i a t i o n t o g i v e t h e pre t r e a t m e n t l e v e l of dna s y n t h e s i s i n e a c h a n i m a l . i n t h e s e a n i m a l s i4c-thymidine w a s u s e d f o r a n a l y s i s of t h y m i d i n e i n c o r p o r a t i o n i n t o dna 1 . 5 h o u r s a f t e r i r r a d i a t i o n . the d o u b l e l a b e l l i n g allowed a n a l y s i s of e f f e c t s of r a d i a t i o n i n e a c h a n i m a l (11). 3 i n t a c t regen. regen. regen. a n a l y s i s of dna dna was e x t r a c t e d a c c o r d i n g t o schmidt-tannhluser a s m o d i f i e d by munro & f l e c k ( 1 2 ) and q u a n t i f i e d a c c o r d i n g t o burton ( 3 ) o r i s o l a t e d and q u a n t i f i e d a c c o r d i n g t o kasche amneus ( 7 ) . i n c o r p o r a t i o n of l a b e l l e d thymidine was a n a l y s e d by l i q u i d s c i n t i l l a t i o n (mark i1 s c i n t i l l a t i o n c o u n t e r ) and t h e s p e c i f i c a c t i v i t y of dna w a s c a l c u l a t e d a s t h e amount of r a d i o a c t i v i t y p e r mg of dna p e r i n j e c t e d amount o f r a d i o a c t i v i t y . the e f f e c t of r a d i a t i o n d o s e w a s c a l c u l a t e d a s t h e r a t i o between t h e d i f f e r e n c e i n dna s p e c i f i c a c t i v i t y i n i r r a d i a t e d and non i r r a d i a t e d a n i m a l s and t h e r a d i a t i o n dose. 24 25 2 0.24 -f 0 . 0 8 24 25 4 2.30 1.12 4 8 24 25 3 0.86 z 0.16 0.4 8 8 24 25 4 0.62 0.19 3.2 results t a b l e 1. thymidine i n c o r p o r a t i o n i n t o l i v e r dna. s e r i e s i. e f f e c t of p a r t i a l hepatectomy and r a d i a t i o n d o s e . i r r a d i a t i o n performed 8 h o u r s a f t e r p a r t i a l hepatectomy. mean s t a n d a r d d e v i a t i o n , r e l a t i v e v a l u e s . l i v e r i r a d i a t i o n i3h-thymidine / k i l l i n g \number i3h-spec. / e f f e c t of s t a t u s 1lhours a f t e r / h o u r s a f t e r if 1;;i;;ty i n i r a d i a t i o n d o s e x l o 3 p a r t .hep. p a r t .hep. a n i m a l s dna 8 i 24 i 25 i 4 10.54 0.121 i p.1 regeni i thymidine i n c o r p o r a t i o n i n c r e a s e d t e n f o l d i n l i v e r t i s s u e 24 h o u r s a f t e r p a r t i a l hepatectomy (table 1). the v a r i a t i o n of t h y m i d i n e i n c o r p o r a t i o n b e t ween d i f f e r e n t a n i m a l s w a s h i g h . i r r a d i a t i o n 8 h o u r s a f t e r s u r g e r y d e c r e a s e d thymidine i n c o r p o r a t i o n 24 h o u r s p o s t o p e r a t i v e l y . the e f f e c t of t h e r a d i a t i o n 45 d o s e w a s h i g h e s t a t 4 gy. i n c r e a s e d r a d i a t i o n d o s e (table 1) reduced t h y m i d i n e i n c o r p o r a t i o n . a n i n c r e a s e d t h y m i d i n e i n c o r p o r a t i o n i n t o l i v e r t i s s u e made i s c h e m i c d u r i n g i r r a d i a t i o n was n o t found (table 2 ) . the c a t h e t e r s i n t h e a r t e r i a l and p o r t a l s y s t e m s , n e c e s s a r y f o r a d m i n i s t r a t i o n of t h e s t a r c h p a r t i c l e s , d e p r e s s e d t h y m i d i n e i n c o r p o r a t i o n ( t a b l e 2 ) a s h a s been n o t e d b e f o r e (11). t a b l e 2 . thymidine i n c o r p o r a t i o n i n l i v e r dna on t h e second day a f t e r p a r t i a l hepatectomy. s e r i e s 11. e f f e c t of d i g e s t i b l e s t a r c h p a r t i c l e s d u r i n g i r r a d i a t i o n 8 h o u r s a f t e r p a r t i a l hepatectomy. 3h-thymidine w a s a d m i n i s t e r e d i . p . 24 h o u r s a f t e r p a r t i a l hepatectomy, and t h e r a t s were k i l l e d 25 h o u r s a f t e r p a r t i a l hepatectomy. s t a r c h p a r t i c l e s r a d i a t i o n dose h o u r s a f t e r gy p a r t . h e p . 7 8 + 7 8 c a t h e t e r s i n a. g a s t r o d . v . i l e o c o l i c a number of 3h-spec. a c t i v i t y i n dna anima 1 s m sd 4 2.43 0.61 4 1.67 1.13 7 0.62 _+ 0.26 1 ) 7 0.85 0.36’’ r a d i a t i o n 14c-thymidine h o u r s a f t e r p a r t . hep. dose h o u r s a f t e r gy p a r t . h e p . 26.5 3 25 26.5 7 25 26.5 1) d i f f e r e n c e n o t s i g n i f i c a n t . student’s t t e s t . k i l l e d 14c-spec. e f f e c t of 1 4 c / 3 ~ i n h o u r s a c t i v i t y r a d i a t i o n l i v e r dna a f t e r i n dna d o s e x 1 0 3 m -+ sd p a r t . h e p . m -+ sd 27.5 1.2320.421’ ) 0.5620. 27.5 0 . 7 4 4 . 2 7 1 ) 0.16 . 0 .39&0.09 3 1 27.5 0.59*0.212) 0.09 ‘0.344to.104) the d e p r e s s i v e e f f e c t o f r a d i a t i o n on t h y m i d i n e i n c o r p o r a t i o n w a s less pro nounced on t h e second day a f t e r p a r t i a l hepatectomy and o n e hour a f t e r i r r a d i a 46 t i o n (table 3 and 4) t h a n on t h e second day a f t e r p a r t i a l hepatectomy and 16 h o u r s a f t e r i r r a d i a t i o n ( t a b l e 1 and 2 ) . however, t h e d i f f e r e n c e between thymi d i n e i n c o r p o r a t i o n i n i r r a d i a t e d and n o n i r r a d i a t e d a n i m a l s i n t h e one h o u r group w a s s i g n i f i c a n t b o t h a t 3 and 7 gy ( t a b l e 3 ) . the s i g n i f i c a n c e was s l i g h t l y im proved when t h e r a t i o between 3h-thymidine i n c o r p o r a t i o n b e f o r e i r r a d i a t i o n and t h e "c-thymidine i n c o r p o r a t i o n a f t e r i r r a d i a t i o n was c a l c u l a t e d f o r e a c h a n i m a l ( t a b l e 3 ) . the temporary e x c l u s i o n of blood by s t a r c h p a r t i c l e s d e c r e a s e d t h y m i d i n e i n c o r p o r a t i o n i n t o dna as s e e n from a n a l y s e s 1 . 5 h o u r s a f t e r i n j e c t i o n ( t a b l e 4 ) . the 1 4 c s p e c i f i c a c t i v i t y of dna d e c r e a s e d from 1.00 t o 0.70 o r 30% and t h e r a t i o l 4 c i 3 h i n e a c h a n i m a l from 0 . 9 4 t o 0.58 o r 39%. m i la'' 6 8 6 9 8 9 t a b l e 4 . thymidine i n c o r p o r a t i o n i n l i v e r dna on t h e second day a f t e r p a r t i a l hepatectomy. s e r i e s i v . immediate e f f e c t of r a d i a t i o n a n d / o r d i g e s t i b l e s t a r c h p a r t i c l e s . a l l a n i m a l s had c a t h e t e r s i n t h e g a s t r o d u o d e n a l a r t e r y and i l e o c o l i c v e i n and were p r e l a b e l l e d w i t h 3h-thymidine 24 h o u r s a f t e r p a r t i a l hepatectomy. dna m 2 sd 1.00+0.36 0.7020.31 0.8020.82 0.4020.21 0.89?0.53 0.31?0.22 t a r c h a r t i c 3 3 7 d i n e p a r t .hep. 26.5 26.5 25 2 6 . 5 25 26.5 25 26.5 7 25 26.5 l i l l e d irs a f t e r iar t . hep . 27.5 27.5 2 7 . 5 27.5 27.5 27.5 lumber i14c-spec. 1 i 4 c l 3 h i n )f l a c t i v i t y i n dna m + sd 40 0.36?0.16 89 0.45?0.14 31 0.34?0.18 % ._ 00 6 1 50 38 48 36 the d e c r e a s i n g e f f e c t on t h y m i d i n e i n c o r p o r a t i o n a s a r e s u l t of temporary i s c h e m i a w a s o b v i o u s a l s o i n i r r a d i a t e d a n i m a l s . the mean i 4 c s p e c i f i c a c t i v i t y d e c l i n e d from 0.80 t o 0 . 4 0 , i . e . a n e x t r a 40% a t 3 gy a n d from 0.89 t o 0 . 3 1 o r a n e x t r a 58% a t 7 gy r e s p e c t i v e l y when s t a r c h p a r t i c l e s were p r e s e n t d u r i n g i r r a d i a t i o n . the c o r r e s p o n d i n g i 4 c l 3 h r a t i o s d e c l i n e d from 0.47 t o 0.36 and from 0.45 t o 0.34 a t 3 and 7 gy r e s p e c t i v e l y . thus, i m m e d i a t e l y a f t e r i r r a d i a t i o n t h e temporary i s c h e m i a a c t s s y n e r g i s t i c a l l y w i t h t h e i r r a d i a t i o n i n d e p r e s s i n g t h y m i d i n e i n c o r p o r a t i o n . the mean e f f e c t of t h e temporary i s c h e m i a c a u s e d by t h e s t a r c h p a r t i c l e s i n n o n i r r a d i a t e d and i r r a d i a t e d r a t s was a 43% d e c r e a s e of thymidine i n c o r p o r a t i o n (table 4 ) . discuss i o n the r e s u l t s show n o p r o t e c t i v e a c t i o n i n terms of t h y m i d i n e i n c o r p o r a t i o n 47 from temporary i s c h e m i a d u r i n g t h e f i r s t h o u r s a f t e r i r r a d i a t i o n . on t h e c o n t r a r y , t h e e x c l u s i o n of blood seemed t o a c t s y n e r g i s t i c a l l y w i t h t h e r a d i a t i o n i n d i s t u r b i n g c e l l u l a r dna s y n t h e s i s . the d i s t u r b a n c e s s h o u l d , however, be of o p p o s i t e c h a r a c t e r . r a d i a t i o n l e a d s t o i n c r e a s e d amounts o f dna damaging oxygen f r e e r a d i c a l s . blood e x c l u s i o n s h o u l d l e a d t o a low oxygen t e n s i o n and d e c r e a s e d amounts o f f r e e r a d i c a l s . both e f f e c t s seem t o push t h e l i v e r c e l l s o u t of o r d e l a y t h e i r e n t r a n c e i n t o t h e s-phase. it h a s been shown, however, t h a t t h e p r o t e c t i v e a c t i o n o f blood e x c l u s i o n and low oxygen t e n s i o n d u r i n g i r r a d i a t i o n i s q u i t e e v i d e n t a t l a t e r s t a g e s of r e c o v e r y ( 5 , 6 ) . thus, c e l l metabolism i s n o t permanently damaged and t h e c e l l s s h o u l d r e t u r n t o t h e c e l l c y c l e and s y n t h e s i z e dna. i n t h e p r e s e n t e x p e r i m e n t t h e c e l l s o f t h e 24 h o u r r e g e n e r a t i n g l i v e r s w e r e p r o b a b l y r e t u r n i n g t o t h e c e l l c y c l e and t h e s-phase a f t e r b e i n g i r r a d i a t e d d u r i n g i s c h e m i a 1 6 h o u r s e a r l i e r . the s i m i l a r v a l u e s of dna s p e c i f i c a c t i v i t y i n p r o t e c t e d and n o n p r o t e c t e d a n i m a l s i n d i c a t e d t h a t t h e c e l l s i n p r o t e c t e d l i v e r s had overcome t h e a d d i t i o n a l d e c r e a s e c a u s e d by t h e t e m p o r a r i l y low oxygen t e n s i o n . 3 p r e l a b e l l i n g o f dna by h-thymidine and comparisons of 3h/14c-ratios i n dna was p r e v i o u s l y shown t o improve t h e s i g n i f i c a n c e o f r e s u l t s ( 1 1 ) . the v a l u e of d o u b l e l a b e l l i n g w a s n o t s o c l e a r i n t h i s s t u d y . the p e r i o d between i n j e c t i o n of 3h-thymidine and 14c-thymidine was r a t h e r l o n g , i . e . 2.5 h o u r s and t h e i r r a d i a t i o n i n t h e meantime makes h a n d l i n g of t h e a n i m a l s n e c e s s a r y . both may i n f l u e n c e t h y m i d i n e i n c o r p o r a t i o n i n t o dna. the o b s e r v e d d e c r e a s e o f thymidine i n c o r p o r a t i o n i n t o dna d u r i n g a temporary t o t a l b l o c k of l i v e r blood s u p p l y h a s i m p l i c a t i o n s i n t h e u s e o f s t a r c h p a r t i c l e s f o r t h e l o c a l a c c u m u l a t i o n o f d r u g s ( 1 3 ) . drugs t h a t are e n e r g y dependent and have t o b e m e t a b o l i z e d t o be e f f e c t i v e r e q u i r e u n i n t e r r u p t e d blood f l o w . t h i s would n o t be a problem when s t a r c h p a r t i c l e s are used t o a c c u m u l a t e d r u g s w i t h a f f i n i t y t o c e l l membranes and d e l a y e d c e l l u l a r u p t a k e . references 1. 2 . 3. 4. 5. altman, k . i . , g e r b e r , g . b . & okada, s . : the l i v e r . i n r a d i a t i o n b i o c h e m i s t r y , vol 11, ed. altman, 1.1. & g e r b e r , g . b . ap 1970, pp. 160-194. barbason, h . : i n f l u e n c e of x i r r a d i a t i o n on t h e d i f f e r e n t s t a g e s of t h e c e l l cycle i n r e g e n e r a t i n g l i v e r . virchows arch. b c e l l p a t h . 16:363-369, 1974. b u r t o n , k . : a s t u d y of t h e c o n d i t i o n s and mechanisms of t h e diphenylamine r e a c t i o n f o r t h e c o l o r i m e t r i c e s t i m a t i o n of dna. biochem. j. 62:315-323, 1956. f o r s b e r g , j . o . : t r a n s i e n t blood f l o w r e d u c t i o n induced by i n t r a a r t e r i a l i n j e c t i o n o f d e g r a d a b l e s t a r c h m i c r o s p h e r e s . experiments on r a t s . acta c h i r scand 144:275-281, 1978. f o r s b e r g , j . o . , j u n g , b . & l a r s s o n , b . : mucosal p r o t e c t i o n d u r i n g i r r a d i a t i o n of e x t e r i o r i z e d r a t ileum. e f f e c t of hypoxia induced by s t a r c h micro s p h e r e s . acta rad onc 17:485-496, 1978. 48 6. 7. 8 . 9. 10. 11. 1 2 . 13. 1 4 . f o r s b e r g , j . o . , j i b o r n , h. & j u n g , b . : p r o t 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k.: i n t r a a r t e r i a l and i n t r a p o r t a l i n v i v o c a t h e t e r i z a t i o n o f t h e r e g e n e r a t i n g r a t l i v e r . e f f e c t s upon body and l i v e r w e i g h t s and dna s y n t h e s i s . uppsala j med s c i , 1983. munro, h. & f l e c k , a . : recent developments i n t h e measurement of n u c l e i c a c i d s i n b i o l o g i c a l m a t e r i a l . a n a l y s t 91:78-88, 1966. t u n a , r . , f o r s b e r g , j . o . & agerup, b.: enhanced u p t a k e of a c t i n o m y c i n d i n t h e dog k i d n e y by s i m u l t a n o u s l y i n j e c t i o n of d e g r a d a b l e s t a r c h m i c r o s p h e r e s i n t o t h e r e n a l a r t e r y . cancer 5o:l-5, 1982. walters, r . a . & enger, m . d . : e f f e c t s of i o n i z i n g r a d i a t i o n on n u c l e i c acid s y n t h e s i s i n mammalian c e l l s . i n : adv rad b i o l vol 6 , ae' 1976, pp. 1-48. address f o r r e p r i n t s : l. lewan, ph.d. department of zoophysiology helgonavagen 3b sweden s-223 62 lund 4-832859 49 adipocytes express tissue factor and fvii and are procoagulant in a tf/fviia-dependent manner article adipocytes express tissue factor and fvii and are procoagulant in a tf/fviia-dependent manner desir�ee ed�ena, grigorios panagiotoub, dariush mokhtaria,b, jan w. erikssonb, mikael åberga and agneta siegbahna adepartment of medical sciences, clinical chemistry, uppsala university, uppsala, sweden; bdepartment of medical sciences, clinical diabetes and metabolism, uppsala university, uppsala, sweden abstract background: tissue factor (tf) combined with its ligand fvii initiates blood coagulation and intracellular signaling. obese and type 2 diabetic subjects have increased tf expression in their adipose tissue and an increased risk for thrombotic complications. here we address the role of tf/fvii on adipocyte functions. materials and methods: subcutaneous fat was obtained by means of needle aspiration from healthy volunteers, and adipocytes were isolated after collagenase digestion. 3t3-l1 fibroblasts kept in culture were differentiated into adipocytes by addition of ibmx, dexamethasone, rosiglitazone, and insulin to the media. proteins and mrna were analyzed by western blot and rt-pcr. coagulation activity was determined by a colorimetric fx-assay. lipolysis was measured as free glycerol using a colorimetric method. glucose uptake was evaluated by scintillation counting of d-[u-14c] glucose. results: in isolated human primary adipocytes we found expression of tf and fvii. tf expression was confirmed in 3t3-l1 adipocytes, and both cell types were found to be procoagulant in a tf/fviiadependent manner. fxa was generated without fviia added to the coagulation assay, and active siteinhibited fviia blocked fxa formation, supporting our finding of fvii production by human primary adipocytes. there was no evidence for a role of tf in either lipolysis or glucose uptake in our experimental settings. conclusion: human primary adipocytes express active tf and fvii, and the tf/fviia complex formed on the adipocyte surface can activate substrate fx. whether the tf/fviia complex conveys signaling pathways leading to biological functions and has any biological activity in adipocytes beyond coagulation remains to be elucidated. article history received 30 may 2019 revised 5 july 2019 accepted 15 july 2019 keywords adipocytes; coagulation; fvii; lipolysis; tissue factor introduction obesity is an increasing global problem and a major risk factor for development of type 2 diabetes (t2d) (1,2). t2d is associated with a plethora of cardiovascular and thrombotic complications, including atherosclerosis, myocardial infarction, ischemic stroke, and peripheral vascular disease (3–5). dysregulation of hemostasis is linked to the development of t2d and considered a contributor to its cardiovascular comorbidities. previous studies report evidence for involvement of both coagulation and fibrinolysis pathways (3,6–8). tissue factor (tf), also known as coagulation factor iii, is a transmembrane protein and the main initiator of blood coagulation. after vascular injury, tf binds and activates coagulation factor vii (fvii) into fviia, and thrombin formation is subsequently initiated. tf is normally expressed on cells outside the vasculature, and activation of the coagulation cascade by aberrant expression of tf promotes thrombotic episodes in patients with a variety of clinical disorders (3,9–12). for instance, obese subjects have increased plasma concentrations of tf and fviia and increased expression of tf in their adipose tissue. moreover, obesity has been linked to increased plasma tf activity (3,13–15). these observations may explain the development of the hypercoagulable state and related complications that are associated with excessive body fat accumulation. in addition to its role in coagulation, tf/fviia also mediates intracellular signaling related to other physiological mechanisms including angiogenesis, migration, tumor growth, apoptosis, and inflammation (16–19). we have previously shown that tf/fviia signaling augments cytokineinduced pancreatic beta-cell death, which is proposed to contribute to diabetes (20), while others have coupled tfmediated signaling to high-fat diet-induced obesity, adipose tissue inflammation, and insulin resistance in mice (12,16,21,22). obesity is characterized by insulin resistance, and, apart from its role in glucose uptake, insulin also has anti-lipolytic effects (19). partly due to insulin resistance, obese and t2d individuals exhibit higher lipolysis rates, leading to increased free fatty acids (ffa) concentrations in plasma (23). moreover, plasma ffa correlates with adipose contact agneta siegbahn agneta.siegbahn@medsci.uu.se department of medical sciences, clinical chemistry, uppsala university, uppsala, sweden. � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 3, 158–167 https://doi.org/10.1080/03009734.2019.1645248 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1645248&domain=pdf&date_stamp=2019-09-09 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2019.1645248 http://www.tandfonline.com tissue tf mrna levels (15), which may indicate a possible role for tf in lipolysis. in the present study, we investigated the impact of tf/fviia complex formation on several adipocyte functions in model systems of isolated human primary adipocytes and murine 3t3-l1 adipocytes. materials and methods reagents collagenase type ii and isoproterenol used in human adipocyte experiments, 3-isobutyl-1-methylxanthine (ibmx), dexamethasone, insulin, and par1 (tfflr) and par2 (sligrl) agonists used in 3t3-l1 adipocyte experiments were all from sigma-aldrich sweden ab. tnf-alfa, interferon-gamma, and il1beta were from r&d systems, usa. insulin used in human adipocyte experiments was from novo nordisk a/s, denmark. human recombinant active site-inhibited fviia (fviiai) and recombinant human and murine fviia were kind gifts from lc petersen, novo nordisk. fx was from enzyme research lab, usa. rosiglitazone was from cayman chemical, usa. taqman probes were from applied biosystems, usa. the following antibodies were used: gapdh 14c10, beta actin 8h10d10, cleaved caspase-3 d175, phsl s563, and hsl from cell signaling technology, usa; tf n1c3 from genetex usa; tf af2339 and tf af3178 from r&d systems, usa; irdye 680cw and irdye 800cw from li-cor biosciences, uk. human adipocyte isolation isolation of human primary adipocytes from healthy volunteers was essentially performed as previously described (24). briefly, subcutaneous fat was obtained from the lower abdomen by needle aspiration after dermal local anesthesia with lidocaine (xylocain, astrazeneca, s€odert€alje, sweden). the study was approved by the regional ethics review board uppsala (dnr 2013/183 and 2013/494), and all participants provided written informed consent. adipocytes were isolated after collagenase type ii digestion in hanks’s medium: medium 199 with hanks’s salts, l-glutamine, 5.6mm glucose and hepes supplement (life technologies) supplemented with 4% bovine serum albumin (bsa) and 150nm adenosine with ph 7.4 at 37 �c in a shaking water bath for 60min. following collagenase treatment, adipocytes were filtered through a 250lm nylon mesh, subsequently held at 37 �c and washed four times in hanks’s medium before lipolysis and glucose uptake assays were performed. 3t3-l1 cell culture, differentiation, and dream 3t3-l1 cells (atcc) were grown to confluence in growth medium: dmem glutamax with 4.5 g/l glucose and pyruvate supplement (life technologies) supplemented with 10% fetal bovine serum (fbs), 100 u/ml penicillin, and 100 mg/ml streptomycin in a humidified chamber at 37 �c and 5% co2. differentiation was induced by culturing in growth medium supplemented with 2 lm rosiglitazone, 0.5 mm 3-isobutyl-1methylxanthine (ibmx), 0.25 lm dexamethasone, and 170 nm insulin. after 48 h, medium was changed to growth medium supplemented with 170 nm insulin. adipocytes were fully differentiated after 8–10 days, and density-based separation followed by re-plating of enriched adipocytes in monolayer (dream) was performed, as described by kajimoto et al. (25). mda-mb-231, hcc1937, and katoiii cell culture mda-mb-231 human breast cancer cells, hcc1937 human breast cancer cells, and katoiii human gastric carcinoma cells were grown in rpmi medium (life technologies) supplemented with 10% fbs, 100 u/ml penicillin, and 100 mg/ml streptomycin in a humidified chamber at 37 �c and 5% co2. sirna transfection in 3t3-l1 adipocytes dreamed 3t3-l1 adipocytes were re-plated in 24-well plates in growth medium. silencerv r select validated sirna toward tf and scramble rna (10nm ambion/life technologies, usa) were transfected into the adipocytes using lipofectamine rnaimax (invitrogen ab, stockholm, sweden) as described by the manufacturer. experiments were performed 72h post sirna transfection. mrna analysis human primary adipocytes were isolated as described and washed three times in phosphate-buffered saline (pbs). total rna was extracted using trizol according to the manufacturer’s protocol (invitrogen). mda-mb-231, hcc1937, and katoiii cells were washed three times in pbs and total rna isolated using rneasy mini kit according to the manufacturer’s protocol (qiagen, hilden, germany). cdna was obtained using oligo(dt) (invitrogen). reverse-transcriptase pcr (rt-pcr) analyses of tf, fvii, and the housekeeping gene beta-actin were performed on cdna with platinum taq dna polymerase as described by the manufacturer (invitrogen) with 40 amplification cycles. primers and protocol have previously been published (26). samples were separated by 1% agarose gel electrophoresis, and bands were visualized by gel red and uv using a quantity one flour-s multi-imager (bio-rad laboratories, hercules, ca, usa). realtime quantitative pcr (qrt-pcr) analyses of par2, antithrombin iii (at-iii), tissue factor pathway inhibitor (tfpi), and the housekeeping gene b2-microglobulin (assay-ondemand, applied biosystems) were performed on cdna and run on an abiprism 7500 (applied biosystems). western blot 3t3-l1 and human primary adipocytes were lysed in 2% sodium dodecyl sulphate (sds) sample buffer supplemented with 5% beta-mercaptoethanol. proteins were separated on a bolt mini-gel and transferred to immobilon-fl pvdf membranes (2.5 h at 120 v). the membranes were subsequently blocked 1 h in odyssey blocking buffer (li-cor biosciences) and incubated overnight at 4 �c in odyssey blocking buffer with primary antibodies diluted 1/1000. after incubation, membranes were washed 3 � 5 min in tbs with 0.01% tween-20 and incubated 40 min with secondary antibodies upsala journal of medical sciences 159 (1/10,000 dilution) conjugated to ir-dyes 680 or 800 and washed as above. proteins were visualized with the odyssey infrared imaging system (li-cor biosciences) and quantified using odyssey v3.0 software. coagulation activity assay the coagulation activity assay was performed in isolated human primary adipocytes of a 5% lipocrit in 96-well plates and sirna transfected 3t3-l1 adipocytes in 24-well plates. cells were washed three times in hepes-buffered saline with 0.1% bsa (hbsa). human primary adipocytes were pre-treated with fviiai for 5min or left untreated. thereafter, cells were incubated with or without 10nm hfviia (murine fviia for 3t3-l1 adipocytes), 300nm fx, and 10mm cacl2 for 2h at 37 �c. fxa formation was stopped in hbsa supplemented with 25mm edta and 4mm chromogenic substrate (s-2765) (chromogenix, usa) added for 15min at 37 �c. medium from 3t3-l1 cells was moved to 96-well plates for analysis. a standard curve was included in every experiment, ranging from 0.1pg/ml to 1000pg/ml of re-lipidated htf (siemens, usa), and a trend line was calculated from the standard curve. tf concentration was estimated based on the calculated trend line. absorbance was read at 405nm. all experiments were performed in duplicate. lipolysis in human primary adipocyte the lipolysis assay was essentially performed as described previously (27,28). in brief, isolated adipocytes were in a 2%–3% lipocrit suspension and kept in vials with hanks’s medium (4% bsa, 150 nm adenosine with ph 7.4) in a gently shaking water bath at 37 �c. cells were pre-incubated for 30 min with 10 nm human recombinant fviia. lipolysis was evaluated at a basal level and in response to isoproterenol (1 lm for 2 h) with and without insulin (100 lu/ml) preincubation for 10 min. glycerol released to the medium was measured by colorimetric absorbance read at 540 nm with free glycerol reagent (sigma-aldrich sweden ab) in a tecan magellan plate reader and used as lipolytic index. duplicate measurements were performed for all conditions. lipolysis in 3t3-l1 adipocytes lipolysis assay in 3t3-l1 adipocytes was performed after dream in all experiments. experiments were conducted using the abcam lipolysis assay kit (ab185433) as described by the manufacturer with adjustments for volumes suitable for 24well plates. briefly, cells were washed and kept in buffers supplied in the kit. for the different experiments, 10nm of mfviia or 25lm of par1 or par2 agonists was added for 30min followed by incubation with 5 nm isoproterenol for 3 h. glycerol released to the medium was measured by colorimetric absorbance read at 570 nm. for lipolysis estimations with long-term cytokine exposure, cells were dreamed and allowed to settle for 24h. thereafter, cells were pre-stimulated with fviia (10nm, 30min) and subsequently kept for 48h in growth medium supplemented with a mixture of tnf-alfa (10 ng/ml), il-1 beta (5 ng/ml), and ifn-gamma (5ng/ml). lipolysis assay was thereafter performed as described above. signaling 3t3-l1 adipocytes were dreamed and allowed to settle overnight. cells were washed and kept in buffers provided in the lipolysis assay kit. cells were left untreated or stimulated as follows: 5 nm isoproterenol for 3 h, 1 nm insulin for 10 min followed by 5 nm isoproterenol for 3 h, 10 nm mfviia for 3 h, 10 nm mfviia for 15 min. after incubation, cells were washed with pbs and western blot performed as described above. glucose uptake in human primary adipocytes glucose uptake was essentially performed as previously described (29). isolated adipocytes, lipocrit 5% were suspended in glucose-free krebs-ringer-hepes (krh) medium (4% bsa, 150nm adenosine with ph 7.4) and kept in vials in a gently shaking water bath at 37 �c. cells were pre-incubated with or without fviia (10nm, 30min). cells were left untreated or incubated with insulin (1000 lu/ml, 15min). d-[u-14c] glucose (0.86lm; ge healthcare, buckinghamshire, uk) was added to vials, which were then further incubated for 45min. reaction was stopped by transferring adipocytes in suspension to vials on ice, and adipocytes were separated by centrifugation through silicone oil. glucose uptake was evaluated by triplicate measurements of the adipocyte-associated radioactivity in a beta-counter. statistics graphs and statistics were obtained using the graphpad prism 7.0a software. statistical significances were obtained by comparison to corresponding control using student’s unpaired t test (two-tailed). the bars indicate mean þ sem, and p � 0.05 was considered statistically significant. results tf and fvii are expressed in human adipocytes, and tf can be effectively down-regulated in 3t3-l1 adipocytes first, we wanted to investigate the mrna expression of tf in isolated human primary adipocytes. it has previously been reported that 3t3-l1 adipocytes and adipose tissue from mice express fvii (30), and therefore we wanted to study the fvii mrna expression in human adipocytes. to confirm the method, tf and fvii mrna levels were studied in mda-mb231, hcc1937, and katoiii cells using rt-pcr and previously published primers (26). the cell lines were used as positive controls on the basis of their documented high tf expression and the known ectopic production of fvii by katoiii cells. mda-mb-231 cells were used as negative control for fvii expression. all three cell lines expressed tf. hcc1937 and katoiii cells expressed fvii, whereas mda-mb-231 lacked fvii expression (figure 1(a)). expression of tf and fvii mrna was then studied in human primary adipocytes from five different subjects using the same protocol. strong bands were detected for tf, and 160 d. ed�en et al. weaker but distinct bands were detected for fvii in all five subjects (figure 1(b)). we also performed mrna analysis for par2 and b2-microglobulin, using qrt-pcr, and found that adipocytes express par2 (ct mean value: 31.9±0.3 for par2 as compared to 19.5±0.2 for b2-microglobulin, n¼ 5; supplementary figure 1a, available online). an additional qrt-pcr analysis for at-iii, tfpi, and b2-microglobulin in human primary adipocytes revealed a gene expression of these anti-coagulant factors (ct mean values: at-iii 32.3±0.7, tfpi 26.5±0.3 as compared to b2microglobulin 20.0±0.3, n¼ 3; supplementary figure 1b, available online). tf was studied on the protein level in human primary adipocytes obtained from two donors. western blot was performed on the lysed adipocytes, and tf expression was evaluated with two different antibodies against tf: tf n1c3 from genetex and tf af2339 from r&d systems. tf protein was detected with both antibodies (figure 1(c)). tf was also detected on the protein level in differentiated 3t3-l1 adipocytes using western blot. tf protein levels were effectively down-regulated by sirna transfection with lipofectamine on dreamed 3t3-l1 adipocytes. the tf/gapdh quota on measured band intensities in tf sirna-treated cells had a mean of 0.03 compared with scr sirna-treated cells with a mean of 0.22 and control cells with a mean of 0.37 (n¼ 3). translated to percentages, tf sirna treatment down-regulated tf with 87% ±0.2% as compared with scr sirna-treated cells (p< 0.001) and 92% ±0.3% compared with untreated control cells (p< 0.001) (figure 1(d)). adipocytes are procoagulant in a tf/fviia-dependent manner in both human primary adipocytes and 3t3l1 adipocytes having shown that adipocytes express tf, we continued to study the function of tf by assessing their coagulation activity in a fxa substrate assay. both human primary adipocytes and dreamed 3t3-l1 adipocytes were coagulant active in response to fviia (figure 2(a,b), respectively). in human primary adipocytes, there was coagulation activity without addition of fviia. addition of fviiai together with fviia decreased the coagulation activity with 70% in comparison to the group only receiving fviia (p< 0.01) (figure 2(c)). tf down-regulation with sirna transfection using lipofectamine in 3t3-l1 adipocytes decreased the coagulation activity with 45% compared with untreated adipocytes (p< 0.001) and with 40% compared with cells treated with scr sirna (p< 0.01) (figure 2(d)). figure 1. tf and fvii expression in adipocytes. (a) tf and fvii mrna expression were evaluated with rt-pcr in mda-mb-231, hcc1937, and katoiii cells to confirm the method. all cell lines expressed tf. hcc1937 and katoiii expressed fvii, whereas mda-mb-231 did not. beta-actin was included as a positive control. (b) human primary adipocytes were obtained from healthy volunteers. tf and fvii were expressed on mrna level, detected with rt-pcr using the same protocol as in (a) (n ¼ 5). (c) human primary adipocytes were obtained from healthy volunteers. tf expression was evaluated on protein level using western blot and two different antibodies towards tf. the figure shows one representative western blot for each antibody; adipocytes in shown experiment were from two different donors. (d) 3t3-l1 adipocytes were dreamed and cells left untreated or treated with tf sirna or scrambled sirna as control for 72 h. samples were lysed in sds-sample buffer and protein levels evaluated with western blot (figure shows one representative blot). tf sirna transfection resulted in a down-regulation of tf with 87% (p < 0.01) compared to scr sirna and 92% (p < 0.01) compared to control. n ¼ 3. error bars represent sem. ��� ¼ p � 0.001. upsala journal of medical sciences 161 https://doi.org/10.1080/03009734.2019.1645248 https://doi.org/10.1080/03009734.2019.1645248 https://doi.org/10.1080/03009734.2019.1645248 lipolysis in human primary adipocytes is not affected by fviia stimulation next, we investigated the role of tf in lipolysis in human primary adipocytes. cells were left untreated or stimulated with 10 nm fviia for 30 min. standard protocols for stimulation and inhibition of lipolysis using isoproterenol and insulin, respectively, were utilized (27,28). isoproterenol stimulation resulted in increased lipolysis as compared to untreated cells, whereas insulin counteracted the effect of isoproterenol stimulation. fviia stimulation did not affect the basal or isoproterenol-stimulated lipolysis, and neither did it alter the anti-lipolytic effect of insulin (figure 3). lipolysis in 3t3-l1 adipocytes is not affected by fviia stimulation, tf down-regulation, and par1 or par2 agonists to further investigate the possible effects of tf/fviia in lipolysis we used 3t3-l1 adipocytes. isoproterenol stimulation resulted in lipolysis in all experiments similar to the effects seen in the human primary cells. pre-stimulation with fviia (10 nm, 30 min) did not alter the lipolysis rate either at the basal level or in response to isoproterenol (figure 4(a)). we further investigated the effect of tf down-regulation using sirna transfection and also the effect of par1 and par2 stimulation in lipolysis. since both the binary complex tf/fviia and the trinary complex tf/fviia/fxa may trigger signaling through proteolytical activation of par1 and par2, we stimulated the tf sirna and scr sirna-transfected cells with par1 and par2 agonists or left the cells untreated. there was no difference between tf-depleted cells and cells with intact tf (figure 4(b,c)). pre-stimulation with the par agonists one by one (25 lm, 30min) did not alter the lipolysis rate either on the basal level or in response to isoproterenol (figure 4(c)). data from figure 4(b,c) are from the same set of experiments and can be directly compared with one another. long-term cytokine treatment results in lipolysis, but combination with fviia stimulation does not alter the lipolysis rate the adipose tissue of t2d patients and pre-diabetics is likely to be exposed to pro-inflammatory cytokines (6,31). since figure 2. coagulation activity in adipocytes. (a) human primary adipocytes were isolated, and coagulation activity was measured as fxa generation after stimulation with 10nm fviia in three distinctive subjects. the table presents active tf in pg/ml based on calculation from standard curve. three distinctive experiments. (b) coagulation activity was measured as fxa generation after stimulation of 3t3-l1 cells with 10nm mfviia. the table presents active tf in pg/ml based on calculation from standard curve. three distinctive experiments. (c) human primary adipocytes were isolated and pre-stimulated with 100nm fviiai for 5 min or left untreated. incubation was with or without 10nm fviia. negative control incubated without fviia and fx. graph represents absorbance values. fxa generation was 70% (p < 0.001) less in cells treated with fviiaiþ fviiaþ fx compared with cells treated with fviia þ fx and 90% less (p < 0.001) in cells treated with fviiai þ fx compared with cells treated with fx. n ¼ 3. error bars represents sem. (d) tf was down-regulated using sirna in 3t3-l1 adipocytes, and coagulation activity was evaluated as described. graph represents absorbance values as percentage of control. fxa generation was 45% (p < 0.001) less in cells with down-regulated tf compared with untreated control cells and 40% (p < 0.01) less compared with cells treated with scrambled sirna. n ¼ 3. error bars represent sem. �� ¼ p � 0.01; ��� ¼ p � 0.001. 162 d. ed�en et al. cytokines are known to stimulate lipolysis (32), we addressed the question if fviia in combination with the pro-inflammatory cytokines tnf-alfa, ifn-gamma, and il1-beta would exert additional effects on lipolysis. 3t3-l1 adipocytes were dreamed, allowed to settle for 24 h, and then stimulated with cytokines with or without fviia pre-stimulation (10 nm, 30 min) or only treated with fviia for 48 h before the lipolysis assay was performed. one group was left untreated and served as a negative control, and one group was stimulated with isoproterenol (5 nm, 3 h) and served as a positive control. long-term cytokine stimulation of cells led to an increase of glycerol release to the same extent as 3 h of isoproterenol treatment when compared to untreated cells. fviia did not alter the lipolysis rate either at the basal level or in response to the cytokine treatment (figure 5). hormone-sensitive lipase (hsl) is phosphorylated by isoproterenol but unaffected by fviia stimulation lipolysis can be initiated by different ligands and g-proteincoupled receptors, but the signaling pathways eventually converge in the phosphorylation of hormone-sensitive lipase (hsl). phosphorylated hsl catalyzes the hydrolysis of diacylglycerol to monoacylglycerol that is further hydrolyzed to glycerol and free fatty acids (33). in dreamed 3t3-l1 adipocytes, isoproterenol increases the phosphorylation of hsl compared with untreated control cells (p < 0.01), and insulin counteracts the effect of isoproterenol. there was no difference in hsl phosphorylation in mfviia-stimulated cells as compared to that of untreated cells (figure 6). figure 3. lipolysis in human primary adipocytes. adipocytes were isolated from healthy volunteers and kept in a 2%–3% lipocrit suspension in hanks’s medium in a shaking water bath holding 37 �c. cells were stimulated with fviia (10 nm, 30 min) or left untreated for control. lipolysis was evaluated on basal level and in response to isoproterenol stimulation (1 um, 2 h) with and without insulin (100 lu) added 10 min before isoproterenol. media were collected, and glycerol released to the media was measured by colorimetric absorbance read at 540 nm with free glycerol reagent and used as lipolytic index. fviia did not alter lipolysis rate in any condition. isoproterenol increased lipolysis in cells without and with fviia compared with untreated cells and fviia-treated cells, respectively (p < 0.001). insulin pre-stimulation on isoproterenol-treated samples decreased lipolysis in absence and presence of fviia compared with samples only treated with isoproterenol (p < 0.001) and samples treated with fviia þ isoproterenol (p < 0.01), respectively. all experiments were performed in duplicate. n ¼ 4. error bars represent sem. ��� ¼ p � 0.001 compared to corresponding control group; ## ¼ p � 0.01 compared to corresponding isoproterenoltreated group; ### ¼ p � 0.001 compared to corresponding isoproterenoltreated group. figure 4. lipolysis in 3t3-l1 adipocytes. (a) 3t3-l1 adipocytes were dreamed and incubated for 72 h. after 72 h cells were pre-stimulated with fviia (10 nm, 30 min) or left untreated. lipolysis was stimulated using isoproterenol (5 nm, 3 h), or cells were left untreated. glycerol released to the media was measured by colorimetric absorbance read at 570 nm with abcam colorimetric method and assessed as index of lipolysis. fviia stimulation does not alter basal or isoproterenolstimulated lipolysis. isoproterenol stimulation increases lipolysis in absence and presence of fviia compared with untreated and fviia-treated cells, respectively (p < 0.001), n ¼ 4. error bars represent sem. (b,c) 3t3-l1 adipocytes were dreamed and cells treated with tf sirna or scrambled sirna as control for 72 h. after 72 h cells were pre-stimulated for 30 min with 25 lm par1 or par2 agonist or left untreated before lipolysis assay was performed as in a. b and c represent data from the same experimental sets and are directly comparable. (b) tf down-regulation does not alter basal nor isoproterenol-stimulated lipolysis. isoproterenol stimulation increased lipolysis in cells treated with scr sirna and tf sirna compared with respective cells with no isoproterenol stimulation (p < 0.01). (c) neither par1 nor par2 agonists alter basal nor isoproterenol-stimulated lipolysis (compared with respective cells without par agonist stimulation in b), and there is no difference between tf-depleted cells and cells with intact tf. isoproterenol stimulation increases lipolysis rate in par1-treated (p < 0.01 for tf sirna-treated and p < 0.05 for scrambled sirna-treated) and par2-treated (p < 0.01) cells compared with respective cells with no isoproterenol stimulation. n ¼ 4. error bars represent sem. � ¼ p � 0.05 compared to corresponding control group; �� ¼ p � 0.01 compared to corresponding control group; ��� ¼ p � 0.001 compared to corresponding control group. upsala journal of medical sciences 163 glucose uptake in human primary adipocytes is not affected by fviia stimulation lastly, we investigated the effect of tf/fviia on glucose uptake in human primary adipocytes. isolated cells were left untreated or pre-treated with fviia and incubated with or without insulin (1000 mu/ml). insulin stimulation increased glucose uptake (p < 0.01), but pre-treatment with fviia did not alter basal or insulin-stimulated glucose uptake (figure 7). discussion in the present study we showed that both isolated human primary adipocytes and 3t3-l1 adipocytes express tf and are procoagulant. we also showed that isolated human primary adipocytes express fvii. however, tf/fviia signaling pathways are not involved in the lipolysis or glucose uptake of these cells. previous studies have demonstrated that human adipose tissue and murine adipocytes express tf (15,22,34), but to the best of our knowledge this is the first study providing evidence that human primary adipocytes themselves have high expression of tf. the most well-known function of tf/ fviia is the initiation of coagulation (35). both human primary adipocytes from the subcutaneous adipose depots and 3t3-l1 adipocytes clearly proved able to form fxa in response to fviia stimulation. it has been suggested that the anatomical location of adipose tissue predisposes to different types of thrombotic event after injury (36). subcutaneous adipose tissue (sat) is associated with venous thrombosis figure 5. lipolysis in 3t3-l1 adipocytes after cytokine treatment. cells were dreamed and left overnight. on the following day, cells were either left untreated, stimulated with fviia (10 nm, 48 h), treated with a cytokine mix (tnf-alfa 10 ng/ml, il-1-beta 5 ng/ml, and ifn-gamma 5 ng/ml, 48 h), or pretreated with fviia (30 min, 10 nm) before addition of the cytokine mix. lipolysis assay was performed as described in methods. fviia does not affect glycerol release on basal levels or in combination with cytokines. a 48 h treatment with cytokines (in absence or presence of fviia) results in glycerol release from 3t3l1 adipocytes to the same extent as isoproterenol stimulation (5 nm, 3 h) in contrast to untreated cells (p < 0.001). n ¼ 4. error bars represent sem. ��� ¼ p � 0.001 compared to corresponding control group. figure 6. hsl signaling in 3t3-l1 adipocytes in response to isoproterenol and fviia. 3t3-l1 adipocytes were dreamed and left overnight. on the following day, cells were washed and kept in lipolysis assay buffer provided in the abcam lipolysis kit. cells were stimulated with 1 nm insulin for 10 min before addition of 5 nm isoproterenol for 3 h, one group only stimulated with isoproterenol and two groups stimulated with 10 nm of mfviia for 3 h or for 15 min. one group was left untreated and used as control. isoproterenol increased phosphorylation of hsl compared to untreated control cells (p < 0.01), and insulin counteracts the effect (p < 0.05 compared with isoproterenol-treated cells). fviia stimulation does not change phosphorylation of hsl compared to untreated control cells in any of the tested settings. n ¼ 6. error bars represent sem. �� ¼ p � 0.01 compared to untreated; # ¼ p � 0.05 compared to isoproterenoltreated group. figure 7. glucose up-take in human primary adipocytes. adipocytes were isolated from healthy volunteers and kept in a 10% lipocrit suspension in krh media (4% bsa, 150 nm adenosine with ph 7.4). cells were stimulated with fviia (10 nm, 30 min) or left untreated for control. glucose uptake was evaluated on basal level and in response to insulin (1000 mu/ml, 15 min). d-[u-14c] glucose was added and incubated 45 min. reaction was stopped on ice and adipocytes separated by centrifugation through silicone oil. glucose uptake was evaluated by measuring the adipocyte-associated radioactivity in a beta-counter. graph represents counts per minute as percentage of untreated. fviia did not alter glucose uptake on basal level or in response to insulin. insulin increased glucose uptake in absence (p < 0.01) and presence of fviia (p < 0.05) compared with untreated and fviia-treated cells, respectively. n ¼ 4. error bars represent sem. all experiments performed in duplicate. �� ¼ p � 0.01 compared to untreated; � ¼ p � 0.05 compared to fviia-stimulated group. 164 d. ed�en et al. and shows stronger relationship with functional measurements of hypercoagulability after injury as compared to visceral adipose tissue (vat) (36). many studies have pointed to the association between adipose tissue and hypercoagulability (37, and references therein), and the present study furthers this association by providing evidence for human as well as murine adipocytes to initiate coagulation activity. in a study comparing proteomics in vat from pre-obese non-diabetics with pre-obese t2d patients it was found that at-iii was increased in t2d subjects (38). in our study we used primary adipocytes from the subcutaneous depots of healthy volunteers and found mrna expression of both at-iii and tfpi in these adipocytes. even though sat and vat have distinct characteristics it cannot be ruled out that t2d would increase the at-iii expression in sat as well. most likely, the presence of at-iii and tfpi in sat suppresses the coagulation when initiated, and this may explain why there are not even more clinical events in the form of thrombosis. when we performed the coagulation assay in human primary adipocytes without adding fviia we still recorded fxa formation at almost the same rate as when fviia was added. addition of active site-inhibited fviia (fviiai) to isolated human primary adipocytes significantly reduced the generation of fxa. fviiai is a derivative of fviia where the catalytic site is rendered inert (39). fviiai retains its ability to bind tf, and a conformational change in the protein gives it an increased affinity and prolonged dissociation half-life as compared to fviia. in contrast to tf/fviia, the tf/fviiai complex does not induce coagulation activity. we propose that the dampening effects by fviiai on the procoagulant activity, induced in the absence of recombinant fviia, is explained by our finding that human primary adipocytes have their own expression of fvii. the predominant source of fvii under normal physiologic conditions is hepatocytes, but they are not the only known fvii-producing cells. during inflammation and atherosclerosis it has been found that monocytes/macrophages express fvii (40–42). ectopic expression of fvii has also been found to be frequent in various cancers, where the signaling promotes cell migration and invasion activities (26). it has previously been reported that 3t3-l1 adipocytes and murine adipose tissue express fvii (30), but we now provide evidence for fvii expression in isolated human primary adipocytes as well. the coagulation process in vivo is readily initiated at picomolar levels of fviia, and only few tf molecules are needed on the cell surface for this step (43,44). about 90% tf protein down-regulation efficiency in the 3t3-l1 cells resulted in reduction of the coagulation activity by 45%. the discrepancy between tf down-regulation and the remaining tf coagulation activity is best explained by the described potency of the coagulation system. lipolysis is typically initiated by activation of beta-adrenergic receptors on the surface of the adipocyte. this indirectly leads to increased production of cyclic adenosine monophosphate (camp) and protein kinase a (pka) and, further downstream, to activation of lipases (33). one of these lipases is hormone-sensitive lipase (hsl) that needs to be phosphorylated for lipolysis to occur. insulin binding to the insulin receptor results in downstream activation of pi3k/akt that inhibits pka activity, resulting in decreased lipolysis (33). tf/fviia signaling is able to activate both pi3k/akt signaling, and activation of par2 can cause pka activation (45,46). using quantitative real time pcr (qrt-pcr), we found human primary adipocytes to express par2 (supplementary figure 1a, available online). we therefore speculated that tf/fviia signaling could affect either the proor the anti-lipolytic pathway. however, we failed to see any effect of tf/fviia signaling on lipolysis in any of the experiments performed. the endogenous fvii produced by the human primary adipocytes was enough to stimulate coagulation activity to almost the same level as when the cells were treated with 10 nm fviia. however, coagulation is initiated at picomolar concentrations of fviia, whereas tf/fviia signaling has been shown to be dose-dependent up to 50 nm concentration of fviia (43,44,47). it is therefore unlikely that the fvii produced by the adipocytes would be enough to stimulate signaling to such an extent that it would mask any effects of the fviia stimulation in our lipolysis experiments. if the lack of effect on lipolysis by fviia stimulation did depend on a saturation of fviia, there should have been an effect on lipolysis in the experiments with tf knock-down. addition of the cytokine mix for 48 h increased the glycerol release to the same extent as 3 h of isoproterenol stimulation, but fviia had no additional effect. no increase in hsl phosphorylation due to the cytokine treatment was recorded by western blot analyses performed on lysates from these experiments, but we did notice an increase in cleaved caspase 3 in the cytokine-treated group (supplementary figure 2, available online). the western blot data suggest that the increased glycerol release observed in the cytokine-treated groups might be caused by apoptosis rather than lipolysis. however, it has previously been reported that il-1beta and tnf-alfa can induce lipolysis and that tnf-alfa in adipose tissue contributes to insulin resistance, which indirectly further increases lipolysis (reviewed in [32]). in perspective of these results, it is possible that the long-time stimulation in our experiments leads to a miss of the phosphorylation of hsl or that lipolysis in this setting occurs via some other signaling pathway. regardless of the reason for the increased glycerol release in the cytokinestimulated groups, fviia addition had no effect. we have previously shown that tf/fviia signaling affects glucose-stimulated insulin secretion in human pancreatic islets, and thus there is a connection between tf/fviia and glucose signaling (20). in the present study, we investigated whether fviia stimulation would affect the uptake of glucose in human primary adipocytes. however, tf/fviia had no effect on glucose uptake in the adipocytes. together with the lipolysis results, this does not support a direct interaction between coagulation activity and insulin action in adipose cells. in conclusion, we have demonstrated that human primary adipocytes and 3t3-l1 adipocytes express active tf, human primary adipocytes express fvii, and, moreover, we found that the tf/fviia complex formed on the adipocyte surface can activate the macromolecular substrate fx. in comparison, upsala journal of medical sciences 165 https://doi.org/10.1080/03009734.2019.1645248 https://doi.org/10.1080/03009734.2019.1645248 https://doi.org/10.1080/03009734.2019.1645248 tf/fviiai formed on human adipocytes and depletion of tf in 3t3-l1 adipocytes significantly decreases fxa formation. quite in contrast, tf/fviia did not exert biologically relevant effects on lipolysis or glucose uptake, nor were there any effects of fviia when added in combination with cytokines. whether the tf/fviia complex affects signaling pathways in adipocytes leading to biological activity beyond initiation of coagulation remains to be further investigated. acknowledgements maria j. pereira is acknowledged for her laboratory expertise and advice. assel sarsenbayeva is acknowledged for assisting with isolation of biopsies. disclosure statement no potential conflict of interest was reported by the authors. funding this study was supported by grants from stiftelsen olle engkvist byggm€astare, hj€artoch lungfonden, vetenskapsrådet, and erik, karin och g€osta selanders stiftelse. notes on contributors desir�ee ed�en, phd student at the department of medical sciences, clinical chemistry, uppsala university. grigorios panagiotou was a research physician at the department of medical sciences, clinical diabetes and metabolism, uppsala university, during this project and now works as a physician in newcastle, uk. dariush mokhtari was a researcher at the department of medical sciences, clinical chemistry and later clinical diabetes and metabolism, uppsala university. he is currently positioned at the swedish medical product agency. jan w. eriksson, professor at the department of medical sciences, clinical diabetes and metabolism, uppsala university. mikael åberg, researcher at the department of medical sciences, clinical chemistry, uppsala university and head of scilifelab clinical biomarkers facility. agneta siegbahn, senior professor of clinical coagulation science at the department of medical sciences, clinical chemistry, uppsala university. references 1. seidell jc, halberstadt j. the global burden of obesity and the challenges of prevention. ann nutr metab. 2015;66(suppl 2):7–12. 2. kahn se, hull rl, utzschneider km. 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al. neutrophil elastase activates protease-activated receptor-2 (par2) and transient receptor potential vanilloid 4 (trpv4) to cause inflammation and pain. j biol chem. 2015;290:13875–87. 47. pendurthi ur, allen ke, ezban m, rao lv. factor viia and thrombin induce the expression of cyr61 and connective tissue growth factor, extracellular matrix signaling proteins that could act as possible downstream mediators in factor viia x tissue factor-induced signal transduction. j biol chem. 2000;275:14632–41. upsala journal of medical sciences 167 abstract introduction materials and methods reagents human adipocyte isolation 3t3-l1 cell culture, differentiation, and dream mda-mb-231, hcc1937, and katoiii cell culture sirna transfection in 3t3-l1 adipocytes mrna analysis western blot coagulation activity assay lipolysis in human primary adipocyte lipolysis in 3t3-l1 adipocytes signaling glucose uptake in human primary adipocytes statistics results tf and fvii are expressed in human adipocytes, and tf can be effectively down-regulated in 3t3-l1 adipocytes adipocytes are procoagulant in a tf/fviia-dependent manner in both human primary adipocytes and 3t3-l1 adipocytes lipolysis in human primary adipocytes is not affected by fviia stimulation lipolysis in 3t3-l1 adipocytes is not affected by fviia stimulation, tf down-regulation, and par1 or par2 agonists long-term cytokine treatment results in lipolysis, but combination with fviia stimulation does not alter the lipolysis rate hormone-sensitive lipase (hsl) is phosphorylated by isoproterenol but unaffected by fviia stimulation glucose uptake in human primary adipocytes is not affected by fviia stimulation discussion acknowledgements disclosure statement references in vitro fertilisation in domestic mammals—a brief overview review article in vitro fertilisation in domestic mammals—a brief overview ylva sjunnesson department of clinical sciences, reproduction, the centre for reproductive biology in uppsala (cru), swedish university of agricultural sciences (slu), uppsala, sweden abstract many factors influence the final oocyte maturation, fertilisation, and early embryo development, and there are both similarities and differences between species. when comparing the advancement of assisted reproductive technologies (arts), the development in the bovine species is not far behind the medical front, with around one million in vitro-produced bovine embryos each year. this rate of progress is not seen in the other domestic species. this review aims to give an overview of the development and specific difficulties of in vitro embryo production in various domestic animal species, with the main focus on cows, pigs, and cats. in production animals, the aim of arts is commonly to increase the genetic progress, not to treat reproductive failure. the arts are also used for preservation of genetic diversity for the future. however, specifically for oocyte maturation, fertilisation, and early embryonic development, domestic mammals such as the cow and pig can be used as models for humans. this is particularly attractive from an animal welfare point of view since bovine and porcine oocytes are available in large numbers from discarded slaughterhouse material, thereby decreasing the need for research animals. both for researchers on the animal and human medical fronts, we aim for the development of in vitro production systems that will produce embryos and offspring that are no different from those conceived and developed in vivo. species-comparative research and development can provide us with crucial knowledge to achieve this aim and hopefully help us avoid unnecessary problems in the future. article history received 11 october 2019 revised 11 november 2019 accepted 21 november 2019 keywords embryo; in vitro maturation; in vitro production; oocyte; species comparison introduction it is easy to assume that in the beginning of our embryonic development we animals are all very much alike. in some aspects this is true, but there are major differences between species, and even between mammals. in many ways, cows and humans are similar in the advancement of assisted reproductive technologies (arts) and in vitro fertilisation (ivf), but many species still lag far behind. this review attempts to give an overview of the development and specific difficulties of in vitro embryo production, including ivf, in various domestic animal species, with the main focus on cows (bovines), pigs (porcines), and cats (felines). commonly in production animals, the focus is not to overcome reproductive failure but to enhance the genetic progress, both by selection of the most genetically valuable individuals and by increased number of offspring from these individuals. with species-comparative knowledge, our possibilities to understand the mechanisms behind various aspects of the final oocyte maturation, fertilisation, and early embryonic development in vitro will increase. ivf-associated arts such as artificial insemination, which is used successfully and to a very large extent in developed countries, sperm capacitation, intracytoplasmic sperm injection (icsi), embryo transfer (et), and cloning fall outside the scope of this review and are only briefly mentioned. an overview of procedures the procedures of in vitro oocyte maturation (ivm), in vitro fertilisation (ivf), and the first days of in vitro embryo culture (ivc) are collectively termed in vitro embryo production (ivp). in common domestic animals we usually retrieve immature oocytes from the ovaries, either after normal castration where ovaries are removed, in ovum pick-up (opu) procedures in live animals, or after the death of the animal (due to slaughter, euthanasia, or after fatal trauma). in experimental settings or within some commercially ivp companies, in vivo maturation procedures may occur. since animal ovaries are not considered fit for human consumption in most countries, they are a waste product in the meat industry and can easily be retrieved fresh in large quantities from consenting abattoirs. routine castrations or neutering/spaying of pets are performed on the explicit wishes of the owners and according to recommendations of veterinarians due to various reasons that can include prevention of unwanted offspring as well as changes in temperament or behaviour contact ylva sjunnesson ylva.sjunnesson@slu.se department of clinical sciences, reproduction, the centre for reproductive biology in uppsala (cru), swedish university of agricultural sciences (slu), po box 7054, se-750 07 uppsala, sweden � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 2, 68–76 https://doi.org/10.1080/03009734.2019.1697911 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1697911&domain=pdf&date_stamp=2020-05-21 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2019.1697911 http://www.tandfonline.com connected to hormonal actions in an intact animal (1). normal routine castrations are performed daily in most veterinary clinics, and also here the ovaries are discarded as waste and can be collected for ivp purposes (2). this relatively easy access to large quantities of ovaries without having to use research animals makes species like the cow and pig perfect candidates for ivp research, spanning from testing of in vitro media and methodological improvements to controlled trials of newly emerging risk substances like environmental pollutants found in humans and our environment (3,4). however, with the use of these discarded ovaries there are no possibilities to control cyclicity or follicular growth that would be possible in research animals. due to the presence of continuous follicular waves in most domestic animals (5), the ovaries will always contain cumulus oocyte complexes (cocs). therefore, the approach is usually to retrieve relatively immature cocs and perform the final maturation in vitro. the selection process starts with aspiration of follicles of a certain size range depending on species, commonly by hand with needles and syringes. the follicles aspirated should not be too small, since they would contain cocs that are too immature and difficult to mature to mii stage (6,7). on the other hand, the follicles should not be too large since they could contain cocs that are too mature, with already-expanded cumulus cells or atretic oocytes, that would make them unsuitable to include in the process (figure 1). after the aspiration or opu all cocs are collected, and only cocs of good quality are selected for the ivm process (figure 1). the ideal bovine coc is light and transparent, and has a compact multilayered cumulus investment and a homogeneous ooplasm (8). in both pigs and cats the cocs are darker than in the bovine, but otherwise the same criteria are used. during ivm, media are supplemented with lh and fsh, and in some cases oestradiol, growth hormones, and insulin. after ivm the medium is changed to promote sperm capacitation and fertilisation. the duration of fertilisation ranges between a few hours and up to one day, depending on species, and is followed by ivc where the medium is promoting the early embryo development. embryos are commonly evaluated and classified according to the international embryo technology society (iets) guidelines (9). a common time point for transfer back to a recipient animal is in the stages of morula or early blastocyst, but later blastocyst stages as well as earlier embryos could also be transferred (figure 2). in order to avoid the risk of transferring diseases, embryo transfer teams (mainly working with bovines) must also follow the recommendations of the iets (10). history of ivp in domestic mammals already in the 1950s, live births after ivf were demonstrated in rabbits and, one decade later, in mice (table 1). in the beginning, successful ivp systems were usually in vivo-based co-culture systems where maturation/fertilisation and/or figure 1. bovine oocytes. (a) immature oocytes of acceptable quality for ivp. (b) immature oocytes of unacceptable quality for ivp (b1: partially nude oocyte; b2: completely nude oocyte with pale cytoplasm; b3: over-expanded oocyte). (c) oocytes matured in vitro where the majority have good expansion of cumulus cells. upsala journal of medical sciences 69 culture took place in oviducts from rabbit or sheep and later on after co-culture with oviductal cells. gradually, supporting cells were excluded as formulas for the media improved. chemically defined media have given valuable insights to our understanding of the impact of various substances on oocyte maturation, fertilisation, and early embryo development and the possibility to avoid unknown macromolecules. moreover, potential contaminants from e.g. serum in culture conditions are of great importance (33). arts have over the years developed from invasive surgical procedures to ultrasound-guided techniques in many species. ivm is still a major hurdle to overcome in most species, and another difficulty has been the capacitation process. different species, as well as male individuals within the same species (34), have differences in substances and concentrations of these that can induce capacitation. heparin, serum albumin, epinephrine, penicillamine, hypotaurine, coffein, bicarbonate, and calcium are some of the substances that can induce capacitation (35,36). it is quite obvious that there is a large step from maturing and fertilising oocytes in vitro to actually being able to produce live offspring after both procedures since this seems to take over 20years in most species (table 1). it is worthy of note that, even though cases of successful ivf with live offspring have been reported in dog and horse, such reports have been few and have proven difficult to repeat. ivp in cattle today the cattle ivp industry is expanding at a high rate and does not show any signs of slowing down. according to the figure 2. in vitro-produced bovine embryos day 8 after fertilisation. (a) expanded blastocysts. (b) mix of blastocysts, embryos in earlier developmental stages, and non-fertilised oocytes (1: blastocysts; 2: expanded blastocysts; 3: hatching blastocyst, 4: hatched blastocyst). (c) hatched blastocysts of good quality. the arrow indicates an empty zona pellucida after hatching. table 1. examples of years of early attempts of ivm, ivf, and ivc in selected species. species ivm (ref.) ivf (ref.) birth of live offspring after ivf (ref.) birth of live offspring after ivm and ivf (and in some cases ivc) (ref.) cattle 1965 (11) 1977 (12) 1982 (13) 1990 (14) pig 1965 (11) 1975 (15) 1986 (16) 1989 (17) sheep 1965 (11) 1959 (18) 1986 (16) 1986 (16) rabbit 1955 (19) 1954 (20) 1959 (21) 1983 (22) mouse 1965 (11) 1968 (23) 1970 (24) 1970 (24) cat 1989 (25) 1989 (25) 1988 (26) 1997 (27) dog 1976a (28) 1976a (28) 2015a (29) horse 1981a (30) 1989a (31) 1991a (32) alimited success. 70 y. sjunnesson international embryo technology society (iets) almost one million transferable in vitro-produced cattle embryos were produced all over the world in 2017 (table 2). unfortunately, not all countries contribute to this report, and these numbers are therefore under-estimated. there has been a remarkable increase since 2016 when only about half a million bovine embryos were produced. most of the transfers were carried out in brazil, closely followed by the usa. in europe, there were some 50,000 transfers of ivp embryos carried out, and most of them were done in russia followed by the netherlands, spain, germany, and france. the total number of embryo transfers (both of ivp and in vivo-derived embryos) all over the world was over 1.5 million in 2017 (37). bovine ivp systems are working very well, but there is always room for improvement. approximately 90% of the aspirated oocytes reach mii stage, and some 80% of these become fertilised in vitro. after culture in vitro around 50% of the fertilised oocytes become blastocysts (38). the success story of the cattle ivp could have been very different due to the appearance of the large offspring syndrome (los) in the late 1980s and the following decade. after transfer of ivp embryos, alarming reports started to emerge, beginning in the farming press, where some of the calves born had malformations and were too large, which led to difficult calvings (33,39). naturally, this became a serious problem for animal welfare, and researchers searched for the background mechanism and a solution. even at present, the full mechanisms are unknown, but the addition of serum to the culture medium has been identified as a likely factor behind the los in both sheep and cows (40). today bovine serum albumin and amino acids are used during culture, instead of serum, with better results. it seems likely that the effects of the serum were through epigenetic changes (41). one of the lessons to be learned from the los legacy is that seemingly minor changes in medium composition may have a pronounced impact on the offspring. opu has been modified for successful use in cattle. the technique in cattle is supported by the possibility of the operator to move an ovary to the right position via a gloved hand in the rectum of the cow. it is the major technique to retrieve oocytes for ivp and et (37). in the swedish university of agricultural sciences (slu), uppsala, extensive studies have been carried out to investigate the animal welfare effects of repeated opu sessions on heifers (42), the only negative experience seemingly being the routine administration of the epidural anaesthesia needed. even though many techniques used in humans have worked very well in cattle, the icsi procedure is still far from successful, possibly because no spontaneous activation of the oocyte occurs after injection of a spermatozoon (43). however, it could also be attributed to other factors such as the darkness of the ooplasm, the large sperm heads, and the toughness of the oolemma. this is in contrast to other forms of micromanipulation that seem to work nicely in cattle. micromanipulation of bovine embryos for sexing and/or genotyping is becoming more and more common, with around 10,000 embryos tested in 2017 (37). the number is likely to have increased since 2017 when more relevant genetic information for selection of the best possible traits in cattle rapidly became available. this possibility to make fast genetic progress also increases the use of ivp in cattle. ivp in pigs the production of pig embryos in vitro still has many problems to overcome before it can be said to be efficient. one of the main problems is inherent to ivf itself, which often results in polyspermic fertilisation. when the sperm concentration is decreased, there is just a minor effect until the concentrations are so low that there will be no penetration of spermatozoa at all. a failure of the pig oocyte maturation in vitro is probably connected to the polyspermic situation (44), but other factors such as an altered zona reaction (45), and even temperature during ivf (46), have been proposed. the pig ivp has a two-day maturation process, and it is likely that there are many conditions having an impact on oocyte maturation that we are not aware of as yet. ivm in pigs gives relatively high rates of oocytes matured to mii stage, between 75% and 85%, but unfortunately the polyspermic rates can reach 50–70% (47). this was observed in the first experiment using single-layer colloid centrifugation before pig ivf was performed in the ivp unit at slu, uppsala (48). to overcome the extreme fertilising efficiency of the colloid centrifugation-selected spermatozoa, the concentrations of spermatozoa for fertilisation had to be lowered from the standard 5 � 105 spermatozoa/ml to 4 � 103 spermatozoa/ ml. since polyspermic porcine embryos can cleave and develop to the blastocyst stage, as normal embryos (49), polyspermia has to be assessed by investigation of pronuclei. table 2. summary of ivf in common domestic mammals. species main purpose of ivf approximated number of in vitro-produced embryos in 2017 examples of specific characteristics bovines genetic progress 1,000,000a most techniques used in humans work well except icsi porcines research and genetic modifications limited number and not possible to estimate prone to polyspermia in vitro, and gametes are highly sensitive to low temperatures felines model for endangered feline species very limited number and not possible to estimate induced ovulators equine increasing reproductive potential among selected individuals 500a ivf is not successful, but icsi and cloning are performed canine research extremely limited number and not possible to estimate oocytes are in mi stage at ovulation and difficult to mature in vitro ovine genetic progress 100a surgical ovum pick-up and transfer are needed areference (37). upsala journal of medical sciences 71 this has to be done via staining or high-g centrifugation in most domestic animals since the ooplasm is too dark to visualise the pronuclei in light microscopy. if the oocytes are monospermic, around 80% can reach the blastocyst stage (47). recent research within this field is mostly focussed on selecting spermatozoa in various ways to avoid polyspermy (50). icsi is used with some success in pigs (51), thereby bypassing the polyspermy problem entirely. apart from the polyspermy problem, pig oocytes and embryos are very sensitive to low temperatures. if temperature drops below 25 �c the oocyte maturation process permanently stops in most cases (52). with pig embryos, it is not possible to store them at a low temperature before transfer since they are all irreversibly damaged if temperature drops below 10 �c (53). furthermore, porcine oocytes and embryos, compared with other mammalian species, are very sensitive to cryopreservation, possibly due to the high amount of lipids present (54). the advantages of an efficient pig ivp are many. the genetical and technical progress could of course be significantly secured and enhanced, but the pig also has a high value as a model for human situations. despite many difficulties, ivp in pigs is continuously explored. research in uppsala led to the first production of piglets after fertilisation and culture in chemically defined media where the effects of various components during fertilisation were assessed (55). ivp in cats no doubt, the domestic cat population as a whole has no problem to reproduce. however, the domestic cat is an excellent model for other feline species, and almost all large cats are threatened by extinction at least to some extent. only a few domestic kittens have been born after ivf (26,27,56). when a genetically important animal unexpectedly dies, gametes can be collected and either directly subjected to ivp or frozen for future use. for cats, between 40% and 80% of the cocs mature to mii phase, and the proportion of cleaved embryos developing in vitro can reach 70% (56) but is usually lower. in slu uppsala, we have used the domestic cat as a model to increase the number of spermatozoa that can be salvaged from the testicles after removal. the standard procedure is to collect only spermatozoa from the cauda epididymis since this is where the most mature spermatozoa can be found (57). using an ivp system, we found that spermatozoa from the corpus epididymis had fertilising capacity and blastocysts were produced, even if it was at a lower rate (2). with this feline model we saw around 8% parthogenetically activated oocytes, but none continued to the blastocyst stage (unpublished data). since the parthogenic embryos can be similar to normal embryos, they cannot be separated by simple microscopy. ivp in horses, dogs, and sheep even though this short review cannot cover all domestic mammals where ivf has been performed or attempted, some common species have to be mentioned. ivf methodology in the horse has so far never been well established, but icsi and cloning have been done with good results (58). the reason for the failed ivf in the horse has been extensively discussed by leemans et al. (59) and is believed to be due to the failure to capacitate stallion spermatozoa. the interest for ivf and related technologies for the horse is increasing within certain sports and breeds where money is available and reproductive biotechnologies are allowed. unfortunately, mares seem to be poor responders to superovulation (60), and only one or few oocytes can commonly be retrieved per cycle; this, together with the lower access to slaughterhouse ovaries, makes progress in this field slow. the total number of in vitro-derived embryos transferred in the horse is currently low (table 2). however, the numbers can be expected to rise in the coming years as icsi, and possibly also cloning, become more established. dogs pose a special case since their oocytes are ovulated in the mi stage and several days are required for them to mature to the mii stage in the oviduct. in vitro maturation has never been truly established, but after in vivo maturation and in vivo culture the first—and, so far, only—puppies were born after ivf in 2015 (29). the main hurdles for dog ivp are the maturation process and the fact that dogs are monooestroual breeders, which means that the oestrus is followed by a long pause, resulting in a bitch normally only having one to three oestrous periods during one year. in vitro production of sheep embryos is well established, but success rates are lower than for bovines (61). many factors regarding sheep ivp correlate well to cows as they are both ruminants. unfortunately, surgical opu and transfer are still necessary in sheep due to their anatomy, and this makes the procedure far more invasive than in bovines. the commercial activity of ovine in vitro-derived embryo transfer is very low (table 2). animals as models for humans in many cases animals can be used as models for humans with regard to the final oocyte maturation, fertilisation, and early embryo development in vitro. depending on which factors are to be investigated, different species might be of interest. thus, rodents are not always the most suitable models for humans, especially when considering oocyte maturation and fertilisation (62). one example is the oocyte transcriptome where human oocytes are more closely related to cow oocytes than to mouse oocytes (63). there are also many other specific characteristics making the human, the cow, and the pig much more similar compared with rodents, for example the timing of the early embryo development in vitro (4). in the ivp unit for pigs in slu uppsala, the impact of stress was investigated by adding plasma from stressed or non-stressed sows to the ivp media (64,65). plasma was collected from the corresponding time points around ovulation in vivo, and stress was simulated by adrenocorticotropic hormone injections. in these studies, stress seemed to have a major impact on spermatozoa function at the time of 72 y. sjunnesson fertilisation, possibly by prematurely induced acrosome reactions (65). in the slu uppsala ivp unit for cows, several studies have been conducted with focus on the impact of insulin during ivm (66). these studies were mainly focussed on the metabolic imbalance seen in dairy cattle after calving but are also of comparative interest to metabolic disorders in humans. we observed a negative impact of insulin in ivm on blastocyst development, evidenced by an effect on the gene expression patterns as well as morphologically. as a final example, los should be mentioned. since los can be induced by the addition of serum to culture medium, it has been proposed as a model for beckwith–wiedemann syndrome in human (67). so, hopefully this major setback can be made useful in future research. future development with the arrival of new and technically relatively simple tools for gene edition—crispr-cas9 and talen systems (for reviews, see 68,69)—there is an increased interest in genetic modifications of animals aimed for both production and research. given the new possibilities for genetic modification and the physiological similarities between humans and pigs, the pig is regarded as an excellent model for human diseases, studies of gene functions, as well as pharmacological studies (68,69). potentially, the genetically modified pig could also be a donor of tissues and organs for xenotransplantation (47). as regards bovines, genomic analyses have become a tool for selection of the most valuable individuals, already at embryo stages. this is one of the reasons for the massive increase in ivp within bovine production industry last years. the combination of embryo biopsies for genomic selection and cryopreservation of embryos has led to a very efficient procedure to improve genomic merit rapidly (70), a process that previously took several years. the genome-editing technologies mentioned above have also been adapted to cattle with good results. these technologies might help us understand and improve genetic traits in cattle as well as producing healthy cows that could provide us with bio-medical proteins in their milk (71). as with the pig, the cow oocyte maturation and early embryo development in vitro are valuable and sensitive models for humans, e.g. for investigations of the reproductive impact of environmental pollutants (3). confocal microscopy is a method explored in the ivp facilities at slu, uppsala, and used for basic research and morphological characterisation of oocytes and embryos. using fluorescent staining, we can easily assess various structures (figure 3) and relate this phenotypic information to e.g. gene expression. as cows are increasingly kept in warm parts of the world, we have to expect to see impaired reproductive performance in production animals not suited to hot environments. to perform ivp and transfer morula or blastocyst stages could be a future way to bypass heat stress, since the bovine embryo goes from being very sensitive to heat stress up to the four-cell stage, to largely resistant by the morula stage (72). in many species, ivp media consisting of completely defined reagents or semi-defined reagents (usually including serum albumin) have been developed. this is attractive from a research perspective where all components can be accounted for and their functions assessed (55) but also from a biohazard point of view (33). frozen in vitro-produced embryos are now transported all over the world. this is very positive from an animal welfare point of view, since it minimises the need for transportation of live animals to enhance the genetic progress, thereby increasing our possibilities to feed the growing human population. however, to ensure that this process does not contribute to the spreading of diseases, biosecurity must never be at risk. the ivp systems must be enhanced and developed for more species for us to be able to preserve genetic diversity. when developing new methods, a better follow-up of the impact on the offspring is also needed to avoid situations like los. animal welfare should always be assessed when figure 3. three-dimensional images of embryos produced by confocal microscopy. (a) an in vitro-produced hatching bovine blastocyst 8 days after fertilisation. visualised are nuclei (red) and neutral lipid droplets (green). (b) an in vitro-produced expanded porcine blastocyst 7 days after fertilisation. visualised are f-actin (green) and active mitochondria (orange). upsala journal of medical sciences 73 introducing new technologies, including changes in medium compositions. the ultimate goal is the development of ivp systems that will produce embryos and offspring that are no different from those conceived and developed in vivo. we still have far to go, but comparative research and development across species in both animal and human embryology can provide us with the crucial knowledge needed. we can learn a lot from such comparative studies and thereby hopefully avoid unnecessary problems in the future. acknowledgements the author would like to thank previous and present co-workers and collaborators and would also like to apologise to all authors not cited due to space restrictions. the author would also like to specifically acknowledge the centre for reproductive biology in uppsala (cru), which has been the origin for the collaboration between uppsala university and the swedish university of agricultural sciences within the field of human and animal ivf. disclosure statement the author reports no conflicts of interest. funding funding for the work cited where the author has been involved was received from: the swedish research council formas [swedish research council for environment, agricultural sciences and spatial planning, project numbers 221–2007-374, 221–2010-549, and 942–2015-00476], the swedish research council [vr, project number 2017–02116], carl tryggers stiftelse [cts 17:413], and stiftelsen nils lagerl€ofs fond [ksla, gfs2017-0032]. the platform for developmental biology slu financed equipment used to provide the images in this article. notes on contributor ylva sjunnesson, dvm, phd, associate professor, director of the centre for reproductive biology in uppsala (cru). references 1. wongsaengchan c, mckeegan d. the views of the uk public towards routine neutering of dogs 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in pigs. j anim sci biotechnol. 2018;9:5. 70. sirard ma. 40 years of bovine ivf in the new genomic selection context. reproduction. 2018;156:r1–7. doi:10.1530/rep-180008 71. yum sy, youn ky, choi wj, jang g. development of genome engineering technologies in cattle: from random to specific. j anim sci biotechnol. 2018;9:16. doi:10.1186/s40104-018-0232-6 72. hansen pj. reproductive physiology of the heat-stressed dairy cow: implications for fertility and assisted reproduction. anim reprod. 2019;16:497–507. doi:10.21451/1984-3143ar2019-0053 76 y. sjunnesson https://doi.org/10.4161/epi.24655 https://doi.org/10.1126/science.1258096 https://doi.org/10.1530/rep-18-0008 https://doi.org/10.1530/rep-18-0008 https://doi.org/10.1186/s40104-018-0232-6 https://doi.org/10.21451/1984-3143-ar2019-0053 https://doi.org/10.21451/1984-3143-ar2019-0053 abstract introduction an overview of procedures history of ivp in domestic mammals ivp in cattle ivp in pigs ivp in cats ivp in horses, dogs, and sheep animals as models for humans future development acknowledgements disclosure statement references use of in vitro fertilization—ethical issues review article use of in vitro fertilization—ethical issues kjell asplund department of public health and clinical medicine, umeå university, umeå, sweden abstract this report is an ethical analysis based on both facts and values. in in vitro fertilization (ivf), there is an intricate interaction between rapid scientific development and changing societal values. in most countries, the ethical discussion is no longer on whether or not ivf in itself is ethically justifiable. therefore, in this review, i discuss other ethical aspects that have emerged since ivf was first introduced, such as upper age limits, ‘ownership’ of gametes and embryos, ivf in single women and samesex couples, preimplantatory genetic testing, social egg freezing, commercialization, public funding, and prioritization of ivf. despite secularization, since religion still plays an important role in regulation and practices of ivf in many countries, positions on ivf among the world religions are summarized. decision-making concerning ivf cannot be based only on clinical and economic considerations; these cannot be disentangled from ethical principles. many concerns regarding the costs, effects, and safety of ivf subtly transcend into more complex questions about what it means to society to bear and give birth to children. article history received 18 september 2019 revised 21 october 2019 accepted 21 october 2019 keywords age limits; ethics; in vitro fertilization; infertility; preimplantatory genetic testing; public funding; prioritization introduction ethics is about the systematic reflection on human values and actions. simplistically, the two principal components of an ethical analysis are facts and values. when facts and values change, the ethical analysis must be revised. in vitro fertilization (ivf) is an illustrative case of this dynamic process. in some aspects, the expansion of scientific information has made decision-making easier; it has left less room for opinions and speculations, and facilitated evidencebased regulations and clinical recommendations. on the other hand, rapid technological development has implied a number of new ethical challenges. since medical ethics has a history that goes back to the hippocratic principles, it would be easy to conclude that values are, or should be, unwavering. it is then thought-provoking to view ivf as an example of how radically societal values and attitudes have changed over the last four decades. given the changes in facts and values, the prerequisites for an ethical analysis of ivf are under ongoing evolution. in most countries, the ethical discussion is no longer on whether or not ivf in itself is ethically justifiable. therefore, the major focus of this article is on new ethical questions that are invoked by biotechnological development or changes in societal values. the focus of this article is on ivf practices and ethical discussions in europe and english-speaking countries. this is where most of the scientific literature on ethical issues on ivf originates, but it inevitably means that this article is ethnocentric. several examples of ethical analyses are taken from my work as chair of the swedish council on medical ethics, which advises the swedish government and parliament on medical ethics issues. i will discuss ethical aspects on upper age limits, ‘ownership’ of stored gametes and embryos, ivf in single women and same-sex couples, preimplantatory genetic diagnosis, social egg freezing, egg sharing, surrogacy, commercialization, public funding, and prioritization of ivf. finally, despite secularization, since religion still plays an important role in the regulation and practices of ivf in many countries, positions on ivf among the world religions are summarized. brief historical notes the ethical controversy over ivf had already started when the results of the first animal ivf experiments were published in the mid-1930s (the results were later contested). while some commentators saw it as a promising development to help infertile couples, others were critical, saying that the scientists were playing god (1). it is interesting to note that the animal experiments evoked eugenic questions that were in focus at the time. in a 1936 article in new york times, it was stated: ‘advocates of “race betterment” might urge such procedures for men and women of special aptitudes, physical, mental, or spiritual’ (cited by biggers (1)). in the press, there was speculation, repulsive in nature, on both surrogacy and contact kjell asplund kjellasplund1@gmail.com department of public health and clinical medicine, umeå university, reimersholmsgatan 59, 11740 stockholm, sweden � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 2, 192–199 https://doi.org/10.1080/03009734.2019.1684405 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1684405&domain=pdf&date_stamp=2020-05-21 http://orcid.org/0000-0001-6710-4152 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2019.1684405 http://www.tandfonline.com a world where men were not needed for reproduction: ‘the mythical land of the amazons would then come to life’ (1). in 1978, when the first child was born after ivf in the uk, the negative ethical comments of the 1930s were reiterated with more fervour. the protests seem to have been particularly strong when attempts were made to introduce ivf in the usa. john biggers, one of the ivf pioneers, recalled memories from a public hearing arranged by the state of virginia: ‘the entire meeting turned out to be a shouting match, the two groups [“pro-lifers” and ivf proponents] hurling insults at each other’ (1). in the 40 years since the first successful human ivf, enormous experience and scientific knowledge have accumulated. few of the fears of negative effects have been met. values have changed, partly because more facts are available, but also due to general trends in values in many populations, secularization perhaps being the most important. today, the principal resistance to ivf as such rests mainly in catholic and orthodox christian settings. the philosophical argument that being born is better than not being born has also been invoked in the ivf debate (the opposing view is abundantly present in philosophy as well as religion, for example elaborated by the philosopher david benatar in his book better never to have been: the harm of coming into existence (2)). age limits upper-middle-age women who give birth to a child after ivf are often subject to public opinion on how ‘unfitting’, ‘unnatural’, and even ‘repulsive’ this is. table 1 summarizes the principal arguments for and against a fixed upper age limit for ivf in older women. in the public debate, the age limit discussed is usually in the 40–50-year age range. thus, the arguments concern both facts and values. the likelihood of outcomes such as successful pregnancy or the death of the mother before the child is able to support itself are examples of fact-based arguments, as are the consequences of the outcomes. the most obvious value-based arguments are the right of children to be brought up under safe conditions and women’s reproductive autonomy. some arguments are based on both facts and values. are older women as good mothers as younger women? the answer may be value-based, but it may also be based on the scientific information available. there have been reports that children born to mothers above the age of 35 or 40 do not have increased physical or mental vulnerability, but a modest increase in vulnerability has also been reported at maternal ages above 35 (3). yet, at maternal ages above 35 or 40 years, child vulnerability still seems to be consistently lower than that in children born to very young mothers (3). compared to upper age limits for the mother, there has been little discussion on age limits for the father. men are, on average, older than women when they become parents and have a somewhat shorter life expectancy than women. therefore, the argument of being able to take responsibility for a child until adulthood would apply equally, if not more, to men as to women. after considering basic ethical principles and the large variations in individual prerequisites, both medically and socially, the swedish council on medical ethics has recommended that there should be no strict upper age limit for publicly funded ivf (4). instead, individual assessments should be made, taking into consideration biological rather than chronological age. the needs of the child are emphasized: at least one parent should be young enough to take responsibility for the child until adulthood. when political decisions are made, other ethical values and non-ethical factors may play a major role. in 2016, public healthcare providers in sweden agreed to apply a national upper age limit for publicly funded ivf (5). the age limit was set at 40 years in women and 56 years in men. stored embryos could be used up to a maternal age of 45. a major reason for this decision was to eliminate the then-existing regional variations in age limits. the principles of fairness, cost-effectiveness, and simplicity in decision-making seem to have overridden other ethical principles, most importantly reproductive autonomy and non-discrimination by age. in swedish private practice with out-of-pocket payments, more variable age limits are applied, usually well above 40 years. in most countries, there are no national age limits for ivf, and practices vary. in the uk, for instance, public funding is decided locally by clinical commissioning groups (ccgs), with different practices across the country. there are, however, healthcare professionals’ guidelines that recommend that women up to the age of 40 should be offered three cycles of ivf and women up to the age of 42 one cycle of ivf (6). single women and same-sex couples the major ethical questions about providing ivf or other forms of assisted reproduction for single women and same-sex table 1. four common arguments supporting and four arguments against an upper maternal age limit for ivf. supporting age limit against age limit � a child has a right to a safe childhood and adolescence. this right may be encroached upon when the mother has an age-related risk of disease and possibly death. � the woman and the foetus are at increased risk with advancing maternal age. � with advancing maternal age, the chance of successful ivf decreases; if there is public financing of ivf, this argument includes cost-effectiveness and prioritization deliberations. � if donated eggs are used, limited availability of eggs may justify that younger women with strictly ‘medical’ infertility and a higher chance of successful pregnancy are prioritized over women with age-related infertility. � reproductive autonomy: a woman should have the right to choose when she wants to have a child. � with secular trends of improved physical health in upper middle age, the prerequisites for being a healthy and active mother have improved. chronological age has therefore become less decisive when parenthood is considered. other factors, such as the presence of chronic lifethreatening disease or severe alcohol and drug abuse, are more crucial. � middle-aged women are often psychologically mature and have a stable social and economic situation; the preconditions for care of a child are usually good. � an upper age limit means formalized age discrimination. upsala journal of medical sciences 193 couples have concerned the welfare of the child. ethical arguments for and against ivf in singles and homosexuals that have been discussed are those of fairness, non-discrimination, reproductive autonomy, and children’s well-being. a systematic literature review (with methodological problems in most of the included studies) compared adolescents born through ivf for single women or women in same-sex partnerships with naturally conceived adolescents. there were no differences in psychological adjustment or parent–adolescent relationships (7). not surprisingly, legal restrictions on ivf and other forms of assisted reproduction for singles and same-sex couples vary between countries. in a survey of legislation in the 28 eu countries published in 2014, medically assisted reproduction (including ivf) for single women was permitted in 11 countries and not allowed in 11 countries, whereas the legal status was undefined in the remaining 6 countries (8). since 2014, some countries with a previous ban have modified the legislation to permit ivf for single women. it is difficult to discern a distinct pattern of countries permitting or prohibiting ivf as to traditional values, level of secularity, or extent of self-expression in the population [see world values survey (9)]. some regulatory differences exist even between countries where assisted reproduction is permitted for single women. in sweden, a child born through gamete donation has the right to know how it was conceived and who is the genetic father and mother; this is based on the principle of autonomy. some swedish women prefer to have ivf performed in denmark, where the anonymity of the sperm donor is ensured. for female same-sex couples, there is usually no specific legislation; when ivf for single women is allowed, this would also cover female same-sex couples. in a 2013 statement by its ethics committee, the american society for reproductive medicine called for programmes providing ivf and other fertility services to ‘treat all requests for assisted reproduction equally without regard to marital/partner status or sexual orientation’ (10). in most countries where surrogacy is allowed (or not prohibited by law), ivf with sperm from a homosexual father-to-be may be used for conception of the surrogate mother. who ‘owns’ stored gametes and embryos? humans have always strived for a form of immortality through our genetic offspring (11). gamete storage has now enabled us to plan prospectively, allowing for untimely demise or social reproductive choices. the aim is that our genetic ‘identity’ should continue after our death. aside from what the deceased person may have wanted, it is often a wish of the family to immortalize their loved one. for the family, use of stored gametes (or embryos) may be a relief from part of the sorrow (11). thus, when gametes are collected and stored before cancer treatment and the patient dies, should the remaining partner have a right to use them for ivf? in practice, this issue occurs when the remaining partner is a woman, but hypothetically a remaining male partner could request to use an egg from a deceased female partner to engage a surrogate mother. would a person who stores sperm, eggs, or embryos choose to reproduce after death? anyone who has decided to store them for future reproduction could perhaps be presumed to have consented. usually, however, the consent does not involve the event that the person dies. use of gametes from deceased individuals is currently prohibited by law in france, germany, sweden, and other countries, even when there is written consent from the deceased. in the uk, sperm from a deceased person can be used for ivf if there is written consent before death. in the usa, practices vary from clinic to clinic, and written consent is not always required (12). postmortem sperm retrieval (pmsr) raises significant ethical and legal concerns, including issues of implied/presumed consent and the designation of sperm as property. in many countries, the legal situation is not clear. most commentators seem to agree that a core principle is not to reproduce anyone without his or her permission (12). thus, consent should be obtained. in countries where the use of gametes from deceased individuals is prohibited (see above), any use of pmsr would require a modification of the legislation. grieving family members are not always in the position to make rational choices. therefore, some experts have recommended compulsory wait times (up to one year) before using the retrieved sperm for conception. if a couple who have decided to store embryos for future use splits up and only one of the partners wants to use them (in a hypothetical case, the male partner may want to use the embryos for surrogacy), who ‘owns’ the embryos and has the right to decide (it would be more appropriate to talk about disposal than strict ownership)? a uk case attracting much public attention may illustrate the ethical and legal complexity. embryos were stored before oophorectomy in a woman with ovarian cancer (13). when the couple split up half a year later, the man wanted the embryos to be destroyed. the clinic informed the woman that uk laws requested consent from both partners for storage to continue. the woman brought the case to court, all the way to the grand chamber of the european court of human rights. the courts, both british and european, decided against the woman’s wishes (but the decisions were not always unanimous). the legal—and ethical—dilemma was: how to weigh the need for consent from both partners against the right to a family life as enshrined in the european convention of human rights. the grand chamber decided that the right to a family life could not override the male partner’s withdrawal of consent (13). thus, the principle of consent has strong legal support of the european court. ivf and preimplantatory genetic testing (pgt) the fact that ivf may be used for purposes other than the treatment of infertility evokes additional ethical questions and dilemmas. 194 k. asplund in families in which a child with a severe monogenetic disease has previously been born or if there is a high risk of aneuploidy, preimplantation genetic testing (pgt; previously pgd, preimplantation genetic diagnosis) offers a way to escape a pregnancy with a severely diseased child. a possible late-term abortion or the birth and early death of an infant may be avoided. for early-onset severe or lethal monogenetic diseases and structural chromosome rearrangements, the use of pgt is relatively non-controversial ethically. other applications of pgt have raised ethical concerns. the most obvious question is: what diseases and aneuploidies should be assessed? increasingly, pgt is being considered for late-onset, variably penetrant, and less severe conditions. even more ethically challenging: could testing be based on non-disease characteristics with genetic influences, such as intelligence and beauty (what has been called ‘preimplantatory genetic profiling’)? the concern that such testing would lead to choosing a child to order, as a commodity that has been designed simply to meet the needs and desires of the parents (‘catalogue babies’), was raised already when pgt was introduced in the 1990s (14). this prospect has created fears that the increasing frequency of ‘genetic profiling’ will move toward a modern eugenics movement (15). pgt can also be used to select embryos by sex and thus reach what has been referred to as ‘family balancing’. particular concern has arisen when ivf has been used for selection by sex in populations where male offspring are favoured over female offspring for cultural and economic reasons. in the uk, the use of pgt for sex selection of embryos for non-medical reasons is explicitly forbidden (16). highly publicized cases of ‘savior siblings’ have concerned the use of preimplantation tissue typing (ptt) to select embryos that could produce children suitable to become donors of stem cells or tissues for siblings who suffer from severe diseases (17). the merits of saving a sibling can be rationalized and commended. however, in other organ and tissue donation, treatment of a person solely for the purpose of becoming a donor is rejected. in ptt, questions on consent and the protection of children’s autonomy become paramount. the regulation of pgt and ptt varies between countries, often also between regions in the same country. in most eu countries, pgt is allowed, either by legislation or because there is no explicit prohibition (18). the most detailed regulation is probably in the uk, where the human fertilization and embryology authority (hfea) lists more than 600 genetic conditions for which pgt is allowed. these conditions have also been approved for use in cases involving ptt. however, unlike pgt, ptt requires additional approval on a case-bycase basis for specific patients (19). there are attempts to harmonize the use of pgt in eu countries. the european court of human rights has sanctioned italy and latvia for refusing access to pgt. the court refers to the right to bring a child into the world who is not affected by the illness that they carry (20, 21). in other countries, such as canada and the usa, there is no national regulation of pgt. it has been argued that not regulating pgt also involves taking a moral position (22). storage of oocytes for social reasons since the success rate of ivf declines rapidly in ages above 35 years when the woman uses her own eggs, social egg freezing (oocyte cryopreservation) has been introduced as a means to preserve and store oocytes retrieved at an earlier age. stored oocytes are used in ivf at a time when the social circumstances for having a child would be preferable (23). ethical considerations that have evolved in the discussions on social oocyte cryopreservation have concerned reproductive autonomy, risks involved in egg retrieval, undue hope, risk of failure, and the need for truly informed consent. to this end, the pros and cons of an upper age limit for ivf have been added (see above). most countries do not have national regulations on social oocyte cryopreservation. one exception is israel, where the procedure has been regulated and authorized for public support. the main justification has been that of promoting individual autonomy (24). when the swedish council on medical ethics reviewed social egg freezing, the medical problems with delayed motherhood were weighed against reproductive autonomy. the council found no convincing ethical arguments for a ban on social egg freezing, but concluded that the costs should not be covered by public funding (4). egg sharing the term egg sharing is used when a woman who is already having ivf donates some of her eggs to other women. this may be done as an entirely altruistic act, but the eggs may also be donated to the clinic where she is having treatment in return for free or discounted treatment. if so, an indirect form of economic incentive is introduced. this is accepted in some countries, whereas other countries have taken a universal position against commercialization of organ or tissue donation, including donation of gametes; only modest reimbursement for expenses is allowed. in the uk, where egg sharing in return for free treatment is allowed, the human fertilization and embryology authority emphasizes that the woman should never be put under any pressure to share her eggs. she should be informed that egg sharing is a big decision with serious implications, and she should receive professional counselling before going ahead. in the uk, as in some other countries, this includes information about the right of children conceived by egg sharing to know about their genetic origin when they turn 18 (25). surrogacy the term surrogacy has in itself a judgmental connotation (and the term surrogate children is even more objectionable). but since the terminology battle now seems to be over (even among healthcare professionals), i am, somewhat reluctantly, using surrogacy here. upsala journal of medical sciences 195 surrogacy may either be partial, when pregnancy is initiated through insemination, or full, involving eggs from another woman than the surrogate mother. in full surrogacy, eggs and sperm may come from the intended parents or from external donors. full surrogacy thus involves ivf with no genetic link to the surrogate mother but with genetic link(s) to two, one, or none of the intended parents. the ethics of surrogacy, whether partial or full, is covered by an abundance of analyses and debate articles (26). much of the ethics debate is on altruistic versus commercial surrogacy, autonomy versus exploitation of women, human dignity, medical risks, balancing interests of the persons involved and the long-term well-being of surrogate mothers, children, and their families. however, the ethical debate has only rarely distinguished between partial and full surrogacy. most intended parents seem to prefer to maximize the genetic linkage to the extent that is medically feasible. ivf then becomes the method of choice for conception, and this may be regarded as catering for the principle of autonomy. an additional possible argument for full surrogacy is that the psychological impact would conceivably be less if the surrogate mother knows that she is not genetically related to the child she is carrying and will be separated from. commercialization of ivf with the increasing demand for ivf, the economic impact of the ivf sector has come into focus highlighting the possible negative aspects of the commercialization of ivf. ethical questions that are often raised in the debate include equity, possible exploitation of need and hope, consent that is truly informed, and the many components of marketing ethics. the ‘ivf industry’ has been seen as an example of what social scientists describe as an increasing trend toward a market-driven construction of health, medicine, and the human body (27). most of the public debate on commercialization of ivf has not, however, concerned ivf as such but the reimbursement of gamete donors (egg donors in particular), the selling of embryos, and the use of ivf for commercial surrogacy. public funding of ivf and prioritization whether or not ivf should be funded publicly is, to a large extent, a matter of priority ethics. the swedish model for priority-setting in healthcare may serve as an example. it is based on three ethical principles (28). the human dignity principle this is basically a principle of equal value, equal human rights, and non-discrimination. all people have human dignity simply by being human and not for what they have or do. access to healthcare services should not depend on sex, social or economic background, religion, sexual orientation, or cognitive function, among others. according to this principle, singles and homosexuals should have the same right to ivf as infertile heterosexual couples in need of sperm or egg donation. regional and socioeconomic differences in access to public funding of ivf are not in accordance with the principle of human dignity. it has been argued that the right to health, as defined by the world health organization, includes the right to have children. even if this perspective is accepted, it does not necessarily translate into a right to public funding of ivf (29). the needs and solidarity principle resources should be allocated to patients who have the greatest need. need is assessed by (a) the severity of the health problem; (b) the potential health improvement that would be brought about by a healthcare intervention; and (c) the scientific support for a favourable benefit–risk ratio. the solidarity component means that special attention should be paid to those who cannot themselves express their needs, such as children, people with dementia, or patients with severe mental disorders. the principle of needs and solidarity contrasts with requests for healthcare, often not equalling needs. the main ethical debate in the treatment of infertility concerns need. there are many individual variations in how involuntary childlessness is perceived, and these may be affected by societal norms, attitudes of next-of-kin and friends, how childlessness is presented in the media, and other factors. decisions on public funding of ivf are, however, not made at the individual level but at group levels. the cost-effectiveness principle according to this principle, the healthcare system has a duty to utilize its resources as effectively as possible, and there should be a reasonable balance between the costs and effects of an intervention. this principle is subordinate to the other two principles. the cost-effectiveness of ivf primarily depends on three factors: (a) treatment success rates; (b) multiple pregnancies; and (c) the cost of treatment (29). the cost-effectiveness has been estimated to be notably lower in older than in younger women (30), so the (subordinate) cost-effectiveness principle may conflict with the human dignity principle. a weakness in the cost-effectiveness studies is that costs are usually measured per successful outcome such as live births or ‘takehome babies’. in other medical areas, costs are usually expressed as costs per quality-adjusted (qalys) or disabilityadjusted life years (dalys) gained. the different denominators make it difficult to prioritize ivf versus other healthcare interventions (horizontal priority-setting). the three principles for priority-setting provide an ethical platform, but they do not resolve all issues regarding the prioritization of ivf. a frequent question is: should women and men with an established medical cause of infertility have a higher priority for assisted reproduction than those with unexplained infertility? since the medical preconditions may differ, the need can, at least to some extent, be expressed in 196 k. asplund term of effects, adverse effects, and the strength of scientific documentation. but usually, needs in terms of distress and suffering do not differ by cause of infertility; the existential burden remains the same. therefore, it would not be acceptable to prioritize ivf solely based on the presence or absence of an established medical cause of infertility. it has also been argued that women with age-related infertility have a greater need for ivf since they are more likely to be permanently infertile and their time to become pregnant is running out. public funding of ivf varies between countries. in north america, payment for ivf is usually through private health insurance or out-of-pocket funds. in the usa, high costs have generated ‘reproductive tourism’ to countries with lower fees but also with safety concerns (31). in many european countries, there are programmes for public funding but with considerable variations in the number of cycles that are funded, age limits, and the proportion of the total cost that is paid for, among other factors (29,32). public funding is often restricted to ivf performed in public hospitals and clinics (29). in public funding, equity is an important aspect of the human dignity principle. it has repeatedly been shown that socioeconomically disadvantaged groups have less access to assisted reproduction services than more privileged groups. socioeconomic disparities persist after adjusting for several confounding factors, including age at first birth and geographic remoteness (33). in finland, where ivf is publicly funded whether performed in public or private clinics, socioeconomic inequities have been observed in private but not public clinics (34). ivf and religion while academic bioethics usually considers both facts and values when performing ethical analyses, religions rely principally on values. religious beliefs may be decisive when infertile women and men consider the ivf option. they may also impact law-making and other regulations in a country. ivf and procedures associated with ivf are summarized by religion in table 2. by necessity, the table gives a very simplified view. within one religion, different sects have diverse interpretations and have reached varying conclusions. some of the information in the table is from unofficial internet sources; the summary should therefore be regarded as provisional. the text below comments on the religions’ positions on ivf. the roman catholic church opposes ivf. in 2007, pope benedict xvi declared that ivf and other forms of assisted reproduction are unworthy methods of conception, since they separate the procreative goal of marital sex from the goal of uniting a married couple. an additional reason for the resistance against ivf is that some embryos (beginnings of a new lives) are discarded (35). the position of the eastern orthodox churches on ivf seems to be somewhat less restrictive than that of the catholic church. under some circumstances, the eastern orthodox church permits the use of parents’ gametes for ivf, fertilising only as many embryos as will be implanted, thus avoiding scenarios under which embryos are discarded (36). among the protestant churches, there is no common statement on ivf. in most protestant countries, ivf as such is no longer disputed, but some of the applications are questioned. for instance, the church of england has expressed profound concern at offering fertility treatments to single women and gay couples (36). followers of the jewish faith are encouraged to have children (‘be fruitful and multiply, fill the earth and subdue it’, genesis 1:28). ivf is allowed, and in israel it is encouraged. certain aspects of assisted reproduction are still controversial among orthodox jews, for example the collection of sperm (the ‘spilling of seed’ is prohibited) and donation of gametes and embryos (36). islamic jurisprudence states that any act is permissible unless prohibited by a text in the quran. islam affirms the importance of marriage, family formation, and procreation. according to sunni islam fatwas (religious opinions/rulings), all forms of assisted reproduction are allowed as long as the sperm and oocyte are those of the husband and his wife (37). a third party cannot be involved in the conception; that is, gamete or embryo donation is not allowed. most sunni muslims accept surrogacy, provided that the gametes come from the prospective parents (37). shi’a principles and practices are similar to those in the sunni fatwas, except that shi’as permit gamete donation, the rationale being that it does not involve sexual intercourse with someone outside the family. gestational surrogacy using ivf is accepted by shi’a muslims (36). table 2. ivf by religion and cultural tradition. religion/cultural tradition ivf ivf for singles gamete donation embryo donation surrogacy through ivf christianity catholic no no no no no orthodox yes/no no no no no protestant yes yes/no yes/no yes/no no judaism yes debating yes yes/no yes/no islam sunni yes no no debating yes shi’a yes no yes yes yes hinduism yes no sperm only, conditional yes yes buddhism yes no yes yes yes chinaa yes debating no no no japana yes no sperm only no no aemerging from legislation. modified and expanded from sallam and sallam (36). see the text for additional restrictions and other comments. upsala journal of medical sciences 197 hinduism is liberal with most assisted reproduction procedures, but demands that the egg and sperm come from a married couple. there are, however, exceptions: the sperm may also come from a close relative of an infertile man (36). buddhism is very permissive regarding ivf. it does not restrict the use of ivf to married couples, and sperm donation is permitted. in chinese and japanese societies, there are influences from several religions, such as buddhism, confucianism, taoism, and shintoism. in addition, japan is a highly secularized country, and in china it is common that several beliefs are practiced at the same time (it has been suggested that this should not be called ‘religion’ but rather ‘cultural practices’, ‘thought systems’, or ‘philosophies’ (36)). it is thus difficult to estimate to what extent religious beliefs have influenced the regulation of ivf and other forms of assisted reproduction in these countries. in china, ivf as such is permitted, but sex selection without medical indication, surrogacy, and gamete and embryo donation are prohibited (36). japanese law permits ivf and sperm donation but not oocyte donation and surrogacy (36). after several landmark rulings by japan’s supreme court, the japan society of obstetrics and gynecology has allowed unmarried couples access to ivf. permission does not include single women or same-sex couples. concluding remarks this review has focussed on the individual woman and her partner. it should be added that, in some countries, ivf is used as a part of national policy to increase birth rates. south korea (38) and israel (39) have been exemplified as such pronatalist countries. if a pronatalist policy involves ivf promotion combined with a ban on abortions, as in south korea, women’s reproductive health and rights are at stake (38). this review has also aimed at showing that decision-making concerning ivf cannot be based only on clinical and economic considerations; they cannot be disentangled from ethical principles, nor from social, political, and philosophical considerations. as noted by mladovsky and sorenson (29), many concerns regarding the costs, effects, and safety of ivf subtly raise more complex questions about what it means to society to bear children. disclosure statement no potential conflict of interest was reported by the authors. notes on contributor kjell asplund, md, phd, professor emeritus of medicine, umeå university, sweden. former director general of the national board of health and welfare and chief medical officer of sweden. former chair of the swedish council on medical ethics. orcid 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[cited 2019 oct 31]. available at: http://yris.yira.org/essays/2385. upsala journal of medical sciences 199 https://eclj.org/eugenics/echr/-italie-et-la-convergence-des-droits-de-lhomme-et-des-biotechnologies https://eclj.org/eugenics/echr/-italie-et-la-convergence-des-droits-de-lhomme-et-des-biotechnologies https://www.hfea.gov.uk/donation/donors/egg-sharing/ https://www.hfea.gov.uk/donation/donors/egg-sharing/ http://www.vatican.va/roman_curia/congregations/cfaith/documents/rc_con_cfaith_doc_19870222_respect-for-human-life_en.html http://www.vatican.va/roman_curia/congregations/cfaith/documents/rc_con_cfaith_doc_19870222_respect-for-human-life_en.html http://www.vatican.va/roman_curia/congregations/cfaith/documents/rc_con_cfaith_doc_19870222_respect-for-human-life_en.html http://yris.yira.org/essays/2385 abstract introduction brief historical notes age limits single women and same-sex couples who ‘owns’ stored gametes and embryos? ivf and preimplantatory genetic testing (pgt) storage of oocytes for social reasons egg sharing surrogacy commercialization of ivf public funding of ivf and prioritization the human dignity principle the needs and solidarity principle the cost-effectiveness principle ivf and religion concluding remarks disclosure statement references vol_116_002_sups_a_545494 90..99 upsala journal of medical sciences. 2011; 116: 90–99 review article toll-like receptors (tlrs) and mannan-binding lectin (mbl): on constant alert in a hostile environment ingrid-maria bergman linnaeus university, school of natural sciences, kalmar, sweden abstract in the beginning were neither b cells nor t cells nor antibodies, but innate immune defense alone. the primary functional theme of innate immunity is the distinction between self and non-self, which is maintained by a vast number of cellular and subcellular components. in this context, the immense importance of the toll-like receptors (tlrs) is well established. positive (darwinian) selection seems to be acting on the ligand-binding domains of these molecules, suggesting a selection pattern similar to that previously observed in the mhc proteins. in sharp contrast to tlrs, the biological significance of mannanbinding lectin (mbl) is controversial, and, concerning humans, it has been suggested that low concentration of mbl in serum represents a selective advantage. in this mini-review, based on a doctoral thesis, evolutionary aspects of tlrs and mbl are discussed. key words: innate immunity, mbl, molecular biology, positive selection, recognition, tlrs innate immunity—a prerequisite for survival the vertebrate immune defense has two arms: the innate and the adaptive immune systems. these are ideal partners because of their different recognition strategies, which have complementary strengths and weaknesses (1). through the use of pattern recognition receptors (prrs), the innate immune defense is highly efficient at distinguishing self from non-self, but has—due to its non-clonal nature and despite safety measures like complement control proteins (2) and the activities of sialic acid-specific lectins (siglecs) (3) and signaling regulatory protein a (sirpa) (4)—the potential to cause significant collateral damage to self tissue. the adaptive immune defense, relying on clonally expressed receptors, is highly effective in targeting the immune response against infection while sparing uninfected tissue; however, the cells of the adaptive immune defense cannot by themselves reliably determine the origin of the antigen they are specific for. by joined forces, the evolutionary ancient and immediate innate immune defense and the younger, highly specific, but temporally delayed adaptive immune defense maximize the survival potential of their host (1). in bony fish—the most diverse group of vertebrates (5)—several innate immunity components are more active and diverse than their mammalian counterparts (6). among piscine toll-like receptors (tlrs), there are examples of duplicated genes and wider ligand repertoires compared to the mammalian equivalents (7). two mannan-binding lectins (mbls) have been detected in rainbow trout, but there are indications that these may be members of a larger trout mbl family (8). certain complement factors are more diverse in fish than in mammals (6), and the molecules of the alternative pathway of complement display five to ten times higher titers in fish (9). these differences between mammalian and piscine innate immunity may represent a compensatory strategy, correspondence: ingrid-maria bergman, phd, linnaeus university, school of natural sciences, se-39182 kalmar, sweden. fax: +46-480-447340. e-mail: ingrid-maria.bergman@lnu.se (received 22 november 2010; accepted 1 december 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.545494 since the adaptive immune response in fish is limited and slow. also, the adaptive immune defense generally develops late in marine species, making the innate immune defense their only protection against pathogens during the first two or three months after hatching. the lower anti-pathogen activity of the innate immune defense in mammals can be interpreted as an evolutionary shift in function, in which communication with the adaptive immune defense and maintenance of homeostasis is becoming increasingly important (6). the innate immune defense has previously been considered unsophisticated and non-specific, a ‘little sister’ of the more important adaptive immune defense. however, the growing awareness of the immense importance of tlrs has promoted innate immunity research and led to new insights concerning its elaborate nature and specificity, stressing its role as the primary defense system in all species, including those which possess an adaptive immune defense. in mammals, tlrs and mbl occupy central positions at the border between innate and adaptive immunity: complement, initiated by mbl, co-ordinates adaptive immune functions (10), and dendritic cells, the most specialized antigen-presenting cells, are known to express tlrs (11). this mini-review, based on a doctoral thesis, aims to describe evolutionary aspects of tlrs and mbl and contribute to the discussion on how natural selection is shaping these molecules for their roles in host defense. who’s there? the recognition strategies of the innate immune defense the main task of the innate immune defense is to discriminate between self and non-self. this is achieved through three strategies of recognition: recognition of microbial non-self, recognition of missing self, and recognition of altered self (figure 1) (12). recognition of microbial non-self is based on the interaction between prrs—such as tlrs and mbl—and micro-organism-associated molecular patterns (mamps). mamps are conserved structures, invariant in a particular class of microorganisms, which are not present within the host. furthermore, these structures are essential for the viability/adaptive fitness of the micro-organism and thus not easily discarded (13). secreted prrs, like mbl, bind to microbial cells and flag them for phagocytosis or elimination by the complement system, while membrane-bound prrs, like tlrs, activate signaling pathways that induce antimicrobial effector mechanisms and inflammation (12). intercellular as well as intracellular cross-talk between tlrs and complement is known. binding of the target effector cell activation of immune response activation of nk cells assisted apoptosis effector cell target target effector cell inhibitory receptor recognition of altered self recognition of missing self recognition of microbial non-self activating receptor a b c figure 1. three recognition strategies used by the innate immune defense. a: recognition of microbial non-self induces immune response. b: natural killer cells interact with target cells through activating and inhibitory receptors. when both types of receptors are engaged, the inhibitory receptors are dominant and the natural killer cell is not activated. however, if self marker molecules are missing, the natural killer cell is released from its state of inhibition. c: expression of markers of altered self flags the cell for destruction. tlrs and mbl: on constant alert in a hostile environment 91 complement-derived anaphylatoxins c5a and c3a to their receptors—c5ar and c3ar, respectively— modulates tlr4, tlr2/6, and tlr9 signaling (14). moreover, pentraxin 3 (ptx3), a prr belonging to the pentraxin family, is produced by a variety of cells and tissues in response to tlr signaling and modulates complement activation through interaction with c1q and factor h (15). at the intracellular level, the complement receptor-3 (cr3), an integrin, can be transactivated by tlr2 via an insideout signaling pathway which is distinct from the myeloid differentiation primary response protein 88 (myd88)-dependent pro-inflammatory signaling pathway. conversely, cr3 can initiate tlr2 and tlr4 by promoting the recruitment of the adapter molecule myd88-adapter-like (mal) (16). crosscommunication between tlrs and complement may serve to avoid misinterpretation of signals from non-dangerous non-self and help in the fine-tuning of the subsequent immune response to a particular microbe (17). the missing self recognition strategy is based on molecular markers expressed on healthy cells: if these markers are missing, the cell is targeted for destruction. the major histocompatibility complex i (mhci) proteins, constitutively expressed on all nucleated cells but often down-regulated as a result of viral infection, serve as self markers and ligands for inhibitory receptors which block the lytic activity of nk cells. conversely, recognition of altered self is based on markers expressed only by abnormal or damaged cells, which thus are flagged for elimination (12). toll-like receptors discovery the first tlr was cloned and characterized in 1997 by ruslan medzhitov and co-workers: under the hypothesis that the expression of co-stimulatory molecules and cytokines by antigen-presenting cells was induced by a non-clonal component of immunity, human tlr4 was discovered. it was also found that this molecule could induce activation of the nuclear factor kb (nfkb) pathway and expression of the costimulatory molecule b7.1, which is necessary for activation of naive t cells (18). tlrs have previously been considered solely dedicated to host defense, but evidence is emerging that these receptors may also be involved in central nervous system development and maintenance (19,20). advances made recently in the understanding of toll-like receptor biology in host defense and disease have been reviewed by kawai and akira (21). evolutionary perspectives tlr4 is the most ancient tlr, dating its origin before vertebrate life (22). today’s mammalian tlr family can be subdivided into two groups, which are the result of a gene duplication event prior to the divergence of invertebrates and vertebrates: the tlr1 family, consisting of tlrs 1, 2, 6, and 10, and a second group, comprised of all other tlrs. orthologs of tlr1, tlr6, and tlr10 seem to be present exclusively in mammals (23). tlr10 separated from the tlr1/6 precursor about 300 million years ago, roughly at the time of divergence from birds, while tlr1 and tlr6 appeared approximately 170 million years later (22). following duplication, gene conversion events between tlr1 and tlr6, limiting the divergence between these two genes, have been suggested (24). in mice and humans, remnants of a second, disrupted tlr2-like gene is located in tandem with the functional version of tlr2. this indicates the occurrence of a gene duplication after which one of the gene copies has developed into a pseudogene. since there are two tlr2 genes in chicken, the duplication event probably occurred before the divergence of mammals and birds (25). the nematode caenorhabditis elegans possesses a single tlr (figure 2) (26). on the other hand, the genome of the purple sea urchin encodes 222 tlrs (27), demonstrating that in the absence of adaptive immunity a large repertoire of prrs may provide a survival advantage in a pathogen-rich environment (28). the genome of the florida lancelet is thought to harbor roughly 50 tlr genes (29), while at least 20 are known in the south african clawed frog (30). seventeen different tlrs are known in bony fish (7). the orthologs of tlr6 and tlr10 in mammals are absent, while tlr14—which has been found only in aquatic animals, suggesting an aquatic pathogen (31)—and tlrs 18–23 have been identified (7). some piscine tlrs appear to have a different ligand repertoire and functional role compared to their mammalian counterparts: piscine tlr3 has been shown to respond to viral as well as bacterial mamps, while tlr4 may be acting as an inhibitor of the myd88-dependent signaling pathway in zebrafish (7). tlrs also exhibit a different diversity pattern in fish compared to mammals: in rainbow trout, a soluble tlr5—possibly amplifying flagellininduced signaling (32)—is present together with the membrane-bound version, while two tlr4 genes and two tlr8 genes have been found in zebrafish, and two splicing isoforms of tlr9 are present in gilthead seabream and large yellow croaker (7). in comparison, soluble versions of tlr2 (33) and 92 i.-m. bergman tlr4 (34), both with negative regulatory function towards their respective membrane-bound versions, are known in mammals. in the chicken genome, ten tlr genes have been identified (figure 2), one of which–chtlr15—is specific for chicken (25). ten functional tlrs are known in humans and pigs (35). in mice, tlrs 11–13 have been identified (36), but mouse tlr10 is non-functional, due to a retroviral insertion (37). tlr11 is present as a pseudogene in humans (38). controlling the double-edged sword due to the potent nature of tlr signaling, tlr activation is a double-edged sword, being essential for provoking the innate immune response and enhancing the adaptive immune response, but simultaneously opening a door for pathogenesis. negative regulation of tlr signaling is achieved at multiple levels through a large number of molecules (21). during acute bacterial infection, soluble tlrs can function as decoy receptors, preventing over-activation of the membrane-bound versions. once ligand-receptor interaction has occurred, intracellular regulators further control tlr signaling (39). furthermore, tlr signaling is also controlled by membrane-associated regulators, e.g. single immunoglobulin interleukin-1 receptor-related molecule (sigirr) (39), through mirnas (40), by degradation of tlrs and production of anti-inflammatory cytokines as well as through apoptosis (39). in addition to this, codon usage offers an alternative, more indirect mechanism for regulation. it has been shown that in most human tlr genes, the codon usage pattern deviates from the general one in humans, dictating a low level of protein expression (41). we have recently reported a similar difference between the general codon usage pattern in pigs and that in tlr1, tlr2, and tlr6 (42), the alternative codon usage pattern also extending to tlr10 (unpublished data). it is likely that this specific tlr codon usage pattern—mirroring the conditions in humans—results in protein expression at a low level. moreover, at least in piscine tlrs, the leucinerich repeats (lrrs) may have a role not only in ligand recognition but also in the limitation of tlr activation, since a mutant zebrafish tlr3 construct, lacking lrrs and most of the endosomal domain, showed 100 times higher reporter gene expression compared to the wild-type tlr3 construct (43). south african clawed frog: 20 tlrs b and t lymphocytes present purple sea urchin: 222 tlrs complement present caenorhabditis elegans (nematode): 1 tlr florida lancelet: 50 tlrs chicken: 10 tlrs pig: 10 tlrs man: 10 tlrs mouse: 13 tlrs fruit fly: 9 tlrs origin of life figure 2. schematic and simplified overview of the development of the immune defense. numbers of toll-like receptors vary between species and tend to decrease during the course of evolution. proteins belonging to the complement system are present in the purple sea urchin. the south african clawed frog possesses an adaptive immune defense based on b and t lymphocytes. tlrs and mbl: on constant alert in a hostile environment 93 comparisons to drosophila toll and plant tir seen from an evolutionary point of view and based on the detailed structure of the extracellular domain, tlrs can be divided into vertebrate-like tlrs (v-tlrs) and protostome-like tlrs (p-tlrs) (27). applying this approach, the drosophila toll family (figure 2) includes eight p-tlrs (toll included) and one v-tlr (toll-9) (29). phylogenetic analysis of the toll/interleukin-1 receptor (tir) domains in the drosophila toll family reveals that these—toll-9 being an exception—are more closely related to one another than to tlrs in mammals. thus, it is likely that the two groups of receptors—toll and the drosophila toll-related receptors except toll-9 on the one hand, and mammalian tlrs on the other—have evolved independently while carrying out different main functions (44). the difference is further emphasized by the fact that the physical interaction between mammalian tlrs and mamps (21) occurs in a completely different manner compared to the binding of the endogenous ligand spätzle by toll (45). flagellin-sensitive 2 (fls2) in arabidopsis thaliana, thale cress, is a transmembrane protein involved—like tlr5—in the recognition of flagellin. fls2 is equipped with an extracellular lrr domain, but the low degree of sequence similarity between fls2 and human tlr5 suggests that these proteins arose independently. resistance (r) proteins containing lrr domains fused to tir domains are present in plants, and r proteins resembling either cd14, a co-receptor in the tlr4 receptor complex, or tlrs are also known (46). in contrast to animal tirs, plant tir domains may interact directly with pathogen effectors, while a role in signaling for plant tir domains remains to be established. however, some tir domain-containing r proteins are found in the nucleus and may have roles as transcription factors. again, these differences in the use of the tir domain may imply convergent (47) or parallel (28) evolution rather than conservation from an early ancestor of both animals and plants. mannan-binding lectin mbl: an initiator of the complement cascade the role of the complement cascade in the human immune defense has recently been reviewed by zipfel (10). mbl and ficolins initiate the lectin activation pathway of complement in co-operation with mblassociated serine proteases (masps) (48). the lectin pathway is the most recently described of the main complement activation pathways (49) but possibly the most ancient: a glucose-specific lectin which interacts with masps has been purified from the sea pineapple (50), and an ortholog of the mammalian mbls is present in lamprey (51). ficolin-3 has been found to be the most potent of the lectin pathway initiators in humans, followed by ficolin-2 and mbl (52). mbl is similar in structure to c1q, a subunit of the classical pathway c1 complex, and mammalian mbl—but not mbl in chicken and fish—is thought to have the same ability as c1q to stimulate phagocytosis through the c1qrp receptor. the function of mbl/masps is equivalent to that of the c1 complex: c4 and c2 are cleaved, and the classical/lectin pathway c3 and c5 convertases—c4b2a and c4b2ac3b, respectively—are formed (48). recently, a novel mbl/ficolin-associated protein, representing a short splice variant of the masp1 gene and denoted map-1, has been detected in humans. this protein is expressed in myocardial and skeletal muscle and thought to inhibit the complement system by preventing the cleavage of c4 (53). the mbl molecule mbl belongs to the protein family of collagenous lectins, which includes multifunctional proteins with defensive and housekeeping roles (48). the functions of collagenous lectins and tlrs are known to intersect: mbl has been found to inhibit mouse macrophage tumor necrosis factor a (tnfa) production—which is a consequence of tlr signaling leading to activator protein 1 (ap-1) activation (54)—by shielding blastomyces dermatitidis epitopes (55), while in-vitro experiments have shown a dampening effect of surfactant protein a (sp-a) on pulmonary inflammation caused by zymosan, probably through the binding of sp-a to tlr2 (56). in most mammals, two forms of mbl—mbl-a and mbl-c—are present. these are encoded by separate genes, denoted mbl1 and mbl2. duplication of the mbl gene probably occurred after the divergence of birds and mammals, since only one mbl gene is present in chicken. in humans and chimpanzee, mbl1 is a pseudogene (48). the basic subunit of mammalian mbl is a trimer composed of three identical monomers, each consisting of four distinct domains: a cysteine-rich n-terminal domain, a collagen-like domain, a neck region, and a c-terminal carbohydrate recognition domain. the collagen-like domain is composed of numerous g-x-y motifs (x and y representing any amino acid), typical of a helix structure. if a glycine in a g-x-y motif is replaced by a more bulky amino acid, the formation of the trimer triple helix is disturbed. this, in turn, impairs the functionality of the molecule, since this is dependent on oligomerization of the basic trimers (57). the binding of the 94 i.-m. bergman functional mbl molecule to the pathogen is, in turn, dependent on mbls’ steric specificity and the spatial organization of the carbohydrate ligands on the pathogenic surface; conversely, self tissue is spared by these requirements (50). in serum, mbl exists in complex with masps 1–3 and small mbl-associated protein (smap), the latter representing a short splice variant of the masp2 gene and playing a regulatory role in the lectin pathway. when mbl binds to a cell surface displaying a fitting carbohydrate pattern, the proenzyme forms of masps are cleaved and become proteolytically active (58). differences between species the abundance, forms, and functions of the collagenous lectins vary considerably between species. in humans, one mbl and three ficolins are expressed, while there are two ficolins and two mbls in most other species. furthermore, in mbl and ficolins, the recognition domains are aggregated at one pole, which creates their sertiform (‘bunch of tulips’) formation. this facilitates multivalent binding to monosaccharides on microbial surfaces. in contrast, in the bovine-specific conglutinin (cg) and collectin46 (cl-46), the recognition domains are distributed at diametric poles, leading to a cruciform which facilitates agglutination of targets; however, these cruciform molecules have not been shown to activate complement. moreover, unlike other collagenous lectin trimers, those of bovine-specific collectin-43 (cl-43) and human collectin liver 1 (cl-l1) do not assemble into higher-order oligomers. differences like these may reflect co-evolution with relevant pathogens (48). tlrs and mbl: finding mr darwin—and not finding him toll-like receptors the weaknesses of some widely used statistical methods aiming to identify positive (darwinian) selection by comparisons of numbers of non-synonymous and synonymous single nucleotide polymorphisms (snps) have been pointed out by hughes and co-workers (59). if a protein is evolving under positive selection, then a pattern of dn>ds (i.e. the proportionate number of non-synonymous differences, dn, exceeding the proportionate number of synonymous differences, ds) is expected, as has been found regarding the peptide-binding region (pbr) of the mhc molecules. mhc proteins present peptide antigens to antigen-specific t cells. since mhc molecules encoded by different alleles present different antigens, the advantage of a heterozygote in terms of increased peptide-presentation capacity maintains mhc polymorphism. thus, from a biological point of view, positive selection repeatedly favoring amino acid changes in the pbr is reasonable. however, this pattern may not be characteristic for positive selection in general. the methods based on comparisons of non-synonymous and synonymous snps often fail to rule out alternative hypotheses, in particular the relaxation of purifying selection and the effects of population bottle-necks (59). furthermore, the dn/ds statistic, originally developed for quantification of selective pressure in divergent species, has also been scrutinized and challenged. even though it is traditionally used to quantify selection pressure within populations, it has been shown to be inadequate when applied in this manner or for closely related taxa (60,61). it is known that many pathogens, among them yersinia pestis, express lipid a structures which do not fully stimulate tlr4 (62), demonstrating that at least some mamps do evolve in response to selective pressure. moreover, there is accumulating evidence of a species-specific component in tlr function, which, in turn, might reflect co-evolution with relevant micro-organisms (63). bovine tlr2/ tlr1 (63) and chicken tlr2-type2/tlr1-type2 (64) both display broader ligand specificities than the corresponding receptor complexes in most mammals. tlr2 in ruminants seems to be subject to a lower degree of selection compared to tlr2 in other mammals (63), and polymorphism in bovine tlr2 is known to occur mainly between breeds, the genotype in a particular breed probably reflecting the microflora at hand (65). furthermore, certain wild-derived inbred strains of mice differ in tlr3 genotype from classical laboratory strains (66). our own findings, showing that wild boars and domestic pigs display different polymorphic patterns in the tlr1, tlr2, tlr6 (42), and tlr10 (unpublished data) genes, parallel these observations and further emphasize that different microbial environments are involved in shaping the differences in genotypes observed between populations/strains. to summarize, positive selection acting on the ligand-binding domains of tlrs appears reasonable from a biological point of view. indeed, the pattern observed in an analysis of 22 mammalian tlr2 sequences parallels that of mhc: amino acids responsible for the binding of mamps evolve under positive selection, while regions important for heterodimerization—especially the tir domain—are subject to purifying selection (63). moreover, a similar pattern has been reported concerning bovine tlr4 (67). furthermore, biased distributions of non-synonymous snps, compatible tlrs and mbl: on constant alert in a hostile environment 95 with different modes of selection acting on different parts of the genes, have been reported regarding porcine tlr genes (68,69). also, in bony fish, certain sites in the lrr domains of tlr9 seem to evolve under positive selection (70). barreiro and co-workers (71) reported evidence for population-specific events of positive selection on the tlr10-1-6 gene cluster, strong purifying selection acting on the intracellular tlrs, and more relaxed selective constraint on cell surface tlrs in humans. taken as a set, human tlrs have evolved under purifying selection according to this study (71). in most tlrs, wlasiuk and co-workers detected positive selection between humans and chimpanzees, as opposed to purifying selection within species (72). also, a comparison between human and rhesus monkey orthologous gene pairs revealed significantly lower dn—and thus greater functional constraint— on the tir domain compared to lrrs in tlr4, tlr5, tlr7, tlr8, and tlr9, but no positive selection was reported (23). bovine tlr10 seems to evolve under strong purifying selection, indicating an important function for the corresponding receptor. this is interesting, since tlr10 is the only member of the tlr family for which no ligand is known. it remains to be defined what kind of functional constraint might be important enough to justify strong purifying selection (73). in contrast, relaxed selective constraint, indicating some degree of redundancy, has been suggested for human tlr10: in the largescale screening project undertaken by barreiro and co-workers, tlr10 was found to be by far the most variable of the human tlr genes (71). due to markedly different physiologies, the pathogenic load and the balance between commensals and pathogens are likely to differ between ruminants and nonruminant species. thus, species-specific function and co-evolution with relevant micro-organisms might explain why bovine and human tlr10 seem to be subject to different modes of selection. our own findings regarding polymorphic patterns in the tlr1, tlr2, tlr6 (42), and tlr10 (unpublished data) genes in pigs point in the direction of purifying selection: visualizations of the amino acid positions corresponding to detected non-synonymous snps, using the crystal structure determined for the human tlr1-tlr2-lipopeptide complex (74) combined with protein sequence alignments, indicated that the majority of the variable positions are distinct from the ligand-binding site and the dimerization interface. it has been argued that positive selection detectable as a pattern of dn>ds, indicating repeated amino acid changes at a limited set of codons, is likely to be very rare. comparisons of dn and ds would not detect single non-synonymous changes at single codons, whole or partial gene deletion, or changes in the patterns of gene expression; however, such punctual events are known to produce adaptive genotypes and likely to be more common (59). moreover, it has been pointed out that the highly mutable cpg positions are much more common in replacement sites in codons than in introns, rendering the fundamental assumption of equal mutation rates in different parts of the genome unjustifiable. thus, the intensity of purifying selection on coding sequences may be greater than previously inferred (75). in agreement with this, roach and co-workers found no support for positive selection in the vertebrate tlr phylogeny through evaluation of synonymous/non-synonymous substitution ratios (36). in conclusion, despite their potential as a parallel to the mhc proteins as far as patterns of selection are concerned, a conservative approach regarding the extent to which positive selection is acting on and has been detected in tlrs still seems appropriate. mannan-binding lectin in the coding sequence of the human mbl2 gene, three non-synonymous snps, all with decreasing effects on mbl concentration, have been identified. two of these snps implicate the replacement of a glycine residue in a g-x-y motif with an aspartic acid (the b allele) and a glutamic acid (the c allele), respectively. furthermore, three promoter polymorphisms affecting mbl serum concentrations are known. low-producing mbl alleles are present at varying frequencies in different human populations (76). mbl deficiency has been found to increase susceptibility to many infectious diseases (77), but also, in contrast, to increase resistance against leishmaniasis (78) and tuberculosis (tb) (79). however, in the case of tb, the existing view has been challenged. in a recent meta-analysis carried out by denholm and co-workers (80), no significant association was found between mbl2 genotype and pulmonary tb infection. however, the majority of the analyses did not report mbl2 haplotypes inclusive of promoter polymorphisms. mbl serum concentrations were consistently elevated during tb infection, but this increase was also of a degree consistent with the acute-phase reaction. if indeed mbl deficiency does not protect against tb, it is challenging to propose a different disease that may have promoted the high frequency of low-producing mbl alleles observed in some human populations (80). it is believed that during evolution, the human mbl1 gene has been turned off by the same molecular mechanisms causing the variant mbl2 alleles, i. e. 96 i.-m. bergman mainly through mutations affecting the glycines in the g-x-y motifs in the collagen-like domain. this is consistent with the hypothesis that low mbl levels represent a selective advantage (81). in the coding sequence of porcine mbl1, a non-synonymous snp, implicating the replacement of the glycine in g-x-y motif 16 with a cysteine, has been detected. this snp is present at different frequencies in various domestic pig populations (82) and is assumed to affect mbl-a concentration in serum (83). the existence of this snp in porcine mbl1 may be an indication of an on-going evolutionary process favoring lowproducing mbl alleles, mirroring what might be the case in humans. however, the hypothesis of selection for low-producing mbl alleles in human populations is not undisputed: balancing selection, implying an advantage for heterozygotes (84), as well as neutral evolution and redundancy (85) has been suggested. the study by verdu and co-workers (85) is based on a large-scale sequencing project, and data have been analyzed through several methods. however, bearing in mind the importance of biological reasoning for conclusions about modes of evolution, it seems wise not to dismiss the study by seyfarth and co-workers (81), since it is based on comparisons between functional and non-functional mbl1 genes in humans and other primates. concluding remarks there seems to be a number of genes encoding defense and/or immunity proteins which have evolved rapidly since the divergence of humans and chimpanzees (86). however, enard and co-workers have recently shown (87) that positive selection acting simultaneously on the same genes in humans and other primates is not uncommon and not restricted to a particular function, e.g. immune capacity. on the other hand, independent selective sweeps in human, chimpanzee, and orangutan were inferred for the tlr6-1-10 gene cluster (87). also, immune-related genes are over-represented when recently occurring selective events in human populations are considered. the invention of farming—which led to a massive increase of human population sizes, possibly facilitating the spread of certain infectious agents—and exposure to new zoonoses following the domestication of animals may have posed strong challenges on the human immune system (86). recently, the tir protein family has been pointed out as an example where the existence of a limited number of protein domains with relevant functions has constrained evolution, resulting in the emergence of proteins with identical architectures but lacking orthologous relationship and performing different functions in different lineages, the drosophila toll proteins and the vertebrate tlrs being examples. there are several examples of prr domain architectures which have emerged multiple times, suggesting that parallel evolution may be a common phenomenon in innate immunity evolution (28). concerning to what extent positive selection is acting on tlr genes, evidence is somewhat contradictive. this might, at least in part, be explained by differences between species and by which snps are present in the particular animal materials used to produce data sets. however, the biological function of tlrs does not exclude the possibility that they are parallel to the mhc molecules as far as patterns of selection are concerned. in general, the commonly accepted arms race model of interaction between pathogens and their hosts needs to be complemented by a conception of adaptive evolution as consisting of a series of leaps, as it seems obvious—from a theoretical point of view (59) and as a result of laboratorybased research (72)—that adaptive evolution often is episodic. acknowledgements i would like to express my gratitude to professor kristina nilsson ekdahl, linnaeus university, kalmar, and uppsala university, uppsala, sweden, and professor göran andersson, swedish university of agricultural 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and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 obstructive sleep apnea syndrome (osas) and hypertension: pathogenic mechanisms and possible therapeutic approaches wang zhang & liang-yi si to cite this article: wang zhang & liang-yi si (2012) obstructive sleep apnea syndrome (osas) and hypertension: pathogenic mechanisms and possible therapeutic approaches, upsala journal of medical sciences, 117:4, 370-382, doi: 10.3109/03009734.2012.707253 to link to this article: https://doi.org/10.3109/03009734.2012.707253 © informa healthcare published online: 25 sep 2012. submit your article to this journal article views: 2376 view related articles citing articles: 33 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2012.707253 https://doi.org/10.3109/03009734.2012.707253 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2012.707253 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2012.707253 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2012.707253#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2012.707253#tabmodule upsala journal of medical sciences. 2012; 117: 370–382 review article obstructive sleep apnea syndrome (osas) and hypertension: pathogenic mechanisms and possible therapeutic approaches wang zhang & liang-yi si department of geriatrics, the first affiliated hospital, third military medical university, chongqing, china abstract obstructive sleep apnea syndrome (osas), a chronic condition characterized by collapse of the pharynx during sleep, has been increasingly recognized as a health issue of growing importance over the last decade. recently emerging evidence suggests that there is a causal link between osas and hypertension, and hypertension represents an independent risk factor in osas patients. however, the pathophysiological basis for patients with osas having an increased risk for hypertension remains to be elucidated. the main acute physiological outcomes of osas are intermittent hypoxia, intrapleural pressure changes, and arousal from sleep, which might induce endothelial dysfunction, sympathetic activation, renin–angiotensin–aldosterone system activation, lipid metabolism dysfunction, and increased oxidative stress. this brief review focuses on the current understanding of the complex association between osas and hypertension. key words: approach, hypertension, obstructive sleep apnea syndrome, pathogenic mechanism introduction sleep-related breathing disorders lead to concomitant alterations in the central nervous and cardiovascular systems, and such alterations often induce additional health issues. sleep-disordered breathing represents a wide spectrum of sleep-related breathing abnormalities. obstructive sleep apnea (osa) is a condition in which there is repetitive partial or complete collapse of the pharynx during sleep. osa associated with excessive daytime sleepiness is commonly called obstructive sleep apnea syndrome (osas). osas is the most common sleep-disordered breathing abnormality (1,2) and often results in apnea or hypopnea, which can lead to snoring. our understanding of osas has evolved from initially regarding it as merely an annoying social situation to the recognition that osas may act through various mechanisms to increase cardiovascular risk and lead to increased morbidity and mortality (3). in particular, there is evidence that untreated osas may contribute to the pathophysiological mechanisms underlying the origin and/or development of hypertension, cardiac ischemia, myocardial infarction, congestive heart failure, and stroke (4). of the different possible consequences of osas in patients, the most widely recognized may be the development of systemic hypertension. while many reviews have described the association between osas and hypertension, the diagnosis, prevalence, etiology, and new mechanisms linking osas to hypertension are outlined in this review. definition and diagnosis of osas osas symptoms, including habitual and intermittent snoring, recurrent arousal during sleep, excessive daytime sleepiness, and witnessed apneas suggest the occurrence of osas. however, the gold standard diagnostic test for osas is the overnight in-laboratory polysomnography. polysomnography uses multi-channel continuous recordings for electrocardiography, electromyography, electroencephalography, electro-oculography, nasal airflow, correspondence: liang-yi si, department of geriatrics, the first affiliated hospital, third military medical university, chongqing 400038, gaotanyan street, p. r. china. fax: +86 (0)23-68754150. e-mail: wz1974@medmail.com.cn (received 15 march 2012; accepted 25 june 2012) issn 0300-9734 print/issn 2000-1967 online � 2012 informa healthcare doi: 10.3109/03009734.2012.707253 snoring sounds, blood oxygen saturation, thoracic and abdominal impedance belts for respiratory effort, and intra-esophageal pressure. the apneahypopnea index (ahi), defined as the average number of apneas and hypopneas per sleep hour, has always been used to evaluate osas severity. an apnea is defined as the complete airflow cessation for at least 10 seconds (5). hypopnea may happen during sleep or awake states and fluctuate in severity of episodes of shallow breathing or an abnormally low respiratory rate. according to the american academy of sleep medicine task force recommendations, osas is defined as an apneahypopnea index (ahi) >5, along with excessive daytime somnolence (6). another diagnosis standard of osas is the number of apneas (where airflow stops completely) and obstructive hypopneas (> 50% reduction in respiratory flow or > 30% reduction linked to more than 3% desaturation and/ or microarousals) lasting more than 10 seconds per hour. a threshold of 15 events per hour of recording is applied for osas diagnosis (7). osas is further subclassified into mild osas (ahi = 5–15), moderate osas (ahi = 16–30), and severe osas (ahi >30). a habitual snorer is a subject who always snores at night, while the ahi is <5 (6). epidemiology of osas the prevalence of osas varies among study populations, due to the use of different variables, and is influenced by the criteria used to define osas as well as by the population characteristics. the first large epidemiologic polysomnographic study of osas was done in 1995 (8). later, many studies reported the adult prevalence of osas in many different countries and among different ethnic groups (9–15). the overall estimated prevalence of osas is in the range of 3%–7% in adult men and 2%–5% in adult women (16), with certain subgroups of the population bearing higher risk. these subpopulations include overweight or obese people and middle-aged and older subjects. notably, the results that there was no substantial difference in osas prevalence in north america, europe, australia, and asia clearly suggest that osas is common not only in developed countries, but also in developing countries. in 2008, the american heart association and the american college of cardiology distributed a joint scientific statement pointing out that 85% or more of humans with clinically significant osas have not been diagnosed (17). the referral populations of osas patients may represent only the tip of the iceberg of osas prevalence. evidence connecting hypertension to osas osas is widespread in the middle-aged and older population, while hypertension is also highly prevalent among the middle-aged and older population. this raises the possibility of considerable co-morbidity between hypertension and osas. a lot of experimental and clinical evidence has indicated that the extent of the co-morbidity of hypertension and osas is actually substantially greater than expected. in animal studies, direct evidence of the relationship between osas and hypertension has been well-established. experimental osas resulted in acute transient increases in nighttime blood pressure (bp) and eventually produced sustained daytime hypertension (18). acute normalization of blood pressure reduced sleep apneas in rats, even in the context of lifelong hypertension (19). troncoso brindeiro et al. developed a model of sleep apnea by exposing rats to chronic intermittent hypoxia (cih) during sleep and found that this protocol increased blood pressure (20). in humans, a large number of studies have sought to determine the presence and extent of a causal relationship between osas and hypertension, independent of frequently co-occurring and possibly confounding variables including age, body weight, and body mass index. the most convincing evidence for the support of a causal relationship between osas and hypertension has come from numerous epidemiological data collected in community populations. early studies indicated that hypertension was found in about 50% of osas patients (21), while about 30% of hypertensive patients also have osas (22–25). the wisconsin sleep cohort study analyzed data on sleep-disordered breathing, blood pressure, habitus, and health history, at baseline and after 4 years of follow-up, in 709 wisconsin state employees, using attended full polysomnography (26–28). relative to the reference category of an apnea-hypopnea index of 0 events per hour at baseline, the odds ratios for the presence of hypertension was 2.03 (95% confidence intervals 1.29–3.17) in subjects with an apneahypopnea index of 5.0 to 14.9 events per hour, suggesting that the presence of hypertension was independent of known confounding factors and that sleep-disordered breathing is likely to be a risk factor for hypertension and subsequent cardiovascular morbidity in the general population (26). moreover, the non-dippers exhibited a blunting of the sleep-related fall in blood pressure and an increased variability in blood pressure associated with sleepdisordered breathing (29). these results have been partly or completely confirmed by additional independent studies (30–35). osas and hypertension 371 some researchers have concluded that an increase in diastolic blood pressure might be the earliest hypertensive change associated with osas, evidenced by the fact that diastolic blood pressure was higher early in the course of osas (36), and diastolic and systolicdiastolic hypertension were the prominent types of hypertension observed both by clinical and ambulatory measurements (37). on the contrary, sin et al. demonstrated that the systolic blood pressure was significantly higher in patients with osa than in patients without osa, indicating a high prevalence of systolic hypertension in patients with osa (38). in addition, systemic hypertension was associated with a greater exacerbation of blood pressure variability in osas patients during sleep (39). the baroreflex sensitivity, an index of the cardiovascular control, was lower during wakefulness and rapid eye movement sleep in untreated osas patients than in normal subjects, and negatively correlated with the increase of blood pressure after apneas (40). therefore, it seems that hypertension, increased blood pressure variability, and decreased baroreflex sensitivity may tightly correlate with osas and contribute to the increased cardiovascular risk of osas. the similar risk factors in osas and hypertension the pathophysiological pathways linking risk factors between osas and hypertension intersect with upper airway dilator muscle activity abnormalities, reduced arousal from sleep, reduced lung volume, and impaired ventilatory control stability (41). community-based studies have identified some major risk factors for osas, including age, gender, and obesity. in parallel, these factors are also risk factors of hypertension (1,5). we summarize the similar risk factors between osas and essential hypertension in figure 1, and discuss them below. age the prevalence of osas increases with age (42-44). in older persons (‡ 65 years), the prevalence of osas is 2to 3-fold higher than that in middleaged individuals. however, the clinical and prognostic impact of osas in elderly patients appears lower than in young and middle-aged patients (11,41,45). several studies have been conducted to identify the cause of the relationship between age and osas, but it seems that no consensus has been reached. eikermann et al. reported that increasing age was correlated with both pharyngeal collapsibility and increase in pharyngeal resistance independent of body mass index (bmi) and gender during sleep, suggesting that the increased prevalence of osas in elderly individuals may mainly involve the dysfunction of muscles surrounding the parapharyngeal area (46). the prevalence of hypertension also increases with age (47). however, there is no satisfactory answer to the concurrent impact of age on osas and hypertension. obesity during the past three or four decades, the united states has witnessed a dramatic increase in the number of overweight or obese individuals, which has become a public health issue (48,49). epidemiologic studies from europe and north america have clearly identified body weight as the strongest risk factor for osas. respiratory function was certainly affected by obesity, and obesity could induce the collapse of upper airways during sleep (50). in addition, the frequency of respiratory events during sleep rises when body weight increases. an early report indicated that excess body weight was present in >60% of the sleep apnea patients (51). in the wisconsin cohort study, a 10% weight gain predicted an approximately 32% increase in ahi and was associated with a 6-fold increased risk of osas, whereas a 10% weight loss predicted a 26% decrease in ahi (44), suggesting that the incidence of osas increases along with the increased incidence of obesity. longitudinal data from the cleveland family study demonstrated that ahi was significantly associated with bmi and waist–hip ratio (52). recently, it was demonstrated that among americans aged 30–69 years, approximately 40% of adults with sleep-disordered breathing had bmis ‡ 25 (53), indicating obesity being a major risk factor for osas hypertension age gender race alcohol use obesity systemic inflammation endothelial dysfunction oxidative stress other factors similar epidemiolog ical factors similar biochemic al factorssimilar biochemic al factors figure 1. convergence of epidemiological and biochemical variables in patients with osas and essential hypertension. 372 w. zhang & y. liang development of osas. obesity is associated with anatomic changes that predispose to obstruction of the upper airway during sleep. these alterations may generate excess adiposity around the pharynx. thus, the upper airway could be narrowed by the increases of adipose tissue in the neck and around the upper airway (54,55). moreover, central obesity is associated with decrease of lung volume (56), which may cause an increase in pharyngeal collapsibility due to a loss of caudal traction of the upper airway (57–59). however, in the asian population, where visceral obesity is less prevalent, the prevalence of osas is not proportionately reduced. this phenomenon suggests that there may be a craniofacial effect interacting with body habitus (60). with obesity, the extra bulk of adipose tissue around the neck narrows the airway. a narrowed airway would increase the chances of airway collapse and closure during sleep. mortimore and colleagues demonstrated that truncal and upper body obesity may be superior predictors of osas compared to body mass index, because of more fat deposition in the upper airway or pharynx (61). flemons et al. also showed that increased neck circumference, which may be a marker for localized obesity, increased the risk of osas (62). several imaging studies of patients with osas showed larger lateral parapharyngeal adipose tissues and pharyngeal walls in the neck and upper airway compared with non-obese controls (63). however, there have also been some inconsistent reports. using magnetic resonance imaging, schafer et al. reported that ahi significantly correlated to the amount of intraabdominal fat, whereas there was no association between ahi and the size of parapharyngeal fat pads or subcutaneous fat of the neck region (64). the discrepancies may arise from different sets of populations and need to be further investigated. this increasing prevalence of obesity and obesityrelated hypertension has not only been described in the western developed countries but also in developing countries such as china and india (65). although obesity undoubtedly is a major risk factor of hypertension and osas, respectively, there is a lack of solid and clear clinical and experimental evidence for the in-depth mechanisms underlying the involvement of obesity between hypertension and osas. from a clinical viewpoint, nevertheless, a pragmatic approach to treating osas and preventing hypertensionrelated events would be desirable by treating obesity or reducing body weight. ethnicity most of the clinical population-based studies on osas prevalence were conducted in usa, europe, and australia. recently, in asian countries, including china, india, and korea, several studies have been undertaken to characterize the burden of osas (13,14,34,66–75). the prevalence of osas in asians is comparable to that documented in published reports of european and american populations. on the contrary, some studies suggest that the situation of osas may differ by race. the overall prevalence of sleep-disordered breathing was approximately equal in caucasians (30%) and african-americans (32%), but the severity was higher in the african-american population (76). the odds ratio for severe sleep-disordered breathing was 2.55 for african-americans compared to caucasians, even after adjustment for bmi, sex, and age. in the cleveland family study, redline et al. found that african-americans with sleep-disordered breathing were younger than caucasians with sleep-disordered breathing. in addition, they showed that the association of body mass index with osas was stronger in caucasians than in african-americans (77). in the same subjects, brachycephaly was found to be associated with an increased ahi in caucasians but not in african-americans (78). similarly, a cross-sectional study of new zealand maori (polynesian) and european (caucasian) men showed that small reductions in mandibular prognathism and a wider bony nasal aperture represent major factors associated with osas in polynesian men, whereas in the caucasian group osas was associated with a larger neck circumference and a reduced retropalatal airway size (79). in addition, bmi was a stronger predictor of osas severity in caucasian men compared with that in polynesian men (79). ethnicity is associated with hypertension prevalence and is an important independent contributor to osas prevalence (47,80–82). the prevalence of osas in japanese hypertensive patients was around 10% (83), which was one-third that of the western hypertensive participants of the new york sleep heart health study (84). however, in the largest cross-sectional study involving a total of 6132 participants in china, the prevalence of hypertension in patients with osas was 56.2% (85), which is comparable to western countries. the discrepancies among these results may need to be confirmed in other trials with larger number of patients. various categories of hypertension in osas in normal subjects, blood pressure decreases during sleep by 10%–20% of the awake value and increases promptly on waking. an absence of this nocturnal dip in blood pressure correlates directly with the amount of deep sleep and inversely with indices of sleep osas and hypertension 373 fragmentation (86). thus, there is great interest in the clinical observation and pathophysiological mechanisms underlying dipping and non-dipping patterns of ambulatory blood pressure profiles. in osas patients, various categories of hypertension were characterized. nocturnal hypertension a nighttime fall of blood pressure (dipping) is normal. on the contrary, reduced nocturnal bp (non-dipping) or even higher nocturnal bp than daytime bp is an undoubted risk factor for hypertensive patients due to the end-organ damage and subsequent cardiovascular events (87–89). a blunted nocturnal bp dipping phenomenon is common in hypertensive patients (90). the differences between non-dipping and dipping may be related to the following: 1) alterations in the autonomic nervous function, 2) congestive heart failure, 3) chronic kidney disease, and 4) osas. strikingly, the nocturnal bp profile described in studies of 24-hour blood pressure measurements in osas patients is similar to that in non-dipping hypertensive patients. in the wisconsin sleep cohort study, young and colleagues (28) found a dose-response relationship between sleep-disordered breathing and 24-hour blood pressure, independent of known confounding factors. baguet et al. found that 42% of apneic patients had clinical hypertension (37). in the study, 42% of the osas patients showed office hypertension, while 58% of subjects had daytime hypertension, and 76% of patients had nighttime hypertension. also, diastolic, systolic, and mean blood pressure values during sleep were significantly related to apnea-hypopnea index and age (91). another study revealed that blood pressure night/day ratios in patients were associated with severity of osas (92). moreover, a casual relationship between the increasing respiratory disturbance index and the average 24 hour systolic blood pressure was only observed in non-dipping individuals and hypertensive osas subjects (93). the repeated end-apneic arousal and/ or hypoxic asphyxia and the subsequent sleep fragmentation contributed to nocturnal and diurnal elevation of bp (94). furthermore, the hypertension is associated with a greater exacerbation of shortterm variability during sleep in osas patients (39). moreover, the relationship between osas and nocturnal hypertension might differ between males and females. interestingly, portaluppi et al. studied 100 new cases of hypertension in men and found that non-dipping individuals exhibited a blunting of nocturnal blood pressure dipping phenomenon and an increased variability in bp associated with osas (29). this suggested that hypertensive non-dipping individuals had a high probability of coexisting sleepdisordered breathing. however, a significant relationship of nighttime/daytime blood pressure difference and ahi existed in men but not in women (95). osas-related hypoxemia and hypercapnia, sleep fragmentation, increased sympathetic activity, chemoreflex activation, and nighttime blood pressure surges may be involved in the increased peripheral vascular tone (nocturnal hypertension) and subsequent cardiovascular events in osas patients (figure 2). cardiovascular events of relevance include increased incidence of platelet activation, obstructive cardiomyopathy, cardiac arrhythmias, myocardial ischemia or/and infarction, and sudden cardiac death (4,17,96–99). osas may also increase the incidence of hemorrhagic and ischemic stroke (100–102) and subsequent neurological injury and cognitive impairment (103,104). resistant hypertension osas is an important secondary cause of resistant hypertension and is particularly common in patients with resistant hypertension (105). resistant hypertension, also called refractory hypertension, occurs where high blood pressure is not returned to within normal ranges with use of three drugs, including diuretics. a significant gender difference is typical, with osas being more prevalent and more severe in male than in female patients (106). two cross-sectional studies demonstrate that the more severe the osas, the less likely blood pressure could be controlled despite increasing the number of antihypertensive agents (107,108). osas was extremely common in subjects with resistant hypertension, and a significant correlation between plasma aldosterone concentration and osas severity was observed in subjects with resistant hypertension but not in control subjects (109), suggesting that aldosterone excess may contribute to osas severity. osas nocturnal hypertension cardiovascular events figure 2. a schematic link between osas and nocturnal hypertension leading to cardiovascular events. 374 w. zhang & y. liang masked hypertension masked hypertension is defined as a normal clinic blood pressure and elevated out-of-clinic blood pressure, assessed using self-monitoring of blood pressure by the patients at home (110,111). masked hypertension includes stress-induced hypertension, morning hypertension, and nocturnal hypertension, which is defined as a sleeping blood pressure >120/ 70 mmhg. recently, baguet et al. showed a large incidence of masked hypertension, as determined by ambulatory bp monitoring (abpm), in patients with osas who were considered normotensive. the authors concluded that masked hypertension is frequently underestimated in osas and is nearly always present when clinic blood pressure is >125/ 83 mmhg (112). a recent study revealed that among apparently normotensive male osas patients, masked hypertension is present in one-third of patients and there is a progressive impairment of arterial stiffness in osas patients with masked hypertension, indicating that the diagnosis of masked hypertension may be underestimated in osas patients and that osas has an association with arterial stiffness independent of masked hypertension (113). moreover, obesity may be involved in the relationship between osas and masked hypertension. although common etiologies of masked hypertension include stressful living situations, sleep apnea resulting from obesity is also a possible etiology (114). however, the relationship between masked hypertension and osas needs more study. pulmonary hypertension the first report regarding osas and pulmonary hypertension was in 1988. no significant correlation existed between pulmonary arterial pressure and the apnea index (115). however, while intravascular pulmonary arterial pressure decreased during apneas and increased at the resumption of breathing, transmural pulmonary arterial pressure showed a progressive increase during apneas that decreased once ventilation had been resumed, suggesting pulmonary arterial pressure might be related with osas (116). the pulmonary artery hypertension was linked to the presence of an obstructive ventilatory pattern, hypoxemia, and hypercapnia, while the severity of osas plays only a minor role (117). in a study that recruited 92 consecutive patients, osas was found to be an important independent risk factor for pulmonary hypertension (118). mechanism underlying the link between osas and hypertension the main acute physiological consequences of osas are intermittent hypoxia, intrapleural pressure changes, and arousals, which might induce endothelial dysfunction, sympathetic activation, lipid metabolism dysfunction, increased oxidative stress, etc. (figure 1). all of these consequences could increase the artery tone and arterial stiffness, and thereby increase the risk of systemic hypertension and further cardiovascular diseases such as stroke and atherosclerosis. intermittent hypoxia intermittent hypoxia, particularly if associated with episodes of intermittent reoxygenation, is one of the most important features in osas (119). intermittent hypoxia causes remarkable blood oxygen desaturation, commonly within a cycle time of less than 1 min. to a certain degree, the intermittent hypoxia induced a repetitive hypoxia/reoxygenation cycle in osas, resembling ischemia-reperfusion injury, by promoting the production of reactive oxygen species (ros), activating systemic inflammation, and ultimately impairing endothelial function. apart from the induction of systemic inflammation and oxidative stress, intermittent hypoxia increased the plasma level of vasoconstrictive endothelin1 (120). additionally, intermittent hypoxia has been proposed to enhance peripheral chemoreceptors and the sympathetic nervous system activity (121). moreover, hypoxia-induced cellular responses, such as apoptosis (122) and autophagy (123,124), have to be considered. endothelial dysfunction endothelial dysfunction is a systemic pathological state of the vascular endothelium and can be broadly defined as an imbalance between vasorelaxation and vasoconstriction substances produced by the endothelium (125). endothelial dysfunction is an early marker of vascular damage that precedes clinically overt vascular disease and might be an important promoter of cardiovascular events in patients with osas (4,126). the strong association between osas and cardiovascular diseases may be due to significant endothelial dysfunction in osas patients and therefore is accumulating evidence for the association of osas and the impaired endothelial function/reduced endothelial repair capacity. osas and hypertension 375 a previous study showed that even mild osas was associated with reduced endothelium-dependent vasodilatation (127). greater impairment of endothelial function was undoubtedly associated with more severe osas (128–137). studies on hypertensive patients with osas revealed that there indeed is an association between osas and endothelial dysfunction. furthermore, in normotensive patients with osas, endothelium-dependent vasodilatation, measured by forearm blood flow, was impaired (138,139). nitric oxide (no), the most important vasodilatory molecule synthesized by the endothelium, decreased in patients with osas (138,140,141). in addition, the level and activity of endothelial progenitor cells, a population of rare cells that circulate in the blood with the ability to differentiate into endothelial cells, decreased in patients with osas (134,142–144). many above-mentioned signaling pathway proteins, including tumor necrosis factor, interleukins il-1, il-6, and il-8, nuclear factor kb (nf-kb), the renin–angiotensin system, and hypoxia-inducible factor-1 (hif-1) participate in the molecular mechanisms underlying the osas-induced endothelial dysfunction (4,145). these signaling proteins will be discussed in the next section. inflammation and oxidative stress the evidence that osas is associated with increased oxidative stress and inflammation is based on findings from both animals and humans. promoting inflammation and oxidative stress is a major cause of endothelial dysfunction by decreasing no availability and reducing endothelial vasodilatation capacity. cells exposed to intermittent hypoxia demonstrated selective activation of the pro-inflammatory transcription factor nf-kb, whereas the adaptive regulator hif-1 was not activated (146). in addition, elevated nf-kb activity induced by chronic intermittent hypoxia was accompanied by increased expression of inducible nitric oxide synthase (inos) level, a putative and important nf-kb-dependent inflammatory and oxidative protein (147). moreover, nf-kb could further stimulate the production of pro-inflammatory mediators such as il-8 and intercellular adhesion molecule 1 (icam-1) (4). tnf-alpha levels are independently associated with excessive daytime sleepiness, and il-8 has shown elevated levels in patients with osas compared with controls (148). a recent study raised the possibility that the obese osas patients might have elevated tnf-alpha levels compared to bmi-matched controls (149). these factors may contribute to the hypertension in osas. c-reactive protein (crp) plays an important role in hypertension (149,150) and is correlated with osas. shamsuzzaman et al. reported that plasma crp levels were significantly higher in patients with osas than in controls (150), suggesting that the severity of osas is proportional to the crp level, which was confirmed later by numerous cross-sectional, case-control, and non-randomized interventional studies (151-155). apart from these systemic investigations, cellular studies on lung might also provide some association between inflammation and osas (156,157). management and treatment of osas non-drug therapy weight loss is an effective treatment of obesity in osas patients. weight loss improves significantly sleep apnea and has favorable effects on blood pressure and baroreflex sensitivity (158). the improvement of obstructive sleep apnea after weight loss might be related to improvement in pharyngeal and glottic function (159). avoidance of alcohol is to be recommended as deterioration of osas occurs by alcohol abuse (160,161). continuous positive airway pressure (cpap) therapy the most common and highly efficient therapeutic procedure of eliminating airway obstruction is continuous positive airway pressure (cpap) therapy, a form of treatment first described in 1981 by sullivan et al. (162). this therapy exerts a blood pressure-lowering effect, reduces nocturnal sympathetic nerve traffic, blunts blood pressure surges, decreases nocturnal blood pressure surge, and improves cardiovascular prognosis in many osas patients (163–165). the benefit was larger in patients with more severe sleep apnea than in those who had less severe apnea, but was independent of the baseline blood pressure (166). however, there are also some studies reporting negative effects (167,168). lack of concordance may be due to heterogeneity of selected subjects and study design. thus, three meta-analyses were employed to summarize findings of intervention studies in cpap treatment. bazzano et al. reported that mean net change in systolic blood pressure for those treated with cpap application of 2–24 weeks (818 patients) compared with controls was – 2.46 mmhg (p < 0.05) (169). another meta-analysis included 12 trials (572 patients) and reported a significant –1.69 mmhg decrease in mean bp, with similar reductions in sbp and dbp (p < 0.05) (170). on the contrary, alajmi et al. reported that the effects of cpap on bp in osas patients 376 w. zhang & y. liang (572 patients) were modest and not statistically significant (p = 0.23) (171). the authors considered that in unselected patients with sleep apnea, cpap may have modest effects on bp, whereas they could not exclude the possibility that certain subgroups of patients may have more robust responses to cpap (171). obviously, although cpap has been the firstline therapeutic strategy for osas, the beneficial effect of cpap remains an open question. conclusions over the last decade, there has been increased interest in osas-related research and increased understanding of the osas-related cardiovascular complications. because osas is associated with hyper-tension and hypertension associated end-stage organ diseases such as stroke, coronary heart disease, and arrhythmia, the employment of cpap is highly encouraged as cpap therapy seems to assist blood pressure control at least in those with severe apnea, resistant hypertension, and daytime sleepiness. this field continues to be updated monthly. a number of key questions, however, remains to be resolved. more clinical research is warranted to characterize more fully the underlying mechanisms and to develop practicable strategies for osas treatment. declaration of interest: this work was supported by chongqing natural science grant (cstc2009bb3571). we thank medjaden bioscience limited for assisting in the preparation of this manuscript. the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. parati g, lombardi c, narkiewicz k. sleep apnea: epidemiology, pathophysiology, and relation to cardiovascular risk. am j physiol regul integr comp physiol. 2007;293:r1671– 83. 2. weiss jw, liu md, huang j. physiological basis for a causal relationship of obstructive sleep apnoea to hypertension. 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meta-analysis of randomized controlled trials. lung. 2007;185:67–72. 382 w. zhang & y. liang rationale for a swedish cohort consortium article rationale for a swedish cohort consortium johan sundstr€oma,b , cecilia bj€orkelundc, vilmantas giedraitisd, per-olof hanssone, marieann h€ogmana , christer jansona, ilona koupilf,g, margareta kristensonh, ylva trolle lagerrosi,j, jerzy leppertk, lars linda, lauren lissnerl, ingegerd johanssonm, jonas f. ludvigssonn,o, peter m. nilssonp, håkan olssonq , nancy l. pedersenn , andreas rosenbladk, annika rosengrene, sven sandinn,r,s, tomas sn€ackerstr€omb, magnus stenbeckt, stefan s€oderbergu, elisabete weiderpassn,v,w,x , anders wanhaineny, patrik wennbergz, isabel fortieraa, susanne hellerb, maria storg€ardsb and bodil svennbladb adepartment of medical sciences, uppsala university, uppsala, sweden; buppsala clinical research center (ucr), uppsala, sweden; cdepartment of public health and community medicine/primary health care, institute of medicine, sahlgrenska academy, university of gothenburg, gothenburg, sweden; ddepartment of public health and caring sciences, uppsala university, uppsala, sweden; edepartment of molecular and clinical medicine, institute of medicine, sahlgrenska academy, university of gothenburg, gothenburg, sweden; fdepartment of public health sciences, stockholm university, stockholm, sweden; gdepartment of public health sciences, karolinska institutet, stockholm, sweden; hdepartment of medical and health sciences, division of community medicine, link€oping university, link€oping, sweden; idepartment of medicine, unit of clinical epidemiology, karolinska institutet, stockholm, sweden; jdepartment of endocrinology, metabolism and diabetes, karolinska university hospital huddinge, huddinge, sweden; kv€asterås centre for clinical research, uppsala university, uppsala, sweden; ldepartment of public health and community medicine/epidemiology and social medicine, institute of medicine, sahlgrenska academy, university of gothenburg, gothenburg, sweden; mdepartment of odontology, school of dentistry, umeå university, umeå, sweden; ndepartment of medical epidemiology and biostatistics, karolinska institutet, stockholm, sweden; odepartment of pediatrics, €orebro university hospital, €orebro university, €orebro, sweden; pdepartment of clinical sciences, skane university hospital, malm€o, lund university, lund, sweden; qdepartment of clinical sciences, cancer epidemiology, lund university, lund, sweden; rdepartment of psychiatry, icahn school of medicine at mount sinai, new york, ny, usa; sseaver autism center for research and treatment at mount sinai, new york, ny, usa; tdepartment of clinical neuroscience, karolinska institutet, stockholm, sweden; udepartment of public health and clinical medicine, and heart center, umeå university, umeå, sweden; vdepartment of research, cancer registry of norway, institute of population-based cancer research, oslo, norway; wgenetic epidemiology group, folkh€alsan research center, faculty of medicine, helsinki university, helsinki, finland; xdepartment of community medicine, university of tromsø, the arctic university of norway, tromsø, norway; ydepartment of surgical sciences, uppsala university, uppsala, sweden; zdepartment of public health and clinical medicine, family medicine, umeå university, umeå, sweden, and; aaresearch institute of the mcgill university health centre, montreal, canada abstract we herein outline the rationale for a swedish cohort consortium, aiming to facilitate greater use of swedish cohorts for world-class research. coordination of all swedish prospective population-based cohorts in a common infrastructure would enable more precise research findings and facilitate research on rare exposures and outcomes, leading to better utilization of study participants’ data, better return of funders’ investments, and higher benefit to patients and populations. we motivate the proposed infrastructure partly by lessons learned from a pilot study encompassing data from 21 cohorts. we envisage a standing swedish cohort consortium that would drive development of epidemiological research methods and strengthen the swedish as well as international epidemiological competence, community, and competitiveness. article history received 30 november 2018 accepted 30 november 2018 keywords common infrastructure; epidemiological research; pilot study; rare outcomes; swedish cohort consortium background united we stand, divided we fall at the turn of the millennium, it was recognized that candidate gene association studies generated a large amount of non-replicable results. this rapidly led to a common understanding among genetic epidemiologists of the need for very large sample sizes in order to generate robust results. these insights seem not to have disseminated consistently to non-genetic epidemiology. effectively, the scientific literature is flooded with underpowered studies, and it is becoming increasingly recognized that these studies cannot be replicated, casting doubts on the credibility of research results in general (1). hence, there is an urgent need for data resources where research results can be replicated in an independent sample. further, for optimized return on investment of taxpayers’ and other funders’ money and maximized benefit to patients and populations, it is essential that researchers other than those who originally assembled a database can use and re-use those data (2). these insights contact johan sundstr€om johan.sundstrom@medsci.uu.se � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 1, 21–28 https://doi.org/10.1080/03009734.2018.1556754 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1556754&domain=pdf http://orcid.org/0000-0003-2247-8454 http://orcid.org/0000-0002-6392-6092 http://orcid.org/0000-0002-8794-9635 http://orcid.org/0000-0001-8057-3543 http://orcid.org/0000-0003-2237-0128 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1556754 http://www.tandfonline.com point, as the way forward, to increasing access to individual cohorts and leveraging integration of data across studies to obtain the statistical power required to answer contemporary research questions. why another cohort consortium, and why in sweden? the recognition of the need to collaborate has led to development of consortia of cohorts in the last decade. many swedish population-based cohorts already participate in a multitude of international cohort consortia. some consortia are defined by access in the participating cohorts to specific exposures (e.g. the emerging risk factors collaboration, the international database on ambulatory blood pressure in relation to cardiovascular outcomes, or research on european children and adults born preterm) (3–5); others are defined by access to specific outcomes (e.g. the european prospective investigation into cancer and nutrition) (6); while yet others include broad selections of cohorts with access to very general exposures (e.g. the ncd risk factor collaboration) (7). hence, these consortia are by design limited in terms of inclusion and exclusion criteria for the participating cohorts and data content. sweden is a country with unique opportunities for epidemiological research. together, the governmental agencies national board of health and welfare (sos) and statistics sweden (scb) host a multitude of individual-level data of great importance for epidemiological researchers (exemplified in table 1 and on www.registerforskning.se). using the 12-digit personal identity number, unique to all swedish citizens, information from the registers can be uniquely linked (8). because most swedish prospective population-based cohorts have access to outcomes collected in these registries in a structured way across the whole nation, swedish cohorts have close to complete follow-up for all its participants except for those who emigrate. all events that are severe enough to result in e.g. death, an admission or visit to any hospital (diagnoses classified using the international classification of diseases [icd] system), a surgical procedure (classified using the nordic medico-statistical committee classification of surgical procedures [ncsp] system), or a filled drug prescription (classified using the anatomical therapeutic chemical classification [atc] system) are collected in the same way in all cohorts. hence, a cohort consortium consisting exclusively of swedish prospective population-based cohorts will bring to the table a unique possibility to study uncommon exposures and outcomes, classified prospectively. other prerequisites for success the prospective population-based cohort study in a rich official registry setting—where a defined, prospectively examined group of people is followed over time based on a personal identity number for register linkages—is a highly valuable observational study design. sweden has a large number of carefully collected population-based cohorts that have been followed for decades. we have ongoing recruitment into several high-quality cohorts. as mentioned, we have a variety of national socio-demographic and medical registries, covering the whole population since many decades. in addition, we have a large number of leading epidemiological researchers in the country, with a combined knowledge spanning most current research fields. the swedish government has also recently provided strategic targeted funding to epidemiological research. however, swedish cohort research is poorly coordinated today. many research projects are underpowered by using only one cohort at a time, leading to uncertain results with little benefit to patients and the public. furthermore, rare diseases and exposures are impossible to study in individual cohorts due to lack of statistical power and are therefore discriminated. hence, the stage is set for an initiative to unite all swedish cohorts in an infrastructure for cohort collaboration. pilot study in order to gain experience from administering a collaborative cohort project, we undertook a study to identify risk factors for subarachnoid haemorrhage (sah) among more than one million cohort participants in 21 cohorts (9). sah was chosen because it is comparatively rare and most cohorts would be too small to study this condition. below, each step is first described and then commented. eventually, the study was completed and presented at a scientific meeting (9). the time invested in the different processes is illustrated in figure 1. the table 1. examples of swedish official registries. registry contents swedish total population registry (14) place of residency; country of own and parents’ birth; marital status; date of death or emigration swedish censuses socio-economic group; education; income; sick leave swedish national insurance agency sick leave; pensions swedish education registry highest education swedish 9th grade registry junior high school grades swedish multi-generation registry (15) number of children and siblings; identity of parents if born after 1932 and alive in 1961 swedish medical birth registry (since 1973) (16) numbers of pregnancies and births; pregnancy outcomes swedish prescribed drug registry (since 2005) (17) pharmacy-expedited drug prescriptions swedish inpatient registry (since 1964, with complete national coverage since 1987) (18) diagnoses of all hospitalizations; surgical and other procedures swedish cancer registry (since the 1950s) (19) all cancer diagnoses swedish cause-of-death registry (20) causes of death, including contributing factors swedish out-patient registries (day-care surgery since 1997, all others since 2001) all diagnoses. hospital-based mandatory; primary care voluntary 22 j. sundstr€om et al. http://www.registerforskning.se pilot study helped us chart and quantify the multitude of obstacles involved in cohort research in sweden in general, and especially in a collaborative cohort project. the lessons learned from that project were essential for the development of the current proposal. inclusion of cohorts the first invitation to participate in the study was sent in february 2011 to 35 epidemiologists and data managers representing 29 swedish cohorts. because we did not know of all potential cohorts at the start, additional cohorts were invited and included until 2014. ultimately, 21 cohorts participated in the study. requirements for cohort data were stated in a research proposal, with very few inclusion (data on systolic and diastolic blood pressure and smoking status) and exclusion (previous sah) criteria. each cohort had to be enriched with data from official registries hosted by the national board of health and welfare (sos) or statistics sweden (scb). as it turned out, all but five cohorts were enriched with such data. reasons for cohorts declining participation in the project were of different kinds; most often the cohort holders did not have the time, or data were deemed unreliable, incomplete, or unsuitable for the study by the cohort holders. many cohorts required applications on their own forms, which was very time-consuming to comply with. some cohorts did not have a steering group or any formal procedures for approval of projects, which tended to hamper communication. ethics approval the primary application to the ethics review board was submitted when all cohorts were identified, and cohort holders had agreed to participate in the project. in the first round of comments, the ethics review board required details on inclusion criteria for all cohorts, descriptions of the data extraction procedure from each cohort, details on the information given to all participants in each cohort at enrolment and examples of informed consent forms, and descriptions of the informed consent procedures in each cohort. after submission of these complementary documents to the ethics review board (figure 2), they found the information to the figure 1. time (h) spent on processes in the pilot study. sap: statistical analysis plan. figure 2. resubmission to the ethics review board. upsala journal of medical sciences 23 participants about the present study insufficient. the ethics review board required an advertisement in daily news press, with instructions on how to opt out of the project. this was done. the ethics review board approved the study after 6 months of correspondence. none of the 949,683 cohort participants opted out of the study. one of the great benefits of cohort studies is the possibility to study many outcomes, including ones that were not identified at the time of the design of the cohort. the ethical and societal gains from that possibility (the eliminated need for conducting a new cohort study to answer every new research question) need to be better recognized and balanced against the limited potential harms. table 2. proposal for a swedish cohort consortium (cohorts.se). component description data management some swedish cohorts are kept at the highest possible standard, with secure storage of data in modern data formats, including backups, controlled data access, and access logs. many also have curated data with complete and documented procedures for data curation as a result of e.g. logical checks, monitoring, adjudication, or outlier detection; and with data curation traceable through an auditing feature in the data warehouse. unfortunately, many valuable cohorts are kept under suboptimal circumstances in some or all of these respects. sharing experiences and tools for efficient data management among cohort owners is an important feature of a cohort infrastructure. the maelstrom research offers guidelines (23) and a suite of open source software (24) (opal, onyx, mica, and agate) to support management of cohort data. optimally, all cohorts in the infrastructure would run a local instance of opal in conjunction with their other software stack (24). opal can be used to achieve data management and harmonization. it is integrated with r, and, using the datashield approach, such an infrastructure could support advanced statistical data analysis across cohorts without having to share or disclose individual-level data (11). cataloguing cohort steering groups hold detailed knowledge and documentation on the cohorts’ design, sampling procedures, data collection methods, losses to follow-up, data quality, and other cumulative knowledge about the data gathered through a multitude of research projects. given the complexity in the structure of the existing datasets and the rich content of the data, the experience of the cohort holders is invaluable for effective use of the data. this experience needs to be condensed into a useful metadata repository. in cohorts.se, the maelstrom approach for study and variables cataloguing is proposed. a key activity in joint cohort projects is searching for harmonization potential among cohorts with similar data. a prerequisite for this is a metadata catalogue containing structured details about the cohort design and the variables and samples collected in the cohort study. in cohorts.se, it is proposed to use the mica software offered by maelstrom. mica is a software application for web portals for individual cohorts or for consortia. when used in conjunction with the maelstrom component opal, for data management, mica also allows authenticated users to perform distributed queries on the content of study databases hosted on remote servers and retrieve summary statistics (24). a pilot catalogue including 6 studies and 10,000 variables is accessible on www. maelstrom-research.org/mica/network/cohorts.se enrichment in order to stay relevant, epidemiology needs collection of new cohorts, and re-investigation and expansion of existing cohorts. cohorts.se proposes to customize and use the maelstrom’s onyx software to support high-quality collection of new cohorts. onyx is a web-based application that manages participant logistics at cohort assessment clinics, including appointment management, controlling stage availability and dependencies, consents, questionnaires, sample collection (barcode scanning), linkage to sensory equipment such as ecgs or scales, producing personalized reports for participants, and exporting encrypted data to multiple destinations. in addition, cohorts.se will aid in variable selection for new investigations of cohorts, guided by knowledge of usefulness of existing variable lists in other cohorts in cohorts.se. cohorts.se is also proposed to provide standardized integration of commonly used data sources like public national registries. project management for researchers to conduct projects using the consortium, some information needs to be readily available, including cohort metadata, variable lists, definitions of variables, and standardized application forms for research proposals. we propose that cohorts.se provides basic support for submission of research proposals, coordinates decisions to join a collaborative project, coordinates data access, provides statistical support as needed, and facilitates critical steps in the publication process. further, cohorts.se should facilitate compliance with legal and ethical requirements, and ensure complete anonymization of data before delivery to researchers, facilitate meta-analysis or, where relevant, use federated analysis such as datashield. harmonization in order to make joint analyses, variables from different cohorts need to be measured on the same scale. using the maelstrom approach, harmonization of variables is facilitated and documented in stored scripts (25). harmonized data can then be analysed within the maelstrom system, or exported for analysis on other analysis platforms. the harmonization process is unique for each research project, but parts will be re-usable in subsequent projects. these parts will grow over time with shorter and shorter time needed for reprocessing. power calculations cohorts.se should be able to provide data useful for statistical power calculations for all cohorts storing data on an opal server connected to mica. at the moment, user-friendly tools for determining adequate sample size for more complex analyses that include clustered data, family designs, or multiple interactive effects are not readily available. power calculations require detailed knowledge about the structure and contents of data in specific cohorts, including information on missing data for combinations of variables. assuring that the existing data are adequate for a proposed analysis will likely be a common question. statistical analysis analysing harmonized data from different cohorts today often means transferring and pooling of individual data into a single large database, but that practice may be infeasible because of time, ethical, or legal issues. datashield offers a possibility to analyse data as if they were pooled, though still stored at local opal servers behind firewalls (11). cohorts.se can set up analysis servers at several participating universities, permitting different analyses of the joint cohort data from multiple access points. local cohort operations the backbone of cohorts.se is the participating cohorts and all persons involved in the management of the cohorts. for a successful infrastructure, incentives must be developed for the cohort holders to keep their cohorts safely stored in modern formats on adequate hardware, accessible for research projects, up to date in the cohorts.se catalogue, sufficiently staffed with data management personnel, and governed by a functioning steering committee or other body. within cohorts.se, a charter with recommendations of levels of governance, maintenance, data storage, and data curation will be proposed. 24 j. sundstr€om et al. http://www.maelstrom-research.org/mica/network/cohorts.se http://www.maelstrom-research.org/mica/network/cohorts.se data processing agreements personal data cannot legally be transferred between entities without a written agreement between the data controller (the sending part) and the data processor (the receiving part). such agreements were set up between each cohort and uppsala clinical research center (ucr). it is the responsibility of the data controller to set up such agreements, but ucr did this as an extra service. many cohort representatives were entirely unfamiliar with the legal requirements. in some cases, ucr had not received copies of the signed agreements from the cohort representatives after two reminders, although ucr had signed them and received the data. in some cases, the cohorts had developed their own data transfer agreement forms, with unknown relationships to the legal data controlling entity (typically a university). data transfer the project used the ucr data transfer system, which complies with swedish and eu data security laws (21, 22), and ensures that data are handled to the highest possible standard, with access restrictions and data encryption in transit and at rest. contact between data managers for each cohort and the data manager at ucr was essential for secure data transfer in accordance with ucr standard operating procedures. in some cases, no cohort data manager was available, and the cohort holder or a research assistant made the data transfer. it is of high importance to identify the data manager early in the process for most effective communication. one researcher sent the data using hotmail, which led to a notification by the quality assurance team at ucr. a federated system such as datashield (11) without the need to physically send data would better safeguard participants’ integrity. enrichment with registry data five of the 21 cohorts were not enriched with data from scb and sos at the time of the invitation to the study. for these five cohorts, the ucr project manager applied for selected variables from sos and scb. an application was written to sos, and the same form was used for the application to scb. the ethical approval together with a protocol synopsis were attached as well as a list of needed variables. the five cohort datasets were sent with personal identification numbers to sos. sos delivered the enriched datasets to scb. scb enriched the datasets further and sent them pseudonymized (without personal identification numbers) to the research group at ucr. this procedure was very time-consuming. one reason was that sos and scb appeared to have no direct communication between each other. a federated system such as datashield (11) (and the system proposed in an accompanying article in this issue of the journal) with participation of sos and scb, would be preferable for optimizing speed and integrity. data harmonization the final database required harmonization of a dozen variables. this harmonization is a crucial step in a project where the aim is to analyse pooled data. the harmonization work (processing study-specific data under a common format) was performed at ucr, and took a very long time for several reasons besides the difficulties related to data management and processing. the documentation for many of the variables (data dictionaries) was incomplete, or even non-existent, for many of the cohorts. the relevant staff at the cohorts was not always identified at the start, and finding the right person could be cumbersome. even when the right person was identified, responses by mail or telephone were sometimes very slow. preliminary results were presented to the cohort holders and data managers at a teleconference meeting. misunderstandings of the research proposal were sorted out. solving those issues was quite swift, and the database was considered final soon after the meeting. using dedicated tools such as maelstrom (23) for data descriptions and dictionaries and for harmonization is likely to considerably speed up the process. presenting descriptive analyses at a teleconference at an early stage is also likely to speed up harmonization—inspection of variable distributions stratified by cohorts gives opportunities for cohort representatives to identify mistakes or misunderstandings. statistical analysis a statistical analysis plan was presented in 2014, specifying that associations of risk factors with sah should be analysed using cox proportional hazards models, accounting for the clustered data structure using shared frailty models. during the analysis work, we decided to change to poisson models instead. reasons for this were that two time scales were of interest (calendar time and age) and that cohorts collected decades apart were difficult to model using cox regression. other peculiarities in the data observed at the time of analysis were that not all ages were present in all calendar periods; and problems with correctly identifying events and event-free time because registries used to capture the outcome did not cover all of sweden until 1987, and because some cohorts with very long follow-up needed to account for several icd code versions and had not done so initially. it is a good idea to study descriptive statistics for each cohort in parallel with statistical analysis plan development and share those data with the cohorts, both in order to check harmonization of key variables and to make sure that the plan fits the data. principles for missing data imputation should be determined at the outset, but may need to be refined once data are in. good communication between principal investigator/statistician/data manager is important. proposal we have led an initiative for a national collaborative infrastructure, the swedish cohort consortium (cohorts.se). the full proposal can be found on www.cohorts.se, with contributing cohorts and upsala journal of medical sciences 25 http://www.cohorts.se researchers listed on https://snd.gu.se/sv/catalogue/keyword/ cohortsse; table 2 provides a condensed outline. the long-term scientific goal is to facilitate greater use of swedish cohorts for world-leading research; excellence would mainly be achieved by making cohort data discoverable and more accessible and supporting collaborations between cohorts. important components of such an infrastructure would build on open access and open science and involve data curation and management, cataloguing study designs and variables content, developing common procedures for access to data, harmonizing variables to support research projects, linking cohorts and official registries using novel techniques, achieving statistical analysis including a method for distributed data analysis eliminating the need to send data between cohorts, and implementing collaborative web interfaces for researchers, data managers, statisticians, and publication managers. the proposed undertaking is not trivial. even with appropriate approaches such as these, the terminologies, procedures, technologies, and methods used vary markedly between cohorts. because of this complexity and the heterogeneity of information collected from pre-existing cohorts, integration of the information presents major challenges. achieving scientifically valid harmonization requires secure data environments and specialized expertise and resources. our initiative aims to fill this need. ethical considerations ethical, legal, and social implications of collaborative cohort analysis arise from the fact that cohorts are initiated with different goals, and that participants in different cohorts therefore will have been given different information about the intended use of the data. the consent procedures may vary widely across cohorts. permission from an ethics review board is a valid proxy for consent from the cohort participants when applied to secondary analyses of the data (10), and handling of all collaborative projects in the infrastructure at an ethics review board experienced in such research, such as the procedure in the uk biobank, would be a great advantage. other ethical issues may result from an increased accessibility of the cohorts to the research community, including risks of breaches of security and privacy. cohorts.se aims to develop appropriate means of data access that ensure privacy and secure data handling. in order to protect the privacy of individuals, but at the same time utilize the strength that some individuals participate in multiple cohorts, a novel secure technique has been proposed for joining cohorts using the personal identity number (see accompanying paper by sn€ackerstr€om and johansen in this issue of the journal). use of datashield (11) also minimizes the risk. the main ethical gains from facilitating collaborative cohort analysis include benefits to patients and populations in the form of more precise, timely, and reliable research findings due to better-powered studies, and research findings that would be impossible to obtain without collaborative analysis. ethical gains from increasing the accessibility to and use of cohort data are that study participants’ data are better utilized and their donation therefore becomes more valuable, with higher benefit to patients and populations. other ethical advantages may include safer management of sensitive person data and higher equality in access to necessary support functions. today, the management of some cohorts is very vulnerable, with insecure backup routines and key knowledge about the cohort maintained by single persons. in cohorts.se, data management will be significantly improved compared to the standards in place at the weakest environments today. we propose common data and material transfer agreements for all cohorts, saving time and ensuring that all cohorts are treated equally. we also propose transparent and clearly stated common rules for liabilities, access rights, and limits of use based on international charters for sharing and access to data. potential gains to society less waste in research cohorts.se will facilitate modern data curation and management, which will allow us to increase the level of data security, ensuring future-proof storage of valuable swedish cohorts. there is a clear threat of total oblivion for older cohorts with low to minimal use and vulnerable management structures. cohorts.se will catalogue all cohorts in a detailed and structured way, which will facilitate use of valuable cohort data that are known only to a smaller circle of researchers today and therefore under-used. a pilot catalogue including six studies and 10,000 variables is accessible on www.maelstrom-research.org/mica/network/cohorts.se. table 3. international infrastructures with which collaboration may be sought. name website the asia-pacific cohort studies collaboration www.apcsc.net emerging risk factors collaboration www.phpc.cam.ac.uk/ceu/research/erfc prospective studies collaboration www.ctsu.ox.ac.uk/research/meta-studies/psc/psc-website monica risk, genetics, archiving and monograph (morgam) www.thl.fi/morgam biomarker for cardiovascular risk assessment in europe (biomarcare) www.biomarcare.eu national cancer institute (nci) cohort consortium http://epi.grants.cancer.gov/consortia/cohort.html european cohort consortium (follow-up to bbmri-lpc) www.bbmri-lpc.org research on european children and adults born preterm (recap) https://recap-preterm.eu/ the maelstrom software suite (23) is used for several of these collaborations, including the nci cohort consortium, bbmri-lpc, and recap. using the same suite of tools for cohorts.se will facilitate collaboration with those international consortia. some swedish cohorts are already catalogued in maelstrom, including ulsam, epihealth, lifegene and twingene. the continuation of the bbmri-lpc is uncertain, but there are initiatives to transform it into a european cohort consortium, which will most likely be open for inclusion of more swedish cohorts. some of the other networks that use maelstrom are described on https://www.maelstrom-research.org. 26 j. sundstr€om et al. https://snd.gu.se/sv/catalogue/keyword/cohortsse https://snd.gu.se/sv/catalogue/keyword/cohortsse http://www.maelstrom-research.org/mica/network/cohorts.se http://www.apcsc.net http://www.phpc.cam.ac.uk/ceu/research/erfc http://www.ctsu.ox.ac.uk/research/meta-studies/psc/psc-website http://www.thl.fi/morgam http://www.biomarcare.eu http://epi.grants.cancer.gov/consortia/cohort.html http://www.bbmri-lpc.org https://recap-preterm.eu/ https://www.maelstrom-research.org cohorts.se will reduce the time wasted waiting for results. as an example, the 14,000,000 person-years of follow-up in the pilot study of this infrastructure (9) would take uk biobank (500,000 screened subjects) 28 years to acquire. this means that cohorts.se will allow world-leading research that may be impossible to achieve elsewhere today. a common infrastructure for all cohorts can facilitate access to data about the cohort participants held by statistics sweden and the national board of health and welfare, and potentially reduce the very long (currently up to 2 years) waiting times for data at these governmental agencies. combining multiple cohorts permits better-powered solutions for any research question that is today explored in single cohorts, generating more precise results. but it is especially valuable as it permits timely and adequately powered research on rare diseases, rare exposures, and extreme levels of exposures. building competence, community, and competitiveness cohorts.se will provide collaborative interfaces for researchers, data managers, and biostatisticians, including national meetings and educational efforts. we have good reason to believe that this will drive development of epidemiological research methods, increase the quality of research projects, maximize the prospects of getting research projects funded by international funding agencies, and in the long run secure excellent research environments and influx and retention of excellent researchers. this has been the case in norway, where a structured collaboration between cohorts over two decades has played an important role in developing epidemiological research environments and projects and facilitating cohort collaborations (12). further support for the forecast that a cohort collaboration infrastructure will drive research excellence comes from the emerging risk factors collaboration. this task has led to a very strong development of cohort research methods, the legacy of which includes important publications in leading medical journals, a large suite of statistical software developments, shared freely on their website (13), as well as personal experience, competence, and networks among the leading researchers. this proposal has been developed at two national workshops among cohort researchers taking part in the pilot study of cohorts.se, and subsequently in writing groups composed of researchers from all parts of sweden. participants in these activities have reported the networking with new acquaintances with similar interests as very fruitful. the popularity of this proposal is also reflected in the fact that since the initiation of the pilot study the number of cohorts in the initiative has more than doubled, with a total of more than 40 cohorts at seven universities participating in the proposal. cohorts.se aims to eventually embrace all interested prospective population-based swedish cohorts. the long-term strategic importance of cohorts.se for the swedish medical research community is great. it will add lasting value to existing cohorts, registries, and researcher groups, providing an opportunity for sweden to obtain a leading position in epidemiology and further attract leading international researchers. it can facilitate swedish participation and leadership in international cohort collaborations, such as those in table 3. the importance to the pharmaceutical industry of uncovering the pathophysiology of uncommon diseases may also be vital. conclusion in sum, coordination of all swedish prospective populationbased cohorts in a common infrastructure would enable more precise research findings and facilitate research on rare exposures and outcomes, leading to better utilization of study participants’ data, better return of funders’ investments, and greater benefit to patients and populations. we envisage a strong standing swedish cohort consortium that would drive development of epidemiological research methods, and strengthen the swedish epidemiological competence, community, and competitiveness. note on corresponding author johan sundstr€om is professor of epidemiology at uppsala university, and a cardiologist at uppsala university hospital. acknowledgements susanne heller, maria storg€ards, and bodil svennblad contributed equally. disclosure statement the authors report no conflicts of interest. orcid johan sundstr€om http://orcid.org/0000-0003-2247-8454 marieann h€ogman http://orcid.org/0000-0002-6392-6092 håkan olsson http://orcid.org/0000-0002-8794-9635 nancy l. pedersen http://orcid.org/0000-0001-8057-3543 elisabete weiderpass http://orcid.org/0000-0003-2237-0128 references 1. baker m. 1,500 scientists lift the lid on reproducibility. nature. 2016;533:452–4. 2. walport m, brest p. sharing research data to improve public health. lancet. 2011;377:537–9. 3. emerging risk factors collaboration, di angelantonio e, gao p, pennells l, kaptoge s, caslake m, et al. lipid-related markers and cardiovascular disease prediction. jama. 2012;307:2499–506. 4. li y, wei ff, thijs l, boggia j, asayama k, hansen tw, et al. i. ambulatory hypertension subtypes and 24-hour systolic and diastolic 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register. eur j epidemiol. 2017;32:765–73. 21. personuppgiftslag (1998:204). 22. general data protection regulation (gdpr). available from: http:// eur-lex.europa.eu/eli/reg/2016/679/oj. 23. fortier i, raina p, van den heuvel er, griffith le, craig c, saliba m, et al. maelstrom research guidelines for rigorous retrospective data harmonization. int j epidemiol. 2016;46:103–5. 24. doiron d, marcon y, fortier i, burton p, ferretti v. software application profile: opal and mica: open-source software solutions for epidemiological data management, harmonization and dissemination. int j epidemiol. 2017;46:1372–8. 25. fortier i, doiron d, little j, ferretti v, l’heureux f, stolk rp, et al. is rigorous retrospective harmonization possible? application of the datashaper approach across 53 large studies. int j epidemiol. 2011;40:1314–28. 28 j. sundstr€om et al. http://www.phpc.cam.ac.uk/ceu/research/erfc http://www.phpc.cam.ac.uk/ceu/research/erfc http://eur-lex.europa.eu/eli/reg/2016/679/oj http://eur-lex.europa.eu/eli/reg/2016/679/oj abstract background united we stand, divided we fall why another cohort consortium, and why in sweden? other prerequisites for success pilot study inclusion of cohorts ethics approval data processing agreements data transfer enrichment with registry data data harmonization statistical analysis proposal ethical considerations potential gains to society less waste in research building competence, community, and competitiveness conclusion note on corresponding author acknowledgements disclosure statement references proceedings of the upsala medical society: how it all started 150 years ago full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 proceedings of the upsala medical society: how it all started 150 years ago bo s. lindberg to cite this article: bo s. lindberg (2015) proceedings of the upsala medical society: how it all started 150 years ago, upsala journal of medical sciences, 120:2, 65-71 to link to this article: https://doi.org/10.3109/03009734.2015.1027430 © informa healthcare published online: 27 apr 2015. submit your article to this journal article views: 349 view related articles view crossmark data citing articles: 3 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://doi.org/10.3109/03009734.2015.1027430 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1027430 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1027430 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1027430&domain=pdf&date_stamp=2015-04-27 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1027430&domain=pdf&date_stamp=2015-04-27 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1027430#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1027430#tabmodule upsala journal of medical sciences. 2015; 120: 65–71 proceedings of the upsala medical society: how it all started 150 years ago bo s. lindberg malma ringväg 41a, 75645 uppsala, sweden key words: history, journal, medicine upsala läkareförening (the upsala medical society) was founded in 1832 on the initiative of professor israel hwasser, who was the chairman of the society until his death in 1860. the aims of the society were to promote medical science and create an atmosphere of friendship and collegiality among members of the faculty of medicine, medical students, and medical practitioners. an important function was the library of foreign books and journals that the society brought together. at regular meetings, a student was elected for each occasion as a vice chairman, and he would read a paper about a topic of interest, such as a case of a disease, or report on an article from the medical literature. during the first 30 years, no minutes from these lectures were kept, but during the early 1860s short records of the content of these lectures were written. the first lecture relating to own research was given in 1864 by the young medical student olof hammarsten, later a famous professor of medical and physiological chemistry. the theme for his discourse was bile acids, a subject for which he was to become world famous. as more original research was presented at the meetings, it became natural to try to disseminate the contents of the lectures to people outside the society and uppsala. in 1865, the professor of surgery, carl benedict mesterton, therefore suggested that the proceedings from the sessions of the society should be printed, and after a short deliberation the first volume of proceedings of the upsala medical society—upsala läkareförenings förhandlingar— was published in september 1865. it was distributed to 73 private persons and book-sellers, all of whom were subscribers. thirty-three foreign hospitals and medical associations abroad were sent free copies, and 11 other medical journals received a copy in an exchange agreement which proved to be very valuable. the first volume comprised just over 460 pages, but later the content of volumes expanded to more than 700 pages. the editor of the newly started ‘journal’ was robert fristedt (figure 1). a selection of the content during the years in the very first issue, the newly appointed professor in physiology, frithiof holmgren, reported a great discovery concerning the physiology of the senses: the retinal current. in the following decades, a lot of important articles about the senses were published. these are dwelt upon in a separate chapter in this supplement. other important contributors to the journal at that time, e.g. olof hammarsten, johan anton waldenström, and ivar sandström, also have their own chapters. regular authors from the beginning were august almén (professor of medical and physiological chemistry), frithiof holmgren, and edward clason (professor of anatomy). they contributed to the first issue with 8, 13, and 13 articles, respectively. correspondence: bo s. lindberg, malma ringväg 41a, 75645 uppsala, sweden. e-mail: bo.sven.lindberg@gmail.com (received 26 february 2015; accepted 28 february 2015) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1027430 http://informahealthcare.com/journal/ups mailto:bo.sven.lindberg@gmail.com august almén reported in 1867 on a method to detect sugar in the urine, later known as almén’s test (1). it relies on the reducing properties of glucose and other reducing sugars. the reagent is an alkaline bismuth solution. when heated in the presence of a reducing sugar, the bismuth is reduced to a black precipitate. the article was published in swedish. abroad, his method seems to have been unknown. in an 1883 review of test methods for sugar in the urine, george oliver states that the fehling test with cuprous oxide is the best available but does not even mention almén (2). in 1907 olof hammarsten compared almén’s test with some other sugar tests and found it superior (3). almén’s method was used in sweden for almost 100 years, until the test strips for glucose were introduced in 1957. a drug that for the first time gave the practitioner an effective remedy against insomnia was chloral hydrate. it was first synthesized in 1832, but not introduced into medicine until 1869, when mathias liebreich discovered its effectiveness in inducing sleep. chloral hydrate was used for short-term treatment of insomnia and as a sedative before minor surgical or dental operations. it was displaced in the late nineteenth century by bromide and in the mid-twentieth century by barbiturates. august almén, fredrik björnström (later professor of psychiatry at the karolinska institute), and gustaf kjellberg (the first professor of psychiatry in uppsala) wrote in proceedings of upsala medical society in 1869–1870 on the new drug only a few months after liebreich’s article, das chloralhydrat, ein neues hypnotikum, was published in berlin. almén was an analytical chemist, and he reported regularly in the journal on analyses of spring water from the many active mineral wells in sweden. he also was very interested in the quality of pharmaceuticals, mostly vegetables, and he reported on the yearly updates of the statutes for the pharmacies in the country. this latter interest made him leave the chair as professor, and he became the general director of the royal medicinal board in 1883. as professor of medical and physiological chemistry, almén was succeeded by olof hammarsten, one of the most famous scientists in uppsala around the turn of the nineteenth century. sanitary conditions and infectious diseases in 1857–1863 louis pasteur had demonstrated that fermentation is caused by the growth of micro-organisms. in the following years the role of these organisms in various diseases was discussed in the whole medical world, which figure 1. robert fristedt. fristedt (1832–1893) was professor in pharmacology and natural history. he was the editor of upsala läkareförenings förhandlingar from the very beginning 1865 to his death in 1893. photo: heinrich osti, 1877. uppsala universitetsbibliotek. kartoch bildenheten, id 12141. 66 b. s. lindberg was mirrored in the proceedings of the upsala medical society. at that time, many expeditions to remote parts of the world were undertaken. the famous swedish–finnish explorer, adolf nordenskjöld, went to spitsbergen in 1868 on a scientific expedition on the iron steamer sofia. the young physician carl hippolyt nyström (father of the surgeon gunnar nyström) was invited to act as the ship’s doctor. after a correspondence with pasteur, he decided to investigate the fermentation and putrefaction of meat at spitsbergen. his aim was to investigate if germs were as common in this remote and cold area of the world as in more inhabited countries. he found that, with what he said was a modern word, ‘protorganisms’ were present even at spitsbergen (4). the following year, in 1869, the swedish chemist henrik gahn in uppsala patented a preserving remedy which was called ‘aseptin’. it consisted of boric acid. gahn was a relative of the anatomist edward clason and had close connections to many of the professors at the university. the professor of pathology and editor of the proceedings, per hedenius, had suggested the brand name of aseptin. carl benedict mesterton, professor of surgery, recommended in advertisementsa dilution ofboric acid spiced with a decoct of clove called ‘amycos’ as an excellent antiseptic agent,less toxicthancarbolicacid.theprofessorofcivillaw,viktornordling,wasamemberoftheboardofhenrikgahnaseptinamycoscompanyinuppsala.pureorsimpleaseptinwasusedtopreservemilk,bread,andpreparedmeat,anddouble aseptin (two parts boric acid and one part potash alum) was used to prepare raw meat and fish. carl hippolyt nyström reported in the proceedings 1871–1872 on a trial with aseptin. he concluded that aseptin prevents the invasion of bacteria in liquids and meat and that it could put a stop to the putrefaction of meat at a concentration of 8% (5). in 1865, uppsala had 13,500 inhabitants. the population then increased rapidly, and in 1900 the town had 21,500 inhabitants. the sanitary conditions did not increase in parallel with the population growth. for many years a frequent issue at the meetings of the society and in its proceedings was constitutio epidemica. typhus, smallpox, malaria, diphtheria, and dysentery were serious infections contracted by the inhabitants of uppsala. in 1884 there was an epidemic of typhoid fever in uppsala. the source of infection turned out to be a stock of cattle just outside the town, the owner of which was the professor of civil law, viktor nordling. the case was very awkward for him as he was also the chairman of the town council. a delegation from upsala medical society with magnus blix as its chairman carried out an investigation and published a report in proceedings of the upsala medical society in 1884 (6). nordling was prosecuted in court on the charge of having violated the sanitary regulations. he was convicted and forced to resign from all his official tasks. the first great epidemic of cholera occurred in sweden in 1834. the disease then recurred in nine big waves until 1872. in 1892 there was a big outbreak in hamburg. the professor of medicine, salomon eberhard henschen, gave a lecture in the society, then published with the title: ‘how should we protect ourselves against cholera?’ (7). he stressed the importance of quarantine and that travellers from areas with cholera should be kept isolated until they were proved not infected. with this article henschen started a fierce dispute with august almén, who now was general director of the royal medicinal board. the nordic directors had agreed on common rules for all scandinavian countries with regard to quarantines. they had agreed that there was no need for medical expertise in the decisions about who must stay in quarantine and who should not. this could be decided by customs officers—an agreement henschen said was erroneous. customs officers without medical education did not have the competence to judge if a person was sick or not, particularly in early or abortive cases. such rules would only lull the community into a false sense of security. at the end of the nineteenth century, tuberculosis was one of the most common causes of death. for that reason, the public reacted euphorically to the discovery of the tuberculosis bacillus in 1882 by robert koch, since it aroused hopes for a cure. until that time, the only effective remedy for an infectious disease was quinine for malaria. at the tenth international medical congress held in berlin in 1890, koch introduced a cure for tuberculosis, which he called tuberculin. the public trusted the famous physician and reacted enthusiastically. tuberculin was tested in many countries, even in sweden by, among others, henschen in uppsala. he reported on his trial in the 1891–1892 volume of proceedings of the upsala medical society, having found, as others had, that tuberculin could cause severe adverse effects such as bleedings from the kidneys (8). he strongly advised against its use as a remedy, and instead recommended isolation of the contagious patients. once again, he came in conflict with august almén, when he suggested it should be mandatory to register the sick persons, as was the case in denmark and norway. almén thought that a register would further stigmatize the diseased, and not until 20 years later did it become mandatory to trace contagious persons and register them. pharmaceutical frauds in times when safety regulations of pharmaceutical preparations were still rudimentary, many authors warned colleagues in proceedings of upsala medical society about falsifications. the pharmacist edward august andberg proceedings of the upsala medical society 67 reported on falsified castoreum in 1865–1866, and ryno hoffstedt reported on falsified wax in 1871–1872. august almén wrote about hops adulterated with absinth in 1874–1875, and his wife’s nephew carl thore mörner (later professor of medical and physiological chemistry) wrote as a young medical student in 1888–1889 about falsified gummi resina asa foetida. asa foetida, an oleo-gum-resin, was traditionally used to treat various diseases, including asthma, gastrointestinal disorders, and intestinal parasites. in swedish it was called ‘dyvelsträck’. mörner wrote in the 1898–1899 volume about some examples of misuse of chemicals. one of these was the use of aseptin to preserve meat. the henrik gahn aseptin-amycos company had advertised that aseptin was quite harmless. mörner, on the contrary, showed that it might cause serious adverse effects and that it had been prohibited in many countries, e.g. germany (9). mörner collaborated with the physiologist thorsten thunberg, and they gave a speech on pharmaceutical fraudulence at the upsala medical society’s meeting of 30 october 1903 (10). together with fredrik clason, medical practitioner in uppsala and son of edward clason, they started ‘the office for information on advertisements on drugs’ in 1903. at the turn of the century, the statutes for pharmacies from 1683 were still applicable. the trade in what was called patent drugs and humbug drugs had spread into the pharmacies, and there was no general agreement on what was a genuine pharmaceutical drug. the office published a series of booklets about fraudulent drugs. their merciless but amusing disclosure of the contents of these drugs contributed to the new chemists shop license statute in 1913, which demanded lists of content of all pharmaceutical products. the office relinquished its activity when it had attained its purpose. perhaps most amusing of all articles in the proceedings is august almén’s juicy murder of ‘liebig’s meat extract’. justus von liebig was regarded as one of the great chemists of the nineteenth century, particularly in agricultural chemistry where he had identified nitrogen as essential to plant life. he also established the first laboratory-based school of chemistry at the university of giessen (now named justus liebig university giessen). nowadays he is considered a founder of organic chemistry, and the journal he created in 1832, annalen der pharmacie (today’s european journal of organic chemistry), is still very important in the field. in the 1860s there was a shortage of food, particularly bread and meat, in many countries, which caused riots. in south america there was a surplus of meat, but at that time it was not possible to transport the meat to the starving populations of europe. liebig had devised an efficient method of producing beef extract from cattle carcasses from south america. in 1865, he founded the liebig extract of meat company, marketing the extract as a cheap, nutritious alternative to real meat. in the swedish newspaper aftonbladet from february 1867, an uncritical article about the liebig’s meat extract was published. almén tested liebig’s extract and found it contained very little of fats and proteins. it was just a mixture of salts, spiced water, and some nitrogen-containing substances such as creatine and creatinine, but these are without nutritive value and normally excreted via the kidneys. the nutritive value of the liebig’s extract was almost nil. almén condemned the company for duping consumers in its advertising, particularly the claim that 34 pounds of meat were used to make a single pound of extract. such advertising, he argued, misled poor people into thinking they were consuming 34 pounds’ worth of beef nutrients with each jar. such a mixture of salts could be bought for 1% of the price of liebig’s extract. justus von liebig responded to almén’s criticism in a letter to the lancet, which almén commented upon in proceedings of upsala medical society, but he concluded in his article: ‘mundus vult decipi, ergo decipiatur’, which means: ‘the world wants to be deceived, so let it be deceived’ (11). surgery and obstetrics on 8 december 1869, the swiss surgeon jacques reverdin gave a lecture at a meeting of the société impériale de chirurgie de paris entitled greffe épidermique. he had successfully transplanted small pieces of skin, of partial or full thickness, removed from a healthy area, and seeded in a granulated wound surface to be covered. he published his experiences in 1872 in a book entitled de la greffe épidermique, but the new method seems to have become internationally known before the book was printed. carl benedict mesterton described skin transplantations in six patients in the year before. in only one case was the transplant rejected due to a purulent infection (12). his article was translated into english, published in british medical journal the same year, and mentioned in reverdin’s book. karl gustaf lennander succeeded mesterton in 1891 as professor of surgery. the disadvantage with the reverdin technique mesterton had used for skin transplantation was that the small pieces of skin were often detached from the wound after a while. the german surgeon carl thiersch in leipzig introduced another method at a congress in 1874 (his father-in-law was justus von liebig, whose meat extract almén had criticized). 68 b. s. lindberg he used skin grafts consisting of thin strips or sheets of epithelium with the tops of the dermal papillae cut off with a sharp knife. lennander started to use thiersching, as it has been called. in 1889, while not yet professor, he reported on his experiences. in most cases, the long-term results were good. remarkable is that he transplanted a piece of mucous membrane from the inside of the lower lip to the conjunctiva in two patients, one of whom had a sarcoma of the conjunctiva, the other with an ulcer on the nose and the eye-lid (13). lennander was the first in scandinavia to perform an appendectomy. the patient was a 28-year-old student with purulent peritonitis after appendicitis. after five months, the patient could be discharged from the hospital (14). appendicitis then became one of lennander’s main interests. in 1892 he published the results from his first 34 patients with only one deceased (15). at that time, it was not obvious that the treatment should be surgical. many internists argued for a conservative treatment. because of this opposition, lennander was keen on reporting the long-term results of the operation. his pupil and later professor in surgery in uppsala, gunnar nyström, reported in his thesis the results from 460 cases of non-purulent appendicitis from 1891, when the first patient was operated upon, until the end of 1905. only five patients had died during the hospital stay (16). in 1909, another of lennander’s pupils, knut harald giertz, defended his thesis on perforated appendices with peritonitis. of 553 patients, 20.5% died, and, because of the high mortality, he stressed the importance of early operation when appendicitis is suspected (17). in order to facilitate the access to the appendix and other organs in the right lower part of the abdomen, lennander introduced in 1897 a paramedian incision. its advantage was that the iliohypogastric nerve could be spared. it is sometimes still called the lennander incision (18). lennander is famous for his studies of the sensory nerves of the peritoneum. he found that there is a distinct difference in the sensitivity of the parietal and visceral peritoneum; however, he did not publish his findings in proceedings of the upsala medical society but in centralblatt für chirugie in 1901 (19). he also tried to prevent deep thrombosis after abdominal surgery. he had found that a dependent position of the legs may give rise to thrombosis of the veins. in these cases, therefore, the pelvis alone must be kept low, while the legs should be raised and maintained at a higher level than the buttocks. he also recommended massage of the legs and that the patient should lift and actively move his legs regularly when lying in the bed. infusions of solutions of sodium chloride should be given (20). at that time patients had to stay in bed for three to five weeks after an operation. the fact that early mobilization could prevent thrombosis was first proposed in scandinavia by the finnish gynaecologist edvard björkenheim at the eighth meeting of the nordic surgery society in 1909, but his message was forgotten until the 1940s. blood transfusions during the 1870s many attempts were made to save the lives of patients by means of transfusions of blood. in the tenth volume of proceedings of the upsala medical society 1874–1875 several articles were published on the subject. carl henrik björck reported on a case of chronic pneumonia treated with blood transfusion, and oscar viktor peterson reported on a case of transfusion with blood from a lamb and a case of sudden death where blood transfusion was tried. fredrik belfrage wrote about five cases of carbon monoxide poisoning treated with transfusion. johan anton waldenström tried in vain to get hold of a sheep when he was to give a transfusion to a 41-year-old woman who was bleeding heavily after a delivery. instead, blood from two powerful men was used. the patient displayed all the now classical signs of strong transfusion reactions and died within a few days. it was still 25 years before blood groups were discovered and safer therapy became available. proceedings of the upsala medical society and the outside scientific world proceedings of the upsala medical society was meant to be an organ for the upsala medical society relating what was discussed at its meetings. for many years, it was a window to the surrounding scientific world. the contents of medical journals from abroad, mainly germany, were reported. but it became evident that the communication was just one-way. as it was printed in swedish, the proceedings could not be read by more than a few. some great scientific discoveries remained unknown abroad for many years. not until holmgren published an account in german of his detection of the retinal current in 1879–1880 did this pioneering work become known abroad. what has been called the last anatomical discovery, the parathyroid glands, was published in the proceedings in swedish in 1879–1880 under the title ‘om en ny körtel hos menniskan och åtskilliga däggdjur’ by ivar sandström. for detailed information on his remarkable achievements you are referred to henry johansson’s chapter in this proceedings of the upsala medical society 69 issue (21). its importance remained mainly unknown during his lifetime. disappointment with the lack of acknowledgement may have contributed to his unhappy life and suicide. another example of late recognition is magnus blix’s discovery of the cold-, warm-, and pressure points in 1882. they became known in the scientific world outside sweden only when he published his findings in german in 1884. independently of each other, alfred goldscheider (professor at the university of berlin in 1884) and henry donaldson (at johns hopkins university in 1885) reported that the skin was not continuously sensitive but instead had specific loci for specific tactual senses. in many books on the history of medicine, blix now is said to share the priority with them, but in fact he published his findings two years before them. disputes about priorities were common even in the nineteenth century. one example is a letter that hermann cohn, ophthalmologist in breslau, wrote to the upsala medical society in 1878. he complained about an article holmgren had published on colour blindness and the distance between the pupils. holmgren refuted cohn’s allegation and expressed his regret that the struggle for priority caused too hasty publications (22). discoveries published in swedish always run the risk of being overlooked. this may have been the reason why frithiof holmgren founded skandinavisches archiv für physiologie in 1889. it was meant to be an organ for all physiologists in scandinavia. later, several other more specialized journals were established, but proceedings of the upsala medical society still received many manuscripts from the members of the society. in the 1920s, when hugo laurell was the editor, it started to publish in english, german, and french. later, even articles in german were not always paid attention to when anglo-saxon journals dominated. current contents, index medicus, and similar databases were not commonly used until the 1950s. thus, it was not easy to keep oneself always updated on the increasing flow of publications. concepts of the respiratory dead space were introduced during the latter part of the nineteenth century and have been continuously elaborated until the present time. a crucial stage in this development was the concept of the ‘volumen inefficax’ by henrik enghoff in 1931 (23) and his suggestion in 1938 that the pco2 of the arterial blood could be substituted for the alveolar pco2 (24). his contribution was overlooked at the time but, when rediscovered almost 10 years later, it was immediately hailed as a milestone in the understanding of respiratory function. enghoff was the first to demonstrate through arterial blood samples that the dead space varied during different physiological conditions. only at a later date was it realized that this essential advance had been made by enghoff. such appears to be the fate of so much work published in european journals. whatever the cause, there is no reference to enghoff in an article in american journal of physiology in 1946 by r. l. riley, j. l. lilienthal, d. d. proemmel, and r. e. franke, who in most people’s eyes laid the post-war foundations of quantitative interpretation of changes in alveolar pco2 and po2. modern clinical respiratory physiology and respirator treatment rest on enghoff’s theoretical framework on the functional dead space (25). declaration of interest: the author reports no conflicts of interest. the author alone is responsible for the content and writing of the paper. references 1. almén a. alkalisk vismutlösning såsom reagens på socker-haltig urin. upsala läkareförenings förhandlingar. 1866–1867;2:472–81. 2. oliver ga. on bedside urine testing: qualitative albumen and sugar. hk lewis, london: 1883. 3. hammarsten o. om värdet af det alménska och det worm-müllerska sockerprofvet vid urinundersökningar. upsala läkareförenings förhandlingar. 1906–1907;12:149–65. 4. nyström ch. om jäsningsoch förruttnelseprocesserna på spetsbergen. upsala 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studie über 533 fälle aus der chirurgischen klinik zu uppsala vom 1.9.1888-31.7.1907. dissertation uppsala: 1909. 18. lennander kg. om buksnitt genom endera rectusskidan med förskjutning af den mediala eller laterala kanten af m. rectus. upsala läkareförenings förhandlingar. 1897–1898;3:322–6. 19. lennader kg. über die sensibilität der bauchhöle und über lokale und allgemeine anästesie bei bruchund bauchoperationen. zentralblatt fur chirurgie. 1901;8:209–23. 20. lennander kg. om möjligheten att förekomma trombos i de nedre extremiteternas vener efter bukoperationer, jämte några ord om efterbehandlingen efter operationer för varicösa vener. upsala läkareförenings förhandlingar. 1899;4:329–36. 21. johansson h. the uppsala anatomist ivar sandström and the parathyroid gland. ups j med sci. 2015;120:72–7. 22. holmgren f. die cohn’sche sache. upsala läkareförenings förhandlingar. 1878–1879;14:537–97. 23. enghoff h. zur frage des schlädlichen raumes bei der atmung. skandinavisches archiv für physiologie. 1931;63:15–74. 24. enghoff h. volumen inefficax, bemerkungen zur frage des schädlichen raumes. upsala läkareförenings förhandlingar. 1938;44:191–218. 25. nunn jf, holmdahl mh. henrik enghoff and the volumen inefficax. ups j med sci. 1979;84:105–8. proceedings of the upsala medical society 71 http://www.ncbi.nlm.nih.gov/pubmed/384640?dopt=abstract ss1 a selection of the content during the years sanitary conditions and infectious diseases pharmaceutical frauds surgery and obstetrics blood transfusions proceedings of the upsala medical society and the outside scientific world declaration of interest references prevalence and risk factors for age-related cataract in sweden original article prevalence and risk factors for age-related cataract in sweden magnus hugossona and curt ekstr€omb adepartment of ophthalmology, university hospital, uppsala, sweden; bdepartment of neuroscience, ophthalmology, uppsala university, uppsala, sweden abstract background: cataract is a major cause of visual impairment worldwide. there is a paucity of prevalence studies from sweden. therefore, we report the prevalence of cataract and its risk factors in a population-based study of older adults in sweden. methods: the tierp glaucoma survey was conducted in the municipality of tierp, sweden, including 760 subjects aged 65–74 years. the presence of cataract was determined based on retroillumination, with lens opacities evident on slit-lamp examination. to assess risk factors for cataract, odds ratios (ors) were calculated, adjusted for age and gender. results: a total of 234 individuals were found to have cataract, 12 of whom had undergone cataract surgery. the prevalence adjusted for nonparticipation was 31.5% (95% confidence interval [ci] 29.4–33.6), 35.2% (95% ci 28.7–41.8) in females and 26.2% (95% ci 19.8–32.6) in males. cataract was associated with age �70 years (or 1.93; 95% ci 1.41–2.64), female gender (or 1.54; 95% ci 1.12–2.11), and myopia (or 2.3; 95% ci 1.16–3.56), while pseudoexfoliation, smoking, diabetes, hypertension, and ischaemic heart disease were not. conclusion: nearly one-third of the sample were estimated to have lens opacities, or had undergone cataract surgery, making cataract a frequent disorder of older age. the study provided further evidence that increasing age, female gender, and myopia are associated with cataract. article history received 28 april 2020 revised 18 june 2020 accepted 23 july 2020 keywords cataract; epidemiology; population survey; prevalence; risk factor introduction cataract remains a major cause of visual impairment worldwide (1). however, in many countries cataract surgery is easily available. in fact, cataract surgery is one of the most common surgical procedures performed in western countries. in sweden, with a population of 10 million inhabitants, approximately 110,000 cataract surgeries are performed annually according to the swedish cataract register (2). cataract is a major burden for health-care providers around the world. from a public health perspective, it is important to know the prevalence of undiagnosed lens opacities. identifying modifiable predictors for the development of cataract will make it possible to initiate strategies to delay the need for cataract surgery. from a societal perspective, it is also important to reduce the costs for health care, especially with an ageing population in mind. prevalence studies on cataract and its risk factors have been widely reported from various parts of the world in the past decades. the results of three of these studies on people in the age span of 65–74 years are presented in table 1. clearly, in population studies on lens opacities, prevalence rates are a function of the examination methods and diagnostic criteria used. several classification and grading systems for lens opacities have been developed, including the wisconsin cataract grading system (6) and the lens opacities classification system iii (locs iii) (7). in both systems, photographs were taken of the lenses and were compared to a set of standard colour transparencies of lens opacities. in other grading systems, the findings at biomicroscopy were categorized according to a written description of lens changes (3). previous studies have identified several risk factors associated with cataract, including smoking (8,9), diabetes (10), sunlight exposure (11), high body mass index (12), steroid use (13), increasing age (14,15), female gender (16,17), myopia (17,18), and pseudoexfoliation in the anterior eye segment (19,20). in a study on the tierp population, pseudoexfoliation was found to be a predictor for cataract surgery (21). the aim of this investigation was to report the prevalence of age-related cataract and its risk factors in a population survey in sweden. methods the tierp glaucoma survey the tierp glaucoma survey was a population-based study on residents in the municipality of tierp, south-central sweden, conducted in 1984–86. its target population comprised 2,429 people, aged 65–74 years. the size of the sample was limited contact curt ekstr€om curt.ekstrom@neuro.uu.se department of neuroscience, university hospital, se-751 85 uppsala, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 4, 311–315 https://doi.org/10.1080/03009734.2020.1802375 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1802375&domain=pdf&date_stamp=2020-10-22 http://orcid.org/0000-0002-8265-6518 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1802375 http://www.tandfonline.com to about one-third of the target population. of the eligible number of 838 residents, 760 (91%) underwent a detailed eye examination, as described elsewhere (22). the characteristics of participants and non-participants are presented in table 2. the study was primarily designed to address the distribution and determinants of open-angle glaucoma. however, a great amount of information was collected, including data on cataract. briefly, an interview was first held, covering medical and family history. information was also obtained from medical records. after perimetry, the pupils were dilated to a diameter of at least 3 mm and biomicroscopy undertaken. the presence of cataract was ascertained based on retroillumination using indirect ophthalmoscopy with lens opacities evident on slit-lamp examination. a grading of the amount of opacities in six stages was also performed. individuals with definite lens opacities in either eye or those who had undergone cataract surgery in one or both eyes were classified as having cataract. the first stage of lens opacities, described as ‘early senile changes’ in the framingham eye study (3), was not accepted as definite cataract in the present study. the study was approved by the human subjects committee at the faculty of medicine, uppsala university and adhered to the tenets of the declaration of helsinki. informed consent was obtained from all participants. statistical methods prevalence estimates were adjusted for non-response by applying the frequency of cataract among those who presented for the examination to those who did not, with age and gender strata. confidence intervals (cis) for prevalence rates were calculated using the normal approximation to the binomial distribution. risk factors for cataract expressed as odds ratios (ors) were estimated using 2 � 2 tables. control for age and gender was performed according to mantel–haenszel. to simultaneously assess several predictors affecting the risk for cataract, multiple logistic regression analyses were also employed with cataract as the dependent variable. results prevalence of cataract a total of 234 individuals were found to have cataract, 12 of whom had undergone cataract surgery. the prevalence adjusted for non-response was 31.5% (95% ci 29.4–33.6), 35.2% (95% ci 28.7–41.8) in women and 26.2% (95% ci 19.8–32.6) in men. another 116 people had early lens changes in either eye, not classified as cataract. the distribution of cataract by age and gender is shown in table 3. cataract was strongly related to increasing age in both women and men. prevalence of past cataract surgery twelve subjects had a history of cataract surgery, equal to an unadjusted prevalence of 1.6% (95% ci 0.7–2.5). aphakia in one or both eyes was present in 11 subjects and pseudophakia in either eye in two subjects. in addition, traumatic aphakia was observed in two subjects. of these, one had age-related cataract in the unaffected eye and was included among the cataract cases. risk factors for cataract to assess risk factors for cataract, ors were calculated, adjusted for age and gender. table 4 provides ors for potential risk factors. high age, female gender, and myopia increased the risk for cataract, while pseudoexfoliation, current smoking, diabetes mellitus, systemic hypertension, and ischaemic heart disease did not. the variables mentioned in the previous passage were tested in logistic regression models. the ultimate model included cataract, age as a continuous variable, gender, and myopia. for gender and myopia, the result was almost identical to that of the stratified analyses. every year of advancing age increased the risk for having cataract with 16% (or 1.16; 95% ci 1.10–1.23). females ran a 52% higher risk than males. there was no indication of interactions in the models. table 1. prevalence of cataract in subjects 65–74 years of age in the framingham eye study, the beaver dam eye study, and the blue mountains eye study. study ref. method prevalence (%) framingham (3) slit-lamp, ophthalmoscopy total 18.0a females 19.3 males 16.0 beaver dam (4) photography total 74.3b females 76.3 males 71.4 blue mountains (5) photography total 68.4b females 73.2 males 62.2 aonly individuals with visual acuity of 20/30 or worse in either eye were examined; including subjects with a history of cataract surgery. bright eye only. table 2. participation in the population survey in tierp, by age and gender. females (n ¼ 429) males (n ¼ 409) examined examined age yes (%) no (%) yes (%) no (%) 65 � 69 years 209 (93.3) 15 (6.7) 195 (90.7) 20 (9.3) 70 � 74 years 187 (91.2) 18 (8.8) 169 (87.1) 25 (12.9) 65 � 74 years 396 (92.3) 33 (7.7) 364 (89.0) 45 (11.0) table 3. prevalence of cataract in the population survey in tierp, by age and gender.a females (n ¼ 396) males (n ¼ 364) age prevalence 95% ci prevalence 95% ci 65 � 69 years 26.3% 20.3–32.2 22.1% 16.2–27.9 70 � 74 years 45.9% 37.8–52.0 30.8% 23.8–37.7 65 � 74 yearsb 35.2% 28.7–41.8 26.2% 19.8–32.6 difference (females � males): 9.0% (95% ci �0.2 to 18.1). aincluding past cataract surgery. badjusted for nonparticipation. ci: confidence interval. 312 m. hugosson and c. ekström discussion to the best of our knowledge, only one population-based study has previously been reported on cataract prevalence in sweden, namely the sk€ovde cataract study (23), with the tierp study being the second. the study in sk€ovde used the locs iii grading system, including people 70–84 years of age. a study like the one in sk€ovde was also conducted in oulu county, finland on people aged �70 years (24). furthermore, a case–control study, with controls chosen from the population register, on the association of cataract with diabetes type 2, was undertaken in laxå, sweden (25). in this study, a previous version of the locs iii system was used. the prevalence of cataract in tierp was low compared with the population surveys of beaver dam, wisconsin, us and blue mountains, australia (table 1). however, in subjects 70–74 years of age, the prevalence observed in tierp was rather close to the prevalence in oulu (38.2 and 44.6%, respectively). the age-specific prevalence in the framingham eye study was lower than that in tierp (18.0 and 31.5%, respectively). a likely explanation for the difference can be the more conservative inclusion criteria in the framingham study; only subjects with a visual acuity of 20/30 or worse were classified as having cataract. in the laxå study, the prevalence of cataract in the control group of people 65–74 years of age was 72.7% in females and 78.3% in males. although the estimates were based on only 24 and 36 cases, respectively, the prevalence in laxå seems to have been much higher than that in tierp. the reported prevalence rates of cataract vary for several reasons. firstly, there is no clear-cut definition of when a lens is opaque enough to be classified as cataract. in the present study, a subject was thought to have cataract if lens opacities were observed on retroillumination using indirect ophthalmoscopy, and the findings were evident on slit-lamp biomicroscopy. it is likely that this method underestimates the frequency of cataract compared with photographic methods. a standardized lens opacity grading system, like locs iii, should be preferred in population surveys on cataract. secondly, the age of the examined population differs between studies. age is an important risk factor for the development of cataract, as shown in this study. thirdly, inclusion of subjects with a history of cataract surgery may have an impact on prevalence rates. in the present report, the rates presented for the studies in framingham, tierp, oulu, and laxå included patients who had undergone surgery, while the rates for beaver dam and blue mountains did not. fourthly, an unequal distribution of risk factors for cataract such as exposure to sunlight, smoking habits, and diabetes may affect prevalence rates. finally, the population survey in tierp was conducted in 1985–86, while some of the studies referred to in this report were performed more recently. it is not impossible that lifestyle factors have changed over time and thereby modified the prevalence rates. consistent with previous research, age was found to be a major risk factor for the development of cataract in both females and males. accumulated oxidative stress to the lens proteins over the years has been suggested as the primary explanation (26). furthermore, we confirm findings from previous studies that females are at a higher risk of cataract than males (16,17). it has been suggested that oestrogen plays a role in protecting the lens from oxidative stress (27–29). in this context, decreasing oestrogen levels after menopause would increase the risk for cataract. on the contrary, other studies have concluded that postmenopausal hormone replacement therapy increases the risk for undergoing cataract surgery (30). the association of age-related cataract with myopia is well-known (17). in most cases, the development of myopia is thought to be a consequence of a shift in refraction in subjects with nuclear lens opacities. however, follow-up studies have demonstrated the possibility of a causal relationship between myopia and cataract, in particular posterior subcapsular cataract (18). with an increasing number of myopic individuals in many countries (31), it is possible that cataract may affect more people and appear earlier in life in the coming years. in the present study, myopia increased the risk for having cataract 2-fold. unfortunately, adjustment for nuclear opacities could not be performed, which is likely to have biased the result. cigarette smoking (8,9) and diabetes (10) are established risk factors for lens opacities. nevertheless, we were unable to confirm the effect of smoking and diabetes. the sparse number of exposed cases is a likely explanation. furthermore, we could not verify a relationship between cataract and pseudoexfoliation, reported in other population studies (19,20). other known risk factors like sunlight exposure (11), high body mass index (12), and steroid use (13) were not examined. compared with the population studies in framingham, beaver dam, and blue mountains, the tierp glaucoma survey was a small study, limiting its statistical power. however, the high participation rate was a strength of the study. the lack of a standardized grading system of cataract, leading to table 4. odds ratios for cataract in the population survey in tierp, adjusted for age and gender. no. of cases characteristics (n ¼ 234) orm-h 95% ci age �70 yearsa no 98 1.00 yes 136 1.93 1.41 � 2.64 female genderb no 95 1.00 yes 139 1.54 1.12 � 2.11 pseudoexfoliation, either eye no 189 1.00 yes 45 1.12 0.75 � 1.68 myopia, either eye no 209 1.00 yes 25 2.03 1.16 � 3.56 current smoker no 206 1.00 yes 28 0.73 0.46 � 1.17 diabetes no 201 1.00 yes 33 1.25 0.79 � 1.99 hypertension, treated no 159 1.00 yes 75 1.26 0.90 � 1.78 ischaemic heart disease no 195 1.00 yes 39 1.18 0.77 � 1.80 aadjusted for gender. badjusted for age. ci: confidence interval; orm-h: mantel–haenszel adjusted odds ratio. upsala journal of medical sciences 313 potential misclassification of disease, was a limitation of the study. another weakness was that the type of cataract was not registered in the population survey. there are evidence that cortical and nuclear lens opacities do not share the same modifiable risk factors (17,32). to estimate the lifetime risk of cataract surgery in this cohort of older adults, a follow-up study is in progress. conclusion nearly one-third of the participants in the tierp glaucoma survey were found to have lens opacities, or had undergone cataract surgery, making cataract a frequent disorder of older age. nevertheless, the age-specific prevalence was low compared with studies using standardized grading systems based on photography. the study provided further evidence that increasing age, female gender, and myopia are associated with cataract. disclosure statement the authors report no conflicts of interest. funding the swedish medical research council, crown princess margaretha’s foundation for the visually impaired, the glaucoma research fund at the department of ophthalmology, uppsala university hospital, and uppsala county council provided financial support for this study. notes on contributors magnus hugosson is a md at the department of ophthalmology, university hospital, uppsala, sweden. curt ekstr€om, md, phd, is a senior researcher at the department of neuroscience, ophthalmology, uppsala university, uppsala, sweden. orcid curt ekstr€om http://orcid.org/0000-0002-8265-6518 references 1. khairallah m, kahloun r, bourne r, limburg h, flaxman sr, jonas jb, et al. number of people blind or visually impaired by cataract worldwide and in world regions, 1990 to 2010. invest ophthalmol vis sci. 2015;56:6762–9. 2. national quality register for cataracts. 2019 aug 8. available at: 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17b-estradiol on activity, gene and protein expression of superoxide dismutases in primary cultured human lens epithelial cells. curr eye res. 2018;43:639–46. 30. ejdervik lindblad be, håkansson n, philipson b, wolk a. hormone replacement therapy in relation to risk of cataract extraction: a prospective study of women. ophthalmology 2010; 117:424–30. 31. holden ba, fricke tr, wilson da, jong m, naidoo ks, sankaridurg p, et al. global prevalence of myopia and high myopia and temporal trends from 2000 through 2050. ophthalmology 2016;123: 1036–42. 32. arnarsson a, jonasson f, sasaki h, ono m, jonsson v, kojima m, et al. reykjavik eye study group. risk factors for nuclear lens opacification: the reykjavik eye study. dev ophthalmol. 2002;35: 12–30. upsala journal of medical sciences 315 abstract introduction methods the tierp glaucoma survey statistical methods results prevalence of cataract prevalence of past cataract surgery risk factors for cataract discussion conclusion disclosure statement references uppsala biobank—the development of a biobank organization in a local, regional, and national setting article uppsala biobank—the development of a biobank organization in a local, regional, and national setting anna beskowa,b adepartment of immunology genetics and pathology, uppsala university, uppsala, sweden; buppsala biobank, uppsala clinical research center, uppsala, sweden abstract a biobank is generally in an international setting considered as a sample collection with linked data. in sweden we have a lot of sample collections, but the definition of a biobank has changed, and it has become an organization that administrates many sample collections as well as an infrastructure to support research. uppsala biobank was started in september 2008 as a joint biobank organization between uppsala county council and uppsala university. at the start there were 138 registered biobanks in uppsala for these two principals. the decision was to have only one biobank, where all previous biobanks would be transformed to be sample collections. uppsala biobank has gone from the wish to centralize biobanking administration to be a research infrastructure, a national model for hospital-integrated biobanking, a support structure for biobanking activities in the health care region, and the local competence center for all biobank issues in uppsala. article history received 26 june 2018 revised 11 november 2018 accepted 11 november 2018 keywords biobank; cancer; infrastructure; organization background in 2003 sweden got its first biobank act, act (2002: 297) on biobanks in health care. according to that a biobank is defined as a collection of biological material stored for one or more purposes and information on this material. the legislation together with guidelines from the national board of health and welfare (sosfs 2002: 11 and sosfs 2004: 2) regulate biobank operations in sweden. biobanks may consist of one or more sample collections. the collections of samples have different primary purposes, with collection for health care and diagnostics purposes being most common, followed by collection of samples for research. the biobank act applies to human material such as blood, saliva, urine, and different kinds of tissue samples. the donor must give an informed consent that biobank samples are stored and used for approved purposes. to be able to use samples collected within health care for research, an ethical approval and a written consent are needed. collaboration between the swedish county councils the legislation was not easily adoptable to general health care and research activities within the health care providers. the need for consent processes and the need to document access to diagnostic samples for research made it difficult for the 21 swedish counties to set up their own solutions. unique solutions for each county/health care provider also made it difficult for researchers to understand how to access samples. the swedish biobank project started, and it led to the formation of the national biobank council with a strong national network including six regional biobank centers and 21 biobank coordinators working together. biobanking—a hot topic in the world meanwhile, the notion of biobanking was growing globally. the international society for biological and environmental repositories (isber) was formed in 1999 (www.isber.org). some years later a european chapter was formed (https:// esbb.org/). the european commission acknowledged that the competition globally in medical research using biobanks was increasing. large countries such as usa and china with larger populations for biobanking had an advantage that the european countries had to face together. the preparatory phase of bbmri (esfri roadmap) started in 2008 with the goal to form an eric to make it possible for researchers to collaborate within the eu to increase the population base. start of uppsala biobank in 2007 a prospect for a common biobank between karolinska institutet and stockholm county council— stockholm biobank—was presented. the idea to have a joint biobank was that this would make it easier for the users, who usually work for both principals, to join resources and work more efficiently together. stockholm biobank as it was contact anna beskow anna.beskow@uppsalabiobank.uu.se uppsala biobank, uppsala clinical research center, dag hammarskj€olds v€ag 38, se-751 85 uppsala, sweden. � 2018 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 1, 6–8 https://doi.org/10.1080/03009734.2018.1547992 http://www.isber.org https://esbb.org/ https://esbb.org/ http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1547992&domain=pdf http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1547992 http://www.tandfonline.com then presented has not yet been formed, even though efforts have been made. the idea was picked up by professors christer sundstr€om and ulf gyllensten in uppsala. they convinced uppsala academic hospital and the medical faculty at uppsala university to join forces and start uppsala biobank to become the single joint human biobank. uppsala biobank started on 1 september 2008 and had four aims: 1) to administrate uppsala biobank; 2) to build a stable and long-term organization; 3) to be a competence center for biobank matters; 4) and to build an infrastructure for research. administration of uppsala biobank the first decision of the local biobank council was that there would be only one biobank in uppsala, which meant transforming all previous 138 biobanks into sample collections. it took four years for one person to perform this task, working half-time (1). now uppsala biobank consists of approximately 170 research sample collections and eight sample collections for diagnostic purposes (e.g. clinical pathology, clinical microbiology, and clinical genetics). uppsala biobank administrates about 100 biobank applications every year. on average half of them are applications to access samples from clinical pathology, and more than half are applications to collect and access samples for clinical trials. less than 10% become new sample collections. each sample collection has a responsible person linked with a written agreement between the director of the biobank and the person responsible for the sample collection. the agreement includes rights and responsibilities. the rights give the person responsible for the sample collection the disposition right to the samples and clarify the responsibility regarding safe keeping of the samples and data to secure quality and integrity according to the legislation. a stable and long-term organization there is a signed agreement between the principals, region uppsala represented by uppsala academic hospital and uppsala university represented by the medical faculty. the agreement describes the organization, mission, responsibilities, finances, and steering of uppsala biobank. each principal equally funds uppsala biobank. region uppsala is the legal principal of the samples, and uppsala university is the research principal. a competence center for biobank matters uppsala biobank activities include information and education of researchers, staff, and students. questions are either sent to the biobank by e-mail or telephoned. in 2017 more than 500 questions were received, and they ranged across the whole spectrum of biobanking. uppsala biobank has a web page that was released in a new and updated version in april 2017 (www.uppsalabiobank.uu.se). an infrastructure for research in 2009 the swedish government announced investments in strategic research projects and infrastructures. uppsala university filed several applications including biobank activities. together with umeå university, uppsala university applied for a cancer research infrastructure: uppsala–umeå comprehensive cancer consortium, u-can. the goals of u-can include formation of a clinical database and a biobank with samples collected routinely from cancer patients to create the substrate for high-quality cancer research (2). the requirement to collect samples in routine health care was challenging, and there was a need to form collaborations between all actors affected to find a long-term solution. the requirements on the collection of samples were to work in the hands of the routine staff with systems already in use. it also had to be possible to collect samples 24 h a day, seven days a week in order to avoid exclusion of acute cases. moreover, it must be possible to include not only cancer but also other diagnoses. the solution means that biobank samples can be ordered in the electronic health record as an analysis. when ordering other analyses, a package of biobank samples can be ordered at the same time. labels are printed, and diagnostic samples and samples for research are taken simultaneously. samples are sent to the clinical chemistry laboratory, where they are registered, sorted according to sample type, centrifuged and aliquoted with a liquid handler. samples are frozen and transported to uppsala biobank for long-term storage in low-temperature freezers. all information and data about the sample and donor are sent automatically to the biobank laboratory information management system (biobank lims). the solution is called health care integrated biobanking (hib), and now 45 projects collect samples at 77 different clinics. samples are also taken at primary care centers and sent to the hospital for biobanking. the health care region health care integrated biobanking has now spread throughout the uppsala–€orebro health care region. the region includes seven county councils (uppsala, €orebro, dalarna, g€avleborg, v€armland, v€astmanland, and s€ormland). apart from uppsala, also €orebro, g€avleborg, and v€armland have started hib in 2014, and dalarna as well as v€astmanland will start shortly. s€ormland is planning to start in 2019. the uppsala–€orebro region collaborates within the regional biobank council. in order to work efficiently the decision was made to share a common regional biobank lims. the regional biobank lims is hosted and developed by uppsala biobank. uppsala biobank, with the support of the principals, is investing in an automated solution for long-term low-temperature storage (-80 �c). uppsala biobank will also support the region with automation for storage and withdrawals if needed, e.g. the county of g€avleborg has decided to send their samples to uppsala for storage and withdrawals. upsala journal of medical sciences 7 http://www.uppsalabiobank.uu.se the national contribution in 2010 the swedish research council founded a national biobank infrastructure called bbmri.se to be the european counterpart to bbmri.eric. karolinska institutet was the host of the infrastructure, which was purely based on university collaboration. the national biobank infrastructure already in place through the national biobank council was a collaboration between all county councils and the universities, but this infrastructure was not included. bbmri.se aimed to increase the access to samples owned by the county councils, but without including them. lack of collaboration between the two national infrastructures made the swedish research council stop the funding of bbmri.se in 2016. concurrently, the uppsala biobank model of hib was spread not only to the uppsala–€orebro health care region but also to the rest of sweden. setting up hib at several hospitals in sweden has been possible through funding from swelife and the health care principals to support local and national biobanking projects. now 19 hospitals in sweden have hib in production, and several more are about to start. since biobanking is something that the government sees as a strategic area to invest in, the swedish research council opened up for a new application for a national biobank infrastructure. the requirement was that the principal’s universities with medical faculties and the corresponding county councils collaborated and applied together. in 2016 a joint steering board with the deans from the medical faculties and the research directors from the corresponding county councils was formed to collaborate around the new national biobank infrastructure, biobank sweden. the basis for biobank sweden comes from the former national biobank council with already existing biobanks and network. an application was sent to the swedish research council in march 2017 for funding of some of the development needed of the infrastructure to better support research on biobank samples in sweden and european collaboration through participation in bbmri-eric. the application was granted funds for 2018–2019. conclusions in september 2018 uppsala biobank celebrates its tenth anniversary. during these 10 years the biobank has been built, starting from 138 separate biobanks with no coordination, to an organization fulfilling all four missions set for the local biobank responsibility. uppsala biobank has also taken and takes responsibility for the development of the regional and national biobank infrastructure and has shown that collaboration and openness are important features for success. international collaborations using biobank samples will hopefully further strengthen swedish research. declaration of interest the author reports no conflicts of interest. notes on contributor anna beskow holds a phd in medical genetics (2003) and is the head of uppsala biobank since the start in 2008. references 1. engelmark mt, beskow ah. analysis of the research sample collections of uppsala biobank. biopreserv biobank. 2014;12:325–31. 2. glimelius b, melin b, enblad g, alafuzoff i, beskow a, ahlstrom h, et al. u-can: a prospective longitudinal collection of biomaterials and clinical information from adult cancer patients in sweden. acta oncol. 2018;57:187–94. sword 8 a. beskow abstract background collaboration between the swedish county councils biobankinga hot topic in the world start of uppsala biobank administration of uppsala biobank a stable and long-term organization a competence center for biobank matters an infrastructure for research the health care region the national contribution conclusions declaration of interest notes on contributor references distinctive alteration in the expression of autophagy genes in drosophila models of amyloidopathy and tauopathy original article distinctive alteration in the expression of autophagy genes in drosophila models of amyloidopathy and tauopathy mehrnaz haghia, raheleh masoudia and seyed morteza najibib,c adepartment of biology, college of sciences, shiraz university, shiraz, iran; bcenter for molecular protein science, lund university, lund, sweden; cdepartment of statistics, college of sciences, shiraz university, shiraz, iran abstract background: alzheimer’s disease (ad) is one the most common types of dementia. plaques of amyloid beta and neurofibrillary tangles of tau are two major hallmarks of ad. metabolism of these two proteins, in part, depends on autophagy pathways. autophagy dysfunction and protein aggregation in ad may be involved in a vicious circle. the aim of this study was to investigate whether tau or amyloid beta 42 (ab42) could affect expression of autophagy genes, and whether they exert their effects in the same way or not. methods: expression levels of some autophagy genes, hook, atg6, atg8, and cathepsin d, were measured using quantitative pcr in transgenic drosophila melanogaster expressing either ab42 or tau r406w. results: we found that hook mrna levels were downregulated in ab42-expressing flies both 5 and 25 days old, while they were increased in 25-day-old flies expressing tau r406w. both atg6 and atg8 were upregulated at day 5 and then downregulated in 25-day-old flies expressing either ab42 or tau r406w. cathepsin d expression levels were significantly increased in 5-day-old flies expressing tau r406w, while there was no significant change in the expression levels of this gene in 5-day-old flies expressing ab42. expression levels of cathepsin d were significantly decreased in 25-day-old transgenic flies expressing tau r406w or ab42. conclusion: we conclude that both ab42 and tau r406w may affect autophagy through dysregulation of autophagy genes. interestingly, it seems that these pathological proteins exert their toxic effects on autophagy through different pathways and independently. article history received 6 january 2020 revised 11 june 2020 accepted 16 june 2020 keywords alzheimer’s disease; amyloid beta; autophagy genes; drosophila melanogaster; tau introduction alzheimer’s disease (ad) is characterized by progressive memory impairment and dementia. this neurodegenerative disease is also one of the main causes of death in the elderly population (1). neurofibrillary tangles (intracellular inclusion of hyperphosphorylated tau) and ab plaques (extracellular inclusion of ab42 peptide) are two major markers in the brain of ad patients (2). the mechanism underlying ad pathology is not fully understood. some hypotheses point to these aggregations as the reason for developing ad symptoms. on the other hand, recent investigations have shown that aggregate formation is a defense mechanism against soluble and aggressive forms of ab and hyperphosphorylated tau (3,4). although these aggregates may exert protection against their soluble oligomer forms, they block axonal/dendritic transport (5) and cause damage to mitochondrial complexes (6,7), leading to increased levels of reactive oxygen species (ros) in the brain of ad patients. autophagy (self-eating) is one of the pathways responsible for clearing these aggregations in order to prevent their accumulation (8). to eliminate the aggregates, they are transferred via a dynein/dynactin motor complex to a perinuclear aggresome. then, they are encapsulated by neurofilaments, and the aggresome is formed. ultimately, aggregates are cleared when the aggresome is fused to a lysosome (9). hook protein is an adaptor facilitating the association of motor complex with its cargos (10). many stimuli can trigger the initiation of autophagy by ulk1/atg1 protein kinase complex (11). this serine/threonine kinase phosphorylates beclin1/atg6 in the beclin1-vps34 pi3 kinase complex involved in nucleation of autophagosome formation (phagophore). following atg6 phosphorylation, vps34 pi3 kinase can convert phosphatidylinositol (ptdins or pi) to pi 3-phosphate (pi3p), which is essential for expansion of the autophagosome membrane (12,13). next, elongation phase and phagophore formation occur (14). two ubiquitinlike pathways, atg5–atg12 and the lc3 conjugation systems, are involved in this phase. lc3 (i)/atg8 is cleaved by atg4 at the carboxyl-terminal site. later, lc3 is converted to its active form, lc3ii, through covalent bonding to phosphatidyl ethanolamine, which assists the fusion of the autophagosome membrane with the lysosome (15,16). ultimately, the internal contact raheleh masoudi rmasoudi@shirazu.ac.ir department of biology, college of sciences, shiraz university, shiraz, iran supplemental data for this article can be accessed here. � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 4, 265–273 https://doi.org/10.1080/03009734.2020.1785063 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1785063&domain=pdf&date_stamp=2020-10-22 https://doi.org/10.1080/03009734.2020.1785063 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1785063 http://www.tandfonline.com components are degraded by lysosome hydrolases such as cathepsin d (17–19). hyperphosphorylated tau and ab aggregates can affect mitochondrial complexes and its electron chain, leading to increased levels of ros and oxidative stress (6,7). interestingly, it has been shown that ros is involved in the induction of autophagy in ad. lipinski et al. reported that ros can enhance autophagy through increase in type iii pi3 kinase activity. they also confirmed that, unlike in normal aging, autophagy genes are transcriptionally increased in ad patients (20). on the other hand, many studies have shown that ros can lead to autophagy impairment by various pathways either directly or indirectly. ros directly inhibits lc3 lipidation and also its translocation to the phagophore membrane (21). ros increases nf-jb and nitric oxide (no) synthase which, in turn, enhances no levels (22). no can inhibit jnk1 via s-nitrosylation at c116, leading to reduction of bcl2 phosphorylation, which ultimately increases its interaction with beclin1. this event disrupts the formation of the hvps34/beclin1 complex, and autophagy is eventually impaired (23). it seems that presence of protein aggregates and autophagy dysfunction in ad create a vicious circle. the aim of this study was to investigate the effect of pathological tau r406w and ab42, two major causes of aggregate formation in ad, on the expression of autophagy genes, atg6, atg8, hook, and cathepsin d. these genes are involved in different stages of autophagy. considering that ad is an age-dependent disorder, the expression levels of autophagy genes were assessed at two different time points in ab42 or tau r406w transgenic drosophila melanogaster. tau r406w is an autosomal dominant mutation that causes tau-positive frontotemporal dementia in human. its expression in drosophila melanogaster, using a binary system, provides a proper tauopathy model (24). materials and methods chemicals rna extraction and cdna synthesis kits were purchased from cinnagen and parstous companies, iran, respectively. brilliant ii sybr green qpcr master mix was provided by biofact, germany. other materials used in this study were provided from cinnagen, iran and merck, germany. fly strains drosophila melanogaster stocks were raised in standard rolled oats-agar medium at 22 ± 1 �c, 60–70% humidity, and 12-h light/12-h dark circadian cycle. mapt r406w for tauopathy (expression of this protein in transgenic fly is discussed in wittmann et al., in 2001) (24) and ab42 for amyloidopathy (bloomington stock no. 33769) (25) were expressed in neurons using dmel\p{gawb}elavc155 (bloomington stock no. 458) driver. while expressed tau remains in the cytoplasm, presence of a signal peptide in the ab42 construct (26) causes ab42 transportation to the endoplasmic reticulum (27). to investigate the pathogenesis of ab42 and tau r406w, gmr-gal4 driver (bloomington stock no. 8605) (28) was applied to express these proteins in the fly eyes. flies expressing uas-tau r406w were from feany’s lab (harvard medical school, boston, ma, usa), and all other lines were from bloomington drosophila stock centre. all crosses and their counterpart controls (parental lines) were applied in triplicate. all transgenic stocks were outcrossed to w1118 for several generations to obtain the identical genetic background of all lines, prior to the tests. climbing assay for climbing assay analysis, nine groups of flies, with 10 flies per group (mix of both genders), were prepared for every single genotype. all 10 flies were transferred into a vial, and the vial was tapped gently. after each tap, flies were observed for the first 10 s to record the number of flies that were able to climb above the 8-cm marked line on the vial. this assay was repeated five times for each group with a 2min interval between each measurement (29,30). drosophila eye analysis transgenic (gmr-gal4/tau r406w and gmr-gal4/ab42) and control (gmr-gal4/+) flies from at least three independent crosses (9 flies per group, per cross) were processed for light microscopy and image analysis with three experimental repeats. imaging was performed using a nikon 80i light microscope to observe degeneration phenotype in the fly eyes. a new plugin (fleye) in imagej software was used to analyze the fly eyes. regularity in the ommatidia was represented by probability parameter (pp) including: pp0, green; pp1, blue; pp2, yellow; pp3, orange; and finally pp4, red. change in the colour from green to red represents the degree of reduction in eye regularity (28). quantitative real-time pcr first filial generation (f1) and their parental lines (as control) were collected at 5 and 25 days after eclosion. for rna extraction, flies were kept in acetone and stored in a �20 �c freezer overnight (31,32). then, flies were frozen at �80 �c. after 10 min, flies were shaken harshly in the tube in order to separate the heads. rna was extracted from 100 heads on liquid nitrogen by rnx plus kit following the manufacturer’s protocol. rna concentration was measured using nano drop (thermo fisher scientific), and 3 mg of total rna was reversetranscribed to cdna. amplification of cdna was performed using biofact syber green master mix in an abi 7500 pcr machine. all primers were designed as the exon–exon junction primer. rpl32 was applied as reference gene to determine the relative expression levels of hook, atg6, atg8, and cathepsin d using a 2(-delta delta c(t)) method (33). each sample was run in triplicate. the sequences of primers are given in supplementary table s1. 266 m. haghi et al. https://doi.org/10.1080/03009734.2020.1785063 statistical analysis here, we applied r (version 3.6.1) and spss (version 19) programmes to perform statistical analysis. the number of samples in biological studies is usually small, and the assumptions of parametric statistical models cannot be achieved. one solution is to use alternative non-parametric statistical models, which have lower power compared to the parametric versions. another solution is to use bayesian inference, which is more powerful to infer reliable and reproducible results (34–36). in this study, because ab42 and tau r406w have two subgroups (cross and control), we used the bayesian hierarchical mixture model for testing. our hypotheses were that there are differential gene expression and climbing ability in two biologic conditions (ad and control) at different time points (5 and 25 days). the proposed model exploits available position-specific read counts, minimizing required data preprocessing and making maximum use of available information. our analysis has been done by stan language that is using a state-of-the-art algorithm known as hamiltonian monte carlo (hmc), which builds upon the metropolis–hastings algorithm by incorporating many theoretical ideas from physics. specifically, we used the rstanarm package in r software, which is a powerful package for bayesian hierarchical models by stan_lme4 for estimating the model parameters. in this paper, we considered a full multilevel model for group/subgroup�time� (genes) that means we assume random intercept and slope for each category. we had to have an estimation of different intercepts and slopes for each experimental condition, which was clearly needed based on the raw data (see figures 1 and 2). the number of samples is selected to be 4000 (2000 for warmup), and four independent chains ran for the sake of convergency and posterior sampling evaluation. the median estimation and the highest posterior density (hpd) intervals with probability of 0.95 are reported based on 2000 samples from posterior distribution of model parameters. the bayesian contrast estimations are evaluated by the emmeans package in r. besides the bayesian model, we have also presented our data as fold changes when comparing crosses and their counterpart controls. independent samples t test was employed to compare the mean values. p values less than 0.05 were considered as statistically significant. results decrease in the climbing ability of ab42 or tau r406w transgenic flies locomotive defects can be examined in transgenic flies as one of the ad symptoms. here, we tested negative geotaxis ability as a behavioural assay to show that the expression of our target genes can affect the natural tendency of flies to move against gravity (29,30). to assess the climbing ability, 5and 25-day-old transgenic flies were examined. it was found that the ability of climbing in both transgenic flies was remarkably decreased compared to their controls. the raw data and bayesian estimation of difference between controls and transgenic flies are depicted in figure 1 (for more details see table 1; and also supplementary table s2). there was 0.32and 0.42-fold decrease (p values � 0.001) in 5-day-old ab42 or tau r406w-expressing flies, respectively; 25-day-old flies expressing ab42 or tau r406w showed 0.44and 0.53-fold reduction (p values �0.001) in the climbing ability, respectively. for more details see supplementary figure s1 and supplementary table s3. eye degeneration was observed in flies expressing either tau r406w or ab42 to screen the pathogenesis of our genes of interest (ab42 and tau r406w), we investigated the degeneration of drosophila retina as a model system by expressing the transgenes using the gmr-gal4 driver. as can be seen in figure 3, there was more irregularity (yellow and red colors) in the eye ommatidia of transgenic flies compared to parental lines figure 1. (a) representing the raw data of the percent of flies (control and cross) above the target line in three independent biological repeats for 5and 25-dayold flies and the fitted bayesian hierarchical model lines for each group and subgroup and their shaded 95% highest posterior density (hpd) region based on 2000 posterior samplings of model parameters. (b) bayesian estimation of contrast between medians (control–cross) and their 95% hpd region based on 2000 posterior samplings of each contrast. because zero is not included in any of the reported hpd’s intervals, there is significant difference between the control and cross negative geotaxis ability with 5% error type (a ¼ 0.05). upsala journal of medical sciences 267 https://doi.org/10.1080/03009734.2020.1785063 https://doi.org/10.1080/03009734.2020.1785063 https://doi.org/10.1080/03009734.2020.1785063 https://doi.org/10.1080/03009734.2020.1785063 with regular eye ommatidia (blue and green colour). interestingly, both ab42 and tau r406w transgenic flies demonstrate irregularities in their eyes. quantitative real time pcr alteration in the expression of hook, as a pre-autophagy gene as mentioned earlier, hook is a mediator to facilitate interaction between motor proteins and their cargo as a preautophagy protein (37). in the ad brain, reduction in hook3 expression and increase in hook2 mrna levels have been observed. interestingly, when hook3 is knocked down, there is an increase in ab production (38). while there are three forms of hook in humans, only one form of hook has been reported in drosophila. here, we assessed the effect of ab42 and tau r406w on the expression levels of dhook (drosophila hook) using transgenic flies. as can be seen in figure 2 and from bayesian contrast estimations in table 2, although there was no significant change in the levels of dhook expression in 5-day-old flies expressing tau r406w, a prominent increase was observed in the levels of this gene in 25-day-old flies. it seems that tau r406w exerts its effect on the hook expression at a later time point of the life cycle of this fly. regarding ab42-expressing flies, there was a significant decrease in the levels of hook expression in both 5figure 2. representing the raw data of autophagy-related gene expression in three independent biological repeats of ab42 or tau r406w transgenic flies and the fitted bayesian hierarchical model lines for each group and subgroup shaded by their 95% hpd region based on 2000 posterior samplings of model parameters. table 1. bayesian contrast estimation of medians (control–cross) for climbing assay in 5and 25-day-old flies. time point fly type contrast estimate (control–cross) lower hpd (2.5%) upper hpd (97.5%) 5 days ab42 29.92a 27.55 32.12 tau r406w 39.49a 37.13 41.67 25 days ab42 34.83a 32.44 37.04 tau r406w 42.53a 40.15 44.74 athe 95% highest posterior density (hpd) intervals for each contrast are reported. therefore, there is a significant difference between control and cross with 5% error type i (a ¼ 0.05) in all of the cases thus marked. 268 m. haghi et al. and 25-day-old flies. for more details see figure 4; and also supplementary table s4. regarding differential expression, there was about a 2.26fold increase in the hook expression in 25-day-old flies expressing tau r406w (p ¼ 0.018). the reduction in the expression of hook was around 0.5and 0.59-fold for 5and 25-day-old ab42-expressing flies, respectively (p ¼ 0.001 and 0.002). more details are in the supplementary data, supplementary figure s2 and supplementary table s5. alteration in the expression of autophagy genes expression of autophagy genes in fly is influenced by various factors like age (39) and oxidative stress (40). on the other hand, it has been shown that suppression or enhancement of some autophagy genes like atg8 can increase the sensitivity to aging and oxidative stress (41). here, the expression levels of three autophagy markers including atg6, atg8, and cathepsin d were assessed to track the autophagy process in drosophila models of ad. transgenic flies expressing either tau r406w or ab42 were applied as tauopathy or amyloidopathy models at two different ages, 5and 25-day-old flies. this could provide some information on how time might affect the expression of some autophagy genes in the presence of two different types of amyloid-like aggregates. atg6, a core protein in the nucleation stage of autophagy one of the main proteins for phagophore formation is atg6. according to the bayesian model analysis, our data showed (figure 2 and table 2) that atg6 was upregulated in 5-dayold flies expressing either ab42 or tau r406w while the mrna levels of this gene had a considerable decrease in 25day-old transgenic flies (for more details, see figure 4; and also supplementary table s4). it seems that both tau r406w and ab42 aggregates exert the same effect on the expression levels of this gene at different time points. figure 3. eye degeneration has been observed in flies expressing tau r406w or ab42. a and b, respectively, are analyses for eye ommatidium regularity in 5and 25-day-old flies by imagej, and columns a, b, and c are gmr-gal4/+, gmr-gal4/tau r406w, and gmr-gal4/ab42. (pp ¼ probability parameter) table 2. bayesian contrast estimation for medians of genes expressions (control–cross) in 5and 25-day-old flies and different genes and fly types. gene type time point fly type contrast estimate (control–cross) lower hpd (2.5%) upper hpd (97.5%) hook 5 days ab42 0.512a 0.3964 0.625 tau r406w �0.0002 �0.114 0.128 25 days ab42 0.647a 0.525 0.764 tau r406w �1.354a �1.475 �1.235 atg6 5 days ab42 �3.414a �3.530 �3.296 tau r406w �5.353a �5.470 �5.231 25 days ab42 0.569a 0.444 0.681 tau r406w 0.829a 0.716 0.951 atg8 5 days ab42 �2.164a �2.290 �2.046 tau r406w �5.229a �5.343 �5.106 25 days ab42 0.452a 0.335 0.567 tau r406w 0.685a 0.567 0.803 cathepsin d 5 days ab42 0.058 �0.065 0.173 tau r406w �1.196a �1.309 �1.078 25 days ab42 0.683a 0.564 0.800 tau r406w 0.858a 0.743 0.977 athe 95% highest posterior density (hpd) intervals for each contrast are reported. therefore, there is a significant difference between control and cross with 5% error type i (a ¼ 0.05) in all of the cases thus marked. upsala journal of medical sciences 269 https://doi.org/10.1080/03009734.2020.1785063 https://doi.org/10.1080/03009734.2020.1785063 https://doi.org/10.1080/03009734.2020.1785063 https://doi.org/10.1080/03009734.2020.1785063 https://doi.org/10.1080/03009734.2020.1785063 there was a 4.45and 6.31-fold increase in the levels of atg6 in 5-day-old flies expressing ab42 or tau r406w, respectively (p � 0.001). in contrast, 25 days after eclosion these transgenic flies showed a significant decrease (p � 0.001), around 0.58and 0.85-fold, in the atg6 expression levels compared to their counterpart controls. for more details see supplementary figure s2 and supplementary table s6. atg8, as the main gene in the elongation phase atg8 is the marker protein in the elongation phase of the autophagosome formation. this unique protein undergoes two processing steps. in the first step, atg8 is cleaved by atg4 and the next step is mediated by the atg5–atg12–atg16 complex to get conjugated with phosphatidylethanolamine. these two steps are necessary for elongation and closure of the autophagic membrane (42). the expression of this gene showed the same pattern as the atg6. there was a significant increase in the expression levels of atg8 in 5-day-old transgenic flies expressing either tau r406w or ab42, followed by a prominent decline in the levels of atg8 mrna expression in those flies 25 days after eclosion (for more details on the bayesian model see figures 2 and 4, and table 2; and also supplementary table s4). therefore, it seems that both forms of aggregates have similar impact on the atg8 mrna levels. in the fold change analysis, 5-day-old ab42 or tau r406w transgenic flies showed a 3.15and 6.28-fold increase in the levels of atg8 (p � 0.001), while there was a significant decline (p � 0.001) in the expression levels of this gene (about 0.45and 0.70-fold, respectively) in 25-day-old flies expressing ab42 or tau r406w (see supplementary figure s2 and supplementary table s7 for more details). expression of the lysosome enzyme cathepsin d, involved in the final step of autophagy in the central nervous system, the activity of cathepsin d is essential to control neuronal homeostasis, cell migration, and interneuron communication. cathepsin d-mediated proteolysis plays a significant role in neuronal survival by accomplishing the degradation of aggregated proteins that reach the lysosomes via autophagy (43). there is evidence to reveal that this protein is involved in amyloidogenic processing of the amyloid precursor protein (app), as a critical component of a, b, and c secretase (44). we found that the mrna levels of cathepsin d have different patterns in 5-day-old flies expressing tau r406w or ab42. according to the bayesian model, there was no significant change in the levels of cathepsin d mrna in flies expressing ab42, while tau r406w-expressing flies showed a prominent increase in the mrna levels of this gene at day 5 after eclosion. interestingly, at day 25, both transgenic flies showed a figure 4. bayesian estimation of median contrasts (control–cross) for gene expression in 5and 25-day-old flies. hook gene has different expression pattern in ab42or tau r406w-expressing flies either 5 or 25 days old, while atg6 and atg8 mrna levels changed with similar pattern in both transgenic lines either 5 or 25 days after eclosion. 270 m. haghi et al. https://doi.org/10.1080/03009734.2020.1785063 https://doi.org/10.1080/03009734.2020.1785063 https://doi.org/10.1080/03009734.2020.1785063 https://doi.org/10.1080/03009734.2020.1785063 https://doi.org/10.1080/03009734.2020.1785063 https://doi.org/10.1080/03009734.2020.1785063 https://doi.org/10.1080/03009734.2020.1785063 remarkable decline in the expression levels of cathepsin d mrna (figures 2 and 4, and table 2; and also supplementary table s4). the fold change analysis showed that there was a 2.24fold increase (p ¼ 0.007) in the levels of cathepsin d expression in 5-day-old flies expressing tau r406w. in 25-day-old ab42 or tau r406w-expressing flies, there was a 0.74and 0.85-fold decrease, respectively (p � 0.001). more details are included in supplementary figure s2 and supplementary table s8. discussion according to the amyloid cascade, proposed by hardy and higgins in 1992, amyloid is the main culprit in ad pathology (44). other events in ad, including tau hyperphosphorylation and subsequent neuronal death, were supposed to be downstream events of the amyloid pathway (45–47). therefore, most treatments for ad have been based on the removal of ab (reviewed in 46). recently, there is increasing evidence to support the idea that pathological tau can exert its own toxic effect independently (48–50). on the other hand, there are some studies showing that aggregations of ab (senile plaque, sp) and tau (neurofibrillary tangles, nft) are, in fact, a defensive mechanism against ab monomers and the soluble pathological tau (3,51). however, these aggregates, in turn, block axonal/dendritic transport (5) and can lead to mitochondrial damage and an increase in the levels of ros (6,7). in order to shed light on the mechanism underlying the relation between protein aggregates and autophagy dysfunction in ad, here, we investigated the differential expression of autophagy genes in ab42-expressing flies as an amyloidopathy model and flies expressing tau r406w as a tauopathy model (24). we used tau r406w transgenic flies as they have been reported to exhibit ad-like phenotypes (52). on the other hand, previous studies have shown that tau wildtype transgenic flies have no aggregate formation (53). therefore, to clarify the effect of the aggregates of this protein on autophagy genes, this model was more suitable. our climbing assay and fly eye analysis showed that ab42 and tau r406w can cause defects in climbing ability and eye regularity in transgenic flies. these data also showed that tau can exert its toxicity independent of ab42. neurons are highly dependent on the autophagy to remove protein aggregates such as sp and nft (8). defects in this pathway clearly lead to neurodegenerative disorders such as ad. there is strong evidence to confirm the association of autophagy dysregulation with ad (54). herrmann and colleagues showed that hook3 was co-localized with tau aggregate and retained in those aggregates (38). surprisingly, in our current research, there was a remarkable increase in the levels of dhook expression in 25-day-old flies expressing tau r406w. it is possible that hook is the adaptor for tau aggregates in the autophagy pathway and that is why the expression of hook is increased in tau r406w-expressing flies. however, the expression of this gene is downregulated in ab42-expressing flies. this may suggest that either the transportation of ab aggregates is mainly managed by another adaptor or ab42 can affect autophagy through hook downregulation. in summary, it seems that although both tau r406w and ab42 alter the expression of hook, their effects occur via different mechanisms. several studies have shown that beclin1 (atg6), a main protein to initiate autophagy, is downregulated in ad, while some other evidence argues that autophagy genes are increased during ad (20,55,56). despite all the controversies, it is clear that autophagy is deregulated in this disease. atg6 is downregulated in early and late stages of ad (55). however, it has been shown that atg8 expression is increased in ad patients (56,57). according to our results, while atg6 and atg8 showed upregulation in their mrna levels in 5-day-old flies expressing either tau r406w or ab42, a remarkable downregulation was observed for both genes at day 25 after eclosion. it appears that in earlier stages (5 day after eclosion) autophagy genes are increased in order to clear the aggregates. however, ultimately autophagy is decreased at later time points, probably due to an increase in the ros production (6,7) or other mechanisms. association of autophagosome with lysosome is the last step of autophagy. finally, its internal components are degraded by lysosome hydrolases like cathepsin d (17). cathepsin d is the only proteolytic enzyme the expression of which, in different tissues, is regulated in response to growth factors, cytokines, and vitamins (58). cathepsin d-mediated proteolysis is essential to neurons because it degrades unfolded/oxidized protein aggregates that continuously reach the lysosomes via autophagy or endocytosis (43). many proteins produced in neurons are physiologic substrates of cathepsin d and will be abnormally accumulated if they are not efficiently degraded (e.g. app, a-synuclein, and huntingtin). therefore, dysfunction of cathepsin d in the lysosomal system is closely related to the mechanism underpinning neurodegeneration (43). in 2006, urbanelli et al. demonstrated that there was a decrease in the cathepsin d mrna and protein levels in ad (59). however, in 1995, cataldo et al. demonstrated intense cathepsin d immunoreactivities and lowered cathepsin d mrna levels in degenerating neurons (60). the upregulation of cathepsin d synthesis and accumulation of hydrolaseladen lysosomes indicated an early activation of the endosomal-lysosomal system in vulnerable neuronal populations, possibly reflecting early regenerative or repair processes (60). urbanelli and colleagues provided evidence of altered balance of the cathepsin d expression in skin fibroblasts from patients with sporadic or familial forms of ad (59). in particular, they showed that the expression of this gene is downregulated at both the transcriptional and translational levels and its processing is altered in ad fibroblasts (59). high levels of the constitutively active form of ras in normal or ad fibroblasts induce cathepsin d downregulation. furthermore, the p38 mapk signalling pathway also appears to downregulate the cathepsin d levels. urbanelli et al. proposed that the impairment of lysosomes in ad can be one of the main factors for the progression of the disease (59). upsala journal of medical sciences 271 https://doi.org/10.1080/03009734.2020.1785063 https://doi.org/10.1080/03009734.2020.1785063 https://doi.org/10.1080/03009734.2020.1785063 https://doi.org/10.1080/03009734.2020.1785063 finally, in 2018 chai et al. investigated the cathepsin d immunoreactivities in the temporal and parietal cortices of well characterized ad brains. their results showed an increase in the cathepsin d immunoreactivities in ad tissues and its correlation with neuropathological nft scores, and phosphorylated pser396 tau burden (17). our data showed that there was a significant increase in the mrna levels of cathepsin d in tau r406w-expressing flies just 5 days after eclosion. this increase could be due to regenerative or repair processes occurring at earlier stages of the disease (60). however, in 25-day-old transgenic flies, this gene showed significant reduction. following the increase in tau r406w and ab42 aggregations at later time points (25day-old flies), probably due to an increase in ros production, cathepsin d is downregulated (43). moreover, cystatin c, as an inhibitor of cathepsins (cysteine protease), is upregulated by ros and has been shown to be co-localized with the ab42 peptide (61,62). conclusion in summary, our results suggest that both ab42 and tau r406w can affect the autophagy pathway through gene expression dysregulation. interestingly, they showed a similar effect on the genes involved in the nucleation and elongation steps of autophagy. however, tau r406w and ab42 exert different effects on the expression of the pre-autophagy gene, hook, and a gene involved in the last step of autophagy, cathepsin d. we conclude that although both tau r406w and ab42 can alter the process of autophagy during ad, it seems that they act independently, through different mechanisms. therefore, ad treatment involving the removal of merely ab42, without considering pathological tau, will not be sufficiently effective in ameliorating ad symptoms. acknowledgements we would like to show our gratitude and appreciation to dr. m. haddadi for providing the desired drosophila stock and sharing his pearls of wisdom with us during the course of working with the flies. we also express our sincere thanks to dr m. ebrahimi for assistance with fleye plugin. disclosure statement no potential conflict of interest was reported by the author(s). funding this research was financially supported by shiraz university research council. seyed morteza najibi was supported by a swedish research council grant (2016–06947) and a grant from essence@lu. notes on contributors mehrnaz haghi, phd student of molecular and cellular biology, department of biology, shiraz university, shiraz, iran. raheleh masoudi, phd of molecular genetics, assistant professor in department of biology, shiraz university, shiraz, iran. seyed morteza najibi, phd of statistics, researcher 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al. cathepsin d expression is decreased in alzheimer’s disease fibroblasts. neurobiol aging. 2008;29:12–22. 60. cataldo am, barnett jl, berman sa, li j, quarless s, bursztajn s, et al. gene expression and cellular content of cathepsin d in alzheimer’s disease brain: evidence for early up-regulation of the endosomal-lysosomal system. neuron 1995;14:671–80. 61. mcgrath lt, mcgleenon bm, brennan s, mccoll d, mcilroy s, passmore ap. increased oxidative stress in alzheimer’s disease as assessed with 4-hydroxynonenal but not malondialdehyde. qjm. 2001;94:485–90. 62. christen y. oxidative stress and alzheimer disease. am j clin nutr. 2000;71:621s–9s. upsala journal of medical sciences 273 http://www.r-project.org/ abstract introduction materials and methods chemicals fly strains climbing assay drosophila eye analysis quantitative real-time pcr statistical analysis results decrease in the climbing ability of aβ42 or tau r406w transgenic flies eye degeneration was observed in flies expressing either tau r406w or aβ42 quantitative real time pcr alteration in the expression of hook, as apre-autophagy gene alteration in the expression of autophagy genes atg6, a core protein in the nucleation stage of autophagy atg8, as the main gene in the elongation phase expression of the lysosome enzyme cathepsin d, involved in the final step of autophagy discussion conclusion acknowledgements disclosure statement notes on contributors references upsala j m e d sci 88: 9-15, 1983 cord blood platelet aggregation; quality control by a two-sample technique g u n n a r ahlsten, u w e ewald a n d t o r s t e n t u v e m o from the department of p a e d i a f r i t s , university hospital, uppsala, sweden abstract t w o blood s a m p l e s w e r e t a k e n f r o m t h e c o r d s a t 17 n o r m a l d e l i v e r i e s 2-4 min a n d 5-8 min a f t e r b i r t h , r e s p e c t i v e l y . t h e d i f f e r e n c e in p l a t e l e t c o u n t b e t w e e n e a r l y a n d l a t e s a m p l e s in p l a t e l e t r i c h p l a s m a w a s <5% i n n i n e c o r d s ( g r o u p a), a n d g r e a t e r in e i g h t c o r d s ( g r o u p 8). p l a t e l e t a g g r e g a t i o n s t u d i e s on t h e e a r l y a n d t h e l a t e blood s a m p l e s s h o w e d c o n s i s t e n t r e s u l t s w i t h i n e a c h c o r d in g r o u p a b u t n o t in g r o u p 8. t h e c o r r e l a t i o n s b e t w e e n t h e r e s p o n s e s w e r e high f o r g r o u p a. t h e a g g r e g a t i o n r e s p o n s e s w e r e a l s o s l i g h t l y b u t s i g n i f i c a n t l y h i g h e r in t h e l a t e s a m p l e s in t h i s g r o u p (p<o.oi in g r o u p a; n s . in g r o u p 8). t h e d i f f e r e n c e s b e t w e e n r e s p o n s e s i n e a r l y a n d late blood s a m p l e s c o u l d n o t b e e x p l a i n e d by a c i d b a s e d i s s i m i l a r i t i e s . t h e v a r i a b i l i t y in c o r d blood p l a t e l e t a g g r e g a t i o n r e s u l t s c a n b e g r e a t l y r e d u c e d by p l a t e l e t c o u n t i n g in p r p of t w o i n d e p e n d e n t blood s a m p l e s , a c c e p t i n g onfy s a m l e s w i t h c o n c o r d a n t p l a t e l e t c o u n t s ( ~ 5 % d i f f e r e n c e ) . introduction platelet a g g r e g a t i o n of c o r d blood is o n e of t h e tools u s e d f o r i n v e s t i g a t i o n of h a e m o s t a s i s in t h e newborn. d e c r e a s e d a g g r e g a b i l i t y of p l a t e l e t s in r e s p o n s e to a d p , c o l l a g e n a n d e s p e c i a l l y e p i n e p h r i n e c o m p a r e d w i t h t h a t i n a d u l t s h a s b e e n r e p o r t e d i n n o r m a l c o r d blood (1,6,10). d i f f e r e n c e s in t h e c o n d i t i o n s of t h e m o t h e r s , a n d in t h e p r e g n a n c i e s , t h e t y p e of o b s t e t r i c a n a l g e s i a a n d t h e d r u g i n t a k e i n f l u e n c e t h e r e s u l t s profoundly (1,3,4,9,11). t e c h n i c a l d i f f e r e n c e s , s u c h as t h e sodium c i t r a t e c o n c e n t r a t i o n i n r e l a t i o n t o t h e h a e m a t o c r i t , m a y play a r o l e (12). d i s c r e p a n c i e s b e t w e e n r e s u l t s in d i f f e r e n t s t u d i e s m a y b e d u e to p a r t i a l a c t i v a t i o n a n d / o r a g g r e g a t i o n of p l a t e l e t s d u r i n g t h e c o u r s e of d e l i v e r y or d u r i n g t h e p r o c e s s of p l a t e l e t p r e p a r a t i o n . a c t i v a t i o n in t h e v e s s e l o r d u r i n g blood c o l l e c t i o n a r e e x p e c t e d to r e d u c e t h e p l a t e l e t c o u n t in w h o l e blood. p l a t e l e t a c t i v a t i o n d u r i n g t h e p r e p a r a t i o n p r o c e d u r e , e.g. d u r i n g c e n t r i f u g a t i o n will o n l y b e d e t e c t e d if t h e p l a t e l e t s a r e c o u n t e d in 9 t h e f i n a l p l a t e l e t r i c h p l a s m a (prp). a s t h e n o r m a l v a r i a t i o n in t h e n u m b e r of p l a t e l e t s in p r p v a r i e s w i t h i n w i d e r a n g e s , p a r t i a l a c t i v a t i o n a n d / o r a g g r e g a t i o n r e s u l t i n g in a d e c r e a s e d p l a t e l e t c o u n t will n o t b e disclosed in a s i n g l e s p e c i m e n , b u t will probably r e s u l t in a d i f f e r e n c e in p l a t e l e t c o u n t b e t w e e n t w o s p e c i m e n s f r o m t h e s a m e c o r d . t h e a i m of t h i s s t u d y w a s t o e x a m i n e t h e r e l a t i o n b e t w e e n a c c o r d a n c e in p l a t e l e t c o u n t a n d a c c o r d a n c e in a g g r e g a t i o n r e s p o n s e s in t w o i n d e p e n d e n t blood s a m p l e s f r o m t h e s a m e c o r d in a well-defined g r o u p of n o r m a l m o t h e r s a n d t h e i r h e a l t h y infants. subjects a n d methods f o r incfusion in t h e s t u d y , t h e m o t h e r s had to b e h e a l t h y non-smokers w i t h n o r m a l p r e g n a n c i e s e n d i n g in a n o r m a l v a g i n a l d e l i v e r y at g e s t a t i o n a l w e e k s 38-41. f u r t h e r c r i t e r i a w e r e t h a t o n l y n 2 0 0 2 a n d / o r p u d e n d a l n e r v e block w e r e g i v e n as o b s t e t r i c a n a l g e s i a , a n d t h a t n o d r u g s h a d b e e n t a k e n d u r i n g t h e l a s t m o n t h p r i o r to delivery. t h e m o t h e r s w e r e e s p e c i a l l y q u e s t i o n e d c o n c e r n i n g i n t a k e of non-steroid a n t i i n f l a m m a t o r y drugs. s e v e n t e e n i n f a n t s w i t h a p g a r s c o r e s 28 a t 1 a n d 5 rnin a n d a b i r t h w e i g h t a p p r o p r i a t e f o r g e s t a t i o n a l a g e w e r e a c c e p t e d . w i t h o u t d i s t u r b i n g t h e n o r m a l d e l i v e r y , t w o i n d e p e n d e n t blood s a m p l e s w e r e d r a w n f r o m t h e c l a m p e d c o r d by p u n c t u r i n g t h e u m b i l i c a l vein 2-4 a n d 5-8 rnin a f t e r d e l i v e r y , t h e s e c o n d s a m p l e c l o s e r to t h e p l a c e n t a . with a 16c s i l i c o n i z e d c a n n u l a , 9 m l of blood w e r e a s p i r a t e d i n t o a s y r i n g e c o n t a i n i n g 1 m l of s o d i u m c i t r a t e (3.2%). t h e s a m p l e w a s a c c e p t e d only if i t w a s o b t a i n e d e a s i l y w i t h i n 20 sec. a f t e r blood s a m p l i n g , a l l handling of t h e t w o s a m p l e s f r o m t h e s a m e c o r d w a s s i m u l t a n e o u s . t h e p l a t e l e t r i c h p l a s m a ( p r p ) w a s p r e p a r e d by c e n t r i f u g a t i o n f o r 15 m i n a t 140c at room t e m p e r a t u r e . t h e p l a t e l e t c o u n t of t h e p r p w a s d e t e r m i n e d on a t h r o m b o c o u n t e r c ( c o u l t e r e l e c t r o n i c s , h i a l e a h , f l o r i d a , usa). a p l a t e l e t suspension of 300 x lo9/l w a s o b t a i n e d by d i l u t i n g t h e p r p w i t h p l a t e l e t p o o r p l a s m a (ppp), which w a s y i e l d e d a f t e r spinning down t h e r e s t of t h e s a m p l e f o r 10 min a t 2 7 5 0 6 . p l a t e l e t a g g r e g a t i o n w a s p e r f o r m e d in a p a y t o n d u a l c h a n n e l a g g r e g o m e t e r at 3 7 0 c , w i t h m a g n e t i c s t i r r i n g a t 900 r p m within 30-90 rnin p o s t p a r t u m . a f t e r a s t a b l e base-line had b e e n e s t a b l i s h e d f o r half a m i n u t e , a g g r e g a t i o n w a s induced in 0.25 m l of t h e p l a t e l e t suspension by a d p (sigma, s t o c k h o l m , s w e d e n ) 1.1 o r 3.3 u m o l / l , c o l l a g e n (hormon c h e m i e , munich, g d r ) 1.0 o r 5.5 mg/l, o r e p i n e p h r i n e (aco, s t o c k h o l m , s w e d e n ) 8.3 ug/l -final c o n c e n t r a t i o n in t h e c u v e t t e . l i g h t t r a n s m i s s i o n c h a n g e s ( a o d ) w e r e r e c o r d e d a n d e x p r e s s e d in p e r c e n t of t h e p r p p p p l i g h t t r a n s m i s s i o n d i f f e r e n c e . m a x i m a l a g g r e g a t i o n , a n d f o r c o l l a g e n a l s o t h e d u r a t i o n of t h e l a g p h a s e (sec), w e r e m e a s u r e d at a l l recordings. q u a l i t a t i v e l y t h e r e c o r d i n g s w e r e a n a l y z e d a c c o r d i n g t o t y p e of c u r v e , i.e. r e v e r s i b l e or i r r e v e r s i b l e a g g r e g a t i o n . a s t h e a m o u n t s of p l a t e l e t suspension o b t a i n e d a f t e r p r e p a r a t i o n w e r e too s m a l l in s o m e cases, a l l s t i m u l i c o u l d n o t b e t e s t e d in a l l cords. a l l a g g r e g a t i o n s p e r f o r m e d in t h e 17 c o r d s a r e i n c l u d e d in t h e following d e s c r i p t i o n of t h e r e s u l t s . m e a n s a n d s t a n d a r d d e v i a t i o n s a r e given. s i g n i f i c a n c e a n a l y s i s 10 w a s p e r f o r m e d by p a i r e d t-tests. results p l a t e l e t c o u n t t h e p l a t e l e t c o u n t i n p r p v a r i e d w i d e l y f r o m o n e c o r d to a n o t h e r (365-1140 x lo9/l), b u t t h e r e w a s n o s y s t e m a t i c d i f f e r e n c e b e t w e e n t h e e a r l y a n d t h e l a t e s a m p l e s ( t a b l e 1). t h e v a r i a t i o n i n t h e d i f f e r e n c e in p l a t e l e t c o u n t b e t w e e n t h e e a r l y a n d t h e l a t e s a m p l e , e x p r e s s e d as p e r c e n t of t h e c o u n t in the e a r l y s a m p l e , w a s a l s o c o n s i d e r a b l e ( 25.5 +21.8 %). in n i n e of t h e 17 c o r d s t h i s d i f f e r e n c e w a s less t h a n 5% ( t a b l e i). in t h e f o l l o w i n g p r e s e n t a t i o n w e h a v e d i v i d e d t h e 17 c o r d s a r b i t r a r i l y i n t o t w o g r o u p s g r o u p a, w i t h a d i f f e r e n c e in p l a t e l e t c o u n t b e t w e e n the e a r l y a n d t h e l a t e c o r d blood s a m p l e of <5%, a n d g r o u p b, w i t h a d i f f e r e n c e of >5%. t a b l e i . platelet c o u n t in p r p of e a r l y a n d l a t e c o r d blood s a m p l e s . ( m e a n +sd) c o r d no. e a r l y s a m p l e s l a t e s a m p l e s d i f f e r e n c e / g r o u p e a r l y s a m p l e % i 2 3 4 5 6 7 8 9 10 i 1 12 13 14 15 16 17 890 440 850 i140 740 625 390 700 652 700 700 777 533 520 660 490 1035 885 380 840 960 740 660 475 750 659 670 680 77 1 479 525 740 365 995 0.6 13.6 1.2 15.8 + o 5.6 + 21.8 + 7.1 + 1.1 4.3 2.9 0.8 10.1 + 1.0 + 12.1 25.5 3.9 a b a 0 a b b b a a a a b a b 0 a t o t a l 697 5 2 0 1 681 5 187 g r o u p a (n=9) 763 5 149 752 a 140 g r o u p b (n=8) 622 2 235 601 5 2 1 0 p l a t e l e t aggregation q u a l i t a t i v e d e s c r i p t i o n e p i n e h r i n e (8.3 ug/l) p r o d u c e d n o v i s i b l e r e a c t i o n s (n = 8). c o l l a g e n 1.0 m g / l (n = 16) p r o d u c e d n o r e s p o n s e i n i 1 c o r d s a n d i r r e v e r s i b l e a g g r e g a t i o n i n one. in t h e r e m a i n i n g f o u r c o r d s r e v e r s i b l e a g g r e g a t i o n w a s o b t a i n e d . c o l l a g e n 5.5 mg/l (n = 12) c a u s e d r e v e r s i b l e a g g r e g a t i o n i n o n e case a n d i r r e v e r s i b l e 1 1 aggregation in i 1 cases. a d p 1.1 pmol/l (n = 15) produced reversible aggregation in 12 cases. in one cord no r e a c t i o n was seen. in t h e remaining t w o cords, both in group b, reversible aggregation was noted in t h e e a r l y sample, while in t h e l a t e sample no significant aggregation was s e e n . a d p 3.3 v m o l / l (n = 17) produced reversible aggregation in 13 c o r d s and irreversible aggregation in four. t h e r e was no d i f f e r e n c e between t h e e a r l y and t h e l a t e s a m p l e with r e s p e c t to q u a l i t a t i v e responses t o any of t h e stimuli t e s t e d in group a, while in group b t h e r e was slight inconsistency f o r a d p 1.1 pmol/l. q u a n t i t a t i v e results the number of s a m p l e s studied was sufficient for s t a t i s t i c a l evaluation in t h e case of a d p 1.1 and 3.3 u m o l / l and of collagen 5.5 mg/l. in group a t h e e a r l y and t h e l a t e s a m p l e s from t h e s a m e cord showed very similar peak aggregation responses t o t h e s e stimuli (fig.l), t h e k values (slopes) f o r t h e regression lines q u i t e c l o s e t o 1 (0.81-1.01) and c o r r e l a t i o n c o e f f i c i e n t s relatively high (r = 0.85-0.95). t h e r e was also a s t r o n g c o r r e l a t i o n between t h e e a r l y and l a t e samples with r e s p e c t to t h e lag phases in response t o collagen 5.5 mg/l (r = 0.99). group a r = 0.85 25 %f, k = 1.02 ~ 0 0 25 50 aod% 3 3,urnol/l 100 100 50 group a . r = 0.89 k . 0 8 6 50 i " 0 0 50 100 aod% 0 5 5 m g i i group a r = 0.95 0 l 50 k = o 8 1 100 aody. early samples relationship b e t w e e n maximal aggregation (aod %) in e a r l y and l a t e blood samples in response to a d p 1.1 pmol/l, a d p 3.3 p m o l / l and collagen 5.5 mg/l. group a (b) and group b (0). the line r e p r e s e n t s t h e equation x = y. a s a l s o s e e n in fig.1, t h e relations between t h e e a r l y and t h e late s a m p l e s w e r e less c o n s i s t e n t in group b with k values varying from 0.59-2.25 and r = 0.53-0.98. t h e r e was a small but s y s t e m a t i c d i f f e r e n c e in t h e maximal aod b e t w e e n t h e e a r l y and l a t e samples in group a, with a significantly lower peak aggregation in t h e early s a m p l e f o r a l l t h r e e t e s t e d stimuli (table 2, fig.1). a similar significant d i f f e r e n c e , with a s h o r t e r l a g phase f o r t h e l a t e sample, was a l s o found in response to collagen 5.5 mg/l in group a (p<o.oi) but not in t h e t o t a l group. no significant d i f f e r e n c e b e t w e e n t h e early and t h e l a t e samples was found in group b (p>o.1 f o r a l l stimuli) (table 2). 12 table 2. aggregation responses (aod) t o adp and collagen sign i f icant). (mean +sd)(n.s. = non stimuli maximal aggregation @od) early sample l a t e sample adp 1.1 p m o l / l early sample l a t e sample a d p 3.3 p m o l / l early sample l a t e sample collagen 5.5 m g / l t o t a l (n= 12) 14.9 54.5 p<0.02 18.055.3 (n=17) 38.921 6.0 n.s. 40.9217.6 (n= 12) 77.3515.0 n.s. 80.3217.8 group a (n=8) 1 4.954.5 p<o.o1 18.625.4 (n=9) 33.9l12.5 p<o.o1 39.4212.0 (n=9) 77 .82 i 6.7 p<o.o1 84.3514.3 group b (n=4) 15.055.1 n.s. 16.855.7 (n=8) 44.651 8.4 n.s. 42.5523.1 a c i d base data are given in table 3. p c 0 2 was significantly lower in the l a t e samples. table 3. acid-base data for a l l samples and for groups a and b. (mean +sd)(n.s. = non significant). ph t o t a l (n= 15) group a (n=8) group b (n=7) pco2 t o t a l (n= 15) group a (n=8) group b (n=7) early sample l a t e sample early sample l a t e sample early sample l a t e sample early sample l a t e sample early sample l a t e sample early sample l a t e sample before aggregation 7.4320.06 n.s. 7.4350.05 7.4320.05 n.s. 7.4320.05 7.4320.07 n . s. 7.4350.06 3.7020.39 3.5950.43 3.7620.34 ~ ~ 0 . 0 5 3.6750.33 3.6350.45 3.5020.53 p<o . 00 i p<o.o1 a f t e r aggregation 7.5820.09 n.s. 7.5950.10 7.5920.07 n.s. 7.5920.07 7.5720.1 i n.s. 7.5850.13 2.720.47 2.5920.48 2.6920.37 n.s. 2.5820.3 3 2.75l0.60 2.6020.64 p<0.02 p<o.o1 13 discussion d u r i n g t h e f i r s t m i n u t e s a f t e r b i r t h t r e m e n d o u s biological c h a n g e s o c c u r in t h e c h i l d a n d t h e p l a c e n t a , m a n y of which m a y b e e x p e c t e d to i n f l u e n c e t h e v e r y c o m p l e x f a c t o r s underlying p l a t e l e t a g g r e g a o i l i t y . if p l a t e l e t a c t i v a t i o n o c c u r s d u r i n g d e l i v e r y , t h e n u m b e r of c i r c u l a t i n g p l a t e l e t s m i g h t b e d e c r e a s e d . p l a t e l e t a c t i v a t i o n c a n a l s o t a k e p l a c e d u r i n g blood s a m p l i n g a n d p r e p a r a t i o n of p l a t e l e t suspensions (8). t h i s i s e v i d e n t f r o m o u r f i n d i n g s of d i f f e r e n c e s in p l a t e l e t c o u n t s . in t w o i n d e p e n d e n t blood s a m p l e s t a k e n at a n i n t e r v a l of 3-5 min t h e r e w a s no s y s t e m a t i c d i f f e r e n c e b e t w e e n t h e e a r l y and t h e l a t e s a m p l e s b u t a r a n d o m l y d i s t r i b u t e d d i f f e r e n c e b e t w e e n -26 a n d +22 %. t h e cut-off p o i n t w a s set a t 5 % f o r t w o reasons: 1. t h e d i s t r i b u t i o n of t h e d i f f e r e n c e s w a s c o m p l e t e l y s y m m e t r i c a l b u t s t i l l s i g n i f i c a n t l y d i f f e r e n t f r o m a n o r m a l d i s t r i b u t i o n . n i n e cases w e r e c l u s t e r e d a r o u n d a m e a n of -1% ( r a n g e -5 to +3%). t h e r e m a i n i n g e i g h t cases w e r e e v e n l y d i s t r i b u t e d in t h e r a n g e s + 5 t o 30% ( f o u r c a s e s ) a n d -5 t o -30% ( f o u r cases). t h i s f i t s w i t h t h e h y p o t h e s i s t h a t t h o s e c l u s t e r e d a r o u n d t h e m e a n r e p r e s e n t d i f f e r e n c e s b e t w e e n t w o biologically i d e n t i c a l s p e c i m e n s ( g r o u p a), while t h e o t h e r s r e p r e s e n t d i f f e r e n c e s b e t w e e n o n e s p e c i m e n w i t h a r e d u c e d n u m b e r of p l a t e l e t s a n d a n o t h e r w i t h t h e o r i g i n a l p l a t e l e t c o u n t . 2. t h e cut-off l i m i t s had t o e x c e e d t h e e m p i r i c a l c o u n t i n g e r r o r w i t h t h e c o u n t e r u s e d in t h e h o s p i t a l (1-3%). t h e v e r y high c o r r e l a t i o n s b e t w e e n t h e a g g r e g a t i o n r e s p o n s e s in t h e e a r l y a n d t h e l a t e s a m p l e s in g r o u p a, o b t a i n e d at q u a n t i t a t i v e analysis, a r e in a g r e e m e n t w i t h t h e c o n c l u s i o n t h a t no s i g n i f i c a n t c h a n g e in p l a t e l e t a g g r e g a b i l i t y had t a k e n p l a c e . t h e m o r e i n c o n s i s t e n t r e s u l t s in g r o u p b s u g g e s t , on t h e c o n t r a r y , t h a t c h a n g e s in p l a t e l e t a g g r e g a b i l i t y had o c c u r r e d in o n e or b o t h s a m p l e s . t h i s f u n c t i o n a l a l t e r a t i o n m i g h t b e d u e to a r e l e a s e of s m a l l a m o u n t s of adp f r o m a c t i v a t e d p l a t e l e t s , r e s u l t i n g in a t e m p o r a r y u n r e s p o n s i v e n e s s of s o m e of t h e p l a t e l e t s t o a g g r e g a t i n g a g e n t s (7). t h e i n t e r e s t i n g f i n d i n g of a s m a l l b u t s y s t e m a t i c d i f f e r e n c e , w i t h h i g h e r p l a t e l e t a g g r e g a b i l i t y in t h e l a t e s a m p l e , w a s only m a d e in g r o u p a (0% d i f f e r e n c e in p l a t e l e t count). t h i s i n d i c a t e s t h a t t h e s e s u b t l e q u a n t i t a t i v e c h a n g e s in p l a t e l e t a g g r e g a b i l i t y are only d e t e c t a b l e in s a m p l e s n o t e x p o s e d t o a c t i v a t i o n . platelet c o u n t i n g in p r p f r o m t w o i n d e p e n d e n t blood s a m p l e s t h u s s e e m s t o e f f e c t i v e l y d i s c l o s e p l a t e l e t a c t i v a t i o n a n d i s r e c o m m e n d e d f o r q u a l i t y c o n t r o l . t h e r e a s o n f o r t h e m o r e pronounced a g g r e g a b i l i t y in t h e late s a m p l e s c a n n o t b e e x p l a i n e d by t h e s e d a t a . acid-base c h a n g e s c o u l d n o t a n s w e r f o r t h e d i f f e r e n c e , as t h e only s i g n i f i c a n t d i s c r e p a n c y o b s e r v e d in p c 0 2 b e t w e e n e a r l y a n d l a t e s a m p l e s would b e e x p e c t e d t o i n f l u e n c e p l a t e l e t a g g r e g a b i l i t y in t h e o p p o s i t e d i r e c t i o n (2). d i f f e r e n t physiological p r o c e s s e s involved i n t h e n o r m a l d e l i v e r y , expulsion of t h e p l a c e n t a a n d c o n s t r i c t i o n of t h e u m b i l i c a l v e s s e l s m i g h t r e s u l t i n t h e r e l e a s e of s u b s t a n c e s i n f l u e n c i n g p l a t e l e t f u n c t i o n , e.g. c a t e c h o l a m i n e s ( 5 ) , 5 h y d r o x y t r y p t a m i n e (5-ht) (14) a n d p r o s t a g l a n d i n s (13). 14 this s t u d y , in c o n t r a s t t o previous i n v e s t i g a t i o n s in t h i s field, c o m b i n e s e x t r e m e l y s t r i c t c r i t e r i a f o r inclusion in t h e m a t e r i a l with a two-sample t e c h n i q u e f o r exclusion of a c t i v a t e d samples. using t h i s t e c h n i q u e c o r d blood a g g r e g a t i o n s t u d i e s in newborn i n f a n t s c a n b e possible to p e r f o r m for s c i e n t i f i c purposes. acknowledgement this s t u d y w a s s u p p o r t e d by a g r a n t f r o m s t i f t e l s e n s a m a r i t e n t o t o r s t e n tuverno. references i. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. corby, d.g. & schulman, i.: t h e e f f e c t s of a n t e n a t a l d r u g a d m i n i s t r a t i o n on a g g r e g a t i o n of p l a t e l e t s of newborn infants. j p e d i a t r 79:307-313, 1971. foley, m.e. & mcnicol, g.p.: an in v i t r o s t u d y of acidosis, p l a t e l e t f u n c t i o n , and p e r i n a t a l c e r e b r a l i n t r a v e n t r i c u l a r h a e m o r r h a g e . l a n c e t i:1230-1232, 1977. haslam, r.r., e k e r t , h. & gillam, g.l.: hernorrage in a n e o n a t e possibly d u e to m a t e r n a l ingestion of s a l i c y l a t e . j p e d i a t r 84:556-557, 1974. h a t h a w a y , w.e., mahasandana, c. & makowski, e.l.: c o r d blood c o a g u l a t i o n s t u d i e s in i n f a n t s of high-risk p r e g n a n t women. am j o b s t e t g y n a e c o l 121:51-57, 1975. l a g e r c r a n t z , h. & b i s t o l e t t i , p.l.:catecholamine r e l e a s e in t h e newborn i n f a n t a t birth. p e d i a t r r e s 11:889-893, 1977. mull, m.m. & h a t h a w a y , w.e.: a l t e r e d p l a t e l e t f u n c t i o n in newborns. p e d i a t r r e s p a c k h a m , m.a., ardlie, n.g. & mustard, j.f.: e f f e c t of a d e n i n e compounds on p l a t e l e t a g g r e g a t i o n . am j physiol 217:1009-1017, 1969. salzrnan, e.w., lindon, j.n. & rodvien, r.: c y c l i c amp in human blood p l a t e l e t s : r e l a t i o n t o p l a t e l e t prostaglandin s y n t h e s i s induced by c e n t r i f u g a t i o n o r s u r f a c e c o n t a c t . j c y c l i c n u c l e o t i d e r e s 2:25-37, 1976. s a n t a n g e l o , g., palumbo, a. & maccarrone, n.: fisopatologia e v a l u a z i o n e d i a g n o s t i c a dell' a g g r e g a z i o n e p i a s t r i n i c a in pediatria. minerva p e d i a t r 27: 148-154, 1975. s t u a r t , m.j.: t h e n e o n a t a l p l a t e l e t : evaluation of p l a t e l e t rnalonyl dialdehyde f o r m a t i o n as a n i n d i c a t o r of prostaglandin synthesis. rr j h a e m a t o l 39:83-90, 1978. s t u a r t , m.j., elrad, h., g r a e b e r , j.e., hakanson, d.o., sunderji, s.g. & barvinchak, m.k.: i n c r e a s e d s y n t h e s i s of prostaglandin endoperoxides a n d p l a t e l e t h y p e r f u n c t i o n in i n f a n t s of m o t h e r s with d i a b e t e s mellitus. j l a b clin med 94:12-17, 1979. tsao, c., lo, r. & raymond, j.: c r i t i c a l i m p o r t a n c e of citrate-blood r a t i o in p l a t e l e t a g g r e g a t i o n studies. am j clin p a t h o l 65:518-522, 1976. t u v e m o , t.: r o l e of prostaglandins, prostacyclin a n d t h r o m b o x a n e s in t h e c o n t r o l of t h e umbilical-placental c i r c u l a t i o n . in: p r o s t a g l a n d i n s in t h e p e r i n a t a l period (ed. m.a.heyrnan), pp.91-95. g r u n e & s t r a t t o n inc., new york, n.y., 1980. whaun, j.m.: t h e p l a t e l e t of t h e newborn infant: 5-hydroxytryptarnine u p t a k e and release. thrornb h a e m o s t 30:327-333, 1973. 4:229-237, 1970. a d d r e s s f o r reprints: dr g.ahlsten d e p a r t m e n t of p a e d i a t r i c s university h o s p i t a l 5-750 14 uppsala sweden 15 vol_116_002_sups_a_552812 107..114 upsala journal of medical sciences. 2011; 116: 107–114 original article therapeutic strategy of third-generation autologous chondrocyte implantation for osteoarthritis tomoya kuroda1,2, tomoyuki matsumoto1,2, yutaka mifune1,2, tomoaki fukui1,2, seiji kubo1, takehiko matsushita1, takayuki asahara2,3, masahiro kurosaka1 & ryosuke kuroda1 1kobe university graduate school of medicine/department of orthopaedic surgery, kobe, japan, 2stem cell translational research, kobe institute of biomedical research and innovation, kobe, japan, and 3department of regenerative medicine science, tokai university school of medicine, tokai, japan abstract background. autologous chondrocyte implantation (aci) is considered a promising choice for the treatment of cartilage defects. however, the application of aci to osteoarthritic patients is, in general, contraindicated. the purpose of this study is to evaluate the efficiency of three-dimensionallystructured aci (3d-aci; cares) in a rat model of knee osteoarthritis (oa). methods. oa-like degenerative changes in the articular cartilage were created by transecting the anterior cruciate ligament (aclt) in athymic nude rats. two weeks later, cares was transplanted at the cartilage injury sites created by micro-drilling in the patella groove (chondrocyte-implanted (ci) group: cares collagen with human chondrocytes; collagen group: cares collagen without cells; and sham group: sham operation; n = 15/group). results. reverse transcription polymerase chain reaction (rt-pcr) analysis demonstrated the expression of humanspecific type 2 collagen and sry-type high-mobility-group box 9 (sox9) in the ci group—not in the other groups—throughout the study period. double immunohistochemistryfor human-specific type 2 collagenand human leukocyte antigen-abacavir (hlaabc) at week 4 showed positive staining in the ci group only. macroscopic assessment showed better repair at the cartilage defect sitesinthecigroup,comparedtotheothergroups.histologicalassessmentwithtoluidinebluestainingshowedthatthethicknessof the articular cartilage and semi-quantitative histological scores were higher in the ci group than in the other groups up to week 20. conclusions. we demonstrate, for the first time, that 3d-aci is effective in repairing cartilage defects in a rat model of aclt-induced oa. key words: chondrocyte implantation, geriatrics, knee osteoarthritis, orthopedics introduction knee osteoarthritis (oa), a clinical syndrome with low-grade inflammation caused by abnormal wear of the articular cartilage, is associated with pain, destruction of joints, and/or a decrease in the synovial fluid that lubricates the joints. it is estimated that the number of patients with knee oa is more than 21 million in the us, and symptomatic oa of the knee occurs in 6% of adults aged 30 or older and 13% of people aged 60 or over (1). however, it is also well known that because of the limited capacity of the cartilage for repair, abnormal wear of the articular cartilage is a major clinical problem when the cartilage becomes damaged (2). recently, various therapeutic strategies including bone-marrow stimulation and transplantation of osteochondral autografts or allografts have been developed to restore articular cartilage so as to achieve a permanent repair (3). among them, autologous chondrocyte implantation (aci) is a promising choice for cartilage repair. the classical aci (first generation) correspondence: tomoyuki matsumoto, md, phd, kobe university graduate school of medicine/department of orthopaedic surgery, 7-5-2 kusunoki-cho, chuo-ku, kobe 650-0017, japan. fax: +81-78-351-6944. e-mail: matsun@m4.dion.ne.jp (received 29 september 2010; accepted 3 january 2011) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2011.552812 was originally described in 1994 (4) and approved by the us food and drug administration in 1997 (5). this first-generation aci provided significant and long-term benefits for patients in terms of diminished pain and improved function (6). in order to overcome the hypertrophy or ossification of the patched periosteum and the complex operative technique associated with first-generation aci, a second-generation aci was developed, in which bioengineered bilayer collagen or synthetic membranes were used in order to avoid spill-over and asymmetric distribution of chondrocytes following implantation. in some products, a periosteal flap is not needed. recently, further technological advances have led to a third-generation aci, where chondrocytes are embedded into three-dimensionally constructed scaffolds (i.e. 3d environment) for cell growth. these ‘all-in-one’ grafts do not need a periosteal cover or fixing stitches and can be trimmed to exactly fit into the cartilage defect with fibrin glue (7). indeed, several clinical studies of 3d-aci for cartilage injury have been reported (2,4,8–10). despite the advantages of this new technique in surgical simplicity, shorter operating time, and the possibility of performing the surgery via a mini-arthrotomy or arthroscopy, the product has contraindication for use in patients suffering from oa. however, there are several studies in which favorable clinical outcomes have been reported (7,11). therefore, the purpose of this study is to evaluate the efficiency of 3d-aci using a rat model of knee osteoarthritis produced by transection of the anterior cruciate ligament (acl). materials and methods oa animal model the institutional animal care and committees of riken center for developmental biology approved all animal procedures. female athymic nude rats (f344/n jcl rnu/rnu, japan clea) aged 8–10 weeks, weighing an average of 200 g, underwent acl transection (aclt) as previously reported (12). animals were briefly anesthetized by an intraperitoneal injection of ketamine hydrochloride (60 mg/kg) and with ether. after shaving of the knee joint, a parapatellar incision was made, followed by lateral displacement of the patella, thereby enabling access to the joint space. the acl was then easily visible and surgically excised. the increase in anterior displacement of the tibia in relation to the femur was used to ensure that the ligament was transected. the surgical incision was then closed. at the end of the 2-week post-surgical period, in order to confirm the progression to oa, the knee joints were opened in a parapatellar approach, and the entire knee was examined macroscopically and photographed. then the distal femurs were removed and histologically assessed as described later. preparation of chondrocytes and collagen matrix in this study, to meet the demand supposing a clinical use, we used human chondrocytes. human chondrocytes isolated from a healthy volunteer (28 years old, african-american) were purchased from lonza (basel, switzerland). to expand these cells, 1 � 105 cells were plated in 6-well dishes with standard medium (alpha-mem; gibcobrl, invitrogen japan k.k., tokyo, japan) containing 10% fetal bovine serum (vitromex, san antonio, tx, usa), 2 mm l-glutamine (gibcobrl), 100 units/ml penicillin (gibcobrl), and 100 mg/ml streptomycin (gibcobrl) and incubated at 37�c with 5% co2 in ambient air. the medium was replaced every 3–4 days. after culture for 14 days, the cells were harvested with 0.25% trypsin/1mm edta solution (gibcobrl) and counted. cares (arthro kinetics, krems am de donau, austria) is a 3d mechanically stable transplantation system based on patient-specific autologous cartilage cells. these counted cells were expanded in vitro according to cares manufacturer’s protocol. in brief, they were expanded with standard medium (see above) and subsequently 2 � 107 cells were mixed with 50 ml collagen ms solution, a gel neutralization solution (arthro kinetics, krems am de donau, austria). an appropriate volume of the above solution was pipetted into microtiter plates and incubated for 20 minutes. culture was performed at 37�c, 5% co2, and 95% relative humidity (rh) for 10–14 days. culture medium was removed from the gel every 3–4 days and replaced by fresh and preheated (37�c) medium. preparation of collagen matrix without cells was conducted in the same manner as those with cells, except that no cells were seeded. cell implantation and study groups a parapatellar incision was made, and the knee joint was exposed via lateral dislocation of the patella. to make an appropriate space to mount the chondrocyteloaded collagen implant in the trochlear groove of each femur, a 1.5-mm diameter micro-drill was used. animals in the chondrocyte-implanted (ci) group were treated with several mm of the chondrocyteloaded collagen gel (normal articular cartilage thickness is 0.072 ± 0.013 mm (13)) and fixed with 108 t. kuroda et al. fibrin glue (kaketsuken, kumamoto, japan) to keep the implant stable. animals in the collagen group were treated with fibrin glue-fixed collagen implants without cells. animals in the sham group were treated with fibrin glue only (n = 15 each). after surgery, the rats were allowed to move freely within their cages. no animal was observed to have an abnormal gait or impaired locomotion. rt-pcr analysis of rna isolated from transplantationsites to examine human cell-derived chondrogenesis, mrna expression for chondrogenic markers (humanspecifictype2collagen(col2)andsox9)wasassessed byrt-pcranalysis.atweek4,totalrnawasobtained from the transplantation sites using tri-zol (life technologies, gaithersburg, md, usa) according to themanufacturer’sinstructions.first-strandcdnawas synthesized using the rna la pcrkit ver 1.1 (takara bio. inc., otsu, japan), amplified by taq dna polymerase (advantage-gc cdna pcr kit, clontech and amplitaq gold dna polymerase, applied biosystems). pcr was performed using a pcr thermocycler (mj research ptc-225). and hcol2, hsox9, and rgapdh were amplified by taq dna polymerase (advantage-gc cdna pcr kit, clontech) at the following conditions: 35 cycles of 30-second initial denaturation at 94�c, annealing at 56�c for 1 minute, and 30 seconds of extension at 72�c according to the manufacturer’s instructions. subsequently, pcr products were visualized in 1.5% ethidium bromide stained agarose gels. human chondrocytes (lonza) were used for positive control for human-specific chondrocyte-related genes. primers to avoid interspecies cross-reactivity of the primer pairs between human and rat genes, we designed the following human-specific primers using oligo software (takara bio. inc.). none of the primer pairs showed any cross-reactivity to rat genes (data not shown). . hcol2 primer sequence: sense aag caa gta gcg cca atc t; antisense gga agt agg gtg cca taa cac . hsox9 primer sequence: sense atg ttt ggc ctg aag cag aga; antisense ggc ggt aca ggt cga gca tat a . rgapdh primer sequence: sense ctg atg ccc cca tgt tcg tc; antisense cac cct gtt gct gta gcc aaa ttc g tissue harvesting rats were euthanized with an overdose of ketamine and xylazine. bilateral femurs were harvested and quickly embedded in optimal cutting temperature (oct) compound (miles scientific, elkhardt, in, usa), snap-frozen in liquid nitrogen, and stored at �80�c for histochemical and immunohistochemical staining as described previously (14). rat femurs in oct blocks were sectioned, and 6-mm serial sections were mounted on silane-coated glass slides and air dried for 1 hour before being fixed with 4.0% paraformaldehyde at 4�c for 5 minutes and stained immediately. immunohistochemical staining to detect the chondral restoration by transplanted human cells in the rat tissue at week 4, immunohistochemistry was performed with the following human-specific antibodies; human leukocyte antigen (hla)-abc (becton-dickinson (bd) pharmingen, franklin lakes, nj, usa) to detect various lineages of human cells and hcol2 (bd pharmingen) to detect human cell-derived chondrocytes. staining specificity for human cells without cross-reaction to rat cells was confirmed by histochemical staining for hla-abc and hcol2 using rat and human chondrocytes (lonza) (data not shown). the secondary antibodies for each immunostaining were as follows: alexa fluor 594-conjugated goat anti-mouse igg1 (molecular probes, invitrogen japan k.k., tokyo, japan) for hla-abc staining and alexa fluor 488-conjugated goat anti-mouse igg2a (molecular probes) for hcol2. dapi solution was applied for 5 minutes for nuclear staining. double immunohistochemistry with hla-abc and hcol2 was performed to detect human chondrocytes in the articular cartilage. macroscopic and histological assessment of cartilage regeneration to confirm the sequential recovery from oa-like arthritis, we performed macroscopic and histological evaluation from 0 to 20 weeks after operation. the rats were euthanized at 4, 8, and 20 weeks after the operation. the entire knee was dissected, examined macroscopically, and photographed. the distal parts of the femur were then fixed as described above. each specimen was sectioned (5 mm thick) sagittally and perpendicularly to the defect. the sections were stained with toluidine blue. these sections were obtained from the center of the defect, and as many as ten such sections were chondrocyte implantation for osteoarthritis 109 prepared from each knee. the sections from each animal were examined and scored independently by three blinded observers. we evaluated chondral repair semi-quantitatively using a grading and scoring system, which is a modification of that described by prizker et al. (15). the scale is composed of six histological gradings (0: surface intact, cartilage intact; 1: surface intact; 2: surface discontinuity; 3: vertical fissures; 4: erosion; 5: denudation; 6: deformation) and four histological stages (0: no oa activity; 1: <10% involvement (surface, area, volume); 2: 10%–25%; 3: 25%–50%; 4: >50%) assigning a total score (score = grade � stage) ranging from 1 point (normal articular cartilage) to 24 points (no repair). statistical analysis the results were statistically analyzed using a software package (microsoft office excel, microsoft corporation, washington, usa). all values were expressed as means ± se. multiple comparisons among groups were made using a one-way analysis of variance (anova). post-hoc analysis was performed by fisher’s plsd test. a probability value < 0.05 was considered to denote statistical significance. results confirmation of aclt model of rat osteoarthritis at 2 weeks after the aclt operation, irregular articular surface and osteophyte formation around the margin of the bilateral femoral condyle and the patella groove were confirmed (figure 1a). toluidine blue staining showed gradual reduction of the thickness of the articular cartilage layer in the patella groove (figure 1b). chondrogenesis by transplanted human cells to histologically validate the phenomenon of human cell-derived chondrogenesis at the repair sites, hlaabc staining for various lineages of human cells and hcol2 for human cell-derived chondrocytes was performed using tissue samples obtained at 4 weeks after implantation. transplanted human cells were detected as hla-abc-positive cells. differentiated human chondrocytes derived from the transplanted cells were detected as hcol2-positive cells at the chondral repair site in the ci group, while hcol2positive cells were not identified in the other groups (figure 2a). the number of double-stained cells was higher in the ci group compared to the other groups (ci group 820 ± 52.3/mm2; collagen group a day 0 day 7 day 14 b figure 1. rat osteoarthritis (oa) model with anterior cruciate ligament transection. a: macroscopic appearance of the anterior cruciate ligament transection (aclt) model of rat oa. at 2 weeks post-surgery, the joints have obvious articular surface damage and osteophyte formation around the margin of the medial femoral condyles and the patella groove. b: toluidine blue staining showing the thickness of gradual degeneration of the patellar groove (original magnification �20). 110 t. kuroda et al. 0.0 ± 0.0/mm2; sham group0.0 ± 0.0/mm2, respectively (p < 0.01 for ci group versus the other groups)). rt-pcr analysis of tissue rna, isolated from the transplantation sites, for human-specific chondrogenic markers (hcol2 and hsox9) revealed that expression of hcol2 and hsox9 was detected in the ci group but not in the other groups (figure 2b). macroscopic and histological recovery of regenerated cartilage macroscopic assessment showed better recovery as judged by smooth surface of articular cartilage in the ci group when compared to the other groups at week 4 and 8 (figure 3a). histological assessment with toluidine blue staining (figure 3b) showed that the thickness of the articular cartilage was significantly higher in the ci group than in the other groups at week 4 (ci group 15.3 ± 4.1 mm; collagen group 6.0 ± 1.0 mm; sham group 5.0 ± 2.0 mm, respectively; p < 0.05 for ci group versus the other groups). the differences between the ci group and the other groups also attained statistical significance at week 8 (ci group 27.5 ± 3.6 mm; collagen group 9.7 ± 2.5 mm; sham group 8.3 ± 1.5 mm, respectively; p < 0.05 for ci group versus the other groups). semiquantitative histological scorings at week 4 and 8 were better in the ci group than in the other groups (week 4: ci group 5.2 ± 1.1, collagen group 9.0 ± 2.1, sham group 15.6 ± 1.1; week 8: ci group 1 ± 0, collagen group 7.8 ± 1.6, sham group 14 ± 2.2, respectively; p < 0.05 for ci group versus the other groups). however, at week 20, the surfaces of the articular cartilage did not differ among the three groups (figure 3a). histological assessment with toluidine blue staining at week 20 nevertheless showed that the articular cartilage was thicker in the ci group than in the other groups, although the decreasing trend of the thickness was maintained (ci group 13.0 ± 3.6 mm; collagen group 2.6 ± 1.2 mm; sham group 3.0 ± 1.0 mm, respectively; p < 0.05 for ci versus the other groups) (figure 3b). in line with this, the histological score at week 20 was still better in the ci group than in the other groups (ci group 8 ± 2.8; collagen group 12 ± 2.8; sham group 22 ± 2.8, respectively; p < 0.05 for ci versus the other groups). discussion oa is characterized by the progressive loss of articular cartilage that leads to chronic pain and functional restrictions in affected joints. in the past decades, various surgical procedures including microfracture (16,17), autologous grafting procedures (18,19) and mosaicplasty (20,21) have been developed for the treatment of cartilage defects. however, there are 1 ci collagen sham hcol2 hsox9 hgapdh 2 3 4 5 a b figure 2. cartilage regeneration by transplanted human cells. a: representative double immunostaining for hcol2 (green) and hlaabc (red) using tissue sample of the regenerated articular cartilage at week 4 (original magnification �200).human-derived chondrocytes were identified as double-stained cells with hcol2 and hla-abc at the chondral repair site in the ci group (yellow area), but not in the other groups. b: rt-pcr analysis for human-specific chondrogenic markers of tissue rna isolated from the chondral repair sites. expression of hcol2 and hsox9 was detected in the ci group, but not in the other groups. 1: ci group. 2: collagen group. 3: sham group. 4: chondrocytes (positive control). 5: no rna (negative control). chondrocyte implantation for osteoarthritis 111 4 w ci collagen sham ci collagen sham 8 w 20 w 4 w 8 w 20 w a b figure 3. macroscopic and histological evaluation at weeks 4, 8, and 20. a: macroscopic assessment showing a better recovery as judged by a relatively smooth surface of the articular cartilage in the ci group compared to the other groups at weeks 4 and 8. in contrast, there was no apparent difference in the thickness of the articular cartilage between the three groups at week 20. arrows indicate the defect area. b: histological assessment with toluidine blue staining showing that the thickness of the articular cartilage was higher in the ci group than in the other groups at weeks 4, 8, and 20. however, at week 20, the thickness of the articular cartilage was decreasing in all groups. arrow heads indicate the thickness of the articular cartilage. 112 t. kuroda et al. no treatments that can reproducibly restore a normal articular surface. in recent years, chondrocyte implantation techniques, namely aci, have emerged as a potential therapeutic option. some studies also suggest a certain durability of the aci, even in longterm follow-up studies, possibly because of its ability to produce hyaline-like cartilage and better integration with the adjacent articular surface (22,23). until now, the efficacy of aci in patients suffering from oa has remained unclear. several researchers reported the application of aci to patients older than 45 years or who had failed prior treatments for articular cartilage defects of the knee (24,25). however, in these studies, oa-like changes of the articular cartilage was part of the excluding criteria. in the present study, we used collagen matrix, cares, as a thirdgeneration autologous chondrocyte implant. the cares is a 3d collagen type 1 gel, and the thickness of the transplants can be adjusted to the thickness of the cartilage in each patient. some clinical reports using the cares are encouraging, even in patellafemoral defects (26). however, there are no reports showing the usefulness of cares for cartilage repair in patients suffering from oa. in this study, we used human chondrocytes in athymic nude rats. using this xenotransplantation animal model, to confirm cartilage regeneration and maturation of human chondrocytes, molecular and immunohistological assessments using humanspecific markers were conducted. rt-pcr and immunohistochemistry demonstrated survival of implanted chondrocytes at the defect site of the rat knee. this scientific approach provides us with useful information demonstrating the mechanistic effect of the third-generation aci for oa. as the next step, we are planning to conduct a clinical trial to investigate the proof of concept of cares for use in focal oa patients. in addition, whereas a comparison of the cares technique with standard aci is beyond the scope of the current study, comparative experiments to examine the effects of different aci techniques in oa animal models are warranted. in the present study, we used human chondrocytes isolated from a relatively young volunteer. although we intend to use this system for relatively young oa patients with focal oa regions, implantation of chondrocytes from older patients should also be investigated for the confirmation of the efficacy of the system also in that group of oa patients. there was increased thickness of the articular cartilage and little deformity around the defect site when comparing the ci group with the other groups. this may be taken to indicate that the ci contributes to the stability of the cartilage. up to 8 weeks, the thickness of articular cartilage at the defect sites in the ci group improved even when oa was getting worse due to the instability of the knee joint following acl resection. however, at week 20, there was no increase of the thickness of the articular cartilage in the ci group. in the clinical setting, ligamentous instability is usually included in the excluding criteria of the study design. hollander et al. described that oa did not inhibit tissue regeneration and might even have enhanced it, suggesting that degenerating tissues are primed for repair and require only the appropriate cellular cues and environment for proper regeneration of healthy tissue (27). cartilage degradation in oa is considered to involve a delicate imbalance between anabolic and catabolic processes (28). injured joints without oa may lack the endogenous anabolic pathways. therefore, they must rely entirely on the introduced cells to produce a repair. in other words, joints with oa may repair relatively more easily than without oa. there are some limitations in this study, such as for instance that we used bovine serum to prepare the graft. the use of human serum should preferably be investigated for clinical settings. in addition, we used a 1.5-mm diameter micro-drill in order to stabilize the implant and ensure that the medullary canal space was not reached. however, there might be a possibility that there might be an effect of chondroprogenitor cells on the articular cartilage. hayes et al. described that the unique distribution of sulfation motifs within the microenvironment of superficial zone chondrocytes seems to designate early stages of stem/progenitor cell differentiation and is consistent with the fact that these molecules play a functional role in regulating aspects of chondrogenesis (29). in the present study using a rat oa model, we showed the therapeutic potential of third-generation aci for treating cartilage defects of knee oa. we believe our findings provide new insights into the treatment of oa and broaden the application of third-generation aci for knee oa as a new surgical modality. acknowledgements support was provided by the translational research support program, ministry of education 2007–2011 jst07057014. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. hunter dj. advanced imaging in osteoarthritis. bull nyu hosp jt 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father a child as long as spermatogenesis is present. there are several techniques to recover testicular sperm in patients with noa. however, retrieval of spermatozoa is unfortunately still only successful in a subset of patients with noa, and the most superior sperm retrieval method is still under debate. a more recent technique, microdissection testicular sperm extraction (md-tese) with an operative microscope collecting larger and more opaque seminiferous tubules, is a non-blind sperm retrieval technique with theoretical benefits. the md-tese procedure seems to be feasible, effective, and safe in noa patients but also more technically demanding and time-consuming compared with conventional blind techniques. in the present report, we describe our clinical experience and results from our first 159 md-tese procedures. the probability to retrieve sperm with the md-tese technique is high in noa cases where earlier sperm retrieval with blind methods such as needle aspiration, percutaneous needle biopsy, or conventional tese has failed. article history received 20 november 2019 revised 8 january 2020 accepted 27 february 2020 keywords azoospermia; in vitro fertilization; male infertility; micro-tese introduction the introduction of intracytoplasmic sperm injection (icsi) in 1992 revolutionized the treatment of male infertility (1). the most severe form of male infertility is non-obstructive azoospermia (noa) where spermatogenesis is impaired or totally absent. azoospermia is defined by the complete absence of spermatozoa in at least two semen analyses and is present in approximately 1% of adult men. if testicular spermatozoa can be retrieved, men with noa can achieve biological fatherhood by means of icsi (2). the aetiology of azoospermia is divided into three groups: pre-testicular, testicular (non-obstructive), and post-testicular (figure 1). pre-testicular cases can often be treated successfully with hormones. men with post-testicular (obstructive) azoospermia are usually normogonadotropic with normal spermatogenesis. then, spermatozoa can easily be recovered for icsi by percutaneous epididymal sperm aspiration (pesa) (3) or testicular sperm aspiration (tesa) (4,5). men with testicular azoospermia (noa) have impaired spermatogenesis, and surgical testicular procedures are required to retrieve sperm. there are several techniques available, such as tesa (4,5), percutaneous testicular biopsies (6), and open biopsies (conventional testicular sperm extraction, ctese) (2) (supplementary figure 1; available online). the overall success rate of retrieving sperm with these techniques varies between 25% and 50% in most reports (2,4–6). a more recent technique, microdissection testicular sperm extraction (md-tese), has been shown to achieve significantly better sperm recovery rates compared with ctese (7,8). md-tese is a ‘non-blind’ technique where seminiferous tubules are directly examined throughout the testis using an operative microscope (supplementary figure 1; available online). this is in contrast to tesa, percutaneous testicular biopsies, and ctese, which all are blind techniques. the md-tese procedure seems to be feasible and safe in noa patients but also more technically demanding and timeconsuming compared with ctese (7,9). in sweden, tesa and percutaneous biopsies are traditionally the most common methods to retrieve sperm for icsi in patients with noa. these procedures are carried out in most of the swedish fertility centres because of their simplicity and low costs. sperm retrieval rates (srr) with tesa vary, but, with an optimal technique, srr have been reported as high as 35% (4,10). in 2013 livio fertility center in g€oteborg was the first swedish ivf clinic to introduce md-tese, but recently additional centres have introduced it as well. however, due to the lack of large randomized controlled trials the superiority of this new technique is still questioned. the aim of this report is to describe our own clinical experience and results with md-tese and discuss how this technique is best administered and performed in relation to hormonal treatment of and egg recovery from the female partner. contact g€oran westlander goran.westlander@livio.se livio fertility center, g€oteborg 412 55, sweden. supplemental data for this article can be accessed here. � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 2, 99–103 https://doi.org/10.1080/03009734.2020.1737600 https://doi.org/10.1080/03009734.2020.1737600 https://doi.org/10.1080/03009734.2020.1737600 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1737600&domain=pdf&date_stamp=2020-05-21 https://doi.org/10.1080/03009734.2020.1737600 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1737600 http://www.tandfonline.com microdissection tese, surgical procedure procedures are preferably performed under local anaesthesia. a midline incision is made into the scrotum, and the tunica vaginalis is opened. the testis covered with the tunica albuginea is visualized, and the testis is opened widely in an equatorial plane along the mid-portion. this allows for wide exposure of seminiferous tubules in a physiological approach that follows intratesticular blood flow (11). the remainder of the procedure is performed under an operative microscope at �20–25 magnification. small samples are excised from the tubules. larger and more opaque tubules are more likely to contain sperm (supplementary figure 2; available online). up to 15 biopsies on each side are taken, but this can vary due to testicular size and tubular status. the procedure is terminated when all areas of the visualized parenchyma have been examined under the microscope or when further dissection is thought likely to jeopardize the testicular blood supply. biological search for spermatozoa fragments of testicular tissue are placed in ivf medium in sterile petri dishes and minced using micro-scissors or scalpels. fractions are put in droplets of fertilization medium under oil, and thereafter well-trained embryologists begin their search for spermatozoa. preoperative preparation before surgery, at least two semen analyses have been performed to confirm azoospermia. moreover, serum-fsh, lh, testosterone and shbg concentrations have been analyzed to distinguish between obstructive and non-obstructive azoospermia and to exclude hypogonadism. a physical investigation has been performed measuring testicular volume with an orchidometer, and testicular ultrasonography to exclude scrotal abnormalities such as varicocele, epididymal/ testicular cysts, hydrocele, and testicular tumours. finally, a karyotype has been analyzed in most noa men, and screening for y-chromosomal microdeletions carried out as well. hormonal treatment men with noa suffer from testicular insufficiency. serum fsh concentrations are most often elevated, and serum testosterone is borderline to low. it is controversial whether low testosterone concentrations predict the success of md-tese. medical therapies that increase serum testosterone might also increase the intratesticular testosterone levels, which might be beneficial for spermatogenesis (12). drugs often used are aromatase inhibitors and human chorionic gonadotrophin (hcg) (11). clinical predictors of positive sperm retrieval at present, there is only one good clinical predictor of negative sperm retrieval in men with noa. if screening for y chromosome microdeletions reveals complete deletion in the azf-a or azf-b areas, the likelihood of finding sperm with any surgical sperm recovery technique is negligible (13). a sufficient number of sperm aimed for icsi can be surgically recovered independently of small testicular volumes, high fsh, or low testosterone concentrations (14,15). sperm retrieval rate according to testicular histology the most common histological conditions seen in men with noa are hypospermatogenesis, sertoli cell-only syndrome (scos), maturation arrest, and atrophy (7). hypospermatogenesis is characterized by a reduced number of germ cells, and all stages of spermatogenesis are present. these men have the best probability for a successful sperm recovery by surgical retrieval. md-tese, ctese, as well as sperm aspiration techniques (tesa, fna) all have acceptable positive srr (4,5,7). scos is associated with the absence of germ cells, with normal sertoli cells exclusively lining the seminiferous pre-tes�cular azoospermia tes�cular azoospermia (non-obstruc�ve) noa post-tes�cular azoospermia obstruc�ve azoospermia dysfunc�onal aspermia/azoospermiacongenital congenital acquired acquired chromosomal aberra�ons klinefelters syndrome (47,xxy) transloca�ons /inversions cryptorchidism unknown origin malignant disease -surgery -post irradia�on chemotherapy local trauma orchi�s abuse of anabolic steroides unknown origin hypogonadotropic hypogonadism cys�c fibrosis / cbavd epididymi�s local trauma pelvic surgery vasectomy unknown origin *erec�le dysfunc�on *retrograde ejacula�on *spinal lesion *peripheral neuropathy e�ologies of azoospermia /aspermia figure 1. aetiologies of azoospermia/aspermia. 100 g. westlander https://doi.org/10.1080/03009734.2020.1737600 tubules. however, focal areas with intact tubules can be found especially with the ‘non-blind’ md-tese technique. men with scos have lower srr compared with other histological conditions with srr ranging from 22% to 44.5% (7,15). however, in the current literature, md-tese seems more beneficial with successful sperm retrieval in histological patterns of patchy spermatogenesis such as scos. in patients with uniform histological patterns such as maturation arrest, the outcome of md-tese seems less favourable compared with other retrieval techniques (7). clinical routines and results with the exception of complete y-chromosomal microdeletions in azf-a and azf-b regions, no secure clinical predictors of sperm retrieval have been demonstrated (16). therefore, most patients with noa in our fertility centre are offered operative surgical sperm retrieval. first-line treatment is usually a diagnostic tesa where multiple testicular samples of testicular tissue are collected with a 19-gauge butterfly needle. in case of insufficient amounts of recovered tissue, procedures can be complemented with percutaneous needle biopsies where biopsy cylinders are harvested in different regions of the anterior part of the testis. procedures are performed under local anaesthesia, and srr in our centre are up to 34% in patients with noa (5,10). spermatozoa are cryopreserved if possible. if sperm are retrieved, a subsequent ivf cycle is offered with a repeated tesa procedure, alternatively with thawed testicular sperm. if no sperm are found, patients are offered md-tese in parallel with hormonal stimulation and egg recovery from the female partner. the results after our first 159 md-tese procedures are shown in table 1. twelve repeated cycles were excluded. in one out of 12 repeated procedures, sperm were not retrieved. one patient had two repeated procedures. altogether the 12 repeated cycles resulted in another five deliveries. most cases had undergone a previous unsuccessful sperm retrieval such as tesa or ctese. sperm sufficient for icsi were identified in 51% of cases after md-tese. seventy-six percent of cases where icsi was performed resulted in fertilization and development of at least one good-quality embryo and embryo transfer. a maximum of two embryos were transferred at the cleavage stage and only one embryo at the blastocyst stage. sixty-two first cycle embryo transfers resulted in 22 (35%) ongoing pregnancies/ deliveries. in addition, from the 81 cases with positive sperm retrievals, 14 more ongoing pregnancies/deliveries have been reported from subsequent ivf cycles using thawed testicular sperm or thawed vitrified blastocyst embryos. we do not consider md-tese to be the first line of treatment, with the exception of very small atrophic testes such as in klinefelter syndrome. up to one-third of the patients with noa in our fertility centre have sperm sufficient for icsi with needle aspiration techniques. starting work-up procedures in noa cases with a diagnostic tesa will reduce the number of requested procedures of md-tese, which is more invasive, time-consuming, and expensive compared with the needle aspiration techniques. tesa can easily be repeated in a subsequent ivf cycle if sperm is retrieved and if cryopreservation of testicular sperm is not possible. however, a subsequent md-tese procedure in case of negative sperm retrieval after tesa is well motivated, as more than half of noa patients who fail with diagnostic tesa/ctese are able to have sperm retrieval with a dedicated md-tese (10,15). discussion at present, there are no doubts that md-tese has theoretical benefits compared with the ‘blind’ recovery techniques such as tesa, percutaneous biopsies, and ctese. a tendency towards a higher srr using md-tese has been reported, but good clinical randomized studies are still lacking (7). in our fertility centre more than 170 md-tese procedures have been performed, most of them with a previous ineffective tesa procedure. sperm sufficient for icsi were found in more than 50% of the cases. in 76% of these cases, fertilization and embryo development were normal, resulting in embryo transfer. ongoing pregnancy/delivery rate per embryo transfer was 35% when only the first treatment mdtese/ivf cycles were included. the overall encouraging results, highly comparable with our other conventional ivf results, might be explained by the relatively low age of the female partners. in most ivf cycles, the female age was <40 years in combination with at least a normal egg reserve. two embryos were transferred in a majority of cases, but only three twin pregnancies have been reported. this is most likely explained by the lower implantation rate when using testicular sperm for icsi in men with noa (17). our reported results are similar to those of a systematic review (8), where srr with md-tese technique in eight different studies ranged between 43% and 63%. a recently published meta-analysis reported srr with md-tese in 15 studies between 2000 and 2019. the pooled srr of the series was 45% (95% ci 39–54%). compared with needle aspiration techniques (tesa), md-tese seemed to be superior (18). klinefelter syndrome (ks) is the most frequent group of chromosomal abnormalities in men with noa, with a table 1. all non-obstructive azoospermia patients. number per cent number of patients 159 number of md-tese performed 171 men with retrieved sperm first procedure 81 51 (81/159) cycles leading to embryo transfer 62 77 (62/81) pregnancies per embryo transfer 30 48 (30/62) ongoing pregnancies/deliveries per embryo transfer 22 35 (22/62) cumulative number of ongoing pregnancies/deliveries after md-tese procedures 36 21 (36/171) upsala journal of medical sciences 101 prevalence ranging from 1:500 to 1:700 in newborn males. because of the genetic alteration – men with ks represent a group with one extra x chromosome added to the male karyotype, 46,xy – there is progressive testicular damage leading to impaired or absent spermatogenesis. a meta-analysis from 2017 with 37 studies included (19) reported an overall srr of about 40%. among our 159 patients with md-tese procedures, 19 were diagnosed with non-mosaic ks. our srr (37%) was similar to previous reports. interestingly, in cases where sperm were retrieved, 86% resulted in embryo transfer, and ongoing pregnancy/delivery rates per embryo transfer were high (67%). in subjects with ks, we can report a final live birth rate (lbr) of 32% (6/19) for the couples who initiated ivf in combination with md-tese (table 2). this is higher compared with the meta-analysis from corona et al. (19) reporting an overall lbr of 16% per couple who initiated the assisted reproductive techniques. however, only 19 subjects were enrolled in our report. all children born were healthy with a normal karyotype, which is in line with other reports showing a risk of chromosomal abnormalities similar to that reported in subjects without ks. at present, many published studies have shown high srr in men with ks, and conception appears safe (19). unfortunately, many fertility doctors, not aware of the treatment possibilities for ks men, recommend treatment with donor sperm instead of referring couples to centres performing md-tese and ivf. policies differ between clinics in the treatment of couples where md-tese is involved. the first and probably most common policy is to perform a diagnostic md-tese combined with cryopreservation of testicular tissue in the case of sperm retrieval (20). no parallel hormonal treatment and oocyte retrieval of the female partner end in less emotional and financial implications when sperm recovery is unsuccessful. however, in severe cases of noa, only occasional spermatozoa are found after dissection of tubules. in subsequent ivf cycles, a high risk of not finding sufficient sperm after thawing has been reported (17). another option is to cryopreserve oocytes prior to mdtese. if sperm are retrieved after md-tese, oocytes can be thawed and microinjected with testicular sperm. in case no sperm are retrieved, oocytes might remain cryopreserved and subsequent treatment with donor sperm is possible if accepted by the couple. however, oocyte freezing prior to md-tese has financial implications, and the optimal number of vitrified oocytes recommended prior to md-tese has been a matter of debate (21). the third alternative is to run a parallel ivf cycle along with the md-tese procedure (10). sperm retrieval is performed in the morning and oocyte recovery later on the same day. fertilizing retrieved fresh oocytes with prepared donor sperm on the same day as md-tese is also an alternative in cases of no sperm retrieval. however, an ethical consideration arises, in not letting the couple know if sperm from the male partner or a donor will be used for fertilization on the day of md-tese and oocyte retrieval. conclusion in conclusion, there is evidence to suggest that md-tese may improve sperm retrieval in men with noa, but goodquality randomized studies are still lacking. complications from md-tese are rare (<10%) and usually minor (18). the efficacy of md-tese with high srr is due to the new ‘nonblind’ technique making it possible to find tubules with spermatogenesis under an operative microscope. on the other hand, md-tese procedures are more time-consuming and require the purchase of an operating microscope. to date, md-tese seems to be the best method for the intraoperative identification for sperm-producing tubules in noa men, and hopefully, the procedure can be offered to more patients with noa, especially in the nordic countries where only a few centres have the clinical experience of the technique. however, we do not necessarily recommend md-tese to be the only method of sperm retrieval in men with noa. up to one-third of patients with noa will, at a lower cost, achieve sperm recovery by less complicated and invasive needle aspiration and percutaneous biopsy techniques. disclosure statement there are no conflicts of interest. notes on contributor g€oran westlander, md, phd, medical director, livio fertility centre, g€oteborg, sweden. references 1. palermo g, joris h, devroey p, van steirteghem ac. pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. lancet. 1992;340:17–8. doi:10.1016/0140-6736(92)92425-f 2. devroey p, liu j, nagy z, goossens a, tournaye h, camus m, et al. pregnancies after testicular sperm extraction and intracytoplasmic sperm injection in non-obstructive azoospermia. hum reprod. 1995;10:1457–60. doi:10.1093/humrep/10.6.1457 3. craft i, shrivastav p, quinton r, duke v, kirk j, bouloux p, et al. treatment of male infertility. lancet. 1994;344:191–2. doi:10.1016/ s0140-6736(94)92792-8 4. lewin a, reubinoff b, porat-katz a, weiss d, eisenberg v, arbel r, et al. testicular fine needle aspiration: the alternative method for sperm retrieval in non-obstructive azoospermia. hum reprod. 1999;14:1785–90. doi:10.1093/humrep/14.7.1785 table 2. md-tese in patients with klinefelter syndrome. number per cent number of patients 19 number of procedures 20 men with retrieved sperm first procedure 7 37 (7/19) cycles leading to embryo transfer 6 86 (6/7) pregnancies per embryo transfer 6 100 (6/6) ongoing pregnancies/deliveries per embryo transfer 4 67 (4/6) cumulative number of ongoing pregnancies/deliveries after md-tese procedures 6 30 (6/20) 102 g. westlander https://doi.org/10.1016/0140-6736(92)92425-f https://doi.org/10.1093/humrep/10.6.1457 https://doi.org/10.1016/s0140-6736(94)92792-8 https://doi.org/10.1016/s0140-6736(94)92792-8 https://doi.org/10.1093/humrep/14.7.1785 5. westlander g, hamberger l, hanson c, lundin k, nilsson l, s€oderlund b, et al. diagnostic epididymal and testicular sperm recovery and genetic aspects in azoospermic men. hum reprod. 1999;14:118–22. doi:10.1093/humrep/14.1.118 6. tuuri t, moilanen j, kaukoranta s, makinen s, kotola s, hovatta o. testicular biopty gun needle biopsy in collecting spermatozoa for intracytoplasmic injection, cryopreservation and histology. hum reprod. 1999;14:1274–8. doi:10.1093/humrep/14.5.1274 7. deruyver y, vanderschueren d, van der aa f. outcome of microdissection tese compared with conventional tese in noa: a systematic review. andrology. 2014;2:20–4. doi:10.1111/j.2047-2927. 2013.00148.x 8. bernie am, mata da, ramasamy r, schlegel pn. comparison of microdissection testicular sperm extraction, conventional testicular sperm extraction and testicular sperm aspiration for noa: a systematic review and meta-analysis. fertil steril. 2015;104:1099–103. doi:10.1016/j.fertnstert.2015.07.1136 9. ishikawa t, nose r, yamaguchi k, chiba k, fujisawa m. learning curves of microdissection testicular sperm extraction for nonobstructive azoospermia. fertil steril. 2010;94:1008–11. doi:10. 1016/j.fertnstert.2009.03.108 10. westlander g, schmidt j, westin c. microdissection testicular sperm extraction (md-tese) – a new sperm recovery technique helping men with non-obstructive azoospermia. lakartidningen. 2019;116:fl9i. 11. dabaja aa, schlegel pn. microdissection testicular sperm extraction: an update. asian j androl. 2013;15:35–9. doi:10.1038/aja. 2012.141 12. reifsnyder je, ramasamy r, husseini j, schlegel pn. role of optimizing testosterone before microdissection testicular sperm extraction in men with non-obstructive azoospermia. j urol. 2012; 188:532–7. doi:10.1016/j.juro.2012.04.002 13. krausz c, hoefsloot l, simoni m, t€uttelmann f. eaa/emqn best practice guidelines for molecular diagnosis of y-chromosomal micro deletions. state of the art 2013. andrology. 2014;2:5–19. doi:10.1111/j.2047-2927.2013.00173.x 14. ramasamy r, lin k, gosden lv, rosenwaks z, palermo gd, schlegel pn. high serum fsh levels in men with non-obstructive azoospermia does not affect success of microdissection testicular sperm extraction. fertil steril. 2009;92:590–3. doi:10.1016/j.fertnstert.2008.07.1703 15. berookhim bm, palermo gd, zaninovic n, rosenwaks z, schlegel pn. microdissection testicular sperm extraction in men with sertoli cell-only testicular histology. fertil steril. 2014;102:1282–6. doi:10. 1016/j.fertnstert.2014.08.007 16. tsujimura a. microdissection testicular sperm extraction: prediction, outcome, and complications. int j urol. 2007;14:883–9. doi: 10.1111/j.1442-2042.2007.01828.x 17. vernaeve v, tournaye h, osmanagaoglu k, verheyen g, van steirteghem a, devroey p. intracytoplasmic sperm injection with testicular spermatozoa is less successful in men with non-obstructive azoospermia than in men with obstructive azoospermia. fertil steril. 2003;79:529–33. doi:10.1016/s0015-0282(02)04809-4 18. khatibi a, eriksson m, gejervalll a-l, petzold m, strandell a, svensson m, et al. effectiveness and safety of microdissection testicular sperm extraction in infertile men with non-obstructive azoospermia. g€oteborg: region v€astra g€otaland, sahlgrenska university hospital, hta-centrum: 2019. regional activity based hta 2019. p. 108. 19. corona g, pizzocaro a, lanfranco f, garolla a, pelliccione f, vignozzi l, et al.; on behalf of the klinefelter italian group (king). sperm recovery and icsi outcomes in klinefelter syndrome: a systematic review and meta-analysis. hum reprod update. 2017;23: 265–75. doi:10.1093/humupd/dmx008 20. klami r, mankonen h, perheentupa a. microdissection testicular sperm extraction in finland – results of the first 100 patients. acta obstet gynecol scand. 2017;1:53–8. doi:10.1111/aogs.13243 21. pedersen j, jessen e, milton am, blach k, fedder j. the potential of sperm retrieved by micro-tese in fertilizing vitrified and warmed oocytes. abstract. nordic fertility society (nfs) 2019 congress, g€oteborg, sweden. upsala journal of medical sciences 103 https://doi.org/10.1093/humrep/14.1.118 https://doi.org/10.1093/humrep/14.5.1274 https://doi.org/10.1111/j.2047-2927.2013.00148.x https://doi.org/10.1111/j.2047-2927.2013.00148.x https://doi.org/10.1016/j.fertnstert.2015.07.1136 https://doi.org/10.1016/j.fertnstert.2009.03.108 https://doi.org/10.1016/j.fertnstert.2009.03.108 https://doi.org/10.1038/aja.2012.141 https://doi.org/10.1038/aja.2012.141 https://doi.org/10.1016/j.juro.2012.04.002 https://doi.org/10.1111/j.2047-2927.2013.00173.x https://doi.org/10.1016/j.fertnstert.2008.07.1703 https://doi.org/10.1016/j.fertnstert.2008.07.1703 https://doi.org/10.1016/j.fertnstert.2014.08.007 https://doi.org/10.1016/j.fertnstert.2014.08.007 https://doi.org/10.1111/j.1442-2042.2007.01828.x https://doi.org/10.1016/s0015-0282(02)04809-4 https://doi.org/10.1093/humupd/dmx008 https://doi.org/10.1111/aogs.13243 abstract introduction microdissection tese, surgical procedure biological search for spermatozoa preoperative preparation hormonal treatment clinical predictors of positive sperm retrieval sperm retrieval rate according to testicular histology clinical routines and results discussion conclusion disclosure statement references analgesic effects of dexmedetomidine and remifentanil on periprocedural pain during percutaneous ablation of renal carcinoma article analgesic effects of dexmedetomidine and remifentanil on periprocedural pain during percutaneous ablation of renal carcinoma egidijus semenasa, maria l€onnemarkb, p€ar dahlmanb, michael hultstr€oma,c and mats erikssona asection of anesthesiology and intensive care, department of surgical sciences, uppsala university, uppsala, sweden; bsection of radiology, department of surgical sciences, uppsala university, uppsala, sweden; csection for integrative physiology, department of medical cell biology, uppsala university, uppsala, sweden abstract background: percutaneous ablation of renal carcinoma is frequently a favourable treatment alternative, especially in elderly patients suffering from co-morbidities. also, it is less resource-demanding than conventional surgery of renal carcinoma, and one may, therefore, assume that the incidence of this procedure may increase. analgesia is necessary during this intervention. the aim of this study was to explore the possibility of analgosedation and its relation to patient comfort and safety during percutaneous ablation of renal carcinoma. methods: forty-six patients, sedated with dexmedetomidine and remifentanil, supplemented with infiltration anaesthesia (lidocaine 1%), underwent percutaneous (radiofrequency or microwave) ablation of renal carcinoma in this prospective study. results: the patients expected pain intensity around the numerical rating score (nrs) 4.5 (interquartile range [iqr] 3.5–5.5), which was slightly lower than pain experienced during the procedure nrs 5 (iqr 2–7; p ¼ 0.49). eight percent of the patients needed supplementary morphine during the ablation procedure. sedation score did not differ during ablation, at arrival to or discharge from the recovery ward. median periprocedural treatment time was 12 minutes (iqr 12–16). treatment time did not correlate with experienced pain (r2¼0.000074, p ¼ 0.96). the median length of stay in the recovery room was 120minutes (iqr 84–154). there were seven serious adverse events. conclusions: this proof-of-concept study has shown that analgosedation during percutaneous ablation of renal carcinoma can be performed with a generally tolerable degree of patient satisfaction. however, pain occurs and should be managed adequately. patient safety must be a major concern for the anaesthetic care. article history received 30 october 2019 revised 15 january 2020 accepted 19 january 2020 keywords analgesia; dexmedetomidine; microwave ablation; radiofrequency ablation; remifentanil; renal carcinoma introduction the number of discovered cases of renal cancer is increasing, the most rapid increase being in small tumours (1). this seems to be due to more widespread imaging, where crosssectional imaging techniques are used. as a consequence of earlier diagnosis, in combination with new treatment options, mortality in renal cancer seems to be decreasing (2). optimal treatment may be dependent on both the type and the stage of renal cancer (3). advantages of radiofrequency ablation include the rare occurrence of deterioration of renal function with an overall 5-year survival rate of 58–85%, according to meta-analyses (4). radiofrequency ablation is indicated in t1 tumours (i.e. the cancer cells are only growing in the most superficial layer of tissue, without growing into deeper tissues) in patients with advanced age and significant co-morbidities, including reduced renal function as well as single kidney. major complication rates (e.g. relevant deterioration of renal function) are reported in 0–14% of interventions (4). in a retrospective study, 19 patients with renal cell carcinoma were treated with radiofrequency ablation, while 21 patients were treated with partial nephrectomy. length of hospital stay, mean procedural time, and blood loss were lower in patients treated with radiofrequency ablation. elevated serum creatinine concentrations were noted in patients undergoing partial nephrectomy, but not in those undergoing radiofrequency ablation. regarding the occurrence of metastasis, there were no differences (5,6). since radiofrequency ablations are performed in the radiological department, the anaesthetic procedure is more timeconsuming and space-occupying than in a regular operating room, designed for preand postanesthetic management. also, the distance between the radiological department and the surgical unit may cause problems when assistance is needed in an anaesthetic emergency. furthermore, there is an advantage to the radiologist if the patient is cooperative and able to follow instructions. thus, we decided to evaluate whether sedation in combination with local anaesthetic contact mats eriksson mats.eriksson@surgsci.uu.se section of anesthesiology and intensive care, department of surgical sciences, uppsala university hospital, 751 85 uppsala, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 1, 52–57 https://doi.org/10.1080/03009734.2020.1720047 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1720047&domain=pdf&date_stamp=2020-02-19 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1720047 http://www.tandfonline.com could be a favourable approach. initially, we used midazolam in combination with remifentanil for this purpose. however, we had some complications in the form of mental confusion, too heavy sedation, agitation, etc. therefore, we decided to change this concept and instead use dexmedetomidine and remifentanil during percutaneous ablation of renal cell carcinoma. this drug combination has proven efficacy in several surgical procedures, including awake craniotomy (7). initially, this regimen was, because of safety concerns, always commenced in the presence of both an experienced anaesthesiologist and a well-educated anaesthetic nurse. as we found this routine satisfactory, we decided to evaluate this procedure in a proof-of-concept study. the primary aim of our study was to evaluate overall patient comfort, focussing on the possibility to substitute anaesthesia with analgosedation during percutaneous ablation of renal carcinoma. the secondary aims were to: � evaluate anaesthetic consumption as well as analgesic requirements during the first 24 hours after the procedure. � evaluate patient safety during the perand postprocedural phases of percutaneous ablation of renal cell carcinoma in analgosedated patients. � describe patient characteristics and periprocedural circumstances for reference and guidance in similar interventions, either therapeutic or diagnostic. materials and methods fifty-one patients planned for percutaneous ablation of renal carcinoma were screened for participation in this study. all patients gave their oral and written consent to participate in this study, which was approved (2013/409) by the regional ethical review board at uppsala university, sweden. according to eudralex, we do not see this study as a clinical trial, which would need approval by the swedish medical products agency. this study was registered at clinicaltrials.gov on december 13, 2013. in two patients, general anaesthesia was considered a better option, and one patient refused to participate in this study. two patients were excluded because of other treatment options. demographic data of the 46 (22 female) remaining patients were as follows: age (mean ± sd) was 66 ± 12 years; mean weight 79 ± 16 kg; mean height 170 ± 9 cm. the majority of patients (37/46, 80%) had limited co-morbidities and were considered to be classified as asa 2, according to the american society of anaesthesiologists (asa) physical status classification system; some were classified as asa 1 (n ¼ 7), and only 2 were asa 3. inclusion criteria were: � patients of both sexes, aged 18–80 years, able and willing to participate in this study. � renal cancer for which radiofrequency or microwave ablation was planned. � signed informed consent form. exclusion criteria were: � patient refusal. � pregnancy. � known allergy to dexmedetomidine and remifentanil. � atrioventricular block grade ii or iii or other significant cardiac conduction dysfunction. � history of stroke. � low blood pressure not responding to treatment. protocol before the onset of the procedure, patients were asked to assess expected maximal pain intensity during the radiofrequency/microwave ablation, according to a 11-point numerical rating scale (nrs), where ‘0’ is no pain and ‘10’ is the worst imaginable pain (8). the patients were informed that pain should be expected during the procedure and that analgesic drugs would be given on demand. in order to avoid bias due to expected different procedural pain levels (radiofrequency versus microwave ablation), all patients received identical information. ethical considerations were based on our clinical experience from radiofrequency/microwave ablation and included an algorithm aiming to avoid unnecessary pain. during the postprocedural period in the recovery ward, patients were asked, approximately 15 min after arrival, to arbitrarily assess maximal pain intensity during the ablation and also to assess overall satisfaction, as a ‘satisfaction score’, with the sedation technique (1, dissatisfied; to 5, satisfied). since the patients were transferred from the roentgenological department to the recovery ward, a time period of 30–45 min between discontinuation of dexmedetomidine and remifentanil and first estimation of pain was realistic. when leaving the recovery ward, the patients were, once again, asked to assess the overall satisfaction with the sedation technique and also to assess the maximal pain intensity during the recovery phase. sedation during the percutaneous ablation procedure, at arrival to the recovery ward, and at discharge, was evaluated according to the ramsey sedation scale score (9), which describes a patient as follows: 1. anxious and agitated, or restless, or both 2. co-operative, oriented, and calm 3. responsive to commands only 4. exhibiting brisk response to light glabellar tap or loud auditory stimulus 5. exhibiting a sluggish response to light glabellar tap or loud auditory stimulus 6. unresponsive nineteen patients asking for premedication received paracetamol 1 g orally, approximately 1 h before start of the ablation procedure. no other analgesic was given before the radiofrequency ablation. conventional anaesthetic variables (five-lead electrocardiogram, non-invasive blood pressure, heart rate, transcutaneous oxygen saturation, end-tidal carbon dioxide) were monitored. after administration of 0.5 mg of atropine, all patients received a continuous infusion of upsala journal of medical sciences 53 dexmedetomidine, starting at 0.4 lg kg�1 h�1 and remifentanil administered by a target-controlled infusion, where the initial plasma level was set to be 0.5 ng ml�1. when necessary, infusion rate of remifentanil was changed at the investigators’ discretion, in order to achieve a comfortable sedation for the patient. dexmedetomidine infusion was kept stable throughout the procedure. local anaesthetic (8–10 ml of lidocaine 1%) was infiltrated in the kidney at the radiologist’s discretion during the treatment. an anaesthetic nurse and/or anaesthetist was present during the entire procedure. serious adverse events (saes) were defined as complications that were either fatal, life-threatening, requiring hospitalization, resulting in persistent significant disability, or requiring intervention to prevent permanent damage. percutaneous ablation procedure the procedure was performed under ct-guidance (somatom definition flash, siemens, forchheim, germany). in 42 sessions one tumour was ablated, and in 4 sessions two tumours were ablated. when an optimal needle path was identified, the skin and needle track were infiltrated with 8–10 ml local anaesthesia. prior to ablation, core biopsies with a 1.2 mm cutting needle were taken (promag ultra, argon, angiotech, gainesville, fl, usa; or quick core biopsy needle, cook medical, bloomington, in, usa). tumours were ablated using either a radiofrequency (rf) ablation system (cool-tip rf ablation system e series, medtronic, boulder, co, usa; n ¼ 37) or two different microwave (mw) ablation systems (emprint ablation system medtronic, boulder, co, usa, n ¼ 5; or microthermex, bsd medical, bountiful, ut, usa, n ¼ 4). the choice of electrodes or probes was based on the manufacturers’ recommendations. hydrodissection was used in 27 patients. statistics after validation and, when needed, confirmation of data was obtained from patients and co-workers in this study, the data were entered into an excel spread sheet, serving as a database, from which calculations were performed. values are, as considered appropriate, expressed as mean ± standard deviation (sd) and/or median, first (q1) and third (q3) quartiles, respectively. paired wilcoxon signed rank test was used for evaluating possible differences in expected versus periprocedural pain perception. a 40-patient sample size was calculated to achieve 85% power for the paired wilcoxon signed rank test of expected versus experienced pain to detect a 2 nrs mean difference at the 5% significance level. p < 0.05 was considered significant. results tumoral growth in 16 patients the renal carcinoma was exophytic, in 13 patients the tumour was endophytic, and in 17 cases it was both exophytic and endophytic. three patients had bilateral renal tumours, and in two cases a suprarenal gland was involved. in one patient the procedure was interrupted because of severe pain. expected and experienced pain the patients expected pain around nrs 4–5 (mean: 4.6 ± 1.9; median 4.5, iqr 3.5–5.5), which was slightly lower than how they assessed their pain during the procedure (mean: 5.1 ± 2.8; median 5, iqr 2–7), although the difference was not statistically significant by mann–whitney (p ¼ 0.49). four patients (8%) needed supplementary morphine during the roentgenological interventional procedure. anaesthetic consumption and effect the average total dexmedetomidine given was 55 ± 20 mg, and remifentanil 371 ± 288 mg with an average maximal target concentration on target-controlled infusion for remifentanil of 2.92 ± 1.15 mg. this corresponded to 0.7 ± 0.2 mg kg�1 dexmedetomidine, and 4.8 ± 3.4 mg kg�1 remifentanil. or, taking treatment time into account, dexmedetomidine 0.06 ± 0.038 mg kg�1 h�1 and remifentanil 0.37 ± 0.28 mg kg�1 h�1. treatment time treatment time varied between 4 and 36 min. average treatment time was 14.13 ± 6.7 min, median 12 (iqr 12–16) min. treatment time did not correlate with experienced pain (r2 ¼ 0.00007398, p ¼ 0.9553) (figure 1). treatment options and pain treatment options were either rf or mw. the majority, 37 patients, received rf, while 9 patients received mw. there was no difference between the two treatment groups in experienced pain level, average rf ¼ 4.94 (sd 3.04) versus mw ¼ 5.36 (sd 2.58) (figure 2). recovery maximal pain during the recovery phase was lower than both expected and experienced pain during the periprocedural phase (mean: 2.9 ± 2.5; median: 2.5, iqr 0–5). sedation score (8) during the percutaneous ablation, at arrival to the recovery ward and at discharge from the recovery ward, did not differ (all occasions: median: 2, iqr 2–2). four patients (8%) experienced postoperative nausea and vomiting, which needed treatment with metoclopramide, ondansetron, betamethasone, or a mixture thereof, during the post-interventional phase. thirty-five patients (73%) experienced pain (nrs > 0) during the recovery phase. morphine, usually at low dose (mean: 3.5 mg; median: 1 mg; iqr: 0–7) was administered to 25 of these patients. ketobemidone at 2 mg was given to 2 of these 35 patients. paracetamol was given to 18 patients, who 54 e. semenas et al. had not received paracetamol as premedication. eleven patients were given clonidine intravenously. fifteen patients needed mixtures of these analgesic drugs during the recovery phase. three patients needed supplementary morphine in the ward, and 15 of the 46 patients were given further paracetamol in the ward. one patient received ibuprofen for pain relief in the ward. mean length of stay in the recovery ward was 143 ± 168 min (median: 120 min; iqr: 84–154). one patient (see below) stayed overnight. mean satisfaction score was 3.7 ± 0.7 (median satisfaction score was 4 at arrival to the recovery ward; iqr: 3–4). patient satisfaction at discharge from the recovery ward was in the same range. mean satisfaction score was 4.4 ± 0.6 (median satisfaction score was 5; iqr: 4–5). adverse events there were six procedural complications (saes) in the form of pneumothoraxes (although no drainage was needed, this was considered potentially life-threatening) and five reported post-procedural adverse events, one of which was a perioperative cardiac arrest, which was successfully resuscitated. this sae was, at least partly, explained by the fact that a high dose of remifentanil was administered shortly before the termination of the intervention. after this incident, our handling routines of extensive pain were somewhat figure 1. treatment time did not correlate with experienced pain (r2¼0.00007398, p ¼ 0.9553). figure 2. pain median radio frequency ablation (5, iqr 2–7.4) versus microwave ablation (5, iqr 4.25–6.75). upsala journal of medical sciences 55 modified, and use of local anaesthetic was further encouraged. the other aes that required treatments were severe nausea (n ¼ 2), pain in shoulder (n ¼ 1), and pruritus (n ¼ 1). discussion the possibility to perform therapeutic and/or diagnostic interventions is not limited to interventional radiology; neurosurgery, endoscopy, and vascular surgery are other fields where analgosedation may be favourable both to severely disabled patients as well as from a health-economical perspective. there is no reason to assume that interventional activities will be limited to the present fields. therefore, it is our intention that the present study will contribute to the narrative of future investigations, aiming to explore new options in the field of sedative comfort without interfering with long-term results. anaesthetic experience (general anaesthesia versus conscious sedation) during percutaneous ablation of hepatocellular carcinoma has been retrospectively investigated (10). the choice of the anaesthetic technique depended not only on the tumour, but also on patient factors, since this was a cohort with frequent comorbidities. although some patients scored high pain levels during the renal intervention, the overall patient comfort in this prospective investigation seems satisfactory, since there were no significant differences in expected versus experienced periprocedural pain. also, the need for supplementary morphine during the procedure was 8%, suggesting that the vast majority of patients found the administered amounts of dexmedetomidine and remifentanil sufficient. postoperative pain was experienced in nearly three out of four patients, which is not surprising due to the fact that short-acting drugs were used. however, the total amount of opioids given at the recovery unit was not high. dexmedetomidine is a potent alfa-2 agonist, having anxiolytic, analgesic, and sedative properties. it is an efficient adjunct to regional anaesthesia and reduces the need for opioids. furthermore, dexmedetomidine may decrease the inflammatory response to renal ischaemia, which may be an advantage in this context (11). the other drug used was remifentanil, with favourable pharmacodynamic properties in this setting, since it is metabolized by esterases, making it minimally altered by age or renal or hepatic dysfunction. remifentanil has analgesic properties with minimal effect on cognitive function (12). this combination may be advantageous for the purpose of analgosedation, where unconsciousness is not desirable and muscle relaxation is not necessary. our use of dexmedetomidine and remifentanil in the roentgenological department is an example of the known demand for sedation during regional anaesthetic procedures outside the operating room (13). in the future we can expect that different methods of applying sedation will be used, since there is sometimes a shortage of qualified anaesthetists, bringing up a discussion whether non-anaesthesiologists can be responsible for qualified sedation. against the background of this study, such a concept seems questionable, since remifentanil and dexmedetomidine are both examples of potent drugs. these challenges are mastered through the use of well-educated staff familiar with drugs and equipment (e.g. target-controlled infusion technique), to facilitate safety and efficacy during such procedures (14). however, quick access to a physician capable of managing life-threatening events is a must. pneumothoraxes may occur during percutaneous ablation of renal carcinoma. spontaneously breathing patients may be less prone to develop intrapleural inflation of gas, if the visceral pleura is perforated, compared to general anaesthesia, where intermittent positive pressure ventilation is applied, and, especially, when nitrous oxide is used. according to our experience, the incidence of pneumothoraxes in this study is in the same range as in fully anaesthetized patients. this study has several limitations. the most obvious one is the fact that this was not a randomised controlled trial. in a retrospective study performed between 2010 and 2015, including 51 patients, kim et al. (15) found that general anaesthesia seems superior to conscious sedation for local tumour control. although their findings may favour general anaesthesia, it should be remembered that some patients are not suitable for general anaesthesia. also, the mean age of their patients was 57 years, compared to ours, which was 66 years. furthermore, their mean treatment time of sedated patients was significantly shorter compared to anaesthetized patients, but in the same range as in our study. another drawback of our study, as well as the study by kim et al. (15), is the limited number of patients, as well as the fact that only a small number of roentgenological or anaesthetic staff was involved. preoperative paracetamol was administered to 40% of the patients, which might have reduced pain perception in some cases. ketobemidone was administered to two patients suffering from nausea and emesis in an attempt to reduce this discomfort. also, ours was a single-center study with a heterogeneous population, and our results should, therefore, be interpreted with care from a global aspect. conclusions sedation with dexmedetomidine and remifentanil during percutaneous ablation of renal carcinoma appears to be efficacious, although several patients reported high pain scores. somewhat surprisingly, the patients assessed the procedure as overall acceptable. postoperative pain is a frequent problem, especially during the first two hours after the radiological intervention, and prophylactic analgesic regimen should be applied. however, care must be taken to avoid overdosage of opioids in this population. analgosedation during ablation of renal carcinoma should be performed only by dedicated and well-trained anaesthetic staff. a large, preferably multi-center, prospective, randomized controlled trial, comparing analgosedation versus general anaesthesia during percutaneous ablation of renal carcinoma focussing on long-term therapeutic effects, safety, patient comfort, optimal dosage, and health economy would be 56 e. semenas et al. advantageous. a non-opioid premedication is also recommended in order to limit the need for extra analgesics preand postoperatively. acknowledgements the authors are indebted to mrs. m. hoerling for linguistic revision of this manuscript. disclosure statement the authors have no conflicts of interest. funding this study was sponsored by uppsala university, sweden. no other financial sources have contributed to this study. notes on contributors egidijus semenas, md, phd, consultant in anaesthesia and intensive care, department of surgical sciences/anaesthesiology and intensive care, faculty of medicine, uppsala university, uppsala, sweden. maria l€onnemark, md, phd, assoc. prof. consultant in urogenital and interventional radiology, department of surgical sciences, uppsala university, sweden. p€ar dahlman, md, phd, consultant in urogenital and interventional radiology, departement of surgical sciences, uppsala university, sweden. michael hultstr€om, md, phd, faha, associate professor, specialist in anaesthesiology and intensive care medicine. unit for anaesthesiology and intensive care medicine, department of surgical sciences, uppsala university, uppsala, sweden, and unit for integrative physiology, department of medical cell biology, uppsala university, uppsala, sweden. mats eriksson, md, phd, is associate professor of anesthesia & intensive care, departement surgical sciences at uppsala university, sweden. references 1. znaor a, lortet-tieulent j, laversanne m, jemal a, bray f. 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https://doi.org/10.1016/j.carj.2017.07.003 abstract introduction materials and methods protocol percutaneous ablation procedure statistics results tumoral growth expected and experienced pain anaesthetic consumption and effect treatment time treatment options and pain recovery adverse events discussion conclusions acknowledgements disclosure statement references vascular permeability—the essentials full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 vascular permeability—the essentials lena claesson-welsh to cite this article: lena claesson-welsh (2015) vascular permeability—the essentials, upsala journal of medical sciences, 120:3, 135-143, doi: 10.3109/03009734.2015.1064501 to link to this article: https://doi.org/10.3109/03009734.2015.1064501 © informa healthcare published online: 29 jul 2015. submit your article to this journal article views: 2747 view related articles view crossmark data citing articles: 98 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2015.1064501 https://doi.org/10.3109/03009734.2015.1064501 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1064501 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1064501 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1064501&domain=pdf&date_stamp=2015-07-29 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1064501&domain=pdf&date_stamp=2015-07-29 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1064501#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1064501#tabmodule upsala journal of medical sciences. 2015; 120: 135–143 review article vascular permeability—the essentials lena claesson-welsh uppsala university, department of immunology, genetics and pathology, rudbeck laboratory, uppsala, sweden abstract the vasculature, composed of vessels of different morphology and function, distributes blood to all tissues and maintains physiological tissue homeostasis. in pathologies, the vasculature is often affected by, and engaged in, the disease process. this may result in excessive formation of new, unstable, and hyperpermeable vessels with poor blood flow, which further promotes hypoxia and disease propagation. chronic vessel permeability may also facilitate metastatic spread of cancer. thus, there is a strong incentive to learn more about an important aspect of vessel biology in health and disease: the regulation of vessel permeability. the current review aims to summarize current insights into different mechanisms of vascular permeability, its regulatory factors, and the consequences for disease. key words: edema, histamine, junctions, pore, vascular permeability, vegf introduction the main function of the vasculature is to serve as a blood conduit to ensure efficient oxygenation of tissues, followed by the return of the deoxygenated blood to the lungs. the vasculature is also pivotal for a range of other homeostatic functions relating to the circulation such as hemostasis, lipid transport, and immune surveillance (1). endothelial cells are the main constituents of blood vessels. they rest on a basement membrane with their basolateral side and face the blood with their apical/luminal side. in the healthy individual, the vasculature is stable, and endothelial cell survival is continuously maintained (2). during particular physiological responses such as embryo development, ovulation, and regrowth of the endometrium, or in conjunction with injury or disease, there is a need for new vessels to form. in fact, the growth of all new tissues, whether healthy or not, is accompanied by blood vessel formation. a main underlying mechanism is the relative hypoxia in the growing tissue (3). during embryogenesis, vessels form de novo in a process denoted vasculogenesis, while angiogenesis implies vessel formation from the pre-existing vasculature. endothelial cells in different vessels and in different organs have distinct functions and morphologies (4). in certain organs, such as the brain and in endocrine winner of the rudbeck award, 2014, at the medical faculty of uppsala university for her distinguished research on the formation and function of blood vessels under normal and pathological conditions. correspondence: lena claesson-welsh, uppsala university, department of immunology, genetics and pathology, rudbeck laboratory, dag hammarskjöldsv. 20, 751 85 uppsala, sweden. e-mail: e-mail: lena.welsh@igp.uu.se (received 9 june 2015; accepted 13 june 2015) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1064501 http://informahealthcare.com/journal/ups mailto:e-mail: lena.welsh@igp.uu.se organs, endothelial cells present certain morphological features that reflect the need for communication between the organs and the circulation. in the brain, the vasculature forms a particularly strong barrier, the blood–brain barrier (bbb) (5), to protect the brain parenchyma from detrimental edema. in hormoneproducing organs, such as the endocrine pancreas, endothelial cells display specialized fenestrae on their surface. these are diaphragm-covered ‘holes’ in the plasma membrane, which allow extremely rapid exocytosis of hormones (6). in most organs, the endothelial cells form a dynamic barrier between the blood and the tissue. in resting conditions, the vasculature continuously leaks solute and small molecules but restricts extravasation of larger molecules and cells. in many diseases, including cancer and chronic inflammatory conditions, the vascular barrier disintegrates and leakage increases and may become chronic. the leakage of larger molecules and cells results in edema, inflammation, and often disease progression. this review will discuss the current knowledge of how different types of vascular permeability are regulated, how regulation is lost in diseases, and, finally, how insights into regulatory mechanisms can be exploited therapeutically. mechanisms in permeability traditionally, the term ‘vascular permeability’ implies the basal vascular sieving of solute and small molecules, which occurs in an unstimulated setting. molecules smaller than 40 kda may extravasate spontaneously (7), whereas larger molecules require the active disruption of the vascular barrier in order to extravasate to the surrounding tissue. such induced leakage takes place preferentially in post-capillary venules (8,9), but capillaries and larger venules may also leak (10). the mechanism underlying vascular leak may be different in different organs and depend on the specialized vasculature. however, two main models have been proposed. one depends on formation of transendothelial channels from vesicles or vacuoles, the vesiculo-vacuolar organelle (vvo), and the other involves endothelial junctions that can be transiently dissolved and allow extravasation. the actin cytoskeleton may have a critical role in gap formation. moreover, the specialized junction in the brain vasculature, instrumental in the bbb, and the features of fenestrated endothelium will be described. the involvement of these different mechanisms may depend on the vessel type, the organ, the kinetics of the transport, and the nature of what is transported across the vascular wall—solute, molecules, or cells (figure 1). the vesiculo-vacuolar organelle the vvo has been described and interpreted mainly using electron microscopy analyses, which have shown that vvos are prominent structures in both tumor-supplying and normal vessel endothelial cells (11,12). based on the use of various tracers, for example electron-dense ferritin, vvos have been implicated as the primary pathway for macromolecular extravasation (9). there is general consensus on the notion that vesicular transport across the endothelium (transcytosis) is an important mechanism for delivery of macromolecules to tissues. during transcytosis, caveolae, specialized regions in the plasma membrane (pm), ‘pinch off’ from the pm to form discrete vesicular carriers that shuttle to the opposite side of the endothelium where vesicles fuse with the pm, and discharge their cargo into the perivascular space. endothelial transcytosis may occur in specialized vascular beds or under particular physiological conditions. transcytosis has been described in the brain vasculature, and it is elevated under conditions at which the bbb is broken due to pericyte deficiency (13). vvos may be one possible mechanism for transcytosis. vesicles and vacuoles that make up the vvo were originally thought to derive from caveolae. a main protein in caveolae is caveolin-1. while caveolin-1 luminal side tissue side ec solute cells molecules fenestrae transcellular gap paracellular opening figure 1. different mechanisms for extravasation of solute, cells, and molecules. specialized capillaries in endocrine organs have pores, fenestrae, in the plasma membrane. fenestrae allow rapid exchange of solute and molecules such as hormones. transcellular gaps provide a route for inflammatory cells, which, however, also may exit through paracellular junctions. disintegration of junctions allows extravasation of molecules. 136 l. claesson-welsh knock-out mice lacked caveolae and showed reduced permeability to macromolecules, the vasculature still contained vvos (14). the origin of the vvo is therefore presently not known. a challenge in further analyses of vvos is that they cannot be studied by conventional light microscopy. moreover, there is at present no genetic loss-of-function model in which to study vvos. paracellular junctions endothelial cell–cell junctions are organized into adherens and tight junctions. the main component of adherens junctions is vascular endothelial (ve)cadherin, a transmembrane protein which forms homophilic complexes between endothelial cells (15). adherens junctions dissolve in response to a number of stimuli, including vascular endothelial growth factors (vegfs) and inflammatory cytokines such as histamine and bradykinin, to allow extravasation of macromolecules (see below). these stimuli cause ve-cadherin dissolution through a triggering event that may involve hyperphosphorylation of ve-cadherin (figure 2). ve-cadherin is abundantly phosphorylated also in the basal, unstimulated state, through flow-mediated activation of c-src, which triggers ve-cadherin phosphorylation directly or indirectly (16). the triggering event causing ve-cadherin internalization remains to be identified, and mechanisms different from a direct phosphorylation of ve-cadherin by c-src have been suggested (17). in a physiological setting, the dissolution of adherens junctions is transient, and the junctions will soon close again in part due to ve-cadherin recycling and reappearance on the cell surface (18). there are very few reports showing junctions in their open state or the kinetics of opening and closure in vivo (19). in diseases characterized by excess vascular permeability (also denoted vascular leak), the regulation of junction dynamics is lost and the junctions remain open. this is denoted chronic permeability (20). the actin cytoskeleton retraction of the endothelial cell body has been implicated in mediating increased vascular permeability (8). thus, the action of intracellular motor proteins causes cells to contract in a manner that facilitates opening of paracellular junctions. however, the cell retraction hypothesis has been challenged, and the cell shape changes observed have been attributed to a natural recoil process occurring when cell–cell junctions are disassembled (21,22). the role of the actin cytoskeleton needs to be further studied. endothelial fenestrations endothelial cells in many vessels form an uninterrupted vasculature. in certain organs, however, the endothelial cells display specialized structures to facilitate rapid transport across the endothelium; see tse and stan (6) for a detailed description. one example is the fenestrated endothelium that is present in vessels in endocrine glands, digestive tract mucosa, and the kidney peritubular capillaries. here, endothelial cells are equipped with fenestrae, circular pores, covered by a diaphragm. a key protein in the diaphragm is plasmalemmal vesicle protein-1 (pv1), organized in radial fibrils. gene targeting of pv1 does not prevent formation of fenestrae as such but results edema degraded vec p p, p p, p p p, p p, p pvec vec junction closed regulators: vegf histamine bradykinin junction open ve-cadherin, microsphere leaky blood vessel ba figure 2. opening of adherens junction in molecular extravasation. panel a outlines schematically how ve-cadherin (vec) engaged in hemophilic interactions at adherens junctions is regulated by hyperphosphorylation, correlating with internalization and degradation of vecadherin. ve-cadherin may also recycle; see text. panel b shows leaky mouse tracheal vasculature after tail-vein injection of vegf and fluorescent microspheres (white), followed by whole-mount immunostaining for ve-cadherin (red). vegf-induced vascular leakiness leads to edema. vascular permeability—the essentials 137 in loss of the diaphragm and severe leakage of plasma proteins (23). there are naturally occurring fenestrae without diaphragm, i.e. in the kidney glomerulus (6). the sinusoidal endothelium in the liver and the bone marrow also shows fenestrae without a diaphragm. these fenestrae, also denoted ‘gaps’, are heterogeneous but of larger diameter than the endocrine vessel fenestrae. how these large openings still maintain the vascular barrier is unclear. the blood–brain barrier the bbb is a unique barrier with the purpose of preventing the brain from exposure to the blood and the adverse consequence of edema, which may be detrimental for the tightly enclosed brain. the brain vasculature has, in addition to adherens junctions, also high-resistance tight junctions and an abundant basement membrane. perivascular components such as astrocytes, pericytes, and neurons participate functionally in creating the bbb (5). a potentially unique feature of the bbb is the transendothelial vesicular transport of a range of nutrients and metabolic waste products (24). there is keen interest from the pharmaceutical industry to find strategies to interrupt the bbb for drug delivery. there is still limited information on to what extent the bbb can be transiently opened in response to growth factors and inflammatory cytokines (see below) (25). comparative information on molecular mechanisms in central nervous system and peripheral permeability is also lacking. vascular permeability to solute, molecules, and cells vascular permeability to solutes and small molecules occurs constitutively and appears not to require an active process. it is likely that the constant sieving of solute is important in maintaining the interstitial pressure in the tissue. it also serves to maintain the immune surveillance function of the lymphatics. interstitial fluid collected by the lymphatics is carried via lymphatic capillaries to lymph nodes where foreign antigens will be exposed to the immune system (26). plasma contains three main molecular constituents: albumin, globulins, and fibrinogen (27). extravasation of macromolecules serves diverse purposes, for example to maintain the balanced blood and interstitial pressures, to act in immune surveillance, and to carry other molecules, such as hormones and lipids, across the vessel wall. extravasated fibrinogen, processed to fibrin, may form a provisional matrix on which new blood vessels extend (28). extravasation of inflammatory and immune cells serves specific purposes in different pathologies. these cells are a prerequisite for healing of an acute disease process but may also propagate a chronic disease and interfere with recovery. overall, studies on the regulation of vascular permeability often suffer from the lack of physiological read-outs. it is clear, however, that permeability to solutes, molecules, and cells to some extent is differently regulated. transient opening of paracellular junctions is the favored model for molecular extravasation. junctional gaps appear to be required also for extravasation of inflammatory cells; however, the preferred route of exit for leukocytes and immune cells has been difficult unequivocally to sort out (29). inflammatory cells adhere to the endothelium through binding to specific adhesion molecules on the endothelial surface. the cells can then transmigrate directly through the thin endothelial wall, or pericellularly through endothelial junctions (30-32). the route of choice might depend on the stimulus, type of leukocyte, and vascular bed. interestingly, expression of a fusion protein between ve-cadherin and a-catenin in mice resulted in a complete sealing of junctions to macromolecular extravasation (33). inflammatory cell extravasation was, however, not completely restricted. indeed, the extent of immune cell extravasation appeared not to be affected (33). it is possible that different inflammatory cells extravasate through different mechanisms or that the cells are sufficiently plastic to adapt to the possibilities offered in the particular situation. finally, exit of inflammatory cells may be differently regulated in acute and chronic inflammation. regulation of vascular permeability vascular permeability can be influenced directly by molecules that cause the barrier to disintegrate, whether it is a transvessel pore or a junction that needs to be opened. the relative extent of permeability can also be indirectly regulated by the blood pressure and the resulting blood flow. an increase in blood flow, e.g. as a consequence of vasodilation (34,35), will increase vascular permeability. molecular regulators of vascular permeability include growth factors and inflammatory cytokines. angiogenic growth factors vegf was originally denoted vascular permeability factor (vpf) implying its essential role in regulation of the vascular barrier (36). vegf is produced by all nucleated cells in the body; its expression is upregulated in hypoxia (37). vegf binds to two structurally 138 l. claesson-welsh related receptors denoted vegfr1 and vegfr2 (38). both receptors, but preferentially vegfr2, have been implicated in regulation of permeability and angiogenesis. the role of vegfr1 is more unclear, and it may serve primarily as a negative regulator of vegfr2. for details, see koch et al. (38). binding of vegf to vegfr2 leads to receptor dimerization, activation of the intracellular tyrosine kinase activity, and tyrosine phosphorylation of both the receptor itself and intracellular substrates for the kinase, so-called signal transducers. a number of phosphorylation sites in vegfr2 have been identified (39). several of these phosphorylation sites have been studied in loss-of-function analyses by phenylalanine knock-in, in vivo and/or in vitro. the most interesting site at this point appears to be the y949 site in the vegfr2 kinase insert. it serves as a binding site for an adaptor molecule, t cell-specific adaptor (tsad), which binds to the cytoplasmic tyrosine kinase c-src. silencing or gene inactivation of tsad makes endothelial junctions unresponsive to vegf, resulting in loss of vegf-induced vascular permeability (40). several studies from the david cheresh lab implicate c-src in phosphorylation of the critical adherens junction protein ve-cadherin (41,42). according to the model, c-src-induced phosphorylation of ve-cadherin promotes dissolution of ve-cadherin contacts between cells, followed by internalization and degradation or recycling of vecadherin (18). the other vegfr2 phosphorylation sites induce signaling pathways that also contribute to vascular permeability regulation. these sites include y1173 (y1175 in the human vegfr2), which binds phospholipase cg (43), as well as the adaptor molecule shb (44), and y1212 (y1214 in the human vegfr2), which binds the adaptor nck (45). for details on their downstream pathways, the reader is referred to a previous study (46). whether other growth factors for which there are receptors on endothelial cells, such as placenta growth factor (binding exclusively to vegfr1) or fibroblast growth factors (fgfs, binding to fgfr1 and fgfr2), mediate acute or chronic vascular permeability has not yet been addressed in detail. inflammatory cytokines the two best-studied inflammatory cytokines in vascular permeability are histamine and bradykinin. histamine is produced by mast cells and binds to g-protein coupled h1 and h2 histamine receptors (gpcrs) on endothelial cells (47). bradykinin is cleaved from kininogen; it acts via gpcrs b1 and b2 (48). although other mechanisms have not been excluded, it is quite well established that both histamine and bradykinin mediate activation of the serine/ threonine kinase akt, which phosphorylates and thereby activates endothelial nitric oxide synthase (enos) (49-52). thereby, p-enos catalyzes the generation of no. no is a potent regulator of the vascular tone; it mediates vasodilation by stimulating soluble guanylyl cyclase and increasing cyclic gmp in smooth muscle cells (53). akt is not the only kinase that can phosphorylate and activate enos, but it is the best-studied pathway. another target effect of nitric oxide is s-nitrosylation of beta-catenin that will cause its dissociation from ve-cadherin and consequently the disassembly of adherens junctions (54). the enos-no pathway is implicated also in vegf-regulated vascular permeability since ablation of enos expression blocks the vegf response (51). figure 3 depicts a schematic summary of signal transduction pathways regulating disassembly of adherens junctions. vascular permeability and disease vascular permeability and cancer tumor vasculature displays a spectrum of morphological and functional abnormalities including loss of vessel hierarchy, increased tortuosity, poor perfusion, instability, and increased vascular leakage (55). to a considerable extent, the tumor vessel phenotype is a consequence of hypoxia-driven persistent vegfproduction (3). anti-angiogenic treatment e.g. using pve-cad ve-cad junction closed processes that open junctions junction open axl vegfr2 tsad src pi3k akt no enos vasoconstriction rac no py cell retraction vascular permeability figure 3. signal transduction regulating opening of adherens junctions. three main pathways are depicted: 1) vegf-induced activation of c-src leading to ve-cadherin (ve-cad) hyperphosphorylation (py); 2) activation of enos leading to no generation and effects on adherens junctions; and 3) activation of small gtpases such as rac followed by rearrangement of the actin cytoskeleton and cell retraction. for details, see text. vascular permeability—the essentials 139 vegf-blocking antibodies or vegfr kinase inhibitors therefore induces a more normal tumor vessel morphology and attenuates the exaggerated permeability (56). the therapeutic benefit of anti-angiogenic treatment in prolonging progression-free and overall survival depends on the cancer diagnosis, and i refer to in-depth recent reviews on this important matter (57). to what extent the potential benefit of anti-angiogenic therapy on growth of the primary tumor and suppression of metastatic spread primarily depends on suppression of vascular permeability, or whether other effects of the treatment e.g. on neo-angiogenesis in the tumor are more important, has not been clarified. the excess tumor vascular permeability has a range of deteriorating effects on the tumor microenvironment including increased interstitial pressure leading to impaired therapeutic delivery (58). moreover, the leaky vasculature may facilitate both leukocyte infiltration into the tumor and escape of tumor cells into the blood to establish distant metastases. vascular permeability and myocardial pathology tissue damage in myocardial infarction (mi) is triggered by tissue ischemia as a consequence of vessel occlusion and poor blood flow. this in turn leads to an acute increase in vascular permeability and tissue edema, impairing the ability of the heart to pump efficiently. moreover, the increased permeability is manifested as increased infiltration of inflammatory cells in the acute phase after vessel occlusion (20,59). one of the first cell types to enter the infarcted myocardium is the neutrophil (60). neutrophils contribute to tissue damage, e.g. by producing several enzymes that produce reactive oxygen species (ros) and other tissue-damaging metabolites such as nitrosylated products. such enzymes include nicotinamide adenine dinucleotide phosphate-oxidase (napdh oxidase) and myeloperoxidase (mpo) (60). elevated mpo levels predict the risk of heart disease in subgroups otherwise associated with low risk (61,62). elevated mpo levels also independently predict the early risk of future cardiovascular events in patients with acute coronary syndromes (63,64). vascular permeability in retinal disease the vasculature in the eye is protected by the blood– retinal barrier (brb), which is maintained by tight junctions between retinal capillary endothelial (rce) cells and retinal pigment epithelial (rpe) cells, which form the inner and outer brb, respectively (65). rce cells possess intercellular tight junctions, which are formed by rce and glial cells (66). loss of normal brb function is a common feature of many retinal degenerative disorders including age-related macular degeneration, diabetic retinopathy, and retinal vein occlusions (67). age-related macular degeneration patients present focal ischemia in the outer retina with associated inflammation, which induces vegf production and angiogenesis resulting in hyperpermeable vessels. prolonged elevation of blood sugar concentrations in diabetic patients causes endothelial apoptosis, basement membrane thickening, and pericyte loss, accompanied by increased vegf synthesis and vascular permeability. retinal vein occlusions can be attributed to hemodynamic disturbance (increased coagulation, impaired flow properties) resulting in ischemia and increased vegf synthesis (see stewart (67) for details). a common aspect of many eye diseases is therefore ischemia, increased vegf production, and excess vascular permeability (68).the excess permeability has been attributed both to the overstimulated, abnormal vasculature and to changes in the phosphorylation of tight junction proteins such as occludin and zona occludens protein 1 (zo1) (69). vascular permeability and lymphatics the blood and lymphatic vasculatures constitute two parallel circulatory organs, connected by the emptying of lymph into the left jugular vein. blind-ending lymphatic capillaries collect interstitial fluid by pumping the liquid, which will pass lymphatic valves that close to prevent ‘back-flow’. tissue edema facilitates the draining of the interstitial fluid through the initial lymphatic vessels by pulling on the vessels through their tissue-anchored filaments (70). in pathologies such as cancer, lymphatic vessels are often collapsed due to the excessive interstitial pressure and edema, implying that the lymphatic vasculature is dysfunctional (70). several cell types in the cancer produce lymphatic growth factors, including vegfc that binds the lymphatic receptor tyrosine kinase vegf receptor 3 (vegfr3) (71). similar to the overstimulated and dysfunctional blood vasculature, the lymphatics may undergo neo-angiogenesis in cancer, which would facilitate draining of the tumor edema, on the one hand, but also, on the other hand, provide a route for spread of the cancer via the lymphatics. however, the relationship between vascular permeability and lymphatic function (collection and propagation of liquid, formation of new lymphatic vessels, and intraand extravasation) is to a large extent unexplored. perspectives excess vascular permeability resulting in edema and swelling of the tissue (in latin: tumor) was noted 140 l. claesson-welsh already in the encyclopedia de medicina by aulus cornelius celsus (25 bc–50 ad) as one of the four cardinal signs of inflammation (tumor, rubor, calor, dolor). a focus of interest today is whether specifically suppressing excess vascular permeability is therapeutically beneficial in a range of diseases. thereby, tissues engaged in the disease would be less edematous and the interstitial pressure would be lower, allowing more efficient delivery of conventional therapeutics, such as chemotherapy to treat cancer. a more efficient delivery of chemotherapeutics, perhaps at a lower, less toxic, dose, is obviously of considerable interest clinically. it would be expected that the barrier presented by non-leaky vessels would provide better perfusion and thereby facilitate tissue homeostasis and promote healing. acknowledgements i thank my co-workers for their dedicated work, in particular dr xiujuan li and dr naoki honkura for preparation of the confocal image for figure 2 and dr narendra padhan for assistance in drawing the figures. funding: work in the author’s laboratory was supported by grants from the swedish cancer society (can2013/661), the swedish research council (2010-2521), worldwide cancer research (131295), and the knut & alice wallenberg foundation. declaration of interest: the author reports no conflicts of interest. references 1. lanitis e, irving m, coukos g. targeting the tumor vasculature to enhance t cell activity. 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http://www.ncbi.nlm.nih.gov/pubmed/22212972?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23031671?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23031671?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/10758225?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/10758225?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24561443?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24561443?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17522591?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17522591?dopt=abstract abstract introduction mechanisms in permeability the vesiculo-vacuolar organelle paracellular junctions the actin cytoskeleton endothelial fenestrations the blood&ndash;brain barrier vascular permeability to solute, molecules, and cells regulation of vascular permeability angiogenic growth factors inflammatory cytokines vascular permeability and disease vascular permeability and cancer vascular permeability and myocardial pathology vascular permeability in retinal disease vascular permeability and lymphatics perspectives acknowledgements declaration of interest references a priming dose of intravenous ketamine-dexmedetomidine suppresses fentanyl-induced coughing: a double-blind, randomized, controlled study upsala journal of medical sciences. 2014; 119: 333–337 original article a priming dose of intravenous ketamine-dexmedetomidine suppresses fentanyl-induced coughing: a double-blind, randomized, controlled study amin j. saleh, liangbin zhang, sally m. hadi & wen ouyang department of anesthesiology, the third xiangya hospital, central south university, changsha, hunan, p.r. china abstract objective. this study was designed to investigate whether a priming dose of ketamine-dexmedetomidine can effectively suppress fentanyl-induced coughing (fic). methods. altogether 400 patients of asa i and ii, aged 18–70 years, undergoing various elective surgical procedures, were randomly allocated into four groups of 100 patients each. patients in the placebo group received volume-matched normal saline 0.15 ml/kg + normal saline 0.05 ml/kg. one group of patients was given ketamine 0.15 mg/kg + normal saline 0.05 ml/ kg (ket), and another group dexmedetomidine 0.5 mg/kg + normal saline 0.05 ml/kg (dex). finally, one group of patients received ketamine 0.15 mg/kg + dexmedetomidine 0.5 mg/kg (ketodex). after fentanyl administration, the onset time and severity of cough for 1 min were recorded. cough severity was graded as mild (grade 1–2), moderate (grade 3–5), or severe (grade >5). result. the incidence of fic was 53%, 34%, 20%, and 9% in the placebo, dex, ket, and ketodex groups, respectively. the incidence of cough was significantly lower in the ketodex group. likewise, the onset time of cough was significantly delayed in the ketodex group. only nine patients in the ketodex group had either mild (6%) or moderate (3%) cough, with none suffering from severe cough. conclusion. a priming dose of ketodex effectively suppressed the cough reflex induced by fentanyl and delayed the onset time of cough. therefore, treatment with ketodex may be a clinically useful method for preventing fic. key words: anesthesia, fentanyl-induced coughing, dexmedetomidine, ketamine introduction during anesthesia induction and surgery, patients may experience some psychological and sympathetic adverse effects. opioids are often used to allay anxiety and to decrease pain associated with surgery (1,2). fentanyl, in particular, is often used as a premedicant agent during the induction of general anesthesia to prevent elevation of blood pressure during intubation in the operation room. the use of fentanyl is due to its rapid onset, short duration, intense analgesia, ease of titrability, reduced cardiovascular depression, and low histamine release (3). however, intravenous bolus administration of fentanyl often elicits cough, and the frequency can reach 65% (4,5). thus, fentanyl-induced coughing (fic) is often observed after intravenous bolus administration of fentanyl during anesthesia induction. however, fic is undesirable during anesthesia induction, especially in some patients suffering from coexisting diseases including increased intracranial pressure (icp), cerebral aneurysm, brain trauma, brain hernia, dissecting aortic aneurysm, open eye injury, reactive airway disease, or pneumothorax (6). furthermore, severe fic at the induction time can cause multiple conjunctival and periorbital petechiae (7) and lead to upper airway correspondence: w. ouyang, department of anesthesiology, the third xiangya hospital, central south university, changsha 410013, p.r. china. e-mail: ouyangwen133@vip.sina.com (received 12 september 2014; accepted 18 september 2014) issn 0300-9734 print/issn 2000-1967 online � 2014 informa healthcare doi: 10.3109/03009734.2014.968270 http://informahealthcare.com/journal/ups mailto:ouyangwen133@vip.sina.com obstruction that might require immediate intervention (8). therefore, effective ways to prevent fic are clinically important. the mechanism of fic is still unclear. previous studies with various pharmacological interventions have been conducted to reduce the incidence of fentanyl-induced cough using readily available anesthetic adjuncts (9) such as n-methyl-d-aspartate (nmda) receptor antagonists, benzodiazepines, lidocaine, propofol, a2 agonists, atropine, priming and slow injection of fentanyl, inhalation of selective b2-adrenergic bronchodilators, salbutamol, beclomethasone, or sodium chromoglycate (10-16). several studies have been conducted on dexmedetomidine, alone, or in combination with other agents to reduce the incidence of fentanyl-induced cough (17,18). because of its unique highly selective a2-adrenoceptor agonist mechanism, dexmedetomidine is regularly used in clinical practice to blunt the cardiovascular response to tracheal intubation (19-23). on the other hand, intravenously injected ketamine, an nmda antagonist, has well-known potent analgesic and bronchodilatory effects (24-29). however, the suppression of fic with priming doses of intravenous ketamine and dexmedetomidine (ketodex) has not been studied. the aim of the present study was to investigate whether ketodex could effectively prevent or suppress fic. materials and methods patient selection after the study had been approved by the ethical committee of third xiangya medical hospital of central south university, changsha, hunan, p.r. china, written informed consent was obtained from all participants before they were enrolled into this randomized, prospective, double-blind, placebocontrolled study. altogether 400 american society of anesthesiologists (asa) physical status grade i and ii patients, aged between 18 and 70 years, undergoing general anesthesia for various elective surgeries, were recruited. the exclusion criteria were those older than 70 years or younger than 18 years, body weight exceeding 20% of the ideal body weight, impaired kidney or liver functions, a history of bronchial asthma and chronic obstructive pulmonary disease, a history of smoking, respiratory tract infection, hypersensitivity to local anesthetics, hyperactive patients on medication containing angiotensinconverting enzyme inhibitors, and patients on anesthetic premedication. procedures none of the patients received any premedication before surgery. after the patient’s arrival at the operating room, peripheral venous access was established on the right or left antecubital vein for fluid and drug administration with a 20-g intravenous cannula. electrocardiography, non-invasive blood pressure, and pulse oxygen saturation were monitored. dexmedetomidine (200 mg/2 ml) was diluted with normal saline to obtain a concentration of 4 mg/ml, and ketamine (100 mg/1 ml) was diluted with normal saline to obtain a concentration of 10 mg/ml. patients were randomly assigned to four groups of 100 patients each, using computer-generated random numbers, to receive dexmedetomidine 0.5 mg/kg + normal saline 0.05 ml/kg (dex group), ketamine 0.15 mg/kg + normal saline 0.05 ml/kg (ket group), or ketamine 0.15 mg/kg + dexmedetomidine 0.5 mg/kg (ketodex group). the placebo group received normal saline 0.15 ml/kg + normal saline 0.05 ml/kg. the above-mentioned drugs were administered intravenously, in 10 ml isotonic saline. dexmedetomidine 0.5 mg/kg or its corresponding normal saline (0.05 ml/kg) for all groups was infused at a steady rate of 2 ml/min, over 5 min. ketamine 0.15 mg/kg or its corresponding normal saline 0.05 ml/kg was administrated over 10 s, 1 min before the fentanyl bolus injection (3.0 mg/kg within 5 s). after the fentanyl injection any episode of cough was classified as coughing. the onset time (from the end of bolus administration to the beginning of coughing), frequency, and severity of cough for 1 min were recorded, by a blinded observer, who was unaware of the type of medication given to the patients. syringes for injection were labeled with specific numbers, keeping the identity of the group treatment concealed from all research staff until the end of the study. recorded numbers of episodes of coughing were classified based on the number of coughs observed per min. cough severity was graded as mild (grade 1–2), moderate (grade 3–5), or severe (grade >5). later, general anesthesia was induced with a sleeping dose of propofol (1–2 mg/kg), and then anesthesia was maintained with infusion of propofol and/or inhalation agent and air/oxygen mixtures. statistical analyses statistical analyses were carried out using spss statistical package, version 13.0 (spss, inc., chicago, il, usa) for windows. results are expressed as the mean ± sd, number, proportion, or percentage. frequencies of coughing and proportions of sex 334 a. j. saleh et al. and asa classes were compared using the chi-square test or fisher’s exact test with bonferroni correction. one-way analysis of variance was used to compare age and weight among the four groups. a value of p < 0.05 was considered to be statistically significant. results a total of 427 patients were assessed for eligibility, and 27 (6%) patients were excluded. among them were 17 (4%) patients who did not meet the inclusion criteria, 8 (2%) other patients declined to participate, and 2 (0.5%) were excluded for other reasons. those who withdrew did not differ significantly in any characteristics from those who continued the study. therefore, a total of 400 (93%) patients were enrolled in the study, and there were 100 patients in each group. the demographic data were comparable for age, weight, gender, and asa status in the four groups (table i). following an intravenous fentanyl bolus, 53 patients had cough in the placebo group, 20 in the ket group, 34 in the dex group, and only 9 of the patients in ketodex group (table ii). the incidence of cough was significantly lower (p < 0.05) in the ketodex group when compared with the other groups. the onset time of cough was significantly delayed in the ketodex group. only nine patients in the ketodex group had either mild (6%) or moderate (3%) cough, with none suffering from severe cough (table ii). no patient in any of the groups suffered from hypoxemia, apnea, truncal rigidity, nausea, vomiting, or other adverse effects after the bolus injection of fentanyl. discussion cough reflexes commonly occur after fentanyl administration during induction of general anesthesia. thus, fic is then frequently observed but has so far received little attention, and therefore fic may continue to be a troublesome factor in the delivery of safe and effective patient care (7). multiple mechanisms have been proposed to be responsible for the occurrence of fic at induction. thus, fentanyl has been shown to inhibit central sympathetic outflow causing vagal predominance, inducing cough and reflex bronchoconstriction (6,30,31). several recent studies have been conducted to investigate different pharmacological drugs with regard to their potency to reduce the adverse effects of fic (3-5,8-12). in our study, the dose (3 mg/kg) of fentanyl was selected because it fitted to the common administration in our daily clinical practice. interestingly, our study demonstrated that a priming dose of intravenous ketodex reduced the incidence of fic to 9% in compared to 53%, 34% and 20%, in the placebo, dex and ket groups, respectively. of nine patients who had coughed in the ketodex group, none of them experienced any degree of severe coughing 1 min after fentanyl administration. patients in the placebo group showed a higher table i. demographics of four patient groups (n = 100). group placebo ket dex ketodex age (y) 39.1 ± 15.1 38.8 ± 17.3 40.2 ± 14.2 39.6 ± 15.6 gender (m/f), n 61/39 58/42 66/34 63/37 weight (kg) 58 ± 6 61 ± 6 60 ± 3 62 ± 1 asa (i/ii), n 94/6 93/7 98/2 96/4 values are means ± standard deviation. no statistically significant differences were observed in this table. asa = american society of anesthesiologists physical status; ket = ketamine group; dex = dexmedetomidine group; ketodex = ketamine-dexmedetomidine group. table ii. incidence of coughing and its severity after fentanyl injection. severity of cough (%) groups incidence (%) onset (s) mild moderate severe total no cough (%) placebo 53/100 (53) 15.7 ± 3.9 25 (25) 17 (17) 11 (11) 53 47 ket 20/100 (20) 18.6 ± 3.3 10 (10) 6 (6) 4 (4) 20 80 dex 34/100 (34) 18.2 ± 3.2 16 (16) 11 (11) 7 (7) 34 66 ketodex 9/100 (9) 25.8 ± 3.8b 6 (6)b 3 (3) 0 (0)a 9 91 the dose of fentanyl was 3 mg/kg for an injection duration of <5 s. severity of coughing was graded as mild (grade 1–2), moderate (grade 3–5) and severe (grade >5) based on the number of coughs. the onset time (mean ± sd) was the median value of patients who coughed. ap < 0.01, bp < 0.05. ketodex suppresses fentanyl-induced coughing 335 incidence rate (53%) than patients in the ket (20%) or the dex (34%) groups. there was a relative risk reduction for the ket group in the incidence of fic (20%), but the difference did not attain statistical significance. in addition, the onset time of cough was delayed in the ketodex group. the relatively slow time course for the fentanyl injection (5 s) may be considered to be a possible reason when compared with injection times (<2 s) used in previous studies (16,17,32-35). in our study, no patients received any premedication before surgery. this may be the reason why more of the patients in the placebo group experienced coughing (53%). yeh et al. demonstrated that ketamine (0.15 mg/kg) could suppress fic (12). the activation of nmda receptors in the larynx, lung, and airways can trigger airway constriction. therefore, the direct bronchodilating and inhibitory effect of ketamine on bronchomotor tone may be attributed to its nmda receptor antagonism to attenuate the fentanyl cough reflex (23,36). in another study, he et al. demonstrated that dexmedetomidine (0.5 mg/kg and, to a higher extent, 1.0 mg/kg) reduced the incidence of fic (18). dexmedetomidine is a highly selective a2-adrenergic agonist with sedative and analgesic properties. the combination of ketamine and an a2-adrenergic agonist has bronchodilatory effects on airway smooth muscles. the rapid response of the cough reflex after fentanyl injection suggests that a pulmonary chemoreflex is the likely mechanism, mediated by either irritant receptors or by vagal c-fiber receptors in close proximity to pulmonary vessels (5,12). the relevant limitation of our study is that the priming dose of ketamine (0.15 mg/kg) in the ket and ketodex groups was low, so perhaps the relatively high incidence rate in both groups could have been different if we had used a different dose. in conclusion, the present study has shown that a priming dose of intravenous ketodex could suppress fic caused by fentanyl within 5 s during general anesthesia induction. therefore, ketodex could be a clinically effective choice for attenuating fic. acknowledgements the authors thank dr duan kai ming for support in conducting this 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http://www.ncbi.nlm.nih.gov/pubmed/20885016?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20885016?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21935685?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21935685?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21935685?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21935685?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23385035?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23385035?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7617171?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7617171?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7617171?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7617171?dopt=abstract abstract introduction materials and methods patient selection procedures statistical analyses results discussion acknowledgements declaration of interest references johan anton waldenström, a versatile doctor and pioneer: professor of medicine and practising surgeo full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 johan anton waldenström, a versatile doctor and pioneer: professor of medicine and practising surgeon anders waldenström to cite this article: anders waldenström (2015) johan anton waldenström, a versatile doctor and pioneer: professor of medicine and practising surgeon, upsala journal of medical sciences, 120:2, 90-94 to link to this article: https://doi.org/10.3109/03009734.2015.1027427 © informa healthcare published online: 10 apr 2015. submit your article to this journal article views: 210 view related articles view crossmark data https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://doi.org/10.3109/03009734.2015.1027427 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1027427 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1027427 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1027427&domain=pdf&date_stamp=2015-04-10 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1027427&domain=pdf&date_stamp=2015-04-10 upsala journal of medical sciences. 2015; 120: 90–94 johan anton waldenström, a versatile doctor and pioneer: professor of medicine and practising surgeon anders waldenström norrlands universitetssjukhus, umeå, sweden key words: doctor, medicine, surgeon johan anton waldenström was born in 1839. his father was erik magnus waldenström, a district medical officer in luleå whose district encompassed the entire upper norrland. erik magnus was the first doctor in his family. he studied in lund where he also obtained his doctorate of medicine (md). immediately afterwards, in 1819, he moved to luleå, where he remained until his death. he is described as a forceful man, as a doctor, as a local politician, and as the head of his family. one might question a doctor being called forceful, but it was not uncommon for him to undertake taxing home visits by sledge and could well be away from home for several weeks at a time. one such journey was estimated at between 300 and 400 kilometres! this man was remarkable for his striving to provide his sons with an academic education. he fathered 15 children, all of whom but one reached adulthood. he paid for the academic education of all his 10 sons, which was considered a great achievement for a district medical officer whose salary was so small it was necessary to supplement it with a family-run farm. as an example of their limited means, it can be mentioned that johan anton had to share his school books with his brother paul petter, who although he was one year older was in the same class. they stuck together like two peas in a pod throughout their studies. in their last years of upper high school, they studied together at the lyceum in uppsala. at university paul petter studied theology and johan anton medicine. in order to make the most of their meagre funds, they travelled together to uppsala by steamer at the beginning of the academic year, taking with them as many necessities as possible including food which was made to stretch as far as possible. naturally they boarded together. they listed all their expenses, and this was checked by their strict father. the circumstances are well described in numerous letters, often weekly and signed ‘your ever-loving father’ and beginning with ‘my dear boys’. now and again a letter began curtly, ‘my boys’; ‘and it hurt johan and me [paul petter] so hard that we closed our books and we were unable to study for the remainder of the day’. the use of language and the meaning of words have truly changed over the years, and it demonstrates how hard it can be to interpret the essence of old texts without understanding the language of the times. in the summers they had to return to their parents’ home in luleå and help with the farming. this was particularly disliked by paul petter, who considered such labour to be unworthy of a student! having finished school, their father believed that they should take what we now call a sabbatical year at home in order to consider what line of study to follow. this too was financed by their father. both the brothers worked hard at their studies and could by no means be called ‘jolly’ students. the one who became more generally well-known of the two was paul petter who, as a result of differences of opinion about free communion and the significance of the creed, came into such conflict with the church, and specifically the swedish evangelical mission, that he decided to help form an independent mission society foundation. thus the mission covenant church of sweden was founded. in addition to his work within the mission covenant church, correspondence: anders waldenström, norrlands universitetssjukhus, umeå, sweden. e-mail: anders.waldenstrom@medicin.umu.se (received 25 february 2015; accepted 26 february 2015) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1027427 http://informahealthcare.com/journal/ups mailto:anders.waldenstrom@medicin.umu.se he was a senior lecturer in gävle and a member of parliament. he was famous for his rhetoric and repartee. he was at one time sweden’s most read author. alongside his brother, johan anton pursued an interesting and remarkable career as a doctor and scientist. he was considered not quite as brilliantly intelligent as his oneyear-older brother, but he was extremely industrious, meticulous, and with such sound judgement that the faculty awarded him the helmfeldtska and hwasserska bursaries on completion of his medical studies in 1867. during his last summer of studies he had a temporary post at sätra brunn and showed such accomplishment that the faculty wanted him to continue within academia. the bursaries would finance one year overseas for further studies in europe. this journey was planned by him and his tutor and took him amongst other places to würzburg, dresden, vienna, and prague. in würzburg he came into contact with von recklinghausen, and in vienna he studied under billroth. he returned to uppsala full of impressions, experience, and new scholarship. but maybe most important of all was that he had also acquired good self-esteem. on finishing his studies he had been afflicted with self-doubt and a fear that he would not be a good enough doctor. a righteous man, he felt that he would be unable to improve his knowledge to be able to serve his patients in the best way possible. he expressed this in a prayer at the beginning of his inauguration speech, many years later. he most probably began to write the speech the day that he fell ill with an illness which led to his death 8 days later. the speech began with the prayer: ‘true belief is the armour which alone can protect us against all temptations in times of happiness and failure’. after this he praised in turn his majesty the king, his excellency the archbishop, the university vice-chancellor, and the members of the greater university council, all other university lecturers, and finally the university’s student body. what is interesting is how this 15-page, unfinished text shows the traditions of the times in this context. he declares his respect for his colleagues and asks them for their support and approaches the students in a way that one perhaps would not imagine belonged to that age: ‘this very word professor is seen by many as a vast dividing wall, which makes the distance between the teacher and the taught far too great. away with it! it should rather be the opposite—let the scholar approach the teacher: for to whom can a disciple turn more safely for advice and information . . .’ on returning to uppsala from his foreign travels he was awarded the post of assistant surgeon at the akademiska hospital. however, he soon applied for the post of ‘doctor for the town and its poor’. at the same time he started a private practice in the town. this quickly flourished, and he sought larger premises. he left his post at the akademiska hospital in order to take up the position of town doctor. this gave him a low salary but good opportunities to see and help patients. his great interest in teaching, based on his experience in würzburg, led to his founding the waldenströms polyclinic where students could train in a polyclinic and perform house visits. this was totally new in sweden and was highly appreciated, but it finished when johan anton died. possibly it demonstrates that the form of training is not always paramount, rather the person who does the teaching, i.e. the lecturer’s skilfulness. it is, however, obvious that the training gave social insights and experience from authentic home environments that were valuable for the students in their future careers as doctors. after a dispute with a colleague about the most suitable timing to operate for cataracts, he decided to write his thesis on the subject. he took his doctorate of medicine with his thesis, written very quickly and entitled: ‘när bör den hårda åldersstarren opereras’ [‘when should the hard cataract of the elderly be operated upon?’]. he was given a high grade and became an associate professor of surgery and obstetrics. for the last years of his life he was the permanent stand-in for the professor of practical medicine and became a professor in that subject on 18 october 1879. on 15 november 1879 he was forced to suspend his daily surgery because of serious symptoms in his abdomen which he recognized from an earlier attack of perityphlitis, i.e. acute appendicitis. a few days later he suffered symptoms that can be interpreted as peritonitis. he realized that time was running out. since he was accustomed to opening up both the abdomen and the bowel, he tried to persuade his assistant to operate on him, giving detailed instructions about how to proceed. he had earlier stated that a doctor’s duty is to operate on a patient, even when the risks are great, if the patient will most certainly die without an operation. despite this he could not manage to prevail on anyone to operate on him, and he died. he wanted his body to be examined after death by his colleague and friend fritjof holmgren, and this took place. his diagnosis and a previous episode of the same disorder were confirmed. it may seem ironic that he would succumb to an illness that he himself well could have been the first to cure surgically, had it happened to somebody near him other than himself. as ill luck would have it, he was buried in uppsala cathedral on the very day that should have been his inauguration as professor, 23 november 1879. the funeral was attended by an enormous number of people. shops were closed, and people assembled to honour the dead man. it is said that not since that of linnaeus had so many people been present at a funeral in uppsala. one hundred and fifty wreaths were apparently laid on his grave. why would a doctor be honoured in johan anton waldenström, a versatile doctor and pioneer 91 such a way? probably it was because his patients realized he was clever and never lost interest in them, in combination with his winning personality. moreover, one can assume that he was liked and respected by his colleagues for his teaching ability and his skills in medicine and science, if one can believe the words spoken at the time of his funeral, being aware of hyperbole on such occasions. according to one colleague, in another context, his humility, positive attitudes in the presence of his patients, and most of all his tireless interest in his patients night and day made him popular among patients not only from the town but also from its outlying districts and even from many distant parts of the country. there is an anecdote about a private trip he made on a steamboat with friends in norrland. people in the area heard about it and flocked around in small boats in order to climb aboard to consult the doctor. some examples of observations and scientific work serve to illustrate his versatility and broad interests. his scientific production spanned observations within diverse areas of medicine such as eyes, surgery, gynaecology, internal medicine, and pathology. his contemporary colleague professor hedenius supposedly said, ‘that no other, even equipped with waldenström’s unusual work capacity, would have been able to embrace all the special research fields of practical medicine’. besides, ‘[he] always championed a definite opinion, which he could defend, often victoriously and always having the last word’. his first case study is also historically the most remarkable since it describes for the first time a successful operation for gastric volvulus which involved laparotomy and sigmoidostomy, rinsing the abdominal cavity and wound with carbolic acid according to lister’s directions, and resulting in a positive outcome. the operation was performed on 24 march 1879 and is described in upsala läkareförenings förhandlingar 1878–1879, vol. xiv: ‘volvolus flexura sigmoideae—laparo—colotomia—hälsa’ by a. f. lindstedt and j. a. waldenström. this is number 4 under the title ‘6 fall av tarmförträngning’ [‘six cases of intestinal stricture’]. lindstedt was the assisting student, and it was he who took notes. the medical record of the operation, the epicrisis, and comments were written by waldenström. the pre-eminence of this operation in world literature was established initially by the london professor, v. zachary, in 1939. there is a very detailed case history and surgical notes, well worth reading; however, there is no room for them in this article. rather i include extracts and comments showing the knowledge of the times, ways of expressing oneself, and hopes for the future. one case concerns a 52-year-old man with all the signs of intestinal obstruction who arrived on 23 march 1879. his status is punctiliously described: ‘in the afternoon a puncture was effected through the abdominal tissues revealing swirls of intestines, some faecal–foul smelling gas was released through three-quarter casing.’ for reasons unknown, the patient was moved the following day from the akademiska hospital to a private abode where an operation was initiated in the morning, using chloroform as an anaesthetic. an 11-cm incision was made just to the right of the linea alba, starting 5 cm above the symphysis. ‘the wound revealed three stretched swirls of the large colon, the serous coating of which was much spotted. because of the gaseous bloating of the said intestinal swirl, no hand could be inserted in the abdominal cavity through the incision for fear of the intestine bursting, so the upper swirl, which turned out to be the transverse colon, was pulled out and punctured’. after this it is noted that he examined the abdominal cavity with his hand, palpating its organs. ‘then when the coil of intestine which was outside the cavity (flexura iliaca coli) had been twisted a little to the left, its string-like attachment disappeared . . . the abdominal wall had contracted so much that when the intestine was to be returned to the abdominal cavity, it was impossible. this necessitated having to evacuate the flexura iliaca coli from most of its contents. to this end, a 2-cm incision was made in the intestinal wall in the direction of its teniae. genom den sålunda erhållna öppningen bortskaffades ungefär en 1/2 kanna tjockflytande mörka faeces. sårränderna, omsorgsfullt rentvättade med karbollösning, förenades med catgutsuturer (enligt joberts metod).’. . .’sedan så mycket som möjligt av den i bukhålan nedrunna karbollösningen uppsugits med en skaftad svamp, förenades åter ränderna af buksnittet medels 4 suturae clavatae samt ytliga och djupa suturae nodosae af karboliserad tråd. operationen varade i 1 1/2 timme.’ after this there is a detailed description of the course of events day by day, including a complication which is judged to be pneumonia. despite this the patient is discharged on 6 may with a clean bill of health. i would like to end this version of the case history with a comment quite typical for waldenström. ‘då den sjuke oaktadt använd medicin icke haft öppning sedan 18 dagar, återstod ingenting annat än att öfverlemna honom åt sitt oundvikliga öde eller att på operativ väg söka häfva hindret. visserligen var 18 dagars förstoppning och deraf följande nedsättning af krafterna en för en gynnsam utgång af operationen betänklig omständighet, men å andra sidan utgjorde frånvaron af kräkningar samt ömhet i buken ett gynnsamt tecken till, att den sjukes bukhinna icke var särdeles irritabel. jag beslöt mig derföre för en operation, äfven om, såsom jag först förmodade, en elakartad nybildning i tarmen kunde vara med i spelet. jag anser det nemligen orätt numera, att, när en patient går en oundviklig död till mötes, uppskjuta ett operativt ingrepp, som möjligen kan rädda den sjuke, derför att möjlighet 92 a. waldenström förefinnes, att sjukdomen är af den beskaffenhet, att den icke kan genom operation undanröjas, men hvilket man icke kan före operationen bestämdt afgöra. erfarenheten har redan visat, att bukhålan kan, om nödiga försigtighetsmått iakttagas, öppnas och undersökas utan den ringaste fara.’ this shows a true surgical mind, particularly the ‘least danger’, and even for a cardiologist it sounds like an underestimation of the risks. in the field of gynaecology, he was particularly interested in ovaries and ovarian cysts and their pathogenesis. he studied the subject as a pathologist, but describes the procedure of ‘fem ovariotomier och en paraovariotomi’ [‘five ovariectomies and a unilateral ovarectomy’], all of which were successful and can be counted among his most important works, including ‘om behandlingen af för ymniga menses’ [‘on the treatment of excessive menstruation’]. in this context the description he gave in ‘transfusion med anledning af blödning efter förlossning’ [‘transfusion because of haemorrhage after delivery’] can also be mentioned. it concerns a 41-year-old woman who is haemorrhaging since he has not been able to remove the whole placenta. having tried to pull out the remaining parts with his fingers, he has to resort to breaking them up. at this point the uterus contracts so strongly that the bleeding ceases. the woman, however, is exhausted, and waldenström deems transfusion to be necessary. at times her eyes darkened and the radial pulse could not be detected. ‘med anledning häraf beslöt jag mig för att göra transfusion på patienten. först försökte jag erhålla ett får, men då detta ej lyckades, erhöll jag blod från tvänne starka karlar. blodet defibrinerades, silades tvänne gånger genom linne, allt under det att detsamma hölls vid en temperatur, som varierade mellan 35–37�c. 160 gram injicerades.’ five days later the patient died, and since the post mortem could not give clear cause, waldenström wrote: ‘på grund af hvad symptomerna under lifvet jemförda med de vid obduktionen funna förändringarna gifva vid handen, måste man antaga, att patienten aflidit af kraftuttömning, och att den gjorda blodinsprutningen icke haft förmåga att underhålla lifsverksamheten tills en för lifvets bestånd tillräcklig qvantitet blod blifvit återbildad.’ blood transfusion in those days was hardly standard practice. blundell described in 1818 a blood transfusion with human blood where the recipient survived. in 1873, i.e. the year before the case in point, sir thomas smith in london described a successful transfusion with defibrinated blood to a newly delivered infant. there were examples of case reports, but more often the result was negative. nonetheless waldenström attempted this treatment in an otherwise hopeless case. interestingly enough he mentions that the donor blood came from ‘a brace of strong men’ perhaps in the hope that the donors’ strength would be communicated through the blood. on the subject of surgical techniques, waldenström gives a highly precise description of how a tracheostomy should be performed. this too can be found in upsala läkareförenings förhandlingar. he points out what a vast difference there is in doing a tracheostomy on a corpse compared to a living patient and how important it is to fixate the bronchus during the procedure. in a lengthy medical history with comments, he deals with a case of onychomycosis. in another case he describes a young man who has caught a cold, becomes more and more sleepy with the supervention of neurology, and whose case concludes in purulent meningitis and death. waldenström claims, ‘att en snufva, som alltid anses för en särdeles obetydlig åkomma, kan gifva upphof till en purulent meningitis, måste vara mycket sällsynt, alldenstund detta icke finns angifvet i den litteratur, som stått till mitt förfogande, och på grund häraf är det anförda sjukdomsfallet särskildt anmärkningsvärdt.’ finally i want to mention the description of a 49-year-old woman who in 1878 suffered severe peritonitis ‘hvarifrån hon endast långsamt tillfrisknade. patienten hade nu fallit och slagit sacrum mot en trappa och som följd fått svårighet att urinera. vid undersökning ingick ‘touchering per vaginam’ where a tumour was discovered in the posterior fornix. it was thought to be a calcified fibromyoma which was inhibiting the release of urine and so it was decided to attempt to redress the tumour. ‘efter några ögonblicks sakta tryckning på svulsten i riktning uppåt och något bakåt hördes ett skrapande ljud, som äfven uppfattades af patienten själf. jag uttog genast instrumentet, patienten reste sig upp och yttrade, att hon nu erfor samma känsla af välbefinnande, som då hennes luxerade arm för några år sedan reponerades.’ this medical case history unfortunately had an unhappy ending, since the patient soon afterwards experienced severe abdominal pains again which were diagnosed as peritonitis. she died 6 days later. at the post mortem, they found a dermoid cyst with teeth and bones (that presumably had caused the grating sound). it is unclear if the rupture of the cyst happened at the time of the fall or when the repositioning was attempted, but waldenström commented, ‘alldenstund den häftiga smärtan i buken utgjorde det första symptom af den peritonitis, för hvilken patienten dukade under, och då smärtan inställde sig omedelbart efter undersökningen, så måste det av mig gjorda försöket att reponera svulsten antagas hafva varit vållande till bukhinneinflammationens uppkomst. att detta lindriga ingrepp kunde hafva en sådan verkan, är icke förklarligt på annat sätt, än att en del af cystan genom fallet brustit och benet i dess vägg frakturerade samt att vid undersökningen något af dess innehåll utprässats i bukkaviteten.’ johan anton waldenström, a versatile doctor and pioneer 93 when johan anton died, he left his wife elisabeth, née gibson, and son henning (later to become professor of orthopaedic surgery and my paternal grandfather) as well as an unborn son, also johan anton, who became the senior physician at the surgical clinic in falun. the widow married again, this time to jonas waern, professor of medicine at the karolinska institute and step-father to henning and johan. thus johan anton was the first of four professors of medicine directly descended from erik magnus as a result of his ambition to afford his sons an academic education. acknowledgements warm thanks for help with the literature are extended to professor bo lindberg, uppsala. declaration of interest: the author reports no conflicts of interest. the author alone is responsible for the content and writing of the paper. further reading 1. släkten waldenström codex. 1984. isbn 91-86664-01-8. 2. en svensk pionjär inom bukkirurgin. barthold carlsson, göteborg. 3. upsala läkarförenings förhandlingar bd vi–xv. 4. pioneers in acute abdominal surgery. oxford: 1939. 94 a. waldenström ss1 acknowledgements declaration of interest further reading phosphohistidine phosphatase 1 (phpt1) also dephosphorylates phospholysine of chemically phosphoryla full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 phosphohistidine phosphatase 1 (phpt1) also dephosphorylates phospholysine of chemically phosphorylated histone h1 and polylysine pia ek, bo ek & örjan zetterqvist to cite this article: pia ek, bo ek & örjan zetterqvist (2015) phosphohistidine phosphatase 1 (phpt1) also dephosphorylates phospholysine of chemically phosphorylated histone h1 and polylysine, upsala journal of medical sciences, 120:1, 20-27, doi: 10.3109/03009734.2014.996720 to link to this article: https://doi.org/10.3109/03009734.2014.996720 © informa healthcare published online: 09 jan 2015. submit your article to this journal article views: 897 view related articles view crossmark data citing articles: 11 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2014.996720 https://doi.org/10.3109/03009734.2014.996720 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2014.996720 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2014.996720 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2014.996720&domain=pdf&date_stamp=2015-01-09 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2014.996720&domain=pdf&date_stamp=2015-01-09 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2014.996720#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2014.996720#tabmodule upsala journal of medical sciences. 2015; 120: 20–27 original article phosphohistidine phosphatase 1 (phpt1) also dephosphorylates phospholysine of chemically phosphorylated histone h1 and polylysine pia ek1, bo ek2 & örjan zetterqvist1 1department of medical biochemistry and microbiology, uppsala university, uppsala, sweden, and 2department of chemistry, uppsala university, uppsala, sweden abstract background. phosphohistidine phosphatase 1 (phpt1), also named protein histidine phosphatase (php), is a eukaryotic enzyme dephosphorylating proteins and peptides that are phosphorylated on a histidine residue. a preliminary finding that histone h1, which lacks histidine, was phosphorylated by phosphoramidate and dephosphorylated by phpt1 prompted the present investigation. methods. histone h1 and polylysine were phosphorylated at a low concentration (3.9 mm) of phosphoramidate. their dephosphorylation by recombinant human phpt1 was investigated by using a deae-sepharose spin column technique earlier developed by us for studies on basic phosphoproteins and phosphopeptides. determination of protein-bound, acid-labile phosphate was performed by a malachite green method. mass spectrometry (ms) was used to investigate the occurrence of n-e-phospholysine residues in a phosphorylated histone h1.2 preparation, and to measure the activity of phpt1 against free n-w-phosphoarginine. results. histone h1.2,which lackshistidine, was phosphorylated byphosphoramidate on severallysine residues, asshownbyms. phpt1wasshowntodephosphorylatephosphohistoneh1ataratesimilartothatpreviouslydescribedforthedephosphorylation of phosphohistidine-containing peptides. in addition, phosphopolylysine was an equally good substrate for phpt1. however, no dephosphorylation of free phosphoarginine by phpt1 could be detected. conclusion. the finding that phpt1 can dephosphorylate phospholysine in chemically phosphorylated histone h1 and polylysine demonstrates a broader specificity for this enzyme than known so far. key words: histone h1, phosphohistidine phosphatase, phospholysine, phospholysine phosphatase, php, phpt1, protein histidine phosphatase introduction the discovery of protein phosphorylation on histidine (1) was made 52 years ago (2). most of the work relatedtophosphohistidinehassofarbeenperformedon the bacterial phosphoenolpyruvate-glucose phosphotransferase system (3) and two-component systems (4).ineukaryoticcells,protein-boundphosphohistidine constitutes a significant amount of the total phosphoamino acid (5). still, only a very low number of phosphohistidine-containing proteins have been identified in contrast to the great number of proteins phosphorylated on serine, threonine and tyrosine (2,6,7). theproteinsphosphorylatedonhistidinearetoalarge extent represented by catalytic intermediates of metabolic enzymes, such as the extensively studied nucleoside diphosphate kinase (ndpk) that transiently forms 1-phosphohistidine (8,9) and atp-citrate lyase and succinyl-coa synthetase (succinate thiokinase) that similarly form 3-phosphohistidine in their active sites(10-15). theslow progressineukaryoticphosphohistidine research can be explained by the lability of the n–p bonds of phosphohistidines at the acid conditions thatareroutinelyusedinmostphosphoproteinresearch (5,16-18). however, recent developments of specific antibodies in combination with mass-spectrometric correspondence: pia ek, department of medical biochemistry and microbiology, uppsala university, uppsala, sweden. e-mail: pia.ek@imbim.uu.se (received 18 october 2014; accepted 4 december 2014) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2014.996720 http://informahealthcare.com/journal/ups mailto:pia.ek@imbim.uu.se methods adapted to the detection of phosphohistidine (19) give hope for a change in the near future. in 2002, a new tool for studies of eukaryotic histidine phosphorylation was obtained in the form of a 14 kda phosphohistidine phosphatase (phpt1) that was independently discovered and characterized by ek et al. (20) and klumpp et al. (21). the latter group named the enzyme protein histidine phosphatase (php). no activity of this enzyme could be detected toward a set of o-phosphorylated peptides containing phosphoserine, phosphothreonine, or phosphotyrosine (20). the activity was independent of divalent cations, and okadaic acid did not inhibit the enzyme (20,21). by mutational studies of the recombinant human enzyme, his-53 was shown to be essential for the phosphatase activity toward phosphohistidine (22). the 3d structure of the enzyme was determined both by x-ray crystallography (23) and nmr (24). atp-citrate lyase and the b-subunit of g-protein were soon identified as potential physiological substrates (25,26). convincing evidence of a physiological role for phpt1 has been obtained from studies of the potassium channel kca3.1 that is phosphorylated by nucleoside diphosphate kinase b (ndpk-b) on histidine residue 358 (27,28). in these experiments, ion transportation was activated by the phosphorylation and deactivated by a phpt1-dependent dephosphorylation. similar results have been obtained for the calcium channel trpv5, although a direct proof of the formation of phosphohistidine in this case is still lacking (29). the interesting possibility of ndpk acting as a protein kinase with phpt1 as the balancing phosphoprotein phosphatase has been discussed in a review by wieland et al. (30). furthermore, evidence for a possible role of phpt1 in cytoskeletal reorganization (31,32) and in hepatocellular carcinoma cell proliferation (33) has been presented. in several of the experiments above, the possibilities to modulate the expression of the phpt1 gene have been crucial. another approach to study the substrate specificity of phpt1 was described by attwood et al. in their study of chemically phosphorylated histone h4 peptides containing his-18 and his-75 (34). these phosphopeptides were good substrates of phpt1. in parallel, our group designed a new method for the determination of phosphohistidine phosphatase activity based on highly basic substrates, such as chemically phosphorylated histone h4 and ac-vrlkhrklrpna, a peptide representing the amino acid sequence around his-358 of kca3.1 (35). during preliminaries to the latter work several histone preparations were tested beside histone h4. commercial preparations of histone h1 from calf thymus, in spite of their lack of histidine, were phosphorylated to the highest degree of all histones tested and were dephosphorylated by phpt1 at high rates. these unexpected results have been investigated in the present study. materials and methods materials human recombinant phosphohistidine phosphatase (phpt1) was expressed and purified as described by ma et al. (22). calf thymus histone h1 (iiis) and polylysine were from sigma-aldrich (stockhom, sweden). calf thymus histone h1 was also from calbiochem(stockhom,sweden),abcam(cambridge, uk), santa cruz (heidelberg, germany), and signalchem (stockhom, sweden). trypsin was from promega (stockhom, sweden). n-w-phosphol-arginine was from sigma-aldrich. non-radioactive phosphoramidate was synthesized by applying the method described for [32p]phosphoramidate by buckler and stock (36). malachite green reagent was biomolgreen from ah diagnostics (stockholm, sweden). deae-sephacel was from ge health care (uppsala, sweden). micro bio-spin columns were obtained from biorad (stockholm, sweden). methods protein phosphorylation and dephosphorylation. histone h1 and polylysine were chemically phosphorylated by 3.9 mm phosphoramidate as described for histone h4 (35). thus, 50 mg of histone h1, 100 mg of 30 kda polylysine, or 100 mg of 90 kda polylysine, each dissolved in 25 ml 10 mm hcl, were separately mixed with 25 ml 25 mm tris/hcl (ph 8.5) and 1 ml 0.2 m phosphoramidate to give ph 7.0 and incubated for at least 24 h. tween 20 was then added to give the final concentration 0.04% (w/v), and the incubation was interrupted, either by freezing at 80�c or by centrifugation for 2 min at 900 g on a 200-ml deae-sephacel spin column equilibrated in 25 mm tris/hcl (ph 8.5) as described (35). the eluted volume was immediately centrifuged on a second deae-sephacel column, and the eluted volume from the latter column was used for dephosphorylation experiments and analysis of the acid-labile phosphate bound to the basic protein, as described (35). biomolgreen was used as the malachite green reagent. dephosphorylation was performed at ph 7.5 and 30�c by incubating a 50-ml aliquot of the eluted volume from the second spin column with 5 pmol of phpt1 in 1 ml 25 mm hepes (ph 7.5) for indicated times. the incubation was interrupted by an immediate centrifugation at 900 g on a deaesephacel spin column to bind phpt1 and any released orthophosphate. the eluted volume from phpt1 also dephosphorylates phospholysine 21 this column was analyzed for remaining acid-labile, protein-bound phosphate as described above. histone h1 and polylysine concentrations were estimated by uv spectrometry at 280 nm and 215 nm, respectively. investigation of phpt1 activity against n-w-phosphoarginine. n-w-phosphoarginine (250 mm) was incubated for 40 min with recombinant phpt1 in the same buffers and volumes as described above for phosphohistone h1 and phosphopolylysine. n-wphosphoarginine and arginine were analyzed by ms, as described below. mass-spectrometric analysis. chromatographically purified histone h1 from signalchem was chemically phosphorylated as described above for the h1 preparation from sigma-aldrich, and then diluted with nine volumes of 0.1 m ammonium hydrogen carbonate and digested with 1 mg trypsin/100 mg histone for 3 h. the digestion was interrupted by freezing the sample at –20�c. a 1-ml thawed sample was run on a thermo orbitrap velos (termo scientific, stockholm, sweden) in 0.1% (w/v) formic acid, using a gradient from 4% to 30% acetonitrile in 120 min, followed by a steep gradient to 48% in 10 min, and finally by a washing step at 80% acetonitrile. ms1 resolution was held at 60,000 (full width at half maximum), and fragments were measured between 300 and 1800 da. fragmentation was generated with collision-induced dissociation (cid). the lc-column was a thermo easy column c18 (100 mm � 75 mm and 3 mm beads) from termo scientific. buffer a was 0.1% formic acid in h2o, and buffer b was 0.1% formic acid in acetonitrile. the instrument was using lock-mass for improved mass accuracy. the resulting raw-format files were analyzed by xtandem over the web. beside the default settings, phosphorylated serine, threonine, and lysine were chosen as possible modifications. for ms analysis of phosphoarginine and arginine, a thermo velos equipment was used in infusion mode. a syringe pump delivered 3 ml/min of 50% methanol (no acid) into which the sample was diluted with four partsofsolvent.theinstrument’sstandardcapillaryinlet was used, and when the analysis seemed stable several manualrecordingsof1minweremade.thespectrawere averaged, and the 255.08 da peak was used for comparison of the plus with the minus dephosphorylation samples.tostrengthenfurthertheidentificationofphosphoarginine, a brief fragmentation was also recorded showing the characteristic loss of hpo3 (80 da). results phosphorylation of histone h1 in the pre-study of commercial histones mentioned in the introduction, the chemical phosphorylation by phosphoramidate was found to be highest for histone h1 from calf thymus. under the conditions used, all histone h1 preparations could be chemically phosphorylated to a level of 1–1.5 mol phosphate/mol protein. by amino acid analysis, performed by the amino acid analysis center at uppsala university, sweden, the histidine content of the histone h1 preparation from sigma-aldrich (iiis) was found to be less than 0.2 mol/mol protein and could thus not account for the observed level of acid-labile phosphorylation. this histone is obtained from the supernatant after precipitation of nuclear fraction with 5% trichloroacetic acid (37). the amino acid analysis data were compatible with reported primary structures of histones h1.2–5. therefore, this histone h1 preparation was judged to be suitable for the present phosphorylation/dephosphorylation experiments. although the content of arginine was found to be 4.4 mol/mol protein, the possibility of arginine phosphorylation by phosphoramidate can probably be excluded (38). since, however, a phosphorylation of peptide-bound lysine residues by phosphoramidate has been demonstrated (38), a phosphorylation of some of the numerous lysine residues (e.g. 59 lysine residues/mol protein in histone h1.2) was considered possible. compared to this high number of lysine residues, the phosphorylation of histone h1 is quite low. this can be explained by the initial concentrations used for phosphoramidate (3.9 mm) and histone h1 (0.05 mm), which are much lower than used for histidine-containing peptides (20,34) and lysine-containing peptides (38). the low initial concentration of phosphoramidate was, however, a deliberate choice in order not to exceed the binding capacity of the deae-sephacel spin columns used as described under methods. dephosphorylation by phpt1 of chemically phosphorylated histone h1 all preparations of phosphorylated histone h1 could be dephosphorylated by phpt1. one set of experiments is described in figure 1. the initial rate of the phpt1-catalyzed dephosphorylation was 1.0 ± 0.1 mol/s per mol of enzyme (mean and sd of seven separate experiments). this rate is in the same order of magnitude as previously described for the dephosphorylation of chemically phosphorylated histone h4 (35) and a chemically phosphorylated histidine-containing peptide (20). in the absence of phpt1, phosphorylated histone h1 was stable under the conditions used, i.e. the mean remaining phosphohistone h1 for seven separate experiments at 0, 5, and 10 min was 101% ± 2%. other commercial histone h1 preparations, phosphorylated to about 22 p. ek et al. the same degree, were dephosphorylated by phpt1 at similar initial rates (data not shown). phosphorylated polylysine and its dephosphorylation by phpt1 to confirm that protein-bound lysine can be n-e-phosphorylated by phosphoramidate and then dephosphorylated by phpt1, polylysine (30 kda) was investigated. when phosphorylated under conditions identical to those used for histone h1, a phosphorylation of 1.5–2 mol/mol polylysine was usually obtained. phosphopolylysine was dephosphorylated by phpt1 at a rate that was 70% ± 8% (n = 3) of that of phosphorylated histone h1 (figure 1). in the absence of phpt1, phosphopolylysine was stable under the conditions used, i.e. the mean remaining phosphopolylysine for three separate experiments at 0, 5, and 10 min was 100% ± 1%. to exclude the possibility that n-terminal n-aphospholysine in the 30 kda polylysine was the only substrate of phpt1, polylysine with a greater mean molecular size (90 kda) was investigated together with the 30 kda polylysine. under the conditions of this experiment, performed three times, the phosphorylation was 6.0 ± 2.6 and 1.6 ± 0.5 mol/mol protein for the 90 kda and 30 kda polylysine, respectively. upon extended incubation of both types of phosphorylated polylysines with phpt1, all phosphate was released (data not shown). this shows that beside any n-terminal phospholysine, several lysine residues were n-e-phosphorylated and could be dephosphorylated by phpt1. phpt1 and phosphoarginine no dephosphorylation of n-w-phosphoarginine by phpt1 could be detected, although the incubation time was 4-fold that under which phosphohistidine h1 and phosphopolylysine were significantly dephosphorylated. the intensity reported for the phosphoarginine peak was 1.33 � 106 for the control without dephosphorylation and 1.52 � 106 after attempting dephosphorylation, while the reported intensity for arginine was negligible. ms-sequencing of chemically phosphorylated histone h1: identification of phosphorylated lysine residues in order to control that the phosphorylation of histone h1 really occurred on lysine residues, the calf thymus histone h1 obtained from signalchem was phosphorylated to 1.5 mol phosphate/mol protein by phosphoramidate as described above and subjected to ms-sequencing after trypsination. this chromatographically purified histone h1 preparation was chosen in order to minimize the risk of interference with the lc-ms/ms. from the ms-data, this histone h1 was interpreted by the xtandem program to be histone h1c, which corresponds to histone h1.2 according to an alternative nomenclature (39). the high reliability of this interpretation is apparent from the log(e) for this assignment, which was –683 and defined as ‘the base-10 log of the expectation that any particular protein assignment was made at random (e-value)’. sequence data are given in figure 2. seven out of the 59 lysine residues were identified as targets for the chemical phosphorylation and are highlighted. 0 20 40 60 80 100 120 0 2 4 6 8 10 12 p ro te in -b o u n d a c id -l a b il e p h o s p h a te ( mm ) time (min) figure 1. decrease in phosphate in phosphoramidate phosphorylated histone h1 (&) and 30 kda polylysine (^) during incubation with phpt1. the concentration was 1 mg/ml of phosphohistone and 2 mg/ml of phosphopolylysine. an amount of 5 pmol phpt1 was added per 51 ml incubation volume. the reaction was performed at ph 7.5 and 30�c during indicated times and was interrupted by centrifugation of 50 ml of the reaction mixture through a spin column containing 200 ml of deaesepharose equilibrated in 25 mm tris/hcl ph 8.5. the protein-bound, acid-labile phosphate in the final eluate was analyzed as described under methods. each time point was analyzed in duplicate. 1 msetapaapa aappaektpv kkkaakkpag arrkasgppv 41 selitkavaa skersgvsla alkkalaaag ydveknnsri 81 klglkslvsk gtlvqtkgtg asgsfklnkk aatgeakpka 121 kkagaakpkk aagaakktkk atgaatpkkt akktpkkakk 161 paaaavtkkv akspkkakaa kpkkaaksaa kavkpkaakp 201 kvakpkkaap kkk figure 2. amino acid sequence and phosphorylated sites of bovine histone h1.2 as determined by mass spectrometry. lysine residues are marked red, and those identified as targets for the phosphorylation by phosphoramidate (i.e. seven residues) are highlighted. out of the 305 peptides that were used to identify the protein, 14 peptides were reported to contain phospholysine. phpt1 also dephosphorylates phospholysine 23 of the 305 tryptic peptides that were used to identify the protein, 14 were reported to contain phospholysine, and, of these, two contained phospholysine as the c-terminal residue (both representing lys-46). this seems to be incompatible with the substrate specificity of trypsin, and the interpretation of lys-46 as a target for phosphorylation may therefore be questioned. however, a phosphorylation of at least six different lysine residues seems to have been verified. since only a fraction of each target lysine would be phosphorylated at the low phosphoramidate concentration used, this number is compatible with the total phosphorylation of 1.5 mol/mol protein. ms spectra figure 3 shows the fragmentation pattern of a phospholysine-containing peptide representing residues 82–90 of histone h1.2. the peptide was selected as doubly charged, and if one follows the y-ion series an almostcompleteladderisfound(onlythec-terminalk is missing). the bound phosphate seems to be quite stable, and the expected phospho-fragments with higher mass are all present both as doubly and singly charged, implying that the phospholysine is capable of carrying a plus charge even if phosphorylated. the lysine-bound phosphate is removed between peaks 741.3 and 533.3 in the y-series, showing that the phosphorylated residue is lys-85. on top of the figure the small insert depicts the yand b-ions series in a miniformat, where the size of each staple is proportional to the abundance of the corresponding fragment in the main spectrum. the main spectrum also shows peaks corresponding to 18 da-losses of both yand b-ions. discussion the present study shows that phosphohistidine phosphatase (phpt1) can dephosphorylate a chemically phosphorylated histone h1 that does not contain histidine residues. this is explained by the finding that some of the lysine residues of histone h1 were phosphorylated during the incubation with phosphoramidate and became good substrates of phpt1. itisknownthatpeptide-bound lysineresiduescanbe n-e-phosphorylatedbyphosphoramidate(38).asseen from the sequencing of the phosphorylated histone h1.2 by ms (figure 2), several lysine residues were identified as targets for the phosphoramidate. kowalewska et al. found that fragmentation of phospholysine peptides by cid gave a too low number of fragments to establish the phosphorylation site and suggested that this was caused by extensive neutral losses of hpo3 (80 da) and water (18 da) (38). however,interpretationofthespectruminfigure3suggests that not all phospholysine-containing peptides show this extensive loss of hpo3 and water since it showed a sufficientnumberofphosphorylatedfragmentstoallow the identification of the phosphorylated site. it is worth noting that most of the lysine residues of histone h1, that were identified as targets for phosphoramidate, are also subject to natural posttranslational modifications (39,40). therefore, it is reasonable to suppose that some of these sites may be l l l k 533.3 7 4 1 .3 7 9 1 .3 9 1 1 .4 7 7 3 .4 8 9 3 .5 8 7 8 .4 7 2 3 .46 7 4 .4 6 9 2 .4 5 9 5 .2 5 7 9 .3 5 6 1 .3 4 9 4 .7 4 6 6 .3 4 4 6 .2 3 3 3 .1 2 8 4 .2 2 3 4 .1 2 1 6 .1 4 2 7 .7 4 9 2 .2 5 0 3 .7 4 5 6 .2 svskg 100 80 60 40 20 0 0 200 400 600 800 1000 –0.4 0.4 error (da)m/z r i figure 3. ms/ms spectrum showing the fragmentation pattern of one of the peptides obtained after trypsin treatment of histone h1 from signalchem. the y-ion series is shown in red, and the b-ions series is in blue. also shown is yand b-ions fitting with the loss of 18 da in violet and turquoise, respectively. other observed but unspecified mached ions are grey. the small inset at the top shows only the yand b-ions, with the length of the staples representing the intensity of the ions. 24 p. ek et al. accessible also to enzyme-catalyzed phosphorylations, e.g. by histone h1 kinases, such as those described by smith and co-workers (41-44) and sikorska et al. (45). the same sites, when phosphorylated, may then be accessed also by phpt1. one prerequisite for this to happen would be that lysine-phosphorylated histone h1, usually located in the nuclei, and phpt1, usually located in the cytosol, can meet at least temporarily in the same cell compartment. interestingly, it has been shown by immunohistochemical methods that part of the phpt1 can be located also in the nuclei (www.proteinatlas.org/search/phpt1) (46). phpt1 could also dephosphorylate phosphopolylysine at a rate similar to that of the phosphorylated histone h1. whether this means that a phospholysine residue itself, rather than the amino acid sequence around the phospholysine, defines the substrate specificity of phpt1 may be a subject of future investigations. of interest in this context is the finding by attwood et al. that the kinetics of the dephosphorylation of short phosphohistidine-containing peptides depends on the amino acid sequence around the phosphohistidine (34). a free a-amino group of an n-terminal amino acid may be phosphorylated by phosphoramidate (47), although to a lesser extent than the e-amino group of lysine residues (38). we therefore cannot entirely exclude the possibility of an n-terminal phosphorylation of histone h1 or polylysine. however, n-ephosphorylation actually occurred of histone h1, as judged from the ms-spectra. a similar phosphorylation occurred of polylysine, as judged from the finding that the phosphorylation increased almost four times when a 90 kda polylysine was compared to a 30 kda polylysine as phosphate acceptor. the finding that phpt1 shows activity toward phospholysine in addition to phosphohistidine adds to the fact that phpt1 can also dephosphorylate free phosphoramidate (20,34). this activity towards phosphoramidate actually made attwood and wieland suggest, in a recent review, that phpt1 (php) ‘may not be quite so specific for phosphohistidine’ (48). the enzyme is, however, not active against all n–p bonds, since it did not cleave n-w-phosphoarginine under conditions used in the present study. this result raises the question of whether phpt1 is in fact similar to, or even identical with, the partially characterized 13 kda phosphoamidase described in 1999 by hiraishi et al. (49). according to these authors, the latter enzyme dephosphorylated ndpk that was autophosphorylated on a histidine residue. this is, however, in contrast to a result by klumpp et al. who, in their studies on the dephosphorylation of atp citrate lyase, did not obtain any dephosphorylation of autophosphorylated ndpk by phpt1 (25). therefore, the current evidence suggests that the two enzymes are not identical. other phosphatases, with activity against phospholysine, have also been reported. comprehensive reviews on this matter have been published (50,51). in addition to the 13 kda phosphatase, hiraishi et al. have studied a bovine liver 56 kda phosphatase that is active toward free phosphohistidine and phospholysine (52). the corresponding human enzyme has been cloned and further characterized, and the chromosomal location of its gene was determined to be 10q26.13 (53). according to a blast/align search at www.ncbi.nlm.nih.gov its amino acid sequence shows no similarity to that of phpt1. in addition, the phpt1 gene has a different chromosomal location, i.e. 9q34.3 (20). wong et al. (54,55) investigated the presence of phospholysine phosphatase activity in soluble rat tissue extracts and in a partially purified form by using phosphorylated polylysine as the probe. interestingly, the activity against this substrate in extracts from rat liver corresponds to the activity of phpt1 in pig liver cytosol, as measured with a phosphohistidine peptide as the substrate (20). to our knowledge, further purification and characterization of the enzyme described by wong et al. have not been reported, and its identity with phpt1 is thus unknown. in conclusion, the results of the present study suggest that phpt1 would be active against several phospholysine-containing proteins in vivo. with the exception of histone h1, which is already shown to be enzymatically phosphorylated in vivo and in vitro (41-44), such proteins remain to be identified. we assume that the well-characterized phosphohistidine phosphatase phpt1 will become a valuable tool in the investigation of the role also of phospholysine in eukaryotic regulatory protein phosphorylation. the value of this tool may add to future characterizations of putative lysine kinases and to new phosphoproteomic approaches of the type recently outlined for phosphorylated lysine peptides (56). acknowledgements gunilla pettersson and elvy netzel are gratefully acknowledged for performing some of the experiments in this work. funding the work was supported by the medical faculty of uppsala university. phpt1 also dephosphorylates phospholysine 25 http://www.proteinatlas.org/search/phpt1 http://www.ncbi.nlm.nih.gov declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. boyer pd, deluca m, ebner ke, hultquist de, peter jb. identification of phosphohistidine in digests from a probable intermediate of oxidative phosphorylation. j biol chem. 1962; 237:pc3306–8. 2. kee jm, muir tw. chasing phosphohistidine, an elusive 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its dephosphorylation by phpt1 phpt1 and phosphoarginine ms-sequencing of chemically phosphorylated histone h1 identification of phosphorylated lysine residues ms spectra discussion funding declaration of interest references neoplastic pericarditis as the initial manifestation of a papillary thyroid carcinoma full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 neoplastic pericarditis as the initial manifestation of a papillary thyroid carcinoma nikolaos tsoukalas, ioannis d. kostakis, stamatina demiri, georgios koumakis, vasileios barbounis, kalypso barbati & anna efremidis to cite this article: nikolaos tsoukalas, ioannis d. kostakis, stamatina demiri, georgios koumakis, vasileios barbounis, kalypso barbati & anna efremidis (2013) neoplastic pericarditis as the initial manifestation of a papillary thyroid carcinoma, upsala journal of medical sciences, 118:3, 196-198, doi: 10.3109/03009734.2013.801541 to link to this article: https://doi.org/10.3109/03009734.2013.801541 © informa healthcare published online: 23 may 2013. submit your article to this journal article views: 437 view related articles citing articles: 2 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.801541 https://doi.org/10.3109/03009734.2013.801541 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.801541 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.801541 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.801541#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.801541#tabmodule upsala journal of medical sciences. 2013; 118: 196–198 case report neoplastic pericarditis as the initial manifestation of a papillary thyroid carcinoma nikolaos tsoukalas1,3, ioannis d. kostakis2, stamatina demiri3, georgios koumakis3, vasileios barbounis3, kalypso barbati4 & anna efremidis3 1department of medical oncology, ‘401’ general military hospital, athens, greece, 2second department of propedeutic surgery, ‘laiko’ general hospital, national and kapodistrian university of athens, medical school, athens, greece, 3second department of medical oncology, ‘agios savvas’ anticancer hospital, athens, greece, and 4department of pathology, hellenic red cross hospital ‘korgialeneio-benakeio’, athens, greece abstract neoplastic pericarditis represents approximately 5%–7% of the cases with acute pericarditis and is rarely the initial manifestation of malignancy. the most common cause is lung cancer, followed by breast cancer, lymphomas, leukemia, and esophageal cancer. neoplastic pericardial disease is extremely rare in thyroid cancer, especially as the first manifestation. here, we present a papillary thyroid carcinoma that was manifested with pericarditis and cardiac tamponade in a 49-year-old female. key words: cardiac tamponade, neoplastic pericarditis, pericardial effusion, thyroid cancer introduction neoplastic pericarditis represents approximately 5%–7% of the cases with acute pericarditis (1-4). theoretically, any malignant tumor may cause pericarditis/pericardial effusion (1,2) through direct extension or metastasis via lymphatic or blood vessels into the pericardium (3,5). however, the most common malignancy causing pericarditis/pericardial effusion is lung cancer, followed by breast cancer, lymphomas, leukemia, and esophageal cancer. primary neoplasms of the pericardium, such as mesothelioma, are very rare (1-3,5). only few cases of neoplastic pericarditis in patients with thyroid cancer have been reported in the literature. here, we present a case of papillary thyroid carcinoma that was manifested with pericarditis and cardiac tamponade. case report a 49-year-old female, who was a heavy smoker, but with negligible previous medical history, was admitted to a hospital due to stridor, dyspnea, cough, and hoarseness. physical examination revealed no pathological signs, blood tests were normal, and the patient was treated with corticosteroids without a definite diagnosis. ten days later, she developed epigastric pain that radiated to the left scapula and the left supraclavicular fossa. she was admitted to a hospital, where an echocardiogram and a chest ct scan revealed the presence of pericardial effusion (figure 1). during her hospitalization, she developed cardiac tamponade, for which she urgently underwent pericardiocentesis. blind biopsies were taken from the pericardium and the upper lobe of the left lung, which showed malignant cells forming papillae and invading lymphatic and blood vessels (figure 2a). however, it was not possible to identify the primary origin of the neoplasm, despite the thorough investigation with abdominal ct scan and technetium-99m-mdp bone scan. in addition, blood tests were normal, apart from the values of cea (20.6 ng/ml (normal value: <4.7 ng/ml)) and ca-125 (341.4 iu/ml (normal correspondence: nikolaos tsoukalas, md, msc, medical oncologist, department of medical oncology, ‘401’ general military hospital, 10-12 gennimata n. street, 11524 ampelokipi, athens, greece. fax: +30 2107494095. e-mail: tsoukn@yahoo.gr (received 16 march 2013; accepted 29 april 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.801541 http://informahealthcare.com/journal/ups mailto:tsoukn@yahoo.gr value: <34 iu/ml)). the patient received one cycle of chemotherapy with carboplatin 5auc, docetaxel 60 mg/m2, and bevacizumab 5 mg/kg for a neoplasm of unknown primary origin, which improved her symptoms. afterwards, she was admitted to our department for further investigation and treatment. the physical examination revealed a palpable nodule of the right lobe of the thyroid gland, and the ultrasound demonstrated diffuse heterogeneity of the same lobe. nevertheless, the ultrasound failed to reveal any specific suspicious area, and the serum concentration of thyroglobulin was normal (17.7 ng/ml (normal value: <78 ng/ml)). the cytological examination after a blind fine-needle aspiration biopsy of the right lobe of the thyroid gland revealed the presence of a papillary thyroid carcinoma. subsequently, a cervical mri was performed, demonstrating extensive lymphadenopathy. the patient underwent total thyroidectomy and bilateral lymph node dissection. the histological examination verified the presence of a stage ivc (t3n1bm1) papillary carcinoma with high-grade (grade iii) malignancy and sporadic anaplastic changes, lymphovascular and perineural invasion, and metastatic infiltration of the cervical lymph nodes (figure 2b). a reexamination of the pericardial fluid from the pericardiocentesis revealed the expression of thyroglobulin by the malignant cells. an i131 scan was also performed, but without demonstrating abnormal i131 uptake. the patient’s treatment was continued with the use of the same chemotherapy regimen for a total of six cycles without any major adverse events and with excellent tolerance. after the completion of chemotherapy, a fdgpet scan was performed with negative results. afterwards, the follow-up was based on physical examination, blood tests, and imaging examinations. unfortunately, the disease relapsed six months later with a very aggressive clinical course due to lung metastases and cardiac tamponade. it was not possible to administer any anticancer treatment at that time, and she passed away due to respiratory and cardiac failure. discussion as mentioned previously, approximately 5%–7% of pericardial effusions have a malignant origin (1-4). most patients with neoplastic pericardial disease have an already diagnosed malignancy (3). acute pericarditis or pericardial effusion is the initial manifestation of malignancy in about 4% of the cases with pericardial disease (1,3), but this percentage rises to approximately 20% in large, symptomatic effusions without obvious origin (1,5), as in our case. risk factors for malignant etiology are cardiac tamponade at presentation, as in our case, recurrent or incessant pericarditis, no response to non-steroidal anti-inflammatory drugs, and a history of malignancy (1-3). cytological examination of pericardial fluid or pericardial biopsy is required for a diagnosis to be made (1-3,5). however, pericardial effusion is detected in only a b figure 2. histological images with malignant cells from the papillary thyroid carcinoma. a: biopsy of the pericardium (hematoxylin and eosin stain, 100�); b: histological examination of the thyroid gland (hematoxylin and eosin stain, 200�). figure 1. chest ct scan in which the pericardial effusion is revealed. thyroid cancer presented with neoplastic pericarditis 197 12%–25% of patients with metastasis to the pericardium, but cardiac tamponade, which developed in our patient, is relatively rare among them (5). neoplastic pericardial disease is extremely rare in thyroid cancer, especially as the initial manifestation. several cases have been reported in the literature (6-16). neoplastic pericarditis (6,9), massive pericardial effusion (13), or cardiac tamponade (7,8,10,12,16) have been reported as the first manifestation of a previously occult thyroid cancer. on the other hand, malignant pericardial effusion (14,15) or cardiac tamponade (11) have also been diagnosed in patients with already diagnosed thyroid cancer. most cases have pertained to papillary carcinoma (7,9-11,14-16), as in our patient, whereas a follicular (13) and a mucinproducing carcinoma (8) have also been described as causes of neoplastic pericardial disease. conclusions neoplastic pericardial disease is most commonly caused by lung cancer, followed by breast cancer, lymphomas, leukemia, and esophageal cancer (1-3,5), and is rarely the first manifestation of malignancy (1,3). however, thyroid cancer, and especially papillary carcinoma, should be included in the differential diagnosis of pericarditis/pericardial effusion. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. imazio m, brucato a, derosa fg, lestuzzi c, bombana e, scipione f, et al. aetiological diagnosis in acute and recurrent pericarditis: when and how. j cardiovasc med (hagerstown). 2009;10:217–30. 2. imazio m, spodick dh, brucato a, trinchero r, markel g, adler y. diagnostic issues in the clinical management of pericarditis. int j clin pract. 2010;64:1384–92. 3. imazio m, brucato a, mayosi bm, derosa fg, lestuzzi c, macor a, et al. medical therapy of 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www.ncbi.nlm.nih.gov/pubmed/11332365?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15698739?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15698739?dopt=abstract abstract introduction case report discussion conclusions declaration of interest references serum gamma glutamyl transferase and alanine transaminase concentrations predict endothelial dysfunc full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 serum gamma glutamyl transferase and alanine transaminase concentrations predict endothelial dysfunction in patients with non-alcoholic steatohepatitis huseyin arinc, bahadir sarli, ahmet oguz baktir, hayrettin saglam, erkan demirci, yasemin dogan, serkan kurtul, hatice karaman, abdulsamet erden & ahmet karaman to cite this article: huseyin arinc, bahadir sarli, ahmet oguz baktir, hayrettin saglam, erkan demirci, yasemin dogan, serkan kurtul, hatice karaman, abdulsamet erden & ahmet karaman (2013) serum gamma glutamyl transferase and alanine transaminase concentrations predict endothelial dysfunction in patients with non-alcoholic steatohepatitis, upsala journal of medical sciences, 118:4, 228-234, doi: 10.3109/03009734.2013.814734 to link to this article: https://doi.org/10.3109/03009734.2013.814734 © informa healthcare published online: 08 jul 2013. submit your article to this journal article views: 799 view related articles citing articles: 18 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.814734 https://doi.org/10.3109/03009734.2013.814734 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.814734 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.814734 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.814734#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.814734#tabmodule upsala journal of medical sciences. 2013; 118: 228–234 original article serum gamma glutamyl transferase and alanine transaminase concentrations predict endothelial dysfunction in patients with non-alcoholic steatohepatitis huseyin arinc1, bahadir sarli1, ahmet oguz baktir1, hayrettin saglam1, erkan demirci1, yasemin dogan1, serkan kurtul1, hatice karaman2, abdulsamet erden3 & ahmet karaman3 1department of cardiology, kayseri education and research hospital, kayseri, turkey, 2department of pathology, kayseri education and research hospital, kayseri, turkey, and 3department of gastroenterology, kayseri education and research hospital, kayseri, turkey abstract purpose. cardiovascular diseases are the leading cause of death in patients with non-alcoholic steatohepatitis (nash). we aimed to investigate the presence of endothelial dysfunction and whether serum concentrations of liver enzymes may reflect the severity of such an endothelial dysfunction in patients with nash. methods. fifty patients with nash diagnosed by liver biopsies and 30 healthy controls were included. blood samples after fasting were harvested for measurements of glucose, insulin, cholesterol, triglyceride, and liver enzymes. all patients underwent transthoracic echocardiography and brachial and carotid artery doppler ultrasonography to evaluate flow-mediated dilatation (fmd) and carotid artery intima-media thickness (cimt). results. patients with nash had impaired fmd (4.9 ± 2.8% to 9.3 ± 4.4%, p < 0.001) and higher cimt (0.79 ± 0.16 mm to 0.64 ± 0.11 mm, p < 0.001) when compared with healthy controls. linear regression analyses revealed that serum concentrations of gamma glutamyl transferase (ggt) and alanine transaminase (alt) were associated with fmd and cimt. conclusions. patients with nash have impaired fmd and increased cimt when compared with healthy controls. in patients with nash, serum concentrations of ggt and alt might have a predictive value for fmd and cimt. key words: alanine transaminase, endothelial dysfunction, gamma glutamyl transferase, non-alcoholic steatohepatitis introduction non-alcoholic fatty liver disease (nafld) is one of the most common causes of elevated liver enzymes and chronic liver disease in western countries (1). patients with elevated liver enzymes in the absence of alcohol consumption and secondary causes of liver disease are described as having nafld (2). non-alcoholic steatohepatitis (nash) is a more severe form of nafld. it is characterized by steatosis, lobular inflammation, ballooning, and fibrosis in liver biopsies, which is the gold standard for diagnosis of nash (3). the prevalence of cardiovascular disease and death from cardiovascular causes is increased in patients with nafld (4). carotid intima-media thickness and presence of coronary plaques were also increased in patients with nafld (5,6). although the association between cardiovascular diseases and nafld is well known, data concerning the role of endothelial dysfunction in patients with nash—a more severe subgroup of nafld—are limited. endothelial dysfunction is associated with an increased risk of myocardial infarction, coronary revascularization, and cardiovascular death (7). measurements of flow-mediated dilatation of the brachial correspondence: bahadir sarli, md, kayseri education and research hospital, department of cardiology, 38010, kayseri, turkey. fax: +90 352 3207313. e-mail: drsarli@yahoo.com (received 21 march 2013; accepted 6 june 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.814734 http://informahealthcare.com/journal/ups mailto:drsarli@yahoo.com artery with two-dimensional and doppler ultrasonography have been used for the diagnosis of endothelial dysfunction, and a high correlation with angiographic results has been demonstrated (8). in this study we aimed to investigate the presence of endothelial dysfunction and the role of liver enzymes in predicting endothelial function in patients with nash. methods study population fifty patients previously diagnosed with nash (27 male and 23 female, mean age 42 ± 9 years) by liver biopsies in the gastroenterology department of kayseri education and research hospital between may 2010 and april 2012 were included. the indication for liver biopsy in these patients had been elevated liver enzymes in blood tests over 6 months. all patients underwent transthoracic echocardiography and brachial and carotid artery doppler studies after careful physical examination. also, blood samples after fasting were harvested for biochemical analyses, including lipids, glucose, insulin, creatinine, aspartic acid transaminase (ast), alanine transaminase (alt), and gamma glutamyl transferase (ggt) concentrations (olympus au-640 analyser, mishima olympus co. ltd, shizuoka, japan). all blood tests were repeated three times, and mean values were used for statistical analyses. to diminish any confounders that might influence endothelial function, patients with established coronary artery disease (cad), peripheral arterial disease, hypertension, hyperlipidemia, diabetes, a history of smoking, and use of anti-hyperlipidemic drugs were excluded. thirty healthy volunteers, ageand sex-matched (19 male and 11 female, mean age 41 ± 6 years), were selected as a control group. flow-mediated dilatation and carotid intima-media thickness endothelial dysfunction was evaluated using brachial artery flow-mediated dilatation (fmd). brachial artery fmd was measured with a high-frequency (7.0–13.0 mhz) ultrasound scanning probe to obtain longitudinal images of the brachial artery at a point 5 to 10 cm proximal to the antecubital fossa (siemens medical sol, mountain view, ca, usa). all scans were performed by the same sonographer. twodimensional images were obtained at baseline with doppler ultrasound scanning to assess arterial diameter. angle correction software was used during doppler imaging to approximate a 20-degree angle of incidence to blood flow. increased shear stress was achieved by producing reactive hyperemia. a pressure cuff placed on the right forearm was inflated up to 50 mmhg higher than systolic blood pressure for 5 minutes in order to induce ischemia by occluding arterial flow. arterial diameter measurements were repeated within 60 seconds of cuff deflation. arterial diameter was measured from the right arm at the intima-media interface of the clearest echocardiography line. images were acquired with electrocardiogram gating, with measurements made in end diastole corresponding to the onset of the r wave. measurements were reported as % change in diameter. carotid artery intima-media thickness measurements were performed in the supine position with the neck extended and the chin turned away from the side being examined. the right and left common carotid arteries were imaged proximal to the bulb in multiple longitudinal planes for clearest resolution of the intima-media thickness (imt) of the far wall. the mean imt was obtained by manually tracing the intima-media in the far wall of the artery. measurements were performed on three end diastolic images and averaged. statistical analysis all analyses were carried out using spss 15.0 for windows (spss inc., chicago, il, usa). continuous variables were given as mean ± standard deviation; categorical variables were defined as percentages. the variables were investigated using the kolmogorov–smirnov test to determine whether or not they were normally distributed. the t test was used to compare continuous variables between the two groups. non-parametric values were compared with mann–whitney u test. chi-square test was used to compare categorical data. a multiple linear regression model was used to identify the statistical significance of relationships between selected variables and fmd and carotid artery intima-media thickness (cimt). the model fit was assessed using appropriate residual and goodness-of-fit statistics. a twotailed p value < 0.05 was considered as statistically significant. results fifty patients with nash and 30 healthy volunteers were included (table i). there were no differences between patients and controls in terms of age and gender. moreover, concentrations of serum glucose, total cholesterol, low-density lipoprotein (ldl) cholesterol, high-density lipoprotein (hdl) cholesterol, triglyceride, and creatinine were similar. however, serum concentrations of ggt, ast, alt, and insulin were elevated in the nash patients. likewise, the endothelial dysfunction in nash 229 homeostasis model assessment (homa) index and body mass index were higher in patients with nash when compared with controls (table i). doppler studies revealed that patients with nash had an impaired flow-mediated dilatation when compared with the controls (table i). furthermore, carotid artery sonographic imaging showed that cimt was higher in patients with nash. linear regression analysis showed that serum ggt and alt concentrations were significantly associated with fmd (ggt: coefficient b: –0.36, p = 0.007; and alt: coefficient b: –0.43, p = 0.002) and cimt (ggt: coefficient b: 0.40, p = 0.021; and alt: coefficient b: 0.38, p = 0.027) (tables ii and iii). also, correlation analyses showed that there was a statistically significant correlation between serum concentrations of ggt and alt and fmd and cimt, respectively (figures 1 and 2). discussion non-alcoholic steatohepatitis is a more severe subgroup of nafld which ranges from simple steatosis to steatohepatitis, advanced fibrosis, and cirrhosis. nafld is defined histologically in liver biopsies by exhibiting macrovesicular steatosis, lobular inflammation, balloon degeneration of hepatocytes, and zone 3 pericellular fibrosis (9). despite sampling variability and inter-observer discordance, a liver biopsy is the gold standard for diagnosis of nash (10). patients with nash are more prone to develop cad than patients with simple steatosis; however, it is still unclear whether liver enzymes could be used as specific markers of endothelial dysfunction in these patients. table i. comparison of baseline demographics, laboratory features, and doppler studies of patients with non-alcoholic steatohepatitis and controls. nash (n = 50) controls (n = 30) p value age (years) 42 ± 9 41 ± 6 0.686 gender (male/female) 27/23 19/11 0.414 bmi (kg/m2) 30 ± 4 28 ± 3 0.004 glucose (mg/dl) 90 ± 12 92 ± 8 0.662 total cholesterol (mg/dl) 221 ± 36 205 ± 38 0.065 ldl cholesterol (mg/dl) 126 ± 27 119 ± 39 0.359 hdl cholesterol (mg/dl) 42 ± 6 43 ± 7 0.782 triglyceride (mg/dl) 172 ± 57 174 ± 33 0.835 creatinine (mg/dl) 0.8 ± 0.3 0.9 ± 0.3 0.325 ggt (u/l) 55 ± 18 36 ± 20 <0.001 ast (u/l) 48 ± 10 30 ± 6 <0.001 alt (u/l) 61 ± 22 45 ± 13 0.001 insulin (mu/ml) 13 ± 3 5 ± 2 <0.001 homa-ir 3.3 ± 1.5 1.5 ± 0.3 <0.001 ejection fraction (%) 65 ± 6 66 ± 5 0.183 ba baseline diameter (mm) 40 ± 6 39 ± 4 0.172 fmd (%) 4.9 ± 2.8 9.3 ± 4.4 <0.001 cimt (mm) 0.79 ± 0.16 0.64 ± 0.11 <0.001 ast = aspartic acid transaminase; alt = alanine transaminase; ba = brachial artery; bmi = body mass index; cimt = carotid intima-media thickness; fmd = flow-mediated dilatation; ggt = gamma glutamyl transferase; hdl = high-density lipoprotein; ldl = low-density lipoprotein. table ii. linear regression analysis showing relationships between several variables and flow-mediated dilatation of brachial artery. coefficient b p value age –0.09 0.298 bmi 0.01 0.958 ast –0.09 0.335 alt –0.43 0.002 ggt –0.36 0.007 insulin –0.05 0.610 homa index –0.09 0.414 total cholesterol 0.16 0.846 230 h. arınc et al. elevated serum ggt enzyme activity has been widely used as a marker of alcohol abuse and liver dysfunction including nafld. the concentration in serum of ggt is a highly sensitive laboratory measurement for the presence of nafld (11,12). banderas et al. showed that serum ggt and triglycerides and obesity were predictors of nafld in patients with the metabolic syndrome (13). alanine aminotransferase is a simple marker for nafld and has been shown to be associated with liver fat accumulation as measured by magnetic resonance (mr) proton spectroscopy (14,15). dixon et al. found that alt was an independent predictor for the presence of nash and advanced pericellular fibrosis in liver biopsies of 105 obese patients undergoing obesity surgery (16). accumulating data suggest that ggt and alt also might be markers of endothelial dysfunction and atherosclerosis besides their role in nafld. the serum ggt concentration was shown to be significantly associated with endothelial dysfunction, extensive atherosclerotic cardiovascular involvement, and adverse cardiac events and mortality during the course of acute coronary syndromes (17-19). several studies investigating the role of ggt in stable cad, acute coronary syndrome, and st segment elevation myocardial infarction revealed that ggt is an independent predictor of shortand long-term mortality (19,20). similarly, serum alt concentrations were also found to be associated with endothelial dysfunction as measured by fmd and cimt in patients with diabetes (13,21). however, in patients with nash relations between alt and endothelial dysfunction have previously not been studied. brachial artery fmd is a simple and readily available tool for assessing endothelial function (22). impaired fmd is an early sign of subclinical atherosclerosis and may be useful in identifying patients carrying a high risk of complications of atherosclerotic vascular diseases (23). it is well known that, in patients with nafld, cad is the leading cause of table iii. linear regression analysis showing relationships between several variables and carotid artery intima-media thickness. coefficient b p value age 0.09 0.415 bmi 0.09 0.401 ast 0.18 0.133 alt 0.38 0.027 ggt 0.40 0.021 insulin 0.03 0.821 homa index 0.09 0.536 total cholesterol 0.01 0.902 12.00 10.00 8.00 6.00 r = 0.948 p < 0.001 r = 0.883 p < 0.001 a b 4.00 2.00 0.00 12.00 10.00 8.00 6.00 4.00 2.00 0.00 20.00 40.00 60.00 80.00 20.00 40.00 60.00 80.00 100.00 f lo w m e d ia te d d il a ta ti o n ( % ) f lo w m e d ia te d d il a ta ti o n ( % ) gamma glutamyl transferase (u/l) alanine transaminase (u/l) figure 1. in patients with non-alcoholic steatohepatitis, serum gamma glutamyl transferase and alanine transaminase concentrations were negatively correlated with flow-mediated dilatation of the brachial artery. a: graphics showing correlation between gamma glutamyl transferase concentration and flow-mediated dilatation. b: graphics showing correlation between alanine transaminase concentration and flow-mediated dilatation. endothelial dysfunction in nash 231 that, followed by hepatic malignancy and cirrhosis (24). in patients with nafld, impaired fmd of brachial artery and increased cimt have been reported in several studies (25,26). however, cimt and fmd have previously not been studied in patients with nash. in our study serum concentrations of both ggt and alt were found to be predictors of endothelial function in patients with nash. several mechanisms have been employed in this association linking ggt and alt with both nash and endothelial dysfunction. first, increased serum concentrations of ggt and alt reflect oxidative stress, which may play a role in the development of both nash and endothelial dysfunction. ggt acts as a mediator in the transportation of extracellular glutathione into most types of cells. production of free radicals leads to reduction in glutathione and induces ggt to protect glutathione levels (27). serum ggt concentrations usually increase as a response to pre-existing oxidative stress. results from the cardia study showed that a high serum ggt concentration is a result of pre-existing oxidative stress and ggt is expressed as a cellular defender against oxidative stress (28). high serum concentrations of ggt documented in patients with coronary artery disease are thought to be a result of oxidative stress active in the formation of atherosclerotic plaque (29-31). however, oxidative stress also plays a major role in the progression of nafld to nash (32). lipid peroxidation products and 8-hydroxydeoxyguanosine were demonstrated in both plasma and liver biopsies of patients with nash, indicating contribution of oxidative stress to progression of liver disease (33). additionally, elevation in alt concentrations represent fat deposition in liver and other organs with non-adipose tissues, and an increase of intracellular triglyceride in these tissues might be a mediator for production of reactive oxygen species which may further lead to organ dysfunction in both liver and vascular endothelium (34). in our study, increased serum concentrations of ggt and alt in patients with nash and impaired fmd suggest that pre-existing oxidative stress plays a role in the development and progression of both nash and endothelial dysfunction. moreover, high serum ggt and alt concentrations might also reflect the inflammatory state which may contribute to the development of endothelial dysfunction and fibrosis in nash (35,36). results of a previous study conducted by lee and colleagues showed that ggt is a component of subclinical inflammation, which is also involved in the pathogenesis of nash and atherosclerosis (37,38). in a previous study which included patients with the metabolic syndrome, kerner et al. showed that high serum alt concentrations were correlated with high c-reactive protein levels. the authors concluded that hepatic a 1.1 1 0.9 0.8 0.7 0.6 0.5 20.00 40.00 60.00 c a ro ti d i n ti m a -m e d ia t h ic k n e s s ( m m ) 80.00 r = 0.870 p < 0.001 gamma glutamyl transferase (u/l) b 1.1 0.9 1 0.8 0.7 0.6 0.5 c a ro ti d i n ti m a -m e d ia t h ic k n e s s ( m m ) 20.00 40.00 60.00 80.00 100.00 r = 0.855 p < 0.001 alanine transaminase (u/l) figure 2. in patients with non-alcoholic steatohepatitis, serum gamma glutamyl transferase and alanine transaminase concentrations were highly correlated with the carotid intima-media thickness. a: graphics showing correlation between serum gamma glutamyl transferase concentration and carotid intima-media thickness. b: graphics showing correlation between serum alanine transaminase concentration and carotid intima-media thickness. 232 h. arınc et al. inflammation as a result of hepatic steatosis might be a potential contributor to low-grade systemic inflammation seen in patients with nash (35). this study has some limitations. first, the number of patients included is relatively small to draw definite conclusions from. however, we included a homogeneous, biopsy-proven nash population. second, the absence of measurements of oxidative and inflammatory markers is also a limitation. nevertheless, we conclude that patients with nash have impaired fmd and increased cimt when compared with healthy controls. in patients with nash, serum concentrations of ggt and alt might have a predictive value for fmd and cimt, and these biomarkers might therefore be useful for the demonstration of endothelial dysfunction. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. torres dm, harrison sa. diagnosis and therapy of nonalcoholic steatohepatitis. gastroenterology. 2008;134:1682–98. 2. adams la, angulo p. recent concepts in non-alcoholic fatty liver disease. diabet med. 2005;22:1129–33. 3. matteoni ca, younossi zm, gramlich t, boparai n, liu yc, mccullough aj. nonalcoholic fatty liver 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www.ncbi.nlm.nih.gov/pubmed/21200101?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21655948?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21655948?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11950797?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11950797?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/11818477?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15499043?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15499043?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15499043?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21041983?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21041983?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15694941?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/15694941?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12881453?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/12881453?dopt=abstract abstract introduction methods study population flow-mediated dilatation and carotid intima-media thickness statistical analysis results discussion declaration of interest references trust versus concerns—how parents reason when they accept hpv vaccination for their young daughter full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 trust versus concerns—how parents reason when they accept hpv vaccination for their young daughter maria gottvall, maria grandahl, anna t. höglund, margareta larsson, christina stenhammar, bengt andrae & tanja tydén to cite this article: maria gottvall, maria grandahl, anna t. höglund, margareta larsson, christina stenhammar, bengt andrae & tanja tydén (2013) trust versus concerns—how parents reason when they accept hpv vaccination for their young daughter, upsala journal of medical sciences, 118:4, 263-270, doi: 10.3109/03009734.2013.809039 to link to this article: https://doi.org/10.3109/03009734.2013.809039 © informa healthcare published online: 19 jun 2013. submit your 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gottvall1*, maria grandahl1*, anna t. höglund1, margareta larsson2, christina stenhammar1, bengt andrae3 & tanja tydén1 1department of public health and caring sciences, uppsala university, uppsala, sweden, 2department of women’s and children’s health, uppsala university, uppsala, sweden, and 3centre for research and development, uppsala university/county council of gävleborg, gävle, sweden abstract background. from spring of 2012, human papillomavirus (hpv) vaccine against cervical cancer is offered free of charge to all girls aged 10–12 years through a school-based vaccination programme in sweden. the aim of this study was to explore how parents reason when they accept hpv vaccination for their young daughter and also their views on hpv-related information. methods. individual interviews with parents (n = 27) of 11–12-year-old girls. the interviews were recorded, transcribed verbatim, and analysed using thematic content analysis. results. three themes emerged through the analysis: trust versus concern, responsibility to protect against severe disease, and information about hpv and hpv vaccination is important. the parents expressed trust in recommendations from authorities and thought it was convenient with school-based vaccination. they believed that cervical cancer was a severe disease and felt a responsibility to protect their daughter from it. some had certain concerns regarding side effects and vaccine safety, and wished for a dialogue with the school nurse to bridge the information gaps. conclusions. trust in the recommendations from authorities and a wish to protect their daughter from a severe disease outweighed concerns about side effects. a school-based vaccination programme is convenient for parents, and the school nurse has an important role in bridging information gaps. the findings from this qualitative study cannot be generalized; however, it can provide a better understanding of how parents might reason when they accept the hpv vaccination for their daughter. key words: decision-making, hpv vaccination, parents, school-based vaccination, school nurses introduction several countries have included human papillomavirus (hpv) vaccine against cervical cancer as part of the general child vaccination programmes for girls between 10 and 14 years old (1,2). the rationale for publicly funded vaccination programmes is to achieve a high coverage, in order to attain herd immunity. school-based vaccination programmes generally have a higher uptake than other programmes (3). in sweden, from spring of 2012, the quadrivalent hpv vaccine is offered free of charge to all girls aged 10–12 years through a school-based vaccination programme. information about hpv vaccination and informed consent is distributed to the parents by the school nurses. this information is standardized by the authorities, and its distribution is mandatory (4). complementary information about hpv and hpv vaccine is optional and can be distributed verbally or in writing. catch-up vaccination is offered to all *m.go. and m.gr. contributed equally to this work. correspondence: maria grandahl, department of public health and caring sciences, box 564, 751 22 uppsala, sweden. fax: +46 (0)18 471 6675. e-mail: maria.grandahl@pubcare.uu.se (received 25 january 2013; accepted 23 may 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.809039 http://informahealthcare.com/journal/ups mailto:maria.grandahl@pubcare.uu.se girls born 1993–1998. hpv vaccines have been available since 2006 at a market cost of about 330e for three doses, and since may 2007 reduced by the government subsidy to 200e. this opportunistic vaccination reached an uptake of about 25% of the age groups 13–17, strongly associated with parents education: higher chance of vaccination with higher parental education (5,6). several aspects affect parents’ attitudes towards the hpv vaccination for their daughters (7–11). recommendations from physicians (8–11), belief in the effectiveness of hpv vaccine or vaccines in general (8–10), belief in protection against cancer (10-12), care for daughters’ health (9), and whether the parents had previously accepted childhood vaccinations (9,10) are examples of such factors. a survey of swedish parents (n = 13,946) of 12–15-year-olds in 2007 found that parents’ main concern about the hpv vaccine was whether the vaccine had any adverse effects and that information about hpv vaccine safety and efficacy was important to parents (13). barriers to vaccinating daughters against hpv are fear of side effects (8,9,12,14), the long-term safety of the vaccine (11), daughter’s young age and a wish to wait until daughter is older (9–11). some studies report worries of increased sexual risk-taking as a barrier (9,13), and some not (15,16). vaccination against hpv is a new addition to the swedish school-based vaccination programme. it is the first time a vaccine is offered against a sexually transmitted infection (sti). the vaccine uptake can be affected by parents’ attitudes, which may be different beforetheintroductionofaprogrammecomparedtothe day when they are to fill in a consent form for the vaccination.knowledgeofwhatfactorsactuallyaffected parents’ decision regarding hpv vaccination for their young daughter in a publicly funded school-based programme is limited (9). therefore, the aim of this study was to explore how parents reason when they accept hpv vaccination for their young daughter and also their views on hpv-related information. material and methods design an explorative qualitative study design was adopted using individual interviews with parents who had accepted hpv vaccination for their 11–12-year-old daughters. informants inclusion criteria were parents who had accepted hpv vaccination for their 11–12-year-old daughters. the parents were recruited from three strategically chosen municipalities in mid-sweden where the vaccination programme had already been initiated in the schools. in several other municipalities the vaccination programme started one term later. school nurses (n = 100) from the three municipalities distributed an invitation letter about the study to all parents of 11– 12-year-old girls (n = 1,888) in their schools. a total of 29 parents who had agreed to vaccinate their daughter volunteered to participate. two parents were not interviewed due to practical issues. characteristics of the participants (23 women, 4 men) are presented in table i. procedure school nurses who agreed to assist with the recruitment of participants distributed an invitation letter to all parents of 11–12-year-old girls in their schools. parents interested in participating in an interview were asked to contact the researchers for more information and to make an appointment for the interview. the parents determined the time and place of the interview, and all interviews took place 1–4 weeks after the parents had decided to vaccinate their daughter. some girls had recently received their first injection, and some were to get it within a few days of the interview. interviews took place at the parents’ or researchers’ work-place, at parents’ home, at a café, or at a library. every interview started with verbal information about the study and acknowledging that participation was voluntary. the parents were asked to sign a consent form and to fill in a short questionnaire with demographic questions. all interviews were table i. characteristics of the participants. age mean 44 (range 36–52) sex 23 women/4 men civil status single 2 living with partner 6 married 19 highest education university/college 22 high school 4 vocational training/education 1 country of birth sweden 24 other european country 2 non-european country 1 more than one child 25 (93%) 264 m. gottvall et al. audio-recorded and lasted an average of 20 minutes. the parents were offered a movie ticket in return for their participation. data collection continued until little new information emerged from the interviews. the interviews were conducted by m.go., m.gr., c.s., and t.t. between march and april of 2012 and were then transcribed verbatim. all interviewers are experienced in qualitative interviewing and are registered nurses and midwives with experience in clinical work, for example in paediatric, adolescent, and maternal health. instrument a semi-structured interview guide with two main open-ended questions was used for the study: ‘how did you (and your partner) reason before making a decision about the hpv vaccination for your daughter?’ and ‘what did you think about the information you received from school?’ when necessary, the interviewer asked for clarifications or follow-up questions such as: ‘could you tell me more about this?’ pilot interviews were conducted in february 2012 with three mothers of girls aged 12– 16. these interviews did not indicate any need for changes in the interview guide. data analysis the interviews were analysed with thematic content analysis as described by burnard et al. (17). the interviews were read several times, and notes summarizing what was said were made in the margin. these notes were the initial codes, which were then collected from all the interviews and reviewed, removing duplicates. the number of codes was then reduced further into categories and themes, by grouping together overlapping or similar codes. thereafter, the transcripts were read again and data that fit under a certain category were labelled accordingly. the initial analysis was conducted by m.go. and m.gr. to check for validity, four researchers (a.t.h., t.t., m.l., and c.s.) read three to six of the transcripts each, to identify categories. these category systems were similar to the initial category system. the categories and themes were then discussed with all authors until a consensus was reached. examples of the analytical process are presented in table ii. to obtain trustworthiness in the study, the quality criteria for qualitative studies as outlined by guba and lincoln were considered; credibility, dependability, conformability, and transferability (18). since little is known about swedish parents’ decision-making about hpv vaccine for their daughter the inductive approach was used and data were analysed without any predetermined theory. ethical requirements the study was approved by the regional ethical review board in uppsala, sweden (d.nr. 2012/48). results the analysis resulted in three main themes describing factors that had influenced parents’ decision to accept hpv vaccination for their daughter: trust versus concern, responsibility to protect from severe disease, and information about hpv and hpv vaccination is important. each theme consists of two to four categories (figure 1), which are presented below and illustrated by quotes. trust versus concern trust in authorities. parents expressed a trust in vaccine recommendations from authorities and experts and said that the hpv vaccination was an offer they had decided to accept. many had accepted the other vaccines offered in the child vaccination programme and were positive to vaccines in general. they believed the authorities make decisions that are good for the people; therefore, a vaccine included in the school-based vaccination programme is likely to be reliable. it has been discussed and investigated and they have finally decided that this is what people must do, so i feel that we must, in any case, trust that the recommendations are right. (mother, age 51, interview #1). it was also expressed that school-based vaccinations are very convenient and accessible for the parents. they believed that the vaccine coverage would table ii. examples of the analytical process. interview transcript initial coding framework category we will probably know more about the side effects in 10 years. that we don’t know today. and that you might be able to discern in 5–10 years while the pandemic side effect narcolepsy you could have discerned earlier. (interview 1) takes time to see all the side effects. compares it with the swine flu. unknown side effects worry hpv vaccination—how parents reason 265 increase through this system since it makes it easier for parents to accept the vaccine merely by filling in ‘yes’ on a piece of paper. several parents opined that the children trust the school nurse who administers the vaccination and feel comfortable with their classmates around them, further simplifying matters for parents. parents could, however, also see a risk that peer pressure could be an issue for those who want to say no to the vaccination. parents also viewed schoolbased vaccination as efficient from a socioeconomic perspective, since the vaccination took place during school hours and parents did not have to be present. it becomes more accessible, it rolls along by itself, automatically without having to make an appointment, driving yourself there . . . it is really good for us parents with limited time and so on . . . it’s great . . . smoother. (father, age 45, interview #12). trust in vaccine effectiveness and safety. even though many parents felt they had limited knowledge about the vaccine, they expressed trust in the vaccine’s effectiveness and safety. parents believed that the vaccine was well-tested in other parts of the world and that a large number of girls had already been vaccinated without severe side effects. i was not particularly well informed but i think side effects are important, it seems pretty clear since there are so many people who have been vaccinated that the side effects should have been evident . . . so for me, it was obvious to vaccinate. (mother, age 43, interview #14) however, all parents were not convinced about the vaccine’s effectiveness even though they trusted its safety and thus had accepted the vaccination. unknown side effects worry. a worry about unknown side effects was expressed, and parents compared it to the mass swine flu vaccination in 2009–2010, which caused narcolepsy in several swedish children. many had not been worried about unknown side effects in vaccines before the swine flu vaccination. however, they estimated the risk for serious side effects of the hpv vaccine as being lower than the potential positive effects of the vaccine. vaccinations are good and bad, think about the swine flu from recent memory. the hysteria and how it was after, so one can feel that it has become difficult with vaccinations . . . but now i have become more observant about what i am going to put in her. (mother, age 48, interview #11) concern about commercial interests. some parents were concerned about the underlying purpose of profitmaking by the pharmaceutical industry. they discussed whether one could trust the vaccine trials or if the vaccine company could have influenced it. the industry maybe has other purposes than to help people; they earn money too . . . they earn money in the first place. (father, age 43, interview #2). figure 1. themes and categories that emerged through the analysis. 266 m. gottvall et al. responsibility to protect from severe disease concern about daughters’ future health. parents had accepted hpv vaccination for their daughter to preserve her future health and to protect her from cancer. they felt that vaccination against cancer was an offer they had to accept. i mean, a flu if you are normal, that you can overcome, but cervical cancer, that maybe you can’t overcome. it is such a serious disease . . . if i say no to the vaccine and she gets sick, i would never be able to forgive myself. (mother, age 46, interview #20). a common reason for accepting the hpv vaccination for their daughter was that they themselves or someone close to them had a history of an abnormal pap-smear, cervical cancer, or other cancer diagnosis. they had experienced the negative consequences of cancer and, therefore, felt that it was important to provide the best possible protection for their daughter. i have myself had cervical cancer, so i think there is even more reason that my daughter will be vaccinated. there was no doubt, just a yes. (mother, age 44, interview #19). herd immunity. most parents had their daughter vaccinated against hpv for her future health, but some also felt a responsibility to vaccinate her out of concern for others. they stated that in sweden many childhood infections have been eliminated through the general vaccination programme which gives protection even for unvaccinated children. therefore, some saw it as egoistic and irresponsible not to agree to vaccinations. i think that it’s a social responsibility since many of the diseases that we are vaccinated against under the general vaccination programme can cause a great havoc in our population and to not participate in the vaccination programme, i think, is irresponsible towards others. (mother, age 46, interview #10). information about hpv and hpv vaccination is important a wish for more information and a dialogue with the school nurse. the information from the school was satisfactory according to many of the parents, but some requested further information about the virus, including the seriousness of cervical cancer, and the risks and benefits of the vaccination. due to their limited knowledge about the virus and the vaccine, they requested a dialoguewiththeschoolnurseinadditiontothewritten information they had received from the school. one parent requested more neutral information that addresseduncertaintieswiththevaccine.someparents also requested better information for their daughters, whotheybelievedhadnotreceivedenoughinformation regarding the vaccine at school. i thought it was a pretty hard decision. i thought that i got quite insufficient information in the papers that came home from school . . . and the worst part, i think, is that when you have a school nurse who is going to vaccinate hundreds of children, and who is not well informed . . . because if you put a name and telephone number on a paper, then you should be able to answer parents’ questions. (mother, age 48, interview #11). i think that it would have been great if someone from the health care field could have come to a parent meeting . . . so that as a parent, one had the opportunity to ask questions . . . one of these papers can easily become lost in the backpack. (mother, age 41, interview #18). scary information. all parents had agreed to vaccinate their daughters, but two of the young girls refused vaccination because of fear after having read scaremongering leaflets. several other girls had also heard scaremonger rumours and were worried about serious side effects of the vaccination. their parents therefore felt unsure of the decision they had made and were uncertain of which sources to trust. my daughter and her friend came home and were a little sad and wondered if there was rat poison in the vaccine. (mother, age 38, interview #17). another barrier the parents mentioned was the daughters’ fear of injections. however, this was not a reason to reject the vaccination. rather, they felt a need to discuss with their daughter more deeply the value of the vaccination and explain the vaccination procedure in order to make her more comfortable with the injection. it is just this fear of needles, if the injection hurts; so it can be good to prepare your daughter that it will hurt. can you use the numbing cream or routines around the vaccination itself? (father, age 43, interview #2). the daughter is too young to understand complex information. many parents wanted to talk to their daughter about other preventive methods for cervical cancer, such as condom use and pap-smear exam, but few thought the time around vaccination was a good time to do so. they were of the opinion that their daughter was too young to understand such information. they also felt it was difficult to talk about and did not know when was a good time to talk about the sexual transferability of the virus and protection against sexually transmitted infections (sti). however, some parents hpv vaccination—how parents reason 267 believed their daughter might understand more than they were aware of, and one parent believed that the age of 11–12 was a good age to talk about such issues. many parents had informed their daughter briefly about the vaccination but most did not involve her in the decision-making. i think that it might be too early in the middle school, but definitely in secondary school . . . seventh grade. i think so and then maybe continuously, it is a very important question . . . it is not everyone that gets this information at home for various reasons. (mother, age 44, interview #27) discussion discussion of the findings the parents expressed trust in recommendations from authorities and believed it was important to protect their daughter against a severe disease. they thought it was convenient with school-based vaccination and appreciated that the vaccination was administered by the school nurse. however, they had concerns about the vaccine safety and side effects and, thus, requested additional and more adequate information about hpv and hpv vaccine. to our knowledge, this is the first study to explore how parents reason when they accept hpv vaccination for their young daughter, as part of the swedish school-based vaccination programme. for the parents, the fact that the vaccine was included in the school-based vaccination programme was a sign that the vaccine was safe and well-tested. other studies have also found the importance of a vaccine being recommended by authorities and medical specialists (9,11). a school-based vaccination programme helped convince the parents of the safety of the vaccine, and also made the vaccination convenient and accessible for parents. school-based vaccination programmes are a way to facilitate vaccinations for all children regardless of socio-economic background (19). another finding was that, although the parents had trust in authorities, most of them brought up the swine flu vaccination in 2009–2010, when the vaccine was recommended for everybody in sweden. subsequently, this vaccination caused unforeseen cases of narcolepsy in children (20). this incident seemed to have affected most of the interviewed parents and made them more critical of new vaccines and concerned about unknown side effects. they considered the risks versus benefits carefully before making a decision about vaccinating their children. despite this, the hpv vaccine coverage during the first year of the implementation of the school-based vaccination programme in sweden was relatively high, specifically 79% (21). in line with previous studies, the parents considered cervical cancer to be a serious disease and out of concern for their daughter’s health, wished to protect her from it (9,11). several of the parents in this study had experience of cancer either directly, or through family or friends. a previous study had a similar finding among parents accepting hpv vaccine for their daughter (11). parents felt a responsibility to protect their daughter, but also a responsibility to protect others through containing the spread of the disease. scaremongering did not appear to have had a major impact on the decision-making for the parents in our study, but it had affected the decision in some families where the daughter became frightened and did not want to be vaccinated. scare campaigns could be met with adequate and transparent information to enable parents to make an informed choice about the hpv vaccine (22). few of the parents in this study had discussed the virus and other future preventive methods, e.g. condom use, with their daughter. they felt that the girls were too young to discuss this subject. challenges with information regarding hpv have previously been pointed out (23). our study found that parents believe their children trust the school nurse. this underlines the importance of school nurses as key persons in spreading information about hpv and hpv prevention to school children. it is, therefore, of great importance that the school nurse is well educated in the area to be able to respond to questions from parents and to inform them as well as their children about hpv and hpv prevention. the school nurse is the hub for the vaccination in a school-based programme and can contribute to a well-functioning vaccination programme with a high coverage and satisfied parents. discussion of methods the intention was to recruit a broad and varied sample through the choice of municipalities and schools in different socio-economic regions. to simplify participation, the parents were allowed to decide the time and place of the interview. the reason for choosing only those parents who accepted vaccination was to get a genuine understanding of their reasoning before the decision. the sample consisted mainly of female parents with a post-secondary education. this over-representation of well-educated parents could be a selection bias, and it is possible that the results would have been different with more participants having a lower education level. further studies among 268 m. gottvall et al. parents with low education level and also among those who declined vaccinations for their daughters are needed. as with all qualitative studies, the results cannot be generalized but can provide a better understanding of which factors influence parents’ decision to accept hpv vaccination for their daughter and how they view the information they received. the analysis process was systematic and rigorous, all data transcripts were thoroughly analysed, and the main findings as well as contrary findings have been presented in the results section supported with illuminating quotes to ensure credibility. even though the results cannot be generalized, the authors believe that they can be transferred to other groups of well-educated parents. to check for validity and to avoid lone researcher bias (17), several researchers individually read the transcripts to identify categories. these category systems were then compared to the initial analysis. all researchers took part in discussing the categories and themes until a consensus was reached. conclusion parents accepted hpv vaccination since they trusted the recommendations from authorities and wanted to protect their daughter from a severe disease. this outweighed the concerns they had about side effects and commercial interests. the parents thought it was convenient with a school-based vaccination programme and identified the school nurse as an important source of information about hpv and hpv vaccination. the findings from this qualitative study cannot be generalized; however, they can provide a better understanding of how parents might reason when they accept the hpv vaccination for their daughter. acknowledgements this study was funded by the swedish cancer society, the uppsala-örebro regional research council, the medical faculty at uppsala university, the solstickan foundation, and the swedish society of nursing. we are grateful to the parents who took part in the interviews and to the school nurses who helped with the recruitment. declaration of interest: in 2010, m.go. and t.t. lectured about their own research on events for school nurses organized by sanofi pasteur (msd). the authors have no other potential competing interests to declare. the authors alone are responsible for the content and writing of the paper. references 1. mosina l, martin r, eckert l. hpv vaccination in europe: experience from national immunization programmes who. 2010. 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"is cancer contagious?": australian adolescent girls and their parents: making the most of limited information about hpv and hpv vaccination. vaccine. 2010; 28:3398–408. 270 m. gottvall et al. www.ncbi.nlm.nih.gov/pubmed/17679241?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17679241?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17679241?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21211477?dopt=abstract http://www.lakemedelsverket.se/alla-nyheter/nyheter-2012/lakemedelsverket-samordnar-forskning-kring-vaccinsakerhet-och-narkolepsi-/ http://www.lakemedelsverket.se/alla-nyheter/nyheter-2012/lakemedelsverket-samordnar-forskning-kring-vaccinsakerhet-och-narkolepsi-/ http://www.lakemedelsverket.se/alla-nyheter/nyheter-2012/lakemedelsverket-samordnar-forskning-kring-vaccinsakerhet-och-narkolepsi-/ http://www.smittskyddsinstitutet.se/nyhetsarkiv/2013/stor-andel-flickor-vaccinerade-sig-mot-hpv/ http://www.smittskyddsinstitutet.se/nyhetsarkiv/2013/stor-andel-flickor-vaccinerade-sig-mot-hpv/ http://www.smittskyddsinstitutet.se/nyhetsarkiv/2013/stor-andel-flickor-vaccinerade-sig-mot-hpv/ www.ncbi.nlm.nih.gov/pubmed/20199758?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20199758?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20199758?dopt=abstract abstract introduction material and methods design informants procedure instrument data analysis ethical requirements results trust versus concern trust in authorities trust in vaccine effectiveness and safety unknown side effects worry concern about commercial interests responsibility to protect from severe disease concern about daughters' future health herd immunity information about hpv and hpv vaccination is important a wish for more information and a dialogue with the school nurse scary information the daughter is too young to understand complex information discussion discussion of the findings discussion of methods conclusion acknowledgements declaration of interest references the precision hypertension care (physic) study: a double-blind, randomized, repeated cross-over study article the precision hypertension care (physic) study: a double-blind, randomized, repeated cross-over study johan sundstr€oma,b, lars linda, shamim nowrouzia, per lytsya , kerstin marttalaa, inger ekmanb, patrik €ohagenb and ollie €ostlundb adepartment of medical sciences, uppsala university, uppsala, sweden; buppsala clinical research center (ucr), uppsala, sweden abstract high blood pressure is the leading risk factor for premature deaths and a major cost to societies worldwide. effective blood pressure-lowering drugs are available, but patient adherence to them is low, likely partly due to side effects. to identify patient-specific differences in treatment effects, a repeated cross-over design, where the same treatment contrasts are repeated within each patient, is needed. such designs have been surprisingly rarely used, given the current focus on precision medicine. the precision hypertension care (physic) study aims to investigate if there is a consistent between-person variation in blood pressure response to the common blood pressure-lowering drug classes of a clinically relevant magnitude, given the within-person variation in blood pressure. the study will also investigate the between-person variation in side effects of the drugs. in a double-blind, randomized, repeated cross-over trial, 300 patients with mild hypertension will be treated with four blood pressure-lowering drugs (candesartan, lisinopril, amlodipine, and hydrochlorothiazide) in monotherapy, with two of the drugs repeated for each patient. if the study indicates that there is a potential for precision hypertension care, the most promising predictors of blood pressure and side effect response to the drugs will be explored, as will the potential for development of a biomarker panel to rank the suitability of blood pressure-lowering drug classes for individual patients in terms of anticipated blood pressure effects and side effects, with the ultimate goal to maximize adherence. the study follows a protocol pre-registered at clinicaltrials.gov with the identifier nct02774460. article history received 17 june 2018 revised 2 july 2018 accepted 5 july 2018 keywords hypertension; blood pressure; randomised clinical trial; precision medicine background high blood pressure is the leading risk factor for premature deaths worldwide (1) and accounts for more than 10% of all health care costs in some developed countries, with a mere fifth of that attributable to medication costs, and four-fifths to complications (2). less than half of those who have high blood pressure are aware of it, and, of those receiving blood pressure-lowering drug treatment, only one-third have their blood pressure at target levels (3). effective blood pressure-lowering drugs are available, with the first-line drug classes—angiotensin-converting enzyme (ace)-inhibitors, angiotensin receptor blockers, calcium channel blockers, and thiazide diuretics—considered equally effective on average. most of their protective effects depend on the amount of blood pressure reduction achieved, and there are few class-specific protective effects (4). they do, however, have class-specific side effects. poor adherence is likely the main cause of poor blood pressure control, with as little as 1 in 2 patients estimated to be adherent to prescribed treatment in developed countries (5). because poorly controlled blood pressure is such an important burden on patients and health care systems, improving adherence is imperative. the concept of precision medicine promises maximization of treatment benefit and minimization of treatment harm using subtle characteristics (typically involving, but not limited to, gene variants) of patients with the same trait to guide treatment decisions. if the promise holds true widely, hypertension would be an optimal field, with a handful of first-line drug classes that all have similar average blood pressure-lowering effects, and each has common side effects that hamper adherence. for hypertension, the concept needs answers to several important questions. the most important question is if there is a clinically relevant and repeatable between-person difference in blood pressure response or side effects to a given drug (6). if there is not, then there is no potential for precision medicine. analysing variance components in parallel group trials (which cannot disentangle all of the relevant variance components) (7), a clinically irrelevant between-person difference in blood pressure response has been suggested to be the scenario for ace-inhibitors (8), but this is unconfirmed and unknown for other drug classes. it is also unknown if between-person differences in blood pressure response are different for different drugs (9,10), and magnitudes of withinand between-person variations in side contact johan sundstr€om uppsala university, department of medical sciences, uppsala, sweden. e-mail: johan.sundstrom@medsci.uu.se � 2018 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 1, 51–58 https://doi.org/10.1080/03009734.2018.1498958 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1498958&domain=pdf http://orcid.org/0000-0003-1949-6299 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1498958 http://www.tandfonline.com effects are particularly unknown. resolving these issues is imperative for the possibilities of precision blood pressurelowering treatment, and the present study, the precision hypertension care (physic) study, aims to answer those questions. proper methods for that purpose have been proposed (7,11) and are used in this study. if this study indicates that there is a potential for personalized blood pressure-lowering treatment, we will explore the most promising predictors of blood pressure and side effect response to the drugs, using peripheral blood analyses in combination with clinical data. if feasible, we will then explore the potential for development of a biomarker panel to rank the suitability of each of the blood pressure-lowering drug classes for the individual patient in terms of both anticipated blood pressure effect and side effects, with the ultimate goal to maximize adherence. objectives primary objective the primary objective is to establish the potential for precision hypertension care, by investigating if there is a consistent between-person variation in blood pressure response to the common blood pressure-lowering drug classes of a clinically relevant magnitude, given the within-person variation in blood pressure. secondary objectives a. to investigate the between-person variation in side effects of blood pressure-lowering drugs in contrast to the within-person variation in side effects. b. to investigate patient treatment preferences when presented with: i) their individual ratings for each drug class; ii) a combination of their individual ratings and blood pressure effect of each drug class. c. to develop a prediction model based on biomarkers and clinical characteristics for patient treatment preferences. d. to develop and evaluate a point-of-care tool using this prediction model, in order to inform treatment decisions in the clinical setting. tertiary objectives a. to evaluate betweenand within-person variation in response in mean and standard deviation of daytime, nighttime, and 24-h blood pressure and heart rate to the common blood pressure-lowering drug classes. b. to evaluate betweenand within-person variation in self-reported symptoms and quality of life in response to the common blood pressure-lowering drug classes. c. to evaluate betweenand within-person variation in pulse wave characteristics and estimated central blood pressure in response to the common blood pressurelowering drug classes. d. to evaluate betweenand within-person variation in dna methylation, rna, proteins, and metabolites in response to the common blood pressure-lowering drug classes. e. to investigate associations of dna genomic variation, dna methylation, rna, proteins, and metabolites with blood pressure effects and self-reported symptoms and quality of life. f. to investigate the associations of self-reported symptoms and quality of life collected using an electronic diary with patients’ treatment preferences and adverse events reported in the clinic. study design rationale to identify the patient-by-treatment interactions and hence investigate the patient-specific differences in treatment effects, a repeated cross-over design, where the same treatment contrasts are repeated within each patient, is needed. this has been convincingly suggested for decades (12), but such designs have been surprisingly rarely used given the current focus on precision medicine. in the physic trial, each patient is given all four first-line hypertension drugs, one at a time, and in addition repeats two of the treatments. this allows us to quantify whether there is consistent preference of one treatment over another within a blinded patient over different treatment periods. separation of period effects and drug effects is key. without pairwise repetition of treatments, a trial cannot assess whether a patient that preferred a certain treatment over another did so because of the treatment or because of an external factor such as the season or normal day-to-day variation. it would also have been impossible to assess whether a variation in treatment effects was due to differences between drugs in on-treatment within-patient variation, such as patients having larger day-to-day or season-to-season variations when on one of the drugs. the study needs to be double-blind, and the order of the treatment periods randomized, in order to avoid obvious sources of bias. evidence-based titration and target doses have been chosen for all four drug classes included in this protocol (13). within each drug class a compound that fitted the gelatin capsules was chosen. the suggested treatment duration per treatment arm is based on recent guidelines (14). the study is outlined in table 1, examples of treatment sequences illustrated in figure 1, and the sequence of events described in table 2. the first patient was randomized in the first quarter of 2017; last patient out is expected in the fourth quarter of 2021. study visits and periods visit 1. the screening visit (visit 1) will occur at least two weeks before randomization (visit 2). written informed consent is 52 j. sundstr€om et al. obtained, eligibility criteria (described in table 3) checked, and assessments are made of weight, height, waist, hip circumference; a physical examination is carried out, concomitant medication is noted, office blood pressure is measured, and blood is sampled. the patient fills out two questionnaires covering medical history and beliefs about medicine. all questionnaires in the study are summarized in table 4. written lifestyle advice (physical activity, weight loss, dietary changes, stress management, etc.) in accordance with national guidelines (sbu 2007) is provided to all participants. placebo run-in period. after the screening visit, the patient stops taking any concomitant blood pressure-lowering drugs and starts on a placebo run-in period of two weeks to establish an untreated baseline to be used for characterization of the study sample. visit 2. at visit 2, eligibility criteria are checked once again. assessments made at this visit include fasting venous and capillary blood sampling, questionnaires, and pulse wave analysis, and 24-h blood pressure and ecg monitoring is initiated. if all criteria are met, the patient is randomized and given the first study drug. the study drug is to be taken once daily. the day after visit 2, the patient returns the 24-h blood pressure and ecg equipment to the clinic. treatment period. between visits 2 and 3 patients are dosing study drug at home, and fill out the side effects questionnaire at home at specific time points. visit 3. at visit 3 of each treatment period (which occurs after 4–6 weeks on the target dose of the study drug, 7–9 weeks after the previous visit 3) the patient comes fasting to the clinic for blood sampling, body weight, concomitant medication, and adverse events (ae) checks, office bp measurement, and initiation of 24-h blood pressure and ecg measurement. the patient fills out two paper questionnaires: the side effect questionnaire (identical to the questionnaire figure 1. study design. each patient is randomly assigned to a treatment sequence created from one of the six possible combinations, by random permutation of the order in which the active treatments are given (the figure is a mere illustration of a few examples of possible sequences). hence, 1/6 of the patients receive acei and arb in two periods each, 1/6 of patients repeat the acei and ccb periods, 1/6 of the patients repeat the acei and diuretic periods, 1/6 repeat the arb and ccb periods, 1/6 repeat the arb and diuretic periods, and 1/6 repeat the ccb and diuretic periods. block randomization is used to ensure that approximately the same number of patients are assigned to each of the six combinations of active treatments. acei ¼ angiotensin-converting enzyme inhibitor; arb ¼ angiotensin receptor blocker; ccb ¼ calcium channel blocker; diuretic ¼ thiazide diuretic. blank arrows indicate placebo run-in and wash-out. table 1. study outline. setting patients diagnosed with hypertension with a systolic blood pressure between 140 and 159 mmhg within five years before the start of the trial. patients must be pharmacologically untreated or use blood pressure-lowering monotherapy at the time of recruitment to the present study. design double-blind, randomized, repeated cross-over, single-centre study. number of patients (planned) 300 patients included to obtain 240 evaluable patients. interventions candesartan: 8 mg weeks 1–2; 16 mg weeks 3–8 lisinopril: 10 mg weeks 1–2; 20 mg weeks 3–8 amlodipine: 5 mg weeks 1–2; 10 mg weeks 3–8 hydrochlorothiazide: 12.5 mg weeks 1–2; 25 mg weeks 3–8. treatment duration the study consists of six active treatment periods: four classes of blood pressure-lowering drugs will be tested, treatment with two classes are repeated for each patient. patients will be assigned a treatment order based on block randomization ensuring that the same number of patients will repeat each treatment. active drug is given during 6–8 weeks of each treatment period (titration dose week 1–2, target dose week 3–8). placebo will be administered during 2 weeks of placebo run-in (between visit 1 and 2) and during 7 days’ wash-out between treatment periods. this gives a total duration of treatment of 47–59 weeks for each included patient. outcome the primary analysis of individual variation in treatment differences in visit 4 daytime (10.00–20.00) ambulatory systolic blood pressure will be performed using a random effects model with correlated random intercept and treatment contrasts, allowing correlation between all random effects, and with treatment period as a fixed effect. all available periods with >90% treatment adherence from all randomized patients will be used. upsala journal of medical sciences 53 for home use) and a treatment preference rating questionnaire. the patient returns all unused study drug (ensuring that they have full coverage of study drug until the completion of visit 4) and receives the next treatment kit, which starts with a 7-day placebo wash-out period before the next active drug treatment period starts. in rare cases, if a patient due to unbearable side effects of a specific treatment so wishes, bailout visits 3 and 4 can be carried out and the treatment period terminated prematurely so that the next treatment period can begin. visit 4. at visit 4, the day after visit 3, the 24-h blood pressure and ecg measurement ends and the patient returns the equipment to the clinic. the treatment periods and visits 3 and 4 are repeated for a total of 6 times. visit 5. visit 5 occurs 1–2weeks after the sixth and last visit 4. at visit 5, the patient is asked by an independent physician to do an overall treatment preference rating including all treatment periods, based on a summary of the patient’s own reported side effects, treatment preference ratings for all treatment periods, and ambulatory daytime blood pressures. all data in the summary have been validated by data management personnel, signed by the pi and locked before being presented to the patient. as a final step, the independent physician breaks the randomization code for each completed patient, and prescribes the drug preferred by the patient for continuous use. statistical considerations analysis population intention-to-treat analyses will be used for all side effects, including all patients taking at least one dose of the study drug in a new treatment period. as-randomized analyses will be used for blood pressure endpoints, including all patients having had a treatment adherence of more than 90% during that treatment period. table 2. sequence of events. visit 1 placebo run-in (�2 weeks) 2 treatment period 5dosing at home 3 4 week –1 –(–2) 1 1–7 7 8 day –7 –(–14) 1 2–54 55 56 informed consent � inclusion/exclusion criteria � � anthropometrya � physical examination � office bpb � � � concomitant medication � � � safety samplesc � � �d dna genomic samplese � medical history qg � beliefs about medicine q � placebo dosing � � randomization � study drug dosingh � � � � staff train patients in electronic diaryf � self-reporting of symptoms in electronic diaryi � � persyve q � � pulse wave analysis � capillary blood sampling and dried blood spots � venous blood samples for dna methylation, rna, other biomarkersj � � ae/sae recordingk � �q treatment preference rating ql � 24-h bp/ecgm �n � � return of unused study drug by patients � supply next study drug and 7-day placebo � overall treatment preference ratingo � prescription of preferred drug for continuous useo � a measured using standard field protocols. b prior to measurement patient will rest sitting for 10 min. measurement twice within 5 min in-between measurements using a sphygmomanometer. c the following safety samples will be analysed: hb, hba1c, sodium, potassium, calcium, creatinine, asat, alat, c-gt, and pregnancy test. d last safety samples are taken and analysed at visit 3, in the last treatment period only. e whole blood sample taken in two 7/6 ml edta vacutainersv r . f patient fills in the electronic diary (persyve q) under supervision of study staff. g q covering demographic items, medical history, smoking, and physical activity. h dosing once daily in all treatment periods. i when patients are dosing at home (between visit 2 and 3) symptoms will be recorded in persyve q using an electronic diary. patients will report spontaneously but will also be prompted via sms at three occasions: after 3, 5, and 7 weeks of dosing. j details specified in a laboratory manual. k adverse events recorded by clinical staff at visits 2 and 3 in each treatment period (observations, open questions, and reporting of symptoms spontaneously mentioned by patients). persyve q completed before the open ae question. l at visit 3 in each treatment period patients will fill out a treatment preference rating q consisting of five questions covering willingness to pay, treatment preference, safety, and motivation. m 24-h bp/ecg at the end of each treatment period, between visits 3 and 4. applied at visit 3 and removed the same time at visit 4. n 24-h bp/ecg also at the end of placebo run-in to get a baseline assessment. o overall patient preference rating and prescription of preferred study drug for continuous use. q ¼ questionnaire. 54 j. sundstr€om et al. primary analyses: individual variation in treatment differences the primary analysis of individual variation in treatment differences in visit 4 daytime systolic blood pressure will be performed using a random effects model with correlated random intercept and treatment contrasts, allowing correlation between all random effects, and with treatment period as a fixed effect. all available periods with >90% treatment adherence from all randomized patients will be used. the global hypothesis—that there is a contribution from patient-specific differences in relative treatment response to the variation in on-treatment blood pressure in the analysis set—will be tested using a likelihood ratio test based on the maximumlikelihood fit of that model compared to a model with random intercept (only) and treatment contrasts and treatment period as fixed effects. since variance parameters must be non-negative, the null hypothesis lies on the border of the parameter space of the larger model. hence the usual assumption of chisquare distribution in the likelihood ratio test is not valid, and the test statistic will be compared to a reference distribution obtained by parametric bootstrapping to obtain a p values. p < 0.05 will be considered statistically significant. appropriate methods will be used to present estimates of treatment contrast variances, covariances, and residual variance. homoscedasticity of the residual variance will be investigated. a detailed statistical analysis plan for pre-defined analyses will be finalized before unblinding. problems with model convergence or other changes to the analysis will be recorded in a statistical report. for each pair of treatment regimens (say x and y), descriptive plots and tables will be produced based on the patients that had repeated periods with both treatment x and y. a pre-defined pairing of the periods for each patient, into what amounts to two cross-over trials in the same patient, will be used to produce two independent measurements of the treatment contrast x–y within each patient. corresponding analyses will be performed for other blood pressure measurements. similar analyses, adapted for type and distribution of the variable, will be performed for visit 3 measurements of other outcome variables, as appropriate. predefined analyses will be described in the sap. no formal adjustment for multiplicity will be performed in the primary analyses. secondary analyses: exploratory biomarker analyses if the primary analyses indicate that there may be a substantial between-person variation in effect of treatment on blood pressure, exploratory analyses will be conducted developing prediction models for individual effects on blood pressure. prediction models for side effects will be pursued irrespectively of results of the primary models. models will be developed using dna, rna, and other biomarkers to predict individual treatment effects on blood pressure and side effects. the institute of medicine guidelines for development and validation of omics-based tests will be used (15). because of the limited sample size, the cross-validation approach will be prioritized over training/test set sample division approaches. similar models as in the primary analyses will be used, typically involving mixed models with fixed effects for most of the independent variables, and random effects across patients and other structural elements. significance level correction for multiple testing approaches such as 5% fdr will be applied. table 3. inclusion and exclusion criteria. inclusion criteria � male or female aged �40 years and �75 years � previously diagnosed with hypertension with systolic blood pressure between 140 and 159 mmhg within 5 years prior to the start of the trial � pharmacologically untreated or using blood pressure-lowering monotherapy at visit 1. no blood pressure-lowering medication taken during the placebo run-in period � office systolic blood pressure between 140 and 179 mmhg and diastolic blood pressure at or below 109 mmhg at visit 2 � patients must give informed consent to participate in the study exclusion criteria � medical history, clinical signs, or laboratory results indicating secondary hypertension, including primary aldosteronism or renal artery stenosis � evidence of serious hematological, respiratory immunological, renal, hepatic, gastrointestinal, endocrinological, metabolic, neurologic, malignant, psychiatric, or other diseases as revealed by medical history, physical examination, and/or laboratory assessments � active gout � previous or present arterial occlusive diseases such as myocardial infarction (mi), stroke or acute arterial insufficiency (unstable angina pectoris or transient ischemic attacks [tia]), or heart failure (nyha class iii or iv, or left ventricular systolic dysfunction irrespectively of function class) � moderate or severe aortic or mitral insufficiency � renal failure, including hemodialysis or kidney transplant(s) � atrial fibrillation in need of rate control � symptomatic hypotension, defined as weakness or syncope upon rising to an erect position associated with a decrease in systolic blood pressure � diabetes requiring insulin or oral glucose-lowering drugs � any history of serious abnormal drug reaction to active or inactive compounds in the study drugs, including angioedema � any condition associated with poor compliance including alcoholism or drug dependence � patients who will not comply with the study protocol as judged by the investigator � women who are pregnant or lactating or not using appropriate contraception for at least 3 months prior to visit 1. acceptable contraceptive methods are: combined (estrogen and progesterone containing) hormonal contraception associated with inhibition of ovulation (oral, intravaginal, transdermal), progesterone-only hormonal contraception associated with inhibition of ovulation (oral, injectable, implantable), intrauterine device, intrauterine hormone-releasing system, bilateral tubal occlusion, and vasectomized partner � continuous use of concomitant medication that can interfere with study medication, i.e. digitalis glucosides, sotalol, cholestyramine, colestipol, nsaid, lithium, carbamazepine, cyp3a4-inhibitors, cyp3a4inducers, dantrolene, diuretics, aliskiren, gold, sympathomimetics, tricyclic antidepressants, antipsychotics, anaesthetics, and potassium supplements � clinical laboratory assessment outside normal range at visit 1 and judged clinically significant by the investigator � previous randomization in present study table 4. questionnaires. at visit 1 the patients complete a questionnaire covering demographic items, medical history, smoking, and physical activity. at visit 1 the patients complete a ‘beliefs about medicine’ questionnaire. at visit 2 after placebo run-in and at visit 3 after each treatment period, patients fill out a modified section 2.1 of the ‘persyve’ questionnaire on paper in the clinic. patients are also prompted via sms to fill out the questionnaire at home at specific time points—at week 3, week 5, and week 7 of each treatment period, on paper or using an electronic diary (provided by symptoms europe ab, filled out via smartphone). at visit 3 in each treatment period patients will fill out a ‘treatment preference rating questionnaire’ consisting of five questions covering willingness to pay, treatment preference, safety, and motivation. at visit 5 patients fill out an ‘overall treatment preference rating’. upsala journal of medical sciences 55 in further exploratory analyses we will use instrumental variable regression techniques combining genomic dna with dna methylation/rna/protein/metabolite data (mendelian randomization) to address the causal role of the identified biomarkers for blood pressure response and side effects, which is unimportant for prediction purposes, but key for further investigation of new druggable targets. determination of sample size statistical power for detecting between-patient variation in treatment effects was simulated using r v. 3.2.2 (r foundation for statistical computing, vienna, austria), and packages lme4 v. 1.1–10 (16) and pbkrtest v. 0.4–4 (17). altogether 500 trials were simulated for each alternative hypothesis, and for each simulated trial a p values was computed using 1000 bootstrap iterations for the reference distribution of the test statistic (table 5). p < 0.05 was considered a significant outcome. the trials were simulated without period effects, and the analysis model did not include period as a factor. the validity of the method was investigated by simulating power under the null hypothesis. the on-treatment standard deviation of betweenand within-patient variation in systolic blood pressure was assumed to be 14 and 12mmhg, respectively, based on repeated measurements about 1 month apart in 21 patients. two scenarios were simulated. in the first, normally distributed uncorrelated individual treatment effects were added to between one and four of the treatment arms, with a standard deviation of 5, 7.5, or 10 mmhg. with n ¼ 240 patients, the trial has a power of 82% for detecting individual treatment effects if only one treatment arm differs from the others by having an individual variation in effect with sd ¼7.5 mmhg, and a very high power if two or three treatments have patient-specific effects on that level. the second scenario is intended to describe a simplified on/off switch for effect. we assume that for one of the treatments 20%, or 50%, of the patients experience an effect that is 10, 20, or 30 mmhg higher (or lower) than the remaining patients. the trial has 90% power to show if a subgroup of 20% of patients has an effect that differs by 20 mmhg on one of the treatments. in terms of sd of treatment effects, the power is similar to the simulations under normal distribution (the sd is the subgroup effect times 0.5 for the 50% case and times 0.4 for the 20% case). study conduct permissions the study was approved by the ethics committee of uppsala university (dnr 2016/135) and the swedish medical products agency (dnr 5.1–2016-25102; eudract 2015–003049-24). the study follows a protocol pre-registered at clinicaltrials.gov with the identifier nct02774460. the study is conducted in accordance with the protocol, applicable regulatory requirements, gcp, and the ethical principles of the declaration of helsinki as adopted by the 18th world medical assembly in helsinki, finland, in 1964 and subsequent versions. written informed consent will be obtained from all patients prior to enrolment in the study. randomization and blinding the study will be patient-, investigator-, study nurse-, ucr study team-, and sponsor-blinded. clinical examinations, interpretations of 24-h blood pressure and ecg measurements, and assessments of side effects, treatment preferences, and other questionnaires will be performed by persons unaware of the treatment allocation. the order of the different active treatments is randomized for each patient. the patients are assigned consecutive randomization numbers according to their inclusion order, and clinical staff will record the numbers being used. the randomization list is prepared at uppsala clinical research center (ucr). the investigational products are manufactured and packaged in inconspicuous blister packaging and labelled by apotek table 5. summary of power simulation. simulation model (mmhg): sbpi ¼ intercepti þ indtreata � treatai þ indtreatb � treatbi þ indtreatc � treatci þ indtreatd � treatdi þ ei; period between-patient variation assumption: intercepti � n 0; 14ð þ within-patient variation assumption: ei;period � nð0; 12þ null hypothesis with n ¼ 240 (for validation purpose, should be 5%) treatai ¼ treatbi ¼ treatci ¼ treatdi ¼ 0 5.2% normally distributed individual treatment effects with sd ¼ x mmhg treatai � n 0; að þ; treatbi � n 0; bð þ; treatci � n 0; cð þ; treatdi � n 0; dð þ n ¼ 240 x ¼ 5 x ¼ 7:5 x ¼ 10 a ¼ b ¼ c ¼ 0; d ¼ x 29% 82% >99% a ¼ b ¼ 0; c ¼ d ¼ x 46% 97% >99% a ¼ 0; b ¼ c ¼ d ¼ x 57% >99% >99% a ¼ b ¼ c ¼ d ¼ x 64% >99% >99% a fraction p of patients have x mmhg uniformly higher bp on treatment d (note that sd treatdið þ ¼ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi p � 1�pð þ p � x, maximum sd¼x/2 at 50% fraction) treatdi � bð1; pþ � x n ¼ 240 x ¼ 10 x ¼ 20 x ¼ 30 p ¼ 20% 12% 90% >99% p ¼ 50% 27% >99% >99% 56 j. sundstr€om et al. produktion och laboratorier ab (apl) in accordance with good manufacturing practice (gmp) and local regulatory guidelines. data management and monitoring electronic case report forms (ecrf) are used for all data collection, in accordance with a pre-specified data management plan. in accordance with gcp principles, monitoring of the study is arranged by the sponsor, and carried out by ucr, in accordance with a pre-specified monitoring plan. this study uses two study databases, database #1 for the majority of the study, and database #2 for visit 5. on an individual patient level, after the last visit 4, database #1 is validated by data management personnel, signed by the pi, and locked, before being presented to the patient by an independent physician. after each patient has provided a new overall treatment preference rating, the independent physician signs the ecrf, breaks the randomization code for that patient, and enters the ecrf data into database #2. upon study completion databases #1 and #2 are merged. perspective in the physic trial, we use the only design that can study the potential for precision medicine for blood pressure-lowering treatment (12). the crucial disentangling of all variance components has not been possible in previous studies of this research question (8–10). we reproduce a previous trial (9), removing several important sources of bias, increasing the sample size 5-fold, and using ambulatory blood pressure measurements that further increase power. the concept of n-of-1 trials to guide treatment decisions has been advocated (18), and it is akin to the physic study design but on an individual level (19). because the evaluation of blood pressure effects should not be done until after a month on the target dose, n-of-1 trials of four classes of blood pressure-lowering drugs would be prohibitively long if all treatment contrasts were to be repeated in an individual patient. hence the appeal of a biomarker panel to guide treatment choice. importantly, our study design has the power to rule out any potential for biomarker-based precision hypertension care, in which case research resources are better spent elsewhere. it can also inform on the sensibleness of n-of-1 trials in hypertension, or, simply put, the usefulness of trying another drug if a patient experiences side effects or questionable blood pressure effects of the first drug. disclosure statement the authors report no conflicts of interest. notes on contributors johan sundstr€om, md phd, specialist physician at uppsala university hospital, professor of epidemiology at uppsala university; principal investigator and designer of the trial. lars lind, md phd, specialist physician at uppsala university hospital, professor of medicine at uppsala university; co-principal investigator and co-designer of the trial. shamim nowrouzi, md, resident in internal medicine at uppsala university hospital; co-investigator and co-designer of the trial. per lytsy, md phd, specialist physician, associate professor, medical advisor at the swedish agency for health technology assessment and assessment of social services; co-designer of the trial. kerstin marttala, rn, research nurse at uppsala university hospital; study nurse and co-designer of the trial. inger ekman, project manager at uppsala clinical research center; co-designer of the trial. patrik €ohagen, biostatistician at uppsala clinical research center; co-designer of the trial. ollie €ostlund, phd, biostatistician at uppsala clinical research center; co-designer of the trial. orcid per lytsy http://orcid.org/0000-0003-1949-6299 references 1. lim ss, vos t, flaxman ad, danaei g, shibuya k, adair-rohani h. a comparative risk assessment of burden of disease and injury attributable to 67 risk factors and risk factor clusters in 21 regions, 1990-2010: a systematic analysis for the global burden of disease study 2010. lancet. 2012;380:2224–60. 2. hodgson ta, cai l. medical care expenditures for hypertension, its complications, and its comorbidities. med care. 2001;39:599–615. 3. rahimi k, macmahon s. blood pressure management in the 21st century: maximizing gains and minimizing waste. circulation. 2013;128:2283–5. 4. neal b, macmahon s, chapman n. effects of ace inhibitors, calcium antagonists, and other blood-pressure-lowering drugs: results of prospectively designed overviews of randomised trials. blood pressure lowering treatment trialists’ collaboration. lancet. 2000;356:1955–64. 5. world health organization. adherence to long-term therapies: evidence for action. geneva, switzerland: world health organization; 2003. 6. bell kj, hayen a, macaskill p, craig jc, neal bc, irwig l. mixed models showed no need for initial response monitoring after starting antihypertensive therapy. j clin epidemiol. 2009;62:650–9. 7. senn s. mastering variation: variance components and personalised medicine. stat med. 2016;35:966–77. 8. bell kj, hayen a, macaskill p, craig jc, neal bc, fox km, et al. monitoring initial response to angiotensin-converting enzyme inhibitor-based regimens: an individual patient data meta-analysis from randomized, placebo-controlled trials. hypertens. 2010;56:533–9. 9. dickerson je, hingorani ad, ashby mj, palmer cr, brown mj. optimisation of antihypertensive treatment by crossover rotation of four major classes. lancet. 1999;353:2008–13. 10. laverman gd, de zeeuw d, navis g. between-patient differences in the renal response to renin-angiotensin system intervention: clue to optimising renoprotective therapy? j renin angiotensin aldosterone syst. 2002;3:205–13. 11. senn s, rolfe k, julious sa. investigating variability in patient response to treatment-a case study from a replicate cross-over study. stat methods med res. 2011;20:657–66. 12. senn s. individual therapy: new dawn or false dawn. ther innov regul sci. 2001;35:1479–94. 13. james pa, oparil s, carter bl, cushman wc, dennison-himmelfarb c, handler j, et al. 2014 evidence-based guideline for the management of high blood pressure in adults: report from the panel members appointed to the eighth joint national committee (jnc 8). jama. 2014;311:507–20. upsala journal of medical sciences 57 14. mancia g, fagard r, narkiewicz k, redon j, zanchetti a, b€ohm m, et al. 2013 esh/esc guidelines for the management of arterial hypertension: the task force for the management of arterial hypertension of the european society of hypertension (esh) and of the european society of cardiology (esc). eur heart j. 2013;34:2159–219. 15. committee on the review of omics-based tests for predicting patient outcomes in clinical trials; board on health care services; board on health sciences policy; institute of medicine. evolution of translational omics: lessons learned and the path forward. washington, dc: national academies press (us); 2012. 16. bates d, m€achler m, bolker b, walker s. fitting linear mixed-effects models using lme4. j stat soft. 2015;67:48. 17. halekoh u, højsgaard s. a kenward-roger approximation and parametric bootstrap methods for tests in linear mixed models – the r package pbkrtest. j stat soft. 2014;59:1–30. 18. scuffham pa, nikles j, mitchell gk, yelland mj, vine n, poulos cj, et al. using n-of-1 trials to improve patient management and save costs. j gen intern med. 2010;25:906–13. 19. araujo a, julious s, senn s. understanding variation in sets of nof-1 trials. plos one. 2016;11:e0167167 58 j. sundstr€om et al. abstract background objectives primary objective secondary objectives tertiary objectives study design rationale study visits and periods statistical considerations analysis population primary analyses: individual variation in treatment differences secondary analyses: exploratory biomarker analyses determination of sample size study conduct permissions randomization and blinding data management and monitoring perspective disclosure statement notes on contributors references activated pancreatic stellate cells can impair pancreatic islet function in mice full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 activated pancreatic stellate cells can impair pancreatic islet function in mice guangxiang zang, monica sandberg, per-ola carlsson, nils welsh, leif jansson & andreea barbu to cite this article: guangxiang zang, monica sandberg, per-ola carlsson, nils welsh, leif jansson & andreea barbu (2015) activated pancreatic stellate cells can impair pancreatic islet function in mice, upsala journal of medical sciences, 120:3, 169-180, doi: 10.3109/03009734.2015.1032453 to link to this article: https://doi.org/10.3109/03009734.2015.1032453 © informa healthcare published online: 08 apr 2015. submit your article to this journal article views: 1100 view related articles view crossmark data citing articles: 21 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2015.1032453 https://doi.org/10.3109/03009734.2015.1032453 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1032453 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1032453 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1032453&domain=pdf&date_stamp=2015-04-08 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1032453&domain=pdf&date_stamp=2015-04-08 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1032453#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1032453#tabmodule upsala journal of medical sciences. 2015; 120: 169–180 original article activated pancreatic stellate cells can impair pancreatic islet function in mice guangxiang zang1, monica sandberg1, per-ola carlsson1,2, nils welsh1, leif jansson1 & andreea barbu1,3 1department of medical cell biology, uppsala university, uppsala, sweden, 2department of medical sciences, uppsala university, uppsala, sweden, and 3department of immunology, genetics and pathology, uppsala university, uppsala, sweden abstract background. pancreatic or islet fibrosis is often associated with activated pancreatic stellate cells (pscs). pscs are considered not only to promote fibrosis, but also to be associated with glucose intolerance in some diseases. we therefore evaluated morphological and functional relationships between islets and pscs in the normal mouse pancreas and transplanted islets. methods. immunohistochemistry was used to map the presence of pscs in the normal mouse pancreas and islets implanted under the renal capsule. we isolated and cultured mouse pscs and characterized them morphologically by immunofluorescence staining. furthermore, we measured their cytokine production and determined their effects on insulin release from simultaneously cultured islets. results. pscs were scattered throughout the pancreas, with occasional cells within the islets, particularly in the islet capsule. in islet transplants they were found mainly in the graft periphery. cultured pscs became functionally activated and produced several cytokines. throughout the culture period they linearly increased their production of interleukin-6 and mammalian keratinocyte-derived chemokine. psc cytokine production was not affected by acute hyperglycemia. syngeneic islets cocultured with pscs for 24–48 h increased their insulin release and lowered their insulin content. however, short-term insulin release in batch-type incubations was unaffected after 48 h of co-culture. increased islet cell caspase-3 activation and a decreased islet cell replication were consistently observed after co-culture for 2 or 7 days. conclusion. activated pscs may contribute to impaired islet endocrine function seen in exocrine pancreatitis and in islet fibrosis associated with some cases of type 2 diabetes. key words: beta-cell replication, insulin release, pancreatic islets, stellate cells introduction pancreatic islets constitute complex organs distributed within the pancreas of almost all vertebrates. most interest has been directed towards endocrine cells in generalandbeta-cellsinparticular.however,isletsalso containacomplexstroma,withmanycellscontributing to the unique microenvironment needed for optimal endocrine function. the connective tissue within the isletsincludesacapsuledelineatingitfromtheexocrine tissue, as well as an extracellular matrix (1). pancreatic stellate cells (pscs) are matrixproducers distributed throughout the endocrine and exocrine parts of the gland (2). stellate cells occur in many organs in the body (3), and they have been intensively studied in the liver where they can be identified by the presence of the intermediary filaments vimentin and desmin, and possess vitamincorrespondence: andreea barbu, department of immunology, genetics and pathology, rudbeck laboratory, uppsala university, 75185 uppsala, sweden. fax: +46186110222. e-mail: andreea.barbu@igp.uu.se (received 10 december 2014; accepted 18 march 2015) this is an open-access article distributed under the terms of the cc-by-nc-nd 3.0 license which permits users to download and share the article for noncommercial purposes, so long as the article is reproduced in the whole without changes, and provided the original source is credited. issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1032453 http://informahealthcare.com/journal/ups mailto:andreea.barbu@igp.uu.se a-containing lipid droplets (4). upon stimulation by e.g. cytokines, stellate cells become activated and express nestin and in particular a-smooth muscle actin (a-sma) (5). activated pscs have morphological characteristics of myofibroblasts and pericytes and contribute to fibrogenesis in chronic pancreatitis as well as to the desmoplastic reactions in pancreatic cancer (6-9), both of which have been implicated in impaired glucose metabolism (10,11). despite the importance of pscs in pancreatic pathology their role in impaired glucose tolerance and type 2 diabetes is not well understood. interestingly, there is islet fibrosis, without any significant exocrine engagement in type 2 diabetes (12). it has been suggested that local cytokine production in the islets from macrophages/lymphocytes is responsible for this (12). it may therefore be that pscs contribute to impaired islet function, not only in exocrine pancreatic disease but also in certain types of diabetes. furthermore, pscs are likely to contribute to the frequently seen graft fibrosis occurring after islet transplantation, which is of importance for islet graft dysfunction (13). however, pscs may to some extent be advantageous in this context, since they are known to modulate immune reactions and to improve islet transplant survival, maybe by stimulating graft revascularization (7,14). it has also been suggested that they may constitute a progenitor pool of endocrine cells (15). thus, in view of the possible and ambiguous effects of pscs on islet function, we performed this study to evaluate in more detail the distribution of these cells in the normal mouse pancreas and in transplanted islets and to characterize functional interactions between beta-cells and culture-activated pscs in vitro. material and methods chemicals all chemicals and reagents were from sigma-aldrich (st. louis, mo, usa) unless given otherwise. animals and cells all animal experiments were approved and performed according to the guidelines and regulations of the local committees for animal care at uppsala university. male c57bl/6 or c57bl/6 (nu/nu) mice (taconic, ry, denmark) were used in all experiments. the animals were housed in the animal department at the biomedical centre (uppsala, sweden) and had free access to pelleted food and tap water. mouse pancreatic b-tc6 cells (atcc; manassas, va, usa) were maintained in dulbecco’s modified eagle medium (dmem; gibco, grand island, ny, usa) with 10% fetal calf serum, 2 mm l-glutamine, 100 u/ml benzylpenicillin, 0.1 mg/ml streptomycin (roche diagnostics scandinavia, bromma, sweden) in humidified air and 5% co2 at 37 �c. medium was changed every third day, and the cells were used in passage 3–8. islet isolation, culture, and transplantation c57bl/6 mouse pancreatic islets were isolated and transplanted as previously described in detail (16 (17)). pancreatic islets were prepared by collagenase digestion, as previously described, subsequently handpicked with braking pipettes under a stereo microscope (leica m50, leica microsystems, germany), and maintained free-floating in groups of 150 islets at 37�c (air/co2, 95:5) in 5 ml rpmi 1640 culture medium (sigma-aldrich, st. louis, mo, usa) supplemented with 2 mm l-glutamine, 11 mm glucose, 10% (vol/vol) fetal calf serum, and 0.1 mg/ml streptomycin. syngeneic, adult male, c57bl/6 mice were used as recipients. before transplantation the animals were anesthetized with an intraperitoneal injection of avertin. the left kidney was exposed through a flank incision, and 250 islets were implanted under the renal capsule (17). the animals were then observed until fully recovered from anesthesia, and were kept in separate cages for 2 or 4 weeks. maintenance of the animals and all experiments were approved by and performed according to the guidelines and regulations of the uppsala ethical committees for animal research (permit number: c107/11). isolation and culture of mouse pancreatic stellate cells pscs were isolated by a modification of the method described by apte et al. (18). briefly, pancreatic tissue from mice was minced and digested with 0.1% collagenase a (sigma-aldrich, st. louis, mo, usa) and 0.1% dnase in hanks’s balanced salt solution (hbss; sva, uppsala, sweden) for 10 min. digested tissue was then filtered through a 100-mm nylon cell strainer (bd falcon, franklin lakes, nj, usa). cells were washed and resuspended in hbss. the cell suspension was centrifuged into a 30% (wt/vol) solution of nycodenz (axis-shield, oslo, norway) at 1,400 g for 20 min. pscs separated into a grainy band just above the interface of the nycodenz cushion and the hbss. this band was harvested, and the cells were washed and resuspended indmem containing 10% fbs, 4mmglutamine, and antibiotics (penicillin 100 u/ml and streptomycin 100 mg/ml). cells were maintained at 37�c in a 170 g. zang et al. humidified atmosphere of 5% co2/95% air. the culture medium was replaced the day after initial seeding and subsequently each third day. the purity of the isolated pscs was determined by staining for desmin, vimentin, glial fibrillary acidic protein (gfap), and sma. only isolations with purity >95% were used for further experiments. staining of cells and sections the following antibodies and dilutions were used: pdx-1 primary antibody (sc-14664, santa cruz biotechnology, santa cruz, ca, usa; 1:100, goat polyclonal), cleaved caspase-3 primary antibody (9661, cell signaling technology inc., danvers, ma, usa; 1:200, rabbit polyclonal), desmin (cm036, biocare medical, concord, ca, usa; 1:100, for immunohistochemistry, mouse monoclonal), desmin primary antibody (5332, cell signaling technology inc.; 1:50, for immunofluorescence, rabbit monoclonal), secondary antibody fitc-conjugated donkey antirabbit igg (h+l) (711-095-152, jackson immunoresearch lab., bar harbor, me, usa; 1:500), vimentin (5741, cell signaling technology inc.; 1:100, rabbit monoclonal), secondary antibody fitc-conjugated donkey anti-rabbit igg (h+l) (711-095-152, jackson immunoresearch lab.; 1:500), anti-a-sma primary antibody (sc-32251 santa cruz biotechnology; 1:100, mouse monoclonal), secondary antibody alexa fluor 594 donkey anti-mouse igg (h+l) (invitrogen, eugene, or, usa; 1:500). b-tc6 cells, islets, paraffin-embedded pancreas, and islet-graft containing kidneys were stained as previously described (19). for quantification of pscs we counted the fraction of the area occupied by desmin-positive cells in pancreatic sections or islets implanted under the renal capsule. a square grid (121 intersections) was randomly placed over the sections, and the number of intersections located over desmin-positive cells in both endocrine and exocrine pancreas as well as in islet grafts was estimated. a minimum of 1,210 intersections were counted in each sample. for morphologic characterization, isolated pscs were seeded and cultured in culture slides (bd biosciences, erembodegem, belgium) for 2 or 10 days, washed in pbs, fixed in ice-cold acetone for 15 min at room temperature (rt), and subsequently blocked in pbs supplemented with 3% bsa for 20 min at rt, then incubated with primary antibodies in blocking solution for 16 h at 4�c. thereafter the slides were washed in pbs and incubated with secondary antibodies in pbs 1% bsa for 1 h at rt. nuclear staining was performed by incubation with hoechst 33258 (invitrogen), 1 g/ml, for 30 min at rt. for lipid droplet determination, slides were further incubated for 30 min at rt with nile red (sigma-aldrich, st. louis, mo, usa) solution at a final concentration of 10 g/ml. cells were washed in pbs and analyzed using fluorescence microscopy (zeiss axioplan 2 microscope; carl zeiss, göttingen, germany), using an axiocam hrm camera and an axiovision imaging software. co-culture of pscs and islets following isolation, islets were cultured for 24 h before they were included in any experiments. islets were cultured with or without culture-activated pscs on cover slips. a total of 1 � 105 pscs were seeded in a six-well plate (cover slip ø 25 mm) and 40 islets, pre-incubated for 24 h in medium rpmi 1640, were added 24 h later. all co-culture experiments were performed in medium rpmi 1640 as outlined above for islet cultures. the islets were harvested after 2 or 7 days. in some experiments the removed islets were fixed in methanol for 2 h. they were then blocked with 0.5% pbs, 0.5% fcs, 0.2% triton-x followed by applying a primary antibody against caspase-3 at 4�c overnight. the islets were then washed with pbs, incubated with goat anti-rabbit secondary antibody (a11008, invitrogen; dilution 1:500) for 1 h, washed and mounted with dapi. to study cell proliferation a total of 1 � 105 pscs or 5 � 104 b-tc6 cells were seeded in six-well plates with ø 25 mm cover slips as given above and cultured for 24 h. after this, b-tc6 cells on cover slips or islets were further cultured, with or without pscs, for 24 or 72 h. edu (5-ethynyl-2�-deoxyuridine; 10 mm; invitrogen) was added to the medium for 2 h (b-tc6 cells) or overnight (islets). after this, cells were fixed in 4% formaldehyde for 15 min and permeabilized in 0.5% triton x solution for 20 min. islets were stained for pdx-1 at 4�c overnight, washed with pbs, and further incubated with rabbit anti-goat secondary antibody (a11080, invitrogen; dilution 1:500) for 1 h. finally, they were treated for 30 min with click-it reaction cocktail, mounted with immunofluorescence mounting, as outlined in the description of click-it edu imaging kits (c10339, invitrogen). edu incorporation is shown as the percentage of positive nuclei of the total, for b-tc6 cells, and as percentage of positive nuclei of the pdx-1-positive cells, for islets. insulin release from islets co-cultured with pscs as given above, 40 islets were co-cultured with pscs on cover slips. the rpmi 1640 medium was collected pancreatic stellate cells and pancreatic islets 171 after 12, 24, 36, and 48 h, and its insulin content analyzed by application of the protocol for mouse/rat insulin assay kit (k152bzc-1; meso scale diagnostics, gaithersburg, md, usa) measured on a meso scale diagnostics instrument. for measurement of total insulin content, the islets were harvested and homogenized by sonicating 20 islets in 100 ml redistilled h2o and subsequently mixed with 135 ml 95% acid ethanol. this solution was incubated at –20�c overnight and then centrifuged for 5 min at 10,000 g. the insulin concentration of the supernatant was analyzed following the same protocol as given above. to evaluate possible effects of substances released from culture-activated pscs into the medium we performed studies in which conditioned medium from 48-h cultured pscs was added (5% [vol/vol]) to islet culture medium. islets were further incubated in this mixed medium for 24 h. medium was then removed, and both this and the islets were analyzed for their insulin content as given above. further, isolated mouse islets were cultured alone, pre-cultured for 48 h with culture-activated pscs, and then incubated in krbh during 2 consecutive h at 1.7 and 16.7 mm glucose, respectively. insulin release into the medium during each of these hours was then measured. detection of proinflammatory cytokines released from cultured pscs to determine the effects of the inflammatory reactions present in the diabetic pancreas on the psc’s cytokine expression, isolated pscs in passage 3–8 were either untreated or cultured for 24 h in the presence of one of the following cytokines: 50 u/ml interleukin-1b (il-1b; 200-01; peprotech, rocky hill, nj, usa), 20 ng/ml tumor necrosis factor-a (tnf-a; 310-01a, peprotech), 10 ng/ml interleukin-6 (il-6; 216-16, peprotech), 1,000 u/ml interferon-g (ifn-g; 300-02, peprotech). then the culture medium was collected, and its cytokine contents were analyzed according to the manufacturer’s instructions for mouse proinflammatory 7-plex assay ultra-sensitive kit (k15012c-1, meso scale diagnostics). this allowed us to assess the concentrations of interleukin1b (il-1b), interleukin-6 (il-6), interleukin-10 (il-10), interleukin 12p70 (il-12p70), interferon-g (ifn-g), tumor necrosis factor-a (tnf-a), and mammalian keratinocyte chemoattractant (mkc). cytokines in pscs measured with quantitative real-time pcr cultured pscs were treated with 1.67 mm for 1 h and then for another hour with 16.7 mm glucose in krbh buffer. in separate experiments other pscs were incubated with 11 mm, 25 mm, or 50 mm glucose in the normal culture medium for 96 h. total rna of pscs was extracted according to the procedure of the rneasy mini kit (74104; qiagen, hilden, germany) using on-column dnase digestion with rnase-free dnase set (79254, qiagen). onestep quantitative real-time rt-pcr was performed with quantitect� sybr�green rt-pcr-kit (204243, qiagen) on a lightcycler� real-time pcr machine (lightcycler 2.0; roche). primer sequences are shown in table i. cycle threshold (ct) values were determined with the lightcycler software v3.5 (qiagen). primers for b-actin were used as internal standards. products were analyzed using melting-curve analysis and gel separation. statistical calculations all values are given as means ± sem. probabilities (p) of chance differences between the groups were calculated with anova variance analysis or student’s unpaired t test as given in the text. results stellate cells in pancreas and islet grafts desmin-positive pscs were found throughout the mouse pancreas, associated with intraand interlobular connective tissue in the exocrine parenchyma but also within and surrounding the islets (figure 1a). in islets syngeneically transplanted under the renal table i. primer sequences used in the semi-quantitative real-time pcr studies. b-actin r cac tat ttg gca acg agc gg b-actin f tcc ata ccc aag aag gaa ggc il-1b r acg gat tcc atg gtg aag tc il-1b f gag tgt gga tcc caa gca at il-6 r aat taa gcc tcc gac ttg tga ag il-6 f ctt cca tcc agt tgc ctt ctt g il-10 r ggg cat cac ttc tac cag gta a il-10 f ctg gac aac ata ctg cta acc g ifng r cag cca gga aca gcc atg ag ifn-g f cct ggg gcc tag ctc tga tnf-a r cga tca ccc cga agt tca tnf-a f act gcc aga aga ggc act cc r = reverse; f = forward; il-1b = interleukin-1b; il-6 = interleukin6; il-10 = interleukin-10; ifn-g = interferon-g; tnf-a = tumor necrosis factor-a. 172 g. zang et al. capsule, scattered desmin-positive cells were found, especially in the periphery of the grafts. they were mainly located in the connective tissue capsule overlying the endocrine cell aggregates (figure 1b). there were no differences between the total fractional area occupied by stellate cells in the exocrine pancreas or endogenous and transplanted islets (figure 1c). we separately counted this fraction in the perior intra-insular parts of endogenous or transplanted islets. the peri-insular region was defined as being associated with the capsule surrounding the islets, whereas the intra-islet region was totally within the islets. in endogenous islets, pscs were present to the same degree in these regions (figure 1c). in islet grafts, however, there was a marked enhancement of the area of peri-insular pscs, whilst these cells were very few within the transplanted islets (figure 1c). isolation of pancreatic stellate cells we could routinely isolate and culture pscs with a purity of more than 95%. immediately after isolation they contained cytoplasmic lipid droplets (figure 2a). after culture for a few days the pscs became activated, as represented by morphological changes with disappearance of lipid droplets and a more fibroblastoid appearance (figure 2b). moreover, pscs were immuno-positive for desmin (figure 2c, d), vimentin (figure 2e), and a-sma (figure 2d, e). up to 3 days after isolation, desmin expression was doubled by the peri-nuclear presence of lipid droplets in the cytoplasm, as assessed by nile red staining (figure 2c). at later stages (10–14 days exocrine pancreas a c b d e s m in p o s it iv e a re a i n t o ta l a re a ( % ) endogenous islets transplanted islets *** ** 4 3 2 1 0 t ot al a re a p er i-i ns ul ar s tr om a in tr ais et figure 1. desmin staining (brown) of endogenous pancreas and implanted mouse pancreatic islets. transplants were implanted under the renal capsule 4 weeks before study. a: there are stellate cells within the exocrine parenchyma and within the islets in the endogenous gland, with especially prominent cells seen in the islet capsule. b: stellate cells are also associated with the capsule overlying the transplant. a few scattered cells are also seen within the graft. scale bars 100 mm. c: the fractional area of desminpositive cells in exocrine pancreas and within (intra-islet) or around (peri-insular) endogenous islets and transplanted islets. values are means ± sem for 5–6 experiments. ** p < 0.01, and *** p < 0.001 compared with the value for endogenous islets (student’s t test). a c d e desmin nile red merge/hoechst desmin a-sma merge/hoechst vimentin a-sma merge/hoechst b figure 2. isolation and identification of mouse pancreatic stellate cells. a: pancreatic stellate cell from mouse cultured for 24 h. there are numerous lipid droplets in the cytoplasm. b: activated pancreatic stellate cells have lost lipid droplets and attained a fibroblastoid appearance. c–e: isolated pscs were cultured on culture slides, and their protein expression was analyzed as described in ‘materials and methods’. c: desmin (green) and nile red (denoting lipid droplet presence) (red) fluorescence in pscs cultured for 2 days after isolation. d: desmin (green) and a-sma (red) immunoreactivities in pscs cultured for 14 days after isolation. 20� magnification. e: vimentin (green) and -sma (red) immunoreactivities in pscs for 14 days after isolation. scale bars 50 m (a and c) and 100 m (b, d, e). in all micrographs hoechst 33258 was used for nuclear staining (blue). pancreatic stellate cells and pancreatic islets 173 of culturing) desmin and vimentin expression was paralleled by a strong -sma immunoreactivity (figure 2d, e). co-culture of islets and pancreatic stellate cells when culturing stellate cells and islets together for 2 days there were no adverse morphological effects on either cell type. there was a marked increase in medium insulin concentration after co-culture periods between 24 h and 48 h when compared with culture of the same number of islets alone (figure 3a). pscs cultured alone as expected released no insulin (figure 3a). medium insulin accumulation was unaffected when freshly isolated islets were exposed to pscs for 12 h (figure 3a). on the other hand, islet insulin content was decreased after 48 h of co-culture with pscs (figure 3b). additional experiments were performed after 48 h of coculture, when glucose-stimulated insulin release was measured in a batch-type incubation system. pscs did not significantly affect insulin release in these experiments (data not shown). however, when we considered insulin secretion as percentage of the intracellular insulin content, we found a higher glucose-induced insulin release as well as elevated basal insulin secretion, although the latter did not attain statistical significance (p = 0.067) (figure 3d). when islets were cultured in medium preconditioned with pscs for 24 h there was an increase in insulin release to the culture medium similar to that seen when co-cultured with pscs (figure 3c). however, islet insulin content remained unchanged (data not shown). effects on beta-cell replication and cell death b-tc6 cells were harvested during passage 3–8 and then cultured with or without attached pscs in the culture dish. there was a clear and consistent reduction in edu-staining in pdx-1-positive cells (figure 4a), and this was also seen when mouse a stellate cells m e d iu m i n s u li n ( n g /i s le t) is le t in s u li n ( n g /i s le t) islets islets + stellate cells islets p = 0.06 islets + stellate cells 1.67 mm glucose 16.7 mm glucose islets islets + stellate cells islets islets + stellate cells islets + 5 medium * * * * * *140 120 100 80 60 40 20 0 m e d iu m i n s u li n ( n g /i s le t) in s u li n r e le a s e (% o f in s u li n c o n te n t) 0 50 100 150 200 250 0 2 4 6 8 10 12 14 16 18 1600 1200 800 400 0 24 h 36 h 48 h12 h0 h b c d figure 3. mouse islet insulin release after co-culture with mouse pancreatic stellate cells. a: insulin release into the medium at different time points after co-culture of isolated mouse islets and mouse pancreatic stellate cells. b: islet insulin content in isolated mouse pancreatic islets after 48 h of co-culture with mouse pancreatic stellate cells. c: insulin release from isolated mouse islets cultured alone or with either 5% (vol/ vol) medium conditioned by 24 h of culture with mouse stellate cells. d: insulin release into the medium, presented as fraction of total insulin content, from isolated mouse pancreatic islets co-cultured with isolated mouse pancreatic stellate cells. a total of 40 islets and 105 pancreatic stellate cells were present in each culture dish. during the release experiments islets were incubated in krbh 1 h each at 1.7 and 16.7 mm glucose. insulin concentrations were measured with either meso scale immunoelectrodetection (a, b) or elisa (c, d). values are means ± sem for 4–9 experiments. * p < 0.05 compared with islets alone. 174 g. zang et al. islets were similarly cultured together with pscs (figure 4b). the number of caspase-3-positive islet cells was higher when islets and pscs were co-cultured for 7 days compared with that of islets cultured alone (figure 5). effects of exogenously added glucose and cytokines on pscs cytokine production when pscs were incubated for 1 hat a low and then for 1 h at a high glucose concentration (1.67 or 16.7 mm) we observed no differences in the cytokine expression for tnf-a, ifn-g, il-1b, il-6, or il-10 (table ii). when similar experiments were performed but different glucose concentrations (11, 25, or 50 mm) were maintained for 96 h there was a tendency towards an increase inil-1b inthe pscscultured with 50 mm and a decrease in tnf-a for those cultured with either 25 or 50 mm glucose (table ii). pscs produced high amounts of il-1b, tnf-a, and mkc, and measurable quantities of il-6 and il-12p70 (figure 6). pscs were exposed to different cytokines for 24 h, and addition of il-6 mainly increased the medium concentrations of il-1b and tnf-a (figure 6a), whilst addition of ifn-g increased il-6 medium concentrations (figure 6b). addition of il-1b increased il-6, il-10, and mkc (figure 6c), whereas the combined supplementation with ifn-g and il-1b increased concentrations of il-6, il-10, tnf-a , and mkc (figure 6d) in the culture media. when concentrations of cytokine released into the medium by cultured pscs were followed for 33 days we found a linear increase in il-6 and mkc concentrations, and the increase in concentration was most pronounced in the former (figure 6e). concentrations of the other cytokines were low (figure 6e). discussion pscs have been estimated to constitute nearly 4% of the total number of cells in the normal pancreas (18). their number though is markedly increased at least in animal models in which islet fibrosis seems to play a role in the development of type 2 diabetes. this assumption has been based on previous studies in which psc inactivation was paralleled by reduced islet fibrosis and increased insulin content (20). this is, to our knowledge, the first study with primary islet cells, showing a direct effect of isolated culture-activated pscs on the function and proliferation of insulin-producing cells. we have applied protocols enabling us to detect, isolate, and culture a b mono-culture co-culture mono-culture co-culture e d u p o s it iv e b -t c c e ll s ( % ) *** *** 0 10 20 30 40 50 e d u p o s it iv e i s le t c e ll s ( % ) 0 0.5 1 1.5 2 2.5 3 3.5 1 day 3 days figure 4. effects of mouse pancreatic stellate cells on beta-cell replication. a: b-tc6 cells cultured alone or co-cultured with isolated mouse pancreatic stellate cells for 24 h. b: isolated mouse pancreatic islets cultured without or with isolated mouse pancreatic stellate cells for 72 h. at least 3,000 cells/experimental group were counted for each observation. values are means ± sem for 4–5 experiments. ** p < 0.01, and *** p < 0.001 when compared with mono-cultures. mono-culture co-culture c a s p a s e -3 p o s it iv e i s le t c e ll s ( % ) *** 0 5 10 15 20 25 30 2 days 7 days figure 5. effects of mouse pancreatic stellate cells on islet cell death. fraction of caspase-3-positive cells in islets cultured alone (mono-culture) or after co-culture with isolated pancreatic stellate cells for 2 or 7 days. at least 3,000 cells were counted for each experimental group. values are means ± sem for 4 experiments. *** p < 0.001 when compared with the corresponding monoculture value. pancreatic stellate cells and pancreatic islets 175 pscs and have confirmed that activated pscs produce cytokines, both during basal conditions and especially so after cytokine stimulation. co-culture of activated pscs and isolated islets affected insulin release in short-term batch-type experiments when insulin secretion was calculated as a percentage of insulincontent,reducedinsulincontent,andincreased accumulation of insulin in the medium during prolonged culture periods. islet cell death increased, whereas beta-cell replication decreased. thus, activatedpscsmayplayaroleinthelong-termimpairment of glucose tolerance associated with exocrine pancreatic diseases. however, we cannot quantify how much ofthenegativeeffectoffibrosiscomesfrompscsperse, but our data demonstrate that these cells, when activated, can promote beta-cell dysfunction and death, highlighting the need for further studies of these interactions. furthermore, our results in the mouse insulinoma b-tc6 cell line confirm some of the recently published results suggesting negative functional effects of pancreatic stellate cells on other immortalized insulin-producing cell lines (21,22) as well as in animal models of type 2 diabetes (22). immediately after isolation pscs could be identified by their lipid droplets and positive staining for vimentin, desmin, and gfap (5,23). isolated pscs were negative for platelet-derived growth factor receptors (data not shown), thereby confirming that they were not pericytes (24). pscs in culture became activated as evidenced by the disappearance of lipid droplets and expression of a-sma. all these findings are corroborative of previous studies (5,18). pscs were found throughout the pancreas, and we, in accordance with previous investigators (5,23), chose to use desmin as the staining of choice for their detection after comparing several other markers. in pancreatic islets pscs were mainly associated but not limited to the islet capsule. the capsule composition is species-dependent (25), and cells making up the capsule have been supposed to derive from fibroblasts. in view of our present findings it may be that pscs also participate in the formation of this structure. it should be noted that psc activation is prevented by somatostatin (26) produced in islet dcells in rodents, which are preferentially located in the islet mantle (27). it can be conceived that high local somatostatin concentrations would maintain pscs in the islet periphery quiescent. interestingly, the presence of a continuous interstitial matrix connection between the endocrine and exocrine pancreas has been previously suggested, which is lost due to fibrosis in rodent models and humans with type 2 diabetes mellitus (28). this organized, fibrillar collagen was closely associated with pericytes, which were proposed to be able to differentiate into myofibroblasts/pscs (28). indeed, when activated pscs were suppressed by conophylline in gk rats, a type 2 diabetes model, islet fibrosis was reduced (20), supporting a role for pscs in this process. this interesting concept is worthy of further investigations. after syngeneic transplantation under the renal capsule desmin-positive cells were found, mainly in the renal capsule over the graft, and their combined table ii. cytokine expression in pscs as determined by semi-quantitative real-time pcr. pscs were either incubated for 2 h in krbh buffer (1 h 1.67 mm followed by 1 h 16.7 mm glucose concentration or without any glucose at all for 2 h) or incubated for 96 h at different glucose concentrations. incubation protocol 2 h krbh buffer 96 h culture with rpmi media no glucose 1 h each of 1.7 mm + 16.7 mm glucose 11 mm glucose 25 mm glucose 50 mm glucose ifn-g /b-actin mrna expression (arbitrary units) 13.10 ± 0.30 12.49 ± 0.78 18.91 ± 0.94 19.28 ± 0.61 19.03 ± 0.59 tnf-a /b-actin mrna expression (arbitrary units) 5.15 ± 0.33 4.78 ± 0.38 18.29 ± 0.22 17.71 ± 0.50 17.42 ± 0.39 il-1b /b-actin mrna expression (arbitrary units) 6.05 ± 0.14 5.56 ± 0.25 16.89 ± 0.56 16.94 ± 0.41 18.20 ± 0.45 il-6/b-actin mrna expression (arbitrary units) 1.33 ± 0.20 1.23 ± 0.38 6.70 ± 0.20 6.45 ± 0.27 6.56 ± 0.32 il-10/b-actin mrna expression (arbitrary units) 8.97 ± 0.15 8.76 ± 0.07 nd nd nd relative mrna expression was calculated using the formula: relative expression = 2-dct, where dct = ct gene of interest – ctb-actin. values are means ± sem for 4–5 experiments. ifn-g = interferon-g; tnf-a = tumor necrosis factor-a; il-1b = interleukin-1b; il-6 = interleukin 6; il-10 = interleukin-10; nd = not determined. 176 g. zang et al. area was approximately the same as in the endogenous islets. this location is somewhat surprising, since a pronounced central fibrosis is usually seen in intrarenal grafts, probably due to hypoxia (29). the present findings do not support a major role for pscs in islet graft fibrosis. this is in contrast to the role played by pscs in both experimental animals and man in fibrosis seen in chronic pancreatitis and the desmoplastic reactions associated with pancreatic adenocarcinomas (2). in the latter condition pscs contribute to the tumor-promoting and immunosuppressive environment (6-8). as will be discussed in more detail ifn-γ tnf-α mkcil-10 il-12 il-1β ifn-γ tnf-α mkc il-10 il-6 il-12 p70 il-1β ifn-γ + il-1β pbs a b c d e il-6 pbs pbs il-1β pbs ifn-γ *** p70 ifn-γ tnf-α mkcil-10 il-12 il-6 p70 tnf-α mkcil-10 il-12 il-6il-1β p70 tnf-α mkcil-10 il-12 il-6 p70 m e d iu m c y to k in e c o n c e n tr a ti o n ( p g /m l) ** * * ** *** *** *** *** ** ** ** *** 10,000 1000 100 10 1 0.1 0.01 m e d iu m c y to k in e c o n c e n tr a ti o n ( p g /m l) 10,000 100,000 1000 100 10 1 0.1 0.01 m e d iu m c y to k in e c o n c e n tr a ti o n ( p g /m l) 10,000 100,000 1000 100 10 1 0.1 0.01 m e d iu m c y to k in e c o n c e n tr a ti o n ( p g /m l) 10,000 100,000 1000 100 10 1 0.1 0.01 c y to k in e c o n c e n tr a ti o n i n m e d iu m ( p g /m l) 0 500 1000 1500 2000 2500 3000 3500 4000 4500 3 5 7 9 13 18 22 33 dsys figure 6. medium release of interferon-g (ifn-g), interleukin-10 (il-10), interleukin 12p70 (il-12p70), interleukin-1b (il-1b), interleukin 6 (il-6), tumor necrosis factor-a (tnf-a), and keratinocyte-derived chemokine (mkc) from pscs after incubation for 24 h either alone or with added cytokines. the latter consisted of il-6 (a: 10 ng/ml), ifn-g (b: 1,000 u/ml), il-1b (c: 50 u/ml), or ifn-g + il-1b (d: 1,000 u/ml + 50 u/ml). e: cytokine concentrations in medium from mouse pancreatic stellate cells after different times of culture. values are means ± sem for 3–7 experiments. note logarithmic scale in a–d. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the corresponding control value. in e, values for mkc and il-6 are higher (p < 0.01) at all times compared with their respective value at day 3. pancreatic stellate cells and pancreatic islets 177 below, both these conditions with pancreatic fibrosis are associated with an increased incidence of type 2 diabetes (10,11), as is islet fibrosis in itself (12). co-cultures of pscs and islets were performed to evaluate possible direct interactions affecting endocrine function. after >12 h of co-culture a marked increase of insulin released into the medium was detected, and it was associated with a decrease in islet insulin content. under normal physiological conditions pancreatic beta-cells maintain a remarkably stable balance between insulin secretion and insulin production. whenever glucose stimulates insulin release, there is a rapid and corresponding increase in proinsulin biosynthesis that efficiently replenishes intracellular insulin stores (30,31). however, it is possible that chronic exposure to paracrine interactions with activated pscs evokes a continuous stimulation of insulin release, which is only partially compensated for by a sustained insulin biosynthesis. this, in turn results in a decrease of the intracellular insulin stores. that the increased medium insulin release was due to beta-cell death, with leakage of insulin, seems unlikely, since the culture conditions were optimized for islets and we could detect no increased cell necrosis with propidium iodide staining. an increased apoptosis was seen as evidenced by caspase-3 staining, but this should only marginally affect the insulin concentrations. therefore it seems as if activated pscs stimulate insulin release from beta-cells. this was at least partially due to a factor released from the pscs, since pscs-conditioned medium had similar effects. when we performed acute batch-type release experiments there were no differences in insulin release between islets cultured alone or co-cultured islets and pscs after 48 h of preculture. however, it should also be considered that pscs-mediated elevation of insulin release into the medium resulted in a significant reduction in the insulin content of pancreatic islet cells. thus, when insulin secretion was considered as a percentage of the intracellular insulin content, we found that pscs significantly potentiated glucose-induced insulin release as well as elevating basal insulin secretion. viewed in this way, co-culture of beta-cells with activated pscs over a 48-h period in vitro enhanced rather than inhibited glucose-induced insulin release, which points out a possible additional mechanism by which activated pscs present in the fibrotic pancreas can contribute to beta-cell exhaustion in the pathogenesis or amplification of type 2 diabetes. in the co-culture experiments we found an increased apoptotic cell death and a decreased replication rate in the islet cells. the latter was seen also in beta-cell lines. this is an interesting finding since, as mentioned above, both chronic pancreatitis and pancreatic adenocarcinomas are associated both with the presence of activated pscs with increased matrix formation and an increased incidence of type 2 diabetes (10,11). the reasons for the glucose intolerance have been suggested to be the fibrosis per se, with destruction of the pancreatic architecture, especially in chronic pancreatitis (11), as well as production of various tumor-derived factors exerting diabetogenic effects in the pancreas and liver (32). our present findings open up the possibility that also an increased beta-cell death and defect replication may be involved in the type 2 diabetes pathogenesis during these conditions. this is in line with some studies in chronic pancreatitis in man (33) and pancreatic cancers (34). islet fibrosis per se has been suggested to play an important role also in type 2 diabetes in man and experimental animals (12,35). in a secreted proteome analysis of quiescent and activated human pscs many pro-apoptotic proteins were expressed, whereas those stimulating proliferation mainly affected tumor cells rather than islet cells (36). interestingly, it has previously been demonstrated that activated rat pscs produce connective tissue growth factor (ctgf), which binds to integrin 51 and exerts a profibrogenic effect (37). the diabetic pancreas is characterized by immunological (type 1 diabetes) or inflammatory (type 2 diabetes) reactions in which cytokines are important players, facilitating functional impairment and cytotoxic effects on pancreatic islet cells. therefore we found it of interest to evaluate pscs’ cytokine production following a hyperglycemic challenge as well as in the presence of cytokines proven to be present around and within the diabetic endocrine pancreas. in our hands, pscs produced large amounts of il-1b, tnf-a , and measurable quantities of il-6 and il-12p70. this is in line with previous studies on cultured stellate cells from other organs. furthermore, we demonstrate here, for the first time in stellate cells, high expression of mkc, a functional homolog of human interleukin il-8, which is central for induction of th1 responses. a continuous increase in il-6 and mkc secretion was observed, whilst other cytokine concentrations did not change significantly, which is in line with previous studies (38,39). the cytokine release was not affected by an acute 1-h exposure to hyperglycemia, besides a marginal effect of very high (50 mm) glucose concentrations on il-1b. the rationale for studying this was previous observations that both hyperglycemia and hyperinsulinemia stimulate activation and proliferation of pscs through erk 1/2 phosphorylation in vitro (40). moreover, when pscs were exposed to exogenously added high doses of cytokines we found that il-6 and tnf-a had only minor effects 178 g. zang et al. on their cytokine secretion. exogenous il-1b increased release of both il-6 and mkc, whereas a combination of il-1b and ifn-g increased il-12p70, il-6, and mkc secretion. all of these cytokines may therefore, in an autocrine fashion, act in synergy to promote pancreatic fibrosis (2) and contribute to the impaired islet endocrine function and islet cell survival associated with some cases of type 2 diabetes. to summarize we report a direct effect of cultureactivated pscs on the viability and proliferation rate of mouse islet cells in in vitro co-culture experiments, paralleled by a stimulated insulin release both at basal levels and after a glucose challenge. our data suggest a possible additional mechanism by which activated pscs present in the fibrotic pancreas can contribute to beta-cell exhaustion. this could be involved in the pathogenesis or amplification of type 2 diabetes and opens up for new treatment scenarios based on modulation of the activation of pscs. acknowledgements the skilled assistance of ing-britt hallgren is gratefully acknowledged. leif jansson and andreea barbu contributed equally to this work. funding: this work was supported by the swedish research council (521-2011-3777), the juvenile diabetes research foundation, an efsd/novo nordisk grant, the swedish diabetes association and the family ernfors fund. the study was also supported by grants in the name of michael welsh, uppsala university (swedish diabetes association, swedish research council and swedish cancer association). declaration of interest: there 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discussion acknowledgements declaration of interest references surface glycans contribute to differences between seminal prostasomes from normozoospermic and oligozoospermic men article surface glycans contribute to differences between seminal prostasomes from normozoospermic and oligozoospermic men bojana milutinovi�c, sanja go�c , ninoslav miti�c , maja kosanovi�c and miroslava jankovi�c university of belgrade, institute for the application of nuclear energy, inep, zemun, serbia abstract background: extracellular vesicles (evs), released from the plasma membrane or intracellular compartments, have a specific composition related to their parent cells, but they can, additionally, be modified by the extracellular environment. although glycans are known to contribute to ev composition and may have biomedical importance as biomarkers and recognition signals, they have not been extensively investigated. in this study, seminal prostasomes, i.e. evs from seminal plasma (sp) of normoand oligozoospermic men, were analyzed in order to detect possible changes in their surface glycans under altered physiological conditions. methods: prostasomes were isolated from pooled sp by differential centrifugation and gel filtration, followed by glycobiochemical characterization using lectin/immune-transmission microscopy and ionexchange chromatography. results: within the frame of overall similarity in protein composition, surface glycans specifically contributed to the differences between the examined groups of prostasomes in terms of presentation of sialylated and mannosylated moieties. these changes did not affect their anti-oxidative capacity, but implied a possible influence on the accessibility of galectin-3 to its ligands on the prostasomal surface. conclusions: subtle differences in the presentation of surface molecules may be helpful for differentiation among vesicles sharing the same physical properties. in addition, this may point to some unexpected regulatory mechanisms of interaction of distinct populations of vesicles with their binding partners. article history received 11 december 2018 revised 1 march 2019 accepted 5 march 2019 keywords glycosylation; prostasomes; seminal plasma; sialic acid introduction extracellular vesicles (evs) are nano-sized membraneenclosed structures released by all cells in the extracellular environment (1–4). evs’ ability to carry molecules, to pass through biological fluids, cross barriers, and target specific cells provides them with a great potential to be used as next-generation natural nano-tools (5,6). diversity in the composition of lipids, proteins, and nucleic acids associated with evs is related to parent cells or tissues, and, in this way, they act as specific biogenetic markers, at the same time carrying markers common to all evs (7,8). however, it is possible that evs could also be modified by the extracellular environment, but data concerning externally adsorbed components including glycoproteins are scarce (9). thus, both influences pervade evs’ composition, leading to the formation of a distinct molecular pattern of their luminal/internal cargo, the membrane itself, as well as their surface cargo (9). some of these patterns were shown to be changed under different physiological and pathophysiological conditions (2) and, consequently, may have significant biomedical importance. this study is a comparative analysis of evs isolated from seminal plasma (sp) of normoand oligozoospermic men, focused on their glycosylation. it is known that a majority of evs in sp are prostasomes, evs secreted by the prostate (10). they were originally examined in prostatic fluid and subsequently in sp (10–12). in contrast to accumulated experimental data on prostasomal ultrastructure and composition of proteins and lipids (10), glycans, i.e. complex oligosaccharides, have not yet been studied in detail. an altered glycoproteome of sp has already been observed and related to different sperm count and fertility status in comparison to sp obtained from ejaculate with normal parameters (13,14). however, few studies have addressed the diversity of prostasomes in related conditions. in general, quantitative rather than qualitative differences in the examined prostasome parameters were found in sp from infertile subjects or those with abnormal semen parameters in comparison to values for sp of healthy men. thus, in one study, the number of vesicles and associated enzymatic activity was found to be very low in azoospermia (15). differences in levels of expression of some common proteins were reported recently, most of which were overexpressed in prostasomes from contact miroslava jankovi�c miraj@inep.co.rs institute for the application of nuclear energy, inep, university of belgrade, banatska 31b, 11080 zemun, serbia � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 2, 111–118 https://doi.org/10.1080/03009734.2019.1592266 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1592266&domain=pdf&date_stamp=2019-05-31 http://orcid.org/0000-0002-6104-693x http://orcid.org/0000-0003-2756-6517 http://orcid.org/0000-0002-1143-1805 http://orcid.org/0000-0002-7889-5110 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2019.1592266 http://www.tandfonline.com normozoospermic men compared to those from non-normozoospermic (16). thus, we aimed at resolving potential differences in surface glycans of prostasomes from selected sp samples. as recognized components of lipids and proteins, surface glycans are presumed to contribute significantly to different functional activities of prostasomes. as in the case of cell–cell and cell–molecule interactions where carbohydrate recognition plays a crucial role, glycans on prostasomes could constitute molecular requirements for their action as a communication tool and harbor biomarker structures (17). according to biogenesis data, the prostasomal membrane is kindred to the plasma membrane of the ancestral epithelial cell (18) and, in this way, may be a reflection on the vesicle surface of an altered status of prostate physiology, as already proposed for reflection on vesicle cargo (16,18). materials and methods material monoclonal anti-cd63 antibody (clone ts63) was from abcam (cambridge, uk) and biotinylated goat anti-galectin-3 (gal-3) antibodies from r&d systems (minneapolis, mn, usa); 3,30,5,50-tetramethylbenzidine (tmb), bovine serum albumin (bsa), d-lactose, and methylalpha d-mannopyranoside were from sigma (st. louis, mo, usa). biotinylated goat antimouse igg, biotinylated plant lectins, concanavalin a (cona), sna (sambucus nigra agglutinin), and the elite vectastain abc kit were from vector laboratories (burlingame, ca, usa). sephadex g 200 and sephadex deae a-50 were from pharmacia ab, uppsala, sweden. the silver stain kit and sdspage molecular mass standards (broad range) were from bio-rad (hercules, ca, usa). nitrocellulose membrane and pierce ecl western blotting substrate were from thermo scientific (rockford, il, usa). bca protein quantification kit was from abcam (cambridge, uk). microwell plates were from thermo scientific (roskilde, denmark). lymphocyte separation medium lsm-b was from capricorn scientific gmbh (ebsdorfergrund, germany). human semen samples this study was performed on leftover, anonymized specimens of human semen taken for routine analysis, and since existing human specimens were used it is not considered research on human subjects. the study was approved by the institutional ethics committee according to the guidelines (no.# 02-832/1), which conform with the helsinki declaration, 1975 (revised 2008). sperm parameters were assessed according to the recommended criteria of the world health organization (released in 2010), concerning numbers, morphology, and motility. sperm cells and other debris were removed from the ejaculate by centrifugation at 800 � g for 20min. isolation of prostasomes from human seminal plasma two pools of human sp of normozoospermic (n) and two pools of human sp of oligozoospermic (o) men were used for isolation of prostasomes. each pool contained 10 individual seminal plasma samples. prostasomes were isolated from sp according to a modified protocol of carlsson et al. (19) in that cd63-immunoreactivity was used as an indicator of the presence of evs. ion-exchange chromatography (iec) of seminal prostasomes a deae sephadex a-50 column (10 ml bed volume) equilibrated with 0.05 m tris–hcl buffer, ph 7.6, was used for iec of evs as described before (20). briefly, after loading of prostasomes (1 ml), non-bound material was washed away with equilibration buffer, followed by step-by-step elution with 0.05 m tris–hcl buffer, ph 7.6, containing 0.05 m nacl, 0.1 m nacl, 0.2 m nacl, and 1 m nacl. fractions (1 ml) were collected, immobilized on microwell plates, and subjected to a lectin-binding assay using cona and sna to monitor elution of charge-resolved populations (20). initially, specificity of the lectin-binding was confirmed by inhibition using 0.2 m lactose (for sna) and 0.2 m methylalpha d-mannopyranoside (for cona). sds-page proteins were resolved on 10% separating gel with 4% stacking gel under denaturing and reducing conditions (21) and stained with silver nitrate, using a silver stain kit (bio-rad) according to the manufacturer’s instructions. the gel was calibrated with sds-page molecular weight standards (broad range). western and dot blot samples were transferred onto nitrocellulose membrane by semi-dry blotting using a trans-blot sd (bio-rad). the conditions were: transfer buffer, 0.025 m tris containing 0.192 m glycine and 20% methanol, ph 8.3, under a constant current of 1.2 ma/cm2 for 1 h. the membrane was blocked with 3% bovine serum albumin (bsa) in 0.05 m phosphate buffer saline (pbs), ph 7.2, for 2 h at room temperature, and then incubated with anti-cd63 antibody as described below. for dot blot, 3 ml of each corresponding fraction was applied to the nitrocellulose membrane, dried, and blocked as described above. immunoblot analysis for immunoblot analysis, proteins on the membrane were incubated with anti-cd63 antibody (20 lg/ml) overnight at 4 �c. after a washing step, bound antibody was detected by incubation with biotinylated goat anti-mouse igg for 30 min at room temperature. the membrane was rinsed, and the hrpo mixture from the elite vectastain abc kit was added. after another washing step the blots were visualized using pierce ecl western blotting substrate according to the manufacturer’s instructions. 112 b. milutinovi�c et al. transmission electron microscopy (tem) and lectin-tem tem was performed as described previously (20). for lectintem, samples were applied to the formvar-coated, 200mesh, cu grids by grid flotation on 10 ll sample droplets, for 45 min at room temperature. this was followed by steps of: fixation (2% paraformaldehyde, 10 min); washing (pbs, 3 � 2 min); quenching (0.05 m glycine, 10 min); blocking (2% bsa, 30 min); washing (0.1% bsa/pbs, 6 � 2 min); binding of either (a) biotinylated lectins (sna, 0.4 lg/ml and cona, 0.62 lg/ml, 1.5 h at room temperature) or (b) biotinylated anti-gal-3 igg (0.5 lg/ml of 1% bsa/pbs, 1.5 h at room temperature); binding of streptavidin-gold (1:100 in 0.1% bsa/ pbs, 1 h at room temperature); washing (0.1% bsa/pbs, 3 � 2 min); post-fixing (2% glutaraldehyde, 5 min); and final wash with dh2o. grids were then air-dried and images were collected using a philips cm12 electron microscope (philips, eindhoven, the netherlands). preparation of polymorphonuclear neutrophils (pmn) from whole blood pmn were isolated from a healthy blood type o þ donor. whole blood was collected in edta-containing tubes and left to sediment for 2 h at room temperature. the supernatant and upper sediment were layered on 3 ml of lymphocyte separation medium and centrifuged at 1800 rpm for 30 min. the pellet, containing pmn and erythrocytes, was treated with isotonic 0.15 m nh4cl containing 4 mm nahco3 lysing solution, ph 7.3, for 10 min on a rotating mixer. the residual neutrophils were washed in 0.05 m pbs, ph 7.2, and pelleted by centrifugation at 1800 rpm for 10 min. isolated pmn were counted manually using a neubauer chamber. determination of reactive oxygen species (ros) production in pmn pmn activation assay was performed as previously described (22). briefly, 50 ml of cell suspension (250,000 cells), 10 ml of the corresponding sample (total protein concentration 100 mg/ml), 10 ml of luminol, and 10 ml of phorbol myristate acetate (pma) (40 mg/ml) were added. to monitor baseline ros production, the samples were substituted with 0.05 m pbs, ph 7.2. chemiluminescence was measured using a wallac 1420 multilabel counter (perkinelmer, monza, italy) over 30 min with a 5-min counting delay and expressed as arbitrary units (au). results are presented as the mean value with standard deviation (sd) of three separate experiments. statistical analysis was carried out using paired student’s t test, with mean values considered significantly different when p < 0.05. results electron microscopy there were light and dark vesicles ranging from 50 to 150 nm in both groups of samples. however, in contrast to seminal prostasomes of normozoospermic men (figure 1(a)), those of oligozoospermic men contained more amorphous substance with one or several vesicles appearing immersed in them (figure 1(b)). sds-page when isolated seminal prostasomes were resolved electrophoretically, complex patterns of differently abundant and partially overlapping protein bands ranging from 35 kda to over 250 kda were observed (figure 2(a)). in addition, there was comparable immunoreactivity to cd63, the common marker for evs (figure 2(b)). iec the surface composition of examined seminal prostasomes was initially inspected by iec (figure 3). there were no striking differences within the samples. thus, representative elution profiles of charge-resolved prostasomes of normozoospermic and those of oligozoospermic men, as detected by sambucus nigra agglutinin and cona, suggested a major population released with 1 m nacl (constitutively present in all four pools) in both samples. it was positive for cd63 by dot blot, indicating a concentration of negatively charged vesicles in it. the sna-binding pattern of a population of prostasomes from normozoospermic men eluted with 1 m nacl revealed a relatively sharp peak exhibiting a hint of unseparated populations in its trailing edge, whereas at least two partially separated subpopulations were observed figure 1. transmission electron microscopy of seminal prostasomes. inserts show enlarged the typical appearance of seminal prostasomes from normozoospermic men (n) (no protein in the background) and seminal prostasomes from oligozoospermic men (o) (protein material in the background with vesicles seemingly immersed). upsala journal of medical sciences 113 among prostasomes of oligozoospermic men. in contrast to this, cona-binding patterns seemed to be more heterogeneous, appearing as several more or less separated subpopulations. in addition to vesicles-enriched populations (cd63-positive), both sna and cona revealed lower-charge cd63-negative ones (occasionally present). in general, there was a broad elution profile across these populations, especially for prostasomes of oligozoospermic men. thus, in prostasomes of oligozoospermic men, there were cd63-negative populations eluted in the flow-through fraction as well as with 0.05 m, 0.1 m, and 0.2 m nacl. the population eluted by 0.2 m nacl reacted with sna and cona, whereas the others reacted with cona only. in prostasomes of normozoospermic men, one cd63-negative population was separated in the flow-through fraction, and it was cona-reactive. lectin-tem the subtle differences implied by iec between prostasomes of normozoospermic men and those of oligozoospermic men were further analyzed using lectin-tem. native samples were analyzed by means of both techniques, but, in contrast to iec, figure 3. surface glycosylation of seminal prostasomes: ion-exchange chromatography. two pools of each group of isolates were subjected to ion-exchange chromatography on deae a-50 column eluted with 0.05 m tris–hcl buffer, ph 7.6 (a), containing: 0.05 m nacl (b); 0.1 m nacl (c); 0.2 m nacl (d), and 1 m nacl (e). elution was monitored by sambucus nigra agglutinin (sna) and concanavalin a agglutinin (cona) binding reactivity. n pool 1 (thick line) and n pool 2 (thin line) had protein concentration 313 mg/ml and 473 mg/ml, respectively. o pool 1 (thick line) and o pool 2 (thin line) had protein concentrations of 318 mg/ml and 581 mg/ml, respectively. asterisk denotes cd63-positive fractions as determined by dot blot. (n ¼ seminal prostasomes from normozoospermic men; o ¼ seminal prostasomes from oligozoospermic men). figure 2. protein composition of seminal prostasomes. seminal prostasomes isolates were resolved on 10% sds-page under reducing and denaturing conditions and stained with silver or transferred onto a membrane and subjected to immuno-blot. a: a representative total protein pattern of seminal prostasomes with three characteristic bands (12) in the region of 90–150 kda (asterisk). b: cd63, an evs-associated marker gave characteristic smeared band above 31 kda. the numbers indicate the position of molecular mass standards (kda). (n ¼ seminal prostasomes from normozoospermic men; o ¼ seminal prostasomes from oligozoospermic men). 114 b. milutinovi�c et al. which concentrates different vesicles according to their general negative charge, lectin-tem is expected to give more insight into presumed heterogeneity among particular vesicles based on homogeneity/intensity of staining of selected glycans on their surface. in the preparation of prostasomes from normozoospermic men, sna bound to all vesicles, having a rosette-like appearance, whereas not all prostasomes of oligozoospermic men were stained. in contrast to vesicles, no staining of surrounding proteins was observed (figure 4(a)). as for cona only some vesicles were stained in the preparation of prostasomes of normozoospermic men (figure 4(b)). in contrast to that, almost all vesicles in the preparation of prostasomes of oligozoospermic men were stained. in addition, in prostasomes of oligozoospermic men, there was staining of some amorphous material as well. in the context of surface composition, the evs marker gal3 was also examined (figure 4(c)). it was observed in both sample groups by immune-tem, and there were no striking differences as regards its antigenic moieties in prostasomes of normozoospermic men compared to those of oligozoospermic men. ros production in pmn the influence of differences in surface composition on known antioxidative activity of prostasomes was checked using an assay on pmn. ros production increased markedly after stimulation with phorbol myristate acetate, reaching its maximum after 30 min (figure 5(a)). in the presence of populations of prostasomes of normozoospermic men and those figure 4. surface glycosylation of seminal prostasomes: lectinand immune-transmission electron microscopy. a: lectin-tem using sna. inserts show enlarged characteristic pattern of sna-reactivity to each sample group. b: lectin-tem using cona. inserts show enlarged characteristic pattern of cona-reactivity to vesicles in each sample group. in o, staining of some proteinaceous material was also observed (arrowheads). c: immune-tem using anti-galectin-3 antibodies. inserts show enlarged characteristic pattern of anti-gal-3-reactivity to each sample group. micrographs show most characteristic patterns obtained. although differences in the reactivity of particular vesicles could be noticed, it does not affect the general reactivity of the sample (as in iec when taking all vesicles into account). (n ¼ seminal prostasomes from normozoospermic men; o ¼ seminal prostasomes from oligozoospermic men). upsala journal of medical sciences 115 of oligozoospermic men eluted with 1m nacl, ros production was lower, but still present (figure 5(a)). at maximum ros production time (30 min) this decline was statistically significant for all tested samples (figure 5(b)). discussion in agreement with data on disparate sp compositions in normoand oligozoospermic men (13,23–25), and presumed influence of extracellular environment on evs, the results obtained indicated distinct differences in surface glycans of related seminal prostasomes. altered presentation of sialylated and mannosylated structures was clearly observed when corresponding samples were analyzed using tem, whereas both iec and total protein patterns were not so decisive/comprehensive. it is known that evs comprise a continuum of particles of different size, morphology, and density (1). these parameters have been shown to vary considerably between ev preparations (also influenced by the techniques used), making ev heterogeneity in individual preparations intrinsic and general (26). since we approached ev surface glycosylation, these parameters were found not relevant for our examination. consequently, glycosylation was analyzed on prostasomes isolated from pooled sp samples in order to average individual sample heterogeneity, whereas surface glycosylation as revealed by iec was taken as a reference for comparison. thus, when resolved on iec, prostasomes in both sample groups (also in each of their pools) concentrated in the highest-charge cd63-positive population. it exhibited broad elution profile that indicated two differently abundant subpopulations being in agreement with previous data on their existence (10). cona-binding patterns which follow sna-binding patterns in the highest-charge population (prostasome-associated) are assumed to reflect the specificities of the extracellular environment where they are released. additional heterogeneity, especially in prostasomes from oligozoospermic men, seen as cona-binding to lower-charge populations (non-prostasomal), could be due to separation of co-isolated proteins/protein complexes, which represent non-specific/common contaminants as shown previously (20). in this study that was supported by their variable presence in all examined sp pools. indeed, when prostasomal surface glycans were visualized by lectin-tem, another aspect of iecassumed heterogeneity between the examined samples clearly emerged. subtle differences regarding distribution of sna-binding sites, but pronounced ones regarding those of cona, were revealed in prostasomes from normozoospermic compared to prostasomes from oligozoospermic men. sna, specific for sialylated structures, bound only vesicles (with no respect to sample examined), whereas cona specific for various mannose-containing sialylated and non-sialylated nglycans additionally bound to material known as amorphous substance (27,28). this was more pronounced in prostasomes isolated from sp with abnormal semen parameters. separation of prostasomes, in the highest-charge iec fraction, is in accordance with data indicating that they display a net negative surface charge (29). in relation to this, it is known that prostasomes inhibit production of reactive oxygen species by stabilizing the plasma membrane (22,30) and that this effect is expected to rely on surface charge. in general, anionic nanoparticles do not pass through the cell membrane, i.e. they prevent membrane damage/hinder decomposition (31). when differences in surface glycans (associated with negatively charged prostasomes) were examined to discover whether they have implications on the antioxidative capacity of prostasomes from normozoospermic and oligozoospermic men, the results obtained indicated that ros production in pmn was decreased with no differences between the examined samples. the possibility could not be overlooked that changes in the presentation of surface glycans may have more pronounced effects on other types of known prostasomal activity (32), involving protein–protein or protein–carbohydrate interactions at the level of a particular molecule. in relation to this, gal-3, known as part of the prostasomal proteome (33,34), was found to be present on the surface of vesicles from normozoospermic and oligozoospermic men. gal-3 is a versatile carbohydrate-binding protein involved in basic physiological processes (35,36). however, regardless of any biological consequence of its figure 5. the effect of seminal prostasomes on reactive oxygen species (ros) production in polymorphonuclear neutrophils (pmn). a: kinetics of ros production in pmn after activation with 12-myristate-13-acetate phorbol ester (pma) alone (max) or in the presence of charge-resolved population of n and o, eluted with 1 m nacl. b: comparison of ros production (%) in pmn after 30 min of pma addition, alone or in the presence of charge-resolved population of n and o, eluted with 1 m nacl shows statistically significant decrease for all. the results are mean values of three experiments, and vertical bars present percent of standard deviation. asterisk (�) indicates p < 0.05. (au ¼ arbitrary chemiluminescence units; n ¼ seminal prostasomes from normozoospermic men; o ¼ seminal prostasomes from oligozoospermic men). 116 b. milutinovi�c et al. possibly altered accessibility to binding partners in the context of differently presented glycans, this may represent an interesting and unexpected regulatory mechanism, in general. it has already been shown that the glycans of evs contribute in a specific way to the complexity of the normal sp glycome compared to other sp constituents (37). in this study, a further distinction among prostasomes was visualized at the level of particular vesicles, pointing to molecular arrangements/setup at the ultrastructural level as a naturally occurring process and an overlooked source of heterogeneity. disclosure statement no potential conflict of interest was reported by the authors. funding this work was supported by the ministry for education, science and technological development of the republic of serbia under grant 173010. notes on contributors bojana milutinovi�c, phd, research associate, department for immunochemistry and glycobiology, institute for the application of nuclear energy inep, university of belgrade, serbia. sanja go�c, phd, research associate, department for immunochemistry and glycobiology, institute for the application of nuclear energy inep, university of belgrade, serbia. ninoslav miti�c, phd, research associate, department for immunochemistry and glycobiology, institute for the application of nuclear energy inep, university of belgrade, serbia. maja kosanovi�c, phd, research associate, department for immunochemistry and glycobiology, institute for the application of nuclear energy inep, university of belgrade, serbia. miroslava jankovi�c, phd, principal research fellow, department for immunochemistry and glycobiology, institute for the application of nuclear energy inep, university of belgrade, serbia. orcid sanja go�c http://orcid.org/0000-0002-6104-693x ninoslav miti�c http://orcid.org/0000-0003-2756-6517 maja kosanovi�c http://orcid.org/0000-0002-1143-1805 miroslava jankovi�c http://orcid.org/0000-0002-7889-5110 references 1. raposo g, stoorvogel w. extracellular vesicles: exosomes, microvesicles, and friends. j cell biol. 2013;200:373–83. 2. y�a~nez-m�o m, siljander pr-m, andreu z, zavec ab, borr�as fe, buzas ei, et al. biological properties of extracellular vesicles and their physiological functions. j extracell vesicles. 2015;4:27066. 3. gassart a de, geminard c, fevrier b, raposo g, vidal m. lipid raftassociated protein sorting in exosomes. proteins. 2003;102: 4336–44. 4. colombo m, moita c, van niel g, kowal j, vigneron j, benaroch p, et al. analysis of escrt functions in exosome biogenesis, composition and secretion highlights the heterogeneity of extracellular vesicles. j cell sci. 2013;126:5553–65. 5. fais s, o’driscoll l, borras fe, buzas e, camussi g, cappello f, et al. evidence-based clinical use of nanoscale extracellular vesicles in nanomedicine. acs nano. 2016;10:3886–99. 6. ohno si, drummen gpc, kuroda m. focus on extracellular vesicles: development of extracellular vesicle-based therapeutic systems. int j mol sci. 2016;17:172. 7. simpson rj, lim jwe, moritz rl, mathivanan s. exosomes: proteomic insights and diagnostic potential. expert rev proteomics. 2009;6:267–83. 8. kalra h, drummen gpc, mathivanan s. focus on extracellular vesicles: introducing the next small big thing. int j mol sci. 2016; 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bdepartment of clinical medicine, aarhus university, aarhus, denmark; cthe fertility clinic skive, skive regional hospital, skive, denmark abstract in this review the advantages of the gonadotropin-releasing hormone agonist (gnrha) trigger are discussed beyond those immediately associated with ovarian hyperstimulation syndrome (ohss) prevention. the gnrha trigger concept has sparked the development of novel protocols, enriching the assisted reproductive technology (art) armamentarium for the benefit of present and future patients. thus, gnrha trigger already has a pivotal role, not only for the standard in vitro fertilisation (ivf) patient, but also for patient groups like oocyte donors, cancer patients, patients with poor ovarian reserve, and patients with immature oocyte syndrome and empty follicle syndrome. herein, we discuss the importance of the gnrha-elicited midcycle fsh surge and the potential improvement in oocyte yield and embryo competence. article history received 1 november 2019 revised 11 february 2020 accepted 27 february 2020 keywords cancer; gnrh trigger; hcg; ivf; oocyte donation; ovulation trigger gnrha trigger—improving oocyte yield and embryo competence? in assisted reproductive technology (art), hcg has been extensively used as a surrogate for the midcycle luteinizing hormone (lh). due to its biochemical components and similar biological dynamics of lh, hcg binds to and activates the same receptor as lh, the lh/hcg receptor, thus ensuring excellent exposure of the follicle to lh activity. in contrast, when hcg is used for ovulation trigger there is a complete lack of the midcycle fsh surge and activity as seen during the natural midcycle surge of gonadotrophins (1). until recently, clinicians have been relying solely on lh activitydependent triggering of final oocyte maturation and, thus, have taken it for granted that the natural midcycle fsh surge was biologically redundant. however, since the early days of comparisons between gonadotropin-releasing hormone agonist (gnrha) trigger and hcg trigger in in vitro fertilisation (ivf), studies reported the retrieval of more metaphase ii (mii) oocytes and embryos after gnrha trigger (2–4). thus, kol and humaidan in 2010 questioned this paradigm, suggesting that the complex process of final follicular maturation and ovulation in ivf cycles might benefit from the synchronised interaction of both lh and fsh activity (5). although the exact role of the fsh midcycle surge is not fully understood, it is known to: 1) promote nuclear oocyte maturation (6); 2) favour cumulus–oocyte communication, enhancing the network of gap junctions within the cumulus–oocyte complex (7); 3) stimulate cumulus expansion (8); and 4) favour the release of proteolytic enzymes involved in ovulation (plasmin) (9). as observed previously, the surge of gonadotrophins elicited by a bolus of gnrha differs from that of the natural midcycle of gonadotrophins in duration and profile. it has also been shown that the elicited flare of lh as well as fsh resembles the natural midcycle surge of gonadotrophins and was found to effectively stimulate final oocyte maturation and ovulation (10), leading to the development of competent embryos as recently demonstrated in pgt-a cycles (11). moreover, studies in oocyte donors (12) and oncologic fertility-preservation patients (13,14) demonstrated a significant increase in mii oocytes and the number of good-quality embryos in gnrha-triggered cycles as compared with hcgtriggered cycles. nonetheless, it is worth noticing that others have not corroborated these findings (15–17). based on the previous observations, lamb and co-workers explored the possible benefits of adding fsh to the hcg trigger bolus in a randomised placebo-controlled trial in a total of 188 ivf cycles (18). thus, apart from the hcg trigger bolus, a total of 95 patients received 450 iu of fsh, whereas the remaining 93 patients received a placebo. there were higher retrieval rates (70% versus 57%) as well as fertilisation rates (63% versus 55%) in the fsh group of patients. following the same line, lin et al. (19) retrospectively analysed data from 376 normo-responder patients, undergoing gnrh-antagonist co-treatment, of whom a total of 191 received the so-called ‘dual trigger’ approach (20) compared with 187 patients receiving hcg only. more mii oocytes were contact juan carlos castillo jcastillo@institutobernabeu.com department of human assisted reproduction, instituto bernabeu, av. albufereta 31, 03016 alicante, spain � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 2, 138–143 https://doi.org/10.1080/03009734.2020.1737599 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1737599&domain=pdf&date_stamp=2020-05-21 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1737599 http://www.tandfonline.com retrieved within the dual trigger group (10.53 versus 8.03), and live-birth rates per embryo transfer were higher as well (41% versus 30%), suggesting a beneficial effect of adding fsh activity at the moment of final follicular maturation in ivf. more recently, the same group published a similar study (21), this time focussing on patients with diminished ovarian reserve (n ¼ 427). the dual trigger group had higher oocyte fertilisation rates (73.1% versus 58.6%), clinical pregnancy rates (33.0% versus 20.7%), and live birth rates (26.9% versus 14.5%) when compared with the hcg-only trigger group. of note, the pregnancy loss (17.4% versus 37.0%) and embryo transfer cancellation rate (6.1% versus 15.4%) were lower in the dual trigger group. taken together, the available data show how modifications in the trigger strategy based on physiology may have potential benefits for oocyte/embryo competence. at the time of trigger, it seems that fsh activity enhances a proper resumption of the meiotic processes of the oocyte, thus adding clinical value by improving oocyte recovery and fertilisation, and by improved pregnancy rates. although the specific mechanisms need further exploration, these findings make the introduction of an fsh surge in addition to the surge of lh activity in ivf an attractive option for further improvement of success rates in ivf (5,18). immature oocyte syndrome in the human species, oocytes are created only during a small period of time during foetal life, after which they arrest in meiosis i (mi) until exposed to fsh and lh later in life. generating embryos during ivf depends on multiple variables, and one of utmost importance is obtaining mii oocytes after the ovulation trigger. as noted previously, during ovarian stimulation (os) for ivf, this process was traditionally induced by hcg which binds to the lh receptor acting as a surrogate for the natural midcycle lh surge. in this aspect, immature oocyte syndrome has been defined as a condition with more than 25% immature oocytes at retrieval after os, despite the correct administration and timing of hcg (22). the aetiology of this obscure phenomenon is unknown, but has profound consequences for the couple, including a significant reduction in the chance of conceiving during ivf treatment (23). following a recent case report, describing a successful pregnancy by use of the dual trigger concept (20) in a patient with a history of immature oocyte syndrome (24), a retrospective study by griffin et al. in 2012 reported the results of 27 women with a previous history of immature oocyte syndrome after hcg triggering (25). in the subsequent cycle, patients were triggered with a combination of gnrha and hcg. by such means the number of mii oocytes increased compared with the previous cycle, resulting in the development of more transferable embryos. the odds of a mature oocyte retrieved for patients who received a dual trigger was 2.51 times higher, after controlling for confounding factors. the authors speculated that the retrieval of more mii oocytes might be associated with the presence of a surge of fsh/lh in addition to the hcg activity; yet the ongoing pregnancy rate per transfer was still disappointingly low (17.4%). in contrast, another small-size retrospective study of patients previously having a low proportion of mii oocytes (66%), despite normal response to os, and who were subsequently submitted to a ‘double trigger’, reported not only a significantly higher number of mature oocytes (6.5 versus 3.6), but also an encouraging 50% clinical pregnancy rate (26). the ‘double trigger’ involves co-administration of gnrha and hcg for final oocyte maturation, but at 40 and 34 h, prior to opu, respectively (26). taken together, the presence of an fsh surge in addition to the lh and hcg surge seems to be a valuable tool in the armamentarium for the treatment of patients with immature oocyte syndrome. however, larger studies are needed to validate the reported retrospective results prior to its routine implementation. empty follicle syndrome following hcg trigger empty follicle syndrome (efs) was first described in 1986 in four patients in whom no oocytes were retrieved after apparently normal follicular development and appropriate oestradiol levels after os (27). efs is still a disturbing and challenging situation in ivf clinical practice, and the incidence varies among studies, ranging from 0.59% to 3.5% (28,29). moreover, efs seems to be associated with pcos, gnrh-antagonist co-treatment (30), and diminished ovarian reserve (31). interestingly, revelli et al. suggested that efs might not be a constant condition. thus, in a study of 43 patients undergoing a second stimulation after efs in the first cycle, a total of 37 patients (86%) obtained mii oocytes, although the stimulation protocols were similar, and a hcg trigger was used for the second cycle too (32). this could be caused by cycle-to-cycle variations in oocyte quality. however, a contribution from the statistical phenomenon ‘regression to the mean’ could also play a role as it is defined by the fact that an extreme variable in the first measurement is more likely to be closer to the mean in the next measurement. in contrast, several case reports suggest a different picture. thus, lok et al. described a case presenting with two consecutive efs (33). first, in a long gnrha down-regulation protocol and subsequently in a gnrh antagonist protocol, both were triggered with 10,000 iu urinary hcg. in a second attempt, a single dose of gnrha was used for final follicular maturation. nine mii oocytes were retrieved from 10 follicles, and eight of these fertilised normally. two good-quality embryos were used for fresh transfer, and four embryos were cryopreserved. a similar case has been described by deepika et al. (34), with two consecutive efs cycles with adequate follicular development and hormonal levels, and an uneventful oocyte pick-up after the use of an hcg trigger. in a third attempt, using gnrh antagonist co-treatment, the dual trigger option was chosen. a total of 10 mii oocytes were retrieved, and subsequently two-good quality blastocysts were transferred, leading to one successful live birth. however, the most puzzling case was described by beckfruchter (35). herein, the authors reported a case of a young upsala journal of medical sciences 139 woman with a normal karyotype and primary infertility of 25 months, submitted to ivf. after seven cycles including either a long gnrha down-regulation protocol or gnrh antagonist co-treatment, resulting in normal follicular development and oestradiol levels, very unfavourable outcomes were obtained at retrieval, including four cycles with efs and three cycles with 1–4 immature oocytes only. of note, in these seven cycles rhcg (up to 13,000 iu) was used for trigger. in the final successful cycle, a ‘double trigger’ (gnrha, 40 h prior to opu; and hcg, 34 h prior to opu) was used, 16 mii oocytes were retrieved, and a total of 11 embryos developed; two embryos were transferred, and nine were cryopreserved; the fresh transfer resulted in the term birth of a healthy child. in the cases of lok et al. and beck-fruchter et al. (33,35) it is difficult to clearly distinguish between the effect of the dual trigger concept versus an isolated action of the gnrha bolus. nevertheless, in view of the previous hcg failures, it seems reasonable to assume that some form of endogenous lh and/or the additive effect of the fsh surge could have played a role for the successful outcome. all these observations suggest that efs is a genuine entity. albeit of obscure aetiology, efs may represent a syndrome of impaired granulosa cell function, in which oocyte meiotic maturation is not resumed, cumulus expansion does not ensue, and the immature oocyte–cumulus complexes are resistant to follicular aspiration (35). however, evidence is mainly based on case reports and might suffer from publication bias. for the time being, it cannot be ruled out that in some patients the fsh surge is needed for optimal resumption of the oocyte meiotic processes, including efs cases after hcg trigger. finally, it is important to note that efs can be encountered also after gnrha trigger (28). ovulation trigger in oocyte donation cycles according to the european ivf-monitoring consortium, oocyte donation cycles account for up to 32.4% of all art treatments in some countries (36). the oocyte donation is usually performed in an altruistic manner by young and healthy women. even though important complications account for less than 1% of all oocyte donation cycles (37), moderate to severe ovarian hyperstimulation syndrome (ohss) might occur more frequently than anticipated, 0.87%�9.47% (37,38). as these patients do not proceed to embryo transfer, the incidence accounts exclusively for earlyonset ohss and are related to hcg trigger in the presence of a high ovarian response. the use of gnrh antagonist co-treatment followed by gnrha trigger has been shown to be the most advantageous protocol for the oocyte donor in terms of safety and efficacy. in oocyte donation cycles, outcomes after gnrha trigger are similar to those of hcg trigger. in 2009, galindo et al. reported similar oocyte maturation and fertilisation rates in 212 oocyte donors randomised to receive either gnrha or rhcg for trigger (38). furthermore, although donors at high risk of ohss were excluded from randomisation, nine donors had mild ohss and one donor severe ohss in the rhcg group, whereas no ohss cases were observed in the gnrha trigger group. additional randomised controlled trials have consistently reported similar outcomes. importantly, the pregnancy rates in recipients are similar to those seen after hcg trigger (39,40). further benefits for the oocyte donor population include: shorter duration of the luteal phase, reduced luteal phase discomfort and abdominal distension, and reduced ovary volume. all these added benefits contribute to a more friendly process for the oocyte donor (41–43), and gnrha trigger should be the ‘gold standard’ for the oocyte donor. ovarian torsion ovarian torsion (ot) happens when an ovary twists on its attachment to other structures. the development of an ovarian mass or ovarian enlargement is commonly related to the development of ot and may affect up to 7.5% of women who experience abdominal pain in emergency departments (44). os may result in ovarian conditions that predispose patients to ovarian augmentation and torsion, with potentially significant consequences, as ot may lead to necrosis requiring ovariectomy, if left untreated. in a recent retrospective cohort study (45), the incidence of ot and its subsequent complications of ivf cycles were explored. the analysis included more than 15,000 ivf cycles, using either hcg trigger and fresh embryo transfer or gnrha trigger and elective frozen embryo transfer (efet). as previously reported (46), ot was an infrequent complication (14 out of 15,577; 0.09%). it is worthy of note that of the 14 diagnosed ot cases, a total of 13 were diagnosed in the hcg-triggered fresh embryo transfer group and 1 in the gnrha trigger efet group (0.13% versus 0.018%, p < 0.049). importantly, although the total oocyte number obtained in the gnrha trigger group was higher than in the hcg trigger group, the incidence of ot was lower in the former. the authors correlated the lower ot rate to a lower ohss rate in the gnrha trigger group compared to the hcg trigger group (0.05% versus 2.4%, p < 0.001). in addition, others reported that the ovary will gain its normal volume faster and closer to the baseline prestimulation volume after gnrha trigger (47,48), which may further contribute to a reduction in ot development. fertility preservation in cancer patients according to the global cancer observatory (https://gco.iarc. fr/), breast cancer is the most common malignancy diagnosed in reproductive-age women worldwide, accounting for approximately 30% of all new cases reported in 2018. since diagnostic tools and treatments have improved over the last decade, fertility preservation in this specific group of patients has gained importance. considering the fact that a vast majority of breast cancers are hormone-dependent (49), attention has focussed on the supraphysiological oestradiol levels occurring during os prior to oocyte preservation. additionally, when hcg is used as a trigger agent, its luteotrophic effect will potentiate the function of multiple corpora 140 j. c. castillo et al. https://gco.iarc.fr/ https://gco.iarc.fr/ lutea, further increasing oestrogen levels during the luteal phase. in order to overcome these undesired effects of ovarian stimulation and ovulation trigger in breast cancer patients, os protocols were developed involving the use of aromatase inhibitors (in addition to exogenous fsh) and gnrha trigger. in their first small-size retrospective analysis oktay et al. explored the use of either gnrha trigger or hcg trigger in women with breast cancer who submitted to oocyte fertility preservation before chemotherapy (50). gnrha trigger resulted in a considerable drop in luteal oestradiol levels compared with hcg trigger. furthermore, more mii oocytes were retrieved, leading to the development of more 2pn embryos after gnrha trigger. the same group in an extended analysis (13), including 129 breast cancer patients (46 in the gnrha trigger group versus 83 in the hcg trigger group), confirmed their previous results in terms of more mii oocytes (77.3% versus 67.6%, p ¼ 0.007) and a higher number of cryopreserved embryos (7.7 versus 5.4, p ¼ 0.002) in the gnrha trigger group. more recently, a larger retrospective cohort study (14) included 341 patient who underwent oocyte freezing for fertility preservation (75.3% breast cancer patients) and reported a higher number of mii oocytes and embryos cryopreserved (11.8 versus 9.9, p ¼ 0.04; and 9.2 versus 6.4, p < 0.001) after gnrha trigger. finally, the use of gnrha trigger in the so-called ‘random start controlled ovarian stimulation’ protocol was published, showing that gnrha trigger effectively stimulates a flare of fhs/lh also in the luteal phase (51). the random start protocol has the potential to shorten the time to oocyte retrieval before oncology treatment. in conclusion, on the basis of the reduced luteal oestrogen exposure after gnrha trigger, the reduced risk of ohss, and the improved cycle outcomes, the available evidence supports the use of gnrha trigger in all women with breast cancer undergoing fertility preservation. development of new protocols in humans, the traditional model of follicular growth states that a single cohort of antral follicles develops during the follicular phase of a menstrual cycle. this classical theory of follicular recruitment has been challenged by baerwald et al. by demonstrating the presence of two and even three wave-like changes in folliculogenesis during one single menstrual cycle, of which only one terminated in ovulation (52). this new physiologic knowledge of folliculogenesis alongside previous experiences in the random start controlled ovarian stimulation protocol paved the way for the development of the so-called ‘double stimulation’ protocol. a double stimulation protocol consists of two consecutive ovarian stimulations, the second one performed in the luteal phase, starting immediately after the first oocyte retrieval. this novel approach was initially used with promising results in a group of patients with poor ovarian reserve (53). from the double stimulation, a total of 26 women had 1–6 viable embryos cryopreserved, and 21 women underwent 23 cryopreserved embryo transfers, resulting in 13 clinical pregnancies. importantly, in this novel protocol using gnrha trigger in the follicular as well as the luteal phase of ovarian stimulation, it was found that the pituitary is able to respond adequately to a gnrha trigger during the luteal phase, even in the presence of high circulating progesterone levels. nonetheless, the fsh and lh surge induced by the same dose of gnrha was higher after the first trigger compared with the second trigger. other investigators, exploring the same concept of combined follicular and luteal phase os, socalled duostim, reached the same conclusions (54). it was proposed that duostim would provide a better opportunity of retrieving oocytes in patients with poor ovarian reserve, in a shorter timespan as compared with conventional os, and also suggested its potential use in oncologic patients in need of emergency fertility preservation. finally, there is a recent publication reporting on the concept of double random ovarian stimulation, initiating os regardless of the cycle day, and proceeding immediately with a second stimulation after the first retrieval in oncological patients (55). reason for caution a substantial part of the literature in the field of gnrha trigger for the abovementioned patient sub-groups can be considered low-quality evidence. hence, well conducted prospective trials are awaited in this area of ivf. conclusions gnrha trigger has had a pivotal role in changing ovulation trigger policies worldwide, not only for the standard ivf patient, but also for patient groups like oocyte donors, cancer patients, patients with poor ovarian reserve, and patients with immature oocyte syndrome or empty follicle syndrome. thus, gnrha trigger plays an important role beyond ohss prevention. moreover, the gnrha trigger concept has sparked the development of novel protocols, enriching the art armamentarium for the benefit of present and future patients. disclosure statement not related to this manuscript, th received honoraria for lectures from ferring, ibsa, besins, and merck. ph received unrestricted research grants from msd, merck, and ferring as well as honoraria for lectures from msd, merck, gedeon-richter, theramex, and ibsa. ph and th are listed as inventors in an international patent application (pct/ uk2018/040882). notes on contributors juan carlos castillo is a senior consultant (md phd) at instituto bernabeu, alicante, spain. thor haahr is a medical doctor and a phd student at 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j, dolz m, bonilla-musoles f. successful pregnancy following dual triggering concept (rhcg þ gnrh agonist) in a patient showing repetitive inmature oocytes and empty follicle syndrome: case report. j med cases 2013;4:221–6. 25. griffin d, benadiva c, kummer n, budinetz t, nulsen j, engmann l. dual trigger of oocyte maturation with gonadotropin-releasing hormone agonist and low-dose human chorionic gonadotropin to optimize live birth rates in high responders. fertil steril. 2012;97: 1316–20. 26. zilberberg e, haas j, dar s, kedem a, machtinger r, orvieto r. coadministration of gnrh-agonist and hcg, for final oocyte maturation (double trigger), in patients with low proportion of mature oocytes. gynecol endocrinol. 2015;31:145–7. 27. coulam cb, bustillo m, schulman jd. empty follicle syndrome. fertil steril. 1986;46:1153–5. 28. castillo jc, garcia-velasco j, humaidan p. empty follicle syndrome after gnrha triggering versus hcg triggering in cos. j assist reprod genet. 2012;29:249–53. 29. blazquez a, guill�en jj, colom�e c, coll o, vassena r, vernaeve v. empty follicle syndrome prevalence and management in oocyte donors. hum reprod. 2014;29:2221–7. 30. singh n, dalal v, kriplani a, malhotra n, mahey r, perumal v. empty follicle syndrome: a challenge to physician. j hum reprod sci. 2018;11:274–8. 31. zreik tg, garcia-velasco ja, vergara tm, arici a, olive d, jones ee. empty follicle syndrome: evidence for recurrence. hum reprod. 2000;15:999–1002. 32. revelli a, carosso a, grassi g, gennarelli g, canosa s, benedetto c. empty follicle syndrome revisited: definition, incidence, aetiology, early diagnosis and treatment. reprod biomed online. 2017;35:132–8. 33. lok f, pritchard j, lashen h. successful treatment of empty follicle syndrome by triggering endogenous lh surge using gnrh agonist in an antagonist down-regulated ivf cycle. hum reprod. 2003;18: 2079–81. 142 j. c. castillo et al. 34. deepika k, rathore s, garg n, rao k. empty follicle syndrome: successful pregnancy following dual trigger. j hum reprod sci. 2015; 8:170–4. 35. beck-fruchter r, weiss a, lavee m, geslevich y, shalev e. empty follicle syndrome: successful treatment in a recurrent case and review of the literature. hum reprod. 2012;27:1357–67. 36. de geyter c, calhaz-jorge c, kupka ms, wyns c, mocanu e, motrenko t, et al. art in europe, 2014: results generated from european registries by eshre: the european ivf-monitoring consortium (eim) for the european society of human reproduction and embryology (eshre). hum reprod 2018;33: 1586–601. 37. bodri d, guill�en jj, polo a, trullenque m, esteve c, coll o. complications related to ovarian stimulation and oocyte retrieval in 4052 oocyte donor cycles. reprod biomed online. 2008;17: 237–43. 38. galindo a, bodri d, guill�en jj, colodr�on m, vernaeve v, coll o. triggering with hcg or gnrh agonist in gnrh antagonist treated oocyte donation cycles: a randomised clinical trial. gynecol endocrinol. 2009;25:60–6. 39. melo m, busso c, bellver j, alama p, garrido n, meseguer m, et al. gnrh agonist versus recombinant hcg in an oocyte donation programme: a randomized, prospective, controlled, assessor-blind study. reprod biomed online. 2009;19:486–92. 40. sismanoglu a, tekin hi, erden hf, ciray nh, ulug u, bahceci m. ovulation triggering with gnrh agonist vs. hcg in the same egg donor population undergoing donor oocyte cycles with gnrh antagonist: a prospective randomized cross-over trial. j assist reprod genet. 2009;26:251–6. 41. cerrillo m, rodr�ıguez s, mayoral m, pacheco a, mart�ınez-salazar j, garcia-velasco ja. differential regulation of vegf after final oocyte maturation with gnrh agonist versus hcg: a rationale for ohss reduction. fertil steril. 2009;91:1526–8. 42. hern�andez er, g�omez-palomares jl, ricciarelli e. no room for cancellation, coasting, or ovarian hyperstimulation syndrome in oocyte donation cycles. fertil steril. 2009;91:1358–61. 43. castillo jc, dolz m, moreno j, gij�on l, ferrer r, ferrero e, et al. triggering with gnrh agonist in oocyte-donation cycles: oestradiol monitoring is not necessary during ovarian stimulation. reprod biomed online. 2012;24:247–50. 44. huchon c, fauconnier a. adnexal torsion: a literature review. eur j obstet gynecol reprod biol. 2010;150:8–12. 45. berkkanoglu m, coetzee k, bulut h, ozgur k. risk of ovarian torsion is reduced in gnrh agonist triggered freeze-all cycles: a retrospective cohort study. j obstet gynaecol. 2019;39:212–7. 46. roest j, mous hv, zeilmaker gh, verhoeff a. the incidence of major clinical complications in a dutch transport ivf programme. hum reprod update. 1996;2:345–53. 47. garcia-velasco ja, motta l, l�opez a, mayoral m, cerrillo m, pacheco a. low-dose human chorionic gonadotropin versus estradiol/progesterone luteal phase support in gonadotropin-releasing hormone agonist–triggered assisted reproductive technique cycles: understanding a new approach. fertil steril. 2010;94: 2820–3. 48. abbara a, islam r, clarke sa, jeffers l, christopoulos g, comninos an, et al. clinical parameters of ovarian hyperstimulation syndrome following different hormonal triggers of oocyte maturation in ivf treatment. clin endocrinol. 2018;88:920–7. 49. howlader n, altekruse sf, li ci, chen vw, clarke ca, ries lag, et al. us incidence of breast cancer subtypes defined by joint hormone receptor and her2 status. j natl cancer inst 2014;106: dju055. 50. oktay k, buyuk e, libertella n, akar m, rosenwaks z. fertility preservation in breast cancer patients: a prospective controlled comparison of ovarian stimulation with tamoxifen and letrozole for embryo cryopreservation. j clin oncol. 2005;23:4347–53. 51. ozkaya e, san roman g, oktay k. luteal phase gnrha trigger in random start fertility preservation cycles. j assist reprod genet. 2012;29:503–5. 52. baerwald ar, adams gp, pierson ra. a new model for ovarian follicular development during the human menstrual cycle. fertil steril. 2003;80:116–22. 53. kuang y, chen q, hong q, lyu q, ai a, fu y, et al. double stimulations during the follicular and luteal phases of poor responders in ivf/icsi programmes (shanghai protocol). reprod biomed online. 2014;29:684–91. 54. ubaldi fm, capalbo a, vaiarelli a, cimadomo d, colamaria s, alviggi c, et al. follicular versus luteal phase ovarian stimulation during the same menstrual cycle (duostim) in a reduced ovarian reserve population results in a similar euploid blastocyst formation rate: new insight in ovarian reserve exploitation. fertil steril. 2016; 105:1488–95.e1. 55. sighinolfi g, sunkara sk, marca al. new strategies of ovarian stimulation based on the concept of ovarian follicular waves: from conventional to random and double stimulation. reprod biomed online. 2018;37:489–97. upsala journal of medical sciences 143 abstract gnrha trigger—improving oocyte yield and embryo competence? immature oocyte syndrome empty follicle syndrome following hcg trigger ovulation trigger in oocyte donation cycles ovarian torsion fertility preservation in cancer patients development of new protocols reason for caution conclusions disclosure statement references upsala j med sci 88: 51-59, 1983 urinary adenylate kinase isoenzyme pattern in patients with myocardial infarction gunnar ronquist and goran frithz department of clinical chemistry, university hospital, uppsala, and department of internal medicine, central hospital, eskilsfuna, sweden in memory of the late professor gunnar &-en abstract adenylate kinase was purified in pooled urinary samples from patients with uncomplicated myocardial infarction. the purification procedure included am monium sulfate precipitation and column chromatographic steps. it was neces sary to stabilize the enzyme during purification with 2-mercaptoethanol and amp. polyacrylamidgelelectrophoresis in sodium dodecanyl sulfate revealed the release of 4 different isoenzymes of ak into urine from patients with myocardial infarction. the molecular weights of these isoenzymes were esti mated to be 21,000; 24,000; 33,000 and 36,000, respectively. introduction the measurement of serum enzyme activities is an established laboratory pro cedure supplemental to clinical and electrocardiographic examination for diag nosis of myocardial infarction. the most widely used enzymes in this context include asat [ 8 1 , l d [17] and ck [ 6 1 . since difficulties have arisen in finding an enzyme specific enough for the myocardium, several different enzymes appea ring in the serum have been used as a diagnostic aid for establishing myo cardial infarction [2,16 1 . ah: was found in elevated amounts in serum during the early phase of myo cardial infarction [ 7 ] . different reports on the molecular weight of ak have been given. hence the molecular weight of rabbit muscle ak was 21,000 while that of baker’s yeast was 41,000 [ 4 1 . due to the low molecular weight of the enzyme it was established to be present also at a high rate in urine in con nection with myocardial infarction 1 7 1 . the present communication deals with the purification of ak from large amounts of pooled urines from patients with uncomplicated myocardial infarc tion. 5 1 material and methods abbreviations. adenylate kinase (ak), alanine aminotransferase (alat), aspar tate amino transferase (asat), creatine kinase (ck), diethyl aminoethyl (deae), ethylene diamine tetraacetic acid (edta), lactate dehydrogenase (ld), poly a c r y l a m i d e g e l e l e c t r o p h o r e s i s (page), rotations per minute (r p m), sodium dodecanylsulfate (sds), tris(hydroxymethy1)-aminoethane (tris), ultraviolet ( u w . collection of urine. urine was collected during the first 3 days after admission to the hospital. the diagnosis of myocardial infarction was based upon the following three criteria. a) clinical history typical clinical history with acute central chestpain of at least 15 min duration. b ) positive electrocardiographic findings the appearance of a q-wave and/or s-t elevations followed by subsequent t-inversions in a localized area. c) positive serum enzyme pattern typical enzyme pattern with an increase of asat with lower alat and/or increase of ld in a characteristic time course. at least two of these three criteria should be fulfilled. every effort was performed to rule out irrelevant diseases such as pulmonary embolism, cardiac congestion or liver disease. the urine was voided into plastic or glass bottles and immediately put into refrigerator. dithiothreitol was added to each bottle as an thiolgroup reactant giving a final concentration of 1 mm in the urine. the urine was examined for presence of blood and albumin and if such a contamination was found, the urine was discarded. the urine specimens were kept cool until further handling in the laboratory, which took place within 24 h. only urine that contained ak activity was considered for further hand ling. they were kept frozen for at most 3 months until use. the different lots of urine totally amounting to about 10 1 were thawed over night in room temperature. they were filtered once and concentrated about 25 times by ultrafiltration [i] . the concentrated urine thus obtained was cen trifuged for 30 min in a sorvall refrigerated centrifuge using rotor number s s 3 4 at 6000 rpm resulting in a small pellet that was discarded. the clear supernatant usually amonting to about 400 ml contained about 5 mg nitrogenlml micro-kjeldahl method). purification of the enzyme from urine: 1. precipitation with ammoniumsulfate. amonium sulfate at 0.4 saturation was added and the urine was kept overnight at 4'c.the urine was 60 min at 4oc and at 5000 rprn in the s o m a l l centrifuge. the precipitate centrifuged for 52 was washed once in ammoniumsulfate at 0.4 saturation and the two supernatants were combined. a second precipitation was performed with ammonium sulfate at 0.8 saturation and kept overnight at 4oc. and the precipitate was washed once in ammonium sulfate at 0.8 saturation. the precipitate was dissolved in 10 ml of 0.5mnahc03, ph 7.1, and dialysed for 4 h with two changes of external water that contained 5 mm mgc12 and the 2-mercaptoethanol. 2. chromatography on blue sepharose c1-6b (pharmacia, uppsala, sweden). all separatory steps were performed at 4oc. the bluesepharose was equilibra ted with 0.02 m trishc1, ph 7.5 containing 5mm mgcl and 0.4 mm edta and 2 1j.m 2-mercaptoethanol. about 90 ml of the buffer-equilibrated gel was used each time. centrifugation followed as above 2 the ammonium sulfate-fractioned urinary material representing an amount of about 365 mg was applied to the blue sepharose column. elution started with the 0.02 m trishc1-buffer containing mg added to a final concentration of 5 mm. the next step included the addition of 0.2 m nacl and the final one, 0.4 m nacl (fig. 1). no additional activity was recovered when increasing nacl conc to 0.8 m. the fraction containing ak activity of fig 1 was concentrated by ultrafiltration and dialysed (5 mm mgcl and 2 um 2-mercaptoethanol) before freeze-drying. 3. fractioning on deae-sephacel column (pharmacia, uppsala, sweden). the main fraction containing ak activity of figure 1 was subjected t o further purification on deae-sephacel. about 300 mg of the lyophilized material was applied to a 50 ml column of deae-sephacel by dissolving it in 3 ml of a 0.015 m trishcl buffer ph 8.0 containing 2 1j.m 2-mercaptoethanol and 5 mm amp. gradient elution was carried out by mixing 260 ml 0.015 m tris hc1 ph 8.0 containing 2-mercaptoethanol and amp with 260 ml of the same buffer also con taining 1m kc1. three uv-absorbing peaks were identified, the third of which also contained ak-activity. this active material was dialysed against mgcl and 2-mercaptoethanol and lyophilized as described before. 4. gel chromatography. the lyophilized material amounting to about 225 mg was dissolved in 1.25 ml distilled water and applied to a 25 ml sephadex g25 column and eluted with distilled water. low molecular weight material (salts and amp) was separated from ak. 5. p o l y a c r y l a m i d g e l e l e c t r o p h o r e s i s jnsodiumdodecanylsulfate. 1 mg of purified material was dissolved in 0.5 ml of the gel buffer consisting of 7.8 g of nah2p04 . h 0;25.63 g of na2hp04 . 2h 0; 2 g of sds dissolved in 1 litre distilled water. page in the presence of sds was in accordance with shapiro et a1 [121. 2+ , edta and 2-mercaptoethanol. a m p was 2 2 2 2 53 0.7 e 0 0.5 2 rt) n w 0 5 0.3 m m (31 0 m 6 i o.' 0 20 40 60 80 fraction number fig 1. chromatography of ammonium sulfate-fractionated human urinary material on blue sepharose. material representing an amount of about 360 mg was applied to the column containing 90 ml of buffer-equilibrated gel. elution started with the buffer that was 0.02 m tris-hc1, ph 7.5, containing 5 mm mgc12, 0.4 mm edta, 2 pm 2-mercaptoethanol and 5 mm amp. arrows in figure indicate the additions of 0.2 m, 0.4 m and 0.8 m of nac1, respectively, to the eluent buffer. the electrophoretic mobilities of the ak isoenzymes were plotted against the logarithm of known protein-standard mixture i1 supplied by merck reagents (e. merck ab, stockholm, sweden) following the technique described by weber and osborn [15]. ak activity in urine was determined at 25oc in accordance with a previous report [71. results the specific ak activity of concentrated, filtered and centrifuged -1 urine was 0.45 units x mg n about 4.5 times to 2.1 units x mgn min after the precipitation steps with ammonium sulfate and dialysis prior to separation on blue sepharose. the result of this column chromatography is given in fig.1. x min-l (25oc). this activity increased by -1 -1 54 3 . 8 . * 1 2 e c 0 q) r*i 2 0.8 w v z 6 m m 0.4 6 i h e . v 2 c 10 20 30 40 f r a c t i o n n u m b e r fig 2. deae-sephacel chromatography of urinary adenylate kinase recovered from purification on blue sepharose. about 300 mg of lyophilized material was applied to the 50 ml column of deae-sephacel by dissolving it in 3 ml of 0.015 m tris-hc1 buffer, ph 8.0, containing 2 ~ j m 2-mercaptoethanol and 5 mm a m p . gradient elution carried out by mixing 260 ml of the tris-buffer solution with an equal amount of the same solution also containing 1 m k c 1 . a broad uv-absorbing peak was detected by continuous reading at 280 nm. the ak activity was concentrated in this major fraction of the chromatogram (fig 1). this fraction was subjected to further purification on deae-sephacel (fig 2). three peaks with uv light absorption at 280 nm were recovered and practically all ak activity was found in the third peak. the material of this peak was gel-filtered on sephadex g25 (fig 3 ) . the ak-bearing peak (first peak) was hereby separated from amp (second peak). the material with ak activi ty recovered from sephadex g25 column chromatography was concentrated and analysed on page in the presence of 0.2% sds. four different bands were identified and the molecular weights were estimated as follows: band 1, 21,000; band 2, 24,000; band 3, 33,000 and band 4 , 36,000 (fig 4). 55 10 3 5 e 4 c . a z 3 > y t 1 2 t j a a y 1 5 10 15 20 25 fraction number fig 3 . chromatography of main peak with ak activity (fig 2) on sephadex g25. the lyophilized, active material, about 225 mg, was dissolved in 1 . 2 5 ml distilled water and applied to the column containing 25 ml of sephadex g25. elution was performed with distilled water. discussion we have isolated four different isoenzymes of ak in urine of patients with myocardial infarction. the molecular weights of the isoenzymes ranged between 21,000 36,000. our results agree fairly well with those of russell et a1 [ l o ] who claimed that human heart tissue may contain five ak isoenzymes in the molecular weight range 21,000 31,000. their results further indicated that ak isoenzymes may reflect three orders of specificity, namely a species specificity, an organ specificity and a subcellular specificity. therefore the isoenzymes studied in the present work might all derive from the myo cardial tissue but from different organelles as well as from the cytosol. 56 f i g 4 . sds-page of 1 mg of p u r i f i e d m a t e r i a l w i t h ak a c t i v i t y . the g e l s show p r o t e i n s w i t h known m o l e c u l a r w e i g h t s ( l e f t , p r o t e i n s t a n d a r d m i x t u r e i1 from merck reagents) and t h e d i f f e r e n t isoenzymes of a c t i v e peak 1 ( f i g 3) from sephadex chromatography ( r i g h t ) . s i n c e ak was i s o l a t e d from u r i n e i n i t i a l l y d u r i n g t h e c o u r s e of m y o c a r d i a l i n f a r c t i o n and o n l y from p a t i e n t s d i s p l a y i n g an u n c o m p l i c a t e d p i c t u r e of t h i s a c u t e h e a r t d i s e a s e , w e have r e a s o n s t o b e l i e v e t h a t t h e isoenzymes were n o t d e r i v e d from o t h e r o r g a n s . f u r t h e r , f o r comparison, ak a c t i v i t i e s were measured i n u r i n e s from one p a t i e n t w i t h uncomplicated m y o c a r d i a l i n f a r c t i o n and a n o t h e r w i t h advan ced l i v e r d i s e a s e due t o a l c o h o l i c a b u s e . 57 these measurements were performed in the absence and presence of ~ x ~ o ~ m diadenosine pentaphosphate. an inhibitory action by the additive was typically registered in the urine from the patient with myocardial infarction. such a pattern was not observed in the other patient with liver disease, here instead a slight stimulation was apparent (data not given in results). (cf.ref 11). hence, the patient with uncomplicated heart disease displayed an urinary enzyme with different characteristics vis 5 vis the inhibitor com pared to the patient with liver disease. the isoenzymes studied most probably appear as single polypeptide chains [14] and not in a hybrid dimeric form similar to ck 131 or a tetrameric form like ld [51. the enzyme was labile during purification and it was necessary to supply the suspension medium with amp as well as thiol group reagents to stabilize the enzyme. nevertheless a certain degree of inactivation probably took place during purification (&.ref 9,13). by large scale preparation of puri fied ak from urine of patients with myocardial infarction, enough material has been obtained for the purpose of antibody production for use as a diagnos tic tool. such work is under progress. acknowledgements we are obliged to mr s eklund for skilful technical assistance. this study was supported by grants from the swedish medical research council, project no b81-03x-05916-01a and the siidermanland county council. references 1. 2. 3. 4. 5. 6. 7. 8. bgren, g. & ronquist, g . : incorporation of phosphorous into human erythrocyte ghost fractions. acta chem scand 18:2029-2043, 1964. auvinen, s.: evaluation of serum enzyme tests in the diagnosis of acute myocardial infarction. acta med scand (suppl) 539:l-25, 1972. baillie, e.: ck isoenzymes: part i. clinical aspects. laboratory medicine 10:267-276, 1979. bergman, th.e.: enzyme handbook, vol. i, pp 448-471. springer verlag, berlin, heidelberg, new york, 1969. cohen, l., djordjevich, j. & ormiste, v.: serum lactate dehydrogenase isoenzyme patterns in cardiovascular and other diseases, with particular reference of acute myocardial infarction. j lab clin med 64:355-374, 1964. dreyfus, j.c., schapira, g., resnais, j. & scebat, l.: la creatine kinase serique dans le diagnostic de l’infarctus myocardique. revue fr etud clin biol 5:386-388, 1960. frithz, g . , ericsson, p. & ronquist, g.: serum adenylate kinase activity in early phase o f acute myocardial infarction. upsala j med sci 81:155 158, 1976. karmen, a., wroblewski, f. & la due, j . s . : transminase activity in huan blood. j clin invest 34:126-133, 1955. 58 9. maruhn, d., fuchs, j., mues, g. & bock, k.d.: normal limits of urinary 1 0 . rusell, p.j. horenstein, j.m., goins, l., jones, d. & laver, m.: excretion o f eleven enzymes. clin chem 22:1567-1574, 1976. adenylate kinase in human tissues. i. organ specificity of adenylate kinase isoenzymes. j biol chem 249:1874-1879, 1974. exkretion von adenylatkinasen nach zellschzdigungen. klin wschr 53:617-622, 1975. 12. shapiro, a.l., vinuela, e. & maizel, j.v.: molecular weight estimation o f polypeptide chains by electrophoresis in sds-polyacrylamide gels. biochem biophys res commun 28:816-821, 1967. urine. proc soc exp biol med 167:530-535, 1981. e.f.: primary and tertiary structure of the principal human adenylate kinase. eur j biochem 68:281-290, 1976. tions by dodecyl sulfate-polyacrylamide gel electrophoresis. j biol chem 244:4406-4412, 1969. 16. witteveen, s.a., hemker, h.c., hollar, l. & hermens, w.: quantitation of infarct size by means of plasma enzyme levels. br heart j 37:795-799, 1975. 17. wroblewski, f. & la due, j.s.: lactate dehydrogenase activity in blood. proc soc exp biol med 90:210-213, 1975. 11. sachsenheimer, w., goody, r.s. & schirmer, r.h.: elimination und 1 3 . sumi, h. & toki, n.: inhibitors of acrosin and sh-protease in normal human 14. von zabern, j., wittmann-liehold, b., untucht-grau, r., schirmer, h. & pai, 15. weber, l. & osborne, m.: the reliability of molecular weight determina address for reprints: gunnar ronquist, m.d. department of clinical chemistry, university hospital s-751 85 uppsala 59 de-identified linkage of data across separate registers: a proposal for improved protection of personal information in registry-based clinical research article de-identified linkage of data across separate registers: a proposal for improved protection of personal information in registry-based clinical research tomas sn€ackerstr€om and christian johansen uppsala university, uppsala clinical research center, uppsala, sweden abstract over the last decades the advent of digital documentation has provided research communities with valuable resources of information for clinical research. to utilize the potential of information about patients, their health care, and its outcome that is already available in different registers, the possibility to cross-reference information from different registers is inevitably required. when performing linkage, we are currently forced to disclose information of participating subjects either to the administration of the other register(s) or to the researcher. considering the increased concern of issues around personal integrity, this is a limitation that affects the ethical implications of proposed research and that might in the end affect the willingness of subjects to participate in registers. for this reason we propose a different methodology for performing cross-referencing, one that effectively prevents information leakage between the different organizations participating in linking the data. we believe that it is possible to use commonly adopted technologies within the area of data security and encryption to perform linkage without disclosing any sensitive information between different participants. in this paper we demonstrate how common techniques of encryption could be implemented to achieve that and furthermore significantly simplify discovery and feasibility surveying ahead of studies. article history received 17 june 2018 revised 14 september 2018 accepted 18 september 2018 keywords data linkage; data protection; ethics; registry research background the widespread adoption of electronic documentation in health care has brought a huge potential to perform research by analyzing data from existing sources. ranging from scientific cohorts, clinical quality registers, and healthcare data to general data from personal devices, the potential for novel research is growing beyond what we could have imagined just a short time ago. in sweden the population is furthermore comparatively well covered by registers with high quality and rich content, meaning that individual subjects are often better documented than elsewhere (1). the documentation is, however, fragmented in separate registers of different types, with different purposes and in different legal domains. in order to fully study any specific population one needs to cross-reference data between the registers in question. the procedure of cross-referencing is, however, one that raises significant legal and ethical concerns (1 p. 15). the partitioning of information in separate organizations serves to mitigate the risk of gathering too much information about a person in one single place. linking information between registers again is obviously not uncontroversial and needs to be done with caution. with the adoption of the new general data protection regulation (2) there is an increased awareness of integrity issues, and the continued availability of clinical data requires development of tools that safeguard the integrity of subjects while still allowing research to progress in the interest of the very same subjects. ‘security by design’ is now a mandatory guideline on the design of it systems that manages personal information of any kind—and in particular, sensitive data (3). in clinical research it is common practice that data are used in a de-identified form, either anonymized or with pseudonyms replacing identifiers. this becomes problematic in the case of cross-referencing. in the current situation it is not possible to cross-reference without disclosing the identity of the subject either to the researcher or between the registers themselves, which really puts the ethical issues in focus. within cohorts.se we are therefore proposing the development of technical infrastructure that facilitates distributed analysis and use of safe cross-referencing that avoids leakage of sensitive data between participating organizations. the proposal it should be mentioned that our idea is not primarily a novel technical solution. nor does it require development of new software or use of cutting-edge technology. what we are proposing is rather about using well established technologies in a new area. we will not go into great detail regarding cryptography; it is arguably one of the most complex areas within the field of computer science and is well covered in the literature (4–6). in this paper we will only present some contact tomas sn€ackerstr€om tomas.snackerstrom@ucr.uu.se uppsala university, uppsala clinical research center, 751 85 uppsala, sweden � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 1, 29–32 https://doi.org/10.1080/03009734.2018.1527420 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1527420&domain=pdf http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1527420 http://www.tandfonline.com general principles and concepts that are essential to establish for the sake of the presentation. the area of cryptography is centered around two generic problems in all forms of remote communication: secrets and trust—secrets as in the ability to ensure that only the intended recipient can read your message even though the message must be carried by untrusted messengers or might be eavesdropped on in the transport to the recipient; and trust in the sense that when received there has to be means for the recipient to verify the authenticity of the message, that it actually is from the sender, and that it has not been manipulated in any way. both of these requirements are challenging but currently solvable ones. in cryptography there are two main classes of methods: symmetrical and asymmetrical encryption algorithms. the symmetrical class of algorithms uses a key and some method of substituting characters based on the key. the asymmetrical algorithms use a pair of keys and non-reversible functions to create encrypted messages. one part of the key can decrypt messages if, and only if, they are encrypted with the matching part of the pair. the symmetrical algorithms are significantly easier to compute but require that a key is agreed upon prior to sending the messages. in the case of asymmetrical algorithms the strict relationship between the key pairs can be used to address the question of authenticity of the messages. one part of the key is made public, and the other is kept secret. as messages encrypted by one part can only be decrypted by the other, the fact that one part is public becomes a means to assure whether it was the matching private key that encrypted the message in question. the two methods are usually applied in combination. asymmetrical methods are used to communicate a key that is then used to symmetrically encrypt the messages. we recognize that these attributes could be used to create a system where cross-referencing between different registers can be performed without the need to disclose personal information between any of the participants. we use the aspect of secrets when encrypting the identifiers, making them impossible to reverse, and we use trust in all of the remote communication for different systems to identify themselves to each other (‘authenticate’ in technical terminology). researcher register register param get: get key id:7 get key id:7 param a:b a b id:7 id:7 .... .... .... param id:7 .... [a,b,...] id:7 .... epn: a b issuing service key service 7 in:7 7 a∩b a∩b 1 2 3 3 4 6 7 6 5 5 4 ... .... .... figure 1. illustration of the communication between participants involved when performing data linkage over different organizations. any communication outside of what is illustrated is assumed to be prohibited and prevented by technical means. 30 t. sn€ackerstr€om and c. johansen beyond the question of technology there is also a requirement to create an organizational structure for the administration and management of the required services in the proposed infrastructure. our strategy relies on the ability to separate two required tasks into different administrations. obviously, everything is dependent on the assumption that it can be regulated and that the administrations in question do not violate the rules. one should keep in mind that if we cannot rely on participating administrations to act according to rules there is no way to protect data in any system, existing or in the future. naming may need improvement, but for this discussion we call the services an ‘issuing service’ and a ‘key service’. the issuing service is responsible for overall orchestration of communication between the participating systems. the key service is only involved in creating and guarding the encryption keys during the processing. it is imperative that the key service only deals with the tasks around keys and that the issuing service is truly separated from the key service. to explain the concepts involved we will present a walkthrough of a hypothetical scenario. we want to stress that what we want to achieve is to minimize information leakage between different participating roles and organizations in this, yet providing the researcher with the ability to receive data that can be linked. researcher ‘rachel’ has an idea about studying the correlation between two treatments or how one affects the outcome of the other. for this she needs to combine data from two different registries that both include data that are considered sensitive personal information. register a may not disclose personal data to register b and vice versa. rachel needs to receive data in an anonymized form and yet she needs to be able to link data between the two registers that relate to the same subject. if neither register can send data to each other, nor send it to rachel, it becomes intuitively attractive to designate a third party that could do the reference linkage. this has been suggested before but is really not helping the situation as it only creates a new entity that gets all the combined data instead. this is where the separation of key management as a distinct service, separate from handling of data, becomes a possible solution. in the following section there will be a description of the procedure of providing a dataset to rachel without disclosing any common identifiers, as illustrated in figure 1. in our proposed system rachel creates a request for linkage to the issuing service in step one. we can now assume that there is a degree of administration required to verify ethical approvals as such, but once that is managed the issuing service creates an issue or a ‘job’ for the proposed data linkage and gives it an id (‘in:7’ for issue number 7 in figure 1). in this job the requested registries are identified from what has been requested in the application (and given ethical approval). in the second step the issuing service then requests that the key service creates a key for the job with the specific id and tells the key service which registries are participating in the linkage job. the key service confirms to the issuing service that the key is created, but obviously keeps the key secret. the third step is where the issuing service sends independent requests to the registries in question, presenting information about the id of the job, any parameters provided by rachel (possible selection parameters, columns requested, etc.), and if required a reference to where to acquire the key. each registry individually requests the key for the job providing the reference id they got from the issuing service, indicated as the fourth step. the key service authenticates the register systems by referencing the list of registers that the issuing service provided when requesting the job. if successfully authenticated, the common key is responded to the requesting register. at this point, the register is applying the encryption on all the relevant identifiers of their set of subjects, doing so independently and using the key and an encryption algorithm that was specified by the issuing service at the earlier step three. given that a common algorithm is used, all participating registers will then have created pseudonyms for their identifiers that are unique and similar for any given personal identifier that is common across registries, and that, without access to the key, cannot be reversed using any known computational method. in step six the registries, independently of each other, report back their respective list of encrypted identifiers to the issuing service along with the reference for the job. when all the participating registries have reported back, the issuing service can easily distinguish the set of identifiers that are common for all participating registries (expressed as the intersection of a and b, a \ b). note that the issuing service only gets the encrypted identifiers, but can still distinguish which identifiers are common in the sets it has received, as any given identifier will have the same encrypted representation. the seventh and last step is where the issuing service returns the set of encrypted identifiers that are common for the registries, the intersection, to rachel along with a ticket identifying her as the rightful recipient of the extracted dataset. with that, rachel has a set of identifiers that are unique to her request and that can be used to request the actual data from each register. doing so for all registers gives her tables from each participating registry which has common identifiers for the participating people but that still does not identify them directly. the only caveat remaining is the risk of indirect identification by the actual data, but that problem exists regardless of method for linkage and needs to be considered in the ethics review. it is reasonable to assume that the issuing service will need to be a point where a certain degree of control is performed, thus it is important that it is managed by a capable administration. summing up the rather elaborate description above we would like to point to the key aspects of the proposed solution. first, and most importantly, no information about the content in any of the registries ever leaves the registry itself. the encryption is done by the registry using reference implementations if standardized crypto-algorithms and only encrypted identifiers are communicated externally. there is an ongoing evolution of algorithms, but for this purpose there are alternatives that can be regarded as secure (7,8). second, the upsala journal of medical sciences 31 registries can remain oblivious of the other registries involved. the registry solves its task without communication with any other registry involved. as a matter of fact, it does not need to know which or even how many registries take part in a job. third, the key service never handles any data or receives any identifiers. it only distributes keys to securely authenticated clients. and finally, the system can also be created such that when the issuing service has received a set of encrypted identifiers from all participating registers in the job, it can order the key service and registers to destroy the key, prior to handing the list of encrypted identifiers to the researcher. this means that when the identifiers are sent to the researcher the key that was used in the intermittent process is already destroyed before data are given to the researcher. in this way, one can confidently ensure there is no foreseeable way of reversing the information in the identifier. this will also guarantee that data extracted in different research projects are not accidentally linked by the researcher after it has been handed out. each ‘issue’ will have its unique and highly temporary key. final thoughts although the proposed process might seem complicated when describing the theory, it should be said that all of what is happening is standard procedures and technologies used every day. certificates are used to identify every server on the internet, and encryption is applied to ensure privacy whenever that is required, which is most of the times on the internet. it should also be mentioned that all of the added communication can be automated and performed instantly. the technical overhead is minimal and can be performed in the background. for the user of the web it is not more complicated to access a service over a secure connection than over an unprotected one. everything is set up in the background, and all that the user experiences is the green symbol that indicates that the server is authenticated as a trusted one by some authority the computer trusts. likewise, when rachel presents her request for cross-referencing a set of registers there is obviously a need for an administrative process where her request and her ethical approval have been verified from a bureaucratic point of view. once this has been granted, the rest of the process can be automated to the point where rachel gets the links to where she can download her data. it requires an amount of development to create the systems, but as mentioned it is more about applying technologies and system components that are already available. the automated aspect of the envisioned infrastructure could also enable some sought-after features. one of the most evident obstacles in designing a registry study is the difficulty in evaluating the feasibility of it. often there is a need to know the approximate number of available subjects to assess the possibility of performing the study at all. when cross-referencing is required it is obviously impossible to get an idea about the number of subjects that are common between a set of registers without doing the actual linkage. this means that in the current situation the researcher has to speculate on an idea, go through the process of acquiring ethical approval and the administrative process to get the linked dataset in order to discover whether or not there are enough subjects to perform a study. with an infrastructure such as envisioned above, one could quickly answer such questions without disclosing personal information to anyone. the ability to answer such questions prior to initiating a process of ethical approval would not only give the researchers a tool to investigate the feasibility of their ideas, it would save the ethics committees the work required to decide upon research questions that are not even possible to investigate practically. it would even provide an important piece of information to the ethics processing itself. showing that there is a large intersection between clinical areas is a fairly good indication that it covers an area that has a medical value to investigate. acknowledgements we would like to thank professor johan sundstr€om for being a constant source of inspiration and for encouraging us to write this paper. professor arne andersson is acknowledged for his helpful editorial contributions. disclosure statement no potential conflict of interest was reported by the authors. notes on contributors tomas sn€ackerstr€om is a systems architect at the uppsala clinical research center, sweden. christian johansen is a systems architect at the uppsala clinical research center, sweden. references 1. ros�en m, guldgruvan i ha€lsooch sjukva˚rden €oversyn av de svenska kvalitesregistren. stockholm: sveriges kommuner och landsting; 2010. available from: https://webbutik.skl.se/bilder/artiklar/pdf/7164-613-2.pdf (accessed 24 august 2018). 2. regulation (eu) 2016/679 of the european parliament and of the council of 27 april 2016 on the protection of natural persons with regard to the processing of personal data and on the free movement of such data, and repealing directive 95/46/ec (general data protection regulation); 2016. available from: https://publications.europa.eu/en/publication-detail/-/publication/3e485e15-11bd -11e6-ba9a-01aa75ed71a1/language-en (accessed 24 august 2018). 3. european data protection supervisor. guidelines on the protection of personal data in it governance and it management of eu institutions. available from: https://edps.europa.eu/sites/edp/files/publication/it_governance_management_en.pdf (accessed 24 august 2018). 4. ferguson n, schneider b, kohno t, cryptography engineering: design principles and practical applications. hoboken, nj: wiley; 2010. 5. menezes aj, van oorschot pc, vanstone scott a, handbook of applied cryptography, 5th printing. boca raton, fl: taylor & francis; 1997. available at: http://www.cacr.math.uwaterloo.ca/ hac/. 6. wikipedia. books on cryptography. available from: https://en.wikipedia.org/wiki/books_on_cryptography (accessed 24 august 2018). 7. wikipedia. strong cryptography. available from: https://en.wikipedia.org/wiki/strong_cryptography (accessed 24 august 2018). 8. wikipedia. security level. available from: https://en.wikipedia.org/ wiki/security_level (accessed 24 august 2018). 32 t. sn€ackerstr€om and c. johansen https://webbutik.skl.se/bilder/artiklar/pdf/7164-613-2.pdf https://webbutik.skl.se/bilder/artiklar/pdf/7164-613-2.pdf https://publications.europa.eu/en/publication-detail/-/publication/3e485e15-11bd-11e6-ba9a-01aa75ed71a1/language-en https://publications.europa.eu/en/publication-detail/-/publication/3e485e15-11bd-11e6-ba9a-01aa75ed71a1/language-en https://publications.europa.eu/en/publication-detail/-/publication/3e485e15-11bd-11e6-ba9a-01aa75ed71a1/language-en https://edps.europa.eu/sites/edp/files/publication/it_governance_management_en.pdf https://edps.europa.eu/sites/edp/files/publication/it_governance_management_en.pdf http://www.cacr.math.uwaterloo.ca/hac/ http://www.cacr.math.uwaterloo.ca/hac/ https://en.wikipedia.org/wiki/books_on_cryptography https://en.wikipedia.org/wiki/books_on_cryptography https://en.wikipedia.org/wiki/strong_cryptography https://en.wikipedia.org/wiki/strong_cryptography https://en.wikipedia.org/wiki/security_level https://en.wikipedia.org/wiki/security_level abstract background the proposal final thoughts acknowledgements disclosure statement notes on contributors references sups_a_555369 79..79 upsala journal of medical sciences. 2011; 116: 79 anders fredrik regnell – an early and generous sponsor of our society borne in 1807 as the son of anders rignell, a horse driver and brita persdotter, a servant girl, anders fredrik became motherless at an early age. his father then married a rather wealthy widow and innkeeper, and with his new wife he was busy taking care of business. there wasn�t much time for family life for the young boy, but, he had the fortune of having a good education. curiously, anders fredrik got his last name, regnell, by a misspelling when enrolled in school. anders fredrik regnell became a student in uppsala 1824, passed the preparatory exams for medical studies 1830, med lic 1836 and got his medical doctorate as primus 1837. from 1836 to 1839 he worked in stockholm and for some while as an assistant to anders retzius. he was appointed position as a cholera physician twice. 1839-1840 he worked as a ship’s doctor travelling with the jarrama expedition to the mediterranean regions. returning home from this he was understandably concerned by his own health, with a lung disease causing blood coughing. thus, he decided to emigrate to a warmer and healthier climate. in rio de janeiro he passed the tests of the medical faculty and was approved as a doctor. he then established a successful practice in the small town caldas in minas geiras. although he was something of a lonesome wolf he gave hospitality to many visitors from his old country. as other well-known medici he showed an interest in botany. later in life he collected plants in tropical regions, sending them to different museums in the old world to be characterized by experts. in south america he also showed interest in animal life, geology and meteorology. many great men have been remembered for their knowledge, contributions to science by new discoveries or by their synthesis of evolutions in science. anders fredrik regnell deserves to be remembered also because of his monetary contributions to research and education. due to his earnings as a skilled doctor and to wise investments in different projects e g mining he made a large fortune. donations of impressive magnitude were made to many different organisations. first of all to his alma mater, uppsala university and in fact regnell’s donations were most significant as they, next to those of gustav ii adolf, were the largest ever given to uppsala university. he gave money to the swedish society of medicine and to the swedish museum of natural history in stockholm as well. he also contributed to our society in a very substantial way. among other things we should be greatful to anders fredrik regnell for giving us the possibility to publish this journal at that time under the name of upsala läkareförenings förhandlingar. he was a honorary member of our society, the swedish society of medicine and of the learned societies in uppsala and gothenburg. by one of his compatriots he was characterized as resembling “a cactus; rich in nutrient fluids with magnificent beautiful flowers, but, with a bizarre growth and filled with thorns”. bo lindberg, a former chief physician at our hospital, this spring will publish a biography on the life of anders fredrik regnell. the title of the book is “anders fredrik regnell – läkare, botanist, donator”. our society has contributed economically to the publication of this book, hard copies of which will be purchased by the university library. it will also be available electronically since it will be published under open access premises. i shall read this book with great interest, as the life of anders fredrik regnell deserves to be remembered. torbjörn karlsson chairman of upsala läkareförening issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2011.555369 vol_116_002_sups_a_534827 138..141 upsala journal of medical sciences. 2011; 116: 138–141 original article comparison of two different methods of fiber-optic nasal intubation: conventional method versus facilitated method (nasal-18) ali mohammadzadeh1, mohammad haghighi1, bahram naderi1, amanullah chaudhry2, zahid hussain khan3, mohammad r. rasouli3 & soheil saadat4 1department of anesthesiology, guilan university of medical sciences, rasht, iran, 2department of anesthesiology, razi hospital, rasht, iran, 3department of anesthesiology, imam khomeini medical center, tehran university of medical sciences, tehran, iran, and 4sina trauma and surgery research center, tehran university of medical sciences, tehran, iran abstract objectives. as the conventional fiber-optic nasal intubation technique has several potential difficulties, we compared in this study another technique (nasal-18) with the conventional one in attempting fiber-optic nasal intubation with a possible higher rate of success. methods. a randomized controlled trial was carried out at a teaching hospital. forty-eight patients aged 25–45 years with american society of anesthesiologists (asa) classes i and ii undergoing elective faciomaxillary surgery were allocated to two groups of fiberscopic nasal intubation using either the nasal-18 technique or the conventional method (control). in the nasal-18 group, a nasal tube was gently inserted into the nasopharynx till mark 18, then a fiberscope with 41 cm length was glided over it and advanced through the nasal cavity till the glottis could be visualized. finally the nasal tube was rolled over the fiberscope instead of one-step passage of the nasal tube after visualization of glottis in the controls. times from the start of insertion of the fiberscope into nares till visualization of vocal cord (t1) and from here to complete intubation (t2) were recorded. these times were compared between the two groups. results. t1 values in nasal-18 and control groups were 65.2 ± 33.2 and 151.0 ± 56.5 seconds, respectively (p < 0.0001). t2 durations were measured as 25.1 ± 18.5 and 21.8 ± 10.1 seconds in control and nasal-18 groups, respectively (p = 0.45). the nasal-18 group had a success rate of 83% compared to 66.7% in the control group. conclusions. the nasal-18 method reduces the time needed for successful fiber-optic intubation. this method can be added to the list of techniques in facilitating fiber-optic intubation. key words: facilitating method, fiber-optic intubation, nasal intubation introduction operations that are conducted on or in the vicinity of the jaws, in which the mouth is being used for manipulation or insertion of devices, and where there is limited jaw mobility, make usage of a conventional laryngoscope extremely difficult or even impossible (1–4). even today, years after the first application of the fiberscope, anesthesiologists still encounter difficulty in intubation in 90% of cases (4–7). the conventional fiber-optic nasal intubation method in which the nasal tube is inserted in one step after visualization of the vocal cords by a flexible fiber-optic laryngoscope (8), two major problems are encountered, i.e. visualization of the glottis and the entry point at the level of vocal cords, and insertion of the fiberscope into the trachea (5,8). anesthesia, with or without the use of muscle relaxants, leads to paralysis of oropharyngeal muscles which in turn leads to a backward fall of the correspondence: zahid hussain khan, md, professor of anesthesiology, imam khomeini medical center, tehran university of medical sciences, keshavarz blvd, tehran 1419733141, iran. e-mail: khanzh51@yahoo.com (received 9 august 2010; accepted 21 october 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.534827 oropharyngeal structures such as the tongue and the epiglottis on the posterior wall of the pharynx (7,9). as a result, the space is compromised, with little of it available for passing the tube into the trachea (10,11). some maneuvers such as jaw thrust, head flexion, tongue retraction, and use of intubator airways like berman and ovassapian have been successfully used to overcome the aforementioned problems. by pressing on the arytenoid cartilage, air passage can be occluded, as is in selick’s maneuver (2,6,9,12). use of intubator laryngeal mask airway (ilma) is another way to facilitate fiberscopic intubation (1,11,13). the goal of the nasal-18 or the tube-first approach is to minimize the duration the fiberscope is between the cords, and also to reduce the time required to advance the tube into the larynx. this study was designed to ascertain whether the facilitated method (nasal-18) is superior to the conventional fiber-optic nasal intubation, as judged by the time for glottic visualization (t1) and the final time to intubation (t2). methods this study was carried out from january 2008 to june 2008 at the poursina teaching hospital on 52 patients who were candidates for elective maxillofacial operation of type 1 leforte fracture (miniplate insertion in the upper and lower jaws). their ages ranged from 25 to 45 years, and they all fell within the american society of anesthesiologists (asa) physical status classes of i and ii. patients were excluded if there was a contraindication for nasal intubation. the patients were randomly allocated into two groups of nasal intubation using either the nasal-18 or the conventional method (control) based on a table of randomization. in the control group, the fiberscope was inserted into the nasal cavity via the nostril and advanced through it till the vocal cords were visualized. then, the nasal tube, which had been mounted on the scope beforehand, was glided over the scope and advanced through the vocal cords into the trachea. whereas in the nasal-18 group, or the tube-first approach, the nasal tube was inserted into the nasal cavity and advanced through it till the mark 18 reached the level of alae of the nose. with the tube advanced to the 18 cm at nare, the tube tip stands just above the larynx and breath sounds are audible through the tracheal tube. with additional anesthesia directed at the larynx (topical lidocaine), run the long scope through the tube, visualize the larynx, and pass between the cords towards the carina. the final depth of insertion of the nasal tube should be 26–28 cm at the nare, establishing its correct placement in the trachea as confirmed by bilateral audible breath sounds. standard monitors including pulse oximetry, capnography, electrocardiography, and non-invasive blood pressure measurement were performed prior to the administration of intravenous medications and starting the nasal intubation. all patients received 2 microgram/kg of fentanyl, 3 mg of midazolam, and propofol at the rate of 30–35 mg/kg/min. atropine (0.5 mg) was used to reduce mucus secretions of airways. after shrinkage of the nasal cavity by the instillation of phenylephrine drops (3 drops in each nostril), lidocaine spray (10%) was used to anesthetize the nasal and oral mucosae. with the use of 5 ml of 2% lidocaine transtracheally, the tracheal and bronchial mucosae were anesthetized. nasal intubation with a spiral tube #7 followed. three experienced anesthesiologists in fiber-optic nasal intubation were involved in fibroscopy of patients, and each anesthesiologist performed both techniques. the fiberscope used during this procedure was a scholly type flexilux 2000 with a length of 41 cm and a diameter of 3.7 mm. after dipping the fiberscope into the lubricating jelly, we inserted it into the nasal cavity until it reached the pharynx, and an attempt to locate the vocal cords then ensued. during this period, patients were awake and co-operated well with the anesthesiologists. time was recorded in seconds, from the start of insertion of fiber-optic laryngoscope in the nares till visualization of vocal cords (t1) and from this to successful intubation when the endotracheal tube cuff was inflated (t2). if it was necessary, facilitating techniques such as bronchoscopy, head flexion, and jaw thrusts were utilized. a time period of more than 180 s for the procedure or inability to intubate was considered as failed intubation, and the patient was then intubated using a different modality. statistical analysis was performed using the spss (spss inc., chicago, il, usa) version 12. student’s t test was used to compare t1, t2, age, and weight, and chi-square test was used to compare asa class, sex, and other categorical variables between groups. a p value < 0.05 was considered to be significant. a linear regression analysis was utilized to compare t1 and t2 between control and nasal-18 groups, controlling for the effect of age, sex, asa class, and other variables. ethical considerations approval of the study was obtained from the institutional ethical committee of human research, comparison of fiber-optic nasal intubation methods 139 and written informed consent was obtained from each patient. results a total of 48 patients consented to participate in the study. there were no significant differences regarding age, sex, and asa class between the two groups. as demonstrated (table i) the t1 as well as total time of nasal intubation were significantly shorter in the nasal-18 group than in the control group (p < 0.001), while there was no significant difference between the two groups regarding the t2 duration (p = 0.455). jaw thrust maneuver was used in 25% of the cases, which was the same in both groups (p > 0.05). use of head flexion for facilitation of entry of the endotracheal tube was necessary in 4.2% and 33% of the cases in the nasal-18 and control groups, respectively (p = 0.023). success rates in the nasal-18 and control groups were, respectively, 83% and 67%. in the nasal-18 group, the failures were all related to the time it took us to intubate which exceeded the range specified in the ‘methods’ section (mean 280 ± 30 s), whereas in the control group the failures were assignable to inability to intubate in 63% of the cases and prolonged intubation (t1+t2 > 180 s) in the remainders (mean 480 ± 20). this was higher than the corresponding time in the nasal-18 group (p = 0.002). in the nasal-18 and control groups, 7 and 14 cases, respectively, needed facilitating maneuvers such as jaw thrust and head flexion (p = 0.04). regression analysis indicated an inverse relationship between t1 and asa class (p < 0.05). after adjusting for the effect of other variables, there was a significant difference between the two groups regarding t1 (table ii) but not t2. discussion the incidence of difficulty in passing an endotracheal tube over an orally inserted fiberscope varies considerably between studies, ranging from 0% to 90% (5). nasal fiber-optic intubation can be as difficult as oral fiber-optic intubation. differences in the definition of the difficulty between various studies might have produced these differences in the reported incidences; however, other factors such as a different size of fiberscope, or else the type and size of the endotracheal tube, might also have affected the incidence. the major reason for difficulty in advancing an endotracheal tube over a fiberscope is considered to be deviation of the course of the tube from that of the fiberscope (because of the gap between the two) towards the epiglottis, arytenoid cartilage, pyriform fossae, or esophagus (12,14–16). marfin et al. (5) showed that posterior structures of the laryngeal inlet are the sites of impingement during fiber-optic nasal intubation and suggested anti-clockwise rotation as its solution. use of a spiral flexible tracheal tube, and aligning number 18 on the tube to the level of the nasal ala, had significantly improved the success rate of fibroscopy in our study. we chose number 18 because when this number is reached at the nasal alae, the endotracheal tube has sufficiently advanced to reach a point close to table i. comparison of baseline characteristics, t1, t2, total time, and facilitation maneuvers between two groups, conventional (control) and nasal-18. parameters conventional nasal-18 p value male sex (n) 22 (68.8%) 16 (66.7%) 0.86 asa classes class i 13 (40.6%) 5 (20.8%) 0.11 class ii 19 (59.4%) 19 (79.1%) age (year) 34.5 ± 7.1 35.25 ± 7.9 0.71 weight (kg) 73.9 ± 7.4 74.4 ± 9.0 0.84 t1 (s) 151.0 ± 56.5 65.2 ± 33.2 0.001 t2 (s) 25.1 ± 18.5 21.8 ± 10.2 0.45 total time (s) 176.2 ± 56.3 87.1 ± 41.0 <0.001 success (n) 16 (66.7%) 20 (83.3%) 0.18 jaw thrust (n) 6 (25%) 6 (25%) 1 head flexion (n) 8 (33.0%) 1 (4.2%) 0.02 numerical data have been expressed as mean ± standard deviation. asa = american society of anesthesiologists physical status classification; t1 = time from the start of insertion of the fiberscope into nares till visualization of vocal cord; t2 = time from here to complete intubation as t2. table ii. result of regression analysis for time from the start of insertion of the fiberscope into nares till visualization of vocal cord (t1) as the dependent variable has been summarized. coefficient p value model constant 33.6 – intervention 82.7 <0.001 age (year) 1.5 0.11 weight (kg) �0.6 0.44 asa classes �30.8 0.04 intervention shows the t1 change between two groups, conventional (control) and nasal-18. asa = american society of anesthesiologists physical status classification. 140 a. mohammadzadeh et al. the vocal cords. in the nasal-18 method, the tube acts as a tool that provides good alignment to guide the fiberscope through the nasal cavity to face the vocal cords. this may explain why t1 was shorter in the nasal-18 group, though from this point there was no significant difference between endotracheal intubation times(t2)in thetwo groups.however, thenasal-18 technique probably reduced fiberscope twisting as well as mucus secretions and bleeding as a consequence of milder traumatization of the upper airway tract that could be achieved due to a short t1, and all these put together helped to increase the success rate of nasal intubation. utilization of fiberscope without the use of any facilitating techniques has proven to be a difficult task. in this study we could see that the use of nasal-18 technique allowed us to perform fibroscopy with greater ease and speed. the success rate for a quick and smooth intubation with the nasal-18 method was about 83.3%, while this rate in the conventional method was at 67%. however, the power of this study to detect such a difference was only 16%. a large sample size is needed to compare the difference in more detail. in conclusion, we can state that the nasal-18 technique facilitates the success of fiber-optic nasal intubation in patients. the nasal-18 method was found to be easier for the anesthesiologist and resulted in significantly faster glottic exposure. although this technique needs to be explored by other researchers in larger studies in other craniomaxillofacial anomalies and other difficult cases of airway management, we believe that this technique has the potential ability to be added to the list of routine techniques of fiberoptic nasal intubation. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. asai t. use of the laryngeal mask for tracheal intubation in patients at increased risk of aspiration of gastric contents. anesthesiology. 1992;77:1029–30. 2. aoyama k, takenaka i, nagaoka e, kadoya t. jaw thrust maneuver for endotracheal intubation using a fiberoptic stylet. anesth analg. 2000;90:1457–8. 3. hughes s, smith je. nasotracheal tube placement over the fibreoptic laryngoscope. anaesthesia. 1996;51:1026–8. 4. mason ra. learning fibreoptic intubation: fundamental problems. anaesthesia. 1992;47:729–31. 5. marfin ag, iqbal r, mihm f, popat mt, scott sh, pandit jj. determination of the site of tracheal tube impingement during nasotracheal fibreoptic intubation. anaesthesia. 2006;61:646–50. 6. murashima k, fukutome t. effect of jaw-thrust manoeuvre on the laryngeal inlet. anaesthesia. 1998;53:203–4. 7. nandi pr, charlesworth ch, taylor sj, nunn jf, dore cj. effect of general anaesthesia on the pharynx. br j anaesth. 1991;66:157–62. 8. fulling pd, roberts jt. fiberoptic intubation. int anesthesiol clin. 2000;38:189–217. 9. rogers sn, benumof jl. new and easy techniques for fiberoptic endoscopy-aided tracheal intubation. anesthesiology. 1983;59:569–72. 10. vaughan rs. training in fibreoptic laryngoscopy. br j anaesth. 1991;66:538–40. 11. ayoub cm, rizk ms, yaacoub ci, baraka as, lteif am. advancing the tracheal tube over a flexible fiberoptic bronchoscope by a sleeve mounted on the insertion cord. anesth analg. 2003;96:290–2. 12. schwartz d, johnson c, roberts j. a maneuver to facilitate flexible fiberoptic intubation. anesthesiology. 1989;71:470–1. 13. brull sj, wiklund r, ferris c, connelly nr, ehrenwerth j, silverman dg. facilitation of fiberoptic orotracheal intubation with a flexible tracheal tube. anesth analg. 1994;78: 746–8. 14. ovassapian a, yelich sj, dykes mh, brunner ee. fiberoptic nasotracheal intubation—incidence and causes of failure. anesth analg. 1983;62:692–5. 15. nakayama m, kataoka n, usui y, inase n, takayama s, miura h. techniques of nasotracheal intubation with the fiberoptic bronchoscope. j emerg med. 1992;10:729–34. 16. katsnelson t, frost ea, farcon e, goldiner pl. when the endotracheal tube will not pass over the flexible fiberoptic bronchoscope. anesthesiology. 1992;76:151–2. comparison of fiber-optic nasal intubation methods 141 tf-iups200024 200..203 editorial are we looking under the lamp although we know the lost key is somewhere else? or is it just about the egg? after more than 40 years with in vitro fertilization (ivf), our knowledge about which crucial factors determine human procreation, and how to best assist, is slowly increasing. slowly, too slowly, is the slightly depressing conclusion professor georg griesinger draws in the editorial of this issue of upsala journal of medical sciences. not even the flagship of unbiased evidence in medicine, randomized controlled trial (rct), is left unaffected under his scrutiny. most rcts in assisted reproductive technologies (art) are underpowered, and this problem is rarely solved by meta-analyses (1). but how then do we improve our knowledge about which factors determine human procreation and hence improve diagnostic and treatment modalities? needless to say, rcts and relevant meta-analyses should be the main source of knowledge, but such studies always come after innovative and pilot research, with data suggesting improvements in the outcome of art. such pilot findings may be of the highest quality, like the invention of intracytoplasmic sperm injection (icsi) (2), or the introduction of ultrasound guidance for oocyte collection (3), both methods almost immediately being introduced as game-changers. importantly, in the case of icsi, this method also provided the scientific society with the slightly surprising new information that male infertility in the majority of cases poses no problem when the oocytes are thus fertilized (4). this issue of upsala journal of medical sciences contains reviews of some new means and strategies for ovarian stimulation, i.e. random start (5), duo-stim (6), gnrh agonist trigger (7), and freeze-all (8) – all novelties with the potential of being true game-changers. interestingly, like the new biologic information provided by icsi, the random start of ovarian stimulation, used originally for urgent fertility preservation, also provides new knowledge about the endocrinology behind the recruitment of a dominant follicle in the human follicle phase (7). there is still a need for more carefully performed studies, however, for these novel methods and strategies to be established for appropriate patient populations. questions remaining to be answered are: should all treatments be done with the freezeall strategy, or only those at risk for ohss-complications? which protocol, if any, can safely be used for rescuing the luteal phase after a gnrh agonist trigger? will a double trigger using both a gnrh agonist and hcg injections qualify as the new standard in the antagonist protocol in women with a low risk of ohss? the increasing use of adjuvant therapies in art, most of which prove useless when seriously tested, is discussed in the paper by nardo et al. (9). this is an important field, as the commercialization of art promotes competition between clinics, a competition that often includes offers of various adjuncts. an analogy from the on-going coronavirus pandemic is the call from president trump, on live tv, to use ‘hydroxychloroquine’ as a medication (4 april 2020) to treat the covid-19 infection, much to the obvious embarrassment of his medical experts, as there is currently no evidence for benefits from such treatment. in a brilliant lecture on evidence-based medicine at a recent conference in sevilla, professor hans evers, former chief editor of human reproduction, described eloquently the rise and the fall of one of these adjuvant therapies, the ‘endometrial scratch’. a few years ago, much to the surprise of many experienced clinicians with knowledge of the principles of human reproduction, it was suggested that this deliberate injury to the endometrium could improve implantation. the reports followed a well-known pattern: first ‘pilot’ papers with astonishingly positive results, based on retrospective case series (10). a few supporting papers followed, based on similar ‘preliminary’ data (11). the subsequent mental process in the clinical and scientific community was a feeling of positive surprise and hopefulness that a breakthrough was finally here for the notoriously depressing group of patients with recurrent implantation failure (rif). in early and small publications, most often patients were their own controls, so that all included patients had one or more previous, failed treatments. after any intervention, here scratching, the postintervention treatment will then – by pure chance – result in a pregnancy for some patients. this is a statistical phenomenon named ‘regression to the mean’, which describes the inevitable change to the better for the group as a whole, as no treatment could end worse than the first (12). based on the first positive results, many groups all over the world have introduced the method. the subsequent results are not uniformly positive, but it takes a while before a few negative reports are published (13–15) – it is more appealing to publish breaking news findings than dull negations of first reports, the well-known ‘publication bias’ (16). however, when later, often much later, the results from a correctly performed rct are published (17), the bleak light of monday morning strikes the enthusiastic clinicians advocating the method. the entire process from the first overestimated pilot results, through the intermediate sobering-up phase, to the disappointing rct results follows an established curve called the gartner hype curve or gartner hype cycle. sobering up is, however, not always the case. an interactive mentometer investigation during hans ever’s lecture revealed that two-thirds of the audience had offered, or still did offer, endometrial scratching. as reviewed in the paper by nardo et al. (9), there are numerous examples of similar developments of treatment upsala journal of medical sciences 2020, vol. 125, no. 2, 200–203 https://doi.org/10.1080/03009734.2020.1755398 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1755398&domain=pdf&date_stamp=2020-05-29 https://doi.org/10.1080/03009734.2020.1755398 http://www.tandfonline.com innovations: addition of growth hormone, dhea, corticosteroids, intralipid infusion, acupuncture, etc., all with more or less the same disappointing end results according to current evidence. nevertheless, such negative results add significantly to our understanding of human procreation. most of these methods are in the field of adjuvant therapies or adjuncts, i.e. based on a concept that factors outside the crucial gamete/ embryo/uterus factor play a crucial role. which ‘external’ factors remain to be examined? immunological factors? until recently, it has been believed that the increased incidence of miscarriages, preeclampsia (18), and other complications related to placentation in donor oocyte recipient cycles (19) is caused by immunological incompatibility between the oocyte (embryo) and the recipient (20). new data, results from big data analyses, do, however, question this notion, as they suggest that a likely reason for such complications could be related to the lack of a corpus luteum, given that the endometrial preparation is commonly performed with hormone replacement therapy, also called programmed cycles, in oocyte donor–recipient cycles. results from transfers of frozen–thawed embryos show that such pregnancy complications are increased in programmed cycles also in non-donor treatments, compared with transfers in ovulatory cycles (21). it seems likely that substances produced by the luteal body affect placentation in a way that a mere oestrogen/progesterone-supported endometrium usually does not achieve. these results thus shed new light on the basics of human fertility and art. the results do not rule out the possibility of involvement of immunological factors per se, but they strongly suggest that factors produced by the corpus luteum, apart from progesterone, are important for a normal pregnancy. the conclusions from the lack of positive effects on art outcome from essentially all adjuvant therapies (9), combined with the excellent results of oocyte donation, are that the dominance of the ‘egg factor’ is becoming more evident for each year. the uterus and the endometrium definitely play ‘permissive roles’, i.e. pathological conditions such as intrauterine adhesions or submucous fibroids may reduce ivf results. likewise, a notoriously thin endometrium lowers live birth rates (4). however, when such conditions are absent, does there remain a role for factors such as an unsynchronized endometrium in relation to embryo maturity? although serious scientific and commercial efforts have been put into testing endometrial–embryo synchrony, such as the endometrial receptivity array (era)-test (22), there is little evidence today that this is a factor of true clinical importance. for severe uterine factors, surrogacy or uterine transplantation is the only option. in this issue of upsala journal of medical sciences the use of surrogacy is extensively discussed, focussing on the complicated ethical questions raised (23). when one of the leading geneticists in preimplantation genetic testing for aneuploidies (pgt-a) was asked about the relative importance of the embryo versus the endometrium in human reproduction, his response was 90 to 10 in favour for the embryo, and a similar ratio for the oocyte versus the sperm, as a general estimate of the roles of the major players. current results from pgt-a research support this notion (24). it is likely that with the progress of less invasive pgt-a-related technologies, increased accuracy of selection of the top embryo will take art to higher success rates per transferred embryo. however, the legislation in some countries, like in sweden, still does not allow pgt-a for selection of a euploid blastocyst in the absence of a known serious hereditary condition. the ethical problems associated with pgt-a as a means for embryo selection are discussed by kjell asplund (25). as reviewed by lundin and park, evidencebased assessments of the embryo cleavage and morphology in terms of scoring were introduced fairly late in the history of art, and time-lapse monitoring has yet to prove its benefits (26). will advanced analyses based on machine learning finally prove time-lapse technology to be worth the big costs (27)? most likely, non-invasive pgt-a would provide an optimal means for embryo selection, and its use would reduce the treatment burden, helping with diagnostics in rif cases and for patients with repeated miscarriages. thus, this technology would be of value for a significant subgroup of patients in art, and the ethical issues involved could most likely be dealt with by careful legislation. the ‘egg factor’ is thus dominant as a cause of subfertility and the most frequently limiting factor. recent evidence supports that ambitions to retrieve maximal numbers of oocytes per opu should be encouraged, if needed with gnrh-agonist triggering (28). although earlier data suggested that the pregnancy percentage decreases with oocyte numbers greater than 15–20 oocytes per retrieval (29), this is not true when additional fets are included (28). thus, ‘mild’ ivf stimulation has little role to play in modern ivf. the ‘egg factor’ is still not well known among women in their 20 s. this is shown by studies that describe that young women tend to overestimate the fertility potential of women of 35–40 years of age, as reviewed by delbaere et al. (30). for an increasing number of women, however, the possibility of storing vitrified oocytes for future use is seen as an option. so far, only few reports have been published on the outcome of warming and fertilizing such oocytes later. in this issue of upsala journal of medical sciences a review of pregnancy results in women returning for this purpose is given (31), showing indeed an age-factor for oocytes, with acceptable results for those who had their oocytes frozen before the age of 40, but not later. accumulating true new knowledge in the field of natural and assisted reproduction is a slow process and should be based on rcts and big data analyses. progress is hampered by numerous turns into blind alleys, each of which – if published – nevertheless contributes to current knowledge, as also negative results are important for the full picture. the oocyte is the crucial factor in most cases, which has to be kept in mind, when treatment and laboratory modalities are formed, and when patients are informed about the basic conditions estimating their chances for successful treatment. disclosure statement the authors have nothing to disclose. upsala journal of medical sciences 201 funding the carl von linn�e clinic, uppsala, sweden. notes on contributors jan holte, md, phd, senior consultant and co-founder, carl von linn�e clinic, uppsala science park. chairman of the board, centre for reproductive biology, a joint body between the swedish university of agricultural sciences and uppsala university. affiliated researcher, department of women’s and children’s health, uppsala university. thomas brodin, certified physician 1994. board certified specialist in obstetrics and gynecology 1999. doctor/researcher at the carl von linn�e clinic and the dept of women’s and children’s health, uppsala university, since 2009. orcid thomas brodin http://orcid.org/0000-0002-0696-0294 references 1. griesinger g. is progress in clinical reproductive medicine happening fast enough? ups j med sci 2020;125:65–67. 2. palermo g, joris h, devroey p, van steirteghem ac. pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. lancet. 1992;340:17–8. 3. wikland m, enk l, hammarberg k, nilsson l. use of a vaginal transducer for oocyte retrieval in an ivf/et program. j clin ultrasound. 1987;15:245–51. 4. vaegter kk, lakic tg, olovsson m, berglund l, brodin t, holte j. which factors are most predictive for live birth after in vitro fertilization and intracytoplasmic sperm injection (ivf/icsi) treatments? analysis of 100 prospectively recorded variables in 8,400 ivf/icsi single-embryo transfers. fertil steril. 2017;107:641–8 e2. 5. rodriguez , ka, anastacio a, vonheim e, deen s, malmros j, borgstr€om b. fertility preservation for young adults, adolescents and children with cancer. ups j med sci 2020;125:112–20. 6. vaiarelli a, cimadomo, d, petriglia c, conforti a, alviggi, c, ubaldi n, et al. duostim: a reproducible strategy to obtain more oocytes and competent embryos in a short time-frame aimed at fertility preservation and ivf purposes. a systematic review. ups j med sci 2020;125:121–30. 7. castillo j, haahr t, martinez-moya m, humaidan p. gonadotropinreleasing hormone agonist for ovulation trigger – ohss prevention. ups j med sci 2020;125:131–37. 8. celada p, bosch e. freeze-all, for whom, when and how. ups j med sci 2020;125:104–11. 9. nardo lg, chouliaras s. adjuvants in ivf – evidence for what works and what does not work. ups j med sci 2020;125:144–51. 10. huang sy, wang cj, soong yk, wang hs, wang ml, lin cy, et al. site-specific endometrial injury improves implantation and pregnancy in patients with repeated implantation failures. reprod biol endocrinol. 2011;9:140. 11. narvekar sa, gupta n, shetty n, kottur a, srinivas m, rao ka. does local endometrial injury in the nontransfer cycle improve the ivf-et outcome in the subsequent cycle in patients with previous unsuccessful ivf? a randomized controlled pilot study. j hum reprod sci. 2010;3:15–9. 12. barnett ag, van der pols jc, dobson aj. regression to the mean: what it is and how to deal with it. int j epidemiol. 2005;34: 215–20. 13. yeung tw, chai j, li rh, lee vc, ho pc, ng eh. the effect of endometrial injury on ongoing pregnancy rate in unselected subfertile women undergoing in vitro fertilization: a randomized controlled trial. hum reprod. 2014;29:2474–81. 14. dain l, ojha k, bider d, levron j, zinchenko v, walster s, et al. effect of local endometrial injury on pregnancy outcomes in ovum donation cycles. fertil steril. 2014;102:1048–54. 15. aflatoonian a, research and clinical center for infertility, shahid sadoughi university of medical sciences, yazd, iran, baradaran bagheri r, hosseinisadat r. the effect of endometrial injury on pregnancy rate in frozen-thawed embryo transfer: a randomized control trial. ijrm. 2016;14:453–158. 16. furuya-kanamori l, xu c, lin l, doan t, chu h, thalib l, et al. p value-driven methods were underpowered to detect publication bias: analysis of cochrane review meta-analyses. j clin epidemiol. 2020;118:86–92. 17. lensen s, osavlyuk d, armstrong s, stadelmann c, hennes a, napier e, et al. a randomized trial of endometrial scratching before in vitro fertilization. n engl j med. 2019;380:325–34. 18. schwarze je, borda p, v�asquez p, ortega c, villa s, crosby ja, et al. is the risk of preeclampsia higher in donor oocyte pregnancies? a systematic review and meta-analysis. jbra assist reprod 2018;22:15–9. 19. masoudian p, nasr a, de nanassy j, fung-kee-fung k, bainbridge sa, el demellawy d. el demellawy d. oocyte donation pregnancies and the risk of preeclampsia or gestational hypertension: a systematic review and metaanalysis. am j obstet gynecol. 2016; 214:328–39. 20. savasi vm, mandia l, laoreti a, cetin i. maternal and fetal outcomes in oocyte donation pregnancies. hum reprod update. 2016;22:620–33. 21. von versen-h€oynck f, schaub am, chi y-y, chiu k-h, liu j, lingis m, et al. increased preeclampsia risk and reduced aortic compliance with in vitro fertilization cycles in the absence of a corpus luteum. hypertension 2019;73:640–9. 22. bassil r, casper r, samara n, hsieh tb, barzilay e, orvieto r, et al. does the endometrial receptivity array really provide personalized embryo transfer? j assist reprod genet. 2018;35:1301–5. 23. gunnarsson payne j, korolczuk e, mezinska s. surrogacy relationships: a critical interpretative review. ups j med sci 2020;125:183–91. 24. bellver j, bosch e, espin�os jj, fabregues f, fontes j, garc�ıavelasco j, et al. second-generation preimplantation genetic testing for aneuploidy in assisted reproduction: a swot analysis. reprod biomed online. 2019;39:905–15. 25. asplund k. use of ivf-ethical issues. ups j med sci 2020;125:192–99. 26. lundin k, park h. time-lapse technology for embryo culture and selection. ups j med sci 2020;125:77–84. 27. vermilyea m, hall jmm, diakiw sm, johnston a, nguyen t, perugini d, et al. development of an artificial intelligence-based assessment model for prediction of embryo viability using static images captured by optical light microscopy during ivf. hum reprod 2020. 28. magnusson a, k€allen k, thurin-kjellberg a, bergh c. the number of oocytes retrieved during ivf: a balance between efficacy and safety. hum reprod. 2018;33:58–64. 29. sunkara sk, rittenberg v, raine-fenning n, bhattacharya s, zamora j, coomarasamy a. association between the number of eggs and live birth in ivf treatment: an analysis of 400 135 treatment cycles. hum reprod. 2011;26:1768–74. 30. delbaere i, verbiest s, tyd�en t. knowledge about the impact of age on fertility: a brief review. ups j med sci 2020;125:167–74. 31. wennberg al. social freezing of oocytes: a means to take control of your fertility. ups j med sci 2020;125. doi:10.1080/03009734. 2019.1707332 jan holte and thomas brodin carl von linne kliniken, uppsala, sweden department of women’s and children’s health, akademiska sjukhuset, uppsala university, uppsala, sweden jan.holte@linne.se 202 j. holte and t. brodin https://doi.org/10.1080/03009734.2019.1707332 https://doi.org/10.1080/03009734.2019.1707332 received 9 april 2020; accepted 9 april 2020 � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. https://doi.org/10.1080/03009734.2020.1755398 upsala journal of medical sciences 203 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1755398&domain=pdf&date_stamp=2020-05-29 outline placeholder outline placeholder disclosure statement funding notes on contributors references upsala journal of medical sciences 2021, 126, e5665 http://dx.doi.org/10.48101/ujms.v126.5665 biography contact adam taube adam.taube@statistik.uu.se © 2021 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. observations by a statistical watchdog adam taube department of statistics, uppsala university, po box 513, se-75120 uppsala abstract during more than five decades, the author has kept a critical eye on how statistical methods are (mis-) used in medical research. some areas are presented where serious statistical mistakes are prevalent. two investigations with erroneous conclusions are described in detail. situations where outside authorities have tried to mute medical researchers are also commented upon. the authors own efforts to improve the use of statistical methods and the current situation with easily accessible statistical program packages are described. finally, the importance of continued ‘statistical cleansing’ is stressed. article history received 12 september 2020 revised 21 october 2020 accepted 2 november 2020 published 8 february 2021 keywords selection bias; data torturing; statistical cleansing; multiplicity problems; clinical trials; survival analysis when i started my 60-year random walk in medical statistics, i  happened to read a paper in a swedish medical journal about  ‘science, statistics and psychiatry’. it was written in an  elegant but arrogant manner by a well-reputed medical doctor and contained some serious misunderstandings. the  author seemed to have a high confidence in his own intellectual abilities. obviously, he was one of many doctors at  the time i had heard expressing views like: i don’t apply any  sophistical statistical methods which the reader can’t understand. instead i use my ordinary common sense. that is quite enough. not seldom, this attitude was combined with a certain ‘statistics hostility’, probably a way of compensating for the lack of statistical knowledge. however, arriving at a wrong conclusion by applying an inadequate statistical method in a wrong way does not tell us anything about the method as such. further, it does not demonstrate that there is some sort of gap between using ‘ordinary common sense’ and a correct statistical analysis of a scientific problem. i felt seriously provoked by the article and wrote a paper of my own, presenting some critical arguments. my contribution was formulated in a similar manner as the paper i criticized. i showed the manuscript to the old uppsala-professor gunnar nyström, a retired professor of surgery with an international reputation, a wise man and friend of the family who had recommended me to specialize in medical statistics. he fully agreed on each point in my paper, but was very critical of the arrogant tone in my formulations. intentionally, i had used the same style as the other author, but he was not satisfied: it doesn’t matter in which manner the other author has expressed his views, you have to write as a gentleman! of course, i rewrote my manuscript, which was then accepted in the same journal. it was my first publication in a medical journal and my first effort in ‘statistical criticism’. unfortunately, i have not always been able to follow the advice ‘to write as a gentleman’. sometimes, the temptation to formulate stinging remarks has been too strong, and i have felt that in my job as a statistician on a university level, it is important to keep an eye on how standard statistical methods are used and often misused in medical publications. academic statisticians ought to devote more efforts to this important task. the lack of statistical knowledge or unwillingness to adapt modern statistical methods decreases the scientific quality, and it must be stressed that there are no statistical methods for improving faulty data (‘garbage in–garbage out’). also, the lack of courage to stand up for scientific values is sometimes disturbing. all these make the role of statistical watchdogs important. in the following, i will describe how the work on statistical selection mechanisms made me look for such errors in medical research and then widened my interest to all types of statistical mistakes, which i have found in numerous publications. then, i  describe my own efforts to improve the situation. the problems with improper pressure from outside authorities to prevent certain researchers from publishing what they want are taken up, and finally, some recommendations for future activities to improve the situation are given. my statistical battlefield at the end of the fifties, i collaborated with a physician, willem van der linden, investigating some data where statistical selection mechanisms were involved. in a medical paper, http://dx.doi.org/10.48101/ujms.v126.5665 mailto:adam.taube@statistik.uu.se http://creativecommons.org/licenses/by/4.0/ 2 a. taube the  famous prof. henschen claimed that there was a negative correlation between the occurrence of arteriosclerosis and cancer (1). the basis was to be found in autopsy data. by means of a simple numerical example, willem and i could demonstrate that this pattern could occur simply due to the different death risks, even when the two diseases were not related at all in the population at risk (par), from which the dead patients had come (‘competition among fatality rates’) (2). it was a simple application of ‘berkson’s fallacy’, first described in a paper in 1946 (3). willem and i wrote a short communication on this statistical trap (4) and also a survey of different kinds of statistical selection mechanisms and selective factors in patient data (5). we then continued to publish a number of papers with statistical criticism until willem’s death some years ago. when scrutinizing the statistical analysis in a medical paper, the first step must always be to find out how the data were generated and how they should determine the choice of analysis. this applies both to experimental and nonexperimental studies, but it is perhaps more tricky in the latter case, which will be illustrated here. length of hospital stay sometimes, studies are concerned with a group of patients with a certain diagnosis without further specifications. if, for example, this group consists of individuals available in a hospital during a certain day, or a certain week, the sample can be biased. the longer a patient is hospitalized, the larger is his/her probability of being included in the study. thus, the individuals under study will have an overrepresentation of serious cases. this type of bias was already pointed out by prof. schwarz in 1957 (6) but one can still come across studies where the authors are not aware of this mechanism or just ignore it. even many nonmedical investigations with this type of bias can be found. only some participants are able to answer my studies of various selection mechanisms made it easier for me to identify other situations where the data structure could influence the analysis and sometimes even be used as the basis for misleading results, as in an interview study in africa about women’s age at their first childbirth. the aim was to estimate the mean age for this event. a number of women in the age of 10 to 50 years were asked how old they were when they had their first child. the (arithmetic) mean, directly based on these answers, is of course influenced by the fact that a number of interviewed women haven’t had any children yet. therefore, the analysis must be adjusted in some way to avoid bias. i have come across several studies, about weaning, first intercourse, etc., where this type of bias was not recognized (or ignored?) by the investigators. i was confronted with this problem in ethiopia, where a number of school girls aged between 9 and 17 years were interviewed about their age at menarche, and the aim was to estimate the mean age for this event, an important indicator used in international comparisons. i suggested a new method to adjust for the fact that answers could be expected only from those girls who had started to menstruate (7). later, the theoretical properties of different approaches to this problem were investigated and compared in detail (8). too many and too small comparison groups for the study of prognostic factors in, for example, oncology, it happens that a certain group of available patients (by necessity a limited number) are studied with regard to death or relapse with ‘time to event data’. some already known prognostic variables are studied together with some new (hopefully better) ones. a common approach is to sort the individuals into subgroups with regard to the various background variables. with one dichotomous background variable, there will be two subgroups; with two, there will be four subgroups; with three, eight subgroups; and so on. then, comparisons between the outcomes in various subgroups are performed. in a swedish doctoral work, 49 patients (35 events) were studied with regard to six possible background variables, giving 17 p-values. in a certain category of 25 patients, the subgroup comparisons resulted in 27 more p-values. of course, this gave a number of ‘significant differences’ between various groups due to random variation, but the multiplicity problem with all the p-values was never addressed, nor the fact that some of the subgroups must have contained very few individuals or were perhaps even empty. a recommended rule of thumb is to have at least 10 events per explanatory variable (9). in the same work, another analysis on 26 patients was used for 21 significance analyses and 15 cox regressions. by publishing different analyses of the same data in different journals, the multiplicity problem is sometimes less obvious. it is not surprising that these kinds of studies have revealed extremely few realistic prognostic factors, and it is shocking that such meaningless ‘datatorturing’ can be taken seriously by the medical community and even be published in well-reputed medical journals (10). wrong interpretation of ‘non-significant results’ even if significance testing is a well-established technique among medical investigators, there is one possible misunderstanding which needs special attention, namely the belief that a statistically non-significant result proves that the null-hypothesis is true. a non-significant result simply shows that it was not possible to reject the null-hypothesis due to random variation, too few observations, or something else. when i criticized a certain study, the authors answered: what we find most remarkable is [the fact] that taube hasn’t understood that the null hypothesis is valid which is demonstrated in table 4 which leads to the important conclusion that all these sub-sample populations are parts of one [and the same] single ‘mother population’ (11). it is not difficult to find investigations which have been completely jeopardized due to this misunderstanding of what a statistically non-significant difference really means. observations by a statistical watchdog 3 ignoring the data structure in some studies, it is obvious that the authors base the statistical analysis on wrong assumptions about the structure of their own data. this happened, for example, in an interview study, where a number of persons, say n, were interviewed on three different occasions, and the analysis was performed as if the answers were 3 × n statistically independent observations. this gives an exaggerated view of the sample size and neglects the possible variation between the interview occasions. it would be tempting to continue presenting a list of all the possible mistakes and misinterpretations to be found in some medical papers, but here it is enough to refer to the works by altman and colleagues. they have initiated vivid academic activities in this field in great britain, where critical discussions about statistical methods in medical research seem to be far more established than in sweden (12). in my work with statistical criticism of medical papers, i found that the most serious problems were those connected with the data structure. if the structure is given a suitable model in the analysis, it is not a big issue if, say, minor changes of the analysis method give a confidence interval of 90% instead of the stated 95%. however, if the data character or structure is not taken care of in the subsequent analysis, very serious problems can be expected. for all types of structural problems, the results could be dramatically distorted as will be illustrated in the following two examples, where i happened to take part. it is also interesting to see how the persons involved acted during and after the publication of these investigations. who the researchers were is of no interest here, so no references are given. a misinterpreted clinical trial in the beginning of the nineties, i was contacted by two prominent professors concerning a recent doctoral dissertation from finland, comparing the mortality among prostate cancer patients treated either with estrogens or orchiectomy. independently of each other, they both contacted me for a critical inspection of the work. thus, i spent part of the summer scrutinizing the dissertation and especially the basic, already published, papers included. the project was presented as a randomized trial, comparing three treatment groups a, b, and c, and there were especially some differences in the outcomes in a and c which aroused suspicions. i soon discovered that the basis for the dissertation was not one single trial, but two: first, during several years, a randomized trial comparing a and b, and then, for another and different long period of time, a clinical trial comparing b and c. the two trials did not follow identical protocols, the selection criteria were not the same and the randomization procedures were different. consequently, it was not surprising that the comparisons between the groups a and c gave some ‘mysterious results’ which were quite easy to explain. in the autumn, i presented my findings in a lecture to a number of specialists from the nordic countries. the two professors who had initiated my investigation were very enthusiastic. both stressed how important it was that my findings were published at the soonest. first, i wrote a research report from the department of statistics, which i sent to the author and to the professor in finland, his research supervisor, with polite letters. the latter answered at once, in a hostile way. in summary, he stated that i should refrain from putting my nose into research, which was outside my field of competence. the next step was to write a ‘letter to the editor’ to the actual journal. i thought that this would have more weight, if the two enthusiastic professors also signed it. one of them explained, however, that he didn’t want to disturb his good collaborations with his colleagues in finland and the other gave the cryptic answer: ‘i remain on my own’. in the end, there was only one, a junior physician, who supported me and signed the letter. the editor sent my manuscript to the author in finland who gave a very detailed answer, taking up all my arguments. he concluded that my criticism was fully relevant. after all these, i expected my ‘letter to the editor’ to be published quite soon. nearly 2 years later, i presented this sequence of events in a lecture at a cancer center in seattle, and i finished with a slide: publication when? after the lecture, i was approached by a gentleman from the audience, who was very upset. he was a member of the editorial board of the journal. my ‘letter to the editor’ appeared in the next issue, and i have reasons to believe that this was due to his intervention. from all this, we can conclude that courage and support from professors and supervisors is not always to be expected and that it can take a long time until (if ever) a critical remark can reach the readers. an inadequate ‘survival analysis’ at an international conference, a swedish researcher presented a paper where it was claimed that the survival was longer among those prostate cancer patients who had been operated upon than those who had been treated in other ways. the paper was deemed to be very important, and i guess that it had already been accepted in a well-reputed medical journal, without being thoroughly refereed. the paper was also the most important building block in a forthcoming doctoral dissertation in sweden. i was contacted by the faculty opponent, who was confused by the statistical analysis, but couldn’t exactly point out the possible error. after some study, we found that the basis for the statistical analysis was all prostate cancer patients in a certain geographic region who had passed away within a specified time period. retrospectively, the author had collected clinical data concerning these individuals and could assess whether they had been operated upon or not. after this, he made an analysis of the collected data as if they had come from a prospective cohort study. the remarkable circumstance concerning this socalled survival study is of course that the individuals had to die in order to be included. those patients during the actual period who had survived were not included and the spectacular 4 a. taube results concerning longer survival of the operated patients were not valid. at the public presentation of the dissertation, the opponent raised all these critical comments. there were also several ‘extra opponents’ who had serious objections. in spite of this, the dissertation passed and the paper was published. i wrote a ‘letter to the editor’. this was also signed by several important swedish experts, but obviously, the editor was reluctant to accept our contribution. therefore, i asked prof. marvin zelen in boston, who gladly signed together with some other international experts and the letter was finally published in the journal. one can guess that there were heavy economic interests behind the desire to show that a prostate cancer operation was to be preferred to other alternatives. each year, the journal had a sort of competition among medical students. if they could identify articles, where some specified important medical achievements were presented, they were automatically included in a lottery and could win scholarships for their future medical studies. the (erroneous) result concerning prostate cancer operations was one of the ‘achievements’, which was considered worth finding. later, in a sort of yearbook about the most important recent surgical advancements, the erroneous article from sweden was also included. educational efforts in 1985, i was asked to give a lecture to the editorial staff of läkartidningen on how to present statistical data in a medical paper. in preparing this, i studied all the issues of the journal during the previous year, and there was indeed much to be commented upon. instead of a single lecture, i gave a whole study day. i also wrote a series of critical articles studies in medical statistical routines (13–17). the most important points dealt with the common overuse of ‘significance stars’ and p-values and the quite common, questionable tradition of studying one variable at a time in problems where a multivariate approach was more relevant as some detailed cross tabulations or perhaps regression analysis. probably, this misuse could partly be explained by the fact that the authors were victims of too many elementary medical statistics courses, where they had learned how to handle just one variable, calculating means, standard deviations, and some simple tests between two groups. when time came for multivariate analysis, the course was already over, perhaps after a short presentation of how to calculate a correlation coefficient. i found it somewhat of a paradox that medical doctors, who are forced to a multivariate approach when considering all aspects concerning each single patient, so often studied just one variable at a time when presenting a medical investigation. it should be noted though that in the eighties, multivariate statistical methods were not so easily accessible as they are today. pressure from outside my activities in statistical criticism had some unexpected consequences. i was contacted by medical doctors/researchers who had observed heavy misuse of statistics in their professional environment, but explained that they could not act freely. if they talked about their critical views or published anything about them, they would be victims of various sanctions from above. therefore, they asked me to write about their problems, which i also did in a number of cases. another situation i have experienced several times in connection with my statistics courses for doctoral students in the medical faculty: after the lecture, somebody waits until all the others have left and then asks me about a statistical problem in his/her doctoral project. but i have to promise not to say anything to anybody, since the medical supervisor does not tolerate consultations with outside statisticians. an example of the efforts to mute independent doctors/ researchers: in a large register study of cardiac deaths, a ranking list of the swedish clinics was presented by the national board of health and welfare, socialstyrelsen. in this, one particular clinic was pointed out as especially inefficient. the board was straightforward in presenting this ‘finding’ to the general public. some tabloid papers even warned potential patients not to attend the clinic. two senior doctors at the clinic performed a thorough investigation of all the cases reported from the clinic to the  register and found a number of inadequacies and mistakes,  which fully explained the disastrous statistical results. however, this was never heard of at the official press conference with representatives both from the local staff and socialstyrelsen. before this event, the local doctors had been carefully instructed in a written memorandum from the chief administrator of the county council how to behave: irrespective of whether the medical board is right or wrong, we have to keep a collegial, humble attitude, especially if we present any criticism about them. be responsible, admit that we take it very seriously (the national board of health and welfare is not something you can simply neglect!) and that we will change our routines accordingly, as soon as possible. in a paper about statistical difficulties and mistakes in extensive register studies that i submitted soon after the above event, i included a quotation from the current memorandum, but it was never included (18). it was the only time when an editor has deleted something from my text after i had said ok to the last galley proof. in spite of this negative experience, i must admit that on the whole, i have had great freedom to present and publish my critical views. it is obvious that in a number of situations, the researchers are not free from censorship. therefore, it is highly desirable that people outside the medical establishment can point out (statistical) errors in medical research. i have been able to be critical and publish my arguments because i have not been affiliated with any medical department, especially so during my last 10 years before retirement, when i was employed by the swedish cancer society, where my criticism was very much encouraged. i was surprised and disappointed, however, that i got so little support from my statistical colleagues in the academic world. observations by a statistical watchdog 5 today and tomorrow the negative attitude toward statistical methods seems to have changed. the situation today, 60 years after my first attempts at statistical criticism, is indeed quite different. a number of new statistical methods have been developed and refined approaches in the design of clinical trials are now applied. multivariate methods, like the cox regression, are widely accepted by medical researchers. this development has been possible due to easily available statistical packages. the arsenal of statistical techniques has exploded. unfortunately, this generates a high risk for the misuse of statistical methods. it is not difficult to find studies where the choice of statistical approach seems to be based more on the availability of certain programs than the knowledge of statistical methods and principles. also, during the last decades, a number of national registers have been created, which sometimes are used for statistical analyses made by people with scanty knowledge of statistical principles (19). unfortunately, some of the misbehaviors that i have come across can be expected to occur even in the future, such as the lack of courage among some supervisors/professors, the lack of support from above, and the pressure from certain authorities to prevent medical researchers from publishing what they want. cleansing can never cease when i published a critical comment some years ago about a questionable statistical analysis in a medical paper, the authors answered that professor taube has misinterpreted our paper. it was not intended to be a scientific article, only a report to the socialstyrelsen. this indicates a disturbing and unacceptable dualism in the quality standards of data presentations and conclusions. this attitude is even more directly expressed in a warning on a report from a nutrition institute: the studies between these covers do not have as their object a high scientific level of precision, performed as they are, during a very short term and on small samples. they are meant to provide rough indications and trends of informative value for decision makers and others, who work in a practical local context. such efforts to defend questionable investigations are of course illogical and necessitate continued statistical cleansing. personally, i have great difficulty in accepting that it is more important that data be reliable in a scientific paper than when they are used as a basis for real-life decisions. i can’t help thinking it ought to be the other way around. it is no doubt unethical to perform an erroneous statistical analysis, deliberately or not, but i always hope that mostly such mistakes are not intentional. even so, they can lead to wrong conclusions and inefficient, inadequate, or harmful treatments. therefore, it will always be necessary to keep a critical eye on medical publications. it is an important task for statisticians on an academic level, and their contributions to the critical discussions in various journals would also be recognized as academic merits. sometimes, people have asked me: don’t you get many enemies when you write a critical article? my answer is: yes indeed! but for every enemy, i usually get at least two new good friends. acknowledgment sincere thanks to dr howard fischer for providing most valuable comments on the manuscript. disclosure statement the author reports no conflicts of interest. references 1. henschen f. arteriosclersosens correlation till ålder, kön, nutritionstillstånd, hjärtsvikt, diabetes, gallsten, tuberkulos och maligna svulster. sv läkartidningen. 1959;56:1674–86. 2. van der linden w, taube a. arterioscleros och cancer i obduktionsmaterial. sv läkartidningen. 1959;56:3536–9. 3. berkson j. limitations of the application of fourfold table analysis to hospital data. biometric bull. 1946;2:47–53. doi: 10.2307/3002000 4. van der linden w, taube a. association of cholelithiasis, hiatus hernia, and diverticulosis coli. jama. 1959;180:408. doi: 10.1001/ jama.1959.03000210102018 5. van der linden w, taube a. statistiska selektions-problem i icke-experimentella medicinska material. nord med. 1962;67:589–602. 6. schwarz d, anguera g. une cause de biais dans certaines enquêtes medical: le temps de séjour de maladies à l´hopital. comm. inst. intern. statis. 30 è session. stockholm: almqvist och wiksell; 1957. 7. tekle wold f, sterky g, taube a. the age of menarche in a group of schoolgirls in addis ababa. ethiop med j. 1972;10:159–66. 8. atwood c, taube a. estimating mean time to reach a milestone, using retrospective data. biometrics. 1976;32:159–71. doi: 10.2307/2529346 9. simon b, altman dg. statistical aspects of prognostic factors in oncology. br j cancer. 1994;69:979–85. doi: 10.1038/bjc.1994.192 10. taube a, högberg t. med p: n som i progampaket. läkartidningen. 2002;99:3302–5. 11. taube a. nollhypotesen ‘bevisad’ – vanlig missuppfattning. läkartidningen. 1993;90:684. 12. altman d. statistical reviewing for medical journals. stat med. 1998;17:2661–74. doi: 10.1002/(sici)1097-0258(19981215)17:23 <2661::aid-sim33>3.0.co;2-b. 13. taube a. studier i medicinsk statistisk slentrian. i. att illustrera en åldersfördelning. läkartidningen. 1985;82:2235–6. 14. taube a. studier i medicinsk statistisk slentrian. ii. att rita diagram. läkartidningen. 1985;82:2327–30. 15. taube a. studier i medicinsk statistisk slentrian. iii. det borde (inte) vara stjärnor. läkartidningen. 1985;82:2422–4. 16. taube a. studier i medicinsk statistisk slentrian. iv. varför bara studera en variabel i taget? läkartidningen. 1985;82:2520–1. 17. taube a. studier i medicinsk statistisk slentrian. v. några funderingar. läkartidningen. 1985;82:2585. 18. taube a. missbruk av registerdata i.: osäkra data om infarkt. resultaten övertolkade. läkartidningen. 1996;93:4620–2. 19. taube a. missbruk av registerdata ii.: tretton kirurger gör statistiska snitt. läkartidningen. 1997;94:61–2. http://dx.doi.org/10.2307/3002000 http://dx.doi.org/10.1001/jama.1959.03000210102018 http://dx.doi.org/10.1001/jama.1959.03000210102018 http://dx.doi.org/10.2307/2529346 http://dx.doi.org/10.1038/bjc.1994.192 http://dx.doi.org/10.1002/(sici)1097-0258(19981215)17:23<2661::aid-sim33>3.0.co;2-b http://dx.doi.org/10.1002/(sici)1097-0258(19981215)17:23<2661::aid-sim33>3.0.co;2-b dynamic changes in glycosylation and glycan composition of serum fsh and lh during natural ovarian s full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 dynamic changes in glycosylation and glycan composition of serum fsh and lh during natural ovarian stimulation leif wide & karin eriksson to cite this article: leif wide & karin eriksson (2013) dynamic changes in glycosylation and glycan composition of serum fsh and lh during natural ovarian stimulation, upsala journal of medical sciences, 118:3, 153-164, doi: 10.3109/03009734.2013.782081 to link to this article: https://doi.org/10.3109/03009734.2013.782081 © informa healthcare published online: 25 mar 2013. submit your article to this journal article views: 819 view related articles citing articles: 28 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.782081 https://doi.org/10.3109/03009734.2013.782081 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.782081 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.782081 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.782081#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.782081#tabmodule upsala journal of medical sciences. 2013; 118: 153–164 original article dynamic changes in glycosylation and glycan composition of serum fsh and lh during natural ovarian stimulation leif wide & karin eriksson department of medical sciences, uppsala university, clinical chemistry, university hospital, se 751 85 uppsala, sweden abstract background. glycosylation and glycan composition are of fundamental importance for the biological properties of fsh and lh. the aim of this study was to determine the glycosylation, sialylation, and sulfonation of serum fsh and lh throughout the normal menstrual cycle. methods. serum samples were collected from 79 healthy women with regular menstrual cycles. the mean numbers of anionic monosaccharide (ams), sialic acid (sa), and sulfonated n-acetylgalactosamine (su) residues per fsh and lh molecule were estimated for all sera with methods based on electrophoreses, neuraminidase treatments, and fluoroimmunoassays of the gonadotrophins. results. di-glycosylated glycoforms (fshdi, lhdi) were detected in serum in addition to tetra-glycosylated fsh (fshtetra) and tri-glycosylated lh (lhtri). fshdi exhibited two peaks: one on day 5 to 7 and one, more pronounced, at midcycle. fshtetra plateaued at a high concentration from day 5 to 15, without a midcycle peak. there were lower concentrations of lhdi than lhtri, except at midcycle when the opposite occurred. the mean numbers of sa and su residues per molecule of fsh and lh in serum showed four different patterns during the cycle, all with highly significant (p < 0.0001) differences between levels at different phases of the cycle. the pattern of sa residues on fsh was ‘m’-shaped, and that of su on lh ‘v’-shaped. conclusion. serum fsh and lh governing the natural ovarian stimulation process exhibited dynamic changes of glycosylation and glycan composition. this new information on the fsh and lh molecular structures may lead to more successful mono-ovulatory treatment regimens for ovulation induction in anovulatory women. key words: di-glycosylated fsh, di-glycosylated lh, glycoforms, isoforms, ovulation induction, sialic acid, sulfonated n-acetylgalactosamine introduction human gonadotrophin preparations have been used for the induction of ovulation in anovulatory women during more than five decades. successful treatments were first described by gemzell et al. in 1958 using a crude human pituitary follicle-stimulating hormone (fsh) preparation (1). large pools of pituitaries had been collected at autopsy in hospitals, mainly from elderly men and women. a few years later, donini et al. reported a method for the extraction of gonadotrophins from human menopausal urine, and these extracts, called human menopausal gonadotrophins (hmg), were soon introduced for ovulation induction (2). two decades later, highly purified fsh isolated from menopausalurinewasmarketedforovulationinduction(3).in 1992,recombinanthumanfshpreparationswereintroduced for the same purpose (4). these treatments have been highly successful but also associated with a risk for ovarian hyperstimulation and multifetal pregnancies. although the frequencies of these severe complications havebeenreducedbyusingtheserumoestradiollevelsto monitor the treatments (5-7), later in conjunction with ultrasound and by the use of lower dosages of gonadotrophins, the risk is still substantial (8,9). a principle of step-down protocols has been developed in order to correspondence: leif wide, md, phd, department of clinical chemistry, university hospital, se 751 85 uppsala, sweden. fax: +46 18 611 37 03. e-mail: leif.wide@medsci.uu.se (received 23 january 2013; accepted 28 february 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.782081 mimic the natural ovarian stimulation process more closely (10). after an initial higher dose of fsh, a lower dose of fsh is administered. this is thought to lead to atresia of most of the developing follicles. used for ovulation induction, human pituitaryand urinary-derived preparations from elderly individuals are not physiological. we have shown that the molecular carbohydrate structures of serum fsh and lutenizinghormone(lh)inmenopausalwomenandinmenare different from those in young women (11,12). also, recombinant fsh preparations (13) have a different carbohydrate structure compared with fsh of young women. mono-ovulation is the aim in the treatment of anovulatory women. it has continuously been a desire to trytomimicthenaturalovarianstimulationprocessmore closely to achieve this goal. one prerequisite is then a thorough knowledge about the glycosylation and glycan compositions of serum fsh and lh during the normal menstrual cycle. like other glycoproteins fsh and lh exhibit a very large heterogeneity. the two hormones are synthesized in the pituitary, secreted and circulate in blood as individual spectra of large numbers of isoforms (12,14). the fsh and lh isoforms isolated from pools of human pituitaries differ in their n-glycan compositions (15-18). in addition, fsh has been reported to exist in human pituitary and urinary preparations as two major glycoforms designated tetraglycosylated and di-glycosylated hfsh (19,20). the former glycoform was decorated with two glycans on both the a-subunit and the fsh b-subunit and the latter glycoform with glycans only on the a-subunit. the isoforms can be separated by electrophoresis due to variation in their contents of two terminal anionic monosaccharide (ams) residues: sialic acid (sa)andsulfonatedn-acetylgalactosamine(su).these terminal ams residues are of physiological significance as they are decisive for the half-lives of fsh and lh in human circulation (21,22). both lh and fsh are secreted in a pulsatile manner, and the compositions of the gonadotrophin isoforms in circulation continuously change after each pulse. a consequence of this is that the pituitary effect on the ovary is exerted by continuous qualitativeaswellasquantitativechangesoffshandlh in the circulation. the aim of the present study was to determine the degree of glycosylation, sialylation, and sulfonationofserumfshandlhmoleculesthroughout the normal menstrual cycle. subjects and methods subjects as part of the training in clinical chemistry for medical students at uppsala university hospital, blood samples were taken from students on an ambulatory basis in the morning during the period 2000 to 2011. all female students who agreed to participate in the research project gave a written consent on analytes accepted tobe included and signeda health declaration with information about diseases, medications, alcohol intake, and recent physical activity. information was given about menstrual bleedings (first day of last menstruation, menstrual cycle length, and regularity over the last year) and hormonal contraceptives. the study was approved by the local ethics committee. one serum sample was selected from each of 79 female students, median age 25, range 21–40 y, on the criteria that they were apparently healthy, had regular menstrual cycles with a length of 28 ± 2 days, did not use any hormonal contraception, and did not have the common genetic variant of lh. they all had serum concentrations of fsh and lh within the reference limits for the day of menstrual cycle. the cycle day was adjusted to a 28-day menstrual cycle. information about the first day of next cycle was confirmed for samples taken on cycle days 26–28. no serum samples were obtained for cycle days 2 and 4. fsh and lh serum concentration to exclude individuals with the common variant form of lh, the presence of such forms was first analysed as described (23).the concentrations of lh and fsh in serum samples were measured using time-resolved sandwich fluoroimmunoassays (delfia, perkinelmer-wallac oy, turku, finland), as previously described (24). gonadotrophin values were expressed in iu/l using the international standards for pituitary lh (80/552) and fsh (94/632) as reference standards. the detection limits were 0.02 iu/l, and the interassay coefficient of variation (cv) was less than 3% for both hormones. ams residues per molecule the number of ams residues per molecule was determined by analyses of all serum samples with an electrophoresis technique using a 0.10% agarose suspension in veronal buffer at ph 8.7 (12,25). after electrophoreses fsh and lh activities were measured in 200 ml of the fractions eluted. the area of gonadotrophin activity was resolved into peaks at the positions for different numbers of ams residues per molecule; for fsh varying from 4 to 10 residues and for lh from 1 to 6 residues per molecule. lowand high-glycosylated forms of fsh and lh the fsh molecule can be decorated with a maximum of four n-glycans and the lh molecule with a 154 l. wide & k. eriksson maximum of three. bousfield et al. reported that human fsh exists in the pituitary and urine also as a di-glycosylated form with no glycans on the b-subunit (19,20). estimations of di-glycosylated fsh (fshdi), tetra-glycosylated fsh (fshtetra), di-glycosylated lh (lhdi), and tri-glycosylated (lhtri) were made on the basis of the number of ams residues per molecule. isoforms of serum fsh with 7–10 ams residues per molecule were pooled as variants of fshtetra and those with 4–6 ams residues per molecule as variants of fshdi, with proportional adjustments for the overlapping between the diand tetra-glycoforms. isoforms of serum lh with 4–6 ams per molecule were pooled as variants of lhtri and those with 1–2 ams per molecule as variants of lhdi. fractions of isoforms of lh with 3 ams per molecule were added to the highand lowglycosylatedpoolsinproportiontotheconcentrationsof isoforms in these pools. lowand high-glycosylated isoforms of both hormones differed in sizes and could be separated by gel filtration on sephadex g-100 (unpublished observations). there were no isoforms with zero ams per lh molecule and only an average of 0.88% with one ams per lh molecule, which excluded the likelihood for the presence of mono-glycosylated glycoforms of lh. sa and su residues per molecule determinations included analyses of all serum samples both before (see above) and after neuraminidase treatment with an electrophoresis technique (12,25). the area of gonadotrophin activity after the neuraminidase treatment was resolved into peaks at the positions for different numbers of su residues per molecule. the average numbers of sa and su residues per gonadotrophin molecule in each serum sample were estimated as previously described (12). the method is based upon previous observations (15-18) that negatively charged terminal sa and su residues on the n-glycans determine the variation of the electric charge of human fsh and lh. statistical analyses in order to illustrate the time-related dynamic changes during the cycle, three-day mean values ± sem with one-day step-wise movements throughout the cycle were calculated for different data. the calculation started on day 26 in the previous cycle and the two data-free days 2 and 4 were excluded. data of days 26, 27, and 28 were pooled as the first three-day mean value and of days 27, 28, and 1 as the second mean value, etc. the distributions of data were analysed with d’agostino and pearson omnibus (when more than seven observations) and kolmogorov–smirnov normality tests. when three-day mean values are illustrated in the figures, the ± sem are omitted for means of groups that did not pass a normality test (9 out of 416 three-day mean values; 2.2%). data for serum concentrations of lowand high-glycosylated gonadotrophin forms, numbers of sa and su residues per molecule, and ratios between sa and su residues were compared for different periods of the cycle using twotailed student’s t test. these data passed normality tests. gonadotrophin serum concentrations and ratios between number of sa and su residues per molecule were log transformed before statistical analyses and plotted in the figures as geometric mean ± sem values. a p value less than 0.05 was considered significantly different. results fsh and lh serum concentrations there was a rise of serum fsh during the early follicular phase, from day 27 in the previous cycle to day 5 (figure 1, left panel). the midcycle lh and fsh surges coincided and occurred around day 14. before that the serum lh concentration increased gradually from day 8 to day 12. during the same period the fsh concentration remained at a slightly decreased plateau level. the midcycle surges of fsh and lh were followed by rapid decreases of the serum concentrations of both gonadotrophins. numbers of ams residues per fsh and lh molecule the mean number of ams residues, which is the sum of sa and su residues, during the menstrual cycle was on fsh 6.85 ± 0.022 and on lh 3.13 ± 0.018, and the mean difference 3.71 ± 0.015. there was a highly statistically significant correlation (r = 0.74; p < 10-14; n = 79) between ams residues on fsh and lh. assessments of three-day mean numbers of ams residues per fsh and lh molecule in serum during the menstrual cycle showed that the shapes of the two curves were similar from day 8 to day 25 with a nadir on day 14 and a zenith on day 21 (figure 1, right panel). serum concentrations of fshdi, fshtetra, lhdi, and lhtri the time pattern of the serum fshdi concentration was characterized by a steep rise from day 27 to day 3–6 and then a dip to a decreased concentration lasting from day 7 to day 11 followed by a pronounced midcycle peak (figure 2, left panel; table i). after the menstrual cycle fsh and lh glycosylation 155 midcycle peak there was a rapid decrease to the lowest level on day 17–19. the fshtetra concentration increased to a high plateau level lasting from day 3 to day 15 followed by a slow decrease without any sign of a midcycle peak. both the lhdi and lhtri concentrations showed a pronounced midcycle peak (figure 2, right panel; table i). the concentrations of lhdi showed a larger variation 20 25 lh 20 13 2 16 10 2.5 3.2 4 8 6.3 5 fsh g o n a d o tr o p h in s e ru m c o n c e n tr a ti o n , iu /l 28 4 8 12 16 20 24 28 3.2 6.3 13 2.5 4 5 8 10 16 day of menstrual cycle day of menstrual cycle 0 0 a n io n ic m o n o s a c c h a ri d e r e s id u e s p e r m o le c u le 3.4 2.9 3.0 3.1 3.2 3.3 lh 7.2 6.6 6.7 6.9 6.8 7.0 7.1 fsh 28 4 8 12 16 20 24 28 a b figure 1. a: concentrations of fsh and lh in serum of 79 women with a normal menstrual cycle, plotted as geometric mean ± sem values. b: the number of anionic monosaccharide (ams) residues per molecule of fsh and lh in the same serum samples, plotted as threeday mean ± sem values. data in this figure and in figures 2,3,4,5 have been plotted as three-day moving mean values starting during the last days of the previous cycle and with the first day of the cycle indicated by a vertical hatched bar. 0.5 iu/l fshdi day of menstrual cycle 0.6 0.8 5.0 1.3 1.6 2.5 4.0 3.1 1.0 2.0 fshtetra 28 4 8 12 16 20 24 28 a day of menstrual cycle iu/l lhdi 20 5.0 0.5 10 1.0 2.0 lhtri 28 4 8 12 16 20 24 28 b figure 2. concentrations of fshdi and fshtetra, a, and lhdi and lhtri, b. geometric scales. see also legend to figure 1. 156 l. wide & k. eriksson than that of lhtri during the cycle. the lhdi concentrations were lower than those of lhtri during the follicular and luteal phases of the cycle and higher at midcycle. fshdi and lhdi in serum expressed as a percentage of total gonadotrophin concentrations the three-day mean values of serum lhdi varied from 23.2% to 58.4% and those of fshdi from 19.7% to 48.3% (figure 3). the mean value for the entire cycle period was 36.6% for lhdi and 32.0% for fshdi. there was a similar time pattern of fluctuations in fshdi and lhdi percentages over the menstrual cycle. the percentage of fshdi was significantly (r = 0.69; p < 10-11) correlated with that of lhdi. moreover, percentages of fshdi and lhdi were significantly correlated to the log concentration of lh (for fsh: r = 0.62; p < 10-8; for lh: r = 0.65; p < 10-10). finally, the percentage of fshdi was significantly (r = 0.38; p < 0.001) correlated to the log concentration of fsh. number of ams per glycan on fsh and lh the number of ams per glycan on the fsh molecules was significantly (p < 0.0001) higher at the midcycle phase, with a mean value ± sem of 2.16 ± 0.030 (n = 14), than at the follicular phase, 2.02 ± 0.008 (n = 32), or at the luteal phase, 2.01 ± 0.013 (n = 33). the mean number of ams per glycan on the lh molecule during the menstrual cycle was 1.20 ± 0.002 (n = 79) without any significant variation between different phases of the cycle. table i. serum concentration of fshdi, fshtetra, lhdi, and lhtri at different periods of the normal menstrual cycle. geometric mean values and their 95% limits are shown. cycle days number of women serum concentration, iu/l geometric comparison with previous group p valueamean; range mean 95% limits fshdi 27.0; 26–28 8 0.89 0.59–1.33 4.0; 3–5 8 1.54 1.18–2.01 < 0.05 6.0; 6 5 1.90 1.17–3.10 ns 9.0; 7–11 11 1.30 1.09–1.55 < 0.05 13.9; 13–15 14 2.90 2.06–4.07 < 0.001 18.8; 17–19 8 0.91 0.68–1.24 < 0.0001 22.1; 20–24 12 0.57 0.41–0.78 < 0.05 26.4; 25–28 11 0.96 0.69–1.33 < 0.05 fshtetra 27.0; 26–28 8 1.90 1.29–2.80 4.0; 3–5 8 3.56 2.72–4.66 < 0.01 13.9; 13–15 14 3.52 2.76–4.48 ns 22.7; 20–25 15 1.97 1.63–2.38 < 0.001 lhdi 28.6; 26–1 11 1.04 0.70–1.53 11.3; 10–12 9 2.49 1.58–3.90 < 0.01 13.9; 13–15 14 12.5 8.75–17.9 < 0.01 20.6; 19–22 9 0.79 0.28–2.23 < 0.001 lhtri 1.4; 26–5 19 2.10 1.66–2.66 13.9; 13–15 14 9.24 6.27–13.6 < 0.0001 18.9; 17–21 13 2.55 1.99–3.27 < 0.0001 atwo-tailed student’s t test. menstrual cycle fsh and lh glycosylation 157 numbers of sa and su residues per fsh and lh molecule the time-related change for the number of sa residues on fsh during the cycle was ‘m’-shaped with the lowest levels at midcycle and at day 28 (figure 4, left panel; table ii). the three-day mean number of sa residues varied from 6.28 to 6.73 per fsh molecule. the highest numbers were found at midfollicular phase, day 7–11, and at mid-luteal phase with a plateau from day 17 to day 21. as regards numbers of su residues per fsh molecule during the cycle, there was a fast continuous decrease for 9days,fromcycleday27inthepreviouscycletoanadiron day8followedbyaslowincreasefor17daystoamaximum on day 27 (figure 4, left panel; table ii). the number of su residues was low, and the three-day mean values varied from 0.23 to 0.56 per fsh molecule. there was ahighlysignificantcorrelationbetweenthepercentagesof fshdi and the average number of sa residues per molecule (r = –0.87; p < 10-24) and no significant correlation with the number of su residues (r = 0.03; p = 0.78). the time pattern of numbers of sa residues on lh was characterized by a low plateau level from late luteal phase into early follicular phase and then an increase from the first days of the cycle to a peak on day 12 (figure 4, right panel; table ii). after this peak there was a decrease to day 15 and then a plateau level to day 21, followed by a decrease to the low plateau level during late luteal and early follicular phase. the patternforthe su contentonlh(figure 4,right panel; table ii) was ‘v’-shaped with a high plateau level duringthe late lutealphase intothe first day ofnextcycle and a nadir on day 13–15, that was followed by an increase to a high plateau level from day 19 to day 28. therewasahighlysignificantinversecorrelationbetween the percentages of lhdi and the average number of su 0 10 20 30 40 50 60 70 fshdi day of menstrual cycle f re q u e n c y o f f s h d i a n d l h d i in p e r c e n t lhdi 28 4 8 12 16 20 24 28 figure 3. concentrations of fshdi and lhdi, expressed as per cent of total concentrations of fsh and lh. see also legend to figure 1. 0 m e a n n u m b e r o f re s id u e s p e r m o le c u le 0.2 0.3 0.5 0.4 0.6 6.3 6.4 6.5 6.6 6.7 day of menstrual cycle sa fsh 0 su su 0 0 1.4 lh 0.9 1.0 1.2 1.1 1.3 2.2 m e a n n u m b e r o f re s id u e s p e r m o le c u le sa 1.7 1.8 1.9 2.0 2.1 28 4 8 12 16 20 24 28 day of menstrual cycle 28 4 8 12 16 20 24 28 a b figure 4. numbers of sialic acid (sa) and sulfonated n-acetylgalactosamine (su) residues per molecule of fsh, a, and lh, b. see also legend to figure 1. 158 l. wide & k. eriksson residues per molecule (r = –0.70; p < 10-12) but no statistically significant correlation with regard to the number of sa residues (r = 0.03; p = 0.78). ratio between numbers of sa and su residues per fsh and lh molecule there was an increase of the sa/su ratio for fsh from day 27 in the previous cycle to a peak on day 7– 12 (figure 5; table ii). after that the sa/su ratio decreased to day 14 and then varied during the first part of the luteal phase followed by a decrease from day 21 to day 27. the ratio sa/su values for lh increased from day 1 to a peak value on day 11–13 (figure 5; table ii). after this peak the ratios decreased continuously to a low plateau level from day 24 to day 28. discussion glycosylation of fsh and lh di-glycosylated glycoforms of both fsh and lh, in addition to the fshtetra and the lhtri forms, were detected in serum throughout the menstrual cycle. these four gonadotrophin forms exhibited different table ii. number of sialic acid (sa) and sulfonated n-acetylgalactosamine (su) residues per fsh or lh molecule and ratio of sa/su residues at different periods of the normal menstrual cycle. mean values and their 95% limits are shown. cycle days number of women number of residues per molecule comparison with previous group p valueamean; range mean 95% limits fsh sa 27.0; 26–28 8 6.30 6.14–6.46 9.0; 7–11 11 6.67 6.59–6.75 < 0.0001 13.9; 13–15 14 6.30 6.17–6.44 < 0.0001 18.9;17–21 13 6.71 6.62–6.79 < 0.0001 26.4; 25–28 11 6.29 6.17–6.41 < 0.0001 su 27.0; 26–28 8 0.56 0.49–0.64 8.5; 7–10 9 0.24 0.19–0.29 < 0.0001 14.2; 14–15 11 0.33 0.29–0.37 < 0.01 26.4; 25–28 11 0.54 0.47–0.61 < 0.0001 lh sa 3.2; 1–5 11 1.85 1.75–1.95 12.1; 10–13 10 2.14 2.04–2.25 < 0.001 18.1; 15–21 17 2.00 1.95–2.05 < 0.01 25.6; 23–28 16 1.84 1.77–1.91 < 0.001 su 28.6; 26–1 11 1.34 1.24–1.45 13.9; 13–15 14 0.91 0.85–0.97 < 0.0001 23.8; 19–28 25 1.36 1.30–1.41 < 0.0001 geometric fsh mean 95% limits sa/su 27.0; 26–28 8 11.3 9.71–13.2 9.9; 7–12 16 28.7 24.2–34.0 < 0.0001 14.2; 14–15 11 19.1 16.5–22.2 0.001 25.9; 24–28 14 12.6 11.0–14.3 0.0001 lh sa/su 28.6; 26–1 11 1.39 1.23–1.57 12.1; 11–13 10 2.35 2.01–2.74 < 0.0001 25.9; 24–28 14 1.34 1.20–1.48 < 0.0001 atwo-tailed student’s t test. menstrual cycle fsh and lh glycosylation 159 concentration patterns during the menstrual cycle. fshdi exhibited two peaks—one peak on day 5–7 and one, more pronounced, at midcycle. fshtetra had a high plateau concentration from day 5–15, without a midcycle peak. lhdi had lower concentrations than lhtri, except at midcycle when the opposite occurred. in all graphs data have been presented as three-day moving mean values during the menstrual cycle, starting during the last days of the previous cycle. with this continuous presentation of the results, dynamic time-related changes of data were revealed that had been otherwise hidden when more conventional fixed periods of menstrual cycle phases had been used. n-glycosylation is a post-translational process in which an oligosaccharide is transferred and attached to asparagine on a nascently translated polypeptide (26). the glycosylation of the peptide structures of the subunits of fsh and lh and the following synthesis of the n-glycans to terminal sa and su residues in the gonadotrophin-producing cells of the anterior pituitary have been schematically shown in figure 6. n-glycosylation of the common a-subunit and b-subunits of fsh and lh takes place in the rough endoplasmic reticulum (er) simultaneously in the same compartment. a dolichol (dol)-linked oligosaccharide is attached to an oligosaccharyltransferase (ost) complex, and the oligosaccharide then becomes transferred to a nascently translated polypeptide. the glycosylated a-subunits are associated with glycosylated b-subunits of fsh and lh to form fshtetra and lhtri. furthermore, an fshdi glycoform has been demonstrated in pituitary and urinary preparations, and it consists of an a-subunit with two glycans associated with a non-glycosylated fsh b-subunit (19,20). in our study the three-day mean frequency of fshdi varied in serum from 20% to 48% during the menstrual cycle. these figures are lower than the relative abundance of 60–65% of non-glycosylated b-subunits on fsh reported by bousfield et al. for immunopurified extracts of some pituitaries from 21–43-year-old women (20). the difference between the pituitary and serum values may be explained by a shorter half-life in the circulation of fshdi, due to a lower number of sialic acid residues, compared with fshtetra. the fshtetra and fshdi glycoforms were identified by the number of ams per molecule. similarly, both lhtri and lhdi glycoforms were detected in serum. we suggest that, in analogy with fsh, the lhdi consists of an a-subunit with two n-glycans and a non-glycosylated lh b-subunit. it has been shown that the n-glycan at the position asn30 of the lh b-subunit has a very high abundance of su residues (17). a lack of this n-glycan on the lh b-subunit is compatible with the finding of a negative correlation between the percentage of di-glycosylated lh forms and the average number of su residues per lh molecule. the asn78 glycan on the a-subunit has a low number of su residues (17), and the asn52 glycan on the a-subunit has been shown to be important for signal transduction and for camp and steroid formation (29). it seems less likely that one of these glycans on the a-subunit is missing on the lhdi glycoform. the mean number of ams per glycan on fsh during the follicular and luteal phases of the cycle was 2.02. this number was significantly (p < 0.0001) raised to 2.16 at midcycle, when the concentration of fshdi, with glycosylation on the a-subunit only, was increased compared with that of fshtetra. a possible explanation is that the glycans on the a-subunits of the fsh molecule are more branched than those on the fsh b-subunits. the mean number of ams per glycan on the lh molecule during the cycle was 1.20 without any significant variation during the cycle. this suggests that the branching of the glycans on the lh a-subunits is similar to that of the glycan on the lh b-subunits. how is the formation of the fshdi and lhdi glycoforms regulated? it was recently suggested by bousfield and dias that a possible mechanism for a 10 1.3 lh day of menstrual cycle ratio sa/su residues 1.6 2.0 2.5 20 13 16 32 25 fsh 4 8 12 16 20 24 2828 figure 5. ratios between sialic acid (sa) and sulfonated nacetylgalactosamine (su) residues per fsh and lh molecule. geometric scales. see also legend to figure 1. 160 l. wide & k. eriksson selective formation of non-glycosylated fsh b-subunits could involve inhibition of ost isoforms specific for fsh b-subunits (30). the results of the present study suggest an alternative explanation. the number of ams residues on fsh and lh was highly significantly correlated (r = 0.74; p < 10-14). there was a striking parallelism between the frequencies of fshdi and lhdi glycoforms expressed in per cent of total (figure 3), and the correlation between the two diglycosylated forms was highly significant (p < 10-11). the percentages of the fshdi and lhdi glycoforms were significantly (p < 10-8 and p < 10-10, respectively) cis-golgi medial-golgi: branching and addition of glcnac trans-golgi: golgi rough endoplasmic reticulum -asnasn asn asn ud p ud p udp udp pro -xx x-a rg-s pec ific galnac-4 sulfotrans ferase paps pap cmp cmp so3 -so3asn the subterminal glcnac and galnac residues are substrates for competing enzymes leading to terminal sialic acid or sulfonated galnac residues. fucose key mannose glucose glcnac galnac galactose sialic acid pro-leu-arg n-glycan cmp cmp asn -4 β1-4gal-transferase β1-4 ga lna c-tr ans fera se p p dolichol 52 78 (92) (111) 52 78 7 24 (92) (111) 30 (121) 52 78 (92) tetra-glycosylated fsh (fshtetra) tri-glycosylated lh (lhtri) ost, oligosaccharyltransferase + di-glycosylated fsh (fshdi) ost (121) 52 78 (92) di-glycosylated lh (lhdi) asn-x-thr/ser asn-x-thr/serasn-x-thr/ser ( ? ) α β α β α β α β β1-2 β1-4 β1-4 α 2-3 or α2-6sialyltransferase α2-6sialyltransferase α 2-3/6 α 2-6 figure 6. schematic drawings of the glycosylation of fsh and lh in the rough endoplasmic reticulum and the synthesis of the n-glycans in the golgi of human anterior pituitary gonadotrophin-producing cells. symbol nomenclature according to essentials of glycobiology, 2nd ed. (27). pathways and design from refs. (16,17,26,28).. (cmp = cytidine monophosphate; paps = 3�phosphoadenyl-5�phosphosulfate; udp = uridine diphosphate). menstrual cycle fsh and lh glycosylation 161 correlated with the log concentration of lh in serum. these results suggest that the glycosylation of the two hormones may be restricted by similar mechanisms. the availability of the dol-p and the level of the ost activity have been shown to be able to restrict and regulate the glycosylation process (26). when, during the menstrual cycle, the production rate of the gonadotrophins increases, the available dol-p or ost activity may not increase in parallel. a higher production rate of non-glycosylated b-subunits of both hormones will then be expected. the common a-subunit is produced in large excess to the b-subunits, and enough of di-glycosylated a-subunits may be synthesized. it seems most likely that oestrogens play a major role for the inhibition of the glycosylation process of both fsh and lh throughout the menstrual cycle. glycan composition on fsh and lh glycans on fsh are more branched than those on lh. both tri-antennary and tetra-antennary glycans are produced on fsh in the medial-golgi (figure 6). a sub-terminal n-acetylglucosamine (glcnac) residue is added in the medial-golgi to the asparagine-linked oligosaccharide chains and serves in the trans-golgi as a substrate for two competing enzymes: a b1-4galactosyltransferase starting a pathway to terminal sialic acid, and a b1-4galnac-transferase starting a pathway to terminal su and sa residues (17). the number of terminal sa and su residues per molecule depends on the pathway-substrate concentration and the enzymatic activities along the two pathways. the b1-4galnactransferase recognizes a pro-leu-arg tripeptide on the lh b-subunit which enhances its activity and leads to more sulfonated residues on lh compared to fsh (28,31,32). the tripeptide motif on the lh b-subunit is not present on the fsh b-subunit, and the a-subunit recognition motif is thought to be masked by the b-subunit of fsh (33). therefore, for fsh, the activity of the peptide-specific b1-4galnac-transferase is low, and the sialylation pathway dominates. each one of the four pituitary gonadotrophin glycoforms, designated fshdi, fshtetra, lhdi, and lhtri, is heterogeneous and present in blood as spectra of isoforms varying in their glycan compositions. the glycan compositions vary with respect to branching and terminal ams residues which affect the physical-chemical properties of the isoforms. the biological effect of such spectra of isoforms, for example on their half-life in the circulation, will be a resultant of that of the multiple isoforms. the isoforms that we measure in serum in this study are representative for those reaching the ovary at the moment when the sample was harvested. the individual isoforms secreted from the pituitary have different half-lives in circulation. when the pituitary isoforms are secreted into the bloodstream, they are mixed with those remaining from previous pulsatile secretions having the longest half-lives. the composition of the isoforms secreted from the pituitary is thus slightly different from that of the isoforms circulating in blood. for fsh, the terminal sa residues on the glycans prolong survival implying that the composition of isoforms in blood becomes much more anionic than that of those secreted from the pituitary. the disappearance rate of the lh molecules is regulated both by the terminal sa and su residues on the glycans. lh molecules with two or more terminal su residues are quickly removed from the human circulation suggesting the presence of a mannose/sulfonated n-acetylgalactosamine-specific receptor in the human liver similar to that in rodents (34,35). a consequence of this is that the composition of lh isoforms in blood is usually slightly less anionic compared with that of lh isoforms analysed in age-matched pituitary extracts of men or women (unpublished observation). the number of ams residues per molecule during the menstrual cycle was at minimum at the midcycle for both fsh and lh. this is in agreement with a reported shift to a more basic ph range of fsh and lh isoforms at the midcycle phase compared with follicular and luteal phases of the normal menstrual cycle (36,37). the contents of sa and su residues per molecule of fsh and lh in serum showed four different patterns during the cycle, all with highly significant (p < 0.0001) differences between levels at different phases of the cycle. in a previous study we observed that at the midcycle surge there was a considerable decrease in number of su and increase of sa residues per lh molecule (12). in contrast to lh, the number of sa residues per serum fsh decreased at the midcycle surge. the present investigation on sera from 79 women with a normal menstrual cycle confirmed the previous results but permitted thorough studies of sulfonation and sialylation also during the follicular and luteal phases. this revealed that there were substantial timerelated changes of sialic acid and so3-galnac residues per lh and fsh molecule in serum within both the follicular and luteal phases. the most impressive changes during these phases of the cycle were those of the sa residues per fsh molecule and of the su residues per lh molecule. the values presented for fsh are mean values of fshdi and fshtetra and for lh of lhdi and lhtri, all four glycoforms existing as a large number of isoforms with different glycan compositions. 162 l. wide & k. eriksson relationship between degree of glycosylation and glycan composition of fsh and lh there was a highly significant (p < 10-24) negative correlation between the percentages of fshdi and the average number of sa residues per molecule in our study samples. this is in agreement with a low sa content on fshdi with a non-glycosylated b-subunit (18). as the sa residues are known to prolong the half-life of fsh in the human circulation, the fshdi glycoforms are expected to have a shorter half-life than the fshtetra forms. the average number of sa residues on fsh is low at midcycle and at the last days of the cycle, when the ratios of the concentrations of fshdi to those of fshtetra are high. an expected biological consequence is a shorter half-life of fsh during these two periods of the cycle and a longer half-life around days 8 and 20 of the cycle when the average number of sa per molecule is high. this is in agreement with shorter plasma half-lives reported for fsh molecules released in response to gonadotrophin-releasing hormone at midcycle compared with those released during follicular and luteal phases (38). a shorter half-life in the circulation was also reported for fsh during gnrh receptor blockade in women at midcycle compared with early follicular phase (21). there was a highly significant (p < 10-12) negative correlation between the percentage of di-glycosylated lh forms and the average number of su residues per lh molecule. a low content of su residues on the lhdi is expected to lead to a prolonged half-life for this lh form, as su residues play a major role for the rapid elimination of lh from circulation. at midcycle, the concentration of lhdi was high, the average su content on lh was low, and the sa content on lh was high compared with the rest of the cycle. an expected biological consequence of this is a prolonged half-life of lh during the midcycle phase. it has been reported that the negative feedback effect of oestrogen on gonadotrophin secretion is a direct effect at the pituitary level (39). administration of 17b-oestradiol and progestogen preparations to postmenopausal women has been shown to change the contents of sa and su residues per molecule on both fsh and lh in serum (40). this suggested steroid effects on the enzymes in the trans-golgi of the pituitary gonadotrophin-producing cells. the present demonstration of variations of the serum concentrations of the fshdi and lhdi during the menstrual cycle opens for an additional possible explanation. the number of glycans and their compositions on the di-glycosylated forms differ from those of the trior tetra-glycosylated forms. the changes in serum concentration of the di-glycosylated forms in relation to those of fshtetra and lhtri, respectively, will contribute to the variations observed for mean molecular contents of sa and su during the cycle. simulation experiments were made with different estimations of sa and su contents on the lowand high-glycosylated forms of fsh and lh in combination with the values obtained in this study for the concentrations of lowand high-glycosylated forms. these simulation experiments resulted in an ‘m’shaped pattern for sa residues per fsh molecule and a ‘v’-shaped pattern for su residues per lh molecule during the cycle, similar to the patterns illustrated in figure 4. these results indicate that the variation in the degree of glycosylation in er may have a substantial regulatory effect on the average number of sa and su residues per serum fsh and lh molecules during the menstrual cycle. conclusion as it is known that the glycosylation and glycan composition of the pituitary gonadotrophins regulate their biological properties, we conclude that the changes observed in this study most likely are important for the fine regulation of the natural ovarian stimulation process. this information may be useful when gonadotrophins are used for the inductions of ovulation in anovulatory women. the use of mixtures of fshdi and fshtetra glycoforms with different glycan compositions, perhaps combined also with lhdi and lhtri glycoforms, may lead to more successful mono-ovulatory treatment regimens. declaration of interest: this work was supported by grants from uppsala university. the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. gemzell ca, diczfalusy e, tillinger g. clinical effects of human pituitary 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faseb j. 1994;8:1019–25. 34. fiete d, srivastava v, hindsgaul o, baenziger ju. a hepatic reticuloendothelial cell receptor specific for so44galnacb1,4glcnacb1,2mana that mediates rapid clearance of lutropin. cell. 1991;67:1103–10. 35. roseman ds, baenziger ju. molecular basis of lutropin recognition by the mannose/galnac-4-so4 receptor. proc natl acad sci usa. 2000;97:9949–54. 36. padmanabhan v, lang ll, sonstein j, kelch rp, beitins iz. modulation of serum follicle-stimulating hormone bioactivity and isoform distribution by estrogenic steroids in normal women and in gonadal dysgenesis. j clin endocrinol metab. 1988;67:465–73. 37. wide l, bakos o. more basic forms of both human folliclestimulating hormone, and luteinizing hormone in serum at midcycle compared with the follicular and luteal phase. j clin endocrinol metab. 1993;70:885–9. 38. zambrano e, olivares a, mendez jp, guerrero l, díazcueto l, veldhuis jd, et al. dynamics of basal and gonadotropin-releasing hormone-releasable serum folliclestimulating hormone charge isoform distribution throughout the human menstrual cycle. j clin endocrinol metab. 1995; 80:1647–56. 39. shaw nd, histed sh, srouji ss, yang j, lee h, hall je. estrogen negative feedback on gonadotropin secretion: evidence for a direct pituitary effect in women. j clin endocrinol metab. 2010;95:1955–61. 40. wide l, naessén t, eriksson k. effects of 17b-oestradiol and norethisterone acetate on sulfonation and sialylation of gonadotrophins in post-menopausal women. ups j med sci. 2010; 115:97–106. 164 l. wide & k. eriksson vol_116_002_sups_a_563878 100..106 upsala journal of medical sciences. 2011; 116: 100–106 original article assessment of receptor occupancy-over-time of two dopamine transporter inhibitors by [11c]cit and target controlled infusion olof eriksson1, bengt långström2 & ray josephsson3 1department of radiology, oncology and radiation sciences, division of radiology, uppsala university, uppsala, sweden, 2department of biochemistry and organic chemistry, uppsala university, uppsala, sweden, and 3clinical imaging unit, novartis pharma ag, basel, switzerland abstract introduction. occupancy-over-time was determined for two dopamine transporter (dat) inhibitors through modeling of their ability to displace the pet ligand [11c]cit. the tracer was held at a pseudo steady state in a reference tissue by target controlled infusion. methods. rhesus monkeys (n = 5) were given [11c]cit and studied with a pet scanner. tracer uptake in the reference tissue cerebellum was held at a pseudo steady state by use of target controlled infusion. the pharmacokinetics/pharmacodynamics (pk/pd) of [11c]cit was assessed through the simplified reference tissue model (srtm). bupropion (n = 2) and gbr-12909 (n = 2) receptor occupancies were estimated through modeling of their effects on [11c]cit displacement. results. there was a high uptake of [11c]cit in striatum, which contains a high dat density. the reference tissue cerebellum had a comparatively low uptake. the modeling of [11c]cit pk/pd properties in striatum showed high binding potential (bp = 5.34 ± 0.78). both dat inhibitors caused immediate displacement of [11c]cit after administration. the occupancyover-time was modeled as a mono-exponential function, describing initial maximal occupancy (occ0) and rate of ligand– receptor dissociation (koff). gbr-12909 showed irreversible binding (koff = 0) after an initial occupancy of 76.1%. bupropion had a higher initial occupancy (84.5%) followed by a release half-life of 33 minutes (koff = 0.021). conclusions. the proposed model can be used for assessment of in-vivo occupancy-over-time of dat ligands by use of target controlled infusion of [11c]cit. the concept of assessing drug–receptor interactions by studying perturbations of a pet tracer from a pseudo steady state can be transferred to other cns systems. key words: ccip, [11c]cit, dat inhibitor, srtm, tci introduction positron emission tomography (pet) is a non-invasive imaging modality where a tracer containing a positronemitting nuclide (such as 11c or 18f) can be studied in vivo over time. this study describes how a novel system for target controlled infusion (tci), uipump, can be used for tracer modeling and assessment of acute dynamics of unlabeled pharmaceuticals. the basic programming of uipump has been described earlier (1) as well as some of the possible applications (2,3). [11c]citisatracerforstudiesofaspectsofthecentral nervoussystem(cns),whichbindswithhighspecificity to the dopamine transporter (dat), with low nonspecific binding to other structures (4). the tracer is therefore suitable for studies of the interaction between dat and pharmaceutical compounds that affect the cns. dat is expressed in high densities in striatum, while some other structures in the brain are almost devoid (such as cerebellum). in this study the pharmacokinetic and pharmacodynamic (pk/pd) properties of [11c]cit in striatum (both putamen and correspondence: olof eriksson, preclinical pet platform, dag hammarskjölds väg 14c, 751 83, uppsala, sweden. fax: +46-18-666879. e-mail: olof.eriksson@radiol.uu.se (received 25 october 2010; accepted 12 february 2011) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2011.563878 caudate nucleus) have been assessed by compartmental reference tissue modeling. dat exerts its effect through re-uptake of the neurotransmitter dopamine from the synaptic cleft, thereby regulating the postsynaptic response (5). changes in dat densities have been associated to attention deficient hyperactivity disorder (adhd) (6) and several neurodegenerative disorders, such as schizophrenia (7). it is also a target molecule for dat agonists and antagonists. these classes include both therapeutical drugs and psychoactive drugs (such as cocaine and amphetamine) (8). dat antagonists impair the re-uptake of dopamine, leading to a modulation of the preand postsynaptic function both shortand long-term. these synaptic changes can lead to withdrawal symptoms or abuse problems after long exposure to some dat ligands. this study compares acute ligand–target interactions immediately after administration by modeling the occupancy-over-time based on the ability of the compound to displace [11c]cit from dat. to study the immediate displacement of [11c]cit the pharmaceuticals are administered during a petscan, where tracer levels are kept at a pseudo steady state in a reference tissue (cerebellum) by using tci. apart from designed infusion schemes using tci, pseudo steady state levels of plasma or tissue can also be achieved by a bolus injection followed by constant infusion of tracer, but the time until steady state is usually longer (9–11). occupancy of a ligand to a target is usually determined at equilibrium. the immediate ligand–target pharmacodynamics can be difficult to quantify due to low resolution in the assay. the pk/pd and occupancy of a pharmaceutical can be evaluated based on displacement of a pet tracer. however, in this kind of study the patient is often pretreated (before the scan) with the compound to allow it to reach equilibrium in the target tissue. the initial interactions are therefore often not included. different dat-targeted pharmaceutical compounds have different pharmacodynamics and exert shortor long-term effects. the initial occupancy (during the first seconds, minutes, or hours) may or may not affect the subject. one could for example hypothesize that a very rapid onset of high occupancy, combined with high retention, may trigger up-regulation of dat density and thereby a risk of habituation to the compound. conversely, a low initial occupancy with rapid clearance from dat-rich regions yields a lower risk of causing habituation. therefore, the onset binding and clearance of two compounds to dat were investigated in this regard. bupropion was chosen as a model of a pharmaceutical with low risk of inducing habituation, while gbr-12909 was used as a model for a compound with high risk of inducing habituation (such as cocaine). bupropion is an inhibitor of dat, which has been used in the treatment of adhd and smoking cessation (12). gbr-12909 has high specific antagonistic binding to dat with long duration and has been investigated as a treatment drug for cocaine addiction (13). materials and methods animals five female rhesus monkeys (weight 5.4–9.7 kg; age 15–20 years) were used. the animals were sedated with ketamine (7 mg/kg) before transportation to the pet camera. one venous catheter was applied for tracer administration and one for administration of pharmacological compounds and blood sampling. propofol was administered intravenously until the animal was anesthetized enough for intubation. after intubation the animal was maintained on sevoflurane (3%–8%) mixed with medical air and artificial ventilation. blood samples were taken for estimates of electrolytes, glucose, and hematocrit.blood losses were compensated for with injections of albumin (50 mg/ml). ringer-acetate (0.5 ml/kg/h) was infused during the whole experiment. body temperature, heart rate, ecg, pco2, po2, sao2, and blood pressure were monitored throughout the pet study. experiments were approved by the local ethics committee for animal research and performed in accordance with local institutional and swedish national rules and regulations. radiochemistry [11c]cit was synthesized as described previously (4). the radiochemical purity of each batch was greater than 95%. infusion system the tci system uipump consists of a personal computer and an infusion pump. for this study a programmable univentor 864 infusion pump (agnthos, lidingo, sweden) was used. the uipump program compares the target concentration curve with a model of the compound kinetics after a fast bolus injection and calculates the infusion scheme necessary for compensating the wash-out of the compound to generate a steady state of the compound in a specific tissue. in this study the tci system was used to generate a steady state in the reference tissue cerebellum. receptor occupancy-over-time of two dopamine transporter inhibitors 101 pet protocol the pet imaging studies were performed using a shr 7700 camera (hamamatsu photonics, hamamatsu, japan). the scanner has a 14.3 � 11.4 cm field of view, and the scanner is capable of an axial and trans-axial resolution of 2 mm. all scans were performed in 2d. an 18.5 mbq 68ge rotating point source was used to acquire normalization and blank scans. the attenuation scans were performed in coincidence mode for 20 minutes. dead time correction, random correction, and scatter correction were performed for all scans. images were reconstructed by using 2d filter back projection. all scans were performed with infusion of the radiotracer calculated by uipump, aiming for steady state in cerebellum. the base-line scan was performed over 100 minutes (five animals). the second scan was performed over 110 minutes where a dat ligand, bupropion (n = 2) or gbr-12909 (n = 2), was administered after 60 minutes at a pharmacological dose. all images were analyzed in ida (scanditronix ab, uppsala, sweden). regions of interest (rois) were drawn over striatum and cerebellum in several transversal slices in each subject. each collection of rois was combined into a volume of interest (voi), and time–activity curves (tacs) were generated from each voi. standardized uptake values (suv) values were calculated by using only the amount of radioactivity administrated by the initial short bolus. this procedure resulted in apparently high suv values, as the initial bolus contributed on average only 23% of the total administered tracer volume/activity (the remaining 77% consisting of discrete subsequent infusions). modeling of [11c]cit uptake the [11c]cit uptake in striatum was investigated by implementing the tci system to achieve a pseudo steady state in the reference tissue cerebellum. pk/pd parameters r1, k2, binding potential (bp), and distribution volume ratio (dvr) were determined by fitting the striatal tacs to the simplified reference tissue model (srtm) described by lammertsma et al. (14). cerebellum was used as reference region due to its low expression of dat. the dvr in the reference region was set to 1, assuming bpcerebellum = 0. the model was implemented by the matlabprogram rz (in-house, uppsala imanet ab, uppsala, sweden). assessment of occupancy-over-time the dat inhibitors bupropionhydrochloride (sigmaaldrich, st louis, mo, usa) or gbr-12909 (rbi, natick, ma, usa) were dissolved in 5 ml 0.1 m nacl. they were then administered intravenously as a bolus (5 mg/kg) 60 minutes after administration of radiotracer. srtm is based on the full reference tissue model (15). in the full model the target tissue is described as two compartments, cfree and cbound. several rate constants (k2, k3, and k4) determine the tracer exchange between the compartments. the rate constant k2 determines wash-out from the tissue, while k3 and k4 represent tracer–target binding and release, respectively. in srtm, the ratio k3/k4 is denoted as bp and the two compartments combined into one. the distribution volume ratio (dvr = bp + 1) can be considered to specify the ratio between bound and free tracer. when a dat inhibitor displaces [11c]cit in the striatum, the tracer concentration in the region will decrease due to active transport through the cellular membrane. only free tracer is available for washout through k2. therefore, an increase in washout must be due to an increase in free tracer (or a decrease in bp which determines dvr). decrease in bp is here assumed to equal a decrease in available receptors for the tracer (the apparent k3 becomes lower) due to pharmaceutical binding to dat. the occupancy of all available dat (in %) can then be calculated from the apparent decrease in bp (bpapparent) (equation 1) due to [11c]cit displacement and wash-out. occupancy bp bp bp bp k apparent = − ∗ = ( ) , 100 3 where apparent aapparent / ( )k4 1eq the change in tracer concentration in the striatum can be described by a one-tissue compartment (equation 2). the term a is equal to the plasma concentration of tracer (cp) multiplied by the unknown rate constant k1. cp is assumed to be proportional to ccerebellum and therefore constant during the displacement phase; a is subsequently treated as a constant. dc dt k c t bp = − ∗ + ∗ α α 2 1 21 ( ) ( , ( ) apparent ) where = c k eq p occupancyofeachinhibitortodatwasdescribedas a mono-exponential function (equation 3). the constants occ0 and koff denote initial occupancy (in %) and ligand–target dissociation (in min-1), respectively. there is always an ‘absorption phase’ (usually very short) before the maximal initial occupancy is reached. the absorption can be described by an additional exponential term in the function, but it is not included here for simplicity. 102 o. eriksson et al. occupancy occ e= ∗ ∗o [( off eq k ] t) ( )3 equations 1–3 were combined into equation 4, which was fitted discretely to the experimental data for each dat inhibitor in matlab. best fit was determined iteratively through residual sum of squares. dc dt k c t bp occo e k toff = − + − ∗ ∗ ∗ ∗⎛ ⎝ ⎜ ⎞ ⎠ ⎟ α 2 1 100 100 4 ( ) ( ( ) ( )eq results bolus curve acquisition the uipump bolus input curve was modeled after taccurves over cerebellum from earlier experiments (n = 6) where [11c]cit was administered intravenously (data not shown). the curve in figure 1 is the mono-exponential curve (t1/2 = 13.2 min; plateau at 16.2% of initial value) with best fit to the individual bolus responses in previous experiments. base-line acquisition the bolus input curve was used to program an infusion scheme to reach a pseudo steady state of [11c] cit in the cerebellum. uptake in striatum (figure 2a) increased close to linearly during the entire scan under these conditions, indicating that the interaction between tracer and target was almost irreversible (figure 2b). modeling of [11c]cit uptake results from fitting srtm to striatum using cerebellum as a reference region are presented in table i. each subject was modeled individually. parameters are given as averages ± sem. the estimated k2 rate constant values (mean k2 = 0.10 ± 0.1) confirmed that [11c]cit has close to irreversible binding properties in striatum. bp was high in all subjects (mean bp = 5.34 ± 0.78); especially subject 3 (bp = 8.14) which is the source of the majority of variation in sem. assessment of occupancy-over-time both bupropion and gbr-12909 immediately displaced [11c]cit in striatum (figure 3). there was no decrease in the cerebellum, confirming that the uptake of [11c]cit in the reference tissue was non-displaceable (uptake consists entirely of free or non-specific binding). bupropion and gbr-12909 occupancy could be modeled according to equation 4 above (table ii). gbr-12909 showed lower initial occupancy than bupropion(76.1% compared to 84.5%). the retention of gbr-12909 was very high, however, with a koff = 0 as best fit. this indicates completely irreversible binding to dat, at least within the first hour investigated here. if this assessment is true or a limitation of the model will be discussed below. bupropion–dat dissociation was determined as koff = 0.021, or as having a retention half-life of 33.0 minutes. discussion this study aimed to show that the technique of holding a pet tracer at a pseudo steady state in a reference tissue by tci can be used to determine early changes in receptor occupancy of unlabeled pharmaceuticals at their site of action, as well as quantifying the rate of offset (koff). the pk/pd of the radiotracer [11c]cit was assessed by a simplified reference tissue model. the level of the tracer in the cerebellum was held constant through target controlled infusion by uipump (using a cerebellum input function modeled from several previous experiments). preferably, the target tissue should be held at steady state, but this was not possible in this specific case since there is virtually no clearance of [11c]cit from the striatum due to its very strong binding to dat. the high bp in striatum (5.34 ± 0.78) obtained from the model is consistent with this reasoning. 1.2 1 0.8 0.6 0.4 0.2 0 0 20 40 60 80 100 120 time (min) model [11c]cit response in cerebellum average (n = 6) n o rm a li s e d a c ti v it y figure 1. the response of [11c]cit in cerebellum was modeled from tacs from six previous experiments where the tracer was administered as an intravenous bolus. error bars indicate standard deviation. the model curve was used to calculate the infusion scheme required to achieve pseudo steady state of tracer in cerebellum. receptor occupancy-over-time of two dopamine transporter inhibitors 103 when studying interactions between an unlabeled compound and a target in vivo (for example in an animal model or in patients) with pet, the pk/pd of the compound is essentially determined indirectly through thedisplacementoftheadministeredpettracer.often, the unlabeled compound is administered some time before the tracer to reach equilibrium. in that case, the initial kinetics and dynamics have already occurred at the time of tracer administration and pet scanning. this study showed that all phases of the pk/pd of a compound (including the initial kinetics) can be investigated indirectly through perturbations of a pet tracer at pseudo steady state in a reference tissue. the perturbations of the radiotracer steady state due to an unlabeled compound were quantified as changes due to ligand–target occupancy-over-time. the occupancy model omitted the absorption phase entirely and focused solely on the clearance phase. to study the absorption phase, which can be very rapid, the model probably would need to have resolution and robustness enough to determine large changes during the first minute(s). previous pet studies in humans have determined bupropion occupancy of dat to 26% (steady state dose regimen, 150 mg dose 3 hours prior to scan (16)) and 14% (steady state dose regimen, 150 mg dose 8–15 hours prior to scan (17)). in this study bupropion was found to have a high initial occupancy (84.5%) and a retention half-life of 33 minutes (5 mg/kg dose in rhesus monkey). the clearance would reduce the initial occupancy to the previously reported values in 1–2 hours. gbr-12909 showed high initial occupancy of 76.1% combined with zero release during the first 60 minutes. the occupancy is consistent with a previous pet study in rhesus monkey, where doses of 3 and 10 mg/kg bupropion 90 minutes later produced dat occupancies of 53% and 72%, respectively (18). the very low release seen here indicates a long-term occupancy, which has been previously seen in both rats and rhesus monkeys (13). the optimal model fit for gbr-12909-induced displacement was not ideal. this may be due to the fact that the absorbance phase was omitted from the baseline scan of [11c]cit 80 70 60 50 40 30 20 10 0 0 20 40 60 time (min) s u v striatum cerebellum 80 100 a b figure 2. target-controlled infusion to reach pseudosteady state of [11c]citin cerebellum. as expected, there was high uptake localized to striatum, as shown in a trans-axial projection of a representative subject (a). the pseudosteady state of [11c]citin cerebellum resulted in elevated, close to irreversible striatal uptake (b). error bars indicate sem (n = 5). table i. kinetic parameters for [11c]cit pk/pd in striatum determined from the simplified reference tissue model. the majority of variation in bp and dvr is due to the elevated values in subject 3. parameter subject 1 subject 2 subject 3 subject 4 subject 5 average sem r1 0.58 1.09 1.47 1.17 0.84 1.03 0.15 k2 0.13 0.08 0.12 0.07 0.07 0.10 0.01 bp 3.85 4.18 8.14 4.69 5.87 5.34 0.78 dvr 4.85 5.18 9.14 5.69 6.87 6.34 0.78 dvr ref 1 1 1 1 1 1 104 o. eriksson et al. model, since the fit correlates best with a negative koff. a koff below zero is of course impossible in a biological system, but it indicates that occupancy rose slightly during the first hour after initial displacement. the model for habituation hypothesized in the introduction was based on the initial occupancy (occ0) and the concentrationand time-dependent release (koff). for these two compounds occ0 differed very little. bupropion, which has a steady state dose regimen at low occupancies, was found to have a higher occ0 than gbr-12909. both compounds exert noticeable initial binding to dat. in contrast koff differed greatly between the compounds where bupropion was released with a short half-life (only 25% of the binding remains after approximately 1 hour). gbr-12909 essentially binds irreversibly which could lead to a prolonged dat inhibition resulting in more prominent downstream regulation to the striatal dopamine system than that seen after bupropion administration. in conclusion, the proposed model can be used for assessment of in vivo occupancy-over-time of dat ligands by use of target controlled infusion of [11c] cit. the concept of assessing drug-receptor interactions by studying perturbations of a pet tracer from a pseudo steady state can be transferred to other cns systems. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. eriksson o, wallberg a, syvänen s, josephsson r, långström b, bergström m. a computerized infusion pump for control of tissue tracer concentration during positron emission tomography in vivo pharmacokinetic/pharmacodynamic measurements. bmc med phys. 2008;8:2. 2. eriksson o, josephsson r, långstrom b, bergström m. positron emission tomography and target-controlled infusion for precise modulation of brain drug concentration. nucl med biol. 2008;35:299–303. 3. syvanen s, blomquist g, sprycha m, hoglund au, roman m, eriksson o, et al. duration and degree of cyclosporin induced p-glycoprotein inhibition in the rat bloodbrain barrier can be studied with pet. neuroimage. 200; 32:1134–41. 4. halldin c, farde l, lundkvist c, ginovart n, nakashima y, karlsson p, et al. [11c]beta-cit-fe, a radioligand for quantitation of the dopamine transporter in the living brain using positron emission tomography. synapse. 1996;22:386–90. 5. gulley jm, zahniser nr. rapid regulation of dopamine transporter function by substrates, blockers and presynaptic receptor ligands. eur j pharmacol. 2003;479:139–52. 6. jucaite a, fernell e, halldin c, forssberg h, farde l. reduced midbrain dopamine transporter binding in male adolescents with attention-deficit/hyperactivity disorder: association between striatal dopamine markers and motor hyperactivity. biol psychiatry. 2005;57:229–38. 7. laakso a, bergman j, haaparanta m, vilkman h, solin o, syvalahti e, et al. decreased striatal dopamine transporter binding in vivo in chronic schizophrenia. schizophr res. 2001; 52:115–20. 8. uhl gr. dopamine transporter: basic science and human variation of a key molecule for dopaminergic function, locomotion, and parkinsonism. mov disord. 2003;18:71–80. 9. carson re, channing ma, blasberg rg, dunn bb, cohen rm, rice kc. comparison of bolus and infusion methods for receptor quantitation: application to [18f]cyclofoxy and positron emission tomography. j cereb blood flow metab. 1993;13:24–42. displacement of [11c]cit by bupropion 60 50 40 30 20 10 0 60 70 50 40 30 20 10 0 0 20 40 60 80 100 120 0 20 40 60 80 100 120 time (min) time (min) model striatum cerebellum s u v s u v displacement of [11c]cit by gbr-12909a b figure 3. intravenous administration at 60 minutes of either bupropion 5 mg/kg (a) or gbr-12909 5 mg/kg(b). infusion is aimed at pseudo steady state of [11c]cit in cerebellum. best fit occupancy models for each dat inhibitor are presented as black lines. table ii. occupancy model parameters describing the initial pharmacodynamics of bupropion and gbr-12909. best fit was obtained by minimizing the residual sum of squares (rss). parameter unit bupropion gbr-12909 occ0 % 84.5 76.1 koff min -1 0.021 0.0 model fit (rss) 0.33 1.68 receptor occupancy-over-time of two dopamine transporter inhibitors 105 10. carson re, breier a, de bartolomeis a, saunders rc, su tp, schmall b. quantification of amphetamine-induced changes in [11c]raclopride binding with continuous infusion. j cereb blood flow metab. 1997;17:437–47. 11. carson, re. pet physiological measurements using constant infusion. nucl med bio. 2000;27:657–60. 12. dwoskin lp, rauhut as, king-pospisil ka, bardo mt. review of the pharmacology and clinical profile of bupropion, an antidepressant and tobacco use cessation agent. cns drug rev. 2006;12:178–207. 13. rothman rb,baumann m, prisinzano te, newman ah. dopamine transport inhibitors based on gbr12909 and benztropine as potential medications to treat cocaine addiction. biochem pharmacol. 2008;75:2–16. 14. lammertsma aa, hume sp. simplified reference tissue model for pet receptor studies. neuroimage. 1996;4: 153–8. 15. lammertsma aa, bench cj, hume sp, osman s, gunn k, brooks dj, et al. comparison of methods for analysis of clinical [11c]raclopride studies. j cereb blood flow metab. 1996;16:42–52. 16. learned-coughlin sm, bergström m, savitcheva i, ascher j, schmith vd, långstrom b. in vivo activity of bupropion at the human dopamine transporter as measured by positron emission tomography. biol psychiatry. 2003;54:800–5. 17. meyer jh, goulding vs, wilson aa, hussey d, christensen bk, houle s. bupropion occupancy of the dopamine transporter is low during clinical treatment. psychopharmacology. 2002;163:102–5. 18. villemagne vl, rothman rb, yokoi f, rice kc, matecka d, dannals rf, et al. doses of gbr12909 that suppress cocaine self-administration in non-human primates substantially occupy dopamine transporters as measured by [11c] win35,428 pet scans. synapse. 1999;32:44–50. 106 o. eriksson et al. vol_117_004_sups_a_714416 383..389 full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 underweight and overweight men have greater exercise-induced dyspnoea than normal weight men mirza m. f. subhan, syed a. ali, syed s. i. bokhari, mohammed n. khan & hakimuddin r. ahmad to cite this article: mirza m. f. subhan, syed a. ali, syed s. i. bokhari, mohammed n. khan & hakimuddin r. ahmad (2012) underweight and overweight men have greater exercise-induced dyspnoea than normal weight men, upsala journal of medical sciences, 117:4, 383-389, doi: 10.3109/03009734.2012.714416 to link to this article: https://doi.org/10.3109/03009734.2012.714416 © informa healthcare published online: 30 aug 2012. submit your article to this journal article views: 491 view related articles citing articles: 2 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2012.714416 https://doi.org/10.3109/03009734.2012.714416 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2012.714416 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2012.714416 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2012.714416#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2012.714416#tabmodule upsala journal of medical sciences. 2012; 117: 383–389 original article underweight and overweight men have greater exercise-induced dyspnoea than normal weight men mirza m. f. subhan1, syed a. ali2, syed s. i. bokhari2, mohammed n. khan1 & hakimuddin r. ahmad3 1department of physiology, college of medicine and medical sciences, arabian gulf university, kingdom of bahrain, 2department of biological & biomedical sciences, faculty of health sciences, the aga khan university, karachi, pakistan, and 3faculty of health sciences, jinnah medical college, karachi, pakistan abstract introduction. persons with high or low body mass index (bmi), involved in clinical or mechanistic trials involving exercise testing, might estimate dyspnoea differently from persons with a normal bmi. aims. our objective was to investigate the relationship between bmi and dyspnoea during exercise in normal subjects with varying bmi. material and methods. a total of 37 subjects undertook progressive exercise testing. subjects were divided into three groups: underweight (uw), normal weight (nw), and overweight (ow). dyspnoea was estimated using the visual analogue scale (vas). spirometry, maximum voluntary ventilation (mvv), and respiratory muscle strength (rms) were measured. results and discussion. the intercept of the vas/ventilation relationship was significantly higher in nw subjects compared to uw (p = 0.029) and ow subjects (p = 0.040). relative to the ow group, fvc (p = 0.020), fev1 (p = 0.024), mvv (p = 0.019), and rms (p = 0.003) were significantly decreased in the uw group. the greater levels of dyspnoea in uw subjects could possibly be due to decreased rms. healthy persons should aim to achieve an optimum bmi range to have the lowest exercise-induced dyspnoea. key words: body mass index, dyspnoea, exercise test, respiratory function tests, respiratory muscles introduction dyspnoea is a common symptom in many respiratory disorders, and it is also seen in healthy subjects during exercise (1). increasing grades of dyspnoea in patients have been associated with greater impairment in quality of life (2). after adjusting for age, smoking history, and occupation, dyspnoea has also been shown to be predictive of mortality in elderly people (3). assessments of dyspnoea during exercise have been made in healthy subjects (4), in chronic obstructive pulmonary disease (copd) patients to study the benefits of inspiratory muscle training (5), and in copd patients investigating the effect of therapeutic agents (6). abnormalities in body mass index (bmi) have been shown to affect exercise-induced dyspnoea in obese cardiac patients (7). in this study, obese patients were more breathless compared to overweight (ow) or normal weight (nw) patients, during a progressive exercise test. with regard to underweight (uw) and ow subjects and their estimations of exerciseinduced dyspnoea, no study could be found in the literature. previous studies have also shown that abnormal bmi can be related to pulmonary complications. for example, there is a relationship between low bmi and an increased risk of developing copd (8). similarly, having a low or high bmi has been associated with a greater prevalence of asthma (9). correspondence: dr m. m. f. subhan, department of physiology, college of medicine and medical sciences, arabian gulf university, po box 22979, manama, kingdom of bahrain. fax: +973 17271090. e-mail: feisalmm@agu.edu.bh (received 14 december 2011; accepted 15 july 2012) issn 0300-9734 print/issn 2000-1967 online � 2012 informa healthcare doi: 10.3109/03009734.2012.714416 therefore, is it possible that uw or ow persons, if involved in dyspnoea testing, for clinical trials or for mechanistic studies of the effects of dyspnogenic agents, might estimate dyspnoea differently relative to nw persons? a recent breathlessness survey has suggested that future clinical studies for breathlessness should consider stratification by bmi (10). the purpose of the present study was to determine whether a relationship exists between bmi and the perception of dyspnoea during exercise in uw, nw, and ow subjects. material and methods this was a prospective study, in which healthy subjects underwent exercise and lung function testing. subjects undertook progressive symptomlimited sub-maximal bicycle ergometry during the course of which dyspnoea was estimated each minute using a visual analogue scale (vas). exercise testing was conducted twice, to familiarize the subjects with the procedure; the time period between tests was 1 week. thirty-seven healthy subjects (all male) were studied. all were volunteers and were recruited from the staff and students of the aga khan university (aku), karachi, pakistan, and all were pakistani. all were non-smokers, with no history of cardiopulmonary or other chronic disease. prior to participation, the subjects completed a preexercise testing screening questionnaire and a resting electrocardiogram (ecg). their lung function was assessed by spirometry. based on their bmi, subjects were divided into three groups: underweight (n = 13; < 18.5 kg/m2), normal weight (n = 12; 18.5– 24.9 kg/m2), and overweight (n = 12; > 25.0 kg/m2). mean bmi (± sd) was 22.6 ± 5.2 kg/m2, range 14.5– 35.5 kg/m2. mean age (± sd) was 21.7 ± 4.5 years, range 18–37 years. all subjects were sedentary individuals with minimal levels of habitual activity. experiments were performed at approximately the same time of the day, at least 2 hours after a light meal. informed consent was obtained from the subjects, and the study was approved by the aku human subject protection committee on 15 june 2001 and performed according to the declaration of helsinki. height and weight were taken on a combined stadiometer/weighing scale (seca, hamburg, germany), with subjects wearing light indoor clothing and no shoes. the exercise tests were conducted on an electrically braked bicycle ergometer (tunturi, piispanristi, finland). the subject breathed through a valve box, which allowed inspiration from room air. inspired ventilation (vi) and respiratory frequency were measured using a rotating vane anemometer and ventilation monitor (pk morgan ltd, chatham, kent, uk). the ecg (datascope, mahwah, new jersey, usa) was monitored throughout the test. heart rate and oxygen saturation were monitored throughout the experiment using a pulse oximeter (ohmeda, madison, wisconsin, usa), attached via a finger probe. the pulse oximeter and ventilation monitor were connected to a personal computer which processed and recorded data every 15 s. once attached to the equipment, the subject was given a few min to become acquainted to the apparatus. expired gases were not measured. the vas was used to quantify dyspnoea intensity, and it was drawn as a 100 mm linear line on a card, the extremes of which were labelled ‘not at all breathless’ (0 mm) and ‘extremely breathless’ (100 mm). the subject was given approximately 5 s to mark his response in pencil on the card which was held before him at the end of each minute of the exercise procedure. a separate card was used every min. before the start of each experiment the term ‘breathlessness’ was defined to the subjects as a sense of breathing discomfort, a feeling that breathing was not sufficient for the needs they thought they had. it was emphasized that they should not confuse breathlessness with other sensations associated with exercise, such as leg fatigue. during both exercise tests, subjects were asked to use the same criteria to estimate their dyspnoea. data were recorded for 1 min at rest, and then the subject started unloaded pedalling, for 1 min, at a frequency of between 50–60 revolutions per min. thereafter, the work rate was increased 20 watts per min until a symptom-limited sub-maximum was reached. the test was stopped if the subject indicated chest pain, showed pallor, severe desaturation, a heart rate within 10 beats min-1 of the maximum predicted for that individual (4), indicated greater than 90 mm on the vas, or if there were any significant ecg abnormalities. respiratory muscle strength (rms) was calculated by the addition of the maximum static inspiratory (pimax) and expiratory (pemax) mouth pressures divided by two (11). pimax and pemax were measured at residual volume and total lung capacity, respectively (med graphic profiler, pulmonary diagnostic system; medical graphics corp, st paul, minnesota, usa). tests were conducted according to american thoracic society (ats)/european respiratory society recommendations (12). three measurements were taken, and the highest values were chosen, irrespective of the test. this test could not be performed on three subjects, as they were either on leave or had examinations. although pimax values are 384 m. m. f. subhan et al. negative, for clarity they were reported as positive numbers, as has been done previously (13). spirometry and maximum voluntary ventilation (mvv) were performed on a compact vitalograph electronic spirometer (vitalograph, maids moreton, buckingham, uk). the apparatus was calibrated daily with a 1 litre calibration syringe. tests were conducted according to ats recommendations (14). for spirometry, three to five manoeuvres were performed after adequate rest. the highest lung function values were chosen, irrespective of the test. mvv was measured for 15 s, and the frequency of duration exceeded 80 breaths min-1 in all subjects. mvv was performed twice, and the highest value was chosen. when the subjects’ vas estimation was plotted against vi, a linear vas/vi relationship was observed. correlating vas against ventilation has been previously performed (4,15,16). linear correlation was applied, and from this the x-axis intercept (l min-1) and the slope (mm min l-1) were calculated as done before (4). the x-axis intercept will be referred to as the intercept. as the first test was to familiarize subjects, only exercise and breathlessness data from week two were analysed. heart rate was analysed as the heart rate after the first min of exercise (fc1min), heart rate at 60 watts of exercise (fc60), and maximum heart rate (fcmax). for dyspnoea comparisons between groups, one-way anova was used for analysis. bonferroni’s post hoc analysis was used for comparisons between specific groups. stepwise multiple linear regression analysis of the slope and intercept against predictor variables such as age, height, etc. was also made. all spirometric test data were pooled and analysed using repeated measures analysis of covariance (ancova), including age and height and two dummy variables to describe the three groups. all tests were two-tailed, and the level of probability taken as significance was 5% (p < 0.05). results lung function relative to the ow group, forced vital capacity (fvc) (p = 0.020), forced expiratory volume in 1 second (fev1) (p = 0.024), and mvv (p = 0.019) were significantly decreased in the uw group, while fvc was lower in nw subjects (p = 0.028) (table i). the pimax (p = 0.008), pemax (p = 0.010), and rms (p = 0.003) were also significantly lower for the uw group compared to the ow group. age, height, fev1/fvc % ratio, and peak expiratory flow (pef) did not show any significant changes between groups. results for spirometry and mvv are presented as actual values and not corrected for age and height. one reason for this was that there were no significant differences in age and height between groups, and secondly there are no standardized values for the pakistani population. however, we did analyse the table i. mean (± sd) anthropometric and lung function variables for all subjects in underweight, normal weight, and overweight groups (n = 37). underweight (n = 13) normal (n = 12) overweight (n = 12) p valuea age (years) 21.4 ± 4.2 22.0 ± 4.3 21.7 ± 5.3 0.953 bmi (kg m-2) 17.4 ± 1.1 22.3 ± 2.1 28.7 ± 2.9 <0.0001 height (cm) 174 ± 6 173 ± 4 175 ± 9 0.809 weight (kg) 52 ± 5 66 ± 8 89 ± 15 <0.0001 fvc (l) 4.13 ± 0.65 4.15 ± 0.61 5.10 ± 1.18 0.010 fev1 (l) 3.32 ± 0.60 3.48 ± 0.61 4.12 ± 0.88 0.020 fev1/fvc % ratio (%) 79.4 ± 7.8 83.7 ± 6.3 80.9 ± 3.1 0.226 pef (l min-1) 511.8 ± 123.5 556.9 ± 103.5 588.0 ± 103.4 0.238 mvv (l min-1) 136.2 ± 29.8 143.7 ± 32.0 170.6 ± 41.6 0.048 pimax (cmh2o) b 79.8 ± 34.0 106.3 ± 36.8 144.9 ± 66.4 0.010 pemax (cmh2o) b 80.4 ± 24.3 107.3 ± 19.6 111.9 ± 25.7 0.006 rms (cmh2o) b 80.1 ± 23.5 106.9 ± 25.6 128.5 ± 43.4 0.004 adifference between three groups. bn = 12, 12, and 10 per group, respectively. bmi = body mass index; fev1 = forced expiratory volume in 1 s; fev1/fvc % ratio = ratio of fev1 to fvc; fvc = forced vital capacity; mvv = maximum voluntary ventilation; pef = peak expiratory flow; pemax = maximum static expiratory mouth pressure; pimax = maximum static inspiratory mouth pressure; rms = respiratory muscle strength. body mass index and exercise dyspnoea 385 data as a percentage of caucasian references values, which took into account age and height, and found no disparity between the two methods of analysis in terms of significantly different variables. using ancova, no significant difference was found in the repeated spirometric data between the three bmi groups, taking into account both subject height and age, fvc (p = 0.71), fev1 (p = 0.76), fev1/fvc % ratio (p = 0.93), or pef (p = 0.93). exercise-induced dyspnoea the relationship between vas and vi was significant for all subjects (p < 0.05) and effectively linear for all groups. the mean (± sd) r2 (coefficient of determination), for all experiments was 0.959 ± 0.030 (range 0.882–0.999); r2 was significantly different between groups (table ii). there was a wide range of values for the slope (0.6–12.1 mm min l-1) and intercept (3.4–42.0 l min-1) of the vas/vi relationship in all subjects. the vas/vi intercept (table ii) in the nw group was significantly higher than in both the uw (p = 0.029) and ow groups (p = 0.040). uw maximum work-load (wmax) per kg body mass values were significantly higher than corresponding ow values (p = 0.001). there were no statistically significant differences in vas/vi slope, wmax, maximum ventilation (vimax), fc1min, fc60, and fcmax between the groups tested (table ii). the mean intercept and slope values of the relationships between vas and inspired ventilation for the three groups have been graphically illustrated (figure 1). regression analyses linear correlations of bmi, lung function, exercise, and dyspnoea parameters were made, combining data from all groups (table iii). bmi had significant positive correlations with rms, pimax, mvv, fvc, fev1, and pef and a negative correlation with fc60. the pimax showed positive relationships against fvc and fev1, while it showed a negative correlation with wmax/body mass. the slope of the vas/vi relationship was negatively correlated with rms and r2. the fc60 had significant negative correlations with mvv, pimax, fvc, and fev1. using the stepwise method, significant models emerged for the multiple regression analysis of the vas/vi slope (f3,31 = 29.330, p < 0.00001, adjusted r2 = 0.714) and vas/vi intercept (f1,33 = 218.824, table ii. comparison of mean (± sd) indices of the breathlessness/ventilation relationship and exercise variables between underweight, normal weight, and overweight groups (n = 37). underweight (n = 13) normal (n = 12) overweight (n = 12) p valuea r2 value 0.94 ± 0.03 0.97 ± 0.02 0.96 ± 0.03 <0.050 vas/vi slope (mm min l -1) 2.73 ± 2.99 2.05 ± 1.40 1.96 ± 1.04 0.575 vas/vi intercept (l min -1) 16.1 ± 5.84 22.7 ± 7.54 17.1 ± 3.96 0.022 wmax (w) 113.9 ± 25.0 120.0 ± 29.5 126.7 ± 27.4 0.510 wmax per kg (w/kg) 2.17 ± 0.53 1.80 ± 0.36 1.46 ± 0.39 0.001 vimax (l min -1) 48.0 ± 12.0 45.3 ± 11.2 48.6 ± 8.9 0.725 fc1min (beats min -1) 91.8 ± 12.9 88.0 ± 13.5 91.9 ± 13.6 0.711 fc60 (beats min -1) 123.7 ± 15.4 116.8 ± 12.3 113.8 ± 16.3 0.239 fcmax (beats min -1) 153.0 ± 22.0 145.7 ± 18.8 144.7 ± 19.1 0.533 adifference between three groups. fc1min = heart rate after 1 min of exercise; fc60 = heart rate at 60 watts of exercise; fcmax = maximum heart rate; r 2 = coefficient of determination; vas/vi = relationship of visual analogue scale against inspired ventilation; vimax = maximum ventilation; wmax = maximum work-load; wmax per kg = wmax per kg body mass. -10 0 10 20 30 40 50 60 70 80 90 10 20 30 40 50 60 ventilation (l min-1) v a s ( m m ) uw = under weight nw = normal weight ow = over weight uw ow nw figure 1. a representation of the mean changes in the vas/vi relationship for all subjects in the three bmi groups. there were no differences in the slope values, but the nw intercept value was significantly greater relative to values for uw and ow subjects. 386 m. m. f. subhan et al. p < 0.00001, adjusted r square = 0.865). significant predictor variables for the slope are the fev1/fvc % ratio, vimax, and pemax (table iv). for the intercept, the vas/vi r 2 value was the only significant variable. residuals for the slope and intercept from the regression were plotted against bmi, pimax, and pemax; data points were randomly distributed around zero. discussion the main finding of our study was that uw and ow subjects, relative to nw subjects, showed greater levels of dyspnoea during exercise, due to a lower vas/vi intercept, indicating that these subjects estimated their sensations of exercise-induced dyspnoea earlier than nw subjects. we also found that fvc, fev1, and mvv were significantly decreased in the uw group, relative to the ow group; changes in fvc and fev1 are in agreement with a previous report (17). an earlier study showed a positive correlation between bmi with both fvc and fev1 (18). schachter et al. suggested that an inferior lung function of uw subjects could be due to a decrease in rms (17). fvc, though not fev1, in our ow subjects was significantly greater relative to nw subjects. in contrast, previous data reported no significant differences in fvc and fev1 between ow and nw subjects (17). the present study also showed that both rms and pimax were significantly lower for the uw group compared to the ow group; both values showed a positive correlation with body mass, in agreement with previous work in healthy subjects (19). combined pemax data for uw patients without lung disease and controls have also shown a significant positive correlation with body weight (11). diaphragm muscle mass has been significantly correlated to body mass in normal subjects after autopsy (20). this could be a basis for the significant relationship between bmi with rms and pimax in the present study. the higher rms of the ow subjects may be due to the increase in body mass improving their muscle strength (21) and vice versa in uw subjects. pimax and several spirometric variables were significantly negatively correlated to fc60, indicative that persons with lower exercise heart rates had better lung function. this is supported by work showing an association between physical activity and lung function (22). at any given level of ventilation, uw and ow subjects were significantly more breathless than nw subjects. nw subjects were the least breathless. the relationship between dyspnoea and ventilation was linear, as shown by the r2 value, and had a large inter-subject variation, consistent with previous work (1,4). it is interesting to note that although our vas/vi slope was not significantly affected by changes in bmi, it had a significant negative correlation with rms and pemax, indicating that a decrease in muscle strength was associated with greater exercise dyspnoea. obesity increases dyspnoea (17,23), and reasons for this include an increase in the work of breathing, due to an increased elastic resistance to distension (24), increased respiratory or nasopharyngeal resistance (25), increased pulmonary venous pressures table iii. linear correlation coefficients for selected correlations, using data from all subjects. correlations r value (correlation coefficient) p value bmi versus rms +0.448 0.008 bmi versus mvv +0.419 0.009 bmi versus fvc +0.454 0.004 bmi versus pef +0.333 0.044 bmi versus fc60 –0.339 0.040 pimax versus fvc +0.409 0.018 pimax versus wmax per kg –0.373 0.030 vas/vi slope versus rms –0.377 0.028 vas/vi slope versus r 2 –0.407 0.012 fc60 versus mvv –0.453 0.005 fc60 versus pimax –0.378 0.027 fc60 versus fvc –0.490 0.002 bmi = body mass index; fc60 = heart rate at 60 watts of exercise; fvc = forced vital capacity; mvv = maximum voluntary ventilation; pef = peak expiratory flow; pimax = maximum static inspiratory mouth pressure; r2 = coefficient of determination; rms = respiratory muscle strength; vas/vi = relationship of visual analogue scale against inspired ventilation; wmax per kg = maximum work-load per kg body mass. table iv. multiple regression analysis of the vas/vi slope and intercept, showing significant predictor variables. dependent variable independent variable (predictor) standardized beta coefficient p value vas/vi slope fev1/fvc % ratio 2.684 <0.00001 vimax –1.184 0.004 pemax –0.811 0.029 vas/vi intercept vas/vi r 2 value 0.932 <0.00001 fev1/fvc % ratio = ratio of fev1 to fvc; pemax = maximum static expiratory mouth pressure; r2 = coefficient of determination; vas/vi = relationship of visual analogue scale against inspired ventilation; vimax = maximum ventilation. body mass index and exercise dyspnoea 387 (26), and greater respiratory work (27). a greater work of breathing suggests a decreased ventilatory reserve, which predisposes these persons to respiratory failure and developing dyspnoea. weight reduction in the obese, by diet and exercise or surgery, can improve dyspnoea (26). most of our ow subjects were not obese, and they were unlikely to possess the respiratory and haemodynamic alterations mentioned above, especially since their lung function was better than that of the nw subjects in our study. the current hypothesis for the genesis of dyspnoea is that output of the central motor command from the brainstem to the respiratory muscles is also projected to the sensory cortex (1). human neuroimaging has shown that the insula is essential for dyspnoea perception (28). it has been suggested that the sensory cortex interacts with the insular cortex to modulate the dyspnoea intercept (4). it could be possible that our uw subjects, who had weaker respiratory muscles, had a greater efferent output from the brainstem, thereby increasing their dyspnoea. in ow subjects, the excess body mass could result in a greater work of breathing and more afferent input to the brainstem, which would again result in a greater efferent output from the brainstem. it is unclear as to why the threshold (intercept) and not the gain (slope) are affected by bmi. limitations of the present study are that a larger sample size for each group could have shown more definitive results. the study participants were all men, therefore extrapolating the results to women cannot be done. in the present study we have speculated that changes in the vas/vi intercept in uw subjects were most likely due to changes in rms. an alternative explanation could be that this group was less physically active and conditioned than the other group; however, when exercise heart rates were compared, there were no significant differences. another limitation of this study is that we could not distinguish whether changes in bmi were due to changes in lean tissue mass or fat mass. in addition, most of our subjects were sedentary individuals with poor levels of physical fitness. this might limit generalizing the results with a population having normal or good levels of physical fitness. the ow group had eight overweight and five obese subjects; their mean bmi was 29 kg m-2. for logistical reasons they were combined into one group, due to the small number of subjects in each group and also the fact that four obese subjects had a borderline overweight/obese class i bmi (30–31 kg m-2). vas/vi intercept and slope data were tested and showed no significant differences between these overweight and obese subjects. therefore, it was expected that this combination would have no material effect on the results. it has been recommended that patients with respiratory disease should attempt to keep their bmi in the normal range (29), and our study has shown that this recommendation, with regard to the symptoms of dyspnoea and lung function, should be extended to healthy subjects. additionally, previous work has shown that men with an abnormal bmi are at an increased risk of developing copd (8), were associated with a higher prevalence of asthma (9), and in asthma would have a greater decline in fev1 (30). we strongly agree with a suggestion that future clinical breathlessness studies should consider data stratification by bmi (10). in conclusion, this study has shown that uw and ow healthy subjects showed greater levels of dyspnoea during exercise compared to nw subjects. higher levels of dyspnoea could possibly result from a decrease in rms. the reason for the greater dyspnoea of ow subjects could be an increase in the work of breathing. implications from this study are that uw and ow subjects might be more susceptible to increased dyspnoea if they develop lung disease and, secondly, that caution is needed in clinical or mechanistic trials using subjects with abnormal body mass indices or body fat and varied levels of physical activity. acknowledgements the authors would like to thank salman sabir, department of community health sciences, for statistical guidance. we are also indebted to dr nawal salahuddin, department of medicine, who provided us the facilities to measure respiratory muscle strength in our subjects. both are affiliated with the aga khan university, pakistan. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. adams l, guz a. dyspnea on exertion. in whipp b, wassermann k, editors. exercise, pulmonary physiology and pathophysiology. new york: marcell dekker; 1991. p 449–94. 2. shoup r, dalsky g, warner s, davies m, connors m, khan m, et al. body composition and health-related quality of life in patients with obstructive airways disease. eur respir j. 1997;10:1576–80. 3. tessier jf, nejjari c, letenneur l, filleul l, marty ml, barberger gateau p, et al. dyspnea and 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cederholma and bj€orn zetheliusb adepartment of public health and caring sciences/family medicine and preventive medicine, uppsala university, uppsala, sweden; bdepartment of public health and caring sciences/geriatrics, uppsala university, uppsala, sweden abstract background: fasting insulin resistance indexes are used extensively nowadays. we intended to analyze a new recently presented fasting index, spise (sensitivity formula: 600� hdl-cholesterol0.185/triglycerides0.2/bmi1.338), in comparison with three previously known fasting indexes, regarding correlation with the insulin clamp index, and for the predictive effects of future long-term risks of coronary heart disease (chd) or manifest type 2 diabetes. methods: a total of 1049 71-year-old male subjects from the swedish ulsam study, median follow-up 8 years, were included. all subjects performed the euglycemic insulin clamp, and analyses of four fasting insulin resistance indexes: spise-ir (¼ 10/spise), quicki-ir, log homa-ir, and revised quicki-ir. results: spearman correlation coefficients with the insulin clamp were 0.60–0.62 for all indexes. area under curve at roc analysis was 0.80 for spise-ir, and 0.84 for quicki-ir, log homa-ir, and rev quicki-ir. adjusted hazard ratios per 1 sd index increase for long-term risk chd were similar in all patients: 1.20–1.24 (p ¼ 0.02–0.03). however, comparing the highest quartile (recommended to define insulin resistance) with the lower quartiles, spise-ir was the strongest and the only statistically significant insulin resistance index: hr 1.53 (p ¼ 0.02). adjusted odds ratios per 1 sd index increase for longterm risk of type 2 diabetes were fairly similar (p < 0.001) in all patients: 1.62 for spise-ir, 1.97 for quicki-ir and log homa-ir, and 2.04 for rev quicki-ir, and also when comparing the highest versus the lower quartiles: 2.8–3.1 (p < 0.001). conclusion: spise, easily applicable, performed equally well as other fasting insulin indexes previously recommended for clinical use, regarding correlation with the insulin clamp, and as predictor for future long-term risks of chd or type 2 diabetes. article history received 9 july 2019 revised 11 october 2019 accepted 11 october 2019 keywords coronary heart disease; homa; insulin clamp; insulin resistance; quicki; spise; type 2 diabetes introduction several studies have indicated that increased insulin resistance is a predictor for the development of type 2 diabetes (1–4). it also significantly contributes to accelerated atherosclerosis as a risk factor for coronary heart disease (chd) (5–7). the euglycemic insulin clamp technique is regarded as the reference method for an accurate assessment of in vivo insulin resistance (8–10). however, this method is laborious, expensive, and considered unsuitable for larger-scale or epidemiological studies. other measures at the fasting state have been presented to be more clinically suitable and useful surrogate indexes of insulin resistance/sensitivity, like the homeostasis model assessment (homa-ir) index (11,12), log homa-ir index (13–15), quantitative insulin sensitivity check index (quicki) (16), and revised quicki index (17). however, indexes of insulin sensitivity are also available using 0-h and 2-h glucose and insulin values during the standard 75-g oral glucose tolerance test (ogtt), like the cederholm index (18–20) and the matsuda index (21,22). furthermore, a new fasting insulin sensitivity index, spise (single-point insulin sensitivity index), has recently been introduced by the risc and beta-judo investigators as an easily applicable tool in clinical practice, based on the ratio of triglycerides to highdensity lipoprotein-cholesterol (tg/hdl) and body mass index (bmi) (23). after several repeated regression modelings, the best formula for spise was presented as: 600 � hdl0.185/ tg0.2/bmi1.338. the aim of this study was to evaluate the correlation between the resistance index spise-ir (defined as 10/spise) and the euglycemic insulin clamp, and to estimate the effect of spise-ir as a predictor for risks of future chd and manifest type 2 diabetes. we also made a comparison with the indexes quicki, log homa-ir, and revised quicki regarding their correlations with the insulin clamp and as predictors of these outcomes. material and methods subjects all men born between 1920 and 1924 in uppsala, sweden, were invited to a health survey in 1970 in which 2322 men (82%) participated – the ulsam study (24). after 20 years, at contact jan cederholm jan.cederholm@pubcare.uu.se department of public health and caring, uppsala university, box 564, uppsala, s-75122, sweden � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 4, 265–272 https://doi.org/10.1080/03009734.2019.1680583 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1680583&domain=pdf&date_stamp=2019-12-09 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2019.1680583 http://www.tandfonline.com 71 years of age, 1221 (73%) of the 1681 still-living subjects were invited for reinvestigation in 1991–1995, which constituted the baseline of this study (25). the study was approved by the ethics committee of the faculty of medicine at uppsala university, and it complies with the principles of the declaration of helsinki. written informed consent was obtained from all subjects. patients with data available for all analyzed variables in this study numbered 1049 subjects. this sample was used to analyze the correlation between the euglycemic insulin clamp test and the fasting insulin indexes presented here, and to analyze these indexes as predictors of risk for ischaemic heart disease. a subgroup was also created consisting of 1024 participants, after exclusion of those with manifest diabetes at baseline, and with data available for all analyzed variables. this subgroup was used to analyze the fasting insulin indexes as predictors of risk for development of type 2 diabetes. type 2 diabetes at baseline was defined according to the 1999 world health organization criteria (26). somewhat fewer inclusion criteria regarding the variables applied, as covariates in the regression analyses were used in this subgroup. baseline investigations baseline investigations in 1991–1995 consisted of an euglycemic insulin clamp test, 75-g ogtt, hdl-cholesterol, triglycerides, body weight and height, non-esterified fatty acids (nefa), cystatin c, microalbuminuria, systolic blood pressure under standardized conditions, and smoking present or not (25,27,28). bmi was calculated as weight/height squared (kg/ m2). microalbuminuria was measured as the urinary albumin excretion rate (mg/min). the charlson comorbidity index was included for classification of a range (score) of comorbid diseases and conditions that may affect outcomes in prospective studies (29). glucose tolerance was assessed by 75-g ogtt, separated in time by 1 week from the euglycemic insulin clamp procedure (28). blood samples for fasting concentrations were collected after overnight fasting, and blood samples were also collected at 2-h during the ogtt. concentrations of plasma glucose were analyzed by the glucose dehydrogenase method (gluc-dh; merck, darmstadt, germany). plasma immuno-reactive insulin (iri) was determined with the enzymatic immunologic assay enzymmun (boehringer mannheim, mannheim, germany) (27). these ogtt data were used to detect manifest diabetes at baseline of this study, and also for estimation of ogtt-based insulin resistance indexes in a previous study from our group (20). insulin resistance measures the euglycemic insulin clamp is considered gold standard for measurement of insulin sensitivity (1,8,27,28). insulin was infused at a constant rate of 56 mu/min/m2, calculated to achieve nearly complete suppression of the hepatic glucose output (27,28). the target level of plasma glucose (measured every 5th minute during the 2-h clamp) was 5.1 mmol/l. median was 5.1 mmol/l, 5th percentile 5.0 mmol/l, 95th percentile 5.4 mmol/l, and mean ± sd 5.2 ± 1.3 mmol/l. the insulin sensitivity index at the clamp (m/i) was calculated as glucose disposal rate (mg glucose infused/min/kg body weight) divided by the mean plasma insulin concentration � 100 (mu/l) during the last 60 min of the 2-h euglycemic insulin clamp. the inverse of the sensitivity index m/i is used here as the insulin resistance index, m/i-ir, defined as 10/ (m/i). spise (23) has been developed by the risc and betajudo investigators based on the ratio of triglycerides to hdl-cholesterol (tg/hdl) and bmi, with use of two included studies: the relationship between insulin sensitivity and cardiovascular disease study cohort (n ¼ 1260; age, mean ± sd, 44 ± 8 years) and the beta-cell function in juvenile diabetes and obesity study cohort (n ¼ 29; age, mean ± sd, 15 ± 2 years). all subjects underwent 75-g ogtt and euglycemic clamp tests for calculation of the clampderived sensitivity index m/i value. to refine the tg/hdl ratio, mathematical modelling was applied, including fasting tg, hdl, and also bmi, as compared to the clamp-derived m/ i value, and each of several modelings was scored by the correlation coefficient with m/i and the specificity for insulin sensitivity identification. based on several repeated modelings, the best formula for this new sensitivity index, spise, was then presented as: 600 � hdl0.185/tg0.2/bmi1.338. the inverse of this index, spise-ir, is used in this study as an insulin resistance index, and defined as 10/spise. quicki has been introduced as a fasting index of insulin sensitivity, defined as 1/[log(fasting glucose) þ log(fasting insulin)], where log is the natural logarithm, and glucose is measured in mmol/l and insulin in mu/l (16). the inverse of the sensitivity index quicki is used here as the insulin resistance index, quicki-ir, defined as 1/quicki. revised quicki has been presented as another fasting insulin sensitivity index based on glucose, insulin, and also additionally nefa, and was defined as 1/[log(fasting glucose) þ log(fasting insulin) þ log(nefa)] (17). the inverse of this sensitivity index was used here as the insulin resistance index, rev quicki-ir, defined as: 1/revised quicki. homa-ir was described as a fasting index of insulin resistance by matthews et al. with the formula: fasting glucose mmol/l � fasting insulin mu/l/22.5 (11). homa-ir has been shown to be highly correlated (r ¼ 0.98) with a computerderived homa-ir model (12) in the framingham study (30). only the former has been applied in this study as the basis for the insulin resistance index log homa-ir, defined as the natural logarithmic transformation of homa-ir: log(fasting glucose � fasting insulin/22.5), as previously described by others (13,15). follow-up and outcomes all patients were followed-up after a median of 8 years, with up to a maximum of 10 years (censor date 31 december 2001). all events of fatal or non-fatal chd, defined as icd-10 codes i20–i25, were retrieved by data linkage with the swedish cause of death and hospital discharge registers, a 266 j. cederholm and b. zethelius reliable validated alternative to revised hospital discharge and death certificates (31,32). type 2 diabetes during followup was defined as fasting plasma glucose �7.0 mmol/l in reinvestigations at age 77 years, or as new use of oral hypoglycaemic agents detected by questionnaire or in medical records during the follow-up period. statistical analysis table 1 shows baseline clinical characteristics, given as means (sd) or frequencies (%), and also median values with interquartile range for the insulin indexes. cross-sectional analyses at baseline as table 2 shows, spearman correlation coefficients were used for comparison of m/i-ir with each of the four fasting insulin resistance indexes. ninety-five percent confidence intervals of the coefficients were estimated by bootstrapping with use of the r package rvaidememoire. receiver operating curves (roc) were used for comparison of the highest quartile of m/i-ir (�3.2) with each of the four fasting resistance indexes, as shown in figure 1. we estimated roc area under curve (auc) values for each comparison (table 2). we also calculated the difference between auc for spise-ir and auc for each of quicki-ir, log homa-ir, and rev quicki-ir, and the significance of these differences. all roc data were estimated with the r statistical package proc. longitudinal analyses of chd risk cox regression analysis was used to estimate hazard ratios (hr) with the four fasting resistance indexes as predictors of long-term fatal or non-fatal chd as outcome (table 3). hr with 95% confidence intervals (ci) were given per 1 sd increase of each predictor index, allowing for a direct comparison between the strengths of the four hazard ratios. the z statistic with its p values also indicates the association between predictor and outcome – the higher z value, the stronger association. covariance adjustment was performed with several important conventional cardiovascular risk factors and clinical characteristics: systolic blood pressure, smoking, cystatin c, albuminuria, charlson comorbidity index, and a history of chd. the proportional hazards assumption at cox regression was confirmed with scaled schoenfeld residuals, estimated with the r statistical package cox.zph. the likelihood ratio (lr) test indicates global model fit – the higher value, the better fit. harrell’s c (concordance) measures discrimination between those who will have and those who will not have events – the higher value, the better discrimination, estimated with the r statistical package dynpred. longitudinal analyses of risk for type 2 diabetes multivariable logistic regression was used to analyze the four fasting resistance indexes as predictors of long-term development of manifest type 2 diabetes as dependent outcome (table 4). odds ratios (or) with 95% ci were given per 1 sd increase of each predictor index, making possible a direct comparison between the strengths of the four odds ratios, also indicated by z statistics with p values. adjustments were made for systolic blood pressure, smoking, and charlson comorbidity index. the aic value measures global model table 1. baseline characteristics in 1049 male subjects aged 71 years. clinical characteristics all subjects (n ¼ 1049) glucose 0 min, mmol/l 5.7 ± 1.4 glucose 120 min, mmol/l 8.3 ± 4.1 manifest type 2 diabetes, % 10.2 insulin 0 min, mu/l 12.8 ± 8.3 insulin 120 min, mu/l 70.1 ± 52.5 clamp m/i-ir, 1/(mg/kg/min/ml) 2.7 ± 1.9; 2.1 (1.5–3.2) spise-ir, 1/(kg/m2) 1.4 ± 0.3; 1.3 (1.1–1.5) quicki-ir, 1/(mmol � mu/l2) 4.1 ± 0.6; 4.1 (3.8–4.5) log homa-ir, (l2/mmol � mu) 1.0 ± 0.64; 1.0 (0.64–1.4) rev quicki-ir, 1/(mmol2�mu/l3)a 4.0 ± 0.7; 4.0 (3.6–4.5) systolic bp, mmhg 147.1 ± 19 hypertension treatment, % 33.4 bmi, kg/m2 26.3 ± 3.4 total cholesterol, mmol/l 5.8 ± 1.0 ldl cholesterol, mmol/l 3.9 ± 0.9 hdl cholesterol, mmol/l 1.29 ± 0.35 triglycerides, mmol/l 1.41 ± 0.69 nefa, mmol/la 0.52 ± 0.22 microalbuminuria, lg/min 25.3 ± 95 cystatin c, mg/l 1.24 ± 0.27 smoker, % 20.7 charlson comorbidity index, % level 0 61.0 level 1 20.8 level 2 10.8 level 3 4.5 levels 4–7 2.9 a history of chd, % 8.5 data given as means ± sd or frequencies (%), and for the indexes also as median (interquartile range). arev quicki-ir and nefa were n ¼ 1038 due to missing data for nefa. bp: blood pressure; chd: coronary heart disease; hdl: high-density lipoprotein; ir: insulin resistance; ldl: low-density lipoprotein; m/i: glucose disposal rate divided by mean plasma insulin during the clamp; nefa: non-esterified fatty acids. table 2. spearman correlation coefficients, and roc auc values, comparing m/i-ir and each of the fasting insulin resistance indexes in 1049 71-year-old male subjects. spearman coefficient (95% ci) auc value difference between auc values insulin indexes m/i-ir in all patients q4 of m/i-ir spise-ir z value p value spise-ir 0.62 (0.58–0.65) 0.801 – – – quicki-ir 0.60 (0.56–0.65) 0.844 �0.043 2.3 0.02 log homa-ir 0.60 (0.56–0.64) 0.844 �0.043 2.3 0.02 rev quicki-ir 0.61 (0.57–0.65) 0.847 �0.046 2.6 0.01 difference between auc values compares auc for spise-ir with auc for each of the indexes quicki-ir, log homa-ir, and rev quicki-ir. auc: area under curve (roc; compares the highest quartile q4 of m/i-ir [�3.2] with each of the other indexes); ci: confidence interval (based on bootstrapping; significance level all p < 0.001); ir: insulin resistance; m/i: glucose uptake per insulin unit at the clamp. upsala journal of medical sciences 267 fit – the lower, the better fit. the c statistic measures discrimination between those who will have and those who will not have events – the higher value, the better discrimination. all statistical analyses were performed with r version 3.5.3, 11 march 2019 (33). a two-sided p values <0.05 was considered statistically significant. results baseline characteristics mean ± sd values were 2.7 ± 1.9 units for insulin clamp m/iir, 1.4 ± 0.3 units for spise-ir, 4.1 ± 0.6 units for quicki-ir, 1.0 ± 0.64 units for log homa-ir, and 4.0 ± 0.7 units for rev quicki-ir (table 1). figure 1. roc curves for the highest quartile q4 of m/i-ir (�3.2) versus four fasting insulin resistance indexes, in 1049 71-year-old male subjects. left dashed curve a: rev quicki-ir; left solid curves b and c: quicki-ir and log homa-ir; right dotted curve d: spise-ir. see table 2 for auc values. table 3. the ability of four fasting insulin resistance indexes to predict long-term coronary heart disease, cox regressions in 1049 male subjects aged 71 years, 135 events during median 8 years of follow-up. all patients (n ¼ 1049) q4 versus q1–q3a insulin indexesb hr (95% ci) z valuec p value lr testd harrell’s ce hr (95% ci) z valuec p value spise-ir 1.20 (1.02–1.40) 2.2 0.03 44 0.64 1.53 (1.06–2.21) 2.3 0.02 quicki-ir 1.22 (1.03–1.45) 2.2 0.02 44 0.63 1.02 (0.70–1.51) 0.10 0.9 log homa-ir 1.22 (1.03–1.45) 2.2 0.02 44 0.63 1.02 (0.70–1.51) 0.10 0.9 rev quicki-ir 1.24 (1.04–1.49) 2.4 0.02 44 0.63 1.53 (0.99–2.37) 1.9 0.054 ahr for the highest quartile q4 (n ¼ 264) compared to lower quartiles q1–q3 as reference. cut-off values for q4 (75th percentile) of the indexes expressed per 1 sd were for spise-ir: 4.6; quicki-ir: 7.1; log homa-ir: 2.2; and rev quicki-ir: 5.1. bindexes expressed per 1 sd to allow for direct comparison between hr, adjusted for systolic blood pressure, smoking, cystatin c, albuminuria, charlson comorbidity index, and a history of chd. cz value: a higher value indicates stronger association between predictor and outcome. dlr test measures global fit; the higher value, the better fit. eharrell’s c (concordance) measures discrimination between those who will have and will not have events. ci: confidence interval; hr: hazard ratio. table 4. the ability of four fasting insulin resistance indexes to predict the long-term development of manifest type 2 diabetes, multivariable logistic regressions in 1024 male subjects aged 71 years, no diabetes at baseline 1991–95, 56 events of manifest diabetes during follow-up until 2001. all patients (n ¼ 1024) q4 versus q1–q3a insulin indexesb odds ratio (95% ci) z valuec p value aic valued c statistice odds ratio (95% ci) z valuec p value c statistice spise-ir 1.62 (1.27–2.05) 3.9 <0.001 416 0.69 2.8 (1.6–4.8) 3.6 <0.001 0.68 quicki-ir 1.97 (1.47–2.64) 4.6 <0.001 409 0.72 3.1 (1.8–5.3) 4.0 <0.001 0.70 log homa-ir 1.97 (1.47–2.64) 4.6 <0.001 409 0.72 3.1 (1.8–5.4) 4.1 <0.001 0.70 rev quicki-ir 2.04 (1.48–2.81) 4.4 <0.001 402 0.72 2.9 (1.7–5.1) 3.8 <0.001 0.70 aodds ratios for the highest quartile q4 compared to the lower quartiles q1–q3 as reference. cut-off values per sd for q4 (75th percentile) were for spise-ir: 4.7; quicki-ir: 7.5; log homa-ir: 2.2; rev quicki-ir: 6.5. aic values comparing quartiles were 418, 415, 415, and 409, respectively. bindexes expressed per sd to allow for direct comparison between or, adjusted for systolic blood pressure, smoking, and charlson comorbidity index. cz value: the higher the stronger association. daic measures global fit; the lower value, the better fit. ec statistic measures discrimination. ci: confidence interval. 268 j. cederholm and b. zethelius cross-sectional associations at baseline between clamp m/i-ir and insulin resistance indexes spearman correlation coefficients between insulin clamp m/iir and each fasting insulin resistance index were 0.62 (95% ci 0.58–0.65) for spise-ir, 0.60 (0.56–0.65) for quicki-ir, 0.60 (0.56–0.64) for log homa-ir, and 0.61 (0.57–0.65) for rev quicki-ir, all p < 0.001 (table 2). roc curves for the highest quartile of m/i-ir (�3.2) versus each of the four fasting resistance indexes are given in figure 1. auc values of the roc curves were quite similar: 0.80 for spise-ir, 0.84 for quicki-ir and log homa-ir, and 0.85 for rev quicki-ir, (table 2). differences between auc for spise-ir and auc for each of the other indexes were slightly significant (p < 0.02–0.01). longitudinal prediction of risk for chd the four insulin resistance indexes as predictors for longterm risk of fatal or non-fatal chd estimated at cox regression are shown in table 3. the number of chd events were 135 during median 8 years of follow-up, with 7625 person years at risk (table 3). with each index introduced per 1 sd increase, and adjustments for systolic blood pressure, smoking, cystatin c, albuminuria, charlson comorbidity index, and a history of chd, spise-ir estimated in all patients disclosed a significant hr of 1.20 (1.02–1.40; p ¼ 0.03), with a similar magnitude of hr as the other three fasting indexes, and with similar values of lr test and harrell’s c. however, spise-ir was the only significant index (p ¼ 0.02), and with the highest hr of 1.53, among all four fasting indexes, when analyzing the highest quartile versus the lower three quartiles. quicki-ir and log homa-ir were independent predictors of chd in all patients, with the same hr 1.22 (1.03–1.45; p ¼ 0.02), and the same lr tests and harrell’s c. however, hr for these two indexes were non-significant when comparing the highest quartile versus the lower quartiles. finally, rev quicki-ir had a significant hr 1.24 (1.04–1.49; p ¼ 0.02) in all patients, while hr for quartile 4 versus quartiles 1–3 disclosed an effect of only borderline significance. longitudinal prediction of risk for manifest type 2 diabetes the four fasting resistance indexes as predictors for risk of incident type 2 diabetes during follow-up were analyzed with multivariable logistic regression, with each index introduced per 1 sd increase to allow for direct comparison between strengths of the odds ratios (table 4). in total, 56 new cases of type 2 diabetes were found during follow-up from baseline until 2001, in 1024 participants with no previous diabetes. adjustments were made for systolic blood pressure, smoking, and charlson comorbidity index. spise-ir was strongly significant as predictor in all participants, with or 1.62 (1.27– 2.05; p < 0.001), although slightly lower than or of the other three indexes. global fit was slightly less with a higher aic value; the c statistic of discrimination was 0.69. comparing the highest quartile versus the three lower, or for spise-ir was strongly significant (p < 0.001), similar as for the other three indexes, with a c statistic of 0.68. quicki-ir, log homa-ir, and rev quicki-ir showed similar or in all patients, 1.97–2.04, and when comparing the highest quartile with the lower, 2.9–3.1, with similar aic values. c statistics were 0.68–0.70. discussion the present large cohort study, including more than 1000 participants with data on fasting blood variables and gold standard euglycemic insulin clamp tests, showed cross-sectionally that the newly introduced fasting insulin resistance index spise-ir had a similar degree of spearman correlation with the insulin clamp index m/i-ir as other previously used fasting resistance indexes, quicki-ir, log homa-ir, and rev quicki-ir. a meta-analysis in 2014 based on 120 articles has presented correlations between the insulin index at hyperinsulinemic euglycemic clamp tests and several fasting or ogtt-based insulin resistance indexes (15). among the fasting surrogate indexes, they found the strongest correlation coefficients with rev quicki-ir (r ¼ 0.68), quicki-ir (r ¼ 0.61), and log homa-ir (r ¼ 0.60), and these authors suggested to recommend these three as the most appropriate for use in large-scale clinical studies. however, they had provided no data on the recent spise-ir. thus, our data imply that spiser should be equally appropriate according to its spearman correlation coefficient 0.62 observed here. the usefulness of spise-ir is underlined by the clinical practical advantage of no need to estimate less available data like fasting insulin or nefa variables. an even better correlation between the insulin clamp test and spise-ir was reported in a recent japanese study of 111 healthy non-diabetic men aged 30–50 years, with a spearman coefficient of 0.69 (34). the risc and beta-judo investigators who introduced spise in 2016 have performed roc analyses comparing the insulin clamp test m/i with either spise-ir, the fasting resistance index quicki-ir, or the fasting resistance index homair (23), thus specifying insulin resistance among the total distribution of the insulin index (35,36). they found comparable roc auc values for all three indexes, 0.81–0.83, with no significant difference between them. roc curves in our study comparing the insulin clamp index m/i-ir with the four fasting resistance indexes are presented in figure 1. we found a mainly similar roc auc value for spise-ir in our study, 0.80, and also for quicki-ir, log homa-ir, and rev quicki-ir, 0.84 (table 2). the study groups used by the risc and beta-judo investigators consisted of non-diabetic adults with mean bmi 26 kg/m2 and obese adolescents with mean bmi 38 kg/m2, while our study consisted of 70-year-old male subjects with mean bmi 26 kg/m2. this study also showed that hazard ratios for long-term risk of future chd in all patients were clearly significant and similar for spise-ir and the three other fasting indexes, quicki-ir, log homa-ir, and rev quicki-ir, and with similar harrell’s c measuring discrimination (table 3). however, comparing patients within the highest quartile of a resistance upsala journal of medical sciences 269 index with the patients within the lower quartiles, it was found that spise-ir had a clearly better capability to detect future risk of chd. values within the highest quartile are recommended (35) and often used (36) to characterize insulin resistance among the total distribution of a surrogate insulin index. concerning or for long-term risk of future type 2 diabetes, spise-ir generally had the same highly significant risk effect as the other three fasting indexes in all patients, and comparing the highest quartile with the lower, and with similar c statistic measuring discrimination (table 4). thus, our results are in agreement with the conclusion by the risc investigators that spise-ir is performing equally well and as appropriately as the other previously recommended fasting insulin indexes at the meta-analysis in 2014. spise-ir is easily applicable in clinical practice with its use of an inexpensive routine fasting single-point blood sampling and bmi, and with a simply calculated index. the risc investigators also estimated roc auc values between the insulin clamp test and the ogtt-based insulin sensitivity index matsuda-isi, and found a somewhat higher auc value of 0.86, with a significant difference between auc values for matsuda-isi and spise (p ¼ 0.006). matsuda-isi (¼ 1000/� [0-min glucose � 0-min insulin � 120-min glucose � 120-min insulin]) and the corresponding matsuda-ir (¼ 10/ matsuda-isi) have been used as a well-performing surrogate insulin index in recent years (21,22). however, another ogttbased insulin sensitivity index has been presented by our group, namely the cederholm index (¼ [75,000/120 þ (0-min glucose – 120 min glucose) � 0.19 � body weight kg � 180/ 120]/[(0-min glucose þ 120-min glucose)/2]/log[(0-min insulin þ 120-min insulin)/2]). this index (18,19) and the corresponding resistance index cederholm-ir (¼ 100/cederholm index) have been analyzed by our group and by others in previous studies (20,37,38). matsuda-ir was reported to have a somewhat better simple correlation with the insulin clamp test than cederholm-ir (21). even if our previous survey using exactly the same sample of subjects as in the present study also found this (see the summary presented below), we found a better association between cederholm-ir and the insulin clamp test according to bland–altman plot analysis. a summary is presented below making a comparison between data from our present study and a previous study of our own (20). using the same samples of 1049 and 1024 subjects in both, and with the same follow-up in both, it demonstrates spearman correlation coefficients between m/iir and four insulin resistance indexes, hr per 1 sd insulin index increase for risk of chd, and or per 1 sd insulin index increase for risk of type 2 diabetes. this allows for a direct comparison of the predictive strength of different hr and or, also with similar covariate adjustment in both studies (table 5). it turns out that ogtt-based cederholm-ir clearly is the strongest predictor of future chd and diabetes (p < 0.001), and reasonably more able to measure combined hepatic and peripheral insulin resistance than fasting indexes like homa-ir. spise-ir had a lower correlation with m/i-ir, although similar predictive capacity as matsuda-ir for risks of chd and diabetes according to strengths of hr and or. it cannot be excluded that spise-ir could mainly capture whole-body resistance, capturing combined hepatic and peripheral resistance, although measured at fasting. strengths of this study are the large number of participants performing the euglycemic insulin clamp test, longterm follow-up of events, and outcomes retrieved by register linkages with reliable validated methods in sweden (31,32). using male subjects 71 years of age should mainly exclude the effects of hormonal variation. this age group is also sensible for estimation of risks for chd and diabetes. a limitation may be that unmeasured covariates may have affected the results, although relevant covariates were extensively applied. bmi and blood lipids were not included as covariates in the present study, as these variables are included in the formula for spise. the same results were generally obtained with both quicki-ir and log homa-ir in this study regarding correlations, roc auc values, hr, and or, in accordance with similar mathematical formulas: [log(fasting glucose) þ log(fasting insulin)], and [log(fasting glucose � fasting insulin/22.5)]. we have included both these two surrogate indexes in this article in order to make the comparison between them obvious. however, our opinion is that in future studies it is not necessary to use both of them simultaneously, as they are identical and interchangeable. the finding here, of several insulin resistance indexes to be strong predictors of cvd risk independently of several important conventional cardiovascular risk factors and comorbidities, underlines that insulin resistance also may be associated with other risk factors like endothelial dysfunction (39), chronic subclinical inflammation (40), impaired fibrinolysis, and hypercoagulability (41). conclusion in conclusion, the usefulness of surrogate indexes for insulin resistance in epidemiological studies depends on the strength of their correlation with criterion measures, but also importantly on the degree to which they predict future chd and type 2 diabetes in long-term observational analyses. this large observational study including euglycemic insulin clamp table 5. comparisons of data from the present study and a previous study of our own (20). insulin indexes corr. m/i-ir versus index cox hr (95% ci) p value logistic regression or (95% ci) p value c statistic cederholm-ira 0.71 1.31 (1.15–1.50) <0.001 2.43 (1.87–3.15) <0.001 0.83 matsuda-ira 0.76 1.23 (1.07–1.43) 0.005 1.68 (1.34–2.11) <0.001 0.76 homa-ira 0.60 1.18 (1.02–1.30) 0.03 1.27 (1.05–1.54) 0.01 0.71 spise-irb 0.62 1.20 (1.02–1.40) 0.03 1.62 (1.27–2.05) <0.001 0.69 aprevious study (20). bpresent study; hr and or per 1 sd index increase, similar covariate adjustments. ci: confidence interval; corr.: spearman correlation coefficient; cox hr: hazard ratio at cox regression; log. regr. or: odds ratio at multivariate logistic regression. 270 j. cederholm and b. zethelius tests has demonstrated that the newly introduced index spise-ir as well as previously recommended fasting indexes quicki-ir, log homa-ir, and rev quicki-ir all had a somewhat lower degree of correlation with the clamp resistance index than ogtt-based indexes like cederholm-ir and matsuda-ir. however, all had high roc auc values, with no pronounced difference between them. although the ogttbased cederholm-ir had the strongest effect to predict future risks of type 2 diabetes and chd among all analyzed indexes, spise-ir had a strongly significant effect to predict diabetes and also a significant effect to predict chd, with a mainly similar predicting effect as the other three fasting insulin indexes. finally, this was underlined regarding spiseir when comparing the highest quartile of an index, indicating an insulin-resistant state, with the three lower quartiles. spise-ir turned out to have the strongest hazard ratio for risk chd among the fasting indexes, and was the only one clearly significant (p ¼ 0.02). the hazard ratio for risk of diabetes was also strongly significant (p < 0.001) with highest quartile of spise. thus, this study indicates that spise-ir should be useful and even preferred among surrogate fasting insulin resistance indexes, with the advantage of being easily available in clinical practice, using only fasting blood lipids and bmi, and without the necessity to analyze insulin or nefa variables. likewise, spise-ir should be applicable in clinical studies on treatment of cardiovascular risk factors for risk prediction of chd, type 2 diabetes (34), non-alcoholic fatty liver disease (42), and also possibly useful for complementary evaluation of insulin resistance in obese patients suitable for gastric bypass operations. further clinical controlled studies should be valuable to validate the clinical utility of spise as a biomarker, to be used in prevention of future chd and type 2 diabetes (34), as well as in the prevention of non-alcoholic fatty liver disease (42). acknowledgements the authors are grateful to the ulsam research team and the participating subjects who made this study possible. ulsam studies are supported by research grants from the medical faculty of uppsala university, the swedish medical research council (mfr5446), the uppsala geriatric fund, and the thureus foundation for geriatric research. disclaimer results and views of the present study represent the authors and are not necessarily any official views of the governmental swedish medical products agency, uppsala, sweden, where one of the authors (b.z.) is employed as scientific director of pharmacotherapy. author contributions both authors contributed to the conception of the study. b.z. collected the data. j.c. designed statistical models and performed analyses, and b.z. supported in analysis strategy and interpretation of the 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hemophagocytic lymphohistiocytosis in adults full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 hemophagocytic lymphohistiocytosis in adults maciej machaczka to cite this article: maciej machaczka (2013) hemophagocytic lymphohistiocytosis in adults, upsala journal of medical sciences, 118:3, 201-203, doi: 10.3109/03009734.2013.795634 to link to this article: https://doi.org/10.3109/03009734.2013.795634 © informa healthcare published online: 17 may 2013. submit your article to this journal article views: 688 view related articles citing articles: 2 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2013.795634 https://doi.org/10.3109/03009734.2013.795634 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.795634 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2013.795634 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.795634#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2013.795634#tabmodule upsala journal of medical sciences. 2013; 118: 201–203 letter to the editor hemophagocytic lymphohistiocytosis in adults maciej machaczka1,2 1hematology center karolinska, and 2division of hematology, department of medicine at huddinge, karolinska institutet, karolinska university hospital huddinge, stockholm, sweden dear sir, i read with interest a recent article by antonodimitrakis and colleagues in your journal presenting a case of acquired hemophagocytic lymphohistiocytosis (hlh) in a 60-year-old woman suffering from diabetes mellitus type 2 (1). the reported patient developed a life-threatening hlh associated with a reactivation of an epstein–barr virus (ebv) infection and was successfully treated by means of corticosteroids alone. hemophagocytic lymphohistiocytosis (hlh) is a clinical syndrome of an exaggerated inflammatory reaction triggered by various inherited and/or acquired factors (2). the literature on the topic of hlh in adults is limited; however, acquired (i.e. secondary) forms of hlh (e.g. infection-associated hlh, i-hlh; autoimmune-associated hlh, a-hlh; malignancy-associated hlh, m-hlh) are the most prevalent. antonodimitrakis et al. wrote in their article that ‘the patient’s age showed that a familial form of hlh was unlikely’ (1). familial forms of hlh (fhl) are autosomal recessive disorders related to different mutations in genes encoding proteins required for lymphocyte cytotoxicity (i.e. prf1, unc13d, stx11, stxbp2) (2). although fhl usually arises in infants and the vast majority of hlh cases in adults are acquired, it should be stressed that, albeit rarely, fhl can occur in adulthood, including older individuals (so-called late-onset fhl) (3–5). therefore, the possibility of late-onset fhl in adults with hlh cannot be univocally excluded before tests of nk/t cell degranulation and activity, as well as genetic testing, have been performed. a retrospective study from japan revealed that the frequency of different forms of hlh in adults varied depending on the age bracket (6). among hlh patients aged 15–29 years, i-hlh was the most common (68% of cases). it was caused in equal parts by ebv-hlh (34%) and i-hlh other than ebvhlh (34%). in this age group, the second most common cause of hlh was a-hlh (22%), followed by m-hlh (10%). in the group of patients aged 30–59 years, m-hlh occurred only slightly less frequently than i-hlh (37% and 47%, respectively), followed by a-hlh (9%) and hlh after hematopoietic stem cell transplantation (7%). in the group of patients aged ‡60 years, however, malignancy was the most frequent cause of hlh (68%), followed by i-hlh (26%) and a-hlh (6%) (6). a recent, retrospective, population-based study from our group showed the annual incidence of mhlh in adults to be 1:280,000 per year, or 0.36/ 100,000 individuals per year (7). the results of this study were limited by the small population of the swedish region of northern halland but were strengthened by the long observation period of over 14 years. although m-hlh in east asia is most often associated with nk/t cell lymphoproliferative malignancies, m-hlh in europe tends to develop more frequently in the course of the hematological malignancies (6,7). importantly, it is worth to remember that m-hlh can occur before or during the treatment of known malignancy, or as the first manifestation of an occult malignancy (7,8). therefore an episode of hlh with unclear etiology in the older individual should always serve as a warning signal for a yet undiagnosed malignancy, leading to further careful cancer diagnostics and follow-up, as was correspondence: maciej machaczka, md, phd, hematology center karolinska, m54, karolinska university hospital huddinge, se-141 86 stockholm, sweden. fax: +46 8 7748725. e-mail: maciej.machaczka@ki.se (received 19 march 2013; accepted 10 april 2013) issn 0300-9734 print/issn 2000-1967 online � 2013 informa healthcare doi: 10.3109/03009734.2013.795634 done in the case reported by antonodimitrakis and colleagues. the patient reported by antonodimitrakis et al. underwent repeated biopsies, which failed to show hemophagocytosis in affected organs (1). the diagnosis of hlh can be confirmed in some cases by cytological and/or histopathological evaluation of bone-marrow, spleen, liver, lymph nodes, and cerebrospinal fluid (8,9). cytohistological examination reveals accumulation of lymphocytes and histiocytes (macrophages), sometimes with hemophagocytic activity (figure 1). although examination of bonemarrow slides has the highest sensitivity for the detection of hlh, approximately 20% of patients lack features of hlh on their initial bone-marrow examination (9). hemophagocytic activity is a rather late sign of advanced hlh (7). therefore, further search for hemophagocytic activity of macrophages is encouraged if hemophagocytosis is not proven at the time of hlh onset (8). if the bone-marrow specimen is not conclusive, serial marrow aspirates over time may be helpful as well as a biopsy obtained from other organs (8). all forms of hlh, even when treated in a timely manner, can be fatal. m-hlh has the worst prognosis compared to other forms of hlh (2,6,7). ishii et al. found that 5-year survival reaches only 12% in patients with m-hlh associated with t/nk cell non-hodgkin lymphoma (nhl), 48% in patients with m-hlh associated with b cell nhl, 54% in patients with fhl, and 83%–90% in patients with either i-hlh or a-hlh (6). the therapy of any form of hlh should focus on: 1) suppression of the hyperinflammatory status by destruction of activated cd8+ t lymphocytes and macrophages, and 2) treatment of any existing hlh triggers (2,8). in cases of fhl, an additional aim is the correction of the underlying immune defect by allogeneic stem cell transplantation (allo-sct) (9). treatment of acquired hlh is not standardized so far and remains highly variable across clinical centers. hlh treatment categories include: 1) proapoptotic chemotherapy with etoposide (50–150 mg/m2/dose i. v.), and 2) immunosuppressive drugs, targeting the hyperactivated macrophages (e.g. etoposide, corticosteroids, intravenous immunoglobulin (ivig)) and t cells (e.g. corticosteroids, cyclosporine a (cya)) (2,8). obviously, if possible, treatment of any existing trigger of hlh is mandatory (8). antonodimitrakis et al. successfully treated their patient with corticosteroids only (1). it may be a sufficient approach to employ corticosteroids (with or without ivig) to control hyperinflammation as a frontline therapy in i-hlh and a-hlh (particularly of milder grades) (2). after the improvement of the complete blood count and resolution of coagulopathy, steroids are slowly tapered down to avoid relapses of hlh. corticosteroid-resistant non-responders may benefit from second-line therapies, such as cya (2). if there is no response to the aforementioned drugs (corticosteroids, ivig, cya), use of the hlh-2004 protocol including etoposide is recommended (2,7,8). thus, patients with acquired hlh could be started on therapy without etoposide, as long as treatment adjustments are made rapidly in refractory cases. if hlh is driven by ebv infection, monoclonal anti-cd20 antibodies (i.e. rituximab) that deplete b lymphocytes, the predominant type of cells harboring ebv virus, should be used (10). anecdotal reports have also shown the efficacy of allo-sct in refractory or recurrent acquired hlh (e.g. ebv-hlh, m-hlh) (11,12). since hlh can be encountered by various specialists in the medical field, knowledge of this entity and its diagnostic criteria should be familiar to all physicians. treatment of hlh is difficult, long-lasting, and often associated with a high morbidity and mortality. the article by antonodimitrakis and colleagues is a valuable voice in the debate over adult hlh. declaration of interest: the author reports no conflicts of interest. the author alone is responsible for the content and writing of the paper. references 1. antonodimitrakis p, wassberg c, gerovasileiou s, back j, hällgren r, olsen b. fulminant hemophagocytic lymphohistiocytosis secondary to a reactivated ebv infection: a case report. ups j med sci. 2013;118:42–5. figure 1. may–grünwald–giemsa stain of bone marrow aspirate smear showing four macrophages displaying massive hemophagocytic activity (magnification �400). 202 m. machaczka 2. machaczka m, sydor w, rucińska m, szostek m, musiał j. autoimmune-associated hemophagocytic syndrome/macrophage activation syndrome. in: fang-ping huang, editor. autoimmune disorders—current concepts and advances from bedside to mechanistic insights. rijeka: intech— open access publisher; 2011. p 79–104; isbn 978-953307-653-9. 3. nagafuji k, nonami a, kumano t, kikushige y, yoshimoto g, takenaka k, et al. perforin gene mutations in adult-onset hemophagocytic lymphohistiocytosis. haematologica. 2007;92:978–81. 4. zhang k, jordan mb, marsh ra, johnson ja, kissell d, meller j, et al. hypomorphic mutations in prf1. munc134, and stxbp2 are associated with adult-onset familial hemophagocytic lymphohistiocytosis. blood. 2011;118: 5794–8. 5. sieni e, cetica v, piccin a, gherlinzoni f, sasso fc, rabusin m, et al. familial hemophagocytic lymphohistiocytosis may present during adulthood: clinical and genetic features of a small series. plos one. 2012;7:e44649. 6. ishii e, ohga s, imashuku s, yasukawa m, tsuda h, miura i, et al. nationwide survey of hemophagocytic lymphohistiocytosis in japan. int j hematol. 2007;86:58–65. 7. machaczka m, vaktnäs j, klimkowska m, hägglund h. malignancy-associated hemophagocytic lymphohistiocytosis in adults: a retrospective population-based analysis from a single center. leuk lymphoma. 2011;52:613–19. 8. henter ji, horne a, arico m, egeler rm, filipovich ah, imashuku s, et al. hlh-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. pediatr blood cancer. 2007;48:124–31. 9. henter ji, samuelsson-horne a, arico m, egeler rm, elinder g, filipovich ah, et al. treatment of hemophagocytic lymphohistiocytosis with hlh-94 immunotherapy and bone marrow transplantation. blood. 2002;100:2367–73. 10. balamuth nj, nichols ke, paessler m, taechey dt. use of rituximab in conjunction with immunosuppressive chemotherapy as a novel therapy for epstein-barr virus-associated hemophagocytic lymphohistiocytosis. j pediatr hematol oncol. 2007;29:569–73. 11. ohga s, kudo k, ishii e, honjo s, morimoto a, osugi y, et al. hematopoietic stem cell transplantation for familial hemophagocytic lymphohistiocytosis and epsteinbarr virus-associated hemophagocytic lymphohistiocytosis in japan. pediatr blood cancer. 2010;54:299–306. 12. machaczka m, nahi h, karbach h, klimkowska m, hägglund h. successful treatment of recurrent malignancyassociated hemophagocytic lymphohistiocytosis with a modified hlh-94 immunochemotherapy and allogeneic stem cell transplantation. med oncol. 2012;29:1231–6. hemophagocytic lymphohistiocytosis 203 sups_a_523801 47..51 upsala journal of medical sciences. 2011; 116: 47–51 original article a limited sampling strategy for estimation of the area under the curve (0 to 8 hours) of mycophenolic acid administered three times daily to liver transplant recipients bernardino marcos, lorena bouzas & j. carlos tutor unidad monitorización fármacos, laboratorio central, hospital clínico universitario, instituto de investigación sanitaria (idis), santiago de compostela, spain abstract objectives. gastrointestinal side-effects caused by mycophenolic acid (mpa) are frequent in liver transplant recipients, and in these cases a switch from two to three daily doses is usually recommended. however, a limited sampling strategy for the estimation of mpa area under the curve from 0 to 8 hours (auc(0–8h)) has not been made. design and methods. in 22 liver transplant patients who were administered mpa three times daily, the trapezoidal extrapolated mpa auc(0–8h) values using a sampling time from 0 to 2 hours were calculated. results. a tentative therapeutic range for mpa auc(0–8h) of about 20–40 mg.h/ml is proposed, and in the 13 patients with supratherapeutic values the total leukocyte blood count was significantly lower than in the 9 patients with auc(0–8h) £ 40 mg.h/ ml (p < 0.001). significant negative correlations were found between the total leukocyte blood count and the mpa trough levels (r = �0.458; p < 0.05), auc(0–8h) (r = �0.479; p < 0.05), and auc(0–2h) (r = �0.437; p < 0.05). a significant correlation was found between the trapezoidal extrapolated auc(0–8h) and trapezoidal auc(0–2h) results (r = 0.850; p < 0.001). conclusions. the trapezoidal extrapolated auc(0–8h), and possibly trapezoidal auc(0–2h), may be useful for routine therapeutic mpa monitoring in liver transplant recipients in which the dosing frequency is increased from twice to three times a day. key words: area under the curve, dosing frequency increase, drug exposure, gastrointestinal side-effects, mycophenolic acid introduction mycophenolic acid (mpa) is the active constituent of mycophenolate mofetil (mmf), which is a common component of the immunosuppressive regimens following transplantation. with the increasing use of mpa, the need for more accurate drug dosage has become evident, and the current scientific evidence for its concentration-controlled dosing in solid organ transplantation has been recently reviewed (1). mpa is usually administered in two daily doses, and the full mpa area-under-the-curve (auc) from 0 to 12 hours (auc(0–12h)) is considered the best measure of overall drug exposure; however, it is an impractical monitoring strategy for everyday clinical use (1). limited sampling from the first few hours post-dose makes it possible to predict the full mpa auc, and the most promising results to date regarding therapeutic mpa monitoring come from these limited sampling strategies (1,2). the majority of proposed limited-sampling strategies for estimating mpa auc(0–12h) was developed for kidney transplant recipients, and few algorithms for liver transplant patients are included by bruchet and emson in their recent systematic review (3). the side-effects of mpa mainly involve the gastrointestinal tract (diarrhoea, nausea, abdominal discomfort) and bone-marrow (leucopenia, thrombocytopenia), but do not normally require the immunosuppressive agent to be changed (1,4). diarrhoea and vomiting have a high prevalence among liver transplant patients receiving mmf orally (1,5), and correspondence: j. carlos tutor, pharmd, phd, unidad monitorización fármacos, laboratorio central, hospital clínico universitario, 15706 santiago de compostela, spain. fax: +34 981 950370. e-mail: josecarlostutor@redfarma.org; jocatuva@hotmail.com (received 11 august 2010; accepted 9 september 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.523801 these side-effects usually respond to dose reduction or switching from two to three divided daily doses (5). however, at least to our knowledge, a simplified sampling time profile for the estimation of mpa auc from 0 to 8 hours (auc(0–8h)) in liver transplant recipients treated with mmf three times daily has not been previously reported. the routine application of abbreviated-sampling time strategies to estimate mpa auc is feasible, if all samples can be taken within a 2-hour window (6). the aim of our study was to establish a trapezoidal extrapolated auc(0–8h) using a sampling time of 0 to 2 hours for the therapeutic mpa monitoring in liver transplant recipients, who were administered mpa every 8 hours due to gastrointestinal drug sideeffects using the typical daily dose. the results of the trapezoidal extrapolated auc(0–8h) simplified strategy were compared with those of the trapezoidal auc from 0 to 2 hours (auc(0–2h)), which has been recently used for therapeutic drug monitoring of mpa administered in two daily doses to liver transplant patients (7). patients and methods mpa levels were monitored in 22 maintenance liver transplant recipients (17 males and 5 females) with a mean (± sem) age of 58.1 ± 2.4 years, receiving daily oral treatment with mmf (cellcept�, hoffman la roche, basel, switzerland) at an 8-hour interval in monotherapy (n = 6), or together with cyclosporin (n = 4) or tacrolimus (n = 12). in all of the cases, the reason for switching the mmf dosage from two to three divided daily doses was due to mpa sideeffects involving the gastrointestinal tract (diarrhoea and nausea). after the mpa steady-state was achieved, blood samples were taken in bd vacutainer� tubes containing k3edta as anticoagulant, immediately before the next dose of mmf (c0), and half an hour (c0.5) and 2 hours (c2) post-dose. this study was carried out according to the good practice rules for investigations in humans of the conselleria de sanidade (regional ministry of health) of the xunta de galicia, spain. mpa plasma concentrations were determined in duplicate using the emit� 2000 mycophenolic acid assay in a dimension� xpand plus analyzer (siemens healthcare diagnostics inc., newark, de, usa). in accordance with the procedure developed in kidney transplant recipients by hale et al. (8), the auc from 0 to 2 hours (auc(0–2h)) was calculated using the linear trapezoidal rule, and the auc values were extrapolated from 0 to 8 hours (auc(0–8h)) considering the mathematically estimated concentrations at 6 (c6) and 8 (c8) hours. according to the characteristics of the mpa concentration-time profiles in liver transplant recipients (9), the c6 and c8 concentrations were calculated using the expressions: c6 = 1.25c0 + 0.15, and c8 = c0. the blood counts of total, polymorphonuclear (pmn) and mononuclear (mn) leukocytes, and platelets were carried out in an advia� 2120 hematology system from siemens healthcare diagnostics inc. statistical analysis of the data was carried out using the microsoft excel (v. 5.0) package, and the kolmogorov-smirnov test was applied to check for normality. mpa levels and auc data had gaussian distributions, and consequently pearson’s correlation coefficient, linear regression, and sy.x as measure of dispersion, were used. in other cases the spearman’s correlation coefficient was used. the results are expressed as mean ± sem (median). results the generally considered therapeutic window for mpa auc(0–12h) is 30–60 mg.h/ml (10,11), and consequently, for the maintenance of an analogous daily drug exposure (cumulative 24 hours auc), a tentative therapeutic interval for mpa auc(0–8h) of around 20–40 mg.h/ml may be proposed. the relationship between the trapezoidal extrapolated mpa auc(0–8h) and the trapezoidal auc(0–2h) values is shown in figure 1, and, in accordance with the linear regression equation, the estimated therapeutic range for auc(0–2h) may be about 5–14 mg.h/ml. in the 22 patients studied the mean trapezoidal extrapolated mpa auc(0–8h) was 49.7 ± 4.9 mg.h/ml (range 16.5–92.2 mg.h/ml), and in 13 cases the values were supratherapeutic (>40 mg. h/ml). for the mpa trapezoidal auc(0–2h) a mean value of 18.9 ± 2.0 mg.h/ml (range 7.3–37.9 mg.h/ml) was obtained, and also in 13 cases the values were supratherapeutic (>14 mg.h/ml). however, a modest concordance was observed in the classification of auc(0–2h) and auc(0–8h) values as subtherapeutic, therapeutic or supratherapeutic (figure 1). significant correlations were found between the trapezoidal extrapolated mpa auc(0–8h) and c0 (auc(0–8h) = 9.79c0 + 16.24; r = 0.909; p < 0.001; sy.x=9.9mg.h/ml),c0.5(auc(0–8h)=1.77c0.5+27.38; r = 0.636; p < 0.01; sy.x = 18.3 mg.h/ml), and c2 (auc(0–8h) = 4.42c2 + 18.20; r = 0.753; p < 0.001; sy.x = 15.6 mg.h/ml). similarly, significant correlations were also found between the trapezoidal mpa auc(0–2h) and c0 (auc(0–2h) = 3.03c0 + 8.50; r = 0.689; p < 0.001; sy.x = 6.99 mg.h/ml), c0.5 (auc(0–2h) = 1.06c0.5 + 5.41; r = 0.939; 48 b. marcos et al. p < 0.001; sy.x = 3.31 mg.h/ml), and c2 (auc(0–2h) = 0.99c2 + 11.78; r = 0.415; p » 0.05; sy.x = 8.78 mg.h/ml). with respect to the 13 cases with trapezoidal extrapolated mpa auc(0–8h) > 40 mg.h/ml, in the 9 cases with trapezoidal extrapolated mpa auc(0–8h) £ 40 mg. h/ml the mpa c0 level (1.7 ± 0.2 mg/ml (1.9 mg/ml) versus 4.9 ± 0.5 mg/ml (4.4 mg/ml)) was significantly lower (p < 0.001), and the total leukocyte blood count (7504 ± 687/ml (7600/ml) versus 5131 ± 438/ml (4930/ml)) was significantly higher (p < 0.05). however, for the blood count of pmn, and mn leukocytes, and platelets, the differences were not significant. table i shows the correlation coefficients of the blood count of total, pmn, and mn leukocytes, and platelets with the mpa c0, c0.5, and c2 levels, and auc(0–8h) and auc(0–2h). discussion it has been suggested that concentrations of mpa in the immediate post-dose period may not accurately correlate to total drug exposure, because these concentration–time points do not capture a second peak concentration due to the drug reabsorption into circulation after its biliary excretion (2). however, substantial research has been conducted to develop limited-sampling strategies to estimate mpa auc in kidney, heart, liver, and lung transplantation, and it seems that concentrations in the immediate postdose period can accurately predict total mpa auc(0–12h) (3). the clinical usefulness of the mmf original dosing protocol change from two to three times daily, overcoming the disadvantage of short mpa half-life and improving immunosuppression in haematopoietic cell transplantation, has been the subject of discussion (12–14). with respect to solid organ transplant recipients, treating physicians usually increase the dosing frequency to three times a day if mpa gastrointestinal side-effects are present (5). in these clinical conditions, it may be important to have a simplified strategy available for the mpa auc(0–8h) estimation applicable to the routine drug exposure monitoring. at present, the selection of appropriate target ranges for mpa auc(0–12h) is challenging, as the range of 30–60 mg.h/ml was based on the use of mmf in the early post-renal transplant period with concomitant cyclosporin (3). similarly, the analytical method used for the mpa determination should be considered, as the results produced by the enzyme multiplied enzyme immunoassay (emit) are higher than those of the chromatographic-based techniques, because the antibody used in the immunoassay has cross-reactivity with the pharmacologically active mpa acyl glucuronide metabolite (1,3). in any case, we considered the therapeutic range of 30–60 mg.h/ml for mpa auc(0–12h) due to its wide acceptance (7,10–12,15,16). consequently, for the maintenance of an analogous daily drug exposure, a therapeutic range of 20–40 mg.h/ml may be proposed for the mpa auc(0–8h). the increase of the mmf dosing frequency could statistically maintain higher mpa trough levels and steady-state concentrations (auc/dosing interval) (13), and in 13 cases (59%) 0 20 40 60 80 100 120 0 20 40 60 auc (0-2h)t (µg.h/ml) a u c (0 -8 h )t ( µg .h /m l ) y = 2.09 x + 10.41 r = 0.850 syx = 12.48 figure 1. correlation and regression between the trapezoidal mycophenolic acid (mpa) auc(0–2h) and trapezoidal extrapolated mpa auc(0–8h) in liver transplant recipients treated three times daily with mycophenolate mofetil (mmf) in monotherapy (d), or co-medicated with cyclosporin (.) or tacrolimus (*). the dashed lines correspond to the tentative therapeutic ranges for mpa auc(0–2h) and auc(0–8h). table i. correlation coefficients of the blood count of total, polymorphonuclear (pmn), and mononuclear (mn) leukocytes, and platelets with mycophenolic acid levels and auc(0–2h) and auc(0–8h) (n = 22). total leukocytes pmn mn platelets c0h �0.458 (p = 0.032) �0.399 (p = 0.066) �0.449 (p = 0.036) 0.197 (p = 0.379) c0.5h �0.364 (p = 0.096) �0.201 (p = 0.371) �0.328 (p = 0.136) 0.351 (p = 0.109) c2h �0.312 (p = 0.157) �0.374 (p = 0.086) 0.028 (p = 0.903) 0.020 (p = 0.931) auc(0–2h) �0.437 (p = 0.042) �0.324 (p = 0.141) �0.295 (p = 0.183) 0.238 (p = 0.286) auc(0–8h) �0.479 (p = 0.024) �0.419 (p = 0.052) �0.299 (p = 0.176) 0.205 (p = 0.360) mycophenolic acid area under the curve from 0 to 8 hours 49 the trapezoidal extrapolated mpa auc(0–8h) values were supratherapeutic, suggesting the suitability of a drug dosage adjustment. a modest concordance was found between subtherapeutic, therapeutic, and supratherapeutic auc(0–8h) and auc(0–2h) values (figure 1). co-administration of other immunosuppressant agents may influence mpa exposure, as is clearly shown for cyclosporin with 30%–40% lower dose-normalized mpa exposure (1). however, in the group of patients studied, concomitant cyclosporin administration does not appear to modify significantly the relationship between the mpa auc(0–2h) and auc(0–8h) (figure 1). therapeutic monitoring of mpa in liver transplantation has predominantly used the trough concentration (c0), although additional auc data exist in some studies, suggesting an acceptable correlation of c0 with auc(0–12h) (1) and auc(0–2h) (7). in our study, the higher correlation coefficients were found between mpa c0 and auc(0–8h) (r = 0.909; p < 0.001), and c0.5 and auc(0–2h) (r = 0.939; p < 0.001). it has been reported that in liver transplant recipients treated with two daily doses of mmf, the mpa pharmacokinetic parameters, c0, cmax, and auc(0–12h), were significantly higher in patients with side-effects than in those with an uneventful outcome (16). however, shin et al. (7) recently described no significant relationships among mpa c0 levels or auc(0–2h) with the degree of leucopenia, diarrhoea, or infection. in our group of patients with mpa auc(0–8h) >40 mg.h/ml, the blood count of total leukocytes was significantly lower than in the group of patients with auc(0–8h) £40 mg.h/ml (p < 0.05). similarly, significant negative correlations between the blood count of total leukocytes, with the mpa trough levels, auc(0–8h), and auc(0–2h) were found (table i). regional concentrations of mpa in gastrointestinal epithelial cells, not reflecting systemic exposure, may contribute to undesirable gastrointestinal events (16). in accordance with brunet et al. (17), gastrointestinal side-effects of mpa in liver transplant recipients were associated with high drug levels obtained 40 min after dose, but not with auc(0–12h) or c0. these results may explain the beneficial effect of the increase of mpa dosing frequency, maintaining an analogous daily drug exposure, on the diarrhoea, nausea, and vomiting. trapezoidal extrapolated auc(0–8h) may be used for the routine mpa exposure monitoring in liver transplant recipients treated with this drug three times daily. further studies on a larger number of transplant patients are necessary for the evaluation of the clinical usefulness of the trapezoidal mpa auc(0–2h) in comparison with the estimated auc(0–8h) or auc(0–12h) using limited sampling strategies. acknowledgements we would like to thank to dr s. tomé from the gastroenterology unit for his collaboration. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. kuypers drj, le meur y, cantarovich m, tredger mj, tett se, cattaneo d, et al. consensus report on therapeutic drug monitoring of mycophenolic acid in solid organ transplantation. clin j am soc nephrol. 2010;5:341–58. 2. knight sr, morris pj. does evidence support the use of mycophenolate mofetil therapeutic drug monitoring in clinical practice? a systematic review. transplantation. 2008;85: 1675–85. 3. bruchet nk, emson mhh. limited sampling strategies for mycophenolic acid in solid transplantation: a systematic review. expert opin drug metab toxicol. 2009;5:1079–97. 4. kamphues c, bova r, röcken c, neuhaus r, pratschke j, neuhaus p, et al. safety of mycophenolate monotherapy in patients after liver transplantation. ann transplant. 2009;14: 40–6. 5. drugdex� evaluations. mycophenolic acid. available at: www.micromedex.com/products/drugdex (accessed june 9, 2010). 6. van gelder t, saw lm. the rationale for and limitations of therapeutic drug monitoring of 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c, baiyong s, weixia z, chuan s, et al. monitoring mycophenolic acid pharmacokinetic parameters in liver transplant recipients: prediction of occurrence of leukopenia. liver transpl. 2008;14: 1165–73. 17. brunet m, cirera i, martorell j, vidal e, millán o, jiménez o, et al. sequential determination of pharmacokinetics and pharmacodynamics of mycophenolic acid in liver transplant patients treated with mycophenolate mofetil. transplantation. 2006;81:541–6. mycophenolic acid area under the curve from 0 to 8 hours 51 prevalence and natural history of and risk factors for subaneurysmal aorta among 65-year-old men article prevalence and natural history of and risk factors for subaneurysmal aorta among 65-year-old men knut thorbjørnsena,b, sverker svensj€oa,c, khatereh djavani gidlunda,b, nils-peter gilgend and anders wanhainena adepartment of surgical sciences, section of vascular surgery, uppsala university, uppsala, sweden; bcentre for research and development, uppsala university/county council of g€avleborg, g€avle, sweden; cdepartment of surgery, falun county hospital, falun, sweden; ddepartment of surgery, eskilstuna county hospital, eskilstuna, sweden abstract background: the aims of this study were to determine the prevalence of screening-detected subaneurysmal aorta (saa), i.e. an aortic diameter of 2.5–2.9 cm, its associated risk factors, and natural history among 65-year-old men. methods: a total of 14,620 men had their abdominal aortas screened with ultrasound and completed a health questionnaire containing information on smoking habits and medical history. they were categorized based on the aortic diameter: normal aorta (<2.5 cm; n ¼ 14,129), saa (2.5–2.9 cm; n ¼ 258), and abdominal aortic aneurysm (aaa) (�3.0 cm; n ¼ 233). the saa-group was rescanned after 5 years. associated risk factors were analyzed. results: the saa-prevalence was 1.9% (95% confidence interval 1.7%–2.1%), with 57.0% (50.7%–63.3%) expanding to �3.0 cm within 5 years. frequency of smoking, coronary artery disease, hypertension, hyperlipidemia, and claudication were significantly higher in those with saa and aaa compared to those with normal aortic diameter. current smoking was the strongest risk factor for saa (odds ratio [or] 2.8; p < 0.001) and even stronger for aaa (or 3.6; p < 0.001). men with saa expanding to aaa within 5 years presented pronounced similarities to aaa at baseline. conclusions: men with saa and aaa presented marked similarities in the risk factor profile. smoking was the strongest risk factor with an incremental association with disease severity, and disease progression. this indicates that saa and aaa may have the same pathophysiological origin and that saa should be considered as an early stage of aneurysm formation. further research on the cost-effectiveness and potential benefits of surveillance as well as smoking cessation and secondary cardiovascular prevention in this subgroup is warranted. article history received 17 june 2019 revised 16 july 2019 accepted 22 july 2019 keywords abdominal aortic aneurysm; prevention and control; screening; smoking; subaneurysmal aorta; ultrasonography introduction screening elderly men with ultrasound (us) for abdominal aortic aneurysm (aaa) is recommended by several randomized controlled trials (rcts) (1–4). these studies have demonstrated an approximately 50% reduction in aaa mortality from us-based screening. the largest and most influential rct, the multicentre aneurysm screening study (mass), also demonstrated a reduction in all-cause mortality among those who underwent screening (5). based on these findings, national screening programs have been implemented in sweden, england, and the united states (6–9). the evidence for aaa screening in women is still insufficient; therefore, population-based aaa screening programs for women are not currently implemented in sweden (10). there is an ongoing debate regarding the threshold aortic diameter for continued surveillance (11,12). most screening programs define the minimum aortic diameter for an aneurysm as �3.0 cm and exclude those with aortic diameters below this threshold from further surveillance, whereas some also include individuals with aortic diameters in the range of 2.5–2.9 cm, also called subaneurysmal aortas (saas), in the surveillance program (12). the results from several contemporary studies suggest that this subgroup should be classified as an ‘aneurysm in formation’ and should be treated as such (7,10–13). observational studies indicate that >50% of those with an saa develop a true aaa within 5 years after the initial scan (14–16). more importantly, a considerable proportion of these individuals reach the threshold for surgical intervention within 10–15 years of initial screening (15,16) at an age where they could still benefit from elective aaa repair. the association between aaa and risk factors such as smoking, cardiovascular disease, hypertension, and hyperlipidemia, as well as that between aaa and increased all-cause mortality, is well documented in numerous population-based studies (17–20). there is also evidence indicating that individuals with an aortic diameter between 2.5 cm and 2.9 cm have significantly higher rates of all-cause mortality than contact knut thorbjørnsen knut.thorbjornsen@regiongavleborg.se centre for research and development, uppsala university/county council of g€avleborg, 80188 g€avle, sweden � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 3, 180–186 https://doi.org/10.1080/03009734.2019.1648611 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1648611&domain=pdf&date_stamp=2019-09-09 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2019.1648611 http://www.tandfonline.com those with an aortic diameter <2.5 cm (15,19–24). the risk factors for men with saa have, however, been rather sparsely documented (15). with the implementation of large-scale screening programs, an increasing number of subjects with saa will be detected, and there is clearly a need for more knowledge regarding this subgroup of individuals, as well as regarding the associated risk factors and morbidity. the aims of this study were to determine the saa prevalence among 65-year-old men in middle sweden, to document associated risk factors, and to assess the degree of comorbidities compared with men with aaas and normal aortas. in addition, we determined the natural history of saas after 5 years of surveillance. methods a population-based screening program for aaa among 65year-old men was introduced in the county of uppsala in 2006, and similar programs were gradually launched in the neighboring counties of dalarna, s€ormland, and g€avleborg (figure 1). between 2006 and 2010, all 65-year-old men in the four counties were identified through the national population registry and invited to undergo an us examination of the abdominal aorta. subjects with a history of aaa repair or those who were under surveillance for a known aaa were excluded from invitation. no other exclusion criteria were used. the us examinations were centralized to one hospital in each county with the exception of one county (g€avleborg), where screening was offered at two hospitals. all us examinations were conducted by registered nurses who were specially trained in ultrasonography or ultrasound technicians. all subjects had a single us scan with measurement of the maximum anteroposterior diameter of the infrarenal aorta using the leading-edge-to-leading-edge (lele) method (25). the patients were divided into three groups based on the maximum aortic diameter. an aaa was defined as a maximum aortic diameter of �3.0 cm, an saa was defined as a maximum aortic diameter of 2.5–2.9 cm, and a normal aorta was defined as a maximum aortic diameter <2.5 cm. of those invited, 14,678 subjects were asked to complete a standardized health questionnaire containing questions regarding first-degree relatives with aaa, family and medical history, smoking status (classified as never, former, and current smokers and smoking duration), coronary heart disease (defined as angina pectoris and/or myocardial infarction), diabetes mellitus (including dietary or medical treatment), cerebrovascular disease (transient ischemic attack or stroke), hypertension, hyperlipidemia, claudication, renal failure, and chronic obstructive pulmonary disease (copd). men diagnosed with saa were followed up at 70 years of age with an us of the abdominal aorta as part of a regional protocol. eight men underwent a complementary computed tomography (ct) scan because of an incomplete us scan or iliac aneurysms. using the swedish vascular registry (swedvasc), a nationwide registry with high internal and external validity (26), all men who had already been treated for an aaa were identified and excluded. men under surveillance for a known aaa were identified from local hospital registries. statistical analyses were performed with ibm spss statistics software version 24.0 (ibm, armonk, ny, usa). for comparisons of continuous data, the independent samples t test was used. an uncorrected chi-square test was used to compare three proportions according to associated risk factors. proportions are presented with 95% confidence intervals (ci). to estimate the odds ratio (or) for risk factors associated with saa, the variables with p < 0.1 in a univariate analysis were entered into a multivariable logistic regression model. a value of p < 0.05 was considered statistically significant. the study was performed according to the principles of the declaration of helsinki and was approved by the ethics figure 1. map of sweden showing the geographical area of the four counties in middle sweden. the uptake area comprises: (a) uppsala, population 367,483; (b) g€avleborg, population 285,452; (c) dalarna, population 281,046; (d) s€ormland, population 290,711. population numbers from 2017. upsala journal of medical sciences 181 committee of the uppsala/€orebro region (dnr 2006:112 and dnr 2018/099). as specified by the ethics committee, informed consent was not required. results between 2006 and 2010, a total of 21,938 men were invited, of whom 18,361 were screened (83.7% attendance). of those, 18,317 (99.8%) had an appropriate us measurement and were included in the analysis. twenty-four men were already under surveillance for known aaas, and 62 living men had previously undergone aaa repair. a total of 347 saas were detected (1.9%; 95% ci, 1.7%–2.1%), and 316 men had an aaa (1.7%; 95% ci, 1.5%–1.9%). a normal aorta was observed in 17,654 men (96.4%; 95% ci, 96.2%–98.6%). of 14,678 distributed health questionnaires, 14,620 were completed (99.6%), which formed the cohort for further analyses. the following groups were based on the maximum anteroposterior diameter: normal aorta (<2.5 cm; n ¼ 14,129), saa (2.5–2.9 cm; n ¼ 258), and aaa (�3.0 cm; n ¼ 233) (figure 2). current or former smoking status with a longer smoking duration, coronary artery disease, hypertension, hyperlipidemia, and claudication were substantially more frequently identified among those with saa than among those with a normal aortic diameter. notably, smoking duration was considerably longer in the aaa group than in the saa group (p < 0.001) (table 1); otherwise, the risk factor profile was very similar between the two groups. the distribution of risk factors associated with saa and aaa compared with those associated with a normal aorta is shown in table 1. a multivariable logistic regression analysis (table 2) identified current and ever smoking status, coronary artery disease, hypertension, hyperlipidemia, and claudication as independent risk factors for saa, of which current smoking status yielded the highest or (or 2.8; 95% ci, 2.1–3.7; p < 0.001). in the aaa group, current smoking had a higher or than in the saa group (or 3.5; 95% ci, 2.7–4.7; p < 0.001), and coronary artery disease, hypertension, and hyperlipidemia were also independently associated with aaa. of the 258 individuals with an saa at baseline screening (figure 3), a total of 14 (5.4%; 95% ci, 2.6–8.2) died of nonaaa-related causes within 5 years. a total of six men declined follow-up, and one man moved abroad. one man had undergone elective aaa repair for a large iliac aneurysm and a 4.5-cm aaa after 4.5 years of follow-up and was included in the attenders. a total of 237 (91.9%; 95% ci, 88.6%–95.2%) men were rescanned at 70 years of age. among those re-examined at age 70, 13 (5.5%; 95% ci, 2.6%–8.4%) men had aortic diameters less than 2.5 cm. eighty-nine (37.5%; 95% ci, 31.3%–43.75%) men were classified as still having an saa (2.5–2.9 cm). a total of 135 (57.0%; 95% ci, 50.7%–63.3%) men had reached an aortic diameter of 3.0 cm or greater within 5 years after the initial scan. the annual mean expansion rate was 0.72 mm (95% ci, 0.60–0.84 mm). the frequency of smoking was consistently higher, the smoking duration was consistently longer (p < 0.001), and hyperlipidemia was more frequent figure 2. infrarenal aortic diameters. histogram presenting the distribution of the maximum infrarenal aortic diameter for the screened cohort of 65-year-old men. embedded is a selective histogram of the size distribution of infrarenal aortic diameters �25 mm. 182 k. thorbjørnsen et al. (p ¼ 0.031) among the subgroup of men with saa expanding to aaa within 5 years than among those with saa that did not expand to aaa. regarding the risk factor profile, this subgroup displayed marked similarities with the aaa subgroup at baseline screening (table 3). discussion in this cross-sectional population-based study, the prevalence of saa among 65-year-old men in middle sweden was 1.9%, which was similar to the proportion of men with aaa (1.7%). the prevalence of saa was slightly lower than that observed in other populations (11,13,15). it should be noted that 2.6 cm was used as the lower threshold for this particular subgroup in the gloucester study, with a prevalence of 2.0% compared to 2.5 cm in the present study (13). a higher prevalence was reported by duncan et al. (23), (8.2%), but that study included a wider age range of men (65–74 years). the main finding in the present study is the marked similarity in the risk factor profile of men with saa and men with aaa. the most important risk factor for both saa and aaa is smoking, with the highest or for current smoking, which was also associated with more extensive aortic pathology, especially among men with saa progressing to aaa within 5 years. the observed association between smoking and saa as well as between smoking and aaa is consistent with the findings of previous studies (14,17–19,22). the observed incremental association between smoking duration and disease severity in the present study strongly suggests a dose-response relationship. this indicates that saa and aaa may have the same pathophysiological origin and that saa should be considered as an early stage of aneurysm formation, as suggested by table 1. risk factors associated with subaneurysmal aorta (saa), abdominal aortic aneurysm, and normal aorta in 65-year-old men. risk factor normal aorta (n ¼ 14.129) p value subaneurysmal aorta (n ¼ 258) p value aaa (n ¼ 233) ever smoked 63.0% (62.2–63.8) <0.001 81.0% (76.2–85.8) 0.065 87.1% (82.8–91.5) current smoker 12.7% (12.2–13.3) <0.001 28.7% (23.1–34.2) 0.30 33.0% (27.0–39.1) smoke-years 15.7 (15.4–16.0) <0.001 24.8 (22.5–27.0) <0.001 30.6 (28.3–32.8) pack-years 10.7 (10.4–10.9) <0.001 17.2 (15.2–19.2) <0.001 23.7 (20.9–26.6) first-degree relative with aaa 1.4% (1.2–1.6) 0.22 2.3% (0.5–4.2) 0.39 1.3% (0.2–2.8) coronary artery disease 10.9% (10.4–11.4) <0.001 19.8% (14.9–24.7) 0.11 25.8% (20.1–31.4) hypertension 36.8% (36.0–37.6) <0.001 49.6% (43.5–55.8) 0.20 55.4% (48.9–61.8) hyperlipidemia 23.2% (22.6–23.9) <0.001 36.0% (30.2–41.9) 0.38 39.9% (33.6–46.3) cerebrovascular disease 4.5% (4.2–4.8) 0.002 8.5% (5.1–12.0) 0.72 9.4% (5.7–13.2) claudication 1.2% (1.1–1.4) <0.001 5.0% (2.4–7.7) 0.25 3.0% (0.8–5.2) copd 6.4% (6.0–6.8) 0.52 5.4% (2.6–8.2) 0.12 9.0% (5.3–12.7) diabetes mellitus 12.2% (11.6–12.7) 0.90 12.4% (8.4–16.5) 0.56 10.7% (6.7–14.7) renal insufficiency 0.9% (0.8–1.1) 0.82 0.8% (0.3–1.9) 0.18 0 aaa ¼ abdominal aortic aneurysm; copd ¼ chronic obstructive pulmonary disease. table 2. multivariable logistic regression analysis of covariables associated with subaneurysmal aorta (saa) and abdominal aortic aneurysm (aaa), with the normal aorta group as the reference category. risk factor saa aaa odds ratio 95% ci p value odds ratio 95% ci p value current smokera 2.8 2.1–3.7 <0.001 3.5 2.7–4.7 <0.001 ever smokeda 2.3 1.7–3.1 <0.001 3.6 2.4–5.3 <0.001 10 smoke-yearsa 1.3 1.2–1.4 <0.001 1.6 1.5–1.7 <0.001 10 pack-yearsa 1.2 1.1–1.3 <0.001 1.3 1.3–1.4 <0.001 coronary artery disease 1.4 1.0–2.0 0.04 2.0 1.4–2.7 <0.001 hypertension 1.3 1.0–1.7 0.03 1.6 1.2–2.1 0.001 hyperlipidemia 1.4 1.0–1.8 0.03 1.4 1.0–1.9 0.03 cerebrovascular disease 1.4 1.0–2.3 0.11 1.5 1.0–2.4 0.07 claudication 2.5 1.4–4.6 0.003 1.1 0.5–2.5 0.7 the covariables with p < 0.1 in the univariate analysis were included in the multivariable regression analysis. asmoking covariables were entered separately into the analysis. figure 3. flow chart of the saa cohort. �one man underwent elective aaa repair after 4.5 years of follow-up for a large iliac aneurysm and a 4.5-cm iaaa and was included among the attenders. aaa ¼ abdominal aortic aneurysm; iaaa ¼ intact abdominal aortic aneurysm. upsala journal of medical sciences 183 several authors (11–14,22,27). cohort and observational studies have shown that saa expands to a large extent to true aaa and may rupture over time (5,15,16). svensj€o et al. (14) showed in the uppsala cohort that an infrarenal aortic diameter of 2.5–2.9 cm and smoking were the most important risk factors for the development of aaa within 5 years. the presence of an saa at 65 years of age resulted in a 60-fold increased risk of aaa formation. a meta-analysis from the rescan collaborators concluded that smoking increased the yearly expansion rate of aaas by 35% and doubled the rupture risk. one limitation was that the data were strictly limited to aortic diameters of 3.0–5.4 cm (28). a negative correlation between smoking cessation and the progression of aaas has also been observed (29). smoking cessation is to date the only known effective interventions to prevent small aaas from further expansion and rupture and has been shown to be highly cost-effective (30,31). it is reasonable to assume that subjects with saa could also benefit from targeted smoking cessation programs, and smoking cessation strategies targeting this subgroup should be considered (32). this requires, however, that the saas are detected, which to a great extent is only possible through population screening. one of the consequences of implementing screening programs is the detection of a considerable number of small aaas and saas. there are still uncertainties and substantial variations in the surveillance recommendations for aortas with diameters of 2.5–2.9 cm (saa) (9). based on the design of the rcts, many screening programs consider this subgroup not to be significant and conclude that further surveillance or interventions are unnecessary (33,34), whereas others consider saas to be abnormal and recommend surveillance (11–14,22,27). the 2019 european society for vascular surgery (esvs) clinical practice guidelines on the management of abdominal aorto-iliac artery aneurysms issued a weak recommendation to re-screen men with a saa after 5–10 years (35). in this observational population-based study with a high attendance rate, we report on a cohort with screeningdetected saas, which presented a high rate of progression to true aaas after 5 years of follow-up. thirteen (5.5%) men with a previous saa were classified as having a normal aorta (<2.5 cm) at rescanning. six patients had an aortic diameter of 2.5 and 2.6 cm at baseline screening. the most likely explanation was that the aortic diameter was overor underestimated due to variability in the us measurement technique within the standard deviation (sd), i.e. limits of agreement. a recent study demonstrated that the lele method used in the present study has a variability of 2 mm (25). the remaining seven men were considered to have been misclassified due to measurement error at the baseline screening. in a multicenter observational study by wild et al. (15) that included 1696 individuals with saa from eight european screening programs, 67.7% of the subjects progressed to an aaa after 5 years of surveillance, and a total of 26.2% of this subgroup had reached the threshold for repair of �5.5 cm after 10 years of follow-up. similar findings were also evident in a swedish longitudinal cohort study in which 3268 men were rescanned 5 years after baseline screening at 65 years of age. in total, 52.2% of subjects with saa had progressed to aaa within 5 years (14). a recent publication from the gloucestershire aneurysm screening programme that included 1233 men with saa estimated that 57.6% would progress to aaa 5 years after the initial scan and that 28% would develop a large aaa by 80 years of age (16). longterm follow-up data from the mass randomized trial showed the occurrence of ruptures after 8 years among men initially screened as normal (<3 cm). more than 50% of these ruptures occurred among men with an aortic diameter of 2.5–2.9 cm at the baseline screening (5). ruptured aaa is, to date, more common in subjects older than 75 years of age, and the expectation for intervention in older patients has increased (7,36,37). rapid acquisition of minimally invasive technologies, such as endovascular aneurysm repair (evar), and the ability to intervene have also increased (38). thus, there is an urgent need for the development of evidencebased strategies as to whether the subgroup of individuals with saa should be monitored or not (39). this is even more relevant now that we see an ever-increasing longevity in the population (12,14,20). there is, however, only limited evidence regarding the clinical relevance and cost-effectiveness of surveillance of persons with saa (40). there are also table 3. risk factors for stable saa versus expanding saa during the 5-year follow-up. risk factor saa ! aorta <3.0 cm (95% ci) (n ¼ 102) saa ! aorta �3.0 cm (95% ci) (n ¼ 135) odds ratio (95% ci) p value ever smoked 74.5% (65.9–83.1) 85.2% (79.1–91.1) 1.97 (1.03–3.77) 0.040 current smoker 22.0% (13.0–30.0) 36.0% (27.0–44.0) 2.01 (1.11–3.62) 0.019 smoke-years 20.7 (17.5–23.9) 29.6 (26.6–32.6) 1.03 (1.02–1.05) <0.001 pack-years 15.1 (12.3–17.9) 22.1 (19.1–25.1) 1.03 (1.01–1.05) 0.002 first-degree relative with aaa 1.0% (0.0–2.9) 3.7% (0.5–6.9) 3.89 (0.45–33.78) 0.186 coronary artery disease 18.0% (10.0–25.0) 24.0% (16.0–31.0) 1.45 (0.76–2.76) 0.258 hypertension 46.0% (36.0–56.0) 53.0% (45.0–62.0) 1.34 (0.78–2.24) 0.269 hyperlipidemia 31.0% (22.0–41.0) 45.0% (37.0–50.0) 1.80 (1.05–3.09) 0.031 cerebrovascular disease 8.0% (3.0–13.0) 10.0% (5.0–16.0) 1.36 (0.55–3.38) 0.507 claudication 2.0% (0.0–5.0) 5.0% (1.0–9.0) 2.73 (0.56–13.55) 0.198 copd 5.0% (1.0–9.0) 4.0% (1.0–8.0) 0.90 (0.27–3.04) 0.868 diabetes mellitus 17.0% (9.0–24.0) 10.0% (5.0–15.0) 0.53 (0.25–1.16) 0.107 renal insufficiency 2.0% (0.0–5.0) 0 0.43 (0.37–0.49) 0.102 antiplatelet use 26.0% (1.8–35.0) 33.0% (25.0–41.0) 1.39 (0.79–2.45) 0.255 statin use 31.0% (22.0–41.0) 41.0% (33.0–50.0) 1.55 (0.90–2.66) 0.111 aaa ¼ abdominal aortic aneurysm; saa ¼ subaneurysmal aorta. 184 k. thorbjørnsen et al. psychological aspects that need to be evaluated, to ensure that monitoring of the saa does not do more harm than good. the implementation of population-based aaa screening programs in europe and the usa has coincided with a significant change in the epidemiology of the disease: 1) decreased incidence, mainly due to reduced smoking rates; 2) altered management, most importantly the introduction of evar with improved outcomes and more patients being offered treatment; and 3) increased longevity in the general population (12,14,20,39). model studies have demonstrated that the observed decrease in prevalence is counterbalanced by decreased perioperative mortality and increased longevity, resulting in an unchanged low cost per quality-adjusted lifeyear (qaly) gained (41). this conclusion was confirmed in a recent report from the swedish nationwide aaa screening program, showing a 27% reduction in aaa mortality after 10 years of screening, with an incremental cost-efficiency ratio of e7,770 per qaly gained, corresponding to e49,800 per life saved from rupture. although screening for aaa remains highly cost-effective in a contemporary setting, the effectiveness is less than previous calculations based on older rcts with a higher prevalence of the disease (12). an expanded surveillance program that includes follow-up of saa with smoking cessation and secondary cardiovascular prevention programs in this subgroup might have the potential to further improve the effectiveness of aaa screening programs but needs further evaluation regarding the longterm effects, including health-economy and aspects on quality of life. in conclusion, our findings demonstrated a marked similarity in the risk factor profile between men with saa and men with aaa. smoking was the most important risk factor, and there was an incremental association between smoking duration and disease severity, especially among men with saa progressing to aaa within 5 years. this finding indicates that saa and aaa may have the same pathophysiological origin and supports that saa should be considered an early stage of aneurysm formation. further research on the costeffectiveness and potential benefits of surveillance as well as smoking cessation and secondary cardiovascular prevention programs in this subgroup is warranted. acknowledgements the authors thank the collaborators linda lyttkens, rn, department of surgical sciences, section of vascular surgery, uppsala university, uppsala; ewa pihl, rn, department of surgery, falun county hospital, falun; eva ansgarius, bma, department of physiology, kullbergska hospital, katrineholm; christina sj€ostr€om, rn, department of surgery, g€avle county hospital, g€avle; and karina docter, rn, department of surgery, hudiksvall hospital, hudiksvall. disclosure statement the authors declare that there is no conflict of interest. funding funding was received from: department of surgical sciences, section of vascular surgery, uppsala university; centre for research and development, uppsala university/county council of g€avleborg, g€avle, sweden; and the swedish heart-lung foundation. notes on contributors knut thorbjørnsen, md, consultant general and vascular surgeon, department of surgery, g€avle county hospital, g€avle, sweden. phd student, centre for research and development , uppsala university/ county concil of g€avleborg, g€avle, sweden. department of surgical sciences, vascular surgery, uppsala university, uppsala, sweden. sverker svensj€o, md, phd, consultant general and vascular surgeon, head of vascular surgery, department of surgery, falun county hospital, falun. post-doc, centre for clinical reseearch (ckf), falun, sweden. department of surgical sciences, vascular surgery, uppsala university, sweden. member of the scientific council of the swedish agency for health technology assessment and assessment of social services (sbu). khatereh djavani gidlund, md, phd, consultant general and vascular surgeon, department of surgery, g€avle county hospital, g€avle, sweden. post-doc, centre for research and development, uppsala university/ county concil of g€avleborg, g€avle, sweden. department of surgical sciences, vascular surgery, uppsala university, uppsala, sweden. nils-peter gilgen, md, consultant general and vascular surgeon, head of vascular surgery, department of surgery and urology, eskilstuna county hospital, eskilstuna, sweden. anders wanhainen, md, phd, professor of surgery and research leader, department of surgical sciences, vascular surgery, uppsala, sweden. university, clinical chief of the department of vascular surgery, uppsala university hospital, and guest professor of surgery, department of surgical and perioperative sciences, umeå university, sweden. references 1. ashton h, buxton m, day n, kim lg, marteau tm, scott ra, et al. the multicentre aneurysm 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hospital admissions for england & wales and scotland. eur j vasc endovasc surg. 2012;43:161–6. 37. otterhag sn, gotts€ater a, lindblad b, acosta s. decreasing incidence of ruptured abdominal aortic aneurysm already before start of screening. bmc cardiovasc disord. 2016;16:44. 38. karthikesalingam a, grima mj, holt pj, vidal-diez a, thompson mm, wanhainen a, et al. comparative analysis of the outcomes of elective abdominal aortic aneurysm repair in england and sweden. br j surg. 2018;105:520–8. 39. hamel c, ghannad m, mcinnes mdf, marshall j, earnshaw j, ward r, et al. potential benefits and harms of offering ultrasound surveillance to men aged 65 years and older with a subaneurysmal (2.5-2.9 cm) infrarenal aorta. j vasc surg. 2018;67:1298–307. 40. søgaard r, laustsen j, lindholt js. cost effectiveness of abdominal aortic aneurysm screening and rescreening in men in a modern context: evaluation of a hypothetical cohort using a decision analytical model. bmj 2012;345:e4276. 41. svensj€o s, mani k, bj€orck m, lundkvist j, wanhainen a. screening for abdominal aortic aneurysm in 65-year-old men remains costeffective with contemporary epidemiology and management. eur j vasc endovasc surg. 2014;47:357–65. 186 k. thorbjørnsen et al. abstract introduction methods results discussion acknowledgements disclosure statement funding references sups_a_554719 80..80 upsala journal of medical sciences. 2011; 116: 80 member announcements dear members, we have two forthcoming events that we would like to highlight this time. the first is this year’s martin holmdahl lecture that will take place on march 15 at 6.00 pm in the rudbeck laboratory. the lecture hall will be announced at a later occasion. the speaker is torsten e. gordh and the title of his presentation “a walk on the via dolorosa”. this event is a joint project between selanderska stiftelsen, uppsala medicinhistoriska förening, uppsala medicinska seniorer and the upsala medical society. it is also time for the tradititonal spring meeting of our society. this year we will visit the new “pedagogicum” at blåsenhus. under the guidance of mia lindegren we will learn more about “education in progress” comprising a visit to the “learning lab” and other facilities. it will all take place on april 5 (for further details, see separate announcement and our web page). the address of the learning lab is as follows: kraemers alle’ 1a, plan 2 (infart från norbyvägen vid botaniska trädgården). refreshments will be served and your participation has not to be notified in advance. at this time of the year all members of our society have the privilege of nominating candidates for the rudbeck prize. the name of the candidate and a short justification has to be sent to the chairman of the society but no later than march 1. as in our previous member announcements in this journal we would like to highlight our new web page at www.upsalalakareforening.se. we try to keep it updated as much as possible for your convenience. at this site you will find further details on our activities, on how to become a new member and subscriber of this journal, “veckoprogrammet” and the statutes of the society. further suggestions on how to improve our web site are welcomed and so are viewpoints on how these announcements should be designed. for instance, should we use the swedish language? torbjörn karlsson chairman anna rask-andersen secretary issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2011.554719 time-lapse technology for embryo culture and selection review article time-lapse technology for embryo culture and selection kersti lundin and hannah park reproductive medicine, sahlgrenska university hospital, g€oteborg, sweden abstract culturing of human embryos in optimal conditions is crucial for a successful in vitro fertilisation (ivf) programme. in addition, the capacity to assess and rank embryos correctly for quality will allow for transfer of the potentially ‘best’ embryo first, thereby shortening the time to pregnancy, although not improving cumulative pregnancy and live birth rates. it will also encourage and facilitate the implementation of single embryo transfers, thereby increasing safety for mother and offspring. time-lapse technology introduces the concept of stable culture conditions, in connection with the possibility of continuous viewing and documenting of the embryo throughout development. however, so far, even when embryo quality scoring is based on large datasets, or when using the time-lapse technology, the morphokinetic scores are still mainly based on subjective and intermittent annotations of morphology and timings. also, the construction of powerful algorithms for widespread use is hampered by large variations in culture conditions between individual ivf laboratories. new methodology, involving machine learning, where every image from the time-lapse documentation is analysed by a computer programme, looking for patterns that link to outcome, may in the future provide a more accurate and non-biased embryo selection. article history received 13 january 2020 revised 4 february 2020 accepted 6 february 2020 keywords assisted reproduction; blastocyst transfer; embryo quality; selection algorithms the ivf laboratory a successful in vitro fertilisation (ivf) programme is to a large extent due to the laboratory conditions and the performance of the embryologist(s). a well-functioning and quality-controlled laboratory is crucial. the ivf laboratory today is a highly technical facility, with the most important features being laminar air-flow (laf) benches, incubators, and microscopes, providing dedicated and clean areas for handling, culturing, and assessing gametes and embryos (1). the main tasks in the lab are to optimize—as well as we currently know how—the environment and handling of gametes and embryos, and to score, rank, and select embryos to maximise the possibilities for achieving a live birth. the challenge of in vitro culturing of human embryos is to keep them in an environment as close to their natural environment as possible. the handling of embryos throughout the assisted reproduction technology (art) process usually involves transferring them between dishes and assessing them at specified times (2,3). being outside the incubator will change the culture media (ph, temperature) and thereby the embryo environment. this is believed to create a metabolic stress on the embryo (4–7) which may affect the embryo development and quality. it is therefore important that the time spent handling the oocytes and embryos outside of the controlled incubator environment is minimised. with the increasing implementation of blastocyst culture where the embryos spend a longer time period in vitro, a stable environment will be even more important. also, the static ‘snap-shot’ assessment being performed outside of the incubator once per day in traditional embryo culture means that no information is provided regarding the development between these time points, and significant events may be missed. this would mainly include abnormal cell divisions such as direct cleavage and reverse cleavage (8,9). thereby, using only these short static assessments, some embryos would be incorrectly scored and not properly ranked for quality. a novel technology which enables the integration of more stable embryo culture conditions with embryo assessment is the time-lapse technology. it involves the use of continuous imaging and has been introduced and implemented in art during the last decade. this more exactly timed and electronic documentation of embryo behaviour, possibly in combination with genetic and/or metabolic analyses, is believed to contribute to embryo selection and ranking. however, the introduction of new techniques, or the change of old ones, should always be properly validated. the type of validation needed (randomised, controlled trial [rct], observational studies, meta-analyses, in-house validation) will be dependent upon the magnitude of the change. validation is a time-consuming and expensive process, but is crucial in order to assure safety, efficacy, and reproducibility (10,11). traditional embryo culture in the early days of ivf, a simple culture medium was used, consisting mainly of a buffered salt solution with added contact kersti lundin kersti.lundin@vgregion.se reproductive medicine, sahlgrenska university hospital, g€oteborg, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 2, 77–84 https://doi.org/10.1080/03009734.2020.1728444 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1728444&domain=pdf&date_stamp=2020-05-21 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1728444 http://www.tandfonline.com patient serum. this composition did not support very well the development of embryos for more than 2–3 days (12–14). in order to be able to better sustain and prolong embryo culture in vitro, improvement of media was essential, and the so-called sequential media were developed. the principle of these media is that the developing embryo needs to be provided with appropriate and stage-specific nutrition for each stage of development, similar to the situation as the embryo moves along the fallopian tube towards the uterus. however, with the introduction of closed culture in time-lapse units, the interest in a single medium able to support extended culture was again awakened, the principle now being that if the embryo is provided with all it needs until the blastocyst stage, it will itself utilise the correct nutrients at the correct time (15,16). single media will thus enable an uninterrupted culture for the whole time period (17). in a recent systematic review 20 rcts comparing sequential and single media for blastocyst culture were included. there was no difference in ongoing or clinical pregnancy rates (18). similar findings were reported from a meta-analysis by dieamant et al. (19). so far it is not known which one of the two strategies is the most biologically accurate and appropriate for the embryo, and at present both types of media are in use. clinics will base their choice of culture media on the logistics and the ways of working in the laboratory. thus, in the past, the common strategy was to select the ‘good-quality’ embryos (mainly looking at number of cells, degree of fragmentation, and multinucleation) at the cleavage stage, transfer 2–3 embryos, and cryopreserve the rest of the good-quality embryos, while discarding the non-good’quality’ (judged from morphology and cleavage) embryos. today, following the improved culture conditions in the laboratories as well as the development of more physiological culture media, extended culture until the blastocyst stage is being increasingly practised. this shift from short-term to long-term culture has been gradual, and initially many laboratories, especially in europe, continued to perform the fresh transfer at the cleavage stage (day 2 or 3 post-fertilisation), while excess embryos were cultured to blastocyst stage and cryopreserved. the policy of extended culture of all embryos, both goodand poor-quality, has led to new insights. mainly that although ‘good-quality’ cleavage stage embryos have a higher chance of becoming good-quality blastocysts compared to ‘poor-quality’ early embryos, there is still a > 25% chance that a poor-quality embryo will become a high-quality blastocyst with the same implantation potential as a blastocyst originating from a good-quality embryo (20–22). sadly, this implies that we have for many years discarded embryos that we now know would have had the possibility to develop to high-quality blastocysts with a good potential for implantation and live birth. embryo culture in a time-lapse system several studies have shown that culture and handling of embryos in specialised dishes and closed time-lapse units with image capturing does not seem to impair embryo development compared to sibling oocytes cultured in a conventional setup (23–25). at the same time, there was no difference regarding fertilisation rate, embryo quality at cleavage or blastocyst stage, or ongoing pregnancy rates. however, especially looking from laboratory logistic aspects, there are other advantages with the technology. it provides the possibility to document and assess embryo morphology and timing of developmental events through live image tracking at any time without having to move the embryo or expose it to changes in the environment. it also gives room for increased learning of ‘embryo behaviour’ (such as irregular cleavages) and the possibility to perform studies comparing for example metabolics and environment (oxygen levels, temperature, ph) to timing of specific developmental events, and in the end to success rates. the videos also enable comparisons of embryo development in different settings, such as different culture media and patient populations. in addition, time-lapse documentation may facilitate the training of embryologists in assessing embryo quality, as well as the validation of different scoring systems. if single culture media are used, the embryos can be kept inside the time-lapse system continuously from directly after intracytoplasmic sperm injection (icsi) or fertilisation/denudation has been performed until the day of transfer or cryopreservation. thereby, the logistics of the ivf laboratory are simplified, and embryos can be assessed and graded at any time point during the daily workflow. embryo assessment during the evolution of embryo culture and selection strategies, a number of scoring strategies and algorithms have been developed. most early studies were, however, based on multiple embryos for transfer, a somewhat random choice of variables and cut off levels, and/or simple univariate comparisons (26–30). with time, as more electronic data accumulated, studies on big datasets with more advanced statistical analyses were performed. for early stage transfers, lundin et al. showed, using stepwise logistic regression analysis of 827 day 2/3 transfers, that for icsi embryos, early cleavage was an independent predictor of live birth (31). this was confirmed by van montfoort et al. who, in an analysis of 165 single embryo transfers, found that in addition to cell morphology and cell number early cleavage was an independent predictor for blastocyst development and pregnancy (32). thurin et al. showed, in a multivariate analysis of a selected subanalysis study group comprising 520 transfers, that 4-cell embryos resulted in a statistically higher ongoing implantation rate compared with non-4-cell embryos (33). in 2007, holte and his group prospectively studied 2266 ivf/icsi double-embryo, day 2 transfers with the aim to create an evidence-based morphological embryo scoring model for prediction of implantation. the variables number of cells, variation in blastomere size, and number of mononucleated cells per embryo were found to be significant predictors for implantation. these variables were incorporated into an 78 k. lundin and h. park equation based on a 10-point integrated morphology cleavage (imc) embryo score (34). a follow-up prospective cohort study was performed by the same group (35). with only single embryo transfers included (n ¼ 6252) and live birth rate as endpoint, a slightly revised model was constructed, where number of cells, number of mononucleated cells per embryo, and fragmentation rate were significant predictors, while variation in blastomere size was not significant. similar findings were reached by racowsky et al., where regression analysis of data from the american national database sart was used to standardise embryo scoring and build predictive models for day 2 and day 3 (36). blastomere number was the most powerful predictor for implantation, while the importance of fragmentation was shown to increase for day 3 embryos compared with day 2 embryos. as the practice of extended embryo culture increased, prediction models also for blastocysts were developed. the group of gardner and schoolcraft designed a blastocyst score, which became a ‘gold standard’ for blastocyst assessment. in this system, blastocysts are given a numerical score from 1 to 6 based upon their degree of expansion and hatching status. inner cell mass (icm) and trophectoderm (te) are then scored, from a to c (12). however, the construction of this system is not based on multivariate statistical analyses. in a more recent publication by ahlstr€om et al., the independent ability of expansion stage, inner cell mass, and trophectoderm grading to predict pregnancy outcome was studied (37). the study was a retrospective analysis of 1117 fresh day 5 single blastocyst transfers. live birth outcome was tested to each morphological parameter. all three variables were found to have a significant effect on live birth, but, when adjusted for known confounders, only te remained as a statistically significant independent predictor of live birth. the same group (ahlstr€om et al.) (38), showed in a retrospective study of 1089 patients receiving a frozen–thawed single blastocyst transfer (n ¼ 1089) that when considering pre-freeze morphology, the live birth rate increased significantly for each grade of expansion (or 1.38, ci 1.11–1.72, p ¼ 0.0041), and the probability for live birth was significantly lower for blastocysts of grade b for te compared with grade a for te (or 0.68, ci 0.53–0.87, p ¼ 0.0020) (38). pre-freeze icm morphology did not significantly predict live birth. in addition, the chances for live birth increased for each 10% increase in degree of re-expansion after warming (p ¼ 0.0042). thus, blastocoele expansion and te grade were selected as the most significant pre-freeze morphological predictors of live birth, and degree of re-expansion was selected as the best post-thaw parameter for prediction of live birth. in 2013 van den abbeel et al. showed that in a total of 618 intracytoplasmic sperm injection patients with singleblastocyst transfer on day 5, using a simple logistic regression analysis, all three blastocyst morphology parameters were statistically significantly associated with ongoing pregnancy rates and live birth rates (p < 0.005 for each) (39). however, after multiple logistic regression, only blastocyst expansion stage was a significant predictor of live birth (p ¼ 0.002). time-lapse algorithms the introduction of time-lapse monitoring systems, where images of embryos are captured at time intervals ranging from 5 to 20 min, has resulted in new ways of working and a modified workflow in the ivf laboratory. the technology involves different types of systems; the culture dishes and camera can be placed either as a separate system inside a regular box incubator (open system), or be completely integrated into a smaller, usually bench-top, incubator (closed system). thus, in a closed culture time-lapse setting, the incubator will be an integrated unit with a microscope, enabling both optimised and stable culture conditions as well as direct live viewings and continuous documentation of the embryo development. the embryos are cultured in specifically designed dishes, different for various systems, and assessed from outside the incubator via a screen. the embryos can be monitored in ‘real-time’ and viewed at the end of the culture period as a video sequence, covering the entire time of development. the different time-lapse systems have different software, and they base their scoring on differently constructed algorithms (see overview in lundin and ahlstr€om) (40). it is therefore important to take into consideration that ‘time-lapse’ within art is not a single technology, and a direct comparison of results may not always be possible. the so-called morphokinetic variables are the morphologic and cleavage features documented at exact time points during the embryo development, such as pronuclear appearance and disappearance, cell divisions, and blastocyst formation (see more in guideline by ciray et al.) (41). large datasets including timing of certain development events have been analysed to create algorithms to predict implantation and live birth (42–52). embryos have been classified using individual time-lapse morphokinetic characteristics and related to outcome measured as implantation or live birth. however, so far, the predictive power has been shown to be rather low, with auc levels below 0.8 (43,47–52). in an rct by rubio et al. (n ¼ 857), a statistically increased ongoing pregnancy rate per transfer was found for the timelapse group compared with treatments with embryos cultured traditionally (odds ratio [or] 1.23, confidence interval [ci] 1.06–1.43) (53). however, several problems have been pointed out regarding this study, including different culture conditions for the study and interventions groups, for mixing day 5 and day 3 transfers, and for including both single and double embryo transfers. in another randomised sibling study by yang et al. (n ¼ 600), where only euploid embryos were transferred, there was a significant improvement in ongoing pregnancy rate for the embryos selected on basis of their morphokinetic scores (68.9% versus 40.5%, respectively, p ¼ 0.019) (54). however, also this study had different culture conditions for the groups, making it difficult to determine if the difference observed was due to the selection method or to the culture conditions. upsala journal of medical sciences 79 the most recent cochrane meta-analysis on time-lapse technology included nine rcts (n ¼ 2955 couples) (55). the quality of the evidence ranged from very low to low. the authors conclude that there is insufficient good-quality evidence of outcome differences for embryos cultured or selected in a time-lapse system compared to traditional systems. unfortunately, no data on cumulative live birth have been published as yet. in addition, since embryo development variables are sensitive to environmental conditions, such as type of media, temperature, and gas levels, it has not been possible to extrapolate existing algorithms to other laboratories (56–58), and, so far, no single morphokinetic parameter has been found to be able to predict implantation potential in a multicentre setting (59,60). it is also important to consider that the much-used scoring algorithm, the kid score (known implantation data), is based on cycles where the outcome of all individual embryos is known. this means that all det cycles with a singleton pregnancy have been excluded from the analyses that have provided the algorithm. this has to be considered a serious potential bias when embryo development is compared with outcome, especially since there is believed to exist a ‘cohort’ effect of embryos from the same patient. the question may therefore be put: had the calculated algorithms been different (and more accurate) if these non-implanting embryos had been included? thus, there is currently no conclusive evidence showing that selection through complex scoring systems using additive scores or constructed algorithms is more accurate on a larger scale for finding the embryo with the highest potential for implantation and live birth than the manual/visual selection by the embryologist (59). however, even if the clinical benefit of time-lapse technology embryo culture and selection by morphokinetics is still inconclusive, another possibility is to use it as a deselection tool. for example, it has been demonstrated that certain atypical cleavage patterns, such as the occurrence of direct cleavage to three cells, negatively affects implantation (8,61). these events would in most cases be missed using traditional culture without time-lapse documentation. it is important to note though, that despite the possibility of continuous documentation during the whole culture period, and the development of algorithms to aid in the selection, assessment and selection of embryos is still a manual and subjective intervention being performed by the embryologist. the problems with individual differences in scoring of embryo morphology, and also the annotations of exact times when certain events occur, still remain (62,63). the annotations of the kinetic data also take considerable time, and embryologists in different laboratories may annotate the same events differently, which will in turn influence the calculated score. time-lapse and ploidy a much-debated issue within ivf is the possibility of increasing live birth rates by screening the embryo for aneuploidy before transfer (preimplantation genetic testing [pgt]-a). logically, transferring only euploid embryos should increase live birth rates through increased implantation rates and/or decreased miscarriage rates. however, so far this has been difficult to demonstrate in practice. a few rcts using the modern techniques for pgt-a have been performed. the most recent and largest rct, the star trial, including 661 treatment cycles, found no difference in ongoing pregnancy rate between the intervention group and the control group (64). the study showed ongoing pregnancy rates per intention to treat (itt) of 41.8% (138/330) versus 43.5% (144/331) (p ¼ 0.65) for the intervention group and the control group, respectively. rates per embryo transfer were similarly equal between the groups: 50% (137/274) versus 46% (143/313) (p ¼ 0.32). there was no difference in the miscarriage rates (p ¼ 0.90). a total of 17% of the patients in the pgt-a group did not receive a transfer (compared to 5% in the control group) due to no available euploid blastocysts. the current methods for performing pgt-a are highly invasive, involving removal of cells from the embryos, which could potentially influence the success rates. the time-lapse technology is an expensive tool, and much effort is put into improving its utility. it has been suggested that timing patterns could be indicative of the chromosomal status of the embryo and that algorithms could be developed for prediction of a euploid embryo. several studies have indeed found selected morphokinetic parameters to be associated with ploidy status of the embryo (44,45,65). however, in a large cohort study, rienzi et al. could not find any association between early (up to the 8-cell stage) morphokinetic values and aneuploidy (66). more recently, desai et al. found that, after adjusting for female age, the late kinetic parameters tsb (time for start of blastulation), teb (time for initiation of expansion of the blastocyst), and teb-tsb (the time difference between these two events) were predictive of euploidy (67). the odds of a euploid blastocyst were 1.5 times higher with a tsb < 96.2 h. in addition, deselection of embryos with two or more dysmorphisms, such as multinucleation or irregular cleavage patterns, had a high predictive value. they found no predictive power in any of the early kinetic parameters. the conflicting data between studies may suggest that the same applies for ploidy as for the time-lapse algorithms in general, i.e. the interpretations are sensitive to different patient and laboratory characteristics, and can presumably not currently be used on a universal scale. future developments of time-lapse technology and embryo selection the continuous documentation in time-lapse technology provides huge amounts of data, and only very little of it has been utilised for the existing algorithms. current algorithms are based on annotations being made at specific times, mainly the same few traditional time points that have been used for the traditional manual assessments (41,67). however, projects are currently ongoing using so-called deep machine learning, where large sets of time-lapse videos are analysed, not only looking at the specific predetermined morphokinetic variables, but utilising the complete 80 k. lundin and h. park accumulated data (68,69). the raw time-lapse video sequences are used as input to train and create a model. the model starts by making random predictions, which are compared to the known outcome. the deep learning system has no preexisting assumptions as to which data should be used to build the model, but it analyses all data repeatedly through multiple layers, until a model is created that fits as closely to the known outcome as possible. in this way, subjective assessment by the embryologists are no longer involved. in the study by tran et al., where time-lapse videos from 8836 embryos were used to build and test a model, it was found that the trained model could predict foetal heart pregnancy from time-lapse videos with an auc of 0.93 (95% ci 0.92–0.94) (69). the study included all cycles and embryos (fresh, cryopreserved, donated) handled during the study period and showed as a proof of principle that the deep learning model might be able to predict outcome. future studies could refine the models by including other variables such as day of transfer or patient’s age. it was for example shown by liu et al. that embryos with similar morphology but originating from women of different age show different implantation rates (70). in another study by khosravi et al., >12,000 time-lapse images from 877 good-quality embryos and 887 poor-quality embryos were used to train and implement a machine deeplearning approach to select the highest-quality embryos (68). the model was shown to predict blastocyst quality development with an auc of >0.98 and to outperform the individual embryologists. a decision tree was developed, where the model assessed blastocyst quality integrated with patient age and associated with pregnancy outcome. their analysis showed that chance of pregnancy depended on the blastocyst quality assessed by the model and patient age, varying from 13.8% (age �41 and poor-quality blastocyst) to 66.3% (age <37 and good-quality blastocyst). embryo transfer there is an on-going discussion about the best time for embryo transfer. it is argued, on the one hand, that the blastocyst better represents the correct developmental stage of the in vivo embryo when placed in the uterus, including a better synchrony between the blastocyst and the endometrium (see review by teh et al.) (71). it is also argued that culture to the blastocyst stage allows for the selection of a more viable embryo, thereby resulting in increased implantation rates (72,73). however, on the other hand, assuming that current culture conditions may still be suboptimal, which would increasingly affect the embryo during the extended culture time, it is possible that some embryos may perish during the prolonged time in vitro. it is not known whether a good-quality cleavage stage embryo that survives to the blastocyst stage could have survived if transferred at the cleavage stage. it is estimated that between 25% and 35% of embryos transferred at the cleavage stage implant, while for blastocyst stage transfer it is estimated to be up to 60% (2,3). these estimates were based on traditional morphology assessment, and morphokinetic variables from time-lapse documentation were not taken into consideration. however, looking at cumulative results, including both fresh and frozen-thawed transfers from the same opu, no difference has been shown between early and late transfer, although the early transfer requires a higher number of transfers in total to reach a live birth (74,75). thus, being able to transfer at an earlier stage and still have the advantage of a short time to pregnancy might facilitate the work in the clinic. in the studies by tran et al. and khosravi et al., only day 5 embryos were included, and so far it has not been shown if a similar model would be effective also for early cleavage stage embryos (68,69). being able to better predict the implantation potential of an embryo already on day 2 or 3 might perhaps again change our choice for day of transfer. conclusion there is currently no conclusive evidence that ‘time-lapse technology’, mostly implying either just a closed culture system with continuous documentation and/or a combination of closed culture and morphokinetic algorithms, improves embryo quality, embryo selection, or success rates in ivf. in addition, the algorithms have not been shown to improve outcome. nevertheless, time-lapse technology provides a very useable, although expensive, tool for the laboratory, with safe and stable culture conditions. in addition, it generates large amounts of data that will most probably aid in the selection of embryos. further standardisation and more indepth analyses of the large datasets available from the timelapse documentation may be able to provide us with more targeted and stable algorithms in the future. disclosure statement there are no conflicts of interest regarding this submission. notes on contributors kersti lundin, phd, associate professor at sahlgrenska academy, laboratory director of reproductive medicine, sahlgrenska university hospital, g€oteborg. hannah park, msc, clinical embryologist, laboratory manager of reproductive medicine, sahlgrenska university hospital, g€oteborg. references 1. eshre guideline group on good practice in ivf labs; de los santos mj, apter s, coticchio g, 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2019;22:182–90. 71. teh wt, mcbain j, rogers p. what is the contribution of embryoendometrial asynchrony to implantation failure? j assist reprod genet. 2016;33:1419–30. 72. jones gm, trounson ao, lolatgis n, wood c. factors affecting the success of human blastocyst development and pregnancy following in vitro fertilization and embryo transfer. fertil steril. 1998;70: 1022–9. 73. racowsky c, jackson kv, cekleniak na, fox jh, hornstein md, ginsburg es. the number of eight-cell embryos is a key determinant for selecting day 3 or day 5 transfer. fertil steril. 2000;73: 558–64. upsala journal of medical sciences 83 74. glujovsky d, farquhar c, quinteiro retamar am, alvarez sedo cr, blake d. cleavage stage versus blastocyst stage embryo transfer in assisted reproductive technology. cochrane database syst rev 2016;(7):cd002118. 75. de vos a, van landuyt l, santos-ribeiro s, camus m, van de velde h, tournaye h, et al. cumulative live birth rates after fresh and vitrified cleavage-stage versus blastocyst-stage embryo transfer in the first treatment cycle. hum reprod. 2016;31:2442–9. 84 k. lundin and h. park abstract the ivf laboratory traditional embryo culture embryo culture in a time-lapse system embryo assessment time-lapse algorithms time-lapse and ploidy future developments of time-lapse technology and embryo selection embryo transfer conclusion disclosure statement references tf-iups160037 252..255 review article prevention of unintended pregnancy and use of contraception—important factors for preconception care helena kopp kallnera,b and kristina gemzell danielssona adepartment of women’s and children’s health, division of obstetrics and gynecology, karolinska institutet, danderyd hospital, stockholm, sweden; bdepartment of clinical sciences at danderyd hospital, karolinska institutet, danderyd hospital, stockholm, sweden abstract preservation of fertility and optimizing health before pregnancy is becoming increasingly important in societies where childbirth often is postponed. research shows that as women postpone childbirth they achieve higher levels of education and higher incomes. this leads to advantages for their children and for society. however, as women postpone childbearing they are at risk for contracting conditions which may affect fertility and/or pregnancies, pregnancy outcome, and the newborn child. preconception counseling is therefore becoming increasingly important. women are often unaware of the added health benefits of contraception and have the right to be well informed so they can make decisions to fulfill their reproductive desires. contraception can reduce the risk of unintended pregnancies, ectopic and molar pregnancies, and sexually transmitted infections. in addition, hormonal contraceptives reduce the risk of some types of cancer, dysmenorrhea, heavy menstrual bleeding, and anemia and are a treatment for endometriosis. contraception should increasingly be looked upon as a means of preserving fertility and optimizing health status before a planned pregnancy. thus, effective contraception can provide women with a possibility of achieving their long-term reproductive goals, although childbearing is actually postponed. the most effective contraceptive methods are the long-acting reversible contraceptives, which have been shown to be highly effective especially in young women who have difficulties with adherence to user-dependent methods. therefore, these methods should increasingly be promoted in all age groups. article history received 10 march 2016 revised 24 june 2016 accepted 28 june 2016 keywords added health benefits; combined hormonal contraception; contraception; long-acting reversible contraception; preconception care; unintended pregnancy introduction preservation of fertility and optimizing health before pregnancy is becoming increasingly important in societies where childbirth often is postponed. the average age of first childbirth has steadily been rising in the oecd countries, with first-time mothers’ mean age being 28.2 years in 2014 (1). in the united states the mean age of first-time mothers is 26.3 years (2). postponing childbirth has advantages for women and society. research shows that as women postpone childbirth they achieve higher levels of education and higher incomes (3,4). this leads to advantages for their children and for society (5,6). as women postpone childbirth and have fewer children, they are in need of contraceptives for longer periods. these contraceptives must also be highly effective as women are postponing childbirth during the period in life with the highest fertility. risks of unintended pregnancies do not only include risk of abortion and abortion-related complications but also risk of ectopic pregnancy, which may reduce future fertility, and risk of molar pregnancy that may lead to malignancy. postponing childbirth may also subject women to greater risks of contracting intercurrent conditions such as sexually transmitted infections (stis) and endometriosis, which can affect fertility. other conditions such as heavy menstrual bleeding may lead to anemia and affect women’s health before, during, and after pregnancy. contraception can reduce the risk of unintended pregnancies, ectopic and molar pregnancies, and stis. in addition, hormonal contraceptives reduce the risk of some types of cancer, dysmenorrhea, and heavy menstrual bleeding and are a treatment for endometriosis. women are generally unaware of the added health benefits of contraception. in a swedish study 57% of women could not mention any added health benefits of contraception (7). information and knowledge about the fertilitypreserving capacity of contraceptives and their added health benefits needs to be increased in society. prevention of unintended pregnancy worldwide approximately 40% of pregnancies are unintended, although rates vary across countries and regions (8). unintended pregnancy may result in several outcomes such as birth, miscarriage, induced abortion, ectopic pregnancy, and molar pregnancy. the prevention of unintended pregnancies through the availability of effective contraceptives has changed lives for women, children, and society. contact helena kopp kallner, md, phd helena.kopp-kallner@ki.se department of women’s and children’s health, division of obstetrics and gynecology, karolinska institutet, danderyd hospital, 182 88 stockholm, sweden. � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 4, 252–255 http://dx.doi.org/10.1080/03009734.2016.1208310 http://creativecommons.org/licenses/by/4.0/ prevention of adolescent pregnancy teenage mothers have higher risks of pre-term birth, prenatal and post-partum depression, substance and alcohol abuse, and harsher parenting style (9). in addition, offspring to teenage mothers had worse results in school, higher levels of psychopathology, higher levels of criminal activity, and greater substance abuse levels (9). prevention of adolescent pregnancies should therefore be a high priority in society. in the united states 86% of the decline in teenage pregnancy rates over the last 40 years has been attributed to contraception (10). several studies have examined adherence to different contraceptives among adolescents. young women have more problems with adherence to methods that require daily, weekly, or monthly administration. long-acting reversible methods have been shown to prevent adolescent pregnancies to a larger extent than user-dependent methods (11,12). long-acting methods have also been shown to have lower discontinuation rates (13). in spite of these findings the contraceptive pill is still the most prescribed method among young women in the united states (14) and in europe (15). due to their superior effectiveness in preventing unintended pregnancies, long-acting reversible contraceptives should be promoted among adolescents. prevention of ectopic pregnancy and molar pregnancy worldwide approximately 1%–2% of all pregnancies are ectopic. ectopic pregnancy has a negative effect of future fertility according to several studies (16–19) and is costly for society, with hospital visits, admissions, and absence from work, but also personally for women, with reduced fertility rates. ectopic pregnancy often affects young women who most likely desire preserved fertility (16). ectopic pregnancy due to a planned pregnancy may be considered as inevitable, but ectopic pregnancies may also be due to an unintended pregnancy. in this group of women it is especially important to prevent repeat ectopic pregnancy through adequate contraceptive counseling. all contraceptives protect against conception and therefore decrease the risk of ectopic pregnancy (20,21). with adequate contraceptive counseling, not only abortions would be avoided but also the suffering and costs from an ectopic pregnancy. risk factors for ectopic pregnancy such as pelvic inflammatory disease (pid), usually caused by chlamydia trachomatis or gonorrhea, and endometriosis may also be reduced by contraceptive use. however, the greatest risk factor for ectopic pregnancy is previous ectopic pregnancy, with a recurrent rate of 10%–15% (16,22). ectopic pregnancies can be life-threatening due to intra-abdominal bleeding caused by tubal rupture, tubal abortion, or operative/postoperative complications. it is the most common reason for maternal mortality during the first trimester and is responsible for 9% of pregnancy-related deaths in the us (23). the most effective method for prevention of ectopic pregnancy is the contraceptive implant which prevents ovulation and is a long-acting reversible method. it is unknown what proportion of women receive information on the relative ability of different contraceptives to prevent repeat ectopic pregnancy. molar pregnancy or trophoblastic disease is a rare condition present in 0.57–2 per 1,000 pregnancies (24). molar pregnancy needs to be surgically evacuated and entails a larger risk of hemorrhage compared to surgical evacuation of normal pregnancy. although the condition has a cure rate of approximately 98% in developed countries, it imposes restrictions on women during the follow-up period and may involve aggressive treatment (25). methods for prevention of unintended pregnancy in order to prevent unintended pregnancy, the ‘typical use’ effectiveness rather than the ‘perfect use’ effectiveness of contraceptive methods should be considered in contraceptive counseling. it has been shown that structured counseling and the cost of contraception can influence the contraceptive method that women decide to use (12,26). conventional sources of information on contraceptives such as information from pharmaceutical companies do not contain information on typical use effectiveness, and this may therefore not be common knowledge among providers, nor among women. recently, the importance of long-acting reversible contraceptive methods (larc; implants and intrauterine contraception) in reducing the rate of unwanted pregnancy, abortion, and repeat abortion has been shown (12,27,28). new methods for increasing the effectiveness of short-acting methods such as the pill, patch, and ring have also been explored. a shorter hormone-free interval has been shown to increase the effectiveness of the typical use of the combined hormonal contraceptive pill. in addition, combined hormonal contraceptive pills containing a progestin with a longer half-life have also been shown to increase effectiveness (29). it has been speculated that continuous use of combined hormonal contraceptives may be the most effective way of using these methods. however, no such prospective studies powered for detection of a difference in rates of unintended pregnancies have been published. added health benefits of hormonal contraception the first contraceptive pill was not approved on the indication contraception (which was illegal at that time) but for treatment of menstrual symptoms such as dysmenorrhea and heavy menstrual bleeding. in addition, combined hormonal contraceptives have been shown to reduce the risk of several cancers such as ovarian cancer, endometrial cancer, and colon cancer as shown in several large prospective cohort studies (30–32). use of the intrauterine system containing 52 mg levonorgestrel (release rate 20 lg levonorgestrel per 24 hours), has been shown to reverse precancerous conditions in the endometrium (33). making women aware of the health benefits of hormonal contraception could increase the use of effective contraceptives among women and optimize preconception health status. as women postpone childbirth and subsequently become older when they have their first child, more women are exposed to the risk of cancer, which increases upsala journal of medical sciences 253 with age. the risk of cancer and impairment of fertility may be reduced by contraceptive use. treatment of dysmenorrhea and endometriosis among women and adolescents with severe dysmenorrhea it is estimated that 25%–75% have endometriosis (34,35). the main treatment of endometriosis is progestins, and the firstline recommended treatment is combined hormonal contraception (36). endometriosis is present in 25%–40% of infertile women (37). the aim of the medical treatment with progestins is to achieve anovulation and amenorrhea whereby the inflammatory response, endometriomas, and adhesions are prevented (38). these may otherwise cause infertility from the need of ovarian surgery with reduction in the ovarian reserve and lesions causing tubal infertility. treatment for dysmenorrhea in adolescents should take into account that it may be caused by endometriosis and that endometriosis in adolescents may be aggressive. treatment with progestins can prevent disease progression and future infertility (39). however, contraceptive pills cannot treat infertility caused by endometriosis (40). heavy menstrual bleeding heavy menstrual bleeding is present in 4%–22% of women, depending on which method is used for evaluation of menstrual bleeding, and iron deficiency anemia is present in 5%–10% of women of fertile age (41). the main cause of iron deficiency anemia in fertile women is heavy menstrual bleeding (41). heavy menstrual bleeding increases with age (42). thus, as women postpone childbirth, they become more exposed to the risk of heavy menstrual bleeding and subsequent anemia. women with normal hemoglobin levels may still have iron deficiency. thus, they have depleted the iron depots, most likely due to heavy menstrual bleeding, but are maintaining normal hemoglobin values. recent research shows that women with iron depletion but normal hemoglobin levels may have less resistance to muscular fatigue. suggested neuropsychological effects are, among others, depression and impaired learning and memory abilities (43). the increased need of iron during pregnancy induces iron deficiency anemia in women with reduced iron depots, and iron supplementation is necessary. it has been shown that approximately 10.5% of women in the helsinki area in finland have iron deficiency anemia when they present for delivery (44). iron deficiency anemia puts the mother at higher risk during childbirth, and she also has a higher risk of post-partum infections. anemia may also impact on the ability to breastfeed (45,46). in the finnish register study (44) anemia at presentation for delivery was associated with a higher risk for pre-term delivery, cesarean section, and neonatal intensive care admission of the newborn. post-partum anemia is also linked to post-partum depression, reduced quality of life, and reduced cognitive abilities (46). thus, it is essential for women to enter pregnancy with an optimal iron status in order to maintain health during and after pregnancy. prevention of anemia during pregnancy must be taken seriously, and precautions should be taken early in pregnancy to reduce the risk for adverse pregnancy, delivery, and post-delivery outcomes. when anemia is discovered in early pregnancy a detailed menstrual bleeding history should be taken, and women should be informed about contraceptives which reduce menstrual blood loss in order to optimize future health. conclusion as women postpone childbirth they have an increased need for pre-conception contraceptive counseling. the counseling should focus on the long-term need for effective contraception and avoidance of the consequences of unintended pregnancy such as ectopic pregnancy which reduces future fertility. in addition, pre-conception added health benefits should be stressed. these include prevention of the consequences of heavy menstrual bleeding, anemia, and untreated endometriosis but also of several cancers. during contraceptive counseling the effectiveness of any method in typical use and the superior effectiveness of long-acting methods should be stressed. declaration of interest h.k.k. and k.g.d. have both served on advisory boards for msd, bayer, and gedeon richter. h.k.k. and k.g.d. have both received honorariums for giving lectures for msd, bayer, gedeon richter, and actavis on the topic of contraception and abortion. h.k.k. and k.g.d. have both participated in studies on contraception sponsored by msd and gedeon richter, and, in the case of k.g.d., also bayer. orcid helena kopp kallner http://orcid.org/0000-0001-7184-9165 references 1. 2015. available from: www.oecd.org/els/family/database.htm. 2. mathews tj, hamilton be. mean age of mothers is on the rise: united states, 2000–2014. nchs data brief. 2016(232):1–8. 3. bailey mj, hershbein b, miller ar. the opt-in revolution? 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rl, richter r, bergmann ke, dudenhausen jw. prevalence and risk factors for early postpartum anemia. eur j obstet gynecol reprod biol. 2010;150:126–31. 46. milman n. postpartum anemia i: definition, prevalence, causes, and consequences. ann hematol. 2011;90:1247–53. upsala journal of medical sciences 255 prevention of unintended pregnancy and use of contraception&mdash;important factors for preconception care introduction prevention of unintended pregnancy prevention of adolescent pregnancy prevention of ectopic pregnancy and molar pregnancy methods for prevention of unintended pregnancy added health benefits of hormonal contraception treatment of dysmenorrhea and endometriosis heavy menstrual bleeding conclusion declaration of interest references association of the negr1 rs2815752 with obesity and related traits in pakistani females original article association of the negr1 rs2815752 with obesity and related traits in pakistani females sobia rana and maha mobin molecular biology and human genetics laboratory, dr. panjwani center for molecular medicine and drug research (pcmd), international center for chemical and biological sciences (iccbs), university of karachi, karachi, pakistan abstract introduction: the variant negr1 rs2815752 has recently been linked with obesity in caucasians. however, a very limited number of studies have examined the association of the negr1 rs2815752 with overweight/obesity in non-caucasians with no such study ever performed in pakistani population. therefore, the present study was undertaken to seek the association of the rs2815752 with overweight, obesity, and related traits in pakistanis. subjects and methods: the study involved 112 overweight/control pairs (total 224) and 194 obese/ control pairs (total 388). anthropometric parameters were measured by employing standard procedures. metabolic parameters were determined by biochemical assays. behavioral information was collected through a questionnaire. the rs2815752 was genotyped via taqman allelic discrimination assay. regression analyses were employed to analyze the data in spss software. results: the study revealed significant gender-specific association of the rs2815752 with obesity (or 3.03; ci 1.19–7.72, p ¼ 0.020) and some obesity-related anomalous anthropometric traits (weight, bmi, waist circumference, hip circumference, and abdominal and supra-iliac skinfold thicknesses) in females according to dominant model (h ¼ 0.023). however, no association of the rs2815752 with obesityrelated behavioral and metabolic parameters was observed. conclusion: the negr1 rs2815752 may be associated with obese phenotype and some of the related anthropometric traits in pakistani females. article history received 25 november 2019 revised 13 april 2020 accepted 14 april 2020 keywords genetic association; negr1 rs2815752; neuronal growth regulator 1; obesity; overweight; single nucleotide polymorphism introduction obesity is a metabolic disorder characterized by a chronic imbalance of energy homeostasis due to high energy intake and low energy expenditure, eventually increasing the fat mass to the extent that it becomes harmful to health (1). its prevalence has extended to epidemic proportions worldwide over the past �50 years. it substantially escalates the risk of various non-communicable diseases that in turn lead to high rates of morbidity, mortality, and healthcare costs. moreover, it also significantly increases the economic burden due to its association with unemployment, social disadvantages, and reduced socio-economic productivity. thus, it has become a major health challenge (2). it is a complex and multifactorial malady attributable not solely to multiple developmental, environmental, behavioural, and genetic factors but also to the interactions between these factors (3,4). familial aggregation analysis such as twin and adoption studies has provided compelling evidence for the existence of genetic components in determining obesity and body fat distribution (5). genome-wide association studies (gwas) have identified many genes that confer a predisposition to weight gain (6). the genes near body mass index (bmi; a measure of obesity)-regulating loci are enriched for expression in the central nervous system (cns), indicating that bmi is primarily regulated by processes such as hypothalamic control of eating behaviour, while genes for fat distribution are enriched in local fat depots, indicating that fat distribution is mainly regulated in the adipose tissue itself (7). neuronal growth regulator 1 (negr1) has recently been identified as a new locus responsible for human body weight control in three independent gwas (8–10). the negr1 gene is positioned on chromosome 1p31.1 and is mainly expressed in the hypothalamus (a crucial center for energy balance and regulation of food intake). however, it is also expressed in subcutaneous adipose tissue (sat), heart, and skeletal muscles (11). differential co-expression analysis of obesity-associated networks in human subcutaneous adipose tissue revealed differential expression of the negr1 gene between normal-weight and obese siblings with identification of negr1 as a central hub in an obesity-related transcript network (12). however, in vivo studies have shown varying results for correlation between the expression level of negr1 and manifestation of obesity, perhaps owing to contact sobia rana molecularbiologist1@gmail.com, sobia.rana@iccs.edu molecular biology and human genetics laboratory, dr. panjwani center for molecular medicine and drug research (pcmd), international center for chemical and biological sciences (iccbs), university of karachi, karachi 75270, pakistan supplemental data for this article can be accessed here. � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 3, 226–234 https://doi.org/10.1080/03009734.2020.1756996 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1756996&domain=pdf&date_stamp=2020-06-25 http://orcid.org/0000-0003-2540-5170 http://orcid.org/0000-0003-2312-5706 https://doi.org/10.1080/03009734.2020.1756996 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1756996 http://www.tandfonline.com highly complex regulatory processes of energy homeostasis. the functional inactivation of negr1 in mice led to a small but steady decrease in body mass. in addition, mice carrying a loss-of-function mutation (negr1-i87n) showed decreased food intake but normal energy expenditure, thereby supporting the positive correlation between negr1 expression and obesity (13). however, another in vivo study involving the use of an adeno-associated virus revealed that the decreased expression of negr1 in periventricular hypothalamic areas of rats resulted in escalated bodyweight presumably via increased food intake, but negr1 overexpression did not affect body weight or food intake (14). recently, negr1 has been found to be involved in intracellular cholesterol homeostasis, which signifies its non-cns function associated with human obesity (15). more recently, negr1 deficiency in mice has been found to induce abnormal fat deposition in various peripheral tissues, particularly fat and liver tissue cells, again indicating that the obesityrelated function of negr1 is not restricted to the cns but extends to peripheral tissues as well (16). the rs2815752 variant located upstream of the negr1 gene involving g > a transition has been reported to have one of the biggest effect sizes on bmi values in gwas conducted on caucasians (8–10). a very limited number of studies have examined the association of the negr1 rs2815752 with overweight/obesity in non-caucasians, with no such study ever performed in pakistani population. genetic architecture may considerably differ across populations. thus, genetic association studies should be performed in diverse populations, particularly in under-represented populations, to have a better understanding of genetic variants associated with a trait (17). pakistan has recently attained a 9th position among 188 countries in terms of obesity, and nearly one-third of the country’s population is overweight/obese (18). moreover, the pakistani population, owing to its distinctive features including large size, consanguinity, ethnic diversity, large pedigrees, and rapid nutritional transition, offers many benefits in expounding the pathophysiology of obesity (19). by keeping in view the entire aforesaid scenario, the current study was carried out to examine the association of the negr1 rs2815752 with overweight/obesity and obesity-related multiple traits in a sample of the pakistani population. materials and methods subjects and ethics the study was executed at the international center for chemical and biological sciences (iccbs), university of karachi, pakistan, and after approval from the independent ethics committee (iec) of the institute and advanced studies and research board (asrb) of the university. moreover, the study was performed in accordance with the ethical standards of the helsinki declaration. the study encompassed a total of 612 human subjects of both sexes, whose ages ranged from 12 to 63 years. written informed consent was received from all participants before their participation in the study. the study was based on a case-control design. the total study population of 612 individuals included 112 overweight/control pairs (total 224) that had been age (±5 years)and sex-matched, and 194 obese/control pairs (total 388), age (±5 years)and sex-matched. for inclusion of overweight (ow), obese (ob), and normal weight (nw) subjects in the study, bmi cut-off values set by the world health organisation (�18.5 kg/m2 for normal weight, �25 kg/m2 for overweight, and �30 kg/m2 for obesity) were used for adults (�20-years-old), whereas bmi growth charts (5th to 84th percentile for normal weight, 85th to 94th percentile for overweight, and �95th percentile for obesity) set by centre for disease control and prevention were used for children and adolescents (<20-years-old). the exclusion criteria were: a history of endocrine disorders like pituitary dysfunction, cushing’s syndrome, and hypothyroidism; and also a history of medication such as tricyclic antidepressants, anticonvulsants, phenothiazine, and steroids. the technique of simple random sampling without replacement was employed for the recruitment of subjects. in the sampling procedure, each study subject (obese/overweight/control) was recruited randomly from the general population of karachi in such a way that every individual had an equal chance of being selected, based on the inclusion/exclusion criteria, and no subject was included more than once in the sample. karachi is a cosmopolitan city with a population of varied ethnic backgrounds from all over pakistan. thus, the participants of the study were from diverse ethnic backgrounds including urdu-speaking, punjabi, pashtun, sindhi, balochi, and other ethnicities. collection of behavioural data obesity-related behavioural information including eating timings, diet unconsciousness, and tendency towards fat-dense food was collected from each participant through a questionnaire. collection of anthropometric data anthropometric measurements were performed following standard procedures. height in centimetres and weight in kilograms were measured using a stadiometer (seca 214, germany) and a mechanical column scale (seca 755), respectively. skin fold thicknesses (sft) in millimetres were measured at six different body sites including abdomen, subscapula, supra-ilium, thigh, biceps, and triceps using a skinfold calliper (slim guide, mi). waist and hip circumferences in centimetres were measured using a non-stretchable measuring tape. subsequently, body mass index (bmi), percent body fat (%bf), and waist-to-hip ratio (whr) were derived using the measurements mentioned above. bmi was calculated as a person’s weight in kilograms (kg) divided by the square of his/her height in metres (m2). the %bf was calculated from skin-fold measurements of abdomen, supra-ilium, thigh, and triceps, using specific formulae for males and females: males %bf ¼ 0:29288ð þ � 4 skinfolds sumð þ� 0:0005ð þ � 4 skinfolds sumð þ2 þ 0:15845 � ageð þ�5:76377 upsala journal of medical sciences 227 females %bf ¼ 0:29669ð þ � 4 skinfolds sumð þ� 0:00043ð þ � 4 skinfolds sumð þ2 þ 0:02963 � ageð þ þ 1:4072 waist-to-hip ratio (whr) was calculated as waist circumference (wc) divided by hip circumference (hc). collection of blood sample a venous blood sample in a volume of 5 ml was drawn from each participant of the study after an overnight fast (8–12 h) with a 5 ml syringe. out of this 5 ml sample, 2 ml was collected in an edta-coated vacutainer tube for subsequent dna extraction, while 3 ml blood was collected in another vacutainer tube containing gel and clot activator for subsequent serum isolation. the serum was extracted by centrifuging the tube at 4000 rpm for 10 min. collection of metabolic data blood pressure (systolic and diastolic) was recorded twice from the right arm of the subjects, using a mercury sphygmomanometer (certeza medical, cr-2001, germany) with an accuracy of 1 mmhg. fasting blood glucose (fbg) was determined by a blood glucose monitoring system (abbott, chicago, il). fasting serum insulin concentrations were determined by enzyme-linked immunosorbent assay (elisa) using a commercially available kit (dia source inseasia kit, cat no. kap1251, belgium) on a multiskantm fc microplate photometer (thermofisher scientific, waltham, ma). homeostasis model assessment of insulin resistance (homa-ir) was calculated from fbg and fasting insulin values using the formula: homa-ir ¼ fasting insulin (ml u/ ml) � fasting glucose (mg/dl)/405. homeostatic model assessment of insulin sensitivity (homa-is) was calculated as homa-is ¼ 1 � [fasting insulin (miu/l) � fasting glucose (mmol/l)]. assays related to lipid profile including total cholesterol, high-density lipoprotein cholesterol (hdl-c), low-density lipoprotein (ldl-c), and triglycerides (tgs) were performed by using enzymatic in vitro assay kits (merck, darmstadt, germany) on a roche hitachi chemistry analyzer. very-low-density lipoprotein cholesterol (vldl-c) was calculated by dividing tg by 5. cholesterol-to-hdl-c ratio (chr) was calculated by dividing the concentration of total cholesterol by the concentration of hdl-c. in addition, different anthropometric and metabolic estimations were used to compute various metabolic indices and ratios including visceral adiposity index (vai) (mmol/l), lipid accumulation product (lap) (mmol/l), product of triglyceride and glucose (tyg) index, coronary risk index (cri), atherogenic index (ai), and triglyceride-to-hdl-c ratio (tg/hdl-c). to calculate vai and lap, respective gender-specific formulae were used. to calculate vai and lap, values of tg and hdl-c were converted from mg/dl to mmol/l. males vai ¼ wc � 39:68 þ ð1:88 � bmiþ½ � � tg � 1:03½ � � 1:31 � hdl-c½ � females vai ¼ wc � 36:58 þ 1:89 � bmið þ½ � � tg � 0:81½ � � 1:52 � hdl-c½ � males lap ¼ wc–65ð þ � tg females lap ¼ wc–58ð þ � tg a general formula for both sexes was used to calculate the tyg index: tyg index ¼ ln fasting triglycerides mg=dlð þ½ � fasting glucose mg=dlð þ � 2� to calculate cri, tc was divided by hdl-c, whereas to calculate ai, ldl-c was divided by hdl-c. the tg-to-hdl-c ratio was calculated by dividing the values of tg by hdl-c. genomic dna extraction and snp genotyping extraction and purification of dna from whole blood was performed using a genomic purification kit (ez-10 spin column, cat #bs483, bio basic, markham, canada) following the manufacturer’s recommended protocol. genotyping was performed using taqmanv r predesigned snp genotyping assay (assay id: c_26668839_10, cat no. 4351379, abi, foster city, ca) and taqmanv r genotyping master mix (cat no. 4381656, abi, foster city, ca) on an applied biosystems 7500 real-time polymerase chain reaction machine (thermofisher scientific, waltham, ma). the thermal cycling conditions included an initial step of polymerase activation at 95 �c for 10 min followed by 50 cycles of two steps: denaturation at 95 �c for 15 s, and annealing and extension at 60 �c for 1 min. successful genotyping was performed with 99% genotypic call rates and >95% concordance between duplicate samples. after each run, the post-amplification analysis was done using the software of applied biosystems 7500. we included two negative controls (no template control or ntc) and one positive control (pc) for each genotype, every time the experiment was run. in addition, genotyping of 20% of the samples was repeated to ensure reproducibility. statistical analysis statistical analysis of the data was carried out using the software ibm spss statistics version 21. genotypic frequencies in cases and controls were calculated via chi-square, whereas allelic frequencies were calculated by direct count. genotypic frequencies were reported as counts and percentages. hardy–weinberg equilibrium (hwe) analysis for both cases and controls was performed by using a web-based calculator to check whether the genotypic frequencies are in hwe. logistic regression was performed to examine the association of the variant rs2815752 (g > a) with the risk of overweight/ obesity while considering four models of inheritance (codominant, dominant, over-dominant, recessive). in the case of getting association in more than one model, the degree of dominance index (h) was calculated to select the best-fit 228 s. rana and m. mobin model. age and gender-adjusted odds ratio (or) and 95% confidence intervals (ci) were calculated to determine the risk of overweight/obesity associated with the rs2815752 variant. the association was also investigated separately in mild (overweight) and severe (obese) phenotypes. in addition, analyses were separately conducted in males and females to identify any gender-specific association in the study sample for which only age was adjusted. ordinal regression was also performed on the total sample population to assess the association of the variant with bmi grades, taking bmi grades 0 through 4 as an outcome or dependent variable and the dominant genotype of the variant as an explanatory or independent variable. bmi grade 0 was taken for ‘normal’, grade 1 for ‘overweight’, grade 2 for ‘class i obesity’, grade 3 for ‘class ii obesity’, and grade 4 for ‘class iii obesity’. parameter estimates were then calculated under plum command. fisher’s exact test was applied to compare categorical variables, and mann–whitney u test was applied to compare continuous variables between cases and controls. categorical variables included demographic characteristics and parameters of eating behaviours, while continuous variables included anthropometric and metabolic traits. logistic regression was employed to assess the variant’s association with obesity-linked categorical traits, whereas linear regression was employed to assess the variant’s association with obesity-linked continuous variables, using the best-fit model of inheritance. bmi was additionally adjusted while testing metabolic parameters. statistical significance was taken at a two-sided p-values <0.05 for all the comparisons. false discovery rate (fdr) correction for multiple comparisons was performed using the benjamini–hochberg method. effect size with 95% ci was determined for all variables. power calculation the power calculation was performed employing quanto software. the rs2815752 risk allele ‘a’ frequency (0.64) and the log-additive model were used to calculate power. our study was found to have a power of 80% for the least detectable or of 1.4 with a two-sided type 1 error rate of 0.05. results a comparison between the characteristics of cases and controls is shown in supplementary tables 1–4 (available online). most of the anthropometric and metabolic parameters were found to be significantly abnormal in cases as compared to controls (supplementary tables 1 and 3; available online). no significant differences between cases and controls in terms of parental consanguinity were observed. however, a family history of obesity was found to be significantly higher in cases compared to controls. abnormal eating behaviours like random eating timings and tendency towards fat-dense food were significantly more prevalent in cases as compared to controls. nevertheless, no significant differences were observed between cases and controls in terms of diet unconsciousness. demographic features like percentages of ethnicities are also shown between cases and controls (supplementary tables 2 and 4; available online). genotypic distribution of the negr1 rs2815752 was observed to be in hardy–weinberg equilibrium in both cases and controls (supplementary table 5; available online). the genotypic frequencies did not differ significantly between overweight/obese cases and normal-weight controls (supplementary table 5; available online). a similar observation was seen between obese males and their controls; however, genotypic frequencies were found to differ significantly between obese females and their corresponding controls (table 1). thus, the variant rs2815752 appeared to have a significant gender-specific association with obesity (not with overweight) in females. this association was observed in more than one genetic model (co-dominant and dominant) during age-adjusted regression analysis. the subsequent calculation of the h index indicated a dominant mode of inheritance. thus, according to a dominant genetic model, the ga/aa genotype of the negr1 rs2815752 variant increases 3.03-fold the odds of having obesity in females (table 1). this finding was further strengthened when the association of the variant rs2815752 with bmi grades was assessed by employing ordinal regression. the analysis revealed a significant association between ga/aa genotype and bmi of higher grades. however, after gender stratification, female carriers but not male carriers of the risk genotype (ga/aa) were found to have a significantly greater risk of having higher bmi grades than non-carriers (gg) (table 2). when the association of the variant rs2815752 with the obesity-related anthropometric, metabolic, and behavioural traits was assessed, the variant was found to be significantly associated with some of the anomalous anthropometric traits (weight, bmi, wc, hc, and abdominal and supra-iliac skinfold thicknesses) in females only. on the other hand, no association of the variant was seen with anthropometric traits in males (table 3). furthermore, the lack of association between the rs2815752 and obesity-related metabolic traits was observed (table 4). similarly, we did not find any association of the rs2815752 with aberrant eating behaviours, except a very nominal (p values ¼ 0.048) protective association of the rs2815752 with random eating timings in males. however, no such association was observed in females (table 5). discussion we found that there was a gender-specific association of the rs2815752 with the obese phenotype (but not with overweight) and some obesity-related anomalous anthropometric traits including weight, bmi, waist circumference, hip circumference, and abdominal and supra-iliac skinfold thicknesses in pakistani females according to a dominant mode of inheritance. accordingly, the frequency of the rs2815752 genotypes differed between obese and normal-weight females, with a higher frequency of risk allele (a)-carrying genotypes (ga, aa) and lower frequency of gg genotype in cases as compared to controls. this suggests that the variant may be upsala journal of medical sciences 229 https://doi.org/10.1080/03009734.2020.1756996 https://doi.org/10.1080/03009734.2020.1756996 https://doi.org/10.1080/03009734.2020.1756996 https://doi.org/10.1080/03009734.2020.1756996 https://doi.org/10.1080/03009734.2020.1756996 associated with the extreme phenotype (obesity) but not with the mild phenotype (overweight) in females of our population. similar to our findings, rukh et al. also reported a significant association of the variant rs2815752 with obesity (according to additive model adjusted for age and gender) but not with overweight (20) in a swedish population. in addition, m€agi et al. also reported a strong association of the rs2815752 with higher grade of obesity (bmi � 35.0 kg/m2) in europeans with respect to additive, dominant, and recessive models adjusted for age and gender, but they did not calculate h index for indicating the appropriate mode of inheritance (21). a possible explanation for the observed association of the rs2815752 with obesity but not with overweight could be that the genetic variants that confer susceptibility to weight gain tend to concentrate in extreme phenotypes (bmi � 30) as compared to mild phenotype (bmi � 25) (22). contrary to our findings, a study on germans showed no association of the variant rs2815752 with obesity in gender-adjusted overall as well as gender-specific separate analyses considering the additive model (23). furthermore, a study on chinese children revealed no association of the variant with obesity according to a gender-adjusted log-additive model (24). however, a study conducted on mexican children found a nominal association of the variant with obesity risk, employing an additive model adjusted for age and gender (25). in the current study, the association of the negr1 rs2815752 with many obesity-related anthropometric parameters including weight, bmi, wc, hc, abdominal sft, and supra-iliac sft but not with other anthropometric traits in obese females indicates that this variant contributes to obesity by higher fat deposition in only the waist, hip, and abdomen. the association of the variant rs2815752 with bmi, wc, and hc has also been reported by rukh et al. in a swedish population (20). likewise, a study on south brazilian children also showed a significant association of the variant rs2815752 with bmi and sum of skin-folds (26). in contrast, bauer et al. found no significant association of the negr1 rs2815752 with adiposity measures in dutch females (27). in addition, there was no association of the variant rs2815752 with birth weight and body fat distribution in a meta-analysis conducted by kilpel€ainen et al. (28). in some of our previous studies, we also reported genderspecific association of the other variants such as mc4r rs17782313, fto rs9939609, and lep rs7799039 with overweight/obesity and related anthropometric traits in pakistani females (29–31). in comparison to these previously reported associations, the association of negr1 rs2815752 observed in the current study has a higher impact (effect size) but less strength. as mentioned before, this variant has also been table 1. genotypic frequencies of the negr1 rs2815752 variant among gender-stratified cases (obese) and controls along with assessment of the association of the rs2815752 with obesity. males females genetic model genotype cases, n (%) controls, n (%) adjusted or (95% ci) a adjusted p-valuea cases, n (%) controls, n (%) adjusted or (95% ci) a adjusted p-valuea co-dominant gg 13 (12.15) 10 (9.35) 0.615 7 (8.05) 18 (20.69) 0.042b ga 45 (42.06) 41 (38.32) 38 (43.68) 36 (41.76) aa 49 (45.79) 56 (52.34) 42 (48.28) 33 (37.93) pair-wise comparison gg vs ga 0.84 (0.33, 2.13) 0.720 2.65 (0.98, 7.14) 0.054 gg vs aa 0.67 (0.27, 1.67) 0.394 3.47 (1.28, 9.38) 0.014 b dominant (ga þ aa) 94 (87.85) 97 (90.66) 0.75 (0.31, 1.78) 0.509 80 (91.96) 69 (79.69) 3.03 (1.19, 7.72) 0.020 b gg 13 (12.15) 10 (9.35) 7 (8.05) 18 (20.69) recessive aa 49 (45.79) 56 (52.34) 0.77 (0.45, 1.32) 0.342 42 (48.28) 33 (37.93) 1.75 (0.94, 3.25) 0.076 (gg þ ga) 58 (54.21) 51 (47.67) 45 (51.73) 54 (62.45) over-dominant ga 45 (42.06) 41 (38.32) 1.17 (0.67, 2.02) 0.582 38 (43.68) 36 (41.76) 1.03 (0.56, 1.90) 0.916 (gg þ aa) 62 (57.94) 66 (61.69) 49 (56.33) 51 (58.62) adjusted h index ¼ 0.023 hardy–weinberg equilibrium (hwe): p-value p-value cases 0.597 0.689 controls 0.584 0.177 data represent genotype counts and percentages in parenthesis in cases and controls. data also indicate genotypic frequencies in hwe. association of rs2815752 with obesity in obese cases was tested using chi-square and multinomial regression for co-dominant model and binary logistic regression for other models. aanalysis was performed with adjustment for age. badjusted p-value <0.05 were considered statistically significant. adjusted h index was calculated for indication of appropriate mode of inheritance. aa: mutant homozygous genotype; ci: confidence interval; ga: heterozygous genotype; gg: wild-type homozygous genotype; or: odds ratio. table 2. assessment of the association of the negr1 variant rs2815752 with bmi grades in the sample population using a dominant model of inheritance. all (males and females) males females adjusted adjusted adjusted genotype or (95% ci) p-value or (95% ci) p-value or (95% ci) p-value ga þ aa 1.30 (1.01, 1.67) 0.041a 1.12 (0.79, 1.59) 0.512 1.59 (1.10, 2.28) 0.013a gg 1 1 1 ordinal regression was used to calculate parameter estimates under plum command. bmi grade 0 was taken for normal weight, bmi grade 1 for ‘overweight’, bmi grade 2 for ‘class i obesity’, bmi grade 3 for ‘class ii obesity’, and bmi grade 4 for ‘class iii obesity’. ap-values <0.05 were considered significant. 230 s. rana and m. mobin reported as having one of the biggest effect sizes on bmi values in gwas conducted on caucasians (8–10). pakistan is one of the countries that has entered stage 1 of the obesity transition characterised by a higher prevalence of obesity in women than in men (32). in this context, the observed associations of genetic variants with obesity and obesity-related anthropometric traits in females of our population indicates the existence of sexual dimorphism in genetic susceptibility to obesity and may partly explain the widening gender gap in excess weight as 10% more women gain weight than men in pakistan (18). moreover, gene–environment interactions play an important role in the determination of complex traits like obesity. aberrant lifestyle may be more common in women as compared to men in pakistan (33). thus, the interaction between the genetic variants and aberrant lifestyle may possibly make pakistani females more prone to gain weight. the strategy of stratified analysis can be more powerful than a combined analysis when a variant is restricted to a subgroup or when the extreme difference of a variant’s effect is present in two genders (34,35). gwas have also revealed sexual dimorphic effects in genetic loci for adiposity-related phenotypes, such as waist circumference and waist-to-hip ratio with stronger effects in women (36). obesity is a major risk factor for a host of metabolic disturbances, predominantly in lipid and glucose metabolism. epidemiological evidence indicates that adiposity is associated with aberrant lipid profiles and biomarkers of glucose metabolism (37,38). on the same lines, all the metabolic variables of the current study appeared significantly aberrant in cases as compared to controls. however, there was no association of the negr1 rs2815752 with any of the metabolic parameters in the current study. in a similar manner, a study by haupt and colleagues also showed no association of this variant with biomarkers of glucose metabolism such as fasting glucose and insulin sensitivity (39). moreover, fall et al. in a study on swedish non-diabetic old men also reported no association of the rs2815752 with insulin sensitivity (40). in addition to biomarkers of glucose metabolism, we also sought the association of the variant rs2815752 with parameters of lipid profile (total cholesterol, tg, hdl-c, ldl-c, vldlc, chr, and tg to hdl-c ratio) and other metabolic variables including vai, lap, tyg index, cri, ai, systolic blood pressure, and diastolic blood pressure, which have never been investigated before. the lack of association between the rs2815752 and metabolic traits in the current study suggests that the variant rs2815752 may not have a direct impact on these metabolic parameters, but it may influence these parameters indirectly via influencing bmi. we found no association of the negr1 rs2815752 with aberrant eating behaviours such as tendency towards fat-dense food and diet unconsciousness. the lack of association between the negr1 rs2815752 and eating behaviours may be due to self-reporting bias (under-reporting) of the study participants as relevant data were collected by means of a table 3. assessment of the association of the rs2815752 with obesity-related anthropometric variables in gender-stratified obese cases and controls. variables (unit of measurement) dominant model males females mean (sd) adjusted b (95% ci) adjusted p-value mean (sd) adjusted b (95% ci) adjusted p-value (corrected) weight (kg) gg 83.57 (21.20) �0.848 (�11.139, 9.444) 0.871 62.48 (17.43) 13.102 (3.656, 22.547) 0.033a ga þ aa 82.72 (23.93) 75.55 (22.76) height (cm) gg 171.38 (6.45) �0.746 (�3.835, 2.342) 0.634 156.63 (6.27) 0.857 (�1.54, 3.228) 0.477 ga þ aa 170.63 (7.15) 157.50 (5.59) bmi (kg/m2) gg 28.51 (7.26) �0.163 (�3.472, 3.146) 0.923 25.43 (6.80) 5.043 (1.398, 8.688) 0.003a ga þ aa 28.35 (7.66) 30.46 (8.97) wc (cm) gg 100.33 (16.68) 0.016 (�7.952, 7.983) 0.997 92.08 (20.22) 10.907 (1.828, 19.987) 0.049a ga þ aa 100.35 (18.84) 102.91 (21.50) hc (cm) gg 107.08 (14.20) �0.172 (�6.894, 6.550) 0.960 102.00 (12.26) 7.915 (1.623, 14.207) 0.049a ga þ aa 106.91 (15.65) 109.90 (15.16) whr gg 0.93 (0.07) �0.000 (�0.030, 0.030) 0.987 0.89 (0.10) 0.036 (�0.006, 0.078) 0.099 ga þ aa 0.93 (0.07) 0.93 (0.10) biceps sft (mm) gg 13.91 (8.46) �1.426 (�5.198, 2.345) 0.457 15.39 (9.15) 4.372 (�0.343, 8.400) 0.060 ga þ aa 12.49 (8.68) 19.75 (9.48) triceps sft (mm) gg 25.09 (14.42) �1.216 (�7.588, 5.156) 0.707 23.18 (8.54) 4.382 (0.196, 8.567) 0.060 ga þ aa 23.87 (14.63) 27.55 (10.04) abdominal sft (mm) gg 42.65 (18.48) �2.687 (11.677, 6.302) 0.556 29.93 (12.57) 8.571 (2.445, 14.697) 0.033a ga þ aa 39.98 (21.24) 38.46 (15.22) supra-iliac sft (mm) gg 39.96 (16.91) �4.700 (�13.001, 3.601) 0.266 21.71 (10.10) 5.338 (0.810, 9.866) 0.049a ga þ aa 35.27 (19.55) 27.02 (10.90) thigh sft (mm) gg 37.35 (24.53) �0.893 (�11.704, 9.918) 0.871 32.72 (12.70) 6.765 (0.712, 12.818) 0.058 ga þ aa 36.45 (24.87) 39.48 (14.38) sub-scapular sft (mm) gg 26.61 (12.57) �0.495 (�6.297, 5.307) 0.867 24.11 (11.52) 5.774 (0.194, 11.354) 0.060 ga þ aa 26.12 (13.43) 29.87 (13.37) forearm (mm) gg 281.82 (27.97) �4.182 (�18.783, 10.419) 0.573 241.86 (30.49) 13.995 (�2.116, 30.106) 0.099 ga þ aa 277.61 (33.25) 255.74 (35.66) percentage of body fat gg 28.21 (10.69) �0.997 (�5.664, 3.670) 0.674 28.35 (8.88) 3.672 (�0.421, 7.764) 0.099 ga þ aa 27.22 (10.78) 32.00 (9.86) association with anthropometric traits was assessed by linear regression using dominant model of inheritance. the effect sizes (b) with 95% confidence intervals and p-values were provided after adjustment for age. aan adjusted and corrected p-value <0.05 was considered statistically significant. bmi: body mass index; ci: confidence interval; hc: hip circumference; sd: standard deviation; sft: skin fold thickness; wc: waist circumference; whr: waist-tohip ratio. upsala journal of medical sciences 231 questionnaire. however, there was a weak (p ¼ 0.048) negative (protective) association of the variant rs2815752 with random eating timings in males only. in contrast, micali et al. reported a nominal (p ¼ 0.047) positive association of the rs2815752 with binge-eating (episodes of overeating with loss of control occurring on average once a week over 3 months and accompanied by distress) in male adolescents but not in females (41). the strengths and the limitations of the current study can be indicated at this point. our study was based on a casecontrol design in which cases and controls were matched for age and gender. many of the obesity-related parameters table 4. assessment of the association of the rs2815752 with obesity-related metabolic variables in gender stratified obese cases and controls. variables (unit of measurement) dominant model males females mean (sd) adjusted for age and bmi mean (sd) adjusted for age and bmi b (95% ci) p-value b (95% ci) p-value systolic bp (mmhg) gg 119.57 (11.47) 2.510 (�2.986, 8.006) 0.369 110.72 (12.50) 0.528 (�5.144, 6.200) 0.854 ga þ aa 122.01 (13.60) 116.25 (16.91) diastolic bp (mmhg) gg 77.39 (9.15) 3.334 (�0.938, 7.607) 0.125 73.60 (8.10) 2.125 (�2.463, 6.712) 0.362 ga þ aa 80.65 (10.61) 77.60 (11.53) vai (mmol/l) gg 3.65 (1.79) �0.615 (�1.665, 0.435) 0.249 3.31 (2.35) 0.000 (�0.83, 0.830) 1.00 ga þ aa 3.03 (2.57) 3.60 (1.91) lap (mmol/l) gg 63.87 (42.92) �6.770 (�23.242, 9.703) 0..419 44.69 (36.35) �2.721 (�15.016, 9.574) 0.663 ga þ aa 56.50 (50.32) 60.72 (44.02) tyg index gg 8.91 (0.43) �0.250 (�0.471, �0.028) 0.027 8.55 (0.46) �0.053 (�0.244, 0.139) 0.587 ga þ aa 8.66 (0.57) 8.59 (0.48) fbg (mg/dl) gg 102.26 (14.46) �0.60 (�10.44, 9.24) 0.904 106.40 (19.28) �4.042 (�12.947, 4.863) 0.372 ga þ aa 101.62 (24.38) 105.38 (21.30) insulin (ml u/ml) gg 22.01 (9.12) 1.99 (�4.76, 8.73) 0.562 23.57 (13.57) �1.673 (�6.935, 3.589) 0.531 ga þ aa 23.84 (17.94) 25.12 (13.12) homa-ir gg 5.61 (2.64) 0.48 (�1.57, 2.52) 0.642 6.23 (3.55) �0.546 (�2.128, 1.035) 0.496 ga þ aa 6.05 (5.31) 6.67 (4.04) homa-is gg 0.01 (0.02) �0.001 (�0.004, 0.003) 0.681 0.01 (0.01) 0.001 (�0.003, 0.004) 0.623 ga þ aa 0.01 (0.01) 0.01 (0.01) cholesterol (mg/dl) gg 164.74 (46.57) �11.075 (�28.379, 6.230) 0.208 158.96 (33.16) �8.095 (�23.264, 7.074) 0.294 ga þ aa 153.52 (39.61) 153.25 (36.40) triglycerides (mg/dl) gg 158.26 (69.32) �26.360 (�62.172, 9.452) 0.148 105.16 (41.14) 0.337 (�19.156, 19.831) 0.973 ga þ aa 131.67 (86.85) 112.76 (48.02) hdl (mg/dl) gg 27.43 (6.83) 1.274 (�2.235, 4.773) 0.474 31.52 (7.03) �0.955 (�4.517, 2.606) 0.597 ga þ aa 28.71 (8.18) 30.25 (8.31) ldl (mg/dl) gg 103.56 (40.53) �9.178 (�24.174, 5.818) 0.229 91.64 (31.62) �11.629 (�24.169, 0.912) 0.069 ga þ aa 94.10 (36.30) 86.06 (31.38) vldl (mg/dl) gg 31.51 (13.95) �4.979 (�12.098, 2.140) 0.169 20.88 (8.26) 0.444 (�3.377, 4.264) 0.819 ga þ aa 26.49 (17.17) 22.64 (9.34) chr gg 6.18 (1.78) �0.486 (�1.302, 0.33) 0.241 5.54 (2.37) �0.389 (�1.107, 0.330) 0.287 ga þ aa 5.69 (1.94) 5.33 (1.57) cri gg 6.20 (1.79) �0.579 (�1.313, 0.155) 0.121 5.28 (1.63) �0.149 (�0.768, 0.470) 0.635 ga þ aa 5.62 (1.72) 5.29 (1.47) ai gg 3.84 (1.31) �0.429 (�0.949, 0.090) 0.105 3.06 (1.22) �0.352 (�0.791, 0.087) 0. 115 ga þ aa 3.41 (1.27) 2.94 (0.48) tg/hdl-c gg 6.09 (2.93) �1.056 (�2.788, 0.677) 0.231 3.63 (2.23) 0.096 (0.763, 0.955) 0.826 ga þ aa 5.03 (4.20) 4.00 (2.02) association of the variant rs2815752 with metabolic traits was assessed by linear regression using dominant model of inheritance. the effect sizes (b) with 95% confidence intervals and p-values were provided after adjustment for age and bmi. ai: atherogenic index; bp: blood pressure; chr: cholesterol hdl ratio; ci: confidence interval; cri: coronary risk index; fbg: fasting blood glucose; hdl: highdensity lipoprotein; homa-ir: homeostasis model assessment of insulin resistance; homa-is: homeostasis model assessment of insulin sensitivity; lap: lipid accumulation product; ldl: low-density lipoprotein; sd: standard deviation; tg/hdl-c: triglyceride-to-hdl-c ratio; tyg: product of triglyceride and glucose; vai: visceral adiposity index; vldl: very-low-density lipoprotein. table 5. assessment of the association of rs2815752 variant with eating behaviour in gender-stratified obese/control pairs using a dominant model of inheritance. categorical traits response males females genotype adjusted p-value genotype adjusted gg ga þ aa or (95% ci) p-value (corrected) gg ga þ aa or (95% ci) p-value eating pattern random 19 (82.6%) 106 (55.5%) 0.25 (0.08, 0.77) 0.016 0.048a 13 (52.0%) 89 (59.7%) 1.16 (0.48, 2.79) 0.742 specific 4 (17.4%) 85 (44.5%) 12 (48.0%) 60 (40.3%) diet unconsciousness yes 18 (18.3%) 145 (75.9%) 0.88 (0.31, 2.51) 0.807 0.807 15 (60.0%) 103 (69.1%) 1.44 (0.59, 3.52) 0.420 no 5 (21.7%) 46 (24.1%) 10 (40.0%) 46 (30.9%) tfdf high 10 (43.5%) 66 (34.6%) 0.69 (0.27, 1.71) 0.418 0.627 7 (28.0%) 32 (21.5%) 0.48 (0.17, 1.33) 0.159 mod–low 13 (56.5%) 125 (65.4%) 18 (72.0%) 117 (78.5%) categorical traits were assessed using logistic regression using dominant model. parameters were adjusted for both age and bmi. aan adjusted and corrected p-value <0.05 was considered statistically significant. mod–low: moderate to low; tfdf: tendency towards fat-dense food. 232 s. rana and m. mobin investigated in our study have not been studied before in relation to the negr1 rs2815752. the study employed multiple genetic models for seeking association of the rs2815752 with obesity via regression analysis. the study also applied the calculation of h index in case of getting association in more than one genetic model for an indication of the appropriate mode of inheritance. analyses were adjusted for covariates such as age and/or bmi depending upon the situation. in addition, the study also included corrections for multiple comparisons in order to avoid false-positive results. the limitations of the current study include the likelihood of selfreporting bias in case of behavioural data that were selfreported by the study participants via a questionnaire. in addition, the sample size was considerably reduced after the gender-based stratification, resulting in decreased statistical power (reduced number of participants in the subgroup by sex). thus, the findings may be corroborated by further investigations of this association in a considerably larger sample of pakistani females. in conclusion, our study revealed a gender-specific association of the negr1 rs2815752 variant with obese phenotype and some related anthropometric traits such as weight, bmi, wc, hc, abdominal sft, and supra-iliac sft in females of the pakistani population. on the other hand, there was no association of the rs2815752 with obesity-related metabolic traits and eating behaviour. acknowledgements the authors are thankful to mr. saad mirza, ms. soma rahmani, ms. ayesha sultana, and mr. adil bhatti for their contribution in sample collection. disclosure statement no potential conflict of interest was reported by the author(s). funding this work was supported by a recurring grant given by the international center for chemical and biological sciences (iccbs), university of karachi, pakistan; and by a research grant awarded by the higher education commission, pakistan (ref. no. 5740/sindh/nrpu/r&d/hec/ 2016). the funding bodies did not play any role in the design of the study, sample collection, data collection, data analysis, and interpretation, or in writing the manuscript. notes on contributors sobia rana, ph.d., is an assistant professor at the international center for chemical and biological sciences (iccbs), university of karachi, pakistan. maha mobin, m.phil., is a junior research fellow at the international center for chemical and biological sciences (iccbs), university of karachi, pakistan. orcid sobia rana http://orcid.org/0000-0003-2540-5170 maha mobin http://orcid.org/0000-0003-2312-5706 references 1. church t, martin ck. the obesity 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handling in the isolated perfused rat kidney: demonstration of vasopressin v2-receptor-dependent calcium reabsorption original article electrolyte handling in the isolated perfused rat kidney: demonstration of vasopressin v2-receptor-dependent calcium reabsorption krister bamberga , lena william-olssonb , ulrika johanssonb, anders arnerc , judith hartleib-geschwindnerd and johan s€allstr€ome,f atranslational sciences and experimental medicines, research and early development, cardiovascular, renal and metabolism, biopharmaceuticals r&d, astrazeneca, gothenburg, sweden; bbioscience renal, research and early development, cardiovascular, renal and metabolism, biopharmaceuticals r&d, astrazeneca, gothenburg, sweden; cdepartment of clinical sciences lund, lund university, lund, sweden; dprojects, research and early development, cardiovascular, renal and metabolism, biopharmaceuticals r&d, astrazeneca, gothenburg, sweden; edepartment of medical cell biology, uppsala university, uppsala, sweden; fdepartment of physiology and pharmacology, karolinska institutet, stockholm, sweden abstract background: the most profound effect of vasopressin on the kidney is to increase water reabsorption through v2-receptor (v2r) stimulation, but there are also data suggesting effects on calcium transport. to address this issue, we have established an isolated perfused kidney model with accurate pressure control, to directly study the effects of v2r stimulation on kidney function, isolated from systemic effects. methods: the role of v2r in renal calcium handling was studied in isolated rat kidneys using a new pressure control system that uses a calibration curve to compensate for the internal pressure drop up to the tip of the perfusion cannula. results: kidneys subjected to v2r stimulation using desmopressin (ddavp) displayed stable osmolality and calcium reabsorption throughout the experiment, whereas kidneys not administered ddavp exhibited a simultaneous fall in urine osmolality and calcium reabsorption. epithelial sodium channel (enac) inhibition using amiloride resulted in a marked increase in potassium reabsorption along with decreased sodium reabsorption. conclusions: a stable isolated perfused kidney model with computer-controlled pressure regulation was developed, which retained key physiological functions. the preparation responds to pharmacological inhibition of enac channels and activation of v2r. using the model, the dynamic effects of v2r stimulation on calcium handling and urine osmolality could be visualised. the study thereby provides evidence for a stimulatory role of v2r in renal calcium reabsorption. article history received 6 june 2020 revised 18 july 2020 accepted 28 july 2020 keywords avp; enac; kidney; vasopressin introduction the most profound effect of vasopressin (avp) on the kidney is to increase water reabsorption in the collecting duct mediated through vasopressin v2-receptors (v2r), but there are also data that indicate effects on calcium transport (1,2). the isolated perfused kidney model (reviewed in (3,4)) offers the advantage that test substances and their impact on kidney function can be studied in a more controlled way than in the intact animal. in the isolated organ model, perfusion pressure can be held constant, thereby minimising confounding secondary effects caused by blood pressure alterations occurring in the intact animal. furthermore, the concentration of electrolytes and pharmacological agents in the perfusion medium can be precisely controlled. experiments on isolated kidney preparations can be performed using constant perfusion flow. in such preparations the pressure in the vascular system will change with the vessel tone in the kidney (5,6). therefore, a pressure control system is used in many preparations in order to maintain a stable arterial pressure in the physiological range (3,7). in principle, these systems have a pressure sensor that is connected to a feedback system that regulates the perfusion pump in order to keep the pressure constant. this introduces several technical challenges. the pressure should be measured at the tip of the perfusion cannula in the renal artery, which requires a duallumen cannula. however, given the small size of the artery in mice and rats, it is difficult to place this relatively large cannula in the renal artery. to overcome this, a short section of the aorta can be kept to serve as an adapter for the perfusion cannula (7). another solution is to measure the pressure anywhere in the perfusion system at the level of the kidney. the problem with this approach is that the pressure fall in the fine perfusion cannula is significant and will be flowdependent. consequently, the kidney will be perfused with a contact johan s€allstr€om johan.sallstrom@mcb.uu.se department of medical cell biology, uppsala university, box 571, husargatan 3, se-751 23 uppsala, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 4, 274–280 https://doi.org/10.1080/03009734.2020.1804496 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1804496&domain=pdf&date_stamp=2020-10-22 http://orcid.org/0000-0002-0538-6083 http://orcid.org/0000-0002-2604-2183 http://orcid.org/0000-0003-1386-090x http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1804496 http://www.tandfonline.com different pressure than measured. thus, obtaining a constant perfusion pressure using a feedback system is associated with non-trivial technical problems. in this report, a new approach for perfusion pressure control is presented. in principle a basal pressure-flow calibration curve is constructed at the start of the experiment. the system can thereby automatically correct the flow and maintain the desired pressure at the tip of the perfusion cannula. using this system, the stability of the kidney in terms of electrolyte handling, glomerular filtration rate (gfr), and urine production was optimised, whereupon the effects of vasopressin (avp) on renal calcium handling were studied. the most well-described hormonal regulator of renal calcium handling is parathyroid hormone (pth), which promotes active calcium reabsorption (8). the parathyroid gland, discovered in uppsala in 1877 by ivar sandstr€om (9), secretes pth in response to low serum calcium concentrations. several lines of evidence demonstrate that also avp stimulates calcium reabsorption. earlier studies in brattleboro rats with diabetes insipidus demonstrated that when long-term administration of the selective v2r agonist desmopressin (ddavp) was ceased, the excretion of calcium increased (1). in a more recent clinical trial, patients with central diabetes insipidus (cdi) and nephrogenic diabetes insipidus (ndi) were challenged with ddavp administration (2); cdi patients demonstrated a reduced excretion of calcium, whereas no change was observed in the ndi patients. several channels have been identified for calcium transport along the nephron (10), and mechanistic studies have indicated a stimulatory role of avp both in the cortical thick ascending limb (ctal) (11) and the collecting duct (12). the specific effects of avp on calcium handling in the kidney are, however, not fully understood. the isolated kidney model provides unique possibilities to explore the effects of avp given that it is possible to precisely control the concentration in the perfusate, which is not possible in vivo where systemic variations in hormone levels occur. materials and methods male sprague-dawley rats (cdvr igs, charles river, germany) were used. the animals were anaesthetised using isoflurane, the jugular vein was catheterised, and the abdomen opened via a midline incision. the right kidney was dissected free and the ureter was catheterised using pe10 polyethylene tubing. in order to prevent clotting, a bolus injection of 1500 iu/kg bw heparin (heparin leo, 5000 ie/ml, leo pharma ab, malm€o, sweden) was administered via the jugular vein before the kidney was perfused. to avoid leakage of perfusion fluid, branches of the renal artery such as the suprarenal artery were ligated. a blunt needle was introduced into the right renal artery through the mesenteric artery and secured by silk thread, whereupon the perfusion was started. the renal vein was cut open and the kidney removed and placed in the perfusion system, where it was kept throughout the experiment. all experiments were approved by the local animal ethics committee (reference number n23/13). perfusion system a recirculating perfusion system, modified from a previously described method (13), was used with a constant perfusion pressure (90 mmhg) at 37 �c. all parts of the perfusion system up to the kidney were water-mantled and connected to a constant temperature circulating bath, ensuring constant temperature in the renal parenchyma, which is important given that temperature affects kidney function (14). the kidney was perfused through the renal artery by a computercontrolled peristaltic pump (minipuls iii, gilson inc., middleton, wi, usa). the perfusate was returned to the reservoir by another peristaltic pump (pumpdrive 5001, heidolph instruments gmbh, schwabach, germany). the returning perfusate was filtered using a glass filter (porosity 3 or 4, robu glasfilter-geraete gmbh, hattert, germany) followed by a high-flow syringe filter (acrodisc 32 mm, 5 mm supor membrane, pall life sciences, ann arbour, mi, usa). constant perfusion pressure was maintained by using a microcomputer (described below) that automatically adjusts the peristaltic pump. the reservoir containing 200 ml perfusion buffer was constantly gassed with 95% o2 and 5% co2. the ph in the reservoir was monitored by an electrode and adjusted to physiological levels (ph 7.40) if necessary by addition of small amounts of hcl (1 mol/l). the perfusion buffer was a modified krebs–henseleit buffer with added sodium pyruvate (0.3 mmol/l), sodium l-lactate (2 mmol/l), alfa-ketoglutarate (1 mmol/l), malate (1 mmol/l), urea (6 mmol/l), and 20 amino acids using a stock solution prepared according to taft (4). at the day of the experiment, bovine serum albumin (bsa) (bovine serum albumin lyophilised powder �96%, sigma-aldrich, st. louis, mo, usa), calcium chloride (2.5 mmol/l), fitc-sinistrin (11.25 mg/l; fresenius-kabi, linz, austria), ddavp (400 ng/l; minirinvr , ferring l€akemedel ab, sweden) were added. pressure control system a microcontroller was constructed using the arduino uno device (www.arduino.cc). the pressure signal from the transducer is fed into the controller via a bridge amplifier. the pressure sensor was placed at the level of the kidney. the output from the microcontroller determines the speed of the peristaltic pump. a computer programme on the controller was developed that continuously calculates the difference between the desired perfusion pressure and the actual perfusion pressure, and applies a correction to the pump rate using a proportional–integral–derivative (pid) regulator (arduino pid library) in order to maintain the user-set pressure level. before an experiment was commenced, a calibration function was activated on the controller which runs the pump through a number of pre-defined flow rates. the pressure at each level was recorded by the controller, which generates a calibration curve, using a second order polynomial fit, that is used in the consecutive experiment. this procedure thus allows for a flow-independent control of the perfusion pressure. flow data from the experiments were collected and stored using a powerlab data acquisition upsala journal of medical sciences 275 http://www.arduino.cc system (adinstruments, bella vista, nsw, australia) connected to a personal computer. ten-minute mean values of the collected perfusion data were calculated for further analysis. experimental series three experimental series were performed encompassing five groups. in the first series, the system was optimised by evaluating the effect of 7.5% versus 6% bsa in the perfusate in eight kidneys per group. previous studies (13,15) have demonstrated that an elevated albumin concentration contributes positively to stability and reabsorptive capacity of the isolated kidney. the albumin concentration considered most beneficial regarding reabsorption and stability was chosen. the second series was performed in order to study the effect of v2r stimulation using ddavp in six kidneys using 7.5% bsa (using the previously performed 7.5% bsa experimental group as control). finally, in a third series, the response to the epithelial sodium channel (enac) inhibitor amiloride (amiloride hydrochloride hydrate, sigma-aldrich, st. louis, mo, usa; dissolved in water and administered to the fluid reservoir, 5 mmol/l) in the presence or absence of ddavp was assessed in six kidneys per group. urine was collected and the volumes quantified gravimetrically throughout the experiment every 10 min. in the middle of each 10-min period, a sample (0.5 ml) was drawn from the reservoir. samples of urine and perfusate were analysed for fitc-sinistrin using a spectrophotometer (paradigm detection platform, beckman coulter). electrolytes and glucose were analysed using a blood gas analyser (abl700, radiometer, copenhagen, denmark; or abx pentra 400, horiba medical, kyoto, japan) and osmolality using a micro osmometer (model 3 mo or 2020; advanced instruments, norwood, ma, usa). calculations and statistics all parameters were calculated for each 10-min urine collection period. gfr was calculated as the renal clearance of fitc-sinistrin that has similar properties as inulin but better solubility (16). fractional reabsorption (fr) for sodium, potassium, calcium, chloride, and glucose was calculated as follows: fr ¼ filtered amount�excreted amount filtered amount (1) the filtered amount was calculated as gfr multiplied by the perfusate concentration of the electrolyte, whereas the secreted amount was calculated as urine flow multiplied by the urine concentration of the electrolyte. the results are reported as mean values ± standard error of the mean. single comparisons were performed using student’s t test. multiple comparisons were performed using two-way anova and, when appropriate, followed by fisher’s post hoc test. p < 0.05 was considered statistically significant. calculations were performed using r version 3.6.2 (r foundation for statistical computing, vienna, austria) and excel (microsoft, redmond, wa, usa). results functional optimisation of the isolated perfused kidney renal perfusion was stable throughout the experiment and was not affected by altering the albumin concentration (figure 1). gfr decreased throughout the experiment (approximately 50%) in both groups (figure 1). when using 7.5% bsa versus 6% bsa, gfr tended to be lower, but more stable over time, but the difference did not reach statistical significance. the diuresis was significantly affected by the albumin concentration; kidneys perfused with 7.5% bsa had a diuresis that was more stable over time throughout the experiment and with less than half the urine output compared with kidneys perfused with 6% bsa (figure 1). altering the bsa concentration had a large impact on electrolyte handling (table 1). the fractional sodium, chloride, calcium, and glucose reabsorption was significantly increased when using 7.5% bsa. given that 7.5% bsa contributes to a more stable 4 0 5 0 6 0 7 0 8 0 p e rf u si o n ( m l /m in ) g f r ( µl /m in ) 0 5 0 0 1 0 0 0 1 5 0 0 d iu re si s (µ l /m in ) 0 30 60 90 120 150 180 0 1 0 0 2 0 0 3 0 0 bsa 6% bsa 7.5% * time (minutes) figure 1. perfusion, glomerular filtration rate (gfr), and diuresis in kidneys perfused with 6% or 7.5% bovine serum albumin (bsa), respectively. �p < 0.05 between groups, repeated measures anova. 276 k. bamberg et al. preparation, this concentration was used for the consecutive experimental series. effect of desmopressin (ddavp) on calcium reabsorption in the control group, the urine osmolality gradually fell to about 200 mosm/kg during the experiment, whereas in ddavp-treated kidneys the osmolality increased to about 350 mosm/kg and was sustained at that level (figure 2). the effect on calcium reabsorption was particularly pronounced and displayed a clear temporal correlation to the changes in urine osmolality, whereas sodium reabsorption was not significantly changed (figure 2). when the change in osmolality and urine flow up to the first hour of the experiment was plotted against the corresponding change in calcium reabsorption, the reduction in urine osmolality and the increase in urine flow during the experiment was correlated to a reduced calcium reabsorption (figure 3). the goodnessof-fit of the linear regression was better for urine flow than osmolality. addition of ddavp to the perfusate did not affect gfr or renal perfusion compared with perfusate containing only 7.5% bsa but tended to reduce the urine flow rate (table 1). the ddavp group exhibited a significantly increased fractional potassium reabsorption, but there was a general tendency for an increase in the fractional reabsorption of all measured electrolytes (table 1). effect of epithelial sodium channel (enac) inhibition administration of amiloride caused a pronounced increase in fractional potassium reabsorption, whereas fractional sodium reabsorption decreased (figure 4). the changes were not affected by ddavp. ddavp-treated kidneys displayed an elevated fractional calcium reabsorption compared with the control group both during baseline (0.99 ± 0.00 versus 0.94 ± 0.02; p < 0.05) and during amiloride treatment (0.99 ± 0.00 versus 0.90 ± 0.02; p < 0.05), which agrees with observations in the previous series described above. discussion the experiments performed demonstrate that the presented new approach for pressure control was able to maintain a functional preparation with stable perfusion for up to 3 h. by using the method, the surgical procedure is facilitated since the cannula can be placed directly in the renal artery; only two experiments failed due to problems with surgery. using a higher concentration (7.5%) of albumin promoted fluid and electrolyte reabsorption and contributed to a more stable diuresis over time. addition of ddavp tended to further promote reabsorption of most electrolytes, leading to an electrolyte reabsorption approaching in vivo values (17,18). table 1. three-hour mean values in kidneys perfused with 6% or 7.5% bovine serum albumin (bsa) or 7.5% bsa þ desmopressin (ddavp; 400 ng/l). 6% bsa 7.5% bsa 7.5% bsa þ ddavp perfusion (ml/min) 58 ± 3 60 ± 4 54 ± 2 gfr (ll/min) 899 ± 105 684 ± 90 747 ± 96 urine flow (ll/min) 167 ± 30 61 ± 14a 27.67 ± 7.96 frna 0.88 ± 0.02 0.96 ± 0.01 a 0.97 ± 0.01 frk 0.38 ± 0.02 0.51 ± 0.02 a 0.61 ± 0.03b urine na/k ratio 10.33 ± 1.63 3.61 ± 0.74a 3.34 ± 1.07 frcl 0.86 ± 0.02 0.95 ± 0.01 a 0.97 ± 0.01 frca 0.79 ± 0.02 0.92 ± 0.01 a 0.99 ± 0.00b fr glucose 0.96 ± 0.01 0.97 ± 0.00a 0.98 ± 0.00 urine osmolality (mosm/kg) 241 ± 9 230 ± 9 353 ± 9b ap < 0.05 versus 6% bsa. bp < 0.05 versus 7.5% bsa. ca: calcium; cl: chloride; fr: fractional reabsorption; gfr: glomerular filtration rate; k: potassium; na: sodium. 100 200 300 400 100 200 300 400 * 0.85 0.90 0.95 1.00 0.85 0.90 0.95 1.00 * time (min) 0.90 0.95 1.00 0 90 180 control ddavp 0.90 0.95 1.00 o sm o la lit y (m o sm /k g ) f r c a f r n a figure 2. urine osmolality, fractional calcium (frca), and sodium reabsorption (frna) in kidneys perfused with desmopressin (ddavp; 400 ng/l) compared to control experiments. the bars represent mean values from the period 90–180 min (indicated by the line). albumin concentration in the perfusate was 7.5%. �p < 0.05 versus control. upsala journal of medical sciences 277 ddavp also reversed the fall in urine osmolality that occurred without ddavp, which further contributed to the stability of the system. the applied experimental model is thus considered suitable for studies on electrolyte transport, which is illustrated by the prompt changes in potassium and sodium handling in response to administration of the potassium-sparing diuretic amiloride that inhibits enac. the presented model also demonstrates that it is possible for researchers to design their own experimental equipment fitted to their needs using inexpensive and widely available programmable microcontrollers. other recently published applications include perfusion controllers for hydrogels (19) and temperature regulators for intravital microscopy (20). we have, however, not found any recent developments regarding perfusion systems for kidneys. controlled water reabsorption in the kidney according to physiological needs takes place in the collecting duct where aquaporin-2 (aqp2) channels are incorporated into the apical cell membrane through stimulation of v2r by avp. aquaporins increase water permeability and consequently increase fluid reabsorption to the hyperosmolar interstitium (21). the effects of avp on calcium handling is not that well described, but there is evidence of a stimulatory effect on calcium reabsorption from studies both in rodents (1) and in humans (2). in the present experiments, the kidneys are naturally exposed to the normal physiological levels of avp before the organ was excised from the rat. when perfused without avp, the antidiuretic effect rapidly declined, as demonstrated by the gradually reduced osmolality and increased urine flow. the reduced osmolality due to absence of avp is expected, since it is the normal mechanism for regulation of fluid homeostasis in response to water loading and demonstrates that this mechanism is functional in the isolated kidney. the fall in osmolality was accompanied by a matched decrease in calcium reabsorption that, interestingly, had similar temporal characteristics. given that the effects of vasopressin require aquaporin trafficking (22), the simultaneous effects on water and calcium reabsorption indicate a common tubular target and a related mechanism. incorporation of aquaporins into the apical membrane involves a complex series of events mediated by the g-protein-coupled v2r at the basolateral membrane (reviewed in (22,23)). among the different signalling pathways involved, calcium signalling has osmolality (mosm/kg) f r c a −0.25 −0.20 −0.15 −0.10 −0.05 0.00 0.05 r2= 0.49 urine flow (µl/min) f r c a −0.25 −0.20 −0.15 −0.10 −0.05 0.00 0.05 0 −20−50−100−150 0 20 40 60 80 100 r2= 0.79 (a) (b) figure 3. correlation between change (d) in urine osmolality (a) and urine flow (b) and fractional calcium reabsorption (frca) in kidneys perfused with 7.5% albumin without ddavp. values are calculated as the change from the first 10-min period up to 60 min into the experiment. the values are individual time points derived from the two experimental groups without added ddavp. a straight line was fitted to the data using linear regression, with the square of the correlation coefficient (r2) indicated. 0 20 40 60 80 100 0 .9 0 0 .9 2 0 .9 4 0 .9 6 0 .9 8 1 .0 0 time (min) f r n a baseline amiloride 0 .9 0 0 .9 2 0 .9 4 0 .9 6 0 .9 8 1 .0 0 * 0 20 40 60 80 100 0 .2 0 0 .4 0 0 .6 0 0 .8 0 1 .0 0 time (min) f r k bsa 7.5% bsa 7.5% + ddavp baseline amiloride 0 .2 0 0 .4 0 0 .6 0 0 .8 0 1 .0 0 bsa 7.5% bsa 7.5% + ddavp f r k f r n a amiloride baseline amiloride baseline * * * 120 120 figure 4. fractional reabsorption of sodium (frna) and potassium (frk) in response to amiloride (5 mmol/l) in kidneys perfused with bsa 7.5% or bsa 7.5% þ desmopressin (ddavp; 400 ng/l). the left panel presents the time series data of fractions observed during the 120-min experiment period, whereas the right panel displays mean values at baseline (0–60 min) and at amiloride exposure (60–120 min). �p < 0.05 versus baseline. 278 k. bamberg et al. been shown to have a stimulatory role on aquaporin incorporation (24). the mechanisms for avp-mediated calcium reabsorption are not clear. data on pth-mediated transepithelial calcium transport have been linked to the apical transient receptor potential cation channel subfamily v member 5 and 6 (trpv5/6) in the distal convoluted tubule and the connecting tubule (25). in the collecting duct, further downstream of the tubular system, canonical transient receptor potential channel member 3 (trpc3) may instead have a particular role for calcium reabsorption (26). interestingly, avp has been shown to induce co-localization of aqp2 channels and trpc3 cation channels to the apical cell membrane in the medullary collecting duct. overexpression of these channels in cultured collecting duct cells was also related to increased transepithelial calcium flux (27). accordingly, calcium channels stimulated by avp may, besides being involved in the signalling mechanisms for aquaporin trafficking, directly contribute to calcium reabsorption. in line with this, trpc3 knockout mice displayed an increased urinary calcium concentration compared to their wild-type controls when subjected to water deprivation (28). the correlation observed in the present study between change in osmolality and urine flow and change in calcium reabsorption (figure 3), as well as their temporal relationship (figure 2), consequently further supports that avp increases reabsorption of both water and calcium through incorporation of these channels on the apical membrane of the cells in the collecting duct. the physiological relevance may be to protect against urolithiasis during water restriction, where otherwise calcium levels could be elevated to levels risking precipitation. another indication on the possible role of this channel in pathophysiology comes from a case report where trpc3 expression was found to be upregulated in a patient with williams–beuren syndrome, a rare neurodevelopmental disorder also associated with hypercalcemia (29). however, trpc3 expression was found both in the kidney and the intestinal epithelium. thus, an increased intestinal uptake could also have contributed to the elevated plasma calcium concentrations. to further assess that the perfused kidney model replicates in vivo physiological responses, we assessed the response to enac inhibition by amiloride. administration of amiloride caused a prompt decrease in sodium reabsorption accompanied by an increased potassium reabsorption. these are the expected effects of enac inhibition (30), which reduces potassium excretion by hyperpolarizing the principal cells in the collecting duct, thus reducing the electrochemical driving force for potassium extrusion into the lumen. enac inhibition will also lower the intracellular sodium concentration, thus reducing basolateral potassium entry through the na-k-atpase which also most likely contributes to the reduced potassium secretion (31). given that both enac and trpc3 are expressed in the principal cells (26,32), the normal response confirms that this nephron segment, where the proposed effects of avp on calcium reabsorption occurs, is functional in this model. fractional calcium reabsorption was higher in the ddavp group both during control conditions and during amiloride treatment. the elevated calcium reabsorption by ddavp supports the findings in the previous series and also indicates that the acute effect of ddavp on calcium handling is independent of sodium transport through enac. conclusion in the present report, a new model for pressure control of isolated perfused kidneys has been evaluated and found suitable for studies on electrolyte and fluid transport. the preparation responds to pharmacological inhibition of enac channels as well as activation of v2r. during v2r stimulation, calcium reabsorption was strongly promoted. using the presented experimental model, the dynamic effects of v2r-stimulation on calcium handling and urine osmolality was visualised, which, to the best of our knowledge, has not been previously described. the study thereby provides further evidence for the stimulatory role of v2r in calcium transport. disclosure statement k.b., l.w.o., u.j. and j.h.g. are employed by astrazeneca. j.s. is employed at the swedish medical products agency, se-751 03 uppsala, sweden. the views expressed in this paper are the personal views of the author and not necessarily the views of the government agency. funding the experimental study was performed 2013–2014 and was supported by astrazeneca, sweden. no financial support has been received from astrazeneca for preparation of the manuscript. notes on contributors krister bamberg, phd, director, scientific lead, ckd late stage portfolio at department of translational sciences and experimental medicine, early cardiovascular renal and metabolism biopharmaceuticals research and development, astrazeneca, gothenburg. lena william-olsson, bsc, in vivo biologist at department of bioscience renal, early cardiovascular renal and metabolism biopharmaceuticals research and development, astrazeneca, gothenburg. ulrika johansson, bsc, senior researcher at department of bioscience renal, early cardiovascular renal and metabolism biopharmaceuticals research and development, astrazeneca, gothenburg. anders arner, md, phd, researcher at the department of clinical sciences lund, lund university and professor emeritus at the department of physiology and pharmacology, karolinska institutet. judith hartleib-geschwindner, phd, senior director and global project lead at department of projects, early cardiovascular renal and metabolism biopharmaceuticals research and development, astrazeneca, gothenburg. johan s€allstr€om, phd, researcher at the department of medical cell biology, uppsala university and clinical assessor at the swedish medical products agency. orcid krister bamberg http://orcid.org/0000-0002-0538-6083 lena william-olsson http://orcid.org/0000-0002-2604-2183 anders arner http://orcid.org/0000-0003-1386-090x upsala journal of medical sciences 279 references 1. bouby n, trinh-trang-tan mm, bankir l. stimulation of tubular reabsorption of magnesium and calcium by antidiuretic hormone in conscious rats. study in brattleboro rats with hereditary hypothalamic diabetes insipidus. pflugers arch. 1984;402:458–64. 2. hanouna g, haymann j, baud l, letavernier e. vasopressin regulates renal calcium excretion in humans. physiol rep. 2015;3: e12562. 3. maack t. physiological evaluation of the isolated perfused rat kidney. am j physiol. 1980;238:f71–8. 4. taft dr. the isolated perfused rat kidney model: a useful tool for drug discovery and development. curr drug discov technol. 2004; 1:97–111. 5. marques a, da silva c, de souza p, de 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system pressure control system experimental series calculations and statistics results functional optimisation of the isolated perfused kidney effect of desmopressin (ddavp) on calcium reabsorption effect of epithelial sodium channel (enac) inhibition discussion conclusion disclosure statement references sups_a_524320 8..17 upsala journal of medical sciences. 2011; 116: 8–17 original article preparatory studies of composite mesenchymal stem cell islets for application in intraportal islet transplantation ida rasmusson duprez, ulrika johansson, bo nilsson, olle korsgren & peetra u. magnusson uppsala university, department of oncology, radiology and clinical immunology, division of clinical immunology, the rudbeck laboratory, uppsala, sweden abstract background. low engraftment and adverse immune reactions hamper the success rate of clinical islet transplantation. in this study, we investigated the capacity of human mesenchymal stem cells (mscs) to adhere to human islets of langerhans and their effects in immune modulation and during blood interactions in vitro. methods. composite msc–islets were formed by suspension co-culture, and the phenotype was evaluated by confocal microscopy. islet function was assessed by dynamic insulin release in response to glucose in vitro. mixed lymphocyte–islet reactions (mlir) and the tubing blood loop model were utilized as in vitro tools to analyse the effect of mscs on the innate and adaptive immune reactions triggered by the islets. results. mscs rapidly adhered to islets and spread out to cover the islet surface. insulin expression and secretion were sustained with the msc coating. msc-coated islets showed unaffected reactions with blood in vitro in comparison to control islets. furthermore, mscs suppressed lymphocyte proliferation induced by islet cells in mlir. conclusion. we conclude that it is possible to create composite msc–islets to enable delivery of the mscs by utilizing the adhesive capacity of the mscs. this could have beneficial immunosuppressive effects in optimizing pancreatic islet transplantation. key words: human islets of langerhans, human mesenchymal stem cells, human multipotent stromal cells introduction multipotent mesenchymal stromal/stem cells (mscs) are adherent cells that reside in the stroma, where they produce stromal components (1,2). through their multipotency mscs can differentiate towards fat, bone, or cartilage upon the correct stimuli (1). mscs have been shown to possess an immunosuppressive capacity and to protect against activated immune responses (3–5). mscs can also suppress adaptive immune reactions (6–9) and have shown a dramatic positive effect on severe graft-versus-host disease in humans (10,11). the beneficial effects of mscs make them attractive as tolerance-inducing cells during cell transplantation (3,12,13). transplantation of islets containing insulin-producing beta cells is currently evaluated as a treatment for patients with life-threatening hypoglycaemic episodes and unsatisfactory glycaemic control despite careful insulin therapy (14). one key factor for successful islet transplantation is to use a high number of islets, in part due to loss of islets in response to adverse immune reactions (15–17). the immunosuppressive capacity of mscs, alongside the ability to improve regeneration of damaged tissue (18–21) and to provide a supportive stromal environment, could benefit islet transplantation. mscs have been isolated from pancreatic ductal epithelium and may therefore have a natural role to protect and compose a suitable environment for the endocrine cells in the native pancreas (22). at the same time, the islets provide a surface for adherence of mscs which could improve the survival correspondence: peetra ulrica magnusson, uppsala university, division of clinical immunology, rudbeck laboratory c11, se-75185 uppsala, sweden. e-mail: peetra.magnusson@klinimm.uu.se (received 2 july 2010; accepted 9 september 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.524320 of mscs. in this study, we have analysed the interactions between islets and mscs concerning the potential of mscs to attach to the islets, and the consequences this has on insulin secretion of the islets, blood contact, and immune modulation by the mscs in vitro. delivery of islets and mscs separately during intraportal transplantation will lead to deposition of mscs to other sites than the islets. the possibilities to bind the mscs to the surface of the islets will not only make it possible to target the mscs to the same site as the islets in the clinical intraportal transplantation, but also increase the likelihood of initial contact between the mscs and the islets. here we show that the blood interactions of the msc–islets in the tubing blood loop model have no further negative effect upon the coagulation or the complement system. material and methods isolation of human islets of langerhans human islets of langerhans were isolated by the isolation laboratory of the nordic network in uppsala, sweden, as previously described (23), using a modified semi-automated digestion-filtration method (24–26). pancreases were obtained from brain-dead donors after appropriate consent for multi-organ donation. islet preparations with a purity ranging from 70% to 80% have been analysed in this study. purity was estimated by dithizone staining of endocrine islet tissue. islets were cultured in islet medium: connaught medical research laboratories (cmrl) 1066 culture medium with 10% human abo serum, 2.4 mm l-glutamine, 12 mm hepes, 12 mm nicotinamide, 6 mm sodium pyruvate, 50 mg/ml gentamycine, 20 mg/ml ciproxine, and 0.25 mg/ml fungizone. isolation and expansion of adult human mscs the human mscs used in this study were generously provided by professor katarina le blanc, karolinska institutet, stockholm, sweden. the mscs were isolated and expanded from bone marrow (bm) as previously described (27) following the approval by the ethics committee at huddinge university hospital. the cells were cultured in msc medium consisting of dulbecco’s modified eagle’s mediumlow glucose (dmem-lg), supplemented with 10% heat-inactivated foetal bovine serum (fbs) (from paa, pasching, austria) and 1% antibiotic solution. the cells were classified as mscs based on plastic adherence, differentiation to bone and fat, and expression of surface markers (28). mscs in passages 3–9 from 13 donors were used in this study. adherence and spreading of mscs on human islets approximately 1,000 human islets were mixed with 1 � 105, 2.5 � 105, or 5 � 105 mscs in 1 ml of islet culture medium. to evaluate the degree of coating, the mscs were stained with celltracker green according to the manufacturer’s instructions (molecular probes, eugene, or, usa). the mscs and islets were incubated at 37�c for 3 hours in cell suspension tubes, mixed gently every 30 minutes, and subsequently cultured in islet medium (n = 13). evaluation of the adherence and spreading of the mscs on the islet surface was performed by confocal laser scanning microscopy (zeiss lsm 510 meta, carl zeiss ag, göttingen, germany). to analyse the distribution of mscs, within the islets, islet preparations were fixed for 1 hour at room temperature (rt) in 4% formaldehyde, washed in phosphate buffered saline (pbs) overnight, incubated in 20% sucrose for 12–24 hours, before being embedded in optimal cutting temperature (oct) medium (tissue tech, sakura finetechnical, tokyo, japan) and snap-frozen in liquid nitrogen. sections of 10 mm were counterstained with 4¢,6¢diamidino-2-phenylindole (dapi) (10 mg/ml, fluka, sigma-aldrich, st.louis, mo, usa). measurement of insulin release and insulin dna the functionality of the islets coated with mscs compared with control islets from the same isolation was testedafter48hours.twentyhand-pickedmsc-coated and non-coated control islets were challenged in a dynamic perifusion system, beginning with a low-glucose concentration (1.67 mmol/l), followed by a high-glucose challenge (20 mmol/l) and then re-exposuretothelow-glucoseconcentration.fractions were collected at 6-minute intervals over 126 minutes and analysed for insulin content using commercial enzyme-linked immunosorbent assay (elisa) kits (mercodia, uppsala, sweden). stimulation index was calculated: (average of high-glucose samples)/(average of low-glucose samples). for measurements of insulin/ dna ratio, 20 hand-picked msc-coated and control islets were collected and analyzed for insulin content by elisa (mercodia). quant-it picogreen dsdna assay kit (invitrogen, carlsbad, ca, usa) was used for dna measurements and analysed by a fluoroscan ascent fluorometer luminometer (fl) (thermo electron corp., waltham, ma, usa). immunohistochemistry peripheral blood mononuclear cells (pbmc) were isolated from healthy human volunteers. the blood composite mesenchymal stem cell islets 9 was diluted in pbs, and pbmc were separated on a ficoll gradient (1.077 g/l; axis-shield poc as, oslo, norway). after three washes in pbs, the cells were resuspended in roswell park memorial institute (rpmi) 1640 medium supplemented with 10% human pooled ab serum, 2 mm l-glutamine, 100 u/ml penicillin, and 100 mg/ml streptomycin (complete rpmi medium). to obtain single islet cells the islets were washed twice in pbs and incubated in accutase (sigma-aldrich) for 10 minutes at 37�c. the reaction was stopped by addition of an equal volume of cold fbs, and the cell suspension was carefully vortexed to dissociate the islets. responder pbmc (100,000) and stimulator pancreatic single cells (100,000) were mixed in 200 ml of complete rpmi medium in triplicates, without or with 10,000 (10%) or 1,000 (1%) of mscs. stimulator cells and mscs were irradiated and co-cultured for 5 days. the proliferation of responder pbmc was measured after another 24 hours of tritium-labelled thymidine incorporation (1 mci, radiochemical centre, amersham, uk). the cells were harvested automatically on glass fibre filters, using a tomtec harvesting machine (harvester 96, tomtec, orange, ct, usa). two independent experiments with two islet preparations and in total four different mscs were tested (n = 6). transplantation of composite islets approximately 200 control human islets or an equal number of msc-coated human islets were transplanted under the kidney capsule of c57bl/6/sca nu/nu mice (scanbur ab, sollentuna, sweden). after 2 weeks, the kidneys were removed and stained for insulin (n = 3). to perform transplantation of syngeneic composite grafts, mouse islets were isolated and handpicked from c57bl/6/sca (scanbur, sweden), and approximately 200 islets were coated with 125,000 murine mscs derived from c57bl/6/sca, a kind gift from professor rafi gorodetsky, sharett institute of oncology, hadassah-hebrew university medical center, jerusalem, israel (29). the islets and msc–islets were transplanted under the kidney capsule, and grafts were retrieved 28 days after transplantation (n = 3). animal handling was performed with ethical permission approved by the authorities and according to the ukcccr guidelines for the welfare of animals in experimental neoplasia (30). one-way mixed lymphocyte–islet reactions (mlir) insulin expression in transplanted human islets was analysed in paraffin-embedded tissue. kidney biopsies were fixed in 4% paraformaldehyde for 24 hours and prepared for paraffin embedding. sections were stained for insulin (guinea-pig anti-insulin, dako, glostrup, denmark) following a secondary antibody (goat anti-rabbit envision, dako). finally, the sections were developed in aec (3-amino, 9-ethyl-carbazole, dako) and counterstained with haematoxylin. transplanted syngeneic msc–islets and control islets were prepared as described above including a decalcification step according to manufacturer’s instructions (pargeny, histolab, gothenburg, sweden) of the msc–islet grafts before sectioning and thereafter haematoxylin and eosin staining. the tubing blood loop model untreated control human islets and msc–islets were exposed to fresh human abo-compatible blood using a loop system consisting of polyvinyl chloride (pvc) tubings with a heparinized inner surface (31). the loop system was placed on a rocking apparatus in an incubator at 37�c to simulate a blood-flow inside the pvc tubing. loops with 7 ml of human blood were prepared, and 100 ml of pbs (negative control) or 500–800 coated or uncoated islets in 100 ml of pbs were added (n = 5). the loops were then closed with a polypropylene connector and placed in the 37�c incubator on the rocking apparatus. one ml of blood was collected at 3 different time points (5, 10 and 30 min) in tubes containing 50 ml 0.2 m naedta, ph 7.4, to determine platelet consumption, and plasma was prepared from the remaining blood in the sample for elisa analysis of c3a and thrombin antithrombin (tat) (mercodia, uppsala, sweden). statistical analysis statistics were calculated using the statistica software (statsoft scandinavia ab, uppsala sweden). results were compared using the non-parametric wilcoxon test, and p values £ 0.05 were considered statistically significant. results adherence and spreading of mscs mscs showed a dose-dependent coating of the islets. with addition of 100,000 mscs to approximately 1,000 islets, only a sporadic binding to the islet surface was detected. increased coating was observed 10 i. rasmusson duprez et al. when 250,000 and 500,000 mscs were used (figure 1a). a further increase in the number of mscs did not result in increased coverage. rather there were many single cells in suspension, whereas a proportion of the islets remained free of coating. when pancreatic islets and mscs were incubated together, the mscs adhered to the islets within 1 hour (figure 1b). after 5 hours, the mscs had initiated spreading and covered most of the islet surface. after 24–48 hours, the adherence of the cells was completed with no apparent improvement of coating nor loss of msc attachment for up to 96 hours, which was the selected end-point of the coating experiments (figure 1b). in addition to coating the surface of the islets, mscs were also found within the islet core (figure 1c). insulin secretion in composite msc–islets functionality of the islets was investigated by insulin secretioninadynamicperifusionsystem,wheretheislets were stimulated with different glucose concentrations. the shift from low to high glucose solution stimulates a rapid release of insulin. on average, the coating of islets with mscs did not affect the insulin secretion by the islets (figure 2a, n = 5). the stimulation index was not significantly different between control islets and composite msc–islets (11.0 ± 5.2 for control islets and 9.5 ± 3.6formsc–islets,average± sem,n= 5).additionally, insulin/dna ratio (pmol � l�1 � ng�1) showed no difference between control islets and composite msc– islets, even though the mscs contributed with dna in the insulin and dna ratio of msc–islets (figure 2b, n = 3). human islets or composite msc–islets were transplanted under the kidney capsule of nu/nu mice, and all grafts were positively stained for insulin when retrieved after 2 weeks (figure 2c, n = 3). syngeneic transplantation of mouse msc–islets transplantation of mouse islets, control islets, or msc–islets was performed under the kidney capsule of mice (n = 3). the grafts were removed 28 days after transplantation. by then, the msc–islet grafts were differentiated into bone, and the graft was enlarged in comparison to control (figure 3a, b). the bone formation was further verified after sectioning followed by haematoxylin and eosin staining (figure 3c). mscs and islet cells in mixed lymphocyte–islet reactions (mlir) to analyse the immunological effects of mscs on immune reactions induced by islets, we cultured peripheral blood mononuclear cells (pbmc) with dissociated allogeneic islet cells with or without allogeneic mscs in suspension culture during 6 days and studied lymphocyte proliferation by incorporation of 3h-thymidine during the last 24 hours. mscs and stimulator cells were irradiated not to proliferate, to yield a one-way mlir. the addition of 10% or 1% mscs significantly suppressed proliferation (figure 4). the positive control in which the effector cells reacted against allogeneic pbmc induced a proliferation of 42,493 ± 15,317 counts per minute (cpm) and reaction against islet cells 9,721 ± 3,796 cpm. the results were adjusted to per cent response relative to the respective control without mscs to adjust for donor differences. msc–islets in the tubing blood loop model in vitro studies of islets and msc–islets interactions with abo-compatible blood in tubing loops coated with heparin were investigated. the results showed a similar reduction in platelets (figure 5a) and granulocytes (figure 5b) and increase in lymphocytes (figure 5c) during 30 minutes of incubation in both groups due to infiltration of cells in blood clots. a similar activation of coagulation occurred by control islets and msc–islets as shown by increased levels of thrombin anti-thrombin (tat) (figure 5d). this effect was also shown by activation of the complement by increased levels of c3a (figure 5e). discussion we have evaluated the possibility to coat human islets of langerhans with human mscs and investigated their role in islet function in different in vitro models symbolizing the scenario of clinical islet transplantation. intravenous injections of mscs in rodents show deposit of the injected cells in the lungs (32), indicating that intravenous administration of the cells results in entrapment of the cells. the distribution of mscs depends on the site of infusion (33–35), and injuries can alter the distribution pattern with an increased cell migration to the site of injury (35,36). we incubated mscs with islets and studied adhesion and spreading to the islets for up to 96 hours. in addition to the good capacity to coat islets, mscs were also found within the islet core. coating of islets with mscs did not adversely affect the islet function as confirmed by measuring the composite mesenchymal stem cell islets 11 insulin release elicited by msc–islets after 48 hours of culture. our results show that the interactions between islets and mscs are rapid with a high degree of binding within the first 24 hours. this is compared to coating with aortic endothelial cells (ec) that required up to 7 days for an optimal coverage of the islet surface (23). the addition of mscs to composite ec–islets has previously shown enhanced a. b. c. figure 1. coating of pancreatic islets with mscs. human islets were coated with celltracked mscs (green). a: dose-dependent coating of islets with 100,000, 250,000, or 500,000 mscs after 48 hours of culture. b: the initial adherence and the following spreading of mscs after 1, 5, 48, and 96 hours of culture. one representative experiment is shown. c: mscs within the islet core after 48 hours of co-culture, nuclei in blue. scale bars = 100 mm. 12 i. rasmusson duprez et al. adhesion of the microvascular ec to the islet surface. besides facilitating binding of ec to the islet surface, msc-conditioned medium stimulated proliferation of ec (37). positive effects of mscs on revascularization in pancreatic islets have also been shown in vivo (38). mscs can suppress various immune responses (reviewed in (39)). in this study, we have analysed islets msc-islets 0.0 islets msc-islets 0.1 0.2 0.3 r a ti o i n s u li n /d n a 0.4 0.5 36 0 50 100 150 200 250 in s u li n ( p m o l/ l ) 300 350 400 450 42 48 54 60 66 72 78 time (min) 84 90 96 102 108 114 120 126 a. b. c. islets msc-islets figure 2. insulin secretion and expression in msc-coated islets. a: insulin secretion was measured in a dynamic perifusion system (n = 5) where control islets (black diamonds) or composite msc–islets (light triangles) were initially challenged with low-glucose (1.67 mmol/l) followed by high-glucose stimulation (20 mmol/l, 42–78 min). b: the ratio of insulin versus total dna (pmol � l�1 � ng�1) in islet homogenates (n = 3). c: insulin-containing cells survived in vivo, indicated by insulin-staining of grafts of human islets and msc–islets transplanted under the kidney capsule of nude mice retrieved after 2 weeks (n = 3). a. ** * b. c. figure 3. syngeneic transplantation of mouse msc–islets. a: control mouse islets (*) and mouse msc–islets (**) were transplanted under the kidney capsule of mice and retrieved after 28 days in vivo, showing enlargement of the msc–islet graft (**) in comparison to control islet graft (*) (n = 3). b: close up of the msc–islet graft. c: section of mouse msc–islet grafts after decalcification showing bone formation of differentiated mscs. bar = 100 mm. composite mesenchymal stem cell islets 13 in vitro the potential cellular rejection response that can be triggered by transplanted islets in mlir. mscs significantly reduced proliferation of lymphocytes, with possible importance in co-transplantation of islets with mscs, by inducing a local immunosuppressive milieu surrounding the islets. we dissociated the islets to optimize the contact between lymphocytes and islet cells, since whole islets were unable to stimulate measurable levels of proliferation (data not shown). during the dissociation of islets, naturally other islet cells than beta cells, such as alpha, delta, and pancreatic polypeptide (pp) cells, but also endothelial cells, are released, and they most likely contribute to stimulate proliferation. beta cells express low levels of human leukocyte antigen (hla) class i and do not express hla ii (40). only a small fraction of the non-endocrine cells express hla class dr, mainly macrophages and endothelial cells (41), which may explain the lower proliferation by responder lymphocytes. other factors that can alter the immunogenicity of islets are the production of inflammatory cytokines (42) and the presence of acinar cells in the dissociated islets that can suppress the proliferative response of lymphocytes (43). the effect of composite msc–islets on interactions with blood did not show any increased activation, which is beneficial for intraportal transplantation of composite grafts. mscs could play an important role in suppressing cellular rejection of transplanted islets. fiorina et al. suggested that murine mscs protect the pancreas from autoimmune destruction in a nod mouse model by recruiting the mscs to pancreatic lymph nodes to suppress t cells through the co-stimulatory molecule programmed death-ligand 1 (pd-l1) (44). itakura and colleagues reduced rejection of transplantation of pancreatic islets by co-transplantation of mscs. diabetic rats receiving an intraportal co-infusion of mscs, bone-marrow cells, and islets rejected the islets initially. however, half of them developed stable mixed chimerism and donor-specific immune tolerance, as shown by the engraftment of donor skin and secondset islet transplants and acute rejection of a thirdparty skin (45). undesired differentiation of cells should always be considered in transplantation studies using stem cells. we have chosen to use human mscs and human islets of langerhans. our attempts to use syngeneic models with mouse mscs in composite islets resulted in bone formation under the kidney capsule. this phenomenon was first shown by friedenstein et al. in 1966 (46). human mscs appear to be less inclined to form bone in vivo unless the correct stimuli are present (47). in this study we have not experienced bone differentiation of human mscs transplanted to nu/nu mice. in this study, we wanted to determine the outcome of coating pancreatic islets with mscs by investigating islet function and the effect on innate and adaptive immunity. to conclude, we show that mscs can both cover the surface and colonize the inner core of pancreatic islets. the mscs inhibited lymphocyte proliferation induced by islet cells, whereas the innate immune reactions were unaltered. formation of composite islet grafts also facilitated delivery of mscs together with the islets when transplanted under the kidney capsule. from these data we have shown that the mscs can be bound to the surface of the islets to ensure delivery at the site of transplantation where they may have promising effects on immune regulation to support engraftment of transplanted islets of langerhans. a+bx * *** * 0 20 40 60 80 100 120 p ro li fe ra ti v e r e s p o n s e ( % ) a+ix no mscs +10% msc +1% msc figure 4. mscs suppressed lymphocyte proliferation against dissociated islets in mlir. in one-way proliferative assays between pbmcs and pancreatic islet single cells (a+ix), the addition of 10% (dark grey bars) and 1% mscs (light grey bars) significantly suppressed proliferation, compared to the positive control without mscs (black bars). the relative inhibition was similar to the control where the lymphocytes reacted against allogeneic irradiated lymphocytes (a+bx). the figure shows an average of six different islets/pbmc/msc donor combinations (***p < 0.001; *p < 0.05). 14 i. rasmusson duprez et al. acknowledgements this study was supported by grants from the swedish research council/ssf/vinnova (60761701), the swedish research council (70287302, 90293501), the juvenile diabetes research foundation, barndiabetesfonden (children’s diabetes association), magnus bergvall’s foundation, anders otto swärds foundation, swedish society for medical research, the ernfors family foundation, the novo nordisk foundation, and the national institutes of health (u01ai065192). the authors thank the nordic network for islet transplantation and the isolation team for excellent islet preparations, as well as margareta engkvist and naomi forslund for outstanding technical assistance. i.d., p.m., b.n., and o.k. participated in research design. i.d., p.m., and u.j. participated in the 30 35 40 45 g ra n u lo c y te s ( % ) 50 55 60 65 5 15 time (min) 30 pbs islets msc-islets 5 30 35 40 45 50 l y m p h o c y te s ( % ) 55 60 65 15 time (min) 30 pbs islets msc-islets 5 0 100 200 300 400 c 3 a ( n g /m l ) 500 600 700 800 15 time (min) 30 pbs islets msc-islets t a t ( n g /m l ) 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 5 15 time (min) 30 pbs islets msc-islets 0 5 pbs islets msc-islets 15 time (min) 30 50 100 p lt ( 1 0 9 /l ) 150 200 250 c. e. d. b.a. figure 5. blood interactions in vitro of control islets and msc–islets. control islets (light grey) and human msc–islets (dark grey) were exposed to human abo-compatible blood in the tubing blood loop model, and pbs (black) was used as control. platelet counts (a) and granulocytes (b) decreased, whereas lymphocytes (c) increased. there was no statistically significant difference between control islets and composite grafts. activation of coagulation was investigated by measurements of thrombin anti-thrombin (tat) levels, 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characterization of cells with osteogenic potential from human marrow. bone. 1992;13:81–8. composite mesenchymal stem cell islets 17 is medical urgency of elderly patients with traumatic brain injury underestimated by emergency department triage? article is medical urgency of elderly patients with traumatic brain injury underestimated by emergency department triage? toralph rugea,b, axel c. carlssonc,d, magnus hellstrome, per wihlborga and johan und�enf,g adepartment of clinical sciences malm€o, university hospital of skåne, sweden; bdepartment of emergency medicine, karolinska university hospital, huddinge, stockholm, sweden; cdivision of family medicine and primary care, department of neurobiology, care sciences and society, karolinska institutet, huddinge, sweden; ddepartment of medical sciences, cardiovascular epidemiology, uppsala university, uppsala, sweden; edepartment of surgical and perioperative science, umeå university, umeå, sweden; fdepartment of operation and intensive care, halmstad hospital, halmstad, sweden; ganaesthesiology and intensive care medicine, lund university, lund, sweden abstract background: mortality is high among elderly patients with traumatic brain injury (tbi). recent data suggest that early surgical intervention and aggressive rehabilitation may reduce mortality rates even in elderly patients. our aim was therefore to study the rapid emergency triage and treatment system–adult (retts-a) triage of patients with isolated tbi and examine the differences in acute management according to age. methods: we included 306 adult patients with isolated severe tbi and an abbreviated injury scale (ais) score �3. using a cut-off of 60 years of age, differences in triage priority according to retts-a, time to first computed tomography (ct) scan, length of hospital stay (los), and 30-day survival were studied. results: in patients with an ais score of 3 and 4, we observed that elderly patients had a longer time from admission to first ct scan. in addition, we observed that elderly patients were less often triaged with the highest priority level, despite similar ais scores. los was significantly higher in elderly patients (median 9 days compared with 3 days for younger patients, p < 0.001). finally, age, triage priority, and ais score were independent risk factors for mortality. conclusion: elderly patients with isolated tbi are managed differently than younger patients, which could be due to an under-triage of elderly patients by retts-a. article history received 26 august 2019 revised 13 december 2019 accepted 15 december 2019 keywords elderly patients; emergency department; retts-a; traumatic brain injury introduction traumatic brain injury (tbi) is one of the leading causes of death in the western world, with an incidence between 47 and 849 per 100,000 habitants per year (1,2). elderly patients are particularly vulnerable and are overrepresented among patients with tbi. they also suffer from a higher mortality, prolonged hospitalisation, and significant long-term disability after tbi (2). the main cause of tbi among elderly patients is standing falls, followed by road traffic accidents (3). early identification of severe intracranial injury, such as those needing specific intervention, is important in order to improve outcome. encouraging data from several studies indicate that early surgical treatment and aggressive rehabilitation also improve outcome after tbi in elderly patients (4). therefore, appropriate triage of elderly patients with tbi is critical in order to identify patients who would benefit from early surgical intervention. rapid emergency triage and treatment system–adult (retts-a) is the most common triage system used in sweden. it uses a combination of the patient’s presenting symptoms and signs, in addition to vital sign values, to determine triage priority. the retts-a triage scale levels are classed as red, orange, yellow, green, and blue, in declining level of acuteness (5). current literature indicates under-triage of elderly trauma patients, including those with tbi (6). although an altered level of consciousness is associated with increased mortality (7), most elderly patients have relatively subtle changes of level of consciousness compared with younger patients, despite potentially serious intracranial complications, which may increase the risk of under-triage (8). some of the data presented here were published previously in a smaller pilot study with the main aim to study pre-hospital care of patients with tbi (9). the main finding was that elderly patients had higher mortality and had to wait longer for diagnostic imaging, compared to younger patients. the effect of age on triage priority according to retts-a in patients with isolated tbi has not been studied. our aim was therefore to study the retts-a triage of patients with isolated tbi and examine the differences in acute management according to age. contact toralph ruge thoralph.ruge@med.lu.se department of emergency medicine, karolinska university hospital, huddinge, 171 76 stockholm, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 1, 58–63 https://doi.org/10.1080/03009734.2019.1706674 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1706674&domain=pdf&date_stamp=2020-02-19 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2019.1706674 http://www.tandfonline.com materials and methods study design this was a retrospective cohort study. study setting patients were included from two counties in sweden: v€asterbotten between 2011 and 2012 (lycksele, skellefteå, and umeå), and skåne (university hospitals in malm€o and lund) between 2013 and 2014. patients from v€asterbotten were identified through the injury data base (idb; www. socialstyrelsen.se), and patients from skåne were identified through the patients administrative system (pas; patient medical record). variables were documented according to the swetrau (swedish national trauma register; http://rcsyd. se/swetrau) manual and were subsequently retrieved from the idb and pas. ethical considerations the study was approved by the regional ethical review board in stockholm (2014/1822–31/4). study population inclusion criteria were all patients with an isolated tbi (i.e., no other traumatic injuries), icd 10 codes s06.1–s06.9 or s02.0–s02.1 (s06.0, concussion; s06.1, traumatic cerebral oedema; s06.2, diffuse tbi; s06.3, focal tbi; s06.4, epidural haemorrhage; s06.5, traumatic subdural haemorrhage; s06.6, traumatic subarachnoid haemorrhage; s06.8, other specified intracranial injuries; s06.9, unspecified intracranial injury; s02.0, fracture of vault of skull; and s02.1, fracture of base of skull) and an abbreviated injury scale (ais; ais version 2005 update 2008, https://www.aaam.org/abbreviated-injury-scaleais/) score �3 (10). exclusion criteria were patients with multiple injuries and patients with missing data. for the purpose of the study, patients were divided into those below the age of 60 and those 60 years or older (defined as elderly in this study). data collection the collected variables included age, sex, cause of injury, use of anticoagulants (patients were asked for the use of either warfarin, noak [new oral anticoagulants], or acetylsalicylic acid), triage priority according to retts-a (5), time to first computed tomography (ct) scan (defined as time from emergency department [ed] admission [arrival] to ct), total length of hospital stay (los; measured in number of days from ed arrival to discharge from in-hospital care, only including patients surviving 30 days), and 30-day mortality (counted from ed arrival). rapid emergency triage and treatment system–adult (retts-a) was introduced in sweden in 2005 and is now the most common triage tool in sweden and scandinavia, including also pre-hospital care. retts-a uses a combination of the patient’s presenting symptoms (chief complaint) and the patient’s vital signs in order to determine triage priority. this results in one rettsa triage priority based on the single most deviating vital sign and one based on the severity of the emergency signs and symptoms (ess). the more urgent of the two becomes the patient’s final triage priority. the retts-a triage scale levels are: red, orange, yellow, green, and blue, in declining level of acuteness. vital signs include heartbeat (rate, regular/unregular rhythm), respiratory rate, level of consciousness, and body temperature. medical urgency increases if patient values deviate from the normal range (7). triage was performed by trained nurses at each centre. the lowest three priority levels (level blue, green, and yellow) were combined and redefined as priority level i (not life-threatening), while clinical priority level ii corresponded to retts-a triage level orange (possibly life-threatening), and clinical priority level iii corresponded to retts-a level triage red (life-threatening). the ais scale relies on the anatomical location and severity of injuries. the purpose of the scale is to describe how lifethreatening the anatomical specific injury is as such for the patient, but it does not include an overall evaluation of the patient. the ais scoring system classifies an individual injury by body region according to its relative severity on a 6-point scale (1 ¼ minor, and 6 ¼ maximal). outcomes the primary outcome was triage priority according to rettsa. the secondary outcome measures were time from ed admission to first ct scan, total length of hospital stay (los), and 30-day mortality. statistical analysis data were analysed using spss software, version 18.0. continuous variables at baseline were not normally distributed; therefore, median values and interquartile range were calculated. non-parametric statistical tests were used for univariate analysis, mann–whitney u-test was used to compare differences between two groups, and kruskal–wallis test was used where more than two groups were included into the analyses. the pearson chi-square test and fisher’s exact test were applied when the independent variable was categorical. univariate and multiple linear regression analyses were used to determine the association between the response variable los (days) and patient demographics and clinical factors among patients surviving until hospital discharge. hospital los was highly right-skewed and was therefore transformed and analysed using the natural logarithmic scale in the multiple linear regression in order to obtain the residuals from the normally distributed model. binary logistic regression modelling with stepwise variable selection was performed to identify predictors (tables 3 and 4) of death during in-hospital stay. all tests were two-sided, and p values <0.05 were considered statistically significant. upsala journal of medical sciences 59 http://www.socialstyrelsen.se http://www.socialstyrelsen.se http://rcsyd.se/swetrau http://rcsyd.se/swetrau https://www.aaam.org/abbreviated-injury-scale-ais/ https://www.aaam.org/abbreviated-injury-scale-ais/ availability of data and material the datasets used and/or analysed in the present study are available from the corresponding author upon request. results patient characteristics a total of 330 patients were initially recruited. of these, 24 patients were excluded due to missing data, leaving 306 included patients. patients were divided into two subgroups: those below the age of 60 years (n ¼ 87) and those �60 years (n ¼ 219). there were more men in both age groups, with a higher proportion in the <60 years group (table 1). ais scores were similarly distributed among both groups. the main mechanisms of injury for patients below the age of 60 were standing falls (including falls from <1 to �1 m, 30%) and blunt trauma (20%). for patients 60 years or older, the main mechanism of injury was falls from the same or different levels (70%). the use of anticoagulant drugs (warfarin and acetylsalicylic acid) was more common in patients 60 years or older compared to patients below the age of 60. table 1. patient characteristics. variable age < 60 years age � 60 years p value(n ¼ 87) (n ¼ 219) age (years), median (iqr) 46 (26–56) 81 (72–86) <0.001 male, n (%) 62 (71.3) 124 (56.6) 0.012 ais score, n (%) 0.314 3 52 (59.8) 112 (51.1) 4 22 (25.3) 60 (27.4) 5 13 (14.9) 47 (21.5) triage priority, n (%) 0.013 level i 30 (34.5) 94 (42.9) level ii 28 (32.2) 86 (39.3) level iii 29 (33.3) 39 (17.8) anticoagulants, n(%) <0.001 none 80 (94.1) 112 (51.6) warfarin 2 (2.4) 44 (20.3) acetylsalicylic acid 3 (3.5) 61 (28.1) hospital, n (%) 0.214 skåne 49 (56.3) 106 (48.4) umeå 30 (34.5) 72 (73.0) lycksele 4 (4.6) 17 (7.8) skellefteå 4 (4.6) 24 (11.0) time to first ct scan (min), median (iqr) 80 (45–131) 121 (66–227) <0.001 length of stay (days), median (iqr) 3 (1–10) 9 (3–18) <0.001 values are expressed as median (iqr), or median (%). associations were tested using chi-square or mann–whitney utest. n ¼ 306 for all variables. ais: abbreviated injury scale; iqr: interquartile range; skåne: university hospitals in malm€o and lund. table 2. comparison of triage priority, mortality, time of management, and length of stay by ais score. patients < 60 years patients � 60 years p value triage priority by ais score (n ¼ 306) n n ais ¼ 3, n (%) level i 26 (50.0) 59 (52.7) 0.349 level ii 11 (21.2) 35 (31.2) level iii 15 (28.8) 18 (16.1) ais ¼ 4, n (%) level i 2 (9.1) 17 (23.2) 0.022 level ii 11 (50.0) 31 (51.2) level iii 9 (40.9) 12 (25.6) ais ¼ 5, n (%) level i 2 (15.4) 18 (38.3) 0.073 level ii 6 (46.2) 20 (42.6) level iii 5 (38.5) 9 (17.8) mortality by ais score (n ¼ 306), n (%) ais ¼ 3 1 (1.9) 17 (15.2) 0.013a ais ¼ 4 2 (9.1) 12 (20.0) 0.207a ais ¼ 5 3 (23.1) 13 (27.7) 0.332a time to first ct scan by ais score (n ¼ 306; min), median (iqr) ais ¼ 3 52 94.0 (53.3–144.5) 112 138.0 (75.3–245.3) 0.005 ais ¼ 4 22 52.5 (38.8 –96.3) 60 113.0 (63.8–196.8) 0.002 ais ¼ 5 13 101.0 (58.5–172.5) 47 108.0 (47.0–214.0) 0.713 hospital length of stay by ais score (n ¼ 258; days), median (iqr) ais ¼ 3 51 2.0 (1.0–4.5) 95 8.0 (3.0–14.0) <0.001 ais ¼ 4 20 7.0 (1.5–11.8) 48 9.0 (3.0–17.8) 0.381 ais ¼ 5 10 8.0 (4.8–20.5) 34 12.0 (5.0–25.5) 0.509 values are expressed as number (%), or median (iqr). associations were tested using chi-square or mann–whitney u-test. for length of stay and time to first ct, analyses were only performed for patients who survived. afisher’s exact test. ais: abbreviated injury scale (mann–whitney u-test). 60 t. ruge et al. triage we observed that a greater proportion of patients below the age of 60 were categorised with triage priority iii compared with patients 60 years or older (table 2). among patients with ais ¼ 4 and 5, fewer patients below the age of 60 were triaged with triage priority i compared with patients �60 years (table 2). time from admission to first ct scan the time from ed admission to first ct scan was shorter for patients below the age of 60 and with ais scores of 3 and 4 compared with patients 60 years or older (p < 0.01, table 2). there was no significant difference between the two age categories with respect to time from admission to first ct scan for ais scores 5. we found no association between time from ed admission to first ct scan and hospital los (tables 3 and 4) or mortality (odds ratio ¼ 1.00, 95% confidence interval 0.99–1.01, p ¼ 0.374, data not shown in table 5). hospital los patients below the age of 60 and with ais score >3 had a 4fold lower median los than patients 60 years or older (p < 0.001, table 2). there was no significant difference between the two age categories with respect to hospital los for ais scores of 4 and 5. age, triage priority, and ais score of 5 were independent risk factors for hospital los in both age groups (tables 3 and 4). mortality mortality was higher in older patients for all ais scores; however, this was only statistically significant for patients with ais score of 3 (table 5). mortality was independently associated with age, triage priority, and ais score, but not time from admission to first ct scan (table 5). discussion current literature indicates that there is under-triage of elderly trauma patients, including those with tbi, and that mortality in this group of patients is higher. our present study suggests that elderly patients with tbi are under-triaged when using the rapid emergency triage and treatment system–adult (retts-a). thus, elderly patients less often receive the highest triage priority, despite similar ais scores. moreover, the time from ed admission to ct scan was longer for elderly patients with isolated tbi ais scores of 3 and 4. while mortality was independently associated with age, triage priority, and ais score, mortality was not associated with time from ed admission to first ct scan. there is no widely accepted threshold or cut-off value that constitutes a ‘geriatric’ patient, nor is there a definite cut-off that accurately predicts outcome. various age cut-off values have been proposed, ranging between 45 and 80 years, where most data show that trauma patients over 60 years have an increased mortality compared with younger patients (6). we therefore chose 60 years of age as our cut-off it has been shown that for a given anatomical severity of tbi, elderly patients may present with a higher glasgow coma scale (gcs) than younger patients (8). the reason for this could partly be explained by the fact that the brain undergoes age-related atrophy, allowing a greater haematoma size and more pronounced oedema before clinical signs are recognised (11). this may lead to under-triage of elderly patients and could also explain the poor performance of triage tools in elderly trauma patients (8). tbi in older adults is associated with worse outcome when compared to younger adults (2). our data are in accordance with these findings. we are not able to explain the effect of hospital on los; however, it is likely that the variation in los is dependent on local routines controlling the care of patients with tbi in the different wards included. increased ais score is most often related to affected levels of consciousness in both young and old patients, and triage priority increases with higher ais score (12). however, even table 3. bivariate analysis of test independent variable association with los. variable test type test value p value gender mann–whitney u ¼ �0.74 0.46 age spearman’s rho q ¼ 0.24 <0.001 ais score kruskal–wallis v2(2) ¼ 4.30 0.04 triage priority kruskal–wallis v2(2) ¼ 13.84 <0.001 time to first ct scan spearman’s rho q ¼ �0.09 0.17 anticoagulants kruskal–wallis v2(2) ¼ 2.87 0.24 hospital kruskal–wallis v2(3) ¼ 9.58 0.02 table 4. independent factors associated with length of stay. multiple linear regression on logarithmic transformed response variable length of stay (los).a variable b (95% ci) standard error p value intercept �0.22 0.36 age 0.02 (0.01–0.03) 0.004 <0.001 gender female reference male 0.13 (�0.18–0.45) 0.16 0.41 triage priority level i reference level ii 0.32 (�0.04–0.69) 0.18 0.08 level iii 0.98 (0.52–1.45) 0.24 <0.001 hospital skåne reference umeå 0.48 (0.14–0.83) 0.18 0.01 lycksele 0.52 (�0.09–1.13) 0.31 0.09 skellefteå 0.66 (0.08–1.24) 0.30 0.027 ais score ais ¼ 3 reference ais ¼ 4 0.22 (�0.13–0.56) 0.18 0.22 ais ¼ 5 0.81 (0.41–1.21) 0.20 <0.001 anticoagulants none reference warfarin 0.13 (�0.21–0.48) 0.18 0.46 acetylsalicylic acid 0.06 (�0.25–0.38) 0.16 0.70 time to first ct scan 0.00 (�0.01–0.01) 0.004 0.38 model: f(10,213) ¼ 6.08, p < 0.001, r2 ¼ 22.2%. arespond variables (los) are logarithmic transform. interpretation of b: %dy ¼ 100� b �dx ‘if we change x by one unit, we expect our y variable to change by 100�b percent’ for small b, where b, unstandardised coefficients. ais: abbreviated injury scale; 95% ci: 95% confidence interval; skåne: university hospitals in malm€o and lund. upsala journal of medical sciences 61 though ais score and triage priority are related, they both uniquely contribute to the prediction of mortality. similar to the findings of kehoe et al., we also observed that around 50% of patients with an ais score of 3 in both age groups were relatively unaffected by existing structural damage according to the triage priority (low triage priority) (8). however, we also observed a tendency that elderly patients with ais scores >3 were more seldom triaged with the highest triage priority. on the contrary, we observed that elderly patients were more often triaged with the lowest priority. furthermore, elderly patients had a significantly longer time from admission to first ct scan compared with younger patients with ais scores of 3 and 4. this difference in trauma management could be explained by the inability of the current triage guidelines to detect serious intracranial injury in elderly patients related to a less affected level of consciousness (8). elderly patients have a higher incidence of medical comorbidities, tend to use anticoagulant medication more often than younger patients, and have lower physiological reserves, increasing their susceptibility to even minor trauma and disease (6). however, underlying medication and comorbidities are not considered in most triage tools, including retts-a (13). similarly, age is not included as a core variable in retts-a. among the most commonly used triage systems, only the emergency severity index has been validated in patients >65 years. most other systems have not been tested (14), and triage priority-related mortality is not adjusted for age or gender (13). interestingly, a recent study has shown that age is identified as an independent risk factor for 1and 30-day mortality in patients triaged according to retts-a. this association was more pronounced in patients with lower medical priority (lower triage priority groups) (7,15). compared with patients <50 years of age, patients >80 years had an almost 50-fold increased risk of both 1and 30-day mortality. in that study, age and level of consciousness were the strongest predictors for mortality (7). ed triage tools, including retts-a, rely heavily on vital signs upon arrival. vital sign cut-offs are notoriously unreliable for elderly patients, which may affect the validity of retts-a and similar triage systems in elderly patients (16). taken together, elderly trauma patients are at significant risk of under-triage using retts-a, because of multiple illness and polypharmacy, resulting in delays or incorrect management. older age may be an overly simplistic measure to understand outcomes in geriatric patients, and a better predictor may be the degree of frailty. the most commonly used definition of frailty is the physical frailty phenotype described by fried et al., which is based on criteria related to reduced physical reserves (weight loss, exhaustion, weakness, slowness, and reduced physical activity) (17). however, the use of this definition in elderly trauma patients with affected consciousness might be difficult. therefore, there is a requirement for other instruments to quantify frailty in elderly trauma patients, for example, the trauma specific frailty index (tsfi) (18). the lack of frailty measurement in the current triage system may explain the differences seen in this study. it is well documented that elderly patients with hip fractures benefit from swift acute management in terms of length of hospital stay and mortality, and this is true also for patients 60 years or older with significant comorbidity (19). the present study shows that elderly patients with tbi are not prioritised in an acute setting, wait longer for their initial ct scan and have a longer los and higher mortality. considering recent data showing that aggressive and early treatment of tbi in the aged population may be just as beneficial as in younger patients (4), it seems reasonable to focus more on this patient group. table 5. mortality according to demographic and clinical factors. died, n/total standard error odds ratio (95% ci) adjusted p valuea total 48/306 age 0.01 1.05 (1.02–1.07) <0.001 gender male 28/187 1 female 20/119 0.37 0.79 (0.39–1.62) 0.52 triage priority 0.005 level i 10/100 1 level ii 15/106 0.46 1.18 (0.47–2.92) 0.73 level iii 19/62 0.50 4.62 (1.75–12.20) 0.002 unknown 4/38 0.66 1.45 (0.39–5.30) 0.58 ais score 0.042 ais ¼ 3 18/164 1 ais ¼ 4 14/81 0.42 1.61 (0.71–3.64) 0.26 ais ¼ 5 16/61 0.42 2.78 (1.22–6.29) 0.014 hospital (#) 0.41 skåne 26/155 1 umeå 12/102 0.46 1.12 (0.46–2.78) 0.79 lycksele 3/21 0.74 1.15 (0.27–4.92) 0.85 skellefteå 7/28 0.61 2.82 (0.85–9.34) 0.09 anticoagulants 0.38 none 29/192 1 warfarin 9/46 0.63 0.64 (0.19–2.20) 0.48 acetylsalicylic acid 9/64 0.50 1.35 (0.50–3.69) 0.55 time to first ct scan 0.001 1.00 (0.99–1.01) 0.37 constant 0.000 aadjusted for other variables in the model by logistic regression. nagelkerke pseudo r2 ¼ 24.6%, model v2(12) ¼ 41.1, p < 0.001. ais: abbreviated injury scale; 95% ci: 95% confidence interval. 62 t. ruge et al. our study has some limitations. importantly, only patients with isolated tbi without any other trauma were included. we did not have access to all relevant underlying diagnoses, medication, and comorbidities, as well as decisions regarding withdrawal of care, of the included patients. another limitation is the low number of patients included in the study and its retrospective design. in conclusion, we found that elderly patients with isolated tbi are managed differently than younger patients. the reason for our finding is unclear, and further studies are warranted to study triage according to retts-a in elderly patients. acknowledgements the authors acknowledge the help of p. o. bystr€om. disclosure statement the authors of this manuscript have no conflict of interest to disclose. funding this study was supported by local ‘alf-funding’ from the county of v€asterbotten. notes on contributors johan und�en, phd, is an associate professor at the department of operation and intensive care, halmstad hospital, halmstad, sweden and lund university, lund, sweden. magnus hellstr€om, phd, is a statistician at the department of surgical and perioperative science, orthopaedics, umeå university, umeå, sweden. per wihlborg, is a senior consultant at the department of clinical sciences malm€o, university hospital of skåne, sweden. axel c. carlsson, phd, is an associate professor at the division of family medicine and primary care, department of neurobiology, care sciences and society, karolinska institutet, huddinge, sweden. thoralph ruge, phd, is an associate professor at the 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https://doi.org/10.1186/s13049-016-0213-8 https://doi.org/10.1186/1757-7241-19-42 https://doi.org/10.1186/1757-7241-19-42 https://doi.org/10.1186/s12877-019-1157-4 https://doi.org/10.1186/s12877-019-1157-4 abstract introduction materials and methods study design study setting ethical considerations study population data collection outcomes statistical analysis availability of data and material results patient characteristics triage time from admission to first ct scan hospital los mortality discussion acknowledgements disclosure statement references tf-iups160033 283..288 original article young female cancer patients’ experiences with fertility counselling and fertility preservation—a qualitative small-scale study within the danish health care setting didde hoega, lone schmidta and kirsten t. macklonb adepartment of public health, section of social medicine, university of copenhagen, denmark; bthe fertility clinic, section 4071, university hospital of copenhagen, rigshospitalet, denmark abstract introduction: fertility counselling for young women newly diagnosed with cancer is an important field of preconceptional counselling. this qualitative, small-scale study explored how young women newly diagnosed with cancer experienced specialized fertility preservation counselling and treatment in the public danish health care system. methods: semi-structured, in-depth interviews were conducted with five women below 40 years recently diagnosed with cancer. all women received fertility counselling by a fertility specialist at the fertility clinic, university hospital of copenhagen, denmark before initiation of cancer treatment. participants were interviewed at a place chosen by them, and interviews were recorded and transcribed verbatim. data were analysed using systematic text condensation developed by malterud and inspired by giorgi’s phenomenological analysis. results: none of the participants were aware that chemotherapy could destroy their eggs. the participants described how specialized fertility counselling and fertility preservation contributed to a belief in life after cancer, which gave them hope that they would survive their cancer disease. further, the women described how the possibility of fertility preservation removed a huge concern and enabled them to concentrate on their cancer treatment and on getting better. conclusion: overall, the specialized fertility counselling and treatment to preserve fertility was highly valued. the women felt it gave them a choice about their future fertility. the fertility expert presented the various fertility-preserving scenarios, and the women were content that they had an actual choice. article history received 18 march 2016 revised 12 june 2016 accepted 16 june 2016 key words cancer; fertility preservation; preconceptional counselling; qualitative method; semistructured interviews; systematic text condensation introduction all over the industrialized world, there is a trend among both men and women to delay the birth of a first child (1). previous qualitative interview studies from the nordic countries found that both men and women have various priorities when planning their lives, and having children is one of them. however, many had never reflected on their own reproductive capacity; their fertility is often taken for granted (2–4). unfortunately, some people experience unpredictable events in their lives, which will affect their reproductive capacity. preconceptional counselling is therefore needed in various special situations adjusted to people’s life situations and special needs. a new and important field regarding preconceptional counselling is the specialized fertility counselling among young women, newly diagnosed with cancer. in denmark approximately 900 women below 40 years are annually diagnosed with cancer (5). over the years cancer treatments have become more effective, allowing more people to survive (6). therefore, it is important that the health care system focuses on side effects to cancer treatment, such as infertility. chemoand radiation therapy may have very detrimental effects on the ovarian reserve, leading to premature ovarian insufficiency (poi) and infertility. in order to circumvent these very unwanted side effects, methods have been developed to preserve the fertility in girls and young women with cancer. cryopreservation of embryos, oocytes, or ovarian tissue is a technique currently offered to women to preserve fertility (7,8). several studies indicate that fertility issues are very important to younger women with cancer (9–15). studies from canada, sweden, and the us showed that 50%–60% of women below 40 years with breast cancer or other types of cancer expressed a wish to have one or more children in the future (12,14,15). also, a systematic review found that childbearing after breast cancer is an important issue for survivors (16). previous studies have shown that fertility counselling by a specialist is particularly important for women newly diagnosed with cancer (9–14,17), and that early referral to a fertility specialist who can provide clear consistent advice can contact lone schmidt lone.schmidt@sund.ku.dk department of public health, section of social medicine, university of copenhagen, øster farimagsgade 5, po box 2099, dk-1014 copenhagen k, denmark. � 2016 the author(s). published by informa uk limited, trading as taylor & francis group.this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 4, 283–288 http://dx.doi.org/10.1080/03009734.2016.1204394 http://creativecommons.org/licenses/by/4.0/ help the women make an informed decision about fertility preservation (13). a swedish study found that fertility-related communication is not sufficient, and the women in this study called for more individualized information about their fertility after cancer and information about how cryopreservation of embryos, oocytes, or ovarian tissue is performed (18). a quantitative dutch study argues that more attention should be paid to improve fertility preservation counselling, so that the women understand what fertility preservation involves and feel supported in their decision-making process (19). additionally, it has been shown among childhood cancer survivors that the risk of infertility affects their well-being in adulthood (20). fertility counselling for women with cancer is greatly needed according to earlier studies (9–11,13,14,17,18,21). a newly published review states that limited research has addressed the emotional issues that arise in patients experiencing fertility preservation. furthermore, in most parts of the world the cost of fertility preservation presents a barrier to treatment, if the patient’s insurance does not cover the treatment (22). the aim of this qualitative, small-scale study was to explore how young women newly diagnosed with cancer experienced specialized fertility-preserving counselling. the women had received the specialized fertility preservation counselling within a few days or a few weeks after the cancer diagnosis and before chemotherapy treatment began. in denmark specialized fertility-preserving counselling is offered in a public health care system financed by taxes and without payment by the patients. the programme of ovarian tissue cryopreservation started in the year 1999. since then, almost 800 patients have had tissue frozen, 53 transplantations to 41 patients have been performed, and so far the programme has resulted in 13 children (23). material and methods participants the participants were recruited between february and may 2014 at the fertility clinic, rigshospitalet, university hospital of copenhagen, copenhagen, denmark. inclusion criteria were: women between 18–39 years of age, who had recently been diagnosed with cancer, and who had recently received fertility counselling. the women should be fluent in danish and mentally and physically well enough to participate in an interview. five women were asked to participate in the study, and no one declined. we aimed at recruiting participants with as much variation as possible regarding marital status (married/cohabiting or single), motherhood status (having children or not), and different choices of fertility preservation treatments (cryopreservation of ovarian tissue, vitrification of oocytes, or no fertility preservation treatment). procedures prior to the invitation to participate in this study all women had received specialized fertility preservation counselling by a fertility specialist (k.t.m.). all women had received written information about fertility preservation and individualized oral information during the counselling. all women in the study were invited to participate in the study by k.t.m. all interviews were conducted by the first author (d.h.), and interview techniques and difficulties during data collection were discussed with author l.s. d.h. is a master student in public health science and has a bachelor degree within qualitative sociology. l.s. is a medical doctor with a phd in qualitative health research. k.t.m. is a clinical fertility specialist. the interview took place at a location chosen by the participant: three took place in the participant’s own home, one at the participant’s workplace, and one in a caf�e chosen by the participant. interviews lasted between 45 and 90 min. the interviews were conducted using a semistructured interview guide focusing on the women’s experiences with fertility counselling and fertility preservation. to gain a better and more personal insight into the field of fertility counselling, d.h. observed a fertility counselling session at the fertility clinic, rigshospitalet before conducting the interviews. interviews were recorded and transcribed verbatim, and transcripts were re-checked against the recording. transcripts were anonymized with fictive names. the age of the participants ranged from 25 to 38 years. the participants lived in or near the capital of copenhagen, and all but one were in a relationship. three of the participants had one child, while two did not have any children (table i). analyses the interview text was analysed using systematic text condensation (stc) developed by malterud and inspired by giorgi’s phenomenological analysis. according to giorgi the purpose of the phenomenological analysis is to develop knowledge of how the informants experience their world of life in a certain field, by looking for essences and essential characteristics of the phenomena under study (24). like giorgi’s method, stc implies analytic reduction with specified shifts between decontextualization and recontextualization (25). each translated interview was analysed through the following structured process: first, a general impression of data was obtained by reading the transcript and finding preliminary themes associated with the participant’s experiences with fertility counselling and treatment. then the transcript was read once more with the specific aim to identify meaning units containing information about the research question. the identified meaning units were sorted as thematic code groups across individual participants. the contents of the meaning units were condensed and sorted into subgroups. finally, the meaning units were decontextualized into a consistent statement regarding the participant’s experience. the results are illustrated with quotes from all participants. cut-outs are marked as [. . .]. the transcripts were analysed using nvivo 10.0 software. all authors discussed the interpretations of the findings. we have followed the consolidated criteria for reporting qualitative research (coreq) (26). 284 d. hoeg et al. ethics the study followed the codes of ethics from the helsinki ii declaration. according to danish legislation, interview studies require no approval from an ethical committee. results we identified three themes regarding how the participants experienced the specialized fertility preservation counselling: 1) the feeling of being unable to control anything; 2) the importance of creating opportunities; 3) the feeling of having an active choice among the different scenarios. the feeling of being unable to control anything none of the women knew that chemotherapy could destroy their eggs. one woman described how her dream about children was devastated when she was told that she could lose her eggs as a result of the treatment. truly, a dream was shattered when they told me that my eggs; they could possibly be affected, and that we might not have any children [. . .]. as soon as she said that i could not have children, i was not sick with cancer anymore [. . .] it was completely gone, completely. i had left that behind me [. . .]. (sanne) katrine described how she felt the cancer as something foreign that took control of her and her life. she felt sorrow in the awareness that she might never be able to have any more children. it is absolutely horrible. to lose control is, well . . . actually, it is a bit strange because you take for granted that you can have children and that is not at all something you can count on. [. . .] but that something, you know, like cancer takes the control from you and controls your life for so long [. . .] that is absolutely horrible. (katrine) [. . .] to be sad that you might lose a child you didn’t have . . . well, it sounds completely crazy, but that is how you feel. (katrine) lotte described how having children is an epoch-making life event, which the cancer can extinguish. the fertility issue was a huge issue to her when she got her cancer diagnosis. it became secondary when she started her cancer treatment, because of existential crises and fear of death. [. . .] am i even going to survive all this? how can i then think of children. [. . .] it was the fear of death that took up most of the time or all the time at that period. [. . .] it is fine, then they are preserved [the eggs] . . . but it is not at all certain that i am alive in three years. (lotte) camilla described how she felt overwhelmed with information, so the fertility issue did not differ from the other information. sara described that the oncologist did not talk much about fertility, when she was diagnosed with cancer, and, like camilla, she seemed to experience the confrontation with the fertility issue as secondary. the importance of creating opportunities katrine described how she experienced the fertility-preserving treatment as a positive offer. the fertility preservation was a way to create opportunities, where the cancer had set limits. i have experienced the fertility issue as something very positive. everything else about the cancer [. . .] it has been like limitations and the fertility has been—is possibilities [. . .]. all the talk about it is, after all, to create possibilities for me, where all the other stuff about the chemotherapy was a huge limitation in itself. therefore, i have experienced it as something very, very positive [. . .]. in all the darkness, there has actually been something to light it up. that i think has been extremely positive. (katrine) lotte described how the fertility counselling and treatment contributed to her belief in surviving the cancer. [. . .] so they take the eggs out and they do all that and it costs a huge amount of money. so they must believe that i will survive this somehow—well, since they bother to do all this, right? (lotte) camilla described the importance in self-determination and that the fertility preservation treatment minimized her risk of menopause and infertility as side effects. this gave her a feeling of regaining some control over her body, and made her feel that she had the opportunity to attain a normal functional body again when she had recovered from the cancer. the most important thing is that we decide for ourselves. and then, i think, when you get to the other side have as few implications as possible. to enter menopause prematurely, that is a fairly bothersome implication of having had cancer, so when i hopefully get—or when i am declared well—then we should like to reach some sort of normality that things are as they have always been. (camilla) this indicates that the women felt that fertility counselling and treatment created some new opportunities for them, when they felt unable to control anything, because of the cancer diagnosis and risk of infertility as a side effect. lotte described how the fertility preservation treatment removed a huge concern, which she did not have to think about until after the cancer treatment. one reason given for choosing cryopreservation of ovarian tissue was to lower the risk of early menopause. also cryopreservation of ovarian tissue made the women feel that they would attain some sort of bodily normality again. the women’s argument against receiving fertility preservation was that it meant another demand on their body, besides the cancer treatment. sara described the fertility preservation as a substantial load, which possibly could delay her chemotherapy treatment. lotte described how it is important table i. presentation of study participants with age, number of children, marital status, cancer diagnosis, and selected fertility preservation treatment. name in the study age/children marital status diagnosis fertility-preserving treatment camilla 32 years, one child married breast cancer cryopreservation of ovarian tissue sanne 25 years, no children cohabiting brain tumour vitrification of oocytes katrine 33 years, one child married breast cancer cryopreservation of ovarian tissue sara 29 years, one child cohabiting breast cancer none lotte 38 years, no children single breast cancer vitrification of oocytes upsala journal of medical sciences 285 to consider whether the wish to have children balances the bodily load of fertility preservation. contrary to this, camilla and katrine described how they experienced the fertility preservation as a minor treatment. even though sara rejected fertility preservation she is grateful that she has been made aware of the fertility issue and has been offered advice on possibilities, so that she could make her own decision. the feeling of having an active choice among the different scenarios even though the women have made different decisions concerning fertility preservation, they all described gratitude for the opportunity to preserve their fertility. [. . .] i can say that generally i have been really pleased about it. so i just think that it is important that you [. . .] turn the fertility thing into something positive. not turn it into something negative and say ‘oh no, now i can’t have children’ and then focus on that, but instead say ‘oh yes, i might have children after all’ and focus on this and get this perspective on it. (katrine) just the fact that there has been someone who would give me the opportunity to get to the other side and choose for myself, that has meant a lot—that has truly meant a lot. (sanne) well . . . actually i think that, as a whole, it was fine—positive, kind, and nice people. (sara) the women described it as a good experience and service when the various fertility preservation scenarios were presented to them. they were all pleased and comfortable with the ultrasonography of their ovaries and estimate of their ovarian reserve. they further described the importance of the fertility preservation treatment being their option. [. . .] she explained it all well and put up the different scenarios in a very clear way so there was still something to discuss. but it was an active choice you had to make—that i think was very important. (camilla) the women’s considerations vary in relation to their decision about receiving fertility preservation. regardless of their decision, several women implied that they did not know their cancer and treatment status when they had to consider the fertility preservation treatment. this complicated their decision-making process. they had to make a decision about the fertility-preserving treatment before they knew about the cancer treatment. this analysis showed how the women experienced that the fertility-preserving counselling and treatment gave them the opportunity to regain some of the lost control, by having an active choice among the different scenarios that were listed up for them at the fertility preservation counselling. discussion in this study, it appears that fertility issues are of great importance for women below 40 years diagnosed with cancer, which is in line with results from international studies (9–14,16–18,21). previous studies found that potential loss of fertility could have a profound influence on young women and sometimes be more stressful than the cancer itself (13,18). in this study, one woman described how the fertility issue was so distressing to her that it overshadowed her cancer. a qualitative study from england (13) showed a trend towards childless women worrying about their fertility. their concern influenced their decision about cancer treatment. they were more willing to opt out of the recommended cancer treatment. a quantitative study from canada (12) also found that considerations regarding whether or not to accept fertility preservation were affected neither by the woman’s cancer status nor by whether she already had children. in this small-scale study, we got the impression that women without children were especially concerned about their fertility when they were informed about their cancer diagnosis. further, the participating women with children described how they would have been more concerned about the fertility issue if they had not previously given birth to a child. however, none of the women refused the fertility preservation treatment just because they had a child. only one refused fertility preservation in order to avoid another stressful demand on her body. previous studies have shown that 30%–50% of women decide not to pursue fertility preservation, one reason being that in a majority of countries costs for fertility preservation is covered by the patients themselves. further, women who refused fertility preservation were less likely to regret their decision if they had had pretreatment counselling with a specialist (22). a dutch study found that women affected by cancer welcomed the ability to preserve their fertility despite the cancer diagnosis, and that the women associated the fertility counselling and treatment with positive emotions, such as hope, relief, and a reason to live (9). this agrees very closely with the descriptions found in this study. this study contributes to the existing literature by showing that young women newly diagnosed with cancer experience that specialized fertility preservation counselling and treatment gave them the opportunity to look ahead and imagine a life after cancer. one woman described how the fertility counselling and treatment gave her an indication that the doctors believed she would survive. this indicates that the specialized fertility counselling and treatment contributes to a notion of life after cancer, which gives the women the belief that they will survive the cancer. an american study further found that for younger women the prospect of being able to have children after cancer acts as a powerful stimulus to recover from the disease (27). previous studies found that when women received information about possible fertility preservation treatments they felt they were given a choice on this issue and thus on their own future, which was a positive experience (9,18). in the swedish study, only two women received fertility counselling. the women who did not receive fertility counselling described how they felt they were losing control over their reproductive ability and felt denied the opportunity to participate in decisions concerning future fertility (18). 286 d. hoeg et al. a study from the uk found that even though women were referred to a fertility specialist, it could be difficult to arrange the counselling because of long waiting times (28). all women in our study told that they were referred to the fertility specialist within a very short time; some of them had to consider the fertility preservation treatment before they knew details about their cancer diagnosis and treatment. this complicated their decision-making process. still, all women appreciated that they did not experience any waiting time in the fertility preservation process. generally, findings in this study thus appear to be consistent with the findings of previous studies in relation to young women with cancer and their experiences with fertility-preserving counselling and treatment. strengths and limitations in this study it is a strength that all women had specialized counselling with the same professional fertility expert and that the interviewer, through observation, obtained acquaintance with the field before conducting interviews. further, it is a strength that the participants differed with regard to marital and motherhood status and with regard to which fertility preservation treatment they chose. this we believe has implications on how they experienced the specialized fertility counselling and treatment. all participants were invited by the fertility specialist (k.t.m.) who conducted the fertility preservation counselling. on the one hand this ensured that the women were invited by a health professional they knew beforehand and, further, that women who were very fragile were not invited. on the other hand, this recruitment strategy could imply that the participants felt an obligation to be more positive towards the specialized fertility counselling than they really were. it was emphasized before the interviews were conducted that the participants should feel free to say what they wanted and that participation or not would have no impact regarding their fertility preservation treatment. the main limitation of the study is the inclusion of only five women. the point of saturation during data analyses was not reached. however, the in-depth interviews, lasting 45 to 90 min, contributed with relevant findings to previously published knowledge. in conclusion, we found that specialized fertility counselling and treatment was highly valued because the women felt they were given a choice about their future fertility. the fertility expert presented the various fertility-preserving scenarios in a satisfying way, and the women were content to have an actual choice. further, the specialized fertility counselling contributed to a belief in own survival among the women. declaration of 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different scenarios discussion strengths and limitations declaration of interest funding references tf-iups160012 71..72 editorial remembering claes hellerstr€om and those around him andersson arne editor of upsala journal of medical sciences i can still remember that early morning in may. i had picked up susan bonner-weir, who had come to uppsala to give the kroc lecture of the year. we went to the neuropsychiatric ward at ulleråker hospital, where claes hellerstr€om had spent the last couple of weeks after his devastating stroke four months earlier. i had learnt to interpret his body language in the sense that it was easy to understand when he liked what he heard or saw. there was no doubt that he very much enjoyed seeing susan when sitting in his wheelchair in the garden under some apple trees in blossom. that chat was the last i had with claes. a week later he passed away. on approaching the tenth anniversary of this sad event, we decided to commemorate him with a special issue of upsala journal of medical sciences. at the time of his death claes was one of the two editors of the journal. gunnar ronquist was the other. the two of them had struggled with the revival of the journal (1) and had introduced electronic publishing. when i was asked by gunnar ronquist whether i would like to take over after claes it was a point of honour for me to do so, keeping in mind the long tradition and history of the journal (2,3). at that time i had been following in his footsteps for some 40 years. so why not carry on? in particular i had assisted claes as associate editor of diabetologia for five years in the late 1980s. i can still remember how he spent hours formulating letters to authors—especially the refusals—that started with claes praising the quality of the research reported (almost nobel prize class) and ending with a very sad message that there was at present no space for the submitted article in the journal. we—and i would like to take this opportunity to thank leif jansson and nils welsh for a positive and fruitful collaboration—are very glad to be able to present a commemorative issue, filled with interesting observations and remarks on claes’s long-term relationship with pancreatic islet research. many more could have been included, but contributions from former students (kjell asplund, michael welsh, leif jansson, decio eizirik, and ulf eriksson), hosts for visits 1. l-r: arne andersson, dave sutherland and claes hellerstr€om dave sutherland, an outstanding pioneer in islet transplantation reseach, before the 1990 kroc lecture, with arne andersson and claes hellerstr€om. � 2016 informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution-noncommercial license (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 2, 71–72 abroad (the simon howell group), a thesis respondent (timo otonkoski), collaborators (jens høiriis-nielsen), and old friends (erik gylfe, partik rorsman, and the bonner-weir family) have been most welcome and appreciated. these articles will undoubtedly enhance the reputation of our journal so that it can remain a major distributor of reports in diabetes research (4). by courtesy of our local kroc endowment, a direct result of the personal friendship between claes and the kroc brothers, free copies are available from our department (nils.welsh@mcb.uu.se) on request. this year’s kroc lecture will be given by mark a. atkinson on 18 may. thirty years have passed since paul lacy came to give the first lecture. six years later dave sutherland (see picture above) was given this honour. enjoy your reading. references 1. andersson a, ronquist g, westermark b. on the revival of an old and venerable journal. ups j med sci. 2000;105:3–4. 2. lindberg bs. proceedings of the upsala medical society: how it all started 150 years ago. ups j med sci. 2015;120:65–71. 3. karlsson t. progress and milestones in scientific communication – a 150 years perspective. ups j med sci. 2015;120:63–4. 4. andersson a, lau b€orjesson j. operating in an era of impact factor mania. ups j med sci. 2015;120:124–31. 72 editorial remembering claes hellerstr&ouml;m and those around him references the h1-receptor antagonist cetirizine ameliorates high-fat diet-induced glucose intolerance in male full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 the h1-receptor antagonist cetirizine ameliorates high-fat diet-induced glucose intolerance in male c57bl/6 mice, but not diabetes outcome in female non-obese diabetic (nod) mice ebrahim anvari, xuan wang, stellan sandler & nils welsh to cite this article: ebrahim anvari, xuan wang, stellan sandler & nils welsh (2015) the h1-receptor antagonist cetirizine ameliorates high-fat diet-induced glucose intolerance in male c57bl/6 mice, but not diabetes outcome in female non-obese diabetic (nod) mice, upsala journal of medical sciences, 120:1, 40-46, doi: 10.3109/03009734.2014.967422 to link to this article: https://doi.org/10.3109/03009734.2014.967422 © informa healthcare published online: 07 oct 2014. submit your article to this journal article views: 825 view related articles view crossmark data citing articles: 4 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2014.967422 https://doi.org/10.3109/03009734.2014.967422 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2014.967422 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2014.967422 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2014.967422&domain=pdf&date_stamp=2014-10-07 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2014.967422&domain=pdf&date_stamp=2014-10-07 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2014.967422#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2014.967422#tabmodule upsala journal of medical sciences. 2015; 120: 40–46 original articlediabetes the h1-receptor antagonist cetirizine ameliorates high-fat diet-induced glucose intolerance in male c57bl/6 mice, but not diabetes outcome in female non-obese diabetic (nod) mice ebrahim anvari, xuan wang, stellan sandler & nils welsh science for life laboratory, department of medical cell biology, uppsala university, box 571, se-751 23 uppsala, sweden abstract background. it has been proposed that the histamine 1-receptor (h1-receptor) not only promotes allergic reactions, but also modulates innate immunity and autoimmune reactions. in line with this, we have recently reported that the h1-receptor antagonist cetirizine partially counteracts cytokine-induced beta-cell signaling and destruction. therefore, the aim of this study was to determine whether cetirizine affects diabetes in nod mice, a model for human type 1 diabetes, and glucose intolerance in high-fat diet c57bl/6 mice, a model for human glucose intolerance. methods. female nod mice were treated with cetirizine in the drinking water (25 mg/kg body weight) from 9 until 30 weeks of age during which precipitation of diabetes was followed. male c57bl/6 mice were given a high-fat diet from 5 weeks of age. when the mice were 12 weeks of age cetirizine was given for 2 weeks in the drinking water. the effects of cetirizine were analyzed by blood glucose determinations, glucose tolerance tests, and insulin sensitivity tests. results. cetirizine did not affect diabetes development in nod mice. on the other hand, cetirizine treatment for 1 week protected against high-fat diet-induced hyperglycemia. the glucose tolerance after 2 weeks of cetirizine treatment was improved in high-fat diet mice. we observed no effect of cetirizine on the insulin sensitivity of high-fat diet mice. conclusion. our results suggest a protective effect of cetirizine against high-fat diet-induced beta-cell dysfunction, but not against autoimmune beta-cell destruction. key words: beta-cell function, glucose intolerance, h1-receptor antagonists, high-fat diet, nod mice introduction histamine 1 (h1)-receptor antagonists are commonly used in the treatment of allergic disorders, but histamine may also influence non-allergic inflammatory and autoimmune events (1). for example, in the absence of histamine, achieved by using histidine decarboxylase-knockout mice and a histaminedeficient diet, inflammatory processes in arthritis (2) and colitis (3) are attenuated. in addition, using h1-receptor knockout mice reduced arteriosclerosis, and steatohepatitis-associated inflammation was observed (4,5). also adaptive immunity is influenced by histamine-induced signaling events. indeed, results from h1-receptor knockout mice indicate expansion of th2 cells (6,7), decreases in cytotoxic t-cells (8), and reduced autoimmune encephalomyelitis in an experimental mouse model lacking the h1-receptor (9,10). as type 1 diabetes is an autoimmune disease in which pancreatic beta-cells are destroyed via inflammatory and t-cell-mediated events (11), and as type 2 diabetes is associated with macrophage activation and increased release of pro-inflammatory cytokines (12), it is possible that histamine via activation of the h1-receptor modulates events pertinent to the pathogenesis of both type 1 and type 2 diabetes. this notion is supported by our recent observation that cetirizine, a correspondence: nils welsh, science for life laboratory, department of medical cell biology, box 571, bmc, se-751 23 uppsala, sweden. e-mail: nils.welsh@mcb.uu.se (received 2 september 2014; accepted 15 september 2014) issn 0300-9734 print/issn 2000-1967 online � 2014 informa healthcare doi: 10.3109/03009734.2014.967422 http://informahealthcare.com/journal/ups mailto:nils.welsh@mcb.uu.se second-generation h1-receptor antagonist (13), partially counteracted pro-inflammatory-induced betacell death (14). in line with this, it has also been reported that h1-receptor antagonists can inhibit nf-kb, a transcription factor that plays an important role in expression of adhesion molecules and inflammatory mediators (15-17). in addition, cetirizine suppressed the activation and chemotaxis of t-cells, monocytes, and granulocytes at sites of inflammation, and inhibited production of reactive oxygen radicals and lipid mediators from eosinophils, neutrophils, and mast cells (18-22). therefore, the aim of the present study was to investigate the putative effects of cetirizine on diabetes development in non-obese diabetes (nod) mice, a model for human type 1 diabetes, and glucose intolerance in high-fat diet mice, a model for human glucose intolerance. we observed that cetirizine improved the glucose tolerance of high-fat diet mice, but failed to protect against diabetes in nod mice. methods development of diabetes in nod mice a local colony of non-obese diabetic (nod) mice was kept under standard pathogen-free conditions, with free access to tap water and pelleted food. to study the spontaneous development of diabetes, 20 female nod mice were given cetirizine hydrochloride (sigma-aldrich, st. louis, mo, usa; 25 mg/kg body weight) in the drinking water between 9 and 30 weeks of age. the dose was chosen to be high but far below the sub-lethal dose, which is 55 mg/kg, and the lethal dose, which is 750 mg/kg, in mice (23). twenty control mice were given only drinking water with no extra supplement. the amount of cetirizine in the drinking water was continuously adjusted according to water intake and body weight of the mice. blood glucose levels were measured every week until the first sign of hyperglycemia, then daily. animals were considered diabetic and killed upon two consecutive days of blood glucose >12 mm. the nod mice experiments were approved by the local animal ethics committee. high-fat diet treatment of c57bl/6 mice four-week-old male c57bl/6 mice were purchased from scanbur ab (sollentuna, sweden). when 5 weeks of age, the mice were divided into two groups with 20 mice in each: one given a normal diet (nd) and one given a high-fat diet (hfd). the high-fat diet (hfd) (d12492, research diets, new brunswick, nj, usa) contained 60 kcal% fat, whereas the normal diet (nd) (d12450b, research diets) contained only 10% kcal fat (24). after 7 weeks of diet, both groups were randomly divided into two subgroups, one receiving cetirizine (25 mg/kg body weight) in the drinking water and one group receiving no supplementation. the amount of cetirizine in the drinking water was continuously adjusted according to water intake and body weight of the mice. water intake was not affected by the addition of cetirizine (results not shown). directly before start of cetirizine treatment and after 1 week of cetirizine treatment non-fasting blood glucose levels were determined. for this purpose, blood was withdrawn from the tip of the tail and analyzed using the freestyle mini system (abbot, therasense inc., alameda, ca, usa). the same person collected all blood samples for blood glucose determinations. the high-fat diet experiments were approved by the local animal ethics committee. blood glucose tolerance test after 2 weeks of cetirizine treatment the mice were fasted for approximately 8 h. the mice were then given a single dose of 2.5 g/kg body weight of 30% w/v d-glucose intravenously by injection in the tail vein. blood glucose was determined prior to injection and then at 10, 30, 60, and 120 min after injection. a small drop of blood was withdrawn from the tail and analyzed with the freestyle mini system. insulin sensitivity test mice were given an intraperitoneal injection (1.6u/kg body weight) of the insulin analog actrapid (novo nordisk, bagsværd, denmark). blood glucose was determined on blood samples from the tail, before injection and 15, 60, 120, and 180 min later using the freestyle mini system. statistics the chi-square test was used for analysis of cumulative diabetes incidence. student’s t test was used for comparison of control mice with experimental group when the sample data were normally distributed. non-normally distributed sample data were analyzed using the mann–whitney u test. results cetirizine did not protect against development of diabetes in nod mice the precipitation of overt diabetes in female nod mice usually occurs from approximately 15 weeks of age. to study whether the activity of the h1-receptor cetirizine protects against glucose intolerance 41 affects the onset of disease, we treated female nod mice from 9 to 30 weeks of age with 25 mg/kg body weight per day of cetirizine administered via the drinking water. at 30 weeks of age 10 out of 20 non-cetirizine-treated mice had developed diabetes (figure 1). among the 20 cetirizine-treated mice 12 developed diabetes (p > 0.05) (figure 1). cetirizine did not affect the age at which diabetes precipitated (figure 1). cetirizine ameliorated high-fat diet-induced glucose intolerance in c57bl/6 mice the h1-receptor antagonist cetirizine was given to c57bl/6 mice in the drinking water at 25 mg/kg/day, to determine whether high-fat diet (hfd)-induced glucose intolerance is affected. we found that cetirizine supplemented via the drinking water during the last 2 weeks of a 9-week long hfd period did not significantly affect the weight increase of the normal diet (nd) and hfd mice (figure 2). we also assessed food intake by weighing the cages without mice, before and after addition of new pellets, and observed similar food intake in cetirizine-treated mice as compared to non-cetirizine-treated mice (results not shown). the non-fasting blood glucose levels of hfd mice were higher than those of nd mice after 7 weeks of diet (figure 3). a 1-week treatment with cetirizine resulted in a normalization of the blood glucose levels of hfd mice (figure 3). cetirizine did not affect the blood glucose concentrations of nd mice (figure 3). the glucose tolerance at fasting conditions of the different study groups was assessed by an intravenous glucose injection followed by determinations of blood glucose levels at 0, 10, 30, 60, and 120 min. we observed that cetirizine did not affect the glucose tolerance of nd mice (figure 4a). the glucose concentrations of hfd mice, however, tended to be lower in the cetirizine-treated mice at all time points after the glucose injection (figure 4b). to test whether there was a significant difference in the glucose tolerance, we calculated the area under the curve (auc) and observed a significantly lower auc in the cetirizine-treated mice (figure 4c). cetirizine did not exert any effect on the auc of nd mice (figure 4c). an insulin sensitivity test was also performed after the 9 weeks of hfd and 2 weeks of cetirizine treatment, and we observed that cetirizine did not affect the insulin sensitivity of nd mice (figure 5a). hfd mice displayed a marked reduction in insulin sensitivity as compared to nd mice (figure 5b). the insulin sensitivity of hfd mice was not affected by the cetirizine treatment (figure 5b). control cetirizine 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 time (weeks) c u m u la ti v e i n c id e n c e o f d ia b e te s ( p e rc e n t) 100 90 80 70 60 50 40 30 20 10 0 figure 1. cetirizine does not affect diabetes development in the nod mouse. female nod mice were treated from 9 to 30 weeks of age with 25 mg/kg � day with cetirizine in the drinking water. blood samples were taken at indicated time points for the assessment of hyperglycemia. mice were considered diabetic when the blood glucose values were >12 mm on two consecutive days. at 30 weeks of age, 10 out of 20 control mice and 12 out of 20 cetirizine-treated mice had developed diabetes (p > 0.05 using the chi-square test). 42 e. anvari et al. discussion in nod mice first macrophages and later other immune cells including t-cells infiltrate the pancreatic islets leading to insulitis. as this process progresses with time, beta-cells are destroyed via direct cell-to-cell contacts between the beta-cells and t-killer cells (25). this autoimmune-mediated destruction of beta-cells is a complex process, which at least in part seems to involve the activity of proinflammatory cytokines (26). because histamine, via the h1-receptor, in some cases promotes t-cell th1 polarization, increases dendritic cell-mediated activation of cytotoxic cd8 t-cells, and augments inflammatory events in general (1), we investigated its putative role in diabetes of nod mice using the h1-receptor antagonist cetirizine. in our experimental set-up, in which the mice were given cetirizine in the drinking water from 9 to 30 weeks of age, cetirizine affected neither the overall diabetes incidence nor time of diabetes precipitation. this argues against a major role of immuneand islet cell h1-receptor high-fat diet cetirizine nd control hfd control nd cetirizine hfd cetirizine 5 6 7 8 9 10 11 12 14 age (weeks) w e ig h t (g ) 60 50 40 30 20 10 0 figure 2. weight curve of c57bl/6 mice given a normal diet or a high-fat diet from 5 to 14 weeks of age. after 7 weeks of normal diet (nd, 20 mice) and high-fat diet (hfd, 20 mice), the mice (aged 12 weeks) were divided into four groups with 10 mice in each: nd or hfd, with or without cetirizine (25 mg/kg/day) and followed for another 2 weeks. * * – + – + * cetirizine: nd hfd day 0 day 7 b lo o d g lu c o s e ( m m ) 12 10 8 6 4 2 0 figure 3. non-fasting blood glucose of nd or hfd mice treated with cetirizine for 1 week. the non-fasting blood glucose concentrations were analyzed after 7 and 8 weeks of hfd, directly before start of cetirizine treatment (day 0) and after 1 week of cetirizine treatment (day 7). results are means ± sem; n = 10 for the two nd groups and 8–9 for the two hfd groups. *p < 0.05 versus corresponding nd mice using student’s unpaired t test. cetirizine protects against glucose intolerance 43 activity in the late-stage autoimmune destruction of beta-cells that occurs after 9 weeks of age. however, we cannot exclude that h1-receptor activation affects earlier events in the nod mouse, such as antigen priming and the initial expansion of beta-cell reactive t-cell clones occurring early in regional lymph nodes (27), and that cetirizine supplementation to the drinking water from for example the fourth week might have given a different result. indeed, allergic symptoms in infants have been reported to be associated with the development of type 1 diabetes-related autoantibodies during the first years of life (28). nor can we exclude that doses of cetirizine other than the one presently used might have affected diabetes in nod mice. the transferral of the h1-receptor knockout to a nod background might bring more clarity to the putative role of histamine and the h1-receptor in type 1 diabetes. we here report that the non-fasting blood glucose and the glucose tolerance of high-fat diet c57bl/ 6 mice were improved by a 2-week cetirizine treatment. at first glance, this appears to be at variance with previous studies using h1-receptor knockout mice. a genetic loss of h1-receptor activity promotes hyperleptinemia, visceral adiposity, and moderate insulin resistance in mice given a high-fat diet (5,29). in addition, decreased weight and increased energy expenditure has been observed in mice infused with histamine to the central nervous system (30). this is commonly explained by the leptin–histamine loop, a system in which the leptin-sensing neurons localized in hypothalamic nuclei require histamineinduced signaling for leptin to exert its effects. histamine-facilitated leptin signaling in the central nervous system is known to decrease food intake and increase uncoupling in adipose tissue (5,29,30). accordingly, lack of h1-receptor signaling will result in increased leptin resistance, increased food intake, and decreased energy expenditure. the discrepancy between the present results using the h1-receptor antagonist cetirizine and the h1-receptor knockout mouse may be explained by the low permeability of cetirizine, a second-generation antihistamine, across the blood–brain barrier. thus, it is reasonable to assume that cetirizine affects mainly histamine signaling in the periphery and to a lesser extent the central 20 15 10 5 0 0 min 10 min 30 min 60 min 120 min nd control hfd control nd cetirizine a hfd cetirizine b lo o d g lu c o s e ( m m ) 700 100 200 300 400 500 600 0 hfd control hfd cetirizine nd control nd cetirizine c a u c g t t ( m m /l × m in ) 20 25 15 10 5 0 0 min 10 min 30 min 60 min 120 min b b lo o d g lu c o s e ( m m ) figure 4. glucose tolerance test of nd and hfd mice treated with cetirizine for 2 weeks. after 2 weeks of cetirizine treatment nd (a) and hfd (b) mice were fasted for 8 h and injected intravenously with glucose (2.5 g/kg). blood glucose levels were analyzed at the time points given in the figure. results are means ± sem for 8–10 mice in each group. c: data from figures 4a and 4b were recalculated to area under the curve (auc). *p < 0.05 versus hfd mice without cetirizine treatment using the mann–whitney u test for non-parametric populations. 44 e. anvari et al. leptin–histamine loop. nevertheless, we cannot completely rule out a modest effect of cetirizine on the central leptin–histamine loop. for example, our results might hint at a minor trend to increased body weight in response to cetirizine, and the high dose of cetirizine here used (25 mg/kg) could have facilitated some leakage across the blood–brain barrier. inhibition of the h1-receptor outside the central nervous system improved non-fasting blood glucose and the glucose tolerance of high-fat diet mice. as we did not measure insulin during the glucose tolerance test, it is not clear whether beta-cell function was actually improved. however, our results may suggest that peripheral inhibition of h1-receptor signaling improves beta-cell function, and that this occurs without affecting peripheral insulin resistance. in type 2 diabetes there is a shift in the profile of immune cells, both in islets and in adipose tissue, from an anti-inflammatory to a pro-inflammatory state (31). furthermore, islet macrophages, in response to hyperlipidemia, have been proposed to activate the nlrp3 inflammasome leading to il-1b release, amyloid formation, and beta-cell dysfunction (32). as we have recently observed that cetirizine partially counteracted the effects of the pro-inflammatory cytokines il-1b and ifn-g on beta-tc6 cell death (14), possibly via modulating c-jun n-terminal kinase (jnk) activity and release of the chemokine macrophage migration inhibitory factor (mif), it may be that h1-receptor signaling exacerbates islet inflammation and therefore also beta-cell dysfunction. interestingly, other strategies to reduce systemic inflammation besides using cetirizine, for example by treatment with omega 3-fatty acids or an il-1b neutralizing monoclonal antibody, have in some cases also failed to reduce peripheral insulin resistance (33,34). thus, anti-inflammatory treatments might have less effect on peripheral insulin resistance than on islet dysfunction, a notion that fits well with the results of the present investigation. it is not yet known whether oral antihistamines affect glucose intolerance or type 2 diabetes in human subjects. the present findings open the possibility that targeted inhibition of the h1-receptor expressed on cells in pancreatic islets, and not targeted to adipose/neuronal tissues, can result in improved metabolic control. acknowledgements this work was supported in part by the swedish diabetes association, the family ernfors fund, barndiabetesfonden, and the novo-nordisk foundation. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. neumann d, schneider eh, seifert r. analysis of histamine receptor knockout mice in models of inflammation. j pharmacol exp ther. 2014;348:2–11. 2. rajasekaran n, solomon s, watanabe t, ohtsu h, gajda m, bräuer r, et al. histidine decarboxylase but not histamine receptor 1 or 2 deficiency protects from k/bxn seruminduced arthritis. int immunol. 2009;21:1263–8. 3. bene l, sápi z, bajtai a, buzás e, szentmihályi a, arató a, et al. partial protection against dextran sodium sulphate induced colitis in 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http://www.ncbi.nlm.nih.gov/pubmed/17965772?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17965772?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17965772?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17965772?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/11574888?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/11574888?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/11574888?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/11574888?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21269673?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21269673?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21269673?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21269673?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/12142541?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/12142541?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23121973?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23121973?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23121973?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23121973?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/8616883?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/1981354?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/1981354?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/1981354?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/1981354?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24717804?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24717804?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24717804?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24717804?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/9074948?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/9074948?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/9074948?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/18498541?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/18498541?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/18498541?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/10594545?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/10594545?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7751519?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7751519?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7751519?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17323859?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17323859?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/17323859?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7741033?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7741033?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7584684?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7584684?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/2469708?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/2469708?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/2469708?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15850951?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15850951?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15850951?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/15570044?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/9352590?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/19352320?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/19352320?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20217514?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/20217514?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21466648?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21466648?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/11272151?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/11272151?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/11272151?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/11272150?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/11272150?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/25035952?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/25035952?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24798950?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/24798950?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23328126?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23328126?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23129601?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23129601?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23129601?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/23129601?dopt=abstract abstract introduction methods development of diabetes in nod mice high-fat diet treatment of c57bl/6 mice blood glucose tolerance test insulin sensitivity test statistics results cetirizine did not protect against development of diabetes in nod mice cetirizine ameliorated high-fat diet-induced glucose intolerance in c57bl/6 mice discussion acknowledgements declaration of interest references sups_a_487165 238..244 upsala journal of medical sciences. 2010; 115: 238–244 original article effect of mailed feedback on drug prescribing profiles in general practice: a seven-year longitudinal study in storstrøm county, denmark keld vægter1,2,3, rolf wahlström1,2,4, hans wedel5 & kurt svärdsudd1 1uppsala university, department of public health and caring sciences, family medicine and clinical epidemiology, uppsala, sweden, 2unit of continuous medical education in general practice (fuap), health department, storstrøm county, nykøbing f, denmark, 3centre for development and research in primary care in sörmland, eskilstuna, sweden, 4division of global health (ihcar), department of public health sciences, karolinska institute, stockholm, sweden, and 5nordic school of public health, gothenburg, sweden abstract background. whether written feedback on drug prescribing in general practice affects prescribing habits is controversial. most short-term studies showed no effect. however, the issue has not been tested in long-term studies involving the local general practitioner community. aims of the study. to assess whether prescribing levels in general practice are affected by long-term, unsolicited, systematically repeated, mailed feedback. methods. each of the 94 general practices in storstrøm county, denmark, received semi-annual, mailed feedback about their prescribing volumes and costs within 13 major drug groups, in relation to the levels for all the other 93 practices over a 7-year period in a project initiated by the local general practitioner association. data on the number of defined daily doses (ddds) prescribed per 1000 listed patients in each practice per 6-months, and practice characteristics, were obtained from the pharmaceutical database at the county health department. results. there was a large variation in drug prescribing volume between practices, but little within-practice variation over time. after adjustments for the influence of practice size and other potential outcome-affecting variables, there was no evidence of a general change of prescribing volume over time, no change among practices with a high or a low prescribing level, and no significant change within the various drug groups. conclusions. we found no significant effects on prescribing levels of mailed feedback, even when repeated semi-annually during 7 years and initiated by the local general practitioner community. key words: drug prescribing, general practice, mailed feedback introduction drug prescribing in general practice is subject to attention from all interested parties. in scandinavia, the majority of prescriptions, regardless of medical specialty, are issued by general practitioners (gps) (1,2). significant variations in prescribing habits among gps have been shown (3,4), which to a certain extent may be explained by variations in demographic factors, including patient age, sex, and diagnostic panorama (‘case mix’). there may, however, be as yet unidentified additional reasons for these variations, such as the prescribing habits of individual gps. the ‘optimum’ prescribing profile for a practice is difficult to define. a basic step in the drug prescribing quality assessment process is to become aware of one’s own prescribing profile. this may be accomplished by displaying the prescribing profile of a correspondence: keld vægter, md, department of public health and caring sciences, family medicine and clinical epidemiology section, po box 564, se-751 22 uppsala, sweden. e-mail: keld.vaegter@pubcare.uu.se (received 7 march 2010; accepted 16 april 2010) issn 0300-9734 print/issn 2000-1967 online � 2010 informa healthcare doi: 10.3109/03009734.2010.487165 given practice as compared to the variations among all practices in the area, for instance by mailed feedback. whether this undertaking is enough to start a perpetual rational drug therapy review on the practice level has been debated (4,5). the effect of mailed feedback is controversial. in a number of studies no effect was found (6–9), whereas others have reported limited positive effects in general practice of postal feedback of prescribing profiles for selected drug groups combined with treatment recommendations (10,11). in 1991 an initiative was launched to improve drug prescribing among gps in the former storstrøm county, denmark. during fourteen 6-month periods, mailed feedback to the individual practices was used to encourage gps to review their drug therapy profile and, if needed, reconsider their prescribing practices. in this report the effect of this unsolicited, semiannually mailed feedback on intraand inter-practice variations in drug prescribing was assessed. material and methods setting the former storstrøm county is today part of the larger administrative unit region sjælland. at the time of the study it included the southern part of sjælland, the islands of falster and lolland, and a few other minor islands, and had 257,000 residents. the area is mainly rural with a few small towns and was served by 166 general practitioners distributed across 94 practices. in denmark the general practitioners are private contractors to the county health authority, each taking care of approximately 1500 listed patients. each practice has a specific identification number (pin) within the national health insurance system. all relevant information related to administration and fees in the practices, such as patient demographics, prescriptions (obtained from the danish medicines agency), referrals, and specific services and treatments performed in the practice, is registered in the local county health insurance database. traditionally, most practices in primary health care in denmark have been ‘solo’ practices (run by one gp), but in recent decades the formation of group practices has become increasingly common. in group practices it is not possible to identify the individual gp’s contribution to the common prescribing profile, since the pin refers to the practice as a whole. the population of listed patients in the practice system is stable, with an average annual change between practices of less than 10%. the differences between practice patient populations in terms of age and gender are small (personal communication from the pharmacoeconomic division, danish medicines agency). data collection all prescriptions filled at danish pharmacies, reimbursed as well as non-reimbursed, are registered in a database at the danish medicines agency by practice pin code and anatomical therapeutic chemical (atc) code (12). the registration is almost 100% complete. all prescriptions analysed in this report were fully reimbursed. in 1991 the first steps were taken to establish a ‘gp quality unit’ by collaboration between representatives from general practice and officials from the health administration within the health department of storstrøm county. the aim was to encourage a review among gps of their prescribing habits in order to improve and enhance rational drug therapy. to visualize differences in prescribing habits and to trigger the awareness of the gps, prescribing data on reimbursed pharmaceuticals with the atc codes a02 (antacids), a10 (anti-diabetes drugs), c01 (cardiac drugs), c03 (diuretics), c07 (beta-blockers), c08 (calcium channel-blockers), g03 (reproduction hormones), j01 (anti-bacterial drugs for systemic use), m01 (non-steroid anti-inflammatory drugs), n02 (analgesics), n05 (neuroleptic drugs), n06 (psycho-analeptic drugs), and r03 (anti-asthma drugs) were extracted from the county health insurance database, presented in charts, and mailed to each practice every 6 months. no intervention other than the mailed feedback was made. the feedback diagrams illustrated the prescribing levels of each of the 13 drug groups as number of defined daily doses (ddd) per 1000 listed patients and the practice’s percentile position within the distribution across all practices in the county. the corresponding information on costs per ddd prescribed by the practice was presented in a similar way. an example of the feedback diagrams is shown in figure 1. every 6 months new data were added to the charts and mailed to the practices. if this type of feedback works, the anticipated effect would be a clustering of prescribed ddds towards the mean, i.e. a smaller dispersion between practices and a tendency towards instability of individual practice prescribing patterns over time owing to changing habits. since the initiative for political reasons was launched full scale simultaneously in all practices throughout the county, no control group was available. therefore, the prescribing habits of all practices were followed through the study period. mailed feedback on drug prescribing 239 approval from an ethics committee was not needed since the project did not include direct patient involvement, and no classified information that could reveal patient identity was handled. statistical considerations statistical analyses were performed using the sas software, version 9.1 (13). data were complete. simple (crude) differences between groups regarding continuous variables were tested with student’s t test and differences in proportions with the chisquare test. during the observation period 1992–1998 a few new practices were established. this might have given rise to false low values regarding the numbers of prescribed ddd/1000 patients in the opening period. to avoid this problem, data from the first 6-month period of new practices were excluded. in order to discover and test changing habits four methods were employed. the first focused on intrapractice variation of prescribing habits, where variation would increase in case of changing habits. the prescribing data constitute a time-series of prescriptions issued by the same gp population. the resulting ddds may therefore be auto-correlated, i.e. the value for a specific 6-month period predicts, to a certain extent, the value of the next period. to overcome this problem the sas procedure ‘autoreg’ was used to diagnose and to adjust for auto-correlation. the adjustment included the nearest three 6-month periods, as these were significantly auto-correlated. the resulting adjusted measure of variability, mean square error (mse), may be regarded as a variance adjusted for prescribing trend across time. we used the square root of this measure, which is equivalent to a standard deviation unit, as a measure of prescribing variability within practices over time. the second method was based on the so-called ‘folding rule’ regression analysis. the study period was divided in two sub-periods, and a regression line of prescribed ddds per 1000 listed patients over time was calculated for each practice and for each subperiod, much like a folding rule. the meeting-point, or intersection, between the lines was successively moved across the study period. a significant difference in regression coefficient (‘leaning’) between the two lines would then indicate a change of subscribing habit. in this way the existence of a systematic deviation towards the central part of the prescribing distribution might be detected. thirdly, the change in the distribution of ddd per 1000 listed patients across the fourteen 6-month antibiotics (j01) 1993–1997 d d d /1 0 0 0 li st e d p a tie n ts a u p /d d d semi-annual periods semi-annual periods 0 93–1 94–1 95–1 96–1 97–1 93–2 94–2 95–2 96–2 97–2 93–1 0 4 8 12 16 20 24 94–1 95–1 96–1 97–1 93–2 94–2 95–2 96–2 97–2 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 figure 1. example of feedback information sheet. the left-hand panel presents data regarding the amount of drugs, in this case antibiotics, prescribed. the right-hand panel presents cost data. the dotted line represents the prescribing level of a specific practice relative to all practices in the county. the vertical scale corresponds to the 5th, 25th, 75th, and 95th percentiles of the distribution across all practices. ddd = defined daily doses; aup = costs in danish kroner per ddd. 240 k. vægter et al. periods was tested. if a change of habit had occurred, the distribution would be narrower towards the end of the study period than in the beginning. fourthly, scatter plots of the prescription volumes for each practice over time were produced and scrutinized for change of trend. univariate and multivariate linear regression analyses were used to analyse the influence of various potential determinants, such as age and sex of the listed patients (‘case mix’), age and sex of the gps, practice type, and number of years of experience as a gp, on prescribing variation, measured as mean error, or on practice regression coefficients. all tests were two-tailed. the level of significance was set at p < 0.05. results characteristics of the study population study population characteristics are shown in table i. fifty-four practices were solo practices, and 50 were group practices with an average of 3.2 gps per practice. the 166 gps were on average 50 years old (interquartile range 46–54 years), 81% were men, mean number of years as a gp was 14.5 (interquartile range 8–20 years). there were no significant differences in the distribution of age, sex, and gp experience between gps in solo versus group practices. prescribing habits over time the overall prescribing rate of the 13 drug groups studied increased from 64,870 ddds/1000 patients in the first 6 months of 1992 to 70,360 ddd/ 1000 listed subjects in the last 6 months of 1998, an annual increase rate of 915 ddd/1000 listed subjects. the trends in prescribing rate for the various drug groups are shown in figure 2. the number of ddds per 1000 listed subjects prescribed during the study period increased significantly for antacids (a02), anti-diabetes drugs (a10), beta-blockers (c07), calcium channel-blockers (c08), reproduction hormones (g03), analgesics (n02), psycho-analeptic drugs (n06), and anti-asthma drugs (r03), while it decreased for cardiac drugs (c01) and diuretics (c03), and remained unchanged for antibiotics (j01), non-steroid anti-inflammatory drugs (m01), and neuroleptic drugs (n05). prescribing variation among practices the variations between practices in amount of prescribed drugs within the various atc groups are presented in table ii as semi-annual mean ddd/ 1000 listed patients, the 95th and the 5th percentile of the ddd distribution across practices, and the ratio of these two. there were fairly large variations in prescribing level between the practices, as reflected by the 95th to 5th percentile ratios. the largest differences were seen for neuroleptic drugs (ratio 6.0) and the smallest for anti-asthma drugs (ratio 2.5). there was little variation over time within prescribing practices, as reflected by the root of the mse, a measure of the mean prescribing standard deviation adjusted for the increasing or decreasing trend line over the fourteen 6-month periods (table ii). the root mse was 0.20 units, one-fifth of a standard deviation, with a 95% confidence interval (ci) of 0.18–0.23 for all drug groups combined after adjustment for auto-correlation, indicating only minute deviations from the trend line. among the individual drug groups, analgesics (n02) and calcium channel-blockers (c08) had the lowest variability and anti-diabetes drugs (a10) the highest. the within-practice deviation range was 0.01– 1.08 for individual atc groups. in the folding rule regression analysis across all practices there was no evidence of a deviation of high or low prescribers towards the mean for all gp practices, nor was there any significant change in the ddd per 1000 listed subjects distribution table i. characteristics of the study population. solo practices group practices total n mean or % 95% ci n mean or % 95% ci n mean or % 95% ci n 54 57.4 40 42.6 94 100.0 age, years 54 50.3 48.7–51.9 112 49.9 48.7–51.1 166 50.0 49.1–51.0 male physicians, % 46 85.2 75.4–95.0 88 78.6 70.9–86.3 134 80.7 74.7–86.8 no. of gps in practice 54 1.0 – 112 3.2 2.9–3.4 166 1.77 1.54–1.99 no. of years as a gp 54 15.1 13.0–17.1 112 14.3 12.8–15.7 166 14.5 13.4–15.7 ci = confidence interval; gp = general practitioner. mailed feedback on drug prescribing 241 across time when adjusting for the increasing mean. scrutiny of practice-specific trend lines for the prescribing levels of the various atc groups gave no evidence that high or low prescribing practices tended to change their course (data not shown). the solo practices had larger prescribing variations than group practices (0.30 sd units, 95% ci 0.24–0.35, versus 0.19, 95% ci 0.15–0.23; p < 0.005). among the solo practices there was no difference in prescribing variation between male and table ii. variation of drug prescribing habits, measured as mean standard deviation of prescribed ddd/1000 patients across the followup period. drug group atc code semi-annual meana percentiles ratio 95th/5th root mse 95th 5th mean 95% ci range antacids a02 2475.59 4377.0 1137.4 3.8 0.24 0.21–0.26 0.03–0.66 anti-diabetes drugs a10 2460.11 3889.0 1304.4 3.0 0.30 0.27–0.33 0.03–0.73 cardiac disease drugs c01 4689.73 8289.7 1981.4 4.2 0.30 0.26–0.34 0.03–1.08 diuretics c03 19315.90 31723.8 10143.7 3.1 0.23 0.20–0.25 0.01–0.77 beta-blockers c07 2845.88 4695.4 1366.0 3.4 0.25 0.22–0.28 0.02–0.81 calcium channel-blockers c08 6123.94 10276.1 2747.2 3.7 0.20 0.18–0.22 0.01–0.61 sex hormones g03 4642.50 7545.8 2089.0 3.6 0.22 0.20–0.24 0.03–0.65 antibiotics j01 1433.18 2481.2 645.8 3.8 0.25 0.23–0.28 0.01–0.60 nsaids m01 5107.58 8171.3 2854.1 2.9 0.25 0.22–0.27 0.01–0.63 analgesics n02 5374.84 9979.6 2416.6 4.1 0.20 0.17–0.22 0–0.55 neuroleptics n05 1321.11 2852.4 474.5 6.0 0.23 0.20–0.26 0.01–0.90 anti-depressants n06 3139.88 4501.1 1494.0 3.6 0.21 0.19–0.23 0.02–0.51 anti-asthma drugs r03 9851.54 14575.7 5900.0 2.5 0.28 0.25–0.31 0–0.76 addd/1000 patients. atc = anatomical therapeutic chemical; ci = confidence interval; ddd = defined daily doses; nsaids = non-steroid anti-inflammatory drugs; mse = mean square error. 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 19 92 -1 19 93 -1 19 94 -1 19 95 -1 19 96 -1 19 97 -1 19 98 -1 time, 6-month periods d d d /1 0 0 0 p o p u la tio n c03 diuretics r03 anti-asthma drug c08 calcium channel blockers n02 analgesics m01 nsaids g03 reproduction hormones n06 psycho-analeptic drugs c01 cardiac drugs c07 beta blockers a02 antacids a10 anti-diabetes drugs n05 neuroleptic drugs j01 antibiotics figure 2. levels of prescribed defined daily doses (ddds) per 1000 population per 6 months by drug group. nsaid = non-steroid antiinflammatory drug. 242 k. vægter et al. female gps, but the variation decreased with 0.02 sd units by year of gp age. there were no significant relationships between age, sex, and number of years as gp on the one hand and prescribing volume over time on the other (data not shown). discussion there was a considerable variation in prescribing levels between practices but a considerable stability in the variation of prescribing behaviour over the study period for individual practices, irrespective of analysis method. the mailed feedback had no detectable effect on prescribing behaviour. as expected, there was slightly more variation within solo practices than within group practices because of the counterbalancing effect of accumulated prescribing by of two or more gps in the group practices. the variation decreased somewhat with gp age, and there were no statistically significant gender effects. the analyses were performed on official data, based on filled prescriptions, with little or no data loss. the same authority registered all filled prescriptions, minimizing handling variation. potential disturbing factors, such as auto-correlation, were eliminated during data processing. it was not possible to establish a control group within the county, as the quality improvement initiative (‘gp quality unit’) aimed at covering all practices. comparison with other counties was not possible, since data were not available for areas other than storstrøm county. therefore, the practices served as their own controls over time. data refer to prescribing habits during the 1990s. however, the problem of rational prescribing habits is still prevalent (11). few authors have addressed the issue of stability of prescribing habits in general practice. one of the main reasons may be the lack of comprehensive longterm prescription data registers at prescribing physician level. in a new zealand study from 1992–94 (5), based on reimbursement data, a 9% median intragp variability was found in both volume and total costs from year to year in a regional gp sample (305 gps), and a 16% variation in total costs and 17% in total volume in a national gp sample (74 gps). our finding of no effects of mailed feedback on gps’ prescribing behaviour conforms to what has been shown in a comprehensive 2000 cochrane review (6). similar results have also been shown in the few studies where large data registers were used to collect outcome measures. in a randomized controlled trial in australia (7), no effects were found of unsolicited, posted government-sponsored feedback based on centralized aggregated data on prescribing levels of general practitioners. in a danish randomized, controlled trial it was concluded that postal prescribing feedback in addition to clinical guidelines on the diagnosis and treatment of respiratory tract infections did not influence gps’ prescribing patterns (8,9). however, there are studies that demonstrate some effects. in a canadian trial 54 gps whose prescribing of analgesics was more than two standard deviations above average were randomly allocated to receiving a note on their prescribing volume and a 1-day group education activity, or to receiving a written notification only, or to no intervention (14). those in the first group decreased their prescribing volume by 33%, and those in the second group by 25%, while there was no change in the third group. similar but smaller effects were found in a norwegian study (10) and in a canadian study (11) when written feedback of prescribing profiles was combined with treatment recommendations. in a 2006 cochrane review it vwas found that the combination of audit and feedback had a small to moderate effect on professional practice (15). although mailed feedback only has shown a modest or no effect on doctors’ drug prescribing, it is still widely used in continuing medical education (cme) and in quality assurance and improvement. the method is easy to apply on a large scale and relatively cheap. however, the approach appears to be more effective if combined with other strategies (16), such as audit feedback with peer discussions (17,18). some possible explanations for the lack of success with mailed feedback only in this study might be that the gps may not have paid attention to the diagrams, or they may not have understood the diagrams, or they may have taken the diagrams into consideration but found no reason to chance their prescribing habits. moreover, too much information with poor explanation may have been provided. it is important to note that the establishing of the ‘gp quality unit’, the development of the feedback charts, and the semi-annual mailed prescribing feedback were some of the first, but important, steps in the process of establishing a formal local quality improvement culture within the general practice community of storstrøm county in the early 1990s. the main purpose of the initiative was to initiate reflections on variations in prescribing behaviour and raise awareness about prescribing patterns. the feedback diagrams were not accompanied by any clinically relevant information, and they were based on aggregated prescribing data at the second atc level with no chance of identifying specific substances at the fifth atc level. on the other hand, too detailed prescribing information at this early stage in the mailed feedback on drug prescribing 243 quality assurance process might possibly impede the overall ambition of starting a debate about rational drug prescribing in a broader sense. conclusions no apparent effect of mailed feedback on prescribing habits in general practice was found. other, more activating approaches than postal feedback may be necessary to affect gps’ prescribing behaviour. acknowledgements this work was supported by grants from the health council of storstrøm county, denmark, sörmland county council, nyköping, sweden, and uppsala university, uppsala, sweden. we are indebted to the founders and staff of the gp quality unit of storstrøm county for their efforts in this project. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. swedish pharmacy authority. available at: http://www. apoteket.se/rd/d/2835 (accessed 25 feb 2010). 2. nordic statistics on medicines 1975–1977. in: statistical reports of the nordic countries no 35, 36. helsinki and oslo: nordic council on medicines; 1979. 3. maronde rf, lee pv, mccarron mm, seibert s. a study of prescribing patterns. med care. 1971;9:383–95. 4. oxman ad, thomson ma, davis da, haynes rb. no magic bullets: a systematic review of 102 trials of interventions to improve professional practice. cmaj. 1995;153:1423–31. 5. bishop n, maling t. variability within general practitioner prescribing over time. n z med j. 2000;113:14–16. 6. freemantle n, harvey el, wolf f, grimshaw jm, grilli r, bero la. printed educational materials: effects on professional practice and health care outcomes. cochrane database syst rev. 2000;(2):cd000172. 7. o’connell dl, henry d, tomlins r. randomised controlled trial of effect of feedback on general practitioners’ prescribing in australia. bmj. 1999;318:507–11. 8. sondergaard j, andersen m, stovring h, kragstrup j. mailed prescriber feedback in addition to a clinical guideline has no impact: a randomised, controlled trial. scand j prim health care. 2003;21:47–51. 9. sondergaard j, andersen m, vach k, kragstrup j, maclure m, gram lf. detailed postal feedback about prescribing to asthma patients combined with a guideline statement showed no impact: a randomised controlled trial. eur j clin pharmacol. 2002;58: 127–32. 10. rokstad k, straand j, fugelli p. can drug treatment be improved by feedback on prescribing profiles combined with therapeutic recommendations? a prospective controlled trial in general practice. j clin epidemiol. 1995;48: 1061–8. 11. herbert cp, wrigtht jm, maclure m, wakefield j, dormuth c, brett-maclean p, et al. better prescribing project: a randomized controlled trial of the impact of case-based educational modules and personal prescribing feedback on prescribing for hypertension in primary care. fam pract. 2004;21: 575–81. 12. who collaborating centre for drug statistics methodology. the anatomical therapeutic chemical classification system. oslo: who collaborating centre for drug statistics methodology; 1976. 13. statistical analysis system. available at: http://www.sas.com/ technologies/analytics/statistics/ /factsheet.pdf (accessed 25 feb 2010. 14. anderson jf, mcewan kl, hrudey wp. effectiveness of notification and group education in modifying prescribing of regulated analgesics. cmaj. 1996;154:31–9. 15. jamtvedt g, young jm, kristoffersen dt, o’brien ma, oxman ad. audit and feedback: effects on professional practice and health care outcomes. cochrane database syst rev. 2006;(2):cd000259. 16. wensing m, grol r. single and combined strategies for implementing changes in primary care: a literature review. int j qual health care. 1994;6:115–32. 17. wahlstrom r, kounnavong s, sisounthone b, phanyanouvong a, southammavong t, eriksson b, et al. effectiveness of feedback for improving case management of malaria, diarrhoea and pneumonia—a randomized controlled trial at provincial hospitals in lao pdr. trop med int health. 2003;8:901–9. 18. nilsson g, hjemdahl p, hassler a, vitols s, wallen nh, krakau i. feedback on prescribing rate combined with problem-oriented pharmacotherapy education as a model to improve prescribing behaviour among general practitioners. eur j clin pharmacol. 2001;56: 843–8. 244 k. vægter et al. encounters with adenovirus review article encounters with adenovirus ulf pettersson department of immunology, genetics and pathology, uppsala university, uppsala, sweden abstract in this paper i describe aspects of work on the human adenoviruses in which my laboratory has participated. it consists of two sections—one historic dealing with work performed in the previous century, and one dealing with the application of ‘omics’ technologies to understand how adenovirusinfected cells become reprogrammed to benefit virus multiplication. article history received 25 april 2019 accepted 26 april 2019 key words adenovirus; proteomics; transcriptomics looking backward beginning a life in science in 1965, when i was in in medical school, i contacted my pathology professor, jan pont�en, asking if i could join his research group. jan informed me that he was oversubscribed and instead recommended that i contact his colleague and friend, lennart philipson, who was working further down the hall in the old pathology building in uppsala. lennart had just returned from a postdoctoral training period with igor tamm at the rockefeller. as luck would have it, he accepted me as a student and suggested that i work on the adenovirus (ad) antigens, then known as the a-, b-, and c-antigens. ads had been independently discovered by rowe et al. (1) and by hilleman et al. (2) about 10 years earlier. it was recognized from the start that the viruses replicated with exceptional efficiency and produced large amounts of soluble antigens. philipson, who was closely allied with the institute of biochemistry in uppsala (which under the leadership of nobel laureate arne tiselius had become the mecca of protein separation technology), was hoping to apply state-ofthe-art biochemistry to virus studies. he predicted that ads would be ideal for studies of virus components. at the time of my entry into research, ad was a largely unknown entity. in fact, reprints of all published papers fitted into my briefcase. in 1965, however, a seminal paper appeared in the journal of molecular biology authored by valentine and pereira (3). using electron microscopy, they showed that the adenovirion has a strikingly handsome architecture. an icosahedral capsid, composed of 240 capsomeres, it carries antenna-like projections from each of the 12 corners (figure 1). it became apparent from the structure that the three known soluble a-, b-, and c-antigens correspond to components in the viral shell. the a-antigen makes up the facets, and the b-antigen sits at the vertices. the b-antigen is composed of the vertex capsomere, which is non-covalently linked to an antenna. the last-mentioned is the c-antigen. erling norrby working with another serotype came to an identical conclusion and showed in addition that the b-antigens could form so-called dodecons consisting of 12 penton units (5). in 1966 ginsberg et al. proposed a nomenclature for the antigens (6). antigen a is named hexon, whereas penton is the name of the vertex capsomere consisting of the penton base with the non-covalently attached fiber, the c-antigen. the fiber and the penton base play key roles in the early virus–cell interactions. the knob of the fiber attaches the virus to its receptor. the receptor for most human ad serotypes is car (coxsackie adenovirus receptor). an unexpected discovery was that two unrelated viruses use a common receptor reported before virology became molecular. also the penton base plays an essential role in virus attachment through the presence of an rgd motif which interacts with integrins starting the reorganization of the cell structure. soon after its discovery it was recognized that the ad family comprises several so-called serotypes and that these are associated with different symptoms in infected patients. professor ulf pettersson, winner of the medical faculty of uppsala university rudbeck award 2018 ‘for his pioneering scientific achievements in virology and human genetics’. contact ulf pettersson ulf.pettersson@igp.uu.se department of immunology, genetics and pathology, uppsala university, uppsala, sweden � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 2, 83–93 https://doi.org/10.1080/03009734.2019.1613698 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1613698&domain=pdf&date_stamp=2019-05-31 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2019.1613698 http://www.tandfonline.com today more than 70 human ads are known. in general, they cause mild disease, and subjects are often unaware of their encounters with the virus. the infectivity is low, and it was noted early that widespread infection often occurs in closed communities, e.g. among military recruits. the most common adenovirus infections are associated with mild respiratory symptoms. other types cause keratoconjunctivitis or gastrointestinal and urinary tract infections. in rare cases ads do cause life-threatening disease, primarily in immune-compromised patients. the ad family is, based on antigenicity and dna sequence homology, divided into seven subgenera, named a–g. members of the different subgenera usually cause different symptoms. trentin et al. conducted a systematic study of known human viruses, in alphabetical order (7). they quickly noted that injection of ad 12 into newborn hamsters resulted in tumors at the site of injection within a few months. this finding raised the interest in ads tremendously, and it was quickly demonstrated that ad 12 is able to transform baby hamster kidney cells in vitro (8). the early years studies of the ad proteins were central in the 1960s. the capsid components were fractionated and purified in several laboratories using state-of-the-art biochemical techniques. this work formed the subject of my phd thesis, which i defended in 1970 (9–12). from biochemical studies it became apparent that the virus must contain proteins other than hexons and pentons. amino acid analysis revealed that the composition of the hexon, which should account for 95% of the protein mass of the virus, was similar to that of complete virions except for two amino acids, arginine and lysine. studies by laver et al. (13) and prage et al. (14) showed that ad dna could be isolated as a dna protein complex. further studies revealed that the ad chromatin contains two proteins: one smaller very basic component, and another moderately basic larger component. pioneering studies on the peptide composition of the adenovirion were made by jacob maizel, who had introduced sds polyacrylamide gel electrophoresis as a tool for the characterization of virion components. he identified 10 ad polypeptides in his electropherograms, which with the current nomenclature are named polypeptides ii, iii, iiia, iv, v, vi, vii, viii, ix, and x (15). polypeptides v and vii are highly basic and constitute the ad chromatin together with the very small polypeptide x (also known as polypeptide mm). polypeptide ix is associated with hexons in the facets of the virion, whereas polypeptides vi and viii are located inside the capsid although their precise locations are as yet undetermined. sixty copies of polypeptide iiia are present in adenovirions, probably associated with the inside of the vertices. anderson et al. showed that some of them are synthesized as larger precursors which during capsid assembly are cleaved by an ad-encoded proteinase (16). today it is known that polypeptides iiia, vi, vii, viii, and x are produced as precursors. later work has shown that a few copies of additional proteins are present in the adenovirion, namely iva2 and c-168 (17). moreover, comprehensive studies of the spliced forms of ad mrna give room for additional viral proteins (18). the 1970s was the decade of the ad genome. early work (19,20) had established that ads consist of 13% doublestranded linear dna and 87% protein with a molecular size ranging between 24 and 26 � 106 (19,20). a noteworthy finding was that although the genomes of the human serotypes are very similar in size, they differed dramatically in gc contents, subgroup a being lowest with 47%–49% gc and subgroups c, d, and e the highest with 57%–59% gc (21). garon et al. (22) and wolfson and dressler (23) showed that single strands of ad dna form circles with so-called pan handles due to the presence of inverted terminal repetitions with a length of about 150 bp (24). another unique property of the ad genome is that the ends of the double-stranded dna are covalently linked to a protein, the terminal protein, which plays a role in the initiation of dna replication (25). like some of the other capsid proteins, it is subjected to cleavage during virion maturation. ad encodes its own dna polymerase (26), and when the genome was sequenced it became apparent that it also encodes the terminal protein in a transcription unit which includes genes engaged in the replication of the ad genome. replication of ad dna follows a unique pathway. as a postdoc i began to study replicating ad dna by cscl gradient centrifugation and observed that replicating molecules had a greater density than mature viral dna and that the figure 1. a schematic picture of the adenovirus particle showing the locations of the different capsomeres. modified from (4). 84 u. pettersson difference was eliminated after treatment with a singlestrand specific exonuclease (27). these observations fitted the model published by sussenbach et al. proposing that ad replication occurs by a strand displacement mechanism (28). further studies showed that replication starts at either end of the genome and involves two steps (28). a viral-encoded dna polymerase displaces first one of the strands, which is subsequently replicated in a second step. a puzzling observation is that bacteriophage phi29 employs an identical mechanism for its genome replication. in the early 1970s little was known about viral genome organizations as no dna mapping tools were available. nothing was known about gene numbers and gene locations on the ad chromosome. in the fall of 1971, i attended the dna tumor virus workshop in cold spring harbor. in one of the sessions daniel nathans gave a presentation during which he described how the sv40 genome could be cleaved into a distinct number of fragments by an endonuclease from hemophilus influenzae (now known to be a mixture of hind ii and iii) and that these could be separated by polyacrylamide gel electrophoresis. nathans’s presentation was an eye opener. it clearly showed that the restriction enzymes would be ideal tools for mapping the chromosomes in dna viruses. in the summer of 1971, i had arrived in cold spring harbor as a postdoctoral fellow. one of my colleagues, carel mulder, was looking for an enzyme that would cleave circular sv40 dna at one unique point. herbert boyer and his student yoshimori had generously provided mulder with a tube of an enzyme, now known as ecor1, which had this specific feature. as i happened to have purified ad2 dna in store we decided to collaborate. the results we obtained were clearcut; our friend hajo deluis showed with his electron microscope that the enzyme generated six fragments of different sizes (29), which due to differences in their denaturation profiles could be ordered along the linear genome. richard roberts arrived in cold spring harbor the same year as i did. he set up a ‘factory’ for production of restriction enzymes with different cleavage specificities. using some of these it became possible to cleave the ad genome into sets of overlapping fragments which were ideal tools for gene mapping. from early studies it was known that a distinct set of ad genes are expressed before replication of the genome and another after dna replication had commenced, the so-called early and late genes. transcriptional maps for ad2 were constructed by flint and co-workers (30) and by clark tibbetts and myself (31). the complementary strands of fragments generated by restriction enzymes ecor1, hind iii, and hpa1 were hybridized with mrna isolated either early or late after infection. the results revealed an unexpected complexity with genes located on both complementary strands. the early genes were found in four locations, designated e1, e2, e3, and e4. two were located on the so-called r-strand and two on the l-strand, whereas the bulk of the late mrnas hybridized to the r-strand. ad gene mapping was revolutionized when rna-loop technology was introduced for mapping rna transcripts. the application to ads was pioneered by chow and broker, who constructed complete maps of ad mrnas with this technology (32). the technology paved the way for the discovery of rna splicing in 1977 (33,34). this is one of many iconic discoveries in molecular biology which ad researchers have made (figure 2). in the fall of 1976, while visiting the cold spring harbor laboratory, i attended an improvised seminar given by walter gilbert. he described a revolutionizing dna-sequencing method based on chemical cleavage of end-labeled fragments prior to separation of the resulting fragments by polyacrylamide gel electrophoresis—later called the maxam and gilbert method. although the method was unpublished until a year later (35), gilbert handed out a detailed protocol. i received a copy, which i gave to one of my many talented phd students, g€oran akusj€arvi, when i returned to sweden. ten years later we published the complete 35,937 nucleotides long sequence of ad2 together with richard roberts and his team (36). genes involved in ad oncogenesis have been studied using different approaches. pioneering work was performed the adenoviruses have undoubtedly played a remarkable role in the history of eukaryotic molecular biology. here are a few landmarks: • splicing was discovered in 1977 by chow et al. (32) and by sharp and co-workers (33) through electron microscopic studies of mrna from ad2-infected cells • a general procedure for cell transformation with naked dna was developed by graham and van der eb when they showed that naked ad5 dna has the capacity to infect human and transform murine cells (36,37) • the e2f transcription factor was discovered by joe nevins (82), as an activator of the e2 transcription unit in ad; subsequently it was discovered that it is a key regulator of cell division in normal and tumor cells • a general mechanism in oncogenesis was discovered by harlow and collaborators when they showed that the ad e1 protein binds to the retinoblastoma protein, prb, thereby releasing e2f with uncontrolled cell growth as a consequence (83) • although first discovered in sv40 (84), ad studies (85 ) have revealed that an ad protein sequesters p53, thereby blocking apoptosis and preventing cell death • the ad major late promoter was an invaluable tool for the development of in vitro systems for eukaryotic transcription • ad is an excellent vector for transfer of genetic material in human cells, and oncolytic ads (86) have an established role in cancer treatment figure 2. the iconic status of adenoviruses. upsala journal of medical sciences 85 by graham and van der eb. their first aim was to develop a method for efficient transfer of ad dna to mammalian cells. after systematically testing a multitude of reagents, they discovered that ad dna is efficiently transferred to cells using calcium phosphate precipitation (37). this is a true landmark in the history of eukaryotic molecular biology as the method has proven to have a very broad scope. using the method, they showed that naked ad dna can infect human cells and transform murine cells (38). they showed that the transforming genes are very sensitive to exonuclease treatment, indicating that they are located near one of the ends (39). further on, they managed to transform baby rat kidney cells with a short dna fragment, located at the left-hand end, only representing a few percent of the genome (40). as an extension of the work they succeeded in transforming human cells (41). this was unexpected, since human cells are permissive, allowing the virus to replicate, which leads to cell death. after many trials, they managed to establish the socalled 293 cell line which is transformed by ad5 and only contains a subset of the viral genome, including the lefthand end. through the history of molecular biology it has played an important role as a widely used research tool, among other things allowing mutants with defects in transforming genes to be constructed (42). another approach to identify transforming genes involved studies of ad-transformed cell lines. one method was to search for the presence of viral sequences in cell line 8617, a rat embryo cell line transformed by ad2. dna reassociation kinetics is a powerful method for detection of viral sequences in cells transformed by dna viruses. the technology is simple and involves mixing radioactively labeled viral dna with dna from transformed cells. if viral sequences are present in the transformed cells, the speed of the reassociation of the labeled dna will increase and will reflect the number of viral dna copies in the transformed cell. in collaboration with joe sambrook, i applied the technology to 8617 cells. the results showed, contrary to what was reported, that only one copy of ad2 dna is present per cell (43). further analysis showed that the resident genome is incomplete, lacking sequences from the middle of the genome (44). the study was extended by gallimore et al. employing a bunch of independently ad2-transformed cell lines (45). one of these was shown exclusively to contain sequences constituting 14% of the viral genome located at the left-hand end. results from both approaches demonstrated that that the transforming genes of ad are present in the e1 region of the genome. subsequent studies revealed that region e1 comprises two transcription units, designated e1a and e1b. the early ad mrnas have exceptionally complex structures and are partially overlapping, although they encode unique proteins. a variety of technologies have been used to study them, including northern blotting, s1 nuclease analysis, electron microscopy, and cdna cloning. our contributions stem from a unique cdna library which was constructed during a short visit to the pasteur institute in paris. in 1975 i had my first contacts with recombinant dna. it started with a telephone call from paris to lennart philipson. the reason for the call was that luc montaignier’s collaborator at the pasteur institute, pierre tiollais, had constructed a lambda bacteriophage (46), which could serve as a vector for propagation of foreign dna. at the time, boyer, cohen, and berg had caused a sensation by inserting dna from the mammalian sources in an e. coli plasmid. tiollais wanted to test if dna from a human tumor virus would work. recombinant dna was an extremely controversial subject these days. the asilomar conference took place in 1975, and mayor alfred vellucci in cambridge banned work with recombinant dna in his town. from my visits to cold spring harbor i could see the result in that tom maniatis came to the lab for experiments that were forbidden in his hometown. french law required a high-level safety laboratory for the experiment that tiollais was planning. no such facility existed at the pasteur institute. tiollais knew that sweden at the time had a maximum-security laboratory at the so-called national bacteriological laboratory in stockholm. tiollais therefore came to uppsala with his phd student michel pericaudet. phage constructs with the so-called b and f-fragments of ad2 dna were made in uppsala and then transported to stockholm. the experiment worked, and the recombinant phages replicated normally. the dna was transcribed without generating any ad protein. today the experiment appears trivial. then, however, recombinants between proand eukaryotes generated headlines. pierre wrote a paper, which was submitted to cell. rumors tell that, due to the controversial nature of this kind of work, ben lewin sent the paper for review to more than 50 experts. only one recommended publication, primarily because of ethical concerns. in retrospect it seems obvious that the paper lacked cell qualities. in the end, waclaw szybalski, editor of gene, allowed it to be published in gene (47). some years later, the situation was reversed. in uppsala we were interested in cloning the sites where the viral sequences are integrated in ad-transformed cell lines. now the recombinant dna debate in sweden had reached its peak, and we were unable to do the experiment at home. in the meantime a special self-contained laboratory, called the ‘submarine’, had been constructed at the pasteur. i contacted my friend tiollais who let us come to the pasteur for our experiments. i spent five days in paris mostly making libraries with dna from transformed dna inserted into the french lambda vector. unfortunately, without success. however, something else turned out to be a success. with me to paris i brought a tube with polyadenylated rna from ad-infected cells. benefiting from the collegiality and expertise at the pasteur, i for the first time in my life made cdna constructs and cloned them in e. coli. on my final day in paris, while the taxi was waiting to take me to the airport, i ran into pericaudet who sported a big grin. he advised me to check the bacterial plates, which were loaded with clones. the library was then transferred to sweden where we started to sequence clones from the transforming e1a region. the structures of the two longer e1a messages were unraveled quickly. a manuscript was prepared and submitted to nature, which accepted the paper without revisions. the library turned out to be a goldmine for studies of the ‘zoo’ of early 86 u. pettersson ad mrnas, and my phd students did a great job with cdnas from regions eib, e3, and e4 (44,45,48–51). adenovirus in the ‘omics’ era—a multipronged strategy a transcriptomic view of how host cells are reprogrammed at the turn of the century, after a two decades’ long detour into human genetics, i decided to re-enter the ad field. the plan was to use the emerging ‘omics’ technologies to study how ad-infected cells become reprogrammed during the course of a replication cycle. at the time, microarray analysis had emerged as a powerful technology to study gene expression in eukaryotic cells. no commercial microarray chips were, however, available when we started. instead homemade chips needed to be produced, employing very expensive equipment for spotting dna from clones containing dna sequences with known origins. the method was cumbersome and imprecise, often requiring confirmation of the results by pcr. luckily, a young, very skilled virologist, hongxing zhao, had just completed her postdoc and joined my group. she had the expertise and stamina to get the project rolling. first, we (52,53) and others (54) used microarrays, later switching to cdna (55) sequencing to monitor cellular gene expression. to study changes occurring during the first 2 h of an infection zhao et al. used microarray analysis (56). a dozen up-regulated genes with known functions were identified. eight of these encode transcription factors. a set of five, atf3, atf4, klf4, klf6, and elk 3, are linked to growth inhibition. another set of two, nr4a1 and cebpb, are involved in immune activation. the study was extended to other time points, switching to paired-end sequencing of cdna copies of rna, collected at four different times (57). the results generated a set of cellular gene expression profiles (figure 3). over all, about 7000 cellular genes are expressed at a measurable level at all time points, and a large fraction of the genes is changed more than or equal to 2-fold as compared to their expression in non-infected cells. few changes in rna expression occur early, as only 74 and 200 genes are differentially expressed at 6 and 12 hpi, respectively. most changes take place at 24 hpi, when infection proceeds into the late phase. then about 2200 additional genes become differentially expressed, most of which are up-regulated (figure 3). at 36 hpi fewer additional genes are changed in their expression, and many of them are suppressed. figure 3. the figure illustrates the kinetics of transcription changes. the differentially expressed genes are grouped into 20 clusters (c1–c20). red parts of the arrows indicate transcriptional up-regulation, and the green parts indicate suppression. modified from (57). upsala journal of medical sciences 87 the differentially expressed genes fall into 20 major clusters based on the kinetics of their changes (figure 3). analysis using the database for annotation, visualization, and integrated discovery software show that more than 90% of genes that are transiently up-regulated at 6 hpi are involved in the regulation of cellular proliferation, antiviral response, and cellular signaling. genes in clusters 1 and 2 decay quickly and become suppressed later in infection. genes encoding cytokines, active in immune response and cell growth control, form the most significant group. it is expected that genes showing similar expression profiles are regulated by common transcription factors (tf) or tfs from the same family. thus, the genes in the 20 different clusters have been subjected to analysis for the presence of consensus tf binding sites in their promoter regions. for the genes in cluster 1, nfjb and c-rel binding sites are prominent, as expected, as these are known to be present in genes engaged in immune response or apoptosis. they are hallmarks of most infections and play a critical role in virusinduced cytokine expression. at 12 hpi viral gene expression has begun, redirecting cellular gene expression. genes involved in dna replication are now the most highly up-regulated group together with genes in the post-replicative dna mismatch repair. genes involved in transcription and pre-rna processing are prominent in cluster 6, and genes implicated in cell cycle are significant in cluster 7, including key regulators for cell cycle progression from g1 to the s phase. suppressed genes at this time are involved in antiviral response and in cell growth and proliferation. for the genes in clusters 6 and 7, the presence of the tf binding site for e2f stands out. this is expected as the ad2 e1a protein binds prb, thereby releasing e2f, which activates numerous genes essential for dna replication and cell cycle progression. expression of e2f itself is also up-regulated. among eight e2fs, five show increased expression at 12 hpi, reaching a maximum at 24 hpi before decreasing at 36 hpi. dramatic changes are seen at 24 hpi when infected cells are driven into the s phase. more than 2000 additional genes are now upand down-regulated (figure 3). although most of them are similar in function to those up-regulated at 12 hpi, the number of genes involved in dna metabolism and dna replication increases nearly 10-fold. genes in clusters 13, 14, and 15 include several dna polymerases, replication factors, histones, and many cell cycle regulators. in addition, genes participating in rna processing become up-regulated, like genes required for efficient export of polyadenylated rna and genes encoding components of the exosome complex involved in the degradation and processing of a wide variety of rna species. based on their expression level at 36 hpi, the up-regulated genes at 24 hpi fall into four clusters, 13, 14, 15, and 16. although many functions are shared between them, some differences exist. for the genes in cluster 13, the most significant function is protein translation, rna processing, and cell cycle, whereas the most significant functions of the others are dna metabolism and replication, cell cycle, and stress response. the e2f binding sites are common in clusters 13 and 14, as are the binding sites for gabp, nrf1, and atf/creb. gabp regulates genes that are involved in cell cycle control, protein synthesis, and cellular metabolism. nrf1 activates the expression of some key metabolic genes regulating cellular growth. the most significant tf binding sites for genes in cluster 15 differed in that the atf/creb binding motif becomes more abundant. the atf/creb family has diverse functions, controlling cell proliferation and apoptosis. the number of suppressed genes at 24 hpi also increases. they are involved in either cell motion and structure or growth mediated by growth factors. gene functions involved in cytoskeleton organization are significant in cluster 17, whereas genes implicated in cell adhesion are present in cluster 18. fewer changes in cellular gene expression take place at 36 hpi when the infection has proceeded beyond the late phase and suppression is more pronounced (figure 3). the most significant function of the up-regulated genes is protein translation, including genes for ribosomal proteins, translation initiation factors, and translation elongation factors. genes involved in the generation of metabolites and energy, as well as oxidation/reduction, also become evident. the most outstanding function of the suppressed genes is the cellular macromolecule catabolic process including numerous genes mediating ubiquitination and subsequent proteasomal degradation. another important function is signal transduction, involved in vesicle transport. at 36 hpi, different sets of tf binding sites are significant in the up-regulated genes, including sp1, stra13, and nf-y, in addition to gabp, while the binding sites for e2f become less important. a role for non-coding rnas? it was discovered more than 50 years ago that ad encodes a small abundantly expressed rna first named 5.5 srna, later va rna (58). further studies revealed that va rna in ad2 consists of two species, va rnai and va rnaii (52,53). their positions in the genome were mapped, and sequencing revealed that they are about 160 nucleotides long (59). studies by mathews, shenk, and their co-workers revealed that the more abundantly expressed va rnai plays a critical role in ad replication as it binds to and blocks the doublestranded rna-activated protein kinase. the function of va rnaii, which is absent in some ads, is unknown. va rnai is cleaved by dicer, and the resulting miva rnas are incorporated into risc complexes (60). as many viruses encode their own micrornas (mirnas), we initiated an unbiased search for ad2-encoded mirnas, using deep cdna sequencing of small rnas expressed at different times after infection (56). the results revealed a set of small rnas, constituting more than 1% of the total pool of small rnas. they are between 25 and 35 nucleotides long and map in the region of the va rnai and rnaii genes, although not completely overlapping. a surprising observation is that some of the small rnas are expressed very early before any other viral rnas are expressed. target predictions indicate that the small rnas are involved in signaling pathways. 88 u. pettersson changes in cellular microrna (mirna) expression during the course of an ad2 infection in human lung fibroblasts have also been studied by deep rna sequencing (61). more than 100 mirnas with an altered expression were identified. the changes range from up-regulation of the mirnas known as tumor suppressors (such as mir-22, mir-320, let-7, mir181b, and mir-155) to down-regulation of oncogenic mirnas (such as mir-21 and mir-31) early. late the pattern is the opposite, with down-regulation of tumor suppressor mirnas (such as let-7 family, mir-30 family, 23/27 cluster) and upregulation of oncogenic mirnas (such as mir-125, mir-27, and mir-191). the switch in expression pattern occurs when ad dna replication starts. it thus appears that mirnas are engaged in growth inhibition early and stimulation of growth late. the deregulation of cellular long non-coding rna (lncrna) expression during a human ad infection has also been studied by deep rna sequencing (62). expression increased substantially following the progression of the infection. thus, the expression of more than 50% of some 600 significantly expressed lncrnas is changed more than 2-fold. most of them are up-regulated and activated during the late phase. based on the genomic locations of deregulated lncrnas in relation to known mrnas and mirnas, they are predicted to be involved in many biological processes in the late phase, like growth, structure, and apoptosis. in the early phase wound healing is involved, while cell proliferation, protein synthesis, modification, and transport are scored in the intermediate phase. the most significant functions of cellular rna-binding proteins known to interact with the deregulated lncrnas are splicing, nuclear export, and translation. based on the predictions it seems likely that ads exploit the lncrna network to optimize their reproduction. among eight e2fs, five show increased expression at 12 hpi, reaching their maximum at 24 hpi before decreasing at 36 hpi. mass spectrometry presents another view my scientific career began in protein chemistry. i still have vivid memories of hours spent packing sephadex, agarose, and deae chromatography columns. sequencing, using my countryman edman’s method, was out of the question with the resources we had. mass spectrometry for analysis of small molecules was available but not for molecules as large as polypeptides. in 2002, fenn, tanaka, and wutrich received the nobel prize in chemistry for ‘the development of methods for identification and structure analysis of macromolecules’. from their nobel lectures i became aware of the outstanding potential mass spectrometry offers for studies of proteins. it tempted me to return to my studies of the ad proteins. luckily, i managed to recruit a very skilled postdoc, sara lind (n�ee bergstr€om), who mastered the technology. as a first step, we subjected highly purified ad2 virions to mass spectrometry in a search for hidden proteins and posttranslational modifications (63,64). a number of modifications were detected. the piiia protein is the most highly modified protein with 18 verified phosphorylation sites, three nitrated tyrosine sites, and one sulfated tyrosine. nitrated tyrosines and lysine acetylations are present in hexon and pvi as well. a study of non-structural ad proteins unraveled the primary structures of the proteins predicted to be encoded by the viral genome. twenty-seven non-redundant sites of phosphorylation on seven different non-structural proteins were identified. the most heavily phosphorylated non-structural protein is the dna-binding protein with 15 different sites (65,66). our next step was to use mass spectrometry to monitor changes in cellular protein expression during the course of an ad infection (67,68). state-of-the-art technology, i.e. stable isotope labeling of amino acids in cell culture (silac) combined with nanolc-ms/ms, was used. about 2500 proteins were expressed at a measurable level, and approximately 25% and 35% of these proteins are altered early and late after infection. the proteins fall into different clusters based on their changes in abundance. at 6 hpi, proteins involved in cell-to-cell adhesion, cytoskeletal organization, protein folding, and prevention of protein aggregation or in the nucleolar-cytoplasmic transport stand out. one especially interesting protein, bnip2, interacts with the ad-encoded antiapoptotic 19 kda protein. most proteins, uniquely altered at 12 hpi, are up-regulated, and several different pathways are overrepresented by them. remodeling the cytoskeleton stands out, and many up-regulated proteins are involved in cytoskeletal regulation by rho gtpase as well as inflammation mediated by chemokine and cytokine signaling, and integrin signaling. the toll receptor signaling pathway is deregulated at this time, although the activation increases at 24 hpi. toll-like receptors play critical roles in the innate immune system by recognizing pathogen-associated molecular patterns derived from various microbes (69,70). other altered proteins are involved in the regulation of the glutathione metabolism and detoxification processes, in the organization of the proteasome system, and in signaling pathways such as tgf-beta, rho, and nfjb. the stress response proteins hspa1a/b are mildly altered at 12 hpi before becoming the most highly up-regulated protein at 24 hpi. another deregulated pathway is fructose galactose metabolism. this makes sense as proteins in this pathway are involved in the production of precursors to be used in the glycolysis cycle for energy production. the most highly suppressed proteins at 12 hpi are collagens and proteins related to them. in most cases, the suppression was sustained over time. another suppressed protein at 12 hpi is serpine1, the principal inhibitor of tissue plasminogen and urokinase activators. another interesting pathway overrepresented at 12 hpi is the pdgf pathway. the suppressed plasminogen and the urokinase activators are needed for activation of the pathway, indicating that the cell tries to block activation of the pdgf pathway. the pdgf receptor alpha is suppressed at all time points of the infection, the beta receptor only at late time points. these receptors also bind adaptor molecules which form complexes with other signaling molecules, such as the regulatory subunit p85 of the phosphatidylinositol 30-kinase, and grb2, which binds the nucleotide exchange molecule sos1, thereby activating ras and the erk upsala journal of medical sciences 89 map-kinase pathway. these events apparently take place early in infection before the pdgf pathway is shut down. the activation of pi3k, a consequence of the early virus–cell interactions, leads to actin reorganization via the gtpase activator for the rho, rac, and cdc42 proteins. these proteins are up-regulated in the early phase. at 24 hpi the pentose phosphate pathway is up-regulated at the protein level but not at the mrna level (71). this pathway cooperates with glycolysis for energy production. at late times the virus needs high amounts of nucleotides for its replication, and therefore it requires ribose-5-phosphate. serine synthesized from the glycolytic intermediate 3-phosphoglycerate is converted to glycine, which is an important precursor for purine biosynthesis, via the serine glycine biosynthesis pathway (72). because of its conversion to glycine, serine is also the donor of folate-linked one-carbon units which are required for nucleotide biosynthesis (73). at 6 hpi the serine glycine biosynthesis pathway is activated while the de novo purine biosynthesis and de novo pyrimidine ribonucleotides biosynthesis pathways are activated at all time points, most highly late, like the glycolysis pathway. the fgf and the egf signaling pathways are activated at 6, 12, and 24 hpi but become down-regulated later. the inactivation of these signaling pathways might be caused by the downregulation of their ligands at the mrna level 24 hpi (55). the expression of several proteins involved in the integrin signaling pathway, including several integrins, collagens, and ras family members, increases first, but most genes encoding these proteins are down-regulated at 24 and 36 hpi at the mrna level (71). a number of transcription factor (tf) binding sites have been identified in genes deregulated at the protein level at different time points. myc is highly activated, and its activation increases with time. this explains why the glycolysis and the pentose phosphate pathways are overrepresented in spite of the decreased level of myc mrna. another transcription factor, activated at intermediate and late time points, is e2f. several components in the dna and cell cycle machinery are up-regulated in parallel with their mrnas at 24 hpi, but not at 12 hpi. e2f-activated transcription begins at 12 hpi, while changes at the protein level are noted first at 24 hpi. transcriptomic data reveal an up-regulation of genes controlled by the atf/creb family at 24 hpi (55). these transcription factors control the expression of genes involved in dna and rna metabolism, but also genes involved in the stress response. tf analysis of the proteomic data confirmed the activation of creb1 at late time points. cellular defense against ros is controlled by nrf2, highly activated at all time points. it inhibits lipogenesis, activates the oxidation of fatty acids, simplifies the flux through the pentose phosphate pathway, and increases nadph regeneration and purine biosynthesis. divergent mrna and protein expression the correlation between changes in rna and protein expression in ad2-infected cells is surprisingly low (57). sets of discordantly expressed genes have been analyzed with focus on genes involved in important pathways, like immune response and cell cycle and growth control. nfjb and c-rel binding sites are frequent in promoter regions of the genes that are transiently up-regulated during the early phase, showing that nfjb signaling plays a crucial role in the cellular defense. transcription of most genes involved in this pathway is induced during the early phase, but becomes down-regulated later. rela and ikbkb, an inhibitor of nfjb kinase, have been studied at the protein level. they show, unexpectedly, opposite expression profiles compared to their mrnas. the ikb kinase interacting protein ikbip also shows divergent rna and protein expression profiles. thus, it seems that the nfjb pathway remains activated at the protein level, while transcription is down-regulated. target genes, containing nfjb and crel binding sites in their promoter regions, are, however, suppressed when infection progresses, indicating that the nfjb proteins are functionally inactivated by yet unidentified posttranslational mechanisms. thus, there must be additional regulatory steps involved in the nfjb pathway. jak/stat signaling plays an important role in cellular immune response and growth control. many genes involved in this pathway are deregulated (55,74), including jaks, stats, and their negative regulators, as well as the importin a-5 and the ran nuclear import pathway. most of them are down-regulated transcriptionally. a few proteins in this pathway are detectable at the protein level, and the results are surprising. in contrast to the transcriptional down-regulation of stats, all detected stat proteins—including stat1, 3, and 6—are up-regulated at 24 and 36 hpi. the jak and stat negative regulators are also detectable. expression of the ptpn11 protein is increased at 24 and 36 hpi, although its rna remains unchanged during the infection. at 24 hpi the ptpn1 protein decreases slightly, while its rna level is reduced nearly 4-fold. surprisingly, most stat proteins seem to be inactivated, since no significant up-regulation of genes containing a stat consensus sequence is detected. furthermore, expression of ifna, a well-known target gene in the stat pathway, decreases during the infection. several explanations are conceivable. like most other activators, the functions of stat proteins depend on other site-specific dna-binding proteins, such as p300/cbp, which is sequestered by the e1a protein. in addition, posttranslational modifications, including tyrosine phosphorylation, have an impact on stat function. furthermore, nuclear transport of stat is strictly regulated. this explanation is unlikely, however, since importin a-5 and ran, required for the entry of stat to the nucleus, are up-regulated at both the rna and protein levels at 24 and 36 hpi. apoptosis pathways are extensively deregulated during ad2 infection. the early protein e1a induces p53-mediated apoptosis. this is subsequently counteracted by the adencoded e1b proteins, e1b-55k and e1b-19k. by directly binding to p53, e1b-55k suppresses p53-dependent transcription and promotes p53 degradation through an e4orf6e3 ubiquitin ligase complex (75). the e1b-19k, which is a homolog of bcl-2, binds to bak and abrogates the 90 u. pettersson interaction of bak with bax. it prevents the conformational changes that activate bax and the release of cytochrome c (67). moreover, ad2 e3 gene products inhibit apoptosis induced by death receptor signaling (68). the caspases and the bcl2 family are key players in apoptosis. deregulation of caspase 3 and several genes belonging to the bcl2 family takes place at both the rna and protein levels. at the transcriptional level, six out of eight caspases are down-regulated. only casp3 is detectable at the protein level with an expression profile opposite to that of its rna at 24 and 36 hpi. at 36 hpi, the casp3 rna level had decreased more than 3-fold, while its protein level is increased 1.7-fold. thus, several important players in apoptosis are up-regulated, while their functions must be blocked since apoptosis is efficiently inhibited during an ad2 infection. in fact, several well characterized casp3 targets were up-regulated at the protein level. ad has obviously established efficient ways to counteract the apoptotic pathways by encoding their own antiapoptotic genes, thereby preventing cell death before the replication cycle is completed. epilogue historically, the ad replication cycle is divided into two phases separated by the onset of viral dna replication. based on temporal changes in cellular rna expression patterns, an ad2 infection in primary lung fibroblasts can be divided into four periods. the changes follow a logical pattern. already at 1 hpi a set of transcription factors are upregulated, primarily involved in growth inhibition and cellular defense. a few hours later genes involved in cytokine expression and apoptosis are prominent. at 12 hpi genes involved in the cell cycle, dna synthesis, and replication and rna splicing dominate, while genes engaged in the actin cytoskeleton are suppressed. at 24 hpi the repertoire is enlarged with genes involved in rna processing and translation, while genes involved in growth factor receptors, cytoskeleton organization, and cell adhesion are suppressed. at 36 hpi the repertoire is complemented with genes engaged in energy production and oxidation/reduction, whereas gtpase-mediated signal transduction is suppressed. conclusions drawn from the transcriptomic data are supported and complemented by the proteomic data. differences do, however, exist, as several important deregulated pathways fail to show up in one of the analyses. serine glycine biosynthesis and mannose metabolism are two examples. overall, pathways related to energy and biosynthesis of nucleic acid precursors failed to stand out in the transcriptomic analysis. the pdgf, egf, and fgf pathways are suppressed at both levels, although the proteins decay more slowly than their mrnas. an interesting pathway, activated already at 6 hpi, is the cytoskeletal regulation by rho gtpase. this pathway has been studied during ad infection by other methods (76). after the attachment to its receptor car and the avb5 integrin, virus endocytosis requires reorganization of the host cell actin cytoskeleton. these pathways are interlinked with the toll-like receptor pathway (77,78), which does not show up in the transcriptomic analysis. the proteomic (unlike the transcriptomic) analysis identified an activated myc pathway. stabilization of myc requires the p400 protein, and e1a promotes the association of myc and p400 at myc target genes (79). different studies have characterized virus-induced changes in host cell metabolism (80), and it has been suggested that ad e4orf1 binds to myc and induces the expression of glycolytic target genes and nucleotide biosynthesis from glucose intermediates (81). therefore, the activation of myc by e1a and e4orf1 explains why the glycolysis and the pentose phosphate pathways are overrepresented in spite of the decreased level of myc mrna. a most puzzling observation in our studies concerns the immune defense pathways. some of them are up-regulated early at both the rna and protein levels. rna expression decays at later time points, as would be expected. surprisingly the protein levels are sustained or enhanced late after infection. still the target genes remain suppressed, showing that the proteins are inactivated by hitherto unknown mechanisms. overwhelming evidence shows that non-coding rnas of different kinds play important regulatory roles in mammalian cells. the mirnas act at the posttranscriptional level and regulate translational efficiency. the predicted functions of the mirnas that are deregulated early and late after infection imply that there is a cooperation between the mirnas and the gene products that have been identified in the transcriptomic and proteomic analyses. early mirnas would inhibit cell growth by suppression of oncogenic mirnas and up-regulation of mirnas acting as tumor suppressors. later the situation is reversed, i.e. oncogenic mirnas are up-regulated while tumor-suppressing mirnas are down-regulated. as many as 600 lncrnas seem to be deregulated during an ad2 infection. their predicted functions imply that they could be involved in many steps of the replication cycle, although experimental proof remains to be provided. taken together, available results demonstrate that ad replication involves many more regulatory steps than believed. a conclusion is also that transcriptomic and proteomic studies complement each other and allow a more complete picture of a viral replication cycle to emerge. acknowledgements i am greatly indebted to my exceptionally skilled co-workers, hongxing zhao and sara lind. i thank them for many years—in the case of hongxing zhao nearly two decades—of inspiring and dedicated work. i also thank them for carrying out many experiments presented in this paper and for assistance with this manuscript. warm thanks also to my daughter anna for upgrading the language in my paper and to my friends g€oran akusj€arvi and catharina svensson for valuable comments. last but not least my sincere thanks to the kjell and m€arta beijer foundation and anders wall for three decades of generous financial support. upsala journal of medical sciences 91 funding financial support has been received from the kjell and m€arta beijer foundation and from anders wall. disclosure statement no potential conflict of interest was reported by the author. references 1. rowe wp, huebner rj, gilmore lk, parrott rh, ward tg. isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture. proc soc exp biol med. 1953;84:570–3. 2. hilleman mr, werner jh. recovery of new agent from patients with acute respiratory illness. proc soc exp biol med. 1954;85: 183–8. 3. valentine rc, pereira hg. antigens and structure of the 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processing and machine learning to enable automatic extraction and classification of patients’ smoking status from electronic medical records original article natural language processing and machine learning to enable automatic extraction and classification of patients’ smoking status from electronic medical records andrea caccamisia,b, leif jørgensenc, hercules dalianisb and mats rosenlunda,c adepartment of learning, informatics, management and ethics, karolinska institutet, stockholm, sweden; bdepartment of computer and systems sciences (dsv), stockholm university, stockholm, sweden; ciqvia solutions sweden ab, solna, sweden abstract background: the electronic medical record (emr) offers unique possibilities for clinical research, but some important patient attributes are not readily available due to its unstructured properties. we applied text mining using machine learning to enable automatic classification of unstructured information on smoking status from swedish emr data. methods: data on patients’ smoking status from emrs were used to develop 32 different predictive models that were trained using weka, changing sentence frequency, classifier type, tokenization, and attribute selection in a database of 85,000 classified sentences. the models were evaluated using fscore and accuracy based on out-of-sample test data including 8500 sentences. the error weight matrix was used to select the best model, assigning a weight to each type of misclassification and applying it to the model confusion matrices. the best performing model was then compared to a rule-based method. results: the best performing model was based on the support vector machine (svm) sequential minimal optimization (smo) classifier using a combination of unigrams and bigrams as tokens. sentence frequency and attributes selection did not improve model performance. smo achieved 98.14% accuracy and 0.981 f-score versus 79.32% and 0.756 for the rule-based model. conclusion: a model using machine-learning algorithms to automatically classify patients’ smoking status was successfully developed. such algorithms may enable automatic assessment of smoking status and other unstructured data directly from emrs without manual classification of complete case notes. article history received 24 november 2019 revised 24 june 2020 accepted 30 june 2020 keywords clinical informatics; electronic medical records; machine learning; natural language processing; smoking; text mining introduction the use of electronic medical records (emr) has been increasingly adopted in past decades and is used today in most industrialized countries for documentation of patient care (1). emr data have become an important and integrated part of healthcare to facilitate the sharing of information between healthcare practitioners and document the care of patients, but can also be a source for epidemiological research and real-world evidence (rwe). however, data on many important patient attributes are typically only captured in free text fields as case notes in special sections in the emr systems. such unstructured properties of emr data present an obstacle for rwe and clinical research. the introduction of advanced analytics such as machine learning with text mining methods and algorithms offers the potential for more efficient use of unstructured emr data for medical research (2–4). developing algorithms for automatic classifications of patient characteristics, exposures such as smoking, or disease status in defined categories may allow for easier access to real-world data for studies of safety, effectiveness, and treatment patterns of pharmaceutical product use in routine clinical practice (5). most of the research specifically addressing the problems of classifying smoking status based on secondary data sources was conducted in conjunction with the 2006 ‘smoking challenge’ announced by ‘informatics for integrating biology and the bedside’ (i2b2), a centre for biomedical computing funded by the national institute of health in usa, and by those who continued building on that work (5–7). in addition, a recent us study on dental health records developed a similar model as presented here, but focussing on tobacco consumption (8). however, despite the recent advancements in text mining and machine learning, making use of unstructured information from emrs such as smoking status still presents a challenge for researchers. the aim of this project was to develop a text mining model using machine-learning techniques to classify the smoking status of patients using emr data and to assess the performance attributes of the best machine-learning model compared to a rule-based contact mats rosenlund mats.rosenlund@ki.se department of learning, informatics, management and ethics (lime), karolinska institutet, stockholm, se-171 77, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 4, 316–324 https://doi.org/10.1080/03009734.2020.1792010 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1792010&domain=pdf&date_stamp=2020-10-19 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1792010 http://www.tandfonline.com model. here, we describe the process to derive the best machine-learning model and how it compares with a manual classification. methods the software tools used for pre-processing data were microsoft excel and notepadþþ, while the waikato environment for knowledge analysis software (weka) was used for text mining tasks including text classification and analysis (9). smoking classes we used emr data including patient level smoking information from two observational studies collected during 2014 and 2013 (10,11). the following smoking definitions were applied, which were also the most common classes used in the ‘smoking challenge’ (5): � current smoker: records of explicit statements or details leading to the obvious conclusion that the patient is smoking cigarettes, cigars, or pipe (e.g. statements like ‘10 cigarettes/day’; ‘yes, smokes’). any explicit smoking consumption was enough to be classified as current smoker regardless of quantity or frequency. short answers like ‘yes’ were coded as unknown if the text field was derived from the tobacco–alcohol fields of the emr, since it was not possible to distinguish if it referred to smoking or alcohol habits. � ex-smoker: records which explicitly state that the patient used to smoke but is currently not smoking, regardless of the time since the patient had stopped smoking (e.g. ‘stopped smoking’; ‘smoke free since 1998’; ‘ex-smoker’). � non-smoker: records which explicitly state that the patient is not a smoker (e.g. ‘doesn’t smoke’; ‘nonsmoker’; ‘never smoked’). short answers like ‘no’ were coded unknown if the text field was derived from the tobacco–alcohol fields of the emr, if impossible to distinguish smoking or alcohol habits. � unknown: records which do not fit in any of the previous classes (e.g. ‘didn’t ask’; ‘136/72 pulse 70’). as the purpose of this project was to capture data on smoking habits only, any patient records of snuff without any evidence of smoking was coded as unknown (snuff is a moist tobacco powder commonly used in sweden) (12,13). datasets first, the data were anonymized and cleaned from all patient information other than the smoking status text field. we then created different datasets to train and test the models (figure 1). the first training dataset (‘no-freq’) was derived from 2014 emr data and consisted of 85,509 rows and 3 columns where smoking information was present, with the first column indicating that the row belonged to a smoking status text field, the second containing the actual text typed in the field, and the third described how many times the same sentence appeared in the dataset (sentence frequency). a second training dataset (‘freq’) was generated from the same study data and included 318,858 rows by repeating the rows according to the sentence frequency. this dataset was used to evaluate if sentence frequency may affect the classification when training the models, i.e. how well the classification correctly discriminates between the smoking classes. that may occur in cases where two similar sentences belong to different classes, because using sentence frequency dramatically increases the term-frequency of the words in the most common sentences of the dataset. that in turn influences measures such as the information gain and thus impact the classifiers, particularly for decision trees (14,15). this dataset was used to train all models. the figure 1. description of the workflow used to pre-process data, to obtain coherent and manually classified training-sets and test-set. upsala journal of medical sciences 317 proportion of smokers in our datasets was coherent with national statistics of smoking status in sweden (16). we then created two test datasets based on the 2013 study data. the first test dataset included 8551 rows out of a total of 177,000 rows from that study data and was used to test all derived models. the second test dataset included another subset of 10,000 rows from the same study data and was used to compare the performance of the best performing model and the rule-based model. we decided to use a different test-set (i.e. not a 10-fold cross-validation commonly applied in data mining) because of the availability of a large amount of data allowing us to test the models on unseen data. this test data contained up to 603 characters and an average of 24.5 characters per sentence for the fields with smoking status information, while the original training dataset included up to 84 characters per sentence (average 45), with 90% of the sentences appearing only once. the training dataset was double-coded manually, i.e. an additional annotator went through the file correcting any errors and agreeing with the previous annotator on all proposed changes if in agreement. both test datasets of 8551 entries and 10,000 entries were assessed by another annotator to agree on a gold standard. any mismatches were jointly discussed, and a common agreement was made to achieve a 100% match of those sentences. model development the model development was based on the variation of different macro-settings of four classification algorithms with the tool ‘weka’. we considered the following four classification algorithms: sequential minimal optimization (smo), knn, naïve bayes, and j48. smo is an iterative algorithm used to solve the quadratic programming problem during support vector machine (svm) training, which divides the problem into smaller sub-problems in order to find the hyperplane with the maximum margin (17). the k-nn is a classification algorithm which assigns the class for majority vote depending on the class of the k nearest neighbours to a test sample (18). naïve bayes is a probabilistic classification algorithm based on bayes’ theorem which assumes a high independence between the training-set attributes (19). j48 is the weka implementation of the more known c4.5 classification algorithm. the algorithm creates a binary decision tree checking iteratively for each tree node the information gain ratio of every attribute, in order to evaluate which one to split on (20). smo was chosen because it performed best in the ‘smoking challenge’, and we included k-nn and j48 because they both performed well in that challenge (5,21,22), while naïve bayes was considered because it is an algorithm which is robust to irrelevant features and works well especially in situations with unbalanced classes (23,24). the classifier-specific settings in weka were kept as default, as only the macro-settings (sentence frequency, classifier type, tokenization, and attribute selection) were of interest. all weka settings have been summarized in table 1. in text mining, tokenization is the act of breaking up a sequence of strings into pieces such as words, and we applied two different models for tokenization in order to structure the data by transforming the original text string into a collection of word vectors and binary values (25), one using unigrams (single words) and another combining unigrams and bigrams (single words or two consecutive words). in weka, when the arff file (weka format of input) is loaded, only ‘text’ and ‘smokingstatus’ are present as attributes, and the filter ‘stringtowordvector’ was used, as it scans the rows in the training-set and creates tokens which become the attributes, according to the specified settings. thus, a word vector was created for each training-set row which contained the binary value 1 corresponding to an attribute present in that sentence or 0 otherwise. when the training-set was tokenized and the attributes ready, we created another test selecting a lower number of attributes in the training-set to reduce dimensionality. to select only the attributes with a higher predictive power, the weka filter ‘selectattributes’ was used. the algorithm used for the attribute selection was the ‘infogainattributeeval’ table 1. summary of features and weka settings used to create the models. feature option/setting sentence frequency � yes � no classifier � smo � k-nn � naïve bayes � j48 attribute selection � yes � no tokens � unigrams � unigrams þ bigrams classifiers settings � all classifier specific settings were kept as default except for k-nn k value which was set to 1 � to determine the optimal k, a 10-fold cross-validation was run on both training datasets ‘freq’ and ‘no-freq’, both for unigrams and unigrams þ bigrams, testing k equal to 1, 3, 5, and 10 selectattributes settings � attribute evaluator was set to ‘infogainattributeeval’ � search method was set to ‘ranker’ � the ranker setting threshold was set to 0.0 in order to discard attributes with a negative information gain � all other settings were kept as default stringtowordvector settings � ‘wordstokeep’ was set to 15,000 in order to take into account all the single word tokens as attributes � the tokenizer was set on ‘word tokenizer’ for unigrams or ‘n-gram tokenizer’ with minimum size equal to 1 and maximum size equal to 2 for unigrams þ bigrams, depending on the chosen model � all other settings were kept as default 318 a. caccamisi et al. with the ‘ranker’ function since it demonstrated good performance overall using different classifiers on diverse datasets (26). the ranker setting threshold was set to 0.0 to discard attributes with a negative information gain. after the previous tests were executed in weka, 32 models were produced. to evaluate and compare the models, several statistical measures were used, including positive predictive value (ppv) (true positives/[true positives þ false positives]), sensitivity (true positives/[true positives þ false negatives]), f-score (2 � [ppv � sensitivity]/[ppv þ sensitivity]), accuracy ([true positives þ true negatives]/[true positives þ true negatives þ false positives þ false negatives]), and receiver operating characteristic (roc) area (27). the roc curve is a plot of sensitivity on the y axis and false positive rate (fpr) on the x axis (27). it is an effective method for the evaluation of the performance, where a higher roc area indicates a better performing model. an area under the roc curve equal to 1 is considered the perfect case since it means the fpr is 0 and the sensitivity is 1 (27). finally, in the second part of the study, the error cost was used as discriminating factor, which is defined as the cost assigned to each kind of sentence misclassification based on the expected relative importance. f-score and roc area were calculated by weka and consisted of the weighted average of the individual class measures, while accuracy was calculated dividing the correctly predicted number of records by the total number, independently by the class-specific accuracy. all 32 models were evaluated on the test dataset (4 methods with 8 options) with 8551 entries (figure 2). the test datasets were created semi-randomly, i.e. about 20,000 entries from the original test-set were manually classified and randomly picked by classes and narrowed down to 8551 and 10,000, recreating the same class distribution as in the sentence frequency dataset. this was done to obtain more realistic results that would make the model more relevant for future use (table 2). the models were built with the two best performing classifiers (n ¼ 16). they were then compared using an error cost matrix, in which a weight was given to the different combinations of possible types of misclassification to account for the between-class hierarchy (e.g. an ex-smoker classified as smoker would be considered a less serious error compared to a smoker classified as a non-smoker). we considered the hierarchy of decreasing importance for the error in the following sequence: yes!no and no!yes, followed by no!ex and ex!no, and then yes!ex and ex!yes. a direction of misclassification from any of the smoking classes to unknown was assumed to only decrease the statistical power, while misclassifying an unknown record to any of the smoking classes might introduce bias and hence were assigned a double cost. the cost of each type of misclassification was then multiplied by the values in the defined cost matrix as described in table 3, to obtain the model total cost. the model with the lowest cost or error was considered the best performing model, regardless of its accuracy. the best model and the original rule-based model were compared by testing their individual performance on a final test dataset as described in the workflow in figure 2. the rulefigure 2. evaluation workflow. table 2. training-set class distribution. training-set no-freq training-set freq number percent number percent current smoker 40,743 47.6% 77,401 24.3% non-smoker 8217 9.6% 185,456 58.1% ex-smoker 28,694 33.6% 38,791 12.2% unknown 7855 9.2% 17,210 5.4% total 85,509 100% 318,858 100% table 3. error cost matrix with assigned weights to each type of prediction misclassification. manually classified as predicted as smoker non-smoker ex-smoker unknown smoker 0 20 5 1 non-smoker 20 0 10 1 ex-smoker 5 10 0 1 unknown 2 2 2 0 upsala journal of medical sciences 319 based model was constructed based on expert opinion using a manual classification of all combinations of smoking in the text fields. ethical considerations data for this study received ethics approval from the authorized ethical review board (erb) (dnr.2014/54–31/3 and dnr.2013/267–31/3) (10,11). results the inter-annotator agreement was 99.9% (958 records classified differently out of 104,060) on both the training and test datasets after the individual classification. there were 85,509 text strings containing any information of smoking in the emrs, which were classified as smoking in 40,743 entries, as non-smoking in 8217 entries, and as ex-smoking in 28,694 entries (table 2). there were in total 318,858 entries of smoking-related sentences in the dedicated emr fields, which were classified as 77,401 smoking, 185,456 non-smoking, and 38,791 ex-smoking. the sentence ‘no’ occurred in 93,358 different records of smoking text fields and appeared in an equal number of rows in the second training-set, while only once in the first. the proportion of text strings that could not be manually classified (unknown) was 9.2% for the first training-set and 5.4% for the second. ranking all 32 models run on the test dataset showed that the smo model with no frequency and both unigrams and bigrams as tokens achieved the highest accuracy, followed by the same classifier with the frequency feature combined with both unigrams and bigrams as tokens (figure 3). the third best accuracy was achieved with the k-nn model with no unigrams and bigrams, followed by the same classifier with no frequency but unigrams only. for the j48 models, the highest accuracy was achieved with the features frequency, unigrams/bigrams, and attribute selection, followed by the one with frequency and unigrams only. the naïve bayes models which performed the worst, displayed the highest accuracy with frequency and unigrams/bigrams features, followed by the same features together with attribute selection. models with smo and k-nn classifiers had an average accuracy of 97.22% and 97.19%, f-score of 0.972, and roc area of 0.988 (table 4). models with the j48 classifier displayed a similar roc area result as the smo and k-nn models, but noticeably lower accuracy and f-score. the naïve bayes models presented the lowest results on average on all measures in comparison with the other classifiers. applying the cost matrix described in table 3 to the confusion matrix of the models with the two best classifiers based on the assessment above (i.e. smo and k-nn) demonstrated that the lowest cost of the remaining 16 models was scored by the smo classifier and both unigrams and bigrams as tokens, without any selection based on the information gain (table 5). comparing the performance of the best model and the rule-based model on the final test dataset demonstrated that the machine-learning model achieved a higher ppv (98.10%), figure 3. accuracy of the models. the abbreviation ‘uni þ bi’ refers to the utilization of a combination of unigrams and bigrams as features in the training-set. the abbreviation ‘attr. sel.’ refers to the selection of the most relevant attributes in the training-set. table 4. average of the statistical measures of the models by classifier. model classifier smo naïve bayes k-nn j48 f-score 0.972 0.879 0.972 0.959 roc area 0.988 0.961 0.988 0.989 accuracy 97.22% 87.45% 97.19% 95.72% 320 a. caccamisi et al. sensitivity (98.10%), and f-score (0.98), with an accuracy of 98.14% compared to the rule-based model which had an accuracy of 79.32% (table 6). to maximize the new model performance, the macro-settings which need to be adopted are the svm smo classifier and the unigrams þ bigrams tokenization. sentence frequency and attributes selection did not improve the model. discussion we developed an algorithm to enable automatic classification of smoking status based on patients’ emr data using machine-learning techniques. our results demonstrated better performance compared to a rule-based model, with close to 20% improved accuracy on the same test dataset. thus, our study provides further understanding of how to make use of unstructured emr data for large-scale real-world evidence and epidemiological research by applying more efficient modern machine-learning techniques and more specifically adds to the literature of how to automatically classify smoking status for patients using secondary data sources. most of the previous work addressing the methods for classifying patients’ smoking status has been conducted as a result of the ‘smoking challenge’ (7,21,22,28–32), and by others who continued building on that work (5,6,33). a recent us study on dental health records developed a similar model based on machine learning (8). however, to our knowledge, there is no such prior work performed on swedish data. in accordance with the ‘smoking challenge’, we applied the most frequently used smoking categories, i.e. ‘current smoker’, ‘past smoker’, ‘non-smoker’, ‘unknown’, while some have also considered a more generic class of ‘smoker’ (5). similarly to previous publications on this topic, we took advantage of the most commonly used text mining tool ‘weka’ (9), which is expected to make our results and model development described here more relevant also for others. an svm was also used as classifier in previous smoking classification studies, including the two health information text extraction (hitex) studies (2,5), and in the best model from the ‘smoking challenge’ (5), by clark et al. (30). they achieved 90% accuracy and 0.83 f-score, and 96% accuracy and 0.90 f-score, respectively, while the model in our study achieved an accuracy of 98.14% and an f-score of 0.981. the results obtained by those two models can be directly compared to the ones obtained in the current study since the models entail the same action of classifying a sentence regarding the patients’ smoking status. however, some differences should be acknowledged. for instance, in the two previous models, the text was collected from different parts of the emr and different techniques were used to isolate the sentences relevant for the patients’ smoking status. in this study, however, only the information in the ‘smoking’ or ‘smoking/alcohol’ field of the emr case notes was used. in addition, the text language in the two previous models was english, which may influence the effectiveness of a setting over another—for instance, tokenization with unigrams, bigrams, or trigrams could have a more or less enhanced difference in results depending on the structure of the adopted language. also, the training-set of the two previous models was tokenized using unigrams only, and in the ‘smoking challenge’ the emrs where annotated by two pulmonologists. in the ‘smoking challenge’, 398 annotated emrs of complete case notes were used in the training-set, whereas 85,509 (only smoking text field) were used in our study. the performance of the classifiers used in this work can be compared with the results obtained in previous studies and with the expectations related to their classifier-specific characteristics. smo appeared more valid and reliable as classifier for the automated smoking status classification in this study as well as in the ‘smoking challenge’, with a higher accuracy and f-score because of its characteristics of being able to perform better with a large quantity of input data (34). in fact, svm smo divides the initial quadratic programming problem into the smallest possible sub-problems and solves them analytically one by one. it allows the algorithm to always converge without regard of the dimensionality (17). a recent study on dental health records from the us found that svm performed best to classify patients according to smokers, non-smokers, and unknowns, with a ppv and sensitivity of 98% and f-score of 0.98 (8). in addition, that study included an assessment of the patient’s tobacco consumption, but it was limited to three classifiers instead of four as in our study and there was no consideration of the cost matrix. the k-nn classifier performed better than expected in our study. the k value was set to 1, thus the used classifier was the nearest neighbour (nn). good results achieved using the nn classifier imply the almost total absence of outliers in the table 5. result of the application of the cost matrix to the remaining 16 models. classifier feature model smo k-nn freq unigrams only attribute selection cost 1 � – – � – 1751 2 � – – � � 1983 3 � – – – – 1059 4 � – – – � 1467 5 � – � � – 2139 6 � – � � � 2322 7 � – � – – 1627 8 � – � – � 1921 9 – � – � – 1949 10 – � – � � 1990 11 – � – – – 1970 12 – � – – � 1873 13 – � � � – 2182 14 – � � � � 2327 15 – � � – – 2060 16 – � � – � 2073 table 6. statistical measures of the best model and the rule-based model. measure best model rule-based positive predictive value (ppv) 98.10% 79.90% sensitivity 98.10% 79.30% f-score 0.981 0.756 accuracy 98.14% 79.32% upsala journal of medical sciences 321 training-set (18). this could be due to the orderly structure of the swedish emrs which contain a specific text field for the smoking status (1). on the other hand, naïve bayes was the simplest of the four algorithms used, and it can achieve better results on fewer data (20), especially with unbalanced classes (23), since it is robust to irrelevant features (24). our results, however, illustrate that with a larger amount of data, naïve bayes does not achieve as high accuracy as the other algorithms. j48 performed better than in the ‘smoking challenge’, probably also because of the orderly structure of the swedish emrs with a specific smoking text field (1). on the other hand, when training the algorithm, the quickest classifier was nn (min. 5 s) followed by naïve bayes, svm smo, and j48 (up to 7 days), while the quickest classifier during testing was j48 (1 s minimum) followed by svm smo, nn, and naïve bayes (up to 20 min). all the 32 models we developed using machine learning, even the worst-performing one using the naïve bayes classifier which achieved an accuracy of 85.63%, appeared better than the rule-based sas model which reached a 79.32% accuracy. this confirms that if enough training data are available, a machine-learning classification model generally has the capability of performing better than the analogue rulebased model (35,36). this is mainly due to the increasing difficulty of creating a comprehensive rule-based model as the size and diversity of the dataset increase. however, one of the issues with developing algorithms to capture data from case notes is the risk of grammatical errors and typos in the text and the presence of diverting sentences, e.g. ‘the mother is a heavy smoker’ or ‘the father smoked for 20 years’, which complicates the development of a rule-based approach to define which sentence is related to the current patient. after a first analysis of the 188 sentences misclassified by the best model in this study out of the 10,000 in the ‘final test-set’, the most common mistakes were related to other tobacco types such as snuff. for example, the model appears to give a considerably higher priority to ex-smoker class keywords compared to the unknown class keywords, which is likely caused by the fact that sentences regarding non-cigarette tobacco such as snuff are not always considered unknown since a reference to smoking can be present as well, while keywords used for ex-smokers are mainly adopted for that class only. this means that a sentence referring to a patient who had stopped using snuff in december (original text string: ‘slutade snusa i december’) may be wrongly classified as ex-smoker because of the presence of ‘stopped’, regardless of the presence of any reference to snuff use. that may be caused by low numbers of examples of ex-snuff consumers in the training-set. another situation involves sentences with combinations of tobacco–alcohol text fields with discordant evidence about tobacco and alcohol or reference to alcohol only. correspondingly, if the model gives a higher priority to a keyword referred to the alcohol consumption, compared to the keyword referred to the smoking habit, a sentence such as ‘doesn’t smoke, wine sometimes’ (original text string: ‘r€oker inte, vin ibland’) may be misclassified as ‘smoker’ because of the word sometimes. for the same reason, in cases where there is only a reference to alcohol present in the text field, a sentence such ‘1 glass of wine sometimes in the weekend’ (original text string: ‘1 glas vin ibland på helgen’) may be misclassified as ‘smoker’. additionally, the single word sentence ‘not’ was classified as unknown by the model even though it was classified as ‘no’ in the training-set. this misclassification could be due to different reasons such as the scarce presence of the two words in the training-set, their combination with other words in longer sentences, or because the model considers their absence more relevant than their presence. nevertheless, assessing all possible causes of this misclassification was not within the scope of this work. another example involves sentences with the character ‘/’ between two words. sentences such as ‘party smoker/moderate’, in this case referring to smoking/alcohol habits, may be misclassified as ‘unknown’ as the tokenization process did not remove the ‘/’ character and therefore it was considered as a single word. this misclassification could be avoided by removing the ‘/’ character; however, sentences such as ‘10 cigarettes/day’ are correctly classified independently of the number present in the sentence, precisely because of the presence of this character. the last example involves sentences which would have been interpreted with difficulty even by a human reader. for instance, the exact meaning of the sentence ‘smoked 10–20 cig for 45 years. stopped for 3 years but started again after 2 years and smoked for about 1 year, now stopped for some weeks’ may be difficult to understand, and such sentences may need to be read more than once. with this kind of sentence, the model would make a prediction which in most of the cases is expected to be wrong, due to the presence of repeated contrasting keywords. it is important to consider the impact of such measurement error and misclassification of covariates based on machine learning when used in regression models in epidemiological studies of exposure–disease associations. methods to account for bias due to misclassification of exposure covariates such as smoking have been described by others (37). a sensitivity of 79.3% in a rule-based classification means that in a study of 10,000 patients we would misclassify 621 smokers as non-smokers, including 518 smoking cases and 104 controls misclassified as non-smokers. the improvement by applying our algorithm with a sensitivity of 98.1% means that we would only misclassify 57 smokers as non-smokers, including 48 smoking cases and 10 controls misclassified as non-smokers. assuming a smoking prevalence of 30% and a true odds ratio (or) for smoking-related lung cancer of 9.00 would translate into an or of 8.85 and 7.63 using these models, respectively, which is of similar magnitude as other simulations (38). this was assuming a non-differential misclassification, but if the likelihood of being classified as a smoker depends on disease status, e.g. if lung cancer patients were more likely to report historical smoking, the misclassification would cause overestimation of the relative risk. such fixed-parameter-bias sensitivity analyses are, however, simplistic, and probabilistic bias analysis or bayesian analyses are recommended for risk assessment to account for all sources of uncertainty (39). 322 a. caccamisi et al. limitations of this study include that the algorithms have been applied on a rather short text field where smoking information is entered. moreover, the models were selected only using macro-settings (sentence frequency, classifier type, tokenization, and attribute selection). however, each classifier has its specific parameters which for the purpose of this study were not considered and therefore kept as default values in weka (except for k-nn k value). a selection of the optimal values for these settings (parameters optimization), using for instance a grid-search algorithm, could have further improved the models’ accuracy (37). in addition, multiple local language speaking annotators might have further decreased any possibility of human misclassification in the test-set or in the training-set. thus, further improvements might be possible by finding the optimal classifier-specific parameters for the best performing model. the svm smo classifier used in the best model has as specific settings: the exponent of the polynomial kernel and the complexity value ‘c’ which is set to 1 by default. ‘c’ is a trade-off value between the classifier generalization and the training error. hence, further improvements might be possible through optimization of ‘c’, since the complexity parameter is a positive number between zero and infinite (40). another possibility would be to use innovative techniques like ‘deep learning’, which consists of a convolution of artificial neural networks with a high number of hidden layers (41). that appears promising for data mining, but the scientific body of evidence may still be limited. in conclusion, the machine-learning model performed best when using the svm smo classifier 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31:231–40. doi:10.1016/j.eswa.2005.09.024 41. weiss sm, indurkhya n, zhang t, emerging directions. in: weiss sm, indurkhya n, zhang t, editors. fundamentals of predictive text mining. 2nd ed. london: springer-verlag; 2015. p. 211–13. 324 a. caccamisi et al. http://www.jamia.org http://www.jamia.org https://doi.org/10.5120/17456-8202 https://doi.org/10.3348/kjr.2004.5.1.11 http://www.jamia.org http://www.jamia.org https://doi.org/10.1197/jamia.m2434 https://doi.org/10.1197/jamia.m2442 https://doi.org/10.1197/jamia.m2438 https://doi.org/10.1197/jamia.m2438 https://doi.org/10.1197/jamia.m2440 https://doi.org/10.1093/ije/dyx027 https://doi.org/10.1111/j.0006-341x.2001.00598.x https://doi.org/10.1016/j.eswa.2005.09.024 abstract introduction methods smoking classes datasets model development ethical considerations results discussion disclosure statement references feasibility of genomic profiling with next-generation sequencing using specimens obtained by image-guided percutaneous needle biopsy article feasibility of genomic profiling with next-generation sequencing using specimens obtained by image-guided percutaneous needle biopsy miyuki sonea, yasuaki araia, shunsuke sugawaraa, takatoshi kuboa, chihiro itoua, tetsuya hasegawaa, noriyuki umakoshia, noboru yamamotob, kumiko sunamic, nobuyoshi hiraokac and takashi kubod adepartment of diagnostic radiology, national cancer center hospital, tokyo, japan; bdepartment of experimental therapeutics, national cancer center hospital, tokyo, japan; cdepartment of pathology and clinical laboratories, national cancer center hospital, tokyo, japan; ddivision of translational genomics, exploratory oncology research & clinical trial center, national cancer center, tokyo, japan abstract aims: the demand for specimen collection for genomic profiling is rapidly increasing in the era of personalized medicine. percutaneous needle biopsy is recognized as minimally invasive, but the feasibility of comprehensive genomic analysis using next-generation sequencing (ngs) is not yet clear. the purpose of this study was to evaluate the feasibility of genomic analysis using ngs with specimens obtained by image-guided percutaneous needle biopsy with 18-g needles. patients and methods: forty-eight patients who participated in a clinical study of genomic profiling with ngs with the specimen obtained by image-guided needle biopsy were included. all biopsies were performed under local anesthesia, with imaging guidance, using an 18-g cutting needle. a retrospective chart review was performed to determine the rate of successful genomic analysis, technical success rate of biopsy procedure, adverse events, rate of success in pathological diagnosis, and cause of failed genomic analysis. results: the success rate of genomic analysis was 79.2% (38/48). the causes of failure were unprocessed for dna extraction due to insufficient specimen volume (6/10), insufficient dna volume (2/10), and deteriorated dna quality (2/10). the rate of successful genomic analysis excluding ngs analysis that failed for reasons unrelated to the biopsy procedures was 95.2% (40/42). technical success of biopsy was achieved in all patients without severe adverse events. the rate of success in the pathological diagnosis was 97.9% (47/48). conclusions: image-guided needle biopsy specimens using an 18-g cutting needle yielded a successful ngs genomic analysis rate with no severe adverse events and could be an adoptable method for tissue sampling for ngs. article history received 2 march 2019 revised 7 april 2019 accepted 8 april 2019 keywords biopsy; genomic analysis; needle biopsy; nextgeneration sequencing introduction with the advancement of personalized cancer treatment, the demand for specimen collection for genomic profiling is rapidly increasing (1–3). in clinical practice, individualized treatments, such as molecular targeted therapies, that are selected based on the results of genomic profiling have been indicated for many types of cancer. also, new clinical study designs, such as umbrella trials and basket trials, in which drugs are selected according to a patient’s genomic profile are increasingly conducted (4–6), and the demand for next-generation sequencing (ngs) analysis, which is capable of performing multiple genomic analyses at once, is increasing. percutaneous needle biopsy is recognized as minimally invasive, but the tissue obtained has been considered unfit for genomic profiling because of insufficient specimen quantity compared with surgical specimens. currently, the rate of successful genomic profiling with percutaneous needle biopsy varies from 47% to 100% (7–11). additionally, there are only a few reports with ngs analyses using specimens obtained by percutaneous needle biopsy (10,11). as the role of image-guided percutaneous needle biopsy for ngs genomic profiling has not been established, we aimed to evaluate the feasibility of genomic analysis using comprehensive ngs with specimens obtained by imageguided percutaneous needle biopsy with 18-g needles. materials and methods patients this study was a retrospective observational study employing a medical record survey. selection criteria of the patients are the following: patients who enrolled in a clinical study of genomic profiling using a dedicated cancer gene panel for ngs (trial of onco-panel for gene-profiling to estimate both adverse events and response by cancer treatment [top-gear] study: clinical study registration no. umin000011141) between april 2014 contact miyuki sone msone@me.com department of diagnostic radiology, national cancer center, 5-1-1, tsukiji, chuo-ku, tokyo 1040045, japan � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 2, 119–124 https://doi.org/10.1080/03009734.2019.1607635 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1607635&domain=pdf&date_stamp=2019-06-04 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2019.1607635 http://www.tandfonline.com and february 2017. we included patients undergoing genomic profiling with the specimen obtained with image-guided needle biopsy using interventional radiology technique. patients who were evaluated with out-of-hospital/referred specimens, surgical specimens, and endoscopy specimens were excluded (figure 1). this study is health insurance portability and accountability act compliant, and the institutional review board at our institution waived the approval. all patients provided written informed consent for biopsy procedures. biopsy procedure biopsy sites were selected based on the contrast enhancement on computed tomography (ct) and magnetic resonance imaging (mri) images, mri diffusion restriction, and increased fdg uptake on positron-emission tomography (pet) that were most likely to correspond with high viability of tumor cells. to minimize risk of complications, a puncture route that would not pass through large vessels or organs was planned (figure 2). all biopsies were performed by board-certified interventional radiology (ir) specialists or ir fellows under supervision. puncture was performed under local anesthesia with the guidance of angio-ct (infx-8000c/ aquilion 16; canon medical systems, ohtawara, japan), which is a combined ct and fluoroscopic apparatus, or ultrasonography (us) (ta 510; canon medical systems, ohtawara, japan, or fazone cb; fujifilm, tokyo, japan). images, in which the needle reached the inside of a lesion, were recorded (figure 2). the number of specimens taken was determined according to the size of the specimens obtained and the requirement for the research. the obtained specimens were immediately fixed in 10% neutral-buffered formalin fixative and transported that day to the pathology laboratory. on-site, rapid cytology was not performed. specimen processing and histopathological diagnosis specimens were fixed in neutral-buffered formalin for 24–72 hours and embedded in paraffin. tissue sections of 2–3-mm thickness were prepared for hematoxylin and eosin (he) staining, and 5–10-mm-thick sections were prepared for genomic profiling. in addition to he staining, special stainings, immunohistochemistry, and genomic tests as insurance-approved companion diagnostics were performed as necessary, and pathological diagnosis was rendered by at least two certified pathologists. for patients who consented to the top-gear study before biopsies were taken, dna extraction was performed immediately. in contrast, for patients who consented to and participated in the top-gear study after biopsies were taken, excess specimens harvested during a previous biopsy were used for dna extraction. genomic analysis an ngs apparatus capable of detecting 114 cancer-related gene mutations/amplifications, 12 fusion genes, and 1 gene deletion/polymorphism in a single assay was used for ngs enrolled in top-gear study april 2014-feb 2017 (n= 490) excluded (n= 337) surgical specimen (n= 230) referred biopsy specimen (n= 107) in-house biopsy specimen (n= 143) image-guided needle biopsy by ir (n= 48) excluded (n=95) endoscopy (n= 57) other modalities (n= 38) not analyzed (n= 10) ngs analysis (n= 480) figure 1. flow chart of patient selection. 120 m. sone et al. analysis (ncc-oncopanel ver. 4.0) ncc-oncopanel ver. 4.0 was developed at the authors' institution and is dedicated to the specific research of topgear described in subsection ‘patients’. (12). sequencing libraries were prepared using sureselect xt reagent (agilent technologies, santa clara, ca, usa) and a kapa hyper prep kit (kapa biosystems, boston, ma, usa) and were analyzed on a miseq sequencer (illumina, san diego, ca, usa). bioinformatics analysis was performed, and final decisions for the report were made in conferences by the multidisciplinary team (12). outcomes ascertainment the primary outcome was the rate of successful genomic analysis with specimens obtained by percutaneous needle biopsy. the secondary outcomes were profiling of genetic alterations, technical success rate of biopsy procedures, adverse events evaluated using the common terminology criteria for adverse events v. 4.0, rate of success in pathological diagnosis, and cause of failed genomic analysis. technical success of the biopsy procedure was defined as obtaining tissue sections with imaging confirmation of the biopsy needle within the target. successful ngs analysis was defined as the ability to perform genomic analysis by ngs using dna extracted from the specimen. the causes of failed ngs analysis were categorized as: (i) failure of the puncture of the target site (sampling error); (ii) unprocessed for dna extraction due to insufficient specimen volume; (iii) insufficient dna volume; and (iv) deteriorated dna quality. we also calculated the rate of successful genomic analysis excluding ngs analysis that failed due to reasons unrelated to the biopsy procedures, i.e. reasons (ii) and (iv). statistical analysis categorical variables were estimated as percentages and median values. in order to study whether risk factors existed for the inability to perform genomic analysis, patients were divided into two groups (patients in whom genomic analysis was possible, and patients in whom analysis was not possible), and univariate analysis was performed. patient factors (age, sex, and primary tumor type), tumor factors (site and size), and biopsy procedure factors (needle type, number of specimens collected, and time until dna extraction) were included as variables in the analysis. categorical variables were analyzed using pearson’s chi-square test, continuous variables that showed a normal distribution were analyzed by t test, and continuous variables that did not show a normal distribution were analyzed with the mann–whitney u test. a p value less than 0.05 was considered to be statistically significant. results patient demographics of the 490 patients enrolled in the top-gear study during the study period, 48 were included in this study (figure 1). patient demographics and tumor characteristics are given in tables 1 and 2. feasibility of genomic analysis the success rate of genomic analysis using ngs with biopsy specimens was 79.2% (38/48). a total of 52 mutations, 5 amplifications, and 2 homozygous deletions were identified. figure 2. representative case showing biopsy of a pelvic mass from a woman in her 60s. (a) contrast-enhanced ct of the pelvis demonstrates an enhanced mass (asterisk) between the right external and internal iliac vessels. (b) biopsy was performed using an 18-g cutting needle without penetration of the iliac arteries and veins. table 1. patient demographics. n % gender male 23 48.0 female 25 52.0 age, years, median (range) 54 (23–77) primary site lung 6 12.5 breast 6 12.5 unknown 6 12.5 colon 3 6.3 thymus 3 6.3 bile duct 2 4.2 pancreas 2 4.2 others 20 41.7 upsala journal of medical sciences 121 twenty-six patients had at least one genetic alteration. the causes of failure of analysis (n ¼ 10) were: (i) failure of the puncture of the target site (sampling error) (0/10); (ii) unprocessed for dna extraction due to insufficient specimen volume (6/10); (iii) insufficient dna volume (2/10); and (iv) deteriorated dna quality (2/10). the rate of successful genomic analysis excluding ngs analysis that failed due to reasons unrelated to the biopsy procedures (i.e. reasons [ii] and [iv]) was 95.2% (40/42). of the 6 specimens unprocessed for dna extraction, 3 were due to insufficient volume of the excess specimen, and 3 were due to shortage of the specimen due to requirement of multiple immunohistochemistry tests. the imaging finding of the insufficient dna volume of 2 patients with liver tumor was hypovascularity in the target region. in one patient, who underwent biopsy after medical treatment for liver metastases from bile duct cancer, there was a marked decrease in the enhancement and the size of the tumor on contrast-enhanced ct (figure 3). ages of the specimens of two patients with deteriorated dna quality were 1001 and 1611 days, respectively. when comparing the patients with successful and failed genomic analysis, no statistically significant differences were observed in evaluated variables (table 3). there was a trend correlating failed genomic analysis with tumor size, although it was not statistically significant (p ¼ 0.088). technical results of biopsies the characteristics of the target lesions are shown in table 3. the liver was the most common site (41.7%). the median diameter (largest length in the axial sections) of the target lesion was 35 mm, and 19 lesions (39.6%) were smaller than 30 mm. for the guiding image, us was used in 26 patients (54.2%) and angio-ct in 22 patients (45.8%). all biopsies were performed with 18-g needles with a throw length of 2 cm. automatic biopsy needles (mugnum, bard biopsy systems, tempe, az, usa; pro-mag ultra, argon medical devices, plano, tx, usa) were used in 29 patients (60.4%) and semiautomatic biopsy needles (temno evolution, bd, franklin lakes, ny, usa; bard mission, bard biopsy systems, tempe, az, usa) in 19 patients (39.6%). the co-axial technique was used in 25 biopsies (52.1%). the median number of cores was 3 (range, 1–5). biopsies were technically successful in all patients (100%), and the median procedure time was 20 min (range, 10–50 min). adverse events associated with the procedure included pneumothorax (grade 1) in 1 patient and bleeding (grade 1) in 4. two of the bleeding patients underwent biopsy of the liver and were controlled with a needle-tract embolization. there were no severe adverse events or biopsyrelated deaths. table 2. characteristics of tumors. n % tumor location liver 20 41.7 lung 5 10.4 mediastinum 5 10.4 pelvis 4 8.3 peritoneum 3 6.3 soft part 3 6.3 retroperitoneum 2 4.2 pleura 2 4.2 superficial lymph nodes 2 4.2 diaphragm 1 2.1 bone 1 2.1 tumor size, mm, median (range) 35 (11–180) <30 mm 19 39.6 �30 mm 29 60.4 figure 3. a case of failed ngs analysis. the patient is a man in his 60s with suspected liver metastases from bile duct cancer. (a) contrast-enhanced ct 5 days before biopsy, after the anticancer medical therapy in a clinical trial. all the tumors demonstrated low attenuation representing hypovascular tumors. the largest mass in the posterior segment of the liver (asterisk) was selected for the target site of biopsy. biopsy of five cores from various portions in the tumor was performed under ultrasound guidance. ngs analysis failed, and the pathological diagnosis was necrosis of the tumor. (b) pre-treatment contrast-enhanced ct 2 months before biopsy. the diameters of the liver tumors are larger than that on post-treatment ct (a), and enhancement effects were seen in the periphery of the tumors. 122 m. sone et al. pathological diagnostic yield a histopathological diagnosis was established in 47 patients (97.9%). the diagnoses were adenocarcinoma, 21 patients; epithelioid hemangioendothelioma, 4; thymic cancer, 4; malignant mesothelioma, 2; and other, 17. the tumor cell percentage was measured in 27 patients, and the median was 60% (range, 10%–100%). median dna yield was 0.67 lg. the specimens were fresh (within 7 days from biopsy) in 21 patients (43.8%) and archived (8 days or more after biopsy) in 27 patients (56.2%). the median interval from biopsy to dna analysis was 169 days (range, 10–2068 days). discussion previous studies on genomic profiling of biopsy specimens have provided widely varying results (7–11,13–15). lung cancer was the most commonly reported neoplasm, and egfr, kras, and alk analysis using polymerase chain reaction or fluorescence in situ hybridization was achieved in 67%–100% of the specimens (7–9,13). few reports exist on the use of ngs in percutaneous biopsy specimens. in a retrospective study, young et al. performed ngs analysis on formalin-fixed, paraffin-embedded (ffpe) specimens from fine-needle aspiration in patients with lung (n ¼ 16) or pancreatic (n ¼ 23) tumors, and genomic analysis was successful in all specimens (100%) (10). zheng et al. performed ngs analysis on 1152 ffpe specimens from surgical, fna, and percutaneous biopsy samples, with success rates of 99.3%, 96.9%, and 94.4%, respectively. (11). these prior retrospective studies of ngs reported higher success rates than the present study and identical to the rate without biopsy-unrelated reasons. however, details of the biopsy procedure were not described in these studies. for genomic profiling, which requires the extraction and analysis of nucleic acids, the most important principle is to select a viable, cell-rich area on the imaging. moreover, sites that appear enlarged in comparison to past images are considered to have higher viability (14). intraand inter-tumoral (with multiple organ metastases) genomic heterogeneity as well as temporary changes may affect the results of genomic analysis and the determination of a treatment plan (16,17). however, there is currently no diagnostic imaging method that indicates whether a needle biopsy specimen has been collected from a site having genetic mutations representative of the patient’s status. in the future, as radiogenomics comparing imaging data and the genomic profile of a tumor (18–20) develops, site selection may become more systematic and sophisticated. the required specimen quantity for ngs analysis has previously been determined as a tumor cell percentage of �10% (21,22). if the tumor cell percentage is low, copy number validation detection is difficult, and the effects of artifacts increase (22). in our study, the median tumor cell percentage measured in 27 patients was 60%. this suggests that specimens with a high tumor cell percentage can be obtained by percutaneous needle biopsy planned and navigated by images. in a study of 1564 patients analyzed using ngs, cho et al. reported that analysis was possible in 95.9% (1503 patients) (21), and the recommended parameters for ngs analysis were >1 mm in size and >1 unstained slide in the case of ffpe specimens. regarding differences in the quantity of collected nucleic acid caused by needle diameter, jamshidi et al. reported that the diameter of the needle contributes more to the quantity of collected nucleic acid than does the number of cores sampled; use of an 18-g needle yielded a 4.8–5.7-fold greater quantity of nucleic acid than use of a 20-g needle (23). in our study, use of an 18-g needle with a median of 3 cores yielded satisfactory results of genomic analysis and pathological diagnosis. thus, if the target site is safely accessed, an 18-g needle would be suitable for acquiring samples for genomic analysis using ngs. there are some limitations of this study. first, it was a retrospective study. second, this study involved a small number of patients at a single facility. third, all biopsies were obtained with an 18-g needle, so we did not compare nucleic acid yields with needle size. fourth, surgical specimens were not examined. however, given the standardized technique of biopsy, our results at least have valuable information for the size and the number of cores of specimen. in conclusion, taking image-guided needle biopsy specimens using an 18-g cutting needle yielded a success rate of 79.2% on genomic analysis using ngs; the rate excluding the ngs analysis that failed due to reasons unrelated to the biopsy procedures was 95.2%. since there were no severe adverse events, we propose that image-guided needle biopsy might become an adoptable method for tissue sampling for ngs. by developing imaging-based selection methods of the target biopsy site and image-guided technology for effectively sampling the target site, the percentage of specimens that can undergo genomic analysis is expected to increase, but evaluations based on prospective studies are warranted. disclosure statement the authors declare that they have no conflict of interest. table 3. comparison of patient and tumor characteristics between success and failure in gene profiling. age, years success (n ¼ 38) failure (n ¼ 10) p value median 52 61 0.425 gender male 19 5 0.616 female 19 6 tumor location 0.199 liver 17 3 0.684 non-liver 21 7 tumor size, mm median 36 23 0.088 <30 mm 14 5 0.270 �30 mm 24 5 type of needle automatic 23 6 0.804 semi-automatic 15 4 number of cores median 3 3 0.171 age of specimen, days median 16 249 0.357 upsala journal of medical sciences 123 funding this work was supported by the japan agency for medical research and development (amed) under the practical research for innovative cancer control grant (16ck0106058h0003); the ministry of health, labour and welfare of japan under health and labor sciences research grant (h26-055); and national cancer center under the national cancer center research and development fund (29a-11). notes on contributors miyuki sone is consultant radiologist and the head of interventional radiology center at the national cancer center hospital (ncch) in tokyo, japan. yasuaki arai is the executive advisor to the president at the national cancer center (ncc) in tokyo, japan, and a staff radiologist at ncch. shunsuke sugawara is a consultant radiologist at ncch. takatoshi kubo was a fellow at ncch and currently is a staff radiologist at the university of tokyo. chihiro itou, is staff radiologists at ncch. tetsuya hasegawa is staff radiologist at ncch. noriyuki umakoshi is staff radiologist at ncch. noboru yamamoto is the head of department of experimental therapeutics, ncch and group leader at exploratory oncology research & clinical trial center, ncc. kumiko sunami is a staff physician of department of pathology and clinical laboratories at ncch. nobuyoshi hiraoka is the chief of department of pathology and clinical laboratories at ncch. takashi kubo is a staff member of the division of translational genomics, exploratory oncology research & clinical trial center, ncc. references 1. abi-jaoudeh n, duffy ag, greten tf, kohn ec, clark tw, wood bj. personalized oncology in interventional radiology. j vasc interv radiol. 2013;24:1083–92. 2. romine pe, harkins sk, gray sw. quality in the age of precision medicine: the clinician perspective. j oncol pract. 2016;12:839–43. 3. tam al, lim hj, wistuba ii, tamrazi a, kuo md, ziv e, et al. image-guided biopsy in the era of personalized cancer care: proceedings from the society of interventional radiology research consensus panel. j vasc interv radiol. 2016;27:8–19. 4. gomez-roca ca, lacroix l, massard c, de baere t, deschamps f, pramod r, et al. sequential research-related biopsies in phase i trials: acceptance, feasibility and safety. ann oncol. 2012;23:1301–6 5. lee jm, hays jl, noonan am, squires j, minasian l, annunziata c, et al. feasibility and safety of sequential research-related tumor core biopsies in clinical trials. cancer. 2013;119:1357–64 6. redig aj, janne pa. basket trials and the evolution of clinical trial design in an era of genomic medicine. j clin oncol. 2015;33: 975–7. 7. schneider f, smith ma, lane mc, et al. adequacy of core needle biopsy specimens and fine-needle aspirates for molecular testing of lung adenocarcinomas. am j clin pathol. 2015;143:193–200. 8. solomon sb, zakowski mf, pao w, lane mc, pantanowitz l, dacic s, et al. core needle lung biopsy specimens: adequacy for egfr and kras mutational analysis. am j roentgenol. 2010;194:266–9. 9. tam al, kim es, lee jj, ensor je, hicks me, tang x, et al. feasibility of image-guided transthoracic core-needle biopsy in the battle lung trial. j thorac oncol. 2013;8:436–42. 10. young g, wang k, he j, otto g, hawryluk m, zwirco z, et al. clinical next-generation sequencing successfully applied to fineneedle aspirations of pulmonary and pancreatic neoplasms. cancer cytopathol. 2013;121:688–94. 11. zheng g, tsai h, tseng lh, illei p, gocke cd, eshleman jr, et al. test feasibility of next-generation sequencing assays in clinical mutation detection of small biopsy and fine needle aspiration specimens. am j clin pathol. 2016;145:696–702. 12. tanabe y, ichikawa h, kohno t, yoshida h, kubo t, kato m, et al. comprehensive screening of target molecules by next-generation sequencing in patients with malignant solid tumors: guiding entry into phase i clinical 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d, abtin fg, loh ct, kee st, suh rd, et al. genomic adequacy from solid tumor core needle biopsies of ex vivo tissue and in vivo lung masses: prospective study. radiology. 2017;282:903–12. 124 m. sone et al. abstract introduction materials and methods patients biopsy procedure specimen processing and histopathological diagnosis genomic analysis outcomes ascertainment statistical analysis results patient demographics feasibility of genomic analysis technical results of biopsies pathological diagnostic yield discussion disclosure statement funding notes on contributors references vol_116_002_sups_a_545150 142..147 upsala journal of medical sciences. 2011; 116: 142–147 original article shoulder surface temperature and bone scintigraphy findings in patients with rotator cuff tears yoichi koike1, hirotaka sano2, takeshi kinjyo1, itaru imamura1, onuma masahiro1, masako goto1, masamizu ooyama1, atushi kita1 & eiji itoi2 1department of orthopaedic surgery, japanese red cross sendai hospital, yagiyama honcyo, taihaku-ku, sendai, miyagi, japan, and 2department of orthopaedic surgery, tohoku university school of medicine, 1-1, seiryo, aoba-ku, sendai, miyagi, japan abstract background. complex regional pain syndrome (crps) is one of the serious complications after surgical treatment of a rotator cuff tear. both a measurement of body surface temperature and bone scintigraphy have been used as diagnostic tools for the early phase of crps.unfortunately, few studies have been carried out that applied these methods to the patients after rotator cuff repair. purposes. to clarify both shoulder surface temperature and bone scintigraphy findings in patients with rotator cuff tears. subjects and methods. subjects comprised patients with unilateral rotator cuff tears (five men and five women, mean age 61 years). for measurements of shoulder surface temperature, a thermochron was attached to both shoulders. as for bone scintigraphy, intravenous injection of technetium-labelled hydroxymethylenebisphosphonic acid (99mtc-hmdp)was performed, and then images were taken with a gamma camera. results. during the measurements, the changes in body surface temperature for the affected and healthy shoulders remained within the standard deviation of the reference group. the intensity of radioisotope (ri) uptake for the affected shoulder joint was significantly increased compared to that for the healthy shoulder joint (p < 0.05). conclusion. ri uptake is increased in shoulders with rotator cuff tears, whereas shoulder surface temperature shows no differences on the affected and unaffected sides. key words: bone scintigraphy, complex regional pain syndrome (crps), complications, rotator cuff tear, skin temperature introduction complex regional pain syndrome (crps) is a complication that can occur after surgical treatment of a rotator cuff tear (1,2). primary characteristics of crps are abnormal temperature and bone resorption in the afflicted limb (1,3–5). in the clinical setting, abnormal temperature of the afflicted limb was detected by measuring body surface temperature (3,5), and bone resorption was detected by bone scintigraphy in patients with crps (3,6). when body surface temperature measurements and bone scintigraphy are performed on a patient after rotator cuff tear surgery, one should interpret the findings appropriately based on the proper control data. to obtain the base-line data, both measurements of body surface temperature and bone scintigraphy should be carried out before surgery. base-line data is extremely important to interpret the postoperative data appropriately. unfortunately, to date, bone scintigraphy findings on preoperative patients with rotator cuff tears have not been reported yet. mikayoshi et al. conducted time-dependent measurements over a 24-hour period (7), but there is no report of a follow-up test. based on these backgrounds, we attempted to clarify the correspondence: yoichi koike, department of orthopaedic surgery, japanese red cross sendai hospital, 2-43-3, yagiyama honcyo, taihaku-ku, sendai, miyagi, japan, 982-8501. fax: +81-22-243-1101. e-mail: yokoike@gmail.com (received 27 august 2010; accepted 29 november 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.545150 shoulder surface temperature as well as the bone scintigraphy findings in patients with rotator cuff tears. participants and methods patients participants were ten patients (five men and five women) who were hospitalized for rotator cuff tear treatment. the mean age was 61 years (range 39–76 years), and the average period of affliction was 6 months (range 3–17 months). five patients had a history of injury, and four had contractures as complications. all patients complained of pain at rest and nocturnal pain. the average dash score (8) was 52 points (range 40–75), and the average joa score (9,10) was 58 points (range 46–70). none of the patients satisfied the crps diagnostic criteria established by the japan ministry of health, labour, and welfare (11). on simple x-ray images, six cases showed some osteosclerosis at the greater tuberosity and three cases showed some osteosclerosis of the glenoid. magnetic resonance imaging demonstrated five full-thickness tears and five partial-thickness tears. seven tears involved only the supraspinatus tendon, and three involved multiple tendons. preoperative ultrasound examinations on all patients confirmed that the rotator cuff tears were unilateral. measurements of shoulder surface temperature thermochrons (maxim integrated products, sunnyvale, ca, usa) (surface thermometers with a diameter of 15 mm and built-in temperature sensor, memory, and battery) were used for measurements (12). the measurement method has been reported previously (2). briefly, thermochrons were attached 5 cm below the anterolateral end of the acromion on both sides. insulating tape (nitoms inc., tokyo, japan) was applied to the surface of the thermochrons, and an adhesive sheet (perme-roll, nitto medical co., osaka, japan) further covered the taped surface. anticipating when the patients would be sleeping, the measurement period was set from 21.00 until 07.00 the next morning. the time between measurements was 15 minutes, resulting in 40 measurements taken during the night. after completing the measurements, all thermochrons were removed, and the data were collected. for the reference group, we used previously reported measurement data from a group of ten individuals without shoulder problems (20 limbs) (2). the reference group included four men and six women (mean age 54 years). there were five cases of lower leg fracture, three cases of foot fracture, and two cases of hallux valgus. bone scintigraphy intravenous injection of 740 mbq technetiumlabelled hydroxymethylenebisphosphonic acid (99mtc-hmdp) was performed slowly, and images were taken 3 hours later with a gamma camera (symbia t2, siemens ag, berlin, germany). the shoulder, elbow, and wrist joints on both sides of a forwardfacing image of the entire body were established as regions of interest (rois). the level of radioisotope (ri) uptake in each roi was reported as a relative intensity normalized against the background uptake level. ten patients (five men and five women; mean age 61 years) who had no complaints regarding the upper limbs (20 limbs) were among those for whom bone scintigraphy had been carried out at this hospital during 2009 and were used as the reference group. diagnoses at the time of bone scintigraphy testing for the reference group were: one case of pubic fracture, two cases of lumbar vertebral compression fracture, one case of septic arthritis of the ankle joint, three cases of ankle osteoarthritis, and three cases of femoral head necrosis. statistics we compared body surface temperatures using means for three groups: the affected side of the affected group, the healthy side of the affected group, and the reference group. in the reference group, both shoulders of ten patients (20 shoulders) were pooled into one group. this is because it has already been reported that there is no statistically significant difference in the body surface temperature between upper limbs in healthy individuals (2). ri uptake intensities in the shoulder, elbow, and wrist joints were compared between the affected and healthy sides in the rotator cuff tear group. in the reference group, the left and right sides were compared for each joint. pasw statistics 18 (spss, chicago, il, usa) was the statistical software used for conducting paired sample t tests. the significance criterion was p < 0.05. ethics the protocol of this study was approved by the ethics board of the first author’s institute (registration number r1000245, approved on 20 june 2009) shoulder surface temperature and bone scintigraphy in patients with rotator cuff tears 143 and was conducted in accordance with the declaration of helsinki. all patients gave informed consent to participate in this study. results time-dependent changes in the mean body surface temperature of the affected group (ten shoulders) are shown in figure 1. from 21.00 to 03.00, the temperature slowly decreased and reached a minimum of 34.1�c at 02.00. thereafter, the temperature increased slowly until 07.00. during this time, the changes in shoulder surface temperature for the affected and healthy sides remained within the standard deviation of the reference group. in comparing mean temperatures, there were no statistically significant differences among the three groups. the mean (standard deviation) shoulder surface temperature in the affected side, the healthy side, and the reference group were 34.4 (0.2) �c, 34.5 (0.2) �c, and 34.4 (0.2) �c, respectively. none of the groups represented statistical significant differences. in the bone scintigrams, we observed increased ri uptake in the affected shoulder joint in nine cases (90%). the intensity of ri uptake for the affected shoulder joint was significantly increased compared to that for the healthy shoulder joint. the mean (standard deviation) intensity of ri uptake in the affected side and the healthy side were 3.0 (1.0) and 2.3 (0.7), respectively (p < 0.05; figure 2). on the other hand, in elbow and wrist joints, there were no significant differences in ri uptake intensity between the affected and healthy sides. moreover, no significant differences were found between right and left sides in the reference group for shoulder, elbow, or wrist joints. case presentation case 1. a 71-year-old male was injured after falling on a snow-covered path and hitting his left elbow 6 months earlier. he noticed pain in the left shoulder after the injury, and became aware of nocturnal pain in the left shoulder 4 months after the injury. physical examination revealed a limited range of motion and muscle weakness in abduction and external rotation of the left shoulder joint. there were positive signs of impingement (13), and a block test (14). preoperative joa and dash scores were 60 out of 100 and 45, respectively. no obvious changes of osteoarthritis were observed on plain x-rays (figure 3a). a fullthickness rotator cuff tear was observed on mri (figure 3b, arrow). an increase of ri uptake in the left shoulder joint was observed in the bone scintigram (figure 3c, arrow). however, there were no remarkable left–right differences in the ri uptake for elbow or wrist joints. case 2. a 51-year-old male incurred injury 8 months earlier after falling over while riding a motorcycle. he experienced pain from the right shoulder to the forearm after the injury and became aware of nocturnal pain in the right shoulder 6 months after the injury. physical examination revealed a limited range of motion in abduction, external rotation, and extension of the right shoulder joint. preoperative joa and dash scores were 62 out of 100 and 50, respectively. no obvious arthrotic changes were seen on plain x-rays. a full-thickness rotator cuff tear was observed 36 35 33.5 34.5 35.5 34 21 0 time (o′clock) 3 healthy side (n = 10) affected side (n = 10) reference group (n = 20) 6 t e m p e ra tu re ( ºc ) figure 1. time-dependent changes in the mean shoulder surface temperature.the changes in body surface temperature for the affected (bold line) and healthy shoulders (thin line) remained within the standard deviation (sd) of the reference group (dotted line with sd bars). 144 y. koike et al. shoulder joint * 3.0 4.0 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 elbow joint 2.0 wrist joint 1.0 0 affected group (n = 10) reference group (n = 10) affected side healthy side right side left side figure 2. the intensity of radioisotope(ri) uptake. the intensity of ri uptake for the affected shoulder joint is significantly increased compared to that for the healthy shoulder joint. in elbow and wrist joints, there are no significant differences in ri uptake intensity between the affected and healthy sides. in the reference group, no significant left–right differences are seen for the shoulder, elbow, and wrist joints. (*affected side 3.0 ± 1.0 versus healthy side 2.3 ± 0.7; p < 0.05). a b c figure 3. case 1: a 71-year-old male with a rotator cuff tear in the left shoulder. no obvious changes of osteoarthritis are observed on plain x-rays (a). a full-thickness rotator cuff tear is observed on the coronal t2 wi mri (b, arrow). an increase of ri uptake in the left shoulder joint is observed in the bone scintigram (c, arrow). however, there are no left–right differences in ri uptake for elbow or wrist joints. shoulder surface temperature and bone scintigraphy in patients with rotator cuff tears 145 on mri. an increase of ri uptake in the right shoulder joint was observed in the bone scintigram. however, there were no left–right differences in ri uptake for elbow or wrist joints. in spect (single photon emission computed tomography) imaging carried out at the same time, ri uptake was observed in the greater tuberosity of the humerus, the coracoid process of the scapula, and the glenohumeral joint (figure 4). discussion the shoulder surface temperature in a healthy human changes over time and is influenced by daily fluctuations in core body temperature (15,16). multiple factors, including heat generation associated with muscle contraction, have been reported as external factors affecting body surface temperature (17,18). changes in body surface temperature occurring in each of the upper limbs could give rise to left–right differences in temperature. however, when multiple measurements are taken across time and means are then compared, no left–right differences are observed (2). abnormal shoulder surface temperatures are triggered by various shoulder joint diseases. in adhesive capsulitis and crps, the surface temperature of the afflicted shoulder is significantly reduced compared to the healthy side (19,20). in the present study, there was no significant difference between the surface temperature of the affected shoulder compared to that of the healthy shoulder or in the reference group. these results were consistent with those reported previously by miyakoshi et al. (7), who reported that the body surface temperature of a shoulder with a rotator cuff tear was not different from that of a healthy shoulder. from the clinical point of view, the results of the present study could be interpreted as follows: if a temperature abnormality is observed after rotator cuff surgery, the onset of the temperature abnormality is probably after the surgery. bone scintigraphy excels in detecting bone resorption associated with fractures, bone tumours, osteomyelitis, disuse atrophy, and other conditions (6). increased ri uptake in the cancellous bone of the afflicted limb is a characteristic feature of crps, and early bone resorption can be objectively detected using scintigraphy (3). a b c d figure 4. case 2: a 51-year-old male with a rotator cuff tear in the right shoulder. no obvious osteoarthritis is observed in x-rays (a). a fullthickness rotator cuff tear was observed on the coronal t2 wi mri (b, arrow). an increase of ri uptake in the left shoulder joint is observed in the bone scintigram (c, arrow). in spect images, ri uptakes are evident in the greater tuberosity of the humerus, the coracoid process of the scapula, and the glenohumeral joint (d, arrows). 146 y. koike et al. the increased uptake that occurs in the shoulder joint has previously been observed in patients with adhesive capsulitis (21). interestingly, it has been reported that the cause of adhesive capsulitis is related to the incomplete onset of crps (22,23). waldburger et al. speculated that the increased ri uptake that occurs in adhesive capsulitis reflected the onset of crps (21). it is possible that the increased ri uptake in patients with a rotator cuff tear in the present study also reflects the latent onset of crps. alternatively, it is possible that arthritis, osteophyte formation, or treatments prior to ri examination affected this. clinically, this increase in ri uptake might be seen in patients after shoulder joint surgery. in such cases, the increase of ri uptake might be misdiagnosed as crps onset after surgery. however, sufficient attention should be paid to whether increased ri uptake could be observed in the joint preoperatively. in summary, we reported findings on shoulder surface temperature and bone scintigraphy in patients with rotator cuff tears. the surface temperature of shoulders with rotator cuff tears was not different from that of the contralateral normal shoulders or shoulders in the healthy reference group. this suggests that if a temperature abnormality is observed after rotator cuff surgery, the onset is likely a postoperative one. bone scintigraphy shows an increased ri uptake in shoulders with rotator cuff tears. careful attention should be paid when interpreting ri images, so as not to misdiagnose that these findings were caused by crps in patients after rotator cuff surgery. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. steinbrocker o. the shoulder-hand syndrome: present perspective. arch phys med rehabil. 1968;49:388–95. 2. koike y,sano h, imamura i, goto m, ooyama m, kita a. changes with time in skin temperature of the shoulders in healthy controls and a patient with shoulder-hand syndrome. ups j med sci. 2010;115:260–5. 3. atkins rm. complex regional pain syndrome.j bone joint surg br. 2003;85:1100–6. 4. bickerstaff dr, charlesworth d, kanis ja. changes in cortical and trabecular bone in algodystrophy. br j rheumatol. 1993;32:46–51. 5. harden rn, bruehl sp. diagnosis of complex regional pain syndrome: signs, symptoms, and new empirically derived diagnostic criteria. clin j pain. 2006;22:415–19. 6. zyluk a. the usefulness of quantitative evaluation of three-phase scintigraphy in the diagnosis of post-traumatic reflex sympathetic dystrophy. j hand surg br. 1999;24:16–21. 7. miyakoshi n, itoi e, sato k, suzuki k, matsuura h. skin temperature of the shoulder: circadian rhythms in normal and pathologic shoulders. j shoulder elbow surg. 1988;7: 625–8. 8. schuind fa, mouraux d, robert c, brassinne e, rémy p, salvia p, et al. functional and outcome evaluation of the hand and wrist. hand clin. 2003;19:361–9. 9. hirooka a, yoneda m, wakaitani s, isaka y, hayashida k, fukushima s, et al. augmentation with a gore-tex patch for repair of large rotator cuff tears that cannot be sutured. j orthop sci. 2002;7:451–6. 10. the journal of the shoulder joint editorial committee: manual for evaluation of the shoulder (joa score). the shoulder joint (katakansetsu). 2005;29:747–8 (japanese). 11. sumitani m, shibata m, sakaue g, mashimo t; japanese complex regional pain syndrome research group. development of comprehensive diagnostic criteria for complex regional pain syndrome in the japanese population. pain. 2010;150:243–9. 12. masaki m, koshimoto c, tsuchiya k, nishiwaki a, morita t. body temperature profile of the korean field mouse apodemus peninsulae during winter aggregation. mammal study. 2005;30:33–40. 13. jobe fw, bradley jp. the diagnosis and nonoperative treatment of shoulder injuries in athletes. clin sports med. 1989;8: 419–38. 14. caliş m, akgün k, birtane m, karacan i, caliş h, tüzün f. diagnostic values of clinical diagnostic tests in subacromial impingement syndrome. ann rheum dis. 2000;59:44–7. 15. kräuchi k, wirz-justice a. circadian rhythm of heat production, heart rate, and skin and core temperature under unmasking conditions in men. am j physiol. 1994;267:r819–29. 16. scales we, vander aj, brown mb, kluger mj. human circadian rhythms in temperature, trace metals, and blood variables. j appl physiol. 1988;65:1840–6. 17. bogh m, minors ds, waterhouse jm. can insulated skin temperature act as a substitute for rectal temperature when studying circadian rhythms? chronobiol int. 1994;11: 332–9. 18. rietveld wj, minors ds, waterhouse jm. circadian rhythms and masking: an overview. chronobiol int. 1993; 10:306–12. 19. jeracitano d, cooper rg, lyon lj, jayson mi. abnormal temperature control suggesting sympathetic dysfunction in the shoulder skin of patients with frozen shoulder. br j rheumatol. 1992;3:539–42. 20. vecchio pc, adebajo ao, chard md, thomas pp, hazleman bl. thermography of frozen shoulder and rotator cuff tendinitis. clin rheumatol. 1992;1:382–4. 21. waldburger m, meier jl, gobelet c. the frozen shoulder: diagnosis and treatment. prospective study of 50 cases of adhesive capsulitis. clin rheumatol. 1992;11:364–8. 22. müller lp, müller la, happ j, kerschbaumer f. frozen shoulder: a sympathetic dystrophy? arch orthop trauma surg. 2000;120:84–7. 23. wiffen f. what role does the sympathetic nervous system play in the development of ongoing pain of adhesive capsulitis? journal of manual & manipulative therapy. 2002;10:17–23. shoulder surface temperature and bone scintigraphy in patients with rotator cuff tears 147 low copulatory activity in selectively bred sardinian alcohol-nonpreferring (snp) relative to alcoho full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 low copulatory activity in selectively bred sardinian alcohol-nonpreferring (snp) relative to alcohol-preferring (sp) rats oskar karlsson, giancarlo colombo & erika roman to cite this article: oskar karlsson, giancarlo colombo & erika roman (2015) low copulatory activity in selectively bred sardinian alcohol-nonpreferring (snp) relative to alcohol-preferring (sp) rats, upsala journal of medical sciences, 120:3, 181-189, doi: 10.3109/03009734.2015.1010666 to link to this article: https://doi.org/10.3109/03009734.2015.1010666 © informa healthcare published online: 02 mar 2015. submit your article to this journal article views: 361 view related articles view crossmark data https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2015.1010666 https://doi.org/10.3109/03009734.2015.1010666 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1010666 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1010666 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1010666&domain=pdf&date_stamp=2015-03-02 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1010666&domain=pdf&date_stamp=2015-03-02 upsala journal of medical sciences. 2015; 120: 181–189 original article low copulatory activity in selectively bred sardinian alcoholnonpreferring (snp) relative to alcohol-preferring (sp) rats oskar karlsson1,2,3, giancarlo colombo4, erika roman5 1center for molecular medicine, department of clinical neuroscience, karolinska institute, k8, 171 76 stockholm, sweden, 2drug safety and toxicology, department of pharmaceutical biosciences, uppsala university, box 591, 751 24 uppsala, sweden, 3department of environmental health, harvard school of public health, boston, ma 02115, usa, 4neuroscience institute, national research council of italy, section of cagliari, i-09042 monserrato (ca), italy, and 5unit of neuropharmacology, addiction and behaviour, department of pharmaceutical biosciences, uppsala university, box 591, 751 24 uppsala, sweden abstract background. there is a growing consensus that similar neural mechanisms are involved in the reinforcing properties of natural rewards, like food and sex, and drugs of abuse. rat lines selectively bred for high and low oral alcohol intake and preference have been useful for understanding factors contributing to excessive alcohol intake and may constitute proper animal models for investigating the neurobiological basis of natural rewarding stimuli. methods. the present study evaluated copulatory behavior in alcohol and sexually naïve sardinian alcohol-preferring (sp) and -nonpreferring (snp) male rats in three consecutive copulatory behavior tests. results. the main finding was that, under the conditions used in this study, snp rats were sexually inactive relative to sp rats. to gain more information about the sexual behavior in sp rats, wistar rats were included as an external reference strain. only minor differences between sp and wistar rats were revealed. conclusions. the reason behind the low copulatory activity of snp rats remains to be elucidated, but may in part be mediated by innate differences in brain transmitter systems. the comparison between sp and wistar rats may also suggest that the inherent proclivity to excessive alcohol drinking in sp rats may mainly be dependent on its anxiolytic properties, as previously proposed, and not changes in the reward system. key words: addiction, drugs of abuse, ejaculation, natural reward, sardinian alcohol-preferring and non-preferring rats, sexual behavior, wistar rat introduction there is a growing consensus that similar mechanisms are involved in the reinforcing properties of natural rewards, like food and sex, and drugs of abuse (1-5). the demand for an improved understanding of the basal functioning of the brain reward system, especially with regard to motivational aspects and choice of reinforcer, has recently been emphasized (1,4,5). the mesocorticolimbic dopamine system with cell bodies in the ventral tegmental area and projections to areas including the nucleus accumbens, amygdala, and the prefrontal cortex is highly conserved in evolution, a natural constituent of vital life sustaining behaviors (6), and a key component in reward and addiction processes (2,3,7). it is well acknowledged that sexual activity (8) and intake of palatable food (9) and drugs of abuse including alcohol (10) result in elevated levels of extracellular dopamine in areas associated with the mesocorticolimbic dopamine system, including the nucleus accumbens. in studies of basic mechanisms of eliciting reward-driven behavior correspondence: associate professor erika roman, department of pharmaceutical biosciences, unit of neuropharmacology, addiction and behaviour, uppsala university, po box 591, 751 24 uppsala, sweden. e-mail: erika.roman@farmbio.uu.se (received 17 december 2014; accepted 16 january 2015) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1010666 http://informahealthcare.com/journal/ups mailto:erika.roman@farmbio.uu.se it is important to differentiate the appetitive phase (seeking the reinforcer) from the act of consumption. natural and sexual selection processes are likely to be involved in the development of appetitive and consummatory activity with some key neuronal trajectories shared between species. the development of ratlinesselectively bredfor high and low oral alcohol intake and preference has been a useful strategy for understanding factors contributing to excessive alcohol intake (11-16). these lines were bredforthe samephenotypes,i.e.high orlowvoluntary alcohol intake and preference under the standard, home-cage, two-bottle free-choice paradigm with continuous access to alcohol, water, and food (17-19). one common aim of these selective breeding programs has been to determine behavioral (20), neurobiological (21), and genetic (22) characteristics associated with selection for high and low voluntary alcohol intake and preference. considering the role of the brain reward system in reinforcing properties of natural rewards as well as drugs of abuse it is possible that selective breeding for high alcohol intake affects also other reward-related behaviors. the selectively bred alcohol-preferring and non-preferring lines may therefore represent possible models for studies of similarities between natural rewarding stimuli and drugs of abuse (23-25). here the sardinian alcohol-preferring (sp) and -nonpreferring (snp) rats (16) were used to study some aspects of this fairly unexplored topic. the aim of the present study was to investigate if selective breeding of sp and snp rats has also resulted in differences in copulatory behavior. wistar rats were included as an external reference strain. material and methods animals adult alcohol and sexually naïve sp and snp male rats (kept under specific pathogen and opportunistic free conditions at charles river laboratories, calco, italy; generation s65; n = 15 rats per group) and agematched male wistar rats (sca:wi; scanbur bk ab, sollentuna, sweden; n = 10) were studied. this study was initially designed to compare copulatory activity in sp and snp rats; however, the large degree of sexual inactivity observed in snp rats in several preliminary experiments forced us to include a set of wistar rats as reference strain. wistar rats were preferred over other strains of outbred rats as they constituted the foundation stock from which the selective breeding of sp and snp rats was started (16). it should, however, be noted that the wistar rats used herein were from a different supplier, and vendor-dependent differences between wistar rats exist (26-29). twenty-four female wistar rats (hantac:wh; taconic, ejby, denmark), not related tothe male wistar rats, weighing approximately 300 g, were used as stimulus females. all rats were housed three per cage in transparent cages (59 � 38 � 20 cm) containing wood-chip bedding material and paper sheets as enrichment. the cages were placed in temperature-controlled (22 ± 1�c) and humiditycontrolled (50% ± 10%) housing cabinets with a reversed 12-hour light/dark cycle (lights off between 7.00 a.m. and 7.00 p.m.). the rats were maintained on rat chow (r36; lantmännen, kimstad, sweden) and water ad libitum. the females used as stimuli in the copulatory behaviortestwere broughtinto estrusbyhormonetreatment. they were ovariectomized under isoflurane anesthesia and allowed to recover for 10 days. the hormone treatment consisted of subcutaneous administrations of 25 mg/kg of estradiol benzoate (sigma-aldrich, st. louis, mo, usa) in olive oil 48 h before progesterone, and 1 mg/rat of progesterone (sigma-aldrich) in olive oil 4–6 h before use. prior to the assessment of copulatory behavior, all male rats had undergone behavioral testing (30), which was completed at approximately 20 weeks of age. the sexual behavior test was initiated at 30 weeks of age. all animal experiments were approved by the uppsala animal ethical committee and followed the guidelines of the swedish legislation on animal experimentation (animal welfare act sfs1998:56) and the european communities council directive (86/609/eec). copulatory behavior test male copulatory behavior with receptive females was scored in three tests. the first two tests, which were considered as learning tests, lasted for 15 min, and the animals had two test-free days between each test. the third test, which served to investigate copulatory behavior in sexually experienced rats, was performed one week after the second test. the third test lasted for a maximum of 30 min but was interrupted after scoring the post-ejaculatory interval. the tests were performed in a wooden cage (60 � 35 � 35 cm) with a transparent front, used to score copulatory behavior only, under dim illumination during the dark phase of the light/dark cycle. the male rat was allowed to habituate to the cage for 5 min before the receptive female was introduced. each female was used for two to three males, and the females were alternated between the three copulatory tests. based on established protocols (31-33), parameters according to the ethogram in table i were scored by direct observation by an experienced, blinded observer or calculated based on the scored behaviors. 182 o. karlsson et al. statistical analyses data were tested for normality using the shapiro– wilk’s w test. copulatory behavior data were not normally distributed, and therefore non-parametric statistical methods were used. the learning effect was evaluated by comparing latency and frequency measures over the three tests using the friedman test followed by the wilcoxon signed rank test when appropriate. for these analyses all values were included, and latency measures were set at 900 s if the behavior was not performed. the number of animals performing mounts, intromissions, and ejaculations was evaluated with the chi-square test. the third test was analyzed for performed behaviors, and thus if a behavior did not occur this was considered a missing value. group comparisons of performance in the third test were performed using the kruskal–wallis test and/or the mann–whitney u test. differences were considered statistically significant at p £ 0.05. statistica 10.0 (statsoft inc., tulsa, ok, usa) was used for the statistical analyses. results acknowledging that copulatory activity is a product of two individuals interacting, the behavior of the females was not scored since, after hormonal priming, all females were sexually proceptive and displayed receptive behaviors including lordosis. performance over time the main finding in the present study is a very low copulatory activity in male snp rats (figure 1). thus, fewer snp than sp rats engaged in copulatory behavior in all three tests. in the first and second test, only 2 out of 15 snp males mounted, and in the third test this number was increased to 3 males, which was significantly fewer (chi-square = 8.89, df = 1, p < 0.01; chi-square = 13.39, df = 1, p < 0.001; chi-square = 13.39, df = 1, p < 0.001, respectively) when compared with sp rats. in the second and third test, no snp rats achieved intromission (chi-square = 9.13, df = 1, p < 0.01; chi-square = 17.37, df = 1, p < 0.0001) or ejaculation (chi-square = 10.91, df = 1, p < 0.001). based on this sexual inactivity, snp rats were excluded from further detailed comparison with the sp line. to get information about the copulatory behavior in sp rats, a detailed statistical analysis using wistar rats as reference strain was performed. there were significant differences over time in sp and wistar rats in latency to first mount (sp friedman anova (n = 15, df = 2) = 5.71, p = 0.058; wistar friedman anova (n = 10, df = 2) = 15.08, table i. ethogram of the behaviors scored by direct observation or calculated during the copulatory activity tests. parameters definitions latency mount the time (s) from the introduction of the receptive female until the first mount was observed latency intromission the time (s) from the introduction of the receptive female until the first intromission was observed latency ejaculation the time (s) from the introduction of the receptive female until an ejaculation was observed frequency mount the total number of observed mounts frequency intromission the total number of observed intromissions frequency ejaculation a the total number of observed ejaculations post-ejaculatory interval the time (s) from ejaculation until the next observed intromission mounts + intromissions the sum of observed mounts and intromissions mounts + intromissions/min the sum of observed mounts and intromissions calculated per minute intromission ratio the number of observed intromissions calculated in relation to the total number of observed mounts and intromissions time from first intromission to ejaculation the elapsed time (s) from the first observed intromission until an observed ejaculation mounts + intromissions/ejaculation the sum of mounts and intromissions required to reach ejaculation inter-intromission rate iii the latency (s) from the introduction of the receptive female until ejaculation divided by the total number of intromissions copulatory rate the sum of mounts and intromissions divided by the elapsed time (s) from the first mount until an observed ejaculation athe number of ejaculations was only scored in the first two tests, which lasted for 15 min each. only one sp rat ejaculated twice, and this was in the first test. the third test lasted for a maximum of 30 min but was interrupted after scoring the post-ejaculatory interval. copulatory behavior in sp and snp rats 183 p < 0.001), latency to first intromission (sp friedman anova n.s.; wistar friedman anova (n = 10, df = 2) = 6.59, p < 0.05) and latency to first ejaculation (sp friedman anova (n = 15, df = 2) = 11.38, p < 0.01; wistar friedman anova n.s.) (figure 2). in wistar rats, the latency to first mount and intromission, respectively, was significantly shorter in the second (mount z = 2.29, p < 0.05; intromission z = 2.02, p < 0.05) and third (mount z = 2.80, p < 0.01; intromission z = 2.55, p < 0.05) test relative to the first test. likewise, in the third test, the latency to ejaculation was shorter (z = 2.52, p < 0.05) when compared with the first test in sp rats. moreover, when comparing sp and wistar males, wistar rats had shorter latency to the first mount in the third test (u = 37.5, p < 0.05; figure 2a). the number of intromissions was higher (z = 2.24, p < 0.05) in the third test relative to the first test in wistar rats, with a trend close to statistical significance also for sp rats (z = 1.91, p = 0.056) (figure 3). the sum of mounts and intromissions was higher (z = 2.40, p < 0.05) in the third test compared with the first test in wistar rats, while no such difference was found in sp rats. assessment of copulatory behavior in sexually experienced sp and wistar rats eighty-seven percent of the sp rats and 100% of the wistar rats mounted, while the corresponding values for intromissions were 73% (sp) and 90% (wistar), and for ejaculations 53% (sp) and 50% (wistar) (figure 1). more detailed studies of performed behaviors in the third copulatory behavior test in sp and wistar rats revealed a difference in inter-intromission rate (u = 6.0, p < 0.05) (table ii). moreover, there were trends toward longer latency to first mount (p = 0.088) and fewer mounts and intromissions per minute (p = 0.067), and longer post-ejaculatory interval (p = 0.065) in sp rats relative to wistar rats (table ii). discussion the aim of this study was to determine if the response to a natural rewarding stimulus, i.e. copulatory activity, differed in rats selectively bred for opposite alcohol preference and consumption. the main finding was an almost absence of copulatory activity in the n u m b e r o f a n im a ls ( % ) m o u n ti n g 100 90 80 70 60 50 40 30 20 10 0 ** a n u m b e r o f a n im a ls ( % ) e ja c u la ti n g 100 90 80 70 60 50 40 30 20 10 0 c test 1 test 2 test 3 test 1 test 2 test 3 *** *** n u m b e r o f a n im a ls ( % ) in tr o m it ti n g 100 90 80 70 60 50 40 30 20 10 0 ** ** b test 1 test 2 test 3 **** *** snp sp wi figure 1. the percentage number of snp (n = 15) and sp (n = 15) rats mounting (a), intromitting (b), and achieving ejaculation (c) over the three copulatory behavior tests. due to the low number of snp rats engaging in copulatory activity also in the third test, they were excluded from further detailed statistical analyses of copulatory behavior. wistar (n = 10) rats were included as a reference strain. **p < 0.01, *** p < 0.001, **** p < 0.0001 compared to sp rats (chi-square test). 184 o. karlsson et al. alcohol-nonpreferring snp rats compared with -preferring sp rats. even after three tests, no snp rat had intromissions or achieved ejaculation, and based on this finding they are considered sexually inactive (33), also termed non-copulators (34). copulatory behavior, like drug-taking behavior, is comprised of appetitive and consummatory acts (35). the mount and intromission latencies are considered measures of appetitive acts or sexual motivation (33,36). ejaculation represents a consummatory act, while the interpretation of post-ejaculatory interval is less clear (33). non-copulators are males that do not mate even after they are tested repeatedly with sexually receptive females, and are found in several species including rats and mice (33,34,37). the finding that snp rats do not display sexual motivation or engage in consummatory sexual behavior after three tests may suggest characteristics of non-copulators. since the females used were primed and therefore displayed proceptive and receptive behaviors it is considered unlikely that the behavior of the female rats is the cause for the low copulatory activity observed in the snp rats. in addition, since the snp line is maintained over generations, these rats obviously copulate under other circumstances; the mating technique adopted in the breeding procedure includes indeed long periods of time (up to one week) during which male and female snp rats are housed together in the same cage. when compared with wistar rats, sp rats displayed low sexual behavior in initial tests but increased their copulatory activity over successive testing. this pattern of increasing copulatory activity over time has been attributed to distraction or fear generated by the novelty of the test situation (34). beside inherent proclivity to excessive alcohol drinking, sp rats display more anxiety-related behaviors compared with snp rats and, when tested, sometimes also compared with wistar rats (20,30,38-41). in a more complex setting, i.e. the multivariate concentric square field� test, sp rats are characterized by low general activity and exploratory drive, and low risk-taking behavior compared with snp rats (20,30) as well as other selectively bred alcohol-preferring rat lines (20). since voluntarily consumed alcohol ameliorated different anxietyrelated behaviors (41,42), it has been proposed that sp rats consume alcohol for its anxiolytic properties (16,41). when sexually experienced, i.e. in the third test, the copulatory behavior of sp rats was similar to that of wistar rats. thus, explorative strategies and l a te n c y m o u n t (s ) 900 800 700 600 500 400 300 200 100 0 a l a te n c y i n tr o m is s io n ( s ) 900 800 700 600 500 400 300 200 100 0 b l a te n c y e ja c u la ti o n ( s ) 900 800 700 600 500 400 300 200 100 0 c te st 1 te st 2 te st 3 te st 1 te st 2 te st 3 te st 1 te st 2 te st 3 wi ## ## # # # * sp l a te n c y m o u n t (s ) 80 70 60 50 40 30 20 10 0 wi test 3 * sp figure 2. latency to first mount (a), intromission (b), and ejaculation (c) in the three copulatory behavior tests in male sp (n = 15) and wistar (n = 10) rats. the latency to first mount (a) in the third test is also shown in the insert for a better illustration of the difference between sp and wistar rats. values represent individual data points with the median value marked as a line, and in the insert values represent median and quartile range. * p < 0.05 comparing sp and wistar rats (mann–whitney u test); # p < 0.05, ## p < 0.01 compared to the first copulatory behavior test within the respective group (wilcoxon signed rank test). copulatory behavior in sp and snp rats 185 copulatory activity in sp rats display differences as the copulatory behavior in the third test was similar to that of the wistar rats while no attenuation of anxietyrelated behaviors in sp rats occurs with repeated testing [(30), and roman and colombo, unpublished observation]. a number of brain transmitter systems and specific brain areas have been linked to sexual function. the sp and snp rats differ in a number of brain transmitter systems; these differences have been suggested to underlie their opposite alcohol-drinking phenotypes (21). for example, basal dopamine levels were higher in the nucleus accumbens shell in sp than in snp and wistar rats, and in the medial prefrontal cortex in sp rats relative to wistar but not snp rats (39). dopamine denervation of the nucleus accumbens and infusion of the dopamine d2 antagonist raclopride both affect sexual behavior by delaying the initiation of mounting and intromitting without affecting the number of mounts and intromissions (43). also the endocannabinoid system seems to play a role in the regulation of sexual behavior as the endocannabinoid anandamide was shown to induce copulatory behavior in non-copulating animals (44). the sp rats have higher density of the cannabinoid receptor cb1 and higher levels of the endocannabinoids anandamide and 2-arachidonoylglycerol compared with snp rats (45). these innate differences between sp and snp rats could explain, at least in part, the differences in copulatory behavior observed herein. in previous investigations of responses to a natural reward stimulus, such as palatable food, there were no differences in intake and in some cases also preference of intake of a chocolate-flavored drink (46), sucrose (46,47), or saccharin solutions (48) between sp and snp rats. however, in these studies, the high reinforcing properties of these fluids might have overwhelmed any possible existing difference in reward sensitivity between sp and snp rats. in addition, both sp and snp rats initiated and maintained operant selfadministration of sucrose (49). therefore, on the basis of the relatively scarce amount of data available, at present it cannot be ruled out that snp rats are characterized by a generally lower motivational drive (not limited solely to alcohol and sexual activity) than sp rats. the results in the present study differ markedly from those of a previous experiment where only m o u n ts 100 90 80 70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 a wi sp p = 0.056 # # c te st 1 te st 2 te st 3 te st 1 te st 2 te st 3 te st 1 te st 2 te st 3 in tr o m is s io n s m o u n ts a n d i n tr o m is s io n s 0 5 10 15 20 b figure 3. the number of mounts (a), intromissions (b), and sum of mounts and intromissions (c) in the three copulatory behavior tests in male sp (n = 15) and wistar (n = 10) rats. values represent individual data points with the median value marked as a line. # p < 0.05 compared to the first copulatory behavior test within the respective group (wilcoxon signed rank test). 186 o. karlsson et al. modest differences between sp and snp rats were found (38). however, the study by cagiano et al. (38) was designed to study the impact of alcohol exposure during the perinatal period on subsequent behavioral development, and the animals had undergone perinatal treatment with alcohol or sucrose from gestational day 15 to postnatal day 7, which may explain the different outcome. perinatal exposure to both alcohol and sugar-rich diets affect responses to drugs of abuse later in life (50,51), likely through adaptations in the reward-related mesostriatal dopaminergic system that also may impact on copulatory behavior. in conclusion, the present investigation reveals profound differences in copulatory activity between the selectively bred alcohol-preferring sp and -nonpreferring snp rats. sp rats, with an innate proclivity for high voluntary alcohol intake and high levels of anxiety-related behaviors, display sexual behavior similar to that of outbred wistar rats when sexually experienced; snp rats were considered sexually inactive, with characteristics of noncopulating males, suggesting a lower motivational drive. with regard to natural rewards, our results imply different responses following access to palatable food reward and a sexually receptive female in male sp and snp rats. considering the known differences between the different lines of rats selectively bred for high and low alcohol intake and preference it would be of interest in future studies to compare sexual behavior also in other lines. acknowledgements the authors are grateful for the excellent technical assistance provided by ms marita berg and for valuable comments by professor bengt j meyersonduring the preparation of the manuscript. this study was supported by grants (e.r.) from the alcohol research council of the swedish alcohol retailing monopoly, the swedish society for medical research (ssmf), the swedish brain foundation, and the facias, magnus bergvall, fredrik och ingrid thuring and åke wiberg foundations. declaration of interest: the authors declare that the research was conducted 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http://www.ncbi.nlm.nih.gov/pubmed/15976517?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22564493?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22564493?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22564493?dopt=abstract abstract introduction material and methods animals copulatory behavior test statistical analyses results performance over time assessment of copulatory behavior in sexually experienced sp and wistar rats discussion acknowledgements doi25 references vol_116_002_sups_a_548011 115..123 upsala journal of medical sciences. 2011; 116: 115–123 original article critical diaphragm failure in sudden infant death syndrome pontus max axel siren & matti juhani siren jgk memorial research library, helsinki, finland abstract sudden infant death syndrome (sids) is the leading cause of death in infants between the ages of 1 and 12 months in developed countries. sids is by definition a diagnosis of exclusion, and its mechanism of action is unknown. the sids– critical diaphragm failure (cdf) hypothesis postulates that the cause of death in sids is respiratory failure caused by cdf. four principal risk factors contribute to cdf in young infants: undeveloped respiratory muscles, non-lethal infections, prone resting position, and rem sleep. even relatively minor infections can cause an acute and significant reduction in diaphragm force generation capacity that in conjunction with other risk factors can precipitate cdf. cdf-induced acute muscle weakness leaves few, if any pathological marks on the affected tissue.understanding the underlying mechanism of sids may help in formulating new approaches to child care that can help to further reduce the incidence of sids. key words: sudden infant death syndrome (sids), infection, diaphragm, pediatrics introduction it has been recognized since antiquity that seemingly healthy infants can die suddenly and for no obvious reason during their sleep. indeed, the bible makes reference to such an incidence in the first book of kings. one of the first known medical autopsies of an apparent sudden infant death syndrome (sids) case was conducted by samuel fearn and published in the lancet in 1834 (1). in his short communication, fearn asked a simple question that has frustrated generations of doctors and researchers: ‘what was the cause of death?’ over 175 years later the question is still unanswered (2). sids has been the target of an enormous research effort, with over 9,500 scientific articles published on the subject (3), but it remains one of the most mysterious disorders in medicine. our primary research interest, cancer cachexia (4), gave us a singular perspective into sids, and we formulated the sids–critical diaphragm failure (cdf) hypothesis to answer fearn’s original question. it is perhaps surprising that research areas unrelated to sids could contribute meaningfully to the understanding of this complex syndrome, and yet there is a large body of experimental and clinical evidence that suggests that the cause of death in sids is respiratory failure caused by cdf. we argue that four factors contribute to cdf in sids: undeveloped respiratory muscles, non-lethal infections, prone resting position, and rem sleep. infants younger than 6 months have undeveloped respiratory muscles that are susceptible to cdf, because even minor infections can precipitate a significant reduction in the diaphragm force generation capacity. the prone sleeping position significantly increases the diaphragm work-load, and the intercostal muscles that usually provide respiratory support are inactivated in rem sleep. the non-monotonic mortality rate of sids is explained by the passive maternal antibodies that effectively attenuate infections during the first month of life, and rapid development of the infant diaphragm that reaches the same fiber composition and pressure generation capacity as the adult muscle approximately 6 months post partum. correspondence: pontus max axel siren, jgk memorial research library, snellmanin k 15, 00170, helsinki, finland. fax: +358 9 241 40 55. e-mail: pontusmax@hotmail.com (received 2 december 2010; accepted 10 december 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.548011 sids research has identified over 40 potential sids risk factors (5), making the investigation of the syndrome extremely complicated. however, the salient and uncontentious characteristics of sids are well known; it affects fewer than 1 in 1000 live births in the developed world and is associated with sleep, a bell-shaped mortality incidence rate (peak age 2–4 months), premature and low-birth-weight infants, male gender (60% of cases), episodes of non-lethal infections, and the prone sleeping position (6–8). so far, no convincing genetic or congenital mechanism for sids has been identified. sids is by definition a diagnosis of exclusion that has no identifiable cause of death. however, it is generally accepted that sudden death that is not caused by trauma must be due to either respiratory or circulatory failure (9). clinical observations, heartrate, and respiratory recording of infants who subsequently succumb to sids and animal models strongly suggest a respiratory pathway for the syndrome (2). the absence of any obvious defects or abnormalities in the lungs or inspiratory muscles has led to the conclusion that respiratory failure in sids must be caused either by a dysfunctional control system (2,10) or upper airway obstruction (11). however, the dysfunctional respiratory control system hypothesis fails to explain many of the salient features of sids (6), and studies have found no significant respiratory abnormalities in infants who subsequently die of sids (12,13). furthermore, both human and animal studies (14–17) suggest that upper airway occlusion is not a precipitating factor in sids, and obstructive apnea as the cause of sids is arguably doubtful. the respiratory failure hypothesis is still favored by many sids researchers (11), but so far no convincing mechanism has been identified. surprisingly, the possible role of the diaphragm in the etiology of sids has been largely ignored. in october 2010, there were only 41 pubmed hits with the search words ‘sids and diaphragm’ from a total of over 9,500 sids articles (18). yet, the diaphragm is the main respiratory muscle, and as muller and bryan observe ‘respiratory failure is frequently respiratory muscle failure’ (19). diaphragm and survival two muscles are essential for human survival, and while the vital role of the heart is obvious, the diaphragm is often overlooked. the diaphragm is a thin muscle that is shaped like a parachute and that separates the thoracic from the abdominal cavity. it is the main engine of the vital respiratory pump (20), and its contraction is critical for intrathoracic pressure generation (21). anraku and shargall note that ‘in adults, the diaphragm represents less than 0.5% of body-weight, but it is the most important muscle in the human body after the heart’. the incapacitation of supportive respiratory muscles, such as the intercostals, is not lethal, but bilateral diaphragm paralysis usually results in respiratory failure (22). the adult diaphragm can generate indefinitely approximately 40% of its maximum transdiaphragmatic pressure (pdimax) (23), but it is susceptible to critical fatigue in certain conditions. macklem succinctly observes that ‘the inspiratory muscles form a pump just as vital as the heart, and this pump can and does fail in much the same manner as the heart fails’ (20). in adults, respiratory muscle weakness is one of the most common causes of ventilatory failure (24). we argue that the diaphragm is the locus minoris resistentiae in sids. the young infant has undeveloped secondary respiratory muscles, and the diaphragm is responsible for the majority of respiratory work (25). assuming the same relative organ proportions as in the adult, a normal neonate weighing 3000 g would have a diaphragm that weighs approximately 12 g and is approximately 2 mm thick (26). indeed, roughly similar dimensions are observed in preterm infants weighing between 1260 g and 2100 g who have diaphragms that are between 1.09 (±0.08) and 1.74 (±0.04) mm thick (27). an increase in diaphragm size correlates directly with inspiratory force (28), and full-term infants reach adult levels of pdimax at approximately 6 months of age (25). the diaphragm in young infants is also structurally immature and significantly more susceptible to fatigue than a fully developed muscle. a premature infant’s diaphragm consists of only 10%fatigue-resistant type i fibers. the diaphragm of a full-term infant has 25% type i fibers, increasing to 40% at 3 months and to 50%–55% (adult share) (22) by the age of 7–8 months (19).the younger the infant, the smaller the oxidative capacity of the respiratory muscles and the higher the risk for ventilatory muscle fatigue (19). we suggest this may be a reason why low-birth-weight or premature infants have up to four times the risk of sids compared to full-term neonates (8). overall, the respiratory muscles of the young infant appear to be poorly equipped to sustain increased work-loads. muller and colleagues argue that ‘the normal preterm and term infant is very close to the threshold of diaphragmatic fatigue’ (29). indeed, fatigue patterns can readily be observed if the work of breathing is increased in young infants (30). still, some authors have suggested that the infant diaphragm is not at increased risk of fatigue and that it may be even more fatigue-resistant than the adult inspiratory muscle 116 p. m. a. siren & m. j. siren (31). we do not believe this contention is well supported by the existing evidence. however, even those who disagree about the relative fatigue risk of the infant diaphragm agreed that it can and does fatigue under certain conditions. infection-induced acute diaphragm weakness the central argument of the sids–cdf hypothesis is that even minor infections can cause a rapid and significant reduction in diaphragm strength. respiratory muscle performance can decline for two reasons: due to a reduction in total muscle mass or due to a decline in muscle force generation capacity (32). the loss of muscle mass is an incremental process and is usually associated with chronic diseases such as cancer. the reduction of force generation capacity is associated with acute conditions such as sepsis where muscle strength can decline significantly within hours. we propose that an acute decline in muscle forcegenerating capacity is primarily responsible for cdf in sids cases. a large body of evidence strongly suggests that various types of infections, both viral and bacterial, can cause a significant and acute reduction in diaphragm force-generating capacity. importantly, even minor infections can significantly reduce diaphragm strength without affecting muscle mass or histology. acute infections were shown to reduce significantly skeletal muscle strength in 1977 (33). since then, numerous human and animal studies have shown that infections can induce severe diaphragm and secondary respiratory muscle weakness (34,35), whereas the cardiac muscle does not appear to be similarly vulnerable (36). standardized endotoxins are often used to study diaphragm force reduction (37–39), but a broad range of pathogens, both viral and bacterial, has a similar effect. human studies and animal models have shown that significant diaphragm weakness can be induced by endotoxin injection (40), bronchopulmonary infection (41), septic peritonitis (42), parasitic infection (43), and pathogens such as escherichia coli (44), group b streptococcus (45), streptococcus pneumoniae (s) (46), pseudomonas aeruginosa (41), bordetella pertussis (47), phlebovirus (48), as well as factors associated with upper respiratory tract infection (49). a case study of an infant with vulnerable respiratory muscles illustrates the point. the patient exhibited diaphragm muscle dystrophy as the sole anomaly and suffered from episodes of severe respiratory distress that required mechanical ventilation to sustain life (50). interestingly, these episodes occurred only during staphylococcus aureus or haemophilus influenzae infections. the evidence cited above strongly suggests that various pathogens, both viral and bacterial in origin, can precipitate a significant reduction in the ventilatory capacity of the diaphragm. it is well known that systemic infection can lead to severe loss of diaphragm strength and respiratory failure (51). however, also less severe infections can cause a significant reduction in the force-generating capacity of the diaphragm. animal studies have shown that non-lethal sepsis induced by either streptococcus pneumoniae or e. coli with no change in blood pressure, serum electrolytes, or acid status caused a significant impairment of diaphragm function (52). in human adults, mier-jedrzejowicz and co-workers observed already in 1988 that pulmonary function can deteriorate significantly (p < 0.05) after even apparently mild respiratory infections (53). in patients with vulnerable respiratory systems, routine infections have been shown to result in shortness of breath, reduction in vital capacity, and acute hypercapnia (49). supinski and colleagues recently concluded that in vulnerable populations, ‘increasing evidence indicates that even minor infections can produce profound reductions in diaphragmatic force-generating capacity’ (54). a large body of experimental evidence supports this observation (34,41,43,55–58). it is important to note that the degree of diaphragmatic force reduction is determined not only by the intensity of the infectious assault but also by the immune and the developmental status of the host (41,45). fundamentally, terms such as ‘lethal’ or ‘minor’ used to characterize infections are always relative. infections can reducethe force-generatingcapacity of the diaphragm by 50% in as little as 24 hours (54), and lead to severe respiratory muscle dysfunction and respiratory failure (45). in a study on human adults with routine upper respiratory tract infection, diaphragm strength fell significantly (p < 0.01), with the largest decline occurringbetweendays3 and 7 ofclinicalillness (53). full recovery of respiratory pressure took place by day 14. numerous other studies show that a significant reduction in diaphragm strength can occur within 1 to 48 h of infection indicating that a significant decline in the performance of the main respiratory muscle can occur very rapidly (34,41,42,49,51,59). however, infection-induced muscle force reduction is an acute but temporary event, and the diaphragm can recover its normal function relatively quickly. this may explain why near-sids cases do not manifest diaphragmatic weakness (60). animal models using either streptococcus pneumoniae or e. coli endotoxin show that non-lethal sepsis impairs diaphragm function without affecting muscle mass or histology (46,61). supinski and callahan have observed that ‘24 h after administration of endotoxin, diaphragm specific force had fallen by 50% without any reduction in diaphragm muscle mass critical diaphragm failure in sudden infant death syndrome 117 or diaphragm protein content, obvious depletion of major diaphragm protein bands or specific loss of myosin or actin stores’ (57). sepsis reduces muscle strength long before contractile protein levels decline significantly (34). others have reported similar results (62,63), confirming that even though infections can cause acute and severe diaphragm dysfunction, they leave few, if any pathological marks in the affected tissue. these findings are of some importance, because the absence of significant pathophysiological abnormalities in the inspiratory musculature of sids cases has led to the erroneous conclusion that the diaphragm does not play a role in the etiology of the syndrome (10). the one pathological finding that characterizes over 70% of sids cases is intrathoracic petechiae (64). goldwater reported recently that sids cases demonstrate intrathoracic petechial hemorrhages in the thymus (89.5%), pleura (80%), and epicardium (79.9%) (16). sids-related intrathoracic petechiae are not characteristic of asphyxia, strangulation (16), upper airway obstruction (17), or cardiac arrest (64). rather, experimental evidence suggests that an infection together with gasping or a ‘struggle to breathe’ episode is required for the formation of characteristic intrathoracic petechial hemorrhages (17,65). interestingly, the clinical signs of respiratory muscle fatigue are rapid shallow breathing, paradoxic chest wall/ abdominal movements, and respiratory pauses combined with hyperventilation (20,66). the data presented above strongly suggest that even minor infections can cause a significant and rapid reduction in diaphragm force-generating capacity without affecting muscle mass or histology. surprisingly, the pathophysiological implications of infection-induced acute diaphragmatic weakness have not been previously considered in the context of sids even though it is well known that young infants have undeveloped and potentially vulnerable respiratory muscles. mitochondrial dysfunction and the role of melatonin in sids the molecular mechanisms behind infection-induced acute diaphragm weakness have been actively investigated. systemic infection displays consistent similarities to the pathogenesis of sids (67), and can induce a significant (p < 0.001) reduction in the mitochondrial function (e.g. atp-generating capacity) of the diaphragm muscle tissue (68). there is substantial evidence that excessive free-radical generation plays a central role in infection-induced myopathy and causes mitochondrial impairment. infection causes significant changes in oxidative phosphorylation and induces the selective depletion of several electron transport chains in the respiratory muscles (32). newborns and infants born prematurely are especially prone to oxidative stress because they are exposed to high oxygen concentrations and have reduced antioxidant defense mechanisms and high levels of free iron that are required for the fenton reaction (69). existing evidence strongly suggests that infections can produce rapid and profound alterations in the mitochondrial function of the diaphragm and severely disrupt its energy metabolism. melatonin (n-acetyl-5-methoxytryptamine) is a highly conserved small amphiphilic molecule that is biosynthesizedfrom tryptophan.serotonin isconverted to melatonin in the pineal gland by arylalkylamine n-acetyltransferase (aa-nat) and hydroxyindole o-methyltransferase (hiomt) (70). serotonin is a rate-limiting factor for melatonin. melatonin has been shown to protect against oxidative stress in various divergent experimental systems (71). it is a highly effective antioxidant and free-radical scavenger that can significantly attenuate mitochondrial failure and preserve cell function and survival (72–74). in the clinical setting, melatonin has been shown to improve the clinical outcome in the septic newborn (75). gitto and colleagues observe that ‘several clinical studies that used melatonin showed that it reduces oxidative stress in newborns with sepsis, distress or other conditions where there is excessive ros/rns (reactive oxygen species/reactive nitrogen species) production’(69). however, young infants exhibit transient melatonin deficiency for the first 2–4 months of life (76). pineal dysfunction and impaired melatonin metabolism have been associated with sids (77,78). sturner and colleagues examined melatonin levels from the ventricular cerebrospinal fluid (csf) in sids deaths compared to non-sids cases. after adjusting for age differences, melatonin levels were significantly (p < 0.05) lower among the sids infants (91 ± 29 pmol/l; n = 32) than among those dying of other causes (180 ± 27; n = 35) (79). young infants who experienced a life-threatening event (alte) demonstrated significantly (p < 0.05) lower urinary excretion of the main melatonin metabolite 6-sulfatoxymelatonin compared to controls (1588 ng/24 h versus 3961 ng/24 h) (80). serotonergic deficiency has repeatedly been associated with sids (81), suggesting that the role of the tryptophan–serotonin– melatonin pathway in the etiology of sids should be further investigated. the prone sleeping position and sids a change in the recommended sleeping position for young infants from prone to supine has reduced 118 p. m. a. siren & m. j. siren sids deaths worldwide between 40% (argentina) and 83% (ireland) (2). sids can occur in the supine resting position, but any hypothesis regarding the etiology of sids must convincingly explain why the prone sleeping position is such a significant risk factor. the effects of the prone sleeping position on diaphragm function have been considered previously (82). the hypothesis that the prone sleeping position significantly increases the work-load of the diaphragm was tested by rehan and co-workers in a study with 16 healthy infants (83). the study showed that in the prone position the diaphragm is significantly thicker and, therefore, shorter at the end of both expiratory (eev) and inspiratory lung volumes (eiv). the shortening of any muscle produces a marked fall in the maximal tension it can develop (19), indicating that the inspiratory muscle force of the diaphragm decreases as it shortens. indeed, rehan and colleagues note that ‘this degree of diaphragm shortening is similar to that seen with an increase in lung volume of 15%–30% of vital capacity’. such increases in eev can significantly impair diaphragm performance, and, as rehan and colleagues conclude, ‘in adults, diaphragm strength and endurance as well as the efficiency of breathing are reduced by 40%–50% with this magnitude of increase in lung volume’. greenough and colleagues have reported similar results in several clinical studies with premature infants and observe that respiratory muscle strength is significantly reduced in the prone compared to the supine position in (106–108). interestingly, an earlier study that measured infant breathing did not find significant differences in ventilatory performance as a function of sleep position (84). however, the study reported a significant increase (+66%) in rib-cage motion in the prone compared with the supine position. others have observed that due to the high rib-cage compliance in young infants, the thorax motion increases either during deep inspiratory effort or when the intercostals are inactivated (31). as both the sleep position studies were conducted during non-rem (nrem) sleep when the intercostals are active, it is possible that the significantly increased thoracic motion in the prone position was due to increased diaphragm work needed to maintain ventilation pressure at the same level as in the supine position. indeed, in another study with healthy infants sleeping in the prone position, chest wall distortion during rem sleep led to the reduction of the tidal volume, but ventilation was upheld by significantly (p < 0.01) increasing the work-load of the diaphragm (85). we suggest that healthy infants with normal diaphragms can easily tolerate the added mechanical strain that the prone position imposes on the diaphragm. however, for some infants the prone sleeping position combined with an infectious episode can increase the risk of cdf. why is sids associated with sleep? sids deaths often take place between midnight and 6 a.m. (5,86), and there is a close temporal relationship between sids and sleep (87). the original name for sids, ‘cot death’, reflects this basic observation. the reason for the preponderance of sids deaths during sleep is unclear, and while we should remember that newborns sleep up to 20 hours per day, there seems to be a causal link between sleep and sids. a premature infant spends up to 80% and a 3-monthold approximately 38% of his sleep in the rem phase (19). it is well known that the intercostals muscles show both phasic and tonic inhibition during rem sleep that renders them largely or totally inactive, and that this has a significant effect on respiratory function (31,88–90). the intercostals play a central role in stabilizing the thorax, and, as davis and bureau note, ‘chest wall muscles are critical for ventilation in the infant with a pliable chest wall’ (66). others have suggested that the inactivation of the intercostals during rem sleep may be a contributing factor in sids (82). we argue that the inactivation of the intercostal muscles in combination with other factors can increase the risk of cdf. the internal and external intercostal muscles are activated to attenuate diaphragm fatigue (19,91,92). in a study with preterm infants, diaphragm fatigue was followed either by a 50%–150% increase in intercostal activity or apnea that lasted 10–30 s and often required stimulation (93). in full-term infants, diaphragm fatigue provokes similar responses: the intercostal muscles are activated to support the respiratory effort or apnea lasting 5–20 s ensues (followed by intercostal muscle activation) (91). in another study, the diaphragmatic work-load in the young infant increased by over 150% (p < 0.001) during rem sleep (29). diaphragmatic fatigue patterns were observed only in rem sleep together with marked reductions in intercostal activity and rib-cage retraction. in the young infant intercostal muscle support is even more important than in the adult because their rib-cage is mostly cartilaginous and significantly more compliant compared to adults. if the intercostals do not stabilize the rib-cage during inspiration, a significant portion of the power generated by the diaphragm will be wasted sucking in the ribs rather than fresh air (94), and to maintain the same tidal volume the work of the diaphragm has to increase substantially (88). critical diaphragm failure in sudden infant death syndrome 119 this increased respiratory work-load may contribute to diaphragm fatigue. the studies discussed above suggest that rem sleep is associated with increased diaphragmatic work-load and respiratory muscle fatigue in normal infants. importantly, a fatigued diaphragm can be relieved only by decreasing the respiratory workload or by activating the intercostals. if the critically fatigued muscle is not supported by the secondary respiratory muscles, ventilatory efficiency and muscle oxygenation will progressively decline and the risk of respiratory failure increases. the bell-shaped mortality rate in sids one of the more puzzling aspects of sids is the bell-shaped mortality incidence rate. sids seems closely related to the developmental status of the infant, because 95% of the cases occur before the age of 6 months, and sids is rare in infants 1 year and older. paradoxically, sids is also rare in infants younger than 1 month (5). sids clearly has a different type of mortality incidence rate compared to the monotonic distribution in congenital anomalies (6). others have argued that bacterial toxins cause sids and that maternal antibodies can protect the infant during the first months of life (95). we suggest that the bell-shaped mortality distribution of sids is due to the passive immune protection maternal antibodies provide during the first month of life when the respiratory muscles are most vulnerable. the age profile of sids deaths is reciprocal to infants’ serum concentrations of immunoglobulin (96), and maternal antibodies can increase the immune resistance of neonates during the first months of life (97,98). infants have varying levels of passive immune protection because the maternal antibody concentration determines the neonatal titer at birth (99). full-term infants seem to have higher antibody titers than premature ones (100). however, the passive immune protection wanes relatively rapidly in all neonates. a study with 213 mother–infant pairs showed that the mean time to immunity loss for rubella and varicella was 2.1 months and 2.4 months, respectively (101). for measles the maternal antibodies endured for a median of 2.6 months (99). maternal antibodies to the varicellazoster virus (vzv) had a median half-life of 25.5 days in neonates (range 14.6–76.0 days) (102). another study showed that 26% of infants had potentially protective antibody levels against pertussis (whooping cough) at delivery, but by week 6 nearly 90% had lost most of the original maternal antibodies (103). these and other studies (104,105) suggest that neonates can benefit from passive maternal immune factors for 1–2 months post partum but that the temporary immune protection wanes relatively quickly. concluding remarks we propose that the cause of death in sids is respiratory failure caused by cdf. it is well known that young infants have potentially vulnerable diaphragms and that infections can produce severe respiratory muscle dysfunction. we argue that ‘non-lethal’ infections are the main cause of cdf in sids. as such, there is no specific underlying genetic or congenital vulnerability in sids infants, and completely normal infants can die of sids. however, infants with weak or underdeveloped respiratory muscles who spend a large portion of the day in rem sleep and are exposed to infections are at increased risk. dysfunctional melatonin metabolism may also be a contributing factor in sids. by definition, the infections associated with sids are not considered to be lethal, but they can cause a significant and acute loss of diaphragm strength. additional stressors such as the prone sleeping position and rem sleep can move the infant closer to cdf and precipitate respiratory failure. diagnosing cdf-precipitated respiratory failure is challenging because it usually does not affect muscle mass or histology. an obvious question that the sids–cdf hypothesis might raise is that if young infants have vulnerable respiratory muscles and even minor infections can cause significant loss of diaphragm strength, why is sids not more common? diaphragm fatigue is not the same as diaphragm failure, and while young infants are susceptible to cdf, they also have effective defense mechanisms that help preserve respiratory function. first, the immune system actively attenuates the negative effects of infectious assaults. second, the activation of the intercostal muscles can decrease the respiratory burden and support a fatigued diaphragm. third, the diaphragm develops rapidly, and by age 6 months has the same distribution of fatigue-resistant fibers and pressure-generating capacity as the adult muscle. thus, moving from diaphragm fatigue to cdf usually requires a relatively rare combination of events that include an infectious episode, insufficient immune response, undeveloped respiratory muscles, continuous rem sleep, and often a prone resting position. even if all these factors are present, the fatigued diaphragm may be rescued by altering the sleep state and activating the secondary respiratory muscles. the clinical impact of an infection is dependent on its intensity as well as the immune and developmental status of the host. a potent infection can alone be lethal to a young infant, but a less severe infection will 120 p. m. a. siren & m. j. siren be dangerous only in conjunction with other factors. yet, diagnosing an apparently ‘non-lethal’ infection as the cause of death is by definition a contradiction in terms. in a sense, a sids diagnosis reflects the failure to understand that apparently ‘minor’ infections can be fatal to some infants in certain circumstances. if the causes of infant mortality can be accurately ascribed, sids may no longer be needed as a diagnosis.this goal should be pursued in the spirit of fearn’s original question. acknowledgments the authors dedicate this work to the memory of pirkko and jorma gallen-kallela. we also wish to acknowledge the critical role of information technology in enabling and empowering the revolution in global collaborative innovation. our research would not have been possible without it. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. fearn sw. sudden and unexplained death of children. lancet. 1834;23:246. 2. kinney hc, thach bt. the sudden infant death syndrome. n engl j med. 2009;361:795–805. 3. pubmed search 15 october 2010. 4. siren pma, siren mj. systemic zinc redistribution and dyshomeostasis in cancer cachexia. j cachexia sarcopenia muscle. 2010;1:23–33. 5. sullivan fm, barlow sm. review of risk factors for sudden infant death syndrome. paediatr perinat epidemiol. 2001;15: 144–200. 6. guntheroth wg, spiers ps. the triple risk hypotheses in sudden infant death syndrome. pediatrics. 2002;110:e64. 7. mitchell ea. sids: past, present and 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freeze-all, for whom, when, and how paula celada and ernesto bosch instituto valenciano de infertilidad, valencia, spain abstract background: the ‘freeze-all’ practice refers to the cryopreservation of all mature oocytes or viable embryos after ovarian stimulation. the development of the vitrification technique has been crucial to make this approach a reality, since it increases the post-thaw survival rates and permits comparable implantation rates with fresh embryos. nonetheless, as implantation probabilities are comparable to fresh embryo transfer in normo-responder patients, the freezeall strategy has demonstrated no benefits overall. method: narrative review in which we give an overview of this approach, discuss recent advances in the field, as well as for whom, when and how it is recommended to emply the freeze-all technique. results: however, there is some clinical evidence that shows its feasibility. thus, it has been demonstrated that elevation of progesterone at the end of ovarian stimulation decreases the implantation rates after the transfer of day 6 blastocysts in fresh and some uterine pathologies; freeze-all is also the preferred option for patients undergoing pre-implantation genetic testing, since there is an improvement of the results and it allows for inclusion of all blastocysts of the cohort. in high responders, the freeze-all strategy optimizes the response whilst also minimizing the risk of ovarian hyperstimulation syndrome. conclusion: due to the different cases that a reproductive expert might encounter, it is essential to highlight benefits and drawbacks of this practice. article history received 29 january 2020 revised 4 march 2020 accepted 19 march 2020 keywords controlled ovarian stimulation (cos); cryopreservation; embryo implantation; freeze-all; fresh embryo transfer; frozen embryo transfer (fet); in vitro fertilization (ivf); segmentation introduction the success of embryonic implantation relies on a perfect synchrony between embryo status and endometrial receptivity. this process involves uterine, embryonic, and environmental factors that can contribute to a healthy uterine microenvironment (1). improvements in assisted reproduction techniques (art) allow for the development of new tools and technologies to preserve the safety and optimize treatment results. the ‘freeze-all’ practice refers to the cryopreservation of all mature oocytes or viable embryos after ovarian stimulation (os). its application was primarily illustrated in protocols to avoid ovarian hyperstimulation syndrome (ohss) by delaying implantation (2,3). the term ‘segmentation’ of in vitro fertilization (ivf) treatment was used by elective cryopreservation and postponed embryo transfer pioneers to outline the process where ovarian stimulation and oocyte/embryo retrieval are separated from the process of embryo transfer (2). recently, elective frozen embryo transfer (efet) emerged as an improved term that better describes the entire process (4). in recent years, ivf segmentation, i.e. the ‘freeze-all’ strategy, has been proposed to increase cycle outcomes by avoiding the transfer of embryos in the same os cycle, since the endometrium could be less receptive than in natural or artificial endometrial preparations. several studies performed in recent years have suggested that transferring embryos in a later natural or artificial cycle to an endometrium that has not been exposed to high doses of exogenous gonadotropins could be a suitable approach (5–9). indeed, the aforementioned reports have observed that the clinical pregnancy rate per transfer increases in the cryopreservation group. this strategy is available due to the improvement of cryopreservation technology in the last decade, which has reached the extent of 80–100% post-thaw survival rates and comparable implantation rates with fresh embryos (10–12). freeze all for all strategy several studies have been published to support the ‘freezeall’ procedures as a possible strategy in all ivf cycles (10). however, even though there might be a benefit from using this approach, the evidence available does not justify a change. a careful analysis of previous studies shows that these data have a number of limitations. in most cases, the patients included highlighted a trend to high ovarian response, which is associated with higher risk of ohss, and higher probability of having high oestradiol and progesterone levels at the end of stimulation. contact ernesto bosch ernersto.bosch@ivirma.com instituto valenciano de infertilidad, plaza policia local n� 3, valencia, 46015, spain � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 2, 104–111 https://doi.org/10.1080/03009734.2020.1746870 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1746870&domain=pdf&date_stamp=2020-05-21 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1746870 http://www.tandfonline.com in the study by shapiro et al. (7), 70 patients that had submitted to elective cryopreservation showed a significantly better ongoing pregnancy rate (opr) than the 67 who underwent fresh embryo transfer. however, several limitations and biases have been put forward regarding the small number of patients, the high pregnancy rates in the cryopreservation cycles, and the presence of dual triggering (referred as the effect of co-interventions). in a much larger study, roque et al. (13) observed a significantly higher opr in the ‘freeze-all’ cycles than in fresh transfers. however, the study groups were not comparable as patients that received the ‘freeze-all’ approach showed a significantly greater ovarian response and higher progesterone levels on the day of triggering (1.66 ± 0.14 versus 0.70 ± 0.27). this suggests that these patients were subject to this strategy to avoid the negative impact of high progesterone on endometrial receptivity. therefore, the benefit of the elective cryopreservation has been verified in studies including high responders. roque et al. (13) also compared outcomes between fresh embryo transfer and freeze-all cycles in correlation to the number of retrieved oocytes. in the group with 4–9 retrieved oocytes there were no differences in opr between the fresh and freeze-all group (31% and 33%, respectively). however, comparing the fresh and freeze-all groups, a better outcome was obtained in the freeze-all group when large numbers of oocytes were retrieved (34% and 47%, respectively). this may be taken to indicate that when highresponse patients are excluded, there is no benefit of a freeze-all strategy. these results are in agreement with a retrospective study performed in our centre comprising a cohort of 882 women aged 20–44 years undergoing their first or second ivf/icsi cycle. our study highlighted no benefit on live birth rates (lbr) of freeze-all versus fresh transfer in normo-ovulatory women undergoing ivf (14). we excluded patients with a risk of ohss, high responders, and women with high progesterone levels on the day of trigger, since these subgroups have had improved outcomes with frozen embryo transfer (fet). there were no differences between fet and fresh embryo transfers in normal responders (4–20 oocytes) regarding implantation, clinical and ongoing pregnancies, and live births (14). similar results were highlighted recently in two large randomized controlled trials (rct) (15,16). these studies showed that there is no benefit in lbr of the freeze-all strategy when comparing it with fresh embryo transfer in normo-ovulatory women undergoing ivf. more importantly, the chinese study also displayed that the risk of moderate or severe ohss was significantly decreased in the freeze-all group (15). in recent years, 11 randomized trials including 5379 patients who underwent ivf/icsi were analysed in a meta-analysis (4). when the different subgroups were scrutinized, lbr were positively affected only in hyper-responders and in pre-implantation genetic testing (pgt-a) cycles. there was no difference in lbr in normo-responders. when to freeze-all pgt-a programmes recent developments in ivf practices such as extended embryo culture, vitrification, and trophectoderm biopsy, in combination with recently introduced technologies in pgt-a, have improved opr as regards selective transfer of euploid blastocysts (17). two transfer strategies for euploid embryos currently used in clinical environments are the employment of vitrified/warmed (‘freeze-all’) or fresh embryos for the first embryo transfer (et). the freeze-all strategy involves cryopreservation of all embryos after biopsy, while performing a pre-implantation genetic screening (pgs) of the whole cohort (day 5 and day 6 embryos) in preparation for a frozen embryo transfer. the fresh strategy requires expanded blastocysts to be available on the morning of day 5, and culture overnight to await pgs results for a fresh embryo transfer of euploid embryos before noon on day 6. however, there is a negative impact of controlled ovarian stimulation on embryo–endometrium synchrony when transferring embryos on day 6 in a fresh autologous cycle. this is due to the impaired implantation of day 6 blastocysts in fresh transfers (8). in this context, the freeze-all strategy seems to be more suitable as it allows time for pgt-a results to reach the clinician, and the transfer of a euploid embryo would be performed in a subsequent cycle. this hypothesis was confirmed in a rct in which 179 patients underwent ivf treatment and pgt-a (18). opr (80% versus 61%) and lbr (77% versus 59%) were significantly higher in the frozen group compared with the fresh group. thus, freeze-all is the preferred option for patients undergoing pgt-a, since there is an improvement of the results and it allows inclusion of all blastocysts of the cohort. ohss prevention ovarian hyperstimulation syndrome (ohss) is a lifethreatening complication of ovarian stimulation in ivf cycles. elective cryopreservation of all embryos and their subsequent transfer in non-stimulated cycles may be employed to avoid the endogenous hcg increase in fresh transfer cycles, preventing the risk of ohss. one rct of 125 patients showed that after cryopreservation there was a lower incidence of ohss than in controls with fresh embryo transfers (0 events versus 4 events, respectively) (19). another large rct performed in polycystic ovarian syndrome (pcos) patients (n ¼ 1508) confirmed that the frequency of ohss was significantly lower in freeze-all and subsequent fet cycles versus fresh transfer cycles (1.3% versus 7.1%) (20). furthermore, lbr after the first embryo transfer was higher with this strategy when compared with fresh embryo transfer (49.3% versus 42.0%) in pcos patients. indeed, these studies support the recommendation of the american society for reproductive medicine, which states that the freeze-all strategy in high responders optimizes the response whilst also minimizing the risk of ohss (21). upsala journal of medical sciences 105 elevated progesterone during the last 30 years it has been discussed if the success of art could be influenced by the increase in serum progesterone concentrations during ovarian stimulation. there is evidence to suggest that there is a decrease in implantation rates following fresh embryo transfer (22–24), but also studies reporting no association (25,26) it has been repeatedly demonstrated that elevated progesterone negatively affects implantation by impairing endometrial receptivity (27). however, the association between elevated progesterone and embryo quality is still a matter of debate. while it has been generally accepted that there is no negative impact on oocyte quality or opr in recipients of donated oocytes (23,28), a recent retrospective study of 3400 cycles has postulated that there may be a detrimental effect (29). this study concluded that high serum progesterone concentrations at the end of the follicular phase are associated with a decrease in embryo utilization rates and cumulative live birth rates (clbr) after both fresh embryo transfer and use of the freeze-all strategy. the negative influence that elevated progesterone may have on clbr suggests that the freeze-all strategy is insufficient to solve the problem. day 6 blastocysts as mentioned above, there is an impaired implantation of day 6 blastocysts in fresh transfers. however, if day 6 blastocysts are transferred in a freeze–thaw cycle it is more likely to result in pregnancy with no differences of opr between day 5 and day 6 blastocysts (8,30). the embryo–endometrium asynchrony might primarily be implicated in the impairment, with no effect on rapidly developing (day 5) blastocysts and a higher one in (day 6) blastocysts. uterine pathology there are different uterine pathologies that can be diagnosed during ovarian stimulation. they have all been associated with a decrease in fertility. polyps some studies suggest that polyps have a negative effect on fertility because they affect endometrial receptivity (31). if an endometrial polyp is identified during infertility evaluation, hysteroscopic polypectomy prior to treatment is recommended. nonetheless, various strategies have been considered in the case of polyp detection during art. these include embryo cryopreservation, hysteroscopy, and then transfer in a subsequent cycle (32). adenomyosis a recent meta-analysis showed that adenomyosis seems to have a negative impact on art outcome (33). depot gnrha for 3–6 months, administered alone or in combination with cytoreductive surgery, has been the most applied approach; however, there is poor evidence on the specific outcome in art after such treatment. a recent study showed that vitrified embryos and transfer after treatment with a gnrh agonist tended to increase the pregnancy rates (34). hydrosalpinx some meta-analyses have demonstrated impaired pregnancy outcomes in patients with hydrosalpinx. salpingectomy before embryo transfer improved their chance of achieving a birth after ivf treatment (35). random-start ovarian stimulation during the menstrual cycle, two waves of follicular growth may occur (36). new strategies for ovarian stimulation and, in particular, the random-start ovarian stimulation protocols have followed this new perspective on ovarian function. at present, there are few reports on the efficacy of random-start protocols. preliminary results highlighted similar rates of total numbers of oocytes, as well as metaphase ii oocytes obtained and fertilization rates in early follicular and random-start protocols (37). furthermore, there was no difference in the probability of achieving an euploid blastocyst (38). likewise, improvements of embryo and oocyte vitrification have permitted the development of new ideas such as total ‘disarticulation’ between ovarian stimulation and embryo transfer. in cancer patients, random start is currently performed to minimize delays between ovarian stimulation and cancer therapies, with no difference in oocyte yield between conventional and random-start protocols (39). another approach is the dual stimulation. this strategy can be useful in patients with poor ovarian response in order to save time by continuing ovarian stimulation after the first oocyte retrieval, thereby performing two stimulations in the same cycle (38,40). in the event of a ‘non-conventional start’ stimulation, all the oocytes/embryos need to be cryopreserved and transferred subsequently, due to the asynchrony between endometrial receptivity and embryo development. optimizing preservation methods of preservation slow freeze and rapid thaw techniques in cryopreservation made the first successful pregnancies from frozen embryos a reality. however, the formation of crystals due to the solidification of water is the main issue together with altered intracellular morphology. indeed, these technical aspects led to low success rates, and the improvement of elements related to cryopreservation did not improve the freeze-all approach. a crucial step in the field was the development of the vitrification technique, which combines ultrarapid cooling in combination with cryoprotective agents to increase viscosity and decrease the freezing point of the environment. vitrification was found to be effective regardless of the developmental stage of the embryo (12); nonetheless a study by kuwayama 106 p. celada and e. bosch et al. (41) described the cryotopv r , which was later accepted as an efficient approach. vitrification is considered to be superior to slow freezing, and it is now an established protocol for art (42). endometrial preparation for fet adequate endometrial preparation is mandatory for the success of fet. it is still debated which is the best protocol to prepare the endometrium. fet preparation methods can be divided into artificial and natural cycles. in artificial cycles, endometrial proliferation is achieved by oestrogen supplementation. in natural cycles, endogenous oestrogens secreted during the follicular menstrual cycle enhance the development of the endometrium. natural cycles can be achieved with spontaneous ovulation or with ovulation induction. recent reviews and meta-analyses concluded that there is no difference in lbrs following different methods of endometrial preparation for fet (43,44). it is worthy of note, however, that these data are derived predominantly from retrospective studies. in this scenario, the superiority of one protocol over another should be accepted only after performing prospective randomized studies. hormonal replacement treatment (hrt) or artificial cycle to achieve a receptive endometrium, hrt aims to mimic the natural cycle preparing the endometrium in two stages. the first step employs oestrogens to mimic the follicular phase of a natural cycle. in the second step, progesterone is added to oestrogen to mimic the luteal phase. the initiation of orally administered exogenous oestrogen on day one of the cycle is performed to suppress follicle growth and spontaneous ovulation. oestradiol supplementation also results in adequate endometrial preparation. oestrogens may be administered orally, vaginally, and parentally (transdermal route). a cochrane systematic review concluded that the type of oestrogen supplementation and route of administration had no effect on the success rates of fets (45). different oestradiol supplementation schedules have been developed. the most commonly reported optimal doses vary between 4 and 12 mg/d (44). quite in contrast, progesterone administration and dosage are less standardized. some retrospective studies suggest that the route of administration of progesterone is without impact (45,46). however, a recent rct showed that vaginal progesterone alone (200 mg every 12 h) was inferior to protocols containing intramuscular progesterone (47). one retrospective cohort study of 346 women who underwent hrt fet concluded that doubling the standard dosage of progesterone vaginal gel 90 mg (crinone) significantly increased lbr (48). measuring serum progesterone concentrations has received increased interest in recent years. yovich et al. (49) found an optimal mid-luteal progesterone range between 22 and 31 ng/ml (70 and 99 nmol/l). concentrations of progesterone below 22 ng/ml and above 31 ng/ml were associated with decreased implantation rates. a recent prospective study in our centre confirmed that low serum progesterone concentrations on the day of transfer were associated with lower opr (50). interestingly, there was a wide range of progesterone concentrations, even though all of these patients received the same regimen of progesterone (400 mg/12 h from 5 days before embryo transfer). the results revealed a decrease of 20% in opr in women with serum progesterone concentrations lower than 9.2 ng/ml (29 nmol/l) on the day of embryo transfer. recently, alsbjerg et al. (51) obtained results comparable with the study from labarta et al. (50) described above. the alsbjerg group found a decrease of 14% in opr when progesterone was below 35 nmol/l (11 ng/ml) (table 1). in hrt fet cycles there is no corpus luteum and, hence, no endogenous progesterone production. if a pregnancy occurs, oestrogen and progesterone must be continued until placental autonomy is established to replace the absent corpus luteum. natural cycle exposure to oestrogen and progesterone is a consequence of spontaneous follicle development and ovulation. this method is available only for patients with an ovulatory cycle. to assess embryo–endometrial synchronization it is essential to monitor the cycle with several pelvic ultrasound scans to confirm follicular development and ovulation timing. in a natural cycle, ovulation can occur physiologically by the spontaneous onset of a lh surge (natural cycle) or programmed by triggering ovulation exogenously by an injection of hcg (modified natural cycle). the necessity for luteal phase support is still under debate. for natural cycles a rct demonstrated a significantly higher lbr in the group receiving vaginal progesterone (400 mg twice a day from the day of embryo transfer) compared with those who received no progesterone support (52). for modified natural cycles, the results of several table 1. comparison of optimal serum progesterone concentrations (nmol/l) for cryopreserved embryo transfers in artificial cycles in different studies. study patients included luteal phase support p4 measurement optimal p4 values pregnancy outcomes under and over the cut-off yovich et al. 2015 (49) 529 vaginal micronized progesterone (400 mg/8 h) 2–3 days after embryo transfer >50 nmol/l; best range: 70–99 nmol/l cpr: 44% (<50 nmol/l) versus 64% (70–99 nmol/ l) (p ¼ 0.005) labarta et al. 2017 (50) 244 vaginal micronized progesterone (400 mg/12 h) day of embryo transfer >29 nmol/l opr: 32.7% versus 52.8% (p ¼ 0.016) alsbjerg et al. 2018 (55) 244 vaginal micronized progesterone (90 mg/8 h) 9–11 days after embryo transfer >35 nmol/l opr: 44% versus 58% (p ¼ 0.02) cpr: clinical pregnancy rates; opr: ongoing pregnancy rates; p4: progesterone. upsala journal of medical sciences 107 studies are conflicting, and there is considerable heterogeneity. both prospective (53) and retrospective (54) studies failed to show any difference in terms of pregnancy outcome with or without progesterone support. however, another retrospective study suggested that luteal phase progesterone supplementation decreases the miscarriage rate and improves lbr (55). safety of cryopreservation of embryos analyses of reproductive cryopreservation outcomes such as effects on pregnancies and on neonates highlight several consistent findings. perinatal outcomes large for gestational age (lga) and high birth weight some studies and meta-analyses have reported lga after fet even when considering maternal age and birth order (56–58). large epidemiological studies reported an increased risk of higher birth weight (birth weight >4000 g) and very high birth weight (birth weight >4500 g) in babies born after fet when compared with those born after fresh embryo transfer (59–61). the meta-analysis of maheshwari et al. (62) confirmed these results for high birth weight (rr 1.85; 95% ci 1.46–2.33) and very high birth weight (rr 1.86; 95% ci 1.58–2.19). whether the higher risk of lga is related to the freezing/ thawing procedure per se or if other factors are involved remains unknown. maternal bmi and parity were found to have a significant effect on the birth weight (63). pinborg et al. (56) explored the risk of babies with lga in a fet/fresh sibling cohort. the study suggested that children born after fet are at increased risk of lga, even in the same mother. therefore, this cannot be explained considering intrinsic maternal factors only. aspects associated with the freezing/ thawing procedures need to be considered as well. to what extent there is an association between long-term in vitro culture and lga remains unclear. nevertheless, m€akinen et al. (63) described such an association when analysing the birth weight of the children and the length of embryo culture. however, wikland et al. (64) found no increased prevalence of lga after transfer of vitrified blastocysts compared with slow-freeze cleavage stage transfer. the duration of the cryostorage of the vitrified blastocysts does not appear to affect pregnancy and neonatal outcomes (65). nonetheless, the physiological mechanisms associated with the increased birth weight observed after fet need to be elucidated more in detail. recent evidence pointed out a possible epigenetic regulation of the cryopreservation process itself. as an example, genome-wide analysis has revealed differentially expressed mirnas in fet placentae compared with placentae from fresh embryo transfers potentially involved in birth weight increase and perinatal complications (66). small-for-gestational age and low birth weight it has been shown that there is a lower risk to be small for gestational age for babies born after fet compared to those born after fresh embryo transfer (rr 0.61; 95% ci 0.56–0.67) (62). more than 20 studies showed that there is also a decrease of the probability of low birth weight (less than 2500 g) in babies born after fet (62). preterm delivery (delivery at less than 37 weeks) there are several studies showing that babies born after fet possess a lower risk of prematurity (58–61). the recent metaanalysis of maheshwari et al. (62) confirmed these results, showing a reduction on relative risk of prematurity (rr 0.90; 95% ci 0.84–0.97). others as regards some neonatal outcomes such as antepartum haemorrhage, admission to the neonatal intensive care unit, congenital abnormalities, and perinatal mortalities, there are no differences reported between frozen and fresh transfer strategies (62). obstetric outcomes studies from sweden (57) and japan (60) highlighted the increased risk of pregnancy-induced hypertension and preeclampsia in singleton pregnancies following frozen–thawed cycles compared with fresh cycles and spontaneously conceived pregnancies. a large study in a nordic population revealed a consistently higher risk of hypertensive disorders in pregnancies after fet even when compared with fresh cycle pregnancies in the same mother (67). this may be taken to indicate that the association cannot be attributed only to maternal factors. however, when fet was performed in the natural cycle there were no differences in pre-eclampsia or hypertensive disorders between fet and fresh embryo transfer (15). this suggests that the endometrial preparation protocol might have an impact on the obstetric outcomes (4). cost-effectiveness of fet even though the quality of frozen embryos and the implantation probabilities are comparable to fresh embryo transfer (12,68,69), the overall cost of fertility treatment must be considered since fresh transfer is immediate. there is a need to consider the absence of direct costs of cryopreservation, additional medication, and subsequent fet cycles (18). roque et al. (70) have shown that the freeze-all approach has a lower cost than fresh transfer by analysing two scenarios in a cost-effectiveness analysis. another study by papaleo et al. (71) observed no cost difference between the freeze-all approach and fresh blastocyst transfer per live birth. the authors concluded that the cost similarity is due to insignificant additional expenses such as vitrification, endometrial priming, and monitoring versus fewer embryo transfers needed to obtain pregnancy. when studying normal responder patients, a decision tree mathematical model showed that a single freeze-all cycle 108 p. celada and e. bosch possessed an increased cost effectiveness when compared with a single fresh cycle even with the addition of a secondary supernumerary fet (72). on the other hand, in non-pcos women undergoing ivf/icsi, it was highlighted that there is a similar average cost per couple between the freeze-all approach and the fresh embryo transfer. from a patient perspective, other factors might be crucial to decide on a freeze only or fresh embryo transfer (73). conclusions the availability of a freeze-only strategy in the armamentarium of ivf provides a number of opportunities to optimize the overall outcome in several every-day situations in daily practice. while the indiscriminate use with the goal to improve lbr in unselected populations cannot be proven, the appropriate indication of this strategy makes it possible to overcome a handful of obstacles that could lead to suboptimal results in terms of efficacy and safety. it is crucial that every practitioner knows and understands these indications, in order to provide the best possible health care (figure 1). whether future findings will change the current picture is unknown. it cannot be discounted that new insights particularly related to obstetric and perinatal outcome may lead to a decrease in the use of freeze-only, or to moving towards the natural cycle for endometrial preparation. disclosure statement the authors declare no competing financial interests. notes on contributors ernesto bosch is the director of ivi valencia, spain. in january 2000 he joined the team at the human reproduction unit of the instituto valenciano de infertilidad (ivi) in valencia; 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31:1610–20. 66. hiura h, hattori h, kobayashi n, okae h, chiba h, miyauchi n, et al. genome-wide microrna expression profiling in placentae from frozen-thawed blastocyst transfer. clin epigenet. 2017;9:79. 67. opdahl s, henningsen aa, tiitinen a, bergh c, pinborg a, romundstad pr, et al. risk of hypertensive disorders in pregnancies following assisted reproductive technology: a cohort study from the conartas group. hum reprod. 2015;30:1724–31. 68. herrero l, martinez m, garcia-velasco ja. current status of human oocyte and embryo cryopreservation. curr opin obstet gynecol 2011;23:245–50. 69. wong km, mastenbroek s, repping s. cryopreservation of human embryos and its contribution to in vitro fertilization success rates. fertil steril. 2014;102:19–26. 70. roque m, valle m, guimaraes f, sampaio m, geber s. cost-effectiveness of the freeze-all policy. jbra assist reprod. 2015;19: 125–30. 71. papaleo e, pagliardini l, vanni vs, delprato d, rubino p, candiani m, et al. a direct healthcare cost analysis of the cryopreserved versus fresh transfer policy at the blastocyst stage. reprod biomed online. 2017;34:19–26. 72. zarek sm, mumford sl, segars jh, armstron ay. cost effective analysis comparing a freeze-all protocol to fresh blastocyst embryo transfer in normal responders. fertil steril 2014;102:241. 73. le kd, vuong ln, ho tm, dang vq, pham td, pham ct, et al. a cost-effectiveness analysis of freeze-only or fresh embryo transfer in ivf of non-pcos women. hum reprod. 2018;33:1907–14. upsala journal of medical sciences 111 abstract introduction freeze all for all strategy when to freeze-all pgt-a programmes ohss prevention elevated progesterone day 6 blastocysts uterine pathology polyps adenomyosis hydrosalpinx random-start ovarian stimulation optimizing preservation methods of preservation endometrial preparation for fet hormonal replacement treatment (hrt) or artificial cycle natural cycle safety of cryopreservation of embryos perinatal outcomes large for gestational age (lga) and high birth weight small-for-gestational age and low birth weight preterm delivery (delivery at less than 37 weeks) others obstetric outcomes cost-effectiveness of fet conclusions disclosure statement references tf-iups160030 165..169 original article simplified ultrasound protocol for the exclusion of clinically significant carotid artery stenosis dominika h€ogberga,b, demosthenes dellagrammaticasa, bj€orn kragstermana, martin bj€orcka and anders wanhainena adepartment of surgical sciences, section of vascular surgery, uppsala university, uppsala, sweden; bdepartment of surgery, nu hospital organization, trollh€attan, sweden abstract objectives: to evaluate a simplified ultrasound protocol for the exclusion of clinically significant carotid artery stenosis for screening purposes. material and methods: a total of 9,493 carotid arteries in 4,748 persons underwent carotid ultrasound examination. most subjects were 65-year-old men attending screening for abdominal aortic aneurysm. the presence of a stenosis on b-mode and/or a mosaic pattern in post-stenotic areas on colour doppler and maximum peak systolic velocity (psv) in the internal carotid artery (ica) were recorded. a carotid stenosis was defined as the north american symptomatic carotid endarterectomy trial (nascet) >20% and a significant stenosis as nascet >50%. the kappa (j) statistic was used to assess agreement between methods. sensitivity, specificity, positive predictive (ppv), and negative predictive (npv) values were calculated for the greyscale/mosaic method compared to conventional assessment by means of psv measurement. results: an ica stenosis was found in 121 (1.3%) arteries; 82 (0.9%) were graded 20%–49%, 16 (0.2%) were 50%–69%, and 23 (0.2%) were 70%–99%. eighteen (0.2%) arteries were occluded. overall, the greyscale/mosaic protocol showed a moderate agreement with ica psv measurements for the detection of carotid artery stenosis, j¼0.455. the sensitivity, specificity, ppv, and npv for detection of >20% ica stenosis were 91% (95% ci 0.84–0.95), 97% (0.97–0.98), 31% (0.26–0.36), and 97% (0.97–0.97), respectively. the corresponding figures for >50% stenosis were 90% (0.83–0.95), 97% (0.97–0.98), 11% (0.08–0.15), and 100% (0.99–1.00). conclusion: compared with psv measurements, the simplified greyscale/mosaic protocol had a high negative predictive value for detection of >50% carotid stenosis, suggesting that it may be suitable as a screening method to exclude significant disease. article history received 21 march 2016 revised 7 june 2016 accepted 9 june 2016 keywords carotid stenosis; screening; ultrasound introduction carotid atherosclerotic disease accounts for 20% of ischemic stroke and is the most common cause of stroke in middleaged patients (1). several publications indicate that appropriate medical therapy and risk factor control prevent the development of cerebrovascular symptoms due to carotid stenosis (2–5). hence, screening for carotid stenosis has been discussed during the last decade; although current vascular guidelines do not recommend unselected population screening (6–8), there are recommendations for screening high-risk adults with multiple cardiovascular risk factors (9,10). in a recent study of the swedish population we observed that only approximately 40% of individuals with screening-detected asymptomatic carotid stenosis were on preventive medication with statins and/or antiplatelet agents (11). duplex ultrasonography (dus) is the standard diagnostic method for carotid artery stenosis and includes three modalities: b-mode (greyscale), colour doppler evaluation, and velocity measurements. b-mode allows measurements of intima-media thickness (imt) and characterization of atherosclerotic plaque morphology (figure 1). colour doppler allows for visualization of flow abnormalities such as turbulence related to the presence of stenosis, which gives rise to a characteristic ‘mosaic’ pattern (figure 2). however, it is spectral analysis of the doppler waveform together with measurement of blood flow velocity which is the main parameter used for grading the severity of carotid stenosis (12,13). velocity measurements have certain technical aspects that are important for accurate assessment, such as correct positioning of the sample volume, complete sampling through an area of stenosis, and obtaining a correct doppler angle of insonation. a full carotid duplex ultrasound protocol is therefore highly operator-dependent and can be time-consuming. the aim of this study was to examine whether a simplified dus protocol consisting of assessment for presence of stenosis on b-mode and/or mosaic patterns on colour doppler (the greyscale/mosaic method) could be used as a rapid contact dominika h€ogberg dominika.hogberg@vgregion.se department of surgery, nu-sjukvården, n€al, se-461 85 trollh€attan, sweden � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 3, 165–169 http://dx.doi.org/10.1080/03009734.2016.1201177 and reliable screening method for the exclusion of clinically significant carotid stenosis. material and methods a total of 4,748 subjects were included in the study; 4,646 were healthy 65-year-old men attending a population-based carotid screening programme during 2007–2009, taking the opportunity of the abdominal aortic aneurysm screening programme that invites all 65-year-old men for ultrasound screening in uppsala since 2006, with an attendance rate of 86% (14,15). the results of this carotid screening study have been published previously (11). we also included 102 consecutive patients undergoing carotid dus in 2012 for symptomatic cerebrovascular disease to increase the number of patients with carotid artery stenosis, since a greater number of ‘events’ facilitates comparisons between the evaluated methods. the dus examinations were carried out at the vascular laboratory at uppsala university hospital by three experienced (>3 years of experience in vascular ultrasound) ultrasound technicians. examinations were conducted either with an acuson sequoia system (acuson, mountain view, ca, usa), using an l9-4 mhz linear transducer or a philips iu22 system (philips ultrasound, bothell, wa, usa), using an l9-3 mhz linear transducer. a maximum insonation angle of 60� to the vessel was applied in all examinations. the carotid arteries on both sides were evaluated in both transverse and longitudinal projection for the presence of a visible significant narrowing on b-mode greyscale image and the presence of mosaic pattern on colour doppler image. internal carotid artery (ica) peak systolic velocity (psv) was then measured in the narrowest segment of the vessel as indicated by b-mode duplex ultrasound and/or colour flow changes. the degree of stenosis was determined according to the nascet method modified by jogestrand et al. (16–18), which is the standard used for the accreditation of vascular laboratories in sweden (table 1) (18). dus examinations were performed with the standard presets for carotid ultrasound for each machine. the technician made further optimizations in cases of difficult morphology. the vessels were first examined for presence of stenosis signs with greyscale image and colour doppler, and the findings were registered into a standardized form, after which the ica psv measurements were made and registered. both the greyscale/mosaic method and psv measurements were performed by the same technician without blinding. an interobserver variability analysis was performed on subjects from the same ongoing screening programme in which 36 arteries were included with both normal findings and atherosclerosis. greyscale and/or mosaic pattern findings were compared with presence of stenosis determined by psv. statistical analysis was carried out with spss (pc version 20.0, spss, chicago, il, usa) and the vassar stats website for statistical computation (vassarstats.net). the j statistics were used to assess agreement between methods, and 95% confidence intervals (ci) were calculated. the levels of agreement were defined as: j < 0.20 poor, 0.21 < j < 0.40 fair, 0.41 < j < 0.60 moderate, 0.61 < j < 0.80 good, and j�0.80 very good agreement (19). p < 0.05 was considered statistically significant. sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) were calculated with 95% ci. all subjects gave informed consent prior to the investigation. the regional ethical review board in uppsala approved the study (epn dnr 2007/053). results a total of 9,493 carotid arteries in 4,748 subjects were examined. three subjects underwent a unilateral examination only. the screening group consisted of 4,646 65-year-old men in whom the prevalence of ica stenosis was 1.9%. the table 1. standard criteria for grading carotid stenosis in sweden (16–18). systolic maximal velocity degree of stenosis angle <45� angle 55–60� ecst nascet <1.1 m/s <1.3 m/s <50% <20% 1.1–1.6 m/s 1.3–2.2 m/s 50%–69% 20%–49% 1.7–2.0 m/s 2.3–3.1 m/s 70%–79% 50%–69% �2.1 m/s �3.2 m/s 80%–99% 70%–99% no signal no signal occlusion occlusion ecst: the european carotid surgery trial. figure 2. carotid artery with stenosis and mosaic pattern on colour doppler. figure 1. carotid artery with stenosis on greyscale. 166 d. h €ogberg et al. www.vassarstats.net symptomatic group consisted of 56 men and 46 women with a mean age of 67 years (sd ±12) in whom the prevalence of ica stenosis was 17%. an ica stenosis, diagnosed by means of psv measurement (nascet definition), was found in 121 (1.3%) arteries; 82 (0.9%) were graded 20%–49% (mild), 16 (0.2%) were 50%–69% (moderate), and 23 (0.2%) were 70%–99% (severe). eighteen (0.2%) arteries were occluded. the total time taken to examine both carotid bifurcations using the greyscale/mosaic protocol ranged between two and three minutes. moderate agreement was observed between the two methods, with j ¼ 0.455 (95% ci 0.399–0.511), p < 0.001. for the detection of >20% ica stenosis the greyscale/mosaic pattern method had a sensitivity of 91% (95% ci 0.84–0.95) and a specificity of 97% (0.97–0.98). the ppv was 31% (0.26–0.36), and the npv was 97% (0.97–0.97). for the detection of >50% ica stenosis the sensitivity was 100% (95% ci 0.89–1.0), the specificity 97% (95% ci 0.96–0.97), the ppv was 11% (95% ci 0.08–0.15), and the npv was 100% (95% ci 0.99–1.0). the greyscale/mosaic method detected all significant stenoses >50% but missed a few <50% stenoses; however, most of these were borderline with an ica psv of <2.0 m/s (table 2). interobserver agreement was very good, with j ¼ 0.8 for both mosaic and greyscale evaluation and j ¼ 1.0 for psv. discussion recent data (2–4,20) clearly indicate the benefit of current best medical therapy in the management of patients with moderate and severe asymptomatic carotid artery stenosis in order to reduce stroke risk as well as overall cardiovascular risk. in a natural history study nikolaides et al. reported that a considerable number of neurological events occur in patients with low-grade stenosis (<60%, nascet criteria) (21). there is also evidence that best medical treatment (bmt) affects the intima-media thickness progression and stroke risk, which suggests that individuals with mild and moderate stenosis also could benefit from preventive treatment (22). in a recent population study we observed that most men with a screening-detected asymptomatic carotid stenosis had no other clinical manifestation of atherosclerosis. in fact, only about 40% of individuals in the whole group were receiving a statin and an antiplatelet agent at the time of screening (11). as a consequence, carotid artery screening has a potential role in identifying individuals at risk and enabling the institution of best medical therapy and appropriate follow-up for this cohort, including a small number even considered for operative intervention. several issues need to be resolved, however, before screening for asymptomatic carotid stenosis can be advocated. many of those are included in the world health organization guidelines on principles and practice of screening for disease published in 1968 (23); one is the need for a suitable screening method. ultrasound is a non-invasive and readily available diagnostic tool that already serves as an excellent screening method in other clinical contexts (e.g. screening for aortic aneurysms). several definitions and grading methods of an ica stenosis exist, all based on velocity measurements such as ica psv (12). velocity measurements have certain technical aspects that are important for accurate assessment, such as correct positioning of the sample volume, complete sampling through an area of stenosis, and obtaining a correct doppler angle of insonation (�60�). to handle these technical aspects is investigator-dependent (24). while the preoperative diagnostic analysis should be precise because of its utmost importance for the indication for surgery (25–28), a screening duplex scan does not have to be as precise. instead, it should preferentially be a method that excludes healthy individuals as quickly as possible and, most importantly, identifies the few with a potentially clinically significant stenosis, who can later be examined with higher precision. in a screening setting, a simplified carotid protocol as a primary evaluation may reduce the cost associated with the examination and therefore be preferred compared to a preoperative diagnostic tool. in this context, a simplified dus protocol may allow the training of less specialized technicians who might concentrate on screening assessments, whilst more experienced technicians might continue to provide a thorough diagnostic assessment. the present study showed that carotid artery stenosis could be excluded by means of a simplified dus protocol, with a high negative predictive value (npv) of 100%. the overall agreement with ica psv measurements was moderate, and the greyscale/mosaic pattern method tended to overestimate the number of stenoses. the accuracy of detection of mild stenosis (20%–49%) was very good, and the identification of moderate (50%–69%) and severe (70%–99%) stenoses was excellent. to our knowledge there are no recent population-based studies that evaluate a simplified screening method for carotid stenosis using ultrasound. our study, however, is consistent with the study of hallam et al. published in 1989, who showed in a double-blind comparison complete agreement between colour-flow assessment only and a full dus assessment in 91% of cases (29). interestingly, hallam et al. also found the same j value as in the present report evaluating the interobserver variability. another quick carotid scan method was evaluated by lavenson among 500 consecutive carotid ultrasound patients in 2004; they found a sensitivity of 93% and a specificity of 87% when comparing with a complete carotid ultrasound (30). the present study was a prospective population-based study with a predefined protocol in which greyscale/mosaic and psv were measured simultaneously. one important and unforeseen limitation was the low prevalence of significant stenosis in the general population. a group of symptomatic patients was therefore added to the screening cohort to gain more power to the analysis of the simplified dus method. before the use of this method as a routine screening protocol table 2. contingency table of ultrasound outcome. ica stenosis defined by psv measurement (nascet) normal 20%–49% 50%–69% 70%–99% 100% total ica stenosis by greyscale/mosaic measurement yes 238 71 16 23 4 352 no 9116 11 0 0 14 9141 total 9354 82 16 23 18 9493 upsala journal of medical sciences 167 can be proposed, there are a few factors that must be taken into consideration. first, our study was not blinded, and observer bias is highly possible. in addition, all technicians performing the assessments were experienced in carotid ultrasound and thus may have been more likely to recognize the hallmark signs of carotid stenosis during assessment with b-mode and colour doppler. a natural next step in testing this protocol would be to assess how operators with limited previous experience in dus of the carotid arteries perform compared to experienced ultrasound technicians. to minimize the risk of missing a significant carotid stenosis (in particular with unexperienced operators or in difficult anatomy) subjects with uncertain findings should be referred for a complete carotid ultrasound. such a study design would probably be more representative of a screening setting, since many abdominal aortic aneurysm screening programmes use less experienced operators to examine the aorta (14,31,32). it might be concluded that although the greyscale/mosaic protocol showed only moderate agreement with conventional dus assessment for the detection 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https://vascular.org http://www.equalis.se/media/71780/s005_ultraljudsunders&percnt;c3&percnt;b6kning&percnt;20av&percnt;20karotisart&percnt;c3&percnt;a4rerna&percnt;20v1.pdf http://www.equalis.se/media/71780/s005_ultraljudsunders&percnt;c3&percnt;b6kning&percnt;20av&percnt;20karotisart&percnt;c3&percnt;a4rerna&percnt;20v1.pdf http://www.equalis.se/media/71780/s005_ultraljudsunders&percnt;c3&percnt;b6kning&percnt;20av&percnt;20karotisart&percnt;c3&percnt;a4rerna&percnt;20v1.pdf http://www.equalis.se/media/71780/s005_ultraljudsunders&percnt;c3&percnt;b6kning&percnt;20av&percnt;20karotisart&percnt;c3&percnt;a4rerna&percnt;20v1.pdf http://www.equalis.se/media/71780/s005_ultraljudsunders&percnt;c3&percnt;b6kning&percnt;20av&percnt;20karotisart&percnt;c3&percnt;a4rerna&percnt;20v1.pdf http://www.equalis.se/media/71780/s005_ultraljudsunders&percnt;c3&percnt;b6kning&percnt;20av&percnt;20karotisart&percnt;c3&percnt;a4rerna&percnt;20v1.pdf 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http://aaa.screening.nhs.uk/annualreport simplified ultrasound protocol for the exclusion of clinically significant carotid artery stenosis introduction material and methods results discussion disclosure statement funding information references upsala j med sci 88: 17-23, 1983 increased platelet volume in manifest diabetic rats ulf eriksson’, u w e ewald’ and torsten tuvemo’ departments of medical cell biology‘, and pediatrics’, university of uppsala, uppsala, sweden abstract p l a t e l e t f u n c t i o n , e v a l u a t e d as in v i t r o a g g r e g a b i l i t y , h a s b e e n r e p o r t e d to b e dis t u r b e d in d i a b e t e s , b o t h in h u m a n s a n d animals. p l a t e l e t n u m b e r a n d m e a n v o l u m e g r e a t l y i n f l u e n c e t h e s e a g g r e g a t i o n tests. t h e p r e s e n t s t u d y w a s d e s i g n e d t o e v a l u a t e t h e i i n p a c t of t w o d i f f e r e n t d e g r e e s of e x p e r i m e n t a l l y induced g l u c o s e i n t o l e r a n c e on p l a t e l e t n u m b e r a n d m e a n volume. f o r t h i s purpose, w e u s e d m a n i f e s t d i a b e t i c a n d c h e m i c a l l y d i a b e t i c r a t s . in t h e c o n t r o l group, t h e f e m a l e r a t s s h o w e d a s i g n i f i c a n t l y l o w e r n u m b e r of p l a t e l e t s c o m p a r e d to t h e males. t h e c h e m i c a l l y d i a b e t i c r a t s e x h i b i t e d a t e n d e n c y t o w a r d s i n c r e a s e d m e a n p l a t e l e t v o l u m e , w h e r e a s t h e p l a t e l e t v o l u m e of t h e m a n i f e s t d i a b e t i c f e m a l e s w a s s i g n i f i c a n t l y g r e a t e r t h a n a l l o t h e r groups. t h i s i n c r e a s e w a s f o u n d t o b e mainly d u e to a g e n e r a l s h i f t t o w a r d s l a r g e r v o l u m e s of t h e individual p l a t e l e t s of t h e m a n i f e s t d i a b e t i c f e m a l e s . i t i s s u g g e s t e d , t h a t t h e e n l a r g e m e n t of t h e m e a n p l a t e l e t v o l u m e i n d u c e d by i n c r e a s e d s e v e r i t y of t h e d i a b e t i c state m a y r e f l e c t d e c r e a s e d m e a n age of t h e c i r c u l a t i n g p l a t e l e t s . t h i s i m p l i e s s h o r t e r s u r v i v a l t i m e a n d a n i n c r e a s e d t u r n o v e r of t h e p l a t e l e t p o p u l a t i o n in d i a b e t e s mellitus. introduction d i a b e t e s m e l l i t u s in m a n i s a s s o c i a t e d w i t h i n c r e a s e d risk f o r a t h e r o s c l e r o s i s , m i c r o v a s c u l a r c o m p l i c a t i o n s (28), a n d a r t e r i a l t h r o m b o s i s (18). c h a n g e s in t h e h a e m o static s y s t e m w i t h a l t e r e d p l a t e l e t f u n c t i o n h a v e b e e n d e m o n s t r a t e d by d i f f e r e n t in v i t r o tests in d i a b e t i c p a t i e n t s (3) and m i g h t b e involved in t h e p a t h o g e n e s i s of t h i s d i s e a s e (19). d e t e r m i n a t i o n of s u r v i v a l a n d t u r n o v e r i s b e l i e v e d t o a c c u r a t e l y r e f l e c t t h e i n v i v o f u n c t i o n of t h e p l a t e l e t s (15). indeed, r e d u c e d p l a t e l e t s u r v i v a l h a s b e e n n o t i c e d in d i a b e t i c p a t i e n t s (7, 22). e s t i m a t i o n of n u m b e r a n d m e a n v o l u m e should r e f l e c t t h e t u r n o v e r rate of t h e p l a t e l e t s , s i n c e s m a l l a n d l i g h t p l a t e l e t s g e n e r a l l y r e p r e s e n t a n o l d e r p o p u l a t i o n t h a n large and d e n s e p l a t e l e t s (16). d i s t u r b a n c e s , s i m i l a r t o t h o s e f o u n d in h u m a n d i a b e t e s m e l l i t u s , of t h e in v i t r o p l a t e l e t f u n c t i o n h a v e b e e n d e m o n s t r a t e d i n e x p e r i m e n t a l l y d i a b e t i c r a t s (4, 9, 13, 20). c h a n g e s 2 8 3 2 8 5 9 17 a n a l o g o u s t o t h e p r o p o s e d a n g i o p a t h i c a l t e r a t i o n s in h u m a n d i a b e t e s m e l l i t u s , s u c h as c h a n g e d v a s c u l a r s t r u c t u r e (25) a n d v a s c u l a r b i o c h e m i s t r y (1, 30, 31) h a v e a l s o b e e n o b s e r v e d in d i a b e t i c r a t s . f u r t h e r m o r e , a l t e r e d a r a c h i d o n i c a c i d m e t a b o l i s m in t h e v e s s e l w a l l s h a s b e e n d e c r i b e d in e x p e r i m e n t a l d i a b e t e s i n t h e r a t (9-11, 29). a g a i n s t t h i s b a c k g r o u n d , t h e airn of t h e p r e s e n t s t u d y w a s t o e v a l u a t e t h e i m p a c t of t w o d i f f e r e n t d e g r e e s of c h r o n i c g l u c o s e i n t o l e r a n c e o n p l a t e l e t f u n c t i o n by e s t i m a t i n g t h e n u m b e r a n d m e a n v o l u m e of t h e p l a t e l e t s . t h e t w o d i f f e r e n t t y p e s of e x p e r i m e n t a l d i a b e t e s , used in t h i s s t u d y , h a v e b e e n previously c h a r a c t e r i z e d . t h e m i l d e r c h e m i c a l l y d i a b e t i c (cd) t y p e w a s d e s c r i b e d by p o r t h a a n d c o l l a b o r a t o r s (23) a n d t h e m a n i f e s t d i a b e t i c (md) t y p e h a s p r e v i o u s l y b e e n used in s t u d i e s of d i a b e t e s in p r e g n a c y (5, 6). material a n d methods a n i m a l s a f i r s t g r o u p c o m p r i s i n g 19 f e m a l e s p r a g u e d a w l e y r a t s ( a n t i c i m e x ab, s o l l e n t u n a , s w e d e n ) weighing a b o u t 250 g w e r e m a d e m a n i f e s t d i a b e t i c (md) a t 3 m o n t h s of age by a s i n g l e i.v. i n j e c t i o n of s t r e p t o z o t o c i n ( l o t no. 1613e u-9889, kindly d o n a t e d by dr. w.e.dulin, t h e upjohn co., k a l a m a z o o , mi, usa) a t a d o s e of 45 m g / k g body weight. a t t h e t i m e of t h e e x p e r i m e n t t h e a n i m a l s h a d b e e n d i a b e t i c f o r 1 2 m o n t h s a n d e x h i b i t e d h y p e r g l y c e m i a a n d g l u c o s u r i a , as w e l l as a m e a n w e i g h t of 206 2 3 g. a s e c o n d g r o u p of 17 f e m a l e r a t s of t h e s a m e s t r a i n w e r e g i v e n t w o i.p. i n j e c t i o n s o f a b o u t 100 m g / k g body w e i g h t of s t r e p t o z o t o c i n o n t h e s e c o n d a n d t h i r d d a y s of l i f e (23). a f t e r a t r a n s i e n t h y p e r g l y c e m i a l a s t i n g a b o u t o n e w e e k , t h e s e a n i m a l s ( s u b s e q u e n t l y d e n o t e d as cd r a t s ) a c h i e v e d only s l i g h t l y i n c r e a s e d n o n f a s t i n g s e r u m g l u c o s e levels. t h e c d a n i m a l s w e r e 4 m o n t h s old at t h e t i m e of t h e e x p e r i m e n t a n d w e i g h e d 232 2 4 g. a t h i r d g r o u p o f 21 f e m a l e (247 2 4 g) a n d 20 m a l e r a t s (399 l 6 g), o f t h e s a m e s t r a i n , 4 m o n t h s old, s e r v e d as c o n t r o l s ( n ) a n d w e r e n o t g i v e n a n y injections. a l l a n i m a l s w e r e a l l o w e d f r e e access to w a t e r a n d l a b o r a t o r y c h o w (ewos ab, soder talje, sweden). d e t e r m i n a t i o n of p l a t e l e t n u m b e r a n d m e a n v o l u m e blood s a m p l e s , r e p r e s e n t i n g mixed a r t e r i a l v e n o u s blood, w e r e t a k e n f r o m t h e c u t t i p of t h e t a i l s of non-fasting animals. 1 0 m i c r o l i t e r s of f r e e flowing blood w a s o b t a i n e d a n d d i r e c t l y d i l u t e d (unopette, c l a y a d a m s , l a b o r a t o r y i n s t r u r n e n t a t i o n , p a r s i p p a n y , n j , usa). within 20 m i n u t e s of blood sampling, t h e p l a t e l e t n u m b e r w a s d e t e r m i n e d in a n ultra-flo 100 whole blood p l a t e l e t c o u n t e r ( c l a y a d a m s ) by a u t o m a t i z e d p a r t i c l e i m p e d a n c e c o u n t i n g . a p a r t i a l sizing a m p l i f i e r ( n u c l e a r d a t a i n s t r u m e n t s ab, uppsala, s w e d e n ) s i m u l t a n e o u s l y s o r t e d t h e g e n e r a t e d i m p e d a n c e s i g n a l s a c c o r d i n g to t h e individual p u l s e a m p l i t u d e s in 100 c h a n n e l s , r e s u l t i n g in a d i s t r i b u t i o n c u r v e of t h e p l a t e l e t v o l u m e s of e a c h s a m p l e in t h e r a n g e of 3 27 f e m t o l i t r e s (fl., 10-15 liter). cf. fig. 1. a m e a n p l a t e l e t v o l u m e could t h e n b e c a l c u l a t e d f o r e a c h s a m p l e , using t h e r e l a t i v e f r e q u e n c i e s of 8 sub-classes w i t h e q u a l class w i d t h of 3 fl. cf. fig. 2. 18 s t a t i s t i c s a l l d a t a a r e e x p r e s s e d as m e a n s l s.e.m. p r o b a b i l i t i e s (p) of c h a n c e d i f f e r e n c e s b e t w e e n t h e d i f f e r e n t m e a n s w e r e c a l c u l a t e d a c c o r d i n g t o s t u d e n t ' s t w o t a i l e d t-test (21). a p l a t e l e t s ____t erythrocytes+leucocytes 4 i i i i i i i i 3 9 15 21 27 33 39 45 51 f l 1 1 volume f i g u r e 1 . a t y p i c a l d i s t r i b u t i o n c u r v e of c o r p u s c u l a r v o l u m e s in a blood s a m p l e f r o m a n m a l e r a t . t h e r e l a t i v e f r e q u e n c y of i m p e d a n c e p u l s e s i s s h o w n ( a r b i t r a r y s c a l e ) on t h e o r d i n a t e . results t h e d i f f e r e n t l e v e l s of s e r u m g l u c o s e a r e shown in t a b l e 1. t h e r e w a s n o s e x d i f f e r e n c e in t h e n g r o u p in t h i s r e s p e c t ( p > 0.05). o n t h e o t h e r h a n d , b o t h t h e c d ( p < 0.01) a n d md r a t s ( p < 0.001) e x h i b i t e d i n c r e a s e d s e r u m g l u c o s e v a l u e s c o m p a r e d to t h e n f e m a l e s . in p a r t i c u l a r , t h e md f e m a l e s w e r e m a r k e d l y a b n o r m a l w i t h g l u c o s e l e v e l s a b o u t f i v e t i m e s h i g h e r t h a n t h e n f e m a l e s . t a b l e 1. s e r u m g l u c o s e c o n c e n t r a t i o n s , n u m b e r s a n d mean volumes of p l a t e l e t s in t h e d i f f e r e n t e x p e r i m e n t a l groups. n , c d a n d md d e n o t e n o r m a l , c h e m i c a l l y d i a b e t i c a n d m a n i f e s t d i a b e t i c r a t s , r e s p e c t i v e l y . m e a n s 5 s.e.m. significances: a = p < 0.001 v e r s u s n f e m a l e , b = p < 0.01 v e r s u s n f e m a l e . no. s e r u m platelet p l a t e l e t of g l u c o s e n u m b e r volume rats ( m m o l / l ) ( 1 0 ~ 1 ) (fl) n male 20 5.7 2 0.1 948 5 24= 14.07 l 0.1 1 14.08 2 0.06 14.26 5 0.1 1 n f e m a l e 2 1 5.8 2 0.2 c d f e m a l e 17 6.7 l 0.2 md f e m a l e 19 28.5 l 0.7a 682 2 5 4 14.44 2 0.07a 745 2 27 887 2 42 b 19 t h e n u m b e r of p l a t e l e t s in t h e d i f f e r e n t e x p e r i m e n t a l g r o u p s i s shown in t a b l e 1. in t h e n g r o u p , t h e f e m a l e s h a d a s i g n i f i c a n t l y l o w e r n u m b e r of p l a t e l e t s c o m p a r e d to t h e m a l e s ( p < 0.001). c o n s i d e r i n g t h e f e m a l e s , t h e md r a t s s h o w e d s i g n i f i c a n t l y f e w e r p l a t e l e t s c o m p a r e d to t h e c d r a t s ( p < 0.01) b u t n o t in c o m p a r i s o n w i t h t h e n a n i m a l s ( p > 0.05). t a b l e 1 a l s o shows t h e m e a n p l a t e l e t v o l u m e s of t h e d i f f e r e n t g r o u p s of r a t s . in t h e n group, n o d i f f e r e n c e s c o u l d b e found b e t w e e n t h e s e x e s ( p > 0.05). a t e n d e n c y t o w a r d s i n c r e a s e d c d f e m a l e p l a t e l e t volume c o m p a r e d t o t h e n f e m a l e s w a s n o t e d (0.05 < p < 0.10). t h i s t r e n d w a s a l s o o b s e r v e d in t h e md r a t s , who s h o w e d a m e a n p l a t e l e t v o l u m e g r e a t e r t h a n all o t h e r groups, in p a r t i c u l a r c o m p a r e d to t h e n f e m a l e s ( p < 0.001). i n s p e c t i o n of t h e a c c u m u l a t e d m e a n v o l u m e d i s t r i b u t i o n c u r v e s of t h e n a n d md f e m a l e g r o u p s (fig. 2 ) s h o w e d t h a t t h e i n c r e a s e d m e a n p l a t e l e t v o l u m e of t h e md r a t s w a s t h e r e s u l t of a g e n e r a l s h i f t of t h e d i s t r i b u t i o n c u r v e t o w a r d s l a r g e r volumes. 0 3 6 9 12 15 18 21 24 27 f l mean platelet volume f i g u r e 2. a c c u m u l a t e d m e a n v o l u m e d i s t r i b u t i o n of ( f e m a l e ) n a n d md r a t s . t h e r e l a t i v e f r e q u e n c y of i m p e d a n c e p u l s e s a r e shown on t h e o r d i n a t e . n o t e t h e g e n e r a l s h i f t of t h e md p o p u l a t i o n t o w a r d s l a r g e r volumes. discussion t h e d e c r e a s e d n u m b e r of p l a t e l e t s in t h e f e m a l e s c o m p a r e d to t h e m a l e s in t h e c o n t r o l g r o u p h a s n o t b e e n previously d e m o n s t r a t e d in t h e r a t . p l a t e l e t n u m b e r a n d v o l u m e g r e a t l y i n f l u e n c e t h e r e s u l t s of i n v i t r o p l a t e l e t a g g r e g a t i o n s t u d i e s (121, t h e r e f o r e t h e 20 d e m o n s t r a t e d s e x d i f f e r e n c e m a y b e of i m p o r t a n c e f o r t h e e v a l u a t i o n of p l a t e l e t a g g r e g a b i l i t y . in h u m a n s , d i f f e r e n c e s b e t w e e n t h e s e x e s in p l a t e l e t n u m b e r (17, 27), a n d p l a t e l e t a g g r e g a b i l i t y (14, 24) h a v e b e e n r e p o r t e d . t h e r e s u l t s of t h e p r e s e n t s t u d y m a y i l l u s t r a t e t h e n e c e s s i t y to c o n s i d e r t h e s e x as w e l l as b o t h t h e p l a t e l e t n u m b e r a n d m e a n p l a t e l e t v o l u m e in t h e f u t u r e e v a l u a t i o n s of p l a t e l e t f u n c t i o n . t h e m a j o r f i n d i n g in t h i s s t u d y , t h e m a r k e d i n c r e a s e i n ,ml3 p l a t e l e t v o l u m e , would i n d i c a t e a g e n e r a l l y d e c r e a s e d m e a n a g e in t h e p l a t e l e t p o p u l a t i o n of t h e m a n i f e s t d i a b e t i c r a t s (16). t h i s notion is f u r t h e r s u b s t a n t i a t e d by t h e f i n d i n g of a right-hand s i d e s h i f t in t h e d i s t r i b u t i o n c u r v e of t h e p l a t e l e t s . a d e c r e a s e d m e a n a g e would b e t h e r e s u l t of i n c r e a s e d t u r n o v e r a n d s h o r t e n e d s u r v i v a l t i m e of p l a t e l e t s (7, 16, 22). f u r t h e r m o r e , t h e n u m b e r of md p l a t e l e t s t e n d e d to b e d e c r e a s e d , a n o b s e r v a t i o n a l s o in a g r e e m e n t w i t h a n i n c r e a s e d r a t e of p l a t e l e t t u r n o v e r . s i n c e younger a n d l a r g e r p l a t e l e t s are known to b e m o r e m e t a b o l i c a l l y a c t i v e (16), t h i s finding would b e c o n s i s t e n t w i t h b o t h t h e v i e w of a g e n e r a l h y p e r a g g r e g a b i l i t y of t h e p l a t e l e t s in d i a b e t e s ( 3 , 8), a n d i n c r e a s e d p l a t e l e t t u r n o v e r in v i v o (2, 7, 22). a s i m i l a r t e n d e n c y t o w a r d s i n c r e a s e d m e a n v o l u m e of t h e p l a t e l e t s w a s a l s o o b s e r v e d in t h e c d r a t s . c h a n g e s i n p l a t e l e t f u n c t i o n h a v e b e e n n o t i c e d in t h e v e r y e a r l y p h a s e s of h u m a n d i a b e t e s (26). t h e a c c u m u l a t e d d i s t r i b u t i o n c u r v e of p l a t e l e t v o l u m e s in t h e cd g r o u p did n o t s h o w a g e n e r a l s h i f t t o w a r d s l a r g e r v o l u m e s s i m i l a r to t h a t s e e n in t h e md r a t s . t h e r e w a s i n s t e a d r a t h e r a l o c a l i z e d i n c r e a s e in t h e p r o p o r t i o n of l a r g e p l a t e l e t s . t h e p r e s e n t f i n d i n g s of i n c r e a s e d p l a t e l e t v o l u m e s c o r r e l a t i n g w i t h a n i n c r e a s e d s e v e r i t y of t h e d i a b e t i c state s u g g e s t , t h a t a d i s t u r b e d c a r b o h y d r a t e r n e t a b o l i s m m a y d i r e c t l y , o r i n d i r e c t l y , c a u s e a n i n c r e a s e d t u r n o v e r o f t h e c i r c u l a t i n g p l a t e l e t s . t h i s s u g g e s t i o n would s u p p o r t t h e n o t i o n o f i n c r e a s e d p l a t e l e t a g g r e g a t i o n i n d i a b e t e s m e l l i t u s , s i n c e s h o r t e r s u r v i v a l t i m e , d e c r e a s e d m e a n a g e a n d g e n e r a l e n l a r g e m e n t of t h e m e a n p l a t e l e t v o l u m e i m p l i c a t e s i n c r e a s e d p l a t e l e t a c t i v i t y , a n d t h e r e b y i n c r e a s e d a g g r e g a bility. t h e p r e c i s e r e a s o n f o r t h e d y s f u n c t i o n of t h e p l a t e l e t s in d i a b e t e s m e l l i t u s i s at p r e s e n t n o t c o m p l e t e l y u n d e r s t o o d . a n i m a l m o d e l s w i t h d i f f e r e n t d e g r e e s of g l u c o s e i n t o l e r a n c e m a y t h e r e f o r e play a n i m p o r t a n t r o l e in f u t u r e r e s e a r c h on a l t e r e d p l a t e l e t f u n c t i o n i n d i a b e t e s mellitus. acknowledgements t h e a u t h o r s a r e i n d e b t e d to b a r b r o w e s t e r b e r g , a s t r i d nordin a n d e v a t o r n e l i u s f o r e x c e l l e n t t e c h n i c a l help. f i n a n c i a l a s s i s t a n c e h a s b e e n r e c e i v e d f r o m n o v o i n d u s t r i ab, malmo, s w e d e n ( t o dr. t. t u v e m o ) a n d t h e swedish m e d i c a l r e s e a r c h c o u n c i l ( g r a n t no. 12x-109, a n d p o s t d o c t o r a l f e l l o w s h i p no. k82-12p-6346-01 t o dr. u. eriksson). r e f e r e n c e s 1 . a r n q v i s t , h.j. & dahlkvist, h.h.: e f f e c t of e x p e r i m e n t a l d i a b e t e s o n t h e 21 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 11 i n c o r p o r a t i o n of a m i n o a c i d s i n t o p r o t e i n in r a t a o r t a . horm. metab. res. 11: 384 388, 1979. bonne, c., rornquin, n. & r e g n a u l t , f.: modified s e n s i t i v i t y of d i a b e t i c p l a t e l e t s t o prostacyclin. in: 1 0 t h eur. conf. microcirc., c a l g l i a r i 1978, r i b l t h c a a n a t . pp. 98-103. k a r g e r , basel, 1979. colwell, j.a. & halushka, p.v.: p l a t e l e t f u n c t i o n in d i a b e t e s mellitus. br. j. haernatol. 44: 521-526, 1980. eldor, a., merin, s . & bar-on, h.: t h e effect of s t r e p t o z o t o c i n d i a b e t e s on p l a t e l e t f u n c t i o n in r a t s . thromb. res. 13: 703-714, 1978. eriksson, u., a n d e r s o n , a., e f e n d i f , s., elde, r. & h e l l e r s t r o m , c.: d i a b e t e s in pregnancy: effects on t h e f o e t a l and newborn r a t with p a r t i c u l a r r e g a r d t o body weight, s e r u m insulin c o n c e n t r a t i o n a n d p a n c r e a t i c c o n t e n t s of insulin, glucagon a n d s o m a t o s t a t i n . a c t a endocrinol. 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n c r e a s e d p l a t e l e t a g g r e g a t i o n in e a r l y d i a b e t e s mellitus. ann. int. med. 82: 733-738, 1975. s t e v e n s , r.f. & alexander, m.k.: a s e x d i f f e r e n c e in t h e p l a t e l e t count. br. j. h a e m a t o l . 37: 295-300, 1978. ll 28. s t o u t , r.w., r i e r m a n , e.l. & rrunzell, j.d.: a t h e r o s c l e r o s i s a n d d i s o r d e r s of lipid m e t a b o l i s m in d i a b e t e s . in: d i a b e t e s : i t s physiological a n d b i o c h e m i c a l b a s i s (ed. vallance-owen, j.), pp. 125-169. m t m p r e s s , l a n c a s t e r , 1975. 29. t u r l a p a t y , p.d.m.v., l u m , g. & a l t u r a , b.m.: v a s c u l a r r e s p o n s i v e n e s s a n d s e r u m b i o c h e m i c a l p a r a m e t e r s in a l l o x a n d i a b e t e s mellitus. am. j. physiol. 239: e412-e421, 1980. 30. wolinsky, h., c a p r o n , l., c o l d f i s c h e r , s., c a p r o n , f., c o l t o f f s c h i l l e r , 8. & k a s a k , l. h y d r o l a s e a c t i v i t i e s in t h e r a t a o r t a . 11. e f f e c t s of h y p e r t e n s i o n a l o n e a n d in c o m b i n a t i o n w i t h d i a b e t e s mellitus. c i r c . r e c . 42: 831-839, 1978. 31. wolinsky, h., c o l d f i s c h e r , s., c a p r o n , l., c a p r o n , f., c o l t o f f s c h i l l e r , r. & k a s a k , l. h y d r o l a s e a c t i v i t i e s in t h e r a t a o r t a . i . e f f e c t s of d i a b e t e s m e l l i t u s a n d insulin t r e a t m e n t . c i r c . res. 42: 821-831, 1978. a d r e s s f o r r e p r i n t s ulf e r i k s s o n m.d. d e p a r t m e n t of m e d i c a l c e l l biology u n i v e r s i t y of u p p s a l a s-751 2 3 u p p s a l a s w e d e n 23 henrik sjöbring and the concept of individual psychology in psychiatry full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 henrik sjöbring and the concept of individual psychology in psychiatry lars oreland to cite this article: lars oreland (2015) henrik sjöbring and the concept of individual psychology in psychiatry, upsala journal of medical sciences, 120:2, 95-103 to link to this article: https://doi.org/10.3109/03009734.2015.1027428 © informa healthcare published online: 23 apr 2015. submit your article to this journal article views: 344 view related articles view crossmark data citing articles: 1 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://doi.org/10.3109/03009734.2015.1027428 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1027428 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1027428 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1027428&domain=pdf&date_stamp=2015-04-23 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1027428&domain=pdf&date_stamp=2015-04-23 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1027428#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1027428#tabmodule upsala journal of medical sciences. 2015; 120: 95–103 henrik sjöbring and the concept of individual psychology in psychiatry lars oreland department of neuroscience, uppsala university, uppsala, sweden key words: personality, psychiatry, psychology in 1913 henrik sjöbring presented his thesis, ‘den individualpsykologiska frågeställningen inom psykiatrien’ [‘the question of individual psychology in psychiatry’] in upsala läkareförenings förhandlingar (1), the principal aim of which was to construct a system for characterizing the various dimensions of personality. the system presupposed that it is possible to distinguish different fundamental characteristics, or factors, in our innate psychic constitution and that combinations in different degrees of these fundamental characteristics could produce an infinite number of personality variations. extreme positions, above and below the median, could result in mental illness. at that time this stance was new, but it corresponds well with contemporary beliefs. a person’s personality and its importance for illness, particularly mental illness, has always been interesting, both generally and in medicine. for a long time, a barrier to meaningful typology was the inadequate knowledge about the ‘site of the soul’ and later, when the brain’s role in this connection was established, the functioning of the brain. at the start of the last century this issue was beginning to be dealt with in a more modern way, and kraepelin’s classification in 1899 of two categories of ‘madness’, dementia praecox (schizophrenia) and manicdepressive psychosis, was crucial (2-4). the importance of the personality in relation to mental illness is, however, probably most apparent in the case of less severe forms of illness, where a psychotic condition has not been able to eradicate to any great extent the personality’s fundamental characteristics. the development of psychology in the twentieth century has led to many different typologies, of which henrik sjöbring’s ‘individual psychology’ was the first with its systematic and modern approach that foresaw later developments. sjöbring is generally acknowledged as sweden’s most influential twentieth-century psychiatrist and a leading professor in psychiatry. torsten frey wrote in 1964: if asked today to nominate a group of medical scientists, who more than others have made important contributions to man’s global reactions in health and illness, i would propose from britain john hughlings jackson, from france pierre janet, from germany emil kraepelin, from austria sigmund freud, and from russia ivan petrovitch pavlov. for a swedish name in this company i would not hesitate to propose henrik sjöbring (5). however, sjöbring is not particularly well-known internationally, which is probably due to several factors. he usually wrote in swedish and published his important, early articles in less widely spread fora (his thesis, in 1913, was published in upsala läkareförenings förhandlingar (1). in the same journal in 1920 he published ‘förstämningar and förstämningspsykoser’ [‘melancholy and melancholic psychoses’] (6), in 1922 ‘psykologi och biologi’ [‘psychology and biology’] (7), and in 1922 ‘psychic energy and mental insufficiency’ (8); in 1919 ‘psykisk konstitution och psykos’ [‘psychic constitution and psychosis’] was published in svenska läkarsällskapets handlingar correspondence: lars oreland, department of neuroscience, uppsala university, uppsala, sweden. e-mail: lars.oreland@neurosci.uu.se (received 25 february 2015; accepted 26 february 2015) issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1027428 http://informahealthcare.com/journal/ups mailto:lars.oreland@neurosci.uu.se (9). in addition, as has been pointed out in several of his biographies, his texts are somewhat difficult and written in a very abstract way, often without the facilitation of explanatory examples and concretization. it is also said that he was not prepared to compromise and had difficulty in accepting opinions that did not coincide with his own (10-13). this could create problems not least during the time when freud’s ideas from around 1910 had begun to attract many adherents. sjöbring’s brief dismissal of the psychodynamic school is dealt with later in this article in connection with the review of his thesis. a citation from one of his opponents, carl-henry alström in his book from 1945, ytpsykologi kontra djuppsykologi [superficial psychology versus profound psychology] (14) reflects their differences on this issue: the thoroughly dynamic perception of the development of the psychic phenomenon and the whole approach of his [freud’s] comprehensive empirical investigations exposes a neurologist who, given the nature of his work, is forced to think in a distinctly dynamic and logical way. this is undoubtedly also the reason why freud does not use a statistical approach which involves classifying people into predefined constitutional types, and which is always doomed to end in so called ‘buttonology’. hence it is immaterial whether we choose to classify buttons according to qualitative factors, for instance shape (kretschmer), or quantifiable factors such as size (adler) or several quantitative factors, for instance not only size but also the number of holes (sjöbring) (14). biographic details about henrik sjöbring henrik sjöbring was born in 1879 in aringsås, kronobergs county. his progenitor was the parliamentarian per jonsson from hornaryd in the district of växjö, who was married to kerstin jansdotter, who in her turn was the sister of the professor in oriental languages in uppsala from 1830, pehr sjöbring. professor pehr sjöbring had adopted the name sjöbring after his home parish sjösås and the village bringebäck in the district of växjö, where his paternal grandfather was a famer. the parliamentarian per jonsson and his wife kerstin had a son, nils persson, who also became a member of parliament and the speaker for the yeomanry. nils person and his wife from sjösås, lisa jonsdotter, had two sons, isac and pehr, who both took the name sjöbring. isac became the lord of the manor, a farmer, and was the father of henrik sjöbring. the second son, pehr, became the bishop of kalmar. henrik attended school in växjö, and written sources show that his mathematical aptitude was so great that the school master excused him from attending lessons. henrik sjöbring began studying medicine in lund where he completed his preparatory medical studies in 1897. he took his bachelor of medicine in 1901 and qualified as a doctor with a medical degree from the karolinska institute in 1906 (11,15,16). he had probably by figure 1. henrik sjöbring (oil painting). psychiatric clinic, lund. photo: roger lundholm, skånes universitetssjukhus. 96 l. oreland then already established contacts with the psychiatric clinic in uppsala, especially with olof kinberg, a wellknown swedish psychiatrist. olof kinberg was assistant physician at the hospital in stockholm and the uppsala hospital between 1901 and 1906, after which time he started working at the mental hospital in stockholm (långbro). kinberg wrote, in his obituary for sjöbring: ‘sjöbring began his psychiatric career in his early twenties as my locum at what was in those days called the uppsala hospital where he went on to serve for many years’ (16). with short breaks, sjöbring worked as a psychiatrist in uppsala in various capacities from 1906 to the summer of 1929. a brief history of the concept of personality up to 1913 ever since the days of antiquity, there have been systems for classifying people into different ‘types’. from the time of hippocrates we have the terms sanguine, melancholic, choleric, and phlegmatic, resulting from an excess of the bodily fluids: blood, black gall, yellow gall, and phlegm. the hippocratic school of the fifth century bc discussed the philosophy of the character of the people, their national characteristics, and the importance of the physical and the psycho-social environment in the formation of a national character. the insight about a strong inherited component in character or temperament was noted already by galen in the first century ad. galen adhered to the hippocratic ideas about the connection of bodily fluids with the four elements: heat, cold, damp, and dryness. however galen was already aware that these different elements could be combined in different ways. galen related the excess of black gall (mela chole) to melancholic depression and a predisposal to depressed moods. it was believed that black gall derived from unclean blood that was transported to the spleen, which caused a predisposition for what later became known as a splenetic mood or melancholia. the yellow gall (chole) was related to a tendency to be irritable and hot-tempered (choleric), blood (sanguis) to optimism (sanguineous), and phlegm to indifference or being phlegmatic. that this relationship between an excess of any of the four body fluids and the four temperaments later became so well-known, even to the general public, can be attributed to philip melanchton in the mid-sixteenth century, chiefly through the spread of country almanacs. melanchton, the great friend of martin luther, was a professor in greek at wittenberg, but he also wrote books on psychology that were widely read and he was considered to have minted the concept ‘psychology’ (17-20). during the latter part of the eighteenth century franz joseph gall worked in vienna. gall believed that he could trace character and personality by studying the shape of the skull, phrenology. his hypothesis, which gall strenuously tried to verify through measurements, was based on the idea that various spiritual characteristics existed in special areas of the brain, which could be detectable from the bumps at different places on the skull. gall proceeded in a scientifically correct way according to the knowledge of the nervous system that existed at that time, and he was considered by many to be the first to proclaim a direct connection between the body and soul (18). another typology, arguably more based on reality, and which to a significant degree is a precursor to kretschmer’s influential köperbau und character (1921), is carl gustav carus’s attempt to write his constitutional doctrine in his work symbolik der menschlichen gestalt (1853). carus built his theory on the belief that there is a relationship between the body type and mental constitution. the body types were based on the relationship between height and weight. heavy weight could either be attributed to an athletic physique with choleric and strong spiritual energy or obesity usually linked to a less strong will but often more developed intelligence, sensitivity, and humour. leanness could be differentiated as either a spiritual variant (quick, agile, energetic) or a poor variant with asthenic, stingy, and less intelligent traits (18). a prominent name in psychiatry, emil kraepelin (professor in dorpat in 1886, in heidelberg in 1890, and from 1903 in munich) had at the end of the nineteenth century played figure 2. ernst kretschmer and henrik sjöbring on sjöbring’s sofa. photo: psychiatric clinic, lund. henrik sjöbring and the concept of individual psychology in psychiatry 97 with ideas which somewhat resembled those that sjöbring would present at a later date. however, olof kinberg, who had himself auscultated with kraepelin, wrote: ‘but kraepelin did not account for the totality of the human individual but stopped short with the study of those psychic symptoms, which constitute, through analytical thinking, isolated aspects of the psychic experience (perception, imagination, feelings, will, etc.)’ (16). as if by chance kraepelin presented his most thoroughly constructed thoughts about mental illness as a consequence of personality disorders in the eighth edition of his textbook in psychiatry, which was published in the same year as sjöbring’s thesis, namely 1913. in the literature around sjöbring, three people are usually named as having had a great influence on his theories (5,21). theodor lipps was one of the most important german philosophers at the end of the nineteenth century. sjöbring borrowed the belief from lipps that personality is basically an effort to reach a goal (20). the type of goal can be concrete or abstract, or it may vary. the point is that this effort forms a characteristic manifestation in the individual. sjöbring calls this effort ‘activity’. it is thus possible to claim that lipps in this way promoted the importance of the subconscious, which explains why freud was a great admirer of lipps. another person of importance for sjöbring was the french psychologist and psychiatrist pierre janet, professor in comparative and experimental psychology at the college de france and doctor at salpêtrières mental hospital in paris from 1903. janet, who was a pupil of the famous charcot, had described in texts from 1894, 1898, 1903, and 1909 a system that was not wholly unlike sjöbring’s, namely the appearance of three types of personality, which often reappeared in patients with asthenic-compulsory, hysterical-primitive, and manic-depressive disorder (20). for sjöbring, these three personality types came to be characteristic for sub-valid, sub-solid, and sub-stable individuals or patients—terms which will be described in more detail later. the third person of importance in the development of sjöbring’s theory was wilhelm stern, born in berlin in 1871 and employed as a lecturer in psychology at the university of breslau at the time sjöbring was working on his thesis. in 1911 stern published his book die differentielle psychologie in ihren methodischen grundlagen. stern was later awarded a professorship at the university of hamburg where he stayed until 1933, at which time he emigrated to the usa as a consequence of his jewish heritage. in the usa, he became interested in intelligence and the measuring of intelligence, and it was he who introduced the term intelligence quotient (iq). stern developed janet’s ideas about individual psychology on the grounds of different fundamental factors, which he considered had a strong genetic component: ‘inheritance has figure 3. henrik sjöbring at the time of his retirement in 1944. photo from ref. (28). 98 l. oreland by far the greatest, external environmental factors have by contrast a surprisingly meagre effect’ (20). sjöbring’s main achievement is that he constructed his system, based on common-sense analysis, introspection, and clinical observation, of which factors should be of fundamental biological importance, in contrast to stern’s work that was based mainly on theoretical assumptions. the relationship between stern’s and sjöbring’s systems is probably why sjöbring so often refers to stern in his thesis of 1913, and often claims the superiority of his own system (1). sjöbring forms his theory in articles written in uppsala 1913–1919 in his thesis from 1913, which was simultaneously published in upsala läkareförenings förhandlingar, sjöbring explicates his theory of individual psychology (1). it consists of the pattern of a number of mainly inherited personality variables, where each variable is independent from the others with a gaussian distribution from low (sub-) to high (super-), but together representing the individual’s personality. in contrast to stern, however, sjöbring claims that research on individual psychology must be based on ‘a general comprehension of the psychology of personality’, which presupposes, as sjöbring strongly claims, the necessity of perceiving different aspects of personality as qualities which can be combined in various ways with each other. sjöbring expresses himself somewhat brutally (1): ‘on the contrary, in regard to the subject in question, and vastly different (from stern), i assert this in a one, two or multidimensional variation, the importance of which stern has failed to grasp.’ equally modern is sjöbring’s claim that the constitutional (inherited) personality, ‘the particular disposition’, can be expressed in different ways depending upon childhood environment and vital events in life: ‘it is ab origine just a tendency, just a predisposition, which can develop during an individual’s lifetime. under the impact of various environmental influences’. sjöbring was of course aware that his theory of individual psychology could be difficult to use in psychiatric praxis, but he insists that the theory of normal psychology should nonetheless be followed as far as possible, even clinically, and that the transition between healthy and sick be flexible, an idea which appears to have the support of kraepelin in his psychologische arbeiten (4). when sjöbring mentions the ‘abnormal’ personality in psychiatry he, at first, summarily dismisses the ‘psycho-analytical’ school by writing that it wrongly tries to explain the illness through ‘abnormal activities’ taking place because the individual has been taken out of ‘the normal conditions of the personality . . . and in that connection the environment as a factor of influence becomes predominant’. sjöbring suggests, on the other hand, that there are two main reasons for an ‘abnormal’ personality, a degenerative and an organic reason. the degenerative reason implies that the constitutional, i.e. the inherited, personality is ‘abnormal’. it can be latent and appear only later in life, for example as with dementia praecox (schizophrenia). another possibility is that every single personality variable lies within the normal area, but that an unlucky combination causes the patient to become mentally ill. the organic ‘abnormalities’, on the other hand, can be caused by ‘physical trauma, nutritional disorders, or toxic effects’ (1). on concluding his thesis sjöbring looks ahead to his coming work, trying to identify the most meaningful personality variables: ‘from what i have said, it is clear that most essential and necessary for the work to move forward are further pilot studies to try to ascertain the units of disposition. differential psychology has, as yet, hardly managed to do this at any point, and that serves to show the difficulties that need to be overcome’ (1). the work of identifying the most meaningful variables is described in the subsequent works in upsala läkareförenings förhandlingar and presented in a summarized form in the essay from 1919, ‘psykisk konstitution och psykos’ [‘psychic constitution and psychoses’] (9). much later, in 1974, this essay was translated into english and published in the book themes and variations in european psychiatry (22). the four variables suggested by sjöbring as the psychical, constitutional fundaments are ‘capacity’, ‘validity’, ‘stability’, and ‘solidity’, where low and high variables are referred to, respectively, as suband super-. ‘capacity’ is described by sjöbring as ‘the greater or smaller possibilities for a nerve process’. the closest psychic expression for this capacity is intelligence or aptitude. a sub-capable individual can therefore be described as slow, singleminded, and with a low ability to comprehend complicated issues, while a super-capable individual can differentiate, is open-minded, and with an ability to think quickly. ‘validity’ is the degree of mental energy with which the individual is equipped, and it is apparent, for example, in his or her degree of alertness and ability to work. a super-valid individual is vigorous and full of initiative, but also calm and collected. a sub-valid person, at the opposite end of the scale, is pedantic, long-winded, irritable, and tires quickly. ‘stability’ according to sjöbring, is a variable which in his own words he describes as ‘potential’, or with his invention of a new word as ‘baning’, with which he meant the individual’s ability to form circuits in the brain for new tasks, either of a mental or a physical nature. a simpler description could be the ability, without tiring, to execute a routine activity of a mental or physical kind. a super-stable person is objective and skilful in such a situation and performs with henrik sjöbring and the concept of individual psychology in psychiatry 99 minimal emotion. the fourth variable, ‘solidity’, encompasses the ability to retain the mental process in the form of memory and knowledge. the sub-solid personality is mentally and physically labile and has an erratic temperament. the sub-solid individual can also be charming and appears to be interesting and pleasant. the opposite, the super-solid, is mentally stable, objective, reliable, and thoughtful. on meeting such a person the individual may appear to be dry, boring, and difficult to make real contact with. personality and psychiatry after 1919 and sjöbring’s time in lund the person most often named as a competitor, or co-runner, of sjöbring, is ernst kretschmer. kretschmer was professor of neurology in marburg and became known throughout the world for his book published in 1921, körperbau und character, which has been translated into several different languages. in this work kretschmer linked the body type with the personality, a perception that had existed as far back as classical times. individuals with a pyknic physique, being broad and heavy, show an over-representation of cyclothymic traits, something that is often seen with patients who have a tendency towards cyclical, i.e. manic-depressive disorders. individuals with a leptosomic physique, tall and thin, more often show asthenic, schizoid personalities and an over-representation of patients with schizophrenia. kretschmer’s classification was easy to understand, and, besides, he had a simple, lively, and easily comprehensible way of presenting it, which contributed to his becoming famous, even outside his profession. kretschmer was indeed able to show some empirical proof for his classification, but it lacks relevance in modern psychiatry (18,20,21,23). sjöbring’s constitutional theory was much more difficult to comprehend than kretschmer’s, but it was also considerably more profound and, as would be shown, more relevant with regard to biology and psychology. sjöbring was naturally interested in an academic position to be able to pursue his science, and he therefore applied for professorships in lund in 1923, in stockholm in 1929, and in uppsala the same year. however, the evaluation experts on these three occasions found it hard to comprehend the importance of sjöbring’s work. it is easy to understand sjöbring’s frustration, which is clearly expressed in the 20-page appeal he wrote against the negative decision in 1923 (24). the experts, all of whom had considerable clinical experience, expressed the opinion that sjöbring’s work was too remote from clinical reality. sjöbring, who had tried to erect a general system from both his theoretical and common sense principles responded: ‘under such circumstances it seemed to me not so important to document my findings with clinical examples. i will, however, in no way deny that clinical illumination would not have been desirable and that by so doing i would have found it easier to make myself understood and avoided a lot of misunderstanding.’ sjöbring’s weighty language also in general made it hard to make his thoughts understandable: ‘another remark that has been directed against my work is that i often stereotypically repeat myself. the thing is that i have considered myself forced in various essays to refer, as briefly as possible, to my fundamental philosophy and to provide the necessary terminology.’ august wimmer, professor of psychiatry in copenhagen, was one of the experts. sjöbring summarizes his negative criticism in his appeal, ‘. . . that tries to demonstrate how wimmer’s judgement is incompatible with a full understanding of the problems i raise . . .’. wimmer felt that sjöbring stood for scholastic and constructive formalism and compared him with kretschmer, whom he saw as more clinically grounded than sjöbring. in retrospect it is reasonable to suspect that wimmer found it hard to penetrate sjöbring’s writings, which over and above the complicated wording was written in a foreign language (swedish). sjöbring wrote, with a taunt also directed at kretschmer: ‘it is thus not research which is foreign to the clinical reality, but rather a question of a purposeful attempt to reach out from a construction to a natural and reality-based approach. . . . it is, however, so that we cannot assume that the variations of the constitutional fundament may be able to be interpreted decisively from the surface changes.’ bror gadelius, who was the second evaluation expert, was professor of psychiatry at the karolinska institute from 1908 to 1929 and was considered sweden’s leading psychiatrist during that time. gadelius was contemporary with several of sjöbring’s forerunners, for example kraepelin, bleuler, and janet. gadelius was very clinically minded, but also well versed in the world of research. the svenskt biografiskt lexikon writes of gadelius: ‘with all respect for the researcher freud, he rejected the psychoanalytical dogmas (25). he could not comprehend henrik sjöbring’s attempt to penetrate the depths of the fundamental problems of psychiatry’. sjöbring was undoubtedly disappointed that gadelius had not managed to appreciate his work: ‘this report is virtually wholly an inaccurate representation of what i truly believe, and this is particularly so with the basic philosophy itself . . .’. as an example, gadelius criticized sjöbring’s ideas about the presence of an etiological agent causing an ‘organic illness’ with psychiatric symptoms. however, sjöbring’s conjecture that infections in the brain would be significant for mental disorders was criticized not only by gadelius but also by others. at that time reports of residual mental effects of different kinds of encephalitis were rare. however, in sjöbring’s defence it could be stated that his belief in 100 l. oreland etiological agents proved to be foresighted, since research on the connection between cerebral viral infections and both schizophrenia and depressive disorders is of great current interest with a lot of results supporting such a connection. the third expert, teodor nerander, had for a brief period, 1896–1902, been an extraordinary professor of psychiatry and head physician at the hospital in uppsala, and had most probably met sjöbring during his first year at ulleråker hospital, but in 1902 nerander moved to a post as head physician at lund’s hospital. nerander was considerably more careful in his report than the other two experts, and sjöbring agrees with him that one cannot be certain how strongly his constitutional philosophy is conditioned by genetic factors. like wimmer, even nerander comments on the similarity with kretschmer’s theory of body types, which sjöbring naturally opposes. during the spring of 1929 sjöbring was, however, offered the post of temporary professor and temporary head physician in lund in 1929, and in 1930 he was awarded both posts fully. in lund he had a solid base from which he could teach and gather a group of students around him at a clinic, which was the first psychiatric universitybased clinic in sweden. in general it seems that sjöbring succeeded considerably better in person and orally than in his writing. the young doctors and doctoral students around him came to be called the ‘lund school’, and their use and further development of sjöbring’s system, as well as the easily accessible and clinically based theses and reports, helped to make the system known and frequently used by swedish doctors. sjöbring’s four variables, c, v, st, and so (capacity, validity, stability, and solidity) with plus (+) and minus – signs, were often used as a quick characterization of patients. for example, the combination so–st+ indicated a typical violent person—egocentric, easily aroused, and unmoved by the damage he caused. several members of the lund school became prominent psychiatrists. erik essen-möller succeeded sjöbring as professor in lund, and bo gerle and gunnar lundquist became authors of books and influential scientific reports. torsten frey was appointed professor of psychiatry in uppsala 1960–1974 and bengt lindberg professor of psychiatry in gothenburg (1953–1970). in 1956, at the time of sjöbring’s death, there was no comprehensive picture of his ideas or writings, but in 1958 two of his former students, erik essen-möller and carl-erik uddenberg, edited and published his works in the book struktur och utveckling. en personlighetsteori [structure and development. a theory of personality] (26). the book was translated into french in 1963 and into english in 1973. in addition to writing many articles about sjöbring, essen-möller compiled ‘a sjöbring bibliography’ in 1977 (27). there is also a comprehensive festschrift, with contributions by sjöbring’s pupils, edited by bo gerle and published at the time of sjöbring’s retirement in 1944 (28). naturally, the sjöbring variables were used retrospectively to characterize historical and well-known people as examples. for instance gerle besides presenting an instructive scheme, characterized sacha guitry (light-hearted french writer and actor) as v +so–st+, mussolini and göring as v+so–st–, karl isakson (the extremely shy and self-critical swedish painter) as v–so+st+, virginia woolf as v–so+st+, king gustav iii as v–so–st+, and hans christian andersen as v– so–st– (29). a standardized questionnaire with 60 questions, designed to measure the three variables, solidity, stability, and validity, later became widely acknowledged and useful for clinical studies. the fourth variable, capacity, could be measured better using an intelligence test. the questionnaire was developed by sven marke and g. eberhard nyman and is known as the marke–nyman temperament (mnt) scale (30). during the second half of the twentieth century, the relevance of the sjöbring variables was validated through significant congruity with other internationally used scales for estimating mental conditions. thus, the mnt scale was translated into italian by perris (31), into english by coppen (32), into french by pichot and perse (33), and into english for use in the us by barrett (34). besides a number of clinical studies (27), the validity of the mnt scale has also been confirmed by results showing a correlation with biochemical variables, such as cerebrospinal fluid levels of the serotonin metabolite 5-hiaa (35) and with plasma levels of neuropeptide y and cortisol (36). it was also possible to detect a weak, but nonetheless significant, conformity between sjöbring’s variables and kretschmer’s theory of body types. however, over time other scales, more relevant for clinical use and research, have been developed, and the mnt scale has been replaced with other instruments during the twenty-first century (17,37). by the end of the twentieth century sjöbring’s constitutional typography was beginning to be criticized, for other reasons too. sjöbring’s rejection of psycho-social causes of mental illness and his connection with hypothetical processes in the brain, which started to become irrelevant with the scientific advances in brain research, came to be seen as oldfashioned. the lesion doctrine, which sjöbring promoted increasingly, gained little support from researchers in the mid-twentieth century. this was partly because he called it encephalitis, yet his opponents could only rarely detect that a mentally ill patient had suffered from encephalitis, and in general the idea that a lesional disorder could cause mental symptoms was rejected. sjöbring had even specified that, for example, many schizophrenic symptoms could result from a lesion causing a disruption in the intra-cortical connections—a fully modern henrik sjöbring and the concept of individual psychology in psychiatry 101 interpretation that would have been viewed a few decades ago as extremely speculative. as mentioned earlier, the importance of inflammatory brain conditions for many mental illnesses has been revived and is currently of great interest. henrik sjöbring’s old friend, olof kinberg, tried to characterize sjöbring’s personality in an article in svenska läkartidningen, using sjöbring’s own method (16). as could be expected, kinberg found him to be super-capable (c+). sjöbring’s interest for theoretical argument, the world of ideas, and together with a certain aversion to being associated with concrete objects and tasks was deemed to be super-stable (st+). super-stability could also express itself in a perfectly run house and garden and a strong interest in the fine arts. super-stability also meant that sjöbring had no great need of company, but in small groups where he felt comfortable he could be charming, amusing, boyish, and ironic about himself, which made him very popular—even though people felt they could not get very close to him. kinberg is uncertain about sjöbring’s degree of validity. sjöbring had undoubtedly shown great perseverance and stamina in following through his work programme, but his great summary was never completed, despite his having worked on it for 10 years or more. that may be because, according to his friend kinberg, sjöbring modified his ideas time and again and chose not to publish his work in an unfinished state. as far as the fourth constitutional variable, solidity, was concerned, sjöbring considered that he himself had a minor form of sub-solidity, reflected in his great ability to understand other people by identification and empathy (16). it is hard to know what sjöbring was like as a clinician. bo gerle wrote of sjöbring in his obituary in lunds dagblad: ‘one thing that always sparked his indignation was when a mentally sick person was subjected to unnecessary suffering because of the lack of understanding from the people around, or even more so the narrowmindedness of bureaucratic officialdom. in dealing with his patients, sjöbring had an unusual ability to very quickly reach the root causes . . .’ (11). as an anecdote, bo lindberg, in his book about salomon eberhard henschen, described what happened when salomon’s brother, esaias, was sectioned in connection with possible financial catastrophe: ‘esaias described in a letter to salomon what had happened. when he lay on his hospital bed as a result of coughing up blood from his lungs in february 1921, professor bergmark had entered the ward with associate professor sjöbring. sjöbring, whom he had not met previously, sat on a chair and stared at him for half an hour without saying a word, and later wrote his report that classified him as mentally ill’ (38). henrik sjöbring, who is considered by many to be sweden’s most influential psychiatrist of the twentieth century, must have been a very special person, but a persistent impression after having got to know several of the eminent scientists of the early twentieth century, many of whom published their seminal ideas and discoveries in upsala läkareförenings förhandlingar, is that most of them as a rule had personalities far from the usual—and maybe it takes such people to make new and original developments. declaration of interest: the author reports no conflicts of interest. the author alone is responsible for the content and writing of the paper. references 1. sjöbring h. den individualpsykologiska frågeställningen inom psykiatrien. upsala läkareförenings förhandlingar (nf). 1913–1914;19: 1–60. 2. shepherd m. kraepelin and modern psychiatry. eur arch psychiatry clin neurosci. 1995;245:189–95. 3. ljungberg l. emil kraepelin – en psykiatrins föregångsman. forskning och praktik (sandoz). 1988;20:21–7. 4. kraepelin e. psychologische arbeiten. leipzig: wilhelm engelmann; 1895. 5. essen-möller e. the psychology and psychiatry of henrik sjöbring (1879-1956). psychol med. 1980;10:201–10. 6. sjöbring h. förstämningar och förstämningspsykoser [melancholy and melancholic psychoses]. upsala läkareförenings förhandlingar (nf). 1920;25:73–112. 7. sjöbring h. psykologi och biologi [psychology and biology]. upsala läkareförenings förhandlingar (nf). 1922;28:133–62. 8. sjöbring h. psychic energy and mental insufficiency. upsala läkareförenings förhandlingar 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utlåtanden rörande mina specimen till professuren i psykiatri vid lunds universitet. uppsala: almqvist & wiksells boktryckeri ab; 1923. 25. öberg l. bror e gadelius. svenskt biografiskt lexikon. urn:sbl:14626. 26. sjöbring h. struktur och utveckling, en personlighetsteori. in essen-möller e, uddenberg ce, editors. lund: gleerups; 1973. 27. essen-möller e. a sjöbring bibliography. nord psychiat tidskrift. 1977;81:323–38. 28. gerle b. editor. henrik sjöbring den 9 juli 1944 från vänner, kolleger, lärjungar. lund: gleerupska universitetsbokhandeln; 1944. 29. gerle b. sjöbrings psykiatri och det kliniska vardagsarbetet. nord med. 1957;57:3–11. 30. nyman ge, marke s. sjöbrings differentiella psykologi. analys och skalkonstruktion. lund: gleerups; 1962. 31. perris c. scala temperamentale di marke e nyman. cremona: 1970. 32. coppen a. the marke-nyman temperament scale: an english translation. br j med psychol. 1966;39:55–9. 33. pichot p, perse j. analyse factorielle et structure de la personalité. acta psychiatr scand. 1970:177–82. 34. barrett je jr. sjöbring personality dimensions: norms for some american populations. acta psychiatr scand. 1975;52:107–15. 35. banki cm, arato m. amine metabolites, neuroendocrine findings, and personality dimensions as correlates of suicidal behavior. psychiatry res. 1983;10:253–61. 36. westrin a, engström g, ekman r, träskman-bendz l. correlation between plasma-neuropeptides and temperament dimensions differ between suicidal patients and healthy controls. j affect disord. 1998;49:45–54. 37. dåderman a, lidberg l. självbedömningsskalor avslöjar psykopati. läkartidningen. 1998;95:383–90. 38. lindberg bs. salomon eberhard henschen. en biografi [salomon eberhard henschen. a biography]. acta universitatis upsaliensis. uppsala universitet; 2013. p 109. henrik sjöbring and the concept of individual psychology in psychiatry 103 http://www.ncbi.nlm.nih.gov/pubmed/14798574?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/13400310?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/5904522?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/1146593?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/6199807?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/9574859?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/9574859?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/9492483?dopt=abstract ss1 biographic details about henrik sj&ouml;bring a brief history of the concept of personality up to 1913 sj&ouml;bring forms his theory in articles written in uppsala 1913&ndash;1919 personality and psychiatry after 1919 and sj&ouml;bring&rsquo;s time in lund declaration of interest references personality disorder: a disease in disguise review article personality disorder: a disease in disguise lisa ekselius department of neuroscience, psychiatry, uppsala university, sweden abstract personality disorders (pds) can be described as the manifestation of extreme personality traits that interfere with everyday life and contribute to significant suffering, functional limitations, or both. they are common and are frequently encountered in virtually all forms of health care. pds are associated with an inferior quality of life (qol), poor health, and premature mortality. the aetiology of pds is complex and is influenced by genetic and environmental factors. the clinical expression varies between different pd types; the most common and core aspect is related to an inability to build and maintain healthy interpersonal relationships. this aspect has a negative impact on the interaction between health-care professionals and patients with a pd. from being discrete and categorical disease entities in previous classification systems, the current concept of pd, reflected in the newly proposed icd-11, is a dimensional description based on the severity of the disturbed functioning rather than on the type of clinical presentation. insight about the characteristics of pds among medical practitioners is limited, which is partly because persons do not seek health care for their pd, but instead for other medical issues which are obscured by their underlying personality problems. what needs to be emphasized is that pds affect both the clinical presentation of other medical problems, and the outcome of these, in a negative manner and that the integrated effects of having a pd are a shortened life expectancy. accordingly, pds need to be recognized in clinical practice to a greater extent than previously. article history received 12 august 2018 revised 15 september 2018 accepted 17 september 2018 key words icd-11; personality disorders; personality traits; review article introduction in everyday clinical practice persons who think, feel, behave, or relate to others differently than the average person are identified. this deviation from the norm is a central feature in all personality disorders (pds). although using slightly different formulations over the years, pds are roughly characterized by ‘a pervasive pattern of thought, feeling and behaviour that characterize an individual’s unique lifestyle and mode of adaptation, which deviates markedly from the expectations of the individual’s culture’ (1). such characteristics obviously create problems for those who bear them. pds are likely to have an onset in adolescence or early adulthood, appear to be stable over time, and lead to impairment or distress (1,2). this review, which is an overview on pds and the core problems these ultimately lead to, is commenced with some background information about the concept of personality and on the attempts that have been made to understand and to describe different characteristics of personality, how these characteristics can be structured and understood, and about the deviations in normal personality that form the basis for the different types of pd. above all, the paper focuses on problems met in primary and specialist health care. such problems are common, and persons with pds are professor lisa ekselius, winner of the medical faculty of uppsala university rudbeck award 2017 ‘for her extensive and excellent research in basic and clinical psychiatry and for her immense capacity to motivate everyone around her to flourish’. contact lisa ekselius lisa.ekselius@neuro.uu.se department of neuroscience, psychiatry, uppsala university, university hospital, se-75185 uppsala, sweden supplemental data for this article can be accessed here. � 2018 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2018, vol. 123, no. 4, 194–204 https://doi.org/10.1080/03009734.2018.1526235 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2018.1526235&domain=pdf https://doi.org/10.1080/03009734.2018.1526235 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2018.1526235 http://www.tandfonline.com known to be under-treated with respect to physical health (3) and are over-represented in the group categorized as the ‘difficult patient’ (4,5). the present paper argues that all health-care professionals need basic knowledge about manifestations of different personality traits, above all in the form of manifest pds, as we know that such pathology has a negative effect on the interaction between the patient and health-care professionals in terms of communication, clinical assessment, treatment, and outcome (6). the patients’ suffering is considerable, and generally they report a low qol (7,8). having a pd also infers a risk factor for premature mortality (9,10), which affects individuals and incurs a high cost to society (11). a historic perspective of aberrant personalities the large variation in the way individuals think, feel, and behave has been recognized throughout antiquity. the terms for these characteristics have been diverse. for instance, confucius (551–479 bce) used the combination of ‘blood and vital essence’. the greek philosopher and naturalist theophrastus (c. 371 to c. 287 bc) used the term ‘characters’, and in eighteenth-century france the galenus–hippocrates term ‘temperament’ was reinstituted. the term ‘personality’ has been used since the eighteenth century to label distinguishing qualities of a person (12). pathological personalities have also generated interest over the years. since the fourth century bc, philosophers have been trying to understand what it is that makes ‘us’ what we are. theophrastus, a scholar of plato and aristotle, was the first to publish a systematic description of the multifaceted nature of personality types (12). a few hundred years later, aelius galenus (130–200 ad) linked hippocrates’ four humours to personality characteristics in his description of sanguine, phlegmatic, choleric, and melancholic temperaments. he proposed that each of these four body fluids held a combination of two properties split along two axes: temperature (hot/cold) and humidity (wet/dry). the humoral pathology system influenced the view among european doctors until the breakthrough of medical science in the nineteenth century. in the early nineteenth century, franz joseph gall (1758–1828), a german neuroanatomist, thought that some brain areas were associated with specific functions. he also thought that measurements of the skull represented differences in the individual’s personality (13). philippe pinel (1745–1826), a french physician, was the first to include an aberrant personality in the nosology of psychiatry (14). pinel introduced the term ‘manie sans d�elire’ (mania without delusion). during that time, the term ‘mania’ was employed to refer to states of agitation. pinel described a few of his male patients who were disposed to bursts of irrational anger and impulsive violence in response to minor irritation. in the same intellectual environment jean-�etienne dominique esquirol (1772–1840) introduced the concept monomanie raisonnante and the englishman james cowles prichard (1786–1848) used the term moral insanity. these three physicians were obsessed by the practical question at that time whether psychiatry could explain abnormal behaviour in persons lacking acute psychiatric symptoms who had committed a violent crime (14). during the late nineteenth and early twentieth century, several conceptual systems for normal and abnormal personalities emerged as the result of the work of european psychologists and psychiatrists (e.g. ribot, heymans, lazursky, schneider, and sj€obring). th�eodule ribot (1839–1916), a french psychologist, described normal and abnormal characters. he pointed out that a person’s character is stable from childhood into adult life. ribot described three primary personality types: the sensitive, the active, and the apathetic, all three of which were divided into subtypes (15). the dutch scientist gerard heymans (1857–1930) applied empirical methods to the study of personality. he developed the cube of heymans, a description of a personality typology. he defined personality types in three dimensions: ‘activity level’, ‘emotionality’, and ‘primary versus secondary functioning’, with the last-mentioned dimension comparable to ‘extroversion/introversion’. these three dimensions are represented on the x-, y-, and z-axes of the heymans cube, where all combinations of the three dimensions defined eight personality types (16). the contributions of aleksandr lazursky (1874–1917), a russian psychologist, were not widespread because most of his publications were in russian and because of the political climate of the time. his major contribution was the description of the ‘endopsychic’ and ‘exopsychic’ aspects of personality. the endopsychic components represent the psychological functions (e.g. perception, memory, attention, thinking) that are mainly inborn; the exopsychic components are the consequence of the interaction with the outside world. the interplay between these two aspects of personality determines how a person functions in an integrated social context (17). the german psychiatrist kurt schneider (1887–1967) focused on diagnostic issues that included concepts of ‘psychopathy’, which he had broadly equated to pds. based on his clinical views (18), he vaguely defined abnormal personality as a statistical deviation from the norm. he proposed 10 psychopathic personalities, all of which are very similar to those in the current classifications of pds in the dsm-5 and icd-10 (19). henrik sj€obring (1879–1956), a swedish psychiatrist, suggested four constitution factors of the personality: capacity (intelligence), validity (psychic energy), stability (balance in keynote), and solidity (firmness, tardiness, tenacity). by these variables, all persons can be categorized as either normal, super-, or sub-: e.g. subcapable (unintelligent), subvalid (lack of psychic energy), normosolid, superstable, and so on (20). the first modern attempt to determine the structure of human personality was credited to the english scientist sir francis galton (1882–1911). he used a lexical approach to the dimensions of personality based on the assumption that those personality characteristics important to a group of people will eventually be represented in their language (21). this work was continued by several others (22), and the lexical upsala journal of medical sciences 195 hypothesis constitutes the basis for how current approaches describe personality dimensions. it is also important to mention the work of the psychologists raymond bernard cattell (1905–1998) (23) and gordon willard allport (1897–1967) (24) who independently used advanced statistics (e.g. factor analysis) to discern dimensions of personality. modern concepts of personality disorder and personality before discussing this issue, it needs to be re-emphasized that the description of personalities is based purely on observations, or rather expressions, of the individual’s way to think, feel, behave, or relate. as a corollary, it follows that pds are diagnoses based on symptoms described by the persons themselves, by persons in their surroundings, or are objectively observed in study situations. this circumstance accounts for why the validity and reliability of the current diagnostic instruments lack optimality (25). current knowledge on pathological personalities is primarily based on studies from four perspectives, all of which are necessary to create an in-depth template of what characterizes personality pathology. the first perspective is the clinical picture, i.e. the integrated presentation of the clinical symptoms that are either expressed or witnessed. this perspective is what constitutes the basis for the clinical structured diagnosis according to classification systems. the second perspective entails a determination of underlying dysfunctional personality traits as well as dysfunctional limitations on capacity and functionality in the brain’s cognitive, emotional, and impulse control systems. the third perspective relates to the brain’s biological systems and their functions; this third perspective has highly benefited from the rapid development of brain imaging techniques (26). the fourth perspective denotes the underlying genetic contribution to the above-mentioned phenomena (27), which is currently approached in whole-genome association studies (28). not unexpectedly, studies have shown that the aetiology of personality pathology is complex. overwhelming evidence supports the idea that an interaction between genetic and environmental factors is necessary for the development of human personality. the relation between the dimensions of normal personality and pd is not clear, however. even if a pd has been viewed as an overexpression of personality traits to the extent that they lead to clinically significant distress or impairment, it has recently been demonstrated that a moderate-to-sizable proportion of the genetic influence underlying pd is not shared with the domain constructs of normative personality (29). based on the hypothesis that the domains of dysfunction in pds are linked to specific neural circuits, neuroimaging techniques have been used over the past decade to examine the neural integrity of these circuits in personality-disordered individuals. currently, the literature is flooded with information acquired through this approach. most studies are done to explore borderline pd (30). in general, the studies have thus far demonstrated deviations in neuronal circuitry in areas previously found to be active in the symptomatology that characterizes the specific type of pd. even if the results of such studies contribute to an understanding of underlying physiological processes, they are not yet ready to be used in clinical practice. several studies have examined the effects of being exposed to childhood adversities and the risk to develop pd. just to mention one such study, we recently showed that exposure to cumulative childhood adversity was incrementally associated with a diagnosis of pd in young adulthood (31). furthermore, childhood or adolescent psychiatric disorders have been suggested to trigger a chain of behaviours and responses that foster the more persistent psychopathology of a pd (32,33). to determine the importance of genetic and environmental factors in early childhood in personality pathology the relationship between vulnerability to child abuse and antisocial personality patterns in adulthood was investigated (34). it was shown that individuals with a gene polymorphism that resulted in a low activity in monoamine oxidase a (maoa) were more vulnerable to developing an antisocial personality pattern than those who had high activity in the maoa gene, given that they had been exposed to child abuse. this gene–environment interaction has subsequently been confirmed (35). moreover, a similar interaction for the effects of child maltreatment on antisocial behaviour has also been shown for other genes (36,37). there is thus reason to consider genetic and environmental factors as interacting systems of crucial importance in the development of functional and dysfunctional personality traits. classification of personality disorders the differences in the types of aberration in thought, feeling, and behaviour have been the basis for the classification of different pds. the characteristics described by galenus, and later by e.g. pinel and schneider, are very similar to contemporary classification systems. what today are referred to as pds were earlier called ‘pathological personalities’ or ‘persona pathologica’ and were found under that heading in earlier versions of the icd (up to icd-8). these diagnoses were used rarely, in part because of their stigmatizing connotations. up to now, classification of pds has been based on fulfilling a specified number of defined and ‘specific’ criteria for each pd, resulting in a categorical description; if a defined number of these criteria were met, a disorder was acknowledged, else not. over time, there has been an intraprofessional dispute on whether the classification of pds should be based on the defined and specific characteristics or on the severity of the functional aberration. historically, and currently, in the icd10 (which is from 1992) and in the current american dsm-5 (38) (from 2013) classification is based on types of symptom, i.e. characteristics of the clinical presentation, represented by the abovementioned ‘specific’ criteria for each pd. icd-10 describes nine discrete and specific (as well as one unspecified) types of pd (table 1). the dsm-5 (38) identifies 10 pds of similar structure. the dsm system has gone one step further in classification by grouping the different disorders in three clusters based on some overall common features. to illustrate, cluster a contains odd and eccentric personalities, 196 l. ekselius cluster b includes dramatic, impulsive, emotional personalities, and cluster c fearful and anxious personalities. a basic feature common for the different classification systems is that the aberration must be severe enough to cause a functional impairment in everyday life. this is the ‘general criterion’ for all pds and overrides other perspectives. in other words, even the observance of very odd behaviour or feelings is not enough for a clinical diagnosis of a pd unless it can be ascertained that they lead to impairment or distress in everyday life. currently, there is somewhat of a paradigm shift in that more and more arguments speak for the relevance of a dimensional classification of pd based on the severity of symptoms rather than on the specific characteristics (19). studies have, thus, shown that the conceptualization of pds into discrete categories results in an insufficient description of the problem. rather, it seems that within each discrete pd category the level of dysfunction is dimensional and dependent on the number of criteria fulfilled (39). furthermore, only about half of all individuals with diagnosable pd fulfil criteria for a specific pd and are thus given a diagnosis of unspecified pd (40). in addition, the expression of different symptoms evolves continuously across the lifespan (41). general description of personality disorders in icd-11 the new icd-11 classification system, which was released by who in june 2018 (42), means a radical change in the classification of pds and will impact all aspects of health care, as well as influence the way pds are seen (43). after 1–2 years of adaptive work, icd-11 is expected to be operative internationally in 2020–2021. in icd-11 pds have been classified based on the perspectives mentioned above, i.e. according to the severity of suffering, and are divided into three severity groups: mild, moderate, or severe. the degree of severity is determined by the extent of problems in interpersonal relationships or the ability and willingness to perform expected social and occupational roles, or both (see table 2 for a full description). a new feature in icd-11 is the introduction of the term ‘personality difficulty’, which refers to pronounced personality characteristics that may affect treatment or health services but do not rise to the level of severity to merit a diagnosis of pd. dimensions of personality disorders in icd-11 icd-11 has, thus, wiped out all type-specific categories of pd apart from the main one, the presence of pd itself. instead, the type of clinical manifestation is added as a specific ‘postcoordination’ code describing the clinical characteristics in the form of six different personality domains. factor analytic strategies have supported five domains, although clinical reasoning has suggested six domains (44–46). these six clinically derived personality domains do not fully correspond with the different specific pd types in the earlier icd-10 (table 2). the domain traits are not inherently pathological, but rather represent a profile of underlying personality structure (19). they apply equally to individuals without any pd and to those with severe disorder, but in pd they show where the focus of the disorder is apparent. in severe disorder, several table 1. personality disorders in the icd-10 (2). code disorder characteristics in brief f60.0 paranoid excessive sensitivity to setbacks, unforgiveness of insults, recurrent suspicions without justification regarding the sexual fidelity of the spouse or sexual partner, and a combative and tenacious sense of personal rights. f60.1 schizoid withdrawal from affectional, social, and other contacts, preference for fantasy, solitary activities, and introspection. limited capacity to express feelings and to experience pleasure. f60.2 dissocial disregard for social obligations, callous unconcern for the feelings of others. gross disparity between behaviour and prevailing social norms. behaviour not readily modifiable by adverse experience, including punishment. low tolerance to frustration; low threshold for discharge of aggression, including violence; tendency to blame others, all leading to conflict with society. f60.3 emotionally unstable a tendency to act impulsively and without consideration of the consequences; unpredictable and capricious mood. liability to outbursts of emotion and incapacity to control the behavioural explosions. tendency to quarrelsome behaviour and to conflicts with others. two types are distinguished: the impulsive type with emotional instability and lack of impulse control; and the borderline type, with added disturbances in self-image, aims, and internal preferences, chronic feelings of emptiness, intense and unstable interpersonal relationships, and a tendency to self-destructive behaviour, including suicide gestures and attempts. f60.4 histrionic shallow and labile affectivity, self-dramatization, theatricality, exaggerated expression of emotions, suggestibility, egocentricity, self-indulgence, lack of consideration for others, easily hurt feelings, and continuous seeking for appreciation, excitement, and attention. f60.5 anankastic feelings of doubt, perfectionism, excessive conscientiousness, checking and preoccupation with details, stubbornness, caution, and rigidity. there may be insistent and unwelcome thoughts or impulses that do not attain the severity of an obsessive-compulsive disorder. f60.6 anxious [avoidant] feelings of tension and apprehension, insecurity and inferiority. a continuous yearning to be liked and accepted, hypersensitivity to rejection and criticism with restricted personal attachments, and a tendency to avoid certain activities by habitual exaggeration of the potential dangers or risks in everyday situations. f60.7 dependent pervasive passive reliance on other people to make one’s major and minor life decisions, great fear of abandonment, feelings of helplessness and incompetence, passive compliance with the wishes of elders and others, and a weak response to the demands of daily life. lack of vigour may show itself in the intellectual or emotional spheres; often a tendency to transfer responsibility to others. f60.8 other specific eccentric, ‘haltlose’ type, immature, narcissistic, passive-aggressive, psychoneurotic. f60.9 unspecified diffuse symptoms, not fully qualifying for specific pd, but with the general criterion fulfilled. upsala journal of medical sciences 197 domain traits are likely to be associated with the disorder (47). to describe personality functioning, as many domains as necessary can be applied. the descriptions of the six different domain traits below are slightly and only linguistically modified from those in the original icd-11. negative affectivity in personality disorder the core aspect of negative affectivity is the tendency to experience a broad range of negative emotions. common manifestations, not all of which may be present in everyone at a given time, include experiencing a variety of negative emotions with a frequency and intensity out of proportion table 2. personality disorders in the forthcoming icd-11. 6d10 personality disorder description personality disorder is characterized by problems in functioning of aspects of the self (e.g. identity, self-worth, accuracy of self-view, self-direction), and/or interpersonal dysfunction (e.g. ability to develop and maintain close and mutually satisfying relationships, ability to understand others’ perspectives and to manage conflict in relationships) that have persisted over an extended period of time (e.g. 2 years or more). the disturbance is manifested in patterns of cognition, emotional experience, emotional expression, and behaviour that are maladaptive (e.g. inflexible or poorly regulated) and is manifested across a range of personal and social situations (i.e. is not limited to specific relationships or social roles). the patterns of behaviour characterizing the disturbance are not developmentally appropriate and cannot be explained primarily by social or cultural factors, including socio-political conflict. the disturbance is associated with substantial distress or significant impairment in personal, family, social, educational, occupational, or other important areas of functioning. 6d10.0 6d10.1 6d10.2 mild moderate severe description all general diagnostic requirements for personality disorder are met. disturbances affect some areas of personality functioning but not others (e.g. problems with self-direction in the absence of problems with stability and coherence of identity or self-worth) and may not be apparent in some contexts. there are problems in many interpersonal relationships and/or in performance of expected occupational and social roles, but some relationships are maintained, and/ or some roles carried out. specific manifestations of personality disturbances are generally of mild severity. mild personality disorder is typically not associated with substantial harm to self or others but may be associated with substantial distress or with impairment in personal, family, social, educational, occupational, or other important areas of functioning that is either limited to circumscribed areas (e.g. romantic relationships, employment) or present in more areas but milder. all general diagnostic requirements for personality disorder are met. disturbances affect multiple areas of personality functioning (e.g. identity or sense of self, ability to form intimate relationships, ability to control impulses and modulate behaviour). however, some areas of personality functioning may be relatively less affected. there are marked problems in most interpersonal relationships and the performance of most expected social and occupational roles are compromised to some degree. relationships are likely to be characterized by conflict, avoidance, withdrawal, or extreme dependency (e.g. few friendships maintained, persistent conflict in work relationships and consequent occupational problems, romantic relationships characterized by serious disruption or inappropriate submissiveness). specific manifestations of personality disturbance are generally of moderate severity. moderate personality disorder is sometimes associated with harm to self or others, and is associated with marked impairment in personal, family, social, educational, occupational, or other important areas of functioning, although functioning in circumscribed areas may be maintained. all general diagnostic requirements for personality disorder are met. there are severe disturbances in functioning of the self (e.g. sense of self may be so unstable that individuals report not having a sense of who they are or so rigid that they refuse to participate in any but an extremely narrow range of situations; self-view may be characterized by self-contempt or be grandiose or highly eccentric). problems in interpersonal functioning seriously affect virtually all relationships, and the ability and willingness to perform expected social and occupational roles is absent or severely compromised. specific manifestations of personality disturbance are severe and affect most, if not all, areas of personality functioning. severe personality disorder is often associated with harm to self or others and is associated with severe impairment in all or nearly all areas of life, including personal, family, social, educational, occupational, and other important areas of functioning. 6d11 prominent personality traits or patterns description trait domain qualifiers may be applied to personality disorders or personality difficulty to describe the characteristics of the individual’s personality that are most prominent and that contribute to personality disturbance. trait domains are continuous with normal personality characteristics in individuals who do not have personality disorder or personality difficulty. trait domains are not diagnostic categories, but rather represent a set of dimensions that correspond to the underlying structure of personality. as many trait domain qualifiers may be applied as necessary to describe personality functioning. individuals with more severe personality disturbance tend to have a greater number of prominent trait domains. 6d11.0 negative affectivity in personality disorder or personality difficulty 6d11.1 detachment in personality disorder or personality difficulty 6d11.2 dissociality in personality disorder or personality difficulty 6d11.3 disinhibition in personality disorder or personality difficulty 6d11.4 anankastia in personality disorder or personality difficulty 6d11.5 borderline pattern excerpt from reference (41) with permission from who. 198 l. ekselius to the situation: emotional lability and poor emotion regulation, negativistic attitudes, low self-esteem, low self-confidence, and mistrustfulness. patients fulfilling criteria for this disorder were classified as anxious/avoidant in previous classifications. detachment in personality disorder the core aspect of the detachment domain is the tendency to maintain interpersonal (social detachment) and emotional distance (emotional detachment). common manifestations, not all of which may be present in everyone at a given time, include social detachment (avoidance of social interactions, lack of friendships, and avoidance of intimacy) and emotional detachment (reserve, aloofness, and limited emotional expression and experience). this disorder type is like the schizoid type of pd described in icd-10. dissocial or antisocial personality disorder dissocial or antisocial pd is characterized by a gross disparity between behaviour and the prevailing social norms as well as by a callous unconcern for the feelings of others. moreover, this type of pd can be described by a number of other attributes, including a gross and persistent attitude of irresponsibility and disregard for social norms, rules, and obligations; an incapacity to maintain enduring relationships, although having no difficulty in establishing them; very low tolerance to frustration and a low threshold for discharge of aggression, including violence; an incapacity to experience guilt or to profit from experience, particularly punishment; and finally, a marked proneness to blame others or to offer plausible rationalizations for the behaviour that has brought the person into conflict with society. persons with a dissocial pd often have an early criminal record and exhibit conduct problems in childhood or adolescence. the construct of a dissocial pd largely overlaps characteristics of the concept of psychopathy, which is not a term used to define a psychiatric disorder (48). disinhibition in personality disorder the core aspect of disinhibition traits is the tendency to act rashly based on immediate external or internal stimuli (i.e. sensations, emotions, thoughts) without consideration of the consequences. common manifestations—not all of which may be present in everyone at a given time—include impulsivity, distractibility, irresponsibility, recklessness, and lack of planning. patients fulfilling the criteria for this disorder in previous classifications were classified as histrionic, narcissistic, or borderline. anankastia in personality disorder anankastic pd (or obsessive-compulsive pd) is characterized by a narrow focus on orderliness and perfectionism and on right and wrong, although it also implies a need to control one’s own behaviour and the behaviour of others, as well as the need to control one’s environment to ensure conformity to these standards. common manifestations, not all of which may be present in a given individual at a given time, can include conscientiousness (e.g. concern with social rules, obligations, and norms of right and wrong, scrupulous attention to detail, rigid, systematic, day-to-day routines, obsessiveness about hyper-scheduling, emphasis on organization, orderliness, and neatness) and emotional and behavioural constraint (e.g. rigid control over emotional expression, stubbornness and inflexibility, risk-avoidance, perseveration, and deliberativeness). borderline personality disorder the criteria for this disorder are very similar to those for disinhibition. it was included in the new icd-11 at a very late stage of the process (45). the classification may be applied to individuals whose pattern of personality disturbance is characterized by a pervasive pattern of instability of interpersonal relationships, self-image, and affects, as well as marked impulsivity, as indicated by many of the following behavioural patterns: frantic efforts to avoid real or imagined abandonment; a pattern of unstable and intense interpersonal relationships; identity disturbance, manifested in markedly and persistently unstable self-image or sense of self; a tendency to act rashly in states of high negative affect, leading to potentially self-damaging behaviours; recurrent episodes of self-harm; emotional instability due to marked reactivity of mood; chronic feelings of emptiness; inappropriate intense anger or difficulty controlling anger; and transient dissociative symptoms or psychotic-like features in situations of high affective arousal. the condition involves anxiety without an identifiable connection to concrete stimuli and, among other things, has been called ‘annihilation anxiety’, ‘pan-anxiety’, or ‘global anxiety’. the term ‘emptiness depression’ describes general feelings of despair and hopelessness with dominance of depressive thoughts. borderline pd (in icd-10 ‘emotionally unstable pd’) is a dominating diagnosis in outand inpatient psychiatric care (9,10). clinical expressions are more evident in younger ages and tend to decrease with advancing age. borderline pd is associated with high mortality by suicide (9,10,49). there is also a high risk to die prematurely because of impulsive risk-taking, as well as succumbing to violence from others (9,10). recurrent suicidal threats or attempts, when combined with fears of abandonment, are strongly indicative of the diagnosis (50). even if these characteristics make borderline pattern pd easy to identify, the diagnosis is often overlooked. a key reason for this neglect is the perception that the overemotional, sometimes theatrical, and selfinjurious behaviours are signs of wilfulness and manipulations rather than signs of an illness (51). borderline pd occasionally includes depressive and anxiety symptoms and mild irritability. in general, many persons with borderline pd describe recurrent occasions with panic anxiety, which may lead to suspicion of a primary panic disorder or generalized anxiety disorder. likewise, experienced upsala journal of medical sciences 199 social discomfort and fears can arouse suspicion of primary social anxiety disorder. finally, recent studies have suggested that attention deficit hyperactivity disorder (adhd), bipolar disorder, and borderline pd have similar origins or share common pathological mechanisms (52,53). prevalence and longitudinal perspective pds are common in the general population. a recent overview (54), based on 13 studies conducted in the usa and europe, reported prevalence figures varying from 3.9% to 15.5%. in the who world mental health survey, carried out in 13 countries, the prevalence rate was 6.1% (55). the large variation in prevalence may be due to differences in sampling, diagnostic methods, and study settings. furthermore, there may be differences in culture regarding significant personality pathology. persons in contact with the health-care system exhibit higher prevalence of pds as compared with those not in contact. in fact, one-fourth of patients in primary care (56) and about half of those in psychiatric outpatient facilities fulfil the criteria for a pd (57). pds are equally common or more common in men (54) in the general population. in clinical settings, however, pds are more often recognized in women, probably due to the higher rates of help-seeking behaviour in women (9,10). stability over time has long been a basic concept both in the description of personality and of pds. supporting this concept is the observation that there is rank-order stability over time in the expression of personality symptoms (58). on the other hand, an exaggeration in some personality traits over the life course and a decline in others have been observed (41,59). furthermore, it has recently been shown that drugs affecting serotonin uptake can modulate personality traits (60,61). there is less support for the idea that a pd should be regarded as stable over time. actually, modern research has shown that, although a maladaptive personality can be recognized early in life, it evolves continuously across the lifespan and is more plastic than previously believed (41). in addition, in the case of coexisting mental disorders, their contribution to the clinical picture will vary over time with the state of these disorders. in other words, even if personality traits are largely constant across time, there is a tendency that symptoms in persons with a pd change more over time than those without a pd. this change is often in the direction of improvement (62,63). diagnosis, differential diagnosis, and psychiatric comorbidity establishing a formal diagnosis of a pd is an issue for specialist psychiatry, where it must be regarded as a time-process function. the patient history must cover the life perspective to understand the current clinical landscape in context and against a background of the individual’s unique developmental history. general and permanent problems in work, studies, and relationships are often primary and obvious observations. difficulties in interpersonal relations are often visible already at the first patient encounter. those difficulties justify a stepby-step deepening of the formal diagnostic work while initiating treatment efforts. enhanced personal knowledge will also provide a more nuanced image of the patient’s problems as well as adaptive resources. accounts of the current problem and the patient’s current life situation are a natural starting point when collecting data on the clinical history of the patient. special attention must be given to the risks of suicide and violence. the clinical history should be expanded in a piecemeal manner on appropriate occasions. during the diagnostic process, it often becomes clear that the patient presents with criteria for several disorders, both within and outside the pd spectrum. such comorbidity is common (64,65); it is seen across the whole spectrum of pds and other mental disorders and in general represents a broader pattern of symptoms as well as a more severe condition. this is reflected in the observation that the total number of fulfilled criteria for any pd is related to the observed dysfunction and to the reported qol (54). comorbidity between pds and other mental disorders contributes significantly to functional impairment (64) while also increasing the risk of early mortality (9,10). because of this characteristic, it is not uncommon that the person who fulfils criteria for a diagnosis of pd will seek health care for another mental disorder, a fact that might be misleading during the diagnostic process. not too infrequently, there are rapid onset depressive or anxiety states that motivate the care episode during which the coexisting problems related to a comorbid pd are apparent. a more pressing issue is comorbidity of a more enduring character. for example, a coexisting adhd can obscure the clinical symptoms of borderline pd. conversely, a severe and prolonged eating disorder may dominate over an underlying personality pathology. when the symptomatic picture of pd is complex and partially overlapping between different diagnoses, it is seldom possible to distinguish between different underlying pathologies. the differential diagnostic procedure will therefore be more about evaluating the relative influence of the various demonstrable expressed symptoms on the severity of functioning. to optimize diagnostic accuracy self-assessment tools, semi-structured interviews and personality inventories can be used. the scid-ii is such an interview support for personality diagnosis according to the dsm-iv and dsm-5 (66). personality disorders and health the long-term negative effects of having a pd are significant (9,10,41,54). furthermore, because a pd is often overlooked diagnostically, the potential risks for the bearer may go undetected. a certain proportion of those who fulfil the criteria for a diagnosis of pd will ultimately have psychiatric care, while almost everyone eventually comes in contact with primary care or specialized somatic care. given that personalityrelated problems lead to varying degrees of lack of adaptivity in interaction with other people, there are often complications in the contacts with health care and social services. 200 l. ekselius personality traits are well known to impact health-related qol (8) and outcome in health care. the negative consequence of having a high degree of neuroticism has been extensively studied (67). the consequence of having a pd is, however, only well studied in psychiatric care, where a multitude of studies have shown that having a comorbid pd represents a more severe condition with a worse prognosis as stated above. there has been less focus on somatic care. still, it has been shown that having a pd is related to higher rates of pain, greater use of analgesics, and more primary care, taken together suggesting an increase in somatic morbidity (68). it has also been shown that the outcome of treatment for somatic ill health is usually worse in the presence of a pd (68). for instance, having a pd was found to increase the risk of stroke (69) and coronary artery disease (70,71). furthermore, having any pd is strongly associated with severe occupational outcomes, including disability benefits, regardless of disability diagnosis (72–74). the most studied pd in this respect is borderline pd. persons with this pd tend to be impulsive, and where selfharm is common they have an increased tendency to seek health care (75). borderline pd, however, is related to an increased risk for several health problems, and consequently for a greater consumption of health care (76–79). based on previous knowledge that individuals with a pd have a higher mortality rate and a shorter life expectancy compared with the general population (69,80–83), we recently assessed to what extent this was related to type of pd or cause of death (9,10). data from nationwide swedish hospital registers with a follow-up of 25 years were used. overall, all-cause standardized mortality ratios (smrs) were found to increase in all clusters of pds for natural and unnatural causes of death. the overall smr was 6.1 in women and 5.0 in men, figures in line with those previously reported for anorexia nervosa, with higher rates in cluster b and unspecified/other pds. the increased mortality ratio was seen for all somatic causes of death, reflecting the impact of having a pd on somatic health and wellbeing. the smr for suicide was as high as 34.5 in women and 16.0 in men for cluster b disorders. somatic and psychiatric comorbidity increased smrs further. this excess mortality was also observed for most patients diagnosed with pds not severe enough to lead to hospitalization (9). this observation contradicts the idea that only those persons with a pd severe enough to motivate inpatient treatment are burdened by an increased risk in mortality. this has important practical implications in that most patients with clinical problems linked to a pd are only treated as outpatients. on the other hand, the risk of death in those only treated as outpatients was clearly less than in those who received inpatient care, supporting the view of a difference in clinical severity between these groups (47). aspects on handling and treatment given the impact of pds on treatment outcomes in somatic and mental health care, a clinical pattern suggesting the existence of such a disorder should be identified in primary or somatic specialist care (84). treatment is a concern for specialist psychiatry, however. a well-developed liaison psychiatry, a subspecialty of psychiatry, is particularly suitable. even if treatment modalities are not the topic of this survey, some basic principles must be addressed. because pds are deeply ingrained ways of thinking and behaving that evolved as the personality developed, they are considered difficult to treat. in recent years several studies have emerged that have, to some extent, changed this concept (85–88). in general, there are many challenges and no simple solution in the treatment of pds. at the same time, because fundamental problems of pds are related to interpersonal relations, a structured and stable relationship between the patient and the clinician is the basis for any successful approach. this ‘therapeutic alliance’ looms as the strongest predictor of successful outcome of any treatment attempt. the most difficult challenge for the clinician is to achieve this goal. in fact, the pre-existing quality of the patient’s relationships, rather than the type of pd, is the single factor that most affects the quality of this alliance (89). the strength of this alliance is crucial not only to obtain anamnestic information about the true history of the problems encountered but also to motivate and maintain adherence to treatment. although there is only a paucity of randomized controlled studies on the effect of psychotherapy in pd (90), the few studies published suggest that it should be the core treatment (91), leading to individual benefit and a reduction in care costs (92). currently, no pharmaceuticals are registered for use in pds. any attempt to apply a pharmacological approach is therefore an issue for the psychiatric specialist. such an approach should aim to reduce or eliminate specific symptoms seen in other psychiatric disorders, where there is evidence that the drug in question is efficient. irrespective of which drug is used, the clinical effect should be closely monitored. the underlying hypothesis when attempting psychotropic pharmaceuticals in the treatment of pds is the assumption that the features and attributes associated with the clinical expression are linked to biochemical abnormalities and thus can be regulated by psychotropic drugs. for most specific pds, studies on the putative benefit of pharmaceuticals are lacking, and where studies have been done the results are at best modest (93). despite this limitation, most psychiatrists can testify about patients with pds who are prescribed many drugs, often over a long time, and without information about the expected or obtained benefit, or how the treatment was followed up. such polypharmacy, particularly in combination with poor documentation, can put patients at risk of adverse drug events (side effects) and interactions. in other words, drugs should never be the first-line treatment but may be justified as a supplement to other treatment forms in specific situations. conclusion pd frequently goes undetected, in the shade of other health problems or diseases. pd is a predictor of worse health, upsala journal of medical sciences 201 premature death, and more serious life issues. it constitutes a challenge to health-care professionals and, above all, a burden for the patient, the family, and society. pd involves deviations in cognition, affectivity, interpersonal functioning, and/or impulse control. deepened knowledge requires intellectual approaches based on sociodemographic, as well as epidemiological and advanced genetic and imaging techniques. there is a clinical shift from an earlier focus on the characteristics of discrete pd entities to an awareness of the common features of different pds, the suffering of patients, and the many problems they face in interpersonal relationships and daily life. the new icd-11 classification aims to improve the description of the severity of problems encountered by patients. knowledge of the clinical aspects of pds in general health care, vigilance to symptoms of pd, and appropriate diagnosis are all essential for optimal support to affected patients. disclosure statement no potential conflict of interest was reported by the authors. note on the contributor lisa ekselius, md, phd, is a full professor of psychiatry at uppsala university and a senior consultant in psychiatry at the uppsala university hospital. her research is focused on issues related to personality and personality disorders. these include the epidemiology of personality disorders, but also the contribution of personality traits to the 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principles, methods, and processes. washington, dc: american psychological association; 2010. 92. meuldijk d, mccarthy a, bourke me, grenyer bf. the value of psychological treatment for borderline personality disorder: systematic review and cost offset analysis of economic evaluations. plos one. 2017;12:e0171592. 93. duggan l, duggan c, huband n, smailagic n, ferriter m, adams c. the use of pharmacological treatments for people with personality disorder: a systematic review of randomized controlled trials. personality and mental health. 2008;2:119–70. 204 l. ekselius abstract introduction a historic perspective of aberrant personalities modern concepts of personality disorder and personality classification of personality disorders general description of personality disorders in icd-11 dimensions of personality disorders in icd-11 negative affectivity in personality disorder detachment in personality disorder dissocial or antisocial personality disorder disinhibition in personality disorder anankastia in personality disorder borderline personality disorder prevalence and longitudinal perspective diagnosis, differential diagnosis, and psychiatric comorbidity personality disorders and health aspects on handling and treatment conclusion disclosure statement note on the contributor references bok 03-06.indb femoral subsidence assessment after hip 361 received 15 november 2005 accepted 18 august 2006 femoral subsidence assessment after hip replacement an experimental study thomas ilchmann1, christoph eingartner2, katharina heger2, kuno weise2 1department of orthopedics, kantonsspital, ch-4410 liestal, switzerland and 2bg trauma centre, university of tübingen, d-72072 tübingen, germany abstract background: in an experimental set up information is to be gained on the error of measurement for subsidence assessment of the stem after hip replacement. methods: subsidence was measured with a pencil and ruler for four different reference lines and with the computerized ebra-fca method. hip flexion, rotation and abduction were simulated in a standardized way. in a second experimental set up, subsidence was simulated in defined steps. results: in the tilt study, the maximum error of measurement was 7 mm with standard methods and 1.7 mm with the ebra-fca method. in the subsidence study, there was a maximum error of measurement of 1.9 mm with the standard methods. with ebra-fca, the maximum error of measurement was 0.2 mm when taking all radiographs into account. conclusions: the main error of subsidence measurement is caused by tilt of the femur for standard methods and partly can be reduced by ebra-fca. ebra-fca is more reliable than standard methods but might underestimate the actual subsidence in a clinical situation. introduction the assessment of prosthetic migration and subsidence after hip replacement is an established means of quality control. early and continuous motion of the implant against the surrounding bone can predict a higher percentage of late aseptic loosening (1, 2). in cup analysis it was shown that pelvic tilt is the main source for errors of measurement with standard methods (3, 4) which partly can be detected and corrected with the computerized ebra method (5). for subsidence measurements of femoral components various prominent bone markers were proposed. choosing reference lines close to the implant, correcting radiographic magnification and using highly standardized radiographs may improve measurement accuracy (6–8). whether the implantation of metal markers can improve the accuracy of standard measurements is discussed controversially (3, 9, 10). as for the cup, the main source of error of measurement seems to be eventual tilt and rotation of the femur. the ebra-fca method is thought to detect projection differences, thus reducing the error of measurement (6, 9). in an experimental study we tried to gain further information on eventual errors of measurement. the effect of tilt of the femur on the measurements was tested for upsala j med sci 111 (3): 361–369, 2006 362 t. ilchmann et al. ebra-fca and standard measurements. furthermore, subsidence of the implant was simulated to get information about the measurement accuracy in case of implant loosening. material and methods methods of measurement subsidence was measured with pencil and ruler methods in four different ways (3). a line was drawn parallel to the longitudinal axis of the femur through the centre of the medullar canal. a perpendicular line was drawn to the femoral axis, being a tangent to the tip of the major trochanter. another perpendicular line was drawn through the most prominent point of the lesser trochanter. the centre of the femoral head was marked with a template of concentric circles. the tip of the prosthetic shoulder was defined as the most prominent lateral edge of the implant. the distance of the head-centre to the major and lesser trochanter (h.-major, h.lesser) and of the tip of the prosthetic shoulder to the major and lesser trochanter (s.-major, s.-lesser) were measured. variations in the distance as compared to the neutral position were seen as subsidence (figure 1). radiological enlargement was calculated from the measured head-size in relation to the real head-size of 32 mm. all radiographs were measured in random order by one person in the same way. the ebra-fca measurements were made as described by biedermann et al. (6). the radiographs were digitised and the reference lines drawn on the screen. extreme projection differences were to be detected and excluded from analysis. only radiographs with similar projections were to be used by the software for subsidence assessment. each radiograph was marked with a different date of examination, simulating one radiographic examination per day and permitting a change of the order of the radiographs by changing the dates. the data were presented as time-subsidence graphs and plotted in excel files for statistical analysis. 1. tilt study an uncemented staight femoral component (bicontact®, aesculap®, germany) was implanted in a human plastic right (synbones®) femur and in a left femur (sawbones®), respectively. the bones used differed in size and shape and the prosthetic femoral components differed in the position of implantation. a polyethylene cup was fixed in 40° inclination and slight anteversion in the testing machine, simulating the geometry of a standard hip replacement and modular 32 mm metal heads were used. the hip could be flexed and extended, abducted and adducted, internally and externally rotated and fixed in each desired position (figure 2). starting from a zero position, consecutive movements were made in two degree steps up to 16 of degrees flexion and 4 of degrees extension, 10 degrees of abduction and 8 degrees of adduction, 10 femoral subsidence assessment after hip 363 degrees of internal and external rotation and controlled with a goniometer. furthermore, combined movements of flexion and rotation were made up to 8 degrees of flexion and 6 degrees of external rotation. for each position a standardized radiograph of the hip was taken, centred in the middle of the femoral component. a left and a right hip were examined, 36 radiographs were produced for each side. the radiographs were sorted in subgroups of linear increasing flexion, abduction and internal rotation to detect eventual systematic errors due to consecutive tilt. in order to simulate a more clinical situation, subgroups of five radiographs were selected randomly out of the data-pool and measured with all methods. 2. subsidence study the same type of femoral component was fixed with a custom-made device in a right plastic femur. it could be moved in the femoral canal along the femoral figure 1. standard measurements of subsidence. the femoral axis is marked (line 1) and a perpendicular line drawn tangential to the tip of the major trochanter (line 2) and through the most prominent point of the lesser trochanter (line 3). the distances from the head centre (a, b) and from the tip of the prosthetic shoulder (c, d) to line 2 and 3, respectively, are measured. distance a is the head – major trochanter distance (h.-major), b the head – lesser trochanter distance (h.-lesser), c the shoulder – major trochanter distance (s.-major) and d the shoulder – lesser trochanter distance (s.-lesser). 364 t. ilchmann et al. axis by turning a handlebar, simulating subsidence. the amount of subsidence was measured with a slide calliper (figure 3). the implant was moved in 0.2 mm steps up to a maximum subsidence of 7.0 mm. for each step a standard radiograph of the hip was taken with the femur in the same neutral position. all 36 radiographs were measured with all methods. data was collected on a pc and processed with ms excel. in the tilt study, the absolute differences of the measurements of the radiograph in neutral position and in the subsidence study, the absolute difference of actual subsidence were seen as errors of measurement. mean, median and standard deviation of those errors of measurement were calculated. results 1. tilt study in the right femur, the distance of the head centre to the tip of the major trochanter was 8.1 mm in neutral position. the head size was 39.5 mm, meaning an enlargement of 1.2. figure 2. simulation of flexion, abduction and rotation. the angles of the guide wires in relation to the board were measured with a goniometer. each position was fixed with screws when taking a radiograph. femoral subsidence assessment after hip 365 when looking at the radiographs in 0 degrees and 16 degrees of flexion, the measured length of the stem on the radiographs differed by 1.7 mm. the most obvious projection differences were found in the shape of the proximal hole and in the projection of the shoulder of the implant. with the standard measurements the maximum error was 7.5 (sd 2.35) mm and was found for measurements of the head centre in relation to the lesser trochanter (table 1). the main error was found for movements in extension-flexion (mean 4.0 mm, sd 2.90), followed by abduction-adduction (mean 1.0 mm, sd 0.95) and external-internal rotation (mean 0.7 mm, sd 0.42). for the combined movements, the maximum error was 2.6 (mean 1.2, sd 0.99) mm. the error of measurement depended on the chosen reference lines and was minimal for the distance shoulderlesser trochanter (table 1). with ebra-fca, the maximum error of measurement was 0.4 (mean error 0.2, sd 0.13) mm, taking all 30 radiographs in random order. when the radiographs were taken in a consecutive order of tilt, the maximum error of measurement was 1.4 (mean 0.8, sd 0.56) mm for extension-flexion, 0.4 (mean 0.1, sd 0.14) mm for abduction-adduction and 0.7 (mean 0.3, sd 0.19) mm for internalexternal rotation figure 3. the femoral component was fixed with two screws (a) on the measuring frame and could be moved along the axis of the femur. subsidence was simulated by turning the handlebar (b) and measured with the slide calliper (c). a b c 366 t. ilchmann et al. for the right hip. for the combined movements, the maximum error was 1.0 (mean 0.5, sd 0.41) mm. from all radiographs, smaller samples with 2 to 5 radiographs were selected randomly, simulating a more realistic clinical situation. only a few radiographs from the whole series were detected as tilted and excluded by the software. the maximum detected error was 1.7 mm. when four radiographs were taken with consecutive flexion of 0, 6, 12 and 16 degrees, these were defined as comparable by the software and the measured subsidence was 1.6 mm. in the left femur, the stem was inserted more deeply into the femoral canal, the head centre being 1.5 mm below the tip of the major trochanter: again, the radiographic enlargement was 1.2. when looking at the radiographs in 0 degrees and 16 degrees of flexion, the measured length of the stem on the radiographs differed by 0.8 mm. with the standard measurements a maximum error of 4.3 (sd 1.71) mm was found for measurements of the shoulder in relation to the lesser trochanter (table 2). the main error was produced by movements in extension-flexion (mean 1.2 mm, sd 1.10), followed by external-internal rotation (mean 1.7 mm, sd 1.13) and abduction-adduction (mean 1.1 mm, sd 0.66). for the combined movements, the maximum error was 2.8 (mean 1.4, sd 0.88) mm. again, the error of measurement depended on the chosen reference lines and was minimal for the distance shouldermajor trochanter (table 2). with ebra-fca the error of measurement on the left side was less than on the right side. the maximum error of measurement was 0.2 (mean difference –0.03, sd 0.65) mm, taking all 30 radiographs in random order. when the radiographs were taken in consecutive order of tilt, the maximum error of measurement was 0.5 (mean 0.31, sd 0.16) mm for extension-flexion, 0.3 (mean 0.25, sd 0.11) mm table 1. tilt study, right hip: error of measurement with standard methods for all radiographs h.-major h.-lesser s.-major s.-lesser absolute mean 0,81 2,06 1,07 0,72 absolute median 0,57 1,14 0,70 0,66 max 2,92 7,46 4,10 1,96 sd 0,69 2,35 1,09 0,52 table 2. tilt study, left hip: error of measurement with standard methods for all radiographs h.-major h.-lesser s.-major s.-lesser absolute mean 0,64 0,94 0,27 1,44 absolute median 0,61 0,87 0,18 1,26 max 1,50 2,53 1,52 4,27 sd 0,42 0,73 0,33 1,00 femoral subsidence assessment after hip 367 for abduction-adduction and 0.4 (mean 0.19, sd 0.09) mm for internalexternal rotation for the left hip. for the combined movements, the maximum error was 0.5 (mean 0.3, sd 0.19) mm. selecting smaller samples of radiographs, the error was smaller than for the right femur. 2. subsidence study with the standard measurements, the most reliable reference line was from the shoulder of the implant to the major trochanter. the error of measurement was hardly affected by the chosen reference lines and the maximum error was 1.9 mm, using the lesser trochanter as reference (table 3). the maximum error using the ebra-fca method was 0.2 (0 to 0.2, mean 0.06, sd 0.05) mm. when subgroups of three to seven radiographs with increasing subsidence were selected, a maximal error of 0.5 mm was found. an extreme situation was simulated with 5.0 mm initial subsidence on the first radiograph and a stable situation on further 29 radiographs. as there was no projection difference, all radiographs were comparable with ebra-fca and an initial subsidence of 0.8 mm without further progress was calculated. when the test was repeated with a lower number of radiographs, the error of measurement decreased and in the case of 6 radiographs, the measured subsidence with ebra-fca was 2.4 mm. discussion it is recognized that the error of measurement for femoral subsidence depends on the range of motion of the femur but walker et al. (8) thought that the error of measurement might be neglected within a range of 10° of flexion and rotation on the basis of theoretical calculations. we found a maximum error of measurement of 6.6 mm for 10° of flexion for the right hip but for the left hip and other reference lines, the error of measurement was lower. in a clinical situation flexion up to 10° might be found when the patient is developing a flexion contraction due to hip pain. even in the most flexed and rotated position there were no obvious differences between the radiographs, thus in a standard clinical situation no radiograph would have been table 3. subsidence study: error of measurement with standard methods for all radiographs. h.-major h.-lesser s.-major s.-lesser absolute mean 0,44 0,43 0,41 0,43 absolute median 0,40 0,32 0,34 0,33 max 1,59 1,59 1,08 1,87 sd 0,37 0,33 0,26 0,36 368 t. ilchmann et al. rejected due to excessive distortion and even in the case of minor distortion a considerable error of measurement could occur. the difference in the results of the left and the right hip may be explained both by variations in the shape of the femur and in the position of the implant. on the left side, the femoral component was implanted in a more distal position, the head centre was closer to the major trochanter and the shape of the lesser trochanter changed more when rotated (7). for this purpose, biedermann et al. (6) recommended in a theoretical model the major trochanter and the prosthetic shoulder as reference points. on the other hand, the major trochanter might be less reliable due to heterotopic ossifications in clinical analysis (3). for standard measurements, the optimal reference line would depend on the individual situation. ebra-fca detected and excluded very few tilted radiographs out of our series of measurement because of the small projection differences. but, due to the comparing algorithm and the geometry of the reference lines, the ebra-fca measurements were less affected by eventual tilt of the femur and thus more accurate than the standard measurements. as for the cup (5), a systematic error of measurement due to consecutive tilt in one direction (flexion) could not be avoided and in this case the maximum error of measurement was found. in a random combination of radiographs, the error of measurement with ebra-fca was much less than with standard methods. in the subsidence study, all standard measurements were reliable because the shape of the bony structures remained unchanged. in a clinical situation the detection of these structures and correct drawing of the trochanter lines would be even more difficult and the error of measurement will increase. with ebra-fca, linear subsidence could be measured very accurately. in the case of initial high subsidence and a following stable situation of identical radiographs, all radiographs were compared and the mean subsidence was calculated, underestimating the actual subsidence. no measurement exceeded the actual subsidence. there were no false positive results and ebra tends to underestimate the actual subsidence. ebra-fca has a higher specificity than sensitivity for detecting subsidence (1, 9). the pattern of subsidence is of major importance for predicting the late outcome of the implant (1, 2). it was correctly described with ebra-fca and ebra-fca seems to be reliable in detecting stable implants in the mid-term. conclusions. effects of tilt are more difficult to detect on radiographs in the femur than in the pelvis and ebra-fca is less accurate for femoral assessment than ebra is for the cup. biedermann et al. (6) could demonstrate that ebra-fca improves the measurement accuracy as compared to standard methods in the clinical situation. it is reliable for the detection and description of the patterns of subsidence and the measurements are of prognostic value. early implant movements like initial settlement of the stem can not be detected and described with ebra-fca. analysis of single patients and radiographs in the early postoperative phase has to be done with radiostereometry. femoral subsidence assessment after hip 369 references 1. freeman m a, plante-bordeneuve p (1994) early migration and late aseptic failure of proximal femoral prostheses. j bone joint surg br 76: 432–438. 2. krismer m, biedermann r, stockl b, fischer m, bauer r, haid c (1999) the prediction of failure of the stem in thr by measurement of early migration using ebra-fca. einzel-bildroentgen-analyse-femoral component analysis. j bone joint surg br 81: 273–280. 3. malchau h, karrholm j, wang y x, herberts p (1995) accuracy of migration analysis in hip arthroplasty. digitized and conventional radiography, compared to radiostereometry in 51 patients. acta orthop scand 66: 418–424. 4 wetherell r g, amis a a, heatley f w (1989) measurement of acetabular erosion. the effect of pelvic rotation on common landmarks. j bone joint surg br 71: 447–451. 5. ilchmann t, kesteris u, wingstrand (1998) effect of pelvic tilt on radiographic migration and wear measurements after total hip arthroplasty. hip int 8: 16–23. 6. biedermann r, krismer m, stockl b, mayrhofer p, ornstein e, franzen h (1999) accuracy of ebra-fca in the measurement of migration of femoral components of total hip replacement. einzel-bild-rontgen-analyse-femoral component analysis. j bone joint surg br 81: 266–272. 7. braud p, freeman m a (1990) the effect of retention of the femoral neck and of cement upon the stability of a proximal femoral prosthesis. j arthroplasty 5 suppl: 5–10. 8. walker p s, mai s f, cobb a g, bentley g, hua j (1995) prediction of clinical outcome of thr from migration measurements on standard radiographs. a study of cemented charnley and stanmore femoral stems. j bone joint surg br 77: 705–714. 9. biedermann r, stockl b, krismer m, mayrhofer p, ornstein e, franzen h (2001) evaluation of accuracy and precision of bone markers for the measurement of migration of hip prostheses. a comparison of conventional measurements. j bone joint surg br 83: 767–771. 10. luem m (1999) early clinical results using the fca-method on tantalum marked femora. hip int 9: 109. corresponding author: t. ilchmann department of orthopedics kantonsspital rheinstr. 26 ch-4410 liestal switzerland tel: +41-61-9253345 fax: + 41-61-9252808 e-mail: thomas.ilchmann@ksli.ch adjuvants in ivf—evidence for what works and what does not work review article adjuvants in ivf—evidence for what works and what does not work luciano nardoa,b and spyridon chouliarasc,d areproductive health group, daresbury park, uk; bmanchester metropolitan university, manchester, uk; csidra medicine, doha, qatar; dweill cornell medicine, ar-rayyan, qatar abstract the field of assisted reproductive technology is shaped and changed constantly by advances in science and cutting-edge innovations. in a quest to maximise outcomes, add-on interventions are often adopted and utilised prematurely while the principles of evidence-based medicine seem to be less strictly adhered to. in this review we will attempt to summarise the latest evidence about some of the adjuvants. article history received 29 january 2020 revised 1 april 2020 accepted 1 april 2020 keywords adjuvant therapy; ivf introduction known also as treatment add-ons, adjuvants or adjuncts are additional, optional, treatment steps that may be offered on top of standard fertility treatments. the topic of add-ons has been heavily debated as many consider such treatments unfounded. however, one has to agree that the very reason that this kind of treatments exists, and new innovative treatments keep emerging, is the challenge faced by the clinicians along with the frustration experienced by those trying to conceive. when clinics start offering such treatments a pandora’s box opens, as the question then would be where one draws the line. worldwide the in vitro fertilisation (ivf) industry is already worth billions of dollars and is booming so there seems to be no easy answer to this question. as the debate regarding the provision of add-ons is ongoing, several regulatory bodies and professional learned societies have released guidance and statements urging the public to be cautious and to question the use of such treatment modalities. among those bodies are the royal college of obstetricians and gynaecologists (rcog) and human fertilisation and embryology authority (hfea) in the uk, and the australian-based victorian assisted reproductive treatment authority (varta). the issue of add-ons has been under the spotlight even by investigative undercover journalists who have condemned the practice of some private clinics. large randomised trials in ivf are very hard to pursue as patients are very keen to try novel or already established empirical therapies and therefore being randomised to a placebo arm is unappealing for them. for most of the add-ons presented in this publication, the available evidence is suboptimal, and better-designed studies are essential to give conclusive answers. we will therefore refrain from repeating the same argument separately for each treatment modality. we will also not elaborate on strategies used in order to improve success rates such as: freeze-all, routine use of icsi for non-male factor infertility, preimplantation genetic testing for all cycles, continuous monitoring of the embryos (time lapse imaging), testing for endometrial receptivity or endometrial microbiome, and advanced sperm selection techniques such as intracytoplasmic morphologically selected sperm injection (imsi), physiological icsi (picsi), and sperm head’s birefringence. it is our belief that these do not strictly speaking constitute adjuvants to treatment, and the decision regarding their use may be influenced by other factors. instead we will try to focus on what we consider as ‘pure’ add-ons to treatment. ivf laboratory-related adjuvants embryo glue and adherence compounds the idea of a using a substance that could facilitate blastocyst implantation originates in the early days of ivf. fibrin sealants have failed to demonstrate significant improvement in clinical outcomes; however, lately the spotlight has been on a specific culture medium with added hyaluronan, marketed as embryoglue. hyaluronan is a glycoprotein which is well known to provide a high viscosity environment in the uterus. it has also been observed that the synthesis of intrauterine hyaluronan increases before implantation and goes back to near basal levels after. the latest cochrane review of 3898 participants from 17 randomised control trials (rcts) demonstrated moderate-quality evidence for an improvement in clinical pregnancy rates (cpr) and live birth rates (lbr), with an associated increase in multiple pregnancy rate, when transfer medium was supplemented with hyaluronan. the published evidence may be suggestive of a beneficial contact luciano nardo lnardo@reproductivehealthgroup.co.uk reproductive health group, daresbury park, uk. � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 2, 144–151 https://doi.org/10.1080/03009734.2020.1751751 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1751751&domain=pdf&date_stamp=2020-05-21 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1751751 http://www.tandfonline.com effect of the use of hyaluronan-supplemented embryo transfer media (1). assisted hatching assisted hatching, which is effectively the artificial disruption of the zona pellucida, has been used since the early years of assisted reproductive technology (art), but its popularity is fading. chemical, mechanical, laser-assisted hatching, or even drilling of the zona could in theory aid a hardened zona to break and thus facilitate the transfer of metabolites, growth factors, and signals between the embryo and the endometrium. reviews on assisted hatching have reported statistically significant increases in cpr but no evidence for a difference in lbr. the latest meta-analysis of studies on assisted hatching included 36 rcts with 6459 participants and found that assisted hatching gave a significant increase in cpr, but in the 15 rcts that looked at lbr there was no evidence of benefit from assisted hatching (2). artificial oocyte activation (aoa) failure of oocyte activation is thought to be an important cause of fertilisation failure following conventional icsi. artificially inducing oocyte activation by the use of calcium ionophores has been trialled for women with previous low or total failed fertilisation in previous icsi cycle(s). the suggested physiological mechanism of action is by increasing calcium ion concentration around the ooplasm immediately following sperm–oocyte fusion, although oocyte activation is very complex and therefore the understanding is not complete. the most widespread aoa agents used in human art are two ca2p-selective ionophores—ionomycin and a23187 (also known as calcimycin)—although a protocol using strontium chloride (srcl2) has also been reported. there have been several promising reports regarding application of this technology; however, there is significant heterogeneity in the aoa protocols as well as the indications for its use. a systematic review and meta-analysis of rcts concluded that there was insufficient evidence to prove the effectiveness of aoa. the data were considered dissimilar and of low quality, with studies having various risks of bias (3). data regarding the safety of this intervention have been reassuring till now, and its use in the context of scientific trials can be justified. patient-centered adjuvants improvement of blood supply heparin. heparin offers prophylaxis against formation of micro-thrombi at the implantation site by inhibiting clotting factor xa. both unfractionated and low-molecular-weight heparin (lmwh) have been used to promote successful invasion of trophoblasts in the presence of antiphospholipid syndrome. low-molecular-weight heparin’s favourable pharmacological profile is the obvious reason why it is almost exclusively used in the context of art, and therefore we will mostly focus on evidence regarding lmwh. the role of heparin in women suffering from thrombophilia who are undergoing assisted conception treatment is established, but its use in the general ivf population is still a grey area. one meta-analysis that included rcts of women with repeated implantation failure (rif) after �3 failed embryo transfer cycles (4) and a cochrane review that included a rct of women undergoing their first ivf cycle (5) found improvement in lbr (77–79%) with the use of lmwh. however, they both concluded that its routine use cannot be advised as the evidence is seriously limited by inconsistency, imprecision, and potentially the small sample and statistical methodology. in a third meta-analysis authors gave a similar message. they demonstrated significantly higher cpr and lbr in the observational studies but not when sensitivity analyses included women with rif. when the rcts’ data were pooled there was again no evidence of benefit (6). a more recent large retrospective study in women with two or more unsuccessful ivf cycles failed to demonstrate any benefit from the addition of lmwh (7). there is growing evidence that, other than the antithrombotic effect, lmwh may favour reproductive outcomes via different mechanisms of action, such as modulating the decidualization of endometrial stromal cells and thus endometrial receptivity (8) or increasing heparinbinding epidermal growth factor (hbegf) expression, which in turn is required to stimulate early trophoblast differentiation and invasion (9). these anti-thrombin-independent effects of lmwh suggest that we should look at the evidence of heparin use in art with a fresh eye, and therefore larger studies with a different design are desired. since the use of lmwh is not without risk, like bleeding and thrombocytopenia, the use at the moment in the general ivf population or even in women with implantation failure requires appropriate counselling in light of the limitations of available evidence. aspirin. acetylsalicylic acid is one of the most commonly used medications worldwide and usually prescribed for the prevention of cardiovascular disease due to its role as an antiplatelet-aggregation agent. low-dose aspirin also results in vasodilation and increase in the peripheral blood flow by inducing a shift from thromboxane a2 to prostacyclin. evidence on the prophylactic role of aspirin against preeclampsia and intrauterine growth restriction from the clasp study resulted in its use being adopted for treatment of embryo implantation failure. moreover, it has been shown that aspirin improves uterine and ovarian arteries doppler indices, which in turn leads to improved oocyte quality and favours implantation rates (10). a meta-analysis failed to support its routine use for women undergoing ivf (11). similarly, a cochrane review of 13 rcts (12) suggested that administration of aspirin did not improve pregnancy rates for ivf patients. more recently, another cochrane review of studies in women with recurrent miscarriage, whether associated with thrombophilia or not, did not find any benefit of the use of aspirin alone or in combination with lmwh when upsala journal of medical sciences 145 trials with high risk of bias were excluded (13). therefore, routine aspirin use is not advocated. sildenafil. sildenafil citrate acts as a vasodilator by potentiating the effect of nitric oxide on vascular smooth muscle. it was therefore speculated that improving the blood supply and oxygenation of the uterus could enhance implantation rates. vaginal sildenafil citrate (viagra) is predominantly being used for treating cases with thin endometrium. a retrospective study suggested enhancement of endometrial development in 70% of patients, while high implantation and pregnancy rates were achieved in a cohort of poor-prognosis patients (14). in contrast, another study, where sildenafil supplementation was given in women undergoing fresh ivf or frozen embryo transfer, failed to demonstrate an increase in endometrial thickness or blood supply (15). in general, studies which examine sildenafil’s use are underpowered, heterogeneous, and often its use is combined with other medications. its routine use cannot be recommended outside a research setting. antioxidants in humans, excess formation of reactive oxygen species (ros) can induce oxidative stress and lead to cell damage or death. cells have antioxidant mechanisms which regulate this process in order to achieve homeostasis, but often it becomes unbalanced. high levels of free radicals are thought to affect the reproductive function; therefore, antioxidants are increasingly being used over the counter by patients but also recommended by clinicians to improve natural reproductive potential and ivf outcomes. in male infertility their role is relatively well documented, and recently the term male oxidative stress infertility (mosi) has been suggested as a distinct category (16). however, supplementation with antioxidant in women has not always demonstrated similar results (17). the presence of a dominant th1 phenotype is a common finding in women with recurrent miscarriages and implantation failure. an antioxidant diet regime has been shown to improve the cytokine ratio and therefore may indirectly lead to improved reproductive outcomes (18). one of the antioxidants, coenzyme q10 (coq-10), which is an essential component of the inner mitochondrial membrane, has particularly been of interest due to its effect in the energy production of the oocyte. mitochondrial dysfunction is implicated with ovarian aging in both animal and human models. preliminary work suggested that supplementation with coq-10 may reverse this process (19). it has even been shown that coq-10 improves oocyte and cumulus cells quantity and quality (20), while a recent rct has suggested improvement in ovarian response and embryo quality but due to sample size failed to demonstrate improvement in clinical outcomes (21). still, the optimal duration and dosage of coq-10 supplementation remain elusive. finally, it should be stressed that some ros are signalling molecules in cellular signalling pathways, and therefore lowering their numbers may not be beneficial. it seems that both extremes—oxidative and antioxidative stress—are undesired, and blindly prescribing antioxidant supplements without an accurate determination of the oxidative stress may be too simplistic and counterproductive (22). immunotherapy there is no doubt that the immune regulation of the dialogue between the endometrium and the embryo during implantation is fundamental. the so-called immunological paradox of pregnancy though is still not well understood (23). women desperate to find an explanation for their repeated ivf failures are often worried that their bodies may be rejecting embryos or that the gametes could be incompatible. the anxiety surrounding these theories is only enhanced by unfortunate and sometimes irresponsible media publications and by the specific terminology used in reproductive immunology (e.g., killer, necrosis, etc.). natural killer (nk) cells, cytokines, tumour necrosis factor-alpha (tnf-a), and the balance between t-helper cells (th1/th2) all play a major role in the embryo implantation process. tests used to identify irregularities in the regulation of reproductive immunology such as peripheral blood nk (pbnk) cell number and activity and uterine nk (unk) cell number and activity are gaining popularity. the relevance of some of these tests is questionable as there is a lack of consensus on the methods used for measuring and reporting as well as lack of a clear definition of a ‘normal’ range or what constitutes a ‘high’ cell count for either pbnk or unk cells (24). a systematic review found no significant difference in the pbnk cell number and activity in women with or without implantation failure or miscarriage after art. the authors concluded that the prognostic value of routine testing for pbnk cells is unclear. the implantation rates in women with or without elevated unk cells in the two studies included in this review were comparable (25). below we will attempt to further elaborate on available evidence—or lack thereof—for the most commonly prescribed immune treatments. steroids different regimes of oral prednisolone or dexamethasone are being prescribed as an adjuvant to ivf. it has been proposed that glucocorticoids may improve the intrauterine environment due to the anti-inflammatory and immune-suppressive activity by acting as immunomodulators to normalise the cytokine expression profile and by reducing the unk cell count and activity in the endometrium, thus suppressing endometrial inflammation (26). a cochrane review of 14 rcts found no benefit of glucocorticoids on lbr, cpr, and miscarriage rates when used in an unselected ivf/icsi population, except in a subgroup of ivf rather than icsi cycles (or 1.5, 95% ci 1.05–2.13), which is biologically difficult to interpret (27). there have been several studies though looking into more specific groups of women having ivf and treated with steroids alone or combined with other medications, like 146 l. nardo and s. chouliaras women with rif or recurrent miscarriages or even women with abnormal immune testing such as elevated nk cells, or the presence of anti-cardiolipin antibodies, thyroid peroxidase antibodies, and anti-nuclear antibodies. one meta-analysis on the use of corticosteroids in women undergoing ivf with elevated pbnk cells found only one study which demonstrated an increase in cpr with prednisolone (or 1.63, 95% ci 1.00–2.66). the quality of evidence was considered low to indicate improved ivf outcomes (28). another metaanalysis found significant improvement in lbr and reduction of miscarriage rates when women with idiopathic recurrent miscarriages were treated with prednisolone (29). prednisolone used as an adjunct along lmwh in women with one or more previous unexplained failed ivf or icsi cycles has also been studied. both studies suggested an improvement in ivf or icsi outcomes associated with adjuvant therapy (30,31). the interpretation of all the available evidence is complex, but there may be some rationale for the use of oral steroids in women with rif or recurrent miscarriages with an abnormal immunological profile. due to the low cost and perceived safety in short-term use, except from a weak association with cleft palate (32), prednisolone may be offered in selected cases unless future evidence directs otherwise. intralipids infusion of intralipids is used for parenteral nutrition and is a fatty emulsion containing soya bean oil, egg yolk, phospholipids, glycerine, and water. it is also used in emergencies to treat systemic toxicity induced by local anaesthetics. increasingly it is being used for women with failed ivf attempts and abnormal nk cell activity (33). a double-blind randomised control trial of 296 women with secondary infertility, recurrent miscarriage, and elevated pbnk cells showed no increase in cpr in the group of women who were treated with intralipid infusion (34). another prospective trial in a very selected cohort of patients had to be terminated early as the results favoured the group which did not receive intralipids therapy (35). a retrospective study of women who received intralipids infusion due to elevated pbnk cells failed to identify any benefit, and a cost-effectiveness analysis deemed that the treatment was not cost-effective (36). therefore, despite the relatively low cost of the intervention and the good safety profile, the evidence for its use is lacking, and prospective patients should be made aware of this. immunoglobulin theories regarding the potential mechanisms of actions of i.v. immunoglobulin (ivig) include inhibition of pbnk cell numbers and/or activity, correction of abnormal th1/th2 ratio, but also non-specific immunomodulation leading to enhancement of immunological tolerance. a systematic review published in 2013 included 10 studies and a total of 835 women who received ivig as adjuvant treatment to ivf. a significantly higher implantation rate (rr 2.708, 95% ci 1.302–5.629), cpr (rr 1.463, 95% ci 1.075–1.991), and lbr (rr 1.616, 95% ci 1.243–2.101) were reported, as well as a significantly lower miscarriage rate (rr 0.352, 95% ci 0.168–0.738). although this review showed that ivig use was favourable for all reproductive outcomes, the quality of the retrospective studies included was variable, including a mixture of patients with recurrent implantation failure, unexplained infertility and recurrent miscarriages, several dosing regimes, and co-treatment with other add-ons (37). a more recent systematic review and meta-analysis of the available evidence regarding immunotherapy (38) identified 15 suitable rcts, but only 2 of those were rcts regarding ivig in women undergoing ivf treatment with a history of rif. these studies were already included in the previous meta-analysis by li and colleagues 2013 (37); the number of patients was small, and pooling of their results did not demonstrate any difference between the study and the control group in terms of cpr or lbr (39,40). ivig is a pooled blood product, and as such its use is not without risks. the majority of those are mild and transient; however, serious side effects, although rare, have been reported (41). therefore, even without taking into consideration cost-effectiveness, their use cannot be recommended. anti tnf-a an exaggerated th1 response is thought to be detrimental to the process of embryo implantation and has been linked to infertility; therefore, elevated tumour necrosis factor (tnf) levels have been targeted for therapeutic correction in patients with a raised th1/th2 ratio. a potential reproductive benefit of anti-tnf-a agents, adalimumab (humira), etanercept (enbrel), and infliximab (remicaid) has been proposed in infertile women with abnormal immunological parameters. a small retrospective case–control study of women with th1/ th2 cytokine elevation reported a lbr of 73% in 41 patients who received ivig and adalimumab, 50% in 6 patients who received adalimumab alone, and no live birth in 5 patients who received neither (42). anti-tnf-a agents are also being used off-label for the treatment of endometriosis (43), and an interesting recent retrospective study suggested that etanercept may improve cpr but not lbr in women with an endometrioma having ivf (44). any potential advantage from the use of anti-tnf-a has to be balanced against the known adverse effects of immune suppression such as the increased risk of lymphoma, skin cancer, and granulomatous infections. the risk profile of short-term use is less clear, but until welldesigned, appropriately conducted large clinical trials clarify the role and safety of using anti-tnf-a agents as an adjuvant treatment to improve ivf outcome, their use cannot be supported. granulocyte colony-stimulating factor (g-csf) g-csf is a cytokine that stimulates neutrophilic granulocyte proliferation and differentiation. it has been proposed as an important factor for embryo extravillous trophoblast cells invasion and adequate placentation. it has been used for patients with rif but also for those with thin endometrium. upsala journal of medical sciences 147 despite a rapidly increasing interest over recent years, the actions of g-csf are still not clarified. existing studies are very heterogeneous, include different regimes and routes of administration (intrauterine versus subcutaneous), and their interpretation should be carried out with caution. two very recent attempts to systematically review results from these studies reported conflicting results. zhang and colleagues reviewed 10 studies of various design and demonstrated improved cpr with the use of g-csf in ivf cycles regardless of the route of administration for cases with rif (45). on the other hand, achilli and colleagues identified only five suitable studies for their review, which evaluated women undergoing ivf treatment with or without a history of rif. pooling of the results from two studies of the unselected ivf population demonstrated no difference in terms of cpr (or ¼ 0.99; 95% ci, 0.53, 1.84; p ¼ .98). for the other three studies looking in the rif population, the authors did not pool the results due to the different ways of administration (subcutaneous, intrauterine, or unspecified). one of those did not demonstrate any difference in cpr (intrauterine route), but the other two did show improvement with the use of g-csf (38). notably, a retrospective study did not report any adverse safety outcomes in the context of ivf by the use of g-csf (46). whilst this is reassuring, more studies are awaited to shed light on regimes, route of administration, and groups of patients that may benefit from this promising intervention. dehydroepiandrosterone dehydroepiandrosterone (dhea) is an endogenous steroid hormone produced primarily in the adrenal glands, gonads, and brain. it is difficult to distinguish it from its sulphated product, and it is known to decline with advancing chronological age. it is believed that it can enhance follicular function by increasing the production of insulin-like growth factor-1 (igf-1) and augmenting oestradiol production in granulosa cells, acting as a precursor of androstenedione and testosterone in the ovarian theca cells. in addition, it has been suggested that pre-treatment with dhea can reduce embryo aneuploidy, possibly by improving the ovarian micro-environment in which follicular maturation occurs (47). a worldwide survey conducted in 2010 revealed that over one-quarter of specialists add dhea as an adjuvant to ivf treatment for anticipated low responders. a dosing regime of 25 mg three times daily is standard and has been used in most of the available studies which makes evidence less heterogeneous. a plethora of publications on the positive effects of dhea on ovarian response, embryo quality, and pregnancy outcomes in poor responders undergoing ivf are available. nevertheless, there is still no consensus regarding its perceived benefit, and many of the available studies have been subsequently criticised by others. a large rct investigating 12 weeks of dhea supplementation in 104 poor responders compared with 104 controls found an insignificantly higher pregnancy rate in the control group (48). conversely, a cochrane review revealed higher ongoing pregnancy rates and lbr following dhea use; however, the benefit was not statistically significant (or 1.50, 95% ci 0.88–2.56) when studies with high risk of performance bias were excluded (49). the ditto (dehydroepiandrosterone intervention to treat ovarian aging) single-center rct included a small number of subjects (25 versus 27 in each arm) and failed to demonstrate any benefit from the use of dhea in women with diminished ovarian reserve undergoing ivf (50). other attempts for meta-analyses that followed failed to be conclusive. qin and colleagues suggested significantly increased cpr for patients with diminished ovarian reserve who were pre-treated with dhea when data from nine studies of various design were included. there was, however, no significant increase when the data were restricted to rcts (51). a later meta-analysis including nine rcts reported higher cpr, number of retrieved oocytes, and lbr in women who had been pre-treated with dhea prior to ivf. however, the studies included had high risk of bias, and some used alternative dosage for dhea (52). it is interesting that for the anticipated normal responder population a well-designed double-blind rct did not report any difference in number of oocytes or ovarian response after 12 weeks of dhea (53). however, this study did not report on pregnancy outcomes. the absence of significant side effects should not be considered as an argument to recommend routine use of dhea. nevertheless, it is difficult to stop patients self-medicating especially since dhea is readily available online and over the counter. counselling is essential for patients, and it seems that the use of dhea will not stop unless any new solid evidence, which dictates otherwise, becomes available. growth hormone growth hormone (gh) is a requisite of normal puberty, appears to have a role in ovarian function, and is proposed to have a modulatory action on the effect of fsh on granulosa cells by up-regulating the local synthesis of igf-1. this in turn is thought to enhance the effect of gonadotrophin action at the level of both the granulosa and theca cells. although the potential role of gh in art has been recognised and studied for more than three decades, over the recent years a newfound interest has emerged, and gh has again become a popular topic. a cochrane review published in 2010 included 10 rcts with a total of 440 subjects (54). the authors observed considerable heterogeneity in the definitions, protocols, and outcomes. no benefit was seen by the use of gh supplementation in women considered as normal responders, whereas those considered to be poor responders had a significant benefit in both cpr (or 3.28, 95% ci 1.74–6.20) and lbr (or 5.39, 95% ci 1.89–15.35). the reviewers also put a caution on interpreting the result as the rcts included in the meta-analyses were too few in number and too small in sample size to draw a definitive conclusion. the advantage of adding gh seemed to be limited to treatment cycles with gnrh agonist protocol only, and in a later 148 l. nardo and s. chouliaras study no significant benefit was found by adding gh in antagonist cycles (55). in 2015, an updated meta-analysis reported results in line with previous evidence. the results showed a significant improvement in terms of mature oocytes retrieved and number of embryos available for transfer by gh supplementation in poor responders. however, there was no significant difference in cpr (56). in some of the more recent studies participants were classified as poor responders according to approved criteria. in the study by dakhly and colleagues there was no significant difference in cpr in 240 low responders (classified using the bologna criteria) randomised to gh or placebo and undergoing a long down-regulation protocol of ovarian stimulation (57). the latest meta-analysis regarding the use of gh in controlled ovarian stimulation (cos) was published in 2017 (58). it included all previous articles as well as data from the light study which was performed by the same group. the light study was a multicenter, double-blind, placebo-controlled trial performed in 10 centres throughout australia and new zealand. the authors did not use bologna or poseidon criteria for poor ovarian reserve. after 4 years of enrolment, the study was interrupted prematurely without reaching the desired numbers due to difficulties in recruitment. the number of patients reaching an oocyte retrieval was significantly higher in the gh group; however, no differences were reported in the lbr (59). the meta-analysis of 2017 unsurprisingly showed no evidence of an increased chance of live birth by the use of gh, although there was a significant reduction in the duration of stimulation required and a greater number of oocytes were collected than in the placebo arm. gh together with its receptor, gh-r, and related growth factors are also expressed in the human endometrium and may even have an indirect role in the function and maintenance of the corpus luteum. an additional potential benefit at the level of the uterus has therefore been suggested especially among women with recurrent implantation failure or thin endometrium. thus, some of the beneficial effects of gh on assisted reproduction outcomes that have been suggested in the literature may, at least partially, be a result of the action of gh on endometrial receptivity (60). endometrial injury although injury to the endometrium prior to embryo transfer is not strictly speaking an adjuvant to ivf, we have included it in our review as it is an intervention which has gained a lot of interest over the recent years. the theory behind it was that it may positively affect implantation by inducing the release of factors such as cytokines, interleukins, and growth factors in the endometrium. the most common and easily available procedure was via the use of a pipelle known as ‘endometrial scratching’. after many published studies a rct was conclusive in showing that endometrial scratching does not confer any benefit to reproductive outcomes, including lbr (61). an alternative to endometrial scratching which is also commonly practiced by many, is the routine use of outpatient hysteroscopy prior to starting an ivf cycle, which may diagnose subtle pathology but will also act similarly as endometrial injury. a well conducted, adequately powered, multicenter rct failed to demonstrate any improvement in lbr by the use of the intervention for women with recurrent ivf failure (62). conclusions there are obviously more interventions used in ivf cycles that could be described as adjuvants, but our review cannot exhaust them all. platelet-rich plasm injection in the ovaries, mitochondrial replacement therapy (spindle transfer), melatonin, mitochondria dna load measurement, among others, may prove to live up to the hype in the future. for most interventions mentioned in this document (summarized in table 1) lack of satisfactory evidence may be interpreted as evidence of lack of benefit. this argument from ignorance or ‘argumentum ad ignorantiam’ is a famous informal fallacy in logic. but, before we reject any add-on shown not to work or we embrace others where evidence is favourable, we need to ascertain its effect in patients with a specific profile that might be modulated by the treatment under consideration. novel diagnostic technologies are emerging that will enhance our understanding of the nature and determinants of reproductive functions. these technologies pave the way to a personalised approach in medicine and promise to help us identify specific patients who may benefit or be harmed by an intervention. dismissing adjuvants altogether may pose the risk of keeping us stuck in the ‘one-size-fits-all’ clinical paradigm. until then keeping an open mind and being honest and transparent with the patient is the best way forward. disclosure statement the authors do not have any financial interest in commercial products or services related to the subject of this paper. notes on contributors luciano nardo, md mrcog, is a consultant gynaecologist and rcog subspecialist in reproductive medicine and surgery. he is visiting professor at manchester metropolitan university and clinical director at the table 1. two broad categories are discussed, adjuvants that are related to the ivf laboratory per se and involve gametes or embryos, and medical adjuvants which include patient-centered treatments or interventions. type of adjuvant evidence for efficacy laboratory-related adjuvants embryo glue and adherence 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2017;29:119–25. 59. norman rj, alvino h, hull lm, mol bw, hart rj, kelly tl, et al. human growth hormone for poor responders: a randomized placebo-controlled trial provides no evidence for improved live birth rate. reprod biomed online. 2019;38:908–15. 60. altmae s, aghajanova l. growth hormone and endometrial receptivity. front endocrinol (lausanne) 2019;10:653. 61. lensen s, osavlyuk d, armstrong s, stadelmann c, hennes a, napier e, et al. a randomized trial of endometrial scratching before in vitro fertilization. n engl j med. 2019;380:325–34. 62. el-toukhy t, campo r, khalaf y, tabanelli c, gianaroli l, gordts ss, et al. hysteroscopy in recurrent in-vitro fertilisation failure (trophy): a multicentre, randomised controlled trial. lancet 2016; 387:2614–21. upsala journal of medical sciences 151 abstract introduction ivf laboratory-related adjuvants embryo glue and adherence compounds assisted hatching artificial oocyte activation (aoa) patient-centered adjuvants improvement of blood supply antioxidants immunotherapy steroids intralipids immunoglobulin anti tnf-α granulocyte colony-stimulating factor (g-csf) dehydroepiandrosterone growth hormone endometrial injury conclusions disclosure statement references (9) upsala j med sci 111 (2): 243–248, 2006 sapho syndrome with a tumour-like bony proliferative lesion in distal femur. a case report munenori watanuki, masahito hatori and shoichi kokubun department of orthopaedic surgery, tohoku university school of medicine, sendai, japan abstract sapho syndrome is a group of bone and joint abnormalities associated with skin lesions. a 29-year-old male presented with severe acne on his trunk and anterior chest wall, right knee and foot pain. radiographs and magnetic resonance images showed hyperostosis in the sternocostoclavicular region, sclerosis of one-third of the right distal 5th metatarsal bone and bony outgrowth from the medial condyle of the right femur .the histological findings of the biopsy specimen were consistent with those of old osteomyelitis. all fungal and microbacterial cultures were negative. pain and swelling of the right knee and foot repeated remission and aggravation. there were no radiological changes of the above-mentioned lesions noted within 4years follow-up. introduction in 1987, chamot et al. proposed the term, sapho (synovitis, acne, pustulosis, hyperostosis, and osteitis) syndrome, to designate collectively bone and joint abnormalities associated with skin lesions [1, 2]. the common site of skeletal lesions in sapho is the chest wall (clavicle, sternum, and sternoclavicular joints). long bones are occasionally involved. radiographic changes of the long bone lesions generally suggest either infectious osteomyelitis or a neoplasm such as ewing’s sarcoma[3]. we report a case of sapho syndrome having a cauliflower-like bony mass in the left femur together with an anterior chest lesion and sclerosis of the left 5th metatarsal bone. to the best of our knowledge, no cauliflower-like bone protrusion as in the present case has been reported. 243 received 7 june 2005 accepted 15 june 2005 case report a 29-year-old male had severe acne on his trunk in the summer, in 1991. he had anterior chest pain in october 1999. plain radiographs demonstrated hyperostosis of the sternoclavicular joints. the pain was relieved by non-steroidal anti-inflammatory drugs (nsaids). in 2001, he noticed pain in the right knee and foot, and visited another clinic. he was referred to us after plain radiographs had demonstrated bone tumors in his right femur and right 5th metatarsal bone. on examination, acne conglobata was found on his head and many acne scars, on his trunk. a bony mass was palpated on the posterior aspect of the medial condyle of the right femur. the range of motion of the right knee was slightly restricted. a mild swelling was seen on the lateral side of the right foot. no redness, tenderness or local heat was found. laboratory examination revealed a slight elevation of aso titer (272 todd). white blood cell count (8,900/ml) and c-reactive protein level (0mg/dl) were normal. fungal and microbacterial cultures of throat swabs were negative. plain radiographs showed a mixture of sclerotic and erosive lesions in the sternum including the sternoclavicular and 1st sternocostal joints. the distal metaphysis of the right 5th metatarsal bone was slightly enlarged with dens sclerosis. a tumor-like bony lesion was seen on the medial condyle of the right knee (fig 1). all lesions were clearly visualized by computed tomography. the lesion of the right femur was in continuity with the underlying cortex. it had irregular surface and hypertrophied lamellar structures inside (fig 2). magnetic resonance imaging demonstrated low signal intensity on t1 weighted image, heterogeneous intensity on t2 weighted image in the tumor-like lesion. this lesion and the surrounding soft tissues were enhanced after administration of paramagnetic contrast medium (fig 3). bone scintigram showed increased uptakes in the anterior chest wall, right distal femur, and right distal 5th metatarsal bone. bone biopsies of the lesions at the right femur and 5th metatarsal bone were performed. all specimens were negative for neoplastic disease. histological examination of 244 fig 1. plain x-ray examination revealed mixture of hyperostosis of sternocostoclavicular region (a), sclerosis (arrow) of the right distal 5th metatarsal bone (b) and tumor-like bony proliferative lesion (arrow) (c) in his right knee medial condyle. 1a 1b 1c 2a 2 b fig 2. the lesions (arrow) (a: metatarsal lesion, b: distal femoral lesion) were clearly visualized by computed tomography. the tumor of the right femur was in continuity with the underlying cortex. it had irregular surface and hypertrophied lamellar structures inside. the specimens demonstrated thickened sclerotic trabeculae and marked bone marrow fibrosis with some chronic inflammatory cells (fig 4). fungal and microbacterial cultures were negative. the patient was diagnosed having sapho syndrome because of the presence of polyostotic osteomyelitis or bone tumor like lesions, acne, and the histolo1ogical features of the biopsy specimen. the pain in the right knee and right foot disappeared soon after biopsy. however, skin lesions repeated remission and aggravation. the pain and swelling in the right foot recurred after eight months and disappeared two months later. no progression of the bone lesions was found on plain radiograms within 4-years follow-up. 246 fig 3. magnetic resonance imaging demonstrated a femoral lesion (arrow) with low signal intensity on t1 weighted image (a), heterogeneous intensity on t2 weighted image (b) in the tumor. the tumor and the surrounding soft tissues were enhanced after administration of paramagnetic contrast medium (c). fig 4. histological examination of the biopsy specimen demonstrated chronic inflammatory changes, with sclerotic bone trabeculae, prominent bone marrow fibrosis, and scattered lymphocytes. the histological diagnosis was old osteomyelitis. (a: low power view x10, b: high power view x 40) discussion in 1961, windom et al. were the first to describe musculoskeletal problems associated with acne conglobata [4]. in 1987, chamot et al. first proposed an acronym sapho syndrome, to describe a group of bone and joint abnormalities associated with skin lesions [1]. prior to the creation of this acronym, some 250 cases of hyperostosis of the chest wall with pustular eruption in hands or soles had been reported with over 50 designations [5]. most cases have been reported from japan and northern and western europe, with a few cases described from united states, canada, and great britain[3]. in japan, sapho syndrome has been paid little attention, probably due to that bone lesions are usually localized to the anterior chest wall and have diagnosed as sternocostoclavicular hyperostosis without checking skin lesions. mixture of bone sclerosis and atrophy suggestive of osteomyelitis is one of the bony features of sapho syndrome. hypertrophic bone lesions are often seen in the anterior chest wall. when the anterior chest is involved, adjacent ligaments are also involved by the ossifying process, with production of a sclerotic enthesopathy[3]. radiographic changes of the long bone lesions usually suggest either infectious osteomyelitis or a primary bone neoplastic lesion, mainly ewing’s sarcoma [3]. in the present case, the location of the femoral lesion well coincided with the attachment of the medial head of the gastrocnemius muscle. the traction of this muscle is thought to be one factor, causing a cauliflower-like bone protrusion. the patients with skin trouble like acne, when having tumor or osteomyelitis like bony lesions should be suspected to have this syndrome. in 1999, reith reported that histological findings vary depending on the stages of the disease process. early lesions contain acute inflammation, edema, and prominent periosteal bone formation, whereas late lesions demonstrate markedly sclerotic bone trabeculae with prominent marrow fibrosis and only mild chronic inflammation [6]. in the present case, four months’ duration of symptoms and the histological findings are well consistent with late lesions. treatment for sapho syndrome has not been standardized yet. nsaids are commonly used. however, their efficiency is neither consistent nor complete [2]. corticosteroids or methotrexate have proven useful in most severe forms, but the efficacy has been variable [7]. in japan, tonsillectomy has been performed in order to alleviate clinical symptoms associated with the sapho syndrome, as the manifestation may be regarded as diseases of focus tonsillitis. however, tonsillectomy was not found to bring any improvements to deformed joints or to reduce intractable pain of sternoclavicular hyperostosis[8]. for the present case, nsaids was effective with temporally relief of pain. 247 references 1. chamot am, benhamou cl, kahn mf, beraneck l, kaplan g, prost a (1987) acne-pustulosishyperostosis-osteitis syndrome. results of a national survey. 85 cases. rev rhum mal osteoartic, 54: 187-196. 2. kahn mf, khan ma (1994) the sapho syndrome. baillieres clin rheumatol, 8: 333-362. 3. kahn mf, chamot am (1992) sapho syndrome. rheum dis clin north am, 18 : 225-246. 4. windom re, sanford jp, ziff m (1961) acne conglobata and arthritis. arthritis rheum, 4: 632635. 5. gmyrek r, grossman me, rudin d, scher r (1999) sapho syndrome: report of three cases and review of the literature. cutis, 64(4): 253-258. 6. reith jd, bauer tw, schils jp (1996) osseous manifestations of sapho (synovitis, acne, pustulosis, hyperostosis, osteitis) syndrome. am j surg pathol, 20: 1368-1377. 7. hayem g, bouchaud-chabot a, benali k, roux s, palazzo e, silbermann-hoffman o, kahn mf, meyer o (1999) sapho syndrome: a long-term follow-up study of 120 cases. semin arthritis rheum, 29: 159-171. 8. kataura a, tsubota h (1996) clinical analyses of focus tonsil and related diseases in japan. acta otolaryngol suppl, 523: 161-164. corresponding author: masahito hatori, m.d., assistant professor department of orthopaedic surgery, tohoku university school of medicine, 1-1 seiryomachi, aobaku, sendai, japan 980-8574 tel: 81-22-717-7245, fax: 81-22-717-7248 e-mail: mhato@mail.tains.tohoku.ac.jp 248 oxygen sensing; a stunningly elegant molecular machinery highjacked in cancer full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 oxygen sensing; a stunningly elegant molecular machinery highjacked in cancer lena claesson-welsh to cite this article: lena claesson-welsh (2020) oxygen sensing; a stunningly elegant molecular machinery highjacked in cancer, upsala journal of medical sciences, 125:3, 205-210, doi: 10.1080/03009734.2020.1769231 to link to this article: https://doi.org/10.1080/03009734.2020.1769231 © 2020 the author(s). published by informa uk limited, trading as taylor & francis group. published online: 24 jun 2020. submit your article to this journal article views: 287 view related articles view crossmark data https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.1080/03009734.2020.1769231 https://doi.org/10.1080/03009734.2020.1769231 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.1080/03009734.2020.1769231 https://www.tandfonline.com/doi/mlt/10.1080/03009734.2020.1769231 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1769231&domain=pdf&date_stamp=2020-06-24 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1769231&domain=pdf&date_stamp=2020-06-24 review article oxygen sensing; a stunningly elegant molecular machinery highjacked in cancer lena claesson-welsh department of immunology, genetics and pathology, rudbeck laboratory, uppsala university, beijer, and scilifelab laboratories, uppsala, sweden abstract oxygen is of fundamental importance for most living organisms, and the maintenance of oxygen homeostasis is a key physiological challenge for all large animals. oxygen deprivation, hypoxia, is a critical component of many human diseases including cancer, heart disease, stroke, vascular disease, and anaemia. the discovery of oxygen sensing provides fundamental knowledge of a stunningly elegant molecular machinery; it also promises development of new therapeutics for serious diseases such as cancer. as a result of their impressive contributions to our understanding of the mechanisms by which cells sense oxygen and signal in hypoxia, gregg semenza, peter ratcliffe, and william kaelin were awarded the nobel prize in 2019. article history received 15 april 2020 revised 7 may 2020 accepted 8 may 2020 keywords cancer; hypoxia; prolyl hydroxylation; vegf introduction how is oxygen tension sensed, and what is the consequence of oxygen sensing? the oxygen-sensitive signal is generated by enzymes that catalyse hydroxylation of specific prolyl and asparaginyl residues in hypoxia-inducible factor (hif). hif is the key transcription factor that regulates transcriptional responses to hypoxia. hydroxylation of hif at different phylogenetically conserved sites targets it for degradation in normoxia. in hypoxia, hif escapes destruction and forms active transcriptional complexes that control expression of thousands of genes in the human genome. hif is a heterodimer consisting of one a (oxygen-sensitive) and one b (oxygeninsensitive) subunit. there are three a subunits with partly overlapping but also distinct functions. many of the most prominent and well-characterized hif-regulated genes have key functions in oxygen supply and utilisation via erythropoiesis, angiogenesis, haematopoiesis, and metabolic reprogramming (1). in order to coordinate the most efficient use of oxygen by the cell, hifs activate genes that shift energy dependence away from high oxygen demand, towards glycolysis. the genes encoding essentially all glycolytic enzymes are directly upregulated by hifs (2,3). in addition to pathways important for maintaining oxygen homeostasis, hif targets are involved in autophagy, apoptosis, redox homeostasis, inflammation and immunity, stemness and selfrenewal, and metastasis and invasion (figure 1) (2,4–6). below follows an outline of the scientific careers and the contributions of gregg l. semenza, peter j. ratcliffe, and william g. kaelin to our understanding of oxygen sensing. it is very interesting to read the bibliographies of these outstanding scientists as they cover the entire development of the field. moreover, it is particularly impressive that all three, semenza, ratcliffe, and kaelin, have been clinically active at least during parts of their research careers. this may serve as an inspiration for physicians today, to use their deep clinical insights as a foundation for excellent research. it is indeed possible, and important, to engage in both clinical work and in science, and to do both at a top level! gregg l. semenza after graduating from harvard, gregg l. semenza joined the md/phd programme at university of pennsylvania. for his phd, he studied b-thalassemia. he did his residency in paediatrics at duke university. in 1986, semenza started his postdoc training in medical genetics at johns hopkins school of medicine, first working on haemophilia but later shifting his focus to erythropoietin (epo) and developmental regulation of epo expression, which was known to switch from foetal liver to adult kidney. the goal was to identify dna sequences controlling organ-specific epo expression (7). this work led to the identification of the hypoxia-responsive element (hre), regulating expression of the epo gene (8). in parallel, peter ratcliffe’s group (9) as well as other groups (10) had identified the same or overlapping sequences in the mouse and human epo genes. clearly, these studies represented a major breakthrough in the understanding of hypoxia regulation. semenza and colleagues went on to identify hre sequences in a wide range of genes implicated in o2 homeostasis contact lena claesson-welsh lena.welsh@igp.uu.se department of immunology, genetics and pathology, rudbeck laboratory, uppsala university, beijer, and scilifelab laboratories, dag hammarskj€oldsv 20, 751 85 uppsala, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 3, 205–210 https://doi.org/10.1080/03009734.2020.1769231 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1769231&domain=pdf&date_stamp=2020-06-25 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1769231 http://www.tandfonline.com (11). the hre was narrowed down to 33 nucleotides and shown to bind a hypoxia-induced nuclear factor (12). in a subsequent study published in 1993, semenza mentions hif1 for the first time and shows that it binds to dna also in cells not normally expressing epo. this work led to the conclusion that hif-1 and its recognition sequence are common components of a general mammalian cellular response to hypoxia (13). semenza went on to show the oxygen-regulated, rapid kinetics of hif-1 binding to dna (14). subsequently, his group reported on the purification of hif-1 which was identified as a heterodimer composed of hif-1a and hif-1b, with the ability to bind to an intact but not mutated hre (15). in 1995, semenza and co-workers in their elegant and very impressive hallmark paper (16) deduced the structure of the hif-1 subunits as basic-helix-loop-helix proteins containing a pas (per-arnt-ahr-sim) domain. this was done by sequencing tryptic peptides from the purified hif preparation, and this information was used as the basis for cdna cloning. in their outstanding paper, his group also demonstrated that hif-1, at both rna and protein levels (using newly generated antibodies), was induced in cells exposed to 1% o2 but that they decayed remarkably rapidly upon return of the cells to 20% o2 (16). they moreover presented indications for posttranslational modification of hif-1a. in 1997, semenza implicated hif-1 in tumorigenesis for the first time (17). by generating hif-1a knockout mice, the essential role for hif-1a in embryonic development was demonstrated. lack of hif-1a in mice resulted in lethality at e11 with neural tube defects and cardiovascular malformations (18). the authors proposed that the time point of lethality coincided with that of the embryo size exceeding what could be oxygenated by passive diffusion from the mother’s circulation. in 2000, the semenza lab addressed oxygen-dependent ubiquitination in hif-1a regulation (19). semenza went on to describe fih-1 (factor inhibiting hif-1) as a protein binding to and negatively regulating the c-terminal transactivation domain of hif-1a (20). fih-1 was subsequently shown by ratcliffe and others to be an asparaginyl hydroxylase (see below). semenza then largely turned to the role of hypoxia in diseases. one particular interest was the role of the hypoxiaregulated vascular endothelial growth factor (vegf) and dysfunctional angiogenesis in cancer (21)—a research direction shared with ratcliffe (22) and kaelin (see below). together with others, the semenza group identified overexpression of hif-1 in many cancer types and in metastases (23). in cancer, vegf-regulation through hif-1 involves increased hif-1 expression rather than regulation of its half-life (24). a major focus for semenza has thereafter been to screen for hif inhibitors, which has led to the identification of cardiac glycosides such as digoxin. digoxin was tested in a clinical phase ii trial (completed in july 2018) for breast cancer treatment (see clinicaltrials.gov). however, the use of glycosides in cancer therapy has been criticised for its non-specific effects on tumour and normal cells alike and potentially an increased risk for development of invasive cancer. semenza has remained at the johns hopkins where he is the founding director of the vascular programme at the johns hopkins institute for cell engineering. peter j. ratcliffe peter j. ratcliffe received his md from the university of cambridge and st bartholomew’s hospital, london, in 1978. he relocated to oxford university, where he trained in renal medicine, with a particular focus on renal oxygenation. ratcliffe has been a practicing clinician at the john radcliffe hospital, oxford, and has been a nuffield professor of clinical medicine and head of the nuffield department of clinical figure 1. simplified schematics of oxygen sensing and transcriptional regulation. in normoxia the hypoxia-inducible factor (hif)-1a subunit becomes hydroxylated on several residues by prolyl hydroxylase (phd), which uses molecular oxygen as a substrate. hydroxylated hif-1a is recognised by the von hippel lindau (pvhl) complex which catalyses its ubiquitination and degradation. in hypoxia, hif-1a remains stable and translocates to the nucleus to form a complex with the hif-1b subunit to become a transcriptionally active complex, binding to the hypoxia-responsive element (hre). genes are induced that regulate a wide range of processes exploited to serve the progression of cancer. 206 l. claesson-welsh medicine at the university of oxford since 2004. since 2016 he has been the director of the target discovery institute, university of oxford, and clinical research director at the francis crick institute. in 1990, then a wellcome trust senior fellow, ratcliffe set up the hypoxia biology laboratory in the weatherall institute of molecular medicine, oxford. his focus on epo led him on to study epo transcription in different tissues. he discovered that many different cell types could switch on several orders of magnitude higher expression of epo and other genes when deprived of oxygen (25,26). this was dependent on a 30 enhancer in the epo gene, the hre, that several groups, including ratcliffe’s, had identified (9). a 9-nucleotide stretch was later identified in the phosphoglycerate kinase 1 and lactate dehydrogenase a genes and shown to be present in and confer the same properties as the epo gene 30 enhancer (27). ratcliffe’s group went on to dissect the structural features of hif-1a with regard to oxygen regulation and identified a sequence conferring sensitivity to regulation by hypoxia, cobalt ions, or iron chelation, which they suggested mediates regulation through protein stability (28). in their 1999 hallmark paper in nature, a critical role for the von hippellindau (vhl) tumour suppressor (pvhl) in hif-1 regulation was identified (29). ratcliffe showed the critical, direct complex between pvhl and hif-1, demonstrating that in vhldefective cells hif-1a is constitutively stabilised and activated. re-expression of vhl restored oxygen-dependent instability (29). soon after, the ratcliffe and kaelin groups published back-to-back in science that prolyl hydroxylation is the crucial oxygenand iron-dependent posttranslational modification of hif-1a, required for recognition by pvhl (30,31). both studies identified the critical pro564, conserved in hif-1a throughout phylogeny. further, jaakkola et al. extrapolated from the known properties of prolyl-4-hydroxylase to predict that the hif-ph uses o2 as a substrate and 2-oxoglutarate and fe(ii) as cofactors, where fe(ii) can be substituted for by cobalt(ii), leading to enzymatic inhibition (31). a second, independent, prolyl hydroxylation site in hif-1a was identified by ratcliffe soon after (32). both the kaelin and ratcliffe groups contributed in different collaborative efforts to solve the structural aspects of how pvhl recognises hydroxylated proline residues in hif-1a to cause its degradation, showing that the hydroxyproline inserts into a gap within the pvhl hydrophobic core. interestingly, this pvhl gap is a mutational hotspot in cancer (33,34). with an amazing productivity, ratcliffe soon thereafter published another hallmark paper, for the first time identifying hif prolyl hydroxylases (phds) (35). here, in an outstanding contribution from the ratcliffe lab, epstein et al. presented a novel class of prolyl hydroxylases in mammalian cells, targeting hif-1a directly in a manner modulated by oxygen sensing. the foundation for their work was the identification of the c. elegans egl-9 gene product encoding a 2oxoglutarate-dependent dioxygenase as the prolyl hydroxylase for a hif-1a homolog in c. elegans (35). the identification of egl-9 allowed sequence and predicted secondary structure comparisons with mammalian dioxygenases. ratcliffe has thereafter focussed his research activities on the extent, mechanisms, and biological functions of prolyl hydroxylases and related oxygenases with a focus on pharmacological manipulation (see, for example, 36–38). william g. kaelin william g. kaelin jr received his bachelor degree in mathematics and chemistry and subsequently his md at duke university where he graduated in 1982. he did his residency at johns hopkins but went on to the dana farber cancer institute in boston where he joined david livingston to study tumour suppressors, in particular the retinoblastoma tumour suppressor. in 2002, he became a professor at harvard medical school. while retinoblastoma was a major research interest in livingston’s lab, kaelin soon focussed on another tumour suppressor gene, pvhl. in 1995, when he had established his own lab, kaelin and co-workers published the full-length sequence of the pvhl tumour suppressor, a 30-kda cytoplasmic protein (39). in 1996, a very important step was taken towards the connection between pvhl and oxygen sensing (40). an important observation in this study was that pvhl inhibited production of vascular endothelial growth factor (vegf), glucose transporter 1 (glut1), and platelet-derived growth factor b (pdgfb). as vegf, glut1, and pdgfb had been shown by—among others—ratcliffe (41) to be associated with hif, the connection between pvhl and hif was becoming apparent. kaelin’s lab went on to show that a frequently mutated region of pvhl can bind to ubiquitin ligase complexes (42). subsequently kaelin showed that pvhl is required for hif-1 ubiquitination and that the pvhl-ubiquitin ligase complex binds directly to the oxygen-dependent domain of hif-1a, the region targeted for ubiquitination (43). this important insight was validated and extended almost simultaneously by several groups (ratcliffe, poellinger, conaway; for a review, see 44). kaelin concluded that the ‘vascular tumours that characterise vhl disease may be caused by inappropriate accumulation of hif under normoxic conditions, leading to overproduction of angiogenic peptides such as vegf’ (43). kaelin, in collaboration with other groups, then solved the structure of the pvhl–ubiquitin ligase complex (45). very soon thereafter, the ratcliffe and kaelin groups published their papers back-to-back in science, demonstrating that prolyl hydroxylation is the crucial oxygenand irondependent posttranslational modification of hif-1a, required for recognition by pvhl and hif-1 stabilisation (30,31). both the kaelin and ratcliffe groups contributed in different collaborative efforts to solve the structural aspects of pvhl recognition of hif-1a prolyl hydroxylation (33,34); however, ratcliffe was first to publish on the identification of a hif prolyl hydroxylase (35). kaelin followed suit and identified upsala journal of medical sciences 207 human egln1 (the homologue of c. elegans egl-9) as a prolyl hydroxylase binding to and hydroxylating hif-1a peptides (46). after this rapidly developing phase in the understanding of oxygen sensing and the role in physiology and disease, kaelin has continued to investigate hypoxia regulation and therapeutic applications in the treatment of kidney cancer, as vhl mutation or hypermethylation is very common in sporadic renal cell carcinomas. he has used genetic models to address the contribution of hif-2a, compared to hif-1a, in hypoxia regulation in the skin and the liver (47) and subsequently presented the effects of an hif-2a antagonist, pt2399, in preclinical kidney cancer models (48). other interesting papers of relevance to cancer from kaelin’s group include the finding that glutamate, secreted from triple-negative breast cancer, promotes cysteine depletion. the therapeutic potential of targeting dysregulated glutamine metabolism in different human cancer forms including breast cancer has been addressed by many. however, kaelin and co-workers showed that pdh2 (egln1) undergoes oxidative self-inactivation in the absence of cysteine, allowing hif-1a stabilisation (49). kaelin has also addressed the effect of pdh2 inhibition/hif stabilisation in protection against myocardial ischemia-reperfusion injury, which involves a-ketoglutarate-dependent production of hepatic kynurenic acid that mediates cardiac ischaemic protection (50). hif-based cancer therapeutics by now, it is an established dogma that rapid-growing solid tumours with time develop a hypoxic centre into which infiltrating inflammatory and immune cells are attracted. the phenotype of the inflammatory/immune cells may be protumoral, acting to support tumour growth, for example by producing vegf, inducing tumour angiogenesis (51). the vessels in the tumour are, however, as a rule dysfunctional, with collapsed lumen and poor blood flow. they form rapidly but fail to stabilise. therefore, in spite of the high vegf production, the need for oxygenation in the tumour core is not met, hypoxia persists, and the vicious circle towards increased malignancy and spread is propagated (52–54). although major insights have come from research on mouse models, there are overwhelming data on the increased stability of hif-1a or hif-2a expression in human cancer, promoting progression and worsening prognosis in human solid cancers such as breast, prostate, and colorectal cancer (55–57). many attempts have been made to exploit the possibility to inhibit the hif pathway in cancer with the aim to halt growth and dissemination, boost anti-tumour immunity, and to improve the efficiency of therapeutics such as radiotherapy. one recent example of a hif pathway drug is evofosfamide (evo), a hypoxia-activated prodrug which promotes the release of a dna-alkylating agent, bromo-isophosphoramide mustard. evofosfamide recently failed in two phase iii clinical trials, one focussed on pancreatic cancer and the other on advanced soft tissue sarcoma. however, clinical studies with evofosfamide continue, now in combination with immunotherapy (ipilimumab). another recent failure is tarloxotinib bromide, a hypoxia-induced prodrug targeting the epidermal growth factor receptor. even though therapies directly targeting hif this far have failed, many other strategies to exploit oxygen sensing in a tumour context are being developed. in an elegant collaborative approach magnus essand at uppsala university and claire lewis at sheffield university exploited the fact that hypoxic regions in cancer are accompanied by inflammation (58,59). in their approach, macrophages are transduced with regulatory elements consisting of an hre to drive expression of e1a (the transforming gene of adenovirus) for expression of an oncolytic adenovirus. using promoter elements from prostate-specific genes, expression of the oncolytic virus is restricted to prostate cancer epithelial cells. macrophages transduced with these regulatory elements are injected in the circulation of mice where they infiltrate the hypoxic regions of prostate cancer. there, virus will be produced that specifically proliferates in, and thereby kills, prostate cancer cells. with this strategy, using macrophages as a vehicle, the otherwise therapeutically inaccessible hypoxic tumour core can now be reached, resulting in remarkable suppression of tumour growth and metastatic spread (58). the suppressive effect is maintained over long periods of time, resulting in prolonged survival of the mice. whereas untreated mice succumb from their prostate cancer at day 14, mice treated with macrophages equipped with the capacity to produce oncolytic viruses survive until the end of the observation period at day 42 (58). this is indeed a remarkable effect in a mouse tumour model. the strategy to deliver oncolytic virus to the hypoxic tumour core is now being tested in clinical trials. conclusions according to alfred nobel’s will, the nobel prize in physiology or medicine is awarded for a discovery of great benefit to human kind. in 2019 it went to semenza, ratcliffe, and kaelin for their discovery of oxygen sensing, a prize of the ‘classical school’ recognising a fundamental biological principle. while the full potential with regard to cancer therapeutics still is to come, let’s enjoy the stunning beauty of the biological machinery evolved to keep us oxygenated, allowing us to grow, but only as much as is needed! however, as always, questions remain. are there other oxygen-sensing mechanisms? can oxygen-sensing mechanisms be highjacked by the cancer cell? how do we accurately measure hypoxia? the interested reader is referred to the excellent review by macklin et al. (60). disclosure statement no potential conflict of interest was reported by the author(s). funding the author is supported by grants from the swedish cancer foundation [can2016/578], the swedish research council [2015–02375], the knut 208 l. claesson-welsh and alice wallenberg foundation [kaw 2015.0030, 2015.0275], and a fondation leducq transatlantic network of excellence grant in neurovascular disease [17 cvd 03]. notes on contributor lena claesson-welsh, professor of medical biochemistry at the department of immunology, genetics and pathology, uppsala university, director of the beijer laboratory for geneand neuroresearch, president of the european vascular biology organization (evbo), member of the royal swedish academy of sciences, the finnish society of sciences and letters, the science for life laboratory and a knut and alice wallenberg foundation scholar, is an expert on endothelial biology with focus on the molecular mechanisms regulation vascular permeability in health and 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192..195 case report the appearance of newly identified intraocular lesions in gaucher disease type 3 despite long-term glucocerebrosidase replacement therapy nadia sawicka-gutaja, maciej machaczkab, izabela kuli�nska-niedzielac, jadwiga bernardczyk-mellerd, paweł gutaje, jerzy sowi�nskia and marek ruchałaa adepartment of endocrinology, metabolism and internal medicine, poznan university of medical sciences, poznan, poland; bhematology center karolinska and department of medicine at huddinge, karolinska institutet, karolinska university hospital huddinge, stockholm, sweden; cheliodor swiecicki clinical hospital, ophthalmology outpatient clinic, poznan university of medical sciences, poznan, poland; ddepartment of ophthalmology, nzoz ‘ocu service’, poznan, poland; edepartment of obstetrics and women’s diseases, poznan university of medical sciences, poznan, poland abstract background gaucher disease (gd) is an autosomal recessive lipid storage disorder caused by the deficient activity of the lysosomal enzyme glucocerebrosidase. the presence of central nervous system disease is a hallmark of the neuronopathic forms of gd (types 2 and 3). intraocular lesions (e.g. corneal clouding, retinal lesions, and vitreous opacities) have been infrequently reported in gd type 3 (gd3). moreover, there are virtually no published data on the occurrence and natural course of intraocular lesions in gd3 patients treated with enzyme replacement therapy (ert). case presentation we describe the case of a 26-year-old polish male with l444p homozygous gd3 (mutation c.1448t > c in the gba1 gene) who developed fundus lesions despite 10 years of ert. at the age of 23 years, a spectral domain optical coherence tomography (oct) examination was performed which disclosed the presence of discrete lesions located preretinally, intraretinally in the nerve fiber layer, and in the vitreous body. a 3-year follow-up oct examination has not shown any significant progression of the fundus lesions. conclusions to the best of our knowledge, this is the first published report describing the occurrence of newly identified retinal and preretinal lesions occurring during long-term ert in gd3. we recommend that a careful ophthalmic assessment, including a dilated fundus examination, should be included as part of annual follow-up in patients with gd3. further studies are needed to understand the nature and clinical course of these changes and whether or not these intraocular findings have any predictive value in the context of neurologic and skeletal progression in gd3. article history received 9 november 2015 revised 16 february 2016 accepted 22 february 2016 keywords enzyme replacement therapy; gaucher disease type 3; intraocular lesions; neuronopathic; optical coherence tomography; retina introduction gaucher disease (gd) is an autosomal recessive lipid storage disorder caused by the deficient activity of the lysosomal enzyme glucocerebrosidase (1). there are more than 300 known mutations in the gba1 gene (1q21) that can cause gd, of which the c.1226a > g (n370s) and the c.1448t > c (l444p) mutations are the most prevalent (2). decreased glucocerebrosidase activity results in cytomorphologically noticeable lysosomal accumulation of glucosylceramide in cells of the monocyte-macrophage system such as the spleen, liver, and bone marrow (3,4). gd is known for its phenotypic diversity, and the clinical picture can vary from severe, lethal cases diagnosed before or shortly after birth to cases where patients are completely asymptomatic (5–11). three clinical types of gd are distinguished based on the absence (type 1) or presence (types 2 and 3) of neurologic symptoms and the dynamics of developing clinical signs (5–7). globally, the most prevalent form of gd is non-neuronopathic gd type 1 (gd1) with the main symptoms of thrombocytopenia, anemia, hepatosplenomegaly, and bone manifestations (6,8–11). in addition to the symptoms mentioned for gd1, the presence of central nervous system disease is a hallmark of the neuronopathic forms of gd (5,7,12). horizontal supranuclear gaze palsy is one of the earliest signs of the neuronopathic forms of gd (5,7,13,14). other typical ocular manifestations in neuronopathic gd include oculomotor apraxia and convergent squint (7,13,14). intraocular manifestations such as corneal clouding, retinal lesions, and vitreous opacities have been reported in gd type 3 (gd3), but they have not been characterized in detail (15–17). moreover, there are virtually no published data regarding the occurrence and natural course of intraocular lesions in patients with gd3 treated with enzyme replacement therapy (ert). here, we present the case of a young patient with gd3 who developed intraocular lesions despite 10 years of glucocerebrosidase replacement. in addition, we also provide results of his 3-year follow-up examination using spectral domain optical coherence tomography (oct). contact maciej machaczka, associate professor maciej.machaczka@ki.se hematology center karolinska, m54, karolinska university hospital huddinge, se-141 86 stockholm, sweden � 2016 the author(s). this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 3, 192–195 http://dx.doi.org/10.3109/03009734.2016.1158756 case report a 26-year-old polish male was diagnosed with gd at the age of 3 years. his past medical history was significant for splenomegaly, identified at the age of 12 months, and severe pancytopenia. gaucher cells were not found in aspiration biopsies of the bone marrow. he was splenectomized at the age of 3 years due to massive splenomegaly, and the diagnosis of gd was established by the presence of low activity of glucocerebrosidase in peripheral blood leukocytes. further direct dna sequencing revealed the homozygous mutation c.1448t > c in the gba1 gene (i.e. mutated alleles l444p/ l444p), which suggested gd3. at nearly 6 years of age, he developed avascular necrosis of the left femoral head. the patient started intravenous ert with macrophage-targeted recombinant glucocerebrosidase at the age of 8 years, immediately after ert became available in poland. at the age of 20, his ert was discontinued for 4 months during the unintentional world-wide imiglucerase (cerezymevr , genzyme corporation, cambridge, ma, usa) supply shortage (12). this was followed by a rapid increase in plasma chitotriosidase activity (figure 1), pathological bilateral forearm fractures and fractures in the left brachial bone and both fibulae. the fractures healed slowly, caused chronic pain, and created difficulties in walking for the patient. currently, the patient is receiving infusions of imiglucerase at a dose of 56 units/kg of body weight, administered every other week. ocular findings the patient had annual ophthalmologic follow-up examinations from diagnosis. his eye fundus examinations were normal until lesions were first noted at the age of 18 (i.e. 15 years from his diagnosis of gd and 10 years from the start of ert). the fundus lesions were described as white spots located peripherally and in the posterior pole. in january 2012, when the patient was 23 years old, his eye changes were documented in photographs (figures 2 and 3). at that time, the lesions were located on the surface of the retina and in the preretinal part of the vitreous body. oct confirmed the presence of discrete lesions located preretinally, intraretinally in the nerve fiber layer, and in the vitreous body (figure 4). electroretinography (erg) did not show any pathological pattern, thereby indicating normal retinal function. a recent ophthalmologic examination (performed in december 2014) showed a best-corrected visual acuity of 20/ 20 bilaterally and an intraocular pressure of 19 mmhg (normal values: 11–21 mmhg). ocular motility examination demonstrated limitations in abduction of both eyes. dilated fundoscopy revealed optic discs with well-defined borders and pink color in both eyes. the maculae were normal. further examinations showed yellowish-white punctuate deposits in the posterior pole and the periphery of the fundus bilaterally. a 3-year follow-up oct examination was performed in january 2015, and no signs of progression of the fundus lesions were evident. discussion gd affects less than 1/50,000 population but can be found in all ethnic groups around the world. in poland, the overall prevalence of gd is not yet established; however, in sweden the prevalence is approximately 1/170,000 individuals (6), slightly lower than that reported in other western countries but 2.5 times higher than in other nordic countries (1,6). although the neuronopathic forms are the rarer variants of gd (less than 5% of all gd cases), an endemic cohort of swedish patients with chronic neuronopathic gd3 lives in the figure 1. the patient’s plasma chitotriosidase activity measured annually since the age of 14 years. the patient’s ert was discontinued for 4 months at 20 years of age, causing a sharp increase in his plasma chitotriosidase activity. figure 2. photographs of the patient’s right eye fundus. a: posterior pole; b: periphery of the fundus. white spots overlie retinal vessels. upsala journal of medical sciences 193 county of norrbotten in northern sweden (12). it is worth noting that, in both sweden and poland, gd3 comprises approximately 40% of all known cases of gd (12,14). the incidence of vitreous opacities was found to be 3% in a series of 80 patients with gd1 (18). it was postulated that only those gd1 patients who underwent splenectomy have a tendency to form vitreous aggregates. similarly, it was suggested that preretinal and retinal white spots, which occur mostly in splenectomized patients with gd, represent an unusual systemic location of lipid-laden macrophages, socalled ‘gaucher cells’, due to the higher levels of circulating glucosylceramide (15,19,20). such results found on histopathologic examination of the fundus lesions in gd have only been published in japanese (an autopsy study) and only mentioned in the english medical literature (15). in 2004, shrier and colleagues reported a case of vitrectomy in a 20-year-old white woman with gd (20). the patient’s gd type and gba1 mutations were not reported. her vitreous aspirate specimen disclosed some degenerated and a few typical gaucher cells. the entire vitrectomy specimen, analyzed after centrifugation, showed a large amount of glucosylceramide. intraocular lesions have been infrequently reported in patients with gd3 (16,21). in the case presented here, the reported lesions appeared despite long-term glucocerebrosidase replacement therapy. one possible explanation for this finding is the large molecular size of recombinant glucocerebrosidase, which prevents it from crossing the blood–brain barrier, therefore not allowing it to reach the eye, as postulated by coussa and colleagues (21). similar to the patient with gd3 described by coussa et al., our patient had normal visual acuity and erg findings. however, seidova et al. reported preretinal lesions and subtle retinal dysfunction on erg in a splenectomized patient with gd1 (19). there is only one published report, by sheck et al., describing preretinal opacities seen with oct in a 14-year-old girl with gd3 treated with ert (16). oct is a non-contact optical device that provides cross-sectional images and quantitative analysis of the ocular tissues (22). unfortunately, sheck et al. did not provide any information as to whether these preretinal changes were present before the start of ert, and they have not reported any oct follow-up data (16). based on the oct findings, they proposed that intraocular opacities in gd are preretinal, located at the vitreo-retinal interface associated with localized posterior vitreous detachments rather than vitreous opacities as previously suggested (16). in contrast to sheck and colleagues, we have shown with oct the presence of lesions on the surface of the retina and in the vitreous body. recently, mcneill and colleagues published the results of a pilot study in which they suggest that thinning of the retinal ganglion cell layer may be associated with early neurodegeneration in patients with gd1 (23). we speculate whether retinal and preretinal deposits could be an early warning sign of disease progression in gd. it is worth noting that the appearance of the intraocular lesions seen in our patient preceded his severe skeletal complications by 2 years, which supports this hypothesis. one might argue that interruption of imiglucerase therapy alone could cause the aforementioned bone figure 3. photographs of the patient’s left eye fundus. a: posterior pole; b: periphery of the fundus. white spots present along retinal vessels with a distribution of changes similar to that seen in the right eye. figure 4. optical coherence tomography images taken in january 2012 (a, b) showing (1) intravitreal, (2) preretinal, and (3) intraretinal (in the nerve fiber layer) locations of the deposits in the presented patient with gaucher disease type 3. 194 n. sawicka-gutaj et al. fractures. although this may be true with respect to a longlasting interruption of ert, it is doubtful whether missing only eight doses of imiglucerase would result in such severe bone complications so quickly in the absence of obvious skeletal disease progression in this patient. thus, we recommend that a careful ophthalmic assessment including a dilated fundus examination be included as part of annual follow-up in patients with gd3. when intraocular lesions are detectable in gd3, a subsequent oct examination would provide further information on the exact location of the deposits in relation to retinal architecture. to the best of our knowledge, this is the first published report on the occurrence of newly identified retinal and preretinal lesions occurring during long-term ert in gd3. our case report highlights the fact that oct is helpful in the follow-up of intraocular lesions detected in patients with gd3. further studies are needed to explain better the nature and clinical course of these changes. it would be especially valuable to know whether or not these intraocular findings have any predictive value in the context of neurologic and skeletal progression in gd3. acknowledgements nadia sawicka-gutaj and maciej machaczka contributed equally to the writing of this paper. disclosure statement the authors report no conflicts of interest. references 1. zimran a. how i treat gaucher disease. blood 2011;118:1463–71. 2. hruska ks, lamarca me, scott cr, sidransky e. gaucher disease: mutation and polymorphism spectrum in the glucocerebrosidase gene (gba). hum mutat 2008;29:567–83. 3. machaczka m, markuszewska-kuczy�nska a, regenthal s, jurczyszyn a, gałązka k, wahlin be, et al. clinical utility of different bone marrow examination methods in the diagnosis of adults with sporadic gaucher disease type 1. pol arch med wewn 2014;124:587–92. 4. markuszewska-kuczy�nska a, klimkowska m, regenthal s, bulanda a, k€ampe bj€orkvall c, machaczka m. atypical cytomorphology of gaucher cells is frequently seen in bone marrow smears from untreated patients with gaucher disease type 1. folia histochem cytobiol 2015;53:62–9. 5. tylki-szyma�nska a, vellodi a, el-beshlawy a, cole ja, kolodny e. neuronopathic gaucher disease: demographic and clinical features of 131 patients enrolled in the international collaborative gaucher group neurological outcomes subregistry. j inherit metab dis 2010;33:339–46. 6. machaczka m, hast r, dahlman i, lerner r, klimkowska m, engvall m, et al. substrate reduction therapy with miglustat for type 1 gaucher disease: a retrospective analysis from a single institution. ups j med sci 2012;117:28–34. 7. weiss k, gonzalez an, lopez g, pedoeim l, groden c, sidransky e. the clinical management of type 2 gaucher disease. mol genet metab 2015;114:110–22. 8. machaczka m, klimkowska m, h€agglund h. effort bruising disclosing gaucher disease in a 55-year-old non-jewish woman. j inherit metab dis 2009;32:758–61. 9. machaczka m, klimkowska m, regenthal s, h€agglund h. gaucher disease with foamy transformed macrophages and erythrophagocytic activity. j inherit metab dis 2011;34:233–5. 10. machaczka m, klimkowska m. novel heterozygous c.798c > g and c.1040t > g mutations in the gba1 gene are associated with a severe phenotype of gaucher disease type 1. ann hematol 2014;93:1787–9. 11. machaczka m, lorenz f, kleinotiene g, bulanda a, markuszewskakuczy�nska a, raistenskis j, et al. recurrent pulmonary aspergillosis and mycobacterial infection in an unsplenectomized patient with type 1 gaucher disease. ups j med sci 2014;119:44–9. 12. machaczka m, k€ampe bj€orkvall c, wieremiejczyk j, paucar arce m, myhr-eriksson k, klimkowska m, et al. impact of imiglucerase supply shortage on clinical and laboratory parameters in norrbottnian patients with gaucher disease type 3. arch immunol ther exp (warsz) 2015;63:65–71. 13. svennerholm l, dreborg s, erikson a, groth cg, hillborg po, håkansson g, et al. gaucher disease of the norrbottnian type (type iii). phenotypic manifestations. prog clin biol res 1982;95:67–94. 14. tylki-szyma�nska a, keddache m, grabowski ga. characterization of neuronopathic gaucher disease among ethnic poles. genet med 2006;8:8–15. 15. cogan dg, chu fc, gittinger j, tychsen l. fundal abnormalities of gaucher’s disease. arch ophthalmol 1980;98;2202–3. 16. sheck lhn, wilson cj, vincent al. analysis of the pre-retinal opacities in gaucher disease using spectral domain optical coherent tomography. ophthalmic genet 2012;33:253–6. 17. hsing ye, foster a. preretinal and posterior vitreous deposits in gaucher disease. jama ophthalmol 2014;132:992. 18. wollstein g, elstein d, strassman i, seelenfreund m, zylbermann r, zimran a. preretinal white dots in adult-type gaucher disease. retina 1999;19:570–1. 19. seidova sf, kotliar k, foerger f, klopfer m, lanzl i. functional retinal changes in gaucher disease. doc ophthalmol 2009;118:151–4. 20. shrier em, barr cc, grabowski ga. vitreous opacities and retinal vascular abnormalities in gaucher disease. arch ophthalmol 2004;122:1395–8. 21. coussa rg, roos jcp, aroichane m, miron mc, ospina lh. progression of retinal changes in gaucher disease: a case report. eye (lond) 2013;27:1331–3. 22. adhi m, duker js. optical coherence tomography – current and future applications. curr opin ophthalmol 2013;24:213–21. 23. mcneill a, roberti g, lascaratos g, hughes d, mehta a, garwayheath df, et al. retinal thinning in gaucher disease patients and carriers: results of a pilot study. mol genet metab 2013;109:221–3. upsala journal of medical sciences 195 the appearance of newly identified intraocular lesions in gaucher disease type 3 despite long-term glucocerebrosidase replacement therapy introduction case report ocular findings discussion acknowledgements disclosure statement references ujms 110 (3) bra upsala j med sci 110 (3): 241–244, 2005 pyogenic liver abscesses secondary to carcinoma of the sigmoid colon: a case report and clinical features of 20 cases in japan takashi yokota1, kazutsugu iwamoto2, yuko watanabe2, hidemi yamauchi2 and shu kikuchi2 and masahito hatori3 1department of surgery, national sanatorium tohoku shinseien, 2department of surgery, national hospital organization, sendai medical center and 3department of orthopaedic surgery, tohoku university school of medicine, sendai, japan. abstract we report a case of liver abscesses associated with sigmoid colon cancer in an 81-yearold woman. the patient was referred to our hospital because of a tumorous lesion of the sigmoid colon. five days before the scheduled operation, she presented abdominal pain, fever and chill. imaging scans revealed multiple liver abscesses in both lobes, which were successfully treated with intravenously administered antibiotics. two weeks later, the patient underwent laparoscopic-assisted sigmoidectomy. nineteen cases of liver abscess associated with colonic cancer have been reported during the past ten years in japan, and we report the clinical features of these cases in this paper. an aggressive search for the underlying cause of pyogenic liver abscess should be an integral part of the definitive treatment of this disease. introduction we report a case of liver abscesses associated with sigmoid colon cancer in an 81year-old woman. the patient was referred to our hospital because of a tumorous lesion of the sigmoid colon. five days before the scheduled operation, she presented abdominal pain, fever and chill. imaging scans revealed multiple liver abscesses in both lobes, which were successfully treated with intravenously administered antibiotics. two weeks later, the patient underwent laparoscopic-assisted sigmoidectomy. nineteen cases of liver abscess associated with colonic cancer have been reported 241 received 25 april 2005 accepted 18 may 2005 key words: liver abscess, colon cancer during the past ten years in japan, and we report the clinical features of these cases in this paper. an aggressive search for the underlying cause of pyogenic liver abscess should be an integral part of the definitive treatment of this disease. (key words: liver abscess, colon cancer) case report an 81-year-old woman had felt lower abdominal pain four weeks before admission to our hospital, and a diagnostic work-up, including barium enema and colonoscopy, was performed elsewhere. a double-contrast barium enema was performed and revealed a space-occupying lesion in the sigmoid colon (fig. 1a). examination of biopsy specimens taken from the sigmoid tumor by colonoscopy disclosed fragments of moderately differentiated adenocarcinoma (fig. 1b). five days before the scheduled operation, she presented abdominal pain, fever and chill. physical examination revealed sweating, tachycardia, a temperature of 40.7 c, and severe tenderness of the right upper abdomen. laboratory tests showed hemoglobin of 12.0 g/dl, white blood count of 1400 and crp of 15.8 mg/dl. a computed tomography scan revealed liver abscesses in both lobes (fig. 1c). the patient received treatment with wide-spectrum antibiotics, which was effective. her clinical condition improved, and the results of laboratory tests returned to normal. follow-up imaging scans showed disappearance of liver abscesses (fig. 1d). two weeks later, the patient underwent laparoscopic-assisted sigmoidectomy. her postoperative course was uneventful, and she remained free of recurrence during one-year followup period. discussion the common pathogenetic mechanisms by which bacteria can form liver abscess are 1) ascending biliary infection, 2) portal bacteremia, 3) septicemia, 4) direct extension from intraperitoneal infection, 5) direct trauma to the liver, and 6) secondary infection of metastatic cancer (7, 8). we speculated that the route of infection in our patient was portal bacteremia because there was no evidence of ascending biliary infection and no other inflammatory lesions in the intraabdominal cavity. since our patient had no macroscopic intestinal perforation, invading bacteria would have found their portal entry through a mucosal barrier break near the tumor (2). in our computer-assisted search of the literature including papers published over the past 10 years in our country, we found 19 reports of colonic carcinoma as an underlying disease of a pyogenic abscess of the liver in the absence of hepatic metastases (1-7). these patients included 11 men and 9 women with a mean age of 64 years. the sites of primary colon lesions were the sigmoid colon in 11 patients, rectum in 4 patients, ascending colon in 3 patients and transverse colon in 2 242 patients. abscesses were involved in 9 cases in the right lobe of the liver, in 4 cases in the left lobe, and in 4 cases in both lobes. a solitary abscess was found in 10 cases and multiple abscesses in 8 cases (2 cases unknown). our patient did well with medical therapy alone, a result consistent with many reports that pyogenic liver abscesses can be effectively managed by antibiotics and that surgery is rarely indicated. actually, only 5 patients were operated on; 3 patients underwent drainage and 2 patients partial resection of the liver because liver metastases were suspected (1, 4). ultrasound-guided percutaneous drainage of the abscesses was performed in 8 patients, and the other 7 patients were given only medication. in a report by tanizaki et al. (4), open surgical drainage is not recommended because of the risk of dissemination of carcinoma cells, since liver metastasis could be one of the causes of liver abscess. systemic antibiotic treatment, combined with simple aspiration, has been successfully used for treatment of pyogenic bacterial abscesses. it is interesting to note that in half of these 20 cases, the presence of colon cancer 243 fig 1. a: double-contrast barium enema image showing a space-occupying lesion in the sigmoid colon (arrow). b: colonoscopy disclosed a sigmoid colonic tumor. biopsy showed moderately differentiated adenocarcinoma. c: computed tomography (ct) scan showing one of the liver abscesses (in the right lobe) before the operation (arrow). d: six days after the operation, ct showed progressive improvement of the focal liver lesions together with subjective and clinical improvement. c a b d was discovered only after clinical symptoms of liver abscess had appeared, and association with colon cancer was established first in retrospect. although liver abscesses due to colonic carcinoma are very uncommon, having treated a patient with such a condition, we would like to draw the attention of surgeons to this possibility. references 1. nomi s, fujiwara h, oka k (1994) a case of rectal carcinoma considered as pyogenic liver abscess j jpn soci coloproctology 47:71-74 2. masamune j, midorikawa h, satake k. (1992) a case of sigmoid colon cancer accompanied by liver abscess. j jpn soci coloproctology 45: 219-223 3. nakamura t, tsuchiya y, umehara y. (1996) a case of cancer of the sigmoid colon accompanied by a liver abscess j jpn pract surg soci 57: 2250-2253 4. tanizaki h, kawano n, watanabe h. (2002) a case of solitary liver abscess complicated by early sigmoid colon cancer. j jpn surg associ 63:449-453 5. nobusawa s, matsumoto a, endo h. (2000) pyogenic liver abscesses secondary to carcinoma of the sigmoid colon. a case report and review of the literature. j jpn surg associ 61:730-733 6. sakurai k, miyake h, fujisaki s. (1998) a case of rectal cancer with solitary liver abscess. j jpn soci coloproctology 51:242-247 7. tamaki y, higashi f, kunisho t. (1993) three cases of liver abscess associated with neoplasmic lesion. j wakayama med soc 44: 629-633. 8. sigal t, ahuva gi-s, hana m, et al: pyogenic liver abscess: warning indicator of silent colonic cancer. dis colon rectum 38: 1220-1223, 1995 9. amedeo l, alberto g, maurizio p, et al: right colon adenocarcinoma presenting as bacteroides fragilis liver abscesses. j clin gastroenterol; 14: 335-338, 1992 corresponding author: takashi yokota, m.d. department of surgery, national sanatorium tohoku shinseien, tome, miyagi 989-4601, japan _:0228-38-2121, fax: 0228-38-3765, e-mail address: hki06124@nifty.com 244 (3) hofvander 27-34 27 upsala j med sci 111 (1): 27–34, 2006 violations against children in a national and international perspective yngve hofvander department of women´s and childrens health, uppsala university in his writings, rosén does not at all discuss violations against children, although this certainly was common in his time. actually, children have been beaten and maltreated since times immemorial in their homes, as servants and in schools (1). flogging used to be a corporal punishment in schools and was found to be a valuable disciplinary and pedagogic means. in the swedish school laws from 1611 it was stated, however, that due consideration should be given to those children who were intellectually or physically defect. in 1918 corporal punishment or abusive treatment became forbidden against older children but for younger children this could continue until 1958! not until 1979 sweden introduced as the first country in the world a law forbidding corporal punishment at home and in 1990 sweden ratified the un convention of the rights of the child. the article 19 in this document states:“…to protect the child from all forms of physical or mental violence, injury or abuse, neglect or neglient treatment, maltreatment or exploitation including sexual abuse…” however, article 14 neutralises to some extent this strong statement by saying that …”state parties shall respect the rights and duties of the parents to provide direction to the child in the excercise of his or her right”. in other words the parents may still have the right to corporal punishment if considered fit according to tradition or religion. maltreatment of children has been much in focus (mass media) in recent years. in 1999 in sweden no less than 879 abused children 0-6 years were reported to the police and 5040 in the 7-14 age group (1). most of these were not severe – but probably a large number are never reported. however, it would seem that it has decreased compared to the 1970s corporal punishment from about half of all children to about 1/5. in absolute number this is of course alarmingly many children, some of whom may retain painful memories to later in life. received 17 october 2005 accepted 28 october 2005 sexual intercourse with children (usually defined as less than 12 years) has over the centuries been considered the same as rape and being a very serious crime, resulting in death penalty, at least until the end of the 18th century (2). in these days there was a very marked male dominance and the head of household had a legitimate power over wife, children and servants, which also included the right for corporal punishment. sexual abuse of young girl servants was certainly common, impossible to report – and resulting in expulsion and not seldom illegitimate abortion or infanticide with catastrophic consequences for the mother(2). implementation of the convention of the rights of the child in article 24.3 is stated that …” states parties shall take all effective and appropriate measures with a view to abolish traditional practices prejudicial to the health of children”. practices which are listed which should be reviewed in the light of the convention include “ all forms of genital mutilation and circumcision, binding, scarring, burning, initiation ceremonies e.g. forced holding under water, preferential care of male children, lack of care of disabled children, food taboos, beating children, early marriage” – et cetera… this horrible list of abuse could be made much longer! in general we believe that parents always do the right thing for their children, the best they can and always to the benefit of the children. is that true? i am inclined to answer in the negative – it is not, there are many examples that show that parents may cause harm to their children to serve other purposes, mainly their own, but also purpose of religion, of tradition or a mixture of the two (3). in the following a number of examples are given on violations against children´s integrity and which may be causing much suffering and harm (3). foot binding in china, about 1000 years ago, parents started to bind girls´ feet to prevent them from growing to more than about 10 cm, starting at the age of about 5 years and continuing for 2-3 years. this caused much pain, often infection and the girl became unable to walk and work. the custom started in the royal courts, became a fashion spreading to other sectors among the well-to-do, a sign of beauty, submission and chastity. the parents could thus offer for marriage a girl who was definitely a virgin, thereby resulting in a substantially higher bride price for the benefit of the parents (4). foot binding was forbidden during the 20th century but it is still possible to see elderly women in the streets demonstrating what happened with them in the past. 28 selective abortions in most cultures there has been and still is a preference for boys. in china and india this has taken extreme forms. the physiological sex ration at birth is 100 girls to about 104 boys. since the 1960s – at the time when the onechild policy was put into action in china – the ratio has become more and more skewed, to some 117 boys ( table 1) and for multipara and in some remote areas even up to 135 to 100. (5). ultrasound investigation for foetal sex determination is readily available although forbidden, but there are many ways to get around this. recently the one-child policy seems to have become less rigidly upheld. circumcision of girls – female genital mutilation (fgm) fgm is practiced to a horrifying great extent and shows no tendency to decrease with the exception of small areas where intensive educational and motivational programmes have started. it is basically an african phenomenon and is practiced in varying extent in those countries that form a belt across africa (table 2). in somalia, sudan and ethiopia practically all girls undergo fgm, at least in the traditional society. but also in well educated circles fgm is considered to be a decree in the koran. in a study in somalia (6), a country where the tradition is strongest, among girls where 43 % had secondary school or higher education, no less than 70 % believed that this was a decree in the koran. about 2 million girls undergo fgm annually and more than 130 million currently living girls and females have experienced this mutilating operation (7). tens of thousands of them are presently living in the us and in europe, constituting new medical, ethical and legal problems in these countries. it is illegal in these western coun29 table 1. development of sex ratio at birth in china. the normal biological sex ratio is 100 girls to 102-104 boys (5). years girls boys <1980 100 106 1981 100 109 1986 100 111 1889 100 114 2000 100 117 (130-135) tries to perform the operation and to restore the circumcision after delivery. in sweden it is also illegal to perform the operation abroad. however, so far not a single case has been brought to court in any western country, respect for the parents or other relatives has been given as the reason. instead the mutilated girl is sacrificed! (8) the operation takes different forms of severity. the most simple is excision of the prepuce and part or all of the clitoris. this may be extended to comprise the labia minora. in the most severe form , infibulation, also part or all of the external genitalia are excised and the orifice of the vaginal opening is stitched ( traditionally with a thorn) or scraped, leaving only sufficient room for urine and menstrual blood. a wide variety of immediate and long term complications have been reported – bleedings, infections, sexual problems, delivery complications and infertility (9). it is indeed understandable that marital problems will arise when the bride is infibulated and the vaginal orifice has to be cut open with a knife by a piece of glass! and it is indeed difficult to understand how this mutilating practice, severely affecting half the population in many countries, is allowed to continue, showing only modest signs of decreasing frequency. the international organisations including the ngos have been remarkably inactive, in view of the enormous suffering that is affecting millions of girls and women. however, suffering from the female genital area is usually silent and accepted in the name of chastity and marital honour! in sweden with some 30.000 immigrants from somalia, it is estimated that possibly up to some 50 girls annually are subjected to fgm, the operation being performed country percent fgm somalia sudan 80-100 ethiopia egypt liberia kenya 40-80 nigeria guinea bissau 30 table 2.rate of female genital mutilation (fmg) in selected african countries (7). 31 abroad (martina frank, risk, personal communication). in a number of cases this has been suspected but so far none has been brought to court (risking a sentence of up to 10 years). male circumcision – male genital mutilation, mgm male circumcision is usually associated with a practice originating from the tales in the old testament – abraham going into a covenant with god, prescribing that he had to circumcise himself ( 90 years old!) and all his male household and in exchange should get a multitude of off springs which should populate the land. also we have learnt that jesus was brought to the temple on his 8th day to be circumcised. since then circumcision is one of the traditional rites in jewish life, at least until recently. however, circumcision was practiced in egypt long before that, its background being obscure. the jewish circumcision is in minority, though, as is seen in table 3. in the moslem sphere it is estimated that some 12 million boys are circumcised annually, some time during the period 2 – 14 years. the claim is that this is a command in the koran (which is not true) or in the hadiths (the tales and sayings from the prophet’s time). the operation is often done by laymen, barbers and similar, is cruel and painful, sometimes made as a mass operation, e.g. in the market place, the boy dressed up like a prince, the whole event continued with a big fiesta, the boys being given sweets and gifts – a party which may cost several times more than the operation itself (8). in sweden it is estimated that some 3000 boys are circumcised annually. a number of severe complications have been reported including in recent years one death (10) . background target group no circ/yr religious jews 100.000 religious moslem 12 million traditional africa/s sahara 9 million neonatal usa+ 1.2 million table 3. estimated number of male circumcisions performed annually for religious, traditional or other reasons assuming 80% in the first 3 groups and about 60 % in the 4th group (10). less than 1000 of these operations are performed by the county council health service. we know only little where the remaining are done, by whom and how (11). in 2001 the swedish parliament issued a law, stating that the operation must be done by a doctor and appropriate anesthetics given. an exception was made for the jewish circumcision done by a lay person – a doctor or a nurse had to be present and give the anesthetics. as is obvious from the above the large majority of circumcisions are performed without any control at all from the health service and thereby the law has not much effect as it cannot be implemented. in the traditional african setting, mainly south of the sahara and in west africa, it is estimated that some 9 million mgm are being done annually as an initiation rite to manhood. again these operations are done by lay people such as barbers, are done en masse (“the boy shall go out in the bush”) and are an introduction to big parties. understandably the complication rate may be high. in 1995 in the cape province in south africa 34 boys had died, 12 had got the whole penis amputated and 743 boys had to be admitted to hospital for treatment of septic infections (12). finally, in the usa and other anglo-saxon countries about 1 million circumcisions annually are done, practically all in the neonatal period and in hospital or other safe settings. this tradition started (in germany!) with the idea that it should prevent or stop masturbation. little by little up to more than one hundred indications were added – ranging from asthma to epilepsy. by 1960 about 80 % of all boys underwent circumcision. the figure now is about 57 % and continues to decrease, particularly in areas where the public health service is not paying for the operation (8). a large number of medical professional organisations have stated over and over again that circumcision does not bring with it any medical advantages ( ’’… data are not sufficient to recommend routine neonatal circumcision”) (10). lately a number of studies seem to have indicated that the hiv rate should be lower in circumcised african men, presumably because the hardened surface at the glans penis better would withstand penetration of the hiv. however, still of course the hiv infection rate also in circumcised men is alarmingly high and constitutes an enormous medical and social disaster in africa. circumcision and the convention of the rights of the child there is a un committee located in geneva, consisting of representatives from 18 different countries which shall implement the convention. hearings are arranged about every 3-5 years. genital mutilation – although performed on a massive scale on more than 20 million boys and girls annually and being a medically unnecessary operation – has not been a prominent issue in these hearings. mgm, although listed in the implementation handbook to be reviewed (13) apparently has been totally removed from the agenda and no explanation is given for that (attorney steven svoboda, nocirc, los angeles, personal communication). contributing to that may be what is stated in 32 article 14 in the convention …”freedom to manifest one´s religion or beliefs may be subjected only to such limitation as are prescribed by law, a text which could be interpreted as contrary to the text in article 24.3, which is quoted above. over and over again in the convention is stated that all action taken in relation to the child always shall be “for the best of the child”. with due respect for tradition and religious preferences and wants and considering that the child has no say and the risk for complications which may have life long effects then it is indeed strange that the swedish government has not acted to the effect that this violation against the child must be abolished. references 1. jansson s. children and maltreatment (in swedish). report on corporal punishment and other maltreatment in sweden at the end of the 20th century. sou 2001:18, isbn 91-38-21411-3 2. bergenheim a. brottet, offret och förövaren: vetenskapen och det svenska rättsväsendets syn på sexuella övergrepp mot kvinnor och barn 1850-2000. ( in swedish). carlsson, stockholm, 2005 3. hofvander y. the world´s children – the children´s world. acta paediatr 93:1414-9, 2004 4. fang hsy, yu fyk foot binding in chinese women, canad j surg 1960;3:195-202 5. plafker t sex selection in china sees 117 boys for every 100 girls. brit med j 2002;324:1233 6. dirie m, lindmark g. female circumcision in somalia and women´s motives. arch obstetr gyn scand 1991;70 (7-8):58-65 7. female genital mutilation. report of a who technical working group meeting. geneva: 17-19 july, 1995. geneva, who, 1996 8. the 8th international symposium on circumcision and human rights. an anthropological, medical, legal and ethical analysis. 2-4 september 2004. to be published. 9. almroth l genital mutilation of girls in sudan. doctoral dissertation. karolinska institute, stockholm, 2005. 10. hofvander y circumcision in boys: time for doctors to reconsider world hospitals and health service vol 38, no 2; 2002 11. hofvander y circumcision of boys in sweden performed within the county health service system. report (in swedish). uppsala county council, 2004. 12. sidley p botched circumcision leads to arrest for murder. bmj 1966;313:647 13. implementation handbook for the convention of the rights of the child. unicef, new york, 1998 corresponding author: yngve hofvander department of women´s and children´s health university hospital se-751 85 uppsala, sweden 33 34 knowledge about the impact of age on fertility: a brief review review article knowledge about the impact of age on fertility: a brief review ilse delbaerea , sarah verbiestb and tanja tyd�enc amidwifery education, vives university of applied sciences, kortrijk, belgium; bcenter for maternal and infant health, school of medicine, university of north carolina at chapel hill, chapel hill, nc, usa; cdepartment of women’s and children’s heath, akademiska sjukhuset uppsala university, uppsala, sweden abstract delayed childbearing is currently a major challenge in reproductive medicine as increased age has an important impact on successful conception, both in natural and in assisted reproduction. there is a lack of knowledge about the impact of age on fertility, even in highly educated populations. a number of initiatives have been taken to increase fertility awareness. health care providers have been encouraged to talk with patients about their reproductive life plan (rlp) for almost a decade based on recommendations from the centres for disease control and prevention. this concept has been explored successfully in swedish contraception counselling. a growing number of online interventions aim to raise fertility awareness. these websites or interactive tools provide relevant information for individuals and couples as they consider whether they want children, when they should have them, and how many they may wish to have. these interventions are important, because research depicts that knowledge helps people in their decision-making process. with new fertility preservations such as egg freezing now available, additional education is needed to be sure that women and couples are well informed about the cost and low success rates of this intervention. article history received 18 november 2019 revised 17 december 2019 accepted 18 december 2019 keywords age; awareness; egg freezing; knowledge; fertility introduction having children is valued very highly in all societies. ninety percent of people in western countries want children, generally between one and three (1–4). in higher-income countries, people have children because of ‘their contribution to life satisfaction’ of the couple, ‘development as a person’ in the parents, and ‘for giving and receiving love’. people in lower-income countries may also depend on children to contribute to the financial security of the family (1,5,6). the decision to have children or not may be moderated by individual, societal, and economic factors. interviews with professional women and men who did not have children found that they did not generally give much thought to their fertility, although there were variances between women and men. they believed that fertility problems could be addressed by assisted reproductive technologies (art) or families could be formed through adoption. postponed parenthood was described as an adaptation to societal changes and a contemporary lifestyle with many competing priorities (7). reproductive life planning is a simple concept that can be very complex. people who have experienced instability in their life, live with interpersonal violence, and/or live in poverty with limited options may not believe they have the ability to plan anything in their lives. other people may feel ambivalent about ‘wanting’ a child for many reasons. some people hold religious beliefs that run counter to the idea of planning. a person may wish to become a parent but not have a partner in their life. reproductive plans often change over time due to life circumstances, including relationship changes. family-friendly countries offer parental leave, subsidized childcare, and work places with flexible hours for parents so they can spend more time with their children as a way to create more equity for all people who may wish to become parents (8). many countries, however, offer only limited support, which can make it more difficult for some people to balance family, professional, and financial needs. this brief review aims to continue the conversation on this important topic by describing the impact of delayed parenthood on fertility and reviewing fertility awareness among university and medical students. this paper shares several successful interventions and offers recommendations for future work. advanced maternal age and infertility the number of women in high-income countries who have delayed childbearing has increased over the past decades, with the average age of having a first child being 30 years in europe (9). there are a variety of reasons for this, including investment in education, development of a professional career, and/or difficulties in finding the right partner. delayed contact ilse delbaere ilse.delbaere@vives.be midwifery education, vives university of applied sciences, doorniksesteenweg 145, kortrijk, 8500, belgium � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 2, 167–174 https://doi.org/10.1080/03009734.2019.1707913 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1707913&domain=pdf&date_stamp=2020-05-21 https://orcid.org/0000-0002-8522-2512 https://orcid.org/0000-0002-2491-1170 https://orcid.org/0000-0002-2172-6527 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2019.1707913 http://www.tandfonline.com childbearing is more common among women with higher education. as a consequence of delaying parenthood, there has been an increase in the number of women experiencing problems in getting pregnant, as well as higher risks for pregnancy and childbirth complications. when a woman is younger than 30, she has an 85% chance to conceive within 1 year. at the age of 30, there is a 75% chance to conceive in the first 12 months. this chance declines to 66% at the age of 35 and 44% at the age of 40. this is due to the effect of aging on the ovary and eggs. furthermore, older women are more likely to experience a miscarriage than younger women (27% of pregnancies end in a miscarriage at age 40 compared to 16% at age 30 or younger) (10). advanced maternal age is associated with prolonged time to conceive, and postponed parenthood may affect the desired family size. using a computer simulation programme, habbema et al. calculated the recommended age to start a family for women, depending on the number of children they wanted and to what extent women were prepared to undergo fertility treatment. the model predicts that if a couple wanted a 90% chance to realize their ideal family without in vitro fertilization (ivf), couples with a desire for a one-child family should start at the latest at age 32 of the female partner. when a two-child family is desired they should start when the woman is 27, and when couples want three children they should start at age 23 (11). a computer simulation was also used in research by leridon (10) to assess whether assisted reproduction could compensate for the effect of age on fertility. unfortunately, this was not the case (10). fertility knowledge among men and women of fertile age, with focus on university students in 2004, lampic, skoog svanberg, and tyd�en started to investigate swedish university students’ attitudes about having children and their knowledge about fertility (1,6,12). although the majority of women wished to have two or three children, six out of ten said they would consider having an abortion if confronted with an unplanned pregnancy ‘right now’. important prerequisites for women’s decision to have children were to be sufficiently mature, have a stable partner to share parenthood with, have completed studies, and have financial security. women worried about problems with combining work and having children, including being less competitive in the labour market. slightly more than half of the women wanted to have their last child between ages 35 and 44 years. one-fourth of doctoral students overestimated a woman’s ability to become pregnant between 35 and 40 years of age, and about half had overly optimistic perceptions of the chances to have a baby by means of ivf. women were less certain than men of being able to achieve the desired number of children (1,6,12). sobotka commented on these studies, highlighting that it is a challenge for health care providers to educate university students about fertility issues when they want to postpone parenthood (13). the importance of this education was highlighted, and engaging men is important as male infertility is on the rise (14). finding a partner to share parenthood with can be hard for women who intend to invest time in their own career (15,16). the swedish fertility awareness questionnaire has been used in university populations in many countries with similar findings as in sweden (17). the swedish surveys investigated fertility knowledge with eight questions: 1. at what age are women the most fertile? 2. at what age is there a slight decrease in women’s ability to become pregnant? 3. at what age is there a marked decrease in women’s ability to become pregnant? 4. a young woman (<25 years) and a man have unprotected intercourse at the time of ovulation—how large is the chance that she will then become pregnant? 5. a woman and a man regularly have unprotected intercourse during a period of 1 year—how large is the chance that she will become pregnant if she is 25–30 years old? 6. how large is the chance that she will become pregnant if she is 35–40 years old? 7. how many couples in sweden are involuntarily childless? 8. for couples that undergo treatment with ivf, what is their chance, on average, of having a child? to summarize in brief: men and women had misconceptions about age and fertility and overestimated the success rate of having a child through ivf. in the case of infertility, women were more likely than men to consider adoption, but both genders were much more likely to choose ivf over adoption, indicating an importance of genetic parenthood. as a consequence of these misconceptions, young men and women of reproductive age would benefit from evidence-based education on fertility issues to help them make informed decisions regarding reproductive planning. two recent reviews investigating knowledge about age-related fertility decline concluded that campaigns about age and fertility should be targeted both to people of reproductive age and to health care providers. interventions and campaigns are warranted, especially those targeting men and people with low education. they should be customized to meet individual needs (18). fertility awareness among health care providers and medical students not only is there a lack of fertility awareness in the general population of university students, there is a lack of knowledge among medical students and health care providers as well. medical students’ intentions for future parenthood and their knowledge about fertility have been investigated in several studies. yu et al. used some of the swedish fertility questions in a study of us obstetrics and gynaecology (ob/ gyn) residents. nearly half of the residents were misinformed about fertility decline, and three out of four overestimated the effectiveness of ivf treatment. interestingly, 80% 168 i. delbaere et al. believed that they should initiate discussions about agerelated fertility decline with patients. the findings indicated a need for improved education on age-related fertility decline in ob/gyn residency programmes (19). in an austrian study, fertility awareness was compared between medical students and non-medical students (20). although medical students had a higher awareness of the impact of age on fertility than non-medical students, the general knowledge of fertility was low also among medical students. similar results have been found in american, serbian, ukrainian, and saudi arabian samples (21–25). more than 95% of serbian students, regardless of gender, wanted to have children; most indicated three as the desired number of children. however, their knowledge about fertility was inaccurate as well (22). among ukrainian students, six out of ten respondents reported there was a pronounced decline in female fertility after the age of 45 years (23). sz€ucs found better results in a sample of hungarian, serbian, and romanian female university students in health sciences. they had significantly more knowledge about the menstrual cycle compared to other university students (26). authors across these studies call for more attention to reproductive health and fertility awareness in medical education. current research indicates that future health care providers are not sufficiently prepared to address fertility-related issues with their clients. clinical setting-based interventions to increase fertility knowledge as more women postpone childbearing until their late 30s and early 40s, it is clear that women of all ages would benefit from a better understanding of the biomedical limitations and other issues related to reproductive health that may help or hinder their reproductive plans. working with young adults to create a reproductive life plan is one strategy (27). a reproductive life plan (rlp) provides an opportunity for both men and women to reflect upon their interest and hopes for becoming parents and to receive services that align with their wishes, including contraceptives, health education, and/or preconception care. a person’s immediate plan for becoming pregnant or not can also influence clinical care (e.g. prescription of certain medications). all people should receive care for conditions that could reduce fertility, and preventive health care services. in an article on clinical content of preconception care, jack et al. offered the following recommendation: routine health promotion activities for all women of reproductive age should begin with screening women for their intentions to become or not become pregnant in the shortand long-term and their risk of conceiving (whether intended or not). providers should encourage patients (women, men, and couples) to consider a reproductive life plan and educate patients about how their reproductive life plan impacts contraceptive and medical decision-making. every woman of reproductive age should receive information and counselling about all forms of contraception and the use of emergency contraception that is consistent with their reproductive life plan and risk of pregnancy. (28) we believe that it is relatively easy to follow these recommendations when women seek family planning counselling as long as appointment times are long enough to allow for conversation. in sweden, midwives are responsible for approximately 90% of contraceptive counselling, and the counselling is free of charge. rlp counselling has been evaluated in studies conducted in sweden (29–32), in the us among physicians (33–35), and in iran at a health centre for women receiving maternal and child health care (36). the evaluations pointed out that while rlp counselling had no effect on effective contraception use, knowledge of fertility and awareness of preconception health increased (30–32). after the counselling, a higher proportion of the male swedish participants expressed the goal to have children in their life (31). the expressed age for having the last child among female university students was lowered (30). these effects were not found in iran (36). participants experienced rlp counselling as predominantly positive (30–33,36). mittal et al. used the rlp counselling in a small pilot study with women with chronic diseases. understanding the risks of pregnancy associated with diabetes, hypertension, and obesity increased, as did knowledge about how a reproductive plan could help them think about future decisions (34). stern and robbins explored administrators’ and health care providers’ experience of using rlp counselling in clinical settings (29,37). nearly 60% of health centres reported having written protocols for rlp, and 87% of providers reported frequent use of the rlp during family planning counselling with female clients (37). midwives found the rlp counselling to be a positive experience, in that the counselling was rewarding and easy. they were aware of the importance of approaching this topic carefully and thoughtfully as there are many individual and societal factors that influence a person’s ability to create an rlp (29). one key lesson from the swedish experience is that the support of senior midwife leaders and clinic administrators is important for advancing and sustaining rlp counselling (38). the interest in rlp counselling is growing across sweden, with many coordinating midwives asking the researchers for training and information. in denmark, at the university hospital in copenhagen, a fertility assessment and counselling (fac) clinic was begun in 2011 as an addition to family planning clinics, which were established in the 1970s. delayed childbearing, low fertility rates, and increasing use of social egg freezing prompted the introduction of this new service. the state-funded fac clinic offered free counselling using a risk assessment score sheet which included measurement of anti-m€ullerian hormone, ovarian and pelvic sonography in women, and sperm analyses in men. a fertility expert provided a physical exam and 30-min consultation to all patients. a campaign was launched to make women and men aware of this new clinic (39). after 1 year, a qualitative study explored women’s perceptions about the extent to which the fertility assessment intervention influenced their decision and choices in family planning. most women indicated that their knowledge on upsala journal of medical sciences 169 fertility-related issues increased after they attended the counselling. this knowledge helped them to make informed decisions or to seek fertility treatment. interestingly, older women who became aware of their closing fertile window stated that they felt more ready go ahead and get pregnant and no longer ruminating on pros and cons of having children (16). after 2 years the first 570 women who received care responded to an email questionnaire regarding subsequent pregnancies. the mean age was 35 years at inclusion, and 38% were single. most of them (68%) tried to conceive within 2 years after attending the fac clinic; three-quarters of these had achieved a pregnancy, 21% were still trying, and 5.4% had given up (40). two-thirds (65%) of the women who had low risk scores conceived spontaneously within 12 months. the presence of at least one high risk score reduced the odds of achieving a pregnancy within 12 months by 75%. one-third of the pregnancies in the 2-year follow up were achieved by medically assisted reproduction. the researchers concluded that the fac clinic concept was feasible and provided an important service to help support women in achieving their reproductive life plan (40). future studies should evaluate the cost-effectiveness of the fac clinic. however, addressing pregnancy intention in a primary care setting may be perceived as implicit persuasion for some women, as has been showed in qualitative research on latina and black women (41). garbers et al. (42) conducted community-based participatory research to investigate the experience of pregnancy intention screening in communitybased settings in black and latina women aged 15–49. three themes emerged from this research: personal agency, judgement and shame, and expertise versus authority. the community advisory board that was collected for this study recommends among others that health care providers should initiate pregnancy intention screening in a non-judgmental way and should be supportive of the agency of the patient. also, body language and terminology used are important within the consultation. online fertility education websites rather than doctors have been claimed to be the main source of fertility-related information for people aged 15–45 (5). unfortunately, not all information provided by ‘dr google’ is trustworthy. there is still a need for easily accessed and attractive information from reliable sources (43). this is because online interventions seem to have a positive impact on health behaviours, and online information may thus be a cost-effective manner to disseminate fertility awareness (44–46). there are several good online interventions that have been developed to increase fertility awareness. the fertility status awareness tool (fertistat) by bunting and boivin, for example, can be used by couples or individuals to calculate the fertility status based on some questions (47). in the usa, a new online resource, the in vitro fertilization success estimator tool, released by the centres for disease control and prevention helps people estimate the chance of having a live birth using ivf. both patients and clinicians may use this tool to enhance counselling and communication (48). additional resources and information related to art success rates can also be consulted on the website. ekstrand et al. developed a website with rlp information. the website was translated into english, french, arab, greek, spanish, and somali to facilitate the use among women who could not read swedish. a majority of midwives identified the quiz about fertility as a helpful tool (49). midwives in primary health care testing this website found it useful and referred clients to read more on their own. one midwife noted that the tool ‘is easy to display, easy to understand, easy to recommend the woman to look into and read on her own’ (50). an online intervention in the uk that provides fertility information to adolescents aged 16–18 years and university students aged 21–24 years significantly increased fertility knowledge for university students and also reduced the threat of infertility for university students and adolescents. participation in the study was associated with an increase in feelings of anxiety but a decrease in physical stress reactions. adolescents had more optimal fertility plans compared to emerging adults due to being younger (51). other online tools with the aim to increase fertility awareness and promote preconception health include: � yourfertility.org.au (australia): a comprehensive website with an interactive tool to calculate the best period to conceive, information on the impact of age, lifestyle, and weight on fertility and medical issues impacting fertility. � nhs.uk/live-well/infertility (uk): an informative overview of causes, diagnosis, and treatment of infertility, but also of risk factors and how to preserve the fertility. � myfertilitychoices.com (canada) provides information on fertility testing, preservation of fertility, treatment, and family-building options. there are decision-making resources to facilitate fertility choices. personal stories can be shared on the website, and experts can be contacted with questions. � reproduktivlivsplan.se (sweden) is an online version of the rlp counselling described above (30). people can indicate whether they want to have children or not, and, if they do, whether they want them within 1 year or later. as such, tailored information is provided according to the situation of the couple. furthermore, information about fertility and lifestyle issues is provided, and visitors of the website can test their knowledge. � showyourlovetoday.com (us) provides information to young adults about health and wellness, including information about fertility awareness. � on a website from finland, you can take a reproductive health test and a reproductive health quiz. it is offered in finnish and english: https://repro.tamk.fi/app/select. � other tools are currently in development. in belgium, results of a study on fertility awareness in a flemish population encouraged the researchers to develop an interactive website to inform people about their 170 i. delbaere et al. https://repro.tamk.fi/app/select reproduction. it can be consulted soon at www. klaarvoorkinderen.be. the recent european congress on preconception health and care in denmark (september 2019) was a ‘fertile’ breeding ground for new ideas. several researchers who designed some of the online tools described above worked together to develop a fertility education poster with very clear information for those who want to have children in the future (figure 1) (52). egg preservation one option that women who wish to postpone pregnancy have is to freeze their eggs (oocyte banking)—at least for those who can afford it; egg preservation is costly and often not covered by health insurance plans. some large companies are offering egg freezing as a fringe benefit for their employees. there are many emerging questions as to who benefits from postponing family creation. is it the companies who will have hardworking employees without children? is it the clinics who offer this technical reproductive method? is it the women who can invest in career goals while trying to find a suitable partner? is it the scientists who profit from the fees? in spite of the many unanswered questions, there is a rapidly growing interest in egg freezing that should be carefully monitored and examined over time. one out of 10 female university students (average age 23 years) who attended a clinic for contraceptive counselling in sweden in 2014 had considered freezing their eggs (53). in italy, one-third of female university students had heard about the possibility of fertility preservation through egg figure 1. fertility education poster. source: harper (52). upsala journal of medical sciences 171 http://www.klaarvoorkinderen.be http://www.klaarvoorkinderen.be freezing. of these, 20% were in favour of egg freezing, and half of the women thought that the cost for this procedure should be borne entirely by the woman herself (54). among women in the uk being counselled for age-related egg freezing, the average age was 36.7 years; this suggests that the women were not fully aware of the decline in egg quality from the age of 30 on (55). a qualitative study with women on the waiting list for oocyte banking showed that these women were financially independent and lived in single-person urban households. they opted for oocyte banking because they wished to share parenthood with a future partner rather than becoming a single parent. although women found the costs of the intervention high, they were willing to invest their money to increase their chances for shared parenthood (56). baldwin and culley found that women requesting egg freezing were generally satisfied with the treatment they received from clinics. however, they expressed a desire for more detailed information about potential outcomes from egg freezing (55). while oocyte banking may be an option for women with a strong wish for a child in the future, the current technique is an unreliable insurance. if eggs are frozen before the age of 35, women have a 30–40% chance to conceive when there are 10 eggs available. however, research points out that women interested in elective egg freezing are often at the end of their reproductive life spans (late 30 s to early 40 s). these women want children, but have no partner with whom to build a family (57). although the chances to have a child are rather low, the procedure is expensive (between 1500 eand 3000 eper cycle) and not affordable for everyone. future directions in light of this research, it is important to chart a careful and comprehensive course for the future. education about human reproduction, including fertility, should be widely available beginning in adolescence. people need clear information about their reproductive health, including menstrual cycles and medical conditions and behaviours that can potentially compromise fertility. there are a number of new tools available to support this education. more research is needed to evaluate the efficacy of these tools, including any unintended consequences, as well as to design and test strategies for use in schools, universities and other settings. likewise, health care providers (including midwives and nurses) should receive education about fertility, both during their medical education and through continuing education opportunities. as ivf, oocyte banking, and other technologies continue to develop, providers need to be informed about the efficacy, costs, and success rates so as to provide accurate information to their patients. education and counselling around reproductive life planning and fertility should be approached carefully and thoughtfully, always thinking about the unique needs and situation of the client. conversations on this topic may be upsetting and frustrating for people who may not be able to act on the advice given. providers require training so they can approach these conversations without bias and in a way that supports each person’s needs and goals. the family planning national training centre (fpntc) developed a client-centered reproductive goals & counselling flow chart to improve health care providers’ ability to have clientcentered conversations about fertility (58). services to support reproductive goals, including access to contraceptive methods, preventive health care services, specialty care, relationship support, violence prevention, economic advancement, fertility monitoring, and fertility assistance, must be available to all people, not just those who can afford them. men must receive counselling and be engaged in conversations around fertility and parenthood. research needs to be undertaken to listen to men on this topic and work with them to design messages and identify the best approach for having this conversation. stories that only highlight the ‘miracle babies’ conceived and birthed by older couples may sell magazines but can also do harm by creating false expectations. society at large also plays a critical role in supporting new families so parents can care for their children but also have the opportunity to succeed in achieving their educational and career goals. while countries with family-friendly policies have taken many steps in this direction, ongoing public discussions and research are needed to identify additional strategies that can support gender equity and reproductive autonomy and support for all people. moreover, all deserve to understand human reproduction, including how to manage their fertility. at the same time, providing this information can be very challenging as it can be perceived as judgmental and upsetting and must be offered within the context of many desires—educational and professional success, relationship with a trusted partner, a sense of readiness for the responsibility, and hoping to have a child/children in their lives. further, not everyone has the resources, health, and opportunities to plan for the future. efforts need to be made to not only provide information but to address these larger issues using an equity lens so that everyone can benefit and thrive. disclosure statement professor tanja tyd�en participated in one meeting at bayer’s advisory board in sweden in 2017. ilse delbaere and sarah verbiest have no declaration of interest. notes on contributors ilse delbaere, phd, is a registered midwife, researcher and lecturer at vives university of applied sciences, kortrijk, belgium. sarah verbiest, drph, msw, mph, is a public health social worker, clinical associate professor and director of the jordan institute for families at the school of social work at the university of north carolina at chapel hill, nc, usa. tanja tyd�en, phd, is a registered nurse midwife, and senior professor at uppsala university, uppsala, sweden. orcid ilse delbaere https://orcid.org/0000-0002-8522-2512 sarah verbiest https://orcid.org/0000-0002-2491-1170 tanja tyd�en https://orcid.org/0000-0002-2172-6527 172 i. delbaere et al. references 1. lampic c, svanberg as, karlstr€om p, tyd�en t. fertility awareness, intentions concerning childbearing, and attitudes towards parenthood among female and male academics. hum reprod. 2006;21: 558–64. 2. virtala a, vilska s, huttunen t, kunttu k. childbearing, the desire to have children, and awareness 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http://ec.europa.eu/eurostat/documents/3217494/6776245/ks-05-14-031-en-n.pdf/18bee6f0-c181-457d-ba82-d77b314456b9 http://ec.europa.eu/eurostat/documents/3217494/6776245/ks-05-14-031-en-n.pdf/18bee6f0-c181-457d-ba82-d77b314456b9 counselling; should this be offered to women and men of reproductive age? hum rep. 2015;30:9–15. 40. petersen kb, maltesen t, forman jl, sylvest r, pinborg a, larsen ec, et al. the fertility assessment and counseling clinic—does the concept work? a prospective 2-year follow-up study of 519 women. acta obstet gynecol scand. 2017;96:313–25. 41. donnelly kz, foster tc, thompson r. what matters most? the content and concordance of patients’ and providers’ information priorities for contraceptive decision making. contraception. 2014; 90:280–7. 42. garbers s, falletta ka, srinivasulu s, almonte y, baum r, bermudez d, et al. “if you don’t ask, i’m not going to tell you”: using community-based participatory research to inform pregnancy intention screening processes for 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fertility and the effectiveness of in vitro fertilization. fertil steril. 2013;100:523–9. 47. bunting l, boivin j. development and preliminary validation of the fertility status awareness tool: fertistat. hum reprod. 2010; 25:1722–33. 48. assisted reproductive technology (art). reproductive health. cdc. available from: https://www.cdc.gov/art/ivf-success-estimator/ index.html 49. ekstrand ragnar m, niemeyer hultstrand j, tyd�en t, larsson m. development of an evidence-based website on preconception health. ups j med sci. 2018;123:116–22. 50. andersson tyd�en km. t. implementation of reproductive life planning (rlp) in primary health care supported by an evidencebased website. eur j contracept reprod health care 2019;28:1–7. 51. boivin j, koert e, harris t, o’shea l, perryman a, parker k, et al. an experimental evaluation of the benefits and costs of providing fertility information to adolescents and emerging adults. hum reprod. 2018;33:1247–53. 52. harper j. launch of a global fertility education poster campaign. global women connected; november 2019. available from: http:// www.globalwomenconnected.com/2019/10/fertility_ed/?sfns=mo 53. stenhammar c, ehrsson yt, åkerud h, larsson m, tyd�en t. sexual and contraceptive behavior among female university students in sweden—repeated surveys over a 25-year period. acta obstet gynecol scand. 2015;94:253–9. 54. tozzo p, fassina a, nespeca p, spigarolo g, caenazzo l. understanding social oocyte freezing in italy: a scoping survey on university female students’ awareness and attitudes. life sci soc policy. 2019;15:3. 55. baldwin k, culley l. women’s experience of social egg freezing: perceptions of success, risks, and ‘going it alone’. hum fertil (camb). 2018. doi:10.1080/14647273.2018.1522456 56. de groot m, dancet e, repping s, goddijn m, stoop d, van der veen f, et al. perceptions of oocyte banking from women intending to circumvent age-related fertility decline. acta obstet gynecol scand. 2016;95:1396–401. 57. inhorn mc, birenbaum-carmeli d, westphal lm, doyle j, gleicher n, meirow d, et al. patient-centered elective egg freezing: a binational qualitative study of best practices for women’s quality of care. j assist reprod genet. 2019;36:1081–90. 58. client-centered reproductive goals & counseling flow chart. do you think you might like to have (more) children at some point? available from: https://www.fpntc.org/resources/client-centeredreproductive-goals-counseling-flow-chart 174 i. delbaere et al. https://www.cdc.gov/art/ivf-success-estimator/index.html https://www.cdc.gov/art/ivf-success-estimator/index.html http://www.globalwomenconnected.com/2019/10/fertility_ed/?sfns=mo http://www.globalwomenconnected.com/2019/10/fertility_ed/?sfns=mo https://doi.org/10.1080/14647273.2018.1522456 https://www.fpntc.org/resources/client-centered-reproductive-goals-counseling-flow-chart https://www.fpntc.org/resources/client-centered-reproductive-goals-counseling-flow-chart abstract introduction advanced maternal age and infertility fertility knowledge among men and women of fertile age, with focus on university students fertility awareness among health care providers and medical students clinical setting-based interventions to increase fertility knowledge online fertility education egg preservation future directions disclosure statement references sups_a_517875 52..59 upsala journal of medical sciences. 2011; 115: 52–59 original article symptomatic improvement after catheter ablation of supraventricular tachycardia measured by the arrhythmia-specific questionnaire u22 milos kesek1,3, folke rönn1,3, titti tollefsen2, niklas höglund1,3, ulf näslund1,3 & steen m. jensen1,3 1departments of cardiology and 2thoracic surgery, heart centre, university hospital, umeå, sweden, and 3department of medical sciences, umeå university, umeå, sweden abstract introduction. the main indication for ablation of supraventricular tachycardia is symptomatic relief. generic measures of quality of life are not suitable for direct evaluation of arrhythmia-related symptoms, and a specific tool is needed. the questionnaire u22 quantifies symptoms associated with arrhythmic events. it uses discrete 0–10 scales for quantification of influence of arrhythmia on well-being, intensity of discomfort, type of dominant symptom, and a time aspect that summarizes duration and frequency of spells. we evaluated u22 in a well defined group of patients with paroxysmal supraventricular tachycardia, undergoing an intervention with a distinct end-point and a high success rate. methods. symptoms in patients with accessory pathway and atrioventricular nodal re-entrant tachycardia scheduled for ablation were measured with u22 and sf-36 on admission. the evaluation was repeated after 6 months. results. altogether 58 patients successfully ablated in 2006–2008 completed the four forms (u22 and sf-36 at base-line and follow-up, 210 ± 35 days after ablation). the score for well-being (0–10; 10 being best) increased from 5.9 ± 2.6 to 7.9 ± 1.9 (p < 0.0005). the score for arrhythmia as cause for impairment in well-being (0–10; 10 being highest) decreased from 7.5 ± 2.8 to 2.0 ± 3.1 (p < 0.0005). the time aspect score (0–10) decreased from 4.7 ± 1.5 to 1.4 ± 1.8 (p < 0.0005). the two sf-36 summary measures pcs and mcs increased from 46.9 ± 9.4 to 48.4 ± 10.7 and from 44.9 ± 12.5 to 49.1 ± 9.9 (p = 0.04 and 0.002). conclusion. after successful ablation of accessory pathway and atrioventricular nodal re-entrant tachycardia, the u22 protocol detected a relevant increase in arrhythmia-related well-being. modest improvement in general well-being was detected by the sf-36 protocol. key words: arrhythmia symptoms, catheter ablation, quality of life, supraventricular tachycardia, symptom-specific protocol introduction the main indication for ablation of supraventricular tachycardia (svta) is symptomatic relief (1). the success rate, judged immediately after the intervention, is known to be high (2–4). it has been shown, however, that even after a primarily successful ablation of accessory pathway many patients continue to suffer from arrhythmia symptoms (5). a more appropriate evaluation of procedural success therefore requires measurement of the symptoms at follow-up. the sf-36 (medical outcomes study 36-item short-form health survey) questionnaire and other general protocols measure quality of life and not the paroxysmal symptoms related to arrhythmia. several arrhythmia-specific questionnaires have been described. the symptom checklist—frequency and severity scale (6,7) has been used in ablation of svta, in revised versions in atrial fibrillation studies (8,9), and in a recent retrospective survey of late outcome after svta (10). the different implementations of the checklist are less well documented correspondence: milos kesek, department of cardiology, heart centre, university hospital, s-901 85 umeå, sweden. fax: +46 90 127630. e-mail: milos.kesek@medicin.umu.se (received 17 february 2010; accepted 19 august 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.517875 in the literature. other questionnaires have been applied in atrial fibrillation (symptom specific checklist with seven aspects of arrhythmia (11), symptom severity questionnaire measuring five symptom parameters (12), a bedside-oriented questionnaire with classification into four groups (13)). none of these approaches fulfil the requirement of being well described and commonly accepted for use in different arrhythmia types. u22 (umea 22 arrhythmia questions) is a clinically oriented questionnaire, developed for evaluation of intermittent symptoms related to arrhythmia (see appendix). the questionnaire quantifies multiple self-perceived symptom aspects associated with the arrhythmic events. in measurements on patients with svta scheduled for catheter ablation, u22 indicates a prominent discomfort during arrhythmia. patients with svta that undergo catheter ablation are thus highly symptomatic, although their general wellbeing is only modestly decreased when measured by sf-36 (14). the svta ablation has a distinct end-point and a high success rate, making this patient group suitable for testing the questionnaire. in the present study, we investigate whether the arrhythmiaspecific symptom questionnaire u22 is better suited than the generic questionnaire sf-36 for measurement of the clinical improvement after an ablation of svta. material and methods questionnaires the arrhythmia-specific symptoms were measured with u22 (swedish form) (14). the appendix describes the questionnaire. sf-36, a well documented instrument (15), was used as a generic measure of quality of life. it quantifies the mental and physical well-being in eight subscales with a range of 0–100, together with a physical and a mental summary score. patients patients with svta (accessory pathway (ap) and atrioventricular nodal re-entrant tachycardia (avnrt)), admitted for scheduled routine catheter ablation at the heart centre, university hospital, umeå, sweden during 2006–2008, were invited to answer the base-line u22 and sf-36 forms. next day the diagnosis was established invasively and treated by catheter ablation. the evaluation with u22 and sf-36 was repeated 6 months after the ablation. the answers were prospectively entered into a database and retrieved for the subsequent analysis. analysis first-time interventions for the target diagnoses were selected. the catheterization reports from all ablations were reviewed by an experienced operator, blinded with respect to the u22 results (s.m.j.), and each procedure was evaluated for primary outcome. ablation success in ap was defined as absence of delta wave and retrograde conduction block in the accessory pathway and in avnrt as non-inducibility of tachycardia. the data were analysed in spss, release 13 (spss inc., chicago, illinois). the u22 and sf-36 scores were compared pairwise at base-line versus at follow-up in individual patients. data are presented as mean ± sd. differences between continuous variables were examined by paired and unpaired t test. correlations (spearman’s rho) between individual patients’ scores at base-line and follow-up were used for studying common properties of the responses in patients with varying symptomatology and perception of discomfort. two-tailed fisher exact test was used for testing differences in 2 � 2 table proportions. a p value < 0.05 was considered significant. the study was approved by the ethics committee at the umeå university faculty of medicine. results effect of ablation on u22 and sf-36 between april 2006 and may 2008, 141 patients underwent 156 ablations for svta (1.1 procedure/ patient). from 63 patients undergoing first-time ablation for ap or avnrt, all four forms were available (u22 and sf-36 at base-line and at the follow-up, 210 ± 35 days after ablation). table i summarizes the background data. in 58 of these subjects the ablation was primarily successful, as judged by the blinded analysis. the results are summarized in table ii (see appendix for definition of the u22 scores). according to the verbal definitions underlying the numerical score values, the arrhythmias were highly symptomatic at base-line, and a marked decrease in the symptoms was seen after a successful ablation. the u22 score for relevance of answers was high both at base-line and follow-up. the u22 score for general well-being (question 1) increased from 5.9 ± 2.6 at base-line to 7.9 ± 1.9 at follow-up (table ii). the symptoms in supraventricular tachycardia: u22 53 score for patients’ self-perceived improvement in well-being measured at follow-up (question 2) was 8.2 ± 2.1. the two sf-36 measures physical component summary (pcs) and mental component summary (mcs) increased after ablation from 46.9 ± 9.4 to 48.4 ± 10.7 and from 44.9 ± 12.5 to 49.1 ± 9.9 (p = 0.04 and 0.002). for the profile of the eight sf-36 subscales, see figure 1. correlation between u22 scores before ablation and at follow-up the u22 score for well-being at follow-up correlated to the same score before the ablation (spearman’s rho 0.59; p < 0.0005). similarly, a correlation was found in the time aspect score (rho = 0.32; p = 0.03). no significant correlations in the scores before the ablation and at follow-up were found for arrhythmia affecting the well-being (question 11), discomfort during a spell (question 12), intensity of dominant symptom, and width of symptoms (rho = 0.21, 0.12, 0.03, 0.06; p = n.s. for all). the score for well-being at follow-up was correlated to the pre-ablational symptoms of irregularity and anxiety during a spell (questions 15 and 21) (rho = -0.31 and -0.28; p = 0.03 and 0.04). an absence of correlation was noted between the patients’ retrospective estimation of improvement in well-being after the ablation (question 2follow-up) and the improvement computed from the two forms as (question 1follow-up – question 1baseline) (rho = 0.09; p = n.s.). medication at base-line, 71% of the patients did take some prescribed medication for the arrhythmia (57% in the ap group and 80% in the avnrt group). at follow-up after a primarily successful ablation, 27% remained on medication (9% in the ap group and 38% in the avnrt group). in the two groups of patients on no antiarrhythmic medication at follow-up (73%) and patients on continuing anti-arrhythmic medication (27%), the u22 score for well-being was 8.0 ± 1.8 table i. demographic data. patients with all 4 forms (n = 63) patients with 1–3 missing forms (n = 78) ap avnrt ap avnrt n 26 37 35 43 age mean (sd), years 43.9 (17.9) 57.1 (14.0) 42.3 (18.4) 53.3 (17.4) men, n (%) 17 (65) 14 (38) 27 (77) 18 (42) all four requested forms (u22 and sf-36 at base-line and follow-up) were available from 63 of the 141 patients undergoing first-time ablations for ap or avnrt. five of the 63 ablations with complete data were not primarily successful, and these patients were subsequently excluded. ap = accessory pathway; avnrt = atrioventricular nodal re-entrant tachycardia. table ii. u22 measures at base-line and follow-up. question base-line follow-up p (t test) well-being q01 5.9 ± 2.6 7.9 ± 1.9 <0.0005 arrhythmia events affecting well-being q11 7.5 ± 2.8 2.0 ± 3.1 <0.0005 discomfort during arrhythmia spell q12 8.1 ± 2.2 2.3 ± 3.2 <0.0005 symptom widtha 2.5 ± 1.7 0.9 ± 1.4 <0.0005 intensity of dominant symptom 9.9 ± 0.3 3.9 ± 4.6 <0.0005 incidence of symptoms q08 2.5 ± 1.1 0.9 ± 1.3 <0.0005 time aspectb q08, q10 4.7 ± 1.5 1.4 ± 1.8 <0.0005 relevance scorea q19 9.6 ± 1.5 9.5 ± 1.7 n.s. data from 58 first-time ablations, classified as primarily successful. asee appendix for definition. bsee appendix and table iii for definition. q01, q08, q10, q11, q12, q19 = u22 questions 1, 8, 10, 11, 12, 19. 54 m. kesek et al. versus 7.4 ± 2.2 (p = n.s. for the comparison). the scores for arrhythmia as a cause for impaired wellbeing were 1.5 ± 2.8 versus 3.3 ± 3.5 (p = 0.06), discomfort during attack 1.8 ± 2.9 versus 3.5 ± 3.7 (p = 0.09), dominant intensity 3.1 ± 4.5 versus 5.6 ± 4.8 (p = 0.09), width of symptoms 0.6 ± 1.1 versus 1.3 ± 1.8 (p = 0.1), and time aspect 2.2 ± 2.1 versus 0.9 ± 1.6 (p = 0.02). the sf-36 measures pcs and mcs were 50.6 ± 9.2 versus 42.8 ± 12.9 and 51.0 ± 7.8 versus 44.9 ± 13.6, respectively (p = 0.02 and 0.05). discussion we have evaluated u22 as a tool for measurement of arrhythmia-related symptoms in a well defined group of patients with paroxysmal supraventricular tachycardia who were undergoing an intervention with a distinct end-point and a high success rate. the scores quantifying the arrhythmia symptoms decreased notably. the improvement in general well-being was smaller. u22 thus seems to be more suitable for measuring the effect of ablation than a general questionnaire, like sf-36. in contrast to sf-36, the scales of u22 have a clinical arrhythmia-related meaning, making the questionnaire useful for clinical evaluation of changes in individual patients. an important application for the u22 questionnaire may be in arrhythmias with more diffuse symptomatology and interventions with less well defined endpoints, situations occurring in patients with atrial fibrillation (16,17). correlation between u22 scores before ablation and at follow-up the absence of correlation between base-line and follow-up in the scores describing the arrhythmia is reasonable, in view of a primarily successful ablation. surprisingly, the retrospective estimation of improvement in well-being (question 2) was not related to the computed difference in well-being between baseline and follow-up. a patient’s retrospective estimate of improvement after ablation therefore seems doubtful as a measure of ablation success. sf-36 the profile of the sf-36 subscales at base-line in our group resembles closely that of another recent scandinavian group of patients with svta (18). both groups have a similar profile to an earlier material (6), but on a higher subscale level (figure 1). a relation between general quality of life and the symptom-specific measures can be expected, since the arrhythmia-related symptoms are severe enough to motivate the patients to undergo ablation. this relation is rather weak in our material, where a marked decrease of symptom-specific measures is followed by a modest increase in the sf-36 measures. this is presumably due to the relatively high level of quality of life measured by sf-36 already at base-line, in spite of a marked arrhythmia-related symptomatology. a surprisingly large proportion of our patients remained on medication at follow-up, in spite of a primarily successful ablation. it has been shown that, table iii. computation of the time aspect from the symptom duration and incidence scores. q08 how often never on rare occasions a few times a month a few times a week daily all the time 0 1 2 3 4 5 q10 how long seconds 0 0 1 2 3 4 5 minutes, less than a quarter of an hour 1 0 2 3 4 5 6 a quarter of an hour to one hour 2 – 3 4 5 6 – one to four hours 3 – 4 5 6 7 – longer 4 – 5 6 7 8 9 all the time 5 – – – – 9 10 the time aspect is found by looking up the row with relevant symptom duration score (q08) and column with relevant symptom incidence score (q10). for most combinations the time aspect is a simple addition of the two scores (both with range 0–5). the combination of incidence ‘never’ and duration ‘minutes’ is, however, assigned time aspect value 0. the entries marked as ‘–’ are coded as missing values as they correspond to largely inconsequent combinations of the two scores: incidence ‘never’ with durations ‘a quarter of an hour’ or more; incidences ‘few times a week’ or less with duration ‘all the time’; incidence ‘all the time’ with durations ‘a quarter of an hour’ to ‘four hours’. q08 = u22 question 8; q10 = u22 question 10. symptoms in supraventricular tachycardia: u22 55 after a primarily successful ablation of accessory pathway, 39% ofthe patients continuedtoreport arrhythmia symptoms and 8% remained on anti-arrhythmic treatment (5). corresponding figures for the patients with accessory pathways in the present material are very similar, while an even larger proportion of patients withavnrtremainedonanti-arrhythmicmedication. we lack information from ecg (electrocardiographic)-monitoring that would confirm or exclude a relation of the measured symptoms to an arrhythmia. therefore the reason behind the continuing need for anti-arrhythmic medication cannot be determined. our avnrt patients had a mean age of 57 years, 13 years more than the ap group. these middle-aged and elderly patients are often treated with beta-blockers or ca-antagonists for hypertension. this is one possible explanation for the large proportion of patients that remain on medication after ablation of avnrt. limitations the study describes the pattern of paroxysmal symptoms, measured by u22 in a group of patients before and after an ablation. however, we have no data on the validity and reproducibility of the questionnaire. for computation of specificity and sensitivity of the symptom measurement it would be necessary to relate the described symptoms to incidence of ecgverified arrhythmia. however, no continuous rhythm monitoring was performed in the study. reference values for the u22 scores from some type of control group would be desirable. u22 aims at a type of specific symptom-focused measurement that makes it difficult to construct a proper control group with normal reference values. the u22 questions make sense for patients with arrhythmia symptoms or those that have been cured from an arrhythmia. to achieve meaning in normal controlsthe questionswould have to be rephrased. in the present form it is, however, reasonable to view the u22 scores of the subgroup free from medication at follow-up as representing a reference level after a successful treatment, since 1) the indication is based on a combination of symptoms and invasive electrophysiological diagnosis, and 2) the treatment can be considered as successful in patients that have been primarily successfully ablated and have no anti-arrhythmic medication at follow-up. the values of the sf-36 parameters pcs and mcs in this group are very close to 50 (normal). 83 76 72 73 65 87 82 77 57 69 61 72 77 7371 80 20 40 60 80 100 pf rp bp gh vt sf re mh kesek 2009 (svta at follow-up) kesek 2009 (svta preablation) bubien 1996 (mainly svta) walfridsson 2009 (svta) figure 1. sf-36 subscales. the figure shows the sf-36 profiles at base-line and follow-up, labelled with mean values of the eight subscales in the study group. for comparison, the profiles of an older and a recently published group of patients with svta (6,18) are shown (the mean values were derived from the published graph and the number of patients). at base-line, the present study group is very close to the recent material by walfridsson et al. (18). the profile is similar, but at a higher absolute subscale level than in the older material of patients by bubien et al. (6). this may reflect a development towards ablation in patients with fewer symptoms. (pf = physical functioning; rp = rolephysical; bp = bodily pain; gh = general health; vt = vitality; sf = social functioning; re = role-emotional; mh = mental health; svta = supraventricular tachycardia.) 56 m. kesek et al. a large proportion of patients had to be excluded from the analysis, due to failure to return all the four forms. the number of forms requested from each patient was demanding. two additional factors may have contributed to the high exclusion rate: the base-line forms were not collected by specifically assigned staff, but as a part of normal patient admission routines; and in case of missing follow-up forms, no reminder was sent out. the patients that were excluded due to missing forms were slightly younger, with a higher proportion of men (table i). the english version of u22 was translated from swedish with the intention of corresponding closely with the original meaning. a translation nevertheless introduces a source of error into any comparisons. comparison of treatments based on different translations of a questionnaire should therefore be accompanied by some evaluation, at least by a comparison of the initial levels in similar populations. conclusion the u22 questionnaire detected the expected symptomatic improvement in patients ablated with primary success for ap and avnrt. a prominent increase could be seen in measures of arrhythmia-related wellbeing. in comparison, the improvement observed in the generic sf-36 questionnaire was relatively small. acknowledgements the study was supported by governmental research funds from the swedish nhs (alf medel) and the heart foundation of northern sweden (norrländska hjärtfonden). declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. hlatky ma, wang p. improvement in quality of life after radiofrequency ablation. pacing clin electrophysiol. 2006;29: 341–2. 2. calkins h, yong p, miller jm, olshansky b, carlson m, saul jp, et al. catheter ablation of accessory pathways, atrioventricular nodal reentrant tachycardia, and the atrioventricular junction: final results of a prospective, multicenter clinical trial. the atakr multicenter investigators group. circulation. 1999;99:262–70. 3. kobza r, kottkamp h, piorkowski c, tanner h, schirdewahn p, dorszewski a, et al. radiofrequency ablation of accessory pathways. contemporary success rates and complications in 323 patients. z kardiol. 2005;94:193–9. 4. kesek m. ablation procedures in sweden during 2007: results from the swedish catheter ablation registry. europace. 2009; 11:152–4. 5. oddsson h, walfridsson h, edvardsson n. perception and documentation of arrhythmias after successful radiofrequency catheter ablation of accessory pathways. ann noninvasive electrocardiol. 2001;6:216–21. 6. bubien rs, knotts-dolson sm, plumb vj, kay gn. effect of radiofrequency catheter ablation on health-related quality of life and activities of daily living in patients with recurrent arrhythmias. circulation. 1996;94:1585–91. 7. anselme f, saoudi n, poty h, douillet r, cribier a. radiofrequency catheter ablation of common atrial flutter: significance of palpitations and quality-of-life evaluation in patients with proven isthmus block. circulation. 1999;99: 534–40. 8. dorian p, jung w, newman d, paquette m, wood k, ayers gm, et al. the impairment of health-related quality of life in patients with intermittent atrial fibrillation: implications for the assessment of investigational therapy. j am coll cardiol. 2000;36:1303–9. 9. jenkins ls, brodsky m, schron e, chung m, rocco t jr, lader e, et al. quality of life in atrial fibrillation: the atrial fibrillation follow-up investigation of rhythm management (affirm) study. am heart j. 2005;149:112–20. 10. meissner a, stifoudi i, weismuller p, schrage mo, maagh p, christ m, et al. sustained high quality of life in a 5-year long term follow-up after successful ablation for supra-ventricular tachycardia. results from a large retrospective patient cohort. int j med sci. 2009;6:28–36. 11. erdogan a, carlsson j, neumann t, berkowitsch a, neuzner j, hamm cw, et al. quality-of-life in patients with paroxysmal atrial fibrillation after catheter ablation: results of long-term follow-up. pacing clin electrophysiol. 2003;26:678–84. 12. oral h, pappone c, chugh a, good e, bogun f, pelosi f jr, et al. circumferential pulmonary-vein ablation for chronic atrial fibrillation. n engl j med. 2006;354: 934–41. 13. dorian p, cvitkovic ss, kerr cr, crystal e, gillis am, guerra pg, et al. a novel, simple scale for assessing the symptom severity of atrial fibrillation at the bedside: the ccs-saf scale. can j cardiol. 2006;22:383–6. 14. kesek m, tollefsen t, höglund n, rönn f, näslund u, jensen sm. u22, a protocol to quantify symptoms associated with supraventricular tachycardia. pacing clin electrophysiol. 2009;32:s105–8. 15. ware je jr, gandek b. overview of the sf-36 health survey and the international quality of life assessment (iqola) project. j clin epidemiol. 1998;51:903–12. 16. kesek m, höglund n, rönn f, tollefsen t, jensen s. arrhythmia-specific symptoms measured by the u22 protocol in catheter ablation for atrial fibrillation (abstract). cardiology. 2009;113suppl 1:38. 17. kesek m, tollefsen t, höglund n, rönn f, näslund u, jensen s. symptom measurement in three groups of patients with atrial fibrillation (abstract). scand cardiovasc j. 2008; (42 suppl):29. 18. walfridsson u, stromberg a, janzon m, walfridsson h. wolff-parkinson-white syndrome and atrioventricular nodal re-entry tachycardia in a swedish population: consequences on health-related quality of life. pacing clin electrophysiol. 2009;32:1299–306. symptoms in supraventricular tachycardia: u22 57 appendix u22 questionnaire the u22 instrument aims at quantifying prevalence and severity of arrhythmia-associated symptoms (14). it consists of two related questionnaires to be answered at base-line and follow-up after an intervention, respectively. questions about general well-being, the influence of arrhythmia on the well-being, and the intensity of discomfort and specific symptoms during a spell are answered by choosing one of 11 alternatives, aligned in a horizontal line of 10 cm length, with verbal descriptions of the end-points (anchors). the subject is instructed to select the answer by placing an x in the circle that best reflects how he feels. the answers are translated to a discrete numeric rating scale with range of 0–10 (nrs-10) (numerical rating scale, a discrete analogy to the common vas [visual analogue scale]). the scores give an estimate of well-being, the effect of arrhythmia events on the well-being, discomfort during a spell, and the maximal symptom intensity. a symptom width score is computed as the number of dominant symptoms (the symptoms rated as having equal, maximal intensity). the symptom width score is set to 0 if the subject disagrees to some degree with all symptom descriptions, i.e. all the scores from questions 11–22 are <5. the subject’s understanding and relevancy of the answers is tested by question 19 about itching, a symptom irrelevant in the context of arrhythmias. a relevance score is computed as (10 – scale 19). the incidence and duration of the events is estimated by questions 8 and 10 with 6 non-linear answer alternatives, quantified as 0–5. a time aspect score with a range of 0–10 is computed by adding the two answer scores (table iii). base-line form (english translation of the original swedish forms used in the study) the answer choices are stated within the square brackets. 1. on the whole, how have you felt over the past month? [miserable–very well, 0–10] (2 – not applicable in base-line form) 3. do you take any prescribed medications for your heart rhythm problems? [no; yes] 4. how effective is the medication(s) in treating your heart rhythm problems? [very much worse–very much better, 0–10] 5. how much are you bothered by side-effects of the medication? [not at all–very much, 0–10] (6, 7 – not applicable in base-line form) 8. how often do you experience problems with heart rhythm? (despite taking any medication) [never; rarely; a few times a month; a few times a week; daily; all the time] 9. do the spells start suddenly? [no; yes] 10. how long does a spell usually last? [seconds; more than a minute but less than 15 min; 15 min to 1 hour; 1 to 4 hours; more than 4 hours; all the time] 11. how much do the spells affect your well-being? [not at all–very much, 0–10] 12. how bothered are you while you are experiencing a spell? [not at all–very much, 0–10] 13. my heart beats extremely fast when i’m having a spell [not at all–very true, 0–10] 14. my heart pounds very heavily when i’m having a spell [not at all–very true, 0–10] 15. my heart beats very irregularly when i’m having a spell [not at all–very true, 0–10] 16. i faint or feel extremely dizzy when i’m having a spell [not at all–very true, 0–10] 17. i have terrible pain when i’m having a spell [not at all–very true, 0–10] 18. i feel extremely tired when i’m having a spell [not at all–very true, 0–10] 19. i feel like my skin itches all over when i’m having a spell [not at all–very true, 0–10] 20. i feel extremely short of breath when i’m having a spell [not at all–very true, 0–10] 21. i become very frightened when i’m having a spell [not at all–very true, 0–10] 22. someone close to me becomes very frightened when i’m having a spell [not at all–very true, 0–10] 58 m. kesek et al. follow-up form. the follow-up questionnaire is equivalent to the base-line form, with an addition of three questions that explore the type of remaining symptoms (question 6) and the present general well-being and arrhythmia symptomatology in comparison to base-line (questions 2 and 7). questions 7–22 in the follow-up form are only answered if the patient in question 6 indicates any remaining problems with the heart rhythm. if the patient has no such problems, no further answers are requested, and on analysis the scores for questions 8 and 10–22 will be set to 0, while question 7 will be set to 10. 2. compared to the time before the treatment, do you now feel: [very much worse–very much better, 0–10] 6. have you experienced any problems with the heart rhythm after the treatment? (please disregard the first 3 months after the treatment.) [no; yes, of the same type as before the treatment; yes, of different type] 7. how bothersome are the spells in comparison with those before the treatment? [very much more–very much less, 0–10] symptoms in supraventricular tachycardia: u22 59 tf-iups160043 208..210 commentary who benefits from putting family life into ice? outi hovatta karolinska institutet, department of clinical science, intervention and technology, karolinska university hospital huddinge, stockholm, sweden article history received 22 july 2016; accepted 27 july 2016 the first methods for fertility preservation (fp) were sperm freezing followed many years later by ovarian tissue freezing and oocyte vitrification. fp before cancer treatment is necessary because gametes are often destroyed at the same time as the cancer therapy kills malignant cells. it has been regularly offered to male cancer patients in the form of sperm cryopreservation. in female patients, slow freezing of ovarian tissue containing the oocytes was initiated in 1996 (1). it has since then been used for hundreds of young women, and after re-transplantation of the tissue back to the woman’s ovary after her cancer treatment tens of healthy children have been born (2). there are also genetic disorders in which fp is needed, such as turner syndrome (3). ovarian tissue has to be biopsied under general anaesthesia. but getting mature oocytes instead of tissue is a simpler procedure. oocytes are picked up using trans-vaginal ultrasound-guided needle aspiration after the woman’s hormonal stimulation, which is routine procedure for in vitro fertilization (ivf). ovarian tissue freezing is the only method available for prepubertal girls, but it is not needed for age-related decrease in fertility. human sperm has been frozen for about 60 years with the aim to use it later on for having children. spermatozoa are very small cells, and it was technically relatively easy to cryostore them. but human eggs, oocytes, are very large cells. the diameter of a mature human egg is 120 lm. they are extremely sensitive to damage during freezing. but cryostorage of human eggs has also become feasible. the first attempts, using the conventional slow freezing method with dimethylsulphoxide as the cryoprotectant, were successful already 30 years ago and resulted in pregnancies and births (4,5), but the efficacy was low. the main cause of damage in slow freezing of cells is that ice crystals break intracellular organelles and cellular membranes. vitrification is a cryostoring method in which low temperatures are achieved by adding high concentrations of cryoprotecting substances that form a glass-like amorphous mass when cooled to subzero temperatures. hence, dangerous sharp ice crystals are avoided. the cooling rate has to be fast in order to avoid the toxicity of the cryoprotectants. vitrification of human oocytes then evolved (6). it proved soon more successful than slow freezing (7–11). the feasibility was shown in large oocyte donation programmes. today, vitrification of oocytes is the most favoured method of fertility preservation. hence, it is easy to understand that it is also offered to women who want to postpone their childbirths to higher age than is physiologically feasible. it is called egg freezing for non-medical reasons, or social egg freezing. egg freezing for non-medical reasons, with the indication to postpone childbirths, is today offered by several private ivf units for women who are willing to buy these treatments. in the usa, big companies, such as google and facebook, are offering it for their employees. results from transfer of vitrified thawed eggs vitrification of non-fertilized oocytes has been used in units that offer treatment using donated oocytes. the pregnancy rates have been as high as those using fresh oocytes (12–14). a contributing factor to the high pregnancy rates was probably the better quality of endometrium, the cycles in which cryostored oocytes or embryos were used. in light of this, vitrification of oocytes from healthy young women should not be a problem. the reported clinical pregnancy rate per embryo transfer—36.5% when the best embryo was transferred three days after fertilization (15)—is comparable to fresh ivf cycles in most clinics. there are several factors influencing the success rate of vitrification as a method. the laboratory should have extensive experience with the technique before applying it to women. it is important to take into account the known factors that influence the quantity and quality of oocytes. age is the most important one, but also hereditary and other health factors contribute. at which age should eggs be stored? the optimal age for egg storing is not easy to set. thinking of biology, it should be done as early as possible. but it should always be the young woman’s own voluntary informed choice. young women are more prone to the most severe complication of oocyte collection, ovarian contact outi hovatta outi.hovatta@ki.se senior professor of obstetrics and gynaecology, karolinska institutet, department of clinical science, intervention and technology, karolinska university hospital huddinge, se 141 86 stockholm, sweden � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 4, 208–210 http://dx.doi.org/10.1080/03009734.2016.1219432 http://creativecommons.org/licenses/by/4.0/ hyperstimulation syndrome (ohss) caused by the hormones given to mature the eggs. ohss is potentially life-threatening. it should definitely be a part of counselling. the age of 25 years has been mentioned in literature, but today most women at that age do not yet have any plans for their families. at the age of 36 the situation is much more clear, but the number of potentially collectable oocytes in the ovaries is already then clearly lower even though most frequently very sufficient for a spontaneous pregnancy. the optimal timing was studied by mesen et al. (16), and at the age of 34 years satisfactory numbers of eggs were still obtained. the average age of the women seeking age-related oocyte cryopreservation in an ivf unit in the uk was 36.7 years, which can be regarded as late (17). the number of oocytes that can be collected decreases with age. it has been calculated that seven eggs have to be stored in order to achieve one pregnancy (18). on the basis of the likelihood to get pregnant after ivf, we know that female fertility significantly decreases after the age of 38 years. this age would obviously be too late a time for oocyte cryostoring. the window appears to be between 25 and 36 years, favouring the earlier rather than the later age. however, few 25-year-old women think of their fertility in the future. awareness of limited fertility period among women is not optimal there are many social reasons for women postponing their childbearing in the western world (19). one important reason is the absence of a partner at an appropriate age. extensive individualized counselling is needed to prevent women and their male partners from ‘sleepwalking into infertility’ (20–23). among the reasons, education and career goals have been mentioned. to avoid unnecessary medical procedures, it would be good politically to organize better possibilities for women both to have children and to have working conditions equal to those of males. male partners do not always appear to be aware of the fertile period among women. many men underestimate the impact of advanced female age on fertility and overestimate successful outcome of medically assisted reproduction technology treatment (24,25). in a study among single women seeking treatment with the use of donated sperm, 91% stated that they would have preferred to have the child together with a partner, and 66% had been in a relationship where they desired to have a child. however, in 40% of these cases the man did not want a child (yet) or did not want more children (had a child/children from previous relationship) (26). in summary, it seems that even more men compared to women are more unaware of risk factors for decreasing fertility (including the risk factor of advanced female age) and often desire family formation at more advanced ages. that supports the finding of salomon et al. that single motherhood by choice is women’s plan b as many had had a partner who did not (yet) desire a child (26). increased reproductive equity between women and men increased equity has been seen as one of the factors favouring oocyte cryopreservation (16,17). it probably does. but at the same time, other means are effective. making women socially equal would have much fewer side effects for women. such social and political decisions could be shared parental leaves and equal salaries. conclusions fp for age-related decrease in female fertility is increasingly needed. cryostorage of eggs using vitrification is the method of choice today. it is not risk-free. the efficacy is satisfactory, but it is not at all certain that pregnancies are achieved and infants born after every procedure. the age window for social fp is 25–34 years. to prevent all unnecessary complications of medical procedures it would be important to offer counselling to all women regarding decreases in fertility, and even more important to offer equal working conditions for women (shared parental leaves, equal salaries, high-quality day care) who wish to have their children at the physiologically optimal age. these are important tasks for politicians. references 1. hovatta o, silye r, krausz t, abir r, margara r, trew g, et al. cryopreservation of human ovarian tissue using dimethylsulphoxide and propanediol-sucrose as cryoprotectants. hum reprod. 1996;11:1268–72. 2. donnez j, dolmans mm. fertility preservation in women. nat rev endocrinol. 2013;9:735–9. 3. borgstr€om b, hreinsson j, rasmussen c, sheiki m, fried g, keros v, et al. fertility preservation in girls with turner syndrome – prognostic signs for presence of follicles in ovarian tissue. j clin endocr metab. 2009;94:74–80. 4. chen c. pregnancy after human oocyte cryopreservation. lancet. 1986;327:884–6. 5. van uem jf, siebzehnr€ubl er, schuh b, koch r, trotnow s, lang n. birth after cryopreservation of unfertilized oocytes. lancet. 1987;329:752–3. 6. kuleshova l, gianaroli l, magli ferraretti a, trounson ac. birth following vitrification of a small number of human oocytes: case report. hum reprod. 1999;14:3077–9. 7. cobo a, garcia-velasco ja, domingo j, remohi j, pellicer a. is vitrification of oocytes useful for fertility preservation for age-related fertility decline and in cancer patients? fertil steril. 2013;99:1485–95. 8. quaas am, melamed a, chung k, bendikson ka, paulson rj. egg banking in the united states: current status of commercially available cryopreserved oocytes. fertil steril. 2013;99:827–31. 9. glujovsky d, riestra b, sueldo c, fiszbajn g, repping s, nodar f, et al. vitrification versus slow freezing for women undergoing oocyte cryopreservation. cochrane database syst rev. 2014:cd010047. 10. sol�e m, santal�o j, boada m, clua e, rodr�ıguez i, mart�ınez f, et al. how does vitrification affect oocyte viability in oocyte donation cycles? a prospective study to compare outcomes achieved with fresh versus vitrified sibling oocytes. hum reprod. 2013;28:2087–92. upsala journal of medical sciences 209 11. paramanantham j, talmor aj, osianlis t, weston gc. cryopreserved oocytes: update on clinical applications and success rates. obstet gynecol surv. 2015;70:97–114. 12. potdar n, gelbaya ta, nardo lg. oocyte vitrification in the 21st century and post-warming fertility outcomes: a systematic review and meta-analysis. reprod biomed online. 2014;29:159–76. 13. galliano d, garrido n, serra-serra v, pellicer a. difference in birth weight of consecutive sibling singletons is not found in oocyte donation when comparing fresh versus frozen embryo replacements. fertil steril. 2015;104:1411–8.e1–3. 14. de munck n, belva f, van de velde h, verheyen g, stoop d. closed oocyte vitrification and storage in an oocyte donation programme: obstetric and neonatal outcome. hum reprod. 2016;31:1024–33. 15. de munck n, santos-ribeiro s, stoop d, van de velde h, verheyen g. open versus closed oocyte vitrification in an oocyte donation programme: a prospective randomized sibling oocyte study. hum reprod. 2016;31:377–84. 16. mesen tb, mersereau je, kane jb, steiner az. optimal timing for elective egg freezing. fertil steril. 2015;103:1551–6.e1–4. 17. baldwin k, culley l, hudson n, mitchell h, lavery s. oocyte cryopreservation for social reasons: demographic profile and disposal intentions of uk users. reprod biomed online. 2015;31:239–45. 18. lockwood g, johnson mh. having it all. reprod biomed online. 2015;31:1–5. 19. birch petersen k, hvidman hw, sylvest r, pinborg a, larsen ec, macklon kt, et al. family intentions and personal considerations on postponing childbearing in childless cohabiting and single women aged 35–43 seeking fertility assessment and counselling. hum reprod. 2015;30:2563–74. 20. lemoine me, ravitsky v. sleepwalking into infertility: the need for a public health approach towards advanced maternal age. am j bioeth. 2015;15:37–48. 21. garcia d, vassena r, prat a, vernaeve v. increasing fertility knowledge and awareness by tailored education: a randomized controlled trial. reprod biomed online. 2016;32:113–20. 22. ter keurst a, boivin j, gameiro s. women’s intentions to use fertility preservation to prevent gae-related fertility decline. reprod biomed online. 2016;32:121–31. 23. yu l, peterson b, inhorn mc, boehm jk, patrizio p. knowledge, attitudes, and intentions toward fertility awareness and oocyte cryopreservation among obstetrics and gynecology resident physicians. hum reprod. 2016;31:403–11. 24. virtala a, vilska s, huttunen t, kunttu k. childbearing, the desire to have children, and awareness about the impact of age on female fertility among finnish university students. eur j contracept reprod health care. 2011;16:108–15. 25. lampic c, svanberg as, karlstr€om p, tyd�en t. fertility awareness, intentions concerning childbearing, and attitudes towards parenthood among female and male academics. hum reprod. 2006;21:558–64. 26. salomon m, sylvest r, hansson h, nyboe andersen a, schmidt l. sociodemographic characteristics and attitudes towards motherhood among single women compared with cohabiting women treated with donor semen – a danish multicenter study. acta obstet gynecol scand. 2015;94:473–81. 210 o. hovatta who benefits from putting family life into ice? results from transfer of vitrified thawed eggs at which age should eggs be stored? awareness of limited fertility period among women is not optimal increased reproductive equity between women andmen conclusions references (8) ljungman 87-96 upsala j med sci 111 (1): 87–96, 2006 pain in pediatric oncology: do the experiences of children and parents differ from those of nurses and physicians? gustaf ljungman1, anders kreuger1, torsten gordh2, stefan sörensen3 1department of women’s and children’s health, children's university hospital, uppsala, sweden 2department of surgical sciences, university hospital, uppsala, sweden, 3clinical research center, central hospital, västerås, sweden abstract diagnosis and treatment of pain are central components in the care of children with cancer. the aim of the present study was to compare the viewpoints of children and parents with those of professionals, on different aspects of pain in children with cancer. information was collected through questionnaires and interviews. in particular, we focused on the extent and causes of pain, strategies to reduce procedural pain, pain evaluation, and attitudes to pain treatment. we found that both families and professionals shared the opinion that pain was a common symptom during different phases of cancer treatment but, surprisingly, professionals regarded it as more frequent than families. the groups agreed that treatment related pain is the most critical problem, followed by procedure and cancer related pain. concerning strategies to decrease procedural pain, there was a high concordance in views between groups. nurses and physicians more often claimed that failing pain treatment was associated with psychological factors such as high levels of anxiety in parents and children, loneliness, and lack of preparation. the self-report, according to both parents and professionals, is a feasible procedure even in young children from 4 years of age. both groups asserted that parents were better in ascertaining the extent of their child’s pain. in conclusion, although the families and professionals in this study have many comparable views concerning pain in children with cancer, divergences also exist. to acquire a more accurate picture of the situation we must focus on the views of the children first, and then those of parents and professionals. a tendency to overestimate the problems was observed in professionals. hopefully this reflects a keen awareness of the current situation. 87 received 10 october 2005 accepted 28 october 2005 introduction although there have been improvements in pain treatment in the last decades, pain and underestimation of pain are still common problems in pediatric oncology (1-6). it has also been shown that fear of pain is a major concern of children with cancer (7,8). different studies concerning pain assessment have compared the perspectives of children, parents and medical professionals (9-14). sometimes discrepancies between perspectives have been shown. a proposed reason for this has been inaccuracy in measurement (10,12). when a child’s rating does not agree with an observer’s rating, it has often been concluded that the child is providing an inaccurate assessment (14), or is unable to understand the required task. when the parent’s rating show discrepancies compared to that of a trained observer, it has been presumed that the parents are biased (15). it has been suggested that each mode of assessment may target a different aspect of pediatric pain, and that it may be more useful to understand the factors that contribute to differences among ratings, rather than to outrule data obtained from a certain individual (11,13). previously, problems in pain diagnostics and management have often been investigated from the perspective of medical professionals. in analogy with the discussion on assessment of pain, we consider it important to describe the current pain problems in pediatric oncology from different perspectives so as to form a truer picture of the situation. in a previous publication (4), we presented the views of nurses and physicians in sweden on the prevalence of pain in children with cancer and problems associated with pain evaluation and pain treatment. in a follow-up investigation (5), we summarized children’s and parents’ responses to the same questions. the comparison of these experiences from different perspectives is so important and extensive that we chose to present it as a separate article. this also gave an opportunity to add a minor questionnaire study to make the comparison more complete. the aim of the present study was to compare the viewpoints of children and parents with those of professionals, on different aspects of pain in children with cancer. in particular, we focused on the extent and causes of pain, strategies to reduce procedural pain, pain evaluation, and attitudes to pain treatment. materials and methods nurses and physicians 1. the views of nurses and physicians on different aspects of pain in children with cancer were investigated through a nationwide survey using postal questionnaires. the results and details of the questionnaire have been published (4). of the 47 pediatric departments in sweden, 35 take an active role in the treatment of children with cancer. one physician and one nurse from each of these 35 departments responded (response rate 100%). 88 2. furthermore, a minor complementary questionnaire survey was conducted to investigate the views of medical professionals, at the tertiary care pediatric oncology unit in uppsala, with respect to 10 questions on pain evaluation. in total 25 responses were obtained; 3 physicians and 22 nurses (response rate 83%). this latter questionnaire was designed to allow a comparison with the views of children and parents on the same issues. children and parents the experiences of children and parents were investigated in an interview study (5). all children admitted during a six-month period to the pediatric oncology ward in uppsala, and their parents, were invited to participate in the interview study. for inclusion, the child had to be in the treatment period from one month after diagnosis to three months post-treatment. sixty-six children and their families met the inclusion criteria and interviews were made with 55 (83%) of them. children under the age of 10 years were interviewed concurrently with their parents, though it was made clear that we primarily were interested in the views of the children. for children 10 years and older, interviews were conducted separately. in this way, we intended to obtain two different views of the same problem for comparison. all the answers from children were compared with those from parents. if significant differences were absent; data are presented as overall percentage scores reflecting the answers of both children and parents (children<10 years) and children (from 10 years) together. we found that the views of children and parents generally were comparable. therefore, it was appropriate to combine the attitudes of children and their parents and compare them with the attitudes of professionals. informed consent was obtained from children and parents and the local research ethics committee approved the study. the statistical differences were analyzed with �2-test for non-parametric data and student’s t test for parametric data. the significance level was set at p<.05. results extent and causes of pain as shown in figure 1, intensive pain during the last 3 months was seen either often or very often in approximately 5% according to both families and professionals. about 12% of the families as opposed to 40% of the nurses and physicians believed that, in spite of treatment, moderate or intensive pain occurs often to very often. the difference between the groups was greatest when they were asked whether more effective treatment was possible. the groups were similar as to the prime cause of pain, with treatment related pain being the dominant cause followed by procedure related and cancer related pain (figure 2). 89 strategies to reduce procedural pain the reports from families and professionals on procedural strategies were highly similar (table 1). emla “ (eutectic mixture of local anesthetics, astra, södertälje, sweden) was usually used before venipuncture/cannulation of a vein and before introducing a needle in a subcutaneously implanted intravenous port. general anesthesia was used in 90 extent of pain often/very often 17 <0.001 12 0.001 6 ns 72 40 5 0 10 20 30 40 50 60 70 80 more effective pain treatment possible moderate/intens. pain in spite of treatment intensive pain during last 3 months % p % nurses and physicians children and parents causes of pain greatest problem 49 ns 38 ns 13 ns 41 34 17 0 10 20 30 40 50 treatment related pain procedure related pain cancer pain % p % nurses and physicians children and parents figure 1. extent of pain. percent responses of children and parents (n=55) and nurses and physicians (n=70) regarding the extent and causes of pain. figure 2. causes of pain. percent children and parents (n=55) and nurses and physicians (n=70)giving the following answers concerning which cause of pain is the greatest problem. 91 approximately half of the children when performing a lumbar puncture and conscious sedation in the other half. before performing a lumbar puncture in conscious sedation, emla was used very often, and in two thirds of the cases an additional s.c. injection of local anesthetics was given. bone marrow aspirations and biopsies were usually performed with the child under general anesthesia. there were differences in views of families and professionals on various psychological explanations of inadequate pain treatment. generally, professionals estimated the importable 1 procedure strategies . percent children and parents(c) (n=55) stating the presence of strategies compared with nurses and physicians (n+p) (n=70). % c % n+p emla before venipuncture 98 100 sedation before venipuncture 19 emla before introducing a needle in an iv port 96 89 sedation before introducing a needle in an iv port 7 general anesthesia in lp (lumbar puncture) 46 50 sedation in lp 54 50 emla before lp 82 94 sc injection of analgesics before lp 67 59 general anesthesia in bma (bone marrow aspiration) 86 84 general anesthesia in bmb (bone marrow biopsy) 98 91 causes of failing pain treatment often/very often: 70 6 0.028 18 <0.001 4 0.001 5 <0.001 19 59 25 0 10 20 30 40 50 60 70 anxiety in parent anxiety in child loneliness lack of information/preparation % p % nurses and physicians children and parents figure 3. explanations of failing pain treatment. the views of families (n=55) and professionals (n=70) on different psychological explanations of failing pain treatment. table 1. procedure strategies. percent children and parents(c) (n=55) stating the presence of strategies compared with nurses and physicians (n+p) (n=70). tance of anxiety in parents and children, loneliness, and lack of information/preparation higher than did children and parents (figure 3). pain evaluation a high degree of accord exists between parents and professionals as to pain evaluation (table 2). parents argued that a child can accurately give self-reports already from 3-4 years of age (mean ± 95% confidence interval; 3.5 ± 0.4). this belief on the part of parents found strong support from nurses and physicians (mean ± 95% confidence interval; 4.27 ± 0.45). attitudes on pain treatment according to children and parents, opioids were never refused for fear of addiction. in contrast, 42% of professionals claimed that children occasionally refused the use of opioid analgesics for fear of addiction and 2% stated that refusal occurred often. in the group of professionals, 17% claimed that opioids were sometimes refused because patients’ relatives were skeptical, but no one in this group said that relatives refusal occurred often or very often. discussion previously problems in pain diagnostics and management have often been investigated from the perspective of medical professionals. in this paper we describe the pain problems in pediatric oncology from different perspectives; children’s and parents’ views are compared with those of health professionals so as to form a more complete picture of the situation. we found that both families and professionals shared the viewpoint that pain was a common symptom during different phases of cancer treatment, but professionals considered it even more common. the groups agreed that treatment related pain was the greatest problem, followed by procedure and cancer related pain. concer92 table 2 views on pain evaluation . parents (p) expressing agreement to statements (for all children (n=54), and for children <5 years (n=19) compared with children ≥5 years (n=35)) compared with nurses and physicians (n+p) (n=25). * = p< .05 often / very often correct % p %n+p % <5yrs % ≥ 5yrs 1. to evaluate how much pain a child has, i get the most important information through: a) asking how much pain there is. 68 67 31 84* b) observing the behavior of the child. 81 88 90 77 2. i think that parents are better to judge how much pain their child has than nurses and physicians. 72 64 84 65 3. i find it difficult to know: a) when a child is in pain. 15 16 21 12 b) how much pain a child has (when in pain). 48 44 63 40 4. i think that the pain evaluation routines are such that effective pain treatment can be provided. 83 88 74 89 table 2. views on pain evaluation. parents (p) expressing agreement to statements (for all children (n=54), and for children <5 years (n=19) compared with children ≥5 years (n=35) compared with nurses and physicians (n+p) (n=25). * = p< .05 ning strategies to reduce procedural pain, no difference was observed between groups. nurses and physicians more often claimed that inadequate pain treatment was related to such psychological factors as high levels of anxiety in parents and children, loneliness, and lack of preparation. the use of the self-report technique, according to both parents and professionals, is possible in children from the age of 4 years. moreover, both groups suggested that parents were better in determining the pain intensity their child was experiencing. a potential problem is that we, in most questions, compare the views of children and parents in one region of sweden with experiences of nurses and physicians from the whole country. however, according to a previous national study (4), principles of pain management do not vary much in different regions of the country many of the questions administered to professionals were used in an earlier study (16) and were tested in a pilot study. to study child/parent attitudes, a slightly modified version of the questionnaire questions was developed and assessed in a small pilot study. this probably served to increase the validity and reliability of our investigation. the high response rate in the questionnaire studies and few dropouts in the interview investigation also increase the validity and reliability. extent and causes of pain we found highly significant differences as to how commonly pain occurs despite treatment, and how often more effective pain treatment could be possible. this probably reflects an overestimation and self-criticism on behalf of nurses and physicians. on the other hand, it might reflect an underestimation from children and parents due to denial and perhaps an overly optimistic belief that the medical profession does everything in its power to alleviate their pain and suffering. congruence between the groups was noted in their reports on causes of pain; a finding that confirms the results of previous investigations in children with malignancies (1-4,7). strategies to reduce procedural pain the reports on strategies from children and parents concur with those from professionals and thus increase the validity of the instrument. when pain treatment failed, nurses and physicians estimated that a number of different psychological explanations were more important than did the families. one interpretation of this could be that professionals believe that psychological factors are of great importance for the experience of pain, whereas children and parents see inadequate pain relief merely as a failure of medication. the occurrence of pharmacological under-treatment and inadequate pain treatment resulting from unsatisfactory analysis of pain quality were not further investigated in this study. pain evaluation about 75% of parents and 66% of professionals thought that parents are more capable of determining their child’s pain than are professionals. this finding is not 93 consistent with that from a previous study where it is concluded that parent ratings may not provide a good indication of the amount of overt distress or the child’s pain experience, and that nurse ratings of acute pain may most closely approximate objective assessment of pain and distress behavior (13). an explanation to this inconsistency could be that our questions did not discriminate between acute pain and longer term pain, and that our results may reflect views on the latter rather than on the former. parents and professionals agree that a child can produce reliable self-reports from as early as 4 years of age. this is in concordance with the fact that there are no reliable subjective scales for children under this age (18,19). pain is by definition a subjective experience (20). the self-report technique is therefore the gold standard in pain assessment (19). however, a prerequisite for the use of this technique is an, at least, elementarily developed language, thus limiting the use in younger children. attitudes and information children and parents claimed that they never refused opioids for fear of addiction, but professionals reported some views to the contrary. perhaps this reflects a change of attitudes due to education and information. in conclusion, although the families and professionals in this study have many comparable views concerning pain in children with cancer, divergences also exist. to acquire a more accurate picture of the situation we must focus on the views of the children first, and then those of parents and professionals. in our study, nurses and physicians seeme acknowledgments this project was financially supported by the children’s cancer fund, the foundation of mary béve, västerås county research fund, lions cancer fund at the university hospital of uppsala, and the swedish medical research council (project number 9077, t gordh). references 1. miser a, dothage j, wesley r, miser j (1987) the prevalence of pain in a pediatric and young adult cancer population. pain 29: 73-83. 2. mcgrath p, hsu e, cappelli m, luke b, goodman j, dunn-geier j (1990) pain from pediatric cancer: a survey of an outpatient oncology clinic. j psychosoc oncol 8: 109-24. 3. elliott s, miser a, dose a, betcher d, o´fallon j, ducos r, shah n, goh t, monzon c, tschetter l (1991) epidemiologic features of pain in pediatric cancer patients: a co-operative community-based study. clin j pain 7: 263-68. 4. ljungman g, kreuger a, gordh t, berg t, sörensen s, rawal n (1996) treatment of pain in pediatric oncology: a swedish nationwide survey. pain 68: 385-94. 5. ljungman g, gordh t, sörensen s, kreuger a (1999) pain in pediatric oncology: interviews with children, adolescents, and their parents. acta paediatr 88: 623-30. 6. zernikow b, meyerhoff u, michel e, wiesel t, hasan c, janssen g, kuhn n, kontny u, fengler r, gortitz i, andler w (2005) pain in pediatric oncology-children's and parents' perspectives. eur j pain 9: 395-406. 94 7. enskär k, carlsson m, golsäter m, hamrin e, kreuger a (1997) life situation and problems as reported by children with cancer and their parents. j pediatr oncol nurs 14: 18-26. 8. hedström m, ljungman g, von essen l (2005) perceptions of distress among adolescents recently diagnosed with cancer. j pediatr hematol oncol 27: 15-22. 9. iafrati n (1981) pain on the burn unit: patient vs nurse perceptions. j burn care rehab 2: 259-61. 10. teske k, daut r, cleeland c (1983) relationships between nurse's observations and patients' selfreports of pain. pain 16: 289-96. 11. van der does aj (1989) patients' and nurses' ratings of pain and anxiety during burn wound care. pain 39: 95-101. 12. choiniere m, melzack r, girard n, rondeau j, paquin m (1990) comparisons between patients' and nurses' assessment of pain and medication efficacy in severe burn injuries. pain 40: 143-52. 13. manne s, jacobsen p, redd w (1992) assessment of acute pediatric pain: do child self-report, parent ratings, and nurse ratings measure the same phenomenon. pain 48: 45-52. 14. moinpour c, donaldson g, wallace k, hiraga y, joss b (1990) parent/child agreement in rating child mouth pain. adv pain res ther 15: 69-78. 15. gauvain-piquard a, rodary c, rezvani a, lemerle j (1989) pain in children aged 2-6 years: a new observational rating scale elaborated in a pediatric oncology unit preliminary report. pain 31: 177-88. 16. rawal n, hylander j, arnér s (1993) management of terminal cancer pain in sweden: a nationwide survey. pain 54: 169-79. 17. cornaglia c, massimo l, haupt r, sizemore w, benedetti c (1984) incidence of pain in children with neoplastic diseases. pain 2: s 28. 18. beyer j, wells n (1989) the assessment of pain in children. pediatr clin north am 36: 837-54. 19. mcgrath p (1987) an assessment of children’s pain: a review of behavioral, physiological and direct scaling techniques. pain 31: 147-76. 20. merskey h (1991) the definition of pain. eur j psychiatry 6: 153-59. corresponding author: gustaf ljungman md, phd department of women’s and children’s health children's university hospital se-751 85 uppsala, sweden phone: +46-18-6115586 fax: +46-18-500949 gustaf.ljungman@kbh.uu.se 95 96 vol_117_001_sups_a_640412 41..46 full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 apparent clearance of valproic acid in elderly epileptic patients: estimation of the confounding effect of albumin concentration natalia lampon & j. carlos tutor to cite this article: natalia lampon & j. carlos tutor (2012) apparent clearance of valproic acid in elderly epileptic patients: estimation of the confounding effect of albumin concentration, upsala journal of medical sciences, 117:1, 41-46, doi: 10.3109/03009734.2011.640412 to link to this article: https://doi.org/10.3109/03009734.2011.640412 © informa healthcare published online: 29 dec 2011. submit your article to this journal article views: 689 view related articles citing articles: 9 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2011.640412 https://doi.org/10.3109/03009734.2011.640412 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2011.640412 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2011.640412 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2011.640412#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2011.640412#tabmodule upsala journal of medical sciences. 2012; 117: 41–46 original article apparent clearance of valproic acid in elderly epileptic patients: estimation of the confounding effect of albumin concentration natalia lampon & j. carlos tutor unidad monitorización fármacos, laboratorio central, hospital clínico universitario, instituto de investigación sanitaria (idis), 15706 santiago de compostela, spain abstract background. valproic acid (vpa) apparent clearance (cl) estimated from total serum concentrations is analogous in elderly and non-elderly adult patients. as drug–protein binding decreases in old age, the aim of our study was to evaluate the confounding effect of the serum albumin concentration on the vpa apparent cl in elderly patients. methods. in 102 epileptic out-patients treated with vpa in monotherapy, serum total steady-state trough concentrations (css) were determined. css concentrations were normalized for a 42 g/l albumin concentration (cssn), and the apparent cl and normalized apparent cln were calculated. results. a poor concordance of 53% was found in the classification of css and cssn levels of vpa as subtherapeutic, therapeutic, or supratherapeutic dose. in the elderly (‡65 years) and non-elderly adult patients, the vpa apparent cl was similar; however, normalized apparent cln was significantly lower in older patients (p < 0.01), with a 40% median decrease. conclusions. total vpa concentrations should be interpreted with caution, mainly in older patients, in which determination of unbound or normalized total drug concentrations may be clinically useful. normalization of total concentrations permits an estimation of the masking effect of serum albumin concentrations on the vpa apparent cl in elderly patients. key words: albumin, apparent clearance, elderly patients, unbound fraction, valproic acid introduction although valproic acid (vpa) was initially approved for the treatment of refractory absence seizures, it is now recognized that it has a broad-spectrum antiepileptic activity (1). similarly, vpa may be effective in a variety of psychiatric and neurological diseases such as bipolar disorders, schizophrenia, depression, neurological pain, migraine headaches, alzheimer’s disease, and other neurodegenerative illnesses (2). vpa is well absorbed from all oral dosage forms, with a bioavailability greater than 90%. the drug, structurally related to free fatty acids, is highly ionized at physiological ph and therefore presents to a high degree bound to plasma proteins, primarily to albumin (3–5). in conditions of hypoalbuminemia (even moderate) there is a significant increase in the free fraction (percent unbound) of the drug; likewise, for total serum concentrations up to 75 mg/ml, vpa is about 90% bound to albumin, but above this concentration binding saturates with an increase of the free fraction (4). the effect of age on the vpa pharmacokinetics has been widely studied, although there is clearly a need for improved methodology in the assessment of agerelated changes in pharmacokinetics (6). the apparent clearance (cl) calculated from total serum vpa concentrations is similar in elderly and non-elderly adult patients (7–10); however, in a clinical context these findings must be interpreted with caution, considering that vpa plasma protein binding decreases in old age, with an accompanying increase of the free fraction of the drug (5,8,11). correspondence: j. carlos tutor pharmd phd, unidad monitorización fármacos, laboratorio central, hospital clínico universitario, instituto de investigación sanitaria (idis), 15706 santiago de compostela, spain. e-mail: josecarlostutor@redfarma.org (received 28 september 2011; accepted 8 november 2011) issn 0300-9734 print/issn 2000-1967 online � 2012 informa healthcare doi: 10.3109/03009734.2011.640412 for highly protein-bound drugs, such as vpa, diminished plasma protein binding is associated with a decrease of the serum total concentration (protein-bound plus free drug), and consequently with an increased apparent cl; however, it would be misleading to assume that the amount of drug eliminated per unit of time is also increased (12). the lower total concentration of the drug is associated with a lower protein-bound concentration and increased free fraction, although the unbound concentration and the amount of drug eliminated per unit of time remain unchanged. therefore, the pharmacological effect in these conditions will be analogous to that produced by the higher total drug concentration obtained under normal protein binding conditions for the same daily dose (12). the decrease of serum albumin concentrations in old age is well established (13), and the main aim of our study was to evaluate the confounding effect of albumin levels on the calculated apparent cl of vpa from the serum total concentrations (css) in elderly epileptic patients. consequently, the normalized total vpa concentrations (cssn) for a 42 g/l serum albumin concentration, which corresponds to the percentile 50 of the reference range for individuals between 25 and 55 years of age (13), were estimated as previously described (14), and the corresponding normalized apparent clearances (cln) were calculated. patients and methods 102 epileptic outpatients (53 males and 49 females) with a mean age (± sem) of 40.2 ± 2.3 years (range 6–94 years) and orally treated with vpa in monotherapy were studied. none of them took drugs known to influence vpa–protein binding. in all cases the daily vpa administration was carried out in multiple doses with the same dosing interval. blood samples were taken before breakfast and the morning dose, which had not been modified for at least 3 months prior to the study, and therefore the serum levels of vpa correspond to the trough steady-state concentrations (css). this study was carried out according to the good practice rules for investigation in humans of the conselleria de sanidade (regional ministry of health) of the xunta de galicia, spain. serum vpa total concentrations were determined in a dimension � xpand analyzer using reagents from siemens health care inc. (newark, de, usa). in cases with albumin concentrations lower or greater than 42 g/l (609 mmol/l), total vpa css levels were normalized using the expression (14): cssn = acss/6.5, where 6.5 corresponds to the vpa unbound fraction for albuminemia of 42 g/l, and a is the estimated unbound fraction for a particular albumin concentration using the hyperbolic equation of parent et al. (15): a (%) = ae-bx, where x corresponds to albumin concentration (mmol/l), and the constants a = 130.69 and b = 4.96 � 10-3. for experimental total css concentrations of vpa greater than 75 mg/ml, the normalized cssn values were corrected as previously described (14). the vpa apparent cl or cln values were, respectively, calculated from the serum vpa css or cssn concentrations using a conventional pharmacokinetic procedure (12): cl (or cln) = [(dose/dosing interval)/css (or cssn)]. as in previous studies (9,10), trough rather than average vpa concentrations were used, and consequently the reported cl and cln represent overestimates of actual values. serum activities of alanine aminotransferase (alt), aspartate aminotransferase (ast), and gammaglutamyl transferase (ggt) and concentrations of albumin, bilirubin, and creatinine were determined in an advia 2400 chemistry system (siemens health care diagnostics inc.). the platelet count was measured in blood samples collected 2–3 hours beforehand in k3edta anticoagulated tubes using an advia 2120 hematology system (siemens health care diagnostics inc.). the liver fibrosis ast to platelet ratio index (apri) was calculated in accordance with wai et al. (16): apri = [ast:url/platelet count (109/l)] � 100, where url corresponds to the ast upperreferencelimitsformenandwomen.glomerular filtration rate (gfr) was estimated from serum creatinine using the chronic kidney disease epidemiology collaboration (ckd-epi) equation (17). statistical analysis of data was performed using the statgraphics package, and the kolmogorov–smirnov test was applied to check for normality. the pearson correlation coefficient and student’s t-test were used when the data had a gaussian distribution. otherwise, the spearman correlation coefficient and mann– whitney u test were used. in accordance with the consensus criteria for determination of drugs and their metabolites in biological samples (18), the accepted level for accuracy is a deviation of no more than 15% from the nominal value. results were expressed as mean ± sem (median). results there was a negative correlation between the serum albumin concentration and age (r = –0.462, p < 0.001), with patients older than 65 years of age presenting a significant decrease of the albumin concentration (p < 0.001) (table i). in a majority of patients (n = 97), estimated gfr values were higher than 60 ml/min/1.73 m2. in all cases the 42 n. lampon & j. c. tutor levels of bilirubin were lower than the upper limit of reference (data not shown). there was a relationship between total css and normalized total cssn vpa serum levels (figure 1). for total trough vpa serum concentrations a therapeutic range of 50–100 mg/ml has been widely accepted (19), and a considerable proportion of cases presenting therapeutic css levels, after its normalization by albumin concentration, present supratherapeutic cssn levels, with a modest concordance of 53% in the classification of css and cssn levels as subtherapeutic, therapeutic, or supratherapeutic. table i. pharmacokinetic and biochemical variables according to age in epileptic patients treated in monotherapy with valproic acid. £19 years 20–64 years ‡65 years n (males/females) 24 (14/10) 57 (29/38) 21 (10/11) age (years) 13.7 ± 0.9 (15.0) 37.9 ± 1.6 (38.0) 76.9 ± 1.7 (76.0) vpa dose (mg/day) 836.7 ± 91.0 (800.0)*** 1208.8 ± 52.5 (1200.0) 1066.7 ± 70.3 (1000.0) vpa css (mg/ml) 66.4 ± 4.6 (67.5) 67.9 ± 2.3 (68.9) 60.4 ± 4.3 (54.4) vpa cl (ml/min) 9.2 ± 0.8 (8.8)*** 12.7 ± 0.6 (11.7) 13.9 ± 2.0 (11.45) vpa a (%) 5.87 ± 0.13 (5.6)* 6.59 ± 0.21 (6.2) 9.92 ± 1.09 (8.5)**** vpa cssn (mg/ml) 76.2 ± 9.7 (59.0) 96.7 ± 10.4 (67.9) 145.3 ± 27.7 (71.1) vpa cln (ml/min) 9.3 ± 0.9 (10.0) 11.4 ± 0.8 (11.1) 8.1 ± 0.9 (6.7)** albumin (g/l) 44.2 ± 1.0 (45.0)* 43.1 ± 1.0 (43.0) 37.0 ± 1.1 (38.0)**** ast (u/l) 14.7 ± 0.7 (14.5) 15.0 ± 1.1 (14.0) 17.6 ± 3.4 (13.0) alt (u/l) 14.9 ± 2.3 (12.0)* 19.9 ± 2.0 (15.0) 17.3 ± 3.7 (13.0) ggt (u/l) 12.7 ± 3.1 (8.5) 17.0 ± 1.9 (12.0) 25.7 ± 6.8 (13.0) apri score 0.28 ± 0.02 (0.24) 0.33 ± 0.03 (0.28) 0.48 ± 0.12 (0.31) ast/alt ratio 1.2 ± 0.1 (1.2)**** 0.9 ± 0.1 (0.7) 1.1 ± 0.1 (1.0)*** gfr (ml/min/1.73 m2) 144.0 ± 3.1 (144.2)**** 102.4 ± 2.6 (102.8) 73.2 ± 4.0 (79.2)**** statistical significance against non-elderly adult patients (20–64 years): *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001. results are expressed as mean ± sem (median). 0 50 100 150 200 250 300 350 400 450 0 25 50 75 100 125 n = 102 r = 0.802 p < 0.001 150 vpa css (mg/ml) v p a c s s n ( mg /m l ) figure 1. relationship between total (css) and albumin normalized total (cssn) concentrations of valproic acid in epileptic patients of <65 (*) and ‡65 (.) years of age. the dashed lines correspond to the limits of the valproic acid therapeutic range (50–100 mg/ml). valproic acid clearance in elderly epileptic patients 43 the different pharmacokinetic variables assayed were similar in patients aged between 20–40 and 41–64 years, and therefore these patients were included in one group of non-elderly adults with age between 20 and 64 years. results obtained for the vpa daily doses, css, cssn, cl, and cln in the studied patients grouped according to age are shown in table i. the estimated vpa unbound fraction was significantly greater in the group of elderly patients. significant differences for total vpa css and cssn concentrations between the different groups of patients were not obtained, and the difference between the medians of css and cssn was greater than 15% of median css only in elderly patients. with respect to the vpa daily dose and apparent cl, these variables were significantly lower in patients aged £19 years (p < 0.005). after normalization of serum vpa levels with the albumin concentration, the apparent cln was significantly decreased by around 40% in elderly patients (‡65 years). no significant correlation was found between the vpa normalized apparent cln and gfr (r = 0.074). the interindividual variation of the vpa apparent cl was significantly higher in patients aged ‡65 years than in the group of nonelderly adults (cv = 64% versus cv = 35%); however, the interindividual variation of the vpa normalized apparent cln was similar in both groups of patients (cv = 50% versus cv = 52%). a dichotomy of the data based on the patients’ sex did not reveal results with additional interest. calculation of apri suggests a low likelihood of significant hepatic fibrosis in our vpa-treated patients, because in 84 cases (83%) the apri score was £0.4, excluding the presence of liver fibrosis with a high level of confidence (16,20), and the apri score was only higher than 1.5 in two cases, in which significant liver fibrosis may be supposed (16). in the group of patients with apri score >0.4 the vpa apparent cln was significantly lower (p < 0.05) than in the group with apri score £0.4 [7.3 ± 1.1 ml/min (6.5 ml/min) versus 10.5 ± 0.5 ml/min (10.8 ml/min)]. likewise, significant negative correlations were found between the vpa apparent cln and the apri (r = –0.258, p < 0.005), and ast/alt ratios (r = –0.387, p < 0.001). the apparent cln was also significantly correlated with serum alt (r = 0.280, p < 0.01) and ggt (r = 0.211, p < 0.05) activities, but not with ast activity (r = 0.076). discussion age-related pharmacokinetic changes are due to multiple factors and present a high interindividual variability. gastrointestinal absorption is not generally altered in elderly; nevertheless, decreased plasma protein binding, increased volume of distribution, reduced efficiency of drug-metabolizing enzymes, and diminished drug renal clearance may lead to a decreased elimination half-life of several drugs in older patients (11,22). in accordance with previously published reports (7–10), the apparent cl of vpa calculated from the total css concentrations was analogous in elderly and non-elderly adult patients (table i); however, the serum concentration of albumin was significantly lower in the group with age ‡65 years than in the other groups that were considered (table i), and therefore an increase of the vpa unbound fraction should be assumed in these elderly patients, as previously described (5,11,21,22). normalization of total vpa css concentrations permit estimation of the total cssn levels (the unbound concentrations remain unchanged) that would be expected if the serum albumin concentration in all patients was 42 g/l (14) and consequently a correction of the confounding effect of serum albumin concentrations on the calculated apparent cl from total vpa levels. at present, the clinical usefulness of measurements of vpa unbound concentrations is a debated subject (19,23); however, decreases in vpa protein binding are followed by reductions in total serum concentrations, whereas unbound concentrations are unchanged, and these reduced total concentrations may be misinterpreted as reflecting an inadequate dosage (24). as previously described for total and unbound vpa serum concentrations (25,26), a nonlinear, hyperbolic relationship was found between total css and total normalized cssn concentrations (figure 1). similarly, a poor concordance of 53% was obtained in the classification of total css and total normalized cssn serum vpa levels as subtherapeutic, therapeutic, or supratherapeutic. the diagnostic efficiency of a laboratory test is the percentage of all results that are true, and as a general rule a test is probably not worth doing if its efficiency is less than 80% (27). consequently the results indicated above suggest an unacceptable diagnostic efficiency of the total css concentrations for therapeutic vpa monitoring in our group of epileptic out-patients. in a significant number of cases (around 47%) the determination of unbound vpa concentrations, or at least estimation of total normalized cssn levels, would be clinically useful. in several cases, with vpa css concentrations in the therapeutic range, the estimated normalized cssn levels were significantly supratherapeutic (>200 mg/ml), explaining the hypersedation and dysarthria observed in these patients. after correction of the masking effect of the albumin concentration, the apparent vpa cln calculated from the total normalized cssn levels was significantly 44 n. lampon & j. c. tutor lower in the group of elderly patients than in the nonelderly adult patients group (p < 0.01). the vpa cln median decrease of about 40% in old age (table i) is analogous to the difference previously calculated on apparent cl of unbound vpa (6,28). also, after normalization of the total vpa css concentrations, the interindividual variation of the normalized apparent cln calculated from cssn was similar in all groups of patients, demonstrating that the higher variability of the apparent cl in the group of elderly patients is mainly due to their lower serum albumin concentrations (table i). a high prevalence of ultrasound-diagnosed nonalcoholic fatty liver disease has been previously demonstrated in adolescents (29) and adults (30,31) chronically treated with vpa. fatty liver disease ranges from simple liver steatosis to steatohepatitis with necroinflammation and liver fibrosis, which can progress to cryptogenic cirrhosis, and significant changes occur in hepatic drug-metabolizing cytochrome p450 (cyp) enzyme families during these progressive stages (32). although the principal pathways of vpa metabolism are glucuronidation and b-oxidation, and cyps pathways account for less than 10% of the dose (33), functional polymorphisms of several cytochrome oxidase isoenzymes may explain part of the inter-individual variability in vpa pharmacokinetics (34). in accordance with previously published results (35), data indicated above for apri suggest a low likelihood of significant liver fibrosis in epileptic patients treated with vpa in monotherapy, even in elderly subjects (table i); however, significant negative correlations were obtained between the apparent cln of vpa and the apri score and ast/alt ratio. induction of cyps and ggt by enzyme-inducing anticonvulsant drugs is widely documented and in epileptic patients treated with vpa in monotherapy and polytherapy with carbamazepine, phenytoin, or phenobarbital; a significant correlation has been previously reported between the apparent cl of vpa and alt and ggt serum activities (33). in our patients that were treated with vpa in monotherapy, significant positive correlations were also found between the apparent cln of vpa and serum ggt and alt. vpa is not an enzyme-inducing agent, and in these patients ggt and alt activities may be associated with over-weight and fat accumulation in the liver (33,35). although in simple steatosis the function of several liver cyps isoenzymes may be impaired, the activity of phase ii conjugation enzymes, such as udp-glucuronyltransferase, would be enhanced (36), with the consequent increase of vpa biotransformation and elimination. the main limitation of our study is that the vpa free fraction was not experimentally determined. endogenous substances such as free fatty acids, uremic compounds, and bilirubin may displace vpa from its albumin binding sites, increasing the unbound fraction and leading to an underestimation of the normalized cssn values. free fatty acids commonly increase after fasting states, and in our study blood samples were taken before breakfast; in none of our patients was a situation of renal failure or jaundice observed. although theoretical estimation of normalized cssn concentrations offers approximate results (14), this pharmacokinetic approach permits evaluation of the confounding effect of the albumin concentration on the apparent cl of vpa in old age. in conclusion, total serum levels of vpa should be considered with caution, principally in older patients, in which determination of the drug unbound concentrations, or at least normalized total drug levels for 42 g/l albumin concentration, may be clinically useful. normalization of total vpa levels permits estimation of a decrease around 40% for the apparent cl in elderly patients. acknowledgements one of the authors (n.l.) received a grant from 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preterm or with low weight implies a risk of infertility and premature loss of ovarian function; a national register study gunilla sydsj€o , marie bladh, katarina rindeborn, mats hammar, heriberto rodriguez-martinez and elizabeth nedstrand faculty of medicine and health sciences, division of obstetrics and gynaecology, department of biomedical and clinical sciences, link€oping university, link€oping, sweden abstract background: being born with non-optimal birth characteristics has several long-term consequences on health in general but also for the individual’s reproductive pattern. in premature ovarian insufficiency (poi) the follicles are depleted or dysfunctional. this results in menopause before the age of 40, and for most of the affected women, it causes infertility. the objective of this study was to evaluate the potential effects of being born with non-optimal birth characteristics on the risk of developing poi. methods: this population-based cohort register study included all women born in sweden between 1973 and 1993 who were followed until the end of 2012 (age at the end of follow-up ranged between 39 and 59). women diagnosed with poi were compared with women without this diagnosis with respect to being born small for gestational age, preterm, or with low birth weight. data on birth characteristics and diagnosis of poi were collected from national registers. results: a total of 1,033,878 women were included. being born small for gestational age was associated with a slightly increased odds ratio of poi with 10%. preterm birth and low birth weight were associated with somewhat increased ors of poi after exclusion of those born small for gestational age. similarly, being born preterm or with a low birth weight was also found to be associated with poi to the same extent. conclusions: being born with non-optimal birth characteristics may increase the risk of premature ovarian insufficiency. article history received 14 february 2020 revised 12 may 2020 accepted 12 may 2020 keywords low birthweight; preterm birth; primary ovarian insufficiency; small for gestational age introduction being born with non-optimal birth characteristics – defined as being small for gestational age (sga), or preterm birth, or with low birth weight (lbw) – has several long-term consequences on health in general but also for the individual’s reproductive pattern (1). organ development during prenatal life is influenced by the intrauterine environment, and adverse conditions during foetal life can result in increased risk for metabolic syndrome, cancer, neurologic disease, and mental health problems (1). there is also evidence that young adults and adults that were born preterm and sga have a lower chance of having children. this lowered chance of reproducing has been speculated to be due to psychological as well as medical reasons (2–4). the medical reasons for these risks include the foetal intrauterine environment and the mother’s health and lifestyle prior to and during pregnancy (5,6). during intrauterine life, different tissues grow due to rapid cell division. since different tissues have different timing, disturbances in the transport of nutrients, growth factors, hormones, and oxygen from the placenta may affect different tissues with different timing. the foetus adapts to growth interference by slowing down the rate of cell division (7), and undernutrition may permanently reduce the number of cells in some organs. other effects of undernutrition include changes in the cell type distribution, hormonal feedback, metabolic activity, and organ structure due to ‘lasting’ memories of undernutrition (7). it has been shown that foetal growth restriction also affects the development of the ovaries and reproductive axis (8). in premature ovarian insufficiency (poi) the follicles are depleted or dysfunctional, resulting in menopause before the age of 40 and for most of the affected women causing infertility (9). in a recent study, we found that poi affects 2% of all women (10). poi can be either primary or secondary, and the causes remain unknown in 90% of the cases. even though most cases of spontaneous poi remain unexplained, the knowledge about its aetiology is increasing. identified underlying factors include chromosomal abnormalities, gene mutations, autoimmunity, metabolic disorders, infections, and the mother’s lifestyle factors such as alcohol and contact gunilla sydsj€o gunilla.sydsjo@liu.se division of obstetrics and gynaecology, department of biomedical and clinical sciences, link€oping university, se-581 85 link€oping, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 3, 235–239 https://doi.org/10.1080/03009734.2020.1770380 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1770380&domain=pdf&date_stamp=2020-06-29 http://orcid.org/0000-0003-4296-4038 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1770380 http://www.tandfonline.com nicotine use. each of these factors can lead to either follicle dysfunction or follicle depletion (11). iatrogenic poi is caused by surgery, chemotherapy, or radiation. improved survival after treatment of malignant diseases in children and young adults has resulted in a rising incidence of iatrogenic poi. even though the exact incidence is uncertain (12), it has been found that 6.3% of women treated for cancer developed poi shortly after the treatment (13). in addition, a further 8% of the study cohort developed poi later in life (12). there is a lack of studies evaluating the potential effects of being born with non-optimal birth characteristics on the risk of developing poi. the aim of this study was to test the hypothesis that women born with non-optimal birth characteristics, that is, preterm, with low birth weight, being large for gestational age (lga) or sga have an increased risk of developing poi later in life. material and methods data were collected from swedish health data registers. the swedish medical birth register was established in 1973 and contains valid data on pregnancies, deliveries, and the newborn. the swedish medical birth register covers �99% of all births in sweden (14). data included in this study were gestational age, delivery diagnoses, and the weight and length of the infants. the national patient register records the inpatient care in sweden and all specialist out-patient care. this register holds admission data, diagnoses, external causes of injury, and surgical procedures. the diagnoses in the national patient register are based on the swedish version of the international classification of diseases (icd). icd-8 (version 8) was used until 1986, icd-9 was used between 1987 and 1996, and icd-10 has been used from 1997 and onwards. the national patient register has been validated for high completeness and accuracy (15–17). in addition, we used data from the swedish prescribed drug register (18), which includes information on all prescribed and dispensed drugs in sweden since 2005. it contains information about the patients, prescriptions, doses, amounts, costs, and prescribers’ institutions. the study has been approved by the regional ethical review board in link€oping (03–556, 07-m66 08–08-m 233–8, 2014–112/31). study design the study population was a cohort of 1,036,918 women born in sweden between 1973 and 1993 and were followed until 31 december 2012. cases with poi were identified from the national patient register. the icd diagnosis codes used to define poi were: 2561, 6159, 627 (icd-8), 256.3 (icd-9), and e28.3 (icd-10). the personal identity number unique for each resident in sweden allowed individual linkages of all women between the registers used. cases with poi were also identified by dispensed systemic hormone replacement therapy for climacteric symptoms by women younger than 40 years of age. this therapy included oral and transdermal products within the act-groups g03ca03 oestradiol (excluding lowdose products and products for local vaginal treatment only), g03ca57 conjugated oestrogens, g03cx01 tibolone, g03fa01 norethisterone and oestrogen, g03fa12 medroxyprogesterone and oestrogen, g03fa15 dienogest and oestrogen, g03fa17 drospirenone and oestrogen, g03fb05 norethisterone and oestrogen, g03fb06 medroxyprogesterone and oestrogen, and g03fb09 levonorgestrel and oestrogen. since these drugs are mainly prescribed to treat menopausal women and cannot be used for contraception, women prescribed these drugs who were younger than 40 years of age were considered to have poi. however, since hormone replacement therapy is also prescribed to patients with iatrogenic poi (e.g. after oophorectomy), all women with a cancer diagnosis were excluded if diagnosed with a spontaneous poi. these women were identified in the national patient registry using the following icd-codes: all diagnoses starting with c in icd-10, codes 140–239 in icd-9, and codes 140–239 in icd-8. furthermore, women who had had a bilateral oophorectomy (icd-10 code laf10) and been prescribed and dispensed hormone replacement therapy were determined to have poi and were thus included in the study (n ¼ 302). furthermore, women having the diagnoses turner’s syndrome (q960-q969), ovarian cancer (c569), and/ or thyroid disease (e210-e213, e060-e069) were excluded (n ¼ 3040) leaving 1,033,878 women in the final study population. exposure variables and definitions infants born preterm were separated from those born at term to assess whether there were any differences in the incidence of poi between these two groups. among those born preterm we further separated those born sga from those born appropriate for gestational age (aga). furthermore, we categorized study infants into quartiles regarding the severity of sga to investigate potential doseresponse associations. sga was defined as birth weight �2 standard deviations (sd) of the mean weight at a specific gestational age according to the swedish standard. large for gestational age (lga) was defined in a similar manner but with the limit >2 sd of the mean weight for gestational age. lbw was defined as birth weight below 2500 g and very low birth weight as a birthweight below 1500 g. moderate preterm birth was defined as being born between gestational weeks 32 and 36, and very preterm birth as being born before gestational week 32. statistical analysis pearson’s chi-square statistic was used to explore the bivariate association between poi and socio-demographic factors and birth characteristics. to investigate whether being born preterm, with low birth weight, sga, or lga was related to an increased risk of poi later in life, we analyzed the data using a single and multiple logistic regression, providing odds ratios (ors) with 95% confidence intervals (cis). in the multiple logistic regression models, data were adjusted for attained educational level (categorized into three groups: 236 g. sydsjö et al. elementary, high school, and university) among the study participants and year of birth. each birth characteristic was modelled separately. a p-value <0.05 was considered statistically significant. the mantel–haenszel test for trend for the different levels of sga was used. all statistical analyses were performed using ibm spss, version 24.0 (ibm spss inc., armonk, ny). results a total of 1,033,878 women born between 1973 and 1993 were included in the final study cohort, and 18,627 (1.8%) of these women had poi. preterm delivery and risk of primary ovarian insufficiency the cohort included a total of 51,924 women born preterm, of whom 960 (1.8%) had poi. that was identical to the proportion of women with poi among women born at term. also, 5.0% of the women not diagnosed with poi were born preterm, while the corresponding number for women diagnosed with poi was 5.2% (p ¼ 0.407) (table 1). the unadjusted or was not significantly increased (hr 1.03, 95% ci: 0.96–1.12) (table 2). however, when adjusted for year of birth and educational level, being born preterm was associated with 11% increased risk of poi (or 1.11, 95% ci: 1.04–1.19) (table 2). when excluding women born sga in the preterm group, the statistical differences remained (or 1.08, 95% ci: 1.01–1.16) (table 2). low birth weight and risk of primary ovarian insufficiency a total of 44,087 women were born with low birth weight, and 952 (2.0%) of them had poi compared with 1.8% in women born with normal birth weight. additionally, 4.7% of the women diagnosed with poi were born with a low birth weight compared with 4.3% among the women born with normal birth weight (p � 0.1) (table 1). lbw was associated with a 15% increased risk of poi in the adjusted analysis (or 1.15, 95% ci: 1.07–1.23). when women born sga were excluded, the difference remained significant though the risk was lower (or 1.10, 95% ci: 1.00–1.22) (table 2). small for gestational age and risk of primary ovarian insufficiency among 211,362 women born sga, with a weight less than 1 sd below the mean (1sd), 39,023 women were born 2 sd below the mean (2sd), and 5,842 were born 3 sd below the mean (3sd) (table 1). being born sga (1sd) was associated with a 5% increased risk of poi (or 1.05, 95% ci: 1.02–1.09). being born sga (2sd) indicated a 10% increased risk of poi (or 1.10, 95% ci: 1.02–1.18). finally, being born sga (3sd) was associated with a 16% increased risk of poi (or 1.16, 95% ci: 1.07–1.38) (table 2). using mantel–haenszel’s test for trend, the likelihood of being diagnosed with poi increases with the severity of the growth restriction (p < 0.001, data not shown). large for gestational age and risk of primary ovarian insufficiency being born lga was not associated with the risk of developing poi (table 2). discussion this study found an association between being born with non-optimal birth characteristics and risk of poi later in life. being born in sga, preterm, or lbw was associated with a somewhat increased likelihood of being diagnosed with poi. table 1. socio-demographic and birth characteristics of the study population by the presence of primary ovarian insufficiency (primary ovarian insufficiency, n ¼ 18,627). no primary ovarian insufficiency primary ovarian insufficiency n % n % p-value year of birth <0.001 1973–1975 235,118 23.2 7977 42.8 1978–1982 218,071 21.5 5050 27.1 1983–1987 226,642 22.3 3225 17.3 1988–1993 335,420 33.0 2375 12.8 education <0.001 elementary 67,560 6.8 991 5.4 high school 442,870 44.9 6632 35.8 university 476,811 48.3 10,889 58.8 missing 28,010 2.8 115 0.6 gestational age <0.001 very preterm 4968 0.5 96 0.5 preterm 45,996 4.5 864 4.6 term 871,826 85.9 15,652 84.0 post term 92,461 9.1 2015 10.8 preterm birth 0.407 no 964,287 95.0 17,667 94.8 yes 50,964 5.0 960 5.2 birthweight <0.001 very low birthweight 4655 0.5 91 0.5 low birthweight 38,557 3.8 783 4.2 normal birthweight 972,039 95.7 17,753 95.3 low birthweight <0.001 no 972,039 95.7 17,753 95.3 yes 43,212 4.3 874 4.7 small for gestational age (1sd) <0.001 no 808,140 79.6 14,354 77.1 yes 207,111 20.4 4273 22.9 small for gestational age (2sd) <0.001 no 977,074 96.2 17,781 95.5 yes 38,177 3.8 846 4.5 small for gestational age (3sd) 0.006 no 1,009,542 99.4 18,494 99.3 yes 5709 0.6 133 0.7 large for gestational age (1sd) <0.001 no 878,038 86.5 16,374 87.9 yes 137,213 13.5 2253 12.1 large for gestational age (2sd) 0.076 no 987,461 97.3 18,157 97.5 yes 27,790 2.7 470 2.1 large for gestational age (3sd) 0.436 no 1,010,548 99.5 18,548 99.6 yes 4703 0.5 79 0.4 delivery method 0.057 normal 859,936 84.7 15,896 85.3 instrumental 52,227 5.1 915 4.9 section 103,088 10.2 1816 9.7 upsala journal of medical sciences 237 to our knowledge, there are no published studies investigating the relation between non-optimal birth characteristics and risk of poi. however, an association of being born lbw or sga with the risk of infertility has been shown (19,20). also, women born with low birth weight have been found to be younger when reaching natural menopause (21). the reason for this is unknown, but intrauterine growth restriction has been related to smaller internal genitalia, reduced ovulation rate, and elevated fsh levels in women (8,22,23). the increased risk of poi among individuals born sga and lbw may be due to the influence of under-nutrition during the intrauterine life, which in turn affects the development or survival of the follicles. this might lead to dysfunction of the follicles or lower numbers of follicles, resulting in an increased likelihood of developing poi later in life. preterm birth might not affect the follicles since the follicles usually develop normally if no under-nutrition is present. however, potential explanations for the associations between non-optimal birth characteristics (sga, lbw, and preterm) and poi is that being born with these characteristics increases the risk of several autoimmune diseases, many of which in turn are associated with poi, for example, primary adrenal failure (addison’s disease), autoimmune thyroid disease (schmidt’s syndrome), and type 1 diabetes (24,25). it is thus possible that non-optimal birth characteristics lead to an increased risk of autoimmune disease, ending in autoimmune poi. poi results in premature cessation of ovulation and infertility. this can lead to involuntary childlessness if it occurs among women who have not yet considered becoming pregnant, especially since women increasingly postpone childbearing. since there is no medical therapy today that is proven to improve the ovarian function and fertility for women with poi, fertility preservation is the only solution if the women want children later in life (26). this must be done before the follicles are depleted, and it may be too late at the time of diagnosis. it is therefore of great importance to identify and follow women with a high risk of poi so that an early diagnosis can be made before it is too late with fertility preservation. it should be emphasized, however, that women with poi may still become pregnant. this study has a methodological strength in using a national population-based design, the high validity and completeness of the databases, the large size of the study cohort, and the long follow-up of the study participants. a limitation is a fact that there probably are women with amenorrhoea who have not been diagnosed with poi, making it likely that we underreported the prevalence in our study. the missing of the outcome is, however, not dependent on the birth characteristics, which means that this issue would dilute associations rather than cause them. moreover, by using poi identified not only from the national patient register but also from the swedish prescribed drug register, we found additional women and reduced the figure missing the diagnosis. it should be emphasized, however, that some of the poi cases identified by means of the swedish prescribed drug register maybe women prescribed hormonal therapy due to causes other than poi, such as in conjunction with the treatment of infertility. another limitation is the lack of some potential confounding factors for which we did not have data, for example, body mass index, smoking or alcohol consumption, or year of diagnosis in the swedish prescribed drug register. however, these factors probably do not cause poi (5) but may affect the timing of the amenorrhoea. we did, however, adjust for some other factors, that is, educational level, and year of birth, which might be more relevant to control for. still, the findings in the current study should be interpreted with caution since it is possible that some of the associations found could be affected by factors not included in the analyses. hence, further studies are needed to validate and to deepen the knowledge on the causes of being diagnosed with poi. in conclusion, this population-based and nationwide swedish cohort study show that being born with non-optimal birth characteristics was associated with a slightly increased risk of poi. these findings not only provide new knowledge of the origin of poi but can also help identify women at an increased risk of poi, who should be informed about the risks and the consequences of poi. an implication is that women born sga may be advised not to postpone their pregnancies too long. disclosure statement no potential conflict of interest was reported by the author(s). table 2. crude and adjusted odds ratios and corresponding 95% confidence intervals (cis) of primary ovarian insufficiency among infants born with different characteristics. crude or 95% ci p-value adjusted ora 95% ci p-value premature delivery (<37 gestational weeks) 1.03 0.96–1.12 0.407 1.11 1.04–1.19 0.001 premature delivery, excluding sga 1.00 0.93–1.07 0.997 1.08 1.01–1.16 0.037 low birthweight (<2500 g) 1.11 1.03–1.19 0.004 1.15 1.07–1.23 <0.001 low birthweight, excluding sga 1.02 0.92–1.12 0.715 1.10 1.00–1.22 0.053 small for gestational age (1sd) 1.16 1.12–1.20 <0.001 1.05 1.02–1.09 0.004 small for gestational age (2sd) 1.22 1.14–1.31 <0.001 1.10 1.02–1.18 0.009 small for gestational age (3sd) 1.27 1.07–1.51 0.006 1.16 1.07–1.38 0.095 large for gestational age (1sd) 0.88 0.84–0.92 <0.001 0.95 0.91–1.00 0.036 large for gestational age (2sd) 0.92 0.84–1.01 0.076 1.00 0.91–1.09 0.908 large for gestational age (3sd) 0.92 0.73–1.14 0.436 0.96 0.76–1.20 0.707 sga, excluding pt and lbw 1.22 1.11–1.35 <0.001 1.01 0.91–1.12 0.841 preterm, excluding sga and lbw 0.98 0.89–1.08 0.663 1.05 0.95–1.15 0.321 lbw, excluding sga and preterm 0.97 0.75–1.24 0.803 1.03 0.80–1.32 0.843 aadjusted for year of birth and educational level. 238 g. sydsjö et al. notes on contributors gunilla sydsj€o is a certified psychotherapist/ma behavioural scientist. phd and professor in psychosocial obstetrics and gynaecology, department of obstetrics and gynaecology, department of biomedical and clinical sciences, link€oping university, link€oping, sweden. marie bladh, ma applied statistics. phd and research assistant/postdoc, department of obstetrics and gynaecology, department of biomedical and clinical sciences, link€oping university, link€oping, sweden. katarina rindeborn, md, department of obstetrics and gynaecology, department of biomedical and clinical sciences, link€oping university, link€oping, sweden. mats hammar, md and phd, senior professor in obstetrics and gynaecology, department of obstetrics and gynaecology, department of biomedical and clinical sciences, link€oping university, link€oping, sweden. heriberto rodriguez-martinez, dvm, msc, phd, european specialist ecar, professor in veterinary medicine reproductive biotechnology-slu 1991, professor in reproductive biology-liu 2010, senior professor, department of biomedical and clinical sciences (bkv), division of children & women health (bkh), obstetrics & gynaecology, faculty of medicine and health sciences, link€oping university, link€oping, sweden. elizabeth nedstrand, md, phd senior consultant, associate professor in obstetrics and gynaecology, department of obstetrics and gynaecology, department of biomedical and clinical sciences, link€oping university, link€oping, sweden. orcid gunilla sydsj€o http://orcid.org/0000-0003-4296-4038 references 1. sydsjo g. long-term consequences of non-optimal birth characteristics. am j reprod immunol. 2011;66:81–7. 2. m€annist€o t, v€a€ar€asm€aki m, sipola-lepp€anen m, tikanm€aki m, matinolli hm, pesonen ak, et al. independent living and romantic relations among young adults born preterm. pediatrics. 2015;135: 290–7. 3. dekeyser n, josefsson a, bladh m, carstensen j, finnstr€om o, sydsj€o g. premature birth and low birthweight are associated with a lower rate of reproduction in adulthood: a swedish populationbased registry study. hum reprod. 2012;27:1170–8. 4. saigal s, day kl, van lieshout rj, schmidt la, morrison km, boyle mh. health, wealth, social integration, and sexuality of extremely low-birth-weight prematurely born adults in the fourth decade of life. jama pediatr. 2016;170:678–86. 5. steiner az, d’aloisio aa, deroo la, sandler dp, baird dd. association of intrauterine and early-life exposures with age at menopause in the sister study. am j epidemiol. 2010;172:140–8. 6. tom se, cooper r, kuh d, guralnik jm, hardy r, power c. fetal environment and early age at natural menopause in a british birth cohort study. hum reprod. 2010;25:791–8. 7. richardson mc, guo m, fauser bc, macklon ns. environmental and developmental origins of ovarian reserve. hum reprod update. 2014;20:353–69. 8. ibanez l, potau n, ferrer a, rodriguez-hierro f, marcos mv, de zegher f. reduced ovulation rate 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clin endocrinol metab. 2006;91:1723–8. 14. cnattingius s, ericson a, gunnarskog j, k€all�en b. a quality study of a medical birth registry. scand j soc med. 1990;18:143–8. 15. ludvigsson jf, andersson e, ekbom a, feychting m, kim jl, reuterwall c, et al. external review and validation of the swedish national inpatient register. bmc public health. 2011;11:450. 16. lagergren k, derogar m. validation of oesophageal cancer surgery data in the swedish patient registry. acta oncol. 2012;51:65–8. 17. falkeborn m, persson i, naessen t, kressner u. validity of information on gynecological operations in the swedish in-patient registry. scand j soc med. 1995;23:220–4. 18. national board of health and welfare, centre for epidemiology [internet]. kvalitetsdeklaration, statistik om l€akemedel år 2017 [quality declaration, statistics of drugs 2017]. stockholm (sweden): national board of health and welfare; 2015 (cited 2020 february 10). swedish; available from: https://www.socialstyrelsen.se/globalassets/sharepoint-dokument/artikelkatalog/statistik/2018-4-7-kvalitetsdeklaration.pdf 19. vikstrom j, hammar m, josefsson a, bladh m, sydsj€o g. birth characteristics in a clinical sample of women seeking infertility treatment: a case-control study. bmj open. 2014;4:e004197. 20. ekholm k, carstensen j, finnstrom o, bladh m, sydsj€o g. the probability of giving birth among women who were born preterm or with impaired fetal growth: a swedish population-based registry study. am j epidemiol. 2005;161:725–33. 21. bjelland ek, gran jm, hofvind s, eskild a. the association of birthweight with age at natural menopause: a population study of women in norway. int j epidemiol. 2019. doi:10.1093/ije/dyz207 22. ibanez l, valls c, cols m, ferrer a, marcos mv, de zegher f. hypersecretion of fsh in infant boys and girls born small for gestational age. j clin endocrinol metab. 2002;87:1986–8. 23. ibanez l, potau n, enriquez g, de zegher f. reduced uterine and ovarian size in adolescent girls born small for gestational age. pediatr res. 2000;47:575–7. 24. sadrzadeh s, painter rc, van kasteren ym, braat ddm, lambalk cb. premature ovarian insufficiency and perinatal parameters: a retrospective case-control study. maturitas. 2017;96:72–6. 25. goswami d, conway gs. premature ovarian failure. hum reprod update. 2005;11:391–410. 26. baker v. life plans and family-building options for women with primary ovarian insufficiency. semin reprod med. 2011;29:362–72. upsala journal of medical sciences 239 https://www.socialstyrelsen.se/globalassets/sharepoint-dokument/artikelkatalog/statistik/2018-4-7-kvalitetsdeklaration.pdf https://www.socialstyrelsen.se/globalassets/sharepoint-dokument/artikelkatalog/statistik/2018-4-7-kvalitetsdeklaration.pdf https://www.socialstyrelsen.se/globalassets/sharepoint-dokument/artikelkatalog/statistik/2018-4-7-kvalitetsdeklaration.pdf https://doi.org/10.1093/ije/dyz207 abstract introduction material and methods study design exposure variables and definitions statistical analysis results preterm delivery and risk of primary ovarian insufficiency low birth weight and risk of primary ovarian insufficiency small for gestational age and risk of primary ovarian insufficiency large for gestational age and risk of primary ovarian insufficiency discussion disclosure statement references what animals can teach us about evolution, the human genome, and human disease review article what animals can teach us about evolution, the human genome, and human disease kerstin lindblad-toha,b adepartment for medical biochemistry and microbiology, uppsala university, uppsala, sweden; bbroad institute of mit and harvard, cambridge, ma, usa abstract during the past 20 years, since i started as a postdoc, the world of genetics and genomics has changed dramatically. my main research goal throughout my career has been to understand human disease genetics, and i have developed comparative genomics and comparative genetics to generate resources and tools for understanding human disease. through comparative genomics i have worked to sequence enough mammals to understand the functional potential of each base in the human genome as well as chosen vertebrates to study the evolutionary changes that have given many species their key traits. through comparative genetics, i have developed the dog as a model for human disease, characterising the genome itself and determining a list of germ-line loci and somatic mutations causing complex diseases and cancer in the dog. pulling all these findings and resources together opens new doors for understanding genome evolution, the genetics of complex traits and cancer in man and his best friend. article history received 22 january 2020 accepted 23 january 2020 keywords canine genetics; comparative genomics; genome sequencing; human genetics introduction human disease—early studies human diseases can largely be divided into infectious diseases and genetic diseases. in many cases diseases arise as a result of both genetic predisposition and environmental factors. in the early years, diseases dependent on a single gene were analysed with laborious methods using large families to see if simple sequence length polymorphism (sslps) markers segregated with disease. these single gene diseases included for example cystic fibrosis (cftr) (1) and huntington’s disease (it15) (2), while down’s syndrome was found to depend on an extra chromosome (trisomy 21) (3) leading to a more complex phenotype related to many genes. more common complex diseases such as diabetes, schizophrenia, and rheumatoid arthritis were far too complex to understand. on top of this, cancer was postulated to have both inherited mutations and mutations arising in the tumour (including the knudsen two-hit hypothesis) (4), making the tumour become increasingly malignant (figure 1). tools have transformed genetics over the past 20 years enormous changes related to genome sequencing and gene mapping have occurred, mostly as collaborative efforts striving to develop new technologies, large-scale resources, and computational approaches. as i will discuss below, while dna was described more than half a century ago, the understanding of how genetic diseases arise is still incomplete, but the field makes continuous progress every day. many of the tools and analysis methods, and some of the knowledge amassed, are also already being used to understand the biology of disease. over time, this will allow more detailed diagnosis with better treatment options, and—in the long-term—personalised medicine. comparative genomics sequencing the human genome the human genome has long been a source of wonder, with only a partial understanding of how it works, despite enormous efforts to generate different types of data. after the discovery of dna in the 1950s, more active research on the human genome and the genes and regulatory elements hidden therein took a long time to follow. in the beginning, studies often focussed on a single gene for a single disease. several types of maps relying on different types of markers (sslps and radiation hybrid markers) allowed the mapping of monogenic diseases such as cystic fibrosis and huntington’s disease. it was recognised that the world of disease genetics would greatly improve if the whole human genome could be sequenced. for much of the 1990s, more than 2,800 researchers in a world-wide consortium worked on sequencing the human genome, with different regions or contact kerstin lindblad-toh kersli@broadinstitute.org, kerstin.lindblad-toh@imbim.uu.se department for medical biochemistry and microbiology, uppsala university, bmc, husargatan 3, 752 37, uppsala, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 1, 1–9 https://doi.org/10.1080/03009734.2020.1722298 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1722298&domain=pdf&date_stamp=2020-02-19 http://orcid.org/0000-0001-8338-0253 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1722298 http://www.tandfonline.com chromosomes divided between labs. this public effort, using sanger sequencing (5) led to the first human draft genome (6), but also coincided with the publishing of a different human genome (7), which used a novel approach: wholegenome shotgun sequencing, where segments are randomly sequenced and then put together as a giant puzzle. both genome assemblies covered most of the genome, but struggled with more complex regions such as complex gene families, difficult repeats, and centromeres and telomeres. thus, the human genome project continued to fill in gaps for an additional 5 years or so. while the human genome was declared finished in 2003 (8), there still remained gaps that were unfilled. in the last couple of years using novel long-read technologies, it has finally become possible to sequence complex regions of the genome such as segmental duplications and repeats, for example in centromeric regions (9), thus enabling new and complete assemblies of chromosomes spanning also repeat regions, from telomere to telomere (10). prior to the human genome sequence, common lore had it that the human genome contained �100,000 protein-coding genes. when first analysed, the human genome was found to contain �50% repetitive sequences, for many years thought of as ‘junk dna’ (11). in the rest of the genome, scientists struggled to identify the protein-coding genes that must be there. they used available rna expression data and known protein coding sequence. this allowed them to extrapolate their findings to �40,000 protein-coding genes, assuming that there were still genes they could not find. difficult regions included complex gene families (such as olfactory receptors) or genes with an especially gcrich sequence. mouse and rat genomes the second mammalian genome to be sequenced was the mouse (12). the mouse is the best laboratory animal model for human disease, as it is small and easy to manipulate in captivity, and hence it was deemed of high importance to generate a reference sequence for it. for the reference the c57bl/6j strain was used, despite the fact that multiple strains exist and are used for different disease studies. intriguingly, the haplotype structure of the mouse genome (as determined by whole-genome sequencing multiple strains) was quite blocky (long stretches of sequences were inherited together when looking at multiple strains), and when multiple strains were analysed and compared to the two founder mice, mus musculus domesticus and mus musculus musculus, it was seen that most laboratory strains were hybrids between those two founder strains (13). this finding agreed with the fact that early mice were used as pet mice based on different phenotypes such as colouring or ‘dancing’ mice in japan and china (m. musculus domesticus) and europe (m. musculus musculus) (14). in addition to studying the genetics in different strains, mice have been used both with knock-out mutations and transgenes. in the current era of crispr editing (15), the analysis of mutations in mice has become even easier. rats are similar to mice as laboratory animals, but their larger size makes them more expensive to house, while their physiology is more similar to humans. the brown norway rat genome was sequenced and published in 2004 (16). following this, the rat has been widely used to map complex traits by a combination of sequencing and mapping strategies. for example, one study of outbred rats identified 355 quantitative trait loci for 122 phenotypes including anxiety, heart disease, and multiple sclerosis (17). dog genome the dog, man’s best friend, was the fifth mammal to be sequenced. at 2.4 gb the dog genome is somewhat smaller than the human genome, based on a lower amount of lineage-specific repeat sequences (334 mb versus 609 mb, respectively) (18). previously, the coding-gene count in mammalian genomes had not been precisely determined, but when using conserved synteny between four mammals— human, mouse, rat, and dog—we could revise the number of mammalian genes to �20,000. this number varies slightly between species, primarily based on lineage-specific gene figure 1. professor kerstin lindblad-toh, winner of the medical faculty of uppsala university rudbeck award 2019, ‘for her excellent research in comparative genomics and for developing the dog as a model for biomedical research’. 2 k. lindblad-toh family expansions and contractions, but almost 14,000 genes are 1:1:1 orthologs across human, mouse, and dog (18). much research has gone into trying to understand the early history of dogs and wolves. different studies propose different times and places for the domestication. while the answer is still out there, it would seem logical if wolves domesticated themselves in multiple places and at different times (10,000–40,000 years ago) (19). while studies are ongoing to try to understand the correlation and adaptations making dogs into dogs, one clear genetic event is the duplication of the amylase (amy2b) gene (20), which has only one pair of copies in the wolf, while most dogs have many, roughly five, pairs of copies. this gene is important for the digestion of starch and can be coupled to the changing diet involving considerably more starch in agrarian societies. the fact that dogs living in the arctic do not have the additional amy2b copies is likely due to their living on a meat-rich diet (21). monodelphis genome and vertebrate evolution following the sequencing of a number of useful placental mammals, we moved outside the placental mammals and sequenced the first marsupial — opossum (monodelphis domesticata) (22). the perhaps most intriguing finding when comparing the opossum to placental mammals was that innovations in protein-coding regions are relatively rare, while 20% of eutherian conserved non-coding elements (cnes) are recent innovations. a substantial proportion of these eutherian-specific cnes have arisen from sequences inserted by transposable elements (repeats), pointing to transposons as a major creative force in the evolution of mammalian gene regulation. the chicken red jungle fowl (gallus gallus), being an important food source, was the first bird to be sequenced and published in 2004 (23). the chicken genome is roughly 1 gb in size, roughly one-third of the human genome, which correlates with a lower number of repeat elements and segmental duplications. in addition to 6 pairs of macrochromosomes, and 1 pair of sex chromosomes (the female is the heterogametic sex), chickens also have 32 pairs of intermediate or microchromosomes. microchromosomes are small, gcrich and gene rich. the analysis of the chicken genome in multiple populations has allowed the identification of both morphological traits (24) and traits related to egg and meat production (25) anolis carolinensis was the first lizard to be sequenced (26). lizards, like chickens, have substantial numbers of microchromosomes, and they both rely on eggs for reproduction. the evolution of the amniotic egg was one of the great evolutionary innovations in the history of life, freeing vertebrates from an obligatory connection to water, and thus permitting the conquest of terrestrial environments. a. carolinensis microchromosomes are highly syntenic with chicken microchromosomes, yet they do not exhibit the high gc and low repeat content that are characteristic of avian microchromosomes. also, a. carolinensis mobile elements are very young and diverse—more so than in any other sequenced amniote genome — which possibly has allowed the novel innovations underlying the rapid radiation of 400 lizard species. these species have radiated, often convergently, into a variety of ecological niches with attendant morphological adaptations, providing one of the best examples of adaptive radiation (27). despite the sequencing of a large number of land-living creatures, it was still a mystery how the first creature crawled onto land. curiously enough, a rarely seen fish, called the coelacanth (latimeria chalumnae), and living in the deep ocean, for example off the east coast of africa, was reported to have four lobe-finned limbs similar to many land-living vertebrates. based on material from a stranded coelacanth, we sequenced the coelacanth genome (28), together with the transcriptome of the lung fish. the lung fish also has four limbs, but as it has an extremely large genome (estimated at 40–100 gb: http://www.genomesize.com), containing a lot of transposable elements (29), we could not afford to sequence it at that time. careful analysis of the genes in coelacanth and lung fish showed that the lung fish was more closely related to land-living animals, supporting the primitive notion that both lungs and legs are a great advantage on land. overall modification of gene-regulatory elements may underlie a significant proportion of phenotypic changes on animal lineages. to investigate the gain of regulatory elements throughout vertebrate evolution we identified a genome-wide set of putative regulatory regions for five vertebrates, including human, and looked for signs of gains. in early vertebrate times regulatory gains occurred frequently near transcription factors and developmental genes, but this trend was then replaced by innovations near extra-cellular signalling genes, and finally, in the last 100 million years (during the mammalian radiation), innovations near posttranslational protein modifiers (30). this suggests that the complexity of regulation and function of protein-coding genes have increased continuously (figure 2). stickleback, cichlids, and herring—good examples of environmental adaptations sticklebacks are small fish that were originally marine. they have colonised and adapted to thousands of streams and lakes formed since the last ice age in north america and europe (31). typical changes of the freshwater adaptations included body shape, length, depth, fin position, spine length, eye size, and armour plate number. an early study generating a genome sequence also involved the sequencing of 20 individuals from locations spanning across both freshwater and saline environments globally (32). the study identified 90 genomic regions that consistently varied between fresh and salt water. we also noted the re-use of globally shared standing genetic variation, including chromosomal inversions, to allow for repeated evolution of distinct marine and freshwater sticklebacks. a later study showed similar patterns of adaptation to salinity for the herring, a common food source in scandinavia, spanning between the brackish baltic and the salty northern upsala journal of medical sciences 3 http://www.genomesize.com atlantic ocean (33). the genome sequence complemented with whole-genome sequencing of many populations identified >500 regions related to adaptations to brackish water. further analysis also identified >100 loci that varied between spring and fall breeding populations (34). these studies also suggest that the adaptations can depend on both proteincoding and regulatory adaptations, and that haplotype blocks spanning multiple genes are selected, suggesting that multiple variants in a region might underlie genomic adaptations. the tilapia, present in the nile, again a common food source, was used as a backbone for the analysis of the adaptations present in hundreds of cichlid species in the african lakes of victoria, malawi, and tanganyika. analysis of four fish from the eastern lineage showed gene duplications, an abundance of non-coding element divergence, accelerated coding sequence evolution, expression divergence associated with transposable element insertions, and regulation by novel micrornas compared to the tilapia and other teleost genomes (35). later studies have also identified strong selection on colour schemes and morphology related to where in the lakes the different species live. deeper sequencing of five lake malawi species followed by genotyping in a diverse collection of �160 species from across africa identified �200 genic and non-genic snps varying across species. we observed segregating polymorphisms outside of the malawi figure 2. vertebrate genome sequencing projects shed light on genome evolution, domestication, and adaptation. many of the first vertebrate whole-genome projects represented model species (e.g. mouse and rat), but over time, additional resources representing natural model species have been added. highlighted in this tree are some of the studies that have been undertaken, within and across lineages, to study the processes of natural adaptation (marked a; for example, stickleback adaptation to extreme aquatic environments), domestication (marked d; for example, genetic signatures separating domestic dogs and wolves), and genome evolution (marked ge; for example, exaptation changes in a regulatory sequence function between human and monodelphis). as well as indicating the genetic distances between representative vertebrate species, this tree also illustrates the time periods when novel regulatory innovations arose. in particular, regulatory elements near transcription factors (red box) and developmental genes (yellow box) evolved quickly in early vertebrate history, followed by cell communication (green box) and protein modification (blue box) in the more recent past. as whole-genome sequencing becomes substantially cheaper and more accessible, the expansion of reference genomes within each clade is set to increase, with the publication of 200 mammals, 300 birds, and more than 100 fish expected by the close of 2020. image adapted with permission from meadows & lindblad-toh, nature review genetics (63). 4 k. lindblad-toh lineage for more than 50% of these loci, suggesting that river cichlids have transported polymorphisms between lakes (36). mammals help annotate the human genome in 2008, sequencing was still relatively expensive, but the whole-genome shotgun approach enabled the use of lowcoverage genome sequencing of mammalian genomes to better understand the human genome. in 2011, we published a paper including 2� sequencing (whole-genome shotgun sequencing where random sequences are generated so that each base in the genome is sequenced on average 2fold) of 18 mammals added to the 11 existing (sanger sequenced at 7�) mammals (37). this project allowed the identification of evolutionary constraint of 12-bp elements, resulting in the identification of >3 million constraint elements, encompassing 4.2% of the genome. the protein-coding sequence only covers �1% of the genome, thus suggesting a flood of novel candidate regulatory elements. the data also allowed us to look at synonymous constraint elements where regulatory elements overlap coding sequence, constraint patterns in promoters, and accelerated regions in humans and primates—hallmarks of positive selection for human adaptations. recent work has shown that human accelerated elements encompass regulatory elements such as well conserved enhancers for developmental genes (38). all is not protein-coding genes in addition to protein-coding genes (1%) other regulatory entities encompass at least three times as much space. these regions include non-coding rna transcripts, such as thousands of long intergenic non-coding rnas (lincrnas) (39) and micrornas (mirnas) (40). lincrnas are rna molecules larger than 200 nucleotides and are more or less conserved across species, presumably varying in the strength of function. still, they have a widespread role in gene regulation and other cellular processes including cell-cycle regulation, apoptosis, and establishment of cell identity (41). mirnas are short (20 to 24 nucleotides), non-coding rna molecules composed of a single-stranded sequence. they predominantly act as negative regulators of gene expression (40), but are functionally involved in virtually all physiologic processes, including differentiation and proliferation, metabolism, hemostasis, apoptosis, and inflammation. to better catalogue the regulatory landscape, the encyclopaedia of dna elements (encode) project (https:// www.encodeproject.org) was formed to map functional noncoding elements. initially, chip-seq was performed to detect the location of individual transcription factor binding sites in many tissues. over time, this analysis has expanded to look at differential methylation and acetylation of genomic bases and their binding proteins. recently, assay for transposaseaccessible chromatin using sequencing (atac-seq) (42) has been developed to detect open chromatin. moreover, the 3 c technology has been developed into hic (43), which can detect topologically associating domains (tads) allowing scientists to infer what portions of the genome are within specific regulatory regions in specific tissues (44). in addition, the gtex effort (https://www.gtexportal.org/home/) works to couple genome variation, such as single nucleotide polymorphisms (snps), to differences in gene expression. more than 240 mammals for single base resolution constraint in mammalian genomes as illumina sequencing became affordable, we put together a project with the goal of detecting human single base evolutionary constraint using >240 mammalian genomes (45). of these, 131 genomes were generated by us using discovar-de novo (46), combined with the 110 mammalian genomes in ncbi in march 2017. as this data set is analysed it will allow the study of: 1) the largest eutherian nuclear genome phylogeny; 2) the ability to perform genotype–phenotype correlations across many mammalian species; 3) the evolution of genome structure; 4) reference genomes that can be utilised for species conservation; and, finally, 5) a detailed map of evolutionary constraint, which can be used with human genome-wide association (gwas) catalogues and other species data sets to investigate patterns of constraint in disease-associated regions in any of the 241 genomes. the data set will also make possible the study of accelerated regions under positive selection in any of the sequenced mammalian genomes. comparative genetics complexity of human disease genetics after the generation of the human genome, lots of effort went into detecting variation in the human genome. snps, indels, and larger structural variations were discovered, and snps were put into genotyping panels to allow for genomewide association studies (gwas) to map disease loci. the initial thoughts were that common diseases were caused by common variants. although tens of thousands of patients and controls have been genotyped for many complex traits, the loci identified have only accounted for a fraction of the heritability of the disease. as an example, 113,075 controls and 36,989 cases with schizophrenia have identified 108 genome-wide significantly associated loci (47). these loci are estimated to explain 30–50% of the heritability for schizophrenia. there could be multiple reasons for why only a smaller portion of the heritability has been detected, such as the need of larger samples sizes, environmental factors, epigenetics or a bigger proportion of rare variants. for some diseases such as autism, where individuals with the disease reproduce less frequently, the fraction of novel mutations or rare variants of high effect may be larger (48). to detect individual rare variants, much larger sample sizes are needed; however, methods of burden testing of specific gene regions or pathways will enable the summing of multiple mutations. while currently the gene plus a unified flanking region is involved in the analysis, tad domains and gtex data should upsala journal of medical sciences 5 https://www.encodeproject.org https://www.encodeproject.org https://www.gtexportal.org/home/ be helpful in performing burden tests on regions distinctly important to the gene. the dog as a model for human disease the strongest reason for sequencing the canine genome was to harness the genetics related to the enormous diversity among breeds (49). pet dogs are special because they share our environment, and also share the same diseases as humans, including autoimmune disease, neurological disease, cardiovascular disease, and cancer. in fact, roughly 30% of dogs get cancer, which is similar to the frequency observed in man (50). on top of this, there has been very strong selection for morphological traits and behaviour, suggesting that rare variants with strong effect may have become more common in certain breeds, leading to disease. the bottlenecks at breed creation may also have allowed drift to make some alleles much more common in some breeds. the recent creation of breeds (in the last 200 years) also means that haplotype blocks are long within breed and short across breeds. this allows for a mapping strategy where first high-risk breeds are used to find the rough locations of disease mutation (by case control gwas), and then other breeds are added in to fine-map the region to find the functional mutation (figure 3) (18). based on this, monogenic traits can be mapped with �20 cases and 20 controls, while complex traits, such as cancers, can be mapped with a few hundred cases and controls. in humans, many thousands of patients and controls are needed. many diseases such as osteosarcoma (51), canine systemic lupus erythematosus (52), and canine compulsive disorder (ccd) have now been identified and, in several cases, been translated to the corresponding human disease. obsessive-compulsive disorders (ocd) shares a common aetiology between dogs and humans ccd shows strong clinical similarities with human ocd; both species perform certain normal behaviour in excess and often repetitively. to investigate the genetic causes of ccd, we first performed gwas in 92 cases and 68 controls of the doberman pinscher breed, identifying a single genome-wide significance locus (53). this locus was near the cadherin 2 (chd2) gene, for which the protein is located in the synapse. secondly, careful reanalysis of the data identified multiple regions of suggestive association as well as regions of fixation in the doberman pinscher breed. thirdly, we performed targeted resequencing of all these regions and identified a number of genes with increased numbers of mutations in cases versus controls (54). many of these genes were active gwas: >170,000 markers (a) (b) (c) (d) ½ m b1m b 10 -1 00 kb fine-mapping: many markers in region dog human 10 -1 00 kb gwas: >1 million markers genome-wide tall short trait white coat color size complex disease 20+20 50+50 >100+>100 1 locus (2 mutations) 10 loci* >10 loci* cases + control number of loci trait – size complex disease – >10,000+10,000 100,000+100,000 – >100 loci >100 loci cases + control number of loci figure 3. genome-wide association (gwas) is easier in dogs than in humans. monogenic traits in dogs can be mapped with fewer snp markers and fewer individuals than in humans. gwas in dogs will utilise the long linkage disequilibrium (ld) within dog breeds, followed by fine-mapping in multiple breeds with the same phenotype (panel a). in humans the ld is short, requiring the use of a lot of snp markers already in the gwas step (panel b). the number of snp markers required for different types of traits in dogs is lower, as is the number of loci contributing to each trait in dogs (panel c), while in humans most traits are more complex and require more samples (panel d). 6 k. lindblad-toh in the synapse. finally, we used the genes and pathways found in dogs, combined them with known functionally important ocd genes from humans and mice, and used the combined gene set to perform targeted sequencing of human ocd cases and controls (55). altogether, we analysed 608 genes in 592 cases and 560 controls and identified four genes as strongly associated (one genome-wide). two of these genes, nrxn1 and htr2a, were enriched for protein-coding mutations in cases, while two genes, cttnbp2 (synapse maintenance) and reep3 (vesicle trafficking), had only regulatory mutations in this study. this might suggest that these two proteins with regulatory mutations have such critical functions that they cannot tolerate coding mutations. now larger gwas studies are being performed in humans, and it will be interesting to see if the link to ccd will be further strengthened. taking the next step sequencing technologies change the way we can analyse most species on earth as the cost of long-read sequencing technologies is finally coming down for generating a high-quality genome (and the generation of population data can be cheaply generated by short-reads), the ability to generate genomes from many species changes dramatically. however, one of the challenges still remaining is access to high-quality dna samples, which is necessary for generating a reference genome with longread sequencing, and also for samples from a sufficient number of individuals to allow the generation of population data from different regions of the world. multiple zoos (i.e. the san diego frozen zoo) have collected samples and cell lines which are potentially useful for generating genome sequences or studying variation, but also potentially for in vitro reproduction for endangered species. one such example is the southern white rhino which is close to extinction, with only two individuals still alive and neither of them able to carry a pregnancy. attempts will now be made for a northern white rhino to be a surrogate mother (56). about a year ago the earth biogenome project (https:// www.earthbiogenome.org) (57) started with the enormous aim of generating high-quality genomes for each of 10–15 million eukaryotic species on earth in the next 10 years. to accomplish this, almost every step of the procedure needs to be scaled up: sample collection, sequencing and assembly, annotation, and standardised analysis, as well as species-specific analysis. on top of this comes the generation and analysis of population data and transcriptomics for annotation of genomes. although this is still a moonshot, we are getting closer. importantly, to save diversity of life on earth, sequencing must be combined with more practical conservation efforts such as protection of habitats and inhibition of poaching. mammalian constraint and its use for understanding disease in many mammals as mentioned earlier, most gwas loci fall outside proteincoding regions of the genome, thus requiring the use of various ways to annotate single variants for the likelihood that they are a causative mutation for disease. several techniques are being developed to address this crucial issue. the 200-mammals-data reaches single base constraint for all bases in the human genome, allowing a triage of each position’s likelihood to be functional without any relationship to in which tissues the affected gene is located. as novel methods such as massively parallel reporter assays and genome editing at large scale (58) become possible, it will allow the comparison of overall evolutionary functional constraints to that of functionality in individual tissues. to add to this, additional annotations of all types of transcripts in many healthy and disease tissues as well as many types of functional annotations (both experimental like chipseq, regulome and hic, or bioinformatic hidden markov models [hmms] (59) and machine learning methodologies) will aid our understanding of how normal and diseased tissues are affected by each gene/mutation. to more clearly understand the tissue-specific effects, single cell sequencing (60) has also become more frequent and can decipher cells in, for example, the immune system and brain, where a large number of different cell types live in close proximity and symbiosis. cultured organelles and spatial transcriptomics (61) in key tissues allow further dissection of transcriptomics and functional effects. utilising the canine model for clinical trials the clinical similarity between disease in dogs and humans has been studied for many years. based on multiple gwas data sets, tumour mutations and expression data have shown also a molecular similarity between dogs and humans in many diseases. based on the shorter life-span in dogs compared to humans, the outcome from clinical trials is likely to be informative more quickly. mouse models on the other hand, may give rapid results, but are often induced and are rarely spontaneous. already, trials for canine als (https://vhc.missouri.edu/small-animal-hospital/neurologyneurosurgery/current-clinical-trials/) and multiple cancers are underway (https://trials.vet.tufts.edu/clinical-trials/?fwp_species=dog&fwp_veterinary_specialties=oncology). also, the analysis of cell free dna (cfdna) and circulating tumour dna (ctdna) for monitoring disease progression in liquid biopsies (62) in the dog should be informative. conclusion during the past two decades, the understanding of vertebrate evolution as well as of the human genome, and consequently human disease, has expanded at an exceptional pace. we have increased our understanding of evolutionary principles and the content of the human genome. loci associated with a specific human disease can be in the hundreds, detected with tens of thousands of individuals, yet explaining only a fraction of the disease risk. thus, we have still only scraped the surface when it comes to understanding human disease. upsala journal of medical sciences 7 https://www.earthbiogenome.org https://www.earthbiogenome.org https://vhc.missouri.edu/small-animal-hospital/neurology-neurosurgery/current-clinical-trials/ https://vhc.missouri.edu/small-animal-hospital/neurology-neurosurgery/current-clinical-trials/ https://trials.vet.tufts.edu/clinical-trials/?fwp_species=dog&fwp_veterinary_specialties=oncology https://trials.vet.tufts.edu/clinical-trials/?fwp_species=dog&fwp_veterinary_specialties=oncology the next decade is likely to generate exponentially more data to help protect endangered species by having both a reference genome and population data. it will also increase the understanding of the human genome, including noncoding mutations and rare variants. this will require both an understanding of every base in the human genome as well as large sample sizes to fully map human disease. increasing use of pet dogs for disease gene identification as well as for clinical trials is likely to help propel the biological understanding into canine and human personalised medicine. acknowledgements i am indebted to all the people in my groups in sweden and the united states, as well as a large number of collaborators around the world for their hard work on these projects. this science would not have been possible without you! i thank elisabeth sundstr€om, ann-catherine lindblad, and sue wincent-dodd for useful commentary and proof-reading of the manuscript and kai siang toh for help with illustrations. disclosure statement no potential conflict of interest was reported by the author(s). funding financial support has been received from the knut and alice wallenberg foundation, nih, cancerfonden, and formas. the author is a distinguished professor at the swedish research council. notes on contributor professor lindblad-toh received her phd in human genetics at the karolinska institute. after a short postdoc at the whitehead institute/mit genome institute, she went on to lead many vertebrate genome projects, as well as canine disease projects. she was one of the founders of scilifelab. she is 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obsessive-compulsive disorders (ocd) shares a common aetiology between dogs and humans taking the next step sequencing technologies change the way we can analyse most species on earth mammalian constraint and its use for understanding disease in many mammals utilising the canine model for clinical trials conclusion acknowledgements disclosure statement references ujms109_2.pdf upsala j med sci 109: 123–130, 2004 a twostage surgery for severe femoral neck deformity due to fibrous dysplasia: a case report satsuki onoda, masahito hatori, norikazu yamada, masami hosaka, shoichi kokubun department of orthopaedic, surgery tohoku university school of medicine, 1-1 seiryomachi, aobaku, sendai, japan 980-8574 abstract a free full-text copy of this article can be found at the web page of upsala j med sci: http://www.ujms.se various kinds of surgical treatments have been reported for varus deformity of the proximal femur due to fibrous dysplasia. we report a case of two-stage corrective osteotomy for severe varus-retroversion deformity of the femoral neck due to monostotic fibrous dysplasia. the patient was an 18 year-old man. on initial examination, the spina malleollar distance was 88 cm on the right side and 83 cm on the left. plain radiography showed prominent varus deformity of his left proximal femur. the morphology was 130 degrees on the right side and 85 degrees on the left. computed tomography revealed 60 degree retroversion of the femoral neck. a two-stage surgery was performed, consisting of curettage and bone grafting followed by corrective osteotomy 16 months later. a 55 degree valgus osteotomy was performed in the subtrochanteric region. after osteotomy and 40 degree internal rotation of the shaft, a 130 degree angle plate was used for osteosynthesis. postoperative radiological examination showed a morphology of 140 degrees and computed tomography revealed a 20 degree retroversion of the femoral neck. no recurrence or varus deformity was seen at four years after surgery. although the leg length discrepancy was 2.5 cm, the patient had no difficulty in one foot standing and no restriction of adl (activity of daily living). the well-known progressive varus shepherd’s crook deformity in the polyostotic form of fibrous dysphasia is associated with limb shortening, limping, and occasionally chronic fatigue fractures with disabling pain (1). various kinds of surgical treatments have been reported for this type of varus deformity (1–5). curettage and bone grafting is one of the most common and simple treatments. however, this method often gives bad results as the grafted bones are absorbed and that the progress 123 received 13 january 2004 accepted 29 january 2004 key words: monostotic fibrous dysplasia, osteotomy, two-stage surgery. of varus deformity van not be prevented (6). we report a case of two-stage corrective osteotomy for severe varus-retroversion deformity of the femoral neck due to monostotic fibrous dysplasia. case report the patient was an 18 year-old man. his chief complaint was left coxalgia. in august 1996, he noticed pain in his left buttock and thigh. in february 1997, he fell to the ground and hit his buttock. he was hospitalized with the diagnosis of left femur trochanteric pathological fracture. he underwent conservative treatment. he was referred to our clinic because of pain when walking. on initial examination, the spina-malleollar distance was 88 cm on the right side and 83 cm on the left with a leg length discrepancy of 5 cm. his left trochanter protruded laterally and the left lower extremity was externally rotated. plain radiography demonstrated a 5cm × 5.5 cm sized radiolucent lesion with ground glass appearance and marginal sclerosis in the left femoral trochanteric region. the femoral calcar was partially interrupted with a very thin cortex. plain radiography showed prominent varus deformity of his left proximal femur. the morphology was 130 degrees on the right side and 85 124 fig. 1. plain radiography showing a 5cm x 5.5cm sized radiolucent lesion with ground glass appearance and marginal sclerosis in the left femoral trochanteric region. 125 fig. 2. three dimensional ct visualizing varus and retroversion of the femoral neck. (a: anterior view, b: lateral view, c: posterior view). a b c 126 fig. 3. microphograph of the lesion showing cellular spindle cell proliferation with woven bones. fig. 4. plain radiograph after surgery showing morphology of 140 degrees. degrees on the left (fig. 1). computed tomography (ct) revealed a 60 degree retroversion of the femoral neck. three-dimensional ct visualized varus and retroversion of the femoral neck (fig. 2). mri showed low signal intensities from the femoral head to the lesser trochanter on t1 weighted images and lobulous high signal intensities on t2 weighted fat suppression images. bone scintigraphy showed increased abnormal uptake only in the left trochanteric region. the histological diagnosis of the open biopsy specimen was fibrous dysplasia (fig. 3). the patient was diagnosed with monostotic fibrous dysplasia occurring in the femoral trochanteric region. operative treatment was chosen because of walking difficulty due to the severe hip deformity, leg length discrepancy and high risk of pathological fracture. a two-stage surgery was performed, consisting of curettage and bone grafting followed by corrective osteotomy after 16 months. a 55 degree valgus osteotomy was performed in the subtrochanteric region. after osteotomy and 40 degree internal rotation of the shaft, a 130 degree angle plate was used for osteosynthesis. postoperative radiological examination showed a morphology of 140 degrees and computed tomography revealed a 20 degree retroversion of the femoral neck (fig. 4). no recurrence or varus deformity was seen at four years after surgery (fig. 5). although the leg length discrepancy was 2.5cm, he had no difficulty in one foot standing and no restriction of adl (activity of daily living). 127 fig. 5. plain radiography showing no recurrence or varus deformity at four years after surgery. discussion pathological fractures and deformities are common in patients with fibrous dysplasia. the dysplastic bone is biomechanically unsound, and fatigue fractures often occur even with normal stresses. fractures that result from fibrous dysplasia are often managed non-operatively, as it is thought that these fractures usually heal without difficulty(1, 6, 7). treatments for lesions in the proximal part of the femur, however, are troublesome. symptomatic involvement of the proximal part of the femur frequently requires surgical intervention to achieve relief of pain and resumption of unrestricted activity. the forces of weight and muscle pull on the mechanically weakened bone in this area, producing an unrelenting propensity for fracture and varus deformity. recurrent fractures and relentless progression of the varus deformity in spite of aggressive orthopedic treatment are characteristic involvements in this disease(1, 8, 9). curettage and bone grafting have been the mainstay of treatment for symptomatic and even asymptomatic lesions of fibrous dysplasia. nakashima et al. reported that six of their eight patients who had lesions of the femoral neck were managed successfully with curettage and bone grafting (3). stephenson et al. noted better results after curettage and bone-grafting in patients who were eighteen years or older (7). however, enneking et al. described that the results of curettage and autogenous cancellous bone-grafting were unsatisfactory, since the grafts were incorporated quickly and eventually were replaced by dysplastic tissue (1). however, they reported good results after fibula graft without curettage. guille et al. reported that curettage and cancellous or cortical bone grafting did not appear to have any advantage compared with osteotomy alone in the treatment of symptomatic lesions of fibrous dysplasia (6). in the present case, the varus deformity was so advanced that fibular grafting could not be chosen. a two stage surgery consisting of curettage and bone grafting followed by corrective osteotomy was chosen because of the severity of the varus deformity, and we obtained good functional results. one of the possible reasons for the poor results after correction of varus deformity is thought to be the high recurrence rate in cases of polyostotic fibrous dysplasia. the present case was a monostotic fibrous dysplasia, which has a lower recurrence rate as compared with the polyostotic type. we allowed 16 months to confirm that there was no recurrence of the lesion after curettage and bone grafting, and then, corrective osteotomy was carried out. we believe the patient’s young age and good stability of the osteotomy design also contributed to good results. references 1. enneking wf, gearen pf(1986). fibrous dysplasia of the femoral neck. treatment by cortical bone-grafting. j bone joint surg am 68: 1415–22. 2. freeman bh, bray ew, meyer lc (1987). multiple osteotomies with zickel nail fixation for polyostotic fibrous dysplasia involving the proximal part of the femur. j bone joint surg am 69: 691–8. 3. nakashima y, kotoura y, nagashima t, yamamuro t, hamashima y(1984). monostotic fibrous dysplasia in the femoral neck. a clinicopathologic study. clin orthop 191: 242–8. 128 4. nagda tv, singh h, kandoi m, samant a, patel br (1997). two stage reconstruction for the shepherd's crook deformity in a case of polyostotic fibrous dysplasia. j postgrad med 43: 83–4. 5. tsuchiya h, tomita k, matsumoto t, watanabe s (1995). shepherd’s crook deformity with an intracapsular femoral neck fracture in fibrous dysplasia. clin orthop 310: 160–4. 6. guille jt, kumar sj, macewen gd (1998). fibrous dysplasia of the proximal part of the femur. long-term results of curettage and bone-grafting and mechanical realignment. j bone joint surg am 80: 648–58. 7. stephenson rb, london md, hankin fm, kaufer h (1987). fibrous dysplasia. an analysis of options for treatment. j bone joint surg am 69: 400–9. 8. keijser lc, van tienen tg, schreuder hw, lemmens ja, pruszczynski m, veth rp (2001). fibrous dysplasia of bone: management and outcome of 20 cases. j surg oncol 76: 157–66. 9. ozaki t, sugihara m, nakatsuka y, kawai a, inoue h (1996). polyostotic fibrous dysplasia. a long-term follow up of 8 patients. int orthop 20: 227–32. corresponding author: assistant professor masahito hatori department of orthopaedic, surgery tohoku university school of medicine, 1-1 seiryomachi, aobaku, sendai, japan 980-8574 tel: 81-22-717-7242, fax: 81-22-717-7248 mhato@mail.tains.tohoku.ac.jp 129 tf-iups160007 73..76 biographical item from phylogeny into ontogeny with claes hellerstr€om kjell asplund riksstroke, medicine, department of public health and clinical medicine, umeå university, umeå, sweden my research career started with snakes. for a year, from 1965 to 1966, claes hellerstr€om and i worked together in herpetology (the branch of zoology that deals with amphibians, including frogs, toads, and reptiles). we joined forces with an sas pilot, captain florin, who, between the flights to los angeles, was chairman of the uppsala herpetological society. in his spare time, he collected rattlesnakes in the californian deserts, transported them in the cockpit of his plane, and delivered them to us. our second supplier was a herpetologist located close to arlanda airport. vipers on demand—as we needed snakes, he went out into the forest and collected them for us. why snakes? in histology textbooks from the 1950s, the pancreatic islets were described as being composed of three cell types: alpha (a), beta (b), and delta (d). it had long been known that the beta cells produce insulin. although a substance with hyperglycemic properties—glucagon—had been detected in pancreatic extracts as early as 1923 (1), it was not until the early 1950s that it was discovered that alpha cells produce glucagon (2). in 1960, claes hellerstr€om and his phd supervisor bo hellman described a silver impregnation technique that made it possible to distinguish between two types of alpha cells, which they termed a1 and a2 cells (3,4). they made the case that the a2 cells were the glucagon producers. the question then remained: what is the function of the a1 cells? this is where phylogenetic studies, including those on reptiles, came into the picture. the a1 cell enigma silver-positive a1 cells were found to be relatively uncommon in mammals; beta cells outnumbered other islet cell types by far. hellman and hellerstr€om had therefore been conducting some of their early silver impregnations in pancreatic islets of chickens and ducks, where alpha cells were more abundant (3). however, even in birds only a minority of the alpha cell population was silver-positive (a1 cells). was it possible they could be more prevalent in animals earlier on the evolutionary chain? in claes hellerstr€om’s phd thesis from 1963, he included not only classical histological studies (on the two types of alpha cells) but also a method to isolate pancreatic islets from mice, a procedure that paved the way to cell physiology studies—a more progressive and successful route (5) compared to traditional histological studies. nevertheless, hellerstr€om recruited me in 1964 as one of his first two research aspirants (the other aspirant is now editor of this journal); i was to help him go back into phylogeny. hellerstr€om, together with his supervisor bo hellman, had already plunged into herpetology. in 1962, they published a paper on the pancreatic islets of bullfrogs (6). now that hellerstr€om was on his own, he took on snakes and turtles. if he could find a species where a1 cells were particularly common, he could get a glimpse of what was the function of these mysterious cells. he could perhaps even isolate them and study their function in vitro. when we dissected the rattlesnakes and the vipers, we were somewhat surprised to see how easy it was to find our orientation. the location of organs was similar to that of mammals, except in a more elongated, giacometti-like style (though i had not encountered giacometti’s art at that time). the second remarkable observation was that we did not need a microscope to see a pancreatic islet. there was a separate, millimeter-sized organ, next to the spleen, a megastar compared to islets in other species. in the microscope, we saw islets dispersed throughout the pancreas but, as shown in figure 1, they appeared to accumulate both in number and in size in the caudal direction, culminating in one or two extremely large islets next to the spleen (7). it seemed that this caudal movement had difficulties in stopping; in the spleen, devoid of surrounding exocrine pancreatic tissue, typical pancreatic islets were also present (figure 2). the hypothesis that a1 cells are more abundant in snakes than in mammals was thereby confirmed (figure 1). half of the islet cell population were alpha cells, most of which were silver-positive, i.e. a1 cells (7). in stark contrast to islets in mammals, only a minority of the islet cells in snakes were beta cells. staining for granules, we found distinct granulation in both types of a cells. when we studied fresh squash preparations of microdissected islet tissue or frozen pancreatic sections in dark-field illumination, the islet cells exhibited a distinct luminescence (7). taken together, it seemed that the a1 cells were producing a hormone different from insulin and glucagon. what was it? at the time, in 1966, we simply did not have the tools available to proceed to explore the a1 cell enigma. also, our contact professor kjell asplund kjellasplund1@gmail.se riksstroke, medicine, department of public health and clinical medicine, umeå university, se-90185 umeå, sweden � 2016 the author(s). published by taylor & francis. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons. org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited upsala journal of medical sciences, 2016 vol. 121, no. 2, 1–000 http://dx.doi.org/10.3109/03009734.2016.1152332 enthusiasm for snakes had started to wane as an epidemic of salmonella struck the animal quarters in the basement of our institution. the culprits proved to be the rattlesnakes. then, captain florin was bitten by his puff-adder, hovered between life and death, and was never able to fly again. this put a decisive end to our interest in snakes. immunohistochemistry provided the answer: somatostatin nearly a decade passed. histology had become an academic discipline that by the end of the 1960s seemed to have entered a scientific cul-de-sac. claes hellerstr€om had arrived at this conclusion early on; he turned from cell morphology to cell physiology. then came immunohistochemistry. it had been conceptualized back in the 1940s. but it was not until the 1970s— when the techniques to produce a wide range of specific antibodies became more readily available—that immunohistochemistry became a frontline technique. it vitalized, even revolutionized, histology. it provided scientists with the tools to reveal the secrets of cells with unknown or unclear functions throughout the body, including a1 cells in pancreatic islets. researchers at karolinska institute found somatostatincontaining cells in a number of endocrine tissues. in 1975, in collaboration with claes hellerstr€om, they published an article in which they reported that a1 cells contained somatostatin (8). the a1 cell enigma was thereby solved. in the mid-1980s, a canadian immunohistochemist demonstrated that somatostatin was present in the pancreatic islets of snakes as well (9). reflections on our phylogeny studies half a century has now passed since we published our snake paper. upon re-reading it, a few spontaneous thoughts come to mind. first, even though both a1 cells and snakes turned out to be scientific dead ends, claes hellerstr€om found the ideal beginner’s project for a researcher-to-be like me. the snakes were thrilling, the project was well delineated and perfectly feasible (the results were published within one-and-a-half years), hellerstr€om gave me a well-defined role in the project, and we made some original (although not very sustainable) observations. second, we published our article in zeitschrift f€ur zellforschung und mikroskopische anatomie. in the mid-1960s, a journal with a grandiose german name was still prestigious. at the time our paper was published, traditional histology remained tainted by its german cultural heritage. in uppsala, student textbooks in anatomy were in german. but zeitschrift f€ur zellforschung und mikroskopische anatomie had started to adapt to the cultural shift in medical sciences and published articles not only in german but also in english (ours was in english). it survived by changing its name to cell and tissue research journal. third, it was fascinating to be part of a soon-to-be extinct species of scientists—those involved in the upfront morphology of pancreatic islets. in our 1966 article, we cited a number of papers dating all the way back to the nineteenth century. when hellerstr€om found out that that vincenzo diamare, who had published a comprehensive study on the comparative morphology of pancreatic islets in 1899 (10), was still alive, we dedicated our paper to him on his 95th birthday. just in time, as it turned out. diamare died later in 1966 (11). despite the fascinating structure of snake islets and the ease with which endocrine tissue can be isolated—it can be done with the naked eye—very few original articles on snake islets have been published in recent decades (only one since the year 2000, according to the web of science). our paper was last quoted in 1992. figure 1. silver impregnation (hellman–hellerstr€om technique) of the pancreas of a rattlesnake. a large islet containing numerous blackened al cells is located close to the spleen. reprinted from reference 6, hellman and hellerstr€om, with permission from springer. figure 2. islet from a viper imbedded in the spleen (aldehyde-fuchsin trichrome staining). the insulin-producing beta cells are stained dark. note the lack of surrounding pancreatic acinar cells and the localization of beta cells in the periphery of the islet. �400 in the original publication. reprinted from reference 6, hellman and hellerstr€om, with permission from springer. 74 k. asplund a fourth note is that our findings on argyrophilic a1 cells were ephemeral. the 1975 immunocytochemistry article on somatostatin described above, with hellerstr€om as a co-author (8), solved a problem that had confused us when we were working with the snakes. when hellman and hellerstr€om first identified silver-positive a1 cells, they described them as being distinctly different from d cells (3,4). we apparently confirmed this in snakes, where we found d cells to be agranular, hinting that this cell type did not produce hormone. as immunohistochemistry came along, it became apparent that a1 cells and d cells were identical—both contained somatostatin. since then, our knowledge about the pancreatic islets as an endocrine organ has grown. not only are there alpha, beta, and delta cells, but another distinct cell type that produces pancreatic polypeptide has also been added to the list (12). whether or not there even are epsilon cells in the adult human pancreas (if so, producing ghrelin) seems to be controversial (13). but the a1 cells have remained unheard of since 1980 (and they do not represent the only dead end in my long publication list). interestingly, lars grimelius, an uppsala pathologist, worked on another silver impregnation method during the 1960s. in pancreatic islets, his method apparently impregnated glucagon-producing a2 cells instead of a1 cells (14), which is somewhat of a paradox. as the grimelius method was then found also to be useful for staining neuroendocrine tissue in other organs, it became routine in histopathology. grimelius’s original description of the method was published in the upsala journal of medical sciences (at the time acta societatis medicorum upsaliensis) and is, by far, the most cited article ever published in this journal (15). into ontogeny during the early 1960s, claes hellerstr€om’s interest in phylogeny was paralleled by his curiosity in ontogeny. together with others in the uppsala group, he published studies on the morphology of pancreatic islets in human fetuses (16) and in neonatal rats (17,18). during the same period, claes hellerstr€om’s interest turned from morphology to function. it is telling that the same year that our morphological paper on the pancreatic islets of snakes was published (1966), hellerstr€om published his first article on the metabolism of isolated mouse islets (19). he saw the limitations of traditional histology and the great potential in the emerging new cell physiology. the turn from phylogeny to ontogeny and from morphology to function was a strategic decision, which soon proved to be rewarding. so, ontogeny and function—how to combine these new research directions? hellerstr€om was a certified physician and sought applications for his research that were more practical than snakes could offer. in the 1960s, pregnancy was often seen as a dreaded complication for women with diabetes. there was a considerable mortality among pregnant mothers, there was a high fetal and neonatal mortality, malformations were common, and delivery was complicated by the excessive size of the babies. early after birth, infants born to mothers with diabetes were prone to develop hypoglycemia. this situation raised a number of basic questions. how do insulin biosynthesis and release mechanisms mature during fetal life? is functional maturation affected by the mother’s hyperglycemia? could experimental studies in the offspring of pregnant rats also provide some insights into how diabetic pregnancy should be managed to avoid harm to the fetus and the newborn? this became the themes of my phd studies under claes hellerstr€om’s tutorship. now that i read those papers half a century later, i am less sure that hellerstr€om (and certainly not i) had these clinically very important questions in mind when the project started. judging from what we wrote in our introduction and discussion sections, we were entirely driven by curiosity in the beginning. as the studies progressed, a shift can be discerned with more and more focus on clinical problems and clinical lessons. by the time i left uppsala in 1972, we had found evidence that, in rats, the mechanisms for insulin biosynthesis and release normally do not mature until after birth (20–24). this would provide protection not only against excessive fetal growth in diabetic pregnancy but also against postnatal hypoglycemia in the infant. in poorly regulated diabetes during pregnancy, fetal exposure to hyperglycemia causes early maturation of the insulin biosynthesis and release mechanisms with high insulin levels in the fetus. we concluded that this would increase the risk of fetal macrosomia (and ensuing complications during delivery) and a high risk of hypoglycemia early after birth. reflections on our ontogeny studies the clinical lesson appeared to be simple: to protect against premature functional development of the pancreatic islets, strict metabolic control must be maintained during diabetic pregnancy. this is self-evident in today’s clinical practice, but such was not the case in the early 1970s. instead, a common strategy was to advise young women with diabetes to avoid becoming pregnant. our observations were certainly not the major contributor to the shift in the perception of how diabetic pregnancy should be managed, but they were one of many pieces of evidence that promoted a shift in the therapeutic paradigm. when i left the department of histology for clinical practice and research, hellerstr€om recruited ulf eriksson to study experimental diabetic pregnancy in more detail; this is described in eriksson’s contribution to this volume (25). a lesson for me on a more personal level concerned claes hellerstr€om’s tutoring style. i was the first phd student to graduate under his leadership. he must have had an intuitive gift for scientific leadership, maintaining a careful balance between giving the student an increasing level of independence and the need for scientific rigor. revisiting my earliest publications, it is telling that i was the sole author of several of them. this illustrates not only the shift in publication practice that has occurred since then, but also hellerstr€om’s modesty and generosity as a tutor. upsala journal of medical sciences 75 i did not fully realize the quality of my early scientific training under claes hellerstr€om’s leadership until i turned to clinical research. in the clinic, there was an older generation of researchers whose scientific training mostly had been learning by doing. but, like me, several of my young colleagues in the university hospital had a phd in preclinical experimental research. we clearly benefited from the elementary scientific training that we had obtained; we came to represent a new generation of clinical researchers. a final reflection is that our work with pancreatic islets from fetal and newborn rats signified yet another foresighted shift in claes hellerstr€om’s scientific focus—from normal to pathological states. in the last work i did under his tutorship, we induced intermittent hyperglycemia in pregnant rats and studied the effects on the fetuses (24). hellerstr€om soon went on to study an abundance of different animal models of diabetes. summing up claes hellerstr€om’s years of transition thus, the years i worked with claes hellerstr€om in the 1960s and early 1970s were transitional, both in scientific paradigms and for hellerstr€om as a researcher. we experienced the end of a long era of german-oriented morphological research. hellerstr€om took a strategic decision to shift from morphological to functional studies and from traditional histology to cell biology. he also abandoned his old interest in phylogeny and focused on ontogeny. and he made a gradual transition from studying normal to pathological states. it was a privilege to accompany him on a small part of his innovative scientific journey, now some 50 years ago. disclosure statement the author report no conflicts of interest. the author alone is responsible for the content and writing of the paper. references 1. kimball c, murlin j. aqueous extracts of pancreas iii. some precipitation reactions of insulin. j biol chem 1923;58:337–58. 2. ferner h. the aand b-cells of the pancreatic islets as sources of the antagonistic hormones glucagon and insulin; the shift of the ab-relation in diabetes mellitus. am j digest dis 1953;20:301–6. 3. hellman b, hellerstr€om c. the islets of langerhans in ducks and chickens with special reference to the argyrophil reaction. z zellforsch mikroskop anat 1960;52:278–90. 4. hellman b, hellerstr€om c. the specificity of the argyrophil reaction in the islets of langerhans in man. acta endocrin 1961;36:22–30. 5. welsh m. ups j med sci. 2016;121:77-80. 6. hellman b, hellerstr€om c. cellular composition of the islets of langerhans in the bullfrog, rana catesbiana. acta anat 1962;48:149–55. 7. hellerstr€om c, asplund k. the two types of a-cells in the pancreatic islets of snakes. z zellforsch mikroskop anat 1966;70:68–80. 8. h€okfelt t, efendic s, hellerstr€om c, johansson o, luft r, arimura a. cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special references to the a1-cells of the pancreatic islets and to the hypothalamus. acta endocrin suppl 1975;200:5–41. 9. buchan amj. an immunocytochemical study of endocrine pancreas of snakes. cell tissue res 1984;235:657–61. 10. diamare v. studii comparativi sulle isole di langerhans del pancreas. mschr anat pshysiol (leipzig) 1899;16:155–209. 11. de girolamo a. in memory of vincenzo diamare. anat anzeiger 1966;119:109–18. 12. brereton mf, vergari e, zhang q, clark a. alpha-, deltaand ppcells: are they the architectural cornerstones of islet structure and co-ordination? j histochemistry cytochem 2015;63:575–91. 13. raghay k, gallego r, scoazec jy, garcia-caballero t, morel g. different ghrelin localisation in adult human and rat endocrine pancreas. cell tissue res 2013;352:487–94. 14. grimelius l. a silver nitrate stain for alpha-2 cells in human pancreatic islets. acta soc med upsaliensis 1968;73:243–70. 15. westermark p. lars grimelius and his silver impregnation method commentaries on the paper in upsala journal of medical sciences with the highest number of citations. upsala j med sci. 2015;120:113–16. 16. bj€orkman n, hellerstr€om c, hellman b, petersson b. the cell types in the endocrine pancreas of the human fetus. z zellforsch mikroskop anat 1966;72:425–45. 17. angervall l, hellerstr€om c, hellman b. development of the endocrine pancreas of rats as manifested by the appearance of argyrophilia and granulation. pathol microbiol 1962;25:389–99. 18. hellman b, hellerstr€om c, petersson b. postnatal growth of the endocrine and exocrine parts of the rat pancreas. its relationship to the metabolism of dna. diabetes 1961;10:470–5. 19. hellerstr€om c. oxygen consumption of isolated pancreatic islets of mice studied with the cartesian-diver micro-gasometer. biochem j 1966;98:7c–9c. 20. asplund k. dynamics of insulin release from the foetal and neonatal rat pancreas. europ j clin invest 1973;3:336–44. 21. asplund k. effects of glucose on insulin biosynthesis in foetal and newborn rats. hormone metab res 1973;5:410–15. 22. asplund k, hellerstr€om c. glucose metabolism of pancreatic islets isolated from newborn rats. hormone metab res 1972;4:159–63. 23. asplund k. effects of postnatal feeding on the functional maturation of the pancreatic islet b-cells of neonatal rats. diabetologia 1972;8:152–9. 24. asplund k. effects of intermittent glucose infusions in pregnant rats on the functional development of the foetal pancreatic b-cells. j endocrinol 1973;59:285–92. 25. eriksson u. ups j med sci. 2016;121:92-112. 76 k. asplund from phylogeny into ontogeny with claes hellerstr&ouml;m the a1 cell enigma immunohistochemistry provided the answer: somatostatin reflections on our phylogeny studies into ontogeny reflections on our ontogeny studies summing up claes hellerstr&ouml;m&rsquo;s years of transition disclosure statement references tf-iups160010 113..119 review article glucagon secretion from pancreatic a-cells linford brianta, albert salehib, elisa vergaria, quan zhanga and patrik rorsmana,b aoxford centre for diabetes, endocrinology and metabolism, radcliffe department of medicine, university of oxford, oxford, uk; bmetabolic research, department of physiology, institute of neuroscience and physiology, university of g€oteborg, g€oteborg, sweden abstract type 2 diabetes involves a m�enage �a trois of impaired glucose regulation of pancreatic hormone release: in addition to impaired glucose-induced insulin secretion, the release of the hyperglycaemic hormone glucagon becomes dysregulated; these last-mentioned defects exacerbate the metabolic consequences of hypoinsulinaemia and are compounded further by hypersecretion of somatostatin (which inhibits both insulin and glucagon secretion). glucagon secretion has been proposed to be regulated by either intrinsic or paracrine mechanisms, but their relative significance and the conditions under which they operate are debated. importantly, the paracrine and intrinsic modes of regulation are not mutually exclusive; they could operate in parallel to control glucagon secretion. here we have applied mathematical modelling of a-cell electrical activity as a novel means of dissecting the processes that underlie metabolic regulation of glucagon secretion. our analyses indicate that basal hypersecretion of somatostatin and/or increased activity of somatostatin receptors may explain the loss of adequate counterregulation under hypoglycaemic conditions, as well as the physiologically inappropriate stimulation of glucagon secretion during hyperglycaemia seen in diabetic patients. we therefore advocate studying the interaction of the paracrine and intrinsic mechanisms; unifying these processes may give a more complete picture of the regulation of glucagon secretion from a-cells than studying the individual parts. article history received 3 february 2016 accepted 16 february 2016 keywords diabetes; electrophysiology; experimental diabetes; glucagon; intrinsic mechanisms; pancreatic alpha-cells; paracrine introduction when banting and best (1) injected extracts from pancreatic tissue intravenously or subcutaneously into diabetic dogs and humans, they observed a marked reduction in blood sugar (reflecting the hypoglycaemic action of insulin). however, this was often preceded by a small transient hyperglycaemia, which was initially attributed to epinephrine. a few years later, this phenomenon was suggested to be due to a glucose-mobilizing substance, later named ‘glucagon’ (2). it was another 30 years before the pancreatic a-cells were identified as being the source of glucagon (3), with hypoglycaemia demonstrated as triggering the release of this hormone (4,5). glucagon is part of a homeostatic hormonal system developed to protect against serious decreases in blood glucose— glucose ‘counter-regulation’. this mechanism is the combination of processes that act to protect against the development of hypoglycaemia and (should this occur) restore normoglycaemia. hypoglycaemia suppresses insulin secretion from bcells and stimulates glucagon secretion from islet a-cells, normalizing blood glucose levels. even small changes in glucagon can greatly increase blood glucose; the addition of minimal doses of glucagon (0.50 ng/kg/min) is known to induce longlasting hyperglycaemia (6). glucagon acts exclusively on the liver, where it stimulates both glycogenolysis (the breakdown of glycogen into glucose) and gluconeogenesis (the formation of new glucose molecules), increasing glucose output within minutes. under certain conditions, glucagon can also stimulate production of ketone bodies in the liver, which during fasting or prolonged hypoglycaemia may substitute partially for glucose in meeting the brain’s energy needs. hormones secreted from pancreatic islet cells play central roles in the whole-body glucose homeostasis. the dysfunction of these endocrine cells contributes to both type 1 and type 2 diabetes mellitus (7), with type 2 diabetes mellitus (t2dm) accounting for 90% of the total diabetes incidence (8). t2dm is a major and increasing health problem (9) with a complex aetiology. both genetic and environmental factors are known to be involved in the disease (10). there is an excessive production of glucose by the liver, which contributes to the fasting hyperglycaemia that is characteristic of this disease. this pathological response is commonly attributed to a combination of insulin resistance and a failure of pancreatic b-cells to release insulin as required (11). however, it is becoming increasingly apparent that impaired glucagon secretion also contributes to the excessive hepatic glucose production in t2dm, exacerbating episodes of hyperglycaemia (12–14). impaired secretion of glucagon in t2dm shows itself in a twofold manner: as well as there being too much glucagon secretion during hyperglycaemia, there is also too little glucagon released to normalize hypoglycaemia (15). this compromised physiological and behavioural response to falling blood glucose concentrations is arguably the more fatal arm of impaired glycaemic control in t2dm, with hypoglycaemia being a major cause of mortality in insulin-treated patients (16). contact linford briant linford.briant@ocdem.ox.ac.uk oxford centre for diabetes, endocrinology and metabolism, radcliffe department of medicine, university of oxford, oxford ox3 7lj, uk this article was originally published with errors. this version has been corrected. please see erratum(http://dx.doi.org/10.3109/03009734.2016.1177959). � 2016 informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited upsala journal of medical sciences, 2016 vol. 121, no. 2, 113–119 http://dx.doi.org/10.3109/03009734.2016.1156789 http://dx.doi.org/10.3109/03009734.2016.1177959 whereas the mechanisms by which glucose regulates insulin secretion from the b-cells have been well established (17–19), factors regulating glucagon secretion from a-cells in response to glucose belong to the most contested aspects of islet cell biology (20–22). the heart of the question is whether glucagon secretion is regulated by an intrinsic mechanism (within the a-cells) or by factors released from other cells within the islets (paracrine mechanisms). in this review, we summarize the intrinsic and paracrine mechanisms contributing to the glucose control of glucagon secretion in pancreatic a-cells. we also attempt to combine the intrinsic and paracrine mechanisms using mathematical models to address the possible cause of impaired glucagon secretion seen in diabetes. paracrine regulation of glucagon secretion since glucose stimulates insulin release, and insulin inhibits glucagon secretion, it is inviting to think that inhibition of glucagon secretion from a-cells is secondary to stimulation of the b-cells. a similar argument could be made for somatostatin, the release of which is also stimulated by glucose. the argument that paracrine factors influence glucagon secretion is supported by reports that isolated a-cells (that no longer have paracrine input) are unable to respond appropriately (i.e. decreasing their activity and glucagon secretion) to increased glucose concentrations; in fact, the reports demonstrate that glucose stimulates glucagon secretion when a-cells are removed from their normal paracrine environment (23,24). insulin was the first paracrine factor from b-cells to provide evidence for this inhibitory action (25). gaba is also released from b-cells (26), and some studies have demonstrated it can inhibit glucagon secretion from a-cells by activation of the gaba(a) receptor (27). zn2þ (co-released with insulin) has been suggested to have an important role in glucagon secretion (28,29), but this has been questioned (30). indeed, in mice where the zn2þ granule transporter is knocked out, there was no alteration in the regulation of glucagon secretion by glucose (31). other studies refute the centrality of the control of glucagon secretion by insulin; they demonstrate that single (isolated) a-cells do respond to high glucose by lowering [ca2þ]i and decreasing glucagon secretion (32,33). furthermore, in 5 mm glucose, glucagon secretion is maximally inhibited in mouse islets—a concentration not associated with any change in insulin secretion (22,34). in fact, insulin actually stimulated glucagon secretion in islets exposed to 6 mm glucose. this may explain the paradoxical stimulation of glucagon secretion that occurs in line with increasing insulin secretion (for glucose concentrations �6 mm in mouse islets). therefore, the inhibition of glucagon secretion in <6 mm glucose is not due to insulin secretion. somatostatin (sst) is a potent inhibitor of insulin and glucagon secretion. it has been proposed to be a paracrine regulator of glucagon secretion (35) with an important role for inhibiting glucagon secretion during hyperglycaemia (36). a-cells in islets express somatostatin receptor 2 (sstr2) (37). glucagon secretion is increased in islets in which the sstr2 is knocked out, highlighting sst as a mediator of the glucose inhibition of glucagon secretion (38). sst exerts its inhibitory effect at the level of membrane potential and cell exocytosis. upon binding to the sstr2, sst activates a g-protein coupled inwardly rectifying kþ channel (girk), which repolarizes the cell membrane and inhibits the firing of action potentials (39). this effect in membrane potential is transient, probably due to the desensitization of the receptors. sst also inhibits a-cell exocytosis by effectively decreasing the intracellular camp level (37,40). with increasing glucose concentrations in the range 0–7 mm, sst release increases in parallel with the decrease in glucagon secretion (22,34). therefore, it may be argued that inhibition of glucagon secretion is caused by sst signalling. however, glucose retains an inhibitory influence on glucagon secretion in the presence of cyn154806, an sstr2-specific blocker (34), suggesting that sst does not regulate glucagon secretion (41). furthermore, a number of different laboratories have demonstrated that blocking the sst signalling pathway in a-cells— either by blocking sst receptors (42) or the associated g-protein cascade (43)—increases glucagon secretion but does not prevent inhibition of glucagon release by glucose. in particular, sst does inhibit glucagon secretion at 0–7 mm glucose, but there is an sst-independent pathway that inhibits glucagon secretion in a-cells. taken together, these studies indicate that glucose exerts regulation of glucagon secretion independently of the paracrine factors, via intrinsic mechanisms. interestingly, the ability of the incretin hormone glp-1 to inhibit glucagon secretion in the perfused rat pancreas was abolished after immunoneutralization of somatostatin, whilst the effect of glucose was unaffected (44), indicating that the significance of paracrine and intrinsic mechanisms in the control of glucagon secretion is variable. intrinsic regulation of glucagon secretion pancreatic a-cells, like b-cells, are electrically excitable. at low concentrations of glucose, when the secretion of glucagon is stimulated, a-cells fire continuous overshooting action potentials (aps) (23,27,45). voltage-gated naþ channels (nav channels) and ca2þ channels contribute to the upstroke of aps (46,47). the discharge of high-voltage aps opens voltagegated ca2þ channels (vgccs) to allow influx of extracellular ca2þ into the cytosol. this results in an elevation of the intracellular ca2þ concentration and provides ca2þ signals that trigger glucagon granule exocytosis—a process that makes hormone-containing granules fuse to the cell membrane to release their cargo. pharmacological studies have confirmed that there are at least four different vgccs (t, l, n, and p/qtype ca2þ channels) expressed in the a-cells (46,48,49). among them, the p/q-type ca2þ channel has been identified as the main vgcc that is involved in glucagon secretion evoked by low glucose despite its relatively low contribution in the transmembrane ca2þ current (�30%) (46). the l-type ca2þ channel, although contributing the majority of ca2þ currents (>50%), plays a minor role in the glucagon secretion under these conditions (50,51). this suggests that there is a 114 l. briant et al. tight coupling of the p/q-type ca2þ channels and the exocytotic machinery of the a-cells—which is likely to be compartmentalized. such arrangement avoids the spill-over of ca2þ from other vgccs and enables glucagon secretion be regulated by modulation of p/q-type ca2þ channel activity. when the circulating glucose level rises, glucagon secretion is suppressed. this is likely to be via the reduction of p/ q-type ca2þ channel activity in a-cells. such inhibitory effect can be achieved by lowering the amplitude or the firing frequency of aps, by influencing membrane depolarization or repolarization, respectively (figure 1). the change of membrane potential is a result of glucose metabolism or transport (via electrogenic sodium-glucose co-transporter 2 transporters (52) as discussed below), which leads to the alteration of membrane ion conductance. a-cells are equipped with atpsensitive kþ channels (katp channels) of the same molecular identity as in b-cells (46,48,51). increasing glucose concentrations result in increased glucose metabolism and atp production, inhibiting the katp channel. this in turn leads to membrane depolarization (figure 1a). consequently, the amplitude of aps reduces due to voltage-dependent inactivation of nav channels. as a result, aps, although still being generated, cannot reach the voltage that is sufficient to open p/qtype ca2þ channels. the result is that secretion of glucagon is reduced. besides restriction of kþ conductance, membrane depolarization can also be induced by influx of positively charged ions. in a-cells, this can occur at the stage of glucose transport. in addition to glucose transporters (gluts), a-cells, in particular in human islets, also express sodium-glucose cotransporter 2 (sglt2) (52). when glucose concentrations are high, sglt2 transports glucose into the a-cell together with naþ with a ratio of 1:1 (53). thus, positive charges can be introduced into cells before glucose undergoes metabolism, which will lead to membrane depolarization. although there are no direct electrophysiological data about the role of sglt2 in a-cell membrane potential, it has been reported that blocking it with dapagliflozin could lift the inhibitory effect of glucose on glucagon secretion (52). the central role of katp channels and sglt2 in the suppression of glucagon secretion from a-cells by reducing action potential height (through membrane depolarization) is contested. there are reports that suggest that glucagon secretion is controlled in a different manner: glucose metabolism increases repolarization of the a-cell membrane (reducing its firing frequency, and therefore secretion of glucagon) by either reducing depolarizing cation conductances or enhancing repolarizing kþ conductances (figure 1b). it has been proposed that a-cell excitability at low glucose concentrations was maintained by a store-operated ca2þ-channel (soc) (21,22,42,54). in high glucose, atp generated from glucose metabolism promotes ca2þ sequestration into the endoplasmic reticulum (er) by activating sarco/endoplasmic reticulum ca2þ-atpase (serca). consequently, the soc is closed and the cell membrane repolarizes. in addition, the increasing atp level due to high glucose can also activate a two-pore domain kþ channel—twik-related acid-sensitive kþ channel 1 (task1 channel), thus providing repolarizing kþ conductance (55). both mechanisms suggest that a rise of glucose concentration/metabolism leads to the inhibition of intracellular ca2þ oscillation and thus ceased secretion. these studies were conducted using dispersed single cells that went through mechanical and/or enzymatic dispersing processes. however, most studies on intact islets indicate that the a-cell ca2þ oscillations persist at high (inhibitory) glucose concentrations (46,56). these divergent results may therefore be a result of isolated/cultured a-cells being different from a-cells in situ in terms of their electrophysiology. for example, naþ currents are reduced 10-fold in dispersed single a-cells (57) compared to those recorded from intact islets (46). combining intrinsic and paracrine mechanisms to understand glucagon secretion it is important to emphasize that these intrinsic/paracrine mechanisms are not mutually exclusive. however, major uncertainty remains about their relative roles and whether they interact. for example, at a glucose level where glucagon is maximally inhibited (5–7 mm), although little insulin is secreted, somatostatin secretion is already being stimulated (34,42). thus it may be that both the aforementioned intrinsic and paracrine effects of sst govern the glucose control of glucagon secretion in low glucose (figure 2). it is interesting to notice that, in the presence of sstr2-specific blockers, even at a very low concentration of glucose (below the (a) (b) figure 1. the glucose control of glucagon secretion from a-cells via intrinsic mechanisms. (a) katp channels and sglt2 depolarize the cell, decreasing action potential height and therefore p/q activity. this results in reduced glucagon secretion. (b) task1 and store-operated channels (soc) have been proposed to increase repolarizations in the cell, decreasing action potential frequency, and therefore glucagon secretion. upsala journal of medical sciences 115 stimulatory level for somatostatin secretion), glucagon secretion is much higher than that measured under control conditions (42,46). such an observation suggests that glucagon secretion is under tonic inhibition by somatostatin. indeed, we have observed that 60% (16 out of 27) of a-cells exhibited transient spontaneous repolarizations (possibly due to the activation of girk channels by sst; see figure 3b2 for an example) when sensing 1 mm glucose. the occurrence of such repolarizations only moderately increased (65%; 9 out of 14 cells) when a high concentration of glucose was applied. we have found that although d-cells are stimulated by glucose, some (<10%) d-cells are spontaneously active already at low (1 mm) glucose (e. vergari and p. rorsman, unpublished observations). unifying these intrinsic and paracrine mechanisms could provide a more complete picture of the regulation of glucagon secretion in health and disease. a pharmacological and electrophysiological dissection of how these paracrine and intrinsic mechanisms interact to regulate glucagon secretion would be technically challenging. therefore we resorted to mathematical modelling, a powerful complement to understanding biological systems (58), which provides a useful tool for investigating complex dynamic systems. hodgkin–huxley-like formalisms of the membrane ion channels in a-cells allow the modelling of the a-cell membrane potential. a number of such mathematical models of acells have been created and used to study the mechanisms regulating glucagon secretion (59–62). we investigated whether katp and girk channel activity could explain glucose counter-regulation. we generated a model of the a-cell membrane potential, described by the following differential equation: cm dvm dt ¼ � lcat � lcal � lna � lka � ldr � lkatp � lgirk � ll: (eq. 1) here we have assumed that the membrane potential dynamics are governed by t-type and l-type calcium currents (lcat and lcal ), a fast-activating na þ current (lna ), potassium currents of the delayed rectifying-type (ldr ) and a-type (la ), and a passive leak current (ll). we also have currents due to a g-protein coupled inwardly rectifying (lgirk ) and atp-sensitive potassium channel (lkatp ). all equations for these currents are as in watts and sherman (59), except lgirk which is given by: lgirk ¼ ggirk ffiffiffiffiffiffiffiffi ½k0� p xlr ðvm � vkþ: (eq. 2) here, ggirk is the maximal conductance density of the girk channel (governed by activation of sstr2), xlr is a gating variable, vk is the potassium reversal potential, and ½ko� is the extracellular potassium concentration (see fink et al. (63)). we mimic changes in girk and katp channel activity (driven by changes in glucose concentration) by modulating the maximal conductance densities of the katp and girk channel, gkatp and ggirk , respectively (figure 3). we find that decreasing gkatp depolarizes the membrane potential and reduces action potential height, as seen experimentally when a-cells are exposed to high glucose (figure 3a1,2). when we force the model with ggirk (to mimic periodic sst release), we see that the model exhibits a spontaneous repolarization, as seen experimentally in a subpopulation of a-cells (that are proximal to d-cells) in response to high glucose (figure 3b1,2). the model can therefore explain the different membrane potential responses to high glucose in a-cells. glucose control of glucagon release and t2dm as previously mentioned, insufficient insulin secretion and insulin resistance are exacerbated in t2dm by too much glucagon secretion during hyperglycaemia and too little during hypoglycaemia (64). however, the contribution of katp channels to impaired regulation of glucagon secretion in t2dm is unclear. it is interesting to note that the inversion of glucagon secretion seen in t2dm can be mimicked in a mouse model with deficient katp channels (kir6.2stop (34)). however, this mouse model revealed that high-glucose stimulation of glucagon secretion must have a katp-channel-independent mechanism. it is of great interest that sst receptor antagonists can correct the defective counter-regulation of glucagon secretion observed in diabetic rats (65,66). this is consistent with reports that in diabetes there is an increase in the number of d-cells (67–70). the resultant hypersecretion of sst may, via binding to sstr2 and activation of girk channels, suppress glucagon secretion. however, this hypothesis remains to be experimentally validated. to mimic this increase in sst release in diabetes, we increased ggirk in the mathematical model (increased sst release would cause hyperactivation of girk channels). we then mimicked exposure to high glucose by decreasing katp, and the model demonstrated an increase in firing frequency, but no change in action potential firing (figure 4a). hence the secretion of glucagon in high glucose would be expected to increase in this situation; the model therefore demonstrates that a combination of overactivity of girk and the depolarization due to katp closure may explain the loss of counter-regulation of glucagon secretion seen in t2dm. figure 2. combining intrinsic and paracrine regulation of glucagon secretion. see main text for details. 116 l. briant et al. based on these simulation data we therefore propose a mechanism by which impaired counter-regulation of glucagon secretion is generated in t2dm (figure 4b): in healthy a-cells, girk channel activity at low glucose is relatively low (due to low basal sst release) and katp activity remains sufficiently high to prevent nav inactivation. as a result, the a-cell fires large-amplitude action potentials, and glucagon secretion is high. when glucose is elevated, katp channel activity becomes completely inhibited and the resultant membrane depolarization (via inactivation of nav and reduced action potential amplitude) leads to suppression of glucagon secretion. in t2dm a-cells, there is a higher kþ conductance due to increased katp channel activity (46) and hyperactivation of girk channels as the consequence of sst over-secretion (as suggested by the ability of sst antagonists to increase glucagon (38,39)). the high resting kþ conductance moves the membrane potential to a state of reduced action potential firing. when such t2dm a-cells are exposed to high glucose, glucose leads to the closure of katp channels but girk channel activity is unaffected, and in these cells the response to glucose is similar to what is seen in b-cells: action potential firing is increased due to the membrane depolarization, but the depolarization is not strong enough to cause a reduction of the action potential height. therefore, in contrast to the non-diabetic a-cells, increasing glucose results in a stimulation of secretion of glucagon in t2dm. these intrinsic and (b)(a) figure 4. the interaction of intrinsic and paracrine mechanisms in the regulation of glucagon secretion in t2dm. (a) the mathematical model was simulated under conditions of increased girk conductance, to mimic the increased sst seen in diabetes. under low glucose, the model fired with low-firing frequency (associated with low secretion of glucagon). when the katp conductance was reduced (to simulated high glucose), the cell depolarized and firing frequency increased. however, there was no significant increase in action potential height. therefore, glucagon secretion would be expected to increase in response to high glucose. this model therefore demonstrates the inverted regulation of glucagon secretion seen in diabetes. (b) in a healthy cell in low glucose, katp activity is high and girk activity is nearly minimal ( ). glucagon secretion is high. if the healthy cell is exposed to high glucose, the katp channel closes, causing glucagon secretion to reduce ( ). in t2dm, sst secretion in low glucose is increased (e. vergari & p. rorsman, manuscript in preparation). this pushes the a-cell over the bell into a state of low secretion of glucagon ( ). high glucose, which closes katp channels, will now reduce the total k þ conductance, pushing the cell through the peak of the bell shape, increasing glucagon secretion ( ). t2dm¼ type 2 diabetes mellitus; katp ¼ atp-sensitive potassium channel; girk¼g-protein coupled inwardly rectifying channel; sst¼somatostatin. figure 3. mathematical modelling of the involvement of katp and girk channels in the response of a-cells to high glucose. the mathematical model was simulated under conditions of a stepwise increase in katp (a) and girk channel (b) activity, to mimic the action of high glucose on a-cell activity through intrinsic (katp) and paracrine (girk) activity. when conductance of the katp channel was decreased in the model (a1), action potential frequency increased but amplitude decreased, as can be seen experimentally when a-cells are exposed to high glucose (a2). b: in some a-cells, exposure to high glucose causes spontaneous repolarizations (#) that are blocked by the sst receptor antagonist cyn (and are therefore thought to be due to periodic sst release; see (46)). we therefore mimicked this by increasing the girk conductance in the model (b1). the model was able to produce spontaneous repolarizations, as seen experimentally (b2). katp ¼ atp-sensitive potassium channel; girk¼g-protein coupled inwardly rectifying channel; sst¼somatostatin. upsala journal of medical sciences 117 paracrine mechanisms can, when taken together, explain the impaired regulation of glucagon secretion seen in t2dm. conclusions we have attempted to illustrate the power of combining experimental data with mathematical modelling. it demonstrated, within certain ranges of glucose concentrations, that both intrinsic and paracrine mechanisms can operate in parallel to regulate glucagon secretion from the pancreatic islet acells. in particular, the analyses underscore the important physiological role played by intra-islet somatostatin signalling. it should be pointed out that, in this study, we have restricted our analysis to the electrical activity. however, in addition to its ability to inhibit action potential firing (which is only transient), sst also exerts a more sustained inhibitory effect on the exocytotic release of glucagon (39). the amount of glucagon actually being secreted will reflect the combination of the effects on electrical activity and exocytosis. the mathematic model used here provides a powerful tool to investigate the cause of dysregulation of glucagon secretion seen in diabetes. disclosure statement the authors report no 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disproportionate loss of large islets in patients with type 2 diabetes. plos one 2011;6:e27445. 69. clark a, wells ca, buley id, cruickshank jk, vanhegan ri, matthews dr, et al. islet amyloid, increased a-cells, reduced b-cells and exocrine fibrosis quantitative changes in the pancreas in type-2 diabetes. diabetes res 1988;9:151–9. 70. brereton mf, vergari e, zhang q, clark a. alpha-, deltaand ppcells: are they the architectural cornerstones of islet structure and co-ordination? j histochem cytochem 2015;63:575–91. upsala journal of medical sciences 119 https://protect-us.mimecast.com/s/y1zjbrt699ndi5 https://protect-us.mimecast.com/s/y1zjbrt699ndi5 glucagon secretion from pancreatic &alpha;-cells introduction paracrine regulation of glucagon secretion intrinsic regulation of glucagon secretion combining intrinsic and paracrine mechanisms to understand glucagon secretion glucose control of glucagon release and t2dm conclusions disclosure statement references cathepsin d improves the prediction of undetected diabetes in patients with myocardial infarction article cathepsin d improves the prediction of undetected diabetes in patients with myocardial infarction matthijs a. veldersa, fredrik calaisb, nina dahlec, tove falld, emil hagstr€omd,e, jerzy leppertf, christoph nowakg, åke tenerza, johan €arnl€ovg,h and p€ar hedbergf,i adepartment of medicine, v€astmanland county hospital, v€asterås, sweden; b€orebro university, faculty of health, department of cardiology, €orebro, sweden; ccentre for clinical research, uppsala university, falun, dalarna, sweden; ddepartment of medical sciences, molecular epidemiology and scilife laboratory, uppsala university, uppsala, sweden; euppsala clinical research center, uppsala university, uppsala, sweden; fcentre for clinical research, uppsala university, v€astmanland county hospital, v€asterås, sweden; gdivision of family medicine and primary care, department of neurobiology, care sciences and society (nvs), karolinska institutet, huddinge, sweden; hschool of health and social studies, dalarna university, falun, sweden; idepartment of clinical physiology, v€astmanland county hospital, v€asterås, sweden abstract background: newer therapeutic agents for type 2 diabetes mellitus can improve cardiovascular outcomes, but diabetes remains underdiagnosed in patients with myocardial infarction (mi). we sought to identify proteomic markers of undetected dysglycaemia (impaired fasting glucose, impaired glucose tolerance, or diabetes mellitus) to improve the identification of patients at highest risk for diabetes. materials and methods: in this prospective cohort, 626 patients without known diabetes underwent oral glucose tolerance testing (ogtt) during admission for mi. proximity extension assay was used to measure 81 biomarkers. multivariable logistic regression, adjusting for risk factors, was used to evaluate the association of biomarkers with dysglycaemia. subsequently, lasso regression was performed in a 2/3 training set to identify proteomic biomarkers with prognostic value for dysglycaemia, when added to risk factors, fasting plasma glucose, and glycated haemoglobin a1c. determination of discriminatory ability was performed in a 1/3 test set. results: in total, 401/626 patients (64.1%) met the criteria for dysglycaemia. using multivariable logistic regression, cathepsin d had the strongest association with dysglycaemia. lasso regression selected seven markers, including cathepsin d, that improved prediction of dysglycaemia (area under the receiver operator curve [auc] 0.848 increased to 0.863). in patients with normal fasting plasma glucose, only cathepsin d was selected (auc 0.699 increased to 0.704). conclusions: newly detected dysglycaemia, including manifest diabetes, is common in patients with acute mi. cathepsin d improved the prediction of dysglycaemia, which may be helpful in the a priori risk determination of diabetes as a motivation for confirmatory ogtt. article history received 10 june 2019 revised 9 july 2019 accepted 26 july 2019 keywords acute myocardial infarction; biomarkers; diabetes mellitus; proteomics introduction cardiovascular disease is a common complication to type 2 diabetes mellitus (dm) (1). newer therapeutic agents can reduce progression of prediabetes to dm (2). moreover, these agents have been shown to improve cardiovascular outcomes in patients with manifest dm and high risk for cardiovascular disease, giving renewed incentive to the identification of dysglycaemia in patients with acute myocardial infarction (3,4). european guidelines recommend screening for dm with fasting plasma glucose (fpg) and glycated haemoglobin a1c (hba1c) (1). in case of continued uncertainty, an oral glucose tolerance test (ogtt) can be offered. ogtt is the only clinical method to identify impaired glucose tolerance and dm in patients with normal fpg. impaired glucose tolerance and dm are strongly associated with cardiovascular outcome in patients with myocardial infarction (1,5–7). the use of ogtt during hospitalization for acute myocardial infarction has been shown to be reliably related to long-term glucometabolic state (8). however, ogtt is more time-consuming and more expensive than screening with fpg and hba1c, which may limit its use in clinical practice. because the pathophysiological processes of progressive insulin resistance in muscle and liver, pancreatic beta-cell insufficiency, as well as glucolipotoxicity are present long before the onset of manifest dm, measurement of biomarker molecules of these processes may enable earlier diagnosis of dysglycaemia and dm (9). the proximity extension assay, a highly specific proteomics technology, facilitates the identification of novel protein markers as it allows the simultaneous contact matthijs velders matthijs.alexander.velders@regionvastmanland.se department of medicine, v€astmanland county hospital, sigtunagatan, 721 89 v€asterås, sweden supplemental data for this article can be accessed here. � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution-noncommercial license (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 3, 187–192 https://doi.org/10.1080/03009734.2019.1650141 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1650141&domain=pdf&date_stamp=2019-09-09 https://doi.org/10.1080/03009734.2019.1650141 http://creativecommons.org/licenses/by-nc/4.0/ https://doi.org./10.1080/03009734.2019.1650141 http://www.tandfonline.com measurement of large amounts of proteins in small biological samples (10). the goal of the current study was to use proximity extension assay to identify proteomic biomarkers of dysglycaemia (impaired fasting glucose, impaired glucose tolerance, or dm) in patients admitted with myocardial infarction, and to investigate whether these markers can be used to predict pathological outcome of ogtt in the normoglycaemic subset of patients. materials and methods cohort characteristics the v€astmanland myocardial infarction study (vamis; clinicaltrials.gov identifier: nct01452178) is a cohort of consecutive patients admitted for acute myocardial infarction in v€astmanland county hospital, v€asterås, sweden from november 2005 to may 2011 (11). of 1459 patients admitted with acute myocardial infarction, 201 patients in whom blood samples for biomarker analyses were obtained >72 h after admission were excluded. other exclusion criteria were dementia or confusion (n ¼ 81), other severe diseases (n ¼ 62), linguistic difficulties (n ¼ 57), or declining participation (n ¼ 50), resulting in a final cohort of 1008 patients. for the current analysis, only patients not previously known to suffer from diabetes mellitus with available fpg and 2-h post glucose load values were selected (n ¼ 626). myocardial infarction was defined according to guidelines at the time of inclusion: a typical rise and/or fall in cardiac troponin i exceeding 0.4 mg/l in combination with ischemic symptoms, new pathological q waves (evidence of loss of electrically functioning cardiac tissue), st-segment elevation or depression, or a coronary intervention (12). written informed consent was obtained from all participants. the ethics committee of uppsala university, sweden approved the study (protocol number 2005:169). the study was performed in accordance with the declaration of helsinki. definition of dysglycaemia ogtt was performed a median of 3 days (interquartile range 3–5) after admission to the coronary care unit. levels of fpg and plasma glucose 2 h after oral administration of 75 g glucose were measured from capillary blood with hemocue 201þ (hemocue ab, €angelholm, sweden). the binary variable ‘dysglycaemia’ was defined as impaired fasting glucose, impaired glucose tolerance, or dm according to world health organization definitions for capillary blood samples (13). thus, patients met the criteria for dysglycaemia if fasting capillary plasma glucose was �6.1 mmol/l or if capillary plasma glucose 2 h after ogtt was �8.9 mmol/l. blood sampling blood samples were taken in 5 ml lithium heparin-coated vacuum tubes. the tubes were centrifuged at 2000 g for 10 min (becton dickinson and co., franklin lakes, nj, usa) or 2200 g for 10 min (vacuette, greiner bio-one gmbh, kremsm€unster, austria) at room temperature. plasma was then reallocated to 5 ml plastic tubes and frozen at �70 �c within 2 h. the plasma samples were stored at �70 �c until analysis. before analysis, the samples were thawed at room temperature, mixed and centrifuged at 3470 g at 4 �c for 15 min, and aliquoted into a microtitre plate using a pipetting robot, the tecan freedom evolyzer (tecan, m€annedorf, switzerland). proteomics measurement of protein biomarkers in plasma was performed using the olink proseek multiplex cvdi 96x96 (olink bioscience, uppsala, sweden) at the clinical biomarkers facility, science for life laboratory, uppsala university, uppsala, sweden, as described previously (10). of 92 biomarkers, 11 proteins in which <80% of patients had a valid measurement were removed from further analysis (heat shock 27 kda protein, pappalysin-1, pentraxin-related protein ptx3, beta-nerve growth factor, magnetosome protein, pselectin glycoprotein ligand 1, melusin, sir2-like protein 2, interleukin-4, caspase 8, and natriuretic peptide b). the data were adjusted for plate effect to remove any influence of drift in measurements between plates. values below the limit of detection were imputed as being the limit of detection normalized for the plate. statistics characteristics were summarized using frequencies and percentages for categorical variables and median and 25th and 75th percentiles for continuous variables. the first part of the analyses consisted of biomarker discovery with a mechanistic purpose. biomarker discovery was performed using logistic regression models, assessing each biomarker separately with dysglycaemia as the dependent variable with adjustment for age and sex. adjustment for multiple comparisons was performed with bonferroni correction. significant biomarkers were assessed in multivariable models including additional adjustment for smoking status (current smokers versus nonsmokers), history of hypertension, family history of firstdegree relatives with dm, body mass index, waist circumference, and storage time. serum creatinine, fpg, and hba1c were also considered as covariates but were excluded due to the risk of being collider-variables (supplementary figure 1, available online). waist circumference and storage time of biomarkers remained in the models due to clinical relevance. for these analyses, missing values in covariates (n ¼ 36 cases with missing values among covariates) were imputed into 20 datasets using multivariate imputation by chained equations with predictive mean matching, including the aforementioned covariates. imputed values were compared with the recorded values to assess for aberrations. the multivariable models were pooled across the 20 imputed datasets according to rubin’s rule. the second part of the analyses evaluated the prognostic value of proteomic markers. to assess whether adding 188 m. a. velders et al. https://doi.org/10.1080/03009734.2019.1650141 proteomic biomarkers to established biomarkers and risk factors improved prediction of dysglycaemia, the dataset was randomly split into a training (2/3) and a test set (1/3). cases with missing values for the established risk factors and fpg or hba1c were excluded prior to splitting (n ¼ 44 for the total population), resulting in 582 eligible patients. in the training set, l1-regularized lasso regression was performed to identify the smallest selection of biomarkers to achieve a risk discrimination comparable to the whole assay, when added to risk factors (age, sex, current smoker, history of hypertension, family history of first-degree relatives with dm, body mass index, waist circumference, and storage time) and established biomarkers (fpg and hba1c). these covariates were forced into the model, with subsequent ten-fold bootstrapped cross-validation. discriminatory ability of the model applying the optimum sparse number of biomarkers was evaluated in the hold-out test set using the area under the receiver operator curve (auc) and pseudo r2. likelihood ratio tests were performed to assess goodness of fit before and after addition of the proteomic markers. finally, these analyses were also performed in a training set of the subset of patients with normal fpg (fpg <6.1 mmol/l), with determination of the discriminatory ability of the model in a test set. in the subset of 415 patients with normal fpg, 26 cases with missing values for the covariates were excluded prior to splitting, resulting in 389 eligible patients. calculations were performed using ibm spss 24 (ibm corp., armonk, ny, usa) and r version 3.4.3 (r foundation for statistical computing; 2016, vienna, austria). results in total, 826 out of 1008 patients did not have previously known dm (81.9%), of which 626 underwent ogtt (75.8%) (table 1). of patients that underwent ogtt, 225 patients (35.9%) had normal glucose tolerance, 55 patients (8.8%) had isolated impaired fasting glucose, 215 patients (34.3%) had impaired glucose tolerance, and 131 patients (20.9%) met the criteria for dm (table 2). among the 415 patients with normal fpg, 225 (54.5%) had normal glucose tolerance, 150 (36.1%) had impaired glucose tolerance, and 40 (9.6%) met the criteria for dm. using individual logistic models including sex and age, nine biomarkers were significantly associated with dysglycaemia after correction for multiple comparisons. four markers remained significant after additional multivariable adjustment (table 3). prediction of dysglycaemia the population was split into a training set, containing 388 patients. with age, sex, smoking status, history of hypertension, family history of first-degree relatives with dm, body mass index, waist circumference, storage time, and established biomarkers (fpg and hba1c) forced into the model, l1-regularized lasso regression selected cystatin-b, cathepsin d, galanin peptides, galectin 3, interleukin-6 receptor subunit alpha, matrix metalloproteinase-1, and renin. in the training set of patients with normal fpg (n ¼ 259), only cathepsin d was selected. results of discriminatory ability testing for these models was performed in the test sets containing the remaining one-third of these populations (table 4). in the total population, the first quartile of cathepsin d (<�0.866), second (�0.866 to �0.205), third (�0.206 to 0.470), and fourth quartiles (>0.470) resulted in dysglycaemia rates of 47.4%, 61.9%, 65.2%, and 81.8%, respectively. using table 1. characteristics of patients according to availability of ogtt data. ogtt (n ¼ 626) no ogtt (n ¼ 200) age, median years (iqr) 68 (17) 78 (18) male gender, % (n) 70.0 (438/626) 56.5 (113/200) body mass index, median kg/m2 (iqr) 26.3 (5.1) 25.0 (5.5) waist, median cm (iqr) 96 (14) 95 (15) current smoker, % (n) 22.8 (143/626) 18.6 (37/199) hypertension, % (n) 49.0 (307/626) 58.0 (116/200) hyperlipidaemia, % (n) 25.4 (159/625) 21.6 (43/199) first-degree relatives with diabetes mellitus, % (n) 23.7 (144/607) 18.5 (31/168) previous myocardial infarction, % (n) 18.8 (118/626) 26.5 (53/200) previous stroke, % (n) 5.1 (32/626) 13.0 (26/200) presentation with st-elevation myocardial infarction, % (n) 37.1 (232/626) 27.5 (55/200) hba1c, median mmol/mol (iqr) 38 (5) 38 (6) serum creatinine, median mmol/l (iqr) 84 (27) 90 (37) fasting plasma glucose, median mmol/l (iqr) 5.7 (1.1) 6.0 (1.4) hypertension and hyperlipidaemia were determined according to patient history. hba1c: glycated haemoglobin a1c; iqr: interquartile range; ogtt: oral glucose tolerance testing. table 2. patient characteristics according to outcome of ogtt. normal glucose tolerance (n ¼ 225) dysglycaemia (n ¼ 401) age, median years (iqr) 65 (17) 70 (15) male gender, % (n) 74.2 (167/225) 67.6 (271/401) body mass index, median kg/m2 (iqr) 26.2 (4.5) 26.3 (5.7) waist, median cm (iqr) 94 (12) 97 (14) current smoker, % (n) 28.0 (63/225) 20.0 (80/401) hypertension, % (n) 41.3 (93/225) 53.4 (214/401) hyperlipidaemia, % (n) 23.1 (52/225) 26.8 (107/400) first-degree relatives with diabetes mellitus, % (n) 18.8 (41/218) 26.5 (103/389) previous myocardial infarction, % (n) 16.9 (38/225) 20.0 (80/401) previous stroke, % (n) 4.0 (9/225) 5.7 (23/401) presentation with st-elevation myocardial infarction, % (n) 38.2 (86/225) 36.4 (146/401) hba1c, median mmol/mol (iqr) 36 (4) 39 (6) serum creatinine, median mmol/l (iqr) 82 (23) 86 (29) fasting plasma glucose, median mmol/l (iqr) 5.3 (0.6) 6.1 (1.1) see table 1 for abbreviations and definitions. table 3. association of individual biomarkers with dysglycaemia after adjustment for clinical risk factors, age, and sex. biomarker odds ratio (95% ci) p value cathepsin d 1.61 (1.32–1.97) 4.10 � 10�6 tumour necrosis factor-related apoptosis-inducing ligand 0.60 (0.48–0.75) 5.42 � 10�6 agouti-related protein 1.50 (1.21–1.87) 3.11 � 10�4 interleukin-6 1.45 (1.20–1.76) 1.70 � 10�4 or (95% ci) per sd increase in protein abundance. adjustment performed for age, sex, smoking status, history of hypertension, family history of first-degree relatives with dm, body mass index, waist circumference, and storage time. upsala journal of medical sciences 189 the same increasing quartiles of cathepsin d, the rates of dm were 9.7%, 16.8%, 16.8%, and 39.6%. in the subset of patients with normal fpg, increasing quartiles of cathepsin d resulted in rates of dysglycaemia of 31.4%, 45.4%, 50.5%, and 63.2%, respectively. rates of dm for increasing quartiles were 4.2%, 9.3%, 10.1%, and 18.4%. discussion in this cohort of patients with acute myocardial infarction, newly detected dysglycaemia, including manifest dm, was highly prevalent. multiplex proteomics identified several proteomic markers associated with previously undetected dysglycaemia in this high-risk population. proteomic biomarkers provided an improved prediction of dysglycaemia when added to clinical risk factors, fasting plasma glucose, and hba1c. cathepsin d had the strongest association with dysglycaemia and was the only proteomic marker to predict dysglycaemia in patients with normal fasting plasma glucose. the highest and lowest quartiles of cathepsin d corresponded with high and low risk of dm, potentially aiding in the a-priori risk determination of dm as a motivation for confirmatory ogtt. cathepsin d is an aspartic endopeptidase with the primary biological function of protein degradation in an acidic milieu of lysosomes. it has been studied extensively from the perspective of its role in cancer development and as a suggested tumour marker (14). furthermore, cathepsin d enzymatic activity induces hydrolytic modification of lipoprotein, including low-density lipoprotein, contributing to the accumulation of modified low-density lipoprotein in arterial intima (15). higher levels of cathepsin d have been observed in diabetes complicated by diabetic ulcers and retinopathy (16,17). cathepsin d has also been associated with insulin resistance in two community cohorts, although causality could not be shown with mendelian randomization (18). additionally, it has been suggested as a marker of b-cell function (19). animal models support a role for cathepsin d in lysosomal/autophagic-induced cell death as a major driver of b-cell death and dysfunction in response to glucolipotoxicity in type 2 diabetes. in these models, the glucagon-like peptide 1 agonist exendin-4 was shown to protect b-cells from death by increasing autophagic flux and restoring lysosomal function (20). clinical studies have shown that the glucagon-like peptide 1 agonist liraglutide as an adjunct to diet and exercise reduces the risk of progression from prediabetes to diabetes in obese patients, with improvement of measures of insulin resistance and b-cell function (2). additionally, glucagon-like peptide 1 agonists reduce cardiovascular and overall mortality in patients with type 2 dm (3). whether patients with higher cathepsin d levels have a larger benefit of glucagon-like peptide 1 agonists remains unknown. three other biomarkers (agrp, trail, and interleukin-6) were strongly associated with dysglycaemia in the current material. agrp is a powerful orexigenic (appetite-inducing) peptide involved in the regulation of energy homeostasis. acute activation of agrp neurons causes insulin resistance through impairment of insulin-stimulated glucose uptake into brown adipose tissue. agrp neurons integrate numerous signals of the periphery, including levels of glucose, insulin, and ghrelin (21). inhibition of p2y6 signalling in agrp neurons has recently been shown to reduce food intake and improve systemic insulin sensitivity in obese mice, potentially providing a novel therapeutic target for the treatment of obesity and dm (22). to our knowledge, agrp has not previously been associated with impaired glucose tolerance and dm in patients with myocardial infarction. trail, a type ii transmembrane protein and member of the tumour necrosis factor-ligand family, has been studied extensively in the setting of dm and obesity (23). lower levels of trail were associated with dysglycaemia, which is in accordance with a previous study comparing healthy controls with patients with newly detected but unmedicated diabetes. initiation of treatment was found to elevate levels of trail measured after 6 months (24). interleukin-6 is a pleiotropic cytokine involved in the immune system, metabolism, and numerous other functions. it has previously been identified as a predictor of type 2 dm and associated cardiovascular events (25). table 4. performance of prediction models of dysglycaemia. total population, test set (n ¼ 194) auc pseudo r2 likelihood ratio test, p valuea clinical risk factors and fpg 0.846 0.433 – clinical risk factors and hba1c 0.748 0.218 – clinical risk factors, fpg, and hba1c 0.848 0.438 reference clinical risk factors, fpg, hba1c, and proteomic markers (cystatin-b, cathepsin d, galanin peptides, galectin 3, interleukin-6 receptor sub-unit alpha, matrix metalloproteinase-1, and renin) 0.863 0.469 5.34 � 10�4 patients with normal fasting plasma glucose, test set (n ¼ 130) clinical risk factors and fpg 0.687 0.151 – clinical risk factors and hba1c 0.701 0.175 – clinical risk factors, fpg, and hba1c 0.699 0.176 reference clinical risk factors, fpg, hba1c, and proteomic markers (cathepsin d) 0.704 0.190 1.22 � 10�3 clinical risk factors included in the model: age, sex, smoking status, history of hypertension, family history of first-degree relatives with dm, body mass index, waist circumference, and storage time. alikelihood ratio test assessing change in goodness of fit after addition of proteomic markers to model with clinical risk factors and established markers. auc: area under the receiver operator curve; fpg: fasting plasma glucose; hba1c: glycated haemoglobin a1c. 190 m. a. velders et al. proteomic markers improved the prediction of dysglycaemia, selecting cathepsin d, cystatin-b, galanin peptides, galectin-3, interleukin-6 receptor sub-unit alpha, matrix metalloproteinase-1, and renin. however, cathepsin d was the only useful proteomic marker for the prediction of dysglycaemia in patients with normal fpg. indicated by the lower prediction model auc, it is more difficult to predict a pathological ogtt in patients with normal fpg. both hba1c and fpg had limited performance in these patients, which is in accordance with previous observations (26). as such, ogtt remains an essential test to identify reduced glucose tolerance and dm in patients with myocardial infarction and normal fpg. however, higher quartiles of cathepsin d showed a consistent increasing prevalence of dysglycaemia and manifest dm, which may be helpful to identify patients at high apriori risk of dm. as the proteomic technology does not permit an absolute quantification of the biomarkers, additional laboratory analyses of absolute values are needed to confirm these results, and to determine whether cathepsin d can be used to guide decision-making for the need of further confirmatory testing with ogtt. increased precision of the measurement of cathepsin d could increase the predictive value. however, cathepsin d assays are currently limited by lack of standardization and are associated with higher costs than ogtt. several other limitations should be noted. the current findings are based on a single cohort with limited size including caucasian, predominantly male, patients with acute myocardial infarction. as such, findings need to be validated in other cohorts, as well as other settings such as in primary care. the proximity extension assay chip focused on proteins associated with cardiovascular disease and/or inflammation. an assay targeted directly at diabetes candidate proteins may have revealed additional findings. another limitation is storage of plasma samples for up to 10 years before being analysed, so we cannot exclude the possibility that different protein stabilities might have influenced the analysis. storage time is known to influence concentrations of certain proteins in proteomic analyses, but how this influenced the cathepsin d concentrations is unclear (27). however, the plasma samples were collected and stored consistently, which should have minimized any pre-analytical bias, and analyses were corrected for storage time. as the blood samples were taken within 72 h after acute myocardial infarction, we do not know to what extent, or which, biomarkers exhibited an acute phase expression. in conclusion, dysglycaemia is very common in patients with acute mi, and proteomic biomarkers improve the prediction of undetected dysglycaemia over clinical risk factors and established biomarkers. cathepsin d showed the most promise of these proteomic biomarkers due to its strong association with dysglycaemia and its predictive ability in patients with normal fpg. disclosure statement e.h. has received institutional research grants from amgen, sanofi; consulting fees from amgen, novonordisk, sanofi; and speaker fees from astrazeneca, amgen, boehringer ingelheim, novonordisk, sanofi. j.€a. has received lecturing fees from astrazeneca. the remaining authors report no conflicts of interest. funding this work was supported by the regional research council uppsala€orebro, sweden under grant [rfr-743621]. notes on contributors matthijs a. velders, md, phd, is a postdoctoral researcher and resident in cardiology and internal medicine at the department of medicine, v€astmanland county hospital, v€asterås, sweden. fredrik calais, md, phd, is a senior consultant cardiologist at €orebro university, faculty of health, department of cardiology, €orebro, sweden. nina dahle, md, is a resident in primary care, and a phd student at the centre for clinical research, uppsala university, falun, dalarna, sweden. tove fall, phd, is an associate professor at the department of medical sciences, molecular epidemiology and scilife laboratory, uppsala university, uppsala, sweden. emil hagstr€om, md, phd, is a senior consultant cardiologist and an associate professor at the department of medical sciences, molecular epidemiology and scilife laboratory, and the uppsala clinical research center, uppsala university, uppsala, sweden. jerzy leppert, md, phd, is a professor of family medicine at the centre for clinical research, uppsala university, v€astmanland county hospital, v€asterås, sweden. christoph nowak, phd, bm bch, dipl-psych, is a post-doctoral researcher at the division of family medicine and primary care, department of neurobiology, care sciences and society (nvs), karolinska institutet, huddinge, sweden. åke tenerz, md, phd, is a senior consultant endocrinologist at the department of medicine, v€astmanland county hospital, v€asterås, sweden. johan €arnl€ov, md, phd, is a professor of family medicine at the division of family medicine and primary care, department of neurobiology, care sciences and society (nvs), karolinska institutet, huddinge, sweden, and the school of health and social studies, dalarna university, falun, sweden. p€ar hedberg, md, phd, is an associate professor at the centre for clinical research, uppsala university, v€astmanland county hospital, and a senior consultant in clinical physiology at the department of clinical physiology, v€astmanland county hospital, v€asterås, sweden. references 1. piepoli mf, hoes aw, agewall s, albus c, brotons c, catapano al, et al. 2016 european guidelines on cardiovascular disease 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intern med. 2011;22:8–12. 27. enroth s, hallmans g, grankvist k, gyllensten u. effects of longterm storage time and original sampling month on biobank plasma protein concentrations. ebiomedicine 2016;12:309–14. 192 m. a. velders et al. http://www.who.int/iris/handle/10665/66040 http://www.who.int/iris/handle/10665/66040 abstract introduction materials and methods cohort characteristics definition of dysglycaemia blood sampling proteomics statistics results prediction of dysglycaemia discussion disclosure statement funding references sups_a_526723 18..25 upsala journal of medical sciences. 2011; 116: 18–25 original article effect of renal sympathetic nerve on adrenergically and angiotensin ii-induced renal vasoconstriction in normal wistar-kyoto rats mohammed h. abdulla1, munavvar a. sattar1, nor a. abdullah2, abdul hye khan3, kolla r. l. anand swarup1, hassaan a. rathore1, raisa n. kazi1, fathihah basri1 & edward j. johns4 1school of pharmaceutical sciences, universiti sains malaysia, minden, penang, malaysia, 2department of pharmacology, faculty of medicine, universiti malaya, kuala lumpur, malaysia, 3cardiovascular research center, medical college of wisconsin, milwaukee, usa, and 4department of physiology, aras windle, university college cork, college road, cork, ireland abstract background. this study examined the effect of renal sympathetic innervation on adrenergically and angiotensin ii (ang ii)induced renal vasoconstriction in wistar-kyoto (wky) rats. methods. forty-eight wky rats were treated with either losartan (10 mg/kg/day p.o.) or carvedilol (5 mg/kg/day p.o.) or a combination of them (10 mg/kg/day + 5 mg/kg/day p.o.) for 7 days. on day 8, the rats were anaesthetized, and renal vasoconstrictor experiments were carried out. a group of rats was subjected to acute unilateral renal denervation during the acute study. changes in the renal vasoconstrictor responses were determined in terms of reductions in renal blood flow caused by ang ii, noradrenaline (na), and methoxamine (me). results. in normal animals, losartan decreased (p < 0.05) the renal vasoconstrictor response to ang ii but not to na or me. carvedilol treatment, however, blunted (p < 0.05) the renal vasoconstrictor responses to ang ii and adrenergic agonists. combination of losartan and carvedilol blunted (p < 0.05) the renal vasoconstrictor response to ang ii but augmented the responses to na and me (all p < 0.05). interestingly, when denervated rats were treated with the same combination, there was a reduction (p < 0.05) in the renal vasoconstrictor responses to ang ii and adrenergic agonists. conclusions. data suggest that the renal sympathetic nerve contributes to adrenergic agonist-mediated renal vasoconstrictions in normal rats. the data further indicate an interactive relationship between renin-angiotensin and sympathetic nervous systems in modulating adrenergically and ang ii-induced renal vasoconstriction in wky rats. key words: a1-adrenoceptors, carvedilol, losartan, renal haemodynamics, wistar-kyoto rats introduction angiotensin ii (ang ii) has been shown to increase vascular sensitivity to noradrenaline in rats as well as in isolated vessels (1), hence suggesting that ang ii and noradrenaline exert synergistic actions on vasculature. it has also been reported that the blockade of endogenous ang ii by angiotensin ii receptor type 1 (at1) blockers could alter vascular reactivity to exogenous noradrenaline (2). one of the most important sites of physiological action of ang ii is the renal vasculature, on which it has a direct and potent vasoconstrictor action (3). it has been reported that a certain degree of reninangiotensin system (ras) activity is necessary to optimize the release of noradrenaline from renal correspondence: mohammed h. abdulla, school of pharmaceutical sciences, universiti sains malaysia, 11800 minden, penang, malaysia. fax: +6 016 635 496 1. e-mail: mdapharm78@yahoo.co.uk (received 20 june 2010; accepted 21 september 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.526723 sympathetic nerve terminals (4). it has also been mentioned that noradrenaline released from renal sympathetic nerves initiates renin release by stimulation of b1adrenoceptors on renin-containing juxtaglomerular granular cells (5). the renin released subsequently forms ang ii that exerts preand postsynaptic action on ang ii receptors (4). as reported in dog, dependence of the renal vasoconstrictor response to ang ii on an intact renal innervation, indeed, suggested a role of this peptide in catecholamine release, and it is believed that the renal vasculature is particularly sensitive to the release of catecholamine through an action of ang ii (3). moreover, there is evidence that the elevated vascular noradrenaline sensitivity by ang ii may be attributed to a decline in catecholamine clearance because it has been reported that ang ii diminishes neuronal catecholamine uptake (6). in the present study, an effort has been made to gain insight into the role of renal nerves on the renal vascular responsiveness to ang ii and adrenergic agonists in normotensive wistar-kyoto (wky) rats. the renal vascular tone and responsiveness were assessed under basal conditions and in the presence of acute stimulation with noradrenaline (na), methoxamine (me), and ang ii that were infused directly into the renal artery. series of experiments were carried out in rats treated with or without ras and sympathetic nervous system (sns) blockers. the blockade was caused either by pharmacological or surgical procedures, and the influence of these blockades was assessed in terms of their role in modulating adrenergically and ang ii-induced renal vasoconstriction. therefore, the hypothesis tested in this study was that the renal haemodynamic effects of ang ii and adrenergic agonists are influenced by the presence of an intact renal innervation. methods animals experiments were conducted on age-matched male wky rats (n = 48) collected from animal care facility, universiti sains malaysia, penang, malaysia. animal handling and all procedures on animals were carried out in accordance with the guidelines of the animal ethics committee, universiti sains malaysia, penang, malaysia and had their approval. the animals were fed standard rat chow with free access to tap water and kept on a 12-h light:12-h dark cycle. following a week of acclimatization, animals were randomly assigned into eight groups, namely control (ct), denervated (dnx), losartan (l), denervated losartan (dnx-l), carvedilol (cv), denervated carvedilol (dnx-cv), losartan+carvedilol (lcv), and denervated (losartan+carvedilol) treated (dnxlcv) wky rats (all n = 6). rats treated with carvedilol (dilatrend�, roche, basel, switzerland) received the drug at a dose of 5 mg/kg/day (p.o.) for 7 days (7–9). losartan (cozaar�, msd, nj, usa) was given at an oral dose of 10 mg/kg/day as previously mentioned (10–13). a group of rats received losartan (10 mg/kg/ day, p.o.) along with carvedilol (5 mg/kg/day, p.o.) (7,11). following the 7-day treatment period, the overnight fasted animals were subjected to renal vasoconstrictor study. haemodynamic studies surgical procedures on rats were similar to those described previously (14–16) with some modifications. the overnight (10–12 h) fasted rats were anaesthetized with 60 mg/kg (i.p.) sodium pentobarbitone (nembutal�, ceva, libourne, france). the trachea was cannulated to facilitate respiration. the right carotid artery was catheterized (pp50, portex, kent, uk) and connected to a fluid-filled pressure transducer (p23 id gould, statham instruments, nottingham, uk) linked to a computerized data acquisition system (powerlab�, adinstruments, sydney, australia) for continuous monitoring of mean arterial blood pressure (map) and heart rate (hr) throughout the experiment. the left jugular vein was cannulated to infuse maintenance dose of anaesthetic whenever needed. subsequently, via a ventral mid-line incision, the left kidney was exposed followed by cannulation of the left iliac artery (pp50, portex, uk) for continuous infusion of normal saline at 6 ml/h throughout the experiment. the iliac artery cannula was advanced through the abdominal aorta until its tapered tip faced the origin of the left renal artery to allow optimum administration of drugs into the renal artery (11,15–17). the renal artery was cleared to allow placing of an electromagnetic flow probe (ep 100 series, carolina medical instruments, king, nc, usa). the probe was connected to a square-wave electromagnetic flow-meter (carolina medical instruments, king, nc, usa) which was further linked to a computerized data acquisition system (powerlab�, adinstruments, sydney, australia). upon completion of the surgery, the animal was stabilized for an hour before starting the acute renal vasoconstrictor experiment. acute renal denervation acute unilateral renal denervation of the left kidney was performed according to previous studies (7,12,13). the renal artery was carefully stripped off from the surrounding covering tissues followed by cutting of the effect of renal sympathetic nerve on renal vasoconstriction 19 renal nerve and coating the remaining tissue with 10% phenol in absolute alcohol (7,18,19). the denervation was tested by placing a silver wire electrode to the coeliac ganglia at which electrical stimulation (15 v, 1– 4 hz, 0.2 ms) was applied from a stimulator for 10 s. if there was a significant fall in renal blood flow (rbf), more dissection and cutting of renal nerves were done until no change in the rbf was observed during stimulation (18,19). experimental protocol acute renal vasoconstrictor experimental protocol involved the administration of vasoactive agents that led to constriction of the renal artery, hence reduction in the rbf. bolus doses of noradrenaline (na) (sanofi winthrop, surry, uk) (25, 50, 100, and 200 ng), methoxamine (me) (wellcome, london, uk) (0.5, 1, 2, and 4 mg), and angiotensin ii (ang ii) (ciba-geigy, basel, switzerland) (2.5, 5, 10, and 20 ng) were injected in ascending and descending orders into the intra-renal infusion line in order to ensure their delivery into the renal artery. a series of time control experiments were carried out to examine any time-dependent changes in the experimental protocol. in this series of experiments, map and rbf were measured in a way that matched exactly the protocol which is used with drug administration. the intra-renal administration of agonists was carried out very carefully with a goal of producing renal effect without causing any significant change in the systemic blood pressure. a 10-min interval was allowed for recovery after each agonist treatment phase. map and rbf were monitored continuously throughout the experiment; however, only the values that were measured at the beginning of the renal vasoconstrictor experiment with each of the agonists (na, me, and ang ii) were considered as the basal map and rbf values for each of the corresponding agonists. statistical analysis and presentation of data the vasoconstrictor responses caused by ang ii and adrenergic agonists were taken as the average values caused by each dose of the agonist administered and applied in ascending and descending orders. the overall mean response for each agonist was taken as the average value of the vasoconstrictor responses (drop in rbf) obtained at each dose. the data on the drop of rbf were expressed as the percentage. drop in relation to the baseline value. all data were expressed as mean % reduction ± sem of renal vasoconstrictor responses elicited by all the doses of ang ii, na, and me, and compared between ct, dnx, l, dnx-l, cv, dnx-cv, lcv, and dnx-lcv-treated wky rats. data were analysed by either twoor one-way anova followed by bonferroni post-hoc test. the differences between the means were considered significant at 5% level. results the base-line map and rbf as well as renal vascular resistance (rvr) or hr in dnx, l, dnx-l, cv, dnx-cv, lcv, and dnx-lcv-treated rats were not different from ct (table i). the map recorded before and after administration of the agonists was similar, and this trend was observed throughout the experiment. as an example, figure 1 illustrates this phenomenon and shows the map and rbf responses to the injection of the highest dose of ang ii (20 ng) in rats either treated or untreated with carvedilol. table i. base-line values of body weight, mean arterial blood pressure (map), renal blood flow (rbf), renal vascular resistance (rvr), and heart rate (hr) in ct, dnx, l, dnx-l, cv, dnx-cv, lcv, and dnx-lcv. all data are expressed as mean ± sem. treatment n body weight (g) map (mmhg) rbf (ml min-1 kg-1) rvr (mmhg min kg ml-1) hr (bpm) ct 6 277 ± 8 110 ± 3 9.0 ± 1.0 13.4 ± 2.1 285 ± 12 dnx 6 281 ± 10 109 ± 7 10.1 ± 2.6 11.4 ± 2.2 312 ± 17 l 6 270 ± 7 110 ± 10 8.6 ± 3.1 8.9 ± 0.1 301 ± 13 dnx-l 6 278 ± 8 109 ± 9 11.8 ± 1.5 14.3 ± 4.1 299 ± 14 cv 6 288 ± 9 111 ± 4 11.6 ± 4.9 11.6 ± 4.6 258 ± 19 dnx-cv 6 290 ± 9 108 ± 8 9.8 ± 3.2 10.9 ± 3.1 300 ± 20 lcv 6 272 ± 5 115 ± 6 10.6 ± 2.5 11.4 ± 2.1 258 ± 19 dnx-lcv 6 284 ± 8 118 ± 12 11.2 ± 1.2 11.2 ± 0.7 237 ± 11 bpm = beats per minute. 20 m. h. abdulla et al. renal vasoconstrictor responses noradrenaline (na). noradrenaline caused a dosedependent reduction in the rbf. the renal vascular response to na was smaller (p < 0.05) in cv-treated rats as compared to ct (cv 14.2% ± 3.0% versus ct 32.2% ± 3.0%). quite in contrast, there was no change in the renal vasoconstrictor response to na in l or dnx-l-treated rats compared to ct (l 34.4% ± 3.5% and dnxl 32.7% ± 2.4% versus ct 32.2% ± 3.0%). in rats treated with a combination of losartan and carvedilol (lcv), the reduction in rbf in response to na was greater than ct (lcv 37.3% ± 3.7% versus ct 32.2% ± 3.0%; p < 0.05). dnx-lcv rats had a markedly lower (p < 0.05) renal response to na compared to ct (dnx-lcv 16.2% ± 1.5% versus ct 32.2% ± 3.0%) (figure 2). finally, ct, dnx-l, and dnx-lcv rats had lower overall mean renal vasoconstrictor responses to na compared to dnx (ct 32.2% ± 3.0%, dnx-l 32.7% ± 2.4%, and dnx-lcv 16.2% ± 1.5% versus dnx 39.0% ± 4.0%; all p < 0.05) (figure 2). ang ii -80 -70 -60 -50 -40 -30 -20 -10 0 10 20 0 20 40 60 80 100 120 140 160 180 200 220 240 260 time (sec) r b f % c h a n g e f ro m b a se lin e control carvedilol ang ii 140 130 120 110 100 90 80 70 m a p ( m m h g ) figure 1. time course of the renal vasoconstrictor response to ang ii. map changes (top) and rbf response (bottom) following the administration of a bolus injection (20 ng) into the renal artery. the delay of about 30 s at the beginning of the response is due to the travel time in the cannula before reaching the renal vasculature. stippled lines denote ± sem, n = 5 rats. effect of renal sympathetic nerve on renal vasoconstriction 21 methoxamine (me). intra-renal arterial bolus doses of this agonist resulted in dose-dependent reductions in rbf (figure 3). the magnitude of rbf reduction in cv was lower than ct (cv 9.6% ± 2.4% versus ct 15.7% ± 4.1%; p < 0.05). there was, however, no difference in the renal response to me between l or dnx-l, and ct (l versus ct: 19.5% ± 3.4% versus 15.7% ± 4.1%, and dnx-l versus ct: 17.5% ± 3.1% versus 15.7% ± 4.1%). the vasoconstrictor responses to me in lcv were greater than ct (lcv versus ct: 22.0% ± 2.2% versus 15.7% ± 4.1%; p < 0.05). the renal responses to me in dnx-lcv, however, were significantly lower than ct (dnxlcv versus ct: 8.7% ± 1.0% versus 15.7% ± 4.1%; p < 0.05) (figure 3). further, the vasoconstrictor response to me in ct, dnx-l, and dnx-lcv were lower than dnx (ct versus dnx: 15.7% ± 4.1% versus 24.7% ± 3.5%, dnx-l versus ct: 17.5% ± 3.1% versus 24.7% ± 3.5%, and dnx-lcv versus dnx: 8.7% ± 1.0% versus 24.7% ± 3%; all p < 0.05) (figure 3). -70 -60 -50 -40 -30 -20 -10 0 0.5 1 2 4 methoxamine (µg) % d ro p in r b f * * * ct l cv lcv -70 -60 -50 -40 -30 -20 -10 0 0.5 1 2 4 methoxamine (µg) % d ro p in r b f * * * * ct dnx dnx-l dnx-cv dnx-lcv # # # # figure 3. renal vasoconstrictor responses to graded doses of me in ct, dnx, l, dnx-l, cv, dnx-cv, lcv, and dnx-lcv. *p < 0.05 compared to ct. #p < 0.05 compared to dnx. data were analysed by two-way anova followed by bonferroni post-hoc test, n = 6 rats. -80 -70 -60 -50 -40 -30 -20 -10 0 25 50 100 200 noradrenaline (ng) % d ro p in r b f * * * ct l cv lcv -80 -70 -60 -50 -40 -30 -20 -10 0 25 50 100 200 noradrenaline (ng) % d ro p in r b f * * * * ct dnx dnx-l dnx-c dnx-lcv # # # figure 2. renal vasoconstrictor responses to graded doses of na in ct, dnx, l, dnx-l, cv, dnx-cv, lcv, and dnx-lcv. *p < 0.05 compared to ct. #p < 0.05 compared to dnx. data were analysed by two-way anova followed by bonferroni post-hoc test, n = 6 rats. 22 m. h. abdulla et al. angiotensin ii (ang ii). ang ii also caused a doserelated reduction in rbf (figure 4). it was found that the level of renal vasoconstriction as assessed from the reduction of rbf was lower in l, dnx-l, and cv compared to ct (l versus ct: 8.1% ± 0.7% versus 28.5% ± 2.3%, dnx-l versus ct: 6.7% ± 0.7% versus 28.5% ± 2.3%, cv versus ct: 12.4% ± 1.6% versus 28.5% ± 2.3%; all p < 0.05). a similar trend was also observed in the lcv and dnx-lcv groups compared to ct (lcv versus ct: 21.3% ± 2.1% versus 28.5% ± 2.3%, and dnx-lcv versus ct: 6.1% ± 2.1% versus 28.5% ± 2.3%; all p < 0.05) (figure 4). finally, dnx-l and dnx-lcv rats had lower (p < 0.05) responses to ang ii compared to dnx (dnx-l versus dnx: 6.7% ± 0.7% versus 31% ± 2.9%, and dnx-lcv versus dnx: 6.1% ± 2.1% versus 31% ± 2.9%; all p < 0.05) (figure 4). discussion this study investigated the role of the renal sympathetic nervous system on ang ii and adrenergic agonist-induced vasoconstrictor responses in the renal vasculature of wky rats. this was done by studying the modulation of adrenergically and ang iiinduced renal vasoconstrictions in the presence or absence of a blockade of ras, sns, or both. the at1-receptor blocker losartan was used to block ras, while carvedilol treatment as well as surgical denervation were used to block the sns. as reported earlier, the dose of losartan used in this study has been shown to be sufficient to inhibit ang ii action on at1-receptors (11). the dose of carvedilol used in this study was based on earlier reports from others (8,9) and from our previous study, where carvedilol markedly blunted phenylephrine-mediated vasoconstrictor response (11). in the present study, we found that in wky rats blocking of the sympathetic innervations to the kidney augmented the renal responses to na and me, possibly due to enhancement of the receptors sensitivity to the given agonists (7,12,20), but it did not cause any significant change in the vasoconstrictor response to ang ii. it is suggested that the released noradrenaline in neurons interacts with a1-adrenoceptors, and their chronic activation in turn results in the downregulation of at1-receptors (21). with this finding, it appears that in the absence of endogenous noradrenaline the postsynaptic at1-receptors in the renal vasculature of the wky rats were not affected and, therefore, mediated the vasoconstrictor action of ang ii. the unaffected ang ii-mediated renal vasoconstriction in the absenceofrenal nerve can be furtherexplainedbyan earlier finding that ang ii per se facilitates sympathetic neurotransmission via prejunctionally located at1-receptors (22). these presynaptic effects of ang ii are found in renal vasculature (23,24). these observations led us to suggest that in the renal vasculature there is, indeed, a relationship between at1 and adrenoceptors in mediating actions of ang ii and adrenergic stimuli. -70 -60 -50 -40 -30 -20 -10 0 2.5 5 10 20 angiotensin ii (ng) % d ro p in r b f * * * * # # # ct dnx dnx-l dnx-cv dnx-lcv-70 -60 -50 -40 -30 -20 -10 0 2.5 5 10 20 angiotensin ii (ng) % d ro p in r b f * * * ct l cv lc v figure 4. renal vasoconstrictor responses to graded doses of ang ii in ct, dnx, l, dnx-l, cv, dnx-cv, lcv, and dnx-lcv. *p < 0.05 compared to ct. #p < 0.05 compared to dnx. data were analysed by two-way anova followed by bonferroni post-hoc test, n = 6 rats. effect of renal sympathetic nerve on renal vasoconstriction 23 in the present study it was also observed that the blocking of adrenergic receptors by carvedilol blunted the renal vasoconstrictor action of both ang ii and adrenergic agonists. ang ii has been shown to influence the expression of a1-adrenoceptors in rat vascular smooth muscle (25) and, hence, suggests that indirect effects of ang ii could be mediated in part by increased expression of a1-adrenoceptors (25). although studying the expression of receptors in the renal vasculature was beyond the scope of this study, based on the changes observed in renal blood flow we suggest that in wky rats blocking of a1adrenoceptors perhaps resulted in the reduction of at1-receptors sensitivity to exogenous ang ii. this observation further indicated a possible interdependency in the actions of adrenergic stimuli and ang ii in vascular smooth muscle and strengthens our view of an interactive relationship between at1 and adrenoceptors in the renal vasculature. collectively, these observations indicated an interactive relationship between ras and sns in these rats, and we have earlier reported such interactive relationship between these two systems in a rat model of essential hypertension, spontaneously hypertensive rat (shr) (7). there was a marked attenuation in the renal vasoconstrictor responses to ang ii in normal rats treated with losartan. however, there was no change in the renal vasoconstrictor responses to na and me, and this indicates a weak influence of the blockade of the postsynaptic at1-receptors on a1-adrenoceptormediated renal vascular responses in normal wky rats. this finding was in accord with an earlier report demonstrating that losartan dose-dependently decreased the renal vasoconstriction response to renal sympathetic nerve stimulation but not to exogenously administered noradrenaline (4). this study showed that carvedilol treatment attenuated the renal vasoconstrictions induced by intrarenally administered ang ii. indeed, this observation indicated an adrenergic influence to the renal response toangiiinwkyratsandisinagreementwithanearlier study in anaesthetized rabbits (3). in line with these observations, we suggest an interactive relationship between postsynaptic a1-adrenoceptors and at1receptors in the renal vasculature of wky rat, as blocking of the former by carvedilol influenced the sensitivity of the latter to ang ii. our suggestion of a possible interactiverelationshipbetweenrasandsnsisfurther supported by an earlier study in human that suggested a cross-talk between at1and a1-adrenoceptors, and also showed that in the presence of an increased sympathetic tone carvedilol provides at1-receptor blockade via its a1-adrenoceptor-blocking effects (26). in conclusion, this study showed that there is an interactive relationship between at1 and a1-adrenoceptors in modulating renal haemodynamic responses to ang ii and adrenergic agonists in wky rats. further, we found that in wky rats this relationship between at1 and a1-adrenoceptors is influenced by the renal nerve. acknowledgements this work is supported by several research grants from universiti sains malaysia and ministry of science and technology, malaysia to munavvar a. sattar. mohammed h. abdulla is a recipient of vice chancellor’s award and usm fellowship from the institute of graduate studies of universiti sains malaysia. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. marano g, argiolas l. postjunctional regulation by angiotensin ii of alpha 1-adrenoceptor-mediated pressor responses in the rat. eur j pharmacol. 1994;261:121–6. 2. raasch w, dominiak p, ziegler a, dendorfer a. reduction of 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mathy mj, pfaffendorf m, van zwieten pa. sympatho-inhibitory actions of irbesartan in pithed spontaneously hypertensive and wistar-kyoto rats. fundam clin pharmacol. 2003;17:83–91. 23. wong pc, hart sd, timmermans pb. effect of angiotensin ii antagonism on canine renal sympathetic nerve function. hypertension. 1991;17:1127–34. 24. wong pc, bernard r, timmermans pb. effect of blocking angiotensin ii receptor subtype on rat sympathetic nerve function. hypertension. 1992;19:663–7. 25. hu zw, shi xy, okazaki m, hoffman bb. angiotensin ii induces transcription and expression of alpha 1-adrenergic receptors in vascular smooth muscle cells. am j physiol. 1995; 268:h1006–14. 26. batenburg ww, van esch jh, garrelds im, jorde u, lamers jm, dekkers dh, et al. carvedilol-induced antagonism of angiotensin ii: a matter of alpha1-adrenoceptor blockade. j hypertens. 2006;24:1355–63. effect of renal sympathetic nerve on renal vasoconstriction 25 (5) upsala j med sci 111 (2): 209–214, 2006 evaluation of dade behring n latex cystatin c reagent on abbott ci8200 mats flodin, lars-olof hansson and anders larsson department of medical sciences, university hospital, uppsala, sweden abstract plasma cystatin c has been shown in several studies to be superior to plasma creatinine for estimation of glomerular filtration rate (gfr). reporting cystatin c results in ml/min using conversion formulas for transforming cystatin c expressed as mg/l to gfr expressed as ml/min has greatly facilitated the clinical use of the marker. at our hospital we have an increasing demand for cystatin c and at present we perform over 1,400 cystatin c analyses a month. the test is available at all hours. this in combination with the volume emphasises the need to have the assay close to the routine chemistry instrument to reduce handling time per test and time to report test results. we have thus evaluated the dade behring n latex cystatin c assay (dade behring, deerfield, il, usa) on architect ci8200 (abbott laboratories, abbott park, il, usa). the nephelometric method on the prospec (dade behring) and the turbidimetric method on architect ci8200 showed very good agreement (y = 1.0072x + 0.0042; r2 = 0.987). accordingly, running the cystatin c analyses on a chemistry instrument (architect ci8200) would be proper to increase the availability of the analysis and reduce turnaround times. introduction in the last decades, serum or plasma creatinine has become the most commonly used marker of glomerular filtration rate (gfr) (1,2). despite the common use, creatinine has limitations as marker for renal function. gfr is often calculated from plasma creatinine using the cockcroft-gault (3) or the modification of diet in renal disease (mdrd) study equations (4). these equations consist of several parts, which makes them susceptible to errors especially if the calculation is performed manually. creatinine is influenced by factors such as age, gender, muscle mass, physical activity and diet (5). it is also insensitive for detecting small decreases in gfr, in the so-called creatinineblind gfr area, due to the non-linear relationship between plasma creatinine concentration and gfr (6). thus, there is a need for better gfr markers. our laboratory has been using plasma cystatin c as a marker for gfr since 3 years. 209 received 19 october 2005 accepted 21 october 2005 cystatin c is a polypeptide with a molecular mass of 13 kda and a probable ellipsoid shape with axes of about 30 and 45 å (7). a recent meta-analysis has indicated that cystatin c is superior to plasma creatinine (8) as a marker of renal function. one remaining problem in the use of cystatin c as a gfr marker is the lack of a procedure to transform cystatin c concentrations in mg/l to gfr values in ml/min. to provide clinicians with reliable and readily available gfr data based on single plasma measurements of cystatin c, we report cystatin c both in mg/l and as a “cystatin c calculated gfr” in ml/min since 3 years. this in combination with a 24 h availability of cystatin c has led to a very rapid increase in cystatin c requests during the last 3 year-period. at present we perform over 1,400 cystatin c analyses and there is still a continuous increase. to meet this increasing demand we wanted to move the test from the prospec to the architect ci8200. the aim of this study was to compare the results obtained with the nephelometric assay performed on prospec with the nephelometric assay on architect ci8200 using the same reagent to see if we reliably could move the assay from one instrument to the other. materials and methods patient samples and assays the comparison was performed with 202 consecutive routine requests for cystatin c analysis. plasma cystatin c measurements were first performed by latex enhanced reagent (n latex cystatin c, dade behring, deerfield, il, usa) using a behring bn prospec analyzer (dade behring) and calibrators from dade behring. the test was performed according to the recommendation of the manufacturer. the total analytical imprecision of the method was 4.8 % at 0.56 mg/l and 3.7% at 2.85 mg/l. the results from the instrument was then used to calculate and report a “cystatin c calculated gfr” using the formula y = 77.24x-1.2623(9). the study was approved by the local ethical board at uppsala university (01-167). plasma cystatin c measurements on architect ci8200 was performed using the following instrument settings: primary wavelength 572 nm, sample blank and spline calibration method. 145 ml reagent 1 (3 ml supplement reagent (dade behring) and 42 ml diluent (dade behring)) was mixed with 20 �l reagent 2 (undiluted n latex cystatin c, dade behring) and 15 �l sample diluted 1:50 with dade behring diluent. the sample dilution was performed automatically by the instrument. statistical calculations statistical analysis was performed utilizing excel 2000 (microsoft corporation, seattle, wa, usa). results imprecision data for the architect ci8200 the total imprecision of the instrument was analyzed at 0.96 mg/l (cv 4.19%; n=22) and 1.96 mg/l (cv 5.43%; n=22). the imprecision was also tested by running 80 sam210 ples in duplicate. the cv for these duplicate samples was 3.98% (mean 1.48 mg/l; range 0.58-3.84 mg/l). correlation between cystatin c analyzed on prospec and architect ci8200 cystatin c values analyzed on both instruments showed strong agreement (r2 = 0.987, fig. 1). the linear regression analysis showed a slope very close to 1.00 and an intercept close to 0 (y = 1.0072x + 0.0042). the bias plot (fig. 2) displayed a good agreement between the two methods within the studied range. hemolytic, icteric and lipemic samples were included in the comparison. the results from these samples also showed good agreement between the two methods. discussion glomerular filtration rate (gfr) is generally accepted as the best overall index of renal function and is an important marker for renal disease. reduced gfr influences the metabolism and clearance of many pharmaceuticals used today. thus, in many cases the recommended dose has to be adjusted depending on the patient´s gfr. for 211 fig 1. correlation between cystatin c in 202 patient samples analyzed on a prospec (x-axis) and architect ci8200 (y-axis). the filled line indicates the linear correlation between the two methods and the dotted line indicates the 45o angle. instance, antibiotics and cytotoxic drugs are usually prescribed according to gfr. there is thus a need for gfr markers. inulin, iohexol and 51cr-edta clearances are considered the golden standards for gfr measurements. the disadvantage with these assays is that they are cumbersome, costly and slow which may delay the start of treatment. assays such as plasma creatinine and cystatin c can provide rapid test results. creatinine often overestimates gfr in patients with slight reductions in gfr. it is also difficult to evaluate creatinine in elderly patients with low muscle mass. these patients may have creatinine values in their normal range due to the combination of low muscle mass and reduced gfr. in sweden, calculation of gfr, using the cockcroft-gault equation (3), is generally performed manually in the wards. such manual calculations are time consuming and therefore costly. this led to the development of formulas to automatically convert cystatin c in mg/l to a calculated gfr in ml/min (9,10). to increase the quality of gfr measurements our hospital has tried to replace plasma creatinine measurements with plasma cystatin c for patients that require a more exact gfr quantification e.g. for prescribing pharmaceuticals that are eliminated through the kidneys. the rapidly increasing cystatin c volumes illustrate the clinical value of the assay. we also expect an increased use of cystatin c as a risk marker for cardiovascular disease and mortality in the future (11,12). to meet this demand it would be advantageous to move the assay to a chemistry instrument to improve the handling of the samples. we here report the first method using dade behring reagents on a chemistry instrument (architect ci8200). the close correlation between the two assays shows that the same formula for calcula212 fig. 2. bias plot for cystatin c analyzed on a prospec and architect ci8200 (n=202). 213 tion of gfr in ml/min can be used. references 1. schwartz gj, haycock gb, edelmann jr, cm, spitzer a (1976). a simple estimate of glomerular filtration rate in children derived from body length and plasma creatinine. pediatrics 58: 259-63. 2. kemperman fa, krediet rt, arisz l (2002). formula-derived prediction of the glomerular filtration rate from plasma creatinine concentration. nephron 91: 547-58. 3. cockcroft dw, gault mh (1976). prediction of creatinine clearance from serum creatinine. nephron 16: 31-41. 4. levey as, bosch jp, lewis jb, greene t, rogers n, roth d (1999). a more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation: modification of diet in renal disease study group. ann intern med 130: 461-70. 5. hsu cy, chertow gm, curhan gc (2002). methodological issues in studying the epidemiology of mild to moderate chronic renal insufficiency. kidney int 61: 1567-76. 6. sherman ds, fish dn, teitelbaum i (2003). assessing renal function in cirrhotic patients: problems and pitfalls. am j kidney dis 41: 269-78. 7. bode w, engh r, musil d, thiele u, huber r, karshikov a, brzin j, kos j, turk v (1988). the 2.0 a x-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases. embo j 7: 2593-9. 8. dharnidharka vr, kwon c, stevens g (2002). serum cystatin c is superior to serum creatinine as a marker of kidney function: a meta-analysis. am j kidney dis 40: 221-6. 9. larsson a, malm j, grubb a, hansson lo (2004). calculation of glomerular filtration rate expressed in ml/min from plasma cystatin c values in mg/l. scand j clin lab invest 64: 25-30. 10. grubb a, björk j, lindström v, sterner g, bondesson p, nyman u (2005). a cystatin c based formula without anthropometric variables estimates glomerular filtration rate better than creatinine clearance using the cockcroft gault formula. scand j clin lab invest 65: 153-62. 11. jernberg t, lindahl b, james s, larsson a, hansson l-o, wallentin l (2004). cystatin c: a novel predictor of outcome in suspected or confirmed non st-elevation acute coronary syndrome. circulation 110: 2342-8. 12. shlipak mg, sarnak mj, katz r, fried lf, seliger sl, newman ab, siscovick ds, stehmanbreen c (2005). cystatin c and the risk of death and cardiovascular events among elderly persons. n engl j med 352: 2049-60. corresponding author: anders larsson department of medical sciences, university hospital, s-751 85 uppsala, sweden telephone: 46-18-6110000 fax: 46-18-552562 e-mail: anders.larsson@akademiska.se 214 ujms109_3.pdf upsala j med sci 109: 239–246, 2004 mast cells in testicular lesions sharanamma m kollur,1 v.l.pattankar,2 imad abdien el hag,1 central laboratories, paras central hospital,1 aljouf, saudi arabia and department of pathology, m.r.medical college,2 gulbarga, india. abstract a free full-text copy of this article can be found at the web page of upsala j med sci: http://www.ujms.se the study was performed on orchidectomized tissue and testicular biopsies sent for histopathological examination which included; 9 cases of orchitis, 6 pyocele, 9 haematocele, 13 seminomas, 5 embryonal cell carcinoma, 2 teratocarcinoma, 2 lymphoma, 4 yolk sac tumor, 17 infertility lesions and 6 normal. toluidine blue stained sections were examined under high power magnification (hpm) and the number of mast cell present in 10 consecutive fields was counted. there was a considerable variation in the number and distribution of mast cells in various testicular lesions. mast cells were observed mainly in the areas of inflammatory infiltrate, granulation tissue and immature fibrous tissue. in infertility, interstitium and tubular walls were the areas of predilection for the presence of mast cells. the highest number of mast cells was noted in infertility (23/hpm), compared to inflammatory/reactive lesions (19/hpm) and testicular neoplasms (2/hpm). the highest and the lowest mast cell concentration were observed in infertility and testicular tumours compared to inflammatory/reactive lesions, respectively. the role of mast cells in the pathogenesis of infertility and testicular tumourogenesis requires further investigation. introduction the mast cell is a connective tissue cell which possesses cytoplasmic granules that stain metachromatically with a variety of stains including cresyl violet, toluidine blue and giemsa. it is found around the blood vessels, nerves, glandular duct (1) as well as in inflammatory and neoplastic foci (2–10). mast cells contain important biologically active substances e.g. heparin, histamine and 5-oh-tryptamine (5ht). these substances are involved in various inflammatory and immunological reactions (11). distribution of mast cells in different lesions involving lymph nodes 239 received 15 april 2004 accepted 3 june 2004 key words: mast cells, testicular lesions. (12), breast (6), uterus (13), cervix (14), and prostate (15) were studied, as well as its presence in wound healing (16), pterygium (17), atherosclerosis (18) and other disorders (2, 6, 7, 9). the presence of mast cells in normal testis of man and other species has been reported by various authors (1). however information about mast cell distribution in various testicular lesions is scanty. in this study, the distribution of mast cells in different testicular lesions was studied and their possible role in the pathogenesis of some testicular lesions is conveyed as well. materials and methods paraffin embedded orchidectomized tissue or testicular biopsies diagnosed at the department of pathology, m.r.medical college, gulbarga, india, were used in this study. the study was approved ethically. categories the cases were divided among the following categories: normal testicular tissue, 6 cases, specific and non-specific orchitis, 6 cases, pyocele and haematocele, 15 cases, testicular tumours, 26 cases (13 seminomas, 2 lymphomas, 4 yolk sac tumours, 2 teratocarcinomas, 5 embryonal cell carcinomas), and 17 cases of infertility lesions. mast cell staining and counting sections of 4–5 microns thick were stained by hematoxylin and eosin (h & e) as routinely and also with 1% aqueous toluidine blue for mast cell. h & e stained sections were used to confirm the histological diagnosis whereas, toluidine blue stained sections were used to identify and to count the mast cell. the number of mast cell present in 10 consecutive high power fields was counted in all the sections. the mast cell count was expressed as mean and range of mast cells per hpm. mast cell counts in various testicular lesions were tabulated and compared. the anatomical distribution of mast cell was also observed. statistical analysis mann-whitney non-parametrical test was used for statistical analysis. results mast cell count the range and the mean number of mast cells per high power magnification (hpm) in normal testicles and various testicular lesions were shown in the table1. there was considerable variation in the number and distribution of mast cells in different testicular lesions. the least number of mast cells was observed in neoplastic (2/hpm) lesions, although not statistically significant but lower than their respective number in normal testicular tissue (8/hpm). the highest number of mast cells was seen in haematocele (30/hpm).the next highest was noted in testicular lesions of 240 infertility (23/hpm) followed by pyocele, tubercular orchitis and non-specific orchitis showing a mean of 20, 15, and 12 per hpm, respectively. the mean number of mast cells in testicular tumours (2/hpm) was markedly lower than that of inflammatory and reactive lesions (orchitis, pyocele, haematocele) (19/hpm). the number of mast cells in infertility (23/hpm) was significantly higher than that of the latter group. p<0.05 (table.2) mast cell distribution in haematocele and pyocele the mast cells were seen mainly in the tunica and the interstitium. they were concentrated in the areas of granulation tissue and immature connective tissue. in orchitis, they were seen mainly among other inflammatory cells and around epithelioid cell granulomas. in neoplastic lesions, when present they were mainly in the tunica and practically absent in the tumor parenchyma. the interstitium and seminiferous tubular walls were the areas of predilection for the presence of mast cells in cases of infertility. discussion the present study is a preliminary approach to comment on the mast cell distribution in various common testicular lesions. the mast cells with a battery of crucial chemical mediators and substances in their typical metachromatic granules are known to play a role in health and various disease states in man. because of the 241 table 1. mast cell count (mean and range) in various testicular lesions. number of mast cell per hpm category mean range normal 08 06–10 haematocele 30* 07–44 pyocele 20* 10–28 infertility 23* 06–43 tubercular orchitis 15 06–25 non-specific orchitis 12 03–20 testicular tumours 02 00–05 * p < 0.05 as compared to normal. table 2. mast cell count (mean and range) in various group of lesions number of mast cells per hpm category mean range normal 08 06–10 inflammatory/reactive 19* 03–44 infertility 23* 06–43 neoplastic 02 00–05 * p < 0.05 as compared to normal and neoplastic lesions. 242 fig.1. testicular atrophy. a) increased mast cells seen in the interstitium. (toluidine blue, x 400) b) mast cell in the tubular wall (arrow head). (toluidine blue, x 1000) a b multifarious role of mast cells in oedema formation, angiogenesis and fibrogenesis, it is logical to infer that mast cell alteration could be found in various inflammatory and neoplastic disorders (2, 3, 6, 7, 8, 9). therefore our findings of increased mast cells in various testicular inflammatory/reactive lesions namely, pyocele, orchitis (specific/non-specific) and haematocele are not surprising. the accumulation of mast cells within and around the tumour areas is well known (19–23). the interaction between the mast cell and tumour cell may play a role in the pathophysiology of host reaction to neoplasia. the exact functional significance of such phenomenon is a subject of controversy as in some studies mast cells have been shown to be cytotoxic for some tumours, while in other studies mast cell products was found to promote tumour growth and metastasis (24). in the present study the mast cells were markedly reduced or absent in testicular tumours. similar observation was made in cervical (14) and prostatic neoplasia (15). this is in contrast to their prevailing presence in other tumours such as squamous cell carcinoma of oesophagus (19), lung adenocarcinoma (20), brain tumors (21), melanocytic tumors (22) and breast cancer (23). since many years, it has been observed that, the testicle is an immunologically privileged site. an antigen within it fails to elicit immune response (25). it turns out that, cells in this site, namely sertoli cell, differ from other cells of the body in that they express high levels of fas-l. thus, any cell that expresses fas, perhaps may be killed when they enter this site 243 fig.2. non-specific orchitis. note the presence of mast cells among inflammatory cellular infiltrate and in the premature fibrous tissue. (toluidine blue, x 200) and the mast cell expresses this fas (26). would it be the mechanism behind the reduced number of mast cell in testicular neoplasms, compared to the other sites neoplasms? this needs precise investigation. mast cell count can be used as a prognostic marker in tumours and their number may indicate poor prognosis (19, 20). in view of reduced number or absent mast cell in testicular tumours, a prognostic role is not valid but, they can have a role in differentiating between some neoplastic and inflammatory/reactive testicular lesions. our findings of significantly increased number of mast cells in testicular atrophy (23/hpm), compared to normal (08/hpm) are very interesting. the present results are similar to those published by others (27–29) who highlighted the increased number of mast cells in testicular lesions associated with infertility. meineke et al (29) observed, as we did, a shift of mast cells from interstitium to tubular wall. hashimoto et al (27) used a mast cell blocker for the treatment of patients with idiopathic infertility. an infertile male with idiopathic azoospermia was treated with administration of a mast cell-blocker, tranilast (a.g. scientific, inc., san diego, usa) for one year. the patient was found to have sperms within his ejaculate. however, the ultimate goal of pregnancy was not achieved. recently, hibi et al (30) reported a more significant effect of tranilast on patients with idiopathic oligozoospermia. the number of sperms was significantly increased and pregnancy was achieved in three patients. therefore, we believe that, mast cell may play a role in the pathogenesis of infertility, which has to be elucidated. the cases in the present study though not very large, appear to be quite sufficient to draw logical conclusions which might prove to be of additional value. in conclusion, the number of testicular mast cells was significantly increased in various testicular inflammatory/reactive lesions and lesions associated with infertility, whilst their number in neoplasms was exceptionally reduced. the role of mast cell in the pathogenesis of infertility and testicular tumourogenesis needs further research. references 1. selye h (1965). the mast cells. washington butterworths: 1–67, 281–300. 2. claman hn (1990). mast cells and fibrosis. rheum dis clin north am 16: 141–151. 3. pesci a, majori m, piccoli ml, casalini a, curti a, franchini d, gabrielli m. 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(2001). the treatment with tranilast, a mast cell blocker, for idiopathic oligozoospermia. arch androl 47: 107–111. corresponding author: dr. sharanamma m kollur p o box 961 sakaka al-jouf saudi arabia email: sharanamma@hotmail.com 245 tf-iups160016 196..197 letter to the editor dysfunction of small airways and prevalence, airway responsiveness and inflammation in asthma: much more than small particle size of pet animal allergens dear editor, we read with interest the excellent article from patelis et al. (1) showing that sensitization to small aeroallergens, produced by common pets, was associated with local airway and systemic inflammation, airway responsiveness, and higher prevalence of asthma. we believe that the topic of ‘animal allergy’ may be of great interest not only for clinicians but also for emotional implications in all pet-owner patients, especially in children and in atopic individuals who wish to own a pet. the love for animals in general, and for pets in particular, is increasing worldwide. therefore, it is necessary to evaluate the role of exposure to pet allergens related with the risk factors inducing bronchial asthma, which may also involve small airways. we were not surprised by the findings of patelis et al. (1) considering the well-known capacity of smaller pet-allergencarrying particles to reach peripheral airways and induce significant bronchoconstriction (2). in addition, the high rate of pet ownership in northern europe may further increase the direct exposure to pets. however, in our opinion, some important limitations to the conclusions of this research have not been included in the list of weaknesses already acknowledged by the authors. first of all, it has been shown that dysfunction of the small airways must be considered in a distinct asthma phenotype (3) characterized by several clinical characteristics such as poor disease control, frequent exacerbations, and high degree of severity of bronchial hyper-responsiveness. it is important to underline that several non-allergenic and allergenic agents are able to reach peripheral airways, inducing inflammatory events similar to those determined by pet allergens. outdoor air pollutants, such as pm2.5 and indoor cigarette smoke, represent the most common non-allergenic agents which determine a ‘synergistic interaction’ with allergic sensitization when these compounds are inhaled together with allergens. moreover, they can also determine directly airway inflammation and asthma exacerbation in already diagnosed asthmatics (4,5). apart from pets, other allergenic sources can release allergens in ‘respirable’ (0.5–3 lm diameter) form such as dust mites (6), moulds (e.g. alternaria and aspergillus spp.) (7), and pollens (e.g. parietaria, grasses, birch, olea europaea, artemisia) (8). in particular, pollen grains can induce so-called ‘thunderstorm asthma’ (9,10). in wet conditions, just before the onset of a thunderstorm, atmospheric pollens can release pauci-micronic particles containing allergens. the inhalation of these particles may induce severe asthma exacerbations in some highly sensitized individuals that, in turn, may even require emergency department visits or hospitalization. in conclusion, we agree that about 20% of pet-allergencarrying particles are characterized by aerodynamic properties of reaching small airways, and that these particles are likely to induce intensive inflammatory events. however, many other allergenic/non-allergenic agents are able to induce similar responses in association or not with pet allergens. in geographical areas with a lower rate of pet ownership (and a prevalent ‘indirect’ modality of exposure to pets), previously described agents are likely to be more decisive in inducing asthma with prevalent small airways involvement. finally, dysfunction of small airways is now hypothesized also as a ‘remodelling phenotype’. in other words, individual subjects react to different environmental stimuli with a prevalent ‘peripheral’ response. considering the relevant implications of pet–human relationships, especially in children, these considerations must be taken into account in order to avoid unjustified overestimation or generalization of the role of pet allergens in inducing a higher degree of asthma severity. acknowledgements all authors contributed equally in the writing and revision of the manuscript disclosure statement all authors declare that they have no conflict of interest. funding information the study was carried out without any financial support. references 1. patelis a, dosanjh a, gunnbj€ornsdottir m, borres m, h€ogman m, alving k, et al. new data analysis in a population study raises the hypothesis that particle size contributes to the pro-asthmatic potential of small pet animal allergens. ups j med sci. 2016;121:25–32. 2. zeidler mr, goldin jg, kleerup ec, kim hj, truong da, gjertson dw, et al. small airways response to naturalistic cat allergen upsala journal of medical sciences, 2016 vol. 121, no. 3, 196–197 http://dx.doi.org/10.3109/03009734.2016.1173745 exposure in subjects with asthma. j allergy clin immunol. 2006;118:1075–81. 3. lipworth b, manoharan a, anderson w. unlocking the quiet zone: the small airway asthma phenotype. lancet respir med. 2014;2:497–506. 4. fan j, li s, fan c, bai z, yang k. the impact of pm2.5 on asthma emergency department visits: a sytematic review and meta-analysis. environ sci pollut res int. 2016;23:843–50. 5. price d, bjermer l, popov ta, chisholm a. integrating evidence for managing asthma in patients who smoke. allergy asthma immunol res. 2014;6:114–20. 6. custovic a, woodcock h, craven m, hassal r, hardley e, simpson a, et al. dust mite allergens are carried on not only large particles. pediatr allergy immunol. 1999;10:258–60. 7. afanou ka, straumfors a, skogstad a, navak ap, skaar i, hjeliord l, et al. indirect immunodetection of fungal fragments by field emission scanning electron microscopy. appl environ microbiol. 2015;81:5794–803. 8. bacsi a, choudhury bk, dharajiya n, sur s, boldogh i. subpollen particles: carriers of allergenic proteins and oxidases. j allergy clin immunol. 2006;118:844–50. 9. d’amato g, cecchi l, liccardi g. thunderstorm-related asthma: not only grass pollen and spores. j allergy clin immunol. 2008; 121:537–78. 10. d’amato g, liccardi g, frenguelli g. thunderstorm-asthma and pollen allergy. allergy. 2007;62:11–16. gennaro liccardi department of chest diseases, division of pneumology and allergology, high speciality a. cardarelli hospital, naples italy postgraduate school of respiratory medicine, department of systems medicine, university of rome tor vergata, rome, italy gennaro.liccardi@tin.it antonello salzillo and amedeo piccolo department of chest diseases, division of pneumology and allergology, high speciality a. cardarelli hospital, naples italy luigino calzetta and paola rogliani department of systems medicine, university of rome tor vergata, rome, italy postgraduate school of respiratory medicine, department of systems medicine, university of rome tor vergata, rome, italy received 13 january 2016; accepted 29 march 2016 � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. http://dx.doi.org/10.3109/03009734.2016.1173745 upsala journal of medical sciences 197 dysfunction of small airways and prevalence, airway responsiveness and inflammation in asthma: much more than small particle size of pet animal allergens acknowledgements disclosure statement funding information references vol_116_002_sups_a_519446 124..128 upsala journal of medical sciences. 2011; 116: 124–128 original article kinetics of inflammatory markers following cancer-related bowel and liver resection sándor márton1, jános garai2, valéria molnár2, vera juhász1, lajos bogár1, tamás köszegi3, boglárka falusi1 & subhamay ghosh1 1university of pécs, department of anaesthesiology and intensive therapy, pécs, hungary, 2university of pécs, department of pathophysiology and gerontology, pécs, hungary, and 3university of pécs, department of clinical chemistry, pécs, hungary abstract background. macrophage migration inhibitory factor (mif) was originally described as a cytokine that inhibits migration of macrophages at the site of inflammation. subsequently it was also identified as a stress-induced hormone released from the anterior pituitary lobe in response to some pro-inflammatory stimuli like endotoxins and tumour necrosis factor (tnf-a). aim. to compare postoperative changes in serum mif levels of patients undergoing bowel and liver resections. it has clinical relevance to describe the kinetics of this crucial mediator of systemic inflammation in surgery. methods. a total of 58 patients were studied over 4 years. group a (28 patients) underwent only hepatic resection without enterotomy. group b (30 patients) had bowel resection with enterotomy. mif, il-1b, il-8, prealbumin, albumin, a1-glycoprotein, fibrinogen, and c-reactive protein levels were measured preoperatively, immediately following surgery, and postoperatively for three consecutive days. to evaluate organ functions, multiple organ dysfunction score was used. results. a significantly higher level of mif (4,505 pg/ml) was found in group a when compared to that of group b immediately following surgery. other parameters monitored in this study were not statistically different between the two groups. conclusion. higher elevations in mif levels with liver resections, compared to bowel resections, might be attributable to mif release from damaged liver cells. the presumably minimal endotoxin exposure during bowel surgery was either insufficient or inefficient to induce relevant mif elevations in our patients. to fully delineate implications of this finding further studies are needed. key words: c-reactive protein, endotoxin, macrophage migration inhibitory factor, procalcitonin, tumour necrosis factor introduction macrophage migration inhibitory factor (mif) was the first representative of potent immunomediator cytokines that have been described from the beginning of the 1960s (1). its release from activated t lymphocytes was first considered as its sole source, and its action was thought to be limited to prevention of macrophage migration away from the site of inflammation. mif is now considered to be a stressinduced cytokine hormone produced by several immune cell types, as well as by the anterior pituitary gland, and its most important role is in the counterregulation of the anti-inflammatory effects of glucocorticoids (2). the stress-induced mif secretion from the adenohypophysis is primarily increased by proinflammatory stimuli, i.e. endotoxin, tumour necrosis factor, and also by glucocorticoids (3). in-vivo models have shown that the level of hypophyseal mif fell significantly after intraperitoneal lipopolysaccharide injection accompanied by an correspondence: dr subhamay ghosh, department of anaesthesiology and intensive therapy, university of pécs, ifjuság u. 13, 7624 pécs, hungary. fax: +36 72 536 441. e-mail: gh_o_sh@yahoo.ca (received 27 may 2010; accepted 23 august 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.519446 increase of mif mrna synthesis and raised mif levels in the serum (4). an interesting study showed that endotoxin-related mortality was increased by mif in mice, while administration of anti-mif antibodies revealed the contrary (5). in hypophysectomized mice calandra et al. attributed bacterial infection-induced mif production primarily to macrophages (6). a systematic analysis of mif expression following administration of lipopolysaccharide revealed that within 6 hours of bacterial inoculation mif production increased not only in the pituitary and the macrophages, but also in the lungs, adrenal glands, spleen, and the liver of rats (7). clinical studies have shown that mif has a primary role in endotoxaemia-induced toxic reactions, in sepsis (8), and acute respiratory distress syndrome (ards) (9). in critically ill patients, suffering from septic shock, an increased level of mif was found, but this was lower in the survivors compared to the nonsurvivors (10). surgical trauma is associated with an increased level of adrenocorticotropic hormone (acth) and cortisol (11). in patients suffering from blunt trauma mif levels were higher in the non-survivor than in the survivor groups (12). even higher serum levels of mif were found by satoshi et al. in patients undergoing liver resection for treatment of hepatic neoplasm (13). in our prospective, descriptive clinical study we investigated the changes of mif levels and other indicators of systemic inflammation in patients undergoing bowel resection with presumably higher endotoxin levels (14,15), compared with patients having hepatic resection without enterotomy. based on our hypothesis, we expected a significant increase in mif levels in both groups. following that, we correlated it with early postoperative morbidity and mortality. methods patients following the approval of the local ethical committee, 58 patients were studied between october 2004 and december 2008. group a included cancer patients who had liver resection without enterotomy (28 patients). in group b, patients underwent bowel resection with enterotomy (30 patients). based on our exclusion criteria, patients undergoing emergency operations without bowel preparation, inoperable carcinoma, and patients with chronic organ failure based on acute physiology and chronic health evaluation (apache ii) scores were excluded from the investigation. methods in all the patients, anaesthesia was induced with propofol (2 mg/kg) and fentanyl (0.15 mg/kg); muscle relaxation was achieved with atracurium (0.5 mg/kg, then 0.1 mg/kg in repeated boluses pro re nata). anaesthesia was maintained with sevoflurane in a n2o/o2 2:1 mixture. prior to surgery an epidural catheter was placed, under local anaesthesia, in accordance with the dermatomes of the operative site to administer 0.2% ropivacaine and fentanyl (5 mg/ml) in a continuous epidural infusion. both groups received a single i.v. dose of prophylactic antibiotics, cefuroxime and metronidazole (1.5 g and 0.5 g, respectively). the levels of mif, tumour necrosis factor (tnf-a), interleukin (il)-lb, il-8, prealbumin, albumin, fibrinogen, and c-reactive protein (crp) were preoperatively documented at 60 minutes prior to the procedures (t0), postoperatively at 60 minutes immediately after the surgery (t1), and on three consecutive postoperative days at 24, 48, and 72 hours respectively (t2, t3, t4). to evaluate disease progression and the extent of organ failure the multiple organ dysfunction score (mods) elaborated by marshall et al. (16) that considers the functional parameters of six organ systems was used. the score was determined on admission and later in the first 3 days. performance of the respiratory system was assessed by the pao2/fio2 ratio, and for the cardiovascular system the pulse rate (p), the central venous pressure (cvp), and the mean arterial pressure (map) were monitored. hepatic and renal functions, along with the haematopoietic system, were evaluated from the serum bilirubin, creatinine levels, and the platelet count, respectively. central nervous system (cns) function was assessed with the glasgow coma scale. mods, which ranges between normal function (0 points) and severe multiorgan failure (4–20 points), was calculated as a sum of the individual organ functions. cytokine assays serum mif levels were measured by an enzymelinked immunosorbent assay (elisa) with a duo set elisa development system from r&d systems, minneapolis, usa, according to manufacturer’s instructions. every sample was diluted in the respective elisa assay buffer. serum il-1b, il-8, and tnf-a were determined by an automated chemiluminescent immunoassay analyser, dpc (now siemens) immulite 1000, deerfield, usa. for procalcitonin assays, the immunochemiluminescent kit of brahms diagnostica, berlin, germany was used in a berthold lb inflammatory markers in abdominal surgery 125 9507 luminometer with two programmable injectors. albumin levels were measured by the bromo-cresolgreen dye binding method (hitachi 917, roche), while prealbumin and a1-glycoprotein concentrations were assayed by immunoturbidimetry (hitachi 704, orionpharma). c-reactive protein concentrations were determined using a beckman immage nephelometer. plasma fibrinogen levels were assayed by direct determination of fibrinogen (system ca 1500) using stago thrombin reagent. statistical analysis the data are shown in median and interquartile ranges. the statistical analysis was carried out using the statistical program for social sciences for windows (spss), using mann-whitney and kolmogorovsmirnov and analysis of variance (anova). the correlation of the two variables was tested using spearman’s rank correlation analysis. the temporal changes of the examined parameters were analysed by the wilcoxon test. the level of significance was set at p < 0.05. results the demographic characteristics of the patients and the details of the operations are summarized in table i. the average duration of the surgical procedure was significantly longer in group b compared to that of group a. there were no differences concerning intraoperative transfusions. macrophage migration inhibitory factor in both groups the mif preoperative values (mif0) were found to be within the normal range, and there was no significant difference between the two groups. immediately following surgery the mif values (mif1) measured in those patients undergoing hepatic resection were significantly higher (4,505 pg/ml) compared to the bowel resection group (1,774 pg/ml) shown in table ii. on the first postoperative day normal values of mif were measured in both groups, and these values did not change on the second or the third postoperative days as shown in figure 1. procalcitonin on the first postoperative day, both groups showed higher procalcitonin levels (pct2) than the values reported in literature (17). it slowly declined on subsequent days (pct3, pct4), but these changes were not significant compared to the initial values in these groups. tumour necrosis factor in group a the average tnf-a level was found to increase moderately but gradually; on day 3 decreased levels were observed in group a: tnf-a0: 5.1 pg/ml (4–19 pg/ml); tnf-a3: 8.5 pg/ml (4.9–9.32 pg/ ml). in group b, there was also a gradual increase, with highest levels on day 3: tnf-a0: 5.1 pg/ml (4–6 pg/ml); tnf-a3: 8.9 pg/ml (7.3–16.2 pg/ml). no significant difference was found between the two groups concerning daily tnf-a levels. no correlation was observed in groups a and b between the high mif1 and tnf-a levels. table i. demographic data of patients and multiple organ dysfunction scores (mods) in the two groups. group a (n = 28) group b (n = 30) p age (years) 53 (34–65) 63 (22–77) ns gender (m/f) 19/11 21/7 ns duration of operation (min) 160 (90–270) 240 (60–440) <0.001 body weight (kg) 71 (54–85) 80 (63–118) ns height (cm) 169 (154–183) 173 (160–194) ns mods preoperatively (t0) 2 (1–3) 2 (1–3) ns mods postoperatively (t1) 2 (1–3) 1 (1–3) ns mods day 1 (t2) 2 (1–3) 1 (1–3) ns mods day 2 (t3) 2 (1–3) 2 (1–3) ns mods day 3 (t4) 2 (1–4) 3 (0–3) ns our data are presented as median (interquartile range). for the statistical analyses the mann-whitney test was used. the duration of operation was significantly higher in group b. table ii. macrophage migration inhibitory factor (mif) values in the two groups. groups group a group b anova p mif0 1137.87 ± 741.53 1462.94 ± 1276.49 ns mif1 4505.87 ± 551.72 1774.13 ± 1125.39 0.001 mif2 1661.75 ± 869.88 1506.28 ± 1097.42 ns mif3 1558.50 ± 1113.55 1490.97 ± 1358.18 ns mif4 1302.33 ± 741.25 996.24 ± 913.85 ns values are means ± sd. for the statistical analyses the anova test was used. 126 s. márton et al. c-reactive protein in the first 24 hour the crp level was within the normal range (crp0, crp1), but all subsequent values fell in the pathological range (crp2, crp3, crp4). other parameters in both groups a gradual decrease of albumin and prealbumin levels were observed without significant difference between groups. similarly, no significant difference was observed regarding the il-lb, il-8, a1-glycoprotein, and fibrinogen levels. organ dysfunctions data obtained with mods are summarized in table i. there was no statistically significant difference between the two groups. there was no correlation between mif levels and early postoperative morbidity and mortality (spearman’s rho = 0.21; p = 0.24). discussion sympathetically activated stress reactions might contribute to multi-organ failure in the postoperative period of major abdominal operations. in the pathogenesis of this process, pro-inflammatory cytokines (tnf-a, il-1, il-6) along with systemic endotoxaemia may play a pivotal role. the secretion of mif has been shown to induce production of these critical pro-inflammatory cytokines and has been detected in several organs. the main sites of mif production are the immune cells, i.e. monocytes, macrophageactivated t lymphocytes, and also the anterior pituitary. the most frequent causes of mif release are pro-inflammatory stimuli, especially endotoxins, tnf-a, and interferon-g. systemic endotoxaemia may follow complement-driven systemic activation of the inflammatory cascade that damages the microcirculation, leading to ischaemia and reperfusion injuries, and may also emerge from bacterial translocation due to tissue hypoperfusion of the bowel wall. in our prospective, descriptive study we assessed the induction of macrophage migration inhibitory factor levels in cancer patients undergoing bowel resection (elective enterotomy) in comparison with cancer patients undergoing liver resection (without enterotomy). gando et al. reported the highest mif levels at 24 hours following hepatolobectomy, and this was explained by stress-induced hypothalamohypophyseal activation and also by the liberation of endotoxins (13). in our study, in group a, a significantly higher level of mif was detected immediately after the procedures that normalized on the first postoperative day. no association was observed between elevated mif levels and early postoperative group ba m a cr o p h a g e m ig ra tio n in h ib ito ry f a ct o r (p g /m l) 10000 8000 6000 4000 2000 0 –2000 mif0 mif1 mif2 mif3 mif4 * figure 1. serum mif levels preoperatively (mif0), immediately postoperatively (mif1), and on the three consecutive days (mif2, mif3, mif4). the data were plotted as a ‘box plot’. the minimum–maximum and interquartile ranges are shown on the figure. the difference in the two groups was verified by the mann-whitney u test. *p < 0.006. inflammatory markers in abdominal surgery 127 complications. there was no correlation of immediate postoperatively elevated mif levels with the kinetics of cytokines and acute phase proteins measured in this study. it has been reported that the most effective stimulus of pct release is bacterial endotoxin, hence similar pct levels of the groups here might reflect comparable endotoxin exposure (17). the surgical stimulus activates the hypothalamohypophyseal-adrenal system, resulting in increased plasma acth, glucocorticoid, and mif levels (18). with the help of immunohistochemical analysis, mif has been expressed in hepatocytes in the areas extending out from the central veins to the portal tracts (19). hira et al. have shown mif expression to play a crucial role in hepatocellular carcinoma (20). consistent with the notion, since both of our experimental groups comprised cancer patients and the preoperative mif values did not differ, we assume that highly elevated immediately postoperative mif levels of patients undergoing liver resection are explained by tissue damage-related release of mif from hepatocytes rather than by inflammatory stimulus-driven secretion. this hypothesis is further supported by our findings that the operations lasted significantly longer in group b, hence we assumed that the inflammatory response is greater and the mif secretion was significantly lower. despite the longer operation time, the surgical insult corresponded well in the two groups, therefore a similar increase of the pct and tnf-a levels, in accordance with previous reports including ours, could be attributed to surgical tissue damage (17,21). our study was limited to a certain extent by the unavailability of the liver function tests, but it serves as a pilot investigation for further studies to delineate the postoperative kinetics of mif in different surgical procedures. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. bloom br, bennett b. mechanism of a reaction in vitro associated with delayed-type hypersensitivity. science. 1966; 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bdivision of orthopedic surgery, nakadori general hospital, 3-15, misono-cho, minami-dori, akita 010-8577, japan abstract background: multiple factors are involved in the development of atypical femoral fractures, and excessive curvature of the femur is thought to be one of them. however, the pathogenesis of femoral curvature is unknown. we evaluated the influence of factors related to bone metabolism and posture on the development of femoral curvature. methods: a total of 139 women participated in the present study. curvatures were measured using antero-posterior and lateral radiography of the femur. we evaluated some bone and vitamin d metabolism markers in serum, the bone mineral density (bmd), lumbar spine alignment, and pelvic tilt. results: we divided the women into two groups, curved and non-curved groups, based on the average plus standard deviation as the cut-off between the groups. when univariate logistic regression analysis was performed to detect factors affecting femoral curvature, the following were identified as indices significantly affecting the curvature: age of the patients, serum concentrations of calcium, intact parathyroid hormone, pentosidine, homocysteine and 25-hydroxyvitamin d (25(oh)d), and bmd of the proximal femur (p < 0.05) both in the lateral and anterior curvatures. when we used multivariate analyses to assess these factors, only 25(oh)d and age (lateral and anterior standardized odds ratio: 0.776 and 0.385, and 2.312 and 4.472, respectively) affected the femoral curvature (p < 0.05). conclusion: femoral curvature is strongly influenced by age and serum vitamin d. article history received 4 november 2015 revised 28 march 2016 accepted 27 april 2016 keywords 25-hydroxyvitamin d; atypical femoral fracture; curved femur; osteomalacia; women introduction although bisphosphonates (bps) are the gold standard for osteoporosis pharmacotherapy, several adverse effects related to their long-term use have recently been reported, such as osteonecrosis of the jaw (onj) and atypical low-energy subtrochanteric and diaphyseal femoral fractures due to markedly suppressed bone turnover (ssbt) (1,2). these fractures are typically diagnosed as atypical femoral fractures (aff). however, many patients have been reported with atypical femoral fractures (aff) in the absence of bp therapy (3), although the fracture type was consistent with the criteria of aff suggested by a task force of the american society for bone and mineral research (asbmr) (4). the detailed cause of aff has not been clarified, and multiple factors are thought to be involved in its development. sasaki et al. were the first to report that excessive curvature of the femur may be one of the associated factors, and this was also stated in a review by a task force of asbmr (4,5). however, the pathogenesis of femoral curvature in elderly women is unknown. the aim of this study was to evaluate the influence of factors related to bone metabolism and posture on the development of femoral curvature in elderly women. material and methods subjects a total of 139 women, with a mean age of 75.4 years (53 to 93), all being outpatients visiting a single institution for the treatment or examination of osteoporosis between april 2014 and march 2015, were included in this study. of the 139 patients 64 had received osteoporosis treatment prior to evaluation: bisphosphonate, vitamin d3, selective estrogen receptor modulator, teriparatide, and denosumab were prescribed in 34, 19, 13, 2, and 2 patients, respectively. in addition, a history of fragility fracture was present in 29 patients: vertebral fracture, 15 patients; femoral neck or trochanteric fracture, 13 patients; and distal radius fracture, 4 patients. we excluded patients unable to walk by themselves. clinical evaluations curvature of the right femur was measured with antero-posterior (ap) and lateral views as the angles between two linear lines drawn along the proximal and distal portions of the femoral shaft, using a method to measure femoral curvature contact hiroyuki tsuchie, md tuchikiti@yahoo.co.jp akita university graduate school of medicine, department of orthopedic surgery, 1-1-1 hondo, akita 010-8543, japan � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 3, 170–173 http://dx.doi.org/10.1080/03009734.2016.1185200 http://creativecommons.org/licenses/by/4.0/ established by sasaki et al. (5). we performed some serum laboratory examinations of bone metabolic markers, bone quality markers, and vitamin d metabolism markers: alkaline phosphatase (alp), calcium (ca) adjusted for serum albumin, inorganic phosphorus (ip), intact procollagen i n-terminal propeptide (p1np), tartrate-resistant acid phosphatase 5b (tracp5b), pentosidine, homocysteine, intact parathyroid hormone (pth), 25-hydroxyvitamin d (25(oh)d), and 1,25hydroxyvitamin d3 (1,25(oh)2d). alp, ca, and ip were measured using standard laboratory procedures. tracp5b was measured by an enzyme immunoassay method, p1np and intact pth were measured by an electrochemiluminescence immunoassay method, 25(oh)d and 1,25(oh)2d were measured by a radioimmunoassay method, and pentosidine and homocysteine were measured by a high-performance liquid chromatography method. we measured the bone mineral density (bmd) on ap views of the lumbar spine from l2 to l4 and the femoral neck (discovery, hologic inc., ma, usa). on lateral standing x-rays of the spine including the sacrum and pelvis, we measured the lumbar lordosis angle from l1 to l5 and the pelvic inclination angle to check the lumbar spine alignment and pelvic tilt (table 1) (6). statistical analysis all values are expressed as the mean ± standard deviation (sd). we divided the women into two groups, curved and noncurved groups, based on the average plus standard deviation as the cut-off between the groups for lateral and anterior curvature respectively: the lateral curvature was 6.3 degrees and anterior curvature was 12.3 degrees. because there are many evaluation items, we extracted some factors likely to have associations with femoral curvature by univariate logistic regression. multivariate logistic regression analysis was used to examine the factors of femoral curvature. probability (p) values less than 0.05 were considered statistically significant. results for lateral curvature, the curved group included 25 patients with a mean age of 82.7 years, and the non-curved group included 114 patients with a mean age of 73.8 years. for anterior curvature, the curved group included 19 patients with a mean age of 84.9 years, and the non-curved included 120 patients with a mean age of 73.9 years. the number of patients who were non-curved based on both definitions was 107, the number of patients who were curved based on the lateral but not anterior curvature definition was 13, the number of patients who were curved based on the anterior but not lateral curvature definition was 7, and the number of patients who were curved based on both lateral and anterior curvature definitions was 12. when univariate logistic regression analysis was performed to identify factors affecting femoral curvature we found that the age of the patients, serum concentrations of ca, intact pth, pentosidine, and 25(oh)d, and bmd of the proximal femur, both with regard to lateral (table 2) and anterior curvatures (table 3), differed between women belonging to the non-curved and curved groups. homocysteine, however, only did so with regard to the lateral curvature (table 2). we used multivariate logistic regression analysis to exclude a mutual influence of different factors, and then only 25(oh)d and age affected the femoral curvature (tables 4 and 5). discussion we evaluated some factors that may affect the development of femoral curvature, such as the bone turnover, bone quality, vitamin d metabolism, bmd, and posture, and only 25(oh)d and the age were selected as indices significantly affecting the curvature. osteomalacia is a condition causing the deposition failure of calcium and phosphorus in osteoids after epiphyseal line closure. the causes are broadly divided into failure of vitamin d action and hypophosphatemia; failure of vitamin d action includes the deficiency of vitamin d, abnormal vitamin d metabolism, and abnormalities of the vitamin d receptor (7). published data indicate that the presence of vitamin d deficiency should be suspected when serum 25(oh)d is lower than 20 ng/ml (8,9). in this study, curved group patients with both lateral and anterior curvature showed low serum 25(oh)d, at about 16 ng/ml. although there is a report that vitamin d becomes lower as age increases (10), the serum 25(oh)d concentrations showed significant correlations with femoral curvature both anteriorly and laterally in the present study, even when we eliminated the effect of age. it might be anticipated that vitamin d deficiency, namely osteomalacia, strongly affects the curvature of the femur. the asbmr task force 2013 revised case definition of aff is as follows: 1) the fracture is associated with minimal or no trauma, as in a fall from a standing height or lower; 2) the fracture line originates at the lateral cortex and is table 1. characteristics of study patients. total number 139 age (years) 75.4 ± 10.0 ap curvature (degrees) 2.98 ± 3.32 lateral curvature (degrees) 9.79 ± 2.48 laboratory examinations alp (iu/l) 270 ± 198 ca (mg/dl) 9.15 ± 0.33 ip (mg/dl) 3.50 ± 0.43 p1np (lg/l) 53.5 ± 33.5 tracp5b (mu/dl) 280 ± 125 pentosidine (lg/ml) 0.054 ± 0.030 homocysteine (nmol/ml) 10.0 ± 5.9 intact pth (pg/ml) 44.0 ± 20.2 25(oh)d (ng/ml) 22.6 ± 8.3 1,25(oh)2d (pg/ml) 51.1 ± 18.6 bmd (g/cm2) lumbar spine 0.722 ± 0.146 proximal femur 0.486 ± 0.114 lumbar lordosis angle (degrees) 23.1 ± 18.6 lumbo-sacral angle (degrees) 25.6 ± 10.0 steroid usage: number 5/139 average 4.4 ± 3.4 vitamin d usage: number 19/139 values are expressed as number of patients, or mean ± sd with ranges. 1,25(oh)2d: 1,25-hydroxyvitamin d3; 25(oh)d: 25-hydroxyvitamin d; alp: alkaline phosphatase; ap: antero-posterior; bmd: bone mineral density; ca: calcium; ip: inorganic phosphorus; p1np: intact procollagen i n-terminal propeptide; pth: parathyroid hormone; tracp5b: tartrateresistant acid phosphatase 5b. upsala journal of medical sciences 171 table 2. univariate logistic regression analysis of lateral curvature. variables non-curved curved standardized or 95% ci p number 114 25 age (years) 73.8 ± 9.8 82.7 ± 6.9 3.263 1.767–6.024 <0.001 laboratory examinations alp (iu/l) 268 ± 212 279 ± 117 1.053 0.768–1.567 0.797 ca (mg/dl) 9.18 ± 0.31 9 ± 0.39 0.563 0.352–0.9 0.017 ip (mg/dl) 3.52 ± 0.43 3.45 ± 0.44 0.848 0.542–1.325 0.468 p1np (lg/l) 53.3 ± 33.5 54 ± 34.2 1.019 0.662–1.57 0.93 tracp5b (mu/dl) 280 ± 123 280 ± 141 1 0.648–1.541 0.999 pentosidine (lg/ml) 0.049 ± 0.02 0.077 ± 0.053 2.158 1.363–3.416 0.001 homocysteine (nmol/ml) 9.0 ± 3.4 14.3 ± 11 2.606 1.479–4.592 <0.001 intact pth (pg/ml) 41.6 ± 18 55.0 ± 25.9 1.792 1.197–2.682 0.005 25(oh)d (ng/ml) 24 ± 8 16.4 ± 6.5 0.263 0.135–0.512 <0.001 1,25(oh)2d (pg/ml) 51.9 ± 19.1 47.4 ± 16.1 0.767 0.475–1.239 0.279 bmd (g/cm2) lumbar spine 0.73 ± 0.144 0.683 ± 0.155 0.715 0.452–1.13 0.151 proximal femur 0.503 ± 0.109 0.411 ± 0.106 0.433 0.269–0.697 <0.001 lumbar lordosis angle (degrees) 23.4 ± 19.1 21.4 ± 16.3 0.891 0.582–1.364 0.62 lumbo-sacral angle (degrees) 25.8 ± 10.5 25.0 ± 7.5 0.93 0.604–1.43 0.74 steroid usage (mg) 0.04 ± 0.26 0.72 ± 2.25 11.53 0.918–144.8 0.058 vitamin d usage (number) 16 3 0.835 0.224–3.117 0.958 values are expressed as number of patients, or mean ± sd with ranges. 1,25(oh)2d: 1,25-hydroxyvitamin d3; 25(oh)d: 25-hydroxyvitamin d; 95% ci: 95% confidence interval; alp: alkaline phosphatase; bmd: bone mineral density; ca: calcium; ip: inorganic phosphorus; or: odds ratio; p1np: intact procollagen i n-terminal propeptide; pth: intact parathyroid hormone; tracp5b: tartrate-resistant acid phosphatase 5b. table 3. univariate logistic regression analysis of anterior curvature. variables non-curved curved standardized or 95% ci p number 120 19 age (years) 73.9 ± 9.7 84.9 ± 5.4 5.413 2.391–12.26 <0.001 laboratory examinations alp (iu/l) 252 ± 135 381 ± 404 1.546 0.990–2.415 0.055 ca (mg/dl) 9.18 ± 0.31 9 ± 0.43 0.573 0.342–0.962 0.036 ip (mg/dl) 3.52 ± 0.43 3.41 ± 0.44 0.754 0.451–1.258 0.279 p1np (lg/l) 53.3 ± 33.1 54.6 ± 37.2 1.04 0.644–1.68 0.872 tracp5b (mu/dl) 279 ± 120 285 ± 159 1.045 0.648–1.686 0.856 pentosidine (lg/ml) 0.05 ± 0.02 0.082 ± 0.058 2.168 1.361–3.452 0.001 homocysteine (nmol/ml) 9.6 ± 5.8 11.9 ± 6.4 1.317 0.895–1.938 0.163 intact pth (pg/ml) 42.2 ± 19.4 54.1 ± 23 1.633 1.064–2.506 0.025 25(oh)d (ng/ml) 23.6 ± 8.1 16.8 ± 6.6 0.328 0.166–0.649 0.001 1,25(oh)2d (pg/ml) 51.3 ± 19.1 49.3 ± 15.2 0.893 0.537–1.486 0.189 bmd (g/cm2) lumbar spine 0.728 ± 0.149 0.679 ± 0.121 0.698 0.418–1.167 0.171 proximal femur 0.497 ± 0.113 0.422 ± 0.097 0.517 0.313–0.851 0.01 lumbar lordosis angle (degrees) 23.0 ± 18.2 23.2 ± 21.3 0.998 0.617–1.613 0.979 lumbo-sacral angle (degrees) 25.7 ± 10.1 24.9 ± 9.6 0.921 0.57–1.488 0.737 steroid usage (mg) 0.16 ± 1 0 – – � vitamin d usage (number) 17 2 0.713 0.151–3.367 0.943 values are expressed as number of patients, or mean ± sd with ranges. 1,25(oh)2d: 1,25-hydroxyvitamin d3; 25(oh)d: 25-hydroxyvitamin d; 95% ci: 95% confidence interval; alp: alkaline phosphatase; bmd: bone mineral density; ca: calcium; ip: inorganic phosphorus; or: odds ratio; p1np: intact procollagen i n-terminal propeptide; pth: intact parathyroid hormone; tracp5b: tartrate-resistant acid phosphatase 5b. table 4. multivariate logistic regression analysis of lateral curvature. variables standardized or 95% ci p age (years) 2.312 1.142–4.681 0.02 laboratory examinations ca (mg/dl) 0.649 0.344–1.223 0.187 pentosidine (lg/ml) 1.445 0.833–2.509 0.193 homocysteine (nmol/ml) 1.202 0.749–1.928 0.444 intact pth (pg/ml) 1.209 0.713–2.050 0.479 25(oh)d (ng/ml) 0.776 0.426–1.414 0.004 bmd (g/cm2) proximal femur 0.776 0.426–1.414 0.404 values are expressed as number of patients, or mean ± sd with ranges. 25(oh)d: 25-hydroxyvitamin d; 95% ci: 95% confidence interval; bmd: bone mineral density; ca: calcium; or: odds ratio; pth: parathyroid hormone. table 5. multivariate logistic regression analysis of anterior curvature. variables standardized or 95% ci p age (years) 4.472 1.770–11.3 0.002 laboratory examinations ca (mg/dl) 0.65 0.331–1.278 0.187 pentosidine (lg/ml) 1.551 0.926–2.596 0.096 intact pth (pg/ml) 1.145 0.658–1.994 0.632 25(oh)d (ng/ml) 0.385 0.165–0.898 0.027 bmd (g/cm2) proximal femur 1.111 0.564–2.19 0.763 values are expressed as number of patients, or mean ± sd with ranges. 25(oh)d: 25-hydroxyvitamin d; 95% ci: 95% confidence interval; bmd: bone mineral density; ca: calcium; or: odds ratio; pth: parathyroid hormone. 172 h. tsuchie et al. markedly transverse in its orientation, although it may become oblique as it progresses medially across the femur; 3) complete fracture extends through both cortices and may be associated with a medial spike, whereas incomplete fracture only involves the lateral cortex; 4) the fracture is not comminuted or is minimally comminuted; and 5) localized periosteal or endosteal thickening of the lateral cortex is present at the fracture site (‘beaking’ or ‘flaring’) (4). we previously reported a case of osteomalacia with marked femoral curvature being consistent with the aff definition (11). although plain radiographs showed areas of endosteal thickening and horizontal lines resembling fractures over the outer cortical bones of the femoral diaphysis in this case, these constituted a looser zone, a conventional x-ray sign of osteomalacia. osteomalacia not only affects the femoral curvature, considered as one of the causes of aff, but it may also have been present in previously reported aff cases with femoral curvature. saita et al. reported that the fracture sites of aff are associated with weight-bearing lower limb alignment (12), and we assumed that posture also affected it. spino-pelvic alignment of lumbar kyphosis with posterior pelvic tilt on standing requires hip joint extension, knee joint flexion, and ankle joint dorsiflexion to maintain postural balance. thus, the femur is positioned obliquely to the ground, not vertically, and excessive muscular force on the thigh may be required. although we considered that an increased load on the thigh might cause a curved femur, we could not show any relation between the curved femur and posture change, such as lumbar kyphosis and pelvic tilt. bone quality markers, such as pentosidine and homocysteine, can be used to evaluate the deterioration of bone collagen indirectly (13). deterioration of bone collagen causes a deterioration of bone quality, and the bone strength also declines. we suspected that bone quality affects femoral curvature, but we were unable to demonstrate such a relation. saita et al. indicated that steroid use may be related to aff (14), and we could also consider the influence of bone quality. although there was no relation between the presence of curved femur and steroid usage in this study (tables 2 and 3), we could not sufficiently evaluate this because steroid users only numbered 5 people. we have to perform further detailed studies with a larger number of patients treated with steroids. in conclusion, to the best of our knowledge, the present study is the first to examine the influence of factors related to bone metabolism and posture on the development of femoral curvature in elderly women. such curvatures were strongly influenced by low serum vitamin d concentrations. further extended and detailed studies have to be performed in order to clarify the relation between aff and femoral curvature. disclosure statement the authors report no conflicts of interest. references 1. bamias a, terpos e, dimopoulos ma. avascular osteonecrosis of the jaw as a side effect of bisphosphonate treatment. onkologie. 2010;33:288–9. 2. odvina cv, zerwekh je, rao ds, maalouf n, gottschalk fa, pak cy. severely suppressed bone turnover: a potential complication of alendronate therapy. j clin endocrinol metab. 2005;90:1294–301. 3. tan sc, koh sbj, goh sk, howe ts. atypical femoral stress fractures in bisphosphonate-free patients. osteoporos int. 2010;22:2211–12. 4. shane e, burr d, ebeling pr, abrahamsen b, adler 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fracture. osteoporos int. 2006;17:986–95. 14. saita y, ishijima m, mogami a, kubota m, baba t, kaketa t, et al. the incidence of and risk factors for developing atypical femoral fractures in japan. j bone miner metab. 2015;33:311–18. upsala journal of medical sciences 173 factors related to curved femur in elderly japanese women introduction material and methods subjects clinical evaluations statistical analysis results discussion disclosure statement references tf-iups160042 289..294 original article swedish parents’ interest in preconception genetic carrier screening maria ekstrand ragnara,b, tanja tyd�enb , ulrik kihlbomc and margareta larssona adepartment of women’s and children’s health, uppsala university, sweden; bdepartment of public health and caring sciences, uppsala university, sweden; ccentre for research ethics and bioethics, uppsala university, uppsala, sweden abstract introduction: genetic technologies advance rapidly. it is possible to undergo genetic carrier screening before pregnancy to examine genetic risks to future offspring. we aimed to investigate parents’ interest and motives towards preconception genetic carrier screening (pcs) as well as factors associated with interest in pcs. material and methods: our study sample consists of 777 parent couples within the longitudinal swedish pregnancy planning study. women responded to questionnaires at three occasions: in early pregnancy, late pregnancy, and one year after childbirth. male partners responded to one questionnaire one year after childbirth. results: one-third of the parents were positive (30% versus 34% of women and men, respectively), less than a third were negative (26% versus 28%), and 45% versus 38% were uncertain about whether to consider pcs before a future pregnancy. no differences in pcs interest were found between women and men (p ¼ 0.091), but a higher proportion of women were concerned about negative consequences (53% versus 46%, p < 0.003) and were ‘opposed to such a way of child selection’ (31.8% versus 25.2%, p ¼ 0.002). factors associated with pcs interest were experiences of prenatal diagnostics and positive attitudes towards finding out or choosing sex of one’s child (women), and prenatal diagnostics, self-rated poor health, and pregnancy planning (men). conclusion: both women and men had relatively high uncertainty towards pcs, but women were more concerned about negative consequences. the future extent of the clinical utility of pcs is currently unknown, but parents’ interests and doubts are important aspects to consider. article history received 25 april 2016 revised 6 july 2016 accepted 26 july 2016 keywords interest; motives; parents; preconception genetic carrier screening introduction genetic technologies are advancing rapidly. with safer, faster, and cheaper tests, the possibility for healthy couples to undergo preconception carrier screening (pcs) for autosomal recessive conditions has increased markedly in recent years (1). in order for an autosomal recessive disorder to develop (for example cystic fibrosis (cf), spinal muscular atrophy (sma), thalassemia, and sickle cell disease), the abnormal gene must be present in both biological parents, and, if so, there is a 25% risk of them having a child with the autosomal recessive disease they both carry. it has been estimated that the birth prevalence of severe recessive disorders is between 0.25% and 0.5%, which means that approximately 1–2 in 100 couples are at risk of having a child affected with a recessive genetic condition (2). healthy carriers usually do not know of their carrier status unless they have a known family history of recessive genetic disorder, or if they have had an affected child. preconception carrier screening (pcs) is usually defined as the detection of carrier status before pregnancy, in couples or persons who do not have a known increased risk of being carriers, to determine the risk of having a child with a recessive genetic disorder (1). identifying carriers of autosomal recessive disorders before pregnancy has the potential to benefit prospective parents by making them aware of the possible genetic risks to a future child, and of the reproductive options available. these options include not only prenatal diagnosis, followed (or not) by pregnancy termination in case of an affected fetus or by coming to terms with the risk, but may also include the possibilities of using preimplantation genetic diagnosis, donor sperm or oocytes, seeking adoption—or refraining from having children (3,4). traditionally, pcs has been limited to a small number of specific tests and generally offered to certain (often ethnic) high-risk populations (5). however, advancements in genomics have opened new possibilities for carrier screening in whole populations without a prior risk or family history; in the us for example, the american college of medical genetics recommends all couples regardless of ancestry or geographic origin to be offered screening for sma (6), and the american college of obstetricians & gynecologists recommends all women of reproductive age (regardless of ancestry or geographic origin) to be offered screening for cf (7). besides this, the rapid development in genetic technologies enables broader test panels to be used. this allows screening for the carrier status of a large number of inherited conditions at one go (8–10). the new technology has also opened up for contact maria ekstrand ragnar maria.ekstrand@kbh.uu.se department of women’s and children’s health, uppsala university, uppsala, sweden. � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 4, 289–294 http://dx.doi.org/10.1080/03009734.2016.1218575 http://creativecommons.org/licenses/by/4.0/ commercial companies to offer a wide array of genetic screening tests and services, either directly to the consumer, or via referrals from private health care providers (5). previous research has shown generally accepting views towards pcs. however, this research has mostly been limited to single-gene conditions and performed among target populations for certain conditions—for example, individuals already affected with a genetic recessive disorder and their parents and relatives (11). there is still limited knowledge about public perceptions towards population-based pcs and pcs using expanded panels. however, one study found that public opinions towards expanded pcs were multifaceted, deriving from limited knowledge, feelings of not being at risk, worries that testing might cause unnecessary stress, and/or financial concerns regarding cost of testing (12). pcs is not yet in practice in sweden, and couples without a family history of recessive (and/or other) genetic disorders are currently not offered screening within the public health care system. however, anyone can order pcs tests through international commercial companies online. thus, little is known about interest in pcs among potential parents-to-be. this study aimed to investigate parents’ interest in pcs, stated motives why or why not to consider such screening, and factors associated with interest in pcs. we hypothesized that interest in pcs would be associated with parents having undergone prenatal diagnostics, having requested information about the sex of the fetus prior to delivery, and/or having responded favorable to a hypothetical question about fetal sex selection. furthermore, we postulated that interest in pcs would be associated with a high degree of pregnancy planning, self-rated poor health, previous miscarriage(s), and socio-demographic background characteristics such as a high level of education. to date, there has been no exploration of the factors associated with pcs interest or factors that are likely to motivate individuals to consider pcs. materials and methods setting, participants, and procedure this study was part of the swedish pregnancy planning (swepp) study—a longitudinal cohort study examining lifestyle and health in connection to pregnancy and childbirth among nearly 5,000 women and nearly 1,000 partners, previously described by stern et al. (13). in sweden, antenatal care is offered free of charge to all women. as long as the pregnancy is normal, antenatal care is provided exclusively by midwives. the study invited a total of 215 antenatal clinics, of which 153 (71%) agreed to participate. the antenatal clinics ranged from small to large, in the countryside and in the cities, in nine swedish counties (about 2,500,000 inhabitants). pregnant women were recruited in consecutive order at registration in the antenatal clinic after having received verbal and written information about the study by their midwife. of 5,494 women invited, 4,969 agreed to participate. the women received questionnaires to fill out at the clinic or at home and return by post in a prepaid envelope. the women filled out one questionnaire (q1) in early pregnancy (n ¼ 3,389) and one (q2) around gestational week 34 (n ¼ 2,584). a third questionnaire (q3) was sent out 12 months post-partum to 2,000 women who had replied previously to both q1 and q2. along with q3, a partner questionnaire (q3p) containing a selection of questions from q1–3 was enclosed (n ¼ 2,000), and the women were asked to hand it to their partner/the other parent (of the child that was born 12 months earlier). in total 817 partners/parents filled out and returned the q3p. recruitment took place during the period september 2012 through july 2013, and data collection was completed in march 2015. our study sample consists of 777 parent couples (n ¼ 777 women, and n ¼ 777 men) who had responded to all questionnaires (q1–q3 and q3p) including the two study-specific questions about pcs of relevance for the study aim. we chose to exclude female partners (n ¼ 15) since recessive inherited conditions concern biological parents only. ethics the study was approved by the regional ethical review board in uppsala, sweden (reference number 2010/085). participation was voluntary, and participants were informed that the care given at the antenatal clinic was not related to their participation in the study. informed consent was obtained from all participants. questionnaires q1 contained 148 items, whereas q2, q3, and q3p contained 114 and 156 items, respectively, most of which were multiple choice questions, covering a range of pregnancy-related topics. researchers, clinicians, and laypeople reviewed the questionnaires, and a pilot study was conducted, after which some items were adjusted. in the present paper we use questionnaire items relevant to the aim of this study: questions about pcs, socio-demographic background characteristics, questions about personal health, health of the child, and reproductive history. questions about socio-demographic background characteristics covered: the woman’s age, partner’s age, country of birth, level of education, occupation, and household income. questions about personal and child health covered: selfreported health status, smoking, and whether or not the child had any congenital conditions. reproductive health and history covered: parity, miscarriages, ivf treatment, level of pregnancy planning, intentions of future childbearing, experience of prenatal diagnostics, knowledge about the sex of the fetus prior to delivery, and attitudes towards fetal sex selection. the level of pregnancy planning was measured using a single item, ‘how planned was your current pregnancy?’, and a five-step likert scale with response alternatives ranging from ‘very planned’ to ‘very unplanned’, previously described by tyd�en et al., backhausen et al., and stern et al. (13–15). the questions about pcs were introduced with a short text as follows: some congenital disorders can be passed on. the mutated gene(s) might be carried by one or both parents. 290 m. ekstrand ragnar et al. both parents could be carriers without being or becoming affected by genetic disease, but if a child inherits the gene from both parents there is an increased risk that the child will be affected. in the future, couples will be able to test genetic carrier status before pregnancy occurs. participants were thereafter asked whether or not they would consider taking such a test (‘yes/no/don’t know’), what motive(s) would be of importance to them in their decisionmaking, and whether they believed such a test could lead to negative consequences if it were offered to prospective parents in general (‘yes/no/don’t know’). the statements about motives for or against pcs were developed based on the literature and covered topics commonly discussed in the context of preconception carrier screening. the motives statements were: ‘i do not want the future child to suffer’, ‘i am opposed to such a way of child selection’, ‘it would be an act of responsibility’, ‘it is expected of me by others’, ‘i do not want my child to suffer’, ‘i want to exclude the risk of having a child with a severe genetic disorder’, ‘other motive(s)’. data analysis data were entered and analyzed using ibm spss statistics version 22 (ibm, armonk, ny, usa). categorical data are presented as frequencies and percentages, and continuous data by range, means, and standard deviations. differences regarding women’s and men’s perceptions of and stated motives for preconception genetic screening were analyzed using mcnemar–bowker’s test for categorical variables. for all statistical analyses, a two-sided p value <0.05 was considered significant. a binary logistic regression was used to analyze the association between the dependent variable (interest in pcs) and the independent variables. some variables were recoded and dichotomized. data regarding household income were collapsed into two categories; (�39,999/>40,000 swedish krona per month), as was occupation including studies (<50%/ �50%). the level of pregnancy planning was categorized as ‘planned’ (very or fairly planned) or ‘unplanned’ (neither planned nor unplanned, fairly or very unplanned), level of education as ‘high’ (university/college) or ‘low’ (no complete education, elementary school or high school/vocational education), self-rated health as ‘good’ (very/fairly good) or ‘poor’ (neither good nor bad, fairly/very poor), and interest in pcs as ‘yes’ or ‘no/uncertain’. results background characteristics, including participants’ reproductive health and history and interest in pcs, are presented in table 1. one-third of the parents were positive towards pcs (30% versus 34% of women and men, respectively), 25% versus 28% were negative, and 45% versus 38% were uncertain about whether they would consider a genetic test before a future pregnancy. no differences were found between women and men regarding interest in pcs (p ¼ 0.091). a higher proportion of women than men were concerned about negative consequences if pcs were to be offered to prospective parents (53%, n ¼ 409 of women versus 46%, n ¼ 356 among men, p < 0.003). comparison within the couples regarding motives for and against pcs are presented in table 2. a higher proportion of women compared to men ticked avoiding the (future) child to suffer (p < 0.000), and ‘opposed to such a way of child selection’ (p ¼ 0.002). binary logistic regression analysis showed that increased age, being born outside sweden, self-rated poor health, having undergone prenatal diagnostics, wanting to know the sex of the fetus prior to delivery, and having positive attitudes towards fetal sex selection was positively associated with women’s interest in pcs. among the partners, self-rated poor health, having had a planned pregnancy and having undergone prenatal diagnostics was associated with interest in pcs. see table 3. discussion interest and motives our most interesting result was the varying interest among parents towards pcs; about one-third would like to undergo pcs before a future pregnancy, a little less than a third would not, and 45% of women versus 38% of men were uncertain towards such screening. pcs is a new concept in sweden and is neither publicly debated nor offered as standard care. even though available through commercial companies, there is no direct-to-consumer marketing on the swedish market at present. consequently, it is reasonable to believe that knowledge and awareness regarding pcs among the general public, as well as among swedish health care providers, is low. still, a third of the parents in our study claimed to be willing to undergo pcs. however, it is possible that even more parents would have been willing to consider pcs, had they been more aware about the concept, and especially if pcs was endorsed by health care professionals, commercially marketed, and/or offered within the public health care system. it has been argued that such ‘routinization’ could diminish potential controversies and even justify the introduction of new medical or genetic technologies (16). as a comparison, screening for down syndrome is nowadays widely accepted among swedish parents-to-be and more or less viewed by many as part of standard care. although one-third of the parents showed interest in pcs, the majority was negative or hesitant. low interest or uncertainty must, however, not be explained by lack of knowledge alone. it could also—unrelated to one’s pre-existing knowledge—indicate a general resistance against mass screening for carrier status, or reluctance to take part in seeking information that such screening would entail. in our study, 31.8% of women and 25.2% men motivated their non-interest in pcs with being ‘opposed to such a way of child selection’, and one in five had no demand for receiving information about carrier status. with expanded test panels, and variation in penetrance and severity of the traits that one can be tested for, risk estimation will become quite difficult, both for the prospective parents as well as for health care upsala journal of medical sciences 291 professionals counseling them (17). so, even if pcs promises to enhance reproductive autonomy through offering more reproductive options (18), people may instead feel burdened when facing decisions with uncertain but significant longterm effects on their lives. worries about child selection and/ or doubts about receiving information about potential carrier status are of course important aspects to consider if, or when, pcs is further implemented in sweden. we found no difference between women and men regarding their interest in pcs. however, a higher proportion of women, compared to men, were concerned about negative consequences if pcs were to be offered to prospective parents in general. advancements in genetic technologies and pcs often raise ethical concerns and have been frequently discussed in the literature. besides issues focusing on informed consent and autonomy (1,17), the ethical discussion includes, for example, concerns about a supposed renaissance of ‘eugenics’ (aiming at improving the genetic quality of the human population) (19), medicalization (potentially eroding people’s confidence in the solidity of their health) (20), and discrimination (including carrier stigma, insurance discrimination, and avoidance of the birth of a child with a potentially severe disorder) (18,21,22). the most common motives for pcs, ticked by nearly half of the respondents, were not wanting a future child to suffer or not wanting to face the risk of having a child with a severe genetic disorder. very few stated that their motivation for opting for pcs would be feeling expectations from others. this finding was to be expected as no pcs programs have been implemented in sweden so far. however, implementation of new genetic screening practices may generate various kinds of pressure among targeted users (23–25). hence, such a development could of course be imaginable, should pcs ever become part of swedish standard care. factors associated with pcs we found no clear pattern of factors associated with pcs interest, except for joint experiences of prenatal diagnostics table 1. background characteristics among women (n ¼ 777) and male partners (n ¼ 777). categorical data are presented as frequencies and percentages, n (%), and continuous data are presented as means, standard deviations (sd), and range. women male partners variable n value n value mean age (years) 759 29.78 775 35.30 sd 4.639 5.584 range 17–47 16–58 household income (�39,999/>40,000 sek/month), n (%) 751 478/273 (63.6/36.4) 770 439/331 (56.5/42.6) occupation <50%/�50%, n (%) 777 307/470 (39.5/60.5) 777 140/637 (18.0/82.0) education 766 775 no completed education/elementary school, n (%) 23 (3.0) 33 (4.3) high school/vocational education, n (%) 285 (37.2) 401 (51.7) university/college, n (%) 458 (59.8) 341 (44.0) born outside sweden, n (%) 773 66 (8.5) 772 56 (7.3) self-rated health good/poor, n (%) 775 599/176 (77.3/22.7) 773 517/256 (66.9/33.1) smoking, yes/no, n (%) 776 32/744 (4.1/95.8) 774 57/717 (7.4/92.7) nullipara/multipara, n (%) 510 111/399 (21.8/78.2) – parity 500 1, n (%) 241 (48.2) – 2–3, n (%) 207 (41.4) – �4, n (%) 52 (10.4) – miscarriage, yes/no, n (%) 769 177/592 (23.0/77.0) – ivf, yes/no, n (%) 777 33/744 (4.2/95.8) 777 33/744 (4.2/95.8) prenatal diagnostics, yes/no, n (%) 777 314/463 (40.4/59.6) 777 314/463 (40.4/59.6) congenital condition (in child born 12 months ago), yes/no, n (%) 777 8/769 (1.0/99.0) 777 8/769 (1.0/99.0) pregnancy planning 776 775 unplanned n (%) 156 (20.0) 142 (18.3) planned n (%) 620 (80.0) 633 (81.7) wanting to know sex of child, yes/no, n (%) 758 375/383 (49.5/50.5) 760 395/365 (52.0/48.0) sex selection attitudes 760 positive, n (%) 34 (4.5) – negative/uncertain, n (%) 726 (95.5) – interest in pcs 777 777 yes 233 (30) 261 (33.6) no 198 (25.5) 219 (28.2) uncertain 346 (44.5) 297 (38.2) want more children, n (%) 776 776 yes 424 (54.6) 338 (43.6) no 203 (26.2) 278 (35.8) uncertain 149 (19.2) 160 (20.6) table 2. motives for and against pcs, comparison within couples (n ¼ 710). women/male partners n ¼ 710/710 (%) mcnemar p value not wanting (future) child to suffer 364/286 (51.3%/40.3%) 0.000 exclude the risk of having a child with a severe genetic disorder 321/326 (45.2%/45.9%) 0.811 an act of responsibility 165/194 (23.2%/27.3%) 0.058 expected by others 4/12 (0.6%/1.7%) 0.057 opposed to such a way of child selection 226/179 (31.8%/25.2%) 0.002 not wanting to know/such information 152/155 (21.4%/21.8%) 0.894 other motives 48/60 (6.8%/8.5%) 0.261 292 m. ekstrand ragnar et al. and positive attitudes towards the possibility of finding out— or even choosing—the sex of one’s child (among the women). in contrast to our hypothesis, level of education or history of miscarriages did not seem to influence parents’ interest in pcs; however, poor health and high degree of pregnancy planning did (by male partners). pcs interest was also more likely among women with non-swedish origin. the varying associations to pcs interest are not easily explained, but probably reflect the diversity in pcs attitudes among the parents in our study. clinical implications the future extent of the clinical utility of pcs in sweden is currently unknown. it is likely that awareness about pcs will soon increase among the general public, resulting in increased screening demand among some, and a remaining reluctance among others. diverse opinions among the target group will require great efforts to individualize counseling, provide complete and transparent information, and to ensure autonomy for prospective parents in their reproductive choices. primary care professionals, midwives, and gynecologists will have an important role to play in helping potential parents navigate the rapidly changing landscape of genetic technologies and screening services. no doubt, another issue for reflection will be how to prioritize resources within preconception care. if a growing number of people request pcs in the future, this will probably have noticeable effects in various areas, including economic aspects, health care delivery, and genetic information services, as well as insurance issues, to mention a few. preconception genetic screening options will continue to advance. understanding the role of women and men’s interests and doubts, as well as health care professionals’ perceptions of pcs, is vital in order to prepare for future developments of pcs in sweden. strengths and limitations our study included a large sample of both women and men recruited via antenatal clinics from different settings, in both rural and urban areas in sweden. as shown previously, our female study population is representative for women attending antenatal care in sweden, except for women born outside sweden that are underrepresented (13). regarding the male partner, we had to rely on the participating women for distribution of questionnaires, and consequently we have limited data on the external dropout rate and characteristics of those who chose not to participate. since the community awareness regarding pcs is likely to be low, people in general may have difficulties relating to hypothetical questions about pcs. an introductory text explaining the core concept of pcs was therefore presented to the respondents in the questionnaire, directly followed by the questions and the response alternatives. the introductory text was purposely rather brief. the text included neither severity nor onset of potential recessive disorders, nor was a distinction made regarding different forms of pcs (such as screening for single genes or using expanded panels). this could affect the way respondents interpreted and responded to the questions. the present study design does not allow any further exploration of what kind of concerns respondents might have had towards pcs, nor does it enable deeper understanding of the motives for or against pcs. this needs to be further explored, preferably by using a qualitative study design among potential parents-to-be. conclusion both women and men had relatively high uncertainty towards pcs, but women were more concerned about negative consequences. factors associated with interest in pcs were, among others, experiences of prenatal diagnostics and positive attitudes towards finding out or choosing the sex of one’s child. the future extent of the clinical utility of pcs is currently unknown, but patients’ interests and doubts are important aspects to consider. acknowledgements we thank andreas rosenblad for statistical advice and jenny stern, jennifer drevin, and maja bodin for distribution of questionnaires and data collection. disclosure statement the authors report no conflicts of interest. funding medical faculty, uppsala university, uppsala, sweden. table 3. characteristics associated with parents’ interest in pcs—a binary logistic regression analysis (n ¼ 1,554). variables women (n ¼ 777) partners (n ¼ 777) or 95% ci p value or 95% ci p value age 0.95 0.91–0.99 0.04 1.02 0.99–1.05 0.14 country of birth (swedena/outside sweden) 1.98 1.08–3.64 0.02 1.22 0.67–2.23 0.51 education (higha/low) 1.05 0.71–1.56 0.78 1.18 0.85–1.65 0.31 household income (higha/low) 0.77 0.53–1.13 0.18 0.91 0.65–1.27 0.58 self-rated health (gooda/poor) 1.56 1.04–2.33 0.02 1.50 1.08–2.08 0.01 pregnancy planning (planneda/unplanned) 1.02 0.66–1.56 0.92 0.59 0.38–0.91 0.01 prenatal diagnostics (yesa/no) 0.57 0.40–0.81 0.00 0.63 0.46–0.87 0.00 wanting to know sex of child (yesa/no) 0.56 0.39–0.80 0.00 0.79 0.58–1.09 0.16 gender selection (positivea/negative attitude) 0.26 0.12–0.57 0.00 previous miscarriage (yesa/no) 0.83 0.55–1.26 0.39 areference category. upsala journal of medical sciences 293 orcid tanja 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genet med. 2013;15:482–3. 294 m. ekstrand ragnar et al. http://www.acog.org/&sim;/media/for&percnt;20patients/faq179.pdf http://www.acog.org/&sim;/media/for&percnt;20patients/faq179.pdf swedish parents&rsquo; interest in preconception genetic carrier screening introduction materials and methods setting, participants, and procedure ethics questionnaires data analysis results discussion interest and motives factors associated with pcs clinical implications strengths and limitations conclusion acknowledgements disclosure statement references vol_116_002_sups_a_545898 148..154 upsala journal of medical sciences. 2011; 116: 148–154 original article a preliminary investigation on the possible association between diminished copper availability and non-alcoholic fatty liver disease in epileptic patients treated with valproic acid natalia lampon & j. carlos tutor unidad monitorización fármacos, laboratorio central, hospital clínico universitario, instituto de investigación sanitaria (idis), santiago de compostela, spain abstract background. patients treated with valproic acid (vpa) present a high incidence of non-alcoholic fatty liver disease (nafld) (around 61%). several recent studies suggest that low copper stores could be associated with nafld, and a significant decrease of copper availability in vpa-treated patients has been described. design and methods. in 101 adult epileptic patients treated with valproic acid in monotherapy (n = 75) and polytherapy (n = 26) the copper availability was evaluated using the specific oxidase activity of ceruloplasmin (activity per unit mass of enzyme protein) and the copper/ceruloplasmin ratio. copper deficiency was supposed in the cases in which this biochemical variable was smaller than the lower reference limit (333 u/g). results. the differences between the groups of patients with ceruloplasmin oxidase activity smaller or greater than 333 u/g for the serum levels of aminotransferases, gamma-glutamyltransferase, butyrylcholinesterase, cholesterol, triglycerides, and c-reactive protein, and the apri and fib-4 liver fibrosis scores were not statistically significant. most patients (93%) had low apri and fib-4 scores, suggesting absence of significant liver fibrosis. conclusions. the results obtained do not confirm the hypothesis of an association between diminished copper availability and nafld in patients treated with valproic acid. key words: apri, butyrylcholinesterase, copper deficiency, fib-4, non-alcoholic fatty liver disease, valproic acid introduction valproic acid (vpa) has been widely used since the late 1960s for the treatment of generalized and partial seizures, and it is still the antiepileptic drug with the broadest spectrum (1). new roles for this old drug were later confirmed, due to its beneficial effects in bipolar disorders, schizophrenia, depression, neurological pain, migraine headaches, and a number of neurodegenerative diseases (2,3). it has been highlighted that on rare occasions serious complications may occur in some vpatreated patients, including pancreatitis, coagulopathies, induced hepatotoxicity, and encephalopathy, although there is still a lack of knowledge about the incidence of these special side effects (1). type i vpa-associated hepatotoxicity consists of a dose-dependent elevation of serum liver enzymes, which normalize after drug discontinuation, and type ii consists of a rare idiosyncratic vpa-associated hepatotoxicity that is normally lethal (1). vpa has a significant steatogenic effect in hpg2 cell cultures (4), and development of fatty liver is one of the clinical findings during treatment with this drug (5). using abdominal ultrasound, luef et al. demonstrated that non-alcoholic fatty liver disease was present in 61% of vpa-treated patients (6), with this high prevalence being confirmed in a more recent study (7). impaired hepatic fatty acid oxidation directly inhibiting mitochondrial b-oxidation correspondence: j. carlos tutor, pharmd, phd, unidad monitorización fármacos, laboratorio central, hospital clínico universitario, 15706 santiago de compostela, spain. e-mail: jocatuva@hotmail.com; josecarlostutor@redfarma.org (received 26 november 2010; accepted 2 december 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.545898 enzymes, or sequestering co-factors involved in this metabolic pathway, has been proposed as a possible mechanism for vpa-induced steatosis(4,5,8). chronic treatment with enzyme-inducing antiepileptic drugs (carbamazepine, phenobarbital, or phenytoin) does not produce copper deficiency (9). however, the administration of vpa produces a significant increase of copper biliary excretion in rats (10), and previously published results suggest that diminished copper availability is substantially prevalent among epileptic patients treated with vpa (11). in accordance with aigner et al., a significant proportion of non-alcoholic fatty liver disease patients should be considered copper-deficient (12), and reduced copper availability may be involved in the development of non-alcoholic fatty liver (13), a hypothesis put forward at an earlier date by geubel et al. (14). in our study, an investigation was carried out in epileptic patients treated with vpa on the possible relationship between copper availability and the serum activities of several liver enzymes, whose increase in non-alcoholic fatty liver disease is widely documented (15–22). as previously reported, copper deficiency was supposed in cases with a smaller ceruloplasmin-specific oxidase activity (activity per mass unit of enzyme protein) than the estimated lower limit of reference (11). patients and methods a group of 101 epileptic out-patients (56 males and 45 females) with a mean age (±sem) of 43.7 ± 2.5 years (range 17–84 years) and treated with vpa in monotherapy (n = 75) or polytherapy with associated phenytoin, carbamazepine, and phenobarbital (n = 26) were studied. in all cases the daily drug administration was carried out in multiple doses. the blood samples were drawn before the breakfast and the morning dose of antiepileptic drugs, which had not been modified for at least 3 months prior to the study. consequently the serum levels of vpa correspond to the trough steady-state concentrations (css). none of the patients was receiving a copper dietary supplement, and pregnant women or those taking oral contraceptives were excluded. this study was carried out according to the good practice rules for investigation in humans of the conselleria de sanidade (regional ministry of health) of the xunta de galicia, spain. serum copper concentration was determined by atomic absorption spectrometry (perkin elmer mod 5100pc, waltham, usa) with carbon red atomization. immunoreactive ceruloplasmin was determined in a bn prospec nephelometer (siemens health care diagnostics inc., newark, usa). the oxidase activity of ceruloplasmin was determined as previously described at 30�c using o-dianisidine dihydrochloride as a substrate (23), and the ceruloplasminspecific oxidase activity was calculated using the expression: specific oxidase activity (u/g) = 1000 � oxidase activity (u/l)/immunoreactive ceruloplasmin (mg/l). serum vpa concentrations were determined in a dimension xpand analyzer using reagents from siemens health care inc. serum activities of alanine aminotransferase (alt), aspartate aminotransferase (ast), gamma-glutamyltransferase (ggt), alkaline phosphatase (alp),and butyrylcholinesterase (che), and concentrations of glucose, cholesterol, and triglycerides, were determined in an advia 2400 chemistry system (siemens health care diagnostics inc.). the platelet count, mean platelet volume (mpv), and platelet size distribution width (pdw) were measured in blood samples collected 2–3 hours beforehand in k3edta anticoagulated tubes using an advia 2120 hematology system (siemens health care diagnostics inc.). high-sensitivity c-reactive protein (crp) was determined in a bn ii nephelometer (siemens health care diagnostics inc.). the ast-to-platelet ratio index (apri) was calculated in accordance with wai et al. (24): apri = (ast:url/platelet count (109/l)) � 100, where url corresponds to the ast upper reference limits for men and women. the 4-variables liver fibrosis index (fib-4) was calculated in accordance with sterling et al. (25): fib-4 = age � ast/platelets (109/l) � alt1/2, where age is expressed in years and the ast and alt activities in u/l. statistical analysis of data was performed using the statgraphics package, and the kolmogorovsmirnov test was applied to check for normality. pearson’s correlation coefficient was used when the data had a gaussian distribution. otherwise, spearman’s correlation coefficient was used. the results were expressed as mean ± sem (median). results the estimated lower reference limit for the specific oxidase activity of ceruloplasmin (2.5 percentile of the control group) was 333 u/g (11). table i shows the results obtained for the different biochemical variables assayed in the patients treated with vpa in monotherapy, grouped according to their ceruloplasmin-specific oxidase activities (smaller or greater than 333 u/g). the differences between the mean (median) were not statistically significant, copper availability and nafld in epileptic patients treated with valproic acid 149 with the exception of the data obtained for the copper/immunoreactive ceruloplasmin ratio, which was significantly lower in the group of patients with ceruloplasmin-specific oxidase activities <333 u/g. analogous results were obtained for the patients treated with vpa in polytherapy (table ii). in both cases, significant differences were not achieved after a dichotomy of the data according to the patient’s sex. no significant correlations were obtained for the different variables assayed with the specific oxidase activity of ceruloplasmin or the copper/immunoreactive ceruloplasmin ratio, which have a significant correlation coefficient between them (r = 0.550; p < 0.001). in our study, thrombocytopenia was defined as a platelet count of less than 150 � 109/l, and 11 patients (10 in monotherapy and 1 in polytherapy ) had thrombocytopenia, with a platelet count of 115 ± 9.7 � 109/l (range 42–146 � 109/l), mpv of 9.5 ± 0.4 fl (range 7.6–11.5 fl), and pdw of 54.8 ± 2.4% (range 39.8%–63.8%). in accordance with previously published data on the discrimination of hypo-productive or hyper-destructive thrombocytopenia using mpv and pdw platelet indices (26,27), the decreased platelet count may be due to a hyper-destruction of peripheral blood platelets in these patients. furthermore, in the five patients (three in monotherapy and two in polytherapy) with a platelet count greater than 400 � 109/l, the calculation of the residual pdw according to osselaer et al. (28) suggests that the discrete thrombocytosis of 491.8 ± 37.7 � 109/l (range 409–601 � 109/l) was secondary or reactive. no significant correlations of the platelet count with the daily dose or serum levels of vpa were found. figure 1 shows the distribution of the values obtained for the apri and fib-4 liver fibrosis indices in 50 healthy controls and the patients who were studied. the correlation coefficient of the apri index with the platelet count was r = –0.764 (p < 0.001), and with ast activity r = 0.635 (p < 0.001). the correlation of fib-4 score with platelet count (r = –0.606; p < 0.001) was also greater than with ast (r = 0.348; p < 0.005). the first-order partial correlation coefficient of the apri index with platelet count (maintaining ast as a constant) was r = –0.898 (p < 0.001), and with ast activity (maintaining platelet count as a constant) r = 0.849 (p < 0.001). the partial correlations of the fib-4 score with the platelet count was r = –0.607 (p < 0.001), and with ast r = 0.350 (p < 0.005). discussion it has been previously described that non-alcoholic fatty liver disease is present in 61% of the patients table i. results obtained in the patients treated with valproic acid in monotherapy according to its ceruloplasmin-specific oxidase activity. specific oxidase activity <333(u/g) specific oxidase activity ‡333(u/g) n (males/females) 28 (18/10) 47 (21/26) vpa css (mg/l) 63.2 ± 4.0 (67.9) 63.5 ± 2.7 (60.4) vpa dose (mg/24h) 1170.0 ± 93.1 (1200.0) 1241.9 ± 86.1 (1000.0) specific oxidase activity (u/g) 278.5 ± 6.8 (276.1)a 430.5 ± 13.0 (405.4) copper/ceruloplasmin ratio (mg/mg) 3.2 ± 0.1 (3.2)a 3.9 ± 0.1 (3.8) glucose (mg/dl) 94.5 ± 5.1 (89.0) 88.4 ± 2.0 (87.5) cholesterol (mg/dl) 191.0 ± 10.2 (199.0) 175.5 ± 8.2 (171.0) triglycerides (mg/dl) 134.5 ± 13.2 (108.0) 107.7 ± 8.9 (99.0) ggt (u/l) 31.6 ± 8.8 (12.6) 17.2 ± 2.5 (10.0) alp (u/l) 161.2 ± 11.3 (132.0) 162.1 ± 3.7 (143.0) che (u/l) 7061.6 ± 407.4 (7250.0) 7005.9 ± 229.4 (7050.0) ast(u/l) 14.6 ± 1.1 (13.5) 16.7 ± 1.9 (13.0) alt(u/l) 19.2 ± 1.6 (20.0) 22.1 ± 3.9 (14.5) ast/alt ratio 0.8 ± 0.1 (0.8) 0.9 ± 0.1 (0.7) crp (mg/l) 3.4 ± 1.2 (1.4) 6.6 ± 2.6 (1.4) apri score 0.37 ± 0.06(0.30) 0.35 ± 0.05(0.30) fib-4 score 0.89 ± 0.12(0.80) 0.84 ± 0.12(0.60) asignificance: p < 0.01. 150 n. lampon & j. c. tutor treated with vpa (6,7), and around 38% of these patients may have a diminished copper availability (11), a particular condition that would be involved in the development of liver steatosis (12–14). a relevant proportion of non-alcoholic fatty liver disease patients had low copper stores (12,13), which is associated with more pronounced insulin resistance and other clinical features of the metabolic syndrome, and might be a typical feature of pathophysiological relevance to its hepatic manifestation as non-alcoholic fatty liver disease (12,13). patients with serum copper levels in the currently applied ‘normal’ range may include a significant proportion of cases with copper stores that can be considered depleted (12). the specific oxidase activity of circulating ceruloplasmin, which in adults is not influenced by nondietary factors such as age, gender, or hormone use, is a more sensitive indicator of copper status than either serum copper, ceruloplasmin, or erythrocyte superoxide dismutase (29,30). in the vpa-treated patients with ceruloplasmin-specific oxidase activity below the lower limit of reference (333 u/l) a reduced copper content of the circulating ceruloplasmin due to the diminished liver copper availability should be supposed (11). recently, it has been reported that the severity of non-alcoholic fatty liver disease correlates with high-sensitivity crp values (31). in our patients, a significant relationship of the specific oxidase activity of ceruloplasmin with crp levels was not attained, indicating that the specific oxidase activity is not influenced by the inflammatory status. significant increases of the serum ast, alt, ggt, and che activities in groups of patients with non-alcoholic fatty liver disease are well documented (15–22), as well as its correlation with the grade of steatosis and fibrosis assessed by liver ultrasound (17,19,20). in particular, increased serum che is considered a useful marker of liver steatosis (21,32), associated with adiposity, insulin resistance, lipid profile (22,33), and fatty liver infiltration degree (34). the serum levels of ast, alt, ggt, and che, and ast/alt ratio, apri and fib-4 indices were analogous and without significant differences between the groups of vpatreated patients with ceruloplasmin-specific oxidase activities lower or greater than 333 u/l (tables i and ii). these results do not confirm the hypothesis of a significant association between copper deficiency and fatty liver disease in the patients treated with vpa. it has been hypothesized that elevated che activities could lead to a decrease of acetylcholine which has anti-inflammatory actions and consequently could trigger the onset of low-grade table ii. results obtained in the patients treated with valproic acid in polytherapy according to its ceruloplasmin-specific oxidase activity. specific oxidase activity<333(u/g) specific oxidase activity ‡333(u/g) n (males/females) 10 (7/3) 16 (10/6) vpa css (mg/l) 49.4 ± 4.1 (48.1) 49.1 ± 6.3 (41.5) vpa dose (mg/24h) 1900.0 ± 362.5 (1500.0) 1733.3 ± 244.5 (1500.0) specific oxidase activity (u/g) 256.0 ± 19.5 (279.8)b 454.3 ± 18.0 (439.3) copper/ceruloplasmin ratio (mg/mg) 3.3 ± 0.2 (3.2)a 3.9 ± 0.2 (3.9) glucose (mg/dl) 85.4 ± 1.7 (86.0) 98.9 ± 7.8(87.5) cholesterol (mg/dl) 215.4 ± 19.6 (194.0) 222.8 ± 23.5 (209.0) triglycerides (mg/dl) 126.8 ± 17.6 (130.5) 130.2 ± 25.0 (102.0) ggt (u/l) 65.0 ± 19.3 (50.5) 70.6 ± 18.2 (44.0) alp (u/l) 145.2 ± 21.1 (147.0) 201.2 ± 34.1 (164.5) che (u/l) 7198.6 ± 845.9 (7308.0) 7638.0 ± 798.0 (7147.0) ast(u/l) 17.6 ± 4.4 (14.5) 15.9 ± 1.7 (15.0) alt(u/l) 27.4 ± 9.2 (19.0) 21.1 ± 3.1 (16.0) ast/alt ratio 0.8 ± 0.1 (0.7) 0.9 ± 0.1 (0.8) crp (mg/l) 38.7 ± 26.0 (8.9) 27.0 ± 14.7 (4.9) apri score 0.33 ± 0.07(0.30) 0.25 ± 0.04 (0.20) fib-4 score 0.69 ± 0.12(0.70) 0.63 ± 0.15 (0.50) asignificance: p < 0.05. bsignificance: p < 0.001. copper availability and nafld in epileptic patients treated with valproic acid 151 systemic inflammation (35,36). significant increases of serum che (37,38) and crp (39,40) have been reported in epileptic patients treated with different anticonvulsant drugs. in our patients having crp concentrations lower than 10 mg/l (subclinical chronic inflammation), a significant positive correlation between serum che and crp was found (r = 0.410;p < 0.001); however, in the cases with crp greater than 10 mg/l (acute inflammation conditions) the correlation coefficient between these two variables was negative, although due to the low number of patients considered, statistical significance was not achieved (r = –0.405;p = 0.084). similarly, in the group of patients with crp levels lower than 10 mg/l the che activity was significantly higher (p < 0.005) than in the patients with crp greater than 10 mg/l (7,400.9 ± 189.7 u/l (7,309.0 u/l) versus 5,846.5 ± 681.2 u/l (6,004.0 u/l)), possibly due to a greater liver injury in these last patients. this is an interesting subject that should be studied in a greater detail. non-alcoholic fatty liver disease ranges from simple steatosis, with a benign course, to non-alcoholic steatohepatitis with necroinflammation and fibrosis, which can progress to cryptogenic cirrhosis and endstage liver disease. as shown in figure 1, most control subjects (98%) and vpa-treated patients (93%) have low scores for the apri (<0.5) and fib-4 (<1.45) indices, suggesting the absence of liver fibrosis in these cases. likewise, in the remaining patients the values obtained for both indices suggest a low likelihood of significant hepatic fibrosis. only a 95-yearold male patient with platelet count of 107 � 109/l, ast 16 u/l, and alt 11 u/l (ast/alt ratio = 1.45), and presenting low serum levels of ggt 12 u/l, che 4,621 u/l, and albumin 31 g/l, had a fib-4 score of 4.3 indicative of severe liver fibrosis, although the apri index was 0.60. the variables ast activity and platelet count are used for the calculation of the apri (24) and fib-4 (25) liver fibrosis indices. with the progression of liver fibrosis, serum ast activity increases due to its release from mitochondria and its diminished clearance; likewise, the platelet count decreases due to a hypo-production of thrombopoietin by the hepatocytes and an increased circulating platelet destruction in spleen, as portal hypertension develops. in the vpa-treated patients studied, the coefficient of determination (estimated from the correspondent first partial correlation coefficients indicated above) shows that the apri score is slightly more dependent on the platelet count (r2 = 0.806) than ast activity (r2 = 0.721). the dependence of fib-4 score on the platelet count (r2 = 0.368) was also higher than with ast activity (r2 = 0.122). considering the mpv and pdw platelet indices of our 11 patients with thrombocytopenia, this fact may be due to a hyper-destruction of the circulating platelets as was indicated above. thrombocytopenia is the most common hematologic adverse effect of vpa, although the exact mechanism has not yet been elucidated (41). our study did not reveal any significant correlation between serum vpa concentration and platelet count, or significant higher vpa levels in the 11 patients with platelet count lower than 150 � 109/l with respect to the remaining patients (55.4 ± 7.7 mg/l (48.1 mg/l) versus 0.0 1.0 2.0 3.0 4.0 5.0 f ib 4 s c o re b 0 0.5 1 1.5 2 a p r i s c o re a controls vpa (m) vpa (p) controls vpa (m) vpa (p) figure 1. distribution of the apri score (a) and fib-4 score (b) in males (.) and females (*) controls and patients treated with valproic acid in monotherapy (vpam) and polytherapy (vpap). the dashed lines correspond to the cut-off values for absence (apri <0.5; fib-4 <1.45) or presence of significant liver fibrosis (apri>1.5; fib-4 >3.25). 152 n. lampon & j. c. tutor 59.4 ± 2.5 mg/l (56.5 mg/l)), suggesting that thrombocytopenia in these cases was not mediated by direct drug toxicity on the blood platelets. however, a vpainduced formation of autoantibodies against platelets is possible (41), and the results obtained for the mpv and pdw indices in our patients are compatible with a hyper-destruction or reduced circulating platelet survival, similar to that seen in immune thrombocytopenia. if this assumption is correct, the apri and fib-4 scores would be falsely increased, at least in some cases, reinforcing the suggestion expressed above that in the studied group of vpa-treated patients there is no significant liver fibrosis. with regard to these results, the recently described in-vitro and in-vivo inhibitory effect of vpa on the hepatic stellate cell activation, and consequently on fibrosis development in chronically injured mouse liver (42), should be considered. unfortunately a diagnosis of non-alcoholic fatty liver disease by ultrasonography was not carried out in our study. in any case, the results we have obtained suggest that the decreased copper availability present in the epileptic patients treated with vpa is not associated with fatty liver disease. copper status is linked to iron homeostasis, and copper deficiency causes hepatic iron accumulation that is frequently associated with hepatic steatosis (12). increased liver iron can promote oxidative stress and insulin resistance; however, these processes may be modulated by haptoglobin polymorphism, and hp2-2 phenotype would be a risk factor for metabolic syndrome and non-alcoholic fatty liver disease (43). the association between copper availability, iron metabolism, haptoglobin polymorphism, and metabolic syndrome requires clarification. declaration of interest: one of the authors (nl) received a grant from the sociedad española de 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chronic administration of valproic acid inhibits activation of mouse hepatic stellate cells in vitro and in vivo. hepatology. 2010;51:603–14. 43. nakagawa t, muramoto y, hori m, mihara s, marubayashi t, nakagawa k. a preliminary investigation of the association between haptoglobin polymorphism, serum ferritin concentration and fatty liver disease. cin chim acta. 2008;398:34–8. 154 n. lampon & j. c. tutor ujms 110 (3) bra upsala j med sci 110 (3): 245–249, 2005 angiomyolipoma in the knee-a case report masahito hatori1, mika watanabe2, shoichi kokubun1 1department of orthopaedic surgery, tohoku university school of medicine 2department of pathology, tohoku university hospital abstract extrarenal angiomyolipoma is an uncommon disease and this may be the first report of angiomyolipoma in the knee, mimicking a malignant sarcoma. a 38-year-old man without tuberous sclerosis presented with a history of increased mass in the knee joint. computerized tomography and magnetic resonance imaging demonstrated a çs x çs x çr cm subcutaneous tumor in the knee joint. the tumor was widely excised. histologically, the tumor was, well circumscribed, and composed of smooth muscle, vascular spaces, connective tissue, and mature fat. there were no signs of recurrence at one year and eight months after surgery. introduction during the 1960’s i made a series of inventions which ultimately led to the developangiomyolipoma is a hamartomaous lesion, usually occurring either within or intimately associated with the kidney; pedunculated or satellite nodules may be found in the adjacent soft tissue and less commonly, in regional lymph nodes [1]. however, it is possible to find it in extrarenal sites, being the liver the most frequent one. the rest of sites recorded in literature are exceptional [2]. we describe a case of angiomyolipoma arising in the subcutaneous region in the knee with the radiological imaging and microscopic features. 245 received 25 january 2005 accepted 1 february 2005 key words: angiomyolipoma, knee case report the patient was a 38 years old man. five years ago, he noticed a small mass in his left popliteal fossa. the mass gradually increased in size. he visited a clinic and underwent surgery. the surgery became limited to incisional biopsy because of bleeding of the tumor. he was referred to us with the suspicion of sarcoma. at the examination, a mass was located in the popliteal fossa of the left knee. it had mild tenderness. no adhesion to the surrounding tissues was found. computed tomography(ct) and magnetic resonance imaging(mri) demonstrated a 4 x 4 x 3 cm sized mass in the subcutaneous region in the medial side of the popliteal fossa (fig 1). the mass was widely excised. at one year and eight months after surgery, there were no signs of recurrence. grossly, the tumor presented as a whitish to slightly brown colored well-circumscribed mass in the subcutaneous region. focal hemorrhage was present. microscopically, the tumor was composed of convolutes of thick walled blood vessels, interlacing bundles of mature smooth muscle cells with a prominent perivascular arrangement and mature adipose tissue showing some variations in the cellular size. these three components were arranged irregularly and intermingled each other. rich vascular channels of various sizes were present, some of which were dilated showing hemangioma-like appearances (fig 2). elastica masson goldner stain clearly showed prominent collagen fibers and focally elastic fibers surrounding smooth muscle cells. immunohistochemically, the spindle cells in the tumor were reactive with hhf-35 and éø smooth muscle actin. these positive spindle cells were found around the adipose tissue. the fat cells were positive for s100 protein, 246 fig. 1a fig. 1b fig. 1 mr images demonstrating a subcutaneous mass (arrow) which was well enhanced after gadolinium injection on t1 weighted images ( a: axial image, b: sagittal image) and the vascular endothelial cells were reactive with cd 34. no apparent reactivity was found for hmb-45. discussion angiomyolipoma occurs most commonly in the kidney. the tumor is more common in women than in men. angiomyolipoma is more often encountered in patients with tuberous sclerosis. approximately one third to one half of cases are associated with tuberous sclerosis; less rarely with lymphangioma and lymphangiomatosis [1]. extrarenal sites of angiomyolipoma include the liver [3, 4], spleen [5], retroperitoneum [6], lymph nodes [3] and spermatic cord [7]. the subcutaneous angiomyolipoma is very rare [8, 9,10,11]. in 1990, fitzpatrick et al. described eight cases of cutaneous angiolipoleiomyoma (“angiomyolipoma”). clinically, the tumors were acquired, solitary, asymptomatic nodules that were always acral in location. patients' ages ranged from 33 to 77 years (median 52.6 years); the male/female ratio was 7:1. signs of tuberous sclerosis or renal angiomyolipoma were absent in all these cases [9]. to our knowledge, this is the first case reported of an angiomyolipoma in the knee joint area in which tuberous sclerosis or renal angiomyolipoma was not seen. diagnostically, ct scan and mri reveal a fatty mass with intermixed soft tissue densities, except in those cases in which absence of fat or hemorrhage obscures the radiological findings. the tumour is composed of fat, blood vessels and smooth muscle. the fat has a characteristic tissue density that often permits easy identification of an angiomyolipoma on ct [12]. already in 1988, uhlenbrock et al. reported that at mr imaging, most angiomyolipomas could be clearly characterized because of the depiction of intratumoral fat especially by the use of fat-suppression techniques [13]. in the present case, ct and mri did not reveal fatty component of the 247 fig. 2a fig. 2b fig. 2 microscopically the tumor was composed of convolutes of thick walled blood vessels, interlacing bundles of mature smooth muscle cells with a prominent perivascular arrangement and mature adipose tissue showing some variations in the cellular size (a : low power view x1 , b : high power view x 10). tumor. recently, ren et al. reported that mri showed hypointensity or hyperintensity on t1-weighted images and heterogeneous hyperintensity on t2-weighted images [14]. in our case, mri clearly demonstrated a tumor in the subcutaneous region with low signal intensities and well enhanced on t1-weighted images and high signal intensities on t2-weighted images. microscopically, the tumor is composed of three different tissue components that vary greatly in distribution: (1)mature adipose tissue, (2) convolutes of thick walled blood vessels, (3) irregular arranged sheets and interlacing bundles of smooth muscle [16]. in some tumors the fat is the predominant component, and in others smooth muscle predominated. using elastic tissue stains it is possible to reveal that some blood vessels have developed an elastic lamina whereas other blood vessels lack it [9]. these unique features of this lesion distinguish it from other lesions such as angiomyoma, angiolipoma, and other mixed mesenchymal tumors [15]. angiomyolipomas show consistent immunopositivity for hmb-45 [16]. hmb-45 reactivity, reported for renal angiomyolipomas, has been suggested as a useful tool in differential diagnosis. buyukbabani et al. conclude that, unlike renal angiomyolipomas, hmb-45 reactivity is not helpful in differentiating cutaneous angiomyolipomas [8]. the cell of origin remains mysterious. barnard et al. analyzed angiomyolipoma by electron microscopy and immunohistochemistry to determine the appearance and nature of cells composing angiomyolipomas. the study demonstrated that the angiomyolipoma is likely derived from a single cell that shares homology with the pericyte [16]. okada et al considered that the immature short spindle and epithelioid cells in angiomyolipoma might be primitive mesenchymal cells having an ability to differentiate toward both smooth muscle and fat cells [4]. despite the atypical features, nearly all angiomyolipomas seem to pursue a benign clinical course. there is no evidence that the presence of regional or systemic lymph node involvement, perirenal satellite tumors, or angiomyolipomas growth in other organs reflects malignant potential. malignant transformation, if it ever occurs, must be exceedingly rare [17, 18]. the present case, initially suspected as a sarcoma by unusual microscopic appearances in an unusual site of the knee, has had an uneventful course after surgery. references 1. meis-kindblom jm, enzinger fm. (1996) angiomyolipoma, in meis-kindblom jm, enzinger fm., color atlas of soft tissue tumors. mosby-wolfe, st louis, baltimore, boston, carlsbad, chicago, naples, new york, philadelphia, portland, london, madrid, mexico city, singapore, sydney, tokyo, toronto, wiesbaden, p 87. 2. coscaron blanco e, gomez gonzalez jl, blanco perez p, canizo alvarez a, benito gonzalez f, flores corral t (2004) [cervicothoracic angiomyolipoma: an unusual tumor located at a site difficult to reach for surgery. acta otorrinolaringol esp 55: 148-151. 3. huang pc, chen jt, ho wl (2000) clinicopathologic analysis of renal and extrarenal angiomyolipomas: report of 44 cases. zhonghua yi xue za zhi (taipei) 63: 37-44. 248 4. okada k, yokoyama s, nakayama i, tada i, kobayashi m (1989) an electron microscopic study of hepatic angiomyolipoma. acta pathol jpn 39: 743-749. 5. tang p, alhindawi r, farmer p (2001) case report: primary isolated angiomyolipoma of the spleen. ann clin lab sci 31:405-410. 6. tseng ca, pan ys, su yc, wu dc, jan cm, wang wm (2004) extrarenal retroperitoneal angiomyolipoma: case report and review of the literature. abdom imaging 29: 721-723 7. castillenti ta, bertin ap (1989) angiomyolipoma of the spermatic cord: case report and literature review. j urol 142: 1308-1309. 8. buyukbabani n, tetikkurt s, ozturk as (1998) cutaneous angiomyolipoma: report of two cases with emphasis on hmb-45 utility. j eur acad dermatol venereol 11: 151-154. 9. fitzpatrick je, mellette jr jr, hwang rj, golitz le, zaim mt, clemons d (1990) cutaneous angiolipoleiomyoma. j am acad dermatol 23: 1093-1098. 10. obata c, murakami y, furue m, kiryu h (2001) cutaneous angiomyolipoma. dermatology. 203: 268-270. 11. val-bernal jf, mira c (1996) cutaneous angiomyolipoma. j cutan pathol. 23: 364-368. 12. bosniak ma, megibow aj, hulnick dh, horii s, raghavendra bn (1988) ct diagnosis of renal angiomyolipoma: the importance of detecting small amounts of fat. ajr am j roentgenol 151: 497-501. 13. uhlenbrock d, fischer c, beyer hk (1988) angiomyolipoma of the kidney. acta radiol 29:523-524. 14. ren n, qin lx, tang zy, wu zq, fan j (2003) diagnosis and treatment of hepatic angiomyolipoma in 26 cases. world j gastroenterol 9:1856-1858. 15. argenyi zb, piette ww, goeken ja (1991) cutaneous angiomyolipoma. a light-microscopic, immunohistochemical, and electron-microscopic study. am j dermatopathol 13: 497-502 16. barnard m, lajoie g (2001) angiomyolipoma: immunohistochemical and ultrastructural study of 14 cases. ultrastruct pathol 25: 21-29. 17. bloom da, scardino pt, ehrlich rm, waisman jå@(1982) the significance of lymph nodal involvement in renal angiomyolipoma. j urol 128: 1292-1295. 18. brecher me, gill wb, straus fh 2ndå@(1986) angiomyolipoma with regional lymph node involvement and long-term follow-up study. hum pathol 17: 962-963. corresponding author: masahito hatori, m.d., assistant professor department of orthopaedic surgery, tohoku university school of medicine 1-1 seiryomachi, aobaku, sendai, miyagi, japan 980-8574 tel: 81-22-717-7242, fax: 81-22-717-72481 email: mhato@mail.tains.tohoku.ac.jp 249 ujms110_1.pdf upsala j med sci 109: 57–68, 2004 changes in graft blood flow early after syngeneic rat pancreas-duodenum transplantation leif jansson,1 birgitta bodin1 and per-ola carlsson1, 2 1department of medical cell biology and 2department of medical sciences, uppsala university, uppsala, sweden abstract a free full-text copy of this article can be found at the web page of upsala j med sci: http://www.ujms.se organ transplantation is associated with changes in graft blood flow, both acutely caused by reperfusion associated phenomena, and chronically due to e.g. denervation. the aim of the study was to investigate regional blood flow early after implantation of a syngeneic pancreas-duodenum transplant in rats, i.e. during reperfusion. warm ischemia time was 1–2 min and cold ischemia 90 min. blood flow values were measured with coloured microspheres both 10 and 30 min after implantation in transplanted rats, and at one time point in control rats. a marked decrease in the blood perfusion of the transplanted duodenum compared to the endogenous intestine was seen at both 10 and 30 min. total graft pancreatic blood flow was increased both 10 and 30 min after implantation, whilst islet blood flow remained unchanged compared to the endogenous gland. we conclude that the blood perfusion of the graft is markedly changed in the immediate post-transplantation period, presumably due to reperfusion. however, islet blood perfusion remains constant, suggesting that islet vasculature is less sensitive to changes induced by the implantation. introduction procurement of organs for subsequent transplantation usually necessitates the use of preservation solutions (1, 2). in combination with hypothermia the electrolyte composition of these solutions allows for survival and preservation of function after implantation. there is nevertheless hypoxia in the organs during preservation, and when blood flow is re-established a reperfusion syndrome of varying magnitude will occur (2). this syndrome is, among other things, characterized by the increased formation of reactive oxygen species (ros), which are known to affect the microcirculation in different organs (3). thus, ros will affect the bioavailability and gen57 received 13 september 2004 accepted 21 october 2004 key words: pancreatic islets, organ transplantation, microspheres eration of several vasoactive mediators, such as nitric oxide, and also serve as modulators of vascular smooth muscle function (3). the pancreas contains both exocrine and endocrine parts, with different capabilities to cope with reperfusion injury after implantation of the whole organ. we have previously shown that the blood perfusion of a transplanted pancreas-duodenum preparation in rats is associated with a slightly increased blood perfusion from the second post-implantation week and onwards, presumably due to the absence of neural modulation of the vascular smooth muscle in the blood vessels (4, 5). the nervous system seems to be of particular importance in the regulation of pancreatic islet blood flow (6). however, previous studies have demonstrated that islet blood flow is also regulated by a complex interplay between substances derived from the islet metabolism per se (7, 8), as well as endothelium-derived factors, such as nitric oxide (9). we have previously demonstrated that alloxan, a substance exerting a major part of its diabetogenic effect through ros (10), has a marked influence on pancreatic islet blood flow, and is one of the most powerful potentiators of islet blood perfusion seen (11). in view of these considerations we performed the present study where we examined the blood perfusion in a transplanted pancreas-duodenum preparation both 10 and 30 min after re-establishing the blood flow. since the endogenous pancreas is intact in the grafted animals, the preparation allows us to simultaneously study the effect of the reperfusion in a denervated, transplanted pancreas and duodenum and compare it to the innervated endogenous organs not exposed to reperfusion. methods animals male, inbred wistar-furth rats weighing 325 g, were purchased from b&k universal (sollentuna, sweden). all animals had free access to tap water and pelleted rat food throughout the experiments. principles of laboratory animal care (nih publication no. 23, revised 1985) were followed. all experiments were approved by the local animal ethics committee at uppsala university. pancreas-duodenum transplantations this procedure has been described in detail elsewhere (4). briefly, the donor was anaesthetised with an intraperitoneal injection of ekviticine (chloral hydrate and pentobarbital; apoteksbolaget, umeå, sweden), and placed on a heated operating table. the whole pancreas, together with approximately 5 cm (1 g) of the duodenum, was dissected free from surrounding tissues. through a catheter in the abdominal aorta the preparation was flushed with 5–7 ml of cold (4°c) uw-solution (viaspan™; du pont pharmaceuticals inc., wilmington, de, usa) at a pressure of approximately 100 cm h 2 o. the warm ischemia time was less than 2 min. the graft was then removed from the animal, together with approximately 1 cm of the aorta 58 which contained the two pancreatic arterial blood vessels, and stored at 4°c for 1.5–2 h (cold ischemia time) before being implanted into the recipient. the recipients were anaesthetised with an intraperitoneal injection of thiobutabarbital sodium (120 mg/kg; inactintm; research biochemicals international, natick, ma, usa) and placed on a heated operating table to maintain body temperature at 38°c. polyethylene catheters, filled with heparinized saline (100 u/ml) were inserted into the ascending aorta, via the right carotid artery, and into the left femoral artery. the former catheter was connected to a pressure transducer (pdcr 75/1; druck ltd., groby, uk), and the mean arterial blood pressure was continuously monitored throughout the experiments. the abdominal cavity was opened and the left kidney was removed, and the pancreas-duodenum graft was anastomosed to the renal blood vessels by a non-suturing cuff technique as previously described (12). the secretions from the graft duodenum were diverted from the abdomen through a drain inserted into the distal end of the graft. blood flow measurements with microspheres the arterial blood perfusion of the whole pancreas, islets, duodenum, colon, adrenal glands, kidneys, lungs and liver was measured with a microsphere technique (13). these experiments were performed in non-transplanted, untreated control rats as well as in transplanted animals. briefly, a total of 1.5–2.0 × 105 non-radioactive microspheres (eztrac™; triton microspheres, san diego, ca, usa), with a diameter of 10 mm, were injected via the catheter with its tip in the ascending aorta during 10 sec. starting 5 sec before the microsphere injection, and continuing for a total of 60 sec, an arterial blood sample was collected by free flow from the catheter in the femoral artery at a rate of approximately 0.4 ml/min. the exact withdrawal rate was confirmed in each experiment by weighing the sample. one injection, with black microspheres, was made 10 min after re-establishing the blood perfusion to the graft. a second injection, with the same amount of green microspheres, was made 30 min after revascularization of the graft. reference samples were collected during both these injections. in non-transplanted control rats, only one microsphere injection (green) was made after approximately the same time as required to perform the second measurement in the transplanted rats. arterial blood was collected from the carotid catheter for determination of blood glucose (at both microsphere injections) and serum insulin concentrations (only after the last injection) as given below. the animals were then killed, and the endogenous and transplanted pancreas were removed in toto, blotted, weighed and treated with a freeze-thawing technique, which visualised the pancreatic islets and microspheres (14). approximately 100 mg each of the colon (descending part) left kidney (thin section through the middle, encompassing both cortex and medulla), middle lobe of the left lung, median lobe of the liver and both the endogenous and transplanted duodenum (around the papilla), were removed and treated in the same way. the microspheres in the organs were then counted in a microscope equipped with both bright and dark field illumination (wild m3z; wild heerbrugg ltd., heerbrugg, switzerland). this enabled us to separate the green and black micros59 pheres, thereby making it possible to perform two blood flow measurements in the same animal. the blood flow values were calculated according to the formula q org = q ref × n org /n ref where q org is organ blood flow (ml/min), q ref is withdrawal rate of the reference sample, n org is number of microspheres present in the organ and n ref is number of microspheres in the reference sample. the number of microspheres in the arterial reference samples were determined by sonicating the blood, transferring samples to glass microfibre filters (pore size <0.2 �m), and then counting the number of microspheres in the microscope referred to above. 60 table 1. body weight, organ weights and hematocrit in untreated control wistarfurth rats or 30 min after syngeneic pancreas-duodenum transplantation. treatment none transplantation no of animals 7 7 body weight donor (g) na 331 ± 5 body weight recipient (g) 308 ± 2 325 ± 5 pancreas weight (mg) endogenous 928 ± 27 930 ± 30 transplanted na 981 ± 28 weight transplanted duodenum (mg) na 410 ± 23 hematocrit 44.7 ± 0.7 41.4 ± 1.0* values are means ± sem for 7 experiments. na denotes not applicable. * denotes p<0.05 when compared to the control rats. fig 1. blood glucose concentrations immediately after implantation of a syngeneic pancreas-duodenum transplant in wistar-furth rats. the value at time 0 is before opening of the abdominal cavity of the rat. values are means ± sem for 7 experiments. * denotes p<0.05 when compared to the values at time 0 and 30 min )anova). measurements of blood glucose and serum insulin concentrations blood glucose concentrations were determined with test reagent strips (medisense™; medisense sweden, stockholm, sweden) before and 5, 10 and 30 min after the transplantation. serum insulin concentrations were measured with elisa (rat insulin elisa; mercodia ab, uppsala, sweden ) in samples taken before and 30 min after pancreas-duodenum transplantation. statistical calculations all values are given as means ± sem. probabilities (p) of chance differences were calculated with students paired t-test or analysis of variance (anova; sigmastat; sspd, erfart, germany) with bonferroni’s post-hoc test. a value of p<0.05 was considered to be statistically significant. results all animals tolerated the surgical procedures without any signs of adverse reactions. mean arterial blood pressure was similar in control animals (105 ± 8 mm hg) and 61 fig. 2. total pancreatic blood flow in untreated control wistar-furth rats and rats receiving a syngeneic pancreas-duodenum transplant. measurements were performed both 10 and 30 min after implantation in the transplanted rats. values are means ± sem for 7 experiments. * denotes p<0.05 compared to the value in the endogenous gland at the same time point (student’s paired t-test). transplanted animals (110 ± 7 mm hg) before re-establishing graft circulation. when the vascular anastomosis were made and blood flow through the graft was reestablished an immediate approximately 20–25% decrease in mean arterial blood pressure was seen, and the value remained lower during the course of the study, i.e. 30 min. non-transplanted control rats deviated <5% during the course of the experiments. the hematocrit was lower in the transplanted rats when compared to control animals (see table 1). 62 fig 3. pancreatic islet blood flow in untreated control wistar-furth rats and rats receiving a syngeneic pancreas-duodenum transplant. measurements were performed both 10 and 30 min after implantation in the transplanted rats. values are means ± sem for 7 experiments. table 2. blood flow measurements were made in untreated control wistar-furth rat or 10 and 30 min after syngeneic pancreas-duodenum transplantation. transplantation treatment none 10 min 30 min colonic blood flow (ml/min × g) 0.61 ± 0.23 0.73 ± 0.13 0.89 ± 0.10 arterial hepatic blood flow (ml/min × g) 0.17 ± 0.04 0.14 ± 0.03 0.21 ± 0.12 renal blood flow (ml/min × g) 2.93 ± 0.45 2.47 ± 0.27 3.82 ± 0.56* pulmonary blood flow (ml/min × g) 0.16 ± 0.06 0.17 ± 0.10 0.14 ± 0.06 values are means ± sem for 7 experiments. na denotes not applicable. * denotes p<0.05 when compared to the control rats. the blood glucose concentration was increased 10 min after reperfusion of the graft, but was similar to the control value after 5 and 30 min (figure 1). glucose concentrations remained stable during the experimental procedures in the non-transplanted control rats (data not shown). serum insulin concentrations in the transplanted rats were 3.15 ± 0.43 ng/ml before and 6.10 ± 0.75 ng/ml 30 min after reperfusion (n=7; p<0.01 with student’s paired t-test). in non-transplanted control rats the values were 3.66 ± 0.39 ng/ml (n=7) at the end of the experiments. total pancreatic blood flow was higher in the transplanted pancreas when compared to the endogenous gland in the same animal both 10 and 30 min after reperfusion (figure 2). no changes in islet blood flow were seen (figure 3). both total pancreatic and islet blood flow were similar in non-transplanted control rats when compared to the values in the endogenous pancreas in the implanted rats (figures 2 and 3). the duodenal blood flow was markedly decreased in the transplanted intestine both at 10 and 30 min post-transplantation when compared to the value in the endogenous intestine in the same animals (figure 4). blood flow to the duodenum in non-transplanted control rats was lower than the value seen in the endogenous 63 figure 4: duodenal blood flow in untreated control wistar-furth rats and rats receiving a syngeneic pancreas-duodenum transplant. measurements were performed both 10 and 30 min after implantation in the transplanted rats. values are means ± sem for 7 experiments. * denotes p<0.05 compared to the value in the control animals (anova). § denotes p<0.001 when compared to the corresponding value in the endogenous duodenum (student’s paired t-test). duodenum 30 min after implantation (figure 4). there were no changes in colonic, arterial liver or pulmonary blood flow either at 10 and 30 min after transplantation (table 2). renal blood flow was higher 30 min than 10 min post-transplantation, but did not differ from the value in non-transplanted control rats (table 2). discussion in several instances previous studies have demonstrated changes in the blood perfusion of newly transplanted organs. thus, total pancreatic blood flow increased 90 min after reperfusion in pig pancreas transplants (15). however, measurements with tissue oximetry revealed that most of this increase was not nutritive, but rather shunt blood flow (15). after several hours total flow decreased below normal levels, but shunt flow still accounted for 50% of total flow. this means that tissue po 2 decreased after reperfusion despite a blood flow increase in this model. in another study on 11 patients undergoing simultaneous pancreas-kidney transplantation a decreased post-transplantation po 2 was also seen (16), whereas blood flow and venous haemoglobin saturation were normal. an immediate decrease in tissue po 2 is seen also after transplantation of livers (17). in a study on pancreas-duodenum grafts in pigs graft blood flow was measured with doppler flow probes over duodenum, tail and head of the pancreas 30 and 90 min after reperfusion (18). duodenal flow was <50% in duodenum grafts when compared to the value in donor duodenum, whereas that in the pancreas was decreased by approximately 30–40% [26]. it should be noted that an endothelin-a receptor antagonist exerted a protective effect against graft pancreatitis in this model (18), and the preservation solution celsior induced increased staining for endothelin-1 in pancreas-transplanted minipigs (19). blood flow was also markedly reduced in the transplanted lung baseline and 3 h after reperfusion in dogs (20). since hypoxic vasoconstriction was unlikely to occur it was speculated that this was due to increased endothelin secretion, decreased no production or altered angiotensin ii metabolism (21). in view of these studies it can be speculated that preservation may change the local production of endothelium-derived factors thereby influencing graft vascular function. in the present study total graft pancreatic blood flow was increased both 10 and 30 min after implantation, whereas duodenal blood flow was markedly decreased. it should be noted that graft handling was optimized with regard to ischemia times and that only a very limited graft pancreatitis occurs during these settings (4). it is also unlikely that transcription of any endothelium-derived factors occurs during the short time span before vascular anastomosis (21). the unchanged islet blood flow argues against any effects of endothelium-derived factors, since islet blood perfusion is much more sensitive to both vasodilatation (7) and vasoconstriction (9) induced by such substances than blood vessels in the exocrine parenchyma (6). both warm ischemia (1–2 min) and cold ischemia times (60–90 min) were low in the present study, thereby minimizing the degree of reperfusion injury. furthermore, 64 we used uw solution, which originally was developed to minimize organ damage during pancreas preservation (22). nevertheless, even this brief period of ischemia will inevitably lead to accumulation of e.g. ros and other vasoactive substances within the grafts (1, 2). indeed, ischemia-reperfusion injuries have been thought to be causative in the development of graft pancreatitis, suggesting that the mediators produced during preservation-induced ischemia are of importance for graft function (23). we therefore deem it likely that the formation of ros in the grafted organs after commencing reperfusion may affect graft vasculature. we have previously seen that the scavenging enzyme superoxide dismutase affects pancreatic blood flow in rats (24), and that the radical generating substance alloxan profoundly influences islet blood perfusion (25). furthermore, intestines are very sensitive to ros, as well as to decreased atp-concentrations (26). it may well be that such substances can induce the observed changes in pancreatic and intestinal blood flow, but this notion awaits further experimental confirmation. an interesting observation was that the islet blood perfusion remains unaffected in the immediate post-transplantation period. this is in line with the consistent findings that the endocrine function of a transplanted whole pancreas is able to immediately reverse hyperglycaemia (4). the reason for the slight increase in blood glucose concentrations seen after 10 min, which is back to normal again after 30 min, is likely to represent a stress reaction to the reperfusion. the hyperinsulinaemia seen after implantation of the pancreas is most likely explained by the fact that the islet mass was doubled by the transplantation. a peripheral insulin resistance due to the surgery in itself is also likely to occur. furthermore, it cannot be excluded that some �-cells in the transplanted pancreas have become injured and passively leak insulin. despite the pronounced effects noted in the grafts, no or only minor effects on the blood flow to other organs were seen, with the exception of the endogenous duodenum and kidney. the blood perfusion in these organs was increased 30 min, but not 10 min, after recommencement of the circulation. the reasons are unknown. local secretion of the proinflammatory cytokine tumour necrosis factor � (tnf-�) may affect remote organs (lungs) after release from liver during reperfusion (27), and the concentration of this substance peaks during the first half hour after reperfusion (28). it seems, however, remarkable if only the duodenum and kidneys would be affected by a tnf-� release from the graft. furthermore, extended cold ischemia time during liver transplantation upregulates the chemokines macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant, which through leukocyte activation may affect blood flow (29). in grafted islets chemokines are also upregulated (30). however, since the preservation period was limited in the present study the latter is unlikely to have occurred. however, this issue with effects of increased cytokines/chemokines in the immediate post-implantation period is worthy of further studies. the major finding in the present study was that re-establishment of the blood flow to a pancreas-duodenum graft leads to immediate and organ-specific changes in graft blood perfusion when compared to the corresponding endogenous organs. 65 thus, total pancreatic blood flow increased, duodenal blood flow decreased, whereas islet blood flow remained unchanged. acknowledgements the skilled technical assistance of astrid nordin is gratefully acknowledged. financial support was received from the swedish research council (72x-109, 72xd15043), the swedish diabetes association, the juvenile diabetes research foundation, the efsd/novo nordisk for type 2 diabetes research grant, the novo nordic research fund, åke wibergs stiftelse, magnus bergvalls stiftelse, barndiabetesfonden, anérs stiftelse, groschinskys minnesfond, svenska läkaresällskapet and the family ernfors fund. references 1. mclaren aj, friend pj (2003). trends in organ preservation. transpl int 16: 701–8. 2. southard jh, belzer fo (1995). organ preservation. annu rev med 46: 235–47. 3. nagel e, meyer zu vilsendorf a, bartels m, pichlmayr r (1997). antioxidative vitamins in prevention of ischemia/reperfusion injury. int j vitam nutr res 67: 298–306. 4. jansson l, korsgren o, wahlberg j, andersson a (1992). whole pancreatic and islet blood flow after syngeneic pancreaticoduodenal transplantation in the rat. transplant proc 24: 877–8. 5. jansson l, wahlberg j, andersson a (1993). differences in the vascular response to terbutaline in the native and transplanted rat pancreas. eur surg res 25: 383–9. 6. jansson l (1994). the regulation of pancreatic islet blood flow. diabetes metab rev 10: 407–16. 7. carlsson po, berne c, jansson l (1998). angiotensin ii and the endocrine pancreas: effects on islet blood flow and insulin secretion in rats. diabetologia 41: 127–33. 8. carlsson po, olsson r, källskog ö, bodin b, andersson a, jansson l (2002). glucose-induced islet blood flow increase in rats: interaction between nervous and metabolic mediators. am j physiol endocrinol metab 283: e457–64. 9. svensson am, östenson cg, sandler s, efendic s, jansson l (1994). inhibition of nitric oxide synthase by ng-nitro-l-arginine causes a preferential decrease in pancreatic islet blood flow in normal rats and spontaneously diabetic gk rats. endocrinology 135: 849–53. 10. malaisse wj (1982). alloxan toxicity to the pancreatic b-cell. a new hypothesis. biochem pharmacol 31: 3527–34. 11. jansson l, sandler s (1992). alloxan, but not streptozotocin, increases blood perfusion of pancreatic islets in rats. am j physiol 263: e57–63. 12. olausson m, mjörnstedt l, lindholm l, brynger h (1984). non-suture organ grafting to the neck vessels in rats. acta chir scand 150: 463–7. 13. carlsson po, källskog ö, bodin b, andersson a, jansson l (2002). multiple injections of coloured microspheres for islet blood flow measurements in anaesthetised rats: influence of microsphere size. ups j med sci 107: 111–20. 14. jansson l, hellerström c (1981). a rapid method of visualizing the pancreatic islets for studies of islet capillary blood flow using non-radioactive microspheres. acta physiol scand 113: 371–4. 15. benz s, wiessner r, obermaier r, pfeffer f, hopt ut (2002). microcirculatory events in ischemia/reperfusion of the pancreas defined by continuous tissue oximetry. transpl int 15: 173–9. 16. benz s, pfeffer f, adam u, schareck w, hopt ut (1998). impairment of pancreatic microcirculation in the early reperfusion period during simultaneous pancreas-kidney transplantation. transpl int 11 suppl 1: s433–5. 17. walsh ts, garden oj, lee a (2002). metabolic, cardiovascular, and acid-base status after hepatic artery or portal vein reperfusion during orthotopic liver transplantation. liver transpl 8: 537–44. 66 18. uhlmann d, ludwig s, escher e, armann b, gabel g, teupser d, tannapfel a, hauss j, witzigmann h (2001). protective effect of a selective endothelin a receptor antagonist (bsf 208075) on graft pancreatitis in pig pancreas transplantation. transplant proc 33: 3732–4. 19. uhlmann d, armann b, ludwig s, escher e, pietsch uc, tannapfel a, teupser d, hauss j, witzigmann h (2002). comparison of celsior and uw solution in experimental pancreas preservation. j surg res 105: 173–80. 20. brinkmann m, borgermann j, splittgerber fh, spillner j, reidemeister jc, kuss o, friedrich i (2002). pulmonary blood flow is inhomogeneously reduced after euro collins-preservation and lung transplantation. ann thorac surg 73: 226–32. 21. unruh h (1993). pulmonary endothelial cell function after modified eurocollins solution infusion. j heart lung transplant 12: 700–5. 22. wahlberg ja, southard jh, belzer fo (1986). development of a cold storage solution for pancreas preservation. cryobiology 23: 477–82. 23. klar e, messmer k, warshaw al, herfarth c (1990). pancreatic ischaemia in experimental acute pancreatitis: mechanism, significance and therapy. br j surg 77: 1205–10. 24. svensson am, sandler s, jansson l (2003). role of superoxide anion in pancreatic islet blood flow regulation in anesthetized rats. eur j pharmacol 459: 59–64. 25. jansson l, sandler s (1986). alloxan-induced diabetes in the mouse: time course of pancreatic bcell destruction as reflected in an increased islet vascular permeability. virchows arch a pathol anat histopathol 410: 17–21. 26. haglund u (1994). gut ischaemia. gut 35: s73–6. 27. colletti lm, burtch gd, remick dg, kunkel sl, strieter rm, guice ks, oldham kt, campbell da, jr. (1990). the production of tumor necrosis factor alpha and the development of a pulmonary capillary injury following hepatic ischemia/reperfusion. transplantation 49: 268–72. 28. tange s, hofer y, welte m, anthuber m, jauch kw, geissler ek, ertel w (2001). local secretion of tnf-alpha from the liver does not correlate with endotoxin, il-6, or organ function in the early phase after orthotopic liver transplantation. transpl int 14: 80–6. 29. kataoka m, shimizu h, mitsuhashi n, ohtsuka m, wakabayashi y, ito h, kimura f, nakagawa k, yoshidome h, shimizu y, miyazaki m (2002). effect of cold-ischemia time on c-x-c chemokine expression and neutrophil accumulation in the graft liver after orthotopic liver transplantation in rats. transplantation 73: 1730–5. 30. cardozo ak, proost p, gysemans c, chen mc, mathieu c, eizirik dl (2003). il-1beta and ifngamma induce the expression of diverse chemokines and il-15 in human and rat pancreatic islet cells, and in islets from pre-diabetic nod mice. diabetologia 46: 255–66. address correspondence to: dr. leif jansson department of medical cell biology; biomedical centre, husargatan 3, box 571; se-751 23 uppsala, sweden telephone: +46 18 4714396. fax: +46 18 556401; e-mail: leif.jansson@medcellbiol.uu.se 67 (10) upsala j med sci 111 (2): 249–256, 2006 ollier’s disease treated with grafting using alpha-tricalcium phosphate cement. a case report 1daizo sasaki, 1masahito hatori, 2yoshinori abe and 1shoichi kokubun 1department of orthopaedic surgery, tohoku university school of medicine, 1-1 seiryomachi, aobaku, sendai, miyagi 980-8574. 2department of orthopaedic surgery, sendai municipal hospital, 3-1 shimizukoji, wakabayashiku, sendai, miyagi 984-8501 abstract ollier’s disease is a rare disorder characterized by multiple enchondromas with a unilateral predominance, especially in fingers in early childhood. we experienced a case of ollier’s disease treated four times with simple curettage from the age of 2 years and alpha-tricalcium phosphate cement grafting at the age of 21 years. the forth curettage was performed when the patient was 15 years old and preoperative x-rays had shown remarkable finger deformities. postoperative casting of the involved joints was necessary to prevent fractures but led to some finger contractures. following x-rays demonstrated incredible improvement of the appearance. this suggests that simple curettage alone at an early stage of ollier’s disease provide cosmetic improvement. at the age of 21 years our patient showed enlargement of the intramedullary finger lesions again. some lesions seemed to be impending pathological fractures. this time we chose alpha-tricalcium phosphate cement to fill the cavities after curettage had been carried out. harvesting a large amount of autologous bone was not required. all activities of daily life could be resumed immediately after surgery and none of the finger joints showed further restriction of motion. x-rays taken three years after the operation showed new bone ingrowth surrounding the material with little evidence of absorption. to our knowledge, this may be the first case of ollier’s disease treated with artificial bone grafting reported in the english literature. 249 received 14 june 2005 accepted 22 august 2005 introduction ollier’s disease is a rare disorder characterized by multiple enchondromas with a unilateral predominace that usually occurs in infancy or early childhood. diaphysis and metaphysis of tubular or long bones, particularly fingers, are usually affected, and the lesions are often associated with remarkable deformities and dysfunctions. we experienced a case of ollier’s disease in a patient with multiple enchondromas in the upper and lower extremities from the age of 2 years. after simple curettage of the finger lesions had been carried out four times, the patient was recently operated on with artificial bone grafting using alpha-tricalcium phosphate cement. to our knowledge, this is the first case of ollier’s disease treated with artificial bone grafting reported in the english literature. in this case report, the clinical results are presented. case report the patient was a 21 year-old male. he had undergone an auriculoplasty of the right coloboma lobuli at the age of 10 years and conservative treatment of spontaneous pneumothorax at the age of 20 and 21 years respectively. his mother noticed deformities of his right index finger when he was 2 years old. bone tumors on the proximal phalanx of the right index finger and second matacarpal bone were found and curetted away. microscopical examination revealed enchondromas. he was diagnosed as having ollier’s dis250 fig 1. preoperative x-ray at the age of 15 years. multiple enchondromas of the right fingers are shown. some lesions are associated with severe deformities (arrow). ease, and underwent curettage of the lesions in the second metacarpal on the right hand at the age of 6 years, the middle phalanx of middle finger and second metacarpal when he was 9 years old, and the proximal phalanx of the thumb and middle / proximal phalanx of the right index finger at the age of 15 years. at that occasion, preoperative x-ray showed radiolucent bone tumors in the first metacarpal and proximal phalanx of the right thumb, middle / proximal phalanx of the right index finger, and the right second metacarpal, and proximal phalanx of the right middle finger, and middle phalanx of the right ring-finger. some of these lesions demonstrated ballooning of the surrounding bone cortices and severe deformity (fig 1). after tumor curettage, casting for 4 weeks was employed to prevent postoperative pathological fractures. at the age of 21 years, he visited our clinic again with the complaint of swelling of the thumb, index / middle / ring-finger of the right hand. there were operative scars on his thumb, index / middle finger and mild restriction of motion in these involved joints. plain x-ray showed intramedullary radiolucent lesions in the proximal phalanx of the thumb and first metacarpal bone, middle / proximal phalanx of the index finger, second metacarpal, and proximal phalanx of the middle finger, and middle phalanx of the ringfinger (fig 2). bone scintigraphy identified high-intensity radionuclide uptake not only in the right hand but also in the right scapula / humerus / radius / rib cartilage and left rib / femur (fig 3). the lesions in the right hand seemed to be impending pathological fractures. he underwent tumor curettage of the right proximal phalanx of the thumb, first 251 fig 2. preoperative x-ray at the age of 21 years. although the severe deformities were apparently improved, intramedullary radiolucent lesions had increased in size. 252 fig 3. bone scintigraph. fig 4. x-ray after curettage of the bone tumors and filling of the cavities with alpha-tricalcium phosphate cement. metacarpal and proximal phalanx of the index finger, and second metacarpal and proximal phalanx of the middle finger. an upper arm tourniquet was used to produce a bloodless field. the cortex over the tumor was fenestrated into a square shape of approximately 5 x 8 mm. careful curettage was then performed using a sharp spoon. the gross appearance of the resected tumor showed cartilage-like tissue. alpha-tricalcium phosphate cement was used to fill the resultant bone cavity and to prevent postoperative pathological fractures (fig 4). the cortical bone piece excised for the fenestration was used to cover the material. the patient could resume all activities of daily living immediately after the wound healing. microscopical examination showed that the specimen consisted of cartilage matrix and multiple chondrocytes, the nuclei of which were not enlarged, and mitoses were not seen. these tumors were diagnosed as ollier’s disease. x-rays taken three years after the operation showed new bone ingrowth surrounding the material with little evidence of absorption (fig 5). none of the finger joints showed further restriction of motion. discussion olleir’s disease was first described by ollier in 1899, in a 14 year-old child with multiple enchondromas showing a marked unilateral predominance [13]. multiple enchondromas tend to occur in infancy or early childhood, while solitary enchondromas are 253 fig 5. present x-ray 3 years after the operation. the figure shows that there is a new bone ingrowth surrounding the alpha-tricalcium phosphate and a minimal absorption of this material. most often seen in individuals from 10 to 30 years of age. although solitary enchondromas usually present with the complaint of pain and, occasionally, an associated pathological fracture, ollier’s disease is initially more often noticed because of the deformity and swelling of the involved limb, especially when fingers are affected. in cases affecting the tubular bones of the upper and lower limbs, patients have unilateral angular deformities, leg-length discrepancy, and joint maladaptation. the pathogenesis is still unknown. recent cytogenetic studies have revealed several chromosomal abnormalities in patients with multiple enchondromatoses, leading to sarcoma transformation [4, 14]. liu et al. have found that approximately 30% of patients (mean age at diagnosis was 40.5 years) with ollier’s disease will develop a malignant bone neoplasm, most probably chondrosarcoma [11]. schwartz et al. have reported that the incidence of secondary chondrosarcoma in patients who have ollier disease is about 25 per cent at the age of forty years [15]. for the hand lesions, malignant transformation is considered to rarely occur [5, 8]. the reported standard treatment is curettage of the tumor and filling of the resultant bone cavity with an autologous bone graft. however, it is often difficult to obtain a sufficient amount of cancellous bone to fill the large cavities after removal of multiple lesions. contractures during casting is another concern, because several joints are usually involved. in cases with severe deformity, the treatment of choice is amputation. blauth and sonnichsen have stated that the defect should be filled with loosely emplaced autologous spongiosa [3]. yahagi, however, has described that in cases of pediatric patients who have excellent ability of bone formation, simple curettage alone can yield good clinical results [18]. in our case, the patient has undergone simple curettage four times so far. although preoperative plain x-rays at the age of 15 years showed remarkable deformities in the fingers, preoperative x-rays at the age of 21 years demostrated incredible improvement of the appearance. judging from this finding, curettage alone can be considered to provide a reasonable cosmetic improvement. therefore, we recommend simple curettage of multiple lesions at an early stage when ollier’s disease presents. the present patient had intramedullary lesions that enlarged again 6 years after the last of the previous operations. we used alpha-tricalcium phosphate cement to fill the cavities caused by curettage of the lesions. there has been no description of artificial bone grafting for ollier’s disease published in the english literature. in cases of solitary enchondroma, some authors have recommended simple curettage without bone grafting and have reported good clinical results, especially in younger patients. however, such patients must undergo casting and wait for 4 to 6 weeks before they can resume unrestricted daily activity, and some patients experience joint contracture of the affected limb [7,16,17]. autologous bone grafting is performed conventionally, but the contribution of cancellous bone to the structural strength in the short term is considered to be very low [9]. recently, bone substitutes have also been used instead of autologous bone grafting. although hydroxyapatite, beta-tricalcium phosphate, and various granular materials have excellent biocompatiblity, these can not be considered to provide sufficient structural strength before consolidation [1,6,12]. pmma cement provides 254 immediate mechanical stability [2], but the heat of polymerization and monomer toxicity are concerns. our present case needed initial mechanical stability to prevent restrictions in the activities of daily living. in addition, such a large amount of autologous bone was not easy to obtain. alpha-tricalcium phosphate cement is an easily injectable material for filling cavities and can be used alone to provide excellent stabilization and allow early mobilization [10]. however, resorption seems to occur quite little. a possible concern is that the remaining material may make it difficult to re-operate, if there is a relapse of the tumor in the future. references 1. asanuma k, masui f, kamitani k, fujii k (2002). clinical application of pure-beta-tcp for bone tumors. j joint surg (jpn) 21: 1501-1506. 2. bickels j, wittig jc, kollender y, kellar-graney k, mansour kl, meller i, malawer mm (2002). enchondromas of the hand: treatment with curettage and cemented internal fixation. j hand surg [am] 27: 870-875. 3. blauth w, sonnichsen s (1986). enchondromatoses of the hand. z orthop ihre grenzgeb 124: 165-172. 4. bovee jv, van roggen jf, cleton-jansen am, taminiau ah, van der woude hj, hogendoorn pc (2000). malignant progression in multiple enchondromatosis (ollier's disease): an autopsybased molecular genetic study. hum pathol 31:1299-1303. 5. dahlin dc, salvador ah (1974). chondrosarcomas of bones of the hands and feet a study of 30 cases. cancer 34:755-760. 6. gaasbeek rd, rijnberg wj, van loon cj, meyers h, feith r (2005). no local recurrence of enchondroma after curettage and plaster filling. arch orthop trauma surg 125: 42-45. 7. goto t, yokokura s, kawano h, yamamoto a, matsuda k, nakamura k (2002). simple curettage without bone grafting for enchondromata of the hand: with special reference to replacement of the cortical window. j hand surg [br] 27: 446-451. 8. goto t, motoi t, komiya k, motoi n, okuma t, okazaki h, takatori y, tange t, nakamura k (2003). chondrosarcoma of the hand secondary to multiple enchondromatosis; report of two cases. arch orthop trauma surg 123:42-47. 9. hasselgren g, forssblad p, tornvall a (1991). bone grafting unnecessary in the treatment of enchondromas in the hand. j hand surg [am]. 16: 139-142. 10. joosten u, joist a, frebel t, walter m, langer m (2000). the use of an in situ curing hydroxyapatite cement as an alternative to bone graft following removal of enchondroma of the hand. j hand surg [br] 25: 288-291. 11. liu j, hudkins pg, swee rg, unni kk (1987). bone sarcomas associated with ollier's disease. cancer 59:1376-1385. 12. marui t, yamamoto t, akisue t, yoshiya s, kurosaka m (2004). use of pure beta-tricalcium phosphate to fill cavities after excision of benign bone tumors. j joint surg (jpn) 23: 231-237. 13. ollier m (1899). de la dyschondroplasie. bull soc chir (lyon) 3: 22-27. 14. ozisik yy, meloni am, spanier ss, bush ch, kingsley kl, sandberg aa (1998). deletion 1p in a low-grade chondrosarcoma in a patient with ollier disease. cancer genet cytogenet 105:128-133. 15. schwartz hs, zimmerman nb, simon ma, wroble rr, millar ea, bonfiglio m (1987). the malignant potential of enchondromatosis. j bone joint surg am 69:269-274. 16. tordai p, hoglund m, lugnegard h (1990). is the treatment of enchondroma in the hand by simple curettage a rewarding method? j hand surg [br] 15: 331-334. 17. wulle c (1990). on the treatment of enchondroma. j hand surg [br] 15: 320-330. 18. yahagi h (1988) surgical treatment for chondromatosis in childhood. j jpn soc surg hand 5: 567-570. 255 corresponding author: masahito hatori, m.d. department of orthopaedic surgery, tohoku university school of medicine 1-1 seiryomachi, aobaku, sendai, miyagi 980-8574 tel: 81-22-717-7242, fax : 81-22-717-7248 e-mail : mhato@mail.tains.tohoku.ac.jp 256 vol_117_004_sups_a_672345 390..398 full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 total cholesterol content of erythrocyte membranes is associated with the severity of coronary artery disease and the therapeutic effect of rosuvastatin yucheng zhong, hongxia tang, qiutang zeng, xiang wang, guiwen yi, kai meng, yi mao & xiaobo mao to cite this article: yucheng zhong, hongxia tang, qiutang zeng, xiang wang, guiwen yi, kai meng, yi mao & xiaobo mao (2012) total cholesterol content of erythrocyte membranes is associated with the severity of coronary artery disease and the therapeutic effect of rosuvastatin, upsala journal of medical sciences, 117:4, 390-398, doi: 10.3109/03009734.2012.672345 to link to this article: https://doi.org/10.3109/03009734.2012.672345 © informa healthcare published online: 25 sep 2012. submit your article to this journal article views: 587 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yucheng zhong1, hongxia tang2, qiutang zeng1, xiang wang1, guiwen yi1, kai meng1, yi mao1 & xiaobo mao1 1department of cardiology, institute of cardiovascular disease, union hospital, tongji medical college, huazhong university of science and technology, wuhan, 430022, china, and 2department of pediatric infectious and immunological diseases, wuhan children’s hospital, wuhan, 430016, china abstract introduction. numerous studies suggest that total cholesterol content of erythrocyte membranes (cem) might play a critical role in atherosclerotic plaque progression and instability. however, the exact role of cem in atherosclerosis remains obscure. our study was designed to investigate the association between cem and the severity of coronary artery disease (cad), and to assess the effect of rosuvastatin on cem levels. methods. cem levels were assessed in 136 participants, including acute coronary syndrome (acs) (non-st-segment elevation acs (nsteacs) and st-segment elevation myocardial infarction (stemi)), stable angina pectoris (sap), and controls. the gensini score was used to estimate the severity of cad. additionally, 54 patients with cad were medicated with rosuvastatin, 5 or 10 mg once daily, and then checked at 6 months. results. the highest level of cem was found in the stemi group, followed by the nsteacs, the sap, and the control groups. gensini score in group iv (cem > 141.6 mg/mg) was markedly higher compared with group i (cem £77.6 mg/mg). gensini scores in group ii (77.6 < cem £111.1 mg/mg) and group iii (111.1 < cem £141.6 mg/mg) were also higher than in group i (all p < 0.001). furthermore, a positive correlation was found between cem levels and gensini score (r = 0.714, p < 0.001). cem levels were dose-dependently reduced by rosuvastatin therapy. conclusions. cem levels are positively associated with the severity of cad, meaning that cem might contribute to the development of cad. importantly, rosuvastatin could decrease cem levels in patients with cad and might effectively help to attenuate the progression of cad. key words: coronary artery disease, cholesterol content, erythrocyte membranes, rosuvastatin introduction it is well known that the accumulation of cholesterol in atherosclerotic plaques can lead to plaque instability and subsequently result in acute coronary syndrome (acs) (1,2). previous studies have suggested that cholesterol within plaques is mainly derived from apoptotic/necrotic foam cells (2). the cholesterol in foam cells is mostly esterified (3). however, the proportion of free cholesterol in atherosclerotic plaques is markedly high (4). therefore, it is logical to suggest that cholesterol present in plaques might be derived from other sources. arbustini et al. and kolodgie et al. observed that erythrocyte membranes were present in the necrotic core of advanced atherosclerotic plaques (5,6). further studies have shown that erythrocyte membranes contribute to a significant increase of cholesterol accumulation in atherosclerotic plaques (7), since these membranes contain large amounts of cholesterol (6). thus, it is reasonable correspondence: dr xiaobo mao, institute of cardiology, union hospital, tongji medical college, huazhong university of science and technology, 1277 jiefang avenue, wuhan, 430022, china. fax: +86-27-85726423. e-mail: maoxiaobo75@yahoo.com.cn (received 1 january 2012; accepted 28 february 2012) issn 0300-9734 print/issn 2000-1967 online � 2012 informa healthcare doi: 10.3109/03009734.2012.672345 to postulate that erythrocytes are an important source of cholesterol in plaques. recently, accumulating clinical studies have shown that the cholesterol content of erythrocyte membranes (cem) in acs patients is significantly increased compared to patients with stable angina pectoris (sap) and is considered a potential marker of coronary artery disease (cad) activity (1,8–13). additionally, cem contributes to necrotic core expansion and then results in atherosclerotic plaque vulnerability. however, the relationship between the level of cem and the angiographic degree of coronary atherosclerosis is a controversial issue. the aim of this study was to investigate the relationship between cem levels and the severity of coronary artery stenosis in chinese patients with cad, as assessed by gensini score, and to estimate the therapeutic effect of rosuvastatin on cem levels in patients with cad. materials and methods participants a total of 106 consecutive patients who were admitted to the cardiology department of union hospital wuhan, hubei, pr china, from april 2010 to october 2010 for assessment of angina pectoris were recruited for the study. of these, 72 had acs (31 st-segment elevation myocardial infarction (stemi) and 41 nonst-segment elevation acs (nsteacs); the latter consisted of non-stemi and unstable angina), and 34 had sap. diagnosis of stemi, non-stemi, unstable angina, and sap was performed according to established guidelines (14–17). in addition, 30 patients with atypical chest pain and normal coronary arteries on angiography were considered as controls. all participants underwent coronary angiography after admission. cardiovascular-interrelated factors, such as age, gender, body mass index (bmi), blood pressure, heart rate (hr), and ejection fraction (ef), etc., were estimated via physical examination, electrocardiogram (ecg), and ultrasonic cardiography (ucg). all participants were divided into four groups according to the quartile of cem levels; the lowest quartile group was considered as control. patients with cad (n = 54), who were not receiving hypolipidemic therapy before admission, were medicated with rosuvastatin, 5 or 10 mg once daily, after initial blood sampling, and then followed for 6 months. the dose of rosuvastatin used was decided by plasma low-density lipoprotein cholesterol (ldl-c) levels (cut point 2.6 mmol/l). after that the patients were allocated into two groups: 5 mg rosuvastatin group (low-dose group, n = 25) and 10 mg rosuvastatin group (highdose group, n = 29). exclusion criteria included patients with a history of excessive alcohol intake, hematological, liver, renal, or thyroid diseases, infectious or autoimmune diseases, familial hyperlipidemia, cancer, and those having undergone surgical procedures in the preceding 3 months. patients who were receiving treatment with anti-inflammatory drugs or hormone replacement therapy were not included in the study. furthermore, patients with abnormal red blood cell (rbc) counts (<4.0 and > 5.5 � 1012/l for men and < 3.5 and > 5.0 � 1012/l for women) and/or abnormal hemoglobin (hb) levels(<120and>160g/lformenand<110and>150g/ l for women) were also excluded from the study. the study was approved by the ethics committee of tongji medical college of huazhong university of science and technology, and all participants gave written informed consent prior to study entry. angiographic analyses angiographic images were visually evaluated by two experienced cardiologists, who were not aware of the patients’ clinical and biochemical results, to assess the extent and severity of cad. a coronary artery was defined as ‘diseased’ in the presence of ‡ 50% luminal narrowing (18). the extent of the atherosclerotic disease in the coronary artery tree was assessed by vessel score (1), and the severity of cad was assessed by gensini score as previously described (19). laboratory analyses from all participants, venous blood samples were obtained after a 12h-overnight fast, prior to coronary angiography. blood specimens for cem were collected in standard vacutainer tubes containing citrate, then centrifuged at 450 g for 10 min at 4�c, and then the plasma and buffy coat were carefully discarded by aspiration. the remaining rbcs were resuspended and washed three times at 450 g for 5 min using 0.15 mol/l sodium chloride, and subsequently lysed in 30 volumes of hemolysis buffer (10 mm tris-hcl, 1 mm edta, ph 7.4). the membranes were separated from the hypotonic solution containing hemolyzed rbcs by centrifugation at 27,000 g for 15 min at 4�c; washings with the hypotonic buffer were repeated at least three times until a white/pink�pale pellet consisting of hemoglobin-free erythrocyte ‘ghosts’ was collected (1,9,20). erythrocyte ghosts, which were redissolved in 1 ml of phosphate-buffered saline (pbs) (ph 7.4), were stored at -60�c until further analysis. membrane protein concentration was measured by the bicinchoninic acid (bca) protein assay kit (thermofisher, rockford, cem and coronary artery stenosis 391 usa) (21), and the sensitivity of the assay was 5 mg/l. rbc membrane lipid extraction was performed followingtheproceduredescribedbyfolchetal.(22),andtotal cholesterol, including free and esterified cholesterol, was determined using a commercial enzymatic assay (diasys diagnostic systems gmbh, holzheim, germany) according to the manufacturer’s instruction (1). the lower limit of detection in blood serum was 30 mg/l, and the manufacturer’s reported intraand interassay precision, expressed as a percentage of the coefficient of variation (cv, %), was < 2% in both cases. in brief, a six-point calibration curve was obtained by diluting the standard solution provided in the kit, the absorbance of each sample was measured against a blank at 500 nm, and the result was plotted against the calibration curve to obtain the quantity of total cholesterol. all samples were measured in duplicate. results were expressed as micrograms of total membrane cholesterol per milligram of membrane protein (mg/mg). all other biochemical measurements, including serum total cholesterol, triglyceride, high-density lipoprotein cholesterol (hdl-c), ldl-c, apolipoprotein a-i (apoa-i), apolipoprotein b (apob), lipoprotein(a), fasting glucose, creatinine, uric acid, and high-sensitivity c-reactive protein (hs-crp), were carried out by the biochemical laboratory of our cardiovascular institute using standard methods. statistical analyses continuous variables are presented as mean ± standard deviation (sd) or as medians and interquartile ranges if the distributions were non-normal, while categorical data are presented as percentages. the shapiro–wilk test was used to assess the normality of distribution of continuous variables. for comparisons of two groups, continuous variables were tested using the independent samples t test for normally distributed data and the mann–whitney u test for non-normally distributed data; the chi-square test was used for categorical variables. when three or more groups were compared, one-way anova (normally distributed data) and kruskal–wallis tests (skewed variables) were used. the spearman rank correlation coefficient was used to assess the relationship between cem levels and other variables. statistical analysis was carried out using spss 13.0 (spss inc., chicago, il). a p value of < 0.05 was considered statistically significant. results baseline characteristics table i summarizes the general characteristics of the study subjects. patients in the sap and nsteacs groups were markedly older than controls. a decreased left ventricular ejection fraction in patients with acs was shown compared with controls, whereas no difference was found between patients with sap and controls. a prevalence of smoking was significantly higher in patients with acs compared to patients with sap. however, there were no differences in hypertension, diabetes mellitus, dyslipidemia, or family history between patients with acs and sap. compared to patients with acs, the use of aspirin and statins was more common in patients with sap before admission. compared with the control group, a significant decrease of hdlc levels and an obvious increase of lipoprotein(a) and hs-crp levels were observed in patients with cad. nonetheless, other biochemical results, including total cholesterol, triglyceride, ldl-c, apoa-i, apob, fasting glucose, creatinine, uric acid, and hemoglobin levels were similar between cad patients and controls. table ii depicts the characteristics of participants classified according to the quartile of cem levels. gensini score in group iv (median 70.3, interquartile range 48.4–99), group iii (42, 24.3–56), and group ii (18.5, 6.9–41.9) were markedly higher than in group i (3, 0–16.6) (all p < 0.001) (figure 1a). ldl-c of groups iii and iv were higher than that of group i, respectively (all p < 0.05). lipoprotein(a) and hs-crp levels in groups ii, iii, and iv were higher compared to group i, respectively (all p < 0.01). total cholesterol content of erythrocyte membranes significantly higher cem levels were observed in the stemi group (142.5, 134.2–163.8 mg/mg, p < 0.001), the nsteacs group (128.5, 115.6– 145.5 mg/mg, p < 0.001), and the sap group (86.1, 72.9–103 mg/mg, p < 0.05) compared with the control group (70.6, 55.7–86.6 mg/mg). importantly, cem levels in patients with stemi were obviously increased compared to patients with nsteacs and sap, and cem levels in patients with nsteacs were markedly higher than in patients with sap (figure 1b). rosuvastatin reduces cholesterol content of erythrocyte membranes after 6 months of therapy with rosuvastatin, 5–10 mg/ day, we found that the cem levels were significantly decreased compared to admission levels (p < 0.001) (figure 2a). the reduction of cem in the high-dose group (33.2% ± 4.98%) was markedly 392 y. zhong et al. t ab le i. d em o gr ap h ic an d cl in ic al d at a o f th e p ar ti ci p an ts at ad m is si o n . s a p (n = 3 4 ) n s t e a c s (n = 4 1 ) s t e m i (n = 3 1 ) c o n tr o ls (n = 3 0 ) p va lu e a ge , ye ar s 6 1 .9 ± 7 .7 6 0 .3 ± 7 .9 5 4 .2 ± 8 .6 5 4 .6 ± 8 .2 0 .0 0 2 a m en /w o m en , n 2 3 /1 1 3 5 /6 2 6 /5 2 4 /6 0 .2 4 6 b m i, kg /m 2 2 4 .1 ± 2 .2 2 4 .1 ± 1 .6 2 4 .6 ± 2 .2 2 4 .0 ± 2 .1 0 .8 0 3 s b p , m m h g 1 3 3 .7 ± 1 4 .6 1 2 7 .7 ± 1 1 .2 1 2 6 .8 ± 1 4 .1 1 2 8 .1 ± 1 0 .6 0 .2 7 9 d b p , m m h g 8 0 .9 ± 9 .4 7 7 .2 ± 7 .4 8 0 .1 ± 9 .2 8 1 .5 ± 8 .5 0 .3 5 5 h r , b p m 6 5 .6 ± 7 .0 6 8 .9 ± 8 .1 7 3 .5 ± 1 2 .5 6 7 .9 ± 6 .0 0 .2 0 6 e f (% ) 6 8 .8 ± 6 .5 6 0 .7 ± 6 .3 5 4 .2 ± 6 .6 6 8 .2 ± 4 .0 0 .0 0 1 a h is to ry o f p c i (% ) 5 2 3 0 0 .7 2 5 h is to ry o f c a b g (% ) 0 0 0 0 0 r is k fa ct o rs h yp er te n si o n , n (% ) 2 3 (6 8 % ) 2 7 (6 6 % ) 1 5 (4 8 % ) 0 0 .2 1 1 d ia b et es m el li tu s, n (% ) 9 (2 6 % ) 1 1 (2 7 % ) 1 0 (3 2 % ) 0 0 .8 4 4 d ys li p id em ia , n (% ) 6 (1 8 % ) 8 (2 0 % ) 5 (1 6 % ) 0 0 .9 3 2 s m o ki n g, n (% ) 1 4 (4 1 % ) 2 5 (6 1 % ) 2 2 (7 1 % ) 1 1 (3 7 % ) 0 .0 1 7 b f am il y h is to ry , n (% ) 7 (2 1 % ) 9 (2 2 % ) 6 (1 9 % ) 0 0 .9 6 4 m ed ic at io n s a sp ir in , n (% ) 2 1 (6 2 % ) 1 1 (2 7 % ) 7 (2 3 % ) 0 0 .0 0 1 b c lo p id o gr el , n (% ) 3 (9 % ) 2 (2 % ) 2 (7 % ) 0 0 .7 9 0 b et ab lo ck er s, n (% ) 1 5 (4 4 % ) 1 2 (2 9 % ) 6 (1 9 % ) 0 0 .0 9 3 a c e in h ib it o rs , n (% ) 9 (2 6 % ) 1 3 (3 2 % ) 8 (2 6 % ) 0 0 .8 2 5 a r b , n (% ) 7 (2 1 % ) 6 (1 5 % ) 5 (1 6 % ) 0 0 .7 8 3 c c b , n (% ) 1 2 (3 5 % ) 1 0 (2 4 % ) 8 (2 6 % ) 0 0 .5 4 2 s ta ti n s, n (% ) 1 6 (4 7 % ) 9 (2 2 % ) 6 (1 9 % ) 0 0 .0 2 1 b n it ra te s, n (% ) 5 (1 5 % ) 4 (1 0 % ) 2 (7 % ) 0 0 .5 4 5 a n gi o gr ap h ic an al ys is 0 .6 1 2 n o n -s ig n ifi ca n t d is ea se 3 (9 % ) 3 (7 % ) 2 (7 % ) 3 0 (1 0 0 % ) 1 -v es se l d is ea se 1 2 (3 5 % ) 1 0 (2 4 % ) 9 (2 9 % ) 0 2 -v es se l d is ea se 1 4 (4 1 % ) 1 5 (3 7 % ) 1 0 (3 2 % ) 0 3 -v es se l d is ea se 5 (1 5 % ) 1 3 (3 2 % ) 1 0 (3 2 % ) 0 l m s d is ea se 2 (6 % ) 5 (1 2 % ) 2 (7 % ) 0 0 .5 5 2 cem and coronary artery stenosis 393 t ab le i. (c on ti nu ed ). s a p (n = 3 4 ) n s t e a c s (n = 4 1 ) s t e m i (n = 3 1 ) c o n tr o ls (n = 3 0 ) p va lu e b io ch em is tr y t o ta l ch o le st er o l, m m o l/ l 4 .3 6 ± 0 .7 7 4 .2 9 ± 0 .7 2 4 .7 2 ± 1 .0 2 4 .4 3 ± 0 .7 3 0 .3 3 5 t ri gl yc er id e, m m o l/ l 1 .6 2 (1 .0 5 – 2 .2 3 ) 1 .3 8 (1 .0 7 – 2 .0 0 ) 1 .6 3 (1 .0 2 – 2 .2 5 ) 1 .6 0 (0 .9 8 – 2 .1 1 ) 0 .9 4 0 h d l ch o le st er o l, m m o l/ l 1 .2 4 ± 0 .2 4 1 .1 2 ± 0 .1 6 1 .0 8 ± 0 .2 1 1 .2 6 ± 0 .2 7 0 .0 2 1 a l d l ch o le st er o l, m m o l/ l 2 .3 6 ± 0 .6 3 2 .3 2 ± 0 .6 0 2 .6 4 ± 0 .8 6 2 .3 1 ± 0 .5 9 0 .3 4 6 a p o a -i , m m o l/ l 1 .0 6 ± 0 .1 7 1 .0 3 ± 0 .1 4 1 .0 2 ± 0 .1 5 1 .1 3 ± 0 .2 1 0 .1 8 9 a p o b , m m o l/ l 0 .9 3 ± 0 .1 5 0 .9 5 ± 0 .1 8 0 .9 6 ± 0 .1 9 0 .9 4 ± 0 .1 8 0 .9 5 8 l ip o p ro te in (a ), m g/ l 3 8 .5 (2 7 .3 – 5 7 .0 ) 5 5 .9 (3 1 .3 – 6 5 .6 ) 5 1 .9 (3 5 .9 – 8 0 .4 ) 3 4 .7 (2 1 .6 – 5 2 .1 ) 0 .0 1 8 c g lu co se , m m o l/ l 4 .9 9 (4 .2 5 – 6 .1 2 ) 4 .9 0 (4 .4 0 – 6 .1 0 ) 5 .3 0 (4 .5 5 – 6 .3 4 ) 4 .8 0 (4 .4 2 – 5 .2 0 ) 0 .3 7 5 c re at in in e, mm o l/ l 7 4 .6 ± 8 .5 7 6 .4 ± 9 .7 7 7 .7 ± 9 .5 7 3 .0 ± 9 .8 0 .5 9 5 u ri c ac id , mm o l/ l 3 3 8 .1 ± 8 1 .9 3 5 1 .1 ± 5 0 .4 3 6 7 .1 ± 7 6 .8 3 3 4 .4 ± 6 1 .7 0 .4 9 4 h em o gl o b in , g/ l 1 3 2 .1 ± 1 1 .4 1 3 7 .8 ± 1 1 .0 1 3 6 .3 ± 1 0 .9 1 3 6 .1 ± 1 1 .0 0 .2 1 3 h sc r p , m g/ l 3 .3 3 (2 .9 2 – 3 .8 5 ) 4 .5 8 (3 .3 6 – 6 .7 1 ) 5 .7 7 (4 .8 6 – 9 .4 3 ) 3 .5 5 (2 .5 9 – 4 .0 4 ) <0 .0 0 1 c v al u es ar e ex p re ss ed as m ea n ± st an d ar d d ev ia ti o n (s d ) o r m ed ia n an d in te rq u ar ti le ra n ge fo r co n ti n u o u s va ri ab le s, an d as % fo r ca te go ri ca l va ri ab le s. a o n ew ay a n o v a . b c h isq u ar e te st . c k ru sk al – w al li s te st . a c e = an gi o te n si n -c o n ve rt in g en zy m e; a p o a -i = ap o li p o p ro te in a -i ; a p o b = ap o li p o p ro te in b ; a r b = an gi o to n in ii re ce p to r b lo ck er ; b m i = b o d y m as s in d ex ; c a b g = co ro n ar y ar te ry b yp as s gr af t; c c b = ca lc iu m an ta go n is ts ; d b p = d ia st o li c b lo o d p re ss u re ; e f = ej ec ti o n fr ac ti o n ; h d l = h ig h -d en si ty li p o p ro te in ; h r = h ea rt ra te ; h sc r p = h ig h -s en si ti vi ty c -r ea ct iv e p ro te in ; l d l = lo w -d en si ty li p o p ro te in ; l m s = le ft m ai n st em ; n s t e a c s = n o n -s t -s eg m en t el ev at io n ac u te co ro n ar y sy n d ro m e; p c i = p er cu ta n eo u s co ro n ar y in te rv en ti o n ; s a p = st ab le an gi n a p ec to ri s; s b p = sy st o li c b lo o d p re ss u re ; s t e m i = s t -s eg m en t el ev at io n m yo ca rd ia l in fa rc ti o n . 394 y. zhong et al. expressed in comparison with the low-dose group (26.7% ± 3.90%, p < 0.001) (figure 2b). correlation analysis we assessed the association of cem levels with the severity of cad and several cardiovascular interrelated risk factors. correlation analysis showed that cem levels were strongly associated with serum levels of lipoprotein(a) (r = 0.312, p < 0.001) and hs-crp (r = 0.835, p < 0.001). however, there were no significant correlations with serum levels of total cholesterol (r = 0.184), triglycerides (r = 0.103), hdl-c (r = 0.060), ldl-c (r = 0.161), apoai (r = �0.060), apob (r = 0.090), fasting glucose (r = 0.099), uric acid (r = �0.053), nor with age (r = �0.128). interestingly, we found a significant positive correlation between angiographic gensini score and cem levels (r = 0.714, p < 0.001) (figure 3). in addition, cem levels were significantly correlated with vessel score (r = 0.471, p < 0.001). our data showed that cem levels in patients with 2-vessel disease (124.6, 102.6–140.0 mg/mg, p = 0.007) and 3-vessel disease (151.6, 136.4–170.7 mg/mg, p < 0.001) were significantly higher compared to patients with slight disease (88.9, 77.6–106.7 mg/mg). cem levels in patients with 1-vessel disease (102.4, 77.3–132.4 mg/mg, p = 0.277) were also increased but not statistically significant. discussion there is increasing evidence that red blood cells, especially their membranes, play an important role in atherosclerotic plaque progression and instability (1,13,23). the stability of the plaque appears to be largely determined by the size of the lipid core (24). table ii. characteristics of all participants classified according to the quartile of cem levels. quartile of cem level (mg/mg), groups i iv 3. £77.6 (n = 34) 77.6–111.1 (n = 34) 111.1–141.6 (n = 34) >141.6 (n = 34) age, years 57.1 ± 9.3 59.0 ± 8.4 57.4 ± 7.8 58.4 ± 9.2 men/women, n 27/7 26/8 27/7 28/6 bmi, kg/m2 24.2 ± 2.5 24.1 ± 1.7 24.0 ± 1.9 24.3 ± 1.8 hypertension, n (%) 17 (50%) 17 (50%) 16 (47%) 21 (62%) diabetes mellitus, n (%) 5 (15%) 6 (18%) 11 (32%) 9 (26%) dyslipidemia, n (%) 4 (12%) 6 (18%) 9 (26%) 2 (6%) smoking, n (%) 14 (41%) 14 (41%) 20 (59%) 22 (65%) family history, n (%) 3 (9%) 3 (9%) 7 (21%) 9 (26%) total cholesterol, mmol/l 4.18 ± 0.72 4.37 ± 0.66 4.60 ± 0.91 4.63 ± 0.94 triglyceride, mmol/l 1.29 (0.96–2.41) 1.52 (0.94–2.03) 1.68 (1.18–2.17) 1.52 (1.08–2.18) hdl cholesterol, mmol/l 1.15 ± 0.29 1.14 ± 0.20 1.11 ± 0.18 1.12 ± 0.22 ldl cholesterol, mmol/l 2.07 ± 0.56 2.40 ± 0.55 2.67 ± 0.78b 2.50 ± 0.71a apoa-i, mmol/l 1.07 ± 0.19 1.10 ± 0.14 1.04 ± 0.15 1.03 ± 0.19 apob, mmol/l 0.89 ± 0.18 0.95 ± 0.15 1.00 ± 0.17 0.91 ± 0.20 lipoprotein (a), mg/l 27.2 (20.5–41.4) 43.8 (32.0–58.3)b 54.6 (32.7–78.4)c 58.1 (42.5–69.3)c glucose, mmol/l 4.65 (4.40–5.76) 4.85 (4.71–6.06) 4.95 (4.45–6.06) 5.47 (4.40–6.88) creatinine, mmol/l 74.4 ± 9.7 76.9 ± 14.5 69.4 ± 10.4 77.3 ± 13.2 uric acid, mmol/l 336.2 ± 65.9 333.2 ± 62.2 331.8 ± 51.9 334.3 ± 81.9 hemoglobin, g/l 134.6 ± 10.8 137.7 ± 13.8 134.7 ± 10.4 143.3 ± 19.8 hs-crp, mg/l 2.87 (2.05–3.17) 3.49 (3.21–3.83)c 4.76 (3.82–5.48)c 8.21 (5.71–9.60)c gensini score 3 (0–16.6) 18.5 (6.9–41.9)c 42 (24.3–56.0)c 70.3 (48.4–99.0)c values are expressed as mean ± standard deviation (sd) or median and interquartile range for continuous variables, and as % for categorical variables. p values were calculated using chi-square test, one-way anova, or kruskal–wallis test. ap < 0.05; bp < 0.01; cp < 0.001, compared with the cem level £77.6 mg/mg group. apoa-i = apolipoprotein a-i; apob = apolipoprotein b; bmi = body mass index; hdl = high-density lipoprotein; hs-crp = highsensitivity c-reactive protein; ldl = low-density lipoprotein. cem and coronary artery stenosis 395 moreover, several studies recently suggested that erythrocytes entering the plaque may induce further expansion of atherosclerotic lipid core and promote plaque peroxidation (6,25). also, another study showed that injection of erythrocytes in aortic atherosclerotic plaques led to a remarkable increase of lipid content in the plaques (26). a clinical role of cem in atherosclerosis was first reported by tziakas et al. (1) who found an elevation of cem in patients with acs and discussed its implication in the pathophysiological progress of cad instability. cem might induce apoptosis of macrophages, formation of foam cells, and growth of lipid core with subsequent plaque rupture or erosion (27). we show in this study that respective cem levels in the stemi, nsteacs, and sap groups were significantly higher compared to the control group. remarkably, the highest level of cem was found in n = 34 34 cem levels c e m l e v e ls ( mg /m g ) p < 0.001 ≤7 7. 6 μg /m g ≤7 7. 6– 11 1. 1 μg /m g ≤1 11 .1 –1 41 .6 μ g/ m g >1 41 .6 μ g/ m g 200 g e n s in i s c o re 150 100 50 0 200 150 100 50 0 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p < 0.001 p = 0.012p < 0.001 p = 0.032 34 34 n = 30 controls sap nsteacs stemi 34 41 31 a b figure 1. a: gensini score in patients within different groups according to the quartile of cem level. horizontal line in the box plots represents the median value; the boxed area is the interquartile range, and the whiskers indicate minimum and maximum. b: cem levels in controls and patients with stable angina pectoris (sap), non-st-segment elevation acute coronary syndrome (nsteacs), and st-segment elevation myocardial infarction (stemi). cem = total cholesterol content of erythrocyte membrane. 200 50 40 30 20 10 0 a 150 100 admisssion 6 months low-dose group (n = 25) high-dose group (n = 25) p < 0.001 p < 0.001 c e m le ve ls ( μg /m g ) b δ c e m /c e m a d m × 1 0 0 % cad (n = 54) 50 0 figure 2. a: cem levels in patients with coronary artery disease (cad) on admission and at 6 months. b: the proportion of cem reduction in patients in the low-dose group and high-dose group. dcem = cemadm � cem6 months; adm = admission; cem = total cholesterol content of erythrocyte membrane. 396 y. zhong et al. the stemi group, followed by the nsteacs, and the sap groups. these results are not in agreement with those of tziakas et al. (1) and xu et al. (12), who concluded that cem could be a marker of acs but not of atherosclerosis. we now propose that cem levels are higher in patients with stemi compared to patients with nsteacs. these data further reinforce the idea that cem is a marker of cad instability. we also report a strong association between cem levels and the severity of coronary artery stenosis in patients with cad, as assessed by gensini score. this was in line with previous studies showing positive correlations between cem levels and the number of diseased coronary arteries. the coronary atheroma burden was revealed in patients with angiographically based cad (9,13). on the contrary, tziakas et al. (1) observed that cem levels were not associated with the extent and severity of coronary artery disease. instead, a significant relationship was noted between cem levels and angiographically complex coronary lesions (1). the reason for the disparity is unclear. it could relate to differences in methods of assessment of the coronary stenosis and the criterion for diagnosis of the diseased artery. thus, the intricate relationship between cem and the extent and severity of cad deserves large-scale clinical trials. our data were consistent with previous studies showing that cem levels correlated with serum hs-crp levels (1,11,12), the latter being regarded as a biochemical marker of inflammation. recently, some studies have revealed that serum crp levels are biochemical markers of acs, and a significant correlation between crp level and the severity of atherosclerosis has been documented (28–30). these results further corroborate the idea that cem is a predictor of cad instability similarly to previously known biomarkers such as hs-crp. additionally, we found no significant correlation between cem levels and circulating lipids, such as serum total cholesterol, triglycerides, hdl-c, ldl-c, apoa-i, and apob, in patients with cad. unexpectedly, a significant, positive relationship was noted between cem and lipoprotein(a) in our study. several lines of evidence indicate that lipoprotein(a) is an independent risk factor for patients with cad, leading to coronary artery disease (31,32). therefore, these findings suggest that the increase in lipoprotein(a) levels might contribute to augmented cem levels in cad, and cem could accordingly participate in the progress of cad. we also demonstrate that cem levels were significantly decreased in patients with cad after 6 months of therapy with rosuvastatin, which was dose-dependent. these results are in accordance with previous studies demonstrating that statin treatment reduces cem levels in patients with cad and hypercholesterolemia (1,12,20). although the mechanism of the unloading effect of statins on cem levels remains obscure, a novel role of statins should be noticed and further validated in a large-scale study. in conclusion, the present study demonstrates that cem levels correlated with the severity of coronary artery disease. we have also found a significant relationship between cem levels and serum hs-crp and lipoprotein(a) levels. rosuvastatin treatment reduced cem levels in patients with cad dose-dependently. altogether, cem may play a pivotal pathogenic role quantitatively and qualitatively in patients with cad. the reducing effect of statins on cem levels is a promising new approach in treatment of patients with cad. however, this study enrolled only a small group of chinese patients, and a large-scale clinical investigation is warranted. the precise mechanisms underlying our findings and their clinical relevance remain to be clarified. acknowledgements this study was supported, in part, by grants from the department of science & technology of hubei province (no. 2010cdb07902). declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. yucheng zhong and hongxia tang contributed equally to the manuscript. 200 n = 106 r = 0.714 p < 0.001 150 100 50 0 0 50 100 cem levels g e n s in i s c o re 150 200 figure 3. correlation between cem levels and coronary angiographic gensini score in patients with coronary artery disease (cad). cem = total cholesterol content of erythrocyte membrane. cem and coronary artery stenosis 397 references 1. tziakas dn, kaski jc, chalikias gk, romero c, fredericks s, tentes ik, et al. total cholesterol content of erythrocyte membranes is increased in patients with acute coronary syndrome: a new marker of clinical instability? j am coll cardiol. 2007;49:2081–9. 2. tabas i. consequences of cellular cholesterol accumulation: basic concepts and physiological implications. j clin invest. 2002;110:905–11. 3. tabas i. cholesterol and phospholipid metabolism in macrophages. biochim biophys acta. 2000;1529:164–74. 4. guyton jr, klemp kf. development of the lipid rich core in human atherosclerosis. arterioscler thromb vasc biol. 1996; 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26(suppl a):37a–40a. 398 y. zhong et al. tf-iups160040 256..263 original article general practitioners’ knowledge, attitudes and views of providing preconception care: a qualitative investigation obiamaka ojukwua, dilisha patela, judith stephensona, beth howdenb and jill shawec ainstitute for women’s health, university college london, london, united kingdom; buniversity of bristol, bristol, united kingdom; cfaculty of health and medical sciences, university of surrey, guildford, united kingdom abstract background: preconception health and care aims to reduce parental risk factors before pregnancy through health promotion and intervention. little is known about the preconception interventions that general practitioners (gps) provide. the aim of this study was to examine gps’ knowledge, attitudes, and views towards preconception health and care in the general practice setting. methods: as part of a large mixed-methods study to explore preconception care in england, we surveyed 1,173 women attending maternity units and gp services in london and interviewed women and health professionals. seven gps were interviewed, and the framework analysis method was used to analyse the data. findings: seven themes emerged from the data: knowledge of preconception guidelines; content of preconception advice; who should deliver preconception care?; targeting provision of preconception care; preconception health for men; barriers to providing preconception care; and ways of improving preconception care. a lack of knowledge and demand for preconception care was found, and although reaching women before they are pregnant was seen as important it was not a responsibility that could be adequately met by gps. specialist preconception services were not provided within gp surgeries, and care was mainly targeted at women with medical conditions. gps described diverse patient groups with very different health needs. conclusion: implementation of preconception policy and guidelines is required to engage women and men and to develop proactive delivery of care with the potential to improve pregnancy and neonatal outcomes. the role of education and of nurses in improving preconception health was acknowledged but remains under-developed. article history received 2 april 2016 revised 8 july 2016 accepted 16 july 2016 keywords general practice; preconception care; pregnancy; pre-pregnancy; qualitative background preconception health and care aims to reduce parental risk factors before pregnancy and improve outcomes through health promotion and intervention (1). optimizing maternal and paternal health before conception can influence a child’s future life-course (2–4). current demographic and epidemiological trends such as obesity, diabetes, and delayed childbearing increase the potential for preconception care to lead to significant health gain (5,6). the importance of preconception care has gained greater recognition with the world health organization (who) global action plan for the prevention and control of noncommunicable diseases 2013–2020 (7). within the uk, government policy aims to reduce perinatal morbidity and mortality through promotion of pre-pregnancy care (8). recommendations incorporate promotion of health-related behaviours including reducing the incidence of neural tube defects through folic acid supplementation; reducing preterm births, low birth weight, and poor neonatal and maternal outcomes through smoking cessation, alcohol reduction, and achievement of healthy weight and nutrition prior to conception; reducing infections through screening and vaccination; and identifying medications and occupational and environmental hazards that could be teratogenic. this is reinforced by the recommendation for couples to plan their pregnancies in order to improve maternal outcomes (9). in order to explore and provide an overview of preconception care in england, we undertook a mixed-methods study which surveyed 1,173 women and their partners attending maternity units and general practitioner (gp) services in london. the survey aimed to explore how they had prepared for pregnancy. we also carried out semi-structured interviews with a sub-sample of this group to investigate reasons and motivations as to why they invested in pre-pregnancy care. the findings reported elsewhere (10,11) showed that two-thirds of women plan their pregnancies, but despite this there was little change in preconception health behaviours such as uptake of folic acid by these women. it was found that prospective parents in the uk have little access to specific preconception care services but that 51% received contact jill shawe j.shawe@surrey.ac.uk faculty of health & medical sciences, university of surrey, guildford, surrey, uk � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 4, 256–263 http://dx.doi.org/10.1080/03009734.2016.1215853 http://creativecommons.org/licenses/by/4.0/ advice from a health professional prior to pregnancy in relation to the importance of healthy lifestyle behaviours such as diet, smoking, and alcohol. a quarter of the women in our study had a medical condition, and 26% were taking regular medications and potentially needed specialist advice before trying to conceive. little is known about the specific preconception interventions that gps provide for women of child-bearing age. as part of the study we interviewed gps with the aim of examining knowledge, attitudes, and views towards preconception health and care in the general practice setting. this paper reports the findings. method qualitative methodology with a qualitative description framework (12) was used to describe phenomena attained from gp’s experiences and their views of the organization of preconception care services. purposive sampling was used to recruit from general practice networks in london. fifteen gps with a remit for obstetrics and gynaecology within their practice were invited by email to participate in the study, and seven agreed to be interviewed. the sample size was based on previous experience within qualitative research in relation to the study scope and a mix of other factors such as the funding and the time available (13). interviews were conducted in private offices within the gp practices and carried out by three researchers (b.h., j.a. s., o.o.) who had a women’s health care focus and were trained in qualitative research interviewing. confidentiality was upheld and written consent obtained before proceeding with the interviews. interviews lasted up to 45 minutes, and the gps received an honorarium as compensation for their time. a topic guide (figure 1) was used during the semi-structured interviews which explored: the gp’s own experiences of how preconception care policy, guidelines, and recommendations are implemented in day-to-day practice; the gp’s understanding of content of preconception care; the nature of current service provision; who in the gps opinion should have responsibility for providing preconception care; and opportunities and barriers to future service delivery. interviews were digitally recorded and transcribed verbatim by o.o., b.h., and d.p. transcripts were imported into nvivo 10 computer software (14), and data workshops were then held with four researchers (b.h., d.p., j.a.s., o.o.). thematic analysis using framework method (15) ensured systematic and rigorous exploration of the text in five stages: (1) familiarization with data, (2) identification of a thematic framework, (3) indexing, (4) charting, and (5) mapping and interpretation. mapping and interpretation was an iterative process. using nvivo 10, we developed a matrix and examined charts, codes, and the full transcripts to develop a description of the phenomena and the concepts that evolved. favourable ethical opinion was given by the national research ethics service (nres) committee london, bromley (rec reference 11/lo/0881) as part of the larger study ‘pre-pregnancy health and care in england’. findings the seven gps (4 male and 3 female) who agreed to be interviewed were all partners in their practices with the exception of one who was a part-time salaried gp. the gps had qualified between 1987 and 2006 and had an average of 13.7 years’ experience since qualification. five gps had previous experience in obstetrics and gynaecology; three had drcog qualifications; four were also gp trainers. seven main themes emerged from the data: (1) knowledge of preconception guidelines; (2) content of preconception advice; (3) who should deliver preconception care?; (4) targeting provision of preconception care; (5) preconception health for men; (6) barriers to providing preconception care; and (7) ways of improving preconception care. knowledge of preconception guidelines gps stated that they had gained their knowledge of the elements of preconception care from clinical experience, from past training, or from patient information sources rather than from guidelines. … no … i didn’t know the specific guidelines, i tend to just kind of, erm, i kind of work from information given to patients really. (gp 5) we found that gps were not familiar with preconception guidelines. they mentioned that there ‘must be’ guidelines available from the national institute for health and care excellence (nice) or the royal college of obstetricians and gynaecologists but confessed they had not always read or were aware of them, as the following comments show: interviewer: can i ask what is your awareness of the professional guidelines of preconception care. respondent: limited. interviewer: right. that is very honest. respondent: i have not read them. (gp 1) i would have thought that was in the nice guidelines, because, obviously, we go via the nice guidelines giving folic acid and immunizations, diets, all the things at the beginning … (gp 2) understanding of preconception care elements of preconception care provided relevance of preconception care in their professional role what is actually provided for general healthy population and specific groups (e.g. women with diabetes) professional guidelines for preconception care awareness of guidelines for own profession, and other professional groups and implementation of guidelines. pre-pregnancy health views on the pre-pregnancy health of women and men in general does the pre-pregnancy health of women need to be intervened? future provision of preconception care barriers to intervening in pre-pregnancy health care opportunities to increase pre-pregnancy health care figure 1. interview topic guide. upsala journal of medical sciences 257 content of preconception advice all gps agreed on the main issues that they would discuss as part of preconception care and mentioned healthy eating, being a healthy weight; starting folic acid supplementation; stopping smoking; and reducing or abstaining from alcohol use. in addition to folic acid, gps also mentioned that they gave advice on other vitamin supplementation if required. this was particularly in the context of vitamin d for specific ethnic populations who were deemed to be more at risk of deficiency. in general, the gps also recommended a multivitamin supplement to their patients. we have quite a high, also got quite, umm, a number of patients who have a low vitamin d. [ … ] so … generally i would be probably be recommending a multivitamin. (gp 4) the gps talked about other relevant elements of preconception care, and it was frequently mentioned that they would check that women were rubella-immune or had had a rubella injection, and one also mentioned asking women if they had had chicken pox. they also said that they would do a risk assessment for mental health issues before pregnancy. we risk-assess in terms of mental health problems, if they have got a history of depression, bipolar, schizophrenia—really any sort of mental health issues. (gp 7) the gps appeared very aware that when prescribing medications to any women of child-bearing age that the safety of the medicine in pregnancy needed to be considered. if you assume all women of child-bearing age are either pregnant or are about to get pregnant, you try to do least or less harm as possible. (gp 1) we found that there was lack of consistency given about the specific content of the advice given in relation to alcohol consumption. before conception some gps advised abstinence, whilst others just a reduction of alcohol to less than a unit per day, and others consumption within sensible limits. gps acknowledged that more consistency in the advice provided by health professionals to women was needed. it should be consistent because i think like with ante-natal care it’s really quite difficult [ … ] i say no, you shouldn’t drink alcohol at all and other people say well, you know, i drank through my pregnancy so it’s ok, you can have a glass now and again. umm, it’s not consistent. (gp 6) who should deliver preconception care? although there was agreement on the important elements of preconception care, views were mixed on the role of gps providing such care. most were comfortable referring women with medical diseases such as diabetes and epilepsy to secondary care for preconception counselling. interestingly, some of the gps did not feel they should be the gatekeepers of preconception care. instead, they felt strongly that prepregnancy health should be a public health issue and that campaigns to increase public awareness would empower women to seek information and facilitate their own reproductive choices. respondent: i think most of it is educational … in certain population groups, but it seems to be more a matter of education, providing information. interviewer: so, would that be education by gps and medics, or where would you put the education? respondent: well, i’m not, it depends on where, is this a job of the medical profession? or is it a job of the education services? they overlap, don’t they? i think education is more important than medicine. (gp 1) again, education isn’t it? yeah. education, education, and education. (gp 3) there was no broad consensus that gps could play a greater role and become more involved in the delivery of preconception care. central to that was the lack of discussion on the provision of a specialized preconception service. instead, many gps discussed the integration of care into the consultations. regarding a potential service delivery model, the most common opinion was that pre-pregnancy care could be delivered by nurses who could provide information when carrying out contraception consultations or whilst undertaking cervical screening, during postnatal review, or when seeing adolescents in schools. … if they come in specifically asking for preconception advice, i usually refer to the nurse, they have another consultation and see actually what the agenda is. (gp 1) because it tends to be more with nurses than it does the doctor, to be honest, because when people make a doctor’s appointment, it’s usually because they’re sick or there’s something going on. but when they see the nurse for a smear or a pill check, that’s when they often sort of indulge, you know, because one of the questions they have to do before they have the smear is, you know, are they planning on getting pregnant, are they on contraception. then they can get that information quite quickly and then be able to have an opportunistic counselling session then probably. (gp 2) targeting provision of preconception care the interviews demonstrated that gps would promote healthy lifestyle behaviour to women attending the surgery that were trying to have a baby or thinking of having one in the future. ok, so that could be anybody who’s contemplating pregnancy at any stage so it might be a conversation from, ummm [ … ], the obvious one you know, i’m thinking about getting pregnant what do you think i should do? or i’m not, or when you’re giving contraceptive advice sometimes it’s actually not now but later i might think about getting pregnant … you know, how long should i take it till i … till i stop my contraception so. (gp 6) the gps targeted care and preconception advice to women with established medical conditions such as diabetes or epilepsy. this involved specific advice about the need for good glucose control before conception for women with diabetes, and the need to review medications that may be teratogenic in other chronic medical conditions. for the diabetics, to make sure their sugar is well controlled and that they might need more interventions when they’re pregnant 258 o. ojukwu et al. in the sense of testing their blood glucose levels when they’re pregnant and they might need to be on insulin … (gp 2) for those with epilepsy we, sort of, give them a neurology review. before you fall pregnant, let’s just see whether we can we change to a medication which is least likely to cause problems [ … ] in terms of diabetes. (gp 7) the gps were more likely to see a woman of child-bearing age to discuss contraception rather than preconception health. gps acknowledged that they did not have consultations that centred on preconception care, and those that did stated this was prompted by patient request for information rather than being initiated by the gp. many gps talked about women attending with concerns and anxiety about becoming pregnant because of their age, for example: so i think it tends to be women who, in my experience [ … ], people who are worried that they’re in their 30s and they want to try and conceive and they’ve waited a long time and they want to make sure that they’re doing everything they can to try and conceive a healthy baby. and i think that kind of anxiety is what brings them to the gp to ask for counselling in the first place, because i don’t think i really get people younger than 30 asking for pre-pregnancy counselling. (gp 2) it was generally reported that this group of women, in their 30s or older, tended to be from a higher socioeconomic class and were more educated on preconception health, health-conscious, and more likely to seek preconception care from their gp. middle-class professional women who are all taking folic acid, they have all been for their rubella check and they are exercising and eating healthily and all that kind of stuff and then are getting ready for pregnancy and their diet, and they are on top of things. (gp 5) in contrast, it was mentioned by a gp serving a student population that students were educated about preconception care and therefore less likely to seek advice from their gp. they are quite informed [about preconception health]. i think a lot them seem to be pretty well informed and do all the right things you know, they may drink within normal limits and they don’t smoke and they exercise. (gp 7) the interviews demonstrated that gps identified women who were perhaps in greater need of preconception care as being younger, often from immigrant populations, and less educated on preconception health and hence less prepared for pregnancy. these women were less likely to seek preconception advice from their gp. even trying to discuss ideas and attitudes towards pre-pregnancy health and care was challenging as women presented during pregnancy. we have got two big housing estates who tend to be unfit and they are still smoking and they are drinking and they often [have] unplanned pregnancies. i mean they would never come to, for pre-pregnancy counselling. it would all be just ‘i am pregnant, doctor, … can i have a termination?’ (gp 7) the ethnic minorities probably, and they’re probably the people who have undiagnosed issues so, umm … whether it’s maybe things like sickle cell or thalassemia, or they’re carriers of genetic diseases, but they don’t really sort of voice these things … or issues with other previous pregnancies. (gp 6) gps associated the trend of women who smoked as having poorer preconception health. on further questioning, they felt that offering pre-pregnancy advice to this particular group of women did not always guarantee a positive change in healthy lifestyle resulting in smoking cessation: we, i mean, we offer advice, but it is up to them to take it on board. i mean you continue to tell them stop smoking and, yeah, live healthily, reduce weight and exercise and, but if they take it on board, because not everyone is willing to accept these things, isn’t it? (gp 3) many gps felt that the women in the ‘poorer’ pre-pregnancy health group did not perceive them as a source of information on preconception health. we’ve got a massive somali and bangladeshi community that are having a lot of babies, but they don’t ever come and see me for advice or anything, they just have them … (gp 2) preconception care for men the gps were asked whether they provided preconception care to men. the general consensus was that preconception care focused on women’s health and was not an issue to raise with men unless subfertility was involved. a handful of patients i see are men that come to see me [because] their partners are trying to get pregnant, it is usually just subfertility. (gp 7) one mentioned that the only time he would see a male in the context of pregnancy was if he was with his partner in order to interpret for her. the only time i would see a man for pre-pregnancy is actually when the women is booking in for an antenatal booking and the partner is coming in to interpret for them. (gp 4) the interviews demonstrated that gps did not offer specific preconception care to the general population of men except rarely in the context of a need for genetic counselling or screening for haemoglobinopathies when couples were planning pregnancy. the gps said that they would include men in the general healthy living advice they were giving to women such as smoking cessation and maintaining a healthy diet. this was extended to asking about the partner’s occupation in respect of any hazards that might affect his fertility. if i’m giving counselling advice to a woman, i often transfer that to their partner as well, so [ … ] only probably with the smoking and the drinking, the only thing i’ll extend to their partner is something along the lines of if they work with chemicals or anything like that. (gp 2) it was felt that the health of women was more important and that more evidence-based information was needed to show the effectiveness of preconception interventions for men before this became more of a focus within consultations. barriers to providing preconception care the barriers to gps providing preconception care included women not planning their pregnancies; lack of perceived upsala journal of medical sciences 259 need, motivation, or attendance for health care; and time and financial constraints. the gps acknowledged that unplanned pregnancies or short inter-pregnancy intervals meant women do not seek advice on preconception health and there is a lack of time to improve health behaviours. she hasn’t sought any pre-pregnancy advice, whether you would think of it as contraception or scheduled information or done any of the things, that she knows she [should] have done, [ … ], so the whole thing about folic acid and all that, talking about lifestyle things … there’s a big hole there. (gp 6) in general, the gps specifically had concerns about the ethnic populations they served in regard to high levels of deprivation, language barriers, and cultural health beliefs that resulted in them not accessing health care before pregnancy. … language may or may not be a barrier. umm … they often don’t seek advice until they are pregnant. they don’t think about things, even if they are diabetic or have risk properties, they don’t tend to see that as an issue, or if they’re on medication [ … ] they’re probably the people who have undiagnosed issues, whether it’s maybe things like sickle cell or thalassemia, or they’re carriers of genetic diseases, but they don’t really sort of voice these things. (gp 6) gps communicated both a lack of motivation from health care professionals to provide preconception care and a perceived lack of knowledge of the need for such a service from patients. … lack of knowledge, education on the patient’s part, both health care professional and patients i think. one, we probably don’t provide enough … and patients don’t seek enough because they don’t understand enough … [of] what’s important. if you’re trying to deliver something that the general population doesn’t want, then you’re probably just wasting your time. (gp 6) there was also concern that preconception care could be seen as a political issue with the ‘nanny state’ dictating personal behaviour. there is something much wider and again anthropological about education and the need to prepare to have a family, all sounds a little bit, too patronizing, doesn’t it? one has to be a little bit careful not to overstep the political mark here. (gp 1) gps frequently perceived a lack of time and lack of financial incentives as a barrier to providing preconception care. gp consultations were already restricted to 10-minute appointments, and preconception care would increase consultation times. if you have got a consultation [ … ] you know you have only got 10 or 15 minutes, if you are dealing with a particular problem then to include other health promotion activities as part of that is going to be a barrier. (gp 5) ways of improving preconception care overall the gps had a variety of opinions as to how preconception care can be improved, but raising awareness through continual education and training of both gps and patients was seen as the main impetus together with media campaigns and financial incentives. specific evidence-based guidelines for use in primary care and leaflets that could be offered to women and men were suggested. i think it would be good to have more information out there, probably a standardized guideline for gp surgeries so what is the minimal we should be offering because i don’t think that we’re very consistent. (gp 6) gps viewed the media as an effective method to empower women to seek advice and improve healthy behaviour before pregnancy. another proposal was a screening programme for preconception health from the age of 15. gps agreed that there were opportunities to increase the uptake of preconception care in primary care. they welcomed using the practice nurse to provide care to all women of child-bearing age during routine contraception checks and cervical screening. 80% of your lists are seen within three years so you are seeing these people all the time. so, i think, there is, primary care is a really good place to do this. so i think the opportunities are great actually. (gp 5) the quality and outcomes framework (qof) was seen as both an opportunity currently and for a future intervention for preconception care. targets such as cervical screening and smoking cessation could be used indirectly as an opportunity for pre-pregnancy health promotion. so inadvertently [ … ], we are always calling patients up all the time at random about their smoking history, making sure they have had their cervical smears [ … ] we are inadvertently but not proactively screening women of child-bearing age to say, you know, come in for [preconception care]. (gp 7) gps suggested contacting at-risk women through disease registers to target those with higher risk of need for preconception care. targeting those, i guess if you do demographic studies on those particular at-risk groups that are more prone to problems and, going through your disease registers [ … ] i guess it would be those groups that you want to target. (gp 7) in addition, it was suggested that incentivizing preconception care through the development of suitable clinical indicators in qof would improve provision. financial incentives would then be paid for preconception care activity incorporated into routine appointments. discussion interviews with gps raised seven key themes: knowledge of preconception guidelines; content of preconception advice; who should deliver preconception care?; targeting provision of preconception care; preconception health for men; barriers to providing preconception care; ways of improving preconception care. gps’ knowledge and awareness of preconception guidelines was low. they tended to rely on knowledge gained from clinical experience, past training, and from patient information sources rather than from guidelines. at the time the interviews were carried out, as in other european countries (16), there were limited specific guidelines available in the uk for preconception care. there were, however, guidelines 260 o. ojukwu et al. within antenatal care management which covered folic acid supplementation, smoking cessation, and alcohol consumption prior to pregnancy. more recently in the uk, nice (8) has produced specific guidelines for women and men planning pregnancy, including guidance for both the healthy population and those with medical conditions, and this may lead to greater awareness amongst health professionals. the gps in our study did not feel they should be responsible for providing routine pre-pregnancy health and care advice; they considered nurses to have a more prominent role in delivery of care. women of child-bearing age accessing the practice nurse for their contraception or routine cervical screening could be targeted, and importantly this concurs with women’s views of when preconception care can be appropriately raised (17). other studies have found low motivation from gps to promote preconception care such as folate supplementation because of their own personal beliefs. in a study of australian gps (18), some gps did not promote use of folic acid as they held the belief that neural tube defects (ntds) were so rare that folate was not needed or that folate did not always prevent ntds anyway. although the gps considered preconception care desirable in order to improve maternal and child health, they stated that there was little demand for the service. the small numbers of patients presenting for advice when trying to conceive have been identified by several other studies (10,18–20), with reasons including a lack of familiarity with the concept of preconception care, not knowing that gps would provide such care, poor knowledge in relation to folic acid supplementation, and concerns of over-medicalization of a normal ‘life’ event. gps mainly provided care to women when prompted by fertility issues such as difficulty in conceiving or when patients asked for advice. otherwise preconception care focused on women with pre-existing health conditions such as diabetes or epilepsy where there was a recognized concern regarding prescription of potentially teratogenic medication. although identified as a priority by the centre for maternal and child enquires to prevent poor pregnancy outcomes (21–23), this targeted approach risks a lack of priority being given to women and men who also may be in high need of preventive care. gps identified that women with ‘poorer’ pre-pregnancy health and of greater deprivation were less likely to attend for health care and more likely to present at the surgery with unintended pregnancies. these women are less likely to take folic acid supplements and are more likely to participate in unhealthy behaviour such as smoking, drinking alcohol, and taking illicit drugs (24–26). gps stated that not only their own awareness and knowledge needed to be improved to enable preconception care counselling, but awareness was needed in the general population by those that required care. other studies (11,18) have found a low knowledge of behaviours related to preconception care and a need to increase general awareness. women who receive preconception counselling are more likely to adopt healthier behaviours before pregnancy, leading to an improved diet, folic acid supplementation, and cutting down or stopping smoking (10,27). this reinforces the potential for the role of gps in improving awareness and uptake of preconception care if interventions in primary care were provided. there was, however, a lack of motivation from gps to provide specialist preconception services within their surgeries. there is limited evidence on the implementation of preconception care strategies in primary care (18), and studies are conflicting. routine provision of preconception care for all women is supported by some (28,29), including starting in adolescence (30), whilst the world health organization (who) suggests targeting specific groups (7). a study (31), in which gps invited women to complete a risk assessment and to attend for preconception care, indicated that only a quarter of the women who became pregnant in the year afterwards had attended. the study protocol, however, excluded many women due to their adverse social circumstances, and it was concluded that it was important to develop ways to increase the range of women invited for the intervention. a study is currently targeting populations from specific neighbourhoods of the netherlands which have high levels of deprivation in order to try to decrease the high levels of perinatal mortality. community and general practice preconception interventions are to be implemented with the aim of achieving positive change in preconception risk behaviours (32). gps have an important role in how preconception care should be delivered and in the allocation of resources to support and deliver this service effectively. a review by shannon et al. (33) identified primary care as the most common setting for preconception health service delivery; however, the authors also concluded that there is no agreed consensus on the best method to deliver care within primary care. it is possible that many strategies acting synergistically are needed to improve service delivery. this is consistent with findings from a qualitative study (11), which showed that women who take a micronutrient supplement are more likely to invest in their preconception health and care. the authors identified women belonging to three groups—the ‘prepared’ group, the ‘poor knowledge’ group, and the ‘absent prepregnancy’ group—and argued that different preconception strategies are needed to target different women. men were found not to attend the surgery for pre-pregnancy advice unless subfertility was a concern. the lack of interest and practice of preconception care for men was consistent in our study and in line with other research (34); the majority of the gps did not view men’s preconception health as a priority. studies show that men have a lower knowledge of preconception care and are less likely to see their gp for preconception interventions (35). this is congruent with men’s limited involvement in reproductive and sexual health for preventive health care in general (36). however, the importance of preconception care for men has been highlighted by a us study (37) which found that 60% of men aged between 15 and 64 years have a need for preconception care with concerns of obesity, binge drinking, use of illicit substances, and their high risk for sexually transmitted infections. few had attended screening or preventive services, and there was the opportunity to increase uptake of such interventions. there is growing evidence that men’s health at conception plays a significant role in pregnancy upsala journal of medical sciences 261 and neonatal outcomes through direct and epigenetic influence on the health of spermatozoa (38,39). greater attention needs to be given to involving men in preconception care by reviewing factors such as lifestyle choices (8,40), medical conditions and medications (17,41), environmental exposures (40,42), and screening for infection (40). gps discussed barriers to providing care, which centred on need, awareness, and capacity. in agreement with other studies (10,43), limited consultation time and lack of resources were seen as the main barriers to providing care. currently there is no direct financial reimbursement to gps for integrating preconception care into consultations with women of reproductive age. although gps felt that financial incentives were needed to increase the provision of preconception care, evidence for using incentivized frameworks has been found to be debatable. such frameworks have improved asthma and diabetes care in the short term (44), but in a qualitative pilot study, aimed at increasing antismoking advice to smokers by gps (45), it was found that gps changed the way they documented smoking advice to meet the incentives rather than significantly give more advice in clinical practice. strengths and limitations of the study this is, to our knowledge, the first paper that considers gps’ perspectives on providing preconception care to a general population. the study was undertaken as part of a larger publicly funded study of preconception care in england, and it is an exploration of gps’ views rather than an unbiased evaluation of preconception care. the study does not provide information on the frequency with which women receive care from their gps or the effectiveness of preconception care provided. interviews were undertaken by three researchers which may prevent bias from personal interest, but the context of the study and knowledge that they were from the ‘institute of women’s health’ may have influenced the gps’ conversation. the gps appeared to give honest opinions in regard to their knowledge base and views of service provision, for example in confessing to not having read preconception guidelines, which enhanced the trustworthiness of the findings. the study was carried out in a borough of london where a there is a disparity of wealth and a large ethnic minority population and therefore may be different to many other areas of the uk. however, the findings have similarities to those of other uk studies examining the patient perspective of provision and uptake of care (17,19), which agree that gps could provide more information and be more proactive in respect of preconception care provision. in addition, we found that gps were more likely to provide preconception care to women with medical conditions, and this targeted approach highlighted issues similar to those found by mortagy et al. (46) who interviewed gps and secondary care health professionals focusing on women with diabetes. conclusion this qualitative study set out to examine gps’ knowledge, attitudes, and views towards preconception health and care in the general practice setting. the findings provide an insight into how care is currently delivered in primary care, and the gps described diverse patient groups with very different health needs before pregnancy. a lack of both knowledge and demand for preconception care was found, and, although reaching women before they are pregnant was seen as important, it was not a responsibility that could be adequately met by gps. strategies need to be developed to increase awareness in women with ‘poorer’ pre-pregnancy health, who are more likely to have unintended pregnancies, and to find ways to engage men in preconception care. the role of education and of nurses in improving preconception health was acknowledged but remains under-developed in primary care. 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2008;199:s389–s95. 41. agbaje im, rogers da, mcvicar cm, mcclure n, atkinson ab, mallidis c, et al. insulin dependant diabetes mellitus: implications for male reproductive function. hum reprod. 2007;22:1871–7. 42. jensen tk, bonde jp, joffe m. the influence of occupational exposure on male reproductive function. occup med. 2006;56:544–53. 43. jack bw, culpepper l, babcock j, kogan md, weismiller d. addressing preconception risks identified at the time of a negative pregnancy test. j fam pract. 1998;47:33–8. 44. campbell sm, reeves d, kontopantelis e, sibbald b, roland m. effects of pay for performance on the quality of primary care in england. n engl j med. 2009;361:368–78. 45. coleman t, wynn at, stevenson k, cheater f. qualitative study of pilot payment aimed at increasing general practitioners’ antismoking advice to smokers. bmj. 2001;323:432. 46. mortagy i, kielmann k, baldeweg se, modder j, pierce mb. integrating preconception care for women with diabetes into primary care: a qualitative study. br j gen pract. 2010;60:815–21. upsala journal of medical sciences 263 http://www.qsrinternational.com/products_nvivo.aspx http://www.qsrinternational.com/products_nvivo.aspx https://www.nice.org.uk/guidance/ng3 https://www.nice.org.uk/guidance/ng3 https://www.nice.org.uk/guidance/cg137 https://www.nice.org.uk/guidance/cg137 general practitioners&rsquo; knowledge, attitudes and views of providing preconception care: a qualitative investigation background method findings knowledge of preconception guidelines content of preconception advice who should deliver preconception care? targeting provision of preconception care preconception care for men barriers to providing preconception care ways of improving preconception care discussion strengths and limitations of the study conclusion declaration of interest references untitled clinical outcome of fragment fixation for osteochondritis dissecans 201 key words: osteochondritis dissecans, elbow, fragment fixation received 4 march 2008 accepted 13 march 2008 upsala j med sci 113 (2): 201–208, 2008 clinical outcome of fragment fixation for osteochondritis dissecans of the elbow shingo nobuta1, kazuhiro ogawa1, katsumi sato1, tomowaki nakagawa2, masahito hatori3, eiji itoi3 1department of orthopaedic surgery, tohoku rosai hospital, 2department of orthopaedic surgery, iwate prefectural iwai hospital, 3department of orthopaedic surgery, tohoku university, school of medicine abstract background: the choice of surgical or non-surgical treatments for osteochondritis dissecans (ocd) of the humeral capitellum is still controversial. the purpose of this study was to assess the efficacy of fragment fixation for ocd of the humeral capitellum. methods: we reviewed 28 patients with ocd of the humeral capitellum after a mean follow up of 17 months. all patients were men and mean age was 14 years. twentyseven patients had a history of repetitive overuse of the elbow with baseball pitching, one with tennis. mean duration of overuse of the elbow was four years. all patients had elbow pain and difficulty in throwing, with a mean duration of symptoms for 17 months. the mean arc of flexion before surgery ranged from 11 degrees to 126 degrees. radiographs of the elbow showed a radiolucent cystic area of the humeral capitellum in one patient, a non-displaced split type fragment in 12 patients, and a slightly displaced split type fragment in 15 patients. fragment fixation surgery was performed in all patients by lateral arthrotomy including drilling and fixation of the fragment with a double wiring technique using flexible wire or thread under direct vision. sport activities using upper extremities were restricted for four to six months until the lesion healed in radiograph. results: post-operatively, 25 patients had no pain and three decreased pain. average arc of flexion was one to 132 degrees, an improvement of 16 degrees compared with the pre-operative arc. radiographic findings showed complete healing of the lesion in 11 patients, partial healing in 12, unchanged in three, and loose body formation in two. by tivnon’s evaluation of the elbow function, results were excellent in 19 patients, good in five, fair in two, and poor in two. the ratio of complete or partial healing of the lesion was 100 percent in 16 patients in whom the thickness of the lesion was less than 9 mm on pre-operative radiograph, and 58 percent in 12 patients in whom the lesion thickness was 9 mm or more, which showed a significant difference (p<0.01). conclusions: fragment fixation for ocd of the humeral capitellum was effective in patients whose lesion thickness was less than 9 mm. fixation by flexible wire or thread and revascularization by drilling for the fragment were considered to be insufficient for large lesions with a thickness of 9 mm or more. introduction osteochondritis dissecans (ocd) of the humeral capitellum is commonly seen in adolescent athletes who engage in throwing sports and activities. treatment in the 202 shingo nobuta et al. early stage of ocd lesions is conservative and spontaneous healing is possible (1, 2), but surgical treatment should be applied to advanced lesions. surgical treatments for ocd include fragment removal (3–5), fragment fixation (6–9), bone peg grafting (10), closed-wedge osteotomy (11, 12), and cartilage transplantation (13). to obtain healing of the lesion, we chose the simple surgical procedure of fragment fixation for advanced ocd lesions (14). the purposes of this study were to assess the efficacy of fragment fixation for ocd of the humeral capitellum and to investigate the factors influencing results. patients and methods between 1997 and 2006, 28 patients with ocd of the humeral capitellum underwent fragment fixation surgery in our institute, and were followed-up for seven to 36 months (mean, 17 months). all patients were men and mean age at operation was 14 years (12 to 19). twenty-seven patients had a history of repetitive overuse of the elbow with baseball pitching, and one patient with tennis. mean duration of overuse of the elbow was four years (two to six). all patients had elbow pain and difficulty in throwing, with a mean duration of symptoms for 17 months (range, 3–73 months). the mean arc of flexion before surgery was 11 degrees to 126 degrees. radiographic findings were based on the anteroposterior tangential views of the elbow at 45 degrees of flexion. radiographic findings were classified by minami’s classification (15), type 1 includes those elbows where a radiolucent cystic area is seen in the capitellum, type 2 is the split type, where a clear zone or split line is seen between the fragment and the adjacent subchondral bone, and type 3 includes those elbows with a loose body, where a split fragment has completely separated from its bony bed. of the 28 patients in our series, one was classified as type 1, twelve were type 2 with a non-displaced fragment, and fifteen were type 2 with a slightly displaced fragment. there were no cases of type 3 in our series. the mean width of the lesion including the clear zone and fragment was 12 mm (6 to 17), and the mean thickness of the lesion was 8 mm (5 to 14) on pre-operative radiograph. indications for fragment fixation surgery included failure of more than 6 months of non-operative treatment or an unstable displaced fragment on radiograph, or a high signal intensity line between fragment and the adjacent bone in t2-weighted magnetic resonance imaging (8, 9, 16). surgical technique operations were performed under general anesthesia with an air tourniquet applied. the surgical technique was almost similar to the original description by kondo et al. (6). the elbow was approached laterally, and the lateral collateral ligament was released anteriorly and posteriorly to obtain a wide operative field. the capitellum was explored and the articular cartilage and subchondral bone were inspected. the osteochondral fragment was commonly still attached to its bony bed by fibrous tissues. the osteochondral fragment was raised and the subchondral fibrous tisclinical outcome of fragment fixation for osteochondritis dissecans 203 sue was curetted. using a 1.2-mm diameter kirschner wire, drilling from the joint surface was performed for the fragment and bony bed. two 1.5-mm diameter kirschner wires were then inserted from the posterior side of the lateral epicondyle to the fragment. the medial kirschner wire was removed and a 0.4-mm diameter flexible wire (12 patients, from 1997 to 2002) or thread (16 patients, from 2003 to 2006, because of wire breakage in three patients before 2002) was inserted from the lateral epicondyle into the elbow joint. the lateral kirschner wire was removed and an 18-gauge injection needle was inserted into the joint (figure 1a). the wire or thread was inserted through a kirschner wire hole from the joint surface to the lateral epicondyle with the needle as a guide (figure 1b). the soft wire or thread was then tied on the posterior side of the lateral epicondyle, and the fragment was fixed to its bony bed. the same procedure was repeated to fix the fragment with two wires or threads. all small loose bodies and cartilage debris were removed after checking the coronoid and olecranon fossae. after irrigation was performed, a drain was inserted and the ligament, soft tissue, and skin were sutured. post-operative management a plaster splint held the elbow at 90 degrees of flexion for 3 weeks, and flexion-extension isometric exercise was started on the fourth post-operative day. active motion exercise was started after 3 weeks. a wire was removed under local anesthesia at our outpatient clinic at 6 months to one year post-operatively. sport activities were restricted for about six months until the lesion had healed on radiographs. we assessed the elbow function according to tivnon’s evaluation (17). a return to full preoperative sport activity was rated as excellent; a return to the same preoperative sport, but with decreased performance, was rated as good. with a fair rating, the elbow symptom was improved but persistent pain at throwing caused a change to other sports. a poor result meant that the patient was unable to return to sports and required a second operation. figure 1. operative technique. a, two kirschner wires were inserted to fix the fragment and the medial wire was removed, and a flexible wire or thread was inserted into the elbow joint. the lateral wire was removed and an 18-gauge injection needle was inserted into the joint. b, the flexible wire or thread was threaded through the kirschner wire hole from the joint surface to the lateral epicondyle with the needle as a guide. flexible wire or thread was tied, and the fragment was fixed to its bony bed. 204 shingo nobuta et al. we analyzed the data statistically with use of the student’s t-test and chi-square test. p-values less than 0.05 were considered statistically significant. results post-operatively 25 patients experienced no pain and three had decreased pain. post-operative average arc of flexion was one to 132 degrees, an improvement of 16 degrees compared with the pre-operative arc. according to tivnon’s evaluation of the elbow function (17), the results were excellent in 19 patients, good in five, fair in two, and poor in two. the rate of return to the previous level of sports was 86 percent (24 of 28). post-operative radiographic findings were evaluated into four categories according to minami’s criteria (15). complete healing of the lesion was seen in 11 patients, partial healing in 12, unchanged in three, and loose body formation in two. according to the pre-operative radiographic findings, the patient with type 1 showed complete healing. in type 2 with a non-displaced fragment, there was complete healing in seven, partial healing in three, and loose body formation in two (table 1). type 2 with a slightly displaced fragment showed complete healing in three, partial healing in nine, and unchanged in three. the ratio of complete or partial healing of the lesion was 82 percent (23 of 28). the time of healing of the lesion after operation in 23 patients with complete or partial healing was six to 16 months, the mean being eight months. in four patients with fair or poor results, post-operative radiograph showed partial healing in one patient, unchanged in two, and loose body formation in one. the ratio of complete or partial healing of the lesion on the radiograph was 100 percent in 16 patients in whom the thickness of table 1. relationship between pre-operative radiographic thickness and post-operative healing of the lesion pre-operative radiographic thickness of the lesion post-operative healing <9 mm 9 mm≤ complete (n=11) 7 4 partial (n=12) 9 3 unchanged (n= 3) 3 loose body (n= 2) 2 ratio of complete or 100 (16/16) 58.3 (7/12) partial healing (percent) the ratio of complete or partial healing of the lesion was 100 percent in 16 patients in whom the thickness of the lesion was less than 9 mm in pre-operative radiograph, and 58 percent in 12 patients whose thickness of the lesion was 9 mm or more, which showed a significant difference (chi-square test, p<0.01). clinical outcome of fragment fixation for osteochondritis dissecans 205 the lesion was less than 9 mm on pre-operative radiograph, and 58 percent (7 of 12) in the patients whose lesion thickness was 9 mm or more, which showed a significant difference (table 1, p<0.01). there was no correlation between the width of the lesion on pre-operative radiographs and post-operative radiographic healing. age at operation, duration of overuse of the elbow, duration of the symptoms had no correlation with post-operative radiographic healing. there were no infections, no irritation from wire, but wire breakage was seen in three patients at the time of wire removal. discussion the aims of treatment for ocd of the humeral capitellum are a return to the previous level of sports activities and prevention of osteoarthritic changes of the elbow in the future. the early stage of ocd lesions, which are observed as radiolucent cystic areas on radiograph are commonly treated conservatively with prohibition of sporting activities and rest of the arm. however, surgical treatment should be applied to advanced lesions to obtain healing of the lesion. takahara et al. (18) described that ocd of the capitellum can be classified as stable and unstable, and stable lesions can heal completely with elbow rest, whereas unstable lesions require surgery to obtain better results. they reported that unstable lesions had a capitellum with a closed growth plate, fragmentation, or restriction of elbow motion of 20 degrees or more. completely detached lesions or symptomatic loose bodies should be excised and good results are obtained (19, 20). arthroscopic removal of loose bodies, debridement and abrasion chondroplasty have the advantage of providing good results with minimum invasion (21–23). this procedure can provide a prompt return to sports if the remaining defect of the capitellum is small (18). however, several studies showed that patients who had the removal of the osteochondral fragment or loose body with drilling or curettage could not return to their previous level of sports (2, 3, 17). takahara et al. (18) reported that the results of fragment fixation or reconstruction were significantly better than those of fragment removal alone, and that fragment removal is the best option only if the capitellar defect is small. fragment fixation is one of the surgical procedures for a partially attached lesion (19) which is shown as the split type on the radiograph. fixation by wiring with a bone graft (6, 8), herbert screw fixation (7), fragment fixation with a bone graft and dynamic staples (9), and bone peg grafting (10) were reported with approximately 100 percent of healing and return to a previous level of sports. in our series, the rate of healing was 82 percent (23 of 28) and the rate of return to the previous level of sports was 86 percent (24 of 28) which were worse than the previous studies (6–10). in our surgical procedure, regardless of the size or the thickness of the lesion, fibrous tissues between the fragment and the bony bed was curetted, and the fragment was drilled and fixed without a bone grafts. it is commonly thought that the blood supply to the fragment is damaged if the fragment is detached or if the fibrous tissues between the fragment and the bony bed is curetted (9). there has 206 shingo nobuta et al. been no report that mentions the correlation between the size or the thickness of the lesion and the possibility of healing after fragment fixation. in our patients, the ratio of healing was 58 percent in the patients whose lesion thickness was 9 mm or more (table 1). therefore, we believe that fixation by a wire or thread and revascularization by drilling were insufficient for large lesions with a thickness of 9 mm or more. matsuura et al. (24) reported that low-intensity pulsed ultrasound was useful for healing because of increased revascularization. based upon studies of blood supply of the capitellum (25), ocd is a disorder of endochondral ossification caused by vascular insufficiency and induced by repetitive microtrauma to a stressed and vulnerable epiphysis. a bone graft seems to be effective (6, 7), or a combination of the fragment fixation and the low-intensity pulsed ultrasound (24) is thought to be useful to revascularize and heal large ocd lesions. this study has several limitations. first, the follow up period was short (mean, 17 months). accordingly, we were not able to investigate the development of osteoarthritic change of the elbow. second, the importance of drilling the lesion to stimulate revascularization and healing has never been established (19), but we presume that drilling might be of value for revascularization and healing. conclusions fragment fixation for ocd of the humeral capitellum was effective in patients whose lesion thickness was less than 9 mm. fixation by soft wire or thread and revascularization by drilling for the fragment seemed to be insufficient for large lesions with a thickness of 9 mm or more. acknowledgments the authors thank ms. hiromi takeda and ms. yumi watabe for assisting in the manuscript preparation. references bauer m, jonsson k, josefsson po, linden b (1992) osteochondritis dissecans of the elbow; 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a cur-23. rent concepts review. am j sports med 31: 621–635. matsuura t, yasui n, kashiwaguchi s, iwase t (2003) low-intensity pulsed ultrasound for os-24. teochondrosis of the humeral capitellum in baseball players. seikei saigai geka 46: 1173–1177 (in japanese). singer km, roy sp (1984) osteochondrosis of the humeral capitellum. am j sports med 12: 25. 351–360. corresponding author: shingo nobuta, department of orthopaedic surgery, tohoku rosai hospital 4-3-21, dainohara, aoba-ku, sendai, miyagi 981-8563, japan tel: +81-22-275-1111 fax: +81-22-275-7541 e mail: nobutays@jc5.so-net.ne.jp (14) godfried m roomans 155-168 155 upsala j med sci 111 (1): 155–168, 2006 transmission electron microscopy in the diagnosis of primary ciliary dyskinesia godfried m. roomans1, andrejs ivanovs1, eyman b. shebani1,2, marie johannesson2 1 department of medical cell biology, university of uppsala, box 571, uppsala, sweden 2 department of women’s and children’s health, uppsala university, uppsala university hospital, uppsala, sweden abstract primary ciliary dyskinesia (pcd) is an autosomal recessive disease with extensive genetic heterogeneity. dyskinetic or completely absent motility of cilia predisposes to recurrent pulmonary and upper respiratory tract infections resulting in bronchiectasis. also infections of the middle ear are common due to lack of ciliary movement in the eustachian tube. men have reduced fertility due to spermatozoa with absent motility or abnormalities in the ductuli efferentes. female subfertility and tendency to ectopic pregnancy has also been suggested. headache, a common complaint in pcd patients, has been associated with absence of cilia in the brain ventricles, leading to decreased circulation of the cerebrospinal fluid. finally, half of the patients with pcd has situs inversus, probably due to the absence of ciliary motility in hensen’s node in the embryo, which is responsible for the unidirectional flow of fluid on the back of the embryo, which determines sidedness. pcd, which is an inborn disease, should be distinguished from secondary ciliary dyskinesia (scd) which is an acquired disease. transmission electron microscopy is the most commonly used method for diagnosis of pcd, even though alternative methods, such as determination of ciliary motility and measurement of exhaled nitric oxide (no) may be considered. the best method to distinguish pcd from scd is the determination received 12 october 2005 accepted 27 october 2005 of the number of inner and outer dynein arms, which can be carried out reliably on a limited number of ciliary cross-sections. there is also a significant difference in the ciliary orientation (determined by the direction of a line drawn through the central microtubule pair) between pcd and scd, but there is some overlap in the values, making this parameter less suitable to distinguish pcd from scd. introduction primary ciliary dyskinesia (pcd) is a term used to describe a group of ultrastructural, genetically heterogenous autosomal recessive disorders affecting ciliary movement [1]. the first description of a cilia-related syndrome appeared in the beginning of the 20th century. oeri [2] described a patient with sinusitis, bronchiectasis, and situs inversus, and siewert [3] described a patient with bronchiectasis and situs inversus. then, kartagener [4] in more detail described the same triad of abnormalities (sinusitis, bronchiectasis, and situs inversus) in four patients and first recognized a clinical syndrome, which was subsequently named after him. the pathogenesis of kartagener’s syndrome began to come to light in the 1970s. afzelius et al. [5] described the association of sinusitis, bronchiectasis, and situs inversus with an absence of dynein arms in the tails of the immotile spermatozoa in infertile men. eliasson et al. [6] introduced the term “immotile cilia syndrome” to describe all congenital ciliary defects resulting in impaired mucociliary clearance and male infertility. soon it was determined that lack of motility was not absolute. in some patients, ciliary movement was observed; nevertheless, it was not adequate for normal mucociliary clearance. therefore, the syndrome name was changed to “dyskinetic cilia syndrome” [7]. sleigh [8] proposed to use the term “primary ciliary dyskinesia (pcd)” to denote congenital ultrastructural cilia alterations and the term “secondary ciliary dyskinesia (scd)” to describe acquired ultrastructural cilia defects. cilia are eukaryotic cell organelles that play important roles including cell motility, transport of mucus, other fluids, and even other cells, and function in communication with the extracellular environment [9]. there are two types of cilia: motile cilia, which propel a single cell through liquid or move fluid across the surface of a layer of cells, and immotile cilia, which usually serve as sensors [1]. cilia are found in all animals; however, nematodes and arthropods have only immotile cilia on some sensory nerve cells. cilia are rare in plants occurring more notably in cycads. protozoa have only motile cilia and use them for either locomotion or to simply move liquid over their surface. in humans, motile cilia are found lining the upper and lower respiratory tract, sinuses, middle ear, the ependyma of the brain, the ductuli efferentes of males, and the female oviduct, and they are found in the uterus. furthermore, cilia are morphologically similar to flagella of spermatozoa [10]. motile cilia are also present in hensen’s node. they perform there an embryological function in the determination of laterality [11-13]. motile cilia are usually present on a cell surface in large numbers. in contrast to the motile cilia, immotile cilia usually occur as a single cilium per cell. all types of mammalian cells have a single immotile cilium called “primary cilium” that has been neglected for a long time. recent studies let scientists re-evaluate its physiologi156 cal role in cell signalling and the control of cell growth and development [9]. immotile cilia were found in the vertebrate retinal rods and cones as well as in the apical knob on olfactory neurons [14]. ciliated cells are a part of the mucosal pseudostratified columnar epithelium lining the respiratory tract. each ciliated cell carries approximately 200 cilia on its surface. the function of these cilia is to beat in a coordinated manner to provide normal clearance of mucus and other debris from the airways. the ciliary beat cycle consists of three sequential events: effective stroke, recovery phase, and recovery stroke. the normal ciliary beat frequency is 11-16 hz [15, 16]. ultrastructure of cilia cilia are highly complex organelles. more than 250 proteins are involved in the formation of cilia. the majority of these are components of a specific axoneme structure. there are many other proteins that are important for ciliary assembly, initiation, orientation, and control of ciliary activity [17]. the core of the motile cilium is the axoneme, which consists of nine peripheral microtubule doublets surrounding a central pair of singlet microtubules. each of the microtubules is constructed from heterodimers of �and �-tubulin, assembled into the 13 protofilaments of the a microtubule and the 11 protofilamets of the b microtubule (which, in addition, shares two protofilaments with the a microtubule) the microtubules of the central pair are composed of 13 protofilaments and have approximately the same orientation as the central pair of adjacent cilia, which is important in the production of a coordinated ciliary waveform [1, 9, 10, 18] (fig.1 ). each cilium is anchored to a 157 figure 1. transmission electron micrograph (a) of a patient with scd (a 19 year-old boy) with dynein arms on the a microtubulus. the orientation of the central pair is similar in all cilia. (b) of cilia from a nasal biopsy of a patient with pcd (a four day-old baby boy with situs inversus), lacking dynein arms on the a microtubulus. bar = 0.1 µ m (same for both micrographs) basal body in the cytoplasm near the plasma membrane. this structure is composed of nine microtubule triplets. the axoneme is held together by four sets of protein cross-links. a fibrous inner sheath surrounds the central pair of microtubules. central singlets are connected by a bridge. each microtubule doublet is connected to the adjacent doublets by nexin links and to the central pair by radial spokes [16]. other important structural proteins are known as microtubule-associated proteins. the most studied of them is dynein, which forms outer and inner dynein arms (fig. 1a), structures which are absent or abnormally small in most pcd patients (fig. 1b). dynein is a high molecular weight protein that belongs to the group of mechanochemical atpases [10]. outer dynein arm complexes consist of heavy, intermediate, and light molecular weight dynein chains in addition to at least 10 other polypeptides [19]. inner dynein arms have a more complex structure and are composed of heavy, intermediate, and light chains that are evolutionarily distinct from those in the outer dynein arms [20]. outer and inner dynein arms have different functions in the generation of the ciliary beat. the outer dynein arms have an effect on beat frequency, while the inner dynein arms influence the beat waveform [10]. ciliary activity has been shown to occur via atp hydrolysis by dynein heavy chains, which causes a sliding of the a microtubule relative to the b microtubule. this results in bending of the cilia [16]. other microtubule-associated proteins within the ciliary axoneme include proteins associated with the radial spoke complexes, the central pair apparatus, and the nexin links. radial spoke protein 3 is responsible for the attachment of the radial spoke complex to the peripheral microtubule doublet. the proteins calmodulin and light molecular weight dynein have also been identified in the radial spoke complex and have a role in regulating ca2+ influx [21]. genetics of pcd pcd is an autosomal recessive disorder with extensive genetic heterogeneity; nevertheless, a few rare cases of apparently dominant or x-linked inheritance have been reported [22-25]. all affected genes have yet to be identified. there are at least 250 proteins within a single cilium, each encoded by a separate gene, and many other genes participate in the assembly and regulation of cilia [10, 17]. therefore, it is clear that the number of possible candidate genes is extremely large. the first gene in which a mutation was found to be a cause of pcd was dnai1. this gene was isolated by pennarun et al. [26] from chlamydomonas reihardtii, a unicellular alga with two flagella containing an axonemal structure similar to that of human respiratory cilia and spermatozoa flagella. to date, only three autosomal genes have been shown to cause pcd with defects in outer dynein arms. these genes are dnai1, dnah5, and dnah11. dnai1 encodes outer dynein arm intermediate chain. dnah5 and dnah11 encode outer dynein arm heavy chains [2630]. these genes are located on human chromosomes loci 9p13, 5p15, and 7p21, respectively [1]. no mutations have yet been reported in patients with other ultra158 159 structural cilia abnormalities, but the gene mutations mentioned above account only for a few pcd cases. other genes coding for axoneme dyneins, such as dnah7, dnah9, dnai2, and dnal1 were recently excluded as major causes of pcd [3134]. moreover, the foxj1 gene, encoding a transcription factor involved in ciliary development, was also excluded as a common cause of pcd [35]. several genetic loci for pcd have been identified, and the chromosome region 19q13.3-qter is of particular interest. it is a gene rich region and contains a large cluster of zinc finger genes. this is of potential interest as the only human gene unambiguously associated with situs abnormalities so far is zic3, a gene encoding one of the zinc finger transcription factors. the results of linkage analysis in five families of arabic origin provided conclusive evidence for a pcd locus on chromosome region 19q13.3-qter and confirmed this locus heterogeneity. genetic heterogeneity is expected in pcd with regard to its wide range of ultrastructural phenotypes [10]. recently, it was postulated that genes responsible for x-linked retinitis pigmentosa could also be involved in pcd. a non-consanguineous family was described in which a woman with retinitis pigmentosa gave birth to two boys presenting a complex phenotype associating pcd with retinitis pigmentosa. it is already known that photoreceptor and respiratory cilia have common structures. transmission electron microscopy (tem) showed numerous abnormal cilia in which dynein arms and central singlets were missing [36]. clinical aspects of pcd as all tissues where ciliated cells are present might be affected, a huge variance in the severity of the clinical phenotype of pcd and the predominant symptoms at different ages is possible [10]. dyskinetic or completely absent motility of cilia predisposes to recurrent pulmonary and upper respiratory tract infections resulting in bronchiectasis [24]. the first symptoms of pcd in the respiratory tract may present in neonates, such as unexplained tachypnea, pneumonia with no obvious risk factors, and significant rhinitis with intense mucous production. in infants and older children, pcd may present as atypical disease or disease not responsible to treatment, for example, asthma, chronic wet cough with sputum production, already predictable rhinosinusitis, and chronic secretory otitis media. it is important that in young children chronic sinusitis cannot occur by definition because the sinuses do not reach a mature state until 6 years of age. the symptoms in adults are similar to those of older children, but the secretory otitis media is less severe [37]. in pcd, structural and functional abnormalities in respiratory cilia are frequently associated with similar defects in the tails of spermatozoa and the cilia of the epithelium in the ductuli efferentes of the testis. therefore, the diagnosis of pcd in adult male patients is usually made as a result of the clinical presentation of infertility. men have reduced fertility due to sperm with absent motility or abnormalities in the ductuli efferentes. the epithelium in the ductuli efferentes has cilia, which normally assist in the transportation of spermatozoa out from the testes to the vas deferens [38]. there are only very few studies of the role of these cilia in health and disease. phillips et al. [39] described cilia from the ductuli efferentes, which lacked dynein arms, in a patient with kartagener syndrome who had normal motile spermatozoa. as for spermatozoa motility, men with pcd usually have a normal ejaculate volume and sperm counts, but only 0-30% of the sperm are motile, which is not sufficient for successful fertilization [16]. female subfertility and tendency to ectopic pregnancy have also been suggested [18]. it is considered that muscular contractions are the primary force for the transport of ova and zygotes, and the oviduct cilia participate in this process at least partially [38]. however, halbert et al. [40] are of the opinion that the primary means by which the ovum is moved through the oviduct is mucociliary transport. a woman with the complete kartagener’s syndrome can be fertile or sterile, but female fertility is somewhat reduced when the cilia are immotile [38]. the first and before yet the only report of an ectopic pregnancy in a woman with pcd was provided by lin et al. [41]. this patient had tried unsuccessfully to become pregnant for 8 years. a mild degree of right peritubal and periovarian adhesions was noted, but this was not likely the cause of her infertility. after adhesiolysis, she was still unable to conceive spontaneously for 2 years, until a right ovarian pregnancy occurred. the only identifiable and likely cause of the ectopic pregnancy in this patient was tubal dysfunction due to pcd [41]. nearly half of the patients with pcd has situs inversus (the complete reversal of thoracic and abdominal organs) or rarely situs ambiguous (any other abnormalities of laterality). it has recently been proposed that the monocilia in hensen’s node determinate left-right patterning of the body. there is evidence for the role of mechanical fluid flow in left-right asymmetry. the nodal cilia normally create a leftward stream over the embryonic node of a fluid that most probably contains morphogenic factors, which specify the location of thoracic and abdominal contents in accordance with normal situs solitus [11, 12]. inactivation of the genes associated with left-right patterning of the body should make the asymmetry random. it is also generally claimed that approximately a half of patients has situs abnormalities [6, 42]. sometimes situs inversus is associated with complex congenital heart diseases and abnormalities of the gastrointestinal tract [37]. hydrocephalus is a rare but well described feature of pcd. the third and fourth ventricles are connected by the aqueduct of sylvius. computed tomography of the brain at different time-points revealed that in some patients with pcd the aqueduct is present at birth but progressively becomes occluded, implying that the ciliated ependyma of the brain ventricles is important for a normal flow of cerebrospinal fluid in certain directions [18]. it has been reported that two-thirds of the patients with pcd suffer from chronic headaches [43]. the most obvious explanation of this symptom is abnormal circulation of cerebrospinal fluid. 160 other clinical features of pcd include very severe gastrooesophageal reflux, oesophageal and extrahepatic biliary atresia, intestinal malrotation, splenic alterations (asplenia, splenic hypoplasia, and polysplenia), and renal agenesis [37, 44, 45]. these symptoms are rare and unusual manifestations of pcd. for example, intestinal malrotation accompanying kartagener’s syndrome has been previously reported twice. in these two cases, patients presented bilious vomiting and mechanical ileus [45, 46]. all these rare clinical manifestations of pcd represent the result of a single dysmorphogenic process involving the middle structures of embryo and are connected to situs inversus pathogenesis [47]. diagnosis of pcd a throrough medical history and carefully performed physical examination are the keystones in the successful diagnoss of pcd. clinical features of pcd are usually present in the neonatal period. pcd should be considered in pediatric or adult patients with chronic and intractable sinopulmonary infections, as well as in infertile men. since pcd often imitates other diseases, the investigation of individuals in whom this disorder is suspected can be divided into three stages. at first, all differential diagnoses, including cystic fibrosis and immunodeficiencies, must be excluded. therefore, early investigations include a chest x-ray, sweat test, immunoglobulins and subclasses, cystic fibrosis genotype, and age-appropriate lung function tests. after preliminary tests have been completed, ciliary function and structure must be examined. finally, all discovered pathologies must be evaluated [10, 15]. there are a number of ways to assess ciliary function. to screen older children and adults the saccharin test can be performed. a 1-2 mm particle of saccharin is placed on the inferior nasal turbinate 1 cm from the anterior end. the patient must sit quietly with the head bent forward, and must not speak, sneeze, cough, eat or drink for the duration of the test. the time to tasting saccharin is noted and is a measure of nasal mucociliary clearance. normally it should be less then 60 minutes. the ability of the patient to taste saccharin must also be confirmed [15, 48]. analysis of ciliary beat frequency and quality of ciliary movement also provides possibility to estimate ciliary function. ciliary beat frequency can be measured with phase-contrast microscopy. the approximate normal ciliary beat frequency is 11-16 hz. ciliary beat patterns are usually estimated by an experienced observer; however, beat pattern analysis with digital high-speed video imaging system is also possible. if the ciliary beat frequency and beat pattern are normal, then no further investigations are required [15, 49, 50]. tem is an important part of diagnosing pcd. it can be securely omitted only if the ciliary beat frequency is normal with a normal beat pattern [15]. some investigators prefer to take ciliated cells for ultrastructural examination by brush biopsy whereas others strongly recommend taking a biopsy of intact ciliary epithelium attached to the basement membrane. according to holzmann et al. [50], the most 161 trustworthy results are obtained from bronchial biopsies. however, bronchial biopsy is, in comparison with brush biopsy, more traumatic to the patient. alternatively, biopsies can be taken from the middle turbinate or from the bronchi if bronchoscopy is being performed for some other indications [10]. during ultrastructural examination, at least five good cross-sections from each of 10 different non-adjacent cells should be studied, but more should be examined if differences between cilia are noted [15]. short or missing dynein arms have been found to be a significant diagnostic criterion of pcd. in the majority of patients with pcd, there is absence of both outer (mean number per cilium <1.6) and inner (mean number per cilium <0.6) dynein arms. in controls, the mean number per cilium is 7.5-9.0 and 3.0-5.0 for outer and inner dynein arms, respectively. for secondary ciliary dyskinesia (scd), 8.5 outer dynein arms per cilium and 3.0 inner dynein arms per cilium are present. the low number of inner dynein arms might be explained by their low contrast in electron microscopy [42, 51]. recently, some new methods for the diagnosis of pcd have been proposed. nitric oxide (no) is produced from the respiratory tract (it is mainly synthesized in the paranasal sinuses) [52]. patients with pcd have a very low nasal no. nasal no levels are also slightly decreased in patients with cystic fibrosis. on the other hand, patients with asthma and bronchiectasis have increased exhaled no. these observations engender the idea of a new screening test to diagnose pcd. however, it is too early to propose that solely measurement of exhaled no can be used in the diagnosis of pcd [10]. moreover, there is no standardized measurement method for exhaled nasal no. it has been established that the mean no level for the total population was 837 ppb. the mean no level in children is lower than in adults (751 vs. 897 ppb, respectively). recently, an attempt to determine the repeatability of exhaled no measurements and to create a standardized nasal no measurement technique for the diagnosis of asthma was made. it was concluded that exhaled no measurements might provide a useful clinical tool to assess and monitor upper airways, but further investigations are needed [53]. another suggestion for the diagnostics of pcd is not less exciting and concerns the prenatal diagnosis of pcd. it has been reported that slightly enlarged brain ventricles might be used as a prenatal sonographic marker of kartagener’s syndrome, recommended when the foetus has either situs inversus or a sibling with the disease [54]. it is most likely that mild foetal ventriculomegaly might persist in foetus in accordance with hydrocephalus, a very rare manifestation of pcd. diagnostic problems of pcd from radiographic studies, it has been estimated that the prevalence of pcd in the general population is approximately 1:20,000. the number of actually identified cases is an order of magnitude lower [10]. the clinical pcd phenotype is broad and often mimics other chronic airways dis162 eases, such as cystic fibrosis, asthma or immunologic disorders. pcd is the differential diagnosis in the case of neonatal respiratory distress syndrome of unknown cause, neonatal pneumonia with no history of maternal illness or prolonged rupture of the membranes, significant and prolonged nasal discharge, bronchiectasis of unknown cause, severe or atypical asthma, nasal polyps, hydrocephalus, and infertility [15]. ultrastructural alterations in cilia may be both primary and secondary. it is vital to differentiate them to choose the most appropriate treatment. absent or reduced dynein arms, absent radial spokes, translocation of microtubule doublets, and absent central singlet pair are considered primary ciliary defects and the cause of pcd. other abnormalities, such as compound cilia, addition or deletion of peripheral microtubules, disorganized axonemes, ciliary disorientation, discontinuity of axoneme membrane, and some other are non-specific secondary structural abnormalities [55]. secondary defects usually occur due to respiratory infections, mechanical injury of the mucosa, or locally applied drugs. these alterations are reversible [56]. tem usually provides significant data on the ciliary structure and distinguishes primary ciliary defects from non-specific abnormalities called scd. it has been reported that short dynein arms are found to be a significant ultrastructural finding of pcd [57]. in some cases, it is not possible to distinguish primary and scd without in vitro cell culturing [58]. ciliary orientation as a possible diagnostic criterion of pcd pcd patients usually have a disorder of ciliary orientation (cor) [10]. sometimes patients with typical pcd symptoms have a normal ciliary ultrastructure and normal or near normal ciliary beat frequency. a group of these patients were found to have a disorientation of cilia, in which the individual cilia have a normal ultrastructure but their orientation with respect to each other was abnormal [37, 59]. ciliary disorientation has been reported in many cases of scd and in a very few cases as the single abnormality in pcd [60]. cor is an important parameter of mucociliary clearance. only when cilia beat in the same direction a metachronal waveform is possible. this wave sweeps the mucus layer from the airways towards the oropharynx, where it is swallowed [10]. cor has to be measured by tem from cross-sections of ciliary shafts. lines are drawn through the central pairs of a number of cilia arising from a single cell. these lines should normally all be more or less parallel with each other [15]. internationally accepted normal values for cor are ≤20˚. cor values of 20-35˚ indicate increased disorientation. cor values >35˚ represent a random orientation [60]. therefore, the standard deviation (sd) of these angles should be small. disorientation results in a larger standard deviation [15]. it is conceivable that cor values might be affected by the way in which respiratory mucosa is taken for ultrastructural examination. there are two tissue-obtaining 163 techniques mainly used: nasal biopsy and bronchial biopsy. significant differences in the sd of the ciliary angles in the specimens taken from brush biopsies and bronchial biopsies were not found [61, 62]. other factors influencing ciliary beat measurements have been reported. curette biopsies were compared to forceps biopsies and ciliary beat frequency values did not differ for these two biopsy techniques [63]. in a retrospective study on biopsies taken from 15 patients with pcd and 15 patients with scd at uppsala university hospital (shebani et al., unpublished results) the ciliary orientation (cor) was measured by tem from cross-sections of ciliary shafts. micrographs of cilia were taken at magnification of x30,000. a tem image of each patient was transferred into the digital form and processed with the image analysis software leica im 4.0 (leica microsystems imaging solutions ltd., cambridge, uk). only cilia in which two central microtubules could be seen, were used for determination of cor. for each cilium, electronically two lines were drawn. the first line was drawn through the pair of singlet microtubules. the second one was drawn along the vertical axis of the micrograph. the angle of its intersection with the first line had to be acute. the angle is defined by sides that are directed upwards. measuring cor angles in this manner, only acute angles between -90˚ (to the left of the vertical axis) and +90˚ (to the right of the vertical axis) can be obtained. in some cases, the angle of cor was equal with 90˚. in these cases, the sign (or +) of this angle depended on other cor angles on the particular micrograph. if the majority of angles were negative, this angle was regarded as a right negative angle. in the opposite case, the angle of 90˚ was regarded as a right positive angle. in each case, the sd of cor angles was calculated. this represented an index of cor for particular patient. for each group, a mean±sd value was obtained from all the sd values in this group, which represented an index of cor for particular group (pcd or scd). the mean value of cor in pcd group was 43.61±12.85˚ (range: 17.55 to 70.89˚). the mean value of cor in scd patients was 21.79±11.34˚ (range: 10.93 to 43.56˚). this means that pcd patients had significantly more manifested disorientation of cilia compared to scd patients (p<0.0001). nevertheless, there was a noticeable overlap between pcd patients and scd patients, which means that this parameter is not suitable as a diagnostic criterion. the pcd and scd patients were also divided into pediatric patients and adult patients, according to their age (pediatric patients < 18 years of age). differences in the mean values of cor between pediatric and adult patients with corresponding diagnoses as well as between males and females with the same diagnosis were not statistically significant. the number of dynein arms is a better criterion to distinguish between pcd and scd. shebani et al. (unpublished results) found that the average number of inner respectively outer dynein arms per cilia for the pcd patients was 1.4 respectively 1.5, for the scd patients 5.9 respectively 8.1, and healthy controls had 5.2 respectively 7.9 inner respectively outer dynein arms. pcd patients had significantly (p< 164 0.0001) fewer inner and outer dynein arm-like structures compared to scd patients and healthy controls. there was no significant difference between scd patients and healthy controls. figure 2 shows the means of the number of outer dynein arms per cilia after consecutive measurements on 1 to 30 cilia, for a pcd patient and an scd patient. as shown in the graph the measurement on 10-15 cilia is sufficient to make a good estimate of the number of outer dynein arms and a diagnosis. diagnosis of pcd is far from being a matter of mere academic interest. delayed pcd diagnosis leads to inadequate therapy and usually results in bronchiectasis with a marked reduction in quality of the 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tf-iups160017 198..198 letter to the editor author’s response: dysfunction of small airways and prevalence, airway responsiveness and inflammation in asthma: much more than small particle size of pet animal allergens dear editor, we read with interest the letter from liccardi et al. about our article ‘new data analysis in a population study raises the hypothesis that particle size contributes to the pro-asthmatic potential of small pet animal allergens’ (1). we would like to thank them for comments and for pin-pointing some weaknesses. small airways dysfunction can be considered as a distinct clinical phenotype, but we were not able to examine small airway functional impairment in our population with methods such as measurements of fef25–75, impulse oscillometry, or inert gas washout. we can therefore not investigate the prevalence of this phenotype in our population, but we have no reason to believe that this phenotype was over-represented in our study as these subjects were part of a population-based study. furthermore, it is true that various outdoor air pollutants and cigarette smoke can interact with allergic sensitization and influence inflammation in airways. unfortunately, we did not have available data about pollution, but smoking prevalence was relatively low and our measurements were adjusted for smoking history. allergenic sources others than cat and dog can under certain circumstances release allergens in ‘respirable’ form. the amount of allergens in small particle fraction can vary considerably, at least for pollen (2,3). in our study only cat and dog sensitization was significantly associated with a higher prevalence of asthma and more airway responsiveness, and airway and systemic inflammation. the above associations were not observed for other perennial allergens (mite, mold, and mice). so therefore it is unlikely that the associations noted for cat and dog sensitization are due only to continuous exposure. in a previous study (4) comparison of cat with mite allergens showed that the amount of fel d 1 carried by smaller particles is higher than that of der p 2. we were able to demonstrate an association between sensitization to pet allergens and more inflammation, assessed both as feno and s-ecp, and bronchial responsiveness, and therefore it is plausible that sensitization to small-sized petallergen-particles can have an impact in the development of asthma. however, our study was cross-sectional and this hypothesis cannot be tested, but we can only report these associations and acknowledge the limitation of our study and need for longitudinal studies. disclosure statement the author reports no conflicts of interest. references 1. patelis a, dosanjh a, gunnbj€ornsdottir m, borres mp, h€ogman m, alving k, et al. new data analysis in a population study raises the hypothesis that particle size contributes to the proasthamatic potential of small pet animal allergens. ups j med sci. 2016;121:25–32. 2. riediker m, koller t, monn c. differences in size selective aerosol sampling for pollen allergen detection using high-volume cascade impactors. clinical exp allergy. 2000;30:867–73. 3. schappi gf, suphioglu c, taylor pe, knox rb. concentrations of the major birch tree allergen bet v 1 in pollen and respirable fine particles in the atmosphere. j allergy clin immunol. 1997;100:656–61. 4. de blay f, heymann pw, chapman md, platts-mills ta. airborne dust mite allergens: comparison of group ii allergens with group i mite allergen and cat-allergen fel d i. j allergy clin immunol. 1991;88:919–26. antonios patelis medical sciences, uppsala university, respiratory, allergy and sleep research, uppsala, sweden antonios.patelis@medsci.uu.se received 18 march 2016; accepted 29 march 2016 � 2016 the author(s). published by taylor & francis. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. http://dx.doi.org/10.3109/03009734.2016.1173746 upsala journal of medical sciences, 2016 vol. 121, no. 3, 198 author&hx2019;s response: dysfunction of small airways and prevalence, airway responsiveness and inflammation in asthma: much more than small particle size of pet animal allergens disclosure statement references (4) gustafsson 35-44 35 upsala j med sci 111 (1): 35–43, 2006 from rosén von rosenstein to hugo (the human genome organization) karl-henrik gustavson department of clinical genetics,rudbeck laboratory, university hospital,uppsala,sweden introduction the situation of health for children in sweden in the middle of the 1700 century was very bad with an infant mortality of 50-60 %. rosén von rosenstein made outstanding contribution for better child health and sick care through research and teaching, which tremendously improved child care and social conditions in sweden. one of the most important effects of this was a marked falling in infant death rate. he pointed out that a child was not an incomplete variant of the adult and that children had special needs and specific diseases, which required specific therapy and that deficiency states and diseases could be prevented. this is in line with clinical genetics, the ultimate goal of which is the prevention and management of genetic diseases. rosén von rosenstein defended his thesis ‘‘de historiis morborum rite consignandis’’ (on the writing of case reports) in 1730,where he pointed out the importance of a detailed case and family history, which also is basic in clinical genetics. medical genetics in perspective inheritance patterns have been a subject of interest from time immemorial. the oldest pedigree is on horse breeding in mesopotamia 5 000 years ago (fig.1) it was prescribed in talmud that the sons of women, who had given birth to a son who had bled to death after circumcision, as well as the sons of her sister, should exempt received 3 octorber 2005 accepted 17 october 2005 from the procedure. this may well be the first recorded example of genetic counselling. thus, the jews understood already many thousand years ago that haemophilia is a sex linked disorder only affecting males. the austrian monk gregor mendel (fig.2), the father of genetics, could by performing a series of cleverly designed breeding experiments on garden pees formulate the fundamental laws of heredity, published in 1865.his laws on the patterns of inheritance received no recognition until 1900 when they were rediscovered. probably the most important achievement in biologal research of the last century was the identification and deducing of the correct physical structure of dna by james watson and francis crick in 1953.this formed the basis for molecular genetics. in 1956 m.j.tjio and albert levan (fig.3) showed the normal human chromosome number to be 46,which cleared the way for clinical cytogenetics. 36 fig 1. pedigree illustrating horse breeding 5,000 years ago, from kaldeén, mesopotamia. gregor mender_ wikipedia, den fria encyklopedin fig 2. portrait of gregor mendel. in 1959 jerome lejeune and colleagues described the first chromosomal disorder in humans, trisomy 21 in down´s syndrome. this was the beginning of clinical cytogenetics. the development of chromosome banding by torbjörn caspersson and lore zech (fig.4) in 1968 increased the possibility to identify small chromosome abnormalities and by now more than 1000 different chromosome aberrations have been reported. the most impressive advances have been in the area of molecular genetics. during the last 25 years several thousands of genes have been mapped to specific chromosomes, and molecular defects causing a great number of genetic defects and diseases have been identified. in 2003 the human genome project provided the complete human dna sequence. this is providing us with the first holistic view of our genetic heritage. the human genome contains 3 billion base pairs of dna that contains approximately 30,000 protein coding genes. during the course of the last century the disease panorama has very much changed. the importance of infectious diseases and malnutrition has diminished drastically, at least in developed countries, and the importance of genetic diseases has increased. medical genetics was once confined to rare disorders seen by paediatricians and a few other specialists. it has now become a field of central importance for our understanding of most diseases. the diagnosis, prevention and treatment of many disorders have been influenced by molecular medicine. most of the common disorders such as diabetes, allergic diseases, hypertension, and common birth defects as mental retardation, heart malformations, cleft lip and cleft palate have been shown to mainly have a multifactorial etiology and result from interplay of multiple genes with environmental factors. genetic diseases are thus a significant cause of illness and death. those individually rare conditions are in aggregate a large cause of morbidity and mortality. about 8 per cent of all children have a severe genetic disease or birth defect or will during their life develop a severe genetic disorder. accordingly many children are nowadays recog37 fig 3. albert levan at the microscope. nized as suffering from genetic conditions. it has been estimated that about a third of all admissions to paediatric hospitals are due to diseases with a genetic component. 38 fig 4. torbjörn caspersson and lore zech in the chromosome laboratory. 39 major types of genetic disorders genetic disorders can be classified in 5 major categories; chromosome disorders, single gene defects, cancer, multifactorial disorders, and mitochondrial disorders. chromosome disorders chromosome abnormalities are a leading cause of mental retardation and pregnancy loss. in sweden about 60 of 10,000 live-born children have a chromosomal aberration, visualized under microscope. autosomal numerical aberrations are seen in 12 of 10,000 new-born infants. they have as a role an extra autosome, caused by nondisjunction. most common is down´s syndrome with an extra chromosome 21, seen in one of 800 new-borns. autosomal monosomies are almost always lethal. twenty-four of 10,000 new-born infants have a microscopically observable structural autosomal aberration, usually a deletion. with the use of advanced molecular techniques, especially fluorescence in situ hybridisation(fish) and metaphase based comparative genomic hybridisation(metaphase-cgh)of the chromosomes it is possible to visualize genetic alterations directly on interphase nuclei and metaphase chromosomes. they are powerful but relatively labour intensive techniques that allow detection of deletions, duplications, rearrangements and mapping of translocation break points. a new technology, microarray based comparative genomic hybridization (arraycgh), allowed the resolution of the analysis to increase enormously. in this method a chip has been dotted with dna from many thousands of genes. these genes function as probes for detecting which genes are missing or active in different tissues or cells. this method has permitted detection of a large number of micro-deletions and some micro-duplications that are too small to be observed microscopically. the use of cdna clones as array targets has the advantage of assessing both dna copy number and gene expression on the same platform. these techniques have often made it possible to specify the critical region of the chromosome that has to be deleted or duplicated to cause a specific syndrome. many of these microdeletion syndromes have earlier been regarded as monogenic conditions. twenty-six of 10,000 new-born boys and 13 of new-born girls have a sex chromosomal aberration. chromosome abnormalities are seen in 50% of first-trimester and 20 % of secondtrimester spontaneous abortions. monogenic disorders and some ethical considerations monogenic disorders show mendelian inheritance. the majority of monogenic disorders are caused by single base mutations resulting in structurally abnormal proteins. in september 2oo5 the on-line edition of mckusick´s mendelian inheritance in man lists more than 15,000 known human monogenic traits. of these 14,000 are located on autosomes, 800 on the x chromosome and 43 on the y chromosome. the mutated genes have in many cases been mapped to specific locations on the chromosomes, cloned, sequenced and the protein identified. this research has lead to new important knowledge not only in genetics but also in the basic pathophysiology of the diseases. to the monogenic disorders also belongs a group of disorders, called trinucleotide repeat disorders, where the mutation involves the amplification of a dna sequence that contains repeats of three nucleotides. about 20 diseases caused by such repeat expansions are known, among them the fragile-x syndrome, huntington´s disease, myotonic dystrophy and friedreich´s ataxia. the repetitive sequences are also present in the genes of normal individuals but they are amplified many times in the genes of affected persons. the lengths of the trinucleotide repeat tends to increase as the gene passes from parent to offspring, by which the disease gets progressively more severe through successive generations, so called anticipation. the diagnosis of a genetic disorder is only justified if it is of benefit for the patient or the family. the development of dna-based genetic tests is tremendously rapid and the growing gap between genetic testing and treatment possibilities constitutes an ethical dilemma. the molecular genetic research will further accentuate this, but generates also new knowledge and treatment possibilities, which are necessary to correct this imbalance. many children are suffering from monogenic disorders and it is usually important to make an early diagnosis of a genetic disorder. that the disease affecting a child is genetic may, however, have far-reaching consequences for the family, which require careful consideration. it is important for the paediatrician to help the child and the family to adjust to the new circumstances, once the diagnosis of a genetic disease is made. with the development of dna-based genetic testing, pre symptomatic diagnosis has become available for many inherited disorders. by informing individuals that they are carriers or not carriers of a genetic disease-causing mutation, carrier testing can aid in making reproductive decisions. predictive genetic testing is usually appropriate if the child at risk for the disease can be successfully treated early. however, if the disease does not present itself until adulthood and there is no useful treatment to be made, a predictive testing could have deleterious effects. the principles of genetic integrity and the right of self-determination are crucial in these circumstances. if the child is tested then it loses the opportunity to make its own decisions as an autonomous adult. a predictive testing could also have adverse consequences for insurance or employment in the future. who is to be counted as a child in relation to genetic information? it is obvious that a child of preschool age is not competent to make decisions on genetic risk and genetic tests. i believe that it is wrong to adopt a sharp age-related criterion to draw a line between childhood and adulthood. some 15-year-old individuals are as intellectually and emotionally mature as some 18-years-old. cancer 40 mutations of cell regulatory systems, usually gain or loss of chromosomes or chromosomal segments in somatic tissues, with loss of the normal controls over growth and differentiation, are the primary basis of carcinogenesis. familial cancers make up 5-10% of the most common solid tumours with debut in adulthood. however, most solid tumours in childhood as well as leukemias and lymphomas are not hereditary (non-familial). identification of genes involved in cancer and classification according to genetic defects have often been useful to predict response to specific treatments and clinical outcome. thus, dna microarray tools are now extensively used for study of the molecular bases of cancer. gene expression profiles can also be used for identifying subtypes of the cancer to achieve specific and better treatment. multifactorial disorders multifactorial inherited traits and conditions result from interplay of multiple genes with multiple environmental factors, where a combination of genes from both parents, in addition to often unknown environmental factors, produce the trait or condition. many quantitative traits, such as intelligence, height and weight are multifactorial. because they are caused by additive effects of many genes and environmental factors, each of which having a relatively small effect, these traits tend to have a normal distribution in the population. most of the common major malformations, such as cleft lip and/or palate, club foot, neural tube defects are multifactorially inherited. often one gender is affected more frequently than the other in multifactorial traits. there appears to be a different "threshold of expression", which means that one gender is more likely to show the malformation over the other gender. for example, hip dysplasia is many times more common in females than males and pyloric stenosis is more likely in males. most of the multifactorial disorders such as diabetes and psychoses are seen primarily in adolescents and adults. multifactorial traits do recur in families, because they are partly caused by genes. the recurrence risks are usually based on empirical data. the risk to siblings or offspring is lower than mendelian risks, often falling somewhere near 3% when there is one affected person in the family. the risks increase when more family members are affected and if the parents are related. the risk for a multifactorial trait or condition to happen again depends also upon how closely the family member with the trait is related to you. for example, the risk is higher if your brother or sister has the trait or disease, than if your first cousin has the trait or disease. there are often small subsets of the population where the disorder may be caused by a single gene with a large effect and environmental factors with small individual effects and show monogenic inheritance. identifying specific genes responsible for common disorders is an important goal, since only then we can understand the pathogenesis of the disease. mitochondrial disorders human mitochondria are cytoplasmatic organelles which have their own unique dna(mdna). mitochondrial dna has a high mutation rate. each cell contains sever41 al hundreds or more mitochondria. several copies of a small, double-stranded, circular mdna molecule exist within each mitochondrion. the mitochondria are transmitted in the ovum from the mother to all her children (maternal inheritance).through oxidative phosphorylation the mitochondria produce adenosine triphosphate(atp),the energy source essential for cellular metabolism. mitochondria are thus very important for cell survival. different tissues vary in the extent to which they depend on this energy production. mitochondrial diseases are complex multi-system disorders. the phenotypic effect depends on the location and type of mutation and also on the proportion of mutated mitochondria which are involved during the course of the individual. the mutations cause diseases, which in older children and adults, are characterized by neuromuscular symptoms such as ataxia, ophtalmoplegia, myoclonic epilepsy, dementia, stroke-like episodes, heart failure and myopathia. there is a very severe form in infants (pearson´s syndrome), which is characterized by pancreatic insufficiency, pancytopenia, and lactic acidosis. mitochondrial mutations can also be seen in some common human diseases as deafness, non-insulin-dependent diabetes and schizofrenia. concluding remarks although most genetic disorders can still only be treated symptomatically and with supportive care, recent progress in research has lead to effective treatment for many genetic diseases. current research, particularly in the areas of enzyme replacement and gene therapy gives promise for future treatment possibilities. the main goal for clinical genetics is true primary prevention, which is in line with rosén von rosenstein´s goals, presented in his textbook on paediatrics published in 1764. literature cited bradley jb, johnson d, rubestein d (2001) molecular medicine. blackwell science. harper ps (1998) practical genetic counselling.5th ed. butterworth heinemann. jorde lb, carey jc, bramshad mj, white rl (2003) medical genetics.3rd ed. st. mosby. mckusick va (ed.) omim:online mendelian inheritance in man. http://www3.ncbi.nlm.nih.gpv/omim/ rimoin dl, connor jm, peryitz re, korf b (2002) principle and practice of medical genetics.4th ed. churchill livingstone. scriver cr, neal jl, saginur r, clow a (1973)the frequency of genetic disease and congenital malformations among patients in a pediatric hospital. can med assoc j 108:111-15. 42 corresponding author: karl-henrik gustavson department of clinical genetics,rudbeck laboratory, university hospital se-751 85 uppsala, sweden 43 44 array comparative genomic hybridization identifies a heterozygous deletion of exon 3 of the ryr2 gen full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 array comparative genomic hybridization identifies a heterozygous deletion of exon 3 of the ryr2 gene ivone u. s. leong, jennifer sucich, debra o. prosser, jonathan r. skinner, jackie r. crawford, colleen higgins & donald r. love to cite this article: ivone u. s. leong, jennifer sucich, debra o. prosser, jonathan r. skinner, jackie r. crawford, colleen higgins & donald r. love (2015) array comparative genomic hybridization identifies a heterozygous deletion of exon 3 of the ryr2 gene, upsala journal of medical sciences, 120:3, 190-197, doi: 10.3109/03009734.2015.1029101 to link to this article: https://doi.org/10.3109/03009734.2015.1029101 © informa healthcare published online: 02 apr 2015. submit your article to this journal article views: 546 view related articles view crossmark data citing articles: 2 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2015.1029101 https://doi.org/10.3109/03009734.2015.1029101 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1029101 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2015.1029101 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1029101&domain=pdf&date_stamp=2015-04-02 http://crossmark.crossref.org/dialog/?doi=10.3109/03009734.2015.1029101&domain=pdf&date_stamp=2015-04-02 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1029101#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2015.1029101#tabmodule upsala journal of medical sciences. 2015; 120: 190–197 original article array comparative genomic hybridization identifies a heterozygous deletion of exon 3 of the ryr2 gene ivone u. s. leong1, jennifer sucich1, debra o. prosser1, jonathan r. skinner2,3,4, jackie r. crawford2,3, colleen higgins5 & donald r. love1,3 1diagnostic genetics, labplus, auckland city hospital, po box 110031, auckland 1142, new zealand, 2greenlane paediatric and congenital cardiac service, starship children’s hospital, grafton auckland, private bag 92024, new zealand, 3cardiac inherited disease group, auckland city hospital, auckland, new zealand, 4department of child health, the university of auckland, private bag 92019, auckland 1142, new zealand, and 5school of applied sciences, auckland university of technology, private bag 92006, auckland, new zealand abstract background. catecholaminergic polymorphic ventricular tachycardia (cpvt) is a heritable cardiac disorder characterized by life-threatening ventricular tachycardia caused by exercise or acute emotional stress. the standard diagnostic screening involves sanger-based sequencing of 45 of the 105 translated exons of the ryr2 gene, and copy number changes of a limited number of exons that are detected using multiplex ligation-dependent probe amplification (mlpa). methods. in the current study, a previously validated bespoke array comparative genomic hybridization (acgh) technique was used to detect copy number changes in the ryr2 gene in a 43-year-old woman clinically diagnosed with cpvt. results. the cgh array detected a 1.1 kb deletion encompassing exon 3 of the ryr2 gene. this is the first report using the acgh technique to screen for mutations causing cpvt. conclusions. the acgh method offers significant advantages over mlpa in genetic screening for heritable cardiac disorders. key words: acgh, array comparative genomic hybridization, catecholaminergic polymorphic ventricular tachycardia, cpvt, ryanodine receptor 2 gene, ryr2 introduction catecholaminergic polymorphic ventricular tachycardia (cpvt) is an inherited cardiac disorder characterized by life-threatening arrhythmias during adrenergic stimulation, such as during exercise or acute emotional stress (1). those affected by cpvt have structurally normal hearts, and symptoms present at an early age (2); if left untreated the mortality rate of cpvt is between 30%–50% by 35 years of age (2). there are two main forms of the disease: cpvt1 and cpvt2. cpvt1 is an autosomal dominant disorder caused by mutations in the ryr2 gene, which encodes for the cardiac ca2+ release channel (ryanodine receptor isoform 2) (3,4). cpvt2 is an autosomal recessive form of the disease, which is caused by mutations in the casq2 gene that encodes for a ca2+ binding protein (calsequestrin 2) (5,6). both proteins are located in the cell’s sarcoplasmic reticulum. mutations in the ryr2 and casq2 genes account for 50% and 3%–5%, respectively, of all cpvt cases (1,7). a third (and minor) form of cpvt is caused by mutations in the kcnj2 gene (8). correspondence: donald r. love, diagnostic genetics, labplus, auckland city hospital, po box 110031, auckland 1142, new zealand. fax: +64-9-3074939. e-mail: donaldl@adhb.govt.nz (received 13 january 2015; accepted 8 march 2015) this is an open-access article distributed under the terms of the cc-by-nc-nd 3.0 license which permits users to download and share the article for non-commercial purposes, so long as the article is reproduced in the whole without changes, and provided the original source is credited. issn 0300-9734 print/issn 2000-1967 online � 2015 informa healthcare doi: 10.3109/03009734.2015.1029101 http://informahealthcare.com/journal/ups mailto:donaldl@adhb.govt.nz the ryr2 gene contains 105 exons and encodes for one of the largest ion channel protein in the human body. cpvt-causing mutations in the ryr2 gene have been found in 45 of the 105 translated exons (8,9); many of these mutations are substitution mutations. the main mutation screening method involves pcr-based amplification of 40–43 exons of the ryr2 gene, and subsequent bidirectional sanger-based sequencing (tier 1). if no mutations are found in this targeted group of exons then the remaining exons are sequenced (tier 2). in the absence of sequencedetectable mutations, screening methods then move ontothosethatcandetectlarger-scale eventssuchasan exon 3 deletion (the deletion size ranges from 1.1 to 37.7kb;29affectedindividuals)(9-13),asmalldeletion in exon 99 (one affected individual) (10), and duplication/insertioninexon97(oneaffected individual)(14). these larger mutations are usually detected using multiplex ligation-dependent probe amplification (mlpa), and this method is used to complement the sanger-based screening; however, this method only screens for deletion and duplication events in a subset of exons of the ryr2 gene (15). in contrast, array comparative genomic hybridization (acgh) offers enhanced exon coverage to detect exonic duplications and deletions. we have implemented a customdesigned acgh array in our routine diagnostic testing of cardiac referrals to examine copy number changes in thecodingregionsof99genesassociatedwithinherited cardiac and neuromuscular disorders (15-18). in the present study, we screened a 43-year-old woman clinically diagnosed with cpvt using an acghassayanddetecteda1.1-kbdeletionencompassing exon 3 of the ryr2 gene. a custom-designed pcr-based assay was developed to screen for this deletion event in her children. the acgh method has not been previously applied to cpvt screening (15-18). materials and methods genomic dna (gdna)wasextracted fromperipheral bloodedtasamplesusingthegentrapuregenedna extraction kit (qiagen inc., germantown, maryland, usa), according to the manufacturer’s instructions. a roche nimblegen 12x135k custom cgh array (roche nimblegen inc., madison, wisconsin, usa) was used for copy number change analysis. the acgh was designed to examine the coding regions of 99 genes associated with several cardiac and neuromuscular disorders, including genes responsible for long qt syndrome (lqts), brugada syndrome, hypertrophic cardiomyopathy (hcm), dilated cardiomyopathy (dcm), short qt syndrome (sqt), and cpvt (18). see supplementary materials for the full gene list. details about exonic and intronic probe densities and low-density ‘backbone’ probes that screen the human genome have previously been described (15). a total of 500 ng of the proband’s gdna was processed according to the manufacturer’s instructions (nimblegen array user’s guide: cgh and cgh/loh arrays v9.1) (19), and has been described previously (15,17). in brief, a patient’s gdna and promega control dna were fluorescently labeled with cy3 (sample) and cy5 (control) dyes, and subsequently purified via ethanol precipitation. the fluorescently labeled patient sample and the sex-matched control were combined in equimolar amounts and hybridized to one of 12 arrays on the acgh slide for approximately 48 h using a roche nimblegen hybridisation chamber. subsequently, the slides were washed and scanned using a nimblegen ms 200 microarray scanner. the array image files (.tif) were imported into deva v1.2.1 (roche nimblegen inc.) for analysis. the data were filtered using a log2ratio threshold of less than –0.4 over six probes for a deletion event and a log2ratio threshold of greater than 0.4 over 15 probes for a duplication event. all copy number changes that met these criteria were examined further using the ucsc genome browser, human genome assembly ncbi36/ hg18 (released march 2006) (20), to determine the location and significance of the change. all significant copy number changes were pcrverified to determine the exact breakpoint of the deletion. two sets of primers were designed to flank the deletion site detected by the acgh approach (ryr_ex3_del_f 5’ gcgtatcagagtaagctgtgtc 3’; and ryr2_ ex3_del_r, 5’ aactctgtgactttggaaaaggaat 3’). pcr was performed as follows: 1x faststart pcr buffer, 2 mm magnesium chloride, 0.8 mm each of the forward and reverse primer, 0.4 mm dntp, 0.04 u faststart taq dna polymerase (roche), and 50 ng of gdna. the following cycling conditions were used: 95�c for 4 min, 35 cycles of 94�c for 45 s, 60�c for 30 s, 72�c for 2 min 45 s, and a final extension at 72�c for 10 min. pcr products were purified with exosap-it (affymetrix inc, santa clara, california, usa) prior to bi-directional dna sequencing using bigdye terminator v3.1 (applied biosystems by life technologies, carlsbad, california, usa) to determine the exact deletion breakpoint. the sequenced products were purified using the bigdye xterminator purification kit (applied biosystems ltd) and were then subjected to capillary electrophoresis using the applied biosystems model 3130xl genetic analyzer. the analysis of sequence traces was performed using geneious software (biomatters ltd, auckland, new zealand) (21). acgh identifies a heterozygous deletion in the ryr2 gene 191 results clinical history the proband (ii-3) was a 43-year-old woman who was suspected to have cpvt, and she was referred to the cardiac inherited disease group (cidg) of auckland city hospital for further cardiac/genetic investigation (figure 1). the proband had previously experienced syncope with exertion and multiple ventricular premature complexes (vpcs) whilst monitored (figure 2a). an exercise tolerance test (ett) showed runs of polymorphic ventricular tachycardia (vt) with minimal exertion, and echocardiography confirmed a structurally normal heart. she was managed with beta-blockade and an implantable cardioverter-defibrillator (icd). to control further the vt episodes she later underwent a left thoracoscopic sympathectomy and more recently has responded well to flecainide. the family history was significant for the sudden death of the proband’s brother (ii-1) at the age of 15 years. he had suffered repeated syncope with documented polymorphic vt, and was taking betablockers at the time of his death whilst running. the proband’s mother (i-2) died suddenly at age 52 years, and reportedly had a myocardial infarction on autopsy. clinical details were not available for ii-2, ii-4, and ii-5. the proband’s four children (iii-1 to iii-4) also underwent cardiac investigation due to her probable cpvt diagnosis (figure 1). all had echocardiography holters and exercise testing. none had evidence of structural inherited heart disease, but one of the symptomatic fraternal twins (iii-3) at 7 years of age had ventricular ectopy during exercise both on ett and holters. she was managed with betablockers and left cardiac sympathetic denervation, and has had no syncope over the ensuing 6 years. as well as probable cpvt, iii-3 also exhibited symptoms of absence seizures. an electroencephalogram confirmed that ii-3 suffered from primary generalized epilepsy with absence of seizures. ethosuximide was administered for the absence seizures. she also exhibited learning difficulties and poor concentration, and was diagnosed with attention deficit hyperactivity disorder (adhd). ett (bruce protocol, see figure 2b) and holter conducted eight months post-surgery showed a reduction in vpcs. four beats of vt were recently documented on exercise, and flecainide has since been added. there may be early signs of sinus node dysfunction, with the resting heart rate having fallen on the same dose of nadolol over recent years; the repolarization pattern has begun to look normal. she is asymptomatic for the bradycardia at present, and there have been no prolonged pauses on holter. molecular genetic analysis the acgh analysis of the proband identified a heterozygous deletion on chromosome 1 (1q43) that was approximately 816 bp in size (hg18 coordinates chr1:235,560,602-235,561,416) (figure 3). this deletion encompasses the whole of exon 3 of the ryr2 gene with flanking intronic regions (figure 4). sanger-based sequencing was used to confirm the acgh results and also to determine the exact breakpoints of the deletion. primers were designed to flank the region of interest (figure 4a), and subsequent sanger-based sequencing of the resulting smaller amplicon confirmed the acgh results but with greater resolution such that the deletion proved to be 1,126 bp in size (figure 4a). this mutation (c.169-198_c.273+823 del1126) has been previously reported by ohno et al. (12) and szentpali et al. ii iii 1 1 2 2 3 3 4 4 5 ? 1 2 ? ? ? i key unaffected male/female affected/carrier male/female deceased male/female unknown clinical details? figure 1. pedigree of the ryr2 exon 3 deletion carriers. the proband (ii-3) is indicated by the black arrow. family members carrying the ryr2 exon 3 deletion are indicated in solid black. the members with unknown clinical and genetic background information are indicated by ?. 192 i. u. s. leong et al. (figure 5) (13). exon 3 of the ryr2 gene encodes for a highly conserved region of the ryr2 protein, and the mutation is an in-frame deletion of 35 amino acids (p.asn57-gly91). the proband’s four children were also tested for the same mutation using a pcr-based screen, with only the daughter (ii-3) proving to be a carrier of the deletion event; interestingly, this assay preferentially amplified only the deleted ryr2 gene allele in carrier individuals (figure 4b). the outcome of this mutation screen correlated with the clinical phenotypes of the proband’s children. discussion the custom-designed nimblegen 12x135k acgh confirmed the cpvt diagnosis of the proband, and the targeted pcr-based amplification assay confirmed the cpvt diagnosis of her daughter. the microarray was able to detect the deletion of only one exon of the ryr2 gene. the custom-designed acgh described here has been used previously in screening for deletion/ duplication events in a large number of cardiac genes, but the design criteria have also been more generally applied to allow screening for mutations in non-cardiac-related referrals (15-18). missense mutations are common within the ryr2 gene, with many of the mutations located in 45 out of the 105 exons. while deletion and duplication mutations are not common in the ryr2 gene, it is still important to check for copy number changes for this gene as deletion and duplication events have been detected (9-13). these large heterozygous deletions cannot be detected using standard genetic screening such as direct dna sequencing and high-performance liquid chromatography (9). while a avr v5 i ii iii ii v1 v4 v5 v6 v2 v3 avl avf b figure 2. rhythm strips of the proband and her daughter. a: three lead rhythm strip (25 mm/s, 10 mm/mv) taken during exercise of the female proband (ii-3), at age 29 years following presentation with exertional syncope. two minutes into stage 1 of the bruce protocol, there are runs of polymorphic ventricular ectopy and ventricular tachycardia interrupted with occasional sinus beats. b: 12 lead rhythm strip (25 mm/s, 10 mm/mv) taken during exercise of an asymptomatic 7-year-old daughter of the proband (iii-3) in stage 3, 9 min into the bruce protocol. frequent left bundle branch, inferior axis monomorphic ventricular extra beats develop into bigeminy. acgh identifies a heterozygous deletion in the ryr2 gene 193 mlpa is able to detect exonic deletions, the technique can only be applied to a small number of genes and a limited number of exons. the current acgh method has made it more efficient in screening for dosage changes in several patient samples simultaneously across a wide range of suspected genes. the deletion of exon 3 has been reported by five different groups (9-13), and 31 individuals carry this mutation (nine families, including this study). of the 31 individuals, only three appear to have a deletion of the same location and extent as the one reported here (figure 5, table i) (12,13). it is tempting to suggest that the deletion cluster represents recombination between repetitive elements (alu family of repeats) (figure 5) that flank exon 3. interestingly, the three individuals previously reported do not come from the same geographical region as our patients; the two unrelated cases reported by ohno et al. (12) are ethnically different from our proband. nineteen individuals have deletions of similar sizes in this region (figure 5) (10,11), and two individuals have much larger deletions in this region (3.6 kb and 37.7 kb; these two mutations are not shown in figure 5) (9,12). approximately 80% of these cases suffer from ventricular tachycardia, and approximately half experience atrial fibrillation and sino-atrial dysfunction. a quarter have received an icd or pacemaker, and only the two subjects in the current study have received a left thoracoscopic cardiac sympathectomy. the symptoms exhibited by the patients in the study reported here match the other symptoms found in those with the same mutations (i.e. syncope, bradycardia, ventricular tachycardia, and vpcs); however, the two affected members in our study do not yet show clear signs of sino-atrial dysfunction and left ventricular non-compaction cardiomyopathy (lvnc) which were found in the two other studies (12,13). the ryr2 protein is a major ca2+ releasing channel of the sarcoplasmic reticulum (sr) in cardiac muscle and plays an essential role in excitation–contraction coupling and ca2+ homeostasis in sr (22). all mutations found in the ryr2 gene to date have a gain-of-function effect, which causes a lowered threshold for either cytoplasmic ca2+ or sr ca2+ levels (23). this increase in cytoplasmic ca2+ leads to delays after depolarization (dad) (23). the exon 3 region of the ryr2 gene is highly susceptible to large alu repeat-mediated genomic rearrangements (9). in vitro studies of the effect of ryr2 lacking the 23 5, 55 8, 00 0 23 5, 55 8, 50 0 23 5, 55 9, 00 0 23 5, 55 9, 50 0 23 5, 56 0, 00 0 23 5, 56 0, 50 0 23 5, 56 1, 00 0 23 5, 56 1, 50 0 23 5, 56 2, 00 0 23 5, 56 2, 50 0 23 5, 56 3, 00 0 23 5, 56 3, 50 0 23 5, 56 4, 00 0 [l o g 2 ] segments in ratio data from 555109a05_2013-08-08_12-02_area1(cy3/cy5) cgh workflow with segme...all_data hg18:chr1 location: chr1 : 235,557,760 235,564,280 number of plots: search annotation hr1:235560601-:1 0.00 normalized ratio segments 1.20 0.800 0.400 0.0 –0.400 –0.800 –1.20 –1.60 –2.00 figure 3. copy number changes in chromosome 1q43 of the proband. deva software output showing a copy number change (deletion; 10 probes; log2ratio: –0.6726) localized to chromosome 1q43 (235,560,602-235,561,416; hg18 co-ordinates) for the proband (ii-3). 194 i. u. s. leong et al. exon 3-encoded domain in hek293 cells have shown increased ca2+ release into the cytoplasm, which has been attributed to ca2+ overload leading to dads (24). of the five members of the family that were tested here, two members were affected by adhd (one daughter was confirmed to have cpvt, the other was asymptomatic). as the ryr2 gene mutation did not co-segregate with the appearance of adhd, it could not be concluded that the gene is associated with the disorder. however, there have been two reported cases where cpvt patients (substitution mutations in the ryr2 gene) suffer from adhd (25,26). the proband’s daughter (ii-3) was also affected by generalized epilepsy with absence seizures, which could be caused by the ryr2 exon 3 deletion. previous reports have found that approximately half of children with cpvt also present with seizures (27), and half of ryr2 gene mutation carriers a. b. 1,500 2,000 1,000 850 650 500 400 ii3* iii1 iii2 iii3 iii4 exon 3 1,568 bp exon 3 acgh: ~752 bp actual: ~500 bp acgh: 816 bp actual: 1,126 bp control g g g gc c c c c c c c c c c ta a a g g a gc c c c c c c c c c ta g a ag c ii3 figure 4. pcr amplification of the region encompassing exon 3 of the ryr2 gene. a: amplicon sizes of the expected pcr products using dna from an unaffected individual carrying no deletion of exon 3 of the ryr2 gene (left), and for dna with the exon 3 deletion (right). the deletion size according to acgh and the actual deletion size confirmed by sanger-based sequencing are shown (above red lines). the expected pcr amplicon size of the deletion mutant according to the acgh data and the actual product size are both shown. b: 2% agarose gel showing the results of pcr amplification of the genomic region encompassing exon 3 of the ryr2 gene for the proband (ii-3) and her four children (iii-1 to iii-4). chromatogram of the control and the proband showing where the breakpoint is (indicated by the red line). r e p e a tin g e le m e n ts b y re p e a t m a sk e r r e fs e q g e n e s 2 3 5 ,5 6 0 ,4 0 0 2 3 5 ,5 6 0 ,5 0 0 2 3 5 ,5 6 0 ,6 0 0 2 3 5 ,5 6 0 ,7 0 0 2 3 5 ,5 6 0 ,8 0 0 2 3 5 ,5 6 0 ,9 0 0 2 3 5 ,5 6 1 ,0 0 0 2 3 5 ,5 6 1 ,1 0 0 2 3 5 ,5 6 1 ,2 0 0 2 3 5 ,5 6 1 ,3 0 0 2 3 5 ,5 6 1 ,4 0 0 2 3 5 ,5 6 1 ,5 0 0 2 3 5 ,5 6 1 ,6 0 0 2 3 5 ,5 6 1 ,7 0 0 2 3 5 ,5 6 1 ,8 0 0 2 3 5 ,5 6 1 ,9 0 0 2 3 5 ,5 6 2 ,0 0 0 5 0 0 b a se s h g 1 8 ch r1 ( q 4 3 ) p 3 4 .3 3 4 .2 3 2 .3 1 p 3 3 1 p 3 1 .3 1 p 3 1 .1 2 1 .3 2 1 .1 1 3 .2 1 3 .3 p 1 2 2 5 .2 1 q 1 2 3 1 .1 q 3 1 .3 1 q 4 1 1 q 4 3 q 2 1 .1 q 2 5 .3 1 q 3 2 .1 3 2 .2 1 q 4 4 2 3 .3 p ro b a n d s ze n tp a li e t a l., 2 0 1 3 & o h n o e t a l., 2 0 1 4 m a rj a m a a e t a l., 2 0 0 9 m a rj a m a a e t a l., 2 0 0 9 b h u iy a n e t a l., 2 0 0 7 & m e d e ir o sd o m in g o e t a l., 2 0 0 9 r y r 2 e xo n 3 f ig u re 5 . r ep o rt ed d el et io n s in th e r y r 2 ge n e. id eo gr am o f ch ro m o so m e 1 sh o w in g th e lo ca ti o n o f re p o rt ed r y r 2 ge n e d el et io n s en co m p as si n g ex o n 3 (s h o w n in gr ee n , b lu e, an d ye ll o w b ar s) . t h e lo ca ti o n o f th e p ro b an d ’s ex o n 3 d el et io n is sh o w n in re d , an d th e lo ca ti o n an d ex te n t o f a lu fa m il y re p et it iv e se q u en ce s ar e sh o w n at th e b o tt o m o f th e fi gu re . t h es e gr ap h ic s w er e re d ra w n fr o m th e u c s c ge n o m e b ro w se r b y ac ce ss in g th e n c b i3 6 /h g1 8 as se m b ly (h tt p :/ /g en o m e. u sc s. ed u /) . acgh identifies a heterozygous deletion in the ryr2 gene 195 http://genome.uscs.edu/ in a large dutch cohort suffered from seizures (28). knock-in mouse studies conducted by lehnart et al. (29) showed that a ryr2 gene missense mutation found in cpvt patients caused seizures and sudden death in mice, providing evidence that mutations in the ryr2 gene can cause seizures. acknowledgements we thank the family for kindly permitting this report. funding: this study was financially supported by the national heart foundation (new zealand), and the maurice and phyllis paykel trust. the cardiac inherited disease group is supported by cure kids, who also partially fund dr skinner’s salary. dr leong was financially supported during a large part of this work by cure kids, but also by the rutherford foundation as a new zealand postdoctoral fellow. jennifer sucich has been supported by a labplus (auckland city hospital) masters postgraduate scholarship. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. priori sg, napolitano c, memmi m, colombi b, drago f, gasparini m, et al. clinical and molecular characterization of patients with catecholaminergic polymorphic ventricular tachycardia. circulation. 2002;106:69–74. 2. swan h, piippo k, viitasalo m, heikkila p, paavonen t, kainulainen k, et al. arrhythmic disorder mapped to chromosome 1q42-q43 causes malignant polymorphic ventricular tachycardia in structurally normal hearts. j am coll cardiol. 1999;34:2035–42. 3. priori sg, napolitano c, tiso n, memmi m, vignati g, bloise r, et al. mutations in the cardiac ryanodine receptor gene (hryr2) underlie catecholaminergic polymorphic ventricular tachycardia. circulation. 2001;103:196–200. 4. laitinen pj, brown km, piippo k, swan h, devaney jm, brahmbhatt b, et al. mutations of the cardiac ryanodine receptor (ryr2) gene in familial polymorphic ventricular tachycardia. circulation. 2001;103:485–90. 5. lahat h, pras e, olender t, avidan n, ben-asher e, man o, et al.amissensemutationinahighlyconservedregionofcasq2is associated with autosomal recessive catecholamine-induced polymorphic ventricular tachycardia in bedouin families from israel. am j hum genet. 2001;69:1378–84. 6. postma av, denjoy i, hoorntje tm, lupoglazoff jm, da costa a, sebillon p, et al. absence of calsequestrin 2 causes severe forms of catecholaminergic polymorphic ventricular tachycardia. circ res. 2002;91:e21–6. 7. ackerman mj, priori sg, willems s, berul c, brugada r, calkins h, et al. hrs/ehra expert consensus statement on the state of genetic testing for the channelopathies and cardiomyopathies this document was developed as a partnership between the heart rhythm society (hrs) and the europeant ab le i. c li n ic al su m m ar y o f re p o rt ed fa m il ie s w it h r y r 2 ex o n 3 d el et io n . t h e ro w s sh ad ed gr ey re p re se n t p at ie n ts w it h th e sa m e d el et io n m u ta ti o n as th e cu rr en t st u d y. f am il y d el et io n n s yn b ra d y a v b lo ck v t v p c l v n c a f s a d ys fu n ct io n l v d ys fu n ct io n ic d p ac em ak er r ef . 1 c. 1 6 1 -2 3 6 _c .2 7 2 + 7 8 1 d el 1 1 2 6 1 1 3 1 1 1 0 7 4 1 (1 0 ) 2 c. 1 6 1 -2 3 6 _c .2 7 2 + 7 8 1 d el 1 1 2 6 2 1 2 2 1 1 1 3 3 .6 kb d el o f ex o n 3 1 (9 ) 4 c. 1 6 8 -3 0 1 _c .2 7 3 + 7 2 2 d el 1 1 2 8 4 1 2 1 4 1 2 2 (1 1 ) 5 c. 1 6 8 -2 2 8 _c .2 7 3 + 7 9 3 d el 1 1 2 6 2 1 2 2 1 1 1 6 c. 1 6 9 -1 9 8 _2 7 3 + 8 2 3 d el 1 1 2 6 1 1 1 1 1 1 1 (1 3 ) 7 c. 1 6 9 -2 2 9 2 4 _c .2 7 3 + 1 4 6 5 3 d el 3 7 6 8 2 6 2 5 1 2 2 5 1 3 2 1 (1 2 ) 8 c. 1 6 9 -1 9 8 _c .2 7 3 + 8 2 3 d el 1 1 2 6 2 2 2 1 1 1 2 1 1 9 c. 1 6 9 -1 9 8 _c .2 7 3 + 8 2 3 d el 1 1 2 6 2 1 2 2 2 2 c u rr en t st u d y t o ta l 3 1 8 1 2 7 2 5 9 9 1 6 1 6 5 5 3 (% ) (2 5 .8 % ) (3 8 .7 % ) (2 2 .6 % ) (8 0 .6 % ) (2 9 .0 % ) (2 9 .0 % ) (5 1 .6 % ) (5 1 .6 % ) (1 6 .1 % ) (1 6 .1 % ) (9 .7 % ) a f = at ri al fi b ri ll at io n ; 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http://www.nimblegen.com/downloads/support/05434483001_ng_cghloh_uguide_v9p1.pdf http:/genome.ucsc.edu/ http:/genome.ucsc.edu/ http://www.geneious.com/ http://www.geneious.com/ http://www.ncbi.nlm.nih.gov/pubmed/21645850?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21645850?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21645850?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/21645850?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22374134?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22374134?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/22374134?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/19398417?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/19398417?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/16436635?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/16436635?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/16436635?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7867192?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/7867192?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/16272262?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/16272262?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/16272262?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/18483626?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/18483626?dopt=abstract http://www.ncbi.nlm.nih.gov/pubmed/18483626?dopt=abstract abstract introduction materials and methods results clinical history molecular genetic analysis discussion acknowledgements declaration of interest references ujms110_1.pdf long-term health of children conceived after assisted reproductive technology review article long-term health of children conceived after assisted reproductive technology christina bergha,b and ulla-britt wennerholma adepartment of obstetrics and gynecology, institute of clinical sciences, sahlgrenska academy, gothenburg university, gothenburg, sweden; breproductive medicine, sahlgrenska university hospital, gothenburg, sweden abstract the aim of this narrative review is to summarize the present knowledge on long-term outcome of children born after assisted reproductive technologies (art). the main outcomes covered are neurodevelopment including cerebral palsy, cognitive development, attention deficit hyperactivity disorder and autism spectrum disease, growth, cardiovascular function, diabetes type 1, asthma, malignancies, and reproductive health. results have mainly been obtained from systematic reviews/meta-analyses and large registry studies. it has been shown that children born after art, when restricted to singletons, have a similar outcome for many health conditions as their spontaneously conceived peers. for some outcomes, particularly cardiovascular function and diabetes, studies show some higher risk for art singletons or subgroup of art singletons. the fast introduction of new art techniques emphasizes the importance of continuous surveillance of children born after art. article history received 27 january 2020 revised 8 february 2020 accepted 11 february 2020 keywords assisted reproductive technology; long-term follow up; icsi; ivf introduction there are numerous publications on the short-term outcome after assisted reproductive technology (art), including large registry studies and systematic reviews (srs)/meta-analyses. most of them reveal some adverse outcome also for singletons, particularly concerning preterm birth, low birth weight, and birth defects. literature concerning long-term outcome is much more sparse. this is the case, despite the fact that large cohorts of art children and adolescents now exist. while national birth registries, with extensive data on neonatal outcome, are present in several countries, this is not the case for children’s health in general. to catch children/adolescence health, patient or specific disease registries may be used. these registries include, however, only those children with specific diseases, and most severe diseases occur later in life, leaving few events and low power of such studies. the following summary is based on large registry studies and srs where such exist. cohort studies with sometimes a limited number of children have been included as well. the following aspects will be discussed: neurodevelopment including cerebral palsy (cp), cognitive development, attention deficit hyperactivity disorder (adhd) and autism spectrum disease (asd), growth, cardiovascular function, diabetes type 1, asthma, malignancies, and reproductive health. neurodevelopmental health psychomotor and language development, behaviour, and social functioning three srs from the netherlands, denmark, and australia described no differences in psychomotor development, overall social functioning, language development, and behaviour between children born after art and spontaneously conceived controls (1–3). cognitive development a recent sr focussing on cognitive development following art (4) found conflicting results, mainly due to methodological limitations. however, three studies were considered high-quality studies and suggested art to have some negative influence on cognitive development (5–7). in the early study by str€omberg and co-workers (5) including 5,680 children born after ivf (both singletons and multiples), a 4-fold increase in risk of developmental delay was observed as well as an increased risk of needing habilitation services compared with children from spontaneous conception. when comparing only singletons, there was no increased risk. in a dutch study with a limited number of art children, singletons from intracytoplasmic sperm injection (icsi) were shown to have lower scores on tests of intelligence (on average 5–7 iq points lower) compared with spontaneously conceived singletons (6). in a large population-based registry study (n ¼ 30,959 art children) from sweden a small but significantly increased risk of mental retardation was found in art children (relative risk [rr] 1.18, 95% ci 1.01–1.36) (7). when restricting analysis to singletons statistical significance disappeared. further, in a subgroup analysis of singletons after frozen/thawed icsi, there was an increased risk of mental retardation; however, this was based on few children (n ¼ 7). a danish registry study reported comparable risk of mental contact christina bergh christina.bergh@vgregion.se department of obstetrics and gynecology, institute of clinical sciences, sahlgrenska academy, gothenburg university; reproductive medicine, sahlgrenska university hospital, gothenburg se-413 45, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 2, 152–157 https://doi.org/10.1080/03009734.2020.1729904 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1729904&domain=pdf&date_stamp=2020-05-21 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1080/03009734.2020.1729904 http://www.tandfonline.com retardation in ivf-conceived and spontaneously conceived singletons (8). recently, large registry-based studies from sweden and denmark have shown similar school performances of art children and children born from spontaneous conception. the swedish study (9) included 8,323 art singletons compared with 1,499,667 children born after spontaneous conception, representing all art and spontaneously conceived children born in sweden between 1985 and 2001. interestingly, the mean scores were around 10% higher for the art children. however, after adjustment for relevant confounders, results were similar in the two groups. comparisons have also been made between children from ivf and icsi and between children from fresh and frozen cycles (10–12), with similar results. attention deficit hyperactivity disorder and autism spectrum disease adhd was found to be weakly associated with ivf in a swedish study including 28,158 multiples and singletons born after ivf. yet after adjusting for length of involuntary childlessness, or when only singletons were analyzed, the statistical significance disappeared (13). a danish study including 124,269 children born to women with fertility problems reported a higher risk of adhd (hazard ratio [hr] 1.36, 95% ci 1.29–1.45) in these children compared with children born to women without fertility problems. no adjustment was made for multiple gestations in that study (14). in a swedish study, including 30,959 children born after art, no increased risk of asd was found compared to children born after spontaneous conception (7). nor was there an increase in any emotional and behavioural disorder in a large danish study (8). however, in a californian cohort study a higher risk of asd was reported in icsi singletons compared with standard ivf with fresh embryo transfer (et) (adjusted hazard risk ratio [ahr] 1.65, 95% ci 1.08–2.52) (art n ¼ 19,790) (15). no comparison between children from art and spontaneous conception was performed in that study (15). in a recent meta-analysis it was concluded that art was associated with a greater risk of asd in an overall offspring group compared with spontaneously conceived children (rr 1.35, 95% ci 1.09–1.68); however, this was not seen in singletons (16). cerebral palsy (cp) an increased risk of cp was found for singletons in an early swedish study including 5,680 children born after ivf (aor 2.8, 95% ci 1.3–5.8) (5). in a later and larger swedish study, including 31,614 children born after ivf (multiples and singletons), born 1982–2007, there was a higher risk for cp when analysing singletons and multiple birth children together and compared with all children born during the same time period and registered in the medical birth registry (aor 1.81, 95% ci 1.52–2.13). the analysis was based on 138 art children with cp, and adjustments were made for maternal age, year of birth, parity, and smoking. when only singletons were analysed, no significant increased risk was found (17). likewise, in a danish study there was an increased risk for cp among 33,139 ivf children, but this disappeared when adjusted for multiplicity and gestational age (18). another registry-based danish study, however, including 10,329 singletons after fresh cycles and a control group of 4,800 spontaneously conceived singletons randomly selected, proved an increased risk of cp in singletons after fresh transfer (based on 42 cp cases) compared with spontaneously conceived singletons (aor 2.44, 95% ci 1.15–5.22) (19). however, no adjustment was made for gestational age. in a recent australian register study including a limited number of children (n ¼ 2,914) born after art, the prevalence of cp was more than doubled in art singletons born very preterm (<32 gestational weeks) (20). in summary, for most neurodevelopmental health variables conflicting results exist concerning a possible association between art and adverse outcome. most of the identified risk associations disappeared after adjustment for multiple births or were observed only in subgroups of specific ivf treatments such as cryopreservation. for cp, in particular, there is a need of further large studies, including more recent cohorts of art children. cardiovascular function and metabolism the literature on cardiovascular and metabolic risks in art offspring is limited. studies published so far are based on small cohorts with high risk for selection bias among both art children and controls. cardiovascular and metabolic diseases mainly affect adults, while adolescents and young adults conceived after art are not very common in registries covering these diseases. the most recent sr and meta-analysis on cardiovascular diseases included 19 studies with 2,112 ivf/icsi and 4,096 spontaneously conceived offspring (21). it was found that systolic blood pressure (sbp) and diastolic bp (dbp) levels were higher after art than in spontaneously conceived offspring (weighted mean differences [wmd] 1.88, 95% ci 0.27–3.49 for sbp; and 1.51, 95% ci 0.33–2.70 for dbp). however, the higher wmd in sbp and dbp was only observed in the cohorts born 1990–1999 and not for children born later. the same meta-analysis showed comparable body mass index (bmi), low-density lipoprotein, cholesterol, and fasting insulin levels for art and spontaneously conceived children. a swiss study examined 65 art singletons and 57 spontaneously conceived controls at 11–12 years of age and demonstrated adverse vascular dysfunction among art children (22). these differences still existed at re-examination 5 years later when the children were 16–17 years old. further, the art children had significantly higher sbp and dbp (23). in summary, limited data suggest a potential increase in blood pressure in art children as well as suboptimal cardiovascular function. upsala journal of medical sciences 153 diabetes type 1 few studies have been published concerning the risk of developing diabetes type 1 in children conceived by art. according to a danish cohort study from 2016, including 8,490 ivf and icsi children, there was no increased risk of diabetes type 1 (24). in a recent large registry study from sweden, including 47,938 art singletons and 3,090,602 singletons from spontaneous conception, there were 202 children born after art that developed diabetes type 1 (25). the corresponding figure for children born after spontaneous conception was 17,916. this corresponds to 43.4 (art) and 35.5 (spontaneous conception) children when calculated per 100,000 person-years. after adjustment for several confounders, including maternal and paternal diabetes, there was no overall difference between art and spontaneous conception (ahr 1.07, 95% ci 0.93–1.23). however, children conceived after frozen/thawed transfers had a higher risk of developing diabetes type 1, both compared with singletons from fresh transfers and children from spontaneous conceptions (ahr 1.52, 95% ci 1.08–2.14; and ahr 1.41, 95% ci 1.05–1.89). in summary, information on the occurrence of diabetes type 1 in art children is scarce. in general, there are no alarming results for art children, although there might be an increased risk for children born after frozen/ thawed cycles. growth though children born after art are more likely to be born preterm and with low birth weight, catch-up is common during the first year of life, and most studies have shown similar growth patterns in art and children spontaneously conceived. in a recent sr and meta-analysis from denmark (26), weight and height of art singletons compared with spontaneously conceived singletons were summarized. the sr included 20 studies, altogether 3,972 art children and 11,012 spontaneously conceived children, followed up for a maximum of 22 years. there were no significant differences in weight or height. in summary, so far, overall results on growth in children born after art suggest that they do not differ from spontaneously conceived children. respiratory disorders few studies have investigated the risk of asthma in art children. a swedish registry study of 2,628,728 children born in 1982–2007 including 31,918 children conceived by art revealed an increased risk for asthma in children born after art, increasing the absolute risk from 4.4% to 5.6% (27). however, adjustment for the duration of infertility eliminated the effect, suggesting that the main risk factor seemed to be subfertility, included in the characteristics of women in art. a uk prospective study, the millennium cohort study, of 18,818 singletons born after spontaneous conception or different kinds of fertility treatment, recruited at 9 months of age and based on a follow-up survey at 5 and 7 years of age, found a significant association between art and asthma; however, this was based on few children after art (28). in summary, limited data suggest that the main risk factor for the association between asthma and art is parental subfertility, but neonatal morbidity and maternal asthma may act as mediators. malignancies a population-based british cohort study from 2013, including 106,013 art children followed for a mean of 6.6 years, proved no increase in the overall risk of cancer when compared with the expected risk for singleton births (standardized incidence ratio [sir] 0.98, 95% ci 0.81–1.19) (29). another large registry-based cohort study, combining data from four nordic countries (conartas, committee of nordic art and safety) including 91,796 children (singletons and multiples) born after art and 358,419 children born after spontaneous conception, also concluded that there was no increase in overall cancer rates among art children (ahr 1.08, 95% ci 0.91–1.27) (30). children in this cohort were born 1982–2007 and were followed for a mean of 9.5 years. in a large cohort study from the united states, published in 2019, 275,686 children born after art and 2,266,847 children born after spontaneous conception were followed for 4.5 and 4.7 years, respectively (31). per 1,000,000 person-years the incidence of cancer among art was 251.9 cases and among spontaneously conceived children 192.7 cases. in total 321 and 2,042 children with cancer were identified among art and spontaneously conceived children, respectively (ahr 1.17, 95% ci 1.00–1.36). while no overall significant increase in cancer was observed, there was an increase of hepatic cancer and embryonal tumours among art children in two sub-analyses. a recent study from the netherlands, including 24,268 art children and 13,761 children from spontaneous conceptions, but also including a control group of 9,660 children from subfertile couples, did not observe any increase in risk of cancer among the art children (32). the study period was between 1980 and 2001, and the children were followed for a mean of 21 years. more recently, a study from denmark, including 1,085,172 children born between 1996 and 2012 and followed up to 2015, investigated a possible association between use of fertility drugs, assisted reproduction, and cancer in childhood (33). the study included 19,448 ivf, 13,427 icsi, and 3,356 children from cryopreservation, and a control group of 910,291 children born to fertile women. the mean follow-up was 11.3 years. no overall risk of cancer was found among children born to women with any fertility problems, any fertility treatment, use of any fertility drugs, or use of any art. however, an increased risk was observed among children born after cryopreservation of embryos (ahr 2.43, 95% ci 1.44–4.1). this observation was, however, based on only 14 cases. in summary, there is some inconsistency concerning malignancies in children conceived by art. higher risks have mainly been observed in subgroups of patients and for specific types of malignancies. 154 c. bergh and u.-b. wennerholm reproductive health researchers from brussels have published the first studies on the reproductive outcome in art offspring. in one study among 54 young adult men conceived by icsi because of severe male infertility, it was found that sperm parameters were significantly lower in art men than in men after spontaneous conception. there was, however, no clear correlation between semen parameters of the young icsi men and their fathers (34). the same authors showed similar levels of reproductive hormones among art and spontaneously conceived men, and also similar levels of reproductive hormones, antral follicle count, and levels of anti-m€ullerian hormone in women conceived after icsi (35,36). in summary, limited data published on reproductive health in art offspring suggest some deterioration in sperm counts in icsi male offspring, while in female offspring no adverse effects have been identified. comments and conclusions follow-up studies of children born after art have shown that the majority of children are healthy, even though some adverse outcomes have been demonstrated (table 1). the main risk for adverse outcomes in art, which includes icsi and standard ivf techniques as well as cryopreservation techniques, has been associated with the higher rates of multiple pregnancies in art. sweden has been the pioneering country in the world concerning introduction of single embryo transfer as the main strategy to increase the health of children born after art (see www.qivf.se) (37). concerning long-term effects of art on children outcomes, few studies of high quality exist. studies of growth seem reassuring. for childhood cancer, some discrepancies exist, but most large studies do not show any increase in childhood cancer in art children after adjustment for relevant confounders. most studies on neurocognitive development and asd show no increased risks if adjusted for multiple births. school performances of 15–16-year-old adolescents have been investigated in large registry studies from denmark and sweden and have shown better school performance for art children in crude analyses, but after adjustment for relevant confounders, particularly parental education, no differences of clinical importance have been observed. there are some recent concerns regarding cardiovascular parameters. cohort studies with a limited number of children included have detected altered blood vessel structure and increased blood pressure, both sbp and dbp, in art singletons compared with matched controls and further that these differences remain in adolescence. for diabetes type 1, in a recent swedish large registry study, there was no increase in general for art children but a higher risk in children born after the use of cryopreserved embryos. an important bias in all these follow-up studies is the choice of control group, being children from the general population in most studies. it is known that infertility per se is associated with an adverse child outcome, which may well contribute to the findings of a poorer outcome among art children. control groups with subfertile couples are, however, difficult to find. sibling studies (38,39), where the mothers have given birth to both an art and a spontaneously conceived child suggest that both the infertility and the art technique as such contribute to the child outcome. in conclusion, even though many studies on follow-up of art children show reassuring results, information on longterm follow-up is limited. new and advanced techniques are rapidly introduced and implemented, emphasising the importance of continuous surveillance of children born after art. disclosure statement no potential conflict of interest was reported by the author(s). funding the study was financed by grants from the swedish state under the agreement between the swedish government and the county councils, the alf-agreement [alfgbg-70940]. notes on contributors christina bergh is professor in obstetrics and gynaecology at the department of obstetrics and gynaecology, institute of clinical sciences, gothenburg university. table 1. summary of associations between art and children’s health and morbidity (versus spontaneous conception). art ivf icsi cryopreservation disorder general singletons psychomotor and language development, behaviour, and social functioning no association no association cognitive development no association no association no association no association adhd and asd increased risk no association cerebral palsy increased risk conflicting results cardiovascular function, blood pressure increased risk diabetes type 1 no association no association no association increased risk growth no association asthma no association childhood malignancies conflicting results no association increased risk reproductive health some sperm problems in men, no problems in women upsala journal of medical sciences 155 http://www.qivf.se ulla-britt wennerholm is associate professor in 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icsi-conceived young adult men: the first results. hum reprod. 2017;32:439–46. doi:10. 1093/humrep/dew324 36. belva f, roelants m, vloeberghs v, schiettecatte j, evenepoel j, bonduelle m, et al. serum reproductive hormone levels and ultrasound findings in female offspring after intracytoplasmic sperm injection: first results. fertil steril. 2017;107:934–9. doi:10.1016/j. fertnstert.2017.02.102 37. thurin a, hausken j, hillensj€o t, jablonowska b, pinborg a, strandell a, et al. elective single-embryo transfer versus doubleembryo transfer in in vitro fertilization. n engl j med. 2004;351: 2392–402. doi:10.1056/nejmoa041032 38. romundstad lb, romundstad pr, sunde a, von during v, skjaerven r, gunnell d, et al. effects of technology or maternal factors on perinatal outcome after assisted fertilisation: a population-based cohort study. lancet 2008;372:737–43. doi:10.1016/ s0140-6736(08)61041-7 39. henningsen ak, pinborg a, lidegaard ø, vestergaard c, forman jl, andersen an. perinatal outcome of singleton siblings born after assisted reproductive technology and spontaneous conception: danish national sibling-cohort study. fertil steril. 2011;95: 959–63. doi:10.1016/j.fertnstert.2010.07.1075 upsala journal of medical sciences 157 https://doi.org/10.1001/jama.2019.18037 https://doi.org/10.1093/humrep/dew245 https://doi.org/10.1093/humrep/dew245 https://doi.org/10.1093/humrep/dew324 https://doi.org/10.1093/humrep/dew324 https://doi.org/10.1016/j.fertnstert.2017.02.102 https://doi.org/10.1016/j.fertnstert.2017.02.102 https://doi.org/10.1056/nejmoa041032 https://doi.org/10.1016/s0140-6736(08)61041-7 https://doi.org/10.1016/s0140-6736(08)61041-7 https://doi.org/10.1016/j.fertnstert.2010.07.1075 abstract introduction neurodevelopmental health psychomotor and language development, behaviour, and social functioning cognitive development attention deficit hyperactivity disorder and autism spectrum disease cerebral palsy (cp) cardiovascular function and metabolism diabetes type 1 growth respiratory disorders malignancies reproductive health comments and conclusions disclosure statement references ujms109_2.pdf upsala j med sci 109: 131–140, 2004 field distribution of compound muscle action potentials of the calf muscles in rabbits masahito hatori, yoshinori miyasaka, itaru kimura, richard a. smith, shoichi kokubun department of orthopaedic surgery, tohoku university school of medicine, sendai, japan, department of neurology, national nishitaga hospital, sendai, japan, department of orthopaedic surgery, university of tennessee-campbell clinic memphis tennessee abstract a free full-text copy of this article can be found at the web page of upsala j med sci: http://www.ujms.se the purpose of this study is to verify that compound muscle action potentials (cmaps) generated a stationary potential and to examine extension of the cmaps away from the stimulated muscle. a referential derivation and/or bipolar derivation to record the stationary potentials were carried out following tibial nerve stimulation at the popliteal fossa in 10 rabbits. after recording a bipolar and/or referential derivation, wave changes were monitored before and after severing the tibial nerve. the change of the wave by compressing or direct electrical stimulation to the calf muscle was also monitored. in referential derivatives, the stationary waves were observed on top of the skull in all the rabbits examined with the peak latency of the potentials from 3.78 msec to 5.04 msec. in bipolar derivations, the upper limits of the stationary waves recorded were the trunks. the peak latencies were from 2.35 msec to 5.46 msec with an average of 4.12 msec. by analyzing the results from severing the tibial nerves, compressing the calf muscles, and direct electrical stimulation of the calf muscle, the origin of these stationary potentials was determined to be cmaps of the calf muscles. these findings suggest contamination of the stationary potentials originated by cmaps for recording of any evoked potentials when motor nerves are stimulated. introduction human spinal evoked potentials were first recorded by magladery et al. from electrodes in the subarachnoid space after posterior tibial nerve stimulation in 1951 (1 ). 131 received 9 december 2003 accepted 2 february 2004 key words: compound muscle action potential, electromyography, nerve root potential, stationary potential, far field potential, volume conduction. others recorded spinal evoked potentials with epidural (2 ) or surface electrodes (3). the authors recorded evoked potentials from the sciatic nerve and lumbar nerve roots following tibial nerve stimulation in rabbits. the evoked potentials obtained consisted of two components. the first component, observed in all, was found to be the ascending impulse to the spinal cord. the second component was considered to be of two origins. one was recorded at the weak stimulation and considered to be the impulse descending from the spinal cord. the other, recorded at strong stimulation, was a stationary potential regardless of the recording sites along the sciatic nerve. by severing the sciatic nerve distal to the stimulation site, this stationary potential disappeared. therefore we concluded this wave originated from compound muscle action potentials (cmaps) of the calf muscles (4). the purpose of this study was to verify that compound muscle action potentials generate a stationary potential and to examine extension of the cmaps away from the stimulated muscle. materials and methods ten rabbits were studied according to requirements of the ethics committee of tohoku university. the subject was fixed prone on the operating table after tiopental intravenous anesthesia. a 10 cm longitudinal skin incision was made in the popliteal region. the skin and subcutaneous tissues were reflected to expose the sciatic nerve, the tibial nerve and the gastrocnemius. the common peroneal nerve was compressed and severed with a hemostat at a point 0.5 cm distal to its bifurcation from the sciatic nerve in order to prevent contamination of the peroneal nerve innervated muscle’s cmaps. the tibial nerve was electrically stimulated at the popliteal fossa with needle electrodes touching the nerve. the cathode was located 1.5 cm proximal to the anode. the stimulus was 0.1 msec in duration. the intensity was adjusted so as to induce a twitch of the ankle. for recording stationary potentials originating from the calf muscle’s cmaps, multiple electrodes were placed in 5 cm increments along the ipsilateral sciatic nerve, the spinous process and the head (fig. 1). the ‘0’ level represented the intersection of a line drawn through the top of the bilateral iliac crests and a line drawn through spinous process with the other recording sites indicated by a number from the zero level, assigning a [+] sign proximally, and [–] sign distally. at each point, the skin was incised exposing the fascia. surface electrodes were attached to the fascia with conductive jelly. a referential derivation registered the input from each of the 9 or 10 active electrodes, depending on the size of the rabbits, [–2] through [+6] or [+7] on top of the head as g1 of each channel with g2 on the tip of the contralateral ear. a bipolar derivation was carried out connecting two adjacent leads with g1 distal to g2, i.e., [–2] to [–1] through [+6] to [+7]. each test consisted of an average of 10 to 600 summated responses with a frequency response of 10 hz-3 khz. calf muscle cmaps were simultaneously monitored over the ipsilateral calf muscle with flat-surfaced electrodes soaked with conductive jelly to keep the impedance less than 5 k�. the cathode and anode were placed over the calf muscle 132 and the heel respectively. a flat-surfaced ground electrode was placed on the muscle between the stimulating and recording electrodes. after recording a bipolar and/or referential derivation, the changes of the wave were monitored before and after severing the tibial nerve proximal to the stimulating point followed by severing the nerve distal to the stimulating point. the change of the wave when the calf muscle was compressed with forceps was monitored in two rabbits while recording the derivations. in one rabbit, the calf muscle was electrically stimulated with needle electrodes inserted into the muscle. and a referential derivation registered the input from the active electrode ‘+7’ with calf muscle cmaps monitored over the ipsilateral calf muscle in the same manner. a cadwell 5200a was used for stimulation and data production. results referential derivation was carried out in nine rabbits out of ten. in referential derivatives, the stationary potentials could be observed at any recording site in all the 133 fig. 1. recording method. the tibial nerve was stimulated at the popliteal fossa. multiple electrodes were placed in 5 cm increments along the ipsilateral sciatic nerve, the spinous process and the head. the ‘0’ represented the level of a line drawn through the top of the bilateral iliac crests and with the other recording sites indicated by a number from the zero level, assigning a [+] sign proximally and [–] sign distally. a referential derivation registered the input from each of the 11 active electrodes, [–2] through [+6] or [+7] on top the skull, depending on the size of the rabbits, as g1 of each channel with g2 on the tip of the contralateral ear. a bipolar derivation was carried out connecting two adjacent leads with g1 distal to g2, i.e., [–2] to [–1] through [+5] to [+6] or [+6] to [+7], depending the size of the rabbits. 134 fig. 2a. referential derivation (rabbit�). the stationary wave with the peak latency of 3.78 msec was observed from [–1] through [+7]. the amplitude of the wave gradually decreased as it went distally. 135 table 1. upper limits of recording the stationary potentials by referential and bipolar derivations. upper peak upper peak rabit recording recording latency recording latency no. site recording limit (ms) recording limit (ms) � –2~+6 referential +6 4.02 not performed � –2~+7 referential +7 4.16 not performed � –2~+7 referential +7 4.50 bipolar +2 2.53 � –2~+7 not bipolar +3 5.46 performed � –2~+6 referential +6 5.04 bipolar +2 3.02 � –2~+7 referential +7 4.19 bipolar +6 5.37 � –2~+7 referential +7 3.78 bipolar +3 3.78 � –2~+6 referential +6 4.36 bipolar +4 4.70 –1~+7 referential +7 4.11 bipolar +3 4.11 –2~+6 referential +6 4.53 bipolar +3 4.20 4.32 4.12 fig.2b. the simultaneously recorded cmaps of the calf muscles and the stationary potential at the recording site [+7] disappeared after severing the tibial nerve distal to the stimulating site. 136 fig. 3a. bipolar derivation(rabbit �). the stationary wave with the peak latency of 5.46 msec was observed through [+3]. fig. 3b. the simultaneously recorded cmaps of the calf muscle and the stationary potential at the recording site [+3] disappeared after transection of the tibial nerve distal to the stimulating site. 137 fig. 5. in referential derivation, the stationary potential was observed at the skull by direct calf muscle electrical stimulation. this potential didn’t change in shape or amplitude irrespective of transection of the tibial nerve. fig. 4. the shapes of the stationary potential as well as of the cmaps recorded over the calf muscle changed by compressing the calf muscle. rabbits. the potentials were obtained on the head ([+6] or [+7]) in all the subjects. the peak latency of the potential ranged from 3.78 msec to 5.04 msec with the average of 4.32 msec (table 1). bipolar derivation was carried out in eight out of ten rabbits. in bipolar derivations, the upper limits of observation of the stationary waves were from [+2] through [+6]. the peak latency ranged from 2.35 msec to 5.46 msec with the average of 4.12 msec (table 1). the cmaps of the calf muscles and the stationary potential at the recording site disappeared after severing the tibial nerve distal to the stimulating site while no change occurred by cutting the nerve proximal to stimulation (figs. 2, 3). the shapes of the stationary potential as well as of the cmaps recorded over the calf muscle changed by compressing the calf muscle (fig. 4). in referential derivation, the stationary potential was observed at the skull by direct calf muscle electrical stimulation. this potential didn’t change in shape and amplitude irrespective of severing the tibial nerve (fig. 5). discussion in order to record evoked potentials, it is necessary to derive the electric physiology phenomenon generated in the sensory neuron in vivo with electrodes on the surface or inside the body. the living body organization, which lies between an electrode and electric source, is called volume conductor. volume conduction can be spread from a potential source through a conducting medium, such as the body tissues. on the other hand, evoked potential with latency, which does not change even if the stimulation condition and the deriving condition are changed, is called a stationary potential. the stationary potentials are widely distributed in volume conductors, the shape of which is not changed even if the sites of electrodes are changed, and can be recorded far away from the source of potential and is named far field potential (5, 6, 7). the complex waveform of far-field potentials seems to result from a combination of different physiologic mechanisms that are uniquely dependent on the physical relationship between the nerve and volume conductor (8). there are varying opinions about the mechanisms of generation of far field potential. it occurs when an action potential transverses one of the following: � a region where the shape of the volume conductor changes (9,10,11,12);. � a bent segment of an axon (13,14); � a region in a volume where the resistance changes suddenly (15 ). the present experiment reveals that compound muscle action potentials far away from the ordinary recording spots can be recorded as stationary potentials which have the same peak latency through the volume conductor. deriving to the head was especially possible for referential derivation in all subjects. a latency difference between the cmap and the stationary peak suggests that there is a time lag of occurrence of stationary potentials accumulated in the volume conductor. the study on the optimal position of recording electrodes for cmaps 138 has been carried out (16, 17). however, there have been no reports that reproducible stationary waves originated from cmaps can be recorded even away from the stimulated muscles. neither has the description of the recording method of these stationary potentials. this research shows that it is possible to record cmaps from a part far away from the source as far field potentials which have constant latency. references 1. magladery, jw, porter we, park am, teasdall rd (1951). electrophysiological studies of nerve and reflex activity in normal man. iv. the two-neuron reflex and identification of certain action potentials from spinal roots and cord. bull. johns hopkins hosp 88: 499–519. 2. shimoji k, higashi h, kano t, kimura j, kimura a, ishida t, machida m, yamada t (1971). electroencephalogr clin neurophysiol 30: 236–239. 3. cracco rq (1972). the initial positive potential of the human scalp-recorded somatosensory evoked response. electroencephalogr clin neurophysiol 32: 623–629. 4. hatori m, miyasaka y, nobuta s, sakurai m (1989). an experiment of recording nerve root action potentials from the sciatic nerve in rabbits. tohoku arch orthop surg traumat 33: 101–103. (in japanese) 5. jewett dl (1970). volume-conducted potentials in response to auditory stimuli as detected by averaging in the cat. electroencephalogr clin neurophysiol 28: 609–618. 6. jewett dl, williston js (1971). auditory evoked far fields averaged from the scalp of humans. brain 94: 681–696. 7. cracco rq, cracco jb (1976). somatosensory evoked potential in man: far field potentials. electroencephalogr clin. neurophysiol 41: 460–466. 8. kimura j (1984). principles and pitfalls of nerve conduction studies. ann neurol 16: 415–429. 9. kimura j, mitsudome a, beck do, yamada t, dickins qs (1983). field distribution of antidromically activated digital nerve potentials: model for far-field recording. neurology 33: 1164–1169. 10. kimura j, mitsudome a, yamada t, dickins qs (1984). stationary peaks from a moving source in far-field recording. electroenceph clin neurophysiol 58: 351–361. 11. kimura j, kimura a, ishida t, kudo y, suzuki s, machida m, matsuoka h, yamada t (1986). what determines the latency and amplitude of stationary peaks in far-field recordings? ann neurol 19: 479–486. 12. kimura j, ishida t, suzuki s, kudo y, matsuoka h, yamada t (1986). far-field recording of the junctional potential generated by median nerve volleys at the wrist. neurology 36: 1451–1457. 13. desmedt je, huy nt, carmeliet j (1983). unexpected latency shifts of the stationary p9 somatosensory evoked potential far-field with changes in shoulder position. electroenceph clin neurophysiol 56: 628–634. 14. nakanishi t, tamaki m, kudo k (1986). possible mechanism of generation of sep far-field component in the brachial plexus in the cat. electroenceph clin neurophysiol 63: 68–74. 15. nakanishi t (1982). action potentials recorded by fluid electrodes. electroenceph clin neurophysiol 53: 343–345. 16. wee as, ashley ra (1988). effect of thumb position on thenar compound muscle action potential field. electromyogr clin neurophysiol 28: 163–166. 17. wee as, ashley ra (1988). where is the ideal reference site for recording the thenar compound muscle action potential. electromyogr clin neurophysiol 28: 249–252. corresponding author: masahito hatori, m.d. department of orthopaedic surgery, tohoku university school of medicine, 1-1 seiryomachi, aoba-ku, sendai, 980-8574 japan. tel: 81-22-717-7242 fax: 81-22-717-7248 139 untitled delays in primary lung cancer 287 received 6 may 2008 accepted 2 june 2008 upsala j med sci 113 (3): 287–296, 2008 delays in the diagnosis and treatment of primary lung cancer: are longer delays associated with advanced pathological stage? adnan yilmaz1, ebru damadoglu1, cuneyt salturk1, erdal okur2, leyla yagci tuncer1, semih halezeroglu2 sureyyapasa thoracic and cardiovascular diseases teaching and investigation hospital-istanbul, 1department of pulmonology, 2department of thoracic surgery abstract we aimed to investigate the delays from the first symptom to thoracotomy and to examine whether the delays cause the stage advancement in lung cancer. this prospective study included 138 patients with non-small cell lung carcinoma who underwent thoracotomy. clinical files of the patients were analyzed and a questionnaire was created to obtain information from the patients. the mean duration values were 81.3 days for the application interval, 61.3 days for the referral interval, 20.3 days for the diagnostic interval, and 21.9 days for the treatment interval. the application interval was longer than 30 days (patient delay) in 50 patients (37.9 %). the mean interval from the first visit to doctor to thoracotomy was 97.2 days. there was a doctor delay in 102 (73.9 %) patients; a referral delay in 83 patients (60.1 %), a diagnostic delay in 47 patients (36.4 %), and a treatment delay in 96 patients (69.6 %). the mean total duration was 176.2 days. ninety-four patients (71.2 %) had a total delay. mean total delay was 184.5 days in pathologic stage i, 187.3 days in stage ii, 167.7 days in stage iiia, 142.6 days in stage iiib, and 150.3 days in stage iv (p>0.05). delays during the course between the first symptom and thoracotomy in lung cancer patients were a common problem among our patients. prolonged durations in the application and referral of patients are the most significant cause of delays. presence of delay or length of delay did not correlate with pathologic tumour stage in this study. introduction lung cancer remains to be one of the most common and most lethal forms of cancer. despite the advancements in modern diagnostic and therapeutic modalities, the prognosis of lung cancer is still very poor, with an overall 5-year survival rate of about 15 % worldwide (1). only less than 20 % of patients with lung cancer can undergo pulmonary resection, which is the only curative treatment (2). several factors such as age, sex, comorbidity, performance status, histology and stage of the disease affect the prognosis of patients (3). it has been documented that the survival and cure rates are higher in cancer patients diagnosed in the earlier stages (4, 5). however, diagnostic and treatment delays continue to remain very common problems among the patients with lung cancer (5–8). some studies looking at the impact of delays on tumour stage and survival rate have shown that delays affected tumour stage and survival rate (9–11). on the other hand, others have reported that delays 288 adnan yilmaz et al. were not associated with tumour stage and survival rate (5, 7, 8, 12). the aims of this study were to investigate the delays in patients with lung cancer from the first symptom to thoracotomy and to examine whether the delays affect the stage of lung cancer at the time of thoracotomy. materials and methods we investigated all the patients with primary lung cancer referred from the pneumology departments to first thoracic surgery department of sureyyapasa chest diseases and thoracic surgery training and investigation hospital between january 2005 and july 2006. among the patients who underwent thoracotomy for lung cancer, those who had tumour pathology other than non-small cell lung carcinoma, patients who were referred to surgical department directly from other hospitals, the patients who did not accept responding the questionnaire, and those who did not remember some dates in their disease course were excluded from the study. there were 192 patients who underwent thoracotomy for lung cancer in this department during the period study was carried out. however, the study included 138 patients who had the inclusion criteria. consent was obtained from each patient after full explanation of the purpose and nature of the study. because patient medical records were used and a questionnaire was created to obtain data, ethics committee approval has not been obtained. all patients had a preoperative bronchoscopy and computed tomography (ct) of the thorax and upper abdomen. various techniques such as brain ct or mr, radionuclide bone scintigraphy, pet or pet/ct-were carried out in patients with symptoms or signs suggesting extrathoracic metastases. twenty-three patients had pet/ct investigation preoperatively. preoperative surgical mediastinal exploration was performed in those patients who presented with centrally located tumour, enlarged lymph nodes on ct or increased fdg uptake on pet/ct in mediastinal or hilar lymph nodes. clinical files of the patients were analyzed by 2 authors of this study, and a questionnaire was created to obtain data by the interview with the patients. for each patient, the following information was gathered based on these data: (1) sex, (2) age, (3) symptoms, (4) tumour histology, (5) pathological tnm stage, (6) date of initial symptoms, (7) date of first doctor visit, (8) date of admission to pneumology department of our hospital, (9) date of diagnosis, and (10) date of thoracotomy. pathological tnm stage was based on thoracotomy and pathological reports. the following time intervals and delays were determined for each patient: patient’s application interval was defined as the time passed between the onset of symptoms and the first doctor visit. it was calculated in 132 patients who had symptoms. the application interval that exceeded 30 days was considered indicative of a patient’s delay (13). the referral interval was defined as the time from the first doctor visit to admission to one of the pneumology departments of our hospital for the further investigation, and the interval that exceeded 14 days was considdelays in primary lung cancer 289 ered indicative of a referral delay. the diagnosis interval was regarded as the time passed between the admission to our hospital and the pathological diagnosis was made. because 9 patients admitted to pneumology departments with a pathological diagnosis, this interval was calculated in 129 patients in whom the diagnosis was established in our hospital. the diagnosis interval that exceeded 14 days was considered as indicative of a delayed diagnosis. the treatment interval was the time passed from the diagnosis to thoracotomy, and the interval that exceeded 14 days was considered as indicative of a delayed treatment. doctor’s interval was defined as the time from the first doctor visit to thoracotomy and the interval that exceeding 6 weeks was considered as indicative of a doctor’s delay. the total interval was the time between the onset of symptoms and thoracotomy, and if exceeding 72 days it was considered indicative of a total delay (14, 15). figure 1 demonstrates the time intervals and delays used in this study. statistical analysis the data were analyzed using the spss statistical program. time intervals were compared among stages using kruskal-wallis test. proportions were analysed with exact test. a p-value of less than 0.05 was considered significant. figure 1. components of the time from onset of symptoms to thoracotomy and delays. 290 adnan yilmaz et al. results the study included 138 patients with non-small cell lung carcinoma consisting of 133 (96.4 %) men and 3 (3.6 %) women with a mean age of 58.1 years (range 28–78). demographic characteristics of the study population are presented in table 1. the mean duration between the first existence of symptom and thoracotomy was 176.2 days; 81.3 days for application, 61.3 days for referral, 20.3 days for diagnosis and, 21.9 days for treatment ( table 2). duration between the first symptom and thoracotomy was longer than 72 days in 94 patients (71.2 %). of the patients, 50 (37.9 %) had patient’s delay and 102 table 1. demographic characteristics of the study population n % sex men 133 96.4 women 5 3.6 presenting symptom no symptom 6 4.3 cough 77 58.3 chest pain 50 37.9 dyspnea 29 22 hemoptysis 46 34.8 sputum 3 2.3 others 40 30.3 tumour histology squamous 83 60.1 adenocarcinoma 48 34.8 adenosquamous 1 0.7 non-small cell 6 4.4 location of tumour right lung 64 46.4 left lung 74 53.6 pathological stage i 67 48.6 ii 32 23.2 iii a 17 12.3 iii b 19 13.8 iv 3 2.1 delays in primary lung cancer 291 (73.9 %) had doctor’s delay. referral interval was longer than 14 days in 83 patients (60.1 %), diagnostic work ups took more than 14 days in 47 patients (36.4 %) and waiting time for thoracotomy more than 14 days in 96 patients (69.6 %). there were 276 delays occurring in 138 patients in 4 different steps (application, referral, diagnosis, and treatment) during the disease course between the first symptom and thoracotomy. delays were due to a late admission to a doctor after the first symptoms in 50 patients (18.1 %), and a prolonged referral time in 83 patients (30.1 %), a diagnostic interval in 47 patients (17 %) and a waiting time for thoracotomy in 96 patients (34.8 %) (table 3). distribution of intervals with respect to pathological stage is shown in table 4, and patients with delay with respect to pathological stage in table 5. there was no significant difference among stages. discussion this study shows that there are several delays in different steps from the onset of symptoms to thoracotomy among our patients with lung cancer. these delays are due to either patients themselves, as they admitted to a doctor lately (patient’s delay) or prolonged durations in referral of patients by the doctor, diagnostic workups and waiting time for thoracotomy (doctor’s delay). the mean and median application intervals of patient to a doctor after their first symptoms were 81.3 and 18 days, respectively. our median application interval compares favourably with 21 days of koyi and co-workers’ (6) and 30 days in özlu and co-workers’ studies (16) while mean interval compares less favourably with 30.3 days in gonzalez and co-workers’ (17) and, 41 days in salomaa and co-workers’ studies (5). mean total duration of 176 days between the first symptom and thoracotomy in our study is shorter than koyi and co-workers’ series (203 days) and longer than billings and wells’ series (109 days) (6, 7). we found that the rate of doctor’s delay was higher than that of patient’s delay. table 2.values in the intervals interval n mean (days) sd (days) median (days) 95 % ci (days) application 132 81.3 176.8 18 51.1-111.5 doctor’s delay 138 97.2 108.1 56 81.8-118.8 referral 138 61.3 110.2 20.5 42.9-79.7 diagnosis 129 20.3 28.9 11 15.3-25.3 treatment 138 21.9 19.4 19 18.7-25.2 total delay 132 176.2 210.5 98 140.3-212.1 292 adnan yilmaz et al. table 3. distribution of patients in the intervals in respect to delay criteria intervals n % application interval 132 100 ≤ 30 days 82 62.1 > 30 days 50 37.9 doctor’s delay 138 100 ≤ 6 weeks 36 26.1 > 6 weeks 102 73.9 referral interval 138 100 ≤ 2 weeks 55 39.9 > 2 weeks 83 60.1 diagnosis interval 129 100 ≤ 2 weeks 82 63.6 > 2 weeks 47 36.4 treatment interval 138 100 ≤ 2 weeks 42 30.4 > 2 weeks 96 69.6 total delay 132 100 ≤ 72 days 38 28.8 > 72 days 94 71.2 the mean and median durations after the first doctor visit to thoracotomy were 97.2 and 56 days, respectively, while a previous study reported these durations being 56 and 33 days after the first doctor’s visit and 33 and 9 days after the second doctor’s visit (6). analysis of the total number of delays in our study revealed that therapeutic delays were the most common part of doctor’s delay as the mean duration between the diagnosis and thoracotomy was 21.9 days. however, when the total extent of delays is considered, the application and the referral intervals appeared as the main parameters prolonging the duration. the delays in diagnostic workups (mean of total diagnostic time: 20.3 days) occurred in relatively lesser extent among our patients. several study groups have made recommendations for referral and waiting times in the diagnosis and treatment pathways of lung cancer (13–15, 18). according to one brazilian study, an application interval exceeding 30 days is considered as a patient’s delay (13). the british thoracic society (bts) recommends that all patients should be seen for an initial evaluation by a pulmonary physician within 1 week of referral from their primary care physician and, diagnostic testing should be performed within 2 weeks of the decision (14). the swedish lung cancer study group (18) recommends that in 80 % of all patients, diagnostic tests should be completed delays in primary lung cancer 293 table 4. distribution of intervals in respect to pathological stage pathological stage i ii iii a iii b iv interval (days) (days) (days) (days) (days) application † † p>0.05 mean 86.9 85.6 102.3 43.8 40.7 sd 170.9 168.4 282.9 87.8 34.4 median 15.0 23.0 25.0 16.0 60.0 ci, 95 % 43.8-129.9 22.7-148.4 43.1-247.7 1.5-86.1 -44.7-126.0 doctor ‡ ‡ p>0.05 mean 104.3 97.5 65.4 98.6 103.0 sd 118.6 120.0 64.4 82.6 87.5 median 68 55.0 50.0 75.0 56.0 ci, 95 % 75.4-133.2 54.2-140.9 32.3-98.5 58.8-138.5 -114.5-320.5 total   p>0.05 mean 184.5 187.3 167.7 142.6 150.3 sd 202.9 195.3 344.5 103.1 108.2 median 105.0 108.0 81.0 101.0 136.0 ci, 95 % 133.4-235.6 114.4-260.2 -9.4-344.8 92.9-192.3 118.5-419.2 table 5. distribution of the patients having delay in respect to pathological stage pathological stage i ii iii a iii b iv presence of delay n (%) n (%) n (%) n (%) n (%) patient’s delay † † p>0.05 yes 40 (63.5) 18 (60.0) 10 (58.8) 13 (68.4) 1 (33.3) no 23 (36.5) 12 (40.0) 7 (41.2) 6 (31.6) 2 (66.7) doctor’s delay ‡ ‡ p>0.05 yes 17 (25.4) 9 (28.1) 7 (41.2) 3 (15.8) -no 50 (74.6) 23 (71.9) 10 (58.8) 16 (84.2) 3 (100) total delay   p>0.05 yes 19 (30.2) 7 (23.3) 7 (41.2) 4 (21.1) 1 (33.3) no 44 (69.8) 23 (76.7) 10 (58.8) 15 (78.9) 2 (66.7) 294 adnan yilmaz et al. within 4 weeks from consultation by a specialist, and treatment should be started within 2 weeks thereafter. in the canadian recommendations (15), a maximum of 4 weeks’ elapse could be accepted between the first visit to a general practitioner and diagnosis, and the waiting time for surgery should not exceed 2 weeks. salomaa et al (5) reported that about half of their patients fulfilled the criteria of the bts recommendations. sixty-six percent of their patients fulfilled the diagnostic delay criteria, and 49 % the treatment delay criteria of the swedish lung cancer study group. only 26 % could keep the canadian recommendation of 4-week limit. according to the brazilian study criteria, there was a patient delay in 37.9 % of our patients. our criteria for doctor’s, institutional, diagnosis, treatment and total delays were based on the bts and canadian recommendations. the rates of delay were 73.9 % for doctor’s delay, 60.1 % for institutional delay, 36.4 % for diagnosis, 69.6 % for treatment, and 71.2 % for total delay in our series. delays may be due to several factors depending on the characteristics of the region and the population and status of the institution where the data were collected as well as the healthcare system applied. a british study noted that pre-hospital delays varied depending on the severity of symptoms, level of patient education and complex socioeconomic factors, and the main cause of delays was the multiplicity of pre-treatment investigations for histological verification, tumour staging, and assessment of co-morbidity in different institutions while scarcity of thoracic surgeons and limited theatre time were additional contributory factors in the delay in surgical patients (19). a study in turkey associated the delays with prolonged durations in the appointments for imaging procedures as a part of an organisational problem in the health care system (16). multiple appointments for patients requiring frequent hospital visits and long durations between appointments were the explanations of most of the multifactorial delays in a canadian study (8). less than 40% of our patients applied to a doctor later than 30 days after their first symptoms appeared. indeed, this is not completely the result of healthcare system since the primary healthcares is free for all the people but relatively rare behaviour of applying a doctor for mild symptoms among undereducated people. longer referral time seemed as another main problem in delays. this might be due to partly the duration passed between the distance of patient’s lodgement and one of the main referral hospitals for lung cancer in turkey or the long durations spent in the second or even the third opinions taken by the patients and or family after they have learned the suspicious diagnosis of cancer. however, an organisation remains to be established to take at least the chest x-ray at the first doctor visit in symptomatic patients in risk group for this very common disease, and then refer them to the closest centre where the treatment could be achieved. whether the delay advances the tumour stage is an important debate. some studies indicated the delays as affecting the prognosis negatively (9, 10, 20), while others could not show such an association (5, 7, 12, 18). in the report of christensen et al (9), a few months delay had an impact on the perioperative stage of the cancer, and thereby on the prognosis. o’rourke and edwards analyzed 29 lung cancer patients who were awaiting radical (potentially curative) radiotherapy, and found delays in primary lung cancer 295 that 21 % of potentially curable patients became incurable on the waiting list (10). on the other hand, it was shown in some studies that presence of delay (5) or the length of delay (5, 7) did not correlate with the tumour stage. among the patients in advanced stages in our series, there was neither higher incidence of delay nor the longer delays when compared to earlier stages. similarly, absence of delay or shorter delays were not associated entirely with only earlier stages; all the stages included similar distribution in this regard. to sum up, delays in all the steps along the period between the first symptoms to thoracotomy appear to be a common problem among our patients with lung cancer. socio-medical efforts should be spent to lower the durations in the periods between “the symptoms and application to a doctor” and “referral by the doctor to an institution where the diagnostic and therapeutic measures take place”. presence of delay or length of delay does not correlate with tumour stage in patients who underwent thoracotomy; however, the final conclusion on this subject can only be given with the additional results of the studies that compare parameters among the patients in operable and inoperable stages. references 1. american cancer society. cancer facts and figures 2006. atlanta (ga): american cancer society; 2006. 2. scagliotti gv (2001). symptoms and signs and staging of lung cancer. eur respir mon 17: 86–119. 3. birim ö, kappetein ap, van klaveren rj, bogers ajjc (2006). prognostic factors in non-small cell lung cancer surgery. ejso 32: 12–23. 4. robinson e, mohilever j, zidan j, sapir d (1984). delay in diagnosis of cancer. possible effects on the stage of diseases and survival. cancer 54: 1454–60. 5. salomaa er, sallinen s, hiekkanen h, liippo k (2005). delays in the diagnosis and treatment of lung cancer. chest 128: 2282–8. 6. koyi h, hillerdal g, branden e (2002). patient’s and doctor’s delays in the diagnosis of chest tumours. lung cancer 35: 53–7. 7. billings js, wells fc (1996). delays in the diagnosis and surgical treatment of lung cancer. thorax 51: 903–6. 8. liberman m, liberman d, sampalis js, mulder ds (2006). delays to surgery in non-small-cell lung cancer. can j surg 49: 31–6. 9. christensen ed, harvald t, jendresen m, aggestrup s, petterson g (1997). the impact of delayed diagnosis of lung cancer on the stage at the time of operation. eur j cardiothorac surg 12: 880–4. 10. o’rourke n, edwards r. lung cancer treatment waiting times and tumour growth (2000). clin oncol 12: 141–4. 11. korsgaard m, pedersen l, sorensen ht, laurberg s (2006). delay of treatment is associated with advanced stage of rectal cancer but not of colon cancer. cancer detect prev 30: 341–6. 12. aragoneses fg, moreno n, leon p, fontan eg, folque e (2002), the bronchogenic carcinoma cooperative group of the spanish society of pneumology and thoracic surgery (gccb-s). influence of delays on survival in the surgical treatment of bronchogenic carcinoma. lung cancer 36: 59–63 13. silva ppa, pereira jr, ikari fk. minamoto h (1992). cancer de pulmao e retardo diagnostico: analise de 300 cases. rev ass med brasil 38: 145–9. 14. british thoracic socity (1998). bts recommendations to respiratory physicians for organising the care of patients with lung cancer: the lung cancer working party of the british thoracic society standards of care committee. thorax 53 (suppl 53): 1–8. 296 adnan yilmaz et al. 15. simunovic m, gagliardi a, mccready d, coates a, levine m, depetrillo d (2001). a snapshot of waiting times for cancer surgery provided by surgeons affiliated with regional cancer centers in ontario. can med assoc j 165: 421–5. 16. özlü t, bülbül y, öztuna f, çan g (2004). time course from first symptom to the treatment of lung cancer in the eastern black sea region of turkey. med princ pract 13: 211–4. 17. gonzalez jm, de castro fj, barrueco m, cordovilla r, fernandez jl, gómez fp, moreno de vega b, ramos j, serrano ar (2003). delays in the diagnosis of lung cancer. arch bronconeumol 39: 437–41. 18. myrdal g, lambe m, hillerdal g, lamberg k, agustsson th, stahle e (2004). effect of delays on prognosis in patients with non-small cell lung cancer. thorax 59:45–9. 19. devbhandari mp, bittar mn, quennell p, barber p, krysiak p, shah r, jones mt (2007). are we achieving the current waiting time targets in lung cancer treatment? result of a prospective study from a large united kingdom teaching hospital. j thorac oncol 2: 590–2. 20. jensen ar, mainz j, overgaard j (2002). impact of delay on diagnosis and treatment of primary lung cancer. acta oncol 41: 147–52. corresponding author: associate professor adnan yilmaz maltepe zumrutevler atatürk cad. abant apt. no: 30 istanbul/turkey phone: + 90 216 3058324 email: adnandr_63@yahoo.co.uk fertility preservation for young adults, adolescents, and children with cancer review article fertility preservation for young adults, adolescents, and children with cancer kenny a. rodriguez-wallberga,b , amandine anastacioa, emelie vonheimb, sandra deenb, johan malmrosc and birgit borgstr€omd adepartment of oncology-pathology, karolinska institutet, stockholm, sweden; bdivision of gynecology and reproduction, department of reproductive medicine, karolinska university hospital, stockholm, sweden; cdepartment of pediatric oncology, karolinska university hospital, stockholm, sweden; ddepartment of pediatric endocrinology, karolinska university hospital, stockholm, sweden abstract options for fertility preservation (fp) through cryopreservation methods are currently available for young adults, adolescents, and children. guidelines for fp have been provided by international clinical societies, and emergency procedures aimed at fp have been implemented into clinical practice worldwide. in this article, we review the current data on clinical standards of emergency fp in patients who are facing gonadotoxic effects of cancer treatment, and we also describe the methods that are still under development, usually denoted as experimental. in sweden, programmes for fp have been established at large university hospitals, thus covering the whole country. the swedish publicly financed health care covers both assisted reproduction for treatment of infertility and the cryopreservation of gametes or gonadal tissue when there is a medical indication, such as the risk to become infertile due to oncologic treatment; hence the access to fp is ensured for the whole population. at our centre at karolinska university hospital in stockholm, methods for fp have been offered since 1988. in this article, we also review the oncologic indications for fp in our patient cohort of >3000 individuals during the period 1988–2018. article history received 28 january 2020 revised 17 february 2020 accepted 27 february 2020 keywords cancer survivorship; cryopreservation; fertility preservation; gonadotoxicity; premature gonadal failure; young adults and children introduction fertility preservation (fp) is a modern clinical field that combines medical and technical developments in reproductive medicine and their application to patients with serious diseases, such as cancer, or with benign clinical conditions associated with a premature onset of gonadal insufficiency (1). international guidelines have stated that timely information on methods for fp should be provided to all patients of young age when planning gonadotoxic treatments, as well as to the parents or guardians of children in such clinical situation (2,3). cryopreservation of reproductive cells and tissues is a fundamental tool in the development of clinical programmes for fp (4). development of clinically established and experimental methods for fp: first sperm, thereafter embryos, and then oocytes and gonadal tissue historically, efficacious methods for sperm cryopreservation were achieved first, already in the 1950s, facilitated by the usually large number of spermatozoa obtained per sample and the possibility to use them for research (5). thereafter, the development of treatments using assisted reproductive technologies (art), resulting in multiple embryos produced per treatment, also required a further development of methods to cryopreserve supernumerary embryos for later use. these were reported successful for the first time already in 1983 (6). however, for cryopreservation of oocytes, the advances have been slow. this has been due to the technical difficulties that arise as a consequence of the large cell size and water content of the oocytes, which makes the cells more prone to damage during cryopreservation and thawing (7). research in development of efficient methods to cryopreserve oocytes advanced also slowly due to their scarcity. although the first report of successful vitrification appeared in 1997 (8), the clinical validation of cryopreservation of oocytes by vitrification has only recently been achieved (9,10), and the method became clinically established and recognized by the large international reproductive societies in 2013 (1,4). in addition to these well-established methods for fp, today clinically implemented at most fertility centres worldwide practicing art, additional fp methods have been developed through years of experimentation. ovarian tissue cryopreservation for later re-transplantation in humans has been developed through extensive and focussed translational research during the 1990s (11–13). several cryopreservation protocols have been validated, and large programmes of fp have reported their experiences of cryopreservation of contact kenny a. rodriguez-wallberg kenny.rodriguez-wallberg@sll.se division of gynecology and reproduction, department of reproductive medicine, karolinska university hospital huddinge, stockholm se-141 86, sweden � 2020 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution-noncommercial license (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2020, vol. 125, no. 2, 112–120 https://doi.org/10.1080/03009734.2020.1737601 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2020.1737601&domain=pdf&date_stamp=2020-05-21 http://orcid.org/0000-0003-4378-6181 http://creativecommons.org/licenses/by-nc/4.0/ https://doi.org/10.1080/03009734.2020.1737601 http://www.tandfonline.com ovarian tissue for fp of adult women, adolescents, and prepubertal girls (14–22). in december 2019, the practice committee of the american society of reproductive medicine stated that ovarian tissue cryopreservation for fp should no more be considered as experimental (23). for prepubertal boys, methods for cryopreservation of testicular tissue have also been developed and proposed as a method for fp aiming at the preservation of spermatogonia (24–30). however, the absence of mature sperm in the prepubertal testis is recognized as one of the main difficulties, hence the need of further developments, such as the investigation of tissue transplantation and also the development of in vitro culture systems aiming at obtaining fully viable mature gametes for possible fertility treatment in the future (31). although the subject of fp for children may be difficult to approach and it might also be considered ethically debateable, survivors of cancer in childhood have ranked it as an important quality-of-life issue. international guidelines supporting promotion of fp for children have thus been provided (1,2,23,27,32,33). in sweden, a multidisciplinary group including all university hospitals has also provided national guidelines to ensure that healthcare providers become familiar with the available methods for fp and to facilitate access to fp counselling for children and teenagers with cancer (34). at karolinska university hospital in stockholm, methods for fp have been offered since 1988. the patient cohort currently includes >3000 individuals, adults and children, and the cohort is prospectively followed under studies approved by the regional ethics committee and performed according to the principles of the declaration of helsinki. why oncologic treatment is an indication for undergoing fp the swedish cancer statistics indicate that one in three people will receive a cancer diagnosis during their lifetime, and the number of cancer cases is increasing every year (35). although most cancers occur in aged people, cancer can also present in young adults and children. cancer registration data in europe and in the usa have shown increasing rates of survival, above 80%, in particular for several diseases and for the youngest patients, such as those presenting with cancer during childhood or young adulthood (35). survivorship issues have thus come to the frontline as quality of survival is highly relevant for the patients (36–38). in most cancers, the treatment strategies using chemotherapy, radiotherapy, or surgery are likely to cause permanent infertility (figure 1) (32). chemoand radiotherapeutic gonadotoxic effects are dose-dependent, and some of those effects have been well characterized regarding the protocols used. table 1 summarizes radiation doses and radiotherapy protocols that may impact on ovarian and testicular function (33). irradiation of the uterus may also negatively affect the chances to conceive and increase obstetric risks. as regards chemotherapeutic agents, those are usually given as part of a combination regimen. this fact has contributed to the difficulty in achieving a precise gonadotoxic quantification of individual drugs. table 2 shows a list of chemotherapeutic agents according to the currently known gonadotoxic impact in females and males. in female patients, gonadotoxicity is particularly dependent on age, because the number of primordial follicles that constitute the ovarian pool is non-renewable, and it diminishes with age. older women would thus have a higher risk of developing permanent infertility due to follicle depletion, when compared with younger women undergoing a similar treatment. in men, younger age or a prepubertal status does not provide protection against the damage induced by chemotherapeutic drugs (39). banking of frozen sperm sperm cryopreservation is a standard of care for adolescents and young adults facing gonadotoxic treatment such as the one required for treatment of cancer. it is usually feasible from around 12 years of age, when spermarche usually has been achieved and the testis volume has reached 8 ml (40,41). most studies in the field of fp advocate that all patients of reproductive age should be advised to bank frozen sperm prior to initiating gonadotoxic therapy (42). in cases of ejaculation failure, search for spermatozoa in a urine sample could be proposed. testicular sperm extraction can also be offered, aiming to retrieve spermatozoa in young men and adolescents (33). other methods described for retrieval of spermatozoa in adolescents include penile vibratory stimulation and electro-ejaculation. in sweden, the costs for sperm cryopreservation are covered by the tax-funded healthcare system up to a male age of 55 years. in other countries, patients may need to cover the costs of the procedures and a fee for maintaining the frozen samples. the average cost in the united states for banking three samples varies from $25 to $35 per month. economic barriers have been discussed in previous studies as a limitation to patients’ access to fp (2,3). oncologic indications for sperm cryopreservation in the karolinska cohort at the reproductive medicine clinic of karolinska university hospital sperm cryopreservation aiming at fp has been offered since 1988. referrals of patients with oncologic indications can be accepted without any delay, and the patients may approach the fertility laboratory also during weekends. between 1 january 1998 and 31 december 2018, 1749 male patients have been referred for sperm cryopreservation to our centre. of those, 356 presented with benign diseases. figure 2 summarizes the data of 1393 patients referred for oncologic indications that had sperm cryopreserved at our centre during that period. the most frequent oncologic diagnoses in young men and teenagers include testicular cancer and haematologic malignancies. over 80% of patients had two to three samples banked (range 1–7). young adolescents have been able to provide sperm samples for upsala journal of medical sciences 113 cryopreservation from the age of 14 years. the mean age at sperm banking was 31 years (range 14–61). cryopreservation of embryos, oocytes, and ovarian tissue cryopreservation of embryos, oocytes, and ovarian tissue are methods clinically established for female fp. for embryo and oocyte cryopreservation, hormonal stimulation with gonadotropins is needed, to induce multiple follicle recruitment aiming at obtaining more than one mature oocyte per treatment cycle. stimulation protocols using a gnrh antagonist are currently preferred for fp, as these minimise the time required for ovarian stimulation. the protocols can also be applied with random initiation (random start), that is, regardless of the woman’s cycle day. in general, the delay to egg retrieval is shortened to about 2 weeks in most cases (22,43–46). improvements of stimulation protocols for women with breast cancer undergoing fp aiming at an increase of their safety have been proposed, in particular with coadministration of letrozole alongside gonadotropins, together with a gnrha maturation trigger (47). this specific protocol has been demonstrated to reduce the expected several fold increase in systemic oestrogen levels during conventional hormonal stimulation. furthermore, it achieves low systemic oestrogen levels, similar to those found in a normal menstrual cycle, even though multiple follicles develop responding to the exogenous gonadotropins (47). the number of oocytes obtained in cycles with letrozole is similar to that obtained using standard protocols, and their maturity rate does not seem to differ from those obtained with conventional gonadotropin stimulation protocols (46,47). co-administration of tamoxifen has been also proposed for fp of women with breast cancer. however, protocols with letrozole have demonstrated higher efficacy than those with tamoxifen in a prospective controlled study (48). in sweden, protocols with co-administration of letrozole for fp of women with breast cancer have been implemented at karolinska university hospital since 2009 and thereafter at the remaining large university hospitals (46). a recent large figure 1. fertility preservation (fp) strategies according to modalities of cancer treatment. reprinted, with permission from: rodriguez-wallberg and oktay (32). originally published by and used with permission from dove medical press limited. table 1. radiotherapy protocols with high or intermediate impact on ovarian and testicular function. modified from rodriguez-wallberg and oktay (33). radiotherapy protocols and their risk of prolonged azoospermia in men or amenorrhoea in women high risk total body irradiation (tbi) for bone marrow transplant/stem cell transplant testicular radiation dose >2.5 gy in adult men testicular radiation dose �6 gy in prepubertal boys pelvic or whole-abdominal radiation dose �6 gy in adult women pelvic or whole-abdominal radiation dose �10 gy in postpubertal girls pelvic radiation or whole-abdominal dose �15 gy in prepubertal girls intermediate risk testicular radiation dose 1 � 6 gy from scattered pelvic or abdominal radiation pelvic or whole-abdominal radiation dose 5 � 10 gy in postpubertal girls pelvic or whole-abdominal radiation dose 10 � 15 gy in prepubertal girls craniospinal radiotherapy dose �25 gy14a acranial irradiation for the treatment of brain tumours may induce infertility in both female and male patients by disruption of the hypothalamic–pituitary–gonadal axis and disturbance of gonadotropin secretion. 114 k. a. rodriguez-wallberg et al. prospective study including all swedish centres reporting on women with breast cancer undergoing hormonal stimulation for fp indicates that the proposed improvements to antagonist protocols for emergency fp including random-start and co-administration of letrozole are efficacious, as regards treatment outcomes in terms of oocytes and embryos obtained and cryopreserved (46). the safety of hormonal stimulation in women with breast cancer aiming at fp has also been investigated in several studies (46,49–51). a systematic review published in 2017 indicated no increased risks (52). a recent population-based study with matched cohort design including 188 women with breast cancer that underwent fp from 1999 to 2013 in sweden indicated no increased risk of cancer recurrence, when compared with 378 age-matched controls (53). it is recommended that standardized counselling on fp should be provided early in the process of cancer diagnosis and treatment, to allow the patients to make decisions as regards safeguarding of their future fertility potential. for women with a male partner and with enough time to undergo hormonal stimulation, it has been established to present the option to cryopreserve embryos in the first place, mainly due to the lack of confidence in methods for cryopreservation of unfertilised oocytes during many years, as the method of oocyte vitrification was only accepted as clinically developed in 2013 (1). a large prospective study in sweden has identified recent changes in trends regarding patient decisions after standardized counselling for fp (22). in the swedish cohort, an increasing number of women in committed relationships have chosen to cryopreserve unfertilized oocytes, which is important as it provides autonomy to the women (22). in many countries, law constraints currently include the requirement that the two partners are together at the time of the use of the cryopreserved embryos. indeed, this may be detrimental to women having cryopreserved embryos if the couples split later on (22). in cases when hormonal stimulation or transvaginal oocyte retrieval are not suitable, or when there is not enough time, the only method available for fp is cryopreservation of ovarian tissue. this is also the only method that can be offered to prepubertal girls (2,3,22,23,32,33). several clinical programmes of fp have reported ovarian tissue cryopreservation for adult women and girls worldwide (14–22). in scandinavia, programmes of fp include ovarian tissue cryopreservation at nearly all centres (19). oncologic diagnoses in females attempting fp in the karolinska cohort a detailed analysis of the cohort of women and girls that have undergone procedures for fp due to oncologic conditions at karolinska university hospital has been recently reported (22). in total 1254 females had been referred to our centre between 1 january 1998 and 31 december 2018. in 402 cases the indication was a benign condition (22). figure 3 illustrates the most frequent diagnosis in the karolinska cohort, which included 852 women and girls with cancer who underwent procedures for fp during the period 1998–2018, with a mean age of 27.8 years (range 1–43) (22). utilisation rates of sperm, oocytes, and embryos and efficacy of fp in the long term in general, follow-up studies indicate a low utilisation rate of embryos, oocytes, sperm, and gonadal tissue aimed at fp. the most frequent causes of not proceeding to use reproductive cells or tissues is the current lack of a partner, or the oncological recommendation to postpone pregnancy until a long-enough follow-up time after cancer treatment (22). regarding frozen banked sperm, usage rates have been reported to be less than 10% (54). in previous large studies, the utilization rate reported has been as low as 7% (55,56), and large data have been analysed in a systematic review in 2016 (57). these low numbers have brought up the discussion of the cost-effectiveness of sperm banking. even though usage rates are low, it has been argued that there might be other benefits of banking sperm, such as the psychological and emotional benefit of giving the patient hope in a long-term survival. for women, comparable studies of large sample size are notoriously scarce. a previous retrospective study of women that returned for thawing their oocytes or embryos (58) reported an embryo implantation rate of 27% and live birth rate (lbr) of 44% per embryo transfer (58). most studies have been retrospective and report on the outcome of healthy women undergoing elective oocyte freezing. in a spanish cohort, a number of 8–10 mature oocytes was found to be associated with a reasonable chance of success, with a mean of 10 oocytes resulting in 60.5% live birth rate (lbr) in the women that returned. of note, the lbr was reduced when the women were older than 36years (59), and several studies have confirmed an association of older age with poorer outcome as regards retrieved oocyte yields (22,58–60). in the table 2. chemotherapy agents according to their gonadotoxic impact in females (amenorrhoea) and males (azoospermia). chemotherapy agents high risk cyclophosphamide ifosfamide melphalan busulfan nitrogen mustard procarbazine chlorambucil intermediate risk cisplatin with low cumulative dose carboplatin with low cumulative dose adriamycin low risk treatment protocols for hodgkin’s lymphoma without alkylating agents bleomycin actinomycin d vincristine methotrexate 5-fluorouracil radioiodine treatment for thyroid cancer unknown risk paclitaxel and docetaxel for treatment of breast cancer irinotecan trastuzumab imatinib erlotinib bevacizumab upsala journal of medical sciences 115 women that return to attempt pregnancy following fp, a malignant indication of fp has been associated with a reduced lbr, when compared with that of women that have undergone fp for benign indications (22,60). in the karolinska cohort, the rate of utilization of embryos, oocytes, or ovarian tissue in women of childbearing age who had a follow-up of at least 1 year after fp were 29%, 8%, and 5%, respectively, with pregnancy rates of 66%, 54%, and 25%, and lbr of 54%, 46%, and 7%, respectively (22). the efficacy of re-transplantation of ovarian tissue has been compared with the use of vitrified oocytes in a previous spanish study of 44 women that underwent re-transplantation of ovarian tissue versus 49 women who thawed vitrified oocytes (18). the researchers found a trend towards higher pregnancy and lbr following use of thawed oocytes (rr 1.31, 95% ci 0.90–1.92; rr 1.39, 95% ci 0.95–2.03, respectively) (18), although it did not reach statistical significance. future developments gonadal tissue re-transplantation is the currently feasible way to regain fertility following cancer treatment in women that have cryopreserved ovarian tissue and developed ovarian insufficiency. since the first report of a live birth following ovarian tissue re-transplantation in 2004 (61), an increasing number of transplantation procedures have been reported by large fp programmes, and the number of live births following these procedures is increasing (14–22,62–65). however, re-transplantation may be precluded in cases where there is a high risk of reintroducing malignant cells. maturing gametes in vitro will be a probable option for these patients in the future. the methods are currently functioning in animal models, and experiments using human ovarian tissue donated for research have also reported success in follicle growth and maturation (65–68). nevertheless, there are not yet reported human studies of fertilization of in vitro grown oocytes and subsequent embryo development. because research is ongoing and advances are expected, even patients with malignancy compromising the gonads could be offered fp through gonadal tissue cryopreservation, with the aim to wait for the development of in vitro methods that could result in a fertility treatment in the future. this is also the case of prepubertal figure 2. oncologic indications for sperm banking in a cohort of adolescents and young adults at karolinska university hospital 1988–2018 (n ¼ 1393). mds: myelodysplastic syndrome. 116 k. a. rodriguez-wallberg et al. boys, for whom there are not yet methods developed. there is international consensus that fp through gonadal tissue cryopreservation could be offered only within ethical review board-approved research protocols (1,2,23,32–34,66,69). for prepubertal males for whom only spermatogonia can be preserved but mature sperm are not present, there is also a need of further development of methods to obtain fully mature gametes in vitro or through re-transplantation (23–30,68–71). acknowledgements the personnel at the departments of reproductive medicine, oncology, pediatric endocrinology and pediadric oncology of karolinska university hospital are gratefully acknowledged. disclosure statement no potential conflict of interest was reported by the author(s). funding the swedish cancer society, the swedish childhood cancer fund, radiumhemmets forskningsfonder, stockholm county council, and karolinska institutet. notes on contributors kenny a. rodriguez-wallberg, md, phd, associate professor and associate senior lecturer at the department of oncology-pathology of karolinska institutet; senior consultant at the department of reproductive medicine, karolinska university hospital. amandine anastacio, phd, postdoctoral fellow at the department of oncology-pathology of karolinska institutet, stockholm, sweden. emelie vonheim, md, karolinska institutet and university hospital. sandra deen, md, karolinska 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follicles grown in a multi-step culture system. mol hum reprod. 2018;24:135–42. doi: 10.1093/molehr/gay002 69. lee sj, schover lr, partridge ah, patrizio p, wallace wh, hagerty k, et al. american society of clinical oncology recommendations on fertility preservation in cancer patients. jco. 2006;24: 2917–31. doi:10.1200/jco.2006.06.5888 70. goossens e, van saen d, tournaye h. spermatogonial stem cell preservation and transplantation: from research to clinic. hum reprod. 2013;28:897–907. doi:10.1093/humrep/det039 71. oliver e, stukenborg jb. rebuilding the human testis in vitro. andrology. 2019. doi: 10.1111/andr.12710 120 k. a. rodriguez-wallberg et al. https://doi.org/10.1093/humupd/dmp056 https://doi.org/10.1093/humupd/dmp056 https://doi.org/10.1016/j.fertnstert.2013.03.043 https://doi.org/10.1093/molehr/gay002 https://doi.org/10.1200/jco.2006.06.5888 https://doi.org/10.1093/humrep/det039 https://doi.org/10.1111/andr.12710 abstract introduction development of clinically established and experimental methods for fp: first sperm, thereafter embryos, and then oocytes and gonadal tissue why oncologic treatment is an indication for undergoing fp banking of frozen sperm oncologic indications for sperm cryopreservation in the karolinska cohort cryopreservation of embryos, oocytes, and ovarian tissue oncologic diagnoses in females attempting fp in the karolinska cohort utilisation rates of sperm, oocytes, and embryos and efficacy of fp in the long term future developments acknowledgements disclosure statement references tf-iups150017 77..80 upsala journal of medical sciences, 2016 vol. 121, no. 2, 77–80 http://dx.doi.org/10.3109/03009734.2015.1109012 research article claes hellerström and cartesian diver microrespirometry michael welsh department of medical cell biology, uppsala university, uppsala, sweden abstract cartesian diver microrespirometry was introduced by claes hellerström at the department of histology/ medical cell biology at uppsala university, sweden, to determine rates of oxygen consumption in islets of langerhans. the theory behind this method is touched upon and the main findings described. glucose-stimulated beta cell respiration significantly contributes to increased atp generation, which is a prerequisite for stimulated insulin secretion and synthesis. this has had major implications for understanding the beta cell stimulus–secretion coupling. article history received 8 october 2015 accepted 12 october 2015 published online 18 november 2015 keywords cartesian diver, glucose, insulin secretion, islets of langerhans, respiration introduction the majority of our atp production derives from respiration and oxidative phosphorylation. it is thus essential for research on cell metabolism to have access to techniques accurately determining oxygen consumption. the cartesian diver technique was developed in the 1930s and 1940s at the carlsberg laboratory in copenhagen to assess rates of oxygen utilization (1,2). the technique was at that time unsurpassed in sensitivity, allowing accurate measurements of respiration in samples of 10 mg living tissue or less. the name, cartesian diver, was inspired by its similarity to a device putatively developed by cartesius (rené descartes) for demonstrating buoyancy and ideal gas law. accordingly, a small gas-filled glass vessel sinks or floats in a fluid-filled sealed container depending on the external pressure applied. without external pressure, the capillary vessel with gas floats. applying pressure to the sealed container compresses the gas phase in the capillary vessel and thereby increases its density by forcing liquid into it, thus making it sink. although a tool for demonstrating a scientific principle, it had an entertaining value and became a popular toy for distraction among those who could afford it. cartesian diver theory the simple principle of this procedure is that the density of a capillary vessel having an enclosed gas space increases as oxygen is consumed by the biological sample, and oxygen consumption rates can be calculated by monitoring this change in density. the apparatus consists of a capillary vessel holding the biological sample in a suitable buffer solution, a gas phase, and an oil seal at the top to prevent gas diffusion in and out of the vessel (2). the vessel (cartesian diver) is placed in a closed container with a fluid of a given density that is connected to a pressure manometer. the container sits in a water bath that is under precise temperature control. one critical element for the use of the cartesian diver is that there must be no co2 present in the compartment with respiring cells since production of co2 would offset o2 consumption. therefore the vessel will contain a drop of koh to absorb co2 in addition to a drop of buffer containing the respiring tissue in the closed gas compartment below the neck oil seal. a side-drop below the main drop containing respiring tissue may be introduced as well. the side-drop can be mixed with the tissue-containing drop at a given moment, thus allowing a change in the composition of the buffer of the respiring cells. that experimental design allowed the discovery of the stimulatory effect of glucose on beta cell respiration. applying pressure to the sealed container with the diver will make the diver sink to the bottom. when reducing the pressure by adjusting the manometer, a state can be achieved at which the diver is buoyant at exactly the same position inside the container. thus, the density of the diver at such a state of equilibrium will be the same as that of the liquid in the outside container. the manometer recording will reveal the outside pressure required to achieve the same density of the diver as that of the fluid in the container. when the living cells respire, the enclosed gas compartment below the neck oil seal will decrease due to oxygen consumption occurring without parallel release of gaseous co2 due to its absorption in the koh drop. this will cause liquid from the outside container to enter the top of the diver (above the oil seal), making the diver density go up. for the next recording, less pressure must be applied to the outside container by the manometer. the recorded value of the manometer will be different from the previous one, and subsequent recordings will give a plot that indicates the respiratory rate with time. figure 1(c) shows a schematic view of the experimental setup. the cartesian diver is a capillary glass tube with an inner diameter of less than 1 mm. the foot is a solid glass structure with a drop of koh on top. further above is a side-drop that can be mixed with the drop above containing the islets (or contact michael welsh michael.welsh@mcb.uu.se department of medical cell biology, box 571, husargatan 3, 75123, uppsala, sweden � 2015 taylor & francis other micro tissues) in a co2=hco � 3 -free buffer. above is a drop of oil sealing the compartment below, and above the oil seal there is a third gaseous compartment. its volume will vary depending on the applied pressure from outside (the manometer) and the oxygen consumption by the islets. above the top gaseous compartment at the mouth of the diver there is fluid entering from the outer container (figure 1c). the outer container has been sealed and is only in contact with a manometer (figure 1b) that allows applying pressure on the container with the diver. depending on the pressure applied, the diver will sink or float, and the change in buoyancy due to oxygen consumption can thus be recorded at equilibrium at exactly the same position of the diver in the container. for this purpose, a stereomicroscope is used (figure 1b). claes hellerström and the cartesian diver there were several parallel developments that prompted hellerström to adopt the cartesian diver technique from the carlsberg laboratory in the 1960s. one highly debated issue at that time was whether glucose stimulated insulin secretion via a ‘glucoreceptor’ or via some intermediate linked to glucose metabolism. furthermore, the discovery of the microdissection technique using ob/ob mice allowed preparation of viable beta cell-enriched islet material that could be used for functional and biochemical analysis (3). finally, bo hellman and sven brolin with co-workers developed tools to analyze islet material for metabolic enzyme activities and metabolic intermediate levels, including those of atp (4–7). these analyses required complementary determinations of oxygen consumption, and thus times were ready for establishing the cartesian diver technique at the department of histology (now medical cell biology) at uppsala university. the apparatus constructed is shown in figure 1(b). it contained a water bath that kept a very constant temperature of 37 �c. at the top front there were six containers (one is indicated by a star) containing the floatation fluid and divers. these were sealed and at their tops connected with a manometer (right, arrow) that could be used to adjust the pressure applied on the containers and thus the divers. there was also a stereomicroscope (marked by a lightning-bolt in figure 1b) allowing accurate determination of the position of the diver when buoyant in the outer container and thus having the same density as that of the container’s fluid density at the given applied pressure via the manometer. findings the important initial finding was that islet oxygen consumption increased when the glucose concentration of the incubation buffer was increased (8,9). this indicates that glucose not only increases the atp/adp ratio but also atp generation, a finding that was considered intriguing at the time. the prevailing idea was that atp/adp ratios were autoregulated and if atp utilization increased (drop in atp/adp) this would increase oxidative phosphorylation and respiration, whereas if atp/adp increased, this would inhibit respiration. this apparent peculiarity of beta cell glucose metabolism seems to have several metabolic explanations but nevertheless is of paramount importance for understanding the coupling between glucose metabolism and exocytosis. thus, in extension of these important observations of glucose and beta cell metabolism, the atp-dependent k+-channel was discovered, regulating the beta cell membrane potential and thus [ca2+]idependent exocytosis (10). other metabolic substrates found to increase islet oxygen consumption were citrate, oxaloacetate, d-mannose, d-fructose, l-leucine, 2-ketoisocaproic acid, and 2-aminonorbornane-2-carboxylic acid (bch) (11–19). the fact that bch stimulated islet oxygen consumption was particularly intriguing since it was a non-metabolizable leucine analogue and thus its insulin secretory effects were initially considered to support a receptor model for insulin secretion. the observed bch-stimulation of islet oxygen consumption contradicted this notion but argued that increased metabolism was the cause of insulin secretion also in this setting. the increased metabolic flux was eventually found to depend on allosteric activation of glutamate dehydrogenase, which thereby fuels the tricarboxylic acid cycle and mitochondrial oxidative metabolism (20). consequently, no discrepancy remained between glucose/amino acid metabolism and insulin secretion. willy malaisse presented the fuel hypothesis of glucosestimulated insulin release in 1979 (21). during the course of finalizing this herculean effort he and abdullah sener became interested in islet microrespirometry. at that time i was a young phd student at the department of medical cell biology, and hellerström introduced me to this technique. this he did in a very friendly and inspiring manner, and in concert with willy malaisse’s interest in islet oxygen consumption determinations i found myself in a very productive and friendly collaboration with claes hellerström, willy malaisse, and abdullah sener, in which we (hellerström and i) were able to make small contributions to certain aspects of the ‘fuel hypothesis’. i have very fond memories of those collaborative experiments occurring early in my career. the hypothesis was exciting, the technique—albeit ‘old-fashioned’—challenging and exhilarating, and the environment intellectually stimulating. my involvement in the collaboration between claes hellerström and willy malaisse had a major impact on my future development as a scientist. subsequent developments in the field as mentioned above, the field exploded with the characterization and cloning of the atp-dependent k+-channel, which controls the beta cell membrane potential and thus ca2+dependent exocytosis (10). a molecular link between glucose metabolism and insulin granule exocytosis was thus established. a key enzyme relevant for glucose utilization can be found in glucokinase (22). consequently, genetic changes perturbing beta cell glucokinase activity will result in disturbed glucose tolerance or maturity-onset diabetes in the young (23). a significant contribution of beta cell mitochondrial oxidative metabolism to the coupling between ambient glucose and increased atp production has also been established (24). curiously, glucose does not only stimulate insulin secretion via its metabolism but also via a true ‘glucoreceptor’, the sweet taste receptor t1r3 expressed on the beta cell plasma 78 m. welsh figure 1. a: person actively recording respiration by cartesian diver microrespirometry (ms ing-britt hallgren). b: apparatus at the department of histology/medical cell biology, uppsala university, sweden. the star indicates one of six containers with floatation medium that would contain the divers during experimentation. the arrow indicates the manometer that was in contact with the containers. the system was sealed so pressure could be applied to the container while simultaneously recorded. the lightning-bolt indicates the stereomicroscope that accurately could detect the position of the diver when in buoyant equilibrium. c: schematic view of container containing floatation medium and diver. d: original protocol showing manometer recordings with time at equilibrium, representing the change in pressure required to compensate for the consumption of oxygen. three divers with human islets were maintained in a glucose-free medium until stimulated with 16.7 mm glucose by mixing with the side-drops (arrows). respiratory rates could be determined in relation to islet mass (weight) after retrieval of islets at the end of the experiment. upsala journal of medical sciences 79 membrane (25,26). this reinforces the complexity of the stimulus–secretion coupling in beta cells involving both cell surface receptor and metabolic events. a recent development has been the introduction of technology from seahorse bioscience using the xf analyzer to determine accurately the oxygen consumption and mitochondrial coupling in beta cells (27,28). accordingly, oxygen-sensing fluorophores are employed to record oxygen concentrations, and thus changes in respiration with or without cell permeabilization can be assessed. despite the dramatic increase in our knowledge of the underlying events coupling glucose metabolism with insulin secretion, major gaps in knowledge still exist. for example, what processes can explain the increased atp consumption in glucose-stimulated beta cells? these could be biosynthetic needs, changes in ion fluxes, metabolic turnover—particularly in that of lipid metabolism—and the exocytotic process as such. however, no comprehensive effort to assess the relative importance of these possibilities has been made. another lingering enigma is the cause and consequence of the metabolic defects commonly observed in type 2 diabetic beta cells. recent focus has been targeted on understanding the regulation of beta cell proliferation and the exocytotic process, but our gain in understanding disturbances in beta cell metabolism occurring in type 2 diabetes is limited. potential aberrations could lie in glycolysis, mitochondrial metabolism, and lipid metabolism, but our present knowledge is incomplete and would clearly benefit from future studies elucidating this topic (29,30). acknowledgements the author is grateful to professors arne andersson, bo hellman, anders tengholm, and nils welsh for insightful comments. declaration of interest the author reports no conflicts of interest. the author alone is responsible for the content and writing of the paper. the study was supported by the swedish diabetes fund and by the family ernfors fund. references 1. linderstrom-lang k. on the theory of the cartesian diver microrespirometer. cr lab carlsberg, ser chim. 1943;24:334–98. 2. holter h. technique of the cartesian diver. cr lab carlsberg, ser chim. 1943;24:399–478. 3. hellerstroem c. a method for the microdissection of intact pancreatic islets of mammals. acta endocrinol (copenh). 1964;45:122–32. 4. wettermark g, borglund e, brolin se. a regenerating system for studies of phosphoryl transfer from atp. anal biochem. 1968;22: 211–18. 5. andersson a, borglund e, brolin s. effects of glucose on the content of atp and glycogen and the rate of glucose phosphorylation of isolated pancreatic islets maintained in tissue culture. biochem biophys res commun. 1974;56:1045–51. 6. hellman b, idahl la. [control of atp levels in stimulated pancreatic bcells]. acta diabetol lat. 1969;6(suppl 1):597–611. 7. hellman b. methodological approaches to studies on the pancreatic islets. diabetologia. 1970;6:110–20. 8. hellerstrom c. oxygen consumption of isolated pancreatic islets of mice studied with the cartesian-diver micro-gasometer. biochem j. 1966;98:7c–9c. 9. hellerstrom c. effects of carbohydrates on the oxygen consumption of isolated pancreatic islets of mice. endocrinology. 1967;81: 105–12. 10. rorsman p, braun m. regulation of insulin secretion in human pancreatic islets. annu rev physiol. 2013;75:155–79. 11. hellerstrom c. effects of glucosamine on the respiration of pancreatic islet b-cells. acta endocrinologica. 1968;58:558–64. 12. hellerström c, westman s, marsden n, turner d. oxygen consumption of the beta-cells in relation to insulin release. the structure and metabolism of the pancreatic islets ed. oxford: pergamon press; 1970. 13. hellerstrom c, gunnarsson r. [bioenergetics of islet function: oxygen utilization and oxidative metabolism in the beta-cells]. acta diabetol lat. 1970;7(suppl 1):127–58. 14. malaisse wj, sener a, malaisse-lagae f, welsh m, matthews de, bier dm, et al. the stimulus-secretion coupling of amino acid-induced insulin release. metabolic response of pancreatic islets of l-glutamine and l-leucine. j biol chem. 1982;257:8731–7. 15. malaisse wj, sener a, welsh m, malaisse-lagae f, hellerstrom c, christophe j. mechanism of 3-phenylpyruvate-induced insulin release. metabolic aspects. biochem j. 1983;210:921–7. 16. malaisse-lagae f, welsh m, lebrun p, herchuelz a, sener a, hellerstrom c, et al. the stimulus-secretion coupling of amino acidinduced insulin release. secretory and oxidative response of pancreatic islets to l-asparagine. diabetes. 1984;33:464–9. 17. sener a, welsh m, lebrun p, garcia-morales p, saceda m, malaisse-lagae f, et al. mechanism of 3-phenylpyruvate-induced insulin release. secretory, ionic and oxidative aspects. biochem j. 1983;210:913–19. 18. hellerstrom c, andersson a, welsh m. respiration of the pancreatic b-cell: effects of glucose and 2-aminonorbornane-2-carboxylic acid. horm metab res suppl. 1980;(suppl 10):37–43. 19. welsh m, hellerstrom c, andersson a. respiration and insulin release in mouse pancreatic islets. effects of l-leucine and 2-ketoisocaproate in combination with d-glucose and l-glutamine. biochim biophys acta. 1982;721:178–84. 20. sener a, malaisse wj. l-leucine and a nonmetabolized analogue activate pancreatic islet glutamate dehydrogenase. nature. 1980;288:187–9. 21. malaisse wj, sener a, herchuelz a, hutton jc. insulin release: the fuel hypothesis. metabolism. 1979;28:373–86. 22. garfinkel d, garfinkel l, meglasson md, matschinsky fm. computer modeling identifies glucokinase as glucose sensor of pancreatic betacells. am j physiol. 1984;247:r527–36. 23. vionnet n, stoffel m, takeda j, yasuda k, bell gi, zouali h, et al. nonsense mutation in the glucokinase gene causes early-onset noninsulin-dependent diabetes mellitus. nature. 1992;356:721–2. 24. sharoyko vv, abels m, sun j, nicholas lm, mollet ig, stamenkovic ja, et al. loss of tfb1m results in mitochondrial dysfunction that leads to impaired insulin secretion and diabetes. hum mol genet. 2014;23:5733–49. 25. malaisse wj. insulin release: the receptor hypothesis. diabetologia. 2014;57:1287–90. 26. nakagawa y, ohtsu y, nagasawa m, shibata h, kojima i. glucose promotes its own metabolism by acting on the cell-surface glucosesensing receptor t1r3. endocr j. 2014;61:119–31. 27. fred rg, kappe c, ameur a, cen j, bergsten p, ravassard p, et al. role of the amp kinase in cytokine-induced human endoc-betah1 cell death. mol cell endocrinol. 2015;414:53–63. 28. andersson le, valtat b, bagge a, sharoyko vv, nicholls dg, ravassard p, et al. characterization of stimulus-secretion coupling in the human pancreatic endoc-betah1 beta cell line. plos one. 2015;10: e0120879. 29. zou cy, gong y, liang j. metabolic signaling of insulin secretion by pancreatic beta-cell and its derangement in type 2 diabetes. eur rev med pharmacol sci. 2014;18:2215–27. 30. halban pa, polonsky ks, bowden dw, hawkins ma, ling c, mather kj, et al. beta-cell failure in type 2 diabetes: postulated mechanisms and prospects for prevention and treatment. diabetes care. 2014;37:1751–8. 80 m. welsh claes hellerstr&ouml;m and cartesian diver microrespirometry introduction cartesian diver theory claes hellerstr&ouml;m and the cartesian diver findings subsequent developments in the field acknowledgements declaration of interest references upsala j med sci 105: 3 140,2000 morphologic conversion of helicobacter pylori from spiral to coccoid form scanning (sem) and transmission electron microscopy (tem) suggest viability roger willcn,' birgitta carlcn,2 xin wang," nicos papadogiannakis? rolf odselius3 and torkel wadstrom4 'department of pathology, sahlgrenska university hospital s-413 45 goteborg, sweden, 2department of pathology, 'electron microscopy and 4clinical microbiology lund university hospital and 5departnzent of pathology, huddinge university hospital abstract helicobacter pylon is a pathogen associated with type b gastritis, peptic ulcer disease, gastric atrophy, and stomach cancer. h. pylori exists in two morphological forms, spirals and coccoids. the latter has been described as viable but non-cultivable. the role of the coccoid form in the pathogenesis of gastric disease is disputed. some authors consider the coccoid form to be a degenerative or dead form of h.pylori, while others consider it a resting but still metabolically active form. this study reports the conversion fiom spiral to coccoid form ultrastructurally. dense material is accumulated in the periplasmic space, the spiral bacteria bend and the outer membrane is separated from the inner cell wall layer. remodeling of inner structures takes place, ending in the coccoid form of the bacteria with preserved light polyphosphate areas. reduction of surface takes place by production of surface membrane vesicles, which later are squeezed off. the finding of preserved subcellular structures +d intact double membranes in combination with degenerative forms suggests that some of the coccoids are viable. scanning electron microscopy (sem) demonstrates coccoid form of bacteria with slightly ruffled surfaces but no spiral forms. introduction helicobacter pylon is a human pathogen associated with type b gastritis, peptic ulcer disease and gastric atrophy and stomach cancer [32]. an oral challenge with h.pylori caused an infection in human volunteers, monkeys, gnotobiotic pigs, nude and euthymic mice inducing histopathological changes similar to natural type b gastritis in man [32]. 31 h. pylori exists in two morphological forms, spirals and coccoids. the latter have been described as "viable but non-cultivable"(vbnc). the coccoid form of h. pylori may be viable and can revert to cultivable forms in mice [7, 321 but are no longer cultivable on conventional media [3, 5, 9, 10, 16, 301. the role of the coccoid form in pathogenesis of h. pylori-associated gastritis has been disputed. some authors consider the coccoid form as a degenerative or dead form of h. pylori [ 16,21,25], while others consider it as a resting but still metabolic active form [2, 3, 12,22, 29, 321. the aim of this study was to demonstrate the conversion of spiral form of h. pylon to coccoid forms, and to show both degenerative and intact subcellar morphology suggesting that some of the coccoid bacteria may be viable. material and methods bacterial strains and culture h. pylori strain 553193 was a freshly isolate from human gastric biopsies. the strain was grown on gab-camp agar supplemented with horse serum, 10% and incubated for 48 h at 37" c under microaerophilic conditions to obtain a high yield of spiral shaped h.pylori [32]. to obtain viable but non-cultivable h.pylori bacteria, 3-5 day-old agar cultures were harvested and resuspended in 20 ml of ham's f12 medium supplemented with 10% calf serum (flow laboratories, irvine), and incubated in a micro-aerobic environment for 3 days at 37" c and then stored at 4°c. if no growth was observed arer incubation for 5 days on gab-camp agar at 37"c, harvested cells were defined as viable but non-cultivable [32].all bacteria were analyzed for the presence of spiral forms in the coccoid suspension. no spiral forms were found. transmission electron m i c r o s c o p y m ) during the spiral to coccoid conversion period samples were taken and immediately fixed in 2%glutaraldehyde, in 0.1 m sodium cacodylate buffer (ph 7.2), postfixed in 2% osmium tetroxid, in s-colloidine buffer (ph 7.2), dehydrated in graded ethanol and embedded in agar resin 100. semithin section was cut and examined by light-microscopy. a representative area was chosen and ultra-thin sections, 50 nm were cut on a reichert-jung super nova ultramicrotome and contrasted with uranyl acetate and lead citrate. the grids were examined in a philips cm 10 transmission electron microscope at 60 kv. 32 scanning electron microscopy ( s e m ) coccoid helicobacter cells were placed i n a fixative solution containing 2.5%(wt/vol.) glutaraldehyde for 24 h at 22" c. the preparations were rinsed 3 times in soerensen's phosphate buffer, and then dehydrated in a graded alcohol series ending with 99% ethanol containing a molecular sieve, and then critical point-dried in liquid carbon dioxide. the specimens were mounted on metal stubs and sputtered in a polaron e 5400 sputter coated with gold for sem. each specimen was studied in a philips sem 515 unit at a magnification of x 312 to x 5700.photographs of representative findings were obtained. results sem showed rounded coccoid h. pylori after 5 days of cultivation on gab-camp agar. no spiral shaped h. pylori were detected. the coccoids were seen in clusters and some debris was noted (fig 1). fig 1. clusters of coccoid bacteria after 5 days cultivation on gab-camp agar with rounded contours and some debris. no spiral forms are detected. sem x 5,700 (original magnification). in tem transformation of the spiral form of h.pylori to coccoid form occurred via an intermediate u-shaped form. it is initiated by ingrowth of the periplasmic space on one side of the bacteria separating the double membrane into two membranes (fig 2).accumulation of cell dense material could be seen. later the u-shaped form became predominant with enhancement of electron dense material (fig 3). transection of such u-bent bacteria yielded two forks with a light space in-between. the membranes were separated into an inner and outer part, where the forks were either covered by a single layer at the center and double layer at the periphery (fig 4). sometimes at this development stage a part of the spiral form was clearly visible while the other part became more diffuse (fig 5 ) . 31001 26 33 fig 2 fig 3 fig 2.h.pylori spiral form with separation of inner and outer membrane by the periplasmic space with accumulation of dense granular material (arrow). tem 105,0oo(original magnification). fig 3.u-formed spiral bacteria. note cytoplasmic vesicles, polar density and light areas, supposedly polyphosphate-rich areas. diameter 1.34 pm. tem x 21,000 (original magnification). fig 4 fig 5 fig 4.transection of u-bent h.pylori spiral form with a clear space between forks. note remains of inner membranes around light and dense structures. tem x 52000 (original magnification). fig 5.partly formed coccoid h.pylori. condensation of dense material in one part of the bacteria, double membranes and shedding of cytoplasmic vesicles. tem x 52000 (original magnification). 34 fig 6 fig 7 fig 6.nearly finished h.pylori coccoid. note light areas and cytoplasmic vesicles sometimes containing dense material. tem x 52,000 (original magnification). fig 7.fully developed h. pylori coccoid. note double membranes, dense and light areas and part of a flagella (arrow). tem x 105,000 (original magnification). fig 8 fig 9 fig 8.mixture of transected spiral h. pylori (arrow) (diameter 0.41-0.45 pm) and coccoid forms (arrow head) (1.04-1.16 pm). tem x 15,500 (original magnification). fig 9.coccoid h. pylori with morphologically clear degeneration and fragmentation of membranes and central dense area. tem ~73,000 (original magnification). 35 the electron dense material became gradually more rounded in the center of the coccoid-like structure. the center also contained light spaces, supposedly holding phosphate-rich material. during the transformation many cytoplasmic vesicles were produced, sometimes squeezed in the space outside the coccoid bacteria (fig 6). the transformation strongly suggests an dynamic process. the double membrane system remained but periplasmic dense material disappeared subsequently (fig 7). sometimes flagella-like structures were seen in connection with the fd!y developed coccoid form (fig 7). while transection of the spiral form was between 0.4-0. 8 pm, the coccoid form demonstrated a diameter of 0.9-1.4 pm (fig 8). a progressive loosening and detachment of the outer membrane vesicles accompanied this transformation. degenerated coccoid bacteria were also found with fragmentation of membranes and disruption of centrally dense material (fig 9 ). discussion the mode of transmission of h.pylori from man to man is still unclear. two theories exist: one oral-oral and one oral-fecal transmission through contaminated water [6, 1 1, 171. lately also aniatrogenic spread through the gastroscope and vectors like cats and houseflies has been suggested [6]. the spiral form is very sensitive for air drying and therefore the coccoid form of h.pylori has been suggested to represent the transmission form. therefore much efforts have been put into its characterization. the coccoid form is non-cultivable on conventional media and accordingly pro and cons regarding viability have been put forward. the problem of non cultivable but in water viable coccoid forms as vibrio, shigella, legionella, campylobacter and escherichia coli are well known in aquatic and marine environment [ 161. is the coccoid form of h.pylori also an example of this? in egg passages [ 171, a classical cultivation technique, no coccoid bacteria were growing. lately, however, andersen et al [ 11 found by supplementing base broth medium with 2 % newborn calf serum, mg2+, cu”, fez’, zn”, mn2’ and lysed human erythrocytes that cocci reverted to spiral forms at ph 8.0-8. 5 with recovery of urease activity. in order to find out whether and when certain coccoid h.pylori are viable a vast amount of work with many techniques has been used. in order to induce coccoid forms of h. pylon in vitro several methods were scrutinized. 36 different chemicals as bismuth, as well as bile acids, antibiotics, temperature, nutritients, oxygen tension, starvation and aging are some examples [3, 10, 15, 16,21,25]. some antibiotics seem t o create greater changes to the cell wall than bismuth salts and bile acids [25]. in vivo changes and transformation mechanisms are less well known. coccoid h.pylori bacteria seem to be relatively more common in the duodenal mucosa than in the stomach in humans, [26].several tem studies have analyzed the transformation of spiral h. pylon bacteria into a coccoid form [2-5, 8, 10, 121. sem-investigations are few [ i 5 1 . 0 ~ ~ tem findings of coccoid formations are in accordance with others [2-5, 8, 10, 121. dense material is accumulated in the periplasmic space, the spiral bacteria bend and the outer membrane is separated from the inner layer. remodeling of imer structures occurs and formation of a coccoid takes place. benaissa et al[2] described remaining polar membranes and invagination of surface membranes. we also found flagella structures, found by several other authors [3, 101, but denied by others [19,25,29]. the coccoid center seems t o contain light areas. bode et al [3] demonstrated that they contain polyphosphate-rich material by electron spectroscopic imaging, which is used for basic energy requirements. these light areas were found by us and others [2,4, 121. membranes were found to be intact in most coccoids but also degenerative forms were found [101. the formation of membrane vesicles is a striking finding. later in the coccoid development these vesicles are squeezed off. this might be a remodeling with reduction of surface for better survival, morphologic expression of detoxification mechanism or simply a degenerative phenomenon [21]. as the membrane vesicles seem to be a phenomenon characteristic of all coccoid formation we believe that this is a fbnctional rather than a degenerative event. the size of the coccoid seems to vary depending on the method used to produce them. bode et a1 [3] found a mini-form of 0.3 pm when using antibiotics. other methods yielded bacteria of the size 0.8-1.5 pm as judged by published photos [2,3, 101. our coccoid bacteria showed the diameter of 0.9-1.4 pm as measured by a special device installed inside the electron microscope which enabled us to measure the diameter already at the screen. sem showed clusters of coccoid bacteria with slight uneven surfaces. no spiral forms were detected after 5 days cultivation. clustering seems to be a feature in the human stomach [i21 as well as in vitro [ 151. marked differences between the spiral and coccoid form were discovered [8, 161 by chemical and physiological measurements as well as immuno-, dna, rna and atp-analyses. 3 1 a substantial amount of similarities were, however, discovered with intact or similar properties in both forms of h. pylori. thus, dna and rna were sometimes preserved , especially in younger coccoid cultures [16, 18,22, 301 with incorporation of brdu [31] as a sign of protein synthesis [31], a finding however denied by others [21]. some authors have reported decreased rna /dna content with non-random fragmentation but rarely nil-values [ 16,21,23]. levels of increased atp have also been found after addition of fresh medium [30]. specific cellular antigens sometimes remained [28] but a substantial modification in the cell wall of peptido glycans occurred [14]. immunoblot and rapd-fingerprinting report loss of 160 kd, 120 kd (vac a) and a 35 k d cell surface protein while most other protein bands were weakened or preserved at normal levels [25, 30, 311. adherence of h. pylori coccoid to gastric epithelia and glycocalyx was the same for cocccoid and spiral forms as was also staining for 8 types of lectins [27]. cole at al. [13] reported poor adherence to gastric epithelial cells with little or no interleukin-8 secretion. adhesion with pedestal formation was seen between h. pylori coccoid and epithelial cells [ 19, 3 11 as well as in a balb/ca mice h.pylori infection model [32]. plasminogen and lactoferrin bind to the coccoid form of helicobacter, and also to extracellular matrix protein considered to be an important mechanism for tissue adhesion [20]. moran [24] suggests that we can not exclude that two different types of h. pylori coccoid forms exist; one that is degenerative and one viable but not (at that time) cultivable and that the viable form later on can develop to a degenerative form. the possibility that a minority of bacteria, not detected by methods commonly used, can stay in coccoid or resting state can not be ruled out [ 161. this view might explain the contradictory findings present today regarding viability of the coccoid form of h.pylori. the advantage of tem is that single cells are analyzed compared to most other methods and that l l l y intact as well as degenerated bacteria can be recorded. we strongly believe that some of the coccoids are viable due to the fact that subcellar structures and intact membranes were found in our tem and sem studies. we were also able to see coccoid bacteria attached to gastric epithelia in balb/ca mice with subsequent development of gastritis after 16 weeks [32] .cellini et al. [7] observed the same in balb/c mice. this l r t h e r strengthens our view that the coccoid form of h. pylon is viable. 38 acknowledgement this study was supported by grants from the swedish medical research council (16 x 04723), royal physiographic society, lund and founds from the sahlgrenska university hospital, goteborg, sweden. the skilled photographic help of michael pettersson is greatly appreciated references 1. andersen, a. p., elliott, d. a,, lawson, m., barland, p., hutcher, v.b.& puszkin, e.g.: growth and morphological transformation of helicobacter pylori in broth media. j clin microbiol35: 2918-2922,1997. 2. benaissa, m.,babin, p., quellard, n., pezennect, l., cenatiempo, y.& fauchere, j. l.: chmges in he!icobacter pylori dtrastmcture end antigens during conversion fiom the bacillary to the coccoid form. infect immun 64:2331-2335,1996 3. bode, g., mauch, f.& .malferthheimer, p. :the coccoid forms of helicobacter pylori. criteria for their viability. epidemiol infect 1 1 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31.vijayakumaris, s., khin, m.m., jiang, b. & ho, b.: the pathogenic role of the coccoid form of helicobacter pylori. cytobios 82:25 1-260 ,1995. 32.wang, x., stureghd, e., rupar, r., nilsson, h.o., aleljung, p.a., carlen, b., willen,r.& wadstrom t: infection of balb ica mice by spiral and coccoid forms of helicobacter pylon. j. med. microbiol46:657-663 ,1997. 29.segal, e.d., falkow, s.& tompkin, l. s.: helicobacter pylon attachment to gastric cells offprint request to: roger willen, md , ph.d. department of pathology sahlgrenska university hospital s-413 45 , gothenburg, sweden. 40 (11) upsala j med sci 111 (2): 257–262, 2006 osteolipoma arising adjacent to the sternoclavicular joint. a case report 1yutaka yabe, 1jun kumagai, 1noriyuki koizumi, 1masanori kawamura, 2sadahide ono and 3masahito hatori 1department of orthopaedic surgery, iwate prefecture central hospital. 2department of pathology, iwate prefecture central hospital. 3department of orthopaedic surgery, tohoku university school of medicine, sendai, japan abstract a 45-year-old woman presented with one-year history of a mass on her chest. computed tomography and magnetic resonance imaging demonstrated a tumour near the sternoclavicular joint. the tumour was diagnosed as osteolipoma histologically after resection. osteolipoma is a rare tumour and this may be the first report of osteolipoma arising adjacent to the sternoclavicular joint. introduction lipomas are the most common soft tissue tumours and they occur in any location of the body [1, 2]. variants of lipoma have been described according to the type of tissue present: fibrolipoma, myxolipoma, myolipoma, angiolipoma, pleomorphic lipoma [3 ,4], spindle-cell lipoma [5], angiomyolipoma [6], diffuse lipomatosis [7], lipofibromatosis [8]. in contrast to the high incidence of lipomas, metaplastic formation of cartilage and bone is only rarely seen [9]. lipomas displaying osseous or chondrous structures are referred to as osteolipomas or chondrolipomas respectively [10]. we present a case of solitary osteolipoma arising adjacent to the sternoclavicular joint. 257 received 1 july 2005 accepted 12 july 2005 case a 45-year-old woman was referred to our hospital because of a firm tender mass near the left sternoclavicular joint. she had noticed a mass one year before. the mass had slowly increased in size and become painful. on physical examination, the mass was firm, oval round, smooth, 2.5x1.5cm in size, tender and slightly movable. plain radiography showed an ossified mass near the left sternoclavicular joint. computed tomography revealed an ossified component surrounded by a radiolucent zone close to the sternoclavicular joint. continuity between a tumor and underlying bone was not found (fig 1). magnetic resonance imaging revealed the presence of a mass adjacent to the sternoclavicular joint which had high signal intensity on both t1-weighted and t2weighted images with focal areas of low signal intensity on both images. bone scanning with 99m-tc showed no abnormal findings. the patient underwent excision of the mass. the mass was excised en bloc and composed of mature osseous component surrounded by mature fatty component (fig 2). there was no continuity between the mass and the adjacent bone. 258 fig 1. computed tomography revealing an ossified component surrounded by a radiolucent zone (arrow) close to the sternoclavicular joint. continuity between a tumour and the underlying bone was not found. fig 2. cut section of the gross specimen showing mature osseous component surrounded by mature fatty component. histologically, the major part of the lesion consisted of lobules of mature adipose tissue separated by strands of fibrous connective tissue. the adipocytes were uniform in size and shape. no lipoblasts were observed. matured lamellar bone islands with bone marrow were dotted. osteoblastic rimming around the trabecular bones was also observed. there was no nuclear atypia. the tumour was diagnosed as an osteolipoma (fig 3). following surgery, the symptom disappeared and there has been no recurrence of tumour to date. discussion lipomas with ossification are very rare variants of lipomas. in a series of 635 lipomas seen over a 5-year period only 6 cases with ossification were found [11 ,12]. in general, osteolipomas are usually located adjacent to the periosteum and are referred to as parosteal lipomas [11]. osteolipomas independent of bone tissue have been reported [13 ,14]. some authors differentiate osteolipomas from parosteal lipomas with exostosis [15]. in the present case, on operation there was no continuity between the osteolipoma and clavicle although the tumour location was close to the sternoclavicular joint. there are two main theories of the pathogenesis of osteolipoma. most researchers believe that the origin of adipose cells , chondroblasts and osteoblasts is from different 259 fig 3. microphotograph showing that the tumour is composed of lobules of mature adipose tissue separated by strands of fibrous connective tissue and dotted lamellar bone islands with bone marrow (x 20). types of undifferentiated mesenchymal cells [4 ,16]. the hypothesis is that the neoplastic transformation occurs in a mixture of several types that later differentiate into lipoblasts, chondroblasts, or osteoblasts and fibroblasts [4 ,16]. another hypothesis is that only the adipose cells undergo a neoplastic transformation , and that the cartilage and bone is produced by metaplasia of fibroblasts in chondroblasts or osteoblasts [4 ,16]. this case microscopically showed continuity between osseous and adipose tissues. this finding supports the second hypothesis. computed tomography is of great value in evaluating the osteolipoma [15]. in fact, it easily revealed the fatty density of the mass and the presence of the osseous elements [17]. in the present case, ct played a very important role in preoperative radiological examination. it clearly demonstrated an ossified component surrounded by a radiolucent zone corresponding to fatty component. at mri the tumour showed high signal intensity on t1-weighted and t2-weighted images corresponding to the fatty component with focal areas of low signal intensity on both images corresponding to ossified , calcified or fibrous component. it was difficult to evaluate ossification by mri. the presence of osseous component to imply osteolipoma was thought to be better evaluated by ct scans. the preferential sites of osteolipomas are head and neck, followed by extremities [15]. to the best of our knowledge, there has been no report of osteolipoma arising adjacent to the sternoclavicular joint previously. lipomas with osseous changes have the same prognosis as plain lipomas [10]. excision is the treatment of choice, and no recurrences have been reported [18]. references 1. robbins sl (1960) textbook of pathology. saunders philadelphia london 2. mentzel t , fleicher cd (1995) lipomatous tumours of the soft tissues. an update. virchows arch 427: 353-63 3. rosai (1996) j , ackerman’s surgical pathology . 8th ed st. louis: mosby 4. fujimura n , enomoto s (1992) lipoma of the tongue with cartilaginous change : a case report and review of the literature. j oral maxillifac surg 50 : 1015-1017 5. hattori k, hatori m, watanabe m, osanai t, kokubun s. (2004) scrotum-like protrusion of lipoma arising from the proximal thigh report of two cases. ups j med sci 109: 261-265. 6. hatori m. watanabe m, kokubun s. angiomyolipoma in the knee-a case report ups j med sci in press. 7. takamatsu k, hatori m, ehara s, kokubun s. (1997) diffuse lipomatosis: imaging features. tohoku j exp med 183: 151-157. 8. sasaki d, hatori d, hosaka m, watanabe m, kokubun s. lipofibromatosis arising in a pediatric forearm -a case reportups j med sci in press. 9. katzer b. (1989) histopathology of rare chondroosteoblastic metaplasia in benign lipomas. pathol res pract 184: 437-443 10. ec obermann , s bele , a brawanski , r knuechel , f hofstaedter(1999) ossifying lipoma. virchows arch 434 : 181-183 11. allen pw.(1981) tumors and proliferations of adipose tissue. masson new york paris barcelona. 260 12. plaut gs , salm r , truscott de(1959) three cases of ossifying lipoma. j pathol(lond) 78: 292-295 13. shneider j, swoboda r (1986) oropharyngeal lipoma with osseous metaplasia. zentrallbl allg pathol 133: 249-251 14. ohno y, muraoka m, ohashi y, nakai y, wakasa k (1998) osteolipoma in the parapharyngeal space. eur arch otorhinolaryngol 255: 315-317 15. blanshard jd , veitch d (1989) ossifying lipoma. j laryngol otol 103: 429-431 16. piattelli a , fioroni m , lezzi g , rubin c (2001) osteolipoma of the tongue. oral oncol 37: 468-470 17. f.minutoli , mazziotti , m gaeta , s vinci , a blandino(2001) ossifying lipoma of the parapharyngeal space: ct and mri findings. eur. radiol 11: 1818-1821 18. allard rhb , van der kwast wam , van der waal.(1982) oral lipomas with osseous metaplasia. report of two cases. j oral pathol 11: 18-25 corresponding author: masahito hatori, m.d., assistant professor department of orthopaedic surgery, tohoku university school of medicine 1-1 seiryomachi, aobaku, sendai, miyagi, japan 980-8574 tel: 81-22-717-7242, fax: 81-22-717-72481 email: mhato@mail.tains.tohoku.ac.jp 261 262 upsala j med sci 102: 175-184, 1997 image analysis of the duodenal endocrine cells in mice with particular regard to optical densitometry m. el-salhy and j. mahdavi section f o r gastroenterology and hepatology, department of medicine, university hospital, umed, sweden abstract the endocrine cells in the murine proximal duodenum have been investigated by means of immunohistochemistry and computerized image analysis. five endocrine cell types were identified, namely secretin-, gastric inhibitory polypeptide (gip)-, gastrin-cck-, somatostatinand serotonin immunoreactive cells. the number of endocrine cells/mm3 epithelial cells was estimated and the cell secretory index (csi) for different endocrine cell types was determined. furthermore, the optical density of the cellular immunoreactivity and the immunoreactive area in the cell were determined and an index, cell immunoreactivity content was estimated as the optical density multiplied by the immunoreactive area. it has been suggested that the use of this index might better reflect the cellular peptide/amine content than does the csi. serotonin immunoreactive cells were the predominant endocrine cell type, followed by gastrin/cck-immunoreactive cells. the numbers of secretin-, gipand somatostatin immunoreactive cells were almost identical. all endocrine cell types were present both in crypts and in villi, but were, more numerous in the crypts, except for secretin which was more frequent in the villi. introduction image analysis first appeared as a readily available technique in 1963 with the introduction of the quantitative television microscope (qtm). qtm was designed by metals research ltd and was intended for use in metallographical 175 laboratories. a n early application in the biological field was the measuring, of the size of air spaces in the damaged lung and counting of silver grains in autometallograph (7, 9,11,12). the rapid developments in computer hardware and software made computer image analysis an easy, rapid, precise and objective tool for determining the number and volume of biological structures (3-5, 8, 10). in computerized image analysis the image from video-cameras is digitized into picture elements (pixels). the grey level (i.e. the brightness) of all the pixels in the image can be measured in terms of light transmitted or reflected, or as an optical density value. in coloured images, measurements are taken on the individual colour components within the image. thus, computerized image analysis, in addition to traditional morphometry, makes it possible to measure the greyness or colour levels of a stained biological structure. the mouse is a n excellent experimental animal. the endocrine cells in the gut of this animal have not previously been subjected to detailed investigation. apart from somatostatin ( 6 ) and peptide y y (pyy) (1) in the small intestine, no other endocrine cell types have been examined in the gut of normal mouse. the aim of the present study was to quantify duodenal endocrine cells in mice and to use the opportunity offered by computerized image analysis, using optical densitometry to determine an index reflecting the cellular peptidelamine content. material and methods animals ten mice ( 5 females and 5 males) aged 3 months and with an average body weight of 30 g (bom: nmri strain, b/s bomholtgird breeding and research centre, denmark) were used. the mice were housed in cages, 5 mice in each cage, in a room with artificial light from 6 am to 6 pm. the animals were fed a standard pellet diet (astra-ewos ab, sodertdje, sweden) and allowed water ad 176 libitum. they were kept in the laboratory for 2 weeks prior to sacrifice in a c02 chamber, after an overnight fast. the investigation was approved by the local committee on animal ethics, umea university. immunohistochemistry after sacrificing the animals, the proximal part of the duodenum was removed and fixed overnight in 4% buffered formaldehyde and embedded in paraffin wax. the deparaffinized sections were rehydrated, and immersed in 0.01 % hydrogen peroxide in tris-hc1 buffer, ph 7.6 for 10 min in order to inhibit the endogenous peroxidase activity. to occupy the non-specific binding sites the sections were pre-treated with 1 % bovine serum albumin for 10 min. the sections were immunostained by the avidin-biotin complex (abc) method (dako a/s,glostrup,denmark)as described in detail previously (4). briefly, the sections were incubated for 20 h with one of the primary antisera (for details concerning the antisera used, see table 1). they were incubated with biotinylated swine anti-rabbit igg, diluted 1:200 for l h . the sections were then incubated for 1 h with avidin-biotin-peroxidase complex, diluted 1:loo. staining of the sections was performed in 50 ml tris buffer containing 10 pl of 30% h202 and 25 mg diaminobenzidine tetrahydrochloride (dab), for 15 min. the same abc-kit and biotinylated swine anti-rabbit igg was used throughout the study. the incubation was performed at 4°c. equal parts of avidin and biotinylated peroxidase were mixed with the tris-buffer and allowed to stand for 1 h at 4°c. specificity controls were the same as those described earlier (4). briefly, the primary antibodies were replaced by non-immune rabbit serum, and the primary antisera pre-incubated for 24 h with the corresponding or structurally related peptide (50-75 pg/ml diluted antibody) at 4°c. computerized image analysis the quantimet 500mc image processing and analysis system (leica, cambridge, england) linked to an olympus microscope type bx50 was used. the software used in this system was the leica windows-based image analysis tool kit "qwin" (version 1.02) and an interactive programming system quips (version 1.02). table 1. details of the antisera used. all antisera were raised in rabbit. *specific for ccwgastrin c-terminus. **specific for glucagon n-terminus and cross-reacts with both pancreatic and enteroglucagon. antibody against source working code no porcine secretin eurodiagnostica, malmo, 1: 1000 r-787502 dilution sweden bovine gastric eurodiagnostica 1:1600 r-786403 inhibitory peptide b2 ( g i r synthetic human eurodiagnos tica 1 : 10,000 r-783511 gastrin-17 * synthetic human dakopatts, glostrup, 1: 1600 a566 somatostatin denmark porcine pancreatic eurodiagnostica 1 : 2500 b-3 1 glucagon** porcine motilin eurodiagnostica 1 : 5000 r-842206 serotonin eurodiagnostica 1: 600 b 45-1 178 the number of endocrine cells and their secretory index were determined as described in detail previously (2)using an automated standard sequence analysis operation. measurements were made on 40 randomly chosen fields (20 from the crypts and 20 from the villi) for each peptide and animal. these fields were chosen from three to five sections, at least 50 pm apart. the sections were examined with x20 objective and each field observed in the monitor represented 0.034 mm2 area of the tissue. in quantimet 500mc the optical density is sampled and coded as a grey level value in the range 0 to 255. grey level measurements can be calibrated to known standards and quantimet 500mc utilizes a "look-up table" in which each grey level from 0 to 255 is related to its calibrated value. using quips, an automated standard sequence operation was created to measure the optical density in different endocrine cell types. the microscope illumination intensity was constant. the operating parameters of the ccd-video camera were kept constant during all the measurements performed in this study. ten nucleated cells were randomly chosen for each endocrine cell type and animal. quantification was undertaken with a x40 objective and each pixel corresponded to 0.206 pm. in this automated analysis process, the image was converted to a monochrome image (black and white) and the operator calibrated the grey level so that the section background staining was regarded zero. this was done in every section. the optical density was measured in the cell cytoplasm as mean grey, being equal to the sum of the optical density divided by the number of pixels measured, where sum of optical density is the sum of the calibrated optical density values for each pixel within the measured object. in addition to the mean grey, the immunoreactive area was measured in each cell. the cell immunoreactivity content (cic) was calculated as the mean grey for the cell, multiplied by the immunoreactive area in pm2. the data from each field were tabulated, computed and statistically analysed automatically. 179 fig. 1. secretin-immunoreactive cells in the villi (a), and gastridcck immunoreactive cells (b) in the crypts of proximal duodenum of a mouse. x350. 180 immunohistochernistry secretin(fig. l a ) , gastric inhibitory polypeptide (g1p)-, gastridcck(fig. i b), somatostatinand serotonin-immunoreactive cells were found both in the crypts and in villi, though they were more numerous in the crypts-except for secretin-immunoreactive cells, which occurred mostly in the villi. no glucagon or motilin-immunoreactive cells couid be detected in the examined proximal part of the mouse duodenum. the endocrine cells varied in shape, from flaskto basket-shaped and somatostatin-immunoreactive cells often showed a basal process. there was no immunostaining when the primary antibody was replaced by non-immune rabbit serum or when the primary antisera were pre-incubated with the corresponding antigen. pre-incubation of the primary antibody with a structurally related antigen had no effect on the immunostaining. computerized image analysis the results of image analysis of different endocrine cell types are summarized in table 2. it seems that the most abundant cell is serotonin-immunoreactive cell type followed by gastridcck-immunoreactive cells. the numbers of secretin .gipand somatostatin-immunoreactive cells are almost the same. discussion in a previous report (2) the cell secretory index (csi) was suggested a s a parameter reflecting the endocrine cell peptide/amine content, which is a summation of cellular peptide/ amine synthesis and release. csi represents volume of the cellular immunoreactive secretory granules. t h e cellular peptidelamine content is not only reflected by the immunoreactive secretory granule volume, but also by the intensity of immunostaining. in the present 181 table 2. the cells, the cell content (cic) number of various endocrine cell types per mm3 of epithelial secretory index (csi) mm3 per cell and cell immunoreactivity in the duodenum of mice (mean+se) endocrine cell type number of csi cic cells crypts 3060~498 81.lk_11.5 secretin villi 5078~3 14 16 1.693 1.8 total 4036k284 84.827.7 5334.8k3002.6 crypts 691 12443 77.8k4.6 gip villi 53 1 e 4 2 8 71.324.2 total 61 112343 75. e 3 . 3 2294.52 1429.4 crypts 10503~782 11 1 . e 7 . 3 gastrirdcck villi 9481~638 130.828. 5 total 9979~427 12125.8 1924.221633.5 crypts 64602264 83.3k6.1 somatostatin villi 5000k353 80.8k4.7 total 5737~178 83.3~5.1 6441.3k3336.3 crypts 44626k3075 142.522.3 serotonin villi 232462769 151.6k7.6 total 3480 1 ~ 1 9 0 1 142.223.5 1447.2~108.6 study the cic was suggested to be a more accurate parameter, reflecting the cellular peptide/amine content. in this parameter both the area and intensity of immunostaining are taken into account. it is worth noting, however, that the optical density measured here is a relative value, being related to the background staining of the section as zero. there are several possible sources of error in measuring the optical density of immunostaining: those caused by 182 immunostaining and those caused by the equipment used. use of different dilutions of the first, second and third layers, or with dab, as well as using different incubation times or temperatures, would result in different staining intensities. furthermore, amplification, when abc method is used, can vary if different kits, different biotinylated iggs, or different incubation times with avidin and biotinylated peroxidase are used. moreover, the optical density measurements are affected by the image brightness caused by the illumination of the microscope and by the operating parameters of the ccd-video camera. being aware of all these pitfalls and by standardizing all these factors, optical density measurements and cic can be useful in studies on endocrine cells, where comparison between the cell secretory contents in different groups is required. the present study revealed five endocrine cell types in the proximal duodenum of the mouse. these cell types are similar in appearance and distribution to those described in the human duodenum (4,13). the finding that somatostatin immunoreactive cells were more frequent in the crypts disagrees with an earlier report that they occurred mostly in the villi (6). acknowledgement this study was supported by grants from the medical faculty, umeg university and the familial amyloidosis association (famy). references 1. el-salhy, m.& lundqvist, m.: immunocytochemical investigation of polypeptide y y (pyy) and pancreatic polypeptide (pp) in the gastrointestinal tract of rodents. biomed res 5: 401-404, 1984. 2. el-salhy, m., sandstrom, o., nasstrom, e., mustajbasic, m.& zachrisson, s.: application of computer image analysis in endocnne cell quantification. histochem j 29: 2 49-256, 1977 3 . el-salhy, m.& suhr, 0.: endocrine cells in rectal biopsy specimens from patients with familial amyloidotic plyneuropathy. s a n d j gastroenterol 3 1:68-73, 1996. 4. el-salhy, m., suhr, o., stenling, r., wilander, e., grimelius, l.: impact of familial associated polyneuropathy on duodenal endocrine cells. gut 35: 14131418, 1994. 5. el-salhy, m., danielsson, a . , stenling, r.& grimelius, l.: colonic endocrine cells in inflammatory bowel disease. j intern med 342: 413-419, 1977. 183 6. evans, g.s.: the distribution of endocrine cells along mouse small intestine. bombesin v d somatostatin producing cells. virchows arch b cell pathol incl mol pathol 58: 165-72, 1989. 7. gonzalez, r.c. & wintz, p.: (1987). digital image processing. 2nd edn. addison-wesley, reading. 1987 8. kubota, y . , peters, r.e., ottaway, c.a., tubbs r.r., farmer, r.g. & fiocchi, c.: colonic vasoactive intestinal peptide nerves in inflammatory bowel disease. gastroenterology 102: 1242-1251, 1992. 9. rosenfeld, a. & kak, a.: digital picture processing. 2nd edn.academic press,new york, 1982. 10. ronnblom, a., danielsson, a . , el-salhy, m.: intestinal endocrine cells in myotonic dystrophy: an immunocytochemical and computed image analytical study. (submitted for publication) 11. russ, j.c.: computer assisted microscopy: the measurement and analysis of images. plenum pub1 co, new york, 1990. 12. serra ,j. (1984) image analysis and mathematical morphology,vol. 1. academic press, new york, 1984. 13. sjolund, k., sandtn, g., h s a n s o n , r. & sundler, f.: endocrine cells in human intestine. an immunocytochemical study. gastroenterology 85: 11201130, 1983. correspondence to: magdy el-salhy, md, phd, section for gastroenterology and hepatology, department of medicine, university hospital, s-901 85 urn&, sweden. tel: +46-90-7853867 fax: +46-90-120923. e-mail: magdy.el-salhy @ medicin.umu.se 184 vol_116_002_sups_a_551932 129..132 upsala journal of medical sciences. 2011; 116: 129–132 original article cervical myelopathy due to degenerative spondylolisthesis tomoaki koakutsu, junko nakajo, naoki morozumi, takeshi hoshikawa, shinji ogawa & yushin ishii department of orthopaedic surgery, nishitaga national hospital, sendai, japan abstract objective. to investigate clinical-radiological features of cervical myelopathy due to degenerative spondylolisthesis (dsl). methods. a total of 448 patients were operated for cervical myelopathy at nishitaga national hospital between 2000 and 2003. of these patients, dsl at the symptomatic disc level was observed in 22 (4.9%) patients. clinical features were investigated by medical records, and radiological features were investigated by radiographs. results. disc levels of dsl were c3/4 in 6 cases and c4/5 in 16 cases. distance of anterior slippage was 2 to 5 mm (average 2.9 mm) in flexion position. space available for the spinal cord (sac) was 11 to 15 mm (average 12.8 mm) in flexion position and 11 to 18 mm (average14.6 mm) in extension position; 11 cases were reducible and 11 cases were irreducible in extension position. myelograms demonstrated compression of spinal cord by the ligamentum flavum in extension position. compression of spinal cord was not demonstrated in flexion position. c5-7 lordosis angle was lower than control. c5-7 range of motion (rom) was reduced compared to controls. these alterations were statistically significant. conclusions. dsl occurs in the mid-cervical spine. lower cervical spine demonstrated restricted rom and lower lordosis angle. pathogenesis of cervical myelopathy due to dsl is compression of spinal cord by the ligamentum flavum in extension position and not by reduced sac in flexion position. key words: cervical spine, degenerative spondylolisthesis, myelopathy introduction cervical myelopathy results from compression of the spinal cord by various degenerative processes of the spine. kokubun et al. described the following seven spinal factors compressing the spinal cord: developmental stenosis, dynamic stenosis, disc herniation, segmental ossification of the posterior longitudinal ligament (opll), continuous opll, posterior spur, and calcification of ligamentum flavum. these factors were involved in 98% of cervical myelopathy cases (1). while it has long been suggested that degenerative spondylolisthesis (dsl) of the cervical spine may be a spinal factor (2), kokubun et al. (1) did not include dsl as a spinal factor. the number of reports on cervical spondylotic myelopathy due to dsl has not been low (3–7), but no general consensus has been reached on the onset mechanism of spinal cord compression. the purpose of this study is to investigate clinical-radiological features of cervical myelopathy due to dsl. patients and methods over a 4-year period from 2000 to 2003, 448 patients were operated for cervical myelopathy (excluding patients with rheumatoid arthritis, destructive spondylitis, and a past history of spinal trauma). their medical charts and plain lateral functional x-rays of the cervical spine were reviewed, and dsl at the neurologically responsible disc level was observed in 22 patients (4.9%) (dsl group). there were 13 men and 9 women, with a mean ± sd age at the time of surgery of 74.8 ± 7.3 years (range 58–88 years). of the correspondence: tomoaki koakutsu, department of orthopaedic surgery, emergency center, tohoku university hospital, 1-1 seiryo-machi, aoba-ku, sendai, miyagi 980-8574, japan. fax: +81-22-717-7492. e-mail: koakutsu@med.tohoku.ac.jp (received 15 december 2010; accepted 27 december 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2011.551932 patients who underwent surgery for cervical myelopathy during the same period, the same number of patients with dynamic stenosis (posterior slippage) or posterior spur as a spinal factor at the neurologically responsible disc level was randomly extracted as control (control group). for the control group, there were 14 men and 8 women with a mean ± sd age at the time of surgery of 67.3 ± 9.8 years (range 51–80 years). the mean age was significantly higher in the dsl group than in the control group (p = 0.0072, t-test). using plain lateral x-rays of the cervical spine which were taken with a tube-film distance of 1.8 m preoperatively, the following parameters were measured: (1) disc level of dsl. (2) distance of anterior slippage: the vertical distance between the posterior inferior angle of the slipped vertebra and the tangential line of the posterior surface of the caudal adjacent vertebra. (3) space available for the spinal cord (sac): the distance between the ventrolateral border of the vertebral arch of the slipped vertebra and the posterior superior angle of the lower adjacent vertebra. (4) c5-7 range of motion (rom): according to sasaki’s method (8), the difference in the intersection angle of the tangential line of the posterior margin of vertebrae between extension and flexion positions. (5) c5-7 lordosis angle: according to sumi’s method (7), on plain lateral x-rays, the angle formed by the tangential line of the superior margin of c5 and the tangential line of the inferior margin of c7 in the neutral position was measured; lordosis was defined as ‡0� and kyphosis as <0�. when accurate measurements were difficult because c7 overlapped the shoulder, c6 was used instead. parameters 4) and 5) were also measured for the control group and subjected to statistical analysis (t-test, hazard ratio of 1%). for the dsl group, myelography and ct after myelography (ctm) in extension and flexion positions were used to investigate the pathogenesis of spinal cord compression. results disc level of dsl disc level of dsl was c3/4 in 6 patients and c4/5 in 16 patients; anterior slippage was in the mid-cervical spine in all patients. distance of anterior slippage the average distance of anterior slippage was 2.9 ± 0.8 mm (range 2–5 mm) in the flexion position, 1.1 ± 1.1 mm (range 0–3 mm) in the neutral position, and 0.6 ± 0.7 mm (range 0–2 mm) in the extension position. in 11 patients (50%), anterior slippage was reduced in the extension position. seven patients (32%) also had developmental stenosis (anteroposterior diameter of the spinal canal £12 mm). space available for the spinal cord (sac) the average sac was 12.8 ± 1.2 mm (range 11–15 mm) in the flexion position and 14.6 ± 2.4 mm (range 11–18 mm) in the extension position. in the flexion position where distance of anterior slippage distance was the greatest, sac was minimum. c5-7 range of motion (rom) the average c5-7 rom was 7.3 ± 5.5� in the dsl group and 13.4 ± 7.0� in the control group. the rom was significantly smaller in the dsl group (p = 0.0028). c5-7 lordosis angle the average c5-7 lordosis angle was �0.4 ± 8.6� in the dsl group and 6.8 ± 5.9� in the control group. the c5-7 lordosis angle was significantly smaller in the dsl group (p = 0.0030). pathogenesis of spinal cord compression a typical dsl patient is presented in figure 1. in all patients in the dsl group, myelography and ctm did not show spinal cord compression in the flexion position where sac was minimum, but in the extension position where sac was maximum, spinal cord compression caused by the protruded disc and the ligamentum flavum was confirmed. discussion from antiquity, dsl has been known to cause spinal cord compression (2). sasaki (8) examined x-rays of the cervical spine of 500 healthy individuals in different age-groups and reported that age-related degeneration occurred most commonly in c5/6 and that c4/5 rom increased with age in a compensatory manner. hayashi et al. (5) also documented that age-related degeneration lowered the rom of c5/6 130 t. koakutsu et al. and c6/7 but increased the rom of c3/4 and c4/5 in a compensatory manner, resulting in vertebral slippage. in particular, they found that posterior slippage occurred in extension position, but they did not mention anterior slippage. sumi et al. (7) grouped cervical myelopathy patients into three groups based on c5-7 alignment (lordosis group, straight group, and kyphosis group) and confirmed c4 dsl in 41.2% of the straight group and 61.5% of the kyphosis group. suga et al. (6) reported that c4 dsl occurred in the elderly due to decreased c5-7 rom and forwardleaning body posture, and they emphasized the a b c d figure 1. an 84-year-old man with c4/5 myelopathy. a, b, and c: plain x-ray (a: flexion position, b: neutral position, and c: extension position). in the flexion position, 2-mm anterior slippage in c3 and 3-mm anterior slippage in c4 are demonstrated (a). in the extension position, c3 anterior slippage is reduced, and c4 anterior slippage is 1 mm (c). space available for the spinal cord at c4/5 in the flexion and extension positions is 12 and 15 mm, respectively. c5-7 range of motion and lordosis angles are 3� and 4�, respectively. d: mri showed spinal cord compression at c3/4 and c4/5. e and f: ct after myelography (e: flexion position and f: extension position). in the flexion position with maximum anterior slippage and osseous stenosis, deformation due to spinal cord compression is not confirmed (e). in the extension position with minimum anterior slippage and osseous stenosis, findings indicative of spinal cord compression from the posterior direction due to the buckling of the ligamentum flavum are confirmed (f). degenerative cervical spondylolisthesis 131 importance of c4 dsl in elderly patients with cervical myelopathy. in this study, the c5-7 rom and the c5-7 lordosis angle in the patients with dsl were significantly lower than in the patients with other spinal factors, and dsl occurred at c3 and c4. the above findings suggest that dsl occurs in mid-cervical spine due to decreased lordosis angle and rom of the lower cervical spine. as for the clinical state of spinal cord compression due to dsl, penning (3) and epstein et al. (4) stated that spinal cord compression was caused by osseous spinal canal stenosis of the vertebral arch of slipped vertebrae and the posterior superior angle of lower cervical vertebrae in the flexion position where slippage was the greatest. in this study, myelography and ctm did not show findings indicative of spinal cord compression in the flexion position where anterior slippage and osseous spinal canal stenosis were the greatest, but in the extension position, spinal cord compression by the invagination of the ligamentum flavum into the canal was seen without osseous spinal canal stenosis. spinal cord compression involving dsl was considered to be due to the buckling of the ligamentum flavum in the extension position irrespective of osseous stenosis. mri for suspected cervical myelopathy due to dsl should be performed in extension position because spinal cord compression may not be observed when the cervical spine is in the flexion position. in the present series, longterm surgical results have not been observed, but when taking into account the onset mechanism of spinal cord compression, posterior decompression (either laminectomy or laminoplasty) is rational. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. kokubun s, sato t, ishii y, tanaka y. cervical myelopathy in the japanese. clin orthop. 1996;323:129–38. 2. clarke e, little jh. cervical myelopathy; a contribution to its pathogenesis. neurology. 1955;5:861–7. 3. penning l. some aspects of plain radiography of the cervical spine in chronic myelopathy. neurology. 1962;12: 513–9. 4. epstein ja, carras r, epstein bs, levine ls. myelopathy in cervical spondylosis with vertebral subluxation and hyperlordosis. j neurosurg. 1970;32:421–6. 5. hayashi h, okada k, hamada m, tada k, ueno r. etiologic factors of myelopathy. a radiologic evaluation of the aging changes in the cervical spine. clin orthop. 1987;214: 200–9. 6. suga t, miyazaki m, akiyama k, yoshida s, mihara s. cervical spondylotic myelopathy in aged patients. importance of c4 anterolisthesis. rinshoseikeigeka. 2005;40: 653–63. (in japanese). 7. sumi m, kataoka o, sawamura s, ikeda m, mukai h. radiological analysis of cervical spondylotic myelopathy. rinshoseikeigeka. 1998;33:1277–86. (in japanese). 8. sasaki a. radiology of normal cervical spine. j jpn orthop assoc. 1980;54:615–31. (in japanese). e f figure 1. (continued). 132 t. koakutsu et al. ujms 02-07.pdf height loss in women caused by vertebral fractures and osteoporosis 213 received 1 december 2006 accepted 12 february 2007 upsala j med sci 112 (2): 213–219, 2007 height loss in women caused by vertebral fractures and osteoporosis robert eggertsen1, dan mellström2 1centre of bone research at the sahlgrenska academy department of medicine/primary health care and mölnlycke primary health care and research centre, 2department of medicine/geriatrics. göteborg university, sweden abstract background: to investigate women, with height loss of more than three cm, without earlier diagnosed spinal fractures, for osteoporosis and vertebral fractures. methods: consecutively enrolment of women aged 50–85 years with a height loss of three cm or more. 80 women with a mean age of 70 years old (51–84) were recruited. determination was made of bone mineral density (bmd) with dxa technique on hip, lumbar spine and whole body. lateral spine radiographs were performed on thoracic and lumbar spine. results: mean height loss was 5.2 cm. smaller vertebral fractures were diagnosed in 45% (n=36). all had osteopenia or osteoporosis on bmd measurements, and there were significant differences in t-score for hip and lumbar spine and in bmd, for the hip in subjects with and without vertebral compression. conclusions: women with height loss without knowledge of earlier spinal fractures could be suspected for osteoporosis and vertebral fractures introduction height loss is common in elderly people and is rarely investigated for osteoporosis unless symptoms of pain. elderly women, in sweden and norway have the highest known prevalence of vertebral and hip fractures (1,2). the life time risk of sustaining a clinically diagnosed vertebral fracture in swedish women is estimated to 15%, which is only a subset of all vertebral fractures revealed in a radiographic examination (3,4). vertebral fractures are often silent and gradually result in height loss with several centimetres without patients seeking medical advice. therefore this illness will be diagnosed late, and essential treatment could be delayed with high risk of new vertebral or other fragility fractures (5) vertebral fractures increase the risk for mortality and health consumption and might lead to decreased lung function, back pain, depression and decreased adl function, and increased fracture incidence in both men and women (6–8). fractures can arise with different degrees of shorter height. prevalence studies of vertebral fractures have shown frequencies of 20–30% in men and women with mean ages of 63 and 64 years (9) and of 25% in 85-year-old women (10). it is still unclear how usual this is in women with height loss without considerable symptoms, not being investigated and given the right diagnosis. 214 robert eggertsen and dan mellström the purpose with this study was to investigate women, with a history of height loss of three cm or more, for osteoporosis and vertebral fractures. we decided to make determinations with whole body dual-energy-x-ray absorptiometry (dxa), together with conventional x-ray of the spine in order to determine the prevalence of osteoporotic spinal fractures in this group. material and methods the investigation was performed in the area of mölnlycke primary health care centre comprising about 15000 inhabitants. there are about 2300 women between 50–85 years old living in the district and in this age group 100 were recruited with advertisements in the community press. all, that had decreased more than three cm in height since they were 25 years old without earlier medical diagnose, were consecutively enrolled. height at 25 years of age was based on personal knowledge, and the participators answered a questionnaire regarding earlier height, smoking habits, earlier fractures, heredity or use of corticosteroids. control measurements of their height loss together with actual weight and body mass index were done at the primary health centre before inclusion in the study. 120 women were willing to participate after reading the advertisement. 98 fulfilled inclusion criteria of height loss of more than three cm. fourteen subjects could later not take part due to social reasons and of the remaining 84, four could not perform all investigations due to other diseases, why 80 completed the study. measurements of bone mineral density were done with whole body dxa (hologic 4500 a) at the osteoporotic clinic in göteborg with determination of hip, lumbar spine and whole body. x-ray examinations were performed with the thoracic and lumbar spines projections (lateral spinal radiographs) to identify spinal fractures. the results were assessed by experienced radiologists. the criterion for osteoporosis, assessed by world health organisation (who), was used, which means a t-score of < –2.5 standard deviations (s.d.) of a young female population measured in lumbar spine or hip for osteoporosis and a t-score of –1.0 – –2.5 s.d. for the diagnose of osteopenia (11). blood tests for s-calcium were analysed at the clinical chemical laboratory, sahlgrenska university hospital, to detect cases of primary hyperparathyroidism. the ethics committee at the university of göteborg approved the study and informed consent was obtained from each subject before participation. data analysis means and standard deviations were used for descriptive purpose, t-test for unpaired data was used together with pearson’s chi square test and p-values less than 0.05 were considered to be significant. pearson’s correlation coefficient was used for correlation calculations. height loss in women caused by vertebral fractures and osteoporosis 215 table 1. mean values of t-score and bmd (bone mineral density, g/cm2) n t-score s.d. n bmd s.d. hip 77 –1.323 1.122 76 0.815 0.136 back 79 –1.497 1.553 79 0.890 0.174 whole body 77 –2.066 1.147 77 0.924 0.099 results the participation rate was 80 women in the study who all were tested with s-calcium. complete measurements with dxa were performed in 76 subjects. hip prosthesis (3 subjects), personal reasons, other diseases and technical problems made complete investigations impossible for four patients. four of the 80 women had earlier visited a doctor for suspicion of osteoporosis. one had suffered a hip fracture treated with calcium and d-vitamin and three had been given corticosteroids for asthma and rheumatic disease in periods without treatment for osteoporosis or performance of spinal x-ray. none of them had vertebral compressions. the mean age of the group who completed the measurements was 70 years with a range of 51–84 years. ten were smokers. their height loss compared to 25 years old was from three cm to eleven cm with a mean value of 5.2 cm and a median value of 5 cm. x-ray of thoracic and lumbar spine could be performed in 80 patients and compressions of the thoracic and lumbar vertebras were diagnosed in 36 (45%) of the patients. the fractures were in a majority, smaller wedge-shaped ventral compressions. the rest of the subjects (44/80) had in a mostly findings of spondylosis and disc degeneration, explaining their height loss. the mean height loss for subjects with vertebral compressions (n=36) was 5.3 cm (range 3–11 cm) compared with 5.0 cm (3–8 cm) for the other 55% (n=44) without demonstrated vertebral compression. the differences were not significant. t-score and bmd determinations could be performed in 76–79 subjects with lumbar, hip and whole-body dxa (three had hip prosthesis) and the results are shown in table 1. mean values of t-score for all subjects corresponded to osteopenia in accordance with the who criteria (t-score –1.0 – –2.5 s.d.). in the group with x-ray demonstrated vertebral compressions (n=36) 14 subjects had lumbar spine t-scores corresponding with osteoporosis (t-score < –2.5) compared to eight in the group without fractures (n=44). there were ten individuals of the dxa hip measurements with vertebral fractures who were osteoporotic against two without fractures and among the results from dxa whole body determinations there were 14 with osteoporotic values against 13 without vertebral fractures. t-scores for individuals with vertebral compression differed significantly form the group without compression with p-values in back p=0.017. values in hip were also significant (p=0.012) but not in whole body determination (p=0.091) (table 2). the same pattern was demonstrated for bmd values, which showed a significant difference (p=0.037) in the back with and without 216 robert eggertsen and dan mellström vertebral fractures. there were also significant differences in the hip (p=0.014), but not in whole body dxa (p=0.093) (table 3). after age adjustments the differences remained significant regarding t-score but not for bmd in back (table 2, 3). chi-square test for proportions of individuals with and without osteoporosis showed a significant difference for subjects with and without vertebral fractures in hip (p=0.007), back (p=0.031) but not for whole body (p=0.037). . mean values of body mass index (bmi) was 27.8 kg/m2 in the group without vertebral compressions and 26.3 kg/m2 in the fracture group with a difference of 1.5 (p=0.07). mean values of the weight were 71.3 kg and 66.9 kg with a difference of 4.4 kg (p=0.08). mean age of patients with spinal fractures was 71 years and 68 years without fractures, however not significant. the differences in t-score and bmd were significant also after correction for age except for bmd in the spine. correlation between vertebral fractures and osteoporosis, adjusted for age and bmi, was significant in the hip (p=0.021), but not in the back (p=0.067) or in the whole body determinations (p=0.526). mean values of s-calcium for the 80 subjects were 2.43 mmol/l (range 2.22 –2.78). actual reference area was 2.20–2.60 mmol/l, and one individual had a value of 2.78 that was normalised at control. at follow-up later it raised again, and she was after two years operated for a parathyroid adenoma. table 2. mean t-scores with and without vertebral compression with compression without compression p n t-score s.d. n t-score s.d. p age adjusted hip 34 –1.7 1.3 43 –1.0 0.9 p=0.012 p=0.014 back 35 –1.9 1.5 44 –1.1 1.5 p=0.017 p=0.019 whole body 34 –2.3 1.2 43 –1.9 1.1 p=0.091 ns p=0.136 ns table 3. mean bmd with and without vertebral compression with compression without compression p n bmd s.d. n bmd s.d. p age adjusted hip 34 0.77 0.15 42 0.85 0.11 p=0.014 p=0.012 back 36 0.85 0.18 43 0.93 0.17 p=0.037 p=0.073 ns whole body 34 0.90 0.1 43 0.94 0.1 p=0.091 ns p=0.136 ns height loss in women caused by vertebral fractures and osteoporosis 217 discussion this investigation has showed that reason for height loss of three cm or more in women, in 45 per cent in our material, was undiagnosed vertebral compression. this was correlated to osteoporosis and a possibility to suspect this diagnose was their height loss. lower weight and bmi also seemed to correlate with a higher degree of spinal fractures, with values close to significance. the differences of bmd and t-score between subjects with or without vertebral compression were significant but the numerical values were maybe not as large that these values could be used for safe diagnosing of vertebral compressions. completion with x-ray were necessary for definite diagnose. the recruitment of participators was not done randomly from a population but with advertisement that is becoming more usual in clinical research. all women except four, who wanted to take part, had however not been investigated earlier for their height shortening and had also no current history of back pain leading to medical examination, why the group was valuable to investigate. the prevalence of spinal fractures in our material was 45% which seems high compared to other studies where prevalence values from two to 39% have been demonstrated in men and women in different countries ( 1,8, 12–16). however, those studies were prevalence investigations in contrast to our trial comprising women with known height loss. the differences in height between the groups were small and not significant. however, the group without vertebral compressions had spondylosis and other changes that explained their height loss. the compressions were also small, which indicate that they had occurred without symtoms. this means also that it is necessary to make further investigations in those subjects to obtain a right diagnose. should elderly women with height loss be screened for osteoporosis and spinal fractures? height loss up to five cm could also be explained with decreased intervertebral discs, and in our limited material we could find this. as we could demonstrate a high prevalence of spinal fractures correlated to height loss larger investigations should be performed. considering the high costs of osteoporotic fractures and inability with movement that will follow spinal and other fragility fractures, it must be important to identify these patients in the primary health care (17). this is of value as there is an association between vertebral fractures and increased mortality in patients with osteoporosis (18,19). in many treatment studies against osteoporosis, vertebral compression is used as primary endpoint showing that the risk of a new vertebral compression in the year following an incident vertebral fracture is about 20 per cent (7, 20). to screen all women in this age group is however not recommended because it is not possible to identify individuals who will develop a future fracture (21). with limited resources for dxa measurements referral to spinal x-ray should be done in suspected cases. we conclude, that among our group of women with height loss of more than three cm without earlier known osteoporotic disease or fracture, both spinal fractures (46%) and osteoporosis were common. 218 robert eggertsen and dan mellström references 1. o´neill tw, 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20. kindsay r, silverman sl, cooper c, hanley da. barton i, broy sg (2001) risk of new vertebral fracture in the year following a fracture. jama 285: 320–23. height loss in women caused by vertebral fractures and osteoporosis 219 21. marshall d, johnell o, wedel h. (1996) meta-analysis of how well measures of bone mineral density predict occurence of osteoporotic fractures. bmj 312: 1254–59. corresponding author: robert eggertsen md, phd. mölnlycke vårdcentral s-435 21 mölnlycke sweden tel +46 31986773 fax +46 31986774 e-mail: robert.eggertsen@vgregion.se tf-iups160035 211..215 review article the future of preconception care in the united states: multigenerational impact on reproductive outcomes michelle st. fleur, karla damus and brian jack department of family medicine, boston university school of medicine/boston medical center, boston, ma, usa abstract the future of preconception care will require an innovative multigenerational approach to health promotion for women and men to achieve optimal reproductive health outcomes. in this paper we provide a summary of historical trends in perinatal interventions in the united states that have effectively reduced adverse perinatal outcomes but have not improved disparities among ethnic/racial groups. we describe evidence pointing to an enhanced preconception care paradigm that spans the time periods before, during, and between pregnancies and across generations for all women and men. we describe how the weathering, barker, and life course theories point to stress and non-chromosomal inheritance as key mediators in racial disparities. finally, we provide evidence that indicates that humans exposed to toxic stress can be impacted in future generations and that these phenomena are potentially related to epigenetic inheritance, resulting in perinatal disparities. we believe that this expanded view will define preconception care as a critical area for research in the years ahead. article history received 25 march 2016 revised 20 june 2016 accepted 22 june 2016 key words epigenetics; health disparities; perinatal outcomes; preconception care; preconception health introduction given the myriad of factors that influence preconception health (1), the future of preconception care will depend on how well the progress made so far can be leveraged and expanded. it will also rely on how new scientific evidence can be integrated into the content, risk assessment, and mitigation of preconception risks. the strategies and resources used to facilitate the clinical and public health integration and dissemination of preconception care, including the role of group care, the medical home, workplace and school-based health promotion programs, and home visitation, will also impact future progress. although in the past decade preconception health (pch) and preconception care (pcc) have gained momentum, the number and types of effective evidencebased interventions are limited and have remained relatively stable, and, as for all maternal child health (mch) interventions, there is wide global variability, reflecting regional and national specific challenges and resources. some of these key influencing factors include: the priorities placed on women’s, men’s, and families’ health, women’s reproductive rights, political will, access to and support for preventive health services, raising public health awareness and education, health care provider training, reimbursement of services, developing, implementing, and disseminating evidence-based interventions, baseline perinatal outcomes, and population health status. there is also strong support of other key variables that impact pch/pcc with equal if not greater international variability, such as the importance and influence of the social determinants of health, the role of childhood adverse events, toxic stress, and epigenetics. therefore, discussions and predictions about the future of preconception care must integrate all of these issues into a cohesive context, reflecting how each of the factors might influence the future of pcc. in this paper, after a brief summary of historical trends in seminal perinatal outcomes in the united states (usa), we will present an overview of some of these factors that underscore the need for pcc from the lens of the persistent ethnic/ racial disparities and describe how an innovative epigenetic transgenerational approach can portend the future of preconception care. historical trends and ethnic/racial disparities in perinatal outcomes in the usa from 1940 to 2005 the overall infant, neonatal, and post-neonatal annual mortality rates in the usa declined substantially (2). the infant (age less than one year) mortality rate (imr) decreased by 85%, from 47 per 1,000 live births in 1940 to 6.9 in 2005; the neonatal (age less than 28 days) rate decreased by 84%, from 28.8 to 4.54; and the post-neonatal (age greater than 28 days but less than one year) rate decreased by 87%, from 18.3 to 2.34 (3). after a plateauing of imrs in the usa from 2005 to 2010, rates began to fall again, reaching a historical low of 6.0 in 2013 (4). but despite this progress, the usa continues to have the highest imr of all organization for economic co-operation and development (oecd) countries (5). furthermore, ethnic/racial disparities in perinatal outcomes continue to persist in the usa. for example, in 2013 the majority (76.1%) of the nearly 4 million births in the usa were white and 16.1% were black (6), and contact michelle st. fleur stfleurm@bu.edu boston medical center, department of family medicine, dowling 5, 1 bmc place, boston, ma, usa � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 4, 211–215 http://dx.doi.org/10.1080/03009734.2016.1206152 http://creativecommons.org/licenses/by/4.0/ the 2013 imr for black infants was 11.1 per 1,000 births compared to 5.1 for white infants. therefore, black babies were still twice as likely to die in their first year of life compared to white babies. in addition, black women were also almost twice as likely to deliver a low-birth-weight (lbw) baby (�2,500 g) and three times as likely to deliver a very-lowbirth-weight baby (<1,500 grams) than white women (7). socioeconomic factors (8), genetics, health behaviors, experience of stressful life events (9), and toxic stress (10) have all been studied, but despite decades of programs and research, the factors that contribute to this disparity and effective interventions to promote equity remain poorly understood (11). with lbw and short-term gestation being the leading cause of mortality for black infants (3,12), we will not optimize maternal child health (mch) outcomes until we reduce these disparities (13,14). strategies used to improve maternal child health outcomes maternal child health (mch) research over the past 40 years has focused on improving hospital care, prenatal care, and, more recently, preconception care. in an effort to improve mch outcomes, research has continued to go back in time to address the root causes. intrapartum, antenatal, and preconception care have contributed to improved outcomes, but they have been shown only minimally to impact disparities in imr and lbw. between the 1960s and 1990s, the focus was on improving labor and delivery. this included hospital deliveries, medicalization of birth, electronic fetal monitoring, and ultrasound. some outcomes improved, some worsened, but the disparities remained (15). from the 1980s to 1990s the focus then shifted to antenatal care. in 1968, gortmaker et al. examined all births in new york city and found a strong association between preterm deliveries and inadequate prenatal care (16). since then, the association between inadequate antenatal care and low gestation weight is well documented (17–19). as a result, increased efforts were made to improve access to and quality of prenatal care. however, intervention studies did not show improvements in disparities. with preterm premature rupture of membranes (pprom) identified as the leading cause of preterm births (ptb) (<37 weeks of gestation) in black women, and intrauterine infections often being a precursor to pprom, treating infections such as asymptomatic bacteriuria, pneumonia, chlamydia, and bacterial vaginosis should have led to reductions in black–white differences in ptb. yet, the treatment of infections during prenatal care has also had little impact on disparities in the usa (12,20–22). starting in the 1990s to the present there has been a focus on understanding infant mortality disparities through preconception care (pcc). preconception care is defined as a set of interventions that aim to identify and modify biomedical, behavioral, and social risks to the woman’s health or pregnancy outcome through prevention and management prior to a woman becoming pregnant. strong evidence for some risks include folic acid deficiency, type 1 diabetes, smoking, phenylketonuria, and many others (23). however, to date there is no study showing that the ‘package’ of pcc services improves mch outcomes. data exist that we can identify pcc risks, but there are no data that this impacts mch outcomes or disparities (2). it is possible that intrapartum, antepartum, and preconception services are necessary but not sufficient to improve perinatal outcomes among black women in the usa. more recent and emerging data show that factors impacting prior generations can possibly explain perinatal outcome disparities. since 2005, there has been a focus on better understanding risk factors that impacted previous generations and potential transgenerational effects. explanatory mechanisms have included the life course perspective (9), barker hypothesis (24), adverse childhood events (25), toxic stress (10), healthy immigrant effect (26), impact of stress and societal racism (27,28), weathering hypothesis (29), and epigenetics (30) as possible mechanisms. life course theory and the barker hypothesis the life course perspective by lu et al. (31) posits that disparities in birth outcomes are the consequences of differential developmental trajectories that are set by early life experiences and cumulative allostatic load over the life course (31). it is especially important in mch, where one developmental stage often gets disconnected from another. in perinatal health, the focus is so much on events occurring in the 40 weeks of pregnancy that it is forgotten that there are a great deal of life course influences on perinatal outcomes which impact health and illness potential throughout life (31). for example, in explaining the black–white gap in infant mortality, for decades there was a search for maternal risk factors during pregnancy rather than looking at the mothers’ cumulative life course experiences. the danger of focusing solely on risk factors during pregnancy is that since it does not adequately explain the disparities it can misguide public health interventions and policies. the life course perspective assumes that accumulating stress throughout life somehow results in worsening mch outcomes. but evidence is emerging that lifetime stress does not predict lbw or ptb. among 33,452 women in the pregnancy risk assessment monitoring system (prams) data set, black women reported the highest number of stressful events 12 months before delivery when compared to white women. these stressful events included emotional stress, financial stress, partner-related stress, and traumatic stress. but, in stepwise regression, stressful events were not associated with ptb in that generation x (9). is it possible that the stressful events necessary to impact mch outcomes must accumulate over a longer period of time—perhaps even over a generation or more? the ‘barker hypothesis’, which demonstrates that much of an adult’s health is programmed during his or her experience as a fetus and in early childhood, provides additional insights into adverse and disparate perinatal outcomes. a nationally representative sample in the usa found that children born <2,500 g were at increased risk (or of 2.16, p < 0.01) for stroke, myocardial infarction, heart disease, and ptb by 212 m. st. fleur et al. 50 years of age (24). stroke mortality rates among adults in england and wales are higher among people with lower birth weights. the mothers of these low-birth-weight babies were typically poor, malnourished, had poor overall health, and were generally socially disadvantaged. the odds of stroke more than doubled for those with birth weights <2,500 g compared with those weighing 4,000 g (32). therefore it seems that your mother’s experience, not only yours, leads to adult disease and ptb (32). since a stressful intrauterine environment or being delivered prematurely does not change the genetic code, by what mechanism does this lead to adult disease and subsequent preterm birth? for two decades we thought if we could get women universal access to good-quality prenatal care, then we could influence maternal child health disparities. it is recognized now that to expect prenatal care, in less than nine months, to reverse all the cumulative disadvantages and inequities over the life course of the woman is expecting too much of prenatal care, particularly since many predictive adverse exposures begin during childhood. adverse childhood events and toxic stress adverse childhood experiences (aces) are traumatic events (such as physical, emotional, or sexual abuse, parental divorce, or the incarceration of a parent or guardian), occurring before the age of 18, that the person remembers as an adult. aces are common, and they have long-term associations with adult health risk behaviors, health status, and diseases. the ace study was the largest study that examined the association between adverse childhood events and leading causes of death in 18,000 adults (25). these events included seven categories of adverse childhood experiences: psychological, physical, or sexual abuse; violence against mother; or living with household members who were substance abusers, mentally ill or suicidal, or ever imprisoned. the number of categories of these adverse childhood experiences was then compared to measures of adult risk behavior, health status, and disease. they found that more than half of respondents reported at least one exposure, and one-fourth of respondents reported �2 categories of childhood exposures. the number of categories of adverse childhood exposures had a positive graded relationship with the leading causes of death in adults, including ischemic heart disease, cancer, chronic lung disease, skeletal fractures, and liver disease. furthermore, these childhood exposures compromise the cognitive and emotional development of children as well as their capacity, as adults, to care for the next generation. prolonged activation of the stress response systems in the absence of protective relationships can lead to toxic stress. over time this leads to elevated cortisol levels and decreases in synapses between the cortex and limbic system (10). this stress can have intergenerational effects, with emerging research demonstrating changes at a genetic level. this long-term impact of early adversity on parenting perpetuates a cycle of intergenerational transmission of trauma and health risks. there is also evidence that increasing societal stress can impact mch outcomes. one example is the healthy immigrant effect, which is the phenomenon by which immigrants experience more positive health outcomes (including preterm birth) than the native-born population in developed countries (26). is it possible that native-born foreign populations have more stressful lives than immigrants? if so, it is possible that life stress is related to adverse reproductive outcomes (26). other theories have hypothesized the role of preconceptional stress and its contribution to the high rates of preterm birth experienced by black women (33). one such hypothesis includes the weathering hypothesis; this theory holds that social inequalities leads to an earlier and disproportional decline in the health status of minorities, resulting in widening health disparities between blacks and whites (34). in a study by geronimus et al., black women living in low-income neighborhoods in michigan were found to have a 3-fold increase of preterm births and a 4-fold increase in very preterm births (<32 wks) between the ages of 15 and 34 years when compared to white singleton births (34). it appears that either directly or indirectly, through interactions with host genes, stress is an important risk factor for adverse birth outcomes. epigenetics and multigenerational effects on maternal child health there is now evidence that the ‘epigenome’ or pattern of dna methylation that is laid down during early fetal life might provide a mechanism to explain how intrauterine stress could impact adult health status and the propensity for ptb in the next generation. this variation occurs in the absence of alteration of the gene sequence. the degree of methylation affects how easily the enzymes that transcribe the genes can do their job. therefore, the epigenome learns from its experiences. epigenetic tags act as a kind of cellular memory. while there is rapidly accumulating evidence in animal models that epigenetics can influence the health of future generations, there is now emerging evidence that this could be the case on humans as well. the 1944–1945 dutch famine (35,36) provides empirical support for the hypothesis that early-life environmental conditions can cause epigenetic changes in humans that persist throughout life. individuals who were prenatally exposed to famine during the dutch hunger winter in 1944–45 had, six decades later, less dna methylation of the imprinted igf2 gene compared with their unexposed, same-sex siblings. the association was specific for periconceptional exposure, reinforcing that very early mammalian development is a crucial period for establishing and maintaining epigenetic marks. these data are the first to contribute empirical support for the hypothesis that early-life environmental conditions can cause epigenetic changes in humans that persist throughout life. the periconceptual environment may represent a window of vulnerability during which differential methylation could occur in response to severe environmental stress (35). additional human data that lend support to the theory that stressful events can impact birth outcomes in subsequent generations are provided in studies by collins known as the grandmother epidemiologic data set (37) that links upsala journal of medical sciences 213 three generations of african americans in illinois. rates of lbw were associated with worsening maternal grandmothers’ residential environments during her pregnancy with her daughter, who years later delivered a lbw neonate. this association was independent of the living conditions of the daughter during her pregnancy with the infant with low birth weight. this transgenerational effect supports the notion that epigenetic mechanisms are likely to play a role in the pathophysiology of preterm labor (37). in a pregnant mother, three generations (the mother, her daughter, and eggs of the daughter) are directly exposed to the same environmental conditions at the same time. in this way, three generations are simultaneously exposed to the same environmental stressors such as diet, toxins, and stress, among others (37). in order to provide a convincing case for epigenetic inheritance, an epigenetic change must be observed in the fourth generation. data from the newborn epigenetics study cohort in north carolina measured stress in 537 women at 12 weeks’ gestation using the perceived stress scale. dna methylation at differentially methylated regions was measured from peripheral and cord blood. maternal stress was not associated with ptb. however, elevated stress was associated with higher infant dna methylation (2.8% difference, p < 0.01). this provides evidence that maternal stress may be associated with epigenetic changes that are passed on to their daughters (38). another study showed that severe maternal depressed mood was associated with a 3-fold increase in the risk of lbw and that dna methylation levels of the offspring differed significantly by maternal depressed mood, measured by using the centers for epidemiologic studies depression scale (39), providing further human evidence that epigenetic mechanisms could be involved. if some of the social determinants of health (socioeconomic status, ethnicity/race, education, violence, experiences of racism, health behaviors such as dietary practices, and environmental exposures) are found to affect the degree of dna methylation and differences in phenotype, then one of the mysteries of the gene–environment interactions could be solved. overall implications for the future of preconception care to achieve optimal pregnancy outcomes in the future, which will translate to better health throughout life, enhanced preconception care that spans the time periods before, during, and between pregnancies and across generations for all women and men is needed. this expanded view of pcc requires a paradigm shift to an epigenetic translational approach and strategies that integrate evidence-based science about biomedical risks, toxic stress, social determinants of health, combined with an informed public and providers, adequate health care services, and the effective use of tested technologies for dissemination. such a model should also help to promote equity in outcomes since the life course, barker, and weathering theories all point to stress and nonchromosomal inheritance as key mediators in ethnic/racial disparities. epigenetic changes have been shown in humans exposed to toxic stress that can manifest changes that impact future generations, so that a grandmother’s stress can result in a preterm birth of her grandchild. finally, multigenerational data sets that include bio-behavioral risk factors, measures of environmental and social stress, and reproductive outcomes are needed to analyze the complex relationships that must be studied to inform the content and the effective evidencebased pcc interventions that are yet to be identified. acknowledgements parts of this paper were presented at the 3rd congress on preconception health and care held uppsala, sweden on 16–17 february 2016. disclosure statement the authors report no conflicts of interest. funding this work was supported by the w.k. kellogg foundation [grant number p3024018] and national institute on minority health and health disparities (nimhd) [grant number 1 r01 md 006213-01 a1]. orcid brian jack http://orcid.org/0000-0002-6497-2437 references 1. johnson k, posner s, biermann j, cordero j, atrash h, parker c, et al. cdc/atsdr preconception care work group; select panel on preconception care. recommendations to improve preconception health and health care–united 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article high-fat diet in pregnant rats and adverse fetal outcome parri wentzela, ulf j. erikssona and emilio herrerab adepartment of medical cell biology, uppsala university, uppsala, sweden; bdepartment of biochemistry, ceu san pablo university, madrid, spain abstract background: although pregestational obesity has been associated with increased risk of adverse fetal outcome, the mechanisms behind are not known. we aimed to investigate the influence of the maternal metabolic state on fetal outcome in rats exposed to either a high-fat diet (hfd) or a control diet (cd). we also investigated the impact of serum collected from hfd/cd pregnant rats on cd embryonic development in whole-embryo cultures. material and methods: on gestational day 0, 9, 10, or 20 maternal plasma/serum samples were collected as pregnancies were terminated for the estimations of maternal metabolic state and embryofetal development. we measured embryonic gene expression of ros scavenger enzymes as well as genes involved in inflammation in maternal adipose tissue. results: in hfd maternal plasma/serum, concentrations of glucose, b-hydroxybutyrate, branched-chain amino acids, and leptin were increased, whereas those of triacylglycerol, cholesterol, and palmitic, oleic, linoleic, and a-linolenic acids were decreased. gene expression of cuznsod, il-6, il-10, and resistin was increased in hfd maternal adipose tissue, whereas that of cuznsod and mnsod was decreased in hfd-exposed embryos. hfd caused retention of most fatty acids in the maternal liver as well. conclusion: hfd alters the maternal metabolic state, increases fetal resorptions in vivo, and increases the rate of fetal/embryonic malformations both in vivo and in vitro. these findings suggest that metabolic disturbances in hfd pregnant rats have profound adverse developmental effects in the offspring. article history received 27 december 2018 revised 2 april 2019 accepted 4 april 2019 keywords animal model; fetal anomaly; fetal outcome; high-fat diet; pregnancy; rodent introduction maternal nutrition is known to play a key role in fetal health (1). the prevalence of maternal overweight/obesity is currently increasing and is associated with an enhanced risk of congenital anomalies (2), such as skeletal, heart, neural tube, and orofacial defects (3,4). in addition, fetal and neonatal complications are more common (5) as well as adverse metabolic postnatal outcomes in the offspring of overweight/ obese mothers (6), leading to increased risk of obesity, insulin resistance, and type 2 diabetes later in life (7,8). the exact mechanisms behind the obesity-induced congenital anomalies are not known, but it is likely that obesity-induced dysmorphogenesis is the result of an interplay between genetic and environmental factors. the latter factors include metabolic alterations in mother and offspring, alterations that may induce developmental changes via (epi)genetic processes. a number of metabolic alterations have been identified in obese pregnant women. for instance, in obese humans and rodents given a high-fat diet (hfd) during pregnancy and lactation poor glycemic control has been identified (9,10), as well as increased susceptibility to hypertension (11) and changes in hypothalamic gene expression (12). all these changes were associated with adverse outcome in the offspring. furthermore, in rodents hfds have been found to induce obesity, and there was a positive relationship between the level of fat in the diet and body weight gain (13). however, pregnant rats on hfd reduce their caloric intake by reducing their intake of energy-dense diet and deriving more calories from fat (14). in rats, hfd throughout pregnancy reduced the concentration of docosahexaenoic acid (22:6 n-3, dha) and caused a compensatory increase in arachidonic acid (20:4 n-6, aa) in liver lipids of newborn pups (15). other metabolic factors like branched-chain amino acids (16) and oxidative stress (17) were modified in the offspring of hfd pregnancies, illustrating the capacity of maternal hfd to induce epigenetic and teratogenic effects in the offspring (18). indeed, oxidative stress, endoplasmic reticulum (er) stress, and chronic inflammation have also been suggested to be involved in the obesity-induced maldevelopment of the offspring (6,19–21). in order to contribute to the understanding of obesityinduced teratogenesis, we designed an animal model to study the relationship between hfd-altered maternal metabolism and adverse fetal outcome. contact parri wentzel parri.wentzel@mcb.uu.se department of medical cell biology, uppsala university, po box 571, biomedical center, se-751 23 uppsala, sweden supplemental data for this article can be accessed here. � 2019 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences 2019, vol. 124, no. 2, 125–134 https://doi.org/10.1080/03009734.2019.1604588 http://crossmark.crossref.org/dialog/?doi=10.1080/03009734.2019.1604588&domain=pdf&date_stamp=2019-06-04 https://doi.org/10.1080/03009734.2019.1604588 http://creativecommons.org/licenses/by/4.0/ https://doi.org./10.1080/03009734.2019.1604588 http://www.tandfonline.com material and methods animals fifty-eight female rats, 3 weeks old, from a local outbred sprague-dawley strain (biomedical center, uppsala university, sweden), were housed with free access to pelleted food and water in a room maintained at 22 �c with a 12-h light–dark cycle. one half of the rats was fed a high-fat diet (hfd) (35 g% fat, 26 g% protein, 26 g% carbohydrate; d12492, research diets inc., new brunswick, nj, usa), while the other half was maintained on a control diet (cd) (4.3 g% fat, 19.2 g% protein, 67.3 g% carbohydrate; d12450b, research diets inc.). the fatty acid (fa) concentrations in cd and hfd were measured. the highest ratio difference between the two diets (i.e. hfd/cd ratio) was found for myristic acid (14:0, ma), followed by stearic acid (18:0, sa), palmitoleic acid (16:1 n-7, poa), oleic acid (18:1 n-9, oa), palmitic acid (16:0, pa), linoleic acid (18:2 n-6, la), and a-linolenic acid (18:3 n-3, ala) (supplementary table 1, available online). three-week-old female pups were fed hfd or cd ad libitum, and 9 weeks later (i.e. at 12 weeks of age) the hfd and cd female rats were mated overnight with cd males. pregnancy was confirmed by a positive vaginal smear next morning (gestational day 0). on gestational day 0 tail vein blood was collected for plasma (in edta-na2 tubes) and serum samples from nine rats of each group. plasma/serum was separated from fresh blood by centrifugation at 1500 �g for 15 min at 4 �c. gestational day-0 rats were subsequently killed by cervical dislocation after mild ether anesthesia, and samples of maternal liver and adipose tissue were secured (figure 1). during pregnancy rats were maintained on their respective diet. pregnancy was interrupted on gestational day 9 in five cd rats for the production of embryos aimed for wholeembryo culture (wec). in gestational day-10 rats from the two dietary groups, pregnancy was interrupted for embryonic mrna evaluation (six hfd and six cd rats) and collection of maternal liver. also, on gestational day 10, plasma samples (in edta-na2 tubes)/serum samples from maternal tail vein blood were collected and prepared as described above. pregnancy was interrupted in 15 hfd rats and 8 cd rats on day 20 by cervical dislocation after mild ether anesthesia after collection of tail vein blood for plasma/serum samples, as above. fetuses were dissected out from each uterine horn and evaluated with regard to fetal and placental weight (data not shown), malformations, and resorptions. furthermore, maternal liver and abdominal adipose tissue were collected and kept at -80 �c until analysis. in addition, from the same hfd and cd rats (gestational day 20), maternal blood was collected from the abdominal aorta, immediately centrifuged and prepared ad modum new, (22) and stored at �80 �c until used in wholeembryo culture. ethical evaluation all animal procedures were performed according to the guide for the care and use of laboratory animals (nih, 1985) and were approved by the animal ethics committee of the medical faculty of uppsala university. skeletal staining whole 20-day-old fetuses were fixed in 95% ethanol. staining of the skeleton was performed with alcian blue and alizarin red for 24 h at 37 �c, followed by clearing of the soft tissue by submersion in 1% koh for 48 h (23). whole-embryo culture (wec) pregnant cd rats were killed by cervical dislocation after mild ether anesthesia on gestational day 9. embryos in their intact yolk sacs were dissected out and cultured in serum collected from hfd or cd day-20 pregnant rats, diluted to 80% with saline (22). the embryos were incubated in falcon 50 ml tubes (4–5 embryos/tube) in a roller incubator at 60 rev/min for 48 h at 38 �c. after culture, the embryos were dissected out of their yolk sacs and examined and scored under a stereomicroscope. a malformation score of 0 indicated a completely normal embryo; a score of 1 indicated a single minor deviation. a score of 5 denoted one major malformation, whereas a score of 10 indicated an embryo with multiple major malformations such as open neural tube, rotational defects, and/or heart enlargement. furthermore, we determined crown–rump length and somite number of each embryo for estimation of embryonic growth (24). analyses in plasma/serum, diets, and tissues plasma glucose, triacylglycerols (tag), cholesterol, and nonesterified fatty acids (nefa) were determined enzymatically using commercial kits (glucose, tag, and cholesterol: spinreact reactives, girona, spain; nefa: wako chemicals, neuss, germany). insulin was analyzed by an enzyme-linked immunoassay (mercodia, uppsala, sweden), and b-hydroxybutyrate was analyzed in serum (cobas mirav r multichannel analyzer, hoffman-la-roche, basle, switzerland) using a standard reagent kit (hoffman-la-roche; boehringer mannheim, germany; and sigma). figure 1. layout of study. the gestational day (gd) when the different pregnancies are interrupted is displayed in the first column. the number and types of pregnant rats (cd: control diet; hfd: high fat diet) are displayed in the second column. the samples analyzed are displayed in the third column. t1, 2 and 3 denote tables 1–3, whereas f2, 3, 4 and sf2 denote figures 2–4 and supplementary figure 2 available online. 126 p. wentzel et al. https://doi.org/10.1080/03009734.2019.1604588 https://doi.org/10.1080/03009734.2019.1604588 nonadecanoic acid (19:1) (sigma chemical co., st. louis, mo, usa) was added as the internal standard to fresh aliquots of frozen diet, frozen plasma, frozen liver, and frozen adipose tissue, which were used for lipid extraction and purification in chloroform-methanol (2:1) containing 0.005% bht (25). the final lipid extract was evaporated to dryness under vacuum and the residue resuspended in methanol/toluene and subjected to methanolysis in the presence of acetyl chloride at 80 �c for 2.5 h as previously described (26). fatty acid methyl esters were separated and quantified on a perkin-elmer (waltham, ma, usa) gas chromatograph (autosystem) with a flame ionization detector and a 20 mm � 0.25 mm omegawax capillary column (merck, kenilworth, nj, usa). nitrogen was used as carrier gas, and the fatty acid methyl esters were compared with purified standards (sigma chemical co.). quantification of the fatty acids in the sample was performed as a function of the corresponding peak areas compared with that of the internal standard. liver lipid extracts were also used for the analysis of tag and cholesterol as described (27) and phospholipids (spinreact reactives, barcelona, spain). branched-chain amino acids in serum were analyzed chromatographically after deproteinization with sulphosalicylic acid using a biochrom 30 (biochrom, cambridge, uk) with norleucine as internal standard. adiponectin, leptin, and resistin were measured in serum by an enzyme-linked immunosorbent assay (elisa) (emd merck millipore, burlington, ma, usa). sum of fatty acids sum of saturated fatty acids (sfa) and monounsaturated fatty acids (mufa) represents four different fatty acids. sum of polyunsaturated n-6 fatty acids (pufa) and polyunsaturated n-3 fatty acids (pufa) represents seven and five fatty acids, respectively. in addition, we chose to present some of the fatty acids individually, which are important with regard to embryonic development. homa determination homa-ir was estimated in day-0 pregnant rats on cd (n ¼ 9), or hfd (n ¼ 9) by using the formula: homa-ir ¼ ð½glucose� � ½insulin�þ=405 (1) where plasma samples were expressed in mg/dl (glucose) and mu/ml (insulin). gene expression total rna and cdna from each embryo were isolated with a rneasy mini kit (qiagen, hilden, germany), and each embryo was lysed in 350-ml buffers followed by dnase treatment (rnase-free dnase; qiagen). isolated total rna was washed twice with 50 ml rnase-free water, and the accumulated flow-through was collected, yielding the total rna sample. lastly, 1 ml of an rnase inhibitor (rna guard; amersham pharmacia, uppsala, sweden) was added to each sample. one microgram of total rna was used for complementary dna (cdna) preparation. for cdna synthesis, we used firststrand beads (ready to go; amersham pharmacia biotech). the resulting cdna was diluted threefold with rnase-free water. one microliter of cdna purified from each embryo with 10 ng of converted total rna was amplified and measured in duplicate with real-time pcr using the myiq optical thermal cycler (bio-rad laboratories ab, sundbyberg, sweden) with sybrvr green supermix (bio-rad laboratories) used to detect the pcr product. specific primers for cuznsod, mnsod, gpx1, tnf-a, il-6, il-10, adiponectin, and resistin were constructed with the aid of the primer3 free software (available at http://primer3.sourceforge.net/) and subsequently purchased from tib mol-biol (berlin, germany; supplementary table 2, available online). we have previously assessed the stability of expression of various housekeeping genes and found the glucose-6-phosphate dehydrogenase (g6pdh) gene to be constant in day-10 and day-11 embryos (data not shown). therefore, we chose the g6pdh gene as a reference in the rt-pcr protocol regardless of maternal metabolic state. controls were included in each run of the rt-pcr assay. for each primer pair, one sample with no cdna (with only rnase-free water) was included. to exclude the possibility of remaining dna fragments being present in the samples, 10 ng of the total rna of each sample were amplified in the myiq optical thermal cycler. no pcr product was found in the water or the total rna samples. furthermore, we excluded the avian myoblastosis virus-rt enzyme in the cdna preparation and found no amplified pcr product. results were analyzed for each sample with relative quantification comparing the difference between sample and reference crossing point (cp) values. the relative abundance of mrna/g6pdh in each sample was determined using the following equation to yield the ratio (r) sample to g6pdh: r ¼ 2� cpsample�cpg6pdhð þ (2) total rna from freshly frozen samples (in liquid nitrogen) of maternal abdominal adipose tissue was isolated using aurumtm total rna fatty and fibrous tissue kit (bio-rad laboratories, inc., hercules, ca, usa). one ml of purezol was added to each sample and immediately homogenized and then centrifuged for 5 min. then 0.2 ml chloroform was added to the supernatant, followed by incubation at room temperature for 5 min. thereafter the samples were centrifuged again for 15 min, and the upper aqueous phase was carefully transferred to a new tube and an equal amount of 70% ethanol was added and then mixed thoroughly. the mixture was then shifted to the spin column and washed with 700 ml of low-stringency wash solution, treated with dnase 1, washed again with 700 ml high-stringency and then low-stringency solution before finally eluting total rna with elution solution. statistical evaluation the aim of the study was to analyze the effect of hfd on fetal outcome. the rate of adverse outcome (malformations and resorptions) was therefore chosen as primary endpoint. upsala journal of medical sciences 127 http://primer3.sourceforge.net/ https://doi.org/10.1080/03009734.2019.1604588 comparisons between different experimental groups were based on individual rats (maternal parameters) or litter means (fetal parameters), and values are expressed as mean ± sem. statistical comparison between two groups was performed with one-way anova with fisher’s least significant difference (lsd) as post hoc test. the rates of malformations were compared with chi-square statistics. a value of p < 0.05 was considered to denote a statistically significant difference. results at the start of the experiment, the body weights in the 3week-old pups did not differ between the groups. however, after 9 weeks on the hfd or cd, there was a slight weight difference between the hfd and cd groups on gestational day 0 (supplementary figure 1, available online). this difference remained throughout pregnancy (data not shown). plasma/serum components the glucose concentration was higher in hfd rats than in cd rats, whereas the insulin concentration was similar (table 1). homa calculations reveal no difference between the two groups (supplementary figure 2, available online). in both hfd 20 and cd 20 rats the plasma nefa concentrations increased during pregnancy, and they were higher in the hfd 20 rats than in the cd 20 rats (table 1). the plasma tag and cholesterol concentrations increased from day 0 to day 20 of pregnancy in both groups. however, the plasma tag concentrations were lower in the hfd rats than in the cd rats at all time points measured. the plasma cholesterol concentrations in the hfd 0 and hfd 10 rats were lower than in the corresponding cd pregnant rats, whereas on gestational day 20 the hfd cholesterol concentration exceeded that of the cd rats. in the hfd rats the maternal serum b-hydroxybutyrate concentration was higher when compared with that of cd rats on day 10 of pregnancy (table 1). at day 20 of pregnancy, the sum of sfa was lower in hfd rats than in cd rats. in addition, plasma concentrations of myristic (14:0, ma) and palmitic acid (16:0, pa) were lower in hfd rats than in cd rats. however, the concentration of stearic acid (18:0, sa) was higher in the former (table 1). the sum of mufa and the concentration of oleic acid increased from day 0 to day 20 in both groups. moreover, at gestational day 20 the sum of mufa and the concentration of oleic acid in hfd rats were lower than in the corresponding cd rats. the sum of pufa n-6 fa was decreased in hfd 20 rats, when compared with cd 20 rats (table 1). plasma linoleic acid (18:2 n-6, la) concentrations were lower in both day-0 and day-20 pregnant hfd rats than in the corresponding cd rats, whereas there were no differences between the groups with regard to c-linoleic acid (18:3 n-6, gla). in contrast, the aa (20:4 n-6) plasma concentration was higher in the hfd 10 and hfd 20 rats compared with that of the cd 10 and cd 20 rats. in both groups, the sum of pufa n-3 fa increased to the same extent during pregnancy (table 1). plasma concentrations of a-linolenic acid (18:3 n-3, ala) were lower in hfd 0 and 20 rats compared with cd rats on corresponding days. plasma concentration of docosapentaenoic acid (22:5 n-3, dpa) was lower in the hfd group on day 20. in contrast, the concentration of dha (22:6 n-3) in hfd 20 rats was increased compared with the concentration in cd 20 rats. maternal serum concentrations of the branched-chain amino acids valine, isoleucine, and leucine were higher on day 10 in hfd rats than in cd rats (figure 2(a)). table 1. plasma components/fatty acid concentrations in day-0, day-10, day-20 pregnant rats on control diet (n ¼ 9, 6, 8), or high-fat diet (n ¼ 9, 6, 15). diet and day of pregnancy cd 0 cd 10 cd 20 hfd 0 hfd 10 hfd 20 glucose (mg/dl) 123 ± 2 136 ± 3d insulin (mg/l) 0.7 ± 0.1 0.9 ± 0.2 nefa (mmol/l) 332 ± 15 402 ± 37 705 ± 91b,c 323 ± 18 391 ± 32 1082 ± 130b,c,d tag (mg/dl) 47 ± 4 82 ± 5 222 ± 34b,c 37 ± 4d 45 ± 5d 147 ± 25b,c,d cholesterol (mg/dl) 67 ± 4 71 ± 3 101 ± 11b,c 38 ± 4d 49 ± 5d 129 ± 7b,c,d b-hydroxybutyrate (mmol/l) 58 ± 6 109 ± 8d sum sfa 713 ± 14 696 ± 47 1422 ± 98b,c 620 ± 55 646 ± 23 1023 ± 44b,c,d 14:0 ma 9 ± 1 12 ± 1 28 ± 3b,c 5 ± 1 4 ± 1d 8 ± 1d 16:0 pa 391 ± 21 375 ± 30 1032 ± 81b,c 268 ± 28 255 ± 17 580 ± 26b,c,d 18:0 sa 295 ± 11 293 ± 16 350 ± 15b,c 334 ± 29 370 ± 9d 425 ± 20b,c,d sum mufa 244 ± 23 328 ± 29 933 ± 84b,c 201 ± 47 197 ± 32 456 ± 43b,c,d 18:1 (n-9) oa 214 ± 19 284 ± 25 826 ± 71b,c 190 ± 45 185 ± 32 433 ± 41b,c,d sum n-6 fa 991 ± 17 754 ± 38a 1420 ± 81b,c 836 ± 73 881 ± 41 1228 ± 59b,c,d 18:2 (n-6) la 437 ± 16 335 ± 22 894 ± 62b,c 283 ± 33d 268 ± 3 460 ± 38b,c,d 18:3 (n-6) gla 15 ± 4 15 ± 2 22 ± 3c 9 ± 2 13 ± 1 18 ± 3b 20:4 (n-6) aa 500 ± 27 369 ± 18a 388 ± 18b 491 ± 40 538 ± 21d 591 ± 32b,d sum n-3 fa 84 ± 3 91 ± 5 182 ± 11b,c 74 ± 4 75 ± 3 196 ± 10b,c 18:3 (n-3) ala 16 ± 2 12 ± 2 47 ± 5b,c 7 ± 2d 6 ± 1 9 ± 2d 22:5 (n-3) dpa 16 ± 2 12 ± 1 24 ± 2b,c 14 ± 1 14 ± 2 17 ± 3d 22:6 (n-3) dha 44 ± 3 50 ± 4 93 ± 6b,c 50 ± 3 51 ± 3 163 ± 11b,c,d fatty acid concentrations are expressed as mg/l. mean ± sem. ap < 0.05, day 0 versus day 10, for each group. bp < 0.05, day 0 versus day 20, for each group. cp < 0.05, day 10 versus day 20, for each group. dp < 0.05, cd versus hfd at the corresponding day of pregnancy. cd: control diet; hfd: high-fat diet. 128 p. wentzel et al. https://doi.org/10.1080/03009734.2019.1604588 https://doi.org/10.1080/03009734.2019.1604588 maternal leptin concentrations in serum of hfd rats were higher than in cd rats on both day 0 and 10 of gestation (figure 2(b)). there were, however, no differences between hfd and cd rats with regard to maternal adiponectin (figure 2(c)) and maternal resistin concentrations (figure 2(d)) on either gestational day. liver components total lipids were higher in the hfd livers compared with the cd livers at day 0, whereas the tag concentrations in the hfd group were increased on day 0 and 20 compared with the corresponding cd pregnant rats. the liver cholesterol concentration was higher in the hfd 0 rats compared with the cd 0 rats. in addition, the concentration of phospholipids was higher in the hfd 20 rats compared with the cd 20 rats (table 2). the sum of sfa was increased in hfd 0 compared with cd 0 livers, and the same pattern was found with regard to 16:0 pa and 18:0 sa. the sum of mufa and oa (18:0 n-9) were higher in the hfd 0 compared with cd 0 (table 2). the sum of pufa n-6 fas was higher in hfd 0 and 20 rats compared with the cd 0 and 20 rats. the hfd 0 liver concentrations of la (18:2 n-6) and gla (18:3 n-6) were higher than those in the cd 0 rats. in addition, the concentration of aa (20:4 n-6) was increased in the hfd livers at all time points measured. the sum of pufa n-3 fas was increased in hfd 0 rat livers compared with cd 0 rat livers. hepatic concentrations of ala (18:3 n-3) and dpa (22:5 n-3) were increased in hfd 0 livers compared with cd 0. furthermore, the concentration of dha (22:6 n-3) was increased both in hfd 0 and 10 livers compared with the corresponding cd livers (table 2). adipose tissue sums of sfa and of mufa were higher in the hfd group compared with the corresponding cd group on day 0, whereas the opposite was found on gestational day 20 (table 3). the sum of pufa n-6 fas decreased between day 0 and 20 of pregnancy in both groups. likewise, the concentrations of la (18:2 n-6) decreased from day 0 to day 20 in both groups. adipose tissue concentrations of gla (18:3 n-6) were lower in both the hfd 0 and hfd 20 groups compared with the corresponding cd groups. in addition, the concentrations of aa (20:4 n-6) increased from day 0 to day 20 in the hfd group. the sum of pufa n-3 fas increased from day 0 to day 20 of pregnancy in both groups. in addition, the sum of pufa n-3 fas was decreased in hfd rats compared with cd rats at both gestational days. the concentrations of ala (18:3 n-3) were decreased compared with cd concentrations at both day 0 and 20 of pregnancy. in the cd group, the ala figure 2. (a) serum branched chain amino acid concentration (mmol/l) of pregnant rats on control diet (cd, blue bars), or high fat diet (hfd, red bars) on gestational day 10 (n = 6 in both cd and hfd groups). mean ± sem. � = p < 0.05 for cd vs. hfd. (b) serum leptin concentration (ng/ml) on gestational day 0 and 10 of pregnant rats on cd (n = 9 and 6, blue bars), or hfd (n = 9 and 6, red bars). mean ± sem. � = p < 0.05 for cd vs. hfd. (c) serum adiponectin concentration (lg/ml) on gestational day 0 and 10 of pregnant rats on cd (n = 9 and 6, blue bars), or hfd (n = 9 and 6, red bars). mean ± sem. (d) serum resistin concentration (ng/ml) on gestational day 0 and 10 of pregnant rats on cd (n = 9 and 6, blue bars), or hfd (n = 9 and 6, red bars). mean ± sem. � = p < 0.05 for cd vs. hfd. upsala journal of medical sciences 129 concentrations increased from day 0 to day 20. the dpa (22:5 n-3) concentration diminished from day 0 to day 20, to the extent that the levels were non-detectable on day 20, in both groups. the adipose tissue concentrations of dha (22:6 n-3) increased from day 0 to day 20 in both groups (table 3). gene expression the expression of cuznsod in adipose tissue was higher in the hfd group, whereas neither the expression of mnsod nor gpx1 differed between the two groups (figure 3(a)). concerning cytokines, the expression of tnf-a did not differ between the two groups, but the expression of both il-6 and il-10 was higher in adipose tissue of the hfd group than in the cd group (figure 3(b)). the expression of adiponectin did not differ between the two groups, whereas the expression of resistin was higher in the hfd than in cd rats (figure 3(c)). when the mrna levels in day-10 embryos were measured, we found decreased cuznsod and mnsod gene expression in embryos from hfd rats compared to cd rat embryos. there were no differences in embryonic gpx1 expression between the groups (figure 3(d)). fetal outcome the offspring of hfd and cd rats at pregnancy day 20 were studied with regard to fetal and placental weight, malformations, and resorptions. there were no differences in fetal and placental weight (data not shown), whereas we found malformations in 3.8% of the hfd offspring compared with 0.9% in the cd offspring (figure 4(a)). the malformations were tail and ossification defects (resembling human caudal regression syndrome, cf. figure 4(b)). furthermore, we also found table 2. liver lipid/liver fatty acid concentrations in day-0, day-10, day-20 pregnant rats on control diet (n ¼ 9, 6, 8), or high-fat diet (n ¼ 9, 6, 15). diet and day of pregnancy cd 0 cd 10 cd 20 hfd 0 hfd 10 hfd 20 total lipids (mg/g) 47 ± 2 41 ± 4 43 ± 2 98 ± 16d 59 ± 5a 59 ± 3b tag (mg/g) 5 ± 1 7 ± 2 8 ± 2 27 ± 8d 12 ± 4a 16 ± 2b,d cholesterol (mg/g) 1.6 ± 0.1 2.4 ± 0.4a 1.9 ± 0.1 3.0 ± 0.3d 1.8 ± 0.1a 2.3 ± 0.2b phospholipids (mg/g) 10.2 ± 0.2 11.6 ± 1.6 11.1 ± 1.0 9.7 ± 0.4 14.2 ± 0.4a 13.7 ± 0.8b,d sum sfa 10.9 ± 0.3 13.3 ± 1.0 14.6 ± 0.7 21.2 ± 4.4d 15.0 ± 0.8 14.3 ± 1.0b 14:0 ma 0.10 ± 0.01 0.16 ± 0.02 0.45 ± 0.03b,c 0.16 ± 0.04 0.10 ± 0.02 0.11 ± 0.01d 16:0 pa 4.9 ± 0.2 6.9 ± 0.8 8.3 ± 0.5b 10.4 ± 2.4d 7.3 ± 0.9 7.0 ± 0.6b 18:0 sa 5.7 ± 0.1 6.0 ± 0.3 5.6 ± 0.2 10.5 ± 2.8d 7.4 ± 0.3 6.9 ± 0.5b sum mufa 3.9 ± 0.3 5.9 ± 1.4 8.0 ± 0.4 10.7 ± 2.8d 7.0 ± 1.1 6.8 ± 0.8b 18:1 (n-9) oa 3.3 ± 0.3 4.9 ± 1.1 6.5 ± 0.3 10.2 ± 2.7d 6.7 ± 1.1 6.4 ± 0.8b sum n-6 fa 10.6 ± 0.5 10.5 ± 0.6 8.9 ± 0.6 22.1 ± 4.3d 15.9 ± 1.1 14.3 ± 1.6b,d 18:2 (n-6) la 4.1 ± 0.4 4.1 ± 0.3 3.8 ± 0.4 11.9 ± 3.1d 7.2 ± 1.2a 6.3 ± 0.9b 18:3 (n-6) gla 0.09 ± 0.00 0.13 ± 0.05 0.08 ± 0.00 0.22 ± 0.04d 0.13 ± 0.02 0.12 ± 0.01b 20:4 (n-6) aa 5.8 ± 0.2 5.3 ± 0.2 4.8 ± 0.2 7.8 ± 0.6d 7.0 ± 0.2d 6.3 ± 0.6b,d sum n-3 fa 2.1 ± 0.1 2.3 ± 0.2 2.4 ± 0.1 3.2 ± 0.4d 2.7 ± 0.1 2.6 ± 0.1b 18:3 (n-3) ala 0.09 ± 0.02 0.11 ± 0.02 0.13 ± 0.03 0.30 ± 0.11d 0.18 ± 0.03 0.15 ± 0.02b 22:5 (n-3) dpa 0.38 ± 0.02 0.31 ± 0.07 0.22 ± 0.02 0.65 ± 0.10d 0.36 ± 0.03a 0.29 ± 0.04b 22:6 (n-3) dha 1.4 ± 0.1 1.6 ± 0.1 1.6 ± 0.1 2.1 ± 0.2d 2.1 ± 0.1d 1.9 ± 0.1 fatty acid concentrations are expressed as mg/g. mean ± sem. ap < 0.05, day 0 versus day 10 for each group. bp < 0.05, day 0 versus day 20 for each group. cp < 0.05, day 10 versus day 20. dp < 0.05, cd versus hfd at the corresponding day of pregnancy. cd: control diet; hfd: high-fat diet. table 3. adipose tissue fatty acid concentration (mg/g) in day-0 or day-20 pregnant rats on control diet (n ¼ 9, 8), or high-fat diet (n ¼ 9, 15). diet and day of pregnancy cd 0 cd 20 hfd 0 hfd 20 sum sfa 273 ± 5 374 ± 17a 302 ± 23b 257 ± 11a,b 14:0 ma 12.7 ± 0.3 22.2 ± 2.0a 14.8 ± 1.4 11.0 ± 0.6a,b 16:0 pa 230 ± 5 313 ± 13a 229 ± 20 196 ± 9b 18:0 sa 27 ± 1 39 ± 3a 56 ± 4b 48 ± 2a,b sum mufa 332 ± 7 476 ± 23a 434 ± 27b 366 ± 15a,b 18:1 (n-9) oa 268 ± 6 376 ± 17a 386 ± 24b 329 ± 13a,b sum n-6 fa 211 ± 6 180 ± 8 a 224 ± 12 197 ± 7a 18:2 (n-6) la 202 ± 6 170 ± 8a 213 ± 11 184 ± 7a 18:3 (n-6) gla 1.2 ± 0.03 1.2 ± 0.1 0.7 ± 0.1b 0.9 ± 0.1b 20:4 (n-6) aa 4.2 ± 0.2 5.1 ± 0.6 4.4 ± 0.4 6.0 ± 0.3a sum n-3 fa 14 ± 1 21 ± 1a 11 ± 1b 13 ± 0.4a,b 18:3 (n-3) ala 11.6 ± 0.5 17.0 ± 1.1a 8.6 ± 0.7b 9.1 ± 0.3b 22:5 (n-3) dpa 0.6 ± 0.04 0.00a 0.7 ± 0.1 0.00a 22:6 (n-3) dha 1.0 ± 0.1 1.4 ± 0.2a 0.7 ± 0.1 1.2 ± 0.1a values are mean ± sem. ap < 0.05, day 0 versus day 20 for each group. bp < 0.05, cd versus hfd at the corresponding day of pregnancy. cd: control diet; hfd: high-fat diet. 130 p. wentzel et al. resorptions in 32% of the hfd offspring compared with 3.7% in the cd offspring (figure 4(a)). whole-embryo culture day-9 cd embryos were cultured in vitro and harvested after 48 h, corresponding to gestational day 11 in vivo. we found an increased malformation score (reflecting open neural tube, somatic malrotation, and heart defects) in embryos cultured in serum of day-20 pregnant hfd rats compared with embryos cultured in serum of day-20 pregnant cd rats (figure 4(c)). furthermore, we found growth disturbances (decreased crown–rump length, and somite number) in embryos cultured in hfd serum compared with embryos cultured in cd serum (figure 4(c)). discussion the most important findings in the present study were that a high-fat diet during pregnancy in rats alters several maternal plasma/serum components and causes marked fetal maldevelopment. this was further corroborated by the demonstration of an enhanced teratogenic potential of hfd maternal serum used in wec. hfd rats ingested less food than cd rats, which resulted in both groups consuming the same number of calories (11,12). this agrees with previous reports in rats (28) and suggests that the disturbed embryonic and fetal development in the offspring of hfd rats is the result of the type of dietary fa composition rather than a higher maternal energy intake (29–31). the alterations in maternal hfd plasma/ serum were found in all types of nutrients—carbohydrates, proteins, and lipids. thus, we found increased concentrations of glucose, branched-chain amino acids, and b-hydroxybutyrate in hfd maternal plasma/serum. in the hfd rats we also found decreased plasma concentrations of tag and cholesterol, concomitant with decreases of most plasma pufas, which fits with the fact that most pufas in maternal plasma are carried associated to different lipoproteins in their esterified form both in humans (32) and in rats (33). the hfd rats had decreased plasma concentrations of the n-6 pufa la and increased concentration of aa. also, the n-3 pufa ala was decreased. the increased expressions of il-6 and il-10 in the adipose tissue may suggest an ongoing inflammation in the hfd rats. a postulated enhanced secretion of il-6 by hfd adipose tissue would contribute to chronic inflammation and cause increased c-reactive protein (crp) production, conditions that may affect the development of the embryo/fetus. in separate studies, we also evaluated crp effects in adipose figure 3. (a) gene expression of cuznsod, mnsod and gpx1 in adipose tissue of pregnant rats on control diet (cd, n = 8, blue bars), or high fat diet (hfd, n = 15, red bars) on gestational day 20. mean ± sem. � = p < 0.05 for cd vs. hfd. (b) gene expression of tnf-a, il-6 and il-10 in adipose tissue of pregnant rats on cd (n = 8 blue bars), or hfd (n = 15, red bars) on gestational day 20. mean ± sem. � = p < 0.05 for cd vs. hfd. (c) gene expression of adiponectin and resistin in adipose tissue of pregnant rats on cd (n = 8, blue bars), or hfd (n = 15, red bars) on gestational day 20. mean ± sem. � = p < 0.05 for cd vs. hfd. (d) gene expression of cuznsod, mnsod and gpx1 on gestational day 10 in embryos of six pregnant rats on cd (blue bars, n = 30 embryos), or in embryos of six pregnant rats on hfd (red bars, n = 30 embryos). mean ± sem. � = p < 0.05 for cd vs. hfd. upsala journal of medical sciences 131 tissue by immuno-staining for cd68 and found enhanced macrophage infiltration in hfd rats (wentzel et al., unpublished), corroborating the impression of an inflammatory state in the hfd rats. the decreased plasma concentrations of several pufa n-3 fas on gestational days 0 and 20 suggest a decreased transfer of pufa n-3 fas to the embryo and fetuses during hfd pregnancy (34). several of the long-chain polyunsaturated fas (lcpufas) have been shown to affect bone cells, i.e. osteoclasts and osteoblasts, via various cellular signaling pathways or growth factors, thereby affecting bone formation, resorptions, and bone density in animals and humans (35). furthermore, with regard to the reported alterations in both chondrogenesis and osteogenesis caused by diminished pufa n-3 fas (36–38), the possibility of a pufa n-3 fa deficiency-mediated teratogenic insult may be considered, since it is known that maternal lcpufas play an important role in fetal development (39). in contrast to the findings of lowered lipids in hfd maternal plasma, the hepatic concentrations of total lipids, tag, and cholesterol, as well as most individual fas were higher in the pregnant hfd rats than in the pregnant cd rats, findings that are in concert with previous reports in hfd-fed rodents (29). evidently, the hypotriglyceridemia in hfd plasma can be related to increased lipid content in the hfd liver. this accumulation of lipids appears to be a specific hepatic effect rather than adipose tissue process, since the fa profiles found in maternal hfd adipose tissue mainly coincides with the profiles of maternal hfd plasma. it has been shown that hfd decreases hepatic de novo lipogenesis (40) and also decreases the very-low-density lipoprotein secretion rate (40,41). the latter may be the main factor behind the increased liver tag and cholesterol content, as well as the decreased plasma tag found in the hfd rats of the present study. another possibility contributing to the decreased plasma tag concentrations found in our hfd rats could be an increase in adipose tissue lipoprotein lipase (lpl) activity, which is thought to be an important factor for the removal of tag from plasma (42). however, we could not detect any change in adipose tissue lpl activity in our hfd rats compared to the activity in cd rats (data not shown). although plasma post heparin lpl has been shown by xue et al. to be increased in rats fed hfd for 2 weeks, their plasma tag removal was actually decreased, and they were still hypertriglyceridemic without change in hepatic tag secretion (43). it is therefore proposed that the low plasma concentrations of tag and of most pufas in our hfd rats are the result of their retention in the maternal liver. it cannot be excluded that other factors may contribute to such adverse effects of hfd on embryonic-fetal development, i.e. insulin resistance, high levels of branched-chain amino acids and b-hydroxybutyrate, as well as oxidative stress. the higher plasma glucose concentration in the presence of unchanged insulin levels, and increased expression of resistin, il-6, and il-10 in adipose tissue of the pregnant hfd rats, suggests an imminent insulin-resistant condition (supplementary figure 2, available online). it has been shown previously that hfd causes insulin resistance in pregnant rats, which may alter fetal growth (16). the increased serum concentrations of branched-chain amino acids in our pregnant hfd rats may be the result of a decreased catabolism from a high fat intake, as previously proposed in human subjects (44). the increased branched-chain amino acid concentrations could also influence the embryo-fetal development in the hfd pregnancies as previously suggested in experimental studies of diabetes pregnancy (45,46). in line with this reasoning, a strong synergistic association has been suggested between lipids and branched-chain amino acids promoting metabolic disease (16). figure 4. (a) fetal outcome of rats on control diet (cd, n=8, left bar), or high fat diet (hfd, n=15, right bar) on gestational day 20. number and proportion of malformed (malf), resorbed (res) and viable (viab) fetuses. (b) alizarin red and alcian blue stained malformed fetuses from rats on control diet (cd) (left fetus), or high fat diet (hfd) (middle and right fetus). all fetuses show lack of tail, and the two hfd-fetuses (middle and right panel) also demonstrate decreased general ossification. (c) outcome in cd embryos cultured for 48h in serum from pregnant rats on either control diet (cd), or in serum from rats on high fat diet (hfd). 20 cd embryos were cultured in cd serum, and 20 cd embryos were cultured in hfd serum. malformation score (ms), crown-rump length (cr) and somite number (som). mean±sem. � = p < 0.05 for cd vs. hfd. 132 p. wentzel et al. https://doi.org/10.1080/03009734.2019.1604588 furthermore, the increased leptin concentration in hfd rats at pregnancy days 0 and 10, together with other metabolic alterations, may be the cause of embryo-fetal maldevelopment and skeletal malformations in the hfd rats (38,47). hfd indeed hampered the embryo-fetal development, as evidenced by increased resorption and malformation rates on gestational day 20. in previous studies, we have estimated the malformation rate in early hfd pregnancy to be around 10% (gestational day 11, in vivo, data not shown). in the present study, we found around 4% malformations (mainly skeletal anomalies) on gestational day 20, which may indicate that a number of malformed hfd embryos die in utero in late pregnancy and present themselves as resorptions on gestational day 20. the gene expression of cuznsod was increased in maternal hfd adipose tissue, supporting the presence of oxidative stress in tissues involved in adipogenesis (48). in addition, the decreased hfd embryonic gene expression of antioxidant enzymes, both cuznsod and mnsod found here, also suggests that hfd caused embryonic oxidative stress, possibly mediated by embryonic hypoxia (49). this fits with the findings of oxidative stress induced by hfd in other studies (50), which, indeed, may challenge the intrauterine development (17). in this context, it is worthy of note that serum from day20 pregnant hfd rats, in itself, is able to hamper embryonic development during only 48 h of exposure, as shown in wec experiments. two major differences between hfd and cd plasma/serum are increased concentrations of b-hydroxybutyrate and branched-chain amino acids, both of which have been shown to be teratogenic in wec experiments (45,46). the possible teratogenic effect of the increased leptin concentration, and decreased concentrations of sfa, mufa, and n-6 pufa, of the hfd plasma will be subject to future analysis. in conclusion, the present study shows that hfd in our rat model alters the maternal metabolic state, increases fetal resorptions in vivo, and increases the rate of fetal-embryonic malformations both in vivo and in vitro. moreover, hfd decreases embryonic cuznsod and mnsod gene expression and increases gene expression of cuznsod, il-6, il-10, and resistin in hfd maternal adipose tissue. we also found that hfd causes retention of essential fas in the maternal liver, which may be responsible for their decreased availability to the embryo-fetus, actively contributing to its shortand long-term adverse consequences. these findings suggest that the metabolic disturbances in hfd-induced maternal obesity have profound adverse developmental effects in the offspring. acknowledgements the technical assistance of milagros morante is gratefully acknowledged. disclosure statement none of the authors has any conflict of interest to declare. funding milagros morante was supported by grants from the fundaci�on ram�on areces (civp16a1835) of spain. we gratefully acknowledge the support by the foundation for health care research, uppsala university, grant no. 466–851710. none of these foundations had any role in the design of the project, the analysis of data, or the writing of this article. notes on contributors parri wentzel, phd, associate professor at the department of medical cell biology, uppsala university, sweden. ulf j. eriksson, md, phd, professor at the department of medical cell biology. uppsala university, sweden. emilio herrera, phd, professor at the department of biochemistry, ceu san pablo university, madrid, spain references 1. boekelheide k, blumberg b, chapin 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determination gene expression statistical evaluation results plasma/serum components liver components adipose tissue gene expression fetal outcome whole-embryo culture discussion acknowledgements disclosure statement funding notes on contributors references ujms110_2.pdf upsala j med sci 109: 97–114, 2004 the immunoglobulin genes and chronic lymphocytic leukemia (cll) review based on the doctoral thesis gerard tobin department of genetics and pathology, rudbeck laboratory, uppsala university, uppsala, sweden abstract a free full-text copy of this article can be found at the web page of upsala j med sci: http://www.ujms.se the somatic hypermutation (smh) status of the immunoglobulin (ig) v h genes can divide chronic lymphocytic leukemia (cll) into two prognostic subsets, with mutated v h genes display superior survival compared to unmutated cases. biased v h gene usage has also been reported in cll which may reflect antigen selection. in a v h gene analysis of 265 cll cases we confirmed the prognostic impact of the v h mutation status and found preferential v h gene usage in both the mutated and unmutated subset. interestingly, cll cases rearranging one particular v h gene, v h 3-21, displayed poor outcome despite that two-thirds showed mutated v h genes. many of the v h 3-21 utilizing cases expressed � light chains, rearranged a v�2-14 gene, and had homologous complementarity determining region 3s (cdr3s), implying recognition of a common antigen epitope. we thus believe that the cases rearranging the vh 3 -21 gene comprises an additional cll entity. we further analyzed the v h gene rearrangements and, specifically, the heavy chain cdr3 sequences in 346 cll cases to investigate the role of antigens in cll. we identified six new subgroups with similar hcdr3 features and restricted vl gene usage as in the v h 3-21-using group. our data indicate a limited number of antigen recognition sites in these subgroups and give further evidence for antigen selection in the development of cll. different mutational cutoffs have been used to distinguish mutated cll in addition to the 2% cutoff. using three levels of somatic mutations we divided 323 clls into subsets with divergent survival (<2%, 2–5% and >5% mutations). this division revealed a low-mutated subgroup (2–5%) with inferior outcome that would have been masked using the traditional 2% cutoff. a 1513a/c polymorphism in the p2x 7 receptor gene was reported to be more frequent in cll, but no difference in genotype frequencies was revealed in our 170 cll cases and 97 received 5 october 2004 accepted 13 october 2004 200 controls. however, cll cases with the 1513ac genotype showed superior survival than 1513aa cases and this was in particular confined to cll with mutated vh genes. in summary, we could define new prognostic subgroups in cll using ig gene rearrangement analysis. this also allowed us to gain insights in the biology and potential role of antigen involvement in the pathogenesis of cll. chronic lymphocytic leukemia b-cell chronic lymphocytic leukemia (cll) is the most common adult leukemia in western countries; approximately 400–500 cases are diagnosed annually in sweden. cll is characterized by a malignant b-cell proliferation with surface expression of cd5 and cd23, and low levels of surface immunoglobulin (ig) (1). cll is a heterogeneous disease, where some patients survive for many years with minimal or without treatment, whereas others die rapidly of their disease. cll is more common in men than women with a median age of diagnosis of approximately 65 years. in the clinic two prognostic scoring systems are used, rai and binet, but except for these there are few good clinical prognostic markers that can predict survival in cll at an early stage of the disease (2,3). however, molecular genetic studies are providing new insights in this disease. in cll, no single large genetic aberration has been indicated in all cases, although a number of aberrations are evident in up to 80% of cases (4). the most common of these are deletions at 13q14, 11q22–q23 (the atm gene), 17p13 (the p53 gene) and 6q21 and trisomy of chromosome 12. these aberrations can define risk groups, where poor risk groups include cases with del(11q), del(17p) and trisomy 12, while del(13q) is associated with better prognosis (4). the somatic hypermutation status of the immunoglobulin variable heavy chain (igvh) genes has been proven to be a powerful prognostic indicator in chronic lymphocytic leukemia (cll), since cases with mutated vh genes show significantly longer survival than unmutated cll cases (5,6). the analysis of the ig genes was thus suggested as a new important prognostic tool in cll (5–8). the immunoglobulin genes during an immune response the b-cell can secrete large quantities of ig which are a key element of the secondary immune reaction. the ig molecule consists of two identical heavy chains and two identical light chains (� or �) and is expressed on the surface of all mature b-cells. the heavy and light chains each consist of a variable (v) region which is responsible for antigen binding and a constant (c) region which decides the effector function. the ig heavy chain (igh) locus is located on chromosome 14q32 and the two immunoglobulin light chain (igl) loci on chromosome 2 (�) and 22 (�). the igh locus is estimated to be 1100 kb long and consists of clusters of v h , diversity (d), joining (j h ) and constant (c) germline gene segments. the igh locus consists of approx. 95 v h gene segments, of which 51 represent functional v h genes and the remaining non-functional pseudo genes (9). the v h genes can be grouped into seven v h gene families (v h 1–v h 7) based on at least 80% sequence homology within the v h gene family. the igl loci consists of v l and 98 j l gene segments, but in the igl loci no d genes have been identified. there are 36–40 functional v� and 29–33 functional v� genes (10,11). one of the most important stages in the b lymphocyte development process is the rearrangement of the igh/igl genes. the ordered rearrangement of the ig genes initially occurs during the transition from the pro b-cell to the pre b-cell stage and involves rearrangement of the igh locus. during this process, one d and one j h gene segment are initially recombined and thereafter one vh segment is joined to the previously formed dj h complex. this v h dj h rearrangement will then form the v region of the heavy chain ig molecule. the heavy chain ig is expressed on the surface in conjunction with a surrogate light chain to form the pre b-cell receptor (pre-bcr) which is essential for continued survival of the immature b-cell (12). the transition from the pre b-cell stage to the immature b-cell stage is marked by a cessation of the igh gene rearrangement process and activation of the igl gene recombination. joining of a v� gene to a j� gene occurs initially at the igk locus and if the rearrangement is successful it will be expressed along with the heavy chain ig as the b-cell receptor (bcr) on the cell surface. this event marks the transition to the mature b-cell stage with the cell expressing igm and igd. if a v�j� recombination does not render a functional � light chain, an additional igl rearrangement (the second igk locus or the ig� locus) event may be initiated to rescue the cell from elimination (13, 14). the large number of combinatorial events which can occur in the rearrangement of the igh and igl loci is the first stage in generating the large antibody diversity seen in the immune system. during the joining of the different gene segments random nucleotides may be inserted and base-pairs may be deleted in the junctions, for example the d-j h and v h -d junction, which further generates diversity in the antibody repertoire. just considering the combinatorial events of the heavy and light chain gene segments there are over 1.6 million possible combinations which is further amplified via random nucleotide insertions or deletions (15). this is estimated from the following calculation; the possible v h /d/j h gene combinations are 51v h genes × 27 d genes × 6 j h genes = 8,262 and together with a particular v l gene rearrangement, for example a v�j�, this will render 8262 × (40×5) = 1.6 million potential combinations. somatic hypermutation of the immunoglobulin genes recognition of the functional ig molecule with its specific antigen initiates a process of shm which primarily occurs in the germinal center (gc) environment of lymph nodes and follows antigen recognition and co-stimulation by helper t-cells (16). although mainly seen in the secondary lymphoid tissues such as the lymph nodes gcs have also been reported in other sites such as sites of inflammation in autoimmune disease (17, 18). the shm mechanism involves targeted mutations, mainly point mutations but also insertions and deletions, downstream of the v gene promoter of the igh and igl loci. shm occurs at rates of 10–5 to 10–3 mutations per base pair per generation (1 million fold higher than spontaneous mutations) (19). the shm process recognizes certain nucleotide motifs preferentially with a bias for rgyw (r =a/g, y=c/t and w=a/t) and its inverse repeat wrcy (20), and 99 recently a new motif has been proposed utilizing a dgyw/wrch motif where d=a/g/t and h=t/c/a (21). the shms are particularly focused in six regions called complementarity determining regions (cdrs) of which there are three in each v gene rearrangement. the cdrs are hypervariable regions and come together to form the antigen binding site of the ig molecule (figure 1). the heavy chain cdr3 is considered the most variable region between different ig as it spans the v h dj h junction and contributes to the most diversity in the antibody binding site (22). mutations in cdrs may either lead to amino acid replacements (r) and possibly changes in the affinity for the specific antigen or silent (s) mutations which will leave the amino acid unchanged. successive rounds of shm therefore lead to production and selection of cells with higher affinity for the initiating antigen than the original b-cell. in the normal human repertoire 40% of b-cells show evidence of shm and express the characteristic memory marker, cd27 (23). the ig genes and shm in b-cell neoplasms b-cell leukemias/lymphomas arising from the clonal expansion of a single b-cell which has passed the pre b-cell stage will have rearranged igh and igl genes unique to that cell and therefore the igh/igl rearrangements could be used as specific tumor markers. furthermore, the presence or absence of shm will indicate the differentiation stage of the malignant cell at transformation. this analysis is primarily carried out on the vh region of the igh gene rearrangements (24). currently, a cut off of 98% or greater homology to the germline genes is utilized to delineate shm to rule out polymorphisms and taq polymerase errors (25). for example, most mantle cell lymphomas are unmutated indicating that the transformation occurred in the pre-gc stage (26), whereas all multiple myelomas show evidence of extensive somatic hypermutation corresponding to a derivation from a terminally differentiated plasma cells (27). clonal development within a tumor may also be traced by analysis of the ig gene rearrangement in subclones. evidence of ongoing shm may 100 fig. 1. rearranged variable region of the immunoglobulin genes showing complementarity determining regions (cdr) 1, 2 and 3. the cdr’s come together to form the antigen binding site in the immunoglobulin molecule. v=variable, d=diversity and j=joining. be seen, for instance in follicular lymphoma (28–30), the gc b-cell like form of diffuse large cell lymphoma (31–33), burkitts lymphoma (34, 35) and splenic marginal zone lymphoma (36). these latter forms of lymphomas derive from gc bcells, which is in contrast to multiple myeloma and the activated b-cell like form of diffuse large b-cell lymphoma which do not show signs of ongoing shm and therefore originate from a more differentiated, post-gc b-cell (33,37). until recently, the cell of origin of cll was thought to be a naïve pre-gc cell with no evidence of shm (38–40). however, analysis of the v h gene mutation status in cll has demonstrated that roughly half of the patients show evidence of shm in the v h region indicating passage through a gc reaction while the remaining patients show germline configuration of their v h genes (5, 6, 41, 42). this has seen great interest in the field as this division was shown to be diagnostically useful with patients with unmutated v h genes in the tumor clone had having a clinically worse prognosis than patients with somatically hypermutated v h genes (5–8). thus, the division of cll by shm status was suggested as a good prognostic marker in this disease which in general shows a heterogeneous clinical spectrum. differences in the shm status may indicate different subpopulation origins, however immunophenotype and expression profiling has indicated a very similar overall phenotype to memory cells for both the unmutated and mutated cll cases (43–46). cll cells with unmutated v h genes may differentiate through a t-cell independent mechanism independent of the gc. cll cells with mutated v h genes, which in general have a low mutational load, may differentiate through a gc reaction or through a gc pathway outside the lymph node, which are characterized by a low mutation load and home to a similar environment to the unmutated cells. one possible environment which has been postulated is the marginal zone which is known to contain both ig unmutated and mutated b-cells (47–50). lately, the shm in mutated clls has been shown to be representative of the normal shm process since the v h gene sequences display characteristic targeting of the shm hotspots (51). v gene utilization in lymphoproliferative disease an overrepresentation of certain vh genes has been shown in different b-cell malignancies and in autoreactive b-cells in autoimmune diseases (26, 27, 38, 41, 42, 52–54). in cll, the v h 1-69 and v h 4-34 genes have been found preferentially expressed in several studies (5, 8, 38, 41, 42, 55, 56). over utilization of specific v h genes have also been demonstrated in other b cell tumors, such as salivary gland malt lymphoma (v h 1-69) (57), multiple myeloma (v h 1-69, v h 3-9, v h 3-23, and v h 3-30) (27), mantle cell lymphoma (v h 3-21 and v h 4-34) (26, 53) and nodal marginal zone b-cell lymphoma (nmzl) (v h 1-69 and v h 4-34) (54). in cll, differences are also evident within the ig unmutated and mutated cll groups. in the mutated group, restricted usage of the v h 3-07 gene and the v h 4-34 gene has been demonstrated (5, 41). the latter v gene has been associated with both self and non-self antigens as well as has been found overrepresented in both autoimmune disease and different lymphomas (58–62). in the unmutated cll group, 101 biased usage of the v h 1-69 gene has been reported by a number of groups with v h 1-69 representing a large proportion of the unmutated v h gene rearrangements (5, 42, 56, 63, 64). in addition to frequent usage, the v h 1-69+ cll rearrangements show unusual molecular characteristics such as preferential rearrangement of certain d and j genes and a cdr3 longer than normally seen in cll rearrangements not utilizing the v h 1-69 gene (56, 63). frequent use of one allele (51p1) is another feature of these rearrangements (38, 39, 63). a recent paper has also shown that the features of the v h 1-69 rearrangements in cll are distinctly different from that seen in the normal elderly population (64). the finding of preferential v h , d and j h gene usage as well as longer than average cdr3s in v h 1-69+ cll has led to the speculation of a possible antigen component involved in the development of cll with the hypothesis that bcrs encoded by specific v h dj h combinations could generate ig molecules with affinity to similar antigenic epitopes. a p2x 7 receptor polymorphism and cll p2x 7 is one of seven members of the p2x family of nucleotide receptors. it functions as a ligand (atp-adenosine triphosphate) gated ion channel expressed on the plasma membrane and is expressed on many cells in the immune system, such as lymphocytes, macrophages and dendritic cells, as well as smooth muscle cells, fibroblasts, neurons and epithelial cells. p2x 7 is a bifunctional receptor: at tonic low level stimulation of atp it acts as an ion channel, and may provide growth and survival stimulation, while at longer chronic stimulation of atp it forms a large pore which allows molecules of large size (<900da) to pass through and triggers apoptosis (65–67). in both normal b-cells and t-cells a central role for p2x 7 appears to be in apoptosis; however its growth stimulatory properties have been less well defined, but transfection of p2x 7 cdna has been shown to result in increased proliferation of lymphocytes (68). p2x 7 has been indicated to be of importance for development and progression of cll in two recent reports (69, 70). a single nucleotide polymorphism (snp) at nucleotide position 1513 (1513 a→c) was shown to result in an amino acid change from glutamic acid to alanine in the carboxyl terminal tail and resulted in a loss of function in the receptor in normal b-cells and cll cells (70, 71). individuals with the 1513aa genotype had normal function while individuals with the 1513ac genotype have a 50% loss of receptor function and 1513cc individuals have an almost complete loss of function. while affecting the function, the 1513ac genotype does not appear to affect the surface expression of the receptor (71). interestingly, the 1513ac genotype has been reported to be associated with increased incidence in cll patients (42%) compared to healthy controls (11%) (70). furthermore, expression of the p2x7 receptor in cll has been associated with a more aggressive disease which showed higher expression than indolent cases (69). cll utilizing the ig gene v h 3-21 we initially performed v h gene analysis in 119 cll patients to investigate the shm 102 status as well as the v h gene usage with the ig unmutated group comprised 69 cases (58%) and the mutated group 50 cases (42%) (72). there was an overrepresentation of certain v h genes with the v h 1 family gene, v h 1-69, and the v h 3 family gene, v h 3-21, representing 15.7% and 11.2% of the v h genes amplified, respectively. in accordance with previous studies, the v h 1-69+ cases were almost exclusively represented in the ig unmutated group (5, 42, 56, 63, 64). as expected, the cll cases utilizing the v h 1-69 gene showed poor survival as all of them belonged to the unmutated group, but surprisingly the cases utilizing a mutated v h 3-21 gene also had a poor overall survival (median 63 months). in order to further characterize the v h 3-21 group, we extended this study to 265 cll cases and identified 31 v h 3-21 cases with 21 displaying mutated and ten unmutated v h genes (73). we could confirm the worse prognosis of the mutated v h 3-21+ cases (n=21) with a median survival of 72 months and showed that the whole v h 3-21 group, including both mutated and unmutated, had a short median survival of 83 months (figure 2). from a clinical point of view this group is interesting as they display a poor overall survival, particularly considering that two thirds of the cases are mutated and, therefore, should have a good prognosis. we therefore conclude that v h 3-21-utilizing cases do not fit the pos103 fig. 2. survival analysis of 265 cll cases displaying ig v h unmutated (median survival 70 months), ig v h mutated (median survival 146 months) and v h 3-21 cases (median survival 83 months). tulated prognostic classification of mutated and unmutated cll and that v h 3-21 gene usage defines a group with worse survival irrespective of the shm status. at the molecular level the v h 3-21+ cases showed unusual characteristics such as short cdr3s (8 codons) compared to the remaining rearrangements (15 codons) and they also displayed highly homologous cdr3s, seven cases showed identical cdr3s with a conserved amino acid motif (ardangmdv) and a further five cases showed only one amino acid difference from these seven cases. a restricted � expression was revealed in both unmutated and mutated v h 3-21+ cases (28 cases express � and 3 � on immunophenotype), therefore, we sequenced the light chain gene rearrangements and found that 24 out of 28 � expressing cases rearranged the same v� gene, v�2-14. considering the importance of the cdrs in the antigen binding site and in particular the cdr3, our results of restricted cdr3 features and predominant v�2-14 rearrangements indicated that the vh3-21 + cases showed signs of a common antigen recognition site. the v h 3-21 gene has been shown to be utilized in certain autoantibodies produced in rheumatoid arthritis, sjögren’s syndrome and primary biliary cirrhosis and, similarly to the v h 1-69 gene, an association with autoreactivity specificities has been shown for this vh gene (60, 74, 75). however, the v h 3-21/�2-14 antibody specificity and its role in the development of cll have yet to be defined. in the scandinavian population they represent approximately 11% of cll cases, but they have also been identified in a large german cll cohort (76) and in a cohort of australian cll patients (3 vh3-21 gene rearrangements out of 100 cll cases analyzed, own unpublished data). the potential role of antigens in cll there exists several lines of evidence indicating that an antigen component is involved in cll development, such as the overrepresentation of v h 1-69 gene rearrangements in cll and the autoand polyreactive specificities associated with v h 1-69 usage (41, 42, 63, 77, 78) as well as the recent findings of similar cdr3s in the v h 3-21+ cases. however, a definitive antigenic identification has remained elusive. in order to investigate antigen involvement we characterized 407 v h gene rearrangements in 346 cll (79). we carried out alignment analysis of the hcdr3 sequences in all 368 functional rearrangements to study the region which is considered to contribute to the most variability between ig binding sites as it spans the v h /dj/ h junctions. from this analysis we identified seven groups (51 cases) with similar hcdr3s with three or more cases in each group (79). the largest group contained 22 cases utilizing the v h 3-21 gene with very high homology between the hcdr3s as discussed previously. the six additional groups consisted of 29 cases which had within each group high similarities between their hcdr3s, i.e. similar v h , d and j h gene usage, utilization of the same d reading frame and similar lengths of the hcdr3s. additionally, many of these groups displayed similarities in the junctional regions. for example, four cases rearranged the v h 1-69 gene together with a d3-16 and a j h 3 gene, where the d3-16 gene was used in the same reading 104 frame and the cdr3 (18 amino acids) was of identical length in all cases. the v h /d junction consisted of two amino acids and was identical in all rearrangements while four out of five amino acids were identical in the d/j h junction (figure 3). we also performed vl gene rearrangement amplification and sequencing in the six new groups with homologous hcdr3s and could show highly restricted vl gene use within the subgroups, as have found in the v h 3-21+ cases. in the four cases rearranging the v h 1-69/d3-16/j h 3 genes, all rearranged a v�a27 gene. to investigate if the preferential rearrangement of certain v l genes was restricted to cases with homologous rearrangements, we analyzed further cll cases utilizing the same v h gene as in the homologous groups (v h 1-69, v h 4-39 and v h 1-2) but without homology between their hcdr3s. a variety of different v l genes were found rearranged in these cases, for example in the v h 1-69 utilizing cases 14 non-homologous cll cases rearranged ten different v l genes and restriction was only seen in the two v h 1-69 homologous groups. these data indeed indicate that preferential v l gene rearrangement was restricted to cll cases with homologous hcdr3s. the probability of the same v h /d/j h rearrangement occurring twice is in the order of 1/8262 and occurring with one particular light chain rearrangement is less than 1/1.6 million therefore these findings are unlikely to be a random finding. further support was given by an analysis of 227 hcdr3 from the public databases that showed only homology between two sequences as well as the reported finding of no duplicate ig rearrangements in about 10,000 ig sequences obtained from normal tonsils (80–82). previous studies have identified the same v h 1-69/d3-3/j h 6 (63) and v h 4-39/d6-13/j h 5 (83, 84) combinations in cll with similar amino acid motifs in the hcdr3s, and, additionally, we found four v h 1-69/d3-16/j h 3 rearrangements from cll cases in the public databases which were highly homologous to our v h 1-69 group cases. all of these latter findings strengthen our data of groups with restricted rearrangements and homologous hcdr3s in cll. our study reveals indirect signs of antigen selection in subsets of cll with multiple cases showing both highly homologous v h and v l rearrangements, thus suggesting a limited number of antigens involved in the selection of these bcrs. a recently identified v h 4-39/v�o2 utilizing igg+ cll subgroup, that displayed similar hcdr3s to our v h 4-39 group, has been shown to have comparable antigen binding sites to antibodies reacting towards bacterial carbohydrates and certain autoantigens (84). an antibody termed smi, which utilizes the v h 1-69/v�a27 genes and has been isolated from a cll patient, shows reactivity to a variety of self antigens such as human ig, myoglobulin thyroglobulin, actin and ssdna (85). furthermore, an anticardiolipin antibody was identified from the public database with a similar hcdr3 to the v h 1-69/d3-16/j h 3 rearrangements in our study (genbank accession no aal67508).these groups with highly homologous hcdr3 coupled to restricted vl gene usage were confined mainly to the ig unmutated group with poor prognosis. considering the recent data showing more bcr signaling, as measured by syk phosphorylation, in unmutated cases than mutated cases (86), this may indicate that a limited number of antigens may signal through the bcr and play a 105 more important role in tumor progression of the unmutated cases. further studies of bcr signaling within these specific subgroups and the antigenic specificity may shed light on these issues. somatic hypermutation in cll the vh unmutated and mutated groups in cll display differential prognosis in terms of overall survival, which could be confirmed in both of our initial studies (87). additionally, this division indicates that the original b-cells may have undergone different biological pathways during their differentiation before transformation. the classification of shm currently utilizes a border of 2% to delineate cll cells which have or have not undergone the process of shm (5, 6, 88–90). this border was originally set to rule out polymorphisms and taq polymerase error; however the true biological border is unknown. recent reports have suggested other borders as the best prognostic indicator such as 4% or 5% (91, 92). we investigated the mutation border at various levels in 323 cll cases and in our cohort the best statistical discriminator of overall survival was the division into three groups based on <2%, 2–5% and >5% mutations and the median survival for these three groups was 72 months, 97 months and 150 months, respectively. this resulted in division of the ig mutated group into two subsets with the low-mutated (2–5%) cases displaying worse survival compared to the high-mutated (>5%) cases. thus, the low-mutated (2–5%) cases represent a group with poor survival which would have been masked using the 2% cutoff level. the poor prognostic v h 3-21 cases contained mainly lowmutated cases and therefore could be incorporated into the low-mutated group which fits the poor prognosis associated with both of these subsets. only two v h 3106 fig. 3. amino acid alignment of 4 cll v h 1-69 cdr3 rearrangements and an additional 4 cases from the public databases (db1-4). also shown is the cdr3 from an anticardiolipin antibody. a dot indicated homology with the first sequence. 21+ cases showed >5% mutations and one of these cases displayed poor overall survival of 63 months. the utilization of v h gene sequencing in the routine diagnostic setting has not been readily accepted as it is considered technically difficult and expensive to carry out, therefore surrogate markers such as the protein tyrosine phosphatase, zap-70 which can be analyzed by flow cytometry or immunochemistry have been proposed (93, 94). initial studies showed a high correlation between the vh mutation status and zap-70 expression, where cll cases with unmutated vh genes showed higher expression of zap-70, than mutated cases, however, in larger studies discordant result were evident especially in the ig mutated group (93–96). these possible divergent results may underlie a different clinical course for certain patients such as the low mutated (2–5%) cll cases. subdivision of cll into more than two prognostic groups may thus be useful in evaluation of zap-70. the reason for the diverse prognosis between the cll groups with different mutational frequencies is unknown. the degree of shm in the cell may reflect the number of rounds of divisions the cells have undergone in the gc which is supported by a correlation with telomere length and degree of mutations (97). therefore, it is possible that mutated cells which have divided more frequently during the gc reaction (highly mutated, >5%) may be more anergic/resting post gc when they transform to cll and therefore represent more benign tumor cells. alternatively, poor risk genetic aberrations such as p53 mutation/deletions may underlie the worse prognosis in this low mutated (2–5%) cll subset. polymorphism in the p2x7 receptor the loss of function snp (1513 a→c) of the p2x7 receptor gene was analyzed in a cohort of 170 cll cases as well as in 200 healthy age-matched controls (98). the 1513 ac genotype showed a frequency of 23% and 21% in the cll cohort and control population, respectively, whereas the 1513cc genotype was represented in <1% of cll cases and 5% of controls, respectively. this study did not find any association between increased prevalence of the 1513 polymorphism and development of cll, which is in contrast with the report by wiley et al that showed an increased incidence of this snp in cll (70). this difference could be explained by difference in patient groups included, since the study by wiley et al included familial cll cases which may have resulted in an overrepresentation of the 1513ac genotype in their study. in this study the overall survival was found to be significantly different for cll cases with the 1513aa vs. the 1513ac genotype; the median survival for cases with the 1513ac (n=35) genotype was 104 months compared to 72 months in cases with the 1513aa genotype (98). when the ig unmutated and mutated cll cases were associated with p2x 7 genotype data no difference in overall survival was demonstrated between the ig unmutated with 1513aa and the 1513ac genotypes. however, in the mutated group the 1513ac genotype showed significantly different survival compared to the 1513aa genotype; the ig mutated 1513aa cases showed 107 a median survival of 98 months compared to 151 months in ig mutated 1513ac cases. the poor prognostic group utilizing the v h 3-21 gene did not influence the results when excluded from this study. the reason for the better survival associated with the 1513ac genotype in the ig mutated cases is currently unknown. wiley et al has shown that cll cells with the 1513ac allele have a decreased susceptibility to undergo apoptosis and they may therefore have a prolonged survival in vivo, which seems in contrast to the better survival seen in the ig mutated 1513ac individuals in our study. however, the bifunctional role of the p2x 7 receptor in lymphocyte proliferation and in apoptosis makes it difficult to predict which mechanism is playing a more important part. alternatively, cll cells are sensitive to their microenvironment (99) and reduced function of the p2x 7 receptor in the t-cells and/or dendritic cells may therefore influence the survival of the cll cells. the cll cells with mutated vh genes may be more dependent of t-cell interactions after traversing the gc and more susceptible to cells in the microenvironment, whereas cll cells with unmutated vh genes may develop through a t-independent pathway and therefore may be less susceptible to microenvironment changes. future studies are required to investigate the mechanisms involved in cll. currently, a number of reports have been published which do not support this association of the p2x 7 polymorphism with overall survival (100–102). no association was found between the p2x 7 polymorphism and sensitivity to fludarabine, bcl–2 expression and overall survival in a study by starcyznski et al. (101). furthermore, a study by zhang et al study could not reveal any association between the p2x 7 polymorphism, overall survival and v h gene status, while nuckel et al found no association with treatment free survival (100, 102). however, a number of important differences exist between the studies which may explain the divergent results. the cll cases from our study are mainly from referral centers which would tend to deal with more aggressive cases, while the materials in the zhang et al study were collected mainly from non-referral centers and contain more benign cases (102). the median survival for ig mutated cases in our cohort was 111 months (<10yrs) whereas in the report from zhang et al their ig mutated group had a median survival of 25 years (102). also, the report from starcyznski et al and nuckel et al did not show the ig mutated division of the 1513ac genotype in survival, which was the strongest finding in our report (100, 101). an analysis on a larger cll cohort will hopefully resolve the issue of the importance of the p2x 7 polymorphism in cll prognosis. concluding remarks the shm status in cll is a powerful prognostic indicator of survival in this clinically heterogeneous disease, however; 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72: 259–263. 101. starczynski j, pepper c, pratt g, et al. the p2x7 receptor gene polymorphism 1513 a–>c has no effect on clinical prognostic markers, in vitro sensitivity to fludarabine, bcl-2 family protein expression or survival in b-cell chronic lymphocytic leukaemia. br j haematol. 2003;123:66–71. 102. zhang ly, ibbotson re, orchard ja, et al. p2x7 polymorphism and chronic lymphocytic leukaemia: lack of correlation with incidence, survival and abnormalities of chromosome 12. leukemia. 2003;17:2097–2100. about the author gerard tobin received the israel hwasser award from the uppsala medical association for the best thesis in preclinical medicine in the academic year 2003/2004. he is at present working at the department of genetics and pathology, rudbeck laboratory in uppsala university. corresponding address: dr. gerard tobin, dept of genetics and pathology, rudbeck laboratory c11, uppsala, 751 85 sweden. corresponding address: e mail: gerard.tobin@genpat.uu.se 113 phone +46186110213 114 vol_117_004_sups_a_687406 399..401 full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 microorganisms causing pyogenic spondylitis: comparison of community and hospital-acquired types tatsuro sasaji, noboru yamada & kazuo iwai to cite this article: tatsuro sasaji, noboru yamada & kazuo iwai (2012) microorganisms causing pyogenic spondylitis: comparison of community and hospital-acquired types, upsala journal of medical sciences, 117:4, 399-401, doi: 10.3109/03009734.2012.687406 to link to this article: https://doi.org/10.3109/03009734.2012.687406 © informa healthcare published online: 01 jun 2012. submit your article to this journal article views: 852 view related articles citing articles: 1 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009734.2012.687406 https://doi.org/10.3109/03009734.2012.687406 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009734.2012.687406 https://www.tandfonline.com/doi/mlt/10.3109/03009734.2012.687406 https://www.tandfonline.com/doi/citedby/10.3109/03009734.2012.687406#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009734.2012.687406#tabmodule upsala journal of medical sciences. 2012; 117: 399–401 original article microorganisms causing pyogenic spondylitis: comparison of community and hospital-acquired types tatsuro sasaji, noboru yamada & kazuo iwai department of orthopedic surgery, fukushima rosai hospital, 3-numajiri, tsuzura-machi, uchigo, iwaki 973-8403, japan abstract pyogenic spondylitis is a common infectious disease caused by various microorganisms. it is difficult to predict the infecting microorganism at the time of initiation of treatment. pneumonia is generally clarified into community or hospitalacquired types based on where the infection was acquired, and the infecting microorganisms are different for each type. we retrospectively analyzed 20 cases of pyogenic spondylitis treated in our hospital and categorized the cases into community and hospital-acquired types. we also identified the infecting microorganisms and the rate of sepsis in each type. there were 12 cases of community-acquired and 8 of hospital-acquired infection. the major infecting microorganisms responsible for the community-acquired type were gram-positive cocci, and those responsible for the hospital-acquired type were methicillinresistant staphylococcus aureus and gram-negative bacilli. the rate of sepsis was significantly different for both groups: 16% for the community-acquired type and 75% for the hospital-acquired type. the classification of pyogenic spondylitis based on where the infection was acquired may be useful for predicting which microorganisms are responsible for the disease. key words: classification, infecting microorganism, pyogenic spondylitis, sepsis introduction pyogenic spondylitis is a common orthopedic infectious disease. the incidence of pyogenic spondylitis is increasing in japan, which is an aging society (1). treatment of pyogenic spondylitis is generally conservative and comprises of antibiotics, bed rest, and immobilization (2). antibiotics should be selected according to the sensitivity of the infecting microorganisms. the recommended first-line antibiotics include first and second generation cephalosporins and penicillin, to which gram-positive cocci are sensitive. however, the infecting microorganisms are sometimes bacteria other than gram-positive cocci; in such cases, other antibiotics are required. no reports on the classification of pyogenic spondylitis based on the infecting microorganisms are available. for example, pneumonia is generally divided into community and hospital-acquired types based on where the infection was acquired, because the infecting microorganisms are different in each group (3,4). therefore, we hypothesized that the microorganisms causing pyogenic spondylitis would also differ based on where the infection is acquired, namely, whether the infection is community or hospital-acquired. we retrospectively analyzed the microorganisms causing pyogenic spondylitis among the cases treated in our hospital. the purpose of the present study was to clarify the usefulness of a classification based on where the infection was acquired. patients and methods all patients were informed that the data from their cases would be used for the study. correspondence: tatsuro sasaji, department of orthopedic surgery, fukushima rosai hospital, 3-numajiri, tsuzura-machi, uchigo, iwaki 973-8403, japan. fax: +81-246-26-1322. e-mail: taturosasaji@hotmail.com (received 9 april 2012; accepted 17 april 2012) issn 0300-9734 print/issn 2000-1967 online � 2012 informa healthcare doi: 10.3109/03009734.2012.687406 subjects thesubjectsofthisstudyincluded20patientswhowere treated conservatively for pyogenic spondylitis in our hospital between 2007 and 2011. eleven patients with pyogenic spondylitis caused by unidentified microorganisms were excluded. we treated no case of pyogenic spondylitis surgically. the diagnosis of pyogenic spondylitis was established by culture of the infected intervertebral disc, laboratory data, including white blood cell count and c-reactive protein level, and magnetic resonance imaging. we analyzed age, sex, the infectingmicroorganisms,therateofsepsis,anddivided the patients into two types: community-acquired type of pyogenic spondylitis and hospital-acquired type. we analyzed the associated disease in the hospitalacquired type of pyogenic spondylitis. according to the surviving sepsis campaign guideline, the mortality rate of sepsis within the first month of diagnosis was 30% (5). we thought that it was important whether pyogenic spondylitis was accompanied with sepsis or not. we analyzed the infecting microorganisms and merger rate of sepsis in each type. we defined the community-acquired type of pyogenic spondylitis as the disease that occurred in patients at home and the hospital-acquired type as the disease that occurred in patients treated and hospitalized in other departments. sepsis was defined as a systemic inflammatory response syndrome (sirs) due to infection. sirs was diagnosed when two or more of the following criteria were present: high fever or hypothermia, tachycardia, tachypnea, and abnormal white blood cell count (6). statistical analyses for statistical purposes, the data were analyzed using fisher’s exact probability test to compare the merger rate of sepsis between the types. a probability value of < 0.05 was considered significant. statistical analyses were performed using graphpad prism (graphpad software inc., san diego, ca, usa). results atotalof12maleand8femalepatientswithameanageof 70 years (range 16–82 years) were included in the study. theinfectingmicroorganismswerestaphylococcusaureus in 5 cases, methicillin-resistant staphylococcus aureus (mrsa) in 3, streptococcus agalactiae in 3, coagulasenegative staphylococcus in 2, enterococcus faecalis in 2, and staphylococcusintermedius,pseudomonasaeruginosa,bacillus cereus, campylobacter species, and escherichia coli in 1 each. sepsis developed in 8 cases. the communityacquiredtypeofpyogenicspondylitisoccurredin12cases and the hospital-acquired type in 8. the infecting microorganisms in the community-acquired type were staphylococcus aureus in 5 cases, streptococcus agalactiae in 2, coagulase-negative staphylococcus in 2, and staphylococcusintermedius,escherichiacoli,andenterococcusfaecalis in 1 each. the infecting microorganisms in the hospitalacquired type were mrsa in 3 cases and enterococcus faecalis, pseudomonas aeruginosa, bacillus cereus, campylobacter species, and streptococcus agalactiae in 1 each (table i). gram-positive cocci were found in 10 cases of the community-acquired type. gram-negative bacilli were found in 4 cases and mrsa in 3 cases of the hospital-acquired type. sepsis occurred in 2 (16%) cases ofthecommunity-acquiredtypeand6(75%)casesofthe hospital-acquired type (table ii). the rate of sepsis was significantly different between the two types (p = 0.019). the associated diseases in the hospital-acquired type of pyogenicspondylitiswerealcoholichepatitisin3,chronic renal failure treated with hemodialysis in 1, ileus in 1, lung abscess in 1, lumbar injection in another hospital in 1, cardiopulmonary arrest treated in intensive care unit in 1. discussion the treatment of pyogenic spondylitis is generally conservative and comprises of antibiotics, bed rest, andimmobilization(2).appropriateantibioticselection is important until the sensitivity of the infecting microorganism can be established by culture sensitivity testing. staphylococcus aureus is reportedly the most table i. infecting organism in pyogenic spondylitis. infecting microorganism community-acquired type (12 cases) staphylococcus aureus streptococcus agalactiae coagulase-negative staphylococcus staphylococcus intermedius escherichia coli enterococcus faecalis (gram-positive coccus: 10 cases) hospital-acquired type (8 cases) methicillin-resistant staphylococcus aureus enterococcus faecalis pseudomonas aeruginosa bacillus cereus campylobacter species streptococcus agalactiae (mrsa: 3 cases; gram-negative bacilli: 4 cases) table ii. comparison of the rate of sepsis. community-acquired type (12 cases) hospital-acquired type (8 cases) the merger rate of sepsis (sepsis cases) 16% (2 cases) 75% (6 cases) p = 0.019 compared with each type (fisher’s exact probability test). 400 t. sasaji et al. common microorganism causing pyogenic spondylitis (2). on the other hand, microorganisms other than staphylococcus aureus have also been reported to cause this infection (2). it was unclear what factor was responsible for the difference in infecting microorganisms. we classified pyogenic spondylitis based on where the infection was acquired in the present study. the major infecting microorganisms were gram-positive cocci in the community-acquired type and gram-negative bacilli and mrsa in the hospital-acquired type. thus, classification based on where the infection was acquired may be useful for predicting the identity of the infecting microorganisms. since the development of chemotherapy, the prognosis of pyogenic spondylitis is generally good. generalized sepsis indicates a primary source of infection other than the spine (2). however, sepsis occurred in some cases in the present study. mortality associated with sepsis is unacceptably high, and rapid management is critical (5). in the present study, the rate of sepsis was higher in the group with hospitalacquired type of pyogenic spondylitis. immediate prescription of wide-spectrum antibiotics to which gram-negative bacilli and mrsa are sensitive is essential for the hospital-acquired type of pyogenic spondylitis. no reports on the classification of pyogenic spondylitis based on the infecting microorganisms are available. on the basis of present study, we assumed that classification based on where the infection was acquired was useful for predicting the identity of the infecting microorganism and for selecting the appropriate first-line antibiotics therapy. however, the number of cases was small in the present study. further studies with a large number of pyogenic spondylitis patients would be necessary to address this question. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. hiroshi o. epidemiology of pyogenic spondylitis. sekitui sekizui journal. 2008;21:1084–90; (japanese). 2. wood gw ii. infections of spine. in: canale st, editor. campbell’s operative orthopaedics. vol. 2. philadelphia: mosby; 2003. p 2029–60. 3. mandell la, wunderink rg, anzueto a, bartlett jg, campbell gd, dean nc, et al. infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults. clin infect dis. 2007;44(suppl 2):s27–72. 4. american thoracic society; infectious diseases society of america. guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcareassociated pneumonia. am j respir crit care med. 2005; 171:388–416. 5. surviving sepsis campaign. available at: http://www.survivingsepsis.org/bundles/pages/default.aspx. (accessed 9 april 2012). 6. bone r, balk r, cerra f, dellinger rp, fein am, knaus wa, et al. definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. the accp/sccm consensus conference committee. american college of chest physicians/society of critical care medicine. chest. 1992;101:1644–55. microorganisms causing pyogenic spondylitis 401 ujms 110 (3) bra upsala j med sci 110 (3): 251–258, 2005 desmoplastic melanoma arising in the ankle masahito hatori1, mika watanabe2, hiroshi watanabe3, masami hosaka1, shoichi kokubun1 1department of orthopaedic surgery, tohoku university school of medicine, sendai, japan. 2department of pathology, tohoku university hospital, sendai, japan. 3department of dermatology, tohoku university school of medicine, sendai, japan abstract desmoplastic melanoma (dm) is an uncommon but potentially devastating malignancy that can be cured with early recognition and surgery. dm often occurs in elderly men and develops on sun-exposed areas with the head and neck being the most common site of origin followed by the extremities and trunk. we report a rare case of dm occurring in the ankle in an 85-year-old woman. magnetic resonance imaging of the affected ankle showed a 4.3 x 4.3 x 2.5 cm sized mass which was located in the subcutaneous region, attached to lower leg muscles, the fibula, and the calcaneus. no metastasis to the lung, liver, bones or abdominal lymph nodes was found. in spite of below knee amputation, the patient died of lung metastases 10 months after surgery. among several factors causing early death of the patient, a large size, a deep location and rapid growth of the tumor seem to be most important. introduction desmoplastic melanoma (dm) is an uncommon but potentially devastating malignancy that can be cured with early recognition and surgery. dm has clinical as well as histological features that may be subtle and overlooked, or misdiagnosed as other benign or malignant lesions that would require less aggressive therapy for cure [1]. the difficulty in its diagnosis lies in that most patients have amelanotic skin lesions [2]. dm often occurs in elderly men and develops on sun-exposed areas with the head and neck being the most common site of origin followed by the extremities and trunk [3,4,5,6]. although the incidence of malignant melanoma is much lower 251 received 28 january 2005 accepted 17 february 2005 key words: desmoplastic melanoma, ankle, magnetic resonance imaging, s100 in the japanese than in caucasians, the subungual and periungual sites are commonly found in japanese. dm in the foot is, however, very rare [7]. we report a case of dm occurring in the ankle. case report the patient was an 85-year-old woman. she noticed a non-symptomatic mass in her left ankle. three month later, she was referred to us with the suspicion of malignancy. the colour of the skin over the mass looked normal. the mass was hard as a brick and fixed to the base but had mobility to the skin. no lymph nodes were palpable in the ipsilateral popliteal fossa and inguinal region. bone metastasis was ruled out by bone scan. there was no evidence of metastasis to lung, liver or abdominal lymph nodes by computed tomography. computed tomography of the affected ankle showed a iso-density mass, the periphery of which was irregularly 252 1a fig. 1 magnetic resonance imaging demonstrating a mass located in the subcutaneous region, attached to lower leg muscles, the fibula, the calcaneus and the achilles tendon. the tumour had iso-signal intensities on t1 weighted images (a: axial image ) and inhomogenously high signal intensities on t2 weighted images (b: axial image, c: sagittal image). enhanced. magnetic resonance imaging demonstrated a mass located mainly in the subcutaneous region, attached to lower leg muscles, the fibula, and the calcaneus, deviating the achilles tendon medially. the mass was poorly circumscribed and 4.3 x 4.3 x 2.5 cm in size. it had iso-signal intensities on t1 weighted images and inhomogenously high and low signal intensities on t2 weighted images. the tumour was inhomogenously and highly enhanced after gadolinium injection (fig 1). histological examination of open biopsy specimen demonstrated proliferation of short spindle to polyhedral cells in the dense collagenous stroma. these cells had 253 fig. 1 magnetic resonance imaging demonstrating a mass located in the subcutaneous region, attached to lower leg muscles, the fibula, the calcaneus and the achilles tendon. the tumour had iso-signal intensities on t1 weighted images (a: axial image ) and inhomogenously high signal intensities on t2 weighted images (b: axial image, c: sagittal image). 1b small nuclei with moderately atypia showing little pleomorphism. they had narrow cytoplasm with slightly eosinophilic fine-granular appearance. cellular density was moderate and mitotic figure was almost not detected. focally there was a cluster of atypical cells containing brown granules in the cytoplasm, considered to be melanin 254 fig. 1 magnetic resonance imaging demonstrating a mass located in the subcutaneous region, attached to lower leg muscles, the fibula, the calcaneus and the achilles tendon. the tumour had iso-signal intensities on t1 weighted images (a: axial image ) and inhomogenously high signal intensities on t2 weighted images (b: axial image, c: sagittal image). 1c 255 2a 2b fig 2: microphotographs of the open biopsy specimen demonstrating proliferation of short spindle to polyhedral cells in the dense collagenous stroma. these cells had small nuclei with moderately atypia showing little pleomorphism. focally there was a cluster of atypical cells containing brown granules in the cytoplasm, considered to be melanin pigments. (a: low power view, b: high power view). pigments (fig 2). immunohistochemically positive staining for vimentin, s100 protein and �smooth muscle actin in these cells was observed. immunohistochemical staining after melanin removal showed that a few cells were reactive with hmb-45. melanin a antigen, cd 34, cd 68 and desmin were negative. from these histological findings, desmoplastic melanoma (dm) was suspected. below knee amputation 7 cm apart from the tumour-involved skin was performed because of the location and extent of the tumour. histological examination of sentinel lymph nodes revealed no signs of metastasis. macroscopically the tumour, 40 x 25 mm in size, had almost clear margin, and was opaque coloured with focally black-pigmented areas. no direct invasion into the neighbouring calcaneus was found. microscopic examination of the removed specimen revealed similar histological findings as the biopsy specimen except for a higher cellular density and more atypical feature. there was a fascicular pattern of spindle cells between variably increased collagen fibers. occasionally mitotic figures (9 to 10 cells per 10 hpf) were observed. in the black pigmented area, clusters of melanin-containing polyhedral cells were revealed. histologically the tumour cells had invasive growth pattern along the septum of the fatty tissue in the dermis. there were no abnormal melanocytes observed in the epidermis covering the tumour. immunohistochemically the tumour cells had the same results as the biopsy specimen. ki 67 positivity was counted in 60 %. the histological diagnosis was desmoplastic malignant melanoma. six months after surgery, lung metastases were found. the patient died of lung metastases 10 months after surgery. discussion dm is a rare and atypical form of melanoma. establishing a correct diagnosis is difficult as dm is often clinically innocuous and unlike other subtypes of malignant melanoma [4]. this type of melanoma has usually invaded into the deep reticular dermis or into the subcutis by the time the biopsy is taken. in 2003, parodi et al stated that histopathological identification of dm is confusing because of the intense fibrous reaction in the dermis and minimal, atypical melanocytic proliferation at the dermal-epidermal junction and often that dm is still misdiagnosed unfortunately as a variety of entities, including simple scar, fibrohistiocytic neoplasms, neural tumours, and superficial fibromatoses-with potentially devastating consequences[2]. histologically, early lesions are characterized by superficial tumour fascicles, and randomly diffuse hypercellularity in the upper dermis identified as elongated hyperchromatic pleomorphic spindle cells with stromal myxoid feature. neuroidal melanocytic structures, invasion of adventitial dermis, islands of inflammation, and epidermal lentiginous melanocytic hyperplasia are often present. the most reliable and characteristic features of an early lesion of dm are aggregates of lymphocytes, tumour cells showing cytological atypia, stromal myxoid appearance, and poor circumscription of the dermal infiltrate [1]. the present case also had these characteristic features except aggregates of lymphocytes. 256 257 in equivocal cases, the use of immunohistochemistry (in particular s-100 and neuron-specific enolase) may be helpful in establishing the diagnosis [2]. in the present case, immunohistochemical staining with s100 protein antibody and vimentin was positive. focally the tumour cells were reactive with éøsma and hhf-35 (muscle specific actin). a few cells were reactive with hmb-45. dm is almost always s-100 positive. mature scars were readily differentiated from dm by light microscopy. in contrast, immature scar and dm had many features in common including hypercellularity, nodular lymphoid infiltrates, myxoid stroma, and atypical nuclei [8]. an s-100 study is often necessary to distinguish between tumour and early scar in margins of resection. s-100 staining has also proved to be a valuable adjunct in determining the extent of the tumour at the peripheral margins, particularly for hypocellular and amelanotic tumors [9]. dm is usually hmb-45 negative. all lesions of 128 cases evaluated by skeleton et al. were negative for hmb-45, a marker for premelanosomes. riccioni et al. reported three cases of desmoplastic melanoma (dm) rich in smooth muscle actin. they thought that actin-rich elements differentiate toward mesenchymal elements, paralleling the phenotypic changes seen in sarcomatoid carcinomas [10]. the clinical behaviour of dm is close to soft tissue sarcoma with high rates of local recurrence, low incidence of lymph node metastases, and propensity to develop lung metastases [4]. skeleton et al. evaluated the relationship of histologic features to disease-free survival of 128 cases. factors that correlated with survival included sex, tumour location, tumour depth, and the presence of stromal mucin. the 5-year disease-free survival rate was 68% for all cases and 61% for lesions more than 4 mm deep. wide excision of the primary lesion with clear margins and close follow-up are necessary. it is important that the lesion will be radically extirpated at the time of initial surgery [1]. recently gyorki et al. described their experience with lymphatic mapping and sentinel lymph node biopsy (slnb) in patients with dm to characterize the biological behaviour of these tumors. slnb detected subclinical metastases of dm to regional lymph nodes [5]. slnb at the time of resection can provide useful information to guide early treatment and, coupled to lymphadenectomy in positive patients, may limit tumour spread and prevent recurrence [6]. because of the high local recurrence rate for dm located in the finger, amputation is recommended in an effort to gain effective tumor control [2]. in the present case, slnb was negative. in spite of curative excision by below knee amputation the patient died of lung metastases 10 months after surgery. among the several factors causing early death of the patient in our case are thought a large size of the tumour (40 x 25 mm ) and its deep location. references 1. wharton jm, carlson ja, mihm mc jr (1999) desmoplastic malignant melanoma: diagnosis of early clinical lesions. hum pathol 30: 537-542. 2. parodi pc, scott ca, de biasio f, pezzini i, pertoldi b, beltrami ca (2003) desmoplastic melanoma of the nail. ann plast surg 50: 658-662. 3. jain s, allen pw (1989) desmoplastic malignant melanoma and its variants. a study of 45 cases. am j surg pathol 13: 358-373. 4. jaroszewski de, pockaj ba, dicaudo dj, bite u (2001) the clinical behavior of desmoplastic melanoma. am j surg 182: 590-595. 5. gyorki de, busam k, panageas k, brady ms, coit dg (2003) sentinel lymph node biopsy for patients with cutaneous desmoplastic melanoma. ann surg oncol 10: 403-407. 6. su ld, fullen dr, lowe l, wang ts, schwartz jl, cimmino vm, sondak vk, johnson tm (2004) desmoplastic and neurotropic melanoma. cancer 100: 598-604. 7. kato t, suetake t, sugiyama y, tabata n, tagami h (1996) epidemiology and prognosis of subungual melanoma in 34 japanese patients. br j dermatol : 134: 383-387. 8. kaneishi nk, cockerell cj (1998) histologic differentiation of desmoplastic melanoma from cicatrices. am j dermatopathol 20: 128-134, 1998. 9. eng w, tschen ja (2000) comparison of s-100 versus hematoxylin and eosin staining for evaluating dermal invasion and peripheral margins by desmoplastic malignant melanoma. am j dermatopathol 22: 26-29. 10. riccioni l, di tommaso l, collina g (1999) actin-rich desmoplastic malignant melanoma: report of three cases. am j dermatopathol 21: 537-541. corresponding author: masahito hatori, m.d., assistant professor department of orthopaedic surgery, tohoku university school of medicine 1-1 seiryomachi, aobaku, sendai, japan 980-8574 tel: 81-22-717-7242, fax: 81-22-717-7248 email: mhato@mail.tains.tohoku.ac.jp 258 upsala j med sci 105: 2, 125-133,2ooo isolated human islets trigger an instant blood mediated inflammatory reaction: implications for intraportal islet transplantation as a treatment for patients with v p e 1 diabetes william bennet’, carl-gustav groth’, rolf larsson2, bo nilsson2 and olle korsgren2. department of transplantation surgery’, karolinska institute, huddinge hospital, huddinge, sweden and the department of clinical immunology and transfusion medicine2, uppsala university hospital, uppsala, sweden. abstract islet transplantation offers a logical means to treat insulin-dependent diabetes. however, for reasons poorly understood, the clinical results with islet transplanta tion have been vastly inferior to those obtained with whole organ pancreas trans plantation. the conventional technique for transplanting isolated islets is by intra portal injection, with the islets being trapped in the liver. human islets exposed to human blood trigged an “instant blood mediated inflam matory reaction”, ibmir, characterised by platelet consumption, and activation of the coagulation and complement systems. the islets became surrounded by clots and infiltrated with leukocytes, and there was evidence of islet damage as reflected in insulin dumping. when heparin and a complement inhibitor (scrl), was added to the system, ibmir was suppressed and islet damage reduced. after intraportal pig-to-pig islet intraportal allotransplantation similar morphological changes was found, corroborating the in vitro findings. thus, ibmir inflicts a significant damage to human islets exposed to human blood and ibmir will also, most likely, enhance the subsequent specific, cell medi ated, rejection. platelet and complement activation seem to be the most important factors in the pathogenesis of ibmir. the results presented strongly suggest that ibmir observed both in vitro and in vivo when isolated islets come in contact with blood could provide an explanation for the unsatisfactory results seen in clinical islet allotransplantation. introduction combined kidney-pancreas transplantation has become a valid therapeutic option for the treatment of type 1 diabetic patients with end-stage diabetic nephropathy [ 171. pancreas transplantation restores metabolic control and improves quality of 125 life. even more importantly, it reduces the high mortality rate usually found in this group of patients [ 191. ten years after kidney-pancreas transplantation 80% of the patients are still alive, in sharp contrast to the 20% survival rate of those receiving a kidney alone. the mortality rate in the patients with combined pancreas and kidney transplantation was reduced, mainly because of the lower risk for car diovascular disease. these findings led to the conclusion that combined pancreas and kidney transplantation should be considered for all patients with type 1 diabetes who have end-stage diabetic nephropathy who qualify for kidney transplantation. pancreas transplantation can, however, only be offered to a sub-population of diabetic patients, mainly due to the surgical risk and the post-operative complica tions. previous experience with islet transplantation has demonstrated that although the procedure is safe, the clinical outcome is markedly inferior when compared with pancreas transplantation. to date, 405 adult human islet transplantations have been performed worldwide. of the 200 recipients treated since 1990, only 8% were insu lin-independent 1 year after transplantation. in contrast, insulin independence was achieved at 1 year in 22 % of the patients in a subgroup of 60 in whom 1) the pre servation time for the islets was <8 h, 2) more than 6,000 isletskg were trans planted, 3) the islets were transplanted to the portal vein, and 4) induction immu nosuppression involved administration of antilymphocytic antibodies. fewer than 10 centers worldwide are actively pursuing islet transplantation at this time. cur rently there are 7 patients who are insulin-independent at l to 4 years after trans plantation. (data from the international islet transplant registry, newsletter 8, 1999). the reasons for the poor results that have been obtained with clinical islet transplantation have remained an enigma for the past 20 years. the histocompa tibility barrier, the underlying autoimmune disease, and the immunosuppressive agents used are the same as in pancreas transplantation. however, one major dis tinction is that while the islets remain protected from the recipients blood by the intact endothelial cell lining of the native blood vessels during pancreas trans plantation, transplantation of islets usually entails the injection of the isolated islets into the portal vein, where the islets come into direct contact with whole blood. in clinical islet transplantation, the total number of islets given to a 70-kg patient is about 600 000 ieq (8,000-9,000 ieq/kg bw). this number of islets has an approximate surface area of 400 cm2. the islets are injected into the portal vein, where the low pressure and slow blood flow provide optimal conditions for interaction with the cells and cascade systems in the blood. although several other transplantation sites have been evaluated in experimental models, intra portal transplantation is the only site established thus far for clinical islet trans plantation. thus far, little attention has been paid to the possibility that islets transplanted into the blood stream may elicit an injurious incompatibility reaction. we have studied this possibility both in a human allogeneic in vitro system and in vivo in a pig islet allotransplantation model. an islet perfusion system was developed that 126 mimics the situation in vivo immediately after transplantation of islet cells into the portal vein and have used this system to examine the effect of whole blood upon transplanted islets. isolated human islets were exposed to fresh, non-anticoagulated abo-compatible human blood in polyvinyl chloride (pvc) tubing loops whose inner surface was coated with covalently bound heparin. when human islets were exposed to human blood in the loop, a series of destructive thrombotichnflamma tory events took place. this inflammatory reaction has not been described in detail previously and therefore lacks a descriptive name. we have chosen to name it an instant blood mediated inflammatory reaction or "ibmir'. the role of platelets in ibmir the most striking event observed was the appearance of macroscopic clotting within 5 min after introduction of human islets into the blood. there was a rapid loss of platelets from the blood concomitant with binding of platelets to the islet surface, followed by a continuous fibrin formation that generated a capsule sur rounding the islets. simultaneous up-regulation of p-selectin and secretion of b thromboglobulin indicated platelets activation. these events point to a platelet driven reaction directed against the islets. the ligands to which the platelets bind are still not identified. the rapid decrease in platelet counts was followed by a drop in pmn and monocyte counts, together with a morphological observation of pmns and monocytes clustering around the islets. after approximately 15 to 30 min, in parallel with the consumption of pmns and monocytes, a disruption of normal islet morphology was observed with the appearance of condensed islet cell nuclei and infiltration of a large number of cd1 lb+ cells. under physiological conditions platelet activation is under rigorous control by endothelial cells that inhibit platelet aggregation by several mechanisms. all these regulatory systems are most likely absent on the islet surface. hence, the islets are unable to regulate platelet activation. the process of ibmir is very similar to the platelet activation that occurs in blood in response to vascular injury, i.e. upon contact with connective tissue in the subendothelial matrix or with a biomaterial (e. g. pvc or titanium) which binds fibrinogen to the surface [9]. when islets are infused into the bloodstream via the portal vein, a similar reaction probably occurs: collagen types i, 111, iv, and v have been reported to surround human islets, and collagen is known to mediate platelet binding and activation [20, 211. the a,b, glycoprotein ia-iia complex, cd36, p65, and gpvi have all been proposed as platelet collagen receptors [ 101. another possibility is that membrane structures or plasma proteins which are ligands for platelet receptors (e.g. fibrionogen or von willebrand factor) bind to the islet surface when it is exposed to blood. our previous studies have not defined which of these mechanisms are involved in triggering the plate ledislet event. preliminary electron microscopy data have shown that the surfaces of islets cultured for 3 4 days show only scarce amounts of connective tissue elements. 127 the role of coagulation in ibmir the coagulation system seems to be activated by the intrinsic pathway when islets are mixed with blood in the in vitro model. it is feasible that the negatively charged islet surface activates this coagulation pathway. if so the intrinsic pathway of activa tion could be a potential activator of the whole reaction, since small amounts of fibrin would be able to bind and activate platelets. platelets have been suggested to activate the intrinsic pathway and factor xi has been reported to be activated in response to collagen [22,23]. coagulation via the extrinsic pathway cannot be com pletely excluded from consideration, even though tissue factor was not expressed by the islets, and the up-regulation of tissue factor on activated monocytes is far too slow a process to explain this very rapid activation of the coagulation system [ l ] . however, the extrinsic activation may be important later in the reaction sequence. for example, monocyte binding to p-selectin on platelets has been shown to upregulate tissue factor after approximately 1 h [5]. there are also reports that immune complexes and complement (e. g. c5a) are able to enhance expression of tissue factor on monocytes [12]. the role of complement in ibmir complement activation via the classical pathway (triggered by immune complexes) and the alternative pathway (triggered by “non-self”) leads to formation of a com plex composed of c5b-9. c5b-9 forms a “channel” through the plasma membrane, leading to cell lysis and eventually cell death. another main function of the comple ment system is to generate the anaphylatoxins c3a and c5a, which are extremely potent proinflammatory mediators that can also potentiate the thrombotic reaction. a marked degree of complement activation also occurred when isolated islets were exposed to human blood, as indicated by an extensive generation of c3a and sc5b 9. this activation was presumably not triggered by the islets themselves but occur red secondarily to the previous events. this assumption was supported by the fact that complement activation began after platelet consumption was almost completed and coagulation activation had already started. a number of observations, however, suggest that the complement system does not directly contribute to the destruction of the islets. other investigators have failed to detect complement activation by islets after exposure to human serum [8], despite the fact that islet cells do not express the complement regulatory proteins daf (cd55) and mcp (cd46) and have only low amounts of protectin (cd59) (unpublished observations). these find ings are in agreement with what we have seen when islets are exposed to whole blood since in the initial phase we have no evidence of complement activation on the islet surface. although complement activation is unlikely to cause direct damage to the islet cells through c5b-9 cytolysis, the generation of proinflammatory pro ducts (sc5b-9, c3a, and c5a) most likely potentiates the inflammatory reaction toward the islets. c3a and c5a are powerful chemoattractants for pmns and mono cytes and can stimulate an influx of these cells to/into the islets [2]. activation 128 through the c5a receptor causes enzyme release (e. g. myeloperoxidase and elastase) by granulocytes and cytokine release (il-1, il-6, il-8 and tnf-a) by monocytes. another important effect of c5a is that it up-regulates complement receptor 3 (cdl 1 b/cd18) on pmns and monocytes. c5a stimulates endothelial cells to release heparin sulfate, upregulate tissue factor, secrete von willebrand fac tor and express p-selectin. these changes favour fibrin deposition, augmented thrombin-mediated platelet aggregation and adhesion of pmns. soluble c5b-9 (sc5b-9) triggers endothelial cells to express tissue factor and a number of adhesion molecules [ 181. in addition, complement activation products, particularly sc5b-9, can further enhance platelet activation [ 151. thus complement activation might induce direct inflammatory effects but also indirect effects mediated by the endo thelial cells in the portal system. insulin “dumping” during ibmir during the first 5 min after addition of the islets to the blood in the loops and in parallel with the binding of platelets to the islets we observed a rapid liberation of insulin, followed by a release at a more moderate rate. release of insulin as a result of glucose stimulation was unlikely in this case, since the glucose concentration in the blood never exceeded 7.4 mmol/l,. complement-mediated damage was a poten tial cause of this rapid release, but both the time course and the lack of complement activation products on the islet surface argued against this possibility. the most likely cause of insulin release are, however, mediators released from activated platelets such as ca2+, atp, and adp, all known to stimulate insulin release. in addi tion to platelets, pmns and monocytes are potential effector cells in islet damage. p-selectin exposed on activated platelets, released paf, and the fibrin lattice can all stimulate monocytes and pmns to release a wide range of tissue-damaging en zymes and radicals known to be toxic to b-cells [6, 13, 161. islet b-cells are highly sensitive to secreted chemokines and to free radicals because of the low levels of radical-scavenging enzymes in the islets [ 1 1, 241. furthermore, the physical entrap ment of the islets by platelets, neutrophils, and monocytes in a macroscopic blood clot may enhance the local action of factors toxic to b-cells. clinical and experimental allogeneic intraportal islet transplantation previous case reports have described a phenomenon similar to what we have obser ved in the in vitro loop model. several years ago, the ricordi group published a report of patients transplanted intraportally with human islets [ 141. liver biopsies obtained from one of the patients two days after transplantation revealed an intra portal thrombosis composed of fibrin strands and leukocytes clustering around the islet cells [14]. the authors referred to this finding as an “islet cell thrombus”. in addition, intense portal inflammation was seen two and five days after transplanta tion. similar findings were observed in the other four patients. they interpreted 129 their findings as part of a process of eliminating contaminating non-endocrine tissue. based on our findings in the loop model we challenge this view and suggest that the observed destructive process is unable to discriminate between endocrine and non-endocrine tissue. after intraportal pig-to-pig islet allotransplantation, we have also found morpho logical changes similar to those reported by sever et al. [14]. liver biopsies from these pigs show islets entrapped in fibrin strands, which embolize in the branches of the portal tree [3]. these in vivo observations clearly confirm our findings from the loop system. the effect of heparin and soluble recombinant crl (scr1) in ibmir addition of 4 iu/ml of heparin to the blood in the loops inhibited the macroscopic clotting and the generation of fxiia-at, fxia-at, and tat. as a result of the decreased fibrin generation, the consumption of platelets, pmns, and monocytes was abrogated. a reciprocal effect was obtained by the addition of the recombinant form of the complement inhibitor soluble complement receptor 1 (scr 1). addition of scrl resulted in a nearly complete inhibition of complement activation, with c3a and sc5b-9 levels similar to the background levels. however, the consumption of platelets, neutrophils and monocytes remained unaffected, as were the macroscopic coagulation and the increase in fxiia-at, fxia-at, and tat. thus, it appears that complement activation was not directly responsible for the platelet activation or for initiating coagulation and cellular activation. furthermore, since the clustering of pmns and monocytes around the islets was not influenced by scr1, the comple ment fragments were apparently not the ligands to which the cells were binding. in contrast, the treatment with a combination of both inhibitors (i. e. heparin and scr l), prevented disruption of islet morphology and leukocyte infiltration of the islets a finding which strongly suggests that the reaction is driven by both cascade systems. despite this preventive effect of the two drugs, a thin layer of platelets and fibrin could still be observed on the islet surface, and the rapid release of insulin from the islets was not diminished. this observation further supported the hypo thesis that the initial event in the reaction was triggered by the binding of platelets to the islet surface and that the insulin release indeed was caused by factors from the adhering platelets. state-of-the-art transplantation in relation to ibmir at present most centers performing allogeneic islet transplantation use low-dose systemic heparin at the time of transplantation. heparin is usually administered as a bolus dose of approximately 75 iekg body weight, corresponding to approximately 5000 ie for a 70-kg person or 1 ie/ml of blood. the initial blood concentration is 130 25% of the concentration we used in the loop model, and given its redistribution in the body fluid and its half-life of 1 to 2 h, the heparin concentration in patients is maintained for only a very short time. the heparin concentration in the clinical set ting is far from the concentration of 4 iwml heparin that we used in the in vitro model, a concentration that prevented coagulation and reduced cell consumption. furthermore, since the addition of heparin alone was unable to prevent extensive platelet and fibrin formation around the islets as well as a marked infiltration of cdl lb' cells in our in vitro system, we conclude that only the use of a high heparin concentration in combination with scrl can effectively preserve islet morphology. such a high concentration of heparin is, however, not acceptable for long-term use in patients, particularly in those with cardiovascular complications often associated with diabetes. it is therefore important that we gain a better understanding of mechanisms involved in this undesirable inflammatory reaction and develop clini cally acceptable methods to prevent to this process. immunosuppression in relation to ibmir immunosuppressive drugs given to prevent t cell-mediated allograft rejection are most likely not capable of preventing ibmir. in addition to the acute thrombotic/ inflammatory destructive reactions which are deleterious to the islets, antigen presentation may also be promoted, leading to an accelerated and reinforced cell mediated immunity at a later stage in the process [4, 7]., c.f. the role of adjuvants in immunization. this could explain why the results after intraportal islet transplanta tion to patients with type 1 diabetes, which occurs across both an autoimmune and an allogenic barrier, are dramatically inferior to the results seen in patients who have undergone autologous islet transplantation, a situation in which no immune barrier exists (data from the international islet transplant registry, newsletter 8, 1999). if ibmir were counteracted, a larger islet mass would engraft and escape subsequent specific immune responses, thereby markedly improving the outcome in clinical islet transplantation. concluding remarks the detrimental effects of ibmir on the clinical outcome of islet transplantation in patients with type 1 diabetes could provide an explanation for the relatively low success rate of this procedure. however, the use of a high number of islets ( > l o 000 ieqkg recipient) and an extensive immunosuppressive protocol may in some patients overcome the effects induced by ibmir. nevertheless, our results strongly suggest that the inflammatory events observed both in vitro and in vivo when isolated islets come in contact with blood could provide an explanation for the un satisfactory results seen in islet allotransplantation. 131 acknowledgement this study was supported by grants from the swedish medical research council (06p 11813 and 16x-12219), the ake wiberg foundation, the magnus bervall foundation, the nordic insulin fund, the tore nilsson fund, the torsten and ragnar soderbergs founda tion, the ernfors family fund, the swedish diabetes association, the swedish society of medicine, the swedish society for medical research and the juvenile diabetes foundation international and the knut and alice wallenberg foundation. references 1. amirkhosravi, a., alexander, m., may, k., francis, d. a., warnes, g., biggerstaff, j.& francis, j. l.: the importance of platelets in the expression of monocyte tissue factor antigen measured by a 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chemokine synthesis by human monocytes. j clin invest 97: 1525-1534, 1996. 1 85: 1619-1 627, 1997. address f o r reprints: olle korsgren, md, phd department of clinical immunology, rudbecklaboratory , university hospital, se-75 1 85 uppsala sweden 133 ujms109_3.pdf upsala j med sci 109: 247–254, 2004 cystatin c vs creatinine as markers of renal function in patients on digoxin treatment pär hallberg,1 håkan melhus,1 lars-olof hansson,1 anders larsson1 1department of medical sciences, clinical chemistry and pharmacology, university hospital, uppsala, sweden abstract a free full-text copy of this article can be found at the web page of upsala j med sci: http://www.ujms.se background: the kidney function is a major determinant of the serum concentration of digoxin as this drug is mainly eliminated unchanged through the kidneys. since digoxin is widely prescribed among the elderly, and the glomerular filtration rate (gfr) declines with age, it is important that the clinician takes the patient’s gfr into account when prescribing digoxin. serum cystatin c has been suggested to be superior to creatinine for estimation of gfr, which may have relevance for the optimization of treatment with digoxin. methods: to evaluate which of the two gfr markers serum creatinine and serum cystatin c that best correlates with serum digoxin, we compared the serum levels of digoxin with the serum levels of creatinine and cystatin c in 149 patients on therapeutic drug monitoring of digoxin at our hospital. results: overall, there was a stronger correlation between serum digoxin concentrations and cystatin c (p=0.00001) as compared to creatinine (p= 0.00003). interestingly, of the patients with a serum digoxin concentration 1.5 nmol/l, 29% had a serum creatinine level within normal limits, as compared to 20% with normal cystatin c levels. conclusions: in this study, serum cystatin c correlated better to serum digoxin than did serum creatinine. with improved gfr monitoring, digoxin concentrations should be better controlled. introduction digoxin is commonly used in the treatment of congestive heart failure and atrial fibrillation. the drug has become a subject of discussion after recent publications that showed a gender-related difference in mortality [1], a gender-related difference in serum (s) digoxin [2], and subsequently an increased mortality for men with s247 received 24 may 2004 accepted 7 june 2004 key words: creatinine, cystatin c, digoxin, glomerular filtraten rate, kidney. digoxin 1.5 nmol/l[3]. this has led to a revised recommendation at our hospital to target s-digoxin to <1.5 nmol/l. in this context, an heightened attention to the patient’s s-digoxin level is warranted. adverse drug events affect millions of patients each year and was responsible for up to 14% of acute hospital admissions at a swedish internal medicine clinic in 2001 [4]. in the elderly, adverse drug events occur in 14.6% to 35%, depending on the population setting and measures employed for their identification [5, 7]. toxic concentrations of digoxin may require hospital treatment and digoxin intoxication is one of the most frequent causes of hospital care due to toxic drug effects[8]. among individuals aged 75 and older, almost 20% were on medication with digoxin in 1996 [9]. despite declining use in the last few years, digoxin is still one of the most frequently prescribed drugs; it was listed twice among the top 200 prescriptions in 2000 [10]. in 1995, it was the drug most often monitored therapeutically [11] because of its narrow therapeutic window and potentially serious side effects. digoxin has a half-life of approximately 1.5 days and the drug is mainly eliminated unchanged in the urine [12]. glomerular filtration rate (gfr) has a major impact on s-digoxin [12]. as geriatric patients often have reduced gfr, monitoring kidney function during digoxin treatment is important. in the last decades, creatinine has become the most commonly used marker of gfr [13, 14]. despite its common use, creatinine has limitations as a renal function marker. creatinine is influenced by factors such as age, gender, muscle mass, physical activity and diet [15]. it is also insensitive for detection of small decreases in gfr, in the so-called creatinine-blind gfr area, due to the non-linear relationship between serum concentration and gfr [16]. thus, there is a need for better gfr markers. several markers such as �-trace protein, cystatin c and � 2 -microglobulin have been suggested as alternatives to creatinine [17–19]. the normal serum level of cystatin c is <1.20 mg/l for patients less than 50 years of age and <1.55 mg/l for patients over 50 years of age, while increasing levels is detected in serum from patients with reduced gfr. cystatin c is a polypeptide with a molecular mass of 13 kda and an ellipsoid molecular shape with axes of about 30 and 45 å [20]. studies on the handling of human cystatin c in the rat have shown that the serum clearance of cystatin c is 94% of that of the generally used gfr-marker 51cr-edta [21]. a recent meta-analysis has indicated that s-cystatin c is superior to s-creatinine as a renal function marker [22]. we have studied the correlation between s-digoxin and the gfr markers s-creatinine and s-cystatin c. the study was performed to evaluate which of the two gfr markers s-creatinine and s-cystatin c that best correlated with s-digoxin. one earlier study of 18 healthy elderly individuals found no correlation between either s-cystatin c or s-creatinine and digoxin clearance, and thus concluded that s-cystatin c did not offer any advantages over s-creatinine in this respect [23]. however, the small number of study subjects may have been insufficient to detect a correlation, and also, actual digoxin treated patients have not been studied. 248 materials and methods patient samples and assays consecutive routine requests (n=163) for therapeutic drug monitoring of s-digoxin were also analyzed for s-cystatin c and s-creatinine. the material consisted of samples from 98 females and 65 males, both inand out-patients treated with digoxin for any medical condition. the mean age was 80 years (range 55–106 years), and the mean dose of digoxin was 0.18 mg/day (range 0.07–0.3 mg/day). s-digoxin was determined on the advia 1650 (bayer corp., tarrytown, ny, usa). s-digoxin values below 0.6 nmol/l were reported as <0.6 nmol/l. s-cystatin c measurements were performed by a latex-enhanced reagent (n latex cystatin c, dade behring, deerfield, il, usa) using a behring bn prospec analyzer (dade behring). the total analytical imprecision (coeficient of variation, cv) of the method was 4.8% at 0.56 mg/l and 3.7% at 2.85 mg/l. s-creatinine measurements were performed by means of the modified kinetic jaffe reaction on the advia 1650 analyzer (bayer corp) and reported as s.i. units (µ mol/l). the total analytical imprecision (cv) of the method was 2.6% at 170 µ mol/l and 2.4% at 740 µ mol/l. all assays were performed independently and without prior knowledge of other test results at the department of clinical chemistry and pharmacology at our hospital. the study was approved by the local ethical board at uppsala university (1–167). statistical calculations data on s-cystatin c, s-creatinine and s-digoxin were normally distributed, and statistical correlation analysis was performed with the pearson product-moment correlation test using statistica 5.1 (statsoft inc., tulsa, ok, usa). digoxin values <0.6 (n=14) were excluded from the statistical analyses, giving a total number of patients of 149. p-values <0.05 were taken as statistically significant throughout the study. results digoxin concentrations when analyzing only those patients who had reached steady-state concentrations of digoxin and in whom serum levels had been measured at through were analyzed (n=94), mean s-digoxin was 1.5 nmol/l (range 0.6-4.0 nmol/l), with higher concentrations among females (1.6 nmol/l) than males (1.4 nmol/l). this difference did not reach statistical significance (p=0.24, unpaired t-test). among these patients, 33% of the digoxin concentrations were 1.5 nmol/l and 9% >2.5 nmol/l. there was no correlation between age and s-digoxin (p=0.86, r=-0.02, n=94). correlation between s-digoxin and s-cystatin c vs s-creatinine overall, s-digoxin correlated stronger to s-cystatin c (p=0.00001, r=0.35, n=149) as compared to s-creatinine (p=0.00003, r=0.34, n=149). the difference was more marked (fig 1 and 2) when only patients who had reached steady-state of digoxin 249 250 fig. 1. correlation between s-digoxin and s-cystatin c for individual patients. fig. 2. correlation between s-digoxin and s-creatinine for individual patients. and in whom serum levels had been measured at through (n=94) were analyzed (sdigoxin vs s-cystatin c, p=0.000001, r=0.45, n=94 s-digoxin vs s-creatinine, p= 0.0003, p=0.37, n=94). the difference was similar in both genders. also, of the patients with a s-digoxin concentration 1,5 nmol/l, 29% had a s-creatinine level within normal limits (69–113 µ mol/l), as compared to 20% with normal s-cystatin c levels. there were also inverse correlations between digoxin dose and s-cystatin c (p=0.007, r=–0.28, n=94), and digoxin dose and s-creatinine (p=0.04, r=–0.22, n=94). the correlation between s-cystatin c and s-creatinine is illustrated in fig 3. discussion gfr is generally accepted as the best overall index of renal function. gfr decreases with age and reduced gfr is the most important complication of renal disease. reduced gfr affects the clearance of many drugs used today, including digoxin, so that in many cases the recommended dose has to be adjusted depending on the patient’s gfr. there is thus a need for robust gfr markers. inulin, iohexol and 51cr-edta clearances are considered the golden standards for gfr measurements [22, 24]. the disadvantage with these assays is that they are cumbersome, costly and slow which may delay the start of treatment. assays such as s-creatinine and scystatin c can provide rapid test results. creatinine in combination with the cock251 fig 3. correlation between s-creatinine and s-cystatin c for individual patients. croft-gault equation is often used to estimate gfr [25]. using actual body weight in the cockcroft-gault equation overestimates the gfr for obese patients [26]. an alternative could be to use lean body mass, but this is not usually available. creatinine often overestimates gfr in patients with slight reductions in gfr [27]. this may cause the prescribing physician to treat the patient with unnecessary high drug doses, which will increase the cost and possibly cause side effects. the inverse correlation between digoxin dose and s-cystatin c and s-creatinine in this study indicates that the kidney function should be considered when digoxin is prescribed. however, the high prevalence of patients with uncontrolled s-digoxin 1.5 nmol/l, and the strong correlations between s-digoxin and s-cystatin c and screatinine indicate that even greater consideration should be taken to gfr function. s-digoxin correlated stronger to s-cystatin c as compared to s-creatinine, which is in agreement with previous studies showing s-cystatin c to be superior to s-creatinine as a marker of renal function [22]. the use of cystatin c has been hampered by the limited availability of the test and the problem of relating cystatin c to an estimated gfr. the introduction of new cystatin c tests that can be applied on widely available chemical analyzers will increase the availability of the test. our hospital has for the last year offered measurements of s-cystatin c as a stat request and also reported cystatin c in mg/l as well as converted to gfr (ml/min). this has improved the clinical usefulness of the assay and we have noticed a rapid increase in the number of requests for cystatin c. we conclude that greater consideration should be taken to gfr when prescribing digoxin, and that 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2003. the reliability of different formulae to predict creatinine clearance. j intern med 5: 563–573. corresponding author: anders larsson department of medical sciences, uppsala university hospital, s-751 85 uppsala, sweden telephone: 46-18-6110000 fax: 46-18-552562 e-mail: anders.larsson@akademiska.se 253 (9) funkquist 97-108 upsala j med sci 111 (1): 97–108, 2006 growth and breastfeeding among low birth weight infants fed with or without protein enrichment of human milk e-l. funkquist, t. tuvemo, b. jonsson, f. serenius, k. hedberg-nyqvist department of women’s and children’s health, uppsala university, uppsala, sweden abstract the effect of protein enrichment of mother’s milk on growth of low birthweight infants needs further exploration in order to optimize feeding strategies. the aim of this study was to describe feeding and growth of infants weighing <1,900 g at birth, up to a corrected age of 18 months, with or without protein-enriched breastmilk. a retrospective, descriptive, non-experimental design was used to describe the growth of 52 low birthweight infants. data on their growth and feeding were collected from medical records at hospitals and child health care clinics. despite more severe morbidity, the infants given protein-enriched milk showed similar growth as the other study infants. standard deviation score for length at birth correlated positively with delta standard deviation score for length, from discharge to 12 and from discharge to 18 months corrected age. duration of ‘full’ breastfeeding had a significant impact on subsequent improvement in sds for weight. at discharge a smaller proportion of singletons fed with protein enriched milk were breastfed ‘fully’. infants who established breastfeeding at an early post-menstrual age were born with more optimal weight standard deviation score and had a better weight gain after discharge. we conclude that protein-enriched breast milk enables low birthweight infants requiring especially intensive care to attain growth at discharge comparable to that of healthier infants not given enriched milk. low standard deviation score for length at birth may predict poor growth after discharge. however duration of ‘full’ breastfeeding had a significant impact on subsequent improvement in sds for weight. therefore it is important that mothers of lbw infants are given sufficient support of lactation and breastfeeding. 97 received 17 october 2005 accepted 26 october 2005 introduction the question ‘how should infants with lbw be fed’ is of major interest, as the nutriments given to these infants may predetermine subsequent health [1]. there is a general consensus that the milk from an infant’s mother is the best possible. nevertheless, due to preterm infants’ greater protein requirement, it is generally agreed that human milk needs enrichment with proteinand also sodium, phosphate and calciumto be the best choice for these infants [2, 3]. two more facts support the latter conclusion: a) an association between protein intake and weight gain has been noted in preterm infants [4] and b) variability in protein concentration in mothers´ milk [5, 6]. the aims of feeding lbw with protein-enriched breastmilk are (a) to promote catch-up growth (including head growth) in order to reduce the risk of impaired psychomotor development, and (b) to reduce the risk of the metabolic syndrome [7]. nevertheless, some other facts complicate this question; first of all, lbw infants do not constitute a homogeneous group. it includes sga infants, aga infants, preterm infants and infants with or without severe morbidity. one can assume that these differing categories constitute subgroups as regards nutritional requirements [8]. second, even though protein enrichment affords short-term growth improvement, no long-term benefits have been demonstrated [8-10]. one danish study suggested that unfortified human milk in daily amounts of around 200 ml/kg might be sufficient for preterm infants; in that study, infants given only their own mothers’ unfortified milk were heavier at discharge but had length and head circumference measurements similar to infants fed with preterm formula [11]. finally, in developing countries exclusively breastfed premature infants with vlbw have shown weight gain comparable to intra-uterine growth rates [12]. early proactive enteral feeding has been associated with a reduction in mean days to reach full enteral feeding and days to regain birthweight [13]. despite this, policies for the introduction and 98 list of abbreviations: aga appropriate for gestational age bf breastfeeding cpap continuous positive airway pressure e-group enrichment group ga gestational age lbw low birthweight vlbw very low birth weight md median non-e group non-enrichment pi ponderal index pma postmenstrual age sds standard deviation score delta sds difference between two defined standard deviation scores sga small for gestational age advancement of enteral and oral feeding have been restrictive, and recommendations for maximum amounts around 150 ml/kg and day still appear to be common [14, 15]. in short, although extensive research has been conducted into the association between feeding of mother’s milk and growth of lbw infants, gaps in our knowledge base persist and there is disagreement regarding strategies for optimization of these infants’ growth. the objective of this retrospective, non-experimental study is to describe breastfeeding and growth up to a corrected age of 18 months by infants weighing less than 1900 g at birth and fed breastmilk with or without protein enrichment. methods a retrospective, descriptive, non-experimental design was used to describe the growth of lbw infants born at the neonatal units of two swedish university hospitals, university hospital, uppsala (a) and norrland university hospital, in umeå (b). the design and procedures were approved by the research ethics committees of the medical faculties at uppsala university and umeå university. relevant background data on mothers and information on infants’ growth and feeding during their stay in hospital were extracted from the hospital medical records. data on breastfeeding, complementary feeding and growth after discharge from hospital were obtained from child health care medical records and by a questionnaire to mothers. materials the sample comprised all infants born consecutively in hospital a from december 2000 and february 2002, and in hospital b between august 2000 and february 2002. infants transferred to another hospital before discharge home were excluded. criteria for inclusion in the study were birthweight below 1,900 g and admission to a neonatal unit. the infants should have been free from congenital abnormality or serious illness having a severe impact on feeding tolerance, such as necrotizing enterocolitis, severe cardiac illness, or chromosomal abnormality. furthermore, their mothers were required to be swedish speaking and intending to breastfeed. four infants at hospital a were excluded from the study for other reasons; a pair of twins because of metabolic disease in one twin, another infant because the mother was suffering from a serious illness and one infant because of intraventricular hemorrhage grade iii. this generated a sample of 52 infants (35 from hospital a and 17 from hospital b). gestational age at birth was based on ultrasound examination at 16-18 weeks’ gestation. the infants were deemed to be sga if they had a birthweight below -2 sds according to marsál [16]. niklasson’s adjusted swedish reference standards for size at birth (unpublished) were used to evaluate growth up 99 to 40 weeks pma and niklasson’s swedish reference standards to for growth after 40 weeks pma [17]. pi index was calculated as weight (g) x 100/length3 (cm) [14, 18]. feeding regimens at both hospitals, preterm infants received donor breastmilk which was gradually replaced by own mother’s milk. in cases of insufficient maternal milk, preterm infants were given formula at around 36 postmenstrual weeks. full-term infants received formula before milk production was established. at hospital a the infants were fed every 2 hours as long as they weighed less than 1,500 g and at hospital b until they weighed 1,200 g. other infants were fed every 3 hours. at hospital a supplement was administered by tube or cup and at hospital b by tube or spoon. in order to minimize the interruption of growth already started in fetal life, hospital a followed a nutrition policy stipulating that all sga infants were prescribed a total volume of 100 ml/kg and day on the day of birth, 150 ml/kg on day 1, and 200 ml/kg on day 2. the latter daily volume was maintained until the infant reached what was regarded by the attending neonatologist as adequate catch-up growth. for aga infants the policy was to commence with 65 ml/kg and day on the day of birth, gradually increasing to 170 ml/kg and day on day 9. at hospital b, the nutrition policy for aga infants in hospital a was applied to both aga and sga infants. most mothers lived-in and roomed-in at the neonatal unit and breastfed their infants for at least a couple of days before the infants’ discharge. when breastfeeding was initiated and there were signs of suckling, infants at hospital a were testweighed before and after feeding to determine the amount of milk the infant had ingested. at hospital b the daily amount was assessed by observing the infants’ suckling behaviour. at both hospitals, bottle feeding was introduced only for exceptional reasons. regimens for enrichment the attending neonatologist assessed infants’ need of an enriched breastmilk. at both hospitals the product used was enfamil human milk fortifier. altogether 22 infants were given this product in breast milk (17 at hospital a and 5 at hospital b) from an age between 8 and 35 days, at a median pma of a 32.6 weeks. (the egroup received enriched milk and non-e group infants were not given enriched milk). the dose was gradually increased during 9 days. the enrichment was gradually set out when the infant started to breastfeed or when the breastmilk was replaced by formulafeed. duration of treatment with enrichment in group e was 30.5 days (md) ranging from 10 to 88 days. breastfeeding definitions the breastfeeding definitions used were those currently applied at swedish child health care centers: 100 101 full breastfeeding: infants’ predominant source of nutrition is breastmilk. infants may be given vitamins, minerals and medicines. no other food-based fluids are allowed. from the age of 4-6 months, infants may ingest semi-solid foods, but no non-human milk. partial breastfeeding: infants take both breastmilk and non-human milk, with or without semi-solid foods. statistical analyses the statistical package for social sciences (spss version 11.0) was used for the statistical analyses. the chi-square test and fisher’s exact test, and mann-whitney u-test were used for inter-group comparisons. spearman’s correlation analyses was used to analyse differences in the impact of certain factors on outcome variable. linear regression analysis was used for the exploration of factors contributing to the explanation of infants’ growth. results characteristics of infants in the e and non-e groups. the infants who were assessed by the attending neonatologist as being in need of protein enrichment of breastmilk differed from infants who were not given enriched milk. they were of a younger gestational age and were both lighter and smaller. a greater proportion of these infants also needed more advanced neonatal care and treatment for apnea. the proportion of infants who were growth retarded from birth was the same in both groups (table 1). feeding and breastfeeding. the amounts of milk consumed by the e-group were smaller after one week, but not after two (table 2). most infants in both groups were breastfed. no significant inter-group was evident regarding breastfeeding outtable 1. infant gestational age, weight, length, and head circumference at birth, sga, twin, ventilator treatment, cpap, oxygen, and theophyllamine variable unit enrichment (n=22) no enrichment (n=30) pvalue ga at birth md (range) 30.0 (25.0-33.0) 32.6 (26.7-39.9) 0.000 birthweight, g md (range) 1.236 (713-1.868) 1.663 (947-1.886) 0.000 birth length, cm md (range) 38.5 (32-43.5) 42 (35.5-46) 0.000 birth head , cm md (range) 28 (23.5-31) 30 (25.5-32.3) 0.001 sga n 10 14 ns twin n 3 10 ns ventilator n 9 4 0.049 cpap n 20 16 0.006 oxygen n 15 10 0.024 theophyllamine n 17 7 0.000 table 1. infant gestational age, weight, length, and head circumference at birth, sga, twin, ventilator treatment, cpap, oxygen, and theophyllamine come, with frequencies of ‘partial’ and ‘full’ breastfeeding of 86% and 59% in group e, and 93% and 80%, respectively, in the non-e group. however, when twins were excluded from the sample, a larger proportion of infants in the non-e group were fully breastfed at discharge (table 3). there were no significant differences between the groups regarding duration of full and partial breastfeeding (table 4). growth. the e-group infants lost more weight and regained their birthweight later (table 5). at the time of discharge they were significantly heavier and had larger head circumference than the non-e group infants (table 6); there was no significant difference in lenght. at this time there was no difference in pma, even though infants given enriched breast milk had reached a higher postnatal age. however, at 40 weeks there were no differences between the groups in any of the anthropo102 table 2. total amount of milk at age 7 and 14 days variable unit enrichment no enrichment p-value n=22 n=28*ml/birthweight, kg day 7 n md (range) 131 (40-213) 162 (21-230) 0.007 n=22 n=22**ml/birth weight, kg day 14 n md (range) 183 (45-276) 191 (97-276) ns * two infants had reached full breastfeeding ** eight infants had reached full breastfeeding table 3. breastfeeding in singletons at discharge variable unit enrichment (n=19) no enrichment (n=20) p-value ‘partial’ breastfeeding n (%) 16 (84) 19 (95) ns ‘full’ breastfeeding n (%) 11 (58) 18 (90) 0.031 weaned n (%) 3 (16) 1 (5) table 4. breastfeeding duration in (months) after discharge in singletons, duration in months variable unit enrichment (n=19) non enrichment (n=20) p-value ‘full’ breastfeeding md (range) 3 (0-10) 5 (0-12) ns ‘partial’ breastfeeding md (range) 6 (0-18) 7 (0-13) ns table 5. lowest weight, weight loss percent, regain of birth weight and age in days when infants attained ‘full’ enteral feeding variable unit enrichment (n=22) non enrichment (n=30) p-value lowest weight, days md (range) 4 (2-7) 4 (0-7) ns weight loss, % md (range) 11.9 (2.2-21.3) 7.2 (0-18.3) 0.015 days to regain birthweight md (range) 12.5 (3-31) 10 (0-16) 0.007 days to full enteral feeding md (range) 7 (0-31) 3.5 (0-17) 0.011 table 2. total amount of milk at age 7 and 14 days table 3. breastfeeding in singletons at discharge table 4. breastfeeding duration (in months) after discharge in singletons, duration in months table 5. lowest weight, weight loss percent, regain of birth weight and age in days when infants attained ‘full’ enteral feeding metric variables, nor of proportionality in terms of pi, with a median (range) pi for the e-group of 2.6 (2.2-3.1) and 2.6 (1.9-3.2) for non-e group. on the other hand, when pi values for aga and sga infants were compared, the latter continued to be thin, with consistently lower pi at 40 weeks than to the aga infants (md 2.4 vs 2.6, p < 0.030), 2 months (2.6 vs 2.8, p < 0.045), 12 months (2.2 vs 2.3, p < 0.020) and 18 months (1.9 vs 2.1, p < 0.03). no differences were observed between infants given enriched milk and the ‘control’ infants, regarding sds for weight, length, or head circumference at 2, 4, 6, 12 or 18 months corrected age (table 7). but when the two groups were compared regarding changes in growth in terms of changes in sds (delta sds), several differences were identified. the non-e group infants showed greater improvement in weight gain and head growth from discharge to 2 and 4 months corrected age: no such differences in length were noted (table 8). factors explaining growth. in regression analyses (controlling for the following 103 table 6. age at discharge, weight, length, and head circumference at discharge and at 40 weeks variable unit enrichment (n=22) non enrichment (n=30) p-value pma at discharge, weeks md (range) 37.4 (35.9-42.7) 37.1 (33.6-40.7) ns age at discharge, days md (range) 52 (25-107) 28 (6-89) 0.000 weight at discharge, g md (range) 2.435 (1.810-.825) 2.232 (1.740-2.800) 0.025 length at discharge, cm md (range) 46 (43-48) 45.75 (43.5-49) ns head at discharge, cm md (range) 33.5 (31.5-35.5) 32.5 (30-35) 0.009 weight at 40 weeks, g md (range) 2.938 (2.040-3.840) 3.010 (1.870-5.015) ns length at 40 weeks, cm md (range) 48.75 (41-53) 49 (46-54) ns head at 40 weeks, cm md (range) 35.8 (33-37.5) 35.2 (32-37.3) ns table 7. median weight, length, head circumference sds at birth, discharge, 40 week pma and the corrected age of 2, 4, 6,12 and 18 months (head from 40 week pma) variable birth discharge 40 weeks 2 months e non-e e non-e e non-e e non-e sds weight range -1,6 -3,3 to 1,1 -1,7 -3,9 to 0,0 -1,9 -3,4 to -0,4 -2,0 -3,2 to -0.8 -1,4 -3,2 to 1,5 -0,9 -3,9 to 1,8 -0,4 -2,6 to 1,3 0,1 -3,5 to 2,4 sds length range -1,5 -4,2 to 1,8 -1,1 -4,3 to 1,4 -1,7 -4,4 to 0,3 -1,4 -3,3 to -0,1 -1,5 -5,4 to 1,0 -0,8 -2,7 to 0,6 -1,2 -3,7 to 1,1 -0,6 -3,3 to 0,5 sds head range 0,1 -1,3 to 2,3 -0,2 -1,9 to 1,0 -0,2 -1,4 to 1,7 0,2 -3,2 to 2,0 4 months 6 months 12 months 18 months e non-e e non-e e non-e e non-e sds weight range -1,1 -3,1 to 1,2 -0.2 -3,2 to 2,1 -1,0 -2,9 to 1,2 -0,7 -3,1 to 1,6 -0,7 -2,5 to 1,1 -0,9 -2,6 to 1,9 -0,8 -2,4 to 1,2 -0,6 -2,3 to 1,8 sds length range -0,9 -3,5 to 0,8 -0,3 -3,1 to 1,2 -0,5 -3,7 to 1,3 -0,4 -2,6 to 1,4 -0,4 -3,3 to 1,8 -0,2 -2,4 to 1,6 -0,5 -3,72 to 1.9 -0,2 -2,4 to 1,6 sds head range -0,1 -1,5 to 2,9 0,5 -1,5 to 3,2 0,2 -1,6 to2,2 0,1 -1,9 to 1,9 -0,1 -1,9 to 1,1 -0,1 -2,0 to2,3 -0,4 -2,1 to 1,7 -0,7 -1,9 to 1,6 table 6. age at discharge, weight, length, and head circumference at discharge and at 40 weeks table 7. median weight, length, head circumference sds at birth, discharge, 40 week pma and the corrected age of 2, 4, 6,12 and 18 months (head from 40 week pma) independent factors: head circumference at birth, weight at birth, ga at birth, pma at discharge, oxygen treatment in days, teofyllamin treatment, and enrichment in breastmilk) enrichment in breastmilk no longer came out as a significant factor for head circumference or weight at discharge. significant factors explaining weight at discharge were ga at birth (p < 0.000), weight at birth (p < 0.000) and pma at discharge (p < 0.000). head circumference at discharge was explained by ga at birth (p < 0.000), head circumference at birth (p < 0.02) and pma at discharge (p < 0.000). furthermore, the following variables also emerged as significant for study infants’ growth. length sds at birth correlated positively with d sds for length from discharge to 12 months (p < 0.023) and 18 months (p < 0.022); head circumference at 40 weeks correlated positively with d head circumference from discharge to 12 months (p < 0.037) and 18 months (p < 0.016). ga at birth correlated positively with length sds at birth, but did not correlate with d sds for length later on. duration of ‘full’ breastfeeding correlated positively with d sds for weight at discharge to 6 months (p < 0.025) and discharge to12 months (p < 0.045), and duration of ‘partial’ breastfeeding months correlated positively with d sds for weight at discharge to 6 months (p < 0.042) and discharge to18 month (p < 0.048). the factor ‘months of ‘full’ breastfeeding’ also emerged as significant for the explanation of delta sds for weight in a regression analysis. independent factors included in this analysis were: sds for weight at birth, ga at birth, oxygen treatment in days, teofyllamin treatment, gender, twin, months of ‘full’ breastfeeding, and enrichment in breastmilk. months of ‘full’ breastfeeding explained d weigth sds from discharge to 6 months (p < 0.005), to 12 months (p < 0.007) and to 18 months ( p < 0.033). infants with early ‘full’ breastfeeding. during the process of data analysis, infants with early attainment of full breastfeeding (at a pma of less than 36 weeks) were explored separately. this group consisted of 14 infants, 4 of whom had a birthweight below 1.500 g. (table 9). compared with the other study infants, these infants had a higher median weight sds at birth (-0.9 vs –2.3, p < 0.020). however, at time of discharge, no difference were seen in median weight sds (-1.8 vs – 2.0). at pma 40 weeks the early breastfed infants were significantly heavier (md 3.215 vs 2.887, p < 0.017), higher median weight sds (-0.5 vs -1.4, p < 0.008). the ‘early full breastfed’ infants continued to show superior weight gain up to 2 months corrected age, at which they had achieved a higher weight gain (md 5.223g vs 104 table 8. median changes in weight, length, and head circumference in sds from discharge to 2, 4, 6, 12, and 18 month (head from 40 week pma) variable 2 months 4 months 6 months 12 months 18 months e non-e e non-e e non-e e non-e e non-e delta sds weight 1,5 2,2 (1 0,9 1,6 (2 0,9 1.4 0,9 1,3 0,8 1,5 delta sds length 0,6 0,9 0,9 1,0 0,9 0,8 1,2 1,0 0,8 1,2 delta sds head -0,1 0,5 (3 -0,1 0,8 (4 0,2 0,1 -0,2 0,1 -0,6 -0,4 (1 p < 0.036 (2 p < 0.038 (3 p < 0.012 (4 p < 0.011 table 8. median changes in weight, length, and head circumference in sds from discharge to 2, 4, 6, 12, and 18 month (head from 40 week pma) 4.655g, p < 0.012), and a greater increase in sds (md 0.5 vs -0.5, p < 0.030). when the groups were compared regarding growth in terms of changes in sds from discharge to 2 months corrected age, the infants with ‘early full’ breastfeeding had a higher d weight sds (2.3 vs 1.6, p < 0.047). furthermore, these infants were fully breastfed for a significantly longer period than the other study infants (md 5.5 months vs 3 months, p < 0.041). discussion this study examined the impact on growth of infants with a birthweight below 1,900 g who were given (or not given) protein enriched breastmilk. those infants prescribed enrichment differed from the others at birth by lower ga, lower weight, length, and head circumference; they required more intensive care, lost more weight, and the increase in enteral feeding was slower than in the non-e group. despite more severe morbidity, the infants given protein-enriched milk showed similar growth as the other study infants. even regarding pi there was no difference between the groups. during the follow-up period up to 18 months corrected age, there were no inter-group differences in weight, length, head circumference or pi. on the other hand, when aga and sga infants were compared, the latter had lower pi values. according to lubchencko’s curves, a pi of less than about 2.45 at 40 weeks is below the 25th percentile, and 2.60 at the 50th percentile [14, 18]. the pi levels measured in this study demonstrated that the sga infants were thinner than the aga infants at 40 weeks and subsequently. the reason why the infants in the non-e group showed greater improvement in 105 table 9. infants exclusively breastfeed <36 weeks pma variable unit n<14 ga at birth md (range) 31.2 (28.6-34.0) birthweight, g md (range) 1.640 (1.125-1.868) birth length, cm md (range) 42 (38-43.5) birth head, cm md (range) 29.3 (26-31.5) sga n 3 twin n 3 ventilator n 3 cpap n 11 oxygen n 7 theophyllamine n 6 enrichment n 3 pma ‘full’ bf, weeks md (range 35.1 (32.6-35.9) pma at discharge, weeks md (range) 36.1 (33.6-37.0) weight at discharge, g md (range) 2.119 (1.740-2.590) length at discharge, cm md (range) 45.5 (43-47.5) head at discharge md (range) 32.5 (30-33.5) table 9. infants exclusively breastfeed <36 weeks pma weight gain and head growth from discharge to 2 and 4 months corrected age may be attributed to the effect of enrichment treatment on the e group; the latter infants gained weight during a longer period of hospital stay, the non-e group with a shorter duration of stay had their catch-up growth after discharge from hospital. lbw infants with controlled nutrition in hospital using enriched breastmilk may gain weight slower after discharge, when on demand feeding has been established. when twins were excluded, a smaller proportion of e-group infants were fully breastfed on discharge, compared with the non-e group. it is conceivable that this could be attributed to their lower ga and greater morbidity, necessitating a longer stay in hospital, thus contributing to maternal stress with possible consequent impairment of lactation. another explanation could be the non-verbalized message to the mothers, imparted by adding enrichment to their milk, implying its inadequacy. one further hinder to the establishment of breastfeeding in the e group could be programming for malnutrition in lbw infants, leading to flagging interest in and slower progress with oral feeding, in combination with infant satiety, because of the slower gastrointestinal passage of proteinenriched milk. the subgroup of 14 infants who reached ‘full’ breastfeeding at a low pma had higher sds for weight at birth than the other infants; at discharge this difference had disappeared. one possible explanation for why they took to breastfeeding so early may be that, unlike the infants with lower weight sds at birth, they were not programmed for low energy intake and were therefore more interested in oral feeding. although there is a selection bias, the authors consider the discovery of this fact worth presenting. the finding that a smaller proportion of infants who received enriched milk breastfed ‘fully’ is worrisome, as breastmilk feeding is especially important for vlbw infants because of its impact on cognitive development [19]. breastmilk feeding may also be important for infants at risk of developing the metabolic syndrome, as duration of breastfeeding is evidently associated with reduced risk of high blood pressure [20] and obesity in adult life [21]. one factor that emerged as significant for explaining infant length at 12 and 18 months corrected age was sds for length at birth. this finding agrees with the common observation that a certain proportion of premature infants with lbw and with poor growth already started in fetal life will continue to show poor growth after discharge from hospital, regardless of type of nutrition given during their stay in hospital. however duration of ‘full’ breastfeeding also had significant impact on subsequent improvement in sds for weight. it is therefore essential that appropriate policies and practices for the establishment and maintenance of lactation and breastfeeding in these infants are applied, and that mothers-infants at risk of breastfeeding failure are given sufficient support by adequately trained professionals, in hospital and after discharge. when breastfed infants fail to grow satisfactorily, enrichment can be given as a complement to breastfeeding, intraorally via a syringe or by cup feeding, in order to not jeopardize breastfeeding by introduction of bottle feeding, whether in hospital or after discharge. an optimal design would have been to randomize infants weighing less than 1,900 g to treatment/non-treatment with enriched milk, but for ethical reasons such a study was inconceivable. in this study, data on infants without and with protein enrichment could be 106 obtained in a quasi-experimental situation, using retrospective data. this made it possible to describe adequate growth in lbw infants not given protein enriched breastmilk. conclusion protein enrichment of breastmilk enables lbw infants needing more intensive care to attain growth at discharge, comparable to growth observed in infants with lower degree of morbidity who did not receive protein enrichment. it appears that infants with low sds for length at birth will continue to show poor growth after discharge from hospital regardless of the type of nutrition given during their hospital stay. however duration of ‘full’ breastfeeding had a significant impact on subsequent improvement in sds for weight. therefore it is important that mothers of lbw infants are given sufficient support of lactation and breastfeeding. acknowledgements this study was supported by the vårdal foundation, the gillbergska foundation, the goljes memorial fund, procter & gamble, and swedish paediatric nursing cooperation. the authors would like to express their gratitude to maria haglund and magnus näslund for valuable support with the data collection. references 1. lucas a. (2005) long-term programming effects of early nutrition -implications for the preterm infant. j perinatol 25 suppl 2:s2-6 2. schanler rj. (2001) the use of human milk for premature infants. pediatr clin north am 48:207-19 3. hay ww, jr. (1994) nutritional requirements of extremely low birthweight infants. acta paediatr suppl 402:94-9 4. simmer k, metcalf r, daniels l. the use of breastmilk in a neonatal unit and its relationship to protein and energy intake and growth. j paediatr child health 1997;33:55-60 5. polberger s, lonnerdal b. (1993) simple and rapid macronutrient analysis of human milk for individualized fortification: basis for improved nutritional management of very-low-birth-weight infants? j pediatr gastroenterol nutr 17:283-90 6. velona t, abbiati l, beretta b, gaiaschi a, flauto u, tagliabue p, et al.(1999) protein profiles in breast milk from mothers delivering term and preterm babies. pediatr res 45:658-63 7. griffin ij. (2002) postdischarge nutrition for high risk neonates. clin perinatol 29:327-44 8. kuschel ca, harding je. (2004) multicomponent fortified human milk for promoting growth in preterm infants. cochrane database syst rev cd000343 9. wauben ip, atkinson sa, grad tl, shah jk, paes b. (1998)moderate nutrient supplementation of mother's milk for preterm infants supports adequate bone mass and short-term growth: a randomized, controlled trial. am j clin nutr 67:465-72 10. lucas a, fewtrell ms, morley r, lucas pj, baker ba, lister g, et al. (1996) randomized outcome trial of human milk fortification and developmental outcome in preterm infants. am j clin nutr 64:142-51 11. faerk j, petersen s, peitersen b, michaelsen kf. (2000) diet and bone mineral content at term in premature infants. pediatr res 47:148-56 12. ramasethu j, jeyaseelan l, kirubakaran cp. (1993) weight gain in exclusively breastfed preterm infants. j trop pediatr 39:152-9 13. caple j, armentrout d, huseby v, halbardier b, garcia j, sparks jw, et al. (2004) randomized, controlled trial of slow versus rapid feeding volume advancement in preterm infants. pediatrics 114:1597-600 107 14. merenstein gb, gardner sl. (2002) handbook of neonatal intensive care. 5 ed. st. louis, missouri: mosby. 15. rennie jm, roberton nrc. (2002) a manual of neonatal intensive care. 4 ed. london: arnold. 16. marsal k, persson ph, larsen t, lilja h, selbing a, sultan b. (1996) intrauterine growth curves based on ultrasonically estimated foetal weights. acta paediatr 85:843-8 17. niklasson a, ericson a, fryer jg, karlberg j, lawrence c, karlberg p. (1991) an update of the swedish reference standards for weight, length and head circumference at birth for given gestational age (1977-1981). acta paediatr scand 80:756-62 18. lubchenco lo, hansman c, boyd e. (1966) intrauterine growth in length and head circumference as estimated from live births at gestational ages from 26 to 42 weeks. pediatrics 37:403-8 19. anderson jw, johnstone bm, remley dt. (1999) breast-feeding and cognitive development: a metaanalysis. am j clin nutr 70:525-35 20. lawlor da, riddoch cj, page as, andersen lb, wedderkopp n, harro m, et al. (2005) infant feeding and components of the metabolic syndrome: findings from the european youth heart study. arch dis child 90:582-8 21. koletzko b, broekaert i, demmelmair h, franke j, hannibal i, oberle d, et al. (2005) protein intake in the first year of life: a risk factor for later obesity? the e.u. childhood obesity project. adv exp med biol 569:69-79 biographical notes for eva-lotta funkquist eva-lotta funkquist works as paediatric nurse in a neonatal unit at a university/regional hospital. she is doctoral candidate at the department of women’s and children’s health at the university of uppsala, sweden. the main focus of her research is breastfeeding, and feeding and caregiving procedures. biographical notes for torsten tuvemo professor torsten tuvemo holds the chair in paediatrics at uppsala university. his research has been focused on paediatric endocrinology and human growth. biographical notes for björn jonsson björn jonsson is a medical statistician. his field of research is childhood growth. he is former head of statistics at pharmacia peptide hormones. biographical notes for fredrik serenius fredrik serenius is associate professor of paediatrics at umeå university. he is a neonatologist whose main field of research has been perinatal epidemiology. biographical notes for kerstin hedberg nyqvist kerstin hedberg nyqvist is assistant professor of nursing at the department of women’s and children’s health at uppsala university, combined with clinical work at the neonatal unit of the children’s hospital, uppsala, a university/regional hospital. her teaching and research work is focused on paediatric and neonatal nursing. corresponding author: eva-lotta funkquist rn, mns, doctoral candidate department of women’s and children’s health university children’s hospital 751 85 uppsala sweden eva-lotta.funkquist@kbh.uu.se 108 vol_116_002_sups_a_547636 155..159 upsala journal of medical sciences. 2011; 116: 155–159 original article acute renal failure in severe pancreatitis: a population-based study hung-yuan lin3, jiun-i lai1,2, yi-chun lai1,2, po-chou lin1,2, shih-chieh chang1,2 & gau-jun tang1,2 1national yang-ming university school of medicine, taiwan, republic of china, 2department of medicine, national yang-ming university hospital, taiwan, republic of china, and 3shih-hsin university, department of information management, taiwan, republic of china abstract introduction. acute pancreatitis (ap) is a common illness with varied mortality and morbidity. patients with ap complicated with acute renal failure (arf) have higher mortality than patients with ap alone. although arf has been proposed as a leading mortality cause for ap patients admitted to the icu, few studies have directly analyzed the relationship between ap and arf. methods. we performed a retrospective study using the population-based database from the taiwan national health insurance research database (nhird). in the period from 1 january 2005 to 31 december 2005, every patient with ap admitted to the icu was included and assessed for the presence of arf and mortality risk. results. in year 2005, there were a total of 221,101 admissions to the icu. there were 1,734 patients with ap, of which 261 (15.05%) patients also had a diagnosis of arf. compared to sepsis and other critical illness, patients with ap had a higher risk of having a diagnosis of arf, and patients with both diagnoses had a higher mortality rate in the same icu hospitalization. conclusion. ap is associated with a higher risk of arf, and, when both conditions exist, a higher risk of mortality is present. key words: acute renal failure, intensive care, severe acute pancreatitis introduction acute pancreatitis (ap) is a relatively common medical illness with a wide range of morbidity and mortality, with an estimated incidence of 35–80 cases per 100,000 each year (1). severe acute pancreatitis (sap), as proposed by bradley, known as the atlanta classification, is established by either an apache ii score >8, ranson score >3 (2), the presence of more than one organ failure, or local complications (3). sap has been associated with an increased mortality, estimated from 7% to 47% (4,5). acute renal failure (arf) in the setting of ap has been shown to have a 10-fold increase in mortality (74.7% versus 7%) in a study of 563 patients (6). a similar result was reported in another study, showing 71.2% mortality versus 6.8% in sap patients with or without arf (7). in agreement with the above studies, a recent cross-sectional study reported a 5-fold increase (66.6% with arf versus 14.5% without arf) in mortality in sap patients, providing an updated assessment of the clinical standpoint (8). to our knowledge, few studies directly pin-pointed the relationship between sap and arf in icu patients (8), although both conditions are common etiologies in the icu and pose diagnostic and therapeutic challenges. in taiwan, the national health insurance (nhi) program was started in 1995 and covers nearly all inhabitants. because all claims data of in-patients are available to researchers in electronic form, it is possible to conduct a large-series retrospective study to investigate the prevalence of ap and arf and related mortality data in icu patients in taiwan (9). in light of the above background, we conducted a study using the population-based database of the nhi program in correspondence: shih-chieh chang, md, department of medicine, national yang-ming university hospital,no. 152, xin-min road, yilan, taiwan, 26042. fax: +886-3-935-1838. e-mail: dtsurga9@yahoo.com.tw (received 29 november 2010; accepted 6 december 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.547636 taiwan, aiming to: 1)assess the prevalence and characteristics of ap patients admitted to the icu, and 2) identify the prevalence of arf coexisting with ap, and determine if a relationship exists that can serve as future treatment guide. we then further analyze the statistical significance of the data distribution and identify risk factors leading to increased mortality. this population-based study method is a novel approach in assessment of patients with pancreatitis admitted to icu, and we anticipate the results to provide clinical significance in treatment planning for intensivists and internists. methods database pooled data obtained from the taiwan national health insurance research database (nhird) in the period from 1 january 2005 to 31 december 2005 were used for analysis in this study. the nhird is a nation-wide database including all in-patient medical benefit claims for the taiwanese population, with an inclusion rate of over 96% of the population. the database includes registries of contracted medical facilities and board-certified physicians, monthly records of in-patient claims summaries, and other in-patient hospitalization details. each individual operation’s procedure codes and diagnosis codes are included with compliance to classification using the international classification of disease, 9th revision, clinical modification (icd-9-cm). this study was approved by the ethics committee of the authors’ institution on the basis that no disclosure of any patient’s privacy or individual data could be made public due to the encoding nature of the database. study sample we included every icu hospitalization episode from 1 january 2005 to 31 december 2005. the database included a total of 221,101 patients. we then selected patients with a diagnosis icd-9 code compatible with acute pancreatitis (icd-9 code 577.0) and acute renal failure (584.x; x = 0–9). age, gender, icd-9 classification codes, and mortality data were recorded (figure 1). each admission episode was recorded for the main diagnosis icd code using the international classification of disease, 9th revision, clinical modification (icd-9-cm). the nature of the claims database was that laboratory information was not included, therefore only the admission date, discharge date, patient profile, and icd-9 diagnosis codes were available; there was no way of knowing if the coding was correct for each patient. however, according to the national health insurance policy, a protocol existed of peer review from another independent physician for the claims. if the diagnosis or claim was found at fault, the claim would be revoked; therefore, an internal validation system exists for the accuracy of each claim reviewed. we rely on this internal control to assume accuracy of our database and the study results. statistical analysis in this study, mysql 4.1 (1995–2008 mysql ab, 2008–2009 sun microsystems, inc.), was used as database software for data linkage and processing. descriptive data were presented, including number of patients and percentages. multivariate logistic regression was used to assess hazard ratios and risk (spss software, version 14.0, spss corp., chicago, il, usa). results are displayed as coefficients, odds 221,101 patients admitted to icu from 1 jan 2005 to 30 dec 2005 1,734 patients admitted to icu due to acute pancreatitis 261 patients with both pancreatitis and acute renal failure 1. test for a higher prevalence in ap patients compared with a. all non-ap patients b. non-ap patients with another specific diagnosis 2. test for higher mortality in ap patients having arf, compared with ap patients without arf figure 1. study design. 156 h.-y. lin et al. ratios, and 95% confidence intervals. a p value £ 0.05 (two-tailed) was considered as statistically significant. results statistics of icu admissions in year 2005 during the period of 1 january 2005 to 31 december 2005, there were a total of 221,101 valid icu admissions. the gender ratio was male/female: 60.5%/39.5%. the age distribution is summarized in table i. during the period of 1 january 2005 to 31 december 2005, there were a total of 1,734 icu admissions which included the diagnosis of acute pancreatitis (icd-9 code:577.0). the gender ratio was male/ female: 66.9%/33.1%. the age distribution and mortality rate for each age-group are summarized in table ii. acute pancreatitis is associated with an increased incidence of acute renal failure we included every icu admission to assess patients with diagnosis of acute renal failure (defined as cases with icd-9 coding of 584.x; x = 0–9), diagnosis of acute pancreatitis (icd-9 code:577.0), and patients with both diagnoses. our results showed 261 patients having both diagnosis of ap and arf (male/female ratio: 74.33%/35.67%). we then compared the probability of arf occurring in patients with and without ap. using logistic regression, the incidence of arf coexisting with ap was 15.05% (261/1734), with an odds ratio of 4.82 when compared to nonap patients (coefficient value 1.582; odds ratio 4.862; p < 0.01). to avoid confounding factors and to further validate this finding, we separately tested patients with ap against non-ap patients admitted due to another diagnosis for the incidence of arf. we selected the five leading diagnoses for icu admission: ischemic heart disease (icd-9 code: 410–414), lungrelated disorders (including respiratory failure) (icd-9 code: 518), pneumonia (icd-9 code: 486), cerebrovascular disease (including hemorrhage and infarction of cerebral arteries) (icd-9 code: 431– 434),and sepsis (icd-9 code: 038). we included non-ap patients who had either one of the above diagnoses, and classified them into five groups according to each diagnosis. we then tested the ap group against each group separately. using multivariate logistic regression, the ap group showed a significant increase in risk of having a diagnosis of arf when compared against the other groups (table iii). our results show that patients with ap had a higher risk of having a diagnosis of arf in the same icu hospitalization, when compared with either all non-ap patients or non-ap patients with an individual diagnosis. the presence of acute renal failure is associated with increased mortality in icu patients admitted due to acute pancreatitis we assessed whether ap coexisting with arf was associated with increased mortality. patients with both diagnosis of ap and arf had a mortality rate of 23.76% in the same hospitalization when compared to patients with ap alone without arf, who had a mortality rate of 8.08% (odds ratio 3.752 (2.640– 5.331); p < 0.05) (table iv). mortality was defined table i. age distribution of icu admissions in 2005. age number percentage (%) 18 and under 16,571 7.5 19–28 8,685 3.9 29–38 11,831 5.3 39–48 16,599 7.5 49–58 26,140 11.8 59–68 29,984 13.6 69–78 48,533 22.0 above 78 62,758 28.4 total 221,101 100 table ii. age distribution of icu admissions of acute pancreatitis in 2005. age number prevalence (%) mortality rate (%) 18 and under 26 1.5 3.8 19–28 43 2.5 2.3 29–38 179 10.3 7.3 39–48 347 20.0 10.4 49–58 263 15.2 6.1 59–68 211 12.2 8.1 69–78 302 17.4 9.9 above 78 363 20.9 18.5 acute renal failure in severe pancreatitis 157 as death from any cause in the same course of icu hospitalization. discussion severe acute pancreatitis (sap) in the intensive care unit poses a therapeutic challenge with significant mortality (10). the pathophysiology began as severe systemic inflammatory response in the early stages and necrosis of the pancreas later on (10). it is in the systemic inflammatory process that hypovolemia ensues, either from wide-spread vasodilation or fluid sequestration, with tissue hypoperfusion and ultimately causing organ damage and failure (11). several studies have described organ failure accompanying pancreatitis, with renal failure always one of the culprit organs (5,10,11). acute renal failure has been shown to increase mortality in sap patients (4,5,10), and the progression of acute renal failure should alert the physician of progressive disease. in our study, a total of 1,734 patients were admitted to the icu due to acute pancreatitis (0.78%). the prevalentage-groups were age 39–48 (20.01%) and age over 78 (20.93%),which correlated with the risk factors such as increased alcohol consumption in the mid-40s age-group (12). the prevalent age-groups (age 39–48 and age >70) also presented with the highest mortality (10.4% and 18.5%, respectively). the prevalence of acute renal failure coexisting with acute pancreatitis was 15.05%, significantly higher than non-pancreatitis patients. in non-pancreatitis patients, patients with sepsis had the highest incidence of acute renal failure (13.2%). compared to pneumonia, cerebrovascular disease, myocardial infarction, and respiratory failure, sepsis involves more wide-spread inflammation and organ damage and probably has a more similar pathophysiology with pancreatitis patients with coexisting acute renal failure. commonly used prognostic scoring systems for pancreatitis include the ranson criteria, apache ii score, and the atlanta classification which is usually used for standardizing clinical trials using the above classifications (13). in the ranson criteria an increase in blood urea nitrogen was indexed as a scoring point, while in the apache ii system serum creatinine was included as one factor (14). our study validates this concept that acute renal failure is associated with extremely high mortality in sap patients, and also shows that severe acute pancreatitis itself is associated with higher incidence of acute renal failure. the exact mechanism for pancreatitis-related renal failure is not yet well settled, but studies have shown that systemic inflammation, cytokine production, free radicals, and other factors influencing microcirculation play a role (15). in a recent study (8), the investigators assessed multivariate risk factors that would predict outcome. age, history of kidney disease, and abdominal compartment syndrome were found to be independent prognostic factors (8). the study, using a different approach from that of ours, concluded an increased mortality and duration of icu stay. the results corroborate our study with its lack of information about patient outcome being there with deprived of definitive predictive power. combination of that study and ours would provide strong evidence of increased mortality effect of arf on sap patients. our study provides a novel approach to investigate icu patients by using a large-scale population-based study. because of the near universal inclusion of the taiwanese population in the national health insurance, the nhird reflects a ‘real world’ database table iii. risk of acute renal failure in non-pancreatitis patients with separate diagnoses compared to pancreatitis patients. disease group coefficient odds ratio percentage with acute renal failure p value 1 ischemic heart disease �2.213 0.109 (0.089–0.134) 2.5% <0.05 2 lung-related disorders (including respiratory failure) �0.979 0.376 (0.308–0.457) 8.3% <0.05 3 pneumonia �1.081 0.339 (0.278–0.414) 7.3% <0.05 4 cerebrovascular disease (including hemorrhage and infarction of cerebral arteries) �2.493 0.083 (0.066–0.103) 1.9% <0.05 5 sepsis �0.432 0.649 (0.534–0.789) 13.2% <0.05 table iv. mortality risk in pancreatitis patients with and without acute renal failure. number mortality number mortality rate (%) patients with pancreatitis and acute renal failure 261 62 23.8 patients with pancreatitis without acute renal failure 1,473 119 8.1 158 h.-y. lin et al. representing statistical significance and near minimal sampling error or selection bias. our weakness lies in misclassification errors, which is under control owing to the peer review validation of all claims as previously mentioned. another limitation is that the nhird database only includes claims data and relevant treatment data, while laboratory data are not included. this poses a significant limitation to our study that the exact apache ii score or other scoring system of each patient could not be obtained. we could only rely on the assumption that for a patient to be admitted to the icu, an apache ii score of more than 8 should be common; indeed, according to our peer validation, the claim of a patient admitted to the icu with an apache ii score less than 8 would very likely be revoked. therefore, by definition of the atlanta criteria, patients admitted to the icu with the diagnosis of acute pancreatitis should classify as severe acute pancreatitis (sap). also, the nature of the database precluded the assumption of a causal relationship. however, this study still brings important messages with strong statistical significance owing to its large number of cases and its near total sampling with little room for selection and sampling bias. we can judiciously conclude that acute pancreatitis patients admitted to the icu (very possibly mostly sap patients) have a higher risk of having an acute renal failure, and having both diagnoses may lead to higher mortality. in conclusion, our study of 1,734 patients with acute pancreatitis admitted to the icu revealed that acute renal failure was more frequently encountered in this critical illness associated with higher mortality. our results imply that deterioration of renal function in sap patients should be closely monitored and the possibility of renal damage should be prevented. our study provides a novel approach to studying icu patients using a population-based database. although large case numbers and real-world descriptions are the strengths of our study, further controlled studies with complete clinical characteristics are needed for demonstration of a definite causal relationship. declaration of interest: this study had no specific funding source. all authors declare no conflicts of interest for this work. hung-yuan lin and jiun-i lai contributed equally to this work. references 1. steinberg w, tenner s. acute pancreatitis. n engl j med. 1994;330:1198–210. 2. ranson jh, rifkind km, roses df, fink sd, eng k, spencer fc. prognostic signs and the role of operative management in acute pancreatitis. surg gynecol obstet. 1974; 139:69–81. 3. bradley el 3rd. a clinically based classification system for acute pancreatitis. summary of the international symposium on acute pancreatitis, atlanta, ga, september 11 through 13, 1992. arch surg. 1993;128:586–90. 4. halonen ki, leppaniemi ak, puolakkainen pa, lundin je, kemppainen ea, hietaranta aj, et al. severe acute pancreatitis: prognostic factors in 270 consecutive patients. pancreas. 2000;21:266–71. 5. compañy l, sáez j, martínez j, aparicio jr, laveda r, griñó p, et al. factors predicting mortality in severe acute pancreatitis. pancreatology. 2003;3:144–8. 6. kes p, vucicević z, ratković-gusić i, fotivec a. acute renal failure complicating severe acute pancreatitis. ren fail. 1996; 18:621–8. 7. herrera gutiérrez me, seller pérez g, de la rubia de gracia c, chaparro sánchez mj, nacle lópez b. [acute renal failure profile and prognostic value in severe acute pancreatitis]. med clin (barc). 2000;115:721–5. 8. li h, qian z, liu z, liu x, han x, kang h. risk factors and outcome of acute renal failure in patients with severe acute pancreatitis. j crit care. 2010;25:225–9. 9. national health insurance research database. available at: http://w3.nhri.org.tw/nhird//en/background.html. 10. wilmer a. icu management of severe acute pancreatitis. eur j intern med. 2004;15:274–80. 11. sigurdsson gh. intensive care management of acute pancreatitis. dig surg. 1994;11:231–41. 12. moore aa, gould r, reuben db, greendale ga, carter mk, zhou k, et al. longitudinal patterns and predictors of alcohol consumption in the united states. am j public health. 2005;95:458–65. 13. carroll jk, herrick b, gipson t, lee sp. acute pancreatitis: diagnosis, prognosis, and treatment. am fam physician. 2007;75:1513–20. 14. knaus wa, draper ea, wagner dp, zimmerman je. apache ii: a severity of disease classification system. crit care med. 1985;13:818–29. 15. zhang xp, wang l, zhou yf. the pathogenic mechanism of severe acute pancreatitis complicated with renal injury: a review of current knowledge. dig dis sci. 2008;53: 297–306. acute renal failure in severe pancreatitis 159 tf-iups160022 295..298 original article ethical issues in preconception genetic carrier screening ulrik kihlbom center for research ethics and bioethics, uppsala university, uppsala, sweden abstract population-based preconception genetic carrier screening programmes (pcs) with expanded panels are currently being developed in the netherlands. this form of genetic screening for recessive traits differs from other forms of genetic testing and screening in that it is offered to persons not known to have an increased risk of being carriers of genetic traits for severe recessive diseases and in that they include tests for a large number of traits, potentially several hundred. this raises several ethical issues around justice, consequences, and autonomy. it will be argued that most of these ethical problems call for cautious reflection when setting up pcs and similar programmes within preconception care. it is moreover argued that it is ethically problematic to have an official aim and failing to mention possibly legitimate public aims that actually drive the development of pcs. article history received 17 march 2016 revised 29 april 2016 accepted 9 may 2016 keywords ethics; justice; medicalization; preconception genetic carrier testing; reproductive autonomy introduction this article introduces and discusses some pertinent ethical issues of population-based preconception genetic carrier screening programmes (pcs). the aim is to provide a conceptual framework that may enhance ethical debate and reflection of preconception care in general and pcs in particular. after a brief description of pcs, three ethical grounds for the widely accepted criteria for screening programmes are distinguished, and a number of issues are identified under these headings. preconception genetic carrier testing has for some years targeted groups, families, or individuals, such as the ashkenazi jews and the population at cyprus, who have a known increased risk for being carriers of severe recessive autosomal diseases (1). in these cases, couples belonging to such subpopulations are tested for single or a few recessive traits, and there are no longer any major ethical controversies about whether or not to have the screening programmes. the pcs that are currently being developed in the netherlands differ from earlier screening programmes in two ways (2). firstly, they target couples wanting to have a child and without known increased risk for recessive autosomal diseases, i.e. the general population. secondly, these programmes test individuals for a large number of traits. pcs is not yet in practice in european health care even though available through commercial companies. pilot programmes are currently being developed in at least two university hospitals in the netherlands. the pcs programme being developed at the university hospital in groningen includes a panel of 50 recessive traits for severe and rare diseases with early onset (2). potentially, test panels can be further expanded to several hundred recessive traits. couples for whom the results of the test show that both partners are carriers of such a trait have different options when it comes to family planning. they can decide to live with the 25% risk of having a child with the disease and choose not to do anything. options to avoid the risk range from non-medical options like refraining from having children, adopting children, or even changing partner, to the use of reproductive technologies like in vitro fertilization (ivf) and preimplantation diagnostics, prenatal diagnostics, or sperm/egg donation. discussion currently, there are no medical consensus statements or professional society guidelines regarding the use of pcs. if and when such guidelines are put into place, one might expect that they will not diverge much from the criteria used in order to assess other forms of screening programmes. wilson and jungner proposed 10 criteria for screening programmes almost 50 years ago (3). a more contemporary set of criteria adapted to genetic screening, suggested by andermann, is the following (4): 1. the screening programme should respond to a recognized need. 2. the objectives of screening should be defined at the outset. 3. there should be a defined target population. 4. there should be scientific evidence of screening programme effectiveness. 5. the programme should integrate education, testing, clinical services and programme management. 6. there should be quality assurance, with mechanisms to minimize potential risks of screening. contact ulrik kihlbom ulrik.kihlbom@crb.uu.se uppsala universitet medicinska fakulteten, centre for research ethics & bioethics, uppsala university box 564, uppsala, 751 05 sweden � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 4, 295–298 http://dx.doi.org/10.1080/03009734.2016.1189470 http://creativecommons.org/licenses/by/4.0/ 7. the programme should ensure informed choice, confidentiality and respect for autonomy. 8. the programme should promote equity and access to screening for the entire target population. 9. programme evaluation should be planned from the outset. 10. the overall benefits of screening should outweigh the harm. (4) exactly how these criteria should be interpreted and weighted against each other must, presumably, be a question to be answered in each particular case. the ethical grounds that reasonably justify having these screening criteria rather than others can be summarized as three broad normative ideas: � justice � consequences � autonomy considerations of justice seem clearly to be behind criterion 8, but can also be viewed as the basis of criteria 4 and 5 as effectiveness in health care is important for the capacity to allocate recourses in a fair way. the appeal to consequences justifies primarily criteria 1, 3–6, 9, and 10. thus, in the case of screening programmes, the consequences that may provide good reasons for implementation concern satisfying needs in terms of promotion and maintenance of health and well-being, avoidance and reduction of suffering, and keeping societal costs low in an effective and good way. criterion 7 obviously concerns respect for and promotion of autonomy. the ethical discussion on pcs and reproductive ethics circles around one or several of these three normative ideas that are three of the most central ideas in ethics. the following is not intended as a full list or exploration of ethical issues that can be related to pcs. the vast discussion on genetic results and integrity, abortion, or duties to future generations will be omitted. some of the issues will only be mentioned below; the few discussed in some detail are those less discussed in the literature and most relevant for pcs and preconception care in general. justice one of the debates related to justice is the issue of prioritization. how should resources within preconception care be prioritized in relation to other health care resources (5–8)? another issue of justice is that of discrimination and stigmatization, both with regard to those who will test positive as carriers, and with regard to people who will actually develop the disease that the screening programme tests for. some authors highlight the risk that carriers of a recessive trait can have loss of self-worth and be subject of stigmatization (9–11). a more extensive ethical debate concerns the socalled expressivist argument. this argument claims that a preconceptional or prenatal measure that can be used to avoid the birth of children with certain traits expresses negative views to and about people with those traits (12–15). the expressivist argument and concerns for discrimination are obviously more relevant to pcs with test panels that include traits for diseases that allow a person to live a life of some length and quality. there are, surely, very severe early-onset recessive diseases that are incompatible with living with some quality of life. however, if the test panels were expanded so that they also include disease traits linked with variable penetrance, late onset, and substantial quality of life, then such pcs programmes certainly face this objection. consequences the objections concerning discrimination and stigmatization also relate to the possible consequences of pcs, the argument saying that there is a risk that screening programmes like pcs lead to discrimination, stigmatization, and even eugenics (15). the main problem of evaluating these kinds of ‘slippery slope’ arguments is not their relevance—discrimination, stigmatization, and even eugenics are bad consequences—but their plausibility. there is little evidence provided in the literature that there is a significant risk that something like pcs would lead to, for example, stigmatization. a related concern regarding pcs and preconception care in general is that these practices lead to an increased medicalization of social life (16). medicalization is a term with several usages in the literature. it was introduced in sociology to capture social processes in which medical concepts such as illness and health are applied to aspects of social life previously understood as being outside the realm of medicine (17). it was argued that medicalization of socially deviant behaviour, such as homosexuality and different forms of addiction, increased social control and thereby also individualized the problems that otherwise might be regarded as social. medicalization has since then also been used to denote more normal parts of human life such as child-birth, ageing, and death, as well as more preventive areas of medicine and public health. the last-mentioned is highly relevant to pcs and to preconception care in general, the idea being that the amplified social emphasis on health, in an increasing number of aspects of life, makes people live their lives in accordance with prescriptions from health care professionals. in sociology, the concept of medicalization is used as a way to criticize these social processes (although it should be pointed out that these critics do not think that this is something intended by health care professionals, and neither do they identify health care as the main driving force of medicalization). however, a simple answer to the question whether or not medicalization is desirable seems not to be available. the medicalization of birth, for instance, has had many good consequences in terms of saved lives and reduced suffering. there might also be some bad consequences involved, but an evaluation of medicalization processes must be contextualized and empirically well informed. in the case of pcs and preconception care, it would be most interesting (albeit requiring a lot of effort) to have more thorough empirical prospective studies of the way in which such practices influence how people perceive the planning of pregnancy and the making of a family. 296 u. kihlbom autonomy a concept related to medicalization is that of routinization. this concept has been especially discussed in the context of reproductive medicine and in relation to informed choices and autonomy. that a practice becomes routine within medicine and is considered as standard medical care seems to affect the normative perceptions people have regarding that practice (18,19). a well-known example is that of ultrasound screening including tests for trisomy chromosomal disorders that has become routine in many countries. in these countries most women undergo ultrasound testing, and studies indicate that many women think of such tests as ‘the responsible’ choice that also ‘protects the health’ of their child, although abortion is the only alternative to having the child (20,21). this has led these authors and many others to question the extent to which women have an autonomous choice in these contexts. the implementation of pcs could lead to a similar form of criticism. also here it should be pointed out that health care it is not necessarily the driving force behind the shift in normative expectations. couples and their social environment may well be essential factors in this development, which implies that any effort to adjust should take into account health care professionals and patients as well as their partners. the nature of pcs adds further complexity with regard to informed choice and autonomy. with expanded test panels, the risk estimation will become quite hard both for couples and for those health care professionals counselling them. many of the traits tested for are very rare but also very severe, so the weighing of the severity of the outcome versus its probability will be difficult. moreover, reduced penetrance and variable expressivity may bring further uncertainty into the decision-making matrix. for example, for some recessive traits, not all individuals with the genetic trait will develop the features of the disease (reduced penetrance), and those features may be different for different individuals (variable expressivity). furthermore, with an increased number of traits on the test panel, there might be more than one disease to consider. these are not necessarily insurmountable problems, but they will require a cautious and reflective development of the decision-making procedures around pcs. autonomy or consequences and autonomy—what are the legitimate aims of pcs? related to both the ethical notion of consequences and that of autonomy is the debate concerning the aims of reproductive screening programmes. in fact, the debate can be said to concern the question whether reproductive autonomy should be the primary goal or whether a more pluralistic account including public health considerations is a proper aim. the pluralist aim would include reproductive autonomy as well as improving population health by reducing the prevalence of disability and disease in the newborn population, and reducing future health and social welfare costs. the most cherished aim of screening programmes within reproductive health care is no doubt enhancement of reproductive autonomy (22–25). for instance, de jong et al. say, when discussing prenatal genetic screening, that: enabling meaningful reproductive choice with regard to parenting or avoiding a child with a serious disorder or disability is (or should be) the very aim of offering testing for fetal abnormalities. (22) specifically discussing pcs, de wert et al. claim that: [t]here are good moral reasons for regarding the enhancement of reproductive autonomy rather than prevention as the primary objective both of individual preconception genetic counselling and of pcs. (23) there are a number of problems with this view. first, public health considerations are in fact among the actual motives for having pcs programmes and preconception care in general. second, such motives can be perfectly legitimate aims for a carrier screening. third, there is no necessary opposition between public health aims and reproductive autonomy. finally, there is the corollary problem of having an official goal and other actual goals of the preconception programmes. these problems will be addressed in order. it seems quite obvious that public health considerations are among the actual motives for developing pcs. non-medical indications for offering pcs are not in question so far. the traits considered for being included in pcs are all recessive traits for severe diseases. having recessive traits such as, for example, height and eye size on the test panel would arguably enhance reproductive options, but no one has seriously proposed such a test panel, and for good reasons. neither is anyone questioning the criteria put forward by andermann stating that: ‘there must be a proven positive balance of benefits over harms for those participating’ (4). the harms and benefits considered regarding this criterion must, arguably, also include suffering and well-being of possible future beings. to avoid suffering, pressure, anxiety of couples, and costs of society seems, hence, to be actual motives behind pcs, as well as for other public health interventions. such aims can also be perfectly reasonable for preconception screening programmes, as well as the aim of promoting reproductive autonomy—at least within publicly funded health care systems where it seems simply unethical not to consider societal costs, how other patient groups are affected, and the general public health. the main worry of bringing in societal concerns among aims of pcs and other reproductive screening programmes is that this would pave the way for eugenics: promoting informed choice is commonly recognized as the chief purpose and benefit of prenatal screening, its very presence being viewed as a key way in which the process can be distanced from eugenics. (23) however, as others have pointed out, eugenics does not necessarily issue from the state, nor involve coercion (26,27). in a liberal society where pcs has become a routine, and if social norm pressure was put on couples to make certain choices, informed choice is no guarantee against eugenics. through the routinization of pcs, couples may not really consider this as being offered an additional reproductive choice but at as a standard part in preconception care. moreover, public health aims do not stand in any necessary opposition to the promotion of reproductive upsala journal of medical sciences 297 autonomy—quite the contrary. if the public health motives for setting up the pcs programme were openly declared together with a clearly expressed offer to make informed choices, then couples would have a set of values to contrast their own values against and form their own view. it is presumably easier for a couple to assume responsibility regarding their choice if it is clear that also health care professionals assume responsibility regarding their values and aims. to state public health aims openly is not the same as being directive in the sense of telling people what to do. lastly, there are several ethical problems with having an official aim and another set of actual aims that drive preconception care. health care runs the risk of being accused of deception, which in turn may lead to lack of trust in preconception care. furthermore, it constrains public debate by making it harder to debate the legitimacy of the aims. suppose, for instance, that there were economic incentives for university hospitals for developing pcs programmes. the legitimacy of such incentives cannot be ruled out a priori, but such motives need to be discussed openly. in conclusion, pcs raises several ethical issues that call for reflection on how pcs programmes should be implemented. there are issues around justice that concern prioritization, discrimination, and stigmatization. among the relevant consequences of pcs, medicalization is of primary interest. if pcs becomes established as a part of basic health care, some aspects of private life will become part of health care, and that needs to be discussed by the parties involved. issues around autonomy and informed consent are central in the discussion of pcs, and the phenomenon of routinization needs to be considered. most of these problems seem not to constitute decisive reasons against pcs, but are highly relevant to how the screening programme is set up and how the consent process is designed. however, a clear ethical problem is to have a discrepancy between an official aim and what really drives the development. acknowledgements the author wishes to thank members of the audience at the 3rd european congress on preconception health and care, 18 february 2016, uppsala, sweden, for valuable comments. disclosure statement the author reports no conflicts of interest. the author alone is responsible for the content 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http://www.rug.nl/news-and-events/news/archief2016/nieuwsberichten/umcg-dragerschapstest?lang&equals;en http://www.rug.nl/news-and-events/news/archief2016/nieuwsberichten/umcg-dragerschapstest?lang&equals;en http://www.rug.nl/news-and-events/news/archief2016/nieuwsberichten/umcg-dragerschapstest?lang&equals;en http://www.hgalert.org/topics/geneticdiscrimination/eugenics.htm http://www.hgalert.org/topics/geneticdiscrimination/eugenics.htm ethical issues in preconception genetic carrier screening introduction discussion justice consequences autonomy autonomy or consequences and autonomy&mdash;what are the legitimate aims of pcs? acknowledgements disclosure statement references tf-iups160024 174..178 original article associations between urinary kidney injury biomarkers and cardiovascular mortality risk in elderly men with diabetes aleksandra tonkonogia, axel c. carlssona,b, johanna helmersson-karlqvistc, anders larssonc and johan €arnl€ova,d adepartment of medical sciences, cardiovascular epidemiology, uppsala university, uppsala, sweden; bdivision of family medicine, department of neurobiology, care sciences and society, karolinska institutet, huddinge, sweden; cdepartment of medical sciences, biochemical structure and function, uppsala university, uppsala, sweden; dschool of health and social studies, dalarna university, falun, sweden abstract aim: three urinary biomarkers, kidney injury molecule-1 (kim-1), neutrophil gelatinase-associated lipocalin (ngal), and cystatin c, have been suggested as clinically relevant highly specific biomarkers of acute kidney tubular damage. yet, the utility of these biomarkers in the prognostication of diabetic nephropathy has been less studied. therefore, we aimed to investigate the longitudinal association between these urinary biomarkers and cardiovascular mortality in patients with diabetes. methods: the study sample consisted of participants with diabetes in the community-based uppsala longitudinal study of adult men (n¼91; mean age 77.8 years). during follow-up (median 8.3 years, interval 0.7–13.4 years), 33 participants died of cardiovascular causes. results: in a multivariable cox regression model adjusting for age, glomerular filtration rate, and urinary albumin/creatinine ratio, higher urinary kim-1/creatinine was associated with an increased risk for cardiovascular mortality (hr per sd increase 1.51, 95% confidence intervals 1.03–2.24, p¼0.03). neither urinary ngal/creatinine nor urinary cystatin c/creatinine were independently associated with an increased cardiovascular mortality risk. conclusion: in elderly men with diabetes, higher urinary kim-1/creatinine was associated with an increased long-term risk of cardiovascular mortality independently of established markers of diabetic nephropathy. our data provide support for kidney tubular damage as an important aspect of diabetic nephropathy that merits further investigation. article history received 10 january 2016 revised 15 may 2016 accepted 16 may 2016 key words biomarkers; diabetes; epidemiology; kidney; kim-1 introduction the prevalence of diabetes mellitus and diabetic nephropathy is escalating globally (1). the underlying pathology leading to diabetic nephropathy is multifactorial, and tubular damage appears early in disease development and may precede glomerular damage (2). yet, to date, the role of kidney tubular damage in diabetic nephropathy is incompletely understood. the currently used established kidney disease biomarkers for diagnosing diabetic nephropathy, urinary albumin/creatinine ratio and creatinine-based estimated glomerular filtration rate, have limited sensitivity and specificity as they are influenced by many extra-renal factors. therefore, further exploration of specific tubular damage biomarkers in diabetic nephropathy may prove to be clinically important. urinary kidney injury molecule-1 (ukim-1) (3,4), neutrophil gelatinase-associated lipocalin (ungal) (5,6), and cystatin c (ucystc) (7,8) have all been suggested as highly specific biomarkers for acute tubular damage; however, the aforementioned biomarkers may also indicate chronic tubular damage. yet, data on long-term prognostic importance of these biomarkers in patients with diabetes are limited. kim-1 is a transmembrane protein, almost exclusively expressed on the apical surface of proximal tubule cells as a response to injury, and its expression does not disappear until the damage is totally resolved (3). ngal is a protein which is a part of the lipocalin superfamily (5), expressed in the thick ascending limb of henle’s loop and collecting ducts as a response to injury. cystatin c is freely filtered through glomeruli (9) and thereafter reabsorbed and degraded in the proximal tubuli. thus, only trace amounts can be found in the urine from healthy kidneys (10). in the present study, we hypothesized that chronic kidney tubular damage may increase the risk of macrovascular complications in patients with diabetes. accordingly, we aimed to investigate the association between three kidney tubular damage biomarkers and the risk for cardiovascular mortality in a study population of elderly men with diabetes. material and methods the uppsala longitudinal study of adult men (ulsam) was used as study samples. the design has been described previously (http://ww.pubcare.uu.se/ulsam) (11–13). the analyses were based on the fourth examination cycle in 1997–2001, when the men were approximately 77 years old (n¼838). contact aleksandra tonkonogi aleksandra.tonkonogi@gmail.com malma backe 5k, 75647 uppsala, sweden � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 3, 174–178 http://dx.doi.org/10.1080/03009734.2016.1192704 http://ww.pubcare.uu.se/ulsam http://creativecommons.org/licenses/by/4.0/ complete data for the subgroup of individuals with diabetes were available for 91 participants. a written informed consent was given by all participants, and the study protocol was approved by the ethics committee of uppsala university. medical information participants with diabetes at baseline were defined using the following criteria: use of diabetes medication including oral medication or insulin, or a fasting glucose �7.0 mmol/l. information about smoking habits and ongoing medications was obtained using questionnaires. height (cm), body (kg), waist circumference (cm), bmi (kg/m2), hip circumference (cm), and waist–hip ratio were measured. blood pressure (mmhg) was determined in the right arm with the subject in supine position after 10 min of rest. clinical and biochemical analyses all blood samples, venous, were drawn in the morning after an overnight fast (14). twenty-four-hour urinary samples were collected. urinary kim-1 was analyzed (the cleaved extracellular part which can be found in the urine) using a commercial elisa kit (dy1750 r&d systems, minneapolis, mn, usa). urinary ngal was analyzed using an elisa kit (dy1757, r&d systems, minneapolis, mn, usa). urine albumin was measured by nephelometry (urine albumin, dade behring, deerfield il, usa), using a behring bn prospecv r analyzer (dade behring). creatinine in the urine was measured by means of a modified kinetic jaffe reaction on an architect ci8200v r analyzer (abbott, abbott park, il, usa), and the aforementioned values were used to calculate urinary albumin–creatinine ratio (uacr). the analysis of urinary cystatin c was carried out using a mindray bs-380 (shenzhen mindray biomedical electronics, shenzhen, china) with reagents from gentian (moss, norway). serum cystatin c was analyzed with a latex-enhanced reagent (n latex cystatin c, dade behring, deerfield, il, usa) with a behring bn prospec analyzer (dade behring). hba1c(%) was analyzed using a high-pressure liquid chromatograph with a gradient system (bio-rad laboratories). hdl-cholesterol (mmol/l) was measured by separating the particles by precipitation with chloride and phosphotungstate. total cholesterol (mmol/l) in serum was analyzed using enzymatic techniques, the il test cholesterol trinder’s method 181618-10 in a monarch apparatus (instrumentation laboratories, lexington, usa). plasma glucose was measured using the glucose dehydrogenase method (gluc-dh, merck, darmstadt, germany). estimated glomerular filtration rate (egfr) was calculated using serum cystatin c and by applying the formula y¼77.24x – 1.2623. for one of the cox proportional hazard models albuminuria was dichotomized at 3 g/mmol creatinine. all urinary biomarkers have been presented as a ratio to urinary creatinine to compensate for varying urine concentrations between the patients. outcome information about death due to cardiovascular disease was obtained from the swedish cause-of-death register and defined as international classification of diseases (10th revision) codes i00–i99. statistical analysis to investigate associations between cardiovascular mortality and the novel kidney injury biomarkers multivariable cox proportional hazard models were used. model a: crude; model b: adjusted for uacr; model c: adjusted for egfr; and model d: adjusted for uacr and egfr. additional cox proportional hazard analyses were carried out as secondary analyses, one including ukim-1/cr and known cardiovascular risk factors (systolic blood pressure, hypertension treatment, total cholesterol, hdl, lipid-lowering medication, smoking, previous cardiovascular event, hba1c, diabetes medication, uacr, egfr, and atrial fibrillation). moreover, cox proportional hazard models with all the three kidney injury biomarkers and uacr and egfr and a model with kim-1/cr as a dichotomous variable, below or above 175 ng/mmol, microalbuminuria, and chronic kidney disease ckd were carried out. results baseline characteristics can be seen in table 1, which gives us information about levels of the urinary biomarkers, smoking habits, relevant medication, and much more. during follow-up (median 8.3 years, interval 0.7–13.4 years) 33 participants died of cardiovascular causes (incidence rate of 4.7 per 100 person years at risk). in cox proportional hazard analyses, adjusting for age and established kidney disease biomarkers (egfr and uacr), it was found that higher ukim1/cr was significantly associated with an increased risk for cardiovascular mortality (table 2). table 1. baseline characteristics. variable mean sd a: continuous variables ukim/cr (ng/mmol) 121.4 10.2 ungal/cr (ng/mmol) 2050.4 261.4 ucyst/cr (ng/mmol) 14.0 0.003 age (years) 77.8 0.17 systolic blood pressure (mmhg) 152 4.43 total cholesterol (mmol/l) 5.15 0.14 hdl(mmol/l) 1.16 0.04 bmi (kg/m2) 27.3 0.53 hba1c (mono s, %) 5.8 0.12 gfr-cyst (ml/min/1.73 m2) 73.5 2.5 uacr (mg/mmol) 1.5 0.33 n % b: categorical variables smoking 4 4 hypertension treatment 61 67 lipid-lowering treatment 23 25 diabetes treatment 47 52 previous cvd 7 8 atrial fibrillation 12 14 microalbuminuria 33 36 chronic kidney disease 20 22 bmi: body mass index; gfr: glomerular filtration rate; hdl: high-density lipoprotein; previous cvd: previous cardiovascular disease events; uacr: urinary albumin–creatinine ratio; ucystc/cr: urinary cystatin c–creatinine ratio; ukim-1/cr: urinary kidney injury molecule-1–creatinine ratio; ungal/cr: urinary neutrophil gelatinase-associated lipocalin–creatinine ratio. upsala journal of medical sciences 175 in secondary analyses, ukim-1/cr was still associated with cardiovascular mortality when established cardiovascular risk factors (systolic blood pressure, hypertension treatment, total cholesterol, hdl cholesterol, lipid-lowering medication, smoking, previous cardiovascular disease, hba1c, diabetes medication) were added to model d with adjustments for both uacr and egfr (hr per standard deviation (sd) increase of ukim-1/ cr 1.82, 95% ci 1.13–2.95, p¼0.01). higher ucystc/cr was associated with an increased risk for cvd mortality in ageadjusted models, but this association was attenuated and no longer statistically significant after adjustment for both uacr and egfr (table 2). urinary ngal/cr was not associated with increased cvd mortality in any model (table 2). in a model including all three urinary biomarkers as well as egfr and uacr, only ukim-1/cr and egfr remained associated to cardiovascular mortality (hr per sd increase of egfr 0.58, 95% ci 0.37–0.89, p¼0.01; and for ukim-1/cr 1.54, 95% ci 1.03–2.30, p¼0.03). discussion in the present study, higher ratios of ukim-1/cr were associated with an increased risk of cardiovascular mortality even after adjusting for established kidney disease markers and cardiovascular risk factors. neither urinary ngal/cr nor urinary cystatin c/cr were independently associated with cvd mortality risk in multivariable models. our data provide additional support for kidney tubular damage as an important aspect of diabetic nephropathy that merits further investigation. there are a few previous community-based studies that have reported an association between tubular damage biomarkers and cardiovascular mortality (15–18). however, previous studies investigating the association between the urinary kidney biomarkers and mortality risk in patients with diabetes are scarce and inconsistent. in one study, kim-1/cr did not provide additional prognostic value for 4-year mortality risk after being adjusted for established cvd risk factors in 978 patients with type 2 diabetes (16). in contrast, a recent study in pima indians with type 2 diabetes showed that higher urinary kim-1 was associated with increased long-term risk of mortality but not end-stage renal disease (19). we are not aware of any previous studies reporting the association between urinary kim-1 and cardiovascular mortality in patients with diabetes. perhaps differences between previous studies and our study may be due to differences in outcome (total versus cvd mortality), length of follow-up, sampling procedure (spot sample versus 24-hour collection of urine) or differences in age, ethnicity, and gender distribution of the study samples. several mechanisms may explain the development of tubular damage in diabetes. first, tubular hypertrophy has been observed already after days of increased glucose levels (2). tubular cells are particularly vulnerable since their glucose uptake is insulin-independent (2). furthermore, hypertrophied cells are maintained in the g1 phase of the cell cycle (20) and are consequently more vulnerable to apoptotic stimuli. hyperglycemia also activates many growth factors. for example, tgfb, which can be stimulated by advanced glycation end-products and angiotensin ii (21,22), has been shown to be involved in the development of tubular hypertrophy (21). it has also been shown that higher urinary albumin may directly damage the tubuli through induction of pro-inflammatory and pro-fibrotic changes (23), as well as apoptosis of the tubular cells. tubular hypoxia has also been shown to be associated with nephropathy, and has been proposed to be a fundamental factor underpinning development of chronic kidney disease and end-stage renal disease (24). yet, we cannot say if hypoxia is mirrored by the kim-1 levels in the individuals with diabetes in the present study. the causal link between chronic tubular damage and the development of cardiovascular disease is less clear. when kidney function declines, calcium, potassium, urea, and various harmful proteins accumulate; however, this may be more relevant in later stages of kidney disease. accumulation of harmful substances may in turn lead to accelerated atherosclerosis and hence cvd. furthermore, tubular damage may result in an increased reabsorption of sodium and water in the proximal tubuli (2), which may lead to hypertension. neither urinary ngal nor urinary cystatin c could be linked to an increased risk of cardiovascular mortality. this may partially be explained by the fact that ngal expression is not only limited to the kidneys as is the case with kim-1. ngal is also a biomarker for inflammation. it is noteworthy that cystatin c is taken up by the proximal tubule cells and then degraded (7,8). thus, findings of cystatin c in the urine may mirror tubular dysfunction rather than specific tissue damage. the increased levels of urinary kim-1, as seen in patients with diabetes, may also be the result of reverse causation, meaning that cvd itself results in higher levels of ukim-1. it has been shown that patients with prevalent congestive heart failure have higher levels of kim-1 compared to healthy controls (25). diabetic nephropathy is a common and detrimental complication of diabetes leading to increased morbidity and table 2. the association between urinary biomarkers and the risk for cardiovascular mortality: multivariate cox proportional hazard regression. variable model a hazard ratio (95% ci) model b hazard ratio (95% ci) model c hazard ratio (95% ci) model d hazard ratio (95% ci) ukim-1/cr 1.58 (1.13–2.22) 1.51 (1.04–2.19) 1.50 (1.08–2.10) 1.52 (1.03–2.24) p 5 0.008 p 5 0.032 p 5 0.016 p 5 0.034 ungal/cr 1.07 (0.78–1.48) 0.99 (0.71–1.39) 1.07 (0.76–1.48) 0.98 (0.68–1.43) p¼0.66 p¼0.95 p¼0.71 p¼0.93 ucystc/cr 1.26 (1.00–1.60) 1.16 (0.86–1.56) 1.09 (0.85–1.40) 1.02 (0.75–1.39) p 5 0.049 p 5 0.033 p¼0.50 p¼0.89 p values: significant values in bold. model a: no adjustments; model b: adjusted for uacr; model c: adjusted for egfr; model d: adjusted for uacr and egfr. 176 a. tonkonogi et al. mortality, mainly due to cvd. therefore, it is of great clinical importance to evaluate novel kidney injury biomarkers, which may signal kidney damage at an earlier stage than microalbuminuria and reduced gfr. interestingly, previous studies have reported that elevated levels of kim-1/cr correlate with the progression from normoalbuminuria to microalbuminuria (14) and that urinary kim-1/cr is associated with insulin resistance even prior to the development of diabetes (26), indicating that urinary kim-1/cr could be an early marker for diabetic nephropathy. it is possible that kim-1 could be used to monitor the progression or regression of nephropathy since levels of kim-1 in the urine decrease as a response to raas blockade (27). however, it is not fully known if reduced levels of kim-1 will actually lead to decreased cardiovascular risk (15). importantly, the clinical relevance of kim-1 screening in patients with diabetes remains to be established, and further large-scale studies are needed. the main strength of our study is the longitudinal study design with up to 13 years of follow-up. the main limitation is the small study sample with few events during follow-up, which led to limited statistical power to detect modest associations between kidney biomarkers and outcome. yet, regardless of the limited statistical power, ukim-1/cr predicted cardiovascular mortality in all models. as the study participants were elderly men, extrapolations of our findings to women and other age groups and ethnicities have to be done cautiously. we did not distinguish between type of diabetes; however, diabetes type 2 is dominating among 77-year-old men in the community, and we suggest that the results should be interpreted as if all individuals had type 2 diabetes. in conclusion our data put forward urinary kim-1/creatinine as a promising biomarker for diabetic nephropathy and provide additional support for the importance of early tubular damage as part of the underlying pathology of diabetic nephropathy. acknowledgements aleksandra tonkonogi and axel c. carlsson contributed equally to the writing of this paper. disclosure statement the authors report no conflicts of interest. funding information this study was supported by the swedish research council, swedish heart-lung foundation, the marianne and marcus wallenberg foundation, dalarna university and uppsala university. the funding sources did not play any role in the design and conduct of the study collection, management, analysis, and interpretation of the data and preparation, review, or approval of the manuscript. dr €arnl€ov is the guarantor of this work, had full access to all the data, and takes full responsibility for the integrity of data and the accuracy of data analysis. references 1. ritz e. kidney damage in metabolic syndrome: nip it in the bud. am j kidney dis. 2009;53:726–9. 2. thomas mc, burns wc, cooper me. tubular changes in early diabetic nephropathy. adv chronic kidney dis. 2005;12:177–86. 3. huo w, zhang k, nie z, li q, jin f. kidney injury molecule-1 (kim1): a novel kidney-specific injury molecule playing potential double-edged functions in kidney injury. transplant rev (orlando). 2010;24:143–6. 4. li w, yu y, he h, chen j, zhang d. urinary kidney injury molecule1 as an early indicator to predict contrast-induced acute kidney injury in patients with diabetes mellitus undergoing percutaneous coronary intervention. biomed rep. 2015;3:509–12. 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urinary tubular injury biomarkers, kidney injury molecule-1, and n-acetyl-beta-dglucosaminidase. kidney int. 2011;79:464–70. 15. carlsson ac, larsson a, helmersson-karlqvist j, lind l, ingelsson e, larsson te, et al. urinary kidney injury molecule-1 and the risk of cardiovascular mortality in elderly men. clin j am soc nephrol. 2014;9:1393–401. 16. conway br, manoharan d, jenks s, dear jw, mclachlan s, strachan mw, et al. measuring urinary tubular biomarkers in type 2 diabetes does not add prognostic value beyond established risk factors. kidney int. 2012;82:812–18. 17. helmersson-karlqvist j, arnlov j, carlsson ac, harma j, larsson a. increased urinary cystatin c indicated higher risk of cardiovascular death in a community cohort. atherosclerosis. 2014;234:108–13. 18. helmersson-karlqvist j, larsson a, carlsson ac, venge p, sundstrom j, ingelsson e, et al. urinary neutrophil gelatinase-associated lipocalin (ngal) is associated with mortality in a community-based cohort of older swedish men. atherosclerosis. 2013;227:408–13. 19. fufaa gd, weil ej, nelson rg, hanson rl, bonventre jv, sabbisetti v, et al. association of urinary kim-1, l-fabp, nag and ngal with incident end-stage renal disease and mortality in american indians with type 2 diabetes mellitus. diabetologia. 2015;58:188–98. 20. phillips ao. the role of renal proximal tubular cells in diabetic nephropathy. curr diab rep. 2003;3:491–6. 21. chen s, hong sw, iglesias-de la cruz mc, isono m, casaretto a, ziyadeh fn. the key role of the transforming growth factor-beta upsala journal of medical sciences 177 system in the pathogenesis of diabetic nephropathy. ren fail. 2001;23:471–81. 22. gilbert re, cooper me. the tubulointerstitium in progressive diabetic kidney disease: more than an aftermath of glomerular injury? kidney int. 1999;56:1627–37. 23. waanders f, van timmeren mm, stegeman ca, bakker sj, van goor h. kidney injury molecule-1 in renal disease. j pathol. 2010;220:7–16. 24. mimura i, nangaku m. the suffocating kidney: tubulointerstitial hypoxia in end-stage renal disease. nat rev nephrol. 2010;6:667–78. 25. carlsson ac, larsson a, helmersson-karlqvist j, lind l, ingelsson e, larsson te, et al. urinary kidney injury molecule 1 and incidence of heart failure in elderly men. european j heart fail. 2013;15:441–6. 26. carlsson ac, calamia m, riserus u, larsson a, helmersson-karlqvist j, lind l, et al. kidney injury molecule (kim)-1 is associated with insulin resistance: results from two community-based studies of elderly individuals. diabetes res clin pract. 2014;103:516–21. 27. nielsen se, rossing k, hess g, zdunek d, jensen br, parving hh, et al. the effect of raas blockade on markers of renal tubular damage in diabetic nephropathy: u-ngal, u-kim1 and u-lfabp. scand j clin lab invest. 2012;72:137–42. 178 a. tonkonogi et al. associations between urinary kidney injury biomarkers and cardiovascular mortality risk in elderly men with diabetes introduction material and methods medical information clinical and biochemical analyses outcome statistical analysis results discussion acknowledgements disclosure statement funding information references untitled eosinophilic granuloma of the pelvis 209 key words: eosinophilic granuloma, pelvis, children, mri, treatment received 4 march 2008 accepted 31 march 2008 upsala j med sci 113 (1): 209–216, 2008 eosinophilic granuloma arising from the pelvis in children: a report of three cases akira ando1, masahito hatori1, masami hosaka1, yoshihiro hagiwara1, atsushi kita2, eiji itoi1 1department of orthopaedic surgery, tohoku university graduate school of medicine, 1-1 seiryomachi, aobaku, sendai, japan 980-8574, 2department of orthopaedic surgery, japanese red cross sendai hospital, 2-43-3 yagiyamahoncho, taihakuku, sendai, japan 982-8501 abstract eosinophilic granuloma (eg) is a benign tumor-like condition which is characterized by a clonal proliferation of langerhans-type histiocytes and defined as a local form of langerhans cell histiocytosis (lch). the radiographic appearances of eg are quite different depending on the phase of the disease and the site of involvement. a status of eg in the bone is divided into acute and chronic phases. radiologically acute phase of eg is difficult to differentiate from a malignant bone tumor such as ewing’s sarcoma or acute osteomyelitis. chronic phase of eg may mimic a chronic osteomyelitis or a benign bone tumor. we report 3 children’s cases of eg in the pelvis which showed quite different radiological features and clinical courses. a 6-yearold boy (case 1) had an osteolytic lesion with slightly defined margins in the right acetabulum. a 4-year-old boy (case 2) had a radiologically similar-looking lesion in the left acetabulum. these lesions resembled radiologically chronic osteomyelitis (brodie’s abscess) or a benign bone tumor and healed spontaneously after biopsy. a 2-year-old boy (case 3) had an osteolytic lesion with ill-defined margin in the ilium. it was difficult to differentiate from a malignant tumor such as ewing’s sarcoma, or acute osteomyelitis. the lesion became enlarged after needle biopsy. in spite of an additional curettage, the osteolytic lesion remained in the left pelvis in 1 year. treatment for eg is controversial. curettage of the affected site and bone grafting is usually accomplished. however, some eg heal spontaneously. it is of great importance to understand the natural course of eg and this knowledge will give us the opportunity to avoid unnecessary treatment. eg with poor osteolytic margins may progress further after biopsy. eg with well-defined margins may heal spontaneously after biopsy only. introduction eosinophilic granuloma (eg) is a benign tumor-like condition which is characterized by a clonal proliferation of langerhans-type histiocytes in the bone or lung [1, 2]. the causes and pathogenesis of eg are unknown. in spite of extensive genetic studies or virologic analyses, apparent genetic error or infectious agents have not been found [3, 4]. the radiographic features of eg is quite different depending on the phase of the disease and the site of involvement [3, 5]. a status of eg in the 210 akira ando et al. bone is divided into acute and chronic phases [5, 6, 7]. the acute phase of eg is a destructive, osteolytic lesion and has poor margins. therefore it is difficult to differentiate from a malignant tumor such as ewing’s sarcoma, or acute osteomyelitis [5]. on the other hand, the chronic phase of eg is a well-defined lesion, and the radiological features are similar to those of chronic osteomyelitis (brodie’s abscess) or a benign bone tumor [5, 8]. eg is a local condition of langerhans cell histiocytosis (lch) and make up 80% of lch in children [5]. the systemic condition of lch is a multisystem lifethreatening disorder. for example, hand-schüller-christian disease is defined as a triad of exophthalmos, diabetes insipidus and osteolytic lesions of the skull, and letterer-siwe disease shows hepatosplenomegaly, lymphadenopathy, skin rash, fever, anemia and thrombocytopenia [1, 4]. though these three diseases are quite different in disease expression pattern and their prognosis, the histology is similar to each other and indistinguishable [4]. the incidence of eg is estimated at 0.05–0.5 per 100,000 and 90% of eg occurs under the age of 15 years old [3, 4, 5]. male has twice as high incidence rate as female [8]. flat bones are involved in 70% of cases and long bones in 30% [8]. among the flat bones, the skull is most frequently involved, and the pelvis, vertebrae, mandible, and ribs are followed in decreasing order [1, 5]. we report 3 children’s cases of eg in the pelvis which showed quite different radiological features and clinical courses. case reports case 1 a 6-year-old boy was referred to our hospital with pain in the right thigh and limping for 2 months. physical examination indicated tenderness over the right hip joint without swelling. the motion of the joint was slightly restricted due to pain. plain radiographs of the pelvis showed a slightly defined osteolytic lesion in the right acetabulum (figure 1a). blood test demonstrated normal white blood cell (wbc) count (7,800 /μl), eosinophils (4.7%), and slight increased erythrocyte sedimentation rate (esr) (21 mm/h). computed tomography (ct) showed a 3.0 × 2.0 cm osteolytic low density lesion in the right acetabulum with a slightly defined margin (figure 1b). magnetic resonance imaging (mri) revealed that the mass had lower signal intensities on t1-weighted images and higher signal intensities on t2-weighted images than the bone marrow (figure 1c, d). the right ilium also had lower signal intensities on t1-weighted images (figure 1c). the mass extended into the lateral soft tissue of the pelvic bone on t2-weighted images (figure 1d). based on these examinations, an initial radiological diagnosis was acetabular osteomyelitis and an open biopsy was performed through the anterior approach. histological examination revealed mixed cellular infiltrate with histiocytes, eosinophils, lymphocytes, and macrophages, which led to the diagnosis of eg (figure 1e). because pain was relieved in a week after the biopsy, no further treatment was performed. the plain radiograph showed the signs of healing in 3 months after the biopsy (figure 1f), and complete healing was observed in a year. eosinophilic granuloma of the pelvis 211 case 2 a 4-year-old boy was referred with a month history of pain and limping in the left lower limb. physical examination revealed tenderness over the left hip joint without swelling. the motion of the joint was not restricted. blood test showed normal wbc count (8,520 /μl), eosinophils (5%), and slightly increased esr (35 mm/h). plain radiographs showed a 3.0 × 2.5 cm osteolytic lesion with slight marginal sclerosis in the left acetabulum (figure 2a). a complete bone scan with 99mtechnetium showed an increased uptake in the left acetabulum (figure 2b). the lesion and the surrounding area in the pelvic bone had lower signal intensities on t1-weighted images (figure 2c). the lesion and lateral soft tissue of the pelvic bone had higher signal intensities on t2-weighted images (figure 2d). a temporary diagnosis was osteomyelitis of the left acetabulum. ct-guided needle biopsy was performed through the posterolateral aspect of the lesion. histological examination showed characteristic appearances of eg (figure 2e). his pain rapidly subsided and limping disappeared in a couple of weeks after the biopsy. there was no abnormality radiologically after one year of the biopsy (figure 2f). case 3 a 2-year-old boy was referred with a limping in the left leg for a month. on physical examination, motion of the joint was not restricted and neither swelling nor tenderness was observed. wbc and esr increased (11,460 /μl and 30 mm/h, respectively). eosinophils were within the normal range (3%). plain radiograph figure 1 case 1, a 6-year-old boy of eg in the right acetabulum. a: anteroposterior radiograph of the pelvis showing a slightly defined osteolytic lesion in the right acetabulum (arrow). b: coronal ct demonstrating an osteolytic lesion with well-defined margins (arrow). c: coronal t1-weighted images. the lesion and the right ilium (arrow) showed lower signal intensities. d: coronal t2-weighted images. the lesion and the lateral soft tissue of the pelvic bone (arrow) demonstrated higher signal intensities. e: histological examination indicating a mixed cellular infiltrate with histiocytes, eosinophils, lymphocytes, and macrophages. arrows show eosinophils. f: three months after the open biopsy. radiographic signs of healing appeared. 212 akira ando et al. of the pelvis revealed an osteolytic lesion with ill-defined margins in the ilium (figure 3a). ct showed an osteolytic lesion in the ilium extending into the medial retroperitoneal space (figure 3b). the left ilium showed lower signal intensities on t1-weighted images (figure 3c) and higher signal intensities on t2-weighted images which extended into the medial retroperitoneal space and the lateral gluteal muscles (figure 3d). bone scan with 99mtechnetium showed a massive uptake in the left pelvis (figure 3e). an initial radiological diagnosis was ewing’s sarcoma and ct-guided needle biopsy was performed through the posterior aspect of the left ilium. the histological examination demonstrated a cellular lesion consisting of numerous histiocytes and eosinophils. the lesion was well-defined after the biopsy, but gradually enlarged in a month (figure 3f). ct also confirmed enlargement of the lesion, but the adjacent sacrum was not involved (figure 3g). mri demonstrated that the mass and soft tissue abnormalities became enlarged (figure 3h, i). curettage was carried out immediately. his pain and limping subsided gradually after the operation. however, the plain radiograph taken after 1 year later showed that the osteolytic lesion of the left pelvis remained (figure 3j). discussion the radiographic feature of eg is completely different depending on the phase of the disease and the site of involvement [3, 5]. a status of eg in the bone is divided figure 2 case 2, a 4-year-old boy of eg in the left acetabulum a: anteroposterior radiograph of the pelvis showing a slightly defined osteolytic lesion in the left acetabulum (arrow). b: bone scan with 99mtechnetium demonstrating an increased uptake in the left acetabulum. c: coronal t1-weighted images. the lesion and the left ilium (arrow) showed lower signal intensities. d: coronal t2-weighted images. the lesion and lateral soft tissue of the pelvic bone (arrow) demonstrated higher signal intensities. e: histological examination indicating characteristic appearances of eg. arrows show eosinophils. f: one year after needle biopsy. a radiograph showed normal appearances. eosinophilic granuloma of the pelvis 213 into acute and chronic phases [5, 6, 7]. the acute phase of eg shows osteolysis with poorly defined margins and the chronic phase of eg shows well-defined margins. case 3 is consistent with the acute phase and case 1 and 2 are the chronic ones. in the pelvis, the lesion may present as poorly defined areas of osteolysis in the acute phase that become progressively circumscribed as they mature in the chronic phase [4]. figure 3 case 3, a 2-year-old boy of eg in the left ilium a: anteroposterior radiograph of the pelvis showing an osteolytic lesion with ill-defined margins in the ilium (arrows). b: axial ct demonstrating an osteolytic lesion in the ilium extending into the retroperitoneal space (arrows). c: axial t1-weighted images. the left ilium showed lower signal intensities. d: axial t2-weighted images. the medial retroperitoneal space (arrow) and lateral gluteal muscles (arrowheads) showed higher signal intensities. e: bone scan with 99mtechnetium demonstrating an increased uptake in the left pelvis. f: anteroposterior radiograph of the pelvis in a month after the needle biopsy. the lesion was well-defined but enlarged. g: axial ct demonstrating the enlarged lesion without extension into the adjacent sacrum. h, i: axial t1 and t2-weighted images. the mass and surrounding soft tissue abnormalities enlarged. j: one year after the thorough curettage of the lesion. the osteolytic lesion of the left pelvis remained. 214 akira ando et al. mri is a sensitive, but nonspecific modality to detect bone marrow involvement and a soft tissue mass [4, 5, 9]. t1-weighted images are useful for demonstrating bone marrow involvement and t2-weighted images for indicating a soft tissue mass [4, 5, 9]. these changes are considered as edema of bone marrow and soft tissues [4]. edema of adjacent bone marrow and soft tissue is particularly seen in the acute phase lesions [5]. chronic phase lesions show decreased signal intensity on t2-weighted images, indicating re-ossification of the osteolytic lesions with resolution of the soft tissue mass [4]. the acute phase is defined as the tumor invading the surrounding soft tissue but the chronic phase is localized as itself [8]. case 3 showed an extensive, irregular soft tissue mass in the medial retroperitoneal space and the lateral gluteal muscles. on the other hand, the mass in the case 1 and 2 was localized. vertebral lesions show a symmetrical flattening of the vertebra with intervertevral disc space preservation, which are called as “vertebra plana”, and easily diagnosed only by plain radiographs [4]. however, it is very difficult to diagnose from radiological examinations in the pelvis. plain radiograph or mri is a very sensitive modality to detect the lesions, but it is not specific because of the radiological diversity of eg. we could not distinguish eg from malignant bone tumors such as ewing’s sarcoma or acute osteomyelitis in the case 3 and a chronic osteomyelitis or benign bone tumors in the case 1 and 2. therefore, not only plain radiograph and mri but also open or needle biopsy is indispensable to arrive at the correct diagnosis of eg. in spite of good prognosis of eg, various therapeutic approaches have been proposed. the effectiveness of curettage and bone grafting, local injection of corticosteroids, irradiation, and chemotherapy has been reported [1, 10–13]. because the incidence of eg is low (1% of total primary bone tumor) and the trend to spontaneous healing is high, it is very difficult to assess the true efficacy of these therapies. treatment of eg affecting the pelvis is usually accomplished by curettage of the affected site and bone grafting [4]. on the other hand, spontaneous healing of eg after open or needle biopsy has been reported by several authors [2, 4, 5]. case 1 and 2 showed a pain relief in a few weeks after biopsy and spontaneous healing in a few months. in this respect, a careful observation after open or needle biopsy is thought to be one of the treatment options for eg. some of eg may resolve spontaneously, and the other will persist or expand after biopsy [12]. case 3 showed an osteolysis with ill-defined margins and large soft tissue mass which extended into medial retroperitoneal space and the lateral gluteal muscles. this case did not resolve spontaneously. howard et al. reported a similar case of eg in the pelvis as the case 3 which showed prominent osteolysis with ill-defined margins and large soft tissue edema extending into both side of the bone on t2-weighted images. this case did not show any tendency to resolve after biopsy, and additional curettage was carried out [14]. eg with acute radiological features such as osteolysis with illdefined margins and large soft tissue edema on t2-weighted images may progress after open or needle biopsy. the chronic form of eg with well-defined margins and localized soft tissue mass on t2-weighted images may resolve spontaneously. we need further attention for eg especially with acute radiological features. eosinophilic granuloma of the pelvis 215 references plasschaert f, craig c, bell r, cole wg, wunder js, alman ba (2002) eosinophilic granuloma: 1. a different behavior in children than in adults. j bone joint surg br 84: 870–872. muscolo dl, slullitel g, ranalletta m, aponte-tinao la, ayerza ma (2003) spontaneous 2. remission of massive solitary eosinophilic granuloma of the femur. j pediatr orthop 23: 763– 765. stull ma, kransdorf mj, devaney ko (1992) langerhans cell histiocytosis of bone. 3. radiographics 12: 801–823. hoover kb, rosenthal di, mankin h (2007) langerhans cell histiocytosis. skeletal radiol 36: 4. 95–104. arouz em, saigal g, rodriguez mm, podda a (2005) langerhans’ cell histiocytosis: pathology, 5. imaging and treatment of skeletal involvement. pediatr radiol 35: 103–115. kilborn tn, teh j, goodman tr (2003) pediatric manifestations of langerhans cell histiocytosis: 6. a review of the clinical and radiological findings. clin radiol 58: 269–278. david r, oria ra, kumar r, singleton eb, lindell mm, shirkhoda a, mandewell je (1989) 7. radiologic features of eosinophilic granuloma of bone. am j roentgenol 153: 1021–1026. monroc m, pointe hd, haddad s, josset p, montagne jp (1994) soft tissue signal abnormality 8. associated with eosinophilic granuloma: correlation of mr imaging with pathologic findings. pediatr radiol 24: 328–332. schepper ama, ramon f, marck e (1993) mr imaging of eosinophilic granuloma: report of 11 9. cases. skeletal radiol 22: 163–166. yasko aw, fanning cv, ayala ag, carrasco ch, murray ja (1998) percutaneous techniques for 10. the diagnosis and treatment of localized langerhans-cell histiocytosis (eosinophilic granuloma of bone). j bone joint surg am 80: 219–228. camargo op, oliveira nrb, andrade js, filho rc, croci at, barros tep (1992) eosinophilic 11. granuloma of the ischium: long-term evaluation of a patient treated with steroids. j bone joint surg am 74: 445–447. greis pe, hankin fm (1990) eosinophilic granuloma: the management of solitary lesions of 12. bone. clin orthop 257: 204–211. sessa s, sommelet d, lascombes p, 13. prévot j (1994) treatment of langerhans cell histiocytosis in children. experience at the children’s hospital of nancy. j bone joint surg am 76: 1513– 1525. howard cb, nyska m, porat s, bessorai r, anir a, meller i (1996) solitary eosinophilic 14. granuloma of pelvis in children: a report of three cases. arch orthop trauma surg 115: 216– 218. corresponding author: masahito hatori e-mail: mhato@mail.tains.tohoku.ac.jp phone: +81-22-7177245 fax: +81-22-7177248 (6) upsala j med sci 111 (2): 215–226, 2006 a morphometric study on the endometrium of rat uterus in hypothyroid and thyroxine treated hypothyroid rats 1i.m.inuwa and 2m.a. williams 1dept of human and clinical anatomy. sultan qaboos university. oman 2dept of biomedical science, sheffield university, england abstract hypothyroidism increases the rate of pregnancy loss. other manifestations include menstrual disorder, and infertility. serum levels of gonadotropins are low in hypothyroid patients. though studies of uterine ultrastructure are well established as approaches to investigating the pathophysiology of infertility, they have scarcely been extended to the study of hypothyroid related infertility. the present study investigates the effect of hypothyroidism on the ultrastructure of uterine epithelium. three groups of wistar rats were studied. two groups were initially made hypothyroid using methimazole, and the third group was an untreated control. one hypothyroid group was given daily injections of thyroxine for six weeks. the uteri were removed in all three groups, and processed for transmission electron microscopy and morphometry. it was found that absolute epithelial cell volume was decreased in hypothyroidism. the volume of the nucleus had decreased though its relative volume in the cell had increased. the height of the luminal epithelium in hypothyroid rats also decreased by (33.8%) as compared with controls. basement membrane thickness was significantly increased in hypothyroidism. the changes were all substantially abrogated by the administration of thyroxine. this study suggests that thyroid hormones might be importantly concerned in the maintenance of the normal structure of uterine epithelial cells. introduction it is well known that human hypothyroidism is almost always associated with sub fertility (1). hypothyroid women are known to have difficulty in getting pregnant (2-3). furthermore, should a hypothyroid individual get pregnant, successful conclusion of pregnancy is difficult there being a high risk of both abortion, and still birth in such mothers (4-5). gonadotropin profiles in hypothyroid women have been extensively 215 received 2 december 2004 accepted 17 june 2005 investigated (6-7). most findings have demonstrated a decrease in the serum level of fsh and lh (8-9). based on this observation. it has generally been believed that the reason for infertility in hypothyroid women was anovulation, as there is poor ovarian stimulation by the pituitary gland (10). however, until recently the structure of the hypothyroid uterus had been little studied. the study of uterine epithelial morphology has importance since for implantation to occur, there has to be a process whereby the fertilised ovum and the surface epithelium interact. this necessitates the epithelium being adequately prepared for pregnancy. indeed, surface epithelial structure had been shown to determine the success of implantation (11). it has been reported previously that methimazole-induced hypothyroidism caused gross changes to the histology of the uterus (12). these alterations could functionally translate into an inability of the uterus to maintain pregnancy, and hence explain why abortion is common in hypothyroidism. the aims of this study were to establish via morphometric methods whether hypothyroidism had any effect on the ultrastructure of rat uterine luminal epithelium. material and methods animal model hypothyroid wistar rat was used as a model for the study. hypothyroidism was created by oral administration of 0.02% methimazole in the drinking water (12-13) two groups (a and b) were initially made hypothyroid, with one group, (b), receiving exogenous thyroxine intraperitoneally after hypothyroidism was established. a third control group. (c), of normal rats was maintained on tap water. daily vaginal smears were obtained from rats in all groups to determine the stage of the oestrus cycle. the animals were six weeks old at the start of treatment and were killed after a further six weeks. each rat was killed by an intraperitoneal overdose of anaesthetic (xylazine and ketamine hydrochloride in ratio of 1:2 v / v) before removing the uterine horns. tissue processing one horn from each rat was randomly selected and cut transversely into three equal portion. each portion was further chopped into pieces of about 1 mm3 and then fixed in 2.5% phosphate buffered glutaraldehyde (ph 7.24) for 1 8 hours at 4°c. the fixed pieces were briefly washed in phosphate buffer ( ph 7.2 ) dehydrated in ascending concentrations of ethanol up to and including dried absolute ethanol. the dehydrated tissues were then immersed in propylene oxide for 20 minutes with one change followed by infiltration in a mixture of araldite and propylene oxide (ratio 1:1) for 30 minutes. the infiltrated tissues were then dropped into pots containing fresh 100% araldite resin and left to polymerise at 60° for 48 hours (14). the tissues were embedded in this way so as to ensure randomness in orientation of the tissue when sections were obtained. this was achieved by letting the tissue sink to the bottom of the embedding moulds unaided. blocks were selected, and trimmed, and 216 semi-thin sections (t = 0.5�m) obtained on a reichert-jung ultramicrotome (jx 301). these were stained with toluidine blue and examined under a light microscope (leitz laborlux k). a ribbon of ultra-thin sections was then cut using a diamond knife (diatome mx 3776). the sections were expanded with chloroform vapour, and those producing silver-grey interference colour were picked up on 200�m mesh copper grids (gilder), stained with uranyl acetate, followed by lead citrate (15).electron micrographs were taken on cut film with a philips 301 transmission electron microscope operated at an accelerating voltage of 60 kv. micrographs used for morphometric estimates were taken at a nominal magnification of x1300. a micrograph of a cross-grating replica (2160 /mm) was included in each negative series as a magnification standard. sampling protocol a systematic random sampling procedure was carried out in order to obtain a representative sample of tissue from each group of animals (16). six animals were used in each group. in an earlier pilot experiment, using a two-way anova we had established that there was no significant difference in structure between the two horns of the same rat, and between the various regions of the same horn (12). one horn from each rat was randomly selected and cut transversely into three equal portion. each portion was further chopped into pieces of about 1 mm side and processed into araldite blocks for tem. from each animal, three blocks were selected by lottery (one block per portion). from each block a ribbon of silver sections was obtained and examined under a transmission electron microscope. a raster of systematic random fields of view was obtained on the microscope and six micrographs per block were taken. from each rat six micrographs were selected by lottery (two from each block), and measurements carried out. morphometry morphometric analysis was performed on electron micrograph negatives viewed under a projecting microscope at x13 magnification. an overlay screen bearing an array of points was randomly applied onto the projected image, and the number of points hitting various components of the cell were counted and used to estimate volume densities (vv). epithelial cell height was measured on profiles showing the whole epithelium from basement membrane to the luminal surface taking into consideration the micrograph magnification. the volume-weighted mean volume of epithelial cell nucleus was estimated b\ the point-sampled intercept method (17-18). a random array of test points was superimposed on the electron micrograph negatives. where a point hit a nuclear profile, a line which crossed the profile was drawn through the point at a randomly selected angle. this was repeated for all nuclear profiles hit by the test points. the part of the line which intercepted the nuclear membrane was measured (l).each intercept was then raised to the power of 3 (l3) and the mean (l3) was calculated. this mean nuclear intercept length was used to estimate the nuclear volume thus; 217 the major (a) and minor (b) axes of the epithelial nuclear profiles were also measured, and the mean diameter (d) obtained from the axial ratio of the nucleus was also calculated (19). absolute volume v, of the average epithelial cell was estimated using the volume density, vv, and volume weighted mean volume of the nucleus using the formula; harmonic and arithmetic mean thickness of epithelial basement membrane as measured using the orthogonal intercept method (20-21). micrograph negatives containing slices of the luminal epithelium were projected at a final magnification of x12744. this image was overlaid with random test lines that made chance intersections with the basement membrane surface. taking the plasmalemmal side of the basement membrane as the horizontal, a line was drawn at 90˚ (orthogonal) through the intersection point towards the outer edge of the basement membrane. the lengths of these orthogonal intercepts were measured using a logarithmic ruler. these, together with the number of observations, were used to estimate ‘harmonic mean’ thicknesses of the basement membrane, t h (20-21). all variances were computed and expressed on a ‘between animal’ basis. analysis of differences in absolute cell and organelle volumes, epithelial height, and basement membrane thickness between animal groups was performed using the non-parametric mann-whitney-u test. in each case, the null hypothesis was rejected if the probability of no difference was found to be less than 5% (i.e. p < 0.05). all statistical computations were made using the instat statistical package, version 1.15 (graphpad softwares, 1990) run on a personal computer. results ultrastructure of normal uterine horn luminal epithelium in the rat luminal epithelial cells in uterine horn of control rats were more or less uniform. they were columnar shape sitting on a thin basement membrane, and having clear cut cell borders as shown in figure 1a. the nucleus, placed one third of the way up the cell, was ovoid and mainly euchromatic with a prominent nucleolus. it was about one fourth 218 219 of the length of the cell (axial ratio 3.1). cytoplasm was rich in rough endoplasmic reticulum, with a few lipid droplets situated mainly in the infranuclear region. the luminal surface had numerous slender microvilli with a few electronlucent vesicles just beneath them. giant autolysosomes were present occasionally in the infranuclear regions of the cells. ultrastructure of hypothyroid rat uterine horn luminal epithelium as shown in figures 1b and 2b epithelia from uterine horn in hypothyroid rats were also columnar and lay on a thicker basement membrane than that below normal epithelia (fig 2a). nuclei were also ovoid, mainly euchromatic and with a prominent nucleolus. the nuclei were about one half of the cell height, and were situated in the bottom halves of the cells. lipid droplets were abundant in the cytoplasm and were situated both in the supranuclear and infranuclear regions. rough endoplasmic reticulum was scanty. microvilli appeared shorter than in euthyroid epithelia. all the epithelial structural changes observed were less pronounced in hypothyroid rats given thyroxine. figure 1. (a) uterine horn luminal epithelium in control rat. notice the giant autolysosomes (long arrow) typical of oestrus stage, and a thin basement membrane (arrow head). nucleus (n). (b) uterine horn luminal epithelium in hypothyroid rat. the cells were shorter, with a much thicker basement membrane (arrow head) as compared to euthyroid epithelia. autolysosomes are absent estimated parameters of normal rat uterine horn luminal epithelium the mean absolute volume of a luminal epithelial cell (cv % in parentheses) was 1235.50 (17.5) �m3 with a mean height of 35.14 �m (18.2). volume weighted mean volume of the nucleus was 222.39 (21.6) �m3 and the nuclear/cytoplasmic ratio was 220 variable control mmi mmi+t4 epithelial height ( m) 35.14* (18.2) 23.42 (12.4) 27.81* (18.5) nuclear/cytoplasmic ratio 0.22* (19.9) 0.35 (15.7) 0.26* (19.5) nuclear profile diameter ( m) 7.18 (16.7)* 6.27 (20.9) 6.47 (14.3) nuclear profile axial ratio 3.1 (14.8) 2.9 (27.9) 2.8 (18.5) ‘harmonic mean’ basement membrane thickness (nm) 143.86* (25.5) 252.93 (18.1) 184.52* (16.9) arithmetic mean basement membrane thickness (nm) 187.53* (3.9) 272.07 (9) 232.96* (8.6) *p<0.05 vs hypothyroid rats tabell 1. mean and coefficient of variation (cv%) of volumetric parameters of different compartments of the uterine horn luminal epithelium in euthyroid (control), hypothyroid (mmi) and thyroxine treated hypothyroid rats (mmi+t4). figure 2. (a) high power view of the basement membrane (bs) in euthyroid rat luminal epithelium. notice the lamina lucida (open arrow) and the reticular lamina (rt) lipid droplet (l). (b) high power view of the basement membrane (bs) in hypothyroid rat luminal epithelium. notice the increase in thickness. 0.22 (19.9). mean volume fraction of nucleus in the cell was 0.18 (21.6) whilst that of mitochondria in cytoplasm was 0.061 (19.5), and lipid droplets 0.03 (33.8). harmonic basement membrane thickness was 143.86 (25.5) nm. mean nuclear diameter was 7.18 (16.7) �m with an axial ratio of 3.1 (14.8) (see tables 1 and 2) changes in parameters of the luminal epithelium in hypothyroid rats changes in volumetric and other parameters in hypothyroid rat uterine horn luminal epithelia are illustrated in tables 1 and 2. the absolute volume of the average epithelium was reduced by 72.4% in hypothyroid (mmi) animals as compared to euthyroid (control) animals, whilst in hypothyroid rats given thyroxine the decrease was by 48%. nuclear volume decreased by 60.1% and 39.3% in hypothyroid, and hypothyroid rats given thyroxine respectively. however, the volume density of nucleus in the cell was increased significantly in hypothyroid rats as compared with euthyroid ones. total volume of lipid droplets, was increased by 4.5% in hypothyroid as compared with euthyroid rats. there was no increase in lipid volume in hypothyroid rats given thyroxine. mitochondrial volume density was unchanged in all three groups, though its absolute volume had decreased by 72.8%. in hypothyroid (mmi), and by 48% in hypothyroid rats given thyroxine. the difference in parameters between hypothyroid rats given thyroxine (mmi+t4) and those not given (mmi), was significant at p<0.05 (see table 1). nuclear-cytoplasmic ratio was significantly higher in hypothyroid rats as compared with euthyroid ones. basement membrane thickness was increased by 75.8% in hypothyroid, as compared with euthyroid rats. this increase however, was by 28.3% in hypothyroid rats given thyroxine when compared with euthyroid animals. height of luminal epithelium had decreased by 33.3% in hypothyroid (mm1) as compared with euthyroid (control) rats. in hypothyroid rats given thyroxine (mmi+t4) the decrease 221 compartment volume densities vv absolute volumes v ( m 3 ) control mmi mmi+t 4 control mm! mmi+t4 nucleus 0.18* (21.6) 0.26 (18) 0.21* (23) 222.39* (21.6) 88.55 (17.2) 134.90* (23.3) mitochondria 0.061 (19.5) 0.060 (15.1) 0.060 (12.5) 75.36* (19.5) 20.43 (15.1) 39.18* (12.5) lipid droplets 0.029* (33.8) 0.11 (13) 0.053* (18.6) 35.83* (33.8) 37.46 (13) 34.04* (18.6) whole cell 1235.50* (17.5) 340.57 (14.4) 642.38* (15.1) * = p<0.05 vs hypothyroid rats. tabell 2. mean and coefficient of variation (cv%) of morphometric parameters for uterine horn luminal epithelium in euthyroid (control) hypothyroid (mmi) and thyroxine treated hypothyroid rats (mmi+t4). was by 20.8%. the diameter of epithelial nuclei was greater in euthyroid than in hypothyroid rats (p<0.05) though the mean nuclear axial ratio was not significantly different in all three groups (see table 2). discussion this study has demonstrated the occurrence of significant structural change in the uterine horn luminal epithelium in hypothyroidism. such change was decreased by thyroxine administration. the improvement in epithelial morphometric parameters effected by exogenous thyroxine administration to hypothyroid rats in this study. suggests that in healthy animals the presence of the hormone helps maintain normal epithelial structure. this correlates with the clinical experience which demonstrated that thyroxine administration helped correct menstrual irregularities in hypothyroid women (22-23) similarly, in pregnant hypothyroid women thyroxine administration improves the outcome. the decrease in cell size, nuclear volume. and mitochondrial volume in hypothyroid animals could be thus a direct effect of thyroxine deprivation, particularly since thyroid hormone is believed to exert its influence on cells by facilitating the transcription of dna and hence new protein synthesis. indeed, the rat uterus has been shown by evans et al. (25) and mukku et al. (26) to contain thyroid hormone receptors suggesting that it is a specific site for thyroid hormone action. absence of the hormone could thus slow down the epithelial metabolic processes resulting in a decrease in cell and nuclear volumes as well as a decrease in mitochondrial volume. the fact that this study demonstrates in hypothyroid uterine epithelia a decrease in nuclear size apparently without alteration of shape further suggests that there may be a decrease in nucleic acid transcription in the nuclei of these cells. an increase in nuclear-cytoplasmic ratio coupled with a decrease in total cell volume in hypothyroid animals suggests that the cytoplasmic volume was far more reduced than was the nuclear volume. as the basement membrane is composed of collagen (including type iv), and a number of glycosaminoglycans, an increase in its thickness suggests that hypothyroidism influences the turnover of these components. harvey et al. (27) have shown that hypothyroidism is associated with a significant decreased collagen breakdown. others (28-30) have reported increased deposition of collagen type iv in tissues from hypothyroid individuals. the apparent increase in collagen and glvcosaminoglycan deposition in hypothyroidism is thus probably not due to an increase in synthesis, but to a decreased breakdown of these substances (31). since the thickness of basement membrane is known to be inversely proportional to the rate of diffusion of substances across that membrane (32), the increase would be expected to impair diffusion to the basal aspect of the cells. this would be of particular relevance with respect to large molecular weight substances such as hormones, enzymes etc. impaired diffusion of such control substances is likely to result in altered metabolism and probably impaired morphology, which might explain the generalised decrease in cell size observed 222 here. similarly, an increase in basement membrane thickness might well adversely affect specific epithelial function as in the case of medical conditions such as glomerulonephritis. in fact, there are also reports in the literature of hypothyroidism being associated with basement membrane damaging glomerulonephritis (33-34). hypothyroidism is known to increase the serum levels of lipids and cholesterol in humans. it also causes excessive deposition of fat under the skin (35). the present study shows that extra lipid accumulation occurs within the uterine epithelium suggesting that hypothyroidism may influence generally cellular lipid metabolism. the fact that the observed changes in epithelial structure in this study were similar to those found during dioestrus stage of the oestrus cycle in rats by spornitz et al. (36) further suggest that hypothyroidism affects ovarian steroid hormone metabolism. therefore, alteration in steroid hormone levels during hypothyroidism is especially likely since the dioestrus stage in rats is characterised by low steroid hormone levels, and the hypothyroid rats in this study had a preponderance of dioestrus vaginal smears (12). however, it could also be claimed that the changes we have seen might have been due to another indirect effect of hypothyroidism on gonadotropins, since it has been shown that hypothyroidism leads to a decrease in the serum levels of the pituitary gonadotropins fsh and lh (8, 37-38). this could in turn lead to a decrease in ovarian stimulation and hence a decrease in ovarian steroid hormone levels, causing a decrease in uterine epithelial stimulation resulting in the structural changes observed in this study. in conclusion, it seems reasonable to suggest that though the rarity of pregnancy in hypothyroid women has generally been passed off as due to a high incidence of anovulation (39), the observations made here suggest, that in addition epithelial structural change might be present contributing an adverse affect by making interaction with fertilised ovum less successful. similarly, altered steroid hormone metabolism in hypothyroidism could disturb an existing pregnancy. study of ovarian histochemistry in hypothyroidism, particularly investigation of the activity of steroid synthesising enzymes are indicated and these may prove important since progesterone deficiency is well known to result in infertility, or during pregnancy to abortion. acknowledgements this work was carried out with sponsorship from the university of sheffield. we wish to thank messrs john proctor and mick turton for the micrography work. references 1. hemady zs, siler-khodr m, najjar s (1978) precocious puberty in juvenile hypothyroidism. j paed: 92: 55-59. 2. boyland p, drury mi. 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(1989) in principles and techniques of electronmicroscopy (biological applications). 3rd ed. london: macmillan press. 15. reynolds es. (1963). the use of lead citrate at high ph as an electron-opaque stain in electronmicroscopv. j cell biol: 17: 208 16. williams ma. (1977) quantitative methods in biology in practical methods in electron microscopy. volume 6 (ed. glauert am) amsterdam: north holland publishing company. 17. gundersen hjg, jensen eb. (1985) stereological estimation of the volume-weighted mean volume of arbitrary particles observed on random sections. j microsc: 138: 127-142 18. mayhew tm. (1989) stereological studies on rat spinal neurones during postnatal development: estimates of mean perikaryal and nuclear volumes free from assumptions about shape. j anat: 162: 97-107 19. warren ma, bedi ks. 1982) synapse-to-neuron ratios in the visual cortex of adult rats undernourished from about birth until 100 days of age. j comp neurol: 210: 59-64 20. jensen eb, gundersen hjg, and osterby r. (1979) determination of membrane thickness distribution from orthogonal intercepts. j. microsc. 115:1933 21. hirose k, osterby r, nozawa m, gundersen hjg. (1982) development of glomerular lesions in experimental long-term diabetes in the rat. kidney int: 2 1: 689-695 22. hembree wl, vandewiele rl, (1 978) in the thyroids (ed. werner, s c lngbar. s h) 4th ed. hargerstown, maryland: harper and rowe; 915 23. poretsky l, garber j, kleefield j. (1986) primary amenorhhoea and pseudo-prolactinoma in a patient with primary hypothyroidism. am j med: 181:,180-182 24. decherney a, polan ml, (1984) evaluation and management of habitual abortion. brit. j hosp med. 3: 26 1-262. 25. evans rw, farwell ap, braverjvian le. (1983) nuclear thyroid hormone receptors in the rat uterus. endocrinol: 113: 1459-1463. 26. mukku vr, kirkland jl, hardy m, stancel gm. (1983) evidence for thyroid hormone receptors in uterine nuclei. metab: 32: 142-145. 27. harvey rd, mchardy kc, reid 1w, paterson f, bewsher pd, duncan a, robins sp (1991) measurement of bone collagen degradation in hyperthyroidism and during thyroxine replacement therapy using pyridinium cross-links as specific urinary markers. j clin endocrinol and metab.; 72: 1189-1194 28. abou-rabia n and kendall md. (1994) involution of the rat thymus in experimentally induced 224 hypothyroidism. cell and tis res.: 277: 447-455 29. lopes ac, furlanetto r, sasso ws, didio lja. ( 1993) subcellular alterations of cardiac fibers in rats subjected to hypothyroidism. j submicroscopic cytol and pathol. 25: 263 -266 30. kaya b, kaya h, rehman bu, arslan a, rehman mu (1993) the histopathological evaluation of the effects of thyroid dysfunctions on gingival tissues. cerrahpasa tip fakultesi dergisi: 24: 121-131 31. schiller s, slover ga, dorfman a. (1962). effect of thyroid gland on metabolism of acid mucopolysaccharide in skin biochimie et biophysica acta; 58: 27 32. ganong wf. (1989) in review of medical physiology (ed. wf ganong) 4th ed. london: prentice-hall international.: 4-5 33. madden bp, walker f, gaffney e, and keogh ja. (1989) a case of iga nephropathy associated with vitiligo, primary hypothyroidism and primary adrenocortical insufficiency. irish j med sci. 158: 153-154 34. mattoo tk and akhtar m (1990) familial glomerulopathy with proximal tubular dysfunction: a new syndrome? paed. nephrol: 4: 223-7 35. degroot u, stanbury jb, (1975) in the thyroid and its diseases 4th ed. london: john wiley & sons.: 4 19-425 36. spornitz um, rnderjknecht bp, edelmanl a, scheidegger b, cairoli f. (1994) ultrastructure as a basis for dating of rat endometrium. anat. rec.: 238: 163-176 37. kirby jd, jetton ae, cooke ps, hess ra, bunick d, ackland jf, turek fw, schwartz nb. (l992) developmental hormonal profiles accompanying the neonatal hypothyroidism induced increase in adult testicular size and sperm production in the rat. endocrinol; 131: 559-565. 38. valle lb, oliveira-filho rm. romaldini jh. lara pf.(1985) pituitary-testicular axis abnormalities in immature male hypothyroid rats. j steroid biochem: 23: 253-257. 39. goldsmith re, sturgis sh. lerman j, (1952) the menstrual pattern in thyroid disease. j clin endocrinol and metab. 12: 846. corresponding author: dr im inuwa department of human and clinical anatomy. sultan qaboos university muscat. oman tel: (00968) 24141176 fax:(00968) 241431175 email: ibrahim1@squ.edu.om 225 226 sups_a_528465 26..33 upsala journal of medical sciences. 2011; 116: 26–33 original article two conformational forms of target-bound ic3b that distinctively bind complement receptors 1 and 2 and two specific monoclonal antibodies ulf r. nilsson1, lillemor funke1, bo nilsson1 & kristina n. ekdahl1,2 1division of clinical immunology, rudbeck laboratory c5, uppsala university, sweden, and 2department of natural sciences, linneaus university, kalmar, sweden abstract introduction. the complement system is an essential part of the immune system of vertebrates. the central event of the complement activation cascade is the sequential proteolytic activation of c3, which is associated with profound alterations in the molecule’s structure and conformation and is responsible for triggering most of the biological effects of complement. material and methods. here, we have studied the conformation of c3 fragments deposited onto an igg-coated surface from human serum during complement activation, using a set of unique monoclonal antibodies (mabs) that are all specific for the c3dg portion of bound ic3b. results. we were able to identify two conformational forms of target-bound ic3b: the first recognized by mab 7d18.1, and the second by mab 7d323.1. the first species of ic3b bound recombinant complement receptor 1 (cr1), while the second bound cr2. since cr1 and cr2 are expressed by different subsets of leukocytes, this difference in receptor-binding capacity implies that there is a biological difference between the two forms of surface-bound ic3b. conclusion. we propose that mabs 7d18.1 and 7d323.1 can act as surrogate markers for cr1 and cr2, respectively, and that they may be useful tools for studying the immune complexes that are generated in various autoimmune diseases. key words: complement c3, complement receptors, immunoglobulin g (igg), monoclonal antibodies, neo-epitopes introduction the complement system is an essential part of the innate immune system with the capacity to discriminate between self and non-self. it is also an important player in pathological processes, since it also recognizes altered self in clinical settings such as reperfusion injury and transplantation. the complement system is a removal system, in that it eliminates micro-organisms (bacteria, viruses, parasites), immune complexes, and apoptotic cells from the body. complement factor c3 is the key component in this system, and its cleavage to c3b by labile composite enzymes (convertases) elicits inflammation and cell lysis via target-bound c3 fragments (the anaphylatoxins c3a and c5a) and the generation of the membrane attack complex, c5b-9. during its cleavage to c3b and ic3b, the c3 molecule undergoes several profound structural and conformational changes. these alterations in structure and conformation have recently been elucidated in a series of reports on the 3-d crystal structure of the molecule (1–5). after cleavage of c3 by the convertases, the so-called thiol ester domain (ted) is totally dislocated and moved into a position that allows covalent binding of the molecule to a target surface. digestion of c3b to ic3b by factor i affects the molecule further by releasing the c3f fragment and displacing the c3c domain from the ted. finally, factor i further cleaves ic3b, generating the fragments c3dg (ted) and fluid-phase c3c (figure 1). the conformational changes that occur during the activation of c3 are reflected in alterations in the correspondence: kristina n. ekdahl, division of clinical immunology, rudbeck laboratory c5, uppsala university, sweden. e-mail: kristina.nilsson_ekdahl@klinimm.uu.se (received 20 september 2010; accepted 28 september 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.528465 exposure of neo-epitopes on the surface of the c3 molecule. immunization of rabbits and mice with the native molecule as antigen tends to produce antibodies directed toward both the fluid-phase and the target-bound molecules. in an attempt to generate monoclonal antibodies (mabs) that specifically recognize the bound conformations of c3 fragments, we have produced mabs against reduced polypeptide chains of c3. a number of mabs directed against surface-bound c3 fragments (c3b, ic3b, c3dg) have been generated (6), some of whose epitopes in the c3 molecule have been mapped using cdnaexpressed and synthesized peptides (7,8). several of these antibodies were directed against the n terminus of the c3dg fragment and recognized the ic3b and c3dg fragments (9). all of these mabs, bound to epitopes that are not totally exposed in the 3-d crystal structure, indicate that there are still unidentified conformational states that are associated with binding of the molecule to target surfaces (1). binding of c3b to a target surface is associated with exposure of epitopes in c3 that act as ligands for complement receptors 1–4 (cr1–4). two of these receptors, cr1 (cd35) and cr2 (cd21), are short consensus repeats (scr) consisting of 30 (10,11) and 15 or 16 (due to alternative splicing) (12,13) residues, respectively. cr1 is expressed on erythrocytes and various phagocytes, whereas cr2 is specific for lymphocytes and follicular dendritic cells. in particular, b-cells express a large number of cr2 molecules. in the present paper, we have further characterized the reactivity of four c3dg mabs, 7d18.1, 7d84.1, 7d264.6, and 7d323.1, with regard to their binding to different conformational states of surface-bound c3b/ic3b that are associated with the binding of complement receptors cr1 and cr2. a distinctive association was found between the binding of the mabs and the receptors. material and methods serum preparations fresh, normal human blood was obtained from the blood center of the university hospital, uppsala. normal human serum (nhs) was separated from blood by centrifugation at 4000 g for 20 min at +4�c after clotting for 2–3 h at room temperature. all analyses of complement in serum were performed on stored, frozen material after rapid thawing at 37�c. freezing/refreezing and storage of material were done at �70�c. for some experiments, aliquots of serum were heat-inactivated by incubation for 30 min at 56�c. sources and preparation of purified proteins human igg (gamma globulin) was obtained from pharmacia-upjohn ab (uppsala, sweden), and bovine serum albumin (bsa) (fraction v, ria grade) was purchased from the united states biochemical corporation (cleveland, oh, usa). soluble complement receptor 1 (scr1) brl 55730 was a kind gift of dr henry marsh (smithkline beecham pharmaceuticals, king of prussia, pa, usa). culture supernatants from raji cells (burkitt’s lymphoma), kindly provided by dr sara mangsbo, rudbeck laboratory, were used as a source for cr2. c3 and factor i were prepared from plasma according to hammer et al. (14) and fearon (15), respectively. factor h was prepared from human serum essentially according to hammer et al., except that the first step consisted of a euglobulin precipitation (16). c3b was produced by incubating c3 with trypsin, followed by gel filtration to remove c3a, and ic3b was prepared by incubating c3b with factor i, using factor h as co-factor. antibody preparations polyclonal (pab) horseradish peroxidase-(hrp)conjugated anti-human c3c and c3d antibodies, hrp-conjugated anti-mouse-ig and non-conjugated anti-bsa pab, and mouse monoclonal anti-human cr1 and anti-human cr2 antibodies were purchased from dako (glostrup, denmark). figure 1. sequential digestion of c3, generating c3b, ic3b, c3c, c3dg, and c3d. activation of c3 leads to disruption of the thiol ester (te circular), which then establishes covalent bonds (te linear). the interchain disulfide bonds are indicated (s s). neo-epitopes in surface-bound human ic3b 27 mouse anti-human c3 monoclonal antibodies (mabs) 7d18.1, 7d84.1, 7d264.6, and 7d323.1, specific for epitopes in c3dg, were produced and characterized as described previously (6,8). complement activation studies microtiter plates. complement was activated in the wells of 96-well polystyrene microtiter plates (maxisorp) (nunc, roskilde, denmark) that had been coated with igg as described below. the conformation of the deposited c3 fragments was visualized by elisa using polyclonal (pab) and monoclonal (mab) anti-c3 antibodies. each experiment was repeated five to ten times with similar results. furthermore, analysis of the deposited c3 fragments was performed using western blot analysis. diluents for functional studies and elisas. reagents that were tested functionally were diluted in veronalbuffered saline (vbs) consisting of 5 mm veronal, ph 7.5, with nacl (145 mm), ca2+ (0.15 mm), and mg2+ (0.5 mm). some experiments were performed in vbs supplemented with 0.1% (w/v) gelatin (gvb). phosphate-buffered saline (pbs) containing 0.05% (v/v) tween 20 and 0.02% (v/v) antifoam (dispensor-aspirator, pharmacia-upjohn, uppsala, sweden) was used as the washing fluid. antibody dilutions were carried out in washing fluid containing 0.1% (w/v) gelatin. undesired protein adsorption to polystyrene plates was prevented by incubating the microtiter wells with 1% (w/v) gelatin in pbs at room temperature for 30 min. complement-activating target surfaces. plates were incubated for 1 h at 37�c with 0.2 ml pbs/well of monomeric human igg at 80 mg/ml, washed 3� with pbs, blocked 30 min at room temperature with 0.3 ml 1% gelatin, and rinsed with vbs. the igg-coated plates were used immediately or after storage at �70�c, with 0.3 ml vbs being added per well. complement activation. human serum was diluted in vbs from 100% in 3-fold steps and incubated in prewarmed igg-coated microtiter wells for 2.5 to 120 min, at 37�c as described elsewhere (17). the reaction was stopped by washing with washing fluid containing 10 mm edta. the bound c3 fragments were detected using rabbit pabs antic3c and anti-c3d, as well as a panel of four mabs against epitopes in the c3dg region of c3. the bound primary antibodies were detected using hrp-conjugated anti-rabbit immunoglobulins (pabs) or hrp-conjugated anti-mouse immunoglobulins (mabs). binding of complement receptors (cr) 1 and 2 to deposited c3 fragments. the ability of the deposited c3 fragments to act as ligands for cr1 and cr2 was investigated by adding recombinant scr1 or raji supernatants containing soluble cr2 into microtiter wells in which complement activation had been achieved as described above. after incubation at 37�c for 30 min, mabs recognizing cr1 or cr2 were used for detection, in conjunction with hrpconjugated anti-mouse immunoglobulins. factor i-mediated cleavage of bound c3 fragments with cr1 as co-factor. in order to promote the digestion to ic3b or c3dg of the c3 fragments deposited by complement activation in the igg-coated microtiter wells, separate experiments were performed in which complement activation was performed as described above. thereafter, scr1 and heat-inactivated human serum (or buffer as negative control) were added and incubated at 37�c for 30 min to enable the factor i in serum to digest the deposited c3b, with scr1 as co-factor. subsequently, raji supernatants containing soluble cr2 were added to some of the microtiter wells. finally, bound c3 fragments and cr1 and cr2 were detected as described above. western blot analysis of bound c3 fragments human serum (undiluted) was incubated in iggcoated microtiter wells as described above for up to 60 min. after washing, 25 ml 0.1 m methylamine in 0.1 m glycine-naoh buffer, ph 8, was added to each well and incubated at room temperature with agitation for 10 min. thereafter, 25 ml sdselectrophoresis buffer (12 mm tris, 0.4% sds, ph 6.8)wasaddedtoeachwellofthemicrotiterplate,which was then heated to 85�c for 5 min.after the incubation, the supernatants were loaded onto 10% sds polyacryle amidegelelectrophoresis(sds-page)gels.afterelectrophoretic separation, the proteins were transferred to a pvdf immun-blot membrane (biorad, hercules, ca, usa), and the membrane was blocked in 1% bovine serum albumin diluted in pbs-tween. proteins were detected with biotinylated pab anti-c3d and anti-c3c, or mab 7d264.6, followed by streptavidinhrp and staining with 3,3¢-diaminobenzidine tetrahydrochloride (sigma, steinheim, germany) dissolved in pbs (1 mg/ml plus 0.5 ml h2o2/ml). purified c3, c3b, and ic3b were used as controls. 28 u. r. nilsson et al. results epitope expression of human c3 bound to adsorbed human igg incubation of serum in wells coated with adsorbed igg for 60 min at 37�c induced complement activation, leading to the deposition of c3 fragments on the microtiter plate surface; the highest level of binding was seen with undiluted serum, as detected by pabs anti-c3c and anti-c3d and mab 7d323.1 (figure 2a). in contrast, mab 7d18.1 showed the lowest level of binding at the highest serum concentration. the binding profiles of these two mabs, together with those of mabs 7d84.1 and 7d264.6, were further examined after 120 min of complement activation. under these conditions, binding of mab 7d323.1 was further increased, again with the highest level achieved with undiluted serum; the other mabs showed similar binding profiles, peaking at a serum concentration of ~3% (figure 2b). data presented in each panel in figures 2–4 are from one representative experiment out of five to ten experiments which were performed with similar results. receptor ligand function of human c3 bound to adsorbed human igg the c3 fragments deposited on adsorbed igg as a result of complement activation of serially diluted serum were able to act as a ligand for exogenously added cr1 (figure 3a). the cr1-binding epitopes were already exposed on c3 after 2.5 min of incubation, with maximal expression at high serum concentration (30%). after prolonged incubation (60 min), maximal binding of cr1 was seen at a lower serum concentration (3%). the binding profile of cr1 closely resembled that of mab 7d18.1 (figure 3b). surface-bound c3 fragments also bound cr2, but this binding was seen only on surfaces that had been in contact with high concentrations of serum, at both 2.5 and 60 min (figure 3c). the binding of mab 323.1 mirrored that of cr2 (figure 3d). ligand binding of factor i-digested surface-bound c3 fragments c3 fragments were deposited on surface-bound igg from serially diluted serum. after removal of the serum, cr1 was added together with heatinactivated human serum as a source of factor i to enable the digestion of the deposited c3 fragments to yield ic3b and c3dg. cr1 did not bind to the surface-bound digested c3 fragments, but binding was seen in the wells that had been incubated with cr1 and the buffer control (figure 4a). since there was a surplus of cr1 after addition, it was also detected at high serum concentration. after digestion, the binding profile of mab 7d18.1 shifted from its previous maximum at 3% serum to a peak at 30% serum, suggesting a lower density of binding epitopes (figure 4b). in contrast, factor i-mediated cleavage of the deposited c3 fragments did not affect the binding of cr2 (figure 4c) or mab 7d323.1 (figure 4d). 3.00 a 4 9 2 a b 2.50 2.00 1.50 1.00 0.50 0.00 0.01 0.1 1 10 100 serum concentration (%) 0.01 0.1 1 10 100 figure 2. epitope expression of human c3 bound to adsorbed human igg. human serum was added in 3-fold serial dilution from 100% and incubated at 37�c for 60 min (panel a) or 120 min (panel b). bound c3 was detected using pabs anti-c3d (filled triangles) and anti-c3c (open triangles), and mabs 7d323.1 (squares), 7d18.1 (circles), 7d264.6 (open diamonds), and 7d84.1 (filled diamonds). neo-epitopes in surface-bound human ic3b 29 western blot analysis of bound c3 fragments the composition of the deposited c3 fragments was further investigated by western blot analysis. undiluted serum was incubated on igg-coated elisa plates for up to 60 min. after washing, the bound proteins were eluted with methylamine, and the eluted c3 fragments were visualized by western blot analysis using pabs against c3d (molecular weight » 35 kda) and mab 7d264.6 against an epitope in c3dg, both of which also detected the corresponding epitopes in the 110-kda a-chain of c3 and the 100-kda a¢-chain of c3b; we also used pabs against c3c, which detected the 75-kda b-chain and the 45-kda polypeptide chain of c3c, including the corresponding epitopes in the 110-kda a-chain of c3, the 100-kda a¢-chain of c3b, and the 67-kda fragment of ic3b (figure 5). the staining of the anti-c3d pabs in the controls followed the expected pattern. in the eluted samples, we saw weak staining of a band of 67 kda (i.e. the 67-kda band of ic3b), but no band of 40 kda (i.e. free c3dg). however, the most abundant staining with anti-c3d pabs was of high molecular weight material (>150 kda) in the eluates, suggesting that most of the c3 fragments with exposed epitopes recognized by the anti-c3d pabs were covalently bound to other proteins, producing higher molecular weight eluates. the pattern did not alter during incubation for up to 60 min (figure 6a). staining with mab 7d264.6 revealed the presence of the 67-kda fragment of ic3b at all time points, with no further digestion to c3dg over time (figure 6b). there was no indication of heterogeneity in the size of this fragment. staining with pab anti-c3c confirmed the presence of ic3b by detecting the 75-kda b-chain at all time points, as well as the 45-kda polypeptide chain present in c3c and ic3b (figure 6c). discussion in the present work, we have demonstrated a strict relationship between the ic3b binding patterns of 7d18.1 and 7d323.1 and complement receptors 2.50 a c d b 2.00 1.50 1.00 0.50 0.00 2.50a 4 9 2 2.00 1.50 1.00 0.50 0.00 0.01 0.1 1 10 100 serum concentration (%) 0.01 0.1 1 10 100 figure 3. binding of cr1 (panel a), anti-c3 mab 7d18.1 (panel b), cr2 (panel c), and anti-c3 mab 7d323.1 (panel d) to human c3 bound to adsorbed human igg. human serum (in 3-fold serial dilution from 100%) was added and incubated at 37�c for 2.5 min (dashed lines) or 60 min (solid lines). 30 u. r. nilsson et al. a c d b 2.00a 4 9 2 1.50 1.00 0.50 0.00 2.00 1.50 1.00 0.50 0.00 0.01 0.1 1 10 100 serum concentration (%) 0.01 0.1 1 10 100 figure 4. binding of cr1 (panel a) and anti-c3 mab 7d18.1 (panel b) and of cr2 (panel c) and anti-c3 mab 7d323.1 (panel d) to human c3 fragments bound to adsorbed human igg. the coated surfaces were incubated first with human serum in 3-fold serial dilution from 100% at 37�c for 60 min. after washing, heat-inactivated human serum was then added, together with buffer (solid lines) or soluble cr1 to enable digestion of deposited ic3b to c3dg (dashed lines). α−chain mw mw kda 250 150 100 75 50 37 c3 c3b c3c c3dg c3dic3b α′−chain 67 kda 45 kda 45 kda 27 kda c3dg c3d β−chainβ−chain β−chain β−chain figure 5. electrophoretic pattern of polypeptide chains from the various c3 fragments. the molecular weights (mw) of the standards are indicated. neo-epitopes in surface-bound human ic3b 31 cr1 and cr2, respectively. the two antibodies were able to distinguish between two forms of ic3b, which are produced not by structural differences but apparently as a result of conformational changes in the molecule, indicating that the difference in binding of the receptors is related to a conformational change. monoclonal antibodies 7d18.1, 7d84.1, 7d264.6, and 7d323.1 all bound preferentially to bound c3 fragments, but the binding of 7d323.1 was less strictly limited to bound ic3b, since it also recognized the molecule in the fluid phase. the epitopes of all four antibodies are found close to each other in the n terminus of c3dg and have been pinned down to single amino acid residues (8,9,18). all four mabs bind to c3 (929–932), but 7d264.6 also recognizes c3 (929–936), and 7d323.1 binds to rabbit c3, which has a slightly different sequence in this region: hu929r versus ra929n and (18) hu932r versus ra932q, respectively (19,20). thus, the location of the epitopes differs by only a few amino acid residues. all the anti-c3dg mabs were tested for binding to c3 fragments that had been generated and bound to igg adsorbed in microtiter wells after incubation with nhs for 60 and 120 min, and their binding was compared with that of pabs anti-c3c and anti-c3d, which acted as internal standards for the binding of antibodies to the bound c3 fragments. these studies showed that 7d323.1 bound to c3 fragments at both time points and at all concentrations of nhs, while 7d18.1 and the other two mabs bound less intensely at the higher concentrations of nhs. when the binding of mabs was compared with that of fluid-phase recombinant cr1 (cd35) and cr2 (cd21), a close relationship was seen between the binding of the receptors and the mabs. cr1 bound to a maximum level at a fairly low concentration of nhs, and its level of binding decreased at higher concentrations. this binding pattern was similar to that of 7d18.1, 7d84.1, and 7d264.6, as exemplified by the binding of 7d18.1. on the other hand, the binding of cr2 increased with increasing concentration of nhs. this reactivity was mirrored by the binding of 7d323.1. there are two possible explanations for this pattern of reactivity: there may be structural differences between the two forms of ic3b, or there may be conformational changes that are associated with the two different forms of ic3b. in the transformation of c3b to ic3b, factor i cleaves the a-chain of c3b at two sites, finally releasing the c3f fragment. theoretically, these cleavages can occur in a stepwise manner to generate two forms of ic3b, which could therefore differ in their reactivity with the receptors and mabs. in order to rule out this possibility, we analyzed the c3 fragments by western blotting, which revealed that the bound fragments were in the ic3b form and there were no differences associated with the various concentrations that were used, or with the length of the incubation with the nhs. no differences were found in the 45-kda polypeptide of c3c or the c3dg fragment, which are the polypeptides which would be affected if a stepwise generation of ic3b were to occur. a further indication that the surfaces were coated with ic3b was the fact that when the bound ic3b was cleaved to generate c3dg as a result of incubation of the microtiter plates with c3 c3b ic3b 0 2 5 5 10 10 20 20 60 60 mw kda c3 c3b ic3b 0 2 5 5 10 10 20 20 60 60 mw kda c3 c3b ic3b 0 2 5 5 10 10 20 20 60 60 mw kda 250 150 100 75 50 37 250 150 100 75 50 37 250 150 100 75 50 37 a b c figure 6. western blot analysis of c3 fragments eluted from an igg-coated surface exposed to undiluted human serum for 0, 5, 10, 20, or 60 min, detected using: pab anti-c3d (panel a), mab antic3 7d264.6 (panel b), and pab anti-c3c (panel c). reference preparations of c3, c3b, and ic3b were also analyzed, and the molecular weights (mw) of the standards are indicated. 32 u. r. nilsson et al. heat-inactivated nhs and soluble cr1, the binding of cr1 to the surface was lost. this was a finding that is in agreement with the loss of affinity of the receptor for c3 fragments in the case of c3dg. the reactivity with cr2 remained, as did the binding of the mabs, since their epitopes are present in the n-terminal portion of the c3dg molecule. taken together, these results suggest that the differences in binding of the mabs and complement receptors reflect conformational changes in the molecule, and not structural differences. in summary, this study has identified two forms of ic3b that have distinctive binding reactivities with the complement receptors cr1 and cr2. these forms of ic3b were specifically detected by the mabs 7d18.1 and 7d323.1, respectively. the reactivity of these mabs is potentially of great interest, since the binding of ic3b to cr1 and cr2 generates totally different biological responses in the two cases. the mabs can therefore be used as reagents to identify the different conformational and functional forms of ic3b bound to various immune complexes (21). since immune complexes are found in many autoimmune diseases, such as systemic lupus erythematosus (sle) and rheumatoid arthritis, assays employing these antibodies would add a new dimension to the analysis of immune complexes in patients with these types of disease. acknowledgements we are most grateful to mrs margita nilsson and mrs kerstin sandholm for their skillful technical assistance and to dr deborah mcclellan for editorial assistance. this work was supported by grants from the swedish research council (vr) 2009-4675, 2009-4462, and grant # eb003968 from the national institutes of health (usa) and from faculty grants from the linneaus university in sweden. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and 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this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. high risk of cardiovascular side effects after treatment of hodgkin’s lymphoma – is there a need for intervention in long-term survivors? anne anderssona, gunilla enbladb, martin erlansona, ann-sofie johanssona, daniel molinb, björn tavelina, ulf näslundc and beatrice melina adepartment of radiation sciences, oncology, umeå university, umeå, sweden; bdepartment of immunology, genetics and pathology, section experimental and clinical oncology, uppsala university, uppsala, sweden; cdepartment of public health and clinical medicine, umeå university, umeå, sweden abstract background: hodgkin lymphoma (hl) patients have a good prognosis after adequate treatment. previous treatment with mantle field irradiation has been accompanied by an increased long-term risk of cardiovascular disease (cvd). this study identified co-morbidity factors for the development of cardiovascular side effects and initiated an intervention study aimed to decrease morbidity and mortality of cvd in hl survivors. design: hodgkin lymphoma patients aged ≤45 years diagnosed between 1965 and 1995 were invited to participate. in total, 453 patients completed a questionnaire that addressed co-morbidity factors and clinical symptoms. of these, 319 accepted to participate in a structured clinical visit. the statistical analyses compared individuals with cvd with those with no cvd. results: cardiovascular disease was reported by 27.9%. radiotherapy (odds ratio [or]: 3.27), hypertension and hypercholesterolemia were shown to be independent risk factors for the development of cvd. the or for cvd and valve disease in patients who received radiotherapy towards mediastinum was 4.48 and 6.07, respectively. at clinical visits, 42% of the patients were referred for further investigation and 24% of these had a cardiac ultrasound performed due to previously unknown heart murmurs. conclusion: radiotherapy towards mediastinum was an independent risk factor for cvd as well as hypercholesterolemia and hypertension. a reasonable approach as intervention for this cohort of patients is regular monitoring of hypertension and hypercholesterolemia and referral to adequate investigation when cardiac symptoms appear. broad knowledge about the side effects from radiotherapy in the medical community and well-structured information regarding late side effects to the patients are all reasonable approaches as late effects can occur even 40 years after cancer treatment. article history received 26 november 2020 revised 02 february 2021 accepted 14 february 2021 published 15 march 2021 keywords hodgkin lymphoma; survivorship; cardiovascular side effects; intervention introduction the prognosis for hodgkin lymphoma (hl) patients is good, with >85% 5-year overall survival as the result of treatment with different modalities, usually chemotherapy and/or radiotherapy, irrespective of gender and stage (1). until the end of the 1980s, extended field radiotherapy with mantle field, including the upper chest, was commonly used in the treatment of limitedstage hl with prescribed total doses ranging between 38 and 44 gray (gy). chemotherapy was added in more advanced stages (2). the long-term survivors of hodgkin lymphoma have a risk for long-term side effects such as second malignancies (sm) and cardiovascular disease (cvd), which have been studied in several retrospective studies (3–12). ten years after treatment, there is an increasing risk in hl survivors for primarily sm and cvd (13). therefore, for some stages of the disease, radiation doses, field  sizes and chemotherapy before radiotherapy have been reduced without affecting the cure rate (14). with an increasing population of cancer survivors, the morbidity and mortality among these individuals have become more obvious (15). early identification of side effects from previous treatments is needed to reduce the morbidity and mortality in hl long-term survivors. guidelines for prospective surveillance of this patient group suggest baseline visits and individualized follow-up depending on treatment, but patients as well as physicians seem to lack an awareness of these guidelines (12,16,17). the swedish hodgkin intervention and prevention study (ship) recruited patients from three health care regions in sweden. these patients were diagnosed with hl at the age of 45 years or younger between 1965 and 1995. this study presents the state of health and identifies the development of cvd in long-term survivors of hl with the intention to prevent morbidity and mortality as the result of long-term side effects in hl survivors. http://dx.doi.org/10.48101/ujms.v126.6117 mailto:anne.andersson@umu.se http://creativecommons.org/licenses/by/4.0/ 2 a. andersson et al. material and methods individuals diagnosed with hl at the age of 45 years or younger between 1965 and 1995 were identified using the swedish cancer registry. during these years, mantle field irradiation was the standard treatment for many patients with limited disease. from this cohort, individuals alive at the beginning of 2005 and treated in the northern sweden, uppsala, örebro and southern sweden health care regions were invited to participate in the study. after informed written consent had been obtained, a questionnaire was sent to the patients that included questions concerning hl treatment, if they had any ongoing surveillance program within the health care system, state of health, socioeconomic factors, and family history of both cancer and cvd (i.e. coronary artery disease, congestive heart disease and  valvular disease). since we wanted to use binary logistic regression, individuals who were former smokers were categorized as smokers. no pack-years of smoking were calculated. after returning the completed questionnaire, the patients were offered an open clinic visit. one reminder was sent to the patients who did not return the informed consent and/or questionnaire. the open clinic visit included a thorough medical history with a standardized form of questions targeted to detect the family history of cancer or cvd, symptoms of cvd, clinical investigation, electrocardiogram (ecg) and blood sampling (complete blood count, cholesterol, serum glucose, pro b type natriuretic peptide (pro-bnp) and thyroid-stimulating hormone). patients with pathological findings in their medical history, at clinical examination or in laboratory test results were referred to  a specialist physician for consultation, supplementary investigations, treatment and follow-up. follow-up (fu) time was set for each patient as the time between the year of hl diagnosis and the year when returning the survey. for the cohort attending open clinic visit or phone visit, follow-up was set as time between year of hl diagnosis and the year of clinical visit. the study was approved by the ethics committee in umeå (dnr dnr 05-112m). statistical analyses the questionnaires were scanned by it services and system development at umeå university (its) and this was followed by a data cleaning procedure to exclude eventual errors in outliers. data were analysed using ibm spss statistics, version 23. a comparison was performed on individuals participating and not participating in the study with pearson’s chi-squared test to investigate if age at follow-up, gender, region or distance to the hospital were associated with acceptance to the study. patients were grouped according to young, mid-life and retired status – that is, younger than 40, 40–65 and older than 65 years – to investigate whether there were any systematic differences in age categories. binary logistic regression analysis was performed on potential covariates – that is, radiotherapy, hypertension, hypercholesterolemia, diabetes, smoking habits and family history of cvd – to investigate their association to the risk for cvd. smoking habits were categorized into ‘ever smokers’ and ‘never smokers’; former smokers were categorized as ‘ever smokers’. as the covariates are well-known risk factors for cvd, our primary aim was to investigate whether there was an interaction of radiotherapy and other known cvd risk factors for the development of cvd (18). the cumulative risk for cvd, coronary artery disease, valve disease and heart failure was estimated and stratified according to years after hl diagnosis. results using the swedish cancer registry, we identified 6,946 individuals diagnosed with hl between 1965 and 1995. at the beginning of 2005, 1,700 of these individuals were living in  sweden. from this cohort, we invited 742 individuals diagnosed at the three participating university hospitals (umeå, uppsala and lund) at the age of 45 or younger at the time of treatment to participate in the study. from these, 40  (5.4%) were excluded due to death before inclusion (n  =  24), wrong diagnosis (n = 3) or emigrated/no address (n  =  13). of the 702 invited individuals, 504 (71.7%) agreed to  participate in the study and were mailed a questionnaire; 453 (89.8%) returned a completed questionnaire. these 453 patients had a median follow-up of 22.0 years with a total of 10,796 person-years. gender, age at invitation to the study and distance to the hospital were not significantly associated with participation in the study (data not shown). from the cohort of 453 hl survivors, 325 (71.7%) individuals accepted the invitation to a clinical visit. after review of the medical records of the clinical visits, six individuals were excluded from the study because their primary hl diagnosis was after the age of 45. five individuals requested a telephone interview instead of a clinical visit; if applicable, these data were included in the analysis. the remaining 128 individuals declined both a clinic visit and a telephone interview. characteristics of the 319 individuals included in the final analysis are shown in table 1. of the 319 included in the final analysis, 269 (84.3%) received radiotherapy and 89 (27.9%) had cvd at the time of their clinic visit (table 2). of the 265 individuals treated with radiotherapy and/or chemotherapy, 82 (31%) had developed cvd; of the 50 individuals treated with only chemotherapy, seven (14%) had developed cvd (p < 0.01). the overall cumulative incidences of cvd and of different cvd are detailed in figure 1. of the 219 individuals who had irradiation towards the mediastinum and who attended the clinic visits, 92 (41.6% of irradiated patients) were referred for further investigation, either cardiac ultrasound (n = 31) and/or internal medicine/cardiology consultation as the result of clinical or laboratory findings (n = 63). the overall incidence of cvd in the hl cohort increased continuously after treatment and this increase accelerated after 10 years. heart failure and coronary artery disease showed a stable increase during follow-up. the development of valve dysfunction accelerated 10 years or longer after hl diagnosis. the mean age of the first cvd onset was 51 years and the mean latency time from hl diagnosis to cvd was 26 years. binary logistic regression analyses were performed in the cohort of cases with cvd compared with those without cvd. side effects after treatment of hodgkin’s lymphoma 3 in  a univariate model, a significant association was identified with mediastinal radiotherapy (odds ratio [or]: 4.48, 95% confidence interval [ci]: 2.26–8.88), hypercholesterolemia (or: 8.45, 95% ci: 4.61–15.48) and hypertension (or: 3.85, 95% ci: 2.17–6.75). the multivariate model revealed mediastinal radiotherapy, hypercholesterolemia and hypertension as independent risk factors (table 3). the questionnaire answers  revealed 58 individuals who reported valvular disease.  radiotherapy, especially for mediastinum and hypercholesterolemia, was independently associated with a risk for valve disease (or: 6.14, 95% ci: 2.30–16.40 and or: 3.40, 95% ci: 1.70–6.78, respectively). finally, radiotherapy for mediastinum was an independent risk factor for the development of coronary artery disease (or: 4.41, 95% ci: 1.54–12.7) (table 2). treatment characteristics are listed in table 3. from the initial study cohort of 6,946 hl patients, 2,462 died of causes other than hl. of these, 640 (26%) died of heart disease before we initiated the study, with a median time of 6 years from hl diagnosis to death of heart disease. in our living cohort, 44% (319/702) participated in a clinical visit. there were no obvious systematic differences between the invited and the final group for age, gender or geographical area (data not shown). we did not have ethical permission to check medical records for side effects in patients who for some reason did not participate. discussion in this study with a long median fu time from diagnosis of hl to entering the study (22 years), mediastinal radiotherapy was shown to be an independent risk factor for cvd in hl long-term survivors, especially for the development of valvular disease. in this cohort, the well-known risk factors for cvd, hypercholesterolemia and hypertension were also independent risk factors for developing cvd. surprisingly, other risk factors such as smoking and family history of cvd did not increase the  risk of cvd; however, the incidence of cvd developed continuously from the end of treatment. a retrospective dutch study of 1,474 hl survivors treated at 40 years or younger between 1965 and 1995 found that 31% were deceased, but the study had a slightly shorter follow-up time than our study (13). the dutch study collected treatmentrelated data and data concerning risk factors from medical records, questionnaires, general practitioners or attending physicians: 84% received radiotherapy towards the mediastinum alone or in combination with chemotherapy and the frequency table 1. characteristics of the patients attending the clinical visit. variable male n = 164 (51.4) female n = 155 (48.6) all n = 319 mean age at diagnosis, year (range) 26 (3–45) 26 (7–43) 26 (3–45) mean age at follow up, year (range) 52 (18–85) 50 (22–76) 51 (18–85) mean follow up time*, year (range) 25 (12–43) 23 (12–43) 24 (12–43) cvd, n (%) 57 (34.8) 32 (20.6) 89 (27.9) eversmoker, n (%) 67 (40.9) 55 (35.5) 122 (38.2) hypertonia, n (%) 41 (25.0) 25 (16.1) 66 (20.7) hypercholesterolemia, n (%) 46 (28.0) 18 (11.6) 64 (20.1) diabetes mellitus, n (%) 12 (7.3) 5 (3.2) 17 (5.3) family history of cvd, n (%) 65 (39.6) 63 (40.6) 128 (40.1) * follow-up time calculated from the year of hl diagnosis to year at clinical visit/phone visit. cvd: cardiovascular diseases. table 2. treatment characteristics of patients at an open clinic visit. variable male female all n (%) 164 (51.4) 155 (48.6) 319 chemotherapy, n (%) 84 (51.2) 91 (58.7) 175 (54.9) radiotherapy, n (%) 133 (81.1) 136 (87.7) 269 (84.3) towards mediastinum*, n (%) 103 (77.4) 116 (85.3) 219 (81.4) mantle field*, n (%) 64 (48.1) 79 (58.1) 143 (53.2) radiotherapy + chemotherapy, n (%) 56 (34.1) 69 (44.5) 125 (39.2) only radiotherapy, n (%) 80 (48.8) 64 (41.3) 144 (45.1) only chemotherapy, n (%) 28 (17.1) 22 (14.2) 50 (15.7) dose ≥ 40 gy 91 (55.5) 85 (54.8) 176 (55.2) *percent of individuals who received radiotherapy towards mediastinum and mantle field is calculated among individuals who received radiotherapy. figure 1. cumulative incidence of cardiac events. the cumulative incidence of the first event of valve disease, coronary artery disease, heart failure and cardiovascular disease (cvd) overall in 453 hodgkin lymphoma long-term survivors who developed cvd after treatment of hl (both genders, all ages). based on data from the questionnaire, another five individuals did not report the year of onset, and that is why they are missing in the graph. 4 a. andersson et al. of heart failure was 13.3% for patients receiving more than 21 gy to the heart. thus, data from our study are in line with those in that report, although the frequency of multiple cvd was higher in the dutch study compared with our study (44.4% vs. 31.5%) (3,17). this difference could be explained by the fact that our study included only prospective cases living at the time when our surveillance study started. valve disease and coronary artery disease were the most frequent diagnoses as in the present investigation. in a study of 82 hl survivors who underwent screening for valvular disease (19), the mantle-irradiated individuals had a significantly increased risk for valvular dysfunction compared with those receiving chemotherapy. as in our study, this risk increased after 10 years, suggesting that surveillance with echocardiography 10 years after treatment with radiotherapy towards mediastinum should be carried out. a norwegian group presented an echocardiography study of 116 hl survivors treated at the age of <50 years and with a follow-up of median 5 years. they found aortic and mitral valve regurgitation grade >1 in 24% of the patients and therefore suggested echocardiography screening in hl survivors at risk (20). more recent studies have shown that these valve defects over time develop into clinically significant valve disease demanding surgical intervention. current recommendations for childhood cancer survivors who received radiation to the heart exceeding 20 gy include echocardiography and cardiac exercise test once every 5 years during adulthood. we propose a similar approach for adult hl survivors having received radiotherapy to the mediastinum before the age of 45  years (21). an alternative to regular echocardiograms could be regular cardiac auscultation and referral to echocardiography for the individuals presenting with a heart murmur. in our study, 25% of the individuals examined at the offered clinical visit needed referral due to previously unknown heart murmur. economic considerations might play a role in the choice between regular echocardiograms and regular heart auscultation. our study and the dutch study included data on other risk  factors such as smoking habits, hypertension, hypercholesterolemia and family history. in both studies, hypercholesterolemia was shown to affect the development of valve disease and coronary artery disease in hl survivors, obvious targets for intervention. in a hypothetical cohort of hl  long-term survivors treated at 30 years of age and with a 5-year follow-up after mediastinal irradiation, chen et al. found that lipid screening every 3 years was cost-effective and statin treatment for individuals with elevated lipid levels improved survival (22). therefore, patients receiving mediastinal radiotherapy should receive active secondary prevention treatments, blood pressure monitoring and cholesterol testing at a follow-up to lower incidence and mortality of cvd. the benefit from primary prevention of known risk factors of cvd has been shown in a study of 1893 individuals in a community in northern sweden; specifically, the study found that a long-term community-based cvd prevention program was associated with a decreased risk for death from cvd by 26.1% (23). since the frequency of cvd in hl survivors greatly exceeds the frequency in healthy individuals, targeted intense intervention ta b le 3 . u n iv ar ia te a n d m u lt iv ar ia te re g re ss io n a n al ys is . va ri ab le c ar d io va sc u la r d is ea se * (n = 8 9) va lv e d is ea se (n = 5 8) c o ro n ar y ar te ry d is ea se † ( n = 4 6) u n iv ar ia te lo g is ti c re g re ss io n a n al ys is m u lt iv ar ia te lo g is ti c re g re ss io n a n al ys is u n iv ar ia te lo g is ti c re g re ss io n a n al ys is m u lt iv ar ia te lo g is ti c re g re ss io n a n al ys is u n iv ar ia te lo g is ti c re g re ss io n a n al ys is m u lt iv ar ia te lo g is ti c re g re ss io n a n al ys is o r c i ( 95 % ) o r c i ( 95 % ) o r c i ( 95 % ) o r c i ( 95 % ) o r c i ( 95 % ) o r c i ( 95 % ) r ad io th er ap y (n = 2 65 )‡ 3. 27 1. 34 –7 .9 8 13 .1 75 1. 78 –9 7. 5 1. 62 0. 61 –4 .3 2 r ad io th er ap y to w ar d s m ed ia st in u m (n = 2 19 ) 4. 48 2. 26 –8 .8 8 5. 41 2. 49 –1 1. 76 6. 07 2. 34 –1 5. 70 6. 14 2. 30 –1 6. 40 3. 50 1. 43 –8 .5 6 4. 42 1. 54 –1 2. 67 h yp er te n si o n (n = 6 6) 3. 85 2. 17 –6 .7 5 2. 61 1. 33 –5 .1 4 3. 01 1. 62 –5 .6 1 2. 10 1. 03 –4 .2 8 4. 88 2. 63 –9 .0 4 2. 36 1. 05 –5 .3 0 h yp er ch o le st er o le m ia (n = 6 4) 8. 45 4. 61 –1 5. 48 7. 25 3. 69 –1 4. 24 4. 31 2. 32 –8 .0 3 3. 40 1. 70 –6 .7 8 16 .3 9 8. 25 –3 2. 59 13 .3 5 6. 15 –2 8. 99 fa m ily h is to ry o f c v d (n = 1 28 ) 1. 32 0. 80 –2 .1 6 1. 12 0. 62 –2 .0 0 0. 89 0. 50 –1 .6 0 0. 76 0. 40 –1 .4 4 2. 64 1. 44 –4 .8 4 1. 20 0. 55 –2 .6 0 d ia b et es (n = 1 7) 1. 88 0. 69 –5 .1 0 1. 27 0. 35 –4 .6 3 1. 96 0. 66 –5 .7 9 1. 61 0. 44 –5 .9 8 5. 06 2. 02 –1 2. 65 3. 92 0. 98 –1 5. 67 ev er sm o ke r ( 12 2) 1. 30 0. 79 –2 .1 3 1. 30 0. 72 –2 .3 3 0. 98 0. 55 –1 .7 7 1. 03 0. 54 –1 .9 5 1. 41 0. 77 –2 .5 5 1. 13 0. 52 –2 .4 6 n o te : u n iv ar ia te a n d m u lt iv ar ia te r eg re ss io n a n al ys is o n c v d , v al ve d is ea se a n d c o ro n ar y ar te ry d is ea se c o m p ar ed t o c as es w it h n o c v d , b o th g en d er a n d a ll ag es . i n t h e m u lt iv ar ia te a n al ys is , a ll va ri ab le s in t h e ta b le a re in cl u d ed , e xc ep t ra d io th er ap y. *c ar d io va sc u la r d is ea se (c v d ) i n cl u d es c o ro n ar y ar te ry d is ea se , h ea rt fa ilu re a n d v al ve d is ea se . † c o ro n ar y ar te ry d is ea se (c a d ) i n cl u d es m yo ca rd ia l i n fa rc ti o n . ‡ r ad io th er ap y (u n sp ec ifi ed t ar g et ) i s n o t in cl u d ed in t h e m u lt iv ar ia te m o d el in fa vo u r o f r ad io th er ap y to w ar d s m ed ia st in u m . c i: co n fid en ce in te rv al ; o r: o d d s ra ti o. side effects after treatment of hodgkin’s lymphoma 5 ought to be cost-effective even though no health economic studies have been conducted that focus on hl survivors (24). as there is a long latency for cvd as a late complication of hl treatment and assessing cardiovascular morbidity are of great importance, screening and surveillance of hl long-term survivors at high risk of severe cvd need to be considered, a proposal that has been made by earlier studies. for example, van leeuven et al. suggested screening every 5 years starting 5 years after mediastinal radiotherapy in individuals at high risk for cvd and after 10 years in other hl survivors (25). suggested screening methods include cac-score (coronary artery calcium score), computed tomography (ct)-angiography, echocardiography, ecg and monitoring of risk factors for cvd. the ship study recommends prospective clinical visits at regular intervals to detect new heart murmurs, to monitor other risk factors such as hypertension and hypercholesterolemia, and an echocardiography every 5 years could be suggested rather than just a follow-up. we consider these recommendations to be a reasonable intervention level to detect independent risk factors with the aim to lower mortality rates in patients given mediastinal radiotherapy. limitations a limitation of this study is that we had a drop-out of approximately 28%. however, because the participants had been treated a long time ago, this drop-out rate is acceptable as some patients would likely want to avoid conjuring up their memories of treatment. we also did not include the diseased patients as the major aim was to put living individuals on a surveillance scheme. the cohort size limited the possibility to find moderately strong associations. conclusion modern treatment for hl has abandoned the use of extensive field radiotherapy in favour of chemotherapy and new irradiation techniques that lower the risk of long-term side effects from mediastinal irradiation. nevertheless, a number of patients need irradiation to the mediastinum and these individuals will also benefit from surveillance programs developed for the cohorts of patients in the present study. disclosure statement except for daniel mohlin, who received honoraria from roche, merck, bristol-myers, squibb and takeda, all authors report no conflicts of interest. funding this study was funded by grants from the northern sweden cancer foundation, umea, the swedish cancer foundation, stockholm, acta oncologica through the royal swedish academy of science (bm salary support), stockholm and the heart foundation of northern sweden, umea. notes on contributors anne andersson, md, phd in oncology, department of radiation sciences, oncology, umea university, sweden. gunilla enblad, md, phd, professor of oncology, department of immunology, genetics and pathology, experimental and clinical oncology, uppsala university, sweden martin erlanson, md, phd in oncology, department of radiation sciences, oncology, umea university, sweden. ann-sofie johansson, md, phd in oncology, department of radiation sciences, oncology, umea university, sweden. daniel molin, md, associate professor of oncology, department of immunology, genetics and pathology, uppsala university, sweden björn tavelin, bsc, department of radiation sciences, oncology, umeå university, sweden ulf näslund, md, phd, professor, department of public health and clinical medicine, umeå university, sweden. beatrice melin, md, phd, professor in molecular epidemiology of cancer, principal investigator of the study. orcid anne andersson https://orcid.org/0000-0002-9597-6465 gunilla enblad https://orcid.org/0000-0002-0594-72 ann-sofie johansson https://orcid.org/0000-0001-5783-9489 ulf näslund https://orcid.org/0000-0003-4100-8298 beatrice melin https://orcid.org/0000-0002-9982-3757 references 1. socialstyrelsen. cancer i siffror. national board of health and wellfare. 2018:66. available from 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2016;117:691–6. doi: 10.1016/j.amjcard. 2015.11.027 20. galper sl, yu jb, mauch pm, strasser jf, silver b, lacasce a, et al. clinically significant cardiac disease in patients with hodgkin lymphoma treated with mediastinal irradiation. blood. 2011;117:412–8. doi: 10.1182/ blood-2010-06-291328 21. andersson a, naslund u, tavelin b, enblad g, gustavsson a, malmer b. long-term risk of cardiovascular disease in hodgkin lymphoma survivors – retrospective cohort analyses and a concept for prospective intervention. int j cancer. 2009;124:1914–7. doi: 10.1002/ ijc.24147 22. chen ab, punglia rs, kuntz km, mauch pm, ng ak. cost effectiveness and screening interval of lipid screening in hodgkin’s lymphoma survivors. j clin oncol. 2009;27:5383–9. doi: 10.1200/jco. 2009.22.8460 23. blomstedt y, norberg m, stenlund h, nystrom l, lonnberg g, boman k, et al. impact of a combined community and primary care prevention strategy on all-cause and cardiovascular mortality: a cohort analysis based on 1 million person-years of follow-up in vasterbotten county, sweden, during 1990–2006. bmj open. 2015;5:e009651. doi: 10.1136/ bmjopen-2015-009651 24. lindholm l, stenling a, norberg m, stenlund h, weinehall l. a cost-effectiveness analysis of a community based cvd program in sweden based on a retrospective register cohort. bmc public health. 2018;18:452. doi: 10.1186/s12889-018-5339-3 25. van leeuwen-segarceanu em, bos wj, dorresteijn ld, rensing bj, der heyden ja, vogels oj, et al. screening hodgkin lymphoma survivors for radiotherapy induced cardiovascular disease. cancer treat rev. 2011;37:391–403. doi: 10.1016/j.ctrv.2010.12.004 http://dx.doi.org/10.1093/jnci/94.3.182 http://dx.doi.org/10.1016/s0167-8140(97)00125-4 http://dx.doi.org/10.1200/jco.2002.09.038 http://dx.doi.org/10.1001/jamainternmed.2015.1180 http://dx.doi.org/10.1182/asheducation-2016.1.323 http://dx.doi.org/10.1200/jco.2003.07.131 http://dx.doi.org/10.1056/nejmoa1000067 http://dx.doi.org/10.1200/jco.2002.08.021 http://dx.doi.org/10.1182/asheducation-2014.1.488 http://dx.doi.org/10.1182/blood-2016-09-740332 http://dx.doi.org/10.1016/j.amjcard.2009.10.007 http://dx.doi.org/10.1016/j.amjcard.2009.10.007 http://dx.doi.org/10.1016/j.amjcard.2015.11.027 http://dx.doi.org/10.1016/j.amjcard.2015.11.027 http://dx.doi.org/10.1182/blood-2010-06-291328 http://dx.doi.org/10.1182/blood-2010-06-291328 http://dx.doi.org/10.1002/ijc.24147 http://dx.doi.org/10.1002/ijc.24147 http://dx.doi.org/10.1200/jco.2009.22.8460 http://dx.doi.org/10.1200/jco.2009.22.8460 http://dx.doi.org/10.1136/bmjopen-2015-009651 http://dx.doi.org/10.1136/bmjopen-2015-009651 http://dx.doi.org/10.1186/s12889-018-5339-3 http://dx.doi.org/10.1016/j.ctrv.2010.12.004 (12) upsala j med sci 111 (2): 263–268, 2006 ankle dislocation without accompanying malleolar fracture. a case report 1masahito hatori, 1satoshi kotajima, 2richard a. smith and 1shoichi kokubun 1department of orthopaedic surgery, tohoku university school of medicine. 2department of orthopaedic surgery, university of tennessee-campbell clinic abstract dislocation of the tibiotalar joint without associated fracture is rare. we present here a 21-year-old man who sustained open posteromedial dislocation of the left ankle without malleolar facture when he jumped and sprained his right ankle while playing basketball. the most likely mechanism is forced flexion applied to the ankle joint leading to a rupture of the anterior capsule and lateral structures of the ankle followed by an accelerating inversion stress leading to a posteromedial dislocation of the talus from the tibial condyle. transient paresthesia was noted in the area of the superficial peroneal nerve. at surgery, the anterior part of the tibiotalar joint capsule and anterior talofibular ligament were detached from their original sites. the calcaneofibular ligament was also detached with its associated periosteum and a tiny avulsed bony fragment. the articular facets of the tibia and talus were intact. the treatment consisted of wound irrigation, debridement, reduction and capsular suture followed by immobilization with a short leg cast. about 10 degrees of loss in the range of dorsiflexion was observed. the patient achieved good long-term functional results. introduction ankle fracture dislocation is a common injury in orthopaedic practice. ankle dislocation without fracture is, however, extremely rare [1-8]. there have been few reports describing the detailed operative macroscopic findings. we report here a case of open dislocation of the ankle without accompanying malleolar fracture. 263 received 31 august 2005 accepted 19 september 2005 case the patient was a 21-year-old man. when playing basketball practice, he jumped and failed to land properly on the floor and sprained his right ankle. he was transferred to us and the initial examination showed that his right foot was deviated posteromedially. the tip of the left lateral malleolus was protruding from the skin (fig1). the dorsalis paedis 264 fig. 1a,b. macroscopic photos showing severe varus deformity of the ankle with protrusion of lateral malleolus through the skin. 1a 1b artery was palpable. sensory disturbance was found in the dorsum of the foot. plain radiography showed that talus was dislocated posteromedial to the tibia. no fracture was demonstrated on plain radiography (fig 2a). joint laxity of the wrist and ankle was found. surgery was performed on the same day as the injury occurred. the ruptured skin was extended to expose the damaged parts. the anterior part of the tibiotalar joint capsule was detached from the original tibial site. the anterior talofibular ligament was irregularly torn from its attachment to the lateral malleolus. the calcaneofibular ligament was also detached with a tiny avulsed bony fragment and its associated periosteum. the articular facets of the tibia and talus were intact (fig 2b). manual reposition was possible but the joint easily re-dislocated by inversion. after irrigation and debridement of the wound, the torn ligaments, the capsule and the retinaculum were sutured to their original sites. the wound was closed in layers. postoperative casting was applied for six weeks. walking with cast was permitted four weeks after surgery. after removal of the cast, motion exercises of the affected ankle was started. plain radiography taken after nine months showed mild bone atrophy but no arthritic changes or avascular necrosis of the talus. the range of motion of the injured ankle was restricted by 10 degrees as compared with the normal side. the patient had no difficulty in walking on the flat ground. no muscular or sensory disturbances were found. 265 fig. 2a. plain radiographs showing posteromedial dislocation of the ankle joint without fracture (anteroposterior view). discussion ankle dislocation without fracture occurs most frequently in young adult males [2,3,6,8]. moehring et al. reported that all of 14 patients with this injury were young mostly adult males [3]. open fracture is not uncommon as was seen in 13 of 14 injuries by moehring et al, [3] and in three out of three by rivera et al. [5]. most of these injuries occurred by either traffic accident, fall from a height or were sports related [3,8]. in the series reported by moehring et al., the cause of injury in nine of the 14 cases was a motor vehicle accident and in the remainder, sporting events or a fall [3]. larsen et al. reported a 34-year-old male who sustained this injury while playing basketball [2] like the present case. posteromedial dislocation is most frequent [3]. predisposing factors that contribute to the pathogenesis of this lesion are internal malleolus hypoplasia, ligamentous laxity, weakness of the peroneal muscles, and previous ankle sprains [5]. the present case had no such predisposing factors except joint laxity. the most likely mechanism appears to be anterior or posterior extrusion of the talus from the mortise secondary to a force applied to the plantar flexed foot. final displacement is then determined by the position of the foot and the direction of the force applied [8]. in the present case, the patient 266 fig. 2b. plain radiographs showing posteromedial dislocation of the ankle joint without fracture (lateral view). failed in grounding the floor after jumping while playing basketball. he fell forward with his toe on the floor. given this situation, forced flexion was applied to the ankle joint leading to the rupture of the anterior capsule and lateral structures of the ankle. accelerating inversion stress resulted in posteromedial dislocation of the talus from the tibial condyle. there have been few reports to describe the operative findings of this unusual dislocation. our macroscopic examination of the present case at surgery revealed that the joint capsule, anterior talofibular and calcaneofibular ligaments were detached from the tibial side, lateral malleolus original site and calcaneal insertion site, respectively, the findings of which support the mechanism of this injury. it is noteworthy that a small bony fragment with the insertion of the calcaneofibular ligament was avulsed from the lateral wall of the calcaneus, that had not been shown on preoperative plain radiograms. conservative and operative treatments have been applied to this unusual injury. in closed dislocation, if good reduction is achieved, no operative repairs have been necessary in most reported cases. wroble et al. stated that closed reduction is accomplished easily and that optimum treatment appears to be immobilization in a short leg cast for 6 weeks with no weight bearing for the first 3 weeks [8]. in open dislocation, management consists of immediate reduction, debridement and capsular suture and immobilization with a short leg cast [5]. moehring et al reported that 12 of 13 patients with open dislocations underwent lateral ligamentous repair [3]. in the present case, debridement and repair of the ligaments and capsule were carried out because of the open dislocation. after conservative treatment, wroble et al. reported in a long-term follow-up study of eight patients that all results were good to excellent and no patient reported instability. all patients returned to work and sports participation and the range of motion was normal in all but four patients; none of these lacked more than 10 degrees of motion in any plane [8]. in a surgical series, moehring et al. followed up 12 of the 14 patients. two patients had poor results; the remainder (10 of 12) had good and excellent results. no patient had signs or symptoms of instability [3]. segal et al. reported chronic, posttraumatic peroneal tendon dislocation in a case due to forced planter flexion [6]. rivera reported a 10 to 15 degree loss in the range of dorsiflexion in two and paresthesia in the area of the superficial peroneal nerve in one of altogether three patients [5]. the present case has had neither avascular necrosis of the talus nor instability of the ankle joint but transient sensory disturbance of the foot, corresponding to the peroneal nerve innervated area. this nerve dysfunction was thought to be caused by inversion stress of the ankle leading to stretching of the peroneal nerve at fibula head. like other reported cases, the present case had no activities of daily living restriction in spite of subtle ankle rom restriction. prognosis of this rare injury is thought to be favorable if optimal treatment is given. 267 references 1. greenbaum ma, pupp gr (1992) ankle dislocation without fracture: an unusual case report. j foot surg 31: 238-240 2. larsen j, burzotta j, brunetti v (1998) ankle dislocation without fracture in a young athlete. j foot ankle surg 37: 334-348 3. moehring hd, tan rt, marder ra, lian g (1994) ankle dislocation. j orthop trauma 8: 167172 4. mooney jf, naylor pt, poehling gg (1991) anterolateral ankle dislocation without fracture. south med j 84: 244-247. 5. rivera f, bertone c, de martino m, pietrobono d, ghisellini f (2001) pure dislocation of the ankle: three case reports and literature review. clin orthop 382:179-184 6. segal ls, lynch cj, stauffer es (1992) anterior ankle dislocation with associated trigonal process fracture. a case report and literature review. clin orthop 278: 171-176 7. wehner j, lorenz m (1990) lateral ankle dislocation without fracture. j orthop trauma 4:362365 8. wroble rr, nepola jv, malvitz ta (1988) ankle dislocation without fracture. foot ankle 9: 64-74 corresponding author: masahito hatori, m.d., assistant professor department of orthopaedic surgery, tohoku university school of medicine 1-1 seiryomachi, aobaku, sendai, japan 980-8574 fax: 81-22-717-7248, tel: 81-22-717-7242 mhato@mail.tains.tohoku.ac.jp 268 upsala j med sci 100: 19-32, 1995 charge structure of the glomerular capillary membrane m . wolgast inst of phyvnlngy and medical biophysrcj, uppsala. suieden in 1935 teorell (see teorell, 1953,1983) presented his theory about charged membranes; the title of the paper was "an attempt to formulate a quantitative theory of membrane permeability". the theory was based on the ion equilibria resulting from the presence of charged colloids suggested already in 1911 by donnan although this early work seems to have attracted little attention until it was summarized in 1922 by loeb in the book "proteins and the theory of colloidal behavior. it should be emphasized, however, that this so called donnan distribution refers to an equilibrium, whereas teorell elaborated a set of equations also valid for steady state conditions as according to the nernst-planck equation. teorell also considered the electro-osmotic force resulting from the presence of an electric field in charged membranes, a consideration which later would form the basis of the so called membrane oscillator (teorell, 1983). in this hypothesis it was inferred that fluid flow will lead to rearrangement of the ion distribution and thereby the electric field so as to reduce the fluid flow. since this will tend to restore the ion distribution and the electric field, the fluid flow will again increase which, as before, will rearrange the ion distribution and the electric field with consequent reduction in the fluid flow and so forth. 19 fixed charge density of the glomerular capillary membrane where the present article is concerned, the primary aim of our studies on the glomerular capillary membrane performed in the early 1980's was simply to determine its permeability in terms of the hydraulic permeability and its pore structure (ojteg et al, 1987a, ojteg et al., 1987b). however, at this time it was known, both from morphological (rennke et al., kanwar et al., 1980, simionescu and simionescu, 1984) and physiological studies (chang et al., 1975, deen et al., 1980, deen and bridges, 1982, dworkin and brenner, 1985), that the glomerular capillary barrier also possesses negative charges fixed to the membrane; their function was believed to hinder penetration of albumin into the membrane by simple coulomb repulsion between the negative, fixed groups and the strongly negatively charged albumin molecule . in order to quantify this repulsive force, experiments were undertaken to determine the membrane fixed charge density in terms of meq/l. due to the small dimensions of the glomerular capillary barrier, it was most certainly not possible to apply the technique of "slicing up'' the membrane as made by teorell on his relatively thick porous artificial membranes. instead we chose to compare the transport of charged molecular probes with that of their neutral counterparts and where, as predicted by teorell, the equilibrium distribution and hence the flux of all negative ions would be reduced. a second obstacle arises from the fact that the transport of such charged molecules will be determined by so many forces, i.e. by diffusion, ion migration, hydraulic bulk flow and also by the so called streaming potential. fortunately, however, this problem is solved by the present availability of powerful computers. 20 the choice of molecular probes the choice of the probes does constitute a challenge. the most ideal probe would thus be a molecule nearly as small as sodium and chloride; unfortunately, however, the transport of such small ions would be virtually unrestricted and hence very difficult to assess. very large ions on the other hand, will be affected by the charge-induced large electric field in the vicinity of the pore rim, so that the membrane would give the impression of having a very large average charge density. our choice of myoglobin was a compromise between these two extremes in the sense that its transport will be at least somewhat restricted, but that it is still small enough to occupy not more than a small fraction (11%) of the fluid of the pore. a further advantage arises from the fact that it is a relatively compact, spherical molecule and also from the relative ease of changing its valency without changing the molecular size; the latter was determined from filtration through sephadex gels with about the same steric resolution as the glomerular capillary membrane. obviously, accurate determination of the valence of myoglobin, made polyanionic by a chemical procedure, is of key importance. for this purpose we therefore elaborated a method based on the determination of the electric potential difference that develops across a cellophane membrane, with on one side the probe molecule to be tested dissolved into 10 mm nacl and on the other pure nacl of the same ionic strength of 10 mm. since the electric potential will be proportional to t h e concentration of charges in meqfl, knowledge of the concentration of the test molecule in mma, enables its valence to be determined. biological duta in the biological experiments on rat kidneys, the glomerular membrane was found to 21 restrict the transport of the negatively charged myoglobin. more precisely, the fraction of the negative myoglobin in plasma filtered in the glomeruli was estimated at 15% as against 20% for its neutral counterpart, findings which would suggest a fixed charge density of about 35 meq/l a seemingly trivial finding. however, as regards the latter number, it was calculated that the corresponding electroosmotic pressure would be about 50 mm hg, i.e. about the same as the glomerular capillary pressure of 50-60 m m hg. obviously, this might merely be a coincidence, but since the charge-induced electroosmotic pressure in the peritubular capillaries (i.e. the network surrounding the tubules) also equaled their hydrostatic pressure of about 12 mm hg, it would seem less coincidental. a further highly questionable conclusion drawn from the ion equilibria refers to the prediction that, on the plasma side, the intramembranous hydrostatic pressure must be about 20 mm hg lower than in plasma and thus that the membrane must be rigid, since only a rigid structure would allow such sub-pressures. this is in sharp contrast to both biochemical and morphological data, which suggest rather that the membrane is made up of flexible, gel compounds such as the glycosaminoglycans. the gel hypothesis construction of the model in the search for an alternative model, teorell already in the early 1960’s suggested that, in a charged membrane, the electric field rather than a hydrostatic pressure gradient may account for the net driving force for fluid transfer. he also predicted that in order to account for the fluid transport across the glomerular capillary the transmembranous electric potential difference need not to be larger than about 1 mvolt. 22 as to the question of the source of the electric field, he also pointed out that the intracapillary hydrostatic force, due to the action of the heart, might well be converted into such an intramembranous electric field. as regards this "converting" mechanism, a closer examination of the electron microscopical picture of the capillary membrane (see the below figure) indicates that it consists of a matrix of gel compounds, the glycosaminoglycans, supported by a relatively rigid basement membrane made up of collagen filaments (curry, 1984, simionescu et al., 1984). fig. 1. equality of pressure inside tubes surrounded by a rigid basement membrane. for further explanation see text. in order to elucidate the characteristics of such a structure, we may first consider a (fig. la) a capillary formed by a tight and rigid tubing and where the hydrostatic pressure, p, of e.g. 20 mm hg will be the same in all parts of the tubing. if (fig. 1b) a network of flexible fibers is suspended along the wall of the tubing , the pressure both in the lumen and within the network is still uniform. the introduction of semipermeable barrier between the network and the lumen makes no difference, i.e. as 2 3 long as all the elements are flexible. however, if (fig. lc) colloids exerting an osmotic pressure of 20 mm hg, are added to the free fluid phase in the lumen, fluid will be dragged out of the gel with consequent shrinkage of the gel until it eventually collapses. this could be prevented by negative charges fixed to the fiber structure, which by attracting positive, mobile counter ions, may decrease the water activity to the same extent as the colloids in the free fluid phase in the lumen. more precisely, the charges will lead to the formation of a donnan equilibrium across the gel membrane-lumen interphase, where the sum of the positive and negative small ions in the gel will be larger than that in the free fluid space. as an example, if the concentration of na and c1 in the lumen is 150 meq/l and the charge density of the membrane is 25 meq/l, the intramembranous concentrations of na and c1 will be 163 meq/l and 138 meq/l, respectively. since the sum of na and c1 in the membrane thus is 1 meq/l or 1 mosm/l higher than that in the lumen, the osmotic pressure, rtc, resulting from the charges will be about 20 mm hg (19.33 mm hg at 37"c), a force which is able to balance the colloid osmotic pressure. in the next step (fig. l d ) also the barrier facing the interstitium is made permeable. since the pressure within the gel membrane is still 20 m m hg, fluid will leak out with consequent shrinkage of the gel eventually leading to collapse of the structure. again, this could be prevented by introducing 25 meq/l of negative charges fixed to the matrix at this side of the membrane. lastly if, as in the glomerular capillary membrane, the hydrostatic pressure drop is higher than the colloid osmotic force, the charge density on the bowmans space side has to be larger that on the luminal side (table i). the consequent larger osmotic force on 24 this side of the membrane will then drag fluid from lumen through the membrane and out into bowmans space. the driving force is thus an electro-osmotic pressure gradient rather than a hydrostatic pressure gradient. by estimating the hydrostatic forces across to the boundaries of the glomerular capillary membrane (kallskog et al., 1975), we are thus able to predict the intra membranous charge densities at the plasma and bowmans space side of the membrane, respectively and thence also the respective donnan equilibria (wolgast and ojteg, 1988). this is illustrated in table i, which also demonstrates that the difference in the ion equilibria results in a transmembranous electric potential gradient within the membrane, a gradient which will account for the fluid flow. table i. ion distribution, osmotic and hydrostatic pressures and electric potentials across the glomerular capillary membrane as predicted by the gel hypothesis. the concentration of the two myoglobins, c,, in bowmans space were considered to be unknown. parameter plasma m e m b r a n e bowmans plasma side bowm. sp. side space fixed charge density (meq/l) 26.49 36.86 na (meq/l) 150.00 159.50 165.58 146.01 c1 (rneq/l) 142.13 133.66 128.76 146.01 (na+cl) rnosmikg 292.13 293.16 294.33 292.02 albumin (meq/l) 7.87 0.0 0.0 0.0 osmotic pressure (rnrn hg) 20.0 20.0 44.7 0.0 hydrostatic pressure 56.9 56.9 56.9 12.2 electric potential (mv) -0.72 -2.36 -3.36 0.0 myoglobin (0) 1.041 1.041 cbow g w myoglobin (-6) 1.065 0.737 c,; 0.43 c,, single nephron filtration rate, sngfr net driving electroosmotic pressure difference 1% below 22.7 mm hg 40 nl/min 2 5 validification of the gel hypothesis knowing the above-mentioned ion equilibria, we may obviously also predict the intra membranous concentration of a polyanion such as our probe molecule, myoglobin, and thence, knowing also the bulk flow and the electric potential gradient, its net transport. a comparison between this predicted transport with that found experimentally, then showed an excellent agreement, i.e. in support of the theory; it should be emphasized, however, that it does not constitute a proof. functional ultrastructure of the glonierular capillary membrane in the next step the glomerular capillary membrane is considered to be made up of a network of fibers (fig. 2). utilizing neutral probe molecules such as inulin, native myoglobin and native horse radish peroxidase the size of the pores (or rather meshes in a network) could be determined; the results showing a so called "equivalent" pore radius of 40 a. this would then suggest that the width of the meshes in the network is about 80 a. on this fibers we may then distribute the fixed charges, where a charge density of 35 meq/l suggests an average distance between the charges of 40 a. fig. 2. network of fibers assumed to constitute the capillary membrane. 26 an example of such a network is illustrated in fig 2, showing quasihexagonal-shaped meshes with a width of about 80 a as formed by fibers with binding sites located 40 a apart and with one negative fixed charge in between two binding sites. obviously the data do not permit the assessment of the true configuration of the network, but merely that the network shown in the figure is in accordance with the experimental data both with respect to the charge density and its steric structure. it should be remarked in this context, that the distribution of the charges and the size of the meshes are coupled to each other. this is because the repulsion between the charges actually determines the final size of the meshes, i.e. since in the absence of these repulsive forces, the network would collapse (vide supra). however, the electric field of the charges and hence the repulsive force, is also determined by the prevailing ionic strength; this is because the charges at least partially will be shielded by mobile, positive ions like sodium. the structure seen i n fig 2 is thus restricted to physiological ionic strength, whereas an e.g. higher ionic strength would thus result in a more complete shielding with consequent lower repulsion between the charges and hence in smaller meshes. teorell also anticipated that biological membranes would be in a constant movement; because the electric field of a charge will be subjected to random fluctuations. it should thus be realized that the distance between the "neutralizing" sodium ion in a 150 mm saline solution is as much as 20 a and hence that the occurrence of these ions in the vicinity of the charges will show a random fluctuation. if thus one sodium ion (in excess of a chloride ion) is located close to a charge, the electric field outside the charge will be reduced towards zero. if, on the other hand, no sodium ion is located nearby the charge, the extension of the electric field will be very large. in pure water, for instance, the extension of the electric field will be several hundred angstroms. this fluctuating electric repulsion between the charges will then also be followed by fluctuations of the size and form of the meshes; if there are few "neutralizing" sodium ions, the mesh tends to expand and vice versa a "living membrane" in its true sense. 27 the model also anticipates that the average size and form of the meshes will change from one condition to the other. lf, for instance, the plasma colloid osmotic pressure rises, the membrane has to shrink, i.e. since the chargeinduced balancing electro-osmotic pressure has also to be increased. such a shrinkage could be brought about either by a reduction in the size of the meshes or by a reduction of the pore length or, which is most probable, by both these changes. formation of the glomerular capillary membrane the model also allows for a rational explanation as to the formation of the capillary barrier. it is thus difficult to imagine that the capillary endothelial cells are able to form a homogenous membrane penetrated by discrete pores. they may, however, synthesize fibers carrying regularly arranged "binding sites" and charged groups such that, after extrusion from the cells, they are able to form a network like that shown in fig. 2. evidently, aging processes might damage the molecules forming the lattice structure, but this problem could be solved by a slow extrusion of damaged molecules and replacement by new molecules synthesized by the endotheliurn. the self-rinsing ability of the glomerular capillary membrane a charged membrane, positive or negative, might also be expected to possess a rinsing ability. this is illustrated in fig 3, where the mesh in the upper left corner refers to an intact mesh, where the charges are partially shielded by 3 mobile, positive ions. obviously this shielding will reduce the repulsive force such that the width of the mesh becomes 80 a. if then, by accident, a large molecule enters into a pore (the upper right corner), it will knock out these "neutralizing" ions thence leading to a rise in the repulsive force between the fixed charges. the mesh will then widen at the expense of the surrounding meshes (lower panel), whereby the large molecule is free to move in the downstream direction until it will be expelled in bowman's space and subsequently into the urine. 2 8 fig. 3. mechanism for the self rinsing ability of the capillary membrane. for explanation see text. the strength of the force is in fact very large. as an example, if the large molecule occupies 75% of the space of the pore, the pressure in the space between the large molecule and the walls of the pore will be about 112 atmosphere. in conclusion, the concept of charged membranes presented in 1935 by teorell would seem to apply also to the glomerular capillary membrane a s well as, probably, all other capillary membranes. more precisely the membrane is suggested a s consisting of a hydrated gel rather than a rigid structure, the integrity of which is maintained by negative charges fixed to flexible fibers. as a consequence of this gel concept, the fluid transfer will b e governed, not by the starling forces, but rather by an electric field. the gel model would also seem attractive since it explains 1) the ability of the glomerular capillary membrane to change its permeability in response to external forces, 2) its rinsing ability and since 3) it gives a rational explanation for the formation of the membrane by filaments synthesized by the endothelial and epithelial cells on the two sides of the glomerular capillary membrane. 29 references chang, r.l.s., w. m. deen, c. r. robertson and b. m. brenner. 1975. permselecti vity of the glomerular capillary wall. 111. restricted transport of polanions. kidney int 8, 212 218. curry, f. r. e. mechanics and thermodynamics of transcapillary exchange. in: handbook of physiology. the cardiovascular system. bethesda, md. am physiol. soc., 1984, sect 2 , vol iv., part 1, chapt. 8, p 309-374. deen, w. m. and c. r. bridges. 1982. molecular charge of horse radish peroxidase. am j physiol242, f750-f762. deen, w. m., b. satvat and j.m. jamieson. 1980. theoretical model for glomerular filtration of charged solutes. am j physwl238, f126-fl39. dworkin, l. d. and b. m. brenner. biophysical basis of glornerular filtration. in: the kidney: physiology and pathophysiozogy,edited by d. w. seldin and g. giebisch. new york: raven, 1985, p. 397-427. k k l s k o g , o., l. 0. lindbom, h. r. ulfendahl and m. wolgast. 1975. kinetics of the glomerular filtration in the rat kidney. an experimental study. acta physiol scand 95, 295-300. kanwar, y. s., a. linker and m. g. farquhar. 1980. increased permeability of the glomerular basement membrane to ferritin by removal of glycosaminoglycans (heparan sulphate) by enzyme digestion. j cell b i d 86, 688-693. 30 loeb, j. proteins and the theory of colloidal behavior. new york: mcgraw-hill, 1922. ojteg, g., k. nygren and m. wolgast. 1987. permeability of renal capillaries. i. preparation of neutral and charged molecular probes. actu physiol scund 129, 277-286. ojteg, g., k. nygren and m. wolgast. 1982. permeability of renal capillaries. 11. transport of neutral and charged protein molecular probes. actu physiol scand 129, 287-294. rennke, h.g., y. patel and m. venkatachalam. 1985. glomerular filtration of proteins: clearance of anionic, neutral and cationic horseradish peroxidase in the rat. kidney int 13, 324-328. simionescu, m. and n. simionescu. ultrastructure of the microvascular wall: functional correlations. in: handbook or physiology. the cardiovascular system. microcirculution. bethesda, md. am physiol. soc., 1984, sect 2 , vol iv., part 1, chapt 3, p 41-101. teorell, t. history of the physical chemistry of charged membranes. in: structure and function in excitable cells. new york: plenum publishing co, 1983, p. 321-334. teorell, t. 1953. transport processes and electrical phenomena in ionic membranes. progress in biophysics 3, 305-369. w i e d e m a ” , g. die lehre von der elektrizitat. berlin: 1893, fr vieweg und braunschweig. wolgast, m. and g. ojteg. 1988. electrophysiology of the renal capillary membranes: gel concept applied and starling model challenged. am j physiol 254, f750-f762. 31 tf-iups160019 199..201 letter to the editor sids-cdf hypothesis revisited: explaining hypoxia in sids dear editor, the sudden infant death syndrome (sids)–critical diaphragm failure (cdf) hypothesis (1,2) continues to attract attention 5 years after the publication of the original article in this journal, and several colleagues have contributed perspectives and insights to the hypothesis (3–6). the basic premise of the sids-cdf hypothesis is that the diaphragm is a vital organ that must continuously generate adequate force to maintain ventilation and that critical diaphragm failure is a terminal event and the cause of death in sids. respiratory failure is a common cause of death of adults with compromised diaphragm function, and key sids factors have been shown either to reduce the diaphragm force-generating capacity or to increase the respiratory workload of the diaphragm. both can cause hypoxia that can in turn further compromise diaphragm function and initiate a selfreinforcing feedback loop characterized by: weakened diaphragm – hypoxia – weakened diaphragm (figure 1). various types of non-lethal infections are commonly observed in sids victims, and a large body of research shows that diverse infections can cause a rapid and significant reduction in the force-generating capacity of the diaphragm. the prone sleeping position is another well-known risk factor for sids and one that has been especially difficult to explain in the context of sids etiology. however, clinical studies in infants show that in prone position the respiratory workload of the diaphragm is significantly increased. sids typically occurs during deep sleep when the supportive respiratory muscles are partially or totally inactivated, therewith increasing the respiratory load on the diaphragm. gestational prematurity and low birth weight are risk factors for sids, and both affect the developmental and structural maturity and the force-generating capacity of the diaphragm, which achieves structural maturity after 6 months. the non-monotonic death rate in sids-cdf has been another enigmatic characteristic of the syndrome. the sidscdf hypothesis posits that this is due to the passive immune protection provided by maternal antibodies during the first month of life when the respiratory muscles are most vulnerable. however, the passive immune protection quickly wanes 1 month post partum, especially in the absence of breast-feeding. a considerable amount of research has been conducted on disturbances in the serotonergic system of sids victims, and earlier we discussed how the sids-cdf hypothesis would explain the role of the tryptophan–serotonin–melatonin pathway in the etiology of sids (1). altitude has also been shown to be an independent risk factor for sids (7) and is known to increase the respiratory workload on the diaphragm. these and other sids risk factors have been discussed in previous publications (1,2). however, i have not addressed in detail the role of hypoxia in sids, and since its significance has been highlighted in several recent publications, i will briefly review hypoxia in the context of the sids-cdf hypothesis and propose new research avenues and approaches. several studies over the past decades have highlighted hypoxia as a potentially central event in the etiology of sids (8,9). indeed, already in the 1960s researchers suggested a link between sids and hypoxia (10). recently, neary and breckenridge published a review article titled: ‘hypoxia at the heart of sudden infant death syndrome?’ (11). while i agree with the observation that hypoxia plays a central role in sids, i will argue that sids is not a cardiac phenomenon, as neary and breckenridge suggest, but rather a respiratory event caused by the failure of the vital respiratory pump. hypoxia produces reliable biochemical markers that have been measured in sids victims and that suggest a prolonged period of hypoxia before death (8). a particularly compelling study investigated the correlation of sids and the vascular endothelial growth factor (vegf) gene that is highly sensitive to changes in tissue partial oxygen tension. evidence presented by jones and colleagues suggests that sids victims experienced one or more hypoxic events over several hours to days before death, based on the time needed for gene upregulation and subsequent protein expression and release to body fluids (12). what makes the vegf gene study so compelling is that vegf protein up-regulation has been previously demonstrated in experimental models, where animals were subjected to environmental hypoxia or to a variety of pathologic conditions with compromised tissue oxygenation. following the publication of the vegf study, saugstad and rognum emphasized that sids is preceded by hypoxia and urged researchers to: ‘intensify research identifying possible triggering factors inducing hypoxia and the cascade of events leading to sids’ (13). this communication is in part an attempt to address their unanswered 2003 question. the respiratory system consists of two parts: a gasexchanging organ, the lungs, and a pump that ventilates the lungs. the pump is composed of the chest wall, the respiratory muscles, the respiratory centers of the cns, and the nerves connecting the centers to the muscles. cns control of respiration and possible mechanical defects in the respiratory system have been extensively investigated in the context of sids (14,15). however, as we note in our original article, the possibility that sids is caused by critical weakness of the primary respiratory muscle has received very little attention, and as of march 2016, out of approximately 11,000 sids articles in pubmed, only 50 articles contained the search words ‘sids and diaphragm’, and only a few of those actually address diaphragm weakness in sids. fortunately, the diaphragm has been extensively investigated outside sids research, and its dysfunctions and upsala journal of medical sciences, 2016 vol. 121, no. 3, 199–201 http://dx.doi.org/10.1080/03009734.2016.1176972 vulnerabilities are well known. the diaphragm is the primary respiratory muscle, and it is axiomatic that the diaphragm powers the respiratory pump that oxygenates all muscles in the body, including the diaphragm itself. if the diaphragm becomes sufficiently weakened, respiration cannot be maintained (16). acute respiratory failure due to critical diaphragm fatigue is common in patients with neuromuscular diseases and in some elderly patients (17,18). there is a large body of experimental evidence that shows that hypoxia exacerbates diaphragm fatigability and can impair the diaphragm’s ability to generate force. mouse models have shown that acute hypoxic stress results in significant diaphragm muscle weakness (19), and in pigs acute hypoxemia impairs the diaphragm’s capacity to generate force (20). brotto and colleagues note that hypoxia ‘has long been known to dramatically reduce muscle function’ (21), and in human adults it has been shown that hypoxia exacerbates both diaphragm and abdominal muscle fatigability and can contribute to respiratory failure (22,23). the characteristics of the failure of the ventilation pump are hypoxemia, hypoventilation, and hypercapnia (24). patients who suffer from respiratory failure display increased work of breathing, muscle fatigue, and ventilation failure through imbalance between demand and supply (19). white and colleagues note that ‘it is well recognized that patients with respiratory muscle weakness are at risk of hypoxemia and hypercapnia during sleep, especially rapid eye movement (rem) sleep . . . patients with respiratory muscle weakness show nocturnal hypoventilation, with oxygen desaturation particularly during rapid eye movement (rem) sleep . . . we conclude that in patients with muscle weakness nocturnal oxygenation correlates with diaphragmatic strength’ (emphasis mine) (25). as we discussed in our original article in this journal, the young infant has a potentially vulnerable diaphragm. the diaphragm is largely inert during the gestational period and is structurally undeveloped. it is only approximately 2 mm thick, the supportive muscles are undeveloped, and in the prone sleeping position efficiency of the respiratory pump is reduced. indeed, the prone position has been shown to increase hypoxia in infants, with a nadir in oxygenation coinciding with months 2–3 of life that are also the highest-risk periods for sids (26,27). muller and colleagues go as far as to argue that ‘the normal preterm and term infant is very close to the threshold of diaphragmatic fatigue’ (28). it is well known that that diaphragm weakness can cause acute respiratory failure in adults. the evidence discussed here suggests that sids victims experience prolonged and progressive hypoxia. furthermore, it is known that key sids factors, including hypoxia, can both reduce the ability of the diaphragm to generate force and increase the respiratory workload. the sids-cdf hypothesis posits that the weakened diaphragm – hypoxia – weakened diaphragm self-reinforcing feedback loop could compromise ventilation and lead to respiratory failure. this line of investigation offers promising new avenues for research, and the extensive body of research regarding the diaphragm can and should be leveraged in sids research. a specific area of research that could be fruitful is determining what kind of markers hypoxia or hypercapnia lasting several hours (perhaps even days) would leave on the diaphragm of a young infant and then determining if these markers can be found in sids victims. fiber distribution in the diaphragms of sids victims has been regularly investigated, and no abnormalities have been found (personal communication with professor rognum). however, another potential marker to consider is the cross-sectional area of fibers, rather than fiber distribution per se (personal communication professor o’halloran). there are a number of other biomarkers for hypoxia that could be considered (lactate, ammonia, hypoxanthine, xanthine, and urate levels), and indeed stoltenberg and colleagues have shown that sids victims have increased hypoxanthine concentrations in their vitreous humor (29). however, we must consider the possibility that critical diaphragm failure, which is essentially severe muscle weakness, does not leave definitive and easily measured markers in the muscle tissue of sids victims. even if more accurate biomarkers can be established, the real challenge is not showing that sids victims suffer from hypoxia (this has already been established), but rather developing a model to test if a ‘weakened diaphragm–hypoxia–weakened diaphragm’ feedback loop can be caused by a combination of sids risk factors (infections, prone sleeping position, rem sleep, prematurity, absence of breastfeeding, cigarette smoke, altitude, etc.) and if this loop can cause the diaphragm to weaken to such an extent that it can no longer sustain respiration. i hope i have presented enough evidence here to encourage researchers to leverage the existing knowledge of the diaphragm muscle and its dysfunctions and to investigate further this novel and largely unexplored pathway for sids. finally, we should consider if the term sudden is not misleading in the context of sids. it seems that early practitioners sought to express the fact that the deaths were unexpected rather than rapid. evidence reviewed here strongly suggests that sids is in fact characterized by a prolonged struggle to breathe, progressive hypoxia, and rem sleep high altitude prone sleeping position ‘non-lethal’ infections reduced diaphragm force generating capacity hypoxia increased diaphragm work load low birth weight/ prematurity diaphragm weakness critical diaphragm failure respiratory failure figure 1. the sids-cdf hypothesis posits that several factors can affect the diaphragm force generating capacity and increase diaphragm workload resulting in hypoxia. hypoxia can further reduce diaphragm force generating capacity and increase diaphragm workload. in some circumstances this can lead to diaphragm weakness, cdf and ultimately respiratory failure. 200 p. m. a. siren compromised tissue oxygenation. there is nothing sudden about sids. disclosure statement the author reports no conflicts of interest. references 1. siren pma, siren m. critical diaphragm failure in sudden infant death syndrome. ups j med sci. 2011;116:115–23. 2. siren p. the sids–critical diaphragm failure hypothesis revisited. ups j med sci. 2013;118:62–4. 3. goldwater pn. a perspective on sids pathogenesis. the hypotheses: plausibility and evidence. bmc med. 2011;9:64. 4. van kempen ta, deixler e, crook ma. hypophosphatemia as a key factor in sudden infant death syndrome (sids)? ups j med sci. 2013;118:143–4. 5. eisenhut m. features of diaphragmatic myositis in a case of sudden infant death. ups j med sci. 2011;116:220. 6. szczesny p1, poznanski j, paczek l, zielenkiewicz p. hypophosphatemia and sudden infant death syndrome (sids)–is atp the link? ups j med sci. 2014;119:55–6. 7. katz d, shore s, bandle b, niermeyer s, bol ka, khanna a. sudden infant death syndrome and residential altitude. pediatrics. 2015;135:e1442–9. 8. prandota j. possible pathomechanisms of sudden infant death syndrome: key role of chronic hypoxia, infection/inflammation states, cytokine irregularities, and metabolic trauma in genetically predisposed infants. am j ther. 2004;11:517–46. 9. rognum to, saugstad od, oyasaeter s, olaisen b. elevated levels of hypoxanthine in vitreous humor indicate prolonged cerebral hypoxia in victims of sudden infant death syndrome. pediatrics. 1988;82:615–18. 10. stevens lh. sudden unexplained death in infancy: observations on a natural mechanism of adoption of the face down position. am j dis child. 1965;110:243–47. 11. neary mt, breckenridge ra. hypoxia at the heart of sudden infant death syndrome? pediatr res. 2013;74:375–9. 12. jones kl, krous hf, nadeau j, blackbourne b, zielke hr, gozal d. vascular endothelial growth factor in the cerebrospinal fluid of infants who died of sudden infant death: evidence for antecedent hypoxia. pediatrics. 2003;111:358–63. 13. saugstad od, rognum to. sudden infant death syndrome is preceded by hypoxia. pediatr res. 2003;53:881. 14. duncan jr, paterson ds, hoffman jm, mokler dj, borenstein ns, belliveau ra, et al. brainstem serotonergic deficiency in sudden infant death syndrome. jama. 2010;303:430–7. 15. macfarlane pm, mayer ca, litvin dg. microglia modulate brainstem serotonergic expression following neonatal sustained hypoxia exposure: implications for sudden infant death syndrome. j physiol. 2015 dec 12. [epub ahead of print]. doi:10.1113/jp271845. 16. macklem pt. respiratory muscles: the vital pump. chest. 1980;78:753–8. 17. rochester df, esau sa. assessment of ventilatory function in patients with neuromuscular disease. clin chest med. 1994;15:751–63. 18. delerme s, ray p. acute respiratory failure in the elderly: diagnosis and prognosis. age ageing. 2008;37:251–57. 19. o’leary aj, o’halloran kd. diaphragm muscle weakness and increased ucp-3 gene expression following acute hypoxic stress in the mouse. respir physiol neurobiol. 2015;s1569–s9048:30072-0. 20. watchko jf, laframboise wa, standaert ta, woodrum de. diaphragmatic function during hypoxemia: neonatal and developmental aspects. j appl physiol. 1986;60:1599–604. 21. brotto p, van leyen sa, brotto ls, jin jp, nosek cm, nosek tm. hypoxia/fatigue-induced degradation of troponin i and troponin c: new insights into physiologic muscle fatigue. pflugers arch. 2001;442:738–44. 22. verges s, bachasson d, wuyam b. effect of acute hypoxia on respiratory muscle fatigue in healthy humans. respir res. 2010;11:109. 23. zhu x, heunks lm, ennen l, machiels ha, van der heijden hf, dekhuijzen pn. nitric oxide modulates neuromuscular transmission during hypoxia in rat diaphragm. muscle nerve. 2006;33:104–12. 24. roussos c, koutsoukou a. respiratory failure. eur respir j. 2003;22:3–14s. 25. white je, drinnan mj, smithson aj, griffiths cj, gibson gj. respiratory muscle activity and oxygenation during sleep in patients with muscle weakness. eur respir j. 1995;8:807–14. 26. wong f, yiallourou sr, odoi a, browne p, walker am, horne rs. cerebrovascular control is altered in healthy term infants when they sleep prone. sleep. 2013;36:1911–18. 27. fyfe kl, yiallourou sr, wong fy, odoi a, walker am, horne rs. cerebral oxygenation in preterm infants. pediatrics. 2014;134:435–45. 28. muller n, gulston g, cade d, whitton j, froese ab, bryan mh, et al. diaphragmatic muscle fatigue in the newborn. j appl physiol. 1979;46:688–95. 29. stoltenberg l, rootwelt t, oyasaeter s, rognum to, saugstad od. hypoxanthine, xanthine, and uric acid concentrations in plasma, cerebrospinal fluid, vitreous humor, and urine in piglets subjected to intermittent versus continuous hypoxemia. pediatr res. 1993;34:767–71. pontus m. a. siren singapore pontusmax@hotmail.com received 7 march 2016; revised 30 march 2016; accepted 6 april 2016 � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. http://dx.doi.org/10.1080/03009734.2016.1176972 upsala journal of medical sciences 201 sids-cdf hypothesis revisited: explaining hypoxia in sids disclosure statement references tf-iups160041 264..270 original article systematic development of an evidence-based website on preconception care ilse delbaerea, pascale mokangia, kristien roelensb, an de sutterc , xavier gellynckd , dimitri beeckmane, liselot van de wallef, pieter vandenbulckef and hans de steurd adepartment of midwifery, health education, vives university college, kortrijk, belgium; bdepartment of obstetrics and gynecology, ghent university hospital, ghent, belgium; cdepartment of family medicine and primary health care, ghent university, ghent, belgium; ddepartment of agricultural economics, faculty of biosciences engineering, ghent university, ghent, belgium; euniversity centre for nursing and midwifery, department of public health, ghent university, ghent, belgium; fpublic health agency ‘zorg & gezondheid’, flemish government, brussels, belgium abstract introduction: in february 2015, the flemish minister of welfare, public health and family launched a website on preconception care: ‘gezondzwangerworden.be’. the website was developed in response to the lack of comprehensive communication on preconception care and the inadequate intake of folic acid among flemish women. despite the international recommendation to take 400 lg folic acid on a daily basis one month before conception until 12 weeks of pregnancy, studies show a lack of compliance in women wanting to become pregnant. procedure: a compilation of evidence was made through reviewing well-established guidelines on preconception and prenatal care. the quality of guidelines was assessed by means of agree ii. the topics included in the website were selected by an internal committee of 5 experts and an external committee of 16 experts. content validation was carried out by 40 experts in preconception care or related topics. results: the above-described procedure resulted in an evidence-based website with a selection of relevant, validated information for both women and men who plan a pregnancy and professionals who are consulted by these people. evaluation and recommendation: the website is currently attracting a constant number of 100 to 200 visitors a day. the information on folic acid is among the most requested, which is an important finding with regard to the policy objectives on preconception care. more research is needed in order to evaluate the use and effect of the website more thoroughly. article history received 21 april 2016 revised 1 july 2016 accepted 17 july 2016 keywords evidence-based; folic acid; guideline; information; preconception care; prevention introduction in february 2015, the website on preconception care, ‘gezondzwangerworden.be’, was launched by the flemish minister of welfare, public health and family. the website is edited in dutch and holds evidence-based information for both women and men planning a pregnancy and health care providers. since the mrc vitamin study research group in 1991 found that a daily dose of 400 lg folic acid reduced the risk of neural tube defects drastically, periconceptional supplementation of folic acid has been recommended internationally to women planning a pregnancy (1). however, the time-frame in which women are advised to take folic acid differs between countries (2). the ‘superior health council’ in belgium recommends intake one month before pregnancy until pregnancy week 12; nonetheless, a study in 2010 on the practice of periconception folic acid intake in flanders (the northern, dutch-speaking part of belgium) shows that only 36% of women actually pursue this recommendation (3). because of the proven effect of folic acid in the prevention of neural tube defects and recommendations of public and health authorities, on the one hand, and the lack of compliance in women planning a pregnancy, on the other hand, a resolution proposal on folic acid intake before conception and during pregnancy was granted by the flemish parliament on 16 june 2010 (4). subsequently, several stakeholders were involved in a number of meetings to discuss folic acid intake in the periconceptional phase. the public health agency ‘zorg & gezondheid’ and the royal academy for medicine of belgium organized these meetings on the initiative of the flemish minister of welfare, public health and family. after the final meeting on 28 april 2011, a public procurement was released on folic acid intake and preconceptional advice. one of the goals of this procurement was the development of an evidence-based website on preconception care, with a particular focus on folic acid intake. a multidisciplinary team from ghent university worked on contact ilse delbaere ilse.delbaere@vives.be department of midwifery, health education, vives university college, kortrijk, belgium � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 4, 264–270 http://dx.doi.org/10.1080/03009734.2016.1216481 http://orcid.org/0000-0002-2540-8307 http://orcid.org/0000-0002-8908-3310 http://creativecommons.org/licenses/by/4.0/ this procurement from april 2013 to july 2014. the website was developed in a systematic and scientific manner, and numerous experts were involved in the content validation. procedure search for and selection of guidelines the content of the website is based on available guidelines on preconception and prenatal care. we searched in pubmed, cochrane, web of science, google, and google scholar using the following keywords: guideline(s), preconception, preconception care, preconception health, prepregnancy, before pregnancy, and before conception. we also visited international and national websites of professional organizations providing guidelines. few guidelines on preconception care in healthy women were available. some of the prenatal guidelines included information on the periconceptional phase, and some issues of the prenatal guidelines were relevant for preconception care as well. the guidelines perused were national guidelines from domus medica (association of general practitioners) (5) and kce (federal centre of knowledge for public health) (6), as well as international guidelines from cdc (www.cdc.gov/ncbddd/folicacid/recommendations.html), nhg (7), ajog (8), icsi (9), and nice (10). the quality of the guidelines was assessed by three authors by means of agree ii (the appraisal of guidelines for research and evaluation instrument is recognized as the gold standard for practice guidelines evaluation) (11). selection of topics all relevant topics of the selected guidelines were inventoried. the list was reviewed by a multidisciplinary committee of five internal experts (a gynaecologist, a general practitioner, two midwives, and a researcher with expertise on folic acid), and a first selection was made by this internal expert committee (iec) (n ¼ 5). within the iec, there was a consensus to include information on the items summed up in table 1. in the selection of topics, it was our goal to focus on preconception care rather than prenatal or antenatal care. topics considered less relevant for the website on preconception care were: information on breastfeeding, information on salmonella and parvovirus, soil and water pollution, personal hygiene, information on natal and postnatal care, pregnancy-related conditions, breast and abdominal examination, bacterial vaginosis, group b streptococcus, and asymptomatic bacteriuria. subsequently, the list of topics was sent to an external multidisciplinary committee of experts (eec) (n ¼ 16) together with a questionnaire where the experts could indicate if they agreed with the inclusion of information on the topic within the website and where they could mark the level of importance of every topic. furthermore, the experts provided relevant information. sixteen external experts were involved in the selection of topics: six general practitioners with expertise in preconception care, four gynaecologists (two academic and two non-academic), three midwives with expertise in preconception care, one pharmacologist, one nutritional expert, and one staff member of the most important flemish public service for the well-being of children. table 2 depicts the advice of the eec on the degree of importance for each topic to be included on the website intended for the public and professionals. as for healthy nutrition, experts indicated that basic information is known by professionals. information on nutrition advice after bariatric surgery, malabsorption, and vitamin d is needed more for this group. according to the experts, it was necessary to provide information on women at risk of having a child with neural tube defects. the few experts who did not consider it important to inform the public about the risk of toxoplasmosis reported that—in their opinion—the women were already sufficiently aware of the risk; however, most experts considered it necessary to include information on toxoplasmosis prevention. information on smoking was considered very important because of the potential health benefit in smoking cessation. experts advised to refer to smoking cessation guidance on the website. information on physical exercise was considered important in the prevention of obesity with its adverse effect on fertility and pregnancy outcome. experts considered that information on the optimal age for childbearing was not always relevant for this website. they argued that when people seek information on preconception care, they cannot change their age. nonetheless, most experts agreed to include information on maternal age. the arguments of the external experts were further discussed within the internal expert committee (see below). content development based on the final list of topics, evidence was selected from guidelines and reviews, and a first draft text was written. arguments of the external committee and contradictions in table 1. categories selected by the internal experts. categories subcategories nutrition folic acid and other micronutrients fertility and menstrual cycle lifestyle legal and illegal drugs, body mass index, sports and leisure, hyperthermia, travelling, dental hygiene infections toxoplasmosis, listeriosis, stis, hepatitis b, hepatitis c, hiv, cmv vaccinations rubella, varicella, pertussis anamnesis diabetes mellitus, epilepsy, asthma, thyroid dysfunction, hypertension, thrombo-embolic conditions, mental disorders, medication familial anamnesis genetic disorders, congenital malformation social factors domestic violence and information for the partner (drugs, hyperthermia, medication, harmful effects and radiation, stis) other harmful effects of paint, hair dye, and radiation upsala journal of medical sciences 265 http://www.cdc.gov/ncbddd/folicacid/recommendations.html http://www.cdc.gov/ncbddd/folicacid/recommendations.html the guidelines were discussed with the internal expert committee (iec). content validation in total, another 40 experts were involved in the revision of the information on the website (a detailed list of experts can be found on the bottom of the page ‘gezondzwangerworden.be/ over-de-website’). because of the importance of this process, experts were carefully chosen after consultation with the commissioning party (zorg & gezondheid) and the iec. thus, a multidisciplinary group was chosen to revise the part of the contents corresponding to their field of expertise. as such, gynaecologists, general practitioners, and midwives with an expertise in preconception care were involved; as were specialists in smoking cessation, obesity, and genetics; nutritional specialists; pharmacists; members of public services on occupational health, sexually transmitted infections, and hygiene; and delegates of health insurance companies, federations of birth defects, and members of the dutch health council. all experts revised their part of the draft text thoroughly and formulated suggestions and corrections. the content was modified according to the feedback from the experts. in case of uncertainties, the external experts were consulted. again, we kept our focus on information that was relevant for couples who were not pregnant yet, as the majority of flemish women are followed up thoroughly by a health care professional once they are pregnant. in the draft version, we advised that women should stop alcohol intake when they were pregnant. because of this remark, other experts were consulted and more literature was reviewed. this resulted in a stricter message, on alcohol use, within the website (avoid alcohol when you want to become pregnant). feedback sessions with the commissioning party work progress was discussed with the commissioning party (z&g) on a three-monthly basis. choices were motivated and adjusted if necessary. in these meetings, it was decided that the website would have two parallel parts: a part for the lay public and a part for professionals; however, everyone should have access to both parts. the style of writing was adjusted to the target audience, and the content differed according to specific information needs. furthermore, the name of the website was decided in these meetings as well as the structure of the website, the choice of experts, and the choice of eye-catchers on the front page (folic acid, alcohol, and smoking). pilot study a pilot study was carried out by the ‘thinking aloud’ method (12) in a group of 30 first degree midwifery students (all female) on the one hand, and 6 people of reproductive age on the other hand (5 female, 1 male). one researcher led the group discussion, and another researcher noted the remarks. without further information, the participants were asked to look at the front page of the website and talk about their first impression. they were asked what target audience they believed the website was intended for and what idea they had of the purpose of the website. subsequently, they were asked to formulate a preconception-related question. after some explanation about the development of the website, the participants were asked further to explore the website and to search for the answer to their question on the website. they were asked if the website was complete in their opinion, or if it was too exhaustive, if all information was clear, if the website was user-friendly, and if they thought the website was appealing. for the participants of the pilot study, the target group was obvious, the main messages and the purpose of the website were clear, information was found swiftly, and the website was easy accessible, appealing, and structured. there were some remarks about the colour use (some participants thought the pink was too female, other participants thought the colour would not discourage men visiting the website). table 2. advice of external experts for topics to include in each part of the website. number of experts (n ¼ 16) indicating level of importance. part of the website for the public part of the website for professionals very important important less important very important important less important folic acid 13 3 0 9 6 1 smoking 10 6 0 8 8 0 nutrition 6 9 1 3 10 1 toxoplasmosis 5 8 2 5 8 2 listeriosis 3 6 6 3 7 5 alcohol 4 11 1 5 10 1 drugs 5 10 1 5 9 1 physical exercise 2 11 3 1 9 3 low bmi 0 12 1 1 12 1 high bmi 1 14 0 1 11 2 travelling 1 5 7 1 7 6 hyperthermia 0 10 3 0 9 5 oral hygiene 2 9 1 2 8 3 occupational issues 3 8 1 1 10 3 radiation 3 7 1 2 9 4 paint 1 5 4 0 6 7 socio-economic factors 5 7 1 5 9 1 fertility 3 11 2 3 6 4 general anamnesis 2 9 1 4 10 1 266 i. delbaere et al. furthermore, suggestions for the lay-out were made. there were some remarks on grammar, participants found a few inactive links, and the need to explain difficult words in the website was expressed. participants commented on the ambiguous message about alcohol use (to avoid alcohol when you are thinking of getting pregnant and stop drinking alcohol once you know you are pregnant) and pointed out that symptoms of stis were not always systematically given. based on the input from both groups, the website was further refined and improved. the development of the website is schematically depicted in figure 1. results the website the above-described process resulted in the website, ‘gezondzwangerworden.be’ (in english: ‘getting pregnant in good health’) (figure 2). the front page holds information on folic acid, alcohol use avoidance, and smoking cessation. there is a direct link to information for the partner and a section where relevant news is updated regularly. furthermore, there are seven main tab pages: ‘healthy lifestyle’, ‘becoming pregnant’, ‘infections’, ‘medical information’, ‘other’, ‘for the partner’, and ‘for the professional’. the tab page ‘healthy lifestyle’ holds information on healthy nutrition, vitamins (folic acid and vitamin a), vegetarian and vegan nutrition, weight, hand and food hygiene, oral hygiene, alcohol, smoking, drugs, and leisure activities (physical activity, travelling, hyperthermia). the tab page ‘becoming pregnant’ holds information on the menstrual cycle, lifestyle factors influencing fertility, and advice for people failing to conceive. on the tab page ‘infections’, information is included on toxoplasmosis, listeria, rubella, varicella, pertussis, cytomegalovirus, parvovirus (this information was included later), stis, hepatitis c, and tuberculosis. ‘medical information’ holds information on personal anamnesis, medication, and vaccination. on the tab page ‘other’, information is found on the domestic situation (socio-economic issues), occupational situation, cosmetics (hair dye and colourants, deodorants, and other), paint, and radiation. furthermore, the tab page ‘for the partner’ holds information for both the male partner (lifestyle issues, such as alcohol, smoking, and drugs, hyperthermia, medication, harmful factors such as radiation, and information on stis and hepatitis c) and information for the female partner (lifestyle issues and stis). finally, the tab page ‘for the professional’ has the same structure as the information for the public, but more information on medical issues is included: the information is more thorough and has other accents. for the professionals, the link to the source of information (guideline) is included within the text. the public can also look up the sources by means of a header ‘sources’ at the top of the website. next to this header, there is a header ‘news’, where updates on the website are maintained and where relevant news on preconception care is found, a header where links to relevant websites are found, and a header ‘contact’ where visitors are 1. review of guidelines review by internal expert commi�ee (iec) (n=5) agree ii expert assessment 2. selec�on of guidelines 3. development of comprehensive topic list iec (n=3) 4. selec�on of validated, relevant topic list review by iec (n=5) review by external expert commi�ee (eec) (n=16) 5. content development 6. content valida�on review by iec (n=5) review by eec (n=16) 7. development pilot website/study review by iec/external review (n=5/n=40) 8. improvement website midwifery students (n=30) people of reproduc�ve age 9. promo�on ac�on iec (n=3) 10. launch website involved par�es (university and governmental agency) feedb ack sessio ns with gover nment al agenc y (3mont hly basis) figure 1. flow of development process. upsala journal of medical sciences 267 encouraged to contact their professional for questions or to fill in a contact form if they want to provide feedback on the website. the website was launched on 23 february 2015 by the flemish minister of welfare, public health and family, together with a press release. the news was covered in most written media and was mentioned in the television news at noon. the release of the website was announced to professional organizations of a large number of stakeholders (general practitioners, gynaecologists, midwives, health insurance companies). evaluation after one week, the website had attracted 8,850 users (there are approximately 30,000 births in flanders per year to firsttime mothers) of which almost 6,000 users visited the website on the day it was launched (in total we had 11,431 visitors during the first month). most visitors (46%) were referred by news websites, 36% by direct entry, 10% by social media, and 8% by an organic search on search engines. we attracted users from all regions in flanders. the first week, we received 11 contact forms, of which 9 were from professionals. there was some feedback on the content, which was pursued when relevant. some professionals asked for flyers to promote the website amongst their clients. two care clients responded that the website was not easy to use on a smartphone or tablet, a problem which has been solved promptly by the webhost. after the first week and until now, only few contact forms were received. since this first week, which had much media coverage, and until today (a year after the launch) the website has been reaching a constant average of 100–200 visitors a day (more than 4,000 per month). three out of four visitors were new, and 85% of the visitors were women. table 3 shows the age distribution of visitors. people aged 25–34 years represented 62% of the visitors. at the moment, 70% of visitors reach the website via an organic search on the internet (table 4). people got to the website by searching on a variety of terms, such as ‘becoming pregnant’, ‘folic acid’, ‘healthy pregnancy’, ‘cycle’, and a combination of food products and ‘pregnancy’; 15% of visitors came via links from other websites. more than 77% of those visitors followed the link on the website of kind & gezin (kindengezin.be), which is the most important preventive organ for well-being of children in flanders (the flemish counterpart of one—l’office de la naissance et de figure 2. the website ‘gezondzwangerworden.be’. 268 i. delbaere et al. l’enfance). amongst other things, people look at this website to find information on official childcare initiatives. the front page was, as expected, the most visited page of the website. after this, most users looked at the information on folic acid and then went to information on ‘healthy lifestyle’ and ‘becoming pregnant’ or ‘the menstrual cycle’. updates during this first year of existence, a number of updates have been carried out. first, contact information of a patient organization for people with an unfulfilled child wish has been added. second, the information on vitamins and minerals has been updated according to the new nutritional recommendations of the superior health council, and the information on ‘cosmetics’ has been extended due to a new report on anti-transpirants of this council. the advice of the same council to follow strict hygiene measurements in the prevention of cmv was added as well. subsequently, a health care provider noticed the lack of information on parvovirus. after considering the evidence on this topic, information on the website was completed after review of three experts. finally, information on the zika virus was added with referral to an evidence-based and regularly updated website on tropical medicine (www.itg.be). recommendations google analytics provide a service to evaluate the use of a website to some extent; however, the possibilities are limited. it is, for example, not clear how many and to what extent professionals refer to the website. nonetheless, google analytics show that a number of visitors found our website by using a link provided on the website of professionals. on the contact form it is mentioned that specific questions can and should be discussed with the health care provider. until now, the contact form was only used to provide feedback on the website (as is indicated on the website); however, it is not clear to what extent people address preconception care with their health care provider due to information they read on the website. although the page with information on folic acid is one of the most visited pages of the website, it is currently not clear to what extent folic acid supplementation is (more) successful. it would be of interest to monitor folic acid intake for example in birth registers. finally, it would be valuable to publicize the website on a more regular basis, since an increased number of visitors is noticed each time the website is mentioned in the media (e.g. newspaper message). acknowledgements the authors would like to thank all collaborating experts (gezondzwangerworden.be/over-de-website) for their advice. the authors want to thank the participants of the pilot study for their contribution. furthermore, we thank joline goossens for her contribution in the research proposal, and koen boussery and leen claes for their advice on folic acid recommendations. disclosure statement the authors report no conflicts of interest. funding the research was funded by ‘zorg en gezondheid’, a department of the flemish government involved in well-being, public health, and family care. orcid an de sutter http://orcid.org/0000-0002-2540-8307 xavier gellynck http://orcid.org/0000-0002-8908-3310 references 1. mrc vitamin study research group. prevention of neural tube defects: results of the medical research council vitamin study. mrc vitamin study research group. lancet. 1991;338:131–7. 2. shawe j, delbaere i, ekstrand m, hegaard hk, larsson m, mastroiacovo p, et al. preconception care policy, guidelines, recommendations and services across six european countries: belgium (flanders), denmark, italy, the netherlands, sweden and the united kingdom. eur j contracept reprod health care. 2014;44:1–11. 3. hoppenbrouwers k, roelants m, gu�erin c, van leeuwen k, desoete a, wiersema jr. preventie van spina bifida en andere neuralebuisdefecten door foliumzuursuppletie tijdens de zwangerschap. swvg feiten en cijfers. 2010;3. available from: http://steunpuntwvg.be/images/swvg-1-publicaties/2011_06-r17-jong-gezondheid-eerste-levensweken. 4. dehaene vdt, franssen dc, jans v, jans l, crombez dj. voorstel van resolutie betreffende het gebruik van foliumzuur voor de conceptie en tijdens de zwangerschap [internet]. 2010. p. 2009–11. available from: http://docs.vlaamsparlement.be/docs/stukken/20092010/g462-1.pdf. 5. samyn e, bastiaens h, de sutter a, van royen p. preconceptieadvies. 2008. available from: http://www.domusmedica.be/documentatie/downloads/praktijkdocumenten/richtlijnen/ 730-preconceptieadvies-1/file.html. 6. gyselaers w, jonckheer p, ahmadzai n, ansari m, carville s, dworzynski k, et al. what are the recommended clinical assessment and screening tests during pregnancy? kce reports; 2015. available at: https://kce.fgov.be/publication/report/what-are-thetable 3. average age distribution of visitors to the website ‘gezondzwangerworden.be’. age category percentage of users 18–24 years 12.4% 25–34 years 62.4% 35–44 years 13.3% 45–54 years 7.6% 55–64 years 3.3% 65 years or older 1.1% table 4. visitors to the website ‘gezondz wangerworden.be’. channel percentage of users organic search 70.5% referral (by other websites) 14.7% direct entry 14.2% social media 0.6% upsala journal of medical sciences 269 http://www.itg.be http://steunpuntwvg.be/images/swvg-1-publicaties/2011_06-r17-jong-gezondheid-eerste-levensweken http://steunpuntwvg.be/images/swvg-1-publicaties/2011_06-r17-jong-gezondheid-eerste-levensweken http://steunpuntwvg.be/images/swvg-1-publicaties/2011_06-r17-jong-gezondheid-eerste-levensweken http://docs.vlaamsparlement.be/docs/stukken/2009-2010/g462-1.pdf http://docs.vlaamsparlement.be/docs/stukken/2009-2010/g462-1.pdf http://www.domusmedica.be/documentatie/downloads/praktijkdocumenten/richtlijnen/730-preconceptieadvies-1/file.html http://www.domusmedica.be/documentatie/downloads/praktijkdocumenten/richtlijnen/730-preconceptieadvies-1/file.html http://www.domusmedica.be/documentatie/downloads/praktijkdocumenten/richtlijnen/730-preconceptieadvies-1/file.html https://kce.fgov.be/publication/report/what-are-the-recommended-clinical-assessment-and-screening-tests-during-pregnancy#.v01u2fmltgw recommended-clinical-assessment-and-screening-tests-during-pregnancy#.v01u2fmltgw. 7. de jong-potjer l, beentjes m, bogchelman m, jaspar g, van asselt k. nhc-guideline preconception care. huisarts wet. 2011;54:310–26. available from: https://www.researchgate.net/ publication/286329562_nhc-guideline_preconception_care. 8. jack bw, atrash h, coonrod dv, moos mk, o’donnell j, johnson k. the clinical content of preconception care: an overview and preparation of this supplement. am j obstet gynecol. 2008;199(6 suppl 2):s266–s79. 9. akkerman d, cleland l, croft g, eskuchen k, heim c, levine a, et al. health care guideline for routine prenatal care. institute for clinical systems; 2012. p. 1–115. available from: https://www.icsi. org/_asset/13n9y4/prenatal.pdf. 10. uk. ni for health and clinical execellence. antenatal care routine care for the healthy pregnant woman. uk natl heal serv. 2008;guideline:1–56. 11. dans al, dans lf. appraising a tool for guideline appraisal (the agree ii instrument). j clin epidemiol. 2010;63:1281–2. 12. nielsen j, clemmensen t, yssing c. getting access to what goes on in people’s heads? reflections on the think-aloud technique. proc second nord conf human-computer interact nord ’02. 2002. p. 101–10. 270 i. delbaere et al. https://kce.fgov.be/publication/report/what-are-the-recommended-clinical-assessment-and-screening-tests-during-pregnancy#.v01u2fmltgw https://kce.fgov.be/publication/report/what-are-the-recommended-clinical-assessment-and-screening-tests-during-pregnancy#.v01u2fmltgw https://www.researchgate.net/publication/286329562_nhc-guideline_preconception_care https://www.researchgate.net/publication/286329562_nhc-guideline_preconception_care https://www.icsi.org/_asset/13n9y4/prenatal.pdf https://www.icsi.org/_asset/13n9y4/prenatal.pdf systematic development of an evidence-based website on preconception care introduction procedure search for and selection of guidelines selection of topics content development content validation feedback sessions with the commissioning party pilot study results the website evaluation updates recommendations acknowledgements disclosure statement funding references upsala j med sci 100: 151-160,1995 a clinical method for measuring the distribution of segmental flexion mobility in the cervico-thoracic spine staffan norlander, rpt,’ ulnka aste-norlander, rpt: bengt nordgren, md, phd3 and bo sahlstedt, md, phd3 ’research foundation for occupational safety and health in the swedish construction industry,2 us fysioterapi, trosa and departmentv of ‘rehabilitation medicine and diagnostic radiology, akademiska sjukhuset, uppyala universir): uppsala, sweden abstract the aim of this study was to evaluate the validity and the repeatability of a new technique to assess segmental flexion mobility in the cervico-thoracic spine between segments c7 and t5. the new technique is referred to as the cervico-thoracic-ratio (the ctr-technique). the radiological evaluation of skin distraction measurements showed that validity was high for the ctr-technique. a high correlation between vertebral flexion mobility and skin distraction was recognized individu ally and for the whole group. the evaluation of repeatability was found to be high for intratester and fair for intertester repeatability. the ctr-technique may become a valuable complement to other method? for assessing segmental flexion mobility in patients suffering from neck-shoulder pain in clinical practice. introduction a new technique has been described tomeasure the segmental flexion mobility in thecervico-thoracic junction and the upper thoracic spine (7). the measuring technique is referred to as the cervico thoracic-ratio (the ctr-technique). the ctr-technique describes what is defined as relative flexion mobility (ctr%). relative flexion mobility is a calculated ratio based on absolute values of skin distraction between c7 t5. absoluteflexinn mobility is defined as the measured alteration in cm between 3 cm interdistant skin markings marked from c7 down t o t 5 and measured with a tape measure after a maximal forward flexion of the trunk and neck from an upright posture. the distance of 3 cm marked, in the upright posture, has been used as the definition of one motion segment, as the height of one disc and one thoracic vertebral body is approximately 3 cm, according to (5). the validity of the ctr-technique is dependent on an individual and strong reiationship bezween vertebralflexion mobility and skin distraction in the motion segments between c7 and t5, since the method is meant to be used for individual assessment of flexion mobility. several attempts have been made to establish the relationship between spinal mobility and different methods for cliiiical examination. according to (2) and (9) clinical examinations and radiographic measurements showed high validity for measurement of lumbar curvature during stance and trunk forward flexion. 151 according to (1) and (10) different clinical examinations showed poor validity compared with measurements from radiographic pictures. i n conclusion, validity differs for different instruments and methods. the ctr-technique has to be evaluated i n order to become areliable method for clinical examination of the segmental flexion mobility. repeatubiliry is defined as the capability to repeat a measurement. many clinical methods for examination of spinal mobility are known to show low repeatability. different factors affect the repeatability, for example if the mobility is tested passively or actively, or how long the time interval is between repeated measurernents. according to (3)the best repeatability is obtained if the mobility is tested actively and with as short time interval as possible between measurements, the classic " ~ e s retest design". in the ctr-technique mobility is tested actively. repeatability is also known to be higher when it is perfomied by one tester, intratester repeutubility, compared with measurements done by two testers, intertester repeutuhility. in order to get acceptance of the ctrtechnique the repeatability also has to be evaluated. the aim of this study is to evaluate the validity a n d the repeatability of the ctrtechnique as a method to be used in clinical practice for assessment of the segmental flexion mobility between c7 and ts. materials and methods radiological evaluation of validity the validity of the ctr-technique is dependent on a n individual and strong relationship between vertebral flexion mobility and skin distraction i n motion segments between c7 and ts. in order 10 study the individual relationship six different vertebral angles c7 to ts were evaluated against six corresponding skinmarkings for each subject. the analysis of relationship was also evaluated for the whole group of 42 different vertebral angles versus 42 corresponding skinmarkings. seven male subjects with pain in the neck-shoulder region, mean age 40.3, (sd16.0) years participated i n the evaluation of validity. the evaluation was only done on male subjects, since previous studies did not show any significant differences between female and male subjects with reference to the degree of skin distraction, during forward flexion measured according to the ctr-technique(7). procedure. six small metal markings were glued onto the skin with 3 cm intervals, according to the marking procedure described i n the ctr-technique. the markers were glued with the subject in an upright sitting posture and the upper marking p u t over the most prominent part of the spinous process of c7. lateral radiographs were used to obtain overlay measurements of the alteration of vertebral angles and of skin marker (fig. 1). the first radiograph was taken with the subject i n an upright sitting posture and the second in a maximal flexed sitting posture. since the spinous processes could not be demonstrated in the thoracic region without tomography, the angle between the vertebral endplates were used as the independent variable. an aluminium wedge was usedin order to equalize the contrast differences and to visualize the metal markers on the skin. the angles between the endplates and the distances between the markers were measured on the films with the patient i n upright position a n d 152 i n maximal forward flexion. the t6-vertebra was used as a reference vertebra, the cumulative angles of c7 down to ts were measured with a gauge. the diagonal alteration of metal skin markers m 1 m6 were measured with a pak of compasses and a ruler (fig. 1). the measurements were done only once. figure 1 lateral radiographs were used lo obtain overlay measurements of lhecumulative vertebral angles (c7 ts) and the diagonal alterations of the metal markings (m1 m6). statistical analysis. the determination of the relationship between skin distraction and vertebral flexion mobility was done by deciding the highest regression coefficient for five different regression models. both the relationship for the individual and the whole group was evaluated for each model. a computerprogrmi (quest) calculated the equation for linear (y=a+b*x), exponential ( y =aiexp (b*x)), power (y=a*xab), logarithmic (y=a+b*ln ( x ) ) and polynomial models ( y = a b , * x + b,* x2). vertebral angles were used as the independent variable, and skinmarkers as the dependent variable. breakdowns with one-way anova were also used to describe the mean values of the dependent variable skinmarkers as a function of the independent variable vertebral angles. evaluation of repeatability the evaluation of repeatability was done for intraand intertester repeatability. tests were done by two investigators, two trials each. the evaluation was done with a test-retest design on 26 male subjects, mean age 41.2, (sd 9.3) years. procedure . on arrival the subject was instructed to sit in a chair. the first investigator put the markings on the subject according to the examination procedure described in the ctr-technique, and the subject was asked to flex forward as much as possible. the investigator measured and noted all the five alterations. after that the markings were erased. the same procedure was repeated by the second investigator. finally a second trial was repeated by both investigators, altogether four trials. 12-950246 153 statistical analysis. in the evaluation of repeatability a sign-test was used to reveal byslernatic errors. random errors were evaluated by calculating t h e measuring precision and the relative measuring error. both absolute and relative flexion mobility was evaluated. the precision was calculated as the pooled standarddeviations of the differences between trialsor investigators, divided by the extracted square root of two. the relative measuring error, the coefficient of variation (cv) was calculated as the standard deviation divided by the mean, times 100. breakdown with one way anova was used to analyse the degree of conformity between intraand intertester repeatability expressed as r square and p-values. results evaluation of validity the results of the analysis of the relationship between vertebral flexion mobiiity and skin distraction show that the validity of the ctr-technique was individually very high. (table 1). the degree of relationship vaned between different regression models, which implies an individual variation of spinal flexion mobility. the polynomial model showed the highest degree of individual relationship in five subjects, the logarithmic model in two subjects and the power model and the linear model showed equally high values as the polynomial model in one subject each. the different r2 values ranged between 0.68-0.98, which is a very high degree of explanation and all seven subjects showed a statistically significant relationship in at least one of the models (table 1). table 1 results of individual relationships between dependent variable skin distraction and independent variable vertebral angles. i regression models 0.68 0.1 1 0.91 p-value 0.08 0.002 0.004 0.003 0.04 0.52 0.004 exponential 0.89 0.006 0.83 0.13 0.56 0.07 0.97 0.001 power i;i--i...ilogarithmic +polynomial rz p-value .~ 0.6x 0.04 0.95 0.002 0.88 0.007 0.93 0.003 0.44 0.14 0.00 1.0 0.81 0.04 0 61 0.07 0.95 0.002 0 9 3 0003 0.96 0.002 0.56 0.09 0.01 0.81 0.70 0.08 0.68 0.18 0.96 0.008 0.92 0.023 0.94 0.012 0.82 0.07 0.91 0.03 0.98 0.003 for the whole group the polynomial regression model showed the strongest relationship between vertebral flexion mobility arid skin distraction, r2= 0.44 arid p<0.001. (n=42) (fig. 2). the linear model showed r2= 0.39, (p<o.001), (n=42), the exponential model ?= 0.27 (p<o.ooi), (n=42), the power model r2= 0.19 (p<0.005), (n=42) and the logarithmic model showed r2 = 0.28 (p<0.001), (n=42). themean cumulativevertebral angleforc7 was 18" and the alteration of the am1 skinmarker was 5.3 cm. the cumulative flexion mobility for ti and am2 was 13" versus 3.8 cm. t2 and am3 was 8.1' versus 3.0 cm. t3 and am4 was 5.9" versus 2.5 cm. t4 and am5 was 5 . i ' versus 2.1 cm. t5 and am6 was 4.6" versus 1.8 cm (fig. 2). 154 7 -, i y = 2 2525 0 uo81 x + 0 0078 xi 6 i figure 2 regression model describing thc highest relationship between vertebraiangles (c7 t5) and alteration of skin markings (am1 am6) (r = 0.66, p<o.wl) (n = 32). mean values (& sd) for studied variable>. the breakdowns with oneway anova analysis showed a relationship similar to the polynomial model (table 2) between vertebral angles and skin distraction, r2 was 0.41 and p-value 0.001. increasing vertebral angles showed increasing skin distraction. table 2 thc breakdown with one-way anova analysis showcd a similar rclationship between vertebral angles and alterations of sktnmarkings, rz = 0.413 and p< 0.001 (n = 42). vertebral ... skinmarkings (cm) angles ( )" x sd min m a x 0 6 2.335 0.916 0.600 3.200 6 12 2.883 1.389 1.300 5.800 12 18 3.622 1.645 1.300 5.800 i 8 24 5.300 0.781 4.400 5.800 24 30 6.700 6.700 6.700 -. 4 number crf,ioints 17 12 9 3 1 total 3.083 1.546 0.600 6.700 42 evaluation of repeatability absoliiteflex.xionmobilily. theresults of theevaluation showed that repeatability was high fc~rabsolute flexion mobility, for both intraand intertester repeatability, which was important, as the values of relative flexion mobility exclusively depended on the values of absolute flexion mobility. the absolute flexion mobility was measured in cm according to the description of the ctr-technique from motion segment c7 down to t5. the inrrutester repeatability showed no systematic error between the first and second trial either for tester 1 or tester 2, when absohte flexion mobility was 155 evaluated for all motion segments. the sign test did not show any significant differences between trials for the two testers (table 3, i* and 2*).the intertester repeatability, comparing tester 1 with tester 2 showed a small, but significant systematic measuring error between testers in both trials and in all five motion segments, (table 3,3* and 4 9 . tester 2 systematically measured a 2-3 mm shorter absolute flexion mobility for all motion segments, the random errors expressed as measuring precision (mm) and as relative measuring error cv showed that repeatability was high for both intra and intertester repeatability (table 3, 1* and 4*). in general the precision is higher the shorter the distance measured , while the cv is greater. the cv for intratester repeatability ranged from 2.1 4.8% for tester 1 and from 1.9 4.4% for tester 2 for the different motion segments (table 3, i* and 2*). the cv for inlertesterrepeatability ranged from 2.45.7% for trial 1 and from 1.8-4.1 % for trial 2 for the different motion segments (table 3,3* and4*). the breakdown with one way aiiova analysis describing the relationship between repeated trials for absolute flexion mobility showed a very high degree of conformity, r2values ranged 0.995 0.998 and p-values was p<o.ooi, for both intraand intertester repeatability. table 3 results irom evaluation ofrcpedtability of absolute flexion [nobility (n = 26), 1* and 2" = inwitester, 3* and 4* = intertester repeatability. absolute flexion mobility (cm) x sd 3.8 0.2 3.6 0.2 3.8 0.2 3.6 0.2 7.3 0.3 7.1 0.4 7.3 0.3 7.1 0.3 10.6 0.4 10.5 0.5 10.6 0.4 10.4 0.5 14.0 0.5 13.8 0.6 14.0 0.5 13.7 0.5 17.4 0.5 17.1 0.6 17.3 0.5 17.1 0.6 ~. motion segment trial tester random errors systematic measuring measuring errors precision (mm) error ( c v % ) i sign test .~ i * 1.8 4.8 2* 1.6 3* 2.1 5.7 4* 1.5 4.1 i* 2.7 3.6 2* 2.4 3.4 11s 3* 3.1 4.3 ns 4* 2.5 3.5 4" 0.01 1* 2.7 2.5 ns ' 2* 2.5 2.4 2* 11s 3' 4.2 4.0 :1* ns 4* 2.8 2.6 j* 0.0 i i * 3.1 2.3 ' 1 * n.: 2" 3.0 2.2 2* ns 3* 3.6 2.6 3" 0.05 4* ns 4* 3.1 2.2 2.1 i * ns i * 3.6 2* 3.3 1.9 2* n s 3* 4.1 2.4 3* 0.02 4* ns 4" 3.0 1.8 ~ ~ _ _ _ __ .. ~~~ c 7 t 1 1 1 2 2 3 1 4 2 c 7 t 2 1 1 2 2 3 1 4 2 c 7 t 3 1 1 2 2 3 1 4 2 c 7 7 4 1 2 3 4 c 7 t 5 1 2 3 4 total 1 * lntratestcr 2* intratester 3* intertester 4* intertester repeatability tester 1 10.60 4.8 2.8 3. i n b repeatability tester 2 10.40 4.8 2.6 2.9 11s repeatability trial 1 10.51 4.8 3.4 3.8 0.01 repeatability trial 2 10.49 4.8 2.6 2.8 0.01 _ _ _ _ relative,flexion mobiliry. relative flexion mobility expressed as ctr% is based on two values of absolute flexion mobility. this makes relative values afflicted with a somewhat greater measuring error. the intratester repeatability showed no systematic errors between the first and second trial either for tester 1 or tester 2 when relative flexion mobility was evaluated for all motion segments. the sign test did not show any significant differences between trials for the two testers (table 4, 1 * and 2*). theintertesrclrrepeatability, comparing tester 1 with tester 2 , showed a significant systematic measuring error between testers i n both trials for motion segment c7-t 1 and t4 ts (table 4 , 3 * and 4*). the systematic measuring error in absolute flexion mobility between testers resulted i n a calculated ctr value which in average is 0.7% lower for motion segment c7-tl and 0.4% higher for motion segment t4 t5 for tester 2 compared with tester 1. the random errors expressed as measuring precision (%) and as relative measuring error cv showed that repeatability was high for intratester repeatability and fair for intertester repeatability (table 4, 1" and 4*). in general the precision is higher in motion segments c7-t1 andt4 'i's arid so is also the cv compared with motion segments i n between. the cv for intratester repeatability ranged from 2.9-3.9%; for tester 1 and from 2.9 4.4% for tester 2 for the different motion segments (table 4, 1 * and 2*) the cv for intertester repeatability ranged from 5.47.7% for trial 1 and from 3.2-7.7% for trial 2 for the different motion segments (table 4, 3" and 4"). table 4 resultsfromevaluationofrc~catabilitvofrelativeflexionmobilit\;(n=26), i * m d 2 * =intriltcster,3* and 4* = menester repeatability i relative flexion 1 random error3 motion mobilitj ( c i r % ) measuring measuring ~ segment trial tester x s d c 7 1 ' 1 1 1 2 2 3 1 4 2 1'1 -1'2 1 1 ~~~ 2 2 3 1 4 2 t 2 t 3 1 1 2 2 3 1 2 2 3 1 2 2 3 i 4 2 2 i .x 1.2 21.2 1 .0 22.1 o.y 21.3 1.1 20.0 0.9 20.4 1.3 20.0 0.8 20.0 1.4 19.3 1 .o 19.8 i .3 19.6 1 .o 19.6 1.7 19.7 1.6 19.4 1.5 19.5 1 .s 19.3 1.3 19.3 1.7 19.3 1 .0 18.8 1.4 19.6 1 .0 total 1 * intratester 2" intratester '3* intertester 4* intertester repeatability tester 1 repeatability tester 2 repeatability trial 1 repeatability trial 2 20.0 1.5 20.0 i .3 20.0 1.2 20.0 1.3 precision ('70) error ( c v 7 o ) i * 0.9 39 2' 0.8 3.5 3* 1.3 6.3 4* 0.7 3.2 i* 0.6 3.1 2* 0.9 4.4 3* 1.1 5.4 4* 1.3 6.4 1 * 0.6 2.9 2* 0.8 3.8 3* 1 . 1 6.0 4) 1.5 7.7 i * 0.7 3.1 2* 0.7 3.1 3* 1.5 1.7 4* 1.3 6.8 i * 0.7 3.7 2* 0 6 2.9 3* 1 4 7 0 4* i 0 5 1 0.7 3.5 0 . x 3.7 1.3 6.5 1.2 5.9 systematic errors sign test l x n b 2" ns 3 , 0.02 4* 0.01 i* ns 2* 11s 3* ns 4* ns i * 11s 2* ns 1* ns 4" ns 1 * ns 2* 11s ?* ns 4* ns i* ns 2* ns 3* ns 4* 0.02 * ~~ ~ ~~ ~~~~ ~ ns ns 0.01 0.01 157 the breakdown with one way anova analysis, describing the relationship between repeated trials for relative flexion mobility, showed a very high degree of conformity. intratester repeatability showed a r 2=0.79 (p<o.001) fortester 1 andar2 =0.68 (p<0.001) fortester 2. intertesterrepeatability showed a r2= 0.38 (p<0.001) for trial 1 and a 1-2= 0.58 (p< 0.001) for trial 2. not in any motion segment did the variation of repeatability significantly exceed the limits for what is defined as ordinary mobility i n the classification model, described by (7), (fig. 3). mobility profile motion segment ~~ figure 3 the resuits or the iiiuaand inrerterrer repcatability evaluation shown in thc mobility prof1 lz. the variation betwccn trials and testers is kepi w i t h i n the limils for ordinary mobility. discussion the results of this study have shown that the distribution of segmental flexion mobility i n the cervico dorsal region can be examined with the ctr-technique. the validity of skin distraction measure ments in the cervico-dorsal spine was found to be individually very high. in this study it was necessary to use conventional x-ray technique to evaluate the relationship between skin distraction and vertebral motion. it was also necessary to choose the vertebral endplates to study the alteration of vertebral angles since the spinous processes could not be clearly identified in this difficult region. great efforts were made tooptimize the technique. the number of subjects was limited to seven, since the relationship between individual vertebral flexion mobility and skin distraction was found to be convincingly high for our application. the aim with the ctr-technique was assessinent of joint flexion mobility, from a clinical point of view. the aim was not to decide the exact vertebral angle i n degrees. further more the method has shown an important clinical application as an instrument to identify dysfunction in the cervico-thoracic junction correlating to neck-shoulder pain (8). the evaluation of validity was done on patients suffering from neck-shoulder pain and the evaluation of repeatability was done on healthy subjects. this was not a problem, since clinical trial has shown that 158 the ctr-method can distinguish between different flexion mobility in individuals with and without increased risk for neck-shoulder pain (8). the intratester repeatability demonstrated high repeatability and was not impaired by any systematic measuring error.the intertester repeatability demonstrated fair repeatability for both absolute and relative flexion mobility. the measuring procedure was impaired by a sinall but systematically measuring error for both absolute and relative flexion mobility, values between testers showed slightly different characteristics. it was most likely that the marking procedure of the 3 cm interdistant penmarkings contributed to the systematic measuring error. however, for absolute flexion mobility, the relative measuring error must be considered small, only in one trial and for one motion segment did the cv exceed 5 % (table 3). as tester 2 systematically measured a 2-3 mm shorter absolute flexion mobility for all motion segments, the calculation of the ctr resulted in a value which on an average was 0.7% lower for motion segment c7-tl and 0.4% higher for motion segment t4 ts for tester 2 compared with tester 1. this increased cv to 7.7% as the highest for relative flexion mobility. this variation in intertester repeatability for relative flexion mobility was, however. not greater than i t was comprised within the limits for what was defined as ordinary mobility in the classification model described by (7). consequently the systematic measuring error for intertesterrepeatability was less than what was defined as ordinary variation between individuals. as the ctr was a ratio between two absolute flexion mobility values, it was quite obvious that the relative value was impaired by a somewhat greater measuring error compared with absolute values. if the precision in absolute values could be improved by approximately 1 mm i n each motion segment, t h e cv would decline and nor exceed the 5% limit for relative calculations. this could most likely be achieved by using an instrument with fixed 3 cm interdistances for marking instead of an ordinary tape measure. only a few studies have used the coefficient of variation (cv) of their data to describe repeatability and to our knowledge nobody has studied measurements in the thoracic spine according to the c l r technique. the present study was therefore difficult to compare with other studies. however, some studies must be commented on. gill et a1 (4) found that the intratester variation was 1.5% for the low backmeasurements using the modified schober technique, with the subject examined i n a f u l l y flexed sittingposture. merittetal(6)founda6.6% intratestervariation with the modified schobertechnique, examined in a fully flexed standing posture. thus, also other skin distraction methods have shown values of coefficient of variation of the same magnitude. references 1. burdett, r . , brown, k.& fall, m.: reliability and validity of four instruments for measuring lumbar spine and pelvic positions. phys ther 66: 677-684, 1986. fint, mm.: lumbar posture: a study of roentgenographic measurements and the influence of flexibility and strength. research quarterly 34: 15-20, 1963. gajdosik, r.& bohannon, r.: clinical measurements of range of motion. phys ther 67: 1867 1872. 1987. 2. 3. 159 4. 5 . 6. 7. 8. 9. 10. gill, k., krag, mh., johnson, cb., haugh, ld.& pope, mh.: repeatability of four clinical methods for assessment of lumbar spinal motion. spine 13: 50-53, 1988. kapandji, ia.: the physiology of the joints. volume three. the trunk and the vertebral column. churchill livingstone pp 38-39, 1970. meritt, jl., mclean, tj., erickson, rp.& offord, kp.: measurement of trunk tlexibility in normal subjects. reproducibility of three clinical methods. mayo clin 61: 192-197, 1986. norlander, s., aste-norlander, u . , nordgren, b.& sahlstedt, b.: a clinical method for measuring segmental flexion mobility i n the cervico-thoracic spine and a model for classifica tion. the cervico-thoracic ratio, (in press), scand j rehab med. 1995. norlander, s., aste-norlander, u., nordgren, b.& sahlstedt, b.: mobility in the cervico thoracic junction an indicative factor i n neck-shoulder pain. a cross-sectional study, (submit ted), 1995. troup, jdg., hood, ca.& chapman,ae.: measurernentsof the sagittal mobility of the l u m b a r spine and hips. ann phys med 9: 308-321, 1968. stokes, iaf., bevins, tm.& limn, ra.: back surface cutwture and measur-enienr of lumbar spinal motion. spine 12: 355-366, 1987. address for offprints: staffan norlander bygghalsans forskningsstiftelse svardvagen 25 a s-182 33 danderyd, sweden 160 (5) gunnar sedin 45-60 upsala j med sci 111 (1): 45–60, 2006 water and heat – the priority for the newborn infant gunnar sedin, johan ågren department of women’s and children’s health, uppsala university children’s, hospital, perinatal research laboratory, 751 85 uppsala, sweden abstract free full-text copy of this article can be found at the web page of upsala j med sci:http://www.ujms.se. maintaining fluid and heat balance is of vital importance to the newborn infant. at birth, the infant is exposed to a cold and dry environment, and preterm neonates in particular, are then at risk of dehydration and hypothermia. these conditions may have serious consequences and significantly influence mortality and morbidity. preterm neonates have a high rate of water and heat loss mainly as a consequence of their immature skin. the care environment influences the magnitude of water and heat exchange and needs to be individually tailored on the basis of the infant’s clinical status, maturity at birth and postnatal age. this paper reviews data obtained from series of studies on neonatal water and heat exchange using non-invasive measurements of insensible water loss and calculations of different modes of heat exchange. these studies have influenced the way in which newborn infants are being nursed today. introduction long before the emergence of the field of perinatal medicine, humans kept their newborns warm by nursing them close to their own skin covered with clothing or blankets, just as animals in a litter huddle together to keep warm. at the time of nils rosén von rosenstein (1706-1773), promotion of breastfeeding was in focus, and this included keeping both the breasts and the infant warm (1, 2). from the time of rosén, the infant mortality decreased gradually in sweden (3). more than 100 years later (1880), the first known couveuse or incubator to keep an infant warm was constructed by tarnier and used by his co-workers pierre budin and alfred auvard at the paris maternity hospital. this couveuse was heated by letting a stream of air 45 received 17 october 2005 accepted 28 october 2005 pass over a number of containers of warm water and pass through a wet sponge, which might have served as a humidifier, but pierre budin never mentioned anything about humidification in his original text (4). during a long period of great improvements in health care in the 1900s (3), with a gradual decline in infant mortality, also in infants born preterm, new challenges concerning neonatal fluid balance and treatment of neonatal lung disorders appeared. increased knowledge of newly recognised aspects of lung physiology (5, 6) formed the basis of improvements in assisted ventilation of the newborn infant, resulting in fewer severe complications. during 1971, the introduction of intermittent mandatory ventilation (7), continuous positive airway pressure (8) and high frequency positive pressure ventilation (9) contributed to better respiratory support for both the immature and mature newborn infant. to investigate differences in water exchange between the infant and the environment, hey and katz (10) studied insensible water loss (iwl) both in fullterm infants and infants with a low birth weight, but their technique did not allow studies of the most preterm infants. in another investigation of insensible weight loss, fanaroff et al (11) showed that this loss was very high in infants with a birth weight less than 1250 gram. there was no explanation, however, for the marked weight loss in the low birth weight infants. studies published in 1956 by bent friis-hansen (12) on body water compartments during growth showed that the newborn term infant has a water content of 77%. the relative total body water at birth is even higher in preterm infants than in term infants, being 82% at 32 weeks of gestation, 84% at 28 weeks and 86% at 24 weeks. this fact, together with the observation of the shiny, wet skin of a very preterm infant nursed in an incubator, gave us the idea to study the transepidermal water loss (tewl) in infants of different degrees of maturity (13-15). a series of studies was undertaken to delineate the relation between tewl and gestational age at birth, postnatal age and environmental conditions, and to clarify the importance of this water loss for neonatal water and heat balance. to achieve more accurate estimates of water and heat exchange, studies of respiratory water loss were also included in the project. these studies were made possible by the development of a novel technique. with the gradient method (13-15), transepidermal water loss (g/m2/h) could then be estimated by measuring the evaporation rate (er g/m2/h) from the chest, an interscapular skin area and a buttock. tewl at birth and at different postnatal ages there is an exponential relationship between gestational age and tewl at the first day after birth. infants born at 25 weeks of gestation have tewl values up to 15 times higher than those in term infants (16) (fig. 1). the relationship between tewl and gestational age at different postnatal ages during the first four weeks 46 after birth can be described with exponential equations (17, 18) (fig. 2). in the most preterm infants tewl gradually decreases and the difference in tewl between the most preterm and the term appropriate for gestational age (aga) infant dimin47 figure 1. transepidermal water loss (tewl) in relation to gestational age (g.a.). aga = appropriate for gestational age. w = completed weeks of gestation. (from ref 16) ishes with age, but four weeks after birth tewl is still twice as high in the former as in the latter (18) (fig. 2). among small for gestational age infants, tewl is again highest in the most preterm infants, but their water loss from the skin is lower than that in aga infants (19). evaporative water loss in relation to humidity there is an inverse linear relationship between evaporation rate (er) from the skin surface and ambient humidity in both term and preterm infants at different postnatal ages. the evaporation rate is much higher at a low than at a high relative humidity (15, 16, 19) (fig. 3). 48 figure 2. transepidermal water loss in relation to gestational age at birth at different postnatal ages in appropriate-for-gestational-age infants. (from ref 18) 49 insensible water loss from the skin (iwl s ) iwl s in g/kg/day in infants can be calculated from tewl, body surface area and weight (fig. 4). since tewl is strongly related to postnatal age and since the ratio of body surface area to body weight is higher in the most preterm infants, it is obvious that iwl s is considerably higher in preterm than in term infants. accordingly, the environmental conditions (i.e. ambient humidity) have an even greater impact on the iwl s in the tiniest infants, particularly during the first week after birth (19). figure 3. the relation between evaporation rate and ambient relative humidity in preterm newborn infants of different gestational age groups. (from ref 16) tewl during activity and phototherapy there is a pronounced increase in tewl when infants born at term increase their motor activity, but no sweating is observed. if infants are nursed in a warm environment, the body temperature rises and tewl increases markedly when they begin to sweat (20). in infants in thermal balance there is no indication that phototherapy increases the transepidermal water loss (21), respiratory loss or oxygen consumption (22). previous observations of higher losses during phototherapy (23) might have been due to heat stress and not to the radiation from the phototherapy equipment (21, 22). tewl and radiant heaters when very preterm infants are nursed first in an incubator at rh amb of 50%, the evaporation rate from the skin is significantly lower than when the same infants are nursed at a lower humidity under a radiant heater. still, the evaporation rate is lower under the radiant heater than what can be expected from the environmental vapour pressure. as can be seen in figure 5, the measured water loss is lower than the expected water loss at the vapour pressure in the environment (24). thus there is no indication that radiant heat increases water loss from the skin under conditions with a low air velocity close to the infant. however, losses of water and heat from the 50 figure 4. insensible water loss from the skin (iwls) in relation to postnatal age in infants born at a gestational age of 25-27 weeks. data are given for three different ambient humidities. (from ref 28) skin surface can be increased if the normal gradient close to the skin surface is disturbed by movements of air in the nursery created by air condition systems and staff movements, for example. respiratory water loss a flow-through system for measurement of respiratory water loss (rwl), providing data on oxygen consumption and carbon dioxide production, was developed. the system uses a mass spectrometer, specially equipped with a water channel for analyses of gas concentrations (25). the studies showed that rwl in newborn term infants at rest is 4.9 mg/kg/min, i.e. about 50% of the total insensible water loss at an ambient air temperature of 32.5°c and an ambient humidity of 50% (26). rwl depends on the temperature and humidity of the inspired air and on the respiratory rate, tidal volume, dead space ventilation and the ability of the nose to dehumidify and cool expired air (25, 26). it was also found that there was an inverse relationship between rwl and ambient humidity, with higher losses at a low than at a high humidity (26) and that the insensible water loss from the respiratory tract (iwl r ) and the iwl s were of the same magnitude at an ambient humidity of 20 %, whereas iwl s was much lower than iwl r at a high ambient humidity. rwl increases with increasing activity in term infants. at rest both iwl s and iwlr 51 figure 5. the calculated (open symbols) and measured (filled symbols) values for er (g/m2/h) during care under a radiant heater, together with the corresponding regression lines for er on ph 2 o at the air temperatures of the incubator and the radiant heater, in three infants (from ref 24) were 6 g/kg/day and iwls increased very little, whereas iwl r could theoretically increase to 16 g/kg/day if the infant was able to have the highest level of activity for 24 hours. vo2 and vco2 also increased during activity (27) (fig. 6). water supply and water balance nutrition and/or water supplementation should be provided soon after birth. in term infants, water balance can be adjusted by increasing or decreasing the daily prescribed supply. in preterm infants, especially in those who are extremely preterm, who have very high insensible water losses, sometimes leading to hyperosmolality, high levels of water loss have to be avoided. the supply of fluid depends on the insensible water loss and can be kept low in a humid environment. the local guidelines for neonatal water balance are then: day 1: 65 ml/kg day 2: 75 ml/kg day 3: 90 ml/kg days 4-7: increase gradually up to 150 ml/kg. the volume of all fluid given or collected should be noted. the basic prescription of fluid volumes may be changed if the serum osmolality 52 figure 6. respiratory water loss (rwl) at different levels of activity. activity scale: 0 = asleep, no motor activity. 1 = asleep, minor motor activity. 2 = awake, quiet, no motor actitivy. 3 = awake, quiet, active. 4 = awake, crying, active. 5 = awake, furiously crying. (from ref 27) exceeds 300 mosm/l and when weight loss exceeds 10-15%. even in very preterm infants nursed at a high ambient humidity, there is usually an increase in serum osmolality during the first 3 days after birth, accompanied by an increase in sodium concentration (28) (fig. 7) and in such cases the body weight is carefully monitored so that it does not drop more than 15%. 53 figure 7. (a) mean serum osmolality (s-osm ±sd) and (b) mean serum sodium concentration (s-na ±sd) in 11 infants born at a gestional age of 24 weeks, in relation to postnatal age. (from ref 28) water supply and water balance in preterm infants born at 24 and 25 weeks of gestation, tewl during the first week after birth was slightly higher than that found previously in the group of infants born at 25-27 weeks (29). importantly, in infants born at 24-25 weeks and nursed at a high ambient rh, tewl at a postnatal age of 4 weeks was twice as high as had previously been reported for slightly more mature infants. these findings led to a speculation as to whether maintaining infants at a high humidity for a long time might influence the development of their skin barrier. recently, we studied a cohort of very preterm infants born at a mean gestational age of 25 (range 23-27) weeks and nursed at a high humidity (85%) during the first week after birth. at a postnatal age of one week the infants were randomized to continued care at either 75% or 50% rh for the following three weeks, but studied at the same humidity. preliminary results from this study indicate that infants nursed at the lower level of humidity during the second, third, and fourth postnatal week have a lower tewl than those exposed to the higher humidity. the results imply that the environmental humidity can significantly alter the process of postnatal skin barrier formation (30). these data need further assessment before publication but may lead to changes in the way preterm infants are nursed at our institution. mechanisms for transepidermal water transport the high loss of water from the skin surface reflects one aspect of immature organ function in preterm infants. the epidermis is not fully developed in these infants and its outermost layer, the stratum corneum, is thin and lacks the structure and composition needed to provide its barrier function properties. in addition, recent studies on perinatal rat skin have shown that aquaporins (aqp), a group of integral membrane proteins with high water permeability, are abundantly expressed in the immature skin (31, 32). in this experimental model, higher expression of aqp3 in the basal parts of the epidermis was observed in immature than in more mature rat pups, and tewl was much higher in the preterm pups. if these results are valid for the human preterm neonate, further studies on epidermal aquaporins might be of importance for human perinatal physiology, and interventions aimed at aqp inhibition might prove of clinical value. heat and water balance in extremely preterm infants early after birth when extremely immature newborn infants are exposed to a cold, dry environment after birth, they are at risk of having a rapid drop in body temperature unless measures are taken to prevent heat loss (33, 34). furthermore, the extremely preterm infants have only a limited capacity to produce heat. it is therefore of great importance to create a care environment that favours thermal balance in this situation (35). at birth the exchange of heat depends very much on the maturity of the infant, the environmental temperature and relative humidity and the postnatal age, as displayed in figures 8, 9, and 10. different methods to maintain heat have been proposed such as the use of semipermeable membranes to lower the water and heat loss in infants nursed under a radiant heater (36-38). in a short time early after birth a semipermeable membrane, covering the infant, 54 55 figure 8. ambient temperature (tamb) in relation to gestational age at ambient relative humidity of 20%, 40%, and 60%. (from ref 33, 34) figure 9. heat exchange through evaporation, radiation, and convection in relation to gestational age at relative ambient humidities of 20%, 40%, and 60%. (from ref 35) 56 figure 10. heat exchange between the infant and the environment in relation to postnatal age at relative ambient humidity (rh) of 20%, 40%, and 60% in infants born at 25 to 27 weeks of gestation. (from ref 34, 35) can create a humid environment close to the infant’s skin and may lower the evaporative water and heat exchange during this period. an evaporative water loss from the skin will take place and contribute to a high humidity in the air between a membrane and the skin, which can lower evaporative heat loss temporarily (16). this type of care has not been evaluated concerning the fractional contribution of different modes of heat exchange. if the membrane is in direct contact with the skin, a component of heat conduction between the membrane and the skin may take place. all areas of the infant’s skin that are not covered by a membrane will be exposed to room air temperature, air movements, evaporation and the colder walls of the neonatal intensive care unit. this will substantially alter the conditions for the heat exchange. losses through convection can be considerably increased by air movements at low temperature in the vicinity of the very preterm infant (16, 34). these circumstances have further been discussed in a recent publication (39). very preterm infants who are held skin-to-skin by the mother early after birth and are covered with a blanket, can often maintain a normal body temperature in the first week after life if the gestational age at birth is 28-30 weeks (40). it has also been shown that very preterm infants nursed on a heated water-filled mattress and covered with a blanket can maintain their body heat (41). use of a cap on the infant’s head was proposed many years ago by stothers (42) and may still be a way to save heat for the very preterm newborn infant. acknowledgements these studies were supported by grants from the swedish research council, hrh the crown princess lovisa’s fund for scientific research and the åke wiberg foundation. references 1. rosén von rosenstein n (1764) underrättelser om barnsjukdomar och deras botemedel. edition: the swedish academy of science, stockholm. 2. rosén von rosenstein n (1776) the diseases of children and their remedies. edition: cadell t, in the strand, london. 3. the swedish board of health and welfare. national statistics. infant mortality rate 17512004. 4. budin p (1900) edition: le nourrison, paris: dion. 5. comroe jh, forster re, dobois ab, briscoe wa, carlsen e (1955). the lung. clinical physiology and pulmonary function tests. edition: year book medical publishers. chicago. 6. peters rm (1969). the mechanical basis of respiration. an approach to respiratory pathophysiology. edition: j & a churchill ltd, london. 7. kirby rr, robison ej, schulz j, delemos r (1971) a new pediatric volume ventilator. anesth analg 50:533-537. 8. gregory ga, kitterman ja, phibbs rh, tooley wh, hamilton wk (1971) treatment of irds with continuous positive airway pressure. new engl j med 284:1333-1340. 9. jonzon a, öberg på, sedin g, sjöstrand u (1971) high-frequency positive-pressure ventilation by endotracheal insufflation. acta anaest scand: suppl 43. 10. hey en, katz g (1969) 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iii. relation to gestational age. acta paediatr scand 68:795-801. 17. hammarlund k, sedin g strömberg b (1982) transepidermal water loss in newborn infants. vii. relation to post-natal age in very pre-term and full-term appropriate for gestational age infants. acta paediatr scand 71:369-374. 18. hammarlund k, sedin g, strömberg b (1983) transepidermal water loss in newborn infants. viii. relation to gestational age and post-natal age in appropriate and small for gestational age infants. acta paediatr scand. 72:721-729. 19. sedin g, hammarlund k, nilsson ge, strömberg b, öberg på (1985) measurements of transepidermal water loss in newborn infants. clin perinatol 12:79-99. 20. hammarlund k, nilsson g, öberg på, sedin g (1979) transepidermal water loss in newborn infants. ii. relation to activity and body temperature. acta paediatr scand 68: 371-376. 21. kjartansson s, hammarlund k, sedin g (1992) insensible water loss from the skin during phototherapy in term and preterm infants. acta 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extremely preterm infant. in: hansen tn, mcintosh n (eds), current topics in neonatology, w b saunders company, london, pp 50-66. 29. ågren j, sjörs g, sedin g (1998) transepidermal water loss in infants born at 24 and 25 weeks of gestation. acta paediatr 87:1185-1190. 30. ågren j, sjörs g and sedin g (2005) ambient humidity influences skin maturation in extremely preterm infants. pediatr res 57:5 (abstract). 31. ågren j, zelenin s, håkansson m, eklöf a-c, aperia a, nejsum l, nielsen s,sedin g (2003) transepidermal water loss in developing rats: role of aquaporins in the immature skin. pediatr res 53:558-565. 32. ågren j (2003) water transport through perinatal skin. barrier function and aquaporin water channels. acta universitatis upsaliensis. comprehensive summaries of uppsala dissertations from the faculty of medicine 1249. 48 pp. uppsala, sweden. 33. hammarlund k, sedin g (1982) transepidermal water loss in newborn infants. vi. heat exchange with the environment in relation to gestational age. acta paediatr scand 71:191-196. 34. sedin g (2004) physics and physiology of human neonatal incubation. in: polin r, fox w (eds), fetal and neonatal physiology. w b saunders company, philadelphia, pp 570-582. 35. hammarlund k, strömberg b, sedin g (1986) heat loss from the skin of preterm and fullterm newborn infants during the first weeks after birth. biol neonat 50:1-10. 58 36. bell ef, weinstein mr, oh w (1980) heat balance in premature infants: comparative effects of convectively heated incubator and radiant warmer, with and without plastic heat shield. j pediatr 96:460-465. 37. baumgart s, engle wd, fox ww, polin ra (1981) effect of heat shielding on convective and evaporative heat losses and on radiant heat transfer in the premature infant. j pediatr 99:948-956. 38. baumgart s (1984) reduction of oxygen consumption, insensible water loss, and radiant heat demand with use of a plastic blanket for low-birth-weight infants under radiant warmers. pediatrics 74:1022-1028. 39. sedin g (2004) to avoid heat loss in very preterm infants. editorial on 2004308 heat loss prevention (help) in the delivery room: a randomized controlled trial of polyethylene occlusive skin wrapping in very preterm infants. j pediatr 145:720-722. 40. bauer k, pyper a, sperling p, uhrig c, versmold h (1998) effects of gestational and postnatal age on body temperature, oxygen consumption, and activity during early skin-to-skin contact between preterm infants of 25-30-week gestation and their mothers. pediatr res 44:247-251. 41. sarman i, bolin d, holmer i, tunell r (1992) assessment of thermal conditions in neonatal care: use of a manikin of premature baby size. am j perinat 9:239-246. 42. stothers jk (1981) head insulation and heat loss in the newborn. arch dis child 56:530-534. corresponding author: gunnar sedin department of women’s and children’s health, uppsala university children’s hospital, perinatal research laboratory se-751 85 uppsala, sweden phone: 46-18-6115886, fax +46-18-554079 gunnar.sedin@kbh.uu.se 59 60 untitled over-expression of decoy receptor 297 received 25 may 2008 accepted 14 august 2008 upsala j med sci 113 (3): 297–304, 2008 over-expression of decoy receptor 3 in gastric precancerous lesions and carcinoma gang chen, dianzhong luo department of pathology, guangxi medical university, nanning 530021 people’s republic of china abstract introduction: decoy receptor 3 (dcr3), a newly discovered member of the tumor necrosis factor receptor (tnfr) superfamily, is not only upregulated in cancer cells derived from various cell lineages, but also correlates with the overall survival of certain cancer patients. the objective of the present study was to investigate the expression of dcr3 protein in tissue of gastric precancerous lesions and carcinoma. material and methods: the expression of dcr3 protein in tissue of gastric carcinoma (gc, n=79), dysplasia (n=45), intestinal metaplasia (im, n=37) and chronic superficial gastritis (csg, n=42) was investigated by immunohistochemistry. results: expression of dcr3 in gc was significantly higher than that in dysplasia (p<.05); im (p<.05) and csg tissue (p<.001), respectively. it was also found that dcr3 expression in well differentiated gc was significantly lower than that in poorly differentiated specimens (p<.05). moreover, patients in tumor-node-metastasis (tnm) stages i and ii showed significantly lower dcr3 expression compared with that in stages iii and iv ( p<.05). in addition, dcr3 expression in both lymph node metastasis-negative patients and patients without systemic metastasis was significantly decreased in comparison with that in lymph node metastasis-positive patients(p<.05) and patients with systemic metastasis (p<.001). conclusions: dcr3 is over-expressed in human gc and positively correlated with development and metastases of gastric lesions. the dcr3 gene might serve as an important molecular biological indicator in diagnosing and predicating the clinical outcome in gc patients. introduction decoy receptor 3 (dcr3)/tr6/m68 is a soluble decoy receptor in the tumor necrosis factor receptor (tnfr) superfamily, which involves 4 members: dcr1, dcr2, dcr3 and osteoprotegerin (1,2,3). gene amplification as well as over-expression of dcr3 messenger rna (mrna) and protein have been demonstrated in lymphoma, glioblastoma and also in cancer of the lung, brain, pancreas, liver, esophagus, colon and gastrointestinal tract (2,4,5,6,7,8,9,10). more importantly, the dcr3 expression level was associated with lymph node metastasis and pathological sections in gastric carcinomas (9).the serum level of dcr3 was also significantly elevated in a variety of tumors, including cancers of the digestive system, thyroid, lung, breast and ovary (11, 12). all the evidence suggests that dcr3 can not only become a factor responsible for the progression and immune suppression of tumor cells, but also serve as an effecter molecule to modulate pathological and physiological functions. 298 gang chen, dianzhong luo gastric carcinoma (gc) is one of the most common malignant diseases all over the whole world. gastric carcinogenesis is considered as a multistage progressive process. the early indicator for the subject predisposed to gc is abnormal proliferation of gastric epithelial cells, so chronic atrophic gastritis (cag), dysplasia and intestinal metaplasia (im) are considered as key precancerous lesions with strong facilitation to the development of gc (13,14,15,16). however, the pathogenic mechanisms of this process remain unclear. takahama et al (9) evaluated dcr3 expression in gastric cancer by northern blot and in situ hybridization, but, to date no studies have been carried out of its associations with precancerous gastric lesions, namely, in im and gastric dysplasia by immunohistochemistry (ihc). there has been little information available about the protein status of dcr3 in stomach mucosa collected by gastroscopy or gastrectomy. no detailed study comparing the expression patterns of dcr3 in gastric carcinoma with that in precancerous tissue has been available. furthermore, whether over-expression of dcr3 is involved in the initiation or promotion of gc has not been established. therefore, the aim of the present study was to investigate the expression of dcr3 in chronic gastritis, precancerous and cancerous gastric lesions by ihc analysis. materials and methods from our surgical pathology files, csg (n=42), im (n=37), dysplasia (n=45) and gc (n=79) gastrectomy or gastroscopy specimens were selected in this retrospective study. all cases were randomly chosen in the first affiliated hospital, guangxi medical university, people’s republic of china between may 2001 and december 2005. written informed consent to use the samples for research was obtained from the patients and clinicians. all the guidelines for experimental investigation with human subjects required by the institution were followed. histopathological diagnosis for gastric epithelia was made according to the cellular morphological changes and tissue architecture using previously established criteria (17,18). in brief, csg was characterized by an inflammation manifested by mild lymphocyte and plasma cell infiltration; im, confirmed by the presence of goblet cells in gastric mucosa; dysplasia, characterized by nuclear atypia with or without architectural abnormalities in the gastric epithelium without invasion; ultimately, gc is characterized by invasion of neoplastic gastric cells through the basement membrane. carcinomas were classified according to the histological classification of world health organization (who) criteria (19). of 79 gcs, 51 and 28 were histologically graded as well differentiated and poorly differentiated, respectively. all of the 79 gcs in the present study were intestinal-type carcinomas, because in the multistep process of intestinal-type carcinogenesis, the genetic pathway can be divided into three subpathways: an intestinal metaplasia→adenoma→carcinoma sequence, an intestinal metaplasia→carcinoma sequence and de novo (12). the clinical tumor-node-metastasis (tnm) stage was processed according to the pathology tnm (ptnm) classification of who criteria (20). of 79 gcs, 12 were over-expression of decoy receptor 299 stage i, 15 stage ii, 20 stage iii and 23 stage iv.the patients were 123 men and 80 women whose ages ranged from 20 to 82 years (mean 47±12 years). one or 2 of the most representative sections from each case were selected and stained with a polyclonal antibody against dcr3 (dcr3<h-130>:sc-25464) from santa cruz biotechnology®,inc.. dcr3<h-130>:sc-25464 is a rabbit polyclonal antibody raised against amino acids 171–300 of dcr3 of human origin and 1:300 dilution was used. the 79 gc patients had never received any radiation therapy or chemotherapy. the procedure of ihc was done as described previously (7). one hundred cells from 5 representative areas in each lesion were counted. the staining results were evaluated according to the immunodetection of stain intensity and numbers of positive cells by two pathologists (g.c. and dz.l.), who discussed each case until they reached a consensus. stain intensity was up to the standard of the relative stain intensity of most cells. the degree of staining was subdivided as follows: the stain intensity could be from 0 to 3 (0, no staining; 1, focal or fine granular, weak staining; 2, linear or cluster, strong staining; and 3, diffuse, intense staining); and the positive cells in the observed gastric mucosa ranged from 0 to 3 in percentage (0, no staining; 1, < 30%; 2, 30%-70%; and 3 > 70%). the samples were scored by their summation: 0–1 (-); 2–3 (+); 4 (++); 5–6 (+++). any staining score ≥ 2 (+) was considered as positive expression (21). the fisher exact test was used to compare the expression of dcr3 among different groups with spss 13.0 software for windows (munich, germany). a p value of less than .05 was considered statistically significant. multivariate analysis was used for identifying correlation of dcr3 over-expression with patients’ age, gender, clinical and pathologic parameters. a p value of less than .05 (for 1 side) or less than .025 (for 2 sides) was considered statistically significant. results dcr3 expression in different gastric tissue positive immunostaining for dcr3 was observed in the cytoplasm of gastric epithelial cells and cancer cells with different rates in different types of lesions (image 1a, image 1b, image 1c, image 1d). focal dcr3 over-expression was observed in 2 of 42 cases of csg mucosa. it was slightly increased in the im and dysplasia, and significantly increased in gc. over-expression of dcr3 was more often observed in gc than in dysplasia (p<.05), im (p<.05) and csg (p<.001, respectively). however, there was no difference in dcr3 expression when comparing dysplasia, im and csg (table 1). correlation of dcr3 over-expression with gc differentiation dcr3 over-expression was more often observed in poorly differentiated gc than in well differentiated gc (p<.05, table 2). 300 gang chen, dianzhong luo correlation of dcr3 over-expression with clinical stages in addition to its correlation with the grade of differentiation, over-expression of dcr3 also tended to correlate with the clinical stage of gc. dcr3 expression in the clinical tnm stage i and ii was significantly lower than that in stages iii and iv (p<.05, table 2). correlation of dcr3 over-expression with metastasis dcr3 expression in the lymph node metastasis-negative patients was significantly lower than in patients with lymph node metastasis (p<.05). moreover, the rate of dcr3 expression in patients without systemic metastasis was significantly lower than that in the group with systemic metastasis (p<.05, table 2). correlation of dcr3 over-expression with other clinopathologic parameters multivariate analysis revealed no relationship between dcr3 expression in gc and patients’ age, sex or the tumor invasive depth (data not shown). a b c d figure 1. over-expression of decoy receptor 3 (dcr3) in gastric carcinoma. a and b, chronic superficial gastritis (csg) (a, dcr3 negative; b, dcr3 positive, ×400). c and d, gastric carcinoma (gc) (c, dcr3 negative; d, dcr3 positive, ×400). over-expression of decoy receptor 301 table 1. over-expression of decoy receptor 3 (dcr3) in different types of gastric tissue* p compared with dcr3 intestinal metaplasia dysplasia gastric carcinoma csg 2/42(4.86) >.050 >.050 <.0001 im 4/37(10.81) – >.050 <.050 dysplasia 7/45(15.56) – – <.050 gc 27/79(34.18) – – – * data are given as number scored 2+/total tested (percentage); 2+ indicates linear or cluster, strong staining. table 2. correlation between the expression of decoy receptor 3 (dcr3) and different clinicopathological parameters* clinicopathological parameters dcr3 p differentiation poorly-differentiated 14/28(50) <.050 well-differentiated 13/51(25.49) clinical tnm stages i/ii 8/37(21.62) <.050 iii/iv 19/42(45.24) nodal status negative 4/29(13.79) <.050 positive 23/50(46) metastatic status negative 9/55(16.36) <.0001 positive 18/24(75) *data are given as number scored 2+/total tested (percentage); 2+ indicates linear or cluster, strong staining. discussion gastric carcinogenesis is considered as a multistage and progressive process and an early indicator for a patient predisposed to gc is abnormal proliferation of gastric epithelial cells, such as dysplasia and im, which have both been considered as precancerous lesions for gc (13, 14, 15, 16). however, information about the mechanism of gastric carcinogenesis is very limited. studies of dcr3 protein expression levels at different stages of gastric carcinogenesis may help answer why different stages of cancerous development occur. human dcr3, mostly being expressed in many different classes of tumor cells, can combine with the tnf family members fasl (2), light (4, 22) and tl1a (2), 302 gang chen, dianzhong luo thus to block their interaction with their respective receptors. a study of takahama et al demonstrated that dcr3 mrna was over-expressed in 22 (26%) primary gc in 84 gastric carcinomas compared with each noncancerous tissue by northern blot analysis (9). in this study, we showed that dcr3 had higher expression rate (34.18% vs 26%) in gc compared with the csg and precancerous lesions. such a variation between previous studies and the present study may depend on different detecting methods (northern blot vs ihc). however, we found that the positive immunostaining rate of dcr3 was abnormally higher in gc than that in dysplasia, im and csg, which is indicative of abnormally high cell proliferation activity in gc tissue. the sequential increase of dcr3 overexpression suggest that indeed dcr3 might be involved in gastric carcinogenesis. the results may also provide a better understanding of the pathogenesis and perhaps permit more accurate diagnosis of gc. it is known that gc can result from the metaplasia→dysplasia→carcinoma sequence, i.e., csg→im→dysplasia→gc. however, the molecular mechanisms in such a process remain unclear. in the present study, the expression of dcr3 was positively correlated with the degree of malignancy of the gastric mucosa and the development of gastric lesions. with the likelihood of malignant lesions progressed from csg→im→dysplasia→gc, the positive immunostaining rates for dcr3 similarly increased, showing a good linear correlation between dcr3 expression and lesion progression. though there was almost identical immunoreactivity for im and dysplasia, the higher expression of dcr3 in gc can still indicate that dcr3 expression might be related to the hyperplastic status of gastric mucosa epithelial cells. these data were consistent with the views of li et al, who suggested that the degree of dysplasia in esophagus tissue was strongly associated with increased levels of dcr3 activity (7). in our study on dcr3 in hepatocellular carcinoma (hcc) tissue, we also found a similar increase of the dcr3 expression. the present results indicate that an increased expression of dcr3 might be an important molecular event involved in the process of gastric carcinogenesis. this conclusion is consistent with that of takahama et al. suggesting that measurement of dcr3 activity might be a valuable clinical marker in the progression of dysplasia and subsequent development of gc (9). another interesting observation was that the expression of dcr3 directly correlated with the differentiation level of gc. the dcr3 immunostaining rate in well differentiated gc was lower than in poorly differentiated gc. this conclusion differed from that of takahama et al, who found no differences between the histological type, differentiation and the dcr3 expression levels (9). we believe that variations in technique, materials and methods may partly explain this difference. however, this result is consistent with the findings in human malignant gliomas, which showed that expression of dcr3 correlated with the grade of malignancy (8). the present results may be taken to indicate that over-expression of dcr3 reflects the differentiation level of the stomach mucosa and the positive correlation between dcr3 over-expression with degree of differentiation may aid in monitoring the progression of gc. over-expression of decoy receptor 303 our finding of a relationship between the dcr3 expression and the clinicopathological parameters is consistent with the study of takahama et al. showing that the positive immunostaining rate of dcr3 was correlated well with gc clinical tnm stages, lymph node metastasis and systemic metastasis (9). when followed up for 63 months, takahama et al (9) found dcr3 over-expression in cancerous tissue that was associated with a significantly shortened duration of overall survival compared with that in patients whose cancerous tissue expressed a normal level of dcr3. so, the high coincidental expression of dcr3 protein accumulation might be an important event to enhance gc and a useful biomarker to assess risk for the development and aggressiveness of gc. acknowledgments we are grateful to geoffrey desmet and ivan colling in belgium for their kind suggestions in the manuscript and figures preparation. the study was supported by the fund of guangxi scientific research and techno-exploitation project (05112001-4d). references 1 ozören n, el-deiry ws. 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(2000).gastric dysplasia: the padova international classification. am j surg pathol. 24: 167–176. 18 wang ld, shi st, zhou q, goldstein s, hong jy, shao p, qiu sl, yang cs. (1994).changes in p53 and cyclin d1 protein levels and cell proliferation in different stages of human esophageal and gastric-cardia carcinogenesis. int j cancer. 59: 514–519. 19 fenoglio-preiser c, muñoz n, carneiro f. (2000).tumors of the stomach. in: hamilton sr, aaltonen la. world health organization classification of tumours, pathology and genetics of tumours of the digestive system. 1st ed. lyon: international agency for research on cancer. 37–38. 20 uicc. (1997). tnm classification of malignant tumors.5ed. new york: wiley-liss. 21 miao xp, li js, li hy, zeng sp, zhao y, zeng jz. (2007).expression of ornithine decarboxylase in precancerous and cancerous gastric lesions. world j gastroenterol. 13: 2867–2871. 22 zhang j, salcedo tw, wan x, ullrich s, hu b, gregorio t, feng p, qi s, chen h, cho yh, li y, moore pa, wu j. (2001).modulation of t-cell responses to alloantigens by tr6/dcr3. j clin invest. 107: 1459–1468. corresponding author: prof. dr. dianzhong luo. phd md department of pathology, guangxi medical university, nanning 530021 people’s republic of china. fax: 0086-771-5358943 email: luodianzhong@yahoo.com.cn ujms 110 (3) bra upsala j med sci 110 (3): 259–266, 2005 lipofibromatosis arising in a pediatric forearm -a case reportdaizo sasaki1, masahito hatori1, masami hosaka1, mika watanabe2, shoichi kokubun1 1department of orthopaedic surgery, tohoku university school of medicine, 1-1 seiryomachi, aobaku, sendai, miyagi 980-8574 2department of pathology, tohoku university school of medicine, 1-1 seiryomachi, aobaku, sendai, miyagi 980-8574 abstract lipofibromatosis is a rare pediatric tumour described by fetsch et al. in 2000. there have been few reports about this tumour and few descriptions of its imaging features. we report a case of this tumour forming a slowly growing, painless mass arising in the forearm of a ten-month-old boy. this is the first report of lipofibromatosis to present the radiological findings before surgery. plain radiography and computed tomography demonstrated a tumour located just beneath the fascia. ultrasonic study showed a hyperechoic tumour of 33 x 30 x 7 mm in size. magnetic resonance imaging demonstrated a multilobular mass with high signal intensities on both t1 / t2 weighted images. macroscopically, the tumour was poorly marginated. microscopically, the tumour was composed of abundant adipose tissues transversed by fibroblastic bands. the adipose tissue occupied over 70% of the tumour. adipocytes and fibroblastic element infiltrated into the normal skeletal muscle tissues. recognition of this clinical entity is needed, especially in cases of lipomatous tumours arising in pediatric hands and feet. introduction lipofibromatosis is a rare pediatric tumour described by fetsch et al. in 2000 [1]. since then, there have been few reports about this tumour and few descriptions of its image findings. we report a case of this tumour occurring in the forearm of an infant together with the radiological and histological findings. 259 received 28 january 2005 accepted 5 april 2005 key words: lipofibromatosis, lipomatous tumour, infant, radiological finding case report a ten-month-old boy was referred to our clinic with a painless and slow-growing tumour in the right forearm. the tumour, 25 x 20 mm in size, had a smooth surface, clear margin, and was soft. initial plain radiogram showed a mass beneath the fascia (fig 1). ultrasonic study showed a hyperechoic tumour of 25 x 22 x 6 mm in size. during the follow-up for five months, the mass increased in size to 33 x 30 x 7 mm. computed tomography (ct) showed a tumour located just beneath the fascia, the density of which was similar to the subcutaneous fat (fig 2). magnetic resonance imaging (mri) revealed a multilobular mass of homogenous high signal intensities on both t1 / t2 weighted images. 260 fig 1. plain radiogram showing soft tissue swelling (arrow). radiological differential diagnosis was intramuscular lipoma, lipoblastoma, and other kind of lipomatous tumours. an attempt was made at marginal excision, but the tumour was found to be not encapsulated and to have continuity with the adjacent tissues. removal of the tumour together with the surrounding normal muscles was performed. grossly, the tumour, 30 x 25 x 10 mm in size, was a mass yellowish in color, soft in consistency, and poorly demarcated, involving the muscle (fig 4). microscopic examination revealed abundant adipose tissue transversed by fibroblastic bands (fig 5a). the fibroblastic component had relatively high cellularity with an immature appearance, no cellular atypia, and no nuclear pleomorphism (fig 5b). the adipose tissue occupied over 70% of the tumour. the adipocytes did not have lipoblastic appearances. univacuolated cells were present between the fibroblastic fascicles and the mature adipocytes (fig 5c). around the margin, adipocytes and fibroblastic 261 fig 2. ct scan demonstrated a tumour just beneath the muscular fascia (arrow). 262 fig 3. mr images. a) t1 weighted image. b) t2 weighted image. the tumour had homogenously high signal intensities on both t1 / t2 weighted images (arrow). fig 3. mr images. a) t1 weighted image. b) t2 weighted image. the tumour had homogenously high signal intensities on both t1 / t2 weighted images (arrow). b a 263 cells infiltrated into the normal skeletal muscle tissue (fig 5b). immunohistochemical study showed that the fibroblastic cell exhibited immunoreactivity for s-100 (focal), cd99, cd34, alpha sma (focal), hhf-35 (focal, weak), bcl-2 (focal, weak). no reactivity was detected for ema, melan a, hmb-45. adipocytes exhibited a strong immunoreactivity for s-100, but negative reactivities for the other antibodies. the rate of positive immunoreactivity for ki67 was about 7-8% in the areas of fibroblastic components. the tumour was diagnosed as lipofibromatosis. the patient has had no local recurrence for one year after the operation. fig 4. gross appearance of the specimen. 264 b a discussion lipofibromatosis was proposed by fetsch et al. in 2000. they classified it as a tumour consisting of abundant adipose tissue with a spindled fibroblastic element involving the septa of fat tissue. it forms a slowly growing painless mass, especially arising in the hands and feet of children, and in some cases congenitally. the ages ranged from 11 days to 12 years (median age, 1 year) at the time of initial biopsy or resection. there was a more than 2:1 male predominance [1,3]. histopathologically, adipose tissue is an integral component of this tumour, typically comprising more than 50% of the tumour. in the present case, the adipose tissue occupied more than 70% of the tumour. on the other hand, true fibromatoses exhibit a more solid, sheet-like growth of the fibroblastic element and do not contain fat as an integral component [1,3]. fetsch et al. described that lipofibromatosis had been interpreted as a type of infantile or juvenile fibromatosis, a variant of fibrous hamartoma of infancy, a calcifying aponeurotic fibroma, and a fibrosing lipoblastoma. the image findings of this tumour were not described in their report. an extensive survey of the literature revealed that there was only one case report of recurrence presenting an intramuscular, poorly circumscribed, huge mass with calci265 fig 5. histology of the specimen. a) low-power view. abundant fat involving dense fibrous tissue was noted. b) high-power view of mature adipocytes and spindled fibroblastic element. the skeletal muscle was remarkably infiltrated. c) univacuolated cells were noted between them (arrow). c fication, showing heterogeneous mr signal intensities [2]. our case is the first report to present the image findings of lipofibromatosis before operation. the imaging features of the present case were not different from those seen in lipomas, although microscopically the tumour had a highly cellular fibroblastic component. therefore, it seems difficult to diagnose lipofibromatosis by its imaging features alone. fetsch et al. reported that 72% of such patients had regrowth of their tumour or had persistent disease, and most of whom had been managed by incomplete removal. recognition of this clinical entity is needed, especially in cases of lipomatous tumours arising in pediatric hands and feet. references 1. fetsch jf, miettinen m, laskin wb, michal m, enzinger fm (2000) a clinicopathologic study of 45 pediatric soft tissue tumours with an admixture of adipose tissue and fibroblastic element, and a proposal for classification as lipofibromatosis. am j surg pathol 24:1491-1500. 2. herrmann bw, dehner lp, forsen jw jr (2004) lipofibromatosis presenting as a pediatric neck mass. int j pediatr otorhinolaryngol 68:1545-1549. 3. miettinen m, fetsch jf; (2002); lipofibromatosis; in: christopher dm, fletcher k, krishnan u, mertens f; world health organization classification of tumours. tumours of soft tissue and bone; iarcpress lyon; 85. corresponding author: masahito hatori, m.d. department of orthopaedic surgery, tohoku university school of medicine 1-1 seiryomachi, aobaku, sendai, miyagi 980-8574 tel: 81-22-717-7242, fax: 81-22-717-7248 email: mailto:mhato@mail.tains.tohoku.ac.jp 266 vol_116_002_sups_a_526722 133..137 upsala journal of medical sciences. 2011; 116: 133–137 original article lumbar radiculopathy caused by foraminal stenosis in rheumatoid arthritis tomoaki koakutsu, naoki morozumi, yutaka koizumi & yushin ishii department of orthopaedic surgery, nishitaga national hospital, sendai, japan abstract study design. case-series study. objective. to describe the clinical presentation, characteristic findings of imaging studies, and treatment of lumbar radiculopathy caused by foraminal stenosis in rheumatoid arthritis. background. lumbar lesions in rheumatoid arthritis are relatively rare, with a limited number of systemic reports. methods. six patients with lumbar radiculopathy caused by foraminal stenosis in rheumatoid arthritis were treated. the patients were all women with a mean age of 69 years and mean rheumatoid arthritis duration of 15 years. the medical records and imaging studies of all patients were reviewed. results. the affected nerve roots were l4 in four patients and l3 in two patients. foraminal stenosis was not demonstrated in magnetic resonance images in four of the six patients. selective radiculography with nerve root block reproduced pain, manifested blocking effect, and demonstrated compression of the nerve root by the superior articular process of the lower vertebra in all patients. conservative treatment was performed on one patient, and surgery was conducted for the rest of the five patients; radiculopathy was improved in all patients. conclusions. lumbar foraminal stenosis is a characteristic pathology of rheumatoid arthritis, and should be kept in mind in the diagnosis of lumbar radiculopathy. selective radiculography is useful in the diagnosis of affected nerve roots. key words: diagnosis, foraminal stenosis, lumbar spine, radiculopathy, rheumatoid arthritis, surgery introduction although cervical lesions are well known as spinal involvement of rheumatoid arthritis (ra), lumbar lesions are relatively rare, with a limited number of systemic reports (1–3). most of the cases which have been reported so far are spinal canal stenosis complicated with neurological symptoms caused by vertebral collapse secondary to vertebral body lesions (4,5) or by segmental instability subsequent to facet joint lesions (6,7); thus very few reports have been published about foraminal stenosis (8). we retrospectively reviewed six cases of lumbar radiculopathy in ra that resulted from foraminal stenosis to describe the clinical presentation, characteristic findings of imaging studies, and treatment of this condition. particularly, we compared the usefulness in the diagnosis of foraminal stenosis between magnetic resonance imaging (mri) and selective radiculography (srg) with nerve root block (9). patients and methods the subjects were six patients with ra who met the diagnostic criteria of the american rheumatism association and who were treated at our hospital in 2003– 2005 for lumbar radiculopathy caused by foraminal stenosis. the patients were all females, aged 57 to 75 years (mean 69 years) with 2 to 28 years of duration correspondence: tomoaki koakutsu, department of orthopaedic surgery, emergency center, tohoku university hospital, 1-1 seiryo-machi, aoba-ku, sendai, miyagi 980-8574, japan. fax: +81-22-717-7492. e-mail: koakutsu@med.tohoku.ac.jp (received 12 may 2010; accepted 18 september 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.526722 of ra (mean 15 years). according to steinbrocker’s classification (10), ra staging was evaluated as stage 2 in one patient, stage 3 in one patient, and stage 4 in four patients. their functional classification was assessed as class 3 in four patients and class 4 in two patients. mutilating deformities of the limbs were observed in four patients. all hospital and outpatient records as well as the related radiographs (plain x-rays, myelograms, postmyelographic ct scans, mri, and srg) were reviewed to study clinical presentation, characteristic findings of imaging studies, and treatment. lumbar radiculopathy in ra was diagnosed on the basis of neurological examination findings and imaging of the affected nerve roots. severity was rated according to the criteria for evaluation of low back pain proposed by the japanese orthopaedic association (joa score). the therapeutic modality (with or without surgery; surgical procedures) was noted, and recovery rate was determined from preand postoperative joa scores by hirabayashi’s method (11). generally, it is difficult to diagnose ra-specific lumbar lesions from imaging alone, since lesions of spondylosis deformans and/or lesions of osteoporosis associated with ra are also found. however, we defined ra-specific lesions as follows: rheumatoid nodules in the vertebral bodies (4); vertebral collapse without previous trauma (5); disc space narrowing without osteophyte formation; end-plate erosion (2); and segmental instability associated with facet joint destruction (6,7). thus the presence of these lesions had to be confirmed. since coexisting cervical myelopathy may affect neurological diagnosis, the presence of ra cervical lesions (atlantoaxial subluxation, vertical subluxation, and subaxial subluxation) was examined in plain x-rays of the cervical spine. lumbar spinal canal stenosis was assessed by mri or myelography/postmyelographic ct scans. foraminal stenosis was diagnosed on mri (sagittal and transverse sections through the intervertebral foramen) and srg, and their diagnostic usefulness was compared. results all of the patients complained of pain over the buttock to the anterior surface of the thigh. pain was worsened in the sitting and standing positions but was mitigated in the recumbent position. the l2, l3, and l4 dermatomes were suspected to be the responsible nerve roots. however, complications such as cervical myelopathy and articular lesions of ra in the lower limbs made it difficult to identify the responsible nerve roots on the basis of neurological findings alone. from combined neurological and imaging findings, responsible nerve roots were finally identified as l3 and l4 in two and four patients, respectively. radiculopathy was unilateral in five patients and bilateral in one patient. cauda equine syndrome associated with radiculopathy was diagnosed in only one patient. conservative treatment alleviated the symptoms of one patient; five patients were treated by surgical intervention. preoperative joa score was 8.6 ± 6.3 (range 5–13). three patients were treated by posterior decompression for the affected nerve root. a patient with severe low back pain and another patient with segmental instability in multilevels were treated by posterior decompression and posterolateral fusion with instrumentation. although radiculopathy was improved in all patients, one patient was re-operated for radiculopathy that had developed in the contralateral side after the first operation (table i). their clinical courses were monitored postoperatively for 26.0 ± 3.5 months (range 12–48). postoperative joa score was improved to 15.2 ± 7.5 (range 8–20) with a recovery rate of 32.2% ± 3.7% (range 12.5%–52.6%). bone fusion was achieved in all patients who were treated by posterior decompression and posterolateral fusion with instrumentation. plain x-rays of the cervical spine revealed ra lesions (atlantoaxial subluxation, vertical subluxation, and subaxial subluxation) in four of the six patients. plain x-rays of the lumbar spine, ct, and mri found table i. patients’ data. case gender, age (years) duration of ra (years) mutilating classification affected nerve root treatment 1 f, 74 17 + stage iv, class 4 left l4 ! right l4 a decompression 2 f, 69 28 + stage iv, class 4 right l4 decompression 3 f, 57 25 + stage iii, class 3 right l3 decompression and fusion 4 f, 74 2 + stage iv, class 3 right l4 conservative 5 f, 65 26 stage iv, class 3 left l3 decompression and fusion 6 f, 75 3 stage ii, class 3 bilateral l4 decompression a re-operated. 134 t. koakutsu et al. the following ra lesions: vertebral collapse without previous trauma in three patients; disc space narrowing without osteophyte formation in three; end-plate erosion in six; and segmental instability associated with facet joint destruction in four (anterior spondylolisthesis in two; lateral spondylolisthesis in two; includes overlapping lesion). in all patients, severe osteoporosis was evident, although mri revealed no obvious rheumatoid nodules in the vertebral bodies. excluding the one patient with cauda equine syndrome, spinal canal stenosis was mild in the mr images and/or the myelograms. this finding led us to suspect the presence of foraminal stenosis. foraminal stenosis was not demonstrated in mr images (sagittal and transverse sections through the foramen) in four of the six patients. srg showed that all patients had pain radiating towards the sites where pain is usually felt at the time of nerve root puncture (pain reproduction). transiently after nerve root block, the pain dramatically disappeared, leading to clear identification of the affected nerve root. nerve root compression by the superior articular process of the lower vertebra was detected in all patients. representative images are shown in figures 1–3. discussion although the frequency of lumbar lesions in ra is not considered to be as low as previously described, their a d e b c figure 1. a 69-year-old women, with right l4 radiculopathy as revealed by postmyelographic ct scan. a: in the coronal section, bilateral l4 roots were well depicted. b: in the sagittal section, osteoporosis is evident; multiple vertebral collapse and end-plate irregularity are observed despite no history of trauma. c: in the sagittal section through the right foramen, foraminal stenosis is not obvious. d: in the transverse section (l3/4), spinal canal stenosis is very mild. e: in the transverse section (l4/5), prominent l4/5 facet joint destruction and lateral dislocation are noted. lumbar radiculopathy in rheumatoid arthritis 135 natural course has not been elucidated because there have been only a few systemic reports (1–3). this is likely due to the occasional difficulty in differentiation of ra-specific lesions from osteoporotic fractures of the vertebral body and degenerative spondylolisthesis. moreover, complications such as cervical myelopathy and articular lesions of the lower extremities make neurological diagnosis more difficult (2). ra-associated lumbar lesions are roughly divided into vertebral lesions, facet joint lesions, and intervertebral disc lesions. these lesions must be differentiated from spondylosis deformans and osteoporosis lesions. vertebral lesions (defined as rheumatoid nodules of the vertebral bodies) were reported in 1952 by baggenstoss et al. who observed them in autopsy patients (4). facet joint lesions were described by lawrence et al. (6). in the present study, we defined ra lumbar lesions as rheumatoid nodules of the vertebral bodies (4), vertebral collapse without previous trauma (5), disc space narrowing without osteophyte formation, end-plate erosion (2), and segmental instability associated with facet joint destruction (6,7). endplate erosion was obvious in all of the six patients. the occurrence of ra lesions in the vertebral bodies and intervertebral disc (which has no synovial tissue) was attributed to enthesopathy by shichikawa et al. (12). in most of the reported cases of ra lumbar lesions, neurological symptoms are caused by spinal canal stenosis, stemming from vertebral lesions leading to vertebral collapse (4,5) or from facet joint lesions a b figure 3. selective radiculography (srg) (right l4 nerve root). a: posteroanterior view. b: oblique view. the right l4 nerve root is compressed from underneath by the l5 superior articular process (arrow). pain reproduction at the time of nerve puncture and nerve block induction were confirmed. a b figure 2. magnetic resonance imaging (mri). a: in the sagittal section (right l4/5 foramen; t1-weighted image), foraminal stenosis is not obvious (arrow). b: in the transverse section (l4/5 foramen; t2-weighted image) as well, foraminal stenosis is not evident. 136 t. koakutsu et al. leading to segmental instability (6,7). there are very few cases of foraminal stenosis. heywood reported a case of l4 radiculopathy attributable to a l4/5 facet joint lesion (8), yet he did not describe the pathogenesis clearly. myelography and postmyelographic ct scans cannot be used to establish a diagnosis of foraminal stenosis, which can be detected in mri and srg. in four of the six cases of our series, however, mri failed to detect foraminal stenosis. one possible reason is that the nerve root compression is not detected by recumbent mri because the pain was relieved in the recumbent but not the sitting or standing position. in contrast, an invasive examination, srg, detected the nerve root compression attributable to the superior articular process of the lower vertebra in all patients. furthermore, srg was very useful in the functional diagnosis based on pain reproduction at the time of nerve root puncture and blocking effect in ra cases where the neurological diagnosis cannot be easily established. this seemed to be a characteristic feature of ra in terms of arthritis. we assumed that the pathogenesis of foraminal stenosis in the lumbar spine of ra patients is the compression of the nerve root by the superior articular process of the lower vertebra; this is caused by vertical instability of the facet joint when facet joint lesions are associated with disorders in the vertebral body and the intervertebral disc. impaired nerve roots due to foraminal stenosis are most frequently reported to be present at l5 in spondylosis (13), whereas l4 was the most common site for nerve root impairment in ra. with regard to surgical treatment, crawford et al. stated that ra patients who undergo an instrumented lumbar fusion can expect a slightly higher complication rate than patients without ra, which may be related to osteopenia and immunosuppression (3). inaoka et al. performed posterior lumbar interbody fusion on seven ra patients and reported that collapse of graft occurred in one patient, migration of pedicle screw in two, instability of adjacent level in three, and collapse of adjacent vertebra in four (14). fusion surgery might be performed in view of the pathogenesis of lumbar radiculopathy caused by foraminal stenosis in ra, but it was often difficult to employ fusion surgery with instrumentation because most of the patients in our series were in poor general condition or had severe osteoporosis. we performed posterior decompression without fusion in three cases; short-term outcomes were favourable though one patient was re-operated for secondary radiculopathy. symptoms of radiculopathy were reduced by posterior decompression of the affected nerve root with partial resection of superior articular process which served as a compression factor. conclusions lumbar foraminal stenosis is a characteristic pathology of ra, and should be kept in mind in the diagnosis of lumbar radiculopathy. srg is useful in the diagnosis of affected nerve roots. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. yonenobu k, ochi t, fujiwara k, oda t. pathology and management of spinal lesions in rheumatoid arthritis. j jpn orthop assoc. 1996;70:573–82 (in japanese). 2. kawaguchi y, matsuno h, kanamori m, ishihara h, ohmori k, kimura t. radiologic findings of the lumbar spine in patients with rheumatoid arthritis, and a review of pathologic mechanisms. j spinal disord. 2003;16:38–43. 3. crawford ch 3rd, carreon ly, djurasovic m, glassman sd. lumbar fusion outcomes in patients with rheumatoid arthritis. eur spine j. 2008;17:822–5. 4. baggenstoss ah, bickel wh, ward le. rheumatoid granulomatous nodules as destructive lesions of vertebrae. j bone joint surg (am). 1952;34:601–9. 5. fam ag, rubenstein j. rheumatoid burst fracture. arthritis rheum. 1998;41:747–8. 6. lawrence js, sharp j, ball j, bier f. rheumatoid arthritis of the lumbar spine. ann rheum dis. 1964;23:205–13. 7. otani k, kikuchi s, sato k, chiba k. mri findings of pathological lumbar spondylolisthesis due to rheumatoid arthritis; report of a case. rinshoseikeigeka. 1996;31: 1187–90 (in japanese). 8. heywood awb, meyers ol. rheumatoid arthritis of the thoracic and lumbar spine. j bone joint surg (br). 1986;68: 362–8. 9. sato t, hirata s, kanabuchi t. direct oblique approach for puncture of the lumbar and sacral nerve roots. rinshoseikeigeka. 1990;25:221–5 (in japanese). 10. steinbrocker o, traeger ch, batterman rc. therapeutic criteria in rheumatoid arthritis. j am med assoc. 1949;140: 659–62. 11. hirabayashi k, miyakawa j, satomi k, maruyama t, wakano k. operative results and postoperative progression of ossification among patients with ossification of cervical posterior longitudinal ligaments. spine. 1981;6:354–64. 12. shichikawa k, matsui k, oze k, ota h. rheumatoid spondylitis. int orthop. 1978;2:53–60. 13. jenis lg, an hs, gordin r. foraminal stenosis of the lumbar spine; a review of 65 surgical cases. am j orthop. 2001;30: 205–11. 14. inaoka m, tada k, yonenobu k. problems of posterior lumbar interbody fusion (plif) for the rheumatoid sponsylitis of the lumbar spine. arch orthop trauma surg. 2002;122: 73–9. lumbar radiculopathy in rheumatoid arthritis 137 upsala j med sci 99: 357-362, 1994 7.5 common reference intervals for plasma proteins in the nordic countries ole blaabjergl, per hyltoft petersenl, mogens blom2, kerttu irjala3, adam uldal14, hanne cry5, karla mattila3, irma matinlauri3, erik lund6, jens rahbek norgaardl. 1. department of clinical chemistry, odense university hospital, dk-5000 odense c, denmark. 2. department of clinical chemistry, hjgrring sygehus, dk-9800 h j ~ r i n g , denmark. 3, department of clinical chemistry, t u r k u university hospital, sf 20520 turku, finland. 4 . department of clinical chemistry, kas herleu, dk-2730 herleu, denmark. 5. medi-lab, adelgade 5-7, dk-1304 copenhagen, denmark. 6. department of clinical chemistry, vejle sygehus, dk-7100 vejle, denmark. it is a philosophic question whether it is possible to establish common reference intervals valid for different ethnic groups and geographical areas. from a practical viewpoint, however, reference intervals are in current use and seem to be the best tool for a general validation of the first results from persons consulting the health care system when clinical strategies with clear interpretation of results are missing. the reference individuals should be selected and the measurements and calculations should be performed according to ifcc (1, 3) or equivalent (cf. section 7.1). a n important point, however, is the decisions about dwiding and combining different reference intervals as investigated by harris and boyd (2). according to them the problem is mainly a statistical problem, but as discussed in chapters 7.1, 7.2 and 7.3, it is also a question of judgements based on biology. presumptions for sharing common reference intervals three main conditions have to be fulfilled for establishing common reference intervals: i h the analytical quality must be common the biology must be common, and the interpretation of data must be common. 357 the analytical quality must be common in principle it is sufficient just t o have the same analytical quality in all the laboratories involved, but in practice this can only be obtained by using either the same method (calibrator, reagents, equipment, instructions etc.) or by using specific methods with first class calibrators with values traceable t o the highest level of trueness. only the last solution is of interest for plasma proteins. the ifcc/cap/ bcr 470 reference preparation for plasma proteins makes it possible to obtain the needed standardization. the nordic calibrator has concentration values traceable to this reference preparation (cf. chapter 5). the main problem of plasma protein analyses, the unspecific reactions from turbid samples for nephelometric methods can be solved by ultracentrifugation of the samples (cf. chapter 6). so, the analytical prerequisites are available for establishing common reference intervals. the biology must be common we have investigated two ethnic groups living in different geographical areas in the nordic countries. we d d only find a dfference for s-haptoglobin of such an importance that we decided to specify different reference intervals in finland and denmark for this protein, probably due to differences in genetical subtypes of this protein. the results for immunoglobulins indicated variations, which may be of importance, but the tendencies between the two sexes were conflicting and could not justify any splitting up in finnish and danish reference intervals, as the groups used for the comparison were too small in size and only related t o the age group between 30 and 40 years of age. the question about the immunoglobulins may be clarified by swedish investigations where no increase in s-iga with increasing age was found (personal communication). thus, differences in biology may be disclosed by large scale investigations designed according to more specific questions. the interpretation of data must be common we have not used the ifcc recommendations for estimation of the reference intervals as we wanted to use the probit display in order to validate possible combinings of reference intervals for different groups (cf. sections 7.1 and 7.2). the results, however, are valid as the distributions are close to log-gaussian (sections 7.2, 7.3, and 7.4). the comparison between finnish and danish showed that in the nordic countries it could be possible to establish common reference intervals for the seven proteins, with s-haptoglobin and s-iga as exceptions and with the methodological exceptions for s al-antitrypsin (cf. section 7.3). 358 reference intervals for nine plasma proteins group m (all) w ( + e ) m (>50) w i + e ) w ( > 50) w ( > 50) table 7.5.1 reference interval 0.26-0.45 36.6-48.2 p r o t e i n prealbumin transthyretin m ( < 5 0 ) w (-e) albumin * 39.6-51.1 orosomucoid al-acid glycoprotein a,-antitrypsin ** al-trypsin inhibitor haptoglobin *** transferrin if@ **** immunoglobulin a igg immunoglobulin g igm immunoglobulin m plasma proteins (conc. in g/l) (96 %-intervals) i m (all) m ( > 50) d 0.47-2.05 w b 5 0 ) f 0.32-1.90 m (all) w ( > 5 0 ) 6.1-14.9 w (-e) 0.23-0.39 ---t-- w (-el i 0.45-1.08 1 w (-e) m ( < 5 0 ) w (-el w ( + e ) t w ( + e l 6.915.7 0.70-3.65 w (-e) w (-e) w ( + e ) 1 0.55-2.30 i type mz (0.60-0.99) --t i m ( a l l ) = allmen, m ( > 50) = m ( <50) = w ( > 50) = w (-e) = w ( + e) = * ** men over 50 years, men under 50 years, women over 50 years, women under 50 years, not using estrogens, women under 50 years, using estrogens, the reference intervals for s-albumin include an analytic cv, 3.4 %. reference intervals for type mm and for ms and mz (without estrogen the results for s-a,-antitrypsin are only valid when antisera from dak are used. see text and chapters 5 and 6. d = danes, f = finns. slightly lower s-haptoglobin-values in the finnish population, approx. 0.15 g/l. pp). *** **** non-parametric estimates. all individuals sitting at least 15 min before blood sampling in arm-vein. 359 use of different units (grams or moles) together with minor differences in presentation may, however, give a heterogeneous impression. so, even if the same material with the same method for estimation of the reference intervals have been used the list will be different in each of the nordic countries. this situation was not foreseen at any time during the projects, and it was a great surprise to us. the table with reference intervals for the nine plasma proteins are the results estimated by the project group with values traceable to crm 470. discussion there are several week points in the reference intervals produced. analytical problems concerning s-al-antitrypsin a problem with the transfer of values from crm 470 to the nordic calibrator exists (cf. chapter 5). therefore, at present we can only guarantee the reference intervals for this protein when dako antisera are used. for the other eight proteins there should be no problems as long as the patient samples are not turbid. turbid samples for nephelometry should be ultracentrifuged. estimution of the reference intervals the division of reference intervals into two groups for the eight proteins (three for s-orosomucoid) is more or less obvious. in more questionable cases it has been our decision and we are responsible for the conclusion in each case. in all the cases with convincing log-gaussian distributions the division seems to be reasonable, but for s haptoglobin and s-iga the non-log-gaussian distributions point to problems indicating that the &vision is not optimal. a r e the reference intervals common ? the question whether the reference intervals are common for the nor&c countries except from s-haptoglobin and s-iga can only be answered by a new and extended investigation with a special design for the clarification of the weaker points in this work. are hospital employees and their relutives representative ? this question cannot be answered by this investigation, but it should be examined in another project. 360 general discussion the present situation where each laboratory has its own reference interval for each quantity is not justified by the analytical methods in use. even with such a performance due t o the analytical method, the procedure cannot be characterized as professional, as there are no biological reasons justifjmg different reference intervals except from ethnic differences and environmental conditions and this has not been taken into consideration when laboratory-individual reference intervals were established. there are good reasons for establishing common reference intervals in the nordic countries and even with the incomplete attempt described here for the nine plasma proteins the use of common reference intervals is the only way to strive for. the analytical possibilities based on common calibration and specific methods are the logical consequence for the plasma proteins. it is in any case better than to maintain the situation of laboratory-individual reference intervals. acknowledgements we want to thank all the persons who have contributed to this work, volunteers who gave blood to the investigation and the 20 danish laboratories involved in collecting the blood samples and doing extra analytical work. we are specially in debt of gratitude to inger nwgaard, who performed the protein determinations, and the agarose electrophoreses for estimation of m-components, and to h e richter, who performed the registrations and calculations of the many results. further we are grateful to soren blirup and per just svendsen, who performed the measurements of s-crp and for the antisera used in all the determinations delivered by dako . references 1. alstrom t, grasbeck r, lindblad b, solberg he, winkel p, viinikka l. establishing reference values from adults: recommendation on procedures for the preparation of individuals, collection of blood, and handling and storage of specimens. seand j clin lab invest 1993;53649-52. harris ek, boyd jc. on dividing reference data into subgroups to produce separate reference ranges. clin chem 1990;36:265-70. solberg he. approved recommendations (1987) on the theory of reference values. part 5. j clin chem clin biochem 1987:25:645-56. 2. 3. 36 1 upsala j med sci 105: 57-66,2000 very early discharge from hospital after normal deliveries elisabeth darj and boel sthlnacke department of women 's and children's health, section f o r obstetrics and gynaecology, uppsala university hospital, uppsala, sweden abstract the aim of the present study was to investigate, in a descriptive study, how healthy women experienced early discharge from hospital, 6 -12 hours, after normal deliveries. expecting couples were prepared for early discharge during pregnancy. the women delivered at the university hospital in uppsala. all infants had two obligatory medical examinations, one before leaving the hospital and one for metabolic screening after four or five days. both mothers and infants received postpartum care at home by midwives. one hundred and three women participated and answered a questionnaire four to five days after delivery. twenty of the women were additionally interviewed by telephone four months after delivery. the average length of stay at the hospital after delivery was 8 hours. the families had, on average, 1.9 visits at home. all women considered that they had received sufficient care and advice from the midwives, although 40 percent felt uncertainty about something in the postpartum period. questions frequently raised concerned breast feeding. two infants were readmitted to hospital due to mild neonatal hyperbilirubinemia. all but three women wanted to repeat very early discharge after a future uncomplicated delivery. ninety-five percent of the women were still breast feeding after four months. we conclude, that antenatal preparation and a well-organised, adequate postpartum home-care is of the greatest importance, to establish safe early discharge after uncomplicated deliveries. this concept of early discharge from hospital, with midwifery home care provided, is safe and appreciated by the new parents. 57 introduction the length of hospitalisation after both vaginal and caesarean deliveries has declined during recent years. seven years ago, a non-randomised study of mandatory discharge only four hours after child-birth, with a control group remaining in hospital, was performed in denmark ( 5 ) . the early-discharged women, who had home-visits by midwives, were less satisfied with the postpartum period compared with the control group. they experienced less support, mainly concerning breast-feeding and they made 67% more telephone consultations. the women felt that they were not satisfactorily informed about the compulsory early discharge during pregnancy. an australian study (4) evaluated satisfaction with domiciliary midwifery care after early discharge as compared with hospital care. the study showed that healthy women were largely satisfied with the quality of the care programme they had favoured, due to different expectations of postnatal care, regardless of the form of care they chose there have been concerns, though, about whether women can choose freely or not. data from the 1988 national maternal and infant survey (8) suggest that economic factors have become a driving force behind early discharge. of the total number of women, 12.3% were early discharged and left hospital after one night or less. these women had less education, inadequate prenatal care and lack of private insurance. with increasing early discharge from hospital, according to benefits for the families, there have been concerns about safety for the new-born, as expressed in the washington state experience (6). in another large study, 1200 women were followed in a programme for prenatal preparation. risk assessment, postpartum breast-feeding counselling and home visits were studied (7). a moderate reduction of postpartum readmission to hospital was found. no disadvantages for the neonates were shown when adequate postpartum outpatient care was accessible. other descriptive studies of low-risk populations could not demonstrate differences in maternal competence, breast-feeding, neonatal hyperbilirubinemia or readmission of infants to hospital, but implied psycho-social benefits for some mothers and infants (2, 1). there are previous swedish studies of early discharge from hospital, where mother and child leave hospital within the first 72 hours after delivery, and are followed-up by home-care (1 1, 9). we wanted to make an evaluation of the new programme with very early discharge after childbirth, before introducing it on a large scale at the hospital. in sweden almost 100% of the pregnant 58 women are registered in primary prenatal care. ninety-nine percent of all antenatal care is public and free of charge. all women are given individual antenatal care 9-10 times during a normal pregnancy. no pregnancy or birth allowances are given. the mothers have 450 days maternity leave and the fathers have 10 days paid leave in connection with birth, to care for the new-born and for other children in the family. preparation classes, of 6-8 couples, are offered to all pregnant women and their husbands. they meet on four to five occasions. in classes there is structural education about the progress of a normal pregnancy, delivery and the first postpartum period. breast-feeding is discussed and encouraged. the view of breast-milk, as being natural, often uncomplicated, inexpensive and exactly adapted to the babies need, decreasing infection and allergy rates and giving a close body contact between mother and child, is introduced. the aim of this study was to describe how healthy women experienced discharge from labour ward, directly but after birth, after having first rested, initiated breast-feeding, showered, having had a meal, and having prepared to go home. the postpartum care continued in their own home with home-visits by trained midwives. few studies have examined the incidence of breast-feeding, comparing early discharged women with those in regular care. additionally, the frequency of breast-feeding differs greatly between countries. in 1996, data from the national board of health and welfare (lo), 98% of the swedish women were breast-feeding when the baby was one week old. at four months of age, 84% of all swedish children are still breast-fed. while introducing this new concept of very early discharge after child-birth, we think it is important to maintain the high proportion of breast-fed infants and, furthermore, to evaluate how many children or mothers were readmitted to the hospital during the first four months. methods during six months 1996-1997, healthy women at low-risk, medically and socially, were offered the possibility to leave the hospital 6-12 hours after a normal delivery. the study was approved by the ethics committee of the medical faculty at uppsala university. one hundred and three women were recruited after giving informed consent to the study. all participating women had experienced an uncomplicated singleton pregnancy. there were no signs of high blood pressure, intrauterine growth retardation, 59 gestational diabetes, macrosomia, bleeding, premature rupture of membranes or other complicating factors. before the women were included in the study, the delivery should have passed without any complications and at term. the neonates were examined by a paediatrician before leaving the hospital. they were all healthy and of normal weight. the women were supposed to have good social support at home, meaning that the husband, or another relative, stayed at home to manage the household the first 7-10 days. early discharge included continued postpartum care at home, with visits by specially trained midwives, taking care of both the mother and the child. all midwives providing home care were employed by the department of obstetrics and gynaecology at the university hospital. all families had at least one home-visit, but as many as they needed. if a woman did not require firther home-visits, she none the less had a telephone-call every day in the morning from the home-visiting midwives. the midwives followed a structured protocol (table i), being in contact with the mother and the child. table 1. midwifery structured protocol, used at each home-visit after normal deliveries. neonate: mother: general status general status elimination uterus umbilicus perind tear or episiotomia skin, icterus (1-3) bilirubinemia if needed breast-feeding: information: position delivery sucking technique diet sucking willingness exercise pacifier family relations and sexuality intervals between breast-feeding they had the possibility to draw venous blood from the infant if neonatal hyperbilirubinemia was suspected. at the fourth or fifth day, all children were examined a second time by a paediatrician at the hospital and a metabolic screening was performed. the neonatal metabolic screening in sweden includes blood samples for some inborn errors of metabolism and congenital hypothyreoidism. the women were asked at the same time to return the questionnaires in sealed envelopes. after the first week, if everything was normal, the responsibility was transferred, from the department of obstetrics and gynaecology to the public and cost-free child health care programme. uppsala is a university city and most of the families were living near the 60 hospital. after four months, the first 20 women in the study were interviewed by telephone. the questionnaire included demographic questions concerning age, parity, marriage status and profession (table 2). table 2. demographic distribution of 103 women with experience of early discharge after delivery mean (range) f sd women’s age 29.6 years (21-40) f 4.4 men’s age 32.3 years (22-57) f 6.1 children’s birth length 51.1 cm (48-55) f 1.8 hospitalisation 8.0 hours (6-12) f 2.0 children’s birth weight 3696 g (2750-4730) f 425 home visits 1.9 visits (1-4) f 0.8 further questions focused on the information they had received and when. experiences of the home-visit and care and support from the midwives were estimated on a scale 1 7. the women’s worries about the baby, breast-feeding and extra contact with the hospital was registered. there were also open questions to both the mother and father or other relative about the first days at home. a statistical calculation of the study was performed before start. in order to draw general conclusions of the experiences from a whole population choosing very early discharge after child-birth, from the small present descriptive study of 100 women, it was estimated that at least 70% of them (confidence interval 61-79%), should be satisfied with the postpartum care given and wanted to repeat very early discharge after a future uncomplicated delivery results in the study, 20 women (20%) were primiparas and 82 (80%) multiparas. fifty-two women had had their second child, 20 women their third, four women their fourth, one woman her fifth and one woman her eighth child. there were incomplete data from five women in this respect. ninety-seven percent of the couples were married or living together. twenty-eight percent of the women had an academic occupation, while 11% were out of work. the women had received information initially on early discharge, mainly from midwives and doctors in prenatal care units (table 3). 61 table 3. sources of information about early discharge after delivery n (yo) prenatal care, midwife or doctor 85 (82.5yo) relatives or friends 24 (23.3%) preparing visit at the hospital 15 (14.6%) other 2 (1.9%) newspapers 10 (9.7%) most of them (95%) were satisfied with the information they had received. all but one of the women had the intention of breast-feeding the baby, but all of them did so before leaving the hospital. the families had, on average, 1.9 visits at home by a midwife. thirty-six percent had one visit, 40% had two visits, 22% had three visits and 2% had four visits. forty percent of the women felt uncertainty about something in the postpartum period (table 4). the most frequent questions concerned breast-feeding. table 4. insecurity of following alternatives. n (yo) mother: breast-feeding 25 (24%) breast, nipples 17 (16.3yo) bleeding 5 (4.8%) other 4 (3.8%) crying 8 (7.7%) sleeping 5 (4.8yo) infant: behaving at the breast 11 (10.6y0) vomiting 7 (6.7%) breathing 5 (4.8yo) other 12 (11.5%) fourteen percent of the women called a midwife or the maternity ward for advice. all of the participating women (1 00%) assessed that they had received sufficient comfort and care by the midwives. ninety-four percent experienced early discharge to be positive or very positive (scale 6-7) and 93% experienced midwifery home visits as positive or very positive (table 5). table 5. assessment of experience with early discharge and home visits of 103 mothers after delivery (on a scale 1-7) very negative very positive 1 2 3 4 5 6 7 early discharge 1 2 3 11 86 home visits 1 1 5 15 81 62 comments from the three who were not positive to the concept, concentrated on that the mother or the father was tired, they wanted more help, possibly given by hospital staff, they felt insecure or had questions about how to deal with elder brothers or sisters. one woman and two children (3%) required an extra visit to a physician. they were readmitted for treatment. both of the infants had mild jaundice, discovered at home by the midwife, who took blood samples at the home-visit. maximum serum levels of bilirubin were 332 pmol/l and 328 pmoll, respectively. the two children had light therapy and stayed in the hospital for another 39 and 70 hours, respectively. one woman had problems related to breast-feeding. this, was a swedish woman who had been living abroad and registered late, after 36 gestational weeks, at the prenatal unit. she had not been able to attend all prenatal preparation classes. she was readmitted to the hospital, after two days at home, for extra support and stayed in the hospital for four days, until she was comfortable with the breast-feeding. the baby was healthy. answering the questionnaire during the first week after delivery, all women but three expressed the hope that they would be able to leave hospital early after a coming delivery, with domiciliary care. one woman had had her first child, and suspected that next time it could be exhausting with siblings and a new baby at home. the other two women expressed that they needed more rest before going home after childbirth; one had had her first and the other her second child. all women initiated breast-feeding. in our small study of the telephone-interviewed women, all but one, %yo, were breast-feeding after four months and the women were content with the support they had received at the home-visits. after four months, 18 out of 20 fathers had spent some of the first two weeks with the baby and the wife at home, on average 9.2 days. in 30 % another person was staying in the home for the first few days, with or without the husband at home simultaneously. twelve of the twenty telephone-interviewed women had, during the first four months, experienced some kind of difficulty. five had had problems while initiating breast feeding, two had had sore nipples and five had some other kind of problem at home (e.g. jealousy from siblings, fear of thoughts of sexuality). at the interview all but one woman was still breast-feeding. this woman had quit breast-feeding after one month. four months after the delivery all interviewed women wanted, after a normal future 63 delivery, to leave the hospital as early as this time, being supported by a home-visiting midwife. discussion in the danish study (5) the aim of discharging the mothers and new-born very early was cost-saving, but this could not be confirmed in the investigation. others have been able to show cost-effectiveness (2, 9). the danish mothers felt uninformed about the compulsory early discharge and were dissatisfied with the postpartum care. this emphasises the importance of seeing the pregnancy, delivery and postpartum period as linked together and influencing each other. the parents have to know what to expect after the delivery, if it has been uncomplicated. data from the national study in the united states supported a legal decision mandating a minimum of 48 hours hospital stay postpartum (8). the legislation was suggested to secure that discharge from hospital should be individually based on medical and social factors and not on the economic situation. the availability of adequate postnatal care should be general. we think that there is no need of such a law in sweden, but fully agree with an individual evaluation based on medical and social factors, and that sufficient and high-quality care is crucial for accepting this method of postpartum home-care. we do not let the mother and infant leave the hospital if the obstetrician, paediatrician, or midwife has concerns regarding medical or psycho-social safety. there are limitations with descriptive studies such as ours. randomised controlled trials are difficult to perform in this respect and most studies published are descriptive. gagnon et a1 (3) however, in a randomised study, with both groups equally prepared antenatally, found no apparent disadvantages, but observed emotional benefits for the family after early discharge. no control group was identified during our study. however, the university hospital in uppsala regularly distributes questionnaires to women in the maternity ward asking about their satisfaction with the care, before leaving the hospital. newly delivered mothers stay on average 2.3 days in the ward, 97 % and 96 yo of them were generally satisfied with their hospital stay in 1996 and 1997, respectively. in the present study we found the same estimation of satisfaction in mothers who left hospital very early. these figures may reflect the women’s satisfaction according to their own expectation of postpartum care, as others already have shown (4, 7). comments from the participating women in our study were commonly that the 64 fathers and siblings got to know the baby sooner and were more involved in the care. the safety for the neonates was discussed in the washington state experience (6). the authors conclude that new-borns discharged less than 30 hours of age are at risk for rehospitalization within the first month. subgroups at special risk in the report are children of young mothers < 18 years, primiparas or after rupture of membranes. prematures, new-borns with serious medical conditions, multiple births and babies born after caesarean section, were excluded, but there is no information on how the women or families were prepared and risk-evaluated before delivery. thirty per cent were reported to have had complications during delivery and 25% had less than adequate prenatal care. in our hospital these mothers would not have been included in the very early discharge programme. although 40 % of the women study felt uncertainty in some aspect, only 14% made an extra telephone call for advice, but 100% felt that they had received adequate care and appreciated it. they were comforted by the daily visits by midwives or that the midwives called them every morning. experiences with home-visits revealed no disadvantages, concerning readmission rate to hospital, frequency of breast-feeding, when a prenatal risk-assessment was performed. in the study, the two neonates that developed mild jaundice requiring light therapy were both discovered by the midwife and diagnosed by blood tests performed at home. the families appreciated the care provided and 97% wanted to go home early after a normal h t u r e delivery. as very early discharge after labour is encouraged in the scandinavian countries, we emphasise the importance of antenatally preparing the parents for fairly prompt hospital-discharge and, furthermore, giving a structured and adequate home-care by specially knowledgeable midwives. we do not consider it a failure if a child or a woman needs to be readmitted for some days. our conclusion is that a healthy well-prepared woman with a healthy child, born at term after an uncomplicated pregnancy and delivery, can leave the hospital as early as after 6-12 hours, given that there is a well established programme for continued care at home. acknowledgements the authors would like to thank the foundation for development at the university hospital in uppsala, for financially supporting the study. 51oo126 65 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. references bragg, e., rosenn, b., khoury, j., miodovnik, m.& siddiqi t.: the effect of early discharge after vaginal delivery on neonatal readmission rates. obstet gynecol 89: 930-933, 1997. brumfield, c., nelson, k., stotser, d., yarbaugh, d., patterson, p. & spraybeny, n. : 24-hour mother-infant discharge with a follow-up home health visit: results in a selected medicaid population. obstet gynecol88: 544-548, 1996. gagnon, a,, edgar, l., kramer, m., papageorgiou, a., waghorn, k. & klein, m.: a ratidomised trial of a program of early postpartum discharge with nurse visitation. am j obstet gynecol 176: 205-21 1, 1997. kenny, p., king, m.t., cameron, s. & sheill, a.: satisfaction with postnatal care the choice of home or hospital. midwifery 9: 146-153, 1993. kirkegaard, o., engstrom, h., naested, h. & briand, a.: barselperiodens forlerb efter tvungen ambulant fndsel. (danish). ugeskr laeger 154: 1 19 123, 1992. liu, l., clemens, c., shay, d., davis, r. & novack, a,: the safety of newborn early discharge. the washington state experience. jama 278: 293-298, 1997. mandl, k., brennan, t., wise, p., tronick, e. & homer, c.: maternal and infant health. effects of moderate reductions in postpartum length of stay. arch pediatr adolesc med 15 1 : 91 5-921, 1997. margolis, l., kotelchuck, m. & chang, h-y.: factors associated with early maternal postpartum discharge from the hospital. arch pediatr adolesc med 15 1 : 466-72, 1997. odelram, h., nilsson, b., pehrsson-lindell, d.& ljungkvist, e. : early discharge after delivery a safe and cost-effective form of care. (swedish) lakartidningen 95: 3190-3 194, 1998. the national board of health and welfare. : breastfeeding, children born 1996. centre of epidemiology 1997:7, 1997. waldenstrom, u. : early discharge after hospital birth (dissertation) uppsala universitet. acta universitas uppsaliensis 79, 1987. address for reprints: elisabeth darj, m.d., ph.d. department of women’s and children’s health section for obstetrics and gynaecology university hospital, s-75 1 85 uppsala, sweden 66 sups_a_503287 60..71 upsala journal of medical sciences. 2011; 116: 60–71 original article asthma increase among farmers: a 12-year follow-up anna rask-andersen department of medical sciences, occupational and environmental medicine, university hospital, uppsala, sweden abstract respiratory disease is a well known health hazard for farmers, but the long-term prognosis is less well known. this is a 12-year follow-up of an investigation of swedish farmers, most of them dairy farmers. a questionnaire was mailed to all 418 farmers who were alive of the farmers originally participating in 1982. they were invited to an interview, spirometry, and blood sampling. ninety-onepercent(380)ofthefarmers,321menand59women,respondedtothequestionnaire.themeanagewas56yearsforthe men and 55 years for the women. of the group, 10% were smokers, 25% ex-smokers, and 65% had never smoked. the population estimate for asthma in the farmers was 8.9% in 1994 compared to 2% in 1982, and to 5.4%–6.6% in the general population in the region in 1982. of the asthmatic subjects, one-third had positive rast tests (radioallergosorbent tests). almost 90% of the new onset asthma cases since 1982 had non-ige-mediated asthma. most of the ige-mediated asthmatics had had symptoms for many years, while 70% of the non-ige-mediated asthmatic farmers had no or only wheezing with colds 1982. two new cases of hypersensitivity pneumonitis were identified, and 7.3% had experienced inhalation fever during the last 12 years. in general, individuals with asthma and chronic bronchitis who had left farming were in better health in 1994 as compared to 1982. in conclusion, farmers have an enhanced risk to develop asthma increasing with age. asthma in farmers is often non-ige-mediated. key words: agricultural workers’ diseases, alveolitis, allergy, asthma, extrinsic allergic, farmer’s lung, farming, longitudinal studies, occupational disease, occupational exposure introduction due to the large exposure to dust in their work, farmers have a higher morbidity and respiratory disease mortality than expected, and although this has been known for centuries (1) it remains a serious problem. farmers may suffer from a number of different work-related diseases of the respiratory organs, such as asthma, chronic bronchitis, irritation of the upper respiratory tract, inhalation fever (organic dust toxic syndrome (odts)), and allergic alveolitis, known as farmer’s lung (2–5). in a european study of animal farmers from four countries, a doseresponse relationship was shown between number of hours worked daily inside confinement houses and the development of work-related shortness of breath, cough without phlegm, and inhalation fever (6). the long-term prognosis of respiratory symptoms in farmers is, however, less well known. the present study is a 12-year follow-up of an investigation carried out in 1982 of pulmonary diseases and clinical findings of swedish farmers (7). the purpose of the study is to investigate the longterm prognoses of the various symptoms and diseases of the respiratory organs in farmers and the way in which they are related to farm dust exposure, together with the symptoms and survey results noted 12 years ago (precipitins, rast tests [radioallergosorbent tests], and spirometric data). methods study design in 1982 a base-line study, an epidemiologic survey investigating work-related respiratory symptoms and occupational respiratory diseases, was performed correspondence: anna rask-andersen, department of medical sciences, occupational and environmental medicine, university hospital, uppsala, sweden. e-mail: anna.rask-andersen@medsci.uu.se (received 9 june 2010; accepted 10 june 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.503287 among 6,267 farms (with at least one full-time farmer) in three counties of northern sweden (7). the aim of that study was to evaluate the symptoms caused by and consequences of exposure to mould dust in farmers, such as febrile reactions and allergic alveolitis. questionnaires on work-related and other respiratory symptoms, smoking habits, and working conditions were mailed to all farmers on these farms. the response rate was 72%. according to their answers farmers were allocated to four different groups. only full-time farmers between 15 and 65 years of age were included (n = 2176), defined as a farmer working more than 30 hours per week in farming, or farming and forestry in approximately equal amounts, and who did not have any other main occupation. from each of these four groups, a stratified sample of farmers was invited to attend a thorough medical examination. of the farmers selected, 272 farmers participated and underwent clinical examination. an additional 78 farmers were randomly selected and went through the medical examination in order to make full use of the interviewing capacity. also a number of farmers with possible hypersensitivity pneumonitis were interviewed. altogether 390 farmers were interviewed and examined in 1982. for the 12-year follow-up, a questionnaire was sent in 1994 to all 418 farmers who were alive of the farmers originally selected for medical examinations in 1982. the 380 farmers who answered the questionnaire were invited to a medical examination, which included interview by a physician, blood sampling for rast analyses and precipitins analysis, and lung function test. from the results of the questionnaire, the prevalence of asthma, work-related wheezing, chronic bronchitis, inhalation fever, and hypersensitivity pneumonitis was estimated in the whole group of farmers in the three counties. death certificates were requested for those 35 who had died since 1982. the study was conducted in three counties in the middle part of the swedish region norrland. the main production in 1982 was dairy farming (74%), often combined with forestry (33%). questionnaire the questionnaire sought to obtain information about work history, work conditions on the farm, and respiratory symptoms. the same questionnaire as 1982 was used, but some questions regarding what had happened since 1982 were added. these questions concerned items such as if the farmers were still farming, or if they had changed their farming methods. two reminders were sent to non-responders. the asthma question was: ‘do you have asthma?’ causes of death death certificates for those 35 farmers who had died since 1982 were requested from the swedish death register. clinical examination all 380 farmers who had answered the questionnaires except for those who had moved or with serious other diseases were invited to an examination comprising spirometry, blood tests for allergy tests, precipitating antibodies, and total ige, as well as an interview concerning symptoms, exposure, and farming methods. there were 277 (73%) who participated in the clinical examination. interview the interview was carried out using a standardized scheme by a physician specialized in occupational medicine with special experience of work-related respiratory diseases in farmers (the authour). detailed information on symptoms and their workrelatedness was obtained. doctor-diagnosed asthma was used as a definition of asthma. allergy tests serum total ige concentrations and phadiatop were determined with the pharmacia cap system test (pharmacia and upjohn diagnostics, uppsala, sweden). phadiatop tests with serum ige concentrations of 0.35 ku/l or more were regarded as positive. in addition, specific ige reactivity (rast) was measured against two storage mites (acarus siro and lepidoglyphus destructor) and, in farmers with asthma, against cow. serum samples with ige concentrations of 0.35 ku/l or more were regarded as positive. precipitins a blood sample was analysed for precipitating antibodies against a panel of 16 antigens with both an immunoelectro-osmophoresis method and the immunodiffusion method. the antigens were thermoactinomyces vulgaris, saccharopolyspora rectivirgula asthma increase among farmers 61 (micropolyspora faeni), rhizopus, cladosporium, alternaria, and aspergillus fumigatus. measurement of serum eosinophil markers whole blood was drawn in sst tubes and allowed to coagulate at room temperature for 60 min. the serum was then separated by centrifugation. serum eosinophil cationic protein (secp) was measured by a prototype immunofluorometric assay with the pharmacia cap system� (pharmacia & upjohn diagnostics, uppsala, sweden) and expressed in mg/l. pulmonary function tests measurements of forced expiratory volume in 1 second (fev1), forced vital capacity (fvc), vital capacity (vc), fev% (fev1/vc � 100), and peak expiratory flow rate (pef) were conducted by an experienced nurse using a wedge-type spirometer (vitalograph ltd., buckingham, united kingdom). the spirometer was calibrated according to the manufacturer’s instructions at least once daily. the test was conducted with the subject in an upright position and two to three slow vital capacity manoeuvres were followed by three forced expirations. the test that yielded the highest value was accepted. standardized reference values according to hedenström and malmberg were used (8,9). diagnosis after the examinations, a classification of diagnoses such as inhalation fever, allergic alveolitis, work-related wheeze, doctor-diagnosed asthma, and chronic bronchitis was made by two independent physicians based on all the information obtained in the questionnaires and the interviews, but not on the results of the laboratory findings and pulmonary function tests. medical records were obtained for those farmers who had seen a doctor because of respiratory symptoms. statistical analyses the answers to the questionnaires and the results of the tests were entered in a database, and the program spss was used for statistical analysis. student’s t test or one-way anova (analysis of variance) was used to test differences in continuous variables between groups. proportions were tested with chi-square test or fisher’s exact test (in case the expected frequency for any cell is less than 5). ethical considerations the ethics committee of the uppsala university, sweden approved the study. results questionnaires a total of 91% (380) of the farmers, 321 men and 59 women, responded to the questionnaire. the mean age was 56 years (sd 12) for the men and 55 years (sd 10) for the women. of the men, 11% were smokers, 30% ex-smokers, and 59% nonsmokers. among the women, 15% were smokers, 18% ex-smokers, and 66% were non-smokers. difference between men and women was not significant. sixty-seven per cent of the farmers were still farming; 4% worked mainly in forestry, 11% had other occupations, and 18% did not work at all. of those 89 over the age of 65 and thus on a regular state pension in sweden, 57% were still working in farming or forestry, and 42% were working more than 30 hours a week. of the farms where the farmer still was farming, 61% were dairy farms, 25% had meat production, 7% were swine farmers, 3% sheep, and 2% poultry. still, only 16% had grain production as their main production; grain was handled on 79% of the farms and hay on 91%. potato crop was the main production on 6% of the farms. on 43% of the farms forestry was the main production. population estimates the population prevalence estimates for respiratory symptoms in the whole group of farmers in the three counties, based on the answers of the questionnaire, were as follows: of asthma 8.9%, of work-related wheeze 6.0%, of wheeze with cold 11.1%, of chronic bronchitis 7.7%, and of inhalation fever 8.1%. compared to 1982, the prevalence of all obstructive airway symptoms had increased, especially the work-related wheeze and asthma (figure 1). the prevalence of chronic bronchitis had increased from 3% in 1982 to 13.3% in 1994. a calculation of population estimates on the full-time farmers, only, did not change the estimates significantly. of the 36 farmers with asthma in 1994, the highest risk for asthma was found among those who already had some kind of obstructive symptoms in 1982 (figure 2). one-third of the farmers with asthma in 1994 already had asthma in 1982, 12% had work-related wheeze, 30% had wheeze with colds, 62 a. rask-andersen and one-third had no obstructive respiratory symptoms in 1982. in figure 3 the population estimates for asthma prevalence are compared to asthma prevalence of the general population in sweden in studies from other investigators (10,11). as shown in figure 3, the prevalence of asthma in farmers was lower than in the general population in 1982, but higher in the same farmers in 1994. among the 380 farmers that answered the questionnaire, there were two new cases of hypersensitivity pneumonitis. the yearly incidence of hypersensitivity pneumonitis in 1994 is estimated to be in the same range as in 1982, i.e. 2–3/10,000 farmers. causes of death among the 35 (30 men and 5 women) who had died since 1982, the immediate cause of death was heart diseases in 16, cerebrovascular disorders in 4, cancer in 7, respiratory tract disease in 3 and other causes in 5. pulmonary fibrosis was the cause of death in two of the three cases who had died of respiratory disease. asthma was a contributing cause of death in one of the deaths in heart disease and in another death from epilepsy. in total, respiratory tract disease was either immediate or contributing cause of death in five cases. of those five, two of the subjects had pulmonary fibrosis, two had asthma, and one had other lung disease. they were all men except for one woman who died of pulmonary fibrosis. clinical examinations a total of 276 farmers, 237 men and 39 women, attended the interviews and medical examinations, but blood samples were only obtained from 270. the mean age was 56 years (sd 12) in the men and 55 (sd 9) years in the women. of the men, 8% were smokers, 31% ex-smokers, and 60% were non-smokers. significantly more women were smokers, 18% (p < 0.05). ten per cent of the women were ex-smokers, and 72% were non-smokers. interview there were 36 cases of asthma, 27 cases of chronic bronchitis (and no asthma), 29 cases of inhalation fever (organic dust toxic syndrome (odts)), and 177 who had no respiratory symptoms whatsoever. the remainder (67 subjects) had respiratory symptoms that could not be categorized in any of the mentioned entities. there were significantly more smokers among the farmers with chronic bronchitis compared to the other diagnostic groups (p < 0.05, table i). pulmonary function test pulmonary function values decreased more than expected between 1982 and 1994 (table ii). the mean predicted fev1 was 100% in the whole group in 1982 and had decreased more than expected to 94% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% cohort of 2174 farmers in 1982 36 farmers with asthma 1994 p e rc e n t asthma wheeze colds onlyworkrel wheeze no wheezing figure 2. obstructive symptoms population estimates in the whole cohort in 1982 compared to the obstructive symptoms in 1982 in the 36 farmers with asthma 1994. 0% 5% 10% 15% 20% 25% 30% 1982 1994 p e rc e n t asthma workrel wheeze wheeze colds figure 1. obstructive symptoms population estimates in 1982 and 1994. asthma increase among farmers 63 predicted in 1994 (p < 0.01). vital capacity (vc) also decreased more than expected: from 97% predicted value in 1982 to 92% predicted in 1994 (p < 0.01). both fev1 and vc were significantly lower in the symptomatic farmers (asthma, chronic bronchitis, and inhalation fever) compared to non-symptomatic farmers. a total of 7 cases of emphysema were found among the 276 interviewed farmers. six were men and one was a woman, who was an ex-smoker. two of the men were non-smokers, one was still smoking, and the remaining three had quit smoking. allergy tests farmers with doctor-diagnosed asthma had significantly higher prevalence of positive allergy tests as well as significantly higher levels of total ige compared to the other diagnostic groups (table iii). in the asthma group, 28% (10/36) had positive phadiatop and/or positive rast towards storage mites and/or cow dandruff. the farmers with inhalation fever had a higher prevalence of positive precipitins, and farmers with asthma and chronic bronchitis had a trend to lower prevalence compared to the non-symptomatic farmers, although the differences were not significant. of eight cases (two cow dandruff-positive ones not included) with ige-mediated asthma in 1994, 75% (6/8) had asthma in 1982 (table iv). only one of the eight cases of ige-mediated asthma was completely without respiratory symptoms in 1982, and one had wheezing but only on colds. in contrast, of the 27 nonige-mediated asthma cases, only 15% (4/27) had 0 1 2 3 4 5 6 7 8 9 10 1965 1970 1975 1980 1985 1990 1995 percent general population farmers figure 3. prevalence of asthma in farmers compared to the general population. table i. characteristics of the interviewed farmers in diagnostic groups. diagnosis doctor-diagnosed asthma chronic bronchitis inhalation fever (odts) no respiratory symptoms all number n 36 27 29 117 270 sex women/men 7/36 19% 7/27 26% 1/29 3% 16/117 14% 36/270 13% age years mean (sd) 59 ±11 56 ±10 55 ±11 55 ±11 56 ±11 smokers n, % 3 6% 6a 22% 2 7% 13 11% 26 10% ex-smokers n, % 14 44% 6 19% 9 30% 26 22% 74 27% never smokers n, % 19 50% 15 59% 18 63% 78 67% 170 63% ap < 0.01. odts = organic dust toxic syndrome. 64 a. rask-andersen asthma in 1982. thirty-seven per cent (10/27) had no respiratory symptoms whatsoever in 1982, and 33% (9/27) had wheezing but only with colds. fifteen per cent of the non-ige-mediated asthma cases (4/27) had work-related wheezing in 1982. thus, most of the igemediated asthmatics had experienced symptoms for many years, while 70% of the non-ige-mediated asthmatic farmers had none or only wheezing with colds in 1982. most of the new onset asthma cases had nonige-mediated asthma (23/27 = 85%). the farmers with non-ige-mediated asthma had significantly lower fev1 and vc than the igemediated asthma cases: fev1 76% of predicted versus 97% of predicted, p < 0.05; and vc 82% of predicted versus 96% of predicted, p < 0.01. farmers who were still farming and were exposed to farm dust had a trend to lower fev1 (79% of predicted) compared to farmers who had quit farming (91% predicted), although this difference was not significant. vital capacity was almost the same in farmers who farmed compared to farmers who had quit farming, 84% of predicted versus 88% of predicted. eosinophil cationic protein (ecp) was 12.0 mg/l in the farmers with ige-mediated asthma compared to 10.6 mg/l in the farmers with non-ige-mediated asthma, but the ecp values did not correlate with the spirometry values or exposure to farm dust. exposure during the interviews, many farmers told us that their obstructive symptoms had improved since their exposure decreased, when they left dairy farming for beef table ii. results of the pulmonary function test in different diagnostic groups. diagnosis doctordiagnosed asthma chronic bronchitis inhalation fever (odts) no respiratory symptoms all fev1, %, predicted 1982 mean (sd) 93 b ±15 98 ±15 100 ±9 104 ±14 100b ±15 fev1, %, predicted 1994 mean (sd) 82 c ±21 91b ±19 93 ±13 98 ±15 94b ±17 fev1 loss 1982–1994, %, of predicted value mean (sd) 11 a ±16 8 ±9 7 ±8 5 ±8 6 11 fev1 loss 1982–1994 mean annual losses, ml; men mean (sd) 60 a ±49 53 ±24 47 ±31 45 ±27 47 ±32 fvc, %, predicted 1982 mean (sd) 94 ±12 95 ±16 93a ±9 100 ±14 97b ±13 fvc, %, predicted 1994 mean (sd) 85b ±13 90a ±16 89a ±11 95 ±14 92b ±14 fvc loss 1982–1994, %, of predicted value mean (sd) 8 ±12 4 ±6 5 ±6 5 ±6 5 ±8 fvc loss 1982–1994, mean annual losses, ml; men mean (sd) 59a ±48 40 ±29 37 ±36 43 ±30 44 ±34 ap < 0.05. bp < 0.01. cp < 0.001. fev1 = forced expiratory volume in 1 second; fvc = forced expiratory volume in 1 second; odts = organic dust toxic syndrome. table iii. results of allergy and precipitins tests in different diagnostic groups. diagnosis doctordiagnosed asthma chronic bronchitis inhalation fever (odts) no respiratory symptoms all phadiatop + n, % 5a 14% 3 11% 3 10% 4 3% 18 7% rast storage mites + n, % 3 8% 0 0% 3 10% 3 3% 7 3% phadiatop + or rast storage mites + n, % 8b 23% 3 11% 4 13% 6 5% 25 9% rast cow dander + n, % 3 8% allergy tests + (phadiatop+ or any rast+) n, % 10 28% total ige mean (sd) 147c ±239 40 ±39 63 ±115 48 ±77 60 ±114 ecp mg/l mean (sd) 10.9 ±6.0 11.0 ±6.7 8.6 ±4.8 9.6 ±5.4 10.0 ±5.6 precipitins(+), +, and ++ n, % 4 11% 2 7% 8 27% 23 20% 48 18% ap < 0.05. bp < 0.01. cp < 0.001. odts = organic dust toxic syndrome; ecp = eosinophil cationic protein. asthma increase among farmers 65 farming or completely stopped farming. several farmers had been taking asthma medication that they were able to end after the exposure decreased. also, two farmers who had chronic bronchitis for decades had major improvement after being admitted to hospital for some months after work accidents involving fractures. several of the farmers with work-related respiratory symptoms had also tried to reduce their dust exposure by going over to silage instead of hay, using tower silos or baled silage. others had installed automatic feeding. the farmers could often describe what worsened their airway symptoms such as dust from hay or grinding of grain. one farmer, allergic to pollen, tried to harvest the hay as early as possible to avoid worsening of symptoms during the hay harvest. some had stopped with grain completely because of bad revenues and as a side-effect noted that their airway symptoms improved. in 1982, the method of silage in plastic bales had not been introduced in sweden, but they were introduced prior to 1994, with a decrease of haymaking. in the northern part of the region investigated, the climate is so cold that it is hard to make grain farming profitable. instead silage was raised. in addition, the cattle were fed with purchased pellet fodder so the cattle got all the nutrients needed. hypersensitivity pneumonitis two new cases of hypersensitivity pneumonitis were identified. the first case, a 50-year-old woman, fell ill in december 1982, and the cause was mouldy straw. she had a complete recovery. the other case was a 42-year-old man affected in 1984, and the cause was mouldy wood chips for the purpose of heating his dwelling. he had inhalation fever after handling mouldy wood chips earlier, in the 1970s. this time he slowly got progressive dyspnoea. as he did not realize that the cause was the mouldy wood chips, he continued to use the wood chips. he went to his family doctor and received several treatments with different antibiotics. finally, he went to the emergency room and was admitted to intensive care. by then, he had started to suspect the mouldy wood chips, and informed his doctor. dramatically, an arterial blood sample was drawn and the farmer noticed that the blood was blue. pao2 was 6.2 kpa. the farmer stopped using wood chips. he had a complete recovery. discussion in this longitudinal study of respiratory diseases in farmers with a follow-up time of 12 years, asthma prevalence had increased from 2% to 8.9%. that is higher than the asthma prevalence in the general population in the same region in the same age group (50–60 years) investigated with similar techniques during the same time period (12). by contrast, the asthma prevalence in 1982 when the mean age of our farmers was 44 years was lower than that of the general population. there is an increase in asthma in the whole western world, including sweden, but the increase of prevalence of asthma in the farmers of our study increased more than expected showing that farmers have an enhanced risk to develop asthma increasing with age. almost 90% of the new onset asthma cases developing during the follow-up time had non-igemediated asthma. positive rast tests were only found in 28% of the asthma cases, although a rast test specific for farming such as storage mites and cow dandruff was used in addition to a rast panel of common inhalation allergens (phadiatop). farmers with ige-mediated asthma had higher ecp values than farmers with non-ige-mediated asthma (p < 0.001) in accordance with findings of a norwegian study (13). in that study, ecp showed significant associations with airway obstruction and numbers of rast allergen-positive atopic asthmatics, but no such trends could be seen in this study. work-relatedness of respiratory disorders is hard to evaluate in farmers, since they are working and living in the same environment. farmers often do not have weekends off or vacations, so they might not know if the exposures at work worsen their symptoms. however, in this study even senior farmers over the age of 65 years were included. they were on a pension, and many had reduced their activity in farming. thus, the dust exposure was often lower in the senior farmers in 1994 compared to 1982. results of the interviews revealed work-relatedness, with deterioration occurring in conjunction with exposure to dust at table iv. ige-mediated asthma compared to non-ige-mediated asthma. doctor-diagnosed asthma 1994 obstructive symptoms 1982 allergy test positive 1994 allergy test negative 1994 no symptoms 1 10 wheeze colds 1 9 definite wheeze 0 4 asthma 6 4 total 8 27 66 a. rask-andersen work in many farmers, and an improvement was noted in those who had reduced dust levels or stopped working as farmers. pulmonary function values had decreased more than expected between 1982 and 1994 even in nonsymptomatic farmers with 67% non-smokers. especially large declines were seen in the farmers with asthma and chronic bronchitis. several earlier studies have demonstrated that farmers more often have a reduced fev1 and a more rapid decline in lung function than controls (14–16). the mean annual losses in fev1 and fvc were in the same order of magnitude as for workers exposed for 20 years or more in the canadian grain industry (17). cigarette smoking is the leading cause of chronic obstructive pulmonary disease (copd) and chronic bronchitis. as expected, there were significantly more smokers among the farmers with chronic bronchitis compared to the other diagnostic groups, but emphysema was found in two never-smoking farmers. since emphysema is uncommon in non-smokers, it is possible that the exposure to farm dust contributed to the emphysema development. in a swedish linkage of the 1960 census and the causes of death register, the only occupational group showing an increased mortality due to pulmonary emphysema after adjusting for smoking habits was the occupational group containing farmers as its largest subgroup (18). exposure to vapours, gases, dusts, or fumes in the workplace during the longest-held job was associated with a 2-fold increase in risk of copd (or 2.0; 95% ci 1.6–2.5) in an american study (19). in a recent study by the same group it was found that work-place exposures were strongly associated with an increased risk of copd (20). joint exposure to both smoking and occupational factors markedly increased the risk of copd (or 14.1; 95% ci 9.33–21.2). an especially elevated risk for chronic bronchitis in non-smokers has been reported in french dairy farmers (21). as in 1982, inhalation fever and chronic bronchitis are still very common among farmers, whereas hypersensitivity pneumonitis is a rare disease. of the 35 deaths since 1982, a respiratory tract disorder was either immediate or contributing cause of death in 5 cases. of those five, two of the subjects had pulmonary fibrosis, two had asthma, and one had other lung disease as immediate or contributing cause of death. questionnaires to diagnose asthma have been developed and validated (10), but these were not available in 1982. accordingly, therefore we preferred to use the same question as in 1982 (‘do you have asthma?’) to ascertain asthma in the questionnaire. it is certain that there was an increase of symptoms that the farmers or their doctors would call asthma, but it is less certain whether all these farmers would have asthma if strictly diagnosed. it would have been useful with a methacholine test to confirm the diagnosis of asthma, but it was not possible within the scope of this study. the definition of ‘doctor-diagnosed asthma’ was used to classify cases in diagnostic categories after the interviews to compare results of pulmonary function test and allergy tests. since we found a tendency that more farmers with symptoms came to the clinical examinations compared to asymptomatic farmers, and because more farmers had answered the questionnaire than those attending the interviews, we chose to use the questionnaire data for the population estimates. one of the reasons for not attending the interviews was that farmers had moved and could not be present at the test centres. other reasons were high age or other serious disease. it is well established that farmers may suffer from a number of different work-related diseases of the respiratory organs (2,5), and asthma was described in 1924 (22). our results are supported by a number of other studies. in a danish study from 1988, the mean prevalence of asthma in a representative sample of 1,685 farmers was 7.7%, lowest (3.6%) among farmers aged 30–49 and highest (11.8%) among farmers aged 50–69 years (14). logistic regression analysis revealed that age (or 5.8; 95% ci 2.8–12.2) and pig farming (or 2.0; 95% ci 2.0–3.5) were risk factors for self-reported asthma. the prevalence of asthma among farmers and aged matched subjects from the general population was the same in the age group 30–49 years, but significantly higher among farmers aged 50–69 compared with aged matched subjects from the same sample (or 2.25; p < 0.001). also, in a random sample of 7,496 european farmers from denmark, northern germany, switzerland, and spain, all symptoms related to asthma (wheezing, shortness of breath, asthma attacks) and nasal allergies had significantly lower prevalences in farmers than in the general population (6). the prevalence of asthma (2.8%; 95% ci 2.4–3.2 in farmers of all age groups) was significantly (p = 0.001) lower in farmers aged 20–44 years (1.3%; 95% ci 0.9–1.7) compared to the prevalence in an age-matched sample of the general european population (european community respiratory health survey (ecrhs)) (3.2%; 95% ci 2.9–3.9) (6). in crop farmers in the same european countries, 3.2% reported asthma, which was similar to the prevalences found for the general european population and did not suggest a higher prevalence of symptoms of obstructive lung disease among these european farmers (23). no analysis on different age groups of farmers was presented in this study. the analysis of specific crops showed a high asthma increase among farmers 67 risk of bronchial asthma (or 2.1; 95% ci 1.1–3.9) in workers who grew flowers after adjustment for covariates. in a study of farmers in central sweden, the prevalence of doctor-diagnosed asthma was significantly lower (p < 0.001) in farmers below the age of 50 years, 1.9%, compared to 7.2% in the general population, but 9.4% in farmers aged 55–65 years (24). significantly increased mortality from asthma was found among male farmers in a study of swedish official mortality statistics (25). in another swedish study, an increased risk of hospitalization for asthma was found among male farmers (26). in a swedish study in gotland, an island in the baltic sea, the prevalence of asthma had increased significantly during the previous 12 years (5.3% versus 9.8%) as well as the prevalence of asthma among atopic subjects (3.1% versus 4.9%) (16,27). in this study, 461 dairy farmers were investigated in 1996, and 65 (14.1%) of these subjects participated in the study in 1984. as in our study, a number of asymptomatic farmers (6/12) in 1984 had developed respiratory symptoms, and 19/33, who in 1984 had rhinoconjunctivitis only, were reporting symptoms from the lower airways as well. in contrast to the findings in the present study, these findings could not be explained by a shift towards an older and more symptomatic population, since the mean ages were almost the same in 1984 and 1996. the follow-up in gotland was a cross-sectional study not including retired farmers and people that had left farming as in the present study. in an analysis of data from the european community respiratory health survey (ecrhs) in ten european countries as well as usa and new zealand, the highest risk of asthma (defined as bronchial hyperresponsiveness and reported asthma symptoms or medication) attributed to occupation was found for farmers, or 2.62 (95% ci 1.29–5.35) (28). results for farmers were consistent between 12 countries including usa and new zealand participating in the survey. in france, the prevalence rates of asthma were particularly high in former farm workers (29). farmers appeared to have a higher risk of both cumulative (or 2.30; 95% ci 1.00–5.47) and current asthma (or 5.35; 95% ci 1.33–21.50) compared to whitecollar workers adjusted for age, sex, and smoking history. in new zealand, the prevalence for combination of wheeze and non-allergic airway hyperresponsiveness was significantly increased (or 4.16; 95% ci 1.33–13.1) for farmers and farm workers (30). data from finnish surveillance systems for occupational diseases show that the incidence rate of occupational asthma is exceptionally high in farmers and attributed to the custom in finnish farming to brush the cows daily (31). in other cross-sectional studies of farmers, the reported prevalence of asthma has varied between regions and has also varied dependent on types of farming. according to a review of omland, healthy worker selection, heterogeneity in diagnosis, misclassification, age differences, difference in time of study, and small study populations resulting in low statistical power might also be factors explaining why no difference between farmers and the general population has been observed in some studies (2). in a large norwegian study, significant risk factors for current asthma were asthma heredity (or 2.9; 95% ci 2.1–3.9), asthma as a child or adolescent (or 22.2; 95% ci 15.2–32.4), animal production (or 1.6; 95% ci 1.1–2.2), and age 40–69 years (or 1.8 to 4.6; 95% ci 1.1–7.5) (32). in a danish study of 1,901 farming students (of whom 210 were females) and 407 rural controls, female sex (or (males) 0.5; 95% ci 0.3–0.8), asthma in the family (or 1.6–3.4), and smoking (or 1.7; 95% ci 1.2–2.4) were factors significantly associated to asthma (33). the prevalence of asthma-like symptoms was between 5.4% and 21%, but no difference of the prevalence of asthmalike symptoms was observed between farming students and controls. exposure to chemicals is another risk factor for asthma. in a canadian study, exposure to carbamate insecticides was shown to increase the risk for asthma (or 1.8; 95% ci 1.1–3.1) (34). in pig farmers, the use of disinfectants (quaternary ammonium compounds) (or 9.4; 95% ci 1.6–57.2) and aspects of disinfecting procedure were associated with the prevalence of asthma (35). there are at least three kinds of selections in action influencing the prevalence of asthma in farmers. first, farmers’ sons (preferably) inherit the farm as well as the occupation. since children raised on a farm also have a decreased prevalence of atopic diseases (36), the asthma prevalence of young people starting out farming is low. second, the few farm children that have asthma and allergy are not prone to take up farming as farm children start helping out on the farm at an early age and will soon realize that farming is not a suitable occupation for somebody with atopy and allergy towards e.g. cat, dust mites, or pollen. all the common inhalative allergens are found on farms— pollens, animal dandruff, moulds, and mites (37). the healthy worker effect, ‘selection in’, is thus probably particularly strong in farming due to selfselection already before having actually started the job. actually, during our interviews a number of farmers gave comments such as: ‘my son can’t 68 a. rask-andersen take over the farm because he has asthma’. possibly, there is a long-term healthy non-allergic workers effect over multiple generations (6). third, farmers that get asthma might leave farming because of work aggravation and get another job— survivor bias, that is ‘selection out’ from the occupation (38,39). this might be particularly true for younger farmers that have the opportunity to get another job perhaps through retraining. for an older farmer that develops asthma it might be impossible to get another job, especially if he lives in a remote area with few job opportunities. studies from both sweden and finland have showed that farmers have a lower risk of leaving their occupation than do people in other lines of work, but farmers with allergies more often changed occupation than did farmers with other diseases (38,40). farming is not only a way to make a living; it is a life-style. farms pass from generation to generation. a farmer with asthma might not want to leave the farm. studies have shown that farmers retire early less often than those in other occupations (38). therefore farmers try to continue farming until retirement maybe by slightly changed production such as changing from dairy farming to the less dusty beef farming, using silage, or using a respiratory device. these are reasons that might explain the high asthma prevalence of older farmers and the low asthma prevalence in cross-sectional studies of younger farmers as illustrated by our study and others. the lower asthma prevalence in farmers found in a number of studies can be explained by such selection mechanisms. for example, in a dutch study of swine farmers, the same prevalence of asthma was found as in rural controls, but atopy and symptoms of allergy during childhood were less prevalent in the pig farmers (4.6%) compared to controls (14.6%) (35). in a canadian report, four female swine workers consulted a chest physician because of an acute onset of wheezing and cough suggestive of asthma within weeks of commencing full-time employment in intensive swine production facilities (41). all four had quit working in swine production illustrating selection mechanisms that partly explain why the asthma prevalence in farmers may be lower than in the general population. three of these cases fulfilled the diagnostic criteria of asthma consistent with occupational asthma according to american college of chest physicians guidelines (42). the fourth case had normal spirometry and bronchial responsiveness; however, she experienced severe symptoms, requiring medication, and methacholine challenge was conducted several months after exposure cessation. two of the workers were atopics; one had border-line atopy on a skin prick test. usually in studies of occupational diseases, only subjects in the work-force are included, with an age ranging from the upper teens to 65 years (depending on the retirement age in the country). in this study, even farmers over the retirement age limit were included, which rendered important information. one-fourth of the older farmers were still working full time, while the others either farmed less intensively or had completely left farming, and the exposure to farm dust had decreased. during the interviews, it was obvious that farmers with both asthma and chronic bronchitis had improved after reduction of exposure. several farmers that had been on continuous asthma medication had been able to stop medication after quitting farming. changes in exposure level probably explain the lower asthma prevalence in farmers older than 65 years (the retirement age in sweden) compared to farmers aged 55–65 found in another swedish study (24). in our study, positive rast tests were only found in 28% of the farmers with asthma although rast tests specific for farming such as storage mites and cow dandruff were used in addition to a rast panel of common inhalation allergens (phadiatop). also, only 15% (4/27) of the new onset asthma cases since 1982 had ige-mediated asthma. the prevalence of sensitization to storage mites is much lower in the present study compared to studies in gotland carried out in 1983 and 1996, when about 6% of all the farmers and 20%–25% of the asthmatic farmers had allergy to storage mites (16,27). one explanation might be that gotland is an island in the middle of the baltic sea with a milder, more humid climate during the winter compared to the northern parts of sweden in this study, where storage mites probably would freeze to death during cold winters in a drier climate. in the finnish surveillance data, cow dandruff was the most common of the primary causative animal epithelia and barley the most common of the primary causative flours in farmers (31). in finland, farmwomen take care of cows, and animal epithelia were more commonly noticed as the primary cause in female farmers (77%) than in male farmers (62%). the high rate of allergy to cow dandruff in finnish farmers has been explained by the finnish habit of brushing the cows daily. in our study, only 8% of the farmers with asthma had a positive rast to cow dandruff. among swedish farmers, cow dandruff seems to be of less importance as an occupational allergen, although cow was the most common cause of sensitization to animal dandruff (4.2%) in the swedish study on gotland (16). the prevalence of brushing cows comparing swedish and finnish farmers has not been systematically studied. it is not surprising that farmers do develop asthma because of the high exposure to farm dust containing both a number of well known allergens as well as asthma increase among farmers 69 airway irritants. also, there are studies that have shown that perfectly healthy farmers on farms without any mould problems have an increased number of inflammatory cells in their bronchioalveolar lavage (43). the same research group has also shown an increasing number of positive methacholine tests in naive subjects after exposure in swine confinement buildings (44). what is surprising is the paradox that growing up in these kinds of environment decreases the risk of atopic diseases. the protective effect of contact with live-stock and poultry is consistent in several studies (36). in this study, the risk to develop asthma was largest in the farmers with any kind of respiratory symptom since 12 years. even milder respiratory symptoms that do not fit the diagnostic criteria of a specific disease entity, such as work-related wheeze or wheezing with colds, have to be taken seriously. this is supported by other studies of farmers. acute symptoms during work predicted chronic bronchitis in french farmers (45). european animal farmers with nasal irritation during work had a four times increased risk of bringing up phlegm in the winter time (6). studies of occupational asthma in other occupational settings have also shown that the higher the exposure, the greater the risk for developing occupational asthma, and, by implication, lowering the level of exposure reduces the incidence of disease (39). it can be concluded from large well designed studies in different countries using standardized questionnaires, the same sampling methods, and standardized spirometric measurements that there is a significant relationship between bad working conditions (such a bad ventilation or high respirable dust concentrations) and work-related respiratory symptoms (6,23,46). in conclusion, the finding in this study shows that farmers have an increased risk for adult onset asthma, often not ige-mediated, probably attributable to occupational dust exposure. when discussing how high the risk is, one must take into account that young farmers have a low asthma prevalence due to selection and to the fact that most of them have been raised on farms resulting in a lower prevalence of asthma compared to people that have grown up in other environments. however, after decades of exposure to high levels of dust containing multiple agents (bacteria, moulds, endotoxin, allergens, ammonia, and other irritant gases) that may act in an additive or synergistic manner, the result is a high prevalence of respiratory disease even in such an initially healthy population as farmers. for the affected farmers asthma implicates serious problems with decreased life quality due to aggravation of asthma symptoms caused by daily exposure to farm dust in a disease that can be fatal. it was evident, in this study, that after farmers had improved their working conditions or quit farming their asthma improved and even disappeared completely in some farmers. farmers with even mild respiratory symptoms belong to a risk group with probable high exposure to dust. these farmers warrant concern, should be monitored, and should receive advice on how to minimize exposure in their farms preferably by improving farming methods and/or by using respirators. smoking farmers are also a risk group and should receive advice and help to quit smoking. acknowledgements i want to express my sincere gratitude to dr marianne van hage-hamsten for help with the allergy tests, to dr per venge for ecp analyses, to dr lars belin for help with the precipitin tests, and to dr jim dosman for language revision. thanks to karin enlund, åsa lundin, rn, ohn, and dr nina johnston for 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d, nowak d. work-related respiratory disorders and farming characteristics among cattle farmers in northern germany. am j ind med. 1999;36:444–9. asthma increase among farmers 71 ujms109_3.pdf upsala j med sci 109: 255–260, 2004 three cases of malacoplakia of the gallbladder margrét agnarsdóttir,1 roger willén,1 imad abdien el hag2 1pathology & cytology, centre for laboratory medicine, university hospital, s-75185 uppsala, sweden 2paras central hospital, po box 961, sakaka al-jouf, kingdom of saudi arabia. abstract a free full-text copy of this article can be found at the web page of upsala j med sci: http://www.ujms.se malacoplakia is a granulomatous disease with a histiocytic infiltrate containing calcified bodies called michaelis-gutmann bodies considered to represent an abnormal response to infection involving defective lysosomes and abnormal microtubular assembly. the disease most frequently involves urinary and genital tracts, but has also been described from most organs. reports from the gallbladder are extremely rare and as it might simulate specific infection, parasitic infestation as well as malignancy it is of importance for the surgeon and pathologist to be aware of the entity. in this article we present three cases of malacoplakia of the gallbladder, a rare disease in this location. introduction malacoplakia is a granulomatous disease where microscopic examination shows a histiocytic infiltrate (von hansemans cells) with calcified bodies called michaelisgutmann bodies. these can be seen both intra and extracellularily. the etiology of the disease is unclear. it is thought that it arises as a result of an abnormal response to an infection involving defective lysosomes and abnormal microtubular assembly [1]. the first article describing the disease was published in 1902 [2]. the disease most frequently involves the urinary tract although it has been described in many other organs including the male and female genital tract, the gastrointestinal tract, the retroperitoneum, and the skin [3]. very few cases have been reported from the gallbladder [4, 5, 6]. as the entity might simulate specific infection, parasitic infestation and malignancy we thought it of importance to report another three cases. 255 received 31 march 2004 accepted 23 april 2004 key words: malacoplakia, gallbladder. materials and methods the surgical specimens were fixed in formaline and routinely processed. paraffin sections were stained with hematoxylin and eosin (he), periodic acid-schiff stain (pas) for mucin, prussian blue for iron and von kossa’s stain for calcium. 256 fig. 1. case 1. a concentric assembly of calcified bodies, he stain (a). a positive pas staining showing the characteristic michaelis-gutmann bodies (b). a histiocytic infiltrate also containing michaelisgutmann bodies, he stain (c). samples from case 2 were also examined with a transmission electron microscopy (tem). for tem formaldehyde-fixed tissue was transferred to 2 % glutaraldehyde in 0.1m cacodylate buffer with 4% sucrose. (effective osmolar pressure 300mosm/l) the material was dehydrated in graded alcohols and embedded in agar 257 fig. 2. case 2. the histiocytic infiltrate in the wall is partly arranged in a nodular fashion (a). a he stain showing the presence of the michaelis-gutmann bodies (b). an electron microscopic picture confirming the presence of the michaelis-gutmann bodies (c). 100 resin. the sections were cut at 50–70nm, double stained with 5% uranyl acetate and 0.3% lead citrate, and examined in a philips cm12 electron microscope. case reports case 1. a 64-year old man was referred for a cholecystectomy in the year 2003 at the university hospital in uppsala, sweden because of an inflamed and thickened gallbladder. an open cholecystectomy was performed. macroscopically the gallbladder had a thickened wall with small nodules. case 2. a 40-year old woman was referred for cholecystectomy in the year 1993 at soba university hospital in khartoum, sudan because of repeated attacks of calcular cholecystitis. an open cholecystectomy was performed. macroscopically the gallbladder was reddened and thickened with yellowish nodular areas. case 3. a 37-year old woman was referred for cholecystectomy in the year 2004 at the university hospital in uppsala, sweden because of an inflamed and thickened gallbladder which was removed laparoscopically. macroscopically the gallbladder had a thickened wall and a 1,5 cm stone in the collum area. microscopic examination of samples from all the cases showed chronic inflammation with a histiocytic infiltrate partly arranged in a nodular fashion (fig.1c, 2a, 2b). in this histiocytic infiltrate the characteristic michaelis-gutmann bodies was found in the cytoplasm. these stained positively in the pas stain in all cases (fig. 1b, 3) and in case 1 a concentric assembly of calcified bodies was found (fig. 1a). in case 2 their presence was also confirmed with an electron microscopic examination (fig. 2c). a von kossa’s stain for calcium was performed for all cases but 258 fig. 3. case 3. a positive pas stain showing the michaelis-gutmann bodies. unfortunately the staining wasn’t reliable in case 2 and 3. some histiocytes contained iron-pigments. discussion malacoplakia of the gallbladder is a rare disease with only a handful of cases described [4, 5, 6, 7]. characteristically the michaelis-gutmann bodies usually stain positively for periodic acid-schiff stain, van kossa’s stain for calcium and iron [3, 7]. clinically the appearance of the gallbladder might suggest a specific infection as tuberculosis, infestation of parasites e.g. leishmaniasis [8] (especially so in tropical regions) and a malign process. it is therefore of importance for surgeons and histopathologist to know that this entity does exist also in the gallbladder, in order to install proper treatment and to avoid unnecessary extensive surgery. acknowledgements we are grateful to frank bittkowski for his skilled photographic help. grants from the university of gothenburg and uppsala as well as björnssons foundation supported this study. references 1. dasgupta p, womack c, turner a-g, blackford h-n (1999). malacoplakia: von hansemann’s disease. bju int 84:464–469. 2. michaelis l, gutmann c (1902). über einschlüsse in blasentumoren. ztschr klin med 47: 208–215. 3. stanton m-j, maxted w (1981). malacoplakia: a study of the literature and current concepts of pathogenesis, diagnosis and treatment. j urol 125:139–146. 4. hanada m, tujimura t, kimura m (1981). cholecystic granulomas in gallstone disease. a clinicopathologic study of 17 cases. acta pathol jpn mar;31(2):221–231. 5. charpenter p, prade m, bognel c, gadenne c, duvillard p (1983). malacoplakia of the gallbladder. hum pathol 14(9):827–828. 6. hide g, desai s, bloxham c-a (2001). malakoplakia of the gall-bladder: imaging and histological features. clin radiol 56(4):326–328. 7. willén r, stendahl u, willén h, tropé c (1983). malacoplakia of the cervix and corpus uteri: a light microscopic, electron microscopic, and x-ray microprobe analysis of a case. int j gynecol pathol 2:201–208. 8. fahal ah, el hag ia, el hassan am, hashim fa (1995). leishmanial cholecystitis adn colitis in a patient with visceral leishmaniasis. trans r soc trop med hyg may-jun;89(3):284. corresponding author: roger willén md, phd pathology and cytology centre for laboratory medicine university hospital s-75185 uppsala sweden phone: +46-18-6119408 e-mail: roger.willen@akademiska.se 259 bok 03-06.indb mode of growth determines differential expression of prostasomes 293 received 15 february 2006 accepted 31 may 2006 mode of growth determines differential expression of prostasomes in cultures of prostate cancer cell lines and opens for studies of prostasome gene expression lena carlsson1, lena lennartsson2, gunnar ronquist1, anders larsson1, sten nilson2, ove nilsson3 1department of medical sciences, clinical chemistry, university hospital, uppsala, sweden. 2department of oncology, karolinska hospital, stockholm, sweden. 3department of medical cell biology, biomedical center, uppsala, sweden. abstract the exocrine secretion of the acinar gland cells in the human prostate consists of, among other components, a serous secretion and prostasomes. the prostasomes are functionally associated with both reproduction and prostate cancer development and are capable to raise autoantibodies at various pathologies. therefore, we are trying to characterize prostasome antigens by analysing prostasome-producing cell lines of prostate cancers with the cdna microarray technique. to obtain one state with synthesis of prostasomes and another state without synthesis, we checked whether the prostasome differentiation was influenced by the mode of growing the cells, that is, whether the cells had been growing on a solid support or on a flexible one. we studied the expression of prostasomes in the cell lines pc3, du145 and lncap. we grew the cells with the following methods: monocellular layers on microbeads, multicellular spheroids, single cells in suspension cultures, and xenotransplants in nude rats. the presence of prostasomes was examined by elisa, immunocytochemistry or electron microscopy. the results showed that growing the cells on microbeads (solid support) produced a differentiation of prostasomes, while growing the cells in multicellular spheroids (flexible support) did not. thus it should be possible to apply cdna microarray analyses for characterizing the genes which are active at the cellular expression of prostasomes and then deduce the prostasome antigens. introduction the exocrine secretion of the acinar gland cells in the human prostate consists of, among other components, a serous secretion and small bodies (prostasomes) both of which are located within storage vesicles in the supranuclear parts of the cells. the prostasomes are complex structures and exert several abilities, which are associated with both reproduction and prostate cancer development (1). this has made them interesting in our research on immunocontraception (2) and prostate cancer metastases (3). as a further step in these studies, we have work in progress to find ways to map the prostasome genes. the prostasome gene expression can be deduced by, among other methods, cdna microarray analyses. in this case, two states of growth of the prostate cells have to be compared: one state with synthesis of prostasomes and another state upsala j med sci 111 (3): 293–301, 2006 294 lena carlsson et al. without synthesis. as yet, though, no culturing technique of prostate cancer cell lines for achieving this has been reported. various cellular differentiation parameters of cultured cells, however, can be influenced by the mode of growing the cells. for instance, the expression of differentiation markers in cultured cells of the choriocarcinoma cell-line bowo was influenced by the type of support for the growing cells (4), that is, whether the cells had been growing on a solid support or on a flexible one. now, we know that the cells of prostate cancer lines do produce prostasomes when growing as monolayers on the solid support of petri dishes (5) and that these prostasomes have properties similar to those of seminal prostasomes (6, 7). therefore, we have checked whether also the synthesis of prostasomes is dependent upon the type of cell support during culturing, that is, whether the prostasome differentiation could be influenced by the mode of growing the cell lines. we studied the expression of prostasomes in the cell lines pc3, du145 and lncap. we grew the cells with the following methods: 1. monocellular layers on microbeads. 2. multicellular spheroids. 3. single cells in suspension cultures. 4. xenotransplants in nude rats. the presence of prostasomes was examined by elisa, immunocytochemistry or by electron microscopy. the use of electron microscopy was required due to the small size of the prostasomes. materials and methods cell lines the human prostate carcinoma cell lines pc3, du145 and lncap were obtained from the american type culture collection (rockville, md, usa). cells were grown in rpmi 1140 cell culture medium containing 5 μg/ml phenol red and supplemented with 10% heat-inactivated fetal calf serum, 1% glutamine, 100 μg/ml penicillin and 100 ug/ml streptomycin. growth on solid support growth on microbeads the cells were grown on cytodex beads 1, 2 and 3 (pharmacia fine chemicals, uppsala , sweden) in culture dishes (100 mm) of tissue culture plastic (falcon, becton dickinson) at 37oc in humidified atmosphere of 5% co2 in air using the liquid-overlay technique (see later). cell carpets were obtained after about a week. the beads were saved by centrifugation. the cells were released by trypsination, washed twice in pbs and frozen at –70oc. frozen prostate cancer cells were thawed and pooled, and the suspension of disintegrated cells was centrifuged at 1500 x g for 30 min. the supernatant obtained was subjected to a second centrifugation at 6000 x g for 20 min. the new supernatant containing prostasomes was ultracentrifuged at 100,000 x g for 2 h. the pelleted material was suspended in isotonic tris-hcl buffer, ph7.6, and run through a mode of growth determines differential expression of prostasomes 295 sephadex g200 column (amersham biotech., uppsala, sweden) at 6 ml/h, and 2 ml fractions were collected. the isotonic tris-hcl buffer was the eluant, and the eluate was read at 260 and 280 nm. the fractions with initially elevated uv absorbances and with a positive reaction on the anti-prostasome mab 78 were pooled and ultracentrifuged at 100,000 x g for 2 h. isotonic tris-hcl buffer was added to the pellet, and the resulting mixture was regarded as suspensions of prostasomes. for enzyme-linked immunosorbent assay (elisa), plates were coated with the prepared prostasomes diluted in 100 mm nahco3, ph 9.5 (coating buffer), for 2 h at 37º c. the plates were blocked overnight at 4º c with coating buffer containing 3% bsa, then washed 3 times with 200 μl pbs-0.05% tween 20 (pbs-t) and incubated with either a monoclonal mouse anti-prostasome antibody or a polyclonal hen anti-prostasome antibody, both biotin conjugated, for 2 h at 37º c. after 3 washings with 200 μl of pbs-t, the plates were incubated with streptavidin-alkaline phosphatase conjugate (1:500 in pbs) at 37º c for 1 h and then washed 3 times with 200 μl of pbs-t. finally, 200 μl of alkaline phosphatase substrate solution (1mg/ml p-nitrophenyl phosphate in 1m diethanolamine, 0.5 mm mgcl2, ph 9.8) were added and the plates were incubated for 25 min at room temperature in the dark. the reaction was stopped by 50 μl of 5m naoh. absorbance was measured at 405 nm in an elisa reader system (spectra max 250, molecular devices, sunnyvale, ca, usa). growth on flexible support multicellular spheroids liquid-overlay technique was used to obtain the spheroids (8–10). in short, tissue culture dishes were coated with 0.5 ml of 1% agarose (sigma, st louis, usa) that was boiled in sterile rpmi-1640 without serum. cells of pc3 or du145 were plated in rpmi-1640 supplemented with 10% fetal calf serum and 10 mm hepes (sigma). since the cells could not attach onto the agarose layer of the culture dishes, the cells adhered onto each other and formed cell-clumps, that is, multicellular spheroids. the spheroids were fed every 3 days. the size of the cell spheroids increased over time and reached, after about two weeks, a diameter of approximately 1 mm. by then, the cell spheroids were fixed in formalin or bouin´s solution for 2 h. the specimens were embedded without delay according to the conventional techniques for immunocytochemistry or electron microscopy. single-cell suspension cultures single cells were noted in the culture medium of petri dishes. these cells were embedded in paraffin, using small tubes, for conventional immunostaining with the dab technique. xenotransplants cells from monolayer cultures of pc3, du145 or lncap in petri dishes, were scraped from the tissue culture dishes, washed free of growth medium, and injected 296 lena carlsson et al. s.c. into the flank region of nude rats. when the tumours were palpable, they were dissected free after various times to obtain tumours of various sizes. the specimens were processed for immunostaining or electron microscopy. immunocytochemistry the following protocol, rinsings excluded, was used for immunostaining. 1. blocking with 3% bsa and 1% normal horse serum in pbs for 30 min 2. primary antibody, being undiluted mouse monoclonal igg1 mab78 supernatant for 60 min 3. secondary antibody, being biotinylated horse anti-mouse igg, diluted 1:200 (sigma) for 60 min 4. alkaline phosphatase complex (dako pat) for 30 min 5. substrate vector red or dab for appropriate times positive controls were sections of human prostate gland and negative controls were obtained by omitting the primary antibody. figure 1. apical parts of normal prostate epithelial cells. the cytoplasm is filled with numerous storage vesicles containing small vesicles and dark bodies, i.e. prostasomes. mag. 9000 x. mode of growth determines differential expression of prostasomes 297 results ultrastructure of normal prostate cells electron microscopy of normal prostate epithelium shows that the apical parts of the cytoplasm are filled with numerous storage vesicles containing small, dark bodies, i.e. prostasomes (fig. 1). growth in petri dishes the general morphology of the growing monolayers were similar to earlier reports (11–13). at confluence, the cultures showed a continuous carpet of cells, but eventually small grape-like groups of cells appeared on the monolayers. trypsinized monolayers of pc3, du145 and lncap cells, prepared as cytospin specimens and immunostained with monoclonal anti-prostasome antibody, demonstrated that most cells expressed prostasomes and secretion but that scattered cells or small groups of cells did not show any prostasomes. growth on microbeads pc3 cells grown on three types of cytodex beads were tested by elisa for the presence of secretion. binding of anti-prostasome antibody was demonstrated in figure 2. a cell from the prostate cancer cell line pc3 growing at the periphery of a multicellular spheroid. the vacuoles are remnants of dissolved lipid granules, and no storage vesicles containing prostasomes are visible. mag. 6000 x. 298 lena carlsson et al. preparations from cells grown on cytodex 2 and 3, with cytodex 3 having the highest absorbance value (1.45) compared to cytodex 2 (0.38). preparations from cells grown on cytodex 1 beads did not show any significant increase in absorbance value compared to negative controls. multicellular spheroids electron microscopy of the spheroid-like structures demonstrated that none of the cell lines of pc3 (fig. 2), du145 (fig. 3) or lncap cells contained storage vesicles with prostasomes. suspension cultures of single cells immunostaining of paraffin-embedded cells demonstrated that some cells contained secretion, while others did not. xenotransplants most of the mice inoculated subcutaneously with cells of the prostate cancer lines pc3, du145 or lncap developed tumours. the tumours consisted of lobular figure 3. part of the cytoplasm of a cell from the cancer cell line du145 growing in the periphery of a multicellular spheroid. the surface of the spheroid is observed to the left, and an intercellular space to a neighbouring cell is noticed at the right side of the picture. the cytoplasm contains the ordinary organelles and inclusions but no storage vesicles with prostasomes. mag. 13,000 x. mode of growth determines differential expression of prostasomes 299 groups of cells surrounded by strands of host connective tissue, which in addition formed a capsule around the tumour. no storage vesicles containing prostasomes were observed in the cells of the transplanted cell lines. discussion the apical parts of the normal prostate epithelium contain numerous storage vesicles with secretion and prostasomes. also cell lines of prostate cancers, grown on the solid support of petri dishes, are known to produce prostasomes (5). in the present experiments, we grew the prostate cell lines on the solid support of sephadex microbeads and found that the cells also then were expressing prostasomes. however, when the cells were growing in multicellular spheroids or as xenographs and thus being offered only the flexible support of their cell neighbours, we could not demonstrate any prostasomes. thus, the prostasome differentiation of the cancer cell lines pc3, du145 and lncap is influenced by the mode of growth of the cells. cells growing in culture dishes, prepared by the cytospin technique and then immunostained contained secretion and prostasomes, although some single cells did not (5). we assume that these cells had been squeezed-out from confluent monolayers and by loosing the solid support of the petri dish, the cells ceased to make prostasomes. consequently, studies of paraffin-embedded single cells from the cells suspended in the culture medium showed that some cells contained secretion while others did not. thus, there is a gradual slowing down of the secretory activity, when the cells loose their attachment to a solid support and begin living as free-floating cells or as aggregated cells in cell spheroids. xenotransplants of cancer cell lines grown in immune-deficient mice or rats are often considered as counter-parts to poorly differentiated metastases of prostate cancers (14,15). however, poorly differentiated prostate cancer cells in metastases contain cells which still have the ability to produce prostasomes (16, 17), but our xenotransplants did not demonstrate any prostasomes-containing cells. thus, the influence of the mode of growth on the expression of the differentiation markers studied has to be considered when planning experiments involving prostate cancer cell lines. differentially expressed genes related to prostate cancer have been studied in prostate cancer cell lines with various aims (for references, see 18). for instance, by comparing two lncap cell lines, one androgen-dependent and the other androgen-independent, the gene expression changes during prostate cancer progresssion could be evaluated (19, 20). our results disclose that the genes coding for the prostasome synthesis are differentially expressed due to their mode of growth, that is, prostasomes are expressed by cells growing on solid supports but not on flexible supports. thus, for exemple, by comparing a cancer cell line grown on microbeads (solid support) with the same cell line grown as multicellular spheroids (flexible support), it should be possible 300 lena carlsson et al. to study the expression of prostasome genes and co-expressed genes by cdna microarray screening. additional methods to deduce the prostasome gene expression are also available, for instance, mass spectrometry and screening of cdna libraries. each of these techniques has its drawback and advantage. thus, mass spectrometry requires preparation of the prostasomes with the risk of loosing some prostasome components during the procedures. screening of cdna libraries can be time consuming, but the method defines the genes, which correspond to existing monoclonal antiprostasome antibodies. cdna microarray studies are based on whole cells containing unaffected prostasomes and can offer a broad list of genes active at the cellular expression of prostasomes. since we have shown that prostasomes can raise autoantibodies both at immunological infertility and in patients with metastasizing prostate cancer, well-characterized prostasome antigens would be a valuable tool in our current research. it seems that cdna microarray analyses of prostasome genes followed by protein mappings (www.proteinatlas.org) offer a way to obtain specific prostasome polypeptides which can be used, for instance, in studies of anti-prostasome autoantibodies. acknowledgements this study was supported by the swedish medical research council. references 1. ronquist g, nilsson o (2004) the janus-faced nature of prostasomes: their pluripotency favours the normal reproductive process and malignant prostate growth. prostate cancer prostatic dis 7: 21–31. 2. carlsson, l., larsson, a., ronquist, g. and nilsson, b.o. prostasome antigens as targets for sperm agglutinating antibodies demonstrated by 1-d gel electrophoresis and immunoblottings. int j androl 27:360–367, 2004. 3. nilsson bo, carlsson l, larsson a, ronquist g (2001) autoantibodies to prostasomes as new markers for prostate cancer. upsala j med sci 105: 43–50. 4. hohn h-p, grummer r, bosserhoff s, graf-lignau s, reuss b, bäcker c, denker h-w (1996) the role of matrix contact and of cell-cell interactions in chorioncarcinoma cell differentation. eur j cell biol 69: 76–85. 5. nilsson bo, lennartsson l, carlsson l, nilsson s, ronquist g (1999) expression of prostasome-like granules by the prostate cancer cell lines pc3, du145 and lncap grown in monolayer. upsala j med sci 104: 199–206. 6. wang j, lundquist m, carlsson l, nilsson o, lundquist ö, ronquist g (2000) prostasome-like granules from the pc3 prostate cancer cell line increase the motility of washed human spermatozoa and adhere to the sperm. eur j obstet gynecol reprod biol 96: 88–97. 7. carlsson l, nilsson o, larsson a, stridsberg m, sahlén g, ronquist g (2003) characteristics of human prostasomes isolated from three different sources. prostate 54: 322–330. 8. donaldson jt, tucker ja, keane te, walther pj, webb ks (1990) characterization of a new model of human prostatic cancer: the multicellular tumor spheroid. int j cancer 46: 238–244. 9. ballangrud m, yangwh, dnistrian a, lampen nm, sgouros g (1999) growth and characterization of lncap prostate cancer cell spheroids. clin cancer res 5: suppl., 3171s–3176s. mode of growth determines differential expression of prostasomes 301 10. santini mt, 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neoplastic epithelial prostate cells. prostate 35: 36–40. 17. sahlén g, ahlander a, frost a, ronquist g, norlén bj, nilsson bo (2004) prostasomes are secreted from poorly differentiated cells of prostate cancer metastases. prostate 61: 291–297. 18. calvo a, gonzalez-moreno o, yoon cy, huh ji, desai k, nguyen qt, green je (2005) prostate cancer and the genomic revolution: advances using microarray analyses. mutat res 576: 66–79. 19. vaarala mh, porvari k, kyllönen a, vihko p (2000) differentially expressed genes in two lncap prostate cancer cell lines reflecting changes during prostate cancer progression.lab invest 80: 1259–1268. 20. zhao h, kim y, lapoint j, tibshirani r, pollack jr, brooks jd (2005) genome-wide characterization of gene expression variations and dna copy number changes in prostate cancer cell lines. prostate 63: 187–197. corresponding author: b. ove nilsson md, phd department of medical cell biology biomedical center, uppsala university s-751 23 uppsala, sweden e-mail: ove. nilsson@medcellbiol.uu.se untitled serum carbamazepine and its 10,11-epoxy derivative 171 key words: carbamazepine; carbamazepine-10,11-epoxide; renal insufficiency; emit; hplc; therapeutic monitoring. received 16 january 2008 accepted 28 january 2008 upsala j med sci 113 (2): 171–180, 2008 relative proportions of serum carbamazepine and its pharmacologically active 10, 11-epoxy derivative: effect of polytherapy and renal insufficiency maría j. tutor-crespo, jesús hermida, j. carlos tutor unidad monitorización fármacos, laboratorio central, hospital clínico universitario, 15706 santiago de compostela, galicia, spain. abstract background: the proposed action mechanism and pharmacological activity of carbamazepine (cbz) and its major metabolite, carbamazepine-10,11-epoxide (cbze), are the same. the aim of our study was the investigation of the effect of concomitant antiepileptic treatment and renal insufficiency on the relative proportions of serum cbz and cbze. methods: serum trough steady-state cbz and cbze concentrations were determined by high-performance liquid chromatography (hplc) in 140 epileptic patients treated with cbz in monotherapy (n=100) and polytherapy with phenytoin, phenobarbital and valproate (n=40). the levels of cbz were also determined using the dade behring enzyme multiplied immunoassay technique (emit). the glomerular filtration rate (gfr) was estimated from serum cystatin c using the dade behring nephelometric immunoassay. results: the cbze/cbz and cbz+cbze/cbzemit ratios were significantly increased in 7 cases (3 in monotherapy and 4 in polytherapy) with gfr<60 ml/ min/1.73m2 in relation to the patients treated in monotherapy or polytherapy having normal or mildly decreased renal function (p<0.001). conclusions: in patients with moderate to severe renal insufficiency the relative proportion of cbze with respect to the parent drug is significantly increased. in these cases, the cbz concentrations obtained using the emit, or other immunoassays having low cbze cross-reactivity, may have an inadequate diagnostic efficiency. introduction carbamazepine (cbz) is one of the most widely prescribed drugs for the treatment of childhood and adult epilepsies. in humans only 2% of the dose is excreted as unchanged drug, and the biotransformation pattern of cbz is varied and complex (1). the principal pathway of cbz metabolism is oxidation to the pharmacologically active carbamazepine-10,11-epoxide (cbze), and then hydration to inactive transcarbamazepine-10,11-diol (cbzd) (1,2). the metabolisation of cbz to cbze is catalysed by the cytochrome p450 (cyp) isoforms cyp3a4 and cyp2c8, and the hydrolysis of cbze to cbzd is catalysed by a microsomal epoxide hydrolase (3). the corresponding glucuronide derivatives, and also unconjugated cbz, cbze and cbzd, have been characterized in urine from patients treated with cbz (2). the 172 maría j. tutor-crespo et al. renal clearances of cbz and cbze, about 1 ml/min and 8 ml/min respectively, are urine flow dependent, and cbzd clearance is greater than that of creatinine and relatively independent of urine flow (4). these low cbz and cbze clearance values and their urine flow dependence indicate that glomerular filtration is the main mechanism of renal elimination for both compounds (4). concomitant administration of enzyme-inducing anticonvulsant drugs (phenobarbital and phenytoin) or valproic acid, albeit through different mechanisms, significantly increase the relative proportion of cbze in serum (5–7); however, to our knowledge, the possible effect of renal insufficiency on the relative proportions of cbz and cbze has not been still well studied. drug metabolites with pharmacological activity are candidates for monitoring, although in clinical practice the metabolites that are routinely measured are those for which a convenient method is available (8). determination of cbz is usually carried out using different commercial immunoassays, and the active cbze metabolite is not routinely monitored together with cbz as its measurement requires a high-performance liquid chromatography (hplc) method (8). the cross-reactivity against cbze of the different immunoassays currently used for routine cbz determination varies considerably (9–13), and the enzyme multiplied immunoassay technique (emit) has a low cross-reactivity with the cbze metabolite (11,14–16). the main aim of our study was to investigate the comparative effect of concomitant antiepileptic treatment and renal insufficiency on the relative proportions of cbz and its epoxy derivative in serum, and its possible implications on the cbz determination using emit. patients and methods a group of 140 epileptic patients (73 males and 67 females) with a mean age of 33.8±2.0 years (range 3–88 years), and treated with cbz in monotherapy (n=100) and polytherapy (n=40), was studied. in the cases of polytherapy, the concomitantly administered antiepileptic drugs were phenobarbital (n=9), phenytoin (n=14) and valproic acid (n=18). daily cbz administration was always given in multiple doses, and blood samples were taken at least after a 2 month period without any modification of the dosage, and immediately before the first corresponding daily dose. as a result, the cbz and cbze serum concentrations correspond to the steady-state trough levels. the study was carried out according to the good practice rules for the investigation in humans of the conselleria de sanidade-xunta de galicia, and all patients provided their written informed consent to participate. serum levels of cbz and cbze were determined by hplc using an agilent 1100 series chromatographic system. the assays were carried out using an isocratic system with uv detection at 204 nm, a detection limit of approximately 0.5 mg/l for cbz and cbze, and intra-and inter-assay variation coefficients of less than 5%. likewise, serum concentrations of cbz were determined using the emit 2000 carbamazepine assay in a v-twin analyzer (dade behring). serum samples serum carbamazepine and its 10,11-epoxy derivative 173 were stored for less than 7 days at -25 ºc, conditions that provide adequate stability for the parent drug and its epoxide derivative (17). total cbz clearance (clt) was calculated using the expression (18): clt=f(dose/τ)/css, where f is the bioavailability (0.8), τ the dosing interval, and css the steady-state trough serum concentration. cystatin c was determined using the dade behring particle-enhanced nephelometric immunoassay on the bn prospec nephelometer system, and the glomerular filtration rate (gfr) was estimated from serum cystatin c according to hoek et al (19). in order to estimate the gfr from serum creatinine, the sixvariable modification of diet in renal disease (mdrd) formula (20) was used. serum albumin, creatinine and urea concentrations, were determined in an advia 1650 analyzer (siemens medical solutions). the microsoft excel package (v 5.0) was used to statistically analyse the data, and the kolmogorov-smirnov test was applied to check for normality. pearson’s correlation coefficient was used when the data had gaussian distributions; otherwise, spearman’s correlation coefficient was used. the regression study was made using the passing-bablock method, and the ma68 value was used as error of the estimate. according to the consensus validation criteria of analytical methods for quantitative determination of drugs and their metabolites in a biological matrix (21,22), the acceptance criterion for emit accuracy was a deviation of no more than 15% from the nominal (hplc) value. the results were expressed as mean±sem (median). results seven of the 140 patients studied (3 in monotherapy and 4 in polytherapy), with a mean (±sem) age of 78.3±2.7 years (range: 76–88 years), presented an estimated gfr from serum cystatin c <60 ml/min/1.73m2, which in accordance with the national kidney foundation guidelines indicates a moderate to severe kidney function impairment (22). in these 7 cases, the mean gfr value estimated from serum cystatin c (19) was 41.97±6.29 ml/min/1.73m2 (range 19.3–57.5 ml/min/1.73m2), and from serum creatinine using the six-variable mdrd formula (20) was 43.15±8.40 ml/min/1.73m2 (range 12.8–61.0 ml/min/1.73 m2), with a correlation coefficient between them of r=0.963 (p<0.005). as shown in figure 1a, the relative proportion of cbze with respect to cbz was significantly higher in the patients who had gfr values of less than 60 ml/ min/1.73m2, independently of whether they were treated in monoor polytherapy. furthermore, in these patients the cbz+cbze/cbzemit ratios were significantly higher than in the patients with better renal function (figure 1b); however, the cbzhplc/cbzemit ratio does not appear to be significantly altered by the patients’ gfr, as indicated in figure 1c. a highly significant correlation was found for the total number of patients studied between the cbze/cbz and cbz+cbze/ cbzemit ratios (r=0.774, p<0.001). these results are in accordance with the low cross-reactivity of the emit against to cbze previously described (11,14–16). 174 maría j. tutor-crespo et al. figure 2 shows the regression and correlation found between the results obtained by emit for cbz and the results obtained by hplc for cbz (figure 2a) and cbz+cbze (figure 2b). the difference between the means (medians) obtained by emit and hplc for the cbz concentration, 8.20±0.24 mg/l (8.0 mg/l) and 7.66±0.23 mg/l (7.30 mg/l) respectively, is acceptable in accordance with the validation criterion used (21,22). the degree of renal insufficiency does not appear to affect the dispersion between the values for cbz obtained by emit and hplc (figure 2a), although on considering the values of cbz+cbze, the deviation is higher in cases with a gfr<60 ml/min/1.73m2 (figure 2b). the results obtained for the concentrations of cbz and cbze in the patients with gfr greater than 60 ml/min/1.73m2 (n=133) are shown in table 1. in accordance with our previously published results (10,13), the mean cbze/cbz ratios in the patients with concomitant valproic acid (0.40±0.04) or phenobarbital and phenytoin (0.41±0.04) were analogous. the difference between the means (medians) of the concentrations obtained for cbz by emit and hplc is acceptable in accordance with the validation criterion used (21,22); however, the sum cbz+cbze has a difference higher than 15% with respect to the parent drug levels obtained using emit or hplc. as would be expected, the cbze/cbz ratio and cbz clt were significantly higher in the patients treated in poytherapy (p<0.001), and a highly significant correlation was found between both pharmacokinetic variables (r=0.420, p<0.001). in the 7 patients having gfr <60 ml/min/1.73m2, the levels of cbz and cbze were respectively 5.91±1.03 mg/l (5.2 mg/l) and 5.29±1.11 mg/l (6.1 mg/l), with a cbze/cbz ratio of 0.94±0.18 (0.91) significantly greater than those obtained for the patient groups with normal renal function treated in mono or polytherapy (p<0.001). discussion in our patients, the estimation of the gfr was carried out using serum cystatin c according hoek et al (19). cystatin c has been proposed as a good gfr marker, as this low-molecular protein is produced at a constant rate regulated by a “housetable 1. serum levels of carbamazepine, carbamazepine-10,11-epoxide, and total clearance of carbamazepine in the patients with glomerular filtration rate greater than 60ml/min/1.73 m2 n cbzemit (mg/l) cbzhplc (mg/l) cbze (mg/l) cbze/ cbz clt cbz (ml/min) monotherapy 96 8.61±0.26 (8.40) 8.10±0.25 (7.80) 2.06±0.10 (1.90) 0.25±0.01 (0.24) 50.83±1.67 (50.16) polytherapy 37 6.95±0.51 (6.80) 6.40±0.50 (6.30) 2.30±0.14 (2.30) 0.40±0.03 (0.40) 87.33±8.00 (85.50) the results are expressed as mean±sem(median) serum carbamazepine and its 10,11-epoxy derivative 175 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 0 20 40 60 80 100 120 140 160 180 200 gfr (ml/min/1.73m2) c b ze /c b z ra tio a 0.5 1.0 1.5 2.0 2.5 3.0 0 20 40 60 80 100 120 140 160 180 200 gfr (ml/min/1.73m2) c b z+ c b z e /c b z e m it (m g/ l ) b 0.8 0.9 1.0 1.1 1.2 1.3 1.4 0 20 40 60 80 100 120 140 160 180 200 gfr (ml/min/1.73m2) c b z e m it /c b z h p l c c figure 1. relationship of the cbze/cbz, cbz+cbze/ cbzemit, and cbzemit/ cbzhplc ratios with the gfr in epileptic patients treated with cbz in monotherapy (circles) and polytherapy (triangles). 176 maría j. tutor-crespo et al. keeping” gene expressed in all human nucleated cells (23), and freely filtered at the glomerulus (19). the recent results of pirttilä et al, demonstrate an increased cystatin c expression in astrocytes and neurons during human chronic epilepsy (24); however, this unregulated expression of cystatin c does not appear to possess a systemic condition, as in our epileptic patients with gfr <60 ml/min/1.73 m2, the estimation of gfr values from serum cystatin c (19) and creatinine using the six-variable mdrd equation (20) led to analogous results. 0 5 10 15 20 0 5 10 15 20 cbzemit (mg/l) c b z h p l c ( m g /l ) a y=0.945x-0.158 r=0.979 ma68=0.371 0 5 10 15 20 25 0 5 10 15 20 25 cbzemit (mg/l) c b z+ c b ze ( m g/ l) b y=1.187x+0.169 r=0.917 m a68=0.576 figure 2. correlation and regression between the obtained cbz by emit and cbz (a) and cbz+cbze (b) using hplc in epileptic patients treated with cbz in monotherapy (circles) and polytherapy (triangles), with gfr lower (closed) and higher (open) than 60 ml/min/1.73m2. the dashed lines correspond to the cbz therapeutic range limits (4-10 mg/l). serum carbamazepine and its 10,11-epoxy derivative 177 our results show that in the patients with gfr<60ml/min/1.73m2 there is an exponential increase of the cbze/cbz ratio, in parallel with the cbz+cbze/ cbzemit ratio, as renal function decreases, without the cbzemit/cbzhplc ratio being modified in any significant way (figure 1). the increase of the relative proportion of cbze with respect to the parent drug in renal insufficiency is significantly higher than that produced by the concomitant administration of phenobarbital, phenytoin and valproic acid. in the 5 patients having gfr <50 ml/min/1.73 m2, serum concentrations of the cbze metabolite may be similar, or even greater, than those of cbz, in contrast to the patients, both in mono or polytherapy, with normal or mildly decreased renal function in which the cbze/cbz ratio was always <0.7 (figure 1a). the cbze metabolite has been shown to posses similar pharmacological activity and proposed action mechanism as that of the cbz (25), and consequently, the monitoring of the parent drug and its epoxy derivative may be desirable (11). in patients with severely impaired renal function, we should expect that serum levels of cbz obtained using emit may be clinically unacceptable, because therapeutic serum concentrations of cbz would present neurological toxicity as cbze adds to the therapeutic effect of cbz (figure 2b). in the patients having normal or mildly decreased renal function, the cbz results obtained by emit present a lower deviation with respect to the sum cbz+cbze (figure 2b). in these patients, cbz+cbze would be estimated from the cbzemit values using the linear regression equations: cbz+cbze=1.23cbzemit-0.49 (ma68=0.48 mg/l, r=0.961) for monotherapy, and cbz+cbze=1.14cbzemit+1.02 (ma68=0.48, r=0.969) for polytherapy, with a diagnostic efficiency of 93% for the correct classification of the results as subtherapeutic, therapeutic and supratherapeutic levels (data not shown). in conclusion, an increased proportion of the cbze metabolite in relation to the parent drug was obtained in serum from patients with renal insufficiency, which was significantly greater than that produced by concomitant phenobarbital, phenytoin and valproic acid administration. consequently, on the contrary to the case of phenytoin (26), the cbz monitoring using immunoassays may be clinically unsuitable in patients with severely impaired renal function. in these cases, the cbz determination using the dade dimension immunoassay, which has the highest cbze cross-reactivity of all the most frequently used commercial immunoassays (10,11), should be evaluated. acknowledgements one of the authors (mjtc) received a grant from dade behring obtained through the fundación investigación, desarrollo, innovación, complexo hospitalario universitario de santiago (idichus). 178 maría j. tutor-crespo et al. references amore bm, kalhorm tk, skiles gl et al (1997) characterization of carbamazepine metabolism 1. in a mouse model of carbamazepine teratogeneity. drug metab dispos ; 25: 953–62. maggs jl, pirmohamed m, kitteringham nr, park bk (1997) characterization of the metabo-2. lites of carbamazepine in patient urine by liquid chromatography/mass spectrometry. drug metab dispos; 25: 275–80. reith dm, appleton db, hooper w, eadie mj (2000) the effect of body size on the metabolic 3. clearance of carbamazepine. biopharm drug dispos; 21: 103–11. vree tb, janssen tj, hekster ya, 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in serum 26. samples from patients with renal insufficiency: comparison with high-performance liquid chromatography. j clin lab anal; 21: 119–23. corresponding author: j. carlos tutor, pharmd, phd unidad monitorización fármacos, laboratorio central hospital clínico universitario, 15706 santiago de compostela, galicia, spain email: jocatuva@hotmail.com josecarlostutor@redfarma.org upsala j med sci 99: 237-250, 1994 5. traceability of concentration values 5.1 establishment of a new reference preparation for 14 plasma proteins/crm 470 = rpphs lot 5 s. baudner behringwerke ag, research laboratories, 35001 marburg/lahn, frg on january 1, 1990 the committee on plasma protein standardization (pps) of the international federation of clinical chemistry (ifcc) was established. the members of this committee are: j.t. whicher, leeds/great britain (chairman) s. baudner, marburg/germany j. bienvenu, lyon/france s. blirup-jensen, giostrup/denmark r. ritchie, scarborough/usa further scientists working as associated members &d take part in this committee. their task was to develop a new reference preparation for human plasma proteins which could be accepted worldwide and which would substitute the established who international reference preparations which had proved themselves as unsuitable for some modern immunoassays. 5.1.1.1 quality requirements to the new reference material after some prelirmnary discussions it became the definite object of the committee on pps to reconcile the european and american tendencies as a basis recommendation for the standardization of plasma protein determination. such a reference preparation would have t o fulfil the following quality aims and claims: * all serum samples for the pool from healthy blood donors only with complete anamnesis and with established demographic constitution * absence of infectious diseases (certified) * serum pool immunochemically suitable for as many plasma proteins as possible 237 * physico-chemical characteristics of proteins clearly documented suitable for the majority of the commercially available immunoassays method dependent bias very low * longterm stability/long shelflife ( > 5 years) * suitable as an official calibrator * available in large quantities 5.1.1.2 milestones the above mentioned requirements and the studies to obtain the necessary stability data were accepted and initiated by the bureau communitaire de reference of the european communities (bcr, brussels/belgium) and by the college of american pathologists (cap, northfield/usa), two institutions which have supported the ifcc project by financing various steps. blood collection and the necessary characterization of the samples were organized to start in february 1990. the new matrix reference material was produced as a stabilized serum pool in december 1990. the filling and the freeze drying operations were performed in june 1991 after a previous successful trial using a pilot batch l o t 0100/36. the final product was labeled with lot 91/0619. the freeze dried ampoules of the final product were divided into two groups with the following labeling: a. b. for the european users (bcr) commission of the european communities bcr reference material for proteins in human serum crm no 470 cap/ifcc lot 91/0619 for in vitro diagnostic use only/for 1 ml for the american users (cap) college of american pathologists reference preparation for proteins in human serum caf'/bcr/ifcc lot 91/0619 for in vitro diagnostic use/for 1 ml a value assignment was performed for the following 14 plasma proteins: igg, iga, igm, albumin (alb), transferrin (tf) all mentioned in the german quality control guidelines (rilibak) 238 transthyretin (prealbumin/ttr), al-acid glycoprotein (aiag), haptoglobin (hpt), al-proteinase-inhibitor (q-antitrypsin/alat), a2-macroglobulin (mm), ceruloplas min (cer), c3, c4, crp (by spiking). a detailed report of the ifcc committee on pps on the protein serum standard was released by bcr the material was named crm 470, which stands for certified reference material for immunochemical determinations of 14 human serum proteins. 5.1.2 ifcc material lot 91/0619 blood collection human serum obtained from spontaneously clotted blood of healthy blood donors was used to prepare t h e reference preparation for human plasma proteins. the human serum samples were provided by five different european blood centres: two actions for blood donation each from sheffield and danderyd (table 1). all together there were seven different blood collections, carried out by: the karolinska institute, danderyds hospital, danderyd/sweden the national blood transfusion service, sheffield/great britain the centre national de transfusion sanguine, orsay/france der schweizerische blutspendendienst, bern/switzerland die behringwerke ag, marburg/germany haemolytic and lipaemic sera were discarded after an optical control. the preser vatives sohum azide (0.5 g/l) and the proteinase-inhibitor aprotinin (80,000 kiu/l) were added to t h e selected samples. thereafter they were frozen by means of liquid nitrogen and stored at 70°c for further use. small quantities of the samples of every blood donation were stored separately and used for various analytical tests in accordance with t h e "requirements for biological substances/reference reagents" of t h e who. a medical report was available for each blood donor. only serum donations which fulfill the characteristics and requirements for "healthy blood donors" were further processed. the exclusion of monoclonal immunoglobulin classes igg, iga, igm was also ensured. data of concentration for cholesterol, triclycerides, glucose and got were obtained: they were all normal. the phenotypes of al-proteinase inhibitor and haptoglobin were checked. blood group, age, sex and race of the blood donors were listed. serum samples containing factors which could possibly have an influence on the results of an immunoassay like rheuma factors ( > 30 iu/l), haemolytic components (haemoglobin >40 mg/l) were discarded. finally 364 blood donations (donors 20 to 60 years old; 65% male) were found to be suitable and were used for the ifcc reference material. 239 h , p 0 s o u rc e t a b l e 1 c r m 4 70 r p p h s l ot 5 / c ol le ct io n of b lo od d a te l o t n o . 1 . m r 0 1 0 0 /1 2 2 . b e 0 1 0 0 /1 8 3 . s h 0 1 0 0 /3 9 4 . s h 0 1 0 0 /4 0 5 . d s 0 1 0 0 /4 1 6 . d s 0 1 0 0 /4 2 7 . o r o io o /3 a m a r b u r g i g e r m a n y b e r n e i s w i t z e r l a n d s h e f f i e l d / g r e a t b r i t a i n s h e f f i e l d i g r e a t b r i t a i n d a n d e r y d i s w e d e n d a n d e r y d i s w e d e n o r s a y i f r a n c e f e b r u a r y f e b r u a r y f e b r u a r y s e p t e m b e r m a r c h s e p t e m b e r f e b r u a r y m a r c h 5 8 , 1 9 9 0 5 1 2 , 1 9 9 0 5 a, 1 9 9 0 3 9 , 1 9 9 0 6 9 , 1 9 9 0 1 8 2 1 , 1 9 9 0 8 2 0 , 1 9 9 0 b l o o d d on or s 5 5 4 4 5 7 5 6 4 5 5 3 5 4 in d iv id v o l u m e ( 1 ) 9 .4 9 .0 9 .5 1 0 .2 7 .1 1 0 .2 9 .0 11 p o o l p r o te in ( 9 / 1 ) 7 0 .0 7 4 .0 6 9 .1 6 7 .6 6 7 .7 7 1 .3 6 9 .6 e d /s n a i b :t t9 3 -0 0 3 .d o c / s e p td e r 2 , 19 93 5.1.3 ifcc material lot 91/0619 -stabilization, filling operation and lyophilization cholesterol 1.85 1.96 1.91 2.02 2.05 2.06 1.81 5.1.3.1 requirements for stabilization as discussed in a series of committee meetings it was agreed upon that the stabilization procedure had to fulfil especially the following requirements: triglyceride a p o a-i a p o b 0.95 1.50 1.15 1.16 1.50 1.10 0.93 1.43 1.02 1.30 1.43 1.15 1.09 1.43 1.23 0.96 1.50 1.15 0.80 1.50 0.95 * possibly complete elimination of the lipoproteins/lipids vldl, idl, ldl, hdl and cholesterol * no or only minimal blank value no or only minimal matrix effects * no or only minimal denaturation of the protein structure * no or only minimal reduction of the protein structure for the declared proteins cholesterol treatment by aerosila was the option chosen for the stabilization of the reference material. behringwerke ag had t h e task of performing this treatment. triglyceride 0.0157 0.0167 0.0121 0.0121 0.0163 0.0157 0.0096 table 2 crm 470 2 rpphs lot 5 lipiddapolipoproteins before and after stabilization single pool lot no. 1 mr 0100/12 2 be 0100/18 3 sh 0100/39 4 sh 0100/40 5 ds 0100/41 6 ds 0100/42 7 or 0100/38 single pool lot no. 1 mr 0100/12 3 sh 0100/39 4 sh 0100/40 5 ds 0100/41 6 ds 0100/42 7 or 0100/38 2 be 0100/18 not detectable 24 1 5.1.3.2 influence of treatment on the blankvalue llingle pool lot no. 1 mr 0100/12 2 be 0100/18 3 sh 0100/39 4 sh 0100/4q 5 ds 0100/41 6 ds q100/42 7 or 0100/38 the frozen blood donations were sent to marburg at the beginning of december 1990 where they were thawed and then pooled in seven different serum pools according to the place and date of the blood donation. the initial volumes of those single pools vari ed between 7.1 and 10.2 liters. as had been agreed upon the c3 component was trans formed into the stable c3c fragment by treatment with inulin, after which each single serum pool was stabilized by treatment with aerosil@. blank values yield after stabilization initial / final volume protein cone. (volt) / (volt) (1) [g/l) 4.55 / 0.64 5.7 63.2 5.80 1 0.24 6.0 62.2 5.77 1 0.78 6.5 69.8 6.32 / 0.79 7.3 65.3 4.47 / 0.39 5.0 64.6 9.35 1 0.56 7.3 66.5 1.82 1 0.62 5.2 69.6 the success of the stabilization procedure was shown by the data regarding the blank values and yields as well as by the content of lipoproteins before and after stabilization (table 2). the expected results were obtained for all serum pools. the successful removal of the lipoproteins, which tend to produce turbidity and matrix effects, can be proven on the basis of the drastically reduced blank values (table 3): reduction to approximately 10% of the original value in the untreated serum pools, measured under undiluted conditions using a behring laser nephelometer / bln measuring the signals in volt. table 3 crm 470 rpphs lot 5 / blank value and yield 5.1.3.3 influence of treatment on the protein concentration it was necessary to concentrate the volume of the single pools after treatment with the stabilizing aerosilb. a n average loss of volume of approximately 32% for the material had to be taken into account in order to obtain the original plasma protein concentration in the final pools. no great hfference in concentration before and after stabilization was found for the following eight plasma proteins: 242 igg, iga, albumin, transferrin, a2-macroglobulin, transthyretin (prealbumin), acid a 1 glycopro tein, hapt oglobin. a small reduction (< 10%) was observed for al-proteinase inhibitor (a1-antitrypsin) and c3c. a clear reduction (on average 30%) was observed for igm, c4 and ceruloplasmin. such reductions could not be avoided because the mentioned proteins also have a certain affinity t o the stabilizing material, although it is not as strong as that known for lipoproteins. 5.1.3.4 influence of treatment on the protein structure the protein structure representative for the immunochemical reactivity was also investigated using physicochemical procedures. it had t o be proven that there were no structure alterations and no proteolytic digestions during stabilization. this was especially well recognizable on the basis of the completely intact structure for the protein inhibitors a 1-proteinase inhibitor and a2-macroglobulin. the presence of intact protein inhibitors is very important because they represent a natural protection against any proteases possibly present in such preparations. possible alterations of the protein structure were also checked for other proteins (section 5.1.5.4). 5.1.3.5 preservation of the final pool after fulfilling the set quality requirements for each single serum pool, the seven stabilized preparations were mixed into a single final lot (approx. 41 liters) on december 20,1990. for preservation/proteolytic protection * sodium azide (to final concentration of 0.5 g/1 * aprotinin = antagosan of behringwerke ag (to final concentration of 80,000 hu/u were added to the stabilized lot and this was immediately followed by sterile filtration and storage at 20 "c/30 "c on the same day. the internal denomination ifcc isrp lot 01 00/34 was given to the final bulk product (without crp). 1 7 955059 243 6.1.4 ifcc material lot 01 00/36 pilot batch additives sodium h i d e bemamidine chloride an aliquote of 1.0 liter of the final bulk product obtained on december 20, 1990 and stored at 2ooc/30°c, was carefully thawed and used to produce a pilot batch on march 4, 1991. the following substances were added to this small trial batch for further experiments: final concentration mm. 15.4 mmol/l m a . 1 , o mmol/l (< 0.157g/l) t< 1 g/o * a fraction containing crp, lot 22 49 0 (received from prof. pepys/leeds) to obtain a final concentration of ca. 40 mg/l crp * sodium azide solution (200 g/l) to obtain a final concentration of ca. 1 g/1 * benzamidine chloride to obtain a final concentration of 0.157 g/1 * hepes buf€er to adjust the ph value to 7.2 hepes bufer the pilot batch was filtrated under sterile conditions and 14 days later, on receipt of t h e sterility protocol, filled into vials with 1.0 ml which were freeze dried and sealed. t o p h 7.2 the sterility of the reconstituted lyophilisate was tested and once more confirmed. further quality requirements regarding homogeneity, dry weight, rest moisture and especially the blank value of the lyophilisate after reconstitution with 1 ml destilled water were fulfilled within an acceptable range. the extremely low blank value, which can be measured in the reconstituted lyophilisate, indicated that freeze drying had no influence on the optical clarity of the material. table 4 crm 470 a rpphs lot 5 / preservation / stabilizah'on i i aprotinin i max s o , a x , klu*/l 244 the stability testing program of the pilot batch was performed according to the instructions for stability studies and value transfer given by bcr. no significant alterations in protein concentration (measured by ina and rid) and protein structure (physicochemical analysis) were observed during the period of six months. an acceptable short-term stability of reconstituted material up to seven days from the time of reconstitution was also given. results are identical or similar to those of the final product; therefore details for the pilot batch are reported in connection with the description of the certified preparation = crm 470 (identical to rpphs lot 5). sterility (af tor filling) hohogeweity (filling operation) dry weight of lyophilisate rest hoisture b l a m value (blw table 5 y e s , bwieab regulation y e s , bw/eab regulation europaisches &rzneimittel buch* 1.003 mg (n = 5 ) 1.0024 mg (n = 5 0 ) sd = 6.4 mg cv = 0.639 % 73.4 mg (n = 30) 71.8 mg (n = 3 0 ) sd = 2.02 mg sd = 2.4 mg cv = 2.75 % cv = 3.34 % 0.59 % (n = 30) 0.57 % h20 (fischer) sd = 0.14 % sd = 0.11 % cv = 23.7 k cv = 19.3 % (n = 3 0 ) oloof36 by : 0.40 volt 0100137 sy : 0.54 volt (n = 3 0 ) (n = 20) (n = 3 0 ) (n = 20) 0100/36 sl : 0.29 volt 0100/37 sl : 0.46 volt pilot batch 0100/36 sy and crm 470 : rpphs lot 5 / final characteristics pilot batch final product i lot no. 0100/36 sy crm 470 2 rpphs lot 5 ~ ~~~~ s = serum f y = lyophil. f l = liquid * european pharmacopoea 245 5.1.5 ifcc material lot 91 06 19 final product 5.1.5.1 g e n e r a l r e m a r k s the final bulk product lot 0100/34 was thawed, spiked with the selected crp fraction, protected with proportionally increased amounts of the previously reported preservatives (table 4) and filtrated under sterile conditions. vials were filled with 1.0 ml, freeze dried under conditions identical to those of the pilot batch and sealed. the date of the filling operation was used to identify the reference material (isrp lot 91/06 19, i. e. june 19, 1991). the data obtained for characteristics (table 5) like homogeneity, dry weight and rest moisture of the lyophilisate as well as the blank values before and after lyophilization (reconstituted samples) corresponded very well with those of the pilot batch. 5.1.5.2 stability testing by q u a n t i t a t i o n b e f o r e reconstitution the stability studies were performed under the same conditions as for the pilot batch lot 0100/36/sy (sy = serum lyophilized). the studies were performed according to a program proposed by b c r storage t e m p e r a t u r e s 70 "c/20 "c/ + 2 "c to + 8 "c/ + 20 "c (room temperature)/ + 37 "c/ + 45 "c t i m e intervals months: 1/2/3/6/9/12/18/24 (planned on: 36/48/60) (for the pilot batch shorter intervals/days: 1 /3/7/14/2 1 /28/56/ 126/192) immunochemical methods ina = immunonephelometric assay (bna = behring nephelonieter analyzer) rid =radial immunodiffusion (partigen plates of behringwerke ag) after a storage time of 24 months the results indicated vary good stability (table 6 for ina results). exception: crp for samples stored at + 37 " c / + 45 "c. 5.1 5.3 stability testing b y q u a n t i t a t i o n a f t e r reconstitution the stability testing of the reconstituted material also indicated an excellent and stable quality of all proteins in the liquid form independent of storage at + 2°c to + 8°c or at +20 "c. determinations were made daily up to seven days (short period) and to 28 days (longest period) using a new vial each day. results were compared to those obtained using a vial stored at 70 " c . n o significant changes were observed for any of the proteins. 246 conclusion: crm 470 should be used within seven days after reconstitution with 1 ml distilled wa ter when stored at + 2 "c to + 8 "c after each use. the stability over this period of time is very good, if contamination is avoided. table 6 c r m 4 7 0 = rpphs5 1 stability testing i method: ina (bnai storage time: 2 years results in percent of material stored at -7ooc 4 7 0 0 3 c . x l ~ n = 2 reference: n-protein standard serum, ch.-b. 067651 n-protein standard plasma py, ch.-b. 068034 na-latex crp kit, ch.-8. 2361 3 (behringwerke ag) 247 5.1.5.4 stability testing by analysis of protein structure the stability studies regarding the protein structure were performed under the same conditions as those for the quantitation of proteins by rid and i n a i. e. by using samples stored at different temperatures and by testing within the same time intervals. the patterns were compared with those performed immediately after manufacturing. the methods used were: * vertical polyacrylamid gel electrophoresis (page) (p = 8.0%) with and without sodium dodecyl sulfate (sds) to study the structure of various serum proteins. no significant alteration was observed. 4: vertical page (p = 5.5%) for analysis af a2m showed that it mainly existed in the intact (slow) form and no changes to the fast form were observed during the period of testing. * vertical page followed by immunoblotting for crp: the stored samples kept their intact form (exception: + 37 "c/ + 45 "c). * vertical sds page followed by immunoblotting for c3/c3c: the existence of the fragment c3c was confirmed. for c4/c4c: the complete form c4 as well as small amounts of fragment c4c were detected. for a1at the inhibitor-active and intact form corresponded t o the majority of the protein present. * two-dimensional immunoelectrophoresis for c3/c3c: all patterns were similar; no further alteration was noted after the initial fragmentation (nearly 100%) to the stable form of c3c and c3d. for c4: for alat: the majority was present in the intact form together with a small amount of aggregates. for slight degradation of this protein was seen (this is usual after lyophilisation). only one peak corresponding to the intact form of c4 was observed. summary of examination no distinct alterations in protein structure (table 7) were found during the period of stability testing up to 24 months referring the samples stored at 70 "c/20 "c/ + 2 "c to 8 "c/+18 "c to +22 "c. 248 table 7 protein crm 470 rpphs lot 5 for 14 plasma proteins ifcc/bcr/cap material lot no. 91 1061 9 structure state p r o t e i n s t r u c t u r e s 190 i9a igx intact f native intact f native intact; + aggregates crp alb i intact 1 native intact trp i intact 1 native c3c fragment ++--c4 mented slightly frag ttr (pre) 1 intact 1 native &, ag i intact 1 native a pi d t a ) + aggregates inhibitor active + types intact (8-form) inhibitor active slightly altered siae unchanged hpt hp 1-1 hp 2-1 imixture hp 2-2 (n = 364) method for detection * ie; 2-dim.ie ie; 2-dh.ie ie; 2-dim.ie ie; 2-dh.ie; page18 % paa ie; 2-dh.ie; page18 % paa ~ ~~ ie; 2-dim.ie; pageisds + ib ie; 2-dim.ie; pagefsds + ib ie; 2-dim.ie; page18 % paa ~~ ie, 2 d h . ie; pageisds 7 % paa +ib ie; 2-dh.ie; pagef5#5 % paa ie; 2-db.ie ie; 2-dim.ie ie; 2-dim.ie; page18 % paa + ib 2-dim. ie = two-dimensional immunoelectrophoresis page = vertical golyacrylamide eelelectrophoresis paa = polyqcrylqmide bds = sodium dodecyl sulfate ib = immunoblotting bd/pla / b:tb93-016.da sgterrkr 2, 1493 249 exceptions / slight alterations cer for samples stored at +37 "c and +45 "c after 18/24 months crp for samples stored at + 37 "c and + 45 "c after 18/24 months such results of physicochemical analysis confirmed t h e data of the quantitation by im munoassays. 250 (7) upsala j med sci 111 (2): 227–230, 2006 malacoplakia and spermatic granuloma complicating vasectomy 1margrét agnarsdóttir, 2birgitta carlén and 1roger willén 1pathology and cytology, centre for laboratory medicine, uppsala university hospital, s-75185 uppsala, sweden. 2department of pathology, lund university hospital, 22185 lund, sweden. abstract malacoplakia is a granulomatous disease with a histiocytic infiltrate containing calcified structures called michaelis-gutmann bodies. these structures are considered to represent an abnormal response to infection involving defective lysosomes and abnormal microbubular assembly. the disease most frequently involves urinary and genital tracts, but has also been described from most other organs. here we present the first case of malacoplakia only involving the vas deferens. introduction malacoplakia is a granulomatous disease, which most frequently involves the urinary and genital tract but has also been described from most other organs [1,2]. the disease is characterized microscopically by a histiocytic infiltrate (the so-called von hansemanns cells) containing calcified structures named michaelis-gutmann bodies. these can be seen both intraand extracellularily and are considered to represent an abnormal response to infection involving defective lysosomes and abnormal microtubular assembly [3]. the first article describing the disease was published in 1902 [4]. urinary organs as targets usually involve the urinary bladder and testis with sometimes engagement of the epididymis [5]. sole engagement of the epididymis without concurrent involvement of the testis is reported in 9 cases [5]. we have not been able to find a case of malacoplakia solely targeting the vas deferens. we thus find it of interest to report one case, as besides spermatic granuloma the firm fibrous lump palpated clinically might simulate tumour or other forms of specific inflammatory states. materials and methods the surgical specimens were fixed in formalin and routinely processed. paraffin sections were stained with hematoxylin and eosin (he), periodic acid-schiff stain (pas) for mucin, prussian blue for iron and von kossa´s stain for calcium. 227 received 17 may 2005 accepted 17 august 2005 for electron microscopy paraffin-embedded, formalin-fixed material was used. using light microscopy the area in question was marked and the corresponding field on the paraffin block taken out. a very thin slice (0.25mm) was deparaffinized in xylene for 1.5h in a 60oc oven, thereafter rinsed in fresh xylene again and kept in room temperature for another 30 min. the deparaffinized tissue was cut into small cubes and immersed in 100% “blue” ethanol (0.5% methyleneblue) then in 99.5% ethanol followed by propylene oxide for 30 min each. the tissue was then infiltrated in propylene oxide/resin 1:1 for 2h and in pure resin for 1.5h, polymerized over night in a 60oc oven. ultrathin sections were cut and stained with 4% uranyl acetate dissolved in distilled water for 30 min in 40oc, washed with distilled water and then stained with reynolds lead citrate [6] for 2 min in room temperature. the grids were examined in a philips cm10 electron microscope at 60kv. case report a 39-year old man presented with a tumour in the right vas deferens, which he had noted since 1998. before that the patient had undergone ligation of the vas deferens. the tumour was removed in 2004. macroscopically a cystic lesion was present with a diameter of 2 cm. the cyst was filled with a brownish material. microscopic examination showed a benign cyst in the vas deferens covered with a fibrotic capsule. in the cyst wall a spermatic granuloma was observed containing sperms and a granulomatous inflammatory reaction was also present. focally, an area in the outer part of the wall contained granulated histiocytes (fig. 1). these granules stained positive with iron (fig. 2a), pas (fig. 2b) and von kossa´s staining (fig. 2c). electron microscopy confirmed the presence of the characteristic michaelis-gutmann bodies (fig. 3a and 3b). 228 fig 1. the area in the wall of the cyst containing granulated histiocytes. discussion malacoplakia of the vas deferens seems to be a rare disease as we were not able to find a published case solely targeting the vas deferens. the histopathological appearance is characteristic and is easily recognized. as malacoplakia is a disease that can affect 229 fig 2. the michaelis-gutmann bodies staining positive with iron (a), pas (b) and von kossa´s (c) staining. fig 3. an electron microscopic picture showing the characteristic michaelis-gutmann bodies (a and b). many organs it is important to know that this entity exists. not only for histopathologists but also for clinicians as the macroscopic appearance can easily simulate a malignant process and specific infections as e.g. tuberculosis. spermatic granuloma is a well-known complication after the vasectomy procedure [7, 8], but it is also important to notice whether malacoplakia is present as those lesions represent a septic component of the granuloma lesion. acknowledgements we are grateful to frank bittkowski for his skilled photographic help. the uppsala university hospital foundation for clinical research (alf) and the björnssons foundation supported this study. references 1. stanton m-j, maxted w (1981). malacoplakia: a study of the literature and current concepts of pathogenesis, diagnosis and treatment. j urol 125:139-146. 2. mcclure j (1983). malakoplakia. j pathol. aug; 140: 275-330. 3. dasgupta p, womack c, turner a-g, blackford h-n (1999). malacoplakia: von hansemann´s disease. bju int 84:464-469. 4. michaelis l, gutmann c (1902). über einschlüsse in blasentumoren. ztschr klin med 47:208215. 5. dieckmann k-p, henke r-p, zimmer-krolzig g (1995). malacoplakia of the epididymis. report of a case and review of the literature. urol int 55:222-225. 6. reynolds es (1963). the use o lead citrate in high ph as an electron-opaque stain in electron microscopy. j cell biol 17:208-212. 7. gade j ,brasso k (1990). sperm granulomata. ugeskr laeger 152:2228-2284. 8. davis je (1992). male sterilization. curr opin obstet gynecol 4:522-526. corresponding author: roger willén md, phd pathology and cytology centre for laboratory medicine uppsala university hospital s-75185 uppsala sweden phone: +46-18-6119408 e-mail: roger.willen@akademiska.se 230 tf-iups160036 203..203 announcement apology on behalf of the publisher in the previous issue of this journal (121-02), a number of errors were made. � we like to make readers aware that leif jansson and nils welsh contributed as guest editors to the issue � the title of the issue should have been remembering claes hellerstr€om and those around him. unfortunately, this title was not placed on the front cover as it should have been � in addition to this, a number of the ‘flashes’ were erroneously printed from the previous issue (121-01) on the front cover. taylor & francis apologises for these errors. � 2016 informa plc limited. published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 3, 203 http://dx.doi.org/10.1080/03009734.2016.1207330 http://creativecommons.org/licenses/by/4.0/ apology on behalf of the publisher untitled porous titanium granules 217 received 14 december 2007 accepted 18 december 2007 upsala j med sci 113 (2): 217–220, 2008 porous titanium granules for implant stability and bone regeneration – a case followed for 12 years leif holmberg1 lillemor forsgren2, lars kristerson3 1ädelfors kungsgård 1, holsbybrunn, sweden, 2department of dentomaxillofacial radiology, the institute for postgraduate dental education, jönköping university, jönköping, sweden, 3elvabovägen 22, höllviken, sweden. abstract the clinical and radiological results were excellent for each of the nine fixtures individually tested in a case followed for 12 years after implant treatment using split crest technique in combination with porous titanium granules for an augmentation of the dento-alveolar ridge in a severely resorbed maxilla. introduction the split crest technique is used for augmentation of an dento-alveolar ridge with sufficient vertical bone height but insufficient bone width (1, 2). however, as there still may be a problem to achieve a sufficient bone ingrowth around the fixtures in case of a severely resorbed dento-alveolar ridge, we used porous titanium granules, which previously have been used to stabilize hip prostheses (3, 4), to enhance bone regeneration. case report a patient (born in 1922) received in 1995 nine fixtures ad modum brånemark in her maxilla (figure 1). at surgery a split crest procedure was performed in region 17–14 and 21–27 because of a narrow bone ridge. porous titanium granules (natix®, tigran technologies ab, malmö, sweden) mixed with autogenous blood and bone-suction material was gently packed around the fixtures. the procedure was less time-consuming than bone-grafting and the titanium granules were used to fill the space between the buccal and the palatal bone plates after the splitting procedure. nine months later, 9 abutments were placed and a temporary bridge was connected and adjusted to correct occlusal scheme. the temporary bridge was duplicated after three months into a permanent fixed-bridge-work in gold and acrylic resin denture teeth. radiographic and/or clinical examinations were performed at 6 months after fixture installation and again after 1, 2, 3, 5, 7 and 12 years. after 12 years there was a loss of marginal bone height in average of less than 2 mm compared to baseline 1996 (figure 2–4). the fixture stability was excellent 218 leif holmberg et al. figure 1. the location of the nine fixtures ad modum brånemark in the maxilla. figure 2. radiograph demonstrating the bone height after 9 months. figure 3. radiograph demonstrating the bone height after 12 years. figure 4. after 12 years the loss of bone height around the nine fixtures was in average of less than 2 mm. porous titanium granules 219 for each of the nine fixtures individually tested. she had no infections or inflammations despite insufficient oral hygiene the last years and she was pleased with reliability, function and aesthetics (figure 5). comment and conclusion the porous titanium granules were used as a filling material following a split crest procedure in our case of a severely resorbed maxillary dento-alveolar ridge and allowed for cross arch bridge installation. the procedure was less time-consuming than bone-grafting and the stability (tested for each of the nine fixtures individually) was still maintained after 12 years. the clinical results in this single case using titanium granules in a mixture of bone-suction material and blood should be supported by histologic data when available. in conclusion, for patients who have narrow buccolingual ridges, the ridges can be expanded to allow fixture location by the disjunction of buccal and lingual compactplates; and the placement of porous titanium granules in between seems to be an attractive option to improve bone regeneration. refererences 1. simion m, baldoni m, zaffe d. (1992) jaw bone enlargement using immediate implant placement with split crest technique and guided tissue regeneration. int j periodontics restorative dent 12: 463 –447. 2. coatoam gw, mariotti a. the segmental ridge-split procedure. (2003) j periodontol 74: 757– 770. 3. alffram p-a, bruce l, bjursten lm, urban rm, andersson gbj. (2007) implantation of the femoral stem into a bed of titanium granules using vibration. a pilot study of a new method for prosthetic fixation in 5 patients followed for up to 15 years. upsala j med sci 112: 175–81. 4. turner tm, urban rm, hall dj, andersson gbj. (2007) bone ingrowth through porous titanium granulate around a femoral stem. histological assessment in a six-month canine hemiarthroplasty model. upsala j med sci 112: 183–89. figure 5. the patient was pleased with reliability, function and aesthetics. 220 leif holmberg et al. corresponding author: leif holmberg, dds, senior consultant in restorative dentistry ädelfors kungsgård 1 se-570 15 holsbybrunn, sweden e-mail: gulfholm@hotmail.com (10) calissen s109-116 upsala j med sci 111 (1): 109–116, 2006 quality of life varies with pain during treatment in adolescents with cancer maria calissendorff-selder, md, gustaf ljungman, md, phd department of women’s and children’s health, children's university hospital, sweden abstract pain is one of the most feared problems for adolescents with cancer. pain produces stress with negative physiological and psychological effects. therefore, effective pain management during cancer treatment may influence the outcome. this study investigates variations in pain and quality of life during treatment in adolescents with cancer, and whether there is a co-variation between the two. in a prospective longitudinal questionnaire investigation, quality of life in eight adolescents with cancer was assessed with the psychological general well-being index (pgwb) and compared with the patients’ experiences of pain according to repeated structured interviews. pain troubled the adolescents most in the beginning and in the end of the treatment period, but troubled them less in-between. during treatment, quality of life was low in the beginning, higher in the middle and lower in the end. pain co-varied inversely with quality of life and the adolescents thus seemed to have higher quality of life when pain-relieved. this finding emphasizes the importance of pain management in children and adolescents with cancer. introduction pain in children is often underestimated and therefore inadequately treated (1–5). this is also true for children with cancer (6–11). pain in children with cancer can 109 received 10 october 2005 accepted 28 october 2005 often be reduced to one or more of four basic categories: treatment-related (e.g. pain as side-effects of chemotherapy and radiation), procedure-related (e.g. pain due to lumbar puncture, bone marrow aspiration or postoperative pain), cancer-related (e.g. pain due to infiltration of tumor in various organs or tissues) and pain of other origin (12). treatmentand procedure-related pain have previously been shown to be greater problems than cancer pain, and thus most pain experienced by children may be regarded as iatrogenic (10,11). pain is the most feared problem for children on treatment for cancer (13), and therefore deserves attention and further investigation. . pain produces stress with negative physiological (1,2,14) and psychological (15) effects. therefore, effective pain management during cancer treatment may influence the outcome. children with cancer experience variations in pain during treatment (16). pain seems to be a greater problem at diagnosis, and towards the end of treatment, than in-between. reasons for this are that pediatric malignancies often respond rapidly to treatment (12), that the pain experience may be reduced with the increased security and confidence that comes with time, and that this latter effect weans off towards the end when the patients’ medical visits become less frequent (16). clinical observations suggest that the children’s sense of subjective well-being or distress varies with pain. pain management, where pharmacological and/or psychological approaches may be used depending on the individual child’s coping strategies, reduces pain and concurrently distress, which might improve quality of life. quality of life is also affected by other factors such as security and confidence. our hypothesis was that there is an inverse correlation between the experience of pain and quality of life. to our knowledge, this has previously not been studied in adolescents with malignant diseases. the aims of the investigation thus were to study variations in pain and quality of life in adolescents with cancer during treatment, and whether there is a co-variation between the two. patients and methods this study is part of a project, where children and adolescents with malignant diseases admitted, during one year, to the unit for pediatric hematology and oncology at the children’s university hospital in uppsala (a tertiary referral center for pediatric oncology) were invited to join a prospective longitudinal questionnaire investigation about pain, pain management, and quality of life. questionnaires were presented orally to ensure that they were correctly apprehended. inclusion criteria were: 1) age between 13 and 18 years, 2) treatment period from one month after diagnosis to three months post treatment, 3) understanding of swedish sufficient to make the interview possible, 4) not terminally ill and 5) more than one interview possible. children below 13 years of age were excluded because the questionnaire for quality of life was not validated for this age group. eight adolescents were included in this study, five of which were interviewed twice and three, three times. we did not interview during the first month, because 110 the situation for the families is usually psychologically very complex at this time. structured interviews based on a previously used questionnaire (11,16) were performed to investigate the patient’s experience of pain. for assessment of quality of life, a questionnaire with 22 questions, the psychological general well-being index (pgwb) (17), was used. two experienced nurses familiar with problems of pediatric oncology performed the structured interviews under standardized conditions. interviewers were associated with the pediatric oncology ward, but were not primarily medically responsible for the adolescents they interviewed to avoid biases. the pgwb index was used to measure the affective or emotional states of the adolescents, which corresponds to a sense of subjective well-being or distress. the index consists of six subscales that can be put together to construct an overall index, which is sensitive to an individual’s psychological general well-being. the subscales are: anxiety (5 questions), depressed mood (3 questions), positive well-being (4 questions), self-control (3 questions), general health (3 questions) and vitality (4 questions). for each item there are six response options that are scored on a scale from 0 to 5, according to the frequency of the affective experience. a value of 0 is given for the most negative option, and 5 for the most positive option. the score range for the pgwb index is thus 0 to 110 the higher the score, the better the quality of life whereas the range for the subscales is from 0 to 15, 20, or 25. from the 22 items 6 subscale scores without overlapping items and 1 overall pgwb index score can be derived. informed consent was obtained from patients and parents and the local research ethics committee approved the study. for statistics, kendall’s tau_b was used for non-parametric correlations. p values of <.05 were considered significant. linear regression analysis with anova was used to test associations between variables. all statistical analyses and graphical presentations were performed using spss 10.0 (spss inc. chicago il.) results for demographic and interview data see table 1. quality of life measured with the pgwb index was about 70 in the beginning of the treatment period, when intensive pain was more frequent than later (figure 1). in the end of the treatment period the mean pgwb index was slightly lower than the value in the beginning, and intensive pain was again more frequent. in the middle of the treatment period, pgwb index reached its highest values and intensive pain was less common. the inverse correlation (correlation coefficient = .403) between pain and quality of life was significant (p=.018). when the mean pgwb subscales are presented individually (figure 2), they are lower in the beginning of treatment, and except for general health only, there is a peak at four months. the next peak is at nine months, when all of the subscales 111 reach their highest, or nearly highest, values. after nine months, subscales decline and in the end of treatment, subscales differ. anxiety, depressed mood and self-control tend to increase slightly, while positive well-being is unchanged, and general health and vitality decrease considerably in the end of treatment. 112 table 1. demographic and interview data age (yrs) at 1st 2nd 3rd gender diagnosis diagnosis interview* interview* interview* 1 m lymphoma 16.4 1 12 2 f ewing sarcoma pnet 17.7 4 8 3 f osteosarcoma 18.3 12 21 22 4 m nasopharynx cancer 15.6 2 8 5 m acute lymphoblastic leukemia 13.3 5 12 6 f hodgkin’s lymphoma 14.9 2 8 7 m acute lymphoblastic leukemia 14.1 2 5 8 8 m rhabdomyosarcoma 17.3 2 5 9 * months after diagnosis. 302520151050 p g w b ; p a in x 2 0 120 100 80 60 40 20 0 pgwb painx20 figure 1. mean pgwb index and mean pain x 20. pgwb = psychological general well-being. pain value is multiplied by 20 to adjust it to the values of pgwb. 113 in linear regression analysis, variation in pain score predicted 21 % of the variation in the total pgwb index. in the pgwb subscales the corresponding figures were: general health 27.6 %, positive well-being 17.4 %, vitality 14.9%, anxiety 14.2 %, depressed mood 6%, and self-control 3.3%. according to the first interview, two adolescents experienced pain before diagnosis very often, three sometimes, and three seldom or never. in the beginning of the treatment, the adolescents were asked how often they had experienced intensive pain during the three months preceding the interview. three adolescents had experienced intensive pain sometimes, and five had never experienced intensive pain. this pain was caused by cancer, treatment, and procedures or of other origin. when the adolescents were asked the same question later during treatment, pain had decreased for the three adolescents with intensive pain and had not increased for those with less pain, except for one. discussion we found that pain troubled the adolescents most in the beginning and in the end of the treatment period, but less in-between. this is in accord with a previous study demonstrating that pain is a major problem for children with cancer and that it varies during the treatment period (16). during treatment, quality of life was low in the beginning, higher in the middle and lower in the end. pain co-varied inversely with quality of life and the adolescents thus seemed to have better quality of life when pain-relieved. this seems reasonable given that pain has negative physiological month 302520151050 p g w b 30 20 10 0 vitality health selfcont wellbeing depress anixiety figure 2. mean pgwb subscales. pgwb = psychological general well-being. the subscales are: anxiety, depressed mood, positive well-being, self-control, general health, and vitality. (1,2,14) and psychological (15) effects on children. the main limitation of this prospective longitudinal investigation is that it was performed on only eight adolescents and therefore should be regarded as a pilot study. in such a small study group, each individual’s answers considerably affect the results and power is not high, implying that the results must be interpreted with caution. nevertheless, in spite of the few patients, the inverse correlation between pain and quality of life was significant. another aspect is that this material of eight patients comprises seven different diagnoses. however, there are many similarities between different treatment protocols where the general trend during the last decades has been more aggressive, multimodal, high-intensity, long-term therapy regimen (12). during the middle period of treatment the patients seemed less bothered by pain than in the beginning and in the end. the major reason for better pain control and higher quality of life during the middle period of treatment is probably not a change in pain management, but a real decrease in pain thus improving quality of life. this is in agreement with clinical observations, which also suggest that the children’s sense of subjective well-being or distress vary with pain. during this middle period, the adolescents experienced higher quality of life than in the beginning and in the end. this is also in accord with another study implying that quality of life in adolescents with cancer was higher during treatment than after (18). the pgwb index means have been 76 to 84 for community samples (17) and 46 to 50 for mental health patients at intake and in treatment (19) for populations in the ages 14 to 74 years in the usa. the adolescents in our study had a pgwb index mean of about 70 in the beginning of treatment and in the end; the times when they experienced more intense pain. this is lower than pgwb mean values for the community sample mentioned above. during periods when the adolescents experienced less intense pain, the pgwb index mean was considerably higher, 90 to110, which indicates that the adolescents, when pain relieved, experienced higher quality of life. quality of life issues have been much discussed during the last few years, and new measures have been developed. the ideal measure still does not exist, but today there are measures that have been developed especially for children and adolescents with cancer (20,21,22,23). at the time of the study, however, there were no such specific measures that had been validated and translated into swedish. the pgwb index was chosen because it was considered the best choice at the time. it remains to be clarified whether the adolescents are so well pain-relieved that they experience higher quality of life, if they feel less pain because their quality of life is higher, or if other factors are important. factors that could affect pain as well as quality of life are security and confidence and a feeling of being seen and respected as an individual. the clinical impression is that security and confidence generally increase during treatment, but often decrease again in the end of treatment because of less frequent contacts with the oncological team. it is most important to reduce pain and distress as much as possible because of their 114 negative physiological (1,2,14) and psychological (15) influences during cancer treatment. it is also important that the adolescents undergoing cancer-treatment get painrelief to achieve higher quality of life. most of the pgwb subscales vary according to the mean index, but the subscale for general health differs at four months when it returns to the same value as at one month after diagnosis, whereas the other subscales increase. in the end, the considerable decline in general health and vitality, which deviates from the other subscales, could cause the decreased quality of life. the decline in these two subscales might be explained by the long period of illness and treatment that the adolescents have endured. in conclusion, pain and quality of life varied during treatment of adolescents with cancer. pain co-varied inversely with quality of life and the adolescents thus seemed to have higher quality of life when pain-relieved. this has to be further confirmed in larger studies, but the results agree with findings in adult cancer populations (24,25). this finding emphasizes the importance of pain management in children and adolescents with cancer. references 1. anand k, sippel w, aynsley-green a (1987) randomised trial of fentanyl anaesthesia in preterm babies undergoing surgery; effects on stress response. lancet 1: 62-66. 2. anand k, phil d; hickey p (1987) pain and its effect in the human neonate and fetus. new engl j med 317: 1321-1329. 3. schechter n (1989) the undertreatment of pain in children: an overview. pediatr clin north am 36: 781794. 4. schechter n, altman a, weisman s (1990) report of the consensus 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in children and adolescents with cancer. first results of an evaluation of 49 patients with the pedqol questionnaire. klin padiatr. 212: 211215. 22. varni jw, burwinkle tm, katz er, meeske k, dickinson p (2002) the pedsql in pediatric cancer: reliability and validity of the pediatric quality of life inventory generic core scales, multidimensional fatigue scale, and cancer module. cancer 94: 2090-2106. 23. bhat sr, goodwin tl, burwinkle tm, lansdale mf, dahl gv, huhn sl, gibbs ic, donaldson ss, rosenblum rk, varni jw, fisher pg (2005) profile of daily life in children with brain tumors: an assessment of health-related quality of life. j clin oncol 23: 5493-5500. 24. becker n, bondegaard thomsen a, olsen ak, sjøgren p, bech p, eriksen j (1997) pain epidemiology and health related quality of life in chronic non-malignant pain patients referred to a danish multidisciplinary pain center. pain 73: 393-400. 25. wang xs, cleeland cs, mendoza tr, engstrom mc, liu s, xu g, hao x, wang y, ren xs (1999) the effects of pain severity on health related quality of life. cancer 86: 1848-55. corresponding author: gustaf ljungman department of women’s and children’s health children's university hospital se-751 85 uppsala, sweden phone: +46-18-6115586 fax: +46-18-500949 gustaf.ljungman@kbh.uu.se 116 upsala j med sci 102: 185-198, 1997 a survey of sleep habits and sleeping difficulties in an elderly swedish population lena mallon', and jerker hetta' 'sleep disorders unit, department of psychiatry, university hospital, uppsala, sweden and 'psychiatric clinic, falun hospital, falun, sweden abstract a random sample of 876 subjects aged 65-79 years were investigated by means of a questionnaire concerning sleep and related factors. sleep problems were reported by 23.8% of females and 13.3% of males.moderate or major complaints of maintaining sleep were reported by 43.5% of subjects, early morning awakening 33.4% and difficulties falling asleep 31.4%. daytime sleepiness was more common among males, and a relationship between daytime sleepiness and perceived poor sleep was found. daytime napping was common, but not related to poor sleep. the prevalence of regular sleeping pill users was 7.6% for females and 3.0% for males and a relationship between sleep problems, sleeping pill usage and psychiatric symptoms was established. among the regular sleeping pill users 39.1% had possible depression (pd) and 63.0% had possible anxiety disorder (pa). among respondents with sleep complaints 29.8% had pd and 48.7% had pa. sleep problems were also related to impaired physical health. various medical illnesses contributed to sleep complaints among males, and depression affected sleep the most among females. introduction during the last century there has been a progressive increase in the number of aged people in the community. this change in the demographic pattern will have profound medical consequences because a large proportion of these elderly suffer from chronic, disabling health problems, one of which is sleep disturbances. several community based surveys of sleep disturbances have compared the prevalence of insomnia across age and demonstrated that sleep disturbances are common and that the prevalence of insomnia increases with age, especially among women (2, 11-16, 18, 19,26, 27). karacan (1 1) conducted an interview survey in houston usa of 2347 subjects aged 18 years and older. difficulties initiating sleep did not appear to be age related, whereas difficulty maintaining sleep, early morning awakenings and use of hypnotic medication all increased with age both for females and males. welstein (26) published results from a telephone survey in san francisco usa, where 6340 respondents aged 6 to 103 years of age answered questions about sleeping habits. the age effect in the material was significant, supporting the finding of increasing sleep disturbances with 15-980 148 185 increasing age. in lugaresis study (16) from san marino where 5713 subjects aged between 3 and 94 years were interviewed, reports of insomnia increased progressively after the 20th year. mellinger (19) interviewed 3161 adults aged between 18 to 79 years in the national survey of psychotherapeutic drug use, a cross national survey in usa, and the prevalence of "trouble falling asleep or staying asleep a lot the past year" was 15% in the age group 35 to 49 years and 25% in the age group 65 to 79 years. one third of those with serious insomnia were characterized by syndromes resembling either depression or generalised anxiety. recently more interest has been directed on providing information on sleep disturbances in the elderly population and some studies have specifically surveyed sleep disturbances among elderly subjects (3-5, 7, 8, 22). data derived from the activity and ageing survey in nottinghamshire england, an interview study of 1023 subjects aged over 65 years published by morgan ( 2 2 ) , reported prevalence of "current insomnia often or all the time", 22.5%, significantly more among women. symptoms of anxiety rather than depression emerged out as a the more important predictor of poor sleep quality. from an interview survey in canberra australia of 874 elderly aged over 70 years henderson (8) reported population prevalence of "trouble sleeping last two weeks", 18.0% for women and 12.6% for men. gisalson (5) found that the most commonly reported insomnia complaint of 430 subjects aged over 65 years on iceland was habitual difficulties maintaining sleep, 37% of men and 30% of women. occasional or habitual complaints of both difficulties initiating sleep, difficulties maintaining sleep and early morning awakenings was reported by 10.4% of subjects. in a study on the effects of exercise on cardiovascular function in sunnyvale usa bliwise (3) had the opportunity to study sleep habits in a group of 357 healthy adults aged 50 to 65 years. the results of the questionnaire study showed low prevalence of poor sleep. the prevalence of "trouble falling asleep every night or almost every night" was 2.6% for females and 1.1% for males, and "trouble awakening and returning back to sleep" was 3.3% for females and 4.4% for males. despite these low prevalences, about a third of the population reported not well-rested andor not getting the sleep they required. even though these studies show an increase in sleeping difficulties with age, the prevalence figures show considerable variation, and only a few of the studies have investigated health status in relation to sleep. the principal aim of the present study was to investigate sleep habits, the prevalence and types of sleeping difficulties and related symptoms in a geographically-defined population of elderly in sweden. material and methods subjects the investigation was performed during the month of december 1995. a randomly selected sample of 1228 elderly inhabitants, 65-79 years, in the county of dalarna in sweden were asked to participate in a questionnaire survey. 186 sleep questionnaire sleep behaviours and certain variables that might affect sleep. 1. demographic variables (age, gender, residential status, marital status). 2. questions about life style variables (smoking habits, alcohol drinking habits, coffee and tea consumption, car driving). 3. sleep habits. questions about time for going to bed, time to fall asleep, number of nocturnal awakenings, total sleep time, time for arising, day time napping and sleep-requirement expectations. 4. questions adopted from the uppsala sleep inventory (usi) (13-15) concerning the severity of various sleeping difficulties on a five-point 1. 1. scale (1= no problems, 2= small problems, 3= some problems, 4= great problems, 5= very great problems). in the subsequent statistical analysis scores 1 and 2 were considered to represent "no complaints", score 3 "moderate complaints" and scores 4 and 5 "major complaints". 5. questions adopted from uppsala sleep inventory (usi) concerning how often a symptom occurred on a five-point scale (1= never, 2= seldom, 3= sometimes, 4= often, 5= very often). in the subsequent statistical analysis scores 1 and 2 were considered to represent "never", score 3 "occasionally" and scores 4 and 5 "habitually". 6. questions adopted from the basic nordic sleep questionnaire (bnsq) (23) concerning the frequency of a symptom during the last three months on a five-point scale. occurred ( i = never or less than once a month, 2= less than once per week, 3= once or twice per week, 4= three to five times per week, 5= every night or almost every night). 7. questions about physical illness (medical history, hospital admissions, number of illnesses). physical health status was to be rated on a six-point scale (l=excellent, 2=good, 3=quite good, 4= fairly good, 5=poor, 6=very poor). 8. questions about medication (medication currently used, past and present sleep medication usage). 9. measure of mood. the hospital anxiety and depression scale (had scale) (28) consists of 14 questions in which the overall severity of anxiety and depression is rated on a four point scale (0 to 3). seven questions are related to anxiety and seven to depression. it is recommended that scores of 8 or more on a subscale are taken to indicate possible pathology. the survey sleep questionnaire contained 89 questions designed to supply information about typical procedure each subject was mailed a questionnaire, a pre-stamped envelope and a letter explaining the purpose of the study. subjects were encouraged to call the investigator if they had any questions. full anonymity protection was ensured. one month later a follow up letter, along with duplicates of all material, was sent to all non respondents. the study protocol was approved by the ethics committee of the faculty of medicine at uppsala university insweden. 187 statistical methods the statistical analysis was performed on a macintosh computer with the statview software package. the results are presented as means 5 sd. a chi-squared test was used for categorical variables and unpaired t-test for continuous scales. for all results ~ 0 . 0 5 was required for statistical significance. results general results in all 876 subjects answered the questionnaire after one reminder giving a response rate of 71.3%. respondents were 405 men and 471 women and their mean age was 71.2 years (sd=4.0 years, range 65 to 79 years). table 1 gives the age and sex distribution of respondents in the study and the population in the county of dalarna at the time of the study. the younger age groups were slightly overrepresented in the study and the age group of 75 years and older underrepresented. of the males 79.3% lived with someone else compared to 59.6% of females (x2=37.5; p<o.ool), and 26.0% of females were widowed versus 9.1% of males (x2=40.8; p<o.ooi). table 1. age and sex distribution of respondents in the statistical analysis and the population in the county of dalarna at the time of the study. respondents in study population of dalarna county* men (%) women (%) total men (%) women (%) total (%) \ ’-/ age group (in years) 65-69 162 (40) 183 (39) 345 (39) 6 569 (35) 7 454 (33) 14 023 (34) 70-74 140 (35) 156 (33) 296 (34) 6 510 (35) 7 542 (34) 14 052 (34) 75-79 103 (25) 132 (28) 235 (27) 5 740 (30) 7 463 (33) 13 203 (32) *source: swedish central bureau of statistics, yearbook 1995, stockholm 1996. sleep habits time for going to bed was similar between sexes, 10.32f 0.77 hours, but males woke up earlier, 06.74f1.01 hours versus 07.04f 0.86 hours for females ( t = 4.6; p<o.ooi). sleep latencies was significantly longer for females, 41.7+ 59.0 min versus 27.95 36.6 min (t=4.1; p<o.ool), and females also reported a shorter total sleep time 398.8+ 81.5 min versus 416.2f 71.5 min ( k 3 . 3 ; the estimated need for sleep in the total sample was equal between males and females, 434.4f 59.0 min. the ratio of amount of habitual sleep to the amount of estimated need for sleep expressed in p<o.ool). 188 percent (called sleep sufficiency index, ssi), was 91.4% for females and 95.7% for males ( ~ 4 . 2 ; p<o.ool). the number of reported nocturnal awakenings ranged from 0 to 16, with a mean number of 1.9f 1.3 awakenings per night. no difference between sexes was found (table 2). of the study population 47.7% reported waking up at least twice per night and 19.5% reported waking up at least 3 times per night, equal between sexes. table 2. sleep variables (means f s d ) according to gender. variables females males t value time for going to bed 10.27f 0.76 10.37f 0.77 1.9 (hours) time for waking up (hours) 07.04f 0.86 06.74f l.o1 4.6*** sleep latency (minutes) 41.7f 59.0 27.9f 36.6 4.1*** (minutes) (minutes) sleep sufficiency index (%) 91.4f 14.6 95.7+ 13.2 4.2*** no of awakeningstnight 1.9f 1.4 1.8f 1.2 1.5 *p<0.05, **p<o.ol, ***p<o.ool (n=47 1) (n=405) total night sleep time 398.8f 81.5 416.2f 71.5 3.3*** estimated sleep need 434.1f 61.0 434.8f 56.8 0.1 sleep complaints in a categorical (yes-no) question (csp), 18.9% stated that they had sleep problems. females more often than males, 23.8% versus 13.3% (x2=15.1; p<o.ool). subjects living alone did not report sleep problems more frequently than those living with someone else, and no relationship was found between reported sleep problems and coffee or tea consumption, smoking habits or body mass index (bmi). table 3 shows the reported severity of sleeping difficulties according to the usi-question. the most commonly reported sleep complaints were difficulties maintaining sleep (dms). moderate complaints of dms were reported by 34.2% of subjects, followed by moderate complaints of difficulties initiating sleep (dis) 22.9% and moderate complaints of early morning awakenings (ema) 21.7%. the only gender difference was a female preponderance in reports of major complaints of dis, 12.0% versus 4.5% (x2=19.2; p<o.ool). 189 table 3. prevalence of sleeping difficulties according to gender (%). % females % males % total x2 value (n=471) (n=405) (n=876) dificulties initiating sleep moderate complaints 24.6 20.8 22.9 ns (dis) major complaints 12.0 4.5 8.5 19.2, dificulties maintaining sleep 33.9 34.6 34.2 ns (dms) moderate complaints major complaints 10.0 8.5 9 . 3 n s early morning awakenings 20.1 23.5 21.7 n s (ema) moderate complaints major complaints 12.0 11.3 11.7 ns p<o.ool pain was the most commonly reported complaint during initiation of sleep. among females 18.8% and of males 10.0% reported this as a major complaint ( x 2 = 12.9; p<o.ol), followed by major complaints of tension, 11.7% of females and 6.3% of males (x2=7.2; p<o.ol). how many nights per week a sleeping difficulty occurred was measured by using bnsq. fig. 1 shows that more than three out of four elderly, 78.3%, reported waking up at least one night per week and 60.7% reported waking up at least 3 nights per week. no difference between sexes was found. of females 18.1% reported dis at least 3 times per week versus 7.6% of males (x2=19.7; p<o.ool) and 20.1% of females versus 13.8% of males reported ema at least 3 times per week (x2=5.8; p<0.05). 190 fig 1. frequency of sleeping difficulties according to gender (%) m f m f m f dis dms ema daytime symutoms females reported more often than males "never" or only "occasionally" feeling well rested after sleep, 34.4% versus 23.5% (x2=12.0 p<o.ooi). of the males, 26.0% had moderate complaints of daytime sleepiness compared to 17.5% of females ( x 2 = 8.2; peo.ol), and 5.8% of males and 5.1% of females considered this a major problem. subjects with moderate or major complaints of daytime sleepiness had shorter total night sleep time of subjects reporting moderate or major complaints of daytime sleepiness 37.1% reported sleep problems in the categorical item (csp) compared to 13.7% of those reporting no complaints of daytime sleepiness (x2=49.6; peo.001). some 69.5% of males and 50.5% of females reported daytime napping at least occasionally (x2=31.8; peo.ool), and 29.4% of males and 14.7% of females reported napping habitually (x2=21.1; peo.001). there was no difference in total night sleep time or sleep problems (csp) between subjects napping and subjects not napping. 368.4+ 84.8 min versus 418.1+ 69.8 min (t=8.2; peo.001). sleeping pill usage there was a female preponderance in both past and present sleeping pill usage. almost three times as many females as males used sleeping pills at least once per week, 14.7% versus 5.5% (x2=19.4; peo.001). the prevalence of regular sleeping pill users (every night or almost every night) was 7.6% of females and 3.0% of males (x2=8.7; p<0.05). almost twice as many females compared to males had previously used sleeping pills, 19.2% versus 10.4% (x2=12.7; pe0.001). females had also consulted a physician because of sleeping difficulties more often than males, 15.7% versus 9.3% (x2=7.6; pe0.01). (table 4). 191 table 4. prevalence of sleeping pill usage and physician consultations because of sleeping difficulties according to gender (%). % females % males x 2 value sleeping pill usage>l/week 14.7 5.5 19.4*** sleeping pill usage regularly 7.6 3.0 8.7** previous sleeping pill usage 19.2 10.4 12.7*** consulted physician because 15.5 9.3 7.6** of sleeping difficulties *p<0.05 , **p<o.ol, ***p<o.ool (11471) (11405) psvchological status on the had-scale, scores of 8 or more on a subscale was used as an indicator of depression and anxiety respectively. the criterion level indicating possible depression was exceeded by 13.3% of the total sample, equally between sexes. a significant difference between sexes was calculated for anxiety, 23.5% for females and 10.9% for males had scores of 8 or more on the anxiety subscale ( x 2 = 23.8; p<o.ool). as seen in table 5, almost one-third of subjects stating sleep problems i n the categorical item (csp), 29.8%, scored 8 or more on the depression subscale versus 9.4% of subjects without sleep problems (x2=46.6; p<o.ool). almost one-half of subjects with sleep problems, 48.7%, scored 8 or more on the anxiety subscale versus 10.5% of subjects without sleep problems (x2=128.6; p<o.ool). among the regular sleeping pill users (every night or almost every night) 39.1 % exceeded the cut off point indicating possible depression versus 1 1.9% of respondents not using sleeping pills regularly (x2=27.8; p<o.ool). the cut-off point for possible anxiety disorder was exceeded by 63.0% of subjects using sleeping pills regularly versus 15.1% of subjects not using sleeping pills regularly (x2=68.7; p<o.ool). table 5 . prevalence of had score 28 on depression or anxiety subscale (%) among respondents with and without sleep problems (csp) and among respondents using and not using sleeping pills regularly. sleep no sleep x2value regular n o x2 value problems problems sleeping regular (%.> (%i pill usage sleeping (%) pill usage (%i had 29.8 9.4 46.6*** 39.1 11.9 28.7*** depression score 28 had 48.7 10.5 128.6*** 63.0 15.1 68.7*** anxiety score 28 *p<0.05 , **p<o.ol, ***p<o.ool 192 physical health status subjects stating sleep problems in the categorical item (csp) rated their physical health as being poor more often than subjects without sleep problems. only 19.4% of subjects reporting sleep problems in the categorical item (csp) rated their physical health as being excellent or good compared to 52.3% of subjects without sleep problems (x2=83.5; p<o.ool). table 6. prevalence of any major sleep comlaint (asc) among females and males with and without medical health problems (%). ~ asc" asc" males % in) males % females % in) females o/o hyper tension n o yes cardiac disease n o yes angina no yes diabetes n o yes joint pain n o yes asthma no yes gastric symptoms n o yes depression n o yes urinary tract symptoms no yes prostate problems n o 72.0 (275) 14.6 28.0 (107) 21.5 77.5 (289) 14.2 22.5 (84) 26.2** 82.5 (312) 14.4 17.5 (66) 28.8** 89.4 (339) 14.4 10.6 (40) 37.5*** 74.2 (279) 11.5 25.8(97) 29.9*** 93.1 (352) 16.8 6.9 (26) 19.2 76.7 (289) 15.2 23.3 (88) 22.7 95.4 (357) 15.4 4.6 (17) 29.4 84.3 (316) 15.7 15.7 (59) 17.1 73.3 (277) 15.9 69.0 (309) 21.0 31.0 (139) 22.3 85.1 (378) 20.6 14.9 (66) 27.3 87.9 (392) 19.4 12.1 (54) 35.2** 92.1 (408) 20.3 7.9 (35) 31.4 64.2 (281) 18.9 35.8 (157) 26.1 92.3 (408) 20.3 7.7 (34) 32.4 78.6 (348) 19.8 21.4 (95) 26.3 89.4 (390) 16.4 10.6 (46) 56.5*** 79.1 (352) 19.9 20.9 (93) 28.0 yes 26.7 ( i o i j 19.8 ' a x = any major sleep complaint (dis, dms or ema) *p<0.05, **p<o.ol, ***p<o.ool 193 as can be seen in table 6, among males any major sleep complaint (dis, dms or ema) was related to joint pain, diabetes, cardiac disease and angina. among females any major sleep complaint was more commonly reported by females with depression and angina. discussion the results of our study demonstrate that sleep disturbances are common among elderly subjects. almost one-fifth, 18.9%, of subjects reported sleep problems in a categorical (yes-no) question. other surveys have reported prevalence of insomnia from 10-40%, possibly due to various populations and various definition of investigated sleep problem. for example brabbins (4) reported that 35% of persons over the age of 65 living in liverpool reported trouble sleeping the last 4 weeks, weyerer (27) reported 23.3% prevalence of moderate or severe insomnia among persons aged 60-69 years in the upper bavarian field survey and henderson (8) reported the prevalence of insomnia 15.8% in a sample of people aged 70 years and over in canberra and queanbeyan in australia. like previous investigations, females in our sample reported sleep problems more frequently than males, 23.8% versus 13.3%. females went to bed at the same time as males, but waking up time was later. despite this, females rated their total sleep time as shorter than males. these findings indicate that females spend more time awake in bed. females also complained of never or only occasionally feeling well rested after sleep more often than males, 34.3% versus 23.5%. the most commonly reported sleeping difficulty was dms. moderate or major complaints of dms was reported by 43.5% of subjects. the reports of major complaints of dis, dms and ema was rather uniform (8.5%, 9.3% and 11.7% respectively) and the only gender difference was that females reported major complaints of dis more often than males. reports of sleep continuity disturbances were very common in our study. almost one-half of subjects, 47.7%, reported waking up at least twice per night and 19.5% reported waking up at least 3 times per night. nocturnal awakening occurred for over one-half of subjects, 60.7%, at least 3 nights per week. however, only 9.3% of subjects reported major complaints of dms and our interpretation is that these nocturnal awakenings were not always perceived as a sleep disturbance for many of the elderly subjects. these findings underline the importance of distinguishing between the severity and the frequency of a sleep complaint before interpretations are made. we found a male preponderance in reports of daytime symptoms. moderate or major complaints of daytime sleepiness was reported by 3 1.8% of males versus 22.6% of females, which is in agreement with others. for example, asplund (1) reported that of pensioners in northern sweden 32.0% of males and 23.2% of females were often sleepy during the day and schmitt (24) reported that 37.2% of subjects aged 70-79 years in kentucky, usa, complained of excessive daytime somnolence. 194 schmitt found daytime somnolence to be associated with snoring, and we found, like asplund, that daytime sleepiness was related to poor sleep. in our survey we found that daytime napping was not related to sleep problems. this finding supports the hypothesis that napping is an effect of changed distribution of sleep wakefulness in elderly and that it is not necessarily related to sleep disturbances (9, 20). like previous investigators, we found that napping is a common phenomenon. in our sample 69.5% of males and 50.5% of females reported napping at least occasionally. metz (20) reported that 72.7% of males and 56.3% of females reported taking naps among subjects aged 58-95 years, and gislason (5) reported that 50% of males and 3 i % of females aged 65-84 years on iceland slept during the day for an average of 28 min. in our study population 14.7% of females and 5.5% of males used sleeping pills at least once per week, and 7.6% of females and 3.0% males used sleeping pills regularly (every or almost every night). karacan (11) reported that 17% of females and 15% of males aged over 64 years used sleeping pills sometimes, and 9% of females and 8% of males were regular users. morgan (22) reported that 22% females and 1 1.1 % of males aged over 65 years used sleeping pills and gislason (5) reported that 16.7% of females and 12.2% of males aged over 65 years on iceland were habitual users of sleeping pills. in view of these results, our study provides similar or lower prevalence rates of hypnotic usage. there was a relationship between reports of sleep problems and psychological factors. of subjects stating sleep problems in the categorical question, 29.8% scored 8 or more on the had depression subscale, indicating possible depression, compared to 9.4% of subjects without sleep problems. the criterion level indicating possible anxiety disorder was exceeded by 48.7% of subjects reporting sleep problems versus 10.5% of those without sleep problems. sleep disturbances have often been reported to be associated with depression and anxiety disorders. mellinger (19) found that one third of those with serious insomnia in a population aged 18 to 79 years were characterized by syndromes resembling either major depression or generalised anxiety. henderson (8) found insomnia associated with symptoms of both anxiety and depression in subjects older than 70 years, and morgan (22) found symptoms of anxiety rather than depression as predictor of poor sleep quality in subjects aged 65 to 74 years. among the regular sleeping pill users in our study there was a high percentage of subjects scoring 8 or more on the had subscales respectively, 63.0% had possible anxiety disorder and 39.1% had possible depression. subjects with sleep problems in our study also showed evidence of poor physical health status that confirms previous reports of associations between insomnia, self-perceived poor health and physical illnesses. bixler (2), for example, found among adults that subjects with insomnia more frequently reported multiple health problems and bliwise (3) found very low prevalence of poor sleep in his sample of subjects aged 50-65 years with relatively good health. morgan (22) reported from a sample of elderly that subjects with insomnia were significantly more likely to rate their health below average. from a sample of elderly in australia henderson (8) found insomnia to be associated with poor self rated health. in our sample there was a gender difference insofar as medical illnesses among males, but depression among females, was related to any major sleep complaint (dis, dms or ema). 195 our results are based on subjective reports of sleep quality, which is not the same as sleep disorders of clinical significance. webb (25) states that self-reports reflect the person’s view about sleep and its importance, and these conceptions are important to understand because they will determine if the person defines himself as having a sleep problem for which he might seek professional advice or treatment. as a clinician, it is important to understand the relationship between self-reported sleep problems and general health. the present study shows that sleep problems are common among elderly and our data suggest that late life insomnia is not simply due to a normal ageing process. physical and psychiatric disorders must be considered in the evaluation of sleep complaints in the elderly patient. because our data is cross-sectional we are not able to make definitive conclusions about causal relationships. we therefore intend to continue with longitudinal studies and also make structured clinical interviews in an effort to increase knowledge about sleep complaints among elderly. acknowledgements this study was supported by grants from the swedish medical research council (project 06869), the dalarna research institute, the swedish psychiatric association, the marta and nicke nasvells foundation and the swedish lundbeck foundation. references 1 asplund r. daytime sleepiness and napping among the elderly in relation to somatic health and medical treatment. j intern med 1996;239:261-267. 2 bixler e, kales a, soldatos c, kales j , healy s. prevalence of sleep disorders in the los angeles metropolitan area. am j psychiatry 1979; 10: 1257-62. 3 bliwise d l, king a c, harris r b, haskell w l. prevalence of self-reported poor sleep in a healthy population aged 50-65. soc sci med 1992;34(1):49-55. 4 brabbins c j, dewey m e, copeland j r m, davidson i a, mcwilliam c, saunders p, shama v k, sullivan c. insomnia in the elderly: prevalence, gender differences and relationship with morbidity and mortality. int j geriatric psychiatry 1993;8:473-80. 5 gislason t, reynisdottir h, kristbjarnarson h, benediktsdottir b. sleep habits and sleep disturbances among elderlyan epidemiological survey. j intern med 1993;234:3 1-9. 6 gislason t, almqvist m. somatic disease and sleep complaints. an epidemiological study of 3 201 swedish men. acta med scan 1987;221:475-81. 7 habte-gabr e, wallace r b, colsher p l, hulbert j r, white l r, smith i m. sleep pattern in rural elders: demographic, health and psychobehavioral correlates. j clin epidemiol 8 henderson s, jorm a f, scott r l, mackinnon a j, christensen h, korten a e. insomnia in the elderly: its prevalence and correlates in the general population. med j aust 9 hohagen f, kappler c, schramm e, rink k, weyerer s, riemann d, b berger m. prevalence of insomnia in elderly general practice attenders and the current treatment modalities. acta psychiatr scand 1994;90: 102-08. 10 janson c, bjornsson e, hetta j, boman g. anxiety and depression in relation to respiratory symptoms and asthma. am j respir crit care med 1994;149:93. 11 karacan i, thornby j , williams r l. sleep disturbances: a community survey. in: guilleminault l, lugaresi e, eds. sleep/wake disorders: natural history, epidemiology and long-term evolution. new york: raven press 1983;73-85. 12 lack l, miller w, turner d. a survey of sleeping difficulties in an australian population. community health stud 1988;12:200-7. 1991 ;44:5-13. 1995; 162:22-4. 196 13 liljenberg b, almqvist m, hetta j, roos b, agren h. the prevalence of insomnia: the importance of operationally defined criteria. agn clin res 1988;20:293-8. 14 liljenberg b, almqvist m, hetta j, roos b, agren h. age and prevalence of insomnia in adulthood. eur j psychiatry 1989b;3:5-12. 15 liljenberg b, almqvist m, hetta j, roos b, agren h. affective disturbance and insomnia. eur j psychiatry 1989b;3:91-8. 16 lugaresi e, cirignotta f, zucconi m, mondini s, lenzi p l, coccagna g. good and poor sleepers: an epidemiological survey of the san marino population. in: guilleminault l, lugaresi e, eds. sleep/wake disorders: natural history, epidemiology and long-term evolution. new york: raven press 1983;l-12. 17 lundh l-g, broman j-e, hetta j. perfectionism and insomnia. scand j behav ther 18 mcghie a, russel s. the subjective assessment of normal sleep patterns. j ment sci 19 mellinger g d, balter m b, ulenhuth e h. insomnia and its treatment. prevalence and 20 metz m e, bunnell d e. napping and sleep disturbances in the elderly. family practice res 21 moorey s, gear s, watson m, gorman c, rowden l, tunmore r, robertson b, bliss j. the factor stability of the hospital anxiety and depression scale in paients with cancer. br j psychiatry 199 1 ; 158:255-9. 22 morgan k, dallasso h, ebrahim s, arie t , fentem p h. characteristics of subjective insomnia in the elderly living at home. age and ageing 1988;17: 1-7. 23 partinen m, gislason. basic nordic sleep questionnaire (bnsq): a quantitated measure of subjective sleep complaints. j sleep res 1995;4: 150-5. 24 schmitt f a, phillips b a, cook y r, berry d t r, wekstein d r. self reports of sleep symptoms in older adults: correlates of daytime sleepiness and health. sleep 1996; 19:59-64. 25 webb w b. age-related changes in sleep. clin geriatric med 1989;5.259-74. 26 welstein l , dement w c, redington d, guilleminault c, mitler m m. insomnia in the san fransisco bay area: a telephone survey. in: guilleminault l, lugaresi e, eds. sleep/wake disorders: natural history, epidemiology and long-term evolution. new york: raven press 27 weyerer s, dillinger h. prevalence and treatment of insomnia in the community: results from the upper bavarian field study. sleep 1991; 14(5):392-8. 28 zigmond a s, snaith r p. the hospital anxiety and depression scale. acta psychiatr scand 1994;23 31 8. 1962; 108 1642-54. correlates. arch gen psychiatry 1985;42:225-32. j 1990; 1147-56. 1983;73-85. 1983;76:361-70. correspondence to: lena mallon sleep disorders unit department of psychiatry university hospital s-75 1 85 uppsala sweden tel+46 18 66 30 00 f a x + 4 6 1 8 5 1 5 8 10 197 (13) upsala j med sci 111 (2): 269–274, 2006 operative treatment by external fixation for polyostotic fibrous dysplasia in the elbow joint. a case report 1shigetsune matsuya, 1masahito hatori, 1masami hosaka, 1katsu ito, 2osamu dohi, 3mareyuki endo and 1shoichi kokubun 1department of orthopaedic surgery, and 3department of pathology, tohoku university school of medicine, 1-1 seiryomachi, aobaku, sendai 980-8574, japan, 2orthopaedic surgery, tohoku kosai hospital, 3-11 kokubuncho 2 chome, aobaku, sendai 980-0803, japan abstract fibrous dysplasia is a condition in which normal skeletal bone changes into fibroblastic stroma and immature bone. we report our experience of a unilateral external fixator applied for the treatment of polyostotic fibrous dysplasia arising in the elbow. the patient was a 38 year-old man. his main complaint was left elbow pain and a mass, which increased in size. the histological diagnosis from the needle biopsy was fibrous dysplasia. an articulated unilateral external fixator was used for immobilization. the lesions in the left distal humerus and proximal ulna were curetted and cancellous bone was packed into the cavity. an external fixator (elbow fixator, orthofix inc., mckinney, usa) was applied from the proximal humeral shaft to the distal ulnar shaft for postoperative tentative immobilization and preservation of the affected elbow joint movement. at two years after the operation, the range of motion of the affected elbow was 90 degrees in flexion and -35 degrees in extension. the range of motion in pronation and supination of the left forearm was normal. no evidence of recurrence was noted at two years after surgery. the merits of using an external fixator were: an earlier start of range of motion exercises, and a decrease in the risk of tumor dissemination. introduction fibrous dysplasia is a condition in which normal skeletal bone changes into fibroblastic stroma and immature bone. the operative indication for fibrous dysplasia is 269 received 19 september 2005 accepted 4 october 2005 impending fracture or severe pain. tumor curettage and bone grafting is usually performed for the treatment of fibrous dysplasia. reconstruction after massive curettage of a lesion is very demanding. there have been few reports about the surgical treatment for massive osteolysis and multiple bone involvement by fibrous dysplasia in the elbow. we report our experience of a unilateral external fixator applied for the treatment of polyostotic fibrous dysplasia arising in the left elbow involving both the distal humerus and proximal ulna. case the patient was a 38 year-old man. his main complaint was left elbow pain. there was no evidence of mccune-albright syndrome. he felt pain in the elbow already at age 30 when he lifted heavy objects. his left elbow mass gradually increased in size. on initial physical examination, a 5cm x 4cm sized, hard bony mass was observed in the left upper arm lateral epicondyle. the range of motion of the left elbow was 90 degrees in flexion and -30 degrees in extension. pronation and supination were not restricted. plain radiography demonstrated an 8.0 x 6.5cm sized radiolucent lesion with a ground grass appearance and marginal sclerosis in the left humeral condyle. similarly, a 6.5 x 2.0cm sized radiolucent lesion in the left proximal ulna and a 2.0 x 1.0cm sized in the left proximal radius were observed (fig.1). computed tomography (ct) (toshiba medical systems co., ltd., tokyo, japan) showed osteolytic lesions in the left distal 270 fig. 1. histological findings of the needle biopsy. a spindle cell fibrous tissue background and woven bone with a “chinese characters” pattern were observed microscopically. humerus. magnetic resonance imaging (mri) (ge yokogawa medical systems, ltd., tokyo, japan) showed a cystic change of the distal humerus. the distal humeral bone cortex was extremely thin and the cancellous bone changed into lobulus images on t2wi. secondary aneurismal bone cyst (abc) changes were suspected. skeletal scintigraphy revealed high uptake lesions not only in the elbow but also in the left long finger from the metacarpal bone to the distal phalange. the histological diagnosis was fibrous dysplasia with secondary abc (fig.2). operative treatment was chosen because of the enlargement of the tumor size and the high risk of pathological fractures of the humerus and/or ulna. an articulated unilateral external fixator (elbow fixator, orthofix inc., mckinney, usa) was used to preserve the elbow joint function, since internal fixation may lead to tumor dissemination, and post-operative cast fixation can cause joint contracture. at first, we checked the center of the rotation axis of the elbow joint on anteroposterior view and 271 2a 2b fig. 2. plain radiographs of the left elbow. radiographs showed a ground glass appearance and marginal sclerosis. (a: anterior view; b: lateral view) lateral view using an image intensifier. the lateral aspect of the humerus was exposed by blunt dissection in order to prevent damage to the radial nerve. the external fixator was fixed to the humeral normal bone shaft with three screws, kept away from the tumor lesion, as was the ulnar shaft. the lesions were curetted and cancellous bone from the ilium was packed into the cavity. the external fixator was applied. the external fixator’s humeral and ulnar long axis crossed each other at the mechanical axis of the elbow joint. finally, the ball-joints of the fixator were tightened with a wrench (fig.3). postoperatively, range of motion exercises started two weeks after surgery, the external fixator was removed after six weeks, and the screws after two months. no evidence of recurrence was noted two years after surgery. the range of motion of the affected part of the left elbow was 90 degrees in flexion and -35 degrees in extension. the range of motion in pronation and supination of the left forearm was normal. 2a 2b 272 fig. 3. post operative plain radiographs of the left elbow. an external fixator was used for postoperative fixation (a: anterior oblique view; b: lateral view). discussion in fibrous dysplasia the bone matrices change into fibrous tissue or woven bone, and the strength of the bone is weaker than normal. the predominant complaint of fibrous dysplasia is local pain or deformities. additionally, pathological fractures are not rare in patients with fibrous dysplasia (1,2). when the bone does not appear likely to fracture, conservative therapy is chosen for this disease (3-5). in most cases of pathological or impending fracture, surgical treatment is chosen (6-8). the surgical treatment is usually curettage and bone grafting. an external fixator is sometimes employed for the treatment of bone tumors. the indication for its use includes pathological fractures due to bone tumors, and distraction osteogenesis for bone defects after resection or bone grafting. in addition, external fixation is sometimes used after primary oncological surgery, for the treatment of asymmetry (bone lengthening), pseudoarthrosis, infection, correction of angular deformity, and rescue surgery for knee prosthesis that has succumbed to bone reabsorption (9). for distraction oeteogenesis, the ilizalov method of external fixation is frequently used in the management of bone tumors (10,11). we employed a hinged type of external fixation to preserve the elbow joint function. the orthofix elbow fixator, an articulated unilateral external fixation device employed between the humerus and ulna, is designed to permit controlled movement around the center of rotation of the elbow joint. it allows immediate pronation and supination and early flexion and extension of the forearm, thus preventing post-operative stiffness. as far as we know, there are few reports on the use of an external fixator for tumors in the elbow. in trauma cases, external fixation is used for fracture dislocations or complex elbow injuries (12,13), although the number of patients treated with an external fixator is small. this is because the indication for external fixation is limited to cases where conventional techniques have failed or could not be applied (12). a hinged type of external fixator is more suitable for such cases because of the minimal surgical intervention and earlier postoperative joint mobilization compared to the rigid type. even with vigorous curettage followed by bone grafting, the affected elbow joint movements can be moderately preserved, and pronation and supination are not restricted. in the present case, tumor lesions existed in the distal humerus, proximal ulna and proximal radius around the left elbow joint. remarkably, the distal humeral bone cortex was extremely thin and fragile. surgery was indicated because of the potential high risk of pathological fracture in the left humerus and/or ulna. at the operation, the elbow was judged not to be strong enough to hold the bone postoperatively after curettage and bone grafting. we used external fixation for the postoperative temporal fixation (14) and obtained good functional results. the merits of using an external fixation were an earlier start of the range of motion exercises than with cast fixation and a decreased risk of tumor dissemination. the demerits were the inconvenience in daily life and the possibility of pin infection. 273 references 1. enneking wf, gearen pf (1986). fibrous dysplasia of the femoral neck. treatment by cortical bone-grafting. j bone joint surg am 68: 1415-22. 2. parekh sg, donthineni-rao r, ricchetti e, lackman rd (2004). fibrous dysplasia. j am acad orthop surg 12: 305-13. 3. unni kk (1996). conditions that commonly simulate primary neoplasms of bone. unni kk. dahlin's bone tumors:general aspects and data on 11,087 cases. philadelphia. lippincottraven. 367-76. 4. isefuku s, hatori m, ehara s, hosaka m, ito k, kokubun s 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technique of segmental transport. j hand surg [br] 18: 144-6. 11. erler k, yildiz c, baykal b, atesalp as, ozdemir mt, basbozkurt m (2005). reconstruction of defects following bone tumor resections by distraction osteogenesis. arch orthop trauma surg 125: 177-83. 12. stavlas p, gliatis j, polyzois v, polyzois d (2004). unilateral hinged external fixator of the elbow in complex elbow injuries. injury 35: 1158-66. 13. ruch ds, triepel cr (2001). hinged elbow fixation for recurrent instability following fracture dislocation. injury 32 suppl 4: sd70-8. 14. nielsen d, nowinski rj, bamberger hb (2002). indications, alternatives, and complications of external fixation about the elbow. hand clin 18: 87-97. corresponding author: masahito hatori, m.d., assistant professor department of orthopaedic surgery, tohoku university school of medicine 1-1 seiryomachi, aobaku, sendai, japan 980-8574 fax: 81-22-717-7248, tel: 81-22-717-7242 mhato@mail.tains.tohoku.ac.jp 274 ujms109_3.pdf upsala j med sci 109: 165–178, 2004 development and possible clinical use of antagonists for pdgf and tgf-� carl-henrik heldin ludwig institute for cancer research, box 595, biomedical center, s-751 24 uppsala, sweden abstract a free full-text copy of this article can be found at the web page of upsala j med sci: http://www.ujms.se platelet-derived growth factor (pdgf) and transforming growth factor-� (tgf-�) are examples of signaling molecules which control the growth, survival motility and differentiation of cells. pdgf stimulates the growth mainly of connective tissue cells, whereas tgf-� inhibits the growth of most cell types. pdgf and tgf-� exert their cellular effects by vinding to receptors equipped with tyrosine and serine/threonine kinase activities, respectively. both factors have important roles e.g. during the embryonal development and in wound healing. overactivity of pdgf or pdgf receptors contributes to the development of certain diseases characterized by excessive cell growth including fibrotic disorders, atherosclerosis and malignancies. overactivity of tgf-� also contributes to fibrotic conditions, since tgf-� promotes accumulation of extracellular matrix molecules. in cancer, tgf-� is initially a tumor suppressor due to its ability to inhibit cell growth, however, at later stages of tumor progression tgf-� has tumor promoting activity by enhancing the invasive properties of tumor cells and by suppressing the immune system and promoting angiogenesis. the involvement of pdgf in tgf-� in serious diseases makes clinically useful antagonists highly desirable. a low molecular weight receptor kinase inhibitor of the pdgf receptor kinase is now tested clinically, and tgf-� antagonists are under development. the present review discusses the development and possible clinical use of antagonsts for pdgf and tgf-�. introduction the growth, survival, motility and differentiation of cells are regulated in part by signals the cell receives from its environment. such signals are important, e.g. during embryonal development, wound healing, hematopoiesis and during the immune 165 received 18 march 2004 accepted 25 march 2004 response, and may come from interactions with other cells or the extracellular matrix, or from stimulation by specific soluble growth regulatory factors. accumulating evidence suggest that the lack of growth control of tumor cells is due to constitutive activation of components, which in the normal cell is controlled by growth stimulatory factors, or by loss of functional activity of components in growth inhibitory pathways. modulation of signaling pathways involved in cell growth and survival is therefore an interesting strategy to explore in the treatment of cancer. the present review focuses on two important growth regulatory factors, i.e. platelet-derived growth factor (pdgf), a major mitogen for connective tissue cells, and transforming growth factor-� (tgf-�), which inhibits the growth of most cell types. the possibility to use knowledge about the signaling mechanisms of these factors in the development of drugs for the treatment of patients with cancer or other diseases, will be discussed. platelet-derived growth factor mechanism of action of pdgf pdgf is a family of disulfide-bonded dimers of structurally similar polypeptide chains, which occur as homodimers (pdgf-aa, -bb, -cc and -dd) or as a heterodimer (pdgf-ab) (reviewed in 1). the pdgf isoforms exert their cellular effects by binding to structurally similar �and �-tyrosine kinase receptors. ligand binding induces receptor dimerization which is a key event in the activation of the receptors. since the a-, band c-chains of pdgf bind to the �-receptor, whereas the band d-chains bind to the �-receptor, different types of homoand heterodimeric receptor complexes can be formed depending of the stimulating pdgf isoform and depending on which of the receptors the target cell expresses (fig. 1). ligand-induced receptor dimerization brings the intracellular parts of the receptors close to each other, so that the kinase domain of one receptor can phosphorylate specific tyrosine residues in the other, and vice versa. the autophosphorylation serves two important functions; phosphorylation of the activation loop of the kinase domain enhances the kinase activity, and phosphorylation of tyrosine residues outside the kinase domain creates specific docking sites for sh2-domain-containing signaling molecules. about 10 different types of sh2-domain-containing proteins bind to the pdgf receptors. the specificity in binding is determined by the amino acid residues downstream of the phosphorylated tyrosine. the docking of the signaling molecules activates a cascade of signaling pathways, which ultimately leads to initiation of dna synthesis and cell division, survival and migration (reviewed in 2). among the signaling pathways activated after pdgf stimulation of cells, phosphatidylinositol-3’-kinase (pi3-kinase) and phospholipase-c� (plc�) are particularly important for the motility effects of pdgf, and, in the case of pi3-kinase, for the survival effect. important mediators of the mitogenic effect of pdgf are ras, which is activated by the docking of the adapter grb2 in complex with the exchange factor sos1 to the receptor and which activates the erk map kinase pathway, and 166 src, which induces the transcription factor c-myc. it should be emphasized, however, that intracellular signaling occurs in a network of signaling components, with extensive interactions between different signaling pathways. in vivo function of pdgf pdgf has important functions during the embryonal development. the ligand is often produced by specific epithelial cells, and act on neighboring connective tissue cells by paracrine stimulation. the specific functions of individual pdgf isoforms and receptors have been elucidated by knock-out studies in the mouse. thus, inactivation of the genes for the pdgf b-chain (3) or the �-receptor (4), leads to severe defects in the development of the kidneys due to a complete lack of mesangial cells. also blood vessel development is defect leading to bleedings at the time of birth, which often is the direct cause of death; the reason is that in the absence of pdgf-b or pdgf �-receptor, the vascular smooth muscle cells and pericytes do not develop normally (5). knock-out of the a-chain leads to death of the mice at around the age of 3 weeks, 167 fig. 1. five pdgf isoforms induce the formation of three dimeric receptor complexes. the picture schematically illustrates the binding specificities of pdgf isoforms. the �and �-receptors for pdgf each contains 5 immunoglobulin-like domains extracellularly, and a kinase domain intracellularly which is split into two parts by an intervening sequence. due to a defect development of alveolar smooth muscle cell progenitors (6). in addition, the mucosal lining of the gastrointestinal tract is defect with a decreased number of villus clusters (7). knock-out of the �-receptor gives a severe phenotype with cranial malformations and a deficiency in myotome formation (8). in the adult, pdgf promotes wound healing. pdgf stimulates chemotaxis and growth of many cell types involved in the healing process, including fibroblasts, smooth muscle cells, neurotrophils and macrophages. clinical studies have shown that topical administration of pdgf stimulates wound healing in patients with decubitus ulcers (9). the importance of pdgf for the connective tissue compartment is also illustrated by the fact that pdgf regulates the interstitial fluid pressure (10). the exact mechanism involved is not known, but probably involves increased interactions between fibroblasts of the connective tissue and matrix molecules, and a stimulation of contraction of fibroblasts through effects on the actin filament system of such cells. pdgf in disease overactivity of pdgf has been implicated in several types of serious disorders, including malignant diseases, as well as atherosclerosis and fibrotic diseases. in atherosclerosis, injury to the endothelial cell layer leads to deposit of pdgf from platelets, macrophages and other cells adhering to the injured site. pdgf then stimulates migration of smooth muscle cells from the media into the intima of the vessel, where they proliferate. this intimal thickening causes a narrowing of the vessel lumen and is an early stage in the atherosclerotic process, which subsequently is followed by lipid deposition and plaque formation (11). given the important role of pdgf for the growth and development of different types of connective tissue cells, it is not surprising that overactivity of pdgf can contribute to fibrosis. there are evidence for the involvement of pdgf in lung fibrosis, glomerulonephritis, liver cirrhosis and myelofibrosis (reviewed in 12). in malignancies, pdgf has two principally different effects. on one hand, activation of pdgf receptors, by mutational events or by constitutive autocrine stimulation by ligand, may drive the proliferation and survival of tumor cells. on the other hand, pdgf produced in the tumor may act in a paracrine manner on the nonmalignant cells in the tumor, i.e. fibroblast-like cells in the stroma, and smooth muscle cells, pericytes and, possibly, endothelial cells of blood vessels (reviewed in 13). the first demonstration that autocrine stimulation by pdgf can have a transforming effect, came through the discovery that the sis oncogene of simian sarcoma virus is derived from the pdgf b-chain gene (14; 15). subsequent studies have demonstrated that production of pdgf by tumor cells that possess pdgf receptors is common in glioblastoma (16) and sarcoma (17). experimental proof that pdgf overproduction can cause glioblastoma has come from studies showing that such tumors are formed after intracranial injection of simian sarcoma virus into marmosets (18), or a pdgf b-chain containing retrovirus into mice (19). there are some examples of chromosomal translocations in patients which lead 168 to constitutive activation of pdgf receptors. thus, the skin tumor dermatofibrosarcoma protuberance (dfsp) is associated with a translocation which fuses the collagen 1a1 gene with the pdgf b-chain gene (20). this results in the production of large quantities of a fusion protein, which after processing to pdgf-bb causes autocrine stimulation of growth and cell transformation (21; 22). in the case of the rare disease chronic monomyelocytic leukemia (cmml), the part of the �-receptor gene that encodes the kinase domain is fused to certain genes, e.g. the tel gene, that have in common that they encode proteins that can dimerize (23–25). this results in constitutively dimerized and activated receptors, which drive tumor cell proliferation and survival. a similar situation prevails in the case of hypereosinophilic syndrome (hes); in this disease the pdgf �-receptor gene has been fused to the fip1l1 gene, leading to constitutive dimerization and activation of the receptor kinase activity (26). in the gastrointestinal stromal tumor (gist), the �-receptor for pdgf often have point mutations in the activation loop, or elsewhere, causing activation of the receptor kinase which drives tumor cell growth and survival (27). interestingly, gist alternatively can have mutations in the structurally related tyrosine kinase receptor for stem cell factor (28). finally, a subset of gliomas shows amplification of the �-receptor gene, leading to an increased susceptibility to ligand stimulation or activation in the complete absence of ligand (29; 30). tumors of epithelial cell types generally do not express pdgf receptors. however, pdgf produced in such tumors may stimulate non-tumor cells in blood vessels and in the stroma, in a paracrine manner. whereas pdgf does not have as potent effect in the early stages of angiogenesis as the classical angiogenesis factors of the vascular endothelial cell factor or fibroblast growth factor families, pdgf may have important roles at later stages in vessel maturation through its effects on smooth muscle cell development and pericyte recruitment (5; 31). interestingly, pdgf receptor-deficient melanoma cells transfected with pdgf-b or -d were found to grow faster and to have an increased pericyte coverage of their vessels, compared to untransfected melanoma cells (32). another important effect of paracrine pdgf stimulation relates to the regulation of the interstitial fluid pressure (ifp) in tumors. many solid tumors have increased ifp (33), which is an obstacle in tumor treatment, since it leads to an inefficient uptake of chemotherapeutical drugs from the circulation. there is evidence that pdgf, through its action on stromal myofibroblasts, may be one of the factors causing the increased tumor ifp (34). pdgf antagonists for clinical use given the involvement of pdgf overexpression in serious diseases, clinically useful pdgf antagonists are highly warranted. among the different antagonists that have been developed are molecules that prevent ligand activation of receptors, including antibodies, soluble extracellular receptor domains or dna aptamers 169 (reviewed in 35). the advantage of these types of antagonists is that they are often highly specific, however, since they are macromolecules, they are expensive and cumbersome to administrate. another type of antagonists is low molecular weight inhibitors of the pdgf receptor kinase. these are less expensive and easier to administer, but are not absolutely specific. for instance, imatinib (sti571, glivec) inhibits in addition to the �and �-receptors for pdgf, also the kinases of stem cell factor receptor, abl and arg; it is already used clinically in the treatment of chronic myeloid leukemia which is characterized by overactivity of abl (36; 37), and of gist which is characterized by activation of stem cell factor receptor or pdgf �-receptor (38). initial clinical studies in which glivec has been used on patients with malignancies characterized by activation of pdgf receptors, have shown encouraging results. thus, reduction of tumor mass has been reported for patients with gist (28; 38), cmml (39), hes (26) and dfsp (40; 41). however, whereas treatment of nude mice with intracranially growing glioblastoma has been shown to reduce tumor growth (42), it remains to be determined whether patients with glioblastoma benefit from treatment with pdgf antagonists. pdgf antagonists may also potentially be useful in order to inhibit paracrine pdgf stimulation of normal cells in the tumor. thus, pdgf antagonists may have an anti-angiogenic effect, or may enhance the anti-angiogenic effect of e.g. vascular endothelial growth factor antagonists (43). another interesting possibility is to lower the tumor ifp by treatment with pdgf antagonists and thereby achieve a higher uptake and efficiently of chemotherapy. encouraging results have been obtained in animal models, where a significant enhancement of chemotherapy has been observed in the presence of glivec treatment (44). the initial clinical studies using pdgf antagonists have given encouraging results. it will be a challenge to develop selective pdgf antagonists and explore their usefulness for the treatment, not only of malignant diseases, but also for the treatment of atherosclerosis and fibrotic conditions. transforming growth factor-� mechanism of action of tgf-� tgf-� is a disulfide-bonded dimer, and is a prototype for a family of about 30 members in humans, including tgf-�:s, activins, bone morphogenetic proteins (bmp:s) and growth and differentiation factors (gdf:s). tgf-� family members regulate cell proliferation, differentiation and apoptosis, and have important functions during embryonal development (reviewed in 45). they exert their cellular effects by forming a heteromeric complex of type i and type ii serine/threonine kinase receptors. within such complexes, the constitutively active type ii receptor phosphorylates and activates the kinase activity of the type i receptor. important substrates for the activated type i receptor kinase, include members of 170 the smad family of signal transduction molecules. the smad family consists of three subfamilies. receptor-activated smad (r-smads) are phosphorylated by type i receptors in a c-terminal -ser-xaa-ser motif (smad2 and 3 for tgf-� and activin receptors, and smad1, 5 and 8 for bmp receptors). thereafter, r-smads form complexes with the common-mediator smad (co-smad; smad4) which are translocated into the nucleus, where they regulate transcription by direct or indirect binding to the promoter regions of specific genes. among the target genes for r-smad/cosmad complexes are inhibitory smads (i-smads; smad6 and 7), which are induced by tgf-� or bmp stimulation and act in a negative feed back loop to suppress smad signaling. i-smads bind to the receptors and thereby prevent interaction and phosphorylation of r-smads; in addition, smad7 binds the ubiquitin ligase smurf, which thereby is recruited to the receptors and targets them for ubiquitin-mediated degradation in proteosomes (fig. 2) (reviewed in 46). 171 fig. 2. involvement of smad molecules in tgf-� signaling. the picture illustrates that tgf-� induces a heteromeric receptor complex consisting of two type i and two type ii receptors. within this complex the constitutively active type ii receptor (light blue) phosphorylate the type i receptor (dark blue) in a region just upstream of the kinase domain, whereby the type i receptor kinase is activated. the type i receptor then phosphorylates r-smads (smad2 and 3) which then forms complex with the cosmad (smad4); the complex is then translocated into the nucleus, where it in conjunction with other nuclear proteins (yellow) binds to the promoter regions of specific genes. among the target genes are i-smads (smad6 and 7). the induced i-smads act in a negative feedback mechanism by competing with r-smads for binding to the receptors and, in the case of smad7, by bringing the ubiquitin ligase smurf to the receptor. smad molecules have two conserved domains, the c-terminal mh2 domain which is present in all smads, and the n-terminal mh1 domain which is present in rand co-smads, but not in i-smads. the mh1 domain, in certain smads, regulates nuclear import and transcription by direct binding to dna, as well as via binding to other nuclear proteins. the mh2 domain is responsible for interactions with receptors and for intermolecular interactions within smad complexes. it also mediates interactions with cytoplasmic anchors, like sara, and with a number of nuclear transcription factors. in the linker region connecting the mh1 and mh2 domains, there is a py motif which binds smurf ubiquitin ligases. in addition to the activating phosphorylation of r-smads in the c-terminal tail by receptors, the activity of smad molecules are also modulated by phosphorylation in other parts of the molecules by other kinases, including erk map kinase, protein kinase c and calmodulin-dependent kinase ii. thus, smad molecules are involved in a multitude of interactions, which regulate their activation, subcellular localization, stability and nuclear roles as transcription factors (reviewed in 47). the nuclear partners of smad molecules include the histone acetyl-transferase domain-containing co-activators of the p300 and p/caf families. certain smads bind directly to 5'-gaga-3' motifs in dna, but this binding is of low affinity, and interactions with other dna binding transcription factors are important for smad function. smad complexes also repress the transcription of specific genes, e.g. through interaction with co-repressors associated with histone deacetylase activity, such as tgif, ski and snon. although smad molecules have crucial importance in tgf-� signaling, also other signaling pathways are induced after tgf-� stimulation of cells. these include activation of the erk, jnk and p38 map kinase pathways and pi3-kinase (reviewed in 48). the importance of these signaling pathways for the effects of tgf-� on cells, remains to be elucidated, however, evidence has been presented that jnk and p38 activation are important for induction of apoptosis (reviewed in 49). in vivo function of tgf-� all tgf-� family members have roles during embryonal development. knock-out of their receptors or signaling smad molecules generally leads to embryonic lethality (reviewed in 50). in the adult, tgf-� stimulates wound healing through its ability to cause accumulation of matrix molecules; tgf-� both stimulate production of extracellular matrix molecules, and inhibit the degradation of matrix through the production of protease inhibitors. tgf-� in disease overactivity of tgf-� results in fibrotic diseases, including lung fibrosis, glomerulonephritis and liver cirrhosis. whereas pdgf promotes fibrosis by stimulation of 172 connective tissue cells to migrate and proliferate, tgf-�’s involvement in fibrosis is through its ability to cause accumulation of extracellular matrix. in cancer, tgf-� has a complicated role. initially tgf-� is a tumor suppressor because of its abilities to inhibit cell proliferation and promote apoptosis of cells. in certain tumors loss of function mutations in components in the tgf-� pathway are seen. thus, mutations in the tgf-� receptor genes are seen in colorectal cancers (51), and the smad4 gene is mutated in 50% of pancreatic carcinoma (52). however, at later stages of tumor progression, tgf-� has tumor promoter activity by inducing epithelial-to-mesenchymal transdifferentiation and thereby promoting invasiveness and metastasis of tumor cells that have retained responsiveness to tgf-�. moreover, the effects of tgf-� on the non-malignant cells in the tumor, suppressing the immune response and promoting angiogenesis, also promote tumorigenesis (reviewed in 53). the dual role of tgf-� in tumorigenesis was clearly demonstrated in a keratinocyte tumorigenesis model; overexpression of tgf-� in keratinocytes initially inhibited the formation of benign skin tumors in response to exposure to a chemical carcinogen, but the benign tumors that eventually were formed rapidly progressed to invasive spindle-cell carcinomas (54). tgf-� antagonists for clinical use tgf-� antagonists could potentially be useful in the treatment of various fibrotic diseases, as well as in advanced forms of cancer. inhibition of tgf-� by a soluble extracellular domain of the type ii tgf-� receptor in mouse models for breast cancer and melanoma led to a clear reduction in metastasis and inhibition of angiogenesis (55–57). moreover, low molecular weight inhibitors of the tgf-� type i receptor kinase have been developed (58–61), which offers an alternative way of inhibiting tgf-� signaling. however, the dual role of tgf-� in tumorigenesis makes use of tgf-� antagonists complicated. whereas inhibition of tgf-� signaling, e.g. through sequestration of tgf-� or inhibition of tgf-� receptor kinases, could have beneficial effects in fibrotic conditions or late stage cancers, such treatment would be associated with a risk of promoting recruitment of novel tumor cells due to inhibition of the tumor suppressive effects of tgf-�. an interesting and appealing strategy would therefore be to develop selective tgf-� inhibitors, which inhibit only those intracellular pathways that are involved in tumor progression, while leaving those that are involved in the tumor suppressive effects unperturbed. recent work on the transcription factor yin yang-1 (yy1) has illustrated that this may be a possible strategy. yy1 binds to smad molecules and represses several of their responses, however, yy1 does not affect tgf-�or bmp-induced growth inhibition (62). an inhibitor that mimics the effect of yy1 on tgf-� signaling, would thus be an interesting candidate for evaluation in treatment of advanced cancers. however, such a strategy would clearly require additional work to elucidate in detail which pathways are responsible for the various effects of tgf-� on cells. 173 future perspectives since many diseases involve overactivity of various growth regulatory factors, inhibitors of signal transduction are likely to become important future drugs. the first inhibitors of this type are currently used clinically with encouraging results. pdgf antagonists have proven to be useful in certain rather uncommon malignancies that are dependent on a constitutively active pdgf receptor. however, it is possible that pdgf antagonists can be used in a more general manner to target normal cell types in solid tumors, in order to lower the interstitial fluid pressure and thereby enhance the effect of chemotherapy, and to inhibit angiogenesis and thereby starve the tumor cells. moreover, pdgf antagonists can potentially be used also in the treatment of atherosclerosis and fibrotic conditions. which type of antagonist that will be most useful, remains to be determined. the advantage with antibodies, soluble receptor domains or aptamers is the high specificity, on the other hand, the less specific but more convenient low molecular weight inhibitors have shown rather modest side effects. the most noticeable side effect of glivec treatment has been peri-orbital edema (63), maybe reflecting that pdgf has an important role in the control of the interstitial fluid pressure in loose connective tissue. the first generation tgf-� antagonists, a soluble extracellular tgf-� type ii receptor and low molecular weight receptor kinase inhibitors, are now tested in animal models of advanced cancers. initial results have shown positive treatment effects, and have encouraged continued work to produce clinically useful tgf-� antagonists. it remains to be seen, however, if the side effects during treatment with tgf-� antagonists will be tolerable. since tgf-� has an important controlling effect of the immune system, inhibition of tgf-� may be accompanied by excessive immune reactions. moreover, tgf-� inhibits the growth of most cell types; inhibition of tgf-� may thus promote cell growth and development of tumors. an important future task will be to explore the side effects of tgf-� antagonists, and to elucidate whether it is possible to develop selective tgf-� antagonists which inhibit only those responses that cause disease progression. although it is too early to say how useful antagonists for pdgf and tgf-� will be for the treatment of human disease, the results achieved so far are clearly encouraging, and provide a stimulation for further work to develop and explore the clinical utility of such antagonists. acknowledgements i thank ingegärd schiller for skilful secretarial work. references 1. heldin c-h, eriksson u, östman a (2002). new members of the platelet-derived growth factor family of mitogens. arch. biochem. biophys. 398: 284–290. 2. heldin c-h, östman a, rönnstrand l (1998). signal transduction via platelet-derived growth factor 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buchdunger e, zimmermann j, matter a (2002). glivec (sti571, imatinib), a rationally developed, targeted anticancer drug. nat. rev. drug discov. 1: 493–502. about the author professor carl-henrik heldin is director of the ludvig institute for cancer research in uppsala, sweden, and professor of molecular cell biology at uppsala university. professor heldin received the 2003 rudbeck award for his pioneering studies of the structure and function of pdgf (platelet derived growth factor) and its applications to various new treatments of cancer. he has received a number of prestigious scientific awards and is the author of more than 480 research articles and reviews in international journals. corresponding author: professor carl-henrik heldin ludvig institute for cancer research p.o. box 595 se-751 24 uppsala sweden 178 tf-iups160023 216..221 review article preconception health and care (phc)—a strategy for improved maternal and child health anna berglunda and gunilla lindmarkb anational centre for knowledge on men’s violence against women, uppsala university, uppsala, sweden; binternational maternal and child health, uppsala university, uppsala, sweden abstract maternal health status before pregnancy is a decisive factor for pregnancy outcomes and for risk for maternal and infant complications. still, maternity care does not start until the pregnancy is established and in most low-income settings not until more than half of the pregnancy has passed, which often is too late to impact outcomes. in western societies preconception care (pcc) is widely recognized as a way to optimize women’s health through biomedical and behavioural changes prior to conception with the aim of improving pregnancy outcomes. but the content of pcc is inconsistent and limited to single interventions or preconception counselling to women with chronic illnesses. it has been suggested that pcc should be extended to preconception health and care (phc), including interventions prior to pregnancy in order to optimize women’s health in general, and thereby subsequent pregnancy outcomes, the well-being of the family, and the health of the future child. with this definition, almost every activity that can improve the health of girls and women can be included in the concept. in the world health report of 2005 a longitudinal approach to women’s wellness and reproductive health was highlighted, and the world health organization has proposed a more comprehensive maternal and child health care, also including psychosocial issues and intimate partner violence. the present article gives an overview of the recent literature and discusses contents and delivery of pcc/phc in western as well as lowincome countries. the article puts special emphasis on why violence against women is an issue for phc. article history received 23 march 2016 revised 11 may 2016 accepted 16 may 2016 keywords intimate partner violence; maternal and child health; preconception care; provision of care introduction at the beginning of the new millennium, world leaders gathered at the united nations to shape a broad vision to fight poverty in its many dimensions, resulting in the eight millennium development goals (mdgs). the stipulated time for realizing the goals expired at the end of 2015. preliminary results show that the framework has in many ways been successful in focusing on crucial issues for a positive development of global health and welfare. child mortality for underfive-year-olds was reduced from 90 to 43 deaths for every 100,000 children (mdg 4). maternal mortality rate has declined by 45% globally. more women have got access to reproductive care, and the use of contraception has increased among married or co-habiting women (mdg 5). however, reproductive health risks are still unacceptably high in many countries, and reproductive health and rights remain one of the most important issues for future development (1). maternal health status before pregnancy is a decisive factor for pregnancy outcomes and for the relative risk for maternal as well as infant complications. still, maternal health care does not start until the pregnancy is well established and in most low-income settings not until more than half of the pregnancy has passed. the term preconception care (pcc) is widely used for activities intended to address and prevent specific problems related to pregnancy. preconception health and care (phc), on the other hand, can be defined as the provision of biomedical and behavioural interventions prior to pregnancy in order to optimize women’s wellness and subsequent pregnancy outcomes with the aim to improve not only foetal, infant, and maternal health, but also the health of the whole family and the future well-being of the offspring. hence phc includes medical as well as educational and psychosocial issues. with this broad definition, almost every activity that can improve the health of girls and young women can be included in the concept. the present article gives an overview of the recent literature and important issues in the contents and delivery of health care prior to pregnancy in western as well as low-income countries. the article puts special emphasis on intimate partner violence (ipv) and why violence against women is an issue for phc. the content of preconception health services in western societies pcc is widely recognized as a way to optimize women’s health and improving pregnancy outcomes. but although there is a growing body of evidence supporting that very early pregnancy is a critical period for both maternal and foetal health, preconception care is contact anna berglund anna.berglund@nck.uu.se arosv€agen 5, 722 17 v€asterås, sweden � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 4, 216–221 http://dx.doi.org/10.1080/03009734.2016.1191564 http://creativecommons.org/licenses/by/4.0/ inconsistent, and pcc is mostly limited to single interventions or preconception information and counselling to women with chronic illnesses such as diabetes even in countries with comprehensive health care systems (2). such programmes are common in many western countries and have also been assessed as effective. other initiatives are focused on lifestyle changes such as the cessation of smoking and drinking alcohol, or the supplementation with folic acid. in order to address the lack of consensus regarding the content and the delivery of pcc in the netherlands, the health authorities organized an expert meeting. the consensus meeting resulted in the following definition: a set of interventions and/or programmes that aims to identify and enable informed decision-making to modify biomedical, behavioural, and (psycho-) social risks to parental health and the health of their future child, through counselling, prevention and management, emphasizing those factors that must be acted on before conception and in early pregnancy, to have maximal impact and/or choice (3). this definition of pcc is in accordance with the wider concept of phc. health care in order to improve pregnancy outcomes has rarely been provided in poor countries, where women have many health problems. even though maternal and child health is traditionally organized together and often provided by the same professional staff, little or no attention is given to the women before and between pregnancies. in the world health report, make every woman and child count (from 2005), the longitudinal approach to women’s wellness and reproductive health was highlighted, and the world health organization (who) has in many of its programme recommendations proposed a continuum of maternal and child health, and called for a reformulation of interventions from vertical programmes to those offering a wider range of services (4). in countries where many women have poor health and malnutrition, the activities to be considered for preconception care are numerous. in 2012, who organized a meeting to develop a global consensus on preconception care to reduce maternal and childhood mortality and morbidity (5). in a review prepared for the meeting, the list of activities included: � tobacco use prevention and cessation programmes � nutrition programmes � vaccine programmes � fertility and infertility programmes � female genital mutilation programmes � hiv testing and counselling programmes � mental health programmes � substance use programmes � intimate partner and sexual violence programmes � premarital counselling programmes � genetic counselling programmes � maternal and child health programmes � adolescent-friendly services � occupational health programmes most of these programmes exist as projects or vertical interventions also in many low-income countries, but not with a perspective of preconception care. probably, the easiest interventions to introduce are vaccinations such as against rubella, hepatitis, and tetanus, which can be included in general vaccination programmes. also hiv programmes are often given priority in the health care system, although the barriers for women to attend them are high. preconception interventions that involve community participation are much more complicated and difficult to implement. it is often difficult to assess the effects of preventive activities, especially of a general nature. there are many descriptive reports about various projects for preconception information and mostly specific single interventions, but what is the evidence for preconception care being effective in improving pregnancy outcomes? in a recent review only eight randomized studies of moderate to poor quality were found in a literature search, and half of them were single-risk interventions (6). the outcomes assessed were limited to maternal knowledge, self-efficacy and health locus of control, and risk behaviour, with some evidence of improvement. thus the evidence for improved pregnancy outcomes is indirect at best. the best evidence for important benefits of preconception care is for diabetic women, for whom good metabolic control before pregnancy will decrease the risk of malformations (7). a comprehensive phc approach would address both women and men. it should include promotion of a healthy lifestyle before as well as in between pregnancies, such as advice on nutrition to avoid under-nourishment or hazardous overweight and obesity. appropriate spacing of pregnancies is important to restore a good health status after birth and adequate nutrition of the newborn. phc also includes education about use of tobacco, alcohol, and drugs and the adverse effects on health and reproduction; safe sex information and education; and family planning including meaningful education about contraception, and discussions about when to start a family. phc should recognize the importance of avoiding adolescent pregnancies. it is estimated that 60 million adolescents give birth each year worldwide, even though globally pregnancy in adolescence has mortality rates at least twice as high as pregnancy in women aged 20–29 years (8). the issue of gender-based violence and abuse is intertwined in all mentioned aspects and should be appropriately addressed early as well as later (3,5,7). why violence against women is an issue for phc according to the millennium development goals report, progress has been made towards women’s and girls’ equality in education, employment, and political representation over the last two decades. but to achieve universal realization of gender equality and empowerment of women, one of the key areas to address is gender-based discrimination and violence against women and girls (1). prevalence estimates for violence against women in 2013, who published a systematic review of prevalence estimates of intimate partner violence (ipv) against women, and sexual violence victimization from non-partners. in total 77 studies from 56 countries representing nearly 350,000 upsala journal of medical sciences 217 women, 15 years or older, were included in the review (9). the dominating databases were the who multi-country study on women’s health and domestic violence against women, including women in lowand middle-income countries, the international violence against women survey initiated by the un in the 1990s, and genacis, gender, alcohol and culture, with data from the americas, australia and new zeeland, india, central asia, and europe. the results of the review show that 35% of women worldwide had been subjected to physical or sexual violence by an intimate partner, or sexual violence from a non-partner, or both, during their lifetime. among women who have at some stage had a sexual partner (ever-partnered) 30% had experienced physical and or sexual violence in their relationships. the prevalence was highest in the who african, eastern mediterranean, and south-east asia regions, where approximately 37% reported ipv. in the who region of the americas the next highest prevalence was reported, with approximately 30% of women reporting lifetime exposure. prevalence was lower in the high-income part of the region (23%) and in the european and the western pacific regions, where 25% of ever-partnered women reported lifetime ipv experience. globally, 7% of women reported sexual abuse by someone that was not their partner. in highincome countries the prevalence was higher, close to 13%. since sexual activity outside marriage in many cultures is taboo, the authors strongly believe that prevalence might be underestimated. also, there are very few studies on systematic sexual violence in wartime (9). in a prevalence study among the 28 member states in the european union 22% of european women had been subjected to physical and/or sexual violence by a partner after the age of 15. eleven per cent had experienced sexual violence, and 5% had been raped after the age of 15. in addition, 6% had experienced attempted rape (10). comparing prevalence estimates among countries might be hazardous because of differences in legislation and because the definition of violence varies. however, in sweden a national survey reported that 20% of women had experienced rape and/or attempted rape during their lifetime. in total, 14% of the women in the study had been physically abused, 7% sexually abused, and 20% emotionally abused by an intimate partner in a lifetime perspective (11). violence victimization and ill health several prevalence studies have also considered the possible consequences for health of violence victimization. a number of important health problems have been identified among victims of ipv. the who systematic review of reproductive health found that victimized women were 16% more likely to have a low-birth-weight baby, more than twice as likely to have an abortion, and, in some regions, 1.5 times more likely to acquire hiv. depression was twice as common among victims, as compared to women who had not experienced partner violence (9). recently published swedish research shows that violence victimization is associated with mental as well as physical ill health. the association may be even stronger when the perpetrator is a partner and when the victim has been subjected to more than one form of violence, which usually characterizes violence against women in an intimate relationship (11,12). reproductive coercion another aspect of ipv is the possible impact on women’s control of their reproduction. in a systematic review concerning interactions between ipv and use of contraception, the authors found several ipv-related factors that influence women’s abilities to protect themselves against unplanned pregnancies. violence victimization may eliminate the use of contraceptives, earlier experiences of violence associated with a request for contraceptives may discourage any new attempts or simply the fear of violence could hinder women from requesting the use of contraceptives. relationship imbalances might also impact on the victim’s self-efficacy, resulting in the belief that she is unable to make any autonomous decisions. the reviewed studies reported that a considerable number of women experience reproductive coercion, either in the form of birth control sabotage or pregnancy coercion. loss of reproductive control should also take forced abortions into consideration, but here research is very limited. the authors suggest that health personnel, especially within family planning, should include questions about ipv and reproductive coercion in their consultations (13). unintended pregnancy and induced abortion in relation to ipv unintended pregnancy was recently addressed in a european multi-country cross-sectional study among women in antenatal care. approximately one-fifth of all women reported their current pregnancy to be unintended. among women reporting any lifetime abuse, the prevalence of an unintended pregnancy was 25%, and among women reporting recent abuse 39%. women with a history of any lifetime abuse had significantly higher odds of unintended pregnancy, also after adjusting for confounding factors, with an odds ratio of 1.41 (95% ci 1.23–1.60) for any lifetime abuse and 2.03 (95% ci 1.54–2.68) for recent abuse (14). the prevalence of ipv among women seeking termination of pregnancy was compared to that of women seeking contraceptive counselling in a recent swedish study. in total, 29% of women wanting termination of pregnancy and 22% in the family planning group reported ipv. women seeking abortions were significantly more likely to report ipv (adjusted odds ratio 1.6, 95% ci 1.2–2.1), and among women with repeated terminations the prevalence of ipv was 51% (8,15). identifying violence victimization health care has a major role in identifying women subjected to violence. most women find it acceptable or appropriate to be asked (10,16,17). that trustworthy professionals raised the issue of abuse is considered important by victimized women for their future decisions on taking action to improve their 218 a. berglund & g. lindmark situation (18,19). the questions about violence must, however, be put in an empathic way and in private. in western societies we often believe that the only way to avoid recurrent partner violence is to end the relationship, which tends to be complicated and sometimes dangerous. in many places in the world this is even more difficult for cultural reasons. many researchers have suggested that promoting issues of gender equality could be a way of preventing or reducing ipv. there are encouraging examples within programmes for fighting hiv-aids (20,21). timing of phc phc directed towards young people should focus on lifestyle and safe sex issues such as contraception, prevention of sexually transmitted infections (stis), and gender issues including gender-based violence and human rights. that combining education with provision of contraceptives could be effective in reducing unintended pregnancies among adolescents is supported by a cochrane review (22). raising awareness of the hazards of unprotected sex increases the motivation for preventive measures. a model for education about human papilloma virus (hpv) and its association with cervical cancer has been tried in a randomized controlled trial in swedish school health care. the intervention was based on the health belief model with 30minute structured information face-to-face about the risk for disease and means of prevention. at follow-up three months later, awareness of the risks had increased significantly in the intervention group. the intervention also had a favourable effect on the views on prevention of hpv infection, and significantly more young women in the intervention group chose to get vaccinated against hpv (23). reproductive life plan (rpl) is a tool for information and education about reproductive issues. it has been developed to help women to set realistic goals for their reproduction. rpl provides structured ways of discussing matters such as the impact of age on fertility and how to preserve fertility until pregnancy is wanted (24). rpl has recently been introduced and tried in a swedish family planning setting (25). the midwives found rpl easy to use and very helpful in the dialogue with their patients (26). internationally, some examples of premarital counselling have been initiated and sanctioned by national authorities. in china, premarital counselling was mandatory until 2003 and focused especially on genetic counselling and family planning to strengthen the one-child strategy (27). in iran, premarital information to couples about family planning was introduced to limit population growth (28). in other middle eastern countries premarital counselling has mainly focused on avoiding grave forms of thalassemia (29). providers and organization of phc in order to create the best possible health status for all women of reproductive age, health care providers must be aware of the significance of the condition they are treating in case the woman becomes pregnant. chronic diseases often require medication that may have to be changed before or during pregnancy, and the woman must be well informed of this. fragmentation of health care is a problem for many patient groups, but especially when interventions that need the participation of several professionals or a change in life perspective. in many countries the main part of reproductive health care is performed by physicians without contact with the primary level of care. different aspects of reproductive health are also given as vertical programmes by different caregivers. for instance, family planning services are often separated from sti services. the goal of contraception counselling should, however, not exclusively be to prevent unwanted pregnancy, but also to guard fertility until pregnancy is desired. therefore, there are many advantages with integrating the two. information and education about safe sex can naturally be included in testing for sti, as well as in counselling about contraception. abortion counselling and services could also be integrated into the family planning services (30,31). planning for future pregnancies with appropriate spacing would improve if the staff was also skilled in contraception. components of health care during pregnancy such as education about a healthy lifestyle, basic testing for anaemia, and immunization could easily be integrated into comprehensive preconception care and counselling. in most settings worldwide, facilities already exist for antenatal care and well-baby care and could be extended. it would, however, require some reorientation of staff and that primary health care would assume a central position within the system and the community (31). nurse-midwives have a suitable education for taking on all the suggested components in preconception care and counselling. the use of tools such as a reproductive life plan or other educational tools adapted to the health literacy of the target group can be helpful in initiating discussions about sexuality and reproduction (5,23–25). one of the many concerns when considering strategies for the implementation of phc is the resistance from religious, cultural, traditional, and conservative groups in many societies, where even reproductive health care or health information to young and unmarried persons is seen as threatening moral and family values. this applies especially to information about family planning but can also include issues related to pregnancy in general. there are examples that activities have been renamed in respect for religion and traditions, for instance using the term menstrual regulation instead of termination of pregnancy. the swedish example sweden has had a favourable development of antenatal care and reproductive health care for women, resulting in extremely low maternal and perinatal mortality and easily accessible services for family planning and counselling about stis. ever since midwifery was recognized as a medical profession and mandatory training was established in the seventeenth century, swedish midwives have been responsible for upsala journal of medical sciences 219 normal deliveries. in the 1930s when antenatal care was organized it became a task for the midwives outside the delivery wards. social aspects were considered important in swedish antenatal care from the start. the midwives were to make home visits to give advice on hygiene, nutrition, and infant care and to check that the family was prepared for the arrival of the new baby. later psychosocial support for both parents was emphasized, and classes were held to prepare for parenthood. screening for cervical dysplasia to prevent cervical cancer was implemented in the 1960s, and the testing procedure was added to the responsibility of the midwives, as well as testing for sexually transmitted infections. when the law regulating abortion was liberalized in 1975, the subsequent demands for easily accessible contraception counselling led to the inclusion of family planning in the training of the midwives. they were allowed to administer intrauterine devices for contraception, iuds and prescribe oral contraceptives and became skilled and appreciated advisors. education about health issues, such as the risks of smoking and using alcohol during pregnancy, and advice about suitable nutrition and exercise to avoid overweight and diabetes were eventually included in antenatal care. in addition, information about lifestyle factors became a part of contraceptive counselling. youth clinics were organized in the 1970s and are run by midwives. to support the midwives in complicated cases concerning contraception counselling, pregnancy surveillance, or stis a supervising physician was appointed, often a specialist in obstetrics and gynaecology at the regional hospital. the goal of this comprehensive model is to strengthen normality in reproductive processes and to empower patients to make informed choices about their sexual and reproductive health. midwives as counsellors have become very much appreciated, and the vast majority of women in sweden choose to go to the midwife for reproductive services. one of the strengths of the swedish system is the excellent continuity. patients often meet the same staff for issues of safe sex and family planning, pregnancy, sti, and prevention of cervical cancer. another strength with the swedish system is that the services are easily accessible and free of charge. conclusion pcc has mainly been thought of as counselling before or in early pregnancy to improve maternal and infant health. it has often been limited to single interventions or preconception information and counselling to women with chronic illnesses such as diabetes, or focused on lifestyle changes such as cessation of smoking and drinking alcohol, or supplementation with folic acid. recently it has been suggested that the perspective should be widened to the well-being of the whole family and the offspring, using the concept phc instead. phc covers all reproductive life. early preconception counselling should aim to achieve the best possible sexual and reproductive health for both for women and men. it should start early with education about healthy lifestyle habits, safe sex, and pregnancy planning. in the preconception period, when pregnancy is being planned, general as well as specific information adjusted to the prerequisites of the individual couple is needed. during pregnancy health education should be given to both parents-to-be, in combination with support to enhance the transition to parenthood. between pregnancies, phc aims to restore the nutritional and health status of the mother and the nutrition of the infant and raise the issue of spacing of pregnancies. phc should be delivered in a comprehensive model and by staff skilled in sexual and reproductive health. phc should also be free of charge. it is important that gender equality is addressed in the dialogue with both women and men, and that intimate partner violence is recognized as a possible factor in addition to ill health and non-compliance with health advice. to achieve good continuity, we suggest an organization similar to the swedish model with nurse-midwives as the primary deliverers of care. disclosure statement the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. orcid anna berglund http://orcid.org/0000-0003-3786-7321 references 1. united nations. the millennium development goals report. 2015. available from: http://www.undp.org/content/undp/en/home/ 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http://kunskapsbanken.nck.uu.se/nckkb/nck/publik/fil/visa/464/violence_and_health.pdf http://liu.diva-portal.org http://www.cdc.gov/preconception/rlptool.html http://www.euro.who.int/data/assets/pdf_file/0004/69529/e74558.pdf http://www.euro.who.int/data/assets/pdf_file/0004/69529/e74558.pdf preconception health and care (phc)&mdash;a strategy for improved maternal and child health introduction the content of preconception health services why violence against women is an issue for phc prevalence estimates for violence against women violence victimization and ill health reproductive coercion unintended pregnancy and induced abortion in relation to ipv identifying violence victimization timing of phc providers and organization of phc the swedish example conclusion disclosure statement references tf-iups160028 179..183 original article diabetes treatment and hypoglycaemic episodes in elderly patients at nursing homes in uppsala county angelica walfridssona, maja sehlbergb, ulrika gillespiec, jonathan dahlkvistd and hans-erik johanssona,e a€ostervåla primary health care centre, €ostervåla, sweden; buppsala university, faculty of biomedicine, uppsala, sweden; cuppsala university hospital (akademiska sjukhuset), uppsala, sweden; duppsala l€ans landsting, uppsala, sweden; edepartment of public health and caring sciences/geriatrics, uppsala university, uppsala, sweden abstract aim: the aim of this study was to examine the situation for elderly patients with diabetes living in nursing homes with regard to diabetes treatment, clinical variables, and vascular complications associated with diabetes. a second aim was to evaluate if the patients were at risk of hypoglycaemia. methods: this was a cross-sectional study including diabetes patients from all 30 nursing homes in uppsala county, sweden. current antidiabetic medications, hba1c, hypoglycaemic events, and diabetes complications were registered from the medical records. the patients were stratified into a general group and divided into three groups according to hba1c (<52, 52–73, and >73 mmol/mol). results: of 1,350 individuals, 218 patients were identified with diabetes mellitus. the diabetes duration was 11.2 ± 9.4 years and their serum hba1c concentration 56.0 ± 1.2 mmol/mol. hypoglycaemic events were reported in 24% of the diabetic individuals, and 43.1% of them had hba1c <52 mmol/mol (mean value 44.0 ± 1.1 mmol/mol). of these, 36% were taking antidiabetic drugs. another 35.8% of the patients had hba1c values between 52–73 mmol/mol (mean value 60.0 ± 1.1 mmol/mol), and 82% of these patients were taking antidiabetic drugs. almost 80% of the diabetic patients had either microor macrovascular complications, with diabetes duration as an association for both microor macrovascular complications and hypoglycaemic events. conclusions: a reduction of the use of antidiabetic drugs with follow-up of hba1c level should be considered, especially for multimorbid elderly patients with low hba1c and hypoglycaemia. article history received 15 december 2015 revised 29 may 2016 accepted 30 may 2016 key words diabetes medication; elderly patients; hba1c; hypoglycaemia; nursing homes introduction the prevalence of diabetes is increasing worldwide, affecting more than 8% of the adult population. diabetes is one of the leading causes of cardiovascular disease and death (1–3). in general, it is recommended that patients with type 2 diabetes mellitus (t2dm) receive intensive therapy with tight glycaemic control to avoid microand macrovascular complications (4,5). the value of an intensive diabetes therapy for elderly patients, especially with comorbidities, is under much discussion, and recent guidelines are emphasizing avoidance of very tight glucose control and thereby hypoglycaemia (6–8). many of the elderly patients have a limited life expectancy, and studies have shown intensive plasma glucose-lowering treatment to have poor or no effect on the prevention of microand macrovascular complications, especially if the diabetes duration is longer than 8–10 years (9,10). due to the complexity of ageing and declining body functions, often associated with multiple chronic illnesses, e.g. dementia and polypharmacy, these patients run a greater risk of severe hypoglycaemic complications (11,12). hypoglycaemic events may cause emotional stress and also cognitive impairment, cardiac arrhythmias, and even death (13). the main aim of this study was to examine the situation for elderly patients with diabetes in all 30 nursing homes in uppsala county with regard to hba1c values, diabetes treatment, and microand macrovascular complications. patients were divided into three groups according to hba1c value (<52, 52–73, and >73 mmol/ mol). a second aim was to examine differences in diabetes treatment between the groups and the risk for excessive treatment and hypoglycaemia. subjects and methods patients and data collection in 2012 we collected a list of 1,350 patients from 30 different nursing homes in uppsala county, sweden (figure 1). uppsala is the capital of uppsala county and the fourth largest city in sweden. uppsala county has more than 200,000 inhabitants. data were collected from the patients’ medical records in electronic medical records system (cosmic), and medication details were collected from computer based medication system (pascal). we identified all patients with diabetes, both type 1 diabetes mellitus (t1dm) and t2dm. from the patients’ medical files we registered last known contact hans-erik johansson, md, phd hans-erik.johansson@pubcare.uu.se department of public health and caring sciences/geriatrics, uppsala university, uppsala science park, 75185 uppsala, sweden � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 3, 179–183 http://dx.doi.org/10.1080/03009734.2016.1198441 http://creativecommons.org/licenses/by/4.0/ hba1c value, diabetes duration, age, gender, weight, height, plasma creatinine, current antidiabetic medications, microand macrovascular complications, and hypoglycaemic episodes. retinopathy, nephropathy, and neuropathy were designated as microvascular complications, while myocardial infarction, unstable angina pectoris, brain haemorrhage, brain infarction, and transient ischemic attack were designated as macrovascular complications. hypoglycaemic events were defined as plasma blood glucose <4 mmol/l. we categorized hypoglycaemic events as severe or minor events. events leading to hospitalization, events that needed treatment in the emergency room, and events emerging during hospital care were categorized as severe events. hypoglycaemic events only noted in a patient’s medical journal were considered as minor events. the patients were first stratified into a general group and then divided into three groups according to hba1c (<52, 52–73, and >73 mmol/mol). statistical analyses were made to examine the differences between the groups in antidiabetic treatment, medical dosages, and hypoglycaemic complications. patients on oral antidiabetic drugs (oads) as well as rapidacting insulin when needed were recorded as oads. patients on diet treatment with rapid-acting insulin when needed were recorded as diet treatment. ethics the study was approved by the regional ethics review board at uppsala university. statistics results are given as arithmetic means with their standard deviations. comparisons were made using chi-square tests with the yates correction factor for analyses in differences between diabetes treatment and hypoglycaemic episodes within the groups. the differences between medication doses were analysed using independent samples t test. to avoid too small subgroups a cut-off level at hba1c 52 mmol/mol was chosen. a p value <0.05 was considered statistically significant. multivariable analyses taking co-morbidities was not performed. findings of association of hba1c with risk of hypoglycaemia are crude, and they are not adjusted for the influence of differences in other variables, such as age, diabetes duration, or gender. a subgroup analysis on patients on pharmacological treatment for diabetes was performed. statistical software jmp 5.0 for pc (sas corporation, cary, tx, usa) was used. results of 1,350 individuals at 30 nursing homes, 218 patients (137 women, 81 men) were identified with diabetes mellitus (16.1%) (table 1). their mean bmi was 26.3 ± 5.7 kg/m2, and mean age was 84.6 ± 8.0 years. seven patients had t1dm, and 211 patients had t2dm. mean hba1c was 56.0 ± 1.2 mmol/mol (data missing for 18 patients), and average diabetes duration was 11.2 ± 9.4 years. the diabetes duration in the different hba1c groups was 8.6 ± 1.4, 13.0 ± 11.0, and 16.8 ± 10.7 years, respectively. longer diabetes duration was correlated with higher hba1c, (p¼0.006) (figure 2). the creatinine clearance value was 53.9 ± 26.9 ml/min. eighty-two patients (37.6%) were on diet treatment, 60 patients (27.5%) were on oads, 44 patients (20.6%) had all individuals with diabetes at 30 nursing homes (n=218) insulin (n=45) oad´s + insulin (n= 25)diet (n=82) treatment not found (n=6) all individuals at 30 nursing homes (n= 1350) t1dm (n=7) t2dm (n=211) oad´s (n=60) figure 1. design of the recruitment of patients. table 1. clinical characteristics of 218 diabetes patients at 30 nursing homes, and type of diabetes treatment. patients were divided into three groups by their different hba1c levels. groups all hba1c <52 hba1c 52–73 hba1c >73 no hba1c number of patients 218 94 78 28 18 gender (female/male) 137/81 59/35 50/28 17/11 11/7 age (years) 84.6 ± 8.0 84.1 ± 7.8 84.3 ± 9.0 85.0 ± 7.4 bmi (kg/m2) 26.3 ± 5.7 26.8 ± 5.8 26.8 ± 6.0 25.2 ± 5.1 duration (years) 11.2 ± 9.4 8.6 ± 6.4 13.0 ± 11.0 16.8 ± 10.7 hba1c (mmol/mol) 56.0 ± 1.2 44.0 ± 1.1 60.0 ± 1.1 86.0 ± 1.2 creatinine 53.9 ± 6.9 57.0 ± 28.7 54.4 ± 25.9 52.3 ± 27.0 diabetes treatment: unknown 6 3 2 1 dietary (n) 82 57a 12 2 11 oads (n) 61 25 27 5 4 insulin (n) 44 4a 25 12 3 oadsþ insulin (n) 25 5a 12 8 0 data shown are arithmetic means (±sd). astatistically significant difference (p < 0.05) between groups hba1c <52 and hba1c �52 mmol/mol. bmi: body mass index; oads: oral antidiabetic drugs. 180 walfridsson a. et al. insulin treatment, and 25 patients (11.5%) had combined oadsþ insulin treatment. the number of patients with diet treatment was higher (p < 0.05) in the group with hba1c <52 mmol/mol compared to patients with hba1c �52 mmol/ mol. there were no differences between the groups (<52 and �52 mmol/mol), comparing numbers of patients being treated with only oads (p¼0.80). the number of patients with insulin treatment and hba1c �52 mmol/mol was higher (p < 0.05) compared to patients with hba1c <52 mmol/mol. also, the number of patients with combined oadsþ insulin treatment was higher (p < 0.05) for patients with hba1c �52 mmol/mol than for patients with hba1c <52 mmol/mol. of a total of 69 patients on insulin treatment, 9 patients had hba1c <52 mmol/mol, and 54 patients had hba1c �52 mmol/mol (table 2). for three patients hba1c data were missing, and all of them were treated with an insulin mixture, while two of them were also on intermediate-acting insulin. daily doses of insulin were the same in all groups (p¼0.319) (table 2). moreover, there were no differences between the groups regarding daily doses of long-acting insulin (p¼0.365), intermediate-acting insulin (p¼0.271), mixed insulin (p¼0.490), and continuous rapid-acting insulin (p¼0.490). hypoglycaemic events were observed in 52 patients (table 3). a total of 106 hypoglycaemic events were identified. thirty-four patients had severe hypoglycaemic events. ten patients needed hospitalization due to severe hypoglycaemia, eight patients needed to attend the emergency room for treatment, and 16 patients had hypoglycaemic episodes emerging during hospital care while being treated for other reasons. there were notes about low plasma glucose concentrations, <4 mmol/l, in the medical records of 33 patients. for the sake of clarity it has to be mentioned that some patients were diagnosed with hypoglycaemia more than once. the number of patients treated for hypoglycaemic events was lower for patients in the group with hba1c <52 mmol/mol compared to groups with hba1c �52 mmol/ mol. however, there was no significant difference between the groups <52 and �52 mmol/mol regarding hospitalization due to hypoglycaemic events. a separate analysis of patients receiving glucose-lowering pharmacological treatment (n ¼ 130) showed the same pattern regarding hypoglycaemic events as in the general group (table 3), with the exception that hypoglycaemia treated in the emergency room was more frequent in the group with hba1c �52 mmol/mol (table 4). further, 49 patients received insulin on demand, and these patients were involved in 50% of all major or minor hypoglycaemic episodes. insulin-treated patients with hypoglycaemic episodes in the groups were 5/ 9, 27/37, and 18/20, respectively. not surprisingly, there were no hypoglycaemic episodes reported for patients with diet treatment only, except for two patients during hospital care. diabetic vascular complications were registered for 169 patients (table 3). of them, 45 patients had microvascular complications, 74 patients (33.9%) had macrovascular complications, and 50 patients had both microand macrovascular complications. regarding vascular complications there were no statistically significant differences between the groups. discussion this cross-sectional study showed that diet treatment was most frequent in the group with hba1c <52 mmol/mol, and this group had also fewer hypoglycaemic complications. insulin treatment was less frequent in this group, but oads use was surprisingly equal in all groups. diabetes duration was strongly correlated with higher hba1c. a relatively large subgroup with hba1c <52 mmol/mol received pharmacological treatment: 40% had oads and 10% insulin. with age, limited life expectancy, and risk of severe hypoglycaemia taken into consideration, the perspective is that there are no studies showing beneficial effects from tight glycaemic control among these patients (9,10,12,14). a reduction of hypoglycaemic drugs should therefore be considered for this group, with follow-up to find out if similar hba1c levels may still persist. if it proves difficult to maintain a lower hba1c level because of increased risk of severe hypoglycaemia or other complications, an hba1c level up to 70 mmol/mol may be recommended for these multimorbid elderly patients. for patients in the group hba1c 52–73 mmol/mol, more than 80% of the patients received antidiabetic drug figure 2. the correlation between hba1c (mmol/mol) and diabetes duration (years); p ¼ 0.006. table 2. patients treated with insulin as single therapy or in combination with oral antidiabetic drugs. daily doses expressed in international units. the patients were divided into three groups by different hba1c levels. groups all hba1c <52 hba1c 52–73 hba1c >73 number of patients (daily doses, iu): insulin 69 (31 ± 21) 9 (33 ± 21) 37 (26 ± 19) 20 (35 ± 21) long-acting 14 (28 ± 18) 2 (37 ± 35) 7 (28 ± 21) 5 (24 ± 5) intermediate-acting 26 (20 ± 12) 3 (23 ± 11) 15 (16 ± 11) 6 (24 ± 13) mixtures 39 (30 ± 19) 5 (31 ± 17) 18 (24 ± 18) 13 (39 ± 21) rapid-acting 3 (17 ± 8) 1 (8 ± 0) 2 (22 ± 0) 0 data shown are arithmetic means (±sd). of a total of 69 patients with insulin treatment, 9 patients had hba1c <52 mmol/mol and 54 patients hba1c >52 mmol/mol. for three patients hba1c data were missing, and all of them were given an insulin mixture, while two of them were also on intermediateacting insulin. no differences were observed between the groups regarding daily doses of insulin (p ¼ 0.319). iu: international units of insulin. upsala journal of medical sciences 181 treatment, and 50% had insulin treatment. insulin and sulfonylureas are associated with a high risk of hypoglycaemia (7). fifty patients were treated with sulfonylureas, and 13 of them were found in the group with hba1c <52 mmol/mol. further, with higher hba1c there was an increasing number of insulintreated patients with hypoglycaemic episodes (56%, 73%, and 90%) in the three different hba1c groups. we could not find any differences regarding daily dosage/doses of insulin between the groups with different hba1c levels. this finding might indicate excessive treatment, especially for the patients in the group with hba1c <52 mmol/mol. the results in this study are in accordance with findings from a previous study in nursing homes in sweden (15). it was shown that a significant reduction of diabetic medication could be safely administered without increasing the risk for hyperglycaemia. that study was performed at nursing homes with a similar patient population (n ¼ 98), and the diabetes prevalence was 15%, with a mean hba1c value of 57.0 ± 1.2 mmol/mol, and most patients were on antidiabetic drug treatment. several international studies have evaluated intensive diabetes treatment in t2dm patients regarding complications and that there were negligible effects on preventing microand macrovascular complications, particularly after a longer diabetes duration of 8–10 years (9,10,14). in the present study the patients had a diabetes duration of almost 12 years. the veterans affairs diabetes trial has shown that, for patients with similar diabetes duration and when hba1c was lowered to 51 mmol/mol, there was no beneficial effect on preventing diabetes complications (14). studies on elderly t2dm patients have also indicated a risk for negative side effects, such as hypoglycaemia, of tight glycaemic control (12,15,16). hypoglycaemic events have been shown to be frequent among elderly patients with t2dm and may cause brain damage, cognitive impairment, cardiac arrhythmias, and even death (13,17). hypoglycaemia might also mimic cognitive disorders, e.g. confusion and anxiety, as well as other conditions, and cause unnecessary medication of the t2dm patients with sedatives and psychotropic drugs. thus, withdrawal of diabetes medication in the elderly receiving tight glycaemic control is warranted, and it has been shown to be safe and that it decreases the risk of hypoglycaemia (15,16). in this present study we found a total of 106 hypoglycaemic episodes in 52 patients. compared to observations in previous studies, hypoglycaemic events were less frequent, which may indicate that the method for finding hypoglycaemic events was insufficient. it could not be excluded that episodes of hypoglycaemia were not reported, since hypoglycaemia may mimic several other conditions and, furthermore, many hypoglycaemic episodes are nocturnal. the hypoglycaemic events tended to be more severe among patients with higher hba1c, which might reflect a diabetes disease with poor glycaemic control (table 3). table 3. patients with diabetes vascular complications and hypoglycaemic episodes. the patients were divided into three groups by different hba1c levels. groups all hba1c <52 hba1c 52–73 hba1c >73 number of patients 218 94 78 28 hypoglycaemia: patients with no hypoglycaemic events, n (%) 166 (76.1) 82 (87.2)a 54 (69.2) 17 (60.7) patients with hypoglycaemic events, n (%) 52 (23.9) 12 (12.8)a 24 (30.8) 11 (39.3) severe hypoglycaemic events: hypoglycaemia, hospitalization 10 (4.6) 3 (3.2) 2 (2.6) 5 (17.9) hypoglycaemia, emergency room 8 (3.7) 0 6 (7.7) 1 (3.6) hypoglycaemia, during hospital care 16 (7.3) 4 (4.3) 6 (7.7) 5 (17.9) minor hypoglycaemic events: hypoglycaemia, notes in medical records 33 (15.1) 7 (7.4) 17 (21.8) 8 (28.6) vascular complications: no diabetes complications, n (%) 49 (22.5) 27 (28.7) 14 (17.9) 3 (10.7) complications from diabetes, n (%) 169 (77.5) 67 (71.3) 64 (82.1) 25 (89.3) microvascular complications, n (%) 45 (20.6) 14 (14.9) 17 (21.8) 10 (35.7) macrovascular complications, n (%) 74 (33.9) 38 (40.4) 25 (32.1) 7 (25.0) microand macrovascular complications, n (%) 50 (22.9) 15 (16.0) 22 (28.2) 8 (28.6) microvascular complications: retinopathy, nephropathy, neuropathy; macrovascular complication: myocardial infarction, instable angina pectoris, brain bleeding, brain infarction, tia; hypoglycaemic episodes: plasma glucose <4 mmol/l. astatistically significant difference (p < 0.05) between groups hba1c <52 and hba1c �52 mmol/mol. there were no hypoglycaemic episodes reported for patients with diet treatment only, except for two patients during hospital care. table 4. hypoglycaemic episodes in subsets of patients given glucose-lowering pharmacological treatment. the patients were divided into three groups by different hba1c levels. groups all hba1c <52 hba1c 52–73 hba1c >73 number of patients 130 34 64 25 hypoglycaemia: patients with no hypoglycaemic events, n (%) 80 (61.5) 24 (70.6)a 40 (62.5) 14 (56.0) patients with hypoglycaemic events, n (%) 50 (38.5) 10 (29.4)a 24 (37.5) 11 (44.0) severe hypoglycaemic events: hypoglycaemia, hospitalization, n (%) 9 (6.9) 2 (5.9) 2 (3.1) 5 (20.0) hypoglycaemia, emergency room, n (%) 8 (6.2) 0a 6 (9.4) 1 (4.0) hypoglycaemia, during hospital care, n (%) 15 (11.5) 3 (8.8) 6 (9.4) 5 (20.0) minor hypoglycaemic events: hypoglycaemia, notes in medical records, n (%) 33 (25.4) 7 (20.6)a 17 (26.6) 8 (32.0) hypoglycaemic episodes: plasma glucose <4 mmol/l. astatistically significant difference (p < 0.05) between groups hba1c <52 and hba1c �52 mmol/mol. 182 walfridsson a. et al. the accord study and other epidemiological studies have shown increased risk of death with high hba1c levels, but the patients could still not benefit from intensive glucose-lowering therapy. the rate of death was even higher among patients with more intensive glucose-lowering treatment (5,9,18). further, in our study, 49 patients received insulin on demand, and they were involved in 50% of all major hypoglycaemic episodes and 50% of all minor hypoglycaemic episodes. the fact that 130 patients received pharmacological treatment shows that the subgroup given insulin on demand more often suffered from hypoglycaemic episodes. insulin treatment was more common among patients with hba1c �52 mmol/mol. this could possibly, to some extent, explain why hypoglycaemia occurred more frequently with increasing hba1c. longer diabetes duration was correlated with higher hba1c, and these parameters were associated with increased risk for hypoglycaemic episodes and microvascular complications. almost 80% of the diabetes patients had either microor macrovascular complications, and diabetes duration was associated with both hypoglycaemic episodes and microor macrovascular complications. the results from this study suggest that multimorbid elderly patients with low hba1c suffering from hypoglycaemic episodes could benefit from a re-evaluation of their pharmacological treatments, including changes of agents used, dose reductions, or even stopping selected treatments (15). hba1c should be monitored quite frequently to ensure that serum hba1c concentrations are kept within reasonable levels. disclosure statement the authors report no conflicts of interest. funding information this work was funded by research grants from uppsala l€ans landsting. references 1. tao z, shi a, zhao j. epidemiological perspectives of diabetes. cell biochem biophys. 2015;73:181–5. 2. roper na, bilous rw, kelly wf, unwin nc, connolly vm. excess mortality in a population with diabetes and the impact of material deprivation: longitudinal, population based study. bmj. 2001;322:1389–93. 3. gerstein hc, islam s, anand s, almahmeed w, damasceno a, dans a, et al. dysglycaemia and the risk of acute myocardial infarction in multiple ethnic groups: an analysis of 15,780 patients from the interheart study. diabetologia. 2010;53:2509–17. 4. gaede p, vedel p, larsen n, jensen gv, parving hh, pedersen o. multifactorial intervention and cardiovascular disease in patients with type 2 diabetes. n engl j med. 2003;348:383–93. 5. stratton im, adler ai, neil ha, matthews dr, manley se, cull ca, et al. association of glycaemia with macrovascular and microvascular complications of type 2 diabetes (ukpds 35): prospective observational study. bmj. 2000;321:405–12. 6. kirsh sr, aron dc. choosing targets for glycaemia, blood pressure and low-density lipoprotein cholesterol in elderly individuals with diabetes mellitus. drugs aging. 2011;28:945–60. 7. american diabetes association. standards of medical care in diabetes—2014. diabetes care. 2014;37(suppl 1):s14–s80. 8. moreno g, mangione cm, kimbro l, vaisberg e. guidelines abstracted from the american geriatrics society guidelines for improving the care of older adults with diabetes mellitus: 2013 update. j am geriatr soc. 2013;61:2020–6. 9. gerstein hc, miller me, byington rp, goff dc jr, bigger jt, buse jb, et al. effects of intensive glucose lowering in type 2 diabetes. n engl j med. 2008;358:2545–59. 10. patel a, macmahon s, chalmers j, neal b, billot l, woodward m, et al. intensive blood glucose control and vascular outcomes in patients with type 2 diabetes. n engl j med. 2008;358:2560–72. 11. feil dg, rajan m, soroka o, tseng cl, miller dr, pogach lm. risk of hypoglycemia in older veterans with dementia and cognitive impairment: implications for practice and policy. j am geriatr soc. 2011;59:2263–72. 12. thorpe ct, gellad wf, good cb, zhang s, zhao x, mor m, et al. tight glycemic control and use of hypoglycemic medications in older veterans with type 2 diabetes and comorbid dementia. diabetes care. 2015;38:588–95. 13. mellbin lg, ryden l, riddle mc, probstfield j, rosenstock j, diaz r, et al. does hypoglycaemia increase the risk of cardiovascular events? a report from the origin trial. eur heart j. 2013;34:3137–44. 14. duckworth w, abraira c, moritz t, reda d, emanuele n, reaven pd, et al. glucose control and vascular complications in veterans with type 2 diabetes. n engl j med. 2009;360:129–39. 15. sjoblom p, tengblad a, lofgren ub, lannering c, anderberg n, rosenqvist u, et al. can diabetes medication be reduced in elderly patients? an observational study of diabetes drug withdrawal in nursing home patients with tight glycaemic control. diabetes res clin pract. 2008;82:197–202. 16. lofgren ub, rosenqvist u, lindstrom t, hallert c, nystrom fh. diabetes control in swedish community dwelling elderly: more often tight than poor. j intern med. 2004;255:96–101. 17. patrick aw, campbell iw. fatal hypoglycaemia in insulin-treated diabetes mellitus: clinical features and neuropathological changes. diabet med. 1990;7:349–54. 18. gerstein hc, pogue j, mann jf, lonn e, dagenais gr, mcqueen m, et al. the relationship between dysglycaemia and renal risk in diabetic and non-diabetic participants in the hope study: a prospective epidemiological analysis. diabetologia. 2005;48:1749–55. upsala journal of medical sciences 183 diabetes treatment and hypoglycaemic episodes in elderly patients at nursing homes in uppsala county introduction subjects and methods patients and data collection ethics statistics results discussion disclosure statement funding information references tf-iups160038 299..303 original article using the reproductive life plan in contraceptive counselling tanja tyd�ena,b , sarah verbiestc,d, theo van achterberga,e, margareta larssonb and jenny sterna,b adepartment of public health and caring sciences, uppsala university, uppsala, sweden; bdepartment of women’s and children’s health, uppsala university, uppsala, sweden; cschool of social work and the center for maternal and infant health, university of north carolina at chapel hill, chapel hill, nc, usa; dnational preconception health and health care initiative, chapel hill, nc, usa; eku leuven, department of public health and primary care, academic centre for nursing and midwifery, leuven, belgium abstract having children or not is one of the most important decisions that a person will make in his or her lifetime. the reproductive life plan (rlp) is a protocol that aims to encourage both women and men to reflect on their reproductive intentions and to find strategies for successful family planning, for example to have the wanted number of children and to avoid unwanted pregnancies as well as ill-health that may threaten reproduction. the rlp was developed in an american context for promotion of reproductive health in a life cycle perspective. few studies have systematically evaluated the effectiveness of using an rlp protocol in clinical practice. this article describes the application of using the rlp protocol in contraceptive counselling in sweden. article history received 27 june 2016 revised 28 june 2016 accepted 30 june 2016 keywords counselling; preconception care; preconception health; reproduction; reproductive life plan introduction the decision about if and/or when to have children is one of the most important that a person will make in his or her lifetime. yet, many young adults do not talk with their health care providers or even their partners about their future goals for a family. the reproductive life plan (rlp) is a protocol consisting of a set of questions to guide a conversation about if and when a person might want to become a parent. the rlp protocol was developed in the united states to support reproductive health with a life course perspective (1). the rlp may be used by health care providers and others to encourage both women and men to reflect on their reproductive intentions and then access the appropriate services to achieve those intentions. for some this may be the provision of an appropriate contraceptive method, while for others this may lead to the provision of counselling and care to optimize health before pregnancy to improve birth outcomes. regardless of the person’s decision, it is essential that health care providers aim to support patient autonomy and intentions so that pregnancies are wanted, planned, and as healthy as possible. the centers for disease control and prevention (cdc) has recommended the rlp as a tool to improve preconception health and decrease unintended pregnancies and adverse pregnancy outcomes (2). the cdc offers recommendations for health care providers on how to have conversations about reproductive life planning with patients. figure 1 describes key questions for this conversation. the cdc also has a rlp worksheet for women to guide their thoughts about their future goals (3). the rlp opens the door for other important conversations around preconception health and wellness. these conversations can happen in a clinic, but they can also happen in community settings or be done by self-assessment. as the concept of reproductive life planning has matured in the us, a number of organizations have developed tools for women and men (4–6) in addition to the national effort show your love today (7) and the one key questionr initiative (8). a study using reproductive life planning with low-income african-american and hispanic women and men in publicly funded clinics in the us found this approach to be well acceptable among the target group (9). when tested by physicians at a family health centre in the us targeting women with diabetes, hypertension, and obesity, a rlp intervention was shown to increase women’s knowledge about reproductive health. the authors concluded that the rlp was a brief and cost-effective counselling tool for women with chronic diseases (10). a different study, however, found limited efficacy in using the rlp with urban women utilizing national family planning services in the us (11). while the cdc and a growing number of partners recommend the use of rlp, more studies that systematically evaluate the effectiveness of using the rlp in clinical practice need to be conducted. this article describes the application of reproductive life planning in clinics in sweden, highlighting the results and experience in implementing this concept outside the us context. sexual and reproductive health in sweden with almost 10 million inhabitants, sweden is one of the world’s most family-friendly countries. the country offers contact tanja tyd�en tanja.tyden@pubcare.uu.se uppsala university, department of public health and caring sciences, box 564, se751 22 uppsala, sweden � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 4, 299–303 http://dx.doi.org/10.1080/03009734.2016.1210267 extensive parental benefits. parents receive 480 days of parental leave for one child, and for the first 390 days the compensation is 80% of the parent’s original income (12). parents also receive child allowance from the state until the child is 18 years old. this child allowance (12,600 sek/year � us$1,555/year) is equal for all parents, regardless of income. temporary parental benefits can be obtained if a parent needs to stay at home and take care of a sick child up to the age of 12 years. in spite of these benefits, the total fertility rate in sweden is 1.9, which is lower than needed to maintain a steady population level. the mean age of first-time mothers has increased from 24 years to 29 years over the past four decades (13). delayed childbearing is even more pronounced among women with postgraduate education. skoog svanberg et al. (14) demonstrated that more than half of female postgraduate students did not want to have children during their studies, and as many as 66% wished to have their last child after the age of 35. in 2014, almost 3 out of 10 women attending antenatal care in sweden were 35 years or older (13). the postponement of parenthood has been attributed to the following: (1) introduction of contraceptive technology, (2) increased educational levels and women’s labour force participation, (3) norm and value changes, (4) gender equity, (5) changing partnerships and increasing number of people living alone, and (6) housing and economic uncertainties (15). it is well known that advanced maternal age is a risk factor for pregnancy complications such as chromosomal abnormality, preterm birth, being small for gestational age, stillbirth, neonatal death, gestational diabetes, and preeclampsia (16–23). these risks, however, do not appear to be common knowledge. the median age for first intercourse among girls in sweden is 16 years (24). this means that the majority of women are in need of effective contraceptives many years before most intend to begin childbearing. repeated surveys among female university students show that this group of young women has sexual behaviours that may threaten their fertility. the mean number of sexual partners has increased for this population from 4 to 12 over the past 25 years. in 2014, one out of four women receiving contraceptive counselling had a sexually transmitted infection (25). as sexually transmitted infections can lead to infertility and other health problems, health care professionals in sweden are recommended to discuss sexuality and sexual risk-taking with their patients as part of contraceptive counselling (26). contraceptive counselling in sweden is free of charge, with midwives providing approximately 80%–90% of all of this service. midwives can prescribe hormonal contraceptives as well as insert and remove intrauterine devices and implants for healthy women at antenatal/family planning clinics. they also provide care through youth clinics within the primary health care system. the medical product agency in sweden has issued recommendations for contraceptive counselling (26). the aim of these recommendations is to prevent do you plan to have any (more) children at any time in the future? if yes how many children would you like to have? encourages the client to consider that there is a choice about the number of children one has. if yes how long would you like to wait until you or your partner becomes pregnant? encourages the client to vision their own future. what family planning method do you plan to use until you or your partner are ready to become pregnant? gives the client an opportunity to formulate and communicate a personal strategy. how sure are you that you will be able to use this method without any problems? encourages the client to recognize that methods can have problems and to consider matching method choice to personal circumstances. if no what family planning method will you use to avoid pregnancy? gives an opportunity to formulate and communicate a personal strategy to achieve plan. if no how sure are you that you will be able to use this method without any problems? encourages recognition that methods can have problems and to consider matching method choice to personal circumstances. people’s plans change. is it possible you or your partner could ever decide to become pregnant? relays the message that plans can change and that it is okay, but deliberate decisions about becoming pregnant are possible and desirable. figure 1. reproductive life planning questions. 300 t. tyd�en et al. unwanted pregnancies and preserve women’s fertility until a pregnancy is desired. integrating rlp into contraceptive counselling in sweden one opportunity for offering rlp is during contraceptive counselling. in this situation, providers have a golden opportunity to ask women about their intentions to have children or not and about their possibility of conceiving. we used the rlp in a randomized clinical trial (rct) with female university students (27). we chose this group of young women since highly educated women and men tend to postpone having children until ages when their reproductive capacity has started to decrease. they are not sufficiently aware of the age-related decline in fecundity, and this can impact their plans for a future family (14,28,29). the study took place in a university city in sweden in the spring of 2012. swedish-speaking female university students requesting contraceptive counselling at the student health centre were invited to participate. the women received both verbal and written information from the midwives before giving their written consent, and then all women completed a baseline questionnaire. the response rate was 88% (n¼299). all participants received standardized contraceptive counselling including a medical history, measuring blood pressure and weight, and provision of contraceptives (26). women allotted to the intervention group also received the rlpbased intervention. this intervention consisted of a semistructured discussion aimed to encourage the woman to reflect on her rlp. an interview guide based on the rlp as described by moos et al. and the cdc (1,2) and a fact sheet served as guidance for the midwife. the women also received a specially designed booklet with information about, for example, the fertile window, the use of folic acid, and the importance of a healthy lifestyle when planning a pregnancy. telephone interviews were conducted with all participants two months after their clinic visit. at follow-up, women in the intervention group had better knowledge about reproduction compared to the control group, and they wished to have their last child earlier in life than at baseline. nine out of 10 also considered it rather or very positive that the midwife initiated the rlp discussion, and an equal proportion had the opinion that midwives routinely should discuss rlp with their patients. the positive results among female university students inspired us to explore how this intervention would work in everyday practice and in a larger context (30). we partnered with the senior consultant in the regional antenatal care and the co-ordinating midwife in one swedish county. after an informational meeting about reproductive life planning, 53 of 68 midwives in 16 clinics agreed to participate. the midwives received a rlp guide and booklets. three months later, the midwives received a questionnaire about their opinions and experiences (if any) of using rlp with their clients. we also invited them to participate in a focus group interview to explore their opinions and experiences further. we conducted five focus groups with a total of 22 midwives. the questionnaire revealed that 68% of the midwives had used rlp. the most common reason for not having used rlp was not having received enough information. nine out of 10 who had used rlp considered the very idea of rlp as rather/ very good and had rather/very positive experiences of using it. some midwives used the booklet during counselling as a starting-point for discussion, others just handed it out. the booklet was generally considered an asset for the midwife and as an accessible information source for the woman, particularly when time was restricted. the findings from the focus group interviews resulted in four categories. 1. a predominantly positive experience the rlp was experienced as a rewarding and easy way to broaden the counselling. the midwives experienced predominantly positive reactions from women but acknowledged that the counselling required tactfulness and professionalism. 2. the rlp—a health-promoting tool the midwives considered the rlp to give additional value to the contraceptive counselling, by motivating health-promoting actions. they stressed that women need knowledge about fertility and that family planning counselling is a suitable opportunity for rlp discussions. however, it was also stated that the rlp cannot always be prioritized in clinical practice, and there was disagreement on whether it was suitable for all groups of clients. 3. individual and societal factors influence the rlp counselling the midwives believed many factors could influence the rlp counselling. first, they recognized that their own prejudices and their clientele influence which patients receive this service. second, women’s varying individual knowledge, norms, and premises strongly influence the conditions for the discussion. finally, influences by societal norms and the media were acknowledged to be an important factor. for example, women are expected to postpone parenthood, but still have children, and the media strongly influence the public awareness, for instance through very unbalanced reporting about older celebrity mothers. 4. long-term implementation comprises opportunities, risks, and needs the midwives expressed different opinions regarding whether making rlp mandatory and requiring documentation would facilitate or impede its use in practice. it was suggested that more in-depth preparation for all midwives through role-plays and group discussions would improve uptake and enhance provider receptivity. the midwives also suggested that there upsala journal of medical sciences 301 was potential for expanding rlp counselling to other arenas and health care professions. lessons learned efforts to integrate the rlp concept into contraceptive services offered valuable lessons that will be useful in replicating this work more widely in sweden and informing care in other countries. first, firmly anchoring the concept of rlp among the managers is of utmost importance for implementing the rlp in routine clinical practice. our studies were both supported and encouraged by the senior consultant and co-ordinating midwife. further, health care providers require education and training before they start using the rlp tools. as highlighted by the midwives, respectful, unbiased patient engagement requires tactfulness and professionalism. women and men of reproductive age have different backgrounds and reasons for their individual reproductive life plans. the aim of counselling is to aid the client to formulate their plan and find strategies to reach them, not to pressure the clients to change their plan into what the midwife finds appropriate. this is critical for health professionals to understand and apply to their practice. for successful implementation, health care providers need education both to refresh their general knowledge about lifestyles and fertility and to improve and practise communication skills. education and training should be based on scientific literature and best practice strategies. midwives liked the quality of the rlp materials and found them easy to use (30). clients responded positively to the rlp materials and conversation as well (27). our studies were restricted by a short follow-up period and a non-populationbased group of participants. the women included in the rct represented female university students, and the midwives were from one county only. in sweden three research projects on rlp are currently underway: (1) an rct among a general population of women (n¼1,993) visiting 60 clinics for contraceptive counselling, (2) an rct among men (n¼201) visiting two clinics for testing for sexually transmitted infection, and (3) a prospective study examining the long-term implementation of rlp in one county in sweden. further studies need to explore the longterm impact of rlp in a general population, which could be foundational for also examining the economic impact of this work. as e-health is a growing field of health care, it would also be interesting to compare different modes of delivery of the rlp, for example a mobile application or a web page. finally, the rlp deserves to be explored for other target groups and by other health care providers, in different settings and countries. recommendations for implementation of rlp the swedish experience informs several key factors for implementing reproductive life planning conversations into routine contraceptive counselling and general preventive health care. seven key factors that may facilitate or hinder improvement of health care in general, as for example moving rlp into practice, include: (1) the innovation or guideline itself, (2) health professionals, (3) clients/patients, (4) professional interactions, (5) incentives and resources, (6) capacity for organizational change, and (7) social, political, and legal factors (31). in the exploratory study the rlp received rather positive comments on relevance, the quality of materials, and ease of use (30). this suggests that the rlp itself will help the implementation. also the professionals (midwives) involved were often positive towards the rlp, though some reported their own prejudices on whom the rlp is relevant for and doubt on responsibilities as potential barriers. interprofessional and organizational issues that could help or hinder the rlp’s implementation were not assessed in the swedish study; however, the relevance of resources and societal factors were clearly addressed by some of the midwives. they highlighted that the rlp fits in well with contraceptive counselling visits. they also expressed doubts about the integration with current electronical records. the midwives believed that the rlp tool had the potential to counteract social norms towards postponing parenthood. the study among midwives (30) implies that the rlp tool and guidelines need little improvement, though perhaps the instructions should include assessing clients’ basic knowledge about fertility. future implementation efforts should, however, address practical issues of time for using the rlp, integrated documentation of the rlp and other activities during consultations, and integrating this conversation into standard clinical care provision. further, professionals’ beliefs need to be addressed to some extent to ensure an open mind towards providing rlp counselling to all visitors of reproductive ages, not just certain groups. this implies that change methods might have to be added to the midwives’ introduction into the rlp use in order to convince the midwives of the rlp’s relevance to larger groups; a degree of structural redesign at organizational level and agenda setting at health policy level is probably required to embed the rlp into regular care provision (32). finally, it should be noted that further analyses of experiences with introducing the rlp are needed to take these first insights beyond the contexts of swedish health care and use by midwives only. further explorations of the rlp’s implementation should involve other professions, as well as organizational representatives, e.g. health care administrators and change management experts. disclosure statement the authors 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velde e; eshre reproduction and society task force. why do people postpone parenthood? reasons and social policy incentives. hum reprod update. 2011;17:848–60. 16. jacobsson b, ladfors l, milsom i. advanced maternal age and adverse perinatal outcome. obetet gynecol. 2004;104:727–33. 17. cleary-goldman j, malone fd, vidaver j, ball rh, nyberg da, comstock ch, et al.; faster consortium. impact of maternal age on obstetric outcome. obstet gynecol. 2005;105:983–90. 18. luke b, brown mb. elevated risks of pregnancy complications and adverse outcomes with increasing maternal age. hum reprod. 2007;22:1264–72. 19. jolly m, sebire n, harris j, robinson s, regan l. the risks associated with pregnancy in women aged 35 years or older. hum reprod. 2000;15:2433–7. 20. hook eb, cross ph, schreinemachers dm. chromosomal abnormality rates at amniocentesis and in live-born infants. jama. 1983;249:2034–8. 21. nybo andersen am, wohlfahrt j, christens p, olsen j, melbye m. maternal age and fetal loss: population based register linkage study. bmj. 2000;320:1708–12. 22. fretts rc, schmittdiel j, mclean fh, usher rh, goldman mb. increased maternal age and the risk of fetal death. n engl j med. 1995;333:953–7. 23. flenady v, koopmans l, middleton p, froen jf, smith gc, gibbons k, et al. major risk factors for stillbirth in high-income countries: a systematic review and meta-analysis. lancet. 2011;377:1331–40. 24. h€aggstr€om-nordin e, borneskog c, eriksson m, tyd�en t. sexual behaviour and contraceptive use among swedish high school students in two cities: comparisons between genders, study programmes, and over time. eur j contracept reprod health care. 2011;16:36–46. 25. stenhammar c, ehrsson yt, åkerud h, larsson m, tyd�en t. sexual and contraceptive behavior among female university students in sweden repeated surveys over a 25-year period. acta obstet gynecol scand. 2015;34:253–9. 26. medical products agency. antikonception—behandlingsrekommendation. information från l€akemedelsverket. 2014;25:14–28 [in swedish]. 27. stern j, larsson m, kristiansson p, tyd�en t. introducing reproductive life plan-based information in contraceptive counseling: an rct. hum reprod. 2013;28:2450–61. 28. lampic c, skoog svanberg a, karlstr€om p, tyd�en t. fertility awareness, intentions concerning childbearing, and attitudes towards parenthood among female and male academics. hum reprod. 2006;21:558–64. 29. eriksson c, larsson m, tyd�en t. reflections on having children in the future—interviews with highly educated women and men without children. ups j med sci. 2012;117:328–35. 30. stern j, bodin m, grandahl m, segeblad b, ax�en l, larsson m, et al. midwives’ adoption of the reproductive life plan in contraceptive counselling: a mixed methods study. hum reprod. 2015;30:1146–55. 31. flottorp sa, oxman ad, krause j, musila nr, wensing m, godyckicwirko m, et al. a checklist for identifying determinants of practice: a systematic review and synthesis of frameworks and taxonomies of factors that prevent or enable improvements in healthcare professional practice. implement sci. 2013;3:35. 32. kok g, gottlieb nh, peters gy, mullen pd, parcel gs, ruiter ra, et al. a taxonomy of behaviour change methods: an intervention mapping approach. health psychol rev. 2016;15:1–16. upsala journal of medical sciences 303 http://www.cdc.gov/preconception/reproductiveplan.html http://www.cdc.gov/preconception/reproductiveplan.html http://dethrives.com/mlmpw/overview http://dethrives.com/mlmpw/overview http://whb.ncpublichealth.com/manuals/areyoureadysexandyourfuturerevised-9-10-10.pdf http://whb.ncpublichealth.com/manuals/areyoureadysexandyourfuturerevised-9-10-10.pdf http://www.famplan.org/resources/docs/adult_rhp_busy_woman.pdf http://www.famplan.org/resources/docs/adult_rhp_busy_woman.pdf http://www.cdc.gov/preconception/showyourlove/index.html http://www.cdc.gov/preconception/showyourlove/index.html http://www.onekeyquestion.org/ http://www.onekeyquestion.org/ https://www.forsakringskassan.se/privatpers/foralder/nar_barnet_ar_fott/foraldrapenning https://www.forsakringskassan.se/privatpers/foralder/nar_barnet_ar_fott/foraldrapenning using the reproductive life plan in contraceptive counselling introduction sexual and reproductive health in sweden integrating rlp into contraceptive counselling in sweden a predominantly positive experience the rlp&mdash;a health-promoting tool individual and societal factors influence the rlp counselling long-term implementation comprises opportunities, risks, and needs lessons learned recommendations for implementation of rlp disclosure statement references sups_a_528071 34..38 upsala journal of medical sciences. 2011; 116: 34–38 original article longitudinal trends in laboratory test utilization at a large tertiary care university hospital in sweden mirja mindemark & anders larsson department of medical sciences, section of clinical chemistry, uppsala university, uppsala, sweden abstract background. the aim of the study was to describe and evaluate longitudinal trends in laboratory test utilization over a 7-year period from 2002 to 2008. method. retrospective study using test request data from the clinical chemistry and pharmacology laboratory at akademiska sjukhuset, a large tertiary care university hospital in sweden. changes in test utilization, charges, and expenditures during the study period were used as main outcome measures. results. laboratory test utilization increased by over 70%, with a mean annual increase of 9.3% during the study period. after adjustment for inflation, the laboratory expenditures increased by 20.2% during the study period but represented only approximately 2.0% of the hospital’s total expenditure in 2008. the test menu comprised 663 tests in 2008, an increase by 146% from 2002. the mean inflation-adjusted unit price charged per test increased from e34.9 to e37.5 during the study period. the top 10, 20, and 30 tests accounted for, on average, 46.9%, 66.9%, and 75.5% of the total test volume during the study period, and 47.8%, 66.4%, and 75.7% of the total test volume in 2008. in 2008, 10 analyses, i.e. 1.5% of the number of tests on the menu, accounted for almost half the number of generated test results. conclusions. the total number of generated test results increased by over 70% in less than a decade. even so, the laboratory’s share of the hospital’s total expenditure remained low and virtually unchanged. a very small number of tests accounted for a disproportionately large share of the total number of generated test results. key words: clinical chemistry, diagnostic tests, health care costs, laboratory test utilization introduction being the main source of objective data aiding clinicians in 60%–70% of all critical decisions such as diagnosis, treatment, and follow-up (1), laboratory tests are an essential part of an efficient health care system. as the resources of the health care sector are scarce, demands are raised to lower the costs while maintaining the quality of care. the laboratories are often among the first sections to be targeted for budget reductions, as their costs are easily discernible. however, the impact of laboratory tests on health care as a whole is wide-spread, and the monetary value of their effects is difficult to measure. furthermore, it has been demonstrated that a reduction in test utilization produces disproportionately small true cost reductions (2), and it is by no means certain that simply reducing the number of ordered tests will lead to a decrease in the overall health care costs. akademiska sjukhuset is with its 300-year history the oldest university hospital in sweden and also one of sweden’s largest tertiary care medical centers. in 2008 the hospital had approximately 1,100 beds, with 58,000 admissions per year and more than 750,000 out-patient visits annually. akademiska sjukhuset serves a population of 327,000 people in the county of uppsala, as well as the population in the surrounding counties. as accurate and timely information on laboratory test utilization is vital for financial management of correspondence: mirja mindemark, akademiska sjukhuset, entrance 61, 3rd floor, se-751 85 uppsala, sweden. fax: +46 18 611 37 03. e-mail: mirja.mindemark@medsci.uu.se (received 20 september 2010; accepted 22 september 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.528071 laboratories, the aim of this study was to describe longitudinal trends in test utilization from the perspective of the clinical chemistry and pharmacology laboratory at akademiska sjukhuset in uppsala, sweden. materials and methods test utilization data from 1 january 2002 through 31 december 2008 were retrospectively extracted from the laboratory information system. information on expenses, charges, admissions, and outpatient visits was collected for the years 2002 and 2008 individually. in all presentations of costs, e1 is equal to sek10. charges and expenditures were analyzed after adjusting for inflation according to the consumer price index (cpi), and the 2008 value is presented. the cpi, set at 100 for 1980, was 272.85 in 2002 and 300.50 in 2008. tests were counted as follows: all incoming test orders that generated a result, including nonchargeable results such as calculations or missing or ruined samples, were counted. before they were counted, all test orders were broken down into individual analyses, so that the test volume presented here contained no profiles, bundled tests, or test panels. results the variables considered in this study and their inflation-adjusted values for 2002 and 2008, respectively, are presented in table i. a total of 33,846,377 test results were generated in the clinical chemistry and pharmacology laboratory during the 7-year study period. from 2002 to 2008 the number of generated test results grew by 70.3%, with an average increase of 9.30% or 440,352 test results per year (figure 1). on average 4,835,197 tests results were generated annually during the study period. a total of 1,005 send-out tests were ordered during 2002. the corresponding figure was 3,160 in 2008. the mean price charged per test according to the price-list (not volume-adjusted) increased by 7.4%, or 2.6 inflation-adjusted euros, from e34.9 to e37.5 per test, during the study period. the total testing expense (total expense/total number of generated test results) was e2.8 in 2002 and e2.0 in 2008. the 10, 20, and 30 most commonly ordered tests accounted for, on average, 46.9%, 66.9%, and 75.5% of the total number of generated test results during the study period, and 47.8%, 66.4%, and 75.7% of the total number of generated test results in 2008 (table ii). the number of hospital admissions increased by 8.4% from 53,504 to 58,001 between 2002 and 2008. in 2002, there were 291,000 out-patient visits with a physician. during the study period this number grew by 14.2% to 332,243. the number of additional outpatient visits increased by 27.4% from 329,000 in 2002 to 419,213 in 2008. between 2002 and 2008, the number of hospital beds decreased from 1,200 to 1,100, and the total hospital costs increased by 22.6%. the total expenses of the clinical chemistry and pharmacology laboratory amounted to e10.4 million in 2002 and e12.5 million in 2008, representing an increase of 20.2%. the percentage of the hospital’s expenditures that was constituted by the costs of the clinical chemistry and pharmacology laboratory decreased slightly by 0.1% to 2.0% during the study period. discussion the cost of health care in sweden, as well as in many other countries, increases every year (3–5) and more rapidly so than the gross domestic product (gdp) (6). in 2002, the net costs of health care in sweden amounted to e15,459 million (2008 euros) (7). by 2008, the total expenditure had reached e20,640 million (7), an increase of 34% in less than a decade. the gdp, on the other hand, had during the same period only increased by approximately 17% (8). the total costs of health care in sweden, expressed as a percentage of gdp, were in 2005 9.2% (9), i.e. slightly above the 8.9% average of the oecd countries (10), but substantially lower table i. investigated variables and their inflation-adjusted values presented in 2008 euros. 2002 2008 generated test resultsa 3,760,508 6,402,617 analyses offered 309 663 send-out tests ordered 1,005 3,160 mean price charged per testb e34.9 e37.5 total laboratory expenditures e10.4 million e12.5 million total testing expensec e2.8 e2.0 admissions 53,504 58,001 hospital beds 1,200 1,100 out-patient visits with a physician 291,000 332,243 additional outpatient visits 329,000 419,213 total hospital expenditures e513 million e629 million aincluding non-chargeable test results such as calculations and missing or ruined. baccording to price-list, not volume-adjusted. ctotal expense/total number of generated test results. longitudinal trends in test utilization 35 than, for example, the corresponding 15.2% of gdp spent on health care in the us (10). the annual total cost of laboratory testing in sweden is approximated at e0.8 billion (in 2008 euros), about half of which is represented by the costs of clinical chemistry tests (11). of the total health care costs, laboratory expenditures only account for approximately 4% in sweden (12). the corresponding figures are 20% in the united states, 4% in the united kingdom, 5.2% in australia, and 7%–10% in canada (13). though laboratory tests only account for a very small part of the total health care costs, the laboratories are often among the first to be targeted for budget reductions. however, the impact of laboratory tests on health care as a whole is wide-spread, and the monetary value of their effects is difficult to measure. thus, the relation between test utilization, costs, and quality of care is as complex as it is central to health care management. very little has so far been published about changes in test utilization over time. accurate and timely information on trends in test utilization is essential to financial management of clinical laboratories. the aim of this study was therefore to elucidate the changes over time in clinical chemistry and pharmacology test utilization, data that are not readily available. studies such as this provide base-line data, a necessity when planning for future adaptation and improvements of test utilization and laboratory structure. to our knowledge, this is the first swedish study of clinical chemistry and pharmacology test utilization, charges, and expenses. this 7-year analysis was designed to describe and evaluate trends in test utilization, expenditures, and charges at a clinical chemistry laboratory at a large tertiary care university medical center. the study provides insight into the utilization and economics of laboratory testing during the past 7 years, a period that was characterized by +5.9% +5.9% +9.4% +10.5% +13.1% +11.0% 0 1000 2000 3000 4000 5000 6000 7000 2002 2003 2004 2005 2006 2007 2008 year t o ta l n u m b e r o f p e rf o rm e d t e s ts × 1 0 3 figure 1. the total number of performed tests per year during the study period. table ii. the percentage of the total number of tests represented by the 10, 20, and 30 most commonly ordered tests. year total number of ordered tests 10 most commonly ordered tests 20 most commonly ordered tests 30 most commonly ordered tests 2002 3,760,508 40.4% 59.5% 67.6% 2003 3,983,149 47.7% 69.7% 78.7% 2004 4,218,082 47.6% 69.1% 77.6% 2005 4,614,524 48.8% 69.3% 77.1% 2006 5,100,550 48.0% 67.4% 75.8% 2007 5,766,947 47.9% 66.7% 76.2% 2008 6,402,617 47.8% 66.4% 75.7% mean 4,835,197 46.9% 66.9% 75.5% 36 m. mindemark & a. larsson tightened budget control and ever-growing concern about medical costs. the principal findings of this study were substantial increases in the number of generated test results and in the number of tests offered, despite a virtually unchanged share of the hospitals total expenses represented by the costs of laboratory testing. from 2002 to 2008 the work-load, as defined by the number of generated test results, increased by over 70%, with a mean annual increase of 9.30%. this is higher than the average laboratory test growth rate of 7.2% seen in the netherlands during the 1980s (14) but lower than the average increase rate of 12.1% in the us in 1993 (15). other similar studies have presented laboratory test growth rates of 2.3%–13.8% at different times during the period 1970–2005 (15–18), whereas yet others have demonstrated virtually unchanged (19) or declining utilization rates (20), the latter most likely due to interventions aimed at reducing test ordering. the percentage of the total number of generated test results that was represented by sendout tests increased slightly from 0.03% to 0.05% and thus only accounted for a very small portion of the total number of generated test results throughout the study period. as the mean price charged per test is skewed upwards by a relatively small number of highcost analyses, it is not necessarily representative of the general level of unit prices charged, but it is nevertheless suitable for a comparison of trends. the mean price charged per test had increased slightly during the study period but at a rate lower than would be expected considering the inflation rate as defined by the cpi. however, the data available in this study do not allow analysis of the underlying cause of this. as opposed to the mean price charged per test, the total testing expense takes into account the prices charged per test as well as the test utilization volumes. this variable had, unlike the mean price charged per test, decreased from e2.8 to e2.0 during the study period, most likely due to a combination of increased automation and efficiency. the number of different analyses offered by the clinical chemistry and pharmacology laboratory was 663 in 2008, and had thus more than doubled during the study period. despite this, the share of the total number of generated test results that was represented by the 10 most commonly ordered analyses was very stable at approximately 50% throughout the study period, indicating that most of the new tests that were added to the test menu were low-frequency tests. the 10, 20, and 30 most commonly ordered tests accounted for, on average, 46.9%, 66.9%, and 75.5% of the total test volume during the study period, and 47.8%, 66.4%, and 75.7% of the total test volume in 2008. in a study by nexø, the top-20 tests represented more than 80% of all test requests (19), whereas, in a south african study, the 10 and 30 most commonly ordered tests represented 36.3% and 67.8%, respectively, of all ordered tests (17), results similar to those presented in this study. among the 10 most commonly ordered tests in this study, the intergroup order of tests varied minimally during the study period and mainly consisted of the components of the full blood count with the addition of potassium, and occasionally c-reactive protein (crp). among the 11–20 most commonly ordered tests, the extent of intergroup variation was slightly greater than in the top-10 group and increasingly so further to the bottom of the group. the group containing the top 21–30 tests, however, was much less homogeneous than the two previous groups. the continuity of different analyses was still fairly high among the top 21–30 tests, but no test was consequently represented in this group throughout the study period. the data on the top-30 tests could be useful as an indication of where small changes in test utilization may bring about considerable savings, as small technologies are likely to account for far more of the over-utilization than big expensive technologies (21), and low-cost high-frequency tests have been demonstrated to account for the major proportion of laboratory costs (22). the number of admissions and out-patient visits had increased by 20.2% during the study period. assuming the test-ordering pattern to be the same for the admissions and out-patient visits in 2002 and 2008, the increase in the number of admissions and out-patient visits would merely explain about onethird of the increase in test volume. the major part of the increase in work-load could thus most likely be ascribed to intrinsic growth. there are a few potential limitations of this study. firstly, the test utilization pattern at akademiska sjukhuset may not be representative of that in sweden as a whole. despite this limitation, the results warrant evaluation and are indicative of trends in clinical chemistry and pharmacology test utilization in sweden. secondly, the way the tests are counted will admittedly inflate test count, but this should not influence the evaluation of trends in utilization, with which this study is concerned. thus, as filtering of test-ordering data would risk biasing the results, we chose to present the raw, gross-type test-ordering data. furthermore, presentation of test volumes on the basis of individual tests as opposed to test volumes containing panels as bundled tests should facilitate comparisons between laboratories and countries as test panels include different tests in different settings. thirdly, as the total number of generated test results included some non-chargeable results such longitudinal trends in test utilization 37 as calculations, the total testing expense may be slightly under-estimated. however, there is no reason to believe that the number of non-chargeable results varied significantly during the study period, and this would thus not affect the evaluation of trends. lastly, it should be noted, for this type of study in general, that raw numbers of ordered tests do not reflect the quality of the care provided and that they say nothing of whether the tests were appropriately used. the main strength of this study is that the data on the utilization of a substantial number of clinical chemistry and pharmacology tests, representing a large patient population, were evaluated over an extended period of time. the study defines trends and may thus have potential predictive values. conclusion in conclusion, the total number of generated test results increased by over 70% in less than a decade, whereas a very small number of tests accounted for a disproportionately large share of the total number of performed tests. despite the substantial increase in the number of generated test results, the laboratory’s share of the hospital’s total expenditure remained low and virtually unchanged at approximately 2% during the study period. acknowledgements the authors would like to acknowledge the assistance of anders åhrberg and mats flodin in extraction of test utilization data. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. forsman rw. why is the laboratory an 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statistikdatabasen, nationalräkenskaper, bnp från produktionssidan (ens95) efter näringsgren sni 2002. kvartal 1993k1-2010k2, accessed 20101031. 9. organisation for economic co-operation and development. oecd health data 2008. 2008 www.oecd.org. 10. the national board of health and welfare. hälsooch sjukvårdsrapport 2005 (health care report 2005). 11. larsson a, palmer m, hulten g, tryding n. large differences in laboratory utilisation between hospitals in sweden. clin chem lab med. 2000;38:383–9. 12. hansson l-o. organisation, integration och analysis platforms. 2007. 13. plebani m. appropriateness in programs for continuous quality improvement in clinical laboratories. clin chim acta. 2003;333:131–9. 14. zaat jo, van eijk jt, bonte ha. laboratory test form design influences test ordering by general practitioners in the netherlands. med care. 1992;30:189–98. 15. benge h, csako g, parl ff. a 10-year analysis of ‘revenues’, costs, staffing, and workload in an academic medical center clinical chemistry laboratory. clin chem. 1993;39:1780–7. 16. valenstein pn, praestgaard ah, lepoff rb. six-year trends in productivity and utilization of 73 clinical laboratories: a college of american pathologists laboratory management index program study. arch pathol lab med. 2001;125: 1153–61. 17. pretorius c. utilisation of pathology procedures in the south african private pathology sector between 2003 and 2005. s afr med j. 2007;97:51–7. 18. fineberg hv. clinical chemistries: the high cost of low-cost diagnostic tests. in: medical technology: the culprit behind health care costs? washington dc: us department of health, education and welfare; 1979. 19. nexo e. the roles of primary and secondary health services in the production and utilization of clinical chemical blood analyses. a 5-year survey in the county of frederiksborg. ugeskr laeger. 1992;154:1184–8. 20. griner pf. use of laboratory tests in a teaching hospital: long-term trends: reductions in use and relative cost. ann intern med. 1979;90:243–8. 21. moloney tw, rogers de. medical technology—a different view of the contentious debate over costs. n engl j med. 1979; 301:1413–9. 22. newble di, wangel ag, nelson aw. an audit of investigational services within general medical unitsx. aust clin rev. 1981;9–11. 38 m. mindemark & a. larsson sups_a_548609 1..7 upsala journal of medical sciences. 2011; 116: 1–7 review article influence of microenvironment on engraftment of transplanted b-cells per-ola carlsson department of medical cell biology, uppsala university, uppsala, sweden, and department of medical sciences, uppsala university, uppsala, sweden abstract pancreatic islet transplantation into the liver provides a possibility to treat selected patients with brittle type 1 diabetes mellitus. however, massive early b-cell death increases the number of islets needed to restore glucose homeostasis. moreover, late dysfunction and death contribute to the poor long-term results of islet transplantation on insulin independence. studies in recent years have identified early and late challenges for transplanted pancreatic islets, including an instant bloodmediated inflammatory reaction when exposing human islets to the blood microenvironment in the portal vein and the low oxygenated milieu of islets transplanted into the liver. poor revascularization of remaining intact islets combined with severe changes in the gene expression of islets transplanted into the liver contributes to late dysfunction. strategies to overcome these hurdles have been developed, and some of these interventions are now even tested in clinical trials providing a hope to improve results in clinical islet transplantation. in parallel, experimental and clinical studies have, based on the identified problems with the liver site, evaluated the possibility of change of implantation organ in order to improve the results. sitespecific differences clearly exist in the engraftment of transplanted islets, and a more thorough characterization of alternative locations is needed. new strategies with modifications of islet microenvironment with cells and growth factors adhered to the islet surface or in a surrounding matrix could be designed to intervene with site-specific hurdles and provide possibilities to improve future results of islet transplantation. key words: engraftment, implantation site, islet transplantation winner of the eric k. fernström award for young investigators, 2010 at the medical faculty of uppsala university. correspondence: per-ola carlsson, md, phd, department of medical cell biology, uppsala university, biomedical center, box 571, se-75123 uppsala, sweden. (received 2 december 2010; accepted 6 december 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.548609 clinical pancreatic islet transplantation has, since the introduction of the concept, been almost exclusively performed through the intraportal route to the liver based on the pioneering work of the late dr paul lacy (1). early results of allogeneic islet transplantation were poor, with insulin independence 1 year posttransplantation less than 10% (2). however, in more recent years, transplantation of pancreatic islets from deceased donors has become a curative treatment for selected patients with type 1 diabetes (3,4). nevertheless, many problems still remain, including massive early islet cell death (5). the intravascular injection of pancreatic islets has been shown to elicit an instant blood-mediated inflammatory reaction (ibmir) triggered by tissue factor and cyto/ chemokines expressed by pancreatic islets in the whole blood microenvironment of the portal vein (6,7). moreover, the liver tissue has a very low oxygen tension (8,9), high numbers of resident macrophages (kupffer cells) (10), and possibly also high concentrations of immunosuppressive drugs exposing the islet cells following intestinal uptake of the drugs. this means that in most cases at least two donor pancreases are needed to restore glucose homeostasis, which is far more than the alleged 10%–20% of the total islet volume suggested to be enough to maintain normoglycaemia in humans (3). despite the large transplanted islet mass, the functional capacity of the transplanted islets has been shown to correspond to only about 20% of that found in a non-diabetic person (11). within the first years post-transplantation most patients revert to insulindependence if not re-transplanted. however, only low doses of exogenous insulin are then usually needed, and the major problems with hypoglycaemic attacks most often no longer exist. it is presently unknown whether the progressive decline in islet graft function is site-specific or applies also to other implantation sites. challenges for pancreatic islets early after transplantation a thrombotic reaction and complement cascade is elicited when human islets are exposed to blood, which is manifested by islet entrapment in blood clots, leucocyte infiltration, predominantly neutrophil granulocytes, and disruption of the islet morphology (12) (figure 1). this so-called ibmir occurs in clinical islet transplantation as shown by an increase in concentrations of thrombin–antithrombin complex immediately after islet infusion, and the parallel increase in c-peptide release indicates massive bcell death (7). a study by combined positron emission tomography and computed tomography (pet/ct) technique indicates that early islet cell death can be estimated to be approximately 25% in patients intraportally transplanted with islets (13). besides innate immune reactions, islets transplanted to the liver may have restricted survival rates due to the low oxygen tension in the liver parenchyma. experimental studies indicate that approximately 70% of the islets are hypoxic 1 day after intraportal transplantation, as evaluated by the biochemical marker pimonidazole which accumulates in islet cells at oxygen tension levels below 7.5–10 mmhg (14). since this marker is not accumulated in dead or dying cells, the number of hypoxic cells may even be under-estimated. clinically, also high concentrations of immunosuppressive drugs in the portal vein are likely to hamper islet survival by direct toxic effects (15,16). experimentally, several sites besides the liver have been tested for islet transplantation, including the subcutis, muscle, the intraperitoneal site, the renal subcapsular site, the spleen, bone-marrow, pancreas, and the omental pouch. a more thorough characterization of engraftment has recently been performed with regard to implantation of islets into the pancreas or muscle. orthotopic implantation of islets, i.e. into the pancreas, has been described in experimental settings (17,18). although being the most physiological environment for islets, the pancreas has rarely been considered as a potential implantation organ in clinical practice. surgical interventions and injections in the pancreas are difficult, and there is a high risk of acute complications due to leakage of enzymes from the exocrine cells that causes tissue damage and inflammation. clearly, implantation techniques need to be improved to minimize these early risks, e.g. by subcapsular implantation of islets or other techniques. safety issues have also to be carefully investigated in large-animal models. nevertheless, studies in small animals indicate that this site provides good conditions for early survival of implanted islets, with minimal inflammatory and fibrotic components (19). moreover, the oxygenation of the pancreas is much better than the liver, with a mean oxygen tension in the exocrine parenchyma of ~30 mmhg (20). striated muscle has clinically been tested as implantation site for pancreatic islets based on the feasibility of this site for autotransplantation of parathyroid glands and its easy access. in one swedish patient, islets were, due to severe hereditary chronic pancreatitis, autotransplanted intramuscularly with a remaining high and stable c-peptide production (21). however, also this site seems to provide challenges for early survival of the islets based on the extensive fibrosis occurring in the islet grafts (21,22). especially prominent central necrosis parts may occur in islet grafts, if islets are implanted as clusters, and cause central fibrosis (23). 2 p.-o. carlsson challenges for pancreatic islets late after transplantation for pancreatic islets to survive and regain function after transplantation they need to be properly revascularized. experimental studies show that mouse and human islets transplanted intraportally are poorly revascularized, similar to islets implanted subcapsularly to the kidney or spleen (24–26). it is noteworthy that endothelial cells seem to be stimulated to grow towards the implanted islets, i.e. form a dense vasculature in the immediate vicinity of the islets, but few blood vessels enter the endocrine parenchyma. this is reflected in an impaired oxygenation of intraportally implanted islets that remains for at least 1month posttransplantation, and that is accompanied by increased apoptosis rates (olsson r, olerud j, pettersson u, and carlsson p-o; unpublished observation). late maturation of the blood vessel system may occur, since numbers of pimonidazole-positive islets, suggesting prevailing hypoxia, decrease with time, and apoptosis rates become similar to those in endogenous islets at 3 months post-transplantation. consequences of low blood vessel numbers for islets are not restricted merely to impaired oxygen and nutrient transport but also include less influence of paracrine supporting signals from islet endothelial cells on b-cell function and growth. islet endothelial cells may normally support nutrient-stimulated insulin release and b-cell differentiation by their secretion of basement membrane components, especially laminin, and the glycoprotein thrombospondin-1 sequestered to the islet matrix (27,28). their secretion of laminin and hepatocyte growth factor is of importance to maintain b-cell proliferation and increase the b-cell mass when functionally demanded (29,30). impaired drainage of islet hormones induced by a no-flow phenomenon in isolated islets and early after transplantation also predisposes for sequestration of islet amyloid polypeptide (iapp) as amyloid in islets. irrespective of mechanism, amyloid formation has been described to occur in isolated human islets during culture (31) and following experimental transplantation to the liver in mice (32). a recent autopsy report of an islet-transplanted patient who died from a myocardial infarct indicates that amyloid forms following clinical islet transplantation to the liver (33). late failure of islets transplanted to the liver may also early challenges known obstacles for intraportal islet transplantation known obstacles for intrapancreatic islet transplantation known obstacles for intramuscular islet transplantation instant blood mediated inflammatory reaction (ibmir) hypoxia hypoxia lack of different portal drainage extensive fibrosis safety issues acute pancreatitis none so far reported poor revascularization of intact islets islet amyloid formation lipotoxicity changes in gene expression and functionality kupffer cell activation islet toxic effects of immunosuppressive drugs late challenges early challenges late challenges early challenges late challenges figure 1.obstacles for successful pancreatic islet transplantation at different sites. microenvironment and engraftment of transplanted b-cells 3 be caused by insulin-induced intense lipogenesis in nearby hepatocytes. proof of principle of improved long-term graft function has been shown in a rodent model by blocking lipogenesis (34). hepatic steatosis has been reported in the clinical situation after islet transplantation (35,36). there are reports on sitespecific alterations in islet function after intraportal islet transplantation as well, such as defective glucagon response to hypoglycaemia (37,38) caused by increased glucose flux and glucose levels within the liver secondary to increased glycogenolysis caused by systemic hyperglycaemia (39), and substantial changes in the b-cell gene expression and function (40,41). a perturbed glucose-stimulated insulin release in intrahepatic islets has been shown to correlate with defects in glucose oxidation, (pro)insulin biosynthesis, and decreased insulin content and associated with decreases in the islet gene expression of the b-cell differentiation marker pancreatic and duodenal homeobox gene-1 (pdx-1) and key enzymes in glucose transport and metabolism in the b-cells, such as glucose transporter-2, glucokinase, pyruvate carboxylase, and mitochondrial glycerol phosphate dehydrogenase (41). pancreatic islets experimentally transplanted to the pancreas are much better revascularized than intrahepatic islets (19). however, despite being implanted into their normal physiological microenvironment, intrapancreatically transplanted islets display some functional and gene expression changes, although not as profound as those observed in islets implanted in the liver. a characteristic finding for intrapancreatically transplanted islets seems to be their higher insulin release compared with endogenous islets at low glucose concentrations, and their high expression of the lactate dehydrogenase-a gene, which is normally expressed in low levels in b-cells (41). this may explain why animals receiving large numbers of intrapancreatically transplanted islets are slightly hypoglycaemic (18). pancreatic islets transplanted to striated muscle are rapidly and much better revascularized than intraportally transplanted islets (23). this seems to be irrespective of whether the islets are implanted in clusters or as single islets (22) and has been confirmed in patients autotransplanted with islets due to exocrine pancreas disease (23). the functionality of the newly formed capillaries is good, and oxygen tension levels in intramuscular islet grafts were only slightly decreased when compared to native islets (22). despite lack of direct portal drainage, pancreatic islets implanted to muscle seem to have a superior function when compared to intrahepatic islets (23), although a prerequisite for this is the avoidance of cluster formation at islet implantation (42). tentative strategies for islet microenvironment modification to improve islet graft survival and function modifications of islet microenvironment in the liver recent characterization of islet engraftment at the intraportal site has identified numerous problems that need to be targeted to optimize graft survival and function. however, since islets are dispersed and embolized deep into the liver tissue at transplantation, strategies to modify liver microenvironment per se are difficult. instead such modifications must include the systemic blood environment, or modifications of islet tissue or surfaces prior to transplantation. in fact, several strategies in order to intervene with ibmir have successfully been tested experimentally in vitro and/or in vivo and include modifications of blood with thrombin inhibition by megalatran (43), or with low-molecular-weight dextran sulfate (44), or modifications of the islets and their surfaces with nicotinamide (45), heparin (46), or endothelial coating (47). at present, a clinical trial is conducted in the nordic countries investigating the effects of low-molecularweight dextran sulfate on outcome in patients transplanted intraportally with islets. different anti-apoptotic strategies have been tested to improve early islet survival in experimental models. most successful studies include gene transfection strategies difficult to implement in the clinical situation. however, besides this, e.g. different caspase inhibitor therapies of recipients (48), or even of islets for transplantation (49), have proved effective in improving outcome in minimal islet mass models in mice. moreover, merely prolactin or glucocorticoid supplementation of the culture medium has been shown to improve b-cell survival during human islet culture and early after experimental islet transplantation (50–52). also long-acting glucagon-like peptide 1 analogues improve human islet survival in culture (53), protect murine b-cells of intraportal islet transplants from apoptosis (54), and are beneficial for glucose homeostasis in marginal mass islettransplanted mice (55). one of these long-acting glucagon-like peptide 1 analogues, exenatide, has been introduced in clinical islet transplantation protocols at some transplantation centres (56,57). our recent studies indicate that damage to islets at intraportal transplantation may facilitate subsequent islet revascularization, whereas islets with maintained integrity are poorly revascularized and blood-perfused (henriksnäs j, lau j, zang g, berggren p-o, köhler m, and carlsson p-o; unpublished observation). different means to improve islet revascularization have been tested including over-expression 4 p.-o. carlsson of the pro-angiogenic factor vascular endothelial growth factor (vegf) in the islet tissue by virus vectors (58). more recently, a non-viral gene delivery approach has been successfully tested in a small-animal model, where vegf was delivered to intraportally transplanted human islets by ultrasoundtargeted microbubble destruction (59). translation into the clinical situation may, however, be hampered by technique limitations in penetration of ultrasound in larger organs. more feasible in the clinical setting than trying to increase the expression of proangiogenic factors in the islet tissue may be to inhibit angiostatic factors. we recently showed proof of principle by inhibition of the angiostatic factor thrombospondin-1 in islets by a sirna technique, which improved both islet graft revascularization and function (60). unfortunately, at present there are no pharmacological receptor antagonists for thrombospondin-1 available. however, prolactin was found to decrease the expression of thrombospondin-1 in islets in culture and had beneficial effects for islet graft revascularization and function both when injected to recipient animals during the first days post-transplantation, or merely added during culture of islets prior to transplantation (50). cell coating provides interesting aspects to bioengineering of islets and to obtaining multiple effects that can diminish adverse events following transplantation. besides previously mentioned endothelial coating, other cell types might be considered for modification of islet surfaces. as reported in a paper in the present issue of this journal, mesenchymal stem cells (mscs) form an interesting approach to provide local immunosuppressive effects, at least early after transplantation (61). positive effects of mscs on revascularization in experimentally transplanted islets have recently been shown in vivo (62), and these cells can facilitate endothelial coating of the islet surface in vitro as well (63). several other cell types are of interest to include in the islet microenvironment, including endothelial progenitor cells and neural crest stem cells. endothelial progenitor cells seem to be able to diminish innate immune reactions and improve survival, revascularization, b-cell proliferation, and function in islet grafts (64). neural crest stem cells have the ability to increase substantially b-cell proliferation and improve islet graft function (65). modifications of islet microenvironment in extrahepatic sites as described above, extrahepatic sites to some part provide different challenges for survival and regain of function when compared to the liver. there are also site-specific differences to consider. in general, however, in most cases transplantation to these sites does not include infusion of islets into blood, which diminishes ibmir. some innate immune reactions may remain, but the extent of these remains to be elucidated. it can be envisaged that hypoxia in newly transplanted avascular cells provides a limitation also in extrahepatic sites, especially when islets are implanted in clusters. dispersion of islets at transplantation is of great advantage, and techniques for optimal implantation of islets need to be developed. modifications of islet microenvironment can at most of these sites be obtained by the use of scaffolds and biomaterial implanted before or together with the islets in order to provide spatial and functional support for the islets (66). the biomaterials can be biodegradable or not, and can also be modified e.g. by inclusion of oxygen carriers such as polymerized haemoglobin, anti-inflammatory components or cells, and growth factors and cells to stimulate angiogenesis. in this manner, also substances and cells that do not readily adhere to islet surfaces can be used to modify islet microenvironment and create new micro-organs. studies are on-going to optimize such micromilieus. conclusions in recent years, several hurdles that restrict the success of pancreatic islet transplantation into the liver have been identified. possible solutions to some of these problems have also been tested and include interventions to the ibmir and means to improve revascularization. in parallel, there has been an increased interest in change of implantation site in order to avoid the site-specific problems of the intraportal route. most alternative implantation sites have, however, not been well characterized with regard to engraftment of islets, and many unidentified obstacles at alternative locations may remain. 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hughes e, baker m, et al. polymer scaffolds as synthetic microenvironments for extrahepatic islet transplantation. transplantation. 2006;82:452–9. microenvironment and engraftment of transplanted b-cells 7 ujms109_3.pdf upsala j med sci 109: 261–265, 2004 scrotum-like protrusion of lipoma arising from the proximal thigh – report of two cases – koshi hattori,1 masahito hatori,1 mika watanabe,2 toshihisa osanai,3 shoichi kokubun1 department of orthopaedic surgery, tohoku university school of medicine,1 department of pathology, tohoku university hospital,2 department of orthopaedic surgery, yamagata university school of medicine3 abstract a free full-text copy of this article can be found at the web page of upsala j med sci: http://www.ujms.se we report two cases of large lipomas arising from the proximal thigh with an extremely uncommon shape. the tumors were elastic soft and movable. mri examination revealed both tumors with high signal intensity as fatty tissue on both t1 and t2-weighted images. needle biopsy of the specimens revealed lipoma. marginal excision was carried out for both cases because of the patients’ discomfort. no recurrence was found three and five years after surgery, respectively. introduction lipoma is a benign tumor composed of mature white adipocytes and is the most common soft tissue mesenchymal neoplasm in adults (1). we report two cases of lipoma arising from the proximal thigh with an extremely uncommon shape, i.e. a scrotum-like protrusion. [case1] a 45-year-old male presented with a tumor protruding from his proximal thigh. the tumor had been increasing in size and the skin showed erosion from rubbing against the leg and cloth. in 1997, he first noticed a quail-egg-sized subcutaneous tumor. two years later, it had become pendulous and was covered with intact skin. in 2001, it became as large as a fist, and the erosion appeared. he visited a local clinic and was then referred to our hospital. needle biopsy specimen revealed lipoma. 261 received 11 may 2004 accepted 3 june 2004 physical examination: the tumor was 10×9cm large, protruding from the medial side of the right proximal thigh (fig.1). it was elastic soft, movable against both the subcutaneous and deep tissue. there was skin erosion on the distal end of the tumor. the elastic hard portion was palpable deep inside. plain radiographs showed swelling of the soft tissue. the mass showed iso-density to subcutaneous fatty tissue on ct scan. no calcification was seen inside. some septum-like structure was observed. on mri, the tumor had high signal intensity as adipose tissue on both t1 and t2-weighted images. an irregular septum was seen in the center and distal part of the mass, showing the effect of fat suppression. the peripheral portion and septum were enhanced with the injection of gadolinium compound (fig.2). although the tumor was benign, its growth obviously impaired the patient’s quality of life. marginal excision was performed. microscopically, the tumor consisted of large, mature adipose cells separated by fibrous tissue. no nuclear deformity nor lipoblasts were found (fig.3). in the center of the distal half, necrotic adipose tissue was observed. there were no findings suggesting malignancy. the diagnosis was lipoma with degenerative necrosis. the patient had no local recurrence during the three years after surgery. 262 fig. 1. macrophotograph showing a mass protruding from the medial side of the right proximal thigh. [case2] a 50-year-old male visited our clinic because of a subcutaneous tumor protruding and hanging from his proximal thigh. the patient himself noticed the tumor about 20 years previously. the tumor was rice-grain-sized initially and had enlarged gradually. for two years the enlarged tumor had caused annoyance. 263 fig. 2. mri showing a mass with high signal intensities as adipose tissue and irregular septum in the center and distal part of the mass on both t1 (a) and t2-(b) weighted images. fig. 3. microphotograph showing a tumor consisting of large, mature adipose cells separated by fibrous tissues. a b physical examination: the tumor was 12×8cm large, protruding from the medial side of the right proximal thigh. it was elastic soft, and movable against both the subcutaneous and deep tissue. superficial vein dilatation was observed. the tumor revealed neither heat nor tenderness. plain radiographs showed soft tissue swelling. the mass showed iso-density to subcutaneous fatty tissue on ct scan. no calcification was seen inside. no septumlike structure was found. mri examination revealed the tumor with high signal intensity as fatty tissue in the subcutaneous tissues on both t1 and t2-weighted images. the mri findings were similar to those seen in the first case, although no irregular septum was present. histological examination of the specimen taken with needle biopsy revealed lipoma. the tumor was removed in april 1999 because of the patient's discomfort. microscopically, the tumor consisted of large mature adipose cells, mostly non-nucleated. no lipoblast was found. the diagnosis was lipoma with degenerative necrosis. no distinct recurrence was detected during the five years after surgery. discussion most lipomas arise and grow in the subcutaneous region. to the best of our knowledge, there has been no report of a protruding lipoma like the present cases. though they were large, and looked heterogeneous in mri (case 1), pathological examination showed no evidence of malignancy (5, 6, 7, 8, 9). the following diseases should be considered in the differential diagnosis based on the tumor location and/or radiological findings: well-differentiated liposarcoma, accessory scrotum, inguinal herniation. well-differentiated liposarcoma would show marked variation in the adipocytic size, atypical cells, and a number of lipoblasts (1). in the present cases, the tumor had large, mature adipose cells separated by fibrous tissue. neither nuclear deformity nor lipoblasts were found. accessory scrotum is a type of congenital lipoma, found in the perineal region of newborns (2, 3, 4). it was ruled out because of the advanced ages of the patients. inguinal herniation would occur on the inguinal band, and is easily ruled out by mri. there may be two reasons for the very unusual shape of the tumors, i.e. they arose in an area of thin and weak subcutaneous tissue, or, the subcutaneous tissue became weak along with the growth of the tumor. central necrosis usually suggests malignancy. in the present case, malnutrition due to overgrowth and stalk tortion could account for the necrotic change in the center of distal part of the tumor (case 1) and the non-nucleated cells (case 2). no recurrence was seen after marginal resection. references 1. fletcher cdm, unni kk, mertens f (eds); (2002). world health organization classification of tumours. pathology and genetics of tumours of soft tissue and bone. iarc press: lyon 2. morita t. (1991). congenital perineal lipoma with accessory scrotum: a case report. acta urol jpn 37:647–649. 264 3. konya e. (1996). accessory scrotum with lipoma: a case report. acta urol jpn 42:233–236. 4. budhiraja s. (1999). accessory scrotum. urol int :63:210–211. 5. gaskin cm, helms ca. (2004). lipomas, lipoma variants, and well-differentiated liposarcomas (atypical lipomas): results of mri evaluations of 126 consecutive fatty masses.ajr am j roentgenol 182:733–9. 6. ohguri t, aoki t, hisaoka m, watanabe h, nakamura k, hashimoto h, nakamura t, nakata h. (2003). differential diagnosis of benign peripheral lipoma from well-differentiated liposarcoma on mr imaging: is comparison of margins and internal characteristics useful? ajr am j roentgenol 180: 1689–94. 7. galant j, marti-bonmati l, saez f, soler r, alcala-santaella r, navarro m. (2003). the value of fat-suppressed t2 or stir sequences in distinguishing lipoma from well-differentiated liposarcoma. eur radiol 13: 337–43. 8. kransdorf mj, bancroft lw, peterson jj, murphey md, foster wc, temple ht. (2002). imaging of fatty tumors: distinction of lipoma and well-differentiated liposarcoma. radiology 224:99–104. 9. matsumoto k, takada m, okabe h, ishizawa m. (2000). foci of signal intensities different from fat in well-differentiated liposarcoma and lipoma: correlation between mr and histological findings. clin imaging 24:38–43. corresponding author: masahito hatori, m.d. department of orthopaedic surgery, tohoku university school of medicine 1-1 seiryomachi, aobaku, sendai, japan 980-8574 tel: +81-22-717-7242, fax: +81-22-717-7248 mhato@mail.tains.tohoku.ac.jp 265 upsala j med sci 99: 363-372, 1994 7.6 reference intervals for plasma proteins in patients with non-insulin dependent diabetes mellitus peter thye-€&nnl, ole blaabjerg2, per hyltoft petersen2 1. department of endocrinology m , odense university hospital, dk-5000 odense c, denmark. 2. department of clinical chemistry, odense ijnniuersity hospital, dk-5000 odense c, denmark. when reference intervals are established based on a healthy reference population they may have limited application for clinical purposes, as the average age of the reference individuals often is different from the age of patients (6). this is especially important for plasma proteins, where concentrations often are changing with age (cf. section 7.2). furthermore, some chronical diseases can be considered stable (a certain state of health) at another well defined level. it is, therefore, important to establish these reference intervals in order to evaluate the results, when other diseases are t o be examined. in some diseases, such as non-insulin dependent diabetes mellitus (type 2 diabetes), there are abnormalities in the metabolism, which is most pronounced for the glucose metabolism. type 2 diabetes patients are characterized by insulin-resistance in liver and skeletal muscle (1,2>. this insulin resistance is partly compensated by an elevated release of insulin, but the net result is cellular insulin deficiency. insulin in reduced amounts causes changes in most metabolic processes and might influence the synthesis of plasma proteins. the object of this investigation was primarily to establish reference intervals for nine selected plasma proteins in patients with non-insulin dependent diabetes mellitus, and secondly t o get informations which could evaluate the theory that cellular insulin defiency affects protein synthesis. materials non-insulin dependent diabetic patients 60 t o 80 years of age and without diabetic complications were chosen. they had no concurrent diseases and they were all in a stable metabolic situation treated either with diet alone or with diet and antidiabetic drugs/or insulin, and could be considered in a stable metabolic condition. 363 eighty-three patients were primarily included, but 23 of these were later excluded due to missing samples (7), elevated esr (12), m-component (2), and immediate post study disease (3). of the 60 included patients, the hbal,-concentrations were: mean 9.0 % with range 5.4 t o 16.0 %, and esr mean 12.3 with range 3 to 28. methods blood samples were drawn under the same conditions as for the healthy reference samples (cf. section 7.2). analytical methods were performed on a cobas fara (odense) according t o the same instructions and with the nordic calibrator and with antisera from dako. results the results are illustrated as probit-plots and compared to the hstributions of concentration values for healthy individuals includmg the group more than 50 years of age for each plasma protein (cf. section 7.2 and 7.3). further, the median of the healthy group from 60 to 80 years of age is indicated. s-prealbumin cumulated percentage frequency probit i " " i ' " , , , . . i ' " ' 1 -0.70 -0.60 -0.50 -0.40 -0.30 log g/l i ' ' " 1 ' " ' i " ' ' 1 0.2 0.3 0.4 0.5 g/l fig. 7.6.1 distribution of s-prealbumin concentration values for the diabetic population (*i compared to the healthy reference population (straight line). the median o f the healthy 60 80 age group is indicated (m). 364 s-prealbumin the distribution of s-prealbumin is reduced approximately 10 % compared to the healthy reference population (all minus women below the age of 50 not using estrogens) as well as the reference group of the same age group (fig. 7.6.1). this means that type 2 diabetic patients have about 10 percent lower values compared to the healthy population, and that the reference interval for these should be changed accordingly. s-albumin s-albumin for the diabetic population is reduced approx. 8 % compared t o the reference population (all men and women over 50 years and women below 50 using estrogens) (fig.7.6.2). furthermore, the matched reference population of the same age group is not reduced. consequently, the s-albumin concentrations are reduced by approximately 8 percent, and the reference interval for type 2 diabetics should be reduced accordingly. salbutttin cumulated percentage frequency problt 95 1 90 r ~ l ' l ' i ~ l ' l ~ l ' l ~ l p i 1.54 1.58 1.62 1.66 1.70 log g/l i ~ " ' 1 " " " ' " l 35 40 45 50 g/l fig. 7.6.2 distribution of s-albumin concentration values for the diabetic population (.i compared to the healthy reference population (straight line). the median of the healthy 60 80 age group is indicated (m). s-orosomucoid the distribution of s-orosomucoid concentration values is indistinguishable from the healthy reference population (fig. 7.6.3), so the reference interval is valid. 365 s-orosomucoid cumulated percentage frequency problt i i i l i i l -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 iog g/l i i i 1 ' i ' i ' i 0.3 0.4 0.5 0.6 0.8 1.0 1.2 g/l fig. 7.6.3 distribution o f s-orosomucoid concentration values for the diabetic population 1.) compared to the healthy reference population (straight line). the median o f the healthy 60 80 age group is indicated (m). sa1 -antitrypsin cumulated percentage frequency problt l ' l ! l ' l 1 . 1 . 1 i i i i i i i i i i ' i ' i 1 -0.16 -0.08 0.00 0.08 0.16 0.24 0.32 log qll 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.8 2.0 2.2 g/l fig. 7.6.4 distribution of s-a,-antitrypsin concentration values for the diabetic population (.) compared to the healthy reference population (straight line), the median of the healthy 60 80 age group is indicated (m). 366 s-al-antitrypsin s-al-antitrypsin is comparable to the healthy reference population with a slightly broader distribution (fig. 7.6.4). the type 2 diabetics have not been genotyped, so a fraction of 5 to 10 % of the individuals are expected to belong to ms and mz types, who have lower concentration values (cf. section 7.3). this might explain the fraction of approximately 10 % of patients with lower values in the distribution. there is, therefore, no need for a separate reference interval for diabetic type 2 patients for this protein . s-hap toglobin s-haptoglobin cumulated percentage frequency probit 95 ggl 50 40 30 20 l ' l . i " l 0.0 0.5 1 .o 1.5 2.0 gil fig. 7.6.5 distribution of s-haptoglobin concentration values for the diabetic population p) compared to the healthy reference population (curve). the median of the healthy 60 80 age group i s indicated (w). for s-haptoglobin, the distribution of values for the healthy reference population is neither gaussian or log-gaussian (section 7.2). the distribution for type 2 diabetics, however, seems to be close to gaussian (fig. 7.6.5.). compared to the total reference population over 50 years of age the distribution is broader, but as the median for the matched age-group is indistinguishable we have chosen not t o recommend any change in the reference interval for type 2 diabetics. 367 s-transferrin the distribution of s-transferrin concentration values for type 2 diabetics is approximately 5 % towards lower compared to t h e healthy reference population consisting of all except from women below 50 years of age (fig. 7.6.6). the matched age-group, however, has a median close to t h e median for the diabetics. therefore, there is no reason for a separate reference interval for this protein. s-transferrin cumulated percentage frequency problt 99 i fig. 7.6.6 distribution of s-transferrin concentration values for the diabetic population (*) compared to the healthy reference population (straight line). the median of the healthy 60 80 age group is indicated (m). s-iga for s-iga the dstributions of two age-groups (one below and one above 50 years of age) are neither gaussian nor log-gaussian (cf. section 7.2), whereas the type 2 diabetics seem to have s-iga-values distributed close to gaussian (fig. 7.6.7). this distribution is moved towards higher concentration values, with the matched age group of the healthy population is moved accordingly. therefore, there is no indications for choosing a separate reference interval for type 2 diabetics for this protein. 368 s-iga cumulated percentage frequency m r r 99 95 90 80 70 60 50 40 30 20 10 5 99 1 40 20 10 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 @ gil i . 1 . 1 . " . 1 " " 1 ' i . , . . 0.6 0.8 1.0 2.0 3.0 4.0 5.0 gll fig. 7.6.7 distribution of s-iga concentration values for the diabetic population 1.) compared to the healthy reference population (curve). the median of the healthy 60 80 age group is indicated (m). cwnulated percentage frequency paoblt 0.7 0.8 0.9 1.0 1.1 1.2 1.3 log g/l i 1 1 1 i * 1 * ~ * i ' ' * l 5 6 7 8 10 12 14 16 20 gll fig. 7.6.8 distribution of s-igg concentration values for the diabetic population p) compared to the healthy reference population (straight line). the median of the healthy 60 80 age group is indicated (m). 369 s-igg the distribution of s-igg concentration values for type 2 diabetics is neither gaussian nor log-gaussian but has a median close to the distribution of all healthy individuals minus women below 50 years of age and also to the age-matched healthy population (fig. 7.6.8). there is, therefore, no reason for using a separate reference interval for s igg for type 2 diabetic patients. s-igm s-igm cumulated percentage frequency probrr 9 9 1 9 5 1 30 20 10 i i i i i i i i i i i i i i ' 1 ' i ' i . ' ' ' . [ . 1 -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 iogglc 0.4 0.5 0.6 0.70.8 1.0 1.2 1.41.6 2.0 2.5 q/l fig. 7.6.9 distribution of s-igm concentration values for the diabetic population p) compared to the healthy reference population (straight line). the median of the healthy 60 80 age group i s indicated (m). for s-igm the distribution of concentration values for the type 2 diabetics is lower than the healthy reference population (all minus women below 50 years of age) and t h e median is also lower than the age-match reference group (fig. 7.6.9). this means that the diabetic patients have lower s-igm-values in general, and in consequence the lower reference limit should be moved to approx. 0.25 g/l. 3 70 discussion in chronical diseases like type 2 diabetes, the metabolic condition can be considered stable for long periods but at another metabolic level. this steady-state condition may be reflected in the reference intervals, which could be expected to differ from the reference intervals based on healthy individuals. the consequences of well defined reference intervals for specific diseases, would be a better interpretation of laboratory-data, e.g. could the lower intervals for s prealbumin and s-albumin for type 2 diabetes patients lead to more correct interpretation of low concentration values compared to the general reference intervals. thus, extended hospitalization or protein supplementation to diabetic patiens (which is without effect) could be avoided. therefore, the knowledge of these changes may be important for general practitioners and for hospitals in interpretation of protein data. the model should also be applied to other laboratory analyses and to other chronic diseases which can be considered stable for longer periods of time. in this study the decrease in s-prealbumin and s-albumin, without measurable changes in the other proteins (except from s-igm, see below) may be explained by a decreased protein synthesis due to cellular insulin deficiency as insulin is acting as an anabolic hormone (3 5). insulin exerts its effect intracellularly in mainly the liver and skeletal muscle, where most of the plasma proteins are produced in the liver. the type 2 diabetic patient is insulin resistant, i.e. decreased effect of insulin in muscle and liver and compensate by releasing increased amounts of insulin. the increased insulin release, however, does not fully compensate t h e insulin resistance with the result of peripheral insulin deficiency. this insulin deficiency leeds to intracellular lack of nutrients to the metabolic pathways (cellhunger) and corresponds to the normal hunger/starvation situation where decreases in prealbumin, albumin, and transferrin are experienced. there is no concomitant increase in the acute phase proteins, which indicates that a situation of cellular hunger due to insulin deficiency, but not to inflammation exists in type 2 diabetes. the tendency to lower values in this selected patient category for s-igm is difficult to interpretate. 2 5 9 5 5 0 5 9 371 conclusions the reference intervals for s-prealbumin and s-albumin are reduced for this selected group of type 2 diabetic patients compared to the general reference intervals for healthy individuals. this could be due to a cellular insulindefiency as there exists an altered metabolic condition in the liver and skeletal muscle, which affects protein synthesis. acknowledgements the authors would like to acknowledge the expert technical assistance of h e richter. references 1. 2. 3. 4. 5. 6. damsho p, vaag a, hother-nielsen 0, beck-nielsen h. reduced glycogen synthase activity in skeletal muscle from patients with and without type ii (non-insulin-dependent) diabetes mellitus. diabetologia 1991;34239-45. defronzo r, gunnarson r, bjmkmann 0, olsson m, wahren j. effect of insulin on pheripheral and splanchnic glucose metabolism in non-insulin dependent (type 2) diabetes mellitus. j clin invest 1985;76:149-55. kelleher pc, phinney sd, lockwood dh, et al. effects of carbohydrate-containing and carbohydrate-restricted hypocaloric and eucaloric diets on serum concentrations of retinol-binding protein, thyroxin-binding prealbumin and transferrin. metabolism 1983;32:95. ingenhleek y, de visscher m, de nayer p. measurement of prealbumin as index of protein-calorie malnutrition. lancet 1972; july 15:106-8. shetty ps, watrasiewicz ke, jung rt, james wpt. rapid-turnover transport proteins: an index of subclinical protein-energy malnutrition. lancet 1979; august 4:230-2. djurhuus ms, roland a, vadstrup s, hyltoft petersen p, uldall a. reference intervals based on hospitalized "healthy" patients and medical students in relation to analytical bias for serum potassium. scand j clin lab invest 1992;52:305-12. 372 upsala j med sci 100: 33-40, 1995 red blood cells, phase contrast, interference contrast microscopy and microspectrophotometry n. v. b. marsden from the department of physiology and medical biophysics, biomedical center, uppsala university, uppsa fa, sweden abstract following teorell's (1) observation that the ghosts of hypotonically hemolysed erythrocytes reseal, it was shown that during the time they are permeable to hemoglobin, foreign macromolecules (dextran) can enter and that the hemolysed cell can achieve a final colloid-osmotic equilibria1 state containing dextran a n d some residual hb. in this way dextran reduces the hemoglobin loss in hypotonic hemolysis. some hemoglobin loss is, however, inevitable, a s it begins with a non-diffusive bulk outflow, sometimes observable as a jet, during which time a diffusive influx of t h e colloid-osmotic "balancer", dextran, is not possible. finally, a s expected from a process which is for the most p a r t diffusive, transmembrane macromolecular transport is bidirectional; during hemolysis smaller molecules escape to a greater extent than larger ones. when i arrived i n uppsala in the mid 1950's, teorell had already changed the direction of his interests. instead of gastric secretion, he had begun to address yet another problem with clinical implications and one which might be considered to have a simpler cellular basis; in hemolytic diseases the erythrocytes lose their hemoglobin (hb). already in 1952, using hypotonic hemolysis a s his chosen system and thereby concentrating on a single cell model, he showed quantitatively that hypotonically hemolysed erythrocyte ghosts were not simply ruptured sacs but that for some time permselectivity was maintained and that the ghosts behaved a s perfect osmometers (1). although other workers had postulated that the hemolysed erythrocyte was not simply a ruptured sac, this was, a s far as i know, t h e first quantitative demonstration that, even after becoming leaky to macromolecules, the erythrocyte membrane had functionally resealed, even if only temporarily. in the introduction to this paper teorell succinctly explains his aims: "in view of the seemingly very complicated permeability processes in intact erythrocytes, it was thought t o be advantageous to attempt some investigations on hemolysed 3-955162 33 erythrocytes, the so called 'ghosts'. it was believed that these cells, probably devoid of many of the "living" constituents, could serve a s somewhat simplified models capable of shedding some light on the more elementary properties of cell membranes". the work i shall describe was a direct consequence of teorell's observations on hypotonic ghosts and is really an extension of his hypotonic ghost model to study the interactions of these ghosts with macromolecules, in particular the polyglucoside dextran, a substance of high clinical significance. from his findings and our knowledge we knew that hypotonicaily hemolysing erythrocytes were transiently permeable to macromolecules (the escape of hb) for several seconds in the case of each cell, a time period which should allow considerable transmembrane migration, even bidirectional, of macromolecules. had we adequately contemplated and comprehended the implications of such a state, all our subsequent findings, except jet emission, could have been anticipated. the work i shall describe was done in collaboration with two colleagues, firstly iviartin zade-oppen and later gijran ostling. teorell was always aware of the latest useful instrumental advances and for his studies had acquired one of the first microscopes equipped with phase contrast, a n optical system enabling the visualization of very weak phase objects such as hb-free erythrocyte ghosts; for his work on phase contrast, f. zernike was awarded t h e physics nobel prize in 1953. when erythrocytes are hemolysed in a hypotonic electrolyte solution, two classes of cell are visible under phase contrast: 1) bright unhemolysed cells each with a bright halo, and 2) paie gray, nearly hb-free ghosts without a halo or with only a faint one (fig. la). i n this case hemolysis is an essentially all-or-none phenomenon, t h e fraction of ghosts is, allowing for small variations in cell mass, the same as the fraction of hb released from the cells and is therefore a satisfactory measure of the degree of hemolysis (2,3), which in this case can be defined as the fraction of cells which have hemolysed. i n order to investigate this process further, we planned to estimate the residual material content of the ghosts by measuring their refractive indices (ri) by phase contrast. for this purpose we intended to use the polyglucose dextran a s a reference of known ri. initially, trying to make a short cut, we hemolysed the erythrocytes in a hypotonic electrolyte solution containing a known dextran concentration and thus of known ri, arguing that a s the osmolal concentration of the dextran was very low, it would not significantly alter the degree of hemolysis, which in this case would be high due to a low electrolyte concentration. however, although it should not have been, the result was unexpected. instead of two clearly distinguishable classes of cells as in fig, la, the cells now showed great variation, in that the ghosts did not appear to constitute a homogeneous class as in 34 fig. la. phase contrast appearance of erythrocytes hemolysed in a dextran-free hypotonic electrolyte solution. the bright cells, some coarsely crenated, each with a distinct halo, are unhemolysed, whereas the ghosts are dark gray, without haloes or with only faint ones, although they appear dark in this picture, they are described as pale gray in the text, a description which is more accurate in comparison with the background intensity when observed under the microscope. the degree of hemolysis was about 60%. reproduced with the permission of acta physiol scand. fig lb. phase contrast appearance of erythrocytes hemolysed in a dextran-containing hypotonic electrolyte solution. in this picture there are no typical unhemolysed cells as in fig. 1. these cells appear to be all altered and are, in fact, hemolysed cells containing m e r e n t amounts of hb. at this osmolality (electrolyte = 25 mm and dextran [mw = 250,0001 38.4 gd), all t h e cells have lost some hb and most have a contrast varying between the two classes shown in fig. 1. reproduced with the permission of acta physiol scand. fig. la, but now there were no longer any bright haloed cells as in fig. la; all the cells appeared to belong to another, quite different heterogeneous population as shown in fig. l b . this was interpreted as meaning that all, or very nearly all, the erythrocytes 35 had lost part of their hb but there were very few cells which had the appearance of the nearly hb-free pale gray ghosts shown in fig. la. the explanation for this is is that if erythrocytes are hemolysed in the presence of dextran, dextran molecules up to a size of about 300 kda enter the cells as hb escapes (4). the hemolysing cell thus contains hb and dextran, both of which contribute to the internal colloid osmotic pressure. teorell (1) had shown that hemolysing erythrocytes are able to reseal, and t h e most plausible explanation for the dextran effect is that t h e hemolysing cells containing a mixture of hb and dextran are able to reach colloid osmotic equilibrium with the external solution, and at some point resealing presumably occurs in the absence of a swelling pressure and with some hb still retained. a not inconsiderable amount of dextran enters the hemolysing erythrocyte. in the observations reported in ref. no. 4 the residual hb was about 50% and the number of dextran molecules in the erythrocytes was about one-third of the number of residual hb molecules. further, the solvated volume of the intracellular dextran was about 25% of the mean volume of the unhemolysed erythrocytes, and the intracellular dextran concentration about one-third of that outside. we had now come to a n interesting point and posed the question as to whether dextran had in some way reduced the degree of hemolysis, a seemingly unlikely possibility, but in those days when blood conservation was of central interest, one of some significance. however, when t h e experiment was repeated in another way so t h a t t h e erythrocytes were first hemolysed in a hypotonic solution containing only electrolytes, and dextran was added afterwards, there were now again two clearly distinguishable classes a s in fig. la: unhemolysed erythrocytes and ghosts. in other words when the cells were hemolysed in the presence of dextran, the pattern of hemolysis had been perturbed. even in a n electrolyte-free dextran solution, some hb is retained. it should be noted that this effect requires the dextran to have a minimal molecular mass of about 2 kda ( 5 ) . as far as the isoamylose (dextran) series is concerned, it appears that the smallest effective size is probably the eleven glucose residue oligosaccharide (1.8 kda). thus, smaller sugars such as the trisaccharide raffinose (51, or t h e tetrasaccharide stachyose, are without effect, although once the erythrocytes a r e resealed post-hemolytically, a smaller species, e.g. sucrose, is locked within the ghost. fig. 2 illustrates diagrammatically hemolysis in a pure hypotonic electrolyte solution compared with that when dextran is present (6). in the region ba, hemolysis is incomplete and related to the osmolality, whereas in cb, hemolysis is maximal and constant. the effect of dextran is thus to reduce the maximal loss of hb. the conclusion t o be drawn from this figure is that dextran does not affect t h e fractional number of cells hemolysed but only the fractional amount of hb released. 36 thus in the presence of dextran all the cells in bc are hemolysed, but only p a r t of their hb has been lost. so when an erythrocyte population is partially hemolysed in a i 1 1 1 i ' c b a i osm fig. 2. diagram showing the pattern of hb ~ o s s from erythrocytes as a function of the osmolality ( o m ) of the suspending solution i n the absence (0) and presence of dextran, dl, d2 and d3. the latter refer to an ascending concentration of dextran. reproduced with the permission of ups& j. med. sci. hypotonic dextran solution, although only a fraction of the total hb has been released all the cells (region bc) may be hemolysed. in a s much a s dextran reduces the hb loss in hypotonic hemolysis, it is a n inhibitor. however, if the hemolysis curves in the presence and absence of dextran are compared a s shown in fig. 2, the conclusion is that dextran does not reduce the number of erythrocytes which have undergone hemolysis, although t h e hb loss is reduced. this means that dextran does not offer any cellular protection against hypotonic hemolysis; it merely changes the pattern from an essentially all-or-none phenomenon in a hypotonic polymer-free solution to a partial liberation in a dextran-containing hypotonic solution (2). this inevitably introduces a n ambiguity into the definition of the degree of hemolysis, since in the presence of an external polymer the deviation from an "all-or none" hb loss means that the fraction of the cells that have hemolysed no longer even closely reflects the fraction of hb released. as a way round this difficulty, ostling (7) suggested that in the presence of dextran, hypotonic hemolysis could be said to result in "fractional mass hemolysis" and "fractional number hemolysis", each term having a different value; the two terms indicating the fraction of hb lost and the fractional number of cells that had lost some hb, respectively. as judged from the curves shown in fig. 2, dextran reduces the fractional mass hemolysis but leaves the fractional number hemolysis essentially 49 5 5 162 37 unchanged. if, a s usual, osmotic fragility is measured in a polymer-free hypotonic electrolyte solution, there is, of course, no ambiguity. although erythrocytes that have been hemolysed hypotonically in the presence of dextran are, at the termination of their exposure to hypotonicity, apparently intact, in the sense that their membranes are once more impermeable to small molecules and probably, at least temporarily, retain some ionic permselectivity (11, it is quite likely that the membranes are not restored to their original state and thus they may be mechanically more fragile than normal, as is my impression. notwithstanding this, these cells still have a potential experimental usefulness, even though they may have a shorter life in the "agitated" conditions of the circulation. dextran, for example, can be made fluorescent and erythrocytes labelled during hypotonic hemolysis in the presence of fluorescent dextran have been used in micropuncture studies on renal nephrons (h. r. ulfendahl, personal communication). later, other microinterferometric and microspectrophotometric studies confirmed that erythrocytes hemolysed hypotonically in the presence of dextran appeared to constitute a single population of cells clustered around a n average hb content lower than normal. in the region bc of fig. 2 such cells would be described by ostling (7) as having a "number hemolysistt of 100% and a lower "fractional mass hemolysis". cinephotomicrographic studies were also performed with phase contrast and microinterferometry to follow the process of hb escape from individual hemolysing erythrocytes. for this purpose it was of great advantage to be able to induce hemolysis at will. this would have been somewhat cumbersome with hypotonic hemolysis, as it would have required a device for altering the tonicity while the erythrocytes were in a chamber under the microscope. a much simpler possibility seemed to be to use so-called photodynamic hemolysis in which erythrocytes are first sensitized by exposure for a short time in the dark to a n isotonic electrolyte solution containing the dye rose bengal (about 10-4 m). then later when they are illuminated under the microscope, hemolysis begins in a few seconds and continues until all the cells in the field are transformed into ghosts. in this case it was observed that hemolysis began dramatically with t h e emission of hb in a rapidly escaping jet-like cloud (€49) (fig. 3). that this cloud was a jet was demonstrated by the recoil of the emitting cell and also because an adjacent cell, onto which a jet impinged, moved away. the jet phenomenon indicates that hemolysis begins with a non-diffusive bulk outflow of hb. this is in agreement with heedman's report from microinterferometry (10) that initially, hb escapes with a high velocity. although photodynamic and hypotonic hemolysis are different, the existence of jets is well established in the latter; in an elegant study with a video-camera, zade-oppen (personal communication) followed the jet-induced movements of hypotonically hemolysing erythrocytes. further, kcchen (ll), in a very short but highly informative 38 fig. 3. an erythrocyte emitting a jet containing hb during photodynamic hemolysis (interference contrast). the appearance of the jet depends on the m e setting of the microscope, and the amount of material is greatest in the lighter region. reproduced with the permission of ups& j med sci. note, described how he visualized jets from hypotonically hemolysing erythrocytes using bright-field microscopy combined with an extracellular protein precipitant. although the propellant power of these jets is probably rather poor (m. holwill, personal communication), they do provide an example of how small (intracellular) bodies might be moved from one intracellular site to another. since hemolysis begins with a bulk non-diffusive outflow of hb, dextran, which can conceivably only enter by diffusion, will be excluded until the jet outflow has ceased. ostling (7) only observed the entry of dextran after about 20% of the hb had escaped. it was thus not possible to reduce the hb loss below this approximate value, a n d means that in the presence of hypotonic dextran there is always some hemolysis. he also noted molecular sieving; smaller species entered more rapidly than larger ones. in this respect there is, a s might be expected, symmetry as regards transport in both directions across the membrane, since during hemolysis smaller molecules escape to a greater extent than larger ones (ostling et al. (12)). i n this case, whereas hb escaped to a lesser extent than did smaller species, two other molecules, lactate dehydrogenase and catalase, escaped to a lesser extent than the smaller hb. acknowledgements first, i want to express my heartfelt gratitude to professor torsten teorell for his generous hospitality, great help and interest and for his friendship. in addition my thanks are due to two good friends and colleagues, drs martin zade-@pen and goran ostling, for very enjoyable and fruitful collaboration. further, i wish to acknowledge the collaboration of the late dr howard g. davies, who allowed us to use his cinkphotomicrographic equipment a t king’s college, london, and also of professor erik ingelstam and engineer l. p. johansson (now sjovall), late of the institute of optical research, the technical high school, stockholm, for much good advice and help. finally, i want to acknowledge the generosity of the polymer chemistry 39 department of pharmacia ab, uppsala, for characterizing and donating several "narrow" dextran fractions and in particular, for her never-failing willingness to help, even a t short notice, dr kirsti granath. references 1. teorell, t.: permeability properties of erythrocyte ghosts. j. gen. physiol. 35:669-701, 1952. 2. zade-oppen, a. m.: 1st die hamolyse partiell?. folia haematol 7 7 2, 3. zade-oppen, a. m. m. & marsden, n. v. b.: the phase-contrast appearance of hemolysing red cells and the determination of fragility. acta soc med upsal 73: 91-112, 1968. 4. marsden, n. v. b. & ostling, s. g.: accumulation of dextran in human red cells after haemolysis. nature (lond) 184: 723-724,1959. 5. davies, h. g., marsden, n. v. b.., ostling, s. g. & zade-oppen, a. m. m.: the effect of some neutral macromolecules on the pattern of hypotonic hemolysis. acta physiol scand 74: 577-593, 1968. macromolecules reduce hemoglobin loss in hypotonic hemolysis. upsala j med sci 84:155-161, 1979. 7. ostling, s. g.: permeability of human red cells during hypotonic fractional mass hemolysis in dextran. acta univ. upsaliens. (thesis) 88: 1970. 8. marsden, n. v. b. & zade-oppen, a. m. m.: j e t expulsion of haernoglobin during photodynamic haemolysis. acta physiol scand s u p p l 2 7 7 133, 1966. 9. marsden, n. v. b., zade-oppen, a. m. m. & davies, h. g.: jet expulsion of cellular contents from red cells during photodynamic hemolysis. upsala j med sci 86: 1-8, 1981. 10. heedman, p.-a.: hemolysis of individual red blood cells. exper cell res, 14: 9-22, 1958. 11. kochen, j.a.: structural disturbances during cell lysis. am j dis child 12. hjelm, m., ostling, s. g. & persson, a e. g.: the loss of certain cellular components from human erythrocytes during hypotonic hemolysis in the presence of dextran. acta physiol scand 6 7 43-49, 1960. 132-137, 1960. 6. zade-oppen, a. m. m., ostling, s. g. & marsden, n. v. b.: on how 104: 537-538, 1962. 40 upsala j med sci 99: 277-306, 1994 6. the nordic protein project per hyltoft petersenl, ole blaabjergl, kerttu irjala2, art0 ic6n3, kristian bj0r04. 1 . department of clinical chemistry, odense university hospital, dk-5000 odense c, denmark. 2. department of clinical chemistry, t u r k u university hospital, sf-20520 turku, finland. 3. department of clinical chemistry, helsinki university hospital, sf-00290 helsinki, finland. 4. department of clinical chemistry, rikshospitalet, n-0027 oslo i, norway. 6.1 aim of the project when the nordic protein project was decided by the nordic control committee in 1986 and supported by nordkem in 1987, the purpose of the project was to 1. improve analytical quality of specific plasma protein measurements in the nordic countries, and develop a more general model for analytical quality management, which could be applied to other laboratory analytes as well. 2 the basis for the project was the relationship between calibration and control, where the idea was that external control was nothing but a registration of the current situation, as long as calibration was dependent on a variety of commercial calibrators with no or questionable documentation of standardization. the first aim was, therefore t o introduce a common and reliable calibrator (cf. chapter 5.3 and 5.4) available to all participating laboratories and then assess their analytical performance with control samples of the same high quality as the calibrator. in other words: ’you cannot control analytical quality into your method’ the quality depends on calibration, analytical specificity etc. as described below. for proteins (and other components to be measured by immunological methods) the identity between the measurand in calibrator, patient samples and controls is essential. this ideal, however is not possible for plasma proteins, which are present in a variety of forms, so the aim was ’to measure the average composition of proteins from healthy adults correctly’. this concept, looks rather modest, but even at this low level it can be very difficult, as seen from chapter 5.4 and chapter 7, and later in this chapter. 277 specificity and interference are dependent on the analytical principle and on the reagents used in the measurement procedure and the trueness of a measured value is also dependent of these factors. interference is defined as modification of the signal t o be measured, and a variety of blood substances are interfering with the protein molecules and, thereby, modifying the signal but for healthy individuals this effect is assumed t o be comparable in all samples and therefore, negligible for the investigations in this project. the non-specificity due to turbidity in samples, however, is a serious problem for many protein methods, as the reaction to be measured is the increase in turbidity based on the formation of immunocomplexes, whether these are measured photometrically (turbidimetric) or fluorimetrically (nephelometric). this problem with turbid samples is the major specificity problem and, in consequence, the aim was to design a control system, which could separate bias due to unspecificity from bias due t o the calibrator, and pointing t o the methods which are sensible to turbihty, and need clarification of samples before measurements. further, the aim was to assess the analytical performance in the individual laboratories challenging their calibration function as well as the performance in general. elements of analytical quality 1 (-) analytical (-) (-) specifications / \ during the project it became clear that a definition of good and poor quality was needed. the most relevant concept would have been to estimate analytical quality specifications from the clinical outcome of the results. this approach, however, could 278 not be applied as there are no clear recommendations for interpretation of data from measurements of plasma proteins in the nordic countries. interpretations were generally based on the reference intervals and, consequently, a new aim was to define analytical quality specifications according to the concept of sharing common reference intervals. these quality specifications were evaluated a s a general principle a s described in chapter 4. production of common reference intervals could not be directly integrated into the project, but by co-operation with the danish and the finnish protein groups the common reference intervals were established (cf. chapter 7). common reference intervals establishment reference values %ceu 1 specifications / \ [-) [-) based on the extensions of reference intervals and analytical quality specifications the aim developed t o analytical qua& management in the sense that all elements were integrated entirely, where clinical useful reference intervals were backed up by a system of analytical quality improvement in order to establish the base for the common reference intervals and the control system was designed to test whether each laboratory could qualify for the use according to the analytical quality specifications derived for this purpose, i.e. t o share the common reference intervals established. a number of problems which are related to genetical variants and pathological changes are not taken into consideration in the project. the most relevant are: 279 1. the influence of genetic phenotypes on the quantifications e.g. haptoglobin and ccl-antitrypsin 2. varying composition of immunoglobulins e.g. oligoclonal and monoclonal immunoglobulins 3. binding of components to proteins e.g. bilirubin to albumin and haemoglobin to haptoglobin 4. extreme concentrations e.g. detectability for haptoglobin and extreme values of immunoglobulins 280 6.2 analytical quality specifications the analytical quality specifications were achieved according to the concept of sharing common reference intervals, and were based on an average of published reference intervals, assuming log-gaussian distributions. this assumption was confirmed by the actual distributions obtained from the reference values as described in chapter 7. the actual curves for maximum allowable combined analytical bias and imprecision are displayed in fig. 6.2.1. 2 bias % l 4 1 12 10 a 6 4 2 0 maximum allowable combtned bias and imprecision transfer r in 0 5 10 15 20 25 30 imprecision (cv) % fig. 6.2.1. combined analytical quality specifications as used in the beginning of the project. from hyltoft petersen et al. (3) with permission. it is interesting to see that the acceptable variations and errors are very wide for s haptoglobin and the two immunoglobulins iga and igm, with acceptable imprecision about 25 % (with negligible bias). the acceptable values for bias are about half of this value (when imprecision is negligible). most of the proteins have acceptable imprecisions (with negligible bias) of about 10 % and corresponding bias of 4 to 5 %. the most demanding criteria are for s-albumin with maximum allowable values of 4 and 2 % for imprecision and bias, respectively. these criteria are hardly fulfilled by any laboratory, and this protein illustrates that the variation of homeostatic set-points is narrow. 28 i in the beginning of the project the two analytical specifications for bias and imprecision were used separately allowing the maximum of both simultaneously, but at the end where the new specifications were introduced based on the estimated reference intervals (cf. chapter 7) the combined criteria were taken into consideration. these criteria are shown in fig. 6.2.2. bias % allowable combined bias and imprecision for specific plasma proteins 14 1 10 l 2 l 12 transthyretin (prealbumin) al -trypsin inhibitor transthyretin (prealbumin) al -trypsin inhibitor 0 5 10 15 20 25 30 c v % fig. 6.2.2. combined analytical quality specifications as used at the end of the project, based on the estimated reference intervals (chapter 7). the new analytical quality specifications are very close to the first ones. this was not expected as the separation of the reference intervals into subgroups due to the biological differences according to age, sex and the use of estrogens, might have resulted in more demanding specifications (chapter 7). 282 6.3 creation of analytical quality the calibrator. as mentioned under aim of the project, a very important element of the project was t o supply all participants with the same high quality calibrator for daily use in order t o create that element of quality in t h e project. in the first survey, however, t h e laboratories should use their own calibrator for comparison, and the calibrator was sent together with t h e control samples in the first mail. the calibrator is described in chapter 5.3 and t h e traceability to ifcc/cap/bcr 470 is documented in chapter 5.4 (a new production of calibrator is available from the danish society of clinical chemistry). the calibration function. calibration functions are related to analytical principles and to equipments, and each calibration function should fit the points of dilutions of t h e calibrator in an x-y plot or transformations of data. this problem has not been investigated in the project, but beside instructions from producers of equipment and reagents, dako has developed a number of recommendations for analytical performance at different instruments, which are well documented. bcr crm 470 specifications ------ i creation of analxtical b u m performance control of analmrcal i q u f i w 283 analytical procedure, when buying an equipment for measurements of plasma proteins the analytical principle is already decided. some instruments are designed for the purpose whereas, others can be used for kinetic as well as for endpoint measurements. the two dominating analytical principles are turbihmetry and nephelometry, whereas, the gel-methods are too time consuming for routine work. the key reagents for protein measurements are the antisera, where the commercial available products for the main plasma proteins all are of a quality, without cross reacting immunoglobulins. the individual laboratory is without influence on equipment and antibodies (except from the first choice of buying), whereas, it is responsible for the implementation and the current performance. these, include instructions education and training of technicians as well as maintenance and an internal control system for stability and imprecision. 284 6.4 principles for control as outlined above the analytical quality of the measurements are determined by external factors and internal factors. the external factors, equipment, calibrator and reagents, are outside the laboratory's influence. the internal factors are implementation and performance, and here the laboratory is responsible for the process. another approach is to divide the factors into permanent and variable factors. this, makes the concept of control easier to grasp. the permanent factors are the external: equipment (analytical principle), type of calibrator (traceability), and the internal: implementation. the variable factors are the external: batches of calibrator and reagents, and the internal: daily performance. internal control. an average internal control system can only disclose variations and errors in the variable factors, and these will show up as systematic changes (change in concentration level) and changes in random variation (imprecision). (with special designs other problems, like detection limit, changes in specificity can be controlled internally? but this is not done in general). in consequence? the internal control systems are capable of monitoring stability and imprecision only. variable factors internal control control ( g i i = ) ( y ) \ / 1 control of quality / \ control imprjxision variation 285 external control systems. external control systems can only describe the analytical quality at the time of control, and only to a certain level. so, they will see t h e sum of a mixture of permanent and variable factors if it is not designed for separating these factors. in general, however, an external control system should describe the permanent situation. permanent factors external control control [=) 0 calibration \ / ~ / \ specificity 1-b control 0 specificm principle for the control system. in order t o separate the permanent factors from the variable the control is performed with three assessments over two years with the same control materials, and with repeated measurements over four days in duplicates. thereby, the internal factors can be described separately and the estimate of actual bias can be performed with a rather narrow confidence interval. by repeating the assessment three times the permanent bias can be estimated as long as the laboratory has not changed the method. the permanent factors are calibration and specificity and they are challenged by a set of controls where the main specificity problem, turbidity, is approached by a set of two control materials, which are identical except from the lipid fraction (responsible for t h e turbidity) and three clear pools with relative concentrations of all proteins t o be measured of 0.5, 1.0 and 1.5 as described below. 286 6.4.1 principles for c o n t r o l materials control materials designed for assessing the calibration under optimal conditions should be like an ideal sample without any interfering substances, as denaturation of the measurand, unspecific reactions o r interference will invalidate the calibration and result in an estimated bias, due to conditions which have nothing to do with the calibration proper. these other factors should be dealt with separately by samples which are identical, except from the presence of the interfering substance in one of them. in the project the following four control pools were used (1). (clear) i control t control a ( = c o n t r o l t) (clear) i (turbid) control c (= 0 s " c o n t r o l a ) ( c l e a r ) the clear controls a, b, and c are designed for control of calibration at three levels. control a is a pool from healthy individuals and should have a concentration value near the middle of the reference interval (or slightly below, due to the log-gaussian distribution) and control c is constructed to give half the concentration of control a for the proteins under consideration, while control b has 1.5 times the concentration of control a. control t is identical to control a, but is turbid with the lipoproteins present production of the controls. a serum pool collected from 100 volunteers was produced after test of all individuals for antibodies against hiv and hepatitis. from each 0.5 litre blood was drawn and after coagulation and centrifugation the individual sera were frozen at -80 "c and stored at this temperature until all the portions were thawed and pooled to be used as control t. this pool contains all lipoproteins. a fraction of the control t-pool is ultracentrifuged and restored to three different control pools. the procedure for all of these pools is initially a 'normal' centrifugation at 4 "c in order to get rid of cryo-precipitates. then a portion is weighed and 287 ultracentrifuged for 48 hours at 180.000*g. there are three layers: the lipid layer which is sucked of, the infranatant, which consist of all the low molecular weight: constituents of plasma, but without the plasma proteins under consideration, and the lowest layer with all the plasma proteins in a high concentration. controza after removal of the lipid layer the weight is reconstituted with infranatant from an other ultracentrifugation. controzb. after removal of the lipid layer, part of the infranatant is removed until the volume is 2/3 of the original (by weighing, keeping the specific gravity in mind). control c. after removal of the lipid layer, infranatant (from the production of control b) is added to a volume of 2 times the original (by weighing, keeping the specific gravity in mind). ( 100%) a ( 150%) vol. adj. ___t 180.000 g lipid free 40 h supernatant (100%) (50%) control t control control c all control pools are dispensed into cryotubes (1 ml) and frozen at 80 "c, and kept at that temperature until shipping packed with dry ice to each laboratory. the target values were assigned by measurements in four laboratories by mancini technique. (the assigned values were used as the original target values of the calibrator, and they were important for the evaluation until the ifcc/cap/bcr 470 values were transferred, but the old values have no interest now). 288 6.4.2 principles f o r evaluations of d a t a the general evaluations of data were rather traditional with construction of histogrammes of the data from each participant, but with indication of target value and the accept limits for bias accordng t o the analytical quality specifications shown. for the turbid control t, the results were further divided into turbidmetric and nephelometric analytical principles in order to disclose the robustness of the analytical principles to this challenge. two examples, one for the clear control a and one for the turbid control t, both for s-transferrin, are shown in fig. 6.4.2.1 and fig. 6.4.2.2, respectively. further, the results for control t are divided according t o nephelometric and turbidimetric analytical principles and illustrated in fig. 6.4.2.3. it is seen that the turbidimetric methods are more robust. target value local calibrator 1 2 00 2 50 3 00 350 gll s-transferrin clear control (con a) target value 2 04 232 3 w 3 % oil fig. 6.4.2.1. histogrammes of s-transferrin showing the results for the clear control a. upper: results when laboratories used their own calibrator. lower: result when the nordic calibrator was used. from blaabjerg et al. (2) with permission. the individual eualuations were designed in order to separate the elements of analytical variations and errors according t o calibration function, specificity as well as withinand between-run variation. the results were compared to the analytical quality specifications and described graphically in order to support the local trouble shooting process. 289 target value i local calibrator fig. 6.4.2.2. 2 00 2 50 3 00 350 g l l s-transferrin turbid control (con t) target value 2w 2 50 3 do 350 g l l histogrammes of s-transferrin showing the results for the turbid control t. upper: results when laboratories used their own calibrator. lower: result when the nordic calibrator was used. from blaabjerg et al. (2) with permission. nordic calibrator s-transferrin turbid control (con t) target value a enopoint k i m t i c nephelometric turblolmetrlc + gel technioues t : target value w 3 do 0 turbolmtrlc rocket 7 350 g l t fig. 6.4.2.3. histogrammes of s-transferrin showing the results for the turbid control t. same data as i n fig. 6.4.2.2, but separated according to nephelometric and turbidimetric analytical principles. from .blaabjerg et al. (2) with permission. 290 combined evaluation of calibration function and imprecision for each survey, each laboratory, and each protein the results from controls a, b, and c were arranged in a matrix and treated according to a two-way analysis of variance with replicates. first the data were 'normalized' by multiplying results from control c with a factor 2 (which should bring them to the level of control a) and results from control b were multiplied by a factor 2/3 (also t o bring them to the level of control a). this was done according to the relative dlutions of of the three controls so, if the calibration function was correct the between-control variance component should be negligible. the data were treated as a two-way analysis of variance with crossclassification according to controls and days. as mentioned above the between-control component described the proportionality according to the calibration function (as an average). the between-day-component described the between-run variation (reflecting both variations in performance and variations in the average level of calibration), and the replicate-component described the within-run variation. finally, the interaction-component described fluctuations in the calibration function, i.e. the variation in proportionality of results. this design and evaluations allowed validation of the analytical performance in each laboratory within a short period of time (one week) at three occasions (over two years), and should give a firm basis for the individual laboratory t o disclose the weak links of the analytical procedure, and the informations needed for trouble-shooting. evaluation of effect of turbidity for each day the difference between means of control t and control a were calculated and the mean difference from four days was calculate with 90 % confidence intervals. this was an estimate of the effect of turbidity. graphical presentation of data the results for the clear controls were displayed as a difference plot with the measured results minus target values as ordinate and target values as abscissa. the 90 % confidence interval was shown for each point and, further, the accept limits (for sharing common reference intervals) were illustrated as straight lines. an example of s-albumin is shown in fig. 6,4.2.4. 20-955059 29 1 salbumin (bcg-method) difference (measured target) (g/l) 6 4, 2 0, -2 accept limit control c control a accept limit i ........... ................... i a control c control a 1 -2 accept limit -10 -8j it control b 0 20 40 60 8 0 target c o nc e n t r a t i on ( g / l ) fig. 6.4.2.4. difference-plot for s-albumin from a laboratory using a bcg-dye binding method. ordinate: difference between measured value and target value. abscissa: target value. the points for control c, control a, and control b are given with 90 % confidence intervals. further, the dotted area illustrates the acceptance area for sharing common reference intervals. from hyltoft petersen et al. (3) with permission. this example with s-albumin illustrates a calibration function which should have been curved, but was fitted by a linear function (one-point calibration). a further illustration of the combined effects of the laboratory's own calibrator and the turbidity is performed in a double difference plot for the two differences (measured control a minus target value) and (measured control t minus measured control a). the ordinate is the same as for the difference plot in fig. 6.4.2.4 (but now the only point shown is the control a point with 90 % confidence interval) and the abscissa is the 'turbidity-difference (also shown with 90 % confidence interval). the acceptance limits are shown for both differences. the combined results show up as a cross, which for acceptance should be located within the square formed by the two acceptance areas. 292 in the example (fig. 6.4.2.5) for s-haptoglobin the 'cross' is located within the square, and the calibration as well as the turbidity correction are acceptable. + i \ f \ accept limits '4 0 difference (control t control a) turbidity fig. 6.4.2.5. double d i fference-plot for s-hap toglobin. ordinate: difference between measured control a and the target value. abscissa: difference between measured control t and measured control a. the acceptance limits for sharing common reference intervals are indicated. the data were collected and typed by jens rahbeck nargaard in odense and the computations and printouts were performed by a computer group in uppsala, chaired by torsten aronsson. for each survey each laboratory received a printout for each of the proteins with the computations and plots, together with an evaluation as judged from us, fig, 6.4.2.6 a and b on the next pages is an example of such a printout. s-orosomucoid is taken as an example from an indwidual report (fig. 6.4.2.6 a and b). on the first page (a), the calculations are presented. point 1 shows results with the local calibrator and for calibration with the norhc calibrator. mean values and to,,+sem for estimation of the 90 % confidence interval, then withinand between-run coefficients of variation (cvw and cvb) calculated according to a simple nested design (repeated balanced subsampling), further the target value is shown. in point 2, the effect of turbidity in control t (compared to control a) is given, and in point 3, the variations in the three clear controls over four runs are evaluated accordmg to a two way analysis of variance with crossclassification according to controls and runs, with 293 nkk's protein projekt ororomucoid nordic protein project lab.: 2 9 5 2 evaluation o? tre lab data 111111111111111..111111-111 1) individual pools and efcects of recalibration ........................................................................... values directly cal con c con a con b con t hean value 0 . 5 2 8 0 . 2 6 0 0 . 5 0 5 0 . 7 5 9 0 . 5 3 0 to. l*sem 0.016 0.005 0.014 0.023 0.011 var. within run cvwt 0.9 1 . 9 1 . 4 1 . 4 0.9 var. between run cvb i 2.4 0 . 8 2 . 1 2 . 3 1 . 6 recalibrated values hean value --0.409 0 . 7 9 5 1.194 0 . 8 3 4 to. 1*sem --0.007 0.011 0.014 0.012 var. between run cvb i --0.0 ' 0 . 7 0.0 1.0 ___--_-----------------------------____ __---- ------------------ 0.830 0.380 0.760 1 . 1 5 0 0 . 7 6 0 2 ) evaluation 01 bias (uring control a and control ti total bias bias ?or c l w r control interference ( con t taxget) (con a targist) (con t con a ) -------------------------------------------- direct recal. diract recal. direct mean value -0.23 0.07 0 . 2 5 0 . 0 3 0 . 0 3 to.l*sw 0.01 0.01 0.01 0.01 0.00 3) evaluation oi linearity (uring 2 w o n c, 1*con a and 2/3*con b) evaluation sourcer o f variation 6 n i replicatar 0.008 1.6 within run variation between run 0.009 1.8 betvaen run variation between poolr 0 . 0 0 8 1.6 conrtant unlinearity interaction 0.000 0.0 varying unlinearity ------------------------- ........................................................................... _____------_----------------.................... ------------ evaluation o? njalytical periorfunce 1111111111-111111111-1----11111-1-1good reproducibility within run ................ between run linearity constant (between p o o l r ) . varying (interaction)....... calibrator present ................... nordic calibrator............ roburtnerr in relation to turbidity ............. ................ $ .... accept unaccept commentr: fig. 6.4.2.6.a. example ofprint out from a survey. first page. for further explanations, see text. 294 a c c u ra c y o n 3 le v e ls m e a s u r e d t a r g e t 0 . 11 l a b . : 2 9 5 2 c o m p o n e n t: o ro s o m u c o id c o r r e c a e y , t a r g e t 0 . ::i -0. 9 b ia s fo r c le a r c n r o l (c o n a t a rg e t) 8 .\i 0 . -0 . -:; -0. + 4 , in te r fe r e n c e ? & n t c o n a ) subsampling. finally, a valuation of the different factors is performed, with possibilities for writing comments, which could focus on the problems and advise for trouble-shooting o r improvements. on page 2 of the report (fig. 6.4.2.6.b) two hfference-plots one for the local calibrator and one for the nordic calibrator are presented for the three controls with 90 % confidence intervals. the zero-line indicates no bias and the two sloping lines frame the acceptance area. in the example. the bias is reduced by use of the nordic calibrator, but the results are not perfect, as all controls show values above the target, and the differences are rather constant, indicating 'a constant bias in performance' which e.g. could be a problem with a blanc correction or wrong calibration function. below, the double bias-plot for bias due to calibration (own calibrator) for control a and for bias due to poor correction for turbidity is shown, together with the acceptance area. the 'cross' with 90 % confidence intervals repeats the informations above in the report showing acceptable correction for turbidity, but analytical bias due to the local calibrator. after the three surveys the results were cumulated for each laboratory and the final conclusion was given. fig. 6.4.2.7 illustrates such a conclusion for s-orosomucoid (same laboratory as illustrated in fig. 6.4.2.6, run no 2). the report summarizes the 'turbidity robustness' from the three rounds. further, the results for each clear control in each round are shown in a plot combining bias (with sign) and imprecision, and with acceptance-area (hatched area) according t o the analytical quality specifications for using the common reference interval. only values outside the acceptance-area are shown, and in the example, it is clear that t h e laboratory had serious problems in the first round (a1 with a cv of 20 %, b1 just outside the limits, and c1 with a bias of t30 %), but the situation has improved in the two next rounds (only c2 with a bias of + 7.5 %). communication with the participants some of the evaluations done in this project are a little untraditional and it was therefore of importance to keep the participants informed about the project and the types of evaluations. this information was maintained by papers with a short explanations designated blue folders. an example of a front page and a page with information is shown in fig. 6.4.2.8. 296 the nordic protein project march 1994. laboratory no. 2452 final evaluation in the nordic protein project protein: s-orosomucoid (s-a,-acid glycoprotein) control t turbidity robustness controls: contnol m i 1 s t o n t r c l 4 1 acceptable y e s = 1st round x pndround st 3rdround i i c l e a r 1 i lcleari control t control a i contpol t i iturbid) controls a, b, and c in rounds 1,2, and 3 calibration & imprecision +a% no of control values 4 f outside acceptance l i z control c 1 0 s ~ c o n t r o l a i iclear) three controls in 3 runs only points outside t h e acceptance figure are indicated imprecision $/5 (cv %) -10 -9 -8 -7 -6 5 -4 -3 -2 1 0 1 2 3 4 5 6 7 8 9 10 bias' overall evaluation of the three surveys regarding the use of the common reference intervals for this protein: by use of the nordic calibrator or other calibrator traceable to bcr crm 470 you can use the common reference intervals for t h e native population ................................................................... [ 1 you can possibly use the common reference intervals .... you need improvement of your method ................................... dq hy& [ ] ~ + o u m d , , n . u l kind regards t h e nordic protein project fig. 6.4.2.7. example of a cumulated report to a laboratory after the three surveys. for further explanations, see text. 297 n w 0 0 % $ ' r p r o je c t s o n r e f e r e n c e i n t e r v a l s a f in ni sh p ro je ct o n re fe re nc e in te rv al s f or an ds -i gm ln cl ud in gr ef er en ce in te rv al s f or vi du a1 s) an da ge de pe nd en t i nt er va ls fo r c hi ld re n vi du al s) h as b ee n pe rf or m ed .* a d an is hp ro je ct o n re fe re nc e in te rv al s f or th e ni ne se ru m pr ot ei ns is ta ki ng p la ce p ri m o 19 90 . h er e ap pr ox . 8 00 a du lt s ar e p la nn ed to b e in ve st ig at ed . r es ul ts fr om th e tw o m at er ia ls w ill b e co m pa re d an d th e po si bi lit ie sf or sh ar in gs om e of t he re fe re nc e in te rv al s w ith in g re at er g eo gr ap hi ca l a re as w ill b e in ve st ig at ed . f o r c o m m o n i r e f e r e n c e in t e r v a l s k ir jn h et 01 . r ef er en ce ra ng es fo r ir nr nu nq lo hu tin s i @ . i@ , an d ig m in se ru m in n di rk s n nd c hi ld re n q e d i1 2 to ll ye n rs . (f ob ep u bl iv h ed ) t h e n o r d ic p r o t e in p r o je c t i g o a ls i a n a l y t ic a l q u a l it y s ta n d a r d iz a ti o n c o n tr o l st a r t o f p r o je c t y ou w ill re ce iv e t he fi rs t s et of s am pl es in th e pr oj ec t ( ca li br at or a nd c on tr ol s a , b , c , a nd t , f ou r a m po ul s o f e ac h) in ja nu ar y. t he sa m pl es w ill b e se nd fr oz en o n dr y ic e. pl ea se al so fi nd e nc lo se d th e i ns tr uc tio ns fo r p er fo rm an ce an d tim e s ch ed ul e a ne w fo rm ul a f or re gi st ra tio n o f m et ho ds . in th is fo ld er th e ba ck gr ou nd fo r e st im at io n o f t he g oa ls fo r an al yt ic al q ua li ty is gi ve n. 6.5 results 6.5.1 general evaluations for the general evaluations the main object was to assess the three analytical principles (turbidmetric, nephelometric, and gel methodshn relation to calibration and influence of turbidity of samples. fig. 6.5.1.1. histogrammes o f s-igm showing the results for the clear control a. upper: results when laboratories used their own calibrator. lower: result when the nordic calibrator was used. from blaabjerg et al. (2) with permission. the best overview is given by a histogramme for each control sample, and in fig. 6.5.1.1 are shown the results for the clear control a for the use of local calibrator and for the nordic calibrator by measurements of s-igm. these histogrammes illustrate the general result, that under optimal conditions it is possible to obtain acceptable results from all analytical principles by use of a reliable calibrator. practically all results are within the acceptance limits for the use of common reference intervals. this was expected, but the evaluations of the analytical principles under optimal condtions, here the clear control a, are necessary for the further evaluations of influence of turbidity in patient samples on the robustness of the principles. 299 target value : +l local calibrator n o 05 i " 1 5 2 0 2 5 q r l s-igm turbo control icon ti target value ;i nordic calibrator fig. 6.5.1.2. histogrammes of s-igm showing the results for the turbid control t. upper: results when laboratories used their own calibrator. lower: result when the nordic calibrator was used. from blaabjerg et al. (2) with permission. nordic calibrator s igm turbid contnol (con t i target value nephelometric 0 turbldlmetrlc +gel techniwes targel value fig. 6.5.1.3. histogrammes of s-igm showing the results for the turbid control t. same data as in fig. 6.5.1.2, but separated according to nephelometric and turbidimetric analytical principles. from blaabjerg et al. (2) with permission. 300 when the analytical principles are evaluated under 'ideal' conditions it is easier to make conclusions from the result obtained with the turbid control t, as the only additional factor is the turbidity of the sample. thus, fig. 6.5.1.2 illustrates the same informations as in fig. 6.5.1.1, but now the results obtained with local calibrator are complicated by the combined effect of calibrator and turbidity, whereas, the results with the nordic calibrator demonstrate a nearly pure effect of turbidity, as we know that the the calibrator alone would give acceptable results. the combined use of well defined control materials makes the interpretation clear. table 6.5.1.1 quality of calibration and influence of turbidity turbtdimetry acceptable laboratories2 in percentage of all using the same analytical principle protein prealbumin albumin orosomucoid al-antitrypsin haptoglobin transferrin iga igg igm ~ ~ calibrator local nordic 14 % 86 % 29 % 86 % 25 % 69 % 44 % 100 % 8 % 5a % 6 % a7 % 60 % 97 % 60 % 86 % 60 5% 94 9% ~~ turbidity1 number of laboratories 43 % 50 % 57 % 56 % 100 % 81 % 91 % 97 % 69 % 7 12 14 16 27 31 35 35 35 ~ ~~ ~ 1.: measurements directly on turbid samples. 2.: acceptance criteria according to fig. 6.2.2. the effects of turbidity on the different analytical principles are further illustrated in fig. 6.5.1.3, which shows that the gel and turbidimetric methods are rather insensitive to the turbidity. the nephelometric principles are illustrated for both kinetic and endpoint methods, and both distributions are broadened towards higher values indicating the problem. 30 1 s-transferrin concentrations in plasma are higher than s-igm concentrations and in most analytical principles they are analysed in higher dilutions. accordingly the effect of turbidity was expected t o be less distinct, but as seen from figures 6.4.2.1 to 3, there are still effects for nephelometric endpoint principles. table 6.5.1.2 quality of calibration and influence of turbidity nephelometry acceptable laboratories2 in percentage of all using the same analytical principle ~ ~~ protein prealbumin albumin orosomucoid al-antitrypsin haptoglobin transferrin iga igg igm ~ ~~ calibrator local nordic 26 % 70 % 10 % 50 % 56 % 69 % 10 % 52 % 58 % 100 % 6 % 71 % 44 % 100 % 28 % 96 % 44 % 96 % turbidity1 50 % 80 % 19 % 62 % 92 % 47 % 52 % 96 o/o 4 % number of laboratories 10 10 16 21 24 1 7 25 25 25 ~ ~~~ ~ 1 .: measurements directly on turbid samples. 2.: acceptance criteria according to fig. 6.2.2. in table 6.5.1.1 t o 3 the data are summarized for all nine plasma proteins according to the three main analytical principles, turbidimetric, nephelometric and gel methods. it is seen from the tables as expected that a common calibrator can improve the trueness of all analytical principles t o a high degree. further, the problems with turbid samples are demonstrated for s-igm using the nephelometric principles. two proteins, however, demonstrate special problems: s-albumin has a narrow reference interval and the quality specifications are accordingly demanding, which is reflected in a low percentage of acceptable 3 02 laboratories for all methods. further, many of the dye-binding methods have problems with calibration functions as the calibration curve is fitted by a straight line, resulting in bias for all concentration values, except from one point. table 6.5.1.3 quality of calibration and influence of turbidity gel methods acceptable laboratories2 in percentage of all using the same analytical principle protein calibrator turbidity1 local nordic number of laboratories prealbumin albumin orosomucoid al-antitrypsin haptoglobin transferrin iga i& igm 25 % 0 % 25 % 27 % 43 % 50 % 50 % 100 % 100 % 100 % 0 % 50 o/o 45 % 100 5% 100 % 100 % 100 % 100 % 100 % 100 5% 63 % 45 % 100 % 50 % 100 % 100 % 100 % 4 1 8 11 7 2 2 2 1 1.: measurements directly on turbid samples 2.: acceptance criteria according to fig. 6.2.2. s-al-antitrypsin disclose another type of problem which is related to the protein structure in the calibrator, as evaluated in chapter 5.5, but the storing conditions in the different laboratories may not have been optimal, so a random (i.e. not possible to investigate now) effect of minor denaturation could occur. this effect, however, could also origin from difficulties with measuring the protein by turbilmetric and nephelometric principles. 303 6.5.2 individual evaluations evaluation of individual laboratories is by definition individual, and it is more difficult to give an overview of these, as there are only few general aspects which are not reflected in the general evaluations. the most relevant summing up is therefore to count how many laboratories fulfilled the analytical quality specifications over the three surveys as evaluated by the revised quality specifications (fig. 6.2.2) and evaluated according t o the combined criteria for analytical bias and imprecision as illustrated in fig. 6.4.2.7. two types of evaluations are performed. the first is the average of control results inside the figure of combined criteria in percentage, and the second is the percentage of laboratories with 66 % or more acceptable values. table 6.5.2 evaluation of analytical quality according to the quality specifications for sharing common reference intervals by two criteria. 1. average of the percentage of control results with acceptable values 2. percentage of laboratories with 66 % or more acceptable values protein prealbumin albumin orosomucoid al-antitrypsin haptoglobin transferrin iga igg igm criteria 1 50 % 33 % 66 % 26 % 97 % 63 % 95 % 80 % 90 % criteria 2 number of laboratories 45 % 16 % 73 % 10 % 97 % 51 % 98 % 85 % 95 % 20 51 41 48 61 57 66 66 66 the percentage of laboratories which can share common reference intervals is close to 100 % for s-haptoglobin, s-iga, and s-igm, mainly due t o rather loose criteria for sharing common reference intervals for these three proteins (cf. section 6.2), whereas, 304 the percentages for s-igg and s-orosomucoid are reasonable high. s-prealbumin and s-transferrin are close to 50 %, but the percentages for the two proteins s-albumin and s-al-antitrypsin are so low, that it may be questionable whether it is possible t o use common reference intervals. the first is due t o the very demanding criteria, and the other may be related to instability of the calibrator or controls as discussed above. 305 6.6 conclusions the two main problems in protein measurements are calibration and turbid samples. the calibration problems can be solved for all analytical principles by use of the nordic calibrator or some other reliable calibrator with concentration values traceable to the reference preparation for plasma proteins, bcr 470, allowing the majority of laboratories to use the common reference intervals (with some reservations for s-albumin and s-al-antitrypsin). nephelometric analytical principle have problems with turbid samples, but this can be solved by delipidation of turbid samples, preferable by high speed centrifugation, e.g. with a bench-top ultracentrifuge. acknowledgements we want to thank all the people and institutions who have supported the project and all the laboratories which have participated. we are especially greatful to the nordic committee for quality control, which initiated the project, to nordkem, lab quality and the danish society of clinical chemistry for stimulation and economical support. further, we want to thank torsten aronsson, per gidfors and jens rahbek nprrgaard for the very important data-handling of all the results, as well a s spren blirup-jensen and per just svendsen for transfer of concentration values to the calibrator. a special thank t o karin jorgensen and ane richter for skilful laboratory work and handling of data. references 1. blaabjerg 0, blom m, gry h , hyltoft petersen p, uldall a. appropriate sera for assays and calibration of specific proteins scand j clin lnb inuest 1992;52 suppl. 212:13-5. blaabjerg 0, irjala k, hyltoft petersen p, 1ct.n a, bj0ro k. effect of calibrator and specimen turbidity. moodz 1992;2:109-11. hyltoft petersen p, blaabjerg 0, irjala k, i c h a, bjpro k. a model for quality achievement the nordkem protein project. scand j clin lab inuest 1992;52 suppl. 212:lo-2. 2. 3. 306 upsala j med sci 100: 137-142, 1995 the significance of an across-shift decrease in vital capacity-a re-analysis of a study on subjects exposed to diesel exhaust monica dahlqvrst departmen/ ojeniironinenta1 terhnologi and work scienre, royal in.mtute of lechnolog\, sioc kholnz skeden abstract occupational exposure to diesel exhaust may develop acute as well as chronic lung function impairment. in this study, data from an earlier study on a group of subjects working at tunnel construction site were analyzed. the aini of h e analysis was lo exanline the significance of an across-shift decrease in vital capacity with concern to other lung physiological measuremcnts. there were no statistically significant differences, either in the average agc, time of employment, vital capacity before a working shift after two days of no exposure, or the distributions of smoking habits and respiratory symptoms, bctween thc eight workers who had an across-shift decrease in vital capacity and the five workers who had not. subjects with an across-shift decrease in vital capacity had a significantly greater across-shift decrease in residual volume and total lung capacity than subjects without an across-shift dccreasc in vital capacity. thc pathophysiological mechanisms for this across-shift decrease in rcsidual volume is not fully understood. however, an altcrcd defence mechanism in the lung may play a role for a prolonged relenlion time for the particlcs in the inhaled diesel exhaust, resulting in the across-shift decrease in residual volume. the results thus suggest that measurements of across-shift vital capacity is of importance in identifying susceptible subjects with occupational exposure to diesel exhaust. niroduction measurements of lung volumes are powerful measures to examine some features of lung function akeclion. in a number of studies we have observcd acute, temporary effects on lung function in workers exposed to imtating air contaminants (3,6, 14. 15). these temporary effects were correlated with the conccntration of the air contaminants. the measurement of acute elrects from irritants on the lung function is of value as a complement to exposure measurement% i t is also of great irileresl to study the medical importance of acute temporary effects with concern to other physiological lung function measuremcnts. this is not the least important matter since some of the lung function variables are highly intcrrclatcd (2, 11) and thal am acute temporary decrease in lung function has been reported to predict a more accelerated decline in lung function over time (4,5). if an acute temporary effect on lung function cannot be associated with the exposure, i t is possible that subjects who display a decrease are susceptible. by measuring lung function during a working shift or week, such subjecb may be identified and measures can be taken to decrease or stop the exposure. diesel exhaust contains a number of substances that will efrecl the health of exposed human beings if the substances are present in concentrations high enough. the respiratory effects of exposurc to diesel exhaust include irritative effects on mucous membranes and chronic deterioration in lung function (16). although the exhaust has usually been diluted with a factor about 50 timcs or more, acute, temporary lung 137 function effects havc been shown (13). the particles in the diesel exhaust have been reported to induce this acute, temporary decrease in lung function (12, 14). in order to evaluate possible relationships between an across-shift decrease in vital capacity and other lung physiological variables, data from an earlier published study were rc-analyzed (14). material and ?vethods fifteen workers subjects, working at a tunnel construction site, took part in the initial examination with the aim to study hazardous effects on lung f i c t i o n caused by exposure to particles in diesel exhaust (14). they were examined before b working shift, after two days of no exposure, and after two working shifts of exposure to diesel exhaust, with and without exposure to particles, respectively. the particles in the diesel exhaust were removed from the inhaled air by using a particulate respirator or a half mask during one of the working shifts. official leakage value was lower than 0.1%. none of the filters retains gaseous substances to any appreciable extent. the examinations after exposure after a day working shift were carried out at the same time of the day. between 23 and 59 years, with an average age of 39 years. there were 1 nevcr-smoker, 4 ex-smokers and 8 current smokers. the means (standard error of the mean) for pack-years, py, &ram tobacco consumed per day)*(snioking years)/20 were 8 (2), and 11 (4), for the exand the current smokers, respectively. standardized questionnaire, translated kom the mrcc questionnaire (8). technique (2) and provided values for vital capacity and closing volume. total lung capacity was determined by planimetry, according to integration of the nitrogen wash-out curve as described by buist and ross (1). residual volume was calculated by subtracting the vital capacity from the total lung capacity. the volumes were corrected to conditions of body temperature and pressure, saturated with water (btps). standard statislical methods were employed, in conjunction with correlation analyses. the values obtained hefore a working shift were standardized with concern to age, height and smoking habits (7). the results of the previously published study showed an improvement in lung function when the particles from the diesel exhaust were removed from the inhaled air (14). these values were therefore used as tmexposed values in order to minimize the effects of the diurnal variation. the values obtained after a working shift when the workers were exposed to particles were used as exposed vuhes. across-shift change in lung function in each individual was calculated according to the formula: single breath nitrogen wash-out recordings were available in 13 subjects. all subjects were men chronic respiratory symptoms (cough, exertion dyspnea, wheeze) were assessed by means of a recordings on single breath nitrogen wash-out were carried out according to previously described (exposed unexposed) 0.5 *(exposed + unexposed) change = 100 * (%i. since the aim of this study was to evaluate the significance of an across-shift decrease in vital capacity, the across-shift change in vital capacity was dichotomized. a change (calculated according to the aforementioned expression) of < 0%, signifying a decrease in vital capacity, were assigned 1, le., indicating an effect. ln the same manner, a calcuiated change corresponding to l 0% in vital capacity, i.e., no decrease, were assigned 0, i.e., indicating no effect. stated p-values involved two-tailed analysis; differences were considered to be statistically significant at p 5 0.05. the workers, were comparable between the working shifts with and without exposure to particles (14). daily average concentrations of carbon monoxide and nitric oxides, measured in the breathing zone of 138 table 1. daily average concentrations of gaseous and particulate air pollutants (mg/m3) in the breathing zone for workers exposed to diesel exhaust, with and without an across-shift change in vital capacity. i with, n=8 without, n=5 carbon monoxide 10.7 (1.4)l 10.2 (1.4) nitrogen oxide 4.8 (0.5) 4.9 (0.2) nitrogen dioxide 1.9 (0.2) 1.9 (0.2) total dust 2.2 (0.3) 2.6 (0.5) respirable dust 1.3 (0.1) 1.3 (0.1) * mean (standard error of the mean) results daily average values for the concenlrations of air pollutants were about the same magnitude for the eight subjects who had an across-shift decrease in vital capacity and the five subjects who had not, table 1. age, time of employment, standardized vital capacity before a working shift after two days of no exposure did not differ significantly between the workers who displayed an across-shift decrease in vital capacity and those who did not. the distributions of chronic respiratory symptoms and smoking habits were also similar in subjects with and without an across-shift decrease in vital capacity, table 2. table 2. age, time of employment, vital capacity, percentages of current smokers and subjects with chronic respiratory symptoms for diesel exposed workers, with and without an across-shift decrease in vital capacily. time of employment (years) current smokers ’ mean (standard deviation) vital capacity, before a working shift after two days of no exposure. standardized for age, height and smoking habils percentage (number of subjects) subjects with an across-shift decrease in vital capacity had on the average a significantly greater across the across-shift change in vital capacity was not significantly correlated with the daily average week decrease in residual volume and total lung capacity, table 3. concentrations of the nieasurcd air pollutants (p0.6). 139 % mean (standard error of the mean) p<o0.o5 in comparison with subjects without an across-shill decrease in vital capacity p<o.ooi 10 comparison with subjects without an across-shift decrease 10 vital capacity with, n=8 without, n=5 discussion subjects with an across-shift decrease in vital capacity had a significantly greater across-shift decrease in residual volume and total lung capacity, compared to subjects without an across-shift decrease in vital capacity, table 3. the average age, time of employment, vital capacity before a working shift after two days of no exposure (standardized for age, height, and smoking habits) (7), and the distributions of chronic respiratory sympmms and smoking habits were similar between subjects with and without an across-shift dccrease in vital capacity, table 2. although the examined background variables may have a limited value, it is not likely that, for instance chronic respiratory diseases should result in an across-shift decrease in vital capacity. there is still no generally applicable indicator of diesel exhaust exposure available (16). the exposure judgement of diesel exhaust traditionally has been founded on the concentrations of gaseous components, i.e., carbon monoxide and nitrogen dioxide or by measurements of total and respirable dust. particles in the diesel exhaust have bcen demonstrated to induce acute temporary lung function impairment (12, 14). the across-shift change in vital capacity could thus have k n directly related to the concentration of particulate matter, measured as total or rcspirable dust. the associations were weak between the across shift decrease in vital capacity and the daily time-average concentrations of total and respirable dust, however. daily time-average concentrations of total and respirable dust were also almost identical between subjects with and without an across-shift decrease in vital capacity, table 1. despite the limitation of the measured air pollutants in order m describe the level of diesel exhaust exposure it seems not probable that diffcrcnces in exposure should have inlluenced the finding that subjects with an across-shift decrease in vital capacity also had a greater across-shift dccrcase in residual volume and total lung capacity. therefore, it seems likely to assume that the across-shift decrease in vital capacity indicates a higher susceptibility, independently of the exposure level. the data available to us are not sufficient to explain the pathophysiological mechanisms behind the across-shift decrease in residual volume and total lung capacity in subjects with a dccrcasc in vital capacity. a decreased residual volume may, besides a decreased vital capacity and total lung capacity, be found in subjects with a restrictive lung function impairment. occupational exposure to diesel exhaust results mainly in decreased lung volumes, suggesting a restrictive impairment. this sccms to be true for acute as well as chronic effccts on thc lungs (16). short-term exposure to diesel exhaust may also result in a decreased number of alveolar macrophages and a rcduction in the phagocylic activity in humans (9). the altered defence mechanism in the lung may result in a slower clearance and thus a prolongcd tissue interaction with the particles. a prolonged time o f retention of compounds in the lungs has been suggested to pose a health hazard for humans exposed to organic pollutants in the environment (10). the across-shift residual volume total lung capacity closing volume 140 -19.3 (6.5)1.2 7.9 (7.3) -4.9 (0.8)3 4.3 (1.3) -1.2 (8.2) 15.1 (11.0) shift decrease in residual volume and total lung capacity, in subjects with an across-shift dccredse in vital capacity may thus he the result of a prolonged particle deposition in the alveoli. capacity is of importance in identirying susceptible subjects with occupational exposure to diesel exhaust. howcver, the result may be interpreted with caution since the number of studied subjects is pretty small. in conclusion, the findings in the present analysis suggest that measurements of across-shift vital 1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 references buist, a. s . & ross, b.: predicted values for closing volumes using a modified single breath nitrogen test. am rev respir dis 107: 744-752, 1973. dahlqvist, m., alexandersson, r., nielsen, j. & hedenstiema, g.: single and multiple breath nitrogen wash-out closing volume and volume of trapped gas for detection of early airway obstruction. clin physiol9: 389-398, 1989. dahlqvist, m., johard, u., alexandersson, r., nergstrom, b., ekholm, u. milosevich, b., tornling, g. & ulfvarson, u.: lung function and precipitating antibodies in low exposed wood trimmers in sweden. am j ind med 21: 549-559, 1992. dahlqvist, m. & ulfvarson, u.: acute effects on forced expiratory volume in one second and longitudinal change in pulmonary function among wood trimmers. am j ind med 25: 551-558, 1994. dahlqvist, m., tornling, g., plato, n. & ulfvarson, u.: across-week effects on forced expiratory vital capacity is correlated to longitudinal change in pulmonary function. reanalysis of studies on isocyanate-exposed car painters. occup environ mcd (accepted for publication), 1994. dahlqvist, m., ulfvarson, u., bergstrom, b. & ekholm, u.: temporary effects on lung function in welders engaged in cored wire welding. occup hyg 1 : 119-126, 1994. hedenstrom, h., malmberg, p. & fridriksson, h. v.: reference values for lung function tests in men: regression equations with smoking variables. upsala j med sci 91: 299-3 10, 1986. medical research council's committee on the aetiology of chronic bronchitis.: definition and classification of chronic bronchitis for clinical and epidemiological purposes. lancet i: 775-779, 1965. rudell, b., sandstrom, t., stjernberg, n. & kolmodin-hedman, b . : controllcd diesel exhaust exposure in an exposure chamber: pulmonary effects investigated with bronchoalveolar lavage. j aerosol sci 21 (suppl 1): 411-414,1990. sun, j. d., wolff, r. k . , kanapilly, g. m. & mcclellan, r. 0.: lung retention and metabolic fate of inhaled bennzo(a)pyrene associated with diesel exhaust particles. toxicol appl pharmacol73: 48 59, 1984. teculescu, d. b., pham, q. t. & hannhart, b.: tests of small airway dysfunction: their correlation with the "conventional" lung function tests. eur j respir dis 69: 175-187, 1986. ulfvarson, u. & alexandersson, r.: reduction in adverse effect on pulmonary function after exposure to filtered diesel exhaust gases. am j ind med 17: 341-347, 1990. ulfvarson, u., alexandersson, r., aringer, l., svcnsson, e., hedenstiema, g., hogstedl, c., holmberg, r , roscn, g. & sorsa, m.: effects of exposure to vehicle exhdust on health. scand j work environ health 13: 505-512, 1987. ulfvarson, u., alexandcrsson, r., dahlqvist, m., ekholm, u. & rergstrom, b . : pulmonary function in workers exmsed lo diesel cxhausls: the effect of control measures. am j ind med. 19. 283-289, 3991. ulfvarson. u.. alexandersson. r.. dahlavist. m.. ekholm. u.. berestrbm. b. & scullman. j.: temporary health effects from'exposue lo water-borne paints. scandv j work environ health, 18, ulfvarson, u. & dahlqvist, m.: pulmonary function in workers exposed to diesel cxhausl. in p. cheremisinoff (ed) encyclopedia of environmental conlrol technology. gulf publishing, nj, usa. 376-387, 1992. pp 645-671, 1994. address for reprints: monica dahlqvist department of environmental technology arid work science koyal institule of technology s-100 44 stuckholm 141 ujms 110 (3) bra upsala j med sci 110 (3): 267–273, 2005 a low carbohydrate diet in type 1 diabetes: clinical experience – a brief report jørgen vesti nielsen, eva jönsson, anette ivarsson department of medicine, blekingesjukhuset, karlshamn, sweden abstract due to failure to achieve control twenty-two patients with type 1 diabetes with symptomatic fluctuating blood glucose started on a diet limited to 70-90 g carbohydrates per day and were taught to match the insulin doses accordingly. the caloric requirements were covered by an increased intake of protein and fat. the purpose was to reduce the blood glucose fluctuations, the rate of hypoglycaemia and to improve hba1c. after three and 12 months the rate of hypoglycaemia was significantly lowered from 2.9 + 2.0 to 0.2 + 0.3 and 0.5 + 0.5 episodes per week respectively. the hba1c level was significantly lowered from 7.5 + 0.9 % to 6.4 + 0.7 % after three months and was still after 12 months 6.4 + 0.8 %. the meal insulin requirements were reduced from 21.1 + 6.7 i.u./day to 12.7 + 3.5 i.u./day and 12.4 + 2.6 i.u./day after three and 12 months respectively. furthermore the triglyceride level was significantly lowered whereas the levels for total cholesterol and hdl-cholesterol were unchanged. conclusion: the present report shows that a 70-90 g carbohydrate diet is a feasible long-term alternative in the treatment of type 1 diabetes and leads to improved glycaemic control. introduction the diabetes control and complication trial (dcct), in which conventional versus intensive insulin treatment was studied, showed that a reduction of the risk of microvascular complications in type 1 diabetes was directly linked to a lowered hba1c level (1). the incidence of severe hypoglycaemia including coma and seizures, however, was threefold higher in the intensively treated patients, 61.2 versus 18.7 episodes per 100 patient-years (2). the dietary advice in the dcct includ267 received 25 april 2005 accepted 7 june 2005 ed a carbohydrate proportion of 55 % of the calories. in a 2000 kcal diet this amount corresponds to about 275 g carbohydrates, mostly consumed as starches from potatoes, bread and pasta, that in the gut swiftly are broken down to glucose. this quantity of carbohydrates therefore corresponds to about the same amount of glucose within the body and behaves like ingested glucose. patients with type 1 diabetes commonly take fixed pre-meal doses of insulin. some patients change the dose according to the pre-meal glucose level, but normally the dose is taken without regard to the carbohydrate content of the following meal. however, by way of education, patients can learn to estimate the carbohydrate content of a meal and to match the insulin accordingly. in this way patients with type 1 diabetes reduced mean hba1c from 9.5 to 8.5 %. but the high rate of severe hypoglycaemia still persisted, 20 episodes per 100 patient-years (3). the lesson from the dcct is that normal glycaemic control is impossible unless the risk of severe hypoglycaemia can be reduced. a mismatch between insulin and glucose causes unpredictable glucose swings. with large insulin doses this may lead to severe hypoglycaemia. the mismatching is an inherent problem, since first, the assessment of carbohydrates in a meal is hampered by a large error rate (4), and second, the absorption of insulin may vary by up to 30 % (5). the two major determinants of the blood glucose, therefore, cannot be determined with any accuracy. it thus becomes very problematical to match carbohydrates with insulin. large variations in the input -carbohydrates and insulin -may cause large variations in the output -the glucose levels -manifested as hypoand hyperglycaemia. according to basic calculus a reduction in the size of the input reduces the degree of variation in the output. consequently, a reduction of both carbohydrates and given insulin ought to lead to a reduced degree of fluctuations and thus allow the patient to reduce the mean glucose level and the hba1c safely. for motivated patients this principle works well. a carbohydrate content of less than 40 g per day with accurately matched insulin doses seems to normalise the blood glucose levels and diminish the risk of hypoglycaemia in patients with type 1 diabetes who adhere to the program; the plasma lipids are normalised also(6-8). the objection to the regimen is that a reduction of carbohydrates to less than 40 g per day probably is unrealistic in the general diabetes population. such a radical reduction might limit the number of patients willing to try the method. a reduction to 70-90 g per day, however, seems a feasible long-term choice (9). since a reduced amount of carbohydrates with appropriate insulin doses as well as the ability to count carbohydrates are factors important in regulating the blood glucose, we have combined these two principles in the treatment of patients with poor glycaemic control, when treatment according to the guidelines has failed to stabilise the blood glucose. the purpose is to reduce the glucose fluctuation and hba1c. we have done this in the form of educational programs attended by 6-8 patients. the patients had all failed to achieve an – in their own opinion – satisfying glucose 268 control. all such patients have at our clinic for a couple of years received information on the theoretical background for the herein described treatment model. it is then up to the patients themselves to decide whether they wish to make any changes. all the patients in the present report have actively sought the presented treatment model. a clinical chart review has been performed for the first 24 patients attending the program. it is a quality control, and its purpose is to evaluate to what degree initial positive changes are retained one year later even without close follow-up. an approach using a carbohydrate quantity of 70-90 g per day has to our knowledge not been reported before. the purpose of the present report is to describe the method and the results after one year. 269 table 1. changes in the rate of hypoglycaemia, in hba1c, insulin utilization, cholesterol, hdl-cholesterol and triglycerides in 22 patients with type 1 diabetes before, 3 and 12 months after a change to a diet restricted to 70-90 g of carbohydrates per day. start 3 mo p* 12 mo p* hypoglycaemic episodes/week: § 2.9+ 2.0 0.2+ 0.3 <0.001 0.5+ 0.4 0.004 hba1c (%) 7.5 + 0.9 6.4 +0.7 <0.001 6.4 + 0.8 <0.001 meal-insulin(i.u./day) 21.1+ 6.7 12.7+ 3.5 <0.001 12.4+ 2.6 <0.001 total insulin (i.u./kg/24 hours) 0.5 + 0.1 0.4 + 0.1 <0.001 0.4+ 0.1 <0.001 tot-chol (mmol/l) 5.5 + 1.0 5.7 + 0.8 0.3 5.6 + 0.7 0.3 hdl-chol.(mmol/l) 1.4 + 0.2 1.5+ 0.4 0.6 1.4 + 0.3 0.8 triglycerides(mmol/l) 0.8 + 0.2 0.7 + 0.2 <0.02 0.6+ 0.3 <0.02 * p is for the difference within the group from base line. § n=15. hba1c < 5.6 % in nondiabetic persons. = material and methods the patients were outpatients with type 1 diabetes and episodes of hypoand hyperglycaemia that had been treated at our clinic. the first 24 patients, 17 women and 7 men, who attended the program, constitute the present group. the mean duration of diabetes was 18+13 years and the mean age was 51 + 10 years. seven patients were over-weight and had bmi > 27 kg/m2 (range 27-38). eleven patients administered insulin with an insulin pump. another 13 patients used long acting insulin glargin (lantus) twice or once a day; for meal-insulin they used insulin aspart in a pen device (novorapid) that enables delivery of half units. all patients were requested to record the occurrence of hypoand hyperglycaemia as well as other symptoms of poor diabetes control before they started on the new regime as well as 3 months later. about 12 months after the start the patients again in a letter were asked to record the occurrence of symptomatic hypoglycaemia, i.e. not severe but manageable by the patients themselves without help from others. the program began with a 6-hour-meeting followed by, over the next two months, five follow-up meetings lasting approximately two hours. the main issues on the first day were the effect on the blood glucose by the different constituents of the food, the timing of insulin administered and the timing of blood glucose measurements. the patients received a flow sheet wherein they entered the blood glucose levels, the time of each meal and the amount of carbohydrates. further, the time and amount of insulin units taken were entered together with any occurrence of hyperand hypo-glycaemia. the number of glucose tablets needed to correct low blood glucose levels were recorded as well as the extra insulin units needed to correct a too high pre-meal and bedtime glucose level. the regimen: a carbohydrate restricted diet (70-90 g per day) that excluded potatoes, rice, pasta, bread and cereals, but included hard bread and vegetables. intensive glucose monitoring, > 4 times per day, was required; recording of the time of glucose levels, meals, insulin dosage, exercise and correction of too high or too low pre-meal and bed-time glucose levels with insulin or glucose tablets were also required. the target for pre-meal blood glucose was 5.6 mmol/l (plasma glucose 6.0 mmol/l). the patients were instructed to eat three meals a day and abstain from any eating between the regular meals; the interval between meals should be at least four hours – the duration of one dose of aspart. to assist them the patients were given samples of menus and recipes showing the carbohydrate and protein content. the initial distribution of carbohydrate, protein and fat was 20%, 30% and 50 % respectively. sugar or other fast acting carbohydrates were not permitted. during this 2 months period the regimen for each patient was individually tailored. special attention was paid to the occurrence of gastroparesis. even though this condition might be asymptomatic the effects on glucose control usually are obvious very soon. in such cases the autonomous nervous system was examined and the patient prescribed domperidone (motilium) and if needed switched to insulin human (actrapid). after finishing the program the patients were able to handle their own treatment 270 271 without help. only routine visits to the diabetes nurse every 3-4 months were continued or according to the individual patient’s preferences. hba1c and insulin utilization were recorded at start and after three months. about 12 months later they were once more recorded from the chart. since hba1c vary we have calculated average hba1c from the charts for the previous year before start for each individual. hba1c <5.6 % in non-diabetic persons. (chromatography hplc monos column) total-cholesterol, hdl-cholesterol (hdl) and triglycerides (tg) were measured at start and after 3 months. it was then measured again about 12 months later. the results are presented as means with standard deviation. paired t-test is used for comparisons. results two patients left the program after approximately 1 month for personal reasons. blood glucose: the effect of the diet on the glucose fluctuations is illustrated in figure 1. the size of the fluctuations before the change clearly forbids any lowering of the mean glucose levels. for this patient average hba1c the year prior to start was 7.5 %. one year later it was 4.7 %. blood glucose 0 5 10 15 20 25 30 1 61 measurements mmol/l figure 1. blood glucose values in a patient with type 1 diabetes before and a few days after a change to a carbohydrate restricted diet (70-90 g daily) with adapted insulin doses. the measurements cover 12 days with 58 measurements before the change, and 10 days with 70 measurements after the change. the mean for the 58 measurements before the change was 12.9 ± 6 mmol/l. the mean for the 70 measurements after the change was 5.9 ± 2 mmol/l. figure 1. blood glucose values in a patient with type 1 diabetes before and a few days after a change to a carbohydrate restricted diet (70-90 g daily) with adapted insulin doses. the measurements cover 12 days with 58 measurements before the change, and 10 days with 70 measurements after the change. the mean for the 58 measurements before the change was 12.9 ± 6 mmol/l. the mean for the 70 measurements after the change was 5.9 ± 2 mmol/l. hypoglycaemia: nine patients had failed to record the occurrence of hypoglycaemia before start. the following figures are calculated from the records of 15 patients. the rate of symptomatic hypoglycaemia among these was reduced by 94 % after 3 months on the new regimen, and at twelve months by 82 % from base line. (see table 1) hba1c and insulin: table 1 shows that the effect on hba1c and insulin administration was stable from 3 to 12 months in the group. lipids: there was no change except for a significant 16 % lowering of the triglycerides. two patients lost weight due to other illnesses. otherwise, the bodyweight for the normal-weight patients remained stable, while all the overweight patients lost weight. six patients were diagnosed with diabetic gastroparesis and were prescribed domperidon (motilium). discussion by lowering the carbohydrate amount and corresponding insulin doses the blood glucose fluctuations diminished and a better hba1c was achieved. the rate of hypoglycaemic episodes was reduced without any deterioration in the lipids. it is likely that the risk of severe hypoglycaemia has diminished also. it is remarkable that the effect is retained without close follow-up. in the dcct the patients visited the clinic in person once a month and were in contact by phone and fax once a week to get directions from the diabetes team [1]. this in order to maintain a tight control of hba1c. the group presented here achieved and maintained an hba1c close to the one accomplished in the dcct without close contact to the diabetes team. this suggests that the treatment method presented here is a more reliable tool for the patients. this might be due to the improved predictability of the blood glucose as illustrated in the reduced rate of hypoglycaemic episodes. most patients experience more freedom since they do not have to eat at a fixed time. with a correct dose of basal insulin a meal can be postponed or even cancelled without consequences for the glucose levels. we did not study compliance to the diet, but since both hba1c and insulin requirements stayed low, the findings for the whole group further suggest that the selected initial amount of carbohydrates is feasible in the long term in patients with type 1 diabetes. it was still possible for individual patients at their own discretion to pursue a stricter carbohydrate reduction in order to achieve an even better control, and two persons did so. the high carbohydrate dietary advice in type 1 diabetes is based on avoidance of protein and fat in the food, especially saturated fat. the importance of this may however, with respect to the present findings, be reconsidered (10-12). in summary: the approach to therapy reported here is feasible in type 1 diabetes for motivated patients. it improves hba1c and appears to increase safety by lowering the rate of symptomatic hypoglycaemia, and the effect was retained one year later. 272 acknowledgements we are grateful to peter naeser, md, phd, for reading the manuscript and for valuable advice. we thank kamma willumsen, dietician, for help. references 1. the diabetes control and complications trial research group: (1993). the effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. n eng j med 329:977-986 2. the diabetes control and complications trial research group (1997). hypoglycaemia in the diabetes control and complications trial. diabetes 46(2):271-86 3. dafne study group (2002). training in flexible, intensive insulin management to enable dietary freedom in people with type 1 diabetes: dose adjustment for normal eating (dafne) randomised controlled trial. bmj 325:746-749 4. waldron s (1996). controversies in the dietary management of diabetes in childhood and adolescence. br j hosp med 56(8):450-454 5. heinemann l (2002). variability of insulin absorption and insulin action. diabetes technol ther 4(5):673-82 6. bernstein rk (1980). virtually continous euglycemia for 5 yr in a labile juvenile -onset diabetic patient under noninvasive closed-loop control. diabetes care 3(1):140-143 7. bernstein rk (1997). dr. bernstein’s diabetes solution; first edition; little brown and company , new york 8. o’neill df, westman ec, bernstein rk (2003). the effects of a low-carbohydrate regimen on glycemic control and serum lipids in diabetes mellitus. metab synd rel disord 1(4):291-298 9. nielsen jv, joensson e, nilsson ak (2005). lasting improvement of hyperglycaemia and bodyweight: low-carbohydrate diet in type 2 diabetes. – a brief report. ups j med sci 110:69-74 10. manninen ah (2004). high-protein weight loss diets and purported adverse effects: where is the evidence? sports nutrition review journal (1):45-51 11. hooper l, summerbell cd, higgins jtp, et al.(2001) reduced or modified dietary fat for prevention of preventing cardiovascular disease. cochrane database system rev 3:cd002137 12. ravnskov u (1998). the questionable role of saturated and polyunsaturated fatty acids in cardiovascular disease. j clin epidemiol 51(6):443-460. corresponding author: jørgen vesti nielsen consultant physician dept. medicine blekingesjukhuset karlshamn länsmansvägen 1, 374 80 karlshamn, sweden telephone: +46 454 731000 e-mail: jorgen.vesti-nielsen@ltblekinge.se 273 upsala j med sci 105: 67-72,2000 measurement accuracy of aluminium content in bone hans-olov hellstrom,' ulf lindh2a and bengt mjoberg' 'department of orthopedics, uppsala university hospital, s-75185 uppsala, 2department of biomedical radiation sciences, rudbeck h b o r a t o r y , s-75i85 uppsala, 'centre for metal biology in uppsala, rudbeck kbosatoty, s-75185 uppsala, sweden abstract the aluminium content in bone has been related in several ways: to the weight of wet bone, to the weight of dry bone, to the weight of bone-ash and to the calcium content of bone. we determined the accuracy and precision of measurement (using an inductively coupled mass spectrometer) in 30 bone samples taken from one patient. the coefficient of variation of the aluminiudweight-quotient was 12.4 per cent for wet bone, 4.7 for dry bone and 5.0 for bone ash; and the coefficient of variation of the aluminiudcalcium-weight-quotient was 7.5 per cent. thus, the aluminium content in bone seems to be best related to the weight of dry bone. introduction aluminium inhibits bone mineralization ( 14, 24) and the epidemic of fragility fractures has been suggested to be caused by a chronic low-grade aluminium intoxication (10, 1 1 ) . however, in various studies the aluminium content in bone has been related in different ways: to the weight of wet bone (5, 6 , 9 , 11, 13, 18, 19,21,22), to the weight of dry bone (1-4, 7, 12, 16, 17), to the weight of bone ash (8, 15) and to the calcium content in the bone (10, 23). we therefore determined the means and the coefficients of variation (i.e. sd/mean) of the aluminiudweight-quotient for wet bone, dry bone and bone ash, respectively, and of the aluminiudcalcium-weight-quotient in bone samples from one patient to determine the accuracy of measurement of the aluminium content in bone. material and methods an 89-year old woman was amputated above the knee because of knee contracture and arteriosclerotic gangrene of the foot. immediately after the amputation 30 biopsies were taken from the trabecular bone in the femoral and tibia1 condyles. the bone samples were put in sealed polyethylene test tubes, frozen and stored at -20 "c until analysis. 67 the bone samples were randomly divided into three groups: 10 bone samples were weighed while being wet and analyzed, 10 bone samples weighed and analyzed after drying in 120°c for 48 hours, and 10 after dry ashing in 550°c for 18 hours. the bone samples were decomposed using ultra-pure nitric acid in a quartz tube, an internal standard (indium) added, diluted with high purity water (with a resistivity of more than 18 mr-cm), introduced into an inductively coupled plasma mass-spectrometer (perkin elmer elan 6000) and measured for their content of aluminium; in the first group also for their content of calcium. all handling of the samples was done in a clean room. quality control was assessed by the use of a certified reference material (iaea h-8 animal bone) in every fifth sample randomly distributed in the measurement series. the limits of detection for aluminium in bone tissue (dry weight) was assessed by producing a calibration curve. for this purpose the certified reference bone (iaea h-8) was used. to this sample 0, 5 , 10,20,40,80, 160,320,640 and finally 1280 ng of aluminium per g dry bone was added. the standard addition procedure was consequently used to establish the calibration curve making the calculation of the limit of detection possible. results all samples contained aluminium. the means, ranges and the coefficients of variation (cv) of the aluminiudweight-quotients (for wet bone, dry bone and bone ash) and the aluminium /calcium-weight-quotient are given in table 1. the hypothesis that the samples were drawn from normally distributed populations could not be rejected (p > 0.1, kolmogorov-smirnov normality test). table 1. aluminium content in bone mean ( n g / g ) range (ng/g) cv (%) aywet weight 35 1 309-457 12.4 ayash weight 715 657-745 5.0 ai/ca 2 640 2 470-3 130 7.5 aydry weight 48 1 45 1-509 4.7 the coefficient of variation of the aluminiudweight-quotient in wet bone was larger compared to dry bone (p = 0.01, f-test) and bone-ash (p = 0.01, f-test), but there was no significant difference of this coefficient between dry bone and bone-ash (p = 0.8, f-test). the coefficient of variation of the aluminiudcalcium-weight-quotient was in between the others and was not significantly different from any of these (p = 0.2, f-test). the over-all accuracy was i 8 per cent and the precision was 5 5 per cent as assessed by 68 the certified reference material. the limit of detection for aluminium in dry bone, defined as three times sd of the blank signal, was estimated from the calibration curve to 20.9 ng/g (figure 1). 32000 1 30000 28000 h 26000 24000 c 3 x ._ 2 22000 w w = 20000 18000 16000 0 200 400 600 800 1000 1200 ng/g figure 1. digested bone calibration curve using standard addition of 0, 5, 10, 20,40, 80, 160, 320,640 and 1280 ng aluminium per g dry certified reference bone (r > 0.999, p < 0.0001). discussion the larger coefficient of variation of the aluminiudweight-quotient in wet bone compared to dry bone or bone-ash seems to be due to variable content of water in the bone samples. although there is no significant difference between the coefficients of variation of the aluminiudweight-quotient in dry bone compared to bone-ash, the determination of aluminium is easier related to the weight of dry bone because hardly soluble aluminium compounds are formed during ashing. also, relating aluminium to calcium does not seem to be advantageous over relating it to the weight of dry bone. indeed, the introduction of calcium as a parameter may itself add some variation in the determination of the aluminium content in bone. although the accuracy of the aluminium measurements may not be entirely independent of patients’ bone characteristics, our result from one patient probably provides the order of the accuracy between the examined procedures. most investigations have used atomic absorption spectrophotometry (1-3,5-10, 12, 13, 15 19, 21, 22); a few mass-spectrometry (4, 11). the detection limit for aluminium has been reported to be 23-150 ng/g bone tissue when determined by atomic absorption spectrophoto 69 6i001 26 metry (8, 9, 13, 20), while it was 21 ng/g bone tissue when determined by mass-spectrometry in our study. in conclusion, mass-spectrometry produces among the best detection limits for aluminium in bone, and the aluminium content in bone seems to be best related to the weight of dry bone. acknowledgement this study was sponsored by alzheimerfonden. references 1. alfrey, a.c., legendre, g.r. & kaehny, w.d.: the dialysis encephalopathy syndrome. possible aluminium intoxication. n engl j med 294: 184-8, 1976. 2. bouman, a.a., platenkamp, a.j. & posma, f.d.: determination of aluminium in human tissues by flameless atomic absorption spectroscopy and comparison of reference values. ann clin biochem 23: 97-101, 1986. 3. boyce, b.f., fell, g.s., elder, h.y., junor, b.j., elliot, h.l., beastall, g., fogelman, i. & boyle, i.t.: hypercalcaemic osteomalacia due to aluminium toxicity. lancet 2: 1009-13, 1982. 4. bush, v.j., moyer, t.p., batts, k.p. & parisi, j.e.: essential and toxic element concentrations in fresh and formalin-fixed human autopsy tissues. clin chem 41: 284-92, 1995. 5. cournot-witmer, g.c., zingraff, j., plachot, j.j., escaig, f., lefhvre, r., boumati, p., bourdeau, a., garabcdian, m., galle, p., bourdon, r., driieke, t. & balsan, s.: aluminum localization in bone from hemodialyzed patients: relationship to matrix mineralization. kidney int 20: 375-85, 198 1. 6. d’haese, p.c., couttenye, m.m., goodman, w.g., lemoniatou, e., digenis, p., sotornik, i., fagalde, a., barsoum, r.s., lamberts, l.v. & de broe, m.e.: use of the low-dose desferrioxamine test to diagnose and differentiate between patients with aluminium related bone disease, increased risk for aluminium toxicity, or aluminium overload. nephrol dial transplant 10: 1874-84, 1995. 7. dunstan, c.r., evans, r.a., hills, e., wong, s.y.p. & alfrey, a.c.: effect of aluminum and parathyroid hormone on osteoblasts and bone mineralization in chronic renal failure. calcif tissue int 36: 133-8, 1984. 8. hongve, d., johansen, s., andruchow, e., bjertness, e., becher, g. & alexander, j.: determination of aluminium in samples from bone and liver of elderly norwegians. j trace elem med biol 10: 6-1 1, 1996. 9. leflon, p., plaquet, r., moriniere, p. & fournier, a.: determination of aluminium in bone in haemodialyzed patients, using inductively coupled argon plasma emission spectro metry. clin chim acta 191: 31-8, 1990. 10. mjoberg, b.: aluminium-induced hip fractures. a hypothesis. j bone joint surg (br) 71: 538, 1989. 11. mjoberg, b., hellquist, e., mallmin, h. & lindh, u.: aluminium, alzheimer’s disease and bone fragility. acta orthop scand 68: 51 1-4, 1997. 12. o’mahony, d., denton, j., templar, j., o’hara, m., day, j.p., murphy, s., walsh, j.b. & coakley, d.: bone aluminium content in alzheimer’s disease. dementia 6: 69-72, 1995. 70 13. radunovic, a. & bradbury, m.w.b.: determination of aluminium in different tissues of the rat by atomic absorption spectrometry with electrothermal atomization. analyst 1 18: 14. rodriguez, m., felsenfeld, a.j. & llach, f.: aluminum administration in the rat separately affects the osteoblast and bone mineralization. j bone miner res 5: 59-67, 1990. 15. romanski, s.a., mccarthy, j.t., kluge, k. & fitzpatrick, l.a.: detection of subtle aluminum-related renal osteodystrophy. mayo clin proc 68: 419-26, 1993. 16. salusky, i.b., coburn, j.w., nelson, p. & goodman, w.g.: prospective evaluation of aluminum loading from formula in infants with uremia. j pedriatr 116: 726-9, 1990. 17. sedman, a.b., klein, g.l., merritt, r.j., miller, n.l., weber, k.o., gill, w.l., anand, h. & alfrey, a.c.: evidence of aluminum loading in infants receiving intravenous therapy. n engl j med 312: 1337-43, 1985. 18. slanina, p., falkeborn, y., frech, w. & cedergren, a.: aluminium concentrations in the brain and bone of rats fed citric acid, aluminium citrate or aluminium hydroxide. food chem toxic 22: 391-7, 1984. 19. slanina, p., frech, w., bernhardson, a., cedergren, a. & mattsson, p.: influence of dietary factors on aluminium absorption and retention in the brain and bone of rats. acta pharmacol toxicol 56: 331-6, 1985. 20. tang, s., parsons, p.j. & slavin, w.: rapid and reliable method for the determination of aluminium in bone by electrothermal atomic absorption spectrometry. analyst 12 i : 195 200, 1996. 21. van de vyver, f.l., bekaert, a.b., d’haese, p.c., kellinghaus, h., graefe, u. & de broe, m.e.: serum, blood, bone and liver aluminum levels in chronic renal failure. trace elem med 3: 52-61, 1986. 22. van de vyver, f.l., visser, w.j., d’haese, p.c. & de broe, m.e.: iron overload and bone disease in chronic dialysis patients. nephrol dial transplant 5: 781-7, 1990. 23. wyatt, r.m., ryde, s.j., morgan, w.d., mcneil, e.a., hainsworth, i.r. & williams, a.j.: the development of a technique to measure bone aluminium content using neutron activation analysis. physiol meas 14: 327-35, 1993. 24. zhu, j.-m., huffer, w. & alfrey, a.c.: effect of aluminum on bone matrix inductive properties. kidney int 38: 1141-5, 1990. 533-6, 1993. correspondence to: hans-olov hellstrom, m.d. department of orthopedics uppsala university hospital se-751 85 uppsala sweden 71 upsala j med sci 95: 249, 1990 comments. aspects of administration and economical planning of clinical laboratories in sweden carl-henric de verdier department of clinical chemistry, university of uppsala, uppsala, sweden the tendency towards decentralization and delegation of economi cal responsibility is very clear also in sweden in health care organization in general and in clinical laboratories in particu lar. in many administrative areas for health care counties it is planned from 1991 or 1992 not to give the clinical chemical laboratories a fixed annual budget but to distribute economical resources to the clinics and the primary care districts and let them buy laboratory services according to a predetermined price list. this has been called itzero budgetingt1. economists have discussed to what extent the wards and the primary care units should request service from the laboratories of their own public health organization or from independent public or private laboratories. most economists seem to agree that, in general, it is waste of resources not to primarily use the laboratories of the own organization. in determining the prices of the price-list it is important to have the possibilities to charge higher fees for investigations with higher "laboratory quality specifications" or lower detec tion limits. this is an important argument for the work with quality specifications. correspondence: professor carl-henric de verdier department of clinical chemistry, university of uppsala postal address: university hospital s-751 8 5 uppsala, sweden. 249 untitled sperm donation in sweden 305 received 20 august 2008 accepted 29 august 2008 upsala j med sci 113 (3): 305–314, 2008 swedish sperm donors are driven by altruism, but shortage of sperm donors leads to reproductive travelling erling ekerhovd1, anders faurskov2, charlotte werner1 1centre for reproductive medicine, department of obstetrics and gynaecology, sahlgrenska university hospital, göteborg, sweden, 2faurskov fertility and ultrasound, aalborg, denmark abstract background: swedish legislation requires that sperm donors are identifiable to offspring. in denmark sperm donors remain anonymous. the aim of this study was to examine sperm donation in sweden by identifying socio-demographic backgrounds, motivations and attitudes among donors and to describe options and plans of sperm recipients. furthermore, the willingness of swedish health care providers to assist in treatment abroad, where sperm from an anonymous donor were to be used, was assessed. the extent of travelling to denmark for reproductive purposes was also examined. methods: thirty swedish sperm donors completed a questionnaire and were interviewed about their backgrounds, motivations and attitudes. thirty couples where the infertility workup had shown azoospermia were interviewed about their options for achieving parenthood. the willingness to assist in fertility treatment abroad and the extent of reproductive cross border travelling were assessed by interviewing health care providers and by contacting danish clinics. results: almost all donors were caucasian. the main motivation for sperm donors was to help others. owing to shortage of sperm donors many caucasian recipients intended to have treatment abroad. for most non-caucasian recipients sperm from a donor of appropriate ethnicity were not available in sweden. whether the sperm donor was anonymous or identifiable was not of major importance to most sperm recipients. health care providers expressed unanimous willingness to assist in treatment with sperm from an anonymous donor. our inquiry indicated that more than 250 swedish sperm recipients travel to denmark annually. conclusions: identifiable sperm donors are driven by altruistic motives, but shortage of sperm donors leads to reproductive travelling. recruitment strategies to increase the number of sperm donors in sweden are therefore warranted. introduction sperm donation was previously carried out worldwide without any legal restrictions. the sperm donor remained anonymous to recipients as well as to offspring. in 1985 sweden, as the first country in the world, passed a law that gave a child conceived by donor insemination the right to obtain identifying information about the donor upon reaching an age of sufficient maturity (1). the law states that the sperm donor remains anonymous to the recipient couple and vice versa, and it does not oblige the parents to inform the child that she/he was conceived with donor sperm (2). medical files containing information about sperm donations are sepa306 erling ekerhovd et al. rated from official medical records and are kept for at least 70 years (3). the same legislative principles as for donor insemination have been applied to in vitro fertilization (ivf) since july 2005. these principles also include lesbian couples, who are allowed insemination or ivf with donor sperm in sweden since 2005. the danish legislation regulating sperm donation differs from the swedish legislation. in denmark, sperm donors remain anonymous to recipients and offspring, and donor sperm are used to achieve pregnancy by insemination or ivf for single women as well as lesbian couples (4). the consequences of the swedish abolition of donor anonymity have been hotly debated. it has been argued that more sperm donors were recruited following the enactment of the law (5). others have come to the conclusion that the law resulted in travelling to other countries for reproductive purposes partially owing to a decrease in the number of donors, resulting in shortages of donor sperm, and also because many recipients refused to accept identifiable donors (3,6). in recent years a growing number of married as well as co-habitant heterosexual couples, lesbian couples, and single women have tried to achieve pregnancy by means of donor sperm. thus, the need for donor sperm has increased substantially. in the present study we intended to determine why some men become sperm donors in a non-anonymous system by examining the socio-demographic backgrounds, motivations and attitudes of swedish sperm donors. in addition, couples where the infertility workup had shown testicular azoospermia were interviewed regarding their options and plans for achieving parenthood. finally, the willingness of swedish health care providers to assist in fertility treatment abroad and the extent of travelling from sweden to denmark for reproductive purposes were assessed. material and methods between january 2002 and december 2004, 32 consecutively recruited sperm donors at the centre for reproductive medicine, sahlgrenska university hospital, göteborg, sweden, were asked to participate in the study by completing a questionnaire. the questions were of both qualitative and quantitative nature, with both structured and open-end options. the questionnaire was divided into different sections. the initial section sought detailed socio-demographic information plus present education and occupation. the other sections were about recruitment of donors, motivation for becoming a sperm donor, financial aspects of being a sperm donor, and future implications. after completing the questionnaire and returning it through the post, a telephone interview was undertaken to allow the respondents to deepen their answers and comments. between july 2005 and august 2007, 30 couples where the infertility workup had shown testicular azoospermia were interviewed about their options and plans achieving parenthood. all interviews were conducted at the centre for reproductive medicine as part of a standard programme toward the end of the infertility investigation. one to three months earlier the couples had been offered placement sperm donation in sweden 307 on a waiting list for fertility treatment with donor sperm. they had also been informed that the waiting time for donor insemination or ivf with donor sperm was approximately 18 months. the willingness of swedish health care providers to assist in fertility treatment abroad, where sperm from an anonymous donor were to be used, was assessed by contacting 10 swedish outpatient clinics in obstetrics and gynaecology. in each case a doctor or a nurse was asked about the clinic’s policy regarding assistance in fertility treatment of couples and single women who travel abroad for sperm donation. to examine the extent of travelling for reproductive purposes from sweden to denmark 13 clinics in denmark where sperm donation is carried out were contacted. the clinics were asked about the estimated number of swedish recipients of donor sperm during the previous 12 months. all contacts with other clinics were carried out by e-mail as well as with telephone interviews. results socio-demographic backgrounds, motivations and attitudes of sperm donors thirty out of 32 consecutively recruited sperm donors agreed to participate in the study and returned the questionnaires. twenty-eight of the respondents were caucasian (table 1). the two non-caucasian sperm donors were both born in iran and had both been adopted by ethnic swedes when they were less than 2 years old. they had both grown up in sweden. two-thirds of the respondents had been to college or university. only five of the donors were students, all university students about to finish their degrees. approximately two-thirds of the sperm donors were married or lived in stable co-habitation arrangements. twenty-one of the men had become aware of the need for sperm donation through advertisements at blood donor centres (table 2). twenty-three of the sperm donors proved to be blood donors as well. except for one man, all respondents who were married or lived in a co-habitation arrangement had informed their partners about their intention of becoming a sperm donor before they contacted the fertility centre. only two of the men had told a friend or a colleague about their decision. none of the donors had tried to recruit or intended to recruit other men to become sperm donors. most of the donors stated that, in contrast to blood donation, sperm donation was still not socially acceptable and therefore not a subject one discussed with friends or colleagues. concerning motivation for becoming a sperm donor, the vast majority of the men declared that they only wanted to help infertile couples (table 2). none of the respondents stated that their motive for sperm donation was mainly of financial character. most donors responded positively about donating sperm to single women and les308 erling ekerhovd et al. table 1. socio-demographic characteristics of the sperm donors n = 30 age mean (years) 35.8 (sd, range) (± 4.9, 24-46) n % ethnic background caucasian* 28 93.3 iranian 2 6.7 education high school 7 23.3 college 9 30.0 undergraduate degree 12 40.0 graduate degree 2 6.7 occupational status student 5 16.7 unskilled 3 10.0 blue collar 6 20.0 white collar 9 30.0 professional 7 23.3 marital status married 6 20.0 co-habitant 13 43.3 divorced 2 6.7 single 9 30.0 number of children 0 19 63.3 1 2 6.7 2 5 16.7 3 1 3.3 > 3 3 10.0 * 24 born in sweden, 2 born in russia, 1 born in iceland and 1 born in finland sperm donation in sweden 309 bian couples (table 3). two sperm donors expressed that sperm should not be used to impregnate single women. they were both of the opinion that it was essential for young children to be raised by parents of both sexes. four respondents were against sperm donation to lesbian women since the birth of a child, in their views, should be the result of love between a man and a woman. twenty-five of the sperm donors thought that donors should be reimbursed for travel expenses. five men were of the opinion that sperm donors should not be compensated for expenses at all. they all underlined that the word donation implies that money is not involved in the matter and that sperm donation should be an act of pure altruism. twenty-five men answered that they would still be sperm donors even if no monetary compensation were given. twenty-eight of the respondents stated that they would like to know if their dotable 2. recruitment method and motivation among 30 sperm donors n = 30 n % advertisement at the blood donor center 21 70.0 newspaper article 4 13.3 suggested by others 3 10.0 radio 1 3.3 did not know 1 3.3 purely altruistic motives 19 63.3 purely financial motives 0 0.0 altruistic and financial motives 3 10.0 altruistic and non-financial motives 5 16.7 altruistic, financial and other motives 3 10.0 table 3. attitudes among 30 sperm donors regarding sperm donation to single women and lesbians yes no n % n % would you donate sperm if the sperm were going to be offered to single women? 28 93.3 2 6.7 would you donate sperm if the sperm were going to be offered to lesbian single women? 26 86.7 4 13.3 would you donate sperm if the sperm were going to be offered to lesbians? 26 86.7 4 13.3 310 erling ekerhovd et al. nation had led to birth of at least one child (table 4). most of them expressed that this knowledge was important since birth of a child was a confirmation that their donation had been meaningful. secondly, many of the respondents said that they in the future intended to inform their own children about the sperm donation and that this information was especially important if it had resulted in a child. most of the men said they had a positive attitude toward future contact with potential offspring (table 4). however, several of them underlined that it was important to distinguish between the offspring’s legal right to know the identity of the donor and contacts between offspring and donor. decisions about treatment made by couples in which azoospermia had been diagnosed twenty-one of the 30 men where the infertility workup had shown azoospermia were caucasian (table 5). fourteen of the couples in this group accepted the offer to be on the waiting list for fertility treatment with donor sperm. however, 9 of these couples stated that they planned to have fertility treatment with donor sperm abroad during the waiting period for insemination or ivf to be performed in sweden. six couples of non-caucasian origin had decided to go abroad for fertility treatment with sperm from an anonymous donor. three non-caucasian couples expressed that they were unsure of what to do since fertility treatment outside sweden seemed to be too expensive. none of the non-caucasian couples would accept treatment with donor sperm from a caucasian donor. twenty-eight of the 30 couples were of the opinion that only sperm from an identifiable donor of the same ethnicity was acceptable. table 4. attitudes of 30 sperm donors regarding results of sperm donation and possible future consequences of sperm donation yes no n % n % would you like to be informed if the sperm donation results in pregnancy and birth? 28 93.3 2 6.7 does a sperm donor have certain moral responsibilities for the child later in life? 6 20.0 24 80.0 would you accept meeting the offspring when she/he has become an adult? 26 86.7 4 13.3 sperm donation in sweden 311 assistance in sperm donation abroad and travelling for reproductive purposes to denmark all the interviewed health care providers regarded assistance with fertility treatment resulting in sperm donation from an anonymous donor abroad as a natural part of their job. their willingness to assist was described as similar to helping arrange treatment at another domestic clinic. all the nurses and doctors who were interviewed said they gave such assistance without any discussion or disagreement among colleagues. contacts with danish clinics where insemination or ivf with donor sperm is performed confirmed that swedish couples as well as single women often receive fertility treatment in denmark. although data are incomplete, our inquiery indicated that more than 250 swedish couples and single women annually have fertility treatment with donor sperm in denmark. discussion the study shows that recruitment of identifiable sperm donors is feasible and that the number of identifiable donors at present is inadequate. since there is no shortage of danish sperm donors, many swedish recipients have fertility treatment in denmark. table 5. decisions regarding further fertility treatment made by 30 couples where the infertility workup had shown testicular azoospermia n % ethnic background caucasian 21 70 non-caucasian 9 30 caucasian waiting list for sperm donation 5 23.8 waiting list for sperm donation + sperm donation abroad 9 42.9 sperm donation abroad 2 9.5 adoption 2 9.5 no further fertility treatment 2 9.5 undecided about what to do 1 4.8 non-caucasian sperm donation abroad 6 66.7 undecided about what to do 3 33.3 312 erling ekerhovd et al. most of the men who completed the questionnaire had become aware of the need for sperm donation at blood banks. this clearly reflects the recruitment strategy of the fertility centre. in order to increase the number of sperm donors it seems evident that the recruitment programme has to appeal to a large section of the swedish population. posters and pamphlets at blood banks are probably more cost-effective than advertising in the mass media. in addition, blood donors are also often driven by altruistic or humanitarian motives (7). most of our identifiable sperm donors were mainly motivated by the desire to assist infertile couples. this finding is in agreement with a previous swedish study conducted in 1995–1996 (8). the fact that the vast majority of the men responded that they would still be sperm donors even if no reimbursement were given is encouraging. our study differs to some extent from two danish studies which showed that monetary compensation was of importance to many anonymous sperm donors, in addition to altruistic motives (9,10). during the study period a reimbursement of 300 sek (~30 €) for each donation was given to cover transport costs. most swedish sperm donors were willing to provide sperm to lesbian couples and single women. in fact, after the present study had been initiated the swedish legislation was changed so that donor sperm was made available for insemination or ivf of lesbian couples. shortly after this change in legislation we asked 22 ongoing sperm donors at the fertility centre if they would allow their previously cryopreserved sperm to be used for fertility treatment of lesbian couples. twenty of the 22 men responded positively to the enquiry, confirming a positive attitude toward donating sperm to lesbians, as demonstrated in the present study. the two main problems for recipients of donor sperm disclosed in the present study were the long waiting period for fertility treatment with donor sperm and the almost total absence of non-caucasian sperm donors. in göteborg the waiting period for fertility treatment with donor sperm increased in 2005 from 6 months to 18 months when lesbian couples became permitted to have insemination or ivf with donor sperm. in this study many of the couples who accepted placement on the waiting list for treatment with donor sperm stated that they intended to have treatment abroad. being put on the waiting list was regarded by many couples as a fallback position in case they had not already achieved pregnancy when fertility treatment in sweden became available for them. for most couples of non-caucasian origin, fertility treatment outside sweden was the only option, since no donors of suitable ethnicity were available. the possibility of recruiting a friend or a relative of the male partner to become sperm donor was regarded by all the couples as too complicated or unacceptable. the use of sperm from an identifiable donor was not regarded as a problem by most couples. almost all couples answered that they had not yet decided if they would tell their offspring about the mode of conception. previous studies have shown that a majority of parents do not inform their children about the use of donor sperm, even when sperm from an identifiable donor has been used (11–13). in conclusion, the present study showed that swedish sperm donors are driven by altruistic motives and that shortage of sperm donors forces many recipients to sperm donation in sweden 313 go abroad for fertility treatment. reproductive travelling from sweden to denmark can be described as a safety valve that reduces the problems associated with the shortage of sperm donors. recruitment strategies to increase the number of identifiable swedish sperm donors of different ethnicity are urgently needed. acknowledgements this study was supported by grants from the hjalmar svensson foundation, göteborg, sweden. references 1. svensk författningssamling 1984:1140 (the swedish code of statutes). 2 http://www.socialstyrelsen.se/publicerat/2004/8421/2004-1-1.htm 3. bygdeman m (1991) the swedish insemination act. acta obstet gynecol scand 70: 265–6. 4. lov om kunstig befrugtning [law concerning assisted reproduction] nr. 460 paragraph 20 (cf. cf. sundhedsstyrelsens vejledning af 30. september 1997 paragraph 56.) 5. daniels k, lalos o (1995) the swedish insemination act and the availability of donors. hum reprod 10: 1871–4. 6. pennings g (2001) the loss of sperm donor candidates due to the abolition of the anonymity rule: analysis of an argument. j ass reprod gen 18: 617–22. 7. tscheulin dk, lindenmeier j (2005) the willingness to donate blood: an empirical analysis of socio-demographic and motivation-related determinants. health serv manage res 18: 165–74. 8. lalos a, daniels k, gottlieb c, lalos o (2005) recruitment and motivation of semen providers in sweden. hum reprod 18: 212–6. 9. pedersen b, nielsen af, lauritsen jg (1994) psycosocial aspects of donor insemination. sperm donors – their motivation and attitudes to artificial insemination. acta obstet gynecol scand 73: 701–5. 10. ernst e, ingerslev hj, schou o, stoltenberg m (2007) attitudes among sperm donors in 1992 and 2002: a danish questionnaire survey. acta obstet gynecol scand 86: 327–33. 11. lindblad f, gottlieb c, lalos o (2000). to tell or not to tell – what parents think about telling their children that they were born following donor insemination. j psychosom obstet gynecol 21: 193–203. 12. gottlieb c, lalos o. lindblad f (2000) disclosure of donor insemination to the child: the impact of swedish legislation on couples’ attitudes. hum reprod 15: 2052–6. 13. lycett e, daniels k, curson r, golombok s (2005) school-aged children of donor insemination: a study of parents’ disclosure patterns. hum reprod 20: 810–9. corresponding author: erling ekerhovd centre for reproductive medicine, department of obstetrics and gynaecology, sahlgrenska university hospital, 413 45 göteborg, sweden phone: + 46 31 3421000 fax: + 46 31 418717 e-mail: erling.ekerhovd@obgyn.gu.se tf-iups160029 271..275 original article dietary habits, nutrient intake and biomarkers for folate, vitamin d, iodine and iron status among women of childbearing age in sweden wulf becker, anna karin lindroos, cecilia n€als�en, eva warensj€o lemming and veronica €ohrvik national food agency, p.o. box, 75126 uppsala, sweden abstract background: dietary intake and nutritional status are important for pregnancy and pregnancy outcomes. dietary advice on folate, targeted to women of childbearing age, aims at preventing neural tube defects in the offspring. aim: to describe food and nutrient intake and nutritional status among women of childbearing age in sweden in relation to current nutrition recommendations. methods: dietary intake was assessed using a web-based four-day consecutive food record among adults aged 18–80 years—‘riksmaten 2010–11 adults’. in a subsample, biomarkers of folate, vitamin d, iodine, and iron status were assessed. results: women of childbearing age had lower intakes of fruit and vegetables, fish, and whole grains, but higher intakes of soft drinks. macronutrient composition was generally in line with the nordic nutrition recommendations, except for a lower intake of fibre, a higher intake of saturated fatty acids, and added sugars. mean intakes of vitamin d, folate, and iron were below recommended intakes (ri). median urinary iodine concentration (uic) was 74 lg/l, 20% had insufficient vitamin d status, and 3% low folate concentrations with no age differences. furthermore, 29% of women 18–44 years of age had depleted iron stores. conclusions: the dietary pattern among women of childbearing age (18–44 years) was less favourable compared to older women. intakes of some micronutrients were below ri, but no differences in vitamin d, folate, or iodine status between age groups were observed. however, improvements of folate and iodine status among women of childbearing age are warranted. this can be achieved by following dietary guidelines including use of folic acid-containing supplements. article history received 17 march 2016 revised 8 june 2016 accepted 9 june 2016 key words diet; folate; iodine; iron; nutrition status; vitamin d; women introduction dietary intake and nutritional status among women affect pregnancy and pregnancy outcomes. dietary guidelines in sweden targeted to women of childbearing age include advice on folic acid supplementation (1). adequate folate status is important for prevention of neural tube defects (ntd) in the offspring (2). for pregnant women recommended intakes of several micronutrients are increased (3), and sufficient status prior to conception is therefore also important. this is the case for e.g. iodine and iron, for which there are valid biomarkers. iodine and iron deficiencies are still major nutritional problems world-wide (4,5). about 30% of the world’s population is estimated to have insufficient iodine intake, with increased risk of adverse effects on growth and mental development. the prevalence of iron deficiency (without anaemia) varies between 5% and 17% in industrialized countries, and young women are especially at risk. iron deficiency precedes anaemia. anaemia is associated with fatigue, reduced mood and cognitive function, and poor pregnancy outcomes and quality of life (5). in the recent nation-wide dietary survey ‘riksmaten 2010–11 adults’ biomarkers of these nutrients were collected for the first time. in this paper, data on food consumption, nutrient intake, and status for vitamin d, folate, iodine, and iron among swedish women are presented with a focus on the childbearing age (18–44 years). materials and methods subjects in the riksmaten adults 2010–11 survey, a representative sample of 5,000 individuals aged 18–80 years and living in sweden were invited to participate. the data collection took place between may 2010 and july 2011. food consumption was captured using a web-based food record during four consecutive days (6). a total of 1797 subjects (36%) completed the food record, 1005 women (41%) and 792 men (31%). a subgroup of 1008 individuals, constituting one-fifth of the total sample, were invited to take part in a biomonitoring project. statistics sweden (scb) divided the population sample into seven regions according to affiliation to swedish occupational and environmental medicine centres (oemcs). each region included the regional capital (link€oping, lund, stockholm, umeå, uppsala, gothenburg, and €orebro) together contact w. becker wulf.becker@slv.se national food agency, p.o. box, 75126 uppsala, sweden � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 4, 271–275 http://dx.doi.org/10.1080/03009734.2016.1201176 http://creativecommons.org/licenses/by/4.0/ with two randomly selected counties in each region. an equal number of individuals was selected in each region independently of population size. recruitment took place at four different occasions during the year to cover different seasons. twelve individuals per region and occasion were invited to participate. out of the 1008 individuals that were invited (7 regions, 3 places in each (the capital and 2 counties), 4 occasions, and 12 individuals per round), a total of 300 subjects (30%) agreed to participate, with a higher participation rate (33%) among women. see table 1 for details of number of women in different age groups. dietary assessment the participants reported everything they ate and drank for four consecutive days, using a web-based food diary developed by the national food agency (nfa). food intake and status in all seasons and all days of the week were captured by carrying out the study from may 2010 to july 2011 and by randomly assigning starting days to participants. the level of education was somewhat higher among participants than among non-participants, and drop-out was also higher among e.g. subjects born outside sweden. more details on demographic characteristics have been given by amcoff et al. (6). nutritional biomarkers folate. whole-blood samples for erythrocyte folate status were collected in edta tubes and plasma folate samples in pst tubes by nurses at the oemcs. erythrocyte folate concentrations were analysed at the karolinska university hospital, sweden, using an immunoassay kit (roche folate iii, roche diagnostics gmbh, mannheim, germany). plasma folate was analysed at uppsala university hospital, sweden, using a chemiluminescence immunoassay method on an abbott architect ci8200 analyser with reference standard (cat. no. 1p74-35, abbott laboratories, abbott park, il, usa). vitamin d. plasma 25-hydroxy vitamin d (p-25(oh)d) was analysed at vitas, oslo, norway with lc-ms (7). the assay is accredited by the vitamin d external quality assessment scheme. p-25(oh)d was calculated as 25(oh)d2 þ 25(oh)d3. iodine. urinary iodine concentration (uic) was determined in duplicate by the same laboratory technician at the sahlgrenska academy, department of internal medicine and clinical nutrition, gothenburg, sweden. the method we used was a modified sandell–kolthoff method (8), where iodide is used as a catalyst in the reduction of ceric ammonium sulphate (yellow colour) to the cerous form (colourless) in the presence of arsenious acid. the rate of colour disappearance is directly proportional to iodide concentration. ferritin. iron status was measured as the plasma ferritin concentration using a chemiluminescent microparticle immunoassay (cmia) (architectv r , abbott laboratories) at uppsala university hospital, uppsala, sweden. assessment of nutrition status folate. an erythrocyte folate concentration <317 nmol/l and a plasma folate concentration <6.8 nmol/l are considered to indicate insufficient status (3). vitamin d. plasma 25(oh)d concentrations <50 nmol/l) are considered to indicate insufficient vitamin d status (3). iodine. according to the world health organization (who), a median uic of 100–200 lg/l indicates sufficient iodine status in a population (9). iron. ferritin concentrations <12 lg/l in children and adolescents and <15 lg/l in adults are indicative of depleted iron stores (3,10) in the absence of inflammation. statistical analyses all statistical analyses were done in stata (version 11.2 and 12). shapiro–wilks tests were used to determine normality. non-parametric tests kruskal–wallis and wilcoxon rank–sum were used, and anova and t test were used for normally distributed variables. results there were minor, and non-significant, differences between subjects in the subsample and other participants with regard to weight, height, and bmi. intake of foods and nutrients the intake of fruit and vegetables, potatoes, fish, and seafood was lower, while intake of juice, soft drinks, and cordials was higher among women of childbearing age (18–44 years) than among older women (45–80 years) (figure 1). reported energy intake tended to be higher in the younger age groups. the proportion of macronutrients was relatively 0 50 100 150 200 250 g/ d 18–30 yr 31–44 yr 45–64 yr 65–80 yr figure 1. mean intake (g/d) of selected foods among women in riksmaten 2010–11. table 1. number of women completing the food record and for whom analytical data on biomarkers are available. age group food record p-folate ery-folate p-25(oh)d uic p-ferritin 18–30 years 203 28 26 27 26 28 31–44 years 247 38 35 37 37 38 45–64 years 357 54 53 51 50 54 65–80 years 198 30 27 29 27 30 all 1005 150 141 144 140 150 272 w. becker et al. similar across age groups. however, intake of added sugars was higher, and intake of dietary fibre and whole grains was lower in the younger age groups (figure 2; table 2). reported daily energy intake among women in the subsample was higher than among women not providing blood samples (7.8 versus 7.4 mj; p¼0.038). there were no differences in gross dietary composition, e.g. per cent energy from protein, fat, carbohydrate, and alcohol, and dietary fibre (g/10 mj) and sodium (mg/10 mj). folate intake and status women of childbearing age (18–44 years) had a lower folate intake than women aged 45–80 years (p < 0.001) (table 2). increasing intake of fruit and vegetables was associated with a higher folate intake. the mean erythrocyte folate concentration was 470 nmol/l (sd 117) (n¼141), and mean plasma folate concentration was 17.5 nmol/l (sd 11.0) (n¼150). low blood folate concentrations (erythrocyte-folate <317 nmol/l; plasma-folate <6.8 nmol/l) were found in 3% of the women. despite significantly different folate intakes there were no differences in folate status between women aged 18–44 and 45–80 years (figure 3). however, women reporting a fruit and vegetable intake above 500 g/d had a higher folate status (r2¼ 0.26, p ¼ 0.002). vitamin d intake and status the reported mean intake of vitamin d from the diet among women was 6.4 lg/d lower among the younger age groups (p < 0.001) (table 2). however, there were no differences in vitamin d status, assessed with plasma 25(oh)d (figure 4). insufficient vitamin d status (plasma 25(oh)d <50 nmol/l) was found in 20%. table 2. intake of energy, added sugar, whole grains, vitamin d, folate, iron, and heme-iron among women in riksmaten 2010–11. mean per day and per 10 mj. energy whole grains vitamin d folate iron heme-iron age group n mj/d g/d g/10 mj lg/da lg/ 10 mj lg/da lg/10 mj mg/d mg/ 10 mj mg/d mg/10 mj 18–30 years 202 7.6 35 45 5.2 6.7 223 298 8.9 11.9 0.99 1.38 31–44 years 247 7.6 38 52 6.2 8.3 247 334 9.7 12.9 1.21 1.62 45–64 years 358 7.3 40 56 6.6 9.2 263 365 9.9 13.8 1.19 1.62 65–80 years 198 7.1 43 60 7.6 10.7 275 388 9.4 13.3 1.12 1.62 all 1005 7.4 39 54 6.4 8.8 253 349 9.5 13.1 1.14 1.57 aexcluding supplements. nmol/l nmol/l 0 5 10 15 20 0 100 200 300 400 500 (b) (a) 18-30 y 31-44 y 45-64 y 65-80 y 18-30 y 31-44 y 45-64 y 65-80 y figure 3. folate status among women in the subsample. a: mean plasma folate; b: mean erythrocyte folate concentration. 18-30 yr 31-44 yr 45-64 yr 65-80 yr nmol/l 0 10 20 30 40 50 60 70 figure 4. means of plasma 25(oh)d (nmol/l) among women in the subsample. 0 5 10 15 20 25 30 35 40 protein, e% fat, e% sfa, e% added sugars e% fibre, g/10 mj 18–30 yr 31–44 yr 45–64 yr 65–80 yr figure 2. intake of protein, total fat, saturated fatty acids (sfa), added sugars (as e%), and dietary fibre (g/10 mj) among women in riksmaten 2010–11. upsala journal of medical sciences 273 iodine status in the subsample of participants in the riksmaten 2010–11 study the median uic among women (n ¼ 140) was 74 lg/l. altogether 66% had a uic below 100 lg/l. mean creatinineadjusted uic was 102 lg/g. no intakes were calculated due to incomplete values for iodine contents in foods in the nfa food composition database. iron status intakes of total and heme-iron (mg per day and mg per 10 mj and day) were similar among women of childbearing age (18–44 years) and older women (table 2), and intakes did not differ in the subsample. however, there was a difference between these age groups in mean plasma ferritin concentrations, 41 lg/l (sd 34) and 92 lg/l (sd 74), respectively (p < 0.001). among women of childbearing age, 20% had ferritin values �12 lg/l and 29% <15 lg/l, indicative of depleted iron stores. discussion the low participation rate (41%) in the food consumption survey and in the biomarker subsample (33%) may have influenced the results of this survey. however, all age groups were well represented. the level of education was somewhat higher among participants than among non-participants. the reported intake of fruit, berries, vegetables, and whole grains was generally lower compared to dietary guidelines, while the intake of sugary and fatty foods with a high content of saturated fat and salt was high. women of childbearing age reported lower intakes of fruit and vegetables, fish, and whole grains, and higher intake of soft drinks compared to older women. folate reported mean folate intake was generally above the average requirement (200 lg/d), but below recommended intake (300 lg/d), especially for women of childbearing age (400 lg/d). even adjusting for apparent under-reported intakes, few subjects reached the last-mentioned intake. sixteen women of childbearing age reported intake of folic acid supplements (4%) regularly or sometimes, and 117 reported intake of multivitamin supplements regularly or sometimes. among women in the subsample two (3%) reported intake of folic acid supplements and 16 intake of multivitamins. according to data from the swedish birth register, about 15% of women reported use of folic acid supplements during early pregnancy in 2012, compared to about 1% in 1999 (11). in a study of 1098 pregnant women in the south-west of sweden, carried out in 2013, 30% reported that they used folate-containing supplements during the first trimester (12). in an interview study carried out in early 2010 comprising 1000 adults, drawn from a representative sample of swedish households, 36% of the women reported taking supplements, of which 32% contained folic acid (nfa, unpublished). thus, about one-tenth took folic acid-containing supplements. the study also showed that the knowledge of when a woman should start taking folic acid prior to the planning of pregnancy was low. in sweden, the prevalence of children born with ntd has decreased since the 1970s. since 1999, abortions are included in the statistics, and there has been a decrease up to 2013 (11). this is in contrast to data for many european countries, where no clear trend has been observed (13). data on folate status in the riksmaten subsample, however, indicate that many women of childbearing age would benefit from improved status in order further to reduce the risk of ntd. this can be achieved by adhering to the general dietary guidelines and also using folic acid-containing supplements and choosing fortified products. results from calculations based on weekly menus complying with dietary guidelines and including choice of keyhole-labelled food products (14) suggest that such a diet would result in a folate intake of about 400 lg/d for an adult woman (unpublished). however, this represents an ideal food pattern, which implies substantial changes in the typical diet of today. vitamin d mean vitamin d intake was below recommended intake in all age groups, lowest among the youngest women (18–30 years). despite this, there were no age differences in plasma 25(oh)d, and levels indicated a sufficient status (�50 nmol/l) for the majority. under-reporting could be one explanation, also more frequent outdoor activities and thereby contribution from sun exposure. iodine the median uic of 74 lg/l in the riksmaten subsample is below the who range of 100–200 lg/l and would thus indicate an insufficient status (9). the lower limit of 100 lg/l is based on an average urine volume of 1.5 l/d, which then corresponds to an intake of about 150 lg/d, equal to the recommended intake in the nordic nutrition recommendations (nnr) 2012 (3). however, uic is dependent on osmolality, and the daily fluid intake may vary considerably. using a urine volume of 1.5 l the observed uic of 74 lg/l would correspond to 111 lg/24 h, which is above the average requirement (100 lg/d). alternatively, using data for the creatinine-adjusted uic (102 lg/g) and mean 24-h creatinine excretion reported for german adults (15), the median 24-h iodine excretion has been estimated to be around 130 lg. this is still below the recommended intake (3). a decreasing trend in iodine content in swedish milk has been reported, which is in line with data from swedish market basket studies, indicating a decrease in the average iodine supply from food and beverages (16). thus, a general increase in iodine intake is desirable, especially important for women of childbearing age. use of iodized salt in home cooking and food products by manufacturers and increased fish consumption are feasible options. also, efforts to maintain and possibly restore iodine levels in milk and dairy products are warranted. 274 w. becker et al. iron in the riksmaten subsample there was a non-significant tendency for lower dietary intake of total iron and heme-iron in the youngest age group (18–30 years) compared to women aged 45–80 years. plasma ferritin levels were significantly lower among women of childbearing age, and the proportion with ferritin concentrations <12 lg/l and <15 lg/l was 20% and 29%, respectively. in sweden sifted flour was fortified with carbonyl-iron until the beginning of 1995. sj€oberg and hulth�en (17) compared food habits, iron intake, and status in two cross-sectional studies among 15–16-year-old girls and boys, sampled from schools in gothenburg, before (1994, n¼1245) and after (2000, n¼1020) discontinuation of the fortification. among girls the reported intake of both total and heme-iron was lower in the year 2000. prevalence of iron deficiency, defined as serum ferritin <15 lg/l, was higher (45%) in 2000 than in 1994 (37%). there were, however, no data on mean serum ferritin levels. reported levels of total and heme-iron intake are, however, lower compared to the riksmaten sample. in a longitudinal study on swedish adolescents (60 males and 66 females), carried out from 1993 to 1999, changes in iron status were measured from 15 to 21 years of age. in females, median serum ferritin increased significantly, after a non-significant decrease at 17 years, from 27 lg/l at 15 years to 34 lg/l at 21 years (18). in females, prevalence of iron deficiency, defined as serum ferritin <12 lg/l, was 18%, 26%, and 21% at 15, 17, and 21 years, respectively. in the riksmaten subsample the median serum ferritin level among women aged 18–30 years was 36.5 lg/l. thus, it was similar to the value for the 21-year-old women in the study by samuelson et al. (18). taken together, the results from these studies indicate that young women would benefit from a diet with a higher iron density and availability. conclusions young adults are an important target group for public health work, since food habits are established early in life and women of that age might bear children. in the riksmaten 2010–11 study, women of childbearing age reported less favourable food habits and lower intakes of some micronutrients compared to older women. no or only minor differences in vitamin d, folate, and iodine status between age groups were observed. however, iron status was lower among the younger age groups. the present data indicate that folate, iron, and iodine status among women of childbearing age needs improvement. this can be achieved by following dietary guidelines including use of folic acid-containing supplements. acknowledgements we thank ingalill gadhasson, national food agency for sample handling; elisabeth gramatkovski, sahlgrenska academy, department of internal medicine and clinical nutrition for analysis of iodine and creatinine; and peter ridefelt, department of medical sciences, uppsala university for analysis of serum ferritin, crp, and plasma folate. disclosure statement the authors report no conflicts of interest. funding information financial support from the swedish environmental protection agency made the collection of biological samples in the riksmaten 2010–11 survey possible. references 1. nfa. advice on folic acid for women. uppsala: national food agency; 2015 [cited 2016 january 18]. available from: http://www. livsmedelsverket.se/matvanor-halsa–miljo/kostrad-och-matvanor/folsyra-for-kvinnor/. 2. de-regil lm, pe~na-rosas jp, fern�andez-gaxiola ac, rayco-solon p. effects and safety of periconceptional oral folate supplementation for preventing birth defects. cochrane database syst rev. 2015;12:cd007950. 3. ncm. nordic nutrition recommendations 2012. integrating nutrition and physical activity. copenhagen: nordic council of ministers; 2014. 4. andersson m, karumbunathan v, zimmermann b. global iodine status in 2011 and trends over the past decade. j nutr. 2012;142:744–50. 5. beck kl, conlon ca, kruger r, coad j. dietary determinants of and possible solutions to iron deficiency for young women living in industrialized countries: a review. nutrients. 2014;6:3747–76. 6. amcoff e, edberg a, enghardt barbieri h, lindroos ak, n€als�en c, pearson m, et al. riksmaten 2010–2011. food and nutrient intake among adults in sweden. uppsala: swedish national food agency; 2012. [in swedish with english summary]. 7. meyer he, robsahm te, bjørge t, brustad m, blomhoff r. vitamin d, season, and risk of prostate cancer: a nested case-control study within norwegian health studies. am j clin nutr. 2013;97:147–54. 8. andersson m, berg g, eggertsen r, filipsson h, gramatkovski e, hansson m, et al. adequate iodine nutrition in sweden: a cross-sectional national study of urinary iodine concentration in school-age children. eur j clin nutr. 2009;63:828–34. 9. who. urinary iodine concentrations for determining iodine status in populations. who/nmh/nhd/epg/13.1. geneva: world health organization; 2013. 10. who. serum ferritin concentrations for the assessment of iron status and iron deficiency in populations. who/nmh/nhd/mnm/11.2. geneva: world health organization; 2011. 11. socialstyrelsen. birth defects 2013. stockholm: national board of health and welfare [socialstyrelsen]; 2015. [in swedish with english summary]. 12. eriksson l. [use of folic acid supplements among pregnant women during the first trimester]. gothenburg university, department of diet and sports sciences; 2014. [in swedish]. 13. khoshnood b, loane m, walle hd, arriola l, addor mc, barisic i, et al. long term trends in prevalence of neural tube defects in europe: population based study. bmj. 2015;351:h5949. 14. amcoff e, brugård konde å, jansson a, sanner f€arnstrand j. [change to the keyhole effect on nutrient intake]. uppsala: swedish national food agency; 2015. [in swedish]. 15. johner sa, boeing h, thamm m, remer t. urinary 24-h creatinine excretion in adults and its use as a simple tool for the estimation of daily urinary analyte excretion from analyte/creatinine ratios in populations. eur j clin nutr. 2015;69:1336–43. 16. nfa. market basket 2010 chemical analysis, exposure estimation and health-related assessment of nutrients and toxic compounds in swedish food baskets. uppsala: swedish national food agency; 2012. 17. sj€oberg a, hulth�en l. comparison of food habits, iron intake and iron status in adolescents before and after the withdrawal of the general iron fortification in sweden. eur j clin nutr. 2015;69:494–500. 18. samuelson g, l€onnerdal b, kempe b, elverby je, bratteby le. serum ferritin and transferrin receptor concentrations during the transition from adolescence to adulthood in a healthy swedish population. acta paediatr. 2003;92:5–11. upsala journal of medical sciences 275 http://www.livsmedelsverket.se/matvanor-halsa&ndash;miljo/kostrad-och-matvanor/folsyra-for-kvinnor/ http://www.livsmedelsverket.se/matvanor-halsa&ndash;miljo/kostrad-och-matvanor/folsyra-for-kvinnor/ http://www.livsmedelsverket.se/matvanor-halsa&ndash;miljo/kostrad-och-matvanor/folsyra-for-kvinnor/ dietary habits, nutrient intake and biomarkers for folate, vitamin d, iodine and iron status among women of childbearing age in sweden introduction materials and methods subjects dietary assessment nutritional biomarkers assessment of nutrition status statistical analyses results intake of foods and nutrients folate intake and status vitamin d intake and status iodine status iron status discussion folate vitamin d iodine iron conclusions acknowledgements disclosure statement funding information references sups_a_530701 72..76 upsala journal of medical sciences. 2011; 116: 72–76 original article the relationship between socio-demographic characteristics of patients and diagnostic delay in acute pulmonary thromboembolism yilmaz bulbul, sibel ayik, funda oztuna, tevfik ozlu & sibel sahin karadeniz technical university, school of medicine, department of chest diseases, trabzon, turkey abstract background. in pulmonary thromboembolism (pe), delay to diagnosis is very common. in this study, we examined the role of patients and the socio-demographic characteristics in delayed diagnosis of pe. patients and methods. we evaluated 156 pe patients for the dates of symptom onset, the dates of first visit to a health institution and diagnosis, signs and symptoms, and the socio-demographic characteristics. delays were analyzed using the mannwhitney u test, and the predictors were analyzed using logistic regression analysis. results. of the patients, 60.3% visited a health institution within the first day of the symptoms. mean time from symptoms to the first admission to a health institution (patient delay) was 2.04 ± 3.89 days (median 0 day, range 0–30). current smoking, a high level of education, and co-morbidity were associated with longer patient delays. the time interval from first symptom to the diagnosis (total delay) was 7.93 ± 10.05 (median 4 days, range 0–45) days. while hypotension, syncope, and previous surgery/trauma were significantly associated with a shorter total delay, a previous visit to any health institution was associated with longer total delay. conclusion. in conclusion, although some socio-demographic characteristics of patients such as smoking, educational status, and co-morbid diseases were found to be associated with delayed visit to any health institution, our results showed that physician or health system delays were more prominent in delayed diagnosis of pe. key words: delay to diagnosis, delay to presentation, pulmonary embolism introduction pulmonary thromboembolism (pe) is usually considered to be an acute disease. however, because of the non-specific nature of the signs and symptoms of the disease, delays from the onset of symptoms to hospital admission and to diagnosis are very common. median time from symptoms to hospital admission was reported to vary between 4 and 7 days (1,2). in those studies, delays were reported to be associated with the severity of the disease (presence of multiple signs and symptoms, hypotension, or syncope) and the type of risk factors causing pe (1–4). however, most previous studies were designed retrospectively, and the potential role of patients was not studied. the primary aim of this study was to investigate the role of patients and socio-demographic characteristics in delayed visit to a health institution and in delayed diagnosis of pe. materials and methods study design this prospective and observational study was conducted at farabi hospital, a tertiary care hospital with 750-bed capacity, at karadeniz technical correspondence: associate professor dr yilmaz bulbul, karadeniz technical university, school of medicine, department of chest diseases, 61080, trabzon, turkey. fax: +90 462 3257031. e-mail: bulbulyilmaz@yahoo.com (received 27 august 2010; accepted 5 october 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.530701 university, turkey, and was approved by the local ethics committee. study setting and population the study group comprised all adult patients with pe who were diagnosed and followed up by the chest clinic of farabi hospital between january 2007 and december 2008 and who agreed to participate in the study. diagnosis of pe was confirmed using spiral computed tomography (ct) (somatom volume zoom and sensation 16, siemens, erlangen, germany) and perfusion scan (siemens e-cam dual-head, usa). the date of symptom onset, the date of first visit to any health institution (health center or hospital), and the diagnosis were recorded. in addition to sociodemographic data (age, gender, education status, occupation, place of residence, and smoking status), the signs and symptoms (dyspnea, chest pain, hemoptysis, fever, deep vein thrombosis (dvt), syncope, and hypotension), co-morbid diseases, and risk factors of patients with pe were recorded. the time interval from the onset of symptoms to the first visit to any health institution (a health center or a hospital) was defined as ‘patient delay’, and the time interval from symptoms to the diagnosis was defined as ‘total delay’. statistical analysis the mann-whitney u test was used for the comparison of delays with socio-demographic characteristics, signs and symptoms, co-morbid conditions, and risk factors. results are given as mean ± sd and median (range). p values <0.05 were considered to be significant. factors that may be related to delays were analyzed using logistic regression. results during the study period, 200 patients with pe were diagnosed and followed up by our clinic. of the total, 15 were excluded because of questionable date of symptom onset, and 29 were excluded because they were already hospitalized. of the remaining 156 patients who participated in the study, 97 (62.2%) were females and 59 (37.8%) were males. diagnosis was confirmed using spiral ct in 150 patients and lung scan in 6 patients. the mean age was 64.07 ± 15.90 years, and 87 of the patients (55.8%) were older than 65 years. of the patients, 13.2% were currently smokers, 19.1% were ex-smokers, and 67.7% had never smoked. a total of 68 (43.8%) of the patients were living within a city center, and the remainder lived either in a small town (5.2%) or a village (51.0%). the proportion of patients who were from neighboring cities was 51.8%. of the patients 59.6% were illiterate, and the remainder had graduated from either primary school (31.1%) or high school and university (9.3%). the majority of patients stated their occupation as housewife (58.4%), followed by farmer (13.6%) and others (worker, official, etc.). altogether 132 patients (84.6%) had at least one co-morbid disease (cardiovascular diseases 51.2%, malignant diseases 8.3%, pulmonary disease 4.5%, endocrine diseases 3.8%, etc.). of the patients, 30.6% had previous surgery or trauma, and 69.4% had medical risk factors for pe. symptoms commonly seen at first presentation were dyspnea (73.1%), pleuritic chest pain (51.3%), syncope (16.0%), and hemoptysis (10.3%). the rate of hypotension was 15.5%, and the signs and symptoms of deep vein thrombosis were detected in 15%. patient delay after the onset of symptoms, the average delay from symptoms to the visit to a health institution was 2.04 ± 3.89 days (median 0 day, range 0–30). this interval was 4.25 ± 6.74 days (median 2.5 days, range 0–30) in current smokers and 1.74 ± 3.20 days (median 0 day, range 0–30) in those who had never smoked (p = 0.011). after the start of symptoms, patients with high educational status were admitted within 3.04 ± 5.29 days (median 0 day, range 0–30); however, patients with a lower educational status were admitted within 1.34 ± 2.42 days (median 0 day, range 0–11) (p = 0.028). while patients with a co-morbid disease were admitted within 2.25 ± 4.10 days (median 0 day, range 0–30), patients with no co-morbid condition were admitted to a health institution within 0.83 ± 2.09 days (median 0 day, range 0–7) (p = 0.021). other sociodemographic and clinical factors were not found to be significantly associated with the patient delay. among the clinical and socio-demographical factors, univariate logistic regression analysis showed that only current smoking and education were the factors predicting delayed first visit to a health institution (table i). total delay among 156 patients, 94 (60.3%) were admitted to a health institution within the first day of the symptoms; however, only 31 (19.9%) received final diagnosis of delay in pulmonary embolism 73 pe within the first 24 hours. total delay across the group was 7.93 ± 10.05 days (median 4 days, range 0– 45). there was no correlation between sociodemographic factors and delayed diagnosis (total delay) of pe. however, total delay was significantly longer in patients who were previously admitted to any health institution (7 days, range 0–45 versus 2 days, range 0–30; p < 0.001). univariate logistic regression analysis showed that previous hospital or doctor visits were associated with an approximately 11 times longer diagnostic delay than the patients who did not visit a doctor or a hospital (table ii). on the table i. univariate logistic regression analysis of clinical and socio-demographical factors for patient delay in pulmonary thromboembolism. delayed presentation (after 24th hour) variable or 95.0% ci p age > 65 y 1.322 0.693–2.521 0.396 gender 1.192 0.617–2.395 0.601 education status (literacy) 2.036 1.043–3.974 0.037a occupation 0.903 0.627–1.302 0.585 residence (in urban) 1.027 0.536–1.965 0.937 current smoking 3.456 1.266–9.434 0.015a risk factor (surgery or trauma) 0.056 0.260–1.188 0.129 symptoms and signs dyspnea 1.099 0.532–2.273 0.798 pain 0.918 0.483–1.744 0.795 hemoptysis 1.593 0.564–4.494 0.379 syncope 0.423 0.159–1.128 0.086 hypotension 0.569 0.221–1.466 0.243 deep vein thrombosis signs 1.368 0.470–3.986 0.566 a statistically significant. table ii. univariate logistic regression analysis of factors predicting delayed diagnosis (total delay) of pulmonary thromboembolism. delayed diagnosis (after 24th hour) variable or 95.0% ci p age > 65 y 1.015 0.988–1.042 0.286 gender 0.614 0.261–1.442 0.262 education status (literacy) 2.805 1.123–7.008 0.027 occupation 0.442 0.183–1.068 0.070 residence (in urban) 0.677 0.307–1.491 0.333 current smoking 1.722 0.465–6.378 0.416 risk factor (surgery or trauma) 0.259 0.113–0.592 0.001 symptoms and signs dyspnea 1.140 0.477–2.726 0.767 pain 1.358 0.617–2.992 0.447 syncope 0.286 0.113–0.723 0.008a hypotension 0.267 0.105–0.682 0.006a deep vein thrombosis signs 1.683 0.359–7.880 0.509 previous visits to a health institution 11.592 3.816–35.218 < 0.001a a statistically significant. 74 y. bulbul et al. contrary, patients were diagnosed earlier with a previous surgery/trauma (2 days, range 0–30 versus 4 days, range 0–45; p = 0.001), syncope (1 day, range 0–15 versus 4 days, range 0–45; p < 0.006), and hypotension (1 day, range 0–30 versus 4 days, range 0–45; p = 0.004). logistic regression analysis also indicated a clear association between early diagnosis and previous surgery/trauma, syncope, and hypotension (table ii). no statistically significant association was found between total delay and the remaining socio-demographic and clinical factors. delays and mortality in-hospital mortality was 9.6% (15 patients) within the whole group. of the patients who died, 14 visited a health institution within the first day of symptoms, and 1 patient was admitted the following day (p = 0.013). however, total delay and patient delay were not significantly different between patients who died and those who survived. mortality rates were higher in patients with hypotension (25.0% versus 6.1%; p = 0.010) and those with a medical risk factor (15.0% versus 0%; p = 0.030). discussion pe is usually considered to be an acute illness. however, because the signs and symptoms are not specific, patients may experience significant delays from the onset of symptoms to presentation or diagnosis. in previous studies, the mean time from symptoms to presentation was reported to be 2.9–8.4 days, and mean time to diagnosis was 0.9–2.4 days (2,4,5). timmons et al. reported that approximately 50% of patients presented within 24 hours of symptom onset (6). in other studies, 18.0%–30.4% of the patients were found to have visited a doctor/hospital after 1 week of symptom onset (2,5,6). in the current study, 60% of the patients were admitted to a health institution within the first 24 hours; however, less than half of them were diagnosed with pe within the same day. previous studies have investigated some clinical and demographic factors and their association with delay to presentation and to diagnosis. in a previous study, we reported that patients with more severe disease (presence of hypotension and tachypnea) had presented earlier (2). ageno et al. also reported that the severity of the presentation (presence of multiple signs and symptoms) was associated with earlier diagnosis (3). in a recent study by ozsu et al., it was reported that patients who had a surgical risk factor for pe and syncope were diagnosed earlier (4). the role of age, gender, co-morbidity, symptoms, and the location of thrombi in the pulmonary arterial tree were also investigated in the above-mentioned studies; however, the role of physicians, the role of patients, and socio-demographic factors were not studied. patient delay, the time interval from symptoms to first visit to a health institution, was 2 days. interestingly, there was a reverse association between patient delay and educational status. although the role of education in delayed diagnosis of pe was not investigated in previous studies, there are several studies reporting a clear association between education and early presentation/diagnosis of some other diseases such as malignancy and tuberculosis (7–9). patient delay was also longer in current smokers. we think this delay was related to accompanying pulmonary symptoms and diseases due to smoking. in several studies, smoking has been shown to be well correlated with delay in seeking medical help (10,11). contrary to previous studies, we found that presence of a comorbid disease was clearly associated with patient delay (2–4). we think this was mostly related to the design of our study, because we analyzed delays as patient delay and total delay, separately. indeed, while total delay was not affected by the presence of a co-morbid disease, patient delay was associated with co-morbid diseases. total delay, the average time from the start of symptoms to diagnosis, was 7.9 days. as we mentioned above, of this duration, only 2 days were related to patients themselves. we think the remaining 5.9 days were mostly related to the health system and the physicians. actually, logistic regression analysis showed that previous hospital or doctor visits were associated with an approximately 11 times longer diagnostic delay. it is known that, because of the non-specific signs and symptoms of the disease, pe is usually underdiagnosed. it was reported that pe was confirmed ante mortem in only 30% of patients, with the remaining two-thirds diagnosed by autopsy (12,13). in the current study, we also confirmed that the presence of syncope, hypotension, or a prior trauma/surgery shortened the total delay. in an autopsy series, goldhaber et al. reported that the accuracy of pe was far greater in postoperative patients (13). ozsu et al. also reported that patients with a surgical risk factor were diagnosed earlier (4). in the current study, no association was found between the length of delay and the mortality rate. due to rapid technological advances in ct technology, spiral thorax ct is increasingly employed in the diagnosis of pe, and the diagnostic rate of embolism has increased (14). however, although the diagnosis of pe has increased with the use of ct and the associated use of enoxaparin has risen, a parallel reduction in mortality rates has not been reported (4). delay in pulmonary embolism 75 the present study has a number of limitations. firstly, we detected that an important part of delay was associated with the health system and previous doctor visits. therefore, it might be more helpful to investigate systemand physician-related factors. again, our results would be more valuable if we were able to record alternative diagnoses proposed for patients on their first visit to a health institution. we conclude that there was a considerable patient delay from the onset of pe symptoms until the first visit to a health institution and diagnosis. in addition to the socio-demographic characteristics, including smoking status and high educational status, the presence of a co-morbid disease was also associated with patient delay in pe. however health systemand physicianrelated delays were more prominent, and a previous visit to a health institution was associated with approximately 11 times longer delay in diagnosis of pe. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. jimenez castro d, sueiro a, diaz g, escobar c, garciarull s, picher j, et al. prognostic significance of delays in diagnosis of pulmonary embolism. thromb res. 2007;121: 153–8. 2. bulbul y, ozsu s, kosucu p, oztuna f, ozlu t, topbaş m. time delay between onset of symptoms and diagnosis in pulmonary thromboembolism. respiration. 2009;78:36–41. 3. ageno w, agnelli g, imberti d, moia m, palareti g, pistelli r, et al. factors associated with the timing of diagnosis of venous thromboembolism: results from the master registry. thromb res. 2008;121:751–6. 4. ozsu s, oztuna f, bulbul y, topbas m, ozlu t, kosucu p, et al. the role of risk factors in delayed diagnosis of pulmonary embolism. am j emerg med. 2010 mar 8 (epub ahead of print). doi: 10.1016(j.ajem.2009.07.005). 5. elliott cg, goldhaber sz, jensen rl. delays in diagnosis of deep vein thrombosis and pulmonary embolism. chest. 2005; 128:3372–6. 6. timmons s, kingston m, hussain m, kelly h, liston r. pulmonary embolism: differences in presentation between older and younger patients. age ageing. 2003;32:601–5. 7. storla dg, yimer s, bjune ga. a systematic review of delay in the diagnosis and treatment of tuberculosis. bmc public health. 2008;8:15. 8. abdel-fattah mm, anwar ma, mar1 e, el-shazly mk, zaki aa, bedwani rn, et al. patient and system related diagnostic delay in breast cancer. eur j public health. 1999;9:15–9. 9. vineis p, fornero g, magnino a, giacometti r, ciccone g. diagnostic delay, clinical stage, and social class: a hospital based study. j epidemiol community health. 1993;47: 229–31. 10. basnet r, hinderaker sg, enarson d, malla p, mørkve o. delay in the diagnosis of tuberculosis in nepal. bmc public health. 2009;9:236. 11. hansen rp, olesen f, sørensen ht, sokolowski i, søndergaard i. socioeconomic patient characteristics predict delay in cancer diagnosis: a danish cohort study. bmc health serv res. 2008;8:49. 12. kroegel c. advances in the diagnosis and treatment of pulmonary embolism. pulmonary embolism—how can you mend a broken clot? respiration. 2003;70:4–6. 13. goldhaber sz, hennekens ch, evans da, newton ec, godleski jj. factors associated with correct antemortem diagnosis of major pulmonary embolism. am j med. 1982;73: 822–6. 14. kroegel c, reissig a. computed tomography imaging in pulmonary embolism—the other side of medal. respiration 2004;71:444–7. 76 y. bulbul et al. upsala j med sci 99: 113-120, 1994 expression of an endogenous retrovirus (erv3 herv-r) in human reproductive and embryonic tissues-evidence for a function for envelope gene products erik larsson,' ann-catrin andersson' and b. ove nilsson' ' d e artment of pathology, uppsala university hospital, uppsala, sweden and h e p a r t m e n t of human anatomy, biomedical center, uppsala, sweden a b s t r a c t erv3 (herv-r) is a complete human endogenous retrovirus located on the long arm of chromosome 7. it is expressed in several human tissues as ltr env spliced transcripts (9 and 3.5 kb). the highest level of expression is to be found in placenta and virus expression is down-regulated in choriocarcinoma cell lines. b y means of in situ hybridization, the expression of erv3 =was studied in selected human reproductive and embryonic tissues. it is concluded that (a) e r v 3 env is expressed in syncytiotrophoblasts not only in the placenta but also in hydatidiform moles and choriocarcinomas ( irrespective of origin) (b) erv3 expression in placenta correlates to cell fusion but probably not to the fertilization process itself (c) erv3 %is highly expressed in certain cells i n spermatogenesis but not in the sertoli or leydig cells, and finally (d) erv3 env is expressed in certain embryonic tissues such as the adrenal gland and nervous tissues. introduction the presence of retroviral and retroviral-like particles in different reproductive tissues, oocytes and preimplantation embryos has puzzled scientists for some time. there have been claims that a specific expression of retroviruses exists during implantation and early embryogenesis (for early review see 18 and 4). an attractive hypothesis is that endogenous retroviruses (erv) could play a role in these early events. the expression of retroviruses and/or retroviral related sequences in humans has been detected in reproductive tissues particulary the placenta (9, 12, 17). the hypothesis that ervs are involved in specific processes during early embryogenesis has gained new strength since it is now clear that human dna, like the dna of other species, contains several 9 945252 113 integrated retroviruses and retroviralrelated sequences. (for reviews see 9, 12 and 17). there is also a growing body of evidence that ervs could be involved not only in important normal cellular processes like differentiation and specialization but also in tumorigenesis and immunomodulation(6), the latter a s a consequence of a defective cellular control of erv expression. nevertheless, correlation between the expression of erv and any biological function is yet to be discovered. as a first step towards an understanding of the role of ervs, the expression of one erv, i.e. erv3 was studied in human tissues. erv3 is a complete human erv located on the long arm of chromosome 7 . this retroviral genome contains an open reading frame (orf) throughout the u e n e but has in frame termination codons in the ga? p 01 genes, which should preclude the expression of the virus as particles but may produce typical retroviral proteins especially the env encoded p15e and gp70 ( 1 4 ) . we have previously shown, by northern blot analysis, that erv3 env is readily expressed in most human tissues except choriocarcinomas (6). in this communication we have found, by in situ hybridization, that erv3 -is expressed not only in the placenta but also in other human reproductive tissues such as the testis, embryonic tissues and trophoblastic tumours of ovarian origin. materials and methods the following human tissues were used for in situ hybridization (ish): normal placenta throughout a complete gestational period normal endometrium endometrium of pregnancy, decidua and placental tissues from the same patient hydatidiforme mole (two cases) endometrial choriocarcinoma (two cases) ovarian choriocarcinoma (two cases) normal testis (one case) testis with seminoma and testis with choriocarcinomas (two cases e a c h ) normal and neoplastic human tissues were obtained from the department of pathology, university hospital uppsala. repres entative sample sections were fixed, embedded in paraffin and processed for routine histology. the normal fetal material was obtained from patients operated for ectopic pregnancies, where normal fetal material was also observed. 114 in-situ hybridization (ish) ish was performed as described previously(5). briefly, paraffin embedded tissues were sectioned 4 p m thick and mounted on 3 aminopropyltrietoxylsilane-coated slides (sigma). sections were pre-treated with 0.2 m hcl for ten minutes and permeabilized with 2ug/ml proteinase-k (merck) at 37°c for 15 min prior to hybridi zation. tissue sections were hybridized with 35-s labelled riboprobes (promega biotech) transcribed from supercoiled plasmid templates, yielding probes of consistent activity and size. subsequently they were hybridized overnight at 56" c i n 3x standard saline extract and 50% formamide and washed in 2x standard saline citrate and 50% formamide prior to treatment with rnase a (boehringer), (100ug/ml 37°c for 30 min). the application of nt2b photographic emulsion (kodak) diluted 1:l in distilled water was followed by exposure at 4°c for 2-4 weeks. slides were developed and counterstained with mayers hematoxylin and mounted with permount (fischer scientific). the 1.75 kb erv3 env riboprobe transcribed in both directions was used as probes (6). the sense probe was used as negative control. results we have previously stated that erv3 env is expressed in most human tissues especially the placenta(6). these data were obtained by northern blot analysis of rna extracted from whole tissue samples. in this study we have localized the viral expression in individual cells by ish. placental tissues erv3 env expression in the placenta is localised, almost exclusively, to the syncytiotrophoblasts throughout pregnancy. expression is thus localized only to cells undergoing fusion (fig 1). proliferating cytotrophoblasts were either negative or expressed very small amounts of the env gene as did the cells of the intervillous stroma. a sense probe, included as a control, remained negative in the ish assay. decidual and endometrial glands expressed low levels of erv3 transcript. hydatidiform moles and choriocarcinomas hydatidiform moles contain syncytiotrophoblastic cells to a certain extent. however, the syncytiotrophoblasts in these tissues seem to express erv3 env with the same degree as normal placenta. 115 f i g l . localization of e r v 3 hybridization. the hybridization the cyto syncytiotrophoblastic syncytiotrophoblastic cells (left field.) env in term placenta by in situ product i s exclusively localized to cell layer and in particular to the photo bright field and right dark choriocarcinomas from different locations were included i n this s t u d y . t h e s e i n c l u d e d b o t h e n d o m e t r i a l a n d o v a r i a n choriocarcinomas obtained from elderly women to exclude the possibility of ectopic pregnancies. ovarian choriocarcinomas i s extremely rare and we could find only two cases registred in our pathology files during the last decade. two cases of testicular choriocarcinomas were also investigated. the majority of cells in all these cases comprised of cytotrophoblasts but some multinucleated syncytiotrophoblasts were also seen, so considered because of their hcg expression demonstrated immunocytochemically. in all cases, erv3 env expression was detected either in fused cells or weak and scattered in the cytotrophoblasts. the intensity of the hybridization signal in the cells that expressed erv3 env was the same as in the p l a c e n t a . testis a sample from a histologically normal testis removed in the treatment of metastatic prostatic adenocarcinoma, was investigated and cells, probably spermatogonia andlor primary spermatocytes, showed erv3 positivity, while the remainder including sertoli and leydig cells, were negative. thus erv3 expression seems to b e restricted to specific stages of differentiation. both the investigated seminomas were either negative o r had negligible erv3 expression. human embryos 12 weeks of gestation hybridization was performed on sagittal sections of a human embryo. low levels of erv expression were detected in most 116 organs. however high expression was found in the following locations: adrenal glands, rathkes pouch and (probably) the pituitary gland, nerve roots, primitive glomeruli, peripherial ganglia and structures of the primitive skin. nevertheless, this is a preliminary investigation and will be followed by a detailed study involving several fetuses. discussion in this communication we have attempted to address the question of whether the observed high expression of erv3 =in placenta specifically correlates with pregnancy, indicating that the retroviral genome can only be expressed i n syncytiotrophoblasts which are the result of fertilization. our ish data demonstrate, however, that it is probably the fusion process itself and not the fertilization which correlates with erv3 envexpression. our previous reports concerning the lack of erv3 envexpression in the hydatidiform moles and choriocarcinomas are perhaps due to scarcity of such fused cells in these tissues. the cc cell lines were predomonantly composed of cytotrophoblasts but a few syncytiotrophoblasts were also seen. our conclusions concerning erv3 env expression can be further substantiated by the fact that syncytiotrophoblasts, both in ovarian and testicular carcinomas, expressed erv3 env. in these cases it can be excluded that the choriocarcinomas are tumours derived from a fertilized ovum. the correlation between env expression and cell fusion has been shown to exist in the invitro differentiated trophoblasts when isolated trophoblasts from term placentas were allowed to mature in vitro and fuse to form syncytiotrophoblasts (3). in these model systems envexpression increases in proportion to the degree of cell fusion. it is still unclear whether env expression is a consequence of cell fusion or is causatively involved in the fusion process. however, these questions are being addressed at our laboratory by inhibiting the erv3 e n v e x p r e s s i o n by means of antisense techniques in combination with antibodies raised against structural viral proteins. the effects on the fusiogenic property of cytotrophoblasts during in vitro differentiation will be studied. the erv3 -transcripts contain sequences that can encode not only the transmembrane protein p15e and the envelope gp70 but also nonviral sequences which were found to code for a human zinc finger protein related to the drosophila transcription factor kruppel (7). this means that there exists (a) genetic information for proteins with immunosuppressive properties (p15 e) and fusiogenic capacity (gp70) and (b) a kruppel related transcription factor which may 10-945252 1 1 7 function as a suppressor "dna silencer" in the same transcript. this may support the hypothesis that the erv3 locus (or a related l o c u s ) c o u l d b e i n v o l v e d in n o r m a l i m p l a n t a t i o n , immunosuppression and control of trophoblast invasiveness. this is an attractive, yet unproven, hypothesis. this raises the question of the origin of retroviruses i n general and to thegene in particular: these subjects have been elegantly reviewed several times (2, 15, 16). since 10% of the human genome consists of sequences that are the result of reverse transcription, retroelements have contributed significantly to the evolution of human dna. e r v s show striking similarities to invertebrate transposable elements and, according to a recent hypothesis e r v genomes are of two different types. the first evolved from retroelements which through transpositions and recombinations led to the creation of retroviruses by a stepwise process. since retrotransposons miss an gene they can only transpose within individual cells. the addition of an env gene will lead to a retrovirus retroelement that can move to other cells within the same individual or between species ( interspecies transmission) and thus result in the second type of retroelement as a sequelae of an exogenous infection. according to this hypothesis env genes should also exist as "normal" cellular genes under the control of cellular promotors. this has not been shown but early data from studies in mice reflect that env proteins are expressed to a high degree in reproductive tissues especially in the epididymis, without the production of corresponding numbers of retroviral particles( 13). we do not know as yet whether the expression of ervs in human tissues (especially embryonic and reproductive) indicates that they have a function in differentiation or whether they are expressed as a consequence of differentiation. in the latter case, they could be regarded as inert remnants of germ line infections that occurred during evolution and survived in the genome as parasites since they were difficult to eliminate but gave no selective advantage to the host research with human ervs has been hampered by a lack of reagents reactive with human ervs in general and envelope proteins in particular because of constant failure to purify and characterize a human envelope protein. however, we have shown indirectly that the erv3 env gene is translated to proteins. these data were obtained by immunohistochemical investigations using antisera directed against synthetic peptides and fusion proteins, which were based on the sequence of the w( 1 0 ) . in order to study the role of erv e n v s in cell fusion and immunosuppression, i t will be necessary to block the expression of specific e r v s by antibodies, synthetic peptides and/or disrupt selected env genes in suitable cell lines. we think that erv3 is a animal (2). 118 good candidate for such experiments since it is a single copy virus and we have found that it is differentially regulated during induced differentiation of the human monocytic cell line u937 (1). however, there are also several other ervs expressed in the. human reproductive tissue including some of the multicopy genomes (reviewed in12 and 17). even if erv3does not have an effect related envs could be of importance. acknowledgements these studies were financially supported by grants from the swedish cancer society, project number 2037-b93-10 xcc references 1. andersson, a-c., botling, j., oberg, f., nilsson, k . , cohen, m. & larsson e.: expression of an endogenous retrovirus erv3 during induced differentiation of the human monocytic cell line u-937. (manuscript) 2 . baltimore, d.: retroviruses and retrotransposons. the role in shaping the eukaryotic genome.cel1: 40, 481-482, 1985. 3 . boyd, m. t., bax, c.m.r., bax, b.e., bloxam, d.l. & weiss, r.a.: the human endogenous retrovirus erv3 is upregulated i n differentiating placental trophoblast cells. virology 196: 905-909, 1 9 9 3 . 4 . daniel, j. & chilton, n.: viruslike particles in embryos and female reproductive tract. in: johnson development. mammals: 3 (north holland publ. comp. oxford), 1978. 5 . holmgren, l., flam, f., larsson, e. & ohlsson, r.: successive activation of the plateletderived growth factor b receptor and platelet derived growth factor b genes correlates with the genesis of human choriocarcinoma. cancer res. 53: 2927-2931, 1993. 6 . kato, n., pfeifer-ohlsson, s., kato, m., larsson, e., ohlsson, r., & cohen, m.: tissue specific expression of human provirus erv3 mrna in human placenta :two of three erv3 mrnas contain human cellular sequences. j . virolgy 61:2182, 1987. 7. kato, n., shimotono, d . , van leeuwen & cohen, m . : h u m a n proviral mrnas downregulated in choriocarcinomas encode a zincfinger protein related to kruppel. mol cell biol 10: 4401-4405, 1 9 9 0 . 8 . krieg, a. & steinberg, a.: retroviruses and autoimmunity. j. autoimmunity 3: 137, 1990. 119 9 . larsson, e., kato, n. & cohen m.: human e n d o g e n o u s proviruses. curr top microbiol 148:115, 1989. 1 0 . larsson, e., kato, n . , van leeuwen, d. & cohen, m.: coexpression of erv3 env protein and a zinc finger protein related to kruppel in human placenta, endometrium and macrophages. (manuscript). 1 1 . larsson, e., nilsson, o., sundstrom, p. & widehn, s . : morphological and microbiological signs of endogenous c-virus in human oocytes. int j cancer 28: 551-557, 1981. 1 2 . leib-mosch, c., brack, r., werner, t. bachmann, m., faff, o., erfle, v. & hehlmann, r.: endogenous retroviral elements in human dna. cancer res. (suppl i) 50:5636s, 1990. 1 3 . lerner, r., wilson, c.b., del villano, bc., mc conahay, p.j. & dixon, f.: endogenous oncorna virus expression in adult and fetal mice: quantitative histologic and physiologic studies of the major viral glycoprotein gp70. j exp med 143: 151-166, 1976. 1 4 . o’conell, c., o’brien, s . , nash, w. & cohen,.m.: erv3, a full length human endogenous provirus: chromosomal localization and evolutionary relationships. virology 138: 225, 1984. 1 5 . temin, h.: origin and general nature of retroviruses. in: retroviridae 1 (edited by j.a. levy plenum press new york) 1992. 1 6 . varmus, h. & brown, b.: retroviruses. in: mobile dna. (berg, d. & howe, m. eds.) american society for microbiology publ. corp. 17. wilkinson, d., mager, d. & leong, j.c.: endogenous human retroviruses. the retroviridae (j. levy ed. plenum press) in press 1 9 9 4 . 1 8 . yotsuyanagi, y. & scollosi d.: viruslike paricles and related expressions in mammalian oocytes and preimplantation stage embryos. in: van blerkom and motta, ultrastructure of reproduction 219-234 (martiinus nijhoff publ, hague) 1984. 53-108, 1989. please address correspondance to: erik larsson md, ph.d department of pathology university hospital s 751 85 uppsala tel: 46 18 663822 fax: 46 18 552739 120 upsala j med sci 99: 251-258, 1994 5.2 a new international reference preparation for proteins in human serum s. blirup-jensen and p. just svendsen clinical immunochemical department, dako a/s, 2600 glostrup, copenhagen, denmark introduction a new international reference preparation for proteins i n human serum (rpphs) was released jointly by the european bureau communitaire de rdference (bcr) and the college of american pathologists ( c m ) in july 1993. it has been prepared by the committee on plasma proteins standardization of the international federation for clinical chemistry (ifcc) and is approved by the bcr certification committee as a certified reference material (crm 470). crm 470 is intended for use as a serum matrix reference preparation from which values are to be transferred t o working calibrators and controls used in quantitative immunochemical determinations of serum proteins. it should n o t be used directly in routine laboratory assays. during the past 10 years a large number of reference materials for serum proteins have been used worldwide. these materials have values assigned by a variety of methods against several different primary materials. as a result the values for a given protein may vary as much as 50 100% depending upon the reference material used. the variation in analyte values has been obvious in quality control surveys both in the united states and in western europe (4, 15). although the solutions to this problem are complex the use of a single international reference material by all manufacturers and laboratories should reduce the variability to a substantial degree. at the same time a new reference material would be suited to modern technology like turbidimetry and nephelometry and carry assigned values also for analytes previously unaddressed. 25 1 in 1989 the ifcc committee on plasma protein standardization began the process of collecting, preparing, characterizing and calibrating a new international reference preparation for immunochemical measurement of 14 human serum proteins: transthyretin *) al-antitrypsin haptoglobin c3 complement igg albumin ceruloplasmin transferrin c4 complement iga al-acidglycoprotein"' a2-macroglobulin crp igm *) prealbumin **i orosomucoid since t h e release of the world health organization international reference standard for immunoglobulins (67/86) and for six human proteins and the united states national k f e r e n c e preparation (usnrp lot no. 12-0575c) by the centers for disease control much has been learned about the requirements for reference materials to be used in modern optical immunoassay systems (8,9, 10,11,12). ideally, primary reference materials with all values assigned against purified and highly characterized proteins are desirable. however, because of the availability of only a few such proteins and the urgent need for a new international reference material, the committee decided in 1989 to proceed with development of a preparation calibrated against the relevant world health organization (who) materials for international units (iu) and against the best available materials for mass/volume units. methods for purifying transthyretin, al-acid glycoprotein, and transferrin together with a method for dry mass determination of proteins in one electrolyte had previously been developed by a working group on plasma protein standardization of the ifcc (2, 3). using these protocols proteins of the highest purity and with the best physical characteristics available, were prepared by the protein laboratory a t the university of copenhagen (dk) for this purpose. it has been clear for years that the original material which was used for assigning mass values to usnrp for al-antitrypsin has been superseded by modern preparations which give very different calibration values. indeed the use of such preparations has been the major cause of the marked between-calibrant variation for this protein. it was thus decided to use a new preparation of al-antitrypsin prepared by the clinical chemistry laboratory, malmo general hospital, malmo (s) (7). for c-reactive protein the who reference material was used taking 1 iu as equivalent to 1 mg. the united states national reference preparation for serum proteins (usnrp lot no. 12-05750, from the centers for disease control prevention (cdcp) was used for the remaining proteins. 252 below is given a summary of the preparation of and the value assignment to rpphs/crm 470. a detailed description is given in the bcr-report (1). preparation the preparation of the rpphs/crm 470 consisted in brief of: collection of fresh serum from several hundred healthy individuals in 5 european countries. demographic data for each donor were recorded including sex, race, age, weight, blood group and country of origin. the individual collections were tested for the following infectious agents: hiv-1 and hiv-2, htlv-1, hepatitis b surface antigen and hepatitis c virus antibody. furthermore, the collections were tested for the presence of rheumatoid factors, paraproteins and other abnormalities identifiable by serum electrophoresis. phenotyping of each donor for al-antitrypsin and haptoglobin was performed. examination of the individual collections for haemolysis, hyperbilirubinaemia and turbidity, and exclusion of all collections showing abnormalities or possible interfering substances. the remaining collections were pooled, delipidated and conserved with sodium h i d e , aprotinin and benzamidine, and pure c-reactive protein was added. after a sterile filtration, vials were filled (1.0 ml/vial), freeze-dried and sealed. value assignment to rpphs/crm 470 the goal of the value transfer was to assign concentration values (in g/l) to selected proteins in the target material using accepted reference preparations. in order to do this, transfer methods have been selected and transfer protocols have been worked out. reference preparation (transfer method + transfer protocols) =+ target material. the target material is defined as the serum protein matrix with unknown concentration values (in this case rpphs/crm 470). the transfer procedure involved assignment of concentration values to the 14 proteins listed above. the reference preparation is defined as the protein preparation with known concentration values. in this value assignment, only pure protein preparations or internationally recognized serum protein reference materials were used as mentioned above. 2 5 3 the transfer methods were well established and recognized routine methods such as turbidimetry, nephelometry and single radial immunodiffusion. however, slight variations in the assay principle, in the programming of the instruments or in the reagents may lead to different results. this has lead to the necessity of method standardization (14). to minimize these factors precise transfer protocols with detailed parameter settings for a number of major instruments were developed and confirmed in a trial value assignment exercise. a new approach was developed for the value assignment to serum protein preparations (13). by applying strict theoretical considerations and identifying statistically significant and consistent sources of error. two mathematical models have been derived 1) 2) value transfer from one serum protein preparation to another serum protein preparation. (direct value transfer). value transfer from a pure protein preparation to a serum protein preparation. (indirect value transfer). both models are designed as multiple point value transfer, covering the dynamic measuring range for a specific protein. all possible measures are taken to minimize and correct for statistically significant sources of error, e.g. all volumes dispensed are controlled by weighing, and then converted to volume by dviding the weight by the density of the solution. in the first phase, the transfer procedure involves interpolation of the signal obtained for different dilutions of e.g. the reference material r on a calibration curve made from the target preparation t (fig. 1): calibration and interpolation 0 5 10 15 20 concentration in % of c(t) f i g 1. when signals of dilutions of r are interpolated on the "calibration" curve (based on t) the concentrations are obtained as relative concentrations, e.g. as percentages of the concentration i n t. 254 the relative concentrations of r measured in t units are then in the second phase plotted against the weight-corrected dilutions of the reference material (fig. 2) linear regression 0 092 q,4 0,6 098 1 dilution of the ref. ( r ) fig. 3. the relative concentrations o f (r) (in t units) plotted against the mass corrected dilutions of the reference (r). i f there is no matrix effects the regression line passes through the origin (0,o) with a slope equal to the con centration ratio c(r)/c(t). as c(r) is known, c(t) can easily be calculated. if the two materials behave similarly in the assay (i.e. there is no matrix effects) then the slope of the regression line is equal to the ratio between the concentration of the actual protein in the target material and in the reference preparation. this transfer procedure was used with great success, and data from 27 different laboratories in europe, usa, and japan form the basis of the certified values for the new international reference preparation (pphs/crm 470). the standard deviations obtained were remarkably low. this constitutes the best evidence of a very successful value assignment. availability and use of rpphs/crm 470 the new international reference preparation (rpphs/crm 470) is now available as of july 1993 through either bcr or cap. the material has been approved by the u.s. food an drug administration for distribution in the united states. 2 5 5 the ifcc committee intends that rpphs/crm 470 is to be used as a serum-based matrix reference for the transfer of values to tertiary materials (calibrators &d controls) and not for direct use in laboratory assays. the current lot (91/0619) of rpphs/crm 470 should last for several years if used in this way. the ifcc committee strongly recommends that t h e transfer of values from rpphs/crm 470 to other reference or control materials are performed using protocols similar to those used for the assignment to rpphs/crm 470. the protocols include weighing of volumes used for the reconstitutions and dilutions, assaying of several dilutions of the two materials being involved, with replication of samples and runs, and using appropriate methods of statistical analysis including linear regression through the origin (0,o) (5, 6). the use of rpphs/crm 470 will not eliminate all variations in analyte values, but it will definitely minimize them. however, it is important to stress that the use of authorized transfer methods and protocols is very essential for obtaining correct value assignments. it is to be hoped that the use of a common calibrator world-wide for serum protein analysis will result in a demonstrable improvement between laboratories and kits. however, the innate molecular heterogeneity of proteins and the changes which occur in disease will ensure that the problem of accurate protein measurement wiu never be completely solved. it is the intention of the committee to assign values for further proteins to the rpphs/crm 470 as time and funds will allow. acknowledgement we would like to acknowledge all the members of the ifcc committee on plasma protein standardization: j.t. whicher, leeds, u.k.; s. baudner, marburg, germany; j . bienvenu, lyon, france; s. blirup-jensen, copenhagen, denmark r.f. ritchie, scarborough, maine; a. carlstrom, stockholm, sweden; a.m. johnson, greensboro, north carolina; p. just svendsen, copenhagen, denmark and a.m. ward, sheffield, u.k. references 1. baudner s, bienvenu j, blirup-jensen s, carlstrom a, johnson am, millford ward a, ritehie r, svendsen pj, whicher jt. the certification of a matrix reference material for immunochemical measurement of 14 human serum proteins. crm 470. community bureeu of reference, commission ofthe european communities, brussels, 1993; 1-172. blirup-jensen s, svendsen pj. protein standardization i: purification. procedure for the purification of human prealbumin, orosomucoid and transferrin to human protein calibrators. ifcc document, stage 3, draft 2, 1983-06-01, revised 1986-04-28. 2. 256 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. blimp-jensen s, svendsen pj. protein standardization i 1 dry mass determination and characteriza tion. procedure for the determination of the dry mass concentration of a pure protein preparation dissolved in one electrolyte. ifcc document, stage 3, draft 2, 1983-06-01, revised 1986-04-28. bullock dg, dumont g, vassault a, et al. immunochemical assays of serum proteins: a european external quality assessment survey and the effects of calibration procedures on interlaboratory agreement. clin chim acta 1990; 187 21-35. davies ol, goldsmith pl. in statistical methods in research and production. 4th edition, chapters 6 & 7. isbn 0 582 45087x. longman, london and new york. hald a. statistical theory with engineering applications. chapter 18. copyright 1952 by john wiley and sons, inc. reprinted by toppan printing company, ltd., tokyo, japan. jeppsson jo. purification of al-antitrypsin to be published. reimer cb, smith sj, hannon wh, ritchie rf, van es l, becker w, markowitz h, gauldie j, anderson sg. progress towards international reference standards for human serum proteins. j biol stand 1978; 6: 133-8. reimer cb, smith sj, wells tw, nakamura rm, keitges pw, ritchie rf, williams gw, hanson 135, dorsey db. collaborative calibration of three established reference preparations for specific proteins in human sera as secondary standards for iga, igm and igg. j biol stand 1981; 9: 393-400. reimer cb, smith sj, wells tw, et al. collaborative calibration of the u : s national and the college of american pathologists reference preparations for specific serum proteins. am j clin pathol 1982; 77: 12-9. rowe ds, anderson sg, grab b. a research standard for human serum immunoglobulins igg, iga and igm. bulletin of the world health organisation 1970; 42: 535-52. rowe ds, grab b, anderson sg. an international reference preparation for human serum immuno globulins g, a and m: content of immunoglobulins by weight. bulletin of the world health organisation 1972; 46: 67-79. svendsen pj, blimp-jensen s. protein standardization iii: calibration of a reference serum. procedure for value transfer of protein concentrations. zfcc document, stage 2, draft i, 1988-06-30. svendsen, pj, blirup-jensen s. determination of human plasma proteins by turhidimetry and nephelometry. 1991. dako a/s, copenhagen, denmark. isbn 87-984223-0-8. whicher jt. calibration is the key to immunoassay but the ideal calibrator is unattainable. scand j clin lab invest 1991; 51 (suppl. 205): 21-32. 257 ujms 110 (3) bra upsala j med sci 110 (3): 193–216, 2005 inventions leading to the development of the diagnostic test kit industry – from the modern pregnancy test to the sandwich assays leif wide department of medical sciences, clinical chemistry, uppsala university, uppsala and the academic laboratory, department of clinical chemistry and pharmacology, university hospital, 751 85 uppsala, sweden abstract the universities are encouraged by the government nowadays to stimulate innovations and also to provide the proper machinery for assisting the protection and commercialisation of innovations. a better understanding of the innovation process may help to create an atmosphere suitable for inventions at the university. examples can be taken from successful innovations previously made at the university. during the 1960’s i made a series of inventions, which ultimately led to the development of the diagnostic test kit industry. the first, which i made as an undergraduate, was a simple and reliable test kit for diagnosis of pregnancy. this was followed by the solid phase radioimmunoassay and a solid phase assay for vitamin b12; next, the dual specific non-competitive sandwich assay and the in-vitro test for diagnosis of allergy, called rast (radioallergosorbent test). organon in holland with the pregnancy test kit, and pharmacia in sweden with test kits for radioimmunoassay, became pioneers among the diagnostic test kit industries. pharmacia diagnostics later became one of the leading diagnostic test kit companies in the world and has continued to be so in the field of allergy diagnosis. each one of these inventions started with a few unique observations leading to a technical development. the pregnancy test as well as the allergy test emerged from the development of assay methods with unique qualities with the subsequent search for appropriate applications. the foreseeing of a commercial value on a future market was a very important step. this was followed by the search for a suitable industry interested to exploit the invention with its new business opportunity i.e. apply for a patent, produce and market the products, which in my case consisted of the necessary reagents and equipments for particular diagnostic tests. finally, an agreement had to be settled between the entrepreneur and the inventors. this report describes these inventions and particularly discusses some crucial steps of the innovation processes. 193 received 9 may 2005 accepted 20 may 2005 introduction during the 1960’s i made a series of inventions which ultimately led to the development of the diagnostic test kit industry. the first was a simple and reliable test kit for diagnosis of pregnancy. this was followed by the solid phase radioimmunoassay and a solid phase binding assay for vitamin b12; next, the dual specific non-competitive sandwich assays and the in-vitro test for diagnosis of allergy. at that time the university showed no real interest to encourage innovations leading to commercial products. actually, i even experienced critical comments from several university colleagues. this attitude has completely changed during the last 20 years. today, the universities are encouraged by the government to stimulate innovations and also to provide the proper machinery for assisting the protection and commercialisation of innovations. a better understanding about the innovation process may help to create an atmosphere suitable for inventions at the university. examples can be taken from some of those innovations previously created at the university and having led to successful commercial products. the different steps on the road to a successful innovation are seldom described in the scientific literature. the innovation process there is no universal or single innovation process and it may vary considerably for different successful products. however, each one of the inventions that i made in the 1960’s started with a few unique observations leading to a technical development. the pregnancy test as well as the allergy test emerged from the development of assay methods with unique qualities with the subsequent search for appropriate applications. the foreseeing of a commercial value on a future market was the next very important step. this was followed by the search for a suitable industry interested to exploit the invention with the new business opportunity i.e. apply for a patent, start a production and market the products, which in my case consisted of the necessary reagents and equipments for particular diagnostic tests. finally, an agreement had to be settled between the entrepreneur and the inventors. this report will describe these inventions and particularly discuss some crucial steps of the innovation process. the pregnancy test the boyden technique the idea to develop an immunological pregnancy test emerged from some crucial experiments that i did with boyden’s passive haemagglutination inhibition method aiming to measure human growth hormone (hgh) in blood. boyden had found that protein antigens could be adsorbed to the surface of tannic acid treated sheep erythrocytes (1). he then used the salk pattern technique to detect antibodies to the protein (2). when the protein coated cells sedimented in a test tube with a hemi194 spherical bottom in the presence of antibodies they formed a mat pattern, while otherwise the pattern was a ring or dot in the centre. he also found that addition of free antigen neutralized the antibodies and inhibited the mat pattern formation. this formed the basis for a method to detect the antigen in a solution. a unique observation as a student, i met dr carl gemzell during the course in obstetrics and gynaecology at the karolinska institute and he showed me how he determined hgh in blood with a bioassay using hypophysectomized rats. in the autumn of 1959 i told gemzell that i was interested to start some research in parallel with my medical studies. gemzell, who himself had no experience in immunology, asked me if i was interested to learn some immunology and try to measure hgh in blood with an immunoassay in his laboratory at the king gustav v:s research institute at the karolinska hospital. there was a recent short communication (3) on immunoassay of pituitary hgh using the boyden technique and the same method had been used previously for the assay of insulin in buffer solutions. i developed this method for hgh but found that the instability of the hgh-coated tannic acid treated sheep erythrocytes was a great problem in the immunoassay. i solved this by formalin treatment of the cells prior to the treatment with tannic acid and the hormone. formalin treatment of erythrocytes had been used since the 1920’s in different agglutination reactions, but not for the assay of hormones (reviewed in 4). the method worked well with pituitary hgh added to a buffer solution, but not to blood serum or plasma. as the erythrocytes had been stabilized to particles after formalin treatment they were not sensitive to changes in salt concentration and ph like the intact red blood cells. after the failure to develop a method for hgh in blood i tried to measure the hormone in plain urine. the most perfect salk patterns were achieved, but hgh could be detected only after addition of pituitary hgh to the urine. the concentration of endogenous hgh in normal urine was too low. it seems that no one had ever tried to measure any compound in plain urine with the boyden technique. this trivial experiment was the first crucial step in the innovation process of the pregnancy test. i had a perfect method for urine assays and looked for a suitable application: a hormone known to be present in large amounts in urine. the immunological pregnancy test urine samples were sent to the hospital laboratory to test whether a woman was pregnant or not. animals were then used in the laboratory to detect a hormone, chorionic gonadotrophin (hcg), excreted in urine in large amounts early in pregnancy. could the immunoassay be used to measure hcg in plain urine? in february 1960 i decided to abandon the hgh project and started to immunize two rabbits with hcg to develop a pregnancy test and an assay method for hcg in urine. gemzell was in usa when i got the idea to switch to hcg and few weeks later when he returned i had, to his surprise, the first results with the hcg test. gemzell 195 immediately organized that urine samples were obtained from his ward. only urine from pregnant women gave a positive reaction in the test. the first immunological pregnancy test with a high accuracy had been developed. freeze-dried reagents i had a strong desire that my discovery would be widely used and generally replace the animal tests for pregnancy diagnosis. i realized that only a few larger laboratories could prepare the pregnancy test reagents for their own use. to perform the tests in smaller laboratories, out-patient clinics and pharmacies it was necessary to buy the reagents from some manufacturer. it would then be an advantage if the reagents could be freeze-dried. therefore, i made some experiments which were crucial for the innovation. first, i investigated the possibility of freeze-drying the hcg-coated particles and the antiserum in two different bottles for about 20 tests. these reagents gave accurate results and could be stored at different temperatures. then, i obtained ampoules with a hemispherical bottom and freeze-dried the two reagents together in these for single tests. the pregnancy test was now so simple that only urine and buffer had to be added to the ampoule. the ampoules were sealed and could be stored for future use and seemed suitable as ‘test-kits’ for a doctor’s office or a pharmacy. test-kit for a doctor’s office the principle of the immunological pregnancy test developed to a ‘kit’ for a doctor’s office is shown in fig. 1. one drop of a woman’s urine plus 0.5 ml buffer solution is added to an ampoule containing freeze-dried reagents. the freeze-dried 196 figure 1. a test kit for diagnosis of pregnancy based upon immunoassay of hcg in urine. hcg in urine from a pregnant woman binds the antibodies and prohibits the hcg-coated particles to form a mat pattern. the hcg-coated particles were sheep red blood cells treated with formalin and tannic acid and finally coated with hcg. immunogical pregnancy test, 1960 reagents: freeze-dried hcg-coated particles + antibodies against hcg procedure : add one drop of ur ine + 0.5 ml buffersolution mirror 90 minutes later pregnant non-pregnant antibodies against hcg + v v + + the particles covered the particles covered with hcg slide down with antibodies along the glass wall adhere to the and settle as a glass wall and sharp disc or form a mat ring pattern hcg in a urine sample from a pregnant wo man urine sample from a not-pregnant woma n + hcg-coated particles reagents consist of antibodies to hcg and hcg-particles. the hcg-particles are sheep red blood cells pre-treated with formalin and tannic acid and finally coated with hcg. after 90 minutes the test is read by looking in a mirror underneath the test tube at the pattern formed by the particles on the hemispherical bottom of the ampoule. if the woman is pregnant her urine contains the hormone hcg which binds the antibodies, and the hcg-particles slide along the glass wall and settle as a sharp ring or disc. urine from a not-pregnant woman contains no detectable hcg and the antibodies react with and cover the hcg-coated particles. when these antibody-hcg-coated particles sediment they adhere to the glass wall and form a mat pattern. ready to approach a manufacturer in may 1960 i felt that we were ready to approach a possible manufacturer of the reagents for the pregnancy test. the method was remarkably reliable. tests on over 300 morning urine samples had not given any incorrect result. the hcg-coated particles could be made in batches for 20,000 reactions and they were stable for months. the antiserum from one of the rabbits could be used for over one million reactions. the results from quantitative determinations of hcg in urine of 110 women in early pregnancy indicated that such determinations would be of clinical value. i made a draft of a publication with the title: ‘an immunological pregnancy test’ with leif wide and carl gemzell as authors (5). my experience with the freeze-dried reagents was so exciting, that it seemed to be the right time to contact a suitable industry, before finishing the manuscript. organon, a suitable pharmaceutical company gemzell had earlier obtained financial support from n.v. organon in holland and he suggested that he should write a letter to explore their interest. on 1st of june 1960, dr marius tausk, managing director of n.v. organon in holland, received a letter from dr carl gemzell at the karolinska hospital, stockholm, the first paragraph of which read as follows: ‘a young doctor in my laboratory, l. wide, has developed a new pregnancy test which seems to have many advantages. it is an immunological method, takes only one to two hours, gives a quantitative answer and can be performed in every laboratory or even in a doctor’s office. it is also very inexpensive. a pre-requisite condition is that some material can be prepared and ,,,furnished preferably through some pharmaceutical company. this material, as we have found, can be stored and used at convenience….’ (the letter published by m tausk in ref 6.) pregnosticon dr tausk found the letter exciting and appeared in stockholm shortly thereafter. i demonstrated and discussed the pregnancy test with him and two weeks later with mr polderman, who became responsible for marketing the test by organon under the name of pregnosticon. i also showed that the antiserum and the hcg-particles 197 could be freeze-dried separately in bottles for e.g. 20 or 100 tests. these were suitable for laboratories performing larger series of pregnancy tests using test tubes with a hemispherical bottom. by testing a serial dilution of the urine, the concentration of hcg in urine could be estimated and expressed in iu/l. dr tausk was impressed and organon decided to bring it up immediately, and an agreement was proposed which was accepted by us. organon then needed a few weeks for patent application, and after that we could send the manuscript for publication in acta endocrinologica. organon put in large resources on this project and hoped to launch the first kit in 6 months. however, the freeze-drying of the reagents that had gone without any problem for me in laboratory scale turned out to be the main technical difficulty for organon to solve, and the first pregnosticon kit was not launched until spring 1962. pregnancy diagnosis from ancient day to1960 there is no diagnostic problem that has created more ideas for tests than finding out whether a woman is pregnant or not. the oldest recorded pregnancy test is found in egyptian papyri about 3,350 years ago and was based upon the germination of seeds induced by a factor in the pregnant woman’s urine. several hundreds of methods have been described from antiquity to the present day (reviewed in 7, 8, 9,10). there was no simple test for diagnosis of pregnancy in 1960. they were all based upon injecting animals with plain urine or extracts of urine to detect a biological effect in the animal caused by the possible presence of hcg in urine. two rabbits were commonly used for a test and 8 ml of the woman’s urine was injected into their ear veins. two days later the rabbits were killed and their ovaries were inspected for presence of a bleeding. the rabbits often died due to toxic effects of the urine and the test had to be repeated with an extract of the urine. other laboratories used a mouse test with five mice which were injected twice daily for three days and then killed the following day for inspection of the ovaries. toads and frogs, where either the laying of eggs or the expulsion of spermatozoa was observed, were used in many clinical chemical laboratories and by pharmacists. these animals could be used several times. the idea to develop an immunological pregnancy test was not new in 1960. numerous attempts were made from 1902 to the 1950’s using antisera from animals immunized with extracts of human placenta or pregnancy urine, and around 1950, with purified preparations of hcg (references in 4). it can be noted that all the information needed to prepare the reagents for this pregnancy test were available nine years before i made the invention. in 1951 the boyden technique (1), the formalin treatment of erythrocytes (11), purified hcg preparations (12) and specific antisera to hcg (13) had been described in the scientific literature. a scientist who was very close to develop an immunological pregnancy test was helen strausser making a phd thesis in 1958 at rutgers university, n.j., usa on some methodological studies with a modified boyden technique (14). however, she analysed for the presence of hcg only in kaolin concentrates of 100 ml of pregnancy urine and 198 not in plain urine. its potential use as a pregnancy test is not mentioned in the thesis, and the study was never published. from bioassays to household kits for pregnancy diagnosis the pregnancy tests made in rabbits were replaced by the immunological test at the university hospital in uppsala in january 1961, when i moved to uppsala, where gemzell had become the professor of obstetrics and gynaecology. soon thereafter several other hospital laboratories in sweden started with the immunological pregnancy test assisted by the detailed prescriptions that i finally presented in my phd thesis the following year. i reported in this thesis, defended at uppsala university, that the pregnancy test was positive about 21-23 days after estimated day of ovulation and that the test had an accuracy of 99.8 per cent on 2,230 urine specimens (4). pregnosticon was a success for organon and became the most sensitive and accurate pregnancy test during a 20 years period. the variant with both reagents freezedried in the ampoule (see fig. 1) was marketed as pregnosticon-all-in. a ‘household kit’ of pregnosticon was sold to the public under the name of predictor. the women could from now on make the pregnancy tests at home. pregnancy tests performed on a glass plate, with the results given after only 2 minutes, were soon on the market both by ortho in usa and by organon in holland. however, these tests were less sensitive and less accurate. it was not until the 1980’s that pregnancy tests based upon the sandwich technique (see below), some using monoclonal antibodies, replaced the pregnancy tests developed 20 years earlier. clinical significance of quantitative immunoassays of hcg in my phd thesis (4) i described the value of a quantitative immunoassays of hcg in urine in various clinical conditions based upon more than 10,000 assays of the hcg concentration in urine. information about the hcg level in urine was shown to be of importance for diagnosis of ectopic pregnancy, threatened abortion, intra-uterine foetal death, hydatidiform mole and choriocarcinoma. the immunoassays replaced the bioassays for hcg in urine which had been used for many years in particular for the diagnosis of choriocarcinoma (15). quantitative immunoassays of hcg in urine could be made with the pregnosticon kit from organon, which became the first testkit on the market for a quantitative assay of a hormone. solid phase competitive binding assay an experiment giving rise to new inventions two of the other inventions, the solid phase competitive and the non-competitive binding assays, emerged from one particular experiment with the hcg-coated particles used in the pregnancy test. the first crucial observation was that the particle pattern could be transformed from a mat to a ring and then back to a mat and then again to a ring by adding antiserum, hormone, antiserum and hormone in a sequence. this was true even when i washed the particles between the additions of 199 200 + + antibody coated particle + antigen added two alternatives adhere to the glass wall slide down and settle as a sharp disc figure 2. alternative reactions after the addition of excess amounts of antigen (hcg) to the antibodycoated particles used in the pregnancy test. figure 3. the effect of alternately adding polyclonal antibodies against hcg and 125i-labelled hcg to hcg-coated particles. formation of several layers of antibodies and labelled hormone. antiserum and hormone. there were two possible explanations to this phenomenon as shown in fig. 2. i got a chance to explore this with 125i-labelled hcg in 1965 at the department of clinical chemistry, university hospital, uppsala. i found, as is shown in fig. 3, that antibodies and hormone could be added in several layers on the hcg coated particles. next crucial observation was that the amount of labelled hcg that bound to the particles was directly proportional to the amount of antibody added in the previous step. this observation led to a new assay principle: the noncompetitive ‘sandwich technique’ (see below). another observation was that unlabelled hcg competitively inhibited the binding of labelled hcg to the antibody coated particles. the effect of two different amounts of unlabelled hcg is shown in fig. 4. the particles were separated from the solution by centrifugation and then carefully washed several times. this was followed by determination of the radioactivity bound to the particles which decreased in relation to the amount of unlabelled hcg. this competitive binding assay, using antibody coated formalinized sheep erythrocytes and 125i-labelled hormone, was evaluated in our department in 1965 for determination of hcg, luteinizing hormone (lh), follicle-stimulating hormone (fsh) and insulin in human serum. the highly purified human pituitary gonadotrophin preparations used in the assay were isolated by dr paul roos, department of biochemistry, uppsala university. 201 + + antibody-coated formalinized red blood cell a. b . + + unlabelled antigen labelled antigen more of unlabelled antigen labelled antigen figure 4. unlabelled antigen inhibits the binding of labelled antigen to the antibody-coated particles. effects of a smaller (a) and a larger (b) amount of unlabelled antigen. the principle of the competitive binding assays the principle of the competitive binding assay with a graded response, shown in fig. 5, was first developed by arquilla and stavitsky in 1956 for immunoassay of human insulin (16). in their method the signal was the antibody triggered release of haemoglobin from intact erythrocytes attached to insulin. a considerable improvement of the competitive binding assay was made a few years later by roger ekins (17) in london, uk, and rosalyn yalow and salomon berson (18, 19) in new york, usa. they independently developed considerably more sensitive competitive binding assays by introducing radioactively labelled competing analytes used for the assay of thyroxin and insulin, respectively. from formalized erythrocytes to sephadex particles the lack of information about the levels of the protein hormones in blood and urine of the patients had been a frustrating experience for me when i worked as a gynaecologist at the uppsala university hospital. in spite of its great potentials to measure these hormones, the radioimmunoassay described by yalow and berson had not become used in the clinical routine in the hospital chemical laboratories. i assumed that this was most likely due to the cumbersome and laborious chromatoelectrophoretic method they used to separate free and bound labelled hormone (19). the technique i had developed using antibody coated particles facilitated this separation procedure to a simple washing step. however, even if the performance of the radioimmunoassay now was simple, very few hospital laboratories would have the facilities to prepare the reagents needed for the assay. i saw a large potential market for test kits containing the necessary materials for radioimmunoassays. it then seemed desirable to replace the formalized sheep erythrocytes, used as a solid matrix, with some insoluble polymer like cellulose or crosslinked dextran (sephadex). 202 ++ ++ analyte (ligand) labelled ligand binding reagent figure 5. the principle of competitive binding assays using a labelled ligand, described 1956-60 (16-19). the analyte and a fixed amount of labelled ligand compete for a limited amount of binding reagent. the amount of labelled ligand bound is inverse to the amount of analyte. dr jerker porath, at the department of biochemistry in uppsala, had developed a method to couple enzymes to sephadex using an isothiocyanate-derivative (20). i contacted him and obtained activated sephadex to which we coupled antibodies against hormones. these antibody coated particles had a high specific and low non-specific binding of the radioactively labelled antigen but the precision was so poor, that the first preparations could not be used in the immunoassay. eventually i solved this problem by ultrasonic disintegration of the sephadex particles. dr rolf axén, working in porath’s group, suggested that we should investigate cnbr-activated sephadex. already after the first experiment with coupled antibodies could i report to him, that this immunosorbent was perfect for use in the immunoassay. the cnbr-method (21) was simple to perform and i could immediately introduce it into my own laboratory. i started to investigate sephadex and cellulose particles of different sizes for use in the radioimmunosorbent (rist) assay. to pharmacia with inventions and a new business idea in march 1966, i contacted pharmacia in uppsala to present the invention and convince them to enter into a new business area: marketing reagent kits to hospital laboratories for radioimmunoassay of hormones and many other compounds in blood. there were no radioimmunoassay kits on the market at that time. i suggested that they should start with a patent application for the solid phase competitive radioimmunoassay and include the use of cnbr-activated sephadex particles. i represented the inventors and negotiated with gösta virding, managing director of pharmacia. after about six weeks of considerations at pharmacia an agreement was settled between pharmacia and the inventors: wide, axén and porath. the patent application got a priority date of 2 of june 1966, after which we could publish our results (22, 23). in the same month, after pharmacia’s priority date, kevin catt et al in australia submitted a manuscript for publication describing a similar solid phase radioimmunoassay (24). the general principle is shown in fig. 6. 203 solid phase coupled antibody labelled antigen + analyte (antigen) figure 6. solid phase radioimmunoassay. a competitive immunoassay using solid phase coupled antibody and a labelled antigen. the use of a solid phase facilitates the separation procedure of antibody bound and free labelled antigen to a simple washing step. during the negotiations with pharmacia i stressed that the solid phase competitive assay was not restricted to immunoassays and i wanted the agreement to cover non-immunological systems as well. they asked me to confirm this with an example. a few months later i presented pharmacia with a method for the assay of vitamin b12 in blood. it was a solid phase competitive binding assay using co57labelled vitamin b12 as a tracer and intrinsic factor coupled to cnbr-activated sephadex as a binding reagent (25). an agreement was settled between the inventors and the company, and in october 1966 pharmacia applied for a patent on the solid phase vitamin b12-assay. from sandwich assay to allergy test a new assay principle – a non-competitive ‘sandwich technique’ as mentioned above, the experiment with hcg-coated formalinized erythrocytes, anti-hcg and 125i-labelled hcg gave rise to the solid phase competitive binding assays as well as to the non-competitive sandwich assays. the non-competitive method was first used as a very simple method to detect antibodies using a labelled antigen (fig 7a). i then examined a non-competitive assay of antigens using the 204 b) non-competitive assay of antigens using labelled antibodies a) non-competitive assay of antibodies using labelled antigen antibody antigen i figure 7. a) solid phase coupled antigen in excess binds the polyclonal antibodies. labelled antigen in excess binds to the antibodies. unbound labelled antigen is removed by a washing step. b) solid phase coupled polyclonal antibodies in excess bind the antigen. labelled polyclonal antibodies in excess bind to the antigen. unbound labelled antibodies are removed by a washing step. combination of solid phase coupled antibodies and labelled antibodies (fig 7b). the antigen had to have at least two binding sites. in this experiment the antiserum was polyvalent with antibodies to different epitopes on the hcg molecule. the igg fraction of the antiserum was purified and used both for coupling to the solid phase and for labelling. the difference of the dose response curves of competitive and noncompetitive immunoassays, using a labelled antigen and a labelled antibody, respectively, is shown in fig 8. in both cases the bound fraction of the labelled reagent is indicated on the y-axis. the design of a dual specific allergy test the non-competitive assay using labelled antibodies had not been described in the literature. i had observed that the method had a higher sensitivity and a better precision than the competitive immunoassay. it also had a shorter reaction time, as the reagents were added in excess. if the labelled and the solid phase coupled binding reagents were directed to different sites on the analyte, a dual specific assay was obtained. excited over the wide potential of this assay method with its unique qualities, i was looking for an application with a novelty and clinical significance similar to the successful pregnancy test. i got the idea to develop an allergy test early in the autumn 1966 while listening to a dermatologist, lennart juhlin, at a meeting of the society of experimental biol205 1 10 100 1000 10000 1 0 0 0 1 0 0 0 0 100000 1000000 analyte concentration ( log scale ) 1 10 100 75 50 25 0 100nsb 0 8000 6000 10000 4000 2000 cpm analyte concentration ( log scale ) analyte concentration ( log scale ) cpm figure 8. comparison of dose response curves of competitive and non-competitive (sandwich) binding assays. the bound fraction of labelled ligand and labelled binding reagent, respectively, is shown on the y-axis. ogy in uppsala. this society, sponsored by pharmacia and organized by the department of medical chemistry, uppsala university, was a forum to increase interdisciplinary collaboration and exchange of ideas and knowledge in uppsala. juhlin had just returned from a year in philadelphia, usa and gave a lecture on urticaria, histamine and reaginic activity. i asked him during the lecture whether it had been shown that the reaginic activity in allergy was associated with an antibody. he told us that a group in denver (lead by k ishizaka) had recently shown that the reaginic activity was associated with antibodies. these antibodies did not belong to the known immunoglobulin classes �g, �m, �a or �d-globulin (iga, igm, iga or igd) but to a fifth unique immunoglobulin class called �e-globulin (26-28). with this information, i designed during the lecture a dual specific sandwich test for reaginic antibodies, as shown in fig 9. the reaginic antibody in a serum sample was first bound to the specific allergen attached to a solid phase. the allergen specific reaginic antibody was then detected with a labelled antibody specific against the �e-immunoglobulin (ige) class. a prerequisite for the test was that the reaginic antibody could be bound simultaneously both to the allergen and to the labelled antibody. a signal (e.g. radioactivity, fluorescence) from the label bound to the solid phase indicated that the patient had reaginic (ige) antibodies and was allergic against the particular allergen. the merits of the dual specific sandwich technique for this application are illustrated in fig. 10. it could be expected that the serum sample might contain antibodies of other immunoglobulin classes directed to the specific allergen. if the solid phase allergen was in a large excess, these antibodies of other immunoglobulin 206 ++ analyte solid phase capture reagent labelled binding reagent labelled antibody specific for reaginic (ige) antibodies reaginic (ige) antibody specific for the allergen allergen coupled to solid phase applied for the diagnosis of allergy: the radioallergosorbent test (rast) figure 9. general principle of the dual specific non-competitive sandwich assay. the analyte binds to the capture reagent as well as to the labelled binding reagent. the capture reagent and the labelled binding reagent are added in excess. unbound labelled binding reagent is removed by a washing step. a washing step may also be essential after the analyte is bound to the capture reagent. the application for diagnosis of allergy is described in the text. classes would hardly interfere. they would not be detected by the labelled antibody. also the labelled antibody should be added in excess, and the reaginic (ige) antibodies directed to other allergens should then not interfere. however, as a precaution, the reaginic antibodies against other allergens could be removed in a washing step before the addition of the labelled antibody. penicillin allergy on the following day, i met dr juhlin and showed him my schematic drawings of possible methods to detect the reaginic antibodies. juhlin had a particular interest in penicillin allergy and suggested the use of penicilloyl as allergen in the test. we agreed upon collaboration on penicillin allergy, which eventually resulted in several joint publications (29, 30). during the autumn 1966 juhlin sent me blood samples from penicillin hypersensitive patients, and i obtained the penicilloyl which was supposed to be used in the assay. a remarkable coincidence – a rist assay of an atypical myeloma protein was developed a remarkable coincidence then occurred. an atypical myeloma protein that did not belong to the known immunoglobulins a, d, g, and m had been detected and purified by two colleagues in uppsala: gunnar johansson and hans bennich (31). they had been unable to find a normal counterpart to the myeloma protein (called myeloma-ignd) when investigating 300 sera from blood donors and patients with a single radial immunodiffusion (srd) test. when i heard about their efforts which were presented in a seminar, i informed that radioimmunoassays had a much higher sensitivity than the srd test. the chromatoelectrophoresis, used as a separation step in the radioimmunoassay of yalow and berson (19), may not be applicable for the gammaglobulins. however, i had invented, but not yet published, a new principle which i termed radioimmunosorbent 207 igg-antibo dy against the allergen ige-antibody against another allergen ige-antibody against the allergen labelled antibody against the ige class solid phase coupled allergen figure 10. selective detection of ige-antibodies against a particular allergen in the presence of igg-antibodies to the same allergen and ige antibodies to another allergen. (rist) assay, which may be suitable in this case. i invited them to a collaboration in which they should give me the purified myeloma protein and a specific antiserum, and i should label the protein and couple the antibodies to sephadex and develop a rist assay for the myeloma protein. with this rist method, a protein was detected in normal sera and this protein had a position on electrophoresis, gelfiltration and deae-chromatography corresponding to the atypical myeloma protein. it was concluded that myeloma-ignd represented a new class of human immunoglobulin. the study was published in immunology by johansson, bennich and wide (32) with the title: a new class of immunoglobulin in human serum. in this study we found that one of 62 blood donors had an ignd level 15 times higher than the average. the critical reagent for the allergy test was in my laboratory this blood donor showed clinical signs of extrinsic asthma (32). it was a fair chance that the myeloma-ignd belonged to the immunoglobulin class �e (ige) shown to be associated with the reaginic activity (26-28, 33, 34). when i, late february 1967, heard about the asthma diagnosis, i realized that the critical reagent for the allergy test may have been in my laboratory for several months. within a few days i had obtained sera of allergic patients from the allergy outpatient clinic in the hospital and common allergens from the pharmacy for coupling to solid phase: animal dandruff from horse, cat and dog; pollen from birch and grass; and extracts of shellfish and house dust. it was an immediate success for the design of the dual specific allergy test. the antiignd was used as an anti-ige in the assay to detect allergen specific reaginic antibodies, and the results correlated excellently with those of provocation tests with the same allergens. this was a strong support for a similarity between the ignd and the �eimmunoglobulin (ige). the following month specific antisera to myeloma-ignd and �e-immunoglobulin (ige) were exchanged between the laboratories in uppsala and denver (k ishizaka). the two proteins shared major antigenic determinants, and eventually it was reported that the myeloma-ignd and the ignd in normal serum were of the ige-immunoglobulin class (35, 36). to pharmacia with the radioallergosorbent (rast) test i had kept the non-competitive sandwich assays, including the design of the allergy test, as a secret as i regarded it as a patentable invention. by the time i found out that the allergy test functioned with the anti-ignd, i disclosed the method for hans bennich and gunnar johansson. i suggested that bennich, who had the purified ignd protein, should make a larger batch of immunosorbent purified anti-ignd. then, after i had shown that this functioned well in the test, we could offer the allergy test to pharmacia to apply for a patent and to produce and market a diagnostic test kit. an agreement had to be made between the three of us and pharmacia and this should include, in addition to the invention, also the supply to pharmacia of the necessary amounts of myeloma-ignd and anti-ignd to start the production. i suggested the name radioaller208 gosorbent test or rast in analogy with rist (see above). my negotiation with gösta virding started in june 1967 but was not finalized until the beginning of september. a citation classic as soon as the patent application was sent, i started to make a draft of a manuscript by l wide, h bennich and sgo johansson with the title ‘diagnosis of allergy by an invitro test for allergen antibodies’. this was published in the lancet in november 1967 (37) and became, in 1985, a citation classic in current contents (38) as it for the first time described the allergy test as well as the dual specific non-competitive sandwich assay using labelled antibodies. further applications of the sandwich technique the use of a catching antibody and a labelled antibody to measure an antigen was published in 1969 and presented by me at a karolinska symposium on immunoassay of gonadotrophins (39) : ‘the gonadotrophin in the unknown serum is first bound to 209 solid phase catching antibody labelled antibody antigen with t wo epitopes solid phase catching antibody labelled antibody antigen with two epitopes label dual-specific, non-competitive sandwich immunoassay. figure 11. the principle of the dual specific sandwich assay using a catching antibody and a labelled antibody directed against different sites on the antigen. polymer-coupled antibodies. after washing the solid phase, labelled antibodies are added. they bind to the gonadotrophin on the solid phase and the uptake of radioactivity is in relation to the amount of hormone present in the original serum.’ in the same publication, i introduced the term ‘sandwich technique’ as a general term for the noncompetitive assays. the principle of the dual specific sandwich assay using a catching antibody and a labelled antibody directed against different sites on the antigen is shown in fig. 11. the following year i described three different variants of the sandwich technique at a european workshop in edinburgh on radioimmunoassay methods. the sensitivity of the ige assay was reported to be at least 10 times higher with the sandwich technique than with the competitive radioimmunoassay using the same antiserum (40). other names than the sandwich assays were proposed, such as ‘verknüpfungs test’(41), ‘two-site assay’ (42) and ‘immunoradiometric assay’, the latter name originally used for a competitive immunoassay with labelled antibodies (43). today the term sandwich assay is commonly used, and it covers also the non-immunological systems using the same basic principle, e.g. for dna analyses. general remarks the inventors’ choice of a suitable entrepreneur a very important step in the innovation process is the choice of entrepreneur. it was gemzell who choose organon for the immunological pregnancy test, and i had no objection to that, or suggested any alternative. the amount of resources, both number of people and laboratory space that they immediately put into the project, was impressive. organon had an excellent quality control and their product pregnosticon was highly reliable. organon was a possible choice also in 1966 for the new invention, the solid phase radioimmunoassay. however, i had lost the scientific contact with organon and their reply was unfavourable when i asked for a research grant in 1965. pharmacia in uppsala became an alternative. this company, on the other hand, was much smaller and without any experience within the diagnostic area or immunology. pharmacia had the advantage of being close to us, had a tradition of good contacts with scientists at uppsala university, and their knowledge about insoluble polysaccharides could be essential. it was also attractive to favour a swedish industry, and a maximal profit of the invention was never my objective. my co-inventors, axén and porath, had no objection to let pharmacia have the first option. it turned out to be a successful choice. the innovations from the entrepreneurs’ view the entrepreneurs, in this case organon and pharmacia, had to estimate the potential market for the product, how the project could fit into their organization, their possibility to manufacture the product and the chances to obtain a patent protection. 210 the potential market of the pregnancy test must have been obvious for organon in view of the well documented desire throughout ages of developing a simple and reliable test, which hitherto had failed. it was much more difficult for pharmacia to evaluate the potential market of test kits for radioimmunoassay. over six years had elapsed since the first radioimmunoassay methods were described, and they were not used at the hospitals in the medical service. pharmacia had limited experience in the field of immunology. conventional market researches seemed meaningless to do, as there were no similar products on the market, and only a few people could see the future demand. a pioneer research and development in a new area had to be made. i had several long discussions with gösta virding, in which i convinced him about a large fruitful future market for these products, and his never failing support for the ‘wide-project’ became important. there were periods during the development of the projects, both at organon and at pharmacia, when strong opinions expressed that the projects should be abandoned, as resources had to be taken from other areas within the companies. it was then of vital importance for the projects that they were supported by the managing directors, mauris tausk and gösta virding, respectively. the agreements between the inventors and the entrepreneurs it is a very unequal situation when a young inventor from the university negotiates with professional businessmen and lawyers. the agreement with organon in 1960 was too strictly limited to the particular technical performance of the method as we had described it. when in 1964, organon heard of strausser’s unpublished phd thesis from 1958 (14) (see above) they claimed that this anticipated the novelty of the invention and that they therefore reduced the royalty to half and limited the period for payment to ten years. on the other hand, they were willing to extend the agreement to include also developments and improvements of the test made by organon as long as the basic principle of the original test was applied. we had of course taken the latter for granted when the agreement was settled in 1960. the discussions with organon were useful experiences when i later negotiated with pharmacia. pharmacia got the rights to decide the contents of the patent applications. in the agreement with the inventors they defined the invention as it was described in their patent application. i had objections against this way of limiting the definition of the invention to pharmacia’s patent application. i could foresee many products based upon the basic principle of the invention and not covered by the patent applications. one example, which i discussed when the first agreement was signed, was the use of a solid phase coupled second antibody, later called dasp (double antibody solid phase). different variants of the sandwich technique were other important examples, as pharmacia covered the technique with patents only in the allergy field. in1968, i made numerous attempts to persuade pharmacia to apply for patents on the other different applications of the sandwich technique. when they hesitated to do this, i succeeded to get an informal agreement on the use 211 of the basic principle of the non-competitive ‘sandwich technique’. i realized that it was important for the future to describe and define in text and drawings the basic principles of all our inventions. in this work i had valuable support from björn ingelman, a successful inventor at pharmacia. during the negotiations for the rast method i wanted to change a paragraph against which i had objections in the previous agreements. it was about the rights to sell patents, patent applications and licenses to a third part. after several meetings with virding we decided that pharmacia was not allowed to do this with any of the inventions without the permission of the inventors. an independent inventor from the university has his particular view on the exploitation process. an industrial partner may look at the process and at the agreement in a somewhat different way. this difference may not have been clarified and penetrated before the agreements are signed. we experienced this with a shock when the first royalties were paid from pharmacia. we inventors lived with the view and took it for granted that the royalties were based upon the sales to independent external customers. pharmacia regarded their own foreign sales organizations, which were subsidiaries, as customers. the royalties were calculated on pharmacia’s internal price between these subsidiaries and the part of pharmacia with whom we had signed the agreement. this considerably reduced the royalties that we had expected to get. it is advisable for b,oth parties to take the time necessary to agree on all important principles in order to reach a mutual understanding and create a win-win-situation. a lawyer with experience and a special interest in these particular problems may help to avoid future surprises. it was not until the 80’s, thus after 20 years of experience with negotiations that i felt that i could negotiate on more equal terms. all agreements were rewritten and became valid between the inventors and the pharmacia group, including affiliated companies as wallac oy and electro-nucleonics inc. pharmacia obtained the rights to grant patent licenses. we also agreed upon the definitions of the innovations and signed formal agreements on the dasp-principle and the ‘sandwich technique’. some reflections even if the university showed no active interest in commercial innovations in the 1960’s, i experienced there a stimulating atmosphere for creative research. the institutions at which i worked possessed some free research money that could be used for new ideas. the institutions could supply with technical support in the form of technicians as well as instruments, and they were equipped with a workshop where an instrument maker was employed. i experienced a strong support from the staff both at king gustav v:s research institute in stockholm and at the departments in uppsala. all this facilitated a creative research work. examples have been given above on the value of seminars and meetings in local research societies for the exchange of information and start of collaborations. innovations are seldom created on order. the pregnancy test as well as the allergy test started with the development of methods with unique qualities, and then fol212 lowed by the search for appropriate applications. this sequence of ‘having found a solution and then look for its problem’ is not unusual during an innovation procedure. it can, as exemplified above, be rather trivial observations that can lead to a deviation from the original research into new areas. it can be difficult to formulate the claims in a patent application for an invention within a completely new area. the diagnostic test kit area was new for pharmacia’s patent group. they were chemists and came to focus on the chemistry in their patent applications. in 1968, i presented at pharmacia several drafts for patent protections of the general principle of the sandwich technique, covering fields outside allergy. i stressed the high sensitivity, precision and specificity, and furthermore the short reaction time and the possibility to use other solid matrixes than particles, when compared with the competitive immunoassays. we discussed it with a patent bureau and preliminary drafts were made. however, the patent group at pharmacia feared that such an application could interfere with their previous patent for the allergy test and they decided to stop these attempts. as a consequence, pharmacia had no general patent protection when, in 1976, they launched the sandwich assay of hgh using a solid phase catching antibody together with a labelled antibody. epilogue – global growth of diagnostic test kit industry from a project group to pharmacia diagnostics the so called ‘wide-project’ at pharmacia developed slowly during the first year, but after that in an increasing pace. under the successful leadership of carl-erik sjöberg it grew to a diagnostic division within pharmacia and later to the company pharmacia diagnostics. pharmacia became one of the pioneers in the field of reagent-kits for radioimmunoassay in 1971, when marketing a rist-kit for insulin. the following year they were first on the market with tests for total ige and the solid phase assay for vitamin b12. the use of paper discs instead of sephadex particles was an improvement of the sandwich techniques that i presented for pharmacia in 1968 (44). this eliminated the centrifugation step used in the original version. pharmacia became a pioneer on the market with kits for assays based upon the non-competitive sandwich technique. in 1974 the company launched the test for allergen specific ige (phadebas rast) and two years later they introduced the labelled antibody methods for the assays of hgh and total ige. they were all based upon the use of paper discs as the solid phase. pharmacia reached ‘break even’ for the diagnostic products ten years after the start of the project. about ten years later the number of employees had increased to 1,500. when the profits from the diagnostic products rapidly increased, pharmacia showed its gratitude to uppsala university with a generous donation for research. during a long period of time pharmacia diagnostics was one of the leading diagnostic test kit companies in the world and has continued to be so in the field of allergy diagnosis. 213 the diagnostic test kit industry grows fast on a global multi thousand million dollar market the non-competitive sandwich technique combines an ultra-high sensitivity, a high specificity and a short reaction time, which are features of crucial importance particularly in many clinical applications. in 1975 köhler and milstein (45) described a method for in vitro synthesis of monoclonal antibodies. such monoclonal antibodies are particularly useful in sandwich methods and offer advantages for the kit manufacturers by the means by which these antibodies are isolated and produced. large amounts of antibodies can be synthesized against a single antigenic site. a few immunoassay-kit manufacturers realized the advantages of the sandwich technique in the 70’s, but during the following decade the number of such companies increased rapidly. several companies applied for patents on the use of sandwich techniques in the 80’s causing many bitter patent disputes (46). the diagnostic test kit market grew globally and was a multi thousand million dollar market in the 90’s. the sandwich technique is now used in most of the immunoassays and in other fields such as the array-based dna-analysis. the pregnancy tests in use today are also based upon the sandwich technique. the radioisotopes that successfully had been used as labels in the binding assays were to a large extent replaced by enzyme, fluorescence, and chemiluminescent labels. competitive binding techniques are still applied for assay of small size analytes, and the vast majority uses the solid phase separation system. acknowledgements i thank christer bengtsson for making the prototypes of the figures in corel draw. references 1. boyden sv (1951) the adsorption of proteins on erythrocytes treated with tannic acid and subsequent hemagglutination with antiprotein sera. j exper med 93: 107-120. 2. salk je (1944) a simplified procedure for titrating hemagglutinating capacity of influenza-virus and the corresponding antibody. j 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use in assaying insulin. j clin invest 35: 458-466. 17. ekins rp (1960) the estimation of thyroxin in human plasma by an electrophoretic technique. clin chim acta 5: 453-459. 18. yalow rs, berson sa (1959) assay of plasma insulin in human subjects by immunological methods. nature 184: 1648-1649. 19. yalow rs, berson sa (1960) immunoassay of endogenous plasma insulin in man. j clin invest 39: 1157-1175. 20. axén r, porath j (1966) chemical coupling of enzymes to cross-linked dextran (sephadex). nature 210: 367-369. 21. axen r, porath j, ernbach (1967) chemical coupling of peptides and proteins to polysaccharides by means of cyanogens halides. nature 214: 1302-1304. 22. wide l, porath j (1966) radioimmunoassay of proteins with the use of sephadex-coupled antibodies. biochim biophys acta 130: 257-260. 23. wide l, axén r, porath j (1967) radioimmunosorbent assay for proteins. chemical couplings of antibodies to insoluble dextran. immunochemistry 4: 381-386. 24. catt k, niall hd, tregear gw (1966) solid phase radioimmunoassay of human growth hormone. biochem j 100: 31c-33c. 25. wide l, killander a (1971) a radiosorbent technique for the assay of serum vitamin b12. scan j clin lab invest 27: 151-159. 26. ishizaka k, ishizaka t (1966) physicochemical properties of reaginic antibody. i. association of reaginic activity with an immunoglobulin other than �aor �g-globulin. j allergy 37: 169-185. 27. ishizaka k, ishizaka t, lee eh (1966) physicochemical properties of reaginic antibody. ii. characteristic properties of reaginic antibody different from human �a-isohemagglutinin and �dglobulin. j allergy 37: 336-349. 28. ishizaka k, ishizaka t, hornbrook mm (1966) physicochemical properties of reaginic antibody. iv. presence of a unique immunoglobulin as a carrier of reaginic activity. j immunol 97: 75-85. 29. wide l, juhlin l (1971) detection of penicillin allergy of the immediate type by radioimmunoassay of regains (ige) to penicilloyl conjugates. clin allergy 1: 171-177. 30. juhlin l, wide l (1972) ige antibodies and penicillin allergy. in: dash, jones heh (eds) mechanisms in drug allergy. a glaxo symposium. curchill livingstone, edinburgh and london. 139147. 31. johansson sgo, bennich h (1967) immunological studies of an atypical (myeloma) immunoglobulin. immunology 13: 381-394. 32. johansson sgo, bennich h, wide l (1968) a new class of immunoglobulin in human serum. immunology 14: 265-272. 33. ishizaka k, ishizaka t (1966) physicochemical properties of reaginic antibody. iii. further studies on the reaginic antibody in the �a-globulin preparations. j allergy 38: 108-119. 34. ishizaka k, ishizaka t, hornbrook mm (1966) physicochemical properties of reaginic antibody. v. correlation of reaginic activity with �e-globulin antibody. j immunol 97: 840-853. 35. bennich h, ishizaka k, ishizaka t, johansson sgo (1969) a comparative antigenic study of gamma e-globulin and myeloma-ignd. j immunol 102: 826-831. 36. bennich h, ishizaka k, johansson sgo, rowe ds, stanford dr, terry wd (1968) immunoglobulin e: a new class of human immunoglobulin. bull world health organ 38: 151-152. 215 37. wide l, bennich h, johansson sgo (1967) diagnosis of allergy by an in-vitro test for allergen antibodies. lancet ii: 1105-1107. 38. citation classic. (1985) current content 28: 19. 39. wide l (1969) radioimmunoassays employing immunosorbents. in: diszfalusy e (ed) immunoassay of gonadotrophins. acta endocrinol (copenh) vol 63: suppl. 142, 207-221. 40. wide l (1971) solid phase antigen-antibody systems. in: kirkham ke, hunter wm (eds) radioimmunoassay methods. churchill livingstone, edinburgh and london. 405-412. 41. habermann e (1970) ein neues prinzip zur quantitativen bestimmung hochmolekularer antigene (verknüpfungstest) und seine anwendung auf tetanustoxin, serumalbumin und ovalbumin. z klin chem u klin biochem 8: 51-55. 42. addison gm, hales cn (1971) two site assay of human growth hormone. horm metab res 3: 59-60. 43. miles lem, hales cn (1968) labelled antibodies and immunological systems. nature, lond 219: 186-189. 44. wide l, aronsson t, fagerberg e, zetterström o (1972) radioimmunoassay of allergen-specific ige. in: allergology. excerpta medica 251: 85-91. 45. köhler g, milstein c (1975) continuous culture of focused cells secreting specific antibody of predefined specificity. nature 256: 495-497. 46. ekins r (1989) a shadow over immunoassay. nature 340: 256-258. corresponding author: professor leif wide department of clinical chemistry university hospital se-751 85 uppsala, sweden leif.wide@medsci.uu.se 216 vol_115_004_sups_a_503906 221..231 upsala journal of medical sciences. 2010; 115: 221–231 original article metabolic effects of a late hypotensive insult combined with reduced intracranial compliance following traumatic brain injury in the rat konstantin salci1, per enblad1, michel goiny2, charles f. contant3, ian piper4 & pelle nilsson1 1department of neurosurgery, uppsala university hospital, uppsala, sweden, 2department of physiology and pharmacology, karolinska institute, stockholm, sweden, 3department of neurosurgery, baylor college of medicine, houston, usa, and 4institute of neurological sciences, southern general hospital, glasgow, uk abstract introduction. traumatic brain injury makes the brain vulnerable to secondary insults. post-traumatic alterations in intracranial dynamics, such as reduced intracranial compliance (ic), are thought to further potentiate the effects of secondary insults. reduced ic combined with intracranial volume insults leads to metabolic disturbances in a rat model. the aim of the present study was to discern whether a post-traumatic hypotensive insult in combination with reduced ic caused more pronounced secondary metabolic disturbances in the injured rat brain. materials and methods. rats were randomly assigned to four groups (n = 8/group): 1) trauma with hypotension; 2) trauma and reduced ic with hypotension; 3) sham injury with hypotension; and 4) sham injury and reduced ic with hypotension. a weight drop model of cortical contusion trauma was used. ic was reduced by gluing rubber film layers on the inside of bilateral bone flaps before replacement. microdialysis probes were placed in the perimeter of the trauma zone. hypotension was induced 2 h after trauma. extracellular (ec) levels of lactate, pyruvate, hypoxanthine, and glycerol were analyzed. results. the trauma resulted in a significant increase in ec dialysate levels of lactate, lactate/pyruvate ratio, hypoxanthine, and glycerol. a slight secondary increase in lactate was noted for all groups but group 2 during hypotension, otherwise no late effects were seen. there were no effects of reduced ic. discussion. in conclusion, reduced ic did not increase the metabolic disturbances caused by the post-traumatic hypotensive insult. the results suggest that a mild to moderate hypotensive insult after initial post-traumatic resuscitation may be tolerated better than an early insult before resuscitation. key words: hypotension, intracranial compliance, microdialysis, rat, traumatic brain injury introduction traumatic brain injury (tbi) is a major cause of death and disability globally (1,2). outcome for patients with tbi has improved over the past decades due to improved pre-hospital and neurointensive care that has focused on avoiding/reducing secondary insults in these patients (3–8) rather than through use of new pharmacological treatments for tbi. despite promising results from experimental drug trials, none of the clinical trials of new drugs have been able to show a significant effect on outcome for tbi patients (2). it appears that further improvements in care require continued focus on pathophysiological mechanisms responsible for the enhanced vulnerability of the brain to secondary insults after trauma. understanding the effects of different secondary insults requires multimodality monitoring to elucidate each insult’s effects on the tissue and at what time point the different insults are dangerous to the patient. systemic hypotension is an insult that occurs in 15%–35% of patients with severe head injury and is associated with a significant increase in mortality and morbidity (9–16). both early (from injury through correspondence: pelle nilsson, department of neurosurgery, uppsala university hospital, s-751 85 uppsala, sweden. fax: +46 18 55 86 17. e-mail: pelle.nilsson@neurokir.uu.se (received 1 april 2010; accepted 21 june 2010) issn 0300-9734 print/issn 2000-1967 online � 2010 informa healthcare doi: 10.3109/03009734.2010.503906 resuscitation) and late (in the intensive care unit (icu)) post-traumatic systemic hypotension aggravates tbi (14,16). transient hypotension is common in the icu in tbi patients and can be associated with poorer outcome (17). a recent clinical study has shown that post-traumatic hypotension independently increased the risk of mortality, but it did not increase mortality in tbi patients more than it did for non-tbi patients (18). experimental studies have also shown synergistic effects of tbi and early secondary systemic hypotension resulting in increased contusion volume (19) and reduction of high-energy phosphates (atp) (20), cerebral blood flow (cbf) (21), eeg activity (21), and cerebral oxygen delivery (22). in one tbi model, post-traumatic hypotension and hypoxia had different degrees of impact on brain energy metabolism depending on the timing of when the insult occurred (23,24). the status of intracranial pressure/volume dynamics is another factor that influences the effect of secondary insults upon tbi. reduced intracranial compliance (ic), i.e. reduced ability to compensate for additional intracranial volume, is a common clinical situation following a traumatic brain injury. a reduction of ic may under certain circumstances lead to hemodynamic effects followed by metabolic disturbances and ischemia (25–28). it seems that reduced ic after trauma may enhance the effect of various secondary insults and is an important observation with implications for the management of head-injured patients. in order to facilitate basic studies of these mechanisms a rodent model of tbi has been established where it is possible to decrease ic/pressure volume index (pvi) with negligible effects on intracranial pressure (icp) (29). results from this model show that decreased ic, in combination with superimposed icp insults, can lead to metabolic disturbances in the tissue (30). many experimental studies focus on the direct brain tissue effects of tbi in isolation. this pathophysiology is important to study, but it is very difficult to intervene clinically at this phase because of the inevitable delays between the primary insult and initiation of emergency care. experimental models resembling the post-resuscitation phase in treatment are important to study to increase our understanding of the optimal timing and treatment approach to the patient. the aim of the present experimental study was to determine whether a late post-traumatic hypotensive insult caused a secondary metabolic disturbance in the rat brain following focal cortical injury and whether reduced ic had an impact on such a response. materials and methods thirty-two male sprague-dawley rats (360–540 g) (alab, stockholm, sweden) were used and had free access to food pellets and water. anesthesia was induced by placing the rats in a gas mixture of halothane� 3% and o2:n2o (1:1). thereafter, they were intubated and mechanically ventilated (ugo basil rodent ventilator, varese, italy). anesthesia was maintained with isoflurane (1.2%–1.8%) and o2:n2o (1:2). arterial and venous catheters were surgically implanted into tail vessels and the right femoral artery. mean arterial blood pressure (mabp) was continually measured. arterial blood gases were checked regularly throughout the experiment and were maintained within normal levels, i.e. pco2 4.5–5.5 kpa, po2 12–20 kpa, and ph 7.35– 7.45. body temperature was monitored with a rectal probe and kept between 37.0–37.5�c with a heating pad. after catheter preparation the animals were placed in a stereotaxic frame. the skull was exposed and a burr hole (1.5 mm diameter) was made 1 mm caudal to bregma and 1.6 mm lateral to the midline for access to the left lateral ventricle. bilateral craniotomies (6 � 9 mm) were made over the parietal cortex with the center 3 mm caudal to the bregma on the right side and slightly more caudal on the left side due to the ventricular burr hole (29). a two-way single lumen system (pk kit idt-xx, ohmeda pte ltd, singapore), filled with physiological saline, was used for intracranial pressure (icp) measurements. the tubing system was in open communication between the left lateral ventricle and a pressure transducer. an amplifier (transducer interface, harvard apparatus, edenbridge, england) was connected to a computer running labwindows cvi software for continuous online acquisition, display, and storage of the icp signal. a 24-gauge spinal needle was used as a ventricular catheter (outer diameter 0.55 mm). it was stereotactically inserted in the left ventricle. during insertion continuous registration of icp was carried out. intraventricular placement was confirmed by the sudden decrease in icp upon ventricular puncture, a rapid increase in icp due to jugular vein compression, and the presence of cardiopulmonary pulsation on the monitor screen. the ventricle was punctured at a depth of 3.0–3.5 mm from the dura. for icp recording the zero point was adjusted to the level of the external auditory canals. icp was recorded continuously. a microdialysis (md) probe was stereotactically inserted through a small incision in the dura medially in the perimeter of the trauma zone (right side). the md probe was perfused for 2 h before the trauma, and the last three 10-minute md dialysate samples 222 k. salci et al. were averaged to obtain a base-line value. before trauma or sham injury the md probe and icp needle were removed and stereotactically reinserted within 1 and 3 min, respectively. the md probe had a membrane length of 2 mm (mab 6.14.2; metalant ab, stockholm, sweden). mock csf (containing na+ 140 mm, k+ 2.7 mm, ca2+ 1.2 mm, mg2+ 0.9 mm, and cl�147 mm) was perfused through the probe at a flow rate of 2 ml/min using a microinjection pump (cma/100; cma/microdialysis, stockholm, sweden). after 1.5 h of equilibration the md samples were collected in 10-min fractions. trauma was produced by dropping a 21 g weight from 35 cm onto a piston (diameter 4.5 mm) resting on the dura. the piston was constructed to allow a maximum compression of 1.5 mm (29,31). intracranial volume was altered in the different groups by gluing either 0 or 3 layers of rubber film (0.18 mm thick; kofferdamm medium, dental ab, stockholm, sweden) on the inside of the bone flaps before they were glued back in place. histoacryl (b braun melsungen, germany) tissue glue was used for both procedures (29). one layer of rubber film constituted a volume of approximately 10 ml. the bone flaps were replaced under microscopic control within 5–10 min of impact. two hours after impact hypotension was induced by withdrawal of blood through the femoral artery. earlier experiments from our department, using the same degree of tbi, showed that the observed metabolic disturbances after tbi normalized approximately 2 h after impact (31). heparinized syringes were used to draw blood until the mabp reached approximately 50 mmhg and was maintained at this level for 30 min by further withdrawal of blood or re-injection of blood as needed. normally 5–8 ml blood withdrawal was required to obtain mabp of 50 mmhg. the blood was kept warm (37�c). after 30 min of hypotension, reperfusion started with slow re-injection of blood as required to obtain normal mabp. see figure 1 depicting the flow chart of the experimental protocol. at the end of the experiment, the thorax of the animal was opened and the heart catheterized through the left ventricle wall. the right atrium was opened as an outlet. the animal was perfusion-fixated by infusion of 250 ml of physiological saline followed by 250 ml of 4% formaldehyde solution. the animal was decapitated and the brain kept in 4% formaldehyde solution. the brains were cut in coronal sections over the sites of the ventricular needle, md probe, and craniotomy areas, and analyzed under the operation microscope. analyses of the md fractions (random samples) were performed. analyses of energy metabolites (lactate, pyruvate, and hypoxanthine) were made in order to assess the metabolic state. glycerol was analyzed as a marker of cell membrane degradation. lactate, pyruvate, and glycerol were analyzed with an enzymatic colorimetric method using the cma/600 microdialysate analyzer (cma microdialysis, stockholm, sweden). hypoxanthine was measured using highperformance liquid chromatography with uv detection at 254 nm. briefly, a reverse phase 100 � 4 mm nucleosil-100 c18 column (knauer gmbh, germany) was eluted with a mobile phase constituted of 0.01 m sodium phosphate (ph 6) and 6% methanol. the signal of the uv detector (bas, usa) was evaluated with an electronic sp4290 integrator (spectra physics, usa) against freshly prepared standard solutions. study design and statistical methods the animals were randomly assigned to one of the following four groups (n = 8/group): sham injury with 0 or 3 layers of rubber film and hypotensive insult (groups sh0 and sh3), or trauma injury with 0 or induction trauma/sham impact surgical preparation md equilibration md base-line start phlebotomy start reperfusion hypotension 30 minutes minutes–120 –60 0 60 120 180 240 end t1 t2 t3 t4 t5 figure 1. flow chart of the experiment. time periods; t1 pre-impact, t2 post-impact, t3 pre-insult, t4 insult, and t5 reperfusion. md = microdialysis. late hypotension, metabolism, and traumatic brain injury 223 3 layers of rubber film and hypotension insult (groups tr0 and tr3). for statistical analyses the experiment was divided into five time periods: t1: 30 min before trauma (base-line or pre-impact period), t2: 30 min after trauma (post-impact period), t3: 30 minutes before hypotension (pre-insult period), t4: over the 30 min hypotensive insult (insult period), and t5: 30 min after the insult (reperfusion period) (figure 1). for each of the variables the average per animal was calculated for each time period. one animal (sh0) had missing data for the blood pressure and icp at t5 due to sudden death during this period. hypoxanthine values were missing for two animals in group tr0 (one at all time periods and the other at t3, t4, and t5) due to technical problems. some animals had extra md measurements due to the extra time needed from the start of phlebotomy until bp reached near to 50 mmhg, and these extra measurements were deleted from all analyses. the averages for each animal and time period were then analyzed in an ancova model including the factors: treatment-group (the four groups sh0, sh3, tr0, and tr3), time, and the interaction term (treatment-group*time). icp was included as a covariate in the analyses of all variables except icp. the statistical significance of each effect was determined from the model, and the pair-wise comparisons between groups and time periods were also derived from the model. a probability of 0.05 or less was considered as statistically significant. as the residuals for the variables l/p ratio (lactate/ pyruvate ratio), glycerol, hypoxanthine, and lactate were extremely skewed, these variables were transformed to their natural logarithms before they were analyzed. the data were also summarized by means, standard deviations, medians, minimum, and maximum values per treatment group and time. ethics the experimental protocol was approved by the ethics committee for uppsala university. results physiological parameters and macromorphology blood gases, temperature, and bp for each group prior to impact are shown in table i. blood gases and temperature were maintained at these levels throughout the experiment, with the exception that blood gases could not be checked during the hypotensive insult or reperfusion periods, due to practical reasons. there were no signs of hematoma on the coronal sections of the perfused brains. overall effects figures 2–4 and table ii contain the overall results of the analyses for bp, icp, and each of the dialysates. the time effect and the interaction between group and the time of measurement were the most commonly significant results. a significant time effect indicates that the means at each time differed from each other. the time-by-group interaction indicates that the difference in the mean between the different groups depended on time. for icp this was mainly due to a layers–time interaction and for the dialysates mainly due to an injury–time interaction. a more detailed presentation of the results follows below. table iii shows p values for comparisons within and between groups for different time periods (all time periods not shown). trauma effect and the influence of layers the changes in bp are illustrated in figure 2 and table iii. the average bp before impact in the different groups varied between 86 mmhg and 92 mmhg. the impact resulted in a slight (4.5–5 mmhg) but significant bp increase in the trauma injury groups (tr0 and tr3). figure 3 shows icp by time. baseline (pre-impact) icp was 7.75–9.08 mmhg. during the post-impact period there was a slight but significant icp increase in the groups with three layers (table iii). table i. the different groups and physiological parameters prior to impact. group n injury rubber film layers hypotension po2 (kpa) pco2 (kpa) ph temp ( �c) mabp (mmhg) sh0 8 sham 0 yes 17.9 ± 2.3 5.33 ± 0.31 7.39 ± 0.030 37.1 ± 0.14 90.8 ± 5.92 sh3 8 sham 3 yes 19.0 ± 1.7 5.01 ± 0.35 7.39 ± 0.036 37.0 ± 0.14 91.7 ± 10.3 tr0 8 trauma 0 yes 18.4 ± 1.9 5.03 ± 0.75 7.39 ± 0.044 37.1 ± 0.14 86.3 ± 7.74 tr3 8 trauma 3 yes 17.6 ± 1.9 5.05 ± 0.36 7.38 ± 0.023 37.0 ± 0.13 89.4 ± 6.80 values are shown as mean ± sd. group definition: sh/tr: sham or trauma injury (0/3 = no. of layers). 224 k. salci et al. in the groups with zero layers there was no statistically significant difference. the injury itself did not increase icp during the post-impact period. during the preinsult period there was no statistically significant difference in icp between the groups. figure 4 shows neurochemical changes over time. the average base-line values were: lactate 0.12– 0.14 mmol/l; l/p ratio 30–39; hypoxanthine 0.41– 0.79 micromol/l; glycerol 2.32–4.58 micromol/l. the trauma resulted in a multifold increase for all metabolites during the post-impact period in the trauma injury groups, whereas in the sham injury groups a slight increase was seen that reached significance for hypoxanthine (groups sh0 and sh3) and lactate (group sh3) (table iii). the increase during the post-impact period was significantly higher for the trauma injury groups as compared to the sham injury groups. there was no effect of reduced compliance during the post-impact period. hypotensive insult effect and influence of layers the hypotensive insult resulted in a decrease in bp for all groups (figure 2, tables ii and iii). during the hypotensive insult there was a consistent decrease in icp for all groups compared to the pre-insult period (figure 3 and table iii). the reperfusion resulted in the subsequent return to or slight overshot of preinsult icp levels. the effect of hypotension on icp was the same for all groups. the neurochemical changes induced by the hypotensive insult are shown in figure 4. the insult caused a slight but statistically significant increase in l/p ratio for group sh3 and a slight increase in lactate compared to pre-insult values for all groups, reaching statistical significance for all except tr3. during the reperfusion period the l/p ratio decreased for group sh3 to pre-insult values. lactate remained increased during reperfusion in the trauma injury groups. during the reperfusion period lactate remained higher than the pre-insult period for all groups except tr3. for the other dialysates no significant effects were seen. there were no obvious effects related to layers (e.g. compliance level) during the insult or reperfusion periods. discussion the present study shows that a late hypotensive insult, following a focal traumatic brain injury, affects brain 0 20 40 60 80 100 120 t1 preimpact t2 postimpact t3 preinsult t4 insult t5 reperfusion time period b lo o d p re s s u re ( m m h g ) sh0 sh3 tr0 tr3 figure 2. blood pressure during the time periods (mean ± sd of 30 time points/time period). for statistics and significance see tables ii and iii. time periods t1–t5. sh0/3 and tr0/3 = sham/trauma injury with 0/3 layers of rubber film. late hypotension, metabolism, and traumatic brain injury 225 energy metabolism with increases in lactate and l/p ratio but not to the extent that the energy depletion leads to amp or membrane degradation. reduced ic does not lead to a more pronounced disturbance. the results from this study are similar to the metabolic and membrane responses seen after diffuse axonal injury combined with late hypotension and hypoxia (24). these results suggest that both diffuse and focal brain injury have similar temporal patterns for tissue reaction in the acute phase after the trauma. the mild metabolic disturbance from a delayed hypotensive insult compared to the more pronounced changes seen in an early hypotensive insult helps to illustrate the complex dynamics of intracranial pathophysiology in tbi where very short time spans can lead to changes in tissue reactions to a secondary insult. brain tissue survival in traumatic injury is dependent on adequate supplies of oxygen and glucose so that atp production can be maintained, as is the case in all central nervous system injury. the difference in tissue response between early and late insults is probably related to effects of relative ischemia. earlier studies in a tbi model similar to the present one, but without reduced intracranial volume, have shown: increases of extracellular excitory amino acid levels (31), increase of interstitial [k+] and decrease of interstitial [ca2+] (32), and post-traumatic seizures immediately following the trauma (33). all of these disturbances result in an increased energy demand. in this model it has also been shown that there is a decrease in cerebral blood flow directly after the trauma that persists for 80 minutes (34). the combined effects of these disturbances lead to metabolic disturbances that normalize by 120 minutes post trauma (31). in the present model, which is a modification of the original model (31), it has also been shown that metabolic disturbances normalize within 120 minutes (30). during this recovery period the tissue is sensitive to secondary insults. if the injured brain is subjected to icp insults a more profound metabolic disturbance occurs with increases in interstitial levels of hypoxanthine and glycerol in the trauma group with reduced ic (30). the mild disturbances in metabolism seen in the present study show that once the tissue has recovered from the trauma then it can tolerate at least one secondary insult at a time even if ic is reduced. another factor that may influence the present response is that a hypotensive insult of this magnitude will lead to a decrease in cerebral blood volume, which in turn lowers icp. 0 2 4 6 8 10 12 14 16 18 20 t1 preimpact t2 postimpact t3 preinsult t4 insult t5 reperfusion time period ic p ( m m h g ) sh0 sh3 tr0 tr3 figure 3. icp during the time periods (mean ± sd of 30 time points/time period). for statistics and significance see tables ii and iii. time periods t1–t5. sh0/3 and tr0/3 = sham/trauma injury with 0/3 layers of rubber film. 226 k. salci et al. 0 0.1 0.2 0.3 0.4 0.5 0.6a. t1 preimpact t2 postimpact t3 preinsult t4 insult t5 reperfusion time periods l a c ta te ( m m o l/ l ) sh0 sh3 tr0 tr3 0 10 20 30 40 50 60 70 80 90 100 t1 preimpact t2 postimpact t3 preinsult t4 insult t5 reperfusion time period l /p r a ti o sh0 sh3 tr0 tr3 b. figure 4. lactate (a), l/p ratio (b), hypoxanthine (c), and glycerol (d) during the time periods (mean ± sd of 3 samples/time period). for statistics and significance see tables ii and iii. t1–t5: time periods. sh0/3 and tr0/3 = sham/trauma injury with 0/3 layers of rubber film. late hypotension, metabolism, and traumatic brain injury 227 at what level physiological changes become insults is a question that is central in discussions concerning intensive care. that the hypotensive insult in this study is too mild to cause metabolic disturbances in the tissue is not supported by findings from other studies using the same level of hypotension. geeraerts et al. (24) have shown that tbi and a combined secondary insult of hypotension and hypoxia leads to metabolic disturbances. in their study, with two different time points for a 15-minute long combined 0 1 2 3 4 5 6 7 t1 preimpact t2 postimpact t3 preinsult t4 insult t5 reperfusion time period h y p o x a n th in e ( m m o l/ l ) sh0 sh3 tr0 tr3 c. 0 5 10 15 20 25 30 t1 preimpact t2 postimpact t3 preinsult t4 insult t5 reperfusion time period g ly c e ro l (m m o l/ l ) sh0 sh3 tr0 tr3 d. figure 4. (continued). 228 k. salci et al. insult of hypoxia (pao2 5.3 kpa) and hypotension (mabp 40 mmhg), there was an increase of lactate and l/p ratio at both time points. the late insult at 225 minutes post trauma did not affect cerebral metabolism to the same extent, while the early insult at 25 minutes had a more pronounced effect. these studies also showed that the hypotensive insult itself led to metabolic disturbances if it occurred at 25 minutes post sham trauma and that this disturbance was of the same magnitude as that seen with a late insult after trauma. all of these responses show that the insult is sufficient to elicit a metabolic reaction in the tissue. the findings from the present study, earlier studies with this model, and the studies by geeraerts et al. in a different model, but with similar results, shed light on the problems related to development of neurocritical care in tbi. earlier, experimental studies were a way of dealing with the complex pathology related to tbi. most studies have focused on a single or a few phenomena during the acute phase and have used continuous or snap-shot methods to monitor changes. because of practical and economical reasons most experimental studies only cover a few hours of observation. in contrast, clinical studies have had access to continuous monitoring, around the clock and every day of the week, for as long as the patient has been in the icu. however, these studies have relied on paper documentation of physiological parameters which has only given summary information on care and pathophysiological disturbances. with the table ii. probability values for the different effects. dependent group time icp group*time 1. blood pressure 0.4821 <0.0001 0.0929 0.0689 2. icp 0.1577 <0.0001 – 0.0464 3. log l/p ratio 0.2774 <0.0001 0.1229 0.0007 4. log glycerol 0.6700 <0.0001 0.8954 0.0108 5. log hypoxanthine 0.0971 <0.0001 0.0418 <0.0001 6. log lactate 0.1871 <0.0001 0.4357 <0.0001 table iii. comparisons within and between groups (p values from contrasts in ancova model). blood pressure icp lactate l/p ratio hypoxanthine glycerol t2–t1, sh0 0.0788 0.7773 0.0807 0.9197 0.0152 0.0555 t2–t1, sh3 0.3047 0.0004 0.0036 0.4605 0.0007 0.0525 t2–t1, tr0 0.0489 0.4717 <0.0001 0.0001 <0.0001 <0.0001 t2–t1, tr3 0.0315 0.0001 <0.0001 <0.0001 <0.0001 <0.0001 t2–t1, sh3–sh0 0.6418 0.0060 0.3438 0.5463 0.3964 0.9238 t2–t1, tr0–sh0 0.8761 0.4785 <0.0001 0.0065 <0.0001 0.0005 t2–t1, tr3–sh3 0.3818 0.7826 <0.0001 0.0001 <0.0001 0.0009 t2–t1, tr3–tr0 0.8159 0.0198 0.6115 0.3985 0.4634 0.9520 t4–t3, sh0 <0.0001 0.0034 0.0423 0.2167 0.6306 0.6375 t4–t3, sh3 <0.0001 0.0055 0.0010 0.0145 0.5647 0.3480 t4–t3, tr0 <0.0001 0.0487 0.0092 0.5055 0.6444 0.6548 t4–t3, tr3 <0.0001 0.0006 0.5051 0.9506 0.1772 0.4177 t4–t3, sh3–sh0 0.0040 0.9094 0.3348 0.3668 0.4366 0.7324 t4–t3, tr0–sh0 0.0888 0.4823 0.6926 0.6669 0.9819 0.5052 t4–t3, tr3–sh3 0.1017 0.6286 0.0504 0.0650 0.1522 0.1980 t4–t3, tr3–tr0 0.6699 0.2843 0.1640 0.6011 0.5595 0.7783 t1–t4 = time periods; sh/tr = sham or trauma injury (0 or 3 indicates number of layers). late hypotension, metabolism, and traumatic brain injury 229 advent of modern computers it is now possible to achieve detailed documentation of the pathophysiology occurring with the patient over longer periods of time (35). to fully understand the impact of the type of insult studied in this paper one must move on into the clinical setting and use long-term, multimodality, high-resolution monitoring in the resuscitated patients. with this type of approach it will be possible to identify which findings from the laboratory environment can be applied in the clinical setting. acknowledgements the study was supported by the mattson foundation and the swedish research council (grant number 2002/a0799). professor urban ungerstedt (department of physiology and pharmacology, karolinska institute, stockholm, sweden) for supporting the analyses of the microdialysates. declaration of interest: the 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cortical impact trauma in rat. brain res. 1994; 637:227–32. 34. nilsson p, gazelius b, carlson h, hillered l. continuous measurement of changes in regional cerebral blood flow following cortical compression contusion trauma in the rat. j neurotrauma. 1996;13:4:201–7. 35. chambers i, gregson b, citerio g, enblad p, howells t, kiening k, et al.; the brainit group. brainit collaborative network: analyses from high time-resolution dataset of head injured patients. acta neurochir suppl. 2008;102: 223–7. late hypotension, metabolism, and traumatic brain injury 231 (6) tryggve nevius 61-72 upsala j med sci 111 (1): 61–72, 2006 the evaluation and treatment of therapy-resistant enuresis: a review tryggve nevéus department of uppsala university children’s, hospital, 751 85 uppsala, sweden abstract children with enuresis that neither responds to the alarm or to desmopressin medication usually have nocturnal detrusor over-activity combined with high arousal thresholds as a cause for their bedwetting. the evaluation of these children is focused on 1) excluding underlying pathology such as kidney disease, urinary tract infection or neurogenic bladder, 2) looking for concomitant day-time bladder problems or constipation, and 3) detecting possible reasons for failure of alarm treatment. a bladder diary is essential, but blood tests, radiological examinations or invasive procedures are seldom informative. the non-pharmacologic treatment of these children consists of eradication of constipation, if present, and the provision of advice regarding sound drinking and toilet habits. such treatment is essential but not uniformly sufficient by itself. the first-line pharmacologic treatment of therapy-resistant enuresis is anticholinergic medication, although this is, as yet, not evidence-based. anticholinergics can be combined with desmopressin for better efficiency. for children failing all these measures there is still a place for tricyclic antidepressant therapy, provided that adequate safety precautions are strictly observed. background nocturnal enuresis is caused by three interacting pathogenetic mechanisms: the bedwetting child usually has high arousal thresholds (1) and in addition either suffers from nocturnal polyuria (2) and/or nocturnal detrusor over-activity (3). in the first case the child wets his or her bed because the nocturnal urine production exceeds the amount the bladder can hold and he or she is not aroused by the sensa61 received 3 october 2005 accepted 21 october 2005 key words: enuresis, urotherapy, anticholinergics, imipramine tion of bladder distension; we can call this diuresis-dependent enuresis. in the second case the child wets his or her bed because of sudden, uninhibited detrusor contractions that fail to result in sufficient arousal; we can label this phenomenon detrusordependent enuresis. there are also children with combined diuresisand detrusor dependency (4). diuresis-dependent enuresis is usually not combined with overt day-time bladder dysfunction, whereas in detrusor-dependency careful history-taking usually reveals that the child has day-time incontinence, urgency symptoms or concomitant constipation. the link between constipation and bladder problems is often overlooked (5): if the rectum is used as a storage room – as is the case in constipation – it will deform the bladder and lead to detrusor over-activity and/or residual urine. the first-line treatment of enuresis without daytime incontinence is the enuresis alarm or desmopressin (6). children with diuresis-dependent enuresis usually respond to desmopressin (7), which is logical, given the drug’s antidiuretic action, whereas the factors that determine whether alarm treatment will work or not are related primarily to the motivation and ability of the family and child to follow the instructions given by the care-giver, and partly also to the degree of arousal disturbance. this review will focus on the evaluation and treatment of children with therapyresistant enuresis. ”therapy-resistant enuresis” will in this text denote enuresis that has responded neither to the alarm nor to desmopressin treatment. from the arguments above it should be clear that these children usually have detrusor-dependent enuresis. it is also known that, although the old notion of enuresis as a primarily psychiatric disorder has not stood the test of time, neuropsychiatrical disturbances, such as attention deficit hyperactivity disorder, are overrepresented among enuretic children (8). since these children often have difficulties in complying with the alarm treatment they tend to be overrepresented in the group of therapy-resistant children as well. evaluation patient history a detailed bladderand bowel-oriented anamnesis is of paramount importance in these children. signs such as day-time incontinence (present or previous), urgency or a tendency to either void very frequently or postpone micturition as long as possible should be specifically asked for. children with urinary tract infections (utis) or recurrent bacteriuria often suffer from detrusor over-activity or have residual urine. likewise, signs of constipation, such as recurrent stomach pains, infrequent bowel movements, encopresis (hard stools) must be looked for. underlying kidney disease should be suspected in children with long-standing weight loss, unexplained fatigue or excessive thirst. neurological explanations can be suspected in children with developmental delay, gait disturbances or recurrent utis. obviously, it is also important to know whether the child wets his or her bed every 62 night or just occasionally, how difficult he or she is to arouse from sleep at night and if the child has always been a bed-wetter or not. importantly, we also need to know if the alarm was tried in the correct way (i. e. consistently used without interruption for at least six weeks, the parents helping the child to wake up at the signal). in many cases it is found that the family did not receive adequate information and follow-up during the alarm treatment, and then a new session can often be curative. the presence of heavy snoring or sleep apnoeas should be asked for, since in some cases the removal of upper airway obstruction can result in disappearance of enuresis (9). we usually ask the child if he or she – not just the parents – regards the bedwetting as a big problem. if that is not the case, then compliance problems may become an issue and the risk for neuropsychiatric co morbidity is greater. physical examination the evaluation should include a routine neurological examination (hyperreflexia? positive babinski?), inspection of the genitals (phimosis? ectopic ureteral orifice?), inspection of the lower back and palpation of the rectum. during the latter examination sphincter tone can be assessed and the presence of stool in the rectal ampulla – a strong indicator of constipation – be detected. bladder diary all children with therapy-resistant enuresis should be encouraged to complete a bladder diary, documenting voiding frequency, voided volumes and fluid intake for a few days and enuresis/incontinence episodes and bowel movements for two weeks. this gives invaluable information about bladder and bowel function. frequent, small voiding and urgency indicate day-time detrusor over-activity. infrequent voidings and holding manoeuvres such as squatting or standing on tip-toe with legs crossed indicate voiding postponement and should prompt behavioural change. excessive urine production indicates kidney disease or habitual polydipsia. a low defecation frequency obviously gives reason to suspect constipation. furthermore, the ability of the child and family to fill in the bladder diary gives a good indication about their ability to comply with treatment requiring a high degree of cooperation, such as urotherapy, bowel therapy or the enuresis alarm. other investigations if the child has previously been dry it is obvious that a urine test for leukocytes, bacteria and glucose should be performed in order to exclude uti or diabetes mellitus. blood tests, however, are not informative if the above examinations have not given any suspicion of kidney disease. neither is ultrasound or x-ray examinations indicated. cystometry or cystoscopy is only indicated in the rare cases when the history or physical examination gives reason to suspect neurogenic bladder disturbance. in children with heavy snoring or nocturnal apnoeas the help of an otorhinolaryngologist should be sought for sleep registrations and possible surgical correction. 63 flowmetry, with assessment of residual urine volume, is a very useful investigation in these children, since it gives valuable urodynamic examination. it is our opinion that it is an obligatory examination at least in children who have additional day-time incontinence that has not responded to basic urotherapy and in enuretic children who have had utis. this way, the few children who need to be examined under the suspicion of urethral valves or neurogenic bladder disturbance may be discerned. choice of treatment after the evaluation outlined above we usually have sufficient information to discern which patients need further investigation and be able to start treatment of the remainder. reasonable strategies for different patient groups are presented below, whereas the different medications are described in more detail in the following section. children who have relapsed after successful alarm treatment these children should be given the alarm once more. if it worked previously it will probably work again. this time the strategy of over-learning may be used (10), i.e. the child is asked to start drinking extra water at bedtime after primary alarm success is achieved and then continue until 14 consecutive dry nights have been achieved in spite of the extra fluid intake. enuretic children with concomitant day-time incontinence the general rule is to treat day-time incontinence before addressing the enuresis, although this has recently been challenged (11). first-line treatment of the former is bladder training, which will be most effective with the help of a skilled urotherapist. basic urotherapeutic advice that can be given by everyone is that the child should void regularly every second or third hour and that at least half of the day’s fluid intake should take place before the afternoon. constipation, if present, should be treated (see below). if dysuria or unexplained fever is present a urine culture should be obtained and antibiotic treatment be considered. note, however, that this needs to be carefully evaluated. if there is no prompt disappearance of symptoms during antibiotic treatment then the bacteria were not causative. asymptomatic bacteriuria is common in children with bladder problems (12). if the day-time incontinence remains in spite of bladder training anticholinergic medication may be indicated, given twice or thrice daily or as prolonged release tablets, but before this is considered it has to be ascertained that there is no residual urine present. when the day-time incontinence has disappeared the enuresis – if still present – should be treated with anticholinergics (see below), provided that the alarm has not been effective. 64 65 enuretic children with suspected detrusor over-activity but without day-time incontinence the same basic urotherapeutic advice as outlined for the day-time incontinent children should be given, but in our experience this is seldom enough to make the children night-dry. the first-line treatment that we recommend is anticholinergics, given once daily in the evening. note, however, that this is an experience-based, not evidencebased, recommendation (13). if the response is partial desmopressin may be added. enuretic children with constipation as mentioned above, bladder disturbances and constipation is a very common but often overlooked combination. this is especially the case in children with day-time incontinence or both dayand night-time wetting (14), but constipation may be relevant in quite a few children with isolated enuresis as well. in these children the constipation should be addressed first. we recommend that treatment is started with daily mini-enemas for 3 or 4 consecutive days, followed by bulk laxatives of the non-irritant type for at least a month. the child is instructed to drink lots of water but not too much milk, go regularly to the toilet at least once daily (preferably after meals), eat plenty of fibre-containing food, keep an active lifestyle (not just sit in front of the computer or tv all day!) and – last but not least – develop the habit of going directly to the toilet upon first sensation of rectal filling. sometimes the help of an anotherapist is needed to succeed with this treatment. constipated children with enuresis can be assumed to have detrusor over-activity as the direct cause for their bedwetting and anticholinergics will then be the logical treatment if the alarm, basic urotherapy and eradication of the constipation by itself have not made the child dry. however, as anticholinergics often cause constipation as a side effect, these children should continue on laxative treatment as long as they use these drugs. enuretic children with no sign of day-time bladder or bowel disturbance it is unclear at the moment if these children are best helped by anticholinergic medication, assuming that they have isolated nocturnal detrusor over activity, given as a monotherapy or combined with desmopressin at bedtime, or if they should be given tricyclic antidepressant medication with imipramine. the former alternative is not evidence-based in enuresis and the latter is actively disencouraged by many authorities (see below). for this reason we recommend – always provided that first-line therapy has been adequately tested and failed – that even these children are first given anticholinergics (with the addition of desmopressin in the case of partial response), and that we proceed to imipramine only if this fails. enuretic children who have not responded to, or not tolerated, anticholinergics these severely therapy-resistant children deserve the attention of a specialist, usually a paediatric nephrologist or a paediatric urologist with a specific interest and experience of bladder problems. blood tests or invasive examinations such as cystometry are usually not indicated even in this group but the bladder diary, uroflowmetry and measurement of residual urine certainly are. ultrasound of the kidneys and urinary tract should also be considered, looking for bladder wall abnormalities, kidney cysts, ureterocoele etc, although usually nothing abnormal is found. since the response to first-line treatment modalities may change as the patient grows we recommend that new attempts with desmopressin and the alarm treatment are performed every second year or so in these patients. with these considerations taken into account it is our opinion that imipramine should be offered to these children. anticholinergics in enuresis three drugs have been more than sporadically tested in children: oxybutynin, tolterodine and propiverine. the drugs have anticholinergic and smooth muscle relaxant properties. the clinical efficacy is probably equivalent but tolterodine has fewer side effects (15) and is therefore the drug that we most often use in these children. anticholinergics are well-established and evidence-based in the treatment of daytime urinary incontinence when urotherapy alone does not suffice (16). clinical experience indicates that it is also effective in many children with enuresis (4, 17), which is logical, given the overlapping pathogenesis of the two conditions, but, as yet, this has not been proven in randomized, controlled studies. for this reason, we suggest that these drugs only be used by doctors with specific expertise in the field of paediatric bladder disturbances. considerations before starting treatment in children who have had utis residual urine has to be excluded. this is also the case when medication during both evening and morning is considered. the presence of a residual of >20 ml or >15% of the voided volume after repeated voiding is a contraindication for anticholinergic treatment. constipation should also be ruled out or treated before treatment is started. if the child has recently been constipated bulk laxatives should be given as long as the child takes anticholinergic medications. side effects and risks anticholinergic drugs are not highly toxic but side effects are fairly common. these are the most prevalent: 1 dry mouth: this is not a big problem for children, but the family should be informed about the increased risk for caries and the importance of good oral hygiene 2 nausea and vertigo: this problem is much more common in the adult or elderly and only seldom leads to children having to discontinue treatment. 3 aggressiveness or mood change occurs in a significant minority of children and 66 may lead to a need for discontinuation. it is very rare with tolterodine treatment. 4 constipation: this is the major clinical problem, since enuretic and/or incontinent children have a higher risk for constipation from the start. a common symptom of constipation – apart from the ordinary ones such as stomach ache, encopresis – is that an initially good response to the drug is gradually disappearing with an increasing number of wet nights as the weeks and months pass. 5 urinary tract infections: if a child on anticholinergic treatment has a uti the accumulation of residual urine should always be suspected. consequently, these children should discontinue the medication, start antibiotic treatment and undergo measurement of residual urine. the anticholinergic medication can not be started again until the residual urine has disappeared (the help of a urotherapist may be needed to achieve this). dosage the dosage in nocturnal enuresis is 5 mg oxybutynin or 1-2 mg tolterodine given at bedtime, the lower dosage given to children below the age of 7-8 years. our experience is that the dosage of tolterodine, because of its more benign safety profile, can and should often be increased to 4 mg if 2 mg gives only partial response. if the child has day-time incontinence in addition to his or her enuresis the same dose should be given in the morning as well, after the exclusion of residual urine. follow-up children who are given anticholinergic treatment should be told that the pills are only half the treatment. sound regular voiding and drinking habits are just as important and increase the chance that the child can soon manage without medication. if there is no effect after 1-2 months of treatment medication should be discontinued and alternative treatment be considered. if there is a partial response to the drug then dosage could be increased and/or desmopressin be added in standard dosage. if there is a satisfactory response to treatment without significant side effects, the lowest effective dose should be sought and treatment continued. it is our experience that first attempted drug discontinuation is usually unsuccessful if attempted before approximately 6 months have elapsed. discontinuation attempts should be gradual (2.5 mg oxybutynin or 0.5 mg tolterodine reduction per week) and repeated at least every 6 months. residual urine measurements are recommended after 6 months of treatment in every child who has previously had utis or who is given the drug in slow release tablets or twice/thrice per day. as mentioned above, residual urine measurements are mandatory in the case of uti. otherwise, routine measurements are not needed. imipramine the tricyclic antidepressant imipramine has in several placebo-controlled studies shown better effect than placebo against nocturnal enuresis (18-20). this drug was 67 widely used for this indication during the 60s and 70s and is still so in some parts of the world, but its use has been steeply diminishing since the alarm and desmopressin became established. one additional reason for imipramine not being recommended in enuretic children today is that it is, like all tricyclics, cardiotoxic in high doses, and there have been tragic cases when children have died due to imipramine overdose (21). the drug is, however, safe and reasonably free of side-effects when given in the usual anti enuretic dosage and when not given to children with unstable cardiac arrhytmias (22). we have recently shown that more than 40% of children with severe, therapyresistant enuresis become dry when given imipramine in monotherapy and that this proportion increases to two thirds when the drug is combined with desmopressin (23). the reason for the anti-enuretic effect of imipramine is not completely clear. it appears earlier, and requires lower dosage, than the anti depressive effect (24). the central sympathomimetic/noradrenergic action of imipramine, promoting arousal and inhibiting micturition, is a more likely candidate (18, 25). considerations before starting treatment imipramine should only be considered when all of the following conditions are fulfilled: 1 all relevant firstand second-line therapies (the alarm, desmopressin, basic urotherapy and anticholinergics) have been unsuccessfully tested or not tolerated. 2 there is no reason to suspect cardiac arrhythmias, i.e. the child has no history of syncope or sudden palpitations and there are no cases of sudden cardiac death or unstable arrhythmias in the family. if there is any doubt regarding this, an ecg should be performed to exclude long qt syndrome. 3 the family is compliant, well informed and knows that the pills should be kept securely locked. side effects and risks side effects of imipramine given in anti-enuretic dosage occur in approximately 10% of children (23). the most common side effects are slight nausea, sweating or palpitations, but some children may also experience mood changes or anxiety, effects that often lead to discontinuation of the treatment. all side effects can safely be expected to disappear after discontinuation of the drug. as mentioned above, there are definite risks of severe cardiac side effects, including death, if the drug is overdosed. it is also dangerous to give imipramine to any child with long qt syndrome, but it is very unlikely that this arrhythmia is missed if a proper case history is taken and ecg is performed on wide indications. dosage and follow-up imipramine is started in a dosage of 25-50 mg at bedtime, the lower dosage for children below the age of 9. the effect is evaluated after one month. if the result is unsatis68 factory desmopressin, in standard dosage, can be added and/or imipramine dosage can be increased to 50 mg. we never exceed 75 mg even in heavy adolescents. if the child becomes dry on imipramine treatment it is imperative that regular drugfree intervals are provided, to reduce the otherwise high risk of gradually diminishing effect (23). we instruct the family to discontinue medication for at least 14 days every third month, but some children need to have even more frequent drug interruptions. of course, if the dry nights continue during such a medicine-free interval it should be prolonged until it is clear that the enuresis has returned. in our experience the child usually can manage without imipramine after a year or two. other therapies and future prospects urotherapy is the mainstay of treatment of day-time bladder problems in children, and it is not illogical to assume that it should have some effect in enuresis as well. the few studies that have so far been performed on urotherapy used as monotherapy for this indication have not fulfilled criteria to provide proper evidence-based recommendations (26-28). it is the author’s opinion that urotherapy is an essential component in the treatment of therapy-resistant enuretic children but that it is only seldom effective on its own. as mentioned above, eradication of upper airway obstruction can cure a small group of enuretic children (9). the intriguing findings of kurol et al, that the application of orthodontic devices to provide maxillary expansion may be helpful, may reflect similar mechanisms (29). acupuncture has now shown antienuretic effect in a sufficiently large number of studies that the positive effect can be suspected not to be just coincidence or publication bias (30, 31). its specific role in the therapeutic arsenale can, however, not yet be delineated. the potential cardiotoxicity of imipramine makes it a controversial subject and many experts actively disencourages its use. this situation would improve if a noncardiotoxic drug could be found that is as effective against enuresis as imipramine. we have recently, in an open pilot investigation, found indications that the selective nor adrenaline reuptake inhibitor reboxetine may be such a drug, but this needs to be confirmed in proper, randomized trials before 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nevéus t. reboxetine in therapy-resistant anuresis -results and pathogenetic implications. scand j urol nephrol (in press). corresponding author: tryggve nevéus md phd uppsala university children’s hospital 751 85 uppsala, sweden tryggve.neveus@kbh.uu.se phone +46 18 6110000 fax +46 18 6115853 71 72 upsala j med sci 105: 2, 135-150,2000 epidemiological and clinical studies on insulin resistance and diabetes hans lithell, johan sundstrom, johan amlov, kristina bjorklund, arvo h h n i , anu hedman, bjorn zethelius, liisa byberg, lena kilander, richard reneland department of public health and caring sciences/geriatrics, uppsala university, uppsala, sweden abstract in uppsala, extensive epidemiological and clinical studies on insulin resistance and diabetes have been ongoing for the past 30 years. a prospective cohort study of men born 1920-24, living in uppsala county, was initiated during 1969-74 (the uppsala longitudinal study of adult men, ulsam). risk factors for cardiovascular disease were examined in 2,322 men, and re-examinations have been performed every 10 years. at the first follow-up, when the men were 60 years old, insulin resistance was found to be a risk factor for development of hypertension and diabetes. in addition, treatment with antihypertensive medication was an independent risk factor for development of diabetes. these findings resulted in a series of clinical studies on metabolic effects of antihypertensive agents. at the second follow-up, when the men were 70 years old, the development of hypertension and diabetes was once again in focus, but at this time, cross-sectional and prospective studies of other cardiovascular determinants, such as circadian blood pressure pattern, left ventric ular geometry and function, muscle morphology, ion status, fibrinolysis and cogni tive function, were also performed. the cohort has furthermore been linked to the swedish census and hospital discharge and cause of death registries, it has been used for studies on relationships between birth weight and cardiovascular disease, and genetic analyses have been performed, taking advantage of the long observation time obtained in this cohort. the cohort is currently being re-examined for the third time, and will hopefully continue to provide valuable information on the epidemio logy of diabetes and cardiovascular disease in the future. introduction in the late 1960s, an interest in the pathogenesis for type 2 diabetes mellitus emerged. in uppsala, a prospective cohort study was planned and executed during 1969-74 (27). it was based on a cohort of 50-year-old men living in uppsala county, born 1920-24 (the uppsala longitudinal study of adult men, ulsam). of the 2,841 men available, 2,322 (82%) accepted the invitation to participate in a health 135 survey. a large number of investigations were performed. a questionnaire regarding physical exercise habits, smoking habits and family history of cardiovascular di sease was filled out by the subjects. concentrations of fasting lipids, glucose and insulin were measured, and in 1,692 men an intravenous glucose tolerance test (ivgtt) was carried out. at that time there were still no official guidelines on how to investigate glucose intolerance and it was not until 1980 that the first recom mendations to use oral glucose tolerance tests (ogtt) appeared. however, the use of ivgtt turned out to be fruitful in the epidemiological context. it was also de cided that follow-up examinations would be performed every 10 years. this is a brief summary of past and ongoing research that has its basis in this cohort study or has been initiated due to findings in the cohort study. findings at age 60 insulin resistance and hypertension the first follow-up was carried out in 1980-84, when the men were 60 years old. a total of 1,860 men (87.5% of the eligible men from the original cohort) participated in the follow-up examination. this time the focus was on identifying those men who had developed diabetes or hypertension. for development of hypertension, the blood pressure itself was found to be the most important risk factor. in addition, fasting and late (at 60 minutes of the ivgtt) insulin concentrations, body mass index (bmi), abdominal skinfold thickness and heredity for hypertension were identified as contributing factors. in particular, late insulin concentration and in crease in bmi were important risk factors for development of hypertension. we believe this was the first prospective study to demonstrate insulin resistance (as reflected in insulin concentrations) being a risk factor for hypertension (66). insulin resistance and type 2 diabetes mellitus amongst the 1,834 men who were normoglycemic at age 50 and re-investigated at age 60,77 (4.4%) had developed diabetes at age 60. the most powerful risk factors for development of diabetes were a high fasting insulin concentration (an indicator of insulin resistance), a low insulin response to glucose injection at the ivgtt, bmi, systolic blood pressure and a high glucose value (within the normal range). in addition, treatment with anti-hypertensive medication was an independent risk factor (67). findings at age 70 non-dipping and the insulin resistance syndrome non-dipping is a circadian blood pressure pattern that sometimes accompanies states of autonomic failure (41), diabetes mellitus (1 1) and hypertension (69), but also occurs in apparently healthy subjects. whether non-dipping adds to an in creased risk of end organ damage or not has been disputed. a blunted nocturnal 136 blood pressure reduction has been associated with left ventricular hypertrophy (78), renal dysfunction (6), silent cerebrovascular disease (65) and cognitive deterioration (31). in addition, non-dippers have an increased mortality risk compared with dip pers (50). in contrast, some investigators have failed to find a relationship between an impaired nocturnal blood pressure fall and target organ damage in hypertensive individuals (9, 63). in the ulsam cohort, 24-hour blood pressure was evaluated in 1,057 70-year-old men. preliminary data from a cross-sectional investigation in dicate that non-dippers (n=66), in addition to insulin resistance, diabetes and hyper triglyceridaemia, have an unfavourable lipid profile with elevated levels of free fatty acids (ffa) in serum and an increased proportion of palmitic, palmitoleic and oleic acid in serum cholesterol esters. ion status, hypertension and insulin resistance a disturbed magnesium balance, with reduced intracellular ionised magnesium con centration, has been observed in patients with essential hypertension and in subjects with diabetes mellitus (62). magnesium supplementation in humans has led to increased insulin sensitivity, lowered blood pressure and improved lipid status (55, 6 1, 80). increased serum magnesium concentrations during antihypertensive capto pril treatment were correlated to improved insulin sensitivity (1 8). during bendro fluazide treatment serum magnesium decreased significantly and there was an inverse correlation between the changes in serum magnesium and mean fasting glucose concentrations (14). an association between the magnesium and calcium balance in skeletal muscle and blood pressure response to antihypertensive treat ment has been reported (17). the changes in serum magnesium concentrations, and in the ratio between calcium and magnesium concentrations, correlated to changes in insulin sensitivity and to serum triglyceride concentrations during antihyperten sive treatment with different angiotensin converting enzyme (ace) inhibitors ( 15, 19). it has been questioned whether the total magnesium concentration in blood reflects the biological activity of magnesium since only about 60 % of the mag nesium in the blood is in a free unbound state. in fact, atherogenic lipid fractions and some glucometabolic variables were more closely correlated with circulating ionised magnesium than with total magnesium concentration (2 1). the circulating ionised magnesium concentration in patients with essential hypertension and in insulin resistant but otherwise healthy subjects was within the normal range (21). euglycaemic hyperinsulinaemia increases the circulating ionised magnesium con centration while no significant change was observed of the total magnesium level (16,20). relationships between muscle morphology and insulin resistance skeletal muscle accounts for the largest part of glucose uptake when stimulated by insulin (as much as 90%). the capacity of the muscle to utilise glucose depends largely on muscle structure. muscle rich in type i (oxidative) fibres, as well as muscle rich in capillaries, apparently show much higher glucose uptake than muscle containing mainly type iib (glycolytic) fibres. the percentage of type i fibres and 137 capillary density in skeletal muscle increases with increasing level of physical activity. in contrast, type iib fibres are prevalent in obese people. muscle biopsies were taken in 515 men (43%) at age 70 in order to analyse muscle histology. results of this analysis showed a clear association between insulin sensi tivity and muscle structure. insulin sensitivity showed a positive relationship with percentage of type i fibres and capillary density and a negative correlation with per centage of type iib fibres (25). these associations were independent of the level of physical activity and obesity. moreover, diabetic subjects showed a significantly higher percentage of type iib muscle fibres compared to healthy controls. a more intriguing and less studied aspect is the degree to which muscle structure, i.e. fibre composition and capillary density, is related to hypertension. for that pur pose, a group of normotensive (n=113) and hypertensive (n=43) men were com pared in order to test the hypothesis of a relationship between heart rate, develop ment of hypertension and muscle morphology in a state of normal glucose tole rance. these groups did not differ significantly with regard to fibre type distribu tion, but the hypertensive subjects had a significantly lower capillary supply than the controls, when analysed as number of capillaries around different fibre types. capillary rarefaction in the hypertensive subjects in the present study was closely associated with the increase in blood pressure over a 20-year period. heart rate, a marker of the balance between sympathetic and parasympathetic tone, was inverse ly correlated to capillary supply, which supports the hypothesis of involvement of increased sympathetic tone in a reduction of the capillary net in skeletal muscle (26). insulin has also been found to increase the blood flow in skeletal muscle beds through vasodilation. this increase in blood flow is believed to contribute to glucose uptake by the muscle. the magnitude of the increase in blood flow during hyperinsulinaemia, which is endothelium-dependent, depends not only on the degree of vasodilation in large vessels, but also possibly on the number of capil laries present in the muscle bed. preliminary data show that the capillary density is associated to insulin-induced changes in leg blood flow. this indicates that endo thelial function is closely linked to the capillarisation in skeletal muscle. proinsulin in relation to development of type 2 diabetes and cardiovascular disease in longitudinal studies, the fasting concentration of total proinsulin-like molecules predicts the development of type 2 diabetes over periods of follow-up ranging from 2 to 4 years (76). fasting concentrations of specific insulin, intact and 32-33 split proinsulin have been measured in plasma samples, stored frozen since baseline in liquid nitrogen, by the specific two-site immunometric assay technique (68). zethe lius and co-workers have published preliminary results (83) indicating that proinsu lin may be a long-term predictor for diabetes when compared to known baseline risk factors for type 2 diabetes in this cohort (glucose and immunoreactive insulin concentrations, fasting and at 60 min after an intravenous glucose load, respectively, and use of antihypertensive drug treatment). 138 insulin has been shown to be a risk factor for cardiovascular disease (64). the extent to which proinsulin may contribute to the association between insulin and cardiovascular disease is largely unknown. the aim of one of our studies was to identify the longitudinal relationship between proinsulin and coronary heart disease in the ulsam cohort. the diagnosis of incident disease was collected from the official in-patient and causes-of-death registers. in a preliminary report (82), zethe lius and co-workers have shown results indicating that proinsulin is a predictor for cardiovascular disease and may be independent of known confounders. heart failure and the insulin resistance syndrome heart failure is the only major cardiovascular disorder that is increasing in in cidence and prevalence (44), with substantial morbidity and mortality and a huge economic impact on the public health systems of western society (28). in spite of this, the epidemiological data on heart failure are relatively scarce. previous studies demonstrate an association between insulin resistance and heart failure (53, 74) and an impaired insulin-mediated glucose uptake has been shown to be a prognostic fac tor in heart failure patients (56). preliminary results from h l o v and co-workers (84) indicate that factors related to insulin resistance (10, 49, 52, 79), such as an increased heart rate, increased serum concentrations of proinsulin, a high proportion of dihomogammalinolenic acid in serum cholesterol esters and hypophosphataemia are predictors of left ventricular systolic dysfunction in elderly males after 20 years follow-up, independently of myocardial infarction, hypertension, diabetes and the use of cardiovascular medication. left ventricular hypertrophy and the insulin resistance syndrome left ventricular hypertrophy (lvh) is an important risk factor for cardiovascular and all-cause mortality and morbidity (32, 37), and an increased left ventricular relative wall thickness (rwt, wall thickness divided by ventricular diameter) also has an adverse prognosis (32,77). the aetiology of lvh is largely unknown. cross sectional studies (23, 36, 42, 60, 70) have shown that male sex, age, hypertension, obesity, valvular disease, previous myocardial infarction, heredity and alcohol use are important in lvh. recently, associations between lvh and the insulin resist ance syndrome have been found (39, 43, 54), although some studies (51, 71) have found insulin resistance or impaired glucose tolerance to be more closely related to increased rwt than to lvh. in the ulsam cohort, electrocardiographic and echo cardiographic examinations were performed in 583 men at 70 years of age. thus, this cohort is well suited for the much needed further study of the epidemiology of lvh, including detailed cross-sectional and prospective studies of the relationship between aspects of the insulin resistance syndrome and left ventricular geometry, as well as development of new diagnostic electrocardiographic criteria for lvh. in a cross-sectional study of the cohort at age 70 (7 l), we found that several com ponents of the insulin resistance syndrome (hyperinsulinaemic euglycaemic clamp insulin sensitivity index, fasting insulin, 32-33 split proinsulin, triglycerides, ffa, og'it glucose and insulin levels, waisthip ratio, bmi, 24-h blood pressure and 139 heart rate) were related to left ventricular rwt. only 24-h systolic pressure and heart rate were related to left ventricular mass index. several metabolic parameters were adversely altered in left ventricular concentric remodelling, but only 24-h blood pressure and heart rate in lvh. preliminary data from a small study using positron emission tomography to determine myocardial, skeletal muscle and whole-body insulin sensitivity indicate that myocardial insulin sensitivity may be related to rwt and not to left ventricular mass index (73). preliminary studies of predictors for lvh over 20 years indicate that dyslipidaemia and indices of a low dietary intake of linoleic acid and high intake of saturated and monounsaturated fats, as well as hypertension and obesity, at age 50 are predictive of the prevalence of lvh 20 years later, suggesting that lipids may be important in the aetiology of lvh (72). insulin resistance and pai-i elevated levels of plasminogen activator inhibitor-1 (pai-1) are regarded as a com ponent of the insulin resistance syndrome (1). pai-1 is an important factor in the regulation of fibrinolysis and inhibits the degradation of solid fibrin by forming an inactive complex with tissue-type plasminogen activator (t-pa). plasminogen is cleaved by t-pa to form active plasmin that degrades fibrin clots. it has been suggested that pai-1 is one of the factors linking insulin resistance with cardiovas cular disease (29). pai-1 activity was analysed in the ulsam cohort at age 70 (8). bmi, waisthip ratio, blood pressure, fasting glucose, insulin and serum triglyceride concentrations were all positively related with pai1 activity. insulin sensitivity index, measured by clamp, and hdl cholesterol concentrations were inversely related with pai1 ac tivity. in a multivariate regression analysis, bmi, waisthip ratio, serum triglyceride concentrations, and insulin sensitivity were related to the pai1 activity, all indepen dent of each other. pai-1 activity was also higher in men with type 2 diabetes and impaired glucose tolerance than in men with normal glucose tolerance. this dif ference was not explained by variations in bmi, waisthip ratio, serum triglycerides, insulin sensitivity, blood pressure medication or fasting glucose concentrations. insulin resistance and cognitive function in recent years, it has been recognised that cerebrovascular disease is associated not only with stroke and vascular dementia, but also with late-onset alzheimer’s di sease. dementia research has now focused on risk factors associated with early stages of cognitive impairment, from which a further progress to dementia may be prevented. a subgroup of the 70-year-old men (999 out of 1,221) participated in cognitive function testing. the mini mental state examination (12) and the trail making tests (38) were used, tests which are sensitive for subcortico-frontal dys function. results have been previously published in detail (30, 31). in brief, men with diabetes according to an ogtt (n=130) had significantly poorer results on the cognitive tests, even when stroke, hypertension and atrial fibrillation were taken into account. similarly, clamp measurements of insulin sensitivity were related to 140 cognitive function: men in the highest tertile of insulin sensitivity performed sig nificantly better than those in the lowest tertile. studies on low birth weight, insulin resistance and diabetes low birth weight predicts physiological disturbances in adult life, such as raised blood pressure (3, 33, 34), type 2 diabetes (22, 40, 4 3 , impaired glucose tolerance (22, 59) and insulin resistance (40, 46, 58), all constituents of the insulin resistance syndrome. all of these conditions are associated with an increased risk of and mortality from cardiovascular disease, as is small size at birth (5, 35). low birth weight has also been associated with the insulin resistance syndrome itself (4, 75). birth weight, in this cohort, is inversely associated with fasting concentrations of immunoreactive insulin and insulin resistance at age 50 and positively associated with bmi at age 50 (40) and insulin sensitivity at age 70 (46). birth weight was also related to blood pressure at both ages (33, 34) but not with the concentrations of serum triglycerides or hdl cholesterol at age 50 (40). in a recent study we demon strated that low birth weight is associated with physiological disturbances in adult age that are a subset of the disturbances clustered in the general population as the insulin resistance syndrome (7). birth weight was inversely associated with the subscapular/triceps skinfold ratio investigated at age 50 and used as a measure of truncal fat. low birth weight predicted a smaller hip but not a larger waist and thus the inverse relationship of birth weight with the waisthip ratio at age 70 does not reflect an association of low birth weight with central adiposity. birth weight was also inversely associated with the activity of pai-1 and the concentrations of specific insulin and proinsulin at age 70. there were no associations of birth weight with the concentrations of serum triglycerides or hdl cholesterol at age 70. in short, low birth weight predicts high blood pressure, insulin resistance, truncal obesity and high pai-1 activity but not the abdominal obesity or dyslipidaemia present in the insulin resistance syndrome. understanding why low birth weight predicts only some components of the insulin resistance syndrome and not others may depend on identifying a specific physiological pathway that links this subset of disturbances. one possible mechanism could be increased concentrations of or sensitivity to glucocorticoids. supporting this hypothesis are the observations that long-term treatment of glucocorticoids cause accumulation of truncal fat, gluco corticoids increase the production of pal1 (48), and the sensitivity to glucocorti coids could be programmed in foetal life (57). effects of antihypertensive drugs on insulin resistance in eight different intervention studies, the actively treated comparison groups have been treated with a selective beta-blocker (atenolol). the average effect on insulin sensitivity was a reduction by 19% from the basal value after 4-6 weeks placebo 141 treatment. the average effect of two similar diuretics, given in moderate high doses, was a decrease by 19% in insulin sensitivity. in four studies of calcium-channel blockers, no significant effect on insulin sensitivity was found. in the class of ace inhibitors, the results ranged from +16% with captopril to -10% with lisinopril. the average effect was +2%. with two different angiotensin,-receptor blockers, similar non-significant (+2%) effects on insulin sensitivity were demonstrated. the average of three different studies with alpha-1 selective inhibitors was an improvement of insulin sensitivity by 23%. the effect of moxonidine treatment was an improvement of insulin sensitivity by 12%. however, the improvement was restricted to a sub group of very insulin resistant patients, predefined in the protocol, who improved by 21%. moxonidine belongs to a class of centrally acting inhibitors of the sympa thetic nervous system. the agonistic effect these drugs exhibit on the imidazoline i, receptors causes an inhibition of the sympathetic nervous system. candidate genes for insulin resistance for several years, the geriatrics section of the department of public health and caring sciences has actively collaborated with professor oluf pedersen’s research group at steno diabetes centre in gentofte, denmark. the steno group investigates candidate genes for type 2 diabetes. variants of candidate genes are identified either from the literature or in-house at steno. associations between these genetic variants and relevant phenotypes such as type 2 diabetes, insulin resistance, and insulin secretion are then sought in the ulsam material. in ulsam, dna has been collected from >95% of participants and ivgtt, ogtt and hyperinsulinaemic, euglycaemic clamp data are available. in the first study, hepatocyte nuclear factor 4alpha (hnf-4a), the mody1 gene, was investigated, and two single nucleotide polymorphisms that changed the amino acid sequence were found, one previously reported (thrl30ile) and one new (va1255met). in a danish sample, the frequency of the thrl30ile variant was sig nificantly higher in 509 type 2 diabetes patients (4.7%) compared to control sub jects (1.7%, p=0.008). this finding could not be replicated in the swedish ulsam population, however, in which frequencies of the variant were similar in subjects with type 2 diabetes (5.4%) and glucose tolerant control subjects (5.1 %). the novel va1255met variant was originally only found in type 2 diabetes patients (4/477) and not in controls (0/217). interestingly, the va1255met variant could not be found in any of the 894 ulsam men investigated (47). in a second study, the previously identified gly1057asp variant of the insulin receptor substrate-2 (irs-2) gene was analysed in four different groups of glucose tolerant subjects, one of which was derived from ulsam. no consistent effect on insulin sensitivity or secretion was found (2). a third study investigated the influence of two common genetic variants in the ppplr3 gene encoding the glycogen targeting subunit of type-1 protein phos phatase (24). a previous study in pima indians indicated an association between the asp905tyr, arg883ser, or ppl are polymorphisms, and insulin sensitivity (8 1). 142 the arg883ser variant was not included in the daniswswedish study since it could not be found in 82 caucasian volunteers. in ulsam there was no association be tween either the asp905tyr or the pplare variants and diabetes incidence be tween 50 and 70 years of age. there was no association between the asp905tyr variant and insulin sensitivity, but there was a weak correlation between the pplare variant and whole body insulin sensitivity. in view of the multiple tests involved in the study, however, this correlation was not considered significant by the authors (24). discussion the past and on-going research of our group reviewed in this paper demonstrates the usefulness of an epidemiological study as a basis for research. not only can valuable information be obtained from the study itself, but other studies may de velop from some of the, often unexpected, results. in this case, some of the findings resulted in a series of clinical studies on metabolic effects of antihypertensive agents. in addition, the cohort was useful for studies on relationships between birth weight and cardiovascular disease, and for genetic analyses. in the first follow-up of the ulsam cohort (at age 60), two major findings were made. development of hypertension at age 60 had been preceded by high insulin concentrations at age 50. insulin resistance as a predictor of hypertension has been found in some but not all other studies, and the question of whether insulin is causally related to hypertension is still debated. the other major finding was that high fasting insulin concentrations (as indicators of insulin resistance) and low in sulin response to glucose injection at the ivg'it (as a measure of insulin secretion capacity) were strong predictors of future diabetes. the importance of insulin re sistance for diabetes was further manifested in the 20-year follow-up. we used stored, frozen serum samples and analysed for specific insulin, proinsulin and 32 33 split proinsulin. proinsulin (which reflects insulin resistance) was a powerful risk factor for development of diabetes, particularly during the first ten years. at the second follow-up (at age 70), skeletal muscle biopsies were studied in about 500 of the participants. investigations of muscle fibre composition and capil larisation indicated that hypertensive, glucose-tolerant 70-year-old men have a lower percentage of type i fibres and more type i1 fibres than a control group of normotensive men. furthermore, the capillarisation was less pronounced. capillari sation was related to insulin sensitivity and to blood flow changes during hyperin sulinaemia, and ffa concentrations also played an important role in determining the vasodilatory response to hyperinsulinaemia. we demonstrated a relationship be tween heart rate at age 50 and low capillary density at age 70 as well as between heart rate at 50 and development of hypertension during the following 20 years. this indicates a possible influence of high sympathetic tone being involved in both structural changes in skeletal muscle and hypertension. extensive echocardiographic investigations were performed in nearly 600 men. these data have now been studied with regard to metabolic characteristics of dif 143 ferent morphologic measures. several characteristics of the metabolic syndrome and insulin resistance itself correlated to the left ventricular relative wall thickness, in contrast to the findings for left ventricular hypertrophy, with which only 24-h blood pressure and heart rate were correlated. skeletal muscle insulin resistance may thus be important for development of increased relative wall thickness, as supported from the study with positron emission tomography. in this study population, it is interesting that insulin resistance and proinsulin concentrations are significant predictors also for coronary heart disease and left ventricular systolic dysfunction. some of the effect on coronary heart disease may be mediated by pai-1 activity, which is strongly correlated to insulin resistance. a deleterious effect of insulin resistance also on small vessels may explain the correla tion between cognitive function and insulin resistance/diabetes mellitus. the metabolic consequences of antihypertensive medication have been exten sively described in the original publications and will not be commented in detail here. the increased insulin resistance of beta-blockers and diuretics most likely explains the increased risk for diabetes mellitus associated with use of these drugs, as high doses were used in the 1970s. this was recently confirmed in a large study for beta-blockers but not for diuretics (1 3). the doses used in the study are not provided in the report, but it seems likely that they were lower for diuretics in the study than in the 1970s. much of our data (in co-operation with the london school of hygiene and tropical medicine, london, uk) on the relationship between low birth weight and risk for diabetes confirms david barker’s findings (3-5). however, we have demon strated that this increased risk is mediated by an effect of birth weight on insulin resistance and not on insulin secretion. in our population, it seems that only part of the syndrome is related to birth weight, in particular those metabolic variables that are related to cortisol effects. the effect of candidate genes for insulin resistance has been studied in this cohort in co-operation with steno diabetes centre in copenhagen. this project is ongoing. the ulsam population supplements some of the younger populations of cau casian origin in the steno studies. in summary, these studies on insulin resistance and diabetes are based on a pro spective cohort study of medium size. the population is small for genetic studies, but can supplement studies in younger populations in order to describe the different gene-environment interactions at different ages. most of the findings described here are the preliminary results for some of our ph.d. students, whose dissertations will soon be providing more in-depth analysis of the ulsam data. a list of publications and more detailed information on the cohort can be found at the web-site www.pub care.uu.se/ulsam. references 1 . alberti, k. g . m. m. & zimmet, p. z. definition, diagnosis and classification of dia betes mellitus and its complications. part l : diagnosis and classification of diabetes 144 mellitus. provisional report of a who consultation. in: for the who consultation. dia bet med; 1998. p. 539-553. 2. almind, k., frederiksen, s. k., bernal, d., hansen, t., ambye, l., urhammer, s., et al. search for variants of the gene-promoter and the potential phosphotyrosine encoding sequence of the insulin receptor substrate-2 gene: evaluation of their relation with al terations in insulin secretion and insulin sensitivity. diabetologia 42: 1244-1 249, 1999. 3. 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to insulin resistance are predictors of left ventricular systolic dysfunction in a male popu lation after twenty years of follow-up. eur j endocrinol 142 [suppl 1]:17 (abstract), 2000. address f o r correspondence: hans lithell, md, phd department of public health and caring sciences/geriatrics uppsala university box 609 se-75 1 25 uppsala sweden 150 bok 03-06.indb increased elasticity of synovial membrane after immobilization 303 received 17 february 2006 accepted 21 march 2006 increased elasticity of capsule after immobilization in a rat knee experimental model assessed by scanning acoustic microscopy yoshihiro hagiwara1*, yoshifumi saijo2, eiichi chimoto1, hirotoshi akita3, yasuyuki sasano3, fujio matsumoto1, shoichi kokubun1 1department of orthopaedic surgery, tohoku university graduate school of medicine, sendai, 980-8574, japan 2department of medical engineering and cardiology, institute of development, aging and cancer, tohoku university, sendai 980-8575, japan 3division of craniofacial development and regeneration, tohoku university graduate school of dentistry, sendai, japan abstract objectives: the mechanical property of immobilized joints is not well understood. the present study was designed to investigate the tissue elasticity of the anterior and posterior synovial membrane (sm) in a rat immobilized knee model using scanning acoustic microscopy (sam). moreover, the structural characteristics of the sm after immobilization were examined by transmission electron microscopy (tem). methods: thirty rats had their knee joints immobilized with a plate and metal screws. the rats were fixed at 1, 2, 4, 8 and 16 weeks after surgery and the knee joints were sectioned sagittally for sam. selected specimens were processed for tem. a new concept sam using a single pulsed wave instead of continuous waves was applied to measure the sound speed of the anterior and posterior sm, comparing it with the corresponding light microscopic images. results: the sound speed of the posterior sm increased significantly in the 8and 16-week experimental group compared with that in the control group. the sound speed of the anterior sm showed no statistical difference between the experimental and the control groups at any period of immobilization. the posterior sm of the experimental group was different from that of the control group in the ultrastructural characteristics of extracellular matrices. conclusions: our data suggest that the increased elasticity and structural changes of the posterior sm are one of the main causes of limited extension after a long period of immobilization in flexion using sam, which is a powerful tool for evaluating the elasticity of targeted tissues. introduction joint contracture is defined as a decrease in both active and passive ranges of motion (rom) after immobilization. the decreased rom limits the activity of daily living in various aspects. immobilization, which is a major cause of joint contracture, is beneficial for decreasing pain caused by trauma and preventing the joint from damage in the acute phase of arthritis such as pyogenic and rheumatoid arthritis [1–3]. even by extensive rehabilitation or surgical treatment, however, it is difficult to regain the full rom in an established joint contracture after a long period of immobilization [4,5]. upsala j med sci 111 (3): 303–313, 2006 304 yoshihiro hagiwara et al. the components of joint contracture after immobilization are classified into arthrogenic and myogenic ones. the arthrogenic components are lesions of bone, ligaments, capsule and synovial membrane (sm), while the myogenic components are lesions of muscle, tendon and fascia [6,7]. some investigators have attributed contracture to myogenic causes (8), while others attributed it to arthrogenic causes [4,7,9–13]. it is difficult to evaluate such contradictory reports because different animal species and methods were used in their immobilization experiments. among the arthrogenic components, the stiffness of the capsule and sm through synovial atrophy, retraction, fibrosis, and adhesion may contribute to the limited rom [1,7,8,10,14–17]. though increased elasticity of the capsule or sm has been suggested to be a cause of joint contracture [18], it is not known how the elasticity and the structural characteristics of extracellular matrices are affected by immobilization. a scanning acoustic microscopy (sam) using continuous waves characterize biological tissues by determining the elastic parameters based on the sound speed [19]. recent studies on infarcted myocardium [20], atherosclerosis of aorta [21] and carotid arterial plaques [22] have shown that the acoustic properties reflect the collagen types. in the present study, we applied a new concept sam using single pulsed wave, which can make total time for calculation significantly shorter, to examine the elasticity of the anterior and posterior sm (synovial intima and subintima) in the course of knee joint immobilization in a rat experimental model. material and methods animals. the protocol for this experiment was approved by the animal research committee of tohoku university. adult male sprague-dawley rats weighing from 380 to 400 g were used. their knee joints were immobilized at 145° in flexion by rigid internal but extra-articular fixation for various periods (1, 2, 4, 8 and 16 weeks) according to a previously described method (1). the left and right hind legs were immobilized alternately to avoid potential systematic side differences. the surgery was performed under anesthesia with sodium pentobarbital (50 mg/kg) administered intraperitoneally. a rigid plastic plate (pom-n, senko med. co., japan) implanted subcutaneously joined the proximal femur and the distal tibia away from the knee joint and was solidly held in place with one metal screw (stainless steel, morris, j. i., co., usa) at each end. the knee joint capsule and the joint itself were untouched. postoperative analgesia with buprenorphine (0.05 mg/kg) was injected subcutaneously. sham operated animals had holes drilled in the femur and tibia and screws inserted but none of them were plated. the animals were allowed unlimited activity and free access to water and food. the immobilized animals and the sham operated animals made up the experimental groups and the control groups, respectively. thirty rats (1, 2, 4, 8 and 16 weeks) were prepared. each group was composed of 6 experimental and 5 control animals. increased elasticity of synovial membrane after immobilization 305 tissue preparation. the rats were anesthetized and fixed with 4% paraformaldehyde in 0.1m phosphate-buffer, ph7.4 by perfusion through the aorta. the knee joints were resected and kept in the same fixative overnight at 4c°. the fixed specimens were decalcified in 10% edta in 0.01m phosphate-buffer, ph7.4 for 1–2 months at 4c°. after dehydration through a graded series of ethanol solutions, the specimens were embedded in paraffin. the embedded tissue was cut into 5-μm thick sagittal sections from the medial to the lateral side of the joint. standardized serial sections of the medial midcondylar region of the knee were made. the serial sections were prepared for hematoxylin-eosin stain to observe the histological appearance of sm after immobilization. scanning acoustic microscopy. our sam consists of five parts: 1) ultrasonic transducer, 2) pulse generator, 3) digital oscilloscope with pc, 4) microcomputer board and 5) display unit (figure 1). a single pulsed ultrasound with 5 ns pulse width was emitted and received by the same transducer above the specimen. the aperture diameter of the transducer was 1.2 mm and the focal length was 1.5 mm. the central frequency was 80 mhz and the pulse repetition rate was 10 khz. considering the focal distance and the sectional area of the transducer, the diameter of the focal spot was estimated as 20 m at 80 mhz. distilled water was used as the coupling medium between the transducer and the specimen. the reflections from the tissue surface and from interface between the tissue and the glass were received by the transducer and were introduced into a digital oscilloscope (tektronics tds 5052, figure 1. schematic illustration of a new concept scanning acoustic microscopy (sam). a new concept sam can make total time for calculation significantly shorter than a conventional sam by using a single pulsed wave instead of continuous waves. 306 yoshihiro hagiwara et al. usa). the frequency range was 300 mhz and the sampling rate was 2.5 gs/s. four pulse responses at the same point were averaged in the oscilloscope in order to reduce random noise. the transducer was mounted on an x–y stage with a microcomputer board that was driven by the computer installed in the digital oscilloscope through an rs232c. the x-scan was driven by a linear servo-motor and the y-scan was driven by a stepping motor. finally, two-dimensional distributions of the ultrasonic intensity, sound speed and thickness of the 2.4 by 2.4 mm specimen area were visualized with 300 by 300 pixels. the total scanning time was 121 sec. signal analysis. the reflected waveform comprises two reflections at the surface and the interface between the tissue and the glass. the thickness and sound speed were calculated by fourier-transforming the waveform [19]. image analysis. normal light microscopic images corresponding to the stored acoustic images were captured (dmlb 100 hc light microscope, leica wetzlar, germany). a region of analysis by sam was set in the anterior and posterior sm each in each section (figure 2). in the region, the sound speed of sm, excluding meniscus, bone and cartilage, was calculated with a gray scale sam images using commercially available image analysis software (photoshop 6.0, adobe systems inc., san jose, ca) (figure 4). sam images with a gradation color scale were also produced for clear visualization of the sound speed. the optical and acoustic images were compared to ensure morphological congruence in the analysis. transmission electron microscopy. the posterior sm of 8-week experimental and control groups were fixed with a mixture of 0.04% glutaraldehyde and 4.0% paraformaldehyde in 0.1m phosphate-buffered saline, ph 7.4, at 4c° into the intraarticular space for rapid fixation. the skin around the knee was excised and the posterior sm was immersed with the same fixative for 1h at 4c°. after washed thoroughly with dulbecco’s pbs to remove the fixative, the tissue was cut with a razor blade into pieces and post-fixed with 2% buffered osmium tetroxide. the tissues were stained en bloc in aqueous uranyl acetate solution, dehydrated through a graded series of ethanol solutions and embedded in epon 812 resin (taab laboratory equipment ltd). ultrathin resin sections of the specimens were mounted on copper, counterstained with uranyl acetate and reynold’s lead citrate solution, then observed with a hitachi h-9000 electron microscope [23]. statistics. all data were expressed as the mean ± sd. the statistical significance of difference in the results was evaluated by unpaired analysis of variance, and p values were calculated by tukey’s method. a p value less than 0.05 was considered statistically significant. increased elasticity of synovial membrane after immobilization 307 results sam examination. the gradation color images of the posterior sm in the experimental group differed from those in the control group (figure 3). the posterior sm was composed of low sound speed areas (black to blue) in 2-week immobilization (figure 3a). the low sound speed area decreased and high sound speed areas (yellow to red) gradually increased in the posterior sm of the experimental group figure 2. microphotograph of a sagittal section in the medial midcondylar region of a rat knee. squares indicate regions of analysis by scanning acoustic microscopy in the anterior( ) and posterior( ) synovial membrane. (original magnification × 10, hematoxylin-eosin stain) 308 yoshihiro hagiwara et al. with time (figure 3b). the posterior sm remained same in all the control groups (figure 3c). the anterior sm was similar in all the experimental and control groups irrespective of immobilization periods (figure 3d). the sound speed of the posterior sm is shown in figure 4. there was no statistical difference between the experimental and the control groups in 1-, 2or 4-week immobilization (1w: 1560 m/s 18.7 m/s vs. 1543 m/s 16.3 m/s; p = 0.152, 2w: 1552 m/s 26.0m/s vs. 1535 m/s 8.17 m/s; p = 0.207, 4w: 1551 m/s 4.01 m/s vs. 1553 m/s 13.3 m/s; p = 0.698). in 8and 16-week immobilization, however, the sound speed in the experimental group was significantly higher than that in the control group (8w: 1546 m/s 18.7 m/s vs. 1646 m/s 11.8 m/s; p = 6.69 × 10–6, 16w: 1568 m/s 26.5 m/s vs. 1677 m/s 32.8 m/s; p = 1.06 × 10–4) (figure 4a). there was no statistical difference in the anterior sm in all the experimental and the control groups at any period of immobilization (1w: 1563 m/s 22.7 m/s vs. 1556 m/s 13.8 m/s; p = 0.545, 2w: 1562 m/s 12.4 m/s vs. 1565 m/s 11.4 m/s; p = 0.74, 4w: 1559 m/s 10.1 m/s vs. 1554 m/s 30.4 m/s; p = 0.745, 8w: 1550m/ figure 3. gradation color images of scanning acoustic microscopy in the posterior and the anterior synovial membrane (sm). a, 2-week immobilization group (posterior). b, 16-week immobilization group (posterior). c, a representative of the control groups (posterior, 16-week). d, a representative of the control group (anterior, 16-week). e, gradation color table. most low sound speed areas (black to blue) were replaced by high sound speed area (yellow to red) over time in the posterior experimental group. the posterior sm in the control groups remained entirely black to blue throughout the duration. the anterior sm was similar in the experimental and control group irrespective of the immobilization periods. regions enclosed with a dotted line indicate the sm for calculation. increased elasticity of synovial membrane after immobilization 309 s 20.5 m/s vs. 1564 m/s 15.3 m/s; p = 0.263, 16w: 1556 m/s 14.1 m/s vs. 1558 m/s 22.6 m/s; p = 0.846) (figure 4b). tem examination. in the experimental group, the space among collagen bundles and cells were occupied with the high density matrix, which fills the interspace of collagen microfibrils within the collagen bundle. in contrast, the high density matrix surrounding cells, collagen bundles and fibrils were scarce in the control group (figure 5). discussion the arthrogenic component has been considered as an important factor of joint contracture after immobilization (1,7,8,10,14–18). in a study using a rabbit knee contracture model, the mechanical characteristics were quantified by a torque-angular displacement diagram [18]. knees in 9-week immobilization in flexion showed a significantly larger torque in extension in the experimental group than in the control figure 4. sound speed changes of the posterior and the anterior synovial membrane (sm). a, the posterior sm. b, the anterior sm. in the posterior sm, significant difference of sound speed is seen in 8and 16-week immobilization. there was no statistical difference at any period of immobilization in the anterior sm. solid bars = experimental groups, shaded bars = control groups. values are the mean ± sd. * = p < 0.005 versus control, by tukey’s method. 310 yoshihiro hagiwara et al. group even after total extra-articular myotomies. in the same rat model as ours in the present study but immobilized up to 32 weeks, rom in extension still remained restricted even after total extra-articular myotomies [7]. in canine glenohumeral joint immobilized up to 16 weeks, the intra-articular pressure rose higher by injection of hypaque contrast medium and the filling volume was smaller compared with the control group at a rupture of the capsule [24]. these studies suggest that among the arthogenic components, the capsule and sm may mostly contribute to production of joint contracture. connective tissue proliferation in the sm and its adhesion to articular cartilage in the intra-articular space has been considered as pathological features of contracture after immobilization [25–29]. but conflicting studies with it have been reported. no intra-articular connective tissue proliferation occurred after immobilization [1,30–32]. no contact between the connective tissue and articular cartilage was observed [32]. in the same rat model as ours in the present study but immobilized up to 32 weeks, the decrease in the synovial intima length was observed after 4-week immobilization [1]. this study concluded that mutual adhesions of synovial villi rather than the connective tissue proliferation were the major pathophysiological changes leading to contracture. further, the decrease of the synovial intima length was reported to be greater in the posterior sm than in the anterior sm in the same model as ours in the present study [1]. it may be explained as earlier mutual adhesion of synovial villi in the posterior sm under less tension with the knee immobilized in flexion. connective tissue response after immobilization is important to understand the mechanism of the increased elasticity of the posterior sm. some suggestions configure 5. structural characteristics of the posterior synovial membrane (sm). a, 8-week experimental group. b, 8-week control group. compared with the control group, the space among collagen bundles and cells were occupied with the high density matrix, which fills the interspace of collagen microfibrils within the collagen bundle in the experimental group. c, cell; mf, microfibrils. (original magnification × 360,000, scale bar = 0.5 μm). increased elasticity of synovial membrane after immobilization 311 cern changes in the biochemical composition of periarticular fibrous connective tissue (e.g. patellar tendon, ligament and joint capsule) after immobilization. the notable change was a reduction of water and glycosaminoglycans without decreased collagen mass [9,18,32,33]. these changes were expected to alter plasticity and pliability of connective tissue matrices and to reduce lubrication efficiency [32]. in the same rat model as ours in the present study but immobilized up to 32 weeks, the posterior subintimal area of the experimental groups was smaller than that of the control groups through all the immobilization periods [1]. this result may reflect the decreased water and glycosaminoglycans of the sm. our tem observation showed that the space among collagen bundles and microfibrils was occupied with dense matrices in the experimental group, which may reflect the synovial atrophy due to decreased water content but not increased extracellular matrices. further, adhesions of collagen bundles may limit lubrication and increase elasticity. previous studies analyzed the elasticity of the joint as a whole including ligament, capsule and sm with or without muscles [7,15,18,24]. but it was impossible to evaluate the elasticity of the individual arthrogenic components, especially of capsule and sm in those studies. the present study is the first that measured the elasticity of sm in situ by sam in rat immobilized knees and revealed the increased elasticity of the posterior sm, subsequent to the inhibition of extension to cause the joint contracture. one reason why the elasticity is different between the anterior and the posterior sm of 8and 16-week experimental group may be that compared with the posterior sm immobilized rigidly, the anterior sm keeps motion to a larger extent after immobilization with patella while being active. the present study suggested that the increased elasticity and structural changes of the posterior sm are one of the main causes of limited extension after immobilized in flexion. acknowledgements the authors would like to acknowledge their valued input and efforts of mr. katsuyoshi shoji, mrs. michiko fukuyama and miss haruka sasaki and thank dr. hans k uhtoff and dr. guy trudel for their technical advice of making the animal model. references 1. trudel g, seki m, uhthoff hk (2000) synovial adhesions are more important than pannus proliferation in the pathogenesis of knee joint contracture after immobilization: an experimental investigation in the rat. j rheumatol 27:351–357. 2. patridge reh, duthie jjr (1963) controlled trial of the effect of complete immobilization of the joints in rheumatoid arthritis. ann rheum dis 22:91–99. 3. gault sj, spyker jm (1969) beneficial effect of immobilization of joints in rheumatoid and related arthritis: a splint study using sequential analysis. arthritis rheum 12:34–44. 4. peacock ee (1966) some biochemical and biophysical aspects of joint stiffness: role of collagen synthesis as opposed to altered molecular bonding. ann surg 164:1–12. 312 yoshihiro hagiwara et al. 5. damron ta, greenwald ta, breed a (1994) chronological outcome of surgical tendoachilles lengthing and natural history of gastro-soleus contracture in cerebral palsy. clin orthop 301:249– 255. 6. trudel g. 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of normal and immobilized rabbit knees. arthritis rheum 18:257–264. 19. hozumi n, yamashita r, lee ck, nagao m , kobayashi k, saijo y, tanaka m, tanaka n, ohtsuki s (2004) time-frequency analysis driven ultrasonic microscopy for biological tissue characterization. ultrasonics 42:717–722. 20. saijo y, tanaka m, okawai h, sasaki h, nitta s, dunn f (1997) ultrasonic tissue characterization of infracted myocardium by scanning acoustic microscopy. ultrasoud med biol 23:77–85. 21. saijo y, sasaki h, okawai h, nitta s, tanaka m (1998) acoustic properties of atherosclerosis of human aorta obtained with high-frequency ultrasound. ultrasound med biol 24:1061–1064. 22. saijo y, jorgensen s, mondek p, sefranek v, paaske w (2002) acoustic inhomogeneity of carotid arterial plaques determined by ghz frequency range microscopy. ultrasound med biol 28:933–937. 23. sasano y, ohtani e, narita k, kagayama m, murata m, saito t, shigenobu k, takita h, mizuno m, kuboki y (1993) bmps induce direct bone formation in ectopic sites independent of the endochondral ossification in vivo. anat rec 236:373–380. 24. shollmeier g, sarkar k, fukuhara k, uhtoff hk (1996) structural and functional changes in the canine shoulder after cessation of immobilization. clin orthop 323:310–315. 25. schollmeier g, uhtoff hk, sarkar k, furuhara k (1994) effects of immobilization on the capsule of the canine glenohumeral joint. clin orthop 304:37–42. 26. evans be, eggers gwn, butler jk, blumel j (1960) experimental immobilization and remobilization of rat knee joints. j bone joint surg 42a:737–758. 27. enneking wf, horowits m (1972) the intra-articular effects of immobilization on the human knee. j bone joint surg 54a:973–985. 28. thaxter th, mann ra, anderson ce (1965) degeneration of immobilized knee joints in rats. j bone joint surg 47a:567–585. 29. hall mc (1963) cartilage changes after experimental immobilization of the knee joint of the young rat. j bone joint surg 45a:35–44. increased elasticity of synovial membrane after immobilization 313 30. matsumoto f, trudel g, uhtoff hk (2002) high collagen type i and low collagen type iii levels in knee joint contracture: an immunohistochemical study with histological correlate. acta orthop scand 73:335–343. 31. sood sc (1971) a study of the effects of experimental immobilization on rabbit articular cartilage. j anat 108:497–507. 32. akeson wh, woo sl, amiel d, coutts rd, daniel d (1973) the connective tissue response to immobility: biochemical changes in periarticular connective tissue of the immobilized rabbit knee. clin orthop 93:356–362. 33. akeson wh, amiel d and la violette d (1968) the connective tissue response to immobility: an accelerated aging response? exp gerotol 3:289–301. corresponding author: yoshihiro hagiwara 1-1 seiryo-machi, aoba-ku, sendai 980-8574, japan e-mail: hagi@mail.tains.tohoku.ac.jp tel: +81-22-717-7245 fax: +81-22-717-7248 (8) 231 upsala j med sci 111 (2): 231–242, 2006 gene expression analysis of ectopic bone formation induced by electroporatic gene transfer of bmp4 satoshi kotajima, koshi n. kishimoto, munenori watanuki, masahito hatori and shoichi kokubun department of orthopaedic surgery, tohoku university school of medicine, sendai, japan abstract implantation of bone morphogenetic protein (bmp) using a carrier or by bmp gene transfer into rodent muscle can induce bone formation. implanted bmp becomes bioactive immediately after implantation. in bmp gene transfer, there is a time-lag between the secretion of gene products and bone formation. we analyzed the gene expression of chondrogenic and osteogenic specific markers in the process of ectopic bone formation by using semi-quantitative rt-pcr. a plasmid vector containing mouse bmp4 gene (pcaggs-bmp4) was transferred into the gastrocnemius muscles of mice using electroporation. histological examination revealed the process of endochondral bone formation in the pcaggs-bmp4 transferred muscles. as chondrogenic markers, aggrecan gene maximal expression was detected on day 7 and decreased by day 14, and for collagen x the gene maximal expression was on day 10. as osteogenic markers, osteocalcin (ocn), bone sialoprotein (bsp) and osteopontin (opn) gene expressions were clearly detected from day 10 and then increased by day 14. in conclusion, the present study proved that ectopic bone formation by bmp4 gene transfer into the muscle induced endochondral ossification that corresponded well with that to that by implantation of demineralized bone matrix. introduction bone morphogenetic protein (bmp) induces or stimulates bone formation. some isoforms of bmps (bmp2, 4, 6, and 7) differentiate mesenchymal cells into osteoblastic linage cells (1-3). in classical experiments using bmps, a carrier is necessary for bone induction because bmps have a short half-life in vivo (4). appropriate carrier implantation allows gradual bmps release and works as a scaffold for cells (5). received 1 september 2005 accepted 17 january 2006 gene therapy technique is thought to be a potent option for utilizing bmps. when a bmp coding gene is introduced into cells, the cells continuously produce and secrete bmp. adenoviral transfer of a bmp coding gene into rodent muscle showed abundant ectopic bone formation in immunodeficient animals without a carrier (6). we previously reported that electroporatic transfer of a plasmid dna containing mouse bmp-4 induced bone in immunocompetent mice (7). it is difficult to quantify the amount of protein produced by gene-transferred cells in an in vivo animal model. the pharmacokinetics of bmps might be different between protein and gene based experiments. bmps existing in demineralized bone matrix or put in a carrier are abundant at the time of implantation in vivo and decrease gradually. on the other hand, bmp expression, theoretically, increases after gene transfer, continues for weeks and then decreases gradually. the molecular and cellular events involved in endochondral ossification after implantation of demineralized bone matrix were described in detail by reddi (8). by day 3, mesenchymal and inflammatory cells had appeared around the implant. chondroblasts appeared by day 5 and chondrocytes were maximal on day 7. the hypertrophic cartilage matrix began to undergo calcification by day 9. basophilic osteogenic precursors and osteoblasts appeared on day 10. bone formation was confirmed on days 12 to 18. in this cascade of endochondral ossification, each differentiation step of osteogenic and chondrogenic cells was analyzed by using the following markers: osteocalcin (9), osteopontin (9), bone sialoprotein (10, 11), aggrecan (12, 13), and type x collagen (14). however, these markers of gene expression in this cascade were not assessed after bmp gene transfer but in protein and carrier models (15-17). to the best of our knowledge, this paper is the first to confirm the temporal gene expressions of osteogenic and chondrogenic markers in ectopic bone formation caused by the introduction of bmp4 gene into the muscle. materials and methods mice male c57bl/6j mice were purchased from clea japan, inc. electroporation was performed on 8-week-old mice. mice were maintained under specific pathogen-free conditions at the institute for animal experimentation, tohoku university school of medicine. the institutional animal care and use committee approved all the procedures used in this study. plasmid dna the 1.6kb mouse bmp-4 cdna was kindly provided by brigid l. m. hogan (18). it was inserted into multiple cloning sites of a pcaggs expression vector (19) (pcaggsbmp4). pcaggs-gfp, a gfp-containing plasmid, was used as the control. both plasmids were dissolved in distilled water at 2.0�g/�l. 100 microgrms of each was injected into the animals. 232 in vivo electroporation fifty microliters of 0.5% bupivacaine were injected into the left gastrocnemius as a pretreatment (20). in vivo electroporation was performed as previously described (pulse settings: 100v, 50ms, 6pulses, 1hz) (7, 20). pulses were applied through percutaneously inserted electrodes (0.4mm diameter: unique medical imada, natori, japan) just after injection of pcaggs-bmp4 or pcaggs-gfp (50�l each) into the pretreatment site. mice were euthanized by cervical dislocation 3, 7, 10, or 14 days after electroporation. soft x-ray assessment all gastrocnemius muscles were excised after sacrifice and underwent soft x-ray examination at 20.0kv and 2.0ma, for 10 sec (sro-im50, sofron, tokyo) using x-omat tl film (kodak). histology and immunohistochemistry frozen non-decalcified sections (thickness 10�m) of the specimens were made with a cryostat (bright, uk) after the soft x-ray photography. slides were stained with hematoxylin-eosin, alcian blue or von-kossa. bmp4 expression was detected immunohistochemically by rabbit polyclonal anti-bmp-4 antibody (1:100 dilution, overnight at 4°c; santa cruz biotechnology, santa cruz, ca). hrp labeled goat anti-rabbit antibody was used as the secondary antibody (1:100, 2h at room temperature; dako cytomation). rt-pcr analysis a muscular portion of the gastrocnemius with pcaggs-bmp4 (n=6) or pcaggs-gfp (n=6), 10 mm in length containing the center of the electroporated site was dissected (0.12g). total rna was isolated from the dissected muscles with the rneasy fibrous tissue mini kit (qiagen, germany) and reverse transcription-pcr was done with a takara one step rna pcr kit (takara, japan) following the manufacturer's instructions. the examined marker genes and their primers (in 5’ to 3’ direction) were as follows: transgene marker: mouse bmp4 (accession number: x56848) fwd. cccagagaatgaggtgatctcc rev. tggcagtagaaggcctggtag chondrogenic markers: procollagen type xalpha 1 (col10 accession number: z21610) fwd. gccaggtctcaatggtccta rev. gcacctactgctgggtaagc aggrecan (accession number: l07049) fwd. caggtttccccactgtgtct rev. actccagaccctgggaagtt 233 osteogenic markers: osteocalcin (ocn acession number: x04142) fwd. cttggtgcacacctagcaga rev. accttattgccctcctgctt osteopontin (opn accession number: af515708) fwd. tctgatgagaccgtcact rev. tctcctggctctctttggaa bone sialoprotein (bsp accession number: l20232) fwd. aaagtgaaggaaagcgacga rev. gttccttctgcacctgcttc internal control: glyceraldehyde-3-phosphate dehydrogenase (gapdh accession number: m32599) fwd. tgtttgtgatgggtgtgaa rev. atgggagttgctgttgaa total rna (1�g) was incubated for 30 minutes at 50˚c in a total volume of 50�l and then for 2 min at 94˚c, followed by 25 cycles for 30 seconds at 94˚c, for 30 seconds at 60˚c and then for 30seconds at 72˚c. the pcr products were analyzed on 2% agarose gels and visualized with ethidium bromide. the density of each band was quantified using scion image software (http://www.scioncorp.com). we determined the relative gene expression by dividing the densitometry value of the mrna rt-pcr product by that of the gapdh product. statistical analysis all measurements were performed in triplicate for each specimen and the mean value was used for statistical analysis. results were presented as mean ± standard deviation. the significance of differences between control and pcaggs-bmp4 electroporated muscles was determined using mann-whitney u test. values less than p=0.01 were considered significant. results x-ray assessment a radio-opaque area was observed in the gastrocnemius of all animals electroporated with pcaggs-bmp4 after 14days, but not in those electroporated with pcaggsgfp. histological findings mesenchymal cell infiltration was observed 3 days after bmp4 electroporation (figure. 1a). on day 7, the extracellular matrix was found to be stained clearly with alcian blue (figure. 1b, c). on day 10, when hypertrophic chondrocytes and cartilagenous matrix appeared, the intensity of alcian blue staining decreased (figure. 1d, e). the extracellular matrix was not revealed by von kossa staining. on day 14, calcified bone matrix was detected with von kossa staining (figure. 1f). 234 after electroporation of control pcaggs-gfp, mesenchymal cell infiltration was also observed on day 3. calcium deposits between the muscle bundles were sporadically observed on day 7. no cartilage or bone matrix were found at any time point (data not shown). immunohistologic analysis revealed that bmp4 was expressed in muscle fibers on day 3 (figure. 2a), and the intensity of bmp4 expression decreased with time (figure. 2b, c, d). 235 1 a 1 b 1 c 1 d 1 e 1 f fig 1. axial sections of pcaggs-bmp4 electroporated muscles stained by hematoxylin-eosin (a, b, d), alcian-blue (c, e) and von kossa (f). infiltrations of mesenchymal cells were found between muscle fibers on day 3 (a). extracellular matrices stained with alcian blue were identified on day 7 (b, c). hypertrophic chondrocytes and cartilage matrices were observed on day 10 (d, e). calcified bone matrices stained with von kossa were identified on day 14. bone marrow-like cells were found in calcified matrices (f). semi-quantitative rt-pcr bmp4 mrna expression was clearly detected 3 days after bmp4 electroporation. it gradually decreased and was hardly detected on day 14. no bmp4 mrna expression was detected in control (figure. 2e). aggrecan mrna expression was detected in bmp4 electroporated muscles from day 7, and decreased by day 14. no aggrecan mrna expression was detected in control. col10 mrna expression in 236 fig 2. localization of bmp4 expression analyzed by in situ immunohistochemistry at 3 (a), 7 (b), 10 (c) and 14 days (d) after electroporation. bmp4 was highly expressed in muscle fibers surrounding infiltrated mesenchymal cells on day 3 (a). the intensity of bmp4 expression and the number of bmp4 positive cells decreased with time. reverse transcription-polymerase chain reaction (rt-pcr) showed intense mouse bmp4 mrna expression in pcaggs-bmp4 electroporated muscles on day 3 and its expression decreased by day 14. bmp4 mrna expression was not detected in pcaggs-gfp (control) electroporated muscles (e). bmp4 electrtoporated muscles was significantly higher than in the control on day 10 and 14 (figure 3a,b). ocn and bsp mrna expressions were detected in bmp4 electroporated muscles on day 7. they were significantly elevated on day 10 and increased on day 14. no ocn and bsp mrna expressions were detected in control. opn mrna expression was high on day 3, not detected on day 7, detected again on day 10 and increased on day 14 in bmp4 eletroporated muscles. in control, opn mrna expression was not detected after day 7 (figure. 4a, b, c). 237 fig 3. time course of chondrogenic marker genes expression. aggrecan mrna expression was initially detected on day 7, and decreased by day 14, whereas it was not detected in the control group throughout this entire period (a). procollagen type x alpha 1 mrna expression was significant higher than in the control group on day 10 and 14 (b). the i-bars represent the standard deviation and the asterisk (*) indicates significant differences in mrna expression at each time point (p < 0.01). discussion gene transfer using electroporation is safe and inexpensive. in 2002, the present authors reported bone induction by using electroporatic transfer of bmp4, and the rate of bone formation in balb/ca mice was 67% at 14 days after electroporation (7). it is noteworthy that the present experiment demonstrated 100 % bone formation at 14 238 fig 4. time course of osteogenic marker gene expression. osteocalcin and bone sialoprotein mrna expressions were detected in the pcaggs-bmp4 group on day 7, were significantly elevated on day 10 and further increased on day 14. osteocalcin and bone sialoprotein mrna expressions were not detected in the control group (a,b). osteopontin mrna expression was high in both groups on day 3. its expression in pcaggs-bmp4 electroporated muscles was decreased on day 7 and gradually increased from day 10 and to day 14. osteopontin mrna expression in control group was not detectable after day 7 (c). the i-bars represent the standard deviation and the asterisk (*) indicates significant differences in mrna expression at each time point (p < 0.01). days. in this study, we could induce bone formation in all bmp4 transferred c57bl/6j mice. there are three possible reasons why the bone formation rate improved. the first is that the c57bl/6j strain, is reportedly a better responder for bmps than the balb/ca strain (21). the second reason may be that the pcaggs expression vector containing the cag promoter has higher activity in muscle (19). the third is that pretreatment with bupivacaine, which induces muscle necrosis, may have enhanced the efficiency of gene transfer by direct intramuscular plasmid injection (22-24). additionally, the muscle regeneration process after the pretreatment could provide abundant mesenchymal cells, which are essential for ectopic bone formation (25). however, at 14 days, bone induction was also observed in all mice even without bupivacaine pretreatment (data not shown), which lead us to believe that the strain of the mouse and the change of the expression plasmid contributed to stable bone formation. it is well known that transplantation of bmp protein or gene transfer of bmp into the muscles induce bone formation. however, there have been no reports describing in detail the cell differentiation in the process of bone formation. it is thought to be difficult to clearly distinguish each stage of differentiation of osteoblastic or chondroblastic cells under microscopic observation alone. however, we can recognize differentiation stages by serially examining the gene expression changes of osteogenic and chondrogenic markers. aggrecan is an extracellular matrix of cartilage and its gene is reported to be expressed in proliferating chondrocytes (13). col10 gene is reported to be expressed specifically in hypertrophic chondrocytes (26). aggrecan and col10 mrna expressions and histological examination in our experiment indicated that proliferating chondrocytes appeared on day 7 and had differentiated into hypertrophic chondrocytes by day 10. the gene expressions of ocn and opn were reported to correlate with the appearance of mature osteoblasts (27,28). bsp is extracellular matrix protein produced at the phase of osteoblastic differentiation (29). the ocn and opn mrnas expressions in our experiment indicated that differentiation into osteoblasts and bone matrix formation had started by day 10. opn mrna expression was strongly detected on day 3 in both bmp4 electroporated and control muscles. according to a recent report, opn may serve as a molecule that promotes macrophage binding to nextrotic fibers and may be an impotant mediator in the early phase of muscle regeneration (30). the opn mrna expression on day 3, therefore, does not reflect the appearance of osteoblasts but the activation of these inflammatory cells. in conclusion, examining changes of osteoblastic and chondroblastic gene expression enabled us to understand the differentiation stages of 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dt, einhorn ta (2003) fracture healing as a post-natal developmental process: molecular, spatial, and temporal aspects of its regulation. j cell biochem 88: 873-884. 15. clancy bm, johnson jd, lambert aj, rezvankhah s, wong a, resmini c, feldman jl, leppanen s, pittman dd (2003) a gene expression profile for endochondral bone formation: oligonucleotide microarrays establish novel connections between known genes and bmp-2-induced bone formation in mouse quadriceps. bone 3: 46-63. 16. kubota k, iseki s, kuroda s, oida s, iimura t, duarte wr, ohya k, ishikawa i, kasugai s (2002) synergistic effect of fibroblast growth factor-4 in ectopic bone formation induced by bone morphogenetic protein-2. bone 31: 465-471. 240 17. takita h, vehof jw, jansen ja, yamamoto m, tabata y, tamura m, kuboki y (2004) carrier dependent cell differentiation of bone morphogenetic protein-2 induced osteogenesis and chondrogenesis during the early implantation stage in rats. j biomed mater res a 71: 181-189. 18. jones cm, lyons km, hogan bl. (1991) involvement of bone morphogenetic protein-4 (bmp4) and vgr-1 in morphogenesis and neurogenesis in the mouse. development 111: 531-542. 19. niwa h, yamamura k, miyazaki j (1991) efficient selection for high-expression transfectants with a novel eukaryotic vector. gene dec 108 : 193-199. 20. aihara h, miyazaki j (1988) gene transfer into muscle by electroporation in vivo. nat biotechnol 16: 867-870, 1998 21. marusic a, katavic v, grcevic d, lukic ik (1999) genetic variability of new bone induction in mice. bone 25: 25-32. 22. davis hl, demeneix ba, quantin b, coulombe j, whalen rg (1993) plasmid dna is superior to viral vectors for direct gene transfer into adult mouse skeletal muscle. hum gene ther 4: 733740. 23. vitadello m, schiaffino mv, picard a, scarpa m, schiaffino s (1994) gene transfer in regenerating muscle. hum gene ther 5: 11-18. 24. wells dj. (1993) improved gene transfer by direct plasmid injection associated with regeneration in mouse skeletal muscle. febs lett 332: 179-182. 25. gonda k, nakaoka t, yoshimura k, otawara-hamamoto y, harrii k (2000) heterotopic ossification of degenerating rat skeletal muscle induced by adenovirus-mediated transfer of bone morphogenetic protein-2 gene. j bone miner res15: 1056-1065. 26. kong ry, kwan km, lau et, thomas jt, boot-handford rp, grant me, cheah ks (1993) intron-exon structure, alternative use of promoter and expression of the mouse collagen x gene, col10a-1. eur j biochem 213: 99-111. 27. leali d, dell'era p, stabile h, sennino b, chambers af, naldini a, sozzani s, nico b, ribatti d, presta m (2003) osteopontin (eta-1) and fibroblast growth factor-2 cross-talk in angiogenesis. j immunol. 171: 1085-1093. 28. lee k, deeds jd, chiba s, un-no m, bond at, segre gv (1994) parathyroid hormone induces sequential c-fos expression in bone cells in vivo: in situ localization of its receptor and c-fos messenger ribonucleic acids. endocrinology 134: 441-450. 29. young mf, ibaraki k, kerr jm, lyu ms, kozak ca (1994) murine bone sialoprotein (bsp): cdna cloning, mrna expression, and genetic mapping. mamm genome 5: 108-111. 30. hirata a, masuda s, tamura t, kai k, ojima k, fukase a, motoyoshi k, kamakura k, miyagoe-suzuki y, takeda s (2003) expression profiling of cytokines and related genes in regenerating skeletal muscle after cardiotoxin injection: a role for osteopontin. am j pathol. 163: 203215. corresponding author: masahiti hatori, m.d. department of orthopaedic surgery, tohoku university school of medicine 1-1 seiryomachi, aoba-ku, sendai, miyagi, japan phone: +81-22-717-7242, fax: +81-22-717-7248 e-mail: mhato@mail.tains.tohoku.ac.jp 241 242 torsten teorelll905 1992 proceedings from a symposium to commemorate the life and scientific achievements of torsten teorell on the 4th of august 1992 professor torsten teorell died a t the age of 87 after a very active scientific career leading to significant contributions to physiology. on the 11th of december 1992 the department of physiology at uppsala university arranged a small symposium t o commemorate some of torsten teorell's extensive scientific work and during this symposium six papers were presented. it has been suggested that these papers may be of general interest and they are therefore now being published. 3 upsala j med sci 95: 299-300, 1990 list op planned associated projects in the nordkem project 5/89 on medical need for quality specifications in laboratory medicine no. 1. 2. 3. 4. 5. 6. 7 . 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. particidants title hqrder m,olivarius n, jqrgensen pj, diabetes in primary bihlet i, hyltoft petersen p fraser c, hyltoft petersen p antonsen s grodum e, hyltoft petersen p, hangaard j, bollerslev j et al. djurhus s, rohold a, vadstrup s, hyltoft petersen p magid e, hyltoft petersen p et al. heedman p-a, olsson s, larsson 0 ritter b, marsell r, ostergaard h, olander b, akerblom a arends j, hyltoft petersen p, nqrgaard-pedersen b uldall a, blaabjerg 0 tryding n et al. nilsson ehle p westgard j 0, hyltoft petersen p sandberg s kaikola h-l penttila i m lindstedt g lindstedt g lytken-larsen m, fraser c, hyltoft petersen p health care quality specifications for detection limit reference interval for cortisol at crh-test reference intervals for s--potassium evaluation of clinical tests prevalence of idiop. hemochromatosis and hypercholest. 18 y men qual. spec. maternal s-a-fetoproteinanalys planning & implement. of nordic reference s analyt. specificity, tests in primary care qual. spec. s--cholest s--cholest.clin strat, anal. qual.,control system qual. spec. for tests in primary care 1 8 qual. spec. s--a-feto protein qual . spec. thyroid hormones qual. spec. prostate specific antigen qual. spec. hba,, 299 18. xofstad j, momsen g, gade christensen n 19. nilsson ehle h 20. linnet c 21. linnet c 22. dybkaer r , hyltoft petersen p, de verdier c-h et al. 23. stenman u-h 24. brandslund i, borg rasmussen j 25. sorto a, kaihola h-l qual. spec. b--ph,pco,, po2 i ca" qual. spec. iron de ficiency parame ters anal. goals for p- bilirubin (unconj) determinations anal. goals for p- creatinine determ. nomenclature qual. spec. for human choriogonado tropin (hcg) conc a,-antitrypsin in workers in dusty work areas quality goals & orga nization of qc in pri mary care lab. in the associated projects the quality specification work should be performed for each analytical quantity in each specified situation. models for estimation of needed quality and for specification of acceptable analytical performance and critical errors have been developed during the past ten years. these models should be applied to the clinical strategy in the project, whether a screening, a diagnostic situation or a time-related pathological process. each clinical situation should be analyzed concerning the type of model to be used, and if no relevant model is available to develop or elaborate new and useful models. the main project group must be directly involved by providing the associated groups with the available literature and, if needed, to help the groups to find their relevant models or to try to develop new useful models. on the other hand the main project group should try to compile the collected knowledge and make it useful. 300 untitled effects of wrist splinting for carpal tunnel syndrome 181 key words: carpal tunnel syndrome, wrist splint, nerve conduction measurement received 20 february 2008 accepted 31 march 2008 upsala j med sci 113 (2): 181–192, 2008 effects of wrist splinting for carpal tunnel syndrome and motor nerve conduction measurements shingo nobuta1, katsumi sato1, tomowaki nakagawa2, masahito hatori3, eiji itoi3 1 department of orthopaedic surgery, tohoku rosai hospital, 2 department of orthopaedic surgery, iwate prefectural iwai hospital, 3 department of orthopaedic surgery, tohoku university, school of medicine abstract background: carpal tunnel syndrome (cts) is one of the most common disease among the entrapment neuropathies. wrist splinting has been conventionally used for the cts treatment. the purposes of this study were to assess the efficacy of wrist splinting for cts, and to evaluate the value of the motor nerve conduction measurement as a prognostic indicator for cts. methods: two hundred and fourteen hands with cts were treated by wrist splinting, and reviewed after a mean follow up of seven months. severity of symptoms were minimal lesions in 177 hands, intermediate lesions in 33 hands, and severe lesions in four hands. motor nerve conduction measurement was performed in all cases before and after treatment, and distal latency (dl) and amplitude on compound muscle action potential (cmap) from the abductor pollicis brevis (apb) muscle were analyzed. results: according to kelly’s grading of outcome, results were excellent in 41 hands, good in 110 hands, fair in 45 hands, and poor in 18 hands. excellent or good results were obtained in 131 hands (74 percent) with minimal lesions, 20 hands (61 percent) with intermediate lesions, and in no cases with severe lesions. the ratio of excellent or good results was 79 percent in patients in whom dl of pre-treatment apb-cmap was less than 8 milliseconds (ms), and 62 percent in patients whose dl was 8 ms or more, which showed a significant difference. in nine hands whose pre-treatment apb-cmap was unrecordable, the results were good in one hand, fair in five, and poor in three. conclusions: wrist splinting is most effective in cases of minimal or intermediate lesions with dl of apb-cmap less than 8 ms. if relief of symptoms is not obtained after five months of treatment by splinting, that would be the limit of splinting. surgical release is recommended for cases with severe lesions and with unrecordable apb-cmap. introduction carpal tunnel syndrome (cts) is a compression neuropathy of the median nerve at the wrist, and is one of the most common entrapment neuropathies (1, 2). majority of patients with cts were treated conservatively before having surgery of carpal tunnel release (1–5). the conservative treatments for cts have included wrist splinting, steroid injection in the carpal tunnel, and the use of non-steroidal anti-inflammatory agents. the purposes of this study were to assess the efficacy of 182 shingo nobuta et al. wrist splinting for cts, and to investigate the value of the motor nerve conduction measurement as a prognostic indicator for cts treated by wrist splinting. materials and methods between 1998 and 2007, 214 hands in 167 patients (47 bilateral) with cts were treated by wrist splinting in our institute, and reviewed after a mean follow up of 7 months (4–24). diagnosis of cts was established when a patient had pain or paraesthesiae in the median nerve distribution and objective clinical findings of cts including delayed distal motor latency as mentioned later. twenty-four patients (30 hands) were men and 143 patients (184 hands) were women. the average age was 58 years (29–81). the duration of symptoms varied from one to 240 months, the mean being 18 months. two hundred and six hands were idiopathic cts, six had a preexisting colles fracture, and two had kienboeck disease. according to hamada’s classification of severity of symptoms (6), 177 hands had minimal lesions with sensory disorders (grade 1), 33 hands had intermediate ones with some thenar atrophy (grade 2), and four hands had severe ones with marked thenar atrophy and loss of thumb opposition (grade 3). four patients with severe lesions refused surgery of carpal tunnel release and agreed to undergo treatment by wrist splinting. the wrist splint was custom designed and molded using thermoplastic material, and was applied from the volar side with rigid fixation at a neutral position (figure 1 a, b, c). patients were instructed to wear the splint at night and during daytime when symptomatic. patients were followed once per month in our institute, figure 1. a: wrist splint design. custom-molded, thermoplastic material, volar side, and rigid fixation at a neutral position of the wrist (radial view). b: volar view. c: dorsal view. effects of wrist splinting for carpal tunnel syndrome 183 and anti-inflammatory agents or steroid injection were not used in any patient. motor nerve conduction measurement was performed in all hands before and after treatment. patients were examined with the arm in an outstretched position. palmar skin temperatures were not allowed to fall below 32°c. nicolet viking electromyography (nicolet instruments, madison, wi, usa) with a 10-mm silver disc was used. compound muscle action potential (cmap) from the abductor pollicis brevis (apb) muscle was recorded with surface electrodes by supramaximal stimulation of the median nerve at the wrist. distal latency (dl) was measured from the stimulus artifact to the onset of the potential. amplitude was measured from the baseline to the negative peak of the potential. the criterion for the normal value of dl at our department was below 4.8 milliseconds (ms) and the normal value of minimum amplitude was 0.9 millivolts (mv) (7). cts was diagnosed when the dl of apb-cmap was 4.8 ms or more. if apb-cmap was not recordable, cmap from the second lumbrical (sl) muscle was recorded and analyzed. normal value of dl was below 4.0 ms, and minimum amplitude was 0.3 mv in sl-cmap (7). the post-treatment results were evaluated into four categories according to relief of symptoms by kelly et al. (8). excellent was complete relief of symptoms, good meant persistence of occasional minor symptoms, fair was with some constant or annoying symptoms, and poor meant symptoms unchanged or worse. we compared clinical results with preand post-treatment dl and amplitude of apb-cmap. the data were analyzed statistically by the student’s t-test and the chi-square test. pvalues of less than 0.05 were considered statistically significant. results clinical results were excellent in 41 hands (19.2 percent), good in 110 hands (51.4 percent), fair in 45 hands (21.0 percent), and poor in 18 hands (8.4 percent). satisfactory results (excellent or good) were obtained in 131 hands (74 percent) with minimal lesions, 20 hands (61 percent) with intermediate lesions, and no cases with severe lesions (table 1). pre-treatment apb-cmap was recordable in 205 hands and unrecordable in nine hands. in nine hands with unrecordable apb-cmap, one hand was good, five hands were fair, and three hands were poor. in 205 hands with recorded apb-cmap, mean dl on pre-treatment apb-cmap was 6.0 ms (sd 1.3) in hands with excellent results, 7.0 ms (sd 2.1) with good, and 7.8 ms (sd 2.1) with fair or poor, which showed a significant difference between each two of the three groups (table 2). for comparison of results, we divided 205 hands with recorded apb-cmap into two groups: group a, with slightly delayed dl of pre-treatment apb-cmap (< 8 ms); and group b, with largely delayed dl of pre-treatment apb-cmap (≤ 8 ms) (figure 2 a, b). the ratio of satisfactory results (excellent or good) in group a was 78.6 percent, and that in group b was 61.5 percent, which showed a significant difference (p<0.025, table 3). mean amplitude on pre-treatment apb-cmap was 4.7 mv (sd 3.0) in hands with excellent results, 3.7 mv (sd 2.5) with good, and 3.1 mv (sd 2.5) with fair or poor, which showed 184 shingo nobuta et al. a significant difference both between the excellent and good groups, and between the excellent and fair or poor groups (table 2). mean dl on post-treatment apbcmap was 5.2 ms (sd 1.1) in the excellent group, 6.2 ms (sd 1.8) in good, and 8.0 ms (sd 2.1) in fair or poor, which showed a significant difference between each two of the three groups. post-treatment mean dl had shortened compared with pre-treatment dl in cases with excellent and good results, but that was unchanged in cases with fair or poor results (table 2). mean amplitude on post-treatment apbcmap was 5.7 mv (sd 3.3) in the excellent group, 4.5 mv (sd 2.8) in good, and 3.3 mv (sd 2.8) in fair or poor, which showed a significant difference between each two of the three groups. post-treatment mean amplitude was larger than pretreatment amplitude in all groups (table 2). surgery of open carpal tunnel release was performed in 24 hands later, and the results of wrist splinting in these cases were fair in 7 hands and poor in 17 hands. several other factors were examined statistically to determine whether they could predict clinical results. duration of symptoms was 11.2 ±11.7 months in the excellent cases, 16.7 ±29.2 months in good cases, and 25.9 ±47.3 months for the fair or poor cases. duration of symptoms revealed wide ranges and it was difficult to compare these data. age of the patients in the excellent, good, and fair or poor cases were 53.6 ±9.7 years, 57.9 ±11.0 years, and 59.5 ±11.3 years, respectively, which showed a significant difference both between the excellent and good groups (p<0.05), and between the excellent and fair or poor groups (p<0.01). the time of final evaluation of results in the excellent cases was 4.3 ±1.6 months, while that for the good cases was 4.7 ±1.9 months and that for the fair or poor cases was 5.2 ±2.1 months, which showed a significant difference between the excellent cases and fair or poor cases (p<0.05). the time of the start in relief of symptoms in the excellent cases was 1.5 ±0.7 months, while that for the good cases was 2.0 ±1.3 months and figure 2. a: slightly delayed distal latency (dl) of pre-treatment apb-cmap (<8 ms) in group a; b: largely delayed dl of pretreatment apb-cmap (≥8 ms) in group b. apb, abductor pollicis brevis; cmap, compound muscle action potential effects of wrist splinting for carpal tunnel syndrome 185 that for the fair cases was 2.8 ±1.3 months, which showed a significant difference between each two of the three groups (p<0.05). according to the self-report by the patients, symptoms recurred in 11 hands after ending wrist splinting. the time of recurrence after ending was 7 to 36 months, the mean being 22 months. in these 11 recurred cases, 5 cases started wrist splinting again and had relief of symptoms, and 6 cases had surgery for open carpal tunnel release later. discussion cts is usually diagnosed by the characteristic symptoms of paraesthesiae in the table 1. clinical results of 214 hands according to the severity of symptoms number of hands (%) results minimal intermediate severe total excellent 38 3 0 41 (19.2%) good 93 17 0 110 (51.4%) fair 35 9 1 45 (21.0%) poor 11 4 3 18 ( 8.4%) total 177 33 4 214 (100%) table 2. parameters of apb-cmap before(pre-) and at final follow-up (post-) pre-apb-cmap ; dl pre-apb-cmap ; amp. post-apb-cmap ; dl post-apb-cmap ; amp. 6.0 1.3 4.7 3.0 5.2 1.1 5.7 3.3 7.0 2.1 3.7 2.5 6.2 1.8 4.5 2.8 7.8 2.1 3.1 2.5 8.0 2.1 3.3 2.8 * p<0.001 ** p<0.01 *** p<0.05 * ** *** ** ** ** * n.s. * *** * *** * p<0.001 ** p<0.05 * p<0.001 ** p<0.01 *** p<0.01 * p<0.001 ** p<0.05 *** p<0.02 excellent (n=41) good (n=109) fair, poor (n=55) p value dl, distal latency (ms) ; amp., amplitude (mv) ; n.s., not significant 186 shingo nobuta et al. distribution of the median nerve, positive tinel’s sign at the wrist, phalen’s provocation test, and some thenar atrophy accompanying loss of thumb opposition. surgery of open or endoscopic carpal tunnel release is usually indicated for severe lesions, whereas most patients with minimal lesions are treated initially with conservative methods (1–6). the rationale for wrist splinting was originally based on the observation that symptoms with cts improve with rest and worsen with activity (9, 10). synovial inflammation of the flexor tendons can cause increased pressure in the carpal tunnel and contribute to median nerve compression (11). carpal tunnel pressure is elevated during repetitive hand activity, and the pathophysiology of cts is probably related to the duration as well as magnitude of the pressure increase within the carpal tunnel (12). however, lundborg (13) described that because of decreased muscular activity or faulty position during sleep at night, venous return was reduced and carpal tunnel pressure was increased, thus nocturnal symptoms in cts patients were frequent. werner et al. (14) reported that the pathophysiology of cts involved a combination of mechanical trauma and ischemic injury to the median nerve within the carpal tunnel, but the role of tendonitis and tendinosis was not well defined. with regard to the effect of wrist splinting for cts, previous studies showed minimizing carpal tunnel pressure by reducing synovitis through resting of the wrist. luchetti et al. (15) reported that slightly low pressures were found when the wrist was splinted, but critical pressure levels were not prevented by splinting. about the angle of wrist immobilization, gelberman et al. (16) showed that carpal tunnel pressure was lower when the wrist was in a neutral position than when in flexion or extension. flexion of the wrist seems to cause the flexor tendons of the fingers to be displaced against the palmar side of the carpal tunnel, increasing the pressure on both tendons and the median nerve. phalen (11) reported splinting the wrist in slight extension, whereas weiss et al. (17) described that the ideal position for immobilization is closer to neutral because wrist splinting in a functional position of extension did not minimize carpal tunnel pressure. regarding the rigidity of table 3. a relationship between dl of pre-treatment apb-cmap and results groups divided by dl group a, slightly delayed distal latency (dl) of pre-treatment apb-cmap (<8ms); group b, largely delayed dl of pre-treatment apb-cmap ( 8ms). the ratio of satisfactory results (excellent or good) in group a and that in group b showed a significant difference (chi-square test, p<0.025) group a (n=140) group b (n=65) 110 40 30 25 78.6% (110/140) 61.5% (40/65) excellent or good(n=150) fair or poor (n=55) results (hands) ratio of excellent or good results effects of wrist splinting for carpal tunnel syndrome 187 splints, rempel et al. (12) showed that flexible wrist splints failed to control carpal tunnel pressure during activities. in our patients, custom-molded rigid thermoplastic splints were developed because this splint design with a neutral angle was fit to achieve a neutral wrist position, and seemed to be comfortable for wearing at night. concerning splint wearing time, our patients were instructed to wear at night only in principle, and during daytime when symptomatic. walker et al. (18) reported that efficacy of neutral wrist splints was best with full-time wear instructions compared with night-only wear after six weeks of follow-up. kruger et al. (5) and nagaoka (19) also instructed the use of splint at night and during the day as much as possible. however, burke et al. (20) emphasized night-only wear of splints because wearing the splints during the day seemed restrictive, making it difficult to continue wearing them. manente et al. (21) reported excellent effects of night-only wear of an innovative hand brace with stretching the middle and ring fingers for cts. many patients indicated that nighttime symptoms were most troublesome, and were pleased with the relief that these splints afforded. we believe that a practical method of wrist splinting is wearing the splint at night and during the day as much as possible when symptomatic. about the relationship between clinical results and other factors, celiker et al. (22) described that patients with symptom duration more than 9 months did not respond well to treatment in splinting with an anti-inflammatory drug or steroid injection. kruger et al. (5) revealed that optimal results would be obtained if the splint was applied within the first three months of onset. graham et al. (23) reported that symptom duration of less than three months and absence of sensory impairment were predictive of a lasting response to wrist splinting and steroid injections. kaplan et al. (4) showed that five factors of unresponsiveness to treatment by wrist splint and anti-inflammatory medication were age over 50 years, duration over 10 months, constant paraesthesiae, stenosing flexor tenosynovitis, and a phalen’s test positive in less than 30 seconds. however, burke et al. (20) stated that duration of symptoms did not correlate with the symptom relief provided by splinting. weiss et al. (24) reported that patients 40 years of age or younger had a significant decrease in the rate of symptom resolutions with wrist splinting and steroid injection when compared with patients over 40 years of age. in our patients, duration of symptoms revealed a wide range of time and it was difficult to be analyzed. age of our patients correlated with the results. there was a tendency for patients over 59 years to have an unsatisfactory outcome. concerning period of treatment, gerritsen et al. (25) reported that the success rate based on general improvement was 54 percent after three months, and was 75 percent after 18 months of wrist splinting. mean time of final evaluation in our patients was 4.3 months in the excellent group and 4.7 months in the good, and 5.2 months in the fair or poor. thus, we believe that if relief of symptoms is not seen after five months of splinting, this treatment should be discontinued and other treatments should be employed. steroid injection in the carpal tunnel is a commonly used treatment similarly to wrist splinting (1, 2, 4, 11, 22, 24). goodman et al. (26) showed that the early 188 shingo nobuta et al. dramatic relief of symptoms after steroid injection was associated with a decrease in the nerve conduction delay, but this improvement was not maintained. gelberman et al. (1) reported that symptoms recurred in most patients by nine to fifteen months after injection. in contrast, in the present series with wrist splinting, early relief of symptoms could not be obtained and the mean time of the start in relief of symptoms was 1.5 months or more. however, it is noteworthy that recurrence of symptoms was seen in only 11 hands and most cases received long-term relief from splinting. nerve conduction study is accepted as a standard for the diagnosis of cts (27), and delayed conduction time by recording apb-cmap was first reported by simpson (28). concerning nerve conduction study for cts, kruger et al. (5) showed that in patients treated by wrist splinting, motor latency improved in the symptom relief group while it deteriorated in the non-relief group, and that improvement in motor latency was probably associated with patient perception of relief. gelberman et al. (1) stated that poor results were seen in the cases with delayed dl of more than 6 ms and absence of sensory response. in our patients, the ratio of excellent or good results in the group with delayed dl of less than 8 ms was higher than that in the group with delayed dl of 8 ms or more, and post-treatment dl had shortened in cases with excellent and good results, while it was unchanged in cases with fair or poor results compared with pre-treatment dl (tables 2, and 3). it has been said that segmental demyelination of motor fibers causes slowing of motor conduction (29), and that the degree of delayed dl of apb-cmap is based on the severity of demyelination of thenar motor fibers. thenar motor fibers are vulnerable to compression in the carpal tunnel (30). in the treatment of wrist splinting, the delayed dl of 8 ms or more may reflect the irreversible severe demyelination of the median nerve in the carpal tunnel. however, in our patients, unsatisfactory results were obtained in 21 percent of our cases with delayed dl of less than 8 ms, and satisfactory results were gained in 62 percent of cases with delayed dl of 8 ms or more. in these cases, tendonitis and tendinosis seemed to have a more important role than median nerve impairment from a viewpoint of pathophysiology. in eight of nine cases with unrecordable apb-cmap, results were unsatisfactory. axonal degeneration results in the loss of conductive elements, which leads to reduced amplitude of potentials (29). thus, unrecordable apb-cmap indicates severe axonal degeneration of thenar motor fibers, which warrants early surgical release. werner et al. (14) reported that motor and sensory nerve conduction studies were the best means for assessing the function of the median nerve. in nerve conduction study, the most sensitive method is the median sensory conduction measurement (31), but motor conduction study is technically easy to record because of high amplitude (7). in this study, we measured only the motor nerve conduction. we evaluated the results according to relief of symptoms by kelly et al. (8), which is a simple and subjective method of evaluation. in recent years, a selfadministered questionnaire assessing symptom severity and functional status for cts was reported (32). this instrument is highly reproducible, internally consistent, valid, and responsible to clinical change (18). this questionnaire can be used effects of wrist splinting for carpal tunnel syndrome 189 to check whether a certain therapy relieves symptoms and improves the functional status (33). you et al. (27) reported that the primary symptom severity scale correlated more strongly with the nerve conduction measures than did the secondary symptom scale. gerritsen et al. (34) described that final success rate of splinting for cts was 31 percent, and prognostic indicators were a short duration of cts complaints (one year or less) and a score of 6 or less for severity of paraesthesia at night. however, mondelli et al. (33) showed that the degree of improvement in the symptoms and functional status after surgical release could not be predicted from the pre-surgical self-administered questionnaire. in our series using subjective assessment and nerve conduction, a motor nerve conduction measurement was fairly valuable as a prognostic indicator for cts. the combination of electrophysiologic measurement and evaluation of the characteristic symptoms will provide the most accurate information for cts (31). there are some limitations in this study. first, results were evaluated according to relief of symptoms as subjective findings only. second, the follow-up period was variable (from 4 to 24 months). third, this study was not controlled and was retrospective. finally, in our patients, wrist splints were worn basically at night, but during the daytime also in some patients according to their symptoms. conclusions wrist splinting is effective for cts with minimal or intermediate severity with dl of apb-cmap less than 8 ms. if relief of symptoms is not obtained after five months application of splinting, other treatment options should be employed. surgical release is recommended for cases with severe lesions, or with unrecordable apb-cmap. acknowledgments the authors thank ms. hiromi takeda and ms. yumi watabe for their assistance and participants in this study for their time. references gelberman rh, aronson d, weisman mh (1980) carpal-tunnel syndrome-results of a prospec-1. tive trial of steroid injection and splinting. j bone joint surg 62-a: 1181-1184. duncan kh, lewis rc, foreman ka, nordyke m (1987) treatment of carpal tunnel syndrome 2. by members of the american society for surgery of the hand: results of a questionnaire. j hand surg 12-a: 384-391. quin ce (1961) carpal tunnel syndrome: treatment by splinting. ann phys med 6: 72-75.3. kaplan sj, glickel sz, eaton rg (1990) predictive factors in the non-surgical treatment of carpal 4. tunnel syndrome. j hand surg 15-b: 106-108. 190 shingo nobuta et al. kruger vl, kraft gh, deitz jc, ameis a, polissar l (1991) carpal tunnel syndrome; objective 5. measures and splint use. arch phys med rehabil 72: 517-520. hamada y, ide t, yamaguchi t (1986) the management of carpal tunnel syndrome-results of 6. prospective trial of conservative treatment. j jpn soc surg hand 3: 167-170 (in japanese). nobuta s (2002) electrodiagnosis and prognosis of carpal tunnel syndrome. seikei-saigaigeka 7. (orthop surg traumatol) 45: 1051-1057 (in japanese). kelly cp, pulisetti d, jamieson am (1994) early experience with endoscopic carpal tunnel 8. release. j hand surg 19-b: 18-21. roaf r (1947) compression of median nerve in carpal tunnel (letter). lancet 1: 387.9. heathfield kwg (1957) acroparaesthesiae and the carpal-tunnel syndrome. lancet 2: 663-666.10. phalen gs (1966) the carpal tunnel syndrome. seventeen year’s experience in diagnosis and 11. treatment of six hundred fifty-four hands. j bone joint surg 48-a: 211-228. rempel d, manojlovic r, levinsohn dg, bloom t, gordon l (1994) the effect of wearing a 12. flexible wrist splint on carpal tunnel pressure during repetitive hand activity. j hand surg 19-a: 106-110. lundborg g (1988) nerve injury and repair. churchill livingstone, edinburgh, 102-148.13. werner ra, andary m (2002) review; carpal tunnel syndrome: pathophysiology and clinical 14. neurophysiology. clin neurophys 113: 1373-1381. luchetti r, schoenhuber r, alfarano m, deluca s, de cicco g, landi a (1994) serial overnight 15. recordings of intracarpal pressure in carpal tunnel syndrome patients with and without splinting. j hand surg 19-b: 35-37. gelberman rh, hergenroeder pt, hargens ar, lundborg gn, akeson wh (1981) the carpal 16. tunnel syndrome: a study of carpal canal pressures. j bone joint surg 63-a: 380-383. weiss nd, gordon l, bloom t, so y, rempel d (1995) position of the wrist associated with 17. the lowest carpal-tunnel pressure; implications for splint design. j bone joint surg 77-a: 16951699. walker wc, metzler m, cifu dx, swartz z (2000) neutral wrist splinting in carpal tunnel syn-18. drome; a comparison of night-only versus full-time wear instructions. arch phys med rehabil 81: 424-429. nagaoka m (2002) conservative treatment of carpal tunnel syndrome. seikei-saigaigeka (orthop 19. surg traumatol) 45: 1073-1080 (in japanese). burke dt, burke mm, stewart gw, cambre a (1994) splinting for carpal tunnel syndrome; in 20. search of the optimal angle. arch phys med rehabil 75: 1241-1244. manente g, torrieri f, di blasio f, staniscia t, romano f, uncini a (2001) an innovative hand 21. brace for carpal tunnel syndrome: a randomized controlled trial. muscle nerve 24: 1020-1025. celiker r, arslan s, inanici f (2002) corticosteroid injection vs. nonsteroidal anti-inflammatory 22. drug and splinting in carpal tunnel syndome. am j phys med rehabil 81: 182-186. graham rg, hudson da, solomons m, singer m (2003) a prospective study to assess the out-23. come of steroid injections and wrist splinting for the treatment of carpal tunnel syndrome. plast reconstr surg 113: 550-556. weiss apc, sachar k, gendreau m (1994) conservative management of carpal tunnel syndrome: 24. a reexamination of steroid injection and splinting. j hand surg 19-a: 410-415. gerritsen aam, de vet hcw, scholten rjpm, bertelsmann fw, de krom mctfm, bouter lm 25. (2002) splinting vs surgery in the treatment of carpal tunnel syndrome: a randomized controlled trial. jama 288: 1245-1251. goodman hv, foster jb (1962) effect of local corticosteroid injection on median nerve conduc-26. tion in carpal tunnel syndrome. ann phys med 6: 287-294. you h, simmons z, freivalds a, kothari mj, naidu sh (1999) relationship between clinical 27. symptom severity scales and nerve conduction measures in carpal tunnel syndrome. muscle nerve 22: 497-501. simpson ja (1956) electrical signs in the diagnosis of carpal tunnel and related syndromes. j 28. neurol neurosurg psychiat 19: 275-280. kimura j (1984) principles and pitfalls of nerve conduction studies. ann neurol 16: 415-429.29. arita m, masakado y, kimura a, chino n (1998) the usefulness of motor conduction studies at 30. abductor pollicis brevis and lumbricalis in the diagnosis of carpal tunnel syndrome. jpn j rehabil med 35: 541-548 (in japanese). rempel d, evanoff b, amadio pc, krom m, franklin g, franzblau a, gray r, gerr f, hagberg 31. effects of wrist splinting for carpal tunnel syndrome 191 m, hales t, katz jn, pransky g (1998) commentary: consensus criteria for the classification of carpal tunnel syndrome in epidemiologic studies. am j public health 88: 1447-1451. levine dw, simmons bp, koris mj, daltroy lh, hohl gg, fossel ah, katz jn (1993) a self-32. administered questionnaire for the assessment of severity of symptoms and functional status in carpal tunnel syndrome. j bone joint surg 75-a: 1585-1592. mondelli m, reale f, sicurelli f, padua l (2000) relationship between the self-administered 33. boston questionnaire and electrophysiological findings in follow-up of surgically-treated carpal tunnel syndrome. j hand surg 25-b: 128-134. gerritsen aam, de bos ibck, laboyrie pm, de vet hcw, scholten rjpm, bouter lw (2003) 34. splinting for carpal tunnel syndrome: prognostic indicators of success. j neurol neurosurg psychiat 74: 1342-1344. corresponding author: shingo nobuta, department of orthopaedic surgery, tohoku rosai hospital 4-3-21 dainohara, aoba-ku, sendai, miyagi 981-8563 japan e mail : nobutays@jc5.so-net.ne.jp upsala j med sci 99: 373-376, 1994 7.7 reference intervals for sedimentation rate preben wiggersl, jplrgen dalhplj2, ole blaabjer8, per hyltoft petersen3, 1. department of medicine, haderslev sygehus, dk-6100 haderslev, denmark. 2. department of cardiology, odense uniuersity hospital, dk-5000 odense c, denmark. 3. department of clinical chemistry, odense university hospital, dk-5000 odense c, denmark. the erythrocyte sedimentation rate (esr) is used to monitor the activity of the acute phase reactants, which are raised due to inflammation or necrosis of tissues. however, in order to implement the esr it is essential to investigate the variations in a healthy population. materials and method 4,202 consecutive blood samples from blood donors 2,346 males and 1,856 females were investigated using westergren’s method (2) for determination of esr. the donors gave the blood voluntarily without being payed. the age range was 18 t o 65 years; mean 35.9 years for women and 39.3 years for men. 198 donors were investigated twice with an interval of six months. the present results have been published in another form before (1). results the geometric mean was 4.5 mm/hour for women and 2.5 mm/hour for men. the median value increased with age for both men and women. in fig. 7.7.1 the 97% percentile for the esr-values are shown as function of age. intra-individual variation was investigated in 198 subjects using the difference between first and second sampling. the coefficient of correlation was 0.26 for males and 0.58 for females. 373 erythrocyte sedimentation rate (mmlh) 30 -i women --i i i i women men 15 l o 1 men 5 1 o j i i i i 1 1 20 30 40 50 60 70 age (years) fig. 7.7.1 97% percentile for esr as function of age for healthy men and women. discussion the sedimentation rate is often applied as a 'screening test' in order to identify latent diseases, as the expectation is a quantitative connection between the degree of disease and the enhance increase in of the sedimentation rate. however, before the test can be applied, it is necessary to know the distribution of test results in the actual population in order to determine the validity of the test. like all other reference intervals a fraction of 'defined healthy' individuals will be excluded due to test results outside the reference interval. by using a one-tailed model excluding the upper 2% percentage of individuals the 'discharged' fraction is kept at a low level. 3 74 these limits are used as rule-out criteria in the protein investigations (cf. sections 7.2, 7.3 and 7.4). references 1. dalhpj j, wiggers p. the erythrocyte sedimentation rate in clinically healthy individuals. ugeskr k g e r 1990;152:456-9. westergren a. the technique of the red cell sedimentation reaction. h e r rev tuberc 1926;14: 94-101. 2 . 375 upsala j med sci 95: 251-255, 1990 the nordic protein project p. hyltoft petersen,' 0. blaabjerg,' k . bjgro', a . 1ci.11,~ k. ijala4 departments of clinical chemisiry at hospitals in 'odense, 'oslo, 'helsinki and 4 t u r k ~ i the nordic protein project was decided by the nordic committee on quality control in 1986 and supported by nordkem from 1987. the project works in close co-operation with the finnish non profit company, lab-quality and the data group in uppsala. the activities are co-ordinated with the finnish and the danish protein groups and further with the austrian protein group. the project deals with the three main aswects of quality management: qua 1 ity specifications / \ standardizationcontrol of quality quality specifications the analytical quality specifications (analytical goals) develops from the goal that laboratories should be able to share common reference intervals for analytical components (here serum proteins) within regions where the populations are homogenous*. the statistical models are described in two articles assuming gaussian (1) and loggaussian distributions (2). the preliminary goals are based on the finnsh investigation ( 3 ) and on litterature, using the log-gaussian model. 25 1 the analytical quality specifications are given as 'maximum allowable combined bias and imprecision', as percentage bias and cv % to be fullfilled for the concentration levels of both upper and lower reference limits. for s-haptoglobin, s-iga and s-igm the quality specifications are very wide, allowing a bias of + or -10% (when cv = 0 % ) or cv = 25% (when bias is o % ) , whereas the specifications for s albumin are very restrictive with maximum allowable bias of k 2% (cv = 0%) or cv = 4% (bias = 0%). for the remaining proteins (s-transferrin, s-alantitrypsin, s-prealbumin, s-orosomucoid, and sigg) allowable max bias from k 3 to 5% (cv = 0%) or cv from 7 to 11% (bias = 0%). standardization for the standardization a common liquid frozen serum calibrator has been chosen. it is prepared as a serum pool from more than 1000 male blood-donors (all negative for hepatitis antigen and hiv-antibodies). the cryoprecipitates are removed from 4oc and afterwards lipids are removed by ultracentrifugation. the calibrator is stored and mailed at -8ooc ( 4 ) . the frozen pool by centrifugation after thawing at the calibrator is clear, e.g. showed a preparation from 1981 an extinction (measured at 650 nm, undiluted, 10 mm cuvette) of 0.060 in 1981 and 0.079 in 1989. the proteins are genuine, i.e. behave like proteins in a fresh pool, e.g. they have identical electrophoretic mobility (also after eight years). further the production of the calibrator is reproducible and the proteins are stable. since no other calibrator with long term stable and genuine proteins is known, the combined reproducibility and stability is documentated indirectly by the low batch-to-batch variation, with cv below 2 % for batches from 1981, 1982 and 1984. further 6 proteins show deviation of less than + or -2% from 1984 to 1987, whereas the acute phase reactants were lower in the 1987 praparation ( 3 % for haptoglobin, 4 % for al-antitrypsin, and 5% for orosomucoid) . the first three pools were collected during winter and spring, the 252 1987 preparation during late summer and autumn. the concentration values are assigned from ifcc preparations of pure proteins and from the who reference preparation. control of quality control b (= 1.5’control a) (clear) i control t control a (= control t) (clear) i (turbid) control c (= 0.5*control a) (clear) the control samples are 4 serum pools with target values. three controls are clear with different concentrations, to control standardization and linearity at three concentration levels. the fourth is turbid, to control how effective the different analytical principles (and equipments) are in giving a correct measurement for a turbid patient specimen (5). difference difference (measured value target value) (measured value target value) i i i controls c 8 controls c a 8 05 1 0 1 5 0 5 1 0 15 relative concentration relative concentration the results are visualized by bias plots. 253 the steps in the project the procedure involves 3 steps: a ) the first assignment (january 1990) aims to registrate 'the state of art' (which we know is poor) and to evaluate performances in the 75 participating laboratories. b) the second assignment (scheduled to may) includes for each laboratory a) calibrator for daily use in two years b) the same controls as in a to be used i. in internal control of new calibration 11. in control of the established quality 111. repeated control (late 1990). c) the co-ordinated projects on reference intervals in denmark and finland are planned to supply the laboratories with common reference intervals by may and september 1990, respectively. depending on the results, specific reference intervals can be used in the two countries, or hopefully common reference intervals can be used in all the nordic countries. discussion the aim of the project is to improve the quality of serum protein measurements in the nordic countries to a specified level. the quality is obtained by daily use of sommon calibrator of the highest quality and the quality is controlled by selected samples with target values, to control both the standardization and the interferenbce from turbid sera. 254 the quality specifications are based on the assumption of using common reference intervals and these intervals are delivered by co-ordinated projects. other quality problems like measurements of mcomponents are not included in the project. other analytical quality specifications e.g. related to the use of serum proteins in defined clinical strategies, may be evaluated in cooperation with this 'umbrella project' on 'medical need for quality specifications in laboratory medicine'. references: 1. gowans ems, hyltoft petersen pi blaabjerg 0, hsrder m. analytical goals for the acceptance of common reference intervals for laboratories throughout a geographical area. scand j clin lab invest, 1988;48:757-64 2. hyltoft petersen pi gowans ems, blaabjerg 0, h0rder m. analytical goals for the astimation of non-gaussian reference intervals. scand j clin lab invest, 1989;49:727-37 3. irjala k, koskinen p i icen a, palosuo t. reference intervals for immunoglobulins iga, igg and igm in serum in adults and in children aged 6 months to 14 years. scand j clin lab invest, 1990;50:573-7. 4 . gry hi blaabjerg 0, blom hi uldall a, hyltoft petersen p. preparation of a protein calibrator. scand j clin lab invest, 1988;48 suppl 190:157 5. blaabjerg 0, hellsing k, icen k, hyltoft petersen p i reinskou. nkk's project group on plasma proteins. scand j clin lab invest, 1988;48 suppl 190:156. ----------------- *the biological background for sharing reference intervals is investigated in two other projects in finland and denmark. correspondence: p. hyltoft petersen, magister, department of clinical chemistry, university hospital, dk-5000 odense c, denmark. 255 (11) la proos 117-130 upsala j med sci 111 (1): 117–130, 2006 can bone age determination provide criteria for growth hormone treatment in adopted girls with early puberty? a comparison of the greulich-pyle and the tanner-whitehouse 2 methods. la proos 1, t lönnerholm 2, b jonsson 1, t tuvemo 1 department of women´s and children´s health 1,and department of clinical radiology 2, uppsala university, uppsala, sweden abstract in treatment of idiopathic central precocious puberty, gnrh analogues (gnrha) have been accepted as the treatment of choice. since growth velocity may be impaired with gnrha treatment growth hormone (gh) treatment has been added in clinical trials. recently, a study followed adopted girls with early or precocious puberty on gnrha or combined gnrha and gh treatment to final height. it was found that final height was significantly higher in the combined treatment group, although the difference was small. it was seen that patients that were extremely short at arrival and short at start of treatment seemed to be candidates for combined treatment. we have now analysed the data in order to define criteria for the subgroup in need of combined gnrha-gh treatment in order to achieve normal final height, i.e. above -2 sds. bone ages of 46 patients at start of treatment, randomized to either gnrha treatment or gnrha treatment combined with gh, were examined blindly by the same radiologist and the pah calculated. the methods according to greulich-pyle / bayley-pinneau (gp/bp) and tanner-whitehouse (tw2) were used. predictions versus final height data were analysed. the accuracy of fh prediction was greatest for gnrha treated group using the gp/bp method. the gp/bp method gave useful cut off limits for when combined treatment was necessary to possibly achieve normal height. if pre-treatment gp/pah was > 157cm, the patients attained normal height with gnrha treatment only. ten out of 13 (77%) such girls could be correctly identified. using tw2 with 117 received 30 september 2005 accepted 21 october 2005 a cut off of 164 cm, 9 out of 13 could be selected. using a multi regression equation of best fit the number of correctly selected cases for gnrha treatment only, could not be further increased in this group. we conclude that bone age determination and adult height prediction with the greulich-pyle/bayley-pinneau method, provides useful criteria for selecting the subgroup of adopted girls with early puberty where combined treatment with gnrha and gh is not necessary to reach normal final height. introduction since around 1960 more than 40 000 children from developing countries have been adopted in sweden. eventually it was reported from paediatric clinics all over sweden that many adopted girls developed early puberty and subsequently short final height. the reason for this was unknown. in studies on adopted girls from india it was shown that the degree of stunted growth due to under nutrition at arrival, as well as the velocity of subsequent catch-up growth was associated with the age at menarche. the earliest maturation was found among those most stunted at arrival with the most rapid catch up growth (1-3). studies that describe an association between being born small for gestational age (sga), subsequent growth and early maturation point in the same direction (4). during many years these adopted girls with early pubertal development have comprised a major part of the patients observed and in many cases treated for early or precocious puberty in the paediatric clinics all over sweden. similar findings regarding adopted girls have subsequently been reported from the netherlands, belgium, italy, france, denmark and the united states (5-11). the treatment to counteract early puberty was gonadotrophin releasing hormone analogues(gnrha), intranasal preparations first and depot preparations subsequently (12-19). although this treatment effectively blocked the progress of pubertal development, it was found to be associated with a decrease in height velocity as well (20-22). it seemed reasonable to hypothesize that patients with early or precocious puberty, treated with gnrha and simultaneously with growth hormone (gh) to compensate for the possible gh inhibition, would attain a significantly taller final height than patients treated with gnrha alone. an open randomized stratified multicentre study was designed, involving 20 paediatric clinics. half of the 46 patients received gnrha alone, the other half was given gh in addition to the gnrha. the patients were followed to final height, defined as growth velocity less than 1 cm height increase per year (23, 24). as reported elsewhere (24), the patients treated with the combined treatment attained a higher final height compared to the group treated with just gnrha. although statistically significant, the mean gain in final height for the whole group might be seen as limited, in view of the high cost of added gh treatment. the question remains whether any subgroup among the patients would be seen to benefit clearly from the 118 combined treatment. as reported, patients that were short at arrival in sweden and short at start of treatment seemed to benefit especially from added gh treatment. this indicates that the growth and bone maturation status of the patients before start of treatment might provide additional data toward identification of the subgroup that might benefit from combined treatment. thus the bone age determination and predicted adult height data are of interest to examine. the two since long used methods in clinical practice are those according to greulich-pyle (gp) (25) and tanner-whitehouse 2 (tw2) (26), and they were used in this study. the aim of the present paper was to compare the usefulness of the two methods of bone age determination regarding the prediction of post treatment final height and identification of criteria for combined gnrha and gh treatment in this group of patients. patients and methods the inclusion criteria for the clinical trial were a) girls below 9.5 years of age adopted into sweden from a developing country with no sign of puberty at the time of arrival in sweden, b) breast development defined as stage 2 or stage 3 according to tanner (27), c) pubic hair development defined as at least stage 2 according to tanner or an increase in height of at least 6 cm during the last year, and d) a normal serum level of tsh. fifty girls were recruited for the study. 25 girls were randomized for treatment with only gnrha and the remaining 25 for treatment with gnrha and gh. four children were subsequently excluded from the study: two girls because of misdiagnosis of precocious puberty, one girl due to gh-deficiency and one girl because of hepatitis c. of the 46 remaining girls, 22 were in the gnrha group and 24 girls in the gh/gnrha group. during the first two years of treatment, gnrha was administered as nasal spray 6 times daily. after a protocol amendment, 37 of the girls continued for a third year and eleven of them for a fourth year. during these additional years gnrha was given as a subcutaneous implant every 8 weeks. all the 46 patients were followed until they had attained final height. before start of treatment all girls had a radiograph of the least active hand (usually the left) taken. bone age according to greulich-pyle and tanner-whitehouse 2 was estimated blindly by the same experienced paediatric radiologist (tl). reference tables for gp (25) and, tw2 (26) were used. statistics conventional descriptive statistics have been used. means, standard deviations, medians, minima and maxima are given in table 1. pearsonian bivariate correlation and multiple stepwise regression analyses have been applied. a p-value less than 0.05 was considered statistically significant. standard deviation score (sds) for height was calculated as: 119 (observed height – mean)/sd, where mean and sd are the reference values and standard deviations for swedish children of corresponding age and gender to that child. mean adult height for girls was set to 165.9 cm with an sd of 6.29 cm (28). -2sds corresponds to a height of 153.32 cm. results chronological age and height at arrival, chronological age, bone age, height and predicted adult height (pah) at start of treatment and at final height (fh), subdivided according to treatment group and method of bone age determination are presented in table 1. baseline data did not differ significantly between the two treatment groups, as was to be expected, since the groups were randomly selected. mean final height differed 3 cm between the groups, which is approximately 0.5 sds. 13 out 120 table 1. auxological data, bone age and predictions and final height. mean n sd median min max age at arrival, yrs 2.3 45 2.30 1.1 0.1 7.3 height, cm, at arrival 75.8 45 21.71 67.0 46.0 117.0 height sds at arrival -2.5 45 1.96 -2.2 -6.9 0.7 age, bl 8.3 46 0.80 8.4 6.7 9.7 height, cm, bl 131.2 46 6.52 130.8 117.6 145.3 height sds, bl 0.4 46 1.03 0.2 -1.1 2.9 bone age, tw, bl 10.7 46 1.20 10.7 8.1 13.2 bone age, gp, bl 10.0 46 0.90 10.3 7.8 11.8 predicted adult height, tw, bl 163.3 46 4.92 162.6 153.4 172.7 predicted adult height, gp, bl 155.9 46 6.01 155.2 144.0 172.1 final height, cm 157.5 46 6.26 157.3 146.4 171.5 adult height sds -1.4 46 0.99 -1.4 -3.1 0.9 121 of 22 girls not treated with gh, and 21 of 24 of those gh treated, attained a final height above -2sds, indeed one of the three remaining girls was only 148 cm, but the other two very close to 153 cm. figure 1 shows the pah and actual fh for each bone age determination method and each treatment group. it is seen that the gp method is fairly accurate in predicting the fh in the gnrha treatment group, while tw2 overestimates markedly in this group. in the combined treatment group both methods are inaccurate, gp underpredicting and tw2 overpredicting. greulich pyle further analysis of pah according to gp and final height (fig 2), showed that all subjects with a pah >157 cm (19 patients out of 46, 10 in the gnrha group and 9 in the combined group), attained normal final height (>-2sds or 153 cm) irrespective of treatment with gh. all subjects with a gp prediction < 151 cm became short without gh treatment. if gp prediction was between 151.1 and 156.6 cm, 8 out of 2224 2224 2224n = buserelingh + buserelin 180 170 160 150 140 pah, gp final height pah, tw fig1. prediction of adult height according to the gp and tw2 methods at start of treatment in the two treatment groups. relation to final height. 122 pah, gp 180170160150140 f in a l h e ig h t, c m 180 170 160 150 140 treatment group buserelin gh + buserelin fig 2. individual final height in relation to prediction using the gp/bp method. pah tw 180170160150 f in a l h e ig h t, c m 180 170 160 150 140 treatment group buserelin gh + buserelin fig 3. individual final height related to prediction using tw2. 10 attained normal fh with gh treatment, while those who did not receive gh showed no certain trend either way. out of 7 girls who did not receive gh treatment 4 became short and 3 attained normal fh. two of those with short fh had a height on arrival in sweden of <4 sds. tanner – whitehouse 2 only in the group with a tw2 prediction of >164 cm, also 19 out of 46, all individuals attained a normal fh irrespective of treatment with gh (fig 3). height at start of treatment the relationship between height sds at start of treatment and fh is illustrated in fig 4. it was seen that if height at start of treatment is > 0.7 sds, then normal fh is attained irrespective of treatment group. a lower height sds at start of treatment was associated with a lower final height in both treatment groups. height sds at arrival the relationship between height sds at arrival in sweden and their final height is seen in fig 5. in the very few cases where height sds at arrival was above 0 normal fh was attained irrespective of treatment. if height at arrival was below 0 sds, normal fh was only attained in about half of the cases if gh was not added. 123 height sds at baseline 3210-1-2 f in a l h e ig h t 180 170 160 150 140 treatment group buserelin gh + buserelin fig 4. final height (cm) in relation to height at start of treatment (sds). bivariate correlation analyses in bivariate correlation analyses to find predictors of final height, height at start of treatment and predicted height according to the bone age estimations demonstrated correlation coefficients between 0.68 and 0.83 for the two treatment groups (table 2). for the gnrha treated, the highest correlation was found with gp prediction (0.83). for the combined group tw2 prediction demonstrated the highest correlation (0.81). height sds at start of treatment also demonstrated a high correlation to final height, 0.72 for the gnrha group, as good as the correlation between tw2 prediction and final height (0.72). bone age acceleration and age at start of treatment were not significantly correlated to final height in the bivariate analysis. multiple regression analysis multiple regression step-wise forward analyses were carried out in order to search for a set of predictors of possible use for prediction of treatment result on final height. data for each treatment group was analyzed separately. the candidate predictors for inclusion in the analyses were the same as presented in table 2 on bivariate correlation analyses. the best fit for the combined treatment (gnrha+gh) group was found for the following function: fhcm= 7.8 + 0.95 * pahtw + 1.5*(age at baseline – boneagetw), 124 hsds at arrival 20-2-4-6-8 f in a l h e ig h t 180 170 160 150 140 treatment group buserelin gh + buserelin fig. 5. final height in relation to height sds at arrival in sweden. where the coefficient of multiple determination (r2 ) was 0.75. this function indicates the aforementioned overestimation of pah according to tw2 and that the less pronounced the bone maturation is at start of treatment the taller will the final 125 combined treatment predicted value 170160150140 f in a l h e ig h t, c m 180 170 160 150 140 fhcm=7.75+0.95twp+ 1.5(age-ba(tw)) bl, rsq=0.75 fig.6. relation between tw2 prediction and final height with equation of best fit. gnrha predicted value 180170160150140 f in a l h e ig h t, c m 180 170 160 150 140 fhcm=18.0+0.88gpp rsq= 0.70 fig.7. prediction according to gp in relation to final height in the gnrha treated group. height become (fig.6). regarding the gnrha treatment group, the best fit with an r2 of 0.70 was: fh=18 cm+0.88 x gp prediction (fig 7). all girls with a predicted height above 157 cm, according to this function, reached final height above -2 sds. discussion the aim of the present paper was to find a practical method of evaluation of candidates for combined treatment with gnrha and gh, based on growth data and bone age determination. the first task was to examine the bone age determination methods commonly used, and assess their suitability for providing criteria for the subgroup that could really benefit from combined treatment with gnrha and gh, i.e. attain normal height with this more advanced treatment only. it could not a priori be expected that pre-treatment pahs would accurately predict pah in this very special group of patients, as the standards are basically constructed for normal, healthy children. accurate prediction could only be expected when pre-treatment growth potential had not been modified to any great extent by an intervention. however, we found the pre-treatment gp/pah reasonably accurate on the group level, when only gnrha was given, indicating that growth potential was not much modified by the gnrh analogue treatment. when combined treatment was used, the pre-treatment gp/pah under estimated final height, which might indicate that the treatment had increased final height. still, on the group level, tw2/pah was found to largely overestimate the fh in patients with gnrha treatment. even in the combination group, tw2/pah overestimated the fh. the analysis of gp/pah in relation to the fh results show that quite a few cases do not need addition of gh to attain a final height above -2sds. those who definitely need it in order to attain normal final height are those with gp/ pah < 151 cm. in the interval gp/ pah 151cm – 157cm an additional indicator is needed to identify those who would be candidates for combination treatment. girls with a gp prediction above 157 cm or a tw2 prediction above 164 cm attained normal final height irrespective of treatment. as reported earlier, the height sds at start of treatment is strongly correlated to the fh (24), which is to be expected as it is known that the closely preceding measure of the height at pubertal onset (hapo), is known to be significantly correlated to fh (29). that the height sds at treatment start is within the normal range is explained by the effect of the pubertal growth spurt. the height sds at arrival was, at least according to our earlier, more superficial analysis, correlated to fh (24), and could thus provide an additional indicator for the need of optimal treatment. patients with height at arrival <-4sds obviously 126 would need combination treatment in most cases, but height at arrival seems to be of little value as predictor for the whole group (fig 5). the equation of best fit for prediction of fh resulting from the multiple regression analysis, was found to contain gp/pah and tw2/pah as important factors. the r2 values were sufficiently high to possibly be of some clinical use for making a fh prognosis. the correlation between gp/pah and tw2/pah was highly significant (r=0.78) and both predictions were highly correlated to final height (r=0.74). therefore, the inclusion of either prediction in the multivariate analyses could be seen as random. in the case of the group with single treatment the regression analysis only made a correction to the gp/pah prediction formula and in the group with combined treatment the analyses corrected the tw2/pah prediction and added the value of low bone age maturation to give a better final height prediction. it can, however, be argued that 46 subjects is too limited a population on which a prognostic formula is to be based. on the other hand, 46 randomized patients in this very special patient category, all followed longitudinally from start of treatment to final height after around 10 years without drop-outs, may be a study population hard to improve on. we conclude that bone age determination can indeed provide criteria for combination treatment with gnrha and gh of early puberty in transnationally adopted girls. one aim in this study was to define those girls who would attain final height within normal range without expensive treatment with growth hormone. using baseline height sds when the girls already are in puberty, 15 girls out of 46 had a height sds of +0.8 or higher. five of them did not get gh. all these girls attained normal final height. by adding bone age criteria (gp/pah>157 cm or tw2 > 164 cm) a total of 19 girls (41 %) all attained normal height. ten of those with the high gp prediction (>157 cm) and nine of those with the high tw2 prediction were not gh treated. using only height at start of treatment, 5 of the 13 who did not need gh to attain normal height could be selected. by adding a simple gp prediction with cut off of 157 cm 10 out of the 13 could be predicted. even if the equation of best fit was used, this figure, which may be of practical use, could not be further increased. using the same criteria, i.e. gp > 157 cm on the gh treated group showed that all girls fulfilling it ended up well above 153 cm, indeed above 158 cm. in conclusion, these data indicate that in the great majority of cases of adopted girls with early puberty final height after gnrh analogue treatment can be predicted using greulich – pyle / bayley – pinneau prediction. patients in need of more intensive therapy can then be selected. references 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(abstract) horm res 37: (suppl) 4:33. 7. oostdijk w, yap yn, rekers-momberg itm, massa gg, brand r, drop sls. the impact of early puberty on final height in foreign born adopted children in the netherlands. in:oostdijk w:central precocious puberty and gonadotrophin releasing hormone agonist treatment. doctoral dissertation 1996, erasmus university, rotterdam, isbn 90-9009946-8. 8. de monleon jv, geneste b , huet f (1999) puberté précoce chez les enfants adoptés, un risque à ne pas oublier. arch pediatr 6:589-90. 9. baron s, battin j, david a, limal jm (2000) puberté precoce chez les enfants adoptés de pays étrangers. arch pediatr 7:809-16. 10. teilmann g, main k, skakkebaek n, juul a (2002) high frequency of central precocious puberty in adopted and immigrant children in denmark. horm res 58 (suppl 2):135(abstract). 11. mason p, narad c, jester t, parks j (2000) a survey of growth and development in the internationally adopted child. pediatr res 47:209a (abstract). 12. crowley jr wf, comite f, vale w, et al (1981) therapeutic use of pituitary desentization with a longacting lhrh agonist: a potential new treatment for idiopathic precocious puberty. j clin endocrin metabol 52:370-2. 13. comite f, cutler jr gb, rivier j, et al (1981) short-term treatment of idiopathic precocious puberty with long-acting analogue of luteinising hormone releasing hormone. n engl j med 1546-50. 14. mansfield j, beardsworth de, loughlin js, et al (1983) long-term treatment of central precocious puberty with long-acting analogue of luteinizing hormone-releasing hormone. effect on somatic growth and skeletal maturation. n engl j med 309:1286-90. 15. boepple pa, mansfield mj, crawford jf, crigler jr jf. blizzard jm, crowley jr wf (1990) gonadotrophin-releasing hormone agonist treatment of central precocious puberty: an analysis of growth data in a developmental context. acta paediatr scand (suppl) 367:38-43. 16. boepple pa, crowley jr wf (1991) gonadotrophin-releasing hormone analogues as therapeutic probes in human growth and development: evidence from children with central precocious puberty. acta paediatr scand (suppl) 372:33-8. 17. kletter gb, kelch rp (1994) effects of gonadotrophin-releasing hormone analog therapy on adult stature in precocious puberty. j clin endocrin metabol 79:331-4. 18. antoniazzi f, cisternino m, nizzoli g, bozzola m, corrias a, de luca f, de sanctis c, rigon f, zamboni g, bernasconi s, chiumello g, severi f, tato t (1984) final height in girls with precocious puberty: comparison of two different luteinizing hormone-releasing hormone agonist treratments. acta paediatr 83:1052-6. 19. tuvemo t, gustafsson j, proos la; swedish growth hormone group (2002) suppression of puberty with short-acting intranasal versus subcutaneous depot gnrh agonist. horm res. 57:27-31. 20. hermanussen m (1995) growth promotion by oxandrolone in a girl with short stature and early pubertal development treated with growth hormone gonadotropin-releasing hormone analogue, a case study. acta paediatr. 84:1207-10. 21. oostdijk w, drop sls, odink rjh, hummelink r, partsch cj, sipell wg (1991) long –term results with a slow release gonadotrophin-releasing hormone agonist in central precocious puberty. acta paediatr scand 372 (suppl): 39-45. 22. saggese g, pasquino am,bertelloni s, baronelli gi, battini r, pucarelli i, segni m, franchi g (1995) effect of combined treatment with gonadotropin releasing hormone analogue and growth hormone in patients with central precocious pubert who had subnormal growth velocity and impaired height prognosis. acta paediatr 84:299-304. 128 23. tuvemo t, gustafsson j, proos la (1999) growth hormone treatment during suppression of early puberty in adopted girls. swedish growth hormone advisory group. acta paediatr. 88:928-32. 24. tuvemo t, jonsson b, gustafsson j, albertsson-wikland k, aronson as, häger a, ivarson s, kriström b, marcus c, nilsson ko, westgren u, westphal o, åman j, proos la (2004) final height after combined growth hormone and gnrh analogue treatment in adopted girls with early puberty. acta paediatr 93:1456-62. 25. greulich ww, pyle si radiographic atlas of skeletal development of the hand and wrist, 2nd edn. stanford universitypress, stanford, ca. 1959. 26. tanner jm, whitehouse rh, marshall wa, carter bs (1975) prediction of adult height from height, bone age and occurrence of menarche, at ages 4 to 16 with allowance for mid parent height. arch dis child 50:14-26. 27. tanner jm. growth at adolescence. 2nd ed. oxford: blackwell, 1962. 28. karlberg p, taranger j, engström i, lichtenstein h, svennberg-redegren i (1976) the somatic development in an urban swedish community. acta paediatr scand (suppl) 258:7-76. 29. proos la, karlberg j, hofvander y, tuvemo t (1993) pubertal linear growth of indian girls adopted in sweden. acta paediatr 82:641-4. 30. tato l, saggese g, cavallo l, antoniazzi f, corrias a, pasquino am, cisternino m (1995) use of combined gn-rh agonist and hgh therapy for better attaining the goals in precocious puberty treatment. horm res. 44 (suppl 3):49-54. corresponding author: la proos department of women’s and children’s health uppsala university, se-751 85 uppsala, sweden 129 130 sups_a_548877 77..78 upsala journal of medical sciences. 2011; 116: 77–78 letter to the editor unusual scalp crusted scabies in an adult t-cell leukemia/lymphoma patient yi-chun lai1,2, chung-jen teng1,2,3, paul chih-hsueh chen5, tzeon-jye chiou1,4 & chun-yu liu1,3 1national yang-ming university school of medicine, taiwan, republic of china, 2department of medicine, national yang-ming university hospital, ilan, taiwan, republic of china, 3division of hematology/oncology, department of medicine, 4division of transfusion medicine, department of medicine, and 5department of pathology and laboratory medicine, taipei veterans general hospital, taipei, taiwan, republic of china key words: adult t-cell leukemia/lymphoma, crusted (norwegian) scabies, human t-cell lymphotropic virus 1 a 54-year-old taiwanese lady presented with intermittent fever for 1 week. examination revealed hepatosplenomegaly. laboratory studies revealed marked leukocytosis (leukocyte count of 316,000 /ml) and an elevated lactate dehydrogenase of 2,929 iu/l. examination of her peripheral blood morphology disclosed abnormal lymphoid cells with flower-shaped nuclei (figure 1a), and a subsequent serology testing for human t-cell lymphotropic virus 1 (htlv-1) antibody showed a positive result. a bone-marrow biopsy specimen demonstrated marrow infiltration of atypical lymphoid cells (figure 1b), around 20%–30%, which were immunoreactive for uchl-1, cd3, and cd7, but non-immunoreactive for cd20, cd34, tdt, and myeloperoxidase. in addition, multiple intra-abdominal lymphadenopathies were discovered by a computed tomography scan. she was diagnosed with acute type adult t-cell lymphotropic/leukemia (atl) and was treated with a combination of chemotherapy regimen (cyclophosphamide, vincristine, doxorubicin, and prednisolone (chop)), isotretinoin (roaccutane�), and subcutaneous recombinant interferon alfa-2a (roferon-a�). a response of partial remission was achieved after the treatments, and her following blood routines were in stable status. about 14 months later, she came to our emergency room because of general malaise and abdominal fullness for several days. she had a leukocyte count of 19,400/ml and hypercalcemia (free calcium of 2.57 mmol/l) on laboratory investigation. moreover, she presented with scaly crusted skin lesions over the scalp (figure 1c) and external ears (figure 1d), which were initially considered as skin involvement of atl. no burrows were identified between fingers or over wrists or other skin parts. however, microscopic examination of the scraping scales with potassium hydroxide staining disclosed many scabies mites and hatched eggs (figure 1e). norwegian scabies was settled and she was treated with topical antiscabies ointment, gamma benzene hexachloride. unfortunately, her condition deteriorated rapidly with development of sepsis and subsequent acute respiratory distress syndrome. she died of rapid progression of atl 1 week later. htlv-1 infection is not endemic in taiwan (1); the prevalence of atl in taiwan has been reported to be 6% in 317 patients with non-hodgkin’s lymphoma (nhl) during 1983–1988 in northern taiwan (2), and 2.8% in 72 patients with t-cell nhl during 1989–2002 in southern taiwan (3). in htlv-1 correspondence: chun-yu liu, md, division of hematology/oncology, taipei veterans general hospital, no. 201, sec. 2, shi-pai road, taipei 112, taiwan, republic of china. fax: +886-2-28757762. e-mail: liuchunyu_tw@yahoo.com.tw; cyliu3@vghtpe.gov.tw (received 7 december 2010; accepted 13 december 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.548877 non-endemic regions, the diagnosis of atl can be challenging and difficult to establish. crusted scabies has been reported in htlv-1seropositive patients, some of whom had atl (4,5). the scabies skin lesions usually involve limbs and trunk, either localized or diffuse, and only rarely involve the face or scalp area (6,7). the dermatological presentation of scabies seen in our patient was unusual in that only the scalp and ears were predominately involved, and there were no classical ‘burrows’ that were commonly seen on hands and wrists in patients with scabies infections. htlv-1-induced immunosuppression has been linked to the occurrence of crusted scabies in htlv-1 carriers and in atl patients (8). the atypical dermatological presentation of scabies in patients with atl may cause diagnostic confusion with the more commonly seen skin involvement of atl. moreover, the presence of scabies in atl patients may represent a sign of marked immunosuppression and thus indicate a poorer prognosis. the rapid deterioration after the diagnosis of scabies seen in our patient supports this correlation. although atl-associated skin lesions are common in atl patients, crusted scabies should also be considered in the differential diagnosis of either localized or generalized cutaneous eruptions in patients with atl. declaration of interest: the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. lu sc, chen bh. seroindeterminatehtlv-1 prevalence and characteristics in blood donors in taiwan. int j hematol. 2003; 77:412–13. 2. shih ly, liang dc. non-hodgkin’s lymphomas in asia. hematoloncolclin north am. 1991;5:983–1001. 3. lu d, lin cn, chuang ss, hwang ws, huang wt. t-cell and nk/t-cell lymphomas in southern taiwan: a study of 72 cases in a single institute. leuk lymphoma. 2004;45: 923–8. 4. bergman jn, dodd wa, trotter mj, oger jj, dutz jp. crusted scabies in association with human t-cell lymphotropic virus 1. j cutan med surg. 1999;3:148–52. 5. suzumiya j, sumiyoshi a, kuroki y, inoue s. crusted (norwegian) scabies with adult t-cell leukemia. arch dermatol. 1985;121:903–4. 6. takeshita t, takeshita h. crusted (norwegian) scabies in a patient with smoldering adult t-cell leukemia. j dermatol. 2000;27:677–9. 7. duran c, tamayo l, de la luz orozco m, ruiz-maldonado r. scabies of the scalp mimicking seborrheic dermatitis in immunocompromised patients. pediatrdermatol. 1993;10:136–8. 8. del giudice p, sainte marie d, gerard y, couppie p, pradinaud r. is crusted (norwegian) scabies a marker of adult t cell leukemia/lymphoma in human t lymphotropic virus type i-seropositive patients? j infect dis. 1997;176:1090–2. a c d b e figure 1. a: a flower-like nucleated t cell in the peripheral blood smear from the patient. b: pathology of marrow biopsy revealed lymphomatous involvement in marrow space. c, d: crusted scaly lesions at the scalp and posterior auricular skin fold (arrow). e: microscopic examination of a scraping scale shows two hatched eggs (right) and a mite (left) (koh, �400). 78 y.-c. lai et al. upsala j med sci 104: 231-236, i999 malignant fibrous histiocytoma: a method to control intraoperative hemorrhage by clamping the feeding arteries taka4hi yokota, m d , kazutsugu iwamoto, m d , yasuo kunn, m d and hideml yamauchl, m d departnient of surgery, sendai national hospital mi)agrno-ku, sendai 983 8520, japan abstract malignant fibrous histiocytoma (mfh), also referred to in the past as malignant fibrous xanthoma and fibroxanthosarcoma, is a tumor of mesenchymal tissue origin. a case of retroperitoneal mfh was reported. in this paper, we describe a method of hemostasis and intraoperative control of hemorrhage during resection of retroperitoneal mfh by snaring the feeding arteries. the patient was successfully operated on using this technique. introduction malignant fibrous histiocytoma (mfh) is the most common adult soft tissue sarcoma (3, 8, 12, 14, 20), comprising 10% to 21% of all such tumors (16, 17). the tumor most frequently arises from deep fascia or skeletal muscle in an extremity. the thigh is the single most common site of origin followed by an upper extremity, the chest and retroperitoneum (5, is). retroperitoneal tumors account for only 9 to 16% of all reported cases. occasionally the tumor becomes evident as a huge mass in the retroperitoneal space in which case, laparotomy shows extensive tumor involvement of both visceral and parietal peritoneum with diffuse thickening of each. it is sometimes difficult to resect such a tumor due to feeding arteries around the surface or primary involvement of large vessels around the tumor. in this case, we employed temporal occlusion of feeding arteries and large vessels around the tumor using a rubber tube for control of bleeding during resection of a giant retroperitoneal mfh. case report a 62-year old woman was admitted to sendai national hospital in february 1998 with the complaint of constipation with stools and sever left thigh pain of recent onset. she was found to have a large mass in the left retroperitoneal cavity by computerized tomography (ct) scan. physical examination revealed a firm, fixed left lower quadrant mass in the midclavicular line. 23 1 her medical history was unremarkable. results of laboratory studies, including complete blood ' count and blood chemistry, were within normal limits. a ct scanning revealed a large retroperitoneal mass with heterogeneous low-density contents, with extension to the pelvic sidewall and the lower surface of the left kidney. magnetic resonance (mr) imaging performed to characterize the tumor further, confirmed the findings of the ct scanning. the tumor was shown as a heterogeneous, huge mass occupying left retroperitoneal cavity (fig. 1a). mr imaging sagittal cut showed a large mass elevating the left kidney (fig. 1b). figure 1. preoperative mri scan. (a) transaxial view of retroperitoneal mfh (arrow). (b) sagittal view through the central portion of the tumor (arrow). the anterior surface of the patient is to the left. barium enema revealed left lateral deviation and extrinsic compression of the descending colon by a large retroperitoneal mass, but otherwise was normal. abdominal angiography showed a giant abdominal mass to be relatively hypovascular with stretching of the inferior mesenteric 232 artery, fiom which several feeding vessels entered into the tumor. after adequate bowel preparation, the patient was taken to surgery where laparotomy revealed a fixed mass in the left lower quadrant which extended to the base of the mesentery. the mass elevated the left kidney superiorly and the descending colon medially and adhered to the left common iliac artery from which some feeding arteries were seen. the mass was first thought to be unresectable since the operator could not insert even his hand into the space between the mass and parietal wall of the retroperitoneal space due to profuse bleeding from the feeding arteries. the left common iliac artery, inferior mesenteric and left renal arteries were then surrounded with rubber tape just below their parts near the root. the ends of the tape were brought through a rubber tube that was 5 cm long to form a snare. the loop was tightened and secured with a hemostatic clamp to occlude the vessels. as the retroperitoneal mass adhered to the mesocolon, an excision with mesocolon with adequate tissue margin was performed using electrocautery, while keeping the vessels occluded during the excision for on hour. the resultant defect was closed. as the mass was found to invade directly to the left kidney, left nephrectomy was performed. the tumor was easily dissected off the pelvic wall. after removal of the tumor, the vessels were intermittently opened to locate the bleeding vessels and hemostasis was completed. the snare was then removed, after which the abdominal wound was closed in layer. four weeks later, the patient commenced radiotherapy to the tumor bed and a total of 30 gy in 20 fractions were delivered over 30 elapsed days. discussion the most common site of primary involvement of the genitourinary tract by mfh is the kidney. the patient presented in this report underwent wide local excision with left nephrectomy. radical removal of involved organ is recommended for mfh. the great deterrent to resection of a tumor is the risk of hemorrhage due to the organ's vascularity or large vessels around it. the feeding arteries or large vessels around tumors can be clamped without hazard for a long period in spite of the vulnerability of the alimentary tract to anoxia. in this case, clamping of the common iliac, inferior mesenteric and left renal arteries has been successfully used to achieve quick hemostasis in hemorrhagic mfh. it does not, however, allow the control of hemostasis after tumor removal, since the clamp can only be manipulated through a laparotomy. after excision the main bleeders were found to be on the parietal wall of retroperitoneal space, probably being branches of the left common iliac artery. good hemostasis was achieved with this method; there was no major tumor hemorrhage as was suspected preoperatively. the present technique allows intermittent opening of the feeding arteries and control of hemorrhage arising 233 from the abdominal tumor. despite the aggressive surgical approach, local recurrence rates for this disease are still high, ranging 44% to 67% (4, 11, 16). mfh is a radiosensitive tumor, and radiotherapy is a highly effective treatment modality. postoperative radiation therapy can be very successful in preventing local recurrence after surgical excision (7, 9, 10, 19). the metastatic rate for mfh after radical surgery ranges from 23% to 42% (11, 16), and adjuvant chemotherapy is indicated in the management of these tumors. doxorubicin is reported to be the most effective chemotherapeutic agent for soft tissue sarcomas. it is also the most commonly used drug in the treatment of mfh (1, 2, 6). prolonged remissions in patients with unresectable mfh have been reported using combination chemotherapy and radiation therapy (1 3, 15). although retroperitoneal mfh portends a very poor prognosis, wide local resection with adjuvant irradiation and doxorubicin based chemotherapy is the treatment of choice for tumors amenable to surgical intervention. 1. 2. 3. 4. 5. 6. 7. 8. 2 34 references basset, w. b. & weiss rb.: prolonged complete remission in malignant fibrous histiocytoma treated with chemotherapy. cancer tret rev 62: 1405-1406, 1978. clamon, g. h., robinson, r. a. & olberding, e. b.: prolonged remission of metastatic malignant fibrous histiocytoma induced by combination chemotherapy. j surg oncol 26: 113-1 14, 1984. hashimoto, h., daimaru, y., tsuneyoshi, m. & enjoji m.: soft tissue sarcoma with additional anaplastic components. a clinicopathologic and immunohistochemical study of 27 cases. cancer 66: 1578-1589, 1990. kearney, m. m., 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cancer 39: 1672-1585, 1977. 17. weiss, s. w. & enzinger, f. m.: malignant fibrous histiocytoma: an analysis of 200 cases. cancer 4 1 : 2250-2266, 1978. 18. weiss, s.w.: malignant fibrous histiocytoma. a reaffirmation. am j surg pathol 6: 773 784, 1982. 19. wood, w. c., suit, h. d., mankin, h. j. & cohen, am, proppe, k.: radiation and conservative surgery in the treatment of soft tissue sarcoma. am j surg 147: 537-541, 1984. 20. zehr, r. j., bauer, t. w., marks, k. e. & weltevreden, a.: ki-67 and grading of malignant fibrous histiocytomas. cancer 66: 1984-1990, 1990. adress for reprints: takashi yokota, m.d. department of surgery, sendai national hospital, miyagino-ku, sendai 983-8520, japan e-mail address: yokoyoko@jun.ncvc.go.jp f a : 022-291-8114 235 upsala j med sci 102: 199-210, 1997 hyaluronan production in vitro by fetal lung fibroblasts and epithelial cells exposed to surfactants of n-acetylcysteine h. johnsson,’.* p. heldin,* g. sedin’ and t. c. laurent2 departments of ‘pediatrics, and =medical and physiological c h e m i s t q uppsalu university, uppsala, sweden abstract fetal human lung fibroblasts and feline lung epithelial cells were exposed to either a surfactant or n-acetylcysteine in various concentrations for 24-48 hours, after which the hyaluronan concentration in the culture medium was determined. most of the experi ments showed no stimulatory effect of either artificial or natural surfactant on hyaluronan synthesis. n-acetylcysteine 5-1 00 mg/ml induced progressive stimulation of hyaluronan synthesis by human fetal lung fibroblasts, resulting in a maximum hyaluronan concentra tion six times that released by unexposed cells. a slight increase in hyaluronan synthe sis was also observed after exposure of feline fetal lung epithelial cells to n-acetyl cysteine 50-1 00 pg/ml. introduction the hyaluronan concentration in the lung decreases during the last fifth of normal fetal development, with the lowest value immediately before term (1). the production of hyaluronan increases during pulmonary disease in infants and adults, and as an early response to lung injury in animal models (2, 3, 4). it also increases as a reaction to hyperoxia (5,6). the in vifro hyaluronan production is known to be enhanced by several inflammatory mediators (7). in adult patients with inflammatory diseases of the lungs, increased hyaluronan concentrations have been observed both in interstitial lung tissue and in broncho alveolar lavage fluid (8). in the adult respiratory distress syndrome (ards), charac terized by non-cardiogenic pulmonary edema and inactivation of surfactant as a result of leakage of plasma proteins into the air spaces from areas of epithelial disruption (9), accumulation of hyaluronan parallels the development of edema (1 0 , l l ) . 199 in infant respiratory distress syndrome (irds), a frequent complication of preterm, birth, there is a reduction of surfactant activity, due to a combination of surfactant defi ciency and surfactant inactivation by plasma proteins (9, 12). this results in increased permeability of the alveolar wall, hyaline membrane formation, and increased interstitial lung water (13). the hyaluronan concentration in lung extracts has been shown to increase with increasing severity of irds in premature monkeys, and to decrease with surfactant treatment (2). surfactants, which are used to treat infants with irds by direct instillation into the airways, must not be assumed to be inert substances with a singular capacity to alter surface tension and gas exchange properties of the lung (14). arnon e t a / (1 5) reported that surfactant treatment of newborns with irds increased the total white cell count and the number of macrophages in bronchoalveolar lavage fluid, while gerdes ef a/ (16) found no increase in neutrophil elastase in tracheal aspirates after surfactant treatment. the effects of surfactants in cellular cultures have mostly been studied in macro phages and monocytes, where inhibition of effects of inflammatory mediators has generally been found (17,18). a decrease in the phagocytic function of these cells has also been noted in some studies (19,20). fewer reports have dealt with the effects on fibroblasts, but surfactants have been shown in vifro to both stimulate and inhibit fibroblast proliferation (21), to have effects on the surfactant metabolism in exposed cells (22), and to inhibit synthesis of dna and inflammatory mediators in normal human lung fibroblasts (23). surfactants may also affect tracheal epithelial cells by increasing the membrane potential, and by slightly (<lo%) increasing the ciliary beat frequency in a dose-dependent manner (1 8). several surfactants are in clinical use, and these are either animal lung extracts (e.g. curosurfg9; 20) or artificial products (e.9. exosurf@; 24). apart from the surfactant itself, surfactant preparations may contain other components that may have effects of their own. some preparations from animal sources have contained platelet-activation factor (paf), which has been associated with neonatal disease and increased leukotriene production (25). tyloxapol and cetyl alcohol (detergent and spreading agent, respec tively), which are used in exosurf, can function as antioxidants in vifro, and their in vivo instillation is associated with reduction of lung edema, of oxidized tissue products, and of mortality after hyperoxia (1 4). 200 n-acetylcysteine is frequently used in ventilator-treated infants and applied directly into the airways, usually by nebulization. it is primarily used for its mucolytic effect (26), but it may also act as a free oxygen radical scavenger (27), prevent the production of cytokines by stimulated fibroblasts (28), and prolong the human fibroblast life span (29). in the airway, any instilled drug primarily affects the epithelial lining cells, but epithelial cells and underlying fibroblasts closely interact. for example it is proposed that the prenatal steroid-induced production of surfactant in preterm infants may be due to a direct effect of steroids on fibroblasts, with subsequent release of a stimulatory factor that influences the lung epithelial cells (30). in addition, fetal fibroblasts differ in some respects from adult cells; for instance the inhibition of normal hyaluronan synthesis seen after a cell culture has grown to confluence is less pronounced in fetal than in adult cells (31). fetal cells, therefore, may yield in vifro results that more closely reflect the events in the perinatal period in vivo. the aim of the present study was to determine whether addition of surfactant or n acetylcysteine to cultures of fetal lung cells would affect their hyaluronan production. two brief reports have been presented, one preliminary communication based on the first two experiments (32), and a report on the results of surfactant exposure (33). methods three drugs were tested: a) exosurf@, a synthetic surfactant composed of dipalmitoyl phosphatidyl-choline (dppc, colfosceril palmitate) in cetyl alcohol (i-hexadecanol) and tyloxapol [4-(1 ,i ,3,3-tetramethylbutyi) phenol polymerized with formaldehyde and oxi rane], from glaxo wellcome, goteborg, sweden, b) curosurf @(a porcine lung extract containing lipids and surfactant-associated proteins b and c 80 mg/ml in dppc), from serono, geneva, switzerland, and c) acetylcystein nm pharmao (n-acetylcysteine 200 mg/ml in 0.5 mg/ml edta, and ascorbic acid and sodium hydroxide q.s. ad ph 7) from nm pharma, stockholm, sweden. all preparations are in clinical use. 16-980148, two types of cells, obtained from the european collection of cell cultures, salisbury, uk, were used. human fetal fibroblasts (wi -38, from approximately the 12th gestational week, passage (p) 14-1 7) were cultured in dulbecco’s modified eagle medium (dmem), and feline fetal lung epithelial cells (akd, p 24) were cultured in ham’s f12 medium with 1 % non-essential amino acids. to both culture media 4 mm l-gluthamine, penicillin 120 iu/ml, and streptomycin 100 pg/ml were added. cells were grown in incubators in 5% co, at 37oc in a humidified atmosphere. fibroblasts (30-40 000) and epithelial cells (200 000) were first grown in 12-well dishes in 0.5 ml culture medium containing 10% fetal bovine serum (fbs) for 24 hours. following one wash with medium containing o.lor 0.25% fbs (starvation medium), the cells were cultured in 0.5 ml “starvation medium” for 48 hours, in order to obtain basal conditions. then 0.5 ml of fresh starvation medium, containing one of the three tested drugs at indicated concentrations, was added to duplicate or quadruplicate wells. the tested concentrations were: exosurf 5 pg 40 mg/ml, curosurf 5 pg 20 mg/ml, and n-acetylcysteine 5 pg 120 mg/ml. plain starvation medium served as control and me dium containing 10% fbs as reference. the cultures were then incubated for another 24 or 48 hours. the hyaluronan concentrations in the culture media were determined with a radiometric assay kit (ha 50, pharmacia, uppsala, sweden) (34). mean hyaluro nan values 5 sem were calculated for each drug concentration. the values were then divided by the number of cells and the drug exposure time. since the absolute hyaluro nan values differed substantially between the different experiments, the results were finally expressed in percent of the control value (starvation medium only). the coeffi cients of variation (the standard deviation divided by the mean) (35) within groups were calculated. results cells were tested in six instances between december 1993 and april 1995, with some variation in drug concentration, duration of exposure, and % fbs added to the starva tion media (see method). in one experiment fetal feline epithelial cells were used, and in the other five experiments only fetal human fibroblasts. 202 the following letters are used to denote the different experiments: a. fibroblasts, p14, drug exposure 48 hours, 30 000 cells/well. b. fibroblasts, p16, drug exposure 24 hours, 25 000 cells/well. c. fibroblasts, p16-18, drug exposure 24 hours, 30 000 cells/well. d. fibroblasts, p i 7, drug exposure 24 hours, 30 000 cells/well. e. epithelial cells, p24, drug exposure 48 hours, 200 000 cells/well, also indicated by*.) f. fibroblasts, p17, drug exposure 48 hours, 30 000 cellslwell. for each experiment, the mean hyaluronan concentration/cell/24 hour drug exposure was calculated and expressed as percent of the control (starvation medium alone). as an example, the result of the first experiment with exposure to exosurf is shown in table 1. the rest of the results are summarized in figures 1-3. table 1. hyaluronan (ha) concentration in cell culture medium. results from experiment a with human fetal lung fibroblasts, passage 14, exposed to exosurf or fetal bovine serum (fbs) only for 48 hours, 30 000 cells/well. exosurf fbs n halweli halcel ll24h halcel v24h mglml mean f sem mean mean nglml pg % of control 03.38 0.1% 4 433.9 k 17.4 7.2 67.1 06.75 0.1% 4 382.6 f 36.2 6.4 59.1 13.50 0.1% 4 320.3 k 22.5 5.3 49.5 27.00 0.1% 4 295.8 f 14.2 4.9 45.7 0 0.1% 4 647.0 f 21.8 10.8 100.0 0 10% 4 1444.9 f 45.3 24.1 223.3 in general, the results varied considerably between different experiments, with a mean coefficient of variation of 0.17 (exosurf 0.18, curosurf 0.14, n-acetylcysteine 0.19). 203 200 180 /x 20 0 i ~ tpppp 0 10 20 30 40 exosurf mglml figure 1 a. hyaluronan (ha) in culture medium in relation to exosurf exposure. sum mary of 4 experiments (a-d) with human fetal fibroblasts. exosurf pglml figure 1 b. hyaluronan (ha) in culture medium in relation to exosurf exposure. sum mary of 2 experiments, e* with feline fetal epithelial cells, and f with human fetal fibro blasts. after exposure to exosurf, no increase in the hyaluronan concentration in the culture medium, compared to the control (starvation medium alone), was found in three experi ments with fibroblasts (a,c,f) and one with epithelial cells (e). in two experiments with fibroblasts (b,d) a progressive increase to 188% and 141%, respectively, was noted after exposure to exosurf 2-40 mg/ml (fig. i). 204 180 160 $ 140 s 120 5 100 80 2 60 q) 40 c l $ 20 +a -0-b -a-c +d 1 1 _ _ 0 1 ~~~ i ~ i 0 5 10 15 20 curosurf rnglml figure 2 a. hyaluronan (ha) in culture medium in relation to curosurf exposure. sum mary of 4 experiments (a-d) with human fetal fibroblasts. curosurf pg/ml figure 2 b. hyaluronan (ha) in culture medium in relation to curosurf exposure. sum mary of 2 experiments, e* with feline fetal epithelial cells, and f with human fetal fibro blasts. following curosurf exposure, three experiments with fibroblasts (a,c,f) and one with epithelial cells (e) showed no increase in the hyaluronan concentration compared to the control (starvation medium alone). in two experiments with fibroblasts (b,d) maximal increases to 172 and 122%, respectively, were observed with curosurf at 1 and 5 mg/ml. higher concentrations yielded hyaluronan values below the control level (fig. 2). 205 600 2 500 0 400 5 300 c c 0 0 u r d 0 2 200 3 100 0 7 i1 i ~- 0 20 40 60 80 100 120 n -acetylcystei ne mglml +a -0-b +c +d i+f figure 3 a. hyaluronan (ha) in culture medium in relation to n-acetylcysteine exposure. summary of 5 experiments (a-d, f) with human fetal fibroblasts. = 200 e 150 c1 s c 0 e 100 z d n : 50 0 50 100 150 200 n-acetylcysteine pg/ml figure 3 b. hyaluronan (ha) in culture medium in relation to n-acetylcysteine exposure. summary of 2 experiments, e* with feline fetal epithelial cells, and f with human fetal fibroblasts. after exposure to n-acetylcysteine, one experiment with fibroblasts (a) showed no in crease in the hyaluronan concentration compared to the control (starvation medium alone), while in four experiments with fibroblasts (b,c,d,f) a mainly progressive in crease to 185-589 yo was noted with n-acetylcysteine 5-100 mg/ml. in the experiment with epithelial cells (e*), there was an increase to 171-185% after exposure to n-acetyl cysteine 50-100 pg/ml, but only to 118% after 200 pg/ml (fig. 3). 206 discussion our findings do not indicate that hyaluronan synthesis by fetal lung epithelial cells or fetal lung fibroblasts is stimulated by either artificial or natural surfactant in the concen tration ranges 5 pg/ml 40 mg/ml, and 5 pg/ml 20 mg/ml, respectively. this is in accordance with the finding by juul e t a / (2) that in vivo surfactant treatment of irds re duces the hyaluronan concentration in the lung, and with the report by thomassen et al (23) that surfactants inhibit in vitro synthesis of inflammatory mediators in normal human lung fibroblasts. oxidants are involved in the signal transduction pathways for cytokine secretion, and the inhibitory effect of surfactant supports a proposed antioxidant effect (14). progressive stimulation of hyaluronan synthesis by fetal human lung fibroblasts was induced by n-acetylcysteine in the doserange 5-100 mg/ml, and fetal feline lung epithe lial cells responded with increased hyaluronan synthesis after addition of n-acetyl cysteine in the doserange 50-1 00 pg/ml. this could have some clinical implications, since n-acetylcysteine is commonly used in concentrations of 20 200 mg/ml and ad ministered directly as a fluid into the airway or by nebulizer. thus, the treatment may result in in vivo concentrations comparable to those used in our in vitro experiments. in a preliminary communication (32) we reported a moderate increase in the hyaluro nan concentration after in vifro stimulation of fibroblasts with exosurf or curosurf . the present results, based on several additional experiments, show a more varied picture, with no stimulatory effect of surfactant preparations in most of the experiments. the results of addition of curosurf in concentrations above 20 mg/ml have now been omit ted, as the volume of the drug did not allow for sufficient culture medium. our prelimi nary finding of increased hyaluronan concentrations after exposure to n-acetylcysteine has been confirmed in the present study. we conclude that surfactants used for treatment of irds have only minor effects on the hyaluronan production by fetal pulmonary epithelial cells and fibroblasts in vitro, and that exposure to n-acetylcysteine results in increased synthesis of hyaluronan by fetal pulmonary fibroblasts, but only in a moderate increase in fetal epithelial cells. acknowledgements this work was supported by the swedish medical research council (grants 19x 4998, k97-03x, and 03x-4) and the gillbergska foundation, uppsala, sweden. 207 references 1. allen sj, sedin g, jonzon a, wells af, laurent tc. lung hyaluronan during development: a quantitative and morphological study. am j physiol 260 (heart circ physiol 29): h 1449-54, 1991. 2. juul se, kinsella mg, jackson jg, troug we, standaert ta, hodson wa. changes in hyaluronan deposition during early respiratory distress syndrome in premature monkeys. pediatr res 35: 238-43, 1994. 3. nettelbladt 0, tengblad a, hallgren r. lung accumulation of hyaluronan parallels pulmonary edema in experimental alveolitis. am j physiol 257(lung cell mol physiol 1): 4. nettelbladt 0, bergh j, schenholm m, tengblad a, hallgren r. accumulation of hyaluronic acid in the alveolar interstitial tissue in bleomycin-induced alveolitis. am rev respir dis 139: 759-62, 1989. 5. johnsson h, sedin g, jonzon a, laurent tc. hyaluronan and water content in lungs from preterm rabbit pups kept in air or oxygen (abstract). biol neon 62: 180, 1992. 6. johnsson h, sedin g, jonzon a, laurent tc. hyaluronan and water content in the lungs of term rabbit pups kept in air or oxygen (abstract). ped res 35: 280, 1994. 7. elias ja, krol rc, freundlich b, sampson pm. regulation of human lung fibroblast glycosaminoglycan production by recombinant interferons, tumor necrosis factor, and lymphotoxin. j clin invest 81: 325-33, 1988. 8. sahu sc. hyaluronic acid. an indicator of pathological conditions of human lungs? inflammation 4: 107-1 2, 1980. 9. robertson b. new targets for surfactant replacement therapy: experimental and clini cal aspects. arch dis child 75: f1 -f3, 1996. 10. modig j, hallgren r. increased hyaluronan production in lung a possible important factor in interstitial and alveolar edema during general anesthesia and in adult respiratory distress syndrome. resuscitation 17: 223-31, 1989. 11. hallgren r, samuelsson t, laurent tc, modig j. accumulation of hyaluronan (hyaluronic acid) in the lung in adult respiratory distress syndrome. am rev respir dis 12. gregory tj, longmore wj, moxley ma, whitsett ja, reed cr, fowler aa, hudson ld, maunder rj, crim c, hyers tm. surfactant chemical composition and biophysical activity in acute respiratory distress syndrome. j clin invest 88: 1976-81, 1991. 13. jackson jg, mackenzie ap, chi ey, standaert ta, troug we, hodson wa. mechanisms for reduced total lung capacity at birth and during hyaline membrane disease in premature monkeys. am rev respir dis 142: 413-19, 1990. 14. ghio aj, fracia pj, young sl, piantadosi ca. synthetic surfactant scavenges oxi dants and protects against hyperoxic lung injury. j appl physiol 77: 1217-23, 1994. 15. arnon s, griggs j, silverman m. pulmonary inflammatory cells in ventilated preterm infants: effect of surfactant treatment. arch dis child 69: 44-48, 1993. 16. gerdes j, whisett j, long w. elastase activity and surfactant protein concentration in tracheal aspirates from neonates receiving synthetic surfactant. j pediatr 120: s34 s39,1992. 17. geertsma mf, teeuw wl, nibbering ph, van furth r. pulmonary surfactant inhibits activation of human monocytes by recombinant interferon-y. immunology 82: 450-56, 1994. 18. les brown d, pattishall en. other uses of surfactant. clin perinat 20: 761-89, 1993. l379-84, 1989. 139: 682-87, 1989. 208 19. herting e, jarstrand c, rasool 0, curstedt t, hakansson s, robertson b. effect of surfactant on nitroblue tetrazolium reduction of polymorphonuclear leukocytes stimu lated with type la group b streptococci. acta paediatr 84: 922-6, 1995. 20. speer cp, gotze b, curstedt t, robertson b. phagocytic functions and tumor necrosis factor secretion of human monocytes exposed to natural porcine surfactant (curosurf). ped res 1991; 30: 69-74, 1991. 21. benoit j, cormier m, wepierre j. comparative effects of four surfactants on growth, contraction and adhesion of cultured human fibroblasts. cell biol toxicol 4: 11 1-22, 1988. 22. rice wr, sarin vk, fox jl, baatz j, wert s, whitsett ja. surfactant peptides stimulate uptake of phosphatidylcholine by isolated cells. biochem biophys acta 1006: 23. thomassen mj, antal jm, barna bp, divis lt, meeker dp, wiedemann hp. surfactant downregulates synthesis of dna and inflammatory mediators in normal lung fibroblasts. am j physiol 270 (lung cell mol physiol 14): l159-l163, 1996. 24. phibbs rh, ballard ra, clements ja et al. initial clinical trial of exosurf, a protein free synthetic surfactant, for the prophylaxis and early treatment of hyaline membrane disease. pediatrics 88: 1-9, 1991. 25. moya fr, hoffman dr, zhao b, johnston jm. platelet-activating factor in surfactant preparations. lancet 341 : 858-60, 1993. 26. ziment i . acetylcysteine: a drug with an interesting past and a fascinating future. respiration 50 (suppl 1): 26-30, 1986. 27. bernard gr. n-acetylcysteine in experimental and clinical acute lung injury. am j med 91 (suppl 3c): 54s-595, 1991. 28. raes m, renard p, bosmans e, delaive e, remacle j. effects of antioxidants on il 6 secretion induced by il-i in human cultured lung fibroblasts. involvement of nfkb. in: oxidative stress, cell activation and viral infection (eds. c pasquier et al.), pp. 77-90. birkhauser verlag, basel, switzerland 1994. 29. honda s, matsuo m. relationship between the cellular glutathione level and in vitro life span of human diploid fibroblasts. exp gerontol 23: 81-86, 1988. 30. smith bt, post m. fibroblast-pneumonocyte factor. am j physiol 257 (lung cell mol physiol 1): l174-l178, 1989. 31. chen yj, grant me, schor am, schor sl. differeces between adult and foetal fibroblasts in the regulation of hyaluronan synthesis: correlation with migratory activity. j cell sci 94: 577-84, 1989. 32. johnsson h, heldin p, sedin g, laurent tc. surfactant and n-acetylcysteine stimulate the hyaluronan synthesis by human fetal lung fibroblasts (abstract). ped res 36: 19a, 1994. 33. johnsson h, heldin p, sedin g, laurent tc. hyaluronan production in vitro by fetal lung fibroblasts and epithelial cells exposed to surfactants (abstract). biol neonate 71 (suppl 1): 65, 1997. 34. brandt r, hedlof e, asman i, bucht a, tengblad a. a convenient radiometric assay for hyaluronan. acta otolaryngol suppl 442; 31-35, 1987. 35. bland m. an introduction to medical statistics, 2nd ed, p 267. oxford university press, 237-45, 1989. oxford, uk, 1995. correspondence to: hans johnsson, md department of pediatrics, uppsala university children's hospital s-751 85 uppsala, sweden 209 upsala j med sci 99: 307-314, 1994 7. reference intervals for plasma proteins 7.1 principles for estimation of reference intervals per hyltoft petersen and ole blaabjerg department of clinical chemistry, odense university hospital, dk-5000 odense c, denmark 7.1.1 models f o r estimation of reference intervals models for estimation of reference intervals are either parametric or non-parametric and both have advantages and disadvantages. the parametric models are usually based on the gaussian distribution directly or after transformations of data. the advantage of the parametric approach is the power of all estimates and thereby, a relative narrow confidence interval about each reference limit. the weakness is whether the underlying distribution (directly or transformed) is fitted by the parameters (mean and standard deviation) t o a reasonable extent. if not, then the estimates are biased and the reference limits correspondingly wrong. the quality of the parametric fit can be validated by a number of tests for skewness and kurtosis or by anderson-darling-test o r kolmogorov-smirnov-test as described in ifcc’s recommendations (10). the advantage of the non-parametric approach is its independence of the shape of the underlying biological distribution, whereas the disadvantage is a considerable uncertainty of the estimated reference limits. both approaches are vulnerable to so called outliers when sample size is small, and the effect is reduced by increasing the number of reference values for both approaches. ideally, the ifcc-recommendations (10) should have been applied using the computer programme ref-val for the estimations of reference intervals. the model, however, is very flexible of nature and the programme which optimize the fit of any distribution, lacks the graphical presentation (except from a histogramme). the model is optimal for estimation of reference intervals for one single crude sample (or mixed sample), which makes it irrelevant for comparing a great number of distributions. linnet has given some critical remarks to the ifcc recommendations (9). 21-955059 307 due to the nature of our data and the purpose of the study with a considerable number of reference values, combined with the many subsamples to compare, we decided to use the visual presentation as a tool for decisions about combining or separating sub-groups in the material. 3 2 . 3.0 2,6 2.4 2.2 for this purpose harris and boyd (5) have made an interesting and useful concept, directly related to the separation of reference intervals according to subgroups. the idea in their paper is to focus on the fraction outside each reference limit. when reference intervals for two subgroups are considered, both the combined reference interval and each of the two are calculated. then the fractions outside the common reference limits is calculated for each of the subgroups. ideally they should all be 2.5 %, but with increasing deviations between means and standard deviations of the subgroups the fractions decreases or increases for each of the two times two fractions. the limits for these changes have t o be decided (see below). harris et al. have applied the concept with success t o s-creatine kinase for sub-groups according to race and gender (6). 0.52 0.48 0,44 0.40 0.36 0,32 s-transferrin concentration g/l log g/l women (+e) i 4 men women f mean and 0.90 ci i) ' ~8-20'2~-3o'31-4o14l-50 '51-60'6 1-70'71-801 age groups fig. 7.1.1. mean concentrations with 90 % confidence intervals for each subgroup shown on a log-scale in relation to age for s-transferrin. from blaabjerg et al. (1) with permission. 308 further, the best visual presentation of distributions is the probit-plot (2 5, 7, 8) where the cumulated frequencies are plotted on a standard deviate scale as function of the concentration values. when gaussian, the cumulated distributions show up as straight lines. for reference intervals the most relevant transformation of data is to take the logarithm, as many laboratory reference values are distributed close to log gaussian. the probit-plot has many advantages as many distributions can be shown simultaneously in the same plot without confusion and the judgement of the quality of the parametric fit to the data is better by the eye than by any statistical test. further, when deviating from the parametric model, the plot can be used directly for non parametric estimation. detailed descriptions of probit-transformations and instructions for constructions and advises for interpretations are given by gowans et al. ( 3 ) and hyltoft & hprrder (8). 7.1.2 levels of sub-specification. three levels of specification can be applied: * a very detailed where all subgroups are described, i.e. with specified reference intervals for each subgroup. this approach may be relevant for extremely large subgroups, but the uncertainty of each group is considerable as sample sizes decreases. we have decided t o extract informations about biological tendencies from the graphical presentation of each subgroup as mean with a 90 % confidence interval in relation to age. as most of the distributions are log-gaussian these values are presented on a log-scale. * a rather detailed, but with combination of subgroups according to t h e harris-boyd-concept ( 5 ) , using the limits for fractions outside the combined intervals 1.3 % to 4.4 % according to gowans et al. (4). for the smaller sample sizes (below 120 individuals), however, the actual confidence interval calculated according to bliss (2) was used. moreover, a number of choices and decisions had t o be taken, where a single subgroup differed, without biological or other explainable reasons (see below). this level can be considered the basic clinical chemical level. * a third and more practical level may be a further simplification, where more semi-subgroups are combined, or groups are described by a percentage deviation from the main group. this has been adapted by some of the nordic countries as shown in chapter 8, but these are national applications determined from practical o r other reasons. 309 7.1.3 example the chosen example is s-transferrin, where the mean values with 90 % confidence intervals are shown in relation to age in fig. 7.1.1. women below 50 years of age using estrogen show higher values than the rest and should (as the first estimate) be treated as a separate group. for the rest there is a tendency to decreasing values for increasing age. from approx. 2.6 to approx. 2.4 g/l. for practical reasons we had decided to separate at 50 years and as the numbers in the two oldest age groups are small, then the uncertainty is too large for a clear decision about separating in two different reference intervals above and below 50 years. cumulated percentage frequency probit i ' i . i ' i ' i * i ' l ~ i ~ " ~ l " ' ' 1 1,6 1,8 2,o 2,2 2,4 2.6 2,8 3.0 3.5 4.0 ( g / l ) i i i i i 02 0,3 0,4 0 5 0 6 (log g/l) fig. 7.1.2. probit-plot with log-abscissa showing for s-transferrin the distributions for women using estrogen and the remaining group (all others). from blaabjerg et al. (1) with permission. the two hstributions, one for women using estrogens and one for all others, are illustrated in fig 7.1.2, from which it is clear that both hstributions are close t o log gaussian (straight lines) and clearly separated. from fig. 7.1.1, however, there was also a tendency to decreasing values with age for women using estrogens with the age-group 41 to 50 as the lowest. in consequence this group was compared separately to the total group of women using estrogens. in fig. 310 7.1.3 the broken line indicates the total group and the dotted line the age-group 41 to 50 with a total of 24 indviduals. the latter distribution is not log-gaussian (thin straight line) and converges to the total distribution in both ends. the 90 % uncertainty limits for a sample size 24 is shown around the total distribution (double curves). it is seen that the central values are below the confidence interval, but both ends are inside. as we find no biological evidence for separation, we have chosen to use the total reference interval. 99 95 90 80 70 60 50 40 30 20 10 5 1 cumulated percentage frequency probit women (+e) , total women i 0.90 ci for a sample size of 24 fig. 7.1,3. illustration of a comparison between a subgroup and the total group. probit-plot of s-transferrin showing the total group of women using estrogens (dotted line) with a 90 % confidence interval corresponding to the subgroup (here 24) for each percentile. the sub group (women between 41 and 50 years of age) is shown as the broken line and indicated parametrically by the thin straight line). from blaabjerg et al. (1) with permission. however, when the distributions are separated according to high and low doses of estrogens, then the two groups separate clearly as seen from fig. 7.1.4. 31 1 cumulated percentage frequency problt 99 95 90 80 70 60 50 40 30 20 10 5 1, women (all e) i ' i . 1 i . i . i " ' i ' . . ' i . ' . ' i 1.6 1,8 2,o 2.2 2.4 2,6 2,8 3.0 3.5 4,o (q/l) i i i 1 i 0,2 03 0,4 05 0,6 (log g/l) fig. 7.1.4. distributions of s-transferrin for women using high and low doses of estrogens. cumulated percentage frequency paoblt 99 sedimentation rate 95 7 0 60 50 40 30 others i i . i . 1 . i . i * ~ ~ l ' ' ' ' i ~ . ' ' l 1,6 1,8 2,o 2,2 2,4 2,6 2.8 3.0 3.5 4,o ( q / l ) i i i i i fig, 7.1.5. 0.2 0.3 0,4 0,s 0.6 (logg/l) illustration of the discharged values due to elevated b-sedimen tation rate, s-crp or presence of m-component, and compared to the accepted reference distributions. 312 further, the individuals discharged from the main groups due to elevated values of b-sedimentation rate, s-crp or due to the presence of m-components can easily be displayed on t h e probit-plot, and thereby be compared to the accepted reference individuals as illustrated in fig. 7.1.5, even though they are of very limited sample sizes. references 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. blaabjerg 0, blom m, gry h, carlstrom a, hyltoft petersen p, ickn a, irjala k, lund e, matinlauri i, mattila k, n ~ r g a a r d jr, uldall a. f d e s referenceintervaller i norden for 9 plasmaproteiner. klinisk kemi i norden 1993;4 vol. 533-7. bliss ci. statistics in biology. new york, mcgraw-hill. 1967. gowans ems, fraser cg, hyltoft petersen p. a guide to thee use of probit transformation of gaussian distributions. biochem clin 1989:13327-36. gowans ems, hyltoft petersen p, blaabjerg 0, harder m, analytical goals for the acceptance of common reference intervals for laboratories throughout a geographical area. scand j clin lab invest 198848:757-64. harris ek, boyd jc. on dividing reference data into subgroups to produce separate reference ranges. clin chem 1990;36:265-70. harris ek, wong et, shaw st, statistical criteria for separate reference intervals: race and gender groups in creatine kinase. clin chem 1991;37:1580-2. hyltoft petersen p, gowans ems, blaabjerg 0, harder m, analytical goals for estimation of non gaussian reference intervals. scand j clin lab invest 1889;49:727-37. hyltoft petersen p, harder m. influence of analytical quality on test results. in magid e (ed.). some concepts and principles of clinical test evaluation. nordkem, nordic clinicel chemistry project, helsinki finland 199265-87. linnet k. two-stage transformation systems for normalization of reference distributions evaluated. clin chem 1987;33:381-6. solberg he. approved recommendation (1987) on the theory of reference values. part 5. statistical treatment of collected reference values: determination of reference limits. j clin chem clin biochem 1987;25:645-56. 313 upsala j med sci 95: 265-273, 1990 some methodological aspects on quality assurance in laboratory medicine torgny groth unit f o r biomedical systems analysis, uppsala university, uppsala, sweden knowledge-based systems and quality assurance during the last decade database and knowledge-based system technology have been used increasingly for documentation and processing of medical knowledge related to decision making in laboratory medicine. such systems will hopefully lead to more cost-effective diag nostic and therapeutic procedures. it is also obvious that such formal and non-formal descriptions of various aspects of medical decision making will increase the possibilities and facilitate the intricate work to assess quality requirements on laboratory data in a clinical context. as pointed out before (3 , 4 , 7) the discussion on optimal quality of laboratory tests should be broadened to include not only analytical and pre-analytical quality components, but also the equally or more important aspects of e.g. (i) selection and combination of tests; (ii) frequency of specimen collection; (iii) the influence of incorrect conceptual models for in terpretation of test results; and (iv) mode of communication and presentation of results. some examples have previously been presented in detail in refs. (3,4,7) and will not be repeated here. other more recent applica tions developed within the nordic r&d project on knowledge-based systems in medicine may be found in refs. (11,13,14,17,20). it could be expected that these new techniques for data -, and knowledge-processing and communication will help the clinical laboratory profession in setting standards, guiding training, disseminating information, and establishing collaboration between the laboratory and the clinic (9). 265 practical use of analytical quality specifications considering the diversity of approaches advocated for assessing analytical quality specifications, and the circumstance that any quality specification of laboratory results will only be temporary (1) , the "right thing to do" would be to set up a first provisional ifnordic list" as a starting point to a coordinated learning and teaching process on how to apply such quality specifications in practice. analytical quality specifications expressed as "allowable analytical errors" (aae) at specified concentration levels or ranges should then be applied for: 1. method evaluation studies in connection with establishment of an analytical method for routine use (15). the assessment of "stable analytical performancell should lead to an estimation of imprecision and inaccuracy (bias) over the whole measurement ran ge, and be evaluated in comparision with limits set by the allow able analytical error. it is worth emphasizing that, according to the basic principles of metrology, the analytical bias (remaining after all measures have been taken to reduce it) should be corrected for in the routine operation of the method. this could be performed with use of a correction function, estimated by regression analysis of "conventional true values" versus corresponding measured values. 2. internal analytical quality assurance of the routine method to achieve analytical stability within the limits of allowable errors at one or more selected concentration levels. statistical procedures could be designed to assure the specified quality (10,16), and be optimized in terms of "test yield" (as a measure of "internal laboratory costs") and "defect rate" (as a rough measure of "clinical costsit). the term internal analytical quality assurance (iaqa) is preferred to the conventional term iqc to denote this new approach. interactive "iaqa design programs" are available on pcs based on monte carlo simulation of specified measurement and stat istical control procedures under realistic analytical pertur bations, and given analytical quality goals (5,12). 266 figure 1 shows typical output results from such a program. the optimal number of controls (n) for a given situation could be determined from the plot of test yield and defect rate as func tions of n, and the condition that the false accept rate (defect rate) should be below a certain limit, e.g. 0.1%. in the example shown this means that 3 controls should be used, giving a test yield of 89%. predicted outcome of analytical runs is summarized in a decision matrix, from which predictive values for reject and accept signals are calculated for the quality assurance system. * + ficrure 1. typical output results from the iaqa design program as applied to an analytical method with excellent process stability (1% frequency of critically sized systematic shifts). (a) plot of test-yield and defect rate versus number of controls (n); 267 ( b ) chol e2 okt.-~o aae=180.0000 f= 1% systematic; 0 % random control rules: shewhart 1 (3.00*s) rule with control l i m i t s calculated from s ( t ) for with n= 3 shewhart 1(3.00*s) rule predicted outcome o f analytical runs: acceptable acceptable ? -__-___-_______^_--_---_--__-------- accepted 96.3% .o% rejected 1 2.7% .o% not acceptable .1% .9% p r e d i c t i v e value o f reject= . 2 5 p r e d i c t i v e value of accept=1.00 s e n s i t i v i t y = .90 s p e z i f i c i t y = . 9 7 test yield: .90 defect rate: -1% ficrure 1. typical output results from the iaqa design program as applied to an analytical method with excellent process stability (1% frequency of critically sized systematic shifts). decision matrix showing the predicted outcome of analytical runs for a shewhart 1:3s rule with n=3. (b) it has been suggested that the critically sized systematic error ( se,) and the critically sized increase in inherent random error ( re,) (to be detected by the control systems to assure the specified analytical quality) should be calculated from formulas where known analytical bias is subtracted from the allowable analytical error, i.e. se,=(aae-bias)/s-1.65 re,=(aae-bias)/l.96 s it should be noted that this may lead to unreasonable and unnecessary high demands on process capability (very small critically sized errors and even negative values for bias aae 1.65s). 268 i +aae 1aae c" c fisure 2. calculation of process capability, i.e. critically sized systematic error, a s e c , in one case where known bias has not been corrected for (a), and another case where bias is cor rected for in the routine operation of the analytical method (b) , c*=conventional true value; c=local stable level; s=imprecision of the method. 269 the way out of this dilemma (fig 2.) is to follow the metrolo gical principle of correcting measured values for known bias, and design the statistical procedure to detect critically sized errors calculated from a se,=aae/s-l. 65 a rec=aae/1.96's such a procedure will then assure analytical stability within the aae limits. in the lack of generally accepted ''conventionally true values",c*, analytical stability within allowable deviations from illocal stable levelsil,c, should be the goal. another related problem of internal analytical quality as surance to be considered (especially in high production laborato ries) is the of different analyzers which are used to measure the same components. for more details see ref (10). 3 . external analytical quality assessment & assurance. such external programs, administered by a coordinating organi zation, should be complementary to the iaqa procedures (operating at one or two selected concentration levels) by providing an ass essment of analytical performance (estimation of analytical bias) over a wider concentration range. as described and discussed in more detail elsewhere (6,10,16) this could be performed by (i) distribution of a sufficient number of ifaccuracy assessment stan dards" ( 'iexternal calibrators") to each participating laboratory, and by (ii) analysis of measured values versus agreed upon "con ventional true values" with use of e.g. linear regression. the slope (k) and intercept (1) of the regression line could be used to characterize each laboratory in a strict quality assessment/proficiency testing procedure on the basis of allowable analytical error specifications mapped into allowable regions in the k-1 plane. an "eqa&a simulation program" has been used to study various strategies for decreasing interlaboratory variation (6), and for optimizing the statistical design. a new interactive version of this program has been developed (groth and falk, to be publis hed). the input data describe the analytical performance of a set of participating laboratories, and the proposed design of the quality assessment & assurance program. the performance of 270 various statistical procedures for assessment of accuracy and testing the agreement with the quality goals may be investigated as well as the possible effect of various correction procedures. the regression function could be used in the individual labo ratory for routine correction of significant bias, and/or when called for in connection with communication of laboratory results in health care and scientific work. there are certain limitations of the applicability of this approach due to different chemical specificities of the methods used today. some characterization and standardization of methodologies based on their chemical specificity will certainly be necessary in order to take full advantage of the type of external quality assurance programs discussed here. transmission and transferability of laboratory test results communication of laboratory results is still a major problem both in health care and medical research. in the effort by the commission of ec to improve european health care in a coordinated way, standardization and application of information and communi cation technologies are two important large scale activities. the euclides project, one of several projects within the cec aim exploratory project on advanced informatics in medicine, has worked out a proposal for a european standard for transmission of clinical laboratory data, i. e. "patient related data, control sera related data, test related data, and laboratory related supervisory data" (2) . in order to take full advantage of the emerging technology for transmission of laboratory results between health care units and clinical laboratories via networks (or portable medical "smart cards") it is necessary to solve the transferability problem by developing the appropriate quality assurance procedures (10). knowledge-based systems also have great potentials in this area by providing "expert diagnostics and advice" to individual laboratories and health care units concerning analytical problems and how to improve. 27 1 references 1. 2 . 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. barnett rn. quality control in the clinical laboratory. an individual perspective. lab med, 1989; june: 385-387. de moor gje. euclides: a european clinical laboratory data exchange standard. in proceedings of the aim-euroforum conference, sevilla, 1990. groth t. methods and approaches to determine lloptimalll quality for clinical chemical data. scand j clin invest 1980:40 (supp1.155): 47-63. groth t. biodynamic models as they relate to test selec tion. in clinics in laboratory medicine, eds. es benson, d connelly and e burke, vol. 214,701-715, marcel dekker inc., new york and basel, 1982. groth t i falk h and westgard jo. a quality control simula tor for design and evaluation of internal quality control procedures. scand j clin lab invest 1984; 44 (supp1.172): 195-201. groth t. strategies for decreasing interlaboratory varia tion. studies by computer simulation. scand j clin lab invest 1984; 44 (suppl. 171): 331-347. groth t. the role of formal biodynamic models in laboratory medicine. scand j clin lab invest, 1984; 44 (suppl. 171): 175-192. groth t. analytical quality, test quality and quality costs. upsala j med sci 1986; 91: 205-208. groth t. database management and knowledge-based systems in clinical laboratory medicine. in kerkhof, van diejen-visser (eds.) laboratory data and patient care. plenum press, new york 1988; 101-108. groth t. internal and external quality assurance. scand j clin lab invest 1989; 49 (suppl 193): 87-94. groth t. quality assurance by knowledge based techniques. in automation in blood transfusion, eds. smit-sibinga, das & hogman, kluwer academic publishers, boston, 1989; 83-90. groth t. and westgard jo. a second generation simulation program for optimizing quality control design. clin chem 1990; 36: 1003. groth t, hakman m, hedlund a, zaar b. kbsim/fluid therapy: 272 14. 15. 16. 17. 18. 19. 20. a system for optimized design of fluid resuscitation in trauma. comp meth progr biom, 1991; 34: in press. van waeg g and groth t. allopurinol kinetics in humans as a means to assess liver function: design of a loading test. am j physiol 1989; 257: r237-r245. westgard jo. precision and accuracy: concepts and assess ment by method evaluation testing. crc critical reviews in clinical laboratory sciences. 1981; 283-330. westgard jo, groth t and de verdier c-h, principles for developing improved quality control procedures. scand j clin lab invest 1984; 44 (suppl. 172): 19-41. wiener f, groth t and nilsson u, a knowledge-based system for automatic interpretation of an analytical profile of complement factors. j clin lab anal 1989; 3: 287-295. wiener f, groth t and mortimer d, hallquist i and rane a. a knowledge-based information system for monitoring drug levels. comp meth prog biom, 1989; 29: 115-128. wiener f, c-h de verdier and groth t. the use of knowledge based information systems for interpreting specialized clinical chemistry analyses-experience from erythrocyte enzymes and metabolites. scand j clin lab invest, 1990; 50: wiener f, hedlund a and groth t. a knowledge-based in formation system for advice in the crisis management of the burn patient. burn care rehab, 1990; 11: 201 247259. correspondence: torgny groth, associate professor, unit for biomedical systems analysis, uppsala university, box 2103, s-750 02 uppsala, sweden 18 -908573 273 tf-iups160034 222..226 original article advancing preconception health in the united states: strategies for change sarah verbiesta,b,c , erin mcclaina,b and suzanne woodwarda,b anational preconception health and health care initiative, university of north carolina, chapel hill, nc, usa; bcenter for maternal and infant health, university of north carolina, chapel hill, nc, usa; cschool of social work, university of north carolina, chapel hill, nc, usa abstract in january 2015, the us preconception health and health care initiative (pchhc) established a new national vision that all women and men of reproductive age will achieve optimal health and wellness, fostering a healthy life course for them and any children they may have. achieving this vision presents both challenges and opportunities. this manuscript describes the reasons why the us needs to prioritize preconception health as well as its efforts historically to advance change. the authors share lessons from past work and current strategies in the us to reach this ambitious goal. article history received 4 april 2016 revised 2 june 2016 accepted 16 june 2016 key words family planning; infant mortality; interconception; life planning; maternal mortality; preconception; reproductive health; preconception care; preconception health introduction in 2015, the united states‘ preconception health and health care initiative established a new national vision that all women and men of reproductive age will achieve optimal health and wellness, fostering a healthy life course for them and any children they may have. key components of the vision include the addition of men as a point of focus and intervention and the importance of improving the health and wellness of young adults of reproductive age, regardless of their immediate desire for children. the vision also elevates the life course concept, which invites discussion about the lifelong and intergenerational impact of preconception wellness. achieving this vision presents both challenges and opportunities. this manuscript describes the reasons why the us needs to prioritize preconception health as well as its efforts historically to advance change. the authors share lessons from past work and current strategies in the us to reach this ambitious goal. call for action the united states has progressed in reducing infant and maternal mortality rates. despite these improvements, infant and maternal deaths remain high and disparities persist, particularly among non-hispanic black and american indian/ alaska native populations (1,2). the us infant mortality and maternal mortality rates are higher than most industrialized nations, despite significant health care system investments and health expenditures (1,3,4). us women of reproductive age (18–44 years) report a host of challenges to achieving optimal preconception health. for example, only 29.7% of women consume daily folic acid supplementation, over 25% use tobacco, 24.9% are overweight immediately prior to pregnancy, and 22.1% are obese prior to pregnancy. further, 3.8% of women report physical abuse, and 42.9% report that their most recent pregnancy was unintended (5). the introduction of the patient protection and affordable care act (aca) has prompted a sharp decline in the number of us women who are uninsured, yet 12.8 million women remain without health insurance (6). almost one-third (29%) of uninsured women are not eligible for assistance under the aca because they fall into the coverage gap created by their state’s decision not to expand medicaid (13%) or they are undocumented immigrants (16%). many of these 3.6 million women live in the us south—where the majority of the 19 states that opted not to expand medicaid eligibility to people with incomes less than 138% of the federal poverty level regardless of their family or disability status or age are located—meaning that their access to affordable insurance coverage is severely curtailed (6). defined as a set of interventions that aim to identify and modify biomedical, behavioral, and social risks to a woman’s health or pregnancy outcome through prevention and management (7), preconception care is a critical component of a comprehensive women’s health package to improve maternal and infant health outcomes (8). despite the clear benefits of receiving preconception care and managing preconception health, many women in the us are not as healthy as they could be, and the us has not yet achieved its healthy people contact sarah verbiest sarahv@med.unc.edu university of north carolina at chapel hill, campus box 7181, chapel hill, north carolina 27599-7181, usa � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 4, 222–226 http://dx.doi.org/10.1080/03009734.2016.1204395 http://creativecommons.org/licenses/by/4.0/ 2020 objectives for increasing the proportion of women who receive preconception care and practice positive preconception behaviors (9,10). national initiative founded in 2004, the national preconception health and health care initiative (pchhc) is a public–private partnership that aims to foster connection and multiply local impact through national collaborative efforts. the initiative comprises five implementation workgroups focused on consumers, clinicians, public health, policy and finance, and surveillance and data. these efforts are synchronized to address cross-cutting issues through the pchhc leadership team. representatives include federal agencies, as well as national maternal and child health organizations, professional organizations, statelevel maternal and child health leaders, researchers, policymakers, and private foundations. pchhc has directly and indirectly influenced other national projects, strategies, and policies, including the us department of health and human services’ national prevention strategy and the office of population affairs’ quality family planning guidelines (11). in 2006 the centers for disease control and prevention (cdc) and the select panel on preconception health issued national recommendations to advance the field. the recommendations focused on 10 areas, including: individual responsibility across the lifespan, consumer awareness, preventive visits, interventions for identified risks, interconception care, pre-pregnancy checkup, health coverage for women with low incomes, public health programs and strategies, and research and monitoring improvements (7). pchhc has focused on responding to these recommendations for 10 years. to this end, the initiative has convened three national summit meetings, two preconception health select panels, and supported the five workgroups. the initiative has published reports and articles, including four journal supplements (maternal and child health journal in 2006, women’s health issues in 2008, american journal of obstetrics and gynecology in 2008, and the american journal of health promotion in 2013—available at www.beforeandbeyond.org). the initiative has carried out a series of strategic action plans, including the action plan for the national initiative on preconception health and health care: a report of the pchhc steering committee (2012–2014) (12). in 2014, it launched a website for clinicians, including a comprehensive clinical resource guide on preconception health (www.beforeandbeyond.org). in november 2014, the preconception select panel was reconvened to review successes and challenges to the movement and make recommendations for future action. in order to accelerate the implementation of preconception health in the us, the panel highlighted the importance of action in the following areas: (1) addressing the social determinants of health to create improved conditions for all women and men of reproductive age; (2) expanding social marketing and consumer engagement efforts; (3) focusing on the implementation of the aca’s coverage of well-woman visits and clinical preventive service benefits; (4) improving clinical care support, provider and health system changes; (5) increasing the use of health information technology; (6) expanding measurement to increase accountability; and (7) developing resources and partnerships (11). the full report is available online for review. current strategies based on lessons learned, pchhc redoubled its efforts in 2015, with increased focus on measures and metrics, strategies for integrating preconception services into routine clinical care, consumer engagement, and expanded partnerships. this work is built on a platform that calls for a new social movement in the us to address reproductive and social justice issues. a multi-sector approach is essential for fully actualizing the potential of preconception health. some current efforts are described below. measures two major efforts are underway to enhance metrics on preconception health at both the clinic and population health levels. first, the cdc center for chronic disease prevention and health promotion, division of reproductive health, has convened an internal surveillance and data workgroup to develop a set of state-based indicators to monitor improvements in preconception health, evaluate the effectiveness of preconception health programs, and assess the need for new programs. these indicators are the foundation for future national and state report cards, annual reporting, and media and policy-maker attention. the workgroup has reviewed an existing list of 45 indicators (13) as well as new potential indicators for inclusion. they agreed on evaluation criteria and, in partnership with state-level maternal and child health experts, are prioritizing indicators based on these criteria. a final list of indicators will be published at the end of 2016. efforts then will turn to strengthening the capacity of states and territories to develop surveillance summaries with data-to-action efforts. second, at the clinic level, the pchhc clinical workgroup developed a set of preconception wellness metrics in february 2015. with the advent of health care finance reforms in the us, measures were needed to assess preconception health care delivery (14). based on the premise that a woman’s level of well-being at the time of conception reflects her preconception wellness, and grounded in prior research, the workgroup proposed a set of nine preconception wellness measures to be collected at a woman’s first prenatal assessment. these measures include: (1) intended/planned to become pregnant; (2) entered prenatal care in the first trimester; (3) daily folic acid/multivitamin consumption; (4) tobaccofree; (5) not depressed (mentally well/under treatment); (6) healthy body mass index; (7) free of sexually transmitted infections; (8) optimal blood sugar control; and (9) medications (if any) are not teratogenic (14). a complete review of the indicators and methodology has been published (14). integrating preconception into clinical care in tandem with developing clinical measures, the clinical workgroup is focused on identifying promising practices for upsala journal of medical sciences 223 http://www.beforeandbeyond.org http://www.beforeandbeyond.org integrating preconception health into clinic settings where women receive routine care. this is being accomplished by partnering with four clinics and 14 health care systems in collaborative learning networks. through these networks process barriers and facilitators to implementation of preconception health will be identified, and new tools and resources will be developed to support clinicians and systems better. preconception care is not likely to be fully adopted until health system leaders see such care as a driver for achieving the triple aim—improved patient experience, improved population health, and reduced health care costs. ongoing partnership with professional associations, such as the american congress of obstetricians and gynecologists (acog), is essential, particularly in the development of protocols and expectations for quality well-woman care. defined as an annual preventive health assessment, the well-woman visit includes screenings, evaluation, counseling, and immunizations and is now covered by health plans. the recently released american academy of family physicians’ position paper on preconception care (15) offers another example of critical partnerships to promote change so that health systems/clinicians routinely offer woman-centered preconception care services, including connecting women and men with information and resources necessary to act on provider recommendations. two major federal programs, the maternal and child health block grant program (title v) and the family planning program (title x) of the us public health service act, have increased their focus on preventative health, including preconception and interconception care. title v has made the percentage of adolescents and adult women who report an annual visit in the last 12 months key performance measures for states. the national family planning program has expanded its focus beyond providing women with a contraceptive method to providing adolescent and adult women and men with comprehensive reproductive life planning services that include an increased focus on preventing and promoting early management of chronic conditions, such as hypertension, diabetes, and obesity. consumer education recognizing the necessity of pairing clinical care with excellent patient education and resources, the consumer workgroup conducted formative research to understand women’s knowledge, attitudes, and behaviors as these relate to preconception health and reproductive life planning. in 2013, the consumer workgroup launched their flagship program, show your love, a national campaign to improve the health of young adults through optimal preconception health and reproductive life planning. show your love’s target audience is women and men aged 18–44 who are currently planning to become pregnant, as well as those who do not wish to conceive. in june 2016, the consumer workgroup launched a new show your love consumer-facing campaign, beginning with www.showyourlovetoday.com, the first and only national consumer-focused preconception health educational and community-building platform in the us (figure 1). show your love’s website objectives include: (1) enabling young adults to manage their personal and family well-being by making realistic health and lifestyle improvements daily; (2) educating young adults and their loved ones about tools and resources available to support healthy lifestyles; and (3) bridging the gap in access to and communication between young adults and their health care provider by helping them understand critical components about preconception health care, life planning, and health insurance (including preand post-doctor dialogues, information about access to care) and (4) empowering young adults to become health advocates in their communities by getting involved with show your love. to spur action among young adults in low-income, diverse, or higher-risk communities, efforts are underway to partner with organizations across the nation to diversify and expand preconception health messaging to resonate better with adults of various ethnicities, races, languages, cultures, identities, and communities across the us. show your love campaign materials that are relatable to these populations, including videos, public service announcements, posters, digital ads, doctor dialogue toolkits, and healthy habits checklists, will be rolled out by show your love in the fall of 2016. engaging consumers on national, regional, and local levels through a comprehensive public relations/marketing campaign that encompasses digital, social media, event-based, and media relations tactics is a cornerstone strategy. figure 1. show your love today website launch info. 224 s. verbiest et al. http://www.showyourlovetoday.com tied to the launch of the show your love website, the consumer workgroup launched their social media properties on twitter (www.twitter.com/syl_today) and facebook (www.facebook.com/showyourlovetoday), coining the hashtag: #showyourlovetoday. for the first time, pchhc is actively engaging with consumers and social media to share messages and interact on key pulse points and issues that are trending. in addition, pchhc leaders are actively participating in national media interviews and platforms to share research, best practices, and expert insight on actions needed to move the needle on preconception health. health equity it is critical that young women and men not only have knowledge about key preconception messages, but access to resources to act on the messages to take control of their health. there is significant momentum toward addressing health equity in the us—including new calls by the american academy of social work and social welfare and the american public health association, among others, to focus on the social determinants of health and promote increased integration across sectors to foster innovation and address the barriers that keep young people from actualizing protective preconception behaviors and wellness. leaders from the public health workgroup have highlighted the importance of the following strategies to improve preconception health equity: (1) access to comprehensive, quality, culturally appropriate health care services for all; (2) facilitating change through crucial conversations and listening sessions; (3) developing collaborative, comprehensive programs within and beyond public health that support preconception health, reproductive equity, and life planning; and (4) educating and engaging the maternal and child health workforce and consumers on local and national issues that address the social determinants of health (16). moving forward, resources will be developed to support meet-ups, town hall meetings, crucial conversation training, and tools and resources for engaging young adults. at the policy and finance level, pchhc recently launched a preconception policy action network comprising advocacy organizations in women’s and children’s health and reproductive and social justice. this network will focus on increasing knowledge about preconception health and health care and building skills on appropriate and effective framing of preconception health and equity messages. through this work connections will be fostered and new relationships will be built so as to have preconception health-primed colleagues at policy tables across the country. looking forward after decades of steady progress in advancing preconception health, momentum has grown and is pushing toward a tipping point. moving this work forward requires a thoughtful framing of preconception health messages. activating young adults to attend to their health and plan their families is essential and challenging, particularly for those who live in resource-constrained environments. paternalist frames that ignore the context of men’s and women’s lives will not resonate and may in fact cause harm. elevating consumer-generated strategies and messages paired with available and accessible health-promoting resources can make a difference. building engagement and investment from federal, private foundation, and corporate sector funders is essential. resources to expand traditional prenatal and infant mortality prevention strategies to preconception wellness for young adults who are not planning to become pregnant are rare. leaning in to this new paradigm requires funders to consider their strategies and shift their work to support different points of the life course trajectory. resources are necessary to advance research and build evidence-based practice for fully implementing preconception health as well as fully to expand social marketing and health system change strategies. given the complexity of preconception health promotion, partnership across fields and sectors is required. connecting with scientists, clinicians, consumers, policy-makers, community leaders, and activists nationally and internationally will generate new ideas and the spread of knowledge and practice. in the us, there are opportunities to build on current points of focus and energy, including the annual well visit, expanded and improved postpartum care, screening for reproductive intention, the promotion of long-acting reversible contraception, quality measures, and health reform. socially and politically, there are ripe opportunities for crucial conversations on equal rights, equal pay, racism, sexism, civic engagement, and reproductive justice. ultimately, the health of all young adults is linked to their access to education, employment and career opportunities, quality physical and behavioral health services, and safe communities where they can thrive. attention to the health and well-being of adolescents and young adults is critical for the health of our current society and generations to come. acknowledgements the national preconception health and health care initiative leadership team; w.k. kellogg foundation; the centers for disease control and prevention; national center on birth defects and developmental disabilities; and national center for chronic disease prevention and health promotion, division of reproductive health. disclosure statement the authors report no conflicts of interest. orcid sarah verbiest http://orcid.org/0000-0002-2491-1170 erin mcclain http://orcid.org/0000-0002-2521-4342 suzanne woodward http://orcid.org/0000-0002-2117-0004 references 1. association for maternal and child health programs. issue brief: opportunities and strategies for improving preconception health through health reform. march 2015. available from: http://www. amchp.org/transformation-station/documents/amchp%20preconce ption%20issue%20brief.pdf. upsala journal of medical sciences 225 http://www.twitter.com/syl_today http://www.facebook.com/showyourlovetoday http://www.amchp.org/transformation-station/documents/amchp&percnt;20preconception&percnt;20issue&percnt;20brief.pdf http://www.amchp.org/transformation-station/documents/amchp&percnt;20preconception&percnt;20issue&percnt;20brief.pdf http://www.amchp.org/transformation-station/documents/amchp&percnt;20preconception&percnt;20issue&percnt;20brief.pdf http://www.amchp.org/transformation-station/documents/amchp&percnt;20preconception&percnt;20issue&percnt;20brief.pdf http://www.amchp.org/transformation-station/documents/amchp&percnt;20preconception&percnt;20issue&percnt;20brief.pdf http://www.amchp.org/transformation-station/documents/amchp&percnt;20preconception&percnt;20issue&percnt;20brief.pdf 2. macdorman mf, mathews tj. understanding racial and ethnic disparities in u.s. infant mortality rates. national center for health statistics, 2011. available from: http://www.cdc.gov/nchs/data/databriefs/db74.pdf. 3. singh gk. maternal mortality in the united states, 1935–2007. health resources and services administration, maternal and child health bureau, 2007. available from: http://www.hrsa.gov/ourstories/mchb75th/mchb75maternalmortality.pdf. 4. kassebaum nj, bertozzi-villa a, coggeshall ms, shakleford ka, steiner c, heuton kr, et al. global, regional, and national levels and causes of maternal mortality during 1990–2013: a systematic analysis for the global burden of disease study 2013. lancet. 2014;384:980–1004. 5. robbins cl, zapata lb, farr sl, kroelinger cd, morrow b, ahluwalia i, et al. core state preconception health indicators—pregnancy risk assessment monitoring system and behavioral risk factor surveillance system, 2009. mmwr surveill summ. 2014;63:1–62. 6. kaiser family foundation. women’s health insurance coverage. feb 2016. available from: http://kff.org/womens-health-policy/factsheet/womens-health-insurance-coverage-fact-sheet/. 7. johnson k, posner sf, biermann j, cordero jf, atrash hk, parker cs, et al. recommendations to improve preconception health and health care—united states. a report of the cdc/atsdr preconception care work group and the select panel on preconception care. mmwr. 2006;55:1–23. 8. wise ph. transforming preconceptional, prenatal, and interconceptional care into a comprehensive commitment to women’s health. womens health issues. 2008;18:s13–s18. 9. atrash h, jack bw, johnson k. preconception care: a 2008 update. curr opin obstet gynecol, 2008;20:581–9. 10. united states department of health and human services. healthy people 2020. washington, dc: office of disease prevention and health promotion. available from: www.healthypeople.gov. 11. johnson k, balluff m, abresch c, verbiest s, atrash h. summary of findings from the reconvened select panel on preconception health and health care, december 2015. available from: http://www.citymatch.org/sites/default/files/documents/002192_preconception%20 health%20report%20booklet_5th.pdf. 12. floyd rl, johnson ka, owens jr, verbiest s, moore ca, boyle c. a national action plan for promoting preconception health and health care in the united states (2012–2014). j womens health. 2013;22:797–802. 13. council of state and territorial epidemiologists. core state preconception health indicators. available from: http://www.cste.org/ ?page¼preconindicators&terms¼coreþandþstateþandþpreconcep tion. 14. frayne dj, verbiest sv, chelmow d, clarke h, dunlop a, hosmer j, et al. health system measures to advance preconception wellness: consensus recommendations of the clinical workgroup of the national preconception health and health care initiative. obstet gynecol. 2016;127:863–72. 15. american academy of family physicians. preconception care position paper. december 2015. available from: http://www.aafp.org/ about/policies/all/preconception-care.html. 16. verbiest sv, malin ck, drummonds m, kotelchuck m. catalyzing a reproductive health and social justice movement. matern child health j. 2016;20:741–8. 226 s. verbiest et al. http://www.cdc.gov/nchs/data/databriefs/db74.pdf http://www.cdc.gov/nchs/data/databriefs/db74.pdf http://www.hrsa.gov/ourstories/mchb75th/mchb75maternalmortality.pdf http://www.hrsa.gov/ourstories/mchb75th/mchb75maternalmortality.pdf http://kff.org/womens-health-policy/fact-sheet/womens-health-insurance-coverage-fact-sheet/ http://kff.org/womens-health-policy/fact-sheet/womens-health-insurance-coverage-fact-sheet/ http://www.healthypeople.gov http://www.citymatch.org/sites/default/files/documents/002192_preconception&percnt;20health&percnt;20report&percnt;20booklet_5th.pdf http://www.citymatch.org/sites/default/files/documents/002192_preconception&percnt;20health&percnt;20report&percnt;20booklet_5th.pdf http://www.citymatch.org/sites/default/files/documents/002192_preconception&percnt;20health&percnt;20report&percnt;20booklet_5th.pdf http://www.citymatch.org/sites/default/files/documents/002192_preconception&percnt;20health&percnt;20report&percnt;20booklet_5th.pdf http://www.citymatch.org/sites/default/files/documents/002192_preconception&percnt;20health&percnt;20report&percnt;20booklet_5th.pdf http://www.citymatch.org/sites/default/files/documents/002192_preconception&percnt;20health&percnt;20report&percnt;20booklet_5th.pdf http://www.cste.org/?page&equals;preconindicators&amp;terms&equals;core&plus;and&plus;state&plus;and&plus;preconception http://www.cste.org/?page&equals;preconindicators&amp;terms&equals;core&plus;and&plus;state&plus;and&plus;preconception http://www.cste.org/?page&equals;preconindicators&amp;terms&equals;core&plus;and&plus;state&plus;and&plus;preconception http://www.cste.org/?page&equals;preconindicators&amp;terms&equals;core&plus;and&plus;state&plus;and&plus;preconception http://www.cste.org/?page&equals;preconindicators&amp;terms&equals;core&plus;and&plus;state&plus;and&plus;preconception http://www.cste.org/?page&equals;preconindicators&amp;terms&equals;core&plus;and&plus;state&plus;and&plus;preconception http://www.cste.org/?page&equals;preconindicators&amp;terms&equals;core&plus;and&plus;state&plus;and&plus;preconception http://www.cste.org/?page&equals;preconindicators&amp;terms&equals;core&plus;and&plus;state&plus;and&plus;preconception http://www.cste.org/?page&equals;preconindicators&amp;terms&equals;core&plus;and&plus;state&plus;and&plus;preconception http://www.cste.org/?page&equals;preconindicators&amp;terms&equals;core&plus;and&plus;state&plus;and&plus;preconception http://www.aafp.org/about/policies/all/preconception-care.html http://www.aafp.org/about/policies/all/preconception-care.html advancing preconception health in the united states: strategies for change introduction call for action national initiative current strategies measures integrating preconception into clinical care consumer education health equity looking forward acknowledgements disclosure statement references upsala j med sci 99: 259-266, 1994 5.3 preparation of the nordic protein calibrator ole blaabjergl, mogens blom2, per hyltoft petersenl, adam uldal13, hanne g@. 1, department of clinical chemistry, odense uniuersity hospital, dk-5000 odense c, denmark. 2. department of clinical chemistry, hjerring sygehus, dk-9800 h j ~ r r i n g , denmark. 3. department of clinical chemistry, kas herleu, dk-2730 herleu, denmark. 4. medi-lab, adelgade 5-7, dk-2304 copenhagen, denmark. the problems related t o preparation of secondary calibrators for determination of single human serum proteins are numerous. the proteins in the preparation must be genuine and stable for years. moreover, the preparation must be clear for the use in measurements based on turbidimetric and nephelometric principles. it would, further, be an advantage if the calibrator was prepared from serum from a well defined population so it could be repeatable reproduced. commercial available calibrators for specific determination of human proteins are delipidated by treatment by organic solvents or similar materials. this treatment usually denaturates the proteins to a variable extent due to irreversible reactions, and this is often made worse by lyophilizing of the preparation. therefore, the aim of the project was to produce a clear and reproducible liquid calibrator with genuine proteins stable for years. production of calibrator reproducibility: the reproducibility is obtained by collecting blood from more than 1,000 male blood donors . the last few ml of blood in the tube after bleeding is collected, left at room temperature for coagulation for one to five hours, centrifuged (2,50o*g for 15 min), and the serum aliquots pooled. solid nan, was added to a concentration of 0.1 % (15 mmol/l), and stored a t 80 "c. the blood donors were all tested for hiv and hepatitis b antibodies, and after a few days any pool where one sample was contaminated was discharged. 18 955059 259 delipidation: the calibrator was cleared by ultracentrifugation, the lipid fraction was removed and t h e volume restored by 0.9 % naci. this procedure is very gentle and keep t h e proteins genuine without denaturation. when the sufficient amount of serum is collected, t h e serum is thawed and recentrifuged (2,5oo*g for 15 min), carefully mixed, weighed, and then ultracentrifugated for 40 hours at 180,00o*g a t 4 "c. after ultracentrifugation the lipid layer is sucked off and after a new weighing t h e calculated amount of 0.9 % nacl (154 mmol/l with 15 mmol/l nan3) is added in order to restore t h e original weight and t h e lot is frozen and kept at 80 "c. when all lots are ultracentrifuged they are thawed and thoroughly mixed, t h e calibrator is then aliquoted in small portions (2 ml, 5 ml, and 10 ml) in cryotubes, frozen and stored at -80 "c until distribution for determination of t h e concentrations of t h e single proteins. u lt r ac e nt r if u g at i on i chylo+vldl 1 serum (turbid) c a l ib r a t 0 r (clear) fig. 5.3.1. schematic illustration of preparation of calibrator by ultracentri fugation. from blaabjerg et al. (1) with permission. pools: in order to investigate t h e different steps of the procedure and different storage conditions, several pools were prepared from 1981 to 1989 and kept under different conditions. the pools are 1981: ultracentrifuged (u-81) kept at 20 "c (u-81-20) and at 80 "c (u-81-80] and not ultracentrifuged (nu-81) at 20 "c (nu-81-20) and a t 80 "c (nu 8 1-80). 1983: ultracentrifuged (u-83) kept at 80 "c (u-83-80) and 1984: ultracentrifuged (u-84) kept a t 80 "c (u-84-80). 1989: ultracentrifuged (u-89) kept at 80 "c (u-89-80>. not ultracentrifuged (nu-83) kept at 80 "c (nu-83-80). 260 methods for documentation turbidity o f p o o l s : the turbidity of the described batches of pools prepared as the calibrator was measured photometrically. the extinctions of undiluted pools in 10.0 mm cuvettes were determined spectrophotometrically on a beckman du-8 spectrophotometer at 650 nm. the same instrument was used for all measurements. genuine proteins: it is difficult to demonstrate that the proteins are genuine. the pools were compared to commercial available calibrators and a fresh serum pool by agarose electrophoresis followed by immunofiiation with antibodies from dako according to carlstrom and johansson (3) . table 5.3.1 measurements of turbidity in the pools extinction of undiluted pool in 10.0 mm cuvettes at 650 nm time of time of measurements collection of pools pool nov81 nov82 mar83 apr84 oct84 mar89 sep 1981 nu-81-20 0.587 0.759 0.791 1.111 1.948 2.417 nu-81-80 0.587 0.768 0.738 0.688 0.915 1.024 u-81-20 0.060 0.063 0.061 0.095 0.162 0.554 u-81-80 0.060 0.068 0.063 0.069 0.073 0.079 jan 1983 nu-83-80 u 8 3 8 0 0.365 0.049 0.600 0.062 mar 1984 u-84-80 0.060 0.062 au-de 1989 u-89-80 0.044 combined reproducibility and stability: the stability of the preparations during storage as well as the reproducibility of preparing the pools are measured at the same time by the described procedures. this means that the results reflect both aspects. the measurements were performed at two occasions, in 1984 and in 1989. 1984: radial immunodiffusion was performed according to mancini (5). electro immunoassay was performed according to laurel1 (4). turbichmetry was performed according to blom and hjplrne (2) on a gemsaec centrifugal analyzer. nephelometry was performed on a technicon aip nephelometer@ 26 1 1989: measurements were performed on a cobas fara centrifugal analyser for all measurements antibodies from dako were used. from roche according to dako's recommendations. results turbidity of pools: the turbidity of the pools was determined at several occasions and the extensions are shown in table 5.3.1. it is seen from the table that ultracen trifugation is necessary for obtaining stable negligible values. further, the storage at 80 "c keeps the extinction low with insignificant increases for up to eight years. it should be mentioned that nu-83-80 was stored at 20 "c from april 1984, and that the freezer where nu-81-20 and u-81-20 were kept was defect in july 1984 with temperatures up to 10 "c for one week, but these facts do not change the general tendencies. t t t g c d e r n l 1 i i 1 g g 1 2 3 c + a b f d e fig. 5.3.2. comparison of three calibrator preparations (1:1981, 2:1984, and 3:1987) with six commercial protein calibrators (ab,c,d,e, and f) and a fresh serum pool (gj by agarose electrophoresis and immunofixation. arrows indicate changes in electrophoretic mobility compared to the fresh pool. from blaabjerg et al. (1) with permission. genuine proteins: changes in protein mobility can be disclosed by agarose electro phoresis and immunofixation if the mobility of the proteins is changed, this demonstrates changes in electrical charge and is an indication of denaturation. three calibrator pools (from 1981, 1984, and 1987) and six commercial protein calibrators 262 were compared with a fresh serum pool by electrophoresis and immunofixation in 1987 and the results from the four proteins, which are most sensitive t o denaturation are shown in fig. 5.3.2. prealbumin shows changes in electrophoretic mobility in all the commercial calibrators (indicated by arrows) whereas the three preparations (from 1981 to 1987) of the frozen liquid calibrator all show the same mobility as in the fresh serum pool. in three of the commercial calibrators cxl-antitrypsin shows lower concentration, an extra fraction and a broader band, respectively. no changes in mobility or concentration is seen in any of the frozen liquid calibrators. ceruloplasmin may be the best indicator of denaturation and four of the commercial preparations show changed mobility and decreased concentrations. again the frozen liquid pools behave like the fresh serum pool. table 5.3.2 combined stability of proteins and reproducibility of pools the data are normalized according t o protein and method rad. imm. diff elec. imm. ass turbidimetry nephelometry protein 81 83 84 prealbumin 0.978 1.007 1.015 albumin 0.999 0.992 1.010 orosomucoid 0.989 0.995 1.016 a l a n t i t q p i n 1.021 1.000 0.980 haptoglobin 0.993 0.997 1.009 ceruloplasmin az-macroglobulin transferrin 0.992 1.007 1.001 0.998 1.006 0.996 1.007 0.978 1.015 iga 1.012 0.991 0.998 igg igm 81 83 84 1.001 0.985 1.013 1.063 0.966 0.970 1.015 0.980 1.005 1.045 0.974 0.981 1.017 0.978 1.005 1.013 0.991 0.995 1.010 0.982 1.008 0.978 1.000 1.022 0.989 0.997 1.014 1.020 0.996 0.985 1.016 0.91 0.989 81 83 84 1.011 0.999 0.991 1.028 0.987 0.985 1.016 0.983 0.999 1.012 0.983 1.005 1.003 1.005 0.993 1.020 0.984 0.995 1.015 0.993 0.992 1.044 0.972 0.984 81 83 84 1.000 0.998 1.002 0.992 0.996 1.012 changes in haptoglobin may be more difficult t o interpret as the combinations of phenotypes may be different from calibrator to calibrator. combined reproducibility a n d stability: three preparations of calibrator (from 1981, 1983, and 1884) were compared by quantification of eleven proteins by four methods in 1984. the results for the normalized values are shown in table 5.3.2 according to protein and method. 263 the results are all close to 1.000 and scattered with a variation close to the analytical., only for al-antitrypsin (and perhaps igm) the results are consistent for the three methods indicating about 2 % higher values in the 1981 preparation. this, however, does not indicate instability as the highest values are in the oldest preparation. a new investigation in 1989 including a new preparation (collected in 1987), however, indicated lower values for the acute phase reactants. in table 5.3.3 the largest differences found between the 1984 and 1987 preparations are shown. table 5.3.3 combined stability of proteins and reproducibility of pools the data are given as differences in percentage for 1987 minus 1984 protein m e a n difference 95 % confidence interval prealbumin albumin transferrin orosomucoid al-antitrypsin hap toglobin 0.91 % 0.89 % t 0.57 % 0.45 % + 1.80 % 0.52 % 4.8 % 3.5 % 2.1 % 2.24 to +0.42 % 1.89 t o to.11 % +0.12 to + 1.02 % 1.05 to t0.15 % + 1.60 to +2.00 % 2.32 to + 1.28 % 5.2 to 4 . 4 % 4.1 to 2.9 % -2.6 to 1.6 % the results from table 5.3.3 clearly show lower values for the acute phase reactants in the 1987 preparation. this is not a question of instability as the oldest preparation has the highest values. however, it illustrates that minor fluctuations in mean values of t h e population occurs e.g due to infectious diseases, whereby, the biological uncertainty is more important than the statistical uncertainty, due to sample size. discussion it has been discussed for a long time whether protein calibrators should be frozen liquid or lyophilized, and how the delipidation should be performed. the ifcc-group decided on chemical delipidation and freeze drying. the documentation given here, 264 however, demonstrates that ultracentrifugation and storage as frozen liquid pool is very gentle to t h e proteins (compared to older commercial calibrators) and t h a t t h e proteins are stable for several years. for al-antitrypsin, however, t h e quantifications indicate some minor changes in t h e structure, as described in the following two sections and in chapter 6. except from this problem (which is well known for other protein calibrators) t h e frozen liquid calibrator is for years a reliable calibrator with genuine proteins when stored a t 80 "c. acknowledgements we want to thank elsebeth parlev and karin jorgensen for skilful and painstalung work with preparation of t h e pools and measurements of protein concentrations. references 1. 2. 3. 4. 5. blaabjerg 0, blom m, gry h, hyltoft petersen p, uldall a. appropriate sera for calibration and control of specific protein assays. scund j clin lab inuest 1993;53 suppl. 212:13-15. blom m, h j ~ r n e n. profile analysis of blood proteins with a centrifugal analyzer. clin chem 197622657-62. carlstrom a, johansson b-g. agarose gel electrophoresis-immunofixation. scund j immunoz 1983;17 suppi. 10~23-32. laurel1 c-b. quantitative estimation of proteins by electrophoresis in agarose containing antibodies. anal biochem 1966;15:45-52. mancini g, carbonara ao, heremans jf. immunochemical quantitation of antigens by single radial immunodiffusion. immunochemistry 1965;2:235-45. 265 upsala j med sci 96: 47-61, 1991 metabolic control, residual insulin secretion and self-care behaviours in a defined group of patients with type 1 diabetes karin wikblad, kent montin and lars wibell department of internal medicine, centre for caring sciences, uniuersity hospital, uppsala and department of education, uppsala university abstract a population of 185 type 1 diabetes patients (insulin-dependent, iddm), 25-45 years old, was studied retroand prospectively over a 9-year period with the aim of analysing background factors of importance for the ability to perform adequate self-care. expressed as mean hbalc, the metabolic control was slightly improved at the end of the study, when the insulin schedule had been changed in 60 % of the patients to multidose treatment. the degree of metabolic control remained constant over the years. the impact of residual insulin secretion, measured as 24-hour urinary c peptide, was low. patients with less good metabolic control often had a poor educational background and made less use of self-monitoring of blood glucose (smbg); they also experienced difficulties with smbg. the applied knowledge of diabetes also differed between groups with good and poor control. subjectively, most patients considered their metabolic control to be good, irrespective of the hbalc values. when asked about their own diabetes complications, their answers were often discrepant from the medical records. patients with particularly "good" or "poor" metabolic control were on the whole less satisfied with the education and information received than those with intermediate blood glucose regulation. development of strategies for individually adjusted education seems important. introduction during the last decade patients with type 1 diabetes have increasingly been expected to be active participants in their own treatment, and to perform adequate self-care. factors influencing the patient's ability to do this and to achieve satisfactory metabolic control have been subject to vast investigations but are still not clear. studies on self-care performance often deal mainly with psychological influences on the performance (3,5). other studies have medical and metabolic factors in focus, such as the residual insulin secretion, modes of insulin administration, technology for self-determinations of blood glucose, and so on ( 2 , 14-18). sjoberg et a1 (15) suggested that patients without any residual insulin secretion might experience more restrictions of their life-style compared with patients with a measurable amount of c peptide. it has been proposed that changes in the management of type 1 diabetes, with the introduction of flexible multidose insulin regimes (2, 14, 16) and self-monitoring 47 of blood glucose (14, 17-18), may have a positive influence on the patient's daily self-care and on the metabolic control of the diabetes. education of the patients has long been viewed as an important component of the treatment of diabetes (1, 4, 1 l), although the knowledge per se sometimes has only a limited effect on the performance. a number of studies have been focused on the influence of social factors on self-care and metabolic control ( 6 , 8-9, 12-13, 19). some of them have dealt with factors influencing compliance with treatment (8, 12,24), and others have pointed to the importance of social support (9, 12). the selection of patients has varied between different studies reported in the literature, and the degree to which patient materials are described is also highly variable. in the present study an analysis was made of diabetes-related variables in a well-defined group of patients with type 1 diabetes with the aim of identifying factors affecting the patient's ability to achieve adequate self-care. subjects and methods investigated ~ o u d to obtain a defined and representative sample of type 1 diabetic patients with experience of living with diabetes, the following criteria were established: 1. born between 1939 and 1959, 2. duration of diabetes of at least 5 years (onset of diabetes in 1975 or earlier), 3. currently treated with 2 20 u insulin daily. of the 1025 patients with type 1 or type 2 diabetes who in 1984/85 were regularly visiting the outpatient clinic at the university hospital in uppsala, 193 patients fulfilled these three criteria. at the first screening it was discovered that five of the patients had to be excluded because of brain lesions or mental retardation. three other patients declined to participate. thus the final number of patients in this survey was 185. general drocedure for the period 1980-84 a retrospective evaluation was performed. it was possible here to include an assessment of "long-term metabolic control". glycosylated haemoglobin (hba lc) had been measured routinely at all out-patient visits since 1980. the patient files also contained reliable information about the duration of diabetes, the treatment schedule and the size of the total insulin dose, height and body weight, serum creatinine and the presence of albuminuria. most patients also underwent regular screening for retinopathy. in 1985 a cross-sectional study was performed. a questionnaire was mailed to 170 of the 185 patients (12 were excluded because of advanced visual impairment, 2 patients had moved abroad and 1 patient had died). after two reminders it was completed by 139 patients (77 males and 62 females). the 31 "drop-outs" from this inquiry showed a higher percentage of male subjects and a higher mean hbalc than the total material. the cross-sectional study included measurement of c peptide in the urine in those 166 of the 185 patients from whom an apparently reliable 24-h urine specimen could be obtained. in 1985-1988 a prospective evaeuation of the further treatment and metabolic control of the 48 patients was undertaken. drod-outs: during the 5 years of the prospective study three patients died and 11 moved from the area. methods gzycosyzated haemoglobin (here called hbalc) was initially assayed by means of total hbal microcolumns (bio-rad, richmond) and later by hbalc methods (bio-rad, pharmacia hplc system). the methods correlate well in linear regression, with correlation coefficients of 0.96-0.98 during periods of double determinations at the laboratory. thus all hbalc values were converted to hbalc %. the upper normal limit (mean f 2sd) was 6.1 % in subjects with a normal oral glucose tolerance test. hbalc was usually measured 3-5 times yearly. the classification of retinopathic changes (mainly based on photometric examinations performed by the same ophthalmologist) comprised three categories: no important retinopathic changes, simplex retinopathy (background retinopathy) and preproliferative-proliferative retinopathy. renalfirnction was evaluated by means of albustixb strips at ali visits. for the serum creatinine method of the hospital laboratory, the reference limits were 64-106 pnom . the relative body mass index ( bmi) was used to describe the body weight. relative bmi was calculated from an ideal bmi of 20.9 kg/m2 in females and 22.4 kg/rn2 in males, as proposed by west (21). for screening of cpeptide in the urine, a commercial kit (behringwerke) was used.the method was applied semiquantitatively (c peptide below the detection level of ~ 0 . 4 nmov24 h, above the detection level but low, 0.4-3.9 nmov24 h, or within the reference limits of 4.0-50 nmov24 h). the self-care questionnaire was constructed to obtain demographic data, social data and information concerning subjective health perception, self-care ability, the educational level and applied knowledge about diabetes. the questionnaire consisted of 29 multiple-choice questions, 17 open-ended questions and 5 likert scales for self-estimation of physical health. the social data were classified according to the socio-economic classification system (sei) deveioped by statistics, sweden ( a copy of the questionnaire is supplied by the authors on request). statistical methoak: some results are simply presented as group mean values fsd. according to the data level, median values have also been used. for comparison of mean values, the t test was used and proportions were compared with the use of chi-square tests. correlations were calculated by pearson's product-moment correlation coefficient and the spearman-rank correlation. one factor anova (two-tailed) was used for group comparison. results clinical characteristics age and duration in 1985, as implied by the inclusion criteria, all patients were between 26 and 46 years old, with the same mean age of 36 for males (n= 104) and females (n= 81). the mean age at onset of diabetes (total range 1-33 years) was 15.5 k7.7 years in male and 12.3 f 7.9 years in female subjects. the total mean duration of diabetes was 22.1 years k 8.5 (range 10-41 years). 4-91 8571 49 hba 1 c values during the five-year baseline period 1980-1984 the mean hbalc value in the whole material was 8.7 f1.2 %. in 1986 the mean hbalc was unchanged, 8.7 f1.5 %, and in 1988 it was significantly lower, 8.3 f1.2 % (p<o.ol). as shown in table 1, the patients were arbitrarily divided into four groups according to their metabolic control (good, acceptable, unsatisfactory and unacceptable) as evaluated at baseline. in the intermediate range, groups ii and iii, there was a higher proportion of men with better control ( ~ ~ 0 . 0 5 ) . table 1. patients with various degree of metabolic control. classification based on mean hbalc during the five years 1980-1984. hbalc% group male (104) female (81) mean hbalc % of patients (n) % of patients (n) % & s d 17.5 i 17 (18) 20 (16) 6.9f0.65 7.6-8.4 i1 30 (31) 20 (16) 8.0k0.27 8.5-9.4 ill 24 (25) 34 (28) 8.9f0.25 2 9.5 iv 29 (30) 26 (21) 10.1m.68 insulin dose during 1980-1984, the five-year baseline period, 31 patients had changed to a multidose regime from their previous 1-2 daily injections. as seen in table 2, during the 9 years from 1980 to 1988 the number of patients on a multidose schedule increased continuously from 0/182 to 103/170. at the same time the number of patients maintained on one single daily dose decreased from 103 to 18. meanwhile the mean daily total insulin dose seemed to increase slightly. table 2: total insulin doses and number of injections in 185 type 1 diabetic patients. year insulin dose number of patients with ( h i ts/kg/day ) 1 daily dose 2 daily doses multidose 1980* 0.64 103 79 0 1984* 0.70 44 107 31 1986** 0.72 25 86 69 1988*** 18 49 103 *n=182, **n=180, ***n=170 finally in 1988 patients with poor metabolic control more often had a multidose schedule than those with good control (p<o.o5). when a separate analysis was made of the 31 first multidose patients in 1980-1984, both an increase in the total insulin dose (0.70 u/kg/day in 1980 compared to 0.78 in 1984) and a decrease in the mean hbalc, from 9.1% to 8.5%, were seen after the dose intervention (n.s, ~ ~ 0 . 1 ) . a change from 1 to 2 doses of insulin daily had no impact on the mean hbalc level in 44 patients during the baseline period. 50 bodv weight overweight, defined as a standardized bmi exceeding 120 %, is not common in patients with type 1 diabetes, particularly not in males. in 1980, overweight was found in 3% of the men compared to 13% of the women of this material (p<o.001). in 1986 these figures had increased to 12 % and 14 %, respectively, a difference which was at least partly attributable to a more intensive insulin regime. the mean weight increase from 1980 to 1986 in the 69 patients who had changed to 3-4 daily injections was 2.7 kg, whereas the 25 patients remaining on a single daily dose only showed a small mean weight change, +0.8 kg. retinodathv in 1980 126/183 patients were found to be free from clear signs of retinopathy. this proportion decreased to 77/183, 65/177 and 62/170 i n the years 1984, 1986 and 1988, respectively. there was a relation between the development of retinopathy and the previous hbalc level (classified as in table 1), as seen in table 3 a. patients with hbalc < 7.5% had significantly higher percentage of freedom from retinopathy than those with higher hbalc values (p< 0.01). in 1988 proliferative retinopathy was found in 20/51 subjects with hbalc 29.5 %, but only in 2/34 subjects with hbalc < 7.5 % (pd.001). the patients without retinopathic changes had significantly lower mean hbalc values than those with clear changes (p<0.005). table 3 a. percentage number of type 1 diabetic patients “without retinopathy” as related to the mean hbalc level during the five-year period 1980-1984. hbalc n diabetes duration % without clear changes 1980-84 (mearrksd) 1980 1984 1986 1988 g . 5 (i) 34 22.0k7.7 91 62 59 53 7.6-8.4 (a) 47 24.0k8.1 64 36 30 28 8.5-9.4 (hi) 53 24.9k9.9 58 40 36 30 29.5 (iv) 51 2 l.lf7.7 70 37 29 29 nedhrodathv the qualitative test for proteinuria, albustix@, was normal in 149/183 patients in 1980 b u t only in 126/183 five years later, at the end of the baseline period. after 1984 129/178 subjects remained free from proteinuria until 1988, when 121/168 had a negative proteinuria test. the relation between the hbalc level and a negative proteinuria test is shown in table 3 b, which is analogous to table 3 a. patients with hbalc i 8.4% had significantly higher percentage of freedom from albuminuria than those with hbalc > 8.5% (p < 0.01). ninety-seven per cent of the patients with good metabolic control and 90 % of those with unacceptable control had serum creatinine values within the reference limits. 5 1 table 3 b. percentage number of type 1 diabetic patients "without proteinuria" as related to the mean hbalc level during the five-year period 1980-1984. hba l c n diabetes duration % with negative proteinuria test 1980-84 (me&sd) 1980 1984 1986 1988 17.5 (i) 34 22.0+_7.7 97 91 88 88 7.6-8.4 (ii) 47 24.0f8.1 91 77 77 77 8.5-9.4 (iii) 53 24.9k9.9 75 64 66 58 1 9 . 5 (iv) 51 2 1.1k7.7 67 49 55 47 stability of metabolic control (fig.1) in the analysis of the possible effect of metabolic control on the rate of occurrence of late complications of diabetes (tables 3 a and 3 b), and in the study of self-monitoring of blood glucose and other aspects of self-care, it was of interest to assess the degree of fluctuation between good and poor metabolic control. in this respect considerable stability was found. in each of the four metabolic control groups (table l), the mean hbalc value was calculated for each of the years 1980, 1984, 1986 and 1988, and no change was found in any of the groups over this 9-year period (fig 1). if the mean hbalc for 1988 instead of the mean for the period 1980-1984 had been used as a basis of the grouping, some patients would of course have been allocated to a group above or below the previous rating (borderline cases), but only 18 of the 185 patients would have had to be moved two steps or more upwards or downwards on the 4-grade scale. 1 e 9 n 4 __cpz_ groupiii u 3 $ 8 4 groupii 6 1 1980-84 1980 1984 1986 1988 year figure 1: the mean values of hbalc in the four groups of patients with type 1 diabetes (n=34-53) with different degrees of metabolic control, as defined in table 1, in 1980-1984, are shown to the left. the mean hbalc for each of these groups in four different years betwcen 1980 and 1988 is then shown, to illustrate the degree of stability over this 9-year period. residual insulin secretion the %hour excretion of urine c peptide was determined in 90 % of the 185 patients. as expected, with a mean duration of 22 years and a shortest duration of 10 years, the majority of the patients had no or very little urinary c peptide. no or only a small amount (< 0.4 nmov24 h) 5 2 was found in 133 patients. twenty-two patients had a definitr, excretion but so low that it was clearly in accordance with type 1 diabetes. eleven patients had c-peptide excretion of such a magnitude that it was likely to be of clinical importance (2 4.0 nmo1/24 h). the 33 patients with a detectable amount of c peptide in the urine were matched according to sex, age and duration of diabetes with 33 patients with non-detectable levels of c peptide. the two groups each consisted of 18 males and 15 females. the mean age of the two groups of patients was 38 f 5 years and the mean duration of their diabetes was 22 years (22k9 in the c-peptide group and 22k10 in the matched group). the mean hbalc values were only slightly lower in the patients with urinary c-peptide excretion (excretors) than in those without detectable c peptide in the urine (non-excretors) (excretors: 8.7f1.30%; non-excretors: 9.3+1.65%) and there was no correlation between hbalc values and urinary c-yeptide values (r=-0.07). there was no difference between the two groups in the daily amount of insulin administered (0.65 m.18 uikg/day in c-peptide excretors and 0.68f0.17 in non-excretors). neither was there any difference in relative bmi. the urinary c-peptide excretion showed no correlation with the daily amount of insulin (r=0.20), but was weakly correlated with relative bmi (-0.31). there was a tendency (n.s) towards a lower frequency of late complications among excretors than among non-excretors: 15 excretors had retinopathic changes, against 20 non-excretors, and three had proliferative retinopathy, against six non-excretors; for albuminuria the figures were five and ten (p<0.02). the two groups gave similar answers to the self-care questionnaire. social characteristics seventy-four per cent of the patients were married or living with a partner, without any differences between the four hbalc groups. the type of occupation of the partners differed, however, between group iv and the other three groups. fifty-six per cent of the group iv partners were manual workers, compared with 9% of those of group i, 22% of those of group i1 and 21 % of those of group iii (p<o.ol, group i vs group iv). the same pattern was found regarding the patients' educational level (fig. 2 ) , most patients with good control having completed upper secondary school education. self-care characteristics estimation of own metabolic control eighty-five per cent of the patients considered that their own metabolic control was good or very good and 15 % that it was poor or very poor (fig. 3). the correlation between the self-rating of metabolic control and hbalc values was weak (r=0.27), mainly because hbalc did not differ between the the two intermediate groups. self-monitoring of blood glucose (smbg) forty-nine per cent of all males and 29 % of all females reported that they never monitored their own blood glucose. smbg was carried out systematically by 27 % of the patients with good or acceptable metabolic control but only by 16 and 11 %, respectively, of those with unsatisfactory or unacceptable control. problems with self-monitoring were usually of a practical nature, with 5 3 difficulties in organizing the tests and getting reliable results, but psychological pressure and "test hysteria" was also reported. self-monitoring was associated with difficulties for the performers in , 13 % of p u p i (good control), 38 % of group i1 (acceptable control) and 57 % of groups iii and iv. 4 0 30 r 4 u 8 's a +i 0 20 j el 10 2 0 compulsoiy school upper secondary school fig 2. educational background of the patients. very p004 rn 00 el very goodl ' ' ' q o mi 0 4 6 8 10 1 2 1 4 hbalc (%) figure 3: comparison between measured hbalc values and patients' self-rating of metabolic control. percedtion of health, symdtoms and comdlications the self-rating of physical health and mental balance, as perceived by the patient, in relation to the metabolic control is shown in figs 4 and 5. no correlation between hbalc and the subjective assessment of physical health (r=-0.05) or mental balance (r=-0.06) was found. however, both concerning physical health and mental balance, the patients in group i1 ("acceptable") had the highest mean self-ratings and those in group iv the lowest (p<0.05). regarding the patients' opinions as to whether perceived physical unhealth and mental unbalance were diabetes-related, 15 % of the patients in group i who reported physical unhealth and 38 % of 54 the corresponding patients in group lv considered this to be totally diabetes-related. on the other hand 27 % of those in group i who reported mental unbalance and 11 % of the corresponding patients in group iv considered this to be totally diabetes-related. a comparison between the patients' knowledge about their late complications and information collected from the patient files showed a discrepancy (table 4). patients in the group with good metabolic control were more well-infomed about their retinopathy than those in the other groups. 4 6 8 1 0 12 1 4 hbalc (%) figure 4: patients' self-estimation of physical health in relation to measured hbalc values (l=not well at all, 2=not quite well, 3=moderately well, 4=almost well, 5=healthy) 0 1 4 6 8 10 12 1 4 hbalc (%) figure 5: patients' self-estimation of mental balance in relation to measured hbalc values (l=not at all in balance, 2=not very good balance, 3=moderate balance, 4 = g d balance, 5=very good balance) table 4: percentage number of patients with late complications according to information in patient files and the patients' own report (in brackets) (1985) complication group i group i1 group iii group iv n=34 n=47 n=53 n=5 1 58 (41) 63 (40) 34 (14) 49 (23) retinopathic changes 21 (20) 64 (35) albuminuria 1 2 ( 7) 23 (11) 5 5 knowledrre about diabetes used in dailv practice fifty-seven per cent of the patients gave satisfactory answers when asked how they would balance the insulin dose in the event of an infection with fever. thirty-two per cent would never change the insulin dose, 4 % described irrational actions and 7 % did not know what to do. a satisfactory answer was more common (63%) in patients with good metabolic control than in those with unacceptable metabolic control (54%). when asked a similar question about acute gastroenteritis, 42 % gave satisfactory answers, 36 % did not correct the dose, 15 % felt insecure and 7 % described an incorrect action. a comparison between group i and iv showed that 64% of the former patients, with good metabolic control, gave satisfactory answers, compared with 29% of those of the latter group, with unacceptable metabolic control. one-third of the 139 patients seemed to understand how the amount of food interacts with sixty-five per cent of the patients were satisfied with the amount and quality of the education and information they had received about diabetes and its treatment. the patients in the intermediate groups, i1 and 111, were more content than those in groups i and iv (group i: 60%, group 11: 70%, group iii:73% and group iv:54%). the question “do you tell your friends, relatives and colleagues that you are a diabetic?” was answered as shown in table 5. patients with good metabolic control seemed to tell about their diabetes more often than the others. exercise and insulin. table 5: percentage number of patients that told others that they were a diabetic, in each of the four metabolic control groups as defined in table 1. group 1 groupii groupiii gr0upiv tells close friends 80 54 54 66 tells relatives 83 59 54 63 tells colleagues 67 43 59 60 discussion characteristics of the material it was the aim of this study to follow up type 1 diabetic patients regarding their metabolic control over a long period of time (1980-1988). this 9-year study was performed in a well-defined group of patients with type 1 diabetes. by using both retroand prospective data, virtually all the patients could be followed up throughout the entire period. this would not have been possible if the study had had an exclusively prospective design. this long-term investigation showed that new treatment strategies, such as multidose treatment with insulin pens (60% of the patients had changed to multidose) and more intensive patient education, seemed to have had an impact on the metabolic control. a more aggressive treatment of hypertension in diabetes patients during the recent years could have contributed to the decrease, here observed, in the number of patients with albuminuria. 56 the patients of the study group, identified in 1984-1985 in the middle of the study, were between 25 and 45 years old (mean 36 years) and had had diabetes for at least 10 years (mean 22 years). type 1 diabetes was defined as diabetes requiring treatment with at least 20 units of insulin daily. the out-patient clinic, in an unbiased mode, handles a majority of the young adult patients with type 1 diabetes in the area and all patients who fulfilled the criteria were included. thus the study material was representative of type 1 diabetes patients 25-45 years old with a disease duration of 10-40 years, with two further possible selection factors to consider: 1) such patients would be lost as had died at a young age before 1984 or referred e.g. to dialysis 2) patients not steadily living in the uppsala area but having left the city, or moved in, during the retrospective analysis period (1980-1984) would not be included. the results obtained in the present study should also be viewed in the light of the fact that during the years preceding 1980 the diabetes clinic had been reorganized. a diabetes nurse specialist and a dietician had been included in the staff, and both specific therapeutic intervention and diabetes education activities had been introduced. that the care provided had a reasonable standard is illustrated by the fact that all patients in this study had been examined regarding retinopathy. measurement of glycosylated haemoglobin had been used as a means of identifying patients in particular need of attention, although only 1-2 daily doses of insulin had been used before 1981 and, on the whole, only previously pregnant women had been trained to monitor their own blood glucose. as shown in table 2, multidose treatment (3-dose, 4-dose, csii) was successively introduced betwen 1980 and 1988. to an increasing extent the patients were also taught the usefulness of smbg. main findings in the studv hbalc stability. when the mean hbalc value for 1980-1984 in each patient was used for classification into four groups with different degrees of metabolic control, the groups showed remarkable stability over time, as assessed by calculating the mean values for different years in these patient groups (fig. 1). effect of intervention the first 31 patients in whom a change was made to a three-dose regime showed a decrease in mean hbalc from 9.1 to 8.5 %. about one-third of these patients had changed the insulin regime because of poor metabolic control and it was found that a reduced hbalc level was only obtained in the patients with initial values of above 8 %, although most of them found other advantages from a multidose regime (22). in the whole material, the mean hba ic of 8.3 % in 1988, was significantly lower than the mean base-line value of 8.7 %, a reduction which may have reflected the increasing use of a multidose insulin regime (table 2), continuous patient education andor increased smbg. a slight increase in bodyweight was observed in multidose patients but overweight was uncommon in this material. the use of smbg is correlated to good metabolic control. about 25 % of the patients in the groups with good or acceptable metabolic control regularly measured their own blood glucose and used the values for balancing, compared with 11-16 % in the groups with unacceptable or unsatisfactory metabolic control. in the latter groups smbg was associated with more difficulties. 4 t 9 1 8 5 7 1 57 the educational level is an important factor. one major difference between the group of patients with good control and the other groups lay in the educational background, as shown in fig. 2. here it is seen that “good control” patients had completed upper secondary school. about one-fourth of all patients were single, but among the others 56 % of those with the worst metabolic control (gfoup iv) had partners with manual work. this may be compared with the 9 % in group i and thus seems to provide some confirmation of the importance of the educational level. possible value of confidence and a social network. patients with good metabolic control (table 5) more often talked to their friends, relatives and colleagues about their diabetes. this finding may be related to the importance of the amount of education obtained and of the potential ability to receive education. residual insulin excretion does not seem important. as measured by 24 h urinary c peptide excretion, the residual beta-cell function seemed to be of little or no influence on metabolic control in long-standing type 1 diabetes. only 11/185 patients excreted more than 4.0 nmo1/24 h and 133 of the patients excreted less than 0.4 nmol. all 33 c-peptide excretors were compared with and found to be similar to the 33 matched controls. there were no correlations between hbalc, the daily insulin dose and urinary c peptide. patients can experience difficulties with the assessment of health. it seems to be common to underestimate the presence of late complications of diabetes and the degree of metabolic derangement, when the metabolic control is poor, as shown in fig. 3 and table 4. this may be explained by wishful thinking or by absence of a feeling of decreased well-being. jacobsen et a1 (3) pointed out that poor metabolic control may be associated with a higher threshold for physical symptoms caused by hyperglycaemia. the experience of well-being is determined by other factors than metabolic control (23). when patients were asked to estimate their own physical and mental health (figs 4 and 3, virtually no correlation was found between hbalc and the ratings. however, there was a tendency for the patients with acceptable, but not very good metabolic control to experience the highest degree of mental and physical health. factors affecting metabolic control and self-care among all patients who changed to multidose treatment, there was an overrepresentation of patients in groups iii and iv (poor metabolic control). though the change was partly a technical solution to problems of poor metabolic control, there are other important aspects. even if the primary aim was to achieve better metabolic control, it will probably in the long term prove to give the type 1 diabetes patient a greater sensation of freedom. adequate knowledge will probably also increase the patient’s self-esteem. twenty per cent of the patients had detectable levels of c peptide in the urine, but only 6% had levels within the reference limits. this could be compared with findings of sjoberg et a1 (14), who reported that 36% of the patients were urinary c-peptide excretors but that in these patients the duration of diabetes was shorter (9-16 years) and the detection level for urinary c peptide was 0.2 nmov24 hours, which could explain the difference. the authors found better metabolic control, however, in patients with c-peptide excretion and this could not be statistically verified in our study. the only significant difference between excretors and non-excretors in our study was the frequency of late complications, which was lower in the c-peptide excretors. 5 8 it has been found difficult to provide definite proof that good metabolic control in itself is able to prevent the development of late complications. however, this study adds to other circumstantial evidence by finding a significant difference in mean hbalc values between patients without late complications and those with clear retinopathic changes, for example ( ~ ~ 0 . 0 1 ) . this is also in line with the findings in a randomized study (10) on intensified diabetes treatment, in which a relationship was observed between late complications and blood glucose levels. it is quite obvious that patients with only a short period of general education have poorer metabolic control than those with higher education. the reason for this is not clear, but it is possible that the education and information given to the patients regarding their diabetes is on too high a level for those with only compulsory schooling as an educational basis. the knowledge about diabetes among relatives, friends and colleagues of diabetic patients is an important part of the patient's safety-net. that the patient has the courage to inform others about his diabetes may reflect his self-esteem. regarding self-care behaviours such as self-monitoring of blood glucose, we found that the patients with poor metabolic control did not use this aid as often as those with good control and more often reported difficulties in carrying it out. this is in accordance with the finding that only those who used the results of their smbg in balancing the glycaemic control received a better metabolic control. this could of course be due to lack of information. an explanation for the discrepancy between the late complications as reported in the patient files and as described in the patients' own reports could be either that the patients had been told about the occurrence of late complications but had suppressed the information or that the information had been given in a disguised way in order not to worry the patient too much and was hence unclear. also the fewness of the right answers to the question concerning interactions between food, exercise and insulin might have reflected a misunderstanding of the question, but it might also illustrate how problematic it is for the patients to handle the complex interplay between these three variables. the interaction may seem easy to understand for a non-diabetic, but for patients with type 1 diabetes, who know how hard it is to achieve a balance between food, activities and insulin in their daily social life, it is not always easy to manage (20). achievement of good metabolic control is today the best way of delaying the occurrence of late complications (10). when trying to improve the patient's hbalc values, it is important to tell him that the principal aim of this is to prevent complications and not to increase his well-being. the patient with good metabolic control is not 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(change to three doses of insulin per day. a study of metabolic control and of the patient's attitude to treatment and self-deteminations of blood glucose. in swedish) svenska liikaresiillskapets handlingar, hygiea, 95,6: 163, 1986. wikblad, k., wibell, l. & month, k.r.: health and unhealth in chronic disease. scand j caring sci 5: 5-10,1991. wilson, w., ary, d.v., biglan, a., glasgow, r.e., toobert, d.j. & campbell, d.r.: psychosocial predictors of self-care behaviors (compliance) and glycemic control in non insulin-dependent diabetes mellitus. diabetes care 9: 614-622, 1986. address for reprints: karin wikblad centrum for omvhdnadsvetenskap akademiska sjukhuset s-751 85 uppsala sweden. 23. 24. 61 untitled expression of transforming growth factor-β1 221 received 2 november 2007 accepted 16 december 2007 upsala j med sci 113 (1): 221–234, 2008 expression of transforming growth factor-β1 and connective tissue growth factor in the capsule in a rat immobilized knee model yoshihiro hagiwarap1p, eiichi chimotop1 p, ichiro takahaship2p, akira andop1p, yasuyuki sasanop3p, eiji itoip1 p 1 pdepartment of orthopaedic surgery, tohoku university graduate school of medicine, 1-1 seiryomachi, aobaku, sendai, japan 980-8574, p2p division of orthodontics and dentofacial orthopedics, tohoku university graduate school of dentistry, sendai, japan 980-8570, p3 p division of craniofacial development and regeneration, tohoku university graduate school of dentistry, sendai, japan 980-8570 abstract background: contracture is a very common complication of joint immobilization in daily examination, but its cause is still unknown. a fibrotic change of the capsule is suggested to be one of the main causes of the joint contracture. the goal of this study was to analyze the expression pattern of transforming growth factor-β1 (tgf-β1) and connective tissue growth factor (ctgf), which are implicated in fibrosis in the capsule of a rat immobilized knee model. materials and methods: we immobilized the unilateral knee joints of 66 rats in 150 degrees of flexion using a plastic plate and metal screws. sham operated knee joints of 66 rats had holes drilled and screws inserted but none of them were plated. the capsule from the anterior and posterior portion of the knee joints was harvested at 3 days, 1, 2, 4, 8 and 16 weeks after immobilization and the expression patterns of tgf-β1 and ctgf were characterized using in situ hybridization and immunohistochemistry. results: the in situ hybridization demonstrated that the mrnas of both tgf-β1 and ctgf increased continuously during the first 2 weeks after immobilization and then decreased. the response was relatively higher in the posterior capsule than in the anterior one. in contrast, the immunoreactivity of both tgf-β1 and ctgf increased gradually with time. the response was much stronger in the posterior capsule than in the anterior one. conclusions: the capsule has a potency to produce tgf-β1 and ctgf after immobilization. ctgf may play a role in causing and maintaining capsular fibrosis in collaboration with tgf-β1. the fibrotic change in the posterior capsule may have resulted in limited motion in extension in this immobilized knee model in rats. it may be possible to prevent joint contractures by somehow blocking the fibrotic process. introduction a contracture is a very common complication of joint immobilization. the definition of contracture is a loss of passive and active ranges of motion of a joint [1, 2]. though joint immobilization can be beneficial in decreasing pain and possibly joint damage in the acute phase of inflammatory arthritis [3–5], a contracture usually disturbs activities of daily living. management of an established contracture 222 yoshihiro hagiwara et al. includes physical therapy and surgical procedures, with equivocal functional results [6, 7]. the resulting burden of disability to the patients and the financial cost to our society are enormous [8]. despite years of interventions from professionals involved in primary care and rehabilitation [9], the contracture remains a frequent clinical challenge. many experimental studies on joint immobilization have been reported [10–16]. a florid intra-articular connective tissue proliferation invading the intra-articular space has been considered a feature of contracture caused by joint immobilization [10–15]. this proliferation tissue, mainly composed of synovial lining cells, fibroblasts, fibrocytes, monocytes, adipocytes, and extracellular matrix [10–12, 16, 17], is defined as pannus [18]. contact between the pannus and the surface of the articular cartilage has been postulated to prevent cartilage nutrition and/or mediate pathophysiological changes leading to cartilage degeneration and joint stiffness after immobilization [10, 15, 19–22]. however, there have been contradictory reports about phenomena after joint immobilization. some reported that pannus proliferation occurred after immobilization [5, 8, 23, 24] and no contact between pannus and articular cartilage was observed [25]. others showed that capsular stiffness, resulting in degenerative changes such as synovial atrophy, retraction, fibrosis, and adhesion, might contribute more to joint contractures than muscle function [2, 5, 10, 11, 17, 26–28]. transforming growth factor-β (tgf-β) has been demonstrated to have multiple biological functions, especially in wound repair acceleration and experimental organ fibrosis [29]. tgf-β upregulates fibroblast proliferation and extracellular matrix synthesis and reduces matrix degradation after injury [30]. among the three isoforms of tgf-β1, 2 and 3 in mammals, tgf-β1 is most implicated in fibrosis [31, 32]. a previous report described high expression of tgf-β and tgf-β receptors in synovial cells of human adhesive capsulitis using immunohistochemistry [33]. the concentration of tgf-β1 in synovial fluid increased after immobilization in a rabbit immobilized knee model [34]. tgf-β may be one of the factors related to joint contracture. injection of tgf-β into the knee and ankle joints of rats induced transient synovial hyperplasia [35, 36]. subcutaneous injection of tgf-β1 into newborn mice for 3 consecutive days caused reversible granulation tissue formation without persistent fibrosis [37]. other factors besides tgf-β may be involved in causing the joint contracture. subcutaneous injection of connective tissue growth factor (ctgf) in addition to tgf-β in newborn mice produced long-term persistent fibrosis [29]. this result suggests that ctgf may play a role in causing and maintaining skin fibrosis in collaboration with tgf-β. in our previous study, the elasticity increased in the capsule after 8 and 16 weeks of immobilization as assessed by scanning acoustic microscopy [1]. in that context, we hypothesized that fibrosis, which occurred in the capsule after immobilization, might have contributed to the limited extension observed. in this study, we investigated expression of tgf-β1 and ctgf in the capsule during immobilization using in situ hybridization and immunohistochemistry. expression of transforming growth factor-β1 223 materials and methods animals the protocol for the experiments was approved by the animal research committee of tohoku university. adult male sprague-dawley rats (body weight 380–400g) were used. intra-operative and post-operative analgesia with buprenorphine (0.05 mg/kg) was injected subcutaneously. the unilateral knee joints were immobilized at 150 degrees of flexion with an internal but extra-articular fixator for various weeks (3 days to 16 weeks) as previously described [1, 2]. the left and right hind legs were immobilized alternately to avoid potential systematic side differences. a rigid plastic plate (pom-n, senko med. co., japan) implanted subcutaneously joined the proximal femur and the distal tibia away from the knee joint and was solidly held in place with one metal screw (stainless steel, morris, j. i., co., usa) at each end. the knee joint capsule and the joint itself were untouched. sham operated animals had holes drilled in the femur and tibia and screws inserted but none of them were plated. the animals were allowed unlimited activity and free access to water and food. seventy two rats (3 days, 1, 2, 4, 8, and 16 weeks, 6 rats for the immobilized group and 6 rats for the control group at each time point) were prepared for in situ hybridization and sixty rats (1, 2, 4, 8, and 16 weeks, 6 rats for each group at each time point) were prepared for immunohistochemistry. the immobilized animals and the sham operated animals made up the immobilized group and the control group, respectively. tissue preparation the rats were anesthetized and fixed with 4% paraformaldehyde with or without 0.5% glutaraldehyde in 0.1 m phosphate-buffer, ph 7.4 by perfusion through the aorta. the specimens fixed with glutaraldehyde were used for in situ hybridization. the knee joints were resected and kept in the same fixative overnight at 4°c. to keep the morphology intact, the specimens were decalcified as a whole knee joints in 10% edta in 0.01 m phosphate-buffer, ph 7.4 for 2 months at 4°c. the edta solution was autoclaved before use. after dehydration through a graded series of ethanol solution, the specimens were embedded in paraffin. the embedded tissue was cut into 5-μm sagittal sections from the medial to the lateral side of the joint. standardized serial sections were kept in the medial midcondylar regions of the knee. a region of analysis for in situ hybridization and immunohistochemistry was set in the anterior and posterior capsule each in each section (figure 1). preparation of rna probes digoxigenin (dig)-labeled single strand rna probes were prepared using the dig rna labeling kit (roche, mannheim, germany) according to the manufacturer’s instruction. a fragment encoding rat ctgf (876–1555 bp: genbank accession number nm_022266) was obtained from total rna of embryonic rat limbs using 224 yoshihiro hagiwara et al. reverse transcription followed by rt-pcr and subcloned into the pcrii topo (invitrogen; carlsbad, ca). the following oligonucleotide primers were used for the rt-pcr: upstream primer, 5´cgggttaccaatgacaata3´, downstream primer, 5´gtctttctcctggcatctc3´. the cdna was verified by digestion with restriction enzymes and was confirmed by dideoxynucleotide sequencing. fragments of rat mature tgf-β1 (genbank accession number nm_021578) were purchased from riken gene bank (1230 bp, rdb1195, saitama,p pjapan). a fragment (338–885 bp) digested with psti was subcloned into the pbluescript ii (ks+) (stratagene, la jolla, calif., usa). antisense and sense riboprobes were generated by t3/xbai and t7/ecorv for ctgf and t7/bamhi and sp6/ecorv for tgf-β1, respectively.sense and sense riboprobes were generated by t3/xbai and t7/ecorv for ctgf and t7/bamhi and sp6/ecorv for tgf-β1, respectively. in situ hybridization the protocol used in the present study has been reported elsewhere [38] and is only briefly describedp pas follows. the sections were deparaffinized and washed in pbs, ph 7.4, p pand then immersed in 0.2 n hcl for 20 min. after being washed p pin pbs, the sections were incubated in proteinase k (20 μg/ml; p proche) in pbs for 30 min at 37°c. p pthe sections were then dipped in 100% ethanol and dried in air p pand incubated with the antisense probe or the sense control p pprobe (400 ng/ml) in a hybridization mixture for 16 hrs at 45°c.p pthe sections were washed and treated with rnase (type 1a, 20 μg/ml; sigma, st louis, mo) for 30 min at 37°c. after figure 1. microphotographs of a sagittal section of a rat knee. a: the anterior capsule, b: the posterior capsule. squares indicate regions of analysis for in situ hybridization and immunohistochemistry. m: meniscus, i: intra-articular space, f: femur, t: tibia. (scale bars = 300 μm, original magnification × 5, hematoxylin-eosin stain). a expression of transforming growth factor-β1 225 washing, p pthe hybridized probes were detected immunologically using the p pnucleic acid detection kit (roche), counterstained with methylp pgreen, and mounted with a mounting medium.p pfibroblast-like cells in the capsule were counted (two to three areas per section) and positive cells were defined as same signal intensity as those of hyperchondrocyte in the growth plate. the ratios of tgf-β1 and ctgf positive cells to the total number of cells counted (at least 100 cells) at high magnification (× 400) were calculated. two investigators were blinded with regard to the group of knees that were studied. the consistency of the results was verified by assessing interobserver correlations of 10 randomly chosen histological sections according to the previous report [39]. immunohistochemistry the sections were deparaffinized and immersed in 3.0% hydrogenp pperoxide for 10 min at room temperature.p pafter being washed in pbs, endogenous immunoglobulins were blocked by incubation for 30 min with 10% normal goat serum (nichirei, tokyo, japan) in pbs. the slides were washed again in pbs for 30 min and incubated with a polyclonal rabbit anti-mouse ctgf antibody (dilution 1: 100, abcam, cambridge, uk) or rabbit anti-human tgf-β1 antibody (dilution 1: 400, santa cruz biotechnology, heidelberg, germany, cat. no. sc-146) in pbs overnight at 4°c then rinsed in pbs. the slides were incubated with a goat anti-rabbit immunoglobulin antibody (1: 100, hrp conjugated, dako, copenhagen, denmark) for 30 min and the rinsed in pbs. the final detection step was carried out using 3, 3´-diaminobenzidine tetrahydrochloride (dab, sigma-aldrich corp.), 0.1 m imidazole, 0.03% hydrogen peroxidase as the chromogen for 10 min. counterstaining was with methyl green for 5 min. for negative controls, normal rabbit igg (dilution 1: 100, santa cruz biotechnology, heidelberg, germany, cat. no. sc-2027) was used as a primary antibody. cellular staining was graded as positive if specific staining was seen in slides for a given antibody and as negative if no staining or only rare cellular staining was evident [33]. p p fibroblast-like cells in the capsule were counted (two to three areas per section). the ratios of tgf-β1 and ctgf positive cells to the total number of cells counted (at least 100 cells) at high magnification (× 400) were calculated. the immunoreactivity of matrix staining was graded on a scale of 0–3 where: no staining was 0, weak staining 1, moderate staining 2, and strong staining 3 according to the previous report [24]. p p the staining intensity of endothelial cells was defined as moderate staining 2. two investigators were blinded with regard to the group of knees that were studied. the consistency of the results was verified by assessing interobserver correlations of 10 randomly chosen histological sections for the ratio of positive cells according to the previous report [59]. all the specimens were analyzed for immunoreactivity of matrix staining intensity by two independent investigators. 226 yoshihiro hagiwara et al. statistics statistical analysis among groups was performed using the kruskal-wallis test, with bonferroni/dunn post-hoc multiple comparisons. differences between the experimental and control groups were compared at each time point by mann-whitney’s u test. the interobserver coefficients were calculated with spss 15.0j (spss inc., chicago, il, usa). data were expressed as mean ± sd. a value of p < 0.05 was accepted as statistically significant. results interobserver reliability for in situ hybridization, the interobserver correlation coefficients of the total number of cells and positive cells in tgf-β1 were 0.97 and 0.99, and those in ctgf were 0.92 and 0.99, respectively. for immunohistochemistry, the interobserver correlation coefficients of the total number of cells and positive cells in tgf-β1 were 0.99 and 0.88, and those in ctgf were 0.95 and 0.96, respectively. for mean immunohistochemical scores of matrix staining intensity, the kappa coefficients were 0.88 in tgf-β1 and 0.96 in ctgf, respectively. in situ hybridization strong signals were detected in both tgf-β1 and ctgf from 3 days up to 2 weeks in the immobilized group. the signals of both molecules started to decrease after 4 weeks in the immobilized group and finally reached the control level at 16 weeks. very weak signals were observed in all the control groups (figure 2). no hybridization signal was identified with sense probes for both molecules (data not shown). the ratio of tgf-β1 positive cells was significantly greater from 3 days to 2 weeks in the anterior capsule and from 3 days to 4 weeks in the posterior capsule of the immobilized group compared with the control group. the ratio of ctgf positive cells was significantly greater from 3 days to 2 weeks in the anterior capsule and from 3 days to 8 weeks in the posterior capsule of the immobilized group compared with the control group (figure 3). in the immobilized group, there were statistically significant differences with regard to tgf-β1 expression in the anterior and posterior capsules (anterior capsule: 3 days vs. 4, 8, and 16 weeks; 1week vs. 2, 4, 8, and 16 weeks; and 2 weeks vs. 4, 8, and 16 weeks. posterior capsule: 3 days vs. 1, 4, 8, and 16 weeks; 1 week vs. 2, 4, 8, and 16 weeks; and 2 weeks vs. 4, 8, and 16 weeks) and ctgf expression (anterior capsule: 3 days vs. 1, 4, 8, and 16 weeks; 1 week vs. 8, and 16 week; 2 weeks vs. 4, 8, and 16 weeks; and 4 weeks vs. 8 and 16 weeks. posterior capsule: 3 days vs. 2, 4, 8, and 16 weeks; 1week vs. 4, 8, and 16 weeks; and 2 weeks vs. 8, 16 weeks). there was no statistically significant difference between the control group in tgf-β1 and ctgf expression at any time point. expression of transforming growth factor-β1 227 figure 2. expression of tgf-β1 and ctgf in the capsule (in situ hybridization). upper row (a-d) and lower row (e-h) show tgf-β1 and ctgf expression patterns of mrnas, respectively. a and e: the anterior capsule of the immobilized group at 1 week. b and f: the posterior capsule of the immobilized group at 1 week. c and g: the posterior capsule of the immobilized group at 8 weeks. d and h: the posterior capsule of the control group at 8 weeks. solid line square of upper right corner in each figure is a magnification of the dotted square. strong signals for both tgf-β1 and ctgf were observed both in the anterior and posterior capsules of the immobilized group at 1 week. the signals of both molecules were decreased in the posterior capsule of the immobilized group at 8 weeks. weaker signals of both molecules were detected in the control group at 8 weeks. (arrow heads indicate positive cells. scale bars = 50 μm, original magnification × 400). figure 3. the ratio of tgf-β1 and ctgf positive cells in the capsule (in situ hybridization). tgf-β1 in the anterior and posterior capsules are given in a and b, and ctgf in c and d. (*p<0.05 vs. control, p†pp<0.01 vs. control) a b c d e f g h 228 yoshihiro hagiwara et al. immunohistochemistry the ratio of tgf-β1 positive cells was higher at 1 week in the anterior capsule and for 2 weeks in the posterior capsule of the immobilized group when compared with the control group. the ratio of ctgf positive cells was higher from 1 to 4 weeks in the anterior capsule and from 1 to 8 weeks in the posterior capsule of the immobilized group when compared with the control group (figure 4). weak (1) staining of tgf-β1 was observed in the anterior and posterior capsules of the control group at any time point. on the other hand, moderate (2) staining was detected in the anterior capsule of the immobilized group, which gradually increased but not as strong as in the posterior capsule. in the posterior capsule of the immobilized group, moderate (2) to strong (3) staining was observed at 4 to 16 weeks. the immunoreactivity was significantly greater from 1 to 16 weeks in the posterior capsule of the immobilized group compared with the control group (figure 5, 6). regarding ctgf, immunoreactivity was much weaker than for tgf-β1 in the anterior and posterior capsules of the control group at all time points. weak (1) staining was observed in the anterior and posterior capsules of the control group at any time point. in the immobilized group, weak (1) staining was detected in the anterior capsule. in the posterior capsule, moderate (2) staining was detected at 1 and 2 weeks and the immunoreactivity became gradually stronger (3) at 4 to 16 weeks. the immunoreactivity was significantly greater at 1, 4, 8 and 16 weeks in figure 4. ratios of tgf-β1 and ctgf positive cells in the capsule (immunohistochemistry). (*p<0.05 vs. control, p†pp<0.01 vs. control) expression of transforming growth factor-β1 229 figure 6. immunohistochemical scores of matrix staining intensity. (*p<0.05 vs. control, p†pp<0.01 vs. control). figure 5. immunostainings of tgf-β1 and ctgf in the capsule. upper row (a-d) and lower row (e-h) show tgf-β1 and ctgf immunoreactivity, respectively. a and e: the anterior capsule of the immobilized group at 1week. b and f: the posterior capsule of the immobilized group at 1 week. c and g: the posterior capsule of the immobilized group at 8 weeks. d and h: the posterior capsule of the control group at 8 weeks. solid line square of upper right corner in each figure is a magnification of the dotted square. weak (1) immunostainings of both molecules were observed in the anterior and posterior capsules of the immobilized group at 1 week. strong (3) immunostainings of tgf-β1 and moderate (2) to strong (3) immunostaining of ctgf were detected at 8 weeks in the immobilized group. weak (1) immunostainings of both molecules were observed at 8 weeks in the control group. (scale bars = 50 μm, original magnification × 400) a b c d e f g h 230 yoshihiro hagiwara et al. the posterior capsule of the immobilized group compared with the control group (figure 5, 6). there was no staining for any of the molecules in the negative control (data not shown). in the immobilized group, the ratio of tgf-β1 positive cells differed significantly with regard to the duration of the immobilization as follows: anterior capsule; 1 week vs. 8 and 16 weeks. posterior capsule; 1 week vs. 8 and 16 weeks, 2 weeks vs. 8 and 16 weeks. as regards ratios of ctgf positive cells: posterior capsule; 1 and 2 weeks vs. 16 weeks. in the control group, tgf-β1 and ctgf ratios did not differ at any time point. within the two different groups of animals, immobilized and controls, and within the two parts of the capsule, the immunohistochemical scores for staining intensity of the two molecules were similar. discussion the components of joint contracture after immobilization have been classified into arthrogenic and myogenic ones, but the former has been considered as an important etiological factor after prolonged immobilization [28, 40]. the decrease in synovial intima length after immobilization in the same model as ours suggested that adhesion of synovium villi rather than pannus proliferation was the major pathophysiological change leading to contracture, and the posterior part was more sensitive than the anterior one [5]. our previous report has shown that sound speed transmitted through the posterior capsule, which strongly correlates with the capsular elasticity, increased significantly in the immobilized group at 8 and 16 weeks compared with the control group [1]. the sound speed change indicates that the increased elasticity may be one of the causes of limited extension after prolonged immobilization in flexion. difference in elasticity of the anterior and posterior capsules may be explained by the potential remnant mobility of the patella in the medial/lateral directions in the anterior part of the joint even after immobilization. another possible explanation would be a difference in blood flow in the stretched side and compressed side of the joint. we actually observed a limitation in extension, which rapidly progressed for 8 weeks and advanced slowly thereafter in the immobilized group [41]. whatever the reason, the fibrosis occurred mainly in the posterior capsule. tgf-β is known as a multi-functional regulator of cellular activity [42] and is also thought to be a potent regulator of wound healing, immune response and bone remodeling in vivo [43]. tgf-β is also a crucial regulator of ecm deposition, as it controls the expression of components of ecm, which includes type i collagen, type iii collagen and fibronectin in fibroblasts [44, 45]. though three structural isoforms of tgf-β (tgf-β1, 2, 3), encoded by three distinct genes, have been identified in mammals [46], tgf-β1 is the most implicated in fibrosis [31, 32]. a previous study showed high immunoreactivity of tgf-β in synovial cells of human adhesive capsulitis by immunohistochemistry [33]. the concentration of tgf-β1 in synovial fluid increased 7 days after immobilization in a rabbit immobilized knee model [34]. further, intra-articular injection of tgf-β1 neutralizing expression of transforming growth factor-β1 231 antibodies reduced adhesion in an intra-articular adhesion model in rabbits [45] and diminished fibrosis in an animal model of chronic erosive polyarthritis [47]. tgf-β1 may play an initial important role in producing a joint contracture. in our study the ratio of tgf-β1 positive cells was higher from 3 days to 2 weeks in the anterior and posterior capsules as evidenced by in situ hybridization. however, the ratio was higher in the posterior capsule of the immobilized group up to 4 weeks. in the immunohistochemical study we found that the ratio of of cells positive for tgf-β1 was higher in the posterior capsule as compared with that of the anterior capsule. stronger immunoreactivity of tgf-β1 was detected at 4 to 16 weeks in the posterior capsule of the immobilized group compared with the anterior capsule. the difference of prolonged expression of mrna and accumulation of tgf-β1 may be one of the causes of increased elasticity of the posterior capsule of the immobilized group detected by scanning acoustic microscopy [1]. though stronger immunoreactivity of tgf-β1 was observed, only slight synovial hyperplasia was detected in our study. the discrepancy in the expression pattern of in situ hybridization and immunohistochemisry can be explained as follows; in situ hybridization detects instantaneous expression of mrna in cells and immunohistochemistry detects accumulation of proteins in cells and extra-cellular matrices. proteins are produced by mrna as a template and accumulated in extra-cellular matrices. ctgf is a member of the immediate early gene family, which contains six distinct members: cef10/cyr61 (ccn1), ctgf/fisp-12 (ccn2), nov (ccn3), elm1/ wisp-1 (ccn4), cop-1/wisp-2 (ccn5) and wisp-3 (ccn6) [48]. because a tgf-β response element was found in the ctgf promoter [49], ctgf may be a potential downstream mediator for tgf-β signaling in fibroblasts and some of the actions of tgfβ1 in wound healing may be due to ctgf induction and action [50]. ctgf mrna is overexpressed in a large number of fibrotic conditions, including ssc [51], keloid [52], renal fibrosi [53], inflammatory bowel disease [54], chronic pancreatitis [55], lung fibrosis [56] and liver fibrosis [57]. subcutaneous injection of tgf-β1 into newborn mice caused a transient fibrotic response [29], which indicates that other factors besides tgf-β1 may be involved in making persistent fibrosis. subcutaneous injection of ctgf alone had little effect, but co-injection of tgf-β1 and ctgf resulted in persistent fibrosis [29]. this result suggests that ctgf may play a role in causing and maintaining skin fibrosis in collaboration with tgf-β1, which has been named “the 2-step fibrosis hypothesis in systemic scleroderma” [58]. similarly, injection of tgf-β into joints of rats induced synovial hyperplasia but its reaction was reversible [35, 36]. the enhanced expression of mrna and accumulation of proteins as tgf-β1 and ctgf in the posterior capsule of the immobilized group may support the 2-step fibrosis hypothesis. we conclude that both tgf-β1 and ctgf are likely to play an important role in causing and maintaining a joint contracture. it may be possible to prevent joint contractures by somehow blocking the fibrotic process. 232 yoshihiro hagiwara et al. acknowledgments the authors thank mr. katsuyoshi shoji and miss michiko fukuyama for technical advice. references 1. hagiwara y, saijyo y, chimoto e, akita h, sasano y, matsumoto f, kokubun s (2006) increased elasticity of capsule after immobilization in a rat knee experimental model assessed by scanning acoustic microscopy. ups j med sci 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molecular cloning of the large subunit of transforming growth factor type β masking protein and expression of the mrna in various rat tissues. proc natl acad sci usa 87:8835–8839. 44. varge j, rosenbloom j, jimenez sa (1987) transforming growth factor-β causes a persistent increase in steady-state amounts of type i and type iii collagen and fibronectin mrnas in normal human dermal fibroblasts. biochemistry 297:597–604. 45. roberts ab, sporn mb (1993) physiological actions and clinical applications of transforming growth factor-β. growth factors 8:1–9. 46. fukui n, tashiro t, hiraoka h, oda h, nakamura k (2000) adhesion formation can be reduced by the suppression of transforming growth factor-β1 activity. j orthop res 18:212–219. 47. wahl sm, allen jb, costa gl, wong hl, dasch jr (1993) reversal of acute and chronic synovial inflammation by anti-transforming growth factor beta. j exp med 117:225–230. 48. inkinen k, wolff h, lindroos p, ahonen j (2003) connective tissue growth factor and its correlation to other growth factor in experimental granulation tissue. connect tissue res 44:19–29. 49. grotendorst gr, okochi h, hayashi n (1996) a novel tgf-β response element controls the expression of the connective tissue growth factor (ctgf) gene. cell growth differ 7:469–480. 50. grotendorst gr (1997) connective tissue growth factor: a mediator of tgf-beta action on fibroblasts. cytokine growth factor rev 8:171–179. 51. igarashi a, nashiro k, kikuchi k, sato s, ihn h, grotendorst gr (1995) significant correlation between connective tissue growth factor gene expression and skin sclerosis in tissue sections from patients with systemic sclerosis. j invest dermatol 105:280–284. 52. igarashi a, nashiro k, kikuchi k, sato s, ihn h, fujimoto m, grotendorst gr, takehara k (1996) connective tissue growth factor gene expression in tissue sections from localized scleroderma, keloid and other fibrotic skin disorders. j invest dermatol 106:729–733. 53. ito y, aten j, bende rj, oemar bs, rabelink tj, weening jj, goldschmeding r (1998) expression of connective tissue growth factor in human renal fibrosis. kidney int 53:853–861. 54. dammeier j, brauchle m, falk w, grotendorst gr, werner s (1998) connective tissue growth factor. a novel regulator of mucosal repair and fibrosis in inflammatory bowel disease? int j biochem cell biol 30:909–922. 55. di mola ff, friess h, martignoni me, di sebastiano p, zimmermann a, innocenti p, graber h, gold li, korc m, buchler mw (1999) connective tissue growth factor is a regulator for fibrosis in human chronic pancreatitis. ann surg 230:63–71. 56. lasky ja, ortiz la, tonthat b, hoyle gw, corti m, athas g, lungarella g, brody a, friedmann m (1998) connective tissue mrna expression is upregulated in bleomycin-induced lung fibrosis. am j physiol 275:365–371. 57. paradis v, dargere d, vidaud m, de gouville ac, huet s, martinez v, gauthier jm, ba n, sobesky r, ratziu v, bedossa p (1999) expression of connective tissue growth factor in experimental rat and human liver fibrosis. hepatology 30:968–976. 58. takehara k (2003) hypothesis: pathogenesis of systemic sclerosis. j rheumatol 30:755–759. corresponding author: yoshihiro hagiwara department of orthopaedic surgery tohoku university graduate school of medicine sendai 980-8574, japan tel: +81-22-717-7245 fax: +81-22-717-7248 e-mail: hagi@mail.tains.tohoku.ac.jp upsala j med sci 99: 121-130, 1994 inhibition of fibrinogen binding to platelets by mk-852, a new gpiib/iiia antagonist anders larsson' and tomas l. lindahl' 'department of clinical chemistry, university hospital, uppsala and 2department of clinical chemistry, university hospital, linkoping, sweden abstract mk-852 is a newly developed low molecular weight inhibitor of fibrinogen binding to platelets. platelet aggregation and adhesion of platelets to damaged vessel walls are critical events in haemostasis, and uncontrolled aggregation may cause arterial thrombus formation. depending on the location of the occluded vessel, this may result in unstable angina, myocardial infaction or stroke. platelet aggregation requires binding of fibrinogen to the gpiib/iiia receptor on the platelet surface. thus, inhibitors of fibrinogen binding to the receptor may constitute an efficient way of preventing thrombus formation. we have used flow cytometty and fitc-labelled chicken anti-human fibrinogen antibodies to study the in vitro inhibitory effects of mk-852 on fibrinogen binding to platelets. we show that mk-852 is a very efficient fibrinogen receptor antagonist in vitro. flow cytometty is well suited for clinical use and may be used to monitor treatment with mk-852 or other fibrinogen receptor antagonists. introduciion the use of anti-platelet drug has transformed the treatment of ischemic heart disease (2). today, aspirin is routinely given to patients with acute myocardial infarction and unstable angina. however, aspirin does not completely inhibit platelet aggregation. during the last few yean several research groups have instead developed antagonists to the gpiibma receptor. fibrinogen binding to the platelet receptor gpiibmia has a very important role in platelet aggregation, and an inhibitor to this receptor may thus inhibit platelet aggregation and thromb formation. the first antagonist tested in patients was a murine monoclonal antibody known as 7e3 (3). this antibody has later been modified several times to reduce antigenicity (6). the results obtained with this antibody in ischemic heart disease and during coronary angioplasty has been very promising (19). the results 121 have lead to the development of several low molecular weight fibrinogen receptor blockers (l)., some of these antagonists are currently beiig evaluated in clinical studies. in the present work we have used fluorescence-activated flow cytometry (facs) and chicken anti human fibrinogen-fitc to study the inhibitory effects of a new fibrinogen receptor antagonist (mk-852) (20) on adp and immune complex induced platelet activation. flow cytometry gives single-cell data, allowing the detection of a few activated platelets (15). chicken antibodies are superior to mammalian antibodies for the measurement of platelet bound plasma proteins as they do not induce complement activation (10) or platelet activation (1 1). materials and methods reagents. mk-852 was generously provided by merck, sharp & dohme (west point, pa, usa). adenosine5'-diphosphate was obtained from boehringer-mmnheim (mannheim, germany). all other chemicals were of reagent grade, most purchased fiom merck @armstad4 germany). antibodies. fluorescein vitc) labelled chicken anti-human fibrinogen was obtained from biopool al3 (urn& sweden). rabbit anti-human iga was purchased from dakopatts as (glostrup, denmark). blood sampling. venous blood was obtained from healthy volunteers who had taken no medication for at least 10 days. all volunteers gave informed consent before blood sampling. blood was obtained fiom an antecubital vein without a tourniquet and the blood was collected in 5 r d sodium citrate tubes (367704, becton dickinson, rutherford, nj). platelet-rich plasma was isolated by centdbgation at 140 x g for 10 minutes at room temperature. the concentration of platelets, size and purity were analyzed by a coulter stks cell counter (coulter diagnostics, hialeah, fl). ge&ltr&.on of platelets. 1 ml of platelet-rich plasma was separated on a sepharose s-1000 c o l m (1.5~9 cm) equilibrated with hepes buffer. the grst peak contained the platelets. the number of platelets, size and purity were analyzed by a cell counter. preparation of samples forflow qtomeby. 2.5 pl platelet-rich plasma or 10 pl gel-filtered platelets were added to polystyrene tubes containing 50 pl hepes-buffer (137 m o v l nacl, 2.7 mmovl kcl, 1 mmovl mgcl, 5.6 mmol/l glucose, 1 s/l bovine serum albumin, and 20 mmol/l hepes, ph 7.40) containing varying amounts of mk-852. in some experiments adp was added 122 to the tubes to cause platelet activation and in some experiments 5 pl rabbit anti-human iga was added to the tubes to form immune complexes. samples were incubated for 10 min at room tempe rature followed by addition of 50 pl chicken anti-human fibrinogen-fitc, diluted 1 :10 in hepes buffer. samples were incubated for another 10 min at room temperature. the samples were then diluted and fmed with 500 pl ice-cold pbs containing 1% pformaldehyde. washing was tried but caused losses of cells without improving the analysis. therefore washing steps were not used. the anti-fibrinogen antibody was tested at different dilutions and subsequently used at optimal concentrations to achieve maximal binding. flow cytomeq. labelled platelets were resuspended for analysis by epics profile i1 cytometer (coulter electronics, hialeah, fl). the epics profile i1 cytometer was equipped with a 15 mw air-cooled 488 nm argon laser. forward and side scatter and green (fitc) and red (phycoeryhm) signals were acquired with logarithmic amplification, with a 525 nm and 575 nm bandpass filter for collection of fitc and pe signals, respectively. acquisition and processing of data from 10,000 cells were carried out with the epics elite flow cytometry s o h a r e (coulter electronics, hialeah, fl). based on light scattering properties, each cell is represented by a point in a rectangular coordinate system. the instrument was calibrated for fluorescence and light scatter using "immunocheck" beads and "standard brite" beads (coulter electronics, hialeah, fi., usa). based on light scattering properties, each cell is represented by a point in a rectangular coordinate system. a dis crimination h e is placed around the platelet cluster utilising forward and side scatter. analytical markers were set in the fluorescence channel to divide the negative control sample into two fractions containing 95-97% of the platelets and the brightest 3-5% of the platelets. those platelets with fluorescence greater than the marker were identified as positive events. results inhibition of adp inducedjibrimgen binding toplatelets by m-852. the platelets were activated with varying concentrations of adp (0,o.l pmol/l, 1 pmoiil, 10 pmol/l, 100 pmol/l) in the presence of 1 m a of mk-852 or only hepes-buffer. mk-852 was very efficient in inhibiting the adp induced fibrinogen binding (fig. 1). tne fibrinogen binding in the presence of mk-852 was not significantly different fi-om samples containing 10 mmol/l of edta, the binding of fibrinogen to the receptor is ca2+ dependent. edta was used as a negative control as it inhibits the bmding of fibrinogen to gpiibaiia (1 7). 123 -4with mk-852 -0without mk-852 figure 1. inhibition of adp induced fibrinogen binding to platelets by mk-852. the results are expressed as percentage of fibrinogen positive platelets in the presence of varying concentrations of adp. the plasma fibrinogen concentration was 2.4 g/l. platelets were also activated with 100 pmol/l of adp in the presence of varying mk-852 concentrations. 100 pg/l of mk-852 was very efficient in inhibiting fibrinogen binding (fig. 2). the binding was not significantly different ffom samples containing 10 mmovl of edta. 10 pa of mk-852 gave only a limited reduction in the percentage of fibrinogen positive platelets in comparison with only hepes-buffer. however, the reduction in the mean fluorescence intensity was more pronounced. this indicates that there was a reduction in the amount of fibrinogen bound to each platelet with 10 p g l of mk-852. m > ..y v1 0 a i % m $ a i i i 0 10 100 1000 10000 0 1 concentration of mk-852 ( p g l ) fig. 2. inhibition of adp induced fibrinogen binding to platelets by varying concentrations of mk-852. the results are expressed as percentage of fibrinogen positive platelets in the presence of 100 pmovl of adp. 124 displacement of boldfibrinogen by m-852. platelets were activated with 100 p m o l of adp. after 10 min, mk-852 was added in various concentrations to displace bound fibrinogen. bound fibrinogen was detected with chicken anti-human fibrinogen-fitc. 100 p g l of mk-852 was very efficient in inhibiting fibrinogen binding while 10 pa of mk-852 gave only a limited reduction in the percentage of fibrinogen positive platelets in comparison with only hepes-buf€er (results not shown). the results were not significantly different fiom the results obtained when mk-852 was added before adp. thus, mk-852 could displace platelet bound fibrinogen. 2 60 ? s ,% 40 y l .a c 0. 00 . $ 20 * 2 ll. " 0 e efect of jbrinogen concentration on jibrinogen binding. gelfiltered platelets were mixed with various volumes of platelet poor plasma. 20 or 100 pa of mk-852 (final concentration) were added and the platelets were activated with 100 pmol/l of adp. with 20 pa of mk-852 there was a slightly increased percentage of fibrinogen positive platelets when the fibrinogen concentration was increased &om 1.2 gl to 10 a. with 100 p g l of mk-852 there was no difference in the percentage of fibrinogen positive platelets (results not shown). :\. w m i i i ii;; inhibition of immune complex inducedfibrinogen binding to platelets by m-852. rabbit anti human iga was added to prp to form immune complexes and platelet activation was detected with antibodies a-&t fibrinogen. the immune complexes increased the binding of anti fibrinogen. this binding was inhibited by mk-852 in a dose dependent manner (fig. 3). 10 pa of mk-852 gave only a limited reduction in the percentage of fibrinogen positive platelets while i 30 125 the bitmap gating was set to exclude immune complexes (9). however some immune complexes had the same forward and side scatter as platelets and were thus analysed. the immune complexes bind some anti-fibrinogen antibody resulting in an increased percentage of positive events. this probably explains the higher percentage of fibrinogen positive particles in the presence of mk-852 in the experiment with immune complex activation than in the experiment with adp. positive particles could also be detected in platelet poor plasma when immune complexes were present in the samples suggesting that the positive particles were anti-iga-iga complexes. discussion platelets contribute to normal haemostasis by adhering to damaged endothelial surfaces and then aggregate at the site of damage. if the aggregation is not inhibited, a thrombus is formed that may occlude the vessel or cause embolization. platelet adhesion is mediated by several platelet receptors that interact with adhesion molecules in the damaged area. in contrast to adhesion, platelet aggregation is mediated only by the platelet gpiibhiia receptor, which is found only on platelets and megakaryocytes. the number of gpiibhiia receptors in the platelet membrane are extremely large (approximately 30,000-so,ooo), making it one of the most dense receptors for adhesion and aggregation of any cell. when the platelet is activated, there is a structural change in the gpiibxiia receptor (16). upon activation, the receptor will bind several different ligands, including fibrinogen, fibronectin, vitronectin and von willebrand factor. at physiological protein concentrations it is mainly fibrinogen that is bound, which has a dimeric structure that allows the interaction with two platelets leading to platelet aggregation. it has been shown that the binding of fibrinogen to the gpiibhiia receptor is essential for thrombus growth (13). therefore, the blockade of gpifoima might be a superior approach in preventing arterial thrombus formation (2). like other members of the integrin family, gpiib/iiia contains a recognition site for the peptide sequence arg-gly-asp (rgd) (3,4). several substances that block this site have been tested, including monoclonal antibodies (2), polypeptides isolated fiom leeches (14) or snake venoms (5). recently several peptides have been developed that block the rgd site. one of theses inhibitors is mk-852 that currently is being evaluated in a phase ii trial of unstable angina. peptides that block the rgd site have theoretical advantages to monoclonal antibodies (19). the peptides are usually more rapidly cleared which allows for a fmer titration and the rapid clearance is an advantage if bleeding problems occut as the inhibition will cease quickly once the idision is stopped. the antigenicity of a peptide is less than for a monoclonal antibody which will eliminate the problems with human 126 anti-mouse igg antibodies associated with in vivo use of monoclonal antibodies. we used chicken antibodies to detect fibrinogen binding as these antibodies are superior to mammalian antibodies for the estimation of platelet bound plasma proteins. chicken antibodies do not induce complement activation (10,12) or platelet activation (1 1,12). the results show that mk 852 is an effective blocker of adp-induced fibrinogen binding to platelets. 10 pg'l of mk-852 or larger concentrations reduced the binding of fibrinogen to platelets. we also wanted to see if mk-852 could inhibit immune complex mediated fibrinogen binding. circulating immune complexes (cic) can be found in many diseases such as autoimmune diseases, malignancies and infectious diseases (18) and cic are known to play a pathogenic role in autoimmune diseases. there is also an increased risk of thrombosis and thrombocytopenia associated with these diseases (17). we have previously shown that immune complexes containing mammalian antibodies are efficient promoters of platelet activation measured as fibrinogen binding or microparticle formation (8,9). activated platelets are rapidly cleared fi-om the circulation, which may contribute to the thrombocytopenia in these patients. we show that mk-852 can block cic mediated fibrinogen binding to platelets. further studies have to be performed to see if mk-852 also can reduce the incidence of thrombosis and thrombocytopenia in patients with cic. in this study we show that mk-852 is a very efficient fibrinogen receptor antagonist in vitro and that flow cytometry can be used to monitor the inhibitory effects of mk-852 on fibrinogen binding to platelets. mk-852 was kindly donated by merck, sharp & h h m e (west point, pa, usa). this work was supported by the swedish society of medicine, the swedish medical research council (project no. 13x-9875) and rik,s%rbmdet mot reumatism. reference3 alig l, edenhofer a, p, hurzeler h4, knopp d, muller m, steiner b, tneciak a & weller t. low molecular weight, non-peptide 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flow cytometric analysis. platelets 1993; 4: 73-77. phillips dr, char0 if, parise lv & fitzgerald la. 'ihe platelet membrane glycoprotein iib iiia complex blood 1988; 71: 831-843. seymour jl, h e m 1 wj, nevins, b, stults jt & lazarus ra. decorsin, a potent glycoprotein rrpma antagonist and platelet aggregation inhibitor from the leech mhcrubdelfa decora. j biol chem 1990; 265: 10143-10147 shattil s j, cunningham m & hoxie j k detection of activated platelets in whole blood using activation dependent monoclonal antibodies and flow cytometry. blood 1987; 70: 307 315. shattil s j, hoxie j a, cunningham m & brass l f. changes in platelet membrane glycoprotein iibdiia complex during platelet activation. j biol chem 1985; 260: 11 107-14. 128 17. 18. 19. 20. steiner b, cousot d, tneciak a, gillessen d & hadvw p. ca2+-dependent binding of a synthetic arg-gly-asp (rgd) peptide to a single site on the purified platelet glycoprotein iibhiia complex. j biol chem 1989; 264: 13102-13108 theofilopulos a n & dixon f j. immune complexes in human diseases. am j path01 1980; 100: 530-591. topol i3 & plow ef. clinical trials of platelet receptor inhibitors. thromb haemost 1993; 70: 94-98 yuan as, hand el, hichens h4, olah tv, banish a, femandez-metzler c & gilbert jd. determination of mk-852, a new fibrinogen receptor antagonist, in plasma and urine by radioimmunoassay. j pharm biomed anal 1993; 1 1 : 427-434 ofyprint requests to: anders larsson department of clinical chemistry, university hospital, s-751 85 upp~alq sweden 129 upsala j med sci 100: 41-46, 1995 torsten teorell, the father of pharmacokinetics lennart k. paalzow department of biopharmaceutics & pharmacokinetics, uppsala university, box 580, s751 23 uppsala, sweden during t h e summer o f 1937 t o r s t e n t e o r e l l s u b m i t t e d two s c i e n t i f i c p a p e r s (1,2) f o r p u b l i c a t i o n which a p p e a r e d i n a r c h i v e s i n t e r n a t i o n a l e s e t pharmacodynamie e t de t h e r a p i e i n october i n t h e s a m e y e a r . more t h a n 30 years l a t e r t h e s e two a r t i c l e s came t o be c o n s i d e r e d a s two of t h e most i m p o r t a n t c o n t r i b u t i o n s t o drug r e s e a r c h and l e d t o t e o r e l l b e i n g c h a r a c t e r i z e d a s t h e f a t h e r of p h a r m a c o k i n e t i c s . both t h e s e p a p e r s had t h e same t i t l e , " k i n e t i c s of d i s t r i b u t i o n of s u b s t a n c e s a d m i n i s t e r e d t o t h e body", and t h e f i r s t one d e a l t w i t h e x t r a v a s c u l a r modes of a d m i n i s t r a t i o n (l), w h i l e t h e second one d i s c u s s e d t h e i n t r a v a s c u l a r modes ( 2 ) . i n t h e i n t r o d u c t i o n t o t h e f i r s t p a p e r t e o r e l l p o i n t e d o u t t h a t a t t h a t t i m e t h e i n t e r e s t of t h e p h y s i c i a n o r t h e p h y s i o l o g i s t was f o c u s e d more on p r a c t i c a l p o i n t s such a s t e s t i n g t h e p r o p e r dosage o r s u i t a b l e ways of a d m i n i s t r a t i o n , o r on t h e f i n e r mechanism i n v o l v e d i n t h e e f f e c t s produced by t h e drug, whereas v e r y l i t t l e work seemed t o have been devoted t o . t h e k i n e t i c s , i . e . t h e t i m e r e l a t i o n s o f drug a c t i o n . the o b j e c t i v e s of t h e two p a p e r s by t e o r e l l were t o d e r i v e " g e n e r a l mathematical r e l a t i o n s from which it i s p o s s i b l e , a t l e a s t f o r p r a c t i c a l p u r p o s e s , t o d e s c r i b e t h e k i n e t i c s of d i s t r i b u t i o n of s u b s t a n c e s i n t h e body" and t o p r e s e n t t i m e c o n c e n t r a t i o n c u r v e s a s i l l u s t r a t i o n s of t h e r e l a t i o n s d e r i v e d . to d e s c r i b e what happens t o a drug i n t h e body when it i s a d m i n i s t e r e d by an e x t r a v a s c u l a r mode, e . g . by t h e subcutaneous r o u t e , t e o r e l l made a s i m p l i f i e d model of t h e body a s i l l u s t r a t e d i n f i g . 1. blood i s c i r c u l a t e d t h r o u g h o u t t h e body and can be i l l u s t r a t e d 41 by a c i r c l e of a s water p i p e l i n e i n c o n t a c t w i t h t h e t i s s u e s of t h e body. each t i s s u e h a s a c e r t a i n volume, and as s e e n i n t h e f i g u r e a drug i s t r a n s p o r t e d by d i f f u s i o n from t h e subcutaneous t i s s u e t o t h e blood ( a b s o r p t i o n p r o c e s s w i t h t h e r a t e c o n s t a n t k, ) and t h e n c i r c u l a t e d throughout t h e body. the p r o c e s s e s of t r a n s p o r t from t h e blood t o t h e d i f f e r e n t t i s s u e s a r e d e s c r i b e d by t h e r a t e c o n s t a n t i n t o t h e t i s s u e and by a n o t h e r r a t e c o n s t a n t o u t of i t . some o r g a n s , such a s t h e l i v e r and t h e kidney, have t h e c a p a c i t y t o e l i m i n a t e t h e drug and t h e y a r e i l l u s t r a t e d by one r a t e c o n s t a n t o u t from t h e blood ( f i g . 1 ) . f i g . 1. from t o r s t e n t e o r e l l r e f . 1. t e o r e l l t h e n d e r i v e d t h e d i f f e r e n t i a l e q u a t i o n s f o r t h e s e p r o c e s s e s and p r e s e n t e d t h e i r s o l u t i o n s . b y g i v i n g t h e r a t e c o n s t a n t s and volumes c e r t a i n p o s s i b l e v a l u e s , he c a l c u l a t e d t h e t i m e c o u r s e of drug amounts i n t h e body, e x p r e s s e d a s p e r c e n t of t h e dose, a s i l l u s t r a t e d i n f i g . 2 . a s s e e n i n t h i s f i g u r e , t h e r a p i d a b s o r p t i o n a f t e r a subcutaneous dose produces a peak c o n c e n t r a t i o n i n t h e b l o o d 42 t h a t d e c l i n e s e x p o n e n t i a l l y o v e r t i m e , w h i l e t h e c o n c e n t r a t i o n i n t h e t i s s u e , which most p r o b a b l y i n c l u d e s t h e s i t e of a c t i o n , p e a k s l a t e r t h a n t h a t i n t h e b l o o d b u t t h e n d e c l i n e s i n p a r a l l e l w i t h t h e c o n c e n t r a t i o n i n t h e b l o o d . t h e e l i m i n a t i o n c u r v e and t h e amount of d r u g i n t h e s u b c u t a n e o u s d e p o t a r e a l s o i l l u s t r a t e d i n f i g . 2 . f i g . 3 typical case of extravascular adtrii ttistratiotr in the absence of tissue in activation. (k, = 0.2; k2 = 0.01; k, = 0.005; i.e. " blood " volume/" tissue " volume is i : 2; k, = 0 . 0 0 5 ; k, = 0 ) . f i g . 2 . from t o r s t e n t e o r e l l r e f . 1. f u r t h e r m o r e , by p e r f o r m i n g s e v e r a l n u m e r i c a l c a l c u l a t i o n s , t e o r e l l emphasized t h a t a marked change i n t h e a b s o r p t i o n 43 p r o p e r t i e s may b r i n g a b o u t a marked change i n t h e m a g n i t u d e and d u r a t i o n o f t h e b l o o d and t i s s u e c o n c e n t r a t i o n c u r v e s , a n d s t a t e s t h a t " t h e s e and o t h e r c o n c l u s i o n s may have b e a r i n g s upon p r a c t i c a l pharmacology and t h e r a p e u t i c s " . these c o n c l u s i o n s may t o d a y seem s e l f e v i d e n t , b u t o n e h a s t o r e c a l l t h a t a t t h a t t i m e nobody had r e a l l y t h o u g h t t h a t w e c o u l d t r e a t t h e d i s t r i b u t i o n o f d r u g s i n t h e body i n s u c h a s i m p l i s t i c a n d c l e a r way. a l l t h e c o n c l u s i o n s p r e s e n t e d by t e o r e l l a r e c e r t a i n l y s t i l l v a l i d and a t t h a t t i m e t h e y s h o u l d h a v e been a n e y e o p e n e r t o t h o s e who w e r e u s i n g d r u g s b u t who c l e a r l y w e r e unaware o f what c a n happen t o a d r u g i n t h e body. however, t h e s e t w o p a p e r s of t e o r e l l p r o d u c e d t h e o p p o s i t e e f f e c t and t h e r e v i e w e r s o f t h e a p p l i c a t i o n s f o r t h e c h a i r i n p h y s i o l o g y , which t e o r e l l o b t a i n e d i n 1 9 4 0 , r a t h e r c o n s i d e r e d t h e s e two p a p e r s more a s a b u r d e n t h a n a m e r i t . c o n s e q u e n t l y , t h e y w e r e f o r g o t t e n a n d n o t h i n g r e a l l y happened f o r more t h a n 2 5 y e a r s , when german a n d e s p e c i a l l y u.s. s c i e n t i s t s r e d i s c o v e r e d t e o r e l l ' s work. the word p h a r m a c o k i n e t i c s was i n t r o d u c e d by t h e german p r o f e s s o r f . h . dost i n 1953 a n d i n a r e v i e w a r t i c l e ( 3 ) by john g . wagner ( 1 9 8 1 ) on t h e h i s t o r y o f p h a r m a c o k i n e t i c s w e c a n read t h e f o l l o w i n g : " i n 1937, t e o r e l l , a swedish p h y s i o l o g i s t a n d b i o p h y s i c i s t , p u b l i s h e d two r e m a r k a b l e a r t i c l e s which many now a t t r i b u t e a s b e i n g t h e f o u n d a t i o n s of modern p h a r m a c o k i n e t i c s " . thanks t o t h e work o f v a r i o u s g r o u p s , f o r example s i d n e y riegelman, b e r n a r d b . b r o d i e , e i n o n e l s o n , g e r h a r d levy, john wagner a n d o t h e r s i n t h e u.s.a., and by europeans s u c h a s ekkehard xruger-thiemer, e . j . a r i e n s a n d j a c q u e s v a n rossum, p h a r m a c o k i n e t i c s q u i c k l y d e v e l o p e d d u r i n g t h e 1 9 6 0 ' s . one i m p o r t a n t c o n t r i b u t o r y f a c t o r was t h e r a p i d a d v a n c e s i n b i o a n a l y t i c a l t e c h n i q u e s , which made it p o s s i b l e t o a n a l y s e m i n u t e d r u g c o n c e n t r a t i o n s i n plasma, u r i n e and o t h e r t i s s u e s . d u r i n g t h i s p e r i o d t h e s o c a l l e d c o m p a r t m e n t a l models were u s e d f o r t h e m a t h e m a t i c a l i n t e r p r e t a t i o n o f d a t a . the main drawback o f t h i s a p p r o a c h w a s a p p a r e n t a t l e a s t t o t h o s e who were g o i n g t o u t i l i z e t h e f i n d i n g s , i . e . t h e p h y s i c a n s , who h a d d i f f i c u l t i e s i n u n d e r s t a n d i n g t h e meaning o f t h e i n f o r m a t i o n p r o d u c e d . 44 i n t h e b e g i n n i n g of t h e 1 9 7 0 ' s a new e r a of p h a r m a c o k i n e t i c s began when t h e r e s e a r c h became more o r i e n t e d towards a p h y s i o l o g i c a l approach. with t h e i n t r o d u c t i o n of t h e c l e a r a n c e concept by rowland e t a l . ( 1 9 7 3 ) , a b e t t e r u n d e r s t a n d i n g of p h y s i o l o g i c a l f a c t o r s s u c h a s blood flow, h e p a t i c m e t a b o l i c c a p a c i t y and plasma p r o t e i n b i n d i n g was a c h i e v e d ( 4 ) . during a b o u t t h e same time p e r i o d , a n o t h e r t y p e of p h a r m a c o k i n e t i c model appeared i n t h e l i t e r a t u r e . b i s c h o f f and dedrick ( 1 9 6 8 ) d e s c r i b e d what t h e y c a l l e d a p h y s i o l o g i c a l flow model, which t o o k i n t o account t h e i n f l u e n c e of t h e blood flow and b i n d i n g o f d r u g s i n d i f f e r e n t t i s s u e s of t h e body ( 5 ) . the e l e g a n t t h i n g about t h e s e models was t h a t it was p o s s i b l e t o s c a l e them up from a n i m a l s t o humans by changing, f o r example, t h e magnitude of animal t i s s u e blood flows and t h e e l i m i n a t i o n c a p a c i t y t o t h a t found i n m a n . by t h i s way t h e human s i t u a t i o n c o u l d be p r e d i c t e d from t h e outcome of a n a n i m a l e x p e r i m e n t . once a g a i n t o r s t e n t e o r e l l ' s work was r e d i s c o v e r e d , s i n c e it was obvious t h a t t h e model t e o r e l l had used w a s a l s o t h e f i r s t p h y s i o l o g i c a l model, and i n h i s honour a c o n f e r e n c e was a r r a n g e d i n 1 9 7 2 a t t h e f o g e r t y i n t e r n a t i o n a l c e n t e r a t n i h i n bethesda, which he a t t e n d e d and where h i s achievements were r e c o g n i z e d ( 6 ) . during t h e l a s t two decades p h a r m a c o k i n e t i c s h a s advanced e x p l o s i v e l y and t o d a y it i s one of t h e most q u i c k l y d e v e l o p i n g b r a n c h e s of s c i e n c e , e s p e c i a l l y of t h e p h a r m a c e u t i c a l s c i e n c e s , and it p r o v i d e s u s w i t h a fundamental knowledge upon which modern d r u g t h e r a p y r e s t s . p e r s o n a l l y , i know t h a t t e o r e l l was p l e a s e d w i t h t h e f a c t t h a t uppsala u n i v e r s i t y became t h e f i r s t u n i v e r s i t y i n t h e nordic c o u n t r i e s t h a t e s t a b l i s h e d a c h a i r i n p h a r m a c o k i n e t i c s . i t h a s always been a p r i v i l e g e and an honour f o r m e t o h o l d t h i s p o s i t i o n a t a u n i v e r s i t y t h a t i s known a l l o v e r t h e world a s t h e p l a c e a t which t o r s t e n t e o r e l l , t h e f a t h e r o f p h a r m a c o k i n e t i c s , was working. there i s no r i s k t h a t h i s name w i l l be f o r g o t t e n . 45 r e f e r e n c e s . 1. teorell, t.: kinetics of distribution of substances administered to the body. i. the extravascular modes of administration. arch int pharmacodyn et ther 57: 205-225, 1937. 2. teorell, t.: kinetics of distribution of substances administered to the body. 11. the intravascular modes of administration. arch int pharmacodyn et ther 57: 226-240, 1937. 3. wagner, j.g.: history of pharmacokinetics. pharmacol ther 12: 537-562, 1981. 4. rowland, m., benet, l.z. & graham, c . g . : clearance concepts in pharmacokinetics. j pharmacokin biopharm 1: 123-136, 1973. 5. bischoff, k.b. & dedrick, r.l.: thiopental pharmacokinetics. j pharm sci 57: 1347-1357, 1968. 6. teorell, t., dedrick, r.l. & condliffe, p . e . : pharmacology and pharmacokinetics, fogerty international center proceedings no 20, plenum, new york, 1974. 46 upsala j med sci 100: 5-10, 1995 torsten teorell, the teacher and researcher address of welcome martin h:son holmdahl department ?f anaesthesiology, uniuersity hospital of uppsala, sweden dear ann and eva, dear all torsten teorell’s colleagues and friends. we have gathered for this memorial symposium to remind us of torsten teorell’s scientific achievement. let us rather say that we have gathered in grateful remembrance of what teorell meant to students of all levels within the medical faculty of uppsala as a teacher, researcher and friend during more than half a century of busy activity and for each and every one of us as it relates to our own development. memory alive today and for many years to come by refemng to his still valid theories and methods. at this symposium arranged by the oldest research member, karl-johan obrink colleagues are presenting some of those research themes that teorell initiated. we shall review and generally discuss teorell’s scientific contributions. we are particularly pleased that his daughter eva will give us a very close personal view of his creativity. when we gathered during the summer of 1980 for a symposium on ion and water movements in membranes (i) to celebrate the then 75 year old teorell, he himself contributed actively with an elegant review. at that time i had the opportunity as rector (president) of the university to honour my teacher as one of the university’s internationally most renowned researchers during the scientifically thriving period following world war 11. today, i wish to let teorell himself elucidate his own achievements as a most vital and innovative scientist who in 1940 initiated a 30 year headship of the physiological institution, a particularly flourishing period of the old building known as the regnellianum. in teorell’s obituary in august 1992, we could cite from his teacher ejnar hammarsten’s expert opinion, presented when in 1938 teorell applied for, was called to and received the appointment as associate professor of medical chemistry in uppsala, the following prophetic words: the national and international research community will keep teorell’s 5 "a combination of theoretic brilliance and particularly great technical skill are characteristics of teorell. alwdys prepared to use numerous, simultaneously applied, methods, partly developed by himself, he gives a multifacetted illustration of each problem. his solid and diverse approach in an increasingly obvious way has aimed at demonstrating the importance of electric effects on cell processes with regard to generation of information and magnitude, capacity, speed, location and orientation of various cell functions". when teorell later as a teacher of medical chemistry applied for and was appointed professor of physiology, as expected, this did not occur without a struggle. academic quarrels as a rule are not scientifically promoting and the petitions submitted are hardly constructive. however, this time i have found an exception which includes many correct predictions of scientific developments giving a picture of teorell as a scientist and scholar with which we, as his later colleagues, can agree without reservation. most of you are familiar with that background. three prominent physiologists did not wish to nominate teorell for the position, while the fourth, a pharma cologist, put him in the first place. however, even the three physiologists indicated his exceptional qualifications as a primarily biologically and physico-chemically oriented researcher, but considered his expertise to lie outside the area of the subject of physiology. directed towards a major physiological problem, i.e. the mechanism of hydrochloric acid secretion in the stomach. in order to accomplish a profound scientific attack on this problem, i made time consuming preparations designing suitable analytical methods. for the same purpose, in 1930, i undertook an educational trip to a reputable physiologic laboratory in london, because the necessary surgical technique in animals for work on surviving organs could not be studied at any swedish institution. the result of these joint efforts was a medical thesis in 1933 which was of a physiological nature. t h s work was awarded the "laudable" grade by the medical faculty at the karolinska institute. the mechanism of hydrochloric acid generation remained unexplained, but a number of other related questions received renewed elucidation. while working on these physiological problems, the opinion grew increasingly stronger that a researcher who wishes to devote himself to any secretion problems in the body must have fundamental knowledge in basic science, e.g. chemistry and especially physical chemistry, of greater depth than what current physiological education provides. therefore, over several years, to the best of my ability i tried to acquire increased knowledge in these areas. my method has largely included utilization of suitable results from my medical in one petition teorell himself states: "early on, my main interest was 6 thesis and elaboration on these in a physico-chemical direction. in this way many publications have been accomplished which deal with diffusion problems and ion distribution. in spite of their physico-chemical back ground, these works are intimately connected with physiology. they all originate from physiological questions and are intended to be connected to such problems. as this knowledge has increased, quite naturally the involved field of vision has widened. the physiological secretion problems have been found to be parts of a more general problem complex which includes tissue and cell permeability and the related bio-electricity. against this background one should view the ”paraphysiologic” (lundsgaard) publications of recent years which deal with characteristics and electric potentials of cell membranes and surfaces. this is true also for my visit to cambridge in 1937 to study surface chemistry. thus i must conclude that my development and my aims have not directed me away from physiology. on the contrary, my intentions have been to undertake physiological research in the most profound interpretation of this area using methods which sooner or later must become more commonly utilized”. today, the importance of teorell’s physical and chemical basic knowledge in cell biology to explain life processes is obvious and has made him a pioneer within modern physiology or integrated cell and molecular biology as this field is referred to today in the united states. it is an honour to our medical faculty that this was realized already in january 1940, when teorell was considered the most suitable candidate for this prominent position. a special report by arne tiselius contributed significantly to this. in this statement he included opinions which agree completely with the impression that we colleagues had of teorell as our teacher, scholar and researcher. i would wish to quote the following from tiselius’ report. are particularly important for physiology, as demonstrated by teorell, seems to me to be scientifically fundamental to the interested physiologist and extremely desirable as something which will become a necessity in the not too distant future”. there are also other opinions expressed in tiselius’ report which particu larly well represent the picture of the enthusiastic , creative teacher we all knew a t the regnellianum. teorell‘s remarkable printed discussion at a n international symposium is commented upon by tiselius in the following way. ”teorell’s exceptional ability to contribute invaluably to discussions at lectures, symposia and seminars and his creativity, ability to rapidly grasp new concepts and fast moving mind are particularly obvious”. certainly, a doctoral candidate in teorell’s department of physiology who might have lost his track among the many research problems, might have ” a profound knowledge of those components of physical chemistry that 7 wanted to receive a more rigid and structured guidance instead of a wealth of ideas, however, once the work was finished, most of us were extremely grateful to teorell for his stimulating comments and for his confidence in the individual to allow him to run his study himself. appearance in my work on my doctoral thesis. it became clear to me that the term diffusion respiration did not describe the process of gas transport correctly and did not explain the alveolar build up of carbon dioxide during apnoea. teorell immediately led me to the correct solution through his own profound knowledge of diffusion of molecules against a convection gas flow. dealing with gases he directed me to hertz' early works and formulas. this gave my medical thesis ( 2 ) a basic scientific background. were lost for a considerable time, but all of us remember the stimulating discussions at the lunch table with teorell's encouragement and strong belief in independent and serious research activity which sooner or later would always result in rewarding personal satisfaction and new important tasks. he had an unclouded and optimistic view of the importance of research and its value in the 1930s and during the following years of world war 11, at a time not particularly promising for the labour market. surely, he was right in this respect also. it is to the merit of teorell and his most carefully selected collaborators' that during the time immediately after world war i1 our physiological institution developed into the foremost unit for education of researchers at the uppsala school of medicine. from his years in stockholm, teorell also brought great interest in basic science education. popular publications on the application of basic science to biological problems bear clear wimess to its importance. in these publications he revealed a significant educational and stylistic ability. i myself took part in one of teorell's earliest fundamental courses at the beginning of the 1940s. he lectured enthusiastically to the attentive audience in a most fascinating way about cardiovascular physiology. his background was his own experimental investigations of the isolated heart lung preparation, but the peripheral and central regulation mechanisms of the intact animal were never excluded. some thought perhaps that his problem-oriented education did not provide the simple and rigid abc one might have desired, but most were stimulated to independent thinking. in recent years we did not see teorell so often in basic education as his time was more and more involved not with administration but with his own research. professor teorell was diligently working iate at night in his own laboratory. his own spirit influenced the institution and its i myself experienced an example of teorell's important but sudden certainly, some less rigidly controlled doctoral candidates sometimes 8 enthusiastic staff of researchers and teachers. as a consequence, there was never any difficulty in recruiting new research students. finally, i once more wish to stress that teorell as a teacher, scholar and researcher not only provided us with a scientific role model. his entire friendly personality provided a humane example and through teorell the hierarchic etiquette of old times underwent significant changes. teorell was a most stimulating scientist and man. references (1) symposium on ion and water movements in membranes. upsala j med sci 85:193-342, 1980. ( 2 ) holmdahl, m h:son: pulmonary uptake of oxygen, acid-base metabolism and circulation during prolonged apnea. acta c h i r scand, suppi. 212, 1-128, 1956. 9 untitled consumption of omega-3 enriched eggs 315 keywords: apolipoprotein a1, apolipoprotein b, glucose, human, eggs, omega-3 received 15 june 2008 accepted 17 june 2008 upsala j med sci 113 (3): 315–324, 2008 biochemical effects of consumption of eggs containing omega-3 polyunsaturated fatty acids marie öhman1, torbjörn åkerfeldt2, ingela nilsson3, christer rosen4, lars-olof hansson2, martin carlsson3 and anders larsson2 1primary health care, landstinget västmanland, section of clinical chemistry, 2department of medical sciences, uppsala university hospital, uppsala, 3department of clinical chemistry, kalmar county hospital, kalmar, 4källlbergs industri ab, töreboda, sweden abstract today, eggs with an increased content of ω-3 fatty acids are available but there are few publications on the effects of consumption of such eggs on the lipoproteins and acute phase markers in humans. the aim of the present study was to evaluate the effects of consumption of standard eggs and ω-3 enriched eggs on lipoproteins, glucose and inflammation markers. nineteen healthy volunteers consumed one extra egg per day of either standard eggs or ω-3 enriched eggs in a double-blind, cross-over study. the duration of each period was 1 month. the effects of the different egg diets on apolipoprotein a1 and b (apo a1 and b), lipoprotein (a), creatinine, cystatin c, creactive protein, serum amyloid protein a, interleukin 6, triglycerides, glucose, total-, high-density lipoprotein and low-density lipoprotein cholesterol concentrations were analyzed. addition of one regular egg per day to the normal diet had no negative impact on blood lipids or inflammation markers. consumption of ω-3 enriched eggs resulted in higher levels of apoa1, lower apob/apoa1 ratio and lower plasma glucose. these effects have been associated in previous studies with a reduced risk for cardiovascular mortality and diabetes. introduction cardiovascular disease (cvd) is one of the major causes of death in the western world (1). the cvd risk is influenced by several factors including our diet (2). during the past decades, reduction in fat intake has been the main target of national dietary recommendations to decrease risk of cvd. despite a reduction in dietary fat intake in the u.s., the prevalence of obesity and type 2 diabetes has grown dramatically (3,4). however, several studies indicate that the types of fat consumed have a more important role for cvd risk than the total amount of fat in the diet and that replacing saturated fat with unsaturated fat is more effective than simply reducing total fat consumption and that an increased intake of ω-3 fatty acids substantially lowers the risk of cardiovascular mortality (5). there is also a strong link between lipids, lipoproteins and inflammation (6). 316 marie öhman et al. this has become one of the main themes in the pathogenesis of cvd over the past decades. it is proven that fat-tissue synthesizes tumour necrosis factor α (tnf-α) and interleukin-6 (il-6) (7), cytokines that are involved in the inflammatory process and that stimulate c-reactive protein (crp) and serum amyloid protein a (saa) synthesis, but also play a major role in plaque formation in the vascular tree (8). also, the widely used statins reduce low-density lipoprotein (ldl) and increase high-density lipoprotein (hdl) cholesterol (9, 10) therewith reducing inflammation and thus the acute phase response (11). in 1972, the american heart association published dietary recommendations for the public, limiting the egg consumption to no more than three eggs per week. to this date, this is the only food-specific restriction listed in the dietary guidelines (12). such a recommendation assumes that all eggs are the same, which is not correct. it has been shown that it is possible to strongly modulate the lipid content of consumer eggs by modifying the feed given to the hen (13). today, eggs with an increased content of ω-3 fatty acids are available in several markets but there are few publications on the effects of consumption of such eggs on the lipoproteins and acute phase markers in humans. eggs are a relatively inexpensive protein source with a very good amino acid composition for humans (14). the very high nutritional density makes the egg well suited for elderly individuals who often have a low nutritional status. a poor nutritional status is associated with an increased rate of complications and mortality during hospital care (15, 16). the aim of this study was to evaluate the effects of consumption of standard eggs and ω-3 enriched eggs on lipoproteins, glucose and inflammation markers. the study population consisted of healthy individuals over the age of 45 years. materials and methods study population the study was performed in 2005 and included 19 healthy swedish caucasian volunteers (8 males and 11 females). the study design was a double-blind, cross-over study with half of the volunteers starting with ω-3 eggs and the other half with standard eggs. the daily study intake was one egg and the duration of each period was 1 month. after the end of the first study period the study subjects changed to the other egg type for another month. apart from the addition of one egg per day, the volunteers had no other diet restrictions and continued with their normal diet. inclusion criteria for the study were: healthy individuals with an age over 45 years and without medication that could influence inflammatory parameters or blood lipids. exclusion criteria were known malignancy. the volunteers were informed not to use estrogens, steroids (except inhalation steroids), nsaid, statins or fibrates. the study was approved by the ethics committee at uppsala university and all participants gave their informed consent. consumption of omega-3 enriched eggs 317 eggs the eggs were analyzed prior to the study by analycen, lidköping, sweden. the ω-3-eggs were from hens that had been on a feed containing rapeseed oil. the test results showed a higher content of ω-3 fatty acids in the ω-3-eggs (9.3 % alphalinolenic acid (ala); 0.2 % eicosapentaenoic acid (epa); 1.5 % docosahexaenoic acid (dha)) than in the regular eggs (0.7 % ala; 0 % epa; 1.3 % dha). the non ω-3-eggs had higher contents of ω-6 fatty acids (1.8 % arachidonic acid (aa); 17.4 % linoleic acid (la)) than the ω-3 eggs (0.2 % aa; 13.7 % la). sample collection blood samples were drawn from an antecubital vein in the morning after 12-hour (overnight) fasting and collected in vacutainer tubes without additive (for fatty acid analysis) and lithium-heparin (lh pst™ ii, bd vacutainer systems, plymouth, uk). blood samples were collected immediately prior to the start of the study (baseline) and at the end of each period. the samples were immediately centrifuged and frozen at -20°c until analysis. laboratory assays all plasma samples were analyzed for the following parameters: apolipoprotein a1 (total cv 0.9% at 2.25 g/l), apolipoprotein b (total cv 1.2% at 1.73 g/l), lipoprotein (a) (total cv 5.6% at 265 mg/l), creatinine (total cv 4.8% at 94 μmol/l), cystatin c (total cv 4.2% at 0.96 mg/l), c-reactive protein (total cv 0.9% at 22 mg/l), glucose (total cv 1.0% at 4.4 mmol/l), total cholesterol (total cv 0.5% at 5.7 mmol/l), hdl-cholesterol (total cv 0.8% at 2.2 mmol/l), ldl-cholesterol (total cv 1.2% at 2.3 mmol/l) and triglycerides (total cv 1.4% at 2.1 mmol/l). these assays were performed using an architect ci8200® analyzer (abbott, abbot park, il, usa). saa was measured by nephelometry (dade behring, deerfield, il, usa) using a behring bn prospec® analyzer (dade behring). the total analytical imprecision of the method was 5.9% at 12.8 mg/l. il-6 was measured using an enzyme-linked immunosorbent assay (elisa) method (r&d systems, minneapolis, mn, usa). the total analytical imprecision of the il-6 method was less than 7 %. all assays were performed independently without prior knowledge of patient data. determination of the composition of fatty acids in serum phospholipids fatty acids were analyzed as methyl esthers on a hp 5890 series ii gas chromatograph equipped with a fid and a hp-ffap capillary column (30 m x 0.25 mm x 0.25 μm) (17). 318 marie öhman et al. statistical calculations all calculations were performed with the statistical software package (statview, sas institute inc., cary, nc, usa). differences between groups were tested with kruskal-wallis using relative values (%) at the end of each study period in relation to the sample collected just before the initiation of each study period. p-values < 0.05 were regarded as statistically significant throughout the study. results consumption of regular eggs no significant changes were observed for any of the studied parameters when adding a regular egg to the normal diet (table 1). consumption of ω-3 eggs consumption of ω-3 eggs resulted in significant increases in apoa1 (p = .017) and significant decreases in apob/apoa1 (p = .019) and plasma glucose (p = .018) (figures 1–3). consumption of ω-3-eggs resulted in an almost significant increase in saa. table 1. mean values and sem for the analytes at the beginning and end of each consumtion period analyte before omega-3 eggs sem after omega-3 eggs sem before ordinary eggs sem after ordinary eggs sem saa, mg/l 5.52 1.10 6.70 1.10 6.03 1.40 5.60 1.10 cholesterol, mmol/l 6.25 0.30 6.22 0.30 6.25 0.30 6.22 0.30 hdl-c, mmol/l 1.43 0.10 1.49 0.10 1.45 0.10 1.48 0.10 ldl-c, mmol/l 3.92 0.24 3.85 0.26 3.89 0.24 3.88 0.24 apoa1, g/l 1.62 0.05 1.68 0.06 1.67 0.06 1.66 0.05 apob, g/l 1.15 0.06 1.12 0.06 1.13 0.06 1.14 0.06 triglycerides, mmol/l 1.44 0.16 1.42 0.12 1.48 0.14 1.45 0.16 cystatin c, mg/l 0.69 0.05 0.72 0.04 0.76 0.04 0.75 0.05 crp, mg/l 3.00 1.02 2.84 0.73 2.68 0.98 2.96 1.04 glucose, mmol/l 5.40 0.22 5.21 0.17 5.19 0.19 5.43 0.21 il-6, ng/l 17.80 8.34 17.40 8.50 17.10 8.28 18.26 8.41 ldl/hdl 2.92 0.26 2.71 0.22 2.84 0.23 2.80 0.25 apob/apoa1 0.72 0.05 0.68 0.04 0.69 0.04 0.70 0.05 consumption of omega-3 enriched eggs 319 figure 1. change in percentage in apoa1 during consumption of regular eggs (�) and ω-3-enriched eggs (▲) in relation to values at the beginning of the period. the results are presented as mean and standard deviation for each group. figure 2. change in percentage in apob/apoa1 during consumption of regular eggs (�) and ω-3-enriched eggs (▲) in relation to values at the beginning of the period. the results are presented as mean and standard deviation for each group. figure 3. change in percentage in glucose during consumption of regular eggs (�) and ω-3-enriched eggs (▲) in relation to values at the beginning of the period. the results are presented as mean and standard deviation for each group. 320 marie öhman et al. fatty acid composition in serum phospholipids consumption of ω-3 eggs resulted in significant increase in c 18:3 ω-3 (alphalinolenic acid) (figure 4), while consumption of regular eggs resulted in significant increase in c 20:4 ω-6 (arachidonic acid). discussion despite the long-standing interest in the diet-heart hypothesis, the number of cohort studies that have directly addressed associations between dietary fat intake and risk of chd is surprisingly small and the results are not consistent (5). it is often difficult to change the diet of large populations. despite the recommendations of the american heart association to limit the egg consumption to no more than three eggs per week, the mean egg consumption in the us has remained significantly higher. it is much easier to change the composition of daily food products e.g. fortification of food products with vitamins or minerals. similarly, one could probably exchange one type of egg for another with a better lipid composition. in this study we used ω-3 enriched eggs from hens fed rapeseed oil. ω-3 enriched eggs are obtained by giving the layer hens an ω-3 enriched food. such a feed usually contains fish, rapeseed or flax products. the egg fatty acid composition varies between hens fed fish, rapeseed or flax (18). in metabolic studies, different classes of saturated fatty acids have different effects on serum lipid and lipoprotein levels (19). we studied the effect of addition of one egg a day of either standard eggs or eggs enriched with ω-3 fatty acids to the normal diet in a double-blinded cross-over study. the volunteers were informed not to change their normal diet apart from adding an extra egg to the diet. the average egg consumption in sweden is approximately 0.7 figure 4. change in percentage in alpha-linolenic acid (ala) during consumption of regular eggs (�) and ω-3enriched eggs (▲) in relation to values at the beginning of the period. the results are presented as mean and standard deviation for each group. consumption of omega-3 enriched eggs 321 eggs per day. the larger part of this egg consumption is not as whole eggs. it is thus very difficult to accurately define the daily egg consumption in a study population. we thus chose a cross-over setup to compare the effect of consuming the two egg types. analysis of c 18:3 ω-3 (alpha-linolenic acid) was included in the study as an indicator of compliance. consumption of ω-3 eggs resulted in a significant increase in alpha-linolenic acid. we found no significant effect on total cholesterol or triglycerides either in comparison with baseline values or between the two groups of eggs. this is in agreement with previous reports that egg consumption has a very limited effect on total cholesterol in healthy individuals. we found a significant increase in apoa1 and a significantly improved apob/apoa1 ratio in the group that consumed ω-3 enriched eggs. apolipoproteins and the apob/apoa1 ratio are considered to be superior as cvd risk markers to the traditional blood lipid markers (total cholesterol, ldland hdl-cholesterol) (20–22). they have also been shown to be better markers for treatment effects with statins (23, 24). there was a significant increase in saa but not in il-6 or crp in the group that consumed ω-3 enriched eggs. the same group also showed a significant increase in apolipoprotein a1, which is a marker for hdl-cholesterol. saa, but not crp, is bound to the hdl particles. theoretically, the saa increase could thus be secondary to the apoa1 increase and not due to an inflammatory response. we measured the fatty acid composition of serum phospholipids, which reflect weeks-to months qualitative dietary intake of fatty acids (17). we found increased levels of c18:3 ω-3 in the subjects that consumed ω-3 enriched eggs and significantly lower glucose levels. this is in agreement with previous reports on increased insulin sensitivity in rats fed alpha-linolenic acid (25, 26). in conclusion, addition of one regular egg per day to the normal diet had no negative impact on blood lipids or inflammation markers. consumption of ω-3 enriched eggs resulted in higher levels of apoa1, lower apob/apoa1 ratio and lower plasma glucose concentrations. all these changes have in previous studies been associated with a reduced risk for cardiovascular mortality and diabetes. acknowledgements the eggs used in this study were generously provided by källbergs industri ab, töreboda, sweden. the project was supported by grants from the uppsala university hospital research fund. references 1. watkins lo (2004). epidemiology and burden of cardiovascular disease. clin cardiol 27: 2–6. 2. hu fb, willett wc (2002). optimal diets for prevention of coronary heart disease. jama 288: 2569–78. 3. mokdad ah, serdula mk, dietz wh, bowman ba, marks js, koplan jp (1999). the spread of the obesity epidemic in the united states, 1991–1998. jama 282: 1519–22. 322 marie öhman et al. 4. wild s, roglic g, green a, sicree r, king h (2004). global prevalence of diabetes: estimates for the year 2000 and projections for 2030. diabetes care 27: 1047–53. 5. hu fb, manson je, willett wc (2001). types of dietary fat and risk of coronary heart disease: a critical review. j am coll nutr 20: 5–19. 6. mehra vc, ramgolam vs, bender jr (2005). cytokines and cardiovascular disease. j leukoc biol 78: 805–18. 7. drevon ca (2005). fatty acids and expression of adipokines. biochim biophys acta 1740: 287–92. 8. zwaka tp, hombach v, torzewski j (2001). c-reactive protein-mediated low density lipoprotein uptake by macrophages: implications for atherosclerosis. circulation 103: 1194–7. 9. ballantyne cm, andrews tc, hsia ja, kramer jh, shear c, access study group (2001). atorvastatin comparative cholesterol efficacy and safety study. correlation of non-high-density lipoprotein cholesterol with apolipoprotein b: effect of 5 hydroxymethylglutaryl coenzyme a reductase inhibitors on non-high-density lipoprotein cholesterol levels. am j cardiol 88: 265–9. 10. crouse jr 3rd, frohlich j, ose l, mercuri m, tobert ja (1999). effects of high doses of simvastatin and atorvastatin on high-density lipoprotein cholesterol and apolipoprotein a-i. am j cardiol 83: 1476–7. 11. ridker pm, rifai n, clearfield m, downs jr, weis se, miles js, et al (2001). measurement of c-reactive protein for the targeting of statin therapy in the primary prevention of acute coronary events. n engl j med 344: 1959–65. 12. mcnamara dj (1999). eggs, dietary cholesterol and hart disease risk: an international perspective. in egg nutrition and biotechnology. edited by sim js, nakai s and guenter w. new york: cabi publishing; 55–64. 13. farrell dj (1998). enrichment of hen eggs with n-3 long-chain fatty acids and evaluation of enriched eggs in humans. am j clin nutr 68: 538–44. 14. food and agriculture organization of the united nations (1970). the amino acid content of foods and biological data on proteins. nutritional study #24. rome. 15. dennis ms, lewis sc, warlow c, food trial collaboration (2005). effect of timing and method of enteral tube feeding for dysphagic stroke patients (food): a multicentre randomised controlled trial. lancet 365: 764–72. 16. baldwin c, parsons t, logan s (2001). dietary advice for illness-related malnutrition in adults. cochrane database syst rev 2: cd002008. 17. hagfors l, nilsson i, sköldstam l, johansson g (2005). fat intake and composition of fatty acids in serum phospholipids in a randomized, controlled, mediterranean dietary intervention study on patients with rheumatoid arthritis. nutr metab 2: 26. 18. farrell dj (1999). not all ω-3 enriched eggs are the same. in egg nutrition and biotechnology. edited by sim js, nakai s and guenter w. new york: cabi publishing; 151–62. 19. kris-etherton p, yu s (1997). individual fatty acids on plasma lipids and lipoproteins: human studies. am j clin nutr 65: 1628–44. 20. walldius g, jungner i, holme i, aastveit ah, kolar w, steiner e (2001). high apolipoprotein b, low apolipoprotein a-i, and improvement in the prediction of fatal myocardial infarction (amoris study): a prospective study. lancet 358: 2026–33. 21. walldius g, jungner i (2004). apolipoprotein b and apolipoprotein a-i: risk indicators of coronary heart disease and targets for lipid-modifying therapy. j intern med 255: 188–205. 22. sniderman ad, furberg cd, keech a, roeters van lennep je, frohlich j, jungner i, et al (2003). apolipoproteins versus lipids as indices of coronary risk and as targets for statin treatment. lancet 361: 777–80. 23. walldius g, jungner i (2005). rationale for using apolipoprotein b and apolipoprotein a-i as indicators of cardiac risk and as targets for lipid-lowering therapy. eur heart j 26: 210–2. 24. stewart bf, brown bg, zhao xq, hillger la, sniderman ad, dowdy a, et al (1994). benefits of lipid-lowering therapy in men with elevated apolipoprotein b are not confined to those with very high low density lipoprotein cholesterol. j am coll cardiol 23: 899–906. 25. ghafoorunissa, ibrahim a, natarajan s (2005). substituting dietary linoleic acid with alpha-linolenic acid improves insulin sensitivity in sucrose fed rats. biochim biophys acta 1733: 67–75. 26. nettleton ja, katz r (2005). n-3 long-chain polyunsaturated fatty acids in type 2 diabetes: a review. j am diet assoc 105: 428–40. consumption of omega-3 enriched eggs 323 corresponding author: anders larsson department of medical sciences, clinical chemistry and pharmacology, university hospital, s-751 85 uppsala, sweden phone: 46-18-6114271 anders.larsson@akademiska.se upsala j med sci 95: 137-145, 1990 effects of exogenous adenosine in a patient with transplanted heart. evidence for adenosine as a messenger in angina pectoris bo lagerqvist', christer sylvcn3, gunnar helmius', anders waldenstrom' departments of internal medicine' and diagnostic radiology2, university ho vita1 uppsala and department of internal medicine3, huddinge academic h o s p i d , huddinge, sweden abstract in this pilot study some cardiac effects of exogenous adenosine on the denervated heart were studied in a patient with transplanted heart since 3 years. he was instrumented with catheters into the left coronary artery, the coronary sinus and the right ventricle. adenosine was given in increasing doses intracoronarily, into the aorta at the diaphragmal level and into a peripheral vein. when given into the aorta pain was provoked dose-dependently and not different from a reference group. when given intracoronarily no pain was provoked except at the highest dose when a slight discomfort of the chest was provoked. after intravenous injection no pain was provoked in the chest or in adjacent structures. coronary sinus flow increased dose-dependently and not different from the reference group. no increased heart rate response occurred after intravenous or intracoronary injections. extensive degrees of sinus and a v nodal blockade occurred. in conclusion, the results are in keeping with a role for adenosine as a messenger between myocardial ischaemia and angina pectoris and cardiac sympathetic pressure response. the importance of innervation for proper sinus and av nodal function was also illustrated. 137 introduction adenosine has been proposed to be a messenger between myocardial ischaemia and angina pectoris (10, 11, 7). during myocardial ischaemia adenosine is released in large quantities from myocardial cells into the interstitium (3). when adenosine is given as a bolus intravenously or intracoronarily a pain is provoked in the chest with radiations into adjacent parts of the body.this pain is reported by patients with ischaemic heart disease not to be different from their habitual angina pectoris (1 1,7). provoked in areas supplied by the artery. adenosine induced pain response is attenuated by theophylline and increased irrespective of injection site by dipyridamole in keeping with activation of extracellular membrane bound adenosine receptors. when adenosine is given intraarterially pain is also thus a series of experiments has not been able to contradict the hypothesis that adenosine is a messenger between myocardial ischaemia and angina pectoris. as a further attempt to test this hypothesis we found it pertinent to give adenosine to patients with transplanted heart as such a heart is considered to be denervated. it has however been reported that 5 % of acute myocardial infarcts in transplants are accompanied by chest pain ( 6 ) . in this preliminary report we describe effects of exogenous adenosine on pain, heart rate response, sinus and a v nodal blocks and coronary vasodilation in a patient with a transplanted heart. memods the patient being a 59 years old male was transplanted 3 years ago due to intractable heart failure on the basis of congestive csrdiomyopathy. he had experienced four rejections that were successfully treated. current medication was cyclosporine and dipyridamole, and at the time of investigation he was in new york heart association functional class 1 1 1 . no dipyridamole was taken the last 24 hours before the investigation, which was made in connection with routine endomyocardial biopsy and selective coronary angiography. the study was accepted by the ethic’s committee and the patient was fully informed. a wilton-webster coronary sinus flow catheter was introduced into the coronary sinus under fluoroscopic control, and the position was tested by injection of contrast medium. the coronary sinus blood flow was i38 determined by the thermodilution technique (5). a pacing electrode was introduced via the left femoral vein into the apical region of the right ventricle. the electrode was connected with an external pacemaker with an on demand rate of 60/minute. after diagnostic coronary angiography, adenosine was given with a 7 f coronary-catheter in two arterial positions, first in the left coronary orifice, secondly in the descending aorta at the level of the diaphragm. the patient was unaware of which site of injection was used. adenosine dilutions were made from a standard solution of 5.0 mg/ml in saline, prepared by the hospital pharmacy. adenosine was given in 3.0 ml in doses of 0.1, 1.0, 2.5, 5.0 and 10 mg. a constant injection time of 10 seconds was chosen in order to make the results comparable with those of intravenous injection of adenosine. after the intraarterial injections, adenosine was given as a bolus into an antebrachial vein in doses of 2.5, 5.0, 7.5 and 10 mg; the vein was thereafter flushed with 5 ml of saline. in each dose-response series an injection of saline alone was included, single-blind. at onset of pain the patient raised his left hand. when the maximum pain began to subside the hand was lowered half way, and when the pain had disappeared the hand was lowered to the supine position. these time instants were noted on the online recordings of coronary sinus flow and of ecg (chest leads), the speed of which was 25 mm/sec. the magnitude of maximal pain was rated according to the cr-10 scale designed by borg (2). the patient was also asked ro uescribe the !ocaticn and the character of the pain. before the next dose the patient was asked whether any pain or discomfort remained. if the patient did not wish to proceed further, the last dose given was taken as the maximum tolerable. heart rate was calculated before injection and at the highest rate after injection. when there was no obvious increase in heart rate the calculations were made 30-40 seconds after injection or at least 10 seconds after any sinus or av block. in a reference group coronary angiography was performed in 7 male patients with angina pectoris. this group has been examined principally in the same way, as previously described for 6 of the 7 patients (7). s t a t i s t i c s changes were compared to previously (7) reported reterence values. a value 139 differing more than 2 s.d. away from the reference material was considered statistical significant. table 1. pain, coronary sinus blood flow and heart rate response after injection of adenosine in the heart transplanted patient compared to a reference group (mean f s.d.). observed reference group pain threshold (mg adenosine) lntracoronary injection 10.0 2.7 k3.3 lntraaortic injection 1 .o 1.3 ko.9 intravenous injection no pain 3.6 51.3 pain threshold ratio i n t r a c o r o n a r y / l n t r a a o r t i c 1 0 2.1 + 2.5 time(sec) to start of pain (mean of all doses that gave pain) lntracoronary injection 16,2 19.7 k 5.8 lntraaortic injection 14.3 + 2.8 15.9 f 4.2 intravenous injection 18,7 f 4.5 time(sec) to start of coronary sinus blood flow increase (mean of all adenosine injections) lntracoronary injection 2.8 k 1.2 1.8k 1.8 intravenous injection 22.0 _+ 5.7 13.2 i 5.3 coronary sinus blood flow increase in % lntracoronary injection(1 mg) 165 1!3 k 80 intravenous injection (5mg) 112 211 +lo9 increase in heart rate in % (mean of all adenosine injections) lntracoronary injection 3+2 26 +28 lntraaortic injection 5+4 31 +19 intravenous injection 2k4 42 it24 time to maximum heart rate (sec) (mean of all adenosine injections) in tracoronary injection 56+24 40 f 1 7 lntraaortic injection 35+7 24 + 4 intravenous injection 41 +8 40 + 7 140 results provocation of dain when given intracoronary adenosine provoked chest pain only at the highest dose (10 mg) as opposed to the reference group where pain was induced at 2.7f3.3 mg (meanf s.d.). injection of adenosine into the aorta at the diaphragmal level followed the same dose-response relation as in the reference group and the pain was located to the chest and the diaphragmal area. the lowest dose of adenosine that gave pain (pain threshold) when injected into the aorta was of the same magnitude as in the reference group. when the pain thresholds (in mg of adenosine) between intracoronary and intraaortic injections were compared , the difference was more than 3 s.d. higher as when compared to the correspondingly ratio of the reference group.when the ratio of the score of pain induced by intracoronary and intraaortic injections in patient was compared to the corresponding ratio of the reference group the difference was more than 3 s.d.. after intravenous injection of adenosine no chest pain was provoked, but after the two highest dosages our patient reported pain in the forehead. the patient experienced respiratory stimulation at dl dosages also in accordance with previous findings (1,12). coronarv flow. adenosine administration in the coronary artery and the antebrachial vein,but not into the aorta, resulted in increased coronary sinus blood flow with onset shortly after the onset of injection. the response did not differ from the reference values (table 1). the flow responses shown in table 1 are after intracoronary and intravenous injection of 1 mg respectively 5 mg adenosine. these doses were choosen because they were given to all of the tested subjects. sinus and a v nodal conduction and heart rate response. lntra aortic injection did not induce any conduction dcfects even at the highest dose given. after intracoronary injection of adenosine, except at the lowest dose (0.1 mg), sa arrest/blocks were seen. the duration of 141 figure 1:pain responses of different doses of adenosine. pain response is graded according to the cr-10 borg scale 8 6 .: 4 n 2 0 8 reference group ;t heart transplanted patient . . i . , 6 a. lntracoronary injection 1 i 2 0 0 5 1 0 dose of adenosine (mg) b. lntraaortic injection heart transplanted patient c m . n 0 5 1 0 dose of adenosine (mg) 142 bradycardia was proportional to the dose given and amounted to 32 seconds after 1,0 mg adenosine and to 73 seconds at tbe highest dose (10 intravenous injection did not provoked sa arrest/block but resulted in a v block 11-111 with longer duration being 23 seconds at maximum dose and a dose dependent delay of onset (table 2). in the reference group no sa arrest/block developed, and only 2 out of 7 developed av block after intravenous injection of adenosine, both at higher dosages, 10 and 1 5 mg and both with a duration of the block less than 5 seconds. the mean heart rate before each injection was for the transplanted patient 92.9f5.6 m-1 and for the reference group 62.4k10.0 m 1 mg). table 2. av block after intravenous injection and sa arrest/block after intracoronary injection of adenosine.in to the heart transplanted patient adenosine dose (mg) intravenous : 2 . 5 5 . 0 7.5 1 0 . 0 in tracornary: 0.1 1 .o 2.5 5 . 0 1 0 . 0 time after injection to block (sec) 2 0 21 1 8 1 5 5 3 1 0.5 duration of block (sec) 3 1 2 1 7 2 3 0 3 3 4 4 6 5 7 4 after the disappearance of sinus and a v block the heart rate returned to the baseline (table 1). discussion the main finding in this case report on a heart transplanted patient is that higher doses of intracoronary adenosine were needed to produce pain comparable to the reference group. in contrast, intraaortic administration provoked similar degrees of pain in our patient compared to the reference group. this was expected because the neurons responsible for the pain, when adenosine was given into the aorta at the diaphragmal level, should not be influenced by the transplantation operation itself. although the provocation of pain was greatly attenuated when given intracoronarily, 9t-908572 143 even at the highest dose of intracoronary adenosine only a moderate pain was reported and located to the chest. this pain is less likely to have originated from the pulmonary vascular bed as no pain was reported after intravenous injection. the most probable explanation is that the pain originates from the residual atrial tissue with intact original innervation. another possibility for pain induction could be recirculation of adenosine through thebesian veins and subsequent activation of mediastinal neurons. an alternative possibility is activation of cardiac neurons shown to grow into a canine experimental heart transplant model (9). furthermore, about 5 yo of patients with transplanted hearts experienced chest pain in association with acute myocardial infarction (6). our observations therefore strongly corroborate adenosine as a messenger between myocardial ischaemia and chest pain. coronary sinus flow increased to the same degree as in the reference group both after intracoronary and intravenous injections. also the early onset of flow increase was similar to the reference group. these observations indicate that adenosine induced increase of coronary flow is independent of coronary vascular innervation and in keeping with a direct effect of adenosine on the vascular tone (4). the normally seen increase in heart rate of about 25 ?ao following intravenous and intracoronary injections of adenosine were absent in this transplanted patient. this observation indicate that the increased heart rate is mediated via cardiac innervation. this heart rate response was previously reported to be associated with both increased systolic and diastolic blood pressures (8). these observations could possibly relate to the paradoxical increase of heart rate and blood pressure in anterior acute myocardial infarction that consequently could be mediated by ischaemically released adenosine which in its turn sensitizes sympathetic cardiac nerves resulting in a syrnpatho-adrenergic res2onse. acknowledgements this study was supported by the swedish heart and lung foundation, the groschinsky foundation and soderbergs foundations references 1 berne r.m., dimarco j.p. & belardinelli l. drornotropic effects of adenosine and adenosine antagonists in the treatment of cardiac 144 arrhytmias involving the atrio-ventricular node. circulation 69: 11 95-7, 1984. 2 borg g.: a category scale with ratio properties for intermodal and interindividual comparisons. in: psychophysical judgement and the progress of perception (ed h-g. geissler and p. petzold ), pp 25-34. veb deutscher verlag der wissenschaften, berlin, 1982. 3 edlund a . , fredholm b.b., patrinani p., patron0 c. & wennmalm a . , wennmalm m.: release of two vasodilatators adenosine and prostacyclin from isolated rabbit heart during controlled hypoxia. j physiol 340: 487-501, 1983. 4 fredholm b.b. & sollevi a.: cardiovascular effects of adenosine. clin physiol 6 : 1-21, 1986. 5 ganz w., tamura k., marcus hs., donoso r., yoshida s. & swan hjc. measurement of coronary sinus blood flow by continuous thermodilution in man. circulation 44: 181-95, 1971. 6 gao s.z., schroeder j., hunt s., billingham m. & stinson e.: myocardial infarction in cardiac transplant patients. circulation 78: 11-438, 1988. 7 lagerqvist b., sylven c., beermann b., helmius g. & waldenstrom a.: lntracoronary adenosine causes angina pectoris like pain an inquiry intc the nature of visceral pain. cardiovasc res 24:.609-13, 1990. 8 longhurst j.c.: cardiac receptors:their function in health and disease. prog cardiovasc dis 27: 201-22, 1984. 9 mohanty p.k., thames m.d., capehart j.r., kawaguchi a,, ballon b. & lower r.r. afferent reinervation of the autotransplanted heart in dogs. j am coll cardiol 7: 414-8, 1986. 10 sylven c., beermann b., jonzon b. & brandt r.: angina pectoris-like pain provoked by intravenous adenosine in healthy volunteers. br med j 293: 227-30, 1986. 11 sylven c.: angina pectoris. clinical characteristics, neurophysiological and molecular mechanisms. pain 36: 145-67, 1989. 12 watt a.h. & routledge p.a. adenosine stimulates respiration in man. br j clin pharrnac 20: 503-6, 1985. address for reprints: dr anders waldenstrom dept. of medicine university hospital s-751 85 uppsala, sweden 10 908572 145 upsala j med sci 95: 301-303, 1990 summing up torgny groth,' per hyltoft petersen' and carl-henrik de verdier' 'unit f o r biomedical systems analysis and 'department of clinical chemistry, university of uppsala, uppsala, sweden and 'department of clinical chemistry, university of odense, odense, denmark introduction good laboratory practice (glp) is a concept that was formulated already in 1978 by the us food and drug administration (fda) (2) and was taken up in 1981 by the organisation for economic co operation and development (oecd)(5). several recent documents about glp are of interest to the nordic countries (1,3 , 4 , 6). they describe the meaning of this concept which seems to be equivalent to "good laboratory quality management" as this term is defined by professor james 0 westgard in this publication. the term can be split into several different functions. production of analytical results is the predominant function of the clinical chemical laboratory and analytical quality control and analytical quality assurance are therefore major tasks for this type of laboratory. there are, however, also other very important quality characteristics for a clinical laboratory in general and a clinical chemical laboratory in particular: e. g. turnaround time, optimal selection and utilization of tests and interpre tation of results. particularly nowadays as stressed by professor mogens hqrder (the chairman of the nordkem board) and by dr lars mellin it is also important to relate the quality to the costs both internal ones for the laboratory and external ones for the health care organization and the society. the title of the project "medical need for quality specifications in laboratory medicine" would indicate that it is not only technical and analytical chemical considerations that should be made in the formulation of the quality specifications. it is important to work together with clinicians and primary care physicians and to merge the knowledge from all sides to define the clinical situations where the test is needed, to evaluate the 301 need for prompt reporting, to establish guidelines for interpre tation of the result and to assess the effect of analytical and preanalytical errors. an important continuation of this process is to find ways for obtaining the specified quality, whether they are instructions for reducing preanalytical variations or guidelines for improving analytical quality etc. furthermore, control systems for monitor ing quality should be elaborated, either in general terms or as examples. approaches if the project is given this broad definition it will need considerable time to conclude and a priority list is desirable as well as assistance from other groups working with similar problems. there are several problems with proficiency testing as indicated in the communication by professor ronald laessig. in a future world of medicine exchange of information and knowledge will be far more dominating than today. clinical laboratory data must also be transferable from one place to another. that is the reason why the main project group will work with analytical quality specifications for some plasma proteins and low molecular serum hormones as two important applications. it is also important to have ideas about how to recalculate data from one laboratory to another in order to solve the problem of communi cating clinical laboratory data between hospitals or other health care centres. this will be a task for the main project group as well as attempts to coordinate terminology, abbreviations and computing procedures. it is stimulating for the main project group that several research groups working within this area have after request announced their interest to participate as associated project groups (see the list in section 21). we hope that this joint effort will: * promote the work with establishing clinical laboratory quality specifications in the nordic countries. * widen the activities to cover many important areas * add resources and knowledge to assess, if possible, other quality characteristics as turnaround time, test selection and result interpretation. 302 references 1. de verdier c-h. how to make quality from goals to practical application. scand j clin lab invest (1990);50 suppl 202:67-70. 2. department of health and human services (us) food and drug administration: good laboratory practice for nonclinical laboratory studies , code of federal regulations 1987-04-01;title 21, part 58:228-42, 1987, p 228-42. 3 . dybkaer r, martin dv, rowan rm. good practice in decentralized analytical clinical measurement. scand j clin lab invest 1991;51 suppl (in press). 4 . eccls (de verdier ti hjelm) . guidelines on good practice in clinical laboratories. i. clinical chemistry. in press. who/euro publ. 1991. 5. oecd. principles of good laboratory practice. env/ 6. scandinavian society for clinical chemistry. general scandina vian recommendation on quality control an quality assurance in clinical chemistry. scand j clin lab invest (1990);50:225-227. chem/mc/81.14. manuscript. 1981:15-44. (1981);(unpub) correspondence: professor carl-henric de verdier department of clinical chemistry, university of uppsala postal address: university hospital s-751 85 uppsala, sweden 303 tf-iups160027 159..164 review article antimicrobial resistance—a threat to the world’s sustainable development du�san jasovsk�y, jasper littmann, anna zorzet and otto cars react europe, department of medical sciences, uppsala university, uppsala, sweden abstract this commentary examines how specific sustainable development goals (sdgs) are affected by antimicrobial resistance and suggests how the issue can be better integrated into international policy processes. moving beyond the importance of effective antibiotics for the treatment of acute infections and health care generally, we discuss how antimicrobial resistance also impacts on environmental, social, and economic targets in the sdg framework. the paper stresses the need for greater international collaboration and accountability distribution, and suggests steps towards a broader engagement of countries and united nations agencies to foster global intersectoral action on antimicrobial resistance. article history received 11 may 2016 accepted 26 may 2016 keywords antibiotic resistance; antimicrobial resistance; sustainable development; sustainable development goals introduction despite many proposals and initiatives in recent decades, the world has failed to keep pace with microbes becoming increasingly resistant to available treatments, so-called antimicrobial resistance (amr). any antibiotic use contributes to increasing the number of micro-organisms on which drugs have no effect. amr is thus an unavoidable phenomenon that undermines the effectiveness of basic and modern medicine and is affecting people from birth to death. ‘post-antibiotic era’ today, diminishing antimicrobial effectiveness represents a formidable threat to human and animal health and therefore to overall global development. deaths from drug-resistant infections are projected to increase from currently 700,000 to 10 million annually, and cost estimates are as high as us$100 trillion worldwide by 2050 (1). of special concern is the rapid global spread of multi-resistant bacteria, for some of which there is no available treatment. the prospect of the world entering a ‘post-antibiotic era’, where common infections can no longer be cured, is therefore a real possibility. furthermore, unequal access to health care resources has meant that large populations in resource-poor settings never fully entered the antibiotic age to begin with. consequently, universal access to effective antimicrobials represents one of the greatest opportunities to save and improve millions of lives each year (2,3). otto cars, winner of the rudbeck award 2015, at the medical faculty of uppsala university for his long-term, persevering and successful strategic work in paying attention and taking measures to the increase of antibiotic resistance. contact otto cars otto.cars@medsci.uu.se react europe, department of medical sciences, uppsala university, uppsala, sweden � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 3, 159–164 http://dx.doi.org/10.1080/03009734.2016.1195900 http://creativecommons.org/licenses/by/4.0/ global strategy to address the challenge of amr, the world health organization (who) first published a global strategy for its containment as far back as 2001 (4). however, insufficient human and financial resources to implement the strategy meant that its effect fell short of the intended goal. over the past decade, the issue of amr has become more prominent on the global health agenda. the increasing visibility of the consequences of amr, both in terms of health outcomes and economic costs, has led to an increasing number of calls for global collective action to improve access to antimicrobial medicines on grounds of equity, to maintain their effectiveness, and to increase the supply of new products. among the advocates of such action are heads of states and ministers as well as health care professionals and civil society organizations. but despite the growing recognition of the urgency of tackling amr, international policies and global institutional arrangements are still lacking. there have, however, been some positive developments lately. in 2015, the global action plan on antimicrobial resistance was adopted at the 68th world health assembly in geneva (5). later that year, amr was recognized as a threat to the world’s sustainability and development efforts in the resolution entitled ‘transforming our world: the 2030 agenda for sustainable development’, which incorporates the un’s sustainable development goals (sdgs). although omitted from specific sdg targets, amr is included in paragraph 26 which states: ‘[w]e will equally accelerate the pace of progress made in fighting malaria, hiv/aids, tuberculosis, hepatitis, ebola and other communicable diseases and epidemics, including by addressing growing antimicrobial resistance and the problem of unattended diseases affecting developing countries’ (6). the end of antibiotics the startling truth is that for large populations the era of effective antibiotics has already, or will very soon, come to an end. since amr does not stop at national borders, it must be treated as a global challenge (7). work must be undertaken to achieve a balance between supply and demand of urgently needed technologies through novel innovation models, health system strengthening, and massive efforts to change social norms (8). if allowed to take on pandemic proportions, amr would not only risk undoing much of the progress made within the health sector, but also threaten other development achievements such as poverty reduction and economic growth (9). given the scale of the problem of amr and the many areas of society affected by its consequences, solutions to the challenge we face must follow an integrated and comprehensive policy approach. amr must be redefined in a broader context beyond human health, including agricultural and environmental issues, as well as health security, all within the framework of sustainable development, and should also be reflected in the implementation of the sdgs (10,11). analogies with other fundamental global concerns, such as climate change, can help us understand the actual scope and irreversible consequences we face if decisive and far-reaching actions are not taken. the remainder of this paper will discuss how amr may impact the achievement of specific sdgs. implications for sdg 3 sdg 3: ensure healthy lives and promote well-being for all at all ages maternal and child health. several of the adopted targets in the health-dedicated sdg 3 will be impossible to achieve without the availability of effective antibiotics. their central role for the provision of maternal and child health has been recognized in the every woman every child initiative, which included antibiotics in a list of 13 life-saving commodities (12) that contributed to the achievement of the millennium development goals (mdgs) on maternal and child health. especially in lowand middle-income countries (lmics), where the maternal mortality rate is up to 19 times higher than in the rest of the world, the emergence of amr currently jeopardizes the progress made in reducing maternal mortality over the previous 15 years (9). communicable diseases. alarmingly, 214,000 neonatal sepsis deaths annually are directly attributable to drug-resistant pathogens (13). this severely impacts on plans to reduce neonatal mortality to less than 12 per 1,000 live births under sdg target 3.2 (6). in the last 15 years, child mortality has declined by 47%, which is largely due to reductions in deaths from pneumonia, a leading cause of child mortality globally (14). if drug resistance continues to rise, and if access to effective antibiotics is not guaranteed, these health gains could be reversed. sexually transmitted infections (stis) are a major cause of disease globally, with an estimated 500 million new infections every year, causing long-term disability, infertility, and even death (15,16). some of these infections are becoming increasingly drug-resistant. with approximately 106 million newly infected individuals every year, gonorrhoea is one of the most common stis (16,17). it is therefore extremely worrying that some cases of gonorrhoea have become untreatable because of resistance to last-resort antibiotics (18). furthermore, untreated stis are also well-known risk factors for hiv transmission (19). attention should also be paid to emerging resistance to treatments for hiv, tuberculosis (tb), and malaria, which will create significant obstacles for the achievement of sdg target 3.3 (6). in the case of tb, half a million people developed multidrug-resistant tb in 2014, with an estimated death toll of 210,000. extensively drug-resistant tb has been reported by 100 countries (20). in sub-saharan africa, drug resistance to commonly used hiv medicines was detected in almost 60% of cases (21), while drug-resistant malaria is on the rise in southeast asia (22). in summary, drug-resistant pathogens could reverse the recent positive trend of falling global mortality rates from infectious diseases, which have decreased from 23% to 17% of total deaths over the last 15 years (9,23,24). non-communicable diseases. the impact of amr is not limited to infectious diseases. it also has potentially 160 d. jasovsk�y et al. disastrous consequences for the treatment of many noncommunicable diseases (ncds). a clear example is the threat that amr poses to the safety and effectiveness of procedures such as surgical interventions, cancer treatment, and organ transplants (8). for example, up to 50% of pathogens causing surgical site infections are resistant to standard prophylactic antibiotics in the us. similarly, every fourth patient on cancer treatment suffered from infections caused by pathogens that were resistant to commonly used antibiotics (25). immunocompromised patients who have undergone transplantations or chemotherapy (26) or who have aids are more vulnerable to infections (27). access to effective antibiotics is therefore vital to meeting several sdg targets on ncds. universal health coverage. given the disproportionate global distribution of infectious diseases, with the heaviest burden in lmics, the emergence of amr creates bottlenecks for establishing universal health coverage globally, which sdg target 3.8 (6) calls for. arguably, no health system will be sustainable without effective antibiotics (28), as demonstrated by an example where the cost of treating and caring for a single patient with extensively drug-resistant tuberculosis has been estimated at a staggering $482,000 in the us (29). the indisputable, yet difficult-to-measure magnitude with which antimicrobial medicines of lowered quality contribute to global amr rates should also be addressed accordingly as part of an integrated solution (10). implications for other sdgs sdg 1: end poverty in all its forms everywhere from an international development perspective, amr strikes hardest at the poor. rates of resistant bacteria are high, and affordable treatment options are often not available, especially since many patients pay for medicines out-of-pocket. moreover, the spread of diseases is enabled by weak sanitary systems (8). treating drug-resistant infections is costlier, takes longer, and has lower chances of success than treating drug-susceptible infections. as a result of these factors, amr negatively impacts on national economic performance (30), which could ultimately contribute to slowing down progress towards sdg 1. sdg 2: end hunger, achieve food security and improved nutrition, and promote sustainable agriculture the rapid growth of intensive production systems as a response to increased demand for animal protein has been estimated to increase the consumption of antimicrobials in the food animal sector by two-thirds between 2010 and 2030 (31,32). this inherent conflict has to be resolved if we are to meet the indicators of sdg 2 to double the agricultural productivity by 2030 and to ensure the implementation of sustainable food production systems and resilient agricultural practices. to show the implications of agricultural use of antibiotics, researchers recently studied soil samples spanning 100 years. the study showed that levels of antibiotic resistance genes started to increase by the mid-1970s in soils fertilized by manure. the change in types of resistance genes also roughly paralleled the first reports of these genes in clinical isolates from hospitals. perhaps even more interestingly, the level of some resistance genes decreased again following a ban on non-therapeutic antibiotic use, suggesting accumulated soil resistance genes could be reduced by antibiotic stewardship efforts (33). to develop sustainable animal production practices where high productivity is reached without inappropriate use of antimicrobials is of crucial importance (34). there are several examples of high productivity being reached without use of antibiotics for growth promotion or routine mass-medication, and without severe economic losses (35). the key elements here are good animal husbandry practices that prevent disease, combined with commercial disincentives and a legal framework that regulates the use of antibiotics in the animal sector. sdg 6: ensure availability and sustainable management of water and sanitation for all waste products from hospitals, antibiotic manufacturing plants, and agriculture contribute to increased amounts of antibiotic residues and resistant bacteria in aquatic ecosystems (36,37). for example, measurements at a wastewater plant receiving waste from approximately 90 drug manufacturers showed that 45 kg of a common antibiotic were released into a nearby river each day. the concentration of the antibiotic in the river was higher than it would be in a patient who was treated with the drug (38). as antibiotic residues can be carried by water and through sediments and soil, gradients of different antibiotic concentrations will form, and even very low antibiotic concentrations may be enough to select for highly resistant bacteria (39). importantly, lack of access to clean water and sanitation also facilitates the spread of bacterial diseases, leading to increased morbidity and mortality, especially in children. sdg 8: promote sustained, inclusive, and sustainable economic growth, full and productive employment, and decent work for all as mentioned, resistance to antimicrobial drugs already causes an estimated 700,000 deaths annually and—without effective action—is predicted to cause 10 million deaths annually and cost up to us$100 trillion by 2050 (1). losing effective antibiotics for the treatment of infectious diseases adds an extra burden to states’ health expenditure and can lower productivity, household income, and tax revenues and lead to losses in gdp (30). such enormous costs may undermine efforts for sustainable economic growth as emphasized in sdg 8, making amr a critical global development issue (1,30). sdg 12: ensure sustainable consumption and production patterns antibiotics must be looked upon as a scarce and potentially non-renewable global resource. ensuring sustainable consumption and production of this resource, and recognizing upsala journal of medical sciences 161 the linkages between sdg 12 and other goals such as sdg 2, requires a multipronged approach that addresses three fundamental issues. first, access to effective antibiotics could be considered an integral part of the fulfilment of the human right to health. globally, a staggering 5.7 million people die each year from treatable infectious diseases due to lack of access to antibiotics (2). an estimation shows that universal provision of antibiotics could avert approximately 445,000 community-acquired pneumonia deaths in children under five years of age (13). second, constructing a sustainable yet accessible model of antibiotic distribution and consumption requires conservation mechanisms that would reduce the need for antibiotics while ensuring their appropriate use (40). this would among other things include evidence-based rational use programmes and strong surveillance schemes, coupled with a responsive international framework that includes means of implementation for low-resource countries and locally tailored regulations (2). third, greater efforts to promote the innovation of new antimicrobials and diagnostics are needed (41). the research and development of novel antibiotics has for long been de-prioritized due to scientific and financial bottlenecks (42). the latest class of antibiotics was discovered in 1987 (43), and there is a dangerous shortfall of new drugs in the pipeline. considering the enormous global public value of antibiotics, the failed innovation model needs to be driven by public health needs and transformed to include, for example, public–private projects or novel business models as presented in sdg 9. sdg 17: strengthen the means of implementation and revitalize the global partnership for sustainable development global sustainability efforts require partnerships between governments, civil society, and the private sector as called for in the final sdg 17. framing amr as a sustainability issue will therefore urgently require policy and institutional coherence at all policy levels, from global to local (44). the responsibility of governments and their international co-operation constitutes the primary global partnership as reaffirmed in sdg 17. multi-stakeholder partnerships to avoid an amr ‘tragedy of the commons’ situation, antimicrobial effectiveness needs to be recognized as a fundamentally important global public good and governed accordingly. in addition to the global action plan on amr, there is critical need for cross-sectoral global action by countries and other stakeholders to complement it. there have been multiple calls from various bodies including the g7 health ministers, the foreign policy and global health initiative, and an alliance of champion countries for a multi-stakeholder approach to control amr. such an approach could help to ensure that all relevant aspects of amr are addressed through intergovernmental and cross-un agency action based on respective expertise. to successfully initiate and manage such a multi-stakeholder process, the discussion of global co-ordination and leadership redistribution must be initiated early on. it is also vital to consider and emulate lessons learned from comparable global political initiatives in global health and other areas. examples of such initiatives include the global response to the aids crisis where the un general assembly adopted the declaration of commitment on hiv/aids and the high-level meeting on the prevention and control of non-communicable diseases (ncds) that helped to chart a new international agenda on ncds. similarly, the global response to climate change has shown the type of multi-stakeholder partnerships needed to contain amr. mobilized means of implementation for technology and capacity building political will needs to be reflected in significant commitments to mobilizing means of implementation for technology and capacity building for both lmics and high-income countries. here, the role of national actors spanning from governmental entities to un agencies with strong national presence is essential in the process of effective implementation. it is likewise crucial to strengthen knowledge sharing to support access to innovations in the field of amr. these actions must be coupled with international support for implementing effective and targeted capacity building in lmics to support national plans to implement all the sustainable development goals, including national action plans on amr. data, monitoring, and accountability financing such national action plans, especially in lmics, would consequently enable monitoring and accountability mechanisms for data on access to antibiotics, their use and resistance levels, which are key to lasting global collective action. summary and conclusions amr is not a disease for which we should expect ultimately to develop a cure. instead it is a silent pandemic that is here to stay. amr is a phenomenon where micro-organisms adapt through evolution to survive the onslaught of antimicrobial drugs. it undermines the treatment of many common diseases as well as standard surgical procedures. as we have shown above, it is also a challenge to global development at large. antimicrobial effectiveness must be looked upon as a limited global public good on the verge of becoming scarce, and the world has a collective responsibility to preserve it in order to avoid countless future victims of drug-resistant infections (45). there is no single ‘silver bullet’ to address amr. we need an adaptive, multipronged approach that operates across the sdgs, involving many stakeholders. accordingly, we cannot merely look to the biomedical or health sectors for solutions. amr is a systems failure that requires a crosssectoral response. by placing amr within the sdg agenda, we seek to intensify and widen the international political commitment to finding a solution to this emerging threat before it turns into a global crisis. acknowledgements a first version of this paper with a limited selection of references has been published as a development dialogue paper, by the dag hammarskj€old foundation, uppsala, sweden (http://www. 162 d. jasovsk�y et al. http://www.daghammarskjold.se/publication/antimicrobial-resistance-threat-worlds-sustainable-development/ daghammarskjold.se/publication/antimicrobial-resistance-threat-worldssustainable-development/). disclosure statement the authors declare no conflicts of interest. funding information the authors declare no specific funding for this article. references 1. review on antimicrobial resistance. tackling a crisis for the health and wealth of nations. london, united kingdom: review on antimicrobial resistance; 2014. 2. daulaire n, bang a, tomson g, kalyango jn, cars o. universal access to effective antibiotics is essential for tackling antibiotic resistance. j law med ethics. 2015;43(suppl 3):17–21. 3. mendelson m, røttingen ja, gopinathan u, hamer dh, wertheim h, basnyat b, et al. maximising access to achieve appropriate human antimicrobial use in low-income and middle-income countries. lancet. 2015;387:188–98. 4. world health organization. who global 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&equals;1 http://www.who.int/drugresistance/global_action_plan/en/ http://www.who.int/gho/publications/mdgs-sdgs/en/ http://www.who.int/gho/publications/mdgs-sdgs/en/ http://www.reactgroup.org/toolbox/ http://www.reactgroup.org/toolbox/ http://www.who.int/healthinfo/global_burden_disease/en/ http://www.who.int/healthinfo/global_burden_disease/en/ http://apps.who.int/iris/bitstream/10665/137094/1/9789241564809_eng.pdf http://apps.who.int/iris/bitstream/10665/137094/1/9789241564809_eng.pdf http://www.thelancet.com/article/s1473309915005368/fulltext http://www.thelancet.com/article/s1473309915005368/fulltext http://www.who.int/malaria/publications/world-malaria-report-2015/report/en/ http://www.who.int/malaria/publications/world-malaria-report-2015/report/en/ http://www.who.int/tb/publications/global_report/en/ http://www.who.int/tb/publications/global_report/en/ http://www.who.int/malaria/publications/atoz/9789241509442/en/ http://www.who.int/malaria/publications/atoz/9789241509442/en/ http://www.who.int/iris/handle/10665/68357 http://www.who.int/iris/handle/10665/68357 38. larsson dgj, de pedro c, paxeus n. effluent from drug manufactures contains extremely high levels of pharmaceuticals. j hazard mater. 2007;148:751–55. 39. gullberg e, cao s, berg og, ilb€ack c, sandegren l, hughes d, et al. selection of resistant bacteria at very low antibiotic concentrations. plos pathog. 2011;7:e1002158. 40. heyman g, cars o, bejarano m-t, peterson s. access, excess, and ethics–towards a sustainable distribution model for antibiotics. ups j med sci. 2014;119:134–41. 41. hoffman sj, outterson k, røttingen ja, cars o, clift c, rizvi z, et al. an international legal framework to address antimicrobial resistance. bull world health organ. 2015;93:66. 42. zorzet a. overcoming scientific and structural bottlenecks in antibacterial discovery and development. ups j med sci. 2014;119:170–5. 43. silver ll. challenges of antibacterial discovery. clin microbiol rev. 2011;24:71–109. 44. årdal c, outterson k, hoffman sj, ghafur a, sharland m, ranganathan n, et al. international cooperation to improve access to and sustain effectiveness of antimicrobials. lancet. 2015;387:296–307. 45. cars o. securing access to effective antibiotics for current and future generations. whose responsibility? ups j med sci. 2014;119:209–14. 164 d. jasovsk�y et al. antimicrobial resistance&mdash;a threat to the world&rsquo;s sustainable development introduction &lsquo;post-antibiotic era&rsquo; global strategy the end of antibiotics implications for sdg 3 sdg 3: ensure healthy lives and promote well-being for all at all ages implications for other sdgs sdg 1: end poverty in all its forms everywhere sdg 2: end hunger, achieve food security and improved nutrition, and promote sustainable agriculture sdg 6: ensure availability and sustainable management of water and sanitation for all sdg 8: promote sustained, inclusive, and sustainable economic growth, full and productive employment, and decent work for all sdg 12: ensure sustainable consumption and production patterns sdg 17: strengthen the means of implementation and revitalize the global partnership for sustainable development multi-stakeholder partnerships mobilized means of implementation for technology and capacity building data, monitoring, and accountability summary and conclusions acknowledgements disclosure statement funding information references sups_a_526724 39..46 upsala journal of medical sciences. 2011; 116: 39–46 original article significant differences when using creatinine, modification of diet in renal disease, or cystatin c for estimating glomerular filtration rate in icu patients miklós lipcsey1, mia furebring2, sten rubertsson1 & anders larsson3 1section of anaesthesiology & critical care, department of surgical sciences, uppsala university hospital, uppsala, sweden, 2section of infectious diseases, department of medical sciences, uppsala university hospital, uppsala, sweden, and 3section of clinical chemistry, department of medical sciences, uppsala university hospital, uppsala, sweden abstract background. renal dysfunction is associated with increased morbidity and mortality in intensive care patients. in most cases the glomerular filtration rate (gfr) is estimated based on serum creatinine and the modification of diet in renal disease (mdrd) formula, but cystatin c-estimated gfr is being used increasingly. the aim of this study was to compare creatinine and mdrd and cystatin c-estimated gfr in intensive care patients. methods. retrospective observational study was performed, on patients treated within the general intensive care unit (icu) during 2004–2006, in a swedish university hospital. results. gfr markers are frequently ordered in the icu; 92% of the patient test results had cystatin c-estimated gfr (egfrcystatinc) £ 80 ml/min/1.73 m 2, 75% had egfr £ 50 ml/min/1.73 m2, and 30% had egfr £ 20 ml/min/1.73 m2. in contrast, only 46% of the patients had reduced renal function assessed by plasma creatinine alone, and only 47% had egfrmdrd £ 80 ml/min/1.73 m 2. the mean difference between egfrmdrd and egfrcystatinc was 39 ml/min/1.73 m 2 for egfrcystatinc values £ 60 ml/min/1.73 m 2. conclusions. gfr is commonly assessed in the icu. cystatin c-estimated gfr yields markedly lower gfr results than plasma creatinine and egfrmdrd. many pharmaceuticals are eliminated by the kidney, and their dosage is adjusted for kidney function. thus, the differences in gfr estimates by the methods used indicate that the gfr method used in the intensive care unit may influence the treatment. key words: cystatin c, glomerular filtration rate, human, intensive care, kidney, mdrd introduction glomerular filtration rate (gfr) is generally accepted as the best overall indicator of renal function and is therefore an important marker for renal disease. reduced gfr is one of the most important complications in critically ill patients and is associated with increased morbidity and mortality in the intensive care unit (icu) population (1–4). furthermore, reduced gfr influences the clearance of many pharmaceuticals used today. thus, in many cases the recommended dose of a drug has to be adjusted depending on the patient’s gfr. for instance, the dosage of antibiotics and cytotoxic drugs is usually prescribed according to gfr. monitoring renal function is thus very important in the management of intensive care patients, and gfr markers are frequently used for this purpose, necessitating convenient and reliable gfr markers. inulin, iohexol, and 51cr-edta clearances are considered as ‘gold standards’ for gfr measurements in sweden (5–7). the disadvantage with these assays, and creatinine clearance with urine collection, is that they are cumbersome, costly, and associated with long turn-around correspondence: miklós lipcsey, md, phd, section of anaesthesiology & critical care, department of surgical sciences, uppsala university hospital, se-751 85 uppsala, sweden. fax: +46 18 55 93 57. e-mail: mlipcsey@eml.cc (received 8 september 2010; accepted 20 september 2010) issn 0300-9734 print/issn 2000-1967 online � 2011 informa healthcare doi: 10.3109/03009734.2010.526724 times, which may delay initiation and adjustment of treatment. they are thus less suitable for an intensive care unit requiring rapid decisions and actions. thus, endogenous markers are usually preferred in the intensive care setting. the ideal endogenous marker should have a stable production rate, be unaffected by pathological changes, lack protein binding, be freely filtered through the glomeruli, and lack reabsorption or secretion. to date, no such marker has been identified. serum or plasma creatinine is the most commonly used gfr marker (8,9). creatinine is an inexpensive test widely available in clinical chemistry laboratories, but the assay outcome is hampered by the influence of several extrarenal factors such as age, gender, muscle mass, physical activity, and diet (10,11). it is also insensitive for small decreases in gfr, in the socalled creatinine-blind gfr area, due to the nonlinear relationship between plasma concentration and gfr (12). cystatin c is an endogenous polypeptide that is more sensitive than serum creatinine for the detection of small decreases in gfr and is reported not to be influenced by inflammation, liver function, age, gender, muscle mass, physical activity, or diet (13). human cystatin c has a plasma clearance of cystattin c of 94% of the generally used gfr-marker, 51cr-edta in the rat (14). previously, cystatin c assays were hampered by the limited availability of the test, but cystatin c methods have now been developed for clinical chemistry laboratories making the test widely accessible. cystatin c can thus be analyzed with short turn-round times providing rapid test results for intensive care, at costs comparable to plasma creatinine analysis. a recent meta-analysis has shown that cystatin c is superior to plasma creatinine as a marker of renal function (15). another study has suggested that cystatin c is superior to creatinine as a gfr marker in critically ill patients (16). the aim of this study was to assess the prevalence of reduced gfr in this patient group using creatinine and modification of diet in renal disease (mdrd) and cystatin c-estimated gfr, as several pharmaceuticals are prescribed according to renal function, and to investigate the request frequency of laboratory markers in an icu, with special reference to gfr markers. materials and methods study population this study included all patients treated within the general intensive care unit, uppsala university hospital, during the time period 1 january 2004 to 1 september 2006. the total number of tests per year, presented in table i, was based on all requests during the same time period. the table thus also includes requests for patients below 16 years of age and requests without a valid test result (e.g. samples that could not be analyzed due to insufficient sample volumes). for comparison between cystatin c and creatinine results, only valid test results from patients older than 16 years of age were included. blood samples for cystatin c analyses were usually collected early in the morning and not more than once a day. intensive care unit (icu) the study was conducted in a ten-bed general intensive care unit admitting patients from medical and surgical specialties in a university hospital. approximately 1,200–1,500 patients are treated in this unit each year, with a mean admittance time of 2.5 days. the unit has an abl 725 (radiometer, copenhagen, denmark) blood gas instrument for point of care testing in the ward. sample collection the samples for creatinine and cystatin c analyses were collected in gel tubes with lithium-heparin (lh pst� ii, bd vacutainer systems, plymouth, uk). table i. gfr markers, drug levels in plasma among the most frequently ordered tests during the study period 2004–2006 (mean number of tests per year). test n 1. blood gases (abl 725) 50725 2. creatinine 3089 3. blood cell counts 3070 4. c-reactive protein 3063 5. activated partial thromboplastin time 2883 6. prothrombin complex 2797 7. bilirubin, direct method 1972 8. alanine aminotransferase (alt) 1972 17. pt-gfr (cystc-calculated) 875 25. vancomycin 182 28. tobramycin 120 31. digoxin 84 36. gentamycin 56 pt-gfr, patient-glomerular filtration rate. 40 m. lipcsey et al. plasma cystatin c and cystatin c-estimated gfr (egfr) plasma cystatin c measurements were performed by latex-enhanced reagent (n latex cystatin c, dade behring, deerfield, il, usa) using a behring bn prospec analyzer (dade behring). the total analytical imprecision of the method was 4.8% at 0.56 mg/l and 3.7% at 2.85 mg/l. the cystatin c results are reported as a cystatin c-estimated gfr (egfr) in ml/min/1.73 m2 (17). gfr in ml/min/1.73 m2 was calculated from cystatin c results in mg/ml by the equation y = 77.24 � (cystatin c result)-1.2623 (17). the reference value for cystatin c-estimated gfr (egfr) was ‡ 80 ml/min/1.73 m2. plasma creatinine and modification of diet in renal disease (mdrd) formula-estimated gfr (egfr) plasma creatinine was analyzed with a modified kinetic jaffé reaction on an architect ci8200� analyzer (abbott, abbott park, il, usa) and reported as si units (mmol/l). the method is isotope dilution mass spectrometry (idms)-calibrated in collaboration with the swedish external quality assurance organization (equalis, uppsala, sweden). the total analytical imprecision of the creatinine method was 4.8% at both 94 and 337 mmol/l. the reference interval for creatinine concentration in adult males was 60– 100 mmol/l. the reference interval for creatinine concentration in adult females was 50–90 mmol/l. egfrmdrd was calculated from creatinine using the mdrd formula: egfr = 175 � (creatinine (mmol/l)/ 88.4)�1.154 � age (years)�0.203 � 0.742 (if female) (18). the factor for african americans was not used. statistical calculations all calculations were performed with the statistical software package statistica 7.0 (statsoft, tulsa, ok, usa). associations between continuous variables were tested with spearman’s rank correlation analysis (r). p values < 0.05 were regarded as statistically significant throughout the study. results most frequently ordered tests the most frequently ordered test was arterial blood gas analysis. creatinine was the second most frequently ordered test on the list (table i). aminoglycosides, vancomycin, and digoxin were the most frequently ordered drug tests. cystatin c and creatinine plasma levels during the studied time period there were 1,838 cystatin c test results. of these requests 1,151 (63%) were for male patients and 687 (37%) were for female patients. the mean age of the patients was 62 years (62 for both males and females). median cystatin c concentration in the cohort was 1.96 mg/l (interquartile range 1.38–2.81 mg/l), and mean value was 2.23 mg/l. for males the median cystatin c concentration was 1.99 mg/l (interquartile range 1.39–2.81 mg/l), and for females the median cystatin c concentration was 1.90 mg/l (interquartile range 1.36–2.83 mg/l). during the time period there were 7,566 requests for creatinine. of these requests 4,573 (60%) were for male patients, and 2,993 (40%) were for female patients. median creatinine concentration in the cohort was 91 mmol/l (interquartile range 67–153 mmol/l), and mean value was 131 mmol/l. for males the median creatinine concentration was 99 mmol/l (interquartile range 73–166 mmol/l), and for females the median creatinine concentration was 80 mmol/l (interquartile range 60–132 mmol/l). a total of 2,247 out of 4,573 test results for males were above the reference interval (49%); 1,234 out of 2,993 test results for females were above the reference interval (41%). in 88% of the patients, plasma cystatin c values indicated reduced kidney function, whereas only 46% of the patients had reduced kidney function as evaluated by their plasma creatinine levels. egfr calculated from cystatin c values median egfrcystatinc in the study group was 30.3 ml/min/1.73 m2 (interquartile range 17.9– 50.1 ml/min/1.73 m2), and mean value was 37.6 ml/min/1.73 m2. for males the median egfr was 29.7 ml/min/1.73 m2 (interquartile range 18.1– 49.7 ml/min/1.73 m2), and for females the median egfrcystatinc was 31.7 ml/min/1.73 m 2 (interquartile range 17.9–51.4 ml/min/1.73 m2). out of the 1,838 egfrcystatinc results, only 146 results (7.9%) were within the reference range, while 92.1% of the results were below the reference interval. out of the 1,838 egfr results, 454 results (25%) were higher than 50 ml/min/1.73 m2, and 1,279 (70%) were higher than 20 ml/min/1.73 m2. there was a strong negative correlation between age and egfr (r = �0.316, p < 0.0001). both males (r = �0.302; p < 0.0001) and females (r = �0.344; p < 0.0001) showed similar negative correlations (figures 1 and 2). gfr in intensive care patients 41 paired plasma cystatin c and creatinine analyses there were 1,777 test requests that contained both cystatin c and creatinine results performed on the same test tube. of these requests 1,110 were for males and 667 for females. these samples were from 734 different patients (median 2.42 samples per patient). median creatinine concentration in this subgroup was 127 mmol/l (interquartile range 87–207 mmol/l), and mean value was 167 mmol/l. for males the median creatinine concentration was 134 mmol/l (interquartile range 95–212 mmol/l), and for females the median creatinine concentration was 111 mmol/l (interquartile range 76–199 mmol/l). a total of 789 out of 1,110 test results for males 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 age (years) c y s ta ti n c c a lc u la te d e g f r a 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 age (years) c y s ta ti n c c a lc u la te d e g f r b 0 20 40 60 80 100 120 140 0 20 40 60 80 100 120 age (years) c y s ta ti n c c a lc u la te d e g f r c figure 1. a: age versus cystatin c-estimated glomerular filtration rate (egfr) for all patients (n = 1,838). b: age versus cystatin c-estimated glomerular filtration rate (egfr) for males (n = 1,151). c: age versus cystatin c-estimated glomerular filtration rate (egfr) for females (n = 687). 42 m. lipcsey et al. were in the reference interval (71%); 418 out of 667 test results for females were in the reference interval (63%). in total 1,207 out of 1,777 plasma creatinine results were pathological (68%). out of the mdrd-estimated gfr results 47% were £80 ml/min/1.73 m2, 30% were £ 50 ml/min/ 1.73 m2, and 7% £20 ml/min/1.73 m2. median mdrd egfr was 48 ml/min/1.73 m2 (table ii). out of the 1,777 cystatin c-estimated gfr results 93% were £ 80 ml/min/1.73 m2, 74% were £50 ml/ min/1.73 m2, and 23% were £20 ml/min/1.73 m2. median mdrd egfr was 39 ml/min/1.73 m2. the spearman rank correlation between the two methods was r = 0.753 (figure 3). mdrd-estimated gfr yielded higher values than cystatin c-estimated gfr (figure 4). the mean difference between egfrmdrd and egfrcystatinc was 39 ml ml/min/1.73 m 2 for egfrcystatinc values £ 60 ml/min/1.73 m 2. discussion plasma creatinine’s role as gfr marker assumes a steady state condition of creatinine distribution. however creatinine levels in plasma and urine are subject to variations during critical illness for several reasons. critically ill patients often have increased body water volume and decreased muscle mass and may have impaired liver function. other factors that may influence creatinine production are trauma, fever, and immobilization. there is also the problem of tubular secretion of creatinine to some extent while low urine production may cause tubular reabsorption. thus, creatinine is not an optimal marker for detection of acute renal failure in intensive care patients; there is on-going search for better gfr markers, and cystatin c and proanp (atrial natriuretic peptide) have been suggested as alternatives (19). one study that compared cystatin c and creatinine reported that cystatin c and creatinine performed equally well in intensive care patients (20). two other studies showed cystatin c to be superior to creatinine. one of the studies reported that serum cystatin c detected acute renal failure 1 to 2 days earlier than creatinine (21). the other study compared cystatin c and creatinine with 24-h urine collection and creatinine clearance. cystatin c was significantly better than creatinine to detect reduced gfr (22). neither of these studies compared gfr estimated from cystatin c and creatinine with gfr measurement using an exogenous marker such as iohexol, iothalamate, or chromium-51-edta. in this study we show that, apart from arterial blood gas analysis, gfr markers are the most frequently requested tests in the icu. furthermore we show that there are considerable differences between the studied markers creatinine, egfrmdrd, and egfrcystatinc for estimating gfr in icu patients. reduced gfr is very common in this icu population, especially when using cystatin c for estimating gfr. there was a strong negative correlation between age and egfrcystatinc, while the correlation between age and creatinine was less pronounced. this could be expected as both gfr and muscle mass decrease 0 200 400 600 800 1000 1200 1400 1600 0 20 40 60 80 100 120 age (years) c re a ti n in e ( m ic ro m o l/ l ) figure 2. age versus plasma creatinine for all patients (n = 7,566). table ii. percentage of patients with gfr below 20, 50, and 80 ml/min/1.73 m2, respectively, based on egfrmdrd and egfrcystatinc calculations. £ 20 ml/ min/1.73m2 £ 50 ml/ min/1.73 m2 £ 80 ml/ min/1.73 m2 egfrcystatinc 30% 75% 92% egfrmdrd 7% 30% 47% gfr in intensive care patients 43 with age. an increased plasma creatinine due to reduced gfr is thus partly disguised by the reduced muscle mass. cystatin c detected twice as high a frequency of reduced gfr in spite of a high degree of correlation between these two methods. plasma creatinine, egfrmdrd, and cystatin c assays can all provide rapid test results. creatinine often over-estimates gfr in patients with slight reductions of gfr (11). it is also difficult to evaluate creatinine in elderly patients with low muscle mass. these patients may have creatinine values in the normal range due to the combination of low muscle mass and reduced gfr. this is in agreement with the present results with 92% of the cystatin c values indicating reduced kidney function, while only 46% of the creatinine test results indicated a reduced kidney function. a considerable gap between the results, 93% versus 47% for egfr with cystatin c and egfr with mdrd, respectively, persisted even when the two methods were performed on the same plasma sample. in this study we used the reference value ‡80 ml/min/1.73 m2 regardless of patient age as a decreased egfr in the range of 50–80 ml/min/ 1.73 m2 was associated with increased mortality in elderly individuals (23–26). we included plasma creatinine in this comparison as many gfr evaluations in sweden are based on the creatinine concentration. the gfr estimations from plasma creatinine values are performed in the wards either by a rough estimate based on the creatinine -100 -50 0 50 100 150 200 0 20 40 60 80 100 mean of egfrmdrd and egfrcystatin c (ml/min/1.73 m 2) e g f r m d r d e g f r c ys ta tin c ( m l /m in /1 .7 3 m 2 ) figure 4. bland–altman bias plot for egfrcystatinc and egfrmdrd. the results are presented as the mean of the two methods (x-axis) plotted against the difference between the two methods (y-axis) (n = 1,777). c re a tin in e ( m ic ro m o l/l ) y = 68,671 x 0,9805 0 100 200 300 400 500 600 700 800 900 1000 0 1 2 3 4 5 6 7 8 9 10 cystatin c (mg/l) figure 3. correlation between cystatin c and creatinine in individual patients (n = 1,668). 44 m. lipcsey et al. value or by using nomogram for the cockcroftgault equation (8). the equations are very rarely performed utilizing computers. the problems associated with calculating gfr in the wards have led to the development of formulas to automatically convert cystatin c in mg/l to a calculated gfr in ml/min/1.73 m2 (17,27). this is in agreement with guidelines that laboratories should calculate and report an estimated glomerular filtration rate (egfr) using the mdrd formula with every request for plasma creatinine concentration (28). in the icu, mdrd and plasma creatinine concentration detected similar numbers of patients with reduced gfr, and both methods detected a lower number of patients with reduced gfr in comparison with cystatin c-estimated gfr. cystatin c has also been suggested to be superior to creatinine measurements for intensive care use although this has also been questioned (16,29). in this study we compared cystatin c as gfr marker with creatinine concentration and egfrmdrd. the study shows that a higher proportion of the intensive care patients had impaired kidney function when using egfrcystatinc. several of the drugs used in the intensive care unit are eliminated by the kidneys, and their turn-over is thus influenced by the gfr. determination of plasma concentrations of digoxin, gentamycin, tobramycin, and vancomycin are all among the top 40 test requests in the intensive care unit, and they are all influenced by the gfr. even though concentration of these drugs can be measured, initial therapy is started based on gfr estimates. furthermore, levels of several widely used pharmaceuticals with renal elimination are not routinely assessed. examples of such drugs used in critical care are h2-antagonists, beta-blockers, and antibiotics such as penicillins and cephalosporins. inadequate dosage of these pharmaceuticals may lead to insufficient therapy or adverse effects, which highlight the need for optimal gfr markers or the advantage of pharmaceuticals without renal elimination. conclusions the present study shows that gfr is frequently assessed in the icu, and many intensive care patients have reduced gfr, and the study emphasizes the need to monitor gfr in this patient group. the use of cystatin c instead of creatinine will increase the number of patients identified with decreased gfr. since most icus use plasma or serum creatinine for gfr monitoring, they may miss several patients with reduced egfrcystatinc. the discrepancy between the two methods may influence the pharmaceutical treatment of the patients and shows that there is a need to improve gfr measurements in intensive care. without an optimal gfr marker, using drugs that are less gfr-dependent should be considered. with the existing methodological differences in mind, there is a need for further studies that compare gfr estimated from cystatin c and creatinine with gfr measurement with an exogenous marker such as iohexol, iothalamate, or chromium-51-edta in intensive care patients. acknowledgements this study was financially supported by wyeth ab and the uppsala university hospital 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insufficiency. kidney int. 2002;61:1567–76. gfr in intensive care patients 45 11. shemesh o, golbetz h, kriss jp, myers bd. limitations of creatinine as filtration marker in glomerulopathic patients. kidney int. 1985;28:830–8. 12. sherman ds, fish dn, teitelbaum i. assessing renal function in cirrhotic patients: problems and pitfalls. am j kidney dis. 2003;41:269–78. 13. grubb ao. cystatin c-properties and use as diagnostic marker. adv clin chem. 2000;35:63–99. 14. tenstad o, roald ab, grubb a, aukland k. renal handling of radiolabelled human cystatin c in the rat. scand j clin lab invest. 1996;56:409–14. 15. dharnidharka vr, kwon c, stevens g. serum cystatin c is superior to serum creatinine as a marker of kidney function: a meta-analysis. am j kidney dis. 2002;40:221–6. 16. villa p, jimenez m, soriano mc, manzanares j, casasnovas p. serum cystatin c concentration as a marker of acute renal dysfunction in critically ill patients. crit care. 2005;9:r139–43. 17. larsson a, malm j, grubb a, hansson lo. calculation of glomerular filtration rate expressed in ml/min from plasma cystatin c values in mg/l. scand j clin lab invest. 2004;64: 25–30. 18. manjunath g, sarnak mj, levey as. prediction equations to estimate glomerular filtration rate: an update. curr opin nephrol hypertens. 2001;10:785–92. 19. trof rj, di maggio f, leemreis j, groeneveld ab. biomarkers of acute renal injury and renal failure. shock. 2006;26: 245–53. 20. ahlström a, tallgren m, peltonen s, pettilä v. evolution and predictive power of serum cystatin c in acute renal failure. clin nephrol. 2004;62:344–50. 21. herget-rosenthal s, marggraf g, hüsing j, göring f, pietruck f, janssen o, et al. early detection of acute renal failure by serum cystatin c. kidney int. 2004;66: 1115–22. 22. delanaye p, lambermont b, chapelle jp, gielen j, gerard p, rorive g. plasmatic cystatin c for the estimation of glomerular filtration rate in intensive care units. intensive care med. 2004;30:980–3. 23. culleton bf, larson mg, wilson pw, evans jc, parfrey ps, levy d. cardiovascular disease and mortality in a community-based cohort with mild renal insufficiency. kidney int. 1999;56:2214–9. 24. fried lf, shlipak mg, crump c, bleyer aj, gottdiener js, kronmal ra, et al. renal insufficiency as a predictor of cardiovascular outcomes and mortality in elderly individuals. j am coll cardiology. 2003;41:1364–72. 25. jernberg t, lindahl b, james s, larsson a, hansson lo, wallentin l. cystatin c: a novel predictor of outcome in suspected or confirmed non-st-elevation acute coronary syndrome. circulation. 2004;110:2342–8. 26. larsson a, helmersson j, hansson lo, basu s. increased serum cystatin c is associated with increased mortality in elderly men. scand j clin lab invest. 2005;65:301–5. 27. grubb a, nyman u, björk j, lindström v, rippe b, sterner g, et al. simple cystatin c-based prediction equations for glomerular filtration rate compared with the modification of diet in renal disease prediction equation for adults and the schwartz and the counahan-barratt prediction equations for children. clin chem. 2005;51:1420–31. 28. johnson d, usherwood t. automated reporting of gfr— coming soon to a laboratory near you! aust fam physician. 2005;34:925–31. 29. wulkan r, den hollander j, berghout a. cystatin c: unsuited to use as a marker of kidney function in the intensive care unit. crit care. 2005;9:531–2. 46 m. lipcsey et al. bok 03-06.indb radial nerve palsy at the elbow 315 received 19 september 2005 accepted 4 october 2005 upsala j med sci 111 (3): 315–320, 2006 radial nerve palsy at the elbow yoshihiro matsubaral, yoshinori miyasaka2, shingo nobuta3, kazushige hasegawa4 1department of orthopaedic surgery, tohoku university, school of medicine 2department of orthopaedic surgery, miyagino hospital 3department of orthopaedic surgery. tohoku rosai hospital 4department of orthopaedic surgery, ishinomaki red cross hospital abstract we studied the clinical features and images along with surgical findings of 8 cages of radial nerve palsy due to a space-occupying lesion (sol) at the elbow. based on image findings, we examined compressing masses and their extent, we contrasted them with operative findings of the radial nerve, and we surmised the site of impairment. compressing masses were ganglions in 6 cages, an old radial head dislocation in 1 case, and engorged radial recurrent vessels in 1 case. the extent of the sols was roughly 15–40 mm from the interepicondylar line and 0–30 mm from the radiohumeral joint. in operative findings, only the posterior interosseous nerve (pin) was compressed in 5 cases, while both the pin and superficial branch were compressed in 3 cases. no apparent correlation between operative findings and the type of palsy was found. the radial tunnel has yet to be defined clearly, but the radial nerve palsy is readily understandable in cases of sols via the definition of the radial tunnel as the tubular structure from the radiohumeral joint to the outlet of the supinator muscle. introduction the posterior interosseous nerve (pin) innervates the extensor digitorum communis (edc), extensor digiti minimi (edm), extensor carpi ulnaris (ecu), abductor pollicis longus (apl), extensor pollicis longus and brevis (epl and epb), and extensor indices proprius (eip). when palsy results, wrist extension is possible, but it deviates radially and prohibits extension of the thumb and extension of the mp joints from the index to the little finger, exhibiting “drop finger.” with radial nerve palsy due to sol at the elbow, there are cases of pin palsy as well as palsy of the superficial radial nerve. in addition, disorders featuring intense pain because of entrapment of the pin have also been reported to differentiate it from lateral humeral epicondylitisl)2). we studied the clinical features and images along with surgical findings in cases of radial nerve palsy due to sol of the elbow. this work clarified the site of impairment and provided a differential diagnosis while also mentioning the anatomical position of the radial tunnel and its significance. 316 yoshihiro matsubara et al. materials and methods materials were 8 cases seen by our hospital and affiliated hospitals over a 14-year period from january 1990 to december 2003; in these cases, surgery was performed for radial nerve palsy due to sol of the elbow (table 1). cases included 5 males and 3 females; age upon initial examination was 31–66 years of age (mean 48 years of age). the affected side was the right in 5 cases and the left in 3. of the 8 total cases, a nerve conduction study was performed on 3 cases; the amplitude of the compound muscle action potential of the edc decreased with stimulation proximal to the site of impairment in 2 cases, and a conduction delay was seen in 1 case. neurolysis and removal of compressing masses along with release at the arcade of fröhse were performed in 7 cases; in the case with an old radial head dislocation, only neurolysis and release at the arcade of fröhse were performed. cases which hour-glass like fascicular constriction in the nerve were excluded in this study. in l case, tendon transfer was added. the symptoms preceding palsy and type of palsy (hirayama classification3), presence/absence of dyskinesia and paresthesia) table 1. overview of cases case sex age side surgery 1 m 50 r neurolysis + tendon transfer 2 f 31 r neurolysis + tumor removal 3 m 61 l neurolysis + tumor removal 4 f 32 r neurolysis + tumor removal 5 m 45 r neurolysis + tumor removal 6 m 43 l neurolysis + tumor removal 7 m 58 l neurolysis + tumor removal 8 f 66 r neurolysis + tumor removal figure 1. method of measuring the extent of sol (mm). the area proximal to the line is negative. radial nerve palsy at the elbow 317 in these cases were studied. in addition, compressing masses and their extent were examined based on image findings. measurement used the distance from the interepicondylar line (hueter’s line) and the distance from the radiohumeral joint (fig. l). this was contrasted with operative findings for the radial nerve and the lesion was surmised. results compressing masses were ganglions in 6 cases, an old radial head dislocation in 1 case, and engorged radial recurrent vessels in 1 case. symptoms preceding palsy were heaviness of the elbow in 3 cases and awareness of a mass in 2 cases. no cases complained of neck pain, pain similar to lateral humeral epicondylitis, fever, or cold symptoms. the tyre of palsy was type i (drop finger & thumb) in the hirayama classification in all cases. in 7 cases isolated pin palsy was noted, in addition to palsy of the superficial radial nerve noted in 1 case (table 2). the extent of sol and operative findings was roughly 15–40 mm from the interepicondylar line and 0–30 mm from the radiohumeral joint, as shown in table 3. table 2. compressing masses, symptoms, and type of palsy. pin: posterior interosseous nerve, srn: superficial radial nerve case cause of palsy initial symptom type of nerve palsy 1 dislocation of radial head none drop finger & thumb, pin 2 ganglion heaviness of the elbow drop finger & thumb, pin 3 ganglion none drop finger & thumb, pin 4 ganglion heavinell of the elbow drop finger & thumb, pin + srn 5 ganglion none drop finger & thumb, pin 6 ganglion none drop finger & thumb, pin 7 ganglion heaviness of the elbow drop finger & thumb, pin 8 venous plexus none drop finger & thumb, pin table 3. extent (mm) and operative findings case distance from the interepicondylar line (hueter’s line) distance from the radiohumeral joint line perioperative findings (compression site) 1 16–27 0–11 pin, srn 2 16–38 0–22 pin 3 5.7–44 0–29 pin 4 15–44 0–30 pin, srn 5 40–55 15–30 pin 6 26–40 12–24 pin 7 17–30 –5.0–25 pin, srn 8 17–30 5.0–25 pin 318 yoshihiro matsubara et al. in operative findings, only the pin was compressed in 5 cases while both the pin and superficial branch were compressed in 3 cases (table 3). no apparent correlation between operative findings and the type of palsy was found. a typical case is shown in fig. 2. discussion orthopedic disorders causing drop finger include, in addition to pin palsy, cervical myelopathy (radiculopathy) and extensor tendon rupture. in our cases, some were misdiagnosed upon initial visit. examination of the patient’s medical history such as neck pain is fundamental when differentiating pin palsy from cervical disorders, and assessment of deep tendon reflex and paresthesia, particularly in a manual muscle test is critical. in addition, differentiation from extensor tendon rupture can be assessed by dynamic tenodesis. in some cases of rheumatoid arthritis, assessment of range of motion is difficult due to wrist joint deformity; differentiating from pin palsy due to synovial cyst at the elbow is more difficult4). a needle emg is useful in such cases. diagnosis of cases complicated with cervical lesions and pin palsy figure 2. case 7. a 58-year-old man was seen when he noted drop finger on the left hand. there was a 2:p decline in muscular strength in the ecu, edc, and epl. there was no paresthesia. in mrt2 weighted images, a bilobular, hyperintense area ( ) was seen distal to the radiohumeral joint. surgery was neurolysis and tumor removal along with release at the arcade of fröhse. the mass was a ganglion. postoperatively, muscular strength improved to 4:g. radial nerve palsy at the elbow 319 is considered difficult. in the cases of greater decline in muscular strength and muscular atrophy in muscles innervated by the pin, confirrning repeated use of the upper limbs preceding palsy and a history of sharp pain in the elbow, looking for a mass in the radial head and a tinel’s sign, and performing an electrophysiological study are necessary. the radial nerve branches into the motor branch (pin, deep branch) and sensory branch (superficial branch) at the elbow. konjengbam et al.5) stated that based on anatomical findings the region of bifurcation is an average of 8.93 mm distal to the interepicondylar line and an average of 8.47 mm proximal to the radiohumeral joint. the extent of sol in our cases was roughly 15–40 mm and 0–30 mm, respectively, so damage is likely to have been received beyond the region of bifurcation. in operative findings, compression of both the deep and superficial branches was seen in 3 cases although paresthesia was noted in only 1 of the 8 cases. the cause for this is thought to be because compression from the sol was intensified since the pin was fixed by the arcade of frohse6). ogino et al.7) stated that the force of extension on the pin through pro-supination of the forearm may be associated with palsy, although none of our cases complained of exacerbated palsy accompanying forearm movement. radial tunnel syndrome is reported as a pin disorder that causes intense neuralgia but exhibits no palsyl)2). in our cases of radial nerve palsy due to sol, 3 cases complained of heaviness and discomfort of the elbow but no cases complained of chronic pain. the authors previously reported a case of surgery for radial nerve entrapment with synovial osteochondromatosis8). this case was compared to surgical findings for the current 8 cases, but differences in pathology were not noted. although the nerve compression was the same, pathology in some cases exhibited palsy while it caused only pain in others. elucidation of this pathology is a topic for the future. the inlet of the supinator muscle, known as the arcade of fröhse, is a site where the pin is readily compressed. there are views that this site is the radial tunnel, although lister et al.1) and roles et al.2) have described the tubular structure from the radiohumeral joint to the outlet of the supinator muscle as the radial tunnel. we figure 3. radial tunnel. 320 yoshihiro matsubara et al. also propose defining the radial tunnel that they cited as a site prone to radial nerve palsy. this site is a fibrous-osseous canal consisting of muscle, tendons, ligaments, the articular capsule, and the radial head. similar to carpal tunnel, the appearance of palsy is understandable in cases of pinching at the arcade of fröhse as well as sols (fig. 3). conclusions 1. the clinical features and images along with surgical findings of 8 cases of radial nerve palsy due to sol of the elbow were studied and their characteristics and differential diagnosis described. 2. no apparent correlation between extent of sol and the type of palsy was found. 3. the radial tunnel was defined as the tubular structure from the radiohumeral joint to the outlet of the supinator muscle. references l. lister gd, belsole rb (1979) the radial tunnel syndrome. j hand surg 4:52–59. 2. roles nc, maudsley rh (1972) radial tunnel syndrome. j bone and joint surg 54-b:499–508. 3. hirayama t, takemitsu y, tada h, katsuki m, yoshida e (1994) an analysis of the non-traumatic posterior interosseous nerve palsy. j jpn soc surg hand 11(3):385–387 (in japanese). 4. westkaemper jg, varitimidis se, sotereanos dg (1999) posterior interosseous nerve palsy in a patient with rheumatoid synovitis of the elbow; a case report and review of the literature. j hand surg 24-a:727–731. 5. konjengbam m. elangbam j (2004) radial nerve in the radial tunnel: anatomic sites of entrapment neuropathy. clin anatomy 17:2 1–25. 6. morimoto k, furuse k, maeyama i, yamane m (1982) two cases of paralysis of deep branch of radial nerve due to ganglion at the elbow. orthopedics & traumatology. 31: 96–98 (in japanese). 7. ogino t, minami a, kato h, takahara m, kobayashi m, takabatake t (1990) radial nerve palsy and ganglion at the elbow. j jpn soc surg hand 6(6): 1024–1030. 8. miyasaka y, abe y (1996) a case of contracture of elbow joint with synovial osteochondromatosis (pathomechanism of radial tunnel sydrome). j jpn soc surg elbow 3(1): 117–118 (in japanese). corresponding author: yoshihiro matsubara, department of orthopaedic surgery, tohoku university school of medicine l-l seiryo-machi, aoba-ku, sendai, miyagi 980-8574 japan tel: +81-22-717-7245 fax: +81-22-717-7248 e-mail: ymatsu@mail.tains.tohoku.ac.jp upsala j med sci 95: 275-277, 1990 plans for obtaining a “nordic list” of specifications carl-henric de verdier department of clinical chemistry, university of uppsala, uppsula, sweden in several countries work is going on in order to establish numerical values for quality specifications for clinical chemical (and haematological) analyses and investigations. in some countries this work is connected with proficiency testing organized by a controlling authority or a professional society as proficiency test must have specified rules for approval ( 2 , 4 ) . in the nordic countries several different programs for external quality assurance have been running (1). what is acceptable quality is, however, left to the head of the laboratory as a head physician to decide. new trends within laboratory medicine with more decentralized laboratories, more economical responsibility delegated to the laboratory and more competition between laboratories call for guidelines for quality specifications. this new attitude has also influenced the new scandinavian recommendations for quality control and quality assurance (5) as indicated in the third chapter of this report. considerations in the preparation of a nordic list the board of the nordic clinical chemistrv droiect (nordkem) has earlier and in the preliminary work for this project expressed its wish to let the professionals of the clinical laboratories, the doctors in primary care and the clinicians prepare a list of quality specifications. a lot of questions must be ventilated in connection with the preparation of such a list. table 1 presents a checklist which might be of value when preparing a list of quality specifications for the nordic countries. 275 table 1. checklist for the preparation of a nordic list of quality specifi cations for clinical chemical laboratories. the list of quality specifications must be structured around: system--component, testing/evaluation of different methods/investigations clinical situations (concentration ranges) type of quality specification: 1) clinical needs 2) clinical quality specifications 3 ) analytical quality goals (te,) and in the local laboratory 4 ) laboratory quality specifications (see more in chapter 3, fig. 1). the expression of the quality specifications. the 95 % probabil ity range within which results of investigations should be localized. starting from the analytical side that means e g (where the abbreviations have the following meaning: te, total allowable error = aae, se, critical systematic error expressed as a factor times s , re, critical random error and s inherent random imprecision). starting from the side of clinical needs that means e g te, = bias + se,.s + [component of re,] a > d?. . 1.65 (cv,’ + cv,’ + cv,’)” ( 3 ) (where cv means coefficient of variation, b within-individual, p pre-analytical and a analytical sources of variation; 0 is an significant difference in serial results during a monitoring situation). also other ways of calculating clinical needs and clinical quality specifications using e g decision analysis or biomedical models (see chapter 3 ) are of interest. in accordance with “international vocabulary of basic and general terms in metrology” (vim) (6) and other relevant standards and guidelines. * complementation with matrix effects (specified) and unspecific reactions (specified). existing guidelines for the measurement of such effects should be improved. 276 * * 1. presentation of the list in a standardized form and possibly also as a data base. which components should be included? primarily the 40 most frequently analyzed components and some more analytes of principal interest. only investigations that have a significant role in the clinical process. evaluation of the list with quality specifications? submission to the scand j clin lab invest and to the newsletters of the national societies of clinical chemistry in scandinavia and of the nordic society of clinical chemistry? sending draft for consideration to the national societies for clinical chemistry in the nordic countries? discussion at the next nordic congress in 1992? references in de verdier c-hi aronsson ti nyberg a: (eds): quality control in clinical chemistry efforts to find an efficient strategy, helsinki, nordkem, 1984, p 1-241 2. ehrmeyer ss, laessig rh. use of alternative rules (other than the 1 2s) for evaluating interlaboratory performance data. clin chem (1988);34:250-256. 3. fraser cg, hyltoft petersen pi larsen ml. setting analytical goals for random analytical error in specific monitoring situ ations. clin chem (1990);36:1625-1628. 4. laessig rh, ehrmeyer ss. use of computer modelling to predict magnitude of interlaboratory error tolerated by proposed cdc interlaboratory proficiency testing performance criteria. clin chem 5. scandinavian society for clinical chemistry. general scandina vian recommendation on quality control an quality assurance in clinical chemistry. scand j clin lab invest (1990);50:225-227. 6. zender r. measurements in biological systems. metrological principles and terminology. scand j clin lab invest (1989);49 suppl (1988);34:1849-1852. 193 : 3-10. correspondence: professor carl-henric de verdier department of clinical chemistry, uppsala university postal address: university hospital s-751 85 uppsala, sweden. 277 upsala j med sci 95: 257-258, 1990 external quality assurance model for improvement of specificity and assuracy of serum hormone determinations in the nordic countries a. ruokonen, p. hyltoft-petersen and 0. blaabjerg departments o/ clinical clwmistry, oulun yltopisto, finland and odense university hospilal, odense, denmark previous external quality assurance projects of hormone determi nations in the nordic countries and other areas have shown the wide scatter of results without essential improvement during many years. reasons for the wide scatter like wrong calibration, unspecific reactions and various factors causing imprecision have been mainly characterized but until now very little has been done to eliminate them. in this project we try to reduce their influence. the purpose of this project is to: set up a new quality assurance model for improvement of specificity and accuracy of hormone determinations in the nordic countries to test the model for serum thyrotropin (tsh), free thyroxine (ft,) thyroxine (t,) , triiodothyromine (t,) and cortisol (c) determinations to define practical goals of accuracy and specificity for to maintain the quality, if acceptable accuracy and specifi these hormone determinations city are reached by the quality assurance model. control material (quality assurance samples and the calibrator) is frozen human serum and its t, and c concentrations have been determined by gc-ms. quality assurance samples are used to evaluate specificity and calibration bias (at three concentration levels) of the analytical method of each laboratory. if needed, the calibrator is used for recalibration. there are four kinds of quality assurance samples (fig 1). one of the controls is a normal serum pool ( a ) with known con centrations of hormones, another is the same pool ( b ) with no or very low hormone concentrations, the third pool (c) is the same 17 908573 257 as pool a , but it also contains constituents expected to cause nonspecific reactions. samples a and b are used to test accuracy and samples c and d to test specificity. in result reports quality goals have been defined and comments are given on reproducibility, linearity, specificity and bias of the methods of each laboratory. this project is carried out after the protein project. normal serum samples (tsh, ft4, t4, t3. c) i fis 1. like sample a + constituents expected to cause unspecific reactions ( t 4 , rt3, 11-deoxycortisol, tbg, prealbumin) l quality assurance model like sample a but with no or very lou hormone concentrations and no constituents expected to cause unspecific reactions (test of zero or low) samples (a and b) to test accuracy samples (c and d) to test specificity like sample b + constituents expected to cause unspecific reactions (t4, rt3, 11-deoxycortisol, tbg, prealbumin) 1 b l d i correspondence: dr. aim0 ruokonen, department of clinical chemistry, oulun yliopisto, 90220 oulu, finland 258 tf-iups160018 184..191 original article do subfertile women adjust their habits when trying to conceive? lana salih joelssona,b, anna berglundc, kjell wånggrena,d, mikael loode, andreas rosenbladb and tanja tyd�enf adepartment of women’s and children’s health, uppsala university, uppsala, sweden; bcentre for clinical research, uppsala university, county council of v€astmanland, v€astmanland county hospital, v€asterås, sweden; cthe national centre for knowledge of men’s violence against women, uppsala university, uppsala, sweden; ddepartment of clinical science, intervention and technology, karolinska institutet, stockholm, sweden; edepartment of women’s health, fertility unit, €orebro university hospital, €orebro, sweden; fdepartment of public health and caring sciences, uppsala university, uppsala, sweden abstract aim: the aim of this study was to investigate lifestyle habits and lifestyle adjustments among subfertile women trying to conceive. materials and methods: women (n ¼ 747) were recruited consecutively at their first visit to fertility clinics in mid-sweden. participants completed a questionnaire. data were analyzed using logistic regression, t tests, and chi-square tests. results: the response rate was 62% (n¼466). mean duration of infertility was 1.9 years. during this time 13.2% used tobacco daily, 13.6% drank more than three cups of coffee per day, and 11.6% consumed more than two glasses of alcohol weekly. in this sample, 23.9% of the women were overweight (body mass index, bmi 25–29.9 kg/m2), and 12.5% were obese (bmi �30 kg/m2). obese women exercised more and changed to healthy diets more frequently than normal-weight women (odds ratio 7.43; 95% confidence interval 3.7–14.9). six out of ten women (n ¼ 266) took folic acid when they started trying to conceive, but 11% stopped taking folic acid after some time. taking folic acid was associated with a higher level of education (p < 0.001). conclusions: among subfertile women, one-third were overweight or obese, and some had other lifestyle factors with known adverse effects on fertility such as use of tobacco. overweight and obese women adjusted their habits but did not reduce their body mass index. women of fertile age would benefit from preconception counseling, and the treatment of infertility should routinely offer interventions for lifestyle changes. article history received 8 february 2016 revised 3 april 2016 accepted 4 april 2016 keywords alcohol consumption; assisted reproduction; diet; infertility; lifestyle; obesity; pregnancy; tobacco use introduction a healthy lifestyle is known to be of importance for women, especially when they are planning to get pregnant, since negative lifestyle factors may contribute to impaired reproduction and a lower chance of having a healthy child. such negative factors include smoking, drinking alcohol and overuse of other drugs, underweight or overweight, unhealthy diet, harmful infections, exposure to environmental hazards, and an adverse medical history. these issues might be particularly important for women seeking treatment for infertility (1–3). another important factor affecting female fertility is age. women in europe have delayed their childbearing considerably during the past decades, and the mean age for childbearing is currently 30 years or more in almost half of the member states of the european union (4,5). in sweden, the mean age for having the first child was 28.5 years in 2013 compared with 24 years in 1973. the postponement of the birth of the first child is due to different reasons throughout europe. interviews with swedish men and women who had an academic education showed that a majority of them wanted children, but postponed their parenthood as a rational adaptation to changes in society such as having a sound personal economy. participants believed that fertility could be restored by assisted reproductive technologies (art) (6). a consequence of this demographic trend seems to be associated with an increase in the incidence of female subfertility (7). major advances have been achieved in the field of infertility treatment and assisted reproductive technology (art) during the past 25 years, leading to increased pregnancy rates. however, researchers and clinicians are still trying to identify additional factors that might impact on live birth rates. also in art, factors such as nutrition and diet, exercise, and the use of tobacco and alcohol, as well as being overweight or underweight, might have an impact on outcomes (2,8). the effectiveness of preconception care and counseling (pcc) in general, and for couples with a fertility problem in particular, has been demonstrated (9,10). studies have shown that pcc reduces costs, improves reproductive outcomes, promotes healthy pregnancies, and reduces pregnancy complications and poor neonatal birth outcomes. there is a lack of homogeneous guidelines, recommendations, and services for preconception health care in european contact lana salih joelsson lana.salih@kbu.uu.se women’s and children’s health, uppsala university, se-751 85 uppsala, sweden supplemental data for this article can be accessed here. � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 3, 184–191 http://dx.doi.org/10.1080/03009734.2016.1176094 http://www.tandfonline.com/doi/full/10.1185/03007995.2016.1176094 http://creativecommons.org/licenses/by/4.0/ countries (11,12). the european society of human reproduction and embryology (eshre) guideline development group recommends that fertility staff should consider providing women with information about the impact of lifestyle habits and supporting them in making necessary changes (13). some counseling about lifestyle is generally included in initial consultations in fertility clinics. brochures and hand-outs about lifestyle are generally available at fertility clinics in sweden, but this information is usually not handed out or explained to the patients actively. effective strategies and support for making healthy changes are not routinely offered. despite the existing knowledge about the association of risk of fertility problems and adverse pregnancy outcomes with unhealthy lifestyles (14,15), it is largely unknown to what extent subfertile women adjust their habits during the time they are trying to conceive. effective individual counseling to subfertile women demands an understanding about what measures women take to increase the chances of conception. the aim of this study was therefore to investigate lifestyle habits among subfertile women trying to conceive in relation to their background characteristics. it was hypothesized that age, duration of infertility, bmi (body mass index), immigration status, education, occupation, income, and information searching were associated with lifestyle habits and lifestyle adjustments among subfertile women trying to conceive. material and methods design this was a cross-sectional study constituting baseline measurements for a planned longitudinal cohort study. the followup is scheduled to take place 2 years after the return of the questionnaire. participants and recruitment women attending 10 fertility clinics in mid-sweden participated. eligible women (n ¼ 782) were asked to participate in the study at their first visit to the clinic. the time of this first visit to the clinic is defined as ‘baseline year’. data collection started in may 2013 and ended in march 2015 with a planned follow-up 2 years later. being able to read and write swedish and having no previous infertility diagnosis were inclusion criteria for the study. midwives gave oral and written information about the study to eligible women who fulfilled the inclusion criteria. in 19 cases, women were excluded from the study either because they reported having a female partner or did not answer the question about lifestyle changes while they tried to conceive. thirty-five women declined to participate in the study. those who agreed to participate received a questionnaire to complete at the clinic or at home and return it by mail in a prepaid envelope. the midwife registered a telephone number or an email address for participating women, and a reminder was sent by text message or email to those who had not returned the questionnaire within 2 weeks. the completed questionnaires were returned including the signed informed consent, which immediately upon receipt was separated to be archived securely and safely. during the study period, the midwives listed all registrations, numbers of women approached, and their response (accepted/declined). of the 782 eligible women, 747 (95.5%) agreed to participate in the study, and 466 of these (62.4%) completed the study. a flow chart of the procedure is presented in figure 1. eligible cases n = 874 approached n = 782 accepted n = 747 completed the questionnaire n = 466 included cases n = 448 excluded cases n = 19 not returned n = 281 declined n = 35 lack of time n = 5 personal issues n = 11 type of questions n = 4 not specified n = 15 not approached n = 92 forgot to ask n = 20 lack of time n = 19 non-swedish speaking n = 53 figure 1. flow chart of the study procedure. upsala journal of medical sciences 185 of the group of women (n¼281) who had agreed to participate in the study but did not return the questionnaire, 14% (n¼39) were analyzed using information from patient records with regard to age, infertility duration, bmi (weight/ height2 in kg/m2), tobacco use, and alcohol consumption. the results showed no significant differences between this group and the group of women who completed the study. questionnaire the questionnaire (available online) consisted of 71 items and included validated instruments for pregnancy planning, stress, anxiety, postpartum depression, sexual satisfaction, exercise habits, alcohol consumption, tobacco use, and diet (16–22). researchers and clinicians reviewed the questions and conducted a pilot study with 30 women, after which some items were adjusted. the following themes were covered: demographic characteristics, immigrant background, medical and reproductive history, and methods for seeking information about infertility. the questionnaire also included specific questions on the type of lifestyle factors at baseline year and number of lifestyle changes made while the women had been trying to conceive. in the following sections we present items relevant to the aim of the study. background characteristics questions were asked about background characteristics: age; partner’s age; height and self-reported weight at baseline year as well as 1 year earlier; marital status; duration of current relationship; country of birth; parents’ country of birth; level of education; occupation and household income/month; understanding of swedish language; and duration of infertility. background lifestyle and lifestyle changes questions regarding lifestyle covered health-promoting lifestyle changes: intake of folic acid and/or multivitamin supplements; daily tobacco use; weekly alcohol consumption; daily coffee consumption; type of diet; and weekly physical activity graded in number of hours. the participants were also asked to describe any changes to a healthier diet in free text. access to sites for information-seeking and type of professional counseling were also included. questions covered baseline year as well as the time from when they started to plan for pregnancy. ethical considerations the study was approved by the regional ethical review board in uppsala (2012/278). statistical analysis data were analyzed using ibm spss statistics (version 20; ibm corp., armonk, ny). data regarding age, household income, duration of infertility, and current bmi in kg/m2 were treated as continuous variables and are presented as the mean and standard deviation (sd), while the duration of the relationship and frequency of physical activity were treated as ordinal variables and are presented as the median and interquartile range (q1, q3). differences in background characteristics between women with and without lifestyle changes during the time when they were trying to conceive were analyzed using the mann–whitney non-parametric u test for ordinal variables, student’s t test for continuous variables, and pearson’s chisquare test for categorical variables. for all statistical analyses, a two-sided p value <0.05 was considered significant (table 3). logistic regression was used to analyze the effect of background variables (independent variables) on lifestyle changes (dependent variable). results are reported as the odds ratio (or) with 95% confidence interval (ci) (table 4). the variables with significant values in table 3 were included as covariates in the multivariate logistic regression described in table 4. results the mean age of the studied women was 30.2 years (range 19–42 years), and they had a mean duration of infertility of 1.9 years. about half of the women (54.7%, n ¼ 245) reported that they had been in a stable relationship for more than 4 years. in 71.4% of the women (n ¼ 320), the partners jointly took the decision to start trying to conceive; in 25% (n ¼ 112) the woman decided by herself; and in 3% (n ¼ 14) the partner had taken the initiative. the couples had vaginal intercourse on average twice a week, and 67% (n ¼ 300) used ovulation tests with a mean of 18 tests (sd 26.6, range 1–200) per woman. one out of three women (n ¼ 132) had set a time frame for becoming pregnant of 1 year, and 19% (n ¼ 85) had considered the possibility of adoption. fourteen percent (n ¼ 63) were born outside sweden, and 91% stated that their understanding of the swedish language was very good (table 1). lifestyle during the pregnancy planning period almost half of the women (48.2%, n ¼ 216) were taking folic acid supplements at their first visit to the clinics. however, 59.4% (n ¼ 266) had started to take folic acid by the time they started to plan for pregnancy (table 2). daily tobacco use was reported by 13% of the women (n ¼ 59) and was more common among younger women (or 0.93; 95% ci 0.88–0.99) and women with a lower level of education (or 2.98; 95% ci 1.6–5.5), as well as among women with lower household income (or 0.98; 95% ci 0.96–0.99). six out of ten women (61.6%) consumed 0–1 standard glasses of alcohol per week. being older was the only background characteristic of the women who were consuming alcohol weekly (p¼0.001). lifestyle modifications one-third of the women in our study made few (i.e. 0–1) health-promoting lifestyle modifications during the time they were trying to conceive. one out of four reduced their consumption of tobacco, and 37% changed their alcohol-drinking habits (table 2). 186 l. salih joelsson et al. associations between background characteristics and health-promoting actions/lifestyle changes the univariate analyses (table 3) showed association between household income, level of education, duration of infertility, country of birth, parents’ country of birth, bmi, the intensity level of information searching, and taking folic acid supplements (p < 0.05). however, the multivariate logistic regression model (table 4) showed that women with a higher education level and higher level of searching for information about fertility took folic acid more often (or 1.8; 95% ci 1.1–2.9 and or 2.1; 95% ci 1.2–3.8, respectively). the multivariate logistic regression model did not show any significant associations with other variables. age, household income, education level, and occupation in our analysis associated with decrease or stop in tobacco use in the univariate test. the only background characteristic remaining in the multivariate logistic regression model was that change in tobacco use was more common among women with a lower education level (or 2.2; 95% ci 1.3–3.7). the women were divided into three groups according to their bmi (in kg/m2): underweight to normal with bmi <24.9 in 60.9% (n¼273); overweight with bmi 25–29.9 in 23.9% (n¼107); and obese with bmi �30 in 12.5% (n¼56). there was an association between the level of physical activity/ changing to a more healthy diet and bmi, with an overrepresentation among overweight and obese women (p < 0.001). the obese women exercised more and changed to healthy diets more frequently (or 7.43; 95% ci 3.7–14.9), but despite their efforts they showed no significant decrease in weight compared to their reported weight 1 year earlier. the reported level of understanding of the swedish language was the only factor in our analysis that could be associated with a decrease in alcohol consumption (p¼0.019). age, country of birth, parents’ country of birth, understanding of swedish, information-searching intensity, and higher bmi were associated with the number of health-promoting actions taken by the women during the time they were trying to conceive (p < 0.05). table 1. background characteristics and lifestyles of the 448 women included in the study. characteristic n (%) age (years), mean (sd)a 30.2 (4.8) partner’s age (years), mean (sd) 33.4 (6.8) duration of current relationship (years), median (q1, q3) 5 (3, 8) household income/month (sek) mean (sd) 45,664 (21,541) median (q1, q3) 47,000 (30,000, 59,000) currently working (>50%), n (%) 362 (80.8) level of education, n (%) college/university 242 (54.0) non-college/non-university 201 (44.9) body mass index (bmi) in kg/m2, mean (sd) 24.6 (4.9) normal, �25, n (%) 273 (60.9) overweight, 25–29.9, n (%) 107 (23.9) obese, �30, n (%) 56 (12.5) country of birth, n (%) born in sweden 377 (84.2) born outside sweden 63 (14.1) parent (one or two) born outside sweden, n (%) 78 (17.4) understanding of swedish language, n (%) very good 408 (91.1) quite good/neither good nor bad 33 (7.4) duration of infertility (years) mean (sd) 1.9 (1.7) median (q1, q3) 1.4 (1.2, 2.0) taking folic acid supplementation, n (%) 216 (48.2) taking vitamin supplementation, n (%) 119 (26.6) weekly alcohol consumption, n (%) 60 (13.4) amount of alcohol consumption/week, n (%) >1 glass/week 98 (21.9) >2 glasses/week 52 (11.6) using tobacco, n (%) 78 (17.4) using tobacco daily, n (%) 59 (13.2) eating a balanced diet, n (%) 325 (72.5) vegetarian 20 (4.4) gi diet 15 (3.3) lchf 15 (3.3) coffee >1 cup/day, n (%) 225 (50.2) coffee >3 cups/day, n (%) 61 (13.6) weekly physical activity in hours, median (q1, q3) 3 (2, 4) women who sought information about fertility, n (%) 442 (98.7) number of information sites consulted, n (%) 0–1 115 (25.7) 2 134 (29.9) 3–9 198 (44.2) type of information site, n (%)a mother care unit 158 (35.3) blogs 293 (65.4) health care guide (1177)b 245 (54.7) women who obtained professional counseling, n (%) 316 (70.5) type of professional counselor, n (%) midwife 161 (35.9) specialist 138 (30.8) other 30 (6.7) number of negative lifestylesc 0 102 (22.8) 1 176 (39.3) 2 110 (24.6) �3 60 (13.4) amultiple answer possible. b1177: telephone number for health care guide. cnot taking folic acid; using tobacco; consuming alcohol or coffee; bmi �25 kg/m2. gi: glycemic index; lchf: low-carb high-fat diet; sd: standard deviation; sek: swedish krona. table 2. reported health-promoting lifestyle changes during the time when the women were trying to conceive. reported health-promoting lifestyle changes n (%) started taking folic acid supplement yes 266 (59.4) no 182 (40.6) change in coffee-drinking habits quit drinking 17 (3.8) cut down 93 (20.8) no change 338 (75.4) change in alcohol-drinking habits stopped drinking 59 (13.2) reduced drinking 105 (23.4) no change 284 (63.4) change in tobacco usea quit tobacco 60 (13.4) reduced tobacco use 49 (10.9) no change 339 (75.7) change to more healthy diet yes 92 (20.5) no change in diet 356 (79.5) physical activity more 143 (31.9) less 16 (3.6) no change 289 (64.5) number of reported health-promoting lifestyle changes few (0–1) 146 (32.6) medium (2) 128 (28.6) many (3–6) 174 (38.8) atobacco use¼smoking and/or taking snuff. upsala journal of medical sciences 187 discussion this is the first large-scale swedish study focusing on lifestyle behaviors among women with infertility problems. one interesting finding was that approximately 50% of the women in the study retained habits with negative effects on fertility, although a majority made initially one or more health-promoting lifestyle changes. one would assume that women not succeeding to conceive would be motivated to learn more about this and try to avoid less favorable habits. screening for lifestyle factors and correcting those negatively affecting reproductive health should be emphasized in the initial stage of infertility treatment and preferably when women start their planning to become pregnant. obese women have an increased risk of subfecundity and infertility (23), and miscarriage rates and pregnancy complications are higher in this group (24,25). in our sample, as many as one-fourth were classified as overweight and 12.5% as obese, which is similar to the distribution of weight found among women in early pregnancy in antenatal care in sweden (26). a recent study has shown that using motivational interviewing in a weight control program for pregnant women resulted in a significant weight loss in treated women compared to a control group (27). this indicates that a meaningful weight loss can be achieved with effective interventions. in our study, women with a low level of education had changed their tobacco use habits more, probably because they were the ones using more tobacco in the first place. they also tended to be younger and having a lower household income than women who made no changes in tobacco-use habits. this is worrying because the negative consequences of tobacco use, such as an increased risk of infertility, pregnancy loss, preterm delivery, and fetal growth retardation, are well established (28–30). the use of tests for predicting the time of ovulation was extensive among women in our study, possibly because many women have a poor understanding of their menstrual cycle and of the timing of ovulation. it has been suggested that timing intercourse to coincide with ovulation by using ovulation tests might lead to emotional distress; however, the evidence to support this hypothesis is limited (31). another interesting result in our study was that six out of ten women started to take folic acid supplements when they started their attempts to become pregnant, but 11% stopped after some time. other studies have shown that only three out of ten women, regardless of whether the pregnancy was being well planned, complied with the recommendation from public table 3. results for univariate tests with type of health-promoting lifestyle change as outcome in relation to background characteristics. started to take folic acid changes in tobacco use (decrease/stop) change to more physical activity/ healthier diet yes n¼266 (59.4) no n¼182 (4.6) p value yes n¼105 (23.4) no n¼343 (76.6) p value yes n¼186 (41.5) no n¼262 (58.5) p value age (years), mean (sd) 30.6 (4.8) 29.9 (4.9) 0.142 28.9 (4.8) 30.7 (4.8) 0.001 29.9 (5) 30.5 (4.7) 0.204 duration of infertility (years), mean (sd) 1.5 (3.4) 2.1 (1.9) 0.028 1.74 (4.5) 1.76 (2.3) 0.958 2.0 (3.6) 1.6 (2.3) 0.192 household income/month (sek), mean (sd) 48,596 (21,466) 41,321 (20,974) 0.001 38,978 (20,749) 47,719 (21,394) <0.001 43,193 (22,706) 47,434 (20,532) 0.047 level of education, n (%) <0.001 <0.001 0.08 college/university 165 (68.2) 77 (31.8) 36 (14.9) 206 (85.1) 92 (38.0) 150 (62.0) non-college/ non-university 98 (48.8) 103 (51.2) 68 (33.8) 133 (66.2) 93 (46.3) 108 (53.7) occupation, n (%) 0.21 0.005 0.29 working >50% 220 (60.8) 142 (39.2) 75 (20.7) 287 (79.3) 146 (40.3) 216 (59.7) not working 46 (53.5) 40 (46.5) 30 (34.9) 56 (65.1) 40 (46.5) 46 (53.5) current bmi (kg/m2), mean (sd) 24.2 (4.2) 25.3 (5.8) 0.036 24.4 (4.3) 24.8 (5.0) 0.531 26.4 (5.7) 23.4 (3.8) <0.001 bmi (three categories), n (%) 0.049 0.83 <0.001 normal 166 (60.8) 107 (39.2) 66 (24.2) 207 (75.8) 81 (29.7) 192 (70.3) overweight 68 (63.6) 39 (36.4) 23 (21.5) 84 (78.5) 59 (55.1) 48 (44.9) obese 25 (44.6) 31 (55.4) 14 (25.0) 42 (75.0) 42 (75.0) 14 (25.0) country of birth, n (%) 0.001 0.899 0.74 sweden 237 (62.9) 140 (37.1) 87 (23.1) 290 (76.9) 158 (41.9) 219 (58.1) born outside sweden 26 (41.3) 37 (58.7) 15 (23.8) 48 (76.2) 25 (39.7) 38 (60.3) parents’ country of birth (one or both), n (%) 0.008 0.31 0.48 born in sweden 225 (62.5) 135 (37.5) 86 (23.9) 274 (76.1) 154 (42.8) 206 (57.2) born outside sweden (one or both) 36 (46.2) 42 (53.8) 16 (20.5) 62 (79.5) 30 (38.5) 48 (61.5) understanding swedish, n (%) 0.07 0.58 0.31 very good 249 (61.0) 159 (39.0) 94 (23.0) 314 (77.0) 173(42.4) 235 (57.6) quite good/ neither good nor bad 15 (45.5) 18 (54.5) 9 (27.3) 24 (72.7) 11 (33.3) 22 (66.7) information searching <0.001 0.82 0.038 0–1 sites 53 (19.9) 62 (34.3) 29 (27.6) 86 (25.1) 39 (21.0) 76 (29.1) 2 sites 73 (27.4) 61 (33.7) 32 (30.5) 102 (29.8) 52 (28.0) 82 (31.4) � 3 sites 140 (52.6) 58 (32.0) 44 (41.9) 154 (45.0) 95 (51.1) 103 (39.5) p values (bold information indicates significant values) are from chi-square and student’s t tests. bmi: body mass index; sd: standard deviation. 188 l. salih joelsson et al. health authorities of an intake of 400 lg/day of folic acid via supplements for women planning a pregnancy in order to prevent neural tube defects (32–34). obviously more information to all women planning a pregnancy is needed. our results indicate that greater efforts are needed to improve compliance with the recommendations for subfertile women, as, surprisingly, more than half of the women seeking help for infertility do not comply with these recommendations and do not adjust their lifestyle accordingly. a study from the united kingdom showed that only 2.9% complied fully with the recommendations for alcohol and folic acid intake during the 3 months before becoming pregnant (35). low compliance with nutrition and lifestyle recommendations when planning a pregnancy has also been reported in studies from the usa (36) and iran (37). the fast study by homan et al. (38) showed that an individualized assessment of current lifestyle behaviors for couples attending a fertility clinic, followed by continuous support, is likely to achieve healthy lifestyle changes. an individually tailored plan was developed, including strategies to achieve realistic goals in terms of lifestyle changes, as well as referrals to allied health professionals, e.g. dieticians, physiotherapists, and counselors. results from a study in the netherlands suggest that tailored preconceptional dietary and lifestyle counseling is effective among subfertile couples to change unhealthy behaviors within a period of 3 months (39). this indicates that it is possible to change unhealthy lifestyle factors to reduce or even eliminate them in order to enhance the chances of conception. the response rate (62%) was satisfactory compared to other similar studies in sweden (40). it may be worth considering whether the response rate could have been increased if respondents had been offered a free choice between a paper-based and a web-based questionnaire (41). the relatively high proportion of women of non-swedish origin (14%) is considered to be a strength of this study. however, we used a retrospective measure for estimating the incidences of health-promoting actions and lifestyle changes, which could be either overestimated or underestimated. it is well known that people underreport alcohol intake and smoking, and this might have resulted in an overestimation of the women’s positive health behaviors. table 4. results for multivariate logistic regression model with type of health-promoting lifestyle changes as outcome, given as the odds ratio (or) with 95% confidence interval (ci). or 95% ci p started taking folic acid supplements (cases included in analysis: 377 (84%)): duration of infertility in years (mean) 0.921 (0.840–1.009) 0.078 household income/month (1,000 sek) 1.012 (1.001–1.024) 0.040 level of education college/university 1.853 (1.151–2.983) 0.011 non-college/non-university (ref.) bmi normal (ref.) overweight 1.287 (0.752–2.203) 0.357 obese 0.568 (0.293–1.102) 0.094 country of birth sweden 1.828 (0.659–5.074) 0.247 born outside sweden (ref.) parents’ country of birth born in sweden 1.618 (0.664–3.941) 0.289 born outside sweden (one or both) (ref.) information searching 0–1 sites (ref.) 2 sites 1.143 (0.631–2.067) 0.660 �3 sites 2.172 (1.241–3.801) 0.007 decrease or stop in tobacco use (cases included in analysis: 412 (92%): age (years) 0.950 (0.900–1.002) 0.060 household income/month (1,000 sek) 0.991 (0.978–1.005) 0.223 level of education college/university (ref.) non-college/non-university 2.256 (1.352–3.765) 0.002 occupation working >50% of the time (ref.) not working 1.314 (0.705–2.448) 0.390 increase in physical activity and/or change to more healthy diet (cases included in analysis: 407 (91%)): household income/month (1,000 sek) 0.996 (0.985–1.007) 0.451 level of education college/university (ref.) non-college/non-university 1.057 (0.673–1.661) 0.810 bmi normal (ref.) overweight 2.931 (1.789–4.800) <0.001 obese 7.431 (3.700–14.923) <0.001 information searching 0–1 sites (ref.) 2 sites 1.114 (0.614–2.023) 0.722 �3 sites 2.032 (1.184–3.487) 0.010 upsala journal of medical sciences 189 conclusions detecting and changing unfavorable lifestyles is essential for improving spontaneous fertility and art results. one-third of the studied women were overweight or obese, and some had other lifestyle factors with adverse effects on fertility. obese women adjusted their habits, but there was no adequate change in their weight. women of a fertile age would benefit from preconception counseling. infertility treatment should routinely offer interventions for lifestyle changes. disclosure statement the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. funding information grants were received from the foundation family planning fund in uppsala, the uppsala county council, the college of medicine at uppsala university, sweden, v€astmanland county council, sweden, and the uppsala€orebro regional research council, sweden (ltv-218591, rfr387811). references 1. homan gf, davies m, norman r. the impact of lifestyle factors on reproductive performance in the general population and those undergoing infertility treatment: a review. hum reprod update. 2007;13:209–23. 2. anderson k, nisenblat v, norman r. lifestyle factors in people seeking infertility treatment—a review: invited review. aust new zeal j obstet gynaecol. 2010;50:8–20. 3. anderson k, norman r, middleton p. preconception lifestyle advice for people with subfertility. cochrane database syst rev. 2010;(4): cd008189. 4. european commission. directorate-general for employment sa and eo. demography report 2010: older, more numerous and diverse european. issn 1831-9440, demography. luxembourg; 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introduction material and methods design participants and recruitment questionnaire background characteristics background lifestyle and lifestyle changes ethical considerations statistical analysis results lifestyle during the pregnancy planning period lifestyle modifications associations between background characteristics and health-promoting actions/lifestyle changes discussion conclusions disclosure statement funding information references upsala j med sci 99: 377-384, 1994 8. adding new scopes to traditional eqa schemes emphasizing quality improvement e. olafsdottirl, k. hellsing2, h. steensland3, r. tenhunen4, a. uldall5 'department of clinical chemistry, landspitalinn, i01 reykjavik, iceland 2department of clinical chemistry, university hospital, s751 85 uppsala, sweden 3department of clinical chemistry, ullevdl hospital, n-0456 oslo, norway ?kbquality, stationskarlsgatan 44 sf-00520 helsinki, finland 5department of clinical chemistry, herlev university hospital, dk-2730 herlev, denmark the nordic organizers of external quality assessment (eqa) schemes have taken a broader view of the objectives of such schemes than most traditional eqa organizers, as is well illustrated by a number of nordic quality related projects referred to below. the nordic eqa organizers feel an obligation to support actively quality improvements according t o needs of the laboratories. improved analytical specificity and standardization of analytical methods should become an integral part of eqa schemes, changing the emphasis from assessing the quality of analytical work to assuring quality by external guidance through external quality assurance (eqassur) schemes. in preparation of such a change the following elements have to be considered (i) establishment of quality goals and desirable performance standards following the initial nordic clinical chemistry project (nordkem) where quality requirements were assessed (1) it was recommended that each laboratory set their own quality goals and desirable performance standards. in 1989 the nordic federation of clinical chemistry (nfkk) issued recommendations stating in six paragraphs the essential steps to be taken to improve overall quality in the analytical work (2). how to set quality goals is still a matter of discussion since various approaches are possible and different situations call for different quality demands. the most logical approach would be to set quality goals according to medical needs (3), this however is often difficult since the necessary data are still available for only a limited number of analytes. others have argued that quality goals should be based on biological variation (4) where 377 data are not available t o set goals otherwise. the biological variation approach to goal setting is simple and therefore possibly more likely to be accepted by many laboratory directors (5). the medical need will ultimately be the goal in many settings, since hfferent quality may be required for analysis performed for screening purpose, diagnosis, following treatment or recovery etc. labquality is presently working on goal setting based on the principles of biological variation (4) and the "state of t h e art" today. (ii) assessment of the overall analytical performance the first nordic survey of the clinical chemistry was published in 1970 (6) repor ting the "state of the art" at that time. since then several limited nordic surveys have been run by different members of nordic clinical chemistry, assessing specific areas within the field each time. the longstanding work of labquality in finland has thoroughly recorded and published quality performance in finnish laboratories as well as some more selected fields in other nordic laboratories. national and regional eqa in denmark, norway and sweden have good records of laboratory performance in the last two decades. recently norway and sweden have changed their eqa operation considerably. in norway the hospital laboratories have joined labquality and eqa outside hospitals is in the hands of noklus (norsk senter for kvalitetssikring av laboratorieanalyser utenfor sykehus?. in sweden the newly established seqla (swedish external quality assurance in laboratory medicine) organizes eqa in all fields of laboratory medicine, and concurrently nordic co-operation in the eqa field has been reinforced with the establishment of nqlm (nordic external quality assurance programs in laboratory medicine). (iii) conducting specialized eqassur programs, where specific analytical pitfalls are studied eqassur programs are very useful to sort out methods and instruments that are not giving satisfactory performance and can thus be used as an additional way of correcting analytical problems which have been suspected or detected as poor performers by other means (7, 8). it must however be stressed that poor calibrators, poorly kept instruments, reagents and errors in method performance must be ruled out when eqassur programs are used to screen for nonspecific o r poorly performing instruments/methods. 378 (iv) provide metrological recommendations and relevant calibrators metrological recommendations (9, 10) and use of relevant calibrators (11, 12, 13) have been developed as a result of unacceptable performance in eqa schemes. in 1974-76 recommendations of the scandmavian committee on enzymes were published (10, 14) and shortly afterwards most nordic laboratories converted to the new methods for the four enzymes in blood, alat, asat, gt and ck. in 1990 it was recommended that all laboratories in the nordic countries changed over to the eccls methods (15) for these particular enzymes and most a d so. the latest recommendation is to change over to a common protein calibrator, the bcr crm 470, in all nordic laboratories performing protein analysis before the end of 1994. (v) assessment of quality improvement using eqa schemes or ptt eqa schemes could be used to register quality improvements in analytical areas where such improvement is considered particularly important. this has been de monstrated in a number of surveys on protein analysis in the nordic countries (16). the first one carried out in 1977 revealed an unacceptable inter-laboratory variation. later in a nordkem sponsored protein project (17) where the elements of quality policy, quality improvement and quality control were combined a most striking results in quality improvement were observed. in the protein project the use of common calibrator was linked to an eqa scheme and this combination resulted in a significant improvement in performance of the participating laboratories (18). by linlung the protein calibrator directly to the newly released ifcc/cap/bcr crm 470 protein standard a true target value for protein analysis is set. the protein project also included turbid material in order to signal out methods sensitive to interferences. this is in accord with the above mentioned philosophy and will be looked upon as a leading example in widening the scope of eqa schemes. ivi) supporting internal quality control continuously by specially designed e&assur* programs in denmark and finland the national eqa schemes have successfully been used to support continuously t h e internal qc programs of many laboratories (19, 20, 21). the danish society of clinical chemistry has distributed quality control ma 26-955059 379 terial to its members for use in internal qc, the "k-series" and used that as basis for the national eqa scheme, thus interlinking internal and external quality control. labquality has provided similar services by its longterm eqas, where the same control material is distributed over a long time period (20). (vii) monitoring the methods and the laboratory performance through a simplified eqa scheme using detailed information about methods a novel use of eqa schemes is to monitor the performance of methods when de tailed information about the method protocols is available. the coming ec-direc tive on in-vitro diagnostic products seems to advocate using this approach for supplementing market surveillance of essential in-vitro diagnostic products. (viii} proper action taken by the laboratory when deviating findings occur in eqa schemes suited for laboratory licensing or accreditation the emphasis should not only be on performance criteria for reported results, but also on the correct action taken by the laboratory when deviating findings occur. guidelines for acceptance of proficiency testing programs have been issued by welac (22). according to these proficiency testing should "demonstrate and mo nitor competence of the laboratory to carry out specific tests or analyses". it is ho wever of utmost importance to incorporate into written protocols for daily main tainance of quality, t h e actions taken by the laboratory when deviating findmgs do occur (23). (ix) promote the establishment of common reference intervals where relevant in a health care systems where patients are frequently being referred from the family doctor to a specialist or from one hospital to another transferability of clinical chemistry results and common reference intervals are of importance in providing a safer, faster and less expensive service to the patient. all scientific work on patients' results becomes more reliable and meaningful if this becomes a realization. recently thorough and extensive work has been done on establishing common reference intervals for plasma proteins for the nordic population (24). a foreseen difficulty with common reference intervals for plasma proteins has to be overcome when some laboratories start using substance concentrations instead of the now commonly used g/l units for proteins. 380 well founded reference intervals are certainly a quality aspect of laboratory re sults. as such they fall within the scope of eqassur programs. however it is de batable whether the establishment of common reference intervals for homoge neous populations should be a project attempted by eqa organizers. such a project is today a task to be undertaken by collaboration of several laboratories with a support of well designed eqassur programs. (x) promote the use of modern nomenclature and units in accordance with other european eqa schemes the nordic countries have pro moted the use of modern nomenclature and si units (25). still there are problems to be solved under this heading, such as the use of molar quantities for proteins. the board of the danish society of clinical chemistry has recommended the use of molar units for proteins based on the most likely molecular masses. the use of katal for expressing enzyme activities was adopted in sweden, but the other nordic countries still hold on to the international units (iu) for enzyme activities. some laboratories have not converted over to si units for drugs and nomenclature is still to be adapted t o officially agreed vocabulary in many areas. the eqa organizers will however continue their educational role in promoting the issue and encourage discussions about the issues under dispute hopefully until consensus is reached. the proficiency testing in europe and the usa have been reviewed in libeer's book on external quality assessment in clinical laboratories (26). the fundamental differences are in the professional and educational support given in the european eqas versus the requirements of fulfilling minimum standards set by the clinical laboratory improvement act of the us congress. the nordic eqa-organizers participate in the increased european cooperation among their colleagues and have given the educational purpose of eqassur a strong support. *note: external quality assurance (eqassur) seems to be a better term than external quality assessment in the context of quality improvement program. the latter term may be substituted with the designation "proficiency testing" (pt) according to is0 nomenclature (27). 38 1 correspondance to elin olafsdottir department of clinical chemistry landspit alinn 101 reykjavik, iceland or adam uldall department of clinical chemistry herlev university hospital dk-2730 herlev, denmark references 1. 2. 3. 4. 5 . 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 382 hprrder m (ed.) assessing quality requirements in clinical chemistry. the nordic clinical chemistry project (nordkem). scand j clin lab invest 1980;40,suppl 155:l-144. scandinavian society for clinical chemistry. general scandinavian recommendations on quality control and quality assurance in clinical chemistry. scand j clin lab invest 1990;50:225-7. deverdier c.-h. (ed) medical need for quality specifications in laboratory medicine. a nordkem project 1989-1992. + uppsala j med sci 1990:95;162-309. fraser cg, hyltoft petersen p. quality goals in external quality assessment are best based on biology. scand j clin lab invest 1993;53 suppl612:8-9. hyltoft petersen p, groth t, deverdier c-h. principles for assessing analytical quality specifications ("aqspecs") and their use in design of control systems. upsala j m e d sci 1993;98:195-214. stromme jh, eldjarn l. surveys of the routine work of clinical chemical laboratories in 116 scandinavian hospitals. scand j clin lab invest 1970;25:213-22. uldall a, fogh-andersen n, thode j et al. measurement of ionized calcium with five types of instruments. an external quality assessment. scand j clin lab invest 1985;45:255-61. hyltoft petersen p, blaabjerg 0, irjala k, icen a, bj0ro k. a model for quality achievement the nordkem protein project. scand j clin lab invest 1993;53, suppl212:10-2. uldall a, improvements in the routine investigations of urinary calculi. scand j clin lab invest 1984;44, suppl 172:147-56. keiding r, h ~ r d e r m, gerhardt w et al. recommended methods for the determination of four enzymes in blood. scand j clin lab invest 1974;33:287-396. gerhardt w, waldenstrom j, hdrder m et al. sce nordic alpha-amylase study. 1: method selection and calibration study. 11: assessment of proposed calibration procedure. scand j clin lab invest 1985;45:397-404. b i d . 1986;46.465-9. raabo e, kynde k, sandberg-hansen m. evaluation of a calibrator used for isc-calibration of coagulation tests in denmark. eqanews 1992;3 no 2:4. blaabjerg 0, blom m, gry h, hyltoft petersen p, uldall a. appropriate sera for calibration and control of specific protein assays. &and j ctin lab invest 1993;53,suppl212:13-5. scandinavian committee on enzymes. recommended method for the determination of gamma glutamyltransferase in blood. scand j clin lab invest 1976;36:119-25. eccls standards for enzyme determination. klinisk kemi i norden 1990;2 suppl carlstrom a, gavert j, hellsing k et al. improvement of quality of protein. analysis in nordic countries a comparative study. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. hyltoft petersen p, blaabjerg 0, irjala k, i c h a, b j ~ r o k. a model for quality achievement the n o r d m m protein project. scand j clin lab invest 1993;53 suppl. 212:lo-2. gry h, blaabjerg 0, blom m, uldall a, hyltoft petersen p. preparation of a protein calibrator. scand j clin lab invest 1988;48 suppl. 190:157. blom m, brock a, christensen f et al. external quality assessment programs in denmark. scand j clin lab invest 1984;44 suppl 172175-8. gronroos et al. external quality assessment programs in finland 1971-1983. scand j clin lab znuest 1984;44 suppl 172:179-86. blaabjerg 0, elg p, gerhardt w, hellsing k, olafsdottir e, pentilla i, hyltoft petersen p, steensland h, uldall a. a nordic reference serum suitable for use as trueness control in the 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27-955059 383 upsala j med sci 95: 45-52 association between exposure to asbestos and pleural effusion. results from a questionnaire study of 31000 persons per malmberg and gunnar hillerdal national institute of occupational health, s-17184 solna, sweden and institute of pulmonary medicine, university hospital, s-75185 uppsala, sweden abstract all visitors to a general health survey in 1979, 17,140 men and 14,371 women, completed a questionnaire on smoking habits, exposure to asbestos, silica and welding fumes, and diseases such as pleural effusion, pneumonia, cough, asthma and diabetes. seven per cent of the men reported exposure to asbestos, 10% to welding fumes, and 6% to silica. among those who reported work related dust exposure there was a higher proportion of smokers, and smokers exposed to dust smoked more tobacco per day than non exposed smokers. in the group of men 30-59 years of age, who did not indicate exposure to occupational pollutants 2.7% reported previous pleural effusions. however, among asbestos exposed men of the same ages, the prevalence was more than doubled (5.7%, p<o.ol). this finding was highly significant in a logistic regression model where age and smoking habits were included. the data indicate that 10% or more of diagnosed cases of pleurisy could be associated with previous asbestos exposure. introduction beginning in 1963 and ending in 1985, all inhabitants in the county of uppsala aged 15 years or more were invited to a general health survey every second or third year. during the visit blood pressure, hemoglobin, and urine tests were performed and a small size chest roentgenogram was taken. between 50% and 70% of the population participated each time. in 1979, all visitors were asked to complete a questionnaire regarding exposure to asbestos, silica, welding fumes, and occurrence of certain pulmonary and other symptoms and diseases. the aim was to investigate the relation between smoking habits and exposure to some common occupational pollutants occurring before 1979 and future pulmonary diseases in a prospective study. during the study of the questionnaire responses at follow-up time, observations were made which were considered to be of general interest. thus twice as many men who reported exposure to asbestos also reported a history of pleural effusion, compared to men who did not report asbestos exposure. since the survey was performed in 1979, when the relation between pleural effusion and 45 asbestos exposure were practically unknown among practicing physicians, the data should be less influenced by recall bias than would be the case in similar studies performed at later dates. men exposed to all asbestos welding silica never smokers 40.4 30.2 32.2 29.4 ex-smokers 26.2 30.2 28.9 28.4 smokers 33.4 39.6 38.9 42.3 material and methods women a l l 54.5 14.8 30.7 all visitors at the health survey were asked to complete a questionnaire. in the questionnaire a tobacco smoker was defined as a person who had smoked at least one cigarette per day for one year or more (or about 400 cigarettes, or 5 packs of tobacco (75 gram packs) or 200 cigars). an ex smoker was defmed as a smoker who had stopped smoking since at least six months. the answers were coded as "yes", "no" or "missing". smokers were also asked to estimate the average tobacco consumption per day, the year they started to smoke and the year they stopped smoking, if ex smokers. in the calculation of total tobacco consumption one cigarette was assumed to contain one gram of tobacco, a cigar two grams and a parcel of tobacco 75 grams the participants were also asked to report if they had been exposed to asbestos dusts. similarly they were asked to report exposure to silica dust and welding fumes. in a third section of the questionnaire they were asked to indicate if they presently suffered from or had previously suffered from diseases or conditions as listed in table 4. no distinction was made between "no" replies and missing information in the questions regarding dust exposure or diseases. the responses were coded twice for improved accuracy and fed into a computer (microvax 11) with the database program mimerb. the statistical analysis was performed using the sas statistical package@, using the chi2-methcd and logistic analysis of variance (cat-mod procedure). results all 46 questionnaires from 17,140 men and 14,371 women contained identification numbers which could be used for calculation of age and were thus used in the investigation. another 100 questionnaires from men and 70 from women did not contain identification numbers and were excluded. in 70 questionnaires from men and 180 from women information on the smoking habits were missing. the age dismbution is shown in fig. 1. there were more tobacco smokers among men aged 40 or more than among women and male smokers smoked more than femaie smokers (tables 1 and 2, fig. 2); but at younger ages the opposite was found. the average age when smoking was commenced was practically constant for age groups in men 30 years of age or older. men current-smokers ex-smokers nonnon exposed exposed exposed exposed dday 13.6 14.5 14.1 15.9 i :i:m) 10.4 (s.d.) (8.2) (8.2)l (9.1) (11.6)1 (6.4) women smokers smokers current ex 1 p<o.ool for difference between non-exposed and exposed groups. fig. 1. age distribution. three point moving averages of each one year age group. 2'0 4 0 6 0 y e a r s fig. 2. age distribution of current smokers and ex-smokers (plus current smokers) among male and female participants. percent of all participants in each sex and age group. see legend to fig. 1 for further detail. exposure to asbestos, welding fumes and silica are shown in table 3. among men who reported exposure to occupational pollutants there were more smokers, and dust exposed smokers smoked more tobacco per day (table 2) than among age-matched referents. exposure to occupational dusts, particularly asbestos, was reported more often among young men than among older age groups (fig. 3). of the men aged 30 to 59 years, 8.1% reported exposure to asbestos. the number of persons who reported diseases or symptoms are shown in table 4. the occurrences of diseases and symptoms (asthma, pneumonia, pleurisy, rib fractures, diabetes and chronic cough) in different age groups are illustrated in fig. 4-7. as could be expected, the prevalence of pneumonia, pleurisy and diabetes increased with age, but asthma was more common in the younger age groups. 47 table 3. exposure to occupational agents and smoking habits (per cent) neverwomen smokers smokers smokers asbestos welding 10.4 12.1a 11.5 8.3 0.2 silica 5.7 6.2 4.1 0.2 ~ 4 . 0 0 1 for differences between smoking categories. bp<0.01 for differences between smoking categories. men all csa xsb nsc pneumonia 13.6 14.6l 15.8 11.4 pleuraleffusion 2.9 2.9l 4.1 2.3 rib fracture 9.9 11.3l 11.1 7.9 diabetes 1.6 1.3l 2.4 1.3 asthma 3.6 2.g2 3.8 3.0 chroniccough 2.0 3.7l 1.5 0.9 of men aged 30 to 59 years who did not report exposure to occupational dusts 2.7% reported previous pleuritic disease. similar prevalences of reported pleural disease were found in men who reported exposed to silica dust or welding fumes. however, among men aged 30-59 years who reported exposure to asbestos, more than twice as many (5.7%) reported previous pleural effusion (table 5). this difference was not found in men less than 30 years of age. the association between exposure to asbestos and pleurisy was significant ( ~ ~ 0 . 0 1 ) in a logistic regression model which included age and smoking habits. women all cs xs ns 12.1 11.g2 14.3 11.8 2.2 1.9ns 2.4 2.4 3.2 2s2 2.7 3.6 1.3 0.7l 0.8 1.8 3.2 3.lns 3.8 3.0 1.7 3.01 1.5 1.1 table 5. prevalence of positive answers to question: "have you or have you ever had pleuritis", according to age category and dust-exposure in men. percent of group and group size. 15-29 years 30-44 years 45-59 years 60+ years all(n=17140) aexcluding case s no exposure silica asbestosd or weldin$ 0.9% (3807) 0.8% (396) 0.9% (234) 1.7% (4482) 1.9% (644) 4.4% (498) 4,1% (3072) 3.7% (376) 8.2% (268) 5.4% (2942) 7.1% (240) 9.7% (175) 2.8% (14303) 2.8% (1656) 5.4% (1 175) who also reported exposure to asbestos. bincluding mixed exposure. 48 -.-.s i l i c a 8 a s b e s t o s --w e l d i n g ported exposure to occu15 see legends to fig. 1,2 fig. 3. age distribution of re pational dusts among male participants. for further detail. 10 5 a g e 20 3 0 4 0 5 0 6 0 y e a r s ?=* p l e u r i t i s --'d r i b f r a c t u r e 9--- fig. 4. reported frequency of 20"6 earlier or present pleuritis and rib fractures among 1 5 male and female partici pants. see legends to fig. 1,2 for further detail. a g e 2 0 30 4 0 5 0 6 0 y e a r s 5 p n e u m o n i a and pneumonia among 1 0 see legends to fig. 1,2 5 fig. 5. reported frequency of earlier or present asthma male and female partici pants. for further detail. a g e 2 0 30 4 0 5 0 6 0 y e a r s ?='diabetes 20%l among male and female 5 fig. 6. reported frequency of earlier or present diabetes participants. see legends to fig. 1,2 for further detail. a g e l o 12 0 30 4 0 5 0 6 0 y e a r s 4-908571 49 fig. 7. 20961 ?--’ c h r o n i c cough 1 0 5 reported frequency of earlier or present chronic cough among male and female participants. s e e legends to fig. 1,2 for further detail. 2 0 30 4 0 50 6 0 y e a r s discussion the present data confirm some commonly known observations, such as the circumstance that older men smoke more than women whereas the opposite is found among young subjects. among those who reported exposure to silica, asbestos or welding fumes there were more smokers and dust exposed smokers smoked more than age matched controls. these findings agree with other studies (4, 8) and point to an association between occupation and lifestyle characteristics, which should be considered when interpreting data from different occupational settings. young subjects reported present or earlier diagnosis of asthma more often than middle aged subjects. this finding was unexpected. if the incidence of asthma is constant, an increasing proportion of subjects who have or have ever had the diagnosis would be expected with increasing age, rather than the opposite. several explanations are possible, such as a recent change in diagnostic criteria or improved diagnostic capacity. with increasing age, episodes of childhood asthma may have been forgotten or middle aged subjects with a history of asthma may have chosen not to attend the health survey. the finding can therefore only be regarded as circumstantial evidence in the present debate regarding a possible rise in the true incidence of asthma i n recent years. however, the results of the survey do not refute this notion. men who had indicated exposure to asbestos also reported having suffered from pleurisy more than twice as often as men who did not report asbestos exposure. this finding needs to be interpreted with consideration of the representativity of the material and of possible bias in the answers. the health survey was in operation from 1963 to 1985, and the participation was fairly constant through the years, as was the distribution of participation among different age groups. thus, for the age groups 35 to 65 years, the participation figures have been from 60 to 70%. those who are invited for the first time, i.e. at age 15 to 17, have the highest participation rate (70% or more), b u t the second and third time at age 18 to 25 the interest seems to be low. above age 70, again fewer persons will attend. the exact figures from the year 1976 have been published previously (8) and were as follows: 15-44 years of age,65 %; 45 to 64 years, 72%; above age 65, 48%; and total, 64%. according to unpublished studies the main reasons for non-padcipation was either that the 50 subject was sick and already under medical supervision, or especially among young subjects, a feeling of good health, and thus no need for a free medical checkup. between 6-10% of the men indicated exposure to asbestos, silica or welding fumes and about 3% of all men indicated previous or present episode(s) of pleurisy. the observed frequency of reported pleurisy may be influenced by positive and negative selection, but selection can hardly explain the findings. even if a higher proportion of asbestos exposed subjects who had suffered from pleurisy would attend the survey, than in the general population, such a selection bias would not be of sufficient magnitude to explain the figures obtairied. furthermore, men exposed to silica or welding fumes should have similar bias, but these groups did not have an increased prevalence of pleurisy compared to men who did not report exposure to occupational dusts. another possible confounding factor is recall bias. thus if a relationship between asbestos exposure and pleurisy were known to the medical profession or to the lay public, a careful search of previous exposure to asbestos may have been performed by the subjects at the time of the diagnosis. this would increase the likelihood of remembering such exposure when the questionnaire was completed. benign asbestos pleural effusion is now a well-recognized entity (1, 6, 7). this association between asbestos exposure and pleurisy was, however, f i s t reported in the 1970’s (5, 10, 12) and was not commonly known among the medical profession at the time of the study (1979). the association was virtually unknown in the general public at the time. therefore recall bias is an unlikely explanation. there are few studies regarding how many in the general population who have been occupationally exposed to asbestos. in the usa it has been estimated that 14 million living workers have had significant exposure, which would be more than 10 per cent of the adult male population (11). in norway, in a study of 21,453 men above age 40, a mean of 18% had been occupationally exposed to asbestos. in industrialized municipalities, the figure was 9 to 26%, and i n non industrialized 7 to 8% (8). uppsala county is not very heavily industrialized and the figures agree well with the non-industrialized municipalities in norway. for the whole of sweden, the percentage figures would probably be more like those of the usa. if about 3% of men aged 30 60 years report having had pleurisy and twice as many among the ten percent of men who also indicate exposure to asbestos, then about 10% of all reported pleurisy could be related to asbestos exposure. studies where subjects with pleural disease have been subjected to thoracoscopy have indicated similar proportions (2, 3, 9). the figure 10% may underestimate the true proportion of asbestos related pleurisy since this type of pleurisy is often associated with few symptoms. furthermore, a significant number of those who reported pleurisy i n the present study who had not indicated asbestos exposure, may have been exposed to asbestos unknowingly or may have forgotten this exposure at the time of the inquiry. acknowledgements this work was supported by the swedish national work environment fund. we wish to thank mr henrik modin, mrs gun rosin and mrs britt-marie sundblad for excellent assistance in organizing and editing of data. we also would like to express our thanks to mr per nasman at the institute for statistical analysis, university of stockholm for statistical advice. 5 1 references 1. 2 . 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. anonymus: benign asbestos pleural effusions. lancet 1:1145-1146, 1988. aubert, m., parent, b., brambilla, c. & bwa, a.: our experience with thoracoscopy (author's transl). poumon coeur 37:75-77, 1981. boutin, c., cargnino, p. & viallat, j.: thoracoscopy in chronic pleural effusion (author's transl). poumon coeur 3523-29, 1979. covey, l. s . & wynder, e. l.: smoking habits and occupational status. j occup med eisenstadt, h.: pleural effusion in asbestosis (letter). n engl j med 290:18, 1974. epler, g. r., mcloud, t. c. & gaensler, e. a.: prevalence and incidence of benign asbestos pleural effusion jn a working population. jama 247:617-622, 1982. hillerdal, g. & ozesmi, m.: benign asbestos pleural effusion: 83 exudates in 60 patients and a review of the literature. eur j respir dis 71:113-121, 1987. hilt, b., langard, s . , lund-larsen, p. g. & lien, j. t.: previous asbestos exposure and smoking habits in the county of telemark, norway a cross-sectional population study. scand j work environ health 12561-566. 1986. luther, r., wetzer, k., kiihe, w., spormann, h. & bottger, w.: asbestos-induced pleural diseases-thoracoscopic aspects. endoscopy 20:4-6, 1988. mamson, s . : monosymptomatic exudative pleurisy in persons exposed to asbestos dust. scand j respir dis 56:263-272, 1975. nicholson, w. j., perkel, g. & selikoff, i. j.: occupational exposure to asbestos: population at risk and projected mortality 1980-2030. am j ind med 3:259-311,1982. sluis, c. g. & webster, i.: acute pleurisy in asbestos exposed persons. environ res 5:380 392, 1972. 23537-542, 1981. address for reprints: per malmberg national institute of occupational health s-171 84 solna sweden 52 upsala j med sci 103: 231-236, 1998 increased serum concentrations of carbohydrate-deficient transferrin (cdt) in patients with cystic fibrosis anders larsson,' mats flodin' and hans kollberg2 'department of clinical chemistry, university hospital and 2cf-centre, children's university hospital, s-75185 uppsala, sweden abstract carbohydrate-deficient transferrin (cdt) has been reported to be one of the best laboratory markers in serum (s) for detection of alcohol abuse. we have studied s-cdt values in cystic fibrosis (cf) patients and show that cf patients have increased s-cdt values without high alcohol consumption. cf patients have abnormalities in their protein glycosylation and sialylation, which may explain the increased s-cdt values. introduction chronic high intake of alcohol results in damage to many different organs including development of chronic pancreatitis, liver cirrhosis and myopathy alcoholics are overrepresented among patients in the hospitals and several biochemical markers have been developed to diagnose alcoholic abuse (6,17) carbohydrate-deficient transferrin (cdt) has been used for more than a decade as a marker in serum for alcoholic abuse (1 1) transfemn (t) has several isoforms that differ in the degree of sialylation of the two n-linked oligosaccharide chains (8) the least sialylated isoforms of t, with 0 (asialo t) and 2 (disialo t) sialic acids are usually referred to as s-cdt the formation of s-cdt in individuals with high alcohol consumption is believed to be due to inactivation of enzymes in the golgi apparatus ( 3 ) pathological s-cdt values have also been reported in patients with carbohydrate-deficient glycoprotein syndrome and galactosaemia (1 3) cystic fibrosis (cf) is the most common fatal genetic disease in the united states today (9) it occurs in approximately one of every 3,300 births and affects approximately 30,000 children and young adults one in twenty-nine americans is a carrier of the gene. in sweden there are approximately 475 individuals with cf. there are more than 600 different known mutations that cause cf this makes it sometimes difficult to diagnose cf by dna technology the sweat test is 23 1 currently the standard diagnostic test (1 5). this test measures the amount of salt in the sweat and a high level of salt indicates that a person has cf. sweat tests are time consuming and thus costly ’ and individuals with a high alcohol consumption may also have positive test results. previous studies have shown that cf patients have abnormalities in their glycosylation (5), sulfatation (2) and sialylation (4) of membrane and plasma proteins. such changes may interfere with the measurement of s-cdt. we have used high-performance liquid chromatography to quantify the different isoforms of transfemn in healthy controls and patients with cystic fibrosis. the purpose of the study was to evaluate if cf may cause an increase in s-cdt and to see if isoforms of tf could be used to diagnose cf. materials and methods patients twenty cf patients, aged 7 to 37 years were studied. cf diagnosis was established on the basis of typical clinical symptoms and abnormal sweat tests. none of the patients had hepatic disorders. blood was collected in serum tubes without additive (367609, becton dickinson, rutherford, nj). all patients gave informed consent prior to blood sampling. the serum samples were stored at -20°c until tested. analysis of carbohydrate deficient transferrin (s-cdt) automated ion-exchange chromatography twenty cf patients were analysed. determination of s-cdt and transferrin isoforms in serum was performed according to jeppson et al. (7) at the department of clinical chemistry, university hospital, uppsala. the reference interval for the method is <1.2% and the total imprecision for the assay is 3.5% at a s-cdt value of 2,4 %. a group of 149 healthy blood donors and medical students were used as controls. cdtectm s-cdt was also analysed by cdtecttm (pharmacia, uppsala, sweden) in twelve cf patients. pharmacia performed the assay. statisticai analysis differences between groups were calculated by friedman anova. the statistical analysis was performed with statistica 4.5 (statsoft inc., tulsa, ok). p < 0.05 was considered significant. 232 results transferrin isoforms in serum measured by ion-exchange chromatography the distribution of the different serum isoforms in healthy individuals was: disialo t 0.735%*0.238 (mean*s.d.), trisialo t 4.575%*1.3 15, tetrasialo t 84.886%%1.717, pentasialo t 9.794%&1.760. no asialo t was detected in any of the healthy controls. the mean serum values for the different isoforms in cf patients were: disialo t 0.954% (p<.04), trisialo t 2.584% (p<.0004), tetrasialo t 84.118% (p=.65), pentasialo t 12.347% (p< 03). no asialo t was detected in any of the cf patients. cdtectm six male and six female cf patients were analysed. the mean serum values were 18.5 u/l for men and 18.3 u/l for females. two of the boys had serum cdtecttm values above 20 u/l. these two boys were seven and eight years old and came from two different families. serum values for healthy controls were < 20 u/l for men and < 26 u l for women according to the laboratory at pharmacia. discussion transferrin is present in high concentrations in serum. it is a glycoprotein with two bior tri antennary carbohydrate chains, each terminated with two or three sialic acids (8). differences in the number of carbohydrate chains and sialic acids will alter the isoelectric point of the transferrin molecule. the isoforms related to alcohol abuse contain less sialic acids and have a higher isoelectric point than other isoforms of transfemn. ion exchange chromatography separates serum transferrin into isoforms depending on sialic acid content, iron saturation and amino acid content. after saturation of the transferrin molecule with iron, s-transferrin is normally separated into four isoforms according t o their isoelectric point (pi): pi 5.2 (5 sialic acids), pi 5 4 (4 sialic acids), pi 5.6 (3 sialic acids) and pi 5.7 (2 sialic acids). high alcohol consumption will result in an increase of the pi 5.7 fraction and an additional pi 5.9 (0 sialic acids) fraction may appear. the fractions with pi 5 7 and 5 9 are called carbohydrate-deficient transferrin. several methods have been used for quantification of s-cdt: hplc (7), isoelectric focusinglwestern blotting (1 6), anion-exchange chromatography followed by ria (12) or nephelometry (10). s-cdt has a sensitivity of 82% and a specificity of 97% for alcohol abuse (1 1) in selected materials. the sensitivity of s-cdt varies between different studies depending on the examined population, but the specificity of s-cdt is usually high. false-positive results have previously been 233 reported in patients with primary biliary cirrhosis, genetic variants of transfemn, carboi1;rdrate deficient glycoprotein syndrome or galactosaemia (1 3). the formation of s-cdt in individuals with high alcohol consumption is believed to be due to inactivation of enzymes in the golgi apparatus (3). in cf cells there is a defective acidification of the trans-goldtrans-golgi network, of prelysosomes and of endosomes as a result of diminished c r conductance (1). this results in a reduced sialylation of proteins and lipids. these abnormalities can result from defective acidification because vacuolar ph regulates glycoprotein processing. thus, both patients with high alcohol consumption and cf patients have a defective sialylation that may result in increased s-cdt levels. we have studied the levels of s-cdt in serum from patients with cf. cf is the most common life-shortening genetic disorder in populations of european origin. the basic defect in cf is a mutation in the cystic fibrosis transmembrane conductance regulator (cftr) gene on chromosome seven (14), which results in a faulty transport of sodium and chloride and causes the body to produce abnormal exocrine secretion. symptoms come from many organs, especially the airways and the gastrointestinal tract; but also fiom liver, sweat glands and the reproductive system it is sometimes difficult t o diagnose cf. a simple inexpensive serum assay such as s-cdt could potentially be used as a screening assay for cf. we originally used the cdtecttm method. the cdtecttm method measures the absolute amount of s-cdt and not s-cdt as a percentage of total transferrin. a patient with increased serum transferrin values due to iron deficiency will therefore have a higher cdtectm value cf patients may have an iron deficiency. we thus changed method from cdtecttm to ion-exchange chromatography, which also gave significantly elevated serum levels of cdt in cf patients in relation to healthy controls. thus, we have found increased s-cdt levels in cf patients with two different methods. the method will probably influence the results as we found subnormal values of trisialo t in our cf patients and some s-cdt methods also measure trisialo t. all our cf patients denied high alcohol consumption, had no increase of any other serum marker of alcohol abuse (e.g. s-&at, sy-gt, mcv) and had no history of severe liver disease or other diseases than cf (and cf related disorders). some of the patients were below 10 years of age, which makes high alcohol consumption unlikely. conclusion: cystic fibrosis patients have increased s-cdt levels due to defective sialylation and not due to high alcohol consumption. cf patients also have decreased serum levels of trisialo t. further studies have to be performed to evaluate if s-cdt could be used as a screening assay for cf. 234 acknowledgements we want to thank doctor annika holsing for providing serum samples from cf-patients. this work was supported by riksforbundet cystisk fibros. references 1. barasch, j., kiss, b., prince, a,, saiman, l., gruenert, d. & al-awqati, q . : defective acidification of intracellular organelles in cystic fibrosis. nature 352: 70-73, 199 1. 2. cheng, p.w., boat, t.f., cranfill, k., yankaskas, j.r. & boucher, r.c.: increased sulfation of glycoconjugates by cultured nasal epithelial cells from patients with cystic fibrosis. j clin invest 84: 68-72, 1989. 3. chosh, p. & lakshman, m.r.: chronic ethanol induced impairment of hepatic glycosylation machinery in rat is independent of dietary carbohydrate. alcoholism clin exp res 2 1 : 76-8 1, 1997. 4. dosanjh, a., lencer, w., brown, d., ausiello, d.a. & stow, j.l. : heterologous expression of delta f508 cftr results in decreased sialylation of membrane glycoconjugates. am j physiol 266: c360-366, 1994. 5. duthel, s. & revol, a , : glycan microheterogeneity of alpha 1-antitrypsin in serum and meconium from normal and cystic fibrosis patients by crossed immunoaffinoelectrophoresis with different lectins (con a, lca, wga). clin chim acta 2 15 : 173187, 1993. 6. goldberg, d.m. & kapur, b.m.: enzymes and circulating proteins as markers of alcohol abuse. clin chim acta 226: 191-209, 1994. 7. jeppsson, j.o., kristensson, h. & fimiani, c. : carbohydrate-deficient transferrin quantified by hplc to determine heavy consumption of alcohol. clin chem 39: 21 15-2120, 1993. 8. maensson, o., harlin, a,, brandt, r., seppa, k. & sillanaukee, p.: transferrin isoform distribution, gender and alcohol consumption. alcohol clin exp res 2: 17 1017 15, 1997. 9. rosenstein, b.j. & zeitlin, p.l.: cystic fibrosis. lancet 351: 277-282, 1998. 10. schellenberg, f., martin, m., caces, e., benard, j.y. & weill, j.: nephelometric determination of carbohydrate deficient transferrin. clin chem 42: 551-557, 1996. 1 1. stibler, h. : carbohydrate-deficient transferrin in serum, a new marker of potentially harmhl alcohol consumption reviewed. clin chem 37: 2029-203, 1991. 12. stibler, h., borg, s. & joustra, m.: a modified method for the assay of carbohydrate-deficient transferrin (cdt) in serum. alcohol alcoholism suppl 1: 451-454, 1991. 13. stibler, h., von dobeln, u., kristiansson, b. & guthenberg, c.: carbohydrate-deficient 235 transferrin in galactosaemia. acta paediatr 86: 1377-1378, 1997. 14. tsui, l.c.: the cystic fibrosis transmembrane conductance regulator gene. am j respir crit care med 151: s47-53, 1995. 15. warwick, w.j.: cystic fibrosis sweat test for newborns. jama 198, 177-180, 1966. 16. xin, y., lasker, j.m., rosman, a.s. & lieber, c.s.: isoelectric focusing/western blotting, a novel and practical method for quantitation of carbohydrate-deficient transferrin in alcoholics. alcohol clin exp res 15 : 8 14-82 1, 1991. 17. yersin, b., nicolet, j.f., dercrey, h., burnier, m., van melle, g . & pecoud, a.: screening for excessive alcohol drinking. comparative value of carbohydrate-deficient transferrin, gamma-glutamyltransferase, and mean corpuscular volume, arch intern med 155: 1907-19 1 1, 1995. offprint requests to: hans kollberg cf-centre, children’s university hospital, s-751 85 uppsala, sweden 236 (12) gustafsson med fl 131-136 upsala j med sci 111 (1): 131–136, 2006 bile acid metabolism in extrahepatic biliary atresia: lithocholic acid in stored dried blood collected at neonatal screening jan gustafsson1, gunvor alvelius2, ingemar björkhem2, antal nemeth3 department of women´s and children´s health1, uppsala university, uppsala and departments of clinical chemistry2 and pediatrics3, huddinge university hospital, huddinge, sweden. abstract lithocholic acid (lca) is a potent hepatotoxic compound. fetal lca may have a role in the pathogenesis of neonatal cholestasis/extrahepatic biliary atresia (ehba). fetal liver efficiently hydroxylates lca in several positions. this may represent a detox-ification mechanism. in the present study lca, cholic acid (ca) and chenodeoxycholic acid (cdca) were quantitated by gas chromatography-mass spectrometry using selected ion monitoring in small amounts of stored dried blood from six newborn infants with ehba and fourteen con-trols. the blood was collected at neonatal metabolic screening. mean blood levels (±s.e.m.) of lca were 0.11±0.04 µ m in the in-fants with ehba and 0.08±0.02 µ m in the control infants. the correspon-ding levels for ca and cdca were 15.6±3.6 µ m and 7.4±2.5 µ m in the infants with ehba and 1.7±0.3 µ m and 1.8±0.4 µ m in the controls. the increased levels of ca and cdca in the infants with liver disease can be explained by cholestasis. the low blood levels of lca indicate a normal fetal metabolism of this bile acid in ehba. 131 received 4 october 2005 accepted 20 october 2005 introduction lithocholic acid (lca) is a potent hepatotoxic compound causing cholestasis in several species (1). it has been suggested that fetal lca may play a role in the pathogenesis of neonatal choles-tasis/extrahepatic biliary atresia (ehba) (2). fetal lca may have its origin either in primary synthesis in the fetus (3) or it may be a secondary bile acid derived from the mother (4). in the adult liver lca can be metabolized by sulfation, glucuronidation and hydroxylation (3, 5-8). in addition, the metabolism of lithocholic acid into 3dehydrolithocholic acid has been shown in human liver microsomes (9). the capacity for sulfation and glucuro-nidation of lca appears to be low in fetal liver (3, 10), but it has been shown that fetal liver efficiently hydroxylates lca in several positions (11, 12). such metabolism may represent a detoxi-fication mechanism. if the pathogenesis of extrahepatic biliary atresia involves increased levels of lca during fetal life (2, 13), this condition could be associated with impairment in fetal liver hydroxylations of lca which should lead to increased levels of this particular bile acid at birth. the present report describes analysis of levels of lca in small amounts of stored dried blood from newborn infants with extrahepa-tic biliary atresia. the blood was collected at the time of routi-ne neonatal metabolic screening during the first days of life. subjects stored dried blood, obtained at neonatal metabolic screening from six newborn infants, who were later shown to suffer from extrahe-pa-tic biliary atresia, was analyzed as described in methods. none of the patients was shown to have an underlying cause for their liver disease such as viral infection or inborn error of metabo-lism. as controls, 14 newborn infants, born at the same hospitals as the patients, were used. the control infants had been born immediately before or after the diseased infants. for two of the patients four controls were used, whereas for three of the pati-ents material from two controls, respectively, was av-ailable. for one patient there was no material available from corresponding control infants. patient 1 a girl, born after 40 weeks of normal pregnancy. her birthweight was 3790 g. laparotomy was performed due to persis-tent hyperbilirubinemia and extrahepatic biliary atresia was diagnosed. at two months of age the patient was operated with a portoenterostomy (kasai operation) and an external jejunostomy. microscopical examination of liver tissue supported the diagnosis biliary atresia. the condition of the patient deteriorated gradu-ally and she died at the age of 14 months due to liver failure. 132 abbreviations lca, lithocholic acid (3_-hydroxy-5_-cholanoic acid) ehba, extrahepatic biliary atresia ca, cholic acid (3_,7_,12_-trihydroxy-5_-cholanoic acid) cdca, chenodeoxycholic acid (3_,7_-dihydroxy-5_-cholanoic acid) patient 2 a boy, born after 36 weeks of normal pregnancy. his birthweight was 2760 g. extrahepatic biliary atresia was diagnosed when laparotomy was per-formed at two months of age due to persis-tent hyperbiliru-binemia. at 12 months of age the patient was operated with a portoenteros-tomy. microsco-pical examination of the liver tissue was in agreement with bilia-ry atresia. the liver disease of the patient had a relatively slow progress and at 23 months of age the patient was subjected to orthoptic liver trans-plant. patient 3 a boy, born after normal pregnancy with birth weight 3250 g. he developed jaundice from day 5 and later acholic faeces. at 9 weeks, a clinical investigation indicated biliary atresia. portoenterostomy was performed at the age of 10 weeks. preand intraoperative biopsies showed a liver histology typical for extrahepatic cholestasis. after several episodes of cholangitis and one period of rickets the general condition of the patient improved gradually. at 2 years of age the boy had a mild cirrhosis but an almost normal general condition. patient 4 a girl, born small for gestational age after 42 weeks, with a birth weight of 2820 g. immediately after birth she develo-ped cholestatic jaundice. at 5 weeks extrahepatic biliary atresia was diagnosed at laparotomy and a liver biopsy showed advanced cirrhosis. a kasai operation, performed at this age, did not influence the course of the disease. the girl died at 10 months of age, while waiting for orthoptic liver transplant. patient 5 a boy, born after normal pregnancy, with appropriate birth weight, 3435 g. he developed a major gastrointestinal blee-ding at the age of 1 month due to hypoprothrombinemia caused by anicteric cholestasis. extrahepatic obstruction was diagnosed. laparotomy and a kasai operation were performed at 9 weeks of age. liver biopsy showed advanced cirrhosis. the patient failed to thrive in spite of vigorous nutritional treatment. he died due to bleeding oesophageal varices at the age of 11 months, while wai-ting for liver transplantation. patient 6 a boy, born after normal pregnancy, with birth weight 3820 g. immediately after birth he developed cholestatic jaundice. an examination indicated extrahepatic biliary obstruction. laparotomy and a modified kasai operation with biliary fistula were performed at the age of 1 month. during his first year he had frequent attacks of cholangitis. at one year of age the biliary fistula was removed, whereafter the attacks of cholangitis were less frequent. although the boy never became anicteric and the initial postoperative development was unfavou-rable, his hepato-cellular func-tions were satisfactory seven years after the opera-tion. methods [2,2,3,4,4]-2h5 -cholic acid and [2,2,3,4,4]-2h5 -chenodeoxycholic acid were prepared as described previously (14). [24]-14c-lithocho-lic acid was obtained from amersham, buckinghamshire, england. filter paper blood samples, collected 133 according to the swedish routine neonatal screening program from newborn infants, who were later shown to suffer from biliary atresia, and control infants were used for the analyses (c.f. subjects). before analysis, the material was stored for 1-6 years at +4oc (c.f.15). for the analyses, filter paper material corresponding to 25-50 µ l of blood were extracted with absolute ethanol-potassium phosphate buffer, 0.1m, ph 7.4 (1:1, vol/vol) (15). before the extraction deuteriumlabeled ca, cdca and 14c-labeled lca were added to the solution. during the work up pro-cedure the material was sonicated for 2 minutes and then heated at 70o c for 15 minutes. after centrifugation, the supernatant was hydrolyzed at 110o c for 10 hours with 2m potassium hydroxide solution. after an initial ether extraction the solution was acidified with hydrochloric acid and the bile acids were extracted twice with ether. following methyla-tion and trimethylsilylation the bile acids were quantitated by gas chromatography-mass spectrometry. the concentrations of ca and cdca were obtained from isotope dilution-mass spectrometry as described earlier (14). the concentration of lca was determined from isotope dilution-mass spectrometry using [24]-14c -lithocholic acid as internal standard by recording the ions at m/z 372 and 374 respectively. the above procedure quantitates conjugated as well as glucuronida-ted lca and should also include the major part of sulfated lca present in blood (cf. 16). the very small amounts of material available prevented a detailed analysis of the degree of conjuga-tion and esterification of lca. results table 1 shows results of determinations of lca, ca and cdca in stored dried blood from newborn infants with extrahepatic biliary atresia and their corresponding controls. ca dominated in blood of the patients. the total concentration of bile acids was higher in the blood of the patients as compared to the controls. both groups of children had low levels of lca. there was no evidence for presence of c27-bile acids, neither in the samples of the patients nor in those of the controls. 134 table 1 concentrations of bile acids (µmol/l, mean±s.e.m.) in stored dried blood collected at neonatal metabolic screening from six patients with ehba and 14 controls lithocholic acid cholic acid chenodeoxycholic acid patients 0.11±0.04 15.6±3.6 7.4±2.5 controls 0.08±0.02 1.7±0.3 1.8±0.4 table 1. concentrations of bile acids (µ mol/l, mean±s.e.m.) in stored dried blood collected at neonatal metabolic screening from six patients with ehba and 14 controls 135 discussion dried blood, obtained at neonatal screening, is used to detect several metabolic and endocrine diseases. in addition, saved material can be used to diagnose metabolic abnormalities also when the patient is no longer alive (15). we have earlier used such stored material, collected at neonatal screening, to diagnose zellweger syndrome, a peroxisomal disease with defects in bile acid synthesis (15). in that study, c 27 and c 29 -bile acids were shown to be present in stored dried blood from newborn infants with zellweger syndrome (15). in the present study we describe a method for quantitation of lca, based on isotope dilution mass spectrometry, in small amounts of stored dried blood obtained from newborn infants with ehba. lca is a hepatotoxic bile acid, which could have a pathogenetic role in neonatal cholestasis (2, 13). the occurrence of lca in the newborn may be due placental transfer from the mother (4) or possibly to synthesis in the fetal liver (3). normally, fetal liver efficiently hydroxylates lca (11, 12). this may represent a detoxification mechanism (11). if lca is a pathogenetic factor in ehba, increased levels of this bile acid should be expected to prevail in newborn infants with this disease. the relative amounts of the different bile acids in the control infants are in accordance with studies performed with radio-immuno assay in serum of a larger number of newborns (17). the results show the presence of low levels of lca both in newborn infants with ehba and in corresponding controls. the low levels of lca found indicate a normal metabolism of this bile acid in fetuses with ehba, making it unlikely that lca plays a role in the pathogenesis of this condition. the increased levels of ca and cdca found in the blood of the patients is probably a result of cholestasis. the predominance of ca in the blood of the patients may be due to the fact that the samples are taken at an relatively early stage in the course of the disease, since this result is in agree-ment with those reported in cases of intrahepatic and extrahepatic cholestasis with normal hepatic parenchyma (2). the finding is in contrast to what has been reported for older infants with ehba, in which a bile acid pattern with high concentrations of cdca, possibly reflecting an underlying hepatocellular damage, has been described (18). acknowledgements this work was supported by the swedish medical research council. references 1. tuchweber b, perea a, lee d, yousef im (1983) lithocholic acid induced cholestasis in newborn rats. toxicol lett 19:107-112. 2. javitt nb (1983) progress in gastroenterology. cholestasis in infancy. status report and conceptual approach. gastroenerology 70:1172-1181. 3. lester r, st. pyrek j, adcock ew (1983) diversity of bile acids in the fetus and newborn infant. j pediatr gastroenterol nutr 2:355-364. 4. subbiah mtr, hassan as (1982) development of bile acid biogenesis and its significance in cholesterol homeostasis. adv lipid res 19:137-161. 5. matern s, matern h, farthmann eh, gerok w (1984) hepatic and extrahepatic glucuronidation of bile acis in man. j clin invest 74:402-410. 6. trülsch d, roboz j, greim h, czygan p, rudick j, hatterer f, schaffner f, popper h (1974) hydroxylaion of taurolithocholate by isolated human liver microsomes. biochem med 9:158-166. 7. hofmann af (2004) detoxification of lithocholic acid, a toxic bile acid: relevance to drug hepatotoxicity. drug metab rev 36:703-722. 8. araya z, wikvall k (1999) 6alpha-hydroxylation of taurochenodeoxycholic acid and lithocholic acid by cyp3a4 in human liver microsomes. biochim biophys acta 1438:47-54. 9. bodin k, lindbom u, diczfalusy u (2005) novel pathways of bile acid metabolism involving cyp3a4 biochim biophys acta 687:84-93. 10. balistreri wf (1991) fetal and neonatal bile acid synthesis and metabolism clinical implications. j inher metab dis 14:459-477. 11. gustafsson j, andersson s, sjövall j (1987) bile acid metabolism during development: metabolism of lithocholic acid in human fetal liver. ped res 21:99-103. 12. courillon f, gerhardt mf, myara a, daffos f, forestier f, trivin f (1998) serum c24 bile acids in the developing human fetus. biol neonate 73:76-88. 13. jenner re, howard er (1975) unsaturated monohydroxy bile acids as a cause of idiopathic obstructive cholangiopathy. lancet 7944:1073-1075. 14. björkhem i, falk o (1983) assay of the major bile acids in serum by isotope dilution-mass spectrometry. scand j lab clin invest 43:163-170. 15. gustafsson j, sisfontes l, björkhem i (1987) diagnosis of zellweger syndrome by analysis of bile acids and plasmalogens in stored dried blood collected at neonatal screening. j pediatr 11:264-267. 16. van berge-henegouwen gp, allan rn, hofmann af and yu pys (1977) a facile hydrolysis-solvolysis procedure for conjugated bile acid sulfates. j lipid res 8:118-122. 17. barbara l, lazzari r, roda a, aldini r, festi d, sama c, morselli am, collina a, bazzoli f, mazzella g, roda e (1980) serum bile acids in newborns and children. ped res 14:1222-1225. 18. javitt nb, lavy u (1974) serum chenodeoxycholate in neonatal liver disease. in: matern s, hackenschmidt j, back p, gerock w (eds) advances in bile acid research, fk schattauer verlag, stuttgartnew york, 123-126. corresponding author: jan gustafsson md, phd department of women´s and children´s health university children´s hospital s-751 85 uppsala sweden tel. (46) 18 611 58 19 fax (46) 18 611 58 53 136 tf-iups160013 81..95 review article pancreatic islet blood flow and its measurement leif janssona, andreea barbua, birgitta bodina, carl johan drotta, daniel espesa,b, xiang gaoa, liza grapensparra, €orjan k€allskoga, joey laua, hanna liljeb€acka, fredrik palma, my quacha, monica sandberga, victoria str€omberga, sara ullstena and per-ola carlssona,b adepartment of medical cell biology, uppsala university, uppsala, sweden; bdepartment of medical sciences, uppsala university, uppsala, sweden abstract pancreatic islets are richly vascularized, and islet blood vessels are uniquely adapted to maintain and support the internal milieu of the islets favoring normal endocrine function. islet blood flow is normally very high compared with that to the exocrine pancreas and is autonomously regulated through complex interactions between the nervous system, metabolites from insulin secreting b-cells, endotheliumderived mediators, and hormones. the islet blood flow is normally coupled to the needs for insulin release and is usually disturbed during glucose intolerance and overt diabetes. the present review provides a brief background on islet vascular function and especially focuses on available techniques to measure islet blood perfusion. the gold standard for islet blood flow measurements in experimental animals is the microsphere technique, and its advantages and disadvantages will be discussed. in humans there are still no methods to measure islet blood flow selectively, but new developments in radiological techniques hold great hopes for the future. article history received 3 march 2016 accepted 8 march 2016 keywords blood flow measurements; islet blood flow; microspheres; pancreatic islets in 1960 claes hellerstr€om and co-workers published a paper in which they tried to assess the b-cell activity by quantifying the number of erythrocytes in the islets (1). a correlation between islet endocrine activity and blood flow was assumed, but this was difficult to demonstrate due to lack of precision in the method. he did not, however, forget the topic, and 20 years later in 1980 one of the authors (l.j.) of the present review began studying the islet vasculature under the benevolent and extremely stimulating guidance of claes. claes, you were, as always, correct! there is a correlation between islet endocrine activity and blood flow, and you helped to develop this field of research. we aim to give a brief overview of the islet vasculature, and focus on the methods that can be used to measure its blood flow, discussing the advantages and disadvantages with the various techniques. 1. pancreatic islet morphology a. cellular composition in’t veld and marichal have provided an excellent overview of pancreatic islet morphology (2) where further references can be found. in most vertebrates pancreatic islets are distributed throughout the pancreas in cellular aggregates containing from a few up to several thousands of cells. they are demarcated by a connective tissue capsule, the extent of which is species-dependent. most focus on islet function has been directed towards their endocrine cells, in particular the insulin-producing b-cells. these cells constitute 40%–70% of all endocrine cells in the islets, depending on species. common laboratory animals, i.e. mice and rats, have more b-cells (60%–70%) than humans (40%–50%) (3). the other islet endocrine cells are glucagon-producing a-cells, somatostatin-producing d-cells, pancreatic polypeptide-producing pp-cells, and ghrelin-producing e-cells. most islets contain a mixture of these cells. a notable exception is the uneven distribution of ppand a-cells, where the former are found mainly in the caput region, i.e. the former embryonic ventral anlage, whereas a-cells are found mainly in the tail region, corresponding to the dorsal anlage. b-cells are more evenly distributed in the pancreas, even though humans seem to possess occasional b-cell-rare islets. it should be noted that smaller islets contain predominantly b-cells, whilst the other endocrine cell types are mainly found in the larger islets. the reasons for the coexistence of these hormone-producing cells in discrete micro-organs are not known for certain (4), and we will briefly return to this issue later, since we believe that this organization profoundly affects the interactions between endocrine cells and islet vasculature to achieve a fine-tuning of islet hormone release. besides the endocrine cells, the islets contain numerous other cell types, vascular cells, other stromal cells, immune cells, and neural elements (table 1). the present review focuses mainly on the blood perfusion of the islets, and we will therefore discuss in more detail the cells associated with the islet vasculature. nervous elements in the islets consist of nerve cells with axons, referred to as neuro-insular complexes type 1 when there are nerve cells bodies, and type ii when contact leif jansson, leif.jansson@mcb.uu.se department of medical cell biology, biomedical centre, box 571, husargatan 3, se-75123 uppsala, sweden � 2016 informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited upsala journal of medical sciences, 2016 vol. 121, no. 2, 81–95 http://dx.doi.org/10.3109/03009734.2016.1164769 only intra-islet axons are found (5,6). many of these nerves affect not only hormones secretion per se, but also islet blood flow. the schwann cells should be mentioned in this context, since they seem to surround all islets in an envelope, but their functions remain enigmatic (7). it has been suggested that they may participate in regenerative processes after islet transplantation (8). immune cells can be numerous during islet inflammations, i.e. insulitis, but are normally sparse in the islets. interestingly, it has recently been suggested that recruitment of different subtypes of macrophages may influence the vascularization of islets, especially after transplantation (9,10). however, these notions are beyond the scope of this review. among stromal cells fibroblasts contribute by forming the thin network of reticular and collagenous fibers in the islets as well as the capsule surrounding each islet. normally also pancreatic stellate cells with their characteristic lipid droplets can be seen (11). their exact function in the islets is unknown, but their numbers are increased during desmoplastic reactions to pancreatic adenocarcinomas as well as during chronic pancreatitis (12). in both these instances they contribute to the pancreatic fibrosis characteristic of these conditions. we have also noted that their numbers in islets are increased in gk rats, a type 2 diabetes model characterized by islet fibrosis (11). there are myofibroblasts present under these conditions as well (13), but whether they emanate from the stellate cells or other cell types is unknown. another stromal cell in islets is the interstitial cell of cajal, a cell of neural origin. such cells are common in the intestine where they serve as pacemaker cells, being active in regulation of intestinal motility (13). whether such effects occur also in the pancreas is an intriguing possibility, but so far unknown (14). b. morphology of pancreatic islet vasculature this subject has been highlighted in several reviews (2,15–21). the islets contain fenestrated capillaries, which constitute 8%–10% of the islet volume, and are organized into a glomerular-like network, described already in the 1880s (22), i.e. at a time when islet function was unknown. the number of fenestrae is approximately 10 times higher than in exocrine pancreatic capillaries (23) and is induced by the high local production of vascular endothelial growth factor-a (vegf-a) from the islet b-cells (24,25). thus, after transplantation when islets are implanted into organs with continuous capillaries, the ingrowing new microvessels initially form such capillaries, but already after a few days the endothelium becomes fenestrated (26). it is likely that the fenestrations facilitate hormonal passage into the circulation as well as participate in draining extracellular fluid analogous to functions normally performed by lymphatic capillaries (27). besides being fenestrated, islet capillaries are wider than those in the exocrine pancreas, having a 20%–30% larger diameter, and their vascular density is much higher (23). it can be noted that after transplantation the vascular density of the islet grafts in sites such as the liver is less than that in endogenous islets for several months post-transplantation (15,28). pericytes located between the endothelium and the capillary basement membrane can occasionally be found, but they are not particularly numerous (8,29). their functions in islets are little studied and largely unknown. it has been hypothesized that they contribute to angiogenesis or as contractile cells (8), but these notions are mainly conjectural. the larger islets contain one to three afferent arterioles, which originally branch from intralobular arteries in the exocrine tissue. these afferent arterioles sometimes contribute tributaries also to exocrine acini, but many of them supply only the islets (16). smaller islets are directly incorporated into the capillary network of the exocrine pancreas. the organization of larger islets means that there is an anatomical correlate, which enables autonomic regulation of the islet blood perfusion. the diameter of the afferent arteriole is usually 20–30 lm (figure 1), and it contains one, or occasionally two, layers of vascular smooth muscle cells (vsmc), which are surrounded by adventitial tissue containing fibroblasts, but also occasional adipocytes (30). the anatomy of the veins draining the islets is to some extent species-dependent and also depends on the size of the islets. as mentioned above, smaller islets drain with exocrine veins, whereas larger islets have a different organization (16). most common is a direct venous drainage to one to four larger veins with a basket-like network of smaller vessels lying exoc exocrine pancreas blood flow: 0.4-1.0 ml/min x g po2: 40 mmhg capillary pressure: 6-7 mmhg blood flow: 5-6 ml/min x g po2: 40 mmhg capillary pressure: 4-5 mmhg islet figure 1. blood flow values in exocrine and endocrine pancreatic parenchyma. modified from jansson and carlsson (15). table 1. cell types in pancreatic islets. cell types endocrine cells a-cells b-cells d-cells e-cells pp-cells stromal cells fibroblasts myofibroblasts stellate cells cajal cells occasional duct cells vascular cells endothelial cells vascular smooth muscle cells pericytes adventitial stromal cells immune cells granulocytes lymphocytes macrophages dendritic cells mast cells neural cells neurons schwann cells data from (2,20). 82 l. jansson et al. in association with the connective tissue capsule at the interface between the islets and the surrounding exocrine tissue. these veins then empty into intralobular veins and, finally, into the portal vein. however, some of the islets, particularly in larger mammals, have a so-called insulo-acinar portal system. this means that the islet capillaries empty into numerous small venulae, which then anastomose with capillaries in the exocrine pancreas. by such means there are two serially connected capillary systems, i.e. a portal system. in rodents this is of minor importance, but its role in human islets, as well as other primates, is likely to be larger even though this is little studied (16,31). in general, islets are devoid of lymphatic capillaries, whereas the exocrine pancreas contains numerous such vessels (27,32). lymphatics draining the pancreas contain insulin, but at a concentration <30% of that seen in the portal vein (33). to what extent this represents recirculation of insulin is unknown. in islet grafts, lymphatics initially develop, but with time they decline in number (34). these findings have been interpreted to reflect that the highly permeable islet blood capillaries, as well as the small size of the islets, preclude the need for lymphatic drainage. other endocrine organs, such as the pituitary and parathyroid glands contain lymphatics, but they are much larger than those of islets, and the need to prevent prolonged release of hormones into the circulation by the lymphatics is not present. from an evolutionary point of view, the brockman bodies seen in fish, i.e. centimeterlarge accumulations of islet tissue, contain lymphatics (35). 2. functional implications of islet morphology on blood flow regulation all functional evidence suggests that islet blood flow is regulated autonomously from that of the exocrine pancreas and that this regulation occurs at a pre-capillary level, i.e. in the arteriole (36); for a summary see brunicardi et al. (21,37). there is no evidence that post-capillary regulation located to the draining veins occurs (38). in this section we will provide a more detailed overview of the sites that we believe are of importance for islet blood flow regulation. a. arterioles the presence of vsmc in the arterioles provides the basis for the separate regulation of islet blood flow. both arterioles and the endocrine cells themselves receive efferent nerves, particularly sympathetic nerve fibers, but also non-adrenergic, non-cholinergic (nanc) nerves (5,6,39). the latter are presumably purinergic and/or nitric oxide (no)-containing nerves (40). furthermore, afferent sensory nerves affecting blood perfusion (41) can also be found within the islets (42). a summary of the effects of nerves on islet blood flow and insulin secretion is provided in table 2. arteriolar vsmc are also responsive to most locally produced endothelium-derived vasoactive substances but can be affected by substances, including hormones, reaching them through the systemic circulation as well. islet endothelium, mainly in arterioles but also capillaries, produces both no and endothelin-1 (30,43) and probably several other mediators that may directly influence the vsmc. the prime importance of no for islet blood regulation has been repeatedly demonstrated (44,45). during the last few years, increased focus has been directed against the adventitia of resistance vessels, since e.g. adipocytes, macrophages, and other cells found here may also produce substances, such as adipokines, cytokines, and gaseous transmitters, which may affect vsmc through intravessel paracrine mechanisms. we have for instance observed that gk rats, a type 2 diabetes (t2d) model, express more interleukin-6 in their adventitia than normal rats (unpublished observation). a further influence on arteriolar vsmc may be derived from substances produced by adjacent endocrine cells. it should be noted that some arterioles branch at the periphery of the islets, whilst others penetrate into the islet core before forming into capillaries (16,37,46). this means that a variable part of the arteriole is exposed to high concentrations of substances produced within the islets. these substances include islet hormones and factors co-released with endocrine granules, e.g. chromogranins, synaptophysins, and zn2þ (47). furthermore, other substances produced by metabolically active endocrine cells, namely nucleotides such as atp, adp, and adenosine, may also be present in high concentrations close to the vsmc (48). it is easy to envisage that such substances may affect both endothelial cells and vsmc in the arterioles and thereby affect blood flow. however, to what extent they may also affect ionic channels in capillary endothelial cells and then transmit retrograde signals to the arterioles (49) is an intriguing concept, but one not yet fully explored. b. capillaries since capillaries consist of a tube of single endothelial cells surrounded by a basement membrane, they do not in general possess any blood flow regulatory properties. no signs of any pre-capillary sphincters have been noted in either islet arterioles or proximal capillaries, so it seems likely that the flow regulation is indeed mainly restricted to the arterioles. there are occasional pericytes (29,50), but their contractility and participation in flow regulation in mammals are controversial. morphological studies of islet capillaries, as well as studies on isolated islet endothelial cells, have not demonstrated any table 2. effects of different neurotransmitter substances on insulin release and islet blood flow (ibf). insulin ibf muscarinic (m3) receptors þ þa b1 adrenoceptors 0 0 b2 adrenoceptors þ – b3 adrenoceptors 0 þb a1 adrenoceptors – þ a2 adrenoceptors 0 0 b d2 receptors – þb d3 receptors 0 0 data from (5,95,166,167). ainhibitory effect during hyperlipidemia. bnormalizes increased basal islet blood flow in gk rats, a type 2 diabetes model. upsala journal of medical sciences 83 heterogeneity between individual islets. the organization of the islet capillaries is usually described as glomerulus-like, and islets are highly vascular, although not as profound in human as in rodent islets (51,52). most b-cells have at least parts of their cytoplasm facing a capillary, and the endocrine granules are preferentially located in this part of the cytoplasm (53). this seems to be the case also for the other islet endocrine cells, with the possible exception of d-cells. the latter cell type contains long, slender processes which establish contact with other cell types and to some extent also to the islet capillaries (54). this endocrine cell polarity provides the basis for the b-ad flow hypothesis formed on morphological observations and perfusion studies in rodent islets, but has also been suggested to be valid for human islets (55). rodent islets have a b-cell-rich core surrounded by a mantle of non-b-cells. the arterioles are described as entering the islets through a discontinuity in this mantle, and then branch into capillaries in the b-cell-rich core of the islets (figure 2). this would mean that high local insulin concentrations are transported in the blood towards the periphery of the islets, where a-cells would add glucagon and, finally, the d-cells their somatostatin. paracrine/endocrine interactions between the islet endocrine cells are thereby, at least partially, mediated by the flow direction in the islets (56). besides these morphological studies, also functional studies with retrograde perfusions corroborating these notions have been performed (55). it is worthy of note, however, that this theory has been disputed (21). indeed, there have also been suggestions that the blood flow direction instead is from the periphery towards the center or from ‘top to bottom’ without any direct correlate to microregions of different endocrine cells (21,37). in an elegant study nyman and co-workers (46) examined this in detail in mice and found that all of these patterns could be found, but that core-to-mantle constituted approximately 50% of the studied islets. for technical reasons these studies were confined to larger islets. we have in a series of experiments in rats retrogradely injected microspheres and confirmed the findings of nyman and co-workers (unpublished observations). c. veins the morphology of the venous drainage has been mentioned in the preceding section. the morphology of the veins themselves is similar to that of other veins and venulae in the body, and as expected no valves have been described. venous vsmc are sparse, and the vessel size is quite variable. venulae associated with insulo-acinar portal systems are small and have a diameter of only 15–20 lm (57). there is no evidence of any flow control by the activity of venular vsmc (38). an interesting observation is that when islets are transplanted the graft is drained exclusively by larger veins, and no signs of an insulo-acinar portal system can be discerned (58). another issue of importance is that islet inflammation, such as insulitis, which is associated with a mononuclear cellular infiltration, seems to be initiated in the islet periphery (59,60), i.e. at the location of the veins. in the diabetes-prone biobreeding/worcester rat, an animal model of type 1 diabetes, an islet venular defect has also been described (61). moreover, occasional mast cells, which affect venular permeability, can be found at this location (62), and the number of mast cells is increased in t2d (63). after islet transplantation, so-called ‘blood lakes’ are formed, which probably emanate from disrupted veins. they restitute and become replaced by connective tissue a few days post-transplantation (15). d. islet cellular composition affecting vascular morphology the islet vascular architecture referred to above is probably not static, but is likely to vary not only between different islets but also over time and depending upon the functional demands on the endocrine cells (64). it has been known that smaller islets in rodents with diameters <100 lm mainly contain b-cells (65). these islets seem to be incorporated into the exocrine capillary system (66). this implies that the regulation of islet blood flow in smaller islets is probably not autonomic from that of the exocrine pancreas, but is regulated in concert with the latter. it should be noted, however, that the contribution of the smaller islets to the total pancreatic islet volume is small (65), so a majority of islet endocrine cells are located to islets with an autonomous blood flow regulation. human islets sometimes consist of many a-cells, but the blood flow regulation of such islets is at present unknown. an interesting comparative physiological approach in this context is that some birds, e.g. ducks, have islets containing predominantly a-cells, and the vasculature of such islets differs from that of b-cell-containing islets (67). another factor which affects not only the cellular composition per se but also islet blood flow is age. before birth islets are small and very few islet capillaries have developed, but in the post-partum period islet endocrine and vascular mass both rapidly expand (68). nevertheless, the blood perfusion figure 2. estimation of intra-islet insulin concentrations based on previously published data on insulin release (168) and islet blood flow (87). 84 l. jansson et al. in 5-week-old animals is considerably lower than in older animals. for instance, the fractional islet blood flow in 5-week-old rats is only 1%–2%, which increases to 8%–10% after 15 weeks, 15% after 12 months, and >20% at 2 years of age (69–71). similar findings have been made in mice (72). this increase is present also when adjusted for changes in islet volume. we believe that this reflects increased insulin resistance with age, but the exact mechanisms behind the increase are not known, even though it seems likely that no is involved (73). when islet growth is stimulated as after partial pancreatectomy (74,75) and during pregnancy (76) there is usually an increased islet blood perfusion. a controversial issue in the context of islet growth is to what extent new islets may form by ‘budding’ from precursor cells in smaller pancreatic ducts, an issue previously taken for granted (77,78), but lately refuted (79). there are some older studies suggesting that peri-ductular islets derive their arterial blood from ductal blood vessels (80), and it has even been suggested that ductal blood vessels constitute a third capillary network in an intra-pancreatic portal system (81). in that latter study the authors described that the first system would be in islets, the second in exocrine tissue, and, as mentioned, a third in ducts. such islets would probably contribute very little to total islet volume, and therefore be of little importance. e. pancreatic blood flow and heterogeneity between islets when referring to blood flow values in the exocrine and endocrine pancreas we use values obtained by the microsphere technique. the blood perfusion of the pancreatic islets is considerably higher than that of the exocrine gland, and it is, in adult rats, 5%–20% of total pancreatic blood, despite the islet volume constituting only 1%–2% of the pancreas. normally, there is a close correlation between islet blood flow and total pancreatic blood flow (figure 3), but during e.g. hyperglycemia their responses become dissociated (82,83). when correcting for weight, the corresponding value for islet blood flow is 5–6 ml/min�g islets (figure 1), which is one of the higher blood flow values in the body, normally only surpassed by that to the oxygen-sensing glomus cells in the carotid body (84). very high flow values were recorded also in other species such as for instance atlantic hagfish (85), mouse (86), and rabbit (87). there are, however, no differences in blood perfusion between islets in the head region of the gland, i.e. pancreatic polypeptide-rich islets supplied by the superior mesenteric artery, and those in the tail region which are glucagon-rich and receive their blood from the celiac trunk (88). this holds true for the exocrine pancreas as well, which despite its dual arterial supply regulates its blood flow synchronously (89). of interest in this context is whether this high blood perfusion involves all islets, or only some of them, which would then constitute an extremely well-perfused and presumably functionally more active subgroup. indeed, repeated microsphere injections have indicated that microspheres predominantly end up in the same 10% of islets (90), and that these islets further increase their blood perfusion in response to glucose (90). injection of a marker of low oxygenation, pimonidazole, demonstrated that 20%–25% of the islets are normally poorly oxygenated and with less protein biosynthesis than others, but become better oxygenized at increasing demands for insulin release (28). with a keen eye to its limitations the microsphere technique has been used to identify tentatively the most highly perfused islets, and differences in vascular density, glucose metabolism, b-cell proliferation, and insulin release have been identified, suggesting that there is indeed a coupling between islet blood flow and metabolic activity (91,92). it is worthy of note, however, that only insulin has been studied in this context, and it is unknown if such relationships exist also for other islet hormones. the observation of a highly perfused subgroup of islets opens the intriguing notion that islets receiving lower blood flow may constitute a functional reserve, which can increase their flow and thereby their functional activity upon demand. it is tempting to speculate that this may be a safety measure to prevent an excessive insulin release at any given time. it would also provide a twist to the question raised for many years (4), why b-cells are distributed into a vast number of tiny cell aggregates rather than forming a large, compact gland like most other endocrine organs. 3. techniques used to study islet blood flow the high islet blood flow is normally further increased whenever there is a need for increased insulin secretion, and this is assured by complex interactions between several regulatory mechanisms. furthermore, conditions with decreased glucose tolerance, when there is a need for augmented insulin release, are also associated with an increased islet blood flow (89), and such factors ensuring these blood flow responses include: 1. metabolic factors from b-cells: we have noted that especially adenosine and adp seems to be of major importance for this, and directly couple the degree of ibf (µl/min x g pancreas) 10 20 30 40 50 60 70 p b f ( m l/m in x g ) 0,0 0,2 0,4 0,6 0,8 1,0 1,2 figure 3. correlation between islet blood flow (ibf) and total pancreatic blood flow (pbf) on anesthetized normoglycemic sprague-dawley rats. data are based on 36 recent control experiments. (pancreatic blood flow ¼0.0466þ (0.0158 � islet blood flow)). p < 0.001. upsala journal of medical sciences 85 metabolism to the required blood flow increase (93,94). 2. nervous system: the vagus nerve, sympathetic nerves, and sensory nerves all affect islet blood flow, and their effects are summarized in table 2 (95). 3. endothelium-derived constricting and dilating factors: so far approximately 10 different substances in this category have been examined, and the responses are as expected. the pronounced sensitivity for no shows its major importance for islet blood flow regulation (44). 4. hormones: islet hormones, incretins, and adipokines are potential regulators of islet blood flow. with the exception of incretin hormones, there are only sparse effects of hormones on islet blood flow, but incretins seem to potentiate glucose-induced stimulation of islet blood flow (96). these blood flow regulatory mechanisms are very similar to those mediating responses also in other tissues, and especially so to those mediating postprandial reactive hyperemia in the gut (97). however, with regard to the islets it seems as if the major factor affecting islet blood flow is the prevalent blood glucose concentration, and this may be by activating one or several of the factors referred to above (83,90,98). this would then modulate the blood perfusion according to the needs of the insulin-secreting b-cells. to the best of our knowledge there is no similar coupling between the release of the other islet hormones and blood flow. the distribution of the islets within the exocrine parenchyma means that measurements of total pancreatic blood flow is not sufficient, since the fraction of the blood flow being diverted to the islets is unknown. therefore, measurements must separately register endocrine and exocrine blood flow. this precludes the use of several common methods used to assess regional blood circulation, such as those based on the fick principle, or electro-magnetic or ultrasound flow probes placed around arteries or veins in the organ. these techniques can only determine total pancreatic blood flow, and since the pancreas has multiple arterial supplies, both the superior mesenteric artery and the celiac trunk, this necessitates the placement of at least two probes (89). also techniques based on radiological methods such as magnetic resonance imaging (mri) and positron emission tomography (pet), which have the added benefit of being applicable to humans, provide measurements only of total pancreatic blood flow, mainly due to the fact that the resolution is not sufficiently high to allow visualization of individual islets. however, these problems are at present being overcome, and in the foreseeable future radiological techniques may allow visualization of the islet organ to allow not only determinations of islet blood perfusion, but even more importantly to quantify islet mass in patients (99). several techniques have been experimentally applied to assess islet blood flow, almost exclusively in rodents and mainly in rats. the predominant techniques that have been used to investigate the blood perfusion of endogenous islets are different in vivo microscopy applications, microsphere measurements, and most recently hydrogen gas clearance. studies on transplanted islets have applied mainly in vivo microscopy and laser-doppler flowmetry, but also microspheres have been tried (15,100). we will now comment on advantages and disadvantages with these techniques with a major focus on measurements in endogenous islets. a. microsphere measurements of islet blood flow this technique is a variant of the deposition techniques (see below under d). then, instead of chemical substances, small polystyrene particles (commonly referred to as microspheres) are injected into the arterial blood stream. they are distributed into the different capillary beds, where they become entrapped. in this manner, nutritive blood perfusion rather than plasma perfusion is measured, since the microspheres distribute as erythrocytes. the number of microspheres within each organ is proportional to their blood perfusion, and this can be assessed by quantification of their numbers, either directly by counting or by assessing their labels, which can be radioactivity, fluorescence, or different colors (101). the first experiments applying entrapped particles to measure local blood flow were carried out with starch particles in pigs (102), but other materials such as ceramics have been used. the technique in its present form was introduced in 1967, when isotope-labeled plastic particles with a diameter of 50 lm were injected into fetal lambs (102). this demonstrated that, in comparison with antipyrine measurements, these microspheres did not recirculate to any significant extent, distributed in proportion to the blood flow, and did not affect the circulation physiology in the fetuses, i.e. fulfilled the basic criteria of the microsphere technique. the year after, the concept of an arterial reference sample had been introduced (103), which made measurements of cardiac output with this technique possible. since then this application has developed into the gold standard for regional and intra-organ blood flow measurements (101,104–106). the adequate use of microspheres necessitates the fulfillment of several criteria, namely: 1) adequate mixing of the microspheres with blood in the central circulation; 2) complete extraction of microspheres during the first passage through the tissues; 3) flow properties similar to those of red cells; 4) no circulatory artifacts should be induced by the microspheres; 5) the microspheres (or their marker if they are labeled with something) should remain in the tissues; and 6) the measuring accuracy should be sufficient. most islet blood flow studies, which were first performed in the early 1980s (71,83,87) have used polystyrene plastic particles with a diameter of 10 lm (or occasionally 15 lm) stained black or other colors. fluorescence-labeled microspheres have been used as well (91,92), whilst microspheres with radioactive tracers have been rarely used, even though they have been commonly utilized for measurements of other organ blood flow values (107). (i) criteria for the adequate use of microspheres. it is important that the microspheres are adequately mixed with the arterial circulation and occupy the entire vessel profile, so that their disposal into the tissues mimics that of red blood cells. this can most easily be achieved by administering the 86 l. jansson et al. microspheres where arterial blood flow is turbulent, i.e. into the left atrium or ventricle or in the ascending aorta. indeed, the heart is the only choice if myocardial blood flow is to be studied, since the coronary arteries branch immediately above the aortic valves. in studies in rats less variation in the flow determinations were seen after intra-atrial injections (108). however, in small animals such as rats and mice cannulation of especially the left atrium, but also the left ventricle, can be difficult. an alternative experimental maneuver may be to give the microspheres via cardiac puncture, an approach chosen in mongolian gerbils (109). the placement of the catheter within the heart can be determined by pressure recordings during catheter insertion. there is, however, always a risk of complications by e.g. damage to the aortic valves, since the catheters used for microsphere injections should not be too thin. a diameter of 1 mm is usually required in order to avoid impaction of the microspheres. we usually give the injections into the ascending aorta. we advance the catheter down to the plane of the aortic valves, which can be felt, and then withdraw it 1–2 mm. at this site there is no interference with aortic flow or cardiac output. furthermore, the precision of blood flow determinations in the pancreas is similar as when applying intraventricular microsphere injections (110). as a rule of thumb intra-aortic microsphere injections provide adequate precision for blood flow determinations in organs below the diaphragm, whereas for organs above the diaphragm administration into the heart is necessary. another prerequisite for adequate mixing is that the microspheres do not adhere to one another and form larger aggregates in the microcirculation corresponding to large chains of microspheres. the latter was observed in some initial studies where microsphere behavior in the hamster cheek pouch microcirculation was studied with intravital microscopy (111). formation of such aggregates or chains is prevented today by addition of tween to the microsphere solutions. in all experiments applying microspheres it is desirable to investigate the microsphere content in paired organs, such as the kidneys or adrenal glands. since we count the microspheres in a microscope (see below) we prefer the latter for logistic reasons, i.e. there is a smaller number of microspheres to count. the number of microspheres found is usually in the order of >500 per gland, and this should not differ by more than 10% between the glands if adequate mixing has occurred. (ii) complete extraction during first passing. this is of utmost importance, since recirculation of the microspheres will provide erroneous results, with overestimations of blood flow in well-perfused organs. to avoid this, the microspheres should have a size which is larger than the smallest arterioles and capillaries of the organ. initial studies of microspheres in general led to the conclusion that 15 lm microspheres should be chosen, since their general systemic shunting is small. it has also been shown that the distribution of microspheres of this size correlates well with the distribution of labeled red blood cells (112). thus, to minimize shunting of microspheres through capillaries of an organ, a microsphere size as large as possible should preferentially be used. however, this imposes some problems. firstly, it is advantageous to have as many microspheres as possible within the sample to increase the accuracy of the flow measurement (113). furthermore, smaller microspheres end up in smaller arterioles/metarterioles and thereby increase the resolution of the flow measurement (83). many studies on microsphere shunting are from the early days of microsphere usage. these batches were very variable with regard to size; for instance 15 lm microspheres were actually in the range of 15 ± 5 lm (mean ± standard deviation), and even larger size heterogeneity occurred. the result of this was that there was a predominance of larger microspheres in the peripheral tissues, whilst the smaller sizes recirculated. this would of course produce errors especially with regard to intraorgan flow distribution (104). at present microsphere batches are much more homogeneous, and those with a diameter of 10 lm have a standard deviation of approximately 0.2 lm. a simple way of assessing the total degree of shunting of microspheres in the whole body is to measure the amount of microspheres in the lungs. the normal bronchial circulation is <1% of cardiac output, so microspheres exceeding that amount in the lungs represent microspheres shunted in the periphery. with the use of 10 lm microspheres in rats, approximately 2% of the injected microspheres are found at this location (110). in the whole vascularly perfused pancreas approximately 3%–4% of the microspheres enter the portal vein (114). granted, this is in the whole pancreas, and we do not know for sure whether there are any direct shunts through the islets. there are anecdotic reports that this may occur (100). however, no reports from in vivo microscopy of islets have described any shunting of microspheres through or around the islets. in this context we would like to stress that, as outlined in the next section, the microspheres are supposed to mimic red cells, i.e. produce a measure of nutritive blood flow. if there is shunting allowing red cells to bypass the islets, it still means that the microspheres measure the nutritive blood flow. (iii) flow properties similar to those of red blood cells. an issue of importance in this context is so-called ‘skimming’ of microspheres. this was originally described in kidneys, where microspheres traveled centrally in interlobular arteries, and thereby erroneously preferentially accumulated in the outer regions of the cortex (115,116). this is also referred to as steric restriction affecting the distribution of the microspheres. if considering the vascular anatomy in the pancreas, this seems unlikely to occur in this organ, and skimming is not of importance for most organs in the body. the microsphere size we prefer to use, i.e. 10 lm, is similar to the diameter of red cells. the likelihood of skimming increases with size (115), as well as the simple fact that microspheres become too large to enter some arterioles if they are large enough. islet arterioles are usually 30 lm (30), and we have indeed noted a decreased islet blood flow if microsphere size is increased (83). the question is in which arteriolar size microspheres of different diameter become entrapped. initial studies with in vivo microscopy of hamster cheek pouches and bat wings demonstrated that the arterioles with impacted microspheres were surprisingly large, namely in the order of 20–60 lm (111,117,118). it was also noted that some 10 lm microspheres could slip through the upsala journal of medical sciences 87 capillaries (118). when examining rodent pancreases injected with ink, it seems as if 10 lm microspheres will be found in islet arterioles of a size only marginally larger than the microspheres themselves. when microspheres of different sizes were used to determine arteriolar size, there were some uncertainties in the precision of these estimates (119). for reasons provided below, we believe that designed islet microspheres are representative of islet blood perfusion per se, and do not represent microspheres that have erroneously entered islet capillaries. (iv) on circulatory artifacts induced by microspheres. major factors to take into account in this context are the number and size of the injected microspheres. the microspheres that become entrapped in the regional vascular beds act as emboli. this means that, depending on their number and location, they may induce local ischemia and disturb both the microcirculation as well as the general circulation. the number of microspheres that can be injected without causing any undue effects on circulation has been the subject of numerous studies in the past (101,105,113). however, since the size uniformity of microsphere batches has improved markedly in recent years, with absence of erroneously large microspheres, many previously encountered problems have diminished. in early studies of the microsphere technique a total number varying between 60,000 and 320,000 15 lm and 10 lm microspheres, respectively, was reported not to induce any hemodynamic disturbances in rats (120,121). occasionally, but not always, more than one million 10 lm microspheres could be given without adverse effects (121). we are consistently injecting 300,000 microspheres with a diameter of 10 lm in 300-g rats and 100,000 in mice without ever observing any hemodynamic instability. it is, however, essential to monitor basal circulatory parameters and blood gases to detect circulatory alterations. we have evaluated the pancreas and islets microscopically for signs of necrosis caused by embolizing microspheres without noticing any such signs. however, if the number of injected microspheres exceeded one million there was some exocrine ischemic damage, whilst the islets remained unaffected (unpublished observations). it is also relevant to discuss the number of microspheres needed to attain statistical precision in microsphere measurements of blood flow, especially in smaller organs such as islets or organs with low blood perfusion. in a seminal paper in this context it was found that the number of microspheres within organs followed a poisson distribution (113). by applying statistical calculations they determined that in order to obtain a 10% precision in their measurements, all samples, i.e. arterial reference sample and organ samples, should contain at least 384 microspheres (113). this topic has been an issue of much debate (149,150), and the consensus today is that the experimental error can be kept as low as 10%–15% also with a substantially lower number of microspheres in the samples. it was shown empirically in rats that this was the case when samples contained 200 microspheres (122). the application of the technique to ocular structures and the cochlea led to considerably lower numbers of microspheres in the tissue samples, but it was shown empirically and theoretically that the errors in the determinations still remained low (123,124) provided that the flow values exceeded 10 ll/ min (125). in these instances as few microspheres as around 50 were sufficient. when microsphere measurements of islet blood flow were initially performed, these issues were discussed in detail (71,83,87,126). when applying the microsphere technique in rats the reference sample contains >2,000 microspheres, whereas the whole pancreas contains 2,000 microspheres and the islets 200–400 depending on the experimental manipulations. note that the microsphere content in all islets is summarized and the islets are considered to be one organ. the flow value to the whole islet organ is in the order of 60–100 ll/min, i.e. well within the limits to achieve results with statistical significance. (v) microspheres should be retained in the tissue. after injection the microspheres themselves are supposed to remain in the tissue, and not move after their initial impaction. nevertheless, it might happen that microspheres leave the original organ of impact and move into the venous circulation, i.e. a delayed shunting. these microspheres usually end up in the lungs. by such means there is an underestimation of the true organ blood flow. in most cases when one blood flow measurement with microspheres is carried out, the organ is removed quickly (within minutes) after the injection, meaning that there is little time for any further movement to occur. however, if repeated measurements are performed there is an increased risk for this to occur. in the case that microspheres have different labels to enable separation of different batches, this at least makes it possible to compare this over time. thus, the differently labeled microspheres within the lungs provide a measure of the degree of shunting of different microspheres (90). we have performed studies in rodents where we have injected three differently labeled microspheres (different colors) (90). we found no differences in basal blood flow and interpreted this to reflect that there is no real delayed shunting within the pancreas of rodents of 10 lm microspheres. these experiments also lend support to the notion that no circulatory artifacts are induced by 10 lm microspheres in the used doses, even if administration is repeated. another issue which may lead to delayed shunting is if the microspheres induce local ischemia in the tissue and thereby damage the vasculature so they can dislodge from their site of impaction. this has been described mainly for larger microspheres. in the myocardium up to 20% of the microspheres can escape, also through lymph (127,128). however, no signs of ischemia have been detected in the pancreas after microsphere injections in the doses we use in rodents. another, and potentially serious, source of error is the movement of microspheres within the organ, leading to redistribution with time between different compartments. such a redistribution occurs in the intestine, where microspheres initially trapped in submucous capillary plexa can move to more distal mucosal capillaries (129,130). this is caused by diameter changes in the blood vessels anteor post-mortem. in both these referred studies perfusion pressure was varied, e.g. by administration of isoproterenol. obviously, it is difficult to use microspheres to quantitate the blood flow if capillary systems are connected in series with one another. in the liver, microspheres only allow for blood flow measurements in the 88 l. jansson et al. hepatic artery and not that through the portal vein (97). similar difficulties are encountered also when measuring renal medullary blood flow, where the microspheres end up in glomeruli since they precede the medullar vessels (131). islet capillaries are in parallel to those of the exocrine pancreas so this is not a problem for islet blood flow measurements. however, the flow to the exocrine pancreas is underestimated by microspheres, since some islets are part of an insulo-acinar portal system (2), which means that pancreatic acini receiving their arterial blood from this system cannot be measured with microspheres. the exact contribution of the portal system to exocrine flow is unknown. extraction of microspheres within the tissue in proportion to blood flow assumes that the arterioles and capillaries are homogeneous with regard to diameter and do not contain any major thoroughfare channels with much larger diameter than the microspheres. if there are marked variations it may lead to preferential shunting in parts of the tissue, and thereby underestimations of flow. this is the case for many malignant tumors, which contain large and immature blood vessels, which often shunt microspheres (132,133). islets have a homogeneous capillary diameter, but it is usually larger than that of exocrine capillaries (23), suggesting that islet microspheres could move from the islets into acinar capillaries or veins. alternatively, microspheres in an afferent arteriole could potentially move from the arteriole, which is at least partially within exocrine tissue and into an islet. we have performed experiments where we have artificially altered the blood pressure, both in vivo within the autoregulatory capacity of islets (i.e. 70–140 mmhg) (38) and in vitro with different perfusion pressures (114), without observing any signs of this. moreover, we have measured blood flow in islet adenomas in men1knockout mice, but due to the considerations dealt with above we chose to use hydrogen gas clearance instead (29). the possibility of delayed shunting of microspheres through an organ, in particular those with blood vessels with varying diameters, presents certain difficulties in the use of microspheres to assess organ blood flow. thus, newly formed blood vessels in islet grafts are initially immature with wide diameter variations (26), leading to possible shunting or movements of microspheres. the use of other techniques such as laser-doppler flowmetry (15) or hydrogen gas clearance (134) is therefore highly warranted. (vi) measuring accuracy. this is mainly an issue when tracers from the microspheres are extracted from the tissue of interest. microspheres can be purchased labeled with a radioactive isotope, a fluorescent substance, or different colors (101). radioactivity can be measured with conventional techniques, and as long as a whole organ is of interest there are usually no problems, provided that the isotope is stably incorporated into the microspheres. fluorescence or dyes can be extracted from the organs of interest according to the manufacturer’s instructions, usually involving basic liquids, and then quantified spectrophotometrically. with regard to pancreatic islets there is an additional problem, which is that they constitute only 1%–2% of the pancreas. to quantify the number of microspheres within islets we mainly rely on direct microscopic counting of the microspheres. this can be done in histologic sections, but this is extremely cumbersome. it has, though, been tried on islet grafts (135). since the microsphere diameter is fairly large, there is always a risk that the microtome damages them and thereby distorts the tissue architecture. when applying microspheres to islet blood flow measurements, different ways to clear the exocrine tissue (and thereby visualize both islets and microspheres in thick sections) have been applied. we have applied a freeze-thawing technique, which allows us to quantify microspheres and their localization with a high degree of certainty by manual counting (136,137). the islets can usually be identified directly as whitish aggregates of cells, and if stains such as neutral red or dithizone are applied, their identity can be confirmed (71,137). alternatively, the pancreas can be cleared with e.g. methyl salicylate or other substances (71,87,138). it is important to ascertain that all microspheres have been visualized and that intra-islet microspheres are identified with certainty. the correlation between quantifications in cleared tissues and conventional histological sections is excellent (110). an alternative approach would be to isolate the islets with collagenase and then count the number of microspheres being present in the islets, as well as in the remains of the digest. this has been tried, with radioactive microspheres, but the results have been published only in abstract form (107). the major problem with this approach is that only a limited number of islets can be isolated with this technique, usually in the order of 50%–60% of the larger islets, and none of the smaller ones. this means that only a sample of the islets can be used for the determinations, and since there is a heterogeneity in islet blood flow (vide supra) it is not known if this sample is representative. fluorescent microspheres have been used, to isolate microsphere-containing islets, but in this context the exact degree of islet recovery is unknown (91,92). (vii) use of microspheres for other purposes than blood flow measurements. microspheres can be impregnated with e.g. cytostatic drugs or radioactivity and then injected into organs where they become entrapped and then produce high local concentrations of drugs or radioactivity. this approach has especially been used to treat hepatic tumors since they receive almost all their blood from the hepatic artery. if tributaries to this artery are embolized with large microspheres— often sizes of up to 50 lm have been used—the normal liver parenchyma still receives sufficient oxygen and nutrients from the portal vein to survive. such microspheres can be made of starch or albumin, or, in the case of radioactivity, yttriumlabeled polystyrene (139). we have tentatively tried this approach on islets implanted into the liver of mice. such derive their blood from the hepatic artery (140), and we envisaged affecting islet graft cell replication. however, this did not meet with any success (unpublished observation). since islet blood flow in the endogenous pancreas is much higher than that to the exocrine pancreas, such an approach would also be feasible for endogenous islets. b. in vivo microscopy and pancreatic islet blood flow by direct microscopic observations of islets, the earliest published in 1882 by k€uhne and lea, it has been possible not upsala journal of medical sciences 89 only to map the vascular anatomy of the islets as outlined above, but also to make direct observations on microcirculation in islet capillaries (15,21,37,100). confocal microscopy is pivotal to the recent advances in this field, which allows for visualization of the vascular architecture in thick sections and whole islets. furthermore, improved possibilities to stain islet endothelial cells with different fluorescent antibodies and lectins have contributed to the increased use of these techniques (141). there are also transgenic mice whose islet endothelial cells are spontaneously fluorescent (46). one disadvantage with these techniques has been that they are semi-quantitative. possible ways to measure flow include laser-doppler velocimetry, which uses the frequency shift produced by the doppler effect when laser light is reflected from moving blood cells (142). the direct calibration of these values to blood flow is usually difficult to obtain, whereas relative changes in flow can be easily monitored (143). another source of error when applying this technique is that the penetration depth of the laser light into the tissue needs to be evaluated, since blood cell movements in underlying tissues may otherwise disturb the measurements. so far this technique has been applied mainly to transplanted pancreatic islets (15,100,144), since most of the laser probes are too large to place over individual islets without undue disturbances from surrounding tissues. laser speckle is a random interference effect that gives a grainy appearance to objects illuminated by laser light. if the object consists of individual moving scatterers, such as blood cells, the speckle pattern fluctuates. these fluctuations provide information about the velocity distribution of the scatterers, i.e. blood cells (145). with the speckle technique it is also possible to devise a full-field technique that gives an instantaneous map of velocities in real time. this technique has recently been applied to transplanted islets (146), but its full potential has not yet been developed. another way to assess microvascular flow is the dual-slit cross-correlation technique (147), measuring the time it takes for a red blood cell to traverse from a pre-set point to another, which has been applied to islet grafts (10,100). the techniques referred to above are all excellent for evaluations of graft blood flow or in some cases determinations of microcirculation in single islets. the great disadvantage is that they do not provide blood flow measurements per gram of organ weight for the whole islet organ. because there are heterogeneities in islet blood flow, single islet flow determination becomes sensitive to such random variations. a more recent application where the use of in vivo microscopy has been taken one step further is the migration of macrophages and polymorphic neutrophilic granulocytes (pmn) into islets. this has been applied both to transplanted islets and endogenous islets by phillipson and co-workers, and they have been able convincingly to demonstrate that maintenance of normal islet vasculature and presumably b-cell mass and development depend on this (148–150). in view of the importance of inflammatory reactions in both t1d and t2d the true potential of this technique in this context is really staggering. further details on the use of in vivo microscopy can be obtained elsewhere (21,46,100,148,151). c. perfusion of isolated, single islets this technique is based on microdissection of single islets, a technique originally developed by claes hellerstr€om (152), with their arteriolar supply intact. the islet can then be stabilized with holding pipettes, while the arteriole is cannulated with a custom-made glass pipette (30) as previously described for renal glomeruli (153). islets are placed in a chamber on a stage of an inverted microscope where movement and adjustment of concentric holding and perfusion pipettes can be made (figure 4a). the system is then perfused with buffer through the arteriole, and perifused around the islets, at a perfusion pressure of approximately 40 mmhg. the experimental set-up allows clear visualization of the arteriole, with its vascular smooth muscle, and measurements of contraction or dilation of the blood vessel (figure 4b). this enables direct studies of the vascular reactivity of the arterioles, without any confounding factors from other tissues. however, although blood flow regulation of individual islets can be investigated in a unique manner, it is not possible to perform any measurements on islet blood flow. d. flow deposition techniques for studies of islet blood flow these techniques are based on administration of substances which are deposited in a tissue or volume of tissue in proportion to its blood flow. this necessitates the choice of appropriate substances, as well as the possibilities to detect these holding pipette islet pipette arteriole endothelial lining vsm (a) (b) figure 4. a: pancreatic islet being perfused after cannulation with a glass pipette. the islet has a diameter of 250 lm. b: a close-up of the arteriole showing two rows of vascular smooth muscle. the diameter of the arteriole is 34 lm. 90 l. jansson et al. substances in the tissues of interest. a major difficulty is often the detection step, especially when small structures such as endogenous islets are of interest. however, the detection is non-invasive, which would be a major advantage. another possible source of error is the degree of extraction of the tracer. if that is low, it means that the fractional extraction is higher in organs with low flow. this was the case for the initially used tracers such as rubidium-86 and potassium (131,154). different substances labeled with technetium-99 (155) or xenon-133 (156) have been used as well. at least in their present form the resolution of the detectors applied with these techniques is not sufficient to resolve islet blood flow. one more possibility, which so far has not been used for determinations of islet blood flow, is determinations of 14c-antipyrine or 125i-antipyrine in tissues. these substances accumulate in cells in relation to their delivery through blood and can then be determined autoradiographically. the resolution is excellent, but only relative values of blood flow are provided. furthermore, the technique is very time-consuming and labor-intense (116,157). we recently adapted the hydrogen gas clearance technique to measure both endogenous and transplanted islet blood flow (134). this gives the possibility accurately and repeatedly to measure the blood flow to the same islet. however, in order to perform the measurements thin platinum-electrodes must be inserted into the islets. these electrodes detect the hydrogen concentration in the tissue, and we can then measure its outflow after loading the animal with inhaled hydrogen. the outflow rate has been shown to be proportional to the blood flow. the requirements of electrode insertion limit the number of islets available for the measurements to those that can be directly observed. to increase this number we used duct-ligated rats. ligation of the pancreatic duct leads to exocrine atrophy, but does not interfere with islet blood flow (158). these studies verified the actual blood flow values obtained with other techniques in endogenous and transplanted islets, and confirmed the heterogeneity of islet blood flow (134). e. radiological techniques to measure islet blood flow recent rapid advancements in mri and pet techniques make it likely that it will soon be possible to measure blood flow in endogenous islets in humans. indeed, a recent study from turku has demonstrated that some conclusions on islet blood flow may be inferred from studies on pancreatic perfusion (159,160). most studies are variants of flow deposit techniques, where different tracers have been applied, and conclusions on islet blood perfusion are indirect. there has been intense activity to identify optimal markers, and at present there are several available, some based on serotonin (161,162). another potential application is the use of 18f-fluoro-2-deoxy-d-glucose as a marker of inflammation. nuutila and co-workers have shown that this substance is taken up in islets in nod mice (163) and also that this substance can be used to detect inflammations in e.g. endocarditis (164). this substance would then serve as an excellent marker for insulitis, and such preliminary studies have been presented. in a recent study on lada patients scintigraphy utilizing interleukin-2 radiolabeled with (99m)tc ((99m)tc-il-2) was applied to detect insulitis (165), a technique previously used to image chronic inflammatory-mediated disorders. 4. concluding remarks despite technical difficulties it is possible to determine islet blood flow in experimental animals. however, at present there is no method which allows for determinations of islet blood flow in humans. the knowledge gained so far from blood flow measurements in experimental animals has shown that the islets possess a blood flow regulation which is independent of that for the whole pancreas, and uniquely adapted to the metabolic needs of the endocrine cells. the pancreatic islet blood flow is 5–10 times higher than that of the exocrine pancreas, and can be selectively enhanced whenever the need for insulin secretion is increased. there is also compelling evidence that islet blood perfusion is disturbed during conditions with impaired glucose tolerance and overt type 2 diabetes. at present much work is put into efforts to 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[epub ahead of print]. 165. signore a, capriotti g, chianelli m, bonanno e, galli f, catalano c, et al. detection of insulitis by pancreatic scintigraphy with 99mtclabeled il-2 and mri in patients with lada (action lada 10). diabetes care. 2015;38:652–8. 166. gylfe e, tengholm a. neurotransmitter control of islet hormone pulsatility. diabetes obes metab. 2014;16(suppl 1):102–10. 167. pettersson us, henriksnas j, jansson l. reversal of high pancreatic islet and white adipose tissue blood flow in type 2 diabetic gk rats by administration of the beta3-adrenoceptor inhibitor sr59230a. am j physiol endocrinol metab. 2009;297:e490–4. 168. bratanova-tochkova tk, cheng h, daniel s, gunawardana s, liu yj, mulvaney-musa j, et al. triggering and augmentation mechanisms, granule pools, and biphasic insulin secretion. diabetes. 2002;51(suppl 1):s83–s90. upsala journal of medical sciences 95 pancreatic islet blood flow and its measurement pancreatic islet morphology cellular composition morphology of pancreatic islet vasculature functional implications of islet morphology on blood flow regulation arterioles capillaries veins islet cellular composition affecting vascular morphology pancreatic blood flow and heterogeneity between islets techniques used to study islet blood flow microsphere measurements of islet blood flow in vivo microscopy and pancreatic islet blood flow perfusion of isolated, single islets flow deposition techniques for studies of islet blood flow radiological techniques to measure islet blood flow concluding remarks disclosure statement funding information references upsala j med sci 105: 2, 151-160,2000 diet, nutrition and diabetes mellitus bengt vessby, brita karlstrom, margareta ohrvall, anette jmi, agneta anderson and samar basu unit for clinical nutrition research, department of public health and caring sciences, uppsala uni versity and the metabolic unit, uppsala university hospital, uppsala, sweden “does an excess of fat in the diet lead to atherosclerosis? i believe the chief cause of premature development of arteriosclerosis in diabetes, save for advancing age, is an excess of fat; an excess of fat in the body (obesity), an excess of fat in the diet, and an excess of fat in the blood. with an excess of fat diabetes begins and from an excess of fat diabetics die -formerly of coma, recently of arteriosclerosis. ” elliot p . j o s h 1927 abstract nutritional management of diabetes mellitus, and the importance of diet in the development of insulin resistance, have for many years been important areas of research and education at the unit for clinical nutrition research at the department of public health and caring sciences (formerly department of geriatrics) at upp sala university. the research has more recently focussed on effects of dietary fat quality in the development of insulin resistance and in treatment of diabetes, on interaction between dietary fat and physical activity in relation to insulin sensitivity and on the importance of carbohydrate rich foods with low glycaemic index in the diabetic diet. much work has also been directed towards development of educa tional material about nutrition recommendations and dietary treatment in diabetes mellitus. the ultimate goals for all our efforts are to visualize, and promote, the pos sibilities and fundamental importance of lifestyle changes. this includes an im proved diet and increased physical activity, in the prevention and treatment of diabetes mellitus. introduction type 2 diabetes mellitus develops as a consequence of impaired insulin sensitivity, in combination with inadequate glucose induced insulin secretion, in predisposed individuals. the most important risk factors for development of diabetes mellitus, except for genetic predisposition, are excessive energy intake and physical in activity. obesity, and especially abdominal obesity, is associated with an increased risk of developing diabetes which is apparent at moderate degrees of overweight 151 table 1. metabolic disorders increasing the risk for secondary complications in diabetes mellitus blood lipid disorders hypertension decreased fibrinolysis h ypergly caemia overweight hyperinsulinaemia (abdominal obesity) impaired endothelial mediated vasodilation increased clotting tendency and which accelerates drastically with increasing adiposity. the increasing prevalence of obesity all over the world today threatens to become of epidemic pro portion and consequently cause a rapid increase of the incidence of diabetes mel litus with secondary complications such as atherosclerotic cardiovascular diseases (16). it is estimated that the frequency of diabetes mellitus world wide will increase from about 135 million in 1995 to 300 million in 2025. the rapid increase also indi cates that the development is amenable to change, and can be reversed in response to changes in dietary habits and increased physical activity. dietary treatment is the basis for all treatment of diabetes mellitus. nutritional management aims to help optimize glycaemic control and reduce risk factors for cardiovascular disease and other secondary complications. the major cause of death in diabetes is macroangiopathy coronary heart disease, stroke and peripheral atherosclerotic vascular disease with several-fold increased risk compared with the non-diabetic population. this high risk can not be explained simply in terms of the dominating conventional risk factors (high serum cholesterol, hypertension and smoking). table 1 shows some of the metabolic disorders which are shown to, or assumed to, be related to the high risk for atherosclerotic disease in diabetes mel litus. the metabolic disorders in diabetes mellitus, predisposing to development of atherosclerotic cardiovascular disease, are directly or indirectly related to insulin resistance and abdominal obesity. we have access to potent and well accepted drugs for treatment of hyperglycaemia, hypertension and hyperlipidaemia. development of new drugs for treatment of obesity is a high priority for many pharmaceutical companies. however, there are as yet no efficient drugs for weight reduction avail able. drug treatment of hypertension reduces the blood pressure but has no effect on obesity or other aspects of the risk profile. lipid lowering drugs will reduce the lipid levels but do not improve the insulin sensitivity. many patients with diabetes today require polypharmacy with concomitant inconvenience, risk for side effects and economical consequences for the individual and the society. dietary changes and/or an increased physical activity, on the other hand, have the potential to im prove all aspects of the metabolic syndrome, including abdominal obesity and insu lin resistance, and hence reduce the requirement for drug treatment. dietary changes and increased physical activity are thus logical and broad meas ures aimed at prevention and treatment of diabetes mellitus and its complications. 152 however, funding for research in these areas has been scarce. as the incidence of diabetes is increasing, it follows that there will be increasing costs for drug treat ment of diabetes and its complications. hopefully the direction of funding will shift with more focus on research in nutrition and physical activity in the prevention and treatment of diabetes. nutrition, insulin resistance and diabetes research profiles and educational activities nutritional management of diabetes mellitus, and the importance of diet in the de velopment of insulin resistance, have for many years been important areas of re search and education at the unit for clinical nutrition research at the department of public health and caring sciences (formerly department of geriatrics) at uppsala university. from an interest in the effects of dietary fibre in the diabetic diet (1 1,12), the research has recently been more focussed on effects of dietary fat quality in the development of insulin resistance and in treatment of diabetes, on interaction be tween dietary fat and physical activity in relation to insulin sensitivity and on the importance of carbohydrate rich foods with low glycaemic index (see below) in the diabetic diet. much work has also been directed, within the frames of the nutritio nal council of the swedish diabetes association, towards development of educatio nal material about nutrition recommendations and dietary treatment in diabetes mel litus. similar goals have characterised much of our work within the diabetes and nutrition study group of the european assciation for the study of diabetes. dietary fat, insulin sensitivity and diabetes mellitus there are indications from cross-sectional and dietary intervention studies in humans that a high intake of fat may contribute to the development of obesity and diabetes mellitus. there are also studies suggesting that a high intake of fat is asso ciated with impaired insulin sensitivity and an increased risk of developing dia betes, also independent of obesity. this risk may be modulated by the type of fatty acids in the diet . several studies indicate that a high-fat diet may be especially deleterious in physically inactive, sedentary individuals. obese subjects who are physically active do not experience the same risk (for recent reviews see 19,22 ). experimental animals become insulin resistant when fed high fat diets, but this impairment of the insulin sensitivity can be reversed by exchanging part of the fatty acids for long chain polyunsaturated n-3 fatty acids of the kind found in fat fish and fish oil (1 8). the insulin resistance in these animals is associated with a higher pro portion of saturated, and less polyunsaturated, fatty acids in the phospholipids in the skeletal muscle cell membranes and an increased concentration of intracellular tri glycerides. we have shown that healthy 50-year-old men in uppsala, who later developed type 2 diabetes during a 19-year follow-up period, displayed a fatty acid pattern in the serum lipid esters (25) suggesting that they may have eaten a diet with more saturated and less polyunsaturated fatty acids than men of the same age who re mained healthy. a similar picture was observed among 70-year-old men (28) when 153 the fatty acid composition of serum cholesterol esters was related to insulin sensi tivity, as measured by the hyperinsulinaemic, euglycaemic clamp technique. insulin sensitivity was associated with a low proportion of saturated fatty acids (low palmi tic acid, 16:o) and a high content of the main polyunsaturated fatty acid linoleic acid (18:2 n-6) in serum. there were changed proportions of metabolites of linoleic acid suggesting an increased activity of the enzyme d5 desaturase. thus insulin re sistance, and related disorders, are characterizerd by specific changes of the propor tions of the fatty acid pattern of the serum lipids, indicating possible changes of the activities of the enzymes responsible for elongation and desaturation of the fatty acids in the body (23). these enzymes are today recognized to be at least partly regulated by dietary fatty acids (5). the peripheral insulin sensitivity is mainly determined by the degree of insulin stimulated glucose uptake in skeletal muscles. borkman and coworkers (4) were the first to demonstrate an association between the fatty acid composition of the phos pholipids in the skeletal muscle and insulin sensitivity also in humans. in a study in uppsala (28) it was subsequently shown that the proportion of palmitic acid in the skeletal muscle phospholipids of 70-year-old men was strongly and independently related to insulin sensitivity. the fatty acid composition of the skeletal muscle is influenced by the fatty acid composition of the diet, as earlier demonstrated in expe rimental studies in animals. we could recently, in a human study, demonstrate high levels of saturated fatty acids in the muscle of people who had been on a strictly controlled, butter rich diet for three months (b. vessby et al., unpublished observa tions). dietary supplementation with fish oil increased the proportion of n-3 fatty acids in the muscle significantly. the extent to which the variations in the fatty acid composition in the muscle are due to environmental effects, e.g.diet, or secondary to genetic variations in the activities of the enzymes regulating the metabolism of the fatty acids in the body is currently unknown. in addition, it has been suggested that a reduction of the d5 desaturase may be an effect of fetal undernutrition (15) with possible consequences for the fatty acid composition and insulin sensitivity in adult life. if the dietary fatty acid composition is a significant determinant of insulin sensi tivity, as suggested by experimental studies in animals and observational investiga tions in humans, it should be possible to influence insulin sensitivity by changing the fatty acid composition of the diet in intervention studies also in humans. studies in healthy subjects have, however, hitherto uniformly shown negative results, in apparent contrast to the animal data (for a review see 23). in diabetic subjects most studies have focussed on the effects of supplementation with fish oil rich in n-3 fatty acids. no positive effects on insulin action were found. by contrast, early studies by us and others (20,21) showed an occasional deterioration of the blood glucose concentrations after n-3 rich diets, both after dietary supplementation with fish oil (3,26) and after diets rich in fatty fish (27). this may possibly be due to a reduced pancreatic response to glucose, as the peripheral glucose disposal has remained unchanged. the impairment of blood glucose control sometimes seen in type 2 diabetes after addition of n-3 fatty acids to the diet may be related to the 154 metabolic status of the patients and is probably of less importance than the putative beneficial effects of these fatty acids on lipoprotein metabolism, blood coagulation, increased blood pressure and vascular endothelial relaxation. the methodology for controlled dietary studies is complex, the variability be tween individuals with regard to dietary habits is large, and the costs for studies of this kind are high. in a recent multi-centre study (kuopio, aarhus, naples, wol longong and uppsala), known as the kanwu study, the aim was to perform a con trolled randomised trial of adequate sample size and duration to evaluate the effects of a change of dietary fat quality on insulin sensitivity and insulin secretion in healthy humans. the preliminary results indicate for the first time that a change of dietary fatty acids from more saturated to more monounsaturated fatty acids is as sociated with improved insulin sensitivity in humans (24). dietary fat, physical activity and insulin sensitivity the fatty acid compsition of the skeletal muscle is influenced by diet, but also by the degree of physical activity (2) and of the muscle fibre composition, factors which are related to peripheral insulin sensitivity. we have recently shown that the fatty acid composition in the muscle may be modulated by increased physical activity, also with unchanged dietary fat quality ( l ) , indicating that the metabolism and incorporation of fatty acids in the membrane phospholipids are influenced by the physical activity as such. this may be one mechanism, among several, which contributes to the improved insulin sensitivity in physically active subjects. the dif ference in fatty acid composition between trained and untrained subjects, when on a similar diet, with a pattern indicating an improved insulin sensitivity in the former group, is also significant when adjusted for muscle fibre composition (1). we are continuing this research with the aim to study whether an increased degree of oxidative stress, and lipid peroxidation, in connection with repeated, heavy physical strain (overtraining) may contribute to a reduction of the proportion of easily oxidizable, long chain polyunsaturated fatty acids and hence contribute to an impairment of the insulin sensitivity. carbohydrate rich foods in the diabetic diet the main source of energy in the diabetic diet, according to present nutrition recom mendations, is carbohydrate rich foods (7). provided that low glycaemic index foods (with a low blood glucose response after a meal) and fibre rich foods pre dominate, there appear to be few deleterious effects even at a carbohydrate intake corresponding to 5 5 4 0 % of the total energy intake. overweight and obese subjects may actually benefit from the satiety promoting qualities of such a high carbo hydrate diet. high fat diets, regardless of the nature of dietary fat, are energy dense and may therefore promote obesity. although the interest in carbohydrate rich foods was earlier mainly directed towards the potentially beneficial properties of a high content of dietary fibre, much research has recently concerned carbohydrate rich fods with reduced rates of di gestion, so called low glycaemic index foods. the glycaemic index (gi) was intro 155 table 2. serum (s) lipoprotein and serum (s) apolipoprotein concentrations at baseline and after 3 weeks on lowand high-glycemic index diets. baseline low gi change % s-cholesterol 5.79 2 0.78 4.23 * 0.73 (mmol/l) 27*** s-triglycerides 1.80 * 1.00 1.25 * 0.58 (mmov1) 30*** s-hdl cholesterol 1.06 f 0.26 0.88 f 0.28 -17** (mmov1) s-hdl triglycerides 0.10 k 0.05 0.09 * 0.06 1 0 (mmolfl) s-vldl cholesterol 0.56 k 0.48 0.37 * 0.21 -34 (mmov1) s-vldl triglycerides 1.28 * 0.98 0.94 f 0.47 -27 (mmom) s-ldl cholesterol 4.03 * 0.78 2.87 f 0.70 -29*** (mmov1) s-ldl triglycerides 0.42 k 0.10 0.33 * 0.09 -20*** (mmol/l) ldwhdl cholesterol 3.96 * 1. i5 3.66 * 1.57 -8 (mg/dl) (mg/dl) s-apo a-1 125.8 * 16.24 99.3 f 17.95 -21*** s-apo b 104.3 * 16.25 78.9 f 15.61 -24*** high gi change % 4.46 ko.87 23*** 1.22 * 0.57 32*** 0.87 f 0.27 -19** 0.07 k o . 0 4 -35 0.41 ko.27 -27 0.99 k0.57 23 3.13*0.90 22*** 0.34 f 0.09 -18** 3.84k 1.24 -3 102.5 * 15.56 -19*** 84.3 * 14.67 -19*** p 0.002 0.877 0.700 0.086 0.494 0.1 17 0.003 0.573 0.121 0.036 0.006 data are means f sd. p includes values for differences between the lowand high-gi diets. significant changes during the dietary periods when compared with baseline: * pco.05, **p<o.o1,***p<o.001. from reference 10. duced in the early 1980s as a way of ranking foods according to their glycemic effects (8). a low glycaemic response has been reported to facilitate blood glucose regulation and to improve lipid metabolism in diabetes but others have criticized the gi concept for not being applicable to mixed meals , and hence the long term effects of low-gi foods have been questioned. to study further the usefulness of the gi concept in dietary treatment of diabetes, we have performed a series of studies in cooperation with the department of applied nutrition and food chemistry in lund. in a first study we found that diffe rences in postprandial behaviour of starchy foods persisted, also when the foods were incorporated in mixed meals composed in accordance with the current recom 156 mendations for people with diabetes (9). the meals contained an identical nutrient composition and dietary fibre content. these data illustrate the importance of pre served structure in common foods. subsequently we evaluated the effects of varying the gi of carbohydrate rich foods on metabolic control in type 2 diabetic patients within the frame of a con trolled dietary intervention trial. this study showed that a diet characterized by low gi starchy foods lowers the glucose and insulin responses throughout the day and improves the lipid profile and capacity for fibrinolysis (10). this study also showed that a diet composed in accordance with current guidelines for people with diabetes (low in dietary fat, modified fat quality, high in dietary fibre and low glycaemic index) lowers total cholesterol and ldl cholesterol efficiently (table 2). the extent of cholesterol reduction is comparable with that obtained by treatment with potent cholesterol lowering drugs like the hmg-coa reductase inhibitors (17). education and nutrition recommendations b. v. has been the chairman of the nutrition council of the swedish diabetes as sociation since the foundation of the council in 1987 with b. k. as the secretary since several years. some of the main accomplishments by the council were the publications of a handbook of nutrition in diabetes 1988 (14) and of booklets con cerning the practical dietary management of adult diabetic subjects (“mat vid dia betes bra mat for alla” in 1987 and in revised form “bra mat for alla mat vid diabetes och hjiirtkiirlsjukdom” in 1990 and 1994) and children with diabetes (“bra mat for barn mat vid diabetes” in 1997). both of these booklets have been extens ively used in the education of patients with diabetes, of their family members and by health care personnel working with patients with diabetes and related metabolic disorders. they have also been used in education programmes at schools and by the general population with an interest in diet and health. approximately 500.000 copies of “bra mat for alla” has up to now been printed as well as 14.000 copies of ”bra mat for barn” and both are beeing revised and reprinted. one conceptionally important pedagogical principle, which was introduced in these brochures, was the so called “tallriksmodellen” (1 3). this is a model which can be used in planning healthy meals to achieve optimal proportions between diffe rent foods. “tallriksmodellen” is a simple but useful tool which has been internatio nally recognized and adopted in local versions by the diabetic associations in seve ral other countries. we have also been involved in preparing the european “recom mendations for the nutritional management of patients with diabetes” by the diabe tes and nutrition study group of the european association for the study of diabe tes. the first version was published in 1995 (6) and an updated and revised version has been published this year (7). directions f o r future research ongoing studies in our department are directed towards the importance of changes of dietary fat quality for insulin sensitivity and possible mechanisms behind these 157 relationships. one project explores the possibility that conjugated linoleic acid, an easily oxidizable fatty acid present in small amounts in milk fat, may have insulin sensitizing effects as well as a potential to reduce the proportion of fat in the body. other research focuses on the coupling between diabetes, hyperglycaemia, oxida tive stress and diabetic complications and the effects of dietary antioxidants. studies of importance for future principles for dietary treatment in diabetes in clude investigations of the effects on body weight and metabolic status of diets en riched in vegetables, legumes and fruit and further trials to evaluate the usefulness of carbohydrate rich starchy foods with low glycaemic index. the amount and type of carbohydrate rich foods in the breakfast meals seems to be of special importance. the rate of delivery of carbohydrates in the first meal influences the metabolic response not only after breakfast, but also after the following meal, the so called “second meal” effect. the ultimate goals for all our efforts concerning diet and nutrition in diabetes at the unit for clinical nutrition research are to visualize, and promote, the possibili ties and fundamental importance of lifestyle changes. this includes an improved diet and increased physical activity, in the prevention and treatment of diabetes mel litus. references 1. andersson a., sjodin a., hedman a., olsson r., vessby b. fatty acid profile of skeletal muscle phospholipids in trained and untrained young men. am j physiol, 2000; in press. 2 andersson a., sjodin a., olsson r., vessby b. effects of physical exercise on phospholipid fatty acid composition in skeletal muscle and insulin sensitivity. am j physiol. 274 (endocriol. metab. 37): e432-e438, 1998. 3. boberg m., pollare t, siegbahn a., vessby b. supplementation with n-3 fatty acids reduces triglycerides but increases pai1 in non-insulin-dependent diabetes mellitus. europ clin invest 1992;22:645-650. 4. borkman m., storlien l. h., pan d. a., jenkins a. b., chisholm d. j., campbell l. b. the relationship between insulin sensitivity and the fatty acid composition of skeletal muscle phospholipids. new engl j med 1993;328:238-244. 5. clarke s. d. polyunsaturated fatty acd regulation og gene transcription, a mechanism to improve energfy balance and insulin resistance. brit j nutr 2000, 83(suppl. 1 ) : s w soo. 6. diabetes and nutrition study group of the european association for the study of dia betes. recommendations for the nutritional management of patients with diabetes melli tus. diabetes nutr. metab. 8: 186-1 89, 1995 7. diabetes and nutrition study group (dnsg) of the european association for the study fo diabetes. recommendations for the nutritional management of patients with diabe tes. europ. j. clin. nutr. 54, suppl 1 , si-s3,2000 8. jenkins d. j. a., wolever t. m. s., taylor r. h., barker h, fielden h, baldwin j. m., bowling a. c., newman h. c., jenkins a. l., goff d. v. glycemic index of foods: a physiological basis for carbohydrate exchange. am j clin nutr 1981 ;34:362-366. 158 9. j m i a. e., karlstrom b. e., granfeldt y. e., bjorck i. m. e., vessby b. 0. h., asp n. g. l. the influence of food structure on postprandial metabolism in non-insulin-depen dent diabetes mellitus. am j clin nutr 1995;61:837-842. 10. j m i a, karlstrom b, granfeldt y, bjorck i, asp n-g, vessby b. improved glycemic control and lipid profile and a normalised fibrinolytic activity on a low glycemic index diet in non-insulin-dependent diabetes mellitus patients. diabetes care, 1999;22: 10-1 8. 11. karlstrom b., vessby b., asp n.-g., boberg m., gustafsson i.-b., lithell h., werner i. effects of an increased content of cereal fibre in the diet of type 2 (non-insulin-de pendent) diabetic patients. diabetologia 1984;26:272-277. 12. karlstrom b., vessby b., asp n.-g., boberg m., lithell h., berne c. effects of legumi nous seeds in a mixed diet in non-insulin-dependent diabetic patients. diabetic res 13. karlstrom b., vessby b., eliasson m. diet-a balanced approach. in: diabetes 1988 (r. larkins, p. zimmet, d. chisholm, eds), elsevier science publications bv, 1989:923-925. 14. kost och diabetes. en handbok. svenska diabetesforbundets kostrad. lic forlag. solna 1987. 15. ozanne s. e., martensz n. d., petry c. j., loizou c. l., hales c. n. maternal low pro tein diet in rats rogrammes fatty acid desaturase activities in the offspring. diabetologia 16. seidell j. c. obesity, insulin resistance and diabetes-a world-wide epidemic. brit j nutr 2000; 83(suppl. 1):soo-soo. 17. shepherd j., cobbe s. m., ford i., isles c. g., lorimer a. r., macfarlane p. w., mckillop j. h., packard c. j., the west of scotland coronary prevention study group: prevention of coronary heart disease with pravastatin in men with hypercholesterole mia. new engl j med 1995;333:1301-1307. 18. storlien l. h., jenkins a. b., chisholm d. j., pascoe w. s., khouri s., kraegen e. w. influence of dietary fat composition on development of insulin resistance in rats. re lation to muscle triglyceride and w-3 fatty acids in muscle phospholipid. diabetes 19. storlien l. h., baur l. a., kriketos a. d., pan d. a., cooney g. j., jenkins a. b., cal vert g. d., campbell l. b. dietary fat and insulin action. diabetologia 1996;39:621-631. 20. vessby b. n-3 fatty acids and blood glucose control in diabetes mellitus. j int med 21. vessby b. dietary supplementation with n-3 fatty acids in type 2 diabetes. in: dietary lipids and insulin action. eds: klimes, howard, storlien, sebokova. annals new york acad sciences 1993;683:244-9. 22. vessby b. nutrition, lipids and diabetes mellitus. current opinion in lipidology 23. vessby b. dietary fat and insulin action in humans. brit j nutr 2000;83, suppl.1, s 91 s 96. 24. vessby b. et al. for the kanwu study group. efects of dietary fat on insulin sensitivity and insulin secretion the kanwu study. diabetologia 1999;42(uppl i ) , a46(abstr). 25. vessby b., aro a., skarfors e., berglund l., salminen i., lithell h. the risk to develop non-insulin-dependent diabetes mellitus is related to the fatty acid composition of the serum cholesterol esters. diabetes 1994,43: 1353-57. 26. vessby b., boberg m. dietary supplementation with n-3 fatty acids may impair the glu cose homeostasis in patients with non-insulin-dependent diabetes mellitus. j int med 1987;5: 199-205. 1998;41: 1337-1 342. 1991 ;40:280-289. 1989;225 (suppi 1):207-210. 1995;6:3-7. 1990;228: 165-1 7 1. 159 27. vessby b, karlstrom b, boberg m, lithell h, berne c. polyunsaturated fatty acids may impair blood glucose control in type 2 diabetic patients. diab med 199 1 ;9: 1 2 6 1 33. 28. vessby b., tengblad s., lithell h. the insulin sensitivity is related to the fatty acid compositon of the serum lipids and of the skeletal muscle phospholipids in 70 year old men. diabetologia 1994;37: 1044-1050. address f o r reprints: bengt vessby, md, phd unit for clinical nutrition research uppsala university box 609 se-75 1 25 uppsala sweden tf-iups160032 276..282 original article infertile men’s needs and assessment of fertility care randi sylvesta, jeanette krogh f€urbringera, lone schmidtb and anja pinborga ahvidovre hospital, fertility clinic, hvidovre, denmark; buniversity of copenhagen, department of public health, copenhagen, denmark abstract introduction: male infertility is potentially a severe, low-control stressor. there is limited knowledge of the expectations, needs, and assessment of fertility care among men with severe infertility. the aim of this study was to explore experience, expectations, needs, and assessment of fertility care among danish men having severe male-factor infertility. methods: semi-structured qualitative interview study with 10 men with very low sperm quality initiating intracytoplasmic sperm injection (icsi) treatment at the fertility clinic, copenhagen university hospital, hvidovre, denmark. five of the men participated in a follow-up interview after their first icsi treatment. the data collection took place during november 2014 to may 2015. data were analysed using qualitative content analysis. results: two themes were found: ‘the maze’ and ‘desire for care’. it felt like an eternity for the men from the referral until treatment started. the men did not understand the process, and it was like being in a maze. the men saw fatherhood as something to strive for. they felt that they could not do what a man is supposed to do, and they felt pushed aside and that treatment focused on the women. the men appreciated the staff’s kindness and professionalism but desired the staff to address emotional subjects too. conclusion: the process from referral to treatment felt like a maze for these men. they needed the staff to give them the opportunity to speak of the psychosocial consequences of severe male-factor infertility. article history received 23 march 2016 revised 13 june 2016 accepted 17 june 2016 keywords family formation; infertility; men; qualitative interview; reproduction introduction male infertility is the main or contributing cause in half of the couples who seek fertility treatment (1). it is a widespread international problem as 56% of infertile couples in developed countries seek medical fertility care (2). men having reduced semen quality frequently want to know why the sperm count is low, and try to find explanations and consequences of this (3). infertility concerns both the woman and the man in a couple. the role of the man is often reduced to providing a semen sample on time. this applies even to couples where the male is infertile (4,5). until now only limited focus has been on men with severe male-factor infertility and their thoughts and experiences regarding their fertility care. a previous study on fertility patients treated at four different public fertility clinics in denmark (6) found no sex differences in the evaluation of fertility care, except that women were a bit more satisfied than men with how the staff had performed their medical examinations. both men and women in fertility care in denmark gave high ratings on medical and patientcentred care. male infertility is a potential severe low-control stressor (7), and research has shown that many men with low sperm quality would like to talk about it more widely (3). mikkelsen et al. found that 72% of 210 danish men undergoing fertility care lacked information about the psychological consequences of male infertility (8). approximately 8%–10% of danish children are born after fertility treatment, and half of these after assisted reproductive technology (art) treatment. usually ‘take-home baby’ rates are used to assess the quality of fertility care, although the importance of patient satisfaction with information provided by the clinic and the perception of the health professionals are increasingly being recognized as indicators of quality of care (9). a previous cohort study showed that fertility patients’ satisfaction with both medical care and patient-centred care was higher when the couple had achieved a pregnancy or delivery and higher with decreasing occupational social class (6). a qualitative interview study among fertility patients showed that they preferred that their treatment took place in a specialized fertility clinic with only a few staff members, a short waiting time, and in a setting where the plan of treatment was known by the doctor and the couple (7,10). moreover, it has been suggested that a supportive attitude from the health care professionals and the provision of both medical and psychosocial information should be integral aspects of medical care in fertility clinics (5,6,10,11). finally, there is also evidence that contact randi sylvest randi.sylvest@gmail.com fertility clinic, hvidovre, university hospital of copenhagen, kettegård all�e 30, section 455, dk-2650 hvidovre, denmark � 2016 the author(s). published by informa uk limited, trading as taylor & francis group. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. upsala journal of medical sciences, 2016 vol. 121, no. 4, 276–282 http://dx.doi.org/10.1080/03009734.2016.1204393 http://creativecommons.org/licenses/by/4.0/ men, unlike women, rarely express their problems and needs in the health service or are more reluctant to do so (12,13). the aim of this study was to explore experience, expectations, needs, and assessment of fertility care among men with severe male-factor infertility. the study was conducted among men initiating treatment with intracytoplasmic sperm injection (icsi) in order to optimize the treatment and care of this patient group. material and methods setting the study was conducted at the public fertility clinic at hvidovre hospital, university hospital of copenhagen, denmark. all couples initiating art treatment at our clinic are invited to a scheduled information meeting with the presence of a consultant, laboratory specialist, and a fertility nurse. the meeting lasts for two hours and concerns all aspects of their forthcoming treatment including explanation of the icsi procedure and indications for icsi treatment. all men had been referred for specialist andrological examination of their malefactor infertility at an andrological department outside hvidovre hospital prior to icsi treatment in our clinic. this takes in general two to three months and includes ultrasound examination of the testicles, blood hormone and semen analyses, and screening for chromosomal anomalies and y-chromosome deletions. these investigations take place before the infertile couples can start at our clinic. psychosocial support and professional psychosocial services by mental health workers including psychologists are not provided at danish public fertility clinics. specially trained nurses and doctors in reproductive medicine provide the fertility care. psychological counselling in relation to fertility treatment is not mandatory in denmark. the cost of a maximum of three art treatments (except medication) is covered by the national health service. study population and sampling inclusion criteria were men having severe male-factor infertility (�1 million total motile sperm count (tmc) after processing), planning for icsi treatment, and having no mutual children. participants were recruited after they had been informed that the couple needed to undergo icsi treatment. in that way they had been informed about their very low semen quality. all men were selected consecutively from a log book of treatments at the clinic and from the waiting list. this was done to avoid selection of especially good or bad treatment processes. the participants had different levels of education. family formation aspirations potentially could be influenced by different levels of education (6). short-term educational training was defined as vocational training with a year or less of theoretical content, and long-term educational training was defined as four years or more of theoretical training at a university or similar institution. a total of 15 men were contacted by letter and telephone by the researchers (j.k.f. or r.s.). the participants were informed about the aim of the study, that the study included two interviews (one before and one after their first treatment attempt), and that the participants would be anonymous. four of the invited men did not want to participate, and one of the couples achieved a natural pregnancy before starting icsi treatment. finally, 10 men agreed to participate in the study and were interviewed before their first icsi treatment, but only half of the participants wanted to participate in the second interview, which took place after their first icsi treatment. the remaining five did not want to participate because they experienced the treatment as being severely stressful, and some pointed out that they only wanted to be interviewed again when a pregnancy was achieved. the interviews took place between november 2014 and may 2015. they were performed individually to address the emotional aspect of the topic of low sperm quality. depending on the participant’s preference, the interview took place in his own home or at the hospital. in total, 10 men were interviewed at the first interview—seven at the fertility clinic, and three in their own homes. at the second interview, five men were interviewed—four at the fertility clinic, and one in his own home. data collection we developed two semi-structured interview guides. the first interview before treatment initiation focused on family formation intentions, expectations of the icsi treatment, and thoughts regarding having very low sperm quality. the focus in the interviews was the patients’ expectations of the fertility treatment. the second interview after a first icsi treatment attempt focused on family formation, wishes and suggestions for information, and how the clinical staff could improve their way of addressing and taking care of severely infertile men’s needs in fertility treatment. furthermore, very low sperm quality was addressed again. the interview items were constructed in accordance with the experience and knowledge of the researchers and on the basis of previous studies about infertile couples and infertile men (3,4,7,8,10,11). the interviews were conducted according to kvale (14) by one of two authors, r.s. or j.k.f. all interviews were performed by only one interviewer. the interviews were audio-taped and transcribed verbatim including non-verbal expressions like silence and laughter, and transcripts were re-checked against the recording by the first authors r.s. and j.k.f. transcripts were anonymized, and the men were assigned a codename. the duration of the interviews varied from 23 to 80 min with a mean time of 47 min. in general, the atmosphere during the interviews was informal and relaxed. analysis transcripts were analysed with qualitative content analysis as used by graneheim and lundman (15). the text was analysed with the concepts of meaning units, condensed meaning units, codes, and themes. the analysis was performed in four steps: (1) the interviews were read through to obtain an idea upsala journal of medical sciences 277 of the content; (2) the text was divided into meaning units, which were defined as words, sentences, or paragraphs in the text, where the content related to each other and to the aim of the study; (3) the meaning units were condensed and labelled with codes, which were abstracted and compared for similarities and differences; (4) the codes were then distributed into themes. irrelevant text was excluded from analysis. to strengthen trustworthiness, the condensed meaning units, codes, and themes were discussed and reflected on by all the authors throughout the analysis process until agreement was accomplished. examples of condensed meaning units, codes, and themes are given in table 1. quotations were chosen to represent the range of views for each theme. ethics the study followed the principles of the declaration of helsinki ii for medical research. written informed consent was obtained from all participants. interviews were anonymized, and sensitive data were kept in a separate document. according to danish legislation interview studies do not require permission from the ethical committee. results socio-demographic characteristics and reproductive history about the men are shown in table 2. six of the participants had short-term vocational training, and four had long-term vocational training. of the five who participated in the followup interview, there were three with short-term vocational training and two with long-term vocational training. we identified two themes: ‘the maze’ and ‘desire for care’. the maze when the men received information about their very low sperm quality many questions came to their mind along with uncertainty about what comes next, and they lacked information. they felt frustrated because of the fact that they only received written information. they were sent letters and e-mails, but it was one-way communication, and they wanted personal face-to-face meetings and oral information, dialogue, and an opportunity to ask questions. there was a desire for information about the whole process. the men felt frustrated, irritated, and impatient with the process, and they did not understand why the they had to have several sperm tests and other diagnostic procedures. there was a time period when i didn’t feel like i was doing anything else, other than dropping off semen samples. (person 1) it felt like an eternity for the men from the diagnosis time until treatment started. it was like a maze for them, a maze without a map, making it impossible for them to find a path to follow. the staff they met could not give them an overview of the process, of the map. the staff only knew about their own field and not the process for the male patient as a whole. the men wanted to have children now, but it felt like a long ride. before the men received the information of their very low sperm quality they found themselves at a time in life and at a certain age where it felt right to start a family. the men emphasized that it had to be soon in order to not be too old when they had children. they looked forward to being able to play with their children and being an active part of their lives. some men specified that they had found the right woman with whom they wanted to start a family. to make a family of their own would give them another kind of contentment in their lives. when the decision had been made and the desire to start a family was present, they wanted it to happen soon. . . . because one wants it so desperately, then one just wishes it would happen tomorrow. (person 8) the participants described that they had not always thought about it in this way but that it was something that table 1. example of analysis; condensation, code and theme. condensation code theme ‘it’s just one big machine . . . suddenly, there are just so many people involved in the process.’ machinery the maze ‘you stand with a question mark because you can’t get answers to your questions.’ question mark the maze ‘you are just a caregiver . . . it’s not me who is in treatment . . . you just feel like a caregiver.’ side-line desire for care ‘i feel welcome and comfortable.’ expectations of the staff desire for care table 2. socio-demographic characteristics and reproductive history of the study population. alias age vocational training (short-term or long-term) been with partner (years) time trying to conceive (years) male infertility factor/mixed male and female infertility factor pregnant after first treatment (yes/no) 1 38 short 5 2 mixed no 2 38 long 6 2 male no 3 34 long 4 2 mixed no 4 40 short 2 2 male no 5 33 short 6 1 mixed no 6 32 long 5 2 male no 7 36 short 2 2 male no 8 40 short 2 – male no 9 33 short 3 2 male yes 10 40 long 4 2 male no 278 r. sylvest et al. had occurred with time. in the waiting time before treatment started, they felt like they had been forgotten, and that they were wasting time. they felt like they were in a vacuum, a transfer period, where nothing happened, and they experienced that they did not move ahead. . . . we can’t understand at all that it takes such a long time . . . i’m quite clear that they have to go and take some tests . . . but four months? . . . that’s really an unrealistically long time. (person 9) the infertile men wanted an overall plan and an overview of the process at the time of the referral. the men expressed that they wanted detailed information about the treatment plan, the results in the process, and information on progress of the treatment. they wanted a timetable so they could control and manage their lives. the men that had attended the clinic’s information meeting about ivf/icsi treatment before the first interview were more relaxed and calm compared to the men that had not been to the meeting yet. a clear overview over the whole process; that’s what i really got out of the information meeting. (person 2) one of the men also received good information from the gynaecologist, which meant that he felt much more relaxed compared to the others. he did not feel like he was walking in a maze without a map. the men were afraid of becoming a number in a big system without personal contact and empathy, and they considered it of great importance that the staff took time in dealing with the couple. they did not want to be treated like they were on an assembly line. the men felt it was like machinery, where a lot of people suddenly got involved in the project of getting a baby. . . . it’s just one big machine . . . suddenly, there’s just so many people involved in the process. (person 10) the men described a family as something good and wonderful, something to strive for. family was a core value. the surroundings also played a part in the men’s wishes and thoughts about creating a family. they experienced how their acquaintances already had or were starting to have children. the men noticed if other men were fathers. they noticed how the fathers looked after their children and that they had to think of others than themselves. the men had friends that took their children to soccer matches, and that made the men think about their future child’s activities that they looked forward to being a part of. they saw fatherhood as a special and great job in life. they saw it as a huge responsibility, looked forward to it, and had expectations about something amazing waiting for them. becoming a father was both a dream and something unrivalled, important, and significant to experience. . . . the greatest thing. i’d fight for that; it puts a whole new perspective on life. (person 4) the private thing of starting a family became a public experience. the privacy and the romantic of getting pregnant and having a child disappeared. they had to say goodbye to the hope of getting pregnant naturally. . . . unromantic fantasy . . . that one tries for several months, or however long it takes, and then you are happy . . . i mean, suddenly, 30 people are involved in it. (person 10) desire for care before treatment initiation the men expressed a desire for kindness, understanding, professionalism, and empathy from the fertility clinic staff. they felt it was important that the staff did not look down on them but instead embraced them and held their hands. furthermore, they wanted to know how the hormones were going to affect their partners. all the men expressed satisfaction with the information meeting. here they got the information they needed; statistics and numbers were of particular importance. before the information meeting many of them sought information through other sources like google, the clinic’s homepage, the internet in general, or their partners. but after the information meeting they did not have the same need any more, because they had received the information they felt they required. when the men started treatment, they felt very well informed of the treatment and the plan, and they experienced the staff to be very personal and attentive. the men came to rely on the staff, both in relation to getting an understanding of the situation and getting an overview of the process. they did not feel that they were just a number. the staff were aware of how much it meant to the person sitting in front of them. prior to their first treatment the men expressed that they expected involvement in the treatment both as a man and as a couple. they did not wish only to be placed on the sideline. after their first treatment they did, however, consider it natural that the focus was on the women, as it was her body that was being treated and took the biggest strain. she who gets all the tests, and they’ll need to take some hormones . . . she’s the one who has the pressure . . . which i actually think is fine . . . that the staff are aware that she carries most of the burden. (person 9) the men did not consider themselves as emotionally burdened as their partners, and they wished to believe that the ongoing treatment would succeed. they expressed that they did not need psychological help at this point in the treatment. but they did think that maybe it could be relevant with a psychologist for others. instead some of the men argued that it would be nice to hear some positive stories of and from former patients. . . . one who has been throughout the whole process, someone who could say: ‘hey; it’s gonna be all right. of course it’s going to work’. (person 3) but there was still a desire for psychosocial care. there was still an inner and emotional confusion. the men wanted to appear strong and supporting for their partners, but it was also a burden for them. they were nervous about whether the treatment would work and help them. some of them did not feel that the staff were addressing other things like the more emotional aspects of infertility. it’s not because we talk a lot with the staff. they tell us what is happening, and why it is happening, and what we upsala journal of medical sciences 279 need to do; but that’s kind of it (as far as information goes). (person 4) they tried to push it away, minimize it, and focus on the concrete things and their partner. . . . it’s easier to say ‘hey—i don’t really understand what’s going on’, than it is to say: ‘hey, i need your help handling some of these feelings that i just don’t understand’ . . . it’s much more difficult to mention this to them. (person 2) both partners wanted to become parents. they were two persons, a couple, sharing a wish to have a child. the man wanted to shape his part in the treatment himself. he wanted to be active and participate in the things that his partner had to go through. the men wanted to take part in the scanning, the ovum pick-up, and when their partner was going to learn how to inject the medicine. they wanted the staff to speak to both of them and be attentive towards both of them. . . . it was meant for my partner . . . it wasn’t really that i felt excluded, but it was just a symptom that it was not me who was in treatment . . . it’d be great if the doctor could just look us both in the eyes. (person 6) the men saw it as a couple issue and not as a woman issue. they knew that they had to put down a semen test at the right time, and additionally they were passive on the sideline. at the same time they emphasized that they were called in from the waiting room with the use of both their names and the fact that there was a chair reserved for them beside their partner during ovum pick-up. the men saw themselves as being expected, important, and allowed to pose questions. the men emphasized being a part of the conversations and the scanning to support their wives, along with the fact that two people hear and remember information better than one. . . . it’s important, that we’re both involved in it . . . since we are both a part of it—it’s not just one of us. (person 4) the participants pointed out that it was a treatment that meant a lot to them and their partner. discussion the findings of the present study were relevant in a broader perspective and internationally as male infertility is a global reproductive health problem (1,2). the major finding was the feeling of being in a maze. we also found that lack of information from the time of referral can lead to impatience, a feeling of being forgotten and left in a never-ending line. our study showed that information helped the men to see a way out of the maze. oral and written information about the somatic and psychosocial aspects of infertility and treatment was in high demand, a finding that has been highlighted by others (8). the men wanted face-to-face information about their low semen quality at the time of the referral. the information and time-table helped the men to gain control over their lives (3). moreover, our results showed that most men wished to support their partner in this process. when communicating with the couple, the staff should recognize the sense of responsibility the infertile man has towards his partner and try to support his focus on hope and encouragement. the men wished for more than to be at the side-line, but the system placed them there as caregivers though most of them wanted to be involved and included in the process. the present study identified that the men needed the staff’s recognition and acceptance. this finding corroborates previous results that the less the man felt asked about his experience, the less he felt involved as an equal partner (8) and thus highlights that the system needs to see fertility care as a couple issue and not only as a woman issue. in denmark the vast majority of men are present at the birth of their children, and furthermore men have the possibility to take paternity leave and parental leave. the man should already during fertility treatment have a well-defined role as an equal partner, which leaves it natural that the fertility clinic’s staff consider both the man and the woman when they meet with the couple (8). cues to actions among the participants were that the men found themselves at a time in life when it felt right to start a family with their partner and not being too old for having children. similar results were found by eriksson et al. (17), who explored how young adults reflected on future parenthood. the men noticed if other men were fathers. the surroundings also played a part in the men’s wishes and thoughts about creating a family. the men longed for a time when they could join this community of fathers taking their children to activities, a finding supported also by tjørnhøjthomsen (4). our findings confirmed that the men desire kindness, understanding, professionalism, and empathy from the fertility clinic staff. but not all the needs of the men for psychosocial clinical care were met in the clinic. the psychological strains were often unmentioned and unrecognized. in this study the men did not know how to address the emotional aspect of treatment and male infertility. most men would approach emotional questions by addressing something concrete (13), which was also shown in our study. in working with infertile couples it was important to be aware of the fact that many men did not like the role of being a patient (13). the fact that this was not something which was explicitly addressed by male patients might be due to the tendency in men to be reluctant towards issues relating to illness in general (13). in the present study neither the staff nor the men raised the issue of psychological stress with any frequency. our study supported previous studies and thus highlighted the need for an informed psychosocial approach to the clinical care of the infertile man (6,10,11,18). essential to the improved care and counselling of the infertile man was the way the health care system perceives and meets the male patient in open dialogue. this study suggested that infertile men require a greater degree of openness and detail about their condition and its related psychosocial consequences. the data further highlight the fact that the staff were an important source of emotional support since they had frequent contact with the couple. staff with advanced competence in communication were a sound and effective resource in dealing with the men. 280 r. sylvest et al. although many of the men felt that there was a need for more communication about infertility, none expressed the need to be referred to psychosocial counselling at this point. this finding suggested that it is important both to develop ways to help men to engage in counselling and that it is necessary to meet the psychosocial and emotional needs of men. the staff should address that it was normal and acceptable to feel grief in connection with infertility. there was a need for a space where the man’s feelings and needs could be met and accepted. we recommend that more attention should be paid to the provision of comprehensive information about other aspects of reduced sperm quality, including the prior experiences and views of others (e.g. ‘we know from other men . . .’). with the diagnosis of low sperm count followed an uncertainty and fear that the treatment would not succeed. the wish to hear positive stories could be a way to tackle the emotional confusion, insecurity, uncertainty, and the fear of not succeeding that the men experienced. this supports that it was very important that the staff addressed the psychosocial aspects of the treatment and of having a low sperm count. we have had the care of men in infertile couples as one of our focus areas in our clinic. one of our goals is that the staff should be aware of men’s desires for care in order to optimize the care of the infertile men. a national leaflet is under development concerning men’s experiences of ‘‘being in a maze’’, having low semen quality, and starting treatment. as a small adjustment the name of the man is now put on the front page of the clinical file together with the name of the woman to make it easier for the staff to call also the man’s name and not only the woman’s name when the couple are in the waiting room. this is to address that the man is an important part of the couple and takes part in the treatment too. strengths and limitations in accordance with the standards for qualitative health research suggested by coreq (19) and stige et al. (20), analysis and interpretation have been continuously displayed and discussed among the authors and communicated throughout the study. r.s. is experienced in qualitative interviewing specially in family formation. j.k.f. is also experienced in qualitative interviewing and is a fertility nurse with an understanding of reproductive health care which could represent a strength but also a limitation. the men appreciated the fertility nurse expertise, and they took their opportunity to ask questions concerning fertility treatment issues. this implied that there was a need for information prior to treatment and a desire in treatment to talk about psychosocial issues. at the interview before starting icsi treatment, we accomplished data saturation with 10 interviews. the interviews were quite long, and in the follow-up interview another form of relation occurred between the participant and the interviewer. there was in general a relaxed and open-minded atmosphere in the interview situation. dependability was created by using the same interview guide. qualitative studies aim to get a deeper understanding of people’s lived experiences. striving for generalization was neither possible nor desirable. we think that our results could be transferred to other men with low semen quality in fertility treatment. a limitation of the present study was that the men in this study might not be representative of all infertile men who undergo icsi. men are different, culture is different, and the experience of reduced sperm quality is individual. furthermore, participation in this study was voluntary, which meant a risk of selection bias. in the follow-up interview only five of the men wanted to participate, and therefore we did not reach data saturation. one of the men mentioned the fact that the treatment was so stressful for both of them and it was harder than expected, so he and his partner needed to pause from the treatment and focus on their relationship. another stopped halfway through the first course of treatment because of an infection and never started again. three participants pointed out that they only wanted to be interviewed again when a pregnancy was achieved. this highlights that it is a very emotional subject. conclusion the process of going from diagnosis to treatment felt like being in a maze, which affected their personal life. the men had reached a point in their lives when they wanted to start their own family. they saw fatherhood as something to strive for, and now they needed help to conceive, and they were impatient. they wanted detailed information about the treatment plan and the opportunity to speak of the psychosocial consequences of their low semen quality. the men wanted information and face-to-face meetings with professionals. they felt like they were placed on the side-line. fertility care needs to see the treatment as a couple issue and not only as a woman issue. the staff need to focus on involving both the man and the woman in the treatment, and more focus is needed on the men and their psychosocial needs. acknowledgements we would like to thank all the men who participated in the study. disclosure statement the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. r.s. and j.k.f. contributed equally to the writing of the paper. all the authors conceived of the study. r.s. and j.k.f. conducted the interviews and carried out the analysis in collaboration with l.s. and a.p. all authors contributed to the final draft. funding information hvidovre hospital funded this study. references 1. mclachlan ri, de kretser dm. male infertility: the case for continued research. med j aust. 2001;174:116–17. upsala journal of medical sciences 281 2. boivin j, bunting l, collins ja, nygren kg. international estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care. hum reprod. 2007;22:1506–12. 3. madsen sa. men as patients: understanding and communicating with men. trends urology & men’s health. 2015:22–6. 4. tjørnhøj-thomsen t. tilblivelseshistorier. barnløshed, slægtskab og forplantningsteknologi i danmark [genesis. childlessness, kinship and reproductive technology in denmark]. (phd dissertation). copenhagen, denmark: university of copenhagen; 1999. [in danish] 5. schmidt l, holstein be, boivin j, sångren h, tjørnhoj-thomsen t, blaabjerg j, et al. patients’ attitudes to medical and psychosocial aspects of care in fertility clinics: findings from the copenhagen multi-centre psychosocial infertility (compi) research programme. hum reprod. 2003;18:628–37. 6. schmidt l, holstein be, boivin j, tjørnhoj-thomsen t, blaabjerg j, hald f, et al. high ratings of satisfaction with fertility treatment are common: findings from the copenhagen multi-centre psychosocial infertility (compi) research programme. hum reprod. 2003;18:2638–46. 7. schmidt l. psykosociale konsekvenser af infertilitet og behandling [psychosocial consequences of infertility and treatment]. (phd dissertation). copenhagen, denmark: fadl’s publisher’s; 1996. [in danish] 8. mikkelsen at, madsen sa, humaidan p. psychological aspects of male fertility treatment. j adv nurs. 2013;69:1977–86. 9. alper mm, brinsden pr, fischer r, wikland m. is your ivf programme good? hum reprod. 2002;17:8–10. 10. schmidt l. infertile couples’ assessment of infertility treatment. acta obstet gynecol scand. 1998;77:649–53. 11. hammarberg k, baker hw, fisher jr. men’s experiences of infertility and infertility treatment 5 years after diagnosis of male factor infertility: a retrospective cohort study. hum reprod. 2010;25:2815–20. 12. white a. men’s health in the 21st century. int j mens health. 2006;5:1–17. 13. madsen sa. men’s special needs and attitudes as patients. j mens health gend. 2007;4:361–62. 14. kvale s. an introduction to qualitative research interviewing. london, uk: sage publications; 1996. 15. graneheim uh, lundman b. qualitative content analysis in nursing research: concepts, procedures and measures to achieve trustworthiness. nurse educ today. 2004;24:105–12. 16. dancet eaf, van empel iwh, rober p, nelen wldm, kremer jam, d’hooghe tm. patient-centred infertility care: a qualitative study to listen to the patient’s voice. hum reprod. 2011;26:827–33. 17. eriksson c, larsson m, tyd�en t. reflections on having children in the future interviews with highly educated women and men without children. ups j med sci. 2012;117:328–35. 18. van empel i, nelen w, tepe e, van laarhoven e, verhaak c, kremer jam. weaknesses, strengths and needs in fertility treatment. hum reprod. 2010;25:142–9. 19. tong a, sainsbury p, craig j. consolidated criteria for reporting qualitative research (coreq): a 32-item checklist for interviews and focus groups. int j qual health care. 2007;19:349–57. 20. stige b, malterud k, midtgarden t. toward an agenda for evaluation of qualitative research. qual health res. 2009;19:1504–16. 282 r. sylvest et al. infertile men&rsquo;s needs and assessment of fertility care introduction material and methods setting study population and sampling data collection analysis ethics results the maze desire for care discussion strengths and limitations conclusion acknowledgements disclosure statement funding information references upsala j med sci 104: 237-246, 1999 a qualitative study on changes in local brain ph due to discrete cerebral microembolism magnus w. roos department of physiology, uppsala university, bmc, box 572, 751 23 uppsala, sweden abstract in this work autoradiography of ~~c-5,5-dimethyl-2,4-oxazolidinedione (i4c limo) was used to trace changes i n local cerebral ph in embolized awake rabbits. one hour after i.v. injection of 14c-dmo small cerebral ischemic foci were produced in rabbits by injecting plastic beads into the left heart ventricle under short-acting anaesthesia, and after another hour the animals were put to death and their brains processed for autoradiography of 14c-dmo. evidence of acidosis was i n general not found i n the microischemic regions, though there were a f e w possible exceptions. however in the hippocampus a diffuse acidosis involving a large part of the structure, could b e found in 2 of the 4 experiments. this hippocampal phenomenon probably reflected the same process as has been observed using autoradiography of 2 deoxyglucose (reflecting cellular glucose uptake) on the same ischemic model increased 2-deoxyglucose phosphorylation. because the hipocampus is invloved in the memory function and the fact that small infarcts are coupled to dementia, this phenomenon should be drawn into focus for further studies. 237 introduction small subcortical (diameter less than 1.5 cm) cerebral infarctions are common in humans (1, 13). their presence is closely coupled to dementia (2, 24), which in its advanced stages presents with symptoms like gait difficulties and pseudobulbar palsy (3,10,24). for decades, most human subcortical infarctions have been con sidered to be caused mainly by mural changes of the small penetrating cerebral arteries (5, 6, 14, 18, 20). nowadays it seems that small emboli account for a significant number of these infarctions (6, 18). however, since this kind of stroke is not life-threatening, it is difficult to correlate embolism to the individual infarcts using autopsy material. however, there seems to be a correlation be tween dementia and atrial fibrillation (11) a well-known risk factor for cerebral embolism. many aspects of cerebral microembolism have been studied (4, 7, 8, 9, 19, 21, 23, 30, 32, 33, 35 38, 40, 42, 43). however, the rate of follow-up on these stu dies is pore. moreover -the number of emboli in those studies were too large for studying discrete areas of microischemia. however, a model of irreversible cerebral microischemia, employing plastic beads as emboli, has recently been introduced (25). in that study it was found that emboli trapped in the brain stem and diencephalon affected the local tissue metabolism more often than emboli reaching the cortex. further, from 24-h experiments it was concluded that infarcts tend to appear mainly in the subcortical structures. it was suggested that this might be due to the deeper regions having a less efficient collateral vascular network. moreover, it was reported from acute experiments that in some ischemic foci, mainly located in the basal ganglia, there was a central "dip" in the 2-deo~y-d-[1~c]glucose (a tracer of cellular glucose uptake (34)) activity profile, and it was suggested that this dip may be due to central glucose depletion (27) or a depressed cellular function caused by acidosis. the aim of this work was to come closer to an understanding o f the pathophysiological events taking place in the embolized brain of rabbits by using 238 autoradiography on the tissue activity of 14c-5,5-dimethyl-2,4-oxazolidinedione (14c-dmo) to trace changes in tissue ph. the ischemic model used has been characterized in a series of papers (25 29). on an average, about 25 discrete ischemic regions were obtained per brain. materials and methods animals 4 new zealand rabbits, 3-4 kg b.w., of either sex were used. before the experiments the animals were housed in individual cages at a specialized animal department where they were allowed free access to food and water. the experimental design has been approved by the local ethics committee. the 14c-dmo procedure 1~c-5,5-dimethyl-2,4-oxazolidinedione (1%-dmo) has been used for decades to investigate p h in normal as well as ischemic brain tissue (12, 15, 31, 41). some what simplistic, the higher tissue concentration of 14c-dm0 the higher the tissue ph. in this study, 14c-dmo (amersham int.) was used to trace possible emboli-induced tissue acidosis. the rabbits were fitted with an ear venous catheter to allow injections, and were then injected i.v. with about 250 pci 1% dmo. after about 60 min the beads were administered as described below. arterial blood was collected by using a needle and a piece of tubing. about 60 min after the injection of iac-dmo, the rabbits were killed with a large dose of pentobarbital sodium i.v. the heads were then submerged a s quickly as possible in hexane mixed with blocks of carbon dioxide and proceesed for autoradio graphy as described below. no attempt was made to calculate tissue ph. 18-990757 239 embolization procedure black-dyed beads (styrene/2%/divinylbenzene/170-200 mesh, from bangs laboratories, inc.), size range 75-90 pm, were employed as emboli. about 120 mg of the delivered sphere suspension (10 mg/ml) was mixe’d with 12 g macrodexm (60 mg/ml). to allow insertion of a needle into the heart the rabbits were anaesthetized with brietalb (metohexital, 10 mg/ml), 1 ml/kg, administered i.v. during about 20 s. then, by using a syringe and a needle (0=o.8 mm) connected to a piece of tubing about 0.67 ml/kg of the final suspension of beads was administered transthoracically into the left heart ventricle. within 5 min after the brietalm injection, the animals were conscious sitting upright and moving their heads and extremities. a u t o ra d i o g r a p h y the autoradiographic technique was in accordance with ullberg (39). briefly, whole brains were sectioned in situ in 40 pm thick sections, very few sections being lost in the process. the consecutive series of sections was dehydrated through storage at -20°c and then, some days later, put on film at room temperature for exposure at -20°c. the exposed films were then developed, fixed and rinsed. results 4 foci with decreased 14c-dmo activity (suggesting a decreased tissue ph) located in subcortical structures could be detected two of them were found in one of the rabbits and the other two were found in two other animals (fig. 1). thus, these experiments did not indicate that an acidosis was generally present in these small ischemic regions. however, an interesting finding was that in two out of the four animals one of the hippocampi showed a lower i4c-dmo activity than 240 the other (fig. 1). this finding seemed to correspond to the diffusely increased 2 deoxyglucose (a tracer for cellular glucose uptake (34)) previously found in the hippocampi of embolized rabbits (fig. 2). figure 1. a i4c-dmo autoradio gram. a focus of somewhat de creased 14c-dmo activity indi cating a lower tissue ph can bee seen (white arrow). further it can be seen that one of the hippocampi has a lower 14c-dmo activity than the other (black arrows) compare with fig. 2. figure 2. 2-deoxy-d-[14c] glucose autoradiograms from a previous study on con scious rabbits [29]. a) a region with enhanced 2-deo~y-d-[~4c] glucose (2-dg), located in the right caudate nucleus, can be seen. the focus has a central dip in the 2-dg accumulation. b) this figure shows diffusely increased hippocampal 2 dg accumulation (arrow). this region probably does not reflect ischemia, but rather an increased cellular activity. published with permission. 24 1 discussion changes in the p h are considered to reflect alterations in cerebral circulation and metabolism and are believed to be of importance to the final ischemia related damages (67). this work did not supply support for that acidosis is of any general importance in the process of microinfarction. the sparseness (4 foci in a model generating about 25 ischemic regions per brain (28, 29)) of the foci with decreased 14c-dmo activity may, for instance, be explained by the protons from an increased glycolysis being effectively buffered by buffers locally at hand from the onset of the ischemic process and by buffers continuously diffusing into the ischemic regions from the surroundings. a glucose deficiency (27) may also explain why ischemia does not cause acidosis. another reason may be a low sensitivity of the i4c-dmo method used. reconsidering figs. 1 and 2, we see that in the hippocampus the increased 2-deoxyglucose accumulation and the decreased 14c-dmo activity may reflect the same process increased glycolysis. whether these observations reflect the presence of processes affecting important general brain 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on the doctoral thesis the duodenal mucosal bicarbonate secretion role of melatonin in neurohumoral control and cellular signaling markus sjöblom department of neuroscience, division of physiology, uppsala university, uppsala, sweden. abstract a free full-text copy of this article can be found at the web page of upsala j med sci: http://www.ujms.se the duodenal lumen is exposed to aggressive factors with a high potential to cause damage to the mucosa. bicarbonate secretion by the duodenal mucosa is accepted as the primary important defense mechanism against the hydrochloric acid intermittently expelled from the stomach. the present work concerns both the influence of the central nervous system and the effects of the hormone melatonin on duodenal bicarbonate secretion in anesthetized rats in vivo as well as effects of melatonin on intracellular calcium signaling by duodenal enterocyte in vitro examined in tissues of both human and rat origin. the main findings were as follows: melatonin is a potent stimulant of duodenal mucosal bicarbonate secretion and also seems to be involved in the acid-induced stimulation of the secretion. stimulation elicited in the central nervous system by the � 1 -adrenoceptor agonist phenylephrine induced release of melatonin from the intestinal mucosa and a four-fold increase in alkaline secretion. the melatonin antagonist luzindole abolished the duodenal secretory response to administered melatonin and to central nervous phenylephrine but did not influence the release of intestinal melatonin. central nervous stimulation was also abolished by synchronous ligation of the vagal trunks and the sympathetic chains at the sub-laryngeal level. melatonin induced release of calcium from intracellular stores and also influx of extracellular calcium in isolated duodenal enterocytes. enterocytes in clusters functioned as a syncytium. overnight fasting rapidly and profoundly down-regulated the responses to the duodenal secretagogue orexin-a and the muscarinic agonist bethanechol but not those to melatonin or vasoactive intestinal polypeptide. 115 received 26 october 2004 accepted 11 november 2004 introduction the major functions of the gastrointestinal tract are to distribute to the body sufficient amounts of ingested nutrients and of water and electrolytes, and to expel waste products. to achieve these processes in an adequate manner, this tract has to resist the repeated challenges of ingested noxious, toxic and aggressive agents. it also has to stand firm against potentially harmful endogenous factors, such as hydrochloric acid and digestive enzymes, and prevent them from damaging the mucosa and/or entering the body. the lining of the gastrointestinal tract constitutes the body’s largest surface area facing the external environment. the integrity of the mucosa depends on the balance between aggressive luminal factors and mucosal defense mechanisms. changes in this balance may sooner or later lead to gastrointestinal disorders or diseases. the complex way in which this tube, almost nine meters long in humans, maintains its integrity has challenged and fascinated physiologists for centuries. this work deals with mechanisms that regulate the duodenal mucosal bicarbonate (hco 3 –) secretion, one important mucosal defense mechanism. aggressive factors in the duodenal lumen the duodenal lumen is frequently exposed to aggressive factors with a potential to cause damage to the mucosa. these factors can be divided into two groups: endogenous and exogenous. the main endogenous aggressive factor is hydrochloric acid (hcl), secreted by the parietal cells in the stomach. the secretory capacity is about three liters of hcl in 24 hours, with a ph of about one (1). in 1910 karl schwarz published the first clinical observation that gastric acid was associated with gastric and/or duodenal ulcer disease (2). he noted that acid caused mucosal damage and that this damage could be decreased by luminal neutralization. he formulated the famous dictum “ohne saueren magensaft kein peptisches geschwür“ (“without acid gastric juice – no peptic ulcer”). another harmful factor is pepsin, an enzyme essential for digestion of proteins. the proenzyme pepsinogen is secreted by the peptic cells in the stomach. when catalyzed by secreted hcl, pepsinogen is cleaved into the active enzyme pepsin. there are numerous exogenous factors with the potential to increase the sensibility of or cause damage to the intestinal mucosa. infection with the gram-negative bacteria helicobacter pylori (h. pylori) has a strong correlation with the development of gastroduodenal ulcers (3, 4). when the knowledge of the bacteria became clearer a new dictum was formulated: “no bacteria – no ulcer”. eradication of the bacteria in combination with administration of proton pump inhibitors is effective ulcer treatment. however, duodenal ulcers do occur in non-infected patients. one well known group of substances increasing gastrointestinal damage is the nonsteroidal anti-inflammatory drugs (nsaids), which inhibit mucosal prostaglandin synthesis. it has further been shown that cigarette smoke (5, 6) decreases duodenal bicarbonate secretion and that ethanol (7, 8) increase the susceptibility of the gastrointestinal mucosa to damage. 116 duodenal mucosal mechanisms of protection the duodenum is the most proximal part of the small intestine, into which the stomach intermittently expels chyme with a high concentration of hcl. a healthy duodenal mucosal epithelium resists this challenge. the physiological basis of this barrier function involves several factors and mechanisms. duodenal mucosal protection can be divided into three categories: pre-epithelial, epithelial, and sub-epithelial. pre-epithelial protection the pre-epithelial defense mechanism is often referred to as “the first line” of duodenal mucosal defense. the proximal part of the duodenal lumen often attains acidities as high as close to ph 2 (9–11), but in the immediate vicinity of the surface epithelium the ph remains neutral (12). a ph gradient is formed by the secretion of bicarbonate and mucus from the epithelial cells. the viscoelastic mucus gel on top of the epithelial surface and the bicarbonate secreted into the mucus gel thus provide pre-epithelial defense against damage (11, 13). the mucus gel consists of ~5% mucins (glycoproteins) and >90% water (14). the glycoproteins are secreted by exocytosis by the surface epithelial cells and brunner’s glands. together with water, a gel continuously covering the surface epithelium is formed. the lumen facing part of the mucus gel is loosely adherent to the epithelial surface and the thickness of the gel varies along the gastrointestinal tract (15). the gel provides lubrication for food particles and protects the epithelia from mechanical injury (shear stress). the protective role of the mucus per se is unclear, but it has a low permeability to macromolecules (16) and has been reported to delay back diffusion of hydrogen ions (14). the role of bicarbonate is better clarified, and will be described in more detail in the section “regulation of duodenal mucosal bicarbonate secretion”. the pre-epithelial defense can be summarized as the neutralization of acid and inactivation of pepsin at the duodenal mucosal surface. epithelial protection the epithelial defense is often referred to as “the second line”. epithelial cells of the gastrointestinal tract are interconnected via tight junctions, closing the apical spaces between the cells. the duodenal epithelium, when compared with the gastric epithelium, is often referred to as a “leaky” epithelium (11). this is because of its higher permeability to ions, allowing passive transport of electrolytes between the cells. a characteristic property of the intestinal epithelial cells is that the turnover rate is very high. the average enterocyte only stays alive for two to five days (17). irritating compounds in the intestinal lumen can decrease this time. with such a high turnover rate it is very important to maintain the barrier function intact, preventing agents from entering the body. this is accomplished by restitution, which is a process of rapid re-epithelialization that occurs within a time-scale of minutes to hours. the maintenance of epithelial integrity is also strongly dependent on cell proliferation. the proliferative zone of the duodenal epithelium is in the crypt region (17). during cell migration, from the crypt region to the villus tip, the duode117 nocytes differentiates and acquire the functional characteristics of a villus cell, such as expression of glucose transporters and brush-border hydrolases. this process of migration and differentiation takes between two and five days (17). sub-epithelial protection the blood flow is an important part of the sub-epithelial protection, since ion transport and intestinal motility are highly energy-consuming processes. the arteries of the proximal duodenum originate from the celiac trunk and divide into the gastroduodenal and pancreaticoduodenal arteries. the superior mesenteric artery supplies the more distal part of the duodenum. to achieve a rich blood supply, the vessels of the gastrointestinal tract have a large number of collaterals. it is well known that the amount of blood supplying the mucosa is regulated at the level of the arterioles, the resistance vessels. mechanisms of regulation include neural, humoral, metabolic and myogenic factors (18, 19). the blood flow provides the duodenal mucosa with hco 3 –/co 2 (20, 21) and transfers absorbed nutrients, water, metabolic end-products and/or toxic substances to the liver. the enteric nervous system the enteric nervous system (ens) plays a crucial part in the regulation of gastrointestinal functions such as ion transport (secretion and absorption), motility and mucosal blood flow. as part of the autonomic nervous system, the ens is organized in a complex but very sophisticated network and contains as many neurons as the spinal cord (1). a unique feature of the ens is that it can manage its many functions without input from the brain or spinal cord (22). the ens is embedded in the gastrointestinal wall and consists of the myenteric plexus, located between the circular and longitudinal muscle layers, and the submucosal plexus, located in the submucosa. in general, the myenteric plexus controls gastrointestinal motility and the submucosal plexus coordinates ion transport and mucosal blood flow, but there is also extensive intercommunication between these plexa. although the ens can function autonomously, the central nervous system (cns) has a major influence on gastrointestinal functions. the vagal efferents (parasympathetic) project from their nuclei in the medulla oblongata and terminate in ganglia of the myenteric plexus, as described by kirchgessner & gershon in 1989 (23). these authors also demonstrated that almost no vagal efferents terminate in the submucosal plexus or at the epithelial cells. signals from the vagal fibers have to be conveyed in the myenteric plexus. the influence of the sympathetic nervous system is mainly inhibitory. sympathetic efferent neurons project from the spinal cord, relay in the celiac ganglion and terminate in the myenteric and submucosal plexa, as well as in the mucosa (1). the intestine also possesses delicate sensory characteristics. the primary afferent neurons sense the mucosal epithelium and the luminal contents. these neurons can be divided into three classes: i) intrinsic, ii) extrinsic and iii) intestinofugal neurons (24). the intrinsic primary afferent neurons project only a short distance and have 118 their cell bodies and connections in the intestinal wall. the extrinsic primary afferent neurons have their cell bodies in the vagal and dorsal (spinal) ganglia with processes in the epithelium, and carry information to the central nervous system. the intestinofugal neurons have their cell bodies in the gut wall and carry information to prevertebral ganglia. regulation of duodenal mucosal bicarbonate secretion duodenal mucosal hco 3 – secretion has a key role in duodenal protection against pulses of hcl and pepsin that are intermittently discharged from the stomach. one of the unique features of the duodenal epithelium is that it secretes bicarbonate at higher rates than the mucosa of more distal parts of the small intestine. the main physiological stimulant of the hco 3 – secretion is the presence of acid in the duodenal lumen, and the acid-induced hco 3 – response is mediated by enteric nervous pathways, involving release of vasoactive intestinal polypeptide (vip) and acetylcholine (11, 13), as well as by e-type prostaglandins (pgs) released from mucosal cells (25). several compounds, of both the hormonal and non-hormonal type, have been shown to stimulate duodenal mucosal bicarbonate secretion. vip is a peptide which very potently stimulates intestinal secretion, and infusion of vip increases the hco 3 – transport by the duodenal mucosa in all species tested (11, 26–28). other mediators stimulating duodenal bicarbonate transport include cholecystokinin (cck), pancreatic polypeptide and neurotensin (29), glucagons (30), pituitary adenylate cyclase-activating polypeptide (pacap) (28, 31, 32) and angiotensin ii (33). the roles of pgs and nitric oxide (no) in the hco 3 – secretory response to acid have been studied extensively during recent years. two cyclooxygenase (cox) enzyme isoforms, cox-1 (constitutively expressed) and cox-2 (inducibly expressed), are responsible for pg synthesis. the enzyme responsible for the increase in bicarbonate secretion after acid challenge is the cox-1 enzyme (34). although pgs increase bicarbonate secretion, it has been shown that inhibition of pg synthesis with indomethacin also increases the alkaline output, by a mechanism closely coupled to induction of duodenal motility (35, 36). the effects of no on duodenal alkaline secretion are complex. there have been several reports that systemic (iv) no synthase (nos) inhibition with n-nitro-larginine methyl ester (l-name) increases duodenal mucosal bicarbonate secretion (37–40). other studies, however, have shown that both luminal l-name (41, 42) and iv l-name (41, 43, 44) decrease the bicarbonate secretory response to acid. three isoforms of the no-synthesizing enzyme have been found: nnos (neural), enos (endothelial) and inos (inducible). the nnos and enos isoforms are constitutively expressed and are usually named cnos. takeuchi et al. recently suggested that cnos is responsible for no production following duodenal acidification (34). the bicarbonate secretion is inhibited by nsaids as well as by �2-adrenorecep119 tor sympathetic stimuli. h. pylori infected patients with acute or chronic duodenal ulcer disease have impaired alkaline secretion (45), and eradication of the infection at least partly restores the secretion (46). further, fändriks et al. showed that water extracts from h. pylori inhibit duodenal mucosal bicarbonate secretion in the rat (47). central nervous control influence of the central nervous system on duodenal mucosal bicarbonate secretion is well established. the proximal duodenum is densely innervated with vagal fibers, passing from the medulla oblongata in the cns and terminating in the myenteric plexus (48). the myenteric plexus and the submucosal plexus are also innervated by the sympathetic nervous system. whether the alkaline secretion is stimulated or inhibited depends on the input signals to the secretomotor neurons of the submucosal plexus. electrical stimulation, in the peripheral direction, of the cut vagal nerves in cats (49, 50) and in rats (51, 52) increases the bicarbonate secretion. the stimulatory effects are abolished by peripheral hexamethonium. a further indication that the cns influences secretion is that sham-feeding increases the duodenal mucosal bicarbonate secretion in humans (53) and dogs (54). besides exerting neural influence, the cns can also regulate and control the secretions via release of hormones. �-endorphin released from the pituitary gland influences duodenal hco 3 – secretion (55). there are also reports on centrally elicited stimulation of the secretion by some neuropeptides, including thyrotropin-releasing hormone (trh), corticotropin-releasing hormone (crh) and bombesin (11, 55–57), as well as by some benzodiazepines (58). furthermore, an up to a four-fold increase in secretion has been observed after intracerebroventricular (icv) infusion of the � 1 -adrenoceptor agonist phenylephrine (59, 60). this increase was inhibited by the ganglion-blocking agent hexamethonium and by central nervous (but not intravenous) administration of the adrenoceptor antagonist prazosin. duodenal enterocyte ion transport approximately 90% of the intestinal epithelium consists of enterocytes (61). the knowledge about the intracellular signaling and different ion transporters involved in duodenal enterocyte bicarbonate secretion is increasing, but is still incomplete. three major messenger systems have been suggested as being implicated in the intracellular control of hco 3 – transport processes: i) intracellular calcium-induced responses (muscarinic m 3 agonists and cck a ), ii) cyclic amp-activated transport (prostaglandin ep 3 agonists, vip and dopamine d 1 agonists), and iii) cyclic gmpactivated transport (uroguanylin, guanylin and heat-stable entero-toxin). the duodenal enterocytes possess different mechanisms for acid/base transport possibly reflecting the second messenger system activated. hco 3 – and co 2 reach the epithelium via the blood and hco 3 – is imported at the basolateral membrane by na+(n)-hco 3 – cotransport. co 2 diffuses into the enterocytes and hco 3 – is formed intracellularly by carbonic anhydrase conversion of co 2 + h 2 o to hco 3 – + h+. the 120 enterocytes export hco 3 – at the apical membrane by a cl–/hco 3 – exchanger as well as via an anion conductive pathway. it is suggested that the cystic fibrosis transmembrane conductance regulator (cftr) is the ubiquitous membrane spanning channel that transports cl– as well as hco 3 – (62–64). an amiloride-sensitive na+/h+ exchanger extrudes acid both at the apical and at the basolateral membrane. fig. 1 shows a schematic illustration of the hco 3 – transport by duodenal epithelium. as the duodenal epithelium consists of both villus and crypt cells it is of great importance to verify the source of the secretion of hco 3 –. in general, crypt cells are thought to have a secretory function, whereas the cells in the villi are mainly absorptive (65). the earlier hypothesis that intestinal secretions are only of crypt origin while the absorptive functions are restricted to the villi is, however, under reevaluation. suppression of carbonic anhydrase (ca) activity with acetazolamide decreases duodenal mucosal hco 3 – secretion in humans (66), rabbits (20) and the guinea pig (67). in rats, acetazolamide has been reported to decrease bicarbonate secretion (12), but other authors have found no effect (68, 69). furthermore, the ca isoenzyme ii (ca ii), associated with alkaline secretion, is located mainly in the villi and not in the duodenal crypts (70). 121 fig. 1. a schematic illustration of the ion transporters in duodenal enterocytes. the model is based upon in vitro and in vivo experiments from several species. cftr = cystic fibrosis transmembrane conductance regulator. ae = anion exchanger. nhe = sodium hydrogen exchanger. nbc = sodium bicarbonate cotransporter. melatonin melatonin is the major hormone of the pineal gland and was first isolated by lerner and colleagues (71). melatonin is derived from the amino acid tryptophan, which is converted into serotonin. two enzymes then synthesize serotonin into melatonin. the first enzyme, the light sensitive, is n-acetyltransferase (nat) and the second, the terminal and light insensitive, enzyme is hydroxyindole-o-methyltransferase (hiomt) (72, 73). in the presence of light, no melatonin is synthesized in the central nervous system. the physiological functions of melatonin are numerous. among other effects, findings have suggested that melatonin may be involved in the regulation of circadian rhythms (74), scavenging of free radicals (75, 76), alleviation of jet lag (77) and (in non-human mammals) seasonal reproductive behaviors (74). for many years melatonin was thought to be exclusively synthesized by the pineal gland, but it has become well established that active synthesis of melatonin also occurs in extrapineal sources. in 1975, some russian scientists demonstrated melatonin synthesis in human intestinal enterochromaffin cells (78). furthermore, quay & ma (1976) showed the presence of hiomt in the duodenal mucosa, and hong & pang (1995) provided evidence for nat activity in this tissue. these results have recently been confirmed (79). the amount of melatonin in the gastrointestinal tract does not depend on the presence of light and is not reduced by pineal glandectomy (80). distribution of peripheral melatonin the major source of melatonin in the body is the gastrointestinal tract (81–83). huether showed that the total amount of melatonin in the gastrointestinal tract is at least 400 times greater than the amount in the pineal gland at any time of the day and night (81). a similar observation has been made for serotonin (5-ht), of which approximately 95% is found in the alimentary canal (84). melatonin is also present in several other organs, for example the pancreas, liver, bile, urogenital tract, air way epithelium and the retina. melatonin produced by the enterochromaffin (ec) cells in the intestinal mucosa seems to contribute to the circulating blood concentration during the daytime (85), whereas melatonin released from the pineal gland is responsible for the higher concentrations during darkness (86). the pineal gland melatonin is released in a circadian fashion (74, 86), while the melatonin produced in the gastrointestinal tract steadily enters the circulation (87, 88). most of the melatonin released from the gastrointestinal tract into the portal vein is metabolized in the liver. an interesting phenomenon observed is that when the concentrations of melatonin decrease to a daytime level, the hormone escapes liver metabolism (89). it has also been reported that, in both normal (85, 90) and pineal glandectomized rats (91), a high tryptophan (melatonin precursor) diet drastically increases the blood levels and intestinal levels of melatonin. being a non-polar and lipid-soluble hormone, melatonin crosses biological membranes, such as the blood-brain barrier, and acts at melatonin-specific receptors in the central nervous system as well as at such receptors in peripheral tissues. the 122 half-life of melatonin in the peripheral circulation is ~20–40 minutes, depending on the species (92). melatonin receptors melatonin acts principally via high-affinity receptors coupled to hetero-trimeric guanine nucleotide-binding regulatory proteins (g-proteins). three receptor subtypes have been found (93); two of them, the mt 1 and mt 2 melatonin receptors, have been identified in mammals in molecular cloning studies, and a third receptor, named mt 3 , has been found though not yet cloned (93, 94). melatonin receptors are distributed throughout the gastrointestinal tract (82, 83, 95). the signaling properties of the mt 2 receptor are becoming clearer since the recent development of mt 2 -selective ligands. unfortunately, no high affinity mt 1 receptor ligands have yet been discovered. ligands with high affinity for mt 1 receptors are required to further clarify the physiological and pathophysiological roles of the biological actions of melatonin. the two receptor subtypes mt 1 and mt 2 have in common that they inhibit camp formation and stimulate phosphatidylinositol hydrolysis (93). the melatonin receptors mentioned in this work are defined according to the nomenclature and classification of the nomenclature committee of the international union of pharmacology (96). the denomination mt 1 corresponds to that of the recombinant receptor previously termed ml 1a or mel 1a . mt 2 refers to the native functional receptors with pharmacological characteristics similar to those of the recombinant receptor mt 2 previously termed ml 1b or mel 1b . mt 3 corresponds to the pharmacologically defined melatonin receptor subtype, with unknown gene sequence, previously referred to as ml 2 . aims of the investigation the general aim of this investigation was to further elucidate the central nervous and the peripheral regulation of the mucosa-protective duodenal mucosal bicarbonate secretion. one intention was to examine the influence of the hormone melatonin on the secretion in vivo and to study its effect on duodenal enterocyte intracellular calcium signaling in vitro. more specifically, the following aims were addressed: o study the effects of intravenous infusion, intraarterial infusion close to the duodenum, and duodenal luminal administration of melatonin and some melatonin agonists/antagonists on duodenal mucosal bicarbonate secretion in anesthetized rats in vivo. o elucidate the neurohumoral pathways mediating the increase in duodenal mucosal bicarbonate secretion elicited by phenylephrine administered icv. o investigate duodenal mucosal bicarbonate secretion in pineal glandectomized rats in vivo. 123 o study the release of melatonin from the duodenal mucosa. o examine acid-stimulated duodenal mucosal bicarbonate secretion and the role of melatonin. o compare basal and stimulated duodenal alkaline secretory rates in fed animals and in animals fasted for a short period (overnight). o develop a method for isolation of duodenal enterocytes suitable for studies of intracellular signaling. o elucidate the effects of melatonin on intracellular calcium signaling in duodenal enterocytes. materials & methods in vivo experiments animals all experiments on animals were approved by the uppsala ethics committee for experiments with animals. male outbred sprague-dawley rats (190–260 g) or f 1 hybrids of lewis x dark agouti rats (200–300 g) were placed in a conditioning unit under standardized temperature and light conditions (21–22°c, 12:12 h light-dark cycle) for at least four days after purchase. the rats were kept in cages in groups of two or more and had access to tap water and pelleted food (ewos, södertälje, sweden) ad libitum. anesthesia and general surgery the rats were deprived of food for 16–20 h before the experiments, but had free access to drinking water. the experiments were started by anesthetizing the animal with 5-ethyl-5-(1’-methyl-propyl)-2-thiobarbiturate (inactin®), 120 mg/kg body weight intraperitoneally. the animals were anesthetized in the animal department by the person who had previously handled them. subsequently, the rats were tracheotomized with a tracheal tube to facilitate respiration, and the body temperature was maintained at 37–38∞c throughout the experiments by a heating pad controlled by a rectal thermistor probe. a femoral artery and vein were catheterized with pe-50 polyethylene catheters. for continuous recordings of systemic arterial blood pressure the arterial catheter, containing 20 iu/ml of heparin isotonic saline, was connected to a pressure transducer operating a powerlab system. the vein was used for injection of some of the drugs and for continuous infusion of ringer solution ([in mm] 145 na+, 124 cl¯, 2.5 k+, 0.75 ca2+ and 25 hco 3 –) at a rate of 1.0 ml/h. the latter infusion was given to compensate for fluid loss and to avoid acid/base changes during the experiments. blood acid/base balance was checked in 40 µl arterial blood samples taken at the start and end of the experiments. after completion of the operative setup, the abdomen was closed with sutures and the animal was left undisturbed for 40–60 minutes for stabilization of the cardiovascular, respiratory and gastrointestinal functions. 124 duodenal preparation the abdomen was opened by a midline incision and the gastric pylorus was ligated with a suture. to prevent bile and pancreatic secretion from entering the intestine, the common bile duct was always catheterized close to its entrance to the duodenum, with a pe-10 polyethylene tubing. for measurement of duodenal mucosal hco 3 – secretion, a 12 mm segment of duodenum with its blood supply intact, starting 10–12 mm distal to the pylorus and thus devoid of brunner’s glands, was cannulated in situ between two glass tubes connected to a reservoir (fig. 2). fluid (10 ml of 154 mm nacl), maintained at 37°c by a water jacket, was rapidly circulated by a gas lift of 100% oxygen. hco 3 – secretion into the luminal perfusate was continuously titrated with 50 mm hcl at ph 7.4 under automatic control of a ph-stat system. intraarterial infusions to study effects elicited in the duodenal segment and to minimize possible central nervous actions, compounds were administered close to the duodenal segment by intraarterial (ia) infusion. the hepatic artery was cannulated, tied 3–4 mm proximal to its entrance into the liver, and perfused in the retrograde direction at 17 µ l/min (fig. 3). this perfusion results in distribution of the perfusate mainly to the duodenum (via the cranial pancreaticoduodenal artery) and pancreas. the distribution was checked visually at the start and end of the experiments by ia injection of a small amount of isotonic saline. this procedure changed the bright125 fig. 2. rat proximal duodenum was cannulated in situ between two glass tubes connected to the same reservoir containing isotonic saline. the mucosal hco 3 – secreted into the luminal perfusate was continuously titrated under automatic control of a ph-stat system. ness of the duodenal segment. intracerebroventricular infusions compounds were administered by icv infusions in order to study duodenal secretory stimulation elicited in the central nervous system. a metal cannula was inserted into the right lateral cerebral ventricle by using a stereotactic instrument. a skin incision was made over the right parietal bone, and a 1 mm hole was drilled through the bone, 0.8 mm posterior to the bregma and 1.5 mm lateral to the midsagittal suture. a stainless steel cannula was inserted stereotactically and cemented to the skull. artificial cerebrospinal fluid ([in mm] 151.5 na+, 3.0 k+, 1.2 ca2+, 0.8 mg2+, 132.8 cl–, 25 hco 3 –, 0.5 phosphate; ph 7.4) was infused through this cannula at a rate of 30 µ l/h. all agents infused icv had been dissolved in artificial cerebrospinal fluid. the location of the end of the cannula within the icv space was tested at the end of most experiments by adding evans blue solution to the infusate, followed by dissection of the brain. pineal glandectomy 126 fig. 3. for intraarterial infusion close to the duodenal segment, the hepatic artery was cannulated, and tied before its entrance into the liver, and the administered drugs were infused in the retrograde direction. [from flemström et al. (121) with permission]. we modified a method described by hoffman & reiter in 1965 (97). sprague-dawley rats were anesthetized by intraperitoneal injection (0.27 ml/kg body weight) of a solution (hypnorm®) containing fentanyl 0.315 mg/ml, fluanisone 10 mg/ml and midazolam 5 mg/ml, which induced surgical anesthesia for about 30 min. using the stereotactic instrument, the head of the rat was fixed and the scalp was cut anteroposteriorly along the midline. the skin flaps were reflected and the temporal and occipital muscle masses were scraped free. three lines were cut with a dental drill equipped with a fissure bar, the bone flap was raised and the dura mater was cut with a sharp needle. a forceps was put beneath the superior sagittal sinus and the pineal gland (white and 0.5–1.0 mm in diameter) was removed. experiments on the duodenum were not performed until at least one week after pineal glandectomy. pituitary glandectomy pituitary glandectomy was performed at the møllegaard breeding center by personnel with routine experience of this operation. the pituitary gland in male spraguedawley rats (weighing 190–230 g) was removed by suction with a syringe through the ear. the animals were observed for one week following the operation, and absence of gain in weight (indicating lack of growth hormone incretion) was used to confirm the removal of the pituitary gland. the animals were then transported to uppsala together with non-operated animals of the same breed for control experiments. to maintain body acid/base balance the rats were always supplied with drinking water adjusted to ph 3 with hcl. twenty-four hours before the experiments 0.5 mg/kg dexamethasone (decadron®) was injected intramuscularly to compensate for the loss of endogenous glucocorticosteroids. this injection was necessary to keep the rats alive during the experiments. transmucosal electrical potential difference the duodenal transmucosal electrical potential difference (pd) was measured in some experiments and recorded between the duodenal mucosa and posterior vena cava with a high-input impedance voltmeter via matched calomel half-cells. the half-cells were connected to the animal by means of agar bridges (2 m kcl) with their distal ends located in the luminal solution and the posterior vena cava, respectively. section of vagus nerve and sympathetic chain the common carotid arteries were identified and the surrounding nerves were dissected free from the arteries under light microscopy. in one group only the cervical vagal nerves were cut, at the sub-laryngeal level. in a second group the cervical paravertebral sympathetic chain was cut. in a third group all nerves around the carotid arteries (including vagal trunks and sympathetic chain) were ligated and cut at the sub-laryngeal level. melatonin analyses 127 128 arterial blood samples (0.7–1.0 ml) were obtained from the rat tail artery and from the femoral artery. all blood samples were taken between 11 am and 3 pm. the blood samples were left for 30 min at room temperature to coagulate and then centrifuged at 4000 rpm at 4°c for 7 min. the serum was then stored at –20°c until analyzed at nova medical ab, skövde, sweden, using an elisa assay (bühlmann labs., allschwil, switzerland). the detection limit of the assay was 0.05 pmol/ml. the intra-assay and inter-assay coefficients were below 6.6 %. melatonin was also determined by high-performance liquid chromatography (hplc) with electrochemical detection, running chromeleontm software (dionex corporation, sunnyvale, usa) on an ibm-compatible computer. melatonin was separated on a luna c18 column (5 µ m particle size, 150 x 4.6 mm). the isocratically operated chromatographic system was perfused with the following mobile phase: 0.1 m sodium acetate, 0.1 m citric acid, 0.15 mm edta, 30 % methanol, ph 3.7, at a flow rate of 1.0 ml/min. the electrochemical detector potential was adjusted to +900 mv. the total runtime was 15 min. melatonin was eluted at 12 min and 57 sec and 6-fluorotryptamine was eluted at 5 min and 58 sec. samples of 2.0 ml of re-circulating luminal perfusate, from the chamber illustrated in fig. 2, were taken and 1.0 ng of the internal standard 6-fluorotryptamine was added. the samples were then filtered through an acrodisc® lc 13 mm syringe filter with a 0.2 µ m pvdf membrane (pall gelman laboratory, usa) and freeze-dried. the residues were dissolved in 230 µ l hplc mobile phase. duplicate 100 µ l samples of the solution were injected into the chromatographic system (injector mod. 7725i, 100 µ l loop, rheodyne inc., san francisco, usa). melatonin concentrations were calculated on the basis of comparison with the internal standard. the melatonin detection limit of the hplc system was 0.5 ng. the calibration curves for melatonin and 6fluorotryptamine showed linear responses over the studied ranges. triplicates of melatonin standards (0.5, 1.0, 10, 100 and 1000 ng) were injected into the hplc system and the calibration curve equation obtained was y = 3.25x + 0.42, r2 = 0.99. the internal standard was also injected into the hplc system in triplicates. 6-fluorotryptamine (0.05, 0.5, 5.0 and 50 ng) yielded the calibration curve equation y = 18.6x + 0.075, r2 = 0.99. statistical analysis descriptive statistics are expressed as means ± sem, with the number of experiments given in parentheses. rates of alkaline secretion by the duodenum are expressed as microequivalents of base (hco 3 –) per centimeter of intestine per hour (µ eq•cm–1•h–1). the secretion and mean arterial blood pressure (map) were monitored continuously and recorded at 10-min intervals. the statistical significance of data was tested by repeated measures analysis of variance. to test differences within a group a one-factor repeated measures anova was used, followed by fishers’s plsd post hoc test. between groups the results of hco 3 – secretion with drug administration were compared with the secretory rates obtained with control animals infused with vehicle alone or with other compounds. for this comparison, a two-factor repeated measures anova followed by a one-way anova at each time point was used. if the anova was significant at a given time point, a fisher’s plsd post hoc analysis was used. all statistical analyses were performed on an ibm-compatible computer using statview 5.0 software. p values of <0.05 were considered significant. in vitro experiments human biopsies biopsy specimens from the duodenum were obtained from patients undergoing upper endoscopy at the gastroenterology unit, uppsala university hospital and found to have endoscopically normal duodenal and gastric mucosae. results obtained from 17 biopsy specimens from 8 patients are presented. the project was approved by the ethics committee of the medical faculty at uppsala university, and all subjects provided written informed consent. the specimens were taken between 9 am and 10 am with radial jaw (large capacity with needle) single-use biopsy forceps and immediately transported to the laboratory at the biomedical center, uppsala, sweden. rat tissue preparation the experiments were begun before 9 am, and to avoid possible stimulatory effects of anesthetics on intestinal mucus release, the rats were decapitated. a 3-cm segment of duodenum, starting 2–3 mm distal to the pylorus, was promptly excised via an abdominal midline incision and freed from mesentery. the segment was opened along the antimesenteric axis and the luminal surface was rinsed with a normal respiratory medium (nrm) ([in mm] 114.4 na+; 5.4 k+; 1.0 ca2+; 1.2 mg2+; 121.8 cl–; 1.2 so 4 2–; 6.0 phosphate; 15.0 hepes; 1.0 pyruvate; and 10 glucose plus 10 mg/l phenol red, 0.1 mg/ml gentamicin and 2.0% fetal calf serum). the ph was adjusted to 7.40 immediately before use and the temperature was maintained at 37°c. the sheet of duodenal wall was then put on a precleaned glass slide (lumen side up) and the mucosa was gently scraped-off. the depth of mucosal tissue removed by the scraping procedure (and used for experiments) was tested by morphological examination of the remaining tissue (fixed in 10% neutral buffered formalin and stained with hematoxylin-eosin). the duodenal remnant contained some crypt bases and all submucosa containing brunner’s glands. cells originating from the latter glands were thus excluded from the studied preparations. isolation of enterocytes in clusters the scraped-off rat mucosa or the human biopsy specimens were then cut into pieces 0.3–0.8 mm in diameter which were dispersed and briefly shaken in nrm solution also containing 0.5 mm dithiothreitol (dtt). after sedimentation for 2–3 min, the supernatant was removed and the tissue fragments (in the sediment) washed three times in nrm solution (not containing dtt). following brief gassing 129 with 100% o2, the tissue fragments (15–20 µ l) were then exposed to mild digestion for 3 min by inoculation in 10 ml nrm solution containing 0.1 mg/ml collagenase type h (sigma) and 0.1 mg/ml dispase ii (mannheim). digestion was performed at 37°c in a horizontal shaking water bath, and was stopped by adding dtt (to a concentration of 0.3 mm) and the solution was centrifuged (3 min at 1,000 g). the pellet was washed three times by suspension in 10 ml dme/f12 (with 15 mm hepes and 2.5 mm glutamine) followed by centrifugation (3 min at 1,000 g). hco 3 – (1 mm), gentamicin (0.01 mg/ml) and fetal calf serum (2.0%) were always added to the dme/f12 and the ph was adjusted to 7.40. the preparatory procedure yielded clusters (10–100 cells) of interconnected duodenal enterocytes as well as smaller amounts of single cells. the clusters were composed predominantly of cells with morphological characteristic of crypt cells. the viability after the preparation was tested by trypan blue exclusion (>95%). the final pellet was suspended in ~1.0 ml of dme/f12 (with the same additives) solution and immediately put on ice, a procedure found to increase the viability of the enterocyte clusters compared with keeping the cells at 37°c. cell loading and calcium measurements with fura-2 for measurement of the intracellular calcium concentration ([ca2+] i ), 70 µ l of the cell cluster suspension was loaded at 37°c with fura-2 acetoxymethyl ester (2 µ m) for 20–30 min in an electrolyte solution ([in mm] 141.2 na+; 5.4 k+; 1.0 ca2+; 1.2 mg2+; 146.4 cl–; 0.4 phosphate; 20.0 tes; and 10 glucose; ph 7.40) that has been found appropriate for studies of cell aggregates from other tissues (98). probenesid (1 mm), pluronic f-127 (0.02%) and fetal calf serum (2.0%) were present during the loading procedure. the fura-2-loaded cell aggregates were spun down and placed on an uncoated, precleaned circular glass coverslip (ø 25 mm) at the bottom of a temperature-controlled (37°c) perfusion chamber (fig. 4) and fixed on top of the coverslip by a uniformly sized pore polycarbonate membrane filter. the covering filter and the cell preparation were perfused (1 ml/min) with the electrolyte 130 fig. 4. schematic illustration of the temperature-controlled perfusion chamber. solution and receptor ligands to be tested were added by inclusion in the perfusate. changes in [ca2+] i in the fura-2-loaded cells were measured by the dual-wavelength excitation ratio technique by exposure of the cells to alternating 340 and 380 nm light with the use of a filter changer under the control of an incytim-2 system (intracellular imaging) and a dichroic mirror (dm430, nikon). emission was measured through a 510 nm barrier filter with an integrating ccd camera. calibration of the fluorescence data was accomplished in vitro according to the method described by grynkiewicz et al. in 1985. data analyses all statistical analyses were performed on an ibm-compatible computer using statview 5.0 software. when appropriate, statistical significance was calculated using student’s t-test. non-linear curve-fitting of the data was achived by use of sigmaplot for windows 4.01. results and discussion the gastrointestinal epithelium is the largest surface area in the body. facing the external environment, the epithelium is repeatedly challenged by aggressive factors of both exogenous and endogenous origin. today, the duodenal mucosal bicarbonate secretion is accepted as the primary defense mechanism against the hcl that is intermittently expelled from the stomach. the secretory rates of hco 3 – are higher in the duodenum than in the stomach and in other, more distal parts of the small intestine (11). the investigations described in this study have focused on the central nervous regulation on duodenal mucosal bicarbonate secretion as well as the effects of melatonin on this secretion. on the basis of results from in vivo and in vitro studies, physiological processes of potential importance for regulation of the protective alkaline secretion by the duodenal mucosa are suggested. the essence of the results will be discussed below, but the “take-home” message of this work is that intestinal melatonin is an important mediator in the cnsand hcl-elicited stimulation of duodenal mucosal bicarbonate secretion. this in turn suggests that melatonin may be involved in duodenal mucosal protection against acid. intestinal melatonin most probably originates from the enterochromaffin cells, and the released melatonin activates adjacent enterocytes to secrete hco 3 –. with calcium as an intracellular and intercellular messenger, the duodenal enterocytes form a secretory functional syncytium. further, the sensitivity to some peripheral stimulators of duodenal mucosal hco 3 – secretion depends markedly on the feeding status of the animals. the physiological relevance of bicarbonate secretion evidence for hco 3 – secretion originating from the duodenal mucosa was first reported a century ago in a thesis from pavlov’s laboratory in st. petersburg (99). since that time, both in vitro and in vivo studies have shown that the duodenal 131 mucosa secretes hco 3 – at high rates (11). in the normal situation, when the duodenal mucosa is healthy, bicarbonate enters the continuous layer of viscoelastic mucus gel on top of the epithelial surface and maintains the ph in its cell-facing portion at neutrality in spite of high acidities in the duodenal lumen (12, 13, 16). when the bicarbonate secretion is inhibited by nsaids, or when the secretory neurohumoral regulation is malfunctioning, as in h. pylori-infected patients with acute and chronic duodenal ulcer disease, the acid may acidify the epithelial surface and cause mucosal damage. it should be noted that bicarbonate secreted from the duodenal epithelium is not solely responsible for neutralizing the gastric acid expelled into the intestine. it serves as an epithelial protector and together with bicarbonate-rich juices from the liver and pancreas it inactivates proteolytic enzymes, such as pepsin, and neutralizes the gastric acid. overall, acid-stimulated mucosal hco 3 – secretion probably accounts for ~40% of the neutralization of the gastric acid load to the duodenum; pancreatic and biliary hco 3 – accounting for the remaining bulk neutralization (100, 101). melatonin as an intestinal hormone in humans and other mammals, including rodents, melatonin secretion from the pineal gland peaks at darkness (night), independently of species differences in day or night activity (73). melatonin is synthesized from tryptophan, with serotonin as an intermediate precursor, and is released from the pineal gland into the circulation. being a non-polar and lipid-soluble hormone, melatonin crosses the blood-brain barrier and acts at melatonin-specific receptors in the cns as well as at such receptors in peripheral tissues. importantly, melatonin is also produced by the ec cells in the intestinal mucosa (102) and the total amount of melatonin in the alimentary tract is considerably higher (>400) than that in the cns (81). it should also be noted that ec cells are in close contact with fibers from the autonomic nervous system (103). the physiological role of the intestinal source of melatonin has not been fully established. like the ec cell products guanylin (104) and serotonin (84), intestinal melatonin may have a role in the reaction between the mucosa and the luminal contents. luminal perfusion of melatonin induced high rates of duodenal mucosal hco 3 – secretion (fig. 5.) (105) and such an effect was also observed after iv or close ia infusion (60). this may be in line with the proposal that melatonin acts as an intestinal intraluminal hormone, exerting actions in intestinal segments distal to the sites of release (88). it should be noted in this context that the continuous discharge of bile into the duodenum that occurs in the rat is probably a source of intestinal intra-luminal melatonin (106, 107). at least during conditions of intestinal paralysis, the mucus layer on the surface of the duodenal mucosa provides a physical barrier to the migration of macromolecules and some secretagogues, including prostaglandins, to the epithelial surface (16). the high rates of mucosal hco 3 – secretion induced by melatonin would suggest that the mucus layer does not significantly inhibit the migration of melatonin from the luminal fluid to the epithelial surface (105). we demonstrate for the first time that melatonin and melatonin receptor agonists 132 increase the duodenal hco 3 – secretion in rats (60). the secretagogues were administered by ia infusion close to the duodenal segment, a procedure that would minimize central nervous actions. considerably higher doses were required for stimulation when the hormone was given iv, strongly indicating that the stimulation by melatonin is elicited within the duodenum and is not mediated by a primary central nervous action. the secretory responses were inhibited by iv infusion of the predominantly mt 2 -selective melatonin receptor antagonist luzindole (18-fold selectivity mt 2 >mt 1 ). luminal melatonin is a potent stimulator of the hco 3 – secretion by the duodenal mucosa (105). when rats were pretreated with iv luzindole, the effects of luminal melatonin were efficiently abolished (fig. 5). the ganglion-blocking agent hexamethonium (a nicotinic receptor antagonist) reduced the magnitude of the stimulatory effect of luminal melatonin on hco 3 – secretion (105). it should be noted that the hco 3 – secretory rate always remained significantly higher than that in untreated controls. stimulation of hco 3 – secretion by local intestinal melatonin seems to be in line with the finding that melatonin increased the intracellular ca2+ (fig. 6) in isolated duodenal enterocytes (108). taken together, these observations suggest an action of melatonin on receptors at duodenal enterocytes as well as on such receptors in the ens. neither luzindole at a dose that inhibited the stimulation by exogenous melatonin, nor another melatonin receptor antagonist (4-p-pdot) affected spontaneous (basal) hco 3 – secretion. this suggests that endogenous melatonin has no effect on basal secretion. 133 fig. 5. perfusion of the duodenal lumen with melatonin increased duodenal bicarbonate secretion. this secretory response was abolished by pretreatment with luzindole and significantly inhibited by pretreatment with hexamethonium. [from sjöblom et al. (105) with permission]. it should be pointed out that the doses of melatonin required for stimulation of duodenal hco 3 – secretion seem to be much lower (>100-fold) than those tested in animal models of depressive disease (109) or used in humans for treatment of sleep disturbances or depression (110). in spite of the considerably smaller total amounts of melatonin produced in and released from the pineal gland during the daytime, melatonin from the cns may be important in the night-time control of the duodenal alkaline secretion and mucosal protection. the experiments in this study were started at around 9 am and were performed during the daytime when the pineal gland release of melatonin is low. they do not exclude the possibility of an increase in protective hco 3 – secretion induced by the higher levels of melatonin that occur in darkness. recent studies in rats have shown that during the dark-phase, compared with the daylight phase, the frequency of duodenal and jejunal migrating motor complexes was increased by 20% and that this was abolished by the melatonin antagonist s20928 (111, 112). these authors concluded that pineal gland melatonin is involved in the dark-phase physiological control of the preand postprandial changes of intestinal motility. acid-induced secretion the hco 3 – secretion and in particular the secretory response to acid, as stated previously, is the principal mechanism in duodenal mucosal protection against acid expelled from the stomach. in the presence of a low ph in the duodenal lumen, ~ph 5 in rats (12) and ~ph 3 in humans (113), neural reflexes and mucosal production of prostaglandins are stimulated. the results show that the melatonin antagonist luzindole decreases the hco 3 – 134 fig. 6. isolated human duodenal enterocytes in clusters increased their intracellular calcium concentration after perfusion with melatonin. [from sjöblom et al. (108) with permission]. response to acid (105). this suggests that melatonin is involved in mediating the increase in alkaline secretion induced by the presence of acid in the duodenal lumen. it is reported that the surface epithelium and its close luminal vicinity are neutral even when the ph in the duodenal lumen is close to 2 (12, 39). this raises the intriguing question of how acid is sensed by the secreting epithelium. one hypothesis is that there are acid-sensitive neural receptors or cell filaments protruding into the surface gel that sense the luminal ph. holm et al., on the other hand, have recently proposed that the stimulation of alkaline secretion may not be due to h+ itself, but rather to the rapidly diffusible co 2 generated within the mucus gel during the reaction between secreted hco 3 – and h+ ions (114). there is some uncertainty to which extent cells in the villus tip actually secretes bicarbonate. furthermore, at least part of the duodenal alkaline secretion originates from the villi, but the major bicarbonate output is from the crypt region, findings in line with the general theory that crypt cells have a secretory function whereas cells in the villi are mainly absorptive (65). on the basis of recent studies of intracellular ph (ph i ) in apical villus cells in situ, it has been suggested as an additional mucosal protective mechanism that an acidic phi facilitates basolateral uptake of base (hco 3 –), increasing intracellular neutralization (115). this may be an important defense mechanism for the cells in the villus tip covered by a thin and loosely adherent mucus gel. further evidence that would support the intracellular buffering mechanism is that ca ii is located mainly in the villi and not in the duodenal crypts (70). this suggests that h+ ions that enter the enterocyte can directly, together with hco 3 –, be converted into water and co 2 . concerning the deeper part of the villi and the crypt region secretion of bicarbonate probably plays a crucial role in the protection against the acid. central nervous influence of bicarbonate secretion intracerebroventricular infusion of the � 1 -adrenoceptor agonist phenylephrine has previously been shown to increase the duodenal secretory rate in rats (59). in that study the increase in secretion was abolished by intravenous pretreatment with the ganglion-blocking agent hexamethonium and by icv (but not iv) administration of the adrenoceptor antagonist prazosin. centrally elicited stimulation of the secretion has also been observed after administration of some neuropeptides, including trh (57), crh (55) and bombesin (56), and of some benzodiazepines (58). bilateral ligation of the vagal trunks at the sublaryngeal level inhibits the stimulation of duodenal (57) and pancreatic (116) secretion induced by trh given icv. truncal vagotomy alone also abolishes the responses to icv bombesin and icv or iv administration of benzodiazepines, but identical vagotomy does not affect the response to icv phenylephrine. we found that icv phenylephrine stimulated the duodenal bicarbonate secretion (fig. 7) (60). sectioning all nerves around the carotid arteries, in contrast to sympathetic chain ectomy alone or truncal vagotomy alone, markedly inhibited the duodenal secretory response to icv phenylephrine (fig. 8). differences between effects of 135 136 fig. 7. bicarbonate secretion increased significantly after administration of phenylephrine icv. neither pineal nor pituitary glandectomy inhibited the secretory response to icv phenylephrine. [from sjöblom et al. (60) with permission]. fig. 8. pretreatment with iv luzindole and section of both the vagal trunks and the sympathetic chains (at the sub-laryngeal level) significantly inhibited the duodenal bicarbonate secretion occurring in response to icv phenylephrine while cervical sympathectomy did not influence this response. [from sjöblom et al. (60) with permission]. truncal vagotomy alone and of extended peri-carotid nervectomy have been observed previosly in studies of duodenal distension-secretory interactions (35). these differences may reflect intercommunications between the vagal and sympathetic neural pathways at the cervical level (117) and the anatomical mixing of pathways (118). phenylephrine possibly mediates duodenal bicarbonate secretion by a different central mechanism than the other aforementioned neuropeptides and drugs. our results demonstrate that the melatonin receptor antagonist luzindole is a potent inhibitor of the duodenal secretory response to icv phenylephrine (60, 119). central nervous melatonin had no effect on the secretion. it was also established that the basal hco 3 – secretion in both pineal glandectomized and pituitary glandectomized animals was the same as that in untreated controls (fig. 8) (60). further, there were no differences between pineal and pituitary glandectomized rats and rats with these glands intact in respect to the secretory response to icv phenylephrine. exclusion of a role of pituitary hormones was further confirmed by the finding that iv infusion of neither crh, acth nor msh affected the duodenal hco 3 – secretion 137 fig. 9. when the duodenal mucosal bicarbonate secretion was stimulated with icv phenylephrine the total amount of melatonin in the luminal perfusate increased more than 10-fold. animals pretreated with luzindole and given phenylephrine icv did not increase their alkaline secretion, but released the same amount of melatonin from the proximal duodenum. t = experimental time. [from sjöblom et al. (119) with permission]. in intact animals. the release of melatonin from the duodenal mucosa into the luminal perfusate after icv administration of phenylephrine was investigated (119). compared to control animals, phenylephrine induced an approximately 10-fold intraluminal increase in the melatonin level (fig. 9). pretreatment with luzindole almost abolished the marked increase in bicarbonate secretion induced by icv phenylephrine, but did not inhibit the luminal release of melatonin. the blood concentration of melatonin showed a tendency to an increase in pineal glandectomized rats after icv infusion of phenylephrine compared with that in such animals infused icv with vehicle alone (60). the tendency did not attain statistical significance. the combined results strongly suggest that melatonin is released from the intestinal mucosa after icv stimulation with phenylephrine. fasting influence on secretion ever since pavlov presented his classical work at the end of the 19th century, most experimental studies of gastrointestinal physiology and pathophysiology in intact animals have been conducted after an overnight fasting period (120). the presence of food itself has considerable effects on intestinal functions (24). we therefore examined the question whether the fasting procedure per se influenced the duodenal alkaline secretory response to some secretagogues. it was established that feeding induced or very markedly potentiated the response of the duodenal hco 3 – secreting epithelium to some stimuli but not to others (121). the most pronounced difference was noted after administration of orexin-a. orex138 fig. 10. orexin-a (left) stimulates the duodenal mucosal bicarbonate secretion in fed animals but not in those fasted overnight. the hormone melatonin (right) is a stimulant of duodenal mucosal hco 3 – secretion. no significant differences between the fed and fasted animals were observed. [from flemström et al. (121) with permission]. 139 fig. 11. a model illustrating the proposed role of melatonin in the regulation of duodenal mucosal bicarbonate secretion. the intracerebroventricularly infused phenylephrine binds to � 1 -adrenoceptors in the hypothalamus. this activates the paraventricular nucleus, in the hypothalamus, which has substantial projections to the dorsal motor nucleus of the vagus nerve in the medulla oblongata. the vagal nerves, and cervical sympathetic fibers, then project to the enteric nervous system (ens). the activation of the myenteric plexa, via nicotinic receptors, directly or indirectly via the submucosal plexa, innervates enterochromaffin cells in the intestinal mucosa to release melatonin. the melatonin has paracrine secretory actions at adjacent duodenal enterocytes. melatonin also activates secretomotor neurons in the ens, also leading to bicarbonate secretion. binding of melatonin to the duodenal enterocytes increases intracellular calcium. the increase in calcium concentration activates the electroneutral hco 3 –/cl– exchanger. duodenal enterocytes intercommunicate with adjacent enterocytes to form a secretory functional syncytium. [from sjöblom et al (119) with permission]. ins (a and b) were originally discovered in the cns as peptides that increased the appetite for food in animals (122). both orexins are also found in neurons and in neuroendocrine cells of the intestine (123, 124), and orexin immunoreactivity is colocalized with vip and choline acetyltransferase (125). both ox 1 and ox 2 receptors are thus expressed throughout the intestine in different cell types (125). ox 1 receptors are expressed mainly in neurons, while ox 2 receptors are expressed mainly by endocrine cells. the roles played by orexins in the gastrointestinal tract are not well understood. these peptides have been reported both to increase (123) and to reduce (126) the motility in the small intestine. orexins thus probably act at several levels and some of their different actions are very probably mediated via other neurohumoral systems in the intestine. orexin-a caused a robust increase in the hco 3 – secretory rate in fed animals, but did not affect that in animals fasted overnight (fig. 10) (121). similarly, fasting reduced the secretory sensitivity to the muscarinic agonist bethanechol by a dosefactor of ~100. in contrast, the hco 3 – secretory responses to melatonin (fig. 10) and vip were not affected by overnight fasting. this demonstrates that feeding does not cause a general increase in the responsiveness of secretory peptides, but has a more selective action. the mechanisms by which feeding promotes responses to orexin and bethanechol are not clear. however, fasting may inhibit orexin and muscarinic responses by receptor desensitization or by changing the receptor density. enterocyte calcium signaling normally cells of various types keep their intracellular calcium concentration ([ca2+] i ) at a constant resting level (around 100 nm) (127). upon receptor stimulation, extracellular influx or the release of calcium from intracellular storages can increase the intracellular calcium concentration within a very short time. this activation is the first step that finally leads to cellular events. one of the goals in cellular physiology is to understand how intracellular signaling systems regulate different cellular processes. as in other cells and tissues, agonist-induced [ca2+] i signaling is probably of utmost importance in control of various aspects of enterocyte function, but very few studies of [ca2+] i signaling in enterocytes have been reported. small intestinal enterocytes in situ are programmed to a very restricted life span (2–5 days in rodents) (17). in addition, enterocytes in situ rapidly respond to irritating compounds in the intestinal lumen by apoptosis and expulsion. very probably reflecting these physiological characteristics, small intestinal enterocytes appear more difficult than, for instance, gastric parietal cells or pancreatic �-cells to maintain viable after isolation (128). the results demonstrate that clusters of freshly isolated enterocytes from the proximal small intestine can be kept viable, providing a suitable model for studies of agonist-induced [ca2+] i signaling (108). the viability of the enterocytes in clusters, as studied by trypan blue exclusion, was good (>85% after six hours). it may be compared with the viability (10% after 2–4 hours) reported in studies of intracellular ph in acutely isolated villus tips from rat duodenum (129). the findings show further, for the first time, that melatonin has a direct 140 141 action on duodenal epithelium (108). melatonin increases [ca2+] i in duodenal enterocytes from both rats and humans. low concentrations of melatonin, with ec50 17.0 ± 2.6 nm, and of agonists 2-iodomelatonin and 2-ibmt, increased enterocyte [ca2+] i . the receptor antagonists luzindole (mt 2 >mt 1 ) and dh97 (90-fold selectivity mt 2 >>mt 1 ; teh & sugden 1998) abolished the responses to melatonin. in the main type of melatonin-induced signaling pattern, [ca2+] i spiked rapidly and then slowly returned to baseline or almost baseline values (fig. 6). in a smaller number of cells, [ca2+] i tended to remain at a plateau level. the magnitude of the initial rise in [ca2+] i was dependent on the perfusate concentration of melatonin in some enterocytes. in other experiments, there was a rapid down-regulation of the response, similar to the desensitization observed with cck-8 in duodenal enterocytes in primary culture (130). interestingly, there is a dose-dependent increase in mucosal hco 3 – secretion as well as apparent desensitization of the response when melatonin is administered to rat duodenum in situ (60). the latter occurs during infusion of a relatively high dose (2000 nmol•kg-1•h-1) of the compound. the similarity suggest a role of [ca2+] i in mediating melatonin-induced stimulation of the secretion. perfusion with calcium-free solutions abolished the plateau phase but not the initial increase in [ca2+] i in rat duodenal enterocytes. a biphasic ca2+ response to agonists is characteristic of many non-excitable cell types and a substantial amount of evidence indicates that the initial spike in [ca2+] i is the result of release of ca2+ from an intracellular storage site(s), whereas the later sustained phase is due to the influx of ca2+ across the cell membrane. in duodenal enterocytes in primary culture, carbachol (acting at muscarinic m3 receptors) induced biphasic [ca2+] i responses (130), similar to those observed with melatonin. the sustained phase of the rise in [ca2+] i was, as found here with melatonin, attributable to extracellular ca2+. another interesting type of [ca2+] i response to melatonin was observed in the responding preparations. the initial transient increase in [ca2+] i was followed by slow rhythmic oscillations in [ca2+] i of high amplitude which spread throughout the cluster of enterocytes. oscillations (and spread of oscillations) were never observed in the absence of ca2+ in the perfusate, suggesting that influx of ca2+ contributes to the phenomenon. presence of extracellular ca2+ may also be important, however, in maintaining mucosal cell-to-cell communication. the melatonin-induced oscillations observed in clusters of rat as well as human duodenal enterocytes occurred with about the same frequency (~ one period in 5 min) (108). thus, there was no decline but rather a time-dependent gain in amplitude, and oscillations spread within the cell cluster. we used isolated clusters of enterocytes, a preparation that should be devoid of neural tissue (108). pretreatment with the muscarinic antagonist atropine did not affect the basal [ca2+] i or the response to melatonin, further excluding the possibility that melatonin might act at muscarinic receptors at the enterocyte cell membrane. cellular responses depend on the pattern and magnitude of [ca2+] i signaling (131) and as stated previously calcium is one of the major regulators of physiological functions. we have preliminary data that support our theory that the increase in calcium activates enterocyte stimulus-secretion coupling. in clusters of duodenal enterocytes melatonin affects intracellular ph, suggesting activation of enterocyte acid/base transport (sjöblom 2003, unpublished observations). the duodenal secretagogues dopamine, vip and prostaglandin e2 increase camp production (132, 133), and intracellular cgmp is involved in mediating the hco 3 – secretory responses to guanylin and heat stable enterotoxin (st a ) (64). interactions between these pathways and enterocyte [ca2+] i signaling would seem likely. ion transporters duodenal enterocytes export hco 3 – by an apical cl–/ hco 3 – exchanger as well as an anion conductive pathway, very probably the cftr channel. anion-channel dependent transport of hco 3 – may be a property of crypt cells, where the cftr channels are expressed at the greatest levels. the apical transporters in the villus cells, in contrast, constitute the electroneutral anion exchanger. the duodenal transmucosal electrical potential difference was measured in some experiments (60). the pd was recorded between the duodenal mucosa and the posterior vena cava with a high-input impedance voltmeter via matched calomel halfcells. the results demonstrate that melatonin stimulates duodenal mucosal transport of hco 3 – without a significant change in pd, indicating an electroneutral transport process. clinical relevance convincing evidence that melatonin stimulates hco 3 – secretion in the rat has been provided in this work. furthermore, centrally elicited stimulation induces duodenal luminal release of melatonin, most probably from the intestinal ec cells. in enterocytes, both of human and rat origin, melatonin increases intracellular calcium, suggesting that intestinal actions of the hormone may be similar in the two species. circadian rhythms in pain and discomfort are pathological features in gastroduodenal ulcer, and the incidence of gastroduodenal ulcer is reported to show peaks at certain periods of the year (134). melatonin is the major hormone regulating circadian rhythms. interestingly, there is a strong disturbance of melatonin secretion in both the exacerbation and in the remission stage of the disease in patients with duodenal ulcer (135). studies in fasting animals have shown that the gastric secretions of hco 3 – and mucus, both important in mucosal protection, exhibit day and night rhythms with peak times different from those of the mucosa-aggressive h+ secretion (136). this phase shift in secretory rhythms may, in theory, result in circadian variations in mucosal vulnerability to acid injury. conclusions the work presented in this study provides new and interesting knowledge about the central nervous as well as the peripheral regulation of the mucosa-protective bicar142 bonate secretion by the duodenal mucosa. the conclusions are based upon integrative animal experiments in vivo combined with in vitro experiments with tissues of human and rat origin. fig. 11 summarizes the proposed role of melatonin in the regulation of hco 3 – transport by the duodenal epithelium. the main findings are summarized as follows: o melatonin is a potent stimulant of duodenal mucosal bicarbonate secretion and seems to be involved in the acid stimulation of alkaline secretion. o endogenous melatonin is released from the duodenal mucosa after central nervous stimulation with the _1-adrenoceptor agonist phenylephrine and, furthermore, stimulates duodenal mucosal bicarbonate secretion. o intraarterial infusion close to the duodenum is more effective than intravenous infusion of duodenal secretagogues and also minimizes central nervous actions of infused drugs. o overnight fasting, a standard procedure in experimental studies of intestinal function, rapidly and profoundly downregulates the responses to the duodenal secretagogues orexin-a and bethanechol, but not to melatonin or vip. o a new method for isolating viable duodenal enterocytes was established. clusters consisting of 10–50 cells of 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seasonal periodicity in peptic ulcer disease. chronobiol int, 4: 91–99, 1987. 135. malinovskaya n, komarov fi, rapoport si, voznesenskaya la, wetterberg l. melatonin production in patients with duodenal ulcer. neuroendocrinol lett, 22: 109–117, 2001. 136. larsen kr, moore jg, dayton mt. circadian rhythms of acid and bicarbonate efflux in fasting rat stomach. am j physiol gastrointest liver physiol, 260: g610–g614, 1991. about the author markus sjöblom received the israel hwasser award from the uppsala medical association for the best dissertation in basic medicine in the academic year 2003/2004. he is at present carrying out his postdoctoral training at the division of physiology, department of neuroscience, uppsala university. corresponding author: markus sjöblom, ph.d. uppsala university division of physiology department of neuroscience p.o. box 572 751 23 uppsala, sweden e-mail: markus.sjoblom@fysiologi.uu.se 149 untitled multiple primary malignancies and lung cancer 193 key words: primary malignancy, multiple, lung cancer received 3 march 2008 accepted 13 march 2008 upsala j med sci 113 (2): 193–200, 2008 multiple primary malignancies involving lung: an analysis of 40 cases adnan yilmaz, muyesser ertugrul, leyla yagci tuncer, ebru sulu, ebru damadoglu sureyyapasa thoracic and cardiovascular diseases teaching and investigation hospital-istanbul abstract we aimed to assess the incidence of multiple primary malignancies in primary lung cancer patients. we retrospectively evaluated the clinical files of 1038 primary lung cancer patients diagnosed in 2004. forty patients (3.9 %) had multiple primary malignancies. there were 34 men (85 %) and 6 women (15 %). their mean age was 62.4 ± 8.6 years. while 35 cases were smokers, 5 cases were nonsmokers. tumour pathology of the lung was squamous cell carcinoma in 15 cases, adenocarcinoma in 10 cases, small cell carcinoma in 3 cases and non-small cell carcinoma in 12 cases. there were 2 primary tumours in 37 cases and 3 primary tumours in 3 cases. the first detected tumour was located in larynx in 11 cases, in genitourinary system in 9 cases, in intestine in 5 cases, in lung in 3 cases and in other organs in 12 cases. the mean interval between the first and the second tumour was 77 months with a range of 1 months to 32 years. this interval was shorter than 6 months in 4 cases. treatment modality for the first detected tumour was surgery in 35 cases. the last primary tumour was treated with surgery in 12 cases. in conclusion, the development of multiple primary tumours is not a rare phenomenon. patients with a malignancy should be followed for development of a second primary malignancy. the treatment of lung cancer in patients with a previous malignancy should be the same as for lung cancers presenting as the first cancer. introduction multiple primary malignancies are defined as the occurrence of two or more primary malignancies, where each cancer originates in a separate primary site and is neither an extension, recurrence or metastasis (1,2). in 1889 billroth, quoted by hui and associates (2), first described a patient in whom cancer of the stomach was found after the removal of an epithelioma of the external ear. in 1932 warren and gates identified 1259 verified cases of multiple malignancies either reported in the literature or encountered in their own postmortem examinations (3). while observations of multiple primary malignancies were previously considered isolated and exceptional cases, as a result of the improvement in the diagnostic tools, treatment modalities and supportive care, survival time for cancer patients has been prolonged and the number of multiple primary cancers has continued to grow (4). up to 10 % of cancer patients have been 194 adnan yilmaz et al. reported to acquire multiple primary cancers of separate organ sites in the 10 years following the diagnosis of their first cancer (5). lung cancer is one of the most common cancers worldwide. the risk of developing a second lung cancer in patients with non-small cell lung cancer is approximately 1 % to 2 % per patient per year. for small cell lung cancer, it is approximately 6 % per patient per year (6). it was reported that 193 patients with multiple primary cancers involving lung cancer were found among 22,405 cancer cases (7). in this study, we aimed to assess the incidence of multiple primary malignancies in primary lung cancer patients. materials and methods the present study was conducted at sureyyapasa thoracic and cardiovascular diseases training and investigation hospital, located in istanbul. the clinical files of 1038 primary lung cancer patients diagnosed in our center in 2004 were retrospectively evaluated to determine previous malignancies. information recorded at the time of developing last primary cancer included patient characteristics, histology and anatomic localization of the primary cancers, interval between the first and the second primary malignancy, and treatment modalities. multiple primary malignancies were defined as multiple autonomously originating malignancies in an individual patient. each tumour had to be clearly malignant histologically, each had to be geographically distinct, and the possibility that one tumour represented a metastasis had to be excluded (3). in cases of index tumour in lung, the criteria of martini and melamed (8) were used for the diagnosis of second primary cancer. all last tumours had been staged according to tnm staging system (9). the index tumour was defined as the first detected tumour. synchronous primaries include any second malignancy occurring within 6 months of the diagnosis of the index tumour and metachronous primaries are diagnosed after 6 months. results of the 1038 patients with primary lung cancer, 40 (3.9 %) patients had multiple primary malignancies. there were 34 men (85 %) and 6 women (15 %). their mean age at the time of diagnosis of the last tumour was 62.4 ± 8.6 years. while 35 cases were smokers, 5 cases were nonsmokers. the incidence of the patients with multiple primary malignancies was 4.8 % (35/724) among smokers and was 7.7 % (5/65) among nonsmokers (p>0.05). four patients had history of alcohol and 6 patients had positive family history of malignancy. thirty-seven patients (3.6 %) had double primary malignancies and 3 (0.3 %) had triple primary malignancies. the first and the second tumours were synchronous in 4 patients. results are summarized in table 1. index tumour was located in larynx in 11 patients, in genitourinary system in 9 multiple primary malignancies and lung cancer 195 patients, in intestine in 5 patients, in lung in 3 cases and in other organs in 12 cases. while the index primary tumour was treated with surgery in 35 patients, treatment modality was radiotherapy and/or chemotherapy in 5 patients. the mean interval between the first and the second tumour was 77 months with a range of 1 months to 32 years. this interval was shorter than 6 months in 4 cases. the interval between the second and the third tumour in three patients with the triple tumours were 5, 24 and 72 months (table 2 and table 3). the last primary tumour was lung cancer in all patients. tumour type of the lung was squamous cell carcinoma in 15 cases, adenocarcinoma in 10 cases, small cell carcinoma in 3 cases and non-small cell carcinoma in 12 cases. among patients with non-small cell carcinoma, the stage was 6 stage ia, 7 stage ib, 3 stage iib, 3 stage iiia, 12 stage iiib, and 6 stage iv. all patients with stage ia were treated with surgical resection. while 4 patients with stage ib were treated with surgery, 1 patient rejected surgery. there were 2 medically inoperable patients in this group. while 1 patient with stage iib was subjected to surgical resection, 2 patients rejected surgery. among patients with stage iiia, 1 patient was treated with surgery. one patient rejected surgical treatment. because the other had multiple n2 disease, he was treated with radiotherapy. there were 6 patients with stage iv in this series. they were given chemotherapy. discussion the incidence of multiple primary malignancies has increased in recent decades (7,10). the american cancer society, quoted by mydlo and associates (11), has reported that one out of 5 americans will develop cancer in his or her lifetime. furthermore, there is one out of three chances of developing a synchronous, antecedent or subsequent tumour in these patients’ lifetime. according to two previous reports, the incidence of multiple primary malignancies has ranged from 0.4 % to 11.8 % (2,12). this incidence was 2.4 % in buiatti’s report (13), was 2.5 % in cheng’s series (14) and was 11 % in brock’s study (15). in our series, 3.9 % of the patients with primary lung cancer had multiple primary table 1. distribution of multiple primary malignancies patients n % with lung cancer reviewed 1038 100 with multiple primary malignancies 40 3.9 with double primary malignancies 37 3.6 with triple primary malignancies 3 0.3 with synchronous multiple primary malignancies 4 0.4 with metachronous multiple primary malignancies 36 3.5 196 adnan yilmaz et al. table 2. features of the patients with double primary malignancies case no age (years) sex index tumour treatment of index tumour interval cell type of last tumour 1 60 m colon surgery 6 years squamous 2 72 f kidney surgery 4 years adeno 3 58 m larynx surgery 26 years non-small 4 63 m hodgkin rt* and ct** 5 years squamous 5 49 m pancreas surgery and rt 5 years non-small 6 68 m larynx surgery 2 years adeno 7 54 m larynx surgery 18 months squamous 8 66 f breast surgery and ct 5 months adeno 9 72 m colon surgery 6 years adeno 10 71 m prostate ct 5 years non-small 11 65 m larynx surgery 17 years small cell 12 54 m lung surgery 30 months squamous 13 62 f uterus surgery 10 months non-small 14 61 m lip surgery 1 year squamous 15 75 m thyroid surgery 9 years squamous 16 40 f breast surgery 4 years small cell 17 77 m larynx surgery 7 years non-small 18 62 m bladder surgery 23 months small cell 19 50 f colon surgery 11 years adeno 20 69 m skin surgery 9 years non-small 21 66 m lung surgery 8 months squamous 22 51 m larynx surgery 4 years squamous 23 49 m small bowel surgery 25 months non-small 24 64 m bladder surgery 1 months non-small 25 66 m lung surgery 20 months adeno 26 77 m testicular surgery and rt 32 years adeno 27 55 m rectum surgery 12 years squamous 28 62 m larynx surgery 35 months squamous 29 69 m bladder surgery 14 years adeno 30 67 m larynx surgery 10 years non-small 31 64 m prostate ct 5 years non-small 32 59 f uterus ct 1 months squamous 33 59 m larynx rt and ct 1 months non-small 34 56 m muscle surgery and ct 25 months squamous 35 72 m parotid surgery 2 years non-small 36 62 m lip surgery 13 years squamous 37 60 m larynx surgery 17 months squamous *rt: radiotherapy **ct: chemotherapy multiple primary malignancies and lung cancer 197 malignancies. in the present series, the last tumour was lung cancer in all patients. utsumi et al (12) reported that 37 of 313 primary lung cancer patients had a history of previous malignancy. hui et al (2) found that there were multiple primary malignancies in 2.1 % of the patients. in their series, apart from the 5 patients with simultaneous tumour, lung cancer was the index tumour in 8 patients and the second tumour was in 8 patients. it was reported that a total of 193 patients with multiple primary cancers involving lung cancer were detected among 22,405 cancer patients. of these 193 patients, 51 had lung cancer diagnosed before the occurrence of the other cancers and the remaining 142 had other cancers occurring ahead of the lung cancer (7). index tumour was lung cancer in three patients in our series. in this series, the most frequent index tumour was larynx carcinoma, followed by malignancy of genitourinary and digestive systems. laryngeal index tumours have the highest percentage of pulmonary second primaries (16,17). jones et al (14) reported that 47 per cent of 110 laryngeal index tumours have second primaries in lung. there were 37 lung cancer patients with a history of previous malignancy in a previous report. the previous malignancies included 13 gastric cancers and 6 colorectal cancers (12). according to a previous report, the mean interval between the first and the second tumour was 6 years and 8 months with a range of 2–20 years. the interval between the second and the third tumour in 2 patients with the triple tumours were 2 and 6 months (2). in our series, the mean interval between the first and the second tumour was 77 months with a range of 1 month to 32 years. the interval between the second and the third tumour in 3 patients with the triple tumours were 5, 24 and 72 months. the development of multiple primary malignancies may be associated with several factors such as genetic factors, hormones, environmental carcinogens, dietary factors, previous therapy, alcohol and smoking (7,11,12,14,18). liu et al (10) pointed out that smoker patients had a significantly higher risk for the development of multiple primary malignancies involving lung cancer. two previous reports supported that there was a causal association between cigarette smoking and cancer of table 3. features of the patients with triple primary malignancies case no age (years) sex index tumour treatment of index tumour interval second tumour treatment of second tumour interval third tumour cell type of last tumour 38 59 m left cord vocal surgery 42 months right cord vocal surgery 5 months lung adeno 39 77 m larynx surgery 18 years penis surgery and rt* 2 years lung squamous 40 58 m thyroid surgery 8 years bone surgery and ct** 6 years lung adeno * rt: radiotherapy **ct: chemotherapy 198 adnan yilmaz et al. the aerodigestive system, lungs, stomach, liver, kidney, uterine cervix, and bladder (19,20). oral cavity, orohypopharynx and larynx were locations related to smoking and alcohol (18). in our study, most of the index tumours were tumours related to smoking. it was showed that risk of lung cancer was significantly increased in patients treated for hodgkin’s lymphoma and breast cancer (21,22). the risk of developing a second lung cancer in patients who survived resection of a non-small cell lung cancer is approximately 1 % to 2 % per patient per year (6). it is known that genetic factors play an important role in the development of multiple primary malignancies (11,12,15,23). in our series, there were 35 smoker patients. four of 35 patients also drunk alcohol. six patients had a family history of malignancy. one of 6 patients was nonsmoker. among nonsmoker patients, one patient received chemotherapy for breast cancer and one patient was treated with radiotherapy for uterine cancer. treatment of lung cancer in patients with previous malignancies should be the same as for lung cancer presenting as the first cancer (12). surgery should always be the treatment of choice in these patients if the tumour is 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registries. lancet oncol 6: 557–65. heard a, roder d, luke c (2005). multiple primary cancers of separate organ sites: implications 23. for research and cancer control (australia). cancer causes control 16: 475–81. ferguson mk, demeester tr, deslauriers j, little ag, piraux m, golomb h (1985). diagnosis 24. and management of synchronous lung cancers. j thorac cardiovasc surg 89: 378–85. mathisen dj, jensik rj, faber lp, kittle cf (1984). survival following resection for second and 25. third primary lung cancer. j thorac cardiovasc surg 88: 502–10 corresponding author: associate professor adnan yilmaz maltepe zumrutevler atatürk cad. abant apt. no: 30 istanbul/turkey phone: + 90 216 3058324 email:adnandr_63@yahoo.co.uk (7) alina rodrigues 73-86 upsala j med sci 111 (1): 73–86, 2006 parents’ perspectives on research involving children alina rodriguez, ph.d1.1torsten tuvemo, m.d., ph.d2, mats g. hansson, th.d3. 1dept. of psychology, uppsala university, 2dept. of women’s and children's health, uppsala university, 3centre for bioethics at karolinska institutet and uppsala university abstract children's participation in research is essential for the development of safe and ageappropriate treatments. however, children's participation is limited. the aim of this study was to determine (1) mothers' and fathers' views on which agencies/persons should evaluate the level of acceptable risk for children and (2) parents' willingness to allow children to participate in research. medical factors, sociodemographics, and research attitudes were related to willingness. the study used a cross-sectional and longitudinal design with 863 expectant parents (435 women; 428 men) consecutively recruited at gestational week 19 during routine ultrasound examination at 2 hospitals in uppsala county, sweden. 123 women at gestational week 34 were followed-up. parental ratings of agencies/persons' degree of involvement in risk-evaluation for child research participants and parents' willingness to allow children to participate in research were the main outcome measures. most parents believed that more pediatric research was needed. attitudes played a major role in willingness, indicating a potential for information that could modify willingness. over 80% of mothers and fathers rated the attending physician as needing to be "fully involved" in risk evaluation for research participants. parents’ views contradict current trends in research ethics which place evaluation of risk in the hands of regional agencies. instead, the majority of parents would like the decision to be individually based on the attending physicians advise. we conclude that children's participation in research could be improved by actively incorporating the attending physician and by educating the public so that research attitudes can be based on accurate information. 73 received 17 october 2005 accepted 27 october 2005 introduction medical research is the cornerstone for developing safe and effective treatments. the price for the patient is the risk incurred while serving as a research participant. systematic exclusion of children in research unjustly denies them access to benefits of research participation, i.e. evidence-based medicine. only a subgroup of children receives treatments approved for pediatric use (1-3). prevalence of adverse drug reactions to off-label medication are high (4), because doses are estimated by body weight (5-7) under the assumption that children are small adults. however, pediatric pharmachology shows that children react differently than adults to medicines (8-10) and pain management (11). policy statements urging the inclusion of children in research have been published around the globe by major research groups and professional bodies (12). however, there are a number of barriers to research participation, which result in smaller sample sizes and reduced statistical power (13). the international trend is to allocate the decision-making process regarding both research and clinical ethics to external institutional review boards (scientists plus community members) (14,15). however, the parental perspective is lacking. knowledge about parents’ attitudes and willingness to allow children to participate in research can help us guide information to parents. if research is acceptable to parents, then we can expect that childrens' participation will be more likely. individual differences among parents may be important determinants of willingness. for example, socially disadvantaged parents have been more likely to volunteer their children in some studies (16,17), but not in others (18,19). parents with less education have been found to be more negative to research than college educated parents (20). despite some recognition that parents’ attitudes are relevant for their willingness to enroll their child in research, there is a paucity of prospective data adequately addressing this issue in large representative samples. studies addressing parental attitudes are limited by small sample sizes (21,22), often reliance on responses from one parent or do not provide information separately for mothers and fathers (23), and lack in generalizability because refusal and attrition rates are unknown (20,24,25). parental attitudes gathered retrospectively may be unduly biased by the outcome of the research project. it is likely that if parents’ were happy with the outcome, their attitudes about research will be more positive or more negative if they are unhappy. generalizability is also limited when only parents' of a sick child are studied. parents' of sick children may be amidst a medical crisis. thus, their attitudes may not generalize to parents of healthy children, e.g. whose participation is necessary in screening studies. preterm birth on most occasions is unexpected and linked to medical emergencies. thus, it is especially important to learn expectant parents' views, because they may suddenly need to deliberate over enrolling their preterm baby in research. in order to increase generalizability of our results, we solicited expectant parents because they are involved in repeated contacts with medical professionals and have heightened awareness of health risks, but are not involved in a medical crisis. a subgroup of these parents will have to decide in the impending months whether to enroll their infant in research. we solicited both nulliparous and multiparous participants so that the results 74 could be generalized to both parents expecting their first child and to those already having children. the objectives were to study mothers' and fathers' (1) views on which agencies/persons should evaluate the level of acceptable risk to children in research and (2) willingness to allow children to participate in research. we investigate in a large representative sample whether willingness to allow children to be research participants would vary depending on research attitudes, sociodemographic variables, parental age, medical factors (current medical risk status and previous experience with medical care), and current perceived risk. methods participants a total of 863 expectant parents (435 women and 428 men) were consecutively recruited at 19 weeks of gestation (m=19.00, sd=2.37) in conjunction with routine ultrasound examination at the 2 hospitals in uppsala county, sweden during july 2001 to march 2002. the university hospital serves a population of 300 000 local residents and a region of 1.3 million for referred cases and a smaller hospital was located in a neighboring city. all deliveries were scheduled at the university hospital. prenatal health care including ultrasound examination is free of charge in sweden and is used by nearly 100% of the population (26), thus there are no differences among women attending the hospitals in terms of socioeconomic status. of the 551 couples that were approached 79% of the pregnant women and 78% of their partners agreed to participate. less than 1% (0.60%) attended specialized prenatal care due to high-risk status. because the frequency of women experiencing a high-risk pregnancy was low at midpregnancy, we administered a second questionnaire to a subset of the sample. follow-up participants were consecutively chosen from pool of those indicating interest in further participation (79%). we invited 140 women by mail to participate and 123 completed the follow-up questionnaire (88%). all questionnaires were completed by the participants in their home and returned to the researchers in self-addressed stamped envelope. the inclusion criterion was the ability to understand written swedish. the sample consisted mostly of swedes while 4% of the women and 5% of the men were of non-scandinavian origin. all aspects of this study received ethical approval. measures participants rated to what extent agencies/persons should evaluate the level of acceptable risk to children in research. the list consisted of: pharmaceutical industries, ethics committees (including researchers and community members), institutional review boards (irb) including only researchers, elected hospital administrators, elected national government representatives, the national board of health and 75 welfare, researchers involved in the particular study, attending physician, both parents, mother, father, child, and "other," which the participant specified. involvement by each person or agency was rated on a 4-point scale ranging from should not be involved to fully involved. attitudes concerning research participation consisted of 11 items rated on a 5point scale developed in part on previous research (23,27) (items listed in table 3). willingness to allow children to participate in research was tapped by 9 items (listed in table 4) based in part on conditions associated with randomized trials (28-30). two items (bottom of table 4) were rated on a 5-point scale which allowed for uncertainty whereas the rest of the items were answered on a 4-point scale (ranging from very unwilling to very willing) to reflect the real life situation in which parents have to take a stance. we also asked whether approval of both parents was deemed necessary before a child could participate in research. questionnaires were tested in a pilot sample of expectant parents and proved easy to understand and useful. psychometric properties were excellent in the research sample including internal consistency for willingness to participate in research, cronbach a ≥ .75. sociodemographic characteristics among parents included age, educational attainment, income, and subjective social status. the macarthur scale of subjective social status with an easy pictorial format (10-rung "social ladder") taps participants’ perceived social standing by placing an "x" on the rung which they feel they stand in comparison to others in terms of education, income, and profession. subjective social status has been found to be associated to health outcomes more strongly than objective socioeconomic measures (31). we assessed whether participants had previous experience with medical care for a serious condition. perceived risk status was assessed by two items (5-point scale) measuring anticipated need of medical care for the newborn. medical risk was defined as ultrasound examinations showing deviations from normality and receiving information from medical professionals concerning elevated medical risk. additionally, at week 34, absence from work due to disability was included. statistical analyses descriptive statistics are presented for the two outcomes: agencies/persons who should evaluate the risk in research and willingness to allow children to participate in research. changes over time in women’s opinions and differences between partners were evaluated with paired t-tests. associations between predictors and outcomes were analyzed using correlations and multiple regression analyses. results participant characteristics are shown in table 1. family structure consisted of two biological parents for the overwhelming majority. participants were of mainly middle 76 77 class. approximately half of the women were multiparous. only 1% of the fetuses were considered to be at high risk for medical complications at mid-pregnancy, but was significantly higher during late pregnancy. parents' on average perceived their fetuses as having only slight risk of needing medical care at delivery. table 2 presents the percentages of parents' endorsements of agencies/persons they believe should evaluate the level of risk for children in research. the majority of participants would like the attending physician to evaluate whether the amount of risk is acceptable or not. 'both parents' and 'mother' were also ranked highly. less than 10% of participants rated "other," and in most cases parents did not specify. table 1 participant characteristics in means (standard deviation) or percentages where applicable men women (gestational week 19) women (gestational week 34) total sample % cohabitating 98.6% unplanned pregnancy 9% 9% parity 49% nulliparous age, yrs 31.9 (5.3) 29.5 (4.6) yrs post-secondary education 2.6 (2.9) 2.3 (2.4) income per month 2556 € (939) 1998 € (585) perceived social status1 6.3 (1.4) 5.8 (1.3) previous experience w medical care 27% 30% perceived risk status2 2.2 (0.5) 2.3 (0.6) 2.3 (0.6) elevated medical risk to fetus 1% 16.5% elevated medical risk to mother 17% 19.7% number of disability days 25 (44) 1 possible range 1-10, where 10 equals highest social status 2 1 = no risk, 2 = slight risk, 3 = some risks, 4 = rather large risk, 5 = very high risk of medical complications at birth for the newborn. frequencies in % agency/ person mothers fathers uninvolved or slightly involved very or fully involved uninvolved or slightly involved very or fully involved pharmaceutical industries 89 11 92 8 irb (researchers + community mem.) 35 65 36 64 irb (researchers only) 46 54 46 54 elected hospital administrators 95 5 93 7 government representatives 93 7 92 7 national board of health & welfare 57 43 66 34 researchers involved in the project 42 58 47 53 attending physician 2 98 3 97 both parents 11 89 12 88 mothers 12 88 13 87 fathers 37 63 33 67 children 12 88 22 78 frequencies of endorsements in percent for attitude items* table 1. participant characteristics in means (standard deviation) or percentages where applicable table 2. frequencies of parent's endorsements of agencies/persons should evaluate the level of risk children may be exposed to in research table 3 shows the frequency of endorsements for each attitude collapsed into three categories. men and women were very similar in their attitudes. women did not change 78 table 3 frequencies of parent's attititude endorsements disagree slightly agree agree doctors opinions of treatments differ mothers gestational week 19 8 39 53 mothers gestational week 34 9 38 53 fathers 10 37 53 more research is needed in pediatrics mothers gestational week 19 2 16 82 mothers gestational week 34 3 14 83 fathers 5 17 78 not all side-effects are known mothers gestational week 19 16 33 51 mothers gestational week 34 3 13 84 fathers 21 33 46 most treatments have been scientifically tested mothers gestational week 19 9 24 67 mothers gestational week 34 18 28 54 fathers 11 17 72 only scientifically tested treatments should be given mothers gestational week 19 7 16 77 mothers gestational week 34 3 9 89 fathers 7 18 75 it is acceptable to give adult treatments to neonates mothers gestational week 19 49 33 18 mothers gestational week 34 26 38 36 fathers 51 32 17 research participation increases the chances of successful treatment mothers gestational week 19 26 37 37 mothers gestational week 34 22 39 39 fathers 28 35 37 research participation decreases quality of care mothers gestational week 19 91 7 2 mothers gestational week 34 50 40 10 fathers 89 9 2 research participants receive better care mothers gestational week 19 22 38 40 mothers gestational week 34 19 46 35 fathers 21 33 46 research participation is a given at a univ. hosp mothers gestational week 19 40 30 30 mothers gestational week 34 46 32 22 fathers 47 31 22 all research is approved by an ethics committee mothers gestational week 19 9 24 67 mothers gestational week 34 3 12 85 fathers 12 20 68 *participants rated the extent of their agreement with each attitude statement as follows: 1=do not agree; 2=practically do not agree; 3=slightly agree, 4=mostly agree, 5=completely agree table 3. frequencies of parent's attititude endorsements 79 table 4 frequencies of parent's willingness to allow children to be research participants willingness to… mothers gestational week 19 mothers gestational week 34 fathers 1. allow my child to participate in research that may benefit my child's health. unwilling 16 12 18 willing 84 88 82 2. allow my child to participate in research that may benefit other children's health, but not necessarily my own child's health. unwilling 45 39 45 willing 55 61 55 3. try a new and untested treatment, which lacks information on side-effects, when routine treatments do not have the desired effect. unwilling 49 52 52 willing 51 48 48 4. accept participation in research during pregnancy concerning care of newborns. unwilling 76 47 69 willing 24 53 31 5. allow healthy children to participate in clinical research, which can involve small risks for themselves, but which may benefit other children's health. unwilling 60 60 57 willing 40 40 43 6. allow sick children to participate in clinical research, which can involve small risks for themselves, but which may benefit for other children's health. unwilling 43 44 37 willing 57 56 63 7. allow sick children to participate in clinical research, which can involve small risks for themselves, but may benefit for their own health. unwilling 15 15 13 willing 85 85 87 willingness to receive… unwilling unsure willing 8. information about ongoing research concerning treatment alternatives. mothers gestational week 19 3 10 87 mothers gestational week 34 1 9 90 fathers 6 17 77 9. an invitation to participate in a research project that is relevant for my child's treatment. mothers gestational week 19 6 16 78 mothers gestational week 34 4 16 80 fathers 7 25 68 table 4. frequencies of parent's willingness to allow children to be research participants over time, with the exception of an increased proportion at gestational week 34 who ‘slightly agreed’ with the statement that research participation decreases the quality of care. the majority of parents held strong attitudes concerning the need for more pediatric research and the use of only scientifically approved treatments in pediatrics. willingness to allow children to participate in research was normally distributed for both men and women. parity was non significant (t 416 =-.21, ns), thus results are not presented separately by parity. table 4 shows that parents are overwhelmingly willing to receive information and an invitation to clinical research on behalf of their child. results of paired t-tests showed that women were clearly more willing to receive information (t417=3.62, p<.001) and an invitation to clinical research for their children than their part80 table 5 results of stepwise multiple regression analyses for factors predicting willingnes allow own child to participate in research f p< r2 total men only scientifically tested treatments should be given 15.93 .0001 .07 research participation is a given at a univ. hosp 10.49 .001 .11 research participation decreases quality of care 6.23 .01 .13 education 5.46 .02 .15 women (week 19) research participation is a given at a univ. hosp 31.29 .0001 .10 research participation decreases quality of care 24.18 .0001 .17 education 14.42 .001 .21 only scientifically tested treatments should be given 7.25 .001 .23 subjective social status 6.37 .01 .25 research participation increases the chances of successful treatment 5.94 .01 .27 all research is approved by an ethics committee 4.17 .04 .28 more research is needed in pediatrics 3.61 .05 .29 women (week 34) research participation is a given at a univ. hosp 32.24 .0001 .40 only scientifically tested treatments should be given 6.35 .02 .46 risk of complications for newborn 4.86 .03 .48 total number of days on disability 4.78 .03 .57 education 4.55 .04 .66 presently on disability 4.05 .05 .70 table 5. results of stepwise multiple regression analyses for factors predicting willingness to allow own child to participate in research ners (t 414 =3.05, p<.01). as delivery approached, women tended to become even more positive, but the differences were not statistically significant. table 4 indicates parents were more willing to allow their own children than children in general to participate in research. more than half the parents were willing to volunteer their own child to a research project for the benefit of other children. during mid-pregnancy few parents were willing to enroll their infant in a research project. however, a shift in opinion occurred by gestational week 34 when women were more willing to enroll their expectant infants in research (t 120 =3.35, p<.001). to evaluate which factors predicted willingness, we ran stepwise multiple regression analyses. the predictors were attitude questions, socioeconomic indicators, parental age, previous experience with medical care, medical risk indicators, and perceived risk. we predicted willingness to allow their own child to participate in research (items 1-4 and 89 on table 4). the top of table 5 shows that the same attitudes accounted men’s and women’s willingness to allow their child to participate in research, although their importance differed by gender: (1) only scientifically tested treatments should be given, (2) research participation is a given at a university hospital, and (3) research participation decreases the quality of care (negatively related). education also contributed to willing81 table 6 results of stepwise multiple regression analyses for factors predicting willingness to allow children in general to participate in researc f p< r2 total men not all side-effects are known 26.35 .0001 .10 research participation is a given at a univ. hosp 19.83 .0001 .17 only scientifically tested treatments should be given 4.09 .04 .19 research participation decreases quality of care 4.41 .04 .21 women (week 19) only scientifically tested treatments should be given 12.76 .001 .04 research participation is a given at a univ. hosp 11.58 .001 .08 subjective social status 5.12 .02 .10 not all side-effects are known 4.88 .03 .11 women (week 34) only receive approved treatments 6.20 .01 .06 table 6. results of stepwise multiple regression analyses for factors predicting willingness to allow children in general to participate in research ness. more educated parents were more positive to research participation. the bottom of table 5 shows the longitudinal prediction for women who participated in the follow-up. a large portion of variance was explained for this group (70%). the attitude that research participation decreases the quality of care was no longer important, instead, variables pertaining to medical risks or complications during pregnancy explained willingness. table 6 shows results of multiple regression analyses for willingness to allow children in general to participate in research. the same variables were entered into the equation, but few were significant and a much smaller portion of variance could be explained. approval by both parents to allow a child to participate in research was deemed necessary by 98.0% of men, 98.8% of women at week 19, and 99.1% of women at week 34. discussion among studies evaluating ethical issues in pediatrics, this is the largest prospective investigation of both parents' perspectives. the overwhelming majority of parents (women and men alike) would like the attending physician to evaluate the acceptable level of risk, which indicates that parents trust the doctor's expert advise (even more so than their own judgment). both men and women judge mothers alone as highly as both parents together in evaluating level of acceptable risk. results were not specific to parity, i.e. whether participants were first-time parents or already had children. ethicists and policy makers contend that there is a real risk that ethics will lose its critical function if too much power is placed in the hands of the doctor (32). our study indicates the contrary; procedures for ethical decision-making in clinical research involving children should be developed that are more sensitive to the importance of the trust relationships between doctors and parents. from a parental perspective, clinical research ethics should primarily be a concern for the attending physician and the parents, who are able to consider the unique individual circumstances, instead of far-removed public agencies (e.g. government officials). when it comes to their own child and in real life situations parents seem to be in favour of a context-sensitive model of ethics where the distance between the actual case and its ethical deliberation is kept to a minimum (33). including the attending physician makes sense as pediatric expertise is often lacking in irbs and as there are presently no criteria for judging whether the risks of research are reasonable (12). differences in parents' willingness to allow children to participate in research were explained largely by attitudes and to a lesser extent by level of education, rather than socioeconomic status or perceived medical risk. the attitude that participation is a given when attending a university hospital was particularly relevant for both men and women. this is a rather general attitude that is not specific to pediatrics or the expected outcome of research. the number of women who had high-risk pregnancies increased by week 34. indicators of medical risk at this point also predicted willingness, so that the higher the medical risk, the greater the willingness to allow one's child to participate in research. 82 these factors together explained 70% of the variance. it may be that the benefits of research may be perceived as greater for the individual as medical risk increases. willingness to allow children in general was more difficult to predict. only a small portion of the variance could be explained, but attitudes were again significant. these results suggest that parents are willing to take a stance regarding their own children, but are more hesitant when it comes to others. future research should therefore address parents' views about their own children. nearly 78% of men and 82% of women in our sample agree with the statement that more research in pediatrics is needed and over 80% are willing to allow their child to become research participants. however, approximately 50% of parents are unwilling to try a new treatment and only a small minority realizes that most treatments have not been studied in pediatric populations. our results show an inconsistency in parental attitudes to pediatric research and their knowledge about the conditions of pediatric treatment. participation in research may increase if accurate information about medical treatment for children is provided. we found that parents hold altruistic attitudes, which is in line with other research findings (34-37) thus, scientists need not view pediatric research to be “in crisis” due to dwindling participation (38), instead, pediatric researchers should handle parents’ trust in medical professionals with care and take the opportunity to educate the public. previous research has shown that parents have poor understanding of the elements of informed consent even after volunteering their children as research participants, which clearly opposes the intent of the declaration of helsinki (39). our research points to the opportunity of educating parents about research practices while awaiting the birth of their child as we found that parents are willing to receive such information. the context of receiving prenatal care, with repeated contacts with healthcare professionals with whom patients have build a relationship based on trust, makes for an ideal situation. a potential limitation of the study design is that we did not obtain a behavioural measure of willingness, i.e. we do not know to what extent willingness translates into behaviour. naturally, the specifics of each research project will be decisive for parents' permission to be granted. however, our focus on willingness provides important information regarding how open parents are to research projects in general. previous research shows that those initially positive to research are likely to enroll their children (36). parents were not necessarily willing to enroll their child already during mid-pregnancy, but they were willing to learn about research. our finding is particularly important because preterm birth is difficult to predict especially for nulliparous women, information received during pregnancy may help alleviate their distress at the time of preterm delivery. the finding that attitudes, rather than socioeconomic status or perceived risk, played a major role in willingness is encouraging. modifying parents' attitudes through information at prenatal health care clinics and via media, for example, may serve as a catalyst for greater involvement in research. in conclusion, a partnership between parents and researchers is needed to design studies that will be acceptable for parents. this study showed that parents´ views contradict current medical trends which place evaluation of risk in the hands of regional agencies (11), instead, most parents would like this decision to be individually based on the attend83 ing physician’s advise. in other words, parents would like to have individualized decisions for children rather than a formula that could be applied to many. thus, future recommendations should include not only regional overseeing agencies which are important for multi center studies, but also include the provision for local members such as the attending physician and parents to evaluate risk for children. however, this does not mean that parents are willing to relinquish their right to consent to the attending physician, as nearly all participants reported that consent from both parents is necessary. a clear majority of parents in this study felt that more pediatric research was needed and were willing to allow their child to participate. greater participation by children could be achieved through attitudes based on accurate information on the conditions of pediatric treatment and based on responsible research practices, which would ultimately lead to safer medications in pediatrics. acknowledgements this work was funded by the foundation for health care sciences and allergy research grant nr. e2001 046 and an endowment from barnens hus. this research has been carried out within the framework of the research program ethics in biomedicine at uppsala university. gustaf arrhenius, uwe ewald and leif olsen have contributed to valuable discussions. references 1. collier j, (1999) paediatric prescribing: using unlicensed drugs and medicines outside their licensed indications. br j clin pharmacol 48:5-8. 2. rudolf mc, lyth n, bundle a, rowland g, kelly a, bosson s, et al. 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(part i): parental consent for children participating in clinical anesthesia and surgery research. anesthesiology 98:603-8. 40. snowdon, c, garcia, jo, elbourne d (1997) making sense of randomisation; responses of parents of critically ill babies to random allocation of treatment in a clinical trial, soc.sci.med. 45:1337-55 85 corresponding author: alina rodriguez, ph.d. department of psychology uppsala university box 1225, se-751 42 uppsala, sweden fax: +46 18 471 2123, tel.: +46 18 471 7980 alina.rodriguez@psyk.uu.se 86 ujms 110 (3) bra upsala j med sci 110 (3): 217–231, 2005 acute glomerulonephritis associated with streptococcus pyogenes with concomitant spread of streptococcus constellatus in four rural families almroth g1, lindell å1 , åselius h1, sörén l2, svensson l3, hultman p4, eribe erk5, olsen i5. 1departments of nephrology and 2clinical microbiology, university hospital of linköping, 3department of internal medicine, hospital of eksjö, 4department of molecular and immunological pathology, linköping university, sweden and 5department of oral biology, dental faculty, university of oslo, norway. abstract we studied history, renal histopathology and microbiology of an epidemic of acute glomerulonephritis associated with throat infections and uncommon culture results in four neighbour families. a 40-year-old man (index patient) was referred to a university hospital for dialysis and kidney biopsy due to a suspected acute glomerulonephritis. an acute tonsillitis had preceded the condition. penicillin treatment had been started four days before the discovery of renal failure. throat swabs were positive for �-hemolytic streptococci, group c (gcs). gcs were also found in throat cultures from his wife and two of their children. the bacteria were typed as streptococcus constellatus. a third child had s. constellatus expressing lancefield antigen group g. a neighbour and two of his children fell ill the following week with renal involvement. throat swabs from both these children were positive for s. constellatus. his third child had erythema multiforme and s. constellatus in the throat while a fourth child had �hemolytic streptococci group a; streptococcus pyogenes. kidney biopsies on the index patient and his neighbour showed an acute diffuse prolipherative glomerulonephritis compatible with acute post-streptococcal nephritis and microbiological analysis of renal tissue revealed in both cases s. pyogenes and s. constellatus. the received 23 september 2004 accepted 28 february 2005 key words: glomerulonephritis, s. pyogenes, streptococci group c, streptococci group g, s. constellatus families had had much contact and had consumed unpasteurized milk from our index patient’s farm. in four of seven persons in two additional neighbouring families s. constellatus was found in throat swabs during the same month while two persons carried streptococcus anginosus expressing the lancefield c antigen. in conclusion spread of s. constellatus coincided with the occurrence of four cases of acute glomerulonephritis. the two biopsied patients had both s. pyogenes and s. constellatus present in renal tissue. the epidemic either suggested that the outbreak of glomerulonephritis was due to s. pyogenes but coincided with the transmission and colonization of s. constellatus or that the s. constellatus strains were highly pathogenic or nephritogenic and that this organism can be transmitted in such cases. introduction streptococcus pyogenes or �-hemolytic streptococci group a (gas) is recognized as the causative agent of tonsilllitis, impetigo, scarlet fever and septicemia and can induce glomerulonephritis and rheumatic fever as late complications [1, 2]. the relationship between acute glomerulonephritis and infections with gas was first established in the early part of the 20th century, when rammelkamp and coworkers showed that only certain strains of group a streptococci, especially type 12, were able to induce acute glomerulonephritis [3]. cases or epidemics of acute glomerulonephritis are usually associated with pharyngeal infections or skin infections, i.e. impetigo [3, 4]. �-hemolytic streptococci group c (gcs) can cause tonsillitis but very rarely glomerulonephritis [5, 6]. group g streptococci have been considered to be less pathogenic than group c streptococci but have been associated both with glomerulonephritis and renal failure [7, 8]. streptococcus constellatus, belonging to the “milleri” or “anginosus” group of streptococci sometimes express the lancefield c-antigen. these bacteria differ from the “large colony type” gcs (streptococcus zooepidemicus) in forming small, “pin point” colonies and having a different pathogenic potential. they belong to the normal flora of the mouth. they have not, to our knowledge, been associated with tonsillitis or glomerulonephritis but have been shown to occur in pharyngitis and peritonsillitis [9–11]. in this report we describe an epidemic of acute glomerulonephritis coinciding with the spread of s. constellatus in the patients families and in neighbouring families in a rural area of sweden. possible associations of the spread of the s. constellatus and the occurrence of four cases of acute glomerulonephritis, two of them biopsy-verified and one of them with a nephrotic syndrome, are discussed. 218 patients and methods case reports: a 40-year old man-the index patient (a)-was admitted to his local hospital, eksjö, with signs of acute nephritis. due to a high grade of uremia he was transferred the following day to the university hospital of linköping, sweden for kidney biopsy and acute dialysis. four days before arrival, he had started on a course of penicillin due to acute tonsillitis. three further patients (b-d) described below later developed clinical findings indicating an acute glomerulonephritis. the acute renal failure in patient a remitted after treatment, transiently with cyclophosphamide, corticosteroids, plasmapheresis and dialysis. a kidney biopsy showed acute postinfectious glomerulonephritis.the patient was discharged from the hospital with a serum creatinine of 173 �mol/l (normal range 70-115 �mol/l). at the time of discharge a throat culture was positive for beta-hemolytic streptococci, lancefield group c, reported as gcs. the same type of bacteria were also detected in throat cultures from the patient´s wife and two of their children. a third child had �hemolytic streptococci lancefield group g (ggs) in a culture from the throat while a fourth child was culture-negative. patient a´s renal function was subsequently normalized with both normal serum creatinine values and a normal iohexol clearance of 99 ml/min and 1.73 m2 body square area in november 1997. there was no proteinuria but a slight hematuria (++) . the week after the start of patient a´s illness a neighbour of our patient (b) fell ill (figs 1 and 2). he was admitted to his local hospital, eksjö, with renal failure (screatinine 200 �mol/l and urea 35 mmol/l, normal range 3-9 mmol/l). the neighbour had felt ill for about four weeks and had had a slight oedema. one week before 219 a tonsillitis, penicillin nasopharynx culture neg. glomerulonephritis gcs in throat culture 12/5 15/5 16/5 20/5 b tonsillitis, penicillin , throat and nasoph. culture neg glomerulonephritis ggs in throat culture 14/5-15/5 21/5 2/6 c throat pain glomerulonephritis-gcs in the throat april 20/5 d glomerulonephritis gcs in the throat _________________________________________________________________________________________ 20/5 22/5 fig 1. the time points of tonsillitis, throat cultures and glomerulonephritis in patients a–d. admittance he had received a course of penicillin. a throat culture before the course was negative. antistreptolysin o titre (aso) was 200 (normally < 150) and antideoxyribonuclease b (adnase b) titre was high, >1200 (normally < 400). a kidney biopsy was performed, see histopathology below. a throat culture 12 days afterwards was positive for ggs. his wife was healthy. two of the neighbour´s children (c and d) were admitted to the central hospital of ryhov in jönköping. both had fallen ill with renal involvement. the boy (c) had earlier had a sore throat. he had a hypertension with blood pressures of 140/80160/120 mm hg and was transiently treated with metoprolole. creatinine was 71-83 �mol/l and urinary albumin 0.38 g/24 h (normally below 0.1 g/24h). urinary sediment findings were pathologic. aso was elevated to 560 and adnase was also 220 family 1 (farmer family) m 9, f s.constellatus. f 15, g s.constellatus. (lancefield group c) m 14, h no symptoms s constellatus (lancefield group c) m 8, i s.constellatus (lancefield group c) m 40, a nephritis streptococci c f 35, e healthy s constellatus lancefield group c family 2 m 10, c nephritis s. constellatus (lancefield group c) f 12, d nephritis s. constellatus. (lancefield group c) m 7, k multiforme erythema s.constellatus (lancefield group c) m 2, l ill two weeks earlier s. pyogenes (lancefield group a) . m 41, b nephritis (s.constellatus 12 days later, lancefield group g) f 35, j healthy family 3 m 8, o s.constellatus (lancefield group c) m 6, p s.constellatus (lancefield group c) f 2, q s. constellatus (lancefield group c) m 36, m s. anginosus (lancefield group c) f 34, n s. anginosus (lancefield group c) family 4 m 30, r s. constellatus (lancefield group c) f 28, s (s. constellatus 14 months later) fig 2. schematic drawing of family 1 (farmer family). schematic drawing of family 2 (neighbours). schematic drawings of families 3 and 4 (neighbours). high > 1200. complement level c3 was low (0.14g/l), while c4 was normal (0.22 g/l). his twelve year old sister (d) had a nephrotic syndrome with urinary albumin levels of 3.3-9.7 g/l (normal < 0.03 g/l) and a serum albumin of 29-32 g/l (normal > 35 g/l). serum creatinine was 69-98 �mol/l and the blood pressure was elevated to 150/90-150/100 mm hg. in both children beta hemolytic streptococci, reported as gcs, were found in throat cultures. a third child who acquired erythema multiforme also had the same bacteria in the throat, while a forth child had group a streptococci (streptococcus pyogenes). the time points of tonsillitis, throat cultures and onset of glomerulonephritis are shown in fig. 1. the families had had multiple contacts, and their children usually went to school by the same bus. the patient who first fell ill was a farmer, and the families had consumed unpasteurized milk from the farm of patient a. cultures from the milk were, however, negative for gcs and other streptococci. in six of seven persons of two further neighbouring families, ß-hemolytic streptococci, reported as gcs, were found in throat swabs during the same month. five of these persons were treated with penicillin. a throat culure from one of these patients 14 months later revealed beta-hemolytic streptococci group c. microbiology: throat swabs. throat swabs were transported in amies medium and cultured on 5% sheep blood agar containing columbia agar base (difco). �-hemolytic colonies 221 fig 3. pulse field gel electrophoresis of streptococci. lane 1: dna marker � ladder. lanes 2-16: patients dna. lane 17: dna marker s. aureus 8325 (nctc 8523). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1. dna marker λ ladder 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. dna marker s. aureus 8325 fig 2 pulse field gel electrophoresis of streptococci were isolated and agglutinated with latex particles coated with antibodies to lancefield group a, c and g antigens (oxoid ltd, basingstoke, hampshire, england). pulsed field gel electrophoresis (pfge). chromosomal dna from gbs embedded in agarose plugs was prepared as previously described [12]. an agarose slice was incubated overnight at 30°c with 20 u of smai, (mbi, fermentas) in the provided enzyme buffer. plugs were washed in te-buffer (1h 5°c) before mounting into wells of a 1 % agarose gel (pulsed field certified agarose, bio rad) in 0.5x tbe-buffer ph 8.3. electrophoresis was performed on an automated pfge apparatus (gene path strain typing system, bio rad). standard programs for fragment size 50-600 was used. a standard �-ladder (new england biolabs inc, ma, usa) and smai digested s. aureus 8325 was included alongside the samples. the agarose gel was stained with 0.2% ethidiumbromide and washed in tap water visualized and photographed under uv light (fig. 3). pcr analysis of skaa five to ten colonies, picked from a blood agar plate, were resuspended in 50 ul sterile distilled water in a micro test tube and heated for 5 min at 100°c. after centrifugation, 10 ul of the supernatant was used as template in the pcr reactions. reaction conditions were 0.2 mm dntp´s, 20pmol of each primer with 0.5 u taq polymerases buffer with 1.5 mm mgcl 2 (mbi fermenta, labassco) and bovine serum albumin (0.17 mg/ml). the amplification was performed on a ptc-200 thermal cycler (mj research, scandinavian diagnostic services, (sds) falkenberg, sweden) with annealing temperature of 55°c. forward primer used was ska 453-477; 5´-aaccttgccgacccaacct-3´ and reverse primer ska 770-791; 5´-tccattggtaaaatcgtacgg-3´ [13]. the pcr products were separated with electrophoresis on 0.7 % agarose gel. phenotypic typing of group c streptococci further typing of group c streptococci was performed with api strep from biomérieux sa marcy l´étoile, france. renal biopsy examination part of the renal biopsy material obtained from patients a and b was snap-frozen in isopentane-co2 for immunfluorescence studies. cryostate sections, 4-5 �m thick, were fixed in ethanol, incubated with fitc-conjugated rabbit antibodies to human igg, iga, igm, and c3c (dakopatts, sweden), and examined in an incident-light fluorescence microscope. another part of the biopsy material was immersed in histochoice (amresco, solon, il, usa), embedded in paraplast, and cut into 1to 2-�m sections that were stained with periodic acid schiff´ s reagent and with periodic acid silver methenamine. the remaining part of the biopsies was immersed in 2-% glutaraldehyde, embedded in epon 812. semiand ultra-thin sections were prepared, stained with oso4 and examined in a jeol 1200 electron microscope. 222 223 table 1: pulse field gel electrophoresis description of lanes in fig. 2. lane bacteria lancefield group patient 2 s. constellatus g b 3 " g f 4 s. constellatus c d 5 " c c 6 " c k 7 s.anginosus c m 8 " c n 9 s.constellatus c o 10 " c p 11 " c q 12 " c r 13 " * c s 14 " c h 15 " c g 16 " c i 1 dna marker, λ-ladder 17 dna-marker, s. aureus 8325 *) isolated 14 months after the outbreak table 1: pulse field gel electrophoresis – description of lanes in fig 3. tissue preparation and lysis a nucleon kit [14] for the extraction of genomic dna from tissue (nucleon ht, product code rpn 8509), amersham international plc & scotlab ltd 1997) was used. the tissue biopsies were grinded on liquid nitrogen to fine powder in a sterile mortar. grinding occurred in a laminar air hood. grinded tissue was transferred to an eppendorf tube and 350 �l reagent b was added. thereafter 5 �l rnase solution was added to the tube and the contents were incubated for 30 min at 37°c. proteinase k solution (18 �l) was added and the tissue was incubated at 56°c overnight. after centrifugation at 7,500 rpm for 5 min the supernatant was transferred to a new tube. for deproteinization 100 �l of sodium perchlorate solution was added and the content mixed by inverting the tube several times. dna extraction chloroform (600 �l) was added to the supernatant and the content mixed by inverting the eppendorf tube several times. nucleon resin (150 �l) was then added without mixing the content. centrifugation occurred at 2,200 rpm for 1 min. the supernatant was transferred to a new tube and dna was precipitated by adding 2 volumes (700 �l) of cold ethanol and by inverting the tube. the dna was washed by centrifugation at 4,000 x g for 5 min. the supernatant was discarded and 1000 �l of 70% cold ethanol was added. after mixing, another high-speed centrifugation was made and the supernatant was discarded. the dna pellet was dried in a vacuum evaporator for 15 min. thereafter, the dna was dissolved in te buffer. checkerboard dna-dna hybridization a slightly modified procedure of the checkerboard method developed by socransky et al. [15] was used for dna-dna hybridization. each dna sample was pipetted in volumes of 100 �l and transferred to separate eppendorf tubes. one hundred microliter of 0.5 m sodium hydroxide was added to the sample. all samples were placed in a water bath (100°c) and boiled for 5 min. they were then neutralized using 800 �l of 5 m ammonium acetate. the released dnas from the clinical species and the type strains of s. constellatus ncdo 2226t and s. pyogenes ccug 4207t were placed into the lanes of a minislot (immunetics, cambridge, ma, usa) and then deposited on a nylon membrane (boehringer mannheim, mannheim, germany). the membranes were fixed by baking at 68°c for 30 min followed by exposure to ultraviolet light for 30 s. the membrane with the fixed dna was placed in a miniblotter 45 (immunetics) with the lanes of dna at 90°c to the channels of the device. the membranes were prehybridized at 42°c for 1 h in 50% formamide (applichem, darmstadt, germany), 5 x ssc (1 x ssc = 150 mm sodium chloride, 15 mm sodium citrate (applichem), ph 7.0), 10% casein (sigma, st. louis, mo, usa), 5 x denhardt’s reagent (applicchem), 25 mm sodium phosphate (ph 6.5) and 10 mg/ml yeast rna (boehringer mannheim). digoxigenin-labeled, whole genomic probes and hybridization buffer containing 45% formamide (5 x ssc, 1 x denhardt’s reagent, 20 mm sodium phosphate (ph 6.5), and 10 mg/ml yeast rna, 224 20 ng/ml labelled probe, 10% dextran sulphate (sigma) and 10% casein were placed in individual lanes of the miniblotter. the whole apparatus was covered with saran wrap and transferred to a sealed plastic dish with water. the membranes were hybridized overnight with gentle shaking at 42°c. they were washed in a plastic dish to remove loosely bound probe. to detect hybrids, the membranes were blocked with 10% casein in maleate buffer (100 mm maleic acid, 150 mm sodium chloride, ph 7.5) and then incubated with antidioxigenin antibody conjugated with alkaline phosphatase (boehringer mannheim), diluted 1:20 000 in maleic buffer. signals were detected chromogenically using ntb/bcip tablets (boehringer mannheim) overnight. results microbiology pfge typing showed that 13/15 isolates reported as gcs were in fact s. constellatus and 2/15 were s. anginosus (table 1 and fig 3). reagglutination confirmed that 13 of these 15 strains expressed the lancefield c-antigen and that two belonged to group g. eleven of the 13 s.constellatus strains expressed the c antigen. pfge also showed that 10 of the 11 s. constellatus group c were identical (fig 3, lane 4, 5, 6, 9, 10, 11, 12, 14, 15, 16). the eleventh strain of s. constellatus (fig 3, lane 13) obtained from a throat culture 14 months after the outbreak, differed somewhat from the other 10. pcr-analysis of the ska gene showed that this gene was absent in all 15 isolates. the group c strain from the index patient (a) was not subtyped as it had been isolated in a different laboratory (linköping) and was therefore not available for further analysis. epidemiology the time points of tonsillitis and throat cultures are shown in fig. 1. the family trees and the results of the throat cultures with subtyping are shown in fig 2. kidney histopathology the kidney biopsy material from both patients a and b contained renal cortex with approximately 20 glomeruli. histologically, a moderate to severe increase of glomerular endocapillary cells in a diffuse and global pattern was observed. the proliferating endocapillary cells, together with infiltrating polymorphonuclear cells,encroached considerably on the capillary lumina (fig. 4a). in patient b occasional capillary loops contained a hyaline subendothelial material. there was no significant extra-capillary cell proliferation. examination by immunfluorescence microscopy revealed in patient a large amounts of igg and c3c, and moderate amounts of c1 q, outlining the glomerular capillary loops in a granular, "starry sky" pattern. slight amounts of granular igm and iga deposits were also seen along the 225 capillary loops. patient b showed the same pattern of granular deposits with a dominance of c3c along the capillary loops (fig. 4b), moderate amounts of igg, and slight amounts of iga, while there were no deposits of igm and c1q. both patients showed granular deposits of immunoglobulins and complement in the mesangial region. the ultrastructural examination confirmed presence of endocapillary cell hyperplasia, and electrone-dense subendothelial deposits. in some capillary loops in patient b, these electron-dense subendothelial deposits filled a large part of the cap226 fig 4a. irregular deposits of c3c along the glomerular capillary loops and in the mesangial regions, so-called “starry sky pattern”. fig 4b. glomerulus with accentuated lobular configuration due to severe endocapillary cell proliferation. the proliferating cells together with infiltrating polymorphonuclear leukocytes fill out the capillary lumina. illary lumen. both patients had discrete, large, irregular subepithelial electron-dense deposits, so called “humps” (fig. 4c). patient a showed foci of tubulointerstitial inflammation sometimes with tubulitis. renal tissue analysis the kidney biopsies from patients a and b and one of the 6 controls, a patient with iga nephropathy, were positive for s. pyogenes. checkerboard dna-dna hybridization showed that both renal biopsies contained s. constellatus. s. constellatus was positive for the control patients pl 1723-98, pl 10496-98 and pl 12992-98 but negative for the patients pl 400-98 and pl 12732-98. the latter five control-patients were negative for s. pyogenes. their renal tissue contained, according to light microscopy, mainly nephrosclerosis. discussion this study describes an outbreak of glomerulonephritis associated with the isolation of s. pyogenes in two kidney biopsies, but also with identical strains of s. constellatus in throat cultures, and in both cases in renal biopsies, from the patients and members of the families and other families in the neighbourhood (fig. 2). s. constellatus was also present in 3/6 control kidney biopsies. s. constellatus belongs to the normal flora of the 227 fig 4c. large, discrete electron-dense deposits on the subepithelial side of the glomerular basement membrane, so-called “hump”, covered by a glomerular epithelial cell with many endocytic vacuoles. mouth from which it may spread. in the index patient (a) gcs were isolated but subtyping was not performed. s. pyogenes was positive also in a kidney biopsy of one control patient with chronic glomerulonephritis (i.e. iga nephropathy). the remaining 5 specimens were negative for s. pyogenes. s. constellatus has also been found in abscesses and in peritonsillitis and in rare cases in association with pharyngitis [9–11]. the index patient (a) had a serious dialysis-requiring condition and a second patient (b) had a notable renal failure with a serum creatinine of 200 �mol/l. one child had glomerulonephritis with a nephrotic syndrome and another had glomerulonephritis (c, d). s. pyogenes may have been the main cause of glomerulonephritis in both of the kidney-biopsied patients. whether s. constellatus was a concomitant cause of glomerulonephritis cannot be determined at present. other bacteria than group a streptococci have increasingly been recognized as causes of acute postinfectious glomerulonephritis [16, 17]. an association between s. constellatus and glomerulonephritis has so far not been reported. the possibility of an infection with gas or large colony type gcs, preceding the glomerulonephritis in the index patient and the three patients colonized with s. constellatus must be considered (figs 4 a-b) even if throat cultures at the onset of tonsillitis were negative. this possibility is corroborated by the finding of s. pyogenes in the renal tissue of two patients. this possibility might also be supported by the increased levels of ast and anti-dn:ase b in two patients suggesting a recent infection with gas or possibly gcs [18–20]. s. constellatus is not, to our knowledge, known to produce streptolysin o or dn:ase b. glomerulonephritis associated with large colony type gcs (s. zooepidemicus) has been described but is uncommon [5,6]. the mechanism is not known but may be the same as that proposed for glomerulonephritis associated with gas, i.e. nephritogenic proteins (endostreptosin) produced by the bacteria [21]. both of our kidney biopsied patients had a picture of acute diffuse proliferative glomerulonephritis of the kind seen in acute poststreptococcal glomerulonephritis. in acute post-streptococcal glomerulonephritis, nsap (nephritogenic associated protein) with production of streptokinase from the streptococci has been suggested to induce the binding of complement to the glomeruli by itself and by a specific antibody [22–24]. if s. constellatus caused the glomerulonephritis such a mechanism seems unlikely, since the ska gene was not present in the s. constellatus strains isolated here. the mechanisms of acute post-streptococcal glomerulonephritis are however still unclear [3, 13]. recently a bacterial protein h, a surface protein of s. pyogenes has been discussed in the pathogenesis of acute poststreptococcal glomerulonephritis [25]. circulating immune complexes with deposition and induction of complement and inflammatory cells in glomeruli as well as in situ antigen-antibody reactions are considered as the main mechanism of postinfectious glomerulonephritis, and bacterial antigens not present in gas-induced nephritis may have been involved [3, 13]. 228 s. constellatus is not known to induce tonsillitis, but may occur in peritonsillar abscesses and periapical endodontic lesions of asymptomatic teeth [26, 27]. it may also be present in empyema thoracis and lung abscess and has been described in association with endocarditis [28, 29]. glomerulonephritis has been described also in association with endocarditis caused by �-hemolytic (viridans) streptococci [30–32]. such streptococci are however more able to induce chemotaxis of leucocytes than the streptococci belonging to the milleri group in which s. constellatus is included [33]. the presence of s. pyogenes in the biopsy of the control patient with iga nephropathy may be coincidental. a connection between iga nephropathy and post-streptococcal glomerulonephritis in some cases is suggested in the literature [34, 35 ]. this may also be the case in hennoch-schönlein´s purpura, a disease related to iga nephropathy [36]. the epidemic described suggests either that the outbreak of four cases of glomerulonephritis was due to s. pyogenes but coincided with the transmission and colonization of s. constellatus or that s. constellatus strains were highly pathogenic or nephritogenic and that this organism can be effectively transmitted. further studies on the possible role of s. constellatus in the pathogenesis of post-streptococcal glomerulonephritis [37] are warranted. acknowledgements m norgren umeå, sweden is acknowledged for the analysis of pulse field gel electrophoresis and pcr analysis of the ska gene as well as further typing of group c streptococci. i. messel oslo, norway is thanked for the checkerboard analysis. the county council of östergötland, sweden is acknowledged for financial support. 229 references 1. villareal h jr, rico e, zabriskie jb (1979) streptococcus-related acute glomerulonephritis. arch inst cardiol mex 49: 89-102. 2. van buynder pg, gaggin ja, martin d, pugsley d, mathews jd (1992) streptococcal infection and renal disease markers in australian arboriginal children. med j aust 156: 537-540. 3. glassock rj, cohen ah, adler sc (1996) primary glomerular diseases. in: brenner bm, rector fc(eds), the kidney. wb saunders company, philadelphia 1394-1402. 4. earle dp, potter ev, poon-king t, finklea jf, sharrett ar, ortiz j (1970) streptococcal skin infections and epidemic acute nephritis in trinidad. trans am clin climatol assoc 81: 184-195. 5. barnham m, 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(1969) a new nephritogenic streptococcus. j hyg 67: 691698. 7. reid hfm, bassett dcj, poon-king t, zabrieskie jb, read se (1985) groop g streptococci in healthy school-children and in patients with glomerulonephritis in trinidad. j hyg camb 94: 61-68. 8. roth s, andrassy k, schmidt kh, gunther f, ritz e (1999) febrile lady with acute renal failure and desquamating erythema. am j kidney dis 34: 150-154. 9. fujiyoshi t, okasaka t, yoshida m, maishima k (2001) clinical and bacteriological significance of streptococcus milleri group in deep neck abscesses. nippon jibiinkoka gakkai kaiho; 104 (2): 147-156 (japanese, eng. abstr.). 10. fujiyoshi t, inaba t, udaka t, tanabe t, yoshida m, makishima k (2001) [ clinical significance of the streptococcus milleri group in peritonsillar abscesses] [japanese, eng. abstract]. nippon jibiinkoka gakkai kaiho 104: 866-871. 11. whiley ra, hall lm, hardie jm, beighton d (1999) a study of small-colony, beta-haemolytic, lancefield group c streptococci within the anginosus group: description of streptococcus constellatus subsp. pharyngis subsp. nov., associated with the human throat and pharyngitis. int j syst bacteriol 49: 1443-1449. 12. granlund m, öberg l, sellin m, norgren m (1998) identification of a novel insertion element, is1548, in group b streptococci, predominantly in strains causing endocarditis. j infect dis 177: 967-976. 13. johnston kh, chaiban je, wheeler jc (1992) analysis of the variable domain of the streptokinase gene from streptococci associated with post streptococcal glomerulonephritis. in: orefieci g, ed. new perspektives on streptococci and streptococcal infections. stuttgart: gustav fischer verlag: 339-341. 14. extraction & purification protocols (1997) blood & cultured cells. animal tissues & paraffin sections. amersham life science: 18-19. 15. socransky ss, smith c, martin l, paster bj, dewhirst fe, levin ae (1994) “checkerboard” dna-dna hybridization. biotechniques: 17: 788-792. 16. westrhenen r v, j j weening, krediet rt (1998) pneumonia and glomerulonephritis caused by mycoplasma pneumoniae. nephrol dial transplant 13: 3208-3211. 17. meyrier a (2001). postinfectious glomerulonephritis. in: primer on kidney diseases, 3d ed. ed. greenberg a. academic press. san diego and london 190-194. 18. kaufhold a, ferreri p (1993) the microbiologic aspects, including diagnosis, of beta-hemolytic streptococcal and enterococcal infections. infect dis north am 7: 235-256. 19. eriksson b, jorup-ronstrom c, karkkonen k, sjoblom ac, holm se (1996) erysipelas: clinical and bacteriologic spectrum and serological aspects. clin inf dis 23: 1091-1098. 20. jansen tl, janssen m, traksel r, de jong aj (1999) a clinical and serological comparison of group a versus non-group a streptococcal reactive arthritis and throat culture negative cases of post-streptococcal reactive arthritis. ann rheum dis 58: 410-414. 21. cronin wj, lange k (1990) immunologic evidence for the in situ deposition of a cytoplasmic streptococcal antigen (endostreptosin) on the glomerular basement membrane in rats. clin nephrol 34: 143-146. 230 22. peake pw, pussell ba, karpius te, riley eh, charlesworth ja (1991) post-streptococcal glomerulonephritis: studies on the interaction between nephritis strain-associated protein (nsap), complement and the glomerulus. apmis may; 99(5): 460-466. 23. poon-king r, bannan j, viteri a, cu g, zabriskie jb (1993). identification of an extracellular plasmin binding protein from nephritogenic streptococci j exp med 178: 759-763. 24. nordstrand a, norgren m, holm se (1999) pathogenic mechanism of acute post-streptococcal glomerulonephritis. scand j infect dis 31(6): 523-537. 25. berge a, kihlberg bm, sjoholm ag, bjorck l (1997) streptococcal protein h forms soluble complement-activating complexes with igg, but inhibits complement activation by igg-coated targets. j biol chem 272: 20774-781. 26. claridge je 3rd, attori s, musher dm, hebert j, dunbar s (2001) streptococcus intermedius, streptococcus constellatus and streptococcus anginosus (“streptococcus milleri group”) are of different clinical importance and are not equally associated with abscess. clin infect dis 32: 1511-1515. 27. sunde pt, tronstad l, eribe er, lind po, olsen i (2000). assessment of periradicular microbiota by dna-dna hybridisation. endod dent traumatol 16: 191-196. 28. jerng js, hsuch pr, teng lj, lee ln, yang pc, luh kt (1997). empyema thoracis and lung abscess caused by viridans streptococci. am j respir crit care med 156: 1508-1514. 29. baran j jr, abdo wm, merrit kw, khatib r (1998) tricuspid valve endocarditis due to a moderately susceptible streptococcus constellatus; persistent bacteremia and fatal outcome despite penicillin plus gentamicin therapy. scand j infect dis 30: 420-421. 30. arnold sb, valone ia, askenase pw, kashgarian m, freedman lr(1975). diffuse glomerulonephritis in rabbits with streptococcus viridans endocarditis lab invest 32: 681-689. 31. neugarten j, gallo gr, baldwin ds (1984). glomerulonephritis in bacterial endocarditis. am j kidney dis 3: 371-379. 32. neugarten j, baldwin ds (1984). glomerulonephritis in bacterial endocarditis. am j med; 77: 297-304. 33. wanahita a, goldsmith ea, musher dm, clarridge je3d, rubio j, krishnan b, trial j (2002). interaction between human polymorphonuclear leucocytes and streptococcus milleri group bacteria. j infect dis; 185: 85-90. 34. sagel i, treser g, ty a et al. (1973). occurrence and nature of glomerular lesions after group a streptococci infections in children. ann intern med 79: 492-499. 35. okada k, saitoh s, sakaguchi z et al. (1996). iga nephropathy presenting clinicopathologic features of a post-streptococcal glomerulonephritis. eur j pediatr 155: 327-330. 36. masuda m, nakanishi k, yoshizawa n, iijima k, yoshikawa n (2003). group a streptococcal antigen in the glomeruli of children with henoch-schonlein nephritis. am j kidney dis 41: 366370. 37. rincon j, viera nt, romero mj, mosquera ja (2003). increased production of chemotactic cytokines and elevated proliferation and expression of intercellular adhesion molecules in rat mesangial cells treated with erythrogenic toxin type b; its precursor isolated from nephritogenic streptococci. nephrol dial transplant. jun; 18: 1072-1078. corresponding author: g. almroth, md, phd. department of nephrology, university hospital of linköping, s-581 85 linköping, sweden. phone +4613222000, fax +4613224514, e-mail: gabriel.almroth@lio.se 231 upsala j med sci 93: 45-51, 1988 ear oximetry during progressive hypoxia thorarinn gislason,' bo sandhagen' and efi daskalopouloul departments of 'lung medicine and 2clinical physiology, uppsala university, akademiska sjukhuset, uppsala, sweden abstract the biox i11 pulse oximeter for measuring arterial oxygen saturation (sa02) was compared during rest and under progressively hypoxic conditions, with sa02 values based on arterial blood samples. the measurements were performed in 16 subjects undergoing tests of ventilatory response to hypoxia, by a rebreathing method. for each individual subject, there was a linear response relationship (r=0.99), while for all 126 comparative values the regression equation was: y= 0.83 x + 14.7 (r=0.98). the observed ventilatory response was lower when the calculations were based on oximeter readings. we conclude that the oximeter has acceptable correlation between the biox i11 and sa02 measurements for clinical use, especially when sa02 is above 70%. introduction a method for continuous non-invasive monitoring of arterial oxygen saturation (sa02) by oximetry was described more than 50 years ago, but was not generally accepted in clinical practice until recently, because of doubts about the accuracy of the method (1, 8, 9). with the marked improvement in instrumentation, this technique, which is based on transmission of light through the vascular bed of the ear lobe ( 5 ) , is now gaining increasing attention, both for research and clinical purposes (2, 3, 4). comparative studies between oximetry and measurements based on 45 a r t e r i a l s a m p l e s have been l i m i t e d . on t h e o t h e r hand, t h e r e i s i n c r e a s i n g i n t e r e s t i n c o n t i n u o u s m o n i t o r i n g o f b l o o d g a s changes, e s p e c i a l l y d u r i n g i n v e s t i g a t i o n s o f s l e e p r e l a t e d b r e a t h i n g d i s t u r b a n c e s . the aim o f t h i s p r o s p e c t i v e s t u d y w a s t h e r e f o r e t o estimate t h e a c c u r a c y and r e l i a b i l i t y o f a two-wavelength o x i m e t e r f o r d e t e r m i n i n g sa02 a s compared w i t h a r t e r i a l b l o o d samples, t a k e n a t rest a n d d u r i n g p r o g r e s s i v e h y p o x i a . material a n d methods p a t i e n t s : the s t u d y comprised 1 6 c o n s e c u t i v e p a t i e n t s , who had been r e f e r r e d t o t h e department o f lung medicine o f t h e u n i v e r s i t y h o s p i t a l , uppsala, sweden f o r i n v e s t i g a t i o n o f s l e e p r e l a t e d b r e a t h i n g d i s t u r b a n c e s . t h e i r mean a g e was 51 y e a r s ( r a n g e 38-63) ( t a b l e 1 ) . a l l w e r e h a b i t u a l s n o r e r s , and some a l s o complained of d a y t i m e s l e e p i n e s s and w e r e t h u s s u s p e c t e d o f s u f f e r i n g from t h e s l e e p apnoea syndrome ( 6 ) . they a l l had normal serum b i l i r u b i n v a l u e s a n d none had s k i n p i g m e n t a t i o n . e l e v e n o f t h e p a t i e n t s w e r e o b e s e , w i t h a body mass i n d e x ( w e i g h t / ( h e i g h t ) 2 ) 2 28.0 kg/m2 ( 7 ) , f o u r had a n a i r w a y o b s t r u c t i o n and t h r e e showed an i n c r e a s e i n r e s i d u a l volume by more t h a n 2 0 % ( t a b l e 1 ) . the h y p o x i c v e n t i l a t o r y r e s p o n s e t e s t w a s a p a r t o f t h e i r i n v e s t i g a t i o n programme, which had p r e v i o u s l y b e e n a c c e p t e d by t h e e t h i c s committee o f t h e medical f a c u l t y o f uppsala u n i v e r s i t y . e a r o x i m e t r y : oxygen s a t u r a t i o n was measured by a b i o x i11 p u l s e o x i m e t e r (ohmeda, c o l o r a d o , usa) c a l i b r a t e d a c c o r d i n g t o t h e m a n u f a c t u r e r ' s i n s t r u c t i o n s , and i t s normal r e s p o n s e mode was u s e d ( 2 ) . the ear p r o b e o f t h e o x i m e t e r was f i t t e d t o t h e ear l o b e of t h e s u b j e c t a f t e r p r o d u c t i o n o f v a s o d i l a t i o n by r u b b i n g w i t h a l c o h o l . blood sa02: a r t e r i a l b l o o d s a m p l e s (6-8 m l ) w e r e drawn i n t o h e p a r i n i s e d g l a s s s y r i n g e s ( 1 0 m l ) t h r o u g h a n i n d w e l l i n g c a n n u l a i n t h e r a d i a l a r t e r y and p l a c e d i n i c e w a t e r u n t i l a n a l y s e d . the a n a l y s e s w e r e p e r f o r m e d w i t h i n 30 m i n u t e s . the s a m p l i n g p r o c e d u r e l a s t e d 5-10 s e c o n d s . oxygen s a t u r a t i o n was measured p h o t o m e t r i c a l l y w i t h an o s m 2 oxygen s a t u r a t i o n m e t e r (radiometer, copenhagen, denmark). 46 table 1. characteristics of the patients and results of tests. av/asao2 (l/min/%sa02 ) case age bmi vc rv fev1.o based on based on no. years kg/m2 %pred. %pred. %pred. oximetry arterial sa02 1 39 36.0 2 48 22.8 3 38 31.0 4 54 27.1 5 57 32.6 6 41 26.9 7 52 24.5 8 61 31.4 9 63 29.0 10 49 27.8 11 38 39.1 12 39 28.4 13 60 29.0 14 58 30.4 15 43 39.8 16 59 30.5 89 84 108 106 101 112 90 57 80 103 90 102 124 93 65 83 86 122 57 93 89 65 83 92 100 119 127 71 111 103 213 91 91 82 114 117 109 119 95 53 80 113 103 106 134 96 69 80 0.39 0.43 0.12 0.23 0.37 0.74 0.64 0.73 3.78 1.15 0.55 0.85 1.59 0.70 0.53 0.45 0.41 0.62 0.12 0.24 0.46 0.90 0.63 0.89 4.40 1.20 0.65 1.00 1.63 0.85 0.57 0.48 mean 50 30.4 93 101 98 0.83 0.94 sd 9 4.7 17 36 21 0.86 0.99 a b b r e v i a t i o n s : bmi: body mass index; vc: v i t a l c a p a c i t y ; rv: r e s i d u a l volume; fev1.o: forced e x p i r a t o r y volume i n one second. hypoxic v e n t i l a t o r y t e s t : the s u b j e c t s s a t comfortably and b r e a t h e d room a i r f o r approximately 10 m i n u t e s u n t i l a s t e a d y s t a t e v a l u e of sa02 was a t t a i n e d . the hypoxic t e s t was performed by a method s i m i l a r t o t h a t d e s c r i b e d by rebuck and campbell ( 8 ) . b r i e f l y , t h e p a t i e n t s r e b r e a t h e d 8 l i t r e s of room a i r from a b e r n s t e i n s p i r o m e t e r . during t h e r e b r e a t h i n g procedure t h e c o n c e n t r a t i o n of t h e i n s p i r e d oxygen f e l l a s a consequence of oxygen consumption. the v e n t i l a t o r y response was r e g i s t e r e d a s t h e i n c r e a s e i n v e n t i l a t i o n (av) c a l c u l a t e d f o r each 1% r e c o r d e d d e c r e a s e i n sa02 ( asa02 ) . 47 the p a i r s of sa02 v a l u e s w e r e divided i n t o g r o u p s ( 9 0 9 9 % , 80-89% a n d s o o n ) a n d t h e i r d i f f e r e n c e s w e r e f u r t h e r a n a l y s e d . the r e s u l t s are p r e s e n t e d i n table 2 . the v a l u e s d i s p l a y e d b y t h e o x i m e t e r were f o u n d t o be s i g n i f i c a n t l y lower t h a n t h e a r t e r i a l v a l u e s when a b o v e 9 0 % , b u t h i g h e r when below 8 0 % (table 2 ) . table 2 . d i f f e r e n c e s b e t w e e n o x i m e t e r r e a d i n g s a n d a r t e r i a l s a 0 2 i n d i f f e r e n t r a n g e s . n: number o f s i m u l t a n e o u s m e a s u r e m e n t s , d : mean o f t h e d i f f e r e n c e s . seed: s t a n d a r d e r r o r o f t h e estimate. range n d .%ed p 90-99% 7 0 1 . 3 9 1 . 1 7 < o . 0 0 1 80-89% 22 0 . 4 1 2 . 2 8 n . s . 70-79% 2 1 2 . 4 8 2 . 3 4 < o . 0 0 1 60-69% 11 2 . 8 2 2 . 6 8 < o . 0 1 the v e n t i l a t o r y r e s p o n s e t o h y p o x i a (av/asa02) w a s 0 . 8 3 l / m i n / l % sa02 (k 0 . 8 6 ) when based o n o x i m e t e r r e a d i n g s , a n d 0 . 9 4 l / m i n / l % sa02 ( f 0 . 9 9 ) when based o n a r t e r i a l v a l u e s (table 1 ) . the v e n t i l a t o r y r e s p o n s e s based o n a r t e r i a l a n d o x i m e t r i c v a l u e s w e r e h i g h l y c o r r e l a t e d t o e a c h o t h e r ( r = 0 . 9 9 5 ) ; b u t as a c o n s e q u e n c e of t h e r e l a t i o n s h i p s p r e s e n t e d i n table 2 t h e v e n t i l a t o r y r e s p o n s e w a s s i g n i f i c a n t l y h i g h e r ( p < 0 . 0 5 ) when t h e c a l c u l a t i o n s w e r e based o n a r t e r i a l m e a s u r e m e n t s ( p a i r e d t t e s t ) . the d i f f e r e n c e s b e t w e e n a r t e r i a l a n d o x i m e t r i c v a l u e s w e r e n o t c o r r e l a t e d t o a g e , t o degree o f o v e r w e i g h t , o r t o t h e r e s u l t s o f t h e p u l m o n a r y t e s t s (table 1 ) . t h e r e w a s n o e l e c t r o d e d r i f t , t h e ear p r o b e w a s w e l l t o l e r a t e d a n d n o t e c h n i c a l d i f f i c u l t i e s a r o s e d u r i n g o x i m e t r y . discussion i n t h i s s t u d y w e c h o s e t o e v a l u a t e t h e o x i m e t e r r e a d i n g s d u r i n g p r o g r e s s i v e h y p o x i a i n o r d e r t o d e t e r m i n e t h e v a l i d i t y o f o x i m e t r y 48 an a r t e r i a l b l o o d s a m p l e w a s t a k e n a t r e s t j u s t b e f o r e t h e h y p o x i c t e s t b e g a n , a n d a sample was t h e n t a k e n e v e r y m i n u t e d u r i n g a n d o n e 2 . 5 a n d 5 m i n u t e s a f t e r t h e t e s t . when a p p r o x i m a t e l y 5 0 % of t h e a r t e r i a l s a m p l e h a d b e e n d r a w n i n t o t h e s y r i n g e , t h e sa02 r e a d i n g as d i s p l a y e d b y t h e o x i m e t e r w a s recorded. a l l a r t e r i a l s a m p l i n g a n d r e c o r d i n g s w e r e d o n e b y t h e same p e r s o n . s t a t i s t i c a l a n a l y s e s : v a l u e s are p r e s e n t e d as mean ( f s d ) . the s t r e n g t h o f t h e c o r r e l a t i o n b e t w e e n a r t e r i a l sa02 a n d o x i m e t e r r e a d i n g s w a s e v a l u a t e d b y l e a s t s q u a r e l i n e a r c o r r e l a t i o n . s t a t i s t i c a l p r o b a b i l i t y w a s assessed b y s t u d e n t ' s p a i r e d t t e s t 100 90 80 7 0 bo , results a t o t a l o f 1 2 6 p a i r s o f s i m u l t a n e o u s sa02 r e a d i n g s w e r e o b t a i n e d , 1 6 d u r i n g s t e a d y s t a t e , 78 d u r i n g h y p o x i a a n d 32 o n e a n d t h r e e m i n u t e s a f t e r t h e t e s t ( f i g . 1 ) . the r a n g e o f a r t e r i a l s a 0 2 v a l u e s w a s 5 5 9 9 % . t h e r e w a s n o o v e r a l l b i a s , a s t h e mean sa02 v a l u e w a s t h e same ( 8 8 . 7 % ) when m e a s u r e d w i t h t h e o x i m e t e r a s when based on t h e a r t e r i a l samples. f o r e a c h s u b j e c t t h e o x i m e t e r r e a d i n g s w e r e a l i n e a r f u n c t i o n of t h e a r t e r i a l saog, w i t h a mean c o r r e l a t i o n c o e f f i c e n t r o f 0 . 9 9 3 (ko.01) ( r a n g e 0 . 9 8 3 0 . 9 9 9 ) , w h e r e a s f o r a l l v a l u e s t h e r e g r e s s i o n e q u a t i o n w a s y= 0 . 8 3 x + 1 4 . 7 ( r = 0 . 9 7 6 ) ( f i g . 1 ) . y. r 0 88 4, *i f i g . 1 . r e l a t i o n between 1 2 6 sa02 v a l u e s o b t a i n e d s i m u l t a n e o u s l y by oximetry and from a r t e r i a l b l o o d samples. the l i n e a r r e g r e s s i o n e q u a t i o n ( s o l i d l i n e ) and t h e 95% c o n f i d e n c e l i m i t s (broken l i n e s ) a r e shown. 60 70 80 90 100 a r t p r l a l sao, 488857 1 49 during rapid changes ot sao2 (similar to those observed during sleep apnoea) and also to check the reliability of this method during the hypoxic ventilatory test, which is used to estimate the sensitivity of peripheral chemoreceptors (8). the high correlation between arterial and oximetric readings for each individual subject (r=0.993) suggests that the ear oximeter is very accurate in indicating changes in sa02. the total correlation between arterial and oximeter readings is also fully acceptable for clinical purposes and even better than has been found by other authors (3). however, one must be aware of the increasing differences at low sa02 levels ( 2 ) , which may imply that oximetry may lead to an overestimation of sao2 in monitoring of critically ill patients and to a situation in which patients with sleep-related breathing disturbances may have more severe oxygen desaturation than is detected. the commonly found cardiac arrythmias in the sleep apnoea syndrome might also have a disturbing effect on the oximeter reading, a factor which needs to be further investigated. on the basis of calculations from the hypoxic ventilatory response tests, we agree with chapman et a1 (2) that sa02 values below 70% should be omitted . in summary, the biox i11 pulse oximeter has been found to correlate acceptabley with sa02, is easy to apply and is well tolerated by patients. obviously the non-invasive approach in continuous monitoring of blood gases is a rapidly expanding field which must be followed up by comparative studies to clarify the characteristics of these techniques. acknowledgements this work was supported by grants from the swedish national association against heart and chest diseases, stockholm, the king oscar i1 jubilee foundation, stockholm and the bror hjerpstedt foundation, uppsala, sweden. references 1. burki, n.k. & albert, r.k.: noninvasive monitoring of arterial 2. chapman, k.r., d'urzo, a. & rebuck, a.s.: the accuracy and blood gases. chest 83: 666-670, 1983. response characteristic of a simplified ear oximeter. chest 3. chapman, k.r., lie, f.l., watson, r.m. & rebuck, a . s . : range of accuracy of two wavelength oximetry. chest 89:540-542, 1986. 83: 860-864, 1983. 50 4. 5. 6. 7. 8. 9. douglas, n.i. , brash, h.m., wraith, p.k. , calverley, p.m.a., leggett, r.j.e., mcelderry, l. & flenley, d.c.: accuracy, sensitivity to carboxyhemoglobin and speed of response of the hewlett-packard 47201. an ear oximeter. am rev resp dis119:311 313, 1979. farrey, r. j., walker, l.e., jensen, r.l. & walker, j.m. : ear oximetry to detect apnea and differentiate rapid eye movement (rem) and non rem sleep. chest 89:533-539, 1986. guilleminault, c., cumminsky, j. & dement, w.c.: sleep apnea syndromes: recent advances. adv intern med 26:347-374, 1980. key, a., fidanza, f., karvonen, m.j., kimura, n. & taylor, h.l.: indices of relative weight and obesity. j chron dis 25:329-343, 1972. rebuck, a.s. & campbell, e.j.m. : a clinical method for assessing the ventilatory response to hypoxia. am rev resp dis ries, a.l., farrow, j.t. & clausen, j.l.: accuracy of two ear oximeters at rest and during exercise in pulmonary patients. am rev resp dis 132:685-689, 1985. 109:345-350, 1974. address for reprints: dr thorarinn gislason dept. of lung medicine akademiska sjukhuset, s-751 85 uppsala, sweden. 51 upsala j med sci 95: 225-227, 1990 comments. which tests should be decentralized to the primary health care? nils tryding department of clinical chemistry, central hospital, kristianstad and department of community helath sciences, university of l u n d , dalby, sweden in order to find out the need for laboratory investigations to be performed locally or sent to the central hospital we have regular contacts with physicians in primary care. since 25 years we have registered their ordering habits. after discussions we have decided which investigations we at present think are meningful in different clinical situations. with respect to the techniques available today we have suggested which tests that are suitable for local analyses and which should be sent to the hospital laboratory. continuous re-evaluations are made during meetings with local district physicians and during 5-day postgraduate courses for primary health care doctors and clinical chemists which have taken place every half year since 15 years. the present list of laboratory tests physicians would like to have performed locally according to clinical needs includes: b-erythrocyte sedimentation rate (b-esr) b-hemoglobin (b-hb) u-albumin, u-glucose (teststrips) (u-hemoglobin, u-acetoacetate, u-nitrite teststrips) b-glucose b-, u-cells, microscopy b-coagulation factors (prothrombin complex) pt-bleeding time s-potassium s-c-reactive protein (crp) s-creatinine s-alat, s-gt,s-alp, s-bilirubin, s-amylase, s-cat s-ckmb s-cholesterol, fs-triglycerides, b-hbalc, s-theophylline u-dens ity 15 908573 225 u-a 1 bumi n u-human chorionic gonadotropin (u-hcg) f-hemoglobin the list reflects present swedish practice but varies locally. some of these tests are not needed for immediate action. however, some physicians in health care want e.g. serum cholesterol or gt because they want to discuss the test results directly with the patient. factors like distance to the central laboratory, communica tions, number of analyses, training of personnel etc are important. in order to give room for the new analyses we decided to reduce the number of tests with limited value, e.g. blood leucocyte counting. other laboratory investigations that were substantially diminished in number were b-esr, urine microscopy, s-asat, s bilirubin, s-urate and s-sodium. in fact the total number of tests ordered by the physicians on two health centres was reduced by 30-50 per cent after my information and discussions concerning optimal use of clinical chemistry. the format for ordering tests has an impact. there are also very great differences between physicians. continuous, practically oriented education and information from clinical chemists are necessary. we have asked patients and physicians about how soon they want to know the test results. the advantage of the new techniques is the offering of immediate answers when the physician and the patient are in personal contact. the laboratory test results can be evaluated immediately, together with other medical informa tion. after further questions and investigations diagnostic decisions can be made without delay. the patients are saved travelling time. the personnel is saved work with specimen handling for postal transport. the secretaries are saved clerical work. the physicians have the opportunity to make a final decision without extra communication by telephone or letter. undoubtedly all types of people concerned are willing to pay well for this service. however, money is invested well only when the 226 information obtained is clinically relevant and reliable. llclinicalll comes from the greek word kline which means bed. thus literally clinical chemistry means bedside chemistry. thanks to new techniques this is now a reality. there is no definite answer to the question "which tests should be decentralized to the primary health care?" we have here discussed some factors that must be considered. the clinical need for nearer patient testing is definitely the most important. the clinical chemist must take the responsibility for the evaluation of the effects of the new techniques. continuous discussions between clinical chemists and physicians are necessary. there is a great need for education of the personnel in primary health care and regular quality control systems must be established. in summary: the goal must be to do correct tests as well as to do tests correctly. correspondence: professor nils tryding, department of clinical chemistry, central hospital, s-29185 kristianstad and department of community health sciences, university of lund, 5-24010 dalby, sweden 227 upsala j med sci 95: 259, 1990 the regional tkansferability project elin olafsdottir,' torsten aronsson,' torgny groth' and carl-henric de verdie? 'department of clinical biochemistry, landspitalinn, reykjavik, 'department of clinical chemistry and 'unit f o r biomedical systems analysis, university of uppsala, uppsala, sweden analytical data from s-creatinine and s-urate from the uppsala orebro regional quality assurance program have been studied for a period of more than a year with weekly samples of different concentrations. the analytical bias and analytical stability of the about 20 participating laboratories is estimated over time. the systematic error estimated by linear least squares fit of the measured values vs assigned values shows for some of the laboratories an unacceptably large bias outside the reference ranges for a healthy population. analytical stability estimated over different time periods from slope and intercept of regression lines are of different degree of acceptability. steps to reduce the interlaboratory variability will be suggested and the investigation will be extended in order to study the possibility to use transformation functions for transfer of data by collection of data from e g reference populations with defined condition of health from different geographical areas. a report of this study has been prepared and will be published elsewhere. correspondence: professor carl-henric de verdier department of clinical chemistry, university of uppsala postal address: university hospital s-751 85 uppsala, sweden 259 upsala j med sci 95: 305-307, 1990 glossary of terms many of the terms are defined according to an earlier refer ence (1) . value of quantity value of a quantity. the expression of a quantity in terms of a number and an appropriate unit of measurement ( 2 ) a quantity (2). numerical value (of a quantity). the number in the value of true value of a quantity. is the value which characterizes a quantity perfectly defined, in the conditions which exist when that quantity is considered (2). conventional true value (of a quantity). a value of a quantity which , for a given purpose, may be substi tuted for the true value (2). analytical performance characteristics imprecision of the measurement procedure (s). the inherent imprecision of the measurement procedure is estimated from replication studies. by analysis of variance techniques the within-run and the between-run compo nents of variation may be estimated: s, and s, denote the corresponding standard deviations, and s, stands for the total standard deviation of the measurement procedure (1). bias. total the average systematic error of the measurement procedure, as estimated by a comparison of method, e. g. comparison of patient values between the test method and a definitive method or a reference method (1). bias has obtained the meaning of the difference between the conventional true value and the expected value of the local laboratory. error (te)(4) = absolute error of measurement (3). t e is the rrsumfr of the systematic error and a component of the random error. systematic shift (se). a systematic shift is the systematic error that occurs in the measurement procedure. it is expressed direct in units or as a factor times the inherent imprecision of the measurement procedure (1). 20908573 305 increase in random error (re). an increase in the inherent random error of the measuremnt procedure is expressed as a factor by which the inherent imprecision is multiplied to give the imprecision of the measurement procedure under the influence of an analytical dis turbance (1). frequency of errors. refers to the incidence rate of ana lytical disturbances, which occur in addition to the inherent errors. it is here expressed in percent of runs having analytical disturbances (1). analytical quality specifications allowable analytical error (aae) = total allowable error (te,) = analytical quality goal. aae is a 95 % limit of error, i. e. 19 out of 20 test results should have errors less than this amount. aae is a 1.96.~ limit of error, thus a recommended allowable s must be multiplied by 1.96 to express the error specification in the form of aae (1). critical systematic shift (se,). a term in the equation: aae = bias + se,.s + (component of re,) which must be small enough so that the numerical value of aae is not exceeded. critical increase in random error (re,). see above. laboratory quality specification = analytical quality spe cification of the laboratory. this is the specifica tion given by the laboratory to its customers. if not otherwise stated it means that 95 % of the results delivered should be located within the specification limits. quality specifications including both analytical and non analytical components clinical needs. this term is preferred to the term medical needs. it generally includes analytical limits but may also include non-analytical characteristics as turnaround time. clinical quality specifications. this term includes compo nents of variation as preanalytical and ana lytical errors. it also assumes standardized treat ment and care of the patient. 306 non-analytical quality specifications turnaround time ( t,,, ) utilization of analyses (investigations) result interpretation supply of available analyses and other services cost of analyses laboratory quality management = good laboratory practice (glp) quality laboratory practices quality control quality assurance internal quality assurance external quality assurance references 1. westgard j 0, groth t, de verdier c-h. principles for developing improved quality control procedures. in: de verdier c-h, aronsson t, nyberg a, eds. quality control in clinical chemistry efforts to find an efficient strategy. scand j clin lab invest 1984;44 suppl 172: 19-41. 2. zender r.measurements in biological systems. metrological principles and terminology. in: kallner a , bangham d, moss d, eds. improvment of comparability and compatibility of labora tory assay results in life sciences. ifcc master discussion, 1. bergmeyer conference 1988. scand j clin lab invest 1989;49 suppl 193: 3-10. 3. dybkaer r, martin d v, rowan r m eds. good practice in decentralized analytical clinical measurement. scand j clin lab invest 1991;51 suppl. in press. 4. westgard, j. o., barry, p. l . cost-effective quality control: managing the quality and productivity of analytical processes. p. 1-231. aacc press. washington, dc 1986. 307 upsala j med sci 99: 267-270, 1994 5.4 transfer of values from bcr 470 to the nordic protein calibrator ole blaabjergl, mogens blom2, arto icen3, kerttu irjala4, anders carlstrom5, per hyltoft petersenl, 1. department of clinical chemistry, odense university hospital, dk-5000 odense c, denmark. 2. department of clinical chemistry, hjarring sygehus, dk-9800 hjarring, denmark. 3. department of clinical chemistry, helsinki university hospital, sf-00290 helsinki, finland. 4. central laboratory, turku liniuersity hvspital, sf-20520 turku, finland. 5. department of clinical chemistry, danderyd hospital, 4 1 8 2 88, danderyd, sweden. a protein calibrator must contain the measurants in genuine form, be stable, consist of a matrix which very closely resembles the serum samples from patients, and the target concentration values must be traceable t o the highest level of trueness in the hierarchy, whether a reference method or a reference preparation. the nordic calibrator fulfils the three first conditions (chapter 5.3) and with the ifcc/cap/bcr 470 preparation (chapters 5.1 and 5.2 and ref. 1) available in 1993, the task was to transfer the values from this reference preparation t o the nordic protein calibrator. transfer of concentration values ifcc has set up a protocol for correct transfer of values from ifcc/cap/bcr 470 to any secondary calibrator (chapter 5.2) and the transfer was performed in five nordic laboratories according to this protocol. the five laboratories were 1. 2. 3. 4. 5. department of clinical chemistry, odense university hospital, dk-5000 odense c, denmark, using a turbidimetric method (cobas fara@, roche). department of clinical chemistry, hjarring sygehus, dk-9800 hjarring, denmark using a turbidimetric method (cobas fara@, roche). laboratory, helsinki university central hospital, sf-00290 helsinki, finland using a turbidimetric method (hitachi 91 1@,). central laboratory, university hospital of turku, sf-20520 turku, finland using a nephelometric method (bna@, behringwerke). department of clinical chemistry, danderyd hospital, s-182 88, danderyd, sweden using a (cobas fara@, roche). 267 all measurements were performed according to the ifcc recommendations and antisera from behringwerke were used for the bna-measurements whereas, the other laboratories used antisera from dako. the data from all measurements were computed by saren blirup and per just svendsen (both dako) accordmg to the ifcc protocol and the results for individual laboratories together with mean and standard deviation are shown in table 5.4.1. table 5.4.1 target concentration values for the nordic calibrator. values from 5 laboratories and their mean and standard deviation. measurements and calculations according to the ifcc protocol. protein dand prealbumin 0.3219 albumin 44.137 orosomucoid 0.7773 al-antitryps. 1.1150 haptoglobin 1.0273 transferrin 2.5668 igg 10.088 iga 2.0860 igm 0.8354 odens h j m - 0.3360 0.3355 42.161 43.184 0.7791 0.7685 1.1677 1.2101 1.0191 1.0201 2.5488 2.5357 9.473 9.641 2.0600 2.0768 0.8402 0.8269 hels 0.3146 43.453 0.7914 1.1883 1.0317 2.5682 10.041 2.1103 0.8471 turk 0.3329 45.202 0.7887 1.4250" 1.0206 2.5709 10.129 2.2019 0.8879 mean sd - 0.3282 0.0095 43.627 1.1311 0.7810 0.0092 1.1703 0.0407 1.0238 0.0055 2,5581 0.0152 9.874 0.2974 2.1070 0.0561 0.8475 0.0238 cv % 2.89 2.59 1.18 3.48 0.54 0.60 3.01 2.66 2.80 ~ ~ ~~ ~ ~~ ~ ~~~ ~ *: this value is approx. 20 % higher than the others, and has been omitted from the calculations of mean the measurements of target values for haptoglobin and transferrin are very precise with cv-values close to 0.5 %, and for orosomucoid with approx. 1 %. most of the proteins have cv-values between 2 and 3 % which is considered sufficient for the assignment of the values, but could be investigated further. for prealbumin, helsinki has a lower value than the rest. for albumin turku has the highest value. for igg, odense and hjarring have the lowest values. for iga and igm, turku has the highest values. however, there is no clear picture, except from the highest values for iga and igm for nephelometry (turku). regarding al-antitrypsin, there is a discrepancy between turku, with the nephelometric method and antiserum from behringwerke, and the rest, with turbidimetric methods and antiserum from dako, and even among the latter group, danderyd has a value approx 6 % lower than the rest. the nephelometric value has been omitted from the calculation of the mean and the cause of the difference is investigated further in section 5.5. 268 discussion the transfer of values has been performed according to the ifcc protocol and the procedures have been followed with painstaking accuracy, so the results must be considered the best obtainable. except from al-antitrypsin, the results resemble the assignment of values to ifcc/caf’/bcr 470 and the outcome must be considered as the level of the state of the art, although, we hoped for cv-values below 2 % for all the proteins. the immunoglobulins are heterogeneous with more than 1,000,000 forms, so the compositions of the pools may be dfferent although the number of individuals used for the production of the calibrators is very large. this might explain the higher values for iga and igm by nephelometry. but for prealbumin and albumin there is no simple explanation. al-antitrypsin is, by all means, a real problem, and the fraction of z-phenotypes is too small to give such an effect (of 20 %). the explanation must be looked for in the degree and type of denaturation of the protein in the two pools. the procedures for delipidation are different and the storage conditions (freeze dried and liquid frozen) are also hfferent. both may result in varying types and degrees of denaturation which may result in different reactions with antibodies in the measurements, and thereby different types and sizes of irnmunocomplexes, which may be detected differently by turbidimetry and nephelornetry. if so, then the target values cannot be used for the both types of analytical principles. the value assigned to the nordic calibrator, thus, is only relevant for turbidimetric methods. other problems with al-antitrypsin are discussed in chapter 6. from another point of view the acceptability of the assigned values, is t o compare the dispersions to the analytical quality specifications for using common reference intervals for the plasma proteins. if we look at the cv-values, the problem seems to be serious only for albumin, where the acceptable analytical cv is approx. 4 % for using common reference intervals, which should be compared to the obtained of approx. 3 %. this doesn’t leave much to the laboratories’ analytical imprecision. the cv of the transfer, however, is just an estimate of the dispersion of bias-values among the transfer-laboratories and the estimated bias between the two most diverging laboratories is approx 7 %, which is far outside the acceptable 2 %. the same contemplations should be made for prealbumin and for igg, where the acceptable bias is approx. 5 %, but less serious than for albumin. except from al-antitrypsin, the transferred values, however, give the basis for the use of common reference intervals, which in spite of the weak points, is f a r advantageous to the current situation (cf. chapter 7). 269 acknowledgements we are in debt of gratitude to aija helin, kari mattila, marianne kaehne, and inger norgaard for the transfer of values, and to soren blirup-jensen and per just svendsen for computations of these data. further, we want to thank bcr for providing the ampouls of crm 470 free of charge, and the national societies of clinical chemistry and control groups in the nordic countries for economical support. references 1. whicher jt, ritchie rf, johnson am, baudner s, bienvenu j, blirup-jensen s, carlstrom a, dati f, milford-ward a, just svendsen p. new international reference preparation for proteins in human serum (rpphs). clin chem 1994;40934-8. 270 upsala j med sci 91: 218, 1986 improved reliability of hemoglobin assay with hemocue for satellite and physicians office testing marianne falkensson and henning von schenck department of clinical chemistry, linkoping university, linkoping, sweden introduction: filter photometry of cyanomethernoglobin at one wavelength fol lowing dilution of capillary blood with drabkins solution according to the rec mndations of the international comnittee of standardization in hematology (icsh) is flawed by imprecision related to high sample dilution and by possible errors due to sample turbidity. this study reports our experience of hanoglo bindetermination in satellite and physicians office testing, spot, with a new equipment, hemocue. this equipment is expected to deliver mre precise results because sample dilution is unnecessary and turbidity errors eliminated by read ings at two wavelengths in a microcuvet. methcd: we placed a filterphotometer and a hemocue instrument at the emergency unit of our hospital. the personnel was trained for hanoglobindetermination. capillary blood was obtained and split samples analysed as singles in the filterphotometer according to the icsh and in the hemocue according to the manufacturers instructions (leo diagnostics, helsingborg, sweden). accuracy was checked simultaneously in mta-blood analysed in the hemalog 8/90 as ref erence (r). results: the ease of handling the hemsue equipment was appreciated by the users. the regression equation for the filter photometer (f'p) was fp = 1.02 (r) 10 (n=69, r=0.69). the regression equation for the herrccue (hc) was hc = 0.97 (r) 0.5 (n=69, r=0.95). conclusion: the hemocue simplifies hemoglobinometry and clearly enhanced the reliability of the results by delivering mre precise and specific results as judged from the improved coefficient of correlation. it m y thus be recmnded for spot. 218 upsala j med sci 91: 239-243, 1986 autoradiography and histochemistry of mineralized tissues by means of the ullberg freeze-sectioning technique lars hammarstrom department of oral pathology, school of dentistry, karolinska instituter, box 4064, s-14104 huddinge, sweden abstract the present paper is a review of some different applications of whole-body freeze-dried sections to illustrate the usefulness for studie on mineralized tissues. thus the application of autoradiography fluorescence microscopy and enzyme histochemistry and their combinations is described. the whole-body freeze sectioning technique was originally developed for studies on the distribution of labelled d r u g s i n small laboratory animals ( 5). however it has also proven to be very useful for studies on the mineralized tissues since it allows the sectioning of all the tissues of the body, including bone and teeth without decalcification and any other tissue preparation than freezing. this is a review of some results obtained from studies on these tissues. one of the advantages with the method is that all tissues may be analyz ed without any preselection of tissues to be studied and it has lead to many unexpected results. one of the very first findings of this kind was the observation by andre (1) that 3h-labelled tetracycline was accumulated in the teeth and bone. unfortunately very little attention was paid to it at that time. not until 1962 it was reported that the teeth of children given tetracycline were markedly discolored as well a s malformed ( 6 ) . if andre's unexpected observation had been more widely known many of these develop mental disturbances of the teeth may have been avoided. the affinity of the tetracyclines to the mineralized tissues in combination with their property to be fluorescent in ultraviolet light, have made them useful tools for mor phological studies on mineralized tissues and the whole-body sections have also been used for fluorescence microscopy. the fact that the mineralized tissues could be sectioned without pre deding decalcification o r any other preparatory procedure other than freez ing made it possible to study the distribution of isotops with a very short half-life such as fluoride-18. by means of the whole-body technique eriksson and ullberg ( 3 ) showed a very selective accumulation and retention of fluoride in the mineralized tissues. appelgren, eriksson and ullberg ( 2 ) took advantage of the differences in physical half-life between fluoride-18 and calcium-45 and showed in a double isotope autoradiographic study that the accumulation of fluoride in the mineralized tissues is even more selective than that of calcium. they also noted a difference in the distribution of the two elements in the developing enamel. in a later study i found that the uptake of fluoride in the developing mainly occured during the stage of enamel matrix formation while the main uptake of calcium occurred at a later stage (4). this has been confirmed in other studies and there is still a debate about the mechanism of binding of fluoride to the developing enamel. the usefulness of the whole-body sectioning technique is illustrated by some enzyme histochemical studies in which the tissue-specific localization of some isoenzymes is demonstrated. the classic enzyme in studies on mine ralized tissues is alkaline phosphatase. in a whole-body section of a young rat alkaline phosphatase activity can be demonstrated in a number of tissues such as bone, teeth, intestinal mucosa, kidney, adrenal cortex, lung and brain. if a section is heated to 56' for 60 min. most of the alkaline phos phatase is inactivated except that in intestinal mucosa, bone and teeth. fig. 1 whole-body sections of a young rat incubated for alkaline phos phatase showing all the enzyme activity ( a ) and that of the heat inactivated enzyme ( b ) . 240 a detailed study on developing teeth of monkeys showed that the alkaline phosphtase activity in the capillary walls of the pulp was sensitive to vanadate in contrast to the activity in dental hard tissues. the activity in the cells forming the fig. 2 frozen sections through the first molar of a young monkey showing the activity of alkaline phosphatase ( a ) and that of vanadate resistant alkaline phosphatase (b). not the inhibition of the capillary enzyme ac tivity in the pulp. also the tissue specific distribution of isoenzymes of acid phosphatase can be demonstrated by means of the whole-body sections. incubation of a whole-body section of a young rat for acid phosphatase resulted in a mark ed staining of a number of tissues such as bone, teeth, salivary glands, keratinized epithelium, adrenal medulla, liver, kidney and intestinal mucosa. when fluoride was added to the incubation medium most of the acid phos phatase activity in the soft tissues was almost completely inhibited, while there was still demonstrable activity in bone and teeth. however, a more detailed analysis revealed that osteoclastic acid phosphatase was inhibited while the osteoblastic activity of the enzyme was still demonstrable. copper inhibited the activity in the osteoclasts but left the enzyme activity i n most soft tissues apparently unaffected. tartrate inhibited the activity of all the soft tissues, the osteoblasts and the cells of the dental tissues. the only activity that seemed to remain unaffected was the osteoclastic acid phos phatase. we have purified the osteoclastic acid phosphatase , characterized it and produced polyclonal antibodies against it. immunohistochemistry with polyclonal antibodies showed that the enzyme is present in the ruffled border area of the osteoclasts. 24 1 a e g fig. 3 the activity of acid phosphtase in young r a t s . in a and b the sections have been incubated without any inhibitory agent. in c and d copper ( 1 0 mm), in e and f fluoride ( 1 0 0 mm), in g and h t a r t r a t e ( 1 0 0 mm) have been added to the incubation medium. the results are described in the text. 242 isoenzymes of acid phosphtase with inhibitor characteristics similar to t h e ones found in the cells of bone and teeth have also been found in the bacteria that are involved in the degradation dental tissues during the caries process. an interesting; observation was that fluoride inhibits acid phosphtase in bacteria at t h e surface but not t h e acic! phosphatase activity of the bacteria invading the dentinal tubules. there a r e clinical reports saying that teeth in fluoridated areas have small entrance cavities but wide cavities underneath the surface. histochemical studies on frozen sections through dental calculus have shown that alkaline phosphatase in the bac teria located in a v e r y similar to the formative cells of the mineralized tissues, suggesting an enzymatic process r a t h e r than a passive precipitation of mineral. conclusions enzyme histochemical methods for the demonstration of alkaline and acid phosphatase have been applied to the tape-carried freeze-dried whole-body sections. by means of inhibitors tissue specific localization of isoenzymes has been demonstrated. references 1. andre, t . : studies on the distribution of tritium-labelled dihydrostreptomycin and tetracycline in t h e body. acta radiol. suppl. 142, 1956. a comparison of the distribution of radioactive fluorine and calcium by use of double-isotope autoradiography. acta physiol. scand. 53:339-347, 1961. autoradiographic investigations of the distribution of f18 in mice and r a t s . acta odont . scand. 1 6 : 363-374, 1958. 4 . hammarstrom , l . : distribution in developing r a t enamel of imultaneously injected fluoride and calcium. scand. j . dent. res. 79:369-376, 1971. studies on the distribution and fate of s35-labelled benzyl penicillin in the body. acta radiol. suppl. 118, 1 9 5 4 . 7 . wallman, i . s . & hilton, h . b . : teeth pigmented by tetracycline. lancet i:827-829, 1 9 6 2 . 2 . appelgren, l.-e., ericsson, y . & ullberg, s . : 3. ericsson, y . & ullberg, s . : 5 . ullberg, s . : adress for reprints: professor lars hammarstrom, department of oral pathology, school of dentistry, box 4064, s-141 0 4 huddinge 243 upsala j med sci 99: 385-390, 1994 9 discussion and conclusions per hyltoft petersenl, ole blaabjergl, kerttu irjala2, 1. department of clinical chemistry, odense university hospital, dk-5000 odense c, denmark. 2. department of clinical chemisty, turku university hospital, sf-20520 turku, finland. 9.1 discussion of the protein projects the first aim of the nordic protein project was to improve analytical quality of specific protein measurements in the nordic countries. the main problems were a) the variability of commercial protein calibrators and b) the specificity problem of turbid samples. a) the problem of calibration was solved by introdution of the nordic protein calibrator to all the participating laboratories for daily use. this reduced the variability of calibration considerably (except for a,-anti trypsin). here, the ifcc-initiative of producing an international protein reference preparation (ifcc/caf'/bcr 470) was a lucky co-incidence (even though it delayed the project for nearly two years) as the traceability of the concentration values was established. further, the ifcc-preparation was a success, and all nordic countries have decided to use only protein calibrators with concentration values traceable to this, and probably us and the most of europe will do the same. this, makes the nordic calibrator a valuable link in the hierarchical structure of traceable calibrators and concentration values. the success of the project with the introduction of the nordic protein calibrator was therefore, to a large extent supported by the ifcc-reference preparation. on the other hand, the nordic protein project may make the introduction of the ifcc reference-preparation easier, due to the advantages from the project and from the establishment of common reference intervals for the nordic countries. 385 b) the problems with measurement of turbid samples were evaluated in the project and it was found that the turbidimetric analytical principles in current use are robust, whereas, the nephelometric principles are sensible to turbidity, and therefore need clearing best by ultracentrifugation, e.g. by a top-desk ultracentrifuge before measurements. the combined introduction in the project of analytical quality specifications related to the use of common reference intervals and the co-ordination with projects which established these common reference intervals was an important improvement of the total concept of the projects. thereby, another traceability between the nordic protein calibrator (to bcr 470) and the common reference intervals was established. further, the control system was related to a) the two analytical problems (calibrator and specificity) and b) the analytical quality specifications, as derived for the establishment of common reference intervals. these two conditions, were combined in the design of control systems with control samples produced for purpose a) and with the measure of analytical quality according to b). the general evaluations revealed that the calibration with the nordic protein calibrator made the analytical quality for immunoglobulins, orosomucoid and haptoglobin acceptable for the use of common reference intervals in the majority of laboratories. evaluations of the individual laboratories were divided into a) specificity (capability to handle turbid samples) b) calibration function (i) proportionality (ii) reproducibility of the function c) general reproducibility (i) within-run imprecision (ii) between-run imprecision and this was a guideline for internal trouble-shooting. 386 for the two proteins s-albumin and s-al-antitrypsin the aim was only fulfilled to a partial extent. the analytical quality specifications used for s-albumin are very demanding and it looks like the immunological measurement procedures are less reproducible, probably due to t h e immense dilutions which are necessary for getting the measurement signals down to a level where the instruments are able to measure the signals. for the dye-binding methods other problems may be actual, but only the validity of calibration functions was investigated and found insufficient in most laboratories. for sq-antitrypsin the nature of problems was different. the transfer of concentration values from the ifcc reference preparation to the nordic calibrator revealed a difference related to analytical principles of about 20 %, which was related to changes in the structure of the protein in the calibrator as disclosed by different antisera. further, the control project disclosed a variation, which was not consequent but might be explained by a variable denaturation of the protein not disclosed by the comparison of different calibrator-pools produced over eight years. one reason could be the mailing and storage conditions in the participating laboratories in the project compared to the stable -80 "c conditions for the calibrator pools. these facts, reduce the validity of the calibrator and the applicability of the reference intervals for this protein. the communication was mainly one-way, but telephone calls and letters from some of the participants dsclosed general problems like the use of molar units in denmark, without using updated molar weights. the problems specific for the laboratories could often be solved by telephone. whether the populations really are homogeneous for the plasma proteins investigated except from s-haptoglobin and s-iga and thereby gives the basis for common reference intervals, seems t o be confirmed for the non-immunoglobulin proteins, whereas, the immunoglobulins may turn up more deviating in an extended evaluation. this is impossible to answer now, but the reference intervals estimated in these projects are for the time being the best basis for common reference intervals, and much better than if each laboratory established its own intervals. in conclusion, the aim of improvement in protein measurement in the nordic countries was succesful and the aspects of traceability to crm 470 and the introduction of common reference intervals further improved the total quality for the patients. 387 9.2 discussion of general applications the second aim was to develop a more general model for analytical quality management, which could be applied to other laboratory analytes as well. the principles as well as the practical accomplishment show that it is possible for plasma proteins, and the model should be transferable to other laboratory quantities. but a number of specific problems for these, must be considered separately for each analyte. a) b) c) d) e) the general strategy of establishing common reference intervals is applicable to all naturally occurring components, but it might be more relevant to apply a clinical strategy, which should be confirmed by clinical investigations, and the analytical quality specifications should be formulated according to this strategy. the use of a serum pool calibrator may be rather special for plasma proteins and for other quantities, other types of calibrators may be more relevant. the establishment of a common reference basis (a reference method or a stable reference preparation) is essential and should be created on an international level when possible. problems with specification of the structure of the analyte (to be quantitated) are general, but must be solved according to t h e component under consideration. specificity and interference problems must be identified and the most important should be challenged first. the principle, however, of using pairs (or more) of identical preparations, except from the content of suspected component, is valid to all analytes. the design of the control system and the frequency of surveys should be reasonable also for other quantities, as there is no need for frequent control of the permanent factors, and the variable factor cannot be monitored by external systems (unless there is a daily electronic communication) and this should be handled by internal control. our design with few specially produced control samples and the evaluations with analysis of variance as well as different types of difference plots (measured minus target, and measurements of pairs) should be applicable to any other analyte. 388 the workload, however, by establishing a comparable project is considerable, and a lot of experts need to be associated, as well as assistance from a lot of colleagues is needed for the completion of such a project. and it is expensive, the investments by nordkem for meeting activities and publication of the book were 69 500 finnish mark and for labquality the expenses for the calibrator and control samples were about 200 000 finnish mark (which was paid back). further, the nordic societies of clinical chemistry supported the projects with 10 to 20 000 danish kroner each. moreover, all the laboratories and their staffs have invested money and a lot of time, both in the control project proper, but also in the extra work of additional analyses and blood sampling for the reference interval projects. further, a lot of experts are needed for data handling and for computations as well as for advises needed throughout the whole project. to our knowledge, the only project where all the same elements have been integrated to a comparable level, is the us national cholesterol education program. this programme, however, is performed professionally and accomplished on a large scale. here, the overall strategy is clinical, but except from this, the elements are the same as in the nordic protein projects. in conclusion, this type of integration of all the elements of analytical quality with the creation of reference intervals based on a common traceability should be applied to all naturally occurring analytes in order to ensure patients reliable results over time and geography. 389 upsala j med sci 95: 279-285, 1990 measuring cholesterol a handbook for out-patient clinics and laboratory personnel peter nilsson-ehle department of ciinicul chemistry, uniwrsity of lund, sweden introduction elevated serum cholesterol concentrations is a major risk factor for cardiovascular disease. if cholesterol levels are reduced, the relative risk decreases. with the introduction of new, potent hypolipidemic drugs there is now an increasing interest in cholesterol-lowering programs, with or without support of screening by measurements of s-cholesterol in the population in general or in specific risk groups. guidelines for such intervention programs have been issues by international and national authorities. however, it has so far not been possible to determine the cost-effectiveness of such programs. one important component in these considerations is the efficacy of the screening method used to distinguish subjects who should be treated from those who should not, and its ability to properly register alterations in cholesterol levels in individuals and populations as a result of intervention. the quality requirements for biochemical measurements, when applied to health surveys or screening programs, seems somewhat paradoxically to be stricter than when such measurements are used in the traditional situation to confirm a diagnosis of hyperlipidemia or to monitor patients with overt lipid derange ments during treatment. in screening programs, important medical decisions are based almost entirely on the result of a biochemi cal measurement, and the differences which occur after in tervention in subjects with moderate hyperlipidemia are generally quite small and therefore difficult to detect and quantitate. in patients with hyperlipidemia, in contrast, the alterations and differences in lipid levels are much more pronounced and therefore easier to monitor. a l s o , medical decisions are 279 generally based on a more comprehensive evaluation of the , multiple factors that may influence choice of therapy and prognosis in the individual patient. in an attempt to inform about the chatacteristics of the s-cholesterol measurement and to facilitate its use in health surveys and screening, the swedish society for clinical-chemistry has produced a pamphlet (1) which has been distributed to out-patient clinics and health centers throughout sweden. the primary target groups are nurses and technicians responsible for sampling and for cholesterol measurements with traditional methods or with decentralized techniques. the information covers various aspects on biological variation, the sampling procedure, and analytical performance. finally, some calculations on the performance of the s-choleste rol measurement in screening programs and in patient monitoring are presented. the major points are summarized below. much or the information is based on recent investigations (2, 3). analytical variation both standard methods and the dry chemistry techniques have low intra-assay variation for cholesterol (cv <1-2%). the inter-assay variation is clearly dependent on the setting but can be reduced to 2-3% (cv) provided that proper quality assurance programs are carried out. analytical bias samples drawn in edta tubes yield significantly lower choleste rol concentrations than serum samples, while there is no significant difference between heparin plasma and serum. on the average, cholesterol concentrations in edta samples are 4 % lower than in serum. in edta tubes which have not been properly filled the difference can amount to 12-15%. the lower concentrations measured in edta samples can be attributed to the osmotic effect of the salt, which extracts fluid from the red blood cells, thereby diluting the plasma. 280 errors associated with the sampling procedure the random error associated with blood sampling, as assessed by comparing samples obtained by simultaneous venous punctures in the left and right antecubital vein, is 2-3% (cv). however, the sampling procedure is critical because of the heavy impact of posture on the concentration of cholesterol in the cir culation. in sitting and in the supine position, s-cholesterol levels are significantly lower than in standing; the average differences are 7 and 11%, respectively, but in extreme cases the difference may amount to 22%. intraindividual biological variation the variations in s-cholesterol over the day in healthy subjects is <5% (cv). the variation in fasting samples obtained on consecutive mornings is of similar magnitude. in women, there is a small increase in s-cholesterol around the time of ovula tion, but the biological variation over a month is still <6% (cv) discussion and recommendations the technical performance of modern s-cholesterol measurements seems sufficient to meet the high standards of screening activities. the inter-assay variation, which is relevant for patient monitoring, can be kept below 3 % provided that proper quality assurance programs are enforced. this holds true also for the dry chemistry equipment primarily designed for decentralized laboratory work. however, it should be emphasized that proper training and instruction is critical for this setting. because of the marked and potentially unpredictable reduction of s-cholesterol which may occur in edta blood it is strongly recommend that analyses of lipid and lipoprotein concentrations be performed in serum. if an anticoagulant is needed heparin is the substance or choice. the errors associated with the sampling procedure can also be kept within acceptable limits. however, information on the marked effects of posture is essential to warrant an informative result. 28 1 we strongly advocate the recommendations of the international , federation for clinical chemistry, which state that all venous blood sampling should be performed after the subject has been resting in recumbent position for 15 min. the biological variation in young healthy volunteers is about 5% (cv) under various conditions. also including the trends for serum lipid alterations during the menstrual cycle, the biologi cal variation of s-cholesterol is not greater than 6% (cv) . since the concentration of cholesterol in the circulation is not affected by food intake, samples can be taken at any time during the day to be representative. instructions given to the subjects before sampling, in order to minimize biological variation, include abstinence from alcohol the day before sampling, since alcohol intake may markedly increase lipid levels. also, strenuous physical activities should be avoided the day before sampling, since this may decrease s-cholesterol concentrations. besides these short-term influences on serum lipoprotein levels, it is well known that transient alterations in lipid and lipoprotein levels occur in a variety of situations, which may invalidate the result of serum lipid measurements. of special interest in this context is the marked reduction in s-cholesterol which occurs as a result of the acute phase reaction after infections and after myocardial infarction. to ensure a represen tative result, sampling for serum lipids should be postponed till 3 months after the acute phase. the differentiation between analytical error, sampling error and biological variation is necessary to accurately evaluate the contribution of these sources of variation to the total variation of the cholesterol measurement. fig 1 illustrates that the sampling error and the analytical error, when procedures are carried out competently, is minor compared to the biological variation, which sets the limits for the performance of the s cholesterol measurement. under optimal conditions, the total variation for the s cholesterol measurement is around 6,5%. consequently, the average, typical value for an individual, with 95% probability, is in the range 213% (_+2 sd) from the reported value (e g, reported value 6,5 mmol/l corresponds to 6,65 7,35 mmol/l). a change in s-cholesterol must amount to 18% (2,8 x 6,5%) to be 282 detected with 95% probability. to detect an expected decrease in s-cholesterol e g during cholesterol-lowering treatment (one tailed test) the reduction must be at least 15% (2,3 x 6,5%) to be detected with 95% probability. if smaller reductions are to be detected, several samples have to be obtained on different occasions to obtain a more accurate assessment of the typical cholesterol level. for example, by using the mean of 2 (3) determinations it is possible to detect changes of 11 (8,5) % (15/v2 and 15/q3, respectively). it should be pointed out that the performance of the method, especially in the screening situation, is also profoundly influenced also by a possible systematic analytical error (bias, inaccuracy) which will primarily affect the number of individuals correctly assigned to intervention or non-intervention. the interaction between random and systematic errors in the screening situation is discussed in another contribution this volume (hyltoft-pedersen et al.) . comments about 10 000 copies of the pamphlet have been distributed through the central clinical chemistry laboratories in sweden. judging from spontaneous comments from the target groups, the information is well received and contributes substantially to the improvement of laboratory procedures. also, there has been considerable interest from physicians, indicating that it is important to present this type of information to all levels of medical personnel. furthermore, some drug companies and manufac turers of analytical instruments have incorporated the pamphlet into their information with positive response from their customers. 283 biological variation 3,99% variation at sampling 3,04% analytical variation 1,15% i i i1 i i l l biological variation i1 3,99y0 i i i i + analytical variation 5,15% ficrure 1. the contribution of biological variation, sampling error and analytical error to the total variation of a s-cholerol measurement carried out by trained personnel. the numbers have been calculated by addition of random variances, i e (total variation) * = (biological variation) + (analytical error)2 + (sampling error) * 284 references: 1. att mata kolesterol. en handbok for mottagningar och labora toriepersonal. svensk forening for klinisk kemi. almqvist och wiksell, 1990. 2. nilsson-ehle, p i nordin g i nilsson, j e tryding n. 1989. kolesterol svhrare att mata an att sanka? lakartidningen 14, 1263 1269. 3. nilsson-ehle p. 1990. biological and analytical variation of the determination of serum cholesterol levels: implications for screening and patient monitoring. submitted for publica tion. correspondence: peter nilsson-ehle, associated professor, department of clinical chemistry university hospital s-221 85 lund, sweden 285 upsala j m e d sci 91: 222, 1986 evaluation of blood ammonia checker according to daiichis system , maria wettstam-andersson university hospital, department of clinical chemistry, uppsala, sweden introduction p r e s e n t a t i o n o f d a i i c h i s system f o r d e t e r m i n a t i o n o f b l o o d ammonia i n c a p i l l a r y whole b l o o d . a c c o r d i n g t o t h e manufacture, t h e system has a c l i n i c a l use i n s c r e e n i n g f o r hyperammoneniia and s t a t p r o c e d u r e f o r l i v e r coma. the system has been t e s t e d f o r l i n e a r i t y and i m p r e c i s i o n . principals whole b l o o d , 20 v1 i s a p p l i e d t o a r e a g e n t p l a t e w h i c h c o n t a i n s an a l k a l i n e b u f f e r ( s o d i u m c a r b o n a t e , sodium b i c a r b o n a t e ) . the specimen becomes a1 k a l i n e and t h e ammonia i s l i b e r a t e d f r o m t h e b l o o d . the ammonia gas d i f f u s e s t h r o u g h a p o l y p r o p y l e n e f i l m a t t h e b o t t o m o f t h e r e a g e n t p l a t e , and p e n e t r a t e s i n t o a c o l o r d e v e l o p i n g s e c t i o n , w h i c h i s t r e a t e d w i t h a ph b u f f e r ( b r o m c r e s o l g r e e n ) . the degree o f c o l o r development i s p r o p o r t i o n a l t o t h e amount o f ammonia r e l e a s e d . the b l o o d ammonia c o n c e n t r a t i o n w i l l be d e t e r m i n e d r e f l e c t r o m e t r i c a l l y b y i n s e r t i n g t h e t e s t p l a t e i n t o t h e " b l o o d ammonia c h e c k e r " . results a r e f e r e n c e v a l u e o f 71+21,8 u m o l / l (mean + sd) was o b t a i n e d b y a n a l y s e s the l i n e a r i t y o f t h e method was good i n t h e r a n g e 25-270 p m o l / l , o f c a p i l l a r y b l o o d f r o m 22-healthy i n d i v i d u a l ? . t a b l e 1. i m p r e c i s i o n o f t h e method a t d i f f e r e n t c o n c e n t r a t i o n l e v e l s . conc. l e v e l ( u m o l / l ) n x + sd cv % 9 29 +-11.6 40.0 5 7 7 t 6.4 8.3 10* 123 t 13.1 10.7 5** 268 '7 30.0 11.7 5** 323 11.4 3.5 * the i n c r e a s e i n nh ' was a c h i e v e d b y p l a c i n g a c u f f a r o u n d t h e b i c e p s m u s c l e i n f l a t e i t $0 a p r e s s u r e e x c e e d i n g t h e s y s t o l i c p r e s s u r e by 30 mmhg, and p e r f o r m i n g m u s c u l a r e x e r c i s e . whole b l o o d supplemented w i t h ammoniumchloride. ** conclusions the " b l o o d ammonia c h e c k e r " i s a s m a l l e a s y t o use system w h i c h c o u l d be u s e d i n bed s i d e t e s t i n g . t h e r e i s , however, a r a t h e r pronounced d i f f e r e n c e i n venous and c a p i l l a r y b l o o d ammonia c o n c e n t r a t i o n s . due t o t h e h i g h i m p r e c i s i o n i n t h e l o w c o n c e n t r a t i o n r a n g e t h e system i s n o t s u i t a b l e f o r venous b l o o d specimens. 222 upsala j med sci 100: 11-18, 1995 a short survey of the scientific work of torsten teorell karl johan obrink from the department oj'physiology and medical biophysics, biomedical center, uppsala university, uppsala, sweden t'ursten t'eurellwas born in 1905 and already as a schoolboy n', as we used to call him, was interested in chemistry. he had a small laboratory of his own in the basement a t home where he collected minerals. his dream was t o become a mining engineer. he even practised at a railway laboratory when he was 15. his test in the swedish language for the matriculation examination dealt with "the law of mass action", a subject that he later on used i n a work on antigen antibody interactions. however, the 1920's were characterized by economic depression and becoming a mining engineer remained just a dream. instead he enrolled as a medical student at the karolinska institute in stockholm and was soon working as a young assistant in the department of medical biochemistry under prof. einar hammarsten. he was entrusted with the teaching of physical chemistry to the medical students already when he was 21. this was the time when concepts like ph and donnan equilibrium first appeared and when acid-base determinations were for the first time possible by the van slyke apparatus. teorell's father, who noticed his disposition for such matters gave him hober's "die physikalische chemie der zelle und gewebe" as a christmas present in 1926. this book became a bible for the young man. hammarsten, who noticed torsten's ability, set him to work on the topic of how thymonucleic acid and protein were redistributed when a cell was about t o divide. (thymonucleic acid is what we today know as dna). this topic resulted in a few papers, and though it did not then attract his interest i t brought him further in contact with the nucleus-containing red blood cells of the salamanders, t o which he returned at a later stage. whether teorell suffered from stress o r overwork o r something else i do not know, but for some reason he developed a gastric ulcer. and this aroused an interest in gastric acid. hammarsten supported his interest as he himself was studying the intrinsic factor, important for pernicious anemia. at the end of the 1920's a popular hypothesis about the formation of gastric acid was the notion that acid was formed by the hydrolysis of ammonium chloride. teorell 1 1 therefore developed an analytical method for the determination of ammonia in cat experiments, but found no evidence for that hypothesis. in order to improve his experiments he started to work with isolated but perfused stomachs and for the perfusion he needed heart-lung preparations as used by starling. he received a stipend to work at starling's old department at university college in london. this was before the time of heparin so all blood had t o be defibrinated before every experiment. even if he did not succeed in getting the important results he hoped for, he learned physiological techniques thoroughly. however, a few of his discoveries are still important today: the energy demand was calculated t o be 900 gcal per litre gastric juice of which 94 96 could be accounted for by the accumulation of the hydrogen ions. he showed that the gastric mucosa has a negative respiratory quotient, i.e. for the formation of gastric juice the mucosal cells consume not only oxygen but also carbon dioxide. the consequences of the resulting negative bohr-effect still remain to be studied. fig. 1. "he "exchange diffusion" concept for the regulation of the gastric acidity presented in teorell's dissertation (2) teorell's stay in london seems t o have been of fundamental importance for his further scientific career, because there he met two of the contemporary scientific giants namely a.v. hill and f.g. donnan. without doubt they influenced the main topic of his thesis entitled "untersuchungen uber die magensaftsekretion" (2) and that was the so called "back diffusion theory" (fig. 1). by instilling hydrochloric acid into unstimulated stomachs of cats he found that the hydrogen ions disappeared in a diffusion-like manner. this diffusion theory, as an explanation of the various acid concentrations in the stomach, gave teorell a world-wide reputation. on may 9th 1933 med.kand. torsten teorell defended his thesis. the faculty examiner was the professor of chemistry and pharmacy j o h n sjoqvist, who had himself 38 years earlier written a thesis about gastric secretion. his second examiner (according t o the swedish system) was ulf von euler, who quite recently had defended his own thesis and the third examiner was a friend, torsten h t r o m . 12 membrane transport the diffusion of hydrogen ions through the gastric mucosa could be mimicked by model experiments with artificial membranes and that was the start of an interest that occupied teorell for the rest of his life. in 1933 he not only defended his thesis. he also finished his medical studies, became assistant professor in medical chemistry and in addition a serving naval medical officer in the reserve. in this capacity he often had free hours in his cabin and this time he utilized for studies in mathematics. second order differential equations became everyday fare for him and mathematical language came t o characterize his continued scientific research. he became determined t o acquire a broad and deeper knowledge in science and with a rockefeller fellowship he managed to get to the rockefeller institute in new york. there he worked at the department of general physiology with the well-known professor osterhout, at that time working with the “electric” alga nitella. this model later became an often used model for teorell as he found this alga in uppsala quite near his home. a t the rockefeller institute he was introduced into an extremely productive scientific environment, where besides osterhout names such as michaelis (michaelis menten) and wilbrandt may be mentioned. b fig. 2. the multimembrane technique for analyzing concentration profiles within a homogeneous membrane multimembrane technique fig. 3. demonstration of non-straight-line concentration profiles in a membrane by the teorell was interested in the concentration profiles within the membranes and invented an elegant way of studying them by creating the multimembrane technique (fig 2). by putting several cellophane membranes in series and keeping the liquid interspaces between the membranes thoroughly stirred, he could consider this whole membrane battery as a model of a single membrane with the interspaces as locations within the membrane. with this technique he found that the concentration profiles of ions in the membrane could deviate markedly from straight lines (fig. 3). he 13 further found that sometimes the transport of cations was larger than what could be expected from free diffusion and he postulated that the acceleration of cations coul'd depend on "something" in the membrane itself. this "something" was later identified neutral membrone fig. 4. visualization of the effect of fixed charges in a membrane fig. 6. a resulting concentration profile across a charged membrane of the type in fig. 4 as "fixed negative charges" (fig. 4). at this time his work resulted in an important paper that he, with osterhout's and michaelis' blessing, published in the proceedings of the society for experimental biology and medicine in 1935 (3) just before leaving new york. it consisted of only 3 pages and stated that the presence of immobile groups, e.g. carboxyl groups in a protein containing membrane created due t o their indiffusibility a donnan equilibrium at both sides of the membrane thus leading to a double donnan potential. to this was added the henderson-planck diffusic& potential within the membrane. the total membrane potential was thus the sum of three components (fig. 5). this was something quite novel. the concentration profile within a charged membrane appeared t o be quite different from the profile in a non charged membrane. six months later meyer and severs published almost identical ideas (1) and when the three of them met after the war they decided t o call the new concept the "teorell meyer-sievers theory (tms)". the theory mainly explained the electrical membrane potential but later teorell expanded the theory t o include also ion transport, electrical resistance, electrical rectification and other membrane characteristics. in 1951 he presented a complete theory at a meeting in gottingen, which was later published in 1953 (7). 14 fig. 6. a typical teoreil experimental set-up for simultaneous analyses of a multitude of variables r " : f l v l c;' --@" c; a r a * i +0 0 v ( = * p ) fig. 7. a demonstration of the effect of hydraulic pressure across the membrane. the resulting bulk flow through the membrane will cause a change in the concentration profile and, as a consequence, a change in t h e electrical resistance all calculations and theories were testel in elegant experiments with ingenious models, where all variables could be altered at will (fig. 6). for instance fig. 7 illustrates how water movement through a membrane due to hydrostatic pressure differences will affect the electrical conductance i n the membrane. even if the fixed charge concept was an obvious spin-off from his stay in new york so was probably his discovery of the s o called diffusion-effect. in a few words this describes a generalisation of the donnan effect where the restrictions for ion movements also may depend on diffusion potentials. teorell from now on experimented with three simultaneous forces: the chemical potential; the electrical potential and the hydrostatic potential as illustrated in fig. 8. he made many efforts t o visualize the complicated interactions by building 3 dimensional models. in doing so i t became obvious that sometimes negative conductances were present, i.e. unstable or metastable conditions, that proved t o be of great importance for his later work. 15 0 diffusion. electrophoresis 0 c, concentration e , electrical potential difference p , hydrostatic pressure difference u , mobility c, conductance i , current d , membrane thickness : r e i 2 5 fig 8. the three forces responsible for the property of a permeable membrane: the chemical, the electrical and the hydraulic potentials the membrane oscillator so f a r my description has been based on his scientific progress, but now let us return t o a chronological order. after a short sojourn with the well-known immunologist heidelberger, where he described the precipitin reaction with the use of the law of mass action, he obtained a position as associate professor in medical chemistry at uppsala university in 1936. there he came into contact with arne tiselius and his electrophoresis techniques. it was during such experiments that he later discovered that red blood cell ghosts proved t o be ideal osmometers enclosed by natural membrane material. from 1939 and onwards, due to the war, teorell became involved in various military medical problems, several of them together with the young hans ronge. in 1940 he was appointed professor in physiology a t uppsala university and when he returned to full research activities he once more addressed the gastric mucosa and made a number of important discoveries. together with richard wersall he mapped the electrical impedance of the mucosa (5) and in 1951 he made the important discovery that for each hydrogen ion secreted into the lumen a bicarbonate ion is transported t o the blood side (6), a view that today is generally accepted. after he published the comprehensive monograph about transport processes in membranes in 1953, he thought that at that time it was not possible t o progress any further. but then as he told me one of those few golden moments of a scientist's life occurred one late evening in 1953. he was passing an electric current through a simple porous glass membrane and found as expected a water flow in one direction, but then suddenly the water started t o flow backwards and a long series of oscillations of water movements and electrical potentials began. thus was the 16 e voltage difference fig. 9. the visualization of the "mechano-electrical transduction" "membrane oscillator" discovered. this was the first time that it was shown that a pure electrolyte-membrane system could exhibit changes i n potentials and resistances in the same way as was known for nerves. hodgkin, huxley, code, weidmann and others had concentrated on the development of the action potential but now also changes in conductance and volume could be explained. an important consequence was that now it was possible t o explain how a mechanical stimulus could lead t o a n electrical impulse. this new discovery "the mechano-electrical transduction" could explain how reflexes originate (fig. 9). this new field resulted in an important number of publications and when during the 1950's and 1960's analogue computers appeared they became the totally dominant tool o f teorell's interest. he reproduced physiological reactions of all kinds and his activity was almost unbelievable. in 1970-1971 he collected his findings i n an extensive article i n the "handbook of sensory physiology" (9), b u t his experimentation continued although he had deserted the analogue computer for the digital type. he was encouraged in his work by several american and swedish grants but he was disappointed as he did not seem to see any important impact of his work on physiology. he said: "biophysics is too abstract and multifactorial to be used as long as more diffuse and unassailable hypotheses dominate the thinking". by now, however, teorell was a renowned scientist, very well recognised abroad, much more than at home. with an undertone of sadness he concluded that he had not got the credit for the progress of biophysical science, to which he had devoted his life. but instead his fame came in another way. a certain "youthful iniquity" gave him massive publicity. in 1937 he published two articles on the distribution o f drugs in the body from a 2-955162 17 purely mathematical theoretical viewpoint (4). this was t h e start of pharmacokinetics, a subject that has since become an important topic i n medicine and which is dealt with in another article i n this symposium. at that time it was regarded mostly as a negative merit when he applied for the chair of physiology at uppsala university. today, however, he is in fact considered as “the father of pharmacokinetics”. in spite of his interest in many diversified fields of physiology and biophysics it is easy t o find a common line in his research namely that of creating a physical picture of fundamental life processes. he had no definite long-term goal. most important were the experiments themselves and solving problems. those of us who had the privilege of working with torsten teorell are grateful for being able t o have known a great scientist, a great human being and a dear and beloved friend. references 1. meyer, k.h. & sievers, j.f.: several articles in helv chim acta 19:649, 665, 987, 2. teorell, t.: untersuchungen uber die magensaftsekretion. skand arch physiol, 3. teorell, t.: an attempt to formulate a quantitative theory of membrane permeability. proc soc exp biol med, 33:282-285, 1935. 4. teorell, t.: kinetics of distribution of substances administered to the body. arch int pharmacodyn thbrapie, 57:205-225,226-240,1937. 5. teorel1,t. & wersall, r.: electrical impedance properties of surviving gastric mucosa of the frog. acta physiol scand, 10:243-257,1945. 6. teorell, t.: the acid-base balance of the secreting isolated gastric mucosa. j physiol, 114267-276, 1951. 7. teorell, t.: transport and electrical phenomena in ionic membranes. proc biophys (and biophys chem), 3:305-369,1953. 8. teorell, t.: transport processes in membranes in relation to the nerve mechanism. exp cell res, suppl., 593-100,1958, 9. teorell, t.: a biophysical analysis of mechano-electrical transduction. in ”handbook of sensory physiology”, vol. 1. principles of receptor physiology (ed. w.r. loewenstein), pp. 292-339,1971. 1936. 661225-318, 1933. 18 untitled pregnancy-asaociated myocardial infarction 325 received 17 june 2008 accepted 19 june 2008 upsala j med sci 113 (3): 325–330, 2008 pregnancyassociated myocardial infarction: a report of two cases and review of the literature shohreh shahabi1, nicole a. smith2, charu chanana3, j. dawn abbott4, joshua copel5, john f. setaro6 1 department of obstetrics & gynecology and women’s health, montefiore medical center/albert einstein, college of medicine, new york. 2department of obstetrics and gynecology, natividad medical center, salinas ca. 3department of obstetrics & gynecology, bronx lebanon hospital center, new york. 4division of cardiology, rhode island hospital, brown university, providence, ri. 5department of obstetrics and gynecology, yale university school of medicine, new haven, ct. 6section of cardiovascular medicine, yale university school of medicine, new haven, ct. abstract background: myocardial infarction in pregnancy carries high morbidity. spontaneous coronary artery dissection is one etiology of infarction, and up to one third of cases may arise in the third trimester of pregnancy or within three months postpartum. case: we report two cases of spontaneous coronary artery dissection, one at 34 weeks gestation and one postpartum. both patients were diagnosed with angiography and treated medically and one required percutaneous coronary intervention, with good obstetric outcome and return of cardiac function. conclusion: myocardial infarction, and particularly spontaneous coronary artery dissection, should be in the differential diagnosis of pregnant women presenting with cardiac-type symptoms, despite perceived lack of risk factors. angiography will aid in diagnosis, and multiple therapeutic modalities exist. introduction myocardial infarction (mi) during pregnancy and the puerperium is a rare but potentially devastating event. acute myocardial infarction complicates approximately 1 per 10 000 pregnancies. it clearly causes an increment in both maternal (45%) and fetal mortality, especially reaching the highest levels in the third trimester (50%). (1) historically, the etiology of these events has been poorly understood. with increased use of coronary angiography, trends in coronary lesions are beginning to be identified, and risk factors assessed. nonetheless, care of these patients continues to prove challenging, as few guidelines exist to direct management, and mortality rates remain high. we report two cases of mi secondary to spontaneous coronary artery dissection, one intrapartum and the other postpartum, and review the etiology and management of pregnancy-associated mi. 326 shohreh shahabi et al. case 1 a 32 year old caucasian gravida 12 para 2092 at 34 weeks gestation was referred to high risk obstetrics secondary to a history of multiple abdominal surgeries. while eating lunch in the hospital cafeteria prior to her first appointment, she developed the sudden onset of shortness of breath, diaphoresis, and chest pressure radiating to the right arm described as 10/10 in severity. she was immediately transferred to the emergency department where sublingual nitroglycerin provided some relief. her antenatal course was remarkable for chronic hypertension controlled by methyldopa, hydralazine and nifedipine. additional medications included hydroxyzine and famotidine, while allergies include an anaphylactic reaction to labetalol. her history was significant for asthma, gastroesophageal reflux, and migraine headaches, as well as multiple surgeries, including appendectomy, cholecystectomy, ventral herniorraphy, abdominoplasty, two cesarean sections and three dilation and curettage procedures. she reported nine first trimester losses with three partners. she was a smoker and smoked one half pack year, and had a family history of hypertension. on further history, the patient noted that she had experienced intermittent chest pressure during the preceding months. on physical examination, blood pressure was 157/96 in both arms, and pulse was 113. jugular-venous pressure was normal, heart examination showed regular rhythm with a ii/vi systolic ejection murmur, lungs were clear, extremities revealed trace edema, and deep tendon reflexes were normal. physical exam was otherwise unremarkable. all initial laboratories were within normal limits; however, electrocardiogram revealed an ectopic atrial rhythm and hyperdynamic t waves in leads v4 through v6, which normalized after nitroglycerin. a limited echocardiogram demonstrated mild left ventricular enlargement with a focal area of anterior wall hypokinesis and a normal ejection fraction. the patient was transferred to the coronary care unit for blood pressure control, anticoagulation, and overnight desensitization to metoprolol. in addition to her home medications, she received clopidogrel, heparin, aspirin, and nitrates. cardiac biochemical markers rose overnight, with the third set peaking at cpk 517 units/l, ck-mb 57 units/l , and troponin t 119 ug/l .the decision was made to perform cardiac catheterization with preparations for potential complications requiring emergent cesarean section. angiography was then performed via the femoral approach, revealing a proximal dissection of the left anterior descending artery without evidence of coronary artery disease and normal coronary flow. (figure 1) over the next 48 hours the patient was stabilized, her medications were changed to diltiazem, metoprolol, nitrates, aspirin, and heparin infusion. intermittent fetal heart monitoring remained reassuring, and she was transferred to the maternal special care unit for the remainder of her pregnancy. her hospital course was remarkable for repeat echocardiogram showing resolution of the wall motion abnormality, and for consistently reassuring fetal testing. at 38 weeks, a scheduled cesarean section and tubal ligation were performed, to delivery of a 3200g infant with apgar scores of 9 and 9. all hospital medications were continued after delivery, with the pregnancy-asaociated myocardial infarction 327 exception of heparin, which was replaced by clopidogrel. both mother and infant were discharged home on post-partum day four. postpartum evaluation revealed no evidence of any thrombophilia. case 2 a 32 year old caucasian gravida 1 para 1 presented to the emergency department nine weeks post-partum with the acute onset, one hour prior, of substernal chest pain radiating to the left arm. her pregnancy had been complicated only by preterm rupture of membranes with delivery at thirty-two weeks gestation. she had no medical problems or surgical history, did not smoke or use drugs, and her only medication was multivitamins. her father had had coronary angioplasty at age fifty-five. on examination, the blood pressure was 120/60 in both arms, and pulse was 60. the initial physical examination and laboratory analysis were within normal limits, with the exception of a troponin t of 0.5 ug/l (normal less than 0.4 ug/l), and an electrocardiogram showing nonspecific st abnormalities. she was monitored in the emergency room until a second set of cardiac markers returned, this time showing a rise in the cpk from 95 to 428 units/l, in the mb fraction from 2.3 to 65.6 units/l, and an increase in troponin t to 16.5 ug/l. over the same period, t-wave inversions were seen to evolve in v2. therapy with a glycoprotein iib/iiia inhibitor was initiated, and diagnostic angiography was performed, revealing noncritical lesions in the mid left anterior descending and circumflex arteries, as well as focal hypokinesis of the mid anterior and lateral walls, and a mildly depressed ejection fraction of 40 percent. normal flow was seen in both branches of the left coronary artery and she was pain free, therefore no further intervention was performed. treatment was continued with eptifibatide, heparin, clopidogrel, metoprolol, nitroglycerin, and atorvastatin. the third set of markerss peaked at a cpk of 601 units/l, mb fraction of 104 units/l, and troponin t of 22.3 ug/l. despite continued antithrombotic therapy, she had two further episodes of chest pain relieved by nitroglycerin leading to repeat angiography. at that time, a critical lesion of the circumflex artery was identified. intravascular ultrasound (ivus) confirmed a dissection in the media extending to the adventitia, and compromising the vessel lumen. the lesion was stented, and ivus confirmed that there was no residual dissection. the patient did well, and was discharged home on hospital day four, on clopidogrel, ramipril, metoprolol, atorvastatin, aspirin, and folate. a thrombophilia workup was negative for coagulopathy, and a follow-up echocardiogram performed at six weeks showed a normal ejection fraction. a stress echocardiogram done at 6 months showed mild hypokinesis of the distal lateral wall. comment myocardial infarction (mi) during pregnancy or the puerperium is uncommon, but carries a high risk of morbidity and mortality for both mother and fetus. the etiol328 shohreh shahabi et al. ogy of cardiac events in otherwise healthy young women is unclear, making cardiac complications difficult to predict. surveys of case reports suggest that approximately one-quarter of affected women are hypertensive or smoke, but half have been found to have no cardiac risk factors. multiparity and advanced maternal age may be risk factors. atherosclerosis, thrombosis, vasospasm, and dissection have all been identified as precipitating coronary lesions, though the relative frequency of each is not yet well established. regardless of etiology, it is clear that the vast majority of pregnancy-related cardiac events occur in the third trimester or postpartum (2–4). spontaneous coronary artery dissection (scad) was identified as the cause of mi in both of the cases that we review here. in building upon the review by koul, et al. (3), we were able to identify a total of only 22 cases of intrapartum scad, and 54 cases of postpartum scad reported over the last fifty years. overall, eightypercent of scad cases occur in women, and one-third of those arise in the third trimester of pregnancy or within three months postpartum (4–6). the basis of this relationship has yet to be clearly elucidated, but observational studies suggest that hormonal changes associated with pregnancy along with the hemodynamic stresses present during labor and delivery could trigger an intimal disruption(7, 8). maeder et al proposed pregnancy-related hypercholesterolemia as a contributory factor for pregnancy related coronary artery dissection (9). there is one reported case of coronary artey dissection during hemodialysis due to a recent abortion and consequent curettage at first trimester of her pregnancy (10). diagnosis is typically confirmed through angiography, though intravascular ultrasound has proven to be an important adjunct technique. establishing the coronary lesion in pregnancy, which then may direct therapy, will depend upon the gestational age of the fetus. angiographic interventions are becoming more common in pregnancy, though prior to fourteen weeks gestation, the need for angiography must be balanced against the risk of radiation during organogenesis. fortunately, risk is reduced late in pregnancy or postpartum, when most cardiac events occur. preparation for angiography should include all efforts to lessen radiation exposure to the fetus regardless of gestational age. basic preparations include appropriate shielding and minimizing procedure time. additionally, the obstetric team should be readied for emergent cesarean section in the catheterization suite should expeditious delivery become necessary. fewer angiographic interventions are performed during pregnancy, though case reports on the use of intravascular stents in pregnancy have suggested these interventions can be successful (11, 12). in the case of thrombotic lesions, both revascularization and thrombectomy have been employed. medical treatment will depend on the type of lesion seen, as well as on the hemodynamic status of the patient. heparin, beta-blockers, diuretics, nitrates, and low-dose aspirin are considered safe in pregnancy, and can be used with appropriate hemodynamic and fetal monitoring. more experience is also being gained with the use of glycoprotein iib/iiia inhibitors, and thienopyridines, which are frequently used as an adjunct to angioplasty. we used a short course of clopidogrel without pregnancy-asaociated myocardial infarction 329 any complications. glycoprotein iib/iiia inhibitors vary greatly in their half-lives, and those with a shorter duration of action are preferred when delivery is imminent. when angiography is contraindicated, consideration may be given to the treatment of large infarcts with thrombolytics such as streptokinase, though pregnancy is a relative contraindication to systemic thrombolysis. with spontaneous dissection, anticoagulation may be appropriate to prevent thrombus formation at the site of the lesion, though a potential concern that anticoagulation may cause increased bleeding into damaged vessels also exists. coronary artery bypass grafting has also been successfully performed during pregnancy and in the post-partum period (13). coronary bypass typically remains an option of last resort, as cardiovascular surgery during pregnancy carries unique risks. fetal risks of insult or death are greater than with other surgeries, and importantly, these outcomes appear to be unpredictable. however, when surgery is performed in conjunction with delivery, maternal outcomes appear to worsen (14). paez m and collegues report the case of a post-partum woman with dissection of left coronary trunk who was treated with revascularization surgery, ventricular assistance and a successful heart transplant (15). broader recognition of the risk of mi in pregnancy should lead to more rapid diagnosis and treatment. however, our understanding of the etiology and risk factors of this disorder is incomplete, and until further research in this area is performed, cases are unlikely to be prevented. thus, mi should be in the differential diagnosis of every pregnant patient who presents with symptoms suspicious for cardiac event, particularly those in the third trimester or postpartum, despite any perceived lack of cardiac risk factors. references 1 esinler i, yigit n, ayhan a, kes s, aytemir k, acil t(2003) coronary artery dissection during pregnancy.acta obstet gynecol scand 82(3):194–6. 2 roth a, elkayam u (1996) acute myocardial infarction associated with pregnancy. ann intern med 125:751–762. 3 koul ak, hollander g, mostovits n, frankel r, herrera l, shani j (2001) coronary artery dissection during pregnancy and the postpartum period: two case reports and review of literature. catheter cardiovasc interv 52:88–94. 4 engelman dt, thayer j, derossi j, scheinerman j, brown l (1993).pregnancy related coronary artery dissection: a case report and collective review.conn med. 57:135–9. 5 kay ip, wilkins gt, williams ja.(1998) spontaneous coronary artery dissection presenting as unstable angina. j invasive cardiol 10:274–6. 6 almeda fq, barkatullah s, kavinsky cj (2004) spontaneous coronary artery dissection. clin cardiol 27:377–380. 7 madu ec, kosinski dj, wilson wr, burket mw, fraker td jr, ansel gm (1994). two-vessel coronary artery dissection in the peripartum period. case report and literature review. angiology 45:809–16. 8 klutsteinmw, tzivonid, bitrand, mendzelevskibi, ianm, almagory (1997) treatment of spontaneous coronary artery dissection: report of three cases. catheter cardiovasc interv 40:372–6. 9 maeder m, ammann p, drack g, rickli h. (2005) pregnancy-associated spontaneous coronary artery dissection: impact of medical treatment. case report and systematic review. z kardiol. 94:829–35. 330 shohreh shahabi et al. 10 iltumur k, karahan z, ozmen s, danis r, toprak n (2007).spontaneous coronary artery dissection during hemodialysis in the post-abortion period.int j cardiol. 27; [epub ahead of print] 11 togni m, amann fw, follath f.(1999) spontaneous multivessel coronary artery dissection in a pregnant woman treated successfully with stent implantation. am j med 107:407–8. 12 schiff jh, gries a, ehehalt r, elsaesser m, katus ha, meyer fj (2007).a pregnant woman with acute myocardial infarction due to coronary artery dissection: pre-hospital and in-hospital management. resuscitation. 73:467–74. 13 frey bw, grant rj (2006) pregnancy-associated coronary artery dissection: a case report.j emerg med.30:307–10 14 weiss bm, von segesser lk, alon e, seifert b, turina m (1998) outcome of cardiovascular surgery and pregnancy: a systematic review of the period 1984–1996. am j obstet gynecol 179:1643–53. 15 paez m, buisan f, herrero e (2007) spontaneous dissection of the left coronary artery trunk during the postpartum period treated with revascularization surgery, ventricular assistance and a successful heart transplant. acta anaesthesiol scand 51:960–1 corresponding author: shohreh shahabi department of obstetrics & gynecology and women’s health montefiore medical center/albert einstein college of medicine, new york e-mail: chananacharu@yahoo.com upsala j m e d sci 99: 147-154, 1994 decreased serum insulin-like growth factor i during puberty in children with insulin dependent diabetes mellitus (iddm) erik kreyberg n o r m a n n , ’ u w e evald,* k n u t dahl-jnrrgensen,] stig l a r ~ e n , ~ nils n o r m a n 4 and torsten tuvemo’ department of pediatrics, aker university hospital, oslo, norway, 2department of pediatrics, uppsala university children’s hospital, uppsala, sweden, medstat, stremmen, norway and 4hormone laboratory, aker university hospital, oslo, norway abstract previous reports concerning insulin-like growth factor-i (igf-i) in diabetics are conflicting. this study describes igf-i in children with insulin-dependent diabetes mellitus (iddm) and healthy controls i n relation to pubertal development. sixty-six children participated (34 girls and 32 boys) of which 3 3 had iddm. the mean age in the study population was 14.3 years, (range 7.1 to 19.7). serum igf-i was significantly decreased in diabetics. diabetic girls had a mean igf-i of 28.3 (14.4; =sd) nmolll compared with 42.8 (15.0) nmol/l in controls. in diabetic boys the result was 30.0 (16.0) nmola compared with 44.1 (23.4) in controls. growth hormone was measured in only one fasting morning serum sample from each individual. there was no difference between girls, but diabetic boys had higher mean serum concentration of growth hormone than controls (3.5 (4.8) vs. 1 . 8 (1.5) pga respectively). diabetic girls had delayed menarche, corresponding to a slightly delayed bone maturation. introduction diabetics are in some reports found to have lower serum concentrations of insulin-like growth factor i (igf-i) than controls (4). after the first 6-9 months postnatally serum igf 1 is mainly regulated by growth hormone and nutritional status. normally igf-i increases two-three-fold during puberty and declines with aging (3, 5). several studies have been published about serum levels of igf-i in diabetics but few studies were performed in children or adolescents. the results are conflicting. in a review of the literature i n 1990 some studies were reported showing increased values in diabetics, some normal while some showed decreased concentrations of igf-i in serum compared with controls (4). during puberty most diabetics have impaired metabolic control. children with insulin dependent diabetes mellitus (iddm) have been reported to have delayed puberty compared 147 with controls and in some reports they were shorter. the normal growth spurt was retarded (1,13, 14) and igf-i concentrations decreased compared with healthy controls (2, 6). the reports about the relation between the level of igf-i and microangiopathy have been contradictory. nardelli did not find any correlation between retinopathy and levels of igf i, while merimee found the igf-i level significantly elevated in patients with rapid proliferative retinopathy compared with patients with less severe retinopathy ( 8 , 9). because of these contradictory reports, concerning both the igf-i levels in diabetics and the correlation to angiopathy, we have investigated the pubertal development and the hormonal changes, including igf-i, in children with i d d m and a control group of healthy children. patients and methods patients thirty-three insulin-dependent diabetic patients were included, 17 girls and 16 boys. as control they all brought a healthy classmate of the same sex and age. the groups were matched for chronological age, bone age, height, weight and body mass index (table 1). table 1. anthropometric data of boys and girls with and without iddm (=sd). because of the relatively f e w patients in each pubertal group, the total population was divided into only four main groups. these were two female groups based on breast development (b<3; b23) (according to tanner’s staging ( i s ) ) , and two male groups based on the testicular volume (t<15 ml; t215 ml) (12, 15). 148 the mean diabetes duration was 8.0 (range 0.9-15.0) years in girls and 7.0 (1.0-16.5) years in boys. most diabetics were patients at the outpatient clinic of the department of pediatrics, aker university hospital, while some came from suburban hospitals of oslo. the diabetics were all treated with human insulin. some used two or three injections daily with conventional syringe while others had a multiinjection regimen with insulin-pen. the mean daily insulin dose was 46.4 iu in girls and 42.6 iu in boys ( n s ) (0.9 (0.4-2.2) and 0.8 (0.3-1.3) iuikg body weight respectively; n.s.). there was no difference in systolic or diastolic blood pressure between the diabetic girls and boys (1 15/73 and 115/69 mmhg respectively) compared with the controls (1 16/71 and 112/67 mmhg respectively). the patients and their parents gave written consent. the study protocol was approved by the local ethics committee. methods both the diabetic patients and controls met fasting at the outpatient clinic at 8.00 a.m. the blood-samples were taken before breakfast and the morning dose of insulin. they were all examined and grouped according to the pubertal developmental staging of tanner (12). axillary and pubic hair, breast development, time of menarche and the menstrual cycle were recorded. penile development and testicular volume (by pradertm orchidometer) were measured in boys (15). standing height was measured with a holtaintm stadiometer. body mass index (bmi) was calculated from the standing height and body weight. to reduce variation the standing height and blood pressure were measured by the same technician. bone age was estimated according to greulich-pyle (10). to reduce variation the bone age was determined by only one experienced radiologist. h b a l c was measured by hplc (diamatm, normal <6.1%, cv <3%). insulin-like growth factor i (igf-i) was measured with a ria-kit (immuno nuclearm). n o separation from binding proteins was made. growth hormone (gh), testosterone, estradiol, follicle stimulating hormone (fsh), luteinizing hormone (lh), dehydroepiandrosterone (dhea), androstenedione, cortisol and thyroxine were measured by standard ria-methods. stafistical methods all results were expressed as mean values with standard deviation in brackets. linear regression analysis was used to analyse the correspondence between igf-i and pubertal stage in the different groups (7). all tests used in this analysis were carried out two-tailed and the differences considered significant if the p-values were less than or equal to 5%. the groups were compared by using two-way layout analysis of variance. 12-945252 149 table 2. blood glucose and hba,c in children with i d d m and controls (=sd) results fasting blood glucose and h b a l c values are given in table 2. the mean age o f menarche was delayed by 0.5 years in diabetic girls. the hormone data are given in tables 3 and 4. in girls the mean concentration of serum igf-i increased from 19.2 (5.4) nmolfl in the youngest pubertal group (b<3) to 3 3 . 3 ( 1 5 . 5 ) nmol/l in the oldest (b23). the pubertal increase of igf-i in diabetic girls started later than in controls. control girls had higher values than diabetic girls in early puberty. in early pubertal control boys igf-1 concentrations were higher than in boys with diabetes. in boys the difference between the mean values in late puberty was not as large as in early puberty (41.3 (13.4) vs. 5 3 . 5 (16.0) nmol/l and 21.1 (12.1) vs. 36.7 (26.5) nmolfl respectively). these differences were not statistically significant, but grouping all diabetic boys together and comparing them with controls, serum igf-i concentration was significantly lower (30.0 (16.0) vs. 44.1 (23.4) nmol/l; p<0.05). there was no correlation between igf-i and metabolic control measured by hba 1 c. discussion there are several studies reporting that the serum igf-i concentration is decreased in diabetic patients, while some claim the opposite (4). in our study, both diabetic girls and 150 all girls b<3 b23 boys t-45 t>15 vs. controls *=f i i table 3 . serum igf-i and growth hormone (gh) in boys and girls, with and without iddm, at different pubertal stages (=sd). boys had lower serum igf-i than controls, also when divided into groups of pubertal development. this is identical to what nardelli reported i n 1989, who found 0.89 u/ml of igf-i in controls compared with 0.62 u/ml in diabetics (p<o.ol) (9). salardi and coworkers reported in 1986 that serum igf-i was lower i n diabetic prepubertal groups compared with controls. igf-i was not significantly reduced in pubertal groups (1 1). they also found increased nocturnal secretion of growth hormone in diabetics suggesting a block in production of igf-i. in our study we had too few prepubertal children, compared with pubertal, to make any statistical analysis meaningful. by grouping them into one group with pubertal development less than tanner stage 3 and one with stage 3 or more, we also found that the youngest diabetics as well as the more mature ones had lower igf i levels than controls. the secretion of gh is normally pulsatile with the highest values 151 during the night. growth hormone was measured as a single test, random morning fasting gh concentration. this reduces the importance of the result. growth hormone was significantly increased only in our diabetic boys. diabetes girls 8-3 823 boys t45 t>15 controls girls b c 3 bx3 boys ts15 s. controls table 4. fasting morning serum hormone concentrations in boys and girls, at different pubertal stages (=sd). 152 there are previous reports of reduced height and delayed pubertal development in diabetic adolescents compared with controls. most of the diabetic patients reach a normal adult height. they will grow slightly slower but for a longer period because of the delayed pubertal development, thus reaching normal adult stature (1, 2, 6, 1 3 , 14). the girls in our study were significantly delayed in menarche although the pubertal staging was the same in both the diabetic and the control group. the delay is associated with a significantly lower mean value of estradiol in the diabetic group compared with controls (p<0.05). the mechanism behind the reduced serum igf-1 in our adolescent diabetic patients is not completely clear. the study demonstrates that the reduction of igf-1 was present also when pubertal stage and age were taken into consideration. the decreased igf-1 levels were not correlated with poor metabolic control. there is no evidence for intensive exercise in the diabetic children explaining some part of the reduced igf-1. diabetes during puberty can thus be considered an igf-1 deficient state. this deficiency seems to be connected to increased growth hormone concentrations at least in boys, which might be due to lack of negative feed back control. this study was performed before the possibility to measure free igf-i had appeared, so changes i n igf-binding protein levels can not be excluded. the importance of these findings on diabetes regulation and long term complications have to be further studied. references. 1. beal, c. k.: body size and growth rate of children with diabetes mellitus. j pediatr 32:170-9, 1948. 2. clarson, c., daneman, d. & ehrlich, r. m.: the relationship of metabolic control to growth and pubertal development in children with insulin-dependent diabetes. diabetes res 2:237-41, 1985. 3. d'ercole, a. j.: somatomedins/insulin-like growth factors. clinical paediatric endocrinology oxford: blackwell scientific publications, 74-95, 1989. (brooke cgd). 4. flyvbjerg, a , : growth factors and diabetic complications. diabetic med 7:387-99, 1990. 5. furlanetto, r. w. & cara, j. f.: soniatomedin-c/insulin-like growth factor-i as a modulator of growth during childhood and adolescence. horm res 24: 177-84, 1986. 6 . hjelt, k., braendholt, v., kamper, j. & vestermark s.: growth i n children with diabetes mellitus. dan med bull 30:28-33, 1983. 7. kendall, m. & stuart, a , : the advanced theory of statistics. 2nd ed. london: charles griffin &co. ltd, 1979. 8. merimee, t. j., gardner, d. p., zapf. j. & froesch, e. r.: effect of glycemic control 153 on serum insulin-like growth factors in diabetes mellitus. diabetes 33:790-3, 1984. 9. nardelli, g. m., guastamacchia, e., di polo, s., balice, a., rosco, m., santoro, g., lollino, g. & giorgino, r.: somatomedin -c (sm-c). study in diabetic patients with and without retinopathy. acta diabetol lat 26:217-24, 1989. 10. pyle, s.i., waterhouse, a. m. & greulich, w. w.: radiographic standard of reference for the growing hand and wrist. t-he press of case. western: western reserve university, 1971. 1 1 . salardi, s., cacciari, e., ballardini, d., righetti, f., capelli, m., cicogani, a,, zucchini, s., natali, g. & tassinari, d.: relationships between growth factors (somatomedin-c and growth hormone) and body development, metabolic control, and retinal changes in children and adolecents with iddm. diabetes 35:832-6, 1986. 12. tanner, j. m.: growth at adolescence, 2nd ed. oxford: blackwell scientific publications, 1962. 1 3 . tattersall, r. b. & pyke, d. a,: growth in diabetic children. studies in identical twins. lancet ii:llos-o9, 1973. 14. wise, j.e., kolb, e.l. & sauder, s. e.: effect of glycemic control on growth velocity in children with iddm. diabetes care 15:826-30, 1993. 1 5 . zachmann, m., prader, a., kind, h. p., haflinger, h. & budliger, h. testicular volume during adolescence. helvetica paediatrica acta, 29:61-72, 1974. corresponding address: erik kreyberg normann, department of pediatrics, aker university hospital, 0514 oslo 5 , norway. 154 upsala j med sci 100: 47-52, 1995 a daughter's reflections upon her father's creativity eva teorell david bagares gata 12, 9 1 1 i 3 8 stockholm i don't suppose there was anyone who listened so much to father as i did. ever since my earliest years i came t o be the one for whom he "thought aloud". whatever he began with, he always ended on the subject of his research and work, on his "ponderings", to use his own vocabulary. in almost any situation, while handling his ever-present pipe, he would begin to reflect on virtually any subject, fix on some thread and begin to unwind. very soon he completely lost track of both time and space, and for long hours by day and night he would elaborate his ideas widely, associate freely, create thought patterns and make those phenomenal cross-bridgings that were so typical for him. nothing was too great and nothing too small t o consider totally unconstrainedly he would move from the trivial to the extraordinary, and from the deeply complicated to the simple, in the way that is possible only for the truly penetrating mind. he would always point out something unexpected, and to me, even a s a n adult, he was the great storyteller. but the contents of the stories were not the usual ones. here there existed neither good nor bad, no winners or losers. his stories also lacked both a beginning and end, for the content was about the processes that govern nature. what he narrated was the saga of reality. the fact that i so often understood him was certainly due to his exceptional ability to simplify. a few matches, a packet of throat pastilles and a few sketches on a piece of paper sufficed to create the most fascinating puzzle but a puzzle in which the most important parts were always absent figures. but then, with a light gesture, all of a sudden he would sweep away all the hypotheses as if they were merely pieces on a gaming table and say laughingly "or might it be the other way around, let's start all over again in a new direction!" with the compressed pictorial language of a kind of poetry, he was able to shed light on matters from completely new angles and bring underlying structures, hitherto hidden, to the surface. that which is sometimes called the twilight zone between the knowledge of today and that of tomorrow, or the unfilled patches on the map of knowledge, were by no the way he spoke had the same simplicity. 47 means colourless areas to him; on the contrary, here the colours gleamed brightest. to these areas his mind was drawn. that which was already established easily made him feel confined, and he would ascribe it to a weakness of character that he became bored when things proved too easy or when the equation was finally solved. father was born in 1905 and he left us the summer before last, 87 years old. his long life-span coincided with a n era of change in cultural and social life and in technology, that had never previously, in such a short time, been so dramatic. the situation in his childhood and adolescence seems much more remote than only one generation away. at school, swedish and history were among his favourite subjects. even at a n advanced age he could still refer to particular teachers and lessons. perhaps his interest in the arts, formed at this early stage, contributed in developing his very special ability to regard circumstances in a broad context. not least did this apply t o his relationship t o his own tradition. taught by strong personalities, father and his generation were quite aware, perhaps differently from the way we are today, t h a t not only the entire mass of knowledge about which we think, and the entire reference system (we may well call it a world view) against which we measure our observations, are created and furnished by a n infinite series of predecessors and teachers. thus, even from his student days, it was clear to him that tradition, as well as being a natural and necessary area of growth, is a man-made creation, imposed by all the magnitudes and limitations of man. such distancing from one's own tradition is certainly a prerequisite for profound thinkers. father was always cautious about equating the concepts of fact and truth, as we often do today. his life-long studies of the rise and fall of great cultures made it natural for him to be well aware that the degree of truth in the "facts" of today will come to be revised tomorrow, if it hasn't altogether lost its validity. for certain scientists the mathematical talent is connected to a musical one. in father's case his logical discrimination was linked to another sensory faculty he was a visualist, he comprehended, he thought and formulated in images. like the sculptor, who in a simultaneously methodical and playful way, by the interplay between light and shade, might reveal constantly new aspects of a n object, father played with the searchlight over known and unknown problem complexes within his tradition. but even if his ways of visualisation in many respects resembled those of the sculptor, his visions were never represented in so few dimensions as three. nature is dynamic, not static, so the fourth dimension of time was always conceived. i also believe that he would have approved of likening the whole of human knowledge to a growing, constantly self-changing sculptural organism which, when supplied with new knowledge, is shifted t o earlier centres of gravity and balances, 48 and whose consequently new perspectives will expose new dimensions, forces and functions in itself. i once visited one of father's younger colleagues in india. well acquainted with father's work he was an admirer, yet he was also critical. in his opinion father had spread himself over too many areas and, especially, had not, in accordance with the einsteinian ideal, summarized his deep insights (particularly in t h e field of bioelectricity) in one single equation. father himself considered this criticism justified, but if one tries to look upon it from a reverse perspective, the way he always did himself, perhaps the criticism also emphasizes one of his truly great features a s a person and thinker, namely his inability to believe in one, and only one truth and his unwillingness to lock himself up into one single position. he always distrusted that which was too orthodox, and for him it was almost instinctive always to leave space for the unknown. he, who was a gentle person and very tolerant of others, became noticeably irritated when he met those who were convinced that they had found their own way to salvation. when, in the autumn, we relatives went through father's possessions, i found one of his old slide-rules in a drawer, and for a long time i sat in meditation about this simple b u t ingenious little object, which was one of t h e most important mathematical tools of his generation. the speed with which information is obtained today, and the tremendous possibilities of memory storage which have been brought about by transistors and silicon chips, were something father had access to only during t h e last part of his working life and always enthusiastic about new opportunities, he was one of the first to take advantage of them. but we who remember father's working environments from earlier times, from the fifties, also know what the so-called electrical analogue machines of that time looked like they occupied not only cubby-holes but entire rooms, which seemed impenetrable, veritable snakes' nests of cables and plugs. inconceivable as it might seem, these gigantic machines constantly under reconstruction and readjustment were conceived by father as a physiological reality. preceding and parallel to this electronic period was the slide-rule. still with father's in my hand, i wondered whether the computer revolution will bring about essential differences in man's thinking and comprehension. this question, which is certainly one of the most important of our time, i of course cannot answer. but i can illustrate some further features that were typical of father's conceptions a n d formulated during the pre computer age. my father and his generation had to do their calculating manually, and it took weeks or even months. what went on in the heads of these persons during their endless calculations? was it just a loss of time which even though necessary retarded the free play of their intellect? 49 creativity implies the achievement of something. thus, inbuilt into this concept is a time-consuming quality which may be termed maturation. perhaps the prolonged work of calculation also provided a n opportunity for subconscious or marginally conscious analysis of the problems, a possibility of distancing and sorting them, that came to have an impact on the end result? i know that at least a s an artist, the very mechanical work of producing the artistic product, which sometimes can be a rather dull process between the initial intention a n d t h e completed work, often includes a process during which previously undiscovered knots in the sketch are identified and resolved, bringing more clarity to the final work. this maturation, the process of merging pieces into a structured whole, does not take place on a purely conscious level, but is a kind of subconscious crystallisation resulting from long preparatory work and needs time to be achieved. not only was seemingly endless time spent on calculating, but father and his generation weighed and measured too, and this again was done manually and with mechanical tools. could this have contributed to father’s concept of nature and its categories as a physically tangible reality? it was the presence of their concrete character which he saw and encountered all around him, under the microscope or during a walk in the forest, in mathematics, in saline solutions and in ion transport; or when patting one of the animals of which he was so fond it was this experienced reality that inspired him and aroused his curiosity. it is not the inexperienced that fuels our involvement; only the stimuli of perception make us capable of concentration and drive us further and into greater depth. here, it seems to me, lies one of the most important differences between human and artificial intelligence in involvement. however theoretical father became during his life, his intellect developed in relation to his perceptual capacity, a n d this relationship was never lost. it is also my belief that this broad perceptual spectrum influenced and refined his imagination and intuition and that the observations deriving from this perceptual ability differ qualitatively from those made solely by mental activity. the demand of nature to be investigated, together with the subtle instruments of fantasy and intuition that make this call comprehensible, also form part of the thirst for knowledge which, at least hitherto, is reserved for the human mind. here again there are differences between human and artificial intelligence. sometimes i wonder how t h e human mind of tomorrow will be influenced by the fact t h a t so much of reality is now mediated only indirectly in a non-sensory and already pre-treated way via screens. father’s own imagination was such a completely spontaneous asset to him t h a t i believe it belonged t o one of the few phenomena of nature upon which h e never reflected. if a path led to a blind end, his answer would be: “well, then we will have to find a new one, won’t we?”. he never doubted the possibility of tracing t h e 50 mysteries of living nature. there is a great joy and optimism in trusting t h a t new figurations will constantly appear, and these features were also a special part of his creativity. the fact that during the course of his work he increasingly came to leave the love of his youth, pure chemistry, to focus more on chemical and physical functions related to living material, was, i believe, more than a chance occurrence. it was characteristic that he should transcend the subjects themselves and instead concentrate on their interactions in attempts to understand the processes of life. father's scientific devotion was in itself a way of living and i do not think i have ever met anyone so impossible to divide into a professional and a private person. he was a philosopher who, with a sort of humanistic linncan tradition, p u t questions to nature about its construction and processes. he always tried to pose a n d analyse the most valid and important questions, always attempting t o reformulate and focus them more sharply, never to get bogged down by details but always to seek the whole. humbly and with endless patience he would test, and test again, to see whether his hypotheses would explain the response of nature, well aware that every response could be a chimera, and that all human awareness is, and will remain, a n island in a sea of ignorance. on those occasions when nature did confirm his ideas, he looked upon this not so much a s an achievement but a s new footprints guiding him into the unknown. j u s t a few years ago he made this remark about his own contributions: "certainly i felt a tremendous joy on the few occasions when i succeeded in opening slightly t h e door to our lord's creation, discerning something about its principles". and then h e added: "nowadays it seems to me that people are more interested in how the hinges a r e constructed!". these very quick and often deeply humorous switches of perspective were typical. during his long lifetime father, too, came to be exposed to the hardness that is a n intrinsic part of life. perhaps his long study of nature's work on building up and breaking down, its endlessly oscillating transformations, helped to pave the way for the attitude he developed towards the way life strikes, a sort of stoical submission, whose dark undertones, however, never undermined the deep respect for life itself. this conception of life, a s the very opposite to stagnation, got a particularly poignant formulation in a remark he once made to me concerning depression: "never forget that depression is the art of seeing things from one point of view alone, from a fixed standpoint, and that such goes against life's principles of perpetual motion. there is much of course that i don't known about my father, and perhaps it is in these unknown parts of a person, in the hidden recesses, that the true source of creativity lies, in the ultimate solitude when faced with all the questions encountered between birth and death. perhaps from these forever secret and intangible zones stem in common our great scientific, artistic and religious traditions. 5 1 in his last weeks father's constantly alert mind accelerated almost feverishly, and untiringly he continued to communicate his experiences. he was steering towards a "spiral of stars", a galaxy, but was anxious about not being able to keep course, apd in the inimitable way of the old mathematician he calculated a n d recalculated' t h e manoeuvres. "i have to change stations, you see", he explained. he described the clock that had to be set at zero, but first, time had to begin to float backwards. increasingly often he would also return to the mountain of coral that he had to climb, and which at the end was so close that he could see the carved-out ledges intended for his staircase. right up to his last hours he created extraordinary visions and metaphors, this time for the breakdown phase of the natural processes, whose total extent, from micro to macro, from beginning to end, we know so little about. 5 2 upsala j med sci 91: 201-204, 1986 analytical quality bo sorbo department of clinical chemistry, linkoping university, linkoping, sweden we are all aware that the concept of analytical quality implies that labora tory results are reliable, i.e. are precise and accurate. we thus try to min tain a high standard in these respects with internal and external quality con trol and put a lot of effort and mney in these activities. however, we often neglect another aspect of analytical quality, and that is the factor of time. although, we ought to control the turn-around-time of our tests, how often is that done? we must realize that an unacceptable delay in the delivery of our excellent analytical results to the clinical ward will result in bedside cli nical chemistry taking over even if not being precise and accurate. however, returning back to precision and accuracy raises the question: whatcan we demand of bedside clinical chemistry in these respects? there are optimists who claim that it is desirable and possible to perform bedside clinical chemis try with the same precision and accuracy as conventional clinical chemistry in the laboratory but this is not a realistic attitude. on the other hand, our clinical collegues sometimes claim that the high precision obtained in the lab oratory with conventional wet-chemical methods is not required in clinical practice, especially if the results are used for monitoring therapy and not for diagnostic purposes. this is probably true to some extent, but how imprecise may results from bedside clinical chemistry be? the determination of bloodglu cose is probably the method which has received most attention in this respect. it has thus been proposed (5) that a blood glucose value deviating less than 20% from the true values is acceptable from a clinical point of view, but no real arguments for this proposal were presented. if we use tonk's criteria (9) for blood glucoseacv (coefficient ofvariation) of 13% would be permissible. as a working hypothesis i considered the possibility that a result for blood glucose obtained under bedside conditions should be permitted to be within 2 4 cv from the true rrean. looking at the reports from our external qualitycontrol during the last 6 months i found that the cv for normand hyperglycemic 20 1 values was about 5%, giving a permissible range of authors. however, for hypoglycemic values the cv was about 10% and a range of 5 40% is certainly not acceptable even for bedside clinical chemistry. 20% as suggested by other nevertheless, what is the "state of the art" with respect to analyticalquality of bedside clinical chemistry? the results of interest here are not those ob tained by well-trained laboratory technicians when they evaluate the methodsin the laboratory but those obtained by nurses and doctors in the clinical wards. gibb et a1 (6) thus reported that determinations of serum creatine kinase with a seralyzer in a coronary case unit by the medical staff were of the same qua& ity as those obtained in the central laboratory i.e. the cv was 5-9%. on the other hand clark & broughton (3) found that determinations of serum ureawitha seralyzer had a cv of f 4% if performed by trained laboratory technicians, whereas the results obtained by non-laboratory personnel were much more impre cise with a cv of 5 16%. the latter results are probably unacceptable from a clinical pint of view. furthermore, it was found in a recent study from u p sala ( 2 ) that laboratory technicians working in primary care units obtained results for blood hemoglobin of satisfactory precision, whereas non-laboratory personnel produced much inferior results, especially if no external quality control was available. other reports at this meeting provide further evidence for bedside clinical chemistry being of a higher quality if performed by lab oratory technicians and i shall not expand further on this subject. what should then be done in order to improve the quality of bedside clinical chemistry as performed by non-laboratory personnel? firstly, it should be stressed that bedside clinical chemistry should be directed and supervised by a clinical chemistry laboratory. the laboratory should thus in advanceevaluate both the methods and the instruments desired by the wards or primarycareunits. otherwise, the latter may experience unpleasant surprises as illustrated in a recent report ( 8 ) . a special care baby unit intended to determine serumbili rubin in a bedside manner and purchased a bilirubinmter for this purpose. comparison of the results obtained with this instrument with those obtained in the main laboratory of the hospital revealed, however, that the bilirubinmter gave too law results on highly icteric sera. this was due to an inherent non linearity of the instrument, which had totally escaped the attention of the ward personnel responsible for the purchase of the instrument. furthermore, the central laboratory should provide preventive maintenance of the instruments used for bedside clinical chemistry, even if most of the errors are not caused by the instruments but by the operators of the latter. another important responsability of the central laboratory is to provide 202 written instructions for perfo,ming bedside analytical methods which m y be understood by non-laboratory personnel. the written mterial provided by sup pliers of instruments, kits and test strips often leaves much to be desired in this respect. the central laboratory should also be responsible for the training of non-laboratory personnel and furthemre evaluate their ability to perform even simple bedside tests. an ambitious training program for blood glucose determinations with a visually read test strip was recently described ( 1 0 ) . unfortunately i think that this elaborate training program is too compli cated for mst of our laboratories. but another simple measure, which should not be neglected, is to examine the color vision of those who intend to per form test strip analysis by visual reading, as otherwise large errors m y re sult. this applies especially to diabetics who use test strips forself-control ( 7 ) . the necessity of training all personnel performing bedside clinical chem istry has been stressed in the "guide-lines" adopted by the english clinical chemists ( 1 ) . furthermore, they stress the importance of retraining the per sonnel involved. unfortunately, i think it is fair to say that this aspect has not yet received the attention it should in scandinavia. last, but not least comes quality control. the central laboratory shouldinsist on supervising the internal quality control of bedside analytical methods but should also be responsible for an external quality control system. technical problems have earlier prevented external quality control of blood glucose determinations by test strips, but satisfactory control methcds are now avail able. unfortunately, the quality control of the methods used by patients for self-control has often been neglected. i feel often uncomfortable about the unduly enthusiasm showed by clinical collegues, who put reflectometers and test strips in the hands of their patients with very little instructions and no quality control at all. the clinical chemists must thus engage themselves much mre in these matters than they have done up to now. what has been said in the foregoing m y give the impression that i have a neqa tive attitude to bedside clinical chemistry. however, i whole-heartedly share the opinions so ably expressed by two british colleques ( 4 ) : "clinical bio chemistry now has the potential to move nearer the patient. whether clinical biochemists accompany it is largely a mtter for themselves. there will be those who mke things happen, those who watch things happen, and those who wonder what has happened." let us not wonder what has happened to the quality of bedside clinical chemistry! 203 references andersson, j.r., linsell, w.d. & mitchell, f.m.: guidelines on the perform ance of chemical pathology assays outside the laboratory. br. med. j. 282:743, 1981. bengtsson, p.g., ronquist, g. & holmgren, c.: nytt analyssystem for hem globinbesthing fi olika vbrdnivber i ett prin&drdsomr&de. martid ningen 81:642-644, 1984. clark, p.m.s. & broughton, p.m.: potential applications and pitfalls of dry reagent tests. ann. clin. biochem. 20:208-212, 1983. evans, s.e. & buckley, b.m.: biochemists nearer the patient? br. med. j. 287:1399-1400. 1983. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. fairclough, p.m., clen-ients, r.s., filer, d.v. & bell, d.s.h.: anevaluation of patient performance and their satisfaction with various rapid blood glucose measurement systems. diabetes care 6:45-49, 1983. gibb, i., barton, j.r., adams, p.c., pratt, d., dean, c.r. & tarbit, i.f.: rapid measurement of creatine kinase activity in a coronary care unitusing a portable benchtop reflectance photometer. br. med. j. 290:1381-1383, 1985. lombrail, p., cathelineau, g., gervais, p. & thibault, n.: abnormal color vision and reliable self-monitoring of blood glucose. diabetes care 7:318-321, 1984. mount, j.n. & slavin, b.: bedside biochemistry. j. clin. pathol. 37:715-716, 1984. tonks, d.b.: a study of the accuracy and precision of clinical chemistry determinations in 170 canadian laboratories. clin. chem. 9:217-233, 1963. williams, j. & freedimn, 2.: evaluation of hospital staff nurse performance of chemstrip bg testing. diaktes care 7:398-399, 1984. 204 upsala j med sci 92: 75-78, 1987 reatment of severe hypertension with peroral labetalol goran frithz department of medicine, central hospital, eskilstuna, sweden abstract 22 p a t i e n t s w i t h u n t r e a t e d , e s s e n t i a l h y p e r t e n s i o n ( d i a s t o l i c blood-pressure 2140 mmhg) and i n most cases a l s o showing n e u r o l o g i c a l symptoms were g i v e n a s i n g l e o r a l dose o f 400 mg l a b e t a l o l . all p a t i e n t s d i s p l a y e d a g r a d u a l decrease o f t h e blood-pressure down t o a d i a s t o l i c p r e s s u r e about 110 mmhg w i t h r e l i e f o f t h e symptoms. there were no s i g n s o f n e u r o l o g i c a l d e f i c i t due t o t h e reduc t i o n o f blood-pressure. introduction there i s s t i l l disagreement as t o when an e l e v a t e d blood-pressure s h o u l d be regarded as a m e d i c a l emergency and a c u t e t r e a t m e n t i n i t i a t e d . however, most c l i n i c i a n s would agree t h a t a p e r s i s t e n t d i a s t o l i c blood-pressure o f 1 140 mm hg and/or s i g n s o f encephalopathy r e q u i r e a c u t e m e d i c a l i n t e r v e n t i o n . on t h e o t h e r hand, i t is w e l l known t h a t t o o v i g o r o u s t r e a t m e n t and r a p i d r e d u c t i o n o f t h e blood-pressure may r e s u l t i n unwanted e f f e c t s and even permanent s i g n s o f c e r e b r a l damage. l a b e t a l o l , a combined aand p-adrenoceptor-blocking agent, has been used i n t r a v e n o u s l y i n m i n i b o l u s doses or by c o n t r o l l e d i n t r a v e n o u s i n f u s i o n t o t r e a t v e r y severe h y p e r t e n s i o n . it has been shown t h a t l a b e t a l o l i s r a p i d l y absorbed and e x e r t s i t s maximum p h a r m a c o l o g i c a l a c t i o n two h o u r s a f t e r o r a l a d m i n i s t r a t i o n ( 3 ) . s i n g l e o r a l doses have been r e p o r t e d t o cause a marked r e d u c t i o n o f blood p r e s s u r e ( 2 ) . the a c u t e blood-pressure r e d u c t i o n is m a i n l y achieved by an a-receptor-mediated r e d u c t i o n o f t h e t o t a l p e r i p h e r a l r e s i s t a n c e as l a b e t a l o l has b o t h a r e c e p t o r and p r e c e p t o r b l o c k i n g p r o p e r t i e s . the l a t t e r p r o p e r t y c o u n t e r a c t s t h e r e f l e x i n c r e a s e o f h e a r t r a t e caused by t h e blood-pressure decrease (1 ) . t h i s communication r e p o r t s our experience o f 5 y e a r s ' use o f l a b e t a l o l i n severe h y p e r t e n s i o n i n an emergency ward. 75 patients and methods 22 patients, 14 men and 8 women (mean age 53.4 years), were consecutively treated in the emergency ward and intensive care unit. they were all admitted to the hospital with a diastolic blood-pressure of 2 140 mmhg (range 140-160) and neurological symptoms, such as headache, vertigo and dizziness, were present in most cases. six patients had choked optic discs (fundus hypertonicus i v ) . none had a history of cerebrovascular lesion, myocardial infarction o r renal failure and no antihypertensive treatment had been given. a complete physical exami nation was performed and recorded immediately upon the patient’s arrival. the pressure and heart-rate in the supine position were registered every half hour during the first few hours without any treatment. when the blood-pressure was persistently elevated, treatment was initiated with a single oral dose of 400 mg labetalol. in 6 patients, an intravenous injection of 40-80 mg furosemide was also given owing to signs of left ventricular strain. no other medication was given at the same time. the patients were continuously observed for 6-8 hours with repeated registrations of blood-pressure and heart-rate. the blood-pressure was measured with a mercury sphygomanometer and the diastolic pressure was registered as korotkoff phase v . after the initial period, the patients were transferred to an ordinary medical ward for further treatment and examinations. in five patients there were signs of an impaired renal function with elevated serum creatinine levels (range 138-236 pmol/l. normal upper limit < i 2 5 pmol/l). no one was found to have any endocrine disorder. student’s t-test for paired differences was used for statistical evaluation. results and discussion the effect of the treatment upon systolic and diastolic blood-pressure and heart-rate is shown in table i. there was a highly significant reduction of the blood-pressure after one hour (systolic blood-pressure p <0.001, diastolic blood-pressure p < 0 . 0 1 ) . a further decrease was observed up to four hours after tablet intake (tab i). thereafter, the blood-pressure seemed to stabilize at about 110 mmhg diastolic. no significant changes in heart-rate were observed. linear regression analysis showed a significant correlation between the initial diastolic blood-pressure level and the pressure after four hours (r = 0.52, p <0.02). 76 i n a l l b u t one p a t i e n t t h e n e u r o l o g i c a l symptoms had vanished a f t e r 3 hours. three p a t i e n t s complained o f moderate nausea d u r i n g t h e o b s e r v a t i o n p e r i o d . no severe s i d e e f f e c t s were r e g i s t e r e d d u r i n g t h e a c u t e t r e a t m e n t o r subsequently d u r i n g h o s p i t a l i z a t i o n . i n p a r t i c u l a r , t h e r e were no s i g n s o f n e u r o l o g i c a l d e f i c i t due t o an i m p a i r e d c e r e b r a l c i r c u l a t i o n . tab. i. s y s t o l i c (sbp) and d i a s t o l i c (dbp) blood-pressure and h e a r t r a t e (hr) b e f o r e and 1 , 4 and 8 h o u r s a f t e r o r a l a d m i n i s t r a t i o n o f 400 mg l a b e t a l o l . the s i g n i f i c a n c e s a r e i n r e l a t i o n t o t h e i n i t i a l values. hours 0 1 4 8 sbp mmhg 242 f 10 210.7 f 9 172.1 f 8 168 f 6 p <0.001 p <0.001 p <0.001 dbp mmhg 145 * 7 137.8 2 9 114.5 2 6 112 -f 9 p <0.01 p <0.001 p <0.001 hr beat/min 7 2 2 6 7 8 f 4 7 0 2 5 72 f 4 n.s. n.s. n.s. the f i n d i n g s a r e i n accordance w i t h e a r l i e r o b s e r v a t i o n s t h a t l a b e t a l o l is r a p i d l y absorbed and i t s maximum e f f e c t upon i s o p r e n a l i n e i n d u c e d haemo dynamic changes is a t t a i n e d a f t e r about two hours ( 3 ) . the p r a c t i c a l l y unchanged h e a r t r a t e i n c o m b i n a t i o n w i t h t h e decrease o f blood-pressure i n d i c a t e s a f a v o u r a b l e balance between t h e a r e c e p t o r and p r e c e p t o r b l o c k i n g p r o p e r t i e s o f l a b e t a l o l . e x p e r i m e n t a l s t u d i e s have shown a r a t i o o f 1 : 3 between t h e aand p b l o c k i n g e f f e c t a f t e r o r a l a d m i n i s t r a t i o n ( i ) , which is a p p a r e n t l y a l s o f a v o u r a b l e i n t h e a c u t e s i t u a t i o n . the dose o f 400 mg l a b e t a l o l seems t o be adequate f o r t r e a t m e n t o f hyper t e n s i v e emergencies. the m a t e r i a l is t o o s m a l l t o p r i n t r e l i a b l e c o n c l u s i o n s about t h e a d d i t i v e e f f e c t o f i n t r a v e n o u s d i u r e t i c s and i n t h i s study they were n o t used f o r t h e t r e a t m e n t o f h y p e r t e n s i o n per se. s e v e r a l a u t h o r s have warned a g a i n s t r a p i d r e d u c t i o n o f t h e blood-pressure i n severe h y p e r t e n s i o n and t h e t r e a t m e n t s h o u l d c e r t a i n l y o n l y be g i v e n upon s t r i c t i n d i c a t i o n s and i n h o s p i t a l . however, oral a d m i n i s t r a t i o n of l a b e t a l o l produces a g r a d u a l r e d u c t i o n o f t h e b l o o d pressure. i n this s t u d y , t h e dose used d i d n o t g i v e an excessive f a l l o f blood-pressure, which c o u l d l e a d t o a d i s t u r b e d c e r e b r a l c i r c u l a t i o n , w i t h t h e c l i n i c a l hazards t h i s e n t a i l s . acknowledgement mrs k e r s t i n blomberg is thanked f o r s k i l l f u l a s s i s t a n c e and h e l p . 77 references 1 . koch, g.: c a r d i o v a s c u l a r dynamics a f t e r acute and l o n g t e r m adrenoreceptor blockade a t r e s t , supine and s t a n d i n g and d u r i n g e x c e r c i s e . b r . j. c l i n . pharmac. 8 ( s u p p l ) : 101-105, 1979. 2. richard, d.a.: pharmacological e f f e c t s o f l a b e t a l o l i n man. b r . j. c l i n . pharmac. 3 ( s u p p l ) : 721-723, 1976. 3. richard, d.a., maconochie, j.g., bland, r.e., hopkins, r . & m a r t i n , l.: r e l a t i o n s h i p between plasma c o n c e n t r a t i o n s and pharmacological e f f e c t s o f l a b e t a l o l . eur. j. c l i n . pharmac. 11:85-90, 1977. address f o r r e p r i n t s : goran f r i t h z , m.d. department o f medicine c e n t r a l h o s p i t a l 5-631 88 e s k i l s t u n a 78 upsala j med sci 99: 315-338, 1994 7.2 reference intervals based on a danish population ole blaabjergl, inger nprrgaard2, mogens blom2, jens rahbek nprrgaardl, per hyltoft petersenl, hanne gry3, adam uldall4 1. department of clinical chemistry, odense university hospital, dk-5000 odense c, denmark. 2. department of clinical chemistry, hjmrring sygehus, dk-9800 h j ~ r r i n g , denmark. 3. medi-lab, adelgade 5-7, dk-1304 copenhagen, denmark. 4. department of clinical chemistry, kas herlev, dk-2730 herleu, denmark. 7.2.1 need for establishing common reference intervals is there a need for establishing common reference intervals for the quantities in clinical chemistry ? yes, the reference intervals in current use are in principle established by each laboratory using a relevant number of healthy individuals and the present analytical technique. but, experience shows that it is very difficult to get documentation for any laboratory’s reference intervals. the reason is that it is difficult and time consuming to do the work. so often reference intervals are based on an insufficient number of poorly characterized reference indwiduals or they are transferred from an old to a new analytical method when a new equipment, a new calibrator, or a new analytical principle is introduced in the laboratory without the needed investigations. thereby, the reference interval may remain unchanged whereas the analytical quality is drifting, or the traceability of the reference interval weakens considerably. another approach is to use published reference intervals o r to obtain them from other laboratories but, often without sufficient investigation of the transferability. the producer of some kits supply reference intervals together with the kit and this solution may be the best, as long as the producer can keep the stability of standardization and reagents. the quality of calibration and of analytical procedure is a key-point when establishing reliable reference intervals as recently demonstrated by frprlich et al. (3). they compared results from fresh patient sera obtained in two laboratories in the same town using identical equipment and reporting identical reference intervals. out of 15 analytes only four met the demands for sharing common reference intervals as defined by gowans et al. (4). in an investigation djurhuus et al. (2) demonstrated that for danish laboratories the lower reference limit for s-potassium varied from 3.2 to 3.7 mmol/l whereas the results from an external analytical quality control varied by only 0.23 mmol/l for the same laboratories indicating that the analytical technique is better than the estimated reference intervals. 315 concerning plasma proteins the poor standardization, due to insufficient traceability of calibrators, is well known and it has therefore been accepted practice, that each laboratory should establish its own reference intervals. the reliability of these was investigated in denmark in 1990 by an external control survey with an untreated liquid frozen serum pool collected from healthy subjects as control material. the results were compared to the reference intervals for each laboratory and it was anticipated that the result of the control pool should be located centrally in the interval. s-lgm reference intervals in denmark (june 1990) g/l 4.0 3.0 2.0 1 .o laboratory fig. 7.2.1.1. comparison of reference intervals and results from a n external control survey i n denmark in 1990 for s-igm. for each laboratory the reference interval is shown as a bar and the control result is indicated by a cross. in fig. 7.2.1.1 the results for s-igm are illustrated. as the distribution of reference values is log-gaussian the control result should be located just below the central value of the reference interval, and this seems to be the case for many of the laboratories. with a coefficient of variation, cv,, of 28 % for the control results, the figure thus illustrate that the reference intervals were established according to the standardization in each laboratory. the reference intervals, however, vary by a factor of three between the broadest and the most narrow, indicating insufficient number of reference individuals used for estimating the reference intervals. three laboratories are reporting separate intervals for men and women. 316 the results for s-transferrin show a different pattern as illustrated in fig. 7.2.1.2. in this case the cva for the control is approx. 10 %, but the reference intervals are varying considerably, indicating a lack of traceability for most of the laboratories. the control values are above the highest reference limit for six of the laboratories. regarding immunological methods there should be no serious problems concerning analytical specificity of the methods and with an untreated frozen liquid pool the problems are negligible as also indicated by the constancy of the control results. how the reference intervals have been estimated or chosen by the different laboratories has not been investigated but more than half of the laboratories have serious problems with their reference intervals. s-pansferrin reference intervals in denmark [june 1990) pmol/l laboratory fig. 7.2.1.2. comparison of reference intervals and results from an external control survey in denmark i n 1990 for s-transferrin. for each laboratory the reference interval is shown as a bar and the control result is indicated by a cross. the two examples clearly demonstrate the problems related to reference intervals for plasma proteins and indicate the need for establishing better reference intervals. one of the presumptions for this is a general and reliable standardization with calibrators and concentration values traceable to a common reference preparation. with the ifcc/cap/bcr 470 this is now possible and when we have a general basis for calibration it seems logical to establish common reference intervals for populations homogeneous for the quantities and to control the analytical quality according to the accept limits given (4,5). 317 7.2.2 collection of r e f e r e n c e serum samples and m e a s u r e m e n t s p r e s u m p t i o n s f o r establishing common r e f e r e n c e intervals the biology must be common i.e. the population must be homogeneous for the quantity. the sampling conditions must be standadzed. the analytical quality must be acceptable within the quality specifications, i.e. traceable to the same common reference preparation, and with negligible unspecificity. the interpretation of reference values must be the same. 8 r e f e r e n c e individuals assuming that the population of adults born in denmark and still living in denmark is homogeneous for the nine plasma proteins [ s-prealbumin(transthyretin), s-albumin, s-orosomucoid (al-acid glycoprotein), s-al-antitrypsin (q-trypsin inhibitor), s-haptoglobin, s-transferrin, s-iga, s-igg, and s-igm 1 the selection of reference subjects is based on this population using the following additional criteria: hospital personnel and their relatives. 20 hospital laboratories from the whole country. (the population in denmark is considered homogeneous for the proteins as the area is restricted and the population is well mixed with only moderate influx of foreigners until 20 years ago). state of health: the person must feel healthy no chronic disease no intake of regular medications (insulin, penicillin, antihypertensiva etc.) excluded are blood donors restrictions 24 hours before blood collection: no exhausting exercise usual intake of food not more than 12 g of alcohol (approx. one drink). 318 in each of the 20 laboratories blood was collected from a number of persons according to the scheme: b e group men women (-e) women ( t e) 18-20 2 21-30 2 31-40 2 41-50 2 51-60 2 61-70 2 7 1-80 2 2 2 2 2 2 2 2 2 2 2 2 blood sampling was performed according t o the individual laboratory’s routine: rn sitting position 15 min before and during sampling sampling from arm vein (minimum stasis) in vacuum tubes (if possible) sampling volume for serum approx. 40 ml (without stabilizers) and for blood to sedimentation rate measurements approx. 5 ml (with citrate) the sedimentation rate was performed locally within few hours. handling of blood/serum for protein determinations: mixing of serum rn coagulation for about one hour at room temperature centrifugation at 1500*g for 15 min. pipetting of serum and mixing new centrifugation at 1500*g for 15 min. distribution of serum in 8 cryotubes freezing (at -80 o c if possible) storage and transport: m rn 4 tubes sent to the laboratory measuring all samples 2 tubes kept in reserve (stored locally) 2 tubes for local measurements rule out criteria: m it is well known, that a number of conditions influence the concentrations of plasma proteins. some are considered serious, e.g. diseases especially acute phase reactions. known diseases are excluded as indicated above, whereas, others are unnoticed such as minor infections in an early stage or even weeks after infections. these conditions are not recognized but are sought t o be disclosed by 319 measuring sedimentation rate and s-crp and by excluding results from individuals with elevated values of these tests. further, the presence of m components will influence the immunoglobulin concentrations, so results from individuals with detectable m-components are excluded accordingly. the criteria for excluding were s-crp above 10 mg/l, sedimentation rate according to chapter 7.5, and m-components if a distinct band is detected in the fiy-area by agarose electrophoresis. fortunately, the number of rejections were small (7 %) and none of the values gave reason to assume a serious disease. blood donors are excluded due to the risk of abnormal values of s-transferrin and s-haptoglobin. women using estrogens for anti-conception have been included as a special € p u p . measurements of all samples were performed on a cobas fara (roche) according to the method recommendations from dako and with antisera from dako using the nordic calibrator (all values are traceable to ifcc/cap/bcr 470). each result is calculated from the mean of two measurements performed in separate runs. the controls (a, b, and c) from the nordic protein project were used according to the westgurd-rules (mean and range rules). measurements of s-c-reactive protein, s-crp, were performed by sgren blirup jensen and per just svendsen, dako. electrophoreses for disclosing m-components were performed at t h e department of clinical chemistry, hjarring hospital. the data base was designed for the purpose and calculations of mean, standard deviations, skewness and kurtosis for concentrations and log-values as well as for percentiles for the different combinations of individuals were performed by the computer programme spss. reference individuals it was the goal to collect 20*36 = 720 reference samples. but only 553 samples were received from the 20 laboratories. 37 of these had to be excluded due to the rule out criteria (high sedimentation rate, high s-crp, and the presence of m-components. two had raised values of both sedimentation rate and s-crp and one had both a m component and elevated sedimentation rate. in total 9 m-components, 15 elevated s crp, and 16 elevated sedimentation rates were found. 320 the total number of included reference individuals are: age group men women (-e) women (+ e) 18-20 31 26 24 21-30 37 38 34 31-40 33 38 26 41-50 36 40 24 51-60 30 34 61-70 20 27 7 1-80 14 7 the number of indviduals in the older age groups are small and so less representative in the calculations. the general approach for lumping the groups has been men below 50 years of age (137), women below 50 (not using estrogen) (142), women below 50 (using estrogen) (1081, men above 50 (641, and women above 50 (68). the criteria for selection of reference individuals are in close agreement with the recommendations from the nordic recommendations for production of reference values from adults (l), whereas the rule out criteria are specific for estimation of reference intervals for plasma proteins. 7.2.3 biology and reference intervals for eight proteins for each of eight proteins the biological variations according to age and sex as well as to the effect of estrogens are illustrated by their means and the 0.90 (90 %) confidence intervals, ci. the ordinates are logarithmic as most of the distributions are close to log-gaussian. further, the distributions of the concentrations are shown in probit plots according to the subgroupings and finally the 0.95 interfractile reference intervals are given. the plasma protein al-antitrypsin is described separately in chapter 7.3. s-prealbumin (s-transthyretin). the biological distributions according to age and sex are given in fig. 7.2.3.1.a. the figure shows that the group of women below 50 years of age not using estrogens has lower values. the remaining mean-values seem to describe a curve with increasing values up to about 40 years, and then descending again. it is possible t o choose separate reference intervals for each subgroup but, the degrees of freedom are small and according t o the criteria (section 7.1) it is not reasonable to do so. 321 s-prealbumin 0.40-0.40 0.38 0.36-0.44 0.34 0.32 -0.48 0.30 -0.52 0.20 0.26 -0.56 -0.60 concentration 99 95 90 a0 7 0 .~ 60 50 4 0 30 20 10 5 1 women (-) men women f mean and 0.90 ci i 1 i i i i i 1 18-20 21-30 31-40 41-50 51-60 61-70 71-80 age groups fig. 7.2.3.1.a mean concentrations with 90 % ci for each subgroup shown on a log-scale i n relation to age for s-prealbumin is-transthyretin). s-prealbumin cumulated percentage irequency prorlt i " " i " " ~ " ' ~ " " ' i i " " i -0.70 -0.60 -0.50 -0.40 -0.30 log g/l i " " 1 . 0.2 0.3 0 . 4 0.5 g/l fig. 7.2.3.1.b. probit-plot with log-abscissa showing s-prealbumin is-transthyretin) distributions for women not using estrogens and the remaining group (all others). 322 in fig. 7.2.3.1.b the two distributions are clearly straight lines indicating log-gaussian distributions close to be parallel, accordingly the reference intervals are given as 0.23 to 0.39 g/l for women below 50 years of age not using estrogens and 0.26 to 0.45 g/l for the remaining adults. s-albumin. the biological distributions according to age and sex are given in fig. 7.2.3.2.a. the figure shows that the group of women below 50 years of age using estrogens differ with lower values from the other individuals below 50 years. the mean values of the remaining groups seem to descend with increasing age. it is possible to choose separate reference intervals for each subgroup but, we have chosen to cut at the age of 50 and combining the women below 50 using estrogens with the individuals above 50 years of age. s albumin concentration g/l log gtl 48 1.68 381.58 fig, 7.2.3.2.r b p t women (+e) men women i i i i i 1 i 1 18-20 21-30 31-40 41-50 51-60 61-70 71-80 age groups mean concentrations with 90 % ci for each subgroup shown on a log-scale in relation to age for s-albumin. in fig. 7.2.3.2.b the two distributions are clearly straight lines indicating log-gaussian distributions close to be parallel, accordingly the reference intervals are given as 39.6 to 51.1 g/l for men below 50 and women below 50 years of age not using estrogens and 36.6 to 48.2 g/l for the remaining adults. 22-955059 323 salbumin cumulated percentage frequency problt 0.00 -0.04 -0.08 -0.12 --0.16 -0.20 -0.24 -0.28 fig. 7.2.3.2. b. s-orosomucoic fig. 7.2.3.3.a. (s-cx1 women below 50 (+e women above 50 omen below 50 (-e) i ' l ' i ' i ' i . i ' i ' 1 ~ 1 ~ 1 i ' " ' 1 " ~ ~ 1 ' ' ' 1 ' ' 1.54 1.58 1.62 1.66 1.70 log g/l 35 40 45 50 g/l probit-plot with log-abscissa showing s-albumin distributions for men and women not using estrogens, both below 50 years of age and the remaining group (all others). cid glycoprotein) s-orosomucoid concentration g/l log gtl i i 1 .o 0.9 0.8 0.7 0.6 0.5 i 9;: women 1 1 1 1 1 1 ~ ~ 18-20 21-30 31-40 41-50 51-60 61-70 71-80 mean ? 0.90 ci f men 1 women age group mean concentrations with 90 % ci for each subgroup shown on a log-scale in relation to age for s-orosomucoid. 324 the biological distributions according to age and sex are given in fig. 7.2.3.3.a. the figure shows that the women below 50 years of age separate into two groups according to the use of estrogens, both groups are clearly lower than the remaining groups. the men show constancy and women above 50 are at the same level. it is possible t o choose separate reference intervds for each subgroup of women below 50 years of age as illustrated in fig. 7.2.3.3.b. s-orosomucoid cumulated percentage frequency p r w t fig. 7.2.3.3.b. women below 50 +e -e i i l i i [ i -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 log g/l i i i i ' 1 ' 1 ' 1 0.3 0.4 0.5 0.6 0.8 1.0 1.2 g/l probit-plot with log-abscissa showing for s-orosomucoid the distributions for women not using estrogens, for women using estrogens both below 50 years of age and the remaining group (all others). in fig. 7.2.3.3.b two of the distributions are clearly straight lines indicating log gaussian distributions, whereas, the distribution for women using estrogens might be a little more skew. in order t o investigate whether it is reasonable to separate between the women below 50 with and without estrogens the 0.90 confidence intervals for the percentiles (with 107 degrees of freedom) are shown as the hatched area around the line for women using estrogens in fig. 7.2.3.3.c. there is no overlap with the group not using estrogens, which in turn also differ from the remaining group as the degrees of freedom are 141 (not shown in the figure). all three lines are close to be parallel so, the reference intervals are given as 0.40 to 0.95 g/l for women below 50 years of age using estrogens, 0.45 to 1.08 g/l for women below 50 not using estrogens and 0.54 to 1.17 g/l for the remaining adults. s orosomucoid is the only protein where the reference intervals are given for three separate groups. 325 s-orosomucoid cumulated percentage frequency problt fig. 7.2.3.3. c. women below 50 +e -e 99 80 70 5 0 40 30 20 10 5 1 * 0.9 ci i i i , i , , -0 5 0 4 -03 -02 -0.1 00 0 1 log g/l i i i i i 1 . 1 1 , 03 0 4 0506 08 1 0 1 2 g/l probit-plot with log-abscissa for s-orosomucoid showing the same distributions as i n fig 7.2.3.3.b, but with 0.90 ci for percentiles for 107 degrees of freedom around the lin for women using estrogens. s-haptoglobin s-haptoglobin concentration g/l log g/l 0.7 fig. 7.2.3.4.a t t 16-20 21-30 31-4041-50 51-60 61-70 71-80 age @oups mean concentrations with 90 96 ci for each subgroup shown on a log-scale in relation to age for s-haptoglobin. 326 the biological distributions for s-haptoglobin according to age and sex are given in fig. 7.2.3.4.a. the figure shows age to be the dominating factor as the concentrations are increasing with age. accordingly, the values are separated into two groups, one below 50 and one above 50 years of age. s-haptoglobin cumulated percentage frequency probit fig. 7.2.3.4.b. l . l ' l ' l ' l probit-plot with linear abscissa showing for s-haptoglobin the distributions for individuals below 50 and individuals above 50 years o f age. 0.0 0.5 1.0 1.5 2.0 g/l the two groups are neither log-gaussian, nor gaussian, and the distributions are therefore shown with linear abscissa in fig. 7.2.3.4.b. both distributions are curved but the difference seems to be rather constant at all levels. this difference does not seem important due to the broad intervals but, we have decided to keep two separate intervals. for individuals below 50 the reference interval is 0.35 to 1.85 g/l and above 50 years of age it is from 0.47 to 2.05 g/l. s-transferrin the biological variations and the distributions are described in section 7.1 in which s transferrin is used as an example for the presentations and main problems. reference interval for women below 50 years of age using estrogens is 2.25 to 3.85 g/l and for the remaining group 1.94 to 3.26 g/l. s-iga s-iga resemble s-haptoglobin with values increasing with age and no difference due to sex and to estrogen as seen from fig. 7.2.3.5.a. 327 gll 3.c 2.5 2.0 1.5 0.5 0.4 0.3 0.2 0.1 fig. 7.2.3.5.a. fig. 7.2.3.5. b. concen !ration s-iga i t b mean and 0.90 ci i men women i i i 1 i 1 i 1 18-20 21-30 31-40 41-50 51-60 61-70 71-80 age groups mean concentrations with 90 % ci for each subgroup shown o n a log-scale in relation to age for s i g a s-iga cumulated percenrage frequency problt -0.2 0 . 1 0.0 0.1 0.2 0.3 0.4 0 5 0.6 0.7 1 ' 1 . 1 ' . : ' ' ' ' i . i i 0.6 0.0 1.0 2.0 3.0 4.0 5.0 gil probit-plot with log-abscissa showing s-iga distributions for the individuals below 50 and the individuals above 50 years of age. 328 in the probit-plot (fig. 7.2.3.5.b) the two dstributions are not two straight lines on the log-scale. but, by adding 0.50 g/l to all values (5) the distributions seem to be nearly log-gaussian as seen in fig. 7.2.3.5.c. the highest values, however, seem to diverge from the straight line so the calculation of reference intervals has been performed according to non-parametric estimations of the 2% and 97% percentiles. s-iga cunulated percentage frequency problt 0 0 0 0.50 gil added 0 0 0 0 above 50 0 -0.2 0 1 0.0 0.1 0.2 0.3 0 4 0.5 0 6 0.7 loaqk fig. 7.2.3.5.c. i . 1 1 i 1 4 1 0.6 0.8 1.0 2.0 3.0 4.0 5.0 g/l probit-plot with log-abscissa showing the same distributions for s-iga a s i n fig 7.2.3.3.8, and after addition of 0.5 g / l to all values. the question mark indicates the deuiation from a straight line. the reference intervals according to non-parametric estimations for individuals below 50 are 0.70 to 3.65 g/l and for individuals above 50 years of age 0.70 to 4.30 g/l. s-igg the biological distributions according to age and sex are given in fig. 7.2.3.6.a. the figure shows that the group of women below 50 years of age differ with higher values. the remaining mean-values do not describe a clear pattern and it might be possible to separate the women below 50 in two groups according to estrogen. we have decided, however, to distinguish between women below 50 and the remaining group as illustrated in fig 7.2.3.6.b. 329 s-igg 99 95 90 80 70 60 50 40 30 20 10 5 1 concentration gll 14.0 13.0 12.0 11.0 10.0 9.0 8.0 7.0 fig. 7.2.3.6.a fig. 7.2.3.6. b. log g/l 1.15 1.10 1.05 1 .oo 0.95 0.90 0.85 f :men i mean and 0.90 ci 1 i i i i i i 1 18-2021-30 31-40 41-50 51-60 61-70 71-80 age groups mean concentrations with 90 '% ci for each subgroup shown on a log-scale in relation to age for s-igg. s-igg cumulated percentage frequency probit 50 i i i i i i i 0.7 0.8 0.9 1.0 1.1 1.2 1.3 log g/l i i i i . i ' 1 ' 1 ~ 1 " ' l 5 6 7 8 10 12 14 16 20 g/l probit-plot with log-abscissa showing s-igg distributions for women below 50 years of age and the remaining group (all others). 330 in fig. 7.2.3.6.b the two distributions are close to straight lines (with minor deviations in both directions) indicating that they are close to log-gaussian. 1.40.15 1.30.10 1.1 0.05 1.2 1.0 0.00 0.9 -0.05 0.8--0.10 0.7 -0.15 the reference intervals for s-igg are given as 6.9 to 15.7 g/l for women below 50 years of age and 6.1 to 14.9 g/l for the remaining adults. s-igm the biological distributions according to age and sex are given in fig. 7.2.3.7.a. the figure shows that the group of women below 50 years of age differs with higher values. the remaining mean-values seem to be constant with age. concentration f men women mean and 0.90 ci 1 i i i i i i 1 18-20 21-30 31-40 41-50 51-60 61-70 71-80 age groups fig. 7.2.3.7.a mean concentrations with 90 % ci for each subgroup shown on a log-scale in relation to age for s-igm. in fig. 7.2.3.7.b the two distributions are close to straight lines indicating log gaussian distributions. the reference intervals for s-igm are given as 0.55 to 2.30 g/l for women below 50 years of age and 0.39 to 2.08 g/l for the remaining adults. 33 1 s-lgm cumulated percentage frequency probit i i i i i i i i i i i i 1 i 1 i ' i ' 1 ' 1 ' ' ~ i ~ " ' l -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 log g/l 0.4 0.5 0.6 0.70.8 1.0 1.2 1.41.6 2.0 2.5 g/l fig. 7.2.3.7.b. probit-plot with log-abscissa showing s-igm distributions for women below 50 years of age and the remaining group (all others). 7.2.4 distributions of values from the individuals not included due to the rule out criteria for the estimation of reference intervals 37 individuals were ruled out due to high sedmentation rate, high s-crp or the presence of m-component. among the 16 individuals with elevated sedimentation rate two had elevated s-crp as well and one had an m-component. 15 had elevated s-crp (two of these with high sedimentation rate) and 9 dxclosed an m-component (one also with a high sedimentation rate). as the three conditions are expected to be related to changes in plasma proteins it is interesting to investigate whether the results are different from t h e reference individuals. in the following the distributions are shown on probit-scales in relation to the reference distributions. the number of values, and thereby, the degrees of freedom and the reliability is not impressive but as the majority of the values show a tendency, some tentative conclusions may be made. for s-prealbumin (fig. 7.2.4.1) there is a clear tendency to lower values when s-crp is elevated, whereas, the elevated sedimentation rate and the presence of m components seem t o have negligible effects. 332 s-prealburnin cumulated percentage frequency probit 99 95 90 8 0 70 60 50 40 30 20 5 lo m-component '1 '(others) 0 70 -0 60 0 50 0 4 0 030 log g/l i' ' * . i ' ' . , i ' . ' . i 02 03 0 4 0 5 g l l fig. 7.2.4.1. distributions of concentration values for s-prealbumin from the excluded individuals compared to the group 'others'. one woman below 50 and not using estrogens but disclosing high values of both sedimentation rate and s-crp is excluded, all the others are comparable to the distribution of others. salbumin cumulated percentage frequency problt above 50 and +e esr (0) 1 ' i ' i ' i . l . i ' i . i ' i ~ i 1.54 1.58 1.62 1.66 1.70 log gll i ' " ' 1 1 1 ' 1 1 ' 1 1 ' 1 35 40 45 50 g/l fig. 7.2.4.2. distributions of concentration values for s-albumin f r o m the excluded individuals. 333 for s-albumin (fig. 7.2.4.2) distributions for individuals below and above 50 are shown for the groups with high sedimentation rate (8 in each group) as well as for high s-crp (5 below 50 (not using estrogens) and 10 in the remaining group). two indwiduals are excluded from the group of m-component, reducing the number to 7, which should be compared to the lower distribution. there is a tendency to lower values in all groups but, only for the group of men and women (not using estrogens) below 50 years of age there is a more convincing decrease. s-orosomucoid cumulated percentage frequency probit w (women below 50 +e) (others) i i l i i i i -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 log g r i i i i 1 ' i ' i 0.3 0.4 0.5 0.6 0.8 1.0 1.2 g/l rate fig. 7.2.4.3. distributions of concentration vazues for s-orosomucoid from the excluded individuals. for s-orosomucoid (fig. 7.2.4.3) women below 50 are excluded from the groups with m-components and elevated sedimentation rate, leaving 8 and 1 2 individuals in the two groups, respectively. the group with elevated s-crp is separated in women using estrogens (n = 6) and men and women above 50 (n = 7). m-components reveal no effect but elevated sedimentation rate indicates increased values and high s-crp is accompanied by increased s-orosomucid values, most distinct for women below 50 using estrogens. for s-haptoglobin (fig. 7.2.4.4) there is a slight tendency for higher values in all groups, but the distributions are broader than the distributions for the reference values. 334 s-haptoglobin 99 95 90 80 7 0 60 50 40 30 20 l o 1 cumulated percentage frequency probit y above 50 i ' l ~ l ~ i l l 0.0 0.5 1.0 1.5 2.0 g/l fig. 7.2.4.4. distributions of concentration values for s-haptoglobin from the excluded individuals. s-transferrin (see chapter 7.1) resembles s-albumin with slightly decreased values in all groups, less pronounced for sedimentation rate. m-component-group with n = 8 and sedimentation rate n = 13 (women using estrogens excluded), and s-crp n = 7 for women using estrogens and n = 8 for t h e remaining group. s-iga cumulated percentage frequency problt 99 95 90 5 0 40 sedmentationrate ( y ) i -0.2 -0.1 0.0 0.1 0.2 0 3 0.4 0.5 0.6 0.7 log e/l 1 . 1 . i . . . i " " i 1 . 1 0.6 0.5 1.0 2.0 3.0 4.0 5.0 g/l fig. 7.2.4.5. distributions of concentration values for s-iga from the excluded individuals. 3 3 5 concerning s-iga (fig. 7.2.4.5) one individual had a m-component of class iga (and an additional of type g). the m-component group consisted of n = 6 (excluding'2 individuals below 50). for s-crp 12 individuals below 50 were included, and the sedimentation rategroup separated in 10 below 50 and 6 above 50. for the m component group there was a tendency t o lower values and for the sedimentation rate groups higher s-iga values were obtained. 99 95 90 80 70 60 50 4 0 30 20 l o 5 1 women below 50 others crp (0) sedimentation rate (w<50) sedimentation rate (0) 1 1 1 1 1 1 1 i 1 i i ' i ' t ~ l ~ ~ ~ ! ~ ~ 5 6 7 8 10 12 14 16 20 g/l 0.7 0.8 0.9 1.0 1 . 1 1.2 1.3 log g/l fig. 7.2.4.6. distributions of concentration values for s-igg from the excluded indiuiduals. seven of the nine m-components were of class igg, so there is no distribution for m components in fig. 7.2.4.6. both the sedimentation rate group and the s-crp group were separated into two: s-crp: women below 50 n = 9 and the remaining n = 6, and for sehmentation rate for the same groups n = 4 and n = 12, respectively. the values of s-igg for all groups seem to be slightly increased. two m-components of type igm were found in the material and the corresponding s igm values are excluded. the separation into subgroups is the same as for s-igg. the distributions as shown in fig, 7.2.4.7, are close to the the reference groups, except from a few higher values and a tendency to higher values of s-igm in individuals with elevated sedimentation rate. 3 3 6 cumulated percentage frequency probit 99 95 90 80 70 60 sedimentation sedimentation rate ( w c 5 0 ) women below 50 i i i i i i i i i i i i i i ' 1 ' i ' 1 ' 1 " ' l " " l -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 log g/l 0.4 0.5 0.60.70.8 1.0 1.2 1.41.6 2.0 2.5 g/l fig. 7.2.4.7. distributions of concentration values for s-igm from the excluded individuals. as a general conclusion based on the figures showing the distributions of values from the individuals who were ruled out, it is confirmed that relationships exist between the concentrations of the eight plasma proteins and the two acute phase indicators as well as minor m-components. this implies that the same rule out criteria should be used when plasma protein reference intervals for other ethnic groups are considered. discussion the estimations of reference intervals were planned in order to supplement the calibration and control projects by providing the reference intervals needed for the total quality concept, i.e. 1) common reference intervals, 2) analytical quality specifications for sharing these common reference intervals, 3) creation of analytical quality needed, and 4) control of analytical quality according to the purpose. the sample size of reference individuals was, therefore, designed to approx. 800 (41, actually 720 individuals. the obtained number, however, was 516, which is a considerable number, but less than needed for the ideal situation. further, only minor differences between the groups were expected, except from the the estrogen effects. the actual disclosed and unexpected differences, however, varied from protein to protein and thus reduced the certainty of the distributions of each subgroup. 3 3 7 therefore, the conclusions concerning some of the estimated reference intervals are less certain than expected from the design. a new and better design could be described based on the results obtained here, but even with the limitations of the material, the presented common reference intervals are better than any solution with individual reference intervals for each laboratory presupposing the use of a common calibrator. acknowledgements we want to thank all the persons who have contributed to this work, volunteers who gave blood to the investigation and the 20 danish laboratories involved in collecting the blood samples and doing extra analytical work. we are in debt of gratitude to h e richter, who performed the registrations and calculations of the many results. further we are grateful to sprren blirup and per just svendsen, who performed the measurements of s-crp and to dako for the antisera used in all the determinations. references 1. 2. 3. 4. 5. alstrom t, grasbeck r, lindblad b, solberg he, winkel p, viinikka l. establishing reference values from adults: recommendation on procedures for the preparation of individuals, collection of blood, and handling and storage of specimens. scund j clin lab invest 1993;53:649-52. djurhuus ms, rohold a, vadstrup s, hyltoft petersen p, uldall a. reference intervals based on hospitalized 'healthy' patients and medical students in relation to analytical bias for serum potassium. scund j clin lab invest 1992;52:305-12. frolich a, nielsen bf, nielsen j, conradsen 9 mcnair p. do local hospitals meet the analytical goals for the use of common reference intervals? scund j clin lab invest 1994;54:169-76. gowans ems, hyltoft petersen p, blaabjerg 0, horder m, analytical goals for the acceptance of common reference intervals for laboratories throughout a geographical area. scund j clin lab invest 1988;48:757-64. hyltoft petersen p, gowans ems, blaabjerg 0, h ~ r d e r m, analytical goals for estimation of non gaussian reference intervals. scund j clin lab invest 1889;49:727-37. 338 ujms 110 (3) bra upsala j med sci 110 (3): 233–236, 2005 sertoli-leydig cell tumour in a postmenopausal woman showing all facets of the insulin resistance syndrome (irs) eva dahlgren 1, berit gull, roger willén 2, frank sundler 3, thord rosén 4 and per-anders jansson 5 1department of obstetrics and gynecology the sahlgrenska academy at göteborg university, 2department of clinical pathology and cytology, uppsala university hospital, 3department of physiological sciences, neuroendocrine cell biology, 4university of lund, research center for endocrinology and metabolism and 5the lundberg laboratory for diabetes research the sahlgrenska academy at göteborg university, sweden. abstract sertoli-leydig cell tumours are rare sex stromal tumours with an incidence of <0.5% of all ovarian tumours. most frequently this tumour occurs in young women with a history of amenorrhoea, hirsutism and lowered pitch. here, we report on a woman with irs, postmenopausal virilization and increased testosterone levels due to a sertoli-leydig cell tumour. this is the first case to suggest an association between irs and sertoli-leydig cell tumours. furthermore, we highlight the difficulties in detecting this ovarian tumour with sonography. case report a 64-year-old woman with a three year history of hirsutism was referred to the department of ob&gyn. the woman is a iv-gravida, iii-parous with regular periods until menopause at the age of 47. she smoked 15 cigarettes per day. in 1988, three years after menopause, she was diagnosed with and advised of lifestyle changes for central obesity and hyperlipidemia. in 1990 she was prescribed selective beta-blockade due to hypertension. in 1992 type 2 diabetes was diagnosed and she exhibited microalbuminuria, hence, the woman had developed an overt insulin resistance syndrome (irs) (1). she became normoglycaemic after introduction of multiple insulin injection therapy. however, diabetes complications appeared in the next few years. in 1995 mani233 received 3 february 2004 accepted 28 february 2005 key words: sertoli-leydig cell tumour, insulin resistance, metabolic syndrome, exogenous insulin treatment. fest nephropathy and angina pectoris were diagnosed. in 1997, to improve metabolic control, normal insulin (actrapid®) was substituted with insulin lispro (humalolog®). unfortunately, the woman reported worsening of her angina pectoris, and she was referred to the cardiology unit. in 1998, she underwent a coronary artery by pass graft operation, involving five coronary vessels. postoperatively, the woman reported no angina pectoris, quit smoking, and temporarily normalised her metabolic control. hirsutism was first noted in 1998 but worsened in early 2001. she reported daily cutting of facial hair. the endocrine investigation showed a total serum testosterone level that was markedly elevated (19 nmol/l). on suspicion of an ovarian origin for the high testosterone level a gynaecological examination and a vaginal ultrasound examination (aloka ssd 2000) was performed with a normal finding. an mr investigation of the adrenals excluded adrenal pathology and a second gynaecological ultrasound (atl hdi 5000, vaginal probe 7.5 mhz) was performed. the left ovary measured 22x21x23 mm, was a little squared with some mixed echogenicity, but with normal, not increased circulation. the right ovary was found to be round with a diameter of 16 mm with no deviant echogenicity or vascularization. thus, there was some suspicion of hypertrophy of the left ovary which led to a laparoscopic bilateral salpingoophorectomy of a normal left ovary and a right ovary enlarged to three times normal size, rounded, and without macroscopic evidence of tumorous vegetation on the surface. there were no signs of peritoneal carcinosis or ascites. histopathology confirmed a normal left ovary and in the right ovary an encapsulated well differentiated sertoli-leydig cell tumour, reaching 0.1 cm from the surface. the immuno-histochemical study showed a strong reaction for inhibin (fig 1) in sertoli-leydig cell tumours inhibin is positive in 100% of cases. tumour cells were 234 fig 1: overview of the sertoli leydig cell tumour, with sertoli tubules at the bottom and clusters of leydig cells at the top. strong inhibin immunoreactivity in both sertoli as well as in leydig cell components. magnification. x 120. igf-1 receptor negative except for vessel walls. furthermore, insulin receptor markers were negative on the sertoli cells but diffusely slightly positive within the leydig cells (fig 2). the testosterone level was normalised postoperatively (0.60 nmol/l). metabolic control was not improved 6 months postoperatively, i e hba 1 c ≈ 9.0 %. discussion this is the first time a sertoli-leydig cell tumour is described in a postmenopausal woman showing all characteristics of the irs [1]. our patient was treated with insulin as the initial therapy for her pronounced hyperglycaemia. interestingly, chronic hyperinsulinaemia has been reported to cause an increased production of ovarian androgens including testosterone and a decrease in serum sex hormonebinding globulin [2], suggesting a link between irs and hyperandrogenism. is it likely that hyperinsulinaemia caused the sertoli-leydig cell tumour in our patient? since we did not find any impressive staining of insulin receptors in our tumour biopsies, which is in contrast to a previous report [3] one has to be cautious about any statement regarding an association between endogenous/exogenous hyperinsulinaemia and sertoli-leydig cell tumours based on this case report. how235 fig 2: diffuse slight positive immunohistochemical staining for insulin receptor within the leydig cells but no staining of the sertoli cells. magnification. x 120. ever, it should be mentioned the solid theoretical background for the propensity to develop malignancies in subjects with the irs [4-7]. hence, it might be of value to consider this rare tumour in postmenopausal women exhibiting irs and concomitantly receiving multiple injection therapy with high dosages of insulin to avoid hyperglycaemia. the present case also highlights that false positive and false negative findings on ultrasonography of the ovaries may occur even in the hands of an experienced gynaecological ultrasonographer. indeed, the opposite ovary, contrary to the sonographer’s suggestion, was the site for the sertoli-leydig cell tumour. therefore, to exclude a sertoli-leydig cell tumour, we recommend exploration with laparoscopy if the clinical picture implies a hyperandrogenic state, and ultrasound of the ovaries and mr of the adrenals both are normal. references 1. alberti kgmm, zimmet p for the who consultation group (1998). definition, diagnosis and classification of diabetes mellitus and its complications. part 1: diagnosis and classification of diabetes mellitus. diabet med 15: 539-53. 2. barbieri rl, smith s, ryan kj (1998). the role of hyperinsulinaemia in the pathogenesis of ovarian hyperandrogenism. fertil steril 50: 197-212. 3. nagamani m, stuart ca, van dinh t (1989). steroid biosynthesis in the sertoli-leydig cell tumour: effects of insulin and luteinizing hormone. am j obstet gynecol 161: 1738-43. 4. stoll ba (2002). upper abdominal obesity, insulin resistance and breast cancer risk. int j obes relat metab disord 26: 747-53. 5. colangelu la, gapstur sm, gann ph, dyer ar, liu k (2002). colorectal cancer mortality and factors related to the insulin resistance syndrome. cancer epidemiol biomarkers prev 11:385-91. 6. hsing aw, gao yt, chua s jr, deng j, stanczyk fz (2003). insulin resistance and prostate cancer risk. j natl cancer inst 95: 67-71. 7. kurtzhals p, schaffer l, sorensen a, kristensen c, jonassen i, schmid c, trub t (2000). correlations of receptor binding and metabolic and mitogenic potencies of insulin analogs designed for clinical use. diabetes 49: 999-1000 corresponding author: eva dahlgren md, phd department of obstetrics and gynecology su sahlgrenska 435 14 gothenburg sweden phone:+46313421000 e-mail:eva.dahlgren@vgregion.se 236 upsala j med sci 95: 147-155, 1990 perinatal hypoadrenalism in the rat does not alter glucose tolerance and insulin secretory response to glucose mats g. karlsson, ulf j. eriksson, ingemar swenne department of medical cell biology, uppsala university, uppsala, sweden abstract fetal and neonatal hypoadrenalism was induced by treating rat mothers with metyrapone from day 12 of pregnancy to day 7 postnatally. hypoadrenalism in the neonates was indicated by a slight reduction in body weight, adrenal hyperplasia and a tendency towards reduced adrenal corticosterone concentration. an intraperitoneal glucose tolerance test on the 7-day old neonates did not show any disturbancies of glucose disposal or insulin secretory response to glucose. the data suggest that adrenal corticosteroids are not essential for the perinatal development of the b-cell secretory response to glucose. introduction the functional development of the pancreatic b-cell takes place in an orderly fashion during fetal and neonatal life. in the rat fetus b-cells can be identified morphologically on day 12 of gestation (27, 35) but they do not attain functional competence until later in development. glucose-stimulated insulin secretion is absent on gestational day 15 (28), can be demonstrated on day 18 (3, 1 9 ) 1 develops further during the neonatal period (1) but does not reach its full capacity until adult life (11). the factors governing these developmental changes are not fully understood, but glucose itself has been demonstrated to stimulate the development of a glucose-sensitive insulin secretory response (2, 8).howeverl it is also of interest to note that serum concentrations of growth hormone ( 4 , 29, 30), thyroid hormones (7) and corticosterone (16) all increase dramatically during the perinatal period and could potentially modulate b-cell develop 147 ment. growth hormone increases the insulin output of fetal pancreatic islets in vitro but fails to increase the glucose sensitivity of the secretory process or islet cell replication (32).fetal decapitation in utero, which minimizes pituitary influence on pancreatic development, does not retard the develop ment of insulin secretion (17, 20).thyroid hormones are similarly without effect on b-cell maturation in vitro (32) and perinatal hypothyroidism does not affect the development of the insulin secretory response to glucose in vivo (31). so far the possible role of corticosterone in this process has not been investigated. in the present study insulin secretory development is inves tigated in the offspring of rats treated with metyrapone during pregnancy and lactation. this substance crosses the placenta and diminishes both maternal and fetal corticosterone synthesis (10) by inhibition of adrenal hydroxylases ( 5 , 22), thereby inducing a state of hypoadrenalism. materials and methods female sprague-dawley rats from a local colony (biomedicum, uppsala, sweden) were caged overnight with males and vaginal smears were taken on the following morning. day 0 of pregnancy was considered as the day on which a sperm-containing smear was found. in order to induce hypoadrenalism in the offspring, the rat mothers were treated with metyrapone from day 12 of pregnancy to the end of the experiment on day 7 postnatally.two ml of an aqueous solution of metyrapone ( metopironr; kindly provided by ciba-geigy, gbteborg, sweden) was administered with a gastric tube as a once daily dose of 50-70 mg/kg body weight. control animals were not treated with gastric intubation. all animals had free access to drinking water and pelleted laboratory animal chow (r2 pellets; a-lab, sodertalje, sweden) throughout the experi ment. delivery took place spontanously on day 22 of pregnancy in both experimental and control group. on day 7 postnatally a glucose tolerance test was performed on the offspring. the pups were weighed and some removed for sampling of basal serum concentrations of glucose and insulin or for dissection of pancreas and adrenals. the remaining pups received an i.p. injection of 2 g/kg body weight of glucose in a 30% (w/v) 148 aqueous solution. the injection site was sealed with vaseline and the animals returned to their mothers. at 30, 60 and 120 min after the injection animals from each litter were killed by decapitation and blood collected from the severed neck vessels. at the end of the experiment the mothers were weighed, killed by cervical dislocation and blood collected from the cut neck vessels. blood samples were allowed to clot for one hour at + 4 o before centrifugation and separation of serum. serum samples were stored at -20° until analyzed. serum glucose was determined with a glucose oxidase technique using a beckman glucose analyzer 2 (beckman, fullerton, usa) and insulin was assayed radioimmunologi cally (12). corticosterone was assayed with a commercially available radioimmunoassay (radioimmunoassay ltd, cardiff, uk). pancreatic glands of neonates, dissected out immediately after decapitation, were weighed and sonicated in 500 ~1 distilled water. after sonication 200 p l of each sample was added to 2 ml acid ethanol (70% ethanol with 1.5 ml 1 m hc1/100 ml). the samples were thouroughly mixed and incubated overnight at +4o, centrifuged and the supernatants stored at -20° until assayed for insulin. adrenal glands of neonates, dissected out immediately after decapitation , were weighed and sonicated in 500 pl distilled water. aliquots were removed for determination of their dna content (14, 21). the remaining samples were diluted with equal volumes of absolute ethanol and stored at -20° until assayed for corticosterone. results are given as means f s.e.m. and statistical analysis was performed using student's two-tailed t-test for independent observations. results metyrapone-treated pregnant rats had an apparently normal pregnancy and delivery occurred at the same gestational age as in the control rats. litter size was not affected, nor was there any difference in maternal body weight on day 7 postnatally (table 1). the basal serum glucose and insulin concentrations of metyrapone treated rat mothers were not different from those of control mothers. however, the serum corticosterone concentration was 149 table 1. effects of metyrapone treatment during pregnancy and lactation in the rat. controls metyrapone treated maternal body weight (9) serum glucose (mm) serum insulin (pg/l) serum corticosterone (nm) litter size (n) body weight (9) serum insulin (pg/l) adrenal wet weight (mg) adrenal wet weight/dna corticosterone/adrenal wet weight (pmol/mg) insulin/pancreatic wet weight (ng/mg) neonatal ( m g h 1 303 f 6 (8) 6.5 f 0.3 (8) 0.41 f 0.04 (8) 5.4 t 2.4 (8) 11.0 f 1.0 (8) 14.9 f 0.2 (88) 0.40 f 0.04 (27) 0.81 t 0.08 (25) 39 f 3 (25) 57 f 10 (25) 111 f 7 (27) 316 f 8 (11) 6.5 f 0.3 (8) 0.43 t 0.08 (7) 1.3 f 0.2 (8)* 10.2 f 0.2 (11) 14.4 f 0.2 (104)* 0.29 f 0.01 (24)*** 1.32 f 0.12 (24)*** 52 1: 10 (22)*** 37 f 6 (23) 68 f 11 (25)** table 1. pregnant and lactating rats were administered metyra pone, 50-70 mg/kg body weight, by gastric intubation from day 12 of gestation to day 7 postnatally. on day 7 postnatally both mother and pups were killed and the adrenal and pancreatic glands and serum were collected for assay of hormones. values are given as means f s.e.m. for the number of rats (n) indicated. sig nificance of difference between metyrapone-treated rats and their offspring and control animals: * p<0.05; * * p<o.ol; *** p<o.ool. lowered to 24% of the value of the control mothers. the litters of metyrapone-treated mothers suckled well and upon examination their gastrointestinal tracts were filled with milk, indicating that metyrapone-treatment of the mothers probably did not interfere with their ability to feed the offspring.neverthe less, at 7 days of age their neonates had a slightly reduced body weight (table 1). adrenal wet weight was increased in the neonates of metyrapone treated mothers (table 1). the ratio between adrenal wet weight and dna content was also increased, indicating a cellular hypertrophy. there was also a tendency towards a reduced adrenal concentration of corticosterone in the offspring of metyrapone treated mothers, but this did not reach statistical significance. when an i.p. glucose tolerance test was performed on the 7-day old neonates the glucose tolerance was similar in the offspring of metyrapone-treated mothers and controls (fig 1). the basal serum insulin concentration was lower in the offspring of metyrapone 150 1 i i i 1 0 30 6 0 9 0 1 2 0 time ( min ) 15 1 4controls 5 1 o f i i i i 0 30 6 0 9 0 1 2 0 time ( min ) fig 1. effect of metyrapone treatment of rats during pregnancy and lactation on the glucose tolerance and insulin secretory response to glucose in the offspring. rat mothers were treated with metyrapone (50-70 mg/kg body weight/day) from day 12 of gestation to day 7 postnatally when an intraperitoneal glucose tolerance test (2 g glucose/kg body weight) was performed on the offspring and serum concentrations of glucose (lower panel) and insulin (upper panel) measured. values are given as means +s.e.m. for 7-11 animals. significance of difference between the offspring of metyrapone-treated animals and the offspring of control animals: * p<o. 05. treated mothers, but the insulin secretory response to glucose during the test was similar in both groups (fig 1). the pancreatic insulin concentration in the neonates of metyrapone-treated mothers was only two thirds of that of the 151 control group (table 1). discussion treatment with metyrapone during pregnancy and lactation induces a state of hypoadrenalism in both mother and offspring (9, 10, 26). in the present study this was confirmed by the decreased serum concentration of corticosterone in the mothers. despite the hypoadrenalism the rat mothers thrived, did not lose weight and carried their offspring to term without losses. the neonates appeared healthy although signs of hypoadrenalism such as adrenal hyperplasia and a tendency towards decreased adrenal cortico sterone concentration could be demonstrated. serum concentrations of corticosterone in the offspring (data not shown) showed variations in both the experimental and control group. this was considered a stress response due to handling and changes in environmental temperature during the experiment and would not reflect a failure of metyrapone to inhibit fetal adrenal function. indeed, in previous studies of metyrapone-induced fetal hypo adrenalism a decrease of serum corticosterone concentration was not observed despite adrenal hypertrophy and diminished adrenal corticosterone content (26). the peak values of the glucose tolerance test in the neonates were higher than in adult rats of the same strain (6) and the insulin secretory response to the glucose peaks lower than the corresponding response in adults (33). these observations confirm the immaturity of the insulin secretory response to glucose in 7 day-old rats (11) which as a consequence dispose of glucose less efficiently than adults. the insulin secretory response to glucose in vivo is similar to the response in vitro in the offspring (11). the presently demonstrated lack of difference between the offspring of metyrapone-treated rats and the control animals would therefore suggest the adrenal corticosteroids are not essential for the perinatal development of a glucose-sensitive insulin secretion. chronic hypoadrenalism in the adult rat diminishes insulin secretion (25, 34) whereas administration of glucocorticoids to normal adult animals increases insulin output by stimulation of b cell growth (13, 18, 24) and insulin secretion (23, 24). the 152 present finding of lowered serum and pancreatic insulin con centrations could be interpreted to indicate a hypoplasia of the islet organ in the neonates with hypoadrenalism. the total capacity for insulin secretion would thus be diminished although the glucose sensitivity of the b-cell has been reported to remain intact (15). further experiments, including the morphometric analysis of the endocrine pancreas of the neonates, would be needed to clarify this hypothesis. acknowledgements the skilful technical assistance of gina eriksson and parri wentzel is gratefully acknowledged. this work was supported by the swedish diabetes association, the nordic insulin fund, the expressen prenatal research foundation, the ernfors family fund, the juvenile diabetes foundation (grant 185544) and the swedish medical research council (grants 12x-109, 12x-7475, and 12p 6947). references 1. 2 . 3. 4. 5. 6 . 7. 8. asplund, k. : the effect of glucose on the insulin secretion in foetal and newborn rats. in: the structure and metabolism of the pancreatic islets (ed. s. falkmer, b hellman & i.-b. taljedal), pp 477-484. pergamon press, oxford, 1970. asplund, k.: effects of intermittent glucose infusions in pregnant rats on the functional development of the foetal pancreatic b-cells. j endocrinol 59:285-293, 1973. asplund, k., westman, s. & hellerstrom, c.: glucose stimula tion of insulin secretion from the isolated pancreas of foetal and newborn rats. diabetologia 5:260-262, 1969. blazquez, e., simon, f.a., blazquez, m. & foal p.p.: changes in serum growth hormone levels from fetal to adult age in the rat. proc soc exptl biol med 147:780-783, 1974. domingues, o.v. & samuels, l.t.: mechanism of inhibition of adrenal 11-r-hydroxylase by metyrapone (metopiron). en docrinology 73:304-309, 1963. eriksson, u., andersson, a . , efendic, s., elde, r. b hel lerstrom, c.: diabetes in pregnancy: effects on the foetal and newborn rat with particular regard to body weight,serum insulin concentration and pancreatic contents of insulin, glucagon and somatostatin. acta endocrinol 94:354-364, 1980. fisher, d.a., dussault, j.h., sack, j. & chopra, i.j.: onto genesis of hypothalamic-pituitary-thyroid function and metabolism in man, sheep and rat. rec progr horm res 33:59 116, 1977. freinkel, n., lewis, n.j., johnson, r., swenne, i., bone, a.j. & hellerstrom, c.: differential effects of age versus glycemic stimulation on the maturation of insulin stimulus secretion coupling during culture of fetal rat islets. 153 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21 * 22. 23. 24. 25. 26. diabetes 33:1028-38, 1984. goldman, a.s.: experimental model of congenital adrenal cortical hyperplasia induced in utero with an inhibitor of' 11-r-steroid hydroxylase. j clin endocrinol 27:1390-1394, 1967. goldman, a.s. & yakovac, w.c.: experimental congenital adrenal cortical hyperplasia: prevention by of adrenal hyperplasia and clitoral hypertrophy by corticosterone. proc soc exptl biol med 122:1214-1216, 1966. grill, v. & freinkel, n.: generalized dimunition of the response to nutrients as insulin-releasing agents during the early neonatal period in the rat. diabetes 30:56-63, 1981. heding, l.g. determination of total serum insulin (iri) in insulin-treated patients. diabetologia 8:260-266, 1972. hellerstrom, c.: effects of steroid diabetes on the pancrea tic islets of guinea pigs with special reference to the a1 cells. acta soc med upsaliensis 68:l-16, 1963. hinegardner, r.t.: an improved fluorometric assay for dna. anal biochem 39:197-201, 1972. hoet, j.j., grasso, s. & van assche, f.a.: endocrine factors in islet cell maturation. in: early diabetes in early life (ed. r.a. camerini-davalos & h.s. cole), pp 93-101. academic press, new york, 1975. holt, p.g. & oliver, i.t.: plasma corticosterone concentra tion in the perinatal rat. biochem j 108:339-341, 1968. jack, p.m.b. & milner, r.d.g.: effect of decapitation on the devlopment of insulin secretion in the foetal rabbit. j endocrinol 57:23-31, 1973. kern, h. & logothetopoulus, j.: steroid diabetes in the guinea pig. studies on islet-cell ultrastructure and regeneration. diabetes 19:145-154, 1970. kervran, a. & randon, j.: development of insulin release by fetal rat pancreas in vitro: effects of glucose, amino acids, and theophylline. diabetes 29:673-678, 1980. kervran, a. , randon, j. & girard, j.r. : dynamics of glucose induced plasma insulin increase in the rat fetus at different stages of gestation. effects of maternal hypothermia and fetal decapitation. biol neonate 35:242-248, 1979. kissane, j.m. & robins, e.: the fluorometric measurement of deoxyribonucleic acid in animal tissues with special reference to the central nervous system. j biol chem 233:184 188, 1958. klepec, r., milkovic, k., paunovic, j. & milkovic, s.: steroidogenesis of fetal adrenal gland in vitro: influence of metopirone applied in vivo and in vitro. proc soc exptl biol med 150:249-253, 1975. lenzen, s.: the effect of hydrocortisone treatment and adrenalectomy on insulin and glucagon secretion from the perfused rat pancreas. endokrinologie 68:189-197, 1976. like, a.a. & chick, w.l.: pancreatic beta cell replication induced by glucocorticoids in subhuman primates. am j pathol malaisse, w.j., malaisse-lagae, f., mccraw, e.f. & wright, p.h.: insulin secretion in vitro by pancreatic tissue from normal, adrenalectomized and cortisol-treated rats. proc soc exptl biol med 124:924-928, 1967. milkovic, k., perzovic, m. & pavnovic, j.: pituitary and adrenal glands in neonatal rats studied by metopirone (su 75~329-348, 1974. 154 27. 28. 29. 30. 31. 32. 33. 34. 35. 4885). biol neonate 41:32-37, 1982. pictet, r., rutter, w.j. development of the fetal endocrine pancreas. in: handbook of physiology, section 7, volume 1 (ed. d.f. steiner & n. freinkel), pp 25-66. american physiological society, baltimore,l972. rhoten, w.b.: insulin secretory dynamics during development of the rat pancreas. am j physiol 239:e57-e63, 1980. rieutort, m.: pituitary content and plasma levels of growth hormone in foetal and weanling rats. j endocrinol 60:261 268, 1974. strosser, m.t. & miahle, p.: growth hormone secretion in the rat as a function of age. horm metab rss 7:275-278, 1974. swenne, i.: hypothyroidism in the fetal and neonatal rat does not impair the insulin secretory response to glucose. life sci 33:2207-2211, 1983. swenne, i.: glucose-stimulated dna replication of the pancreatic islets during development of the rat fetus. effect of nutrients, growth hormone, and triiodothyronine. diabetes 34:803-807, 1985. swenne, i., crace, c.j. ti milner, r.d.g.: persistent impair ment of the insulin secretory response to glucose in adult rats after a limited period of protien-calorie malnutrition early in life. diabetes 36:454-458, 1987. vance, j.e., kilabchi, a.e., buchanan, k.d. & williams, r.h.: effect of adrenalectomy on glucagon and insulin relase from isolated islets of langerhans. clin res 16:131(abst ract), 1968. yoshinari, m. & daikuko, s.: ontogenic appearance of immuno reactive endocrine cells in rat pancreatic islets. anat embryo1 165:63-70, 1982. adress for correspondance: dr ingemar swenne dept of paediatrics akademiska sjukhuset s-751 85 uppsala sweden 1 5 5 upsala j med sci 92: 277-286, 1987 object size determination at computed tomography anders magnusson from the department of diagnostic radiology, uppsala university, uppsala, sweden a b s tract to investigate whether large differences in attenuation between an object and its background at ct examination alter the apparent size of the object on the image compared with the conditions with a small attenuation difference, phantom experiments were performed. a t the same time the influence of large attenuation differences on the geometric resolution was examined. the results show that neither the size reproduction of the object nor the geometric resolu tion is altered by variations in attenuation differences. especially with large differences in attenuation between the object and background, however, the window centre setting was of greatest importance for correct reproduction of the object. introduction in computed tomography (ct) s h a r p borderlines between s t r u c t u r e s of different density a r e reproduced a s blurred contours. the degree of blurring depends, among other things, on the x-ray beam width, the dimensions and properties of the detectors, and the reconstruction algorithms employed. this blurring is of importance when measuring the size of s t r u c t u r e s in the image (1,3). when an object which differs markedly in attenuation from its background is examined by ct, its apparent size varies with the viewing conditions. a decisive factor in this context is the question of which p a r t of t h e ct-cal culated attenuation profile is utilized to produce the image. this is determined by the setting of the window centre (wc) and window width ( w w ) (fig. 1). when patients have undergone ct examination both before and after bipe dal lymphography, the lymph nodes have appeared larger after the contrast filling, when the image has been viewed with the window setting normally used for evaluation of the retroperitoneal space. this enlargement has considerably exceeded that caused by the contrast filling in itself ( 2 , 4 ) . it is not clear whether the apparent enlargement is solely an effect of the w w and wc settings or whether it is due to an e r r o r of measurement or of 277 image fig.1. the i n f l u e n c e o f d i f f e r e n t window c e n t r e (wc) s e t t i n g s on t h e ct r e p r o d u c t i o n o f an o b j e c t when t h e window w i d t h ( w w ) i s c o n s i d e r a b l y s m a l l e r t h a n t h e a t t e n u a t i o n d i f f e r e n c e i n t h e image. a ) wc a t t h e l e v e l o f t h e background a t t e n u a t i o n and b ) wc a t t h e l e v e l o f t h e a t t e n u a t i o n o f t h e o b j e c t . note t h a t i n t h i s image t h e grey s c a l e i s reversed, so t h a t h i g h a t t e n u a t i o n values are represented by b l a c k and low ones by w h i t e . i w c w w i image reconstruction. an investigation was therefore undertaken to elucidate the way in which the interpretation of the ct image is influenced by the difference in attenuation between an object and its background, different reconstruction algorithms and different window settings. in addition, the geometric resolution with various attenuation differences and window settings was examined. material a n d methods a siemens somatom d r 2 whole-body scanner with a 2 5 6 x 2 5 6 matrix was used for the investigation. the exposure data were 1 2 0 kv, 0 . 5 1 8 as, exposure time 4.5 s , 480 projections and a slice thickness of 8 m m . reconstructions were performed with a zoom factor of 1 . 7 , corresponding t o a normal abdominal examination, giving a pixel s i z e of 1 . 2 7 mm, and a zoom factor of 6 , with a pixel size of 0.36 m m . for the reconstructions both the standard algorithm (kernel 11, which is normally used for abdominal examinations, and an algorithm 278 with strong edge enhancement (kernel 7) were employed. the images were analysed on the standard diagnostic console with different window settings (scale: air -1000 hounsfield units (hu), water 0 h u ) . the term image is defined in this article as the image reproduced on the monitor screen. areas of interest were magnified; measurements were performed directly on the image screen and on the attenuation profiles, with the cursor incorporated in the console. for investigation of the influence of the attenuation difference on the apparent size of the object, a polystyrene phantom with a diameter of 20 cm was used. in i t s centre there was a cylindrical hole 2 3 m m in diameter. the atte nuation of the phantom was -10 h u . the hole was filled with gastrografina ( 3 7 0 mg i/ml) in dilution of 0 . 7 5 and 2 5 % , which gave the hole attenuation values of 75 and 2050 h u respectively. the phantom was placed in the centre of the gantry and the cylindrical hole passed perpendicularly through the entire ct slice. the width of the attenuation profile was measured at l o % , 50% and 90% of the profile height. to examine the effect of different attenuation differences and window settings on the geometric resolution, a phantom of parallel plexiglass rods with a square 5 x 5 mm cross-section was used. the distance between the rods was 1 m m . the plexiglass rods were examined both in air and when surrounded by water. the attenuation value for plexiglass was 120 h u , which means that the difference in attenuation between plexiglass and air was 1120 h u and between plexiglass and water 120 h u . the ct slice was perpendicular to the longitudinal axis of the rods. results the attenuation profiles for the cylindrical hole when it was filled with gastrografin of different concentrations, are given in fig. 2 . when the stan dard algorithm was used for reconstruction, the width of the profile was inde pendent of the difference in attenuation between the object and background (table 1 ) . neither did the zoom factor used in the reconstruction influence the apparent dimensions. reconstruction with the edge-enhancing algorithm result ed in a decrease in profile width by a maximum of 1 mm on examination of the high-density object. the impact of different wc settings on the apparent size of the object in the image when the object-background difference in attenuation was large (2060 h u ) , is illustrated in figs 3 and 4 . when measured on an image of the object, reconstructed with a standard algorithm and a zoom factor of 6 at different wc settings (from 0 to 2300 h u ) and at a constant w w ( 4 0 0 hu), the diameter varied from 26 to 2 0 mm. the t r u e diameter ( 2 3 m m ) was reproduced at a wc setting of between 1000 and 1200 h u , thus at about half the attenuation differ ence between the object and background. 279 100 76 52 28 hu 2400r 100 76 52 28 hu c d fig.2. ( c and d), r e c o n s t r u c t e d w i t h s t a n d a r d ( a and c ) and edge-enhancing a l g o r i t h m s ( b and d ) . a t t e n u a t i o n p r o f i l e s f o r t h e 23 mrn o b j e c t w i t h a t t e n u a t i o n s o f 75 ( a and b ) and 2050 hu table 1 the w i d t h o f t h e a t t e n u a t i o n p r o f i l e i n mm a t 10% ( d ), 50% ( d ) and 90% ( d ) o f i t s h e i g h t . 1 2 3 attenuation zoom 1 . 7 zoom 6 difference standard standard edge en objectalgorithm algorithm hancement background dl d 2 d 3 dl d 2 d 3 dl d2 d3 8 5 h u 25 2 3 21 2 5 2 3 21 2 5 2 3 2 1 2060 h u 25 2 3 21 2 5 2 3 2 1 2 4 2 3 21 280 fig.3. s e t t i n g s and a c o n s t a n t window w i d t h (400 hu). the ct image o f an o b j e c t o f h i g h a t t e n u a t i o n (2050 hu) a t d i f f e r e n t window c e n t r e (wc) wc = 0 ( a ) , 1200 ( b ) and 2000 hu ( c ) . 26 24 22 20 > i i i i fig.4. e f f e c t o f window c e n t r e on t h e o b j e c t diameter. the window w i d t h i s s e t a t 400 hu. the t r u e diameter (23 mm) i s reproduced a t a window c e n t r e o f between 1000 and 1200 hu (shaded column). fig.5. the 23 nun o b j e c t o f h i g h a t t e n u a t i o n (2050 hu) reproduced w i t h a window c e n t r e o f 1000 hu and a window w i d t h o f 4000 hu. a p p a r e n t diameter (mm) \. 24".-. . .......... '. \ w w 400 fig.6. the i n f l u e n c e o f t h e window c e n t r e and window w i d t h ( w w ) s e t t i n g s on 2 0 t h e reproduced diameter o f an o b j e c t w i t h an a t t e n u a t i o n o f 75 hu. 2 2 '. "-.. w w 200 w w 30 w w 73 z 0 100 200 0 1 1 i i window c e n t r e (hu) 18-878573 28 1 hu 130r a 8 20l 0 hu 180 [ c -201 0 8 16 m" d hu 180r fig.7. a t t e n u a t i o n p r o f i l e s f o r two p l e x i g l a s s rods s i t u a t e d 1 mm from each o t h e r , examined i n water ( a and b ) and a i r ( c and d ) , w i t h t h e use o f standard ( a and c ) and edge-enhancing ( b and d ) a l g o r i t h m s f o r r e c o n s t r u c t i o n . when the high-density object was viewed with a large ww, so that the entire attenuation difference between the object and background was covered by w w , the border of the object against the background became diffuse and it was difficult to define any exact measurement points for the diameter in the image (fig. 5 ) . the wc setting also influenced the reproduction of an object which differ ed relatively little in attenuation from its background (by 85 h u ) , but only when w w was narrow (fig. 6). when w w was smaller than the object-back ground attenuation difference, the apparent size of the object varied with different settings of wc, and this size variation increased with decreasing w w . when, on the other hand, ww was larger than the attenuation difference, the size was not affected by changes in wc as long as the entire attenuation profile lay within the w w limits. on examination of the plexiglass phantom with parallel rods with a square cross-section, in air and in water, parts of the attenuation profiles of the two objects coincided under all tested conditions (fig. 7 ) . the lowest attenuation value for the "space" between the rods, when the images were reconstructed 282 f i g . 8 . the image o f t h e two c l o s e l y a d j a c e n t p l e x i g l a s s rods surrounded by a i r , a t a constant window width (400 hu) and w i t h d i f f e r e n t window c e n t r e s : -1000 ( a ) , -850 ( b ) , -700 ( c ) , -550 ( d ) , -400 ( e ) and -250 hu ( f ) . with a standard algorithm, was found to be higher than t h e attenuation value of the background by 30% of the object-background attenuation difference (fig. 7 a and c ) . this finding was independent of the magnitude of the attenuation difference and of the zoom factors employed. on examination both in air and in water, too low values ( 2 0 and 110 h u ) were obtained for the maxima of the profiles, t h u s 1 0 0 and 1 0 h u , respectively, below the t r u e attenuation value for plexiglass ( 1 2 0 h u ) . on reconstruction with an edge-enhancing algorithm, the attenuation values for the "space" were lower than when a standard algorithm was used; t h u s it was now higher than the background value by only 15% of the object-background attenuation difference, both when the examination was performed in air and in water (fig. 7 b and d ) . the position of wc was of importance for the geometric resolution, espe cially on reconstruction with a standard algorithm. when the plexiglass r o d s , surrounded b y a i r , were viewed with a wc of -1000 hu and a w w of 400 h u , they appeared to form one figure (fig. 8 a ) ; when wc was increased stepwise while w w was kept constant, the rods w e r e reproduced a s one unit (fig. 8 b) until the upper limit of w w exceeded the lowest attenuation value for the "spa 283 ce" between the rods. thereafter the space began to appear in grey tones. the space increased in width with increasing wc up to about -500 hu, and parflllel with the increase in width of the space the size of the rods decreased (fig. 8 c and d ) . when wc was so high that the entire w w lay above the lowest attenua tion value for the space, this appeared black (fig. 8 e ) . the same effect was observed when the image of the plexiglass rods surrounded b y water was viewed with varying wc. as long as wc lay within the values for the attenua tion profile, 0-110 h u (fig. 7 a ) , however, w w had to be relatively small no greater than 60 h u at a wc of 0 hu, for the two rods to converge. however, with the window settings that are normally used at abdominal examinations, 400 h u for w w and 100 h u for wc, the rods appeared clearly separated. discussion variation of the attenuation difference between the object and background did not alter the shape of the attenuation profile. similarly, the width of the profile was influenced to only a minor extent b y the use of different algorithms in the reconstruction. the setting of wc, on the other hand, was of great importance for the reproduced size of the object. thus the apparent diameter of the object of high attenuation ( 2 0 5 0 h u ) varied by 6 m m when the image was viewed with different wc settings. baxter & sorensen (1) found that when wc was set at half the attenuation difference between the object and background, the size of the object was reproduced correctly, a finding in accordance with the present results (fig. 4 ) . when wc is set at the level of the background attenuation on examination of an object of high attenuation, the apparent size of the object is greater than its real size, and its greatest part appears completely white, as this part of the attenuation profile lies above the upper l i m i t of w w (fig. 1 a ) . this occurs in clinical examinations when contrast-filled retro peritoneal lymph nodes a r e being evaluated with settings of wc (100 h u ) and w w ( 4 0 0 h u ) that are normal for abdominal examinations, on account of the fact that the attenuation value of the contrast-filled lymph nodes (1000-2000 h u ) is considerably higher than the upper limit of w w . if, on the other hand, wc is chosen according to the attenuation of the contrast-filled lymph nodes, with w w unchanged, only the maximum of the attenuation profile will give an image, which will mean that the apparent size will be smaller than the real one, at the same time as the image will appear i n grey tones (fig. 1 b ) . for t h e object of low attenuation, the w w setting also influenced the appa rent s i z e in those cases where w w was smaller than the object-background attenuation difference (fig. 6 ) . with decreasing w w the object became more and more distinctly outlined in the image, but in parallel with this the size repro duction became more sensitive to changes in wc. when closely adjacent objects of high attenuation were examined and wc was 284 f i o . 9 . ct image of c o n t r a s t f i l l e d lymph nodes. a ) window s e t t i n g normal f o r abdominal examinations. window c e n t r e 70 hu, window w i d t h 400 hu. b ) window c e n t r e 1000 hu, window w i d t h unchanged. set at a level with the attenuation of the background, at the same time as the selected w w (400 h u ) was considerably smaller than the object-background attenuation difference (1110 hu), the geometric resolution was poor. on ct examination of closely adjacent contrast-filled lymph nodes, these nodes, with wc and w w settings normal for abdominal examinations ( 1 0 0 and 400 hu respec tively), may converge and be interpreted as one large single node (fig. 9 ) . on reconstruction with the edge-enhancing algorithm, t h e geometric resolution was improved at low wc values. this means that in clinical examinations of s t r u c t u r e s differing largely in attenuation, an edge-enhancing algorithm should be used. when wc was set at a level corresponding to half the attenuation difference, good resolution was obtained. the maximal attenuation values for the plexiglass r o d s , when examined in air and water and with use of a standard algorithm for the reconstruction, lay 100 and 10 hu, respectively, below the real attenuation value for plexiglass. this is explained by the fact that the spatial resolution for the algorithm used is insufficient for a correct repro duction of the attenuation values of such small objects. conclusions the difference i n attenuation between an object and i t s background has v e r y little influence on the reproduced size of the object and on the geo metric resolution with the ct scanner used for this investigation. for correct reproduction of the size of an object and for good geometric resolution in the monitor image, t h e wc setting is of the greatest import ance. a correct reproduction of the object of interest was achieved when wc was set at a value in the middle of the attenuation values of the object cand t h e background. 285 3. with a w w that is smaller than the object-background attenuation differ ence, good delineating of the reproduced object is achieved. the edge will become sharper the smaller the w w setting used. parallel with a decreasing ww, however, t h e sensitivity t o the setting of wc increases. 4. in the present experiments ideal conditions prevailed, with objects of similar thickness which passes perpendicularly through the ct slice. in practice, however, consideration must also be paid to partial volume effects when measuring the sizes of structures. references 1. baxter, b.s. and sorensen, j . a . : factors affecting the measurement of size and ct number in computed tomography. invest. radiol. 16:337-341, 1981. 2 . koehler, p.r., potchen, e . j . , cole, w.r. & studer, r . : experimental studies of intralymphatic administration of radiotherapy. radiology 90 : 3. koehler, p.r., anderson, r . e . 8 baxter, b . : the effect of computed tomography viewer controls on anatomical measurements. radiology 130: 189 4. steckel, r . j . & cameron, t.p.: changes in lymph node size induced by 495-501, 1968. -194, 1979. lymphangiography. radiology 87: 753-755, 1966. address for reprints d r anders magnusson department of diagnostic radiology akademiska sjukhuset s-751 85 uppsala, sweden 286 upsala j med sci 95: 309, 1990 acknowledgement the project group for the nordkem-project 5/89 wants to express its sincere gratitude to the board of nordkem, not only for granting the necessary economical resources for the project but also for taking active part in planning the pro ject and thus stressing the formulation of the objectives of the project. the project group wants to express its thanks to mrs gunilla stromstedt, department of clinical chemistry, university of uppsala for secreterial help and for practical organization of the seminar at friibergh's herrgdrd, bro on april 23 2 4 1990. it is a pleasure also to acknowledge mrs anette marcusson, department of clinical chemistry, university hospital, upp sala, who with efficiency and accuracy prepared the manu scripts for printing and mrs sinikka savolainen who delt with the project at the nordkem office in helsinki. 309 upsala j med sci 90: 101-106, 1985 peroperative staging of renal carcinoma a methodologic comparison d;ke fritjofsson, anders hemmingsson, per gunnar lindgren and stig reinholdsson departments of urology and diagnostic radiology, uniwrsity hospital, uppsala, sweden abs t rac 1 angiography, computed tomography and ultrasonography were compared with re spect to staging of renal carcinoma in 41 patients with 46 renal tumours. angi ography and ultrasonography gave correct staging in 52 l and 48 %, respective ly, while correct staging was achieved with computed tomography in 80 5 of the tumours. introduction staging is one of the fundamental guidelines for t h e management and prog nostic evaluation of neoplasms, including renal cancer. critical factors for accurate clinical staging of renal carcinoma are extension through the renal capsule with invasion of perinephric fat, invasion of gerota’s fascia or ad jacent organs, thrombotic tumour extension into the renal vein o r inferior vena cava, involvement of regional lymph nodes and general dissemination. urography and angiography currently are basic procedures in the diagnosis of renal carcinoma. assessment of perirenal tumour extension and of local metastas ization with these methods often is uncertain, however ( 6 ) . on the other hand, selective injection of a large dose of contrast medium into the renal artery ( z ) , possibly supplemented with cavography ( 9 ) gives high diagnostic reliabil ity with respect to extension of tumour to the renal vein o r inferior vena cava. in recent years the noninvasive techniques of computed tomography ( 1 , 6 , 7 , 1 2 , 15) and ultrasonography (3,4) have also been used in the diagnosis of renal tumours. the present report concerns the possibilities for correct preoperative (clinical) staging of renal carcinoma by means of renal angiography (ra), com puted tomography (ct) and ultrasonography ( u s ) . in a series of patients, the findings at these examinations were compared with observations made by the surgeon and/or pathologist. 101 material and methods the series comprised 41 patients with renal carcinoma investigated in the period december 1977 june 1902. there were 20 men and 13 women, with mean age 6 0 . 2 (range 34-73) years. five of the patients had bilateral carcinoma. the total of investigated kidneys thus was 4 6 , involving the right kidney in 28 cases and the left in 18. in 38 patients extrafascial nephrectomy o r , when there was bilateral disease, kidney resection was performed. the other three patients died soon after diagnosis and were studied post mortem. f o r staging of the tumours we used a modification of the classification pro posed by robson et al. (ll), as shown in table 1. the study was restricted to clarification of the intraabdominal extent o f t u m o u r , and disseminated or dist ant metastases were disregarded in the present connection. table 1. staging classification of renal adenocarcin oma according to robson et al. (11) stage tumour i within kidney capsule i1 invading perinephric fat (confined to 111 a involving renal vein o r inferior vena i11 b involving regional lymph nodes involving renal vein o r vena cava and i11 a + b regional lymph nodes iv invading adjacent organ(s) gerota’s capsule) cava r a and ct were performed in all cases, and 35 tumours were also studied with us. ra was performed with conventional technique, injecting contrast medium into the aorta and selectively into the renal arteries. evaluation of the renal vein was facilitated by injection of 30-50 m l angiografin (306 mg i/m1) selectively into the renal artery. on any suspicion of tumour thrombus in the renal vein, cavography was performed with standard technique. for ct a delta 50 fs scanner (ohio nuclear) was used, with exposure time 18 s, slice thickness 13 mm and beam width 5 mm. ct scanning was performed over r the kidneys before and after intravenous injection of contrast medium (conray meglumine, 282 mg i/ml). us over the kidneys was performed in the first 2 years with a sono diagriost 102 b 50 (philips) grey-scale apparatus, and after 1979 we used an atl mark i11 dynamic sector-scan apparatus with a 3.0 mhz transducer. i 22 19 i1 5 4 results ~~ ~~ ~ 1 1 1 1 in table 2 the pathologic and angiographic stagings are compared. stage i tumours were correctly evaluated with ra in most patients, while more advanced tumours often were understaged. ra correctly staged the tumour in altogether 52 7; of the cases, with understaging in 41 e and overstaging in 7 e table 2. pathology staging angiographic staging of renal tumours compared with operation/autopsy renal angiography group n i i1 iiia iiib iiia+b iva iva 10 1 1 4 total 46 30 4 7 5 understaging 19/46 (41 76) overstaging 3/46 ( 7 7;) correct staging 24/46 (52 % ) with us the tumour staging was correct in only 48 e of cases (table 3 ) , mainly in stage i. there was 9 7; overstaging and 43 76 understaging. difficult ies in demonstrating local tumour involvement were encountered at us mainly when there was extension of tumour in dorsal direction. ct (table 4) gave correct staging in 80 l of the tumours, but understaging in 11 % and overstaging in 9 l. ct thus was superior to the other procedures in providing information not only of the extent of tumour within the kidney, but also concerning its relations to the perinephric fat and adjacent orqans. more over, ct was better than the other methods in detection o f nodal metastases. in the course o f this study we received a strong impression that ct in many cases can obviate the need for angiography in the preoperative diagnosis and staging of solid renal tumours. 103 table 3. pathology staging ultrasonographic staging o f renal tumours compared with i 17 2 l14 i1 4 1 1 1 2 iva 6 2 2 1 iiia 2 1 1 iiib 4 3 total 35 3 21 6 2 3 understaging 15/35 (43 % ) overstaging 5/35 ( 9 7 6 ) correct staging 17/35 (48 5) 1 table 4. pathology staging computed tomography staging o f renal tumours compared with i 2 2 1 18 i 1 5 1 2 1 3 1 iiia 3 2 1 iiib 4 3 1 3 iiia+b 2 2 iva 10 10 discussion the results of this study showed ct to be the most reliable method for stag ing of renal carcinoma with respect both to perirenal extension of the tumour and to presence of local metastases. understaging was recorded in 41 % of the tumours at ra and in 43 5 at us. the inadequacy of angiography and ultrasono graphy for staging of these renal tumours accords with previous experience (5, 6,lo). f o r evaluation of tumour growth to the liver, real-time us is valuable, and movements of surrounding tissues in relation to the tumour can be assessed, especially on deep expiration and inspiration. ct overstaged the tumour in four cases (9 :a). the reason was that these tu mours lay against adjacent structures and could not be clearly separated from them at ct. enhanced diagnostic accuracy with ct in such cases may be expected from the more modern equipment that permits rapid serial imaging after a bolus injection of contrast medium, thinner slices and higher spatial resolution. turnour thrombi in the renal vein and vena cava can be detected both with ct (3,4,7,14) and with us (3,4). in the present series venous thrombi were found in only five cases, but subsequent experience has confirmed these diagnostic possibilities. venography, however, is often valuable if tumour thrombus in the caval vein is visualized by us o r ct, to define the cranial limit of the thromb us preoperatively (8,13). computed tomography thus permits more accurate staging of renal carcinoma than do renal angiography and ultrasonography as regards both perirenal ex tension o f turnour growth and presence of local metastases. with computed tomo graphy, however, there is a tendency to overstaging. ultrasonography can be useful especially for judging the relation o f the tumour t o the liver when the tumour is located in the upper part of the right kidney. acknowledgement this investigation was supported by grants from t h e swedish cancer society. 1. 2 . 3. references baert, a.l., marchal, g . , staelens, b. & coenen, y.: ct. evaluation of renal space-occupying lesions. fortschr rontgenstr 126:285,1977. bjijrk, f . , erikson, u., falk, j., lindblad, g. & stenport, g.: clinical and histological studies on the effect o f large doses of roentgen contrast media in renal arteriography. ups j med sci 80:46,1975. goldstein, h.w., green, b. & weaver, r.m.jr: ultrasonic detection o f renal tumor extension into the inferior vena cava. am j roentgenol 130:1083,1978. 105 4. 5. 6. 7. 8. 9. 10. 11. 1 2 . 13. 1 4 . 15. green, d. & s t e i n b a c h , h.l.: u l t r a s o n i c d i a g n o s i s o f hypernephroma e x t e n d i n g i n t o t h e i n f e r i o r vena cava. r a d i o l o g y 115:679,1975. l a c k n e r , k . , k o i s c h w i t z , d., m o l i t o r , b., vogel, j. & schmidt, s . : t r e f f s i c h e r h e i t i n d e r d i a g n o s t i k r e n a l e r raumforderungen. computer-tomographie, sonographie, u r o g r a p h i e , a n g i o g r a p h i e . f o r t s c h r r o n t g e n s t r 140:363,1984. love, l . , c h u r c h i l l , r., reynes, c., s c h u s t e r , g.a., moncada, r. & berkow, a . : computed tomography s t a g i n g o f r e n a l carcinoma. urol r a d i o 1 1:3,1979. marks, w.m., k o r o b k i n , m.,callen, p.w. & k a i s e r , j.a.: c t d i a g n o s i s o f tumor t h r o m b o s i s o f t h e r e n a l v e i n and i n f e r i o r vena cava. am j roentgenol 131:843,1978. mccullough, d.l. & g i t t e s , r.f.: vena cava r e s e c t i o n o f r e n a l c e l l c a r c i n oma. j u r o l 112:162,1974. mcmoy, r.m., k l a t t e , e.c. & rhamy, r.k.: use o f i n f e r i o r venacavography i n t h e e v a l u a t i o n o f r e n a l neoplasm. j u r o l 102:566,1969. p i l l a r i , g . , lee, w.j., kumari, s . , chen, m., abrams, h.j., b u c h b i n d e r , m . & s u t t o n , a.p.: c t and a n g i o g r a p h i c c o r r e l a t e s : s u r g i c a l image o f r e n a l mass l e s i o n s . u r o l o g y 17:296,1981. robson, c.j., c h u r c h i l l , b.m. & anderson, w . : the r e s u l t s o f r a d i c a l nephrectomy f o r r e n a l c e l l carcinoma. j urol 101:297,1969. sagel, s . s . , s t a n l e y , r.j., l e v i t t , r . g . & geisse, g.: computed tomography o f t h e k i d n e y . r a d i o l o g y 124:359,1977. s k i n n e r , d.g., p f i s t e r , r.f. & c o l v i n , r . : e x t e n s i o n o f r e n a l c e l l c a r c i n oma i n t o t h e vena cava: t h e r a t i o n a l e f o r a g g r e s s i v e s u r g i c a l management. j urol 107:711,1972. s t e e l e , j.r., sones, p.j. & h e f f n e r , l.t. jr: the d e t e c t i o n o f i n f e r i o r vena cava t h r o m b o s i s w i t h computed tomography. r a d i o l o g y 128:385,1978. s t r u y v e n , j . , b r i o n , j . p . , f r e d e r i c , n. & schulman, c.c.: computed tomo graphy o f t h e k i d n e y . b r j u r o l 49:583,1977. address f o r r e p r i n t s ake f r i t j o f s s o n department o f u r o l o g y u n i v e r s i t y h o s p i t a l 5-751 85 uppsala sweden 106 upsala j med sci 95: 261-264, 1990 quality specifications p. hyltoft petersen' and carl-henric de verdier' deparlments of clinical chemistry at hospitals in 'odense och 'uppsulu use of analytical components in clinical strategies are decided without paying much attention to the quality of analytical performance, e.g. from danish recommendations: 'in non-insuline dependent diabetics the clinical goal is to keep hba,, < 7.5 percent (1)' and 'guidelines for treatment of patients with pcholesterol (total) between 7 and 9 mmol/l ( 2 ) i . influence from both biological and analytical variations seems to be forgotten or ignored, although the significances of these factors have been documented ( 3 , 4 ) . a l s o sampling error and other preanalytical factors should be considered. these are just examples, but they confirm the need for continuation of two previous nordkem projects on quality requirements (5) and quality control (6). clinical strategy sampling and other preanalytical errors -1biological variation analytical quality specifications the needs for analytical quality are different for various clinical situations as demonstrated for tshmeasurements, where the quality needed for basal s-tsh determinations used 'to predict s-tsh response to tfu-ii is extremely demanding (7), whereas the requirements for b-tsh measurements in screening for congenital hypothyroidism are very loose ( 8 ) . in these examples 261 the analytical procedure used for obtaining the quality required for 'predicting the s-tsh response' is very costly and should ' not be used for the screening of all newborns, where a simpler method is sufficient. the analytical quality needed should be specified for each clinical strategy and the most demanding specifications should be aimed at when new methods and equipments are introduced also when control systems is designed (6). in practice costs, turn around-time, and other factors may be forgotten, interfere in obtaining the needed quality must not and should be the goal for later improvements. analytical quality specifications cost+ +others 1 laboratory quality specification from the clinicians point of view the sources of variations and errors may seem less interesting, but for clinical chemists the splitting up of analytical variation and errors into random and systematic deviation is important. this will give the key for improvements of the quality, and by deviding the systematic deviation into bias (the common deviation from the 'conventional true value' during stable performance) and systematic error (the common deviation from accepted bias in the laboratory) the possibilities for improving quality are reformed. analytical imprecision is mainly determined by the analytical principle (e.g. ria), pipetting and the equipment (e.g. photometer). today many instruments meet most requirements for imprecision. this fact, however, does not help the problem with systematic error and bias. systematic error may be intermittent (e.g. occational wrong performance) or persistent (e.g. batch to-batch variations in kits). the bias is often caused by poor standardization or unspecific methods. the analytical quality specifications are different for 262 monitoring, imprecision and systematic errors are the main factors to be considered, whereas bias (and persistant systematic error) is determining in screening and diagnostics recommendations are specified for monitoring in relation to a fixed concentration. unspecific reactions and matrix effects are often ignored in the specifications of required quality. these factors will also be overlooked in the majorities of c o n t r o l programmes, butthey are important and may result in considerable errors, especially in specimens with extreme compositions. and when general analytical bias (inaccuracy) calibration matrix effects unspecific performance reactions (principle. equipment) procedure physlcal (model) unknown aquired components the unspecific reactions are different from component to component so they should be specified for each analytical component, e.g. for s-triiodothyronine it should be specified that a s-thyroxine concentration of 200 nmol/l was not allowed to increase the striiodothyronine result by more than 0.1 nmol/l. contain a list og maximum allowable the analytical quality specifications should therefore, 1) imprecision 2) systematic error 3 ) bias (mainly standardization) 4 ) matrix effects (specified) 5) unspecific reactions (specified). 263 models for evaluation of imprecision, systematic error, and bias are available (3,4,5), but they should be reinvestigated in order to clearify whether they are still valid or they should be improved. moreover, models for matrix effects and for unspecific reactions should be developed in this project on 'medical need for quality specifications in laboratory medicine'. reference: 1. 2. 3. 4 . 5. 6. 7. 8. arbejdsgruppe nedsat af dansk selskab for intern medicin: ikke-insulinkrzvende diabetes mellitus, diagnostic og behandling, 1988. arbejdsgruppe nedsat af dansk selskab for intern medicin: hyperlipidzmi, 1985. lytken larsen m. et al. a comparison of analytical goals for haemaglobin alc assays derived using different strategies. (submitted for publication). hyltoft petersen p. et al. influence of analytical quality and preanalytical variations on measurements of cholesterol in screening programmes. scand j clin lab invest, 1990;50 h0rder m (ed.) assessing quality requirements in clinical chemistry. scand j clin lab invest. 1980;40 suppl. 155. de verdier c-hi aronsson ti nyberg a (eds.). quality control in clinical chemistry efforts to find an efficient strategy. scand j clin lab invest, 1984;44 suppl. 172. wide l, dahlberg pa. quality requirements of basal s-tsh assays in predicting an s-tsh response to trh. scand j clin lab invest, 1980;40 suppl 155:lol-10. hyltoft petersen p et al. studies on the required analytical quality of tsh measurements in screening for congenital hypothyroidism. scand j clin lab invest, 1980;40 suppl. 198:66-72. suppl. 155:85-93. correspondence: p. hyltoft petersen, department of clinical chemistry, university hospital, dk-5000 odense c, denmark 264 upsala j med sci 90: 171-172, 1985 clinical urology and basic renal research introductory remarks a. erik g . persson department of urology, university hospital, uppsala, sweden the past 10 to 20 years in clinical urology have been characterized by re markable evolution of new techniques and by refinements of older methods for diagnosis and treatment. this rapid progress had resulted in more urologic dis orders becoming amenable to treatment and, for certain conditions, shortening of the hospital stay. there are grounds for optimism concerning future develop ments. however, the inevitable involvements with advanced technology and sci entific methods have increased the demands made on the skills of clinicians. because urology is such a comprehensive field for research, it is necessary to focus on areas o f special interest and, because such questions must be pen etrated in depth, research makes heavy demands on resources. moreover, clinical questions as a rule overlap several areas of the research field, thus underlin ing the importance of linking basic biologic research to clinical urology. the aspects which require extensive interdepartmental collaboration include patho logy, virology, haematology, immunology and physiology. in the studies here presented we have used basic biologic methods, but have dealt with clinical questions. collaboration has been particularly close with the renal research groups at the biomedical centre of uppsala university. many o f the investigations in the past ten years have concerned preservation o f kid neys in connection with renal transplantation and acute renal failure. in pract ical terms, these collaborative projects have led to some changes in clinical routines. the pathophysiology of unilateral nephrectomy has increasingly attracted interest in recent years with regard to the possibility of renal qlomerular damage as a result of glorverular hyperfiltration. questions concerning ureteral obstruction and relief of the associated pain, and the basic physiologic regul ation of that pain, have also been studied at our department. the importance o f renal function in the development and maintenance of arterial hypertension is another subject towards the understanding of which considerable effort has re cently been directed. 12-858572 171 some o f the papers presented in this issue are examples of our strivinqs towards expansion o f knowledge in clinical urology with utilization o f basic medical research. address for reprints: a. erik g. persson department o f urology university hospital 5-751 85 uppsala sweden 172 untitled comparison of placental pten and β1 integrin expression 235 key words: pten; β1 integrin; immunohistochemistry; placenta; abortion received 9 january 2008 accepted 14 april 2008 upsala j med sci 113 (2): 235–242, 2008 comparison of placental pten and β1 integrin expression in early spontaneous abortion, early and late normal pregnancy cigdem tokyol1, fatma aktepe1, fatma hüsniye dilek1, mehmet yilmazer2 departments of 1pathology, and 2 gynecology and obstetrics, afyon kocatepe university school of medicine, afyonkarahisar, turkey abstract background: pten seems to play an important role in cell cycle, growth, migration, and death. integrins are cell surface receptors that play a role in the regulation of cell proliferation, differentiation, implantation, and embryogenesis. pten inhibits β1 integrin signaling. the objective of this study is to investigate the expression of pten and β1 integrin in placental tissues of early spontaneous abortion and first and third trimesters of normal pregnancy. method: a total of 43 placental tissue samples were evaluated using immunohistochemistry for pten and β1 integrin. group 1 included placental tissues of volunteer termination of normal pregnancy during the first trimester (5–10 wk gestation). group 2 included placental tissues of normal vaginal delivery at the third trimester of pregnancy (36–40 wk gestation). group 3 included placental tissues of pregnancy termination because of spontaneous abortion during the first trimester (5–10 wk gestation). results: pten expression of villous trophoblast was decreasing as the pregnancy advanced. pten staining of decidual cells was significantly stronger in tissue samples from early spontaneous abortion than in tissue samples from early and late normal pregnancy (p=0.003, p=0.001, respectively). there was no significant difference between β1 integrin expression of villous trophoblast and decidual cells in three groups. conclusion: our findings suggest that altered patterns of pten expression may be associated with abortion, but it seems that β1 integrin does not contribute to this process as a signaling protein. further evaluation is needed to highlight this subject. introduction pten (phosphatase and tensin homolog deleted on chromosome 10) is a tumor suppressor gene which was identified in 1997 (1–3). the pten gene is frequently deleted or mutated not only in prostatic, endometrial, breast, lung, kidney, bladder, testis, head and neck cancers, but also in glioblastoma, malignant melanoma, and lymphoma (4). pten seems to play an important role in cell cycle, growth, migration, and death (5). the pten gene encodes a dual-specifity protein phosphatase and also has extensive homology to tensin, a protein that interacts with actin filaments at focal adhesions. focal adhesions are sites on the plasma membrane at which in236 cigdem tokyol et al. tegrins aggregate (6). integrins are transmembrane glycoproteins made up of α and β chains (7). β1 integrins interact with a number of signal transduction proteins, including focal adhesion kinase as well as cytoskeletal proteins. in this manner integrins mediate processes such as cell migration, spreading, and growth (4,6). pten inhibits the phosphorylation of focal adhesion kinase in response to integrinmediated processes (6). integrins are cell surface receptors that play a role in the regulation of cell proliferation, differentiation, implantation, and embryogenesis (8,9). pten inhibits β1 integrin signaling. a variety of normal cells undergo apoptosis when they lose attachment to an appropriate extracellular matrix. thus, pten induces apoptosis (4). endometrial functions are carried out by mechanisms of proliferation, differentiation, implantation, and apoptosis. it has been shown that endometrial and decidual integrin and pten expression change throughout the menstrual cycle and pregnancy (8,10–14). integrins and pten are also expressed in normal human placental tissue (15,16). the objective of this study is to investigate the expression of pten and β1 integrin in placental tissues of early spontaneous abortion and first and third trimesters of normal pregnancy and evaluating a comparison between them. such markers may be useful in promoting our understanding of a common pathway for spontaneous abortion regardless of the etiology. methods we performed a retrospective study including three separate series of paraffin-embedded placental tissue samples collected from the pathology files of our hospital from 2001 to 2004. the first series of samples (group 1) included placental tissues of 15 women who underwent volunteer termination of clinically normal pregnancy during the first trimester (5–10 wk gestation). the second series of samples (group 2) included placental tissues of 15 women who had normal vaginal delivery at the third trimester of pregnancy (36-40 wk gestation). the third series of samples (group 3) included placental tissues of 13 women who underwent pregnancy termination because of spontaneous abortion during the first trimester (5–10 wk gestation). gestational ages were calculated using the last menstrual period. all spontaneous abortion patients had ultrasonographic evaluation when they presented with vaginal bleeding and uterine evacuation was performed after ultrasonographic evaluation. women with serious systemic disease (diabetes mellitus, thyroid dysfunction, infectious disease… etc.) and anembryonic pregnancies were not included in the study. comparison of placental pten and β1 integrin expression 237 immunohistochemistry the streptavidin-biotin-peroxidase method was performed using the primary monoclonal antibodies against pten ab-4 (clone 17.a, prediluted, neomarkers, usa) and cd29 (i̇ntegrin beta-1) ab-3 (clone 29co3, prediluted, neomarkers, usa). the representative blocks of placental tissues were sectioned and mounted on poly-l-lysin-coated slides. before immunohistochemistry epitope retrieval was performed by boiling the slides in 10 mm citrate buffer, ph 6.0, for 20 min in a microwave oven. slides were cooled at room temperature for 20 min. endogenous peroxidase activity was blocked using hydrogen peroxide for 10 min. tissues were incubated with blocking serum for 5 min to avoid nonspecific background staining and washed in tris buffered saline (tbs). primary monoclonal antibody against pten and β1 integrin was then applied for 30 min and 1 h respectively at room temperature and washed in tbs. linking antibody and streptavidin peroxidase (lab vision) were added consecutively for 10 min and washed in tbs. the peroxidase activity was visualized with 3-amino-9-ethylcarbazole for 7 min. the slides were counterstained in mayer’ s hematoxylin. positive staining for β1 integrin was defined as immunoreactivity at the cell membrane and for pten cytoplasmic staining. positive controls consisted of known positive samples of placenta. pten and β1 integrin expression were evaluated blindly in villous trophoblast and decidual cells. decidual cells were distinguished from intermediate-type trophoblast by the lack of significant nuclear atypia. the degree of positive staining for pten was evaluated using a semiquantitative scale which was described by taniyama et al (17): 1) negative 2) ≤5% immunoreactive trophoblastic/decidual cells 3) 5-50% immunoreactive trophoblastic/decidual cells 4) ≥50% immunoreactive trophoblastic/decidual cells. the degree of positive staining for β1 integrin was evaluated using a semiquantitative scale which was described by manzotti et al (18): 1) negative 2) ≤10% immunoreactive trophoblastic/decidual cells 3) 10–50% immunoreactive trophoblastic/decidual cells 4) ≥50% immunoreactive trophoblastic/decidual cells. statistical analysis the immunohistochemical data are reported as the mean ± standard error of mean (sem). statistical analysis of the data was performed using kruskal-wallis and mann-whitney u tests. bivariate correlation between variables was determined by pearson’ s correlation coefficients. a p value <0.05 was considered significant. results results of the immunohistochemical staining have been summarized in table 1. the staining pattern of pten was cytoplasmic. expression of pten was prominent in villous trophoblasts in early spontaneous abortion and early pregnancy 238 cigdem tokyol et al. (fig.1). there was no pten staining in villous trophoblasts and decidual cells in late pregnancy. pten staining of villous trophoblasts was significantly stronger in tissue samples from early pregnancy and early spontaneous abortion than samples from late pregnancy (p=0.000, p=0.000). pten expression of villous trophoblast was decreasing as the pregnancy advanced. though there was very weak pten staining in decidual cells in early pregnancy, expression of pten was prominent in decidua in early spontaneous abortion (fig.2). pten staining of decidual cells was significantly stronger in tissue samples from early spontaneous abortion than in tissue samples from early and late pregnancy (p=0.003, p=0.001, respectively). staining for β1 integrin revealed positivity around cell membranes in villous trophoblasts (fig.3), and decidual cells (fig.4). although we have observed β1 integrin expression in villous trophoblasts and decidual cells in three groups, there was no statistically significant difference between them. table 1. pten and β1 integrin immunoreactivity in placental tissues tissues pten β1 integrin trophoblast decidua trophoblast decidua normal pregnancy at first trimester (n=15) 3.1(0.3) 1.1(0.1) 2.0(0.2) 2.5(0.2) normal pregnancy at third trimester (n=15) 1.0(0.0)* 1.0(0.0) 2.8(0.3) 2.4(0.3) spontaneous abortion at first trimester (n=13) 2.6(0.3) 2.2(0.3)** 2.1(0.3) 2.6(0.2) sem is reported in parentheses. *p<0.01 for group 2 vs group 1 and group 3. **p<0.01 for group 3 vs group1 and group 2. figure 1. cytoplasmic staining of pten in villous trophoblasts in spontaneous abortion (x200). figure 2. cytoplasmic staining of pten in decidual cells in spontaneous abortion (x200). comparison of placental pten and β1 integrin expression 239 discussion pten has a critical importance during development and embryogenesis. it has been shown that mice with homozygous-targeted deletion of pten gene have abnormal patterning of ectodermal and mesodermal germ layers and defective placentation (19,20). pten is expressed in placental tissue and is essential for embryonic development (21). in a previous study, pten expression was evaluated throughout the menstrual cycle in normal endometrial tissues. it was reported that proliferative endometrium showed cytoplasmic and nuclear pten expression in the surface epithelium. by the midsecretory phase, epithelial pten is exhausted, but increases dramatically in the cytoplasm of stromal cells undergoing decidual change. it was concluded that stromal and epithelial compartments contribute to the hormone-driven changes in endometrial pten expression and inferred that abnormal hormonal conditions may disrupt normal patterns of pten expression in this tissue (13). kayışlı et al evaluated pten expression throughout the menstrual cycle and during early pregnancy. they found higher pten immunoreactivity in endometrial stromal and glandular cells during late secretory and early proliferative phases. they observed a further increase in pten expression in decidual and glandular cells during early pregnancy. they proposed that pten might be one of the signaling proteins that estrogen and progesterone are acting to affect endometrial cell proliferation and/or apoptosis (14). chen et al investigated the possible involvement of the pten gene in the development of gestational trophoblasts and the pathogenesis of hydatidiform moles. they found that in partial and complete hydatidiform moles, the pten protein expression rate was significantly lower than in early placentas. however, partial hydatidiform moles, complete hydatidiform moles, and invasive moles were not significantly different in terms of pten protein expression. their findings suggested that the regulation of pten expression may play an important role in the development of the early gestational trophoblast and in the pathogenesis of hydafigure 3. β1 integrin positivity around cell membranes in villous trophoblasts in spontaneous abortion (x100). figure 4. β1 integrin positivity around cell membranes in decidual cells in late pregnancy (x200). 240 cigdem tokyol et al. tidiform mole, but not in its malignant transformation (22). pten protein may play an important role in trophoblast development. the early trophoblast cells possess the ability to proliferate and invade during embryo implantation, and form the chorion and placenta. these behaviours of the trophoblastic cells are regulated as the placenta maturates. pten protein may ensure normal development of gestational trophoblasts by controlling trophoblast proliferation and invasion (22). chen et al postulated that down-regulated pten protein expression could lead to abnormal trophoblast proliferation, suggesting that lower pten expression is probably responsible for the pathogenesis of hydatidiform moles (22). ishioka et al analysed changes of apoptosis-related proteins induced by hypoxia in trophoblastic cells to clarify the mechanisms of hypoxia-induced apoptosis by using the poweblot, an antibody-based western array. hypoxia induced apoptosis was accompanied by increased expression of pten the bag-1 antisense oligonucleotide did not affect the expression of pten. their findings were important to detect hypoxic stress of placenta, which leads to preeclampsia and other hypoxiarelated obstetric complications (23). few articles examining expression of pten in placental tissues are available in the literature. to gain further insight on this subject, we have explored pten expression in placenta in light of spontaneous abortion. in our study, pten expression of villous trophoblasts was decreasing as the pregnancy advanced. pten expression decreased parallel to the development of placenta. expression of pten in decidual cells was significantly stronger in placental tissues of spontaneous abortion than placental tissues from normal pregnancies at the first and third trimester. the up-regulation of pten expression in decidua may induce apoptosis and this may interfere with trophoblast proliferation and invasion. the abnormal pten expression may be a common pathway for pregnancy loss regardless of the etiology. integrins are adhesion-receptor proteins that mediates cell-cell and cell-extracellular interactions and plays a fundamental role in the regulation of gene expression, cell proliferation, and differentiation. these receptors link to the extracellular matrix proteins and transduce signals from the extracellular environment into the cell, activating cellular transduction pathways after binding with soluble mediators, cytokines, and growth factors (8). during implantation and pregnancy, trophoblastic cells invade the decidua, simulating the process of stromal invasion by malignant cells. this is a complexly regulated process. it has been demonstrated that human endometrial and decidual cells express β1 integrin on their surfaces and this expression is a dynamic process throughout the menstrual cycle. β1 integrin expression in the human endometrium increases after implantation and remains high in the decidua during early pregnancy. it has been suggested that endometrial integrins play an important role in the process of implantation and decidualization (12,24). however, the role of β1 integrin variants in human decidua during early and late pregnancy remains to be clarified (8,10). lessey et al determined that the timing of expression of the α4β1 integrin framed the putative window of implantation and suggest a role in establishment of uterine receptivity (11). comparison of placental pten and β1 integrin expression 241 yoshimura et al investigated the expression of β1 integrin in human endometrium and decidua. they reported that the immunohistochemical distribution of β1 integrin demonstrated predominantly glandular epithelial staining in the proliferative phase, and stromal and glandular staining in the midsecretory phase (12). korhonen et al examined the distribution of the integrin subunits in human first and second trimester and term placentas. they stated that in first and second trimesters villi, β1 integrin subunit was detected in the stromal cells, whereas in the second and third trimesters it was expressed in villous trophoblast. throughout placental development, decidual cells reacted prominently with anti-β1. they also demonstrated that the expression of integrin complexes is modulated during the differentiation of trophoblastic and decidual cells and suggested that integrin-mediated cell-basal membrane interactions may be important for placental development (15). we have examined the expression of β1 integrin in villous trophoblasts and decidual cells of placental tissues from normal pregnancies at the first and third trimesters and spontaneous abortion. there was no significant difference between β1 integrin expression of three groups. our results are not consistent with the findings of korhonen et al (15). this may be because they used immunoflorescence microscopy and a panel of different antibody complexes. there was no significant correlation between β1 integrin expression and pten expression in villous trophoblasts and decidual cells. our findings suggest that altered patterns of pten expression may be associated with spontaneous abortion, but according to our results, it seems that β1 integrin does not contribute to this process as a signaling protein. additional studies in larger series, including both pten antibody and specific β1 integrin antibody complexes will be needed to highlight this subject. references li j, yen c, podsypanina k, bose s, wang si, puc j, miliaresis c, rodgers l, mccombie r, 1. bigner sh, giovanella bc, ittmann m, tycko b, hibshoosh h, wigler mh, parsons r (1997) pten, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostat cancer. science 28: 1943–1947. steck pa, pershouse ma, jasser sa, yung wk, lin h, ligon ah, langford la, baumgard ml, 2. hattier t, davis t, frye c, hu r, swedlund b, teng dh, 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(2003) differential expression of β1c integrin messenger ribonucleic acid and protein levels in human endometrium and decidua during the menstrual cycle and pregnancy. j clin endocrinol metab 88: 720–729. sueoka k, shiokawa s, miyazaki t, kuji n, tanaka m, yoshimura y (1997) integrins and repro-9. ductive physiology: expression and modulation in fertilization, embryogenesis, and implantation. fertil steril 67: 799–811. van der linden pjq, de goeij afpm, dunselman gaj, erkens hwh, evers jlh (1995) expres-10. sion of cadherins and integrins in human endometrium throughout the menstrual cycle. fertil steril 63: 1210–1206. lessey ba, castelbaum aj, buck ca, lei y, yowell cw, sun j (1994) further characterization of 11. endometrial integrins during the menstrual cycle and pregnancy. fertil steril 62: 497–506. yoshimura y, miyakoshi k, hamatani t, iwahashi k, takahashi j, kobayashi n, sueoka k, mi-12. yazaki t, kuji n, tanaka m (1998) role of beta 1 integrins in human endometrium and decidua during implantation. horm res 50 suppl 2: 46–55. mutter gl, lin m-c, fitzgerald jt, kum jb, eng c (2000) changes in endometrial pten expres-13. sion throughout the human menstrual cycle. j clin endocrinol metab 85: 2334–2338. kayışlı ög, kayışlı üa, al-rejjal r, zheng w, lüleci g, arıcı a (2003) regulation of pten 14. 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169–174. di cristofano a, pesce b, cordon-cardo c, pandolfi pp (1998) pten is essential for embryonic 19. development and tumour suppression. nat genet 19: 348–355. suzuki a, de la pompa jl, stambolic v, elia aj, sasaki t, del barco barrantes i, ho a, wakeham 20. a, itie a, khoo w, fukumoto m, mak tw (1998) high cancer susceptibility and embryonic lethality associated with mutation of the pten tumor suppressor gene in mice. curr biol 8: 1169–1178. podsypanina k, ellenson lh, nemes a, gu j, tamura m, yamada km, cordon-cardo c, ca-21. toretti g, fisher pe (1999) mutation of pten/mmac1 in mice causes neoplasia in multiple organ systems. proc natl acad sci 96: 1563–8. chen h, ye d, xie x, lu w, zhu c, chen x (2005) 22. pten promoter methylation and protein expression in normal early placentas and hydatiform moles. j soc gynecol investig 12: 214–217. ishioka s, ezaka y, umemura k, hayashi t, endo t, saito t (2006) proteomic analysis of mecha-23. nisms of hypoxia-induced apoptosis in trophoblastic cells. int j med sci 4: 36–44. shiokawa s, yoshimura y, nagamatsu s, sawa h, hanashi h, oda t, katsumata y, koyama n, 24. nakamura y (1996) expression of beta 1 integrins in human endometrial stromal and decidual cells. j clin endocrinol metab 81: 1533–1540. corresponding author: cigdem tokyol afyon kocatepe üniversitesi ali çetinkaya kampüsü uygulama ve araştırma hastanesi patoloji bölümü afyonkarahisar, turkey ph: 90 272 2142065 fax: 90 272 2133066 e-mail: ctokyol@yahoo.com ujms110_2.pdf upsala j med sci 110: 151–158, 2005 inflammatory activity: capillary electrophoresis provides more information than erythrocyte sedimentation rate anders larsson, lars-olof hansson department of medical sciences, clinical chemistry and pharmacology, university hospital, uppsala, sweden. abstract a free full-text copy of this article can be found at the web page of upsala j med sci: http://www.ujms.se background: a new automated multicapillary zone electrophoresis instrument with improved resolution buffer (capillarys® with hr buffer, sebia, paris, france) for analysis of human plasma proteins was compared with erythrocyte sedimentation rate (esr). esr determinations have been performed for more than eighty years and it is still one of the most frequently used laboratory tests, mainly to monitor the inflammatory response and as a tumour marker. methods: we studied the relationships between esr, capillary electrophoresis and nephelometric determination of fibrinogen and albumin in 503 consecutive patient samples. the samples were analyzed on the capillarys®. the albumin concentration from the nephelometric determination was used for quantification of the individual peaks in the capillary electrophoresis electropherogram. results: we found no significant correlation between presence or size of m-components and esr. there were moderate to strong correlations between esr, fibrinogen and capillary electrophoretic determination of � 1 antitrypsin, � 1 -acid glycoprotein or haptoglobin for the detection of acute phase response. conclusions: we suggest that esr could be replaced by capillary electrophoresis for the assessment of inflammatory conditions and to detect m-components. introduction substantial changes in the levels of several plasma proteins accompany infections or tissue injuries due to trauma, malignancy, or ischemia, as well as inflammatory conditions [1]. the characteristic pattern of these changes is termed the acute phase 151 received 23 november 2004 accepted 3 december 2004 key words: capillary electrophoresis, erythrocyte sedimentation rate, fibrinogen, m-component, inflammation response, which appears to play a significant role in the host defence. the most widely used assays for measurements of the acute phase response are c reactive protein (crp) [2–5] and the erythrocyte sedimentation rate (esr) [6–8]. esr was originally described as a disease marker more than eighty years ago but still retains an important place in medical practice, probably because it is an easily performed and inexpensive test, and a wealth of information about its clinical significance has accumulated over the years. the test measures the distance that erythrocytes have fallen after one hour in a vertical column of anticoagulated blood under the influence of gravity. the accepted upper limits of normal are 15 mm/first hour for males and 20 mm/first hour for females, based on reference values from students in their twenties. values up to 40 mm/first hour are not uncommon in healthy elderly people [9]. elevated esr is a non-specific finding but it is often used as an acute-phase response marker. however, the esr is influenced by several other factors such as anaemia, erythrocyte size, white blood cell count, immunoglobulins, monoclonal gammopathies, renal failure, pregnancy, age, sex, red cell morphology, room temperature and the placement of the esr tube [10, 11]. capillary electrophoresis is an interesting alternative to esr, as this method can differentiate between m-components and acute phase response and can more accurately measure the increment of individual acute phase proteins including fibrinogen which is the protein that has the largest impact on esr in the inflammatory response [12, 13]. any condition that causes a general increase of acute phase proteins (e.g. infectious diseases, polymyalgia rheumatica, temporalis arteritis, diabetes mellitus, heart disease, malignancies, renal failure and pregnancy) will also elevate esr [13–15]. capillary electrophoresis systems have been adapted to allow rapid separation of plasma samples. assay time with the capillarys® capillary electrophoresis system is less than 10 min. with an automatic interpretation program it should be possible to perform capillary electrophoresis determination of acute phase proteins with shorter turn-around times than esr, with concomitant determination and quantification of m-components. we were thus interested to study the correlation between esr performed with seditainer tubes and capillary electrophoresis for quantification of acute phase response in consecutive patient samples. an automatic esr reader was used to minimise variations due to interindividual interpretation of the esr results. materials and methods patients the samples (n=503) were from consecutive patients referred to the department of clinical chemistry, university hospital, uppsala for esr determination. the study was approved by the local ethical committee at uppsala university (01–167). esr and nephelometric determination of albumin and fibrinogen for esr analysis, blood was collected in seditainer tubes (366065, becton dickinson, franklin lakes, nj usa) and analysed by sedimatic 100 (analys instrument 152 ab, bromma, sweden); normal range 1–20 mm/first hour (< 50 yrs), and 1–30 mm/first hour (> 50 yrs). after esr analysis, the tubes were centrifugated for 10 min at 1300 g and room temperature. the plasma was transferred to plastic tubes and frozen at –70°c. plasma fibrinogen and albumin were analysed utilizing a bn prospec™ nephelometer (dade behring, deerfield, il, usa) with reagents from the same manufacturer, including a calibrator related to crm 470. the results were adjusted for the dilution in the seditainer tubes. all assays were performed at the clinical chemistry laboratory, uppsala university hospital. capillary electrophoresis capillary electrophoresis was performed using capillarys™ capillary electrophoresis system (sebia, paris, france) utilizing the new hr buffer and the same samples used as for the nephelometric determinations. the instrument is equipped with eight capillaries allowing a throughput of approximately 60 samples per hour. the protein separation is performed at ph 9.9 in silica capillaries and the proteins are detected at an absorbance of 200 nm. the instrument expresses the size of the individual peaks as absorbance percentage of the whole sample. the nephelometric albumin value was used to convert the percentage values for the a1-antitrypsin, � 1 -acid glycoprotein and haptoglobin peaks to protein concentrations in g/l. statistical analysis statistical analysis was performed with statistica 4.5 (statsoft inc., tulsa, ok, usa) and excel 97 (microsoft corp, seattle, wa, usa). p < 0.05 was considered significant throughout the study. 153 fig 1. correlations between esr and m-component size in samples containing m-components. results esr the 503 samples were collected from a total of 538 samples referred to the laboratory for routine esr analysis. in 35 (6.5%) cases the technician responsible for routine analysis had judged it impossible to perform the assay due to preanalytical errors (insufficient volume or visible clots). the esr showed a broad distribution range but with a large group of samples within the normal range. 197 samples (39%) had esr £10 mm while 74 samples 154 fig. 2. correlations between esr and fibrinogen values in individual patients (n=503). fig. 3. correlations between fibrinogen and � 1 -acid glycoprotein values in individual patients (n=503). (15%) had esr ≥50 and 27 (5.4%) samples had esr ≥110. the capillary electrophoresis detected m-components in several of the patient sera. there was no significant correlation between esr and the presence or size of m-component (fig. 1.). correlations between esr, fibrinogen and electrophoretic determination of acute phase proteins there were significant positive correlations between esr and the other acute phase protein studied: fibrinogen (r2=0.595), � 1 -antitrypsin (r2=0.437), � 1 -acid glycoprotein (r2=0.451) or haptoglobin (r2=0.388). the correlation between fibrinogen and the other acute phase proteins measured in g/l was stronger than the corresponding 155 fig. 4. correlations between fibrinogen and � 1 -antitrypsin values in individual patients (n=503). fig. 5. correlations between fibrinogen and haptoglobin values in individual patients (n=503). correlations with esr: � 1 -antitrypsin (r2=0.610), � 1 -acid glycoprotein (r2=0.604) or haptoglobin (r2=0.630). if the percentage of the individual peaks from the capillary electrophoresis was used without adjustments for albumin concentration the correlations to fibrinogen were less pronounced: a1-antitrypsin (r2=0.348), � 1 -acid glycoprotein (r2=0.350) or haptoglobin (r2=0.363). (fig. 2–6) discussion the major indications for the measurement of acute phase proteins are to monitor disease activity, and sometimes to provide prognostic information. the tests may also occasionally be used to support a clinical diagnosis [16]. esr and crp are the most widely used assays to monitor the laboratory part of the inflammatory process. currently, esr is preferred in the unites states and the crp is preferred in europe [17], while fibrinogen and other acute phase proteins are rarely used. in europe there is a wider use of plasma or serum protein electrophoresis in the southern (e.g. italy) than in the northern parts. esr is influenced by several factors unrelated to the inflammation as mentioned in the introduction. during an inflammatory reaction, esr is mainly influenced by fibrinogen (55%), followed by a2-macroglobulin (27%), immunoglobulin (11%) and albumin (7%) [18]. it usually takes about four to seven days for esr and fibrinogen to respond, while crp reacts within one to two days [19]. in chronic diseases like ra it may be an advantage to have a slow and stable inflammatory marker like fibrinogen, a1-antitrypsin, a1-acid glycoprotein or haptoglobin in addition to crp. there are several factors, other than the acute phase response, that can influence individual plasma proteins. thus, it may advantageous to determine several acute phase proteins as in capillary electrophoresis. low � 1 -antitrypsin may be due to genetic deficiency while increased levels may be caused by liv156 fig. 6. correlations between fibrinogen and � 1 -acid glycoprotein percentage values in individual patients (n=503). er damage or oestrogen therapy. � 1 -acid glycoprotein is influenced by the glomerular filtration rate with increased levels in plasma from patients with kidney damage while haptoglobin is low in plasma from patients with liver cirrhosis or haemolysis. we have previously shown significant correlations between crp, esr and fibrinogen in a small group of ra patients [20, 21]. the crp and fibrinogen, but not esr, showed highly significant correlation with a functional test (mhaq) [21]. crp and fibrinogen have previously been shown to be associated with radiographic evidence of disease progression in ra. there was (at the end of a twelve months period) a better correlation between radiographic progression and crp than with esr [22]. similar results were found in the study by sjøblom et al., who found good correlation of larsen index with fibrinogen and crp, but not with esr [23]. this study confirms the earlier observations of a correlation between esr and fibrinogen [12, 20, 21]. many of the confounding factors of esr could be avoided using fibrinogen or plasma protein electrophoresis. the many factors influencing esr makes it difficult to interpret an esr result. this is probably the cause of the great inter-individual variability in interpretation of esr results previously reported [24]. in contrast to esr, capillary electrophoresis can be analysed retrospectively on stored plasma samples and the assay can be centralised, which is important in clinical studies to eliminate interlaboratory variation. the percentage of preanalytical error for esr (6.5%) is extremely high for a high volume laboratory assay. as insufficient sample and clots have a minor impact on the capillary electrophoresis we also analysed these samples. in a number of these cases, where it was impossible to perform esr, the capillary electrophoresis revealed the presence of a m-component. the high rate of preanalytical errors is just another argument for the replacement of esr by more specific assays. the health care strives to shorten the patient turnaround times (tat) and thus also laboratory test tat. the transit from paper and agarose electrophoreses to capillary electrophoresis has made it possible to shorten tat significantly. the use of lithium-heparin pst tubes eliminates delay due to sample clotting. with pst-tubes, preanalytical automation, capillary electrophoresis, computerised interpretation and an electronic request-result system it should be possible to provide test results within 30 min from the time that the sample arrives to the laboratory. this is superior to esr tat. in the future we believe that plasma protein electrophoresis with capillaries will be an interesting alternative to esr providing shorter turnaround times and more information to the clinician. acknowledgements the capillarys®‚ reagents were generously provided by sebia, paris, france references 1. suffredini af, fantuzzi g, badolato r, oppenheim jj, o'grady np (1999). new insights into the biology of the acute phase response. j clin immunol 19: 203–14. 2. hedlund p (1947). the appearance of acute phase protein in various diseases. acta med scand 124: 579–82. 157 3. mc conkey b, crockson ra, crockson ap (1972). the assessment of rheumatoid arthritis: a study based on measurements of the serum acute-phase reactants. quart j med 16: 115–25. 4. clyne b, olshaker js (1999). the c-reactive protein. j emerg med 17: 1019–25. 5. du clos tw (2000). function of c-reactive protein. ann med 32: 274–8. 6. fåhreus r (1921). the suspension-stability of the blood. acta med scand 55: 1–228. 7. kavanaugh a (1999). the role of the laboratory in the evaluation of rheumatic diseases. clin cornerstone 2: 11–25. 8. wollheim fa (2000). markers of disease in rheumatoid arthritis. curr opin rheumatol 12: 200–05. 9. shearn ma, kang iy (1986). effect of age and sex on the erythrocyte sedimentation rate . j rheumatol 13: 297–302. 10. brigden m (1998). the erythrocyte sedimentation rate. still a helpful test when used judiciously. postgrad med 103: 257–62. 11. zlonis m (1993). the mystique of the erythrocyte sedimentation rate. a reappraisal of one of the oldest laboratory tests still in use. clinics lab med 13: 787–96. 12. crockson ra, crockson ap (1974). relationship of the erythrocyte sedimentation rate to viscosity and plasma proteins in rheumatoid arthritis. ann rheum dis 33: 53–6. 13. sox hc, liang mh (1986). the erythrocyte sedimentation rate. guidelines for rational use. annals intern med 104: 515–23. 14. brigden ml (1999). clinical utility of the erythrocyte sedimentation rate. am fam physician 60: 1443–50. 15. hansson lo, carlsson i, hansson e, hovelius b, svensson p, tryding n (1995). measurement of c-reactive protein and the erythrocyte sedimentation rate in general practice. scand j prim health care 13: 39–45. 16. richardson c, emery p (1996). laboratory markers of disease activity. j rheumatol 23: 23–30. 17. wolfe f (1997). comparative usefulness of c-reactive protein and erythrocyte sedimentation rate in patients with rheumatoid arthritis. j rheumatol 24: 1477–85. 18. stuart j, whicher jt (1988). tests for detecting and monitoring the acute phase response. arch dis child 63: 115–17. 19. blackburn wd (1994). validity of acute phase proteins as markers of disease activity. j rheumatol 42: 9–13. 20. arvidson ng, gudbjörnsson b, hällgren r, larsson, a (1998). concordant message of different inflammation markers in patients with rheumatoid arthritis. upsala j med sci 103: 35–42. 21. arvidson ng, larsson a, larsen a (2002). disease activity in rheumatoid arthritis: fibrinogen is superior to the erythrocyte sedimentation rate. scand j clin lab invest 62: 315–9. 22. larsen a (1988). the relation of radiographic changes to serum acute-phase proteins and rheumatoid factor in 200 patients with rheumatoid arthritis. scand j rheumatol 17: 123–9. 23. sjøblom kg, saxne t, pettersson h, wollheim fa (1984). factors related to the progression of joint destruction in rheumatoid arthritis. scand j rheumatology 13: 21–27. 24. thue g, sandberg s, fugelli p (1994). the erythrocyte sedimentation rate in general practice: clinical assessment based on case histories. scand j clin lab invest 54: 291–300. corresponding author: anders larsson department of medical sciences, clinical chemistry and pharmacology, university hospital, s-751 85 uppsala, sweden phone: 46-18-6114271 anders.larsson@akademiska.se 158 upsala j m e d sci 92: 185-192, 1987 the influence of bilateral electrical preganglionic sympathetic stimulation on intraand extracranial blood flow lars-owe d. koskinen department of physiology and medical biophysics, biomedical centre, university of uppsala, uppsala, sweden abstract the e f f e c t s o f b i l a t e r a l e l e c t r i c a l stimulation (ss) o f t h e c e r v i c a l sympathetic chain on i n t r a and e x t r a cerebral blood flows were studied w i t h t h e labelled microsphere method in t h e rabbit. c o n t r o l blood f l o w was determined before the ss was started. the stimulation frequency was 7 hz, t h e impulse duration 2 ms, t h e intensity 7 v and t h e stimulation t i m e varied between 1 t o 5 minutes before t h e second blood f l o w determination. a r t e r i a l blood gas values and blood pressure were unaffected by t h e stimulation. due t o the ss there were blood flow decrements in the extracranial tissues between 60-96%. the blood flow in t h e eyes, the dura, pineal gland and choroid plexa was markedly reduced during the 55. n o obvious e f f e c t was e l i c i t e d by t h e ss in t h e regional or t o t a l cerebral blood flow. the stimulation t o c o n t r o l blood f l o w r a t i o ranged between 0.92 f 0.08 t o 1.13 2 0.09 in d i f f e r e n t p a r t s o f t h e brain. the conclusions are t h a t ss e l i c i t s vasoconstriction in several extraand intracranial nonneuronal tissues and in the eye. cerebral blood f l o w i s n o t influenced b y the ss. introduction the cerebral vasculature is innervated by several types o f nerves, p a r t i c u l a r l y b y sympathetic nerve fibres emanating f r o m t h e superior c e r v i c a l ganglion. the influence o f these sympathetic nerves on cerebral blood f l o w has been evaluated i n several species and under a variety o f conditions and w i t h c o n f l i c t i n g results ( 9 , 17, 19). in t h e conscious and anaesthetized r a b b i t a t i t s normal blood pressure no obvious tonic influence o f the cervical sympathetic nerves was detected on regional (rcbf) and t o t a l (cbftot) cerebral blood flow, b u t on extracranial blood flows (10-13). the e l e c t r i c a l stimulation o f t h e sympathetic chain unilaterally did n o t markedly influence t h e cerebral blood flow in t h e normotensive c a t ( 3 ) , monkey (1) and r a b b i t (5, 15). in acute hypertension, on t h e other hand, sympathetic stimulation has a clear protective effect, preventing cerebral hyperemia and disruption o f the blood-brain-barrier (4, 8). indeed, in t h e normotensive 185 . . animal an "escape phenomenon" f r o m the sympathetic influence on the cerebral vessels has been proposed. thus, in the r a b b i t a decrease i n cbf was shown early in the course o f stimulation o f the c e r v i c a l sympathetic chain (18) while beausang-linder bc h u l t c r a n t z (5) found no such effect. it has been speculated that, due t o a double (ipsi and contralateral cervical sympathetic) innervation o f the cerebral vessels a more pronounced e f f e c t could be e l i c i t e d by bilateral stimulation. indeed, it was recently reported by busija (6, 7) t h a t the bilateral, but not unilateral, stimulation of t h e c e r v i c a l sympathetic ganglion a t 8 and 16 h z considerably reduced the cerebral blood f l o w i n t h e rabbit. the present investigation was undertaken in order t o elucidate whether 1) b i l a t e r a l preganglionic cervical sympathetic stimulation a t a frequency regarded as being t h e upper p a r t of the physiological range influences r c b f and c b f t o t and 2) whether nonneural intracranial tissues are a f f e c t e d by the stimulation and 3) t o what extent ocular and extracranial tissues are affected. methods seven new zealand albino rabbits of either sex weighing between 1.7-4.0 k g were used. the animal was anesthetized w i t h a 25 % solution o f urethane i.v. in a dose o f 7 ml k g l b.w. the animal was tracheotomized and ventilated by a palmer pump. mean a r t e r i a l blood pressure (map) measurements, w i t h a druck pdcr 75/1 transducer and an servogor 460 recorder, and blood sampling was conducted f r o m a pair of cannulated arteries. the regional blood f l o w was measured by the labelled microsphere method (2, 20). microspheres were injected d i r e c t l y i n t o the l e f t ventricle through a cannula introduced retrogradely via the l e f t brachial artery. spheres, 15 um in diameter (nen, boston, massachusetts, usa), labelled w i t h 95 nb, io3 r u and 141 ce were used . one femoral vein was cannulated and used f o r drug injections. the cervical sympathetic chain was bilaterally i d e n t i f i e d and sectioned about one centimeter below the upper c e r v i c a l ganglion. the distal p a r t of t h e sympathetic nerve was b i l a t e r a l l y isolated, covered w i t h mineral oil, and electrically stimulated w i t h bipolar silver electrodes. c a r e was taken not t o stimulate the aortic depressor nerve. the stimulator (digitimer dssa, welwyn garden c i t y , england) was operated a t a stimulatory frequency o f 7 hz, intensity 7 v and impulse duration 2 ms. the f i r s t microsphere i n j e c t i o n was performed under resting conditions. the nerve was then bilaterally stimulated f o r 3 t o 5 m i n (in one case 1 min) before, during and 1 m i n a f t e r the second microsphere injection. a r t e r i a l blood gases (pa02 and pac02) and p h were measured a t intervals w i t h an abl2 acid-base analyzer (radiometer, copenhagen, denmark). if needed, sodium bicarbonate was given i.v. t o c o r r e c t deviations i n ph. pancuronium bromide (pavulonr, organon, oss, holland) was administered in a dose o f 0.05-0.2 mg/kg in order t o induce 186 skeletal muscle relaxation. the body temperature was recorded by a r e c t a l thermistor and maintained a t about 38-39oc w i t h a heating pad. heparin, 500 i u / k g b.w. was used as anticoagulant. a f t e r the experiment t h e animal was sacrificed by an i.v. i n j e c t i o n o f saturated kci. organs and tissue samples f r o m organs were excised and placed in preweighed plastic tubes. gray m a t t e r f r o m the f r o n t a l and o c c i p i t a l cortex, hippocampal region, caudate nucleus, thalamic region, hypothalamic region, collicule, pons-mesencephalon, medulla oblongata, cerebellum and spinal cord were excised. t o t a l cbf was calculated as including a l l regions except the medulla oblongata, cerebellum and spinal cord. the eye was dissected i n t o t h e retina, choroid, i r i s and c i l i a r y processes. various other tissues were also sampled. the r a d i o a c t i v i t y o f blood and tissue samples was determined b y gamma spectrometry. blood flows were calculated according t o t h e free flow method (2). a l l results are presented as means 2 sem. statistical evaluations o f the means was performed w i t h t h e two-tailed student' s t t e s t f o r paired observations. results in t h e c o n t r o l situation the cardiovascular variables were w i t h i n the normal range, and d i d n o t change during nerve stirnulation (table 1). mydriasis was observed on b o t h sides during the stimulation. there was no difference i n blood f l o w between t h e t w o sides, and t h e duration o f stimulation did n o t influence t h e response. therefore pooled data were used in t h e calculations o f means. table 1 a r t e r i a l blood gases, p h and map before and during b i l a t e r a l c e r v i c a l sympathetic stimulation. pressures are expressed in kpa. mean i sem. control stimulation map pha 11.0 2 0.7 13.7 ? 0.7 4.2 ? 0.2 7.44 i 0.02 11.0 2 0.9 13.8 2 0.8 4.3 t 0.1 7.42 i 0.02 as shown in f i g u r e 1, b i l a t e r a l sympathetic stimulation caused blood f l o w decrements between 60 and 96 % in some extracranial tissues. the most r e a c t i v e tissues were t h e masseter muscle (p<o.ool) and tongue (p<o.ool), while t h e weakest response was detected in t h e p a r i e t a l bone (p<o.ool) and t h y r o i d gland (p<o.ool). the c o n t r o l blood f l o w was in these tissues 1 3 4 ? 38, 66 2 1 9 , 6 2 1 and 33 ? 6 g m i n -1 per 1009, respec tively. a n intermediate e f f e c t o f t h e nerve stimulation was observed i n t h e p a r o t i d gland 187 (p<o.ool) with a baseline blood flow of 63 f 18 g min-l p e r 1009 and in t h e upper incisive (p<o.ool), t h e c o n t r o l blood flow being 15 2 g min-1 p e r 1009. u 0 0 z s p 50 t * * * 25 fig.1. p e r c e n t a g e e x t r a c r a n i a l blood flows during b i l a t e r a l s y m p a t h e t i c stimulation as compared with unstimulated condition. *** p<o.ool. mean f sem. f i g u r e 2 d e p i c t s t h e s t i m u l a t i o n e f f e c t on ocular blood flow and s o m e i n t r a c r a n i a l blood flows. the normal blood flow in t h e r e t i n a w a s 10 2 2, choroid 1067 2 220, iris 8 2 f 1 2 and ciliary processes 123 f 30 mg min-l. the s y m p a t h e t i c stimulation caused a m a r k e d reduction in all p a r t s of t h e e y e (p<o.ool). dural and pineal gland c o n t r o l blood flow w a s 51 7 g min-1 p e r loog and 24 2 4 mg min-l, respectively and d e c r e a s e d significantly (p<o.ool) during n e r v e stirnulation. a 20% d e c r e a s e (p<o.o2) was d e t e c t e d in t h e choroid plexus which had a baseline blood flow of 34 f 4 mg min-l. t 75 b 50 z s u 0 0 25 fig.2. p e r c e n t a g e ocular and s o m e i n t r a c r a n i a l blood flows during b i l a t e r a l s y m p a t h e t i c stimulation as c o m p a r e d with unstimulated condition. (*i ~ ~ 0 . 0 2 , ** ~ ~ 0 . 0 1 and *** p<o.ool. mean f sem. 188 t h e r e w a s no obvious e f f e c t of t h e s y m p a t h e t i c stimulation on t h e t o t a l or regional c e r e b r a l blood flow (table 2). as e x p e c t e d , t h e highest rcbf was found in t h e c a u d a t e nucleus and c o r t i c a l g r a y m a t t e r . c 6 f t o t w a s 57 + 6 during control condition and 58 + 6 g min-1 p e r loog during t h e bilateral s y m p a t h e t i c stimulation. t h e highest stirnulation t o control ratio, 1.13 t 0.09, w a s found in t h e c a u d a t e nucleus and t h e lowest, 0.92 2 0.08, i n t h e hypothalamic region and, 0.92 2 0.10, in t h e cerebellum. table 2 regional c e r e b r a l and spinal cord blood flow before and during b i l a t e r a l stimulation of t h e c e r v i c a l sym a t h e t i c chain. t h e s t i m u l a t i o n t o control blood flow r a t i o is also shown. values in g minp p e r 1009. mean + sem. control stimulation stimulation/control c b f t o t gray m a t t e r white m a t t e r hippocampal region c a u d a t e nucleus t h a l a m i c region hypothalamic region collicles pons-mesencephalon medulla oblongata cerebellum spinal cord (c1-c3) 57 + 6 7 1 + 11 51 2 4 35 + 2 80 + 6 66 + 9 40 2 6 60 2 7 43 2 4 41 + 6 6 1 $ 1 2 21 2 3 58 + 6 74 + 10 47 + 3 35 2 4 90 + 9 64 2 9 36 2 5 59 + 6 45 r 4 40 2 5 52 + 7 22 + 3 1.03 2 0.07 1.10 + 0.10 0.94 + 0.06 0.98 2 0.07 1.13 2 0.09 0.99 ? 0.06 0.92 2 0.08 0.99 + 0.07 1.06 2 0.10 1.01 + o . l l 0.92 2 0.10 1.12 : 0.12 discussion t h e p r e s e n t investigation shows t h a t extraand nonneural i n t r a c r a n i a l tissues are markedly a f f e c t e d by b i l a t e r a l s y m p a t h e t i c stimulation. thus, a l l tissues investigated showed a d e c r e a s e d blood flow during t h e s y m p a t h e t i c stimulation. t h e d e c r e m e n t s e x c e e d e d 50 % in a l l tissues excluding t h e pineal gland and choroid plexus f r o m t h e l a t e r a l ventricle. t h e vasoconstriction in t h e choroid plexus w a s of t h e s a m e magnitude a s t h a t e l i c i t e d by unilateral c e r v i c a l sympathetic stimulation (5). in t h e p r e s e n t study t h e e f f e c t persisted throughout t h e s t i m u l a t o r y period which w a s not t h e case in t h e f o r m e r investigation. t h e r e f o r e t h e b i l a t e r a l stimulation does not s e e m t o b e m o r e 189 e f f i c i e n t i n e l i c i t i n g vasoconstriction b u t probably exerts a more durable effect. the c o n t r o l blood flows o f t h e dura and eye were similar t o those reported b y linder (15), and t h e magnitude o f vasoconstriction was similar t o t h a t during u n i l a t e r a l sympathetic stimulation. taken together these results suggest t h a t b i l a t e r a l sympathetic stimulation has l i t t l e or no additional e f f e c t on tissue blood f l o w as compared w i t h t h e e f f e c t s e l i c i t e d by unilateral stimulation o f t h e cervical sympathetic chain. concerning the cbf, it has been shown that, using stimulus parameters similar t o those in the present study, b i l a t e r a l stimulation o f t h e superior cervical ganglion o f t h e r a b b i t e l i c i t e d about 20 % decrease i n cbf (7). this e f f e c t was stable throughout the stimulatory period which ranged f r o m 1 t o 6 minutes ( 6 , 7). in t h e present study no obvious e f f e c t on r c b f and c b f t o t was observed. however, there were some differences in t h e experimental procedure. in our study preganglionic c e r v i c a l sympathetic stimulation a t 7 h z was used whereas busija stimulated a t a frequency o f 8 h z d i r e c t l y on t h e ganglion. furthermore, our rabbits were anaesthetized w i t h urethane, and pentobarbital-chloralose was used in the other investigation. it is w e l l known t h a t t h e reduction in cbf produced by barbiturate anaesthesia i s greater than t h a t resulting f r o m urethane anaesthesia as compared w i t h the conscious state (11, 16). thus, t h e cbf presented by busija i s much l o w e r than t h a t in the present investigation. the barbiturate anaesthesia may induce a disproportionate r a t i o between cerebral blood f l o w and metabolism which may be influenced by sympathetic stimulation. indeed, a transient e f f e c t o f sympathetic stimulation on cbf has been shown i n the rabbit, and this was a f f e c t e d by pentobarbital anaesthesia (18). on t h e other hand no influence o f the unilateral sympathetic stimulation on t h e cbf, including the escape phenomenon, was detected by beausang-linder and h u l t c r a n t z (5). i t has subsequently been reported t h a t u n i l a t e r a l sympathetic stimulation caused a decrease in t h e cbf in rabbits pretreated w i t h yohimbine (14). thus, an alphaz-mediated mechanism could be involved in t h e e f f e c t s exerted by the sympathetic nerves on cerebral vessel resistance and prolong the transient e f f e c t previously described. i n a single experiment, t h e stimulation duration before microsphere injection was only one minute. n o tendency t o a decreased cbf was found, indicating t h a t an escape phenomenon was n o t o f great importance. in conclusion, profound vascular constriction is e l i c i t e d in a variety o f extraand intracranial nonneural tissues and i n t h e eye during b i l a t e r a l sympathetic stimulation. a n e f f e c t on the cerebral blood flow was n o t detected, indicating t h a t under resting conditions when our experimental procedure i s followed, the b i l a t e r a l sympathetic stimulation does not markedly influence t h e cerebral blood flow during t h e period investigated. 190 acknowledgment i wish t o express m y gratitude t o professor anders b i l l f o r valuable discussions, ms annsofi holst f o r technical assistance, ms monica thore'n f o r drawing the figures and ms ragnhild westerberg f o r secretarial work. this study was supported by grant no. 14x 00147 f r o m t h e swedish medical research council, grant no. roi e y 00475 f r o m the national eye institute, u s public h e a l t h service and by financial a i d f r o m the satra brunn stiftelsen, university o f uppsala. references 1. alm, a.: the e f f e c t o f stimulation o f t h e cervical sympathetic chain on regional cerebral blood f l o w i n monkeys. a c t a physiol scand 93:483-489, 1975. 2. alm, a. & bill, a.: the oxygen supply t o the retina, 11. e f f e c t s o f high intraocular pressure and o f increased a r t e r i a l carbon dioxide tension on uveal and r e t i n a l blood flow i n cats. a c t a physiol scand 84:306-319, 1972. 3. a h , a. & bill, a.: the e f f e c t o f stimulation o f the c e r v i c a l sympathetic chain on r e t i n a l oxygen tension and on uveal, r e t i n a l and cerebral blood f l o w i n cats. a c t a physiol scand 88:84-94, 1973. 4. bill, a. & linder, j.: sympathetic c o n t r o l o f cerebral blood f l o w in acute a r t e r i a l hypertension. a c t a physiol scand 96:114-121,1976. 5. beausang-linder, m. & hultcrantz, e.: early e f f e c t s o f ,cervical sympathetic stimulation on cerebral, ocular and cochlear blood flow. a c t a physiol scand 109:433 437,1980. 6. busija, d.w.: sustained cerebral vasoconstriction during b i l a t e r a l sympathetic stimulation i n anesthetized rabbits. b r a i n res 345:341-344, 1985. 7. busija, d.w.: u n i l a t e r a l and b i l a t e r a l sympathetic e f f e c t s on cerebral blood flow during normocapnia. a m j physioi 250. (heart c i r c physiol 19):h498-502, 1986. 8. edvinsson, l., owrnan, c. & siesjo, b.: physiological r o l e o f cerebrovascular sympathetic nerves in t h e autoregulation o f cerebral blood flow. b r a i n res 117:518 523, 1976. 9. heistad, d.d., busija, d.w. & marcus, m.l.: neural e f f e c t s on cerebral vessels: alteration o f pressure f l o w relationship. fed proc 402317-2321, 1981. 10. koskinen, l.-o.d.: e f f e c t s o f raised intracranial pressure on regional cerebral blood flow: a comparison o f e f f e c t s o f naloxone and t r h on the microcirculation in p a r t i a l cerebral ischaemia. b r j pharrnacol 85:489-497, 1985. 11. koskinen, l.-o.d.: e f f e c t o f l o w intravenous doses o f trh, acid-trh and cyclo(his pro) on cerebral and peripheral blood flows. b r j pharmacol 87:509-519,1986. 12. koskinen, l.-0. & bill, a.: regional cerebral, ocular and peripheral vascular e f f e c t s o f naloxone and morphine in unanesthetized rabbits. a c t a physiol scand 119935-241, 1983. 13. koskinen, l.-0. & bill, a.: thyrotropin-releasing hormone (trh) causes sympathetic activation and cerebral vasodilation in t h e rabbit. a c t a physiol scand 122:127-136, 1984. 191 14. linder, j.: cerebral and ocular blood f l o w during alpha2-blockade: evidence f o r a modulated sympathetic response. a c t a physiol scand 113:511-517, 1981. 15. linder, j.: e f f e c t s o f c e r v i c a l sympathetic stimulation on cerebral and ocular blood flows during hemorrhagic hypotension and moderate hypoxia. a c t a physiol scand 114: 379-386, 1982. 16. linder, j. & beausang-linder, m.: autonomic nervous influence on cerebral and ocular blood flow. abstracts o f uppsala dissertations f r o m the f a c u l t y o f medicine 406, 1981. 17. purves, m.j.: the physiology o f the cerebral circulation. monographs o f t h e physiological society no. 28. cambridge university press, 1972. 18.sercombe r., lacombe, p., aubineau, p., marno, h., pinard, e., reynier-rebuffel, a.-m. & seylaz, j.: is there an active mechanism l i m i t i n g the influence of t h e sympathetic system on t h e cerebral vascular bed? evidence f o r vasomotor escape from sympathetic s t i m u l a t i o n in t h e rabbit. b r a i n r e s 16k81-102, 1979. 19. traystman, r.j.: m i c r o c i r c u l a t i o n o f the brain. in: the physiology and pharmacology o f the microcirculation, vol. 1, (ed. n.a. m o r t i l l a r o ) pp. 237-298. academic press, n e w york, 1983. 20. wagner, jr., h.n., rhodes, b.a., sasaki, y. & ryan, j.p.: studies o f the circulation w i t h radioactive microspheres. invest radio1 4:374-386, 1969. , address f o r reprints: dr. lars-owe d. koskinen department o f physiology and medical biophysics biomedical centre university o f uppsala box 572 s-751 23 uppsala, sweden 192 upsala j med sci 95: 63-74 the influence of body temperature on lkaumatic vasospasm jonas wadstrom and bengt gerdin department of surgery, university hospital, uppsala, sweden the effect of hypothda on traumatically induced sos spasm was studied in campression of a 3.2 rmn segment of the artery for 3 s. ?he internal diameter was continuously measured with the aid of an operating micrcscope during transillumination of the artery. measurements were k q m before spasm induction and continued until the spasm was ocrmpletely resolved. spasm was first induced at nomthennia and then after reduction of the body tempera ture by 1.0"c and 1.75"c. ?he spasm was evaluate3 in terms of its duration, intensity (% reduction of initial diameter) and severity (area under the curve where diameter was plotted against time). ?he results were ccrmpared with those in a control group which was kept nomthermic. reduction of the body temperature caused a significant im=rease in the duration of the spasm andincreased its severity, but did not influits intensity. an in vivo model of the rabbit ear artery. spasm was induced by standanh 'zed microvascular techniques are well established in several surgical disci plines. these procedures can be attended with very specific complications. one of thm is a local increase in vascular tone, which frequently occur^ at the site of a microvascular anastonasis. this sos spasm is sometimes so severe that it can jeopardize the patency of such an anastomosis and the success of the p e e . the pathophysiology of vasospasm occurring after microvasailar procedures is not laxrwn. m e factor which has been suggested as a cause of aggravation of the spasm is local coolirg, this effect of local cooling has been used in an experimentdl model for investigation of vasospasm occutring in connection with mirramscular surgery (10). the iqgrtance of maintaining a normal bcdy 63 temperature during micruvascular surgery in patients in order t o prevent vasospasm has been pointed out by several autho?s (13,16,7,1,2). 'lhis opinion is n o t , however, based on experimentdl o r clinical studies and to our knmledge the effect of general hypothermia on t r a m t i c t so spasm has not y e t been investigated. w e have therefore tested the hypothesis that a decrease in body temperature aggravates traumatically induced ~sospasn. animals en adult long-eared loop rabbits of both sexes, with a body weight of 2.4 4 . 4 kg, w e r e used. they w e r e housed under s'zed e n v i r o m t a l c o d tions with free access t o w a t e r and focd for t w o w e e k s prior t o the expri mts, in accordam=e w i t h the institution's guide for the care and use of laboratory animals. animal metxiration the animals w e r e anesthetized with alphaxolone-alphadolone (althesm, glaxo), 3 w/kg given intravenously and diazepam ( d i a z d s , kabi v i t r u m , ) , 2 mg/kg intramuscularly ( i . m . ) . ixlring this short-lasting anesthesia the sensow nerve running parallel t o the central ear artery w a s cut a t the base of the l e f t ear. ?his made it possible t o perform the rest of the e x p r k t s with only l i g h t sedation, proctuced by 0-2 further injections of 1 w/kg diazepam given intravenously. the rabbits w e r e kept in a specially designed open box. a w a t e r blanket (aquamatic k-20-d, hamilton, c i n c h a t t i , ohio, usa) w a s l a i d over t h e i r backs and was set a t 37°c. the central body temperature was measured continuously with a rectal thenmneter. the expri mtdl set-up is sham in fig. 1. the skin w a s incised along the left e x artery and the w o a d edges w e r e everted with a continuous suture (fig. 1; right inset). the wound w a s moistened w i t h ringer-acetate and covered with p l a s t i c polyethylene f o i l (glad packr, union carbide) in order to maintain a constant environment in the wound. the preparation of the vessel caused an i n i t i a l , transient m s c q a m , which spontaneously resolved i n approxirrately 10 min. to allm the vessel to regain wmplete n o m l i t y a minimum of 30 m h was always a l l w e d to elapse between the i n i t i a l preparation and the conduction of the f i r s t experiment. 64 fig. 1. an artist's view of the e x p e r b t a l set-up. induction of sos spasm vasospasm was induced w i t h a wachenfeldt clipapplyforceps (fig. 2; stille-werner, stockholm, sweden), w i t h an occlusion area of 3.2 x 1.4 rmn = 4.5 rmn2. a t rest it is cl& and exerts a standardized canpression force of 2.45 n a t an area of 4 . 5 m2, i.e. a pressure of 5.45 x lo5 n/m2, between its claws. the ear artery was pinched w i t h the f o r c e p for 3 s. a f i r s t pinch was made on t h e d i s t a l part of the ear, and two further pinches w e r e made p roxifal t o the f i r s t one. measurement of the inner diamter !ihe ear w a s fixed w i t h needles to a specially designed cork board which allowed t h e c e n t r a l ear artery to be transilluminated w i t h cold light abare w i t h an operatmicroscope equipped w i t h a 25 x objective w i t h a focal distance of 100 rmn and a jvc video color camera. the inner diameter of the a r t e r y w a s measured directly on the video monitor. the resolution of the set-up w a s determined a f t e r calibration w i t h a l e i t z m i a x m e t e r ruler and found to be 0.01 rmn. 'ihe f i r s t measutement w a s made in the undistuked (intralux-500, volpi ag, switzerland). the artery w a s then hspe&ed from vessel, whereafter spasn was induced and measulpments w e r e r e p t e d every min 5-908571 65 u n t i l no visible spasm remained, i.e. when there was no narrowing of the traumatized segment compared with the neighbornontraumatizd vessel -t. fig. 2. a schematic view of the wachenfeldt clipapplying forceps. experiments a. exprimentdl group: n=5. one episode of spasm was f i r s t induced a t no& body temperature. the core temperature w a s then lwerd by applying a p l a s t i c bag f i l l e d with ice cubes t o the back of the animal. t h i s 1cwe.mil the body temperature a t a rate of 1.8"c per hour, a t which rate the diameter of the artery m i n e d unchanged. when the core tenperahre had fallen by close t o 1.0 and by 1.75"c episodes of spasm were again induced. blood smples for determination of serum cortisol w e r e taken before reduction of t h e body temperature and just before the f i r s t induction of spasm after the cooling. they were irmdiately centri fuged, frozen to -70°c and l a t e r analyzed with an fua method. b. control group: -5. these experiments w e r e performd as in the experimental group, but the animals w e r e kept nomthermic throughout the study period. 66 variables the fo1lawi.q variables w e r e measured or c a l d a k d and are depicted in fig. 1. %: the initial inner diameter of the vessel. q: the inner d i a m e t e r during imximdl spasm. i+: the findl inner d i a m e t e r when the spasm was resolved and when the inner diameter of the t r a u m a t i z e d vessel segment w a s the same as that of the adjacent vessel segment. to: the time of removdl of the forceps fm the traumatized vessel. %: the earliest time of maximdl spasm; q. +: the duration of spasm; the time of dp. the follawing four variables w e r e thereafter used in the descriptive and statistical andlysis of the vascqam. 1. !the initial .. temperature. i %i before and after reduction of the boay 2. !lk duratiopl of the spasn, tp 3 . ‘ihe intensity of the spasn, given i n percent and defined as ( b 4 4 1 / 9 x 100 4. !the sxerity of the spasn, calculate3 as the area d x t, defined as the integrated decrease in d i a m e t e r f m to t o tf (shaded area in fig. 3 ) . diameter d i d m i i i i i i i i i i i i i t t o m time t f d f fig. 3 . a graph in w h i c h the diameter is plotted over time. the different variables are defined in the materials and methods section. 67 statistical evaluation m e difference between the first, controle spasm and each of the two experi mental spasnrs w a s calculated for each animal, both in the experimental and the control group. the two groups w e r e then campared r q a r d i q these diffe remes by student’s two-sample t test. all data are given as mean ? sem. a difference at the 5% level is regarded as significant and is indicaw by an asterisk in the figures. 39 p t! 5 8 9 ) c 38 9) 4 3 37 0 pi 36 the experiments the rabbits w e r e very calm and relaxed in their boxes and the e x p e r k t a l set-up did not appear to disturb the awake rabbit to any appreciable extent. ?he k d y temperatures of all animals at the different measurment times are shm in fig. 4 . 6 a 1 i t i a 6 1 t 0 1 i i11 experiments 0 cooled a control fig. 4 . body tenperatwe, in dqrees celcius at the the of the experi me.nts in the experimentdl and control groups. the temperature derreased by close to l.o(degrees celcius) in the experiment 2 and 1.75(degrees celcius) in experiment 3 . n=5 in a l l experi ments. 68 at the first level there was a decrease in body tenperature varying from 0.8 to 1.1"c and at the second level the body temperature had dec=reased by 1.1 2.0"c. ?he expcsed left centml ear a r t e r y d d be readily observed with the cold light transillumination. in the vessel the outer and inner diameters were very similar (fig. 5 a), but after induction of t so spasm these diameters w e r e easily separable (fig. 5 b). after a 3 s pinch with the forceps, the central artery reacted with vasospassn. ime constricted sqmnt was limited to the pinched area. spasm w a s maximal within 1 min ard the inner d i a ~ t e r was decreased to approximately 15% of the original value. 'ihis intense constriction remained virtually stable for 5 mint whereafter it slowly resolved. fig. 5. 'ihe appearance of an u n d i s m (a) and a spastic (b) vessel sefpnent. ?he arruws indicate the border of the tunica nwcularis. 69 the internal diameters before induction of sos spasm were similar in the two groups and at all tins (fig. 6). hypothennia caused a significant in the duration of spasm, whereas the duration was similar in the three' different experiments in the control group (fig 7) . furthermore, the severity of the spasm w a s also significantly bcreasd in the hypothennic animals ccwpared with the controls (fig 8 ) . the intensity of the spasm, i.e. the relative decrease in diameter, remained unchangd d u r i n g hypothda (83.8 +5.3% at -1.75"c) ccanpared with the value in the control group (85.8 f3.3%). the serum cortisol levels were not affected by the ice cooling of the animals( 282 f27 ml/l and 286 ? 29 ml/l after). l.ooo 1 0.800 n e! el 0.600 w k a 4 0.400 ;a a 0 t a 6' 1 0 cooled a control t t t 0 1 normo 0.000 j thermia 1 .o"c 1.75"c fig. 6. the internal diameter before induction of spasm. the boay temperature in the cooled group w a s reduce3 by close to 1.0 and 1,75"c i n experiment 2 and 3, respectively. the control group was kept mnwthennic in all three experiments. n=5 in all experi mts 70 35 30 25 20 15 10 5 0 fig. 7. 20 15 10 5 0 fig. 8. t 1 a o t t * 0 1 i t * 0 0 cooled a control a noitx~o1 .ooc 1.75"c thermia ?he duration of spasm. the body temperature in the cooled group was reduced by close to 1.0 and 1.75-c in expriment 2 and 3, respectively. the control group was kept nomthermic in d l three experherb. n=5 in a l l e x p e x k t s . 0 cooled a control t * 0 1 a t * 0 1 a normo1 .ooc 1.75oc thermia the severity of spasm. the boay temperature in the cooled group was reduced by close to 1.0 and 1.75"c in experiments 2 and 3, respectively. the control group was kept nomthermic in all three -bents. n=5 in all experiments. 71 discussion i n this study w e have shown that general h p t h d a aggravates t r a m a t i c vasospasm i n the central ear artery of the rabbit. it has been postulated on theoreticdl grounds that the rabbit ear is a specialized organ f o r tempera ture control (15,14) . this has been studied specifically by h i l l et 61. (6) , who found, however, that a t l o w ambient temperatures there was a n e t heat loss, an3 that a t high temperatures there w a s a n e t heat influx, demon strating that the rabbit ear is not a special organ for temperature control. we therefore believe that the results of our studies on the rabbit ear artery can be generalized to most vascular beds. there are several possible ways i n w h i c h the body temperature might influence t h e duration and s e v e r i t y of vasospasm. v e s s e l contraction induced by reduction of the core and local temperature is known t o be under sympathetic c o n t r o l ( 4 , 1 2 , 1 7 , 9 ) . themdyrmu ' c studies of the noradrenaline receptor binding have sham a higher a f f i n i t y for the receptor a t lower temperatures (11) and that the maximal response to adrenaline and noradrenaline in the rabbit ear artery occurs a t a temperature close t o 25°c ( 8 ) . there is also an increase i n the myqenic tone, w h i c h is temperature4eperdent. this l a t t e r increase seem t o be independent of the adrenergic response but is ca2+ dependent (5,3). these and other mechanisms may contribute t o the aggravated response of traumatically induced vasospasm observed i n the present study. since the i n i t i a l diameters of the vessels w e r e similar a t norm1 and reduced core temperature and since the serum cortisol levels remained unchanged, activation of the sympathetic response is probably not sufficient to explain the results. it is therefore possible that the myqenic temperature depen dence is an important mechanism underlying the aggravation. this question cannot be answered w i t h certainty, however, on the basis of our results. however, the present study has unequivocally shown that even a very s m a l l reduction of the core temperature aggravates t r a m t i c a l l y induced vasospasm. this stresses the importance of careful control of the body temperature i n c l i n i c a l micsrosurgery. the authors are very grateful t o mr. roland pettersson, b.sc., department of b i o s t a t i s t i c s , pharmacia ab, for help w i t h statistical mtters and to m s . gunnel fredrickson for technical assistance. 72 references 1. asp, c., cox, r.g., mayou, b.j. & sengupta, r.: the role of anaesthetic managemnt in enhanci.r~~ peripheral blood flm in patients underyoing free flap transfer. ann. r. all. sung. 67:177, 1985 2. bird, t. m. & s t r u n i n , l.: anaesthetic considerations for microsurgical repair of limbs. can. anaesth. scc. 31:51, 1984 3. bevan, j. a . : vascular myogenic or stretch-depm3ent tone. j. cardiovasc. pharmawl. 7 (supp 3) :s129, 1985 4. grant, r. t.: further observations on the vessels and nerves of the rabbit ear, w i t h special reference to the effects of denervation. c l i n . sic. 2:1, 1935. 5. mker, c. t. & vanhoutte, p. m.: cooling the central ear a r t e r y of the rabbit:lpdrenergic responses. j. pharmacol. j 3 p . therapeut. 245:89, 1988 6. hill, r.w. & veght, j. h.: jackrabbit ears: surface temperatures and vascular responses. science. 194:436, 1976 7. mcdondld, d.j.f.: anaesthesia for m i m a r q w . br. j. anaesth. 57 : 904 , 1985 8. patton, n. j. & wallace, w. f. m.: responses of isolated rabbit ear arteries to intraand extraluminal l,and ~-noradrexdine, badrenaline and histarnine in the temperature range 37-3 "c. irish j. med. sci. 147:313, 1978 9. phelbes, d. b., mtherford, r. b. & boswick, j. a . : control of sos spasm following trauma and microvascular sung-. 4: 109 , 1979 10. fucket, c.l., winters, r.r.w., g r e w , r.k. & goebel, d.: studies of pathologic vasoconstriction sosp spasm) in micravascular surgery. j. hand. sury. 1oa: 343 , 1985 11. raffa, r. b., aceto, j. f. & tallarida, r.j.: -t of t h m dynamic parameters for norepinephrine contraction of isolated rabbit thoracic aorta. j pharmawl. e q . the.rapeut. 235:596, 1985 12. ridel, w . , iriki. m. & s h n , e.: regional differentiation of sympa thetic activity during peripheral heating and cooling in anesthetized rabbits. pfluegers arch. 332:239, 1972 13. fbbins, d.w.: ?he anaesthetic management of patients u n d e q o ~ free flap transfer. br. j. plast. sury. 36:231, 1983 14. schmidt-nielsen, k.: desert animals. bmdon. oxford univ. press, 1964, pp. 133-38 15. schmidt-nielsen, k., dawson, t. j., hammel, h. t., hinds, d. & jackson, d.c.: the jack rabbita study in desert survival. hvalsdets skr. 48:125, 1965 16. shaw, w.w. & hidalgo, d.a. : mi~.osurgery in trauma. new york. f u h u a publishing company, inc., 1987, p. 39 73 17. walther, 0. e., iriki, m. & simon, e.: antagonistic changes of blood flm and sympathetic activity in different mscular beds following central thenml s t h u l a t i o n . pf1uger-s a r c h . 319:162, 1970 address for reprints: jonas wadstrcm, md urnversity h o s p i t a l 5-75185 -la, sweden -t of surgery telephone +46-18-663000 fax +46-18-693606 74 upsala j med sci 95: 229-232, 1990 quality specifications in laboratory medicine c. g. fraser department of biochemical medicine, ninewells hospital and medical school, d u n d e e , scotland since the aims of the nordkem project are: i. to develop a practical procedure for assessing quality 11. to apply this procedure to a selected number of analytes 111. to use the requirements in design of quality assurance requirements, (one at a time) , and procedures, the real crux of the project is the delineation of numerical sualitv croals. the project plans suggested that the first two steps in the definition of quality specifications are to: i. appraise available formulae and then 11. apply these to calculate allowable limits of error. the next step would be to apply or develop other more sophisti cated methods. the third step is the most exciting, in my view. it may be relevant to consider that most of the available formulae have major disadvantages (1). traditionally, goals have been based on: i. fractions of the reference interval (tonks' rule), 11. the opinions of clinicians (barnett's medically significant cv) i 111. the state of the art, iv. views of individuals and groups and v. biological variation (harris's postulate). the first two are very widely used to this day but, i. reference values depend on performance characteristics, population studied and data reduction technique, and the fraction used is empirical, and 11. use of opinions of clinicians as to change satisfies only 229 0.50 of respondents, changes are due to pre-analytical and biological as well as analytical variation and the changes required are thought always to be necessary for p < 0 . 0 5 . in the past, we in dundee have given much support to the postulate of harris that cvanalytical <1/2 cvbiological. the approach is simple to understand. many data on within subject biological variation are available. the estimates seem independent of age of subjects, number of subjects studied, geography, time scale of study, methodology and whether the subjects are healthy or have stable disease ( 2 ) . the model seems applicable in haematology ( 3 ) and to generate a way of defining goals for therapeutic drug monitoring (8). however, it has been more and more realized that the model has some disadvantages. use of the fraction 112 is somewhat empirical and simply means that, if the goal is achieved, then 10% is added to test result variability due to analytical variability; this may be inappropriate in some clinical situations. moreover, certain goals derived by strict use of the model are unattainable in correct practice and unlikely to be achievable in the near future. some new approaches would be advantageous. in the last few years, further thoughts on defining quality goals have been published. these can be classified as: i. repetitions of previous suggestions, 11. further empirical ideas, and 111. goals based upon clinical situations. the third group includes suggestions made by the nordic group ( 5 ) , harris ( 4 ) and ross (7). these are concerned with tests used for the monitoring of patients. it is generally agreed that this requires the most stringent analytical quality and, therefore, the strictest goals. the nordkem project clearly accepts the principle that the most demanding goal should be applied ubiquitously and this is to be commended. it is considered that the approach proposed for the nordkem project is correct. the clinical approach should be used but with within-subject biological variation brought into consideration. the paper by hyltoft petersen et a1 (6) provides a model which is believed to be worthy of further study and emulation. for some time, it has been suggested that goals should be 230 developed for performance characteristics other than imprecision and inaccuracy. the pressing need is for goals for detection limit, and the project may facilitate development of these using models similar to that described above. the most difficult problem (but pressing need) is for us to be able to gain objective knowledge on clinical decision making in real medical situations. if such information was available, current theories and knowledge on analytical and variation are sufficient to enable relevant quality goals to be set. then, appropriate quality assurance techniques could be invoked. references: 1. 2. 3. 4 . 5 . 6. 7. fraser cg. disirable performance standards for clinical chemistry tests. adv clin chem 1983;23:299-339. fraser cg, harris ex. generation and application of data on biological variation in clinical chemistry. crc crit revs clin lab sci 1989; 27~409-437. fraser cg. desirable performance standards for hematology tests: a proposal. am j clin pathol 1987;88:667-669. harris ek. proposed goals for analytical precision and accuracy in single-point testing; theoretical basis and comparison with data from the college of american pathol ogists proficiency surreys. arch pathol lab med 1988;118:416-420. hsrder m, ed assessing quality requirements in clinical chemistry. scand j clin lab invest 1980;40, supplm 155. lytken larsen mi fraser cg, hyltoft petersen p. a compari son of analytical goals for haemoglobin alc assays derived using different strategies. ann clin biochem 1991 (in press). ross jw. a theoretical basis for clinically relevant proficiency testing limits. arch pathol lab med 1988;118: 421-434. 23 1 8. stewart mj and fraser cg. desirable performance standards for assays of drugs. ann clin biochem 1989;26:220-226. correspondence: dr cg fraser, department of biochemical medicine, ninewells hospital and medical s c h o o l , dundee dd1 9sy, scotland. 232 upsala j med sci 99: 271-276, 1994 5.5 evaluation of target values for al-antitrypsin ole blaabjergl, thea kragh olesen2, per hyltoft petersenl, 1. department of clinical chemistry, odense university hospital, dk-5000 odense c, denmark. 2. department of clinical chemisiry, aalborg sygehus, dk-9100 aalborg, denmark. in section 5.4 a discrepancy was disclosed for cxl-antitrypsin regarding the transfer of concentration values from the ifcc reference preparation (crm 470) to the nordic calibrator between the nephelometric method (bna, behringwerke) and the turbidimetric methods (three cobas fara, roche, and one hitachi 911). this difference, about 20 %, could not be neglected and further investigation was therefore needed. differences in the measurement procedures the main difference in the measurement procedures is the analytical principle, nephelometric and turbidimetric for the bna and the other instruments, respectively. both calibrators (crm 470 and the nordic calibrator) are, however, measured in the same runs (in several dilutions and in replicates) so, the difference must be related to the structure of al-antitrypsin in the two calibrators. according to the ifcc-protocol for value transfer antisera from behringwerke are to be used for the bna-measurements and antisera from dako for the other instruments. the possible reasons for the difference are * analytical principle * antisera q structure of al-antitrypsin in the two calibrators therefore, a comparison to fresh human sera was decided with a design where both calibrators were measured according to the two analytical principles using both antisera. 27 1 10, 5 0 -5 fara measured on cobas l nordic calibrator -if i $ 1 1 i 1 i i a fresh serum 20 15, 10 5 on bna nordic calibrator measured 1 0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 dilution 4 b 0 - -5 -10 fig. 5.5.1 differences i n concentrations of al-antitrypsin measured with antisera from behringwerke and from dako {a = conc(beh1 conc(dako}} of dilutions of a fresh human serum (e) and of the nordic calibrator (m), using crm 470 as primary calibrator, plotted as function of dilution. 90 % confidence intervals are indicated. upper: measurements on cobas fara lower: measurements on bna -i 0 i 1 a -.. 41 fresh serum -l 41 272 materials and design of investigation calibrators: crm 470: collected from february to september 1990, lyophilized in june 199 1. the nordic protein calibrator: collected from august to december 1987, ultracentrifuged from february to september 1989, alliquoted in november 1989, stored at 80 " c . date of investigation: 25.-26. august 1994. fresh h u m a n sera: ten fresh sera from healthy volunteers. a fresh serum 'pool', from one healthy volunteer, type mm. instruments: design: bna nephelometer (behringwerke) and cobas fara (roche). both calibrators and the ten fresh sera and the pool were analysed on both instruments and with both antisera in a total of four runs within the same day. the two calibrators and one fresh serum were determined in six dilutions according t o the ifcc-protocol (i.e. that the weight of each dilution step was controlled), and crm 470 was used as the primary calibrator. the fresh pool and the rest of the sera were determined in one dilution. the four runs gave four independent calibration curves. blank reaction: an independant control run with a non-immune gammaglobulin preparation (dako) was performed on both instruments. correction: in the bna-measurements the results for the lowest dilution deviated from the remaining dilutions and these values were omitted. results in fig. 5.5.1 the effect of antisera on measurements of the concentrations of al-antitrypsin in a fresh human serum and the nordic calibrator (using crm 470 as the primary calibrator) are shown as function of the dilution for the two instruments. the ordinate is the difference between concentrations measured by the two antisera in percentage. for the cobas fara (upper figure), the effect of antiserum is small. the effect on the two materials, however, is negative (approx. 2 5%) for the fresh pool and 273 positive (approx. 3 %) for the nordic calibrator. the results from measurements on the bna-instrument (lower figure) show more pronounced differences between the two materials. whereas, the effect of antiserum is negligible (but variable) for the fresh serum, the effect on the nordic calibrator is increasing from negligible to approx. 15 %. for the fresh sera and the fresh pool the results of measurements of a,-antitrypsin (from one dilution) are illustrated in fig. 5.5.2. here, results from both instruments are shown in one figure. on the cobas fara the differences are distributed about zero, and on the bna the differences are from 1 t o + 5 % (mean of approx. 3.5 %). difference (abbeh-abdako) percentage of concentration 10 _1 -5 o l measured on bna -i== i t t me.4sured on cobas fara 100 125 150 7 5 concentration in percentage of crm 470 fig 5.5.2. difference of al-antitrypsin i n fresh sera measured with antisera from behringwerke and dako {a = conc(behring) conc(dako)/’ (as percentage of concentration) measured on b n a (*) and on cobas fara (w), using crm 470 as primary calibrator, and plotted as function of concentration. 90 76 confidence intervals are indicated. discussion evaluation of the results may be a little complicated, but it is clear that the ’signals’ from measurements of al-antitrypsin in crm 470 and the fresh sera and pool are very close in all the combinations with maximum deviations of 5 %. this means that the 274 structure of the protein in crm 470 is very close to the genuine as judged from the antiboles in the two antisera. minor differences can, however, be seen as measurements on the bna of fresh sera results in a difference of approx. 3.5 % when the two antibodies are used. regarding the nordic calibrator, the deviations from the fresh sera is approx. five percent on the cobas fara and variable (up to 15 percent) on the bna. the changes in structure of the protein, therefore, seem to be more pronounced or of an other type, which is disclosed by the two antisera, and t o a greater extent by the nephelometric analytical principle. the changes in al-antitrypsin structure is not seen in the electrophoretic mobility as demonstrated in section 5.3, or in loss of protein, as the concentration in the calibrator is close to the mean value of the reference interval (even the distribution is log gaussian). the changes seem t o be related to some steps in the preparation procedure as a hint was given by preparation of a new calibrator, where the serum pool by accident was filtered after the ultracentrifugation. in this preparation the effect is doubled, with deviations from fresh sera from zero up t o approx. 30 percent (results not shown). it has not been investigated whether commercial calibrators have the same problems, but evaluations of a series of these (cf. section 5.3) may indicate serious problems for al-antitrypsin in these preparations. with the description of the production of crm 470 (cf. sections 5.1 and 5.2 and ref. l), however, the means for producing reliable commercial calibrators are given. the described deviations have some impact on the estimated reference intervals for s-al-antitrypsin: * the reference intervals (cf. chapter 7) are correct for turbidimetric methods using the nordic calibrator and antisera from dako in direct relation to crm 470, i.e. used as calibrator for the actual measurements, the reference intervals are valid as well for measurements on bna with antisera from behringwerke, and by use of the nordic calibrator, the concentration value of s-al-antitrypsin for the calibrator should be approx. 15 % lower for all other calibrators the validity should be tested in direct relation to crm 470 and fresh human sera. * * * 19 9 5 5 0 5 9 275 these results disclose problems with al-antitrypsin in the nordic calibrator and in the majority of commercial calibrators as well, resulting in restrictions for the use of the common reference intervals. conclusions due t o the problems with the structure of al-antitrypsin in the nordic calibrator the estimated reference intervals for this protein are only valid by use of the nordic calibrator, or by direct investigation of the secondary (commercial) calibrator in relation to crm 470 and a number of fresh human sera. acknowledgements we want to thank marianne kaehne for her skilful analytical work. references 1. whicher j t , ritchie rf, johnson am, baudner s, bienvenu j, blimp-jensen s, carlstrom a, dati f, milford-ward a, just svendsen p. new international reference preparation for proteins in human serum (rpphs). cein chem 1994;40934-8. 276 upsala j med sci 91: 223-224, 1986 new seralyzer compared to routine techniques in clinical chemistry bertel berg, bengt olsson and nils tryding department of clinicaf chemistry, central hospital, kristianstad, sweden new seralyzer (ames) is a reflectometer designed mainly for serum and blood analyses with solid-phase chemistry (reagent strips). we have compared seralyzer with the routine methods of our hospital labora tory for 10 components, especially the two recently introduced tests for potassium and theophylline. serum and blood samples were taken at random from the routine series f o r blind analysis with the new seralyzer using well-trained personnel. conclusion: under optimal conditions the new seralyzer instrument gave good accuracy and precision for s-potassium and s-theophylline. ( s e e figures) f o r the other components results were often good, but sometimes accuracy and precision problems occurred (especially for serum enzymes). ( s e e table) 1. s-k seralyzer eppendorf 2. s-theophyuine seralyzer cobas bio "-140 y i 01 x f0.09 r-0,957 "-34 y i 00 x + 3 0 r=0.994 223 ne w se ra ly ze r (s l ) co mp ar ed t o ro ut in e te ch ni qu es ( rt ) in c li ni ca l ch em is tr y co mp on en t n ro ut in e te ch ni qu e co nc en tr at io n y= a+ bx cv cv cv (a t) x= rt be tw ee nme th od wi th in -m et ho d qu al it y co nt ho sp it al l ab or at or y y= se ra ly ze r rt /s er al yz er rt tr ol p ro gr am fo r we st er n sw ed en sk+ sth eo ph yl li ne scr ea ti ni ne 5as at sbi li ru bi n sck bhe mo gl ob in sch ol es te ro l 14 0 34 13 8 28 39 26 89 41 fl am e ph ot om et ry e m 1 t ja ff 6 ki ne ti c sc e di az o me th od sc e co ul te r s + ch ol es te ro l ox id as e me di an a 41 mm ol /l 0. 09 55 p mo l/ l 3. 0 95 p mo l/ l -6 .3 (> 0. 6) l. lk ka t/ l 0. 09 12 k mo l/ l -2 .0 1. 5 pk at /l -0 .0 3 13 5 g/ 1 0. 54 6. 2 mm ol /l 1. 21 b 1. 01 1 . oo 1. 12 0. 95 1. 07 1 . i6 0. 97 0. 89 r 0. 95 7 0. 99 4 0. 98 5 0. 98 6 0. 97 0 0. 99 1 0. 97 5 0. 92 1 3. 7 6. 3 8. 5 8. 2 11 .7 14 .9 2. 7 1 .i me di an me di an 1. 2 2. 1 3. 1 -_ _ 2. 7 5. 4 2. 7 5. 4 2. 8 6. 7 2. 8 13 .2 0. 83 2. 1 2. 6 3. 7 str ig ly ce ri de s 41 gl yc er ol k in as e 1. 9 mm ol /l 0. 07 1. 05 0. 99 3 6. 0 2. 8 7. 3 sur at e 39 ur ic as e 37 0 km ol /l 30 0. 89 0. 98 2 4. 9 2. 0 5. 2 upsala j med sci 100: 125-1 36, 1995 effect of indomethacin on thrombin-induced pulmonary edema in the rat chul min ahn,1,3 h a a n sandler,i thomas wegener2 and tom saldeenl depurtments of 'forensic medicine and 'lung medicine, uppsala univrrsit?;, u p p a l a , sweden. 3department of' internul medicine, yunsei universit?; college of medicine, seoul, korea abstract the preventive effect of indomethacin on thrombin-induced pulmonary edema was studied in rats. administration of thrombin caused a significant increase in lung weight, wet weight to dry weight ratio (wwdw), and relative lung water content. during infusion of thrombin, mean pulmonary arte ry pressure rose and mean systemic artery pressure fell, pa02 decreased progressively and there was a continuous rise in ph and pac02. an inhibitor of cyclooxygenase, indomethacin, at a dose of 1 m g k g body weight, induced a signifi cant further increase in lung weight (p<0.05), and a tendency towards an increase in ww/dw and water content compared with animals given thrombin alone. treatment with indomethacin, however, counteracted the elevated pulmonary artery pressure occurring in the early phase after thrombin infu sion, but not that in the late phase. systemic artery pressure was not affected by indomethacin. the increases in ph and pacoz after thrombin infusion were attenuated and remained stable almost at baseline level after indomethacin administration. tndomethacin did not prevent the hypoxemia indu ced by thrombin infusion. in conclusion, although indomethacin prevented the early increase in pulmonary artery pressure due to thrombin and the decrease in ph and the increase in pac02, it caused lung vascular permeabili ty to protein to increase more than with thrombin alone. introduction in both the dog ( 2 ) and the rat (6), pulmonary microembolism induced by infusion of thrombin during inhibition of fibrinolysis has been found to induce pulmonary insufficiency with similarities to the cli nical adult respiratory distress syndrome. infusion of thrombin results in systemic hypotension, acute pulmonary hypertension, and increased pulmonary vascular permeability to protein with subsequent pulmonary edema (12). the pathophysiology of thrombin-induced microembolism seems to involve the activity of cyclooxygenase-derived arachidonate metabolites (3). evidence for this assumption is mostly based on studies using various cyclooxygenase inhibitors. among these arachidonate metabo 125 lites, thromboxane is known to be associated with sequences of events that follow infusion of thrombin in the rat (20), and with the development of thrombin-induced pulmonary edema in sheep (8,9). lncre ased permeability to protein may also result from entrapment of fibrin (6) and leukocytes (2 1 , s ) in the lung. the cyclooxygenase inhibitor indomethacin is known to inhibit prostaglandin synthesis (24) and the release of thromboxane a2 in guinea pig lung parenchymal strips (26). it also inhibits several leu kocyte activities in vitro such as neutrophil migration (4), aggregation (13), and adherence (251, and also the myeloperoxidase-h~02-c1 system of neutrophils (22). the aim of the present study was to test the effects of indomethacin on thrombin-induced changes in pulmonary artery and systemic artery pressure and in arterial blood gas exchange variables, and on pulmonary edema induced by thrombin, in rats. materials and methods animals fifty-two male sprague-dawley rats (alab, stockholm, sweden) were used. they were divided into three treatment groups: saline controls (n=8), thrombin plus amca (n=21), and thrombin plus amca plus indomethacin (n=23). all rats weighed between 200 and 250 g and had free access to food (ewos rat pellet) and tap water. mate rials bovine thrombin (topostashe') was kindly supplied by hoffman la roche, switzerland. it was dis solved in physiological saline to aconcentration of 100 iu/ml, and kept at 20' c until used in theexpe riment. the fibrinolytic inhibitor tranexamic acid (trans-4-aminomethyl-cyclohexane-carboxylic acid, amca) was purchased from kabi, stockholm, sweden. indomethacin (confortid') was suppli ed by dumex ais, copenhagen, denmark. pentobarbital (inactin') was obtained from byk-gulden, frg. s-2238, a chromogenic substrate for detection of antithrombin activity, was obtained from kabi, stockholm, sweden. metliods the rats were anesthetized intraperitoneally with 125 m g k g of pentobarbital. they were placed in a supine position and tracheostomized. a tracheal cannula (pe 240, clay adams, becton dickson & co., usa) was inserted for airway support and an abdominal cannula (portex; outer diameter 0.80 mm, hythe, kent, england) was inserted into the peritoneal cavity for amca injection and maintenance of anesthesia. all rats breat hed air spontaneously through a tracheostomy, and the body temperature was kept constant at 38" c with an electric pad throughout the experimental period. intravenous injection sites were prepared in one of the saphenous veins, using polyethylene cathe ters (portex; outer diameter 0.80 mm, hythei, kent, england) and 26 gauge needles for injection of indomethacin and thrombin. indomethacin was dissolved in sterile distilled water to a final concentra 126 tion of 2 mg/ml. the solution was slowly administered through a saphenous vein 30 min prior to the infusion of thrombin. pulmonary microembolization was induced essentially as described previously (16) and modified as described below. in brief, 15 rnin prior to thrombin administration rats were injected through the abdominal catheter with 200 m g k g of amca to prolong fibrin entrapment in the lungs and increase pulmonary damage (2). three hundred sixty i u k g of bovine thrombin was injected manually by the intravenous route over a period of 10 min, using a stopwatch and a 1 .o ml disposable syringe. the doses of indomethacin and thrombin used in this study was based on the findings in a preliminary study, in which animals treated with indomethacin did not tolerate the thrombin dose used in previous studies (500 iukg). the throrn bin dose was therefore reduced to 360 iukg. the rats were killed 90 min after termination of thrombin infusion and the lungs were excised, gently cleaned with gauze, weighed, and dried at 37' c in a warm incubator for approximately 72 h. the lungs were reweighed until their weight was constant. calculation of wet weight to dry weight ratio ( w w d w ) and water content the relative lung water content was calculated in the left lung as described previously (17). water con tent (%) = ww d w / w w x 100. pressure measurements pulmonary artery pressure (pap) was recorded via a silasticr catheter (dow corning co., usa; outer diameter 0.025 inch) inserted into the right jugular vein and then gently advanced to the pulmonary artery. systemic artery pressure (sap) was recorded via a polyethylene catheter (pe 50) inserted into the femoral artery. the catheters were connected to statham transducers and the pressure tracing was performed throughout the experimental period. the position of the catheters was checked from the pressure tracings and confirmed when the rats were killed. rats in which catheters were not properly positioned were excluded from further analysis. arterial blood gas studies blood samples of 100 pl were drawn into heparinized microcapillary tubes from the femoral artery and analyzed immediately with a blood gas analyzer (instrumental laboratory system 1302, italy). the analyses were carried out at 37' c. antithrombin activity antithrombin activity was measured in vitro by means of a chromogenic substrate, s-2238. one vial of s-2238 was dissolved in 15 ml sterile water to a concentration of 0.75 nmovl and 50 pl+ of test plasma or standard was diluted with 3.0 ml working buffer solution. bovine thrombin 53(nkat) was reconstitu ted with 1.5 ml sterile water. at assay, 400 pl of diluted test plasma or standard, and indomethacin in different concentrations were incubated at 37' c for 3-6 min. then 100 pl of thrombin was added and incubated at 37' c for 30 min. s-2238 was thereafter added, mixed well, and incubated at 37o c for exactly 30 s. the reaction was stopped by addition of 300 pl of 50 % acetic acid, which was mixed 127 immediately. the absorbance of the sample was read against distilled water in a photometer at 405 nm. statistical analyses statistical analyses of differences between groups were performed with the wilcoxon-white two sample ranks test. two-way analysis of variance was used to determine the significance of changes in mean pap (mpap), mean sap (msap), and arterial blood gas exchange from the baseline value after infusion of thrombin. the baseline value was the value just prior to thrombin infusion. student’s unpaired t test was used for comparisons between groups at equivalent time periods. a p value of < 0.05 was conside red significant in all cases. results changes in lung weight, ww/du: and water content time schedule for experiments time schedule (min) -30 -1 5 0 10 100 saline amca thrombi n sacrifice 200 mg/kg 360 iu/kg i .v. i.p i .v. lndomethacin amca thrombin sacrifice 1 mg/kg 200 mg/kg 360 iu/kg i.v. i.p. i .v. the experiment was performed on 23 rats according to the protocol shown in figure i . the results are presented in figure 2 . rats injected with thrombin + amca showed a significant increase in lung weight, ww/dw, and water content. rats pretreated with indomethacin had a higher lung weight than those not treated with indomethacin (p<0.05). w / d w and the water content in the indomethacin treated rats were slightly higher but not significantly different from those in rats given thrombin alone. 128 3 2.5 2 a v 01 e m 1.5 p = -i 1 0.5 0 lung weight 10 9 6 7 * # 6 3 g 5 4 3 2 1 0 ww/dw * * t water content * * @ saline wntrd saline &thrumbin lndo ((i thrombin fig 2. preventive effect of indomethacin, 1 mgkg, on changes in lung weight, wwldw ratio, and relative lung water con tent induced by thrombin inhsion in rats. * indicatesp<o.o5 vs saline control group. #denotes p<o.oscompared toanirnals given thrombin & amca alone. mean * s.d. hernodynamic changes the percent changes in mpap and msap are illustrated in figure 3. the infusion of thrombin resulted in a large increase in pap (by 87+23 %). the values then fell progressively during the remainder of the experiment. the increase in pap after thrombin infusion was diminished by pretreatment with indo methacin (p<0.05). five and 15 min. after completion ofthrombin infusion these differences were sta tistically significant. thrombin produced an immediate fall in msap. the decreased arterial pressure after thrombin infusion had slightly recovered 5, is, and 30 min after completion of thrombin infusion, but began to decrease again after 30 min and had fallen by about 30 % from the baseline at 90 min. treatment with indomethacin showed a tendency to counteract the thrombin-induced fall in arterial pressure, but the difference was not statistically significant. changes in arterial pressure after thrombin infusion, considered for the whole experimental period, were not affected by treatment of indometha cin. 129 m p a p * t; 90 0 1015 25 40 m 100 min * i i i i i i i i i i i i i i i i i i -30 0 1 0 1 5 25 40 m 100 min fig 3. preventive effect ofindomethacin, 1 mgkg, on changes in mean pulmonary artery pressure (mpap) and mean syste mic artery pressure (msap) induced by thrombin infusion in rats. baseline represents steady-state values before thrombin infusion. * indicates pc0.05 compared with animals given thrombin and amca alone. mean 5s.e. arteriul blood gas studies the changes in arterial blood gas variables are illustrated in figure 4. thrombin caused a progressive decline in ph and paoz, while pac02 increased continuously. the fall in ph and the rise in pacoz after the infusion of thrombin were ameliorated by indomethacin. the ph in the indomethacin group was significantly higher 15 min after completion of thrombin infusion and subsequently. pacoz was significantly lower 60 and 90 min after thrombin infusion in the indomethacin group. however, the thrombin-induced decrease in pa02 was not affected by indomethacin. antithrombin activity in vitro to elucidate the question as to whether indomethacin possessed intrinsic antithrombin activity, this drug was incubated at various concentrations with thrombin in a chromogenic substrate system. as seen in figure 5 , indomethacin had no inhibitory effect at reasonable doses. 130 i ph 7s i + # # * 0 -30 0101525 40 70 100 pac02 l2 i * -3p0101525 40 70 100 14 18 12 11 g 10 9 8 7 0 pa02 hdo 1 i * i i i i i i i i i i i i i i i i l 400101525 40 70 100 1 min min min fig 4. changes in ph, paco? and pa02 after thrombin infusion in rats treated and not treated with indomethacin (indo), 1 mg&g body weight. baseline represents steady-state values before thrombin infusion. * indicates p<0.05 compared with animals given thrombin and amca alone. mean 2 s.e. antithrombin effect of indomethacm 3.5 3 2.5 . e m a 2 ro 0 d 1.5 1 0.5 0.2 2 20 200 2ooo concentration (uglmi) fig 5. antithrombin activity measured in a chromogenic substrate assay ($2238) at various indomethacin concentrations, expressed as the absorbance change at 405 nrn after 3 min of incubation. 131 discuss ion the initial pressure response to the infusion ofthrombin in the present study was analogous to that seen in previous experiments (20,21), i.e. a rapid increase in mean pulmonary artery pressure and a conco mitant decrease in mean systemic artery pressure. pretreatment with indomethacin attenuated the increase in pulmonary artery pressure in the early phase after thrombin infusion. the rise in mean pulmonary artery pressure after infusion of thrombin may probably be due to apro found increase in pulmonary vascular resistance resulting from thrombin-induced release of throm boxane az (20,8). it is therefore conceivable that the improvement of the mean pulmonary artery pres sure after thrombin infusion in the indomethacin group might be due mainly to inhibition of thromboxane synthesis resulting from cyclooxygenase inhibition, and consequently to a decrease in pulmonary vasoconstriction. the reduced mean systemic artery pressure during the infusion of thrombin was slightly improved in the early phase after thrombin infusion by pretreatment with indomethacin. however, indomethacin did not influence the progressive fall in mean systemic artery pressure caused by thrombin. in this pha se the lung weight started to increase, as seen in previous studies (6,lo). the progressive decrease in mean systemic artery pressure probably reflects a progressive distur bance of the systemic circulation secondary to the pulmonary damage. the infusion of thrombin produced a progressive decline in ph and pa02 and a continuous rise in pacoz. as indomethacin attenuated the changes in ph and pacoz but not the pulmonary edema, the changes in ph and pacoz should not be secondary to edema and are probably due to bronchoconstric tion and vasoconstriction induced by cyclooxygenase derivatives. the decline in pa02 after thrombin infusion was not affected by pretreatment with indomethacin and might partly be secondary to the pul monary edema. indomethacin exaggerated the hypotension caused by n-formyl methyl-leucyl-phe nyl-alanine in pentobarbital anesthetized rats (18) and has also been known to enhance bronchocon striction ( 2 3 ) . it is therefore conceivable that hypotension and bronchoconstriction may also lead to increased hypoxemia in the indomethacin-treated group. thrombin infusion following administration of amca caused an increase in lung vascular permea bility to protein, manifested as an increase in lung weight, wwidw and the lung water content. pre treatment with indomethacin aggravated thrombin-induced increase in lung weight. the same effect was observed in sheep with lung injury induced by endotoxin (17). in contrast, indomethacin preven ted an increase in lung vascular permeability to protein caused by thrombin in sheep in another study (3). this discrepancy in the effects may be explained by species differences and different experi mental conditions. why did indomethacin aggravate lung vascular permeability to protein in this study? lipoxygenase metabolites are known to be potent increasers of lung vascular permeability to pro tein, and they seemed to be involved in the late phase of thrombin-induced microembolism in sheep (3, 19). indomethacin may shunt arachidonic acid metabolites into the lipoxygenase pathway, leading to 132 increased synthesis of chemotactic leukotrienes (7, 15). it has also been found to increase leukocyte chemotaxis in carrageenin-induced inflammation ( i 1). this effect may explain the increased lung weight after administration of indomethacin. ibuprofen, on the other hand, another cyclooxygenase inhibitor, does not seem to induce a shift to the lipoxygena se pathway and inhibited therelease of leukotriene b4 from rat neutrophils in vitro (14). also, we found that ibuprofen decreased, and did not increase pulmonary edema in the same model as was used in the present experiment (1). in summary, although indomethacin prevented the early increase in mean pulmonary artery pressu re due to thrombin and had favourable effects on ph and pacoz, it caused the lung vascular permeabi lity to protein to increase more than did thrombin alone. these findings suggest that indomethacin may not be beneficial in the adult respiratory distress syn drome, which is characterized by an increased permeability to protein and pulmonary edema. references 1. ahn, c.m., sandler, h., wegner, t.& saldeen, t.: effect of ibuprofen on thrombin-induced pulmonary edema in the rat. (manuscript) 2. arfors, k-e., busch, c., jacobson, s., lindquist, o., malmberg, p., rammer, l.& saldeen, t.: pulmonary insufficiency following intravenous infusion of thrombin and amca (tranexamic acid) in the dog. acta chir scand 138: 445-452,1972. 3. bizios, r., minnear, el, van der zee, h.& malik, a.b.: effects of cyclooxygenase and lipoxygenase inhibition on lung fluid balance after thrombin. j appl physiol55: 462-471,1983. 4. brown, k.a.& collins, a.j.: action of nonsteroidal, anti-inflammatory drugs on human and rat peripheral leukocyte migration in vitro. ann rheum dis 36: 239-243, 1977. 5. cooper, j.a., solano, s.j.,bizios, r., kaplan, j.e.&malik,a.b.: pulmonary neutrophdkinetics after thrombin-induced intravascular coagulation. j appl physiol57 826-832,1984. 6. diffang, c.& saldeen, t.: effect of brinase (protease 1 from aspergillus oryzae) on the elimina tion of fibrin from the lungs and pulmonary damage in rats with induced intravascular coagulation and inhibited fibrinolysis. thrombos res 9: 61 1-622,1976. 7. docherty, j.c.& wilson, t.w.: indomethacin increases the formation of lipoxygenase products in calcium ionophore stimulated human neutrophils. bichem biophys res commun 148: 534-538,1987. 8. garcia-szabo, r.r., peterson, m.b., watkins, w.d., bizios, r., kong, d.l.&malik, a.b.: thromboxane generation after thrombin. protective effect of thromboxane synthetase inhibition on lung fluid balance. circ res 53: 214-222,1983. 9. garcia-szabo, r.r., johnson, a,& malik, a.b.: thrornboxane increases pulmonary vascular resistance and transvascular fluid and protein exchange after pulmonary microembolism. prostaglandins 35: 707-721,1988. 10. gerdin, b., diffang, c.& saldeen, t.: pulmonary insufficiency in the rat after intra vascular coagulation and inhibition of fibrinolysis. 1. studies on some pathogenetic mechanisms. eur surg res 13:402-413,1981. 133 10-950246 11. higgs, g.a., eakins, k.e., mugridge, k.g., moncada, s.&vane, j.r.: theeffects ofnonsteroidal anti-inflammatory drugs on leukocyte migration in carrageenin-induced inflammation. eur j pharmacol66: 81-86 1980. 12. johnson, a., tahamont, m.v., kaplan, j.e.& malik, a.b.: lung fluid balance after pulmonary microembolization. effects of thrombin vs. fibrin aggregate. j appl physiol52: 1565-1570,1982. 13. kaplan, h.b., edelson, h.s., korchak, h.m., given, w.p., abraham, s.& weissman, g . : effects of nonsteroidal anti-inflammatory agents on human neutrophil functions in vitro and in vivo. biochem pharmacol 33: 371-378,1984. 14. konstan, m.w., vargo, k.m.& david, p.b.: ibuprofen attenuates the inflammatory response to pseudomonas aeruginosain a rat model of chronic pulmonary infection. am rev respir dis 141: 186-192,1990. 15. lonigro, a.j., stephenson, a.h.& sprague, r.s.: role of arachidonic acid metabolites in the pathogenesis of acute lung injury. adv prostaglandin thromboxane leukotriene res 21 : 42 1 428,1991. 16. nettelbladt, o., tengblad, a.& halgren, r.: accumulation of hyduronan (hyaluronic acid) in lung tissue during experimental alveollitis parallels development of interstitial edema. am j phy siol257 379-84,1989,. 17. ogletree, m.l.& brigham, k.l.: indomethacin augments endotoxin induced increased lung vascular permeability in sheep. (abstract). am rev respir dis 119: a303,1979. 18. olsen, u.b.& bille-hansen, v.: exaggerated hypotension by n-formyl-methionylphenylalanine in indomethacin pretreated rats. role of toxic oxygen. agents actions 17: 489-94,1986. 19. perlman, m.b., johnson, a., jubiz, w.& malik, a.b.: lipoxygenase products induce neutrophil activation and increase endothelid permeability after thrombin-induced pulmonary microembo lism. circres 64: 62-73, 1989. 20. sandler, h., gerdin, b.& saldeen, t.: studies on the role of thromboxane in thrombin-induced pulmonary insuffiency in the rat thrombos res 42: 165175,1986. 21. sandler, h.& gerdin, b.: pulmonary insufficiency in the rat after intravascular coagulation and inhibition of fibrinolysis. 111. studies on the role of polymorphonuclear leukocytes (pmnls). actauniversatis upsaliensis 27: vii.1-vii.12 1985. 22. shacter, e., lopez, r.l.& pati, s . : inhibition of the myeloperoxidase-h~o2-cl-system of neutrop hils by indomethacin and other nonsteroidal anti-inflammatory drugs. biochem pharmacol41: 975-84,1991. 23. undem, b.j., pickett, w.c., lichtenstein, l.m.& adams, g.k.: the effect of indomethacin on immunologic release of histamine and sulfidopeptide leukotrienes from human bronchus & lung parenchyma. amrevrespirdis 236 1183-1187,1987. 24. vane, j.r.: inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. nature new biology 231: 232-235,197 1. 25. venezio, f.r., divincenzo, c., pearlman, f.& phair, j.p.: effects of the newer nonsteroidal anti inflammatory agents, ibuprofen, fenoprofen, and sulindac on neutrophiladherence. j infect dis 152: 690-694,1985. 134 26. vincent, j.e., zijlstra, ej.& bonta, i.l.: the effect of inhibitors on the ltds-induced contractions and release of thromboxane az in the guinea pig lung parenchymal strip. agents actions suppl 14: 69-81,1984. acknowledgements we wish to thank rolf wallin, phd, and mrs. tngalisa bjorklund for their skillful technical help. this work was supported by the swedish medical research council and the swedish national association against heart and lung diseases. correspondence to: h a a n sandler department of forensic medicine university of uppsala dag hammarskjolds vag 17 s-75237 uppsala sweden 135 upsala j med sci 95: 287-290, 1990 selecting a sensitive immunoradiometric method of maternal serum alphafetoprotein for prenatal screening of abnormalities in the fetus i . m. penttila,’ e. puhakainen‘ and m. ryynanen’ department of ‘clinicul chemistry und ‘clinical genetics, kuopio university central hospital, sf-70210 kuopio, finland it is well known that the value of alphafetoprotein in maternal serum (s-afp) can be used as a marker of the fetal abnormalities. in many diseases like in anencephalus, spina bifida and congenital nephrosis the value is increased ( 3 , 4 ) , while low values indicate rearrangement or other chromosomal abnormalities like down’s syndrome (5). in the district of kuopio university central hospital the prenatal screening for inherited genetic diseases was started in 1978. during the early years in late 7o’ies we pointed out our interest for high alphafetoprotein (s-afp) values in maternal serum and used an ordinary commercial ria-kit of behrinwerke ag. in 1984 this ria-method was changed to a double-antibody technique with glass-bead coated first antibody. since january 1987 we have utilized the ria-gnost afp of behringwerke. this immunoradiometric assay has better precision for both high and low values than the earlier methods. the screening system was also enlarged to the whole eastern finland. from the year 1988 annually about 13 000 women from the eastern finland have been examined for s-afp concentration during 14th-18th weeks of pregnancy. this means that at least 95 % of pregnant women has been studied for s-afp. reference values for sera were analyzed using samples of 1125 healthy pregnant women presupposing and confirming that the children born had no abnormalities. with this reference population the mean and median values were calculated as well as the discrimination limits for both low and high values. the gestational age of the pregnancy was verified (and corrected if needed) by a routine ultrasound technique. 287 for the measurement of s-afp we used the ria-gnost afp , immunoradiometric method of behringwerke ag. (marburg, f.r.g.) with the sensitivity of 2 ku/1. the measurements were performed by using an automated gamma counter (gamma master, wallac co., turku, finland). the statistical calculations were made by a microcomputer using standard statistical methods. in the table 1 the medians for s-afp during the weeks of 14th to 18th of pregnancy in the maternal serum with the discrimination limits are presented both for high and low values by using the immunoradiometric method (2). at first from 1987 to 1988 with this method the discrimination limits of 0.4 * median and 3 * median were used, while in 1989 the limits were changed to 0 . 5 * median and 2.5 * median for security reasons (diminished need to amniocentesis) without causing any noticeably loss in the detection of fetal abnormalities. the corresponding discrimination limits for high values in amniotic fluid were: means + 5 * s.d. and means o+ 10 * s.d., the highest one indicating a very probable abnormality in the fetus . the precision of the method used was followed by analyzing pooled serum samples, low, normal and high levels, in every series of analyses. the results are presented in table 2. in addition, our results in welcome immunoassay quality assessment programme 1 (wellcome diagnostics, dartford, england) have shown that the method has been well adapted for routine clinical laboratory practice being well comparable to the results of other laboratories in europe. during the last 12 years we have found many kinds of fetal abnormalities like neural tube defects, congenital nephrosis and anencephalus when using the earlier ria-methods. with the new immunoradiometric method also low values have been measured more precisely. when a variation coefficient of about 5.0 % has been achieved it has been possible to find the pregnancies with inherited chromosomal abnormalities like down's syndrome. from the women with the abnormal s-afp values samples from amniotic fluid were taken (only on voluntary basis) to make the final decision of the abnormality by using am-afp values and chromosome analyses from cells in amniotic fluid or from villus biopsies. every fetus, if found, has been carefully studied for 288 final diagnosis. when only s-afp and the age of the pregnant women were used for screening, about 38 % of pregnancies with chromosome abnormalities like down's syndrome were found. in 1991 we will add to our screening programme the measurements of chorionic gonadotrophin (s-hcg-b) and in selected cases also free estriol (s-estriol). this combination has been reported by noergaard-pedersen & al. (1) to increase the detection rate of abnormalities to about 55-60 %. for the general improvement of the diagnostic value of prenatal screening by s-afp, s-hcg-b and perhaps by free s-estriol it would be necessary to start a common nordic project to evaluate the methods available and the best combination of tests, age included. references: 1. 2. 3. 4. 5. noergaard-pedersen b, olesen larsen s, arends j, svenstrup b, tabor a. maternal serum markers in screening for down syndrome. clinical genetics 1990; 37: 35-43. puhakainen e, ryynanen m. the use of a sensitive alpha-fetoprotein method and low maternal serum value in the screening of trisomies during the second trimester of pregnancy. biochimica clinica 1989;13,suppl. 118: 329. ryynanen m. "screening of fetal malformations with the measurement of alpha-fetoprotein of maternal serum in eastern finland1'. publications of the university of kuopio, medicine, original reports 411981, finland, 1-81. seppala mi aula p, rapola, j, karjalainen 0, huttunen n-p, ruoslahti e. congenital nephrotic syndrome: prenatal diagnosis and genetic counselling by estimation of amniotic-fluid and maternal serum alpha-fetoprotein. lancet 1976; 2: 123-125. wald nj, cuckle hs, densem jw, nanchahal k, royston p chard t, haddow je, knight gj, palomaki ge, canick ja. maternal serum screening for down's syndrome in early pregnancy. brit med j 1988; 297: 883-887. 19 -908573 289 table 1. the median values and the discrimination limits of s-afp in maternal serum during 15th to 18th week of pregnancy of healthy women. week of no. of serum afp, ku/1 pregnancy women median 0 . 5 * median 2 . 5 * median 1 4 3 8 1 9 . 0 9 . 5 4 7 . 5 15 7 8 9 2 1 . 0 1 0 . 5 5 2 . 5 1 6 2 1 7 2 3 . 0 11.5 5 7 . 5 1 7 5 4 2 7 . 0 13.5 6 7 . 5 18 2 7 2 9 . 6 1 4 . 8 7 4 . 0 table 2 . the precision of the immunoradiometric methodfor s-afp. in every series of analyses three levels of pooled frozen sera have been analyzed. as an example the results from three different months in 1 9 8 9 are expressed. s-afp, ku/1 coefficient of variation, % month low medium high low medium high 1 1 1 1 9 8 9 8 . 4 7 8 1 5 5 5 . 9 4 . 0 5 . 0 v i l 1 9 8 9 8 . 2 7 9 158 6 . 1 6 . 0 4 . 5 x / 1 9 8 9 9 . 1 8 0 1 5 3 5 . 5 4 . 5 5 . 0 correspondence: professor i.m. penttila, department of clinical chemistry, university of kuopio, s f 7 0 2 1 0 kuopio, finland 290 upsala j med sci 91: 3 7 4 3 , 1986 selective properties of rains of real and complex rf micropulses in slice-selective spin-echo mri b. jung,' a. ericsson,2 and a. hemmingsson* 'department of hospital physics, university hospital, uppsala and 'department of diagnostic radiology, uppsala university, uppsala, sweden arst ract the spin nutation properties o f frequency selective (space selective i n combination with a magnetic f i e l d gradient) trains o f radiofrequency micropulses were studied i n a numeric model. two cases were considered, one simulating the 90 degrees excitation pulse in spin-echo mri, the other t h e 180 degrees spin inversion pulse. image reconstruction according t o the 2 0 fourier transform technique requests t h a t the e f f e c t o f t h e 180 degree pulse i s independent o f t h e i n i t i a l phase o f t h e spin vector relative t o t h e radiofrequency field. i t was found t h a t with a t r a i n o f phase-stable radiofrequency micropulses with an envelope o f the traditional 'sinc' type the result o f the spin inversion pulse was depending on the i n i t i a l phase whereas this was not t h e case for a t r a i n o f phase-shifted (complex) micropulses. the complex t r a i n o f radiofrequency micropulses also had b e t t e r selective properties both f o r excitation and spin inversion than the r e a l one. introduction in magnetic resonaiice imaging (mri) (1, 3, 5, 6, 8) 'selective excitation' stands f o r a space selective process i n which a frequency-selective radiofrequency (rf) pulse is used t o nutate the spins in a defined slice o f t h e object under the influence o f a linear magnetic f i e l d gradient. in the imaging technique applying the 2 0 fourier transform method ( 5 ) , t h e rf pulse and magnetic field gradient sequence may be a 90 degrees excitation pulse combined with zgrad. defocusing and phase-encoding during xgrad+ygrad, and a 180 degrees pulse combined with zgrad where zgrad stands f o r a magnetic field gradient along the major f i e l d axis and xtirad and ygrad are mutually orthogonal gradients, orthagonal t o this' axis. a spin-echo recorded under xl;rad i s then fourier-transformed t o yield the distribution o f spins i n the x-direction. a number o f such sequences with proper y-encoding (via a set o f properly chosen gradients in the y-direction, ygrad) i s recorded and a second fourier transformation yields the two-dimensional 37 zgrao xgrad yljrao __ ,a,. a. r f t i m e fig. 1: simplified schematic timing diagram o f a spin-echo sequence i n 2-d fourier transform mri. spin-density i n the slice (in this example i n t h e xy-plane), defined by the two rf pulses applied under zgrad. , i n such a process the second rf pulse (180 degrees) o f necessity must handle, on an equal footing, elemental macroscopic magnetizations in the xy-plane, that have all possible phases in relation t o t h e rf pulse. when t r y i n g t o simulate numerically t h e behaviour o f a 2-d fourier mri system with respect t o slice definition, object, movement.s and spectrum of larmor frequencies, an e r r a t i c behaviour w i t h sudden narrow drops i n spin-density sensitivity i n the selected slice was found. this motivated a closer look on the phase-dependence o f the space-selective processes. in t h e f i r s t simulations a selective phase-stable rf pulse, proposed by locher (7), was applied, in l a t e r ones the complex selective rf pulse suggested by silver e t al. (9, 10). two situations were considered. in the f i r s t one t h e space selection and the resulting phase f o r nutation o f a macroscopic magnetization in thermal equilibrium, were studied. this case simulates the 90 degrees .excitation. i n the second situation t h e space selection, t h e resulting magnetization i n the xy-plane and the resulting phase f o r inversion o f a macroscopic magnetization in the xy plane were studied. in the l a t t e r case, simulating spin inversion, the calculations covered a l l i n i t i a l phases. method a simplified timing diagram o f a spin-echo sequence i s given i n fig. 1. the rf pulses were considered t o be composed o f 756 micropulses o f suitably selected strengths b u t o f constant amplitude and o f constant phase, with respect t o an assumed external clock, during their time period. they were applied in combination with the magnetic f i e l d gradient zgrad. the following xgrad pulse defscuses the phases and i s applied t o c o r r e c t f o r the phase development du xgrad pulse, under which the echo i s recorded. the ygrao pulse, o f variable amplitude, encodes t h e echo in t h e y-direction. he second the elements, bx and by, o f a complex rf vector were assigned values according 38 002 0 01 000 -001 r f a 0010 " 0.000 pulse number -000: r f b t ' c . i i . o o l t oa01-l v w fig. 2: real pulse t r a i n (panel 2) according t o (7) and complex pulse t r a i n (pane! b) according t o (9, 10). the amplitudes are given i n revolutions per rriicropulse period (z=o) f o r a resulting nutation o f 90 degrees. in t h e complex case this applies for the real component only. both pulse trains are centered around micropulses 128 and 129. panel c shows the phase diagram o f the t h i r d quarter of the rf t r a i n and panel d the f o u r t h quarter. arrows indicate micropulse number. t o the formulae given by (7) and by (9, 10). i n the former, real, case this yielded bx(i)=c*sinc(2*hi/p)*exp(-i*v(2*d*d) by(i)=o (i.e. phase-stable) where 1=-128,128,1 p=128/4 d=9s*p/24* sinc(j)=siri(ei*j3/(pi*j) and pi-3.1 41 6.. . i n the l a t t e r , complex, case the formulae were bl(i)=c'*hel~i *cos(help~) by(i)=c'*helpl "sin(help2) where help1 =2/(exp(i*incr+5/256)+exp(-i*incr+5/256)) helpz=s*ln(helpi) and incr=5/12q~ the constants c and c' were varied in order t o obtain yaripus nutation angles. the calculations were performed in a coordinate system r o t a t i n g i n the appropriate direction in synchronism w i t h the larmor frequencv o f t h e central disc of the slice (i.e. z-0; no z-gradient offset). the <-axis was pargllel t o the main magnetic field, bo. the x-axis was orthogonal t o t h e z-axis and parallel t o the ex of the rf field, and t h e y-axis orthogonal t o t h e other t w o axis and parallel t o the by o f the rf field. the numerical representation o f a l l magnetic field vectors wa,s scaled t o give the precession angle during t h e time period o f one micropulse. the resultant o f the three components (in x-, y and z-direction) was thus expressed as t h e i b c i phase out d 1 phase out fig. 3: resulting macroscopic magnetizations i n the z-direction as a function o f distance from slice cent,re a f t e r nutation o f a (0,0,1) spin vector. results with r e a l pulse t r a i n in panel a and with complex pulse t r a i n i n panel q. the nutation angles i n panel 2 are 60 degrees (. . . .), 90 degrees (o o o 0) and 180 degrees (x x x x), and the r e l a t i v e strengths o f the complex t r a i n in panel b are 50 (. (. . . .), 70 (0 o o o), 140 (+ + + +) and 200 (x x x x) (see t h e t e x t f o r units). panel c shows the resulting phase a f t e r nutation with t h e r e a l pulse t r a i n as a function o f distance from slice centre arid the corresponding results w i t h the complex pulse t r a i n i s displayed in panel g. nutation angles are in panel c 60 degrees (. . . .) and 180 degrees (x x x x) and i n panel d the pulse strengths are 50 (. . . .) and 200 (x x x x). nutation angles or pulse strengths between those given i n panels c and 4 yield results in between the two curves i n the digrams. the 2-distance is measured i n 2-offset revolutions per micropulse period. precession angle during the time period o f one micropulse. the simulation procedure was then merely t o multiply the magnetization vector, currently present, with t h e appropriate r o t a t i o n natrix and t o repeat this procedure 256 times. the nutation o f the c e n t r a l ( i s . z-0) elemental magnetization, when subjected t o the r e a l component o f a micropulse train, i s regarded as t h e nominal f l i p angle o f the pulse. two cases o f i n i t i a l macroscopic magnetization were considered. i n the f i r s t one the i n i t i a l magnetization was the one a t thermal equilibrium, i.e. parallel t o t h e main magnetic f i e l d (bo), in vector notation (o,o,l). i n t h e other case t h e macroscopic magnetization vector was supposed t o initially be nutated t o t h e xy-plane, i n vector notation (sin(v),cos(v),o). (the vector notation is (x,y,z) w i t h x=component o f the macroscopic magnetization in t h e x-direction etc. and v-phase angle relative t o the x-axis i n the xy-plane immediately before the f i r s t micropulse o f the simulated rf pulse train.) relaxation processes were ignored in the simulations which were performed on a pdp 11/40 computer with programs written in fortran iv. source codes are available on request. 40 d '5lpha5e out 1 x y v e c t o r a l 1 0 phase i n oooo 1 xy-vector b oo0.0 i p h a s e in f phase out c 00 05 1 0 phase i n i . t 0.0 05 1.0 p h a s in fig. 4 : macroscopic magnetizations i n t h e xv-plane as a function o f i n i t i a l phase aft,er inversion of (sin(v),cos(v),o) spin vectors in the xy-plane. results w i t h r e a l pulse t r a i n in panel 2 and w i t h complex pulse t r a i n in panel q. z=o (central plane o f the slice, --------), z-0.01 (panel a) and z=0.025 (panel 4) (. . . .), z=0.025 (panel a) and z=o.o3o (panel b) (0 0 0 0). the resulting phases o f t h e macroscopic magnetizations as a function o f i n i t i a l phase a f t e r the same inversion i s shown i n panel c f o r the r e a l pulse t r a i n and in panel d f o r t h e complex pulse train. z=o (central plane o f t h e slice, -------), 2=0.010 in panel c and z=0.025 in panel r! (. . . .), z=0.020 in panel c and z=o.o28 i n panel d (0 o o oj, z=0.025 i n panel c and z=o.032 in panel d (x x x x). in panel 24j.030 (+ + + +). a l l phar .,es are measured in revolutions and t h e z-distance i s given as 2 o f f s e t precession revolutions per micropulse period. results the rf pulse trains are illustrated i n fig. 2. the resulting macroscopic magnetization in t h e 2-direction a f t e r an rf pulse t r a i n applied t o a (0,0,1) macroscopic magnetization vector is given in fig. 3 as a function o f distance from the central plane (z=o). distance i s measured as precession revolutions in the rotating coordinate system. for symmetry reasons only half o f the selected slice is given i n the illustration. the resulting phase a f t e r this procedure i s also given i n fig. 3. the macroscopic magnetization remaining in the xy-plane a f t e r nutation o f a (sin(v),cos(v),o) macroscopic magnetization is given in fig. 4 as a function o f the i n i t i a l phase v and the resulting phase a f t e r this procedure i s also given i n fig. 4. only p a r t o f the results o f the calculations are displayed in the figures. discussion the space selectivity o f both pulse trains i s rather good f o r 90 degrees pulses as illustrated in figs. 3 and 4 . the complex pulse t r a i n yields a somewhat more uniform magnetization along t h e z-axis than does the r e a l one. this e f f e c t i s accentuated when the strengths o f t h e pulse trains are increased t o approach a nutation angle o f 180 degrees. the mysterious property of the complex t r a i n 41 t o a p p r o a c h 180 d e g r e e s inversion a s y m p t o t i c a l l y f o r high p u l s e s t r e n g t h was n o t e d by (9, 10) a n d i s e v i d e n t in fig. 3b. for b o t h t y p e s of yo d e g r e e s pulse t r a i n s t h e r e s u l t i n g p h a s e was almost l i n e a r l y depending on p o s i t i o n (fig. 3c a n d d). as s t a t e d e a r l i e r (7, y , 10) t h i s seems t o b e a p r e r e q u i s i t e f o r p r o p e r r e f o c u s i n g o f t h e s p i n s b e f o r e t h e r e c o r d i n g o f t h e e c h o . the b r e a k s in t h e l i n e a r f u n c t i o n s o c c u r well o u t s i d e t h e s p a c e s e l e c t i v e r e g i o n s . ’ the e f f e c t of t h e spin-inversion p u l s e t r a i n s i s e v i d e n t l y depending on t h e i n i t i a l p h a s e (fig. !+). this e f f e c t is, however, n o t v e r y a c c e n t u a t e d f o r t h e complex t r a i n u n t i l v e r y c l o s e t o t h e s l i c e b o r d e r . for t h e real p u l s e t r a i n t h e e f f e c t o c c u r s c l o s e r t o z=o, a n d , in f a c t , z=o.o25 in fig. 4a r e p r e s e n t s a case when a minimum in t h e xy-vector h a s a l r e a d y o c c u r r e d at, a smaller z value. also t h e r e s u l t i n g s p i n p h a s e i s l i n e a r l y d e p e n d i n g o n t h e incoming p h a s e u p t o v e r y c l o s e t o t h e s l i c e b o r d e r f o r t h e complex r f t r a i n , w h e r e a s t h e r e a l t r a i n g i v e s d i s t o r s i o n s r a t h e r c l o s e t o t h e s l i c e c e n t r e (fig. 4b a n d c). this d i s t o r s i o n r e s u l t s in a bunching o f t h e r e s u l t i n g p h a s e s a r o u n d two p r e f e r r e d values. i t i s believed t h a t t h i s bunching may s e r i o u s l y a f f e c t t h e p e r f o r m a n c e of t h e imaging s y s t e m . in simulations with o t h e r p h a s e s t a b l e pulse t r a i n s , n o t f u r t h e r d e t a i l e d h e r e , t h e bunching o f p h a s e s was f o u n d t o b e a s s o c i a t e d with t h e o c c u r r e n c e of zeroes f o r some initial p h a s e s in t h e m a c r o s c o p i c magnetization in t h e xy-plane a f t e r inversion. an i n v e r s e p r o p o r t i o n a l i t y of r e s u l t i n g p h a s e on i n i t i a l p h a s e was f o u n d f o r z v a l u e s l a r g e r t h a n t h e smallest o n e giving a z e r o ( f o r some i n i t i a l p h a s e s ) in t h e r e s u l t i n g xy-vector. such a b e h a v i o u r i s i n d i c a t e d i n figs. 3 a n d 4 . i t t h u s seems d i f f i c u l t t o h a v e a good s e l e c t i v e s p i n i n v e r s i o n in a n mri s y s t e m provided t h e r f p u l s e i s r e s t r i c t e d t o b e r e a l ( p h a s e s t a b l e ) . simulation e x p e r i m e n t s by (2, 4 ) s u p p o r t t h i s s t a t e m e n t . u n r e p o r t e d r e s u l t s o b t a i n e d by u s s t r e n g t h e n s t h i s conclusion s i n c e i t was invariably f o u n d t h a t spin bunching a n d low i n v e r s i o n e f f i c i e n c y o c c u r r e d b e f o r e t h e b o r d e r s of t h e s e l e c t e d s l i c e . on t h e o t h e r h a n d a f i r s t t r i a l with a t r a i n of complex r f micropulses g a v e a r e s u l t f r e e of most of t h e s h o r t c o m i n g s of t h e r e a l p u l s e t r a i n . a complex r f pulse would a d d t o t h e complexity of a n mri s y s t e m b u t t h e r e should b e no a b s o l u t e t e c h n i c a l o b s t a c l e s t o s u c h a s o l u t i o n . acknowledgement s u p p o r t e d by g r a n t from t h e swedish m e d i c a l k e s e a r c h council, p r o j . no. k84-17x-6676-02a. 42 references 1. damadian, r.: apparatus and method f o r detecting cancer i n tissue. us pat. 3.789.832, f i l e d march 17, 1972. 2. frahm, j. 8 hanicke, w.: comparative study o f pulse sequences f o r selective excitation i n nmr imaging. j magn reson 60:320-332, 1984. 3. hinshaw, w.s.: spin mapping: t h e application o f moving gradients t o nmr. phys l e t t 4 8 ~ : 8 7 8 9 , 1974. 4. joseph, p.m., axel, l . 8 o'donnel, m.: potential problems with selective pulses i n nmr imaging systems. med phys 11:772-777, 1984. 5. kumar, a., welti, d. 8 ernst, r.: nmr fourier zeugmatography. j magn reson 6. l auterbur, p.c.: image formation by induced local interactions: examples employing nuclear magnetic resonance. nature 242:190-191, 1973. 7 . locher, p.r.: computer simulation o f selective excitation i n n.m.r. imaging. phil trans r soo lond 8289537-542, 1980. 8. mansfield, p. 8 grannel, p.k.: nmr 'diffraction' i n solids? j phys soc c: solid state phys 6:l422, 1973. 9. silver, m.s., joseph, r.i., chen, c.-n., sank, v.j. 8 hoult, d.i.: selective population inversion i n nmr. nature 310:681-683, 1984. 10. silver, m.s., joseph, r.i. 8 hoult, d.i.: highly selective pi/2 and p i pulse generation. .i magn reson 59:347-351, 1984. 18:69-83, 1975. 43 upsala j med sci 93: 53-56, 1988 familial ovarian dermoid cysts karl-henrik gustavson' and curt rune' 'department of clinical genetics, university hospital and 'department of gynecology, samariterhemmet hospital, uppsala, sweden abstract a f a m i l y c o n s i s t i n g showed an i n c r e a s e d chromosomal i n s t a b i o f a mother and h e r two daughters w i t h o v a r i a n dermoid c y s t s frequency o f aneuploidy and chromosomal breakage i n d i c a t i n g i t y . introduction there a r e s e v e r a l r e p o r t s on f a m i l i a l occurrence o f dermoid c y s t s o f t h e ovary: (2,3,4,5,8,10,11). these r e p o r t s p l u s o t h e r c h a r a c t e r i s t i c s o f dermoids, such as h i g h percentage o f b i l a t e r a l i t y and low age o f occurrence, p o i n t t o g e n e t i c a l causes i n some o f t h e cases o f t h e disease. no c y t o g e n e t i c s t u d i e s o f f a m i l i a l cases seem t o have been done. t h i s r e p o r t d e s c r i b e s a f a m i l y c o n s i s t i n g o f a mother and two daughters w i t h o v a r i a n dermoid c y s t s who were s u b j e c t e d t o chro mosomal i n v e s t i g a t i o n s . the mother and one daugther showed s i g n s o f chromosomal i n s t a b i l i t y . case reports p a t i e n t 1: ( 1 : l i n t h e p e d i g r e e ) , i s a 54-year-old, 2-gravidae woman. she was operated on f o r a p e r f o r a t e d a p p e n d i c i t i s a t 26 years o f age, as n u l l i g r a v i d a e woman because o f i n t e r m i t t e n t abdominal p a i n . a t o p e r a t i o n , an ovary t h e s i z e o f a s m a l l orange p a r t l y embedded i n adhesions was found. an ophorectomy was p e r formed. the l e f t ovary was normal and h i s t o l o g i c a l examination o f t h e r i g h t ovary r e v e a l e d a corpus luteum c y s t i n a d d i t i o n t o a plum-sized dermoid c y s t t h a t was w i t h o u t h i s t o l o g i c a l s i g n s o f malignancy. p a t i e n t 2: ( i i : 2 i n t h e p e d i g r e e ) , a 24-year-old n u l l i g r a v i d a e woman p r e v i o u s l y h e a l t h y , who had been t a k i n g c o n t r a c e p t i v e p i l l s d u r i n g t h e 18 months preceeding t h e o p e r a t i o n . a r i g h t s i d e d o v a r i a n lump had been found on r o u t i n e examination. a t o p e r a t i o n a dermoid c y s t t h e s i z e o f a s m a l l orange i n v o l v i n g t h e r i g h t ovary was found and removed. the l e f t ovary was normal. h i s t o l o g i c a l examination r e vealed a dermoid c y s t w i t h o u t s i g n s o f malignancy. p a t i e n t 3: ( i i : 3 i n t h e p e d i g r e e ) , a 22-year-old n u l l i g r a v i d a e woman p r e v i o u s l y h e a l t h y , who had been t a k i n g c o n t r a c e p t i v e p i l l s d u r i n g t h e 24 months preceeding t h e o p e r a t i o n . on r o u t i n e examination a l e f t s i d e d o v a r i a n lump had been found. a t o p e r a t i o n , a dermoid t h e s i z e o f a s m a l l orange i n v o l v i n g t h e l e f t ovary was found and removed. the r i g h t ovary was normal. h i s t o l o g i c a l examination r e v e a l e d a dermoid c y s t w i t h o u t s i g n s o f malignancy. methods on each o f t h e t h r e e p a t i e n t s b l o o d samples were taken on two separate occasions f o r c y t o g e n e t i c a l i n v e s t i g a t i o n s . a f t e r c o n v e n t i o n a l c e l l c u l t u r e procedure and 53 v2 i pedigree of the affected family. 0 normal 0 ovarian dermoid cyst giemsa-banding, 100 metaphases were analysed from each p a t i e n t . chromosome breakage a n a l y s i s was performed on 100 metaphases o f giemsa-stained a i r d r i e d s l i d e s from each p a t i e n t . a n a l y s i s o f s i s t e r c h r o m a t i d exchanges a f t e r brdu l a b e l l i n g were performed on c u l t u r e d lymphocytes from t h e p a t i e n t s , as d e s c r i b e d by a l v e s t & jonasson ( 1 ) . results the r e s u l t s o f t h e chromosomal i n v e s t i g a t i o n s a r e shown i n table 1. the t h r e e p a t i e n t s a l l had a normal female chromosomal c o n s t i t u t i o n . there was an i n c r e a s e d frequency o f aneuploidy (4-20:;) and chromosomal breakage i n patients 1 and 3 as compared w i t h c o n t r o l s . the frequency o f s i s t e r chromatid exchanges was n o t increased. d i s c u s s i o n most o v a r i a n teratomas have a normal 46,xx karyotype, suggesting t h a t o v a r i a n teratomas a r i s e from germ c e l l s and most o f t e n through p a r t h o g e n e s i s ( 7 ) . the p r e s e n t study as w e l l as o t h e r i n v e s t i g a t i o n s (2,4,5,8,10,11) have c l e a r l y demonstrated t h e e x i s t e n c e o f f a m i l i a l forms o f o v a r i a n dermoid c y s t s . our study demonstrated an i n c r e a s e i n t h e number o f a n e u p l o i d c e l l s and chromosomal breakage i n lymphocyte c u l t u r e s o f t h e mother and one o f h e r daughters. these f i n d i n g s support t h e t h e o r y t h a t g e n e t i c f a c t o r s a r e o f importance i n t h e de velopment o f o v a r i a n dermoid c y s t s . the s i g n i f i c a n c e o f t h e a s s o c i a t i o n between t h e o v a r i a n teratomas, which a r e p r o b a b l y dominantly i n h e r i t e d , and t h e f o r e mentioned c y t o g e n e t i c a l f i n d i n g s i n c u l t u r e d c e l l s i s u n c e r t a i n . i t i s p o s s i b l e , however, t h a t t h e i n c r e a s e d frequency o f aneuploidy and chromosomal breakage a r e s i g n s o f chromosomal i n s t a b i l i t y t y p i c a l l y seen i n some d i s t i n c t g e n e t i c d i s o r d e r s w i t h a p r o p e n s i t y t o develop neoplasia, such as f a n c o n i ' s anemia, bloom's syndrome, a t a x i a t e l a n g i e t a s i a , xeroderma pigmentosum and gardner's syndrome, considered t o be expression o f t h e u n d e r l y i n g gene d e f e c t ( 9 ) , and t h a t t h e i n creased frequency o f a n e u p l o i d c e l l s i n c u l t u r e d lymphocytes from our f a m i l i a l cases r e f l e c t s a d i s t u r b a n c e o f t h e m i t o t i c and m e i o t i c processes, which m i g h t be o f pathogenetic importance. such a d i s t u r b a n c e o f t h e m i t o t i c and m e i o t i c process i n our f a m i l i a l cases c o u l d be r e l a t e d t o an u n d e r l y i n g gene d e f e c t or due t o m e t a b o l i c and/or hormonal i n f l u e n c e s . i t should be o f i n t e r e s t t o p e r f o r m f u r t h e r s t u d i e s and compare n o n f a m i l i a l w i t h f a m i l i a l cases. we have t h u s s t a r t e d a p r o s p e c t i v e study o f c o n s e c u t i v e l y operated cases o f o v a r i a n teratomas i n t h e uppsala h o s p i t a l region, sweden, i n c l u d i n g chromosomal analyses and dna a n a l y t i c a l procedures o f b l o o d and dermoid t i s s u e . we have a l s o s t a r t e d a r e t r o s p e c t i v e study i n o r d e r t o f i n d o u t t h e frequency o f f a m i l i a l cases o f t h e disease. 54 p at ie n t t ab le 1 . c yt oq en et ic f in di nq s in c u lt u re d l ym p h oc yt es f ro m t h e th re e re la ti ve s w it h o va ri an t er at om as k ar o t e & iis ) a be rr at io ns p e r ce ll s c e f ra gi le s it es (i n no . c h ro m at id a nd 1s ga ps fr ag m en ts to ta l (m ea n p e r o f ce ll s of 1 0 0 ch ro m at id b re ak ag es ce ll ) ex am in ed c el ls ) p at ie n t 1 (k l) 1s t cu lt u re 2n d cu lt ur e p at ie n t 2 (1 1: l) 1s t cu lt ur e 2n d cu lt ur e p at ie n t 3 ( k 2 ) 1s t cu lt ur e 2n d cu lt ur e c on tr ol s 46 ,x x (1 7) 0. 15 0. 01 0. 03 0. 19 0 4 7 ,x x x (l ) 4 8 ,x x x x (l ) 4 5 ,x (1 ) 4 6 ,x x (4 0 ) 0. 08 47 ,x x ,+ 8( 1) 45 ,x (1 ) 45 ,x x ,4( 1) 45 ,x x -1 2( 1) 4 4 ,x x ,l, -l (l ) 44 ,x ,3, -1 8( 1) 0. 03 0. 03 0. 14 8. 5 0 4 6 ,x x (1 9 ) 47 ,x x ,+ 21 (1 ) 46 ,x x (4 3) 47 ,x x ,+ 5( 1) 4 6 ,x x a ne up lo id y 4 % 0. 01 0. 03 0. 30 0. 04 0. 02 0 0 0. 01 7. 6 0 0 0. 03 0. 18 0. 08 0. 56 0 0. 04 0. 01 0. 09 5. 1 0. 05 0. 00 2 0. 01 2 11 .4 0 acknowledgement t h i s study was supported by a g r a n t from t h e cancer foundation o f t h e u n i v e r s i t y h o s p i t a l , uppsala. references 1. 2. 3 . 4 . 5 . 6 . 7 . 8. 9 . 10. 1 1 . alves, p. & jonasson, j.: new s t a i n i n g method f o r d e t e c t i o n o f s i s t e r chromatid exchanges i n b r d u l a b e l l e d chromosomes. j c e l l s c i 3 2 : 1 8 5 1 9 5 , 1978. brenner, s.h. & wallach, r.c.: f a m i l i a l benign c y s t i c teratomas. i n t j gynaecol obstet 2 1 : 1 6 7 1 6 9 , 1 9 8 3 . brown, e.h.: i d e n t i c a l t w i n s w i t h t w i s t e d benign c y s t i c teratoma o f t h e ovary. am j obstet gynecol 1 3 4 : 8 7 9 8 8 0 , 1 9 7 9 . f e l d , d., labes, d. & nathanson, m.: b i l a t e r a l o v a r i a n dermoid c y s t s i n t r i p l e t s . obstet gynecol 2 7 : 5 2 5 , 1966. h o l l a n d e r , h. & masterson, j . : f a m i l i a l c y s t i c teratoma o f t h e ovary. texas rep b i o l med 2 3 : 4 8 3 , 1 9 6 7 . l i n d e r , d., mc caw, b.k. & hecht, f.: pathogenetic o r i g i n o f benign o v a r i a n teratomas. new engl j med 2 9 2 : 6 3 6 6 , p a r r i n g t o n , j.m., west, l.f. & povey, s.: the o r i g i n o f o v a r i a n teratomas. j med genet 2 1 : 4 1 2 , 1984. p l a t t n e r , g., oxhorn, h. & masterson j.: f a m i l i a l i n c i d e n c e o f o v a r i a n dermoid c y s t s . can med assoc j 1 0 8 : 8 9 2 8 9 3 , 1973. sandberg, a.a.: the chromosomes i n human cancer and leukemia. e l s e v i e r new york, amsterdam, 1980. s c h a u f f l e r , g. i n p e d i a t r i c gynecology year book. m e d i c a l p u b l i s h e r s , chicago, 1958. s i p p e l , a.: dermoid o f ovary i n t h r e e s i s t e r s . z e n t r a l b l gynaecol 4 8 : 8 5 , 1 9 2 4 . address f o r r e p r i n t s : k a r l h e n r i k gustavson, department o f c l i n i c a l genetics, u n i v e r s i t y h o s p i t a l , 5-751 85 uppsala, sweden. 56 uppsala j med sci 91: 245-250, 1986 large section cryomicrotomy: a basic method for microdissectioning and in vitro autoradiography of human nervous tissue sten-magnus aquilonius department of neurology, university hospital, s-75185 uppsala, sweden 1 . i n t r o d u c t i o n e v o l u t i o n o f t h e i m p o r t a n t t e c h n i q u e of whole body a u t o r a d i o g r a p h y o f e x p e r i m e n t a l a n i m a l s , i n t r o d u c e d by u l l b e r g i n 1 9 5 4 , h a s been d i r e c t l y l i n k e d t o t h e development of heavy-duty c r y o microtomes ( 1 6 ) . s i n c e t h e n , l a r g e s e c t i o n cryomicrotomy h a s found s e v e r a l new areas of a p p l i c a t i o n . the p r e s e n t p a p e r w i l l i l l u s t r a t e t h e u s e f u l n e s s of t h i s b a s i c methodology as e x e m p l i f i e d by s t u d i e s i n o u r own r e s e a r c h group. r e p o r t s i n c l u d i n g t e c h n i c a l d e t a i l s have r e c e n t l y been pub l i s h e d ( 5 , 1 0 ) . 2 . t o p o g r a p h i c a l anatomy o f human b r a i n and muscle 2 . 1 b r a i n computed tomography ( c t ) i s e s t a b l i s h e d a s a most i m p o r t a n t t e c h n i q u e i n n e u r o r a d i o l o g y . ct o f t h e b r a i n i s m a i n l y r e s t r i c t e d t o n e a r h o r i z o n t a l p l a n e s , a f a c t which c r e a t e s a need f o r ana t o m i c a l knowledge of b r a i n s e c t i o n s which were n o t commonly s t u d i e d p r e v i o u s l y . t h e r e f o r e , w e a p p l i e d l a r g e s e c t i o n c r y o microtomy o f whole human b r a i n and produced a c o l o u r a t l a s o f h o r i z o n t a l c r y o s e c t i o n s ( f i g . 1 a ) which where p h o t o g r a p h e d j u s t thawed a g a i n s t a b l a c k background ( 3 ) . however, t h i s a t l a s d o e s n o t i l l u s t r a t e t h e r e l a t i o n s h i p b e t ween b r a i n and s u r r o u n d i n g t i s s u e s . a h e a d b r a i n a t l a s b a s e d on t h e t e c h n i c a l m o d i f i c a t i o n s of cryomicrotomy worked o u t by rauschning ( t h i s volume) would be a u s e f u l complement t o t h e neuroimaging t e c h n i q u e s , c t , n u c l e a r m a g n e t i c r e s o n a n c e , and . p o s i t r o n e m i s s i o n tomography. 2 . 2 muscle knowledge a b o u t t h e t o p o g r a p h i c a l d i s t r i b u t i o n o f motor end p l a t e s i n human muscle i s i m p o r t a n t b o t h f o r t h e n e u r o p h y s i o l o 16-868573 245 b fig. 1 different applications of large section cryomicrotomy: a) topographical atlas of the human brain (4) adapted to computed tomography. b) distribution of che staining in whole human biceps muscle ( 5 ) . c ) tissue punches within a section through extrapyra midal structures 5 mm behind the anterior commisure (8). d) in vitro autoradiography in a frontal section of a human hemisphere (3) using the muscarinic ligand 3h-qnb. 246 gist carrying out electromyography and for the clinician taking a muscle biopsy. however, due to technical difficulties at sec tioning, available information was based on cholinesterase (che) staining of muscles from small children only. by means of the heavy-d.uty cryomicrotome (pmv 400, lkb 2250, lkb, sweden) it is possible to obtain horizontal cryosections of adult whole human muscle. we have studied the distribution of motor end-plates in biceps brachii (fig. 1 b), tibialis anterior and sartorius muscle from adults by staining longitudinal cryosections of the whole muscles for che (5). the results from a large number of cryo sections from biceps brachii and tibialis anterior muscles were analysed by a computer to reconstruct the topographical end-plate distribution within the whole muscle (2). 3. the "punch-technique" of tissue sampling from cryosections of human brain and spinal cord a defined and reproducable method of tissue dissectioning is a prerequisite for regional neurochemical studies in post-mortem nervous tissue. for this purpose the "punch-technique" of tissue sampling has been rather extensively used in animal (see 14 for review) and human studies (see 5 for review). we introduced this technique (fig. 1 c) in an investigation on the regional distribution of choline acetyltransferase (chat) within the brain of controls and huntington chorea cases (6). thereby, it was possible to obtain the most detailed localization of human brain chat available at that time and to demonstrate the marked reduction of neostriatal chat in huntington's disease, probably corresponding to a pronounced degeneration of choliner gic interneurons. more recently the "punch-technique" of microdissectioning has been used in investigations on the topical localization of chat within the human spinal cord (4). in addition to an area of high activity in the ventrolateral part of the ventral horn traced into the ventral root region and probably corresponding to motor neurons, another area of high chat activity was found in the apical part of the dorsal horn. in motor neuron disease the re ductions in spinal chat are not restricted to the motor neuron areas (9) indicating a more general involvement of cholinergic structures. in view of the rapid development and application in human post-mortem tissue of immunohistochemical methods for the locali zation of neurotransmitter systems tissue sampling by methods 241 like the "punch-technique" might be of less importance in the future. however, biochemical analysis of enzyme activities within well defined tissue specimens will still represent an important complementary technique especially in quantitative investigations. 4. large section cryomicrotomy for in vivo receptor autoradiography in recent years in vitro autoradiographic techniques (17) have been extensively employed for localization of receptor sites within different tissues. so far, the methods have mainly been applied to rather small sections, like in our own studies, the spinal cord (8, 11). however, technical modifications have now been worked out permitting in vitro autoradiographic studies in very large tissue sections (10). 4. 1 muscle after freezing whole human biceps brachii muscles between metal plates and horizontal cryosectioning, in vitro receptor autoradiography using 3h-alpha-bungartoxin (3h-alpha-btx) as ligand for the nicotinic cholinergic receptors has been performed (7). it could be demonstrated that in motor neuron disease 3h alpha-btx binding occurred over the entire muscle, indicating uncovering of extrajunctional sites while in the control the binding was restricted to the motor end-plate region. 4. 2 brain in the last years the development of large section in vitro receptor autoradiography (fig. 1 d) has enabled studies on regional distribution as well as quantitation of different re ceptor sites within the whole human brain (12, 15, see 13 for review). such studies represent important complements to in vivo receptor autoradiographic studies in man by positron emission tomography. of special interest in this connection is the possi bility to perform in vitro large section autoradiography on post-mortem brain sections in parallel to positron emission tomo graphy using the same 11c-labelled ligand. such studies have re cently been carried out in our laboratories using a bensodiaze pine receptor ligand (d'argy et a1 to be published). 5. conclusions in clinical neuroscience large section cryomicrotomy has found valuable applications in topographical neuroanatomy and as a tool in microdissection of tissue for neurochemical analysis. recently, 248 cryomicrotomy-in vitro-autoradiography has been introduced as a most important technique for the localization of neuroreceptors and provides a useful complement to human in vivo studies by positron emission tomography. references 1. aquilonius, s.-m., arvidson, b., askmark, h. & gillberg, p.-g.: topographical localization of end-plates in cryosections of whole human biceps muscle. muscle nerve 5:418, 7982. 2. aquilonius, s.-m., askmark, h., gillberg, p.-g., nandedkar, s., o l s s o n , y, & stblberg, e.: topographical localization of motor endplates in cryosections of whole human muscles. muscle nerve 7:287-293, 1984. 3. aquilonius, s.-m. & eckernas, s.-8.: a colour atlas of the human brain adapted to computed tomography. esselte studium, stockholm, 1980. graphical localization of choline acetyltransferase within the human spinal cord and a comparison with some other species. brain r e s 211:329-340, 1981. section cryomicrotomy in human neuroanatomy and neurochemistry. in: brain microdissection techniques (ed. a.c. cuello), pp. 165 170. john wiley & sons, chichester, 1983. 6. aquilonius, s.-m., eckernas, s . 8 . & sundwall, a.: regional distribution of choline acetyltransferase in the human brain: changes in huntington's chorea. j neurol neurosurg psychiat 4. aquilonius, s.-m., eckernas, s.-a. & gillberg, p.-g.: topo 5. aquilonius, s.-m., eckernas, s . 8 . & gillberg, p.-g.: large 38:669-677, 1975. 7. askmark, h., gillberg, p.-g. & aquilonius, s.-m.: autoradio graphic visualization of extrajunctional acetylcholine re ceptors in whole human biceps brachii muscle. changes in amyo trophic lateral sclerosis. acta neurol scand 72:344-347, 1985. 8. gillberg, p.-g. & aquilonius, s.-m.: cholinergic, opioid and glycine receptor binding sites localized in human spinal cord by in vitro autoradiography. acta neurol scand 72:299-306, 1985. 9. gillberg, p.-g., aquilonius, s.-m., eckernas, s.-a., lundqvist, g. & winblad, b.: choline acetyltransferase and substance p like immunoreactivity in the human spinal cord: changes in amyotrophic lateral sclerosis. brain res 250:394-397, 1982. io.gillberg, p.-g., jossan, s.s., askmark, h. & aquilonius, s.-m.: large-section cryomicrotomy for in vitro receptor autoradio graphy. j pharmacol methods 15:169-180, 1986. il.gillberg, p.-g., nordberg, a. & aquilonius, s.-m.: muscarinic binding sites in small homogenates and in autoradiographic sections from rat and human spinal cord. brain res 300:327-333, 1984. 12.johnstoni m.v., silverstein, f . s . , reindel, f.o., penney, j.b. jr. & young, a.b.: muscarinic cholinergic receptors in human infant forebrain: 3h-quinuclidinyl benzilate binding in homo genates and quantitative autoradiography in sections. dev brain res 19:195-203. 1985. the human brain. tins 6:284-289, 1986. areas by the punch technique. in: brain microdissection tech niques (ed. a.c. cuellof, pp. 1-36. john wiley & sons, 13.palacios, j.m., probst, a. & cortgs, r.: mapping receptors in 14.palkovits, m. & brownstein, m.j.: microdissection of brain 249 c h i c h e s t e r , 1 9 8 3 . n e u r o t r a n s m i t t e r r e c e p t o r s i n h u n t o n g t o n ’ s disease. n e u r o l o g y , 1 5 . p e n n e y , j . b . & young, a . b . : q u a n t i t a t i v e a u t o r a d i o g r a p h y of 3 2 : 1 3 9 1 1 3 9 5 , 1 9 8 2 . 1 6 . u l l b e r g , s . : the t e c h n i q u e of w h o l e b o d y a u t o r a d i o g r a p h y . c r y o s e c t i o n i n g of l a r g e s p e c i m e n s . s c i e n c e t o o l s , the lkb i n s t r u m e n t a l j o u r n a l , s p e c i a l i s s u e 2 2 3 , 1 9 7 7 . r a d i o g r a p h y : 3 h o p i o i d r e c e p t o r s i n r a t b r a i n . b r a i n r e s 1 7 9 : 1 7 . young, w.s. & k u h a r , m . j . : a new m e t h o d f o r r e c e p t o r a u t o 2 5 5 2 7 0 , 1 9 7 9 . address f o r r e p r i n t s : s t e n m a g n u s a q u i l o n i u s d e p a r t m e n t of n e u r o l o g y u n i v e r s i t y h o s p i t a l s-751 8 5 u p p s a l a sweden 250 upsala j med sci 99: 231-236, 1994 4. analytical quality specifications elizabeth m.s. gowansl, per hyltoft petersen2, ole blaabjerg2 1 . clinical biochemistry, telford hospital, telford tf6 6tf, shropshire, england. 2. department of clinical chemistry, odense university hospital, dk-5000 odense c, denmark 4.1 overall goal for analytical quality specifications it has become dogma that reference intervals for biochemical quantities (components) are method dependent and, therefore, are individual to laboratories. this may have arisen largely because a plethora of factors, including laboratory methodology, do affect reference values. the dogma has been further stimulated by external quality assessment schemes and proficiency testing schemes in which considerable differences between analytical methods and commercial kits have not only been tolerated but also stimulated by grouping into so called method dependent or peer groups. there are a few exceptions from this general approach which include the national cholesterol education program in usa and german control schemes (ringversuche and indstand). however, more correct approaches seem to be developing in us, and through working groups under the auspices of european external quality assessment organizers. in consequence the future may show a general and more informed attitude to the question of transferability of data and, thereby, also to the development and use of common reference intervals. it may be that ethnic differences exist for some quantities, as illustrated by harris et al. (2) as well as ageand sex-dependent differences but this does not provide a rationale for method dependent reference intervals. these biological differences, however, should be described in detail so that the information could be shared by all laboratories. the purpose therefore is to evaluate the analytical quality specifications required for the performance needed for using common reference intervals in laboratories where the populations are homogeneous for a quantity irrespective of whether there is one single interval or several are required according to known differences in the populations. 23 i assumptions for the models a number of assumptions have to be fulfilled for establishing common reference inter vals: 1. 2. 3. 4. 5 . 6. 7. 8. the population or well described subsets must be homogeneous for the quantity. the inclusion and exclusion criteria for the reference sample group must be clear. the preparation of reference individuals before sampling must be well defined. the sampling technique must be standardized. handling, preparation, and storage of samples must not influence the quantity, neither the structure nor the concentration or activity. the model for statistical calculations must be in accordance with the actual distribution of data. the number of reference individuals must be sufficient. the assays must be performed with an analytical quality which is better than the quality specifications for sharing common reference intervals as given below. this infers standardization with traceability of concentration values and specific measurement procedures. the models for homogeneous healthy groups many quantities are distributed symmetrically or with a positive skewness, allowing for application of one of two statistical models, namely gaussian and log-gaussian. the evaluation of the gaussian model is simpler and it is, therefore, used for the principal evaluation. ifcc has given recommendations for estimation of reference intervals where a major point is that at least 120 individuals should be used for the calculation of reference intervals in order to keep the uncertainty about these limits low (5). for a gaussian distribution, this corresponds to a 0.90 confidence interval of each limit of 0.24 times the biological standard deviation, or, that the fraction of individuals outside each limit due to the uncertainty of the sample variation with 0.90 certainty is between 0.013 and 0.044 instead of t h e 0.025 which is expected from an interval calculated as mean k l.96*sbiologi~. 232 the basis of the model for evaluation of analytical quality specifications is to estimate the reference intervals based on a greater number of individuals (e.g. 800), so as to make the sample uncertainty negligible and then allow for analytical error instead of sampling error giving the same maximum uncertainty as allowed by the ifcc. the quality specifications based on this concept, the maximum allowable analytical error combined bias and imprecision must not decrease or increase the fraction outside each reference limit more than 0.013 to 0.044. a graphical evaluation (1) reveals a functional relationship between maximum allowable analytical bias and imprecision as shown in fig. 4.1. analytical bias in fraction of biological standard deviation 0.20 0.10 0.00 maximum allowable combination of analytical i " " l " " 1 maximum allowable combination of analytical i \bias and imprecision 0.00 0.25 0.50 0.75 imprecision in fraction of biological standard deviation fig. 4.1. relationship between maximum allowable combination of analytical bias and imprecision according to the described concept. the units on the axes are fractions of the biological standard deviation, sbiologicap from hyltoft petersen and hgrder (4) with permission. the figure shows that the maximum allowable bias is l b ~ l < 0.24*sbiologi~ when analytical imprecision, sa, is negligible, and that the maximum allowable imprecision is sa < 0.55*sbiologid when ba is negligible. for all other combinations, the values have to be interpolated from the curve. these are the analytical quality specifications for sharing common reference intervals when the distribution is gaussian. 233 for log-gaussian distributions, the procedure is t h e same for log-values and the analytical quality specifications are the same on t h e log-level. the computations, however, are somewhat more difficult. therefore, a graph may help in making the estimates by reading off from a curve. this functional relationship is given in fig. 4.2. the most important fact from log-gaussian distributions is that the analytical quality specifications are dependent on the ratio between the upper and lower reference limits, whereby, the specifications are given as a net of curves. moreover, the specifications are given in terms of percentage bias and percentage coefficient of variation. bias: 6% 0 5 10 15 20 2 5 30 35 imprecision: cv% fig. 4.2. approximated maximum allowable combination of analytical bias (%) and coefficient of variation (%) for log-gaussian distributions. the specifications depend on the ratio between the upper and lower reference limits. from hyltoft petersen et al. (3) with permission. when analytical quality specifications are interpolated from the curves (fig. 4.2.), then t h e analytical imprecision should have been subtracted from the dwtribution in order t o get the isolated distribution (see below). based on the concepts of sharing common reference intervals and of allowing for analytical bias and imprecision instead of uncertainty from samples size it is possible to estimate analytical quality specifications for all endogenous quantities. 234 possible modifications even with the best analytical methods, there will be some uncertainty during measurements of the reference values and, in consequence, in the estimate of reference intervals. furthermore, laboratories using common reference intervals will dwclose a t least some inherent imprecision, which must be included in the stated reference interval. it might, therefore, be considered whether a reasonable low imprecision should be included in the estimation of reference intervals. if so, then the analytical quality specifications are slightly changed, and for laboratories with stable perfomance close t o the imprecision obtained during measurements of reference samples, the specification for analytical bias will be of major importance. for quantities with high ratios between the upper and lower reference limits the effect will be negligible as long as cva is below 5% but, for quantities with low ratios, it may be necessary to use the combined analytical and biological cv for the estimation of reference intervals. in chapter 7 this pragmatic approach is applied to s-albumin. references 1. 2. 3. 4. 5 . gowans ems, hyltoft petersen p, blaabjerg 0, h ~ r d e r m. analytical goals for the acceptance of common reference intervals for laboratories throughout a geographical area. scand j clin l n b invest 1988;48:757-64. harris e q wong et, shaw st. statistical criteris for seperate reference intervals: race and gender groups in creatine kinase. clin chem 1991;37:1580-2. hyltoft petersen p, gowans ems, blaabjerg 0, h ~ r d e r m. analytical goals for estimation of non gaussian reference intervals. scand j clin lab invest 1989;49:727-37. hyltoft petersen p, h ~ r d e r m. influence of analytical quality on test results. in magid e (ed.). some concepts and principles of clinical test evaluation. nordkem, nordic clinical chemistry project, helsinki finland 1992:65-87. solberg he. approved recommendation (1987) on the theory of reference values. part 5. statistical treatment of collected reference values: determination of reference limits. j clin chem clin biochem 1987;25:645-56. 235 upsala j med sci 91: 205-208, 1986 analytical quality, test quality and quality costs torgny groth unit of biomedical systems analysis, uppsala university, uppsala, sweden abstract this paper discusses briefly analytical quality and i t s determinants design of quality assurance procedures on the basis of quality specifications and quality-cost rrrodels test quality ar.d medical costs external quality assessment afid harmonization of clinical chemical test results the potential role of computers, database and knowledge-based systems in achieving quality goals in decentralized analytical activities. analytical quality the analytical quality of a laboratory test result is determined not only b y the quality of the analytical method prcper, but also b y t h e calibration procedure and the control procedure. the saying that 'a chain is no stronger than the weakest link', applies also here, and it is necessary to consider all three links when attempling to optimize the performance of the total analytical procedure. analytical quality is generally expressed in terms of analytical imprecision and analytical bias. this may be appropriate during stable analytical performance. however, the existence of analytical dis turbances resulting in e . g . systematic shifts in baseline and increases in the inherent random e r r o r , alsc influences on the analytical quality of reported test results. the need of a quality control procedure depends on the fre quency of occurencc and the magnitude of these disturbances (11). for instance, i f analytical disturbances occur very seldom or never at all, or if the magnitude is negligible compared to allowable analytical e r r o r s , we do not actually need a quality control system. or) the other hand, i f we do not know the nature of analytical disturbances we require an effective quality coritrol procedure to find out. 205 design o f quality assurance p r o c e d u r e s given the limits for allowable analytical e r r o r s we can estimate critically. sized analytical disturbances, i. e. how large systematic shifts and how large increases in the inherent random e r r o r we can accept without jeopardizing the quality goal. recommendations of allowable analytical e r r o r s in various clinical situations have been published ( c f . 5,6), but much work still remains to be done to assess the quality requirements in primary care. such quality goals derived from medical needs form a rational starting point for a quanti tative design of quality assurance procedures ( 8 ) . computer simulation techniques a r e then very helpful to evaluate various statistical control rules and to determine the optimal number of controls and calibration standards for a particular analytical method (1). a suitable optimization criterion would be to minimize the quality-costs of the procedure ( 1 2 ) . this formulation of the problem considers costs related to r e r u n s caused by t r u e and false rejects of analytical r u n s , requests for r e r u n s in connection with false (and true) accepts, in additon to costs for calibration and control material, training of personnel etc. the multi-rule shewhart procedure published as a "proposed selected method" in clinical chemistry (91, was designed to be applicable also in small laboratories without computer support. it is not very likely, how e v e r , that primary care doctors will plot these types of control charts manually. microcomputers a r e required for implementation of working quality control procedures i n these settings (10). more complex trend analysis techniques could also be easily applied for continuous monitoring of critical changes in accuracy and imprecision. colour-coded displays would fascilitate the interpretation and communication of actual control status to non-labora tory personnel. test quality and medicai, costs a clinical chemistry test is not only an analytical determination but also a decision criterion. the quality of a test therefore also depends on the choice of the discriminatory limit. it should be realised that the conventional reference limits for healthy individuals provided by most central laboratories do not generally constitute optimal discriminatory limits. such limits can only be calculated after specification of the relative importance to avoid m i s classification of different types (falsely positive and falsely negative out comes) in order to minimize costs for misclassification. costs and incon veniences related to misclassification may, to the extent they are quanti fiable, be used a s measures of the importance to avoid misclassification. these costs include "financial cost" for personnel, investigations and care etc. , "health costs" related to mortality, morbidity, pain and anxiety in connection with diagnostic investigations and therapy. 206 the optimal value of the discriminatory limit and the predictive value of a test also critically depends on the prevalence of disease in the situation the test is applied. the analytical quality may also influence on the useful ness of the test. computer simulation is a powerful technique for studying this problem complex. this is illustrated in ref. ( 3 ) as applied to screening of patients with hyperparathyroidism with use of serum calcium determina tions. the same technique can also be applied to assess quality requirements in connection with diagnosis and patient monitoring. harmonization of test results an external quality assessment program should lead to some concrete measures to decrease the variation between various laboratories and differences between single instruments within a laboratory. this problem of "harmonization" of reported test results could be approached by correction of systemic differences a s estimated from analyses of "accuracy assessment standards" and comparison with definitive o r reference methods. this problem will grow in importance with the increased decentralisation of labora tory services. the conditions for a successful implementation of such a 'lharmonization program" has been investigated by computer simulation ( 4 ) , and is practiced in full scale in connection, with health screening in france (p. valdiquik , personal communication). database management systems ( 2 ) and data communication technology ( 7 ) are necessary prerequisites for a successful achievement. tools for implementation of quality assurance procedures a s mentioned above microcomputers a r e necessary for implementation of working quality assurance procedures in decentralized laboratory activities. computers w i l l probably be commonly available in primary care centers and the doctor's office for word processing, patient administration e t c . , so there should be no severe economical constraints. first generation quality control packages have been commercially available for some years ( 1 0 ) . the next generation of these packages will provide powerful database management facilities, advanced colour graphics and sophisticated reasoning mechanisms providing advice on how to do in various situations to assure the specified quality. transmission of quality control data between laboratories for ex ternal quality assessment and harmonization programs is another important facility of such a workstation. knowledge-based systems a r e also of great interest for supporting proper test selection and interpretation of test results, considering knowledge on age-, sexand disease related reference values, optimal discriminatory limits, and the influence of d r u g s on analytical determinations. 207 1. 2 . 3. 4 . 5 . 6 . 7 . 8 . 9 . 1 0 . 11. 1 2 . references groth, t . , falk, h . & westgard, j . o . : a quality control simulator for. design and evaluation of internal quality control procedures. scand j clin lab invest 4 4 (suppl. 172):195-201, 1984. groth, t., larsson, o . , aronsson, t. & de verdier, c-h. : data base management systems for evaluation of analytical procedures. scand j clin lzb invest 44 (suppl. 172):209-213, 1984. groth, t . , ljunghall, s . & de verdier, c-h. : optimal screening for patients with hyperparathyroidism with use of serum calcium observa tions. a decision-theoretical analysis. scand j clin lab invest groth, t . : strategies for decreasing interlaboratory variation. studies by computer simulation. scand j clin lab invest 4 4 (suppl. 171):331-346, 1984. hdrder, m. (ed) : assessing quality requirements in clinical chemistry. report by a project group initiated by nordic clinical chemistry project. scand j clin lab invest 40 (suppl 1 5 5 ) : l 1 4 4 , 1980. skendzel, l.p. , barnett, r . n . & platt, r . : medically useful criteria for analytic performance of laboratory tests. a m j clin pathol 83:zoo-205, 1985. valdiguic , p.m., fernet, p. .% verdier, b . : transmission of quality control data. in proceedings of fifth int cont on computing in clinical laboratories, s t u t t g a r t , june 1985. westgard, j . o . , groth, t. & de verdier, c-h.: principles for developing improved quality control procedures. scand j clin lab invest 4 4 (suppl 172) : 1 9 4 1 , 1984. westgard, j . o . , b a r r y , p . , hunt, m., & groth, t . : a multi-rule shewhart chart for quality control in clinical chemistry. clin chem westgard, j . o . & groth t . : computer systems for implementation of internal quality control procedures. scand j clin lab invest 44 (suppl westgard, j.o. & groth, t . : a predictive value model for quality control. effects of the prevalence of e r r o r s on the performance of control procedures. am j clin path 80:49-56, 1983. westgard, j . o . , hyltoft petersen, p. & groth, t.: the quality-costs of an analytical process. development of quality-costs models based on predictive value theory. scand j clin lab invest 4 4 (suppl 4 3 : 6 9 9 7 0 7 , 1983. 27:493-501, 1981. 172):203-207, 1984. 172):221-227, 1984. 208 upsala j med sci 92: 79-83, 1987 two forms of az-antiplasmin: post-traumatic changes in the rat h. hogstorp and g . carlin institute of forensic medicine, wniversity of uppsaia, wppsafa, sweden abstract the plasminogen-binding (pb-ap) and non-plasminogen-binding (npb-ap) forms ofw2-antiplasmin (ap), were assayed in rat plasma by a modified rocket immunoelectrophoretic technique before and up to 48 h after turpentine-induced trauma, using an intermediary gel containing kringles 1-3 from plasminogen. the concentration of pb-ap was signifi cantly elevated by 2 2 % 2 4 h post-traumatically, while npb-ap was de creased at that point in time, leaving the total ap level unchanged. total ap increased by 57 % during the period 2 4 48 h after trauma, mainly on account of increases in the npb-ap form. i t is concluded that the plasma level of ap can remain unchanged in spite of increased fibrinolysis inhibition, owing to a relative increase in the functionally more active pb-ap. introduction the important fibrinolysis inhibitor%-antiplasmin (ap) has been found to exist in a plasminogen-binding (pb-ap) and a non-plasminogen-binding (npb-ap) form in man (1, 2 , 3 , 4 , 5, 1 2 , 1 4 , 15). we have previously reported on the presence of ap in the rat and described some of i t s changes after induction of trauma ( 8 , 9 , l o ) . the purpose of the present investigation was to determine the effects of turpentine trauma on the two forms of the inhibitor in the rat. materials and methods r r chemicals: french turpentine oil (kebo ab, sweden), cnbr-sepharose 4 b , sepharoser 4b (pharmacia fine chemicals, sweden) ; agarose m 79 (lkb , sweden). anti-rat %ap-igg was prepared as described previously ( 8 ) . animals: male sprague-dawley rats (300 380 g) from the anticimex farm, stockholm, were used. they had free access to food (ewos rat pellets) and tap water throughout the experiment. all surgical procedures were carried out under ether anaesthesia. turpentine trauma was induced by an intramuscular injection of 0.5 ml of turpentine into each hind leg. animals, five at each time, were killed 2 4 , 36 and 48 h after the turpentine injection. preparation of plasma: four millilitres of blood was drawn from the aorta into a plastic tube containing 1 ml of 3 . 8 % trisodium citrate solution. the tubes were immediately centrifuged and the plasma was aspirated with siliconised pipettes and stored at -20°c until analysed. plasma from five untreated animals was pooled and used as a control. assay of the different forms ofo&,-antiplasmin: an electroimmunoassay technique mainly as described by wiman et a1 ( 1 5 ) was employed, using an intermediary gel containing kringles 1-3 from human plasminogen coupled to cnbr-activated sepharose 4b (-5 mg kringles/ml settled gel). in this gel the pb-ap form is absorbed, while the npb-ap form runs through and forms rockets in the subsequent anti-%-ap-igg, containing agarose gel. the intermediary agarose gel contained 9 % "kringle-sepharosetf. total ap was measured on the same plate, using pure sepharose 4b in the inter mediary agarose gel to equate any loss of antigen due to unspecific ad sorption. the difference i n height between the rockets was considered to represent pb-ap. pooled normal rat plasma, 0 . 5 1 . 2 5 p l , diluted to 5 1.11 in 0 . 0 2 4 m veronal buffer ph 8 . 6 , was used for standard curves. the samples contained 0 . 8 3 p1 of plasma and were diluted to 5 pl in veronal buffer. all results are expressed in per cent of total ap found in pooled normal rat plasma. statistical analysis: differences between groups were tested by wilcoxon's rank sum test. a p value below 1 % was considered significant. results the results are presented in fig. 1. pb-ap amounted to 6 3 . 1 % of the total ap in control animals. after 24 h pb-ap was significantly increased by 2 2 % to 7 7 + 4 %. a t the same time npb-ap was significantly decreased to 22 + 7 % while the total ap remained unchanged. between 24 and 48 h the total ap increased to 156 %, and this was mainly referable to an increase in the npb form. 80 % 150 100 50 i i 0 24 36 40h fig. 1 total oc2-antiplasmin (01, plasminogen-binding ( a ) and non-plasminogen -binding ( ) forms of* antiplasmin 24, 36 and 48 h after induction of trauma with turpentine. d l u e s are expressed in per cent (mean 2 s d ; n = 5 ) of totalw2-antiplasmin in normal rat plasma. discussion in this study the concentration of the plasminogen-binding form of 5 a p was increased in rat serum 24 h after turpentine trauma. there was in a previous investigation it was found that the fibrinolysis inhibition activity in serum, which involves not only plasmin inactivation but also inhibition of plasminogen activation and changes in plasmin(ogen) , fibrin interactions, was increased by 2 0 % 2 4 h after turpentine trauma even though no changes were observed in the plasmin inhibition activity in plasma or in the immunologically determined w2-antiplasmin concentration ( 9 ) . these results may be explained b y the present finding of a 2 2 % increase in pb-ap but no change i n the total ap provided that both a concomitant decrease i n the non-plasminogen-binding form. 5 t -87857 1 81 forms of w2-ap a r e potent plasmin inhibitors and that the plasminogen -binding form also interferes with plasminogen activation and with the , plasminogen uptake b y fibrin. earlier investigations w i t h use of human 5 a p have yielded results supporting this view (5, 13, 1 4 ) . preliminary studies (11) have indicated that pb-ap is synthesized by the liver, in which case npb-ap should be a metabolite, although the present findings do not preclude the reverse. a known metabolite of ap is i t s complex with plasmin, which in humans and in mice is rapidly removed from the circulation ( 6 , 7 ) . preliminary attempts to measure plasmin-antiplasmin complexes by using lysine sepharose instead of plasminogen kringles i n the intermediary gel failed to show evidence of such complexes in the present study. acknowledgements these investigations were supported by grants from the swedish medical research council, the torsten and ragnar soderberg foundation, the 80th anniversary fund of the trygg-hansa insurance company, the medical faculty of uppsala university and the swedish national association against heart and lung diseases. 1 2 3 4 5 references bagge, l., jacobsson, h . & saldeen, t . : post-traumatic variation of the primary fibrinolysis inhibitor w2-antiplasmin. in: progress in fibrinolysis. pp 307-312, volume v . churchill livingstone, 1981. bagge, l. & saldeen, t . : the primary fibrinolysis inhibitor and trauma. thromb res 13: 1131-1136, 1978. christensen, u . & clemmensen , i. : purification and reaction mechanisms of the primary inhibitor of plasmin from human plasma. biochem j 175:635-641, 1978. clemmensen , i. : different molecular forms of 06 -antiplasmin. in: the physiological inhibitors of blood coagulation 08 fibrinolysis (ed. d . collen, b . wiman & m. verstraete), pp 131-136. biomedical press. elsevier (north holland), 1979. clemmensen, i . , thorsen, s . , mullertz, s . & petersen, l . c . : proper ties of three different molecular forms of theoc2-plasmin inhibitor. eur j biochem 1 2 0 : 105-112, 1981. 6 collen, d . & wiman, b.: turnover of antiplasmin, the fast-acting plasmin inhibitor of plasma. blood 2 : 313-324, 1979. 7 gonias, s.l., fuchs, h . e . & pizzo, s.v.: a unique pathway for the plasma elimination of d. -antiplasmin-protease complexes in mice. thromb haemostasis 48(2) : 208-810, 1982. 82 8 hogstorp, h . , jacobsson, h . & carlin, g. : studies on?-antiplasmin i n 9 hogstorp, h . , jacobsson, h. & saldeen, t. : effect of hepatectomy on the posttraumatic fibrinolysis inhibition and the primary fibrinolysis inhibitor in the rat. thromb r e s 18:361-368, 1980. the r a t . thromb res 21:247-253, 1981. 1 0 hogstorp, h . & saldeen, t. : synthesis ofdc2-antiplasmin by rat liver 11 hogstorp, h . & saldeen, t.: rat hepatocytes synthesize the plasmino gen-binding form of d2-antiplasmin. abstr . xth international congress on thrombosis and haemostasis. san diego july 14, 1985. thrombosis and haemostasis no 1, vol 5 4 , p 1581. cells. thromb res 28:19-25, 1982. 1 2 kluft, c . & los, n . : demonstration of two forms of u2-antiplasmin in plasma by modified crossed immunoelectrophoresis. thromb res 2 1 : 65 -71, 1981. 13 moroi, m . & aoki, n . : inhibition of plasminogen binding to fibrin by oc2-plasmin inhibitor. thromb res 10:581-586,1977. plasmin. biochem j 191:229-232, 1980. 1 4 wiman, b . : affinity-chromatographic purifications of human w2-anti 15 wiman, b., nilsson, t . & cedergren, b . : studies on a form of olz-antiplasmin in plasma which does not interact with the lysine-binding sites in plasminogen. thromb res 28 : 193-200, 1982. address for reprints: herman hogstorp department of forensic medicine dag hammarskjolds vag 17 sweden 5-752 37 uppsala 6-878571 83 upsala j med sci 99: 339-346, 1994 7.3 reference intervals for al-antitrypsin erik lundl, ole blaabjerg2, mogens blom3, per hyltoft petersen2, jens rahbek nmgaard2, adam uldal14, hanne gry5, ivan brandslundl 1. department of clinical chemistry, vdle county hospital, dk-7100 vdle, denmark 2. department of clinical chemistry, odense university hospital, dk-5000 odense c, denmark. 3. department of clinical chemistry, hjvrring sygehus, dk-9800 hjgrring, denmark. 4. department of clinical chemistry, kas herlev, dk-2730 herlev, denmark. 5. medi-lab, adelgade 5-7, dk-1304 copenhagen, denmark. the plasma protein al-antitrypsin (al-proteinase inhibitor) has many phenotypes, which have a major influence on the concentrations as measured in serum. the most common phenotype in denmark is the mm-type, less common are the two heterozygote types ms and mz, and with low frequency the heterozygote sz and the homozygotes ss and zz. as heterozygotes have lower plasma concentrations, we decided to perform the phenotyping of the reference individuals in order to estimate separate reference intervals for type mm and the two heterozygote types ms and mz. it has recently been proposed, that if relevant reference intervals for the different phenotypes existed, and certain quality goals could be met (11, quantification of al antitrypsin could be used to rule out the disease-causing zz and sz phenotypes and also to identify all possible patients heterozygous for the z-allele. it may also be reasonable to discontinue the acceptance of mz as a normal variant, as recent publications points to this phenotype as predisposing to lung disease under certain conditions (3). hence, only the mm’s should determine the ’normal’ reference interval. analytical methods identification of genetically determined variants for al-antitrypsin was done by phenotyping the al-antitrypsin protein in serum by isoelectric focusing. the procedure described by gorg et az. (2) on immobilize@ dryplates premade by lkb, sweden (no 80-1128-29, ph 4.2-4.9) was used. in brief, gels were reswelled in 25 % w/v glycerol (applc. note 345, pharmacia), serum was reduced with dithiothreitol, blocked with iodoacetamide, and 15 yl applied to the gel (4). the electrophoresis was run at 500 v, 2ma for 18 hours, the last 30 minutes at 2500 v, 2ma7 at 12 oc on the 23-955059 339 lkb multiphor electrofocusing unit supplied with the lkb 2197 power supply. the proteins were passively transferred by blotting to immobilontm-p membranes (millipore corp., usa) and the blot-papers stained in coomassie brilliant blue r250, (merck, frg). this procedure was developed to facilitate storage, filing and reading of t h e results. the dried paper blots were interpreted by three persons, independently. this was done using known variants (mm, ms, mz, ss, and zz) as references. measurements of all samples were performed at the same time as the other eight proteins (cf. section 7.2) on a cobas fara (roche) according to the method recommendations from dako with antisera from dako using the nordic calibrator with values traceable to bcr 470. fig. 7.3.1. illustration of the electrophoretic technique used for phenotyping the individuals for estimating of reference intervals for the piasma protein al-antitrypsin. distribution of phenotypes among the 516 individuals accepted as reference population, seven was not phenotyped because of lack of sample or lost sample identification. the distribution of phenotypes in the remaining 509 individuals is shown in table 7.3.1. 3 40 table 7.3.1 2.0 1.8 1.6 1.4 1.2 1.0 a,-antitrypsin phenotypes 0.3 0.2 0.1 0.0 ~ m m m s mz ss sz zz other total men 1 8 1 9 5 0 0 0 5 200 women notusingestrogens 175 18 8 0 1 1 2 205 using estrogens 9 2 6 6 0 0 0 0 104 total 448 33 19 0 1 1 7 509 biological variation and reference intervals the biological variations are illustrated in fig. 7.3.2 with mean values and 90 % confidence intervals for each subgroup (cf. chapter 7.2) using log-concentrations as ordinate and age-groups as abscissa. the women using estrogens are separated from the remaining groups with approx. 30 % higher values, whereas, the rest are disclosing an astounding constancy. i s; t i i men j women 1 i i i i i i i 18-20 21-30 31-40 41-50 51-60 61-70 71-00 age groups fig. 7.3.2. mean concentrations with 90 % ci for each subgroup shown on a log scale in relation to age for aphtitrypsin. 34 1 distributions of the two mm-groups, i.e. women using estrogem (n = 92) and the remaining group (n = 356) are clearly straight lines indicating log-gaussiari distributions with the lines close to be parallel. the two heterogeneous groups (n = 13 for mz and n = 27 for type ms) consist of fewer individuals, so the question of whether they are log-gaussian can not be answered. both distributions, however, show lower values with mz approx. 60 % and ms approx. 80 % of the mm-values. 99 95 90 80 70 60 50 40 30 20 10 5 1 cumulated percentage frequency probit mz ms mm mm i i i i 1 1 1 -0.16 -0.08 0.00 0.08 0.16 0.24 0.32 log g/l 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.8 2.0 2.2 g/l 1 i i i i i i 1 i i ' i ' i ' i fig. 7.3.3. probit-plot with log-abscissa showing a,-antitrypsin distributions for the phenotypes mz, ms, and mm, where the mm-group is separated into women using estrogens and the remaining group. as for s-transferrin (chapter 7.1 and 7.2) t h e group of women using estrogens is separated into 'high dose group' and 'low dose group' indicating negligible effect for the 'low dose group' as illustrated in fig. 7.3.4. the reference interval for women below 50 years of age using estrogens (type mm) is 1.29 to 2.23 g/l and for the remaining group (type mm) 0.97 to 1.68 g/l, whereas, for type ms it is 0.85 to 1.32 g/l and for type mz 0.60 to 0.99 g/l (both without women using estrogens). 342 cumulated percentage frequency probit 99 95 90 80 70 60 50 40 30 20 10 5 1 /mm / women (low e) a r j (high e) mmi i i i i i 1 i i 1 i i i i i ” ’ i i i 1 -0.16 -0.08 0.00 0.08 0.16 0.24 0.32 log g/l 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.8 2.0 2.2 g/l i fig. 7.3.4. distributions of al-antitrypsin for women using high and low dose of estrogens. results for the excluded individuals according to the rule out criteria as described for the other eight proteins 37 individuals were ruled out due to high erythrocyte sedimentation rate, high s-crp or the presence of m-components. details of the groups as for s-transferrin (cf. sections 7.1 and 7.2). the distributions are shown in fig. 7.3.5 on a probit-scale, for type mm only (women below 50 years of age using estrogens and the remaining group). there is a clear increase in all s-al-antitrypsin values for individuals with elevated s-crp. for m components and elevated sedimentation rate there is a tendency towards higher values with broader distributions. discussion the limitations for evaluation of s-al-antitrypsin are the same as for the other eight proteins (cf. chapter 7.2) but, in addition the separation into phenotypes mm, ms, and mz (and others) reduces the number of individuals in the subgroups. therefore, t h e reference intervals for ms and mz are to be considered more as guidelines than exact. 343 cumulated percentage frequency p r m i mm mm others ,a sedimentation rate crp (e) i i i i i i i 1 i i i i i 1 i i i ' i ' i ' i -0.16 -0.08 0.00 0.08 0.16 0.24 0.32 log g/l 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.8 2.0 2.2 g/l probit-plot with log-abscissa showing a,-antitrypsin distributions for the phenotype m m , the ruled out individuals. fig. 7.3.5. due to the difficulties in the traceability of the values to crm 470 as described in chapters 5 and 6, there is an additional problem for s-al-antitrypsin, as the results are dependent on the analytical principles and the antisera used. therefore, the reference intervals are valid for turbidimetric methods and with the use of antisera from dako, only. for other methods and other antisera, the traceability has to be documented (section 5.5). acknowledgements we want to thank all the persons who have contributed to this work, volunteers who gave blood to the investigation and the 20 danish laboratories involved in collecting the blood samples and doing analytical work. we are specially in debt of gratitude to yrsa eriksen and gudrun andersen, who performed the isoelectric focusing, and to inger n~rgaard, who performed the protein determinations and the agarose electrophoreses for estimation of m-components, and to ane richter, who performed the registrations and calculations of the many results. further we are grateful to s ~ r e n blirup and per just svendsen, who performed the measurements of s-crp and for the donation of antisera used in all the determinations delivered by dako . 344 references 1. brandslund i, lund ed, sigsgaard t. is a low serum concentration of al-antitrypsin associated with an increased susceptibility for byssinosis in cotton mill workers? upsele j m e d sci 1993;98:299-310. 2. gorg a, postel w, weser j, weidinger s, patutschnick w, cleve h. isoelectrick focusing in immobilized ph gradients for the determination of the genetic pi (al-antitrypsin) variants. electrophoresis 1983;4:153-7. sigsgaard t, brandslund i, rasmussen jb, lund ed, varming h. low normal al-antitrypsin serum concentrations and mz-phenotype are associated with byssinosis and familial allergy in cotton mill workers. pharmacogenetics 1994;4: 135-41. thymann m. distribution of alpha-1-antitrypsin (pi) phenotypes in denmark determined by separator isoelectric focusing in agarose gel. hum hered 1986;36 19-23. 3. 4. 345 upsala j med sci 96: 2 13-2 18, 199 1 goitre and deaf-mutism abdelaziz elamin department of paediatrics & child health, university of khartoum, box 102, khartoum, sudan abstract the occurrence of congenital deafness, mutism and goitre unassociated with cretinism or mental retardation in euthyroid patients is known as pendreds syndrome. it has been estimated that 4-10 % of children with congenital deafness suffer from this condition. the perceptive hearing loss is considered to be present at birth although it is frequently not recognized for several years. the cause of the hearing defect is a congenital bilateral malformation of the cochlea of the mondini type. the goitre is not recognized clinically at birth or in early childhood. it becomes apparent in the pre-pubertal years when it presents as a colloid enlargement progressing to a nodular goitre. the thyroid defect has been shown to be a partial defect in iodine organification leading to the underproduction of thyroxine and subsequent thyroid hyperplasia. the syndrome is caused by a single mutant recessive gene responsible for both the deafness and goitre. its autosomal mechanism gives an equal incidence in both sexes, unusual in thyroid disease. this article reviews the current aspects of pathogenesis and treatment of this syndrome and reports its occurrence in two sudanese siblings. introduction the association of congenital deafness and goitre was first described by wood in 1824 [ l ] . however, in 1965 fraser [2] in a detailed monograph reviewed from the literature 233 cases of goitre and congenital deafness and suggested the eponym of pendred's syndrome after the british general practitioner vaughan pendred who in 1896 described two deaf-mute sisters with pronounced nodular goitre [3]. in 1927 brain reported cases of five families in which 12 members had congenital deafness and developed goitre during childhood [4]. after making extensive studies on his patients he came to the conclusion that the syndrome is a genetic disorder caused by 15-918573 213 a single mutant recessive gene responsible for both the deafness and goitre. the gene frequency vanes between 1 in 500 to 1 in 1500 and its autosomal mechanism gives an equal incidence in both sexes, unusual in thyroid diseases which is far more common in girls than boys. the responsible gene has high penetrance, but the intenseness of expressivity may vary within the same family and usually only one generation is affected [5]. it has been estimated that 4-10 % of children with congenital deafness suffer from pendred’s syndrome [6,7]. pathogenesis the perceptive hearing loss is considered to be present at birth although it is usually not recognized for several years. the nature of the hearing loss is debatable. earlier workers relate the hearing defect to intra-uterine thyroid hormone deficiency. according to hodges and his associates [s], the fetal thyroid begins to function at the 12th week of fetal life. if the contribution of the fetus thyroid hormone is inadequate because of an inherited defect the normal development of the nervous system will be incomplete with resultant acoustic nerve damage. thould and scowen attributed the nerve deafness associated with goitre to intra-gestational hypothyroidism [9]. the increasing prevalence of goitre and deaf-mutism in regions of endemic goitre support the association between the two conditions [ 101. however, most of the published literature regarded the syndrome as a separate entity not directly related to endemic cretinism which is characterized by profound mental retardation and nerve deafness [ 1 i]. individuals with deaf-mutism and goitre are usually euthyroid and have normal intelligence. the deafness in pendred’s syndrome is believed to be due to a congenital bilateral malformation of the cochlea of the mondini type [ 12 1.51. the increased occurrence of deaf-mutism in places with endemic iodine deficiency is explained by the high frequency of consanguinity and intermarriages in these areas [16]. the goitre is not recognized clinically at birth or in early childhood. it becomes apparent in the pre-pubertal years when it presents as a colloid enlargement progressing to a nodular goitre in the majority of patients. morgans and trotter in 1958 demonstrated a presumed specific enzymatic defect as the cause of goitre in this condition [17]. the thyroid defect has been shown to be a partial defect in iodine organification leading to the under production of thyroxine [18]. this triggers the secretion of thyroid stimulating hormone (tsh) which stimulates thyroid gland hyperplasia to compensate for the defect by maintaining the minimal level of thyroid hormones production to keep the patient euthyroid. the organification defect is best demonstrated by the perchlorate discharge test which is considered diagnostic in children with goitrous deafness. however, some authors reported considerable variations in this test [ 191. 214 treatment in the past, thyroidectomy was the standard treatment because of the size of the goitre and the pressure symptoms it was causing and because of the extreme hyperplasia seen on thyroid tissue which was confused with malignancy. such experience was reported by elman [20], who found, in two of his patients, changes in the thyroid tissue sufficient for the diagnosis of adenocarcinoma. smith [21] reviewed the pathology of the thyroid tissue removed at operation from several patients with this syndrome. one of the 14 cases of his series was considered to have malignant changes. recently, it has been accepted that surgery may not be needed particularly in children. life long thyroid medication is recommended with the implication that the goitre will be greatly reduced in size or may actually disappear. unfortunately, once the deafness is recognized no treatment at present will reverse it. however, in 1973, deol [22] reported a series of fascinating experiments in mice that could be of clinical importance to man. female mice fed propylthiouracil during pregnancy produced deaf offsprings, but when they fed thyroxine together with propylthiouracil from day one of gestation, no hearing defect occurred in the offsprings. furthermore, when the offsprings of a female mouse who received antithyroid drugs during gestation, were fed thyroxine started immediately after birth, no hearing defect occurred. any practical application of these findings can only be evaluated when a group of newborn babies, who have a sibling with pendred’s syndrome, given thyroxine on day one of life and followed prospectively for development of deafness. because of the high penetrance of this syndrome, it might be advisable to give thyroxine to all newborn siblings of patients with pendred’s syndrome as early as possible. case reports two brothers, ahmed s . , 13-year-old, and osman s . , 7-year-old, were referred to the paediatrics clinic of the university hospital in khartoum, sudan because of thyroid swelling. both boys were known to be deaf and mute since the first year of life. the swelling over the neck appeared at the age of 10 years in the elder brother and 8 months before presentation i n the younger brother and was increasing in size. the parents were first degree relatives and they have another 4 normal children, 2 boys and 2 girls. there was no history of deafness in other relatives, but a cousin has had an endemic goitre with no hearing problem. they live in a village on the west bank of river white nile 160 km south of khartoum, an area with a large number of endemic goitre cases. examination revealed nice cooperative mute children with apparently normal intelligence. they did not attend any school because the only school for deaf children is in 215 khartoum, far away from their village. the thyroid swelling was enormous and nodular in the elder boy and diffuse and relatively small in the younger brother. it were not attached to the skin or underlying structures and no bruit could be heard over them. there was no clinical symptom or sign of hypo or hyperthyroidism and the rest of the physical examination was non contributory. height was below the 25th centile of nchs standards [23] and x-ray examination revealed slight retardation in bone age. thyroid function tests showed thyroxine and t3 levels in the low normal range and an elevated tsh level in both brothers. thyroid scan showed diffuse hyperplasia of the gland with no hot or cold areas in the younger child and a solitary cold nodule on top of diffuse hyperplasia in the other. potassium perchlorate discharge test was positive in both patients. the audiogram showed bilateral high tone deafness in both patients. a diagnosis of pendred’s syndrome was made on the basis of the presence of its classical mad namely, goitre, nerve deafness and positive perchlorate discharge test. although the two patients did not show any clinical symptoms of hypothyroidism, the reduction in height and bone age and the results of thyroid function indicated a slightly decompensated gland. both children were started on l thyroxine immediately after confirmation of diagnosis. conclusion pendred’s syndrome accounts for 4-10% of cases of congenital deafness in childhood. it is increasing recognized in places where iodine deficiency is prevalent, although the association is still unclear. the hearing defect appears early in life and is not reversible once established. the goitre usually appears in the prepubertal years and progresses in size thereafter. surgery is not indicated, but thyroxine should be given as soon as the diagnosis is made and continued for life to reduce the size of goitre and to prevent recurrence. because of the high penetrance of this condition and because of possible benefits siblings of known cases should be given thyroxine from the f i s t day of life. genetic counseling should be available to parents who have a child with pendred’s syndrome and to young adults with this condition. . references 1. wood, k.: memoirs of the literary and philosophical society of manchester, second series 9: 83, 1824. 2. fraser, g.r.: association of congenital deafness with goitre (pendred’s syndrome). ann hum genet 28: 201,1965. 216 3. pendered, v.: deaf mutism and goitre. lancet 2: 532,1896. 4. brain, w.r.: heredity in simple goitre. quar j med 20: 303-319, 1927. 5. thieme, e.t.: goitre and deafmutism. ann surg 182: 173-176, 1975. 6. illum, p., kiaer, h.w., hvidberg-hansen, j. & sondergaard, g.: fifteen cases of pendred’s syndrome, congenital deafness and sporadic goitre. arch otolaryingol 96: 297, 1972. 7. batsaki, j.g. & nishiyama, r.h.: deafness with sporadic goitre: pendred’s syndrome. arch otolaryng 76: 401-406, 1962. 8. hodges, r.e., evans, t.c., bradbury, j.t. & keettel, w.c.: accumulation of radioactive iodine by human fetal thyroid glands. j clin endocrinol 15: 661, 1955. 9. thould, a.k. & scowen, e.f.: genetic studies of the syndrome of congenital deafness and simple goitre. ann hum genet 27: 283-288, 1964. 10. bilginturan, n.: congenital deafness and goitre (pendred’s syndrome). turk j pediatr 8: 216,1966. 11. purves, r.d.: deafness and goitre: pendred’s syndrome. nz med j 76: 89-93, 1972. 12. altmann, f.: the inner ear in genetically determined deafness. acta oto-laryng 58 (suppl 187): 1-39, 1964. 13. jensen, j.: malformations of the inner ear in deaf children. a tomographic and clinical study. acta radio1 (suppl286): 1, 1969. 14. hvidberg-hansen, j. & jorgensen, m.b.: the inner ear in pendred’s syndrome. acta oto laryng 66: 129-135, 1968. 15. andersen, p.e.: radiology of pendred’s syndrome. adv oto-rhino-laryng 21: 9-18, 1974. 16. morgans, m.e.& trotter, w.r.: association of congenital deafness with goitre: the nature of the thyroid defect. lancet 1: 607-609, 1958. 17. fraser, g.r., morgans, m.e. & trotter, w.r.: the syndrome of sporadic goitre and congenital deafness. quart j med 29: 279-295, 1960. 18. neipomniszcze, h., coleoni, a.h., degrossi, o.j., scavini, l.m. & curutchet, h.p.: biochemical studies on the iodine organification defect of pendred’s syndrome. acta endocrinol 89: 70-79, 1978. 217 19. thould, a.k. & scowen, e.f.: the syndrome of congenitai deafness with goitre. j endocrinol 30: 69,1964. 20. elman, d.: familial association of nerve deafness with nodular goitre and thyroid carcinoma. new engl j med 259: 219-223, 1958. 21. smith, j.f.: the pathology of the thyroid in the syndrome of sporadic goitre and congenital deafness. quar j med 29: 297-303,1960. 22. deol, m.s.: an experimental approach to the understanding and treatment of hereditary syndromes with congenital deafness and hypothyroidism. j med genet 10: 235, 1973. 23. nchs growth curves for children birth to 18 years; us series 11 (165) dhew publication number (phs) 78-1650. department of health, education and welfare, hattsville 1977. correspondence address: dr. abdelaziz elamin, international chiid health unit, university hospital s-751 85 uppsala, sweden 218 upsala j med sci 95: 157-160, 1990 hypercalcemia in pulmonary tuberculosis l. lind, s. ljunghall department of internal medicine, university hospital, uppsala, sweden abstract tuberculosis can affect calcium metabolism, mainly through an enhanced production of active vitamin d. the incidence of hypercalcemia among unselected patients with active pulmonary tuberculosis was investigated, retrospectively, during a ten-year period. among 67 patients, the mean serum calcium concentration on admission was significantly raised compared to healthy controls (2.51 f 0.16 (sd) vs 2.43 f 0.07 mmol/l; p<o.ool) and 25 % of the patients had hypercalcemia. after one year of successful tuberculostatic treatment the serum calcium values had normalized. introduction active pulmonary tuberculosis can be accompanied by disturbances of calcium metabolism (1, 3, 4, 6, 9, 10). sevral mechanisms could be involved, e.g. increased production of bone-resorbing factors (8), a reduced degradation of prostaglandins (l), but an enhanced synthesis of active vitamin d is generally considered to be most important (3, 5, 6, 7, 9), as also established in sarcoidosis (2, 5, 11). in clinical practice hypercalcemia is not recognized as a common complication of tuberculosis but there is little information about its incidence among unselected patients. material and methods hospital records from patients being treated for pulmonary tuberculosis at the departments of lung diseases at gavle county hospital, sweden, during the years 1976 through 1985, were investigated in retrospect. all of the following criteria were fulfilled by each patient: 1. pulmonary tuberculosis visible at chest x-ray and verified by positive direct sputum examination or positive loewenstein culture. 2. uninterrupted and uncomplicated treatment for one year with isoniazid and rifampicin, supplemented with myambutol for the first three months. 3. healing of the disease after one year as could be judged from chest x-ray, laboratory and clinical findings. 157 4. 5. pretreatment and post-treatment (at 1 1-1 2 months) values of serum calcium and albumin measured in the same autoanalyzer. the patient should not have received vitamin d or calcium supplementation either before or during chemotherapy. sixty-seven patients (mean age 60 k 1 4 years, 39 men and 28 women) fulfilled the criteria. for comparison of serum calcium values a control group, recruited from a health survey, was investigated. serum calcium and albumin were measured in the autoanalyzer (smac, technicon, usa) used in hospital practice. the values of serum calcium, with a reference range of 2.20-2.60 rnmol/l, are all adjusted for albumin. the coefficient of variation for serum calcium is 1-2 %. treatment effects as well as the comparison between patients and the controls were statistically analysed by student's t-test. comparisons of the proportions of hypercalcemia in patients and controls were made by the chi-square test. s e r u m c a l c i u m ( m m o l / l ) 0 000 0 0 0 00 000 000000 0000000 0000 oooo oooo ow occccxx coco coo 0 oooo 3 0 0 0 0 b a controls 158 r e s u l t s among the 67 patients with pulmonary tuberculosis 25 yo were hypercalcemic (more than 2.60 mmolll) before initiation of chemotherapy. only two persons (3 yo) showed hypercalcemia in the control group (pco.001). the mean value for serum calcium in the patients before treatment was 2.51 k 0.16 (sd) mmol/l which was significantly higher than the mean level found in the control population (2.43 f 0.07, peo.001). after one month of treatment serum calcium levels had decreased to 2.45 f 0.1 1 (p<0.05) and a further reduction to 2.39 f 0.10 mmol/l was seen at the end of chemotherapy (pe0.01, see figure). at this time only one patient had a serum calcium value above the reference range. all together 52 of the 67 patients (78 %) displayed a reduction of the serum calcium values during treatment. the changes of serum calcium were also evident when the values were adjusted for the small rise (2 g/1) in serum albumin which occurred during therapy. d i s c u s s i o n our study demonstrates that mild hypercalcemia is a common finding in unselected patients with pulmonary tuberculosis. while other reports found hypercalcemia generally associated with vitamin d supplementation and sunlight exposure, all our patients with high serum calcium levels were hypercalcemic without supplementation. the uniform distribution of calcium levels before treatment suggests that the alterations of calcium metabolism are not restricted to a few hypercalcemic subjects but is a general phenomenon which accompanies pulmonary tuberculosis. serum calcium also decreased uniformly in most patients after prolonged chemotherapy . acknowledgements county hospital, who have given us advice for this study. we are grateful to the colleagues at the department of lung diseases, gavle references 1. 2. 3. 4. 5. abbasi, a.a., chemplavil, j.k., farah, s., muller, b.f. & arnstein, a.r.: hypercalcemia in active pulmonary tuberculosis. ann int med 90: 324-328, 1979. alberts, c. & van den berg, h.: calcium metabolism in sarcoidosis. a follow-up study with respect to parathyroid hormone and vitamin d metabolites. eur j respir dis 68: 186-194, 1986. cadranel, j., milleron, b., garabedian, m. & aroun, g.: serum concentration of vitamin d metabolites in untreated tuberculosis. thorax 40: 639-640, 1985. davies, p.d.o., church, h.a., brown, r.c. & woodhead, j.s.: raised serum calcium in tuberculosis patients in africa. eur j respir dis 71: 341-344, 1987. epstein, s., stern, p.h., bell, n.h., dowdeswell, i. & turner, r.t.: evidence for abnormal regulation of circulating 1,25-dihydroxyvitamin d in patients with 159 pulmonary tuberculosis and normal calcium metabolism. calcif tissue int 36: fuss, m., karmali, r., bergman, p., gillet, c., prigogine, t., schmerber, j., ' brauman, h. & corvilain, j.: hypercalcemia and elevated 1,25-dihydroxyvitamin d levels in a patients with end-stage renal disease and active pulmonary tuberculosis. new engl j med 312: 1520, 1985. gkonos, p.j., london, r. & hendler, e.d.: hypercalcemia and elevated 1,25 dihydroxyvitamin d levels in a patient with end-stage renal disease and active tuberculosis. n engl j med 31 1 : 1683-1 685, 1984. 8. horton, j.e., raisz, l.g., simmons, h.a., oppenheim, j.j. & mergenhagen, s.e.: bone resorbing activity in supernatant fluid from cultured human peripheral blood leukocytes. science 177: 793-795, 1972. 9. narang, n.k., gupta, r.c. & jain, m.k.: role of vitamin d in pulmonary tuberculosis, japl 32: 185-1 88, 1983. 10. need, a.g., philips, p.j., chiu, f.t.s. & prisk, h.m.: hypercalcemia associated with tuberculosis. br med j 1 : 831, 1980. 11. sandler, l.m., winerals, c.g., fraher, l.j., clemens, t.l., smith, r. & oriordan, j.l.h.: studies of the hypercalcemia of sarcoidosis: effect of steroids and exogenous vitamin d3 on the circulating concentrations of 1,25-dihydroxyvitamin d3. quart j med 21 0: 165-1 80, 1984. 541 -544, 1984. 6. 7. correspondence to: professor sverker ljunghall department of internal medicine university hospital s-751 85 uppsala, sweden 160 upsala j med sci 91: 219, 1986 education of untrained hospital staff for decentralizing of reflectometric blood glucose assay marta-lisa malm and henning von schenck department of clinical chemistry, linkoping university, linkoping, sweden to increase efficiency in larger public hospitals reflectometry of blood glu cose has been one of the objects for decentralizing because of fast bedside re sults. here, we report our preliminary experience with teaching hospital staff previously untrained in laboratory procedures. methods: personnel (n=49) were gathered in groups and trained in a 2 hourspro gram. glucmter (ames/bayer, a/b, elkhart ind.) and dextrostix (a wash-out strip, a/b) instructions were given orally and as handouts. the importance of the size of the blccddrop, reaction time and terminating the reaction was em phasized. first, glucose control solutions were used to determine the numberof outliers off the assigned values. precision of replicates (n=5) was assessed by assaying a pool of mta blood with a known glucose content. then, the trainees obtained capillary blood from each other and determined the accuracy of the mean of their duplicates against a split capillary sample measured in an automated hexokinase method. results: the control solution assay showed that the glucose concentration was overestimated by about 10%. the number of outliers was 9/49. the precision of replicates was plotted against relative frequency. this plot demonstrated (less 3 outliers with c.v.% > 2 0 % ) two groups. one with a mean c.v. of 5+2% (n=29)and another with a mean c.v. of 1023% (n=lo). accuracy shmed a regressionequation y (teststrip) = 0.75x (hexokinase method) + 0.5x (n=49, r=0.79,range 4-8ml/l). discussion: training lowered the apparent number of outliers during theongoing program. precision testing spotted analysts with problems. the largest group of analysts had good precision while a smaller group was identified for re training. accuracy data point at difficulties with the wash-out procedure. conclusion: extensive training is necessary for acceptable handling of wash out strips. external control remains imperative for meaningful decentralizing of blood glucose. 219 ujms 110 (3) bra upsala j med sci 110 (3): 237–240, 2005 emergency operation for phlegmonous gastritis takashi yokota1, takayuki yamaki2, rei yashima2, yasuo yamada2, yoichi narushima2, shu kikuchi2, hidemi yamauchi2 and masahito hatori3. 1department of surgery, national sanatorium tohoku shinseien, 2 department of surgery, sendai medical center and 3department of orthopaedic surgery, tohoku university school of medicine, sendai, japan. abstract phlegmonous gastritis is a rare inflammatory lesion in which bacterial infection occurs in the gastric wall. a case of phlegmonous gastritis producing an intramural filling defect in the stomach is presented. endoscopy showed edematous and reddened gastric mucosa with a mass lesion in the gastric body and antrum. an abdominal ct scan showed diffuse and irregular thickening of the gastric wall. at emergency operation, a total gastrectomy with splenectomy was performed. the most important differential diagnosis is carcinoma, especially scirrhous-type gastric cancer. radiographic findings of phlegmonous gastritis resemble those of scirrhous gastric cancer. more frequent recognition of this disease, early diagnosis and prompt institution of treatment is essential. introduction phlegmonous gastritis is a rare inflammatory lesion of the gastric wall. although the disease usually shows characteristic macroscopic and histological findings1, the diagnosis may contribute to problem and it is sometimes difficult to distinguish phlegmonous gastritis from scirrhous gastric cancer. in this report we describe a case of phlegmonous gastritis with an acute clinical course where emergency operation was necessary. case report a 74-year-old woman was sent to the sendai medical center in june 2002 because of abdominal pain and vomiting of one-week duration. on admission, the patient was drowsy. physical examination revealed tenderness in her upper abdomen with sluggish bowel sounds. blood pressure was 130/65 mmhg, and pulse rate was 55 237 received 11 april 2005 accepted 23 may 2005 key words: phlegmonous gastritis, scirrhous gastric cancer beats/min and regular. her temperature was 38.5°c. laboratory examination showed a white blood cell count of 28.3x109/l; hemoglobin of 129g/l; normal serum sodium, potassium and chloride levels; serum calcium level of 5.5 mmol/l; serum phosphorus level of 1.9 mmol/l blood urea nitrogen of 2.9 mmol/l; and a serum creatinine level of 0.8 mg/dl. liver enzymes were normal. the patient was aggressively treated with fluid resuscitation. one day after the start of treatment, serum calcium level had decreased to 2.5mmol/l. an abdominal ct scan showed diffuse and irregular thickening of the gastric wall (fig. 1a). an upper gastrointestinal series showed narrowing of the antrum and pylorus involving almost the entire gastric cardia, mimicking borrmann type 4 gastric cancer of “linitis plastica” (fig. 1b). at gastroscopy, the stomach mucosa had a thinned atrophic appearance, with numerous white nodules scattered irregularly throughout the body and fundus of the stomach. the nodules varied in size, with the largest being approximately 2-3 mm in diameter. a stricture was observed in the stomach body (fig. 1c). the patient’s symptoms worsened during the next 5 days, and she developed nearly complete gastric outlet obstruction secondary to an annular construction of the antrum. no ulceration was seen. examination of a biopsy specimen from the gastric mucosa revealed severe inflammation. since an upper gi series demonstrated typical features of borrmann type 4, we suspected that it was cancerous lesions. an emergency operation was performed. on laparotomy, a total gastrectomy with splenectomy was performed. adhesion between the stomach and spleen was very strong, and we could not separate them. it was easier to resect the stomach and spleen as one organ. the stomach wall was thickened and edematous with diffuse ulceration. microscopic examination showed the gastric mucosa to be infiltrated by lymphocytes. the submucosa was edematous and infiltrated by inflammatory cells with a focal area of necrosis (fig. 1d). discussion very little is known about the pathogenesis of phlegmonous gastritis. in most cases, hemolytic streptococcus is reported to be the offending organism (1). there are three possible routes of spread: 1) direct injury to gastric mucosa due to sharp bones or other hard food, 2) hematogenous spread from some other septic focus such as endocarditis, and 3) lymphatic spread from a septic focus. some researchers produced a submucosal abscess in dogs by introducing streptococci into the mucosal surface after induction of alcohol gastritis, but others failed to produce a similar condition1. until more is known about the mechanism of gastric defense in response to bacterial invasion, the pathogenesis of this disease will remain unclear. patients with this disease present with an acute abdomen, with intense epigastric pain of relatively rapid onset associated with epigastric guarding (2). leucocytosis and nausea and vomiting are present. in the most fulminant varieties, the patient develops severe toxemia and early peripheral circulatory collapse (3). there are few descrip238 tions of radiographic findings of phlegmonous gastritis (4). the common features seem to be a relatively small intramural mass, often in the antrum of the stomach, usually without destruction of the overlying mucosa. the mucosal folds are completely effaced at the site of involvement. there is frequently gastric retention of barium, and complete pyloric obstruction has been reported. the most important differential diagnosis is carcinoma; actually, the preoperative diagnosis in our patient was scirrhous-type gastric cancer in spite of a “negative” biopsy. scirrhous type carcinoma cells invade extensively under the mucosal layer, and results of biopsy examination are sometimes negative. radiographic findings of plegmonous gastritis resemble those of scirrhous gastric cancer. scirrhous gastric cancer is clinically characterized by diffuse infiltration by cancer cells and shows significant thickening of the gastric wall with no prominently elevated or depressed lesions (5). the infiltrative carcinoma may grow either superficially over the surface of the mucosa or permeate the entire thickness of the wall, producing a pattern known as linitis plastica. a submucosal biopsy has potential diagnostic benefit. 239 figure 1a: abdominal ct showed diffuse and irregular thickening of the stomach body wall (arrow). b: barium examination of the upper gastrointestinal tract revealed a marked large irregular filling defect involving the entire gastric wall (arrow). c: endoscopy showed edematous and reddened gastric mucosa with a stricture in the gastric body. d: a gastric section showing diffuse inflammatory swelling of submucosa with involvement of the muscularis. (original magnification, x100) a b dc there is a lack of agreement about the role of surgery in the management of acute phlegmonous gastritis, some authors recommending antibiotic therapy alone and others recommending gastrectomy or gastrotomy for drainage of submucosal abscesses1-3. early diagnosis and prompt institution of treatment with or without surgery are essential for this disease. references 1. miller ai, smith b, rogers ai (1975) phlegmonous gastritis. gastroenterology 68: 231-238. 2. iwakiri y, kabemura t, yasuda d (1999) a case of acute phlegmonous gastritis successfully treated with antibiotics. j clin gastroenterol 28: 175-177. 3. webster vj (1980) necrotizing gastritis and phlegmonous gastritis-are they separate entities? aust nz j surg 50: 194-196. 4. sood bp, kalra n, suri s (2000) ct features of acute phlegmonous gastritis. clin imaging 24: 287-288. 5. yokota t, teshima s, saito t (1999) borrmann type iv gastric cancer: clinicopathological analysis. can j surg 42: 371-376. corresponding author: takashi yokota, m.d. national sanatorium tohoku shinseien hasama, tome, miyagi 989-4601, japan _:0228-38-2121, fax: 0228-38-3765, e-mail address: hki06124@nifty.com 240 upsala j med sci 105: 1-16,2000 from opiate pharmacology to opioid peptide physiology lars terenius experimental alcohol and drug addiction section, department of clinical neuroscience, karolinsku institutet, s i 7 1 76 stockholm, sweden abstract this is a personal account of how studies of the pharmacology of opiates led to the discovery of a family of endogenous opioid peptides, also called endorphins. the unique pharmacological activity profile of opiates has an endogenous counterpart in the enkephalins and j3-endorphin, peptides which also are powerful analgesics and euphorigenic agents. the enkephalins not only act on the classic morphine (p-) receptor but also on the 6-receptor, which often co-exists with preceptors and mediates pain relief. other members of the opioid peptide family are the dynor phins, acting on the k-receptor earlier defined as precipitating unpleasant central nervous system (cns) side effects in screening for opiate activity, a related peptide, nociceptin is not an opioid and acts on the separate nor-receptor. both dynorphins and nociceptin have modulatory effects on several cns functions, including memory acquisition, stress and movement. in conclusion, a natural product, morphine and a large number of synthetic organic molecules, useful as drugs, have been found to probe a previously unknown physiologic system. this is a unique develop ment not only in the neuropeptide field, but in physiology in general. introduction historical background opiates are indispensible drugs in the pharmacologic armamentarium. no other drug family can relieve intense, deep pain and reduce suffering. morphine, the prototypic opiate is an alkaloid extracted from the capsules of opium poppy. the use of opium extract as a euphoriant and as an analgesic has a long history. some of the earliest documentation is from the sumers in the 3rd millennium b.c. and use spread to the egyptians, greeks and disseminated into the whole known “civilized” world at the time, india, china and europe. opium abuse was common in the orient, but also flourished in europe from the 16” century with a culmination of opium “eating” in the 1 sth century. i 1001 26 1 already in 1806, serturner isolated morphine, named after the greek god of sleep, mor pheus, and found it to be the principal analgesic and addictive component in opium. the access of the pure substance, and the following technological invention of the injection syringe would revolutionize the clinical handling of pain, and together with the development of gaseous anes thetics open the way to surgery under humane conditions. morphine has an intricate chemical structure and is impractical to synthesize. it is therefore necessary to cultivate opium poppy for access to morphine and to codeine (a weaker, broadly used analgesic and antitussive agent). the accompanying problems of illegal traffic stem from the fact that opium poppy is cultivated in geographical areas that are hard to access and control. a simple chemical process, acetylation turns morphine into heroin, which no longer has a medical use, but is preferred by the opiate ad dict. morphine is not a safe drug, it is easily overdosed and is strongly addictive. attempts have therefore been made to produce equally efficacious, non-addicting drugs. in the late 1930s it was found that a synthetic atropine analogue, named pethidine was an opiate analgesic. this work was done in germany, which during the 2"d world war became isolated from opium culti vation areas. german scientists synthesized another analgesic, methadone which was found to be equipotent with morphine and orally active. both pethidine and methadone remain clinically important analgesics. methadone is mainly used, however, as a substitute in maintenance ther apy of chronic, relapsing heroin addicts. the early history and the development of opiate phar macology, has been summarized by the leading german scientist, 0. schaumann (1). the prototypic opiate the identification of synthetic compounds with the same activity as morphine was a success for medicinal chemistry. it generated a tremendous amount of work in synthetic chemistry and vir tually thousands of compounds were prepared and assayed for opiate activity. in fact, opiates became prototypes for pharmacologic assay evaluation. besides behavioral tests for analgesia, such as the escape jump or paw-licking of a mouse on a hot plate, it was found that several iso lated organ preparations, notably the electrically stimulated longitudinal muscleherve plexus from the guinea-pig ileum responded to opiates. the correlation between analgesic activity and activity in the ileum was excellent, taking into account pharmacokinetic differences. in fact, such comparisons were instrumental in defining activity at the site of action and indirectly gave a measure of how well an analgesic penetrated the blood-brain barrier. synthetic work would generate compounds that when injected were 1000 to 10 000 times more potent than morphine (e.g. etorphine used by veterinarians for immobilizing large wild animals), but not much more active in the isolated preparation showing that the new compound distributed to the brain very 2 efficiently. another synthetic derivative, fentanyl would find a use as an intravenous anesthetic (and of course at the same time a potent analgesic). the access to in vitro organ preparations for hndamental studies was of principal impor tance. apparently, a whole family of chemically unrelated compounds had a common mode of action in relatively simple systems, and not only in a behavioral assay where the end point seemed more obscure. the in vitro preparations also allowed correlation between chemical structure and biological activity. thus, opiate pharmacology was equally accessible to the es tablishment of structure-activity relationships ( s a r s ) as other fields of pharmacology, such as anticholinergics or antiadrenergics, where in a similar manner, organ preparations were avail able and the large families of compounds generated s a r s . based on s a r s complementary re ceptor surfaces were depicted and used for activity predictions and guidance of the development of new agents. opiates were, however, importantly unique. whereas anticholinergics or an tiadrenergics were known to antagonize acetylcholine and noradrenaline, respectively, there was no endogenous morphine-like substance known at the time. since acetylcholine is the transmit ter released by electric contractions of the guinea-pig ileum preparation, and opiates inhibited the contraction it was assumed that opiates acted by inhibiting acetylcholine release ( 2 ) . this effect, was, however, thought to be a pharmacological artifact and therefore not relevant physiologically. the opiate receptor it was recognized in the late 1960s that hormone receptors were proteins either in the cellular membrane or in a resting state in the cytosol ready to enter the cell nucleus in a complex with the hormone. these receptors could be identified with the use of hormones that had to be labeled with isotopes to very high specific activity since receptor numbers are very small. for protein hormones or other larger molecules labeling was easily done with radioactive iodine. with smaller molecules, receptor ligands had to be labeled with tritium to highest possible (theoreti cal) specific activity, which was not recommended, since such compounds were thought to de compose rapidly. later work would show that this was not a problem if storage conditions (low temperature, no light, as little water as possible) were optimized. almost simultaneously, three groups published the successfd demonstration of specific opi ate receptor binding in rat brain membranes and in the guinea-pig ileum preparation (3-5). inter estingly, in the latter preparation it was found that the receptor was associated with the nerve plexi and not with the muscle, as was predicted from in vitro opiate pharmacology. the recep tors, so defined showed the expected binding properties, i.e. they bound the labeled prototypic ligand and analgesically active opiates (measured as displacement of label) but not inactive 3 compounds. a critical comparison was the demonstration that of two optical antipodes (com pounds that are identical except in their interactions with an asymmetric surface) the active an tipode had affinity, the inactive not ( 6 ) . the binding assay was found to be very robust and suit able for drug screening. the radioactive ligand could either be the morphine derivative dihy dromorphine (3) or the “superanalgesic” etorphine (5) with equal results. as already pointed out etorphine derives its extreme potency from a more favorable kinetics and reaches the central nervous system in a larger proportion than morphine (or its close analogue, dihydromorphine). by receptor autoradiography, it was soon found that the opiate receptor (or more accurately, binding sites) localize in cns areas known to be relevant for the treatment of pain, the dorsal horn of the spinal cord, the periaqueductal gray and the raphe nuclei, but also in other areas such as the striatum or the amygdala, areas not associated with pain. besides, it was obvious that there was no association with any particular cns neurotransmitter (such as acetylcholine as suggested from the work in the guinea-pig ileum). another important route of investigation was opened with the finding that opiate receptors couple to a second messenger pathway, by acting on g-proteins to inhibit the activity of adenyl ate cyclase. thus opiate receptors seemed to belong to a large class of receptors that included receptors for neurotransmitters such as acetylcholine, noradrenaline, dopamine, etc. yet with a unique specificity. thus, from an obscurity as a pharmacological artifact the opiate receptor seemed to share a place with the “real” receptors. it has already been pointed out that opiate overdose can be fatal. it was found quite early that certain semi-synthetic morphine derivatives could antagonize the toxic actions of opiates. the typical substance, nalorphine had some analgesic activity, in pharmacological terms acting as a partial agonist, but also induced unpleasant side reactions precluding its use as a safe analgesic. further chemical work identified naloxone as a pure opiate antagonist with virtually no activity. naloxone must, however, be given intravenously and is very short acting. a closely related con gener, naltrexone, is also a pure opiate antagonist and has the additional advantage of being orally active. naloxone has become a standard agent for treatment of opiate overdose and is also a pharmacologic probe for the opiate receptor. in fact, the definition opiate and opiate receptor is based on the antagonism by naloxone. it can of course be argued that this is a highly artificial definition and as will be apparent, this definition can be challenged. in most tests naloxone alone was reported to be inactive (for a review on opiate antagonists see ref 7). this seemed to confirm the notion that opiates are usehl pharmacologic agents, but in physiology, artifacts. 4 the endogenous ligands testing a hypothesis two groups independently entertained the idea, that there might be endogenous ligands for the opiate receptor. terenius (8) used the binding assay with membranes prepared from rat brains and tritium-labeled dihydromorphine as a probe for the binding sites. brain extracts were pre pared and subjected to chemical fractionation. partition into organic solvents did not recover any material that displaced the labeled probe, not even under alkaline conditions that would have extracted opiates. however, activity was consistently found in aqueous extracts from which salts and larger molecular weight proteins and other macromolecules had been eliminated. separation in aqueous medium on a gel filtration column identified components with low molecular weight, approximately 700-1 000. the activity was destroyed by treatment with proteolytic enzymes. the identity of these components was clear; they were low-molecular weight peptides. john hughes and hans kosterlitz used a different approach (8). kosterlitz had refined the guinea-pig ileum system for the assay of opiates. various fractions of pig brain extracts were added to the preparation. opiate activity was recorded as the suppression of the electrically induced twitch, since this response is quite non-specific and could be due to “poisoning” they added the extra criterion, reversibility by naloxone. initial positive experiments called for large-scale extractions of pig brain. they entered collaborations with a drug company, reckitts & coleman that had access to large-scale extraction facilities. since the company also is the manufacturer of the su peranalgesic, etorphine chemical characterization of some active fractions turned out etorphine, which apparently was around ubiquitously. after such setbacks they eventually succeeded in isolating and sequencing two homologous opioid peptides, leu-enkephalin and met-enkephalin (9). they soon recognized that the enkephalin sequence was also represented in the hormone p lipotropin, of unknown physiologic significance. a fragment of this hormone (called the c fragment, later p-endorphin) had also been isolated (10). this chemical triumph gave the aston ishing result, not only was there one ligand for the opiate receptor, there were several. hughes and kosterlitz named their peptides enkephalins, since they had been isolated from brain (gk. enkefalos). the structures of these peptides are: leu-enkephalin yggfl met-enkephalin yggfm p-endorphin yggfmtseksqtplvtlfknaiiknaykkge (human) this discovery initiated extensive efforts both in the synthesis of analogues and derivatives and in pharmacologic experimentation. the enkephalin sequence was found to be the minimum active sequence. however, being peptides, they were easily degraded particularly by aminopep tidases that eliminated the n-terminal tyrosine, absolutely essential for activity. replacement of 5 gly’ with dala’ and other modifications generated peptides with excellent stability and po tency. at the receptor level, morphine and enkephalins were found to be approximately equipo tent. the natural ligand, p-endorphin was found to be a potent analgesic if introduced in the cns and also long-acting (1 1). the discovery that an alkoloid and small peptides act on the same receptor was a conceptual breakthrough. several neuroactive peptides were known at the time, for instance substance p and neurotensin, but in no case had a non-peptide agonist with activities similar to the peptide been discovered. the discovery of the opioid peptides set an example. natural product and me dicinal chemists screened large numbers of compounds against peptide receptors. progress was initially slow but it would soon turn out that so-called peptidomimetics can be prepared if enough effort is given to the problem. peptides are never going to be the favored chemical structures for drug development, but the possibility to obtain peptidomimetics opens the neuro peptide systems to h t u r e pharmacotherapies. early studies on opiate receptor ligands the discovery of endogenous opioids initiated research in different directions, particularly in relation to their putative hnctional significance. antibodies were generated to the newly discov ered peptides and used for immunohistochemical studies. already the first studies were reward ing (12). enkephalin immunoreactivity was present in areas of particular relevance for pain and in juxtaposition to areas previously known to be rich in opiate receptors, including the dorsal horn of the spinal cord, the raphe nuclei, the periaqueductal gray, and also in large areas of the basal ganglia, the caudate nucleus, globus pallidus etc. of particular interest in relation to the euphorigenic activity of opiates was the rich innervation of the nucleus accumbens, previously known to be central in the brain’s reward system. it had been shown by olds and others after him that a rat would activate electrodes implanted in this structure thereby releasing endogenous neurotransmitters. such rats would neglect eating, grooming and were less interested in the op posite sex. a closer look at enkephalin distribution in the spinal cord and a comparison with the distribution of substance p revealed interesting differences (13). substance p, a peptide with 11 amino acids was previously known to be present in thin primary afferents, c-fibers, known to be essential for nociception, and the first link in pathways eliciting pain. enkephalin nerve termi nals were present in areas overlapping the substance p terminals. the enkephalin neurons were, however, local interneurons available already at the first synapse. studies of the finer architec ture revealed that opiate receptors were present both on the primary afferent terminals and on the second order neurons. these receptors are accessible to local analgesic therapy via intrathe cal or epidural treatment, which became an important therapeutic principle during this time pe 6 riod, for instance in obstetrics. apparently this is an intrinsically very p o w e h l system and the extent to which it is operated by enkephalin release became an interesting area of study, subse quent work would show that the distribution of p-endorphin immunoreactivity was much more limited, essentially only one diffuse projection from cell bodies in the brain stem. the discovery of endogenous opioid peptides raised hopes that they would not be addictive. it seemed counter-intuitive that the body would generate compounds with such activity. how ever, an early study where rats were allowed to self-administer leu-enkephalin via a catheter implanted in the cerebral ventricles revealed that the rats liked to take enkephalin as readily as morphine. in fact it was necessary to protect the rat from overdosing (14). this discovery was disappointing from a drug development perspective, but exciting from a physiological perspec tive. if the opioid peptides have such p o w e h l properties could it be that they are intrinsic parts of the reward system and in fact involved in the regulation of mood? it has previously been em phasized that the opioid antagonists are practically inactive in pharmacologic experiments, thus suggesting that the tonic activity in opioid systems is low. in fact this may be the way natural selection operated. a p o w e h l tonic release of the opioid peptides would not be functional, pain is a useful signal of injury and a feeling of good mood (pleasure) is not functional except as a reward. therefore opioid peptides may only be released under special, perhaps more extreme conditions such as in “battlefield analgesia”. the lack of pain a soldier feels when wounded and transported to hospital behind the front. these types of extreme conditions are ethically hard to address in experiments. a physically exhausting, yet benevolent activity is running. rats given access to a running wheel in their cages will readily run during their active period. running activity is less if the animals are treated with naloxone. if they are habituated to running for several weeks and thereafter the wheel is locked, they will become nervous, aggressive with a peak maximum after 2 to 3 days. in parallel, there is a drop in their secretion of p-endorphin into cerebrospinal fluid from the ele vated levels during running (15). it is interesting that the time course of the phenomenon is similar to that seen during detoxification of a heroin addict. new family members and the family tree as already pointed out, opiates constitute a group of compounds with a consistent activity spec trum. for instance, despite strong efforts it has not been possible to separate analgesic activity from dependence-producing liability. the newly discovered peptides apparently were no excep tion. it was found, however, that certain synthetic analogues produced side effects, from mild dysphoria to psychotomimetic effect. a prototypic compound with such effect is ketocyclaz ocine. to separate this activity martin and collaborators suggested the existence of a receptor 7 distinct from the “morphine”-receptor (preceptor) and named it the k-receptor. there were other differences, for instance whereas morphine causes urine retention, k-agonists induced diu resis (16). the k-receptor remained a pharmacological curiosity, until a new group of opioid peptides were discovered, the dynorphins. dynorphin a, the first member found, has 17 amino acids and contains the leu-enkephalin sequence in its n-terminus. subsequently, two additional members also with the same n-terminal sequence were discovered. immunohistochemical analysis revealed that the distribution was distinct from that of other opioid peptides. dynorphin interneurons in the dorsal horn of the spinal cord are primarily found in deeper laminae than enkephalin interneurons, suggesting a greater role in modulating nociceptive signals from inner organs. in the brain there is a major presence in striatonigral neurons suggesting a role in motor control, in neurons projecting to the nucleus accumbens suggesting effects on reward and in mossy fibers of the hippocampus suggesting a role in learning. the distribution roughly matched that of k-receptors identified by autoradiography. clearly, in brain dynorphin peptides and k receptors are present in structures not primarily related to pain. it is therefore not surprising that k-receptor agonists are analgesic when given intrathecally but not centrally into the brain. advances in molecular biology led to the cloning of the protein precursors to the opioid pep tides. the first identified precursor was found to be pluripotent and contains three hormones, acth (adrenocorticotrophic hormone), msh (melanocyte-stimulating hormone) as well as p endorphinip-lipotropin and was consequently named proopiomelanocortin. the other precur sors, proenkephalin with no less than 7 enkephalin sequences (6 met-enkephalin and 1 leu enkephalin) and prodynorphin (also named proenkephalin b) with 3 dynorphin sequences all containing leu-enkephalin, were close to follow. the chemical complexity is thus bewildering and the number of chemical species that can be derived from these precursors by partial or hll proteolytic processing is large (17). for a long time the opioid receptors resisted attempts at cloning. the first receptor to be eventually cloned was the 6-opioid receptor (18, 19). this receptor had been postulated from experiments with the enkephalin peptides, which besides activity at the p-receptor also had af finity for another receptor less sensitive to naloxone. this receptor seemed uniquely present in the in vitro preparation, the mouse vas deferens. (the name 6is a homonym with d in defer ens). when the sequence of the &receptor was known it became relatively simple to use homol ogy screening to identify and clone the two other receptors predicted from pharmacologic ex periments, the 1.1and ic-receptors. the entrance of molecular genetics had changed the opioid field dramatically. homology screening identified yet another receptor, named orli (orphan receptor l-1) since no natural 8 ligand was known. a few years later, however, a ligand was found and named nociceptin (20) or orphanin fq (21) since rats given this peptide intrathecally respond as if they have been exposed to a noxious stimulus. nociceptin has subsequently been found to have a multitude of actions in the whole cns. it is distributed in the dorsal horn of the spinal cord overlapping with the enkephalins and in the brain, for instance overlapping with dynorphin in the mossy fibers of the hippocampus. the discovery of nociceptin prompted a change in terminology for its receptor, now called the nor-receptor. in summary, there is now a superfamily of structurally related peptides and receptors. pep tides from three families have opioid activity, operationally defined as activity that can be blocked by the antagonist naloxone. nociceptin has no affinity for the opioid receptors but inter acts with the homologous nor-receptor for which the opioids have no affinity. still, nociceptin itself is structurally related to dynorphin a, and the nor-receptor related to the opioid receptors indicating a common ancestral precursor to all these peptides and receptors. since functional activity after all is the most relevant parameter in biomedical research the terminology opioid is likely to stay. another synonym, endorphin (for endogenous morphine) is also frequently used. the following graph summarizes the properties of this family of peptides. enkephalin and p endorphin have morphine-like opioid activity, are potent analgesics and euphoriants, dynorphin is also an analgesic primarily at a spinal level whereas at higher levels it produces unpleasant effects and is not self-administered, and finally nociceptin, a non-opioid peptide acts on its own unique receptor modulating pain and other cns activities. precursor typical peptide preferred receptor proenkephalin leu-enkephalin' p (=opiate), 6 met-enkephalin' proopiomelanocortin2 0-endorphin' p + 6 prodynorphin dynorphins a and b' k ~ r o n o c i c e p t i n ~ nociceptin nor 1. opioid peptides. 2. also precursor to acth and msh acting on non-opioid receptors 3 . also precursor to other peptides with unknown receptors. opioid peptides and pain both from a principal and practical point of view, it would be of interest to determine the role, if any of opioid peptides in the modulation of clinical pain. one experimental approach is to test the influence of naloxone on pain thresholds. under well-controlled conditions it is in fact 9 possible to demonstrate hyperalgesic effects in laboratory animals. in human healthy volunteers, pain thresholds were, however not much affected, partly because realistic pain conditions would be unethical. other approaches have therefore been taken. it seemed possible that opioid peptides would be released into the cerebrospinal fluid (csf) in enough quantities to be measurable. early studies indicated that this might be feasible. in these studies, the earlier described receptor-binding assay was used to identify opioid material in two chromatographic fractions, covering the molecular weight interval 700-1200, i.e. likely enkephalin-related peptides (22). since csf is usually sampled at a lumbar level contributions from release at the spinal level are likely substantial as also proven in an animal model. already the first studies suggested the possibility that severe, chronic pain might in fact be associated with very low levels of opioid peptide secretion. this was confirmed (23) in a larger series of patients with chronic pain (defined as having lasted for at least 6 months). when compared with levels in a group of healthy volunteers, patients with neurogenic pain (causalgia, neuralgia etc.) tended to have lower levels of receptor-active opioids. it was also found that patients with non neurogenic “idiopathic” pain had levels within or higher than those in volunteers. with a sur prising degree of accuracy, the simple binding assay distinguished patients in the two popula tions. during this time period, there was a general shift in the conceptualization of chronic neuro genic pain, from models where the pain was considered a consequence of nociceptive input from primary afferents (afferent pain) to the understanding of chronic pain as a disease state, probably more often of a central origin. intuitively obvious procedures for pain treatment such as sec tioning of afferent nerves were abandoned, since such approaches were frequently not only clinically ineffective but might even aggregate the condition. in fact, pain of neurogenic pain may derive from deafferentation, i.e. a failed neuronal input. it was also known that such pain was resistant to opiate therapy. in short, such chronic pain, which by definition is a therapeutic failure, does not respond to pharmacotherapies known to modulate acute or chronic afferent pain. parallel work suggested other approaches to the treatment of neurogenic pain. an old chi nese procedure for treatment of many ailments including pain, acupuncture was introduced in china as an alternative to conventional analgesic medication during surgery. the results were successful particularly if the acupuncture needles were stimulated electrically. several clinical investigators attempted acupuncture-like techniques in patients with chronic pain. a frequently used technique transcutaneous electric nerve stimulation (tens) with surface electrodes in stead of needles started to gain interest. it would soon be recognized that different stimulation parameters produced different results, clinically some patients responded well on high 10 frequency, low intensity tens whereas others responded better to low-frequency high intensity stimulation. the latter paradigm elicited deeper stimulation and muscle contractions and was therefore considered “acupuncture-like”. the clinical impression was that particularly the neu rogenic cases responded better to low-frequency tens than idiopathic cases. moreover, nalox one antagonized the pain modulating effect of low-frequency tens suggesting that opioid pep tides were released. this was confirmed in a study where individual patients with severe chronic neurogenic pain donated lumbar csf before stimulation and after stimulation. the stimulation gave an increase (24). similar results were obtained in animal models. the historic background, some of the chinese experience with acupuncture and these early results have been reviewed (25). in a later study, a comparison was made between patients given highand low-frequency stimulations, respectively. on this occasion, two distinct peptide species were measured with radioimmunoassay in csf samples taken before and 15 min after stimulation. the results were interesting since they suggested a differential effect. low-frequency stimulation significantly elevated an enkephalin peptide, high-frequency stimulation a dynorphin peptide. the former in teracts primarily with p-receptors (which are highly sensitive to naloxone and with &receptors) whereas the latter interacts almost exclusively with k-receptors with low naloxone sensitivity (26). these results are compatible with the previously described observation that only low frequency stimulation produces naloxone-sensitive pain relief. there are considerable interindividual differences in the amount of analgesic required to pro duce adequate pain relief, for instance during surgery and postoperatively. it has been assumed that everything else being equal, these differences are related to the individual‘s pain tolerance. patient-controlled infbsion of an opiate has been introduced for maximum comfort postopera tively. interestingly, individual patients titrate their drug intake very accurately to a pseudo steady-state plasma level. this titrated level represents an indirect estimate of the pain intensity, in a series of patients who had to undergo major gastrointestinal surgery, a csf sample was taken at the time for the induction of anesthesia, and another sample was taken postoperatively, when the patient was self-administering opiate. the csf taken prior to surgery was used for opioid peptide analysis using a receptor assay and the sample taken post surgery for analysis of the concentration of opiate. opiate consumption was recorded and the plasma concentration monitored at regular intervals. the study showed distinct differences in the opiate steady state levels, despite efforts to follow a standard protocol for premedication, anesthesia etc. moreover, when the plasma (actually calculated csf) opiate concentration was related to csf opioid ac tivity, an inverse relationship was found (27). thus, a patient who demanded more opiate had lower peptide levels and vice versa, indirectly suggesting that the endogenous release of opioid peptides contributes to the overall pain modulation. 1 1 parturition is a natural painand stressfbl process. a series of primagravidae selected for vaginal delivery participated in another study. these women were offered an epidural treatment with opiate to relieve pain. a lumbar csf sample was taken at an early phase of the delivery and analyzed for two peptide markers, p-endorphin and dynorphin a. women who demanded an epidural had significantly lower levels of dynorphin whereas there was no difference between the two groups regarding p-endorphin (28). experiments in rats have confirmed that dynorphin peptides are relevant for pain modulation in parturition. in conclusion, several experiments suggest that there is a significant contribution of the opi oid peptide systems in the modulation of clinical pain. the systems can be activated by stimula tion techniques either physical, acupuncture or electrical. the level of activity, particularly in the enkephalin system can be boosted by inhibition of enzymatic degradation. this offers an al ternative to treatment with analgesic drugs for moderately severe pain (29). other actions although there is little direct evidence, enkephalins and p-endorphin probably play a role in reward, and in the human, in emotions such as euphoria and pleasure. studying subjective expe riences with biological tools is, however, experimentally very difficult. besides, there are more stringent ethical constraints in studying completely normal reactions rather than pathologic de viations. there is, however, indirect evidence that chemically induced “pleasure” may be medi ated via opioid peptides. naltrexone has been introduced as a drug (reviva@) to prevent relapse in alcoholics. it has been shown that this drug reduces alcohol-induced euphoria. an alternative approach to relapse prevention might be the activation of dynorphin systems (for a review see 30). as already mentioned, agents acting on k-receptors produce unpleasant mental side effects. these effects are reported as altered perception or even delusions and hallucinations (mainly auditory) and therefore, k-agonists may be regarded as psychotomimetic. other agents produc ing hallucinations, for instance lsd-25 mainly produce visual hallucinations that are not typi cally recorded in schizophrenia. it therefore seemed possible that schizophrenics might have an abnormal production of psychotomimetic opioids. to test this possibility, two experimental ap proaches were undertaken. a series of 6 patients with chronic schizophrenia and residual symptoms including auditory hallucinations were given naloxone intravenously. several patients responded one case with habitual hallucinations was essentially symptom-free after naloxone (3 1). this finding stimulated a who-multicenter investigation on the efficacy of the orally ac tive naltrexone. naltrexone was found to have a significant effect on residual symptoms in neu 12 roleptic-treated patients, however not to an extent that clinical use was warranted. the other ap proach, chemical analysis of csf opioid peptides confirmed an elevated secretion of opioid peptides, both using a receptor-assay and direct radioimmunoassay of dynorphin peptides (32). taken together, these data indicate that dynorphin peptides, which are intimately associated with dopamine pathways which are the main targets of neuroleptics, in some way are involved in psychosis. it has also been suggested that dynorphin peptides have a modulatory role in certain behav iors. several investigators have shown that k-agonists suppress self-administration of heroin or cocaine or self-selected intake of alcohol in rats. the site of action might be the nucleus accum bens that receives rich innervation of dynorphin fibers. a model where enkephalin and dynor phins have opposite roles on the activity of the mesolimbic dopamine system has been presented (32). concluding remarks the opiates constitute a unique family of substances with a well-defined pharmacologic pro file. this remarkable and unusual family of compounds acts on mainly one receptor, the p receptor. the endogenous peptides, enkephalins and p-endorphin also act on this receptor and share all the classic actions of opiates, potent analgesia, euphorigenic activity etc. these pep tides and their respective physiologic systems can therefore be identified with these specific ac tions; no other neurotransmitter or neuropeptide is so well related to a particular physiologic profile. it has therefore been possible to establish direct links between activity in these systems and the physiologic (or pathophysiologic) condition. other peptides belonging to the same fam ily, dynorphins and nociceptin cannot be attributed to any unique physiologic function yet, they seem to act as neuromodulators at different levels of the cns. the phylogeny of the opioid systems is not well characterized. most studies have been done in mammals. however, also the mollusk, mytilus edulis synthesizes opioid peptides and has an opioid receptor, 95% identical with the human p-receptor. thus from mollusk to man, nature has provided a system for protection against severe “pain” and “stress”. references 1. schaumann, 0. morphin und morphinahnlich wirkende verbindungen. in: handbuch der experimentellen pharmakologie (0 eichler & a. farah) springer-verlag, berlin, gottingen, heidelberg, 1957. 2. paton, w.d.m. the action of morphine and related substances on contraction and on acetyl choline output of coaxially stimulated guinea-pig ileum. brit j pharmacol. 12: 119, 1957 13 3 . 4. 5. 6. 7. 8. 9. 10 terenius, l. stereospecific interaction between narcotic analgesics and a synaptic plasma membrane fraction of rat cerebral cortex. acta pharmacol toxicol32: 3 17-20, 1973. pert, c.b. & snyder, s.h. opiate receptor: demonktration in nervous tissue. science 179: simon, e.j., hiller, m.m. & edelman, i. stereospecific binding of the potent narcotic anal gesic 3h-etorphine to rat-brain homogenate. proc natl acad sci usa 70: 1947-49, 1973. terenius, l. letter: a rapid assay of affinity for the narcotic receptor in rat brain: applica tion to methadone analogues. acta pharmacol toxicol34: 88-91, 1974. martin, w.r. opioid antagonists. pharmacol rev 19: 463-521, 1967. terenius, l. & wahlstrom, a. search for an endogenous ligand for the opiate receptor. acta physiol scand 94: 74-81, 1975. hughes, j., smith, t.w., kosterlitz, h.w., fothergill, l.a., morgan, b.a. & morris, h.r. identification of two related pentapeptides from the brain with potent opiate agonist activity nature 258: 577-79, 1975. bradbury, a.f., smyth, d.g. & snell, c.r. c fragment of lipotropin has a high affinity for brain opiate receptors. nature 260: 793-95, 1976. 101 1-14, 1973. 1 1 . hughes, j. & kosterlitz, h.w. opioid peptides. br med bull 33: 157-61, 1977. 12. elde, r., hokfelt, t., johansson, 0. & terenius, l. immunochemical studies using anti bodies to leucine-enkephalin: initial observations on the nervous system of the rat. neuro science 1 : 349-51, 1976. analysis of peptide pathways possibly related to pain and analgesia: enkephalin and sub stance p. proc natl acad sci usa 74: 3081-85, 1977. 14. belluzzi, j.d. & stein, l. enkephalin may mediate euphoria and drive-reduction reward. nature 266: 556-58, 1977. 15. hoffmann, p., terenius l. & thoren p. cerebrospinal fluid immunoreactive beta-endorphin concentration is increased by voluntary exercise in the spontaneously hypertensive rat. regul pept 28:233-39, 1990. 16. martin, w.r., eades, c.g., thompson, j.a., huppler, r.e. & gilbert, p.e. the effects of morphineand nalorphine-like drugs in the nondependent and morphine-dependent chronic spinal dog. j pharmacol exp ther 197: 517-522, 1976. 13. hokfelt, t., ljungdahl, a,, terenius, l., elde, r. & nilsson, g. immunohistochemical 17. terenius, l. families of opioid peptides and classes of opioid receptors. adv pain res 18. evans, c.j., keith jr., d.e., morrison, h., magendzo, k. & edwards, r.h. cloning of a delta opioid receptor by fhnctional expression. science 258: 1952-55, 1992. 19. kieffer, b.l., befort, k., gaveriaus-ruff, c. & hirth, c.g. the 6-opioid receptor: isolation of a cdna by expression cloning and pharmacological characterization. proc natl acad sci 20. meunier, j.c., mollereau, c., toll, l., suaudeau, c., moisand, c., alvinerie, p., et al. isola tion and structure of the endogenous agonist of opioid receptor like o k 1 receptor. nature 21. reinscheid, r.k., nothacker, h.p., bourson, a., ardati, a,, henninsen, r.a., bunzow, r.j. et al. orphanin fq: a neuropeptides that activates an opioid like g protein coupled receptor. science 270: 792-94, 1995. 22. terenius, l. & wahlstrom, a. morphine-like ligand for opiate receptors in human csf. life sci 16: 1759-64, 1975. 23. almay, b.g., johansson, f., von knomng, l., terenius, l. & wahlstrom, a. endorphins in chronic pain. i. differences in csf endorphin levels between organic and psychogenic pain syndromes. pain 5: 153-62, 1978. 24. sjolund, b., terenius, l. & eriksson, m. increased cerebrospinal fluid levels of endorphins after electro-acupuncture. acta physiol scand 100: 382-84, 1977. therap 9: 463-77, 1985. usa 89: 12048-52, 1992. 377: 532-35, 1995. 14 25. han, j.s. & terenius, l. neurochemical basis of acupuncture analgesia. ann rev pharma 26. han, j.s., chen, x.h., sun, s.l., xu, x.j., yuan, y., yan, s.c., hao, j.x. & terenius, l. col toxicol22: 193-220, 1982. effect of lowand high-frequency tens on met-enkephalin-arg-phe and dynorphin a im munoreactivity in human lumbar csf. pain 47: 295-98, 1991. 27. tamsen, a., hartvig, p., dahlstrom, b., wahlstrom, a. & terenius, l. endorphins and on demand pain relief lancet 1: 769-70, 1980. 28. lyrenas, s., nyberg, f., willdeck-lund, g., lindstrom, l., lindberg, b. & terenius, l. endorphin activity in cerebrospinal fluid prior to elective cesarean section and in early puer perium. ups j med sci 92: 37-45, 1987. peptidase 24.1 1 : structure, inhibition, and experimental and clinical pharmacology pharma col rev 45: 87-146, 1993. 30. gunne, l.m., lindstrom, l. & terenius, l. naloxone-induced reversal of schizophrenic hallucinations. j neural transm 40: 13-19, 1977. 3 1. terenius, l. & nyberg, f. opioid peptides in the cerebrospinal fluid of psychiatric patients prog brain res 65: 20719, 1986. 32. spanagel, r., herz, a. & shippenberg, t.s. opposing tonically active endogenous opioid systems modulate the mesolimbic dopaminergic pathway. proc natl acad sci usa 89: 33. cadet, p. & stefano, g.b. mytilus edulis pedal ganglia express p opiate receptor transcripts 29. roques, b.p., noble, f., dauge, v., fournie-zaluski, m.c. & beaumont, a. neutral endo 1046-50, 1992 exhibiting high sequence identity with human neuronal p l . mol brain res 74: 242-46, 1999. corresponding address: lars terenius expeimental alcohol and drug addictionsection department of clinical neuroscience karolinska institutet se171 76 uppsala, sweden 15 upsala j med sci 92: 287-292, 1987 relations between clinical signs of right and left cardiac decompensation and radiological signs thereof christer sylvkn, ulf hesser, terje ostby, uirika broom6 and lars zackrisson departments of internal medicine and radiology, danderyd hospital, danderyd and department of radiology, s:t gorans hospital, stockholm, sweden abstract ten consequtive patients arriving at the emergency department for severe systemic cardiac decompensation were investigated in res pect of 17 clinical and laboratory parameters indicative of right or left heart failure. investigations were made at arrival to the hospital and after completed in-hospital care. in respect of left heart failure the prescence of rales and signs of interstitial oedema had a similar-sensitivity. both were of diagnostic value. right heart failure was best diagnosed with clinical parameters such as pitting oedema, filling of the jugu lar vein, and liver enlargement. the laboratory parameters were less sensitive and appeared to have not a diagnostic but well a confirmatory value. introduction clinical decision-making in cardiac decompensation both in res pect of left and right ventricular failure is based on symptoms and signs.pertinent findings are often verified by chest x-ray. this implies that chest x-ray is as sensitive or a more sensitive method and gives more reliable results than the clinical examina tion. ultra-sound scanning of the liver may be used to reveal signs of liver congestion. in fact henriksson et a1 reported that the width of the major right hepatic vein was a reliable indica tion of right heart failure. with the aim of evaluating the sensitivity of different parame ters, an attempt was made to study changes in signs of left and right heart failure at clinical examination, at chest x-ray and at ultrasound investigation of the liver before and after therapy. 287 materials and methods ten consequtive patients arriving at the emergency department due to severe cardiac decompensation and systemic congestion were studied.the following clinical signs were recorded: weight (kg) pitting oedema: o=no oedema, l=ankle oedema, 2=leg oedema, but not above the knee and 3=oedema above the knee. pulmonary rales: o=no rales, l=basal rales, z=rales to the apex of the scapulae, 3=rales to the bases of the scapulae. jugular vein(1): its filling was estimated with the patient recumbent at a 30 degree position.more than 2 cm above the upper central edge of the manubrium sterni was estimated as pathologi cal.the height of the filling (cm) was recorded. kussmauls sign(2): the jugular vein was observed at inspiration. o=decreased filling, l=no change in filling, zincreased filling. hepatojugular reflux (3): a smooth but firm pressure was applied over the liver during 10 seconds.the jugular filling was obser ved: o=no change in filling, l=increased distension of the jugu lar vein but no increased filling in height, 2zincreased filling in height which was estimated in cm when possible. liver: the following criteria was used to estimate dhether it was increased: palpable more than one finger below the right arcus. palpable more than 40% of the distance between the apex of the processus xiphoidues and the umbilicus. in this location the li ver is best palpated as no abdominal muscular tissue covers it. based on these two estimates it was decided whether the liver was enlarged or not.o=liver size within the reference limit, l=enlar ged, 2=painful liver at palpation. chest x-ray: films were taken in upright anterior-posterior and lateral views and when possible, lateral decubitus views for es timation of pleural effusion. the films were then evaluated according to milne et a1 for an estimation of vascular pedicle width (vpw) and v azygos width. vpw is formed by the leash of vessels extending from the thoracic inlet to the heart. milne et a1 have shown that a change in vpw is closely correlated to the change in total blood volume and that the width of v azygos cor relates with mean right atrial pressure. heart size was estimated according in the upright position by routine methods. the presen ce of redistribution of pulmonary blood flow, interstitial oede ma, and kerley b-lines were evaluated. 288 ultrasound: the major right hepatic vein was measured in accor dance to henriksson et a1 , at the confluence with v.cava inferi or. a technicare autosector 1, 3.5 mhz transducer was used. statistics: differences before and after treatment were tested by two-tailed paired students t-test. results the age of the patients was 74+9 (58-89) years, 3 being female. table 1 gives the results. in all patients treatment resulted in disappearence of pathological jugular filling, leg oedema and li ver enlargement and a decrease in weight and heart size. pleural effusion decreased in 7/10 and disappeared in 5/10 patients. he patojugular reflux and an inspiratory increase of the jugular filling disappeared in about half of the patients.the per cent change was high and in the range of 80 to 85% for the jugular filling, leg oedema and liver score. in respect of the jugular filling changes were in the range of centimeters. heart size and weight changed in the 10 per cent range. interestingly, the de crese in absolute heart size was more sensitive (higher p value) than the change in relative heart size. sizes of the superior vena cava or the vascular pedicle and the azygos vein on chest x-ray were in the range of 28-43 mm, 63-78 mm and 7-16 mm, respectively. the major right hepatic vein as studied by ultrasound ranged between .6 and 18 =.these three vessels deacreased their size between 8 and 2 5 % with the highest change observed for the azygos vein. compared to the upper refe rence value for these vessels, the change was 40 to 100 per cent. decreased size of the azygos vein was observed in all patients while the major right hepatic vein decreased only in 75% of the investigated patients. rales and x-ray signs of interstitial oedema disappeared in all patients. signs of redistribution remained in half of the pati ents-kerley lines when present disappeared with treatment. discussion significant decreases were observed in all parameters tested (table 1). the significances were larger for the clinical compa red to the x-ray and ultrasound parameters with the possible ex ception of the width of the vena azygos. in respect of normality 289 table 1. meankstandard deviation of initial value or fraction of patients with an initial prescence of a diagnostic sign,per cent decrease, significance of the decrease (p-value) and the fracti’on of patients with decrease or normalisation of the 17 parameters rneasur ed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . initial % dep-value number of value crease patients jugular filling, 4.8k2.2 cm weight, 78217 kg leg oedema, 1.950.9 score liver , 1.610.5 hepatojugular reflux, 1.010.5 inspiratory jugular 1.320.7 f i 1 1 ing , rales, 2.240.8 heart size, ml absolute 162 12444 score score score score relative 8342141 pleural effusion, 1.320.8 cm vascular pedicle, 7529 m superior vena cava, 3728 azygos vein, 1223 right hepatic vein, 10+4 mm mm mm interstitial oedema 9/9 kerley lines 5/9 redistribution 10/10 a1 12 84 88 60 69 95 14 10 54 9 14 25 20 100 100 40 <0.0002 <o .001 <o. 001 <o. 0001 <o. 01 <o. 05 t o . 0002 <o .005 <o. 01 <o. 01 (0.01 <o .05 <o .005 <o .05 <o. 00001 < o . 00001 (0.05 10/10 10/10 10/10 10/10 7/10 5/10 10/10 7/8 7/8 7/10 7/8 7/9 10/10 6/8 9/9 5/9 4/i 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 290 or not, however, a comparison to reference values must be made. the prescence of clinical signs indicate abnormality and their absence no sign of abnormality. the reference values of the labo ratory parameters relates to values found in a group representing the normal population. upper reference limits for the vascular pedicle and the azygos vein have been defined by milne et a1 ( 4 ) and for the major right hepatic vein by henriksson et al. values reported are 58, 8 and 9 mm respectively.the observer variation in the estimation of ves sels at chest x-ray was reported to be 222%. the ultrasound es timate of the major right hepatic vein is probably larger. the vascular pedicle and thus the superior vena cava was pathologi cally wide in all patients at onset of therapy and remained so after normalisation of clinical parameters except in one patient. the azygos vein was likewise pathological in all patients at on set of study.normalisation was obtained in three patients. the major right hepatic vein was within normal limits in 4 / 8 patients at onset of study and in 6 / 8 at the end of the study. the mean decreases in the width of the 3 vessels studied were 7, 3 and 2 mm respectively.in view of an observer variation (milne et all at 2+2% the changes during therapy in this set of patients with se vere systemic heart decompensation are minimal and must be consi dered insensitive. the tendency observed when comparing values before and after treatment, i.e. that clinical signs of right heart failure are more sensitive parameters than those supplied by the laboratory is thus confirmed when the comparison is made to reference va lues. in respect of right heart failure the laboratory parameters appear not to have a diagnostic but well a confirmatory value. such a conclusion is at variance with the report by henriksson et al. they reported efficient separation between the reference group and patients with severe venous congestion in respect of the major hepatic vein. maybe different degrees of vascular fib rosis affect the compliance of the veins, including the hepatic vein, where also hepatic fibrosis may be of importance. the preference of clinical methods for the diagnosis of right heart failure should be compared to the high degree of covaria tion between the presence of rales and interstitial oedema. chest x-ray therefore appears to contribute with useful information and confirmation in respect of left heart failure. however, signs of redistribution did not covary with the prescence of rales, pos sibly due to the fact that this sign indicates slight degrees of left ventricular failure where auscultation may be non 29 1 contributory. the patient selection in this series was based on undisputable ’ clinical signs of systemic heart decompensation. thus the preva lence of the disease was 100 % . in groups with lower prevalence it is not excluded that chest x-ray methods may have better pre dictive values than clinical methods. in conclusion,left ventricular failure, as diagnosed clinically finds its useful confirmation in traditional findings at chest x ray.right ventricular failure on the other hand appears to be best diagnosed and monitored by simple clinical methods. acknowledgement: this study was supported by grants from the swedish association against heart and lung diseases. references 1. dell’italia, l.j., starling m.r. & o’rourke r.a.: usefulness of the physical examination for excluding hemodynamically important right ventricular infarction. circulation 68,suppl iii 239:956,1983. 2. henriksson, l., hedman, a., johansson r. & lindstrom k.: ultrasound assessment of liver veins in congestive heart failure. acta radio1 diagn 23:361-363,1982. 3. maisel, a . s . , atwood j.e. & goldberger a . l . : hepatojugular reflux: useful in the bedside diagnosis of tricuspid regurgita tion. ann intern med 101:781-782,1984. 4. milne, e.n.c., pistolesi, m., miniati m. & giuntini c.: the vascular pedicle of the heart and the vena azygos. radiology 152;l-17,1984. address for reprints: christer sylven, md, dep of medicine, danderyd hospital, s-182 88 danderyd sweden 292 ujms110_2.pdf upsala j med sci 110: 167–172, 2005 a rare primary location of echinococcal disease: report of a case baris zulfikaroglu, mahmut koc, necdet ozalp, m mahir ozmen, department of surgery ankara numune teaching and research hospital, 06100-ankara, turkey abstract a case of primary hydatid disease, a rare location, is presented. the patient was a 20 year old female who presented with complaining of painful mass in the right hypochondrium 2 months before presantation. this cyst was strongly adherent to the two layers of m.rectus abdominis fascias. it was completely removed. no other site of hydatid disease was found and the patient remained well postoperatively. introduction human hydatid disease is caused by the larvae of the genus echinococcus. the four species known to infect man are e.granulosus, e.multilocularis, e.oligarthrus, e.vogeli (1, 2). e.granulosus is by far the most common cause of disease, followed by e.multilocularis, which causes the alveolar form of the disease. primary hydatid cysts usually localize in the liver (80%), and lungs (15%), but they can affect other organs and body tissues (2, 3, 4). we present a case with a rare location of primary hydatid cyst, located at the intrafascial location of the abdominal wall. case report a 20 year-old woman was admitted to our clinic, with the complain of painful mass in the right hypochondrium noticed two months before presantation. on physical examination, the mass was firm, smooth, tender, not fixed to the overlying skin but fixed to deeper tissue. there was no history of trauma. laboratory studies showed a total leukocyte count of 6.8×109/liter, and eosinophil count of 620/mm3; all other biochemical parameters of serum and urine were in the normal range. serological tests for echnicoccus were positive (1:64 by latex test and 1:2560 by indirect 167 received 1 november 2004 accepted 21 december 2004 key words: hydatid disease, rare location, primary 168 fig. 1. the cyst was completely excised. 169 fig. 2. pathologic study of the specimen confirmed the echinococcus. hemagglutination). ultrasonography and a computed tomography scan showed a cyst of 5 cm in the right rectus abdominis muscle. laparotomy was performed via a right subcostal incision on the presumptive diagnosis of hydatid cyst. a cystic mass of 40×50×30 mm was found between the two fascial layers of m.rectus abdominis. routine abdominal exploration was carried out, but no other cyst or abnormality was found in the abdominal cavity. the cyst was completely excised (photograph 1) and soft tissue was washed with 0.5% silver nitrate solution. pathologic study of the specimen confirmed the echinococcus (photograph 2). abdominopelvic ultrasonography, intravenous pyelography, lung tomography, whole body skeletal scanning, and cranial ct performed postoperatively. there was no evidence of any other foci of hydatid disease. the patient was discharged on the 10th postoperative day in a satisfactory condition. patient was administered 10 mg/kg per day oral albendazole therapy 1 week before surgery. albendazole therapy was continued for three sessions, each lasting 4 weeks separated by fourteen days intervals postoperatively (5). follow-up at 3 years, after yearly repeat examination showed no recurrence of hydatid disease. the patient was informed about the case report. discussion these are the first reported cases of intrafascial involvement with e.granulosus. hydatid disease is a parasitic infection caused by several species of the cestode echinococcus, but the most common is echinococcus granulosus. echinococcus is endemic to many parts of the world, in most sheepand cattle-raising areas, including australia, south america, middle east, south africa, eastern europe, and the mediterranean region. although hydatid cysts were known commonly to affect the liver, lung, spleen, it also affects the brain, heart, kidney, ureter, uterus, fallopian tube, mesentery, pancreas, diaphragm, and muscles (3, 4). the clinical presentation of hydatid disease depends on the size and site of the lesion and the accessibility of the organ involved for clinical examination. as it has been studied previously, the most important factor in diagnosing hydatid disease in unusual localizations in the awareness of its possibility and any growing mass in a patient coming from an endemic region should arise the suspicion of hydatid disease (6). diferent serologic tests are being carried out for the diagnosis, screening and post-operative follow-up for recurrence. these include the hydatid immunoelectrophoresis, enzyme-linked immunosorbent assay (elisa), latex agglutination and indirect haemagglutination (iha) test (7). preoperative diagnosis of hydatid cysts could be made by imaging methods (8). in this case, as well as others, ultrasonography was the most helpful investigations. the best therapeutic option of hydatid cysts is principally surgical. however, medical therapy with mebendazole or albendazole should be considered in order to sterilize the cyst, decrease the chance of anaphylaxis, decrease the tension in the cyst wall and to reduce the recurrence rate post-operatively (9). intra-operatively, 170 the use of hypertonic saline or 0.5% silver nitrate solutions before opening the cavities thends to kill the daughter cysts and therefore prevent further spread or anaphylactic reaction. altough we made all necessary laboratory investigations postoperatively, there was no other cyst or abnormality in the abdomen or in any other system of the body in our patient. we assumed that the cyst was primary. we believe that embryo has reached the fascia from the systemic circulation after leaving the intestine and passing through two filters: the liver and the lungs. detailed investigation is essential in any patient with hydatid disease in an unusual site in order to determine whether the cyst is primary or secondary. location of hydatid disease between the two fascial layers of m.rectus abdominis is extremely rare. although hydatid cyst can rarely be found in subcutaneous tissue (1%), to the best of our knowledge, an intrafascial localization of hydatid cyst has never been reported. if the cyst is found in this location, it should be totally excised. references 1. ammann rw, eckert j (1996) parasitic diseases of the liver and intestines-echinococcus. gastroenterol clin north am 25: 655–689. 2. hadni sb, humbert p, paintaud g, auer h, miguet jp (1996) skin localization of alveolar echinococcus of the liver. j am aca derm 34: 873–877. 3. saracho jo, zarza lp, sanchez j (1998) an unusual cause of pulmonary artery thrombosis. chest 114: 309–311. 4. cangiotti l, muisean p, decesare v, pouché a, giulini sm, tiberio g (1994) unusual localizations of hydatid disease: a 18 year experience. giorn chir 15: 83–86. 5. ozmen mm, coskun f (2002) new technique for finding the ruptured bile duct into the liver cysts: scope in the cave technique. surg lap endosc perc tech 12(3): 187–189. 6. enamy h, asadian a (1976) unusual presentation of hydatid disease. am j surg 132: 403–405. 7. abu-eshy sa (1998) some rare presantations of hydatid cyst (echinococcus granulosus): case report. j r coll surg edinb 43: 347–352. 8. brown ra., millar aiw, steiner z, krige jej, burkimsher d, cywes s (1995) hydatid cyst of the pancreas: a case report in child. eur j pediatr surg 5: 121–124. 9. goel mc, agarwal mr, misra a (1995) percutaneous drainage of renal hydatid cyst: early results and follow-up. br j urol 75: 724–728. corresponding author: baris zulfikaroglu md 7.cadde 70 a /14, 06490-bahcelievler ankara-turkey tel: +90-312-2158652 fax: +90-312-3103460 e-mail: zbaris61@hotmail.com 171 upsala j med sci 91: 225-227, 1986 working group for the rational use of urinary test strips anders kallner, rudolf jagenburg, bertel berg and kristoffer hellsing protocol f o r the evaluation of reagent sets (chemicals and fnstruments) with special reference to "spot-tests" the continuous development and marketing of new reagents and instruments makes it desirable to design a protocol which utilizes acceptable and well recognized methods. such an app roach is particularly important since many laboratories are faced with a demand to compare different reagent sets: it would be an advantage also if manufacturers would describe their products in these terms. the working group is in the process of finalazing a report on evaluation of test strips, highlighting also problems of discrimination values in binary and paucinary tests. the following provisonal protocol has been designed to esti mate imprecision and inaccuracy from patient samples as well as from control and reference materials. also, the with-in, between-series and total variation should be estimated. special methods are advised for the estimation of linearity, and the effects of lipemia and hemolysis. special attention should be given to the effect of the sample matrix. several recommendation for evaluation of methods have been published with details on statistical treatment of the results (1-3). 1. imprecision and inaccuracy 1.1. patient samples about 36 samples should be determined in dupli cates on three different occasions. the samples should also be analyzed with an agreeable, well controlled method which agrees as much as possible with a reference methodology. the patient samples should be of different concen trations. 1.1.1. 1.1.2. estimate the regression between the means of the duplicates and the values obtained by that used as a reference. estimate the standad deviation from the results of the duplicate analyses of three levels, subnormal, "decision interval" and increased values. 225 1.1.3. 1.2. 1.2.1. 1.2.2. identify and reexamine all outliners. evaluate deviation in view of matrix on systemic effects. reference material use three levels of concentrations of a refe rence material which is as close as possible to human. the number of analyses on each level should be abut 25. estimate the mean, standard deviation and coefficient of variation for each reference material. use the reference material which is closest to the decision level and determine the concentra tions in 15 different runs. several runs may be carried out per day but they must then be initiated the normal way, including shut-down of the instrument, recalibration etc. in each run 5 values should be determined. 1.2.2.1. calculate the average within-batch variation. 1.2.2.2. calculate the between-batch variation. 1.2.2.3. calculate the total variation. 2. testing other parameters of performance. 2.1. hemolysis (addition of hemolysed erythrocytes). a hemolysate is prepared from washed human erythrocytes which are frozen and thawed several times. a series of dilution is made from this concentrate, using 0.15 mol/l nacl as diluent. suitable concentrations are 320, 160, 80, 40, 20, 10 and 5 g/l. a serum sample is diluted 1:lo with the appropriate hemoglobin sample. these samples as well as a sample diluted with 0.15 mol/l nacl 1:lo are determined in duplicates. 2.2. 2.3. r lipemia (addition of fat emulsion, intralipid is provisionally suggested). the initial concentration of 100 g/l is diluted with 0.15 mol/l nacl to concentrations of 100, 50, 25, 12 and 6 g/l. a serum sample is then diluted 1:lo with these dilutions of the €at emulsion, including a zero sample. samples should be analysed in duplicates. bilirubinemia. a similar test of the influence of bilirubin should be carried out. a suitable test material is still to be found. 226 2.4. 2.5. 2.6. 2 . 7 drugs. drugs interfering with the methods chemically should be systematically tested. the booklet "drug interference and effects in clinical chemistry" (apoteksbolaget, stockholm, sweden) should be consulted. linearity the linearity of the method should be tested over as wide a range as possible. select a high and a low concentration serum and mix them in a suitable manner to obtain at least five points. plot the found vs. the calculated results, if possible describe the performance using reg ression analysis. sample volume. analytical results should be determined using from -10 to +lo% of the recommended volume of sample. erythrocyte, volume fraction. in case whole blood is recommended the effect of varying the evf should be considered within the fysiological range. it is understood that deviations from the outlined method could be necessary due to technical or other circumstances. depending on the reagent set evaluated, other items might also be important to consider. several reagent sets (chemicals and/or instruments) are in the process of being tested in accordance with this protocol. extensive lists of further reference to the literature are fund in these papers. 1. ifcc expert group on diagnostic kits and reagents. a) recommendations for specifications on labelling of clinical laboratory materials. b) guidelines for the evaluation of clinical chemistry kits in ifcc recommendations and related documents volume i, 1978-83 walter de gruyter 1984. isbn 3-11-008766-9. 2. ifcc/who principles and recommendations of evaluation of diagnostic reagent sets used in health laboratories with limited resources. j clin chem clin biochem (1984) 22: 817 with appendicies a-e (glucose, urea, total bilirubin, total protein and albumin). 3 . white gh and fraser cg: the evaluation kit for clinical chemistry: a practical guide for the evaluation of methods, instruments and reagent kits. j autom chem (1984) 6 : 122. 227 upsala j med sci 90: 149-156, 1985 renal lkansplantation in uppsala lars frodin and ulla backman departments of urology and medicine, university hospital, uppsala, sweden abstract the h i s t o r y and p r o g r e s s o f o r g a n t r a n s p l a n t a t i o n i n uppsala a r e r e v i e w e d . renal t r a n s p l a n t a t i o n was begun i n 1969, and t h e programme now c o m p r i s e s 50 t o 60 t r a n s p l a n t s p e r y e a r . s i n c e 1976 t h e o p e r a t i o n i s p e r f o r m e d a t t h e d e p a r t ment o f u r o l o g y . c l o s e c o l l a b o r a t i o n has been e s t a b l i s h e d w i t h o t h e r d e p a r t ments i n t h e h o s p i t a l , e s p e c i a l l y w i t h t h e m e d i c a l n e p h r o l o g y u n i t . the i n d i c a t i o n s f o r a c t i v e management o f u r a e m i c p a t i e n t s have broadened, and m a i n t a i n i n g r e s o u r c e s on a p a r w i t h t h e demands has c o n s t a n t l y been a problem. t h i s r e p o r t c o n c e r n s immunosuppressive t h e r a p y , t r a n s p l a n t a t i o n r e s u l t s and r e s e a r c h c o n n e c t e d w i t h t h e t r a n s p l a n t a t i o n programme and d e a l s b r i e f l y w i t h t h e p r o s p e c t s f o r u p p s a l a a s a t r a n s p l a n t a t i o n c e n t r e i n t h e f u t u r e . introduction c l i n i c a l r e n a l t r a n s p l a n t a t i o n was begun i n t h e e a r l y 1960s. i n uppsala t h e f i r s t r e n a l t r a n s p l a n t was p e r f o r m e d i n june 1969. the f i r s t p a t i e n t was a 30 y e a r o l d man w i t h p o l y c y s t i c k i d n e y d i s e a s e . a l r e a d y i n t h i s case s p e c i a l uppsala t r e a t m e n t was used, a s f r o z e n a u t o b l o o d , w h i c h a t t h a t t i m e was t h o u g h t t o s u p p r e s s immunologic r e a c t i o n s between t h e p a t i e n t and t h e donor organ, was i n f u s e d p r e o p e r a t i v e l y and p o s t o p e r a t i v e l y . this p a t i e n t s t i l l has a f u n c t i o n i n g t r a n s p l a n t and i s i n f u l l t i m e work. the f i r s t r e n a l t r a n s p l a n t a t i o n was p e r f o r m e d by p r o f e s s o r l a r s thorkn, head o f t h e s u r g i c a l department who, t o g e t h e r w i t h k a r l e r i k f j e l l s t r o m , i n c h a r g e o f t h e n e p h r o l o g i c u n i t i n t h e m e d i c a l d e p a r t m e n t and c l a e s hogman, head o f t h e b l o o d bank, i n t r o d u c e d t h e p r o c e d u r e i n uppsala. i n i t i a l l y t h e p a t i e n t s remained f o r a few days i n t h e i n t e n s i v e c a r e u n i t and t h e n r e t u r n e d t o t h e n e p h r o l o g y u n i t . d u r i n g t h e f i r s t p o s t o p e r a t i v e weeks j o i n t d a i l y v i s i t s were made. throughout t h e e v o l u t i o n o f t h i s work, v e r y c l o s e c o l l a b o r a t i o n between t h e i n v o l v e d d e p a r t m e n t s has been t y p i c a l f o r o u r t r a n s p l a n t a t l o n c e n t r e . 149 this collaboration includes surgeons, urologists, nephrologists, clinical im munologists, radiologists, anaesthesiologists and many scientists involved in basic research within the field. evolution and capacity as more and more patients have been accepted f o r dialysis, the numbers of renal transplants have increased over the years. in addition to the centre in uppsala, dialysis units have been established elsewhere within the health s e r vice region (bollnas, ostersund, sundsvall, gavle and vaster&). the total beds for dialysis were 14 in 1 9 6 9 , and the figure for 1984 is 4 6 . diabetics have been accepted for active therapy since 1975, and older patients have increas ingly entered the programme. the need for more transplantations has of course required extension o f re sources surgical, medical and in the auxiliary services. initially the oper ations were done in the department of surgery as part of the routines there. the first surgical post specifically for transplantation was established in 1 9 7 1 , in 1974 a new department o f urology was opened at the hospital and, as most of the surgeons interested in urology moved to this service, a logical consequence was that the renal transplant programme was taken over by the urology department. in 1 9 7 6 , therefore, this department become wholly respons ible for transplantations, and one of the associate professors at the depart ment was delegated to lead the work. as yet, however, no surgeon was working full time with these patients. not until 1983 was the decision made to permit two surgeons to work f u l l time on the transplantation programme. the waiting list nevertheless has continued to grow, and the number of transplantations has not kept pace. the progress that has been made despite this inadequacy would not have been possible without the extraordinary work done by the nephrologists. table i surveys the growth of the transplant and dialysis programmes at uppsala. the number o f transplantations planned for 1985 is 55 60, and extension o f the facilities at the departments of uro logy and nephrology is expected. the numbers of dialyses reflect the growing need f o r active management of renal failure in uppsala region's total popul ation of 1.2 rnillion. donor organs the uppsala transplantation centre has used a low percentage (c. 5 % ) of kidneys from living related donors. initially this was because of the assurned risk of removing an organ from a healthy individual. although gradually we 150 table 1. growth of the renal transplantation and dialysis services in uppsala" no of no of approximate no year transplants dialyses on waiting list 1969 6 1970 11 900 1971 20 1972 17 1 900 1973 21 1974 30 1 500 1975 31 2 400 1976 34 2 500 16 1977 26 3 odd 20 1978 31 4 100 24 1979 30 3 800 15 1980 32 3 100 30 1981 40 3 900 37 1982 42 4 000 37 1983 42 5 500 62 1984 61 6 000 60 total 474 42 600 301 * z no figures available are increasing use of such sources, the percentage of living donors still is relatively low at our centre. a contributory factor is that harvesting has al ways been well organized in the uppsala region, as a result o f close collabor ation with the other departments of surgery and anaesthetics within the region. the doctors at the university hospital and elsewhere in the region have shown a highly positive attitude concerning management of uraemic patients. at many regional meetings arranged by different disciplines, harvesting of kidneys has been a topic of discussion. the linking o f the transplantation programme to urology also has had posit ive effects. many young urologists thus have worked with problems of uraemic patients during training in uppsala, and have learned techniques o f harvesting and transplanting kidneys. vascular access surgery has also been included in their training. many o f these doctors subsequently work in hospitals within the uppsala region and contribute considerably to the harvesting prograwme. is1 uppsala is one of the centres in "scandia transplant", an organization with in which kidneys are exchanged. table 2 shows that uppsala has had the most pos itive exchange balance of all the scandinavian centres. this implies possibil ities for increasing the numbers of cadaver kidney transplantations. use ofmore living, related donors is another possibility. table 2. 31/12 1982 utilization of kidneys in "scandia transplant" from 1/6 1969 to - donor k i d n e y s total exchange centre transferred used locally imported transplanted balance immunosuppressive therapy and results immunosuppressive therapy has undergone several modifications since we began to transplant kidneys in 1969. initially fairly high doses of steroids were given, in accordance with policy in most countries (5). the dosaqe usually was 200-300 mg on the first postoperative day, tapered to a maintenance dose of 5 to 10 mg/day. azathiaprine was always given together with steroids, beginning with an i n travenous dose before transplantation. the initial dose usually was 2-3 mg/kg/ day, with tapering thereafter according to evidence of bone-marrow depression or severe infection. because o f severe side effects, the immunosuppressive therapy was changed in 1980 to the low-dosage schedule introduced by mcgeown in belfast (3). the ster oid d o s e thus was reduced to 20 mg/day, which was maintained for one month and then very slowly reduced to 5 mg/day. with this therapy there was a high 152 i n c i d e n c e o f a c u t e r e j e c t i o n s . the s u r v i v a l r a t e s o f t r a n s p l a n t s and o f p a t i e n t s , however, were h i g h e r t h a n i n t h e e a r l i e r p e r i o d ( t a b l e 3 ) . t a b l e 3. p a t i e n t and g r a f t s u r v i v a l ( a p p r o x i m a t e f i g u r e s ) immunosuppressive t h e r a p y i n d i f f e r e n t p e r i o d s compared w i t h one-year 1969 1980 1980 1983 1983 1984 p r e d n i s o l o n e p r e d n i s o l o n e p r e d n i s o l o n e h i g h dose + l o w dose + l o w dose + a z a t h i o p r i n e a z a t h i o p r i n e c y c l o s p o r i n one-year s u r v i v a l 2-3 mg/kg/day 2-3 mg/kg/day a p a t i e n t s g r a f t s 75 7; 82 % 93 e 37 % 55 z 72 % a f t e r one y e a r with t h i s regimen, we i n t r o d u c e d a m o d i f i e d s t e r o i d sched u l e , s t a r t i n g with 0.6 mg/day. the a i m was t o d i m i n i s h t h e h i g h i n c i d e n c e o f r e j e c t i o n s w h i l e m a i n t a i n i n g good p a t i e n t s u r v i v a l . i n 1983 we were g i v e n t h e o p p o r t u n i t y t o p a r t i c i p a t e i n a m u l t i c e n t r e s t u d y u s i n g c y c l o s p o r i n a as immunosuppressive a g e n t i n s t e a d o f a z a t h i o p r i n e . with t h i s a g e n t a l o w s t e r o i d r e g i m e n c o u l d be used. d u r i n g t h e f i r s t 10 days t h e p r e d n i s o l o n e dose i s t a p e r e d f r o m 100 mg/day t o 20 mg/day and t h e n s l o w l y re duced t o 10 mg/day w i t h i n 3 months. the dosage o f c y c l o s p o r i n a s t a r t s with 1 5 mg/kg/day and i s t h e n reduced b y 2 rng e v e r y 1 4 t h day i n accordance w i t h t h e c l i n i c a l p i c t u r e and t h e l e v e l o f c y c l o s p o r i n a i n plasma. with t h i s r e g i m e n t h e g r a f t s u r v i v a l was c o n s i d e r a b l y p r o l o n g e d ( t a b l e 3 ) . there were a l s o l e s s problems w i t h i n f e c t i o n s and t h e i n c i d e n c e o f g r a f t re j e c t i o n was reduced from about 70 % t o 30 %. the p o s t o p e r a t i v e p e r i o d t h u s b e i n g r e l a t i v e l y u n e v e n t f u l , t h e h o s p i t a l s t a y was s h o r t e n e d t o 3-4 weeks, as compared w i t h 6-8 weeks e a r l i e r . a m a j o r p r o b l e m with c y c l o s p o r i n , however, i s i t s n e p h r o t o x i c e f f e c t , and so f a r t h e l o n g t e r m r e s u l t s a r e unknown. hope f u l l y , some new analogue o f c y c l o s p o r i n w i l l b e f o u n d t o combine t h e same good immunosuppressive a c t i o n w i t h absence o f n e p h r o t o x i c e f f e c t . o t h e r forms o f t h e r a p y have a l s o been t r i e d t o g e t h e r w i t h t h e a b o v e s t a t e d b a s i c regimens. thus i n 1975 we s t a r t e d t o g i v e b l o o d t r a n s f u s i o n s t o p r e v i o u s l y n o n t r a n s f u s e d p a t i e n t s , f o l l o w i n g p o s i t i v e r e s u l t s w i t h t h i s r e g i m e n r e p o r t e d f r o m o t h e r c e n t r e s ( 4 ) . we have a l s o used d o n o r s p e c i f i c b l o o d t r a n s f u s i o n s (dst) t o one h a p l o t y p e mismatched l i v i n g r e l a t e d r e c i p i e n t s . three u n i t s (200 m l ) o f b l o o d f r o m t h e donor were g i v e n t o t h e g r a f t r e c i p i e n t a t 153 intervals of 2 weeks. up to now 1 2 patients have been treated in this way, and only one has been sensitized. this regimen is still in use in combination with conventional immunosuppressive therapy, with azathiprine and cortisone instead of cyclosporin a, in transplantation from living related donor. rejection therapy treatment of acute rejection has been, and still is, increased dose of steroids. the dose usually has been fairly high about 1 g on 3 successive days. in patients treated with cyclosporin a, rejection therapy consists of a reduced steroid regimen (1.25 g methylprednisolone in 4 divided doses given during 4 days). in the early years we also used local irradiation to treat r e jection. as the effect was questionable, however, irradiation was abandoned. in 1982 rabbit antithymocyte globulin (ratg) was introduced for treatment o f steroid-resistant allograft rejection (2). the effect was dramatic, and almost all of the patients responded with reduction o f serum creatinine. this effect ive antirejection treatment, however, increases the incidence of infections and involves risk of malignancy, especially when given in combination with cyclo sporin a . as techniques of immunosuppression have changed, the overall results as measured by patient and graft survival have improved (table 3). many factors other than immunosuppressive therapy have contributed, however. surgical pro cedures have been refined, in particular the management of complications. dia gnostic techniques have progressed. gammacamera scintigraphy, ultrasonography and biopsy with advanced histologic methods have become available, and the entire clinical care has become more proficient. a retrospective survey there fore readily provides an optimistic basis for future prospects. the improve ments have taken place parallel with acceptance of diabetics and elderly pat ients for renal transplantation. research since 1968 research on preservation and ischaemia o f kidneys has continu ously been conducted in uppsala. five postgraduates working at the department o f urology have produced doctoral theses in this field. close and fruitful col laboration has been established with the renal research group at the institute of physiology and at the university's biomedical centre in uppsala. the re search work begun in 1968 was first conducted at the laboratories o f the pharmacia company, which has continued to provide sponsorship. immunologic research at uppsala university has long traditions. collaborat ion with immunologists has intensified in recent years, especially since the appointment o f young surgeons with basic training in immunology to our uro logic department with its transplantation service. close contacts have thus been established between basic research and clinical work. together with the clinicians, the departments of clinical immunology, microbiology and immunology are working on problems relating to the diagnosis, mechanism and therapy of graft rejection. clinical research has focused on follow-up studies of patients with differ ent immunosuppressive regimens, and on evaluation of new diagnostic procedures. this research is done together with nephrologists, radiologists and oncolog ists. uppsala is one o f the five centres collaborating in a study on cyclo sporin a. parallel with this clinical study, uppsala is conducting a special programme, together with sandoz ltd, on the pharmacokinetics of cyclosporin a. most of the aforementioned departments are also in some way engaged in this project. the field of transplantation brings together workers in basic research and clinical departments in a natural way. we are happy that so many scientists at our university show interest in this work. it is only through such collaborat ion that clinical progress can be made in organ transplantation. the future conclusions concerning the immediate future are readily deducible from table 1. the uppsala region needs expansion of the transplantation service. the current programme envisages 55-60 renal transplants per year, and re sources for this volume are being provided. the future, however, is largely dependent on political decisions. a question now being discussed is if uppsala should accept responsibility f o r transplantations within a larger region. this would result in about 85 renal transplants each year. another question is whether transplantation of pancreas, liver and heart should take place in uppsala. there is need for 15-20 transplants of liver, 20 of pancreas and at least 20 o f heart in our present region. while awaiting the political decisions, the doctors are preparing the field by extending re search, acquiring skills in harvesting multiple organs and stimulating inter est among all categories of hospital workers for organ transplantation. 11 -858572 155 references calne, r.y., r o l l e s , k., t h i r u , s., mcmaster, p., craddock, g.n., aziz, s., white, d.j.g., evans, d.b., dunn, d.c., henderson, r.g. & lewis, p.: c y c l o s p o r i n a i n i t i a l l y as t h e o n l y immunosuppressant i n 34 r e c i p i e n t s o f c a d a v e r l c organs: 3 2 kidneys, 2 pancreases, and 2 l i v e r s . the lancet 11: hoitsma, a.j., van l i e r , h.j.j., reekers, p. & koene, r.a.p.: treatment o f a c u t e r e j e c t i o n o f c a d a v e r i c r e n a l a l l o g r a f t s w i t h r a b b i t a n t i t h y m o c y t e g l o b u l i n , transpl proc. i n press, 1984. mcgeown, m.g., loughridge, w.g.g., alexander, j.a., mcevoy, j., kennedy, j. a., douglas, j,, clarke, s.d. & h e w i t t , j . c . : one hundred k i d n e y t r a n s p l a n t s i n t h e b e l f a s t c i t y h o s p i t a l . the lancet 1:648-651, 1977. opelz, g . & t e r a s k i , p . i . : dominant e f f e c t o f t r a n s f u s i o n s on k i d n e y g r a f t s u r v i v a l . t r a n s p l a n t a t i o n 2 9 2 , 153-158, 1980. salaman, j.r.: n o n s p e c i f i c imunosuppression. in: kidney t r a n s p l a n t a t i o n . p r i n c i p l e s and p r a c t i c e . ed. m o r r i s , p.j. 2nd e d i t i o n , p . 163, 1984. 1033-1036, 1979. 1 . 2 . 3 . 4. 5. address f o r r e p r i n t s : l a r s f r o d i n department o f u r o l o g y u n i v e r s i t y h o s p i t a l 5-751 85 uppsala sweden serum pentraxin 3 concentrations in neonates upsala journal of medical sciences. 2014; 119: 62–64 letter serum pentraxin 3 concentrations in neonates anders lannergård1, fredrik rosenström1, erik normann2 & anders larsson1 1department of medical sciences, uppsala university, uppsala, sweden, and 2department of women’s and children’s health, uppsala university, uppsala, sweden dear editor, c-reactive protein (crp) is one of the most commonly used diagnostic biomarkers in the clinical setting for diagnosing and monitoring of inflammatory and infectious diseases. however, in neonates several questions remain, regarding the interpretation of an increased serum crp concentration during the first days of life in otherwise healthy newborns, especially in preterms (1). pentraxins are endogenous substances that neutralize or eliminate a variety of pathogenic agents in concert with the complement system and macrophages. these complex biological responses to tissue injury, necrosis, infections, or immune-related diseases are part of the natural innate defence system (2). the pentraxin family consists of proteins with cyclic multimeric structures. the well-known acute-phase protein crp belongs to the short members of the family, while pentraxin 3 (ptx3) belongs to the long counterparts. ptx3 is released from macrophages and endothelial cells among a great variety of other cell types in which the protein is synthesized and stored. on stimuli from primary inflammatory signal proteins, as interleukin-1 (il-1), tumour necrosis factor-alpha (tnf-a), and microbial lipopolysaccharides (lps) as well as toll-receptor-mediated signals, ptx3 will be synthesized, and the serum concentration will increase up to 100-fold in 6–8 hours. interleukin-6 (il-6) is a weak inducer for ptx3 (3,4). ptx3 synthesis is thus produced close to the site of inflammation and is not dependent on cytokine production or liver function. all subjects in our study were recruited from the department of women’s and children’s health, uppsala university hospital, uppsala, sweden. three groups were included: umbilical venous cord blood collected from term healthy newborns (n = 36), venous blood from term healthy newborns at 3–7 days of age at phenylketonuria (pku) testing (n = 11), and venous blood from very preterm (vpt) newborns at the neonatal supportive care unit (n = 40). the median gestational age and postnatal age were 26 weeks (range 23–31, iqr 4) and 27 days (range 4–103, iqr 26), respectively. the median birth weight was 954 g (range 422–2244, iqr 400). the study was approved by the local ethical review board of uppsala university. serum ptx3 was determined by a commercial sandwich elisa kit (dy1826, r&d systems, minneapolis, mn, usa), as described elsewhere (5). serum concentrations of ptx3 in term healthy newborns were lower than in term healthy newborns at 3–7 days of age and vpt newborns at the supportive care unit (p < 0.0001) (figure 1). vpt newborns showed a lower serum concentration of ptx3 than term healthy newborns at 3–7 days of age (p < 0.0001) (figure 1). to our knowledge, no studies have presented such data before. previous studies on serum amyloid a and high-sensitivity (hs) crp reported gestational age-related differences, with higher concentrations in vpt compared with full-term healthy babies, during the neonatal period (6-8). a similar pattern seems to apply to ptx3. because ptx3 may be a faster acute-phase protein that is not correspondence: dr anders lannergård, md, phd, department of medical sciences, section of infectious diseases, uppsala university, s-751 85 uppsala, sweden. e-mail: anders.lannergard@akademiska.se (received 17 october 2013; accepted 19 december 2013) issn 0300-9734 print/issn 2000-1967 online � 2014 informa healthcare doi: 10.3109/03009734.2013.878770 http://informahealthcare.com/journal/ups mailto:anders.lannergard@akademiska.se liver-dependent, it is probable that it is superior to traditional biomarkers for mirroring rapid inflammatory courses. compelling indications are present that a high serum concentration of ptx3 predicts shock, poor outcome, and organ failure in meningococcal disease and severe sepsis, respectively, in children and adults (9,10). the serum concentration of ptx3 in the cohort should be interpreted with care because a part of the sample was likely to have its origin from infants treated with antibiotics or with inflammatory reactions. it is important to remember that there is an extremely rapid foetal development up to the expected day of delivery. this means that there will be differences between a prematurely born child with a gestation age of 30 weeks from a child with a gestation age of 36 weeks. the cause of the prematurity may also involve inflammatory reactions which will further increase the variability in ptx3 values in samples from prematurely born babies. nevertheless, the prematurely born cohort, despite the existence of possible factors that may influence the serum ptx3 concentration, could be representative for this group of newborn infants. in summary, serum ptx3 may be a promising acute-phase protein for interpretation of affected newborns with symptoms and signs of sepsis. serum ptx3 is measurable in both term and preterm babies in similarity with other acute-phase proteins. considering the promising results from studies in adult patients with sepsis, ptx3 might represent a helpful tool in diagnosis of sepsis in the neonate as well. the predictive value of ptx3 may thus be higher compared with conventionally used acute-phase proteins. however, regarding serum ptx3 concentrations in infants with infection we propose a prospective study on neonatal sepsis with well-defined established criteria. acknowledgements the staff members at uppsala university hospital have skilfully assisted throughout this study. declaration of interest: financial support by the faculty of medicine, uppsala university is gratefully acknowledged. the authors report no conflicts of interest. the authors alone are responsible for the content and writing of the paper. references 1. hofer n, zacharias e, muller w, resch b. an update on the use of c-reactive protein in early-onset neonatal sepsis: current insights and new tasks. neonatology. 2012;102: 25–36. 2. agrawal a, singh pp, bottazzi b, garlanda c, mantovani a. pattern recognition by pentraxins. adv exp med biol. 2009; 653:98–116. 3. mantovani a, garlanda c, doni a, bottazzi b. pentraxins in innate immunity: from c-reactive protein to the long pentraxin ptx3. j clin immunol. 2008;28:1–13. 4. deban l, jaillon s, garlanda c, bottazzi b, mantovani a. pentraxins in innate immunity: lessons from ptx3. cell tissue res. 2011;343:237–49. 5. larsson a, ronquist g, akerfeldt t. lifestyle intervention is associated with decreased concentrations of circulating pentraxin 3 independent of crp decrease. ups j med sci. 2013; 118:165–8. 6. chiesa c, osborn jf, pacifico l, natale f, de curtis m. gestationaland age-specific crp reference intervals in the newborn. clin chim acta. 2011;412:1889–90. 0 2 4 6 8 10 12 term healthy newborns term healthy newborns at 3-7 days of age very preterm (vpt) newborns p t x 3 ( mg /l ) p < 0.0001 p < 0.0001 ns figure 1. serum concentrations in term healthy newborns (n = 36), term healthy newborns at 3-7 days of age (n = 11), and very preterm newborns (n = 40). a kruskal–wallis test was used for continuous three-group comparisons. data were computed by statistica 10 (statsoft, tulsa, ok, usa). a p value of <0.05 was considered statistically significant. median values are shown with filled bars. ptx3 in neonates 63 www.ncbi.nlm.nih.gov/pubmed/22507868?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22507868?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/22507868?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/19799114?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17828584?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17828584?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/17828584?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20683616?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/23837595?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/23837595?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/23837595?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21723856?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21723856?dopt=abstract 7. chiesa c, natale f, pascone r, osborn jf, pacifico l, bonci e, et al. c reactive protein and procalcitonin: reference intervals for preterm and term newborns during the early neonatal period. clin chim acta. 2011;412:1053–9. 8. lannergard a, larsson a, friman g, ewald u. human serum amyloid a (saa) and high sensitive c-reactive protein (hscrp) in preterm newborn infants with nosocomial infections. acta paediatr. 2008;97:1061–5. 9. sprong t, peri g, neeleman c, mantovani a, signorini s, van der meer jw, et al. pentraxin 3 and c-reactive protein in severe meningococcal disease. shock. 2009;31:28–32. 10. mauri t, bellani g, patroniti n, coppadoro a, peri g, cuccovillo i, et al. persisting high levels of plasma pentraxin 3 over the first days after severe sepsis and septic shock onset are associated with mortality. intensive care med. 2010;36: 621–9. 64 a. lannergård et al. www.ncbi.nlm.nih.gov/pubmed/21338596?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21338596?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/21338596?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18510717?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18510717?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18510717?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18510717?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18650775?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/18650775?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20119647?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20119647?dopt=abstract www.ncbi.nlm.nih.gov/pubmed/20119647?dopt=abstract ss1 acknowledgements declaration of interest references upsala j med sci 95: 291-297, 1990 quality control and quality requirements for the measurement of glycated hemoglobin i. penttila,' j. gavert,' a . julkunen' and t. tantanen' 'department of clinical chemistry, university of kuopio, sf-70210 kuopio, finland, 2labquality l t d , aseniamiehenkatu 4 a , sf-00520 helsinki, finland in the follow-up of the treatment of diabetes mellitus the determination of glycated blood proteins, especially blood hemoglobin, has been proved to be very important in estimation of the long-term balance of the disease. many methods have been developed, but its seems quite evident that the hplc-methods which measure the proportion of glycated hemoglobin alc (ghbalc) or the affinity chromatography measuring all glycated forms of hemoglobin (ghb) are the most useful for the routine clinical laboratory work (1,2,3,5). there are many difficulties in organizing the quality control of the determination of glycated hemoglobin. first problem e.g. in finland is that there are at least six commercial methods in use. in table 1 there are presented the methods and their reference intervals for the determination of glycated hemoglobins. the second is the measurement temperature, which must be know when the values for glycated hemoglobins has been calculated by using minicolumn techniques. the best way is to perform the columnphase in a thermostated water bath. the third and perhaps the most difficult problem is to find the ideal control material which fulfills the criteria: the control material should be identical with patient samples, the concentration of the analyte should be at the desired level and the stability of the control material should be long enough. in the university central hospital district of kuopio we have selected an automated hplc-method and measured ghbalc values for the whole district. today we have two different instruments for the measurement of ghbalc: the older one ( 3 ) is the fplc-system (pharmacia, uppsala, sweden) and the newer one an hplc-instrument of shimadzu (6) with an strong ion-exchange 291 column supplied by beckman instruments inc. (palo alto, ca, u.s.a.).to follow-up the analytical level of our method we have used washed and frozen (-70°c) concentrated erythrocytes (1) . the typical variation coefficients within day and also between days have been between 1.5 and 2.0 %. our method measures glycated hbalc and acetyl hbao (3,7), it is well linear between 3 . 0 and 20 % and is not influenced by hbf thus being suitable for analyses during pregnancy. in the meetings with our clinicians it has been stated that the difference of the ghbalc value measured should not exceed 0.5 % from the right value in the normal reference interval of 4.0-6.0 %. this means that the analytical variation should be below 2.5 %. it seems that this can be reached when the method is carefully selected, calibrated and continuously followed. we believe that this these criteria will be obtained in most laboratories where automated hplc-techniques for ghbalc is used.from the clinical point of view it is almost important that on a district, where the diabetics visit to different physicians and thus to many laboratories, an agreement on the principle (one method) is made for clinical use. the more difficult question concerns the right value when performing the quality control surveys in finland and in nordic countries. the controls should contain both the stabile ketoamine form as well as the labile schiff's base so that they correspond as closely as possible the situation in clinical laboratories. the first step in the measurement of ghbal or ghbalc is the elimination the labile schiff's base, which it is not so marked when using affinity chromatography for ghb ( 4 ) . for this reason lyophilized hemolysates may give too good results when using minicolumns and ion-exchange technique. secondly many commercial lyophilized preparations contain components or fractions which render the hplc-techniques (1). in most surveys we have seen (table 2, fig. 1) that the hplc-methods as a whole are not very much better in performance than the manual methods using either affinity or ionexchange minicolumn. it can, however, be calculated that edta-blood samples are better than the hemolysates (fig. 1) and the smallest coefficient of variation is seen in the group of hplc-methods especially when the variation coefficients are 292 table 1. different methods used by participating laboratories in the the surveys of labquality ltd. for the determination of glycated hemoglobins and they reference intervals method component reference measured intervals isoelectric focusing ghbalc 4.0 6.5 % hplcifplc ionex chromat. acetyl hbalc 4.0 6.1 % mini-ionex chromat.1 ( hbf 1 3.8 6.1 % biorad electrophoresis ghbala,b,c 5.5 9.0 % mini ionex chromat. acetyl hbalc 5.6 8.6 % hbf (other hb:s) mini affinity chromatography ghbala,b,c ghbf ghba2 ghbao 4.6 8.2 % 293 table 2. the quality control results from the surveys during the years 1987 to 1989 by labquality ltd. in finland. the number of laboratories using mini-column techniques (ghbal) has decreased, the users of affinity chromatography (ghb) correspondingly increased and those with hplc-techniques (ghbalc) clearly increased. method sample ghbal edta-b10od/l/87 edta-blood/l/88 edta-blood/l/89 hemolysate/l/87 hemolysate/l/88 hemolysate/l/89 ghb edta-b10od/l/87 edta-blood/l/88 edta-b10od/l/89 hemolysate/l/87 hemolysate/l/88 hemolysate/l/89 ghbalc edta-b100d/l/87 edta-b100d/l/88 edta-b100d/l/89 hemolysate/l/87 hemolysate/l/88 hemolysate/l/89 no. mean sd 21 6.1 % 0.45 20 10.5 % 0.77 14 6.5 % 0.89 21 7.5 % 0.89 19 12.8 % 0.49 14 5.3 % 0.74 8 4.9 % 0.51 8 13.7 % 1.07 13 9.95 % 0.52 8 5.0 % 0.64 8 13.6 % 1.57 13 5.3 % 0.94 5 4.7 % 0.49 6 9.8 % 0.35 22 4.8 % 1.04 5 5.1 % 0.11 6 10.5 % 0.45 22 5.1 % 0.77 cv 7.3 % 7.3 % 13.7 % 11.9 % 10.0 % 10.7 % 10.4 % 7.8 % 10.0 % 12.8 % 11.6 % 17.9 % 10.5 % 3.6 % 14.7 % 2.2 % 4.5 % 14.9 % 294 fig. 1. the results from the quality control surveys of glycated hemoglobin by three most common methods in finland. the results are expressed as the mean values in the survey 211989. control survey 2/1989 of labquality z m 2 (3 0 r 10 w i n w 0 (3 t 5 edta-l edta-h hemol-l hemol-h ghbal ghb ghbalc 295 calculated separately for each nordic country. as stated before it seems possible to maintain the intralaboratory variation good enough, eg. below 3.0 %. however, when the interlaboratory variation is much more higher, it seems that there must be troubles either in quality control samples or in the standardization. the great importance of the value of glycated hemoglobin in the treatment of diabetics and on the other hand possible technical difficulties in the measuring procedures, according to our opinion, is a good reason to start a nordic project. references: 1. 2. 3. 4. 5 . 6. ellis g, diamandis ep, giesbrecht e, daneman d, allen lc. an automated "high-pressure" liquid-chromatographic assay for hemoglobin alc. clin chem 1984; 30: 1746-1752. goldstein de, little rr, wiedmeyer h-m, england jd, mckenzie m. glycated hemoglobin: methodologies and clinicalapplications. clin chem 1986; 32: b65-b70. jeppsson j-0, jerntorp p, sundkvist g, englund h, nylund v. measurement of hemoglobin alc by a new liquid-chomatographic assay: methodogy, clinical utility, and relation to glucose tolerance evaluated. clin chem 1986; 32: 1867-1872. john wg. effect of schiff base (labile fraction) on the measurement of glycated hemoglobin by affinity chromatography. clin chem 1984; 30: 1111-1112. klenk dc, hermanson gt, krohn ri, fujimoto ek, mallia ak, smith pk, england jk, wiedmeyer h-m, little rr, goldstein de. determination of glycosylated hemoglobin by affinity chromatography: comparison with colorimetric and ion-exchange methods, and effects of common interferences. clin chem 1982; 28: 2088-2094. parviainen m, julkunen a, penttila i. *'a rapid ghbalc-analysis by using an hplc-instrument of beckman system gold". kliin. lab 1990; 7: 71-73. 296 7. suhonen l, stenman u-h, koivisto v, teramo k. correlation of hbalc, glycated serum proteins and albumin, and fructosamine with the 24-h glucose profile in insulindependent pregnant diabetics. clin chem 1989; 35: 922-925. correspondence: professor i. penttila, department of clinical chemistry, university of kuopio, sf.70210 kuopio, finland 297 upsala j med sci 92: 193-203, 1987 prevalence of sleep apnea s yndrome-estimation by two stage sampling thorarinn gislason’ and adam taube’ ‘department of lung medicine, akademiska sjukhuset and ’department of statistics, uppsala university, uppsala, sweden this article describes stepwise the methodological and statistical considerations made in the planning of an epidemiological survey of the prevalence of the sleep apnea syndrome (sas) in the municipality of uppsala in sweden. polysomnographic studies are required for an unequivocal diagnosis of sas. was decided to investigate men 30 to 69 years old. of taking a simple random sample (srs) was considered, but statistical calcula tions showed that for prevalences between 1-3% this would lead to totally unacceptable results. the investigation had to be confined to 60 subjects, since all-night it initially, the possibility a postal questionnaire, sent to the total population of 35 779 men in this age group, was then considered and, depending on their replies, they would be divided into low-risk and high-risk stratums of sas. then be called from each group for polysomnographic studies. impossible, as the lowest possible standard error was still too large and the samples would contain unacceptably few cases of sas. we therefore decided to concentrate on the highrisk stratum, obtaining an estimated under limit of the prevalence. optimal numbers would this also proved for economical reasons, we could not send a questionnaire to all the 35 779 individuals, but based the investigation on a srs of 4 ooo men. post-stratified in a high-risk and a low-risk group. from the high-risk group. 60 men were then selected for polysomnographic studies. 193 introduction more often than not, medical research reports deal with results obtained from a group of individuals who, by some means, have been selected from a larger population. selection procedure are rarely stated. by tradition it seems, planning discussions are not desired in written reports. since the statistical conside rations in the planning of this survey led to quite a different procedure than we had anticipated, we think it might be of interest to researchers in similar situations if we reviewed the considerations step by step. the reasons for choosing a particular sampling strategy or the occurrence of sleep apnea with concomitant owgen desaturation has been reported frequently in the last two decades (5, 9, 14). loud and disturbing snoring often associated with excessive daytime sleepiness is the main clinical feature, but numerous reports have also described sleep apnea in combination with various diseases (9, 14). as the occurrence of at least thirty apneic episodes during seven hours of steep (9). diagnosis (9, 10). the sleep apnea syndrome (sas) has been defined a whole-night polysomnographic study is necessary to confirm the table 1 population, sample size and criteria in previous studies on the prevalence of sas reference population sample size (n) and criteria bixler et a1 ( i ) volunteers, students, n = 100 without complaints technical staff and friends of sleep disorders ~ ~~~~~ block et a1 (2) medical and nursing staff n = 49 no breathing + patients complaints kreis et a1 (11) patients from a general n = 26 not medically medical service unstable, demented or expecteq to be discharged within 3 day: n = 87 selected on the basil (7) admitted to s . raffaele of questionnaires and clinica franceschi et a1 all patients (n=2518) hospital during 1 year data lavie (12) industrial workers n = 78 males, selected on (n=1502) the basis of questionnaires carskadon et a1 elderly volunteers from n = 40 without serious (3) non-medical sources medical disorders not complaining spontaneously of sleep problems 194 as is shown in table 1, previous investigations into the prevalences of sas have been based on presumably healthy and selected populations (1. 2). in-patient populations (7, 11). a healthy, working male population (12). or apparently healthy, elderly individuals ( 3 ) . both the designs and the results of these studies have shown great variation (table 1). a cross section of a general population. instead, they consist of special categories of individuals who at the time happened to be available for investi gation. in only two studies (7,12) are there defined selection procedures for the individuals, i.e. they are from specified background populations. lavie’s study (12) is the only one in which statistical considerations have been presented. we notice that none of the investigated groups constitute the aim of this paper is to describe the methodological background in an epidemiological study of the prevalence of sas in a defined population (8). one of our goals was to estimate the prevalence of sas with reasonable precision and another was to investigate a comparatively large group of patients with the disease. as will be shown, when resources are limited, these two goals are difficult to reach in one and the same study. we thus had to lower our ambitions. rather than obtaining an estimate of the prevalence of the disease, a procedure was worked out enabling an estimation of the lower limit of the prevalence. 1. limitations of the population and number of investigated persons in almost all studies on sas this syndrome has been found to be much more common among men than among women and also to occur more frequently in older age groups (3, 6, 14). we chose to study only men, aged 30 69 years. the survey was to be carried out in the municipality of uppsala. sweden (total population 150 579). which had a population of 35 779 men aged 30 69 years at the time of commencement of the study, i.e. 1984. we want to estimate the prevalence of sas in the group, i.e no. of sas cases no. of individuals in the population (at the time of the study) p = 13-878572 195 for economic and technical reasons, the clinical part of the investigation had to be limited from the beginning to 60 subjects, since whole-night poly-, somnographic studies are required for diagnosis. lay in the selection of the 60 persons to be investigated so that the results would be as statistically useful as possible. the methodological problem 2. is a simple random samf'le (srs) satisfactory? suppose we draw a srs of n individuals from a population. the parameter p is then estimated by no. of sas cases in sample psrs no. of individuals in sample the estimate psrs of p has the standard error (se) 1 if the population is large . it is thus possible to calculate the magnitude of the standard error for different sizes of n and p. if we assume that the prevalence of sas is between one and three percent, the relation between the number of men studied, n and se(p), has the shape displayed in figure 1. thus if, for example, the true value of p is 0.03 and we require se(p) 0.01, it means that n must be at least >300. or if p = 0.01 and we require se(p) 10.0033, we must have n = 910. if srs is used, we will therefore need between 300 and 900 observations. by selecting only 60 men by means of a simple random sample (srs) from a population, it is even possible that the sample will not contain one single case of sas, as the chance of this is (l-p)60, which for p = 0.02 is 0.29. if we obtained the result that none of the 60 polysomnographic investigations revealed a case of sas, the equation (l-p)60 = 0.05 would give p = 0.049 as an upper 95% confidence limit for the prevalence p. this would not permit any 'since the population investigated is large and the number of observations very small, the so-called finite population correction is neglected in all formulae (4). 196 0.00 1 i i i i i i i i i i 0 100 200 300 400 500 600 700 800 900 1 o o o n figure 1 the standard error of psrs as a function of the number of observations, n. conclusion to be drawn and the appearance of one or two cases of sas would only increase the previous confusion. unfeasible for prevalences of sas lying between 1-3%. thus this simple procedure was found to be in order to obtain a reasonable degree of precision within the limited economic and practical frame, further information about the population had to be utilized in the design. 3. stratification by means of screening questionnaires we considered the possibility of mailing a questionnaire concerning sleep complaints to all the 35 779 men in the population. before a sample was selected, the population could be divided into. say, two strata one small stratum in which sas was highly suspected and one larger one with presumably very few cases of sas. the system of notations is presented in table 2. this would mean that 197 table 2 notations concerning population and sample population sample individuals of sas individuals of sas stratum number of prevalence number of proportion nl pl "l pl high-risk nh ph "h ph low-risk total n p n the prevalence of sas, the parameter p, can be written as a weighted average nh ph pl + nl p = n n on the basis of the two samples, with nl and % observations respect ively, this parameter is estimated by means of the estimator nh ph pl + nl n n pstrat with the standard error it is well known (4) that se (pstrat) is at a minimum when and it can be shown that se (pstrat) < se (psm) when 198 "h even if ph and pl are unknown, there are good chances of selecting so that stratified sampling will give a better estimate than srs. and nl let us study in more detail how this approach works in a hypothetical example (which numerically is very similar to our real situation). that n = 32 000, nl = 28 ooo, nh = 4 ooo, pl = 0.005, and ph = 0.125, which means that p = 0.02. this example might perhaps be considered somewhat optimistic concerning the screening efficiency. we assume 0.02 0.02 0.01 0.01 0.oc 0.oc i i i i i i 10 20 30 40 5 0 6 0 n l l i i i i 1 i n h 60 5 0 40 30 20 10 0 figure 2 the standard error of p for different sizes of nl and strat l nh with p=o.o2 and n + % = 60. in figure 2 we find se (pstrat) as a function of the allocation of observations to the two strata. when (nl = 13 ; nh = 47) 13 < nl < 53 and nh = 60 -nl, the stratification decreases the standard error of the estimate when compared with it can be seen that se (pstrat) = se (pstrat) and when (y = 52 ; % = 8 ) . for all cases where se (psrs). the best possible choice, 199 i.e. when se (pstrat) (nl = 36. n modest decrease (27%) in the standard error (figure 2). furthermore, even this allocation of the samples with the hypothetical prevalences stated will result in an unacceptably small number of sas cases (3 to 4) for further study. thus, within the economic frame of 60 observations we had to abandon our ambition of estimating the parameter p by means of sampling from both strata. is lowest 24), gives however only a h if it had been economically possible to make, say, 250 polysomnographic studies from the said population, the above approach would have given se (pstrat could be expected. an srs of n = 250 observations would have given se (pm) = 0.0089. in order to halve 1 ooo polysomnographic studies, giving se (pm) = 0.0044. ) = 0.0066 for nl = % = 125. among these, only some 16 sas cases se (p) se (pstrat) = 0.0033. we would have had t o perform as against 4. a limited study of the high risk group only since we have it must be true that nl nh pl + n ph p = n nh ph p2n with an equality sign for the case when the stratification is 100% effective in the sense that all sas cases are to be found in the high risk stratum. we will therefore concentrate our resources on esimating the expression alone, which means that we draw all our observations from the high risk nh ph n stratum. thus the estimator nh ph pu = n estimates a lower limit for the parameter p . the standard error of this estimator is 200 suppose the screening procedure which separates the subjects into a high risk and a low-risk stratum is a test with a sensitivity clear that s (13). it is then thus if for example the sensitivity is 100% (i.e. that all sas cases are i n the high risk stratum), our estimator p will estimate the true prevalence p. u in the previously mentioned hypothetical example, the selected prevalence was p = 0.02, but the expected value of p will be somewhat lower, namely u 4 000 n ph 32 ooo 0.125 = 0.0156 nh will have the standard error se (p ) = 0.0056. pu u the estimator since the total number of sas-cases in the said population was assumed to be 32 000 0.02 = 640 and thereof 4 ooo 0.125 in the high-risk stratum, the sensitivity of the screening procedure is 5001640 = 0.78. therefore, we could also write that 500 640 s . p = 0.02 = 0.0156 by ignoring the low-risk stratum. we will thus be estimating a lower limit, which in this example is about 78% of the true sas prevalence. 5. a double-sampling procedure since the cost of each questionnaire was about us$ 1.50, it was considered too expensive to send it to each one of the 35 779 men in the population. was therefore decided to first take a large srs of n' = 4 ooo men, who would it 201 receive a postal questionnaire. this sample would subsequently be stratified (so-called poststratification) (4) on the basis of the answers given, in a ' high-risk group and a low-risk group with ";i and ni persons respectively. the proportion nh/n can thus be estimated by "1;/n'. from the "1; individuals in the high-risk group, we then take a sub-sample ph of n = 60 persons, in order to obtain an estimate of the sas-prevalence in the high-risk stratum. we thus get the estimator "i; p; = p" n' since n' would be as large as 4 ooo, the random variation in the ratio will be of negligible order of magnitude and the standard error of the ";ih estimated under limit will be this means that the data will be analyzed as if the stratification had been made a priori. conclusion the methodological examples given above clearly show the importance of analysing in advance the method to be used in epidemiological studies. materials that are too small cannot elucidate the question of the prevalence of sas because of their statistical and methodological limitations. in our case financial limitations made it necessary to look for the minimal frequency of sas by investigating only those persons who were most suspected of having this condition from the answers to sleep questionnaires. this work was supported by grants from the national swedish association against heart and chest diseases, stockholm. 202 references 1. 2. 3. 4. 5. 6. 7. 8 . 9. 10. 11. 12. 13. 14. bixler. e.o., kales, a., soldatos, c.r. & bueno, a.v.: sleep apneic activity in a normal population. res com in chem path and pharm 36(1):141-152. 1982. block. a.j., boysen, p.g., wynne, j.w., et al.: sleep apnea. hypopnea and oxygen desaturation in normal subjects a strong male dominance. n engl j med 300:513-517, 1979. carskadon, m.a. &dement, w.c.: respiration during sleep in the aged human. j' gerontol 36:420-423. 1981. cochran, william g.: sampling techniques. 3rd ed. chapters 2 and 5. wiley, n y, 1977. coleman, r.m., roffwarg, h . p . , kennedy, s.j., et al.: sleep-wake disorders based on a polysomnographic diagnosis a national cooperative study. jama 247:997-1003, 1982. coleman, r.m., miles, l.e., guilleminault, c., et al.: sleep-wake disorders in the elderly. am ger soc 19(7):289-296, 1981. franceschi. m., zamproni, p.. crippa, d. & smirne. s.: excessive daytime sleepiness: tion. sleep 5(3):239-247, 1982. gislason, t.. et al.: prevalence of sleep apnea syndrome among swedish men an epidemiological study. guilleminault. c., cumminsky, j. &dement, w.c.: sleep apnea syndromes: recent advance. avd intern med 26:347-374, 1980. a 1-year study in an unselected inpatient popula (in manuscript). haponik. e.f.. smith, p.l. & meyers, d.a.: evaluation of sleep disordered breathing is polysomnography necessary? am j med 77:671-677, 1984. kreis..p., kripke, d.f. & anocli-israel. s.: sleep apnea: a prospective study. the western journal of medicine 1399:171-173, 1983. lavie, p.: incidence of sleep apnea in a presumably healthy working population. sleep 6(4):312-318, 1983. lilienfeld, m. & lilienfeld, e.: foundation of epidemiology. 2nd ed. chapter 6, p 151. oxford university press, 1980. weitzman, e.d.: sleep and its disorders. ann rev neurosci 4:381-417, 1981. address f o r reprints: thorarinn gislason department of lung medicine adademiska sjukhuset s-751 85 uppsala, sweden 14-818512 203 upsala j med sci 88: 127-139, 1983 perspectives on serum acid phosphatase in prostatic disease an evaluation of two methods s . dedorson,' a . fritjofsson,' b . j. norlen' ,and g . ronquist' from the departments of urology' and clinical chemistry,* university hospital, uppsala, sweden abstract acid phosphatase in serum was measured in 116 patients with prostatic disease, benign in 59 and malignant in 57 cases. comparisons were made between radioimmunoassay (ria) and an enzymatic method. the correlation coefficient between the respective values was 0.96 in patients with untreated prostatic cancer, indicating that no significant difference between results with the two methods was to be expected. the correlation coefficient between ria values and cancer stage was 0.48, and between catalytic activity and cancer stage it was 0.50. the validy of the two methods consequently was equal. ria, however, was the more sensitive method, giving elevated values in 10 of 11 patients with untreated stage i11 or stage iv prostatic cancer, as compared with only 4 of the same 11 in the enzymatic assay. this seeming paradox most probably was attributable to differing intrinsic properties of the methods when the upper limits of normal range were established. neither ria nor enzymatic analysis discriminated early prostatic cancer (stages i and 11) from benign lesions. introduction measurement of acid phosphatase in serum has been employed for more than 40 years to detect and monitor malignant prostatic disease ( 8 ) . the prostate gland is the main, but not the sole source of this enzyme in man. research on enzymatic assay of acid phosphatase has therefore been focused on making the procedure more prostate-specific. refinements in substrates and inhibitors for the reaction, however, have not so far led to absolute organ specificity. 9-838572 127 the enzymatic activity of the acid phosphatase molecule is sensitive to ph, with optimum at ph 5.5. the molecule dissociates into its two subunits at ph 2.0 or less and also at ph 7 . 4 or higher. the dissociation results in inactivation of the enzyme, and the two subunits immediately begin to aggregate ( 4 ) . inherent difficulties thus limit the usefulness of enzymatic assay of acid phosphatase. a competitive-binding, 'labelled antigen' technique for isotopic assay of pap (prostatic acid phosphatase) was therefore evolved as an alternative procedure (6). such assay offered improvements with regard to prostate-specificity, stability, sensitivity and precision (7). despite controversy on particular aspects of radioimmunoassay for pap, reports have continued to emphasize the advantages of this principle over the enzymatic technique (5,9,13). the aims of the investigation here presented were to measure pap concentrations in sera from patients with benign or malignant prostatic disease and to compare results from radioimmunoassay (ria) with those from a conventional enzymatic procedure for determining pap activity. patients and methods radioimmunoassay all radioimmunoassays of pap were run in duplicate, using 0.1 ml serum samples and a commercially available ria kit (gammadabr, clinical assays, travenol laboratories, cambridge, mass., usa) in accordance with manufacturer's instructions. after incubation and centrifugation, however, it was found necessary to remove traces of supernatant fluid in the tubes, using filter paper, before making counts of the sedimented radioactive material in the tubes. 128 enzymatic assay the catalytic activity of serum acid phosphatase was measured as recommended by the committee on enzymes of the scandinavian society for clinical chemistry and clinical physiology using paranitrophenyl phosphate as substrate with i,(+)-tartrate as inhibitor. "he upper limit of the reference range €or this method is 58 nkat' 1-'. patients the studies were made on 1 1 6 patients consecutively referred to this department of urology because of prostatic disorder. prostatic cancer was found in 57 of the men and benign prostatic disease in 59. table i surveys the numbers of patients and their mean age distribution according to diagnosis. table 1 distribution of patients according to age and diagnosis diagnosis no of mean age patients (years ) prostatic cancer 57 72.6 stage i stage i1 stage i11 stage iv benign prostatic disease hyperplasia prostatitis other benign process 3 1 5 23 16 59 39 1 3 7 79.0 74.4 73.3 68.7 63.3 6 8 . 1 50.8 5 9 . 1 129 in all cases of prostatic cancer the diagnosis was confirmed by transrectal aspiration biopsy for cytological study. intravenous pyelography and x-ray examination of the lungs were also performed. bone scan was done in all but two patients of this group (1 with stage i1 and 1 with stage iv tumour), with skeletal roentgenography when indicated. since papconcentration was the issue under study, it was not included in the clinical staging of malignancy, which otherwise was in accordance with recommendations by the veterans administration cooperative urological research group (12): stage i thus implied clinically unsuspected cancer in a transurethral resection specimen, stage i1 a palpable nodule confined to the prostate gland, stage i11 periprostatic growth of the tumour and stage iv known distant metastases, irrespective of pap level in the serum and findings at rectal examination. of the 5 7 patients with prostatic cancer, 20 were untreated at the time of the study. the treatment in the other 3 7 patients varied orchiectomy, oestrogen/chemotherapy (estramustin, estracytr) or irradiation, alone, in combinations or in succession. the duration of therapy and the response were highly variable at the time of the investigation. in the group of 59 patients with benign prostatic disorders, histological or cytological examination was done only if surgery was performed or if the clinical findings aroused suspicion of malignancy. statistical methods a hewlett-packard 9820 a calculator, model 20 with standard programmes was used for registering data and for the statistical analyses. the reliability of ria was measured as the correlation coefficient between the duplicate sets of values (n/set = 116). the validity of the respective methods was assessed in the patients with untreated prostatic cancer (n=20) as the correlation between cancer stage and pap concentration according to ria or to the enzymatic assay (fig. 3 ) . the validity was additionally calculated as correlation between 130 results with the two methods in the 59 patients with benign prostatic disease (fig.l), in the total 57 with prostatic cancer (fig.2), and in the 20 patients with untreated prostatic cancer (fig.3). for calculation of correlation coefficients and testing of significance, we used the methods described by snedecor & cochran (10). the sensitivity of the method was judged from the proportion of elevated readings among the patients with prostatic cancer. the specificity was calculated as the proportion of non-elevated readings in patients with benign prostatic disease. the significance of differences in sensitivity and specificity was calculated with the z test (one-tailed, with correction for continuity) as described by snedecor & cochran (10). 4.0 3.2 c l c"u n 6 c#z .1.6 a a a i 0.8 0 -0 0 0 i i i i i + 1.27 1 i i 1 12 24 36 48 s-pap ( e n d nkat . 1-l 1 60 fig.1 correlation between values from enzymatic analysis (enz.)and from radioimmunoassay (ria) of serum phosphatase (s-pap) in patients with benign prostatic disease. regression line upper limit of normal range _ _ _ 131 results to determine the upper normal limit for ria results, pap was measured in serum sampled from 58 male blood donors. "he mean value was 1.20 p g l-l, s . d . 0.80. the upper limit of the reference range therefore was set at 2.80 pg 1-l. positive correlation (r=0.20) with the ria values in the patients with benign prostatic disorders, despite the relatively narrow range of readings in that group (fig 1). the correlation between the two variables was appreciably better among the 57 patients with malignant disease (fig 2), and it was particularly high (0.96) in the 20 patients with untreated prostatic cancer (fig 3 ) . the values from enzymatic analysis showed a weak '"1 12 0 0 0 / i i i 1 1 1 0 40 80 120 160 200 s-pap (enz.) nkat e 1 l fig 2.correlatlon between enz. and ria values in all patients with prostatic cancer. regression line upper limit of normal range 132 the c o r r e l a t i o n between t h e d u p l i c a t e r u n s o f r i a ( r = 0 . 9 9 7 ) e x p r e s s e d h i g h r e l i a b i l i t y o f t h e method. the s t a g e of c a n c e r , moreover, showed s i g n i f i c a n t l y (p<o.o5) p o s i t i v e c o r r e t a t i o n w i t h r e s u l t s from e n z y m a t i c a n a l y s i s ( r = o . 5 0 ) and r i a v a l u e s ( r = o . 4 8 ) . between t h e t w o methods, t h e r e f o r e , no d i f f e r e n c e i n v a l i d i t y w a s d e t e c t a b l e . r i a g a v e e l e v a t e d v a l u e s i n 4 o f 59 p a t i e n t s w i t h b e n i g n p r o s t a t i c d i s e a s e ( f i g 1, table 2 ) . the enzyme t e s t s showed no e l e v a t e d v a l u e s i n t h e same g r o u p . the s p e c i f i c i t y , i . e . t h e a b i l i t y t o r e c o g n i z e b e n i g n n a t u r e of p r o s t a t i c l e s i o n s , t h u s w a s lower w i t h r i a ( 0 . 9 3 ) t h a n w i t h e n z y m a t i c a s s a y (1.00), t h o u g h t h e d i f f e r e n c e w a s n o t s t a t i s t i c a l l y s i g n i f i c a n t . 0 n = 20 r = 0.96 p < 0.001 y = 0.10~ +0.23 2 ‘ a i ---_ _ _ i i l 0 i i i i i 1 40 80 120 160 200 s-pap (enz.) nkat . i-’ fig 3 . c o r r e l a t i o n between enz. and r i a v a l u e s i n p a t i e n t s w i t h u n t r e a t e d p r o s t a t i c c a n c e r . r e g r e s s i o n l i n e upper l i m i t o f normal r a n g e 133 u -4 .tj .tj la 0 k a c -4 14 i m m 2 w 0 h m v) (0 m n a, rl p p m pi 2 5 & a, m h r m rl m r (u rl a\ m d +i (u \o 0 * +i r m h a, u c m u 0 -4 .tj m .tj & nl $ d r r l m m a l o + i + i + i m u m r l d m r m m rim+ . . . +i +i + i . . . h h a c 0 cr m rl rl m r rl + i d rl p 4 +i m ln 0 .4 .tj m .tj 0 0 k c v ) m l d -4 a, c v ) a, -4 m a 0 0 cr co + i m m rl ln 134 the means from r i a and from enzymatic assay were almost the same in the patients with stage i or 11 prostatic cancer as in those with benign prostatic disease. the proportion of patients with values above the upper limits of normal was also similar (table 2). both parameters, however, distinguished cancer stages i11 and i v from the benign conditions, especially when r i a was used. thus, elevated values were given by r i a in 9 of the 23 patients with stage i11 cancer, but by the enzyme method in only 3 patients. the corresponding figures in stage iv cancer were 9 and 4 of 16 patients. the sensitivity, i.e. the ability to recognize prostatic disease as malignant, was 0 . 3 3 with r i a and 0.14 with the enzymatic assay when all 57 cancer patients were included in the analysis. this difference was significant (p<o.ol) and was explained solely by the superior sensitivity of r i a in tumours of stage i11 or i v . the enhanced sensitivity of r i a was still more evident in untreated prostatic cancer (table 3 ) . of the 11 patients with stage i11 or stage i v tumour, 10 showed elevated values in r i a , but only 4 in enzymatic assay. table 3 . radioirmolnoassay (ria) and enzyme assay of s-pap in untreated prostatic cancer values above ria enzyme assay no of serum pap upper range limit diagmsis patients ria e n z y z ~ assay [pg-~-') (nkat-1-1) 11 4 1 0 6 2 4 2 + + + + + p r o s t a t i c cancer 20 5.6-7.4 51.8?68.5 stages i and i1 9 2.1-2.0 19.9+12.1 stage i11 7 6.9-6.8 57.4-51.6 stage iv 4 11.2212.6 113.5-125.9 9t -838572 135 discussion ria measures antigen determinants of the analyzed enzyme, whereas the various methods of enzymatic analysis reflect catalytic activity. when ria was introduced for determination of prostatic acid phosphatase in serum it was hoped that this assay, based on an antibody against a purified isoenzyme, would increase diagnostic acuity. such specificity should contribute to early detection of prostatic cancer and also to the more appropriate tumour staging that is essential for optimum choice of treatment and for accurate monitoring of tumour response. these hopes remain widely held, although it is generally recognized that no known isoenzyme of acid phosphatase is tumour-specific or even tumour-associated. the present study showed good correlation between results with ria and those with the enzymatic method of determining serum acid phosphatase in patients with prostatic cancer, particularly before treatment had been instituted. a priori, therefore, no clear difference was to be expected between the two methods in the continued investigations. their validity, accordingly, proved to be similar. in sensitivity, on the other hand, the methods clearly differed. the most probable explanation of this apparent paradox was intrinsic difference in the properties of the methods when the upper normal limits were determined. if ria is a completely organ-specific test, the upper limit of its normal range should be 'absolute'.the catalytic activity in the enzymatic analysis, however, is assumed not to derive exclusively from the prostate. acid phosphatases from other organs may contribute to the results of the analysis, and such extra-prostatic 'contaminating activities' may diminish with advancing age. the mean age was higher in the cancer patients than in those with benign prostatic disease. intergroup comparisons of ria and enzymatic analysis data (table 2) suggest that the extraprostatic contribution to the latter values could have been inversely related to age. use of an age-matched reference group might well have eliminated this discrepancy. the false positive ria results occurred only in patients with benign prostatic hyperplasia. the 10 per cent of results 136 above u p p e r n o r m a l l i m i t i n t h i s g r o u p i s close t o t h e f i g u r e r e p o r t e d b y f o t i e t a l . ( 6 ) . i n d e a l i n g w i t h t h e problem o f f a l s e p o s i t i v e r e s u l t s , these w r i t e r s i n t r o d u c e d +4 s . d . as u p p e r n o r m a l l i m i t , w h i c h r e d u c e d t h e p r o p o r t i o n t o 5 per c e n t b u t decreased t h e s e n s i t i v i t y o f t h e a s s a y i n s t a g e i c a n c e r . i n o u r s e r i e s the t w o a s s a y t e c h n i q u e s d i d n o t d i f f e r i n r e g a r d t o d e t e c t i o n o f e a r l y c a n c e r . among t h e t o t a l 18 p a t i e n t s w i t h s t a g e i or s t a g e i1 c a n c e r , o n l y o n e showed an e l e v a t e d r i a v a l u e (1 o f the 9 w i t h u n t r e a t e d e a r l y c a n c e r ) . w e t h e r e f o r e c o n c l u d e t h a t n e i t h e r a s s a y i s u s e f u l as a s c r e e n i n g p r o c e d u r e f o r e a r l y d e t e c t i o n o f p r o s t a t i c c a r c i n o m a . c o n t r a d i c t o r y f i n d i n g s were r e p o r t e d b y o t h e r a u t h o r s ( 2 , 3 , 6 , 1 1 , 1 3 ) . on t h e other h a n d , b r u c e e t a1 (1) f o u n d e l e v a t e d r i a v a l u e s i n o n l y 8 p e r c e n t o f p a t i e n t s w i t h s t a g e i or 11 p r o s t a t i c c a n c e r . between i n t r a c a p s u l a r and e x t r a c a p s u l a r c a n c e r , however, r i a d i s c r i m i n a t e d r e a s o n a b l y w e l l i n o u r s t u d y . thus i n s t a g e i11 or i v , r i a g a v e e l e v a t e d v a l u e s i n 1 0 o f 11 u n t r e a t e d p a t i e n t s , w h i l e t h e e n z y m a t i c a n a l y s i s showed o n l y 4 w i t h s u c h v a l u e s . r i a t h e r e f o r e appears t o be u s e f u l f o r the s t a g i n g of p r o s t a t i c c a n c e r . s t a n d a r d i z e d methods a r e e s s e n t i a l when pap d e t e r m i n a t i o n i s u s e d i n t h e c l a s s i f i c a t i o n of p r o s t a t i c disease. e x t e n d e d s t u d i e s o f l a r g e series and r e p e a t a n a l y s e s i n i n d i v i d u a l p a t i e n t s , both r i a and measurement o f c a t a l y t i c a c t i v i t y , a r e r e q u i r e d t o c l a r i f y i f the t w o methods c o n t i n u e t o r e f l e c t t h e same c i r c u m s t a n c e s w i t h r e g a r d t o tumour p r o g r e s s i o n , t h e r a p e u t i c r e s p o n s e and p r o g n o s i s . 137 references 1. bruce, a.w., mahan, d.e., morales, a., clark, a.f. & belville, w.d. : an objective look at acid phosphatase determinations: a comparison of a biochemical and immunological methods. rr j urol 51(3), 213-217,1979. 2. carroll, b.j: radioimmunoassay of prostatic acid phosphatase in carcinoma of the prostate. n engl j med 298(16), 912, 1978. 3. cooper, j.f. & foti, a.g.: a radioimmunoassay for prostatic acid phosphatase. natol cancer inst monogr 49, 235-237, 1978. 4. derechin, m., rustum, y.m. & barnard, e.a. : acid phosphomonesterase of human prostate. molecular weight, dissociation and chemical composition. biochim biophys acta 250, 143-154, 1971. 5. dewers, l.m.: prostatic acid phosphatase: assay comparisons. laboratory management 19, 8-16, 1981. 6. foti, a.g., cooper, j.f., herschman, h. & malvaez, r.r.: detection of prostatic cancer by solidphase radioimmunoassay of serum prostatic acid phosphatase. n engl j med 297(25), 1357-1361, 1977. 7. griffiths, j.c.: prostate-specific acid phosphatase:reevaluation of radioimmunoassay in diagnosing prostatic disease. clin chem 26, 433-437, 1980. 8. gutman, e.b., sproul, e.e. & gutman, a.b.: significance of increased phosphatase activity of bone at the site of osteoplastic metastases secondary to carcinoma of the prostate gland. am j cancer 28, 485-495, 1936. 9. lundholm, g.r., stirton, s . , liedtke, r.j. & batjer, j.d.: prostatic acid phosphatase by radioimmunoassay. sensitivity compared with enzymatic assay. jama 244(18), 2071-2073, 1980. 10. snedecor, g.w. & cochran, w.g.: statistical methods. sixth edition. the iowa state university press. ames, iowa, u.s.a., 1967. 11. wajzman, z . , chu, t.m., saroff, j., slack, n. & murphy, g.p.: two new, direct and specific methods of acid phosphatase determination. urology 13(1), 8-11, 1979. group. cancer of the prostate: treatment comparisons. j urol 12. the veterans administration cooperative urological research 98, 516-522, 1967. 138 13. vihko, p., sajanti, e., jznne, o., peltonen, l. & vihko, r.: serum prostate-specific acid phosphatase: development and validation of a specific radioimmunoassay. clin chem 24(11), 1915-1919, 1978. address for reprints : gunnar ronquist, m.d. department of clinical chemistry, university hospital s-751 85 uppsala 10-838572 139 upsala j med sci 87: 143-149, 1982 neonatal convulsions treated with continuous, intravenous infusion of diazepam i. gamstorp and g . sedin department of paediatrics, university hospital, uppsala, sweden abstract eight infants born at term in the years 1 9 7 4 7 6 , with neonatal convulsions due to severe perinatal asphyxia, were treated for 6 1 1 days with continuous intravenous infusion of diazepam. doses of 1 . 0 1 . 5 mg per hour (mg/h) were usually required to stop the convulsions. in one infant 2 . 7 5 mg/h was needed. during the treat ment, all infants had measurable serum concentrations of diazepam, half of them above 3 5 umol/l. the convulsions stopped in all eight infants, and did not return after discontinuation of the infusion. side-effects were noted in all infants, but they were all able to breathe adequately. at follow-up the psychomotor development was normal in all cases and there were no signs of neurological dis orders, except in one patient, in whom mild epilepsy was observed. continuous infusion of diazepam should be given in doses of at least 1 mg/h (corresponding to around 0.3 mg/kg h) to stop convul sions in full-term infants efficiently and should be increased under close supervision and with monitoring of respiration and heart rate until treatment is effective. introduction neonatal convulsions remain a serious problem, and in recent studies (3, 4, 6 , 7 ) the same grave prognosis has been found as in many earlier ones, that is, roughly half of the infants die or be come handicapped and at most half survive without handicap. the main deciding factor for the prognosis is the background of the convulsions., i.e. the severity of the brain injury wnich causes them. it must always be an advantage to the infant if the fits are brought under effective control as early as possible. each fit is often so short that measures started at the onset of a fit may not influence its duration. to prevent fits a continuous intravenous 143 infusion of an anticonvulsive drug can be given. benzodiazepines are effective anticonvulsants, particularly when given intravenously ( 7 ) . their metabolism differs in the neo natal period from that in later life ( 5 ) . the clinical effect of continuous intravenous infusion of diazepam was studied in eight newborn infants with convulsions, and at the same time repeated measurements were made of the serum concentration of diazepam and of its main metabolite, n-desmethyldiazepam. material and methods in an investigation of infants with neonatal convulsions due to perinatal asphyxia, 8 infants 2 girls and 6 boys born at term in 1 9 7 4 7 6 were subjected to a special study concerning the effects of diazepam therapy. the only reason for selecting these patients was that several blood samples for determination of the serum le vel of diazepam and of its metabolite n-desmethyldiazepam could be obtained. in 6 of the infants the convulsions started during the first day of life, in one during the second day and in one during the sixth day. all infants were born within 2 weeks of the expected date and their birth weights varied between 2 . 9 3 0 and 4 . 4 5 0 kg. a standard diazepam preparation (stesolid r i dumex lakemedel ab, helsingborq, sweden, 5 mg/ml) was used and added to the infusion of isotonic glucose solution which the infant needed for other reasons. a new solution for infusion was prepared every 1 2 hours. before the diazepam infusion was finally stopped, peroral pheno barbital therapy was started. during the neonatal period the 8 infants were admitted to a neonatal intensive care unit where they were under the close ob servation of trained nurses and where their problems e.g. re spiration metabolism, nutrition, infections were taken care of. after discharge they were repeatedly examined at the outpatient unit of the department of paediatrics, uppsala, either by a pacdia tric neurologist or by a neonatologist. an eeg was performed at the ac;e of one year before discontinuation of phenobarbital treat ment and again at the age of two years. those infants who showed a comdletely normal development and physical condition were checked after about two years by general paediatricians at child health centres. this examination included careful history-takingl parti cularly concerning development and symptoms suggesting fits, and 144 physical examination with emphasis on the psychomotor development and neurological status. during the diazepam infusion, capillary blood was drawn and kept deep-frozen until analysed. the analyses, which were per formed by gas chromatography ( 2 1 , comprised diazepam and n-des methyldiazepam determinations and were made at the laboratory of dumex lakemedel, helsingborg, sweden. results the initial diazepam dose was 0.5 mg/h ( 6 patients) or 1.0 mg/h ( 2 patients). the lower dose was ineffective and had to be in creased to 0 . 7 2 . 7 5 mg/h for all infants before the convulsions definitely disappeared. in one patient a dose higher than 2.0 mg/h was necessary: 1-1.5 mg/h was the highest dose neededinthe other 7 patients. when the infant had been convulsion-free for 1 2 2 4 hours, the dose was slowly decreased in steps of 0 . 1 0 . 2 5 mg/h with one or two such changes per 2 4 hours. one patient was treated for 3 days, one for 1 1 days, and the rest for 6 8 days. no fits occurred du ring infusion of the highest dose used in each patient, and all patients remained convulsionfree during the dose-decreasing phase and after infusion of the drug had been stopped. all patients showed obvious side-effects they were drowsy and floppy to va rying degrees. they also needed feeding through a gastric tube and initially parenteral fluid and nutrition. all 8 infants breathed spontaneously and assisted ventilation was not needed. in two patients the first measurements of the serum diazepam and n-methyldiazepam concentrations were made within 2 hours after the start of the infusion: in one of them the serum diazepam le vel was 1 . 2 8 umol/l: one of them never received more than 0.7 mg/h and the other two never more than 1 mg/h. in the other 5 one or several values exceeded 3.5 umol/l, and 4 had at least one value above 35 l j m o l / l . the highest diazepam concentrations were 8 0 . 5 pmol/l (on a dose of 1.5 mg/h; birth weight 4 . 4 5 0 kg) and 5 5 umol/l (on a dose of 1.0 mg/h; birth weight 3 . 0 3 0 kg). the highest dose used among these 8 infants, 2 . 7 5 mg/h for 5 hours in a boy weighing 3 . 8 7 0 kg gave a maximum serum concentration of 2 1 . 7 umol/l. the serum le vel of diazepam, at which the convulsions stopped, varied so much that no "anticonvulsant level" can be defined, but it seems 145 10-822857 to be higher than the level of 0.5-1.2 umol/l (150-350 nq/l) which is considered anticonvulsant in older children (3). fig.1 summarizes all serum diazepam concentrations recorded in relation to the diazepam dose, expressed as mg per kg and hour (mg/kg h). from the figure it is obvious that a dose of 0.35 mg/kg h always gave a stable and high serum level. in full-term infants this corresponds to a dose of 1.0-1.5 mg/h. umol/ 1 e tu q a, n tu u 0 c 0 v . cc ci e 3 a, 0 l 10 1 0.1 0.01 , . . . . i *. ' i l i i 1 1 0 0.2 0.4 0.6 0.8 rng/&.h dose of diazepam firj. 1. serum concentration of diazepam (log scale) in relation to the given doses of the drug in all eight infants. in most cases the serum level of n-desmethyldiazepam started to increase about 1 2 hours after the start of the infusion, and a high level, often still increasing, was recorded during the dose decreasing phase. unfortunately the concentration was not always measured after the infusion was stopped. the highest concentration measured was 9.74 umol/l. this was recorded in the patient who ne ver received more than 0.7 mg/h and, during the dose-decreasing phase, when he had been on 0.4 mg/h for 14 hours. patients showing the highest serum levels of diazepam had the lowest levels of n desmethyl-diazepam, and vice versa. no clear relation was observed between the severity of the clinical findings, & the convulsions and the side-effects of the drug, and the serum levels of diazepam and n-desmethyldiazepam. 146 at follow-up, all patients were well, with normal psychomotor development and normal neurological findings. only one patient had convulsions. this patient had required the highest dose and the longest diazepam therapy. when falling asleep he had a series of jerks lasting for at least half an hour, thus quantitatively ex ceeding the usual type of jerks occurring, when falling asleep. he also had epileptic potentials on his eeg, increasing during light sleep and coinciding with his jerks. in spite of the lack of other types of seizures he is considered to have a mild form of epilepsy and is being treated with carbamazepine. no seizures have been re ported in any of the other patients and their eegs are normal. the diazepam dose and serum levels of diazepam and n-desmethyl diazepam are reviewed in a patient (birth weight 3.8 kg) with a mild respiratory disturbance after birth. on the 3rd day of life convulsions started. his convulsions stopped at a dose of 1 . 5 mg/h when this dose had been used for 24 hours. discussion although all eight infants recovered with no or only mild se quelae, it cannot be concluded that this satisfactory outcome is due to the treatment used. it seems reasonable to suppose that an early and efficient treatment of the convulsions is advantageous to the infant. the doses of diazepam used in this study are of the same mag nitude as those used successfully by thong et al. ( 8 ) €or conti nuous intravenous infusion in convulsing infants. the serum con centrations are in reasonable agreement with those recorded by langslet et al. (5) in newborn infants, who received a single intravenous injection of diazepam. as is obvious from the present study a dose of 0.7 mg/h in a full-term baby may in some cases be enough, but a dose of 1 mg/h is more likely to stop the seizures. if the treatment is not successful, the dose can be increased con siderably. in this study the highest dose needed to stop the sei zures was 2 . 7 5 mg/h. the serum concentrations of diazepam and its metabolite n-desmethyldiazepam, in these newborns, were extremely high compared with those reported in older children ( 1 ) . although the infants showed obvious side-effects, lasting several days longer than the duration of the therapy, none of them needed assisted ventilation. 147 t a b l e 1 . t h e a d m i n i s t e r e d d o s e s o f d i a z e p a m ( m q / h a n d m q / k q h ) a n d t h e s e r u m c o n c e n t r a t i o n s o f d i a z e p a m an d n d e s m e t h y l d i a z e p a m i n o n e p a t i e n t . t h e c o n v u l s i o n s s t o p p e d e n t i r e l y a f t e r 2 4 h o u r s i n f u s i o n o f 1 .5 m q / h . d o s e s e r u m c o n c e n t r a t i o n n d e s m e t h y l d i a z e p a m d a t e h o u r h o u r m q / h m q / k q h d a t e h o u r n q / m l u m o l/ l n q / m l um ol /l d i a z e p a m 2 6 .8 0 9 .3 0 -1 6 .0 0 0 .5 0 .1 3 2 6 .8 1 6 .0 0 1 .2 5 0 .3 3 2 7 .8 1 7 .0 0 2 7 .8 1 7 .0 0 1 .5 0 .3 9 3 0 .8 1 0 .0 0 3 0 .8 1 0 .0 0 -1 7 .0 0 1 .0 0 .2 6 3 0 .8 1 7 .0 0 0 .8 0 .2 1 3 1 8 1 0 .0 0 3 1 .8 1 0 .0 0 0 .6 6 .1 6 0 1 .9 1 3 .0 0 0 1 .9 1 3 .0 0 -1 7 .3 0 0 .4 0 .1 1 2 6 .8 2 7 .8 2 7 .8 2 7 .8 2 8 .8 2 8 .8 2 9 .8 2 9 .8 3 0 .8 3 0 .8 3 0 .8 3 1 .8 3 1 .8 0 1 .9 0 1 .9 1 1 .3 0 3 6 4 0 4 .0 0 4 4 6 3 1 6 .0 0 5 3 7 9 1 8 .3 0 8 6 7 3 0 6 .3 0 4 0 3 4 1 8 .3 0 5 6 4 3 0 6 .3 0 9 8 1 1 ? 7 1 6 4 0 6 .3 0 4 8 1 5 1 3 .0 0 1 3 0 4 2 2 0 .0 0 2 4 5 8 0 8 .0 0 1 4 .3 0 5 0 3 7 0 3 .0 0 2 0 3 1 1 2 .0 0 4 8 1 5 1 .2 8 1 5 .6 7 1 8 .8 9 3 0 .4 5 1 4 .1 6 1 9 .8 1 3 4 .4 5 2 5 .1 5 1 6 .9 1 4 5 .7 9 8 .6 3 1 7 .6 9 7 .1 3 1 6 .9 1 6 7 0 .2 5 1 1 5 0 .4 2 (2 5 (0 .0 9 (5 (0 .0 2 < 5 (0 .0 2 5 0 0 .1 8 2 5 0 .0 9 4 0 0 .1 5 6 0 0 .2 2 1 2 6 0 .4 7 3 7 0 .1 4 1 2 3 0 .4 5 (2 5 (0 .0 9 2 3 1 0 .8 5 in 1 . 2 . 3 . 4. 5. 6. 7. 8 . acknowledgements dumex lzkemedel ab, helsinqborq, sweden, kindly supplied the the technical preparation of the manuscript. drug, performed all the chemical analyses and gave generous help references agurell, s. & berlin, a., fernqren, h. & hellstrom, b.: plasma levels of diazepam after parenteral and rectal administration in children. epilepsia 1 6 : 2 7 7 2 8 3 , 1 9 7 5 . arnold, e.: a simple method for determining diazepam and its major metabolites in biological fluids: application in bio availability studies. acta pharmacol toxicol 3 6 : 3 3 5 3 5 2 , 1 9 7 5 . dennis, j. ( 1 9 7 8 ) : neonatal convulsions: aetiology, late neo natal status and long-term outcome. dev med child neurol 2 0 : 1 4 3 1 5 8 , 1 9 7 8 . erikson, m. & zetterstrom, r.: neonatal convulsions. incidence and causes in the stockholm area. acta paediatr scand 6 8 : 8 0 7 8 1 1 , 1 9 7 9 . lanqslet, a., meberg, a., bredesen, j.e. & lunde, p.k.m.: plas makomsentrasjoner av diazepam oq n-desmetyldiazepam hos nyfddte etter intravends, intramuskulaer, rektal og oral administrasjon referat fra nordisk neuropediateres symposium om behandlinq av krampetilstander hos barn i o s l o , juni 1 9 7 7 , pp 9 2 1 , 1 9 7 7 . naqao, m., hayashi, m., komiya, k . & komiya, h.: a study of neonatal convulsions. in: brain and development, fed. e. fuku yama) p 1 6 6 , proc 1 9 t h ann meet of the japanese society of child neurology. tokyo, 1 9 7 7 . rose, a.l.: neonatal seizures. in: topics in child neurology. spectrum publications inc, new york, pp 7 1 8 3 , 1 9 7 7 . thong, y.h. & abrahamson, d.c.: continuous infusion of diazepam in infants with severe recurrent convulsions. med ann dc 4 3 : 6 3 6 5 . received november 2 , 1 9 8 1 . address for reprints: i. gamstorp, md department of pediatrics university hospital s 7 5 0 1 4 uppsala sweden upsala j med sci 87: 223-233, 1982 evaluation of a dynamometer for measurement of isometric and isokinetic torques martin ericsson' , kurt johansson', bengt nordgren' , lars-olof nordesjo' and octavia borges' from the department of medical rehabilitation', university hospital, uppsala, the institute of physics', uppsala university, uppsala and the department of clinical physiology3, gavle hospital, giiule, sweden abstract a modified version of the original cybex i1 dynamometer with a temperature compensated strain gauge transducer has been evaluated. it was found that the calibration constant was dependent of the vertical load of the device. the ra tio (k) between the applied and the recorded torque is angle independent du ring extension and k is equal to 1.18 and during flexion k is angle dependent and can be expressed as k= -.0008 0 + 1.208, where 0 is the angle of flexion. comments on tee model it is important for clinicians involved in rehabilitating physically dis abled patients to be able to measure changes in muscular performance and strength. the muscles are working both isometrically and dynamically. during the early 1960's, j j perrine developed the concept of isokinetic exercise, i.e. dynamic movement with a constant speed, and it was first reported by his lop and perrine in 1967 (1). the cybex i1 dynamometer* has during some years been used clinically for measurement of isometric and isokinetic torques ( 2 , 3 , 4, 5 ) . a modified ver sion of the original device has recently been described (6). in the original device the torque was sensed via an oilcell. however, the output signal of the transducer was not linear, a factor which made a simple correction of the re corded data difficult. therefore a new device has been developed which senses the torque by means of a strain gauge technique.** the aim of this study has been to investigate the calibration conditions of the modified device in order to be able to present valid data. dynamometer in this modified version of the original cybex i1 dynamometer the hydraulic pressure gauge was replaced by a temperature compensated strain gauge transdu * cybex division of lumex, 100 spence street, bay shore, n.y. 11706 ** aeronautical research institute of sweden 223 cer ( 6 ) . a b l o c k diagram of t h e measuring system i s p r e s e n t e d i n f i g u r e 1. s t r a i n gauge amp1 i f 1 er y transducer tektronix ’ goniorneter amp1 i f i e r x cybex i1 . x y recorder bryan 26700 high speed f i g . 1. block diagram of t h e measuring system. i n t h e y a m p l i f i e r l i n e t h e r e i s a c a l i b r a t i o n s i g n a l , which c o r r e s p o n d s t o a t o r q u e e q u a l t o 50 nm. using t h i s s i g n a l as c a l i b r a t i o n t h e r e s u l t i s deno t e d t h e r e c o r d e d t o r q u e . i n t h e p r e s e n t s t u d y t h e s i g n a l from t h e s t r a i n gauge t r a n s d u c e r h a s been c a l i b r a t e d . c o r r e c t i o n f o r t h e g r a v i t a t i o n a l t o r q u e due t o t h e mass of t h e l e v e r arm and t h e l i m b the mechanical arrangement f o r measurement of t h e t o r q u e (m) i s i l l u s t r a t e d i n f i g u r e 2 . knee \--k joint 224 fig. 2 . mechanical arrangement f o r measurement of t h e t o r q u e (m) d u r i n g knee e x t e n s i o n and f l e x i o n a t a c o n s t a n t angular v e l o c i t y . f = t h e f o r c e produced by t h e limb. l1 = d i s t a n c e between t h e c e n t e r of mass of t h e limb and l e v e r arm and t h e a x i s of t h e knee j o i n t . l2 = d i s t a n c e between t h e p o i n t where t h e l e v e r a r m is a t t a c h e d t o t h e l i m b and t o t h e a x i s of t h e knee j o i n t . 8 = t h e f l e x i o n angle; 8 = oo when t h e l e g i s f u l l y extended. mg = t h e weight of t h e l e v e r arm and t h e limb. due t o t h e f o r c e of g r a v i t a t i o n on t h e l e v e r arm and t h e limb a c o r r e c t i o n must be made f o r t h e g r a v i t a t i o n a l t o r q u e (m ) ( 7 ) . using t h e d e f i n i t i o n s i n figure 2 t h e formula f o r t h i s c o r r e c t i o n i s d e r i v e d a s follows: m = f 1 2 ( a ) m = m g l1 cos 8 ( b ) m = m + m e x t e n s i o n of t h e knee j o i n t ( c ) m = m m f l e x i o n of t h e knee j o i n t ( d ) 9 4 r e g 9 r e g g the c o n s t a n t g i s t h e g r a v i t a t i o n a l a c c e l e r a t i o n and i s equal t o 9.81 m / s and m m t h e l i m b r e l a x e d . the r e s u l t of t h i s measurement i s c a l l e d m' is t h e recorded t o r q u e . the g r a v i t a t i o n a l t o r q u e 0 r e g i s measured a t an angle go, i . e . go = 0 w i t h t h e muscles of g g ' the e q u a t i o n m = m ' cos 0 g g a ( e ) i s t h e n used when c a l c u l a t i n g t h e t o r q u e of t h e l e v e r a r m and t h e limb a s a f u n c t i o n of t h e angle 8 i n t h e e q u a t i o n s (c) and ( d ) above. c h a r a c t e r i s t i c s of t h e recorded t o r q u e the recorded t o r q u e of t h e knee j o i n t f o r both e x t e n s i o n and f l e x i o n i s shown i n f i g u r e s 3 and 4 i n which t h e angular v e l o c i t y i s equal t o 1 2 and 150 d e g r e e s / s , r e s p e c t i v e l y . the t o r q u e of t h e g r a v i t a t i o n a l f o r c e of t h e limb f i g . fig. 1 a = a 2 = a3 = a4 = fig. c and l e v e r a r m i s a l s o p r e s e n t e d i n both f i g u r e s and i s denoted (a3 i n 3 and a i n fig. 4 ) . 3 . recorded torque (m t o r q u e of a 5 kg weight hanging a t t h e o u t e r end of t h e l e v e r a r m . e x t e n s i o n of t h e knee j o i n t . g r a v i t a t i o n a l torque of t h e l i m b and t h e l e v e r a r m a t 1 2 o / s . f l e x i o n of t h e knee j o i n t . 4. recorded t o r q u e of a s u b j e c t a t 15oo/s a n g u l a r v e l o c i t y . 5 ) of a s u b j e c t a t 1 2 o / s angular v e l o c i t y . r e g 0 aj and a6 i s t h e g r a v i t a t i o n a l t o r q u e of t h e limb and t h e l e v e r a r m a t 1 2 / s and 150 / s r e s p e c t i v e l y . m = peak overshoot. 0 p 225 fig. 3 . fig. 4. 226 the r e c o r d e d t o r q u e o f a 5 kg weight hanging a t t h e o u t e r end o f t h e l e v e r a r m i s c a l l e d a' i n f i g u r e 3 . from t h a t p a r t of t h e f i g u r e it i s concluded t h a t t h e r e i s a damped frequency of t h e c o n t r o l system e q u a l t o 10 2 hz. t h i s f r e q u e n c y of t h e c o n t r o l system i s a l s o found i f i g u r e 4 . t h i s o s c i l l a t i o n i s damped o u t a f t e r 4-5 p e r i o d s . the same damped o s c i l l a t i o n s c a l l e d a l i n f i g u r e 3 a r e a l s o seen i n t h e r e c o r d e d t o r q u e o f t h e knee j o i n t b o t h i n ex t e n s i o n and i n f l e x i o n movement. when comparing t h e r e c o r d i n g s a' and a3 it i s obvious t h a t it i s d i f f i c u l t t o keep t h e muscles r e l a x e d d u r i n g t h e whole movement. the c o r r e c t i o n t e r m s of t h e e q u a t i o n s c and d s h o u l d t h e r e f o r e be measured w i t h t h e limb r e s t i n g a t a f i x e d a n g l e ( o o ) . the g r a v i t a t i o n a l t o r que m i s t h e n c a l c u l a t e d u s i n g t h e e q u a t i o n e . 9 the r e c o r d e d t o r q u e o f t h e g r a v i t a t i o n a l f o r c e o f t h e l e v e r a r m and t h e limb a r e a l s o shown i n f i g u r e 4 . the a n g u l a r speed i s 1 2 / s i n t h e r e c o r d i n g a 5 and 15oo/s i n a . the peak o v e r s h o o t (m ) o f t h e 10 h z o s c i l l a t i o n i n c r e a s e s when t h e a n g u l a r v e l o c i t y i s i n c r e a s e d . the v a l u e o f t h e second peak i n c r e a s e s from 6 nm a t 1 2 / s t o 1 2 nm a t 150 / s . these l a r g e peaks o r i g i n a t i n g from t h e a n g u l a r v e l o c i t y c o n t r o l system of t h e cybex i1 makes it d i f f i c u l t t o c a l c u l a t e a v a l i d maximum v a l u e of t h e r e c o r d e d t o r q u e , which a l s o h a s been p o i n t e d o u t by winter e t a1 ( 7 ) . t h i s d i f f i c u l t y i s d e m o n s t r a t e d i n f i g u r e 5 , which i s a c a l c u l a t e d u n i t s t e p r e s p o n s e of a t h i r d o r d e r c o n t r o l system. the a p p l i e d t o r q u e m used i n t h e c a l c u l a t i o n c o r r e s p o n d s t o t h e maximum t o r q u e , and t h e f i g u r e shows t h a t t h e r e c o r d e d t o r q u e of t h e system h a s a peak o v e r s h o o t (m ) . concequently t h e mean v a l u e c u r v e can be used g i v i n g t h e a p p l i e d t o r q u e . 0 6 p 0 0 p t i m e f i g 5. a c a l c u l a t e d u n i t s t e p r e s p o n s e of a t h i r d o r d e r system. m = a p p l i e d t o r q u e ; m = r e c o r d e d t o r q u e ; m = peak o v e r s h o o t . p 227 1 t h i s is a l s o e x a m p l i f i e d i n figure 3 i n t h e r e c o r d i n g ( a ) as a dashed l i n e . there i s a l s o i n f i g u r e 3 an o s c i l l a t i o n w i t h a lower frequency which i s e q u a l t o 1 . 6 2 0 . 2 hz. t h i s frequency i s probably n o t due t o t h e c o n t r o l sys t e m b u t t o t h e mechanical arrangement of t h e l e v e r arm. the peaks of t h i s f r e quency a r e marked w i t h a* where t h e y a r e most e v i d e n t , b u t t h e y can a l s o be seen a t some of t h e a -peaks. the same o s c i l l a t i o n i s a l s o seen i f i g u r e s 7 and 8 where a c o n s t a n t t o r q u e i s a p p l i e d t o t h e l e v e r a r m . i t i s t h e r e f o r e con cluded t h a t t h i s frequency i s n o t g e n e r a t e d by t h e muscles. c a l i b r a t i o n 1. extension of t h e knee j o i n t 2 the t o t a l f o r c e a c t i n g on t h e s t r a i n gauges d u r i n g t h e e x t e n s i o n and f l e x i o n movement of t h e knee j o i n t may i n f l u e n c e t h e r e s u l t of t h e measurement. i n t h i s p r e s e n t a t i o n t h e e x t e n s i o n movement t h i s f o r c e i s d i r e c t e d upwards and v a r i e s from 0 n t o 350 n and i n t h e f l e x i o n movement between 0 n t o 300 n. a s p e c i a l c a l i b r a t i o n equipment w a s t h e r e f o r e c o n s t r u c t e d i n o r d e r t o o b t a i n a s i m i l a r v e r t i c a l l o a d as d u r i n g an a c t u a l i n v e s t i g a t i o n of a s u b j e c t . the ex p e r i m e n t a l equipment f o r t h e c a l i b r a t i o n i s shown s c h e m a t i c a l l y i n f i g u r e s 6a and 6b. two d i s c s o f aluminium w i t h t h e r a d i u s e q u a l t o 0.15 m and 0.49 m r e s p e c t i v e l y w e r e used. p c y b e x i t n l e v e r d i_s_c p i g . 6a 228 f i g . 6b f i g . 6. c a l i b r a t i o n s e t u p . the weight used t o produce a c o n s t a n t t o r q u e i s connected t o t h e l e v e r arm v i a a w i r e and a d i s c . the t o t a l f o r c e a c t i n g on t h e l e v e r arm i s e q u a l t o t h e d i f f e r e n c e between t h e weight of t h e d i s c and m a s s ( m ) i n figure 6a and t o t h e sum of t h e masses i n figure 6b. i n t h e experiment shown i n figure 6a t h e v e r t i c a l f o r c e a c t i n g on t h e a x i s of r o t a t i o n i s equal t o t h e sum of t h e weight of t h e d i s c ( 2 5 kg o r 3 kg) and t h e hanging weight ( m ) . i n figure 6b t h e v e r t i c a l f o r c e i s e q u a l t o t h e d i f f e r e n c e between t h e weights of t h e two masses. the r e s u l t s of t h i s experiment a r e p r e s e n t e d i n f i g u r e s 7 and 8 which corresponds t o f i g u r e s 6b and 6a res pec t i v e l y n i736 m = 3 0 k g 1 ~ 0 w 0' 1 8 0 ' 3 6 0' m = 2 0 k g 1 8 0 ' 3 60' 1 4 4 2 2 0' m = l o k g 1 3 4 3 3 0' 180' 3d 0' r = 0 . 4 9 m fig. 7 . recorded t o r q u e f o r d i f f e r e n t weights ( m ) . the c a l i b r a t i o n configura t i o n i s also shown. the t o t a l v e r t i c a l f o r c e a c t i n g on t h e equipment i s given t o t h e l e f t of t h e f i g u r e . angular v e l o c i t y = 12o/s. 229 n m = 5 0 k g i 2 4 5 m = 3 0 k g m = 2 0 k g 149 m = 1 0 k g i 1 4 7 , 8 9 0 6 0 3 0 0 f i g . 8. recorded t o r q u e f o r d i f f e r e n t w e i g h t s ( m ) . the c a l i b r a t i o n c o n f i g u r a t i o n i s also shown. angular v e l o c i t y = 1 2 / s . 0 the r e c o r d e d t o r q u e i n f i g u r e i shows an a n g u l a r dependence and t h e c a l i b r a t i o n must t h e r e f o r e be made under s i m i l a r c o n d i t i o n s as d u r i n g a c l i n i c a l i n v e s t i g a t i o n . the c u r v e s i n b o t h f i g u r e s f o r m = 50 kg i n d i c a t e t h a t a s t h e r e i s no a n g u l a r dependence o f t h e c a l i b r a t i o n c o n s t a n t i n f i g u r e 8, t h e c a l i b r a t i o n s e t u p i n f i g u r e 6 a c o r r e s p o n d s q u i t e w e l l t o t h e c l i n i c a l s i t u a t i o n i n t h e e x t e n s i o n movement of t h e knee j o i n t . the r e s u l t s from f i g u r e 8 a r e g i ven i n table i and t h e d a t a c o r r e s p o n d i n g w i t h i n e r r o r s t o a s t r a i g h t l i n e . i t i s concluded from t a b l e i t h a t t h e r e c o r d e d t o r q u e of t h e e x t e n s i o n movement h a s t o be m u l t i p l i e d by t h e c a l i b r a t i o n f a c t o r 1.18 i n o r d e r t o g i v e t h e ac t u a l t o r q u e of a s u b j e c t . 230 angle 90 70 50 30 1 0 applied t o r q u e ( n m ) 48 96 144 240 recorded t o r q u e (nm) 41 82 38 80 36 79 35 78 34 78 23 21 3 23 21 3 23 21 3 23 21 3 23 21 3 table i . recorded t o r q u e from t h e c a l i b r a t i o n a c c o r d i n g t o t h e s e t u p i n f i g . 6a ( e x t e n s i o n ) . the radium o f t h e d i s c e q u a l s 0.49 m . 2 . f l e x i o n of t h e knee j o i n t the c a l i b r a t i o n s e t u p f o r t h e f l e x i o n movement i s i l l u s t r a t e d i n f i g u r e 9 . f i g . 9. c a l i b r a t i o n s e t u p c o r r e s p o n d i n g t o t h e f l e x i o n movement of t h e knee j o i n t . the r a d i u s o f t h e d i s c i s 0.15 m . t h i s w a s used t o produce a v e r t i c a l downward f o r c e c o r r e s p o n d i n g t o f l e x i o n o f t h e knee j o i n t . the downward v e r t i c a l f o r c e i s t h e sum of ml and t h e weight o f t h e wheel ( 3 kg) minus t h e upward v e r t i c a l f o r c e g e n e r a t e d by m 2 ’ the r e c o r d e d t o r q u e s f o r d i f f e r e n t v e r t i c a l l o a d s o f t h e r o t a t i o n a x i s are 23 1 shown i n figure 10. 1 . e i 0 i 9 0 5 0 r = 0 . 1 5 m f i g . 10. recorded t o r q u e f o r d i f f e r e n t weights ml and m2. the c a l i b r a t i o n con f i g u r a t i o n i s a l s o shown. the t o t a l v e r t i c a l f o r c e a c t i n g on t h e equipment i s given t o t h e l e f t of t h e f i g u r e . angular v e l o c i t y = 1 2 / s . 0 the conclusion from figure 10 i n t h i s case i s t h a t t h e c a l i b r a t i o n con s t a n t i s angular dependent. the r e s u l t of t h e c a l c u l a t i o n of t h e c o n s t a n t where t h e d a t a i n figure 10 was used i s shown i n figure 11. k t 1 l * l o l .oo 0 3 0 6 0 9 0 8 232 fig. 11. c a l i b r a t i o n c o n s t a n t ( k ) f o r f l e x i o n a s a f u n c t i o n o f t h e angle 8. k = a p p l i e d t o r q u e divided by recorded torque. the v e r t i c a l a x i s k i s equal t o t h e a p p l i e d t o r q u e divided by t h e recorded one. the c a l i b r a t i o n c o n s t a n t i s expressed i n numerical v a l u e s a s k = -0.0008 8 + 1.208, where t h e angle of f l e x i o n of t h e knee j o i n t defined i n figure 2 . conclusion i t was found i n t h i s study t h a t t h e c a l i b r a t i o n c o n s t a n t ( k ) f o r t h e dyna mometer used i n our l a b o r a t o r y d i f f e r s between e x t e n s i o n and f l e x i o n movement of t h e knee j o i n t . the c o n s t a n t i s defined a s t h e r a t i o between t h e a p p l i e d and recorded t o r q u e . during e x t e n s i o n k i s angle independent and e q u a l s 1.18, -.0008 9 + 1.208, where 8 is t h e angle of f l e x i o n of t h e knee j o i n t . acknowledgement this work was supported by t h e swedish medical research council grant no. 1156 ( m . e . ) 1. 2. 3 . 4 . 5. 6. 7 . references hislop, h . j . and p e r r i n e , j.j.: the i s o k i n e t i c concept of e x e r c i s e . j am phys ther assoc 47:114-117, 1967. murray, p., gardner, g.m., mollinger, l.a. and sepic, s.b.: s t r e n g t h of i s o m e t r i c and i s o k i n e t i c c o n t r a c t i o n s . knee muscles o f men aged 20-86. phys ther 60-412, 1980. goslin, b.r. and c h a r t e r i e s , j.: i s o k i n e t i c dynamometry: normative d a t a f o r c l i n i c a l use i n lower e x t r e m i t y (knee) c a s e s . scand j rehab med 11:105: 1 0 9 , 1979. knapik, j.j. and ramos, m.u.: i s o k i n e t i c and i s o m e t r i c torque r e l a t i o n s h i p s i n t h e human body. arch phys med rehab 60-64, 1980. scudder, g . n . : torque curves produced a t t h e knee d u r i n g i s o m e t r i c and i s o k i n e t i c e x e r c i s e . arch phys med rehab 61-68, 1980. gransberg, l . , knutsson, e . and l i t t o n , j . e . : a computer programmed sys tem f o r t h e a n a l y s i s of a c t i v e and p a s s i v e i s o k i n e t i c movements. f r o n t i e r s of engineering i n health care 13.3.1, 1980. winter, d . a . , wells, r . p . and o r r , g.w.: e r r o r s i n t h e use of i s o k i n e t i c dynmometers. eur j appl physiol 46:397-408, 1981. address f o r r e p r i n t s : martin e r i c s s o n , m.d. dept of rehab med u n i v e r s i t y hospital 5-750 1 4 uppsala sweden 233 upsala j med sci 94: 101-109, 1989 a prospective area-based study of the outcome of pregnancy in rural tanzania bo moller,' omari lushino,' josephat kabukoba,' festo k a v i ~ h e , ~ mehari g e b r e m e d h i ~ ~ , ~ olav meirik' and gunilla lindmark' departments of 'obstetrics and gynecology, 4pediatrics and 'social medicine, university hospital, uppsala, sweden, an2 'xiringa regional hospital, iringa, and 'tanzania food and nutrition centre, dar es salaam, tanzania abstract a prospective area-based study on the outcome of pregnancy was carried out in the rural village of ilula in tanzania. a coverage of 99% (n=719) regarding the ultimate outcome for mother and child was achieved, including deliveries that took place in hospital ( 9 % ) , at the dispensary (67%) and at home (23%). there were four maternal deaths (6/1,000). the mean birth weight for singletons was 3,070 g and the low birth weight (<2,500 g) rate 13%. from a gestational age of 37 weeks onwards there was a definite slowing of fetal growth. perinatal mortality rate was 82 per 1,000 born, half of the deaths occurring in low birth weight babies. twinning occurred in 3.5% and the mean length of gestation at delivery for these pregnancies was 35.5 weeks. twins constituted 6.8% of newborns but accounted for 23.0% of perinatal losses, making twin pregnancy a major contributor to perinatal mortality. post-term pregnancies carried no significant increase in mortality. it is concluded that reliable area-based data on the outcome of pregnancy in tanzania can be obtained at village level, with good coverage of the study population, by properly instructed and motivated local staff with moderate supervisory support. introduction in obstetric and perinatal epidemiology it is unusual outside the affluent industrialized countries to obtain data from geographically well defined areas. information regarding obstetric outcome in developing countries is generally based on hospital records. in most of these countries hospital care facilities are limited and are used mainly by selected segments of the population and by patients referred from rural areas. 101 such hospital-based data can therefore only be generalized to a limited extent. in the declaration of monastir (3), the need for more complete and precise collection of reliable epidemiological data from non-hospital-based populations was emphasized. the present report is part of a comprehensive investigation aimed at obtaining basic knowledge of factors influencing the course and outcome of pregnancy in a geographically defined rural area in tanzania, and at studying the effect of the existing antenatal care (anc) programme on the outcome of pregnancy and the survival of the infant. in this paper the basic design of the study and the main outcome variables of pregnancy are described and discussed. materials and methods the study was conducted in the village of ilula in iringa district, tanzania. the study asea was carefully chosen in order to represent a situation typical of many other areas in tanzania (18). telephone and electricity are not available in the village. buses run daily to iringa town, 47 km away. the mother and child health care (mch) and delivery services are run by 2 village midwives under the supervision of a rural medical aide ( r m a ) at the lutheran dispensary. village midwives have had 7 years in primary school followed by on the job training and a 9-month mch-aide course, and rmas 3 years of medical and public health training. doctors and facilities for obstetric operations are available in iringa. all pregnant women living in isere and mtua villages, henceforth referred to as ilula, were entered consecutively into the study log-book at delivery (the first 34 women), or when registering for antenatal care (all subsequent women). all were to be interviewed by the female field worker after delivery within 48 hours, at one week and in some cases at 6 weeks. the questionnaires included information about the mother's social and medical history, obstetric history, pregnancy and delivery complications. through strong community co-operation, we feel confident that we obtained over 99% coverage of pregnancies beyond 2 8 weeks of gestation. the collection of data started on june 13, 1983. all deliveries from that day onwards were included. women included in this report are those registred at anc up to november 13, 1985, all of whom were delivered by june 2, 1986. for cases 102 referred to hospital the iringa regional hospital and for follow-up of high-risk children, information was supplemented through enquiries by the field worker after that time. the date of the last menstrual period (lmp) for the calculation of the expected date of delivery was obtained from the antenatal card and checked against statements in the questionnaire. also, the mothers were asked about the "certainty" of their dates and categorized as certain, uncertain, no period before pregnancy, or ignorant. infants delivered at the dispensary were weighed on a stationary balance scale. some babies delivered at home were weighed on a simple, less accurate spring scale, and these readings tended to cluster around the nearest 500 g value. low birth weight (lbw) infants are those with a birth weight of 1,000 to 2,499 g. the classification into livebirths and stillbirths was made by the midwife. r n 6 results coveraqe and antenatal clinic attendance out of 724 registered women, 5 could not be traced. sevenhundred and seven had pregnancies that went beyond 28 completed weeks or resulted in an infant with a birth weight of 1,000 g or more. questionnaires were available for 682 cases, but in a number of instances they were not complete, e.g. the birth weight was not known in 61 cases. knowledge of the ultimate outcome of the pregnancy was obtained in all cases for the 707 deliveries. of the 707 delivered women, only one negated antenatal care. the average number of visits was 6.3 per pregnancy, with a range from 0 to 14 (fig 1). q 3 80 6 ll 8 10 12 14 number of v i s i t s fiq. 1. antenatal care attendance of ilula mothers. " 0 " indicates no visits (n=1) or an unknown number of visits (n=16). 103 place of delivery of the 7 0 7 deliveries, 6 8 % took place at the ilula dispensary, 23% at home and 9% in the iringa regional hospital. of the women who delivered at home, 45% stated that' they did so of their own choice, 40% because they did not leave for the dispensary early enough in labour, and 15% for various other reasons. gestational aqe lmp was reported as certain by 52% of the women while 35% stated the lmp within 10-14 days. a mean gestational age at delivery for singletons of 39.5 (sd 2.4) weeks was found in the "certain lmp" group. for all singleton pregnancies, including women who were uncertain about their lmp but gave a date, it was 39.4 (sd 2.6) weeks. of the simplex deliveries (fig. 2), 16% were preterm. in twin pregnancies the mean gestational age at delivery was 35.5 (sd 3.2) weeks. % loo [ f 6o t f i q . 2. distribution of gestational age at delivery. 16% of deliveries were preterm, 13 "post term". i l 1 30 32 3l 36 38 lo l 2 l l 46 gestational age (weeks) maternal deaths there were 4 maternal deaths (6/1,000), 2 from postpartum haemmorrhage, one from uterine rupture in a woman who had previously had a caesarean section, and one death from septic complications after a caesarean section. table 1. births and perinatal deaths by birth weight f o r singletons and twins births. (ilula. tanzania 1 9 8 3 8 6 . n . 7 0 7 ) . (pmr=perinatal mortality rate: sb=stillborn: end=early neonatal deaths [within 1 week]). for comments on the group "not weighed", see text. s i n g l e t w i n s a l l birth weight births deaths pmr born deaths pmr born deaths pmr rate rate sb end rate per per per 1 , 0 0 0 1 * 000 1 , 0 0 0 1,000-1.999 1 5 1 0 660 12 7 5 8 0 27 6 11 630 2 . 0 0 0 2 . 4 9 9 27 3 111 2 1 5 240 4 8 1 7 170 2.500-5.000 583 14 24 1 3 0 0 5 9 6 10 4 24 not weighed 57 19 333 4 2 5 0 0 6 1 1 4 7 344 all 6 8 2 4 6 67 5 0 14 2 8 0 7 3 2 3 1 29 8 2 104 perinatal mortalitv as shown in table 1, the perinatal mortality rate (pmr) in singleton pregnancies was 67 per 1,000 and in twin pregnancies 280 per 1,000 born children. total pmr was 82. twins constituted 6.6% of newborns but 23% of the perinatal deaths. estimation of the true pmr relative to birth weight is hampered by the frequent reluctancy to weigh a dead child. table 1 shows birth weight specific mortality with unweighed infants separately. in the 1,000-1,999 g group another 4 infants died between the ages of one and 6 weeks, giving a cumulative mortality of 78% at 6 weeks. in the 2,000 to 2,499 group, 8 of 48 were dead at one week and 3 more at follow up at 6 weeks, bringing the losses to 23% at that time. follow up at 6 months of all liveborn with a birth weight of 2,500 g or less revealed 2 more deaths, one at 3 and one at 5 months. of all deliveries, 13% took place after 42 completed weeks. pmr in this group was 50 per 1000, as compared with 40 in women delivered after 37-41 completed weeks. primipara parous all women table 2 . mean birth weight of newborns girls 2,723 2,974 2,931 by sex and maternal boys 2,955 3,104 3,075 parity. total 2,869 3 , 044 3,011 twins included. birth weiqht the birth weight in relation to the parity of the mother and the sex of the child is given in table 2. fig. 3 shows the birth weight distribution for singleton babies. the mean birth weight of singleton newborns was 3,070 (sd 485) g. number r 120 c n fiu. 3. birth weight distribution among 615 singleton deliveries. 105 birth weight ( g ) taking into account the effect of the clustering of weight readings to the nearest 500 g and the fact that some of the 61 unweighed children undoubtedly were lbw, the rate of lbw infants was estimated to be 13 per cent. 9 loo0 3000 ? 3 5 m 2000 i o o o l i . . . . . . . , p i . ' i ! ' . i ; i t i : i , i : . * , ! . i i , . ' ; i . : ; i f , , : 2 ; ; ; j ; ; : , ~ , ! a , t . ! i ; , ,*,, , . . . : * . . ' fiq. 4 . birth weight by a 1 . . , / * . gestational age in 5 2 2 singleton deliveries. , i , , , , , , , i , i in figure 4 the birth weight for gestational age is plotted for 5 2 2 cases with a known date of lmp. observations before 3 7 weeks of pregnancy are few. linear regression for this period showed a mean increase in birth weight near 0 . 2 kg per week. towards the end of pregnancy there was a markedly slower increase in mean birth weight, in total about 0 . 2 5 kg during the five weeks from 37 to 4 2 weeks, or 5 0 g/week. discussion the study area represents a rural area. its development is probably above the average for rural tanzania in some respects, such as communication and education, but nevertheless the ilula situation is representative of many areas in tanzania. as is often the case in tanzanian health institutions, medical supplies are frequently inadequate, emergency transport is sometimes not available and the referral centre is hard pressed for resources (8,19). this clinic was judged to be in a better category, well organized and esteemed by the local society. at the time of the study the registration rate for antenatal care in the iringa region was judged to be 9 7 % by the ministry of health (8). this is much higher than is reported from villages covered by mobile services (15) and also above the national estimate of 95% registration. the mean number of visits was 6.3, which is above the national mean of 4.3 visits. although numbers in this study are small, the case histories for the maternal deaths are typical (4,lo). they suggest that hospital-based reports on maternal mortality in developing countries, including tanzania, do not exaggerate the situation (7,11,16) even when they include many referred cases. at 82 per 1,000 born, the perinatal mortality rate is over ten-fold that of sweden. in table 3, data available for one area in west africa, for saudi arabia and for sweden are shown. table 3 . comparison of some common measures of the outcome of pregnancy in ilula, two other developing countries, and sweden. missing data = -, pmr = perinatal mortality rate, lbw = low birth weight. year pmr mean lbw twin sex p r w maternal ref area birth rate rate ratio para mortality weight m/f rate /100,000 i lula 1985 82 3,011 g 13% 3.5% 119 16% 600 13 14 abi jan 1975 2,953 g 19% 2.0% 102 25% riyadh 1985 40 3,226 g 9% 1.1% 102 19% 21 sweden 2o 1984 7 3,500 g 4% 1.0% 106 41% 2 economic conditions are reflected in the perinatal mortality rates. it can be seen that there are differences in the proportions of primigravidae and in the rates of twins and low birth weight infants, factors that all influence the perinatal mortality rate. the distribution of birth weights in ilula is similar to that reported from previous studies (l), and shows a shift to the left of 500 g when compared, for example with swedish material (20). low birth weight occurs three times as often in ilula as in sweden, and in the face of prevailing conditions, is a major determinant of perinatal mortality. knowledge of the very high perinatal mortality rate for the smallest infants in the rural areas is important as a reference when judging results of different forms of care for these infants. the high twinning rate, one in thirty pregnancies, as is also found in other reports from africa (7,12), contributed heavily both to prematurity and to perinatal mortality. identification of twin pregnancies and development of appropriate methods for the care of low birth weight infants, 107 also at primary levels of care, should be a priority in a strategy for better perinatal survival. information on the last menstrual period is the best estimate of gestational age in the foreseeable future (5), but it has to be realized that it leads to a falsely increased number of pregnancies classified as postterm, i.e. after 42 full weeks. recent studies (2) indicate that expectant management of post term pregnancy is as safe as induction of labour in these women. postterm pregnancy does not seem to be of clinical importance as a risk factor for adverse perinatal outcome, as is also indicated from our data. in singleton pregnancy birth weight for gestational age under optimal conditions increases in a linear fashion (17). in our material there is a levelling off of growth after 36 weeks of gestation. this slowing of fetal growth, which has also been found in other studies (6,9), is pronounced and probably cannot be ascribed entirely to inaccurate determination of gestational age, but seems to be real and a sign of less than optimal conditions for fetal growth. in developing countries, where a large proportion of births take place at home or in small rural institutions, area-based studies with full coverage of the pregnant population are necessary to relate the outcome in the rural areas to the outcome in hospitals. prospective area-based studies are also needed for general and detailed health planning ( 3 ) . possibly the most important experience from this investigation is that such studies can be implemented in rural areas with the help of properly instructed, trained local health personnel who are given the support of the political infrastructure. acknowledgements this study was supported by sarec grants 81/79 and 83/148. we are indebted to unicef, tanzania, for logistical support. mr and mrs saga of ilula dispensary and miss mwasyete are gratefully thanked for their sustained and devoted work. 108 references 1 bantje, h.: long term and seasonal variations of birth weight distribution in tanzania. research paper no. 13, inst of resource assessment, university of dar es salaam , tanzania. oct 1985. 2 cardozo, l., fysh, j. & pearce, m.: prolonged pregnancy: the management debate. br med j. 293:1059-1063 1986. 3 declaration of monastir: ippf medical bulletin vol 19 no 1:l. 1985. 4 fathalla, m.f. the causes of maternal deaths: a global review. who document no. fhe/pmm/85.9.7. 5 fig0 subcommittee: methodology of measurement and recording of infant growth in the perinatal period. november 11-18 1984. 6 gebre-medhin, m., sterky, g., taube, a.: observations on intrauterine growth in urban ethiopia. acta paediatr scand. 7 harrison, k.a.: approaches to reducing maternal and perinatal mortality in africa. in: maternity services in the developing world, what the community needs. rcog london 1980. 8 ministry of health and who. mimeo, joint primary health care review, p 119. dar es salaam, tanzania 1984. 9 lubchenko, l.o. intrauterine growth as estimated from live born birth weight data at 24 to 42 weeks: pediatrics 37:403 408 1966. 10 maine d. maternal mortality: helping women off the road to death. who cronicle 40:5 175-183 1986. 11 mtimavalye l a r, lisasi d, and ntuyabaliwe w k.: maternal mortality in dar es salaam, tanzania, 1974-1977. east african medical journal 57:2 111-117. 1980. 12 nylander, p.p.s.: perinatal mortality in ibadan. african j. med sci. 2:173-178 1971. 13 reinhardt, m.c. a survey of mothers and their newborns in abijan (ivory coast) helv. paediat. acta suppl 41: 1-132 1978 14 serenius, f. child health in saudi arabia. acta paedia scand. suppl 346, 1988. 15 shears, p. and mkerenga, r.: evaluating the impact of mother and child health (mch) services at village level: a survey in tanzania, and lessons for elsewhere. annals of tropical pediatrics. 5:55-59 1985. 672781-789 1978. 16 who: maternal mortality rates. who geneva. 1986. 17 who: recommended definitions, terminology and format for statistical tables. acta obstet gynecol scand 56:247-253, 1978. 18 who task force on "risk approach" for improved maternal and child health and family planning: risk approach for maternal and child health care. world health forum 2:413-422 1981. 19 andersson-brolin, l., staugard, f., cornell, i., et. al. health centres in need of treatment. sida evaluation report: 1987:4 health, tanzania. stockholm, sweden 20 swedish medical birth registration 1980:2 stockholm, sweden. address for reprints: bo mbller, m.d. central hospital s-631 88 eskilstuna sweden 109 upsala j med sci 92: 85-88, 1987 the effect of cysteamine on the somatostatin content of guinea-pig islets birger petersson department of medical cell biology, university of uppsala, uppsala, sweden abstract cysteamine is known to deplete the immunoreactive somatostatin content in different organs from rat and mouse. the aim of the present work was to test if cysteamine also affects the somatostatin content in guinea-pig islets, since guinea-pigs have a carbohydrate metabolism different from that of other labora tory animals. cysteamine was injected to guinea-pigs and the pancreatic islets were isolated four hours later. cysteamine was also incubated in vitro with isolated pancreatic islets from untreated guinea-pigs. cysteamine depleted the somatostatin content in the pancreatic islets in both the in vivo and in vitro studies. introduction it is well known that the ulcerogenic substance cysteamine (2-amino-ethan ethiol), injected in vivo, causes a selective depletion of somatostatin in many organs in both the mouse and rat (7, 6, 3 ) . besides somatostatin, cysteamine also depletes prolactin ( 3 ) . in addition, cysteamine is known to decrease the somatostatin content of pancreatic islets in vitro (4, 5 ) . the guinea-pig is associated with several biochemical peculiarities and both its insulin and glucagon amino acid sequences are different from those of other animals. the general amino acid sequence for somatostatin seems, however, to be conserved in guinea-pigs ( 1 ) . in view of its biochemical peculiarities,it is of particular interest to follow the effect of cysteamine on somatostatin in the guinea-pig. in the present study we therefore measured somatostatin in isolated pancreatic islets of guinea-pigs after administration of cysteamine in vivo and after exposure of the islets to cysteamine in vitro. 85 materials and methods each of six guinea-pigs received a subcutaneous injection of 60 mg/kg body weight of cysteamine (aldrich, beerse, belgium) dissolved in distilled water. the animals were killed four hours after the injections. three control animals remained untreated and were killed together with the injected animals. the pan creatic glands were rapidly removed and islets were isolated by means of a collagenase digestion technique ( 2 ) . groups of 25 islets from each animal were then sonicated and extracted in 250 u l of ice-cold acid alcohol (0.18 mol/l hc1 in 70 % alcohol) at 4oc overnight. the somatostatin content was determined as described below. / effects of cysteamine in vitro were studied in collagenase-isolated islets from nine normal guinea-pig pancreases. groups of 25 islets were exposed for four hours at 3 7 o c to cysteamine dissolved at a concentration of 10 or 100 ug/ / ml in tissue culture medium (parker 199) containing 10 % (v/v) calf serum. the somatostatin content of the islets was determined after sonication and extrac tion in ice-cold acid alcohol as described above. for determination of the somatostatin content, the acid alcohol extracts of the islet homogenates were centrifuged at 2000 rpm for 20 minutes and the soma tostatin content of the supernatant was assayed by a radioimmunological method (kit nr. 038192; immuno nuclear corp. ,stillwater, minnesota 5508, usa) using synthetic somatostatin-i4 as standard. the sensitivity of the method was approx imately 3 pg per tube. probabilities (p) of chance differences between groups were calculated by stu dent's t-test. results are expressed as mean values 2 standard errors of the means (m%em). results four hours after the subcutaneous administration of cysteamine the somato statin content of the pancreatic islets was significantly depleted compared with that in the control animals ( 1 7 6 1 1 pg/islet versus 934 2 47 pg/islet). in vitro incubation of islets with cysteamine for four hours at a concentration of 100 ,ug/ml resulted in a marked depletion of somatostatin, but at 10 /ug/ml cysteamine did not have this effect. the somatostatin values were 331 2 47 pg/ islet with 100 (see figs. 1 and 2 ) . ug/ml, 640 2 116 with 10 / / u g h 1 and 8 7 3 2 139 in the controls discussion the present results show that not only treatment with cysteamine in vivo but also exposure to cysteamine in vitro causes a rapid and marked depletion of immunoreactive somatostatin in pancreatic islets from guinea-pigs. this is in accordance with previous observations in islets from mice and rats (4, 5). 86 control cystearnine fig. 1 . the bars show the somatostatin content of pancreatic i s , e t s from control guinea-pigs a n d from guinea-pigs treated with cysteamine ( 6 0 mg/kg b . w . ) four hours before the animals were k i l l e d . the numbers of batches a r e given within parentheses. means s.e.m. a r e given. xxx: p < 0.001 i n r e l a t i o n t o the control g r o u p . ( 1 7 ) ( 1 7 ) (17) control cysteamine cystearnine 10,uglml 100,uglml fig. 2. the bars show the somatostatin content of pancreatic guinea-pig i s l e t s incubated f o r four hours with and without cysteamine in the medium. the numbers of batches a r e given within parentheses. means 2 s.e.m. a r e given.ns: p > 0.05; xxx: p < 0 . 0 0 1 in r e l a t i o n t o the control g r o u p . 87 other studies indicate that the loss of immunoreactive somatostatin occurs without changes in the pancreatic insulin or glucagon content (6). although the mechanism for the cysteamine action is not known, there is support for the view that the action is intracellular ( 6 ) . since in many aspects the guinea-pig has a metabolism different from that of many other laboratory animals, it is of in terest to note that the depletion of immunoreactive somatostatin by cysteamine is pronounced in this animal, also. acknowledgements supported by grants from the swedish medical research council (project no 2297 1 . references 1. conlon, j.m.: isolation and structure of guinea-pig gastric and pancreatic 2. howell, s.l. & taylor, k.w.: potassium ions and the secretion o f insulin by 3. parsons, j.a., peterson, e.k. & hartfel, m.a.: effects of cysteamine on pi somatostatin. life sciences 35: 213 220, 1984. islets of langerhans incubated in vitro. biochem j 108: 17 24, 1968. tuitary, mttwi5 tumor, and serum prolactin levels measured by rat lymphoma cell bioassay and radioimmunoassay. endocrinology 114: 1812 1817, 1984. 4. patel, y.c., pierzchala, i . , amherdt, m. & orci, l.: effects of cysteamine and antibody to somatostatin on islet cell function in vivo. j clin inv 75: 1249 1255, 1985. 5. petersson, b. & hellerstrom, c.: rapid depletion of somatostatin in isolated mouse pancreatic islets after treatment with cysteamine. acta endocrinol (kbh.) 110: 227 231, 1985. 6. sorenson, r.l., grouse, l.h. & elde, r.p.: cysteamine blocks somatostatin secretion without altering the course of insulin o r glucagon release. dia betes 32: 377 379, 1983. 7. szabo, s. & reichlin, s.: somatostatin in rat tissue is depleted by cyste amine administration. endocrinology 109: 2255 2257, 1981. address for reprints: dr. birger petersson department of medical cell biology p.o. box 571 s-751 23 uppsala 88 upsala j med sci 91: 220, 1986 25 figure measurments and r e g r e s s i o n l i n e f o r 111 b l o o d samples g l u c o s e c o n t e n t i n k 2 0 assayed b y t h e 2 l a b o r a t o r y ( x a x i s ) e and t h e ward ( y a x i s ) . f o r methodow r l o g i c a l d e t a i l s u see t e x t . s 3 the h a t c h e d l i n e i n d i c a t e s a 1:l r e l a t i o n s h i p . the c l 15 y q 10 e r e g r e s s i o n e q u a t i o n 2 which i s s i g n i f i s 5 c a n t l y d i f f e r e n t 2 i i s : y=0.956x + 1.43, v) f r o m t h e 1 : l r e l a t i o n s h i p . bed-side analysis of blood glucose i 5 10 15 20 25 christina pilstrom department of pediatrics, university hospital, uppsala, sweden p a t i e n t s w i t h n e w l y d i a g n o s e d d i a b e t e s a r e t r e a t e d w i t h i . v . i n f u s i o n s o f i n s u l i n . i n o r d e r t o m o n i t o r t h e i n f u s i o n i t i s n e c e s s a r y t o use a r a p i d , r e l i a b l e method f o r g l u c o s e a n a l y s e s . b l o o d g l u c o s e levels o f t h e same samples were measured i n a c l i n i c a l c h e m i c a l l a b o r a t o r y a s w e l l a s i n t h e p e d i a t r i c ward. t o t a l l y i11 c a p i l l a r y b l o o d samples f r o m 1 3 p a t i e n t s were a n a l y z e d i n d e p e n d e n t o f t i m e o f t h e day. the l a b o r a t o r y d e t e r m i n a t i o n s were p e r f o r m e d i n two d i f f e r e n t l a b o r a t o r i e s b y t h e o r d i n a r y l a b o r a t o r y s t a f f w i t h e i t h e r t h e g l u c o s e a n a l y z o r y s i o r a g l u c o s e dehydrogenase method a p p l i e d t o a g r e i n e r g-300. i n t h e ward t h e a n a l y s e s were p e r f o r m e d b y t h e n u r s e s i n c h a r g e u s i n g t h e t e s t s t r i p bm-test-bg and a r e f l e c t o m e t e r (glucocheck 11). because t h e answer f r o m t h e l a b o r a t o r y r e a c h e d t h e ward more t h a n one h o u r a f t e r t h e achievment o f t h e s t r i p m e t h o d r e s u l t . the two measurments a r e c o n s i d e r e d as i n d e p e n d e n t the i n v e s t i g a t i o n shows t h a t t h e use o f glucocheck ii/bm-test-bg f o r bed s i d e b l o o d g l u c o s e m o n i t o r i n g g i v e s r e s u l t s w i t h a p o o r agreement t o t h e r e s u l t s o f t h e same samples f r o m t h e c h e m i c a l l a b o r a t o r y . 220 upsala j med sci 84: 1-2, 1979 electron probe micro-x-ray analyses of electrolyte composition of fluid microsamples by use of a sephadex bead b. ove nilsson and leif ljung a o m the rrprodrrctron r e \ r u t t h unit, biomrdrc t i / crntw uppbuln, sit e d r n obtaining samples of certain body fluids for determination of the ionic content can involve great difficulties if the available amount of fluid is small and difficult to reach by a micropipette. for instance, samples of uterine secretions of rodents can not be obtained without a serious risk of contamination by interstitial fluid or cell sap. in order to circumvent these problems, we have developed a new technique where we use sephadex beads as carriers of the fluid to be analyzed. in short, a hydrated sephadex bead is inserted at the place for study and after an appropriate time for equlibration between fluid and bead, we have recovered the bead in a volatile, hydrophobe silicon oil and then prepared it for micro x-ray analyses in philips 400 electron microscope. this paper aims to give the details of the preparation technique used to obtain a qualitative information, the procedures for quantitative analyses will be reported later. we have taken the secretion of the mouse uterus as a model, but the technique is applicable also for analyses of other body fluids. i a few sephadex g 150 beads (pharmacia fine chemicals ab, uppsala, sweden) are soaked in bidestilled water for about 15 minutes. six to 8 hydrated beads of sizes around 100 um are selected and aspirated in a glass capillary, connected by a plastic tubing to a micrometer-controled syringe (shardlow micrometers ltd, sheffield, england). the capillary is laboratory made with an inner diameter slightly larger than that of the beads. by the syringe, the beads are positioned close to the capillary mouth to minimize the amount of water transferred together with the beads. the transfer of the beads is performed in a way appropriate for the purpose of the analyses. equilibration between the body fluid and the water of the bead probably occurs within some hours, but this has to be tested for each application. when analysing the secretion of the mouse uterus, the x-ray spectra consequtively obtained displayed a similar configuration after a two-hour stay of the beads in the secretion. 1-792854 i the recovery of the beads are made by flushing with a hydrophobe volatile silicone oil (dc 200, dow corning international ltd., brussels, belgium) collecting the beads in a watch-glass. with a preparation microscope, the beads are observed in the oil, and single beads are transferred onto specimen grids which have been submerged into the oil. the grid with the beads is slowly lifted out of the oil bath and left in room temperature for evaporation of the oil. the specimen grids used are nylon grids (agar aids, stansted, england) mounted with a formvar membrane. grids prepared with sephadex beads are carbon coated before the analyses. ' energy dispersive micro-x-ray analyses are performed with an edax-edith equipment attached to a philips 400 electron microscope for 100 s at 40 kv with a fixed intensity and with a take off angle of 30'. the analysing area is slightly smaller than the circular projection of the spherical bead. before the analyses, the preparations are checked and mapped with a scanning electron microscope (figs. 1, 2). 0 1 0 0 s e c 8 7 4 5 5 i n t v s : 2 5 0 0 h s : 1 0 e v i c h e d b x fig. 1. sephadex beads prepared on a nylon grid with a formvar membrane support. x 900. fig. 2. a representative x-ray spectrum from a sephadex bead which was recovered from the uterus of a mouse after a 3-hour stay in the secretion. the animal was in an experimen tal delay of implantation and had been injected with oestro gen 8 h before the preparation. accepted november 28, 1978 address for reprints: ove nilsson biomedical centre box 571 s-751 23 uppsala sweden upsala j med sci 95: 233-244, 1990 medical need for quality specifications within laboratory medicine ronald h. laessig s t a t e l a b o r a t o r y of h y g i e n e , university of wisconsin c e n t e r for health s c i e n c e s , m a d i s o n , wisconsin, u s a motto: management is doing the thing right; leadership is doing the right thing. introduction the continuous and consistent improvement in the quality of laboratory results is well documented. within the past decade, the quality of laboratory results, i.e. reduced intralaboratory coefficients of variation (cv) and bias values of essentially zero for many tests have exceeded the requirements of clinicians who utilize the laboratory results to treat patients. heretofore, laboratorians have concentrated on improving quality of test results; in the future we must concentrate on focusing limited resources where they will be most cost effective. as numerous studies have documented, clinicians and laboratori ans have considerable difficulty when they attempt to translate laboratory attributes of quality, cv, bias, total error, etc., into clinically meaningful criteria, i.e. changes in test parameters which will evoke a change in treatment regimens. the various studies of itmedical usefulness" to date have resulted in a steadily improved mutual understanding of the problems but no truly definitive answers. laboratorians have recently resorted to speaking of clinicians as their llcustomersll. this popular concept is based in the industrial quality assurance doctrines of demming, juran and others. the concept of the physician as a customer implies not only the requirement on the part of the supplier (laboratory) to satisfy the perceived needs of the shooper, but also the idea that the clinician is free to seek the most appropriate service from the most appropriate (or convenient) source. 233 it is most timely for the board of nordchem to sponsor a seminar and study to assess the need for quality specifications which relate to medical usefulness (i.e. clinical needs). we as laboratorians have, for over forty years, and with good reason, focused our efforts reducing the total error in the analysis processes. in view of the obvious benefits to be obtained by reducing error (bias) and improving precision (reducing cv) this waa indeed ildoing the thing right". the very existence of this nordic seminar on quality specifications indicates that it is time for laboratorians to assume the leadership role, that is ifdo the right thing". in today's health care environment, particular ly with respect to diminished resources in all areas, including laboratories, doing the risht thinq will require us to allocate resources, including quality improvement and test development resources, to those areas of greates potential benefit to patient and clinician. as today's seminar clearly demonstrates, this is not a singular activity of laboratorians, or clinicians; it must be a joint effort of both. the effort must be initially channeled into finding the risht thing to be done. this may include developing new tests; increasing accuracy and precision; improving test quality as characterized by turnaround time, sampling techniques or interpretive reporting or any other attributes of ittotal quality". this is leadership; but more basically, this is communications. once new test parameters are defined, once error specifications, based in medical needs are established, laborato rians can manage the process. we can be counted up on to itdo the thing right". this has always been our forte; it will continue to be. setting quality specifications for laboratory tests i would like to share some of our current research (with sharon s. ehrmeyer) with the group. we have, in the united states, inadvertently taken a new and interesting approach to setting quality specifications. this has come about because our federal government has determined that proficiency testing (pt) will become the major criteria by which laboratories are licensed by the federal government. pt is the process whereby an agency of 234 government sends unknown specimens to laboratories who analyze them and report back their results. if the laboratory gets the right answers, it passes, if not it fails. a laboratory must pass proficiency testing if it is to be reimbursed for tests it performs. the governmental regulations have been proposed in final form as of march 14, 1990. these regulations are very relevant to the theme of this nordkem conference in that they are in effect minimum intralaboratory performance standards for us laboratories. we suspect that these standards are not based on medical need, or laboratory capabilities but rather an intuitive approach to achievable levels of quality. however, under the regulatory approach which is utilized, it is a logical assumption that a laboratory will seek to improve its performance to a level commensurate with consistently passing proficiency testing. these criteria then, in effect, have become us national minimum performance standards for clinical laborato ries. material and methods we have demonstrated previously that it is possible to determine the minimum intralaboratory performance levels necessary to meet the requirements specified in the federal rules for pt programs. under the plausible and tractable assumption of gaussian imprecision, these relationships can be established by computer modeling using a monte carlo simulation approach, or by direct calculation based on statistical analysis. in both approaches a laboratory's internal performance characteristics, that is, its unique imprecision (expressed as the standard deviation [sd] or coefficient of variation [cv]) and its bias, i.e., its offset from the target value, must be taken into account when determining the probability of "passing" one or a series of pt challenges. other factors such as clerical errors, shipping problems, matrix effects, grading errors, etc., which can amount to 5 0 % of the apparent causes of pt failures should not be neglected, but a laboratory's analytical prowess is fundamental. we computed the relationship between a laboratory's internal cv and/or bias for a given analyte, and the recently published 235 federal interlaboratory pt criteria. each analyte has its own pt criterion; for the routine chemistry subspecialty, the criteria are identified in table 1. by standardizing bias and sd as a percentage of these criteria, in effect obtaining "z scores", we reduce each analyte to one generic case for analysis. from given (standardized) cv and bias values we induce a probability of producing a laboratory result outside the (standardized) pt criterion. this determines the probability of passing one pt event under the new five sample per shipment format, modeled as a bernoulli trial. table 1 analyte or test (routine chemistry) enzmes alt/sgpt alp amylase ast/sgot ck ck isoenzymes ldh ldh isoenzymes blood qas po2 pco, ph general 1 albumin ' bilirubin, total ' calcium, total cholestrol, total cholestrol, hdl creatinine glucose iron, total total protein triglycerides bun urea uric acid electrolvtes chloride magnesium potassium sodium ~~ ~ criteria for acceptable performance (target value + ) old units si units 20% 3 sd 3 sd 20% 3 sd 3 s d or mb elevated (+ or ) 20% 3 sd or ldhl/ldh2 (+ or -) 3 sd 5 nun hg or 8% 0.04 1 0 % 0 . 3 mg/dl or 2 0 % 5 pmol/l 1.0 mg/dl 0.25 mmol/l 15% 3 sd 0 . 3 mg/dl or 15% 23 pmol/l 6 mg/dl or 10% 0 . 3 3 mmol/l 20% 10% 3 sd 2 mq/dl or 9 % 4 3 ig/l or 9 % 17% 0.71 mmol/l 5% 25% 0.5 mmol/l 4 mmol/l 236 the new regulations specify that "passing performance" in a pt event (one quarterly shipment) requires that four out of the five results for each analyte (80% of the results) must fall within the defined acceptable range (target value k pt limit). the target value can be the mean of the results from a group of participants using the same method or instrument once outliers have been removed, or can be established by definitive or reference methods. alternatively, the target value can be the mean of results from 80% of 10 or more referee laboratories. from table 1, when "grading" pt results, the acceptable range for glucose is the target value ? the performance criterion [ 6 mg/dl ( 0 . 3 3 mmol/l) or lo%] yielding the greater range. in the case of a pt specimen with a target value of 100 mg/dl ( 5 . 5 6 mmol/l) , acceptable performance would be between 90 ( 5 . 0 ) and 110 mg/dl (6.11 mmol/l). if the target value were 5 0 mg/dl (2.78 mmol/l), the acceptable range would be 4 4 ( 2 . 4 4 ) to 5 6 mg/dl (3.11 mmol/l) . failure to pass pt single analyte if a laboratory does not achieve at least 80% acceptable performance on any given analyte ( 4 or 5 correct results) for a pt event, the laboratory is, in effect, put on probation for the entire subspecialty in which that particular analyte is listed. to actually "fail" pt and be subject to "adverse action" (the term used in the regulations), the laboratory must again fail to achieve acceptable performance for the same analyte on one of the next two pt events. hence, by failing to achieve acceptable performance for the same analyte for any two of three consecutive events, the laboratory may fail the entire subspecialty and thereby suspend testing in the subspecialty. a curious anomaly is that a laboratory could vffail" different analytes, e.g., glucose for one pt event, uric acid for the next, and ck for the third, etc., indefinitely without being suspended in the subspecialty of routine chemistry. failure to pass pt multiple analytes in addition to passing the individual analytes, the regulations 237 require a laboratory to achieve an 80% correct response rate over all analytes in a particular subspecialty. to be subject to adverse action, a laboratory must have less than 80% of all results correct for any two of three consecutive pt events. it is obvious that to fail this, a laboratory must also fail at least one analyte, i.e., have 2 or more incorrect out of 5 results. intralaboratorv performance required to pass one pt event for one analvte, zero bias. the right curve in figure 1 shows the probability of failure, i.e., achieving less than 80% correct for a laboratory analyzing only one analyte in any one pt event. the x axis is in units of internal cv or sd as a percent of the pt limit. for example, if the pt limit is lo%, as for glucose (table l), 100 on the x axis denotes a laboratory with an internal cv of 10%. under these circumstances, this laboratory has a 51% probability (y axis) of "failing" the analyte glucose in any one pt event in which it analyzes 5 pt samples. the right curve in figure 1 is based on the assumption that the laboratory has zero bias, i.e., any deviation from the target value is due only to the laboratory's internal imprecision. similarly, for analytes whose performance criteria (table 1) are defined as multiples of the group sd, i-e., 3 group sd for alkaline phosphatase, the 100% point on the graph is equivalent to a laboratory whose internal sd is equal to the entire performance criteria, or 3 group sd. further, if the laboratory's internal cv is 50% of the stated performance limit, the probability of "failing" a single five sample pt event for one analyte drops just below 2%. with an internal cv of 33% or 113 of the pt limit, the laboratory will, in essence, always pass pt. the presence of co-existing bias reduces the "tolerable" cv, as will be shown in subsequent figures. 238 % chance of some pt failures, related to cv one 2 of 5 event, bias=o% of pt limit in oj ln 0 0 10 20 30 40 50 60 70 80 90 100 120 140 160 180 200 internal sd or cv as % of pt limit ficrure 1. intralaboratorv performance recruired to pass one pt event for multiple analvtes. zero bias. figure 1 with the rest of the curves shows the effect of analyzing multiple analytes (i.e., glucose, bun, cholesterol, etc.) on the laboratory's ability to pass pt. in general terms, for any given internal cv, the more analytes tested, the greater the probability that a laboratory will fail one or more analytes. for example, if a laboratory tests two analytes, and its internal cv is 100% of the pt performance criteria for both, the chance of failing at least one analyte increases from 51 to 76%. a laboratory doing 20 analytes, i.e., operating a large, multi channel instrument (dupont aca, tm hitachi , tm smac, tm etc. ) with all the analytes' cvs equal to 100% of the acceptable performance criteria, would virtually be assured of failing at least one analyte on every pt event. by reducing all of these internal c v s to 50%, the probability of a failure for the same 20 test laboratory is reduced to 32%. with all c v s below the 33% level, the chances of failure are nearly zero. obviously, a laboratory's c v s are not consistent across all parameters; some may be 239 considerably less than 3 3 % , and these tests would cause no problems in pt. however, even 2 analytes near 50% of the pt criteria would cause a 4 % probability of a failure, and 2 analytes at the 100% level would portend an 76% chance of a failure. the obvious conclusion is that a laboratory should strive to reduce all its tests' internal cvs to less than 3 3 % of the table 1 performance criteria; but in particular to con centrate on reducing the imprecision of any tests whose in tralaboratory cvs approach 100% of the pt criteria. % chance of some pt failures, related to cv one 2 of 5 event, bias=20% of pt limit 0 10 20 30 40 50 60 70 80 90 100 120 140 160 180 200 internal sd or cv as % o f pt limit fisure 2. intralaboratorv performance reauired to pass one pt event for figure 2 shows the effect of coexisting bias (20% of the pt criterion) for relevant levels of imprecision on the likelihood of a laboratory failing a single pt event. like figure 1, the family of curves represents respectively (from the right to left) the probabilities of failure for 1, 2, 5 , 10, 20 and 2 7 analytes. non-zero bias. 240 the presence of bias increases the likelihood that a laboratory will fail a pt event. in the case of glucose, where the pt criterion is +lo%, a 20% bias is equivalent to a consistent 2 mg/dl (0,11 mmol/l) error. comparing figures 1 through 3 indicates the effect of increasing bias on the likelihood of failing a pt event. for one analyte, with biases of o%, 20% and 50% and a consistent coexisting internal cv of 50% of the pt criterion, the probability of failure increases from 2 % to 4 % to 18%. further, for 20 analytes, the probability of a failure for biases of 0, 20 and 50%, increases from 32% to 51% to over 98%. while a laboratory does not have the same, or for that matter, any bias on every test, it is obvious that the presence of any significant bias seriously impairs the laboratory's ability to pass that analyte in a pt event. a common reason for analytical failures is the introduction of a large bias rather than large imprecision. due to a pipetting error or a reconstitution problem bias may extend to all analytes and impose a large chance of an 80% failure. consequently, to pass pt, a laboratory first needs to minimize the amount of bias, and then reduce its internal cv if possible. yo chance of some pt failures, related to cv one 2 of 5 event, bias=50% of pt limit 0 10 20 30 40 50 60 70 80 90 100 120 140 160 180 200 internal sd or cv as % of pt limit ficrure 3 . 16-908573 24 1 predicted failure rates as a function of cv and bias combina, tions. figure 4 shows the percent probability of failing a pt event for one analyte as a function of both internal cv and bias. the x and y axes depict intralaboratory cv and bias respectively as a percentage of the pt limit. the curves have a negative slope, since the presence of bias reduces the tltolerabletl internal cv consistent with a given percent probability of a laboratory failing a pt event. the rrlnt denotes a 1% probability of failing one pt event. as indicated by the continuous line, all com binations of cv and bias, falling on the line will yield a 1% chance of failure. those below (left) of the line have a lesser chance of failure. likewise, further to the right, subsequent curves denote the probabilities of failure of 3 , 5, etc., percent for increasing values of the cv and bias. typically laboratories using reasonable care in calibration have small biases. those laboratories whose bias exceeds 20% of the performance limit and whose cvs are in the range of 30 to 100% of the performance limit need to reduce both. 0 0 7 0 0) 0 03 0 r. i ._ ._ _e 8 c 0 o m a 5 0 a ._ z 0 i $ 0 i $ i 0 f) 7 0 ~~~~ le-05 0.005 0.1 % chance 1 of pt 3 failure 5 7 10 15 20 30 40 i i i i i i i i i i i i i i i i ( i i 242 minimizins the bias contribution to the probability of failure in txoficiencv testina. bias does not effect the likelihood of failure to nearly the same extent as cvs of equivalent size. for example if a laborato ry reduces its cv to 3 3 % of the value in table 1, for any given analyte, a coexisting bias of up to 40 percent is tolerable. if the bias can be reduced to any value below 20%, it's contribution to the probability of failure to pass pt is almost negligible. most authors do not deal with the concepts of coexisting bias and imprecision, but rather set bias equal to zero. this is a justifiable assumption at least when dealing with pt data. in point of fact, the traditional function of pt programs has been to reduce bias since even with five samples, pt is ineffective in measuring intralaboratory imprecision. conclusion a laboratory readily can predict the probability of passing pt based on a knowledge of its internal imprecision (cv) and bias. our overwhelming conclusion for the proposed 2 of 5 (or 80%) pt rules is that a laboratory with small (<20%) bias can reasonably assure its likelihood of passing pt if, for each analyte, it reduces the imprecision of each analyte to one-third of the federally mandated limit. this "rule of 1/3" when applied to the federal criteria in table 1, represents performance limits for us laboratories. since the ability to receive revenues for tests performed depends on passing proficiency testing, these limits, without respect to medical usefulness or significance, will become the goal of laboratory's quality assurance efforts. while one might dispute the logic of setting performance limits in this manner; there is a valuable lesson to be learned at the same time. external (or interlaboratory) quality assurance programs which closely mimic pt programs in concept and format, can translate analytical goals into performance limits. for example, if a decision could be reached that the total error requirement for glucose tests should be 3 . 3 mg/dl (0.18 mmol/l); a scheme of interlaboratory quality assurance testing could be computer modeled to determine the criteria (in the case of our 243 example +lo% as in table 1) to be used to assure successful , participants that they met the minimum internal performance requirement. the regulatory approach represents only one use of proficiency testing. properly managed within laboratories or by concerned professional groups, it can be a powerful quality assurance tool. as the us standards outlined in this report are implemented beginning january 1, 1991, we will have an excellent opportunity as scientists to evaluate one of the tools which can be used for managing (or leading) the process of searching for meaningful performance goals for our clinical laboratories. correspondence: professor ronald h. laessig, state laboratory of hygiene, university of wisconsin center for health sciences, madison, wisconsin, usa. 244 upsala j med sci 91: 209-213. 1986 round table discussion the discussion was chaired by carl-henric de verdier department of clinical chemistry, university of uppsala compiled by kristoffer hellsing and carl-henric de verdier ragnar stromberg (industry): instruments that should be simple and rapid to use are not simple and rapid to invent. the demands from the users will also be different, since they may have little experience o f laboratory work. the industry therefore has to build in a large amount of security and caluclating ability into the instruments. the methods have to be robust. the industry also has to equip the methods with some kind o f quality assurance system. three levels of decentralization can be recognized: 1. within the hospitals, to satellite laboratories 2 . to the primary health care, general practitioners etc 3 . to the patients themselves in the development of instruments and methods a close col laboration between the clinical chemists and the industry is necessary. this meeting is a good example of such a collabo ration. ulf nicolauson (national board of health and welfare): the board has several assignments. one is to print different types of regulations, rules, advice and information. another assign ment is to provide the hospital and health service administ rations of the counties with planning documentation. in the mid-seventies the board put out its document "public health and sick care at the eighties" (hs 80). three different functions were described, viz. primary medical care, county organized medical care and regional care. hs 80 provides the following rules: the primary medical care should obtain its laboratory service from the laboratory of the near by county hospital. simple tests demanding quick answers may be perfor med by the personnel of the primary care unit, but the metho 209 dology ought to be described by the laboratory staff. these types of advice are still valid although the board also has produced its next document "public health and sick care at the nineties" (hs 90). this document is less detailed in its structure. it states that the expansion of laboratory service according to hs 80 has been realized. the need for new technics and for capital investments is supposed to increase. it is, however, difficult to foresee the consequences of in creasing preventive actions within primary medical care. questions about liability g o back to the security of the patients, but it also includes the security o f the personnel working with the analytical systems near the patient. there must be long term systems for assessing analytical quality and f o r controlling the function of the equipment and the reagents used. the personnel should also have adequate instructions and training in the proper use of the systems for analysis and control. there are different models f o r organization in different areas and counties. the administrative and the medical respon sibility of the laboratory work performed in the primary care may be given to the director of the laboratory organization o r of the near by laboratory. according to another model the administrative responsibility may be given to the head of the primary care unit. a clinical chemist may then be consultant with a medical responsibility for the analytical systems. the administrative organization of the county should provide sche mes in order to clarify these responsibilities. during the last years no case has been brought up to the board about lia bility within the area of clinical chemistry. so the trial of such cases can not give us any guidance. sven lindstedt (clinical chemistry, goteborg): who is res ponsible for the analyses performed in the decentralized situation? we have witnessed several commersial laboratories growe up during the last years. if such laboratories produce values on which a wrong diagnosis is made, who has then the responsibility? one way to handle such a matter is to create laws, send out quality control samples, make an authorization of laboratories etc. in order to take questions like that under consideration a small group has been formed within the swedish society for clinical chemistry. one way of arguing within the group has been to state that we already have a pro fession: clinical chemistry. why not use that, to give the clinical chemist an authorization. with those thoughts in mind the group has taken contact with the national board of health and welfare. mogens horder (clinical chemistry, odense, denmark): cer tainly the speciality clinical chemistry exists. there has never, however, been stated that the subject should work only as a centralized subject. it is necessary that the clinical chemists know the demands put forward by the primary care. in denmark the primary care doctor is responsible for the labora tory activity. there is a discussion going on how much the clinical chemist form the near by laboratory shall be involved and responsible. goran sjonell (general medicine, stockholm): the young doc tor hides himself behind a lot of laboratory tests. it is necessary to learn him, how to work without all these tests or at least with a limited amount of tests. it is important for the industry to take part directly in what is going on in the primary care. it is also important that the clinical che mists and the primary care doctors start a conversation with the patients on the various analyses performed. most people are very interested in those matters. it is not surprising at all that some people send their hair to england for analyses of trace metals. ulf nicolausen: it is also important for the clinical che mists to get a broader view on the situation for the patient, to put the results together with the anamnesis. the quality of the health and sick care organisation shall be the same on all levels. we shall not discuss in terms of low and high technology on different levels in the health care system. simple tests giving a rapid answers demand a very high tech 21 1 nological development. above all, the activity shall not be ruled by the development, but by the demands. sven lindstedt: we ought to know if the decentralisation process is beneficial for the patient. there were no differen ces in measurable parameters like urinary albumin and ophtal miatric status in a follow up study on diabetic patients hand led with home testing or laboratory testing. nils tryding (clinical chemistry, kristianstad): we must know that the indications for the analysis are correct and that the results are interpreted correctly. if the receiver of a laboratory result does not know the physiological and biochemical mechanisms behind the turnover of a substance, then the analysis is of little value. i think that we shall devote ourselves to what we are educated for, viz. helping our clinical collegues to make clinical chemistry data available. mogens horder: we shall develop clinical chemistry in order to answer the questions put in the primary care. the develop ment has to be done in various regions. there are t o o big dif ferences between e g clinical chemistry in the various count ries in scandinavia. it is important that we put the patient in the center, research within primary care has to get higher priority. agneta haggmark (clinical chemistry, stockholm): education o f the medical students is also important. we are trying hard to teach them to request analyses only when needed. if there is an overconsumption of analyses, there must be imperfections in the education given by other specialities. goran sjonell: i agree. the main responsibility is on the clinically working collegues and not on the subject clinical chemistry. by the way, there is a revolution going on wit hin general medicine. earlier we were given two whole days o f the 6.5 year long study to educate the student. this has now been expanded to two weeks. there are countries where the main 212 part of the education is located outside the hospitals. olof hygstedt (clinical chemistry, borbs): the analysis clo se to the patient is as i see it from a non-fasting speci men. it is very difficult to talk in terms of decentralisa tion, if we do not have reasonable good reference intervals for non-fasting, healthy individuals. nils tryding: according to my view there is no use of refe rence intervals, except if you want to perform a screening investigation on a population. f o r a diseased state other intervals can be used. decision limits are not the same as reference limits. ulf nicolausen: in the ending of this discussion i want to give you some words to take with you home. i should like you to get into closer contact with the primary care and start collaboration and cooperation. it is important that we get rid of the old way of thinking, that the clincial chemists mainly are working for the wards of the hospitals and mainly with sophisticated analyses. we must also work in the direction to raise the quality of the service to primary care and to give support to the people in the developmental work for primary care. carl-henric de verdier: before ending the discussion i like to express the interest from clinical chemistry to collaborate with general medicine. we already have committees working wit hin this area and i also see this meeting and today's discus sion as a good example of this kind of collaboration. 14-868572 213 upsala j med sci 91: 45-52, 1986 effect of n-acetylcysteine on fibrin deposition in the rat lung due to intravascular coagulation thomas wegener,1.2. rolf wallin' and tom saldeen' de artments of 'forensic medicine, 'lung medicine, and 'clinical physiology, university of uppsala, sweden abstract intravascular coagulation was induced in rats by i.p. injection of a fibrinolysis inhibitor, tranexamic acid (amca, 200 mg/kg b.w.), and i.v. injection of bovine thrombin (500 nih unitslkg b.w.) and the fibrin deposition in the lungs was assessed with i-labelled fibrinogen. 125 treatment with n-acetylcysteine (nac) partly prevented the deposition of fibrin in the lungs, and the disappearance of fibrinogen from the blood, but did not seem to influence the elimination of fibrin in the lungs. the results indicate that nac may counteract pulmonary damage in this experimental model, by inhibiting intravascular fibrin formation. introduction n-acetylcysteine (nac) has been shown to have preventive or therapeutic effects on two types of experimental pulmonary damage ( 3 , 1 3 ) . bernard et a1 (3) claimed that nac, as a free radical scavenger, has a favourable effect in endotoxin-induced ards (adult respiratory distress syndrome), in sheep. wegener et a1 ( 1 3 ) found that in another ards model in the rat, nac counteracted the pulmonary damage by diminishing the increase in lung weight and reducing of the microscopically observed interstitial and alveolar oedema. in addition, after administration of nac less fibrin was found in precapillary arterioles by a semiquantitative method , suggesting that nac may decrease the formation of fibrin or increase its elimination from the lungs. 45 the aim of this study was t o quantify the fibrin deposition in the rats with pulmonary damage treated and not treated with nac, using a method employing i-labelled fibrinogen (lo), and to determine i whether nac could diminish the trapping of fibrin in the lungs or increase its elimination. 125 material and methods animals. male sprague-dawley rats (alab, sollentuna, sweden), weighing 195 to 210 g, were used. they were allowed free access to food and tap water. substance. human fibrinogen kabi (grade l) and tranexamic acid (trans-4-aminomethyl-cyclohexane-carboxylic acid, abbreviated to amca were kindly supplied by kabi-vitrum ab, stockholm, sweden). r bovine thrombin (topostasine , hoffman-la roche, switzerland) was used. r n-acetylcysteine (fluimucil , 100mg/ml aqueous solution ph 6.5, zambon spa, milano-vincenza, italy) was generously supplied by the producing company. r pentobarbital (inactin , byk-gulden, konstanz, frg) was used for anaesthesia. 125 human fibrinogen was labelled with i, using the iodine monochloride method ( 6 ) . as human fibrinogen has been shown to give the same result as rat fibrinogen in the present context, and has greater stability (2), it was chosen for these experiments. procedure. labelled fibrinogen solution (2.5 mlfkg body weight b.w.) containing about 2.5 mg of protein and 518 kbq/ml was injected into a tail vein in the rat 24 h before the experiment took place. this procedure was carried out under light ether anaesthesia. about one hour before the experiment began, 0.5 g of inactin was dissolved in 10 ml of sodiumchloride (9 mgfml, isotonic solution) and 2.5 ml/kg (b.w.) was injected intraperitoneally (i.p.). sodiumchloride was abbreviated to saline. to ensure a free airway, all animals were tracheostomized. during the experiment the body temperature was kept constant at 38 c with an infrared lamp. pulmonary damage was induced by an i.p. injection of amca, 200 mglkg b.w., followed 10 min later by a lo-minute infusion of bovine thrombin, 500 nih unitslkg b.w., given into a tail vein by means of an infusion pump. thrombin administration must be combined with amca in order to cause pulmonary damage similar to that seen in patients with posttraumatic pulmonary insufficiency ( 1 , 6 , 7 ) . nac 0 46 was administered i.v. twice in the first experiment and once in the second experiment in a dose of 125 mg/kg b.w. the first experiment comprised 15 rats, divided into three groups. 1. labelled fibrinogen + thrombin + amca + nac (n=6) 2. labelled fibrinogen + thrombin + amca + saline ( s ) (n=6) 3. labelled fibrinogen + saline (n=3). saline served as a control fluid to give equal injection volumes. the experiment was carried out as described in table 1. experimental mod& table 1 h -24 0’ 5‘ 15’ 25‘ 45‘ 105’ 125 1. i-fibrinogen nac amca thrombin nac killed 125 2. i-fibrinogen s amca thrombin 5 killed killed 125 3 . i-fibrinogen s s s 5 at 105 min the animals were killed by aortic exsanguination. the second experiment comprised 16 rats, divided into the same three groups as described above; group 1 (n=7) group 2 (n=5) and group 3 (n=4). experimental model. table 2. h -24 0’ 5’ 15‘ 25’ 30’ 1 . lz5i-f ibrinogen nac amca thrombin killed 2. lz5i-f ibrinogen s amca thrombin killed i-fibrinogen s s 5 killed 125 3. at 30 rnin the animals were killed by aortic exsanguination. 3 < blood was collected in plastic tubes containing either citrate or edta buffer. the lungs were quickly removed, perfused with isotonic saline solution, dissected free of connective tissue, cleaned with filter paper 41 and placed in weighed plastic tubes. the radioactivity of the left lung was determined. in the first experiment the right lung from each animal was homogenized. the homogenization procedure was made according to a method described previously by busch et al(5) to determine how much of the labelled substance was precipitated (fibrin). analysis. the fibrinogen concentration in citrate plasma was determined by the method of nilsson and olow ( 9 ) . the fibrinogen values were corrected for the influence of the erythrocyte volume fraction (evf) upon the citrate dilution of plasma. evf: aortic blood was drawn into edta tubes, and evf determinations were made in triplicate in micro-evf tubes after centrifugation at 10 000 x g for 5 min. radioactivity: the radioactivity in weighed samples of tissue, blood and labelled fibrinogen was measured in the plastic tubes using a (edta) gamma spectrometer. calculations. the fibrin content in the lungs was calculated from a formula presented previously by busch et a1 ( 4 ) and modified by diffang et a1 ( 6 ) . in short, f = i / q ( tex organ (mg/g); q is tie fa:tor for converting i radioactivity to mg fibrin, consisting of the mean relative specific radioactivity (cpm x 10 /g) of plasma fibrinogen in control rats; t is the total t ) where f is the amount of fibrin in the 125 3 125 exp i radioactivity in the tissue specimen in the experimental rat (cpm 3 125 x 10 /g); t is the mean i radioactivity in the tissue specimen in control rats (cpm x 10 /g) = (plasma + extravascular radioactivity). c 3 statistical methods. conventional methods as described by snedecor (12) were used. differences between the groups were tested by student’s t-test and to confirm significance at the 5% level also by wilcoxon-white’s two-sample ranks test. the results are given as mean and s.d. and degrees of significance are indicated as follows: *= ~(0.05, **= p<o.o1, ***= p<o.ool. results the amount of fibrin in the whole lung or per g lung tissue was significantly lower (p<o.ool) in both experiments of rats with pulmonary damage that had received nac-treatment than in such rats not treated with nag. the same results were obtained for homogenized lungs in the first 48 experiment although the mean difference was somewhat smaller (table 3 and 4 ) the fibrinogen level in non-nac-treated animals with pulmonary damage was significantly lower (table 3 and 4 ) than in those with pulmonary damage, that were treated with nac. table 3. experiment 1 erythrocyte volume fraction (evf) and blood and lung fibrinogen 1251 radioactivities and fibrin content in the lung (mean values and sd). groups 1 (n=6) 2 (n=6) 3 (n=3) evf 40 5 40 2 36 2 blood cpm x 10 / g whole lung cpm x 1o3/g 17.6 8.1 7.4 2.8 35.2 6.0 22.6 12.7*** 77.4 !' 11.7 9.7 ! 1.4 homogenized lung, 18.4 11.7*** 54.2 10.1 7.3 2.0 cpm x 103/g whole lung fibrin, 0.5 0.5*** 3.0 0.6 0 mg/g lung tissue homogenized lung 0.5 0.5*** 2.1 : 0.5 0 fibrin, mg/g lung tissue fibrinogen in blood, 1.60 : 0.49** 0.55 0.38 2.64 0.12 g/l level of significance: * ~(0.05, * * p<o.oi, * * * p<o.ool table 4. experiment 2 fibrinogen and fibrin content in the lung (mean values and sd) . groups 1 (n=7) 2 (n=5) 3 (n=4) whole lung fibrin mg/g lung tissue 3.09 0.34*** 5.34 f 0.69 0 fibrinogen in blood g/l 1.07 0.22* 0.43 0.69 2.55 0.15 (n=3) level of significance: * p<o.o5, * * p<o.ol, * * * p<o.001 4-868571 49 fig. 1 shows that there is no difference in the elimination rate of fibrin between animals treated or untreated with nac. 6 4 2 fibrin (mg/g) in whole lung 1 * * * 1 0 5 1 0 50 8 0 thrombin infusion time after end of thrombin infusion (min) fig. 1. elimination rate of fibrin. isotonic sodiumchloride + amca + thrombin --n-acetylcysteine + amca + thrombin level of significance * * * p<o.ool discussion in a previous study, wegener et a1 (13) found that nac partly inhibited ards caused by intravascular coagulation, possibly by its scavenging effect and/or, (in this particular ards model), by influencing the fibrin deposition in the lung. a reduction in the deposition of fibrin could have been due to decreased fibrinogen consumption, to increased fibrin elimination or could be secondary to the scavenging effect. our results indicate that nac counteracts fibrin deposition in the lung directly, since the fibrinogen level in the blood in nac-treated animals is higher than in animals not given this treatment and the amount of fibrin in the lungs shortly after thrombin infusion was much less in nac-treated rats. it may therefore be justified to suggest that nac inhibits fibrin formation in the blood. 50 the very rapid effect on fibrin formation in the lungs after the administration of thrombin indicates that the effect of nac on fibrin formation is not secondary to its scravenging effect but more probably due to a direct anticoagulant effect. as the elimination curves for lung fibrin did not differ between rats treated and not-treated with nac (fig. l ) , this substance probably does not influence the removal of fibrin from the lungs. the findings of sim et a1 ( 1 1 ) that a relatively high concentration of nac showed no fibrinolytic activity when added to fresh human fibrin plates supports our opinion that nac does not excert its effect by increasing fibrin elimination. sim et a1 (11) also found nac to be an active inhibitor of thrombus formation in the microcirculation of the hamster cheek pouch. acknowledgements this work was supported by the swedish national association against heart and lung diseases, the swedish medical research council, and ciba-geigy, sweden. references 1. arfors, k.e., busch, c., jakobsson, s., lindquist, o., malmberg, p., rammer, l. & saldeen, t.: pulmonary insufficiency following intravenous infusion of thrombin and amca (tranexamic acid) in the dog. acta chir. scand. 138:445-452, 1972. 2. bagge, l., busch, c., rammer, l. & saldeen, t.: delayed fibrin elimination from the lungs of burned rats with endogenous inhibition of the fibrinolytic system. thrombosis research 2:365-376, 1973. 3. bernard, g.r., lucht, w.d., niedermeyer, m.e., snapper, j . r . , ogletree, m.l. 6 brigham, k.l.: effect of n-acetylcysteine on the pulmonary response to endotoxin in the awake sheep and upon in vitro granulocyte function. j. clin. invest. 73:1772-1784, 1984. 4. busch, c., rammer, l. & saldeen, t.: quantitation of fibrin deposition and elimination in organs of rats injected with labelled fibrinogen and albumin. thromb. diat. haemorrh. 29:94-107, 1973. 5. busch, c. & rammer, l.: quantitation of fibrin deposition and elimination in organs of rats injected with labelled fibrinogen by isolation of the labelled fibrin from water soluble tracer. thromb diat haemorrh 29, 108 1 1 4 , 1973. 6. diffang, c. & saldeen, t.: effect of brinase (protease i from aspergillus oryzae) on the elimination of fibrin from the lungs and pulmonary damage in rats with induced intravascular coagulation and inhibited fibrinolysis. thrombosis research 9: 611-622, 1976. 51 7. gerdin, b., diffang, c. & saldeen, t.: pulmonary insufficiency in the rat after intravascular coagulation and inhibition of fibrinolysis. eur. surg. res. 13:402-413, 1981. 8. helmkamp, r.w., contreras, m.a. & bale, u.f. :1311-labelling of proteins 9. nilsson, i.-m. & olow, b.: determination of fibrinogen and fibrinolytic activity. thromb. diat. haemorrh. (stuttg.) 8:297, 1962. 10. saldeen, t.: quantitative determination of intravascular coagulation in the lungs of experimental animals. scand j haemat 6: 205-215, 1969. 11. sim, a.k., mccraw, a.p. & davies, m . e . : an evaluation of the anti thrombotic potential of three synthetic compounds. (personal communication of data file by zambon s.p.a., bresso-milan, italy, 1976. 12. snedecor, w.: "statistical methods". the iowa state university press, 1965. 13. wegener, t., sandhagen, b. & saldeen, t.: effect of n-acetylcysteine on pulmonary damage due to microembolism in the rat. eur j resp dis. submitted. address for reprints: thomas wegener department of lung medicine uppsala university akademiska sjukhuset s-751 85 uppsala, sweden 52 upsala j med sci 93: 63-69, 1988 discrepances between ct and eeg findings after acute cerebrovascular disease martin knibestol, erik hagg and bengt liliequist departments of clinical neurophysiology, internal medicine and neuroradiology, university hospital, u m e d , sweden abstract combined eeg and brain ct examinations were performed in 33 patients during the 1st and 2nd week after stroke. ct was abnormal in 17 patients (51%) and eeg was abnormal in 24 patients (72%). in 17 patients ct and eeg showed conflicting results: in 5 patients with normal eeg findings ct was pathological, and 12 patients had normal ct but pathological eeg findings. in this latter group, there were 5 particularly interesting cases with normal ct and a prominent unilateral eeg abnormality. recently patients with this combination of findings have been described where further investigations disclosed internal carotid occlusion, which could be treated surgically. it is suggested that eeg should be more extensively used when ct findings are negative after stroke, and if a major unilateral eeg abnormality is encountered in such cases, further investigations with angiography should be considered in order to exclude surgically treatable internal carotid occlusion. introduction in some recent studies (3,9,10, 11, 12) the relative usefulness of computerized tomography (ct) and electroencephalography (eeg) in investigation of patients with acute cerebrovascular disease (cvd) has been discussed. there is general agreement that both methods are useful and may complement each other: ct gives information on the extent and localization of a lesion and can differentiate between cerebral infarction and hemorrhage, whereas eeg reflects mainly the functional state of the brain and correlates better with metabolic and circulatory function. ct is superior for diagnostic purposes, whereas eeg may have greater value for prognostic information concerning survival and neurological recovery (6). it is important to point out, however, that both ct and eeg can be normal in a substantial number of cvd cases, depending on such factors as size and localization of a lesion and its nature (tia, infarction or hemorrhage). furthermore, ct and eeg may not necessarily both be pathological in the same patient. recently yanagihara et al. (15) described in detail three interesting cases with a prominent unilateral eeg abnormality but normal ct findings, and on angiography these patients proved to have occlusion of the carotid system which could be treated surgically with subsequent clinical improvement and normalization of eeg. they noticed that the frequency with which 63 discrepancy between ct and eeg occurs is not known, but suggested that the number could be considerable, and they emphasized that recognition of this kind of discrepancy may help to establish the diagnosis by directing attention to appropriate neurovascular evaluation and treatment. in the present study we performed combined ct and eeg examinations in a series of cvd patients, with particular attention focused on the frequency of major discrepancies between the two methods, including the type described by yanagihara et al. (15). material and methods the clinical material consisted of 33 consecutive patients aged 45-87 years (mean 64), admitted to the stroke unit at the medical clinic, umea university hospital, during a six month period for acute cvd. the patients were included in the study irrespective of the initial severity or course of the disease. four patients were classified as having tia, whereas 29 patients developed completed stroke with neurological deficits of more than 24 hours' duration. of the latter, a definite diagnosis of cerebral infarction or hemorrhage could be established (by typical ct findings) in 11 respectively 3 patients, whereas the remaining 15 were considered to have an infarction (symptom duration more than 24 hours, lack of bleeding on ct and csf spectrophotometry). every patient in the stroke unit was subjected to a standardized scheme of investigation. repeated clinical assessments were performed at admission, after one and four days and at discharge. all data were registered in a protocol for computer use. the patients usually kept their previous medication (mostly antihypertensive drugs, digitalis and oral hypoglycemic drugs) and no special medication was given for their stroke. of clinical data, only the state of consciousness and side and type of hemisymptoms were used in the present study. eeg was recorded on a 16-channel eeg mingograph, using both unipolar and bipolar derivations including longitudinal and transversal montages. at least one ct and eeg examination was performed in 30 and 24 of the patients respectively within four days after admission and in all patients within eight days. an.additional ct and eeg examination was performed in 24 and 28 patients respectively on day 4-1 1 after admission. ct examination was performed with an em1 mark 1 head scanner with a 160 x 160 matrix. an eeg abnormality, when encountered, could usually be classified as either u = unilateral, polymorphic theta-delta activity, or b = bilateral, mostly bursts of bilateral delta rhythms, commonly known as frontal intermittent rhythmic delta activity (firda). the degree or severity of an eeg abnormality was further scaled according to a semiquantitative 5-grade scale, mostly based on the spatial extent of an abnormality, but to some extent also including temporal characteristics (short episodes versus continuous activity) and frequency aspects (content of theta or delta rhythms). an approximate characterization of the 5 grades of eeg abnormality is as follows: grade 0 = normal eeg grade 1 = slight abnormality typically a few episodes of short duration, mainly theta-rhythms, 64 restricted to one lobe grade 2 = moderate abnormality theta-delta activity covering about one half hemisphere, appearing continuously or as frequent episodes of moderate to long duration grade 3 = severe abnormality continuous mainly delta activity covering a whole hemisphere, or frequent bilateral delta episodes occupying nearly 50% of the recordering grade 4 = very severe abnormality continuous delta activity over both hemispheres results the results of the study are summarized in table 1, where the principal clinical features (state of consciousness and hemisymptoms) as well as ct and eeg findings are presented. the clinical findings concerning hemiparesis and the degree of consciousness and orientation were the same during the first four days in all patients except for patient 061 (an initial dysphasia and paresis of the right arm had disappeared after four days) and 120 (disorientation on day 1 , then fully orientated). the data in the table are based on the first ct and eeg examination during the first week after admission, except for one case (case 119) where ct was negative on day 0 and positive on day 5. in the table the patients are arranged in groups of increasing grade of eeg abnormality. eeg was pathological in 24 patients (72,7%) and ct was positive in 17 patients (51 ,5%). ct and eeg were both negative in only four patients (12,1%), implying that the addition of eeg to ct examination increased the incidence of positive findings from 513 to 87,9 yo. of the 12 ct negative cases with pathological eeg, the eeg abnormality was only slight in 3 cases, and in 9 cases of moderate to very severe degree. of the latter, 5 cases had unilateral and 4 cases had bilateral eeg-abnormality. cases there was good agreement between clinical findings and ct and/or eeg findings as far as laterality of the lesion was concerned. of the 22 patients with hemisymptoms and pathological eeg, 14 had unilateral eeg abnormality in agreement with clinical findings, whereas 8 patients had bilateral eeg abnormality, which could give no objective support in lateralizing a lesion. of the 17 patients with hemisymptoms and positive ct, there were 3 patients where the ct lesion was found in the "wrong" hemisphere: however, in these cases the ct findings were classified as an old infarction probably not related to the present illness (two of these were tia cases, one case probable infarction). it can be inferred from table 1 that there was a correlation between the degree of eeg abnormality and the state of consciousness of the patient: there is an increasing incidence of impairment of consciousness with increasing eeg grade. (spearman's rank correlation coefficient = 0.62, p c 0.001). in most patients the impairment of consciousness was of only slight to moderate degree: only one patient was comatose, and had also a grade 4 eeg abnormality. of the 30 patients with hemisymptoms, 27 had pathological ct and/or eeg, and in most of these 5 88857 1 65 table 1. main clinical findings and results of ct and eeg examinations in 33 patients after stroke s efg findinas case type of impairment of hemipositive or side side type no. lesion consciousness symptoms negative sbx!!al 032 069 099 1 24 066 090 114 001 065 pi tia pi pi i i pi tia i 0 0 0 0 r 0 r 0 r 0 r 0 l 0 054 pi l b msu 115 pi + r l u 042 pi r l u 01 2 i r + l b 061 i r + l l u b gm!gi 009 pi 059 pi r b 004 h + r + l l u 079 i l + r r u i2cacu 052 063 098 112 120 136 002 040 038 067 087 113 119 pi pi pi pi pi tia i i tia h h i i + r r l r r l r r r r r l l l l l l r r l r l l l l l l r r u u b u u u u b b u u u u i&xka 053 pi + r b 003 i + r + l b abbreviations: clinical: i = infarction, h = hemorrhage, pi = probable infarction, tia = transitory ischemic attack, r = right side of body, l = left side of body, m: r = right hemisphere, l = left hemisphere, eeg: r = right hemisphere, l = left hemisphere, u = unilateral abnormality, b = bilateral abnormality. the tia patients reported hemisymptoms anamnestically but did not disclose any hemisigns at examination 66 discussion the frequency of positive ct and eeg findings reported after stroke have been highly varying mainly depending on the composition of the clinical materials. ct has been reported positive in 0-1 8 % in tia, 48-98 % in infarction and 77-100 yo in hemorrhage (1, 7,8,12,14), and correspondingly for eeg 66-86 yo, 77-90 % and 100% (9, 10, 11, 12). in our material with a majority of infarction cases the incidence of positive findings was 72,7% for eeg and 51,5% for ct, or as low as 42% for ct if the three cases with old infarctions are disregarded. the frequency of negative ct is particularly high in tia cases, and is generally higher in the first week than in the second week after stroke, particularly during the first 48 hours after onset (2,7,8,11). in our series two tia cases with negative ct contributed to a low figure, but the time of examination probably played no major role, since most patients also had a ct examination during the second week. there was disagreement between ct and eeg findings in 17 patients, i.e. as high perecentage as 51. of these the 5 cases with positive ct and negative eeg findings are of limited interest in the present context: two of these cases had an old infarction, and furthermore, a normal eeg is often reported in deep lesions (3,12). in three of the 12 patients with negative ct and positive eeg findings the eeg abnormality was only slight (grade 1) and of questionable significance in relation to the present disease, since minor episodes of slow wave activity particularly in temporal areas are not uncommon even in the normal population, especially in elderly individuals (5). the remained 9 patients with normal ct and eeg abnormality of moderate to very severe degree, which no doubt indicated substantial interference with cerebral blood flow, related to the clinical symptoms of acute cvd. however, we will not further discuss the 4 cases with bilateral eeg abnormality, since in these cases it is difficult to make precise statements about the localization and extent of circulatory disturbances. the firda pattern has traditionally been regarded by electroencephalogaphers as generated from deep midline structures, but recent studies have shown that it may also appear in diffuse encephalopathies or focal hemispheric lesions (4, 13). the remaining 5 patients had normal ct and a prominent unilateral eeg abnormality, i.e. a combination of findings identical to those of the three cases described by yanagihara et at. (15). thus our findings in a fairly small series of patients support the assumption by these authors that this type of discrepancy between ct and eeg findings after stroke may not be uncommon. correct recognition of this category of patients is important, since at least some of them may have occlusion of the internal carotid arteries which can be successfully treated by surgical intervention (1 5). eeg is in general probably not part of routine investigations in patients after stroke, but the present findings together with those of yanagihara et at. (15) suggest that it should be more extensively used, particularly in patients with normal ct examination. if the eeg examination in such cases shows a prominent unilateral abnormality, further investigations with cerebral angiography should be considered. 67 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. references campbell, j.k., houser, o.w., stevens, j.c., wahner, h.w., baker, h.l. & folger, w.n.: computed tomography and radionuclide imaging in the evaluation of ischemic stroke. radiology 126: 695-702, 1978. davis, k.r., taveras, j.m., new, p.f.j., schnur, j.a. & robertson, g.h.: cerebral infarction diagnosis by computerized tomography. am j roentgenol 124: 643-660, 1975. van der drift, j.h.a., visser, s.l., jonkman, e.j. & v.d. steen, a.: correlations and discrepancies between clinical aspects, eeg and ct brainscan data in ischaemic cerebral disease. eeg and clinical neurophysiology (eds. h. lechner & a. aranibar), pp. 163-172. excerpta medica, amsterdam, 1980. fariello, r.g., orrison, w., blanco, g. & reyes, p.: neuroradiological correlates of frontally predominant intermittent rhythmic delta activity (firda). electroenceph & clin neurophysiol54: 194-202, 1982. hughes, j.r. & cayaffa, j.j.: the eeg in patients at different ages without organic cerebral disease. electroenceph & clin neurophysiol42: 776-784, 1977. kayser-gatchalian, m.c. & neundorfer, b.: the prognostic value of eeg in ischemic cerebral insults. electroenceph & clin neurophysiol49: 608-61 7, 1980. kinkel, w.r. & jacobs, l.: computerized axial transverse tomography in cerebrovascular disease. neurology 26: 924-930, 1976. ladurner, g., sager, w.d., iliff, l.d. & lechner, h.: a correlation of clinical findings and ct in ischaemic cerebrovascular disease. eur neurol 18: 281 -288,1979. logar, c., martischnig, r., enge, s., sager, w.d. & ladurner, g.: zur wertigkeit von eeg and computertomographie bei lschamischer insulten. 2. eeg-emg 10: 161 -1 66,1979. moglia, a., tartara, a., mola, m., manni, r., rognoge, f. & martelli, a.: value of eeg and computerized tomography in ischemic cerebrovascular disorders. eeg and clinical neurophysiology (ed. h. lechner & a. aranibar), pp. 226-230. excerpta medica, amsterdam, 1980. ott, e., lechner, h., marguc, k., bertha, g., aranibar, a., ladurner & sager, w.d.: correlation of eeg and ct findings with cerebral blood flow measurements in patients with cerebrovascular disorders. eeg and clinical neurophysiology (eds. h. lechner & a. aranibar), pp. 143-147. excerpta medica, amsterdam, 1980. ottonello, g.a., regesta, g. & tanganelli, p.: correlation between computerized tomography and eeg findings in acute cerebrovascular disorders. eeg and clinical neurophysiology (eds. h. lechner & a. aranibar), pp. 148-162. excerpta medica, amsterdam, 1980. schaul, n., lueders, h. 8 sachdev, k.: generalized, bilaterally synchronous bursts of slow waves in the eeg. arch neurol38: 690-692,1981. 68 14. sb&rstrbm, c.e.: diagnostic significance of csf spectrophotometry and computer tomography in cerebrovascular disease. a comparative study of 231 cases. stroke 8: 606-61 2,1977. yanagihara, t., houser, o.w. & klass, d.w.: computer tomography and eeg in cerebrovascular disease. arch neurol38: 597-600, 1981. 15. address for reprints: martin knibestal, m.d. department of clinical neurophysiology university hospital sweden 901 85 umea 69 upsala j med sci 99: 347-356, 1994 7.4 comparison between finnish and danish reference values for plasma proteins kerttu irjalal, irma matinlauril, kari mattilal, ole blaabjerg2, mogens blom3, per hyltoft petersen2, jens rahbek n0rgaard2, hanne g r 9 , adam uldall5, inger n0rgaard3 1. department of clinical chemistry, t u r k u university hospital, sf-20520 turku, finland. 2. department of clinical chemistry, odense university hospital, dk-5000 odense c, denmark. 3. department of clinical chemistry, hjorring sygehus, dk-9800 hjprring, denmark. 4. medi-lab, adelgade 5-7, dk-1304 copenhagen, denmark. 5. department of clinical chemistry, kas herlev, dk-2730 herlev, denmark. it is well known that genetic mutations may result in varying concentrations of plasma/serum proteins. further, hormone concentrations and environmental conditions may influence on the plasma protein concentrations. differences in plasma protein concentrations due to ethnic groups, and geography etc. in the nordic countries may be investigated by comparing finnish and danish reference populations with each other. both are of caucasian origin but, from different groups of language and whereas the finnish climate is belonging to the inland type the danish climate is typical coastal. the populations in the other nordic countries, iceland, norway, and sweden, are mainly of the same ethnic group as the danish, but the climate resembles that of finland. the most pronounced differences might, therefore, be disclosed by comparing a danish population with a finnish one. selection of reference individuals ideal, would have been to make a study with the same design in finland, and compare it with the danish study (cf. sections 7.1, 7.2, and 7.3). it would, however, have been very expensive and could be viewed as too much work, considering the uncertainty of the outcome. it was decided t o carry out the investigation with a smaller but well defined group of the finnish population comparable to a subsample of the danish reference group. by investigation of these selected groups major differences were thought to be easily disclosed and even minor variations might be detected. from the turku region in finland serum samples from 29 men and 27 women (not using estrogens) all of the age between 30 and 40 years, and from the danish materials 24 samples from men and 22 samples from women (not using estrogens) in the same age group were available with sufficient serum for the comparison. 347 rule in and rule out criteria for the reference individuals were the same as for the danish investigation and the sampling conditions as well as the preparation of serum samples were identical (cf. section 7.2). analytical design in order to minimize analytical influence on the results of the investigation the samples were measured by the same analytical methods by the same technician in a random set up, where serum samples from finnish and danish subjects were measured at random in the same analytical runs in double determinations performed in separate runs. presentation of data the results are displayed on probit-plots in relation to the relevant distributions from the danish investigation (cf. sections 7.2 and 7.3). where no differences between the two sexes were found the results were combined, otherwise they were separated and the protein distributions are shown for both the sexes from each country. results s-prealbumin (s-transthyretin) cumulated percentage frequency woeit (women below 50 -e) (others) # men women rms ,= 0 danes o l ~ ~ ~ ' l . ~ ~ ' l ' ~ ' ~ l . . ' ~ l i . ' 1 ' i ' . ' . " . ' ' i -0.50 -0.40 -0.30 log g/l -0.70 -0.60 0.2 0.3 0.4 0.5 gll fig. 7.4.1. distributions of s-prealbumin values for the finnish and danish reference individuals with separate curves for the two sexes. the two straight lines indicate the distributions as estimated in the total danish material. 348 concerning s-prealbumin the finnish and the danish distributions showed differences in concentrations for men and women (below 50 years of age and not using estrogen), and the separate distributions are illustrated in fig. 7.4.1. the finnish and the danish distributions are indistinguishable and close to the distributions from the total danish material. therefore, it was concluded that no possible difference was detectable, and the finnish reference interval should be identical to the danish reference interval for s-prealbumin. s-albumin according to the danish investigation no difference due to sex in the relevant age groups were disclosed and the sex-groups are therefore combined in fig 7.4.2. salbumin cumulated percentage frequency probit l ~ l ' 1 ~ ~ ' t ~ l ~ l ' l ' l ' l 1.54 1.58 1.62 1.66 1.70 log g/l fig. 7.4.2. distributions of s-albumin values for the finnish and danish reference individuals. the straight line indicates the distribution as estimated in the total danish material. the distributions for the finnish and the danish groups are indistinguishable but, both with an tendency to slightly lower values. there is, however, no reason for defining another reference interval. 349 s-orosomucoid (s-a,-acid glycoprotein) concerning s-orosomucoid the finnish and the danish investigation showed differences in concentrations for men and women, and the separate distributions are illustrated in fig. 7.4.3. s-orosornucoid cumulated percentage frequency probit women below 50 -e danes 0 women 0 0 others lipper 0.90 ci i i i i i i i 1 -0.5 -0.4 -0.3 -0.2 -0.1 0.0 0.1 log g/l i i i 1 1 1 . 1 1 1 0.3 0.4 0.5 0.6 0.8 1.0 1.2 gll fig. 7.4.3. distributions of s-orosomucoid values for the finnish and danish reference individuals with separate curves for the two sexes. the two straight lines indicate the distributions as estimated in the total danish material. the distributions of s-orosomucoid for finnish and danish women are indistinguishable whereas, the curves for men are different. however, it is the danish subgroup that differs from the danish main group (indicated by the upper 0.90 confidence interval for 23 degrees of freedom), whereas, the finnish values are distributed accordmg to the danish main group. in consequence, there is no reason for choosing a separate reference interval. s-al-antitrypsin ( s-ccl-proteinase inhibitor) according to the danish investigation no differences due t o sex for the relevant age groups were disclosed and the sex-groups are therefore combined in fig 7.4.4. the distributions for the finnish and the danish groups are indistinguishable but, both with a tendency to slightly lower values, there is, however, no reason for defining another reference interval. 3 50 s-nl -antitrypsin 99 95 90 eo 70 60 50 40 30 20 10 5 1 cumulated percentage frequency probll i 1 ' 1 ' 1 ' 1 1 ' 1 . 1 1 i i i i i i i i i ' i ' i i -0.16 -0.08 000 0.08 0.16 0.24 0.32 log g/l 0.7 0 8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.8 2.0 2.2 g/l fig. 7.4.4. distributions of s-a,-antitrypsin values for the finnish and danish reference individuals. the straight line indicates the distribution a s estimated in the total danish material. s-haptoglobin s-haptoglobin is dfferent from the previously described proteins as the distribution are not log-gaussian, so the comparison is more difficult than for the other proteins. s-haptoglobin cumulated percentage frequency probit finns rn danes 0 8 l ' i ' i . i ' i 0.0 0.5 1.0 1.5 2.0 g/l fig. 7.4.5. distributions of s-haptoglobin values for the finnish and danish reference individuals. the curve indicates the distribution as estimated in the total danish material. 351 the distributions of the finnish and danish reference groups are illustrated in fig. 7.4.5 and compared with the curve for the total danish distribution of s-haptoglobin values. the finnish distribution is located with lower values than both the comparable subgroup and the total danish distribution, but as this distribution is not parametric there is no simple way to illustrate the confidence intervals for the percentiles, which should be chosen non-parametric. the difference is about 0.15 g/l along the whole distribution, and we have decided to define the reference intervals for the finnish population accordingly with limits 0.15 g/l below the danish values. s-transferrin according to the danish investigation no differences due to sex for the relevant age groups were disclosed and the sex-groups are therefore combined in fig 7.4.6. s-transferrin cumulated percentage frequency probit finns rn danes 0 i ' i ' 1 ' l ' 1 ' " " " " l ' ' " l 1.6 1.8 2.0 2.2 2.4 2.62.83.0 3.5 4.0 (9/l) i ' l . 1 ~ 1 . ~ 0.2 0.3 0.4 0.5 0.6 llosgil) fig. 7.4.6. distributions of s-transferrin values for the finnish and danish reference individuals. the straight line indicates the distribution as estimated in the total danish material. the distributions for the finnish and the danish groups are indistinguishable with a slight tendency t o splitting up for the lower values, but without any convincing difference. so, there is no reason for defining another reference interval. 3 5 2 s-iga as for s-haptoglobin, the s-iga distributions are not directly parametric but, might be transformed to distributions close to log-gaussian (cf. chapter 7.2). s-iga curnulaled percentage frequency fsobil -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 ioggll i ’ i . i . . * . i ” . . i ’ i . i 0.6 0.8 1.0 2.0 3.0 4.0 5.0 g/l fig. 7.4.7. distributions of s-iga values for the finnish and danish reference individuals. the curve indicates the distribution as estimated in the total danish material (without transformation). the two distributions are clearly overlapping with a slight tendency to higher values for the finns in the middle of t h e distribution. there is, however, no reason for defining another reference interval for the finns. s-igg for s-igg the danish investigation showed differences in concentrations for women below 50 years of age and the remaining group, and the separate distributions are illustrated in fig. 7.4.8. the finnish and the danish distributions for women are indistinguishable and close to the distributions from the total danish material for women, whereas, the finnish men show higher values than the danish men, who in turn are lower than the total material. thus, there are no uniform differences between finns and danes and it was concluded that the finnish reference intervals should be identical t o the danish. 3 5 3 s-igg 95 90 80 70 60 50 40 30 20 10 ~ 5 1 cumulated percentage frequency probll i men women finns m 0 danes o o 99 i men// women below 50 i i i i i i i 0.7 0.8 0.9 1.0 1.1 1.2 1.3 log g/l i 1 i i ' i ' 1 ' 1 ' 1 " ' l 5 6 7 8 10 12 14 16 20 g/l fig. 7.4.8. distributions of s-igg values for the finnish and danish reference individuals with separate curves for the two sexes. the two straight lines indicate the distributions as estimated i n the danish material. s-ism cumulated percentage frequency pro611 lower 0.90 ci upper 0.90 ci men women finns w 0 danes 0 o worn; below 50 i i i i i i i i i i i i i i i . i ' 1 . 1 " ' 1 ' " ' l -0.4 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 0.4 log g/l 0.4 0.5 0.60.70.8 1.0 1.2 1.41.6 2.0 2.5 g/l fig. 7.4.9. distributions of s-igm values for the finnish and danish reference individuals with separate curves for the two sexes. the two straight lines indicate the distributions as estimated in the total danish material. 3 54 s-igm for s-igm the danish investigation showed differences in concentrations for men and women, and the separate distributions are illustrated in fig. 7.4.9. the finnish distribution for men shows lower values and the women higher values than the danish distributions. further, the finnish distributions are close to the confidence intervals (shown as t h e lower for men and the upper for women). the results for the finns thus seem to be contradictory. with the limited number of degrees of freedom, however, it is not possible t o postulate a more pronounced difference in s-igm values between the finnish men and women, so the danish reference intervals are accepted also for the finnish population. discussion the design of this comparison of plasma protein distributions between the finnish and the danish populations, was set up at a limited scale which could only disclose considerable differences with certainty. the results, however, gave no reasons for questioning the agreement for the plasma proteins prealbumin, albumin, orosomucoid, al -antitrypsin, and transferrin . for s-haptoglobin the difference was clear from the present population samples but an investigation on a larger scale might be relevant in order to confirm the difference. unfortunately we did not investigate the frequencies of the different phenotypes in the materials as the lower values for the finns might be related to a higher frequency of phenotype 1-1 than in the danish population, and it might explain the difference. regarding the immunoglobulins, it is well known that the concentrations of these proteins in plasma are related to the environmental conditions. the results for s-igg and s-igm were confusing in the sense that the differences were reverse for the two sexes, making any conclusions based on the present material difficult. in general, we have shown that in different populations it is possible to use the same reference intervals for the plasma proteins. s-haptoglobin was an exception. it is obvious that the use of these common reference intervals are preferable to any sohtion based on laboratory-individual reference intervals in current use (cf. chapter 7.5). 24-955059 3 5 5 acknowledgements we want to thank all the persons who have contributed to this work, volunteers who gave blood to the investigation and the 20 danish laboratories involved in collecting the blood samples and doing the analytical work. we are specially in debt of gratitude to ane richter, who performed the registrations and calculations of the many results. further we are grateful to serren blirup and per just svendsen, who performed the measurements of s-crp and for the antisera used in all the determinations delivered by dako . 356 ujms109_2.pdf upsala j med sci 109: 71–122, 2004 coagulation, inflammation and myocardial dysfunction in unstable coronary artery disease and the influence of glycoprotein iib/iiia inhibition and low molecular weight heparin stefan james department of medical sciences, cardiology, uppsala university hospital, uppsala, sweden abstract a free full-text copy of this article can be found at the web page of upsala j med sci: http://www.ujms.se patients with unstable coronary artery disease (cad) have an increased risk of subsequent myocardial infarction and death. this study evaluated the safety and efficacy of treatment with glycoprotein iib/iiia inhibition in addition to aspirin, low molecular-weight heparin and its influence on coagulation and inflammation. also, early and differentiated risk assessment utilising markers of inflammation, myocardial damage and dysfunction were evaluated. the global utilisation of strategies to open occluded arteries-iv (gusto-iv) trial randomised 7800 patients with unstable cad to 24 or 48 hours infusion of abciximab or placebo in addition to routine treatment with aspirin and heparin or dalteparin. baseline levels of creatinine, c-reactive protein (crp), troponin t (tnt) and n-terminal pro-brain natriuretic peptide (nt-probnp) were analysed. at selected sites, all patients received subcutaneous dalteparin (n=974), in stead of heparin infusion (n=6826). in a sub-population of dalteparin treated patients (n=404), serial measurements of markers of coagulation, fibrinolysis and inflammation were also performed. addition of abciximab to dalteparin as the primary treatment of unstable cad was not associated with any significant reduction in cardiac events but a doubled risk of bleedings. the combination of abciximab with dalteparin seemed as safe when 71 received 12 november 2003 accepted 3 december 2003 abbreviations: acute coronary syndrome (acs), brain natriuretic peptide (bnp), coefficient of variance (cv), coronary artery disease (cad), coronary artery bypass grafting (cabg), creatine kinasemb (ck-mb), c-reactive protein (crp), electrocardiogram (ecg), glycoprotein iib/iiia (gp iib/iiia), global utilisation of strategies to open occluded arteries iv (gusto-iv), interleukin-6 (il-6), low-molecular weight heparin (lmwh), n-terminal pro-brain natriuretic peptide (nt-probnp), myocardial infarction (mi), percutaneous coronary intervention (pci), plasminogen activator (tpa), plasminogen activator inhibitor (pai-1), soluble fibrin (sf), thrombin-antithrombin complex (tat), troponin t (tnt), unfractionated heparin (ufh). used with heparin. despite full dose dalteparin and aspirin there was a simultaneous activation of the inflammation, coagulation and fibrinolysis systems without any influence of the abciximab treatment. elevated levels of crp, tnt, and nt-probnp and reduced creatinine clearance were independently related to short and long-term mortality. the best prediction of high and low risk was provided by a combination of nt-probnp and creatinine clearance. any detectable elevation of tnt and reduced creatinine clearance, but neither elevation of crp nor nt-probnp, were also independently associated to a raised risk of subsequent myocardial infarction. introduction cardiovascular disease is the most common cause of death in the industrialised world, comprising 46 % of all deaths in sweden (1). in spite of the steady decline in the incidence of myocardial infarction in recent years, the incidence of unstable coronary artery disease (cad) has increased considerably(1). chest pain, or other symptoms suggestive of unstable cad, is furthermore one of the most common reasons for admission to the hospitals emergency rooms. these patients constitute a very heterogeneous population regarding the clinical history and the underlying cause of the symptoms. subsequently, also the prognosis varies considerably. early risk prediction is essential for identification of patients at high risk and selection of the most appropriate treatment strategy. moreover, with early risk-prediction, patients at low risk can be identified and costly and potentially hazardous treatments and prolonged hospital stays can be avoided. unstable cad is caused by a complex interaction between the endothelium, platelets and cascades systems of inflammation, coagulation and fibrinolysis, leading to thrombus formation and compromised coronary blood flow. subsequent myocardial damage, caused by downstream embolization of thrombotic material, as well as inflammation and myocardial dysfunction, contributes to an increased risk of serious complications. treatment of patients with unstable cad aims to symptom relief and to limitation of myocardial damage and prevention of recurrent ischemic events at a tolerable level of side effects. despite substantial improvements in the treatment there is still an approximately 7–10% risk of death or myocardial infarction the initial month after the index episode(2–4). therefore, further improvements by new anti-thrombotic and anti-inflammatory agents and by better and more differentiated risk assessment to tailor the treatment to individual patients, is highly warranted. in the present study, the safety and clinical efficacy of the glycoprotein iib/iiia inhibitor abciximab in combination with either the low molecular-weight heparin, dalteparin or unfractionated heparin in patients with unstable cad was investigated. also, the influence of treatment with abciximab on the activation of systems of inflammation, coagulation and fibrinolysis was elucidated. finally, the utility of biochemical markers of inflammation, myocardial damage and myocardial dysfunction for prognostication of the risk for death and myocardial infarction was elucidated. 72 background clinical manifestations and definition of coronary syndromes the coronary arteries continuously supply blood for the myocardium’s demands of oxygen and nutrients. interruption of the blood flow creates a state of ischemia with accumulation of acid waste products and disintegration of cell membranes resulting in ischemic chest pain. the induced changes in electric potentials can be detected with an electrocardiogram (ecg) and biochemical substances released from the injured myocardial cells can be detected in the peripheral blood. the classification of coronary syndromes is based on the acuteness by which they develop; stable coronary artery disease (cad), acute coronary syndromes (acs) and sudden cardiac death. stable cad is often a chronic disease with a deciduous onset and associated with a relatively low risk (figure 1). in contrast, acs starts acutely and has a considerably worse prognosis. acs is further divided into myocardial infarction (mi) and unstable angina (ua). typically, mi is characterised by a sudden onset of severe chest pain in combination with a shortness of breath, 73 fig. 1. atherosclerosis, plaque rupture and thrombus formation. definitions of various types of coronary heart disease. mi = myocardial infarction. nausea and circulatory instability and defined as symptoms or signs of myocardial ischemia associated with a certain degree of elevation of biochemical markers(5). unstable angina on the other hand is defined as a condition of coronary ischemia without elevation of biochemical markers and is clinically characterised by; a) recent onset (<4 weeks) of angina pectoris on minimal exertion, b) worsening of a stable angina with prolonged, more severe or more frequent episodes of chest-pain c) angina at rest or d) angina after a myocardial infarction (6). transient ischemic ecg changes such as st-depression or t-wave inversion are not obligatory but strongly support the diagnosis. based on the patients ecg findings on arrival, mi are further classified as stelevation or non-st elevation mi. st-elevation mi is a special entity associated with the most dramatic symptoms and the strongest need for urgent treatment in order to restore the obstructed blood flow in the diseased coronary artery. patients with a complete bundle branch block on ecg belong to the same high-risk category. considering both the underlying pathophysiology and symptom evolution, non st-elevation mi and unstable angina are parts of a continuum of unstable cad, only separated from each other by the detection of biochemical markers of myocardial damage, above a somewhat arbitrary and variable limit. the focus of interest in the present study is non st-elevation mi and ua. atherosclerosis and plaque formation the predominating underlying cause of acs is atherosclerosis, which starts in early adulthood in the innermost layer of the vessel wall, the intima. several factors accelerate this process, among them smoking, hereditary factors, hypertension, elevated levels of serum lipids and diabetes mellitus. the first visible signs of atherosclerosis are the so called “fatty streaks” consisting of pools of enlarged macrophages containing engulfed oxidised ldl particles that usually develop in response to mechanical stress (7). over time, smooth muscle cells proliferate and tissue matrix is synthesised in the intima driven by a chronic inflammatory process creating a fibromuscular atherosclerotic plaque (8)(figure 1). late in the process, collagen and calcium is often deposited in the plaque as suggested by the term “atherosclerosis”(9). as the coronary plaque grows larger, the blood flow will be obstructed and may not be able to meet to current metabolic demands of the myocardium distal to the coronary stenosis. the latter situation is the pathophysiological background to the clinical entity called “chronic stable angina pectoris”. plaque disruption and thrombus initiation the atheromatous core of a plaque is a soft, gruel-like hypo-cellular material mainly consisting of lipids. the overlying fibrous cap separates the plaque content from the blood. disruption of a plaque is not an uncommon event but still a complex pathological process that is central to the initiation of the acute coronary syndrome (10). importantly, not only the large plaques or tight stenoses create the acute coronary 74 syndrome. two thirds of arteries with a plaque-rupture and an occlusive thrombus have stenoses of 50 percent or less (11). the major determinants of plaque rupture are the size and consistency of the atheromatous core, the thickness of the fibrous cap, the inflammation and repair within the plaque and the phenomenon of plaque fatigue (12). however, also sudden changes in blood pressure and increased shear stress are important contributors. the plaque rupture leads to exposition of highly thrombogenic factors to the coagulation system and platelets, initiating the thrombus formation, which further exaggerate the narrowing of the coronary artery. platelet activation platelets are small cells found in large numbers in the blood and play a pivotal role in the thrombotic process. at the site of arterial injury, subendothelial tissue is 75 fig. 2. platelet activation. exposed to the blood constituents (figure 2). platelets adhere to the exposed collagen, von willebrand factor (13) and fibrinogen by specific cell receptors i.e. glycoprotein (gp) ia/iia and ib-ix-v complex (14). platelet adhesion results in outside-in signalling resulting in a rapid change of shape. thereby, the platelets increase their surface in order to cover the lesion. in addition to adhesion, several independent mechanisms are important platelet activators, such as thromboxane, serotonin, epinephrine, adenosine diphosphate and thrombin (15). simultaneously, they also release vasoconstrictors, chemotactic factors, clotting factors and receptors (i.e. pselectin and cd40 ligand) from their granules, promoting vasospasm, additional platelet aggregation and thrombin generation. release of adenosine diphosphate and thromboxane from �-granules amplifies the process by autocrine feedback loops. activated platelets undergo structural changes for activation of the most numerous receptor found on the platelet surface, the gp iib/iiia receptor which exists at a number of 40.000–80.000 per platelet (16). the activated gp iib/iiia receptor crosslinks platelets by binding to fibrinogen, creating a plateletfibrinogen net over the site of tissue injury. coagulation cascade (proteins indicated with bold text were evaluated in the study) at the site of a plaque rupture, the glycoprotein tissue factor (tf) will be exposed to the blood. tf is the initiator of the coagulation cascade in vivo and is expressed on smooth muscle cells, fibroblasts and macrophages in the vicinity of the vessel wall(17) (figure 3). the coagulation cascade (18) (19) is complex and includes a series of interactions between a large number of pro-coagulant and anti-coagulant proteins interacting with platelets and endothelial cells. tf binds to small amounts of factor vii, which become activated by proteolytic cleavage into its active form, viia. an auto-amplifying system called the “extrinsic system” thereby becomes activated, ending up with the conversion of prothrombin to thrombin. thrombin is considered a key protein in the cascade by its ability to activate other coagulation factors i.e. factor v, viii and xi, and by controlling the final step in the clot formation; conversion of fibrinogen to soluble fibrin. soluble fibrin (sf) is formed when fibrinopeptide a and b are cleaved from fibrinogen by the action of thrombin. sf circulates in plasma with a half-life of 4–6 hours. the level of sf reflects thrombin activity as the basis for fibrin formation and is therefore a sensitive marker of activation if the coagulation cascade (20). monomers of fibrin rapidly polymerise into cross-linked strands which are finally stabilised by factor viii (activated by thrombin) to form the skeleton in the arterial clot together with the platelets. several anti coagulant proteins are involved in the counter regulation of the coagulation system. one of the most important inhibitors is antithrombin, which acts by forming inactivated complexes with thrombin, thrombinantithrombin complex (tat). the inhibition of antithrombin is markedly accelerated in the presence of heparin. thus, tat can be detected in plasma after thrombus formation and is con76 sidered to reflect thrombin production and activity. the half-life of the complexes is thought to be approximately 5 minutes (20). in the present study fibrinogen, tat and sf were used as markers of coagulation activity for the investigation of influences of abciximab on the coagulation system. fibrinolysis (proteins indicated with bold text were used in the study) the fibrinolytic system has a very important role in balancing the coagulation cascade by inhibiting formation of thrombi and dissolving existing clots (21) (figure 3). the key protein, plasmin, acts by degrading fibrin into its soluble degradation components, one of which is d-dimer. plasmin is produced on the fibrin surface in the blood, by cleavage of the inactive pro-enzyme plasminogen. plasminogen, in turn, is activated by plasminogen-activators. the most important plasminogen activator in the blood is tissue plasminogen activator (tpa), which is produced and released into the blood by endothelial cells as a response to various stimuli, for example ischemia. tpa has short half-life of about 5 minutes due a rapid removal from plasma by the liver and by complex formation with plasminogen activator inhibitor 1 (pai-1). tpa antigen concentration therefore reflect the free tpa level as well as the tpa/ pai-1 complex concentration (22). free plasmin in plasma is extremely rapidly inactivated by �2-antiplasmin in order to reduce the potent effects of plasmin in the circulation. thus, pai-1 activity in plasma attenuates endogenous fibrinolysis and 77 fig. 3. coagulation system. increase fibrin concentration (22). pai-1 is produced by endothelial cells and smooth muscle cells in the vessel wall and by macrophages in the liver and spleen as well as by adipose tissue. while the biosynthesis rate of pai-1 is high, its short biological half-life (8–10 minutes) causes low plasma concentrations (23). since pai-1 belongs to a group of acute phase reactants, its activity in plasma can be increased by inflammatory stimuli as well as by high fibrinolytic activity. in the present study tpa and pai-1 were used as markers of fibrinolytic activity. inflammation inflammation plays an essential role in the pathogenesis of atherosclerosis (7). the earliest evidence of the influence of inflammatory mediators are the “fatty streaks”, which are pure inflammatory lesions, containing monocyte-derived macrophages and t-lymphocytes. continued inflammation at every stage of the disease is indicated by an increased number of macrophages and lymphocytes, (24) secreting cytokines and hydrolytic enzymes. sero-epidemiological and pathophysiological studies suggest that infectious organisms such as herpesvirus, chlamydia pneumoniae and helicobacter pylori may play a role in the initiation and progression of atherosclerosis (25). the exact mechanisms for the regulation of the atheroma progression are still however, largely unknown but contains a complex interaction between different cell-types and inflammatory mediators. furthermore, the rupture of a fibrous cap and thus the initiation of the acute coronary syndrome involves inflammatory action (26) as reflected by an increased concentration of activated macrophages and t-lymphocytes promoting degradation of extracellular matrix in the unstable plaque (27, 28). increased concentrations of monocytes (29), lymphocytes (30), inflammatory mediators such as c-reactive protein (crp), (31) interleukin-6 (il-6), serum amyloid protein a (32) as well as cytokines (33, 34) have been reported in patients with ua and myocardial infarctions at the time of hospital admission. interleukin-6 (il-6) is a cytokine with pro-inflammatory and pro-coagulant properties affecting many different cell-types. it also affects endothelial function and platelet production and is the only cytokine known to induce the synthesis of all the acute phase proteins by the liver. the production of il-6 is stimulated by tumour necrosis factor and interleukin-1 (35, 36). c-reactive protein (crp) is an acute phase protein synthesised by, and released from, the liver in response to circulating interleukin-6. crp is a sensitive marker of infection, tissue damage and inflammation (37). its plasma half-life is rapid (approximately 19 hours) and identical under all circumstances. thus, in contrast to other cytokines the concentration of crp in plasma is mainly dependent on its production. the increased inflammatory activity in patients with unstable cad may also, at least partly, reflect an acute phase reaction resulting from myocardial necrosis (38). in unstable cad there is also evidence of a widespread inflammation in the coronary arteries but the actual source of inflammatory mediators is still unknown (39). however, ischemic episodes themselves, as reflected by st-depressions, do not 78 seem to illicit crp elevation in patients with unstable angina (40). furthermore, recent evidence suggests that crp itself might be involved in the pathogenic mechanisms of the myocardial damage (41). crp has been shown to display pro-inflammatory properties (42) by co-localisation with and ligand binding to, complement in the myocardium (43). activated complement may in turn mediate further vascular and myocardial destruction and induce arrythmias (41). in addition, crp induces monocytes to express tissue factor, which may contribute to the development of disseminated intravascular coagulation and thrombosis in inflammatory states (44). fibrinogen is an acute phase protein produced in the liver and exists in the circulation in increased concentrations in various inflammatory conditions. there is a high degree of correlation between levels of fibrinogen and several well-established clinical risk factors for cardiovascular disease (45). since fibrinogen is both an inflammatory and coagulation mediator it is an unspecific marker reflecting increased coagulation as well as inflammatory activity. in the present study the above mentioned inflammatory markers were used for evaluation of a potential influence of abciximab on the inflammation system as well as regarding crp, also for prediction of coronary events. myocardial damage the troponin complex is formed by three different forms of structural proteins (troponin c, i and t) located in the thin filament of the contractile apparatus of both skeletal and myocardial myocytes regulating the calcium dependent interaction between actin and myosin. cardiac isoforms of troponin i and t are expressed solely on myocardial cells and released from the cytoplasma after disintegration of the cell membrane caused by myocardial necrosis. accordingly, measurable levels of troponin i or t are highly specific for myocardial damage indicating even microscopic areas of necrosis irrespective of the cause (46). the initial rise of troponin concentration occurs 3 to 4 hours after the ischemic injury with a persistent elevation up to two weeks after the event. the mb isoform of creatine kinase (ck-mb) is a cardiac specific enzyme also useful for detection and exclusion of myocardial damage. however, the specificity as well as the sensitivity is lower than for troponins. thus, ck-mb is regarded as the second best alternative for diagnostic and prognostic purposes. myocardial dysfunction brain natriuretic peptide (bnp) is a neurohormone synthesised and released from the cardiac ventricles in response to increased wall tension (47). by affecting the nephrons resulting in natriuresis, bnp and other natriuretic peptides are involved in the tight regulation of extracellular volume. the plasma level of bnp is increased in patients with heart failure and increases in proportion to the degree of left ventricular dysfunction (48). bnp levels also increase after mi and in unstable angina pectoris (49). bnp is produced as a pro-hormone, pro-bnp, which is enzymatically cleaved into bnp and the amino terminal portion of the pro-hormone, n-terminal 79 probnp (nt-probnp) (50). it has been shown that mean levels of nt-probnp are similar to mean levels of bnp in healthy people whereas in response to cardiac impairment the absolute and relative increase of nt-probnp exceeds that of bnp up to fourfold (50). risk assessment in patients with acute coronary syndromes clinical factors patients with acs are heterogeneous both regarding the clinical background, extent and severity of the underlying coronary disease. several clinical (51) as well as biochemical and ecg indicators have been described by multivariable analyses (52). age and male gender are among the most important clinical predictors associated with an unfavourable outcome. various indicators of a history of cardiovascular disease, such as diabetes mellitus, heart failure, hypertension and renal dysfunction are also important factors for risk assessment (52). due to the heterogeneity of acspatients and the variable risk and alternative treatment strategies, a better and a more individualised risk assessment than clinical factors can offer, is needed. electrocardiogram st segment depression > 0.1 mv on the ecg on admission has consistently been found to indicate an increased risk of subsequent death or myocardial infarction (53) with a further increased risk in relation to the magnitude of st-depression (54). even st-depressions of 0.05–0.1 mv provide prognostic information regarding future coronary events (55). t-wave inversion is a less specific ecg finding, concerning the diagnosis as well as the prognosis (56, 57). continuous st monitoring with 12-lead ecg or vectorcardiography better reflects the dynamic nature of myocardial ischemia than the occasional ecg recordings and adds to risk assessment(58) and identification of patients who benefit from extended anti-thrombotic treatment (59). coronary angiography coronary angiography provides an anatomic outlining of the coronary arteries and is considered the golden standard for the assessment of the existence, location and severity of the cad. the number of diseased vessels (>50% stenosis) as well as the complexity of the lesions (60) and existence of visible thrombi (61) contribute to an increased risk. furthermore, coronary angiography is a prerequisite for the decision on interventional procedures. however, still coronary angiography is an invasive procedure with an inherent, although low, risk of adverse events with a not negligible cost. therefore, mainly patients with a high likelihood of unstable cad and a moderate-high risk of subsequent events, based on other predictors, are suitable for an invasive assessment. 80 markers of coagulation and fibrinolysis activity activation of the coagulation and fibrinolytic systems, as demonstrated by elevated markers of thrombin generation, thrombin activity and fibrin turnover, have been demonstrated in the acute phase of unstable cad and are associated with an adverse outcome (62–64). also reduced fibrinolytic capacity has been associated with an increased risk of coronary events in community based populations (65) as well as in unstable angina (66). in addition, increased levels of pai have been associated with an increased rate of events in survivors of mi (67). none of these hemostatic markers are however recommended for use as risk predictors in a clinical setting (68). inflammatory markers elevated markers of inflammatory activity are associated with an increased risk for future cardiovascular events in healthy individuals (69,70), as well as in patients with stable (71), and unstable cad (72–75). in patients with unstable cad the erythrocyte sedimentation rate as well as crp and fibrinogen levels were found to be higher in patients developing refractory unstable angina than in patients with an uneventful clinical course (76). elevation of il-6 on admission for acs seems to be associated with an increased risk of long-term mortality (77, 78) and also identifies those who derive the greatest benefit from an early invasive treatment (78). in large epidemiological studies, an increased fibrinogen concentration has been shown to predict future coronary events (79). also in unstable cad, fibrinogen levels have been shown to contribute to prediction of future risk of death and/or mi in short and long term, independent of troponin elevation and ecg changes (72). in unstable cad, crp elevation on hospital admission has been shown to be an independent predictor of mortality (72) (73–75). however, the association between the crp level and the early risk of mi in unstable cad has not been established, as most studies have presented small patient numbers and combined endpoints (table 3) (31, 71, 74, 80). markers of myocardial damage numerous previous studies have shown that troponin elevation is associated with an impaired outcome in patients with unstable cad (81–83). it has been convincingly shown that elevation of troponin t raises the probability of significant coronary stenoses and advanced coronary artery disease i.e. three vessel disease and left main disease (84). also, thrombus formation is more frequent in patients with troponin elevation (84). recently it has been shown that even very low troponin levels just above the detection limit of the assay is associated with an increased risk of mi and death (84). however, few studies have had a sample size and event rate allowing the separate evaluation of the relations to death and mi separately. the raised mortality associated with troponin elevation is evident in shortas well as long-term (73) and independent of inflammatory activity, ecg changes and impaired renal function 81 82 table 3. published studies on the predictive value of crp elevation in unstable coronary artery disease. in order of sample-size. sa denotes stable angina pectoris; ua, unstable angina pectoris; atypical cp, atypical chest pain; ucad, unstable coronary artery disease ie. unstable angina pectoris + non-st elevation mi, myocardial infarction; emerg. revasc., emergency revascularization; refract. angina, refractory angina. d.l, detection limit. (85). identification of patients with troponin elevation is also useful for targeting therapy with low-molecular weight heparin (86), glycoprotein iib/iiia inhibitors (87, 88) and early revascularisation (89) as these patients derive a particular benefit from these treatment modalities. markers of myocardial dysfunction it is well recognised that elevation of bnp indicates a worse prognosis in patients with heart failure and after mi (48). recently it has been shown that elevation of bnp as well as nt-probnp levels obtained after the acute phase (median time 40 hours) in patients with a broad range of acs, independently predicts mortality (90, 91). on multivariate analyses, bnp levels have not been associated with subsequent mi (91). however, the relationship between levels of bnp or nt-probnp and other risk-markers in the assessment of risk in acs patients has not been fully elucidated. renal dysfunction renal insufficiency is associated with a worse prognosis in a wide spectrum of patients with cardiovascular disease, including acs (92–94). a part of the increased risk associated with reduced renal function is attributable to a large number of co-existing conditions such as age, diabetes and hypertension. still however, renal failure itself has consistently been shown to be associated with a worse prognosis(95). it has been proposed that renal dysfunction is a measure of the extent of vascular damage caused by a variety of insults on the endothelium. in many previous studies, patients with renal insufficiency have been excluded. hence, the prognostic value of cardiac troponin and bnp in patients with and without renal dysfunction presenting with suspected acute coronary syndromes have not been defined. risk scores and combinations of risk markers as several clinical, ecg and biochemical factors have been identified as independent markers of increased risk of subsequent cardiac events, the combinations of different markers have been evaluated. several risk-scores have been developed from large clinical trials of acs patients, by adding different indicators after weighing their relative importance (96). thus, by combining bnp, crp and troponin i, there was a near doubling of the mortality for each additional bio-marker that was elevated in a recent study of patients with acs (97). the higher value of the risk-score, the more benefit from early interventional treatment is derived for acs-patients according to the recently presented score from the frisc ii trial (98). before the present study was performed, neither the relations between levels of nt-probnp and levels of markers of myocardial damage, inflammation and clinical risk factors nor the combination these markers for risk assessment had been elucidated. 83 treatment (therapies marked with bold text were evaluated in the study) the aims of treatment in patients with unstable cad are symptom relief, limitation of myocardial damage and prevention of future coronary events i.e. myocardial infarction and death. anti-ischemic drugs reduce oxygen utilisation by decreasing heart rate, contractility and blood pressure and inducing vasodilatation. �-blocking agents exert their effects by inhibiting �-receptors in the myocardium and thereby decreasing oxygen consumption. treatment of acs patients with �-blockers is associated with a 13 % reduction in the progression to acute myocardial infarction (99). nitrates and calcium channel blockers are vasodilating agents relieving pain but not convincingly shown to reduce coronary events (100). platelet inhibition as platelet aggregation plays a central role in the pathogenesis of acs, antiplatelet regiments are essential to reduce thrombus formation and peripheral embolization of thrombotic material in the coronary arteries. aspirin aspirin irreversibly inhibits cyclooxygenase, impairing prostacyclin metabolism and thromboxane a 2 synthesis predominantly in platelets. as a result, platelet activation and aggregation in response to collagen, adp and thrombin is inhibited. however, at higher concentrations, thrombin and adp still can activate platelets in the absence of thromboxane a 2 . a large number of trials have shown that aspirin reduces death and mi in patients in unstable angina, even in low doses (101). a loading dose of 300 mg is therefore recommended in all patients with acs, followed by long term treatment (68). adp receptor inhibitors inhibition of the adp receptor of the platelets results in decreased platelet aggregation. in a large clinical trial including patients with unstable angina confirmed by ecg changes or elevation of biochemical markers, clopidogrel on top of aspirin was found to reduce the incidence of cardiac events significantly during a median follow-up of 9 months (102). particularly, the incidence of mi was reduced. furthermore, treatment with clopidogrel in combination with aspirin is considered a routine treatment at least one month after implantation of a coronary stent due to its superior effect in reducing acute stent-thromboses (103). glycoprotein iib/iiia inhibitors activation of the gp iib/iiia receptor is considered the final common pathway in platelet aggregation. blocking this receptor almost completely abolishes aggregation of platelets. abciximab is the fab fragment of a monoclonal antibody 7e3, binding with strong affinity to the gp iib/iiia receptor. in contrast to other gp 84 iib/iiia inhibitors, abciximab has a long half-life and binds to the vitronectin (� v � 3 ) and other receptors. by interaction with these receptors, abciximab has been suggested to influence also systems of coagulation and inflammation. glycoprotein iib/iiia receptor inhibitors have consistently been shown to reduce the rate of procedure related myocardial infarctions in patients undergoing percutaneous interventions in a large number of trials (104–108). long term mortality is reduced in patients treated with abciximab and heparin in conjunction with coronary stenting as compared to patients on sole heparin treatment (109). furthermore it has been shown that gp iib/iiia inhibitors, on top of aspirin and heparin, have reduced the rate of death and myocardial infarctions in special patient settings with unstable angina and non q-wave myocardial infarctions (110–113). the benefit has been shown to be most pronounced in high-risk patients with elevated troponin levels (87, 88) and in patients treated with early coronary interventions (52, 114). before the performance of the gusto-iv trial, which constitutes the basis of the present study, no gp iib/iiia inhibitor had been evaluated in a pure non-interventional setting. however, in the capture trial the administration of abciximab to high-risk acs patients for 18 to 24 hours before percutaneous intervention reduced the preintervention incidence of mi by 70% (relative risk reduction; 2.1% to 0.6 %, p=0.029) (105). several oral gp iib/iiia inhibitors have been tested in large trials without any evidence of benefit. in fact, the therapies have been associated with an increased incidence of bleedings and a modest increase in mortality (115), illustrating the potential risk with this type of treatment. coagulation inhibitors unfractionated heparin (ufh) is a heterogeneous mixture of sulphated polysaccarides of varying chain length increasing the effects of antithrombin, which most important way of action is inhibition of thrombin. ufh is effective in reducing the incidence of myocardial events in aspirin treated patients with unstable angina, although the number of clinical trials, and the number of patients included in those trials are relatively few (116). nevertheless, in an international perspective, ufh treatment is regarded standard treatment in patients with unstable cad. low-molecular weight heparins (lmwh) are fragments of unfractionated heparin that possess a greater anti-xa activity in relation to anti-iia (anti-thrombin) than ufh. in addition, lmwh, have several potential advantages over ufh (117, 118). the dose response is more predictable and reliable, the immunogenicity is reduced with less frequent thrombocytopenia, and finally there is less rebound effect after discontinuation of therapy. other advantages from a practical point of view are; longer biological half-life enabling easier administration with subcutaneous injections and less need for monitoring the anticoagulant effect. there is convincing evidence that lmwh is more effective than placebo in reducing cardiac events (119). furthermore, lmwh has been shown to be at least as effective as ufh in short-term. dalteparin was shown to be similar to ufh (120), while 85 enoxaparin was significantly more effective than ufh in the reduction of the cardiac events in large trials (121, 122). the direct thrombin inhibitor hirudin has been evaluated in two large scale trials and found to be slightly more effective than heparin in the short-term, without longlasting effects. in spite of treatment with a combination of aspirin and short term lmwh, there is still a 15–25% risk for recurrent ischemic events the first month after the acs (119) (121, 122). thus, there is a compelling need for improvement of long-lasting treatment effects. complications due to antiplatelet and anticoagulation regimens thrombocytopenia may occur as a result of treatment with ufh as well as gp iib/iiia inhibitors. however, the incidence is fairly low and the condition is reversible upon discontinuation of the drug infusion. mild thrombocytopenia (<100.000/ml) has been reported in 2.5–5.6 % and severe (<50.000/ml) in 0.9–1.6% among abciximab and heparin treated patients (123). with “small molecule” gpiib/iiia inhibitors, somewhat lower incidence numbers have been reported (124) (125). combining the anti-platelet properties of a gp iib/iiia inhibitor with the anticoagulant effect of a lmwh have theoretical advantages which may offer clinical improvements in the medical treatment of patients with acs. the risk of bleedings may however outweigh the potential treatment benefits. thus, close monitoring and reporting side effects of new pharmacological agents or combination of agents are therefore important. acute coronary syndrome trials with gp iib/iiia inhibitors in addition to ufh have reported major bleeding levels around 1.4 to 10.8% and minor bleedings in up to 13.1% (126). the intervention rates and definitions for major and minor bleedings have however varied. the safety of abciximab and enoxaparin as well as tirofiban and enoxaparin has previously been evaluated in relatively small series and seemed to be well tolerated with low and similar bleeding rates (127, 128). coronary revascularisation the difference between a treatment strategy including early coronary angiography and revascularisation if suitable, as compared to a conservative medical strategy and revascularisation only in case of recurrent ischemia or ischemia at a pre-discharge exercise test has been evaluated in a number of clinical trials. since the planning of the gusto-iv trial, convincing evidence has emerged that an early invasive strategy is superior for patients with non-st elevation acs. in the frisc ii trial the composite primary endpoint of death or mi at 6 months was reduced significantly from 12.1% to 9.4% (2). at one-year follow-up there was a significant reduction in mortality in favour of the invasive strategy (129). the results were confirmed in the tactics trial, in which the gp iib/iiia inhibitor tirofiban was used as adjunctive treatment in the invasive group (130). also with enoxaparin as adjunc86 tive treatment, the primary composite endpoint was reduced with an interventional strategy in the rita-iii trial (131). depending on the extent and characteristics of the coronary lesions, as identified by coronary angiography, revascularisation may be carried out by either percutaneous coronary intervention (pci) or coronary artery bypass grafting (cabg). in patients with left main disease or three-vessel disease, particularly in combination with left ventricular dysfunction, cabg is the treatment of choice by virtue of the well-documented reduction in mortality (132, 133). due to a rapid progression in technology, including an increased utilisation of coronary stents and potent platelet inhibitors, pci is however becoming increasingly used as the first line treatment alternative. in patients with one or two vessel disease the choice of pci is indisputable but also in patients with three-vessel disease the results appear to be as good as with surgery (134). however, in the present study (gusto iv) a primary medical treatment was evaluated and early coronary angiography was discouraged. aims the aims were to investigate a large cohort of patients with non-st elevation acute coronary syndrome regarding: � safety and efficacy of abciximab combined with dalteparin or unfractionated heparin. � activation of inflammation, coagulation and fibrinolysis and the influence of abciximab when added to dalteparin. � the activation of inflammation and signs of myocardial damage for the separate prediction of death and myocardial infarction. � signs of myocardial dysfunction for the separate prediction of mortality and subsequent myocardial infarction. materials and methods patients the global utilisation of strategies to open occluded arteries-iv (gusto-iv) trial included 7800 patients with acute coronary syndromes between 1999 and 2000 (135) (figure 4) for evaluation of abciximab treatment without early revascularisation. the primary endpoint in the trial was the occurrence, of death (of any cause), or myocardial infarction (mi), within 30 days after randomisation. in scandinavia, switzerland and selected sites in the us., 72 centres recruited all their patients (n=974) to the dalteparin substudy, referred to as the dalteparin cohort (figure 4). the non-dalteparin patients recruited in all other centres of the gusto iv trial (n=6826) are referred to as the ufh cohort. the primary objective of this substudy was to evaluate the rate of side-effects, during the initial seven days in 87 relation to the randomised abciximab or placebo treatment within the dalteparin cohort (randomised comparison) as well as between the dalteparin and ufh cohorts (non-randomised comparison). a special blood-sampling program for repeated analyses of inflammation and coagulation factors performed at 28 selected swedish sites, enrolling 404 patients, which constituted 74.3% of the patients recruited in sweden and 42% of the patients in the dalteparin cohort. study design inclusion patients with an acute coronary syndrome without persistent st segment elevation were recruited. eligible patients were 21 years of age with one or more episodes of angina lasting 5 min within the last 24 hours if they had either a positive cardiac troponin t or i test or 0.5mm of transient or persistent st-segment depres88 fig. 4. study design of the main gusto iv-acs trial with the sub-studies included in the thesis indicated. sion. patients with a history of acute myocardial infarction distinct from the qualifying event within 10 days prior to enrolment were required to have new st-segment depression and ck-mb levels below the upper limit of normal. exclusion exclusion criteria were myocardial ischemia precipitated by a condition other than atherosclerotic coronary artery disease, persistent st-segment elevation myocardial infarction or new left bundle branch block, percutaneous coronary intervention (pci) within previous 14 days, planned pci or coronary bypass surgery within 30 days after enrolment, confirmed hypertension (systolic >180 mmhg or diastolic >100 mmhg), coexistent condition associated with limited life expectancy and a number of factors associated with an increased bleeding risk (135). randomised and concomitant treatment the study was conducted in a double-blind fashion with patients randomly assigned to; abciximab therapy for 24 h (0.25 mg/kg bolus followed by a 0.125 ug/kg/min infusion up to a maximum of 10 ug/min for 24 h) followed by 24 h of placebo infusion, or abciximab therapy for 48 h (same bolus and infusion for total duration of 48 h), or matching placebo (bolus and 48 h infusion). all patients received 150–325 mg of aspirin as soon as possible after randomisation for long term treatment. the patients received a 70 u/kg unfractionated heparin (ufh) bolus (to a maximum of 5,000 u) followed by a continuous infusion of 10 u/kg/hour (to a maximum of 800 u/hour) titrated to maintain the aptt between 50 and 70 seconds for 48 hours or dalteparin (120 iu/kg to a maximum of 10,000 iu) subcutaneously every 12 hours for 5–7 d or until a revascularisation procedure or discharge. concomitant therapy with beta-blockers was strongly recommended. use of all other cardiac medications was left to the discretion of the investigator. coronary angiography was discouraged during or within 12 hours after the completion of study agent infusion unless the patient had recurrent or continuing ischemia at rest associated with ischemic st-t changes that was unresponsive to intensive medical therapy. if percutaneous coronary intervention was required during study drug administration, blinded crossover to active therapy with abciximab was provided. definitions of clinical endpoints all patients had ecg examinations at baseline, 48 hours and at 30 days, as well as blood samples collected at baseline and 8, 16, 24, 36 and 48h after randomisation to be analysed in a central laboratory for ck-mb. additional samples were to be collected at 0, 8, 16, and 24h after any ischemic episode lasting >20 minutes. mi was defined as either a new significant q-wave ( 0.04 s or at least a quarter of the rwave amplitude in two or more contiguous leads) or ck-mb 3 times upper limit of normal. for patients with ck-mb elevation at entry, a new episode of chest-pain in combination with a new ck-mb elevation was required for mi diagnosis the initial 7 days as presented in detail in the gusto iv-acs publication (135). follow89 ing coronary by pass surgery a new significant q-wave was the only criterion. a clinical endpoint committee blinded to treatment assignment adjudicated all suspected cases of myocardial infarction. during 30-days of follow-up, mortality and rate of adjudicated myocardial infarctions were recorded. at 12 months only allcause mortality was collected. bleeding classification bleeding was classified as major, minor or insignificant by the timi criteria (136). major bleeding during baseline hospitalisation was defined as either 1) intracranial haemorrhage or 2) bleeding associated with a haemoglobin drop greater than 50 g/l. minor bleeding was defined as any of the following: 1) spontaneous gross hematuria or hematemesis; 2) observed blood loss with decrease of haemoglobin >3 but 50 g/l. insignificant bleeding was defined as any bleeding not meeting the criteria for major or minor. statistical methods according to the protocol the outcome analyses were made with the abciximab 24 hours and 48 hours treatment groups combined. the safety data were analysed for the abciximab groups separately but since there was no major difference between the abciximab groups these results were also presented with the groups combined. comparisons were then made between the dalteparin and ufh cohorts. comparisons between groups were made with chi2-test. fischer’s exact test was used for groups with small numbers. for continuous skewed variables a non-parametric, mann-whitney, test was performed. differences between groups were presented as odds ratios o.r. with 95% confidence interval. differences in levels of biochemical markers were evaluated with non-parametric tests given that they were not normally distributed. bivariate correlations were calculated with spearman rank correlation coefficients. significance tests of betweengroup comparisons were made with kruskal-wallis or mann-whitney tests and of within-group comparisons between different time-points with wilcoxon signed rank test. also, the change in median levels from the previous sample for the randomised treatment groups was evaluated with kruskal-wallis test. the material was divided into quartiles on the basis of levels of biochemical markers. means were expressed with one standard deviation for continuous variables and medians were shown with 25–75 percentiles for skewed variables. differences in categorical base-line variables between quartiles were evaluated with chi2 tests for trend. differences between mean or median values for continuous variables were evaluated with one way anova or kruskal-wallis tests as appropriate. skewed variables were log transformed for calculation of independent associations between the variables in a multiple linear regression analysis. kaplan-meyer estimates were used for evaluation of the occurrence of the individual endpoints death and myocardial infarction after enrolment. logistic regression analyses were per90 formed for evaluation of the significance of predictors of mi at 30 days and mortality at 30 days and one-year. included variables are specified in detail in the respective paper. laboratory methods venous blood samples were drawn from direct punctures at randomisation, 24, 48 and 72 hours. the blood was collected into vacutainer tubes containing sodium citrate (0.13 m) or edta. plasma was prepared within 30 minutes of collection by centrifugation at 2000 × g at room temperature for 20 minutes. after centrifugation serum was frozen at –20˚c in aliquots and sent for central laboratory analyses of ck-mb levels. one aliquot of the serum samples at baseline was stored at –70˚c and sent in batches of 500 to the department of clinical chemistry, university hospital, uppsala, sweden for analyses of troponin t, c-reactive protein (crp) and nt-probnp. one batch was unfortunately lost during transportation. at selected swedish sites all patients were recruited to a special blood-sampling program for analyses of inflammation and coagulation factors from serum-samples obtained at baseline, 24 hours, 48 hours and 72 hours. troponin t levels were determined by a third generation assay on an elecsys (roche diagnostics) with the detection limit 0.01ug/l and a total coefficient of variance, cv of 8% at 0.05 ug/l and 4.1 to 6.0% between 0.1 to 11 ug/l. crp concentrations were measured with a high-sensitive chemiluminescent enzyme-labelled immunometric assay (immulite crp, diagnostic products corporation) with a detection limit of 0.1 mg/l. and a total cv of 5.6% at 2 mg/l and 5% at 10 mg/l. plasma nt-probnp was determined with a sandwich immunoassay on an elecsys 2010. the analytical range extends from 20 to 35.000 ng/l. at the central lab, the total cv was 3.3% (n=21) at a level of 209 ng/l and 3.0% (n=21) at a level of 7431 ng/l. in a healthy population (n=407) matched to the frisc ii population (2, 3) for age (range 40–75 years) and gender (32% females) the 97.5 percentile was 290 ng/l. serum creatinine at baseline was analysed at the local laboratories for 7706 patients. the creatinine clearance rate was calculated with the cockcroft and gault equation with correction for gender: {(140-age) × (weight (kg)} / serum creatinine (umol/l) (137). measurements of plasma levels of il-6, thrombin-antithrombin (tat) complexes and tissue plasminogen-activator (tpa) antigen were performed using commercial enzyme-linked immunosorbent assay (elisa) kits (quantikine from r&d systems, abingdon, uk, enzygnost tat, behring diagnostics, marburg, germany and imulyse tpa, biopool, umeå, sweden). soluble fibrin (sf) was analysed using a quantitative spectrophotometric assay (berichrom fm from behring diagnostics, marburg, germany). plasminogen-activator-inhibitor (pai-1) activity was determined by chromolize pai-1 (biopool, umeå, sweden). 91 results the main gusto-iv trial baseline characteristics as planned, gusto-iv-acs enrolled 7800 patients between july 21, 1998 and april 21, 2000, in 458 hospitals in 24 countries. western european sites enrolled 48% of the patients, eastern europe 31% and north america 14%. the mean age was 65 years (sd 11) and mean weight 77 kg (sd 14) and 62% were males. most patients had a history of coronary artery disease, including myocardial infarction (31%), and previous revascularisation (16%). all baseline characteristics were balanced among the three treatment groups. at enrolment, 59% tested positive on a qualitative or quantitative assay for cardiac troponin t or i, 80% had st-segment depression and 32% had both st depression and an abnormal cardiac troponin. primary and secondary endpoints the combined primary endpoint of death and mi at 30 days’ follow-up was similar among the three treatment groups: 8.0% with placebo, 8.2% with abciximab 24 hours and 9.1% with abciximab 48 hours (figure 5). moreover, no significant differences were apparent among the three treatment groups at any time point, 48 hours, 7 days and 30 days. the components of the combined endpoint were also evenly distributed among the three treatment groups. within 30 days after enrolment coro92 fig. 5. kaplan-meyer analysis regarding the primary outcome (death or myocardial infarction) in the main gusto-iv trial during the initial 30-days. nary revascularisation was performed in 30% of patients: percutaneous coronary intervention (pci) 19% and bypass surgery 11%. yet, only few patients (2,5%) underwent revascularisation within 48 hours, while on study treatment (pci 1.6%, bypass surgery 0.9%). 93 fig. 6. primary endpoint events (death or myocardial infarction at 30-days) in pre-specified subgroups of the main gusto-iv trial. as might be expected, events (death and myocardial infarction) were more frequent among patients with high-risk characteristics, such as advanced age, st-segment depression, elevated cardiac troponin or diabetes mellitus than among those without such characteristics. however, no significant treatment effect of abciximab was apparent in any of these subgroups (figure 6). the dalteparin substudy of gusto-iv baseline characteristics the randomised treatment groups were well matched regarding all baseline characteristics. however, as compared to the 6826 patients in the ufh treated cohort, the 974 patients in the dalteparin cohort constituted a higher risk population as indicated by higher age and weight, more prior by-pass surgery, more often an evolving myocardial infarction and a positive troponin test at baseline. safety major bleedings (not related to coronary artery by-pass surgery) were rare in all groups, although numerically doubled by abciximab treatment in both the ufh and 94 fig. 8. result of the primary outcome analysis of the dalteparin substudy of the gusto-iv for subgroups based on age. major and minor bleedings presented for the abciximab and placebo groups in the unfractionated heparin and dalteparin cohorts. dalteparin cohorts (table 1). minor bleedings occurred at similar low rates in the dalteparin and the ufh groups but were also doubled by abciximab treatment in both cohorts (table 1). in the dalteparin cohort, major and minor bleedings occurred in 5.0% in the abciximab group as compared to 1.8% in the placebo group (o.r. 2.71; 1.14–6.41). in the ufh cohort the difference between the abciximab and placebo groups were similar (3.8% vs. 1.8%: o.r. 2.17; 1.52–2.99). particularly elderly (> 65 years) patients had an increased risk of major and minor bleedings with abciximab (figure 8). stroke (any type) was an uncommon event in all groups in the trial (table 1). the highest stroke rate was found in the abciximab group (0.6%) of the dalteparin cohort, not statistically different from the placebo group (0%). the ufh treated patients experienced similar stroke rates (0.3% for both abciximab and placebo). intracranial haemorrhage occurred only in one patient in the dalteparin cohort receiving abciximab 24 hours. thrombocytopenia was more common with abciximab than placebo in the whole patient population. severe thrombocytopenia (<100.000/ml) occurred only with abciximab, both in the dalteparin(0.8% vs. 0.0%, n.s.) and in the ufh cohort (1.6% vs. 0.0%, n.s.). moderate thrombocytopenia (<50.000/ml) was also more frequent with abciximab than placebo both in the dalteparin (3.1% vs. 1.5%, ns) and in the ufh cohort (6.3% vs. 0.9%, p<0.001). thus, during abciximab treatment the risk of thrombocytopenia was halved in the dalteparin as compared to the ufh cohort (p<0.01). 95 table 1. safety at 7 days. comparisons between placebo, abciximab (24 hours + 48 hours) and between dalteparin and heparin treated cohorts. dalteparin heparin placebo abciximab placebo abciximab n=328 n=646 n=2270 n=4556 %(n) %(n) %(n) %(n) stroke 0 (0) 0.6 (4) 0.3 (7) 0.3 (14) intracr. haemorrhage‡ 0 (0) 0.2 (1) 0 (0) 0.1 (4) major bleeding§ 0.6 (2) 1.2 (8) 0.2 (5) 0.7 (34)** minor bleeding§ 1.2 (4) 3.7 (24)* 1.5 (35) 3.0 (137)*** insign. bleeding§ 24.7 (81)††† 46.4 (300)***, ††† 5.0 (113) 15.1 (690)*** blood transfusion 4.9 (16)††† 4.3 (28) 1.9 (44) 3.0 (136)* platelet count <100.000/ml 1.5 (5) 3.1 (20)††† 0.9 (21) 6.3 (285)*** <50.000/ml 0.0 (0) 0.8 (5) 0 (1) 1.6 (73)*** platelet transfusion 0.6 (2) 1.7 (11) 0.1 (3) 1.2 (53)*** *=p<0.05, **=p<0.01, ***=p<0.001 abciximab versus placebo. † p<0.05, ††† p<0.001. dalteparin versus heparin. ‡ intracranial haemorrhage, § bleedings according to timi-criteria, not related to coronary artery by-pass surgery. 96 fig. 7. results of the secondary endpoint, the outcome analysis, in the dalteparin substudy of the gusto-iv depicted in kaplan-meyer curves for the unfractionated heparin and dalteparin cohorts respectively. efficacy the secondary endpoint, outcome at 30 days, did not differ significantly between placebo and abciximab in the dalteparin substudy. in the dalteparin cohort the rate of death or mi in the abciximab groups vs. placebo was 9.6% vs. 11.3% (o.r. 0.85; 0.58–1.25) while in the ufh cohort the corresponding event rates were 8.5 % vs. 7.6 % (o.r. 1.12; 0.95–1.34) (figure 7). thus, there was a trend for a higher event rate in the dalteparin cohort than in the ufh cohort (o.r. 1.27; 1.01–1.58). however, when correcting for other known predictors of an adverse outcome in a forward stepwise multiple regression analysis the dalteparin treatment did not remain an independent predictor of death or mi at 30 days. activation of coagulation, fibrinolysis and inflammation in relation to abciximab treatment coagulation levels of coagulation markers, tat and sf, increased in the acute phase. median tat level increased significantly from baseline (3.1 ug/l) to 24 hours and stayed elevated at the same level at 48 hours (3.7 ug/l). at 72 hours the median level had decreased again and was not different from baseline. median sf level also increased from baseline (20.0 ng/l) to 24 hours and remained elevated until 72 hours (23 nmol/l). at all time-points the median levels of the coagulation markers were similar in the three different randomised treatment groups. neither the absolute nor the relative changes in median levels from previous sample differed between the groups at any time-point. fibrinolysis the median tpa level showed a continuous rise from baseline (8.7 ug/l) until the last sample at 72 hours (17.5 ug/l). in contrast, the pai-1 levels remained unchanged during the 72 hours sampling period. neither the median levels at different time-points, nor the changes within the groups differed significantly between the randomised treatment groups. inflammation the median level of il-6 increased from baseline (5.4 ng/l) and reached its peak median level at 24 hours (7.8 ng/l). the median level decreased thereafter and was at 72 hours no longer statistically significant different from baseline. median crp level increased similarly from baseline (4.4 mg/l) to 24 hours (8.7 mg/l) but in contrast to il-6, the median level remained significantly elevated until 72 hours. the median level of fibrinogen increased continuously from baseline (3.3 g/l) until the last sample at 72 hour (3.9 g/l). there were no differences in median levels or changes of levels of the inflammatory markers between the randomised treatment groups. 97 biochemical markers for prediction of coronary events markers of myocardial damage the median time from the onset of the qualifying episode of ischemic chest pain to randomisation was 9.5 (5.0–16.6) hours. troponin t analyses were available from 7115 (91.2%) of the patients. the troponin t-levels ranged from 0 to 17.3 ug/l and the quartile limits were 0.01, 0.12 and 0.47 ug/l. the rate of the primary combined endpoint of the gusto iv-acs study, death or mi, was increasing with higher troponin t quartiles at all time-points of follow-up (p<0.001). also mortality was markedly increasing with increased troponin t quartiles from 1.1% to 7.4% between the first to the fourth quartile at 30 days (figure 9 a). the rate of mi was 98 fig. 9. rate of a) death and b) mi at 48 h, 7 days and 30 days in relation to quartiles of troponin t. the number of patients with events is noted under the bars. increasing from the firstto the second quartile and was at 30 days 2.5% vs. 6.7%. however, no difference was observed between the upper three quartiles (figure 9 b). there was even a trend to a lower rate of mi in the fourth quartile as compared to the third quartile (5.6% vs. 7.2%, p=0.055). in a multiple logistic regression analysis, increasing troponin t quartile was independently related to both death (o.r. 1.63; 1.43–1.87) and mi (o.r.1.23; 1.11–1.37) at 30 days (table 4). any elevation of troponin t (> 0.01 ug/l vs. 0.01) however, provided a larger differentiation of high and low risk regarding both death and mi (o.r. 3.36;2.10–5.34 and 2.48; 1.79–3.42 respectively) markers of inflammation crp analyses obtained at baseline were available from 7108 (91.1%) patients. the range of crp-levels was 0 to 489 mg/l with quartile limits 1.84, 3.96 and 9.62 mg/l. the rate of the primary combined endpoint, death or mi was significantly increasing with higher crp quartiles at 30 days: 7.1%, 7.3%, 8.1%, 10.5%, p=0.001. this difference was entirely driven by the difference in mortality, which was observed already at 48 hours (figure 10a). at 30 days, mortality increased from 2% in the first quartile to 6.3% in the fourth quartile with increased rates also from the first to the second and from the third to the fourth quartiles. however, at no time-point was there any relationship between the rate of mi and the quartiles of crp (figure 10 b). in the multiple logistic regression analysis increasing crp quar99 table 4. multiple logistic regression analyses on troponin t and crp as predictors of death, mi and death or mi at 30 days. death mi death or mi o.r (95% c.i.) p o.r (95% c.i.) p o.r (95% c.i.) p model 1 tnt quartiles* 1.63 (1.43–1.87) <0.001 1.23 (1.11–1.37) <0.001 1.39 (1.27–1.52) <0.001 crp quartiles† 1.19 (1.05–1.35) 0.006 0.94 (0.85–1.04) 0.26 1.00 (0.92–1.09) 0.91 model 2 tnt (ug/l) >0.01 vs. 0.01‡ 3.36 (2.10–5.34) <0.001 2.48 (1.79–3.42) <0.001 2.84 (2.14–3.76) <0.001 crp (mg/l) >1.84 vs. 1.84‡ 1.72 (1.17–2.55) 0.009 0.76 (0.59–0.98) 0.033 0.93 (0.75–1.16) 0.53 model 3 tnt (ug/l) >0.47 vs. 0.47‡ 2.47 (1.86–3.28) <0.001 1.22 (0.79–1.32) 0.89 1.51 (1.23–1.86) <0.001 crp (mg/l) >9.62 vs. 9.62‡ 1.31 (0.98–1.74) 0.07 0.98 (0.76–1.25) 0.85 1.07 (0.87–1.31) 0.55 o.r = odds ratio, c.i. = confidence interval, mi = myocardial infarction. all three models included: age, male gender, body-weight, smoking, previous angina, stroke, heart failure, diabetes mellitus, hypertension, hypercholesterolemia, previous revascularization, previous myocardial infarction, current treatment with �-blockers and ace-inhibitors, aspirin treatment prior to inclusion, st-depression 0.5 mm and randomized treatment (abciximab 24 hours, 48 hours or placebo). tiles independently predicted 30-day mortality (o.r. 1.19; 1.05–1.35), while there was no relationship to subsequent mi (table 4). creatinine clearance patients with renal dysfunction are at high risk partly because of the high prevalence of multiple risk factors. in the gusto-iv population, a reduced creatinine clearance was significantly correlated with a large number of predictors of a worse outcome, such as diabetes, hypertension, age, heart failure, previous myocardial infarction (85) and elevation of crp, troponin t and n-terminal probrain natriuretic peptide (95). still, a creatinine clearance below the 1st quartile (51 ml/min) was independently associated with mortality as well as subsequent myocardial infarction in multivariate analyses (95). 100 fig. 10. rate of a) death and b) mi respectively 48 h, 7 days and 30 days in relation to quartiles of crp. the number of patients with events is noted under the bars. 101 fig. 11. kaplan-meier survival curves regarding probability of (a) death during one-year and (b) myocardial infarction during 30-days of follow-up for patient strata based on quartiles of nt-probnp. hyperlipidemia (including elevated lp(a) lipoprotein levels), the insulin resistance syndrome, and hyperhomocystinemia are other factors contributing to coronary artery disease in patients with renal dysfunction. the chronic anemia and volume overload associated with severe renal dysfunction, may be important contributors to an increased vascular stiffness, the development of heart failure and subsequent mortality. other specific cardiovascular risk factors contributing to the vasculopathy induced by the renal disease include secondary hyperparathyroidism, increased sympathetic-nerve activity caused by afferent renal reflexes, elevated levels of oxidized low-density lipoprotein, endothelial dysfunction and diminished vascular nitric oxide production (138). moreover, a reduced secretion of erytropoetin and insulin-like growth factor in patients with renal dysfunction may also specifi102 fig. 12. multiple logistic regression analyses for the prediction of myocardial infarction at 30 days and death at 1-year follow-up. cally contribute to an increased risk of thrombotic cardiovascular events by an inhibition of vascular repair (139). markers of myocardial dysfunction determinations of nt-probnp levels in serum samples obtained at randomisation were available from 6809 (87.3%) of the patients in the gusto-iv trial. the ntprobnp levels ranged from 5.3 to 35000 ng/l with a median level of 669 (interquartile range 237–1869) ng/l. increasing levels of nt-probnp were independently positively associated with age, female gender, current smoking, diabetes mellitus, hypertension, previous myocardial infarction and heart failure but negatively with body-weight, hypercholesterolemia and the occurrence of st-depression at baseline. nt-probnp levels were also associated with time from symptom onset and the magnitude of myocardial necrosis i.e. troponin elevation. in addition, nt-probnp levels were associated with renal dysfunction and inflammatory activity as reflected by levels of creatinine and crp. there was an increased mortality among patients in increasing quartiles of ntprobnp (figure 11). the kaplan-meier survival curves for the quartiles separated 103 fig. 13. mortality at 30-days follow-up among strata of patients based on quartiles of troponin-t and quartiles of c-reactive protein. 104 a b early. already at 48 hours after randomisation the difference in mortality between the quartiles was statistically significant (p=0.001), with a mortality of 0.2% (n=3), 0.4% (n=6), 0.4% (n=7) and 1.4% (n=23) respectively. the separation of the curves continued throughout the first year after the index event (p<0.001, log rank). thus, at one-year follow-up the mortality was 1.8 % in31, 3.9% in 66, 7.7% in 131 and 19.2% in 327 in the respective quartile. at one year there was an exponentially increasing mortality in the entire spectrum of nt-probnp levels with a mortality of 0.4% in 3 in the lowest decile (<98 ng/l) and 27.1% in 185 in the highest decile (>4634 ng/l). in a multivariable logistic regression analysis, adjusting for a large number of predictors of long-term mortality, increasing quartiles of nt-probnp still independently contributed to the prediction of 1-year mortality (figure 12). the risk of subsequent mi after the index event was also increasing with increasing quartiles of nt-probnp with an mi rate of 2.7% in 46, 5.4% in 91, 5.7% in 98 and 7.5% in 128 (p<0.001) for the respective quartile at 30-days follow-up (figure 11b). however, in a multivariable logistic regression analysis, age above 65 years, previous mi, creatinine clearance < 50.8 ml/min, angina pectoris, troponin t elevation (>0.01ug/l) and st-depression at baseline, but not the level of nt-probnp constituted independent predictors of myocardial infarction at 30 days (figure 12). 105 c fig. 14. mortality at one-year follow-up among strata of patients based on quartiles of nt-probnp and quartiles of creatinine clearance (a) troponin-t (b) and c-reactive protein (c). combinations of biochemical markers as both troponin t and crp were independent predictors of 30-day mortality (table 4), the prognostic value of combining these markers was also evaluated. the highest mortality at 30 days was found in the patients with both markers in the top quartiles, 9.1% and the lowest in the patients with both makers in the bottom quartiles, 0.3% (figure 13). finally, the long-term prognostic value of different combinations of nt-probnp, troponin t, crp and creatinine clearance were evaluated as all these markers were independent predictors of one-year mortality. a very low mortality was found in patients with nt-probnp in the bottom quartile in combination with creatinine clearance in the top quartile (0.3%) or in combination with troponin t or crp in the bottom quartile (1.6%) (figure 14a, b, c). the highest one-year mortality, 25.7%, was found in patients with levels of nt-probnp in the top and creatinine clearance in the bottom quartile. a similar high mortality was found in patients with nt-probnp in combination with troponin t or c-reactive protein levels in the top quartiles, 22.3 or 23.4 %. discussion effect of abciximab and dalteparin treatment there was no benefit with abciximab administered intravenously during the first 24 or 48 hours after enrolment of patients with acute coronary syndromes who did not undergo early coronary revascularisation. these findings are in sharp contrast to earlier investigations with abciximab in patients with refractory angina (105), in patients undergoing percutaneous coronary intervention (pci) (104, 106, 140), and in studies with other parenteral glycoprotein iib/iiia receptor blockers (110, 111, 141). the reasons for these differences are unclear, and several possible explanations have been discussed. the inclusion of patients was based on chest pain and either elevated cardiac troponin levels and/or st segment depression >0.5 mm aiming at patients at a relatively high risk. yet, the observed event rate in the placebo group, and in the trial overall was lower (8%) than expected (11%). it is possible that the minimal duration of chest pain required (5 minutes) and the low level of st-segment depression required, allowed enrolment if relatively low risk patients. however, the event rate was in fact similar or even higher than in other acs-trials when differences in endpoint definitions are taken into consideration (111, 119, 141, 142). in several subgroups of patients the event rate was clearly higher but there was still no effect of abciximab treatment in these groups (figure 2). based on the concept of a dominating role of platelet aggregation and coagulation activation in acs, a combination of a gp iib/iiia inhibitor and a lmwh in the treatment of patients with non-st elevation acs, has several theoretical, in addition to the practical, advantages. still, no treatment benefit of abciximab could be 106 observed in the dalteparin substudy that included a patient population at somewhat higher risk, resulting in significantly higher event rates than in the ufh cohort. the gp iib/iiia inhibitor abciximab, in addition to its anti-thrombotic effect, also has been suggested to suppress the inflammatory response in unstable cad. an anti-inflammatory effect might be related to abciximab cross-reacting with other integrin receptors (143). in the present study however, abciximab did not suppress the activation of inflammation in unstable cad. this is in contrast to the findings in a substudy of epic (144) in patients undergoing percutaneous coronary intervention. in the latter the suppression of inflammatory activity may have been related to the prevention of myocardial damage at the time of the intervention by abciximab therapy. in gusto-iv no reduction in myocardial damage by abciximab was observed, and the rate of coronary interventions was very low, according to the trial design (135). on the contrary, a possible pro-inflammatory effect of abciximab has emerged as one plausible explanation for unexpected lack of clinical benefit with abciximab in the gusto-iv acs (145), as there even was a trend to a raised long-term mortality in the 48-hour abciximab group. in particular, an unfavourable trend with an excess in mortality was observed in patients with signs of inflammation and without evidence of ongoing intracoronary thrombosis, as reflected by elevated crp levels and the absence of elevated cardiac troponin levels at enrolment (145). however, the results of the current study do not support such an explanation, as abciximab did not influence the development of indicators of inflammatory activity. there is evidence that high dose dalteparin in placebo controlled acs trials reduces thrombin generation and activity, as demonstrated by the reduction of the f1+2, tat and sf levels (146). in the present study tat and sf levels still increased during the initial 72 hours of dalteparin treatment, demonstrating an activation of the coagulation system. although there was an activation of the coagulation and fibrinolytic system, there was no attenuation of the elevation of these by abciximab treatment. previously a number of in vivo and in vitro studies have indicated that abciximab, by its blockade of the glycoprotein iib/iiia receptor, might have an indirect inhibiting effect also on the coagulation system (147, 148). however, the present study does not support any effect of abciximab on the coagulation system. abciximab, in contrast to clopidogrel, is ineffective in reducing platelet-leukocyte aggregates and p-selectin expression in vivo and even increases expression of p-selectin, presumably caused by “outside-in” signalling when abciximab binds to the gp iib/iiia receptor (149). formation of platelet-leukocyte aggregates by platelet p-selectin binding to leukocytes at the site of vascular injury increases thrombin generation and might be an important mechanism that contributes to haemostasis and thrombosis (150). however, the present results also refute any procoagulative effect of abciximab, at least in patients treated with dalteparin and aspirin. thus, in a patient population with non-st elevation acs treated with aspirin and 107 low molecular weight heparin, the addition of abciximab infusion does not prevent an initial activation of the inflammation, coagulation or the fibrinolysis systems. safety of abciximab and dalteparin treatment the safety of abciximab in addition to lmwh and aspirin in the treatment of acs has not been investigated previously in a large-scale trial. in the current trial the numbers of major and minor bleedings were overall low and comparable to other trials of anti-thrombotic treatments in acute coronary syndromes (136). there were no significant differences in bleedings in the non-randomised comparison between full dose of dalteparin and a reduced dose of ufh (table 2). however the addition of abciximab was associated with a doubling in major and minor bleedings, both within the ufh and dalteparin cohorts, in the present trial. acute coronary syndrome trials with gp iib/iiia inhibitors in addition to ufh have reported major bleeding levels in up to 10.8% and minor bleedings in up to 13% (123, 126). the safety of abciximab and enoxaparin combined as conjunctive therapy to intervention was tested in the open label nice 4 trial that included 818 patients which did not show an increased incidence of major bleedings or transfusions (0.2% major and 6.8 % minor bleedings, 1.2% transfusions) (151). tirofiban and enoxaparin vs. ufh was evaluated in the acute-ii trial that included 525 patients presenting with unstable angina or suspected myocardial infarctions without st-segment elevation and permitted coronary angiography after 24 hours. the combination treatment was well tolerated with low and similar bleeding rates in the two groups, i.e. major 1.0% vs.0.3% and minor 2.5 %vs. 4.3% (125). after the publication of the present study, a randomised comparison between enoxaparin and ufh in acs-patients treated with eptifibatide has also been published. major bleedings were less frequent (1.8% vs. 4.6%), while minor bleedings were more frequent for enoxaparin than ufh (30.3% vs. 20.8%) (152). taking differences in definitions into account, these results are in accordance with the present findings of a similar rate of major and minor bleedings when ufh or dalteparin was combined with abciximab. the increased numbers of insignificant bleedings (<30 g/l fall in haemoglobin) in the dalteparin substudy was to a large extent attributable to the 108 table 2. independent predictors of bleedings in the gusto-iv acs study. 95% c.i for o.r independent predictors for major and minor bleedings* o.r lower upper age >65 years† 2.81 2.08 3.80 abciximab treatment 1.62 1.37 1.91 pci†† during initial 7 days 1.81 1.96 2.57 * bleedings according to timi-criteria, not related to coronary artery by-pass surgery. † prespecified cut-off for the outcome analyses and the mean age. †† coronary intervention. included variables: age, sex, weight, history of stroke or tia, diabetes, hypertension, smoking, dalteparin cohort, randomised abciximab treatment, clopidogrel, aspirin, oral anticoagulant and other lmw-heparin usage. subcutaneous heamatomas caused by dalteparin injections, which commonly create. insignificant bleedings were particularly common for the patient groups with augmented risk of bleedings, such as elderly (figure 8) and females. as expected thrombocytopenia was more common with the addition of abciximab both to ufh and dalteparin. severe thrombocytopenia was however rare. therefore concerning the risk of thrombocytopenia at abciximab treatment lmwh seems preferable to abciximab. differential risk assessment with biochemical markers troponin and crp in concordance with other studies the current study showed that both troponin t and crp were significant predictors of an adverse outcome in the early phase after an episode of acs (31, 73, 75). in contrast to previous studies, both crp and troponin t levels were available from a sufficiently large number of patients to allow prospective evaluation of their separate relationships to mortality and risk of mi. furthermore, the independent associations to the different outcome events could also be demonstrated in multivariable analyses. increasing troponin t levels were associated with a continuous rise in mortality. in contrast, any detectable troponin t, i.e. above 0.01ug/l, was associated with a raised risk of mi without any further risk at higher troponin t levels. increased crp levels during the acute stage of unstable cad were related to increased mortality in accordance with previous findings (31, 72, 73, 75). in this large patient cohort the relationship to increased mortality was evident early and further accentuated throughout the 30-day follow-up. there was however no association between the crp levels and the risk of mi. no previous study on inflammatory markers in acs has contained a sufficient number of patients enabling separation of the endpoints death and mi. what might be the reason for the relationship between crp and subsequent mortality but not mi in the acute phase of unstable cad, in contrast to the well-established relationship between crp elevation and subsequent coronary events in the chronic phase of atherosclerotic disease (69, 153–155)? in the acute phase of unstable cad the elevation of crp level is transient and to a large extent caused by an acute phase reaction (156). some unstable cad patients might have a hyperresponsiveness of the inflammatory system, which might exaggerate the acute phase reaction and increase the immune system reaction (157). such a mechanism is supported by the observations of co-localisation of crp and activated complement in infarcted myocardium (43). crp may in itself contribute to inflammation by activation of complement that in turn may mediate myocardial damage, induce arrythmias and provoke contractile dysfunction (41). such an interpretation is in accordance with the relationships between the crp level and the occurrence of cardiac rupture, left ventricular aneurysm formation and mortality after acute mi (158). thus, the crp elevation in unstable cad might indicate a different process than the lowgrade crp elevation that is associated to subsequent coronary events among healthy 109 individuals(69, 153, 159) and in the chronic phase of atherosclerosis after myocardial infarction (160). in unstable cad as well as in chronic atherosclerotic disease there is a lasting elevation of the fibrinogen level (156) that might indicate an underlying chronic low-grade inflammatory condition that in both conditions is associated with a raised risk of later mi. nt-probnp in relation to other markers in the present study, from a large cohort of patients of non-st elevation acs, we demonstrated that baseline levels of nt-probnp are independently related not only to age and female gender (161) but also to low body-weight and renal dysfunction. part of this relationship might be explained by an increased sensitivity to volume overload, as bnp levels have been shown to be secreted at a response to volume overload and to raised intra-cardiac pressure (162) irrespective of the cause. the present study also demonstrated that levels of nt-probnp were independently related to clinical factors indicating any kind of cardiovascular damage or dysfunction supporting that elevation of bnp (or nt-probnp) is a general indicator of reduced cardiac performance rather than a specific indicator of systolic dysfunction (163). moreover, our study demonstrated that ongoing myocardial damage (i.e. minimal troponin elevation), time since start of myocardial ischemia and damage and the inflammatory response (i.e. crp elevation) were related to the magnitude of elevation of nt-probnp, further supporting the concept of bnp being a sensitive and rapid marker of reduced cardiac performance. this is in accordance with the recent report that bnp levels increase as a result of temporary occlusion of a coronary artery in conjunction with a coronary intervention (164) even when intracardiac filling pressures remained unchanged (165). in the present study nt-probnp levels were negatively associated with the presence of st-depression (> 0.5mm) at baseline which, however, might be attributable to the fact that st-depression was part of the inclusion criteria and the low level of > 0.5 mm for the definition of st-depression on admission. recently it has been shown that elevation of bnp as well as nt-probnp levels obtained after the acute phase (median 40 to 72 hours after symptom onset) in patients with a broad range of acs independently predict mortality (90, 91). in the present study we extended these results in a considerably larger population of nonst elevation acs, for nt-probnp obtained already on admission, at a median 9.5 hours after symptom onset in accordance with a previous study from our group in an unselected chest pain population (166). thereby, we could demonstrate that ntprobnp predicted one-year mortality in patients with blood samples obtained within 5.0 hours (first quartile) as well as more than 16.6 hours (fourth quartile) after symptom onset. the present study also demonstrated that any elevation of ntprobnp above the 97.5 percentile, 290 ng/l, in a healthy population matched for age and gender, seemed to be associated with an increased risk of death after the index event. despite the fact that the level of nt-probnp was independently related to sev110 eral riskfactors, the nt-probnp level still was the strongest independent indicator of mortality in the multivariate analysis. also elevation of troponin t and crp as well as reduced creatinine clearance rate (85) independently predicted an increased mortality. accordingly, the combination of several of these markers allowed an even better stratification of future risk of fatal events. the combination of quartiles of increasing nt-probnp levels and quartiles of decreasing creatinine clearance rates provided the best prediction of long-term mortality. among patients in every quartile of creatinine clearance, mortality was increased with increasing quartiles of nt-probnp. the combination of quartiles of nt-probnp and either quartiles of crp or troponin t provided a similar prediction of mortality. interestingly however, elevated levels of troponin t seemed to contribute to an increased mortality only in patients with nt-probnp levels in the top quartile. thus, acs-patients without myocardial dysfunction seem to tolerate even moderately large myocardial infarctions without a lethal outcome. on the other hand, acs-patients with renal dysfunction, myocardial damage or increased inflammatory activity in addition to any reduction in cardiac performance, as indicated by a release of nt-probnp, have a high risk of fatal complications to their heart disease. in contrast to st-depression and troponin elevation at baseline, the risk of subsequent mi at 30-days follow-up was not independently predicted by increasing levels of nt-probnp in accordance with previous studies (167). the reason for this finding might be that bnp is a regulatory myocardial hormone which is not involved in the processes related to the rupture of a coronary plaques or formation of coronary thrombi. in contrast, elevation of bnp has been shown to predict sudden death in patients with heart failure (168). thus, the release natriuretic peptides from ventricular myocytes, in response to increased wall-tension due to ischemia or volume overload, might indicate a propensity to develop ventricular arrythmias, ventricular rupture or terminal heart failure rather than myocardial infarction. clinical implications what biomarkers should be recommended in the early evaluation of patients with unstable cad (figure 15)? for prediction of mi troponin elevation is the strongest biomarker and also reduced creatinine clearance is independently associated with subsequent myocardial infarction. however, for prediction of mortality, several clinical as well as ecg and biochemical markers seem to be useful. on multivariable analysis, nt pro-bnp seems to be the strongest biochemical riskmarker. by virtue of being an unspecific marker of reduced cardiac performance nt probnp seems to be very useful for selection of low-risk patients. a level below the 97.5 percentile (i.e. 290 ng/l) of a healthy population is associated with a very low mortality. in fact it also seems very unlikely that the patient has any significant heart disease at these low levels. patients with elevation of nt pro-bnp on the other hand have increased risk of a fatal complication in relation to the level, which may 111 merit further investigation and treatment. it has been shown that carvedilol treatment is particularly effective in patients with heart failure and elevated levels of nt-probnp (169). however, it remains to investigate whether patients with high levels of nt-probnp might derive a particular benefit from ace-inhibition, early coronary interventions, implantable cardioverter-defibrillators (icd) or other therapeutic modalities. patients with any detectable troponin elevation have an increased risk of death and derive a particular benefit from early coronary intervention (89), gp iib/iiia inhibition (88) (87) and extended low molecular weigh heparin treatment (86) (170). also a moderate reduction in creatinine clearance is independently associated with an increased mortality which should be increasingly recognised although not possible to treat. crp elevation is particularly useful for prediction of long-term mortality. there is evidence that statin treatment reduce crp levels among survivors of mi and that statin therapy is particularly beneficial for patients with crp elevation, independent of lipid levels (171). furthermore, st-depression upon admission as well as several baseline factors are useful for prediction of mortality. 112 fig. 15. risk indicators in unstable coronary artery disease. conclusions in patients with non st-elevation acs not scheduled for early revascularisation: � abciximab infusion on top of dalteparin and aspirin treatment was generally well tolerated although abciximab increased the number of bleedings as compared to placebo. in patients treated with abciximab, bleeding-rates were similar if combined with dalteparin than if combined with ufh. � addition of abciximab to standard treatment with dalteparin as the primary treatment of acs, did not reduce the rate of cardiac events at 30-days follow-up. � despite dalteparin treatment there was a simultaneous activation of inflammation, coagulation and fibrinolytic systems not influenced by abciximab infusion. � baseline levels of troponin t and c-reactive protein were independently related to 30-day mortality. any detectable elevation of troponin t, but not of c-reactive protein was also associated to a raised risk of subsequent mi. concerning mortality, the combination of both markers provided a better risk stratification than either one alone. � increasing quartiles of nt-probnp were independently related to short and long term mortality. the combination of nt-probnp and creatinine clearance provided the best prediction of one-year mortality. � a multimarker strategy with creatinine clearance, troponin, crp and nt-probnp together with ischemic ecg changes and clinical background characteristics has the potential to make risk assessment and clinical decision-making individualized and substantially improved. summary patients with acs constitute a heterogeneous population with different clinical history, extent and severity of the coronary artery disease. in the assessment of risk of cardiac events, several clinical as well as ecg and 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(2000). cardiac troponin i for stratification of early outcomes and the efficacy of enoxaparin in unstable angina: a timi-11b substudy. j am coll cardiol. 36:1812–7. 171. ridker pm, rifai n, pfeffer ma, et al. (1999). long-term effects of pravastatin on plasma concentration of c-reactive protein. the cholesterol and recurrent events (care) investigators. circulation. 100:230–5. about the author stefan james received the israel hwasser award from the upsala medical association for the best thesis in clinical medicine in the academic year 2002/2003. he is at present consultant at the dept. of cardiology, uppsala university hospital for correspondence: stefan james, m.d., ph.d. dept. of cardiology, thoraxcenter, university hospital, se-751 85 uppsala, sweden telephone: +46 18 6112818 fax: +46 18 506638 e-mail: stefan.james@akademiska.se 122 upsala j med sci 91: 251-255, 1986 surface cryoplaning a technique f o r clinical anatomical correlations wolfgang rauschning department of orthopaedic surgery, university hospital, uppsalu, sweden during t h e p a s t decade, r a p i d advances in t h e c l i n i c a l d i s c i p l i n e s a n d mile s t o n e achievements i n d i a g n o s t i c imaging such a s x-ray computed tomography, magnetic resonance imaging and u l t r a s o n o g r a p h y , have aroused renewed i n t e r e s t i n g r o s s anatomy a n d d e t a i l e d segmental anatomical c o r r e l a t i o n s . i n o r t h o p a e d i c s u r g e r y , t h e development of new techniques f o r t h e t r e a t m e n t of d i s o r d e r s of t h e j o i n t s and t h e s p i n e such as a l l o p l a s t i c j o i n t replacement, ligament recon s t r u c t i o n , a r t h r o s c o p i c s u r g e r y , decompressive surgery f o r s p i n a l s t e n o s i s o r improved management of s p i n a l f r a c t u r e s a l l c a l l f o r thorough knowledge of f u n c t i o n a l anatomy a n d biomechanics. in a c l i n i c a l a n a t o m i c a l i n v e s t i g a t i o n of p o p l i t e a l c y s t s a n d t h e i r r e l a t i o n s h i p t o t h e gastrocnemio-semimembranosus bursa (4), knee specimens were frozen i n v a r i o u s p o s t u r e s and then s e r i a l l y s e c t i o n e d on a l a r g e , heavy-duty cryomicrotome, devised by ullberg (7,8) f o r a u t o r a d i o g r a p h i c s t u d i e s i n u n d e c a l c i f i e d experimental animals u p t o t h e s i z e o f monkeys. s p e c i f i c r e q u i r e ment i n h e r e n t in s t u d i e s of musculoskeletal anatomy prompted m o d i f i c a t i o n s of t h e o r i g i n a l c r y o s e c t i o n i n g t e c h n i q u e . in t h i s a r t i c l e , a d e s c r i p t i o n of t h e cryoplaning technique i s supplemented w i t h i l l u s t r a t i v e examples of s p i n e anatomy. technique the a c q u i s i t i o n of f r e s h c a d a v e r i c specimens i s t h e prime p r e r e q u i s i t e f o r good anatomical s t u d i e s . since t h e procurement of such m a t e r i a l s from w i l l e d body donor programs is l i m i t e d with r e s p e c t t o number and t h e u s u a l l y high age of t h e deceased, t h e major i t y of specimens i s o b t a i n e d from d e p a r t ments of pathology and f o r e n s i c medicine d u r i n g r o u t i n e a u t o p s i e s . to prevent t h e deformation of t h e topographic r e l a t i o n s h i p s between t h e bone and t h e s o f t t i s s u e s as well as drainage of blood a n d emptying of f l u i d f i l l e d c a v i t i e s , f r e e z i n g o f t h e specimen in s i t u i s e s s e n t i a l . a r t e r i e s and v e i n s a r e more r e a d i l y d i s t i n g u i s h e d i f they a r e f i l l e d with c o l o r e d l a t e x o r dye p r i o r t o f r e e z i n g . while a whole c a d a v e r , p r o p e r l y p o s i t i o n e d and s u p p o r t e d , 25 1 may be frozen in t o t o , in s i t u f r e e z i n g of a j o i n t o r a s p i n e segment i s d i f f i c u l t and time-consuming. the specimen i s prepared by d i v i s i o n of a d j a c e n t s o f t t i s s u e s and i n s u l a t e d a g a i n s t t h e s k i n a n d organs with c e l l u l o s e . crushed dry i c e i s packed i n t o t h e s e spaces a n d l i q u i d n i t r o g e n i s r e p e a t e d l y poured over t h e i c e t o lower t h e temperatureof t h e c r y o g e n i c . dependent on t h e s i z e of t h e specimen, i t t a k e s two t o f o u r hours b e f o r e i t i s frozen s o l i d and can be c u t o u t with a n o s c i l l a t i n g e l e c t r i c saw. for d i r e c t c o r r e l a t i o n s w i t h computed tomographic s c a n s , t h e specimen may be em bedded i n orthogonal styrofoam boxes f i l l e d with precooled carboxymethyl c e l l u l o s e gel (cmciwhich i s f r o z e n t o i c e . scanning may be performed in o p t i o n a l p l a n e s prior t o s e c t i o n i n g ( 5 ) . other specimens may be embedded d i r e c t l y on t h e s t a g e by f i l l i n g t h e box a n d surrounding metal frame with cmc gel a n d f r e e z i n g a t -70' c . t a p e , b u t by r e p e a t e d l y photographing t h e specimen s u r f a c e ( 4 , 5 ) . a t m i l l i m e t e r i n t e r v a l s . a f t e r t e s t s w i t h cinematographic equipment had shown t h a t t h e microtome s l e d g e s t o p s e x a c t l y i n t h e same p o s i t i o n a f t e r each s t r o k e , t h e following p r o v i s i o n s were made f o r s e q u e n t i a l image r e g i s t r a t i o n : a s t a b l e camerastand w i t h a f i x t u r e f o r t h e v e r t i c a l l y moving k n i f e h o l d e r a l s o c a r r i e s a h o r i z o n t a l b a r towhich a n a d j u s t a b l e hinged r u l e r w i t h a m i l l i m e t e r s c a l e i s a t t a c h e d . the r u l e r i s a l i g n e d along t h e lower b a s e l i n e of t h e image frame a n d t h e camera then focused on t h e plane specimen s u r f a c e . as t h e k n i f e i s f e d down during s e c t i o n i n g , t h e camera and s c a l e a r e lowered, rendering i d e n t i c a l alignment a n d m a g n i f i c a t i o n of t h e images throughout t h e specimen. previously embedded specimens a r e t r a n s f e r r e d t o t h e microtome s t a g e . the anatomical images a r e not o b t a i n e d from s e c t i o n s c o l l e c t e d on adhesive modern scanners "zoom" through a n orthogonal block of t i s s u e i n t h e p a t i e n t in s t i l l cameraspthe image frame i s not a c c u r a t e l y r e l a t e d t o t h e t r a n s p o r t s p r o c k e t h o l e s of the f i l m . a 35 mm s i n g l e lens r e f l e x camera was t h e r e f o r e r e b u i l t f o r e x a c t p i n r e g i s t r a t i o n . photographic images a r e taken on 25 a s a c o l o r r e v e r s a l f i l m w i t h f l a t f i e l d l e n s e s a n d e x t e n s i o n t u b e s allowing f o r v a r i o u s m a g n i f i c a t i o n s . automatic e l e c t r o n i c f l a s h e s a r e used f o r i l l u m i n a t i o n . in a d d i t i o n t o i n r e g i s t e r overview images, c l o s e u p s a r e taken with a second, hand-held camera i n a r e a s of p a r t i c u l a r i n t e r e s t . specimens c o n t a i n i n g l a r g e r p r o p o r t i o n s of hard o r b r i t t l e bone (such a s t h e temporal bone) a r e cryoplaned a t a few microns' t h i c k n e s s whereas l a r g e r , s o f t t i s s u e specimens can be s e c t i o n e d a t 30-40 microns' t h i c k n e s s . photographs a r e taken a t i n t e r v a l s ranging from . 1 mm t o 1 . 0 m m . the microtome i s programmed t o c u t a c e r t a i n number of s e c t i o n s of t h e s e l e c t e d t h i c k n e s s a n d s t o p s a u t o m a t i c a l l y i n t h e p o s i t i o n f o r photography. depending o n t h e anatomical r e g i o n , t h e c u t t i n g h e i g h t i n t e r v a l s vary. fig.1. was f r o z e n i n f l e x i o n . therefore t h e upper a r t i c u l a r p r o c e s s e s of c7 l a c k c o n t a c t w i t h t h e i n f e r i o r a r t i c u l a r processes of c6. also t h e e p i d u r a l v e i n s and the venous s i n u s e s i n t h e foramina a r e engorged.the arrow p o i n t s a t a small paramedian d i s c h e r n i a t i o n . g= dorsal r o o t ganglion up= u n c i n a t e process v a = v e r t e b r a l a r t e r y axial anatomy i n t h e lower c e r v i c a l s p i n e (c6-c7 d i s c ) . t h e specimen f i g . 2 . axial anatomy of t h e lumbar s p i n a l canal a t l3-l4. t h e t h e c a l s a c c o n t a i n i n g t h e cauda equina r o o t s i s surrounded by e p i d u r a l f a t and f i r m l y a f f i x e d t o t h e concave p o s t e r i o r d i s c margin. the v e n t r a l i n t e r n a l v e i n s a r e d e s i g n a t e d by arrows. fig.3. s a g i t t a l s e c t i o n t h r o u g h t h e c e r v i c a l s p i n e o f a 63 y.0. male with a f r a c t u r e d i s l o c a t i o n a t t h e c5-c6 l e v e l . note t h e complete dis r u p t i o n of the d i s c , rupture o f the ligamentum flavum and r e t r o p u l s i o n of f r a c t u r e d spondylophytic r i d g e i n t o t h e v e r t e b r a l canal (arrow) 253 exacting care must be taken in the preparation of the specimen surface for photography. especially at higher magnification, the ice crystals on the fresh ly cut specimen surface are disturbing. compressed air of room temperature is used to remove debries and t o thaw the specimen surface. fraying of soft tiss ues bordering pneumatic cavities (e.g. in the skull) is avoided by filling them with water and allowing it to freeze to ice. to prevent recrystallization, the specimen surface is gently wiped with a warm cloth pad slightly soaked in ethy len glykol. applications in research and teaching the great number of atlases on segmental correlative anatomy published in recent years and the rapid advances in diagnostic imaging underscore the need for improved reference material for the interpretation of computed tomo grams. these tomographic scans are taken not only in the axial, sagittal and coronal plane, but increasingly in oblique, nonorthogonal planes. familiarity with the topographic anatomy in these unconventional planes is essential for a correct diagnosis. a systematic assessment of the multiplanar human anatomy in series of closely spaced sections should also encompass the variations of the normal anatomy with respect to shape, dimensions and intrinsic relationships, define borderline cases and abnormalities and should also demonstrate significant pathoanatomical changes typical for each region. macroscopic pathoanatomical changes in experimental animal models ( 3 ) . it a1 so is used to supplement biomechanical studies, when specimens are frozen under a specific load. subsequent cryoplaning then renders a quantitative assessment of the magnitude of dislocation and deformation and morphometric data ( 1 ) . the technique of surface cryoplaning allows to study the normal and pathol ogical anatomy in specimens containing large amounts of undecalcified bone. the undistorted and also the functional relationships of the skeleton to contiguous soft tissues is maintained as well as the natural colors. sequences o f detailed images taken at submillimeter intervals, high magnification and in exact reg istration allow to "zoom" through a block of tissue in a movie mode. cryoplaning is a powerful tool in basic research, notably for quantifying the irrefutable need for an improved knowledge of multiplanar anatomy for better diagnosis and treatment may entail the necessity of incorporating it in undergraduate anatomy curricula. conventional atlases and textbooks can only accommodate a limited number of illustrations (6). new electronic media with the capacity of storing vast numbers of images (2) and facilitating computer aided interactive use would seem to have a greater potential for teaching of complex three dimensional anatomy. 254 conclusion the uppsala t e c h n i q u e f o r s u r f a c e c r y o p l a n i n g o f f r o z e n specimens c o n t a i n i n g l a r g e p r o p o r t i o n s o f u n d e c a l c i f i e d bone, r e n d e r s s e r i e s o f a c c u r a t e l y r e g i s t e r e d p h o t o g r a p h i c images w h i c h i n h i g h d e t a i l and n a t u r a l c o l o r s d e p i c t t h e u n d i s t o r t e d r e l a t i o n s h i p s between s k e l e t a l elements and c o n t i g u o u s s o f t t i s s u e s . i t i s used t o c l a r i f y c o m p l i c a t e d t o p o g r a p h i c and f u n c t i o n a l a n a t o m i c a l r e l a t i o n s h i p s i n j o i n t and s p i n e specimens and f o r d i a g n o s t i c c o r r e l a t i o n w i t h computed tomo g r a p h i c scans. a p a r t f r o m i t s h i g h i m p a c t on t e a c h i n g and e n h a n c i n g d i a g n o s t i c acumen, c r y o p l a n i n g f a c i l i t a t e s m o r p h o m e t r i c s t u d i e s o f p a t h o l o g i c a l c o n d i t i o n s i n d u c e d i n e x p e r i m e n t a l a n i m a l models and a q u a n t i t a t i v e assessment o f p a t h o l o g i c a l c o n d i t i o n s i n t h e m u s c u l o s k e l e t a l system, w h i c h a l s o i n c l u d e s m u l t i p l a n a r 1. 2. 3. 4. 5. 6. 7. 8. r e f o r m a t t i n g and t h r e e d i m e n s i o n a l m o d e l i n g references asplund, s. : b i o m e c h a n i c a l s t u d i e s o f c o n g e n i t a l d i s l o c a t i o n o f t h e h i p . e x p e r i m e n t s i n human a u t o p s y specimens and r a b b i t s . d o c t o r a l t h e s i s a t uppsala u n i v e r s i t y , 1983. glenn, w.v. ,jr. & rauschning, w . : the knee: m u l t i p l a n a r anatomy, m a g n e t i c resonance i m a g i n g and computed tomography. i n s t r u c t i o n a l l a s e r v i d e o d i s c . american academy o f o r t h o p a e d i c surgeons and n a t i o n a l l i b r a r y o f m e d i c i n e , 1986 m i c h e l s s o n , j-e. & rauschning, w. : p a t h o g e n e s i s o f e x p e r i m e n t a l h e t e r o t o p i c bone f o r m a t i o n f o l l o w i n g t e m p o r a r y f o r c i b l e e x e r c i s i n g o f i m m o b i l i z e d j o i n t s . c l i n o r t h o p re1 res 176: 265-272, 1983. rauschning, w . : p o p l i t e a l c y s t s and t h e i r r e l a t i o n t o t h e g a s t r o c n e m i o s e m i membranosus b u r s a . a c l i n i c a l and a n a t o m i c a l s t u d y . d o c t o r a l d i s s e r t a t i o n a t uppsala u n i v e r s i t y , 1979. rauschning, w. : computed tomography and c r y o m i c r o t o m y o f l u m b a r s p i n e s p e c i mens. a new t e c h n i q u e f o r m u l t i p l a n a r a n a t o m i c c o r r e l a t i o n . s p i n e 8 : 170 180, 1985. rauschning, w . : d e t a i l e d s e c t i o n a l anatomy o f t h e s p i n e . i n : m u l t i p l a n a r ct o f t h e s p i n e (ed. s.l.g. rothman & w.v.glenn, j r . ) , pp 33-85. w i l l i a m s and w i l k i n s . b a l t i m o r e . 1985. u l l b e r g , s . : s t u d i e s on t h e d i s t r i b u t i o n and f a t e o f s 3 5 l a b e l l e d b e n z y l p e n i c i l l i n i n t h e body. a c t a radio1 ( s t o c k h ) suppl 118: 1-110, 1954. u l l b e r g , s. : the t e c h n i q u e o f whole body a u t o r a d i o g r a p h y . c r y o s e c t i o n i n g o f l a r g e specimens. s c i e n c e t o o l s , s p e c i a l i s s u e : 2-29, 1977. adress f o r r e p r i n t s and f u r t h e r b i b l i o g r a p h y : wolfgang rauschning, m.d. department o f o r t h o p a e d i c s u r g e r y u n i v e r s i t y h o s p i t a l sweden s751 85 uppsala 255 upsala j med sci 88: 6 1 4 2 , 1983 short communication on cerebrovascular deaths in middle-aged men during a 7-10-year follow-up hans wberg, hans lithell and hans wedstrand from the department of internal medicine, university hospital, and the institute of medical geriatrics, uppsala uniuersity, uppsala, sweden between 1970 and 1973 all men born in 1920-1924 in the city of uppsala were invited to a health examination. i n all 2.322 men participated and 446 men did not participate. those with hypertension, hyperlipidaemia and reduced glucose tolerance were treated. the death causes up to september 1980, during an average follow-up of 8.5 years (range 7-10 years) were examined. the number of cerebrovascular death (cvd) among participants was 9 (0.4 %) and among non-participants 4 (0.9 %). thus the annual incidence of cvd was 45.6 and 105.5 per 100 000 men, respec tively. the corresponding figure in all sweden was 50.2 (1). a s the number of cvd was small the differences i n incidence should be interpreted with caution. the proportion o f different subdiagnoses within the group of cvd was surprising (table below). participants non-participants total subarachnoid haemorrhage 7 encephalomalacia 1 intracerebral haemorrhage 1 total 9 4 13 all except one of the 13 men had had autopsy. the non-autopsied man had no hypertension and a clinical diagnosis o f subarachnoid haemorrhage (sah). the ratio between deaths from sah and other cvd was 3.5:l among the participants. the same ratio in the male 50-59 year age group of the whole of sweden was 1:3.9. there were 7 deaths in sah in uppsala (2.5 were expected) and 2 deaths in other cerebrovascular causes (7.4 were expected). the number of deaths due to sah was significantly (p< 0.05) greater than expected. 61 hypertension was known in two men one with cerebral haemorrhage and one with encephalomalacia. the first man had a known hypertension since the end of the 1950s with an unsatisfactory blood pressure reduction most of the time. one more man, a non-participant, had a cerebral haemorrhage. thus, cerebral haemorrhage is a rare cause of death in middle-aged men if they are treated for hypertension. no participant with sah had hypertension at the health examination. the main observation in the present follow-up was that the ratio between deaths due to sah and due to other cerebrovascular lesions was greater in uppsala than in sweden. i n fact, the incidence of sahs was significantly greater in uppsala than in sweden. the reason for this is not known. i n other mortality statistics, sah has not decreased in spite of a reduced overall incidence of cvd (1, 2 ) . the occurrence o f sah should not be influenced to any great extent by an improved antihypertensive treatment which is considered to be the most likely explanation of the reduction of other cvd. sah is generally caused by rupture of a congenital aneurysm. therefore, it is possible that any further reduction o f cvd in middle-aged men is limited by the lack of reduction of the incidence of sahs. references 1. causes of death 1969-1978. official statistics of sweden, national central bureau of statistics, stockholm. 2 . garraway, w.m., whisnant, j.p., furlan, a.j., phillips 11, l . h . , kurland, l.t. & 0-fallon, w.m.: the declining incidence of stroke. new engl. j . med. 300: 449-452, 1 9 7 9 . address for reprints: dr hans aberg department of medicine university hospital s-75014 uppsala sweden 62 upsala j med sci 96: 103-1 11, 1991 effects of dietary supplementation with vitamin e on human neutrophil chemotaxis and generation of ltb4 riitta luostarinen, agneta siegbahn’ and tom saldeen from the departments of forensic medicine, university of uppsala, dag hammarskjolds vag 17, s-752 37 uppsala, sweden and ‘clinical chemistry, university hospital, s751 85 uppsala, sweden abstract three weeks’ dietary supplementation with a moderate dose of vitamin e (45 iu dl-a-tocopherol acetate daily), in eight healthy volunteers significantly increased the serum vitamin e level from 12.3 k 3.3 to 16.2 & 3.7 mgll (means k sd) and significantly decreased neutrophil chemotaxis from 15 -+ 3 to 4 f 1 pm/h (means f standard error of the means). generation of leukotriene b, was not influenced by vitamin e, suggesting that the decrease in neutrophil chemotaxis was not due to blockage of the lipoxygenase pathway. neither was the plasma malondialdehyde concentration influenced by vitamin e, contradicting the possibility of an antioxidant effect of vitamin e. as one early event in neutrophil chemotaxis is a n increase in intracellular calcium concentration resulting from increased membrane permeability, it is possible that vitamin e influenced chemotaxis by a stabilizing effect on the neutrophil membrane, rather than by its antioxidant effect. vitamin e supplementation could thus be beneficial in pathological conditions with activated neutrophils, such as ischaemic heart disease. introduction t h e principal function of vitamin e is to act as a biological lipid antioxidant that scavenges free radical intermediates arising during peroxidation of unsaturated fatty acids (1). t h e effects of supplementation of the diet with vitamin e in cardiovascular disease are of interest, as many observations point to the involvement of lipid peroxidation in various stages of the pathological process. low density lipoproteins (ldl) are readily peroxidized and 103 taken up by macrophages in atherosclerotic lesions, and peroxidation of ldl is blocked by antioxidants such as vitamin e (20). further, damaged areas show a lower content of endothelium derived relaxing factor, probably as a consequence of degradation of this mediator by free radicals produced by inflammatory cells in the vessel wall ( i 1). extremely low vitamin e levels in the plasma have been noted in stroke, and low antioxidant defences have been found in patients with ischaemic heart disease and other angiopathic disorders (8, 12). recently it has been suggested that activated leucocytes may play a role in the pathogenesis of ischaemic heart disease, at least partly via release of free rdicz!s acd icterxtion =.vi:h the ecdstke!iun (27). neutrophils from rabbits receiving vitamin e have been found to be less adherent than control cells to the endothelial monolayer (14). also, leucocytes from these rabbits seemed to cause less endothelial cell damage in the pulmonary capillaries. it is thus possible that interference with leucocytes might explain some of the suggested cardioprotective effects of vitamin e. in the present investigation we have therefore examined the effects of a moderate dose of vitamin e on neutrophil chemotaxis and on the production of leukotriene b4 (ltb,) by neutrophils. material and methods subjects and study design eight healthy volunteers (5 men and 3 women) with a mean age of 49 k 8 years (mean k sd, range 33 57) were given 45 mg of vitamin e (45 iu dl-a-tocopherol acetate) as tablets once daily for three weeks. the subjects received no non-steroidal anti-inflammatory or any other drugs or additional vitamin supplements for at least ten days prior to blood sampling. they were told not to change their diets or their lifestyle during the experimental period and not to drink alcohol the day before blood sampling. they fasted overnight and were instructed to avoid vigorous physical activity in the morning of blood sampling, which was always performed at about 8 a.m. the volunteers gave their informed consent. the study was approved by the local ethical committee. blood sampling venous blood samples were taken without stasis with the subjects in the supine position. samples were taken before and after 3 weeks of dietary supplementation with vitamin e. 104 neutrophil preparation neutrophils were isolated from heparinized (10 iu/ml) venous blood. the separation was performed by a slightly modified density gradient separation method as previously described (16). t o the stock iso-osmotic percoll (pharmacia ab, uppsala, sweden) was added a solution consisting of one part hanks' balanced salt solution (hbss, ph 7.4) with calcium and magnesium and one part 0.075 mol/l tris-hc1 to obtain the desired densities 1.1005 g/ml and 1.0776 g/ml. the blood was layered on this discontinuous percoll gradient, which was then centrifuged at 400 g for 5 min at 4°c. the platelet-rich plasma supernatant was then removed and centrihgzticn was continue:! fer 20 min a: so0 g at 4°c. the neutrophil band was removed and washed in cold hbss without calcium a n d magnesium with 0.1% fatty acid free bovine serum albumin (sigma, st. louis, mo), and centrifuged at 400 g for 6 min at 4°c. ammonium chloride (0.155 mol/l) lysis of the erythrocytes in the cell pellet was used (a cold solution containing 8.29 g/l nh,ci, 1 g/l khc03 and 0.037 g/l na,edta, ph 7.4). the neutrophils were then washed with the same hbss solution a s above and centrifuged at 400 g for 5 min at 4°c. purified neutrophils were suspended in hbss with calcium and magnesium for the analysis of ltb, and in gey's solution (26) (for random migration) and in gey's solution with 0.2% human serum albumin (for chemokinesis and chemotaxis). the resulting preparation consistently contained more than 95% neutrophils. the viability was more than 95% as determined by trypan blue exclusion. ne utroph il c h emotaxis the migration was assayed by the leading front technique, using a modified boyden chamber (25). the filter pore size was 3 p m (millipore corp. bedford, ma) and the filter was about 150 p m thick. one hundred p l of neutrophils (final concentration 1.5 x 109/l) was placed above the filter and the chemoattractant, formyl methyl leucyl phenyl a l a n i n e (fmlp), was diluted in gey's solution containing 0.2% human serum albumin and added below the filter. fmlp was prepared a s a stock solution in dimethylsulphoxide (dmso) and kept at -20 "c before use. neutrophils were allowed to migrate for 60 min at 37"c, whereafter the filters were fixed, stained and mounted. migration of neutrophils was assayed as the migration distance of the two furthest migrating neutrophils i n the focus of one high power field (12.5 x 24) (17). the results are expressed as ymlh and are the means of readings of three different fields in duplicate filters. neutrophil chemotaxis was defined as the difference between neutrophil migration in response to fmlp (lo-* 105 mol/l, final concentration) and neutrophil migration towards gey's solution with 0.2% human serum albumin, i.e. chemokinesis or control. random migration did not change during the experiment. generation of ltb, by neutrophils one millilitre of the neutrophil suspension (5 x 109/l) in hbss with calcium and magnesium was warmed at 37°c for 2 min and t h e n stimulated with 10 pmol/l (final concentration) calcium ionophore a 23187. the calcium ionophore was dissolved in dmso (final concentration 0.5 %) and stored at -70°c before the experiments and then di!uted ir, hess. the ce!l suspensicn wm iccubatec! far 19 min ir, a shaking water bath and the reaction was stopped by adding 1 ml of ice cold methanol and placing the tube in ice-cold water. the cell suspension was then centrifuged at 2300 g at 4°c for 15 min and the supernatant was stored at -20°c prior to radio-immunoassay for determination of the generation of ltb, by neutrophils. the cross-reactivity with ltb, was loo%, with 20-oh-ltb4 and 6-trans ltb, 0.4% and with ltc4, ltd,, thromboxane b, and 6-keto-pgfl, < 0.05%. vitamin e in serum the serum was stored at -70°c pending measurement of the vitamin e concentration by high performance liquid chromatography (hplc) (10). the assays were kindly carried out by professor juhani hakkarainen at the department of clinical nutrition, swedish university of agricultural sciences, uppsala, sweden. malondialdehyde in plasma lipid peroxidation i n plasma was measured by determining the concentrations of malondialdehyde in plasma by fast performance liquid chromatography (fplc) with a pep-rpctm c-18 reversed phase column after reaction with thiobarbituric acid (22). after blood sampling, 500 p l of the plasma was immediately mixed with 10 pl of 5% butylated hydroxytoluene in ethanol and then stored at -70°c until assayed. statistical methods student's t-test for paired observations was used to compare values in the same subjects before and after dietary supplementation with vitamin e. means +standard deviations (sd) are shown unless otherwise stated; p values < 0.05 were regarded as significant. 106 results neutrophil chemotaxis a significant decrease in neutrophil chemotaxis, from 15 k 3 to 4 f 1 p d h , was observed after addition of vitamin e (45 iu daily) to the diet for 3 weeks (means k standard error of the means; p < 0.05; fig.1). generation of ltbl by neutrophils the generation of ltb4 by neutrophils did not alter significantly after 3 weeks of dietary supplementation with vitamin e (before treatment: 9.0 k 1.5 ag/5x1!y ce!ls; after trectmcr,:: 3.9 f 1.1 ng/5xlch czlk; table i). vitamin e in serum after dietary supplementation with vitamin e for 3 weeks, the serum vitamin e level was increased to 16.2 l3.7 mg/l from an initial value of 12.3 k 3.3 mg/l (p < 0.001; table 1). malondialdehyde in plasma t h e concentrations of malondialdehyde in plasma were 0.21 k 0.05 pmol/l before the addition of vitamin e to the diet and 0.25 k 0.09 ,umol/l after 3 weeks of this treatment. this change was not significant (table 1). discussion the moderate dose of vitamin e (45 iu daily) given in the diet for three weeks in this study resulted in an increase in the vitamin e level in the serum from 12.3 & 3.3 to 16.2 k 3.7 mg/l. many other investigators have used much higher doses. in one study administration of 1600 iu of vitamin e daily for two o r more weeks to volunteers increased the vitamin e level in the serum from about 1 1 mg/l to about 18 mg/l (3). although a much lower dose of vitamin e was used in our study, the increase in the serum vitamin e level was almost the same. there is some evidence that absorption of high levels of a-tocopherol acetate in humans ( d o 0 iu/d) may be restricted by some limitation in the hydrolysis of the ester at this level of intake (2). it is known from earlier studies that changes in the vitamin e level in leucocytes parallel those in the plasma after dietary supplementation (13). after addition of vitamin e to the diet for three weeks the neutrophil chemotaxis was significantly decreased. to the best of our knowledge 8-918572 107 chemotaxis 0 t weeks 0 3 before after vitamin e vitamin e figure 1. the effect of dietary supplementation with vitamin e (45 iu) for 3 weeks on neutrophil ehemotaxis (mean k standard error of the mean, n=8, *p<0.05). fmlp, 10-8 mol/l, was used as a chemoattractant. such a change in chemotaxis after vitamin e supplementation has not been reported previously. t h e mechanism of this vitamin e effect is unclear. the generation of ltb,, the 5-lipoxygenase product derived from arachidonic acid, did not alter significantly, suggesting that the change in neutrophil chemotaxis was not due to blockage of the lipoxygenase pathway. goetzl (9) has shown that physiological levels of a-tocopherol in vitro d o not reduce but rather enhance the lipoxygenation of arachidonic acid in human neutrophils. only higher, unphysiological levels exert a suppressive effect that is consistent with the role of vitamin e as a hydroperoxide scavenger (9). villa et al. (23) found that vitamin e in vitro inhibited not only arachidonate-induced but also ltb4-induced 108 table 1. the effects of 3 weeks' dietary supplementation with vitamin e (45 iu dl-ct-tocopherol acetate daily) on the levels of vitamin e (mg/l, n=8) in serum and malondialdehyde (mda, pmol/l, n=8) in plasma and on the generation of ltb, by neutrophils (ng/5xlw cells, n=7 before and n=8 after). vitamin e mda ltb4 before 12.3 -c 3.3 0.65 f 0.05 9.0 +. 1.6 js.3 16.3) (6.46 8.93) (6.7 1 1.9) after 16.2 f 3.7 *** 0.62 -c 0.07 9.9 f 1.1 (10.0 20.1) (0.42 0.89) (8.4 1 1.0) values are means and sd, range within parentheses. ***p < 0.001 aggregation of human neutrophils, suggesting that the effects of vitamin e on human leucocyte aggregation may reflect non-lipoxygenase related actions. vitamin e quinone, an oxidized form of vitamin e, seems to be just as efficient as vitamin e itself in counteracting platelet aggregation (5, 21). these findings make i t necessary to look for other explanations than the antioxidant theory as the basic mechanism responsible for the action of vitamin e. previous studies have shown that vitamin e in vitro exerts antiaggregatory effects in platelets without inhibiting the enzymes of the arachidonic acid cascade, but that the mobilization of intracellular calcium to the cytoplasm is significantly inhibited by vitamin e (4, 5 , 19). o n e of the earliest events in chemotaxis is a rapid increase i n the intracellular concentration of calcium, which is a consequence both of calcium displacement from membranous stores and of a selective increase in plasma membrane permeability (18). vitamin e has also been found to decrease the liposome membrane permeability (6) and it might therefore make the plasma membrane less permeable to calcium. in a recent study i t was observed that neutrophils from vitamin e-treated rabbits depolarized more and hyperpolarized more rapidly than placebo cells (24). this finding might indicate differences in the early activation of and ionic flux in neutrophils after administration of vitamin e so that they more rapidly return to a less activated state (24). vitamin e, which is highly lipophilic, 109 could thus exert a membrane-stabilizing influence within the membrane lipid bilayer that is not necessarily related to its antioxidant properties. recent findings indicate that activated leucocytes might participate in o r modify certain pathological conditions, e.g. the development of myocardial infarction, reperfusion arrhythmias or loss of endothelium derived relaxing factor after reperfusion (15). if so, the present results might suggest a role for vitamin e in preventing such conditions by decreasing leucocyte activation. however, the results obtained in the present open study need to be confirmed in a placebo-controlled investigation. f z ; x r i n g :he pas sib!^ scr,e;'l;cia! cffcc: of vitamin e in ca;diovasct;!rtr disease is the finding that in a current cross-cultural epidemiological investigation of healthy middle-aged men representing 1 1 european populations, the a-tocopherolkholesterol ratio in the plasma has shown a strong inverse correlation with mortality from ischaemic heart disease (7). acknowledgements this work was supported by grants from the swedish medical research council and the afa fund, sweden. references 1. 2. 3. 4. 5. 6. 7. 8. 9. 110 bieri, j.g., corash, l. & hubbard, v.s.: medical uses of vitamin e. n engl j med 308:1063-1071, 1983. bjorneboe, a., bjorneboe, g.-e.a. & drevon, c.a.: absorption, transport and distribution of vitamin e. j nutr 120:233-242, 1990. boxer, l.a., harris, r.e. & baehner, r.l.: regulation of membrane peroxidation in health and disease. pediatrics 64:7 13-7 18, 1979. butler, a.m., gerrard, j.m., peller, j., stoddard, s.f., rao, g.h.r. & white, j.g.: vitamin e inhibits the release of calcium from platelet membrane fraction in vitro. prostaglandins med 2:203-216, 1979. cox, a.c., rao, g.h.r., gerrard, j.m. & white, j.g.: the influence of vitamin e quinone on platelet structure, function, and biochemistry. blood diplock, a.t.: the role of vitamin e in biological membranes. in: biology of vitamin e, pp. 45-55. ciba found symp 101, pitman books, london, 1 983. gey, k.f.: inverse correlation of vitamin e and ischemic heart disease. int j vitam nutr res suppl30:224-23 1 , 1989. gey, k.f.: on the antioxidant hypothesis with regard to arteriosclerosis. in: scientific evidence for dietary targets in europe (eds. j.c. somogyi & a. trichopoulou), pp. 53-91. biblthca nutr dieta, no. 37, karger, basel, 1986. goetzl, e.j.: vitamin e modulates the lipoxygenation of arachidonic acid in leukocytes. nature 288: 183-1 85, 1980. 551907-914, 1980. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. hakkarainen, j.r.v., tyopponen, j.t., hassan, s., bengtsson, g.s., jonsson, l.s.r. & lindberg, p.o.: biopotency of vitamin e in barley. br j nutr 52:335-249, 1984. harrison, d.g., freiman, p.c., armstrong, m.l., marcus, m.l. & heistad, d.d.: alterations of vascular reactivity in atherosclerosis. circ res 61 horning, e. & lin, s.: analytical studies of human plasma lecithins, cholesteryl esters, free fatty acids and alpha-tocopherol. in: tocopherol, oxygen and biomembranes (eds. c. de duve & 0. hayaishi), pp. 273 282. elsevier, north holland biomedical press, amsterdam, 1978. kitagawa, m. & mino, m.: effects of elevated d-alpha(rrr)-tocopherol dosage in man. j nutr sci vitaminol35: 133-142, 1989. lafuze, j.e., weisman, s.j., ingraham, l.m., butterick, c.j., alpert, l.a. & baehner, r.l.: the effect of vitamin e on rabbit neutrophil activation. in: pinlogy of vitamin e. pp. 130-146. ciha foiind syvp 101, pitman rnoks, london, 1983. mehta, j., nichols, w.w. & mehta, p.: neutrophils as potential participants in acute myocardial ischemia. relevance to reperfusion. j am coll cardiol ringertz, b., palmblad, j., rhdmark, 0. & malmsten, c.: leukotriene induced neutrophil aggregation in vitro. febs lett 147: 180-182, 1982. siegbahn, a., garcia, r.c., kishi, k., nilsson, k. & venge, p.: specific binding of b-cll cell-derived chemokinetic inhibitory factor (cif) to human polymorphonuclear leukocytes. eur j haematol39: 172-179, 1987. snyderman, r. & goetzl, e.j.: molecular and cellular mechanisms of leukocyte chemotaxis. science 2133330-837, 1981. srivastava, k.c.: vitamin e exerts antiaggregatory effects without inhibiting the enzymes of the arachidonic acid cascade in platelets. prostaglandins leukotrienes med 21: 177-1 85, 1986. steinbrecher, u.p., parthasarathy, s., h a k e , d.s., witztum, j.l. & steinberg, d.: modification of low density lipoprotein by endothelial cells involves lipid peroxidation and degradation of low density lipoprotein phospholipids. proc natl acad sci usa 81 :3883-3887, 1984. steiner, m. & mower, r.: mechanism of action of vitamin e on platelet function. ann n y acad sci 393:289-298, 1982. sonnerborg, a., carlin, g., akerlund, b. & jarstrand, c.: increased production of malondialdehyde in patients with hiv infection. scand j infect dis 20:287-290, 1988. villa, s., lorico, a., morazzoni, g., de gaetano, g. & semeraro, n.: vitamin e and vitamin c inhibit arachidonate-induced aggregation of human peripheral blood leukocytes in vitro. agents and actions 19: 127-13 1, 1986. weisman, s.j., lafuze, j.e., haak, r.a. & baehner, r.l.: effect of vitamin e on fmlp-induced activation of rabbit polymorphonuclear leukocytes. inflammation 11:309-321,1987. wilkinson, p.c.: chemotaxis and inflammation, pp. 33-53. churchill livingstone, new york, 1974. wilkinson, p.c.: chemotaxis and inflammation, pp. 168-172. churchill livingstone, new york, 1974. zweier, j., kayburn, b., flaherty, j. & weisfeldt, m.: recombinant superoxide dismutase reduces oxygen free radical concentrations in reperfused myocardium. j clin invest 80: 1728-1734, 1987. (suppi ii):i1-74-11-80, 1987. 11:1309-1316,1988. c o r r e s p o n d e n c e : tom saldeen m.d., ph.d., d e p t . of forensic medicine, university of uppsala, dag hammarskjolds vag 17, s-752 37 uppsala, sweden 1 1 1 upsala j med sci 90: 107-114, 1985 surgery of renal cancer with extensive caval invasion suggestion for a new approach thomas andreen, torkel aberg and h e fritjofsson departments of urology and thoracic surgery, uniuersity hospital, uppsala, sweden abstract r a d i c a l s u r g e r y f o r r e n a l c a n c e r w i t h i n v a s i o n o f t h e i n f e r i o r vena cava can i m p r o v e t h e p a t i e n t ’ s q u a l i t y o f l i f e and, i n some cases, o f f e r l o n g e r s u r v i v a l or even c u r e . with a c a r e f u l l y p l a n n e d s u r g i c a l approach i t i s p o s s i b l e t o re move r e n a l tumours w i t h t h r o m b o t i c e x t e n s i o n t o t h e most p r o x i m a l p a r t o f t h e i n f e r i o r vena cava w i t h o u t n e c e s s i t y f o r c a r d i o p u l m o n a r y bypass and w i t h o u t un due r i s k t o t h e p a t i e n t . i n t h e o p e r a t i v e p r o c e d u r e , good access and v i s u a l c o n t r o l o f t h e p r o x i m a l vena cava and a l l t h e c o n t r i b u t i n g v e i n s seem t o be c r u c i a l l y i m p o r t a n t . introduction r e n a l c a n c e r i s known f o r i t s tendency t o i n v a d e t h e venous v a s c u l a r t r e e . o f a l l r e n a l c a n c e r s , one i n f o u r i n v a d e s t h e r e n a l venous system, a r o u n d one f o u r t h o f t h e i n v a d i n g tumours w i l l i n v o l v e t h e vena cava i n g r e a t e r or lesser degree, and such i n v o l v e m e n t w i l l r e a c h t h e l e v e l o f t h e h e p a t i c v e i n s or h i g h er i n a l m o s t one o f f o u r cases. most a u t h o r s a g r e e t h a t vena c a v a l i n v o l v e m e n t does n o t i n d i c a t e a more a g g r e s s i v e f o r m o f tumour (l,ll), and t h a t t h e p r o g n o s i s depends more on t h e h i s t o l o g i c c h a r a c t e r i s t i c s and l o c a l g r o w t h o f t h e tumour i n t h e k i d n e y and on o c c u r r e n c e o f tumour i n t h e l o c a l lymph nodes or d i s t a n t m e t a s t a s i s . other a u t h o r s have a t t r i b u t e d p r o g n o s t i c s i g n i f i c a n c e t o t h e l e v e l o f vena c a v a l i n v o l v e m e n t ( 1 4 ) . i t i s g e n e r a l l y accepted, however, t h a t r e m o v a l o f n e o p l a s t i c c a v a l thrombus g i v e s good p a l l i a t i o n and i m p r o v e s t h e p r o s p e c t s f o r l o n g t e r m s u r v i v a l ( 2 , 3 , 6 , 8 ) when t h e r e i s no p e r i n e p h r i c e x t e n s i o n o f tumour. as u n t r e a t e d vena c a v a l i n v o l v e m e n t c a r r i e s a n e a r l y 100 p e r c e n t s h o u l d b e a t t e m p t e d i n p a t i e n t s w i t h l o c a l l y r e s e c t a b l e r e n a l c a n c e r who a r e f i t enough f o r p r o t r a c t e d s u r g e r y . m o r t a l i t y w i t h i n one y e a r ( 1 5 ) , r e m o v a l o f c a v a l tumour thrombus the l i t e r a t u r e c o n t a i n s numerous e x c e l l e n t r e v i e w s o f d i f f e r e n t s u r g i c a l 8-858572 107 approaches (1,3,5-10,12,13). in this paper we present our own experience with a partly new approach that provides excellent access to the upper part of the in ferior vena cava, thus permitting good control of bleeding and prevention of fatal pulmonary embolism. material and methods in the past f o u r years four patients were referred to o u r hospital because o f renal cancer with involvement o f the inferior vena cava up to o r past the entry o f the hepatic veins ( f i g . 1). fig. 1. level of caval involvement. 2) cases 1 and 2, up to and above the hepatic veins, 5) cases 3 and 4, up to but not above the hepatic veins. the age range of the two men and two women was 47 to 78 years. all underwent radical nephrectomy and caval thrombectomy. the postoperative observation time is short (at most 15 months), but at the time of writing a l l four patients are alive and well. preoperative evaluation excretory urography, including tomography, was performed preoperatively in 108 a l l cases. this e x a m i n a t i o n was f o l l o w e d by s e l e c t i v e r e n a l angiography, com p u t e d tomography and i n f e r i o r venocavography. bone scans and c h e s t r a d i o g r a p h y were p e r f o r m e d i n a l l cases f o r d e t e c t i o n o f d i s t a n t m e t a s t a s e s . o p e r a t i v e p r o c e d u r e i n t h r e e o f t h e f o u r cases t h e o p e r a t i o n was p r e c e d e d b y i n s e r t i o n o f a double-lumen swan-ganz c a t h e t e r i n t o t h e r e n a l a r t e r y on t h e a f f e c t e d s i d e and t h e b a l l o o n was i n f l a t e d under f l u o r o s c o p i c c o n t r o l a f t e r p r e m e d i c a t i o n . the o t h e r p a t i e n t was n o t c a t h e t e r i z e d . the p r i n c i p l e s o f t h e t e c h n i q u e a r e s c h e m a t i c a l l y p r e s e n t e d i n f i g . 2. the t e c h n i q u e can b e o u t l i n e d as f o l l o w s . a f t e r i n d u c t i o n o f a n a e s t h e s i a t h e p a t i e n t i s p o s i t i o n e d on t h e o p e r a t i n g t a b l e w i t h a s m a l l p i l l o w under t h e l o w e r t h o r a c i c and upper lumbar s p i n e . a m i d l i n e i n c i s i o n i s made from t h e sternum t o j u s t below t h e u m b i l i c u s and t h e abdomen i s c a r e f u l l y e x p l o r e d f o r s i g n s o f m e t a s t a t i c d i s e a s e . the r e n a l turnour i s p a l p a t e d and i t s r e s e c t a b i l i t y assessed. when o p e r a b i l i t y has been a s c e r t a i n e d , t h e a s c e n d i n g o r descending c o l o n , i n c l u d i n g t h e r i g h t or l e f t p a r t o f t h e t r a n s v e r s e c o l o n ( h e p a t i c o r s p l e n i c f l e x u r e ) i s m o b i l i z e d , as i s t h e duodenum. the l i v e r i s m o b i l i z e d by d i v i d i n g t h e d i a p h r a g m a t i c a t t a c h m e n t s , a l l o w i n g t h e l i v e r t o b e r o t a t e d f r o m s i d e t o s i d e on t h e a x i s o f t h e i n f e r i o r vena cava. i n t h i s way good access i s o b t a i n e d t o t h e a n t e r i o r p a r t o f t h e vena cava. the hepatoduodenal l i g a m e n t i s i d e n t i f i e d and e n c i r c l e d w i t h a r u b b e r band. rumel t o u r n i q u e t s a r e a p p l i e d t o t h e c o n t r a l a t e r a l r e n a l v e i n and, d i s t a l l y , t o t h e vena cava. a l l lumbar v e i n s a r e i d e n t i f i e d and d i v i d e d between s i l k l i g a t u r e s . s p e c i a l c a r e i s t a k e n t o i d e n t i f y and d i v i d e t h e second p a i r o f lumbar v e i n s , as o t h e r w i s e t h e y may be a m a j o r s o u r c e o f b l e e d i n g . a median s t e r n o t o m y i s t h e n p e r f o r m e d up t o t h e t h i r d i n t e r c o s t a l space and t h e i n c i s i o n i s c o n t i n u e d 5-7 cm i n t h a t space on t h e r i g h t s i d e , a v o i d i n g t h e p l e u r a . a s t e r n u m r e t r a c t o r i s i n t r o d u c e d and t h e p e r i c a r d i u m opened i n t h e m i d l i n e and sewn t o t h e wound edges. a rurnel t o u r n i q u e t i s a p p l i e d on t h e i n t r a p e r i c a r d i a l p a r t o f t h e i n f e r i o r vena cava. a p u r s e s t r i n g s u t u r e o f 3 / 0 p r o l e n e i s p l a c e d r o u n d t h e r i g h t a u r i c u l a r appendage. the s u t u r e i s drawn t h r o u g h a s m a l l b o r e r u b b e r t u b e . the a u r i c u l a r appendage i s opened and t h e r i g h t i n d e x f i n g e r i n t r o d u c e d t h r o u g h t h e p u r s e s t r i n g s u t u r e , w i t h t h e surgeon r e s p o n s i b l e f o r t h e t h o r a c i c p a r t o f t h e o p e r a t i o n s t a n d i n g t o t h e l e f t o f t h e p a t i e n t . the p r o x i m a l p a r t o f t h e i n f e r i o r vena cava can t h e n b e p a l p a t e d . by i n v e r t i n g t h e a u r i c l e a s f a r down as p o s s i b l e , t h e o r i f i c e s o f t h e l i v e r v e i n s c a n b e r e a c h ed. the i n f e r i o r vena cava i s t e m p o r a r i l y o b s t r u c t e d within t h e p e r i c a r d i u m i n o r d e r t o assess t h e haemodynamic consequences o f t h i s p r o c e d u r e . i f t h e a r t e r i a l p r e s s u r e d r o p s c o n s i d e r a b l y , t h e c a v a l o b s t r u c t i o n i s r e l e a s e d . the a o r t a i s t h e n m o b i l i z e d i n t h e d i a p h r a g m a t i c h i a t u s , t o e n a b l e i t s c l a m p i n g . f i g . 2. schematic v i e w o f t h e anatomic s i t u a t i o n a t o p e r a t i o n o f r e n a l c a r cinoma w i t h tumour i n v a s i o n o f t h e r e n a l v e i n and i n f e r i o r vena cava. rumel t o u r n i q u e t s a r e a p p l i e d on t h e c o n t r a l a t e r a l r e n a l v e i n , l o w e r i n f e r i o r vena c a v a and t h e i n t r a p e r i c a r d i a l p a r t o f t h e i n f e r i o r vena cava. a p u r s e s t r i n g s u t u r e i s p l a c e d r o u n d t h e r i g h t a u r i c u l a r append age w i t h t h e s u t u r e drawn t h r o u g h a s m a l l b o r e r u b b e r tube. 110 a l l v e s s e l s c o n t r i b u t i n g t o t h e b l o o d f l o w i n t h e upper p a r t o f t h e i n f e r i o r vena cava a r e t h e n clamped f i r s t t h e h e p a t i c a r t e r y and t h e p o r t a l v e i n i n t h e hepatoduodenal l i g a m e n t , t h e n t h e v e i n o f t h e c o n t r a l a t e r a l k i d n e y and, f i n a l l y , t h e l o w e r i n f e r i o r vena cava. i n one o f o u r cases t h e i n f r a d i a p h r a g m a t i a a o r t a a l s o was clamped. the t o u r n i q u e t around t h e i n t r a p e r i c a r d i a l p a r t o f t h e i n f e r i o r vena cava i s now drawn t i g h t a r o u n d t h e f i n g e r , and t h e d i s s e c t i o n o f t h e tumour thrombus i s p e r f o r m e d b l u n t l y w i t h t h e f i n g e r as f a r down as i t can r e a c h . a t t h e same t i m e t h e abdominal team makes a 5-7 cm l o n g i t u d i n a l i n c i s i o n i n t o t h e vena cava. the tumour thrombus i s t h e n d i s s e c t e d from w i t h i n t h e v e i n , u s i n g a l t e r n a t i v e l y a s m a l l n e u r o s u r g i c a l d i s s e c t o r and tumour f o r c e p s . a f t e r c o m p l e t e r e m o v a l o f t h e tumour thrombus, t h e o p e n i n g i n t h e vena cava i s t e m p o r a r i l y o c c l u d e d w i t h a l a r g e v a s c u l a r clamp. a n o t h e r clamp i s a p p l i e d a c r o s s t h e e n t r a n c e o f t h e a f f e c t e d r e n a l v e i n . the i n c i s i o n i n t h e c a v a l w a l l i s c l o s e d w i t h a r u n n i n g v a s c u l a r s u t u r e and t h e clamp i s removed. the v a s c u l a r clamps a r e t h e n r e l e a s e d , b e g i n n i n g w i t h t h e a o r t i c clamp, t h e n t h e hepatoduo d e n a l , f o l l o w e d b y t h e clamps o n t h e d i s t a l vena cava and t h e r e n a l v e i n o f t h e h e a l t h y k i d n e y . f i n a l l y t h e rumel t o u r n i q u e t o b s t r u c t i n g t h e vena cava is r e l e a s e d . the a f f e c t e d r e n a l v e i n i s opened and r e m a i n i n g tumour removed, t h e ca v a l w a l l i s s u t u r e d and t h e clamp removed. t h e r e a f t e r a s t a n d a r d e x t r a c a p s u l a r nephrectomy i s performed, i n c l u d i n g d i s s e c t i o n o f t h e h i l a r lymph nodes. a suc t i o n d r a i n i s p l a c e d i n t h e abdomen and two c h e s t t u b e d r a i n s i n t h e p e r i c a r d ium. results the r e s u l t s i n o u r cases a r e summarized i n t a b l e 1. the e x c e s s i v e b l e e d i n g i n case 1 a r o s e f r o m d e f e c t i v e c o n t r o l o f one lumbar v e i n . the p r o l o n g a t i o n o f t a b l e 1. c a v a l i n v a s i o n c u r r e n t e x p e r i e n c e o f t h e o p e r a t i o n f o r r e n a l c a n c e r w i t h e x t e n s i v e case o p e r a t i n g p e r o p e r a t i v e p o s t o p e r a t i v e hosf o l l o w u p no age/sex t i m e ( h r s ) b l e e d i n g ( m l ) p i t a 1 s t a y ( d a y s ) (months) 1 47/m 6.5 20 700 3 1 15 2 58/f 6.0 6 900 11 10 4 64/m 4.0 8 100 11 2 3 78/f 6.0 7 800 10 6 1 1 1 hospital stay was due to subfebrility and general malaise, with liver tests in dicating non-a, non-8 hepatitis. discussion renal cell carcinoma has a tendency to grow along venous channels. electron microscopy has revealed tumour cells t o surround thin-walled blood vessels that often have fenestrations, allowing tumour to prolapse into the lumen ( 4 , l l ) . in most cases there is no actual overgrowth of tumour on the wall of the vena cava, although the tumour thrombus can be strongly adherent to the vessel wall. most authors agree that extension of renal tumour into the inferior vena cava does not necessarily imply a bad prognosis, if radical surgery is successful ( 2 , l l ) . the observation by others (14) that the prognosis worsens with increasing height of thrombus extension into the vena cava can probably be ascribed to the time factor. the higher the level of tumour thrombus, the greater is the prob ability that the kidney tumour has been present for a long time and thus is loc ally advanced. at present there is no effective therapeutic alternative to radical surgery, and we therefore believe that, whenever possible, such surgery should be at tempted in cases of the described type. we have found that a sternum-splitting incision up to and extending into the third intercostal space on the right side, combined with a pursestring suture on the right auricle and introduction of the surgeon's finger into the inferior vena cava, gives excellent proximal vascular control and greatly assists the removal of tumour thrombus. our experience also is that meticulous care in clamping and/or dividing all tributaries to the in ferior vena cava gives an almost blood-free operating field. clamping time has not given rise to problems in our cases. the left kidney has an excellent col lateral venous flow clamping without ill effects. the liver can withstand a 30-minute interruption of blood supply. introduction of a swan-ganz catheter into the renal artery and insufflation of the balloon before surgery facilitates the dissection of the renal vein, as preliminary clamping of the artery is not then required. and the right kidney can tolerate at least 20 minutes of brief total obstruction of the inferior vena cava is tolerated by most pat ients. the arterial blood pressure regularly falls. if the fall is too pro nounced, the pressure is raised again by clamping the infradiaphragmatic aorta. this additional manoeuvre probably is not dangerous, as the liver and the con tralateral kidney are already compromised. i t was necessary in only one of our four cases. no signs of embolism or metastases to the lungs have appeared in any of our patients. 112 i n one o f t h e f o u r p a t i e n t s t h e tumour thrombus extended i n t o t h e h e p a t i c v e i n s , o b s t r u c t i n g t h e b l o o d f l o w o f t h e l i v e r , w h i c h was g r o s s l y e n l a r g e d . a t t h e end o f t h e o p e r a t i o n , however, t h e l i v e r had become m a r k e d l y s m a l l e r . the p r e o p e r a t i v e symptoms o f a b d o m i n a l f u l l n e s s s u b s i d e d a f t e r t h e o p e r a t i o n . references 1. 2. 3. 4. 5. 6. 7. 8 . 9. 10. 11. 12. 1 3 . 14. beck, d.a.: r e n a l c e l l c a r c i n o m a i n v o l v i n g t h e i n f e r i o r vena cava. radio l o g i c a l e v a l u a t i o n and s u r g i c a l management. j u r o l 118:533-537,1977. c h e r r i e , r.j., goldman, d.g., l i n d n e r , a. & de k e r n i o n , j.b.: p r o g n o s t i c i m p l i c a t i o n s o f vena c a v a l e x t e n s i o n o f r e n a l c e l l carcinoma. j u r o l 128: clayman, r . v . , gonzales, r . & f r o l e y , e.e.: r e n a l c e l l c a n c e r i n v a d i n g t h e i n f e r i o r vena cava. j u r o l 123:157-163,1980. c o l v i n , r.b. & d i c k e r s i n , g . r . : p a t h o l o g y o f r e n a l tumors. i n : g e n i t o u r i n a r y cancer. ed. s k i n n e r , d.g. & de k e r n i o n , j.b. . saunders, p h i l a d e l p h i a , ch 4:84,1978. cummings, k.b., l i , w . i . , ryan, j.a., h o r t o n , r. & paton, r . r . : i n t r a o p e r a t i v e management o f r e n a l c e l l c a r c i n o w a w i t h s u p r a d i a p h r a g m a t i c c a v a l ex t e n s i o n . j u r o l 122:829-832,1979. g i t t e s , r.f.: l o c a l l y e x t e n s i v e r e n a l c e l l c a r c i n o m a c u r r e n t s u r g i c a l management o f i n v a s i o n o f vena cava, l i v e r o r bowel. proc 1 s t i n t e r n a t sympos o n k i d n e y tumours. ed. kuss, r., murphy, g.p., khoury, s. & k a r r , j.p.. l i s s , new york:497-507,1982. 910-912,1982. g i u l i a n o , l., m a r t o r a n a , g . , g i b e r t i , c . & p e s c a t o r e , d.: i1 t r a t t a r n e n t o c h i r u r o i c o d e l l ' a d e n o c a r c i n o m a r e n a l e con t r o m b o s i n e o o l a s t i c a d e l l a cava i n f e r i o r e . m i n e r v a c h i r u r g i c a 36:401-408,1981. kearney, g.p, waters, b.w., k l e i n , l.a., r i c h i e , j.p. & g i t t e s , r.b.: s u l t s o f i n f e r i o r vena c a v a r e s e c t i o n f o r r e n a l c e l l carcinoma. j uro klein,f.a., s m i t h , v.m.j. & g r e e n f i e l d , l.j.: e x t r a c o r p o r e a l c i r c u l a t f o r r e n a l c e l l carcinoma w i t h s u p r a d i a p h r a g m a t i c vena c a v a l t h r o m b i . j u r o l 131:880-883,1984. 769-77331981. mccullough, d.l. & g i t t e s , r.f.: vena cava r e s e c t i o n f o r r e n a l c e l l c a r cinoma. j u r o l 112:162-167,1974. s k i n n e r , d.g., p f i s t e r , r.f. & c o l v i n , r.: e x t e n s i o n o f r e n a l c e l l c a r c i n oma i n t o t h e vena cava: t h e r a t i o n a l e f o r a g g r e s s i v e s u r g i c a l management. j u r o l 107:711,1972. s m i t h , b.m., m u l h e r i n , j.l., sawyers, j.l., t u r n e r , b.j., p r a g e r , r.l. & dean, r.h.: s u p r a r e n a l vena cava o c c l u s i o n . p r i n c i p l e s o f o p e r a t i v e man agement. ann s u r g 199:656-668, 1984. sogani, p.c., h e r r , h.w., b a i n s , m.s. & whitmore, w.f.: renal c e l l c a r c i n oma e x t e n d i n g i n t o i n f e r i o r vena cava. j urol 130:660-663,1983. sosa, e.r., muecke, e.c., vaughan, d.e. & mccarron, j . p . : r e n a l c e l l c a r cinoma e x t e n d i n g i n t o t h e i n f e r i o r vena cava. the p r o g n o s t i c s i g n i f i c a n c e o f t h e l e v e l o f vena c a v a l i n v o l v e m e n t . j u r o l 132:1097-1100,1984. ena re 125: on 113 1 5. valvo, j.r., cos, l.r., altebarmakian, v.k., khuri, f.j. & cocket, a.t.k.: surgery and immunotherapy in renal cell carcinoma involving i n f e r i o r vena cava. urology 20:359-364,1982. address f o r reprints: thomas andrken department o f urology university hospital s-751 85 uppsala sweden 114 upsala j med sci 85: 217-224, 1980 excitability of squid axon membrane in the absence of ion-concentration gradient across the membrane susurnu terakawa from laboratory of neurobiology, national institute of mental health, bethesda, maryland, u s a and laboratory of cell biology, national institute of physiological sciences, okazaki, 444 japan abstract a squid axon membrane separating two solutions of the same chemical com position can exhibit electrical excitability. passage of a constant inward current through such a membrane induces oscillatory responses in membrane poten tial. the salts of cobalt, manganese, nickel, and barium are suitable as a constituent in the solution for demonstrating this oscillatory response. introduction in order to construct a physicochemical theory of nerve excitation, it is advantageous to start the reasonings on the basis of experiments carried out under a simple ionic condition. one successful approach along this line was the demonstration of bi-ionic action potentials, namely, excitation processes which involve only two cations (7-9, 15-17). i present here an alternative approach, a study on membrane excitability involving only one species of cation. the absence of ion-concentration gradient under mono-ionic conditions may reduce ionic theory for nerve excitation into a simple form, thus greatly facilitate understanding of nerve excitation from physicochemical points of view. materials and methods a giant axon of the squid (loligo pealei) was excised and mounted in a lucite chamber containing natural sea water. perfusion were introduced into the axon. chamber was replaced with a continuously flowing solution which contained a divalent-cation salt at the concentration of 1 or 2 mm. two glass cannulae for internal next, the natural sea water in the subsequently, the 217 axoplasm was replaced with the same solution as that used externally. finally, both potential recording and current-supplying electrodes were inserted into the perfusion zone through the outlet cannula (see fig. 1). fig. 1 schematic diagram of the experimental set up used. the solution was prepared by adding a small amount of a concentrated divalent-cation solution to a 12% (v/v) glycerol solution. the electrode used for recording the internal potential was a combination of ag-agc1 wire and a thin glass pipette filled with 0.6 m kc1-agar. the electrode used for measur ing the external potential was a calomel half cell. platinum wire electrodes were sometimes used instead of these kc1-containing electrodes. the current supplying electrodes were pieces of platinized platinum wire placed inside and outside of the axon. a constancy of the membrane current was assured by a 1omg resistance placed in the circuit in series to the axon membrane. results when axons were perfused intracellularly and extracellularly with solu tions containing a cobalt salt only, the potential difference across the membrane remained quiescent in the range of -20 to +15 mv. upon application of a constant electric current through the membrane, oscillatory variations of the transmembrane potential were observed. the response shown in fig. 2 (upper trace) was obtained with using solutions containing 1 mm cobalt citrate and 12% glycerol internally and externally. these responses could be induced only when the direction of current was inward. an abrupt rise (an upward deflection in fig. 2 ) in internal potential was accompanied by a large (7-fold) increase in membrane conductance which was measured by super posing small pulses of current on the sustained inward current. the shape and the amplitude of these oscillatory responses varied widely 218 v i 0 1 30 pa/crn2 1 i u . i 5 s fig. 2 oscillatory response obtained with 1 mm cobalt citrate internally and externally. from axon to axon. usually at the beginning of observation on one axon, the amplitude of responses was large, rise and fall of the responses were very quick and the period in which the membrane potential stayed in high level was long. afterward, this period became shorter and the fall of the membrane potential became slow. when the current applied to the membrane lasted for more than 1 min, the responses gradually became smaller in amplitude and shorter in duration. however, large and long responses reappeared after the current had been interrupted and then re-established, suggesting the existence of a kind of refractoriness. later, the frequency decreased again, and even tually oscillatory responses failed to appear upon simple application of in ward current. responses could be induced further by superposing small and short outward current pulses on the sustained inward current. in this case, the amplitude of the small pulses had to be larger than a certain level which might be called a threshold. the conductance change, the refractoriness, and the presence of threshold suggest that the oscillatory variation of the mem brane potential is a phenomenon similar t o a repetitive firing of action potentials. very similar responses in membrane potential described above could be observed with the use of manganese salts in the place of the cobalt salt. in addition to manganese and cobalt salts, barium chloride and nickel 219 chloride could be used to demonstrate oscillatory responses. when a barium chloride solution was used, rise and fall of membrane potential repeated at - . . high frequency (fig. 3 ) . sometimes, the response appeared as a burst of mv 0 -40 -80 spikes; the fig. 3 oscillatory response obtained with 2 mm barium chloride internally and externally. . 11: i 5 s y w m 2 fig. 4 oscillatory response obtained with 2 mm nickel chloride internally and externally. when nickel chloride was used, the duration of oscillatory responses was long and the frequency of them was low (fig. 4 ) . it was difficult to obtain responses of large amplitude by using nickel chloride. it was extremely difficult to obtain oscillatory responses when a solu tion of calcium chloride, magnesium chloride, or strontium chloride was used. in 10 axons examined with each solution, fall of the membrane potential was not large in spite of a strong inward current. very small responses shown in fig. 5 were barely obtained with a 2 mm calcium chloride solution. it was also 220 0 i . i30 5 s pa/cm2 fig. 5 oscillatory response obtained with 2 mm calcium chloride internally and externally. difficult to obtain the oscillatory response when solutions of cupric chlo ride, cadmium chloride, zinc chloride, and ferrous chloride were used. in these cases, however, a stepwise decrease in the inward current induced a mv 0 -1 v fig. 6 response in membrane potential obtained with 2 mm cadmium chloride internally and externally. single response as shown in fig. 6. superposition of a small pulse of out wardly directed current on the sustained inward current also induced a single response. these responses were similar to the deteriorated responses observed in the axons perfused with a cobalt or manganese solution. the strength of current applied to the membrane affected the amplitude and the interval of oscillatory .responses. with higher strength of current the amplitude of responses became larger and the interval between the fall of a response and the rise of the next response became longer. such a relation ship observed from an axon perfused intracellularly and extracellularly with a 2 mm manganese chloride solution is shown in fig. 7 . the thin broken line 22 1 indicates the weakest possible current which would induce the oscillatory response. fig. 7 dependence of the amplitude and the interval of responses on the strength of current. discussion the squid axon is found to be capable of maintaining electrical excit ability under mono-ionic conditions in which the internal and external concent rations of ions are the same. the results obtained and the conditions employed are very similar to those of inanimate membranes such as porous glass membranes (10, ll), sephadex gel membranes (13), polyelectrolyte membrnaes (5, 6 ) , and lipidic membranes (3, 4 ) . the physicochemical processes underlying oscillat ory phenomena in these inanimate membrane systems have been explained satis factorily by several investigators (1, 2, 12, 1 4 ) . a slight modification of the theories proposed for inanimate membranes may be enough to account for the phenomena described here, -provided that interactions such as electrostatic cross-linkage or coordination bonding between divalent cations and the membrane macromolecules are taken into consideration. the approach presented here probably fills the gap between the studies of artificial membranes and those of biological membranes living under normal ionic conditions. the result of detailed studies will be published elesewhere. 222 acknowledgements i thank dr. ichiji tasaki for his continued encouragement and interest in this study. the experimental work was done at the marine biological laboratory woods hole, massachusetts, u.s.a. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. references katchalsky, a. and spangler, r.: dynamics of membrane processes. q. rev. biophys. 1, 127-175. 1968. mears, p. and page, k . r.: rapid force-flux transitions in highly porous membranes. phil. trans. r. soc. lond. a . 272, 1-46. 1972. monnier, a . m.: experimental and theoretical data on excitable artificial lipidic membranes. j. gen. physiol. 51, 26s-36s. 1968. monnier, a . m., monnier, a , , goudeau, h. and rebuffel-reynier, a . m.: electrically excitable artificial membranes. j. cell. comp. physiol. 66, 147-154. 1965. shashoua, v. e.: electrically active polyelectrolyte membranes. nature, 215, 846-847. 1967. shashoua, v. e.: electrically active protein and polynucleic acid membranes. in: the molecular basis of membrane function. (ed. d. c. tosteson), pp. 147-159. prentice-hall inc., englewood, n.j. 1969. tasaki, i., lerman, l. and watanabe, a . : analysis of excitation process in squid giant axons under bi-ionic conditions. her. j. physiol. 216, 130-138. 1969. tasaki, i., takenaka, t. and yamagishi, s . : abrupt depolarization and bi-ionic action potentials in internally perfused squid giant axons. amer. j. physiol. 215, 152-159. 1968. tasaki, i., watanabe, a . and singer, i.: excitability of squid giant axons in the absence of univalent cations in the external medium. proc. nat. acad. sci., u.s.a. 56, 1116-1122. 1966. teorell, t.: a contribution to the knowledge of rhythmical transport processes of water and salts. exp. cell res. suppl. 3 , 339-345. 1955. 223 11. 12. 1 3 . 14. 15. 16. 17. teorell, t.: electrokinetic membrane processes in relation to properties of excitable tissues. i: experiments on oscillatory transport phenomena in artificial membranes. j. gen. physiol. 42, 831-845. 1959. teorell, t.: electrokinetic membrane processes in relation to properties of excitable tissues. 11: some theoretical considerations. j. gen. physiol. 42, 847-863. 1959. teorell, t.: oscillatory electrophoresis in ion exchange membranes. arkiv fiir kemi (ed. roy. swed. acad. sci.), 18, 401-408. 1961. teorell, t.: excitability phenomena in artificial membranes. biophys. j. 2, 27-52. 1962. terakawa, s.: ca-k bi-ionic action potential. biol. bull. 155, 469-470. 1978. watanabe, a., tasaki i. and lerman, l.: bi-ionic action potentials in squid giant axons internally perfused with sodium salts. proc. nat. acad. sci., u.s.a. 58, 2246-2252. 1967. yamagishi, s . : manganese-dependent action potentials in intracellularly perfused squid giant axons. proc. jap. acad. 49, 218-222. 1973. requests for offprints should be sent to: dr. susumu terakawa, national institute for physiological sciences, okazaki, 444 japan. received 80 10 07 224 ujms109_3.pdf upsala j med sci 109: 179–228, 2004 bystander cells and prognosis in hodgkin lymphoma review based on a doctoral thesis daniel molin department of oncology, radiology, and clinical immunology, uppsala university. abstract a free full-text copy of this article can be found at the web page of upsala j med sci: http://www.ujms.se hodgkin lymphoma (hl) is characterised histologically by a minority of malignant hodgkin and reed-sternberg (hrs) cells surrounded by benign cells, and clinically by a relatively good prognosis. the treatment, however, leads to a risk of serious side effects. knowledge about the biology of the disease, particularly the interaction between the hrs cells and the surrounding cells, is essential in order to improve diagnosis and treatment. hl patients with abundant eosinophils in the tumours have a poor prognosis, therefore the eosinophil derived protein eosinophil cationic protein (ecp) was studied. serum-ecp (s-ecp) was elevated in most hl patients. it correlated to number of tumour eosinophils, nodular sclerosis (ns) histology, and the negative prognostic factors high erythrocyte sedimentation rate (esr) and blood leukocyte count (wbc). a polymorphism in the ecp gene (434(g>c)) was identified and the 434gg genotype correlated to ns histology and high esr. the poor prognosis in patients with abundant eosinophils in the tumours has been proposed to depend on hrs cell stimulation by the eosinophils via a cd30 ligand (cd30l)-cd30 interaction. however, cd30l mrna and protein were detected in mast cells and the predominant cd30l expressing cell in hl is the mast cell. mast cells were shown to stimulate hrs cell lines via cd30l-cd30 interaction. the number of mast cells in hl tumours correlated to worse relapse-free survival, ns histology, high wbc, and low blood haemoglobin. survival in patients with early and intermediate stage hl, diagnosed between 1985 and 1992, was generally favourable and comparatively limited treatment was sufficient to produce acceptable results for most stages. the majority of relapses could be salvaged. patients treated with a short course of chemotherapy and radiotherapy had an excellent outcome. in conclusion prognosis is favourable in early and intermediate stages and there 179 received 14 april 2004 accepted 3 may 2004 are possibilities for further improvements based on the fact that mast cells and eosinophils affect the biology and prognosis of hl. introduction thomas hodgkin and the early reports of the disease thomas hodgkin was born in 1798 in tottenham, middlesex, england in a family of quakers. he was educated at the university of edinburgh, as he was unable to study at oxford or cambridge due to his quaker background. in 1832 he published the first article on malignant lymphomas called “on some morbid appearances of the absorbent glands and spleen” (1), where he described seven patients with generalised lymphadenopathy and splenomegaly examined post mortem. at the time thomas hodgkin made these initial studies he was a curator of the museum and inspector of the dead at guy’s hospital in london (2, 3). it was also known in the 19th century that enlarged lymph nodes could be caused by for example cancer or tuberculosis. however, hodgkin suggested that the disease in some cases could start de novo in the lymph nodes, not being the result of disease somewhere else in the body. indeed, the earliest known description of the appearance of enlarged lymph nodes and spleens dates back to 1669, written by malpighi from bologna (4), after his discovery of the blood capillaries in 1661 (2). material from four of the cases t. hodgkin described were preserved at the museum at guy’s hospital and later re-examined. two of the four cases were indeed hodgkin lymphoma, one probably another kind of lymphoma, and the last one an inflammatory lesion, possibly tuberculosis (2, 5, 6). considering the later development in diagnosis of malignant lymphomas and the never-ending story of lymphoma classifications, 50% correct diagnoses must be considered a high percentage, as these diagnoses were made before the disease was even named, and before microscopy was available. important accomplishments by t. hodgkin besides describing lymphomas were, to name a few, the introduction of the stethoscope in great britain, pathologically describing perforated appendicitis and local spread of cancer, describing aortic insufficiency, recommending more fiber and less sugar and meat in the diet, recognising the contagious nature of cholera, and suggesting improvements in the situation of the poor to prevent spreading of the disease (2, 3). later, when t hodgkin failed to get an appointment as consultant at guy’s hospital, after a controversy with benjamin harrison, the hospitals treasurer, his interest turned to philanthropic activities. for example he founded the “aborigines’ protection society” (2, 3). when he visited the holy land with his friend sir moses montefiore, he was struck by dysentery. he died in jaffa in 1866 and his grave is there to be visited by those wishing to honour this man whose name will always be associated with malignant diseases in the lymphoid system. on his gravestone the words “humani nihil a se alienum putabat” (“nothing of humanity was foreign to him”) are inscribed (2, 3). 180 why the disease changes name in the thesis the disease was named hodgkin’s disease in 1865 by sir samuel wilks (7), 13 years before the first histopathological description was published by greenfield (8), and 33 years after thomas hodgkin’s article was published. however, with some exceptions in this thesis i have chosen to refer to the disease as hodgkin lymphoma (hl) (or hodgkin’s lymphoma), which is more appropriate nowadays, when we consider hl to be a lymphoma among others rather than distinguishing between hodgkin’s disease and non-hodgkin lymphomas (nhl) as was done in the past. in the new world health organisation (who) classification the disease is also referred to as hl (9). dorothy reed and carl sternberg describing the malignant cell there are two other people who will always be associated with this disease, namely carl sternberg (1872–1935) and dorothy reed (1874–1964). they have given their names to the hodgkin and reed-sternberg (hrs) cells. these cells are indeed the malignant cells of hl, however they are in a minority in the tumours, a fact that will be further explored in this thesis. carl sternberg was an austrian pathologist who in 1898 described large cells characteristic for hl, however comprising a minority of the cells in the tumours (10). his description of the hrs cells was further refined by dorothy reed, who in 1902 published the article “on the pathological changes in hodgkin’s disease, with special reference to it’s relation to tuberculosis” (11) where she made clear that hl was not a variant of tuberculosis, and provided skilful drawings of the hrs cells. previously the high proportion of tuberculosis in hl patients had lead pathologists to assume that hl might be a variant of tuberculosis. however, reed showed that there was no reaction to tuberculin (11). the never-ending story of lymphoma classifications according to legend only three things in life are certain: that we will die, that we have to pay taxes, and that the lymphoma classification will change. whether this is correct or not, the fact is that the classification of lymphomas have changed a number of times and that the frequency of these changes does not seem to diminish over time, as we shall see. the first recognized attempt to classify lymphoproliferative diseases was made by rosenthal in 1936 (12). he divided hl into four different types, depending on the degree of lymphocytic infiltration. this classification also had prognostic significance. the next classification, by jackson and parker came 11 years later in 1947 and described the three groups paragranuloma, granuloma, and sarcoma (13). this was modified nine years later, when another group of granuloma with more marked fibrosis and better prognosis was described (14). another 10 years passed and the time came for a new classification. in this new classification a great effort was made to correlate the known microscopic features of the disease to prognosis. the result was a division into six different groups (15). from this the rye classifi181 182 cation, which divides hl into four different groups, was developed (16, 17). the groups according to the rye classification are: lymphocytic predominance (lp), nodular sclerosis (ns), mixed cellularity (mc), and lymphocytic depletion (ld). this classification was used for quite a long time; the next classification was published 28 years later (18). in the revised european-american lymphoma (real) classification both clinical and histopathological, as well as new immunophenotypic, cytogenetic and molecular knowledge is taken into account. classical hl (chl) is divided into lymphocyte-rich classical hl (lrchl) (provisional), ns, mc, and ld. chl is separated from nodular lymphocyte predominance (nlphl) (18). however, with the real classification only six years passed before the who classification emerged (9). in this classification (table 1), as in the real classification, chl is separated from nlphl, the histological groups are the same, but lrchl is no longer a provisional entity. the who classification is currently used by haematopathologists, but from past experience, we know that one day it will be changed. the ld subgroup is today very rare as many cases previously diagnosed as ld are now recognised as lymphomas other than hl, with anaplastic or pleomorphic large cell morphology (9, 19). the tumour cell the hrs cells comprise only 0.1–10%, usually less than 3%, of the cell population in chl tumours. morphologically these cells are large, 20–25 �m, and usually have two separate nuclear lobes with prominent eosinophilic nucleoli and pale chromatin. the cytoplasm is slightly basophilic (20). in nlphl the tumour cells are called popcorn or lymphocytic and histiocytic (l&h) cells. this histologic type is not discussed in detail in this thesis. the origin of the hrs cells is usually germinal centre derived b-cells (21–23), however, in rare cases they can be t-cell derived (23–25). the most accurate way of studying the hrs cells is by single cell analysis. in studies of this kind presence of somatic hypermutations of the immunoglobulin (ig) gene and simultaneous lack of ig mrna transcripts have been demonstrated. this is regardless of presence or absence of crippling ig gene mutations caused by an inactivation of the ig promotor. down-regulation of the octamer-dependent transcription factor oct2, its coactivator bob.1, and the transcription factor pu.1 are suggested as reasons for this (23, 26, 27). the subsequent incapacity of hrs cells to produce ig should lead to apoptosis, as is the case for normal b-cells that loose their capacity to express ig (28). however, in some way they escape apoptosis. different ways for the hrs cells to escape apoptosis have been suggested, such as constant nf�b activation (23, 29, 30). this activation can for example be caused by mutations in the i�b family (23, 30, 31). other possible ways of nf�b activation are via the tnf receptor associated factor traf 1 molecule (32), lmp-1, and cd40 (33, 34). of interest for this thesis cd30 can also induce nf�b activity (35, 36). the cd30 ligand (cd30l)-cd30 interaction is thought to be of great impor183 tance for the hrs cells proliferation (37, 38). cd30 is a member of the tumour necrosis factor (tnf)/nerve growth factor (ngf) receptor superfamily and is expressed on virtually all hrs cells (39, 40). other possible ways of escaping apoptosis are somatic mutation of the cd95 gene (41), possibly p53 mutations (42), bcl-2 expression (43), or rb associated mechanisms (44). however, there is no clear evidence for those mechanisms to be relevant in hrs cells escaping apoptosis. a newly described mechanism in escaping fas-mediated apoptosis is through cflip expression (45). several cytokines are produced by hrs cells (46). among these are interleukin-5 (il-5), an eosinopoietic factor (47), gm-csf, also stimulating eosinophils (48), il9, an autocrine growth factor for hrs cells (49, 50), possibly involved in mast cell activation (51), il-13, involved in mast cell activation (52) (fig 1), il-6 (53), il-7 (54), il-10 (55), and tgf-� (56), a factor considered responsible for the fibrotic bands of nshl (57). in addition to the cytokines, several chemokines have been demonstrated to be expressed by hrs cells (46). examples of these are rantes, which is a possible factor for mast cell attraction (58–60) and eotaxin (ccl11), which could be an explanation for eosinophil infiltration (59). however, results concerning eotaxin are contradictory and another study showed no expression in hrs cells, but showed expression in fibroblasts and macrophages, secondary to stimulation by tumour necrosis factor (tnf)-� from the hrs cells (61) (fig 1). other chemokines found to be expressed by hrs are, for example, thymus and activation related chemokine (tarc) (62), mdc (59), and ip-10 (59). the hrs cells great capability to communicate with the surrounding cells indicates that the hrs cells and the bystander cells are bilaterally dependent of, and 184 fig. 1. interactions between hrs cells, eosinophils, and mast cells. stimulate the presence of each other. it is therefore valuable to study the bystander cells in order to fully understand the biology of this tumour. the bystander cells the surrounding numerous benign cells, previously considered innocent in the disease process, dominate the histologic picture. these cells are for example tand b-lymphocytes, plasma cells, neutrophils, histiocytes, eosinophils, stromal cells, and, less known, mast cells (63–66). the t-cells in chl are to a large extent th2 cells and these are often situated around the hrs cells in a rosette-like manner, possibly recruited by mig, ip10, and tarc expressed by the hrs cells (62, 66, 67). the bystander cells could be interpreted as an immunological reaction against the tumour. however, it is now known that these bystander cells communicate with the hrs cells via various interleukins and cytokines (46, 68). it is also plausible that the bystander cells, and their communication with the hrs cells, are necessary for the progression of the tumour (38, 46, 47, 68–70). the well-known difficulties in establishing and culturing hrs cell lines are another indicator of the hrs cells’ dependence on other factors, such as the bystander cells. even though the hrs cells are indeed tumour cells, created by a series of events, which maybe initiated by hereditary factors and one or more viruses, the progression of the hl is, after malignant transformation, dependent of a complex web of interactions between cells of immunological and inflammatory importance. eosinophilic granulocytes eos (aurora in latin) is the goddess of the red sky at dawn in greek mythology (71, 72). every morning she opens the gates of the sky to her brother helios (the god of the sun). in histopathological routine staining the acidic dye eosin, named after her is widely used. the eosinophil is characterised by a bi-lobular nucleus and cytoplasmatic granules, stained by the eosin. being more or less a nymphomaniac, since being cursed by aphrodite after an unfortunate love affair, eos is the mother of many children, including the winds and the stars, among them the morning star, eosphoros (71, 72). the eosinophilic granulocyte is, however, not as quickly passing as the winds, as it remains in tissue for a few days up to weeks (73). also it is not as abundant in normal tissue as the stars in the sky. however, in many cases of hl eosinophils are present in large numbers (63, 64). we have previously shown a relationship between cases with abundant eosinophils in the tumour and poor prognosis (64), and this has been confirmed in a large german study (74), but questioned in a recent smaller study (75). there is also a correlation between abundant eosinophils and ns-histology. the correlation to survival is especially pronounced in ns-histology (74). eosinophilia in the bone marrow of hl patients does not affect prognosis (76), and blood-eosinophilia in hl is associated with a favourable prognosis (77, 78). in other tumours, such as colon cancer (79) and head and neck cancer (80) presence of tumour-associated tissue 185 eosinophilia is associated with a good prognosis. these data suggest a special role of eosinophils in hl tumours. the mechanisms behind the presence of abundant eosinophils in hl tumours can be secretion of il-5 (47) and gm-csf (48, 81) by the hrs cells (fig 1). another possibility is eotaxin, secreted from fibroblasts and macrophages, stimulated by tnf-� from the hrs cells (59, 61). the reason for the poorer prognosis in eosinophil rich cases of hl has been proposed to be the expression of a ligand to the cd30 receptor (cd30l), which is a member of the tnf/nerve growth factor (ngf) superfamily (38). this interaction can stimulate hrs cell proliferation (38, 68, 82) (fig 1). as an explanation for the correlation to ns-histology the eosinophils’ production of transforming growth factor � (tgf�) and subsequent stimulation of fibroblasts have been proposed (83, 84). eosinophil cationic protein (ecp) can also stimulate fibroblasts (85), which could contribute to the ns-histology if high levels of ecp are present in the hl (further discussed below). in physiological conditions the eosinophils are active in the defence against parasites and viruses, but probably not bacteria (86–88). other possible roles for the eosinophils are in wound healing and in the defence against carcinomas, via their cytotoxic granule proteins or interaction with other cells in the immune system (79, 80, 89, 90). the cytoplasmatic granules of the eosinophil contain a number of basic proteins. these proteins are major basic protein (mbp) (91), eosinophil protein x or eosinophil-derived neurotoxin (epx/edn) (92), epo (93), and eosinophil cationic protein (ecp) (94), further discussed below. there are also other proteins present in the granules, such as il-2 (95), il-5 (96), catalase (97), il-6 (98), tnf-� (99), rantes (100), and bacterial permeability increasing protein (bpi) (101). there are also other conditions, even more intimately associated with the eosinophils than hl, such as asthma, allergy, atopic dermatitis, and the uncommon condition hypereosinophilic syndrome (73). possible roles for the eosinophils in these conditions are, for example, tissue destruction and remodelling (102). after completion of their mission the eosinophils are supposed to go into apoptosis (103). eosinophil cationic protein (ecp) ecp was first described in 1977 in uppsala (94). the protein is a single-chain peptide of 133 amino acids, with a molecular weight ranging from 18–22 kda depending on level of glycosylation (104). ecp is a ribonuclease (105, 106) and member of the rnase a superfamily (73, 90). in experiments with artificial cell membranes ecp damages these, therefore ecp has been proposed to be a pore-forming protein (107). the physiological and pathophysiological functions of the protein are not fully understood. ecp has a capacity for parasite killing (87), which is probably one of the functions of ecp in physiologic conditions. it has also anti-bacterial (108), and 186 anti-viral functions (88). ecp also affects human cells. of interest for this thesis, histamine release from mast cells is stimulated by ecp (109–111). this is further discussed below. ecp can affect fibroblast function, as it inhibits proteoglycan degradation (85). this mechanism can stimulate fibrosis, possibly, for example, in creating the fibrotic bands in nshl. ecp also affects coagulation (112), and it affects plasma cells and b-cells, inhibiting ig production by these cells but not being cytotoxic to them (113, 114). mast cells the name mast cells comes from the german mast zellen, which means ‘well fed cells’ (115). they were named so by paul ehrlich, who also discovered eosinophils, and the name refers to their characteristic cytoplasmatic granules (115, 116). initially, mast cells were proposed to contribute to the host defence by phagocytosis (117). however well in accordance with their name, this is not their most important role in the body. instead, the mast cells have important regulatory functions in the inflammatory process (118). they regulate vascular functions (119) and activate other cells (118, 120) involved in this process. the mast cells also have a role in the immune response against parasites and possibly bacteria (121) and in angiogenesis (119, 122). upon stimulation the mast cells release the contents of their granules, which contain a variety of inflammatory mediators (119). subsequently, cytokines and other factors released by the mast cells activate different leukocytes (123). the most important factor in differentiation, migration, survival, and growth of mast cells is stem cell factor (scf) and its receptor kit (116). on the bad side of the coin, mast cells initiate immediate hypersensitivity reactions, by releasing histamine, stimulated by cross-linked ige, and, interestingly, also by ecp secreted by eosinophils (90, 109–111). ecp also stimulates the release of tryptase and the synthesis of prostaglandin d 2 (pgd 2 ), both vasoactive and proinflammatory mediators, in human heart mast cells (110). overall, the mast cell derived mediators in inflammation, for example in allergic reactions, can be categorised into the following three groups: 1. preformed granule mediators: including histamine, heparin, and proteases like tryptase. the proteases are pre-made, stored in granules and released after stimulation of the mast cells. possible functions of the proteases are tissue remodelling, degradation of microbial structures, and activation of other cells. 2. lipid-derived mediators: including leukotrienes (e.g. b 4 , c 4 ), and prostaglandins (e.g. d2). 3. cytokines, such as tnf-�, il-4, il-5, il-6, il-8, and chemokines (116, 123). diseases with known mast cell involvement are asthma, allergy, rheumatoid arthritis, and mastocytosis. mastocytosis is a rare group of conditions, involving different organs (124). the most common form, affecting the skin, is urticaria pigmentosa, which is indolent. there are also forms with an aggressive course, however these forms are almost always without skin involvement. symptoms of mastocytosis, mostly related to the histamine release, are, for example, pruritus, skin irrita187 tion, abdominal pain, headache, diarrhoea, and problems caused by abnormal haematopoiesis (124). mast cells are also present in different tumours (119, 125), including early reports of their presence in hl (65, 126). the reasons for this accumulation have not been investigated in detail (125). in the tumours the mast cells possibly promote angiogenesis and metastasis (127), and tumour growth (128). a well-known effect of the histamine release from mast cells is, as mentioned, pruritus, or itching. as will be described in the prognostic factor chapter pruritus is a feature of some cases of hl, and has also been related to prognosis (129, 130). another uncommon symptom of hl is pain at alcohol intake (131, 132). interestingly, this alcohol-induced pain can be relieved by intake of anti-histamines, suggesting a role of mast cells also in this unusual symptom (133). polymorphisms a polymorphism is a genetic variant more common in a population than could be explained by a mutation. polymorphisms that persist over many generations are commonly maintained because no one of the forms gives an overall survival advantage. the occurrence of single-nucleotide polymorphisms differs between different genes, as some contain many and some contain none (134). in a population, a polymorphism is distributed according to the hardy-weinberg equilibrium (135). the physiological and pathophysiological role of single-nucleotide polymorphisms is very heterogeneous, as some probably are of no relevance at all, while some alter the function of a protein, or the level of protein production, in such a manner that it can cause disease or affect the course of a disease. the knowledge of the role of single nucleotide polymorphisms in malignant disease is limited. polymorphism can never be the single cause of malignant disease, as they are present in a substantial proportion of the normal population. however, there are examples of single-nucleotide polymorphisms correlating with frequency or prognosis of lymphoma (136–138). if the polymorphism is situated in a gene encoding a protein with a pathophysiologic role in a certain disease, and the polymorphism alters the level of production or function of the protein, the polymorphism can, of course, correlate with the presence or progression of the disease. epidemiology and aetiology of hl the age-distribution of hl is characterised by a peculiar bimodal pattern, with a peak at 25–30 years of age and a steadily increasing incidence after 50 years of age (139). it could therefore be speculated that hl in different age groups are different disease entities. there is a difference in histology between the different age groups supporting this idea; ns-histology, the subgroup of special interest concerning eosinophils and mast cells as will be shown in this thesis, is more common in the young adults and mc-histology is more common in the elderly (140, 141). although the incidence of other lymphomas is alarmingly increasing in the western 188 world (142) this is not true for hl. the overall incidence of hl has decreased but the incidence in young adults has slightly increased (142–146). certain circumstances are associated with a higher incidence of hl in young adults and middle aged: high standard of living in childhood, such as single family housing, small family size, and a high level of maternal education (147). the prevalence of hl is higher in western europe and the united states than in asian countries (140). ns-histology and the first peak of the bimodal age distribution in young adults are features of hl in developed urbanised countries, and mcand ld-histology are more common in developing countries. in the developing countries the first peak is in young boys (140). the aetiology of hl is still not elucidated, but there are some clues. at least in young adult patients, an underlying genetic susceptibility is possible, as siblings (148) and, especially, monozygotic twins (149) to hl patients have an increased risk. there are also indications of an underlying infectious agent (150, 151). the only virus that so far has been shown to relate to hl is epstein-barr virus (ebv), the virus causing mononucleosis (152, 153). ebv is associated to mc-histology and to higher age, although not all reports are consistent (154–156). one could speculate on a correlation with asthma and allergy, on the grounds of the presence of eosinophils in the tumours and the correlation to survival (64, 74), however, no clear evidence of such a correlation has been found. although, in very rare cases hl has presented as an endobronchial lesion (157). in many cases hl is associated with an immune-deficiency with a reduced amount of cd4+ lymphocytes in peripheral blood (158). additionally, there have been observations of an immunological impairment in family members of hl patients (159). it has been suggested that a latent membrane protein(lmp-) 1 immune defect contributes to the development of ebv associated hl (160). interleukin(il-) 10, produced by the hrs cells may also have a role in this, as il-10 has inhibitory effects on t-cell mediated immunity (161, 162). there are also promising results from treatment of relapsed patients with ebv positive chl with ebv-specific cytotoxic t-cells (ctl) (163). clinical investigations and staging the staging of lymphomas has proven to be more stable over time than the classification. the different stages are shown in table 2 (164, 165). however, the investigations required for the staging has changed due to advances in radiology, and new knowledge about treatment and prognosis (see below). the currently recommended investigations are, provided that the hl diagnosis has been confirmed in a biopsy: medical history, physical examination; ear-, nose-, and throat-inspection (biopsies if there are suspect changes); laboratory investigations (b-hb, differential wbc, trombocytes, esr, crp, s-na, s-k, s-albumin, scalcium, b-glucose, s-creatinine, s-urate, s-asat, s-alat, s-bilirubin, s-alp, sldh, optional hiv-test); chest x-ray; computed tomography (ct) (or magnetic resonance tomography (mrt)) scan of chest, axillae, abdomen, pelvis, and for exam189 ple the neck region if it is primarily involved; ultrasonography of the abdomen (two abdominal investigations have been required to rule out hl in the abdomen), ctor ultrasound-guided biopsies if any suspect finding is seen, and bone marrow biopsy (not necessary in stage i-iia) (uppsala/örebro region care programme for lymphomas, 2001; treatment of adult patients with early stages of hodgkin’s disease, nordic lymphoma group study, 1999). 18f-fluorodeoxyglucose (fdg) positron emission tomography (pet) is emerging as a very promising investigation to rule out hl, especially in the abdomen (166, 167), for treatment assessment (168), and as a predictor of disease-free survival (169, 170). previously staging laparotomy with splenectomy was recommended, but this investigation has now been abandoned (20, 171–175). staging laparotomy with splenectomy is associated with morbidity and the need for this investigation is diminishing since more and more early stage hl patients receive chemotherapy (20, 132). in the past lymphangiography was frequently used as an abdominal investigation but as ct has become available it has also been abandoned (132). 190 prognostic factors many prognostic factors have been described for hl. however, when important prognostic factors have been described, then treatment has been adjusted and the patients with negative prognostic signs have been treated more aggressively. subsequently, these factors have in many cases lost their prognostic impact (176). this has happened for example with bulky disease (177). the hodgkin-specific and os have also been improved and are now very good for a malignant disease, which gives few events to study (176). this is all very good for the patient but makes studies on prognostic factors in hl difficult. in historical descriptions of hl it is evident that the natural history of the disease is highly variable (132). in some cases the disease remains localised for many years but in others it spreads rapidly causing the patient’s death after only a short period of time (11, 132). however, untreated the disease is virtually always fatal (11, 132). this huge difference in clinical course necessitated, historically as well as today, reliable prognostic factors (37, 132, 178). these are then used in order to group the patients into risk groups to determine appropriate treatment and to compare treatment results. as the prognosis today is so good, many patients are probably overtreated, with a risk of long-term side effects. this necessitates the finding of factors predicting both good prognosis and poor prognosis (37). factors related to the hrs cells, tumour burden, and dissemination of the disease as early as 1902 it was demonstrated that the spread of the disease correlated to prognosis (11). this has lead to staging systems of which the most important is the ann arbor system (164). the correlation between stage and prognosis has been repeatedly confirmed in clinical studies (132, 179, 180); however, nowadays this correlation is diminishing (176). the staging describes foremost the dissemination, but it also gives a hint of the total amount of disease. as time passed new prognostic factors were described. at first other ways of describing the amount of disease other than dividing it into stage i–iv, were introduced. in cotswolds in 1988, the ann arbor staging system was modified with the tumour bulk (181–184), and the number of sites involved (132, 185) added to the staging (165). in stage ii, spread of the disease can vary and the number of involved anatomic sites gives prognostic information (132, 185). tumour bulk (especially in the mediastinum) has historically proven to be important in early stage hl treated with rt alone (186–189), but in advanced stage, treated with chemotherapy, results are more inconsistent (190–193). efforts have been made to more accurately estimate the total amount of tumour cells by measuring the total macroscopic tumour burden and combining it with the tumour cell concentration in the hl involved tissue. this approach predicts prognosis (194–196). the relative tumour burden obtained from radiologically measurable tumour burden normalized to body surface area is also promising (197). soluble cd30 might also correlate with the number of hrs cells and correlates with prognosis (39, 162, 198–200). different factors reflecting the growth characteristics of the hrs cells are prognostic factors (37). 191 expression of caspase 3 in the hrs cells correlates to favourable prognosis (201). this finding indicates that the apoptosis cascade is important for tumour cell killing by chemotherapy. serum lactate dehydrogenase, commonly used as a prognostic factor in lymphomas other than hl, also have prognostic significance in hl, probably reflecting tumour cell turn-over (202). different localizations of the disease can also affect prognosis (203–205). factors related to the cellular composition of the tumours in accordance with the histologic pattern of few tumour cells surrounded by ‘bystander’ cells, morphological patterns of the tumours have been examined in the search for prognostic factors. mostly, this is done in the histological classifications described above. the different classifications have given prognostic information (37). especially the nlphl subtype behaves biologically in a different manner, giving the division between chl and nlphl introduced in the real classification (18). however, as a majority of hl patients are in the ns subgroup, an effort has been made to sub-group these (206, 207). unfortunately the prognostic information yielded could not be confirmed by other groups (208). among the surrounding cells, activated cytotoxic t-cells have been associated with a negative prognosis in one study (209). our group has described the negative prognostic impact of tumour eosinophilia (64). this is further discussed in the chapter dealing with the eosinophilic granulocyte. factors related to inflammatory activity and the release of cytokines and other factors several prognostic factors are related to cytokines and other factors secreted by the hrs cells or the surrounding cells. b-symptoms (37, 132, 210, 211), and high esr (132, 206, 212), have been introduced as prognostic factors. similar to esr, s-albumin (213), b-hb (213, 214), wbc (213), and lymphocytopenia (205, 214) are prognostic factors that can be related to cytokines secreted by the hrs or the surrounding cells. in addition to the b-symptoms, pruritus, or itching, has in some reports been demonstrated to have prognostic relevance (129, 130). this is particularly interesting when examining the role of mast cells in hl, as these cells are known to cause itching via histamine release. soluble vcam-1 and icam-1 have been described as prognostic factors (199, 215). several cytokines have also been studied as prognostic markers with promising results (37). host related prognostic factors several other, patientor host-related, prognostic factors, such as sex ( men having a worse prognosis (132, 216)), and old age (132, 216) have been introduced. patients with immune-deficiency related to hl have a worse prognosis, in a multivariate analysis (217). several prognostic factors are currently used, as will be described. 192 prognostic scores the scotland and newcastle lymphoma group (snlg) index, valid for all stages, was introduced by stephen proctor, newcastle, in 1991 (214, 216, 218). the patient’s age, clinical stage, absolute lymphocyte count, b-hb, and presence of bulky disease are required for calculation of the index. the index is then calculated by an equation involving these parameters, entered as absolute figures or as different scores. this prognostic index has proven accurate in selecting risk groups (219). in advanced stage hl an international prognostic score (ips) has been developed by the international prognostic factors project on advanced hodgkin’s disease and described by hasenclever and diehl (213, 220). the ips is used for patients with advanced hl between 15 and 65 years of age and takes into account seven binary adverse prognostic factors (table 3). it was developed from a database consisting of 5141 patients, of which complete data was available on 1618 patients. still there is a need for new prognostic markers, and scores, to be able to minimise treatment in order to diminish side effects in low-risk patients with a long expected remaining life span. these new prognostic markers are likely to be related to the biology of the disease. as the hrs cells are few and surrounded by numerous bystander cells it is logical to search for new prognostic markers in these interactions and the presence and quantity of the bystander cells. treatment and prognosis treatment of hl is one of the great success stories in non-surgical treatment of malignancies, together with treatment of childhood leukaemias and testicular cancer. apart from these diseases cure in malignant diseases is usually dependent on radical surgery but in these cases cure is reached through oncological treatment, i.e. medical treatment and radiation, alone. from being a disease that was incurable and virtually always ended with the patient’s death, hl has become a highly curable disease (132). radiotherapy for treatment of hl was introduced for the first time as early as in 1902 (221). it was subsequently refined (222), and improved the prognosis of hl dramatically (216, 223). when chemotherapy was introduced the treatment was giv193 en as single drugs, rendering only short remissions. a great break-through in hl treatment, and in oncology, was when combined chemotherapy regimens were introduced, initially the mopp (mechloretamine, vincristine, procarbazine, prednisone) regimen (224–226). the combined chemotherapy led to greatly improved long-term survival. as treatment today has given an overall favourable prognosis, but with a risk of late side-effects, such as secondary tumours, focus has switched from only achieving a cure, to additionally trying to avoid late side-effects (132, 227–233). this is further discussed in the next chapter. in table 4 a review of chemotherapy regimens used in hl is given. treatment in early and intermediate stage (i–iia) hl is also discussed in the next chapter. advanced stage hl is generally treated with a full course of chemotherapy, which often means eight courses, followed by rt if the disease was initially bulky or if there is any residual tumour after the chemotherapy (220, 228, 234). with this kind of treatment at least 80–90% of younger patients reach cr. however, the problem is that only 50–70% of advanced stage patients will remain long-term diseasefree (203, 226, 235), although recently improved results have been shown with the beacopp and escalated beacopp regimens (236). other promising new intensive chemotherapy regimens are stanford v (237, 238) and pvace-bop (219). however, stanford v was inferior to abvd, and the mec combination, in terms of response and ffs in a recent study (239). current treatment in advanced stage (iib–iv) hl in adult patients, 65 years or younger, in the uppsala/örebro region in sweden is: 0–2 risk factors according to ips: abvd × 6–8, 3–5 risk factors: standard beacopp × 6–8, and 6–7 risk factors: escalated beacopp × 6–8. six cycles of chemotherapy is chosen if the patient is in cr after 2 cycles. in patients above the age of 65 the treatment is chop × 6. in all of these groups, chemotherapy is followed by involved field rt, 1.76 gy per fraction to 30 gy, if the disease was initially bulky (care programme for lymphomas in the uppsala/örebro region, 2001). all patients in advanced stage hl should receive prophylactic antibiotic (trimetoprim+sulfonamid), anti-viral (aciclovir), and anti-fungal therapy (fluconazol). children (<18 years) are treated according to a separate programme. treatment and problems in patients with early and intermediate stage hl commonly early stage hl is defined as stage i–iia, and intermediate stage is defined as early stage with certain risk factors that differ between different groups. early stage hl has been treated with extended field rt with good results regarding the number of cr and freedom from failure (132, 223, 227, 228). the approach with extended field rt was first introduced by vera peters in the 1930–40s and then developed by henry s kaplan at stanford in the 1960s (223). the extended (or locally extended) field in patients with supradiaphragmatic disease is commonly mantle-field, including all supradiaphragmatic lymph nodes commonly involved: i.e. the cervical, supraclavicular, axillary, infraclavicular, superior mediastinal, hilar, 194 195 and supracarinal nodes. in patients with infradiaphragmatic disease the most common extended field is inverted y. other extended fields used in many patients are total nodal irradiation (tni), that is mantle plus inverted y, and subtotal nodal irradiation (stni). before the development of modern radiology techniques, laparotomy with splenectomy, i.e. ps, was introduced in order to exclude abdominal disease, to minimise treatment in early stage hl (164, 254) (see also clinical investigations and staging). unfortunately this procedure has led to an increased incidence of fatal pneumococcal infections and is now abandoned (132, 255). early and intermediate stages have also been treated with limited chemotherapy followed by either involved or extended field rt (132, 171–175, 227, 228, 235, 256), with favourable results. for a long time it was debated whether primary com196 bination therapy was beneficial or if those who relapsed after rt could be salvaged with chemotherapy and thus not have a worse prognosis. in a meta-analysis by specht et al (257), this was indeed the case. young patients in early and intermediate stages have a good prognosis with a high short-term survival almost regardless of treatment approach. however, there is a risk of serious side effects from the treatment. most important are the late side effects, for example induced malignancies, such as breast cancer secondary to radiation, acute leukaemias secondary to chemotherapy, lung cancer, and sarcomas (229–233). hypothyreosis is a very common late side effect and should always be 197 assessed in hl controls after treatment (231, 232, 258). patients irradiated to the shoulder region (for example with mantle treatment) often suffer muscular atrophy in that area (231, 232). heart complications are also of importance, especially in elderly patients (259). recently fatigue has been emphasised as a complication to hl treatment (260). the late effects are of outmost importance in young patients with a long expected remaining life time. of the acute side effects the most important are infectious complications (132). a national care programme for hl was introduced in sweden in 1985 (177). the concept of the programme was to give less intensive treatment than internationally recommended at that time, in order to minimise late side effects. prognostic subgroups were considered. the aim was to have no more than 20–30% relapses in any group and a high probability of salvaging relapsing patients. patients with low risk disease were treated with locally extended field rt alone, and patients with high risk disease were treated with a short course (1 cycle of mopp/abvd) of chemotherapy, followed by rt. laparotomy should be performed in some subgroups (see material and methods). an early evaluation after a mean follow-up of 5 years for patients diagnosed 1985–1989 (227) showed that the treatment results were favourable and fulfilled the initial objectives of the care programme. the model with a short course of chemotherapy followed by rt had been used at certain centres in the early 1980s, and is now introduced more and more into trials and as routine treatment by many large co-operative groups, but the long-term results of large patient groups have not been studied. an overview of results in early and intermediate stage hl trials is given in table 5. the idea of this table is to fairly comprehensively illustrate the improvement in treatment results over time and to give a background for the results achieved within the swedish national care programme. current treatment in the nordic countries in adult patients is in chl, stage ia–iia below 70 years of age with no risk factor 2 abvd with subsequent 1.76 gy × 17 to 30 gy involved field rt, and with risk factor 4 abvd followed by involved field rt. risk factors are: esr 50, more than two locals, and bulky disease for supradiaphragmal disease. in cases of infradiaphragmal disease the risk factor are: bulky disease, central or pelvis engagement, stage ii, and esr 50. in patients over 70 years the recommended chemotherapy is chop in the uppsala/örebro region. nlphl patients without risk factor are treated with involved field rt to 30 gy, and in those with risk factors 2–4 abvd followed by involved field rt to 30 gy. the drugs and major side effects of the abvd regimen are listed in table 6. material and methods patients and clinical characteristics a diagnostic tumour biopsy was available from all patients (i–v). before further analysis all cases were re-evaluated according to the real classification (18) (i–iv). 198 clinical and pathological staging was made according to the ann arbor system (164). complete remission (cr) was defined as disappearance of all known disease (i–v). in 54 newly diagnosed patients with hl (mean age 34, male/female ratio 2.4/1) ecp levels in serum (s-ecp) were measured at diagnosis, and a follow-up s-ecp was measured in 17 of these patients (i). forty-three hl patients (mean age 33, male/female ratio 1.2/1), and 70 apparently healthy medical students were analysed in the ecp polymorphism study. forty-two patients (mean age 35, male/female ratio 1.9/1) were included in the cd30l and mast cell study. in 36 of these patients serum tryptase was measured prior to treatment. a material of patients diagnosed between 1989 and 1994 has been collected and characterised. in 123 (mean age 43, male/female ratio 1.4/1) of these patients the material was available and sufficient for immunohistochemical analysis and they were included in the mast cell and prognosis study. three hundred and eight consecutive patients (median age 31, male/female ratio 1.2/1) in early and intermediate stages, age 17–59 years, were registered in the swedish care programme (177, 227) in 1985–1992. all of these were included in the study. treatment and staging according to the national care programme the staging was made according to the ann arbor system (164, 165). however, during the study described in paper v, lymphogram was abandoned as a hl investigation method. all patients were treated according to the recommendations in the swedish care programme for hl (177). in short, the treatment in early and intermediate stages was (227): in cs+ps ia only radiotherapy was recommended, except in bulky disease, when radiation was preceded by one cycle of mopp/abvd. in ps ib one cycle of mopp/abvd was followed by mantle irradiation, and in cs ib 3–4 cycles of mopp/abvd ± radiation was used. patients in cs iia were treated with 3–4 mopp/abvd ± radiotherapy, and in ps iia only radiotherapy, or, if the disease was bulky, 1 cycle of mopp/abvd followed by radiotherapy. in cs iib recommended treatment was 3–4 mopp/abvd ± radiation, and in ps iib one mopp/abvd + radiation. patients in stage iii, after laparotomy staged as ps iii 1 a, were treated with radiation, either alone or preceded by one cycle of mopp/abvd. in advanced stages treatment was three to four courses of mopp/abvd, followed by rt in cases of initially bulky disease, slow tumour regression or if the patient did not reach cr (234). in relapses after rt, treatment was chemotherapy if the relapse was within the treated area. if the relapse was outside the treated area and localized new rt could be given. patients relapsing after chemotherapy were recommended chemotherapy, 199 in early relapses (within one year) and for most late relapses the mime (methylgag, iphosphamide, methotrexate, and etoposide)-regimen was used, with or without subsequent high-dose therapy with stem cell support (264). in recent versions of the care programme treatment recommendations are as described in the treatment chapters in the background section. children were treated according to separate protocols. serum proteins s-ecp was measured by means of a commercially available kit (pharmacia and upjohn diagnostics, uppsala, sweden), immediately after the sampling (i). the 95% range of s-ecp in healthy individuals is 4–16 �g/l (265). serum levels of tryptase, a mast cell specific proteinase, were also measured using a commercially available kit (pharmacia and upjohn diagnostics, uppsala, sweden). immunohistochemistry the tissue specimens were routinely fixed in neutral buffered formalin, embedded in paraffin and sectioned in 3 �m thick sections. prior to the staining the slides were deparaffinised. the eosinophil-specific monoclonal antibodies (mab:s) eg 1 and eg 2 (pharmacia-upjohn diagnostics, sweden) (266) were visualised with the apaap technique. in order to visualise expression of cd30l in mast cells in hl tumours, a doublestaining technique for cd30l and the mast cell specific protease tryptase was developed. the anti-cd30l mab (igg2b isotype, genzyme diagnostics, cambridge, ma) was visualised with the dab method in a ventana machine (ventana bio tek systems, tucson, az, usa) according to the manufacturer’s instructions. after this procedure the slides were washed and microwave-treated, before they were stained with an anti-tryptase polyclonal rabbit antibody (pab) (267) utilising the apaap method. the tryptase-specific mab provided by dr. l.b. schwartz, mcv, richmond, va (268) was visualised with abc/hrp and dab technique. the positive and double-positive cells were counted in 10 randomly selected high power fields (hpf, 500x). an ocular with a lattice square net was used. in ns cases only cellular areas were counted. cell cultures one human mast cell line, hmc-1 (269), and one human basophilic cell line, ku 812 (270) were used. culturing was as previously described (271, 272). human mast cells were obtained by culturing umbilical cord blood cells in complete roswell park memorial institute (rpmi) 1640 medium supplemented with 10% heat-inactivated fetal calf serum (fcs), 50 ng/ml stem cell factor (immunex, seattle, wa, usa), and 10ng/ml il-6 (peprotech, london, uk) (273, 274). mast cell numbers and purity were determined by staining for tryptase. the purity of mast cells was >92%. u-698 was used as a positive control for cd30l (275). u-2932, a non-hodgkin lymphoma derived cell line, found to be cd30-negative (unpublished 200 observations) was also used. the hl-derived cell lines used were hdlm-2 (276), l 540 (277), km-h2 (278), and dev (279). other cell lines used were cl.mc/c57.1, a cloned growth factor-independent mouse mast cell line of balb/c origin (280), mcp5/1, a growth factor-dependent mouse mast cell line (281), and p815, a growth factor-independent mouse mastocytoma cell line (282). reverse transcription polymerase chain reaction (rt-pcr) total rna was prepared using tripure isolation reagent (boehringer mannheim, mannheim, germany). rna from hmc-1 cells was treated with heparinase i (sigma). one microgram of rna was reverse-transcribed by oligo-p (dt)15 priming using amv reverse transcriptase (kit from boehringer mannheim). for human cd30l amplification, a 571 bp fragment starting from position 312, and for mouse cd30l amplification a 554 bp fragment from position 242 were generated. the pcr products were size fractionated on agarose gel, stained and photographed. the identity of the products was confirmed by restriction enzyme cleavage and sequencing. flow cytometry the cells were stained with a mab against cd30l (m81, immunex), kit (yb5.b8, dr ashman, adelaide, australia), or isotype controls. following incubation and washing they were incubated with a fitc-labelled f(ab´)2 fragment of rabbit-anti mouse ig (dako, glostrup, denmark). the cells were then analysed using a facscan (becton dickinson, mountain view, ca, usa). endonuclease restriction digestion the samples were amplified with primers producing a 644 bp fragment. seventeen microliters of the pcr product (non-biotinylated) was incubated with 10u pst i in an appropriate digestion buffer (invitrogen). the samples were digested over night and subsequently analysed on an 1.5 % agarose gel containing ethidium bromide. to verify that the highly homologous dna region of the epx/edn (rnase2) gene had not been co-amplified, the 644 bp pcr fragment was subjected to the ecp gene specific cla i endonuclease digestion (283). this resulted in complete digestion (and two bands of sizes 241 and 403) showing that only the region containing the ecp gene had been amplified (data not shown). calculation of isoelectric point (pi) the pi’s of the ecp-variant proteins were calculated by vectornti software (infomax, north bethesda, md). statistical analysis all statistical analyses were made utilising the statistica software (version 4.5–6.0 statsoft, tulsa, usa). hl-specific survival, os, and rfs were visualised with kaplan-meier graphs and compared with the log-rank significance test. os was 201 defined as time from diagnosis to death of any cause. hl-specific survival was defined as time from diagnosis to death with hl. rfs was defined as time from diagnosis to first relapse. patients who never reached cr (or cru) were not censored and their time of follow-up was zero in this analysis. cox hazards regression model was used to compare the importance of different prognostic variables. differences in proportions were evaluated with the chi2-test, or fisher exact test, if the frequency in any group was too small (<5). to compare the actual distribution of a polymorphism with the calculated hardy-weinberg equilibrium distribution the chi2 test was used. the mann-whitney u test was used as a non-parametric test. to investigate correlations between two continuous parameters the spearman test was 202 used. we also used an analysis of variance (anova), followed by multiple comparison using fisher’s method. results eosinophils in hl tissue eosinophilia and serum ecp s-ecp was measured in 54 newly diagnosed hl patients. mean s-ecp was 25.4 �g/l (range 2.2–71.7 �g/l). in 61% of the patients s-ecp was higher than the upper normal limit (16�g/l). the s-ecp correlated to the number of eosinophils in the tumours (p=0.01) and to ns histology (p<0.05). there was also a correlation between s-ecp and the negative prognostic factor esr (p<0.01), and wbc (p=0.0002), and there was a tendency of an association between s-ecp and bulky disease (p=0.06). the number of eosinophils stained with eg 2 correlated to high esr (p<0.05), and to high leukocyte count (p=0.02). follow-up values of s-ecp were, in most of the cases where it was measured, lower than s-ecp at diagnosis. s-ecp levels according to clinicopathologic characteristics are shown in table 7. ecp polymorphisms as we had shown a correlation between eosinophils, ns-histology, and survival (64), and a correlation between s-ecp, eosinophils in the tumours, ns-histology, and negative prognostic factors, it was logical to study if polymorphisms, possibly affecting the function of ecp, could affect the course of hl. single nucleotide polymorphisms in the gene coding for ecp were found at three different positions; 277(c>t), 434(g>c), and 562(g>c), base numbers referring to gene bank no. nm_002935. polymorphisms at position 277(c>t) and 434(g>c) result in the following amino acid shifts: arg45cys and arg97thr. the polymorphisms 277(c>t) and 562(g>c) were present only in a heterozygous form and 434(g>c) was present in both a heterozygous and a monozygous form. pi calculations of the variant proteins showed that the pi was 10.2 for arg45cys and 10.5 for arg97thr. the original ecp has a pi of 10.7. as 434(g>c) was located at a restriction endonuclease site for the enzyme pst i, inhibiting the dna cleaving activity of the enzyme, the material screened by sequencing could be rflp analysed by pst i digestion. the 434gg form gave complete cleavage, the 434cc did not cleave at all, and the heterozygous form, 434gc, gave both cleaved and uncleaved fragments. the results of this analysis in the seventy apparently healthy subjects studied were in accordance with the sequencing results. of the seventy healthy subjects, 3 (4%) were found to be homozygous for the 434c genotype and 30 (43%) were heterozygous, 434gc. the 277(c>t) polymorphism was found in a heterozygous form in 2 (3%) of the 70 subjects. the frequencies of alleles for both polymorphisms were in hardy-weinberg equilibrium. 203 the prevalence of the 434(g>c) polymorphism was investigated in 43 patients with hl. the results were similar to those of the healthy subjects, with 25 (58%) 434gg, 16 (37%) 434gc, and 2 (5%) 434cc. the 434gg genotype, however, was over-represented in patients with ns histology (72% versus 39% in non-ns patients, p = 0.03), which is seen in table 8. furthermore, the negative prognostic factor esr was higher among patients with the 434gg genotype (p = 0.009). b-hb tended to be lower for the 434gg genotype (p=0.06). 204 fig. 4. a. overall survival according to number of mast cells (p=0.06, n=123). b. disease-free survival according to number of mast cells (p<0.01, n=123). mast cells in hl expression of cd30l on mast cells and stimulation of hrs considering cd30l had been discussed as a mechanism of eosinophils stimulating the hrs cells (38), and that we noted that cells stained for cd30l in hl morphologically resembled mast cells, we decided to explore this further. one way of doing this was to develop a double staining technique for tryptase, detecting mast cells, and cd30l. tryptase-positive cells were visualised in all 42 examined tumours. fifty percent of these expressed cd30l and 66% of the cd30l-positive cells were tryptase-positive cells. double-positive cells were found in 95% of the tumours. more tryptasepositive and cd30l-positive cells were found in ns histology compared to the other histological groups, although the differences were not significant. no double positive cells were found in ld histology. there was a tendency to an association between double-stained cell infiltration and bulky disease (p=0.08). the tryptase levels in serum were within the normal range (284) (mean 5.1 ng/ml), and did not correlate to the number of mast cells in the tumours. expression of cd30l mrna was detected using rt-pcr in the human mast cell line hmc-1 and in two preparations of stem cell factor dependent cord blood cultured human mast cells. cd30l mrna was also found in the murine mast cell lines c57 and p815 and in murine bone marrow derived cultured mast cells, but not in the murine mast cell line mcp5/1, although expression could be induced by activation with ionomycin. expression of cd30l on the surface of hmc-1 cells and in vitro developed human mast cells was measured by flow cytometry. almost all hmc-1 cells were strongly positive for cd30l and cd30l was also shown on the in vitro developed human mast cells. to determine if mast cells can stimulate proliferation of hrs cells via cd30lcd30 interaction co-culture assays were performed. the cd30l positive mast cells induced a dose-dependent increase in [3h]-tdr uptake in the hl cell lines hdlm-2, dev, km-h2, and l-540. the increase in [3h]-tdr uptake that was seen in hrs cell lines could be inhibited by anti cd30l mab. the spontaneous uptake of [3h]tdr in hdlm-2 was also inhibited by this antibody. isotype control antibodies did not affect the proliferative response. the cd30l negative cell line ku812 did not 205 increase the uptake when co-cultured with hdlm-2 cells. instead, a reduction in proliferation was seen. hmc-1 cells additionally induced an increased [3h]-tdr uptake in the non-hodgkin lymphoma cell line u-2932. mast cell infiltration and prognosis tryptase positive mast cells were immunohistochemically visualised in 113 (92%) of the tumours and in every histopathological subgroup. the mean number of mast cells in the tumours was 19.2 (range 0–101) mast cells/10 hpf. seventy-one (58%) tumours had >10 mast cells/10 hpf and 52 (42%) had <10. mast cells were located both in proximity to the hrs cells and in areas of fibrosis. the mast cell infiltration correlated to ns-histology (p=0.008) and there was a tendency to an association between number of tumour mast cells and stage (p=0.07). rfs and os according to number of mast cells in the tumours are shown in figure 4. wbc and b-hb correlated to number of tumour infiltrating mast cells, as shown in table 9. esr also tended to correlate to number of mast cells in the tumours. hl in early and intermediate stages – treatment and prognosis overall 304 (99%) of the patients reached cr (or cru) after treatment. rfs at five years was 80% and at ten years 74% for all patients. in the individual stages the rfs was 79%–95% except in psiii1a where it was 53%. hl-specific five-year survival was 97% and hl-specific ten-year survival 92% (fig 5). os was 93% at five years and 85% at ten years. in total, 74 (24%) patients relapsed a first time, and 20 (6%) a second time, and 18 (6%) died in their disease. rfs and os were significantly worse in patients treated with radiotherapy alone compared to those treated with a short course of chemotherapy followed by rt in the whole material (p=0.0003 and p=0.004 respectively). also when radiotherapy alone was compared to all those treated with chemotherapy (including full chemotherapy) the differences in rfs and os were significant (p=0.006 and p=0.04 respectively). the difference in rfs was also pronounced in patients with bulky disease (p=0.0005). patients treated with one course of mopp/abvd and radiotherapy had an excellent outcome with, at ten years, rfs 95%, hl-specific survival 98%, and os 98%. concerning prognostic factors, in univariate analyses, histology, age, esr, and stage significantly affected the rfs, but not sex, b-hb, or bulky disease. in a multivariate analysis, only stage and mc-histology significantly affected the rfs. if treatment (rt versus chemotherapy±rt) was included in the analysis, treatment, stage, and esr significantly affected the outcome. rfs was not significantly worse in patients with 3 risk factors or more according to the international prognostic score (ips). of the 44 (14%) patients that died, 18 (6%) died from hl. of the 26 patients that died without hl, the cause of death was in eight cases other malignancies, six serious infections, three myocardial infarctions, one pulmonary embolism, one cerebrovascular lesion, one retroperitoneal bleeding, and one suicide. in four cases the 206 cause of death was missing, and in one case probably without hl, but with no autopsy performed. discussion bystander cells in hl the rationale for studying the surrounding cells in hl is quite clear; if the tumour cells do not survive without this inflammatory infiltrate, knowledge about the surrounding cells and their interactions with the hrs cells can give both new biological prognostic factors and possible therapeutic approaches, possibly less toxic than the traditional rt and chemotherapy. the results presented here show that both eosinophils and mast cells have a role in the pathogenesis of hl, and that there are similarities in their biological functions in hl. they should thus be considered not innocent bystander cells but guilty opportunists. eosinophils in hl tissue eosinophilia in tumours other than hl abundance of tissue eosinophils is associated with a better prognosis, assumed to be due to the cytotoxic activity of the eosinophils (79, 80, 285, 286). however, in hl heavy eosinophilia in the tumours is associated with a worse prognosis (64, 74), although this is debated (75, 208, 287, 288). furthermore, hl patients with blood eosinophilia have a better prognosis (77, 78), and patients with bone marrow eosinophilia have a similar prognosis to those without (76). this suggests a profoundly different role of the eosinophils in hl compared to other tumours. a possible explanation of the worse prognosis in patients with abundant eosinophils in their tumours could be that the eosinophils stimulate the proliferation of the hrs via cd30l-cd30 interaction (38, 69, 82, 289). however, we show that the predominant cd30l expressing cells in hl tumours are the mast cells. in paper i there is a tendency that tumours with ns-histology contain more eosinophils. this finding is demonstrated in previous studies (63, 64, 75, 290). the association between the eosinophils and the sclerosis associated with ns histology is not completely elucidated but a number of theories to explain it exist. one possibly contributing factor could be the secretion of tgf� from the eosinophils (83, 84). heavy molecular weight tgf� levels in urine are elevated in nshl patients (83), and much of the tgf� is secreted from eosinophils (84). another possible mechanism is the ability of ecp to inhibit proteoglycan degradation in fibroblasts (85). interestingly, prognosis in patients with ns-histology in general is relatively good (287), but patients with ns-histology and eosinophilia in the tumours have a poor prognosis (64, 74). the eosinophil infiltration, as estimated by eg2 positive cell count also correlates with wbc and there is a strong tendency to an association with esr. this suggests a role for the tumour eosinophils in the inflammatory activity of hl. this is further discussed below. 207 ecp the elevated s-ecp levels in a majority of hl patients and the strong correlation between s-ecp and eosinophil infiltration in the tumours shown in paper i implicate that large amounts of s-ecp is produced by eosinophils in the tumours. it also indicates that the ecp produced by these eosinophils has a role in the disease process. this is supported by the follow-up measurements, although analysed in relatively few patients, where the s-ecp in most cases had diminished as the patients went into remission. s-ecp could thus be used to follow treatment effects, especially for possible treatments directed against the eosinophils. the idea of ecp being important in the disease process is supported by the correlation of ecp and histology. one possible role of the ecp is in the fibrosis, as both s-ecp and tumour eosinophilia are associated with ns-histology. a possible mechanism of enhancement of the fibroblasts function is that ecp inhibits proteoglycan degradation in fibroblasts (85). in paper i, s-ecp tended to correlate to bulky disease. nearly all patients with bulky disease had ns histology. hence, a relationship between many eosinophils, being able to stimulate fibrosis, ns-histology, and large tumours is seen. therefore, it could be speculated that large tumour bulk is caused by eosinophil-driven fibrosis and cd30l-cd30 stimulation of the hrs cells. however, the association between s-ecp and bulky disease could also partly be explained by a proportional relationship between the tumour volume and s-ecp. in previous studies no correlation was seen between eosinophil infiltration and bulky disease (64, 74). the correlations between ecp, especially 434gg ecp (below), and disease activity could hypothetically be explained by an activation of the mast cells or the mast cells expression of cd30l by ecp, secreted from the eosinophils in the tumours. there are earlier studies suggesting a stimulatory effect of ecp on mast cells (109–111). this could also possibly explain why the eosinophils and ecp have a disease-supportive role, so distinctly different from the role in other tumours, where the eosinophils appear to be a defence against the tumour, and the ecp to have mainly a cytotoxic effect against the tumour cells (79, 80, 285, 286). these results also support the idea that ecp is not cytotoxic against the hrs cells. this is also in line with results showing absence of cytotoxic activity of ecp against blymphocytes but inhibition of immunoglobulin production in these cells (113, 114), as hrs cells are b-cell derived and lack ig expression (21–23). the correlation of s-ecp and wbc adds support to the theory of eosinophils and ecp as part in the disease process. secreted growth factors from the tumour eosinophils or hrs could stimulate the bone marrow resulting in a high wbc. the lack of significant correlations between s-ecp levels and survival in this study was probably due to short follow-up and a favourable prognosis. ecp polymorphisms we describe three different polymorphisms, two of which give amino acid substitutions in the ecp protein. the 434(g>c) changes an arginine to a threonine at posi208 tion 97 (arg97thr). the 434(g>c) polymorphism is common, as almost half the population has this substitution either as a heterozygous or homozygous variant. the frequency of 434(g>c) that we found (26%) is almost the same as the frequency shown by zhang and rosenberg (28%) (291). the polymorphism is in hardyweinberg equilibrium, which means that it is randomly distributed in the population. considering the polymorphism is equally distributed in hl as in the normal population the polymorphism does not seem to have any aetiological role in hl. however, it seems to have a role in the pathophysiology of the disease. the finding of a correlation with ns-histology suggests an altered biological function of the protein affecting the development of this specific histologic type. this could hypothetically be explained by an impaired ability of the ecp to affect fibroblasts via inhibition of proteoglycan degradation (85). the correlation to high esr and the tendency to lower b-hb in 434gg patients also indicates a link to the inflammatory process. a correlation between the 434gg genotype and allergic symptoms supports an altered biological function of the ecp depending on genotype (292). the influence on inflammation and on the course of the disease in hl could also be explained by a change in the ability to stimulate mast cells, which we demonstrate have a role in hl. the different forms of ecp could, for example, hypothetically differ in the ability to stimulate cd30l expression in mast cells. the stimulation of mast cells by ecp has been studied (109–111), but not a possible stimulatory effect on cd30l expression. mast cells in hl expression of cd30l and stimulation of hrs we also show that mast cells express cd30l, that they are the predominant cd30l expressing cells in hl, and that they can stimulate hrs cells via cd30l-cd30 interaction. mast cells were visualised in all examined hl tumours. the presence of mast cells in hl tumours has been demonstrated, but the role of these cells had not been examined before this study (65, 126). approximately half of the mast cells expressed cd30l. previously, cd30l has been shown on a number of other cell types, for example subsets of activated t cells, neutrophils, histiocytes/ macrophages, and eosinophils (38, 68, 293), but not on mast cells. in this material mast cells were also the dominating cell type expressing cd30l, showing that the cd30l expression of mast cells is more prevalent than that of eosinophils. we also confirmed the mast cells expression of cd30l by demonstrating that a human mast cell line and in vitro developed human mast cells expressed cd30l mrna and protein. interestingly, mast cells in ldhl did not appear to express cd30l, therefore it can be speculated that this expression is regulated by, for example, t-lymphocyte derived cytokines. however, only two ld cases were examined. when co-cultured with hrs cell lines mast cells were shown to stimulate growth 209 of the hrs cell lines, demonstrated as a dose-dependent increase in [3h]-tdr uptake, and this was abolished by addition of anti-cd30l mab. stimulation of hrs cell lines via recombinant cd30l and cd30l expressed by eosinophils have previously been shown (38, 82). this ability to stimulate the hrs cells via cd30lcd30 interaction taken together with the fact that most cd30l expressing cells in the tumours are mast cells suggest an important role for the mast cells stimulating the tumour cells in hl after being attracted, activated or stimulated to proliferation by the hrs cells. the attraction of mast cells can be via rantes (60) and possibly the activation via il-9, as il-9 has been shown to be expressed by hrs cells (50) and it has also been shown to stimulate murine mast cells. however, stimulation of human mast cells via il-9 has not been demonstrated. preliminary results indicate an up-regulation of the mast cells cd30l expression upon il-9 stimulation (not published). probably the hrs cells and the mast cells can also interact via, for example, il-13 (294). we also showed an inhibition of the spontaneous [3h]-tdr uptake in an hrs cell line by the anti-cd30l mab. this suggests an expression of cd30l on the hrs creating an autocrine loop. we have also detected cd30l mrna and protein in hrs cell lines in preliminary experiments, supporting this theory (not published). mast cells and prognosis our previous findings led to a study of the clinical role and possible relationship to prognosis of mast cells in hl. it is demonstrated that number of mast cells in the tumour predicts a worse relapse-free survival in hl patients, and that higher number of mast cells correlates with ns histology. mast cells were detected in most of the cases examined and in every histopathological subgroup. a cut-off point of 10 mast cells/10 hpf was chosen because of the distribution of mast cell numbers seen in this material and the material in which we studied cd30l. the disease-free survival was significantly poorer in patients with >10 mast cells/10 hpf and the os tended to be worse than in those with <10 mast cells/10 hpf. further attempts of sub-grouping those with >10 mast cells/10 hpf did not yield any further information. in stage iib–iv and bulky disease both disease-free survival and os tended to be worse in patients >10 mast cells/10 hpf. hence, a stimulatory effect of the presence of mast cells, not accentuated by an abundance of these cells is implicated. ns histology correlated to abundant mast cells in the tumours. the number of mast cells also correlated to known prognostic factors, such as high wbc, low bhb. this indicates a role of the mast cells both in the inflammatory reaction of hl and in the progression of the ns-histology. the number of mast cells correlates thus to unfavourable prognosis and might be an explanation to the previously mentioned correlation of itching and bad prognosis (129, 130). these results, taken together with results in paper i–iii demonstrate that 210 the eosinophils and the mast cells are not innocent bystander cells but guilty opportunists. early and intermediate stages treatment and prognosis generally favourable treatment results in patients with early and intermediate stage hl, treated with limited tailored treatment, in an unselected population-based material is shown. the overall hl-specific survival was 92% and os 85% after 10 years. the initial aim of at most 20–30% relapses was obtained in all subgroups except psiii1a, and the aim of salvaging most relapsing patients was also achieved. the treatment recommended in the swedish national care programme was, when it was introduced, in many cases less extensive than internationally recommended. in a comparison with the work of international groups during the same time our results are comparable (table 5). from this comparison we conclude that relatively limited tailored treatment can be given in order to diminish the late side effects without compromising the outcome of the patients. the best results in this material were obtained for patients receiving limited chemotherapy followed by rt. patients with risk factors (bulky disease or b-symptoms) receiving combined therapy had an even better survival than those without risk factors receiving rt alone. in patients with bulky disease treated with rt alone, violating the principles, results were much worse than in those receiving combined therapy. this is in line with the present international trend to treat all patients with early or intermediate stage hl with limited chemotherapy followed by if-rt (171–175, 295). the concept is that addition of a short course of chemotherapy allows limitation of the rt volumes, in order to potentially diminish long-term side effects. however, these principles were introduced prior to this trend. in this material the given rt was extended (mantle) field rt, therefore the results are not directly comparable to results from limited chemotherapy and if-rt. however, several studies have shown that if-rt is as effective as extended field rt when it is preceded by a short course of chemotherapy (296–298). another international trend some years ago was the abolishment of staging laparotomy with splenectomy (171–175). our results indicate that laparotomy can be avoided since the recurrence rates were comparable in csiia and psiia, treated with rt (table 2, paper v). the poor prognosis in patients over the age of 40 is previously described (299, 300). as these patients are not within the group of elderly patients, over 60 years, with established poorer prognosis (228, 301), a possible conclusion could be that patients within different age segments of the population under 60 years could gain from separate treatment strategies. however, it is not possible to draw such a conclusion on these results alone. a possible explanation of the different outcome could be a difference in histopathology, with more mc patients in this group. also, there was no impact of age in the multivariate analysis, in contrast to the impact of mc211 histology. however, it was not possible to histopathologically re-evaluate these cases and it is possible that some of these cases would be classified as lymphomas other than hl today. since these patients were treated changes have been made in the swedish recommendations. staging laparotomy with splenectomy has been abandoned. extended field (mantle and (s)tni) rt and rt alone is no longer recommended (except ifrt in lp-histology, stage i+iia without risk-factors). the recommended chemotherapy was in 1994 changed from mopp/abvd to mopp/abv, and in the nordic trial from 1999 to abvd. there are several ongoing international trials on early and intermediate stages. on the basis of previous eortc studies (table 5) the current h9 trial has been designed, in co-operation with gela (228). standard-treatment in the favourable group is 6 ebvp followed by involved field radiotherapy (if-rt) (36 gy), and this is compared to 6 ebvp + if-rt (20 gy) or no radiotherapy at all. the unfavourable group of patients is randomised between 6 abvd, 4 abvd or 4 baseline beacopp, all followed by if-rt. josting and diehl (172) conclude that recent trials have reported excellent results with combined-modality treatment in favourable (early stage) hl and that in early stage unfavourable (intermediate stage) new chemotherapy regimens can decrease failures and still reduce the need for radiation. in this paper they describe ongoing ghsg trials for these two groups comparing 2–4 abvd + 20–30 gy involved field radiotherapy for favourable (early-stage) and 4 abvd versus 4 beacopp + 20–30 gy involved field radiotherapy for unfavourable (intermediate stage). the aim of both minimising therapy, by for example substituting extended field rt with limited chemotherapy and if-rt, and investigations, by abandoning laparotomy with splenectomy, is to minimise the late side effects in these patients with a long expected remaining life span. these results from a population-based material with relatively long follow-up can give an indication about the long-term results of such an approach. the relatively limited tailored treatment in the swedish care programme thus fulfilled its purpose of producing favourable results, and additionally in an international perspective. patients with risk factors treated with a short course of chemotherapy followed by rt had an excellent outcome. most patients relapsing could be salvaged with chemotherapy. the late toxicity was also limited, further strengthening this approach. however, it is still too early to reliably evaluate the risks of secondary malignancies and cardiac mortality. prognostic factors in paper v, the risk factors that remained after univariate and multivariate analyses were: stage (psiii 1 a versus the others), esr, and mc histology. treatment also affected the outcome if it was included in the analysis, further emphasising the advantage of combined therapy. the ips, initially developed for advanced stages, did not add any prognostic information in this material. the snlg index was not 212 evaluated in this material, but can be considered in future swedish or nordic trials. the risk factors described in the current care programme are, for patients with supradiaphragmal presentation, bulky disease, number of involved sites, and esr. as there is a need for identifying patients where treatment can be further minimised to avoid late side effects, further research is necessary concerning prognostic factors, especially as the ips did not add prognostic information in this group. these prognostic factors are possibly found in the biology of the disease, i.e. in the role of the tumour cells (194), the bystander cells, and their communication with each other (64, 74). conclusions hl patients express high levels of s-ecp, probably originating from eosinophils infiltrating the tumours. high s-ecp correlated to negative prognostic factors and ns-histology. the 434(g>c) polymorphism in the ecp gene is common in the normal population. in hl it correlates to ns-histology and to high esr. mast cells express cd30l in vitro and in vivo, they are the predominant cd30l expressing cells in hl tumours, and they can stimulate hrs cells in vitro via cd30l-cd30 interaction. abundant mast cells in the tumours predict a worse relapse-free survival in hl patients, and a high number of mast cells correlates with ns histology. generally treatment results were favourable in patients with early and intermediate stage hl, treated with limited tailored treatment. risk factors that remained after univariate and multivariate analyses were: stage, esr, and mc-histology. treatment also affected the outcome if it was included. ips could not be used to predict outcome, therefore new prognostic factors would be valuable. these results suggest that the mast cells and eosinophils in hl should not be considered innocent bystanders but guilty opportunists. new prognostic factors are needed in early and intermediate stage hl. knowledge about the interactions between these guilty opportunists and the hrs cells 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(2001). involved field (if) radiotherapy is as effective as extended field (ef) radiotherapy after 2 cycles of copp/abvd in patients with intermediatestage hd. blood 98: 768 (abstr 3199). 299. vlachaki mt, hagemeister fb, fuller lm, besa pc, hess ma, brown b, cabanillas f, cox jd 227 (1997). long-term outcome of treatment for ann arbor stage i hodgkin’s disease: prognostic factors for survival and freedom from progression. int j radiat oncol biol phys 38: 593–9. 300. mauch pm (1996). management of early stage hodgkin’s disease: the role of radiation therapy and/or chemotherapy. baillieres clin haematol 9: 531–41. 301. enblad g, glimelius b, sundstrom c (1991). treatment outcome in hodgkin’s disease in patients above the age of 60: a population-based study. ann oncol 2: 297–302. corresponding author: daniel molin department of oncology, radiology and clinical immunology, oncology akademiska sjukhuset 751 85 uppsala phone: +46 18 611 02 13 daniel.molin@onkologi.uu.se 228 upsala j med sci 91:221, 1986 experiences from a laboratory evaluation of a solid phase analytical system kristoffer hellsing, catarina bernet and annika lindback department of clinical chemistry, university hospital, uppsala, sweden glucochek i1 from medistron, uk, was kindly supplied by orion diagnostica, sweden. this reflectometer was especially construc ted in order to make serial analyses possible. the test strip bg was kindly supplied by boehringer mannheim scandinavia. the edta-blood samples used had been sent to the laboratory for rou tine glucose determinations. imprecision: within-series ( 2 0 samples) imprecision values were in the order of 2-3 cv% within the range 5 15 mmol/l, but for samples with low glucose content ( 1 . 6 and 3.8 mmol/l) the imprecision was 6 . 6 and 9.7 cv% resp. in order to obtain these values a familiarization period for the technician was necessary. comparison: when 2 0 samples ( 3 1 2 mmol/l) were compared with two of our routine methods (glucose dehydrogenase method on grei ner g-300 and glucose analyser from ysi) the tested method gave a reasonable correlation but showed 2 0 % higher values. time factors: the first incubation time (blood on the rea gent strip, varied 1 0 3 0 0 s ) is important. after the recommended time, 6 0 s , there is still an increase of colour development. after an incubation time of about 1 5 0 s a plateau is reached. the second incubation time (after blood has been wiped off, varied 10-330s) is not critical. at 6 0 s the reading is on a slowly de creasing slope which reaches a minimum after 2 3 min. the changes are, however, s o small that even big misreadings are unimportant. influence of hematocrit: it has been claimed (kutter; schnelltest in der klinischen diagnostik, 1983, p. 5 5 ) that the result is inversely dependent of the hematocrit. we could confirm this observation. the decrease was, however, of the same size even using the reference method (glucose analyser). thus, the decrease is not dependent on the reagent strip but rather on the blood as such. 22 1 upsala j med sci 95: 245-248, 1990 general administrative and economical aspects lars mellin national board of health, helsinki, finland introduction because my field of knowledge, at least to some extent, is in administration, my intention is to give an overlook of the health care system in finland. i am doing this, as i hope, to describe a system, that in my opinion gives fairly good preconditions to arrive at a good standard of services in laboratory medicine as well. in this short presentation focus is put upon comments on general organization, on the planning process and allocating of resources, on general expenditure and last but not least important, on late tendencies in development of health care administration. the approach i have described, although done on a very general level, is essential also to understand the role and position of every part of health care services, as laboratory medicine, in the health care system as a national entity and in the overall producing of high standard and optimal services. o r g a n i z a t i o n / a d m i n i s t r a t i o n the health care system in my country has since the seventhies gone through considerable change in many ways. the main target has been focused on developing primary health care (phc) as a system or good standard and availability of services. local authorities (460 in 1989) are mainly responsible for producing health care services. phc-services are handled by about 215 health centers (partly as local authorities joint functions) and hospital services (purely as joint functions; from 1991 on, 'public health a c t , 1972 245 on an uniform base) by 21 hospital care districts’ (in contrary to about 100 hospital districts today). this gives excellent possibilities to achieve good regional cooperation, which, according to legislation, is also compulsory between different levels of service. this also includes the obligation for the hospital district to direct the development and ensure the standard of, for instance, laboratory medicine services in all hospitals and health centers in the (central) hospital district region. planning system and allocation of resources the cabinet approves every year, covering the next five years, when presenting state budget proposal to parliament, the national plan for producing social wellfare and health care services. the provincial authorities (12) approves (and also allocate resources for instance laboratory staff) regional (hospital care) and local (phc, environmental health) plans, designed according to general goals and priorities given in the national plan, within limits of those resources allocated by ministry of social affairs and health to the province in question. local authorities implement the approved plan. the plannig process does not include the private sector, though. expenditure health care share on gnp has during many years been very steady at a level of 6-7 percentage ( % ) . the increase of the amount of elderly, tecnological and methodological development, increasing demands, decentralization etc. may result in an increase in the health care share of gnp to about 10% in the year 2030. the local authorities get state subsidies (reimbursement) and the amount is based on the approved plan and solvency class of the local area (in general 48-50% of all expencies reimbursed on national level). *special hospital care act, 1990 246 tendencies in developing administration in the seventhies and partly in the eighties central state control has been very rigid and planning very prescriptive. there has since a number of years been a strong tendency to increase independence of local authorities. state authorities are not any more to any substantial degree allowed to give instructions or directives to local authorities in other sence than almost pure informative. there is also strong evidence, that local authoriti es very soon can have their state subsidies without any precondi tions or llearmarkingll and also without close supervision. the outcome of this development is an open question. there is much good to say about it. there may on the other hand, f o r instance, be increasing differences in the level of services between local areas and between hospital districts. the subunits are responsible for developing services and there might be vigorous competition of resources even inside health centres and inside hospital districts. the role of state provincial authori ties in securing even overall optimal development will diminish. this may indicate, that conditions for developing laboratory medicine services as well, are increasingly depending on abilities of the laboratory itself and on able leadership. as an administrator and looking on the health care system as a whole, you may notice the "seed of unhealthy inclinationt1 in the overall development due to too much authority (and ability) on the part of individual experts and their ability to influence the political decision makers. the question of assuring minimun amount of all kinds of health care services for the population in all parts of the country is an important one, especially in times of lack of staff. optimal use of resources at least in theory and on a national level it may be in creasingly difficult to achieve optimal use of resources due to increased independence of local authorities. thinking for instance of laboratory medicine, there are some questions in the mind of an administrator, which have partial relevans also to what has been said above. these are of course well known aspect 247 and have also been discussed very much in the context of actual nordkem projects. the following comments have their relevans specially in primary care: right diagnosis with a minimum of tests! all new inventions must not necessarily and straigth away be adopted and used! test but dont't harm! the customer (patient in phc) in focus. individual variations means lack of exact knowledge (continuity in care will help, though). there is little need of a very sophisticated and scientific level of laboratory services in everyday production of primary care services. secure enough but not unnessessary high quality. correspondence: lars mellin, senior medical officer, national board of health, helsinki, finland. 248 upsala j m e d sci 87: 99-109, 1982 the amount of adenine nucleotides and glycolytic intermediates in erythrocytes, liver and muscle tissue correlated with the body weight (age ) in wistar rats magnus h j e l m and costa arturson from the department of chernicul p a t h o l o g y , lnstitiite of c h i l l htwlth tind h o s p i t a l for sick children, london. united kingdom and the biirn center. university h o s p i t a l , uppsalti, s w d e n abstract the c o n c e n t r a t i o n s o f a d e n i n e n u c l e o t i d e s i n t h e l i v e r and s k e l e t a l muscle i n c r e a s e d d u r i n g t h e m a t u r a t i o n p e r i o d ( d u r i n g t h e f i r s t 100 days o f l i f e ) and remained f a i r l y c o n s t a n t i n a d u l t r a t s . the c o n c e n t r a t i o n o f a d e n i n e n u c l e o t i d e s i n e r y t h r o c y t e s d e c r e a s e d w i t h age. the c o n c e n t r a t i o n s o f hexose monophosphates i n c r e a s e d w i t h age i n l i v e r and the o b s e r v e d changes of m e t a b o l i t e s i n t h e d i f f e r e n t t i s s u e s w i t h i n c r e a s m u s c l e t i s s u e and decreased w i t h age i n e r y t h r o c y t e s . i n g age o f r a t s c o u l d n o t be e x p l a i n e d by a changed w a t e r c o n t e n t . introduction i n f o r m a t i o n a b o u t t h e n a t u r e and m a g n i t u d e o f age dependent changes o f t i s s u e m e t a b o l i t e s i s s c a n t y , and as concerns a d e n i n e n u c l e o t i d e s and g l y c o l y t i c i n t e n n e d i a t e s a l m o s t l a c k i n g . thus a number o f i n t r a c e l l u l a r m e t a b o l i t e s : a d e n i n e n u c l e o t i d e s (atp, adp, amp), g l u c o s e 6-phosphate (g-6-p), f r u c t o s e 6-phosphate (f-6-p), were d e t e r m i n e d i n l i v e r and s k e l e t a l muscle and i n e r y t h r o c y t e s , r e s p e c t i v e l y . the amount o f t h e m e t a b o l i t e s was p r i m a r i l y c o r r e l a t e d w i t h t h e body w e i g h t . s t a t i s t i c a l l y s i g n i f i c a n t c o r r e l a t i o n s were t r a n s f o r m e d i n t o c u r v e s showing t h e r e l a t i o n s h i p between t h e amount o f t i s s u e m e t a b o l i t e and age, where age was de r i v e d f r o m t h e mean c o r r e l a t i o n c u r v e between body w e i g h t and age f o r t h e t y p e o f r a t used. material w h i t e male r a t s o f w i s t a r s t r a i n were used. d u r i n g t h e e x p e r i m e n t s a l l r a t s were k e p t s i n g l e i n m e t a b o l i c cages and d a i l y measurements o f body w e i g h t and t h e amount o f f o o d and w a t e r consumed were made. they were f e d on a s t a n d a r d d i e t supplemented w i t h v i t a m i n s , w i t h a c a l o r i c c o n t e n t o f 51 p e r c e n t c a r b o h y d r a t e , 32 p e r c e n t f a t and 17 p e r c e n t p r o t e i n . the a n i m a l s r e c e i v e d food ad l i b i t u m and t h e y had f r e e access t o w a t e r . a l l r a t s were s t a r v e d 24 h o u r s 99 p r i o r t o s a c r i f i c e . the r a t s were divided in two groups: 4 9 animals were used f o r t h e determina t i o n o f i n t r a c e l l u l a r m e t a b o l i t e s i n l i v e r and s k e l e t a l muscle and i n e r y t h r o c y t e s . 17 animals were used f o r the measurement of the a p p a r e n t c e l l water i n the same type o f c e l l s . methods e t h e r a n a e s t h e s i a and removal of t i s s u e rapid, l i g h t e t h e r a n a e s t h e s i a a s s o c i a t e d w i t h minimal d i s t u r b a n c e of t h e r a t s was achieved. the l i v e r was exposed through a m i d l i n e , abdominal i n c i s i o n . small p i e c e s of l i v e r were removed u s i n g precooled s t a i n l e s s s t e e l tongs which were immersed i n l i q u i d n i t r o g e n . tissue from m . quadriceps femoris was taken by t h e same sampling procedure. 25-50 mg of l i v e r and muscle t i s s u e was o b t a i n e d . the time r e q u i r e d f o r t h e i n c i s i o n t o t h e sampling v a r i e d between 15-30 s e conds, on t h e average f a s t e r f o r t h e l i v e r . blood (about 1 m l ) was c o l l e c t e d from t h e h e a r t by a s y r i n g e and immediately t r a n s f e r r e d i n t o 5 ml of 6 % ( v / v ) i c e c o l d p e r c h l o r i c a c i d . in o r d e r t o study t h e accuracy of t h e sampling procedure b i o p s i e s a l s o were s t o r e d i n v i t r o during c e r t a i n time i n t e r v a l s b e f o r e f r e e z i n g t h e specimen i n l i q u i d n i t r o g e n . e x t r a c t i o n of t i s s u e the frozen t i s s u e was prepared f o r a n a l y s i s of t h e d i f f e r e n t s u b s t a n c e s ac cording t o dale ( 5 ) e x c e p t t h a t l i q u i d n i t r o g e n was used i n s t e a d of l i q u i d ox ygen and t h a t t h e p e r c h l o r i c a c i d e x t r a c t of the d i f f e r e n t t i s s u e s was brought t o ph 6 . 5 i n s t e a d of ph 3.5. chemi c a l methods a t p , a d p and amp were determined according t o adam ( 1 ) by enzymatic methods a n d with s p e c t r o p h o t o m e t r i c r e a d i n g s . the methods a r e not a b s o l u t e s p e c i f i c f o r a t p , a d p and amp as a l s o o t h e r t r i , d i and mononucleotides, i f p r e s e n t , may r e a c t . the e r r o r i n t r o d u c e d in t h e a c t u a l values a r e probably of minor import a n c e , however, as t h e amount of atp i n p e r c h l o r i c a c i d e x t r a c t s of l i v e r t i s s u e determined by t h e method given above and c o n t r o l l e d by t h e more s p e c i f i c hexo k i n a s e methods ( 1 7 ) d i d n o t d i f f e r s i g n i f i c a n t l y ( m . 1 0 , n = 1 0 ) . g-6-p and f-6-p were determined e n z y m a t i c a l l y ( 1 4 ) and w i t h f l u o r i m e t r i c r e a d i n g s . glycogen was converted t o glucose ( 2 1 ) a n d t h e glucose c o n c e n t r a t i o n was de termined with t h e glucose o x i d a s e method of hjelm and de verdier ( 1 3 ) . haemo g l o b i n was determined by t h e cyanomethaemoglobin method and h a e m a t o c r i t values were o b t a i n e d with an i n t e r n a t i o n a l micro-capillary c e n t r i f u g e model m b , i n t e r n a t i o n a l equipment company, nedham, hts, mass., usa. the h a e m a t o c r i t values were n o t c o r r e c t e d f o r t r a p p e d plasma ( 7 ) . the amount o f " f r e e w a t e r " was e s t i m a t e d i n l i v e r and m u s c l e t i s s u e s by means o f d e h y d r a t i n g t i s s u e p i e c e s o f a p p r o x i m a t e l y e q u a l s i z e t o c o n s t a n t w e i g h t a t a t e m p e r a t u r e o f +105oc i n a t h e r m o s t a t c o n t a i n i n g a w a t e r a b s o r b i n g medi urn. c a l c u l a t i o n s the s t a t i s t i c a l method used f o r c o r r e l a t i o n s between m e t a b o l i t e s and body w e i g h t was p i e c e w i s e l i n e a r r e g r e s s i o n ( 8 ) . the r e l a t i o n s h i p between age and body w e i g h t e s t a b l i s h e d by t h e l o c a l b r e e d e r of t h e m i s t a r r a t s , was used t o t r a n s f o r m t h e e x p e r i m e n t a l l y o b t a i n e d r e l a t i o n s h i p between t h e c o n t e n t o f meta b o l i t e s and body w e i g h t i n t o one between t h e c o n t e n t of t h e m e t a b o l i t e s and age. results and d i s c u s s i o n 1. m e t h o d o l o g i c a l a s p e c t s on t h e p r o c e d u r e f o r s a m p l i n g o f t i s s u e s the q u i t e c o n s i d e r a b l e change o f t h e c o n c e n t r a t i o n o f a number o f metabo l i t e s i n l i v e r b i o p s i e s w i t h i n seconds i s shown i n f i g . 1. 2oor % loo+ 200 10 20 2ool 100 time, sec 200 r 100 p 200 10 20 time, sec 10 20 time, sec 100 10 20 time ,sec f i g . 1. c o n c e n t r a t i o n s o f atp, adp, amp, g-6-p and f-6-p i n l i v e r b i o p s i e s r e moved u s i n g p r e c o o l e d s t a i n l e s s s t e e l t o n g s ( = l o 0 %) and t a k e n w i t h k n i f e and immersed i n l i q u i d n i t r o g e n a f t e r 3,5, 7, 10, 15 and 20 seconds r e s p e c t i v e l y . 101 the r e s u l t s t r e s s e s t h e i m p o r t a n c e o f an a c c u r a t e s a m p l i n g t e c h n i q u e o f t i s s u e s w i t h a r a p i d t u r n o v e r o f m e t a b o l i t e s i n o r d e r t o o b t a i n r e l e v a n t i n f o r m a t i o n a b o u t t h e c o n d i t i o n s i n v i v o . the i n c o n s i s t e n c y between v a l u e s f o r some t i s s u e m e t a b o l i t e s i n r a t s o f c o m p a r a t i v e w e i g h t s , c o m p i l e s f r o m t h e l i t e r a t u r e i n t a b l e 1, i n d i c a t e s t h a t t h e s a m p l i n g o f t i s s u e s m i g h t n o t always have been ac c u r a t e and t h a t e a r l i e r r e s u l t s c a n n o t d i r e c t l y be used as r e f e r e n c e v a l u e s . t a b l e 1. the average c o n c e n t r a t i o n s o f a d e n i n e n u c l e o t i d e s i n m u s c l e t i s s u e , -issue and e r y t h r o c y t e s i n r a t s and some o t h e r s p e c i e s . (range o f body w e i g h t 100-200). ~~ t i s s u e atp adp amp zptp, adp, amp 1 ) muscle ti s s ue i m a i , r i l e y & berne ( 1 9 6 4 ) pedersen & sachs (1965) scopes & newbold (1968) chaudry, sayeed & baue (1974) berne & rubio (1974) p r e s e n t i n v e s t i g a t i o n l i ve r t i s s ue maass & timm (1964) kolousek, j i r a c e k , z i c h a e t a1 (1965) hems, ross, b e r r y e t a1 (1966) puddu, c a l d a r e r a & m a r c h e t t i (1967) chaudry, sayeed & baue ( 1 9 7 4 ) hirasawa, chaudry & baue (1978) ozawa e t a1 (1981) p r e s e n t i n v e s t i g a t i o n e r y t h r o c y t e s ’ ) * ) kolousek, j i r a c e k , z i c h a e t a1 (1965) p r e s e n t i n v e s t i g a t i o n l ) 6 . 3 0.76 0.29 7.4 4.8 4 . 5 1.3 0.11 5.9 3.6 0.6 0.07 4.3 5.43 1.29 0.21 6.9 4.8 0.9 0.1 5.8 2.3 0.78 3.2 1.71 0.57 2.5 0.94 0.21 0.33 0.90 2.1 1.85 0.82 0.25 2.47 0.73 0.14 2.38 0.55 0.14 3.0 1.2 0.4 1.57 0.17 0.05 1.9 0.4 5.4 3.7 3.3 2.9 3.3 3.1 4.6 1 . 8 moles x m u s c l e t i s s ue p e r m l o f e r y t h r o c y t e s o r p e r gram o f w e t w e i g h t o f l i v e r o r ’) the average h a e m a t o c r i t v a l u e assumed t o be 40 % 102 at p moles 1061g we! weight l i v e , 3.5 3 . 0 2.0 1.0 _ / * i 100 200 300 g body weigh! i a t p , a d p , a m p moles * i06/g of wet weight l l w , 2 . 5 2 . 0 1.5 1.0 l . 0 3.0 100 200 300 g body weigh! 20 30 lo s o 6 0 7 0 a g e , days a t p m101es. 10s/q we! weight muscle i * \ . \ 8 , 3.0 2.0 1.0 f i g . 2 . concentrations of atp and t h e sum of atp, adp and amp i n l i v e r , s k e l e t a l muscle and e r y t h r o c y t e s c o r r e l a t e d with body weight ( a g e ) . re g r e s s i o n l i n e w i t h 95 % confidence i n t e r v a l and 95 % t o l e r a n c e l i m i t f o r t h e measurements i s i n d i c a t e d . :: * ,' ,' 3.0 i \ ;\. erythrocytes \ \ ._ . \ 100 2 0 0 300 9 body weight i atp, a d p , a m p moles .106/rnl of erythrocytes erythrocytes i '--~ 100 2 0 0 300 g body weight 2 0 30 lo 5 0 6 0 i o a g e . days 100 200 300 g body weigh! 2 0 30 lo 50 6 0 7 0 a g e , d a y s 103 2 . t h e c o n c e n t r a t i o n of t i s s u e m e t a b o l i t e s c o r r e l a t e d w i t h body weight ( a g e ) 2.1. adenine n u c l e o t i d e s i n muscle and l i v e r t i s s u e and i n e r y t h r o c y t e s the c o n c e n t r a t i o n of atp i n t h e l i v e r i n c r e a s e d s i g n i f i c a n t l y during t h e ma t u r a t i o n p e r i o d , i . e . during t h e f j r s t 100 days o f l i f e , and remained f a i r l y c o n s t a n t i n a d u l t r a t s ( f i g . 2 ) . p r e l i m i n a r y s t u d i e s i n r a t s showed an i n c r e a s ed number of mitochondria i n l i v e r t i s s u e from animals w i t h a body weight of 400 g compared t o 60 g. the observed i n c r e a s e of t h e c o n t e n t o f atp i n t h e l i v e r of maturing r a t s may a c c o r d i n g l y be d u e t o an i n c r e a s e d number of mitochon d r i a . the observed changes o f a d p and amp and o f t h e sum of adenine n u c l e o t i d e s w i t h age ( f i g . 2 ) could a c c o r d i n g l y be secondary t o keeping t h e over a l l e q u i l i b r i u m o f t h e a d e n y l a t e kinase reaction,(atp x a m p ) / ( a d p ) 2 a t a c o n s t a n t l e v e l . of adenine n u c l e o t i d e s i n muscle t i s s u e changed with age i n a s i m i l a r way a s i n the l i v e r . i t i s , however, n o t known i f t h e number of mitochondria i n muscle t i s s u e v a r i e s with age. the s i t u a t i o n i s a l s o complicat ed by t h e f a c t t h a t muscle t i s s u e i s composed of muscle f i b r e s of d i f f e r e n t t y p e s , which might c o n t a i n the same o r a d i f f e r e n t number of mitochondria per u n i t volume of tissue. t h u s a change of t h e r e l a t i v e frequency o f t h e types of muscle f i b r e s w i t h age m i g h t e x p l a i n t h e observed change of t h e over a l l con tent of atp. work i s i n p r o g r e s s t o c l a r i f y t h i s problem. the c o n c e n t r a t i o n the c o n c e n t r a t i o n o f adenine n u c l e o t i d e s i n e r y t h r o c y t e s decreased slowly d u r i n g the maturation p e r i o d . in human e r y t h r o c y t e s hbf has a h i g h e r a f f i n i t y t o 2 , 3 diphosphoglycerate than hba ( 6 ) . i n t a c t e r y t h r o c y t e s i n neonates c o n t a i n about twice as much of t h e phosphocompound a s i n a d u l t s t o a s s u r e t h e same oxy gen r e l e a s e c a p a c i t y of e r y t h r o c y t e s i n neonates and a d u l t s ( 1 1 , 1 2 ) . the s i t u a t i o n might be analogous f o r t h e binding o f atp t o a f e t a l and a d u l t type of r a t haemoglobin. then t h e d e c r e a s i n g c o n t e n t of atp i n e r y t h r o c y t e s from young r a t s could be e x p l a i n e d by a concomitant d e c r e a s e of f e t a l r a t haemoglobin. however, l i t t l e i s known about r a t haemoglobins a t p r e s e n t and e s p e c i a l l y about t h e binding of o r g a n i c phosphocompounds t o t h e molecule. more experimental work i s necessary t o c l a r i f y t h e s i t u a t i o n . i t does n o t seem l i k e l y , however, t h a t the decreased c o n t e n t of atp i s r e l a t e d t o a changed mean age of t h e c i r c u l a t i n g e r y t h r o c y t e p o p u l a t i o n ( c f . 1 2 ) . n y l a t e kinase reaction,(atp x a m p ) / ( a d p ) t o d e c r e a s e w i t h age in l i v e r and mus c l e tissue, whereas the energy charge o f t h e a d e n y l a t e pool c a l c u l a t e d accord i n g t o atkinson ( 2 ) : ( a t p + 0.5 a d p ) / ( a t p + a d p + amp) s t a y e d c o n s t a n t . no o b vious changes of t h e s e two parameters w i t h age could be observed i n e r y t h r o c y t e s ( f i g . 3 ) . there was a t r e n d f o r t h e apparent e u i l i b r i u m c o n s t a n t , k , f o r t h e ade1 104 o ’ l f i g . 3. the a p p a r e n t e q u i l i b r i u m c o n s t a n t , k , f o r t h e a d e n y l a t e kinase r e a c t i o n ( a t p x a m p ) / ( a d p ) 2 and t h e energy charge of t h e a d e n y l a t e pool i n l i v e r , s k e l e t a l muscle and e r y t h r o c y t e s c o r r e l a t e d with body weight ( a g e ) . the t o t a l v a r i a n c e of a t p , a d p and amp and of t h e a p p a r e n t e q u i l i b r i u m c o n s t a n t , k , around t h e r e g r e s s i o n l i n e s a g a i n s t age showed a c o n s i d e r a b l e v a r i a n c e , i n t h e o r d e r o f 2 10 t o t 15 % i n a l l t i s s u e s s t u d i e d ( f i g . 2 and 3 ) . the t o t a l v a r i a n c e around the r e g r e s s i o n l i n e f o r the energy charge a g a i n s t age i s con s i d e r a b l y less and i n t h e o r d e r of ? 5 % ( f i g . 3 ) . a major p a r t of t h i s v a r i ance could be e x p l a i n e d by f a c t o r s r e l a t e d t o t h e handling o f the specimens and t h e a n a l y t i c a l procedure. i f t h i s assumption i s c o r r e c t t h e i n t e r i n d i v i d u a l ( b i o l o g i c a l ) v a r i a n c e o f t h e energy charge must be extremely low. i t a l s o f o l lows t h a t the c o n s i d e r a b l e i n t e r i n d i v i d u a l v a r i a n c e observed f o r t h e i n d i v i d u a l adenine n u c l e o t i d e s could r e f l e c t a homeostatic mechanism by which the de mand of keeping t h e energy charge w i t h i n a narrow a b s o l u t e l i m i t i s l i n k e d t o a varying i n t e r m e d i a r y energy metabolism, a d j u s t e d t o meet i n d i v i d u a l demands. i t s h o u l d , however, be p o i n t e d o u t t h a t t h e energy charge i s a lumped para meter i n l i v e r and kidney t i s s u e as both l i v e r and muscle c e l l s r e p r e s e n t m u l t i compartment systems with adenine n u c l e o t i d e s p r e s e n t both i n mitochondria and cytoplasma and r e l a t e d t o t h e c e l l membrane. 2 . 2 . hexose monophosphates i n muscle and l i v e r t i s s u e and i n e r y t h r o c y t e s . in fig. 4 the c o n c e n t r a t i o n o f the hexosemonophosphates glucose-&phosphate and fructose-6-phosphate a r e c o r r e l a t e d w i t h body weight. evidently t h e r e i s an i n c r e a s e o f t h e m e t a b o l i t e s i n both muscle and l i v e r t i s s u e , most pronounced i n young r a t s . how t h i s f i n d i n g s should be explained i s u n c e r t a i n . though t h e n u m b e r of measurements i n e r y t h r o c y t e s i s l i m i t e d t h e r e s u l t i n d i c a t e s t h a t t h e r e i s a d e c r e a s e o f t h e amount of t h e hexosemonophosphates, a l s o i n young animals. this might be r e l a t e d t o t h e d i f f e r e n t binding o f the compound t o a f e t a l and i05 a d u l t type of r a t haemoglobin. 0 0 0 0 0 % 0 o o o 0 00 8 0 oo 00 0 0 0 8 i j g-6-p + f-6-p muscle liver erythrocytes moles = 1 0 7 9 tissue x id’ 10 a 6 l 2 100 200 300 loo 500 100 200 300 loo 500 body weight, g body weight, g body weight, g fig. 4. concentration of g-6-p and f-6-p in l i v e r , s k e l e t a l muscle a n d erythro cytes correlated with body weight (age). 2 . 3 . glycogen in l i v e r t i s s u e the amount of l i v e r glycogen has been correlated with body weight a n d the age of the animal i n fig. 5. there i s a pronounced increase of glycogen during the f i r s t seventy days of l i f e a n d then a decrease of the compound. there i s n o obvious explanation f o r the finding. however, i t seems of importance t o con s i d e r t h i s r e s u l t in f u t u r e experimental s i t u a t i o n s where l i v e r glycogen i s de termined e i t h e r in r a t s of d i f f e r e n t age or during longitudinal s t u d i e s . 2 . 4 . water in muscle and l i v e r t i s s u e and in erythrocytes the content o f water i n the three types of c e l l s investigated has been de termined and correlated t o the body weight or aae. i t appears from fig. 6 a n d 7 t h a t the small changes observed are n o t a t a l l of such a magnitude t o explain the changes of metabolites as a r e s u l t of a changed water content of the invest igated c e l l types. 2 . 5 . other f a c t o r s influencing the amount of metabolites in the t i s s u e in ves t i gated other f a c t o r s t h a n those discussed above might be of importance t o explain the present findings, e.g. the e f f e c t of l i g h t e t h e r anaesthesia. this form of anaesthesia might in the absence of a blocking compound have released adrena 106 l i n e , a c t i v a t i n g t h e a d e n y l a t e c y l c l a s e system and with secondary e f f e c t s on t h e i n t e r m e d i a r y metabolism e s p e c i a l l y of t h e l i v e r . i t would be necessary t o c a r r y o u t a d d i t i o n a l work t o e l u c i d a t e t h i s problem. glycogen mg/g w e t w e q h t 1 100 i 60 ! 20 1 liver i i 1 1 . ~~ 100 200 300 400 500 body wecght, g fig. 5 . the amount o f glycogen i n the l i v e r c o r r e l a t e d with body weight ( a g e ) . apparent water content, % liver tissue muscle tissue __il 1 ' i i 100 200 300 400 500 body weight, g mchc 36 i 40 1 mch 22 1 16 1 ~ l 1 1 i i 100 200 300 loo 500 body weight. g f i g . 6 . m c h c , m c v and mch i n e r y t h r o c y t e s c o r r e l a t e d w i t h body weight ( a g e ) . fig. 7 . the a p p a r e n t water c o n t e n t o f l i v e r and muscle t i s s u e c o r r e l a t e d w i t h body weight ( a g e ) . i07 acknowledgement s u p p o r t e d b y t h e swedish m e d i c a l research c o u n c i l ( p r o j e c t nos. 40x-676 and 40y-2370). 1. 2. 3. 4. 5 . 6. 7. 8 . 9. 10. 11. 12. 13. 14. 15. references adam, h . : adenosin-5 t r i p h o s p h a t e and a d e n o s i n 5 d i p h o s p h a t e i n h.-u. bergmeyer ( e d ) . methoden d e r e n z y m a t i s c h e n analyse, v e r l a g chemie gmbh, weinheim 1962, p . 539 a n d 573. a t k i n s o n , d. e . : the e n e r g y c h a r g e o f t h e a d e n y l a t e p o o l as a r e g u l a t o r y p a r a m e t e r . i n t e r a c t i o n w i t h feedback m o d i f i e r s . biochem 7:4030, 1968. berne, r . m. & rubio, r . : adenine n u c l e o t i d e m e t a b o l i s m i n t h e h e a r t . c i r c . res. 34:109, 1974. chaudry, i. h . , sayeed, m. m. and baue, a . e . : e f f e c t o f h e m o r r h a g i c shock on t i s s u e a d e n i n e n u c l e o t i d e s i n c o n s c i o u s r a t s . can j phys pharm 52:131, 1974. dale, r . a . : e f f e c t s o f s a m p l i n g p r o c e d u r e s on t h e c o n t e n t o f some i n t e r m e d i a t e m e t a b o l i t e s o f g l y c o l y s i s i n r a t t i s s u e s . j p h y s i o l 181:701, 1965. garby, l . and de v e r d i e r , c . h . : a f f i n i t y o f human h e m o g l o b i n a t o 2 , 3 d i p h o s p h o g l y c e r a t e . e f f e c t o f hemoglobin c o n c e n t r a t i o n and o f ph. scand j c l i n lab i n v e s t 27:345, 1971. garby, l. and v u i l l e , j.-c.: the amount o f t r a p p e d plasma i n a h i g h speed m i c r o c a p i l l a r y h e m o t o c r i t c e n t r i f u g e . scand j c l i n lab i n v e s t 13:642, 1961. g r o t h , t., f a l k , h. and h j e l m , m.: a programme f o r e s t i m a t i o n o f t r e n d s and r e f e r e n c e i n t e r v a l s o f b i o m e d i c a l v a r i a b l e s . to be p u b l i s h e d 1982. hems, k., r o s s , b. d., b e r r y , m. n. and krebs, h . a . : gluconeogenesis i n t h e p e r f u s e d r a t l i v e r . biochem j 101:284, 1966. hirasawa, h., chaudry, i. h. and baue, a. e . : improved h e p a t i c f u n c t i o n and s u r v i v a l w i t h a d e n o s i n e t r i p h o s p h a t e m a g n e s i u m c h l o r i d e a f t e r h e p a t i c i s chemia. s u r g e r y 83:655, 1978. h j e l m , m.: the c o n c e n t r a t i o n o f some n u c l e o t i d e s and g l y c o l y t i c m e t a b o l i t e s i n human e r y t h r o c y t e s o f d i f f e r e n t ages. f o l i a haemat ( l e i p z i g ) 89:392, 1968. h j e l m , m.: the mode o f e x p r e s s i n g t h e c o n t e n t o f i n t r a c e l l u l a r components o f human e r y t h r o c y t e s w i t h s p e c i a l r e f e r e n c e t o a d e n i n e n u c l e o t i d e s . scand j haemat 6:56, 1969. h j e l m , m. and de v e r d i e r , c.-h.: a m e t h o d o l o g i c a l s t u d y o f t h e e n z y m a t i c d e t e r m i n a t i o n o f g l u c o s e i n b l o o d . scand j lab c l i n i n v e s t 5:415, 1963. h o h o r s t , h. j.: d-glucose-6-phosphate and d f r u c t o s e 6 p h o s p h a t e i n h . u. bergmeyer ( e d ) . methoden d e r e n z y m a t i s c h e n a n a l y s e . v e r l a g chemie, gmbh, weinheim 1962, p. 134. i m a i , s . , r i l e y , a. l. and berne, r. m.: e f f e c t s o f i s c h e m i a on a d e n i n e nu c l e o t i d e s i n c a r d i a c and s k e l e t a l muscle. c i r c kes 9:443, 1964. i08 16. kolousek, j. , j i r a c e k , v. , z i c h a , b. e t . a l . : the i n f l u e n c e o f x r a d i a t i o n , m e t h i o n i n e s u l p h o x i m i n e , b u r n s , and c y t o s t a t i c a g e n t s ts 160 ( s p o f a ) on t h e u r i n a r y e x c r e t i o n o f i n o r g a n i c phosphates and ammonia and on t h e l e v e l o f a d e n i n e n u c l e o t i d e s i n b l o o d and l i v e r o f r a t s . neoplasma ( b r a t i s l ) 12:565, 1965. 17. lamprecht, w. and t r a u t s c h o l d , l . : adenosine-5-triphosphate: d e t e r m i n a t i o n w i t h h e x o k i n a s e and g l u c o s e 6 p h o s p h a t e dehydrogenase i n h.-u. bergmeyer (ed!. methods o f e n z y m a t i c a n a l y s i s . new york. academic p r e s s i n c . 1965, chap. 5, pp 543-552. 18. maass, h. and timm, m. : n u k l e o s i d p h o s p h a t g e h a l t i n v e r s c h i e d e n e n organen d e r r a t t e nach g a n z k o r p e r b e s t r a h l u n g . s t r a h l e n t h e r a p i e 123:64, 1964. 19. ozawa, k . , yamaoka, y . , kimura, k . , kamiyama, y . , sato, m. ukikusa, m. and tobe, t. : c i r c u l a t i n g h e p a t o d e p r e s s a n t f a c t o r s d e c r e a s i n g t h e e n e r g y c h a r g e l e v e l s o f t h e remnant l i v e r a f t e r hepatectomy. e u r . s u r g . res. 13:444, 1981. 20. pedersen, p . l. and sachs, j . : hexosephosphate f o r m a t i o n and t h e r e g u l a t i o n o f g l y c o l y s i s i n muscle. a r c h biochem 112:548, 1965. 21. p f l e i d e r e r , g.: glykogen bestimmung a l s d-glucose m i t hexokinase, p y r u v a t k i n a s e und lactat-dehydrogenase i n h. u. bergmeyer ( e d ) . methoden d e r en z y m a t i s c h e n analyse, v e r l a g chemie gmbh, weinheim 1962, p. 59. f a t t y l i v e r . biochem j 102:163, 1967. c l e . biochem j 109:197, 1968. 22. puddu, p., c a l d a r e r a , c. m. and m a r c h e t t i , m.: s t u d i e s on e t h i o n i n e i n d u c e d 23. scopes, r. k . and newbold, r. p . : postmortem g l y c o l y s i s i n ox s k e l e t a l mus address f o r r e p r i n t s : gosta a r t u r s o n , m. d. b u r n c e n t e r u n i v e r s i t y h o s p i t a l s-750 14 uppsala sweden i09 upsala j med sci 84: 215-227 hepatic triglyceride and lipoprotein lipase activities of post-heparin plasma in normals and hypertriglyceridemics jonas boberg*, merike boberg, richard gross, john d. turner, jan augustin** and w. virgil brown department of medicine, school of medicine, university of california, san diego, la jolla, california, u s a abstract post-heparin plasma hepatic triglyceride lipase (h-tgl) and lipoprotein li pase (lpl) activities were 8-34 and 3.5-21 (range) pmol/ml/hr respectively in males with normal serum lipid concentrations. in females the corresponding values were 4-25 and 4-16 pmol/ml/hr. no significant differences were observed between ages or between the two sexes. male patients with hypertriglyceridaemia had similar activities of h-tgl but significantly lower values for lpl activities than the control males. significant linear correlations were found between serum triglyceride con centrations and lpl activities both for males (coefficient of correlation = r = -0.64) and for females (r = -0.62). introduction several studies (1-3) have demonstrated that the serum triglyceride (s-tg) concentrations in man during fasting conditions to a great part are determined by the removal process of tg rich s-lipoproteins very low density lipopro teins (vldl) and chylomicrons from the blood stream. this removal process is thought to be mediated essentially by lipoprotein lipase (4). in man the act& vity of this enzyme has been demonstrated in adipose (5-6) and muscle tissue (7). although the most common way to determine this enzyme activity in man has been in post-heparin plasma (4). however, recently post-heparin lipolytic act& vity (phla) has been shown to contain at least two triglyceride lipase activi preliminary report given at the american heart association 47th scientific sessions, november 18-2 1 , 1974. *visiting postdoctoral nih research fellow and also supported by the swedish medical research council (60f-4187 and 19r-4187). present address: department of geriatrics, university of uppsala, box 12042, s-750 12 uppsala, sweden **visiting postdoctoral research fellow supported by the "deutsche forschungs gemeinschaft", westgermany (an 4511). 215 ties*. one probably originating from liver and the other from extrahepatic tissues (8). a method for isolation from post-heparin plasma, these two trigly ceride lipase activities and for quantitative determination of the two enzyme activities separately has previously been described (9). this method includes affinity chromatography where the enzymes are partly purified to avoid inter ference from serum lipoproteins and apo-lipoproteins. the present report de monstrates a clinical application of this method in which human subjects with normal serum lipid concentrations and male patients with hypertriglyceridaemia have been investigated. methods subjects and experimental procedure the subjects were 42 males between 20 and 81 years and 38 females between 12 and 78 years of age. in both these groups all subjects had normal s-tg < 2 . 4 8 mmol/l ( 2 2 0 mg/100 ml) and cholesterol <7.8 mmol/l (300 mg/100 ml) con centrations. the subjects were recruited either from the laboratory personel or from volunteers in the community. the latter subjects participated in a pilot study done prior to a health screening survey. all were free living and apparently healthy. they had normal fasting blood sugar and urine analyses. none were on prescription except for digoxin medications. the male patients with hypertriglyceridaemia were those referred to the lipid research clinic or the metabolic unit at the veterans administration hospital, san diego. no pa tients with diabetes mellitus, hypothyroidism, liver or renal disease were in cluded in the study. the procedure was performed in the morning after over-night fast. firstly the subjects were weighed and a short medical history was recorded. a scalp vein needle was introduced into an anticubital vein and a venous blood sample of 15 ml was taken into edta glas tubes. immediately thereafter 6 0 iu per kg body weight of heparin (riker laboratories, 5000 iu/ml) was injected intra venously through the scalp vein needle followed by about 10 ml of saline wash. fifteen minutes after the heparin injection the post-heparin sample was with drawn (30 ml of blood). all blood samples were kept on ice for 1 to 2 hours be fore centrifugation. analytical methods blood samples were centrifuged at 5000 g x min and the blood plasma was re covered into capped vial8. the preheparin samples were kept for 1 to 36 hours at 4oc before serum lipid and lipoprotein analyses were done. post-heparin plae ma samples were frozen in 6 ml aliquots at -7ooc for not more than one month *in this paper these two lipase activities are called hepatic triglyceride li pase (h-tgl) activity and lipoprotein lipase (lpl) activity. 216 d 0 ) u c c m 0 u n rl r l c 3 o h m i a , x t j h u u .d -% rl ? 1 h c i . d r l c h u 0 1 i u e r l x m 1 e i . n e v n -3 m i a u . ' w i- n 7 m i m n f m 3 a ? c n 0 +i r. 7 n 0 n n +i m 0 m u +i m i 0 i m w . * o 0 m w ul 0 0 u m 0 +i n m v 0 m v u o n n n 0 w +i m 0 m hl +i m 0 .h 0 ul m 0 +i w 7 m 0 r. m +i m 0 +i m in 0 m +i 7 hl 0 +i m m n 0 +i c a 0 r. +i c n 0 hl m +i n 0 m v) +i -3 0 0 0 +i m 0 n m v m -3 i m c -3 0 0 m +i m 0 n ul v 0 a i a u m 0 0 w +i m 0 n c v 0 a a n 0 0 +i 7 m 0 n m m v rl m u 0 h 217 before determination of h-tgl and lpl activities were done. serum lipids and lipoproteins were determined according to the stan'dar dized methods routinely used at the lipid research clinic labora tory (10). isolation of h-tgl and lpl activities from postheparin plasma were done with affinity chromatography on sepharose covalently linked with heparin ( 1 1 ) according to a method previously descri bed in detail ( 9 ) . aliquots of the enzyme fractions recovered from the chromatography were incubated with a labelled triglyce ride emulsion (8). labelled free fatty acids released during in cubation for 30 minutes were measured ( 1 2 ) . lipase activity was expressed as umoles fatty acids released per ml of post-heparin plasma per hour of incubation. during the whole investigation a frozen control post-heparin plasma sample was run to check for the interday variations of the procedures. statistical analyses were done according to snedecor (13). results postheparin triglyceride lipase activity in normal subjects. results obtained in normal subjects, males and females, are presented in tables 1 and 2 respectively. the females were not obese and the average s-tg concentration was 0.88 mmol/l with no significant trend o f either increase o r decrease with age. scholesterol concentration (both total and in beta-lipoproteins) showed the increase with age which has been demonstrated several times before. mean value of the whole group was 5 . 3 ? 0 . 2 mmol/l for total and 3.2+0.2 mmol/l for beta-lipopro tein cholesterol concentration. h-tgl activities of the younger females ( ( 3 0 years) were on the average higher than for the groups of older people. however this difference was not significant. no significant change with age was demonstrated for lpl activity. mean values of h-tgl and lpl activities for normal females were 1 3 . 5 and 7.7 umoles/ml/hr respectively. the normal male subjects were slightly heavier than the fe males. similar concentrations of s-tg and total and beta-choleste rol were found for males and females. alfa-lipoprotein choleste rol concentrations were slightly lower in the males compared to the females. h-tgl and lpl activities did not change with age in the normal males. the mean values were 19.6 and 6.6 umol/ml/hr 218 t a b l e 2 . w e i g h t l h e i g h t i n d e x , s e r u m t r i g l y c e r i d e ( t g ) a n d c h o l e s t e r o l c o n c e n t r a t i o n s , b e t a a n d a l f a l i p o p r o t e i n c h o l e s t e r o l c o n c e n t r a t i o n s a n d h e p a t i c t r i g l y c e r i d e ( h t g l ) a n d l i p o p r o t e i n l i p a s e (l p l ) a c t i v i t i e s i n n o r m o t r i g l y c e r i d a e m i c m a l e s . s u b j e c t s ( n ) w e i g h t / s e r u m t g s e r u m c h o l b e t a l i p o a l f a l i p o h t g l l p l a g e h e i g h t c o n c e n e s t e r o l p r o t e i n p r o t e i n a c t i v i t y a c t i v i t y r a n g e s i n d e x * t r a t i o n c o n c e n t r a c h o l e s t e c h o l e s t e u m o l / u m o 1 / y e a r s m m o 1 / 1 * * t i o n r o l e s t i r o l e s t i m l / h r * * m l / h r * * m m o 1 / 1 * m a t e m a t e m m o l / l * m m o l / l * (3 0 (1 1 ) 0 . 8 7 * 0 . 0 2 0 . 9 5 4 . 5 ? 0 . 2 3 1 4 5 (9 ) 1. 03 ?0 .0 5 1 . 0 3 5 .o fo . 2 4 6 6 0 (9 ) 1 . 0 3 * 0 . 0 3 1 . 0 3 5 . 5 + 0 . 5 > 6 0 ( 1 3 ) 1 . 0 3 * 0 . 0 3 1 . 0 2 5 . 2 * 0 . 2 t o t a l ( 4 2 ) 0 . 9 9 ? 0 . 0 2 0 . 9 9 5 . o f o . 2 ( 0 . 8 8 1 . 0 4 ) ( 0 . 8 7 1 . 2 1 ) ( 0 . 8 8 1 . 2 1 ) ( 0 . 9 2 1 . 1 3 ) ( 0 . 9 5 1 . 0 7 ) 2 . 6 * 0 . 4 1 . 4 * 0 . 1 1 9 . 4 3 . 4 + 0 . 3 1 . 3 + 0 . 1 1 9 . 2 3 . 7 ? 0 . 5 1 . 3 + 0 . 2 2 0 . 9 3 . 2 * 0 . 2 1 . 5 + 0 . 1 17 .8 3 . 3 + 0 . 2 1 .4 *0 .1 1 9 . 6 ( 1 7 . 8 2 1 . 2 ) ( 1 6 . 9 2 1 . 8 ) (1 8 . 3 2 3 . 8 ) ( 1 6 . 4 1 9 . 5 ) ( 1 8 . 6 2 0 . 4 ) 6 . 3 ( 5 . 7 6 . 8 ) 6 . 4 ( 5 . 5 7 . 3 ) 6. 2 ( 5 . 6 6 9 ) 7. 3 ( 6 . 5 8 . 2 ) 6 . 6 ( 6 . 2 6 . 9 ) * = m e a n v a l u e s t a n d a r d e r r o r o f t h e m e a n a r e g i v e n . * * = a n t i l o g a r i t h m o f m e a n v a l u e a n d w i t h i n b r a c k e t s a n t i l o g a r i t h m o f m e a n v a l u e s t a n d a r d e r r o r o f t h e m e a n a r e g i v e n . n = n u m b e r o f s u b j e c t s w i t h i n e a c h g r o u p . c i c3 i 10 1 1 normal f €ma,! es i i i i i 1 i l l i 1 10 l p l a c t i v i t y , p m o l / m l / h r normal ma1 € 0 . i i 1 i i i i i i i i 1 i 10 l p l a c t i v i t y , p m . o i / m l / h r fig.1. relationship between post-heparin 1 ipop ro t e in 1 ip as e ( l p l ) activity and ser um triglyceride (tg) concentration in heal thy females with "nor mal" serum lipid values. correlation analysis of logarithmic values of both parameters resul ted in r=-0.62, p<o.ool. fig. 2. relationship between post-heparin lipoprotein lipase (lpl) activity and serum tri glyceride (tg) concentra tion in healthy males with "normal" serum lipid values. correlation ana lysis of logarthmic val ues of both parameters resulted in r=-0.64, p<o .oo 1 . 220 respectively. these values for h-tgl activity are significantly higher for the males than for the females, while no significant difference of lpl activity was found between the sexes. postheparin triglyceride lipase activities in patients with hypertriglyceridemia only male patients with hypertriglyceridemia were investi gated. results for a comparison between normal and hypertriglyce ridemicmales are summarized in table 3 . naturally serum tg and cholesterol concentrations were higher in the hypertriglycerid emics compared to the controls. furthermore the hypertriglycerid emics were heavier and had significantly lower alfa-lipoprotein cholesterol concentrations. h-tgl activity was on the average significantly higher in the patients with elevated s-tg concentra tions than the controls, showing mean values of 2 2 . 8 compared to 1 9 . 6 umol/ml/hr for the controls. lpl activities on the other hand were significantly (p < o . o o l > lower in the patients with hypertriglyceridemia compared t o the controls. the mean values were 4 . 7 for hypertriglyceridemics compared to 6 . 6 umol/ml/hr for the male controls. relationship between s-tg concentrations and h-tgl and lpl activities no significant correlations were found between h-tgl activity and concentrations o f any serum lipid or lipoprotein fraction. between s-tg concentrations and lpl activities significant correlations were found for both female and male normotriglycerid emics (figures 1 and 2 ) . the correlation coefficients were r = 0 . 6 2 and r = 0 . 6 4 . a s shown in figure 3 about one third of the male patients with hypertriglyceridemia fell outside the range of lpl activity for the controls. however, for the rest of the patients there were overlap of the data. a s demonstrated in figure 4 no significant correlations occurred between the two post-heparin triglyceride lipase acti vities. the two enzyme activities seem to vary quite independent lyinboth the female and male controls and also in the patients with high s-tg concentrations. 221 h ) h ) h ) ta bl e 3. an tr op om et ri c da ta , se ru m li pi ds , se ru m li po pr ot ei ns a nd p os the pa ri n he pa ti c (h -t gl ) an d li po pr ot ei n li pa se (l pl ) ac ti vi ti es in m al e no rm o an d hy pe rt ri gl yc er id ae mi cs . ~ ~ ~ ~ ag e we ig ht se ru m t g se ru m ch ol be ta -l ip o al fa -l ip o htg l l p l ye ar s* he ig ht co nc en tr a co nc en tr a pr ot ei n pr ot ei n ac ti vi ty ac ti vi ty in de x* t io n t i on ch ol es ti ch ol c on ma te ce nt ra ti on um ol / um o 1 / mm ol /l ** m o l / l * mm ol /l * mm ol /l * ml /h r* * ml /h r* * no rm ot ri 46 .9 ?2 .9 0. 99 *0 .0 2 0. 99 5. 0* 0. 2 3. 3* 0. 2 1. 4* 0. 1 19 .6 6. 6 gl yc er i (0 .9 51. 07 ) (1 8. 620 .4 ) (6 .2 -6 9) da em ic s n = 4 2 hy pe rt ri 48 .2 ?1 .7 1. 13 *0 .0 2 6. 07 6. 4* 1. 6 3. 1+ 0. 2 0. 9k o. l 22 .8 4. 7 gl yc er i (5 .1 47. 18 ) (2 1. 524 .3 ) (4 .4 -5 . o) da em ic s n = 3 3 tva lu e 0. 36 4. 36 15 .7 2 4. 64 -0 .4 5 -2 .2 3 2. 39 -3 .8 6 st at is ti ca l si gn if ic an ce p >o .o 5 p< o. oo l p< o. oo l p< o. oo l p> o .0 5 ~ ( 0 . 0 5 ~ ( 0 . 0 5 p< o. oo l *= me an v al ue ri th m o f me an v al ue st an da rd e rr or o f th e me an a re gi ve n. * *= an ti lo ga rt hr n of th e me an v al ue an d wi th in b ra ck et s an ti lo ga st an da rd e rr or o f th e me an a re gi ve n. n =n um be r o f su bj ec ts w it hi n ea ch g ro up . c z 0 i 4 i n w 0 z 0 0 w i a 0 c 0 . 0 0 z a 3 w cn 4 0 a i k 20 > c v 4 i 0 normal males 0 hyperlipoprotffn€mfc males 10 c : o o 0 0 o % o w "oo o o t o 0 o 0 8" 0" 0 0 0 0. s= i t a fig. 3. relationship bet ween post-heparin lipopro tein lipase (lpl) activity and serum triglyceride (tg) concentration in normotri glyceridemic and hypertri glyceridemic males. further data of the former group see under figure 2. lpl a c t i v i t y , p m o l l m l l h r fig. 4 . relationship between lipoprotein lipase (lpl) activity and hepatic triglyceride lipase (h-tgl) activity. 223 discussion the present study reports on a clinical application of a method which quantitatively determines two different tg lipase activities in post-heparin plasma. the main advantage with the method used ( 9 ) compared to other methods described (14-16) is that no interference with plasma components like apo lipoproteins or whole lipoproteins can occur. two other methods have been described to estimate h-tgl and lpl activities in human post-heparin plasma ( 1 4 1 6 ) . one of them using an antibody against h-tgl activity which is aimed to completely abolish the contribution of this enzyme to whole tg lipase acti vity in post-heparin plasma ( 1 5 , 1 6 ) . the other method is based on the sugges tion that h-tgl activity is not inhibited while lpl activity is completely inhibited by protamin-sulphate ( 1 4 ) . in both these methods whole post-heparin plasma from the investigated patient is present during the assay, which means presence of the patient’s own apoproteins an lipoproteins which might serve as sustrate for the enzymes competing with the exogenous substrate added to the assay. there might also be inhibitors present in the whole post-heparin plasma ( 1 7 ) . thus the present method seems to have one advantage compared to previously described methods in quantitating h-tgl and lpl in human post-hepa rin plasma. however, the importance of this aspect is not fully evaluated since the in vitro addition of serum lipoproteins did not change the estimated activities of either h-tgl or lpl in the two methods described earlier (14,151. the application of this method in normal males and females and in male patients with hypertriglyceridemia demonstrated no relationship at all between the two post-heparin tg activities h-tgl and lpl. this observation is an addi tional piece of evidence indicating that these two enzyme activities really are two different enzymes. this has recently been demonstrated in a study where the two enzyme proteins have been purified and characterized ( 1 8 ) . h tgl activity was higher in normal males compared to females, and also higher in normal males compared to male patients with hypertriglyceridemia. none of the patients or subjects were missing the h-tgl activity and we have found no evidence for a relationship between this enzyme activity and any physiological or pathophysiological condition. others have reported, however, that this enzyme activity might be low in patients with hypothyroidism ( 1 4 ) . lpl activities in normal males and females occurred within the same ranges and there was no age dependence demonstrable. the range of activity presented here is slightly higher than described by krauss et a1 ( 1 4 ) , who used the me thod dependent on complete protamine-sulphate inhibition of lpl activity. the latter authors also found an age dependent lpl activity in females. a decrease of lpl activity with age in both males and females was demonstrated by hut tunen et a1 ( 1 9 ) who used the selective innnunochemical method ( 1 5 ) . these 224 authors also demonstrated higher absolute values than presented in this study for both h-tgl and lpl activities ( 1 9 ) . the reason for this difference is not known but might be explained either by differences in substrate preparation or differences in the recovery of the enzyme isolation procedures. the isolation procedure used in the present paper probably gives a lower yield than the im munochemical method. there were significant correlations between lpl activities and s-tg concen trations both for normal females and normal males. this is in agreement with two earlier studies ( 1 9 , 2 0 1 where different but still rather specific methods have been used to quantitate lpl activities. earlier it has been shown that there is no correlation between endogenous s-tg production ("turnover") and stg concentration in males with hypertriglyceridemia ( 3 ) . this finding may indicate that the level of s-tg concentration in normals generally are deter mined by the rates of clearance of s-tg (or by the fractional removal rate) evidence for which has been gained earlier with other methods ( 3 , 4 ) . since the enzyme lpl most probably plays an important role in the removal process of the s-tg the significant correlations found in this study between lpl activities and s-tg concentrations fit well with previous studies on serum tg turnover. earlier studies on post-heparin lpl (20) and tissue lpl activities ( 2 1 ) have reported significantly lower values in patients with hypertriglyceridemia. however, the range of values measured for the lpl activities in these patients overlaps that from analyses of subjects with normal serum tg concentrations. overlapping of activities in these two groups occurred also in this report, however, this was slightly less pronounced. lpl activities were significantly lower in patients with hypertriglyceridemia. in about one third of the patients the low lpl activities might be the cause of the hypertriglyceridemia while in the rest of the patients contribution of other factors must be added to explain the cause of the hypertriglyceridemia. references 1 . eaton, r.p., berman, m. & steinberg, d.: kinetic studies of plasma free fatty acid and triglyceride metabolism in man. j clin invest 4 8 : 1 5 6 0 1 5 7 9 , 1 9 6 9 . 2 . havel, r . j . , kane, j.p., balasse, e.o., segel, n. & basso, l.v.: splanch nic metabolism of free fatty acids and production of triglycerides of very low density lipoproteins in normotriglyceridemic and hypertriglyceridemic humans. j clin invest 4 9 : 2 0 1 7 2 0 3 5 , 1 9 7 0 . boberg, j., carlson, l . a . , freyschuss, u., lassers, b.w. & wahlqvist, m.l.: splanchnic secretion rates of plasma triglycerides and total splanch nic turnover of plasma free fatty acids in men with normoand hypertri glyceridemia. europ j clin invest 2 : 4 5 4 4 6 6 , 1 9 7 2 . 3 . 225 4. 5. 6. 7. 8. 9. boberg, j.: mechanisms of hypertriglyceridemia in man. acta universitatis upsaliensis 1971. (abstr, no. 105.) (dissertation.) person, b., bjerntorp, p. & hood, b.: lipoprotein lipase activity in human adipose tissue. metabolism 15: 730-741, 1966. lithell, h. & boberg, j.: a method of determining lipoproteinlipase activi ty in human adipose tissue. scand j clin lab invest 37:551-561,1977. lithell, h. & boberg, j.: determination of lipoprotein lipase activity in human skeletal muscle tissue. biochim biophys acta 528:58-68, 1978. ehnholm, c., shaw, w., greten, h., langfelder, w. & brown v.w.: separation and characterization of two triglyceride lipase activities from post-he parin plasma. in: atherosclerosis iii (ed. g. schettler & a. weizel), pp. 557-560, springer-verlag, berlin, 1974. boberg, j . , augustin, j., baginsky, m., tejada, p. & brown, v.w.: quanti tative determination of hepatic and lipoprotein lipase activities from human post-heparin plasma. j lipid res 18:544-547, 1975. 10. wood, p. et al.: manual of laboratory operations. lipid research clinics program. volume 1 lipid and lipoprotein analyses. dhew publication no. (nih) pp. 76-628, 1974. 1 1 . iverius, p-h.: coupling of glycosaminoglycans to agarose beads (sepharose 12. belfrage, p. & vaughan, m.: simple liquid-liquid partition system for iso 4b). biochem j 124:677-683, 1971. lation of labelled oleic acid from mixtures with glycerides. j lipid res 10:341-344, 1969. 13. snedecor, g.w.: in: statistical methods. the iowa state university press, ames, iowa, 1961. 14. krauss, r.m., levy, r.i. & fredrickson, d.s.: selective measurement of two lipase activities in post-heparin plasma from normal subjects and patients with hyperlipoproteinemia. j clin invest 54:1107-1124, 1974. 15. huttunen, j.k., ehnholm, c., kinnunen, p.k. & nikkilb, e.a.: an immunoche mica1 method for selective measurement of two triglyceride lipases in human post-heparin plasma. clin chim acta 63:335-347, 1975. 16. greten, h., degrella, r., klose, g., rascher, w., de gennes, j.l. & gjone, e.: measurement of two plasma triglyceride lipases by an immunochemical method. studies in patients with hypertriglyceridemia. j lipid rps 17:203 210, 1976. 1 7 . brown, v.w. & baginsky, m.: inhibition of lipoprotein lipase by an apopro tein of human very low density lipoprotein. biochem biophys res comm 46:375-382, 1972. 18. ostlund-lindqvist, a-m.: lipoprotein lipase and salt resistant lipase. acta universitatis upsaliensis 1978. (abstr. no. 301.) (dissertation.) plasma lipoprotein lipase and hepatic lipase in normal subjects and in patients with hypertriglyceridemia. correlations to sex, age and various parameters of triglyceride metabolism. clin sci mol med london 50:249-257, 1976. 20. boberg, j.: heparin released blood plasma lipoprotein lipase activity in patients with hyperlipoproteinemia. acta med scand 191:97-102, 1972. 19. huttunen, j.k., ehnholm, c., kekki, m. & nikkilb, e.a.: post-heparin 226 21. tithell, h., boberg, j., hellsing, k. & vessby, b.: relationship between the lipoprotein lipase activity of human adipose and skeletal muscle tis sue and the elimination rate of i.v. injected intralipid. in: protides of biological fluids (ed. h. peeters), pp. 3 8 9 3 9 2 . pergamon press ltd., oxford, 1 9 7 7 . accepted january 20, 1 9 7 9 address for reprints: jonas boberg, m.d. department of geriatrics university of uppsala p.0.box 12042 s-750 12 uppsala 12 sweden 6-792856 227 upsala j med sci 91: 216, 1986 clinical chemistry in primary health care relationship between hospital laboratories and general practice questionnaire answers f r o m 33 directors of clinical chemistry laboratories bertel berg and nils tryding department of clinical chemistry, central hospital, kristianstad, sweden 1. who has the formal and o r practical responsibility for clinical chemistry in primary health care within the region (for choice of methods, laboratory equipment, quality control, instrument service)? 16 hospital laboratories have formal and practical responsibility, 8 not practical but formal, 7 practical but not formal, 2 neither formal nor practical. 2. number of professional laboratory technicians per region working in primary health care/number of regions. 0 / 4 1/6 l$-2/3 35-4/3 5-75 >8 technicians/l2 regions 3. special systems for sample transport from primary health care to hospital laboratories are established in 27 regions. 4. transport of analytical results from the hospital laboratory: by post and/or by car. 5. postgraduate training in clinical chemistry for primary health care staff is organized in 1 3 regions for doctors (often sporadic) in 11 for laboratory technicians (often sporadic) in 14 regions for other personnel 6. number of hospital personnel posts designated for instruction to primary health care staff/number of regions: 0/15 0.3/1 0.5/7 1/6 2/2 4 post-/i region 7 regions also have instruction for microbiology. 7. joint meetings between personnel in primary health care and clinical chemistry have been held between doctors in 8 regions. 8. will more analyses be performed locally in primary health care with new techniques for clinical chemistry near the patient? most regions are positive to new techniques but all emphasize the need for personnel training and quality assessment. 216 upsala j med sci 84: 61-66, 1979 local traumatization with heat-cutaneous reactions and early effects on serum zinc concentration in rats with alloxan diabetes of very short duration goran hallmans, folke lithner and erik hagg ftom the departments of pufhology und medicine, university of umepi, lime;, sweden abstract local heat trauma was induced in rats with alloxan diabetes of 3 days' duration. the cutaneous reaction, water content of the skin and serum zinc concentration were estimated. petechiae within the area of traumatization were observed more often and were more abundant in the controls than in the diabetic animals. there was no difference between diabetic animals and controls with regard to the increase of water content of traumatized skin or to the water content of non-traumatized skin. there was no difference in serum zinc levels between non-traumatized dia betic animals and controls. after traumatization there was a decrease of serw zinc levels in both animal groups, but to a significantly lower level in the diabetic animals compared to the controls. introduction the skin of the lower extremities in diabetics has an altered reaction to local traumatization with heat and cold (9). this phenomenon is related to the occurrence of late diabetic lesions, such as microangiopathy and neuropathy, but not to the diabetic metabolic derangement per se (9, 11, 12). a similar traumatization with heat has been performed in alloxan diabetic rats of varying age and duration of diabetes (10). within the area of traumatization there was a pronounced reaction consisting of an increased erythema in animals with long duration of diabetes. in short-term alloxan diabetic rats there was only a slight increase of erythema compared to controls, but not until two weeks after the traumatization. i n a similar study, traumatization with local heat was performed on rats with 5-7 weeks duration of diabetes in which evans blue was injected intravenously ( 6 ) . concerning the early reaction, there was no difference between diabetic animals and controls. neither histological nor histochemical studies revealed any differences. however, there was an increased water content of the non-traumatized skin of the diabetic animals to controls. 61 an increased water content in diabetic tissue has been described earlier ( 3 , 4 ) . to determine if the increased water content of diabetic tissue is directly related to the diabetic metabolic derangement per se, we have, in the present study, estimated the water content of tissue in rats with alloxan diabetes of very short duration ( 3 days). decreased levels of zinc in plasma, leucocytes and erythrocytes and in creased levels in urine have been demonstrated in newly diagnosed diabetics (8). in non-diabetic rats there is a pronounced decrease of plasma zinc levels in different inflammatory conditions already after eight hours (1). we there fore have considered it of interest to determine the serum zinc levels in traumatized and non-traumatized animals with or without diabetes. material and methods thirty-two male albino rats of the highly inbred r-strain were used (5). diabetes was induced in 16 animals at the age of 3 months with an intravenous injection of alloxan, 0 . 3 4 mmol/kg, as described earlier (7). n o insulin was given after the injection of alloxan. a l l animals had blood glucose values above 1 3 . 9 mmol/l, polyuria and glucosuria. methods of depilation, anesthesia and determination of water content were described in a previous article (6). traumatization was performed three days after the alloxan injection, the temperature used for traumatization was 60 oc for 5, 10 and 15 seconds. local traumatization was induced in the anesthetized animals by placing the end surface of an electrically heated cylindrical brass rod, 18 mm in diameter, against the skin, as described previously (6, 9). the traumatized areas were inspected after 0.5, 4 and 8 hours and then the animals were killed. both induction and inspection of the skin injury were performed without knowledge of the presence or absence of diabetes. the extent of cutaneous hemorrhages was assessed using four grades: 0, 1+, 2 + and 3 + . serum was collected in weighed, acid-rinsed glass tubes (jena). the samples were dried for 72 hours at 110 oc, ashed for 24 hours at 500 o c and the ash dissolved in 0.5 ml 3 mmol/l hcl overnight. a l l samples were then diluted with 2 ml deionized water. the serum zinc concentrations were determined using a varian atomic absorption spectrophotometer at 2 1 3 . 9 nm. reference samples of zinc in 0.6 mmol/l hcl were used ( 2 ) . the significance of the difference between means was calculated using the student's t-test. differences of extent of petechiae were tested with the non parametric rank sum test of wilcoxon. p < 0.05 was chosen as the level for statistical significance. 62 results the blood glucose level at the beginning of the traumatization was 17.4 0.9 mmol/l for the diabetic animals and 5.2 (mean 0 . 4 mmo1/1 for the controls s.e.). no animal had ketonuria. macroscopic assessment of purpura within the area of traumatization. p 3 . 2 . i ' 0 10s p at all the three degrees of traumatization the controls had extent of purpura 8 hours after the traumatization as compared betic animals (fig. 1). o c o n t r o l s d i a b e t i c a n i m a l s fig. 1. data on local cutaneous p e t e c h i a e with heat 60 o c h o u r s a f t e r t r a u m a t i z a t i o n 05 i i i 8 e x t e n t , g o 01 zation. the extent of the pete 0000 0 0 0 0 0 0 chiae within the traumatized area o e o n was assessed using four grades: ooo o n ... .. 0, 1+, 2+ and 3 + . the p > 0.05 values are omitted. no. of diabetic animals = 8, .. .. :go :at c o o 1 c o o 1 an increased with the dia the effect of traumatization and different 0 periods of traumatization (5, 10 2 + 0 and 15 seconds). the effect of . 000 0 traumatization registered at 0.5, 4 and 8 hours after the traumatiit o m 888" w' 15s 3 + i * + 1 + i 0000 controls = 8. o m 000 go" ... ..a ... 0 ... 0 1 dodo .. .. 0000 ::u determination of the water content of traumatized and non-traumatized skin. the water content of traumatized skin was increased compared to that of non traumatized skin both in diabetic animals and in controls. there was no sig nificant differences between diabetic animals and controls with regard to the increase of water content of traumatized skin, nor with regard to the water content of the non-traumatized skin (fig. 2). determination of serum zinc concentration. there was no difference in serum zinc concentration between non-traumatized diabetic animals and controls (fig. 3). after traumatization the serum zinc levels were decreased both in the controls (p < 0.05) and in the diabetic ani mals (p < 0.001). serum zinc concentration after traumatization was significant ly lower in the diabetic animals compared to that of the controls (p < 0.01). 63 0 nontraurnatized 5 traumatized d w "l 6 7 n s ;p(moii i fig. 2. data on local cutaneous traumatization with heat 60 o c and different periods of trauma tization (5, 10 and 15 seconds). water content of skin determined as the wet weight (ww) and dry weight (dw) using the relation ship z-x-d!? . the determinations were performed on excised pieces from non-traumatized skin and skin traumatized with heat. num ber of animals = n. mean s.e. dw 0: non-traumatized a: traumatized fig. 3 . zinc concentration in serum (pmol zn/l) of controls and diabetic animals with or without local cutaneous trauma tization with heat 60 o c (5, 10 and 15 seconds). eight animals in each group. mean s . e . ns = not statistically signifi cant. a betic animals 64 discuss ion in the present study, the controls had an increased extent of purpura within the area of thermal traumatization compared to diabetic animals. in a similar study we have previously estimated the extent of visible blue spots within the cutaneous area of thermal traumatization upon intravenous injection of evans blue, there being no difference in this respect between d abetic rats and con trols (6) or between diabetic rabbits without ketosis and controls (13). it is possible that, in demonstrating plasma leakage with evans blue we might have concealed an increased extent of purpura. in the present study there was no difference with regard to the water content of non-traumatized skin between the controls and the animals with diabetes of very short duration. thus, the early diabetic state does not seem to alter the water content of skin. an increased water content of different tissues in dia betic animals (3, 6) and man (4) is probably caused by a long-standing diabetic metabolic derangement. the demonstrated decrease of the serum zinc levels after traumatization in our study is in accordance with earlier reports of induced acute inflammatory reactions in non-diabetic rats (1). the more pronounced decrease of serum zinc levels observed in the animals with diabetes of short duration might be ex plained by a difference in function of phagocytic cells. phagocytic cells re lease a leucocytic endogenous mediator (lem). lem initiates a variety of effects including a decrease in the serum zinc levels and an increase in the zinc con centration in the liver (1). references 1. beisel, w.r., pekarek, r . s . & wannemacher, r.w.: homeostatic mechanisms affecting plasma zinc levels in acute stress. 2: trace elements in human health and disease, academic press, 1:87-106, 1976. 2. bergman, b., sjiistrem, r. & wing, k . r . : the variation with age of tissue zinc concentrations in albino rats determined by atomic spectrophotometry. acta physiol scand 92:440-450, 1974. 3. forscher, b.k. & cecil, h.c.: some effects of alloxan diabetes on acute inflammation. j applied physiol 13:278-282, 1958. 4. fuchs, u.: elektronmikroskopische untersuchungen menschlichen muskel kapillaren bei diabetes mellitus. frankfurter zeitschr pathol 73:318-327, 1964. 5. hagg, e.: renal lesions in rats with long-term alloxan diabetes. a semi quantitative light microscopic study with particular reference to the glomeruli. acta pathol microbial scand 82 a:199-210, 1974. 5-792854 65 6. hallmans, g., lithner, f. & hlgg, e.: early cutaneous reactions to local traumatization with heat in alloxan diabetic rats. upsala j med sci 83: 17-21, 1978. 7. korec, r.: experimental diabetes mellitus in rat. publishing house of the slovak academy of sciences, bratislava 1967. 8. kumar, s. & rao, k.s.j.: blood and urinary zinc levels in diabetes mellitus. nutr metabol 17:231-235, 1974. 9. lithner, f.: cutaneous reactions of the extremities to local thermal trauma. acta med scand 198:319-325, 1975. 10. lithner, f. & hlgg, e.: cutaneous reactions of alloxan diabetic rats to local thermal trauma. upsala j med sci 80:99-102, 1975. 11. lithner, f.: skin lesions of the legs and feet and skeletal lesions of the feet in familial amyloidosis with polyneuropathy. acta med scand 199:197 202, 1976. 12. lithner, f.: lesions of the legs in diabetics and in patients with familial amyloidosis and polyneuropathy. acta med scand suppl 589, 1976. 13. lithner, f. & hallmans, g.: cutaneous reactions to local traumatization with heat in alloxan diabetic rabbits with and without ketosis. upsala j med sci 83:23-28, 1978. accepted december 10, 1978 address for reprints: folke lithner, m.d. department of medicine university of umeh s-901 85 umeh sweden 66 transplacental amino acid transfer and its study using positron emission tomography a short review based on a doctoral thesis lars berglund introduction during its life span, the placenta fulfills its purpose of protecting and supporting the growing foetus in a variety of ways. despite its central significance for the well-being of the foetus, our knowledge of its function remains far from adequate, albeit many studies have been conducted. the placenta acts as an immunological barrier, though not completely, separating the maternal immunological system from the foetal transplantation antigens. the mechanisms of maternal immunological acceptance of the foetus are poorly understood, but a prerequisite for this coexistence is the maternal acceptance of the placenta as a "foetal allograft" and is achieved by the partial absence of transplantation antigens on the syncytiotrophoblast (44). the pregnancy is accepted immunologically by the mother, despite the maternal immune system retaining its principal function throughout pregnancy. the placenta, though far less efficient, has been likened to the foetal "lung". the diffusion rate for gases per unit weight of the placenta is approximately one-fiftieth that of the lung (39). this is explained partly by the main difference between the placental exchange between two blood compartments, and the pulmonary exchange between one blood and one gaseous compartment. the respiratory gases, 02, co;! and co, are presumed to cross the placenta by simple diffusion (33). the existence of a facilitated transfer mechanism for co has been discussed ( 5 ) , but not c o n f i i e d (35). the placenta satisfies foetal demands for energy and nutrients such as fat, sugars and amino acids by different transfer mechanisms. foetal fat is produced from free fatty acids transferred by simple diffusion across the placental membranes and by foetal lipogenesis from maternal carbohydrates (39). the principal sugar used by the human foetus is glucose and its transfer shows several characteristics suggesting a carrier transport system responsible for transfer across the haemwhorial placenta. carrier transport systems have been demonstrated in vitro, for the d hexoses, d-pentoses and lactate (30). considerable interest has been shown in the placental transfer of amino acids most of which will be transferred to the foetal circulation. some, however, will be used as a source of energy by the placenta and for the synthesis of placental proteins. placental protein synthesis accounts for 12 16% of amino acids transferred from the mother (6,40). as in the case of sugars, placental transfer of amino acids is effected by transport mechanisms and not by simple diffusion (39, 65). much 7-908572 95 remains to be learnt about these mechanisms. the human placenta after the human placenta has attained its definitive architecture, the maternal surface is subdivided into lobules by septa protruding into the intervillous space. these septa are folds of the basal plate, formed by a combination of trophoblast and decidual cells (41) and d o not seem to play any physiological role (15). one to several foetal primary stem villi correspond to each interseptal, intervillous lobular space. each primary stem villus breaks up just below the chorial plate into secondary stem villi, which in turn divide into tertiary villi which sweep down to attach to the decidua forming the foetal placental lobule (15, lo). the placental villus containing the foetal capillary bed surrounded by the maternal blood is the functional unit in transplacental transfer. the blood constituents must traverse a three-layered membrane in either direction: the foetal capillary endothelium, the mesoderm of the villous core and two types of trophoblast, cytotrophoblast and syncytiotrophoblast both of which have a roie in nutrition including absorbtion, phagocytosis and active transport (58). in the human, the syncytiotrophoblast and cytotrophoblast exhibit striking cytological differences. the syncytiotrophoblast has well developed endoplasmatic reticulum and golgi apparatus and numerous large mitochondria, which per unit volume appear to be more numerous than in the cytotrophoblast (20). the trophoblast represents approximately only 14% of the placental "parenchymal" volume according to morphomemc measurements (4,28). it is metabolically the most active region of the placenta (65), and its oxygen consumption is considerable, probably 25-30% of the total uterine consumption including that of the foetus,placenta and uterine muscle (38). the syncytiotrophoblast shows several features common to cells engaged in energy production and transfer. this is a prerequisite for its production of placental steroids and proteins and for its decisive role in active matemo-foetal transfer. during the second half of the pregnancy, the cytotrophoblast layer gradually disappears although residual cytotrophoblast cells persist till term. compared with the syncytiotrophoblast cells, cytotrophoblast cells are structurally simple with limited golgi apparatus and endoplasmatic reticulum (39). the main significance of the cytotrophoblast cells seems to be as a germinal zone for the syncytiotrophoblast layer. transfer of amino acids. the concentrations of most amino acids in human maternal plasma are reduced during pregnancy compared to the non-pregnant state (63). the total plasma concentration of free amino acid in the foetus is about twice the maternal concentration (7). the amino acid concentration in the trophoblast is even higher compared to the maternal plasma suggesting a transfer process involving active transport from maternal plasma to the trophoblast and passive diffusion along a downhill gradient from the trophoblast into the foetal circulation (64). christensen and streicher (7), were among the first to suggest the existence of active transport systems for the transfer of amino acids across the placenta. enders et a1 (14) determined the specificity of the principal placental transport systems for the neutral amino acids and found that 96 they corresponded approximately to the "a", "l" and "asc" systems of christensen. the "a" system is sodium-dependent and favours short amino acids with linear side chains such as alanine, glycine and the non-metabolizable model amino acid: alpha-isobutyric acid. the "l" system is sodium-independent and is most reactive with the branched chained amino acids such as valine, leucine and isoleucine and with the aromatic amino acids. the "asc" system is also sodium dependent and restricted in its activity to alanine, serine and cysteine (8). the specific canier systems all overlap in their affinity for the different amino acids. methionine is thus transported both with the "a" and "l" systems. control of transfer. the aim of placental transfer control of amino acids is to optimize the foetal amino acid supply to the demands. stulc (57) questioned the necessity of a detailed regulation in view of the stable foetal environment produced by maternal homeostasis. however, the maternal homeostatic mechanisms are too often faced with extraordinary demands, such as during maternal smoking, illness and starvation (18) which may need adaptive measures of placental transfer to ensure optimal foetal supply. local control at the placental cellular level is influenced by the energy supply, since the active component of amino acid transfer is very energy dependent (39). earlier results indicated that active transport across the placenta was dependent upon energy from oxidative sources (11, 53). more recent research indicates that oxidative metabolism was not required, and that the energy demands for both placental transfer and placental protein synthesis were satisfied by anaerobic glycolysis the transport capacity of the placenta is adaptable in vitro, to changes in amino acid availability. preincubation of placental tissue in amino acid free medium increased cellular concentration of the non-metabolizable amino acid alpha-isobutyric acid (52). later, enhancement of amino acid transport under conditions of amino acid shortage has been found to be limited to transport system "a" of christensen in both placental tissue (14) and fibroblasts (17). in vivo studies have revealed more conflicting results. young and widdowson ( 6 2 ) reported that feeding pregnant guinea pigs on a low-protein diet caused retarded foetal growth and enhanced placental transfer and foetal uptake of alpha-isobutyric acid. rosso (45) was unable to confirm the results of young and widdowson. ahokas et al. (1) observed that a protein and energy restricted diet in rats had a variable effect on the transplacental transfer of alpha-isobutyric acid. (34, 43). hormonal regulation. the concentration of amino acids in venous blood displays a circadian rhythm, which is especially evident in the aromatic amino acids phenylalanine, tyrosine and tryptophane (60). this rhythm is not altogether explained by the fluctuations in protein intake and physical activity during day and night and thus suggests a hormonal influence on the amino acid pattern. plasma cortisol levels seem to have an impact, though not decisive, since the rhythm has been shown to persist despite hypophysectomy (59). both cortisone and hydrocortisone increase the uptake of alpha-isobutyric acid into the liver, which might be a reflection of the 97 catabolic action of steroid hormones on skeletal muscles (31). noradrenaline and other catecholamines increase the incorporation of tryptophane into proteins in organ cultures (61). kidman, weiss and kosta (27) studied protein metabolism and amino acid accumulation in the submaxillary gland of the rat and found a higher uptake of alpha-isobutyric acid into proteins during reduced sympathetic activity of the gland. they concluded that adrenergic nerve impulses modulate the transport of amino acids from the plasma to the tissues. conclusive evidence for hormonal regulation of placental transport systems is still scanty. dancis et al. (1 l), however, found that insulin increased amino acid uptake in human but not in guinea pig placental slices. growth hormone, lactogenic hormone, oestradiol, oestrone sulphate and oxytocin produced no measurable response. yudilewich and sweiry (65) summarized the available studies of the effect of insulin on placental transfer and concluded that most investigators were unable to find any effect. the placenta possesses a well developed cholinergic system localized mainly in the syncytiotrophoblast layer. high levels of acetylcholine (ach) and enzymes responsible both for its production and degradation have been found in the placenta (46). evidence for placental cholinergic receptors has been put forward by sastry and sadavongvivad (47). the significance of the placental cholinergic system is not fully understood, but the system might be involved in the regulatory function of amino acid transfer. selective inhibition of choline acetyl transferase, an enzyme involved in the synthesis of ach, lowered both the concentration of ach and alpha-isobutyric acid uptake in the isolated human placental villous (48). barnwell and sastry ( 3 ) found that nicotine, a well known cholinergic blocking agent, produced a concentration dependent and reversible reduction in the uptake of all amino acids examined. they concluded that the placental cholinergic function by decreasing ach release may affect placental amino acid uptake. methods for placental transfer studies in vitro studies of placental tissue and fragments. placental transfer studies in humans have often been based on the incubation of placental slices and tissue fragments in media containing radioactively labelled amino acids. the uptake of natural and non-metabolizable amino acids has been measured. the possibility of adding metabolic inhibitors and hormones to the incubation medium and preincubating the tissue under various conditions such as amino acid shortage have made it possible to characterize the placental amino acid transfer mechanisms. dancis et al. (11) suggested that the transfer of amino acids to the foetus and the establishment of a maternal-foetal gradient involved a prior concentration by the placenta with a release into the foetal circulation. smith et al. (52) noted that placental tissue took up alpha-isobutyric acid actively and that, in an hour, the concentration in the intracellular fluid reached a level five times that of the incubation medium. preincubation of the tissue increased the rate of uptake to a concentration 25 times that in the incubation medium. enders et al. (14) established the existence of specific transport systems for neutral amino acids in the human placenta from tissue studies using a competitive inhibition technique. 98 the wide variation in fluid distribution and high extracellular water content of the placental tissue reduce the precision with which the intracellular uptake of the labelled amino acid can be measured from in vitro tissue incubation studies, making it necessary to estimate the fluid spaces each time placental fragments are incubated (52). the use of isolated cell membrane vesicles as models for transport studies seems to optimize the possibility of controlling the composition of the environment on either side of the membrane. separation of the different membranes permits the study of properties involved in transport across the individual membrane. results suggest that na+ stimulates the transport of valine across the intestinal epithelial brush border membrane but has no effect on transport across the basolateral membranes (22). dicke and henderson (12) investigated the uptake of the non-metabolizable amino acid alpha-isobutyric acid in isolated vesicles of human syncytiotrophoblast membrane from pregnancies complicated by diabetes mellitus, hypertension or by deliveries of small-for-gestational age (sga) neonates. the uptake of alpha-isobutyric acid was significantly less in the sga group. in vitro studies of the intact placenta. several techniques for in vitro studies of placental transfer in various species have been described. schneider, panigel and dancis (49) cannulated the foetal artery and veins of newly delivered placentas and perfused the foetal side with physiological solution. the lobule supplied by the cannulated foetal vessels was identified by the colour change by the infusion medium where upon the intervillus space of the lobule was cannulated from the maternal surface for perfusion. the technique has been used in both human and animal placentae. with the in vitro perfusion technique schneider et al. (50, 51) showed that the human placental membrane exhibited a stereospecific active transport of amino acids with a polarity in active transport directed towards the foetus and a diffusion back to the maternal circulation. the transfer rate of alanine, glycine and lysine was estimated as 2.8 pmol per min for a placenta of 450 g. the inevitable deterioration of the preparation with risk of leakage, the use of artificial solutions in the maternal and foetal circulations and the artificial flow on both sides of the placental membrane are major drawbacks in such a model. james et al. (23), observed that the blood flow in the placenta seems to vary, in that parts of the placenta receive different amounts o f blood at a given time. an in vitro model seems to be best suited for the study of qualitative aspects of placental transfer. care should be taken in interpretating quantitative results and especially in extrapolating the observations to in vivo situations (64). in vivo animal studies. animal models have been employed extensively for in vivo studies of transplacental transfer . the foetus is kept either in utero or ex utero with chronic implantation of catheters after surgical exposure of maternal and foetal vessels . using such a technique, stegink et al. ( 5 5 ) showed that aspartate, like glutamate, but unlike most other amino acids, was not concentrated in the foetal circulation. with the same technique, the foetal plasma concentration of taurine was found to maintain the normal foetal to maternal concentration gradient 99 of 1.31.5 during increasing maternal plasma concentrations (56). ewes have been used frequently but the foeto-placental complex of the ewe shows features which differ from the human complex. in the ewe placenta (58). the maternal and foetal circulations are separated by the endothelium of maternal blood vessel and endometrial connective tissue in addition to the cellular layers separating the two circulations. this implies significant differences for the transfer of substances to the foetal lamb compared with the human foetus. the guinea pig has been used commonly in in vivo placental transfer studies. the preparation and its merits have been described in detail by young (64). after removal of the foetus and cannulation of the umbilical vessels, the foetal circulation is perfused with artificial fluids with the placenta in situ . an intact maternal placental circulation is thus maintained and the study of the transfer of substances is possible independent of foetal influences. the problem of the possibility of impaired maternal circulation and hence a need for pharmacological agents to maintain the maternal arterial pressure is stressed. although the guinea pig placenta, like the human placenta, belongs histologically to the haemochorial type, dissimilarities must be considered, when relating data from transfer studies in the guinea pig to humans. the guinea pig placenta does not show any intervillous space, but has preformed channels for the circulating maternal blood. additional differences in the placental foetal capillary network make the circulatory pattern different from the pattern in the human placenta. the importance of the rodent yolk sack in the transfer of protein molecules to the foetus compared with the vestigial human yolk sack is another difference restricting the use of guinea pigs in obtaining data relevant to human transplacental transfer. in vivo human studies. the study of placental transfer in vivo in the human presents great difficulties due to the inaccessibility of the foetus and placenta. in vivo studies have been done after injecting a substance into the mother near the time of delivery then collecting neonatal and maternal blood samples . christensen and streicher (7) found from studies based on maternal amino acid loading, that high doses of methionine reduced the ratio of glycine between foetal and maternal plasma. they were the fiist to discuss the involvement of an active transfer mechanism of amino acids across the placenta to the foetus. gaul1 et al. (16) cannulated human foetuses after hysterotomy prior to abortion. maternal and foetal blood samples were taken simultaneously at intervals after administering an amino acid load intravenously to the mother. materno-foetal transport of methionine, leucine and ornitine was found to take place against a concentration gradient despite a twoto three fold difference in the initial concentrations. from the above, it is evident that our knowledge of transplacental transfer is based on in vitro studies of intact human placental lobules, placental tissue or its sub-cellular fractions. knowledge of the conditions prevailing in vivo has been extrapolated from results of studies of different animal species performed mostly with invasive techniques. for a better understanding of the in vivo transfer in the intact human foeto-placental complex, a new approach seems necessary. methods 100 positron emission tomography. positron emission tomography (pet) enables non invasive kinetic studies of physiological processes to be performed in vivo in animal and humans. the general application of pet has been reviewed by jones (24). previously pet has been used mainly to investigate physiological and pathological conditions in the central nervous system ( 19, 32), and in the lung and heart (13) pet utilizes positron emitting radionuclides with a short-life such as 15o,13n, andllc with a half life of approximately 2 min, 10 min and 20.4 min respectively. the decay of the radionuclides produces positrons, the antiparticles of electrons. a positron is immediately annihilated by an electron within a few mm from the site of decay, giving rise to two photons which escape in almost opposite directions. the annihilation radiation can be detected by external detectors. coincidental detection in two opposing detectors provides the possibility of locating the event of the decay. in the present series of investigations, 11c was produced by bombardment of n2 with protons at the tandem accelerator laboratory, university of uppsala. the 11c atoms react with 0 2 present in the target gas in minute amounts, forming llc02 mainly which was utilized for chemical synthesis of [11ch+z and -d -methionine (29). a scanditronix pc 384-3b positron emission tomograph (ab scanditronix, uppsala, sweden) was used. it has two detector rings which surround the animal giving three adjacent cross sectional images with a thickness of approximately 11 mm and a spatial resolution of 8 mm. figure 1 . schematic layout of the positron emission tomography system used in the present study. 101 the radioactivity was administered as an intravenous bolus injection. the distribution of radioactivity was recorded initially for 2o-sec-periods, while the subsequent exposure times were progressively increased according to a predetermined program. cross-sectional images of the body organs based on the amount and distribution of radioactivity during each exposure were reconstructed and displayed on a screen. during the experiment, it was possible to delineate a specific region of the image and follow the amount of radioactivity in the region of interest. additional information was obtained by external detection of radioactivity in maternal venous blood samples collected at regular intervals and measured in a well-counter, or by continuous analysis of radioactivity in arterial blood withdrawn through a catheter in the maternal femoral artery and analyzed in a specially designed detector. in some experiments, the radioactivity in the amniotic fluid and urine was measured. the uptake of radioactivity within the tissue was expressed as an uptake index which represents the measured radioactivity per cm3 of tissue in relation to the amount of radioactivity administered per gram of body weight. assuming that tissue density is approximately ig per cm3, an uptake index of one will represent an even distribution of the tracer in the body. the same uptake indices as for the pet measurements were calculated for the samples measured in the well-counter and in the detector for continuous blood sampling. positron emission tomography in the study of transplacental transfer. pet was first introduced in the study of transplacental transfer when tracer amounts of [11ch3]-i methionine or [11ch3]-d methionine were administered intravenously as a bolus dose to six pregnant rhesus monkeys. the distribution in time of radioactivity was followed for up to 60 min by pet in separate regions of interest (roi) in cross sections of the body containing the uterus with at least one placenta and the foetal liver (fig 2). samples of blood, urine and amniotic fluid were collected at regular intervals.materna1 plasma was separated in a high and a low molecular weight fraction by the use of gel filtration. the blood, the urine, the amniotic fluid samples and the high molecular weight fraction obtained from maternal plasma (m.w.>5000) were analyzed for radioactivity. in order to further evaluate pet as a tool in the study of transplacental transfer, the appearance of [ 11ch31-i -methionhe in the amniotic cavity, which includes the amniotic fluid and foetus was measured in six rhesus monkeys after the administration of a bolus tracer dose to the mother. further, in four of the monkeys, the measurements were repeated after seven days on an iso-caloric protein restricted diet. two foetuses died in utero. the transplacental transfer was again measured 14 days after return to normal diet in the two monkeys with live foetuses. two pregnant monkeys on normal diet throughout the period underwent the same scanning protocol. blood samples for amino acid analysis were collected from all the monkeys throughout the period of study. 102 figure 2 pet image of n cross sectionalslice of a pregnant rhesus monkey. the region of interest corresponding to the fetus is seen in the middle, surrounded by the amniotic cavity. the two placentas are seen to the left and above. the uptake of trace amounts of [ 11ch31-i -methionine in the foetus was studied with pet. after 15-20 minutes, the time required to attain a steady state in the distribution of radioactivity in the organs, the whole uterus was exposed to the detectors i n sequential steps. in each step, the radioactivity was recorded for 100-200 seconds. the total amount of radioactivity within the amniotic cavity was calculated and presented as a percentage of the radioactivity given to the mother. the mean radioactivity concentration in the amniotic cavity was obtained by dividing the total amount in the amniotic cavity by its volume defmed by pet. the kinetic information obtained by pet in the investigation of the transport of [11ch3]-f methionine from mother to foetus in the rhesus monkey was evaluated using a simple compartment model (fig 3). 103 figure 3 the compartment model used for the evaluation of kinetic data from pet studies of transplacental transfer of [11ch3]-[ methionine in the rhesus monkey. a catheter was placed in a femoral artery and connected to a reversible syringe pump for continuous monitoring of the radioactivity. [ 11ch31-i -methionine was administered by an intravenous bolus injection. after each investigation, regions of interest (rois), corresponding to placental and foetal tissue were defined from the radioactivity distributions obtained by pet. the radioactivity concentrations in the placental roi ( c t ~ ~ ) , foetal tissue roi ( c f ~ j and arterial blood (ckj were plotted as functions of time and fitted to an equation derived from the compartment model. rate constants were calculated, where kl and k2 describe maternal placental blood flow and k3 and b, the transfer of [ilch+z -methionine into placental tissue and foetus respectively. results the pet image made it possible to identify large structures like the maternal liver, aorta, kidneys, placenta, amniotic fluid, and foetal liver. analysis of the uptake in blood and in the regions of the pet image corresponding to the aorta and placenta revealed higher peak concentrations with slower rate of decline when [ 11ch31-d methionine was used. uptake of radioactivity was more rapid and rose to a higher level in the foetal liver when illch3i-i methionine was used. the concentration of radioactivity in the high molecular weight fraction of plasma was about the same during the first 10 15 min when the two enanthiomeric forms were used but increased when [11ch3]-1methionine was used, whereas the radioactivity remained on a low level when [11ch3] d -methionine was used. in the case of [llch3]-z methionine, the rate of excretion of radioactivity in urine was o.ol%/min of the given dose and 10 20 times higher when [11ch3]-d 104 methionine was given. the study shows that it is possible to identify and delimit the essential maternal and foetal structures in the pet image and to monitor the radioactivity necessary for the estimation of transplacental transfer of methionine. l methionine is the biologically active form which is used in protein synthesis. this was reflected in the pet examinations by: (a) a more rapid clearance from the blood and the placenta, (b) a higher and more rapid uptake in the foetal liver, (c) a higher uptake in maternal high molecular weight fraction of plasma and @) a lower urinary excretion rate when [11ch3]-i methionine was used compared with [11ch+d methionine. the mean reduction in daily energy and protein intake on the restricted diet was 16% (range 12-22) and 54% (range 32-68) respectively. no significant changes were noted in the mean venous serum concentrations of methionine in the mothers during the period of study, although both methionine as well as a majority of the other amino acids showed a tendency towards lower values during protein resmction. in one monkey while on restricted diet, the fraction of radioactivity in the amniotic cavity diminished compared with the fraction while on normal diet. this monkey showed a decrease in the amniotic cavity volume while on restricted diet and its foetus died i n utero after the second investigation. the other three monkeys showed an increased fraction of radioactivity in the amniotic cavity during restricted diet. two of them had an increased amniotic cavity volume and subsequently produced live births; the third displayed a constant amniotic cavity volume and the foetus died in utero after the second pet investigation. the study of transplacental transfer kinetics showed an initial fast rise of radioactivity to a concentration of approximately 20 in the arterial blood and of approximately 3.5 in the placenta. the rise in arterial blood was followed by a rapid decrease to 0.5 in two minutes and 0.1, in ten minutes after the injection . the uptake in the placenta decreased at a slower rate and stabilized at a steady level after three to five minutes. different investigations revealed great variations in the kinetics of the placental uptake curve in the first two minutes after the injection both in different animals and in each of the placentas of the same animal. fitting of data from the pet investigation to equations derived from the model revealed that a four compartment model is useful in the interpretation of results from transplacental studies with pet. the rate of transfer of methionine to the foetus was estimated as 0.8-1.3 nmol/min/g placenta. the transfer of methionine to placental tissue was found to equal the transfer to the fetus. an approximate blood flow through the intervillous space of 128 d m i n was found.the correlation between placental transfer to the foetus and the maternal blood flow in the intervillous space was low. discussion pet has been used in neurological, psychiatric and cardiological research and has contributed to new knowledge in the fields of metabolism, blood flow and pharmacology in normal and 105 pathological conditions. pet will establish a position as a clinical tool in the-diagnosis and ' treatment of intracranial tumors. properties of pet, such as its non-invasive character and ability to provide kinetic information make it an attractive tool for perinatal research and placental physiology. pet as a tracer technique pet offers several advantages in experimental investigations, one being that only trace amounts of the substance under study are needed. hence pharmacological effects of the labelled compound will not occur. in most of our studies, the amount of radioactivity that was administered varied in the range of 50 200 mbq. the specific radioactivity obtained at the end of synthesis of the d/l-methionine was in the order of 370 mbq per pmole. the levels of free amino acids in serum during pregnancy and foetal life in the rhesus monkey have been investigated by kerr (25). h e found concentrations between 32 and 42 pmole/l between 100 and 150 days of pregnancy. these results are in accordance with our own estimations ranging between 10 and 40 pmole/l. the amount of methionine administered during pet investigations was in most cases well below 0.5 pmole. the labelled methionine could therefore be regarded as a tracer for the pool of methionine in the rhesus monkey. incorporation of the tracer it was shown that [lmzh3]-i methionine was incorporated in the high molecular weight fraction of plasma to a much greater degree than [ 11ch3j-d methionine. the difference was visible 20 minutes after the administration of the different tracers. this is in agreement with the concept that the i -forms of the amino acids are the main precursors in protein synthesis. at the same time this fact limits the time during which kinetic information of transplacental transfer of i -methionine can be obtained to 10-15 minutes. after this time an increasing amount of radioactivity seems to be incorporated into peptides and proteins. routes of degradation yielding an increasing amount of radioactivity in the low molecular fractions of plasma even shortly after the administration of radioactivity have also been shown (37). knowledge of the metabolic pathways involved and additional analytical techniques are necessary for a proper interpretation of pet data derived from the metabolized radio labelled molecule. it may seem contradictory that transplacental transfer in one part of the investigation was studied after a steady state had been established in the organs. however, at that point transfer was studied from a static point of view, giving the relative amount of the tracer administered to the mother that was retrieved in the foetus. although some of the radioactivity at the time of the investigation was incorporated into proteins or metabolites, we assumed that the radioactivity reached the foetus bound to methionine and that the measured radioactivity, irrespective of where it was incorporated or not, should be an indicator of the accumulation of methionine in the foetus. it was also assumed that the transfer of methionine was an one-way transfer during the time of the investigation and that the amount of tracer transferred to the foetus was incorporated and trapped in foetal proteins. the assumption was based upon results which showed that the uptake of [11ch3]-i -methionine in the high molecular weight fraction of plasma was not seen until 15 min 106 after administration of radioactivity a period preceded by cellular extraction of the tracer from the blood to prepare for the protein production in the cells.the assumption was further supported by the result from one pet experiment, in which [11ch3]-i -methionhe was administered to the foetus. no radioactivity could be seen in the placenta or in the maternal tissues during the first 15 min. (unpublished observations). practical difficulties complicated the analysis of the pet data. maternal and/or foetal movements were indicated by unexpected irregularities in the uptake curve usually seen simultaneously in all three pet slices. the initially defined roi did not any longer cover the intended structure. this problem was solved by either ending the analysis of data when movements f i s t were discerned, provided the movements took place during later parts of the investigation, or by defining a new roi to be used for the analysis of the remaining part of the experiment. the analysis of rois covering the placenta posed specific problems. it was not always possible to decide whether lack of congruence between the placenta and its roi was merely the effect of maternal movements. in all other rois except that of the placenta there was a good correspondence between the specific tissue and the corresponding roi. this shift between the placenta and its roi, which would normally be looked upon as the effect of maternal movements, might as well be caused by changes in maternal placental blood flow or -volume, possibly due to uterine contractions. previous studies have given both morphological and functional support to the concept of non-uniform blood flow through the intervillous space of the placenta. (23,2). the delineation of the amniotic cavity was facilitated by the difference in radioactivity between the amniotic fluid and uterine wall surrounding the placenta(s) the delimitation of the caudal and cranial parts of the amniotic cavity was obscured by the high uptake in the adjacent maternal bowel and liver and the relative scarcity of amniotic fluid in the rostra1 and caudal regions. this problem was most evident in the study involving resmcted food intake (paper 11). the intention not to incorporate any irrelevant tissue into the amniotic cavity roi, might lead to underestimation of the volume of the amniotic cavity. close monitoring of maternal and foetal movements during the pet experiment together with complete delineation of the placenta by other means than pet, such as x-ray computed transmission tomography and/or ultra sonography should help to elucidate these problems. transplacental transfer. two different approaches to the use of pet in transplacental transfer were developed; one which describes pet in a quantitative situation and the other, where pet is applied to the kinetic situation of transplacental transfer. both methods are hampered by factors such as lack of adequate resolution in the pet and the risk of foetal movements but both possess individual advantages. the measurement of the relative amount transferred to the foetus offers several advantages. the calculations are less complicated and yield results that are easier to interpret, although they reflect only a static aspect of transplacental transfer. it is only necessary to monitor the radioactivity from one source, the foetus, which excludes the use of intraarterial 107 catheters for continuous monitoring of radioactivity in maternal arterial blood, necessary in' compartmental analysis. the limited resolution prevents pet measurement of foetal volume, which would be the most accurate volume to measure. the amniotic cavity volume could be measured by a dilution technique and the foetal volume could then have been subtracted. since the intention was to make serial measurements, amniotic puncture was avoided so as not to induce premature delivery. only minute amounts of radioactivity were found in the amniotic fluid compared with the foetus. the uptake in the amniotic cavity, which was identified in the pet image by the contrast between the low uptake in amniotic fluid and the high uptake in the adjacent tissues, was therefore considered to be approximately equal to that of the foetus. the minute amount of radioactivity measured in the amniotic fluid also poses a problem, in that changes in the volume of amniotic fluid alone will influence the tracer concentrations. consequently, tracer concentration in the amniotic cavity is a less sensitive measure of transplacental transfer. measurement of total radioactivitv in the amniotic cavity would probably relate more closely to transplacental transfer, but measurement of total radioactivity is adversely affected by incomplete delineation of the amniotic cavity in its caudal and cranial poles as discussed above. a compartment model of the placenta and the fitting of experimental data to the equation derived from the model have several advantages. the dynamic events inherent in the transfer process could be described in terms of rate constants which can be used for further description of transfer. results from several investigations can be compared and treated by standard statistical methods. kinetic analysis of transfer with pet necessitates continuous monitoring of radioactivity concentration of maternal arterial blood, which could only be achieved by means of external detection in blood withdrawn continuously through a catheter. percutaneous arterial catheterization has proved difficult to accomplish due to the small diameter of the suitable femoral vessels. surgical exposure of the vessels was refrained from, since this would make repeated puncture of the vessels difficult due to scarring, violating the non-invasive character of the method. the small diameter of the vessels necessitated also the use of very thin catheters which inevitably increased the risk of clotting. apart from difficulties in maintaining the catheter patent during the experiment (see above), the resistance to flow in the catheter will introduce divergences in the sampled radioactivity curve. such errors can be reduced by minimizing the length of the catheter by placing the detector as close as possible to the animal, and by withdrawing blood through the catheter at utmost speed. in practice, the small diameter of the femoral artery sets the limit to the size of the catheter. the external detector which is used today demands an effective length of catheter of approximately 30 cm. a smaller design of the detector would allow closer positioning of the detector to the animal and thus a reduction of the effective length of the catheter. the speed at which blood was withdrawn during the experiments was set at 1 ml per min, which resulted in a blood loss of 20 ml per investigation. the blood loss had to be kept at a minimum, so as not to disturb the physiology 108 of the animal. at the moment, it is not possible to monitor adequately the maternal blood concentration in the rhesus monkey by means of pet. the small cross sectional area of the maternal aorta will give poor counting statistics and will be sensitive to maternal movements. the problem will be solved with a whole-body pet camera. the whole animal can then be examined simultaneously and the uptake from the maternal heart will provide the input function for the radioactivity in maternal arterial blood. a low correlation was found between maternal placental blood flow and transplacental transfer of methionine in the evaluation of the data derived from the compartment model. this observation is in agreement with clapp's (9) statement, that during normal conditions, flow rates through both the uterine and foetal umbilical circulations are relatively unimportant variables in maintaining effective transplacental exchange.these results may be of importance in the evaluation of ultrasonographic monitoring of the umbilical blood flow, introduced to detect intrauterine foetal growth retardation in human pregnancies. young (62) found an increased rate of transfer of amino acids to the foetus during protein restriction, as did ahokas et al. (1). it is evident that neither from the part of the investigation dealing with static transfer, nor in the part describing the compartment model, could it be shown that protein restriction had any specific effect on transplacental transfer, even if a tendency to increased concentration of radioactivity could be shown in the amniotic cavity during protein restriction. the two monkeys that were fed on a protein restricted diet prior to the investigation displayed transplacental transfer rate constants that did not deviate significantly from those observed in the group fed on normal diet. however, the results indicate a mean transfer rate to the foetus of 1.1 nmol/min/g placenta, which is lower than the value of 7.6 nmol/min/g placenta reported by schneider et al. (49) using an in vitro model with an isolated human placental cotyledon. our results indicate a transfer capacity of 38 mg methioninel24h for an ordinary rhesus placenta weighing 165 gram, which is the mean weight of a placenta during the last month of pregnancy (53). holt (21) calculated the amount of amino acid required to maintain adequate growth in infants to be between 33-45 mg methionine/kg/day. although our study and that of holt are not strictly comparable, our estimate of the transplacental transfer rate seems to be similar to that of holt and the amount of amino acids transferred adequate to meet the physiological needs for foetal growth. it was found that the transfer of methionine into the placental tissue equalled that to the fetus. the placenta will use amino acids for incorporation into structural proteins for its own growth and for the production of peptide hormones. it has also been found that the placental metabolic rate is high during in vivo conditions and that the placental consumption of oxygen accounts for half of that of the pregnant uterus and its contents (38,42) future prospects. the main purpose of the present series of investigations was to develope pet as a method for studies of the foeto-placental complex. it should be possible to extrapolate the 109 knowledge acquired about the kinetics of placental transfer in the rhesus monkey to human ' pregnancy. the studies were conducted also as a preparation for the application of the pet technique to studies of human pregnancy. before the pet technique can be applied in the study of human pregnancy, two additional prerequisites must be fulfilled. (a) it should be proven beyond doubt that the technique does not carry any radiation hazard to the mother and her child. (b) a pet camera with a detector opening large enough to contain a pregnant woman must be available. in the case of radiation hazards to the mother and foetus, several issues need further elucidation. the decay of positron emitting radionuclides will yield radiation derived from the positrons during their short penetration into the tissue (&radiation) and from the subsequent annihilation photons . the consequences of tissue interaction with the photons will resemble the consequences of tissue interaction with the &particles, although the absorbtion of photons does not have the same finite range, since photon interaction will give rise to harmful high energetic electrons all along the path of the photons (54). in investigations involving positron emitting radionuclides the absorbed dose cannot be calculated generally, since the dose is dependent on the half life and &energy of the radionuclide and on the distribution and excretion of the labelled ligand in the body. thus the calculation of the effects of radiation must be made separately for each series of investigations. the radiation dose to the foetus from a pet investigation involving the administration of 100 mbq of a 11c-labelled ligand to the mother can be estimated roughly. (a) if the administered ligand is kept solely within the maternal circulation and is not transferred to the foetus, the foetal dose will be exclusively derived from the maternal photon radiation and amounting to about 0.2msv. (b) ligands which cross the placenta and become evenly distributed in the organs of the mother and foetus would increase the b-dose from the positron radiation, while the photon dose would remain evenly distributed. the total radiation dose would amount to 0.4 msv. (c) ligands that cross the placenta and concentrate in the foetus to twice or five times that of an even distribution would yield a foetal radiation dose of 0.6 and 1.2 msv respectively. these figures can be compared to the normal background radiation which, excluding the contribution from radon, is about 1.0 msv/year in sweden and the mean absorbed dose from a radiographic pelvimetry for foeto-pelvic disproportion which is 0.7-1.9 msv (36). a new law on radiation protection will recommend a maximum dose of 5 msv to the foetus during pregnancy. it seems that application of pet in the investigations of human pregnancies in a near future will be both technically possible and ethically justifiable with regard to the radiation hazards involved. our efforts so far have been concentrated on exploring the basic questions concerning the application of the pet technique as a tool for the study of placental physiology. additional work will be needed in order to develop a method for placental blood flow estimations. progress has so far been hampered by the difficulty of establishing and maintaining patency of the maternal arterial catheter necessary for the continuous monitoring of arterial blood radioactivity. arterial 110 catheterization will be greatly facilitated when applied to the human in view of the large size of the arteries in humans compared to the rhesus monkey and may even prove superfluous in view of the possibility in the future of monitoring the maternal arterial radioactivity concentration directly by pet. foetal growth is dependent on energy and nutrient supply. foetal supply involves functions such as foetal blood flow, placental transfer capacity and maternal placental blood flow. with research tools suited for the estimation of placental transfer and maternal placental blood flow, it will be possible to investigate the basic mechanisms relevant for foetal growth, not only in human pregnancies that are compromised by different causes and result in retarded growth, but also in conditions associated with accelerated foetal growth, as maternal diabetes. both carbohydrates, fat and several other amino acids besides methionine relevant for foetal growth can be labelled with 11c and used in pet studies. the equipment employed in the present studies does not allow investigation of small foetal structures. the foetal liver was chosen because it is a homogeneous organ of sufficient size and of great importance for protein synthesis. more modem pet cameras have better resolution, permitting the examination of smaller structures. the pet technique can be used in a wide range of investigations during pregnancy including foetal developmental pharmacology, foetal brain research which might give clues to the etiology of common diseases such as cerebral palsy and in investigations of foetal influence of maternal drug abuse. a new pet center in uppsala is being built and will be ready in 1991. this center will be run jointly by the university of uppsala and the hospital, and will house facilities for nuclide production and analytical chemistry as well as a new whole body and brain camera. the advent of a new pet center will hopefully facilitate retrieval, display and computer manipulation of acquired data from the pet investigations. acknowledgements this investigation was supported by grants from the swedish medical research council (grant no. 07 194-04), from "forenade liv" mutual group life insurance company, stockholm, sweden and from "expressen" prenatal research foundation. references 1. ahokas, r. a., lahaye, e. b., anderson, g. d. & lipshitz, j.: effect of maternal dietary restriction on fetal growth and placental transfer of alpha-amino isobutyric acid in rats. j nutr 2. arts, n. f. t. & lohman, a. h. m.: the vascular structure of the placenta in the rhesus monkey and in man. in: aspects of obstetrics today (ed t.k.a.b. eskes et al. ), pp. 203-208. excerpta medica, 1975. 3 . bamwell, s . l. & sastry, b. v. r.: 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(ed. t. chard), pp. 469-499. w.b. saunders company, london philadelphia toronto, 1986. 45.ross0, p.: maternal-fetal exchange during protein malnutrition in the rat. placental transfer of alpha-amino isobutyric acid. j nutr 107:2002-2005, 1977. 46.sastry, b. v. r., olubadewo, j., harbison, r. d. & schmidt, d. e.: human placental cholinergic system. occurrence, disaibution and variation with gestational age of acetylcholine in human placenta. biochem pharmacol25:425-431, 1976. 47.sastry, b. v. r. & sadavongvivad, c.: cholinergic systems in non-nervous tissue. pharmacol rev 30:65-132, 1979. 48.sastry, b. v. r., barnwell, s. l. & moore, r. d.: factors affecting the uptake of alpha-amino acids by human placental villus: acetylcholine, phospholipid methylation, ca++ and cytoskeletal organization. in: fetal nutrition metabolism and immunology. the role of the placenta. trophoblast research (ed. r. k. miller & h. a. thiede), pp. 81-100. plenum medical book company, new york and london, 1983. 49.schneider, h., panigel, m. & dancis, j.: transfer across the human placenta of antipyrine, sodium and leucine. am j obstet gynecol6:822-828, 1972. so.schneider, h., mohlen, k-h. & dancis, j.: transfer of amino acids across the in vitro perfused human placenta. pediat res 13:236-240, 1979. 5 1. schneider, h., mohlen, k-h., challier, j-c. & dancis, j.: transfer of glutamic acid across the human placenta perfused in vitro. british journal of obstet gynaecol86:299-306, 1979. 52.smith, c. h., adcock 111, e. w., teasdale, f., meschia, g. & battaglia, f. c.: placental amino acid uptake: tissue preparation, kinetics, and preincubation effect. am j physiol 53.smith, c. h. & depper, r.: placental amino acid uptake. 11. tissue preincubation, fluid 3~97-124, 1983. 224:558-564, 1973. distribution, and mechanisms of regulation. pediat res 8:697-703, 1974. 54. stalnacke, c-g.: on the use of 11c-labeled compounds in metabolic studies. an experimental study with (metyl-11c) methionine. acta universitatis upsaliensis. abstracts from the faculty of science, thesis 756, 1984. 55.stegink, l. d., pitkin, r. m., reynolds, w. a., brummel, m. c. & filer, l. j.: placental transfer of aspartate and its metabolites in the primate. metabolism 28569-676, 1979. 114 56.stegink, l. d., reynolds, w. a., pitkin, r. m. & cruikshank, d. p.: placental transfer of 57. stulc, j.: is there control of solute transport at placental level?. placenta 9: 19-26, 1988. 58.wimsatt, w. a.: some aspects of the comparative anatomy of the mammalian placenta. am j 59.wurtman, r. j., chou, c. & rose, c. m.: daily rythm in tyrosine concentration in human 60. wurtman, r. j., rose, c. m., chou, c. & larin, f. f.: daily rythms in the concentrations of taurine in the rhesus monkey. am j clin nutr 34:2685-2692, 1981. obstet gynecol84: 15681594, 1962. plasma: persistence on low-protein diets. science 158:660-662, 1967. various amino acids in human plasma. n engl j med 279:171-175, 1968. 61.wurtman, r. j., shein, h. m., axelrod, j. & laren, f.: incorporation of w-tryptophan into 14c-protein by cultured rat pineals: stimulation by 1-norepinephrine. proc natl acad sci usa 62.young, m. & widdowson, e. m.: the influence of diets deficient in energy, or in protein, on conceptus weight, and the placental transfer of a non-metabolisable amino acid in the guinea pig. biol neonate 27:184-191, 1975. 63.young, m.: transfer of amino acids. in: placental transfer (ed. b. p. chamberlain & a. w. wilkinson), pp. 142-158. pitman, london, 1979. 64.young, m.: placental transfer. i. small animal models. in: animal models in fetal medicine. ( 4 . p. w. nathanielsz), pp. 245-272. elsevier/north-holland biomedical press, amsterdam new york oxford, 1980. 65.yudilevich, d. l. & sweiry, j. h.: transport of amino acids in the placenta. biochim biophys 62:749-755, 1969. acta 822:169-201, 1985. address for reprints: dr lars berglund, department of obstetrics and gynecology, akademiska sjukhuset, s-751 85 uppsala, sweden 115 upsala j med sci 105: 17-30,2000 impact of different inspiratory flow patterns on arterial c 0 2 tension agneta markstrom,'. anders hedlund? michael lichtwarck-aschoff,'. anders nordgren,' and ulf sjostrand' 'department of anesthesiology and intensive care, university hospital, uppsala, sweden, 2department of otorhinolaryngology, university hospital, uppsala, sweden, 31nstitute of anesthesiology and surgical intensive care medicine, zentralklinikum augsburg, frg, 4department of plastic surgery-burns unit, university hospital, uppsala, sweden abstract ventilation with decelerating inspiratory flow is known to reduce the dead space fraction and to decrease paco2 . constant inspiratory flow with an end-inspiratory pause (eip) is also known to increase the removal of co,. the aim of the study was to elucidate the effect of the pauseho-flow period while both the pattern and rate of inspiratory flow was unchanged, and when the lung was ventilated with sufficient peep to prevent end-expiratory collapse. surfactant depleted piglets were assigned to decelerating or constant inspiratory flow with 24 breaths per minute (bpm) o r 1 2 bpm, or to constant flow, without and with an end-inspiratory pause of 25%. by adding an eip the total time without active inspiratory flow of the respiratory cycle was kept unchanged. gas exchange, airway pressures, functional residual capacity (using sulfurhexafluoride) and haemodynamics (thermo-dye indicator dilution technique) were measured. irrespective of ventilatory frequency, pacoz was lower and serial dead space reduced with decelerating flow, compared with constant inspiratory flow. with an end-inspiratory pause added to constant inspiratory flow, serial dead space was reduced but did not decrease paco, . the results of this study corroborate the assumption that total time without active inspiratory flow is important for arterial c0,-tension. introduction examples of newer means of mechanical ventilation are modified forms of pressure-controlled ventilation (pcv), high frequency positive pressure ventilation (hfppv), pressure support ventilation (psv), and airway pressure release ventilation (aprv), (6,10,21,27). under these modes a square wave of pressure is intermittently applied to the airway opening, and alveolar pressures and volumes are established during inspiration. opinions differ as to whether improvements in gas exchange and lung mechanics can be specifically attributed to particular types 2-100126 17 of inspiratory flow pattern (1,3,5,28). in patients, the pathophysiology of acute respiratory failure is complex, having many causes and effects. this diversity is an important consideration when applying mechanical ventilation. physiological and theoretical arguments favour strategies that avoid tidal alveolar collapse (12.26) and maintain transalveolar pressure within normal limits. compared with constant flow inspiration, ventilation with decelerating inspiratory flow is known to reduce the dead-space fraction due to improved distribution of inspired gas (7,9). as the rapid gas inflation gives inspired gas a longer residence time, it presumably enhances intrapulmonary gas mixing (20) and improves co;! exchange (11). volume-controlled ventilation guarantees minute ventilation but allows airway pressure to increase as impedance increases. an end-inspiratory pause (eip) is known to improve ventilation and is commonly used during volume-controlled ventilation (vc-eip) (8,9,17). several studies have shown improved gas distribution and increased removal of c 0 2 when using an end-inspiratory pause, but most studies have failed to show improved arterial oxygenation (28,11,17,). modell and co-workers (20) claim that gas exchange impairment must exist before significant response to flow pattern can be detected. in this study the peep level was set sufficient high to prevent end-expiratory collapse, thereby no improvement in oxygenation was expected. the aim of the study was to elucidate the effect of the pauselno-flow period while both the pattern and rate of inspiratory flow were kept unchanged. in order to arrange similar conditions during the active inspiration we added an eip to the constant inspiratory flow. the respiratory cycle is then divided into 2.5 seconds (s) active inspiration both with and without addition of an eip. by choosing 12 breaths per minute (bpm), the period of no-flow with decelerating inspiratory flow corresponded to a pause of 25% with constant inspiratory flow material and methods twelve swedish landrace piglets, with a mean body mass of 28 kg (+ 4), were subjected to lavage. surfactant was removed (15,22). anaesthesia (18,29) was induced with tiletamine 3 mg x kg-1+ zolazepam 3 mg x kg-1 and xylazine 2.2 mg x kg-l and atropine 0.04 mg x kg-' given intramuscularly, followed 5 min later by a ketamine infusion at 20 mg x kg-l x h l . in addition, 20 mg of morphine was given iv. before the initial tracheotomy, preparation and introduction of intravascular catheters. pancuronium bromide was given as relaxant as a continuous infusion at 0.26 mg x kg-1 x h l . the animals' lungs were ventilated through an 8 mm diameter 18 mallinckrodt endotracheal tube (mallinckrodt inc, glens falls, n.y., usa) with a servo 300 (servo ventilator 300, siemens-elema ab, solna, sweden). to maintain the animals body temperature at 38.0 c (k0.6) a thermostatically controlled heating pad was used. the animals were given 0.45% nacl with 2.5% glucose (rehydrex, pharmacia infusion ab, uppsala, sweden ) at 20 ml x kg-1 x h-1 and a bolus of 15 ml x kg-1 of dextran-70 (macrodex 70, pharmacia infusion ab) to ensure normovolaemia. the investigations were performed at the experimental laboratories of the department of anaesthesiology and intensive care at university hospital, uppsala. the local medical ethics committee for animal experimentation reviewed and approved the protocol. monitoring intravascular catheters were surgically placed for the measurement of central venous, pulmonary arterial (via the external jugular vein) and aortic (via the carotid artery) pressures. the position of each catheter was confirmed by pressure tracing on a bedside monitor (siemens sirecust) and recorded with reference to the mid-thorax and at end-expiration level. arterial and venous blood gases were measured (abl 300/osm 111, radiometer a / s denmark). cardiac output (14,16.23) and extravascular lung water p t v ) , were estimated using a cold system (pulsion medizintecknik kg, germany). airway pressures were obtained from the digital displays of the ventilator. every morning a pre-use hnctional check was performed according to the schedule set out in the operating manual for the ventilator and the ventilator’s pressure and flow transducers had been calibrated with independent devices. the static chest-lung compliance (clt) was calculated according to the formula clt = tidal volume x (end-inspiratory pressure expiratory pressure)-1 , but with appropriate modifications to make allowance for the compressible volume (24,25). when the end-inspiratory occlusion pressure and the total end-expiratory pressure (peep) were measured, the ventilator’s hold function was used for 5 s before the equilibrium values were noted. to measure functional residual capacity (frc), serial dead space (sds), the sf6 tracer gas wash-idwash-out method was used (13). frc was calculated as the total volume of sf6 washed-out divided by the alveolar concentration at the end of wash-in. sds was obtained from the first few expirations during wash-out, and was defined as the volume expired when the sf6 concentration reached 50% of the alveolar concentration in that breath. in our laboratory the coefficient of variation for three sequential measurements in 9 animals for frc, under a broad range of tidal volumes and different flow conditions, was 1 f 0.08%. 19 recruitment immediately after lavage, the surfactant depleted lungs were recruited with i:e 1: 1 and the external peep was set to 25 cmh,o and with a tidal volume to produce a peak inspiratory pressure (pip) of 50 cmh20 during cyclic ventilation for 5 min. ventilatory settings a servo ventilator 300 (siemens-elema ab, solna, sweden) provides both volume controlled ventilation (vc; constant inspiratory flow) and pressure-regulated volume controlled ventilation (prvc; decelerating inspiratory flow). experimental procedure following anaesthesia and preparation, the animals were placed in prone position. bronchoalveolar lavage was performed as described previously (16, 22). after lavage, a recruitment procedure was performed. ventilation during the study was with inspiration-expiration ratio of 1:1, f i 0 2 0.3, and constant tidal volume. peep was set to 13 cm h,o. in this animal lung model several studies have proven that peep of 13 cm h,o is sufficient to prevent alveolar collapse. this peep level was maintained during the whole study. at 24 bpm the animals were normocapnic but hypercapnic at 12 bpm. to study how different inspiratory flow patterns utilized inspiration time, we used recordings from the inspiratory-expiratory flow (see fig 1). the animals were then assigned to either decelerating or constant inspiratory flow without eip at 24 or 12 bpm, or to constant inspiratory flow at 12 bpm with an e p of 25% added . the respiratory cycle with 12 bpm was divided into 2.5 s active inspiration both with and without eip. by adding the pause of 1.25 s (25%) the total inspiration time was 3.75 s, resulting in an i:e ratio of 3:l. each setting was applied for 15 min before measurements. calculations and statistics all ventilatory volumes and derived parameters have been converted to btps conditions. indexed values are related either to body mass, or to body surface area (bsa). differences were evaluated with a non-parametric analysis of variance (friedman test). if significant differences were detected, these differences were evaluated using the paired sign t test. a standard statistics package was used (statview, abacus concepts, berkeley, ca). statistical significance was accepted at * p l 0.05. 20 results the results are presented in tables 1 and 2 and in figures 1 and 2. with all modes under study the tidal volumes remained constant, which produced normocapnia at 24 bpm, though at 12 bpm the animals were hypercapnic. table 1 presents the results for decelerating versus constant inspiratory flow at 24 bpm and normocapnia. in table 2 the results for decelerating versus constant inspiratory flow without and with 25% eip and hypercapnia are presented. during a short period, postlavage with zero peep ventilation and f i 0 2 1.0, p a 0 2 was reduced from 98 a4 to 9 +2 kpa, while venous admixture (qvdqt) increased from 7 to 32.5%. during the postlavage studies with a peep level of 13 c m h 2 0 and i302 of 0.3, the (qvdqt) ranged between 6% and 14%. airway pressure with 24 bpm and decelerating flow, peak inspiratory pressure (pip) was 25 +3 cmh20 and with constant inspiratory flow pip was 28 +3 cmh20. at 12 bpm and decelerating flow, pip was 24+2 cmh20, with constant inspiratory flow 25 a3 cmh20, increasing to 27+3 cmh20 with an eip. paco,, serial dead space, functional residual capacity decelerating flow at 24 bpm yielded a paco2 of 6.3 +2 kpa, and with constant inspiratory flow, 6.9 +1 kpa. at 24 bpm and decelerating flow, serial dead space was 146 +13 ml, and for constant flow inspiration, 156 +14 ml. at 12 bpm and decelerating flow, paco2 was 8.4 +1 kpa, with constant inspiratory flow, 9.1+1 kpa, and 8.9 +1 kpa with an eip. serial dead space (sds) was 141k8 ml for the decelerating flow and it was reduced to the same extent (141 +12) for constant inspiratory flow with eip. for significances see tables 1 and 2. discussion in a previous study (19) we found that paco, was lower with the decelerating inspiratory flow than with constant inspiratory flow. we assumed that this could be related to how the flow patterns distribute and redistribute the gas. the aim of the present study was to elucidate the effect of the pauselno-flow period while both the pattern and rate of inspiratory flow was unchanged, and when the lung was ventilated with a peep level set to prevent end-expiratory collapse. we found that arterial carbon dioxide tension (pacoj was lower and serial dead space reduced with decelerating flow, compared with constant inspiratory flow, irrespective of ventilatory frequency. with an end 21 i total peep icmh201 i 1 3 + 1 functional residual capacity [ml] pa02 [kpa] i pip[cmh201 i 25 k 3 *vc24 10632216 1075 +204 16 +4 18 2 2 mean airway pressure 19+2 *vc24 tidal volume [ml*kg-l] 12 +2 end-inspiratory hold pressure [cmh2o] compliance [rnl*~mh20-~1 24 lt3 sds [ml] s v 0 2 [%i itbv [ml*kg-11 13 21 146lt13 *vc24 156 lt14 55 211 61 +12 19 +3 21 2 3 28 k3 17+2 12 21 24 23 35 lt6 table 1. results for decelerating versus constant inspiratory flow at 24 bprn under norrnocapnia values are means i1 sd. in the paired t-test on mean difference of a given value "pc24" (decelerating flow at 24 bpm) denotes a significant difference vs "vc24" (constant inspiratory flow at 24 bpm). all ventilatory volumes and derived parameters have been converted to btps conditions. indexed values are either vs body mass, or vs body surface area (bsa). statistical significance was accepted at: 'p< 0.05. inspiratory pause (eip) added to constant flow, serial dead space was reduced but no significant difference in paco, was seen. after comments on the inspiratory-expiratory flow recordings and the rationale behind the eip, these findings will be discussed in the following paragraphs. inspiratory-expiratoryflow recordings these pressurelflow recordings (figure 1) illustrate that, with decelerating flow, the pre-set inspiratory pressure remains constant throughout inspiration. at 24 bpm the no-flow period is 0.3 s and at 12 bpm it is 1.25 s. during constant inspiratory flow there is a linear increase in pressure while the tidal volume is delivered. with constant inspiratory flow and an eip, there is also a linear increase in airway pressure which decreases during the pause i.e. no-flow period. the respiratory 22 post lavage total peep [cmh20] pip [cmh20] p c 1 2 *10.05 v c 1 2 v c i 2 e l p *20.05 13a 13k1 13+1 24 22 *vcu 25+3 27 +3 *vci 3 eip i 2210 1057 functional residual capacity lmll mean airway pressure end-inspiratory hold tidal volume [ml*kg-l] pressure [cmh20] compliance [rnl*~mh20-~1 i 10332177 i 1128 2200 *pc12 *vc12 1822 *vc12 1622 1922 *vc12,pc12 13 21 13 21 13 21 23 23 2323 2423 *vc 12 3828 42511 3627 ~ pa02 [kpa] paco2 [kpa] sds [ml] s v 0 2 [%] i 53212 i 53210 i 5529 14 21 13 22 14 21 8.4 21 * v c ~ 9.1 21 8.9 21 141 28 *vc12 154-cl3 141 212 *vc12 itbv [ml*kg-11 ci [l*min-l*(m2)-1] qvdqt [%i do;?i [ml*min-i*(m2)-'1 table 2. results for decelerating versus constant inspiratory flow at 12 bpm without and with eip under hypercapnia. values are means *1 sd. in the paired t-test on mean difference of a given value, "pc12'' (decelerating flowat 12 bpm), denotes significant difference vs "vc12" (constant inspiratory flow at 12 bpm), and vs ''vc12~1p" (constant flow at 12 bpm with end-inspiratory pause of 25%). all ventilatory volumes and derived parameters have been converted to btps conditions. indexed values are either vs body mass, or vs body surface area (bsa). statistical significance was accepted at three levels: *p< 0.05. 21 24 21 23 2023 6 20.8 6.0 20.8 6.0 20.8 12 2 7 14 26 1 1 24 563 2121 5692140 571 2117 cycle at 12 bpm is divided into 2.5 s active inspiration both with and without eip. by adding the pause of 1.25 s (25%) the total inspiration time will be 3.75 s, resulting in an i:e ratio of 3:l. notably: from the figure it is obvious that there was an ongoing flow at expiration i.e. there was an intrinsic peep when an eip was added. rationale for adding the end-inspiratory pause we wanted to elucidate the effect of the pauselno-flow period. for constant inspiratory flow this was only possible if the pause was added to an otherwise unchanged inspiratory phase. to obtain similar pauselno-flow periods, we chose 12 bpm. with this frequency the no-flow period with decelerating inspiratory flow was 1.25 s, corresponding to an eip of 25% with the constant 23 inspiratory flow. by adding the 1.25 s pause after the active inspiration in the constant inspiratory mode, the active inspiration time of 2.5 s was unchanged, but the added pause time of 1.25 s increases the total inspiratory time to 3.75 s, reducing expiratory time. consequently, the inspiratiodexpiration (1:e ratio) increases to 3:1, and also produces an intrinsic peep which increased frc (see table 2). that was why 12 bpm was chosen, as adding a 24 bpm pause had resulted in an unacceptably intrinsic peep. 1 1 0 0 1 2 3 4 5 time (seconds) figure 1 25 20 0, i 15 5 10 f 5 t -0.5 1 j d o 0 1 2 3 4 5 20 0, i -0.5 -1 0 1 2 3 4 5 725 " 4 4 i i 4.5 1 1 0 0 1 2 3 4 5 time (seconds) recordings of ventilatory cycles in one of the piglets with a peep level of 13 cmh20 and tidal volume kept constant. top; decelerating inspiratory flow at 12 bpm (pc,,) and constant flow at 12 bpm without eip (vc,,). centre; pc,, and vc,, with end-inspiratory pause (vc,,+eip). bottom: pc, and vc,,. these pressure/flow conditions illustrate that, with decelerating flow, the pre-set inspiratory pressure is constant throughout inspiration. with pc,,, the no-flow period is 1.25 s and with pc,,, 0.3 s. during constant inspiratory flow there is a linear increase in pressure while the tidal volume is delivered. with constant inspiratory flow and an eip, there is also a linear increase in airway pressure which decreases during the pauseho-flow period. the respiratory cycle at 12 bpm is divided into 2.5s active inspiration, both with and without eip. by adding the pause of 1.25 s (25%) total inspiration time will be 3.75 s, resulting in an i:e ratio of 3.1. 24 pc12 vc12 vc12 + eip flow i figure 2. schematic drawing of inspiratory and expiratory flow curves with pc,, (top), vc,, (centre) and vc 12+eip (bottom). left side of the panel: inspiratory flow. right side: expiratory flow. the time (in seconds) during which flow is active, near no-flow as well as pause time are indicated. the total inspiratory time, and the time during which neither active inspiratory flow nor an inspiratory near no-flow (pc,,) nor an inspiratory pause (vc,,+eip) exists are also given and labelled as: '%without active inspiratory flow". 25 mean airway pressure mean airway pressure is closely related to certain ventilation settings, and influences haemo dynamic function and ventilator-induced barotrauma. ventilation with decelerating inspiratory flow has always a higher mean airway pressure, since a greater proportion of volume and flow is delivered earlier during the inspiration period than with constant inspiratory flow. baker et al (2) hypothesised that the combination of increased mean airway pressure, better intrapulmonary gas distribution, and longer diffusion time all accounted for the improved gas exchange with decelerating inspiratory flow. constant inspiratory flow with an eip will also maintain the lung volume and alveolar pressures. the pause adds directly to the mean airway pressure and this will be further accentuated if the pause also causes intrinsic peep by shortening expiratory time. this study shows that the mean airway pressure is even higher for the constant flow with eip. constant inspiratoryflow with and without an eip irrespective of ventilatory frequency, with decelerating flow pacoz was lower and serial dead space reduced, compared with constant inspiratory flow. this could be due either to early delivery of tidal volume, or to the period of no-flow, or to both. adding an eip to the constant inspiratory flow precluded assessment of the impact of the early delivery of tidal volume with the decelerating inspiratory flow. instead, the intention was to obtain similar active inspiration conditions for both inspiratory patterns. using constant inspiratory flow with eip, serial dead space (sds) was reduced but no statistically significant decrease in paco2 could be demonstrated, compared with constant flow without a pause. this finding was unexpected. one reason for the absence of decreased paco, when eip was added could be related to the fact that the “total time without active inspiratory flow” remains the same as for constant flow without a pause, as illustrated in fig. 2. with decelerating inspiratory flow, flow takes place mainly during the first 0.8 s, thereafter followed by a no-flow period of 1.7 s before expiration for 2.5 s. the “total time without inspiratory flow” was 4.2 s, which was 2.5 s expiration +1.7 s no-flow period. for constant flow without a pause inspiratory flow was ongoing during 2.5 s , followed by a 2.5 s expiration, yielding a ”total time without inspiratory flow” of 2.5 s (expiration). when an eip was added to the constant flow, the total inspiratory time was prolonged resulting in an i:e 3: 1, but the inspiratory flow continues for 2.5 s followed by a pause of 1.25 s during which flow ceases and time of expiration will be only 1.25 s. 26 note that, “total time without inspiratory flow” was exactly the same as for constant flow without a pause, viz. 2.5 s expiration 1.25 s + 1.25 s pause. the above is related to what happens when gas is inspired into the lungs; part of it mixes with the resident gas, while some remains unmixed in the conducting airways (4). this unmixed portion con-stitutes the serial dead space (sds) because it is in series with the mixed gas in the alveolar region and is only partly determined by anatomical factors. the interface of the distal boundary of the dead space moves up the airway during breathholding and down if flow is increased. the sds is reduced when the flow rate at changeover from inspiration to expiration slows down and especially if the expiration phase is allowed to start slowly. time is then allowed for mixing to occur and the interface of gas mixing is allowed to move up the airways. with decelerating inspiratory flow, initial flow is high and diminishes rapidly, the major part of the tidal volume being delivered after only 0.8 s. during the remaining 1.7 s of inspiratory time, convective flow ceases and hence the distal boundary at which convective flow equals diffusion moves up the airway, resulting in increased paco, elimination. this is in contrast to constant flow both with and without eip, where flow is ongoing and tidal volume is not delivered until the end of 2.5 s. in summary, the results of this study corroborate the assumption that total time without active in spiratory flow is important for arterial c0,tension . acknowledgments this study was supported by the swedish medical research council (project 4252), stockholm, and the lions cancer foundation, uppsala, sweden; and the laerdal foundation for acute medicine, stavanger, norway 27 references 1. al-saady, n. & bennett, e. d: decelerating inspiratory flow waveform improves lung mechanics and gas exchange in patients on intermittent positive-pressure ventilation. intensive care med 11: 68-75. 1985. 2. baker, a. b., colliss, j. e. & cowie, r. w: effects of varying inspiratory flow waveform and time in intermittent positive pressure ventilation. 11: various physiological variables. br j anaesth 49: 1221-1234, 1977. 3. bergman, n. a: effects of varying inspiratory waveforms on gas exchange. anaesthesiology 4. bowes, c . l., richardsson, j. d., cumming, g. & horsfield: effect of breathing pattern on gas mixing in a model with asymmetrical alveolar ducts. j appl physiol58: 18-26, 1985. 5. dammann, j. f,. mcaslan, t, c. & maffeo, c.j: optimal flow pattern for mechanical ventilation of the lungs. 2. the effect of a sine versus square wave flow pattern with and without an end-inspiratory pause on patients. crit care med 6: 293-310, 1978. 6. downs, j. b. & stock, m. c:. airway pressure release ventilation: a new concept in ventilatory support. crit care med 15: 459-461, 1987. 7. eriksson, i. & sjostrand, u: effects of high-frequency positive pressure ventilation (hfppv) and general anaesthesia on intrapulmonary gas distribution in patients undergoing diagnostic bronchoscopy. anesth and analg 59: 585593,1980. 8. fuleihan, s . , wilson, r. & pontoppidan, h: effects of mechanical ventilation with end-inspiratory pause on blood gas exchange. anaesthesia and analgesia 55: 122-129, 1976. 9. jansson, l. & jonsson, b. a: theoretical study on flow patterns of ventilators. scand j respir dis 53: 237-246, 1972. 10. katz, j. a. & marks, j. d: inspiratory work with and without continuous positive airway pressure in patients with acute respiratory failure. anaesthesiology 63: 598-607, 1985. 11. knelson, j. h., howatt, w. f. & demuth, gr: effect of respiratory pattern on alveolar gas exchange. j appl physiol29: 329-331, 1970. 12. lachmann, b: open the lung and keep the lung open (editorial). int care med 18: 319-321, 1992. 13. larsson, a., linnarsson, d., jonmarker, c., jonsson, b., larsson, h. & werner, 0: measurements of lung volume by sulphur hexafluoride washout during spontaneous and controlled ventilation. further development of a method. anesthesiology 67: 543-550, 1989. 14. lichtwarck-aschoff, m., zeravik, j. & pfeiffer; u: intrathoracic blood volume accurately reflects circulatory volume status in critically ill patients with mechanical ventilation. int care med 18: 142-147, 1992. 15. lichtwarck-aschoff, m., nielsen, j. b., sjostrand, u.h. & edgren, e.l: an experimental randomized study of five different ventilatory modes in a piglet model of severe respiratory distress. intensive care med 18: 339-347, 1992. 16. lichtwarck-aschoff, m., leucht, s., kisch, h., zimmermann, g., blumel, g. & pfeiffer: u. monitoring of right ventricular function using a conventional slow response thermistor catheter. intensive care med 20: 348-353, 1994. 17. lyager , s: influence of flow pattern on the distribution of the respiratory air during intermittent positive-pressure ventilation. acta anaesthesiol scand 12: 191-21 1, 1968. 18.locher, w., ganter, m. & fassbender, c. p: correlation between drug metabolite concentration in plasma and anesteic action of ketamine in swine. am j vet res 51: 391-398, 1992. 19. markstrom, a. m., lichtwarck-aschoff, m., svensson, b. a., nordgren, k.a. & sjostrand, u. h: ventilation with constant versus decelerating inspiratory flow in experimentally induced acute respiratory failure. anesthesiology 84: 882-889, 1996. 1 103-07, 1979. 28: 390-395, 1967. 28 20. modell, h. i. & cheney, f. w: effects of inspiratory flow pattern on gas exchange in normal and abnormal lungs. j appl physiol46: 1103-07, 1979. 21. montgomery, ab,. stager, m. a,. carrico, c. j . & hudson, l. d: causes of mortality in patients with adult respiratory distress syndrome. am rev respir dis 132: 485-489, 1985. 22. nielsen, j. b., sjostrand, u. h., edgren, e. l., lichtwarck-aschoff, m. & svensson. b.a.: an experimental study of different ventilatory modes in piglets in severe respiratory distress induced by surfactant depletion. intensive care med 17: 225-233, 1991. 23. pfeiffer, u . j . , backus, g . , bliimel, g., eckart, j . , muller, j., winkler, p., zeravik, j . & zimmermann, g : a fiberoptics based system for integrated monitoring of cardiac output, intrathoracic blood volume, extravascular lung water, 02-saturation, and a-v differences. in: lewis, f. r.& pfeiffer, u. j: eds. fiberoptics in critical care monitoring. springer, berlin, heidelberg, new york, london, paris, tokyo, hong kong: pp 114-125, 1990. 24. pierson, d. j . & kacmarek, r . m: foundations of respiratory care. new york: churchill livingstone p. 976, 1992. 25. scott, l.r., benson, m. s. & pierson, d. j: effect of inspiratory flow rate and circuit compressible volume on auto-peep during mechanical ventilation. respir care 31: 1075 1079, 1986. 26. sjostrand, u. h., lichtwarck-aschoff, m., nielsen, j. b., markstrom, a. m., larsson, a , , svensson, b. a., wegenius, g . a. & nordgren, k. a: different ventilatory approaches to keep the lung open. intensive care med 21: 310-318, 1995.. 27. sjostrand, u: high-frequency positive-pressure ventilation (hfppv) a review. crit care med 28. sykes, m. & lumley, j: the effect of varying inspiratory flow on gas exchange during 29. wheland, g . & flecknell, p. a: the assessment of depth of anaesthesia in animals and man. address for correspondence: agneta markstrom, department of anesthesiology, university hospital, s-751 85 uppsala, sweden. fax: + 46 18 32 18 09 8: 345-364, 1980 anaesthesia for open-heart surgery. br j anaesth 41: 374-380, 1969. laboratory animals 26: 153-162, 1992. 29 effect of olsalazine sodium on migrating motor complexes in the upper small bowel of human volunteers marta ryde and sven gustavsson department of human pharmacology, pharmacia ab and department of surgery, uppsala university hospital, uppsala, sweden abstract the effect of a peroral dose of olsalazine sodium on the migrating motor complexes (mmc) in the upper small bowel was studied by manometry in nine healthy volunteers during fasting. recordings were obtained from the duodenum and/or the upper jejunum for 4.0 7.5 h. during total baseline observation periods of 30 h, fourteen mmcs could be identified compared to ten complexes during post dose registrations of 25 h. this gives an average mean interval for baseline pre dose complexes of 2.2 h, and for post dose complexes 2.5 h. if excluding the two subjects, who had the longest interval until the first mmc before dosing and did not present any mmc after dosing, the corresponding figures are 1.9 h and 1.9 h, respectively. although our material is small with respect to the large individual variation in the recordings the results indicate that mmc does occur after an olsalazine sodium dose which is twice the dose normally recommended. besides we can conclude that there is no apparent interference with the time for mmc occurrence in the upper small bowel in fasting humans. introduction in recent studies (1, 2, 6, 9 1 , 5-aminosalicylic acid (5-asa) has been shown to be the main therapeutic compound of sulfa salazine (sasp) in the treatment of ulcerative colitis. in order to avoid some of the side effects encountered during sasp therapy, which emerge from the sulphapyridine component, an azo-compound of two 5-asa molecules was synthesized. this 243 compound, olsalazine sodium (who pi") [disodium 3,3'-azo-bis (6-hydroxy-ben~oate)~ disodium 5,5'-azo-di-salicylatef ads] is , now under evaluation in clinical studies. it has been shown to have a therapeutic effect in patients suffering acute attacks (7, 13), as well as a prophylactic effect against relapses in patients in remission (12). as for sasp the azo-bridge in ads is split by bacteria in the caecum and the liberated 5-asa is then further metabolized to n-acetyl-5-aminosalicylic acid (ac-5-asa) (10). in clinical trials and in early human studies with ads, occasional episodes of diarrhoea have been reported, in some cases already a few hours after the first dose. one of the hypotheses to explain the diarrhoea was disturbance in the migrating motility complexes (mmc) of the small bowel. experi ments with fasted rats showed that ads but not sasp, changed the distribution pattern of a transit marker in the small bowel which could be interpreted as an indirect evidence for a disturbancy of the mmc in rats (5). the aim of the present study was to explore the effect of ads on interdigestive mmc in the upper small bowel of human volunteers. material and methods ma ter ia 1 drug: 250mg ads was filled into hard gelatine capsules no. 1. placebo: starch coloured with riboflavine and indigotin laque colour was dispensed in hard gelatine capsules no. 1. participated in the study. they were all healthy according to medical history, physical examination and laboratory investigation and were not on any medical treatment. they signed an informed consent and the study was approved by the ethics review committe at the medical faculty at uppsala university subjects: nine healthy male volunteers (aged 27-45 years) methods analytical method: blood was collected into heparinized tubes which were kept in an ice bath for half an hour and then centri fuged at 3000 rpm at +4" c. the plasma samples were assayed for content of ads with liquid chromatography and spectrophotometric 244 detection after acidic extraction. for determination of 5-asa and ac-5-asa derivatisation with proprionic anhydride preceded the acidic extraction and liquid chromatography with fluorescence detection. recording equipment: small bowel manometry was used to study the effect of ads on the mmcs in the upper small bowel. a standard triple lumen catheter for pulmonary artery pressure registration (pulmoball, vygon, 1 2 5 cm), with one opening located at the tip, and one 30 cm proximally of the tip, was introduced via the nose of the participating volunteers. the third lumen of the catheter led to a balloon close to the tip of the catheter. by inflation of the balloon, the tip of the catheter passed at least to the horizontal part of the duodenum as checked by fluoroscopy. a continuous perfusion of sodium chloride through both lumens was started. the pressures from these two lumens were recorded via two external transducers (statham-gould, statham instruments inc.) the transducers were connected to a polygraph (pharmacia ab) and the intraluminal pressure was continuously recorded for 4.0-7.5 h. the set-up was calibrated at room temperature. study, and water, but no food, was allowed until the study was finished. after the catheter position was obtained, the volunteer was lying comfortable in a supine position and a baseline registration was started. a mmc complex was identified as an intense spiking activity which apparently differed from the preceding motility pattern and was of about 5 min persistence. after registration of at least one baseline complex showing a normal rest activity, an oral dose of 2 g (5.78 mmol) ads ( 8 capsules a 0 . 2 5 g) was given with 100-150 ml of water, and the registration was continued for another couple of hours. to each of four subjects, 8 placebo capsules were given at least 1 h before the dose, with the same amount of water, in order to document any effect of an intake of only capsules plus water. blood samples were drawn at time 0, 0.5, 1 and 2 h after the study design: the volunteers fasted for about 8 h before the dose to confirm an adequate absorption of the drug. results in all volunteers at least one pre dose mmc complex could be 245 identified. the maximal spiking height was estimated to 60-130 mm hg at the distal location. that the complexes really migrated distally was verified in four of our volunteers, with registration both at the proximal and distal opening, giving a migration velocity of 6.5 cm 15 cm/min. the registrations in the other five subjects were considered to represent classical mmc activity though the registration was made only through one channel since the appearance of the registrations were identical with those for the subjects who actually showed migration. i a a a i a i a + a i + a t a a + i a i a i a i i a k a i i a a+ i i i subject 85002 85004 8 5 0 0 7 85008 85009 8 5 0 0 1 8 5 0 0 3 85005 8 5 5 0 1 4 f a a a i a i i a a a i a a : * a i a i i i a a i f i i i i fig. 1. individual time for appearance of mmcs ( a ) and placebo ( f ) intake related to a 2 g a d s dose in nine healthy male vo unteers. in those individs where a clear migration of the mmcs is seen a double line is drawn k b , ) . the start and the end of the resp. lines indicate the extention of each individual study. the subjects who reported incidences of diarrhoea are marked with *. no obvious effects on the mmcs were seen in the nine subjects after the 2 g oral a d s dose or, in four of the subjects, after eight placebo capsules taken with the same amount of liquid as 246 the dose (fig. 1). a statistical evaluation was not found feasible due to the large individual variation in relation to the number of subjects tested. during total pre dose observation periods of 30.5 h, in the nine volunteers, 14 mmcs could be identified, and during the post dosage registrations of 25 h, ten complexes were recognized (fig. 1). this gives an average mean interval of 2.2 h for baseline complexes, and 2.5 h for post dose complexes. with regard to all nine participants only those with the longest registration time > 140 min, from introduction of the catheter to the first mmc, did not show up with any mmc post dose, all other subjects had at least one mmc post dose. it could be expected that the mere intake of eight capsules could interfere with the mmc but the data on the effect of placebo capsules in four of our subjects does not indicate this. excluding the two subjects, who had the longest (>140 min) pre dose mmc interval, (85005 and 85008, see fig. 11, and who did not present any post dose mmc during another 3-4 h, the corresponding figures were 1.9 h and 1.9 h, respectively. was reached within 1 h (table 1) which is in accordance with findings in earlier studies. 5-asa was not found in any plasma sample and ac-5-asa was found already 2 h after dosage in four out of nine volunteers. in all but two subjects the peak plasma concentration of ads table 1. individual and mean plasma concentrations of olsalazine sodium (ads) and n-acetyl-5-aminosalicylic acid (ac-5-asa) after a 2 g (5.78 mmol single oral dose in nine healthy male volunteers. plasma concentration p.mol/l after dose ~~~ subject ads ac5 asa nr oh 0.5h 1.0h 2.0h oh 0.5h 1.0h 2.0h 8 5 0 0 1 ~ 85002 85003 85004 85005 85007 85008 8500g3 85501 mean s.d. ~0.06 1.6 2.0 1.4 <0.3 < 0 . 3 ~ 0 . 3 0.4 t0.06 2.3 6.9 5.7 to.3 to.3 ~0.3 <0.3 <0.06 3.4 3.8 2.6 <0.3 <0.3 <0.3 0.6 <0.06 2.0 4.4 6.1 to.3 ~0.3 ~0.3 <0.3 t0.06 2.5 3.0 1.6 <0.3 <0.3 to.3 t0.3 <0.06 1.0 4.5 to.3 <0.3 <0.3 <0.3 ~ 0 . 0 6 3.8 4.4 4.1 <0.3 ~0.3 <0.3 0.4 <0.06 2.7 2.9 2.4 ~0.3 <0.3 <0.3 0.3 <0.06 1.4 1.9 2.0 <0.3 t0.3 <0.3 ~0.3 2.3 3.8 3.2 0.96 1.55 1.84 ' i and diarrhoea at 2.5 h, 7 h and 3.25 h, resp. 241 three subjects, ( 8 5 0 0 1 , 8 5 0 0 2 and 8 5 5 0 1 , table 1 1 , experienced diarrhoea 3-7 h after the 2 g dose which is twice the therapeutically used dose at each dosing event. the diarrhoea was preceded by borborygmi and a feeling of abdominal discomfort. in all three subjects with diarrhoea, at least one mmc was seen after dosing indicating that the mmcs were not influenced by the diarrhoea. no correlation between peak serumconcentration of ads or appearance of ac-5-asa with diarrhoea was seen. discussion the main finding in the present study was that ads did not appear to influence the mmc in the upper small bowel since the overall mean pre dose mmc interval was 2 . 2 h and the post dose interval was 2.5 h. the fact that in the four subjects where more than one mmc was identified before dose, two subjects showed up with a longer interval between mmcs after dose, one with about the same and one with shorter interval also supports the conclusion that there is no dramatic effect of ads on mmc frequency in healthy subjects, either increase nor decrease as response to dosing. we have performed our study using a thin swan-gauz type of catheter which has the obvious advantage of being more easily tolerated than conventional manometry tubes. moreover it must be pointed out that, our results have a bearing only on the motility of the upper small intestinal tract. however studies of mmc in the distal small bowel is less suitable since only about 10% of the mmcs in humans pass all the way down to the caecum (11). it has been proposed by thompson et al. (14) that there are no similar distinct activities in the small bowel beside the migrating complexes, which makes us confident that all registrations were mmc complexes though the migration pattern was only identified in four of our subjects. the mean intervals of about 2 h between the predose mmcs found in this study are of the same order as those reported by for instance vantrappen et al. ( 1 5 ) and thompson (14) in healthy volunteers. the max spiking height of the mmcs range 60-130 mm hg in our study, was also similar to the amplitudes 248 reported by for instance thompson (14) and erckenbrecht et al. (3). the migration velocity 6.5 15 cm/min found by us is in accordance with the findings (7.7 cm/min) of vantrappen et al. (15). appearance of ac-5-asa in plasma and the episodes of diarrhoea can not be recognized from the results in this study. however the mean plasma peak concentration of ads i s almost the same as that found after a 1 g dose in our own laboratory and by van hogezand et a1 (8). the low mean peak concentration of ads and the appearance of ac-5-asa in plasma already at 2 h in 4 of 9 subjects (table 1) might indicate an accelerated transit of the drug through the small intestine as a consequence of the simultaneous, continuous infusion of saline. a correlation between peak plasma concentration of ads and the results of this study show that oral administration of ads obviously did not affect the mmcs in the upper small intestine in man. thus, the changes in bowel habits sometimes seen in clinical studies with ads and in three of our volunteers, does not seem to be referred to disturbances in the initiation of interdigestive mmcs. in a study in rats (5) ads was found to change the distribu tion in the small intestine of a transit marker which was taken as an indication of disturbancy of the mmc. this is not sup ported by the results in this study which might either be explained by species variation or, more probably, by the fact that the method used in the animal study was an indirect method. the results found in rats could as well be a result of an increased volume of the content in the small intestine. this is in accordance with the increase in ion and water secretion seen in the ileum in animal studies (4) with ads. acknowledgement analysis of the content of ads, 5-asa and ac-5-asa were performed at the department for bioanalysis at pharmacia ab, head ph.d. n-0. ahnfelt. 16-878573 249 references azad khan, ak., piris, j. & truelove, s. c.: an experi-, ment to determine the active therapeutic moiety of sulphasalazine. lancet 892-895. 1977. campieri, m., lanfranchi, g. a., bazzocchi, g., brignola, c., sarti, f., franzin, g., battocchia, a., labo, g. & dal monte, p. r.: 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hellemans, j. & ghoos, y.: address for reprints: marta ryde department of human pharmacology pharmacia ab 7 5 1 8 2 uppsala 25 1 upsala j med sci 87: 235-242, 1982 a paracorporeal rat heart model for ischemic and reperfusion studies jan hultrnan, jan ola forsberg, hans erik hansson and gunnar ronquist department of anaesthesiology, department of general surgery, department of thoracic and cardiovascular surgery and department of cfinical chemistry, university hospital, uppsala, sweden abstract paracorporeal rat hearts were supplied with blood derived from the abdominal aorta of a supporting rat. the circulatory stability and working capacity of the supporting animal was analyzed in the experimental situation in terms of po2, so2, pc02, hc03, ph and electrolytes, all of which were within the normal range before and during a 60 min period of paracorporeal perfusion. for evalua tion of ischemic damage in this model studies were made on three groups of ex cised hearts. they were subjected to 10, 15 or 20 min of complete global ische mia at 37oc (ambient temperature) and reperfused for 30 min, including ecg, observations of contractility and an analysis of creatine kinase efflux in the coronary effluent. the results showed good reproducibility and the data were in accordance with reports from similar studies on langendorff preparations. the model, which is easily set up, inexpensive and based upon pulsatile blood per fusion, should be more physiologic than the conventional langendorff prepara tion. introduction improved myocardial protection is probably the most important contribution to recent advances in open-heart surgery. the clinical improvements are greatly attributable to extensive experimental laboratory investigations (1, 2, 13, 16) the isolated rat heart model has proved to be very useful in studies of myo cardial ischemia, and experimental experiences have led to clinical innovations (10). an often used rat heart model is the langendorff preparation (15) and its modifications (la), in which an isolated heart is perfused with a crystalloid solution in a non pulsatile way, usually a krebs-henseleit solution. in the clinical situation, however, the conditions are different. after the ischemic period the aortic cross-clamp is released and a pulsatile coronary blood flow is reinstituted. the paracorporeal perfusion technique is known from earlier experiments (6, 17, 20). gamble et al. (6) modified the paracorporeal heart model in the rat 235 but did not use a direct pulsatile retrograde perfusion technique in the aorta of the excised heart. aiming at a situation closer to the clinic we have modi fied the paracorporeal rat heart model. its reliability was evaluated and data on enzyme efflux correlated to ischemic heart damage are presented. material and methods rats non-starved, 300 g male sprague-dawley rats were used. the exoerimental model the paracorporeal rat heart model consists of three main parts (fig. 1): 1. the supporting rat. 2. the excised heart. 3 . the retransfusion system. the ascending aorta of the excised heart was connected to the abdominal aorta of the supporting rat by a tube ("pump tube"). the coronary effluent from the excised heart was collected in a temperated water-jacketed funnel and retrans fused to the supporting rat. after the ischemic period pulsatile blood flow was established from the supporting rat to the excised heart. the temperature of the supporting rat and the excised rat heart was kept at 3 7 o c c l0c. cannubtion obdominol aorto peristaltic roller pump fig. 1. the experimental model the experimental model is composed of three main parts: 1. the supporting rat 2. the excised heart 3 . the retransfusion system 236 surgical and technical procedures administration of inactinr (byk-gulden, germany) , 120 mg-kg stomy the right carotid artery was cannulated for continuous blood pressure re cording via an emt 34 transducer (elema-schbnander, sweden) and a m 34 recorder (elema-schgnander, sweden). the right jugular vein was cannulated for transfu sion. heparin in a dose of 200 iu was given intravenously. the supporting rat was breathing air spontaneously. rats intended for supporting function were anaesthetized by intraperitoneal -1 . after tracheo the abdominal aorta was cannulated and the cannula was fixed to the aorta by silk ligatures. close to the aorta, the cannula was cut and rejoined by a more flexible piece of silicone rubber tubing to permit easy clamping. connections permitted blood sampling and infusion. the body temperature was recorded con tinuously in the abdominal cavity. heart donors were anaesthetized with ether. after 200 iu of heparin intra venously, the blood was rapidly collected from the abdominal aorta. following thoracotomy and ligation of the aortic arch branches the heart-arch preparation was excised. the time required for this procedure was 3-5 min. the ascending aorta was attached to the cannula from the supporting rat. electrodes for bipolar ecg recording were placed at the apex, the right atrium and the aortic arch. during the ischemic period the heart was kept in a water jacketed funnel filled with plain saline at 37oc-38oc. the retransfusion system consisted o f a peristaltic roller pump (multiperpex 2115, lkb products, broma, sweden), a temperature controlled water bath and an air bubble trap. all connections were of silicon rubber tubing. the system was primed with 20 ml of heparinized blood collected from the heart donor and an additional rat. 10 ml of saline was added to the system during the first 7 d n of reperfusion. subsequently no extra volume was added. the retransfusion of blood was adjusted by regulating the height of the bubble trap. the mean arte rial blood pressure (mabp) of the supporting rat was maintained at 95+10 mu hg. experimental protocols to test the endurance to ischemia, excised hearts were subjected to complete global ischemia at 37oc (ambient temperature) for 10, 15 or 20 min. the number of hearts examined in these three groups was 6, 8 and 7, respectively. samples of arterial blood from the supporting rat were obtained before reperfusion was started and 10 and 30 min after the start. sampling of the coronary effluent from the excised hearts was performed via the funnel after 10, 20 and 30 min of reperfusion. no buffer solutions were used. the following recordings were made: 1. supporting rat: mabp and heart rate. 2. arterial line (pump tube): nabp po2’ s02’ pc02’ ph 16-822858 237 hc03-, base excess (be) electrolytes na', k+, ca ++ , and glucose 3. excised heart: ecg (continuous recording) 4. coronary effluent: flow rate creatine kinase (ck) efflux the ck activity was determined according to the recommendations given by the scandinavian committee on enzymes. for background measurements samples were taken from the supporting animal and from the blood prime before the start of reperfusion. for control six excised hearts were reperfused directly after cannulation for determination of ck efflux and to evaluate the influence of enzyme efflux from the supporting rat, another six rats were cannulated as supporting animals, but with no excised heart in the paracorporeal line. the flow through the cannu la was adjusted to 8 ml-min . samples taken from the blood prime and from the paracorporeal line after 10, 20 and 30 min were analysed for ck activity. -1 results supporting rat most rats showed a stable condition during the experiment. their body tempe rature, blood gases, electrolytes and blood pressure had to be within normal range for acceptance as supporting animals. sodium, potassium and calcium concentrations in samples from the arterial line determined in six cases before reperfusion and after 10, 20 and 30 min (ischemic periods 10, 15 and 20 min) were all within the normal range. arterial blood gases analysed before and 10 and 30 min after the start of reperfusion showed only minor variations, indicating high stability in the system (table i). for control, additional blood gas analyses performed on blood samples after 5, 15 and 20 min of reperfusion with the same results. blood glucose values at repeated determinations were within the normal range. table i. blood gas data for the supporting rats before and after 10 and 30 min of reperfusion of excised hearts subjected to 10, 15 and 20 min of complete global ischemia at 37oc. a s there was no significant diffe rence between the three groups, the means and sd were calculated from all supporting rats. significant differences (p<0.05) during support compared with pre-supporting values are indicated with an asterisk. n = number of animals. ph pc02 (kpa) po2 (kpa) be so hc03 (78 ( m o l . 1-5 before reperfusion 7.40-10.03 5.3k0.6 12.4t1.0 -0.1k1.7 95t1 24t1 n 19 19 18 18 18 18 10 min of reperfusion 7.37k0.03 5.0k0.5 13.0k1.5 -3.1t2.0* 96t1 2 . 2 2* n 20 20 20 19 19 19 30 min of reperfusion 7.38k0.03 5.1k0.5 1 2 . 2 t 1 . 2 -2.3k1.6 95k1.3 231.3 n 22 22 2 2 2 1 21 21 238 the hemodilutive effect of added saline resulted in a hb concentration of (sm2.1) at 10 and 30 min of reperfusion, -1 13.6 g.1-l (sitt2.1) and 14.4 gel respectively, as compared with 17.5 g-1 (sm2.0) before transfusion. -1 excised hearts all hearts subjected to 10 min of ischemia regained sinus rhythm and a rate of at least 140 beatslmin after 10 min of reperfusion. after 15 min of ischemia only 113 of the hearts returned to normal rhythm, while the other hearts deve loped serious arrhythmias. after 20 min of ischemia no heart returned to normal rhythm (table 11). table 11. heart activity after 10 min of reperfusion of hearts subjected to 10, 15 and 20 rnin of complete global ischemia at 37oc, verified by ecg and observed contractility. n = number of examined hearts. duration of pre-reperfusion ischemia 10 min 15 rnin 20 min n = 10 n = 12 n = 9 sinus rhythm non-reversible 140 beatslmin 10 4 8 9 heart activity arrhythmias the coronary flow rate in the 15-min ischemic group is given in table 111. it was already fairly high during the first minute and increased to a maximum of 9 ml/min after 4-5 rnin of reperfusion. after 20 rnin a constant flow rate of about 5 ml/min was established. -1 table 111. coronary flow (ml-min ) during reperfusion of 7 excised hearts subjected to 15 min of complete global ischemia at 37oc. sampling periods one min. mea&sd. duration of reperfusion (min) 01 2' 4' 10' 20' 30' flow ml-min -' 6.e1.7 8.7t2.4 9.222.9 7.1k1.9 5.011.8 4.6? 1.5 the ck background activity was found to be stable, with a mean of 8.1 (sd * 2.0 ukat.1 ) in supporting animals (27 animals) and 4.8 (sdt 1.8 ukat-1-l) in the blood prime (27 experiments). in the control group with no heart in the paracorporeal line the background activity was similar. -1 the ck activity in the coronary effluent after 10, 20 and 30 min of reper fusion showed similar patterns depending upon the period of ischemia (table iv). in control samples after 40 and 50 min of reperfusion there was a greater in crease in the accumulation of ck in the 20-min ischemic group compared to the other groups. a significant difference (p<0.05) between the ck activity in 239 samples from h e a r t s s u b j e c t e d t o 10 rnin of i s c h e m i a , compared t o t h e 15and 20 rnin groups, was observed. the d i f f e r e n c e between t h e two l a s t g r o u p s , however, was i n s i g n i f i c a n t . i n t h e c o n t r o l s t h e e f f l u x of ck was low (table iv). -1 table i v . the e f f l u x of c r e a t i n e k i n a s e ( u k a t 1 ) i n t h e c o r o n a r y e f f l u e n t a f t e r 10, 2 0 and 30 rnin of r e p e r f u s i o n o f h e a r t s s u b j e c t e d t o 10, 15 o r 20 min of complete g l o b a l ischemia a t 37oc. to compare, v a l u e s a r e g i v e n from a group of e x c i s e d h e a r t s r e p e r f u s e d d i r e c t l y a f t e r c a n n u l a t i o n and a n o t h e r group w i t h no e x c i s e d h e a r t i n t h e para c o r p o r e a l l i n e , i.e. cr e f f l u x from t h e s u p p o r t i n g r a t . mean 2 sd. n = number of e x p e r i m e n t s . r e p e r f u s i o n of e x c i s e d h e a r t s 10 min 20 min 30 rnin 10 min 1 2 . 3 4 . 1 15. w 4 . 3 1 8 . b 4 . 2 n 6 6 6 15 rnin n 19.&5.7 30.w9.6 34.72 8 . 7 8 8 8 d u r a t i o n 20 min 25.1210.7 3 7 . b 1 3 . 9 4 6 . 2 1 7 . 8 of n 7 7 7 ischemia c o n t r o l group n 6 6 6 c o n t r o l group n 6 6 6 ( r e p e r f u s e d d i r e c t l y ) 7 . % 0 . 7 11.31.1 1 3 . e 1 . 6 (no e x c i s e d h e a r t ) 6.120.9 7 . 4 2 1 . 1 10. if 1.1 discussion i n v e s t i g a t i o n s u s i n g d i f f e r e n t models have i n c r e a s e d t h e !<nowledge concerning h e a r t metabolism. i n many ways t h e langendorff model and i t s m o d i f i c a t i o n s ( 9 , 18) a r e s u p e r i o r t o i n s i t u models. any p e r f u s i o n medium may be a d m i n i s t e r e d and any h e a r t t e m p e r a t u r e , p e r f u s i o n p r e s s u r e and d e g r e e of oxygenation can be m a i n t a i n e d . the p e r f u s a t e i s e a s i l y c o l l e c t e d , measured and examined. . the langendorff p r e p a r a t i o n , however, c o n s t i t u t e s a d e n e r v a t e d h e a r t which i s devoid of normally o c c u r r i n g humoral f a c t o r s , and most o f t e n a non-corpuscu l a r p e r f u s i o n medium i s used. t h i s may i n f l u e n c e n o t o n l y t h e p h y s i o l o g i c a l d i s t r i b u t i o n of oxygen and s u b s t r a t e t o t h e d i f f e r e n t p a r t s of t h e myocardium b u t a l s o t h e removal of m e t a b o l i t e s (6, 2 1 ) . these d i s a d v a n t a g e s can p a r t l y be overcome by t h e u s e o f a mechanical pump and w i t h donor blood a s p e r f u s i o n medium. however, t h e b e s t "pump" q u a l i t i e s i n terms of p u l s e rate and p r e s s u r e a r e o b t a i n e d by t h e most s u p e r i o r pump a v a i l a b l e a n o t h e r h e a r t . thus, t h e p a r a c o r p o r e a l h e a r t model (17, 20) i s a middle c o u r s e optimized i n terms of pump f u n c t i o n and pump flow. the p a r a c o r p o r e a l h e a r t can be i n f l u e n c e d by hormones and s u b s t r a t e s from t h e s u p p o r t i n g r a t , which i s i m p o r t a n t f o r c l i n i c a l a p p l i c a t i o n s . v a r i a t i o n s i n acid-base and e l e c t r o l y t e s can a l s o be c o r r e c t e d by t h e pump rat. 240 to stimulate the clinical reperfusion situation we used a retransfusion sys tem. the model was stable for at least 60 min of paracorporeal circulation but 30 min was enough to cover our interest in early reperfusion after ischemia. a prerequisite for good function of this system was that the supporting ani mal was able to stand the surgical trauma and could increase its cardiac output to compensate for the high demands due to the high flow through the excised heart. these requirements were fulfilled. thus their acid-base status, blood-gas balance and electrolytes were stable both before and during perfusion of the excised hearts. quickly retransfused volumes neither caused deficient reoxygena tion nor signs of "pump" insufficiency in terms of pulse pressure failure in the aortic arch of the experimental heart. concerning the reliability of the model, our findings corroborate the results of other investigators using langendorff preparations. thus, the critical period of complete global ischemia at 37oc was between 15 and 20 min (12). the efflux of ck after different periods of ischemia was also in accordance with other reports (12, 22). the model showed a high degree of reproducibility. heart function in terms of electrical activity and observed contractility correlated well to the ischemic time. the efflux of ck was also proportional to the ischemic time. this model can easily be modified to allow the use of a left ventricular latex balloon for isovolumetric contrac tion studies (4, 14, 1 9 ) . the mechanisms of ischemic heart damage associated with heart surgery have been extensively discussed. increased attention has been paid to the early period of recirculation (7, 11). significant damage to heart muscle seems to occur within a few minutes of recirculation. the role of calcium and eventually further energy content depletion during the reperfusion period has been stressed. furthermore, an adverse effect of molecular oxygen, 'ithe oxygen para dox", has been proposed ( 3 , 12). this may be based upon a possible toxic effect of oxygen-derived free radicals, such as the superoxide (0 -) and hydroxyl (oh') radicals (5). 2 we believe that the presented model, which is simple and inexpensive, is suitable for studies of myocardial ischemia and the reperfusion phenomena. although the present experimental arrangement is still far from the clinical situation, it is closer than the langendorff preparation. acknowledgements the study was supported by grants from the tore nilson fund for medical research, the swedish society against heart and lung diseases, and fortia a b . 24 1 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. references bretschneider, j.h., hubner, g., knoll, d., lohr, b., nordbeck, h. & spieckermann, p.g.: myocardial resistance and tolerance to ischemia. physiological and biochemical basis. j cardiovasc surg 16:241-260, 1975. buckberg, g.d., brazier, j.r., nelson, r.l., goldstein, s.m., mcconnell, d.h. & cooper, n.: studies on the effects of hypothermia on regional myo cardial blood flow and metabolism during cardiopulmonary bypass. i. the adequately perfused beating, bifrillating and arrested heart. j thorac cardiovasc surg 73:87-94, 1977. demopoulous, h.b.: free radicals in medicine and biology. acta phys scand suppl 492 symposia, 1979. fallen, e.l., elliott, w.c. & gorlin, r.: apparatus for study of ventricu lar function and metabolism in the isolated perfused rat heart. j appl physiol 22:836-839, 1967. fridovich, i.: quantitative aspects of the production of superoxide anon radical by milk xanthine oxidase. j biol chem 245:4053-4057, 1970. gamle, w.j., conn, p.a., edalju kumar, a., plenge, r. & monroe, r.g.: myocardial oxygen consumption of blood-perfused, isolated, supported, rat heart. am j physiol 219:604-612, 1970. ganote, c.e., worstell, j. & kaltenbach, j.p.: oxygen-induced enzyme re lease after irreversible myocardial injury. effects of cyanide in perfused rat hearts. h e r j pathol 84:327-350, 1976. gordon, k.m. & visscher, m.b.: the mechanism of failure in the completely isolated mammalian heart. am j physiol 125:461-473, 1939. hearse, d.j., humphrey, s.m. & chain, e.b.: abrupt reoxygenation of the anoxic potassium-arrested perfused rat heart: a study of myocardial enzyme release. j molec cell cardiol 5:395-487, 1973. hearse, d.j., humphrey, s.m. & garlick, p.b.: species variation in myo cardial anoxic enzyme release, glucose protection and reoxygenation damage. j molec cell cardiol 8:329-339, 1976. hearse, d.j.: reperfusion of the ischemic myocardium. j molec cell cardiol 9 : 606-616 , 1977. hearse, d.j. & de leiris, j. (eds.) enzymes in cardiology. diagnosis and research. d.j. hearse and j. de leiris, new york, john wiley & sons, chichester, brisbane, toronto, 417-444, 1979. kirsch, u., rodewald, g. & kalmar, p.: induced ischemic arrest. j cardio vasc surg 63:121-130, 1972. kligfield, p., horner, h. & brachfeld, n.: a model of graded ischemia in the isolated perfused rat heart. j appl physiol 6:1004-1008, 1976. langendorff, 0.: untersuchungen am iiberlebenden sbugetieren. pfliigers archiv 61:291-332, 1895. melrose, d.g., dreyer, e., bentall, h.h. & baker, j.b.e.: elective cardiac arrest. lancet ii:217 1955. mulch, j., schaper, j., gottwik, m. & hehrlein, f.w.: recovery o f the heart after normothermic ischemia. thorac cardiovasc surg 27:145-152, 1979. neely, j.r., liebermeister, h., battersby, e.j. & morgan, h.e.: effect of pressure development on oxygen consumption by isolated rat heart. amer j physiol 212:804-814, 1967. opie, l.h.: effect of extracellular ph on function and metabolism of isolated perfused rat heart. amer j physiol 209:1075-1080, 1965. sarnoff, s . j . , case, r.b., welch, g.h.jr., braunwald, e. & stainsby, w.n.: performance characteristics and oxygen debt in nonfailing, metabolically supported isolated heart preparation. amer j physiol 192:141-147, 1958. taegtmeyer, h., hems, r. & krebs, h.a.: utilization of energy-providing substrates in the isolated working rat heart. biochem j 186:701-711, 1980. waldenstrgm, a.p., hjalmarsson, a.c., jodal, m. & waldenstrgm, j . : signifi cance of enzyme release from ischemic isolated rat heart. acta med scand 201:525-532, 1977. 242 upsala j med sci 84: 3-8, 1979 differential effect of cupric ions on the in vitro utilization of glucose by blastocysts and pancreatic islets claes-goran ostenson, grels naeslund and b. ove nilsson from the department of histology a n d reproduciion r e s e a r c h u n i t , biomedical c e n t r e , v p p s u l a , s\t,eden abstract to evaluate a possible interference of cupric ions on the trophoblast cell metabolism, glucose utilization was studied in mouse blastocysts delayed & utero and later activated by oestradiol for 18 h. isolated pancreatic islets from guinea pigs were used for comparative purposes. the pancreatic islets were not affected when the copper concentration was 0.1 mol/l whereas for the blastocysts a marked effect was found already at a concentration of 0.01 mmol/l. this indicates that copper concentrations even lower than that of the uterine secretion in presence of a cu-iud might affect different enzymes linked with the glucose metabolism of the trophoblast cells. introduction blastocysts of the rat, mouse and rabbit when exposed in vitro to cuprous or cupric ions are detrimentally effected (5,7,8,13,20), although the degree of the damage is modified by the amount of albumin present in the incubation medium (13) making comparisons between concentrations in utero and in vitro difficult. the blastocyst sensitivity to copper develops gradually, probably appearing at the period of transition from the morula to the blastocyst stage (7,23). further more, the influence of the copper ions seems to be reversible since the rat blastocysts transferred from a copper containing to a non-treated uterine horn developed normally ( 6 ) . although the gradual appearance of the copper effect and its reversibility are compatible with many types of adverse mechanisms, a blocking of some vital process such as the uptake or metabolism of nutrients seems likely, since it is known that the achievment of the blastocyst stage is accompanied by a rapid increase in metabolic rate of the zygote ( 4 ) . glucose is one nutrient efficiently utilized by cells. the blastocyst at initiation of implantation increases its capacity of glucose utilization (21). to evaluate a possible interference by copper on the cell metabolism, glucose utilization was studied in isolated mouse blastocysts. isolated islets of guinea pigs were similarly chosen for comparative studies since their rate of glucose utilization is well 3 known from previous studies (25) and since they have a similar size and shape as the blastocyst. materials and methods the blastocysts were obtained from nmri mice, which had been ovariectomized and injected 2.c. with 0.02 ml depo-provera (containing 1 mg medroxiprogesteron acetate; upjohn co., kalamazoo, usa) about a week previously in order to obtain an experimental delay of implantation (3,15). the mice in delay were injected s.c. with 0.1 pg oestradiol18hbefore the experiment. animals were killed by an overdose of mebumal (aco, stockholm, sweden), the uterus flushed with hanks solution containing 2 mg albumin/ml, and the blastocysts collected with a small pipette. the islets were isolated aseptically from pancreatic glands of normal guinea pigs using the collagenase digestion technique according to the method of howell and taylor (14). the islets were then maintained in tissue culture for three days (1) prior to the experimental procedure. glucose utilization was measured using a technique previously applied to isolated islets of langerhans (2,25), in which the formation of 'h-water from 5h-glucose is determined. this reflects glycolysis and the hexose mono phosphate shunt (hmps), but not the use of glucose for glycogen synthesis. batches of 20-30 blastocysts or 15 isolated pancreatic islets were incubated in small vials (16), containing 20 ~1 of krebs-ringer bicarbonate buffer, supple mented with 2 mg/ml bovine plasma albumin, fraction v (armour pharmaceutical co., eastbourne, u.k.), 16.7 mol/l glucose and the labelled substrate 5h glucose (the radiochemical centre, amersham, u.k.) of specific activity 6 ci/mmol. copper chloride was dissolved in redistilled water and added to the buffer in a final concentration of cupric ions of 100 or 10 pmol/l. in some vials cupric ions were omitted. the vials were placed inside scintillation flasks containing 0.5 ml redistilled water and sealed with a rubber membrane. incubation was performed for 60 minutes at +37oc in a shaker and in a gas phase of 95% 02:5x c02. the metabolism of the blastocysts or the islets was then stopped by an injection of 10 111 0.2 further incubation at room temperature was carried out to allow the tritiated water formed by the living material to equilibrate with the water in the scintillation flasks. controls without biological material or pancreatic islets were included in each experiment to allow correction for the blank. the radio activity in the water was then measured by liquid scintillation spectrometry. the recovery of 3h-water during the equilibration period was tested by measure ments of the diffusion of a known amount of 3h-water, and was in this system found to be approximately 70%. 3 3 mol/l hc1 through the rubber membrane. a the rate of glucose utilization was calculated according to a formula given 4 by ashcroft et a1 (2) and expressed as either pmol of glucose utilized per blastocyst per hour or as pmol per ug islet dry weight per hour. results the implanting blastocysts, obtained 18 hrs after the injection of oestrogen, in contrast to pancreatic islets, showed a markedly decreased utilization of glucose when cupric ions were added to the incubation medium (table 1). the basal utilization of glucose (at 16.7 mmol/l), calculated on a dry weight basis, was 113 pmol/pg/h for the pancreatic islets, and 315 pmol/pg/h for the blastocysts, considering that a mouse blastocyst in delayed implantation has an average dry weight of 35 ng (12). table 1 2 + effect of cu guinea pig pancreatic islets determined as the formation of 3h20 from 5h on the in vitro glucose utilization of mouse blastocysts and 3 - glucose concentration of implanting blastocysts pancreatic islets cucl2 ( m o l / l ) ( pmo 1 / b 1 as to cy s t / h) ( e m 0 1 / ij g /h) 11.1 + 1.1 (8) 0 0.01 6.5 2 0.9 (7) 0 . 1 4 . 0 + 0.6 (5) p < 0.01 p < 0.05 113.1 + 10.0 (9) 131.7 f. 8.9 (6) 124.6 2 8.6 (11) the results are given as mean values in each group given in parentheses. s.e.m., and with the number of experiments discussion the degree of development among the uterine zygotes during a normal implanta tion in the mouse differs substantially(l1). since the functional changes in the zygotes are rapid, this developmental variability renders observations on meta bolic changes less valid. in an attempt to obtain a better synchrony of zygote development, the blastocysts were recovered from animals in an experimentally delayed implantation. at implantation, these blastocysts display similar properties as do normal blastocysts. the present results show that the glucose utilization of the blastocysts is markedly affected by cupric ions at a concentration of only 10 pmol/l. this in hibitory level of copper corresponds to the concentration of about 10 pmol/l that cross (8) has found to produce a decrease in membrane ion transport, and to the concentration of about 20 pmol/l which holland and pike (13) have found to influence the in vitro development of mouse embryos. considering that the copper concentration in the uterine secretion from women carrying a cu-iud is 5 approximately 700 pmol/l ( 1 3 ) , the reported inhibition of the blastocyst metabolism in vitro could well be active also in vivo in spite of a protecting effect by droteins in the secretion. the fluxes of glucose after its phosphorylation in a cell are generally mediated via glycolysis, the hexose monophosphate shunt ( h m p s ) , and glycogen synthesis. anaerobic glycolysis is the main pathway, while the contribution of the hmps varies considerably in different cell types, from approximately 0 to at least 30 per cent ( 2 4 ) . the hmf's in b-cells of mouse pancreatic islets accounts for only a few per cent of the total glucose utilization, even at a high glucose concentration (2). in the cells of a blastocyst at an early stage of implantation and with a high synthesizing activity, this percentage may be much higher. the flux of glucose into glycogen is similarly low in the pancreatic islet cells, estimated at less than 10 per cent of the glucose utilization (2). although we have not measured the rate of glycogen synthesis in the tropho blasts, morphological studies have indicated that a rapid increase in glycogen granules occurs in these cells 18 hours after activation of the implanting blastocyst (21). thus, it appears that the w s and the glycogenic pathway are quantitatively qore important in the blastocyst cells than in the pancreatic islet cells. the mechanism of a copper-induced inhibition of the glucose utilization could involve some or all of these metabolic pathways. a plausible mode of interference of cupric ions with glucose metabolicm could be as an antagonist to other divalent ions, 5.s. zinc and magnesium ions, in some of the glycolytic enzymatic reactions (22). a step that might be affected in this way is the enolase reaction, where a part of the tritiated water in the determination of glucose utilization is obtained. a n influence of copper on the hmf's is also possible. earlier findings in erythrocytes implicated a copper-induced decrease of the hmf's activity, caused by inhibition of the enzymes glucose-6-phosphate dehydrogenase and glutathion reductase ( 9 , 1 0 , 1 7 ) . however, it was later shown that copper, at a concentra tion less than 100 umol/l, was actually stimulating the h m p s , and that the decreased amount of reduced glutathion previously mentioned, was due to an accelerated oxidation of this compound (18). indeed, the overall mode of action of cupric ions on the hmps seems to be complex and difficult to fully interpret. glycogen synthetase in human endometrial cells has been reported to be stimulated by copper in utero ( 1 9 ) . if this effect is also present in tropho blast and islet cells, a copper induced increase of glycogen synthesis could contribute to a reduced rate of glucose utilization in our assay, by making less 5h-glucose available to glycolysis. 3 it still remains to be explained why the glucose utilization of the blasto cysts but not of the islets, was affected by cupric ions. it is possible that 6 differences in glucose handling, where the blastocyst may have higher fluxes into glycogen synthesis and the h m p s , are responsible for this opposite reac tion to copper. moreover, it is worthy of note that a pancreatic islet consists of a vasculated, compact mass of a few thousand highly specialized cells, while a blastocyst at this stage only contains a shell of approximately 50 undifferen tiated cells around the blastocele. thus, penetration of cupric ions into the tissue, as well as transport across the cell membranes, may be factors that differ between these two organs. however, it can be concluded that cupric ions markedly affect the glucose utilization of the blastocyst at a copper concen tration that is lower than that of the uterine secretion from a woman with a copper-containing intrauterine device. acknowledgements financial support: swedish medical research council (project 12x-70 to professor ove nilsson, project 12x-109 to associate professor claes hellerstram) and "the expressen prenatal research foundation". depo-provera was kindly supplied by upjohn ab, sweden. technical assistance: mrs barbro einarsson and mrs ing-britt hallgren. references 1. andersson, a.: tissue culture of isolated pancreatic islets. acta 2. aschcroft, s.j.h., weerasinghe, l.c.c., bassett, j.m. & randle, p.j.: endocrinol (kbh) 83: suppl 205, 283-294, 1976. the pentose cycle and insulin release in mouse pancreatic islets. biochem j 126: 525-532, 1972. mammalian embryology (ed. j.c. daniel, jr.), pp. 419-435. academic press, new york, 1978. 4. brinster, r.l.: metabolism of the ovum between conception and nidation. in: mammalian reproduction (ed. h. gibian & e.j. plotz), pp. 229-263. springer-verlag, berlin, heidelberg. 1970. 5. brinster, r.l. & cross, p.c.: effect of copper on the preimplantation mouse embryo. nature 238: 398-399, 1972. 6 . chang, c.c. & tatum, h.j.: a study of the antifertility effect of intrauterine copper. contraception 1: 265-270, 1970. 7. chang, c.c., tatum, h.j. & kincl, f.a.: the effect of intrauterine copper and other metals on implantation in rats and hamster. fertil steril 3. bergstram, s.: experimentally delayed implantation. in: methods in 21: 274-278, 1970. 8. cross, m.h.: effect of copper on isolated rabbit blastocysts. j reprod 9. deiss, a . , lee, g.r. & cartwright, g.e.: hemolytic anemia in wilson's 10. fairbanks, v.f.: copper sulfate-induced hemolytic anemia. arch intern 11. finn, c.a. & mclaren, a . : a study of the early stages of implantation 12. hensleigh, h.c. & weitlauf, h.m.: effect of delayed implantation on fertil 32: 485-489, 1973. disease. ann intern med 73: 413-418, 1970. med 120: 428-432, 1967. in mice. j reprod fertil 13: 259-267, 1967. dry weight and lipid content of mouse blastocysts. biol reprod 10: 315-320, 1974. early blastocysts against the toxic effects of cuprous and cupric i o n s during development in vitro. j reprod fertil 53: 335-339, 1978. 13. holland, m.k. & pike, i.l.: albumin protection of mouse morulae and 7 14. howell, s.l. & taylor, k.w.: potassium ions and the secretion of insulin by islets of langerhans incubated in vitro. biochem j 108: 17-24, 1968. 15. humphrey, k.w.: the induction of implantation in the mouse after 16. keen, h., field, j.b. & pastan, i.: a simple method for in vitro ovariectomy. steroids 19: 591-600, 1967. metabolic studies using small volumes of tissue and medium. metabolism 12: 143-147, 1963. 17. metz, e.n.: mechanism of hemolysis by excess copper (abstract). clin 18. metz, e.n. & sagone, a.l.: the effct of copper on the erythrocyte 19. middleton, j.c. & kennedy, m.: the biological actions of endouterine 20. naeslund, g.: blastocystotoxic effect of copper in vitro. contraception 6: 281-285, 1972. 21. nilsson, b.o., ostenson, c.-g., eide, s. & hellerstrbm, c.: role of glucose in the mouse uterine secretion for the activation of the implanting mouse blastocyst. in manus. advances in pharmacology and chemotherapy (ed. s. garattini, a'. goldin, f. hawking & i.j. kopin), pp. 327-409. academic press. new york, london, 1977. j reprod fertil 32: 429-439, 1973. mcgraw-hill book company, new york, 1968. (the phosphogluconate oxidative pathway, p. 420). hellerstrijm, c.: effects of insulin on the glucagon release, glucose utilization and atp content of the pancreat?c a -cells of the guinea pig. in: glucagon: its role in physiology and clinical medicine (ed. p.p. fog, j.s. bajaj & n.l. fog), pp. 243-254. springer-verlag. new york, 1977. res 17: 32, 1969. hexose monophosphate shunt pathway. j lab clin med 80: 405-413, 1972. copper. contraception 11: 209-225, 1975. 22. oster, g. & salgo, m.p.: copper in mammalian reproduction. in: 23. webb, f.t.g.: the contraceptive action of the copper iud in the rat. 2 4 . white, a . , handler, p. & smith, e.l.: principles of biochemistry. 25. ostenson, c.-g., andersson, a., brolin, s . e . , peterson, b. & accepted january 3, 1979 address for reprints: claes-ggran ostenson department of histology biomedical centre box 571 s-751 23 uppsala sweden 8 upsala j med sci 90: 157-162, 1985 urologic complications in 159 consecutive renal bansplantations j a n wahlberg, gunnar tufveson and lars frodin department of urology, university hospital, uppsala, sweden abstraci the urologic complications were reviewed in 159 consecutive renal transplant ations. there were 23 major complications ( 1 4 % ) in 22 patients. one patient died as a consequence of urologic complications and two other grafts were lost. i n the remaining cases the grafts could be saved by surgical revision o r con servative treatment. the principles of diagnosis and treatment of these complic ations are discussed, introduction although the diagnosis and treatment of graft rejection is the main problem in renal transplantation, surgical complications can create clinical difficult ies. survival o f patients and o f grafts has improved considerably in recent years ( 3 , 1 4 ) , predominantly due to better understanding of transplantation im munology. the incidence of technical complications has not correspondingly de creased. the complications associated with ureteral reconstruction still cause significant morbidity and also some mortality (9,10,11,13). the present study was initiated to review the incidence o f urologic complications in o u r renal transplant patients in the past 3 f years and to evaluate o u r diaqnostic and management procedures. material and methods between january 1981 and july 1984, 159 renal transplantations were perform ed on 144 patients. ten o f the patients received kidneys from living relatives. the first 89 transplantations were made with azathioprine and prednisolone f o r immunosuppression, whereas in the later 70 cases cyclosporin a and prednisolone were used. operative procedures f o r the primary urinary drainage, ureteroneocystostomy ( 1 , 1 5 ) was used in all cases. the bladder was filled with saline solution via a urethral catheter a?d an incision 3-4 cm long was made in the dome of the bladder down to the mucosa. at the distal end of the incision the mucosa was opened and the split end of the graft ureter was anastomosed to the opening in the mucosa, using a running 5-0 vicrylr suture. the muscle incision was closed with interrupted sutures, creating a subrnucosal tunnel to prevent reflux. f o r secondary,neocystostomy the same technique was used, provided that the length of the remaining ureter was sufficient for a tension-free ansstomosis, but in these cases a ureteral stent was left for 3-10 days. if the ureteral length was insufficient for neocystostomy, a ureteropelvostorny was made, utiliz ing the graft recipient's own ureter for urinary drainage. in most of such cases the recipient's own kidney was left behind. percutaneous nephropyelostomy was made under ultrasound guidance. diagnostic procedures the patients were monitored with daily routine laboratory tests, includinq serum creatinine, urea, white blood count, haernoglobin and electrolytes. we have tried to perform ultrasound scanning as a once-weekly routine in the first three postoperative weeks, and additional scans were made if urologic conplic ations were suspected. renal scintigraphy was performed on the first postoper ative day and once weekly for 3-4 weeks. intravenous pyelography o r ultrasound guided antegrade pyelography was added if further inforrnatioi was needed. results among the 159 transplantations there were 23 major urologic complications in 22 patients, giving an overall rate of 14 l. ureteral obstruction occurred on 11 occasiorls in 10 patients, and urinary leakage in 12. these 22 p3tients are individually presented in table 1. one of the 144 patients died of sepsis following urinsry leakage, giving a mortality rate of 0.7 x. three grafts were lost, two because of upper o r lower polar necrosis with urinary leakage, and the third was in the fatal case. the rate o f graft loss thus was 1 . 9 e. to treat the urologic complications, 23 ad ditional procedures were performed (fig. 1). 5econdary.ureteroneocystostomy was successful in seven of nine cases, giving adequate urinary outflow and/or healing of urinary fistula. in one patient a distal ureteral stenosis was corrected by neoimplantation, but ureteropelvost omy was necessary 1 4 years later due to progressive fibrosis of the ureter. 158 t a b l e 1. u r o l o g i c c o m p l i c a t i o n s i n 159 r e n a l t r a n s p l a n t a t i o n s immunotime supa f t e r case age p r e s s t r a n s no ( y r s ) i o n * p l a n t i 33 az 2 1 days 2 52 az 3 days 3 45 az 1 mo 4 33 az 23 mo 5 28 az 1 mo 18 mo 6 53 az 4 days 7 42 az 4 days 8 38 az 3 mo 9 38 az 2 mo 1 0 39 cya 2 days 11 62 cya 2 rno 1 2 38 c ~ a 40 days 13""" 52 az 4 days 1 4 6 1 cya 6 mo 15 54 cya 16 5 9 cya 17 43 cya 18 64 cya 19 35 cya 20 32 cya 2 1 67 cya 22""" 36 cya 30 days 4 days 30 days 6 days 7 days 1 day 7 days 7 days d i a g n o s i s management** f i s t u l a ups o b s t r u c t i o n n e o i m p l a n t a t i o n f i s t u l a tx o b s t r u c t i o n c l o t r e m o v a l o b s t r u c t i o n n e o i r n p l a n t a t i o n o b s t r u c t i o n ups f i s t u l a n e o i m p l a n t a t i o n f i s t u l a n e o i r n p l a n t a t i o n + l o w e r p o l a r r e s e c t i o n o b s t r u c t i o n n e o i m p l a n t a t i o n o b s t r u c t i o n nps f i s t u l a n e o i r n p l a n t a t i o n o b s t r u c t i o n p e l v o p l a s t y f i s t u l a ups f i s t u l a n e o i m p l a n t a t i o n o b s t r u c t i o n nps f i s t u l a tx o b s t r u c t i o n ups f i s t u l a ups f i s t u l a n e o i m p l a n t a t i o n o b s t r u c t i o n nps f i s t u l a ups o b s t r u c t i o n ups f i s t u l a n e o i m p l a n t a t i o n ob s e r v a t i o n outcome t i m e s a t i s f a c t o r y 6 rno s a t i s f a c t o r y 1 " g r a f t l o s s s a t i s f a c t o r y 9 rno o b s t r u c t i o n 17 'i r e c u r r e d s a t i s f a c t o r y 2 'i s a t i s f a c t o r y 30 g r a f t l o s s , 2 " p a t i e n t d i e d s a t i s f a c t o r y 26 i ' s a t i s f a c t o r y 1 5 " s a t i s f a c t o r y 17 s a t i s f a c t o r y 14 " s a t i s f a c t o r y 1 3 " s a t i s f a c t o r y 1 2 " u n s a t i s f a c t 2 o r y g r a f t l o s s s a t i s f a c t o r y 8 mo s a t i s f a c t o r y 6 'i s a t i s f a c t o r y 2 " s a t i s f a c t o r y 7 i ' s a t i s f a c t o r y 6 i ' s a t i s f a c t o r y 3 s a t i s f a c t o r y 4 i' * az a z 3 t h i o p r i n e , cya = c y c l o s p o r i n a ** nps = p e r c u t a n e o u s nephropyelosotomy, tx = t r a n s p l a n t e c t o r n y , ups = u r e t e r o pelvostornv *** = l i v i n g donor i n t h e n i n t h p a t i e n t t h e secondary u r e t e r o n e o c y s t o s t o m y was f o l l o w e d by r e c u r r ence o f u r i n a r y leakage, s e p s i s and death. u r e t e r o p e l v o s t o r n y was s u c c e s s f u l i n a l l seven cases i n w h i c h i t was p e r f o r r n ed. i n s i x o f them t h e r e c i p i e n t ' s own k i d n e y was l e f t b e h i n d , w i t h o u t c o m p l i c a t i o n s . the outcome a f t e r c l o t r e m o v a l i n one case and u r e t e r o p e l v o p l a s t y i n a n o t h e r was l i k e w i s e s a t i s f a c t o r y . f o l l o w i n g c o n s e r v a t i v e t r e a t m e n t , with p e r c u t a n e o u s nephropyelostorny, t h e u r e t e r a l o b s t r u c t i o n s u b s i d e d s p o n t a n e o u s l y i n two o f t h e t h r e e cases and t h e c a t h e t e r c o u l d be removed. i n t h e t h i r d case t h i s c o n s e r v a t i v e t r e a t m e n t was c o n t i n u e d because t h e p a t i e n t r e f u s e d f u r t h e r s u r g e r y . 159 10 from living 138 no further operations \159 1 23 additional procedures transplants /donors patients 3 nephropyelostomy 149 cadaveric / 1 ureteropelvoplasty 159 144 transplants 1 clot removal from ureter 7 pelvoureteric anastomosis 9 repeat ureteroneocystosomy 2 transplantectomy (to own ureter) fig. of renal transplantation -1. additional procedures performed in treatment o f urologic complications discussion in considering the urologic complications of renal transplantation, three figures are particularly important, viz. the rates of mortality and graft loss and the total morbidity. one patient in the present series died as a result of urologic complications. this figure is lawer than in many earlier reports ( 1 0 , 1 1 , 1 3 ) , but is comparable with other authors’ results (2). the rate of graft loss was also fairly low, 3/159. in all three cases there was calyceal leakage due to upper o r lower polar necrosis in grafts with multiple arteries. the lesson learned from these disastrous complications is that early removal of such grafts should be considered. an attempt to save one kidney with lower polar necrosis by ureteral neoimplantation and lower polar resection resulted in re currence of the urinary fistula and, ultimately, death of the patient. i r i contrast to mortality rate and graft loss, the overall complication rate was rather high compared with some other series (2,5,10,12), but was largely similar to figures in two reports ( 9 , l l ) . analysis of the complications in our series revealed inadequate ureteral blood supply as the probable cause in 75 76 of the cases. this stresses the importance of careful handling of the ureteral blood vessels during procurement and transplantation, and consequently these procedures should be entrusted only to surgeons familiar with the techniques. since urologic complications produce significant morbidity, the emphasis should be on prevention. f o r this purpose, a method for preoperative o r intraoperative assessment o f the ureteral blood supply would be of great value. successful visualization of the ureteral blood supply was obtained with sodium fluorescein in the perfusion solution ( 6 ) . we have tried this method in six kidneys, but so 1 6 0 f a r have been u n a b l e t o draw c l i n i c a l l y r e l e v a n t c o n c l u s i o n s f r o m o u r a t t e m p t s . o f t h e r e m a i n i n g cases w i t h a e t i o l o g y o t h e r t h a n u r e t e r a l i s c h a e m i a , two had u r i n a r y l e a k a g e due t o i n s u f f i c i e n c y o f u r e t e r o n e o c y s t o s t o m y and i n one case t h e u r e t e r was i n a d v e r t e n t l y anastomosed t o t h e p e r i t o n e u m . the p a t i e n t w i t h u r e t e r a l o b s t r u c t i o n from b l o o d c l o t s was o f s p e c i a l i n t e r e s t , s i n c e t h e h a e m a t u r i a was caused by an i n f e c t i o n w i t h a d e n o v i r u s t y p e 11 ( 4 ) . o u r s e r i e s c o n t a i n e d one case o f u r e t e r o p e l v i c j u n c t i o n o b s t r u c t i o n p r o b a b l y p r e e x i s t i n g i n t h e g r a f t b u t becoming s i g n i f i c a n t when t h e k i d n e y was i n a new p o s i t i o n . t h i s c o m p l i c a t i o n has been e a r l i e r r e p o r t e d (11). our case was s u c c e s s f u l l y managed w i t h a von l i c h t e n b e r g u r e t e r o p e l v o p l a s t y ( 7 ) . our r e s u l t s i n d i c a t e t h a t i t i s p o s s i b l e t o p e r f o r m a u r e t e r o p e l v o s t o m y and s i m p l y l i g a t e t h e p r o x i m a l end o f t h e p a t i e n t ' s own u r e t e r , l e a v i n g t h e n a t i v e k i d n e y b e h i n d . we have n o t o b s e r v e d i n f e c t i o n i n t h e r e m a i n i n g k i d n e y or o t h e r n e g a t i v e e f f e c t s from t h i s p r o c e d u r e . nonperformance o f nephrectomy m i n i m i z e s t h e o p e r a t i v e trauma a g r e a t advantage i n t h i s g r o u p o f p a t i e n t s . s i n c e u r e t e r a l o b s t r u c t i o n s u b s i d e d d u r i n g nephropyelostomy t r e a t m e n t i n t w o cases, a c o n s e r v a t i v e approach may b e w a r r a n t a b l e i n o b s t r u c t i v e d i s o r d e r s . p o s s i b l y t h i s approach may be t r i e d f o r a l o n g e r p e r i o d t h a n i n o u r s t u d y . s u c c e s s f u l t r e a t m e n t o f u r o l o g i c c o m p l i c a t i o n s u s i n g p e r c u t a n e o u s n e p h r o p y e l o stomy and d i l a t i o n o f u r e t e r a l s t r i c t u r e s has been r e p o r t e d ( 8 ) . we t r i e d d i l a t i o n o f t h e u r e t e r t h r o u g h a p e r c u t a n e o u s nephropyelostomy t u b e i n one p a t i e n t (case 1 6 ) w i t h o u t success. p r o b a b l y , however, t h i s mode o f t r e a t m e n t m e r i t s t r i a l i n most p a t i e n t s w i t h s t e n o s i s o f t h e g r a f t u r e t e r . i n c o n c l u s i o n we, l i k e o t h e r t r a n s p l a n t surgeons, recommend t h a t u r o l o g i c c o m p l i c a t i o n s i n r e n a l t r a n s p l a n t a t i o n be t r e a t e d w i t h o u t d e l a y . i n c o m p l i c a t e d c o n d i t i o n s , such a s r e n a l p o l a r n e c r o s i s w i t h c a l y c e a l u r i n a r y l e a k a g e , t r a n s p l a n t e c t o m y s h o u l d be c o n s i d e r e d a t an e a r l y s t a g e , t o a v o i d e n d a n g e r i n g t h e p a t i e n t ' s l i f e . i f u r e t e r a l o b s t r u c t i o n i s p r e s e n t , a t e m p o r a r y u r i n a r y d i v e r s i o n t h r o u g h a p e r c u t a n e o u s nephropyelostomy i s a d v i s a b l e . the f i n a l c o r r e c t i o n s h o u l d be done l a t e r , w i t h p a t i e n t and g r a f t i n o p t i m a l c o n d i t i o n . m i n o r u r i n a r y l e a k a g e w i t h o u t s i g n s o f i n f e c t i o n can p r o b a b l y h e a l d u r i n g p r o l o n g e d u r i n a r y d i v e r s i o n . we have f o u n d u l t r a s o u n d s c a n n i n g e x t r e m e l y u s e f u l i n t h e management o f u r i n a r y c o m p l i c a t i o n s . the h i g h d i a g n o s t i c a c c u r a c y o f t h i s method, i n combin a t i o n w i t h t h e p o s s i b i l i t i e s f o r p e r f o r m a n c e o f a n t e g r a d e p y e l o g r a p h y and o f c r e a t i n g u r i n a r y d i v e r s i o n , can s o l v e v a r i o u s d i a g n o s t i c p r o b l e m s as w e l l a s a c u t e d i f f i c u l t i e s i n management. 161 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. references alexander, g.p, van geerbruyden, j., moyelk, j. & t o u s s a i n t , j.: r e s u l t a t e t c o n s i d e r a t i o n s b p r o p o s de 50 t r a n s p l a n t a t i o n s r e n a l e s chez l’homwe. p r o c eur d i a l t r a n s p l 155:3,1966. d r e i k o r n , k . , rohl, l . , horsch, r . & r o s s l e r , w.: u r o l o g i c c o m p l i c a t i o n s i n r e n a l t r a n s p l a n t a t i o n . t r a n s p l p r o c xiv:76-77,1982. f r o d i n , l . & bsckman, u.: renal t r a n s p l a n t a t i o n i n uppsala. uppsala j med s c i f r o d i n , l., g r e f b e r g , n. & cars, 0.: acute h e m a t u r i a i n a r e n a l t r a n s p l a n t p a t i e n t caused b y a d e n o v i r u s t y p e 11. i n m a n u s c r i p t . h e l l i n g , t.s., alleman, r.e., thomas, c . y . , mo3re, j.d. & koontz, p.g.: p r e v e n t i o n and management o f u r o l o g i c a l c o m p l i c a t i o n s f o l l o w i n g r e n a l t r a n s p l a n t a t i o n . transp prox xii:695-708,1980. k i r c h n e r , k.h., m a c m i l l a n , r.a., k r e u g e r , r.p. & s e s h a d r i , r.: f l u o r e s c e i n sodium i n j e c t i o i f o r e v a l u a t i o n o f u r e t e r i c v a s c u l a t u r e p r i o r t o c a d a v e r i c r e n a l t r a n s p l a n t a t i o n . t r a n s p l a n t a t i o n 33:loo-101,1982. larsson, a . & f r o \ d i n , l . : management o f h y d r o n e p h r o s i s i n a k i d n e y t r a n s p l a n t p a t i e n t . case r e p o r t . uppsala j med s c i liebermann, r.p., g l a s s , n.r., crumny, a.b., s o l l i n g e r , h.w. & b e l z e r , f . 0.: n o n o p e r a t i v e p e r c u t a n e o u s vanagement o f u r i n a r y f i s t u l a s and s t r i c t u r e s i n r e n a l t r a n s p l a n t a t i o n . surg gynecol o b s t e t 155:667-672, 1982. l o u g h l i n , k . r . , t i l n e y , n.l & r i c h i e , j.p.: u r o l o g i c a l c o m p l i c a t i o n s i n 718 r e n a l t r a n s p l a n t p a t i e n t s . s u r g e r y 95:297-302,1984. malek, g.h., k e h l i n q , d.t., daouk, a . a . & k i s k e n , w.a.: u r o l o g i c a l com p l i c a t i o n s o f r e n a l t r a n s p l a n t a t i o n . j urol 109:173-176,1973. mundy, a.r., podesta, m.l., bewick,m.,rudge, c.j. & e l l i s , f.g.: the u r o l o g i c a l c o m p l i c a t i o n s o f 1000 r e n a l t r a n s p l a n t s . b r j u r o l 53:397-402, 1981. sagalowsky, a . i . , r a u s l e r , c.w., p e t e r s , p.c., dickerman, r.m., g a i l i u n a s , p., helderman, j.h., h u l l , a . r . & a t k i n s , c.: u r o l o g i c c o m p l i c a t i o n s i n 505 r e n a l t r a n s p l a n t s w i t h e a r l y c a t h e t e r removal. j u r o l 1 2 9 9 2 9 9 3 2 , 1 9 8 3 . s t a r z l , t.e., g r o t h , c . g . , putnam, c.w., peun, j., halgrimson,c.g., g l a t mark, a., gecelker,l., b r e t t s c h n e i d e r , l. & s t o n i n g t o n , o.g.: u r o l o g i c a l c o m p l i c a t i o n s i n 216 human r e c i p i e n t s o f r e n a l t r a n s p l a n t s . am surg 172: 1-22,1970. t e r a s a k i , p . i . , perdue, s.t., s a s a k i , n., michey, m.r. & w h i t b y , l . : l m p r o v i n g success r a t e s o f k i d n e y t r a n s p l a n t a t i o n . j a m a 250:1065-1068,1983. woodruff, m.f.a., nolan, b., robson, j . s . & mcdonald, m.k.: r e n a l t r a n s p l a n t a t i o n i n man. e x p e r i e n c e s i n 35 cases. l a n c e t i:6-12, 1969. address f o r r e p r i n t s : jan wahlberg department o f u r o l o g y u n i v e r s i t y h o s p i t a l s-781 85 uppsala sweden 162 upsala j med sci 91: 217, 1986 paper spotted blood glucose for quality control of reflectance meters henning von schenck department of clinical chemistry, linkoping university, linkoping, sweden the usefulness of blood glucose results obtained with various teststrips and reflectance mters rely mainly on the quality of the strip and the skills of the analyst. dependable qualitycontrol,therefore, remains a main task for the large hospital when decentralizing analysis of bloodglucose close to the pati ent or to satellite laboratories. adequate quality control has, however, been inhibited by the lack of blood that is resistant to glucolysis and still appropriate to teststrip chemistry. this study reports the applicability of filter paper sampling of capillary blood to quality control of decentralized glucose determination. methods: capillary blood (50 p) was applied on filter paper. a split sample was assayed with decentralized reflectance meters, i.e.: the dextrostix gluco mter ( a s ) or the reflotest-glucose reflomat and the reflocheckclucose reflocheck system (boehringer mannheim) and the result written on the filker paper. the papers were mailed once weekly to the laboratory. there, the spots were punched out and assayed as detailed in clin chem 35:706-709, 1985 with the routine laboratory procedure using glucose hexokinase. results: the accuracy of the filter paper method was 0.96x + 0.4 (n=68, ~0.98) with intraand interassay c.v. of 1.2-5.2% and 9.9-4.4%, respectively. ana lytical recovery was 80%. the results were stable during 3 weeks. the regres sion equations for the glucmter systems were y= 1.04x 0.3 (n=50, r=c).96) and y= 0.98x + 0.3 (n=46, r=0.98). for the reflocheck y= 0.95x + 0.5 (n=46, ~0.95) and the reflomat systems y= 1.03x + 0.3 (n=39, y=0.96), y= 0.86x + 1 . 1 (n=43, ~0.96) and y= 0.85x + 0.9 (n=43, ~0.95). conclusion: the filter paper method is useful for quality control of decentra lized glucose determination. problem cases can be identified as exemplified by our finding of 3 different regression equations for the same reflectance meter system, one of which gave results very close to the identity line. 217 upsala j med sci 92: 205-213, 1987 positron emission tomography: an animal model of spinal distribution of drugs after intrathecal administration per hartvig’, lars l. gustafssod, kjell bergstrom’, ulf mostrom’, urban ponten3, bo lindberg4 and hans lundqvist’ ‘hospital pharmacy, departments of ’diagnostic radiology, ’neurosurgery and 4primate laboratory, university hospital, ’department of physical biology, gustaf werner institute, university of uppsala, uppsala and 6department of clinical pharmacology, huddinge university hospital at karolinska institute, huddinge, sweden abstract an animal model has been developed in the rhesus monkey for noninvasive monitoring of csf transport of drugs by external detectors i.e. positron emission tomography. the model compromises the cannulation of the subarachnoid space (with a spinal needle), and has been used without any damage to the monkey. with the method it was shown that injection rate had a major influence on the transport rate of 68gac1 in the csf. injection of 0.5 ml over 60 sec gave the highest radioactivity near the injection site, whereas an injection rate of this volume shortly after injection. this method have been of value for the determination of drug kinetics after spinal administration. 3 over 10 sec resulted in high radioactivity more rostrally introduction positron emission tomography (pet) is a noninvasive technique which measures the kinetics of biochemical processes and transport of radiolabelled compounds in vivo (5,6). the technique furnishes cross-sectional images o f the distribution o f radioactivity in the body and can be regarded as an in vivo autoradiographic method. the technique may be potentially useful in measuring cerebrospinal (csf) bulk flow o r the distribution of low molecular radiotracers after spinal (intrathecal o r epidural) administration, since kinetics a r e monitored noninvasively by external detectors. the rostra1 bulk flow of csf has been studied by gamma scintigraphy using radio tracers such as iodinated human serum albumin (4). it is doubtful, however, if albumin will mirror the distribution of small drug molecules owing to differences in molecular weight and physio-chemical properties. 205 an e x p e r i m e n t a l model u s i n g rhesus monkeys was d e v e l o p e d t o s t u d y t h e d i s t r i b u t i o n o f i n t r a t h e c a l l y a d m i n i s t e r e d 68ga b y pet. the o b j e c t i v e s were t o : * e v a l u a t e a model u s i n g p e t f o r t h e measurement o f t h e t r a n s p o r t o f s m a l l r a d i o l a b e l l e d m o l e c u l e s i n csf a f t e r s p i n a l a d m i n i s t r a t i o n * s t u d y t h e i n f l u e n c e o f i n j e c t i o n r a t e on t h e d i s t r i b u t i o n o f t h e r a d i o t r a c e r i n t h e csf. materials and methods animals rhesus monkeys (macaca m u l a t t a ) w e i g h i n g 6.3-10.5 k g from t h e p r i m a t e l a b o r a t o r y o f r e p r o d u c t i v e research, uppsala u n i v e r s i t y were used a f t e r an o v e r n i g h t f a s t . the a n i m a l s were a n e s t h e t i z e d w i t h r e p e a t e d doses o f 50-100 mg o f k e t a m i n e i n t r a m u s c u l a r l y ( k e t a l a r , parke-davis nj, usa) g i v e n e v e r y 30-60 m i n u t e s . diazepam (diazemulsr, k a b i v i t r u m stockholm, sweden) was a d m i n i s t e r e d i n t r a v e n o u s l y as a supplement i n doses o f 5-10 mg e v e r y 30-60 m i n u t e s . a f t e r i n d u c t i o n o f a n a e s t h e s i a , t h e monkey was p l a c e d l e f t s i d e down i n a s p e c i a l l y c o n s t r u c t e d p l a s t i c c r a d l e , w h i c h was k e p t h o r i z o n t a l d u r i n g t h e e x p e r i m e n t . r i n t r a t h e c a l a d m i n i s t r a t i o n the s p i n a l c a n a l was p u n c t u r e d between t h e s p i n a l p r o c e s s e s l3-l4 w i t h a m o d i f i e d s p i n a l n e e d l e 40 mm l o n g and w i t h a dead space o f 50 p1 ( e v e r e t t s p i n a l n e e d l e r , avons m e d i c a l l t d r e d d i t c h , england, 0.7 mm odx90 mm). to s t a b i l i z e t h e n e e d l e d u r i n g t h e e x p e r i m e n t i t was passed t h r o u g h a p l a s t i c s t o p p e r , t h a t was f i x e d t o t h e n e e d l e w i t h a screw and t o t h e s k i n w i t h a t a p e . the escape o f one d r o p o f csf i n d i c a t e d p u n c t u r e o f t h e s u b a r a c h n o i d space. the n e e d l e had a s h o r t b e v e l , w h i c h was p o s i t i o n e d w i t h i t s o p e n i n g p o i n t i n g c r a n i a l l y . b e f o r e t h e i n j e c t i o n o f r a d i o a c t i v i t y , t h e p o s i t i o n o f t h e n e e d l e was v e r i f i e d w i t h f l u o r o s c o p y , a f t e r t h e i n j e c t i o n o f 0.1 m l m e t r i z a m i d e (amipaque , 170 i / m l , nyegaard, oslo, norway). the csf-pressure was m o n i t o r e d b y c o n n e c t i n g t h e s p i n a l n e e d l e t o a gould p r e s s u r e t r a n s d u c e r v i a a s a l i n e f i l l e d p o l y e t h y l e n e c a t h e t e r . r the s i z e o f t h e s p i n a l c a n a l was c a l c u l a t e d b y computed tomography (ct; siemens somatom dr2) i n a monkey w e i g h i n g 9.3 kg. the c r o s s s e c t i o n a l a r e a o f t h e s p i n a l c a n a l was e s t i m a t e d a t d i f f e r e n t l e v e l s by m e a s u r i n g t h e t w o axes i n t h e a x i a l c t scan and u s i n g t h e e l l i p s i s . the volume o f t h e s p i n a l c a n a l was c a l c u l a t e d by m u l t i p l y i n g t h e c r o s s s e c t i o n a l a r e a w i t h t h e l e n g t h measured on t h e l o n g i t u d i n a l scan. csf and b l o o d s a m p l i n g a second s p i n a l n e e d l e ( e v e r e t t s p i n a l n e e d l e , 0.5 mm odx50 mm) was i n t r o d u c e d i n t o t h e s u b a r a c h n o i d space t h r o u g h a l a t e r a l p u n c t u r e a t c1-c2 l e v e l f o r csf 206 sampling ( 1 , 9 ) , a pe 50 polyethylene catheter was connected to the end of this needle through an adapter. blood from a peripheral vein and csf samyles were collected up to 40 and 60 minutes after injection, respectively. radiopharmaceuticals 68gallium as the chloride salt ( 68ge/ ml of 1 m hydrochloric acid. this solution was neutralized with 1 m sodium hydroxide with phenol red as indicator. 68 gacl ) was produced in a tin dioxide open bed 3 68 ga column (new england nuclear medicine, boston, usa) eluted with a few before administration, the solution was filtered through a 0.22 pn membrane filter for sterilization. the radioactive dose varied between 10 and 60 mbq in a 0.5 ml sample. the injections were given in the same animal following each other (with an 2-hour interval) and given at a constant rate by a syringe pump (model 220 sage instruments) over 10 o r 60 sec, respecti;ely (table 1 ) . table 1. maximal radioactive uptake in the spinal canal after intrathecal administration at l3-l4. registration with positron emission tomography. exp radiotracer duration of spinal level maximal time aftet. injection * injection (sec) uptake min 1 1 15 300 8 580 27 80 20 2 68gac13 10 350 6 1 68gac13 60 f: l 6 166 26 3 'uptake = nci/cm dose x bodyweight-' (1 ) instrumental for the imaging of different spinal levels, the cradle was moved to radio logically predetermined positions. imaging o f the monkey was performed at the spinal levels l6, l1 and t8 in a pc 384-38 positron tomograph (scanditronic instrument ab, uppsala, sweden) equipped with two rings of detectors that gave three simultaneous slices of 13 mm thickness each (5). images were recorded for 60 sec throughout the study with the initial kinetics o f radioactivity monitored at the l 1 level. 207 the influence o f the injection rate was studied by using two collimated beta scintillation detectors ( b g o ) positioned at l6 and l1 and with the positron camera at the t8 level. the distribution of radioactivity could thus be monitored at three different levels simultaneously. calculations the radioactivity per cm3 was calculated as a function of time at each spinal level corresponding to l6, l1 and t8 and was corrected for physical decay of the radionuclide from the time of administration. the values were divided by the amount of radioactivity given per gram body weight. a normalized uptake of 1.0 corresponded to the uptake that would have been achieved if the administered radioactivity was equally distributed in the whole body of the monkey assuming 1 cm is equal to 1 9. the standard deviation of measured radioactivity was always below 15%. radioactivity in blood and csf were measured in a well-counter and expressed as uptake values as described above. 3 . results animal model intrathecal drug administration, csf-sampling, anesthesia and positioning of the cradle in the positron camera were tested in 4 animals before the experiments started. satisfactory anesthesia of the monkeys was achieved with ketamine and diazepam with no signs of involuntary movements o r hyperventilation. the animals could be kept in the same position f o r several hours and they did not show neurological sequela. the intrathecal positioning o f the needle was crucial, since a constant csf flow did not ensure a correctly placed needle. this may also occur when the needle is partly localized in the subdural space after disconnection of the arachnoidea ( 8 ) . puncture of the subarachnoidal space at c1-c2 could be carried out without problems and samples o f 100 p o l o f csf were taken every 15 min. the frontal and sagittal diameters at all levels of the spinal canal were less than 8 and 6 mm, respectively (table 2). the estimated volume of the dural sac was 7-9 ml. 208 t a b l e 2. anatomic d i m e n s i o n s o f t h e s p i n a l c a n a l c a l c u l a t e d from c t scans i n a rhesus monkey ( w e i g h t 9.3 k g ) . l e n g t h o f s p i n a l d u r a l sac ( f o r a m e n magnum s 2 ) : 350 mm. e s t i m a t e d volume o f t h e s p i n a l c a n a l ( f o r a m e n magnum s 2 ) : 7-9 ml. d i s t a n c e between t h e p u n c t u r e s i t e (l4-l5) and d i f f e r e n t v e r t e b r a e l e v e l s : l7: 50 mm; l1: 75 mm; t8: 170 mm; c7: 270 mm. v e r t e b r a l l e v e l f r o n t a l and s a g i t t a l d i a m e t e r s (mm) c r o s s e c t i o n a l a r e a ( e l l i p t i c ) n c5 t4 t i 1 l1 l3 l5 6.7 x 4.7 4.0 x 3.7 5:3 x 3.7 6 . 1 x 5.3 6.7 x 5.3 7.5 x 5.3 25 12 15 28 2 8 30 p o s i t r o n e m i s s i o n tomography i n j e c t i o n r a t e h a d a pronounced i n f l u e n c e on t h e s p r e a d o f g a l l i u m ( t a b l e 1 ) and t h e model was t e s t e d b y u s i n g measurements o f t h e csf-pressure and e x t e r n a l m o n i t o r i n g o f r a d i o a c t i v i t y a t s e v e r a l l e v e l s by c o l l i m a t e d d e t e c t o r s . the d i s t r i b u t i o n o f r a d i o a c t i v i t y a t d i f f e r e n t s p i n a l l e v e l s a f t e r i n j e c t i o n t i m e s o f 60 and 10 sec is shown i n f i g . 1 and 2, r e s p e c t i v e l y . i n j e c t i o n o v e r 60 sec gave a l i n e a r i n c r e a s e o f a c t i v i t y a t a l l r e c o r d i n g s i t e s s t a r t i n g i m m e d i a t e l y and c o n t i n u i n g d u r i n g t h e whole i n j e c t i o n p e r i o d ( f i g . 1 ) . the i n c r e a s e i s s t e e p a t l6, i . e . 1 cm c a u d a l t o t h e i n j e c t i o n s i t e . a t t8, 15 c m f r o m t h e i n j e c t i o n s i t e , t h e i n c r e a s e i s s m a l l e r b u t l i n e a r o v e r t h e whole p e r i o d . a f t e r end o f t h e i n j e c t i o n no f u r t h e r i n c r e a s e i n t h e r a d i o a c t i v i t y was measured a t l6. a t l1 and t8 t h e i n c r e a s e i s l i n e a r up t o 8 m i n with an a p p r o x i m a t e d o u b l i n g o f t h e a c t i v i t y w i t h i n 12 and 6 m i n , r e s p e c t i v e l y . b e f o r e i n j e c t i o n t h e r e c o r d e d csf p r e s s u r e was 1 mmhg. i t was 3 mmhg a t 2 m i n a f t e r end o f i n j e c t i o n . 209 3000 8 2000 \ q c 3 0 i 1 0 60 120 240 360 480 600 3000 '0 s : 2000 3 \ 0 s z 0 0 ci 1000 0 i a 0 e i i i i i i , i 7 600 t i e after injection (seconds) fig. 1 . inikial kinetics o f radioactivity (counts p e r second) at vertebrae levels l 6 (0) a n d l 1 (a) monitored by external detectors bgo and radioactivity at t8 (m) registered by positron emission tomography. the dose was given intra thecally in 0.5 r n l o f 68ga-c1 over 60 seconds at l3-l4. 3 1 210 a different pattern is shown with a 10 sec injection (fig. 2 ) . a fast but non linear increase in radioactivity is seen at all recording sites. the caudally located detector at l6 close to the injection site only recorded a small fraction of the total radioactive dose. the measured radioactivity at t8 is three times higher than at l6. after the injection fluctuating value over 200 sec indicate turbulence in the system. at 200 sec a slow increase in radio activity at t8 and a slow decrease at l6 was measured. at l1 the radioactivity increased by 5 0 per cent to constant values after 6 min. recorded csf pressure prior to administration was 8 mmhg. it increased rapidly to a maximal value of 2 5 mmhg and decreased then slowly. the csf pressure was stable at 4-6 mmhg at 6 min after injection. the radioactivity in csf samples taken from c1-c2 remained low throughout the experiment. discussion the present study demonstrates a new method to monitor drug distribution in the spinal canal and is also a new application of pet. the size of the rhesus monkey allows the body to be freely moved in the pet but the animal is still large enough for injections o f rather large volumes (0.5 ml) into the spinal canal. the model and the detection technique were, thus, adequate for studies of distribution after intrathecal administration of the radiotracer 68ga in the spinal canal. the rapid initial distribution after an intrathecal dose was documented with the model. the kinetics of the radioactivity could be monitored at three levels simultaneously by using two external detectors and the pet-camera. together with the sampling o f csf at c1-c2 a good measure was given of the distribution of radioactivity. it was documented that the injection rate higly determined the spread of radioactivity in the csf. a considerable change of volume may occur in the spinal dural sac, which acts as a pressure reducing chamber for the cranial csf (8). changes in the spinal csf space are balanced by a decreased volume of the epidural veins of the spinal canal. the measured diameters of the spinal canal (table 2) indicates that the maximal size of the spinal dural sac can be estimated to 7-9 ml. assuming that the spinal cord diameter is about 2 / 3 to 3 / 4 of that of the spinal canal, a spinal csf volume of 3 5 ml can be calculated which is in accordance to the data of rieselbach(l0). the injected sample ( 0 . 5 ml) thus represents 1 0 1 5 per cent o f the spinal csf space, which in humans would be a slightly larger volume than used to achieve spinal anaesthesia (2). 21 1 the injected volume will displace csf and expand the dural sac. in the lumbar i region this volume would correspond to a column of 3-4 cm and in the thoracic region to 7 9 cm (table 2). due to the direction of the bevel o f the needle the displacement of csf will mainly be cranially. with the rapid injection surprisingly small amounts of the tracer were found even just caudal to the injection site. it can be concluded that very little expansion apparently occured in the caudal dural sac during the rapid injection with concomitant high csf pressure. the cranial csf space on the contrary may accomodate some 10 per cent of the spinal csf volume by changing the blood pool by 0.5% of its intra cranial volume. the high radioactivity observed at t8 at the end of the fast injection cannot be explained simply by displacement o f csf. a considerable turbulence with mixing o f the sample and the csf has to be postulated. turbulence may also explain the early increase in activity detected at t8 during the slow injection. the distribution rates of the radioactivity that are measured during the initial minutes after intrathecal injections of this volume (0.5 ml) are therefore not representative of csf bulk flow. this affects the distribution of the injected tracer within the spinal dural sac during at least the first 5 minutes. the anatomical dimensions of the spinal canal (cf table 2) are smaller than can be resolved in the pet-system used (spatial resolution about 8x8~13 mm). the calculated uptake values (nci/cm /dose/bw) are relative and do not give exact quantitative measurements ( 5 , 6 ) . consequently the measurements from pet images represent concentrations in the spinal canal as a whole and cannot be strictly related to csf or the spinal cord. however, in the case of 68ga almost all radioactivity would be within the csf because of the hydrophilicity of the ion. 3 another limiting factor of positron emission tomography is the observation time. the positron emitting tracers have a rapid physical decay with half-lives of 68ga and "c of 68.7 and 20.4 min, respectively. this makes prolonged studies impossible. more long-lived positron emitters like 73se(tl 7.1 h) can, when they are available, overcome such drawbacks. 2 despite the limitations, pet gave an in vivo quantitation of variations in concentrations at different spinal levels and made it possible to study factors which influence rostra1 transport after intrathecal application. the animal pet model including technique for cannulation of the subarachnoid space may be applied to study the transport of several types o f drugs such as e.g. opioid analgesics, in the csf after intrathecal injection. such studies using this 212 t e c h n i q u e is p r e s e n t e d e l s e w h e r e ( 7 ) . acknowledgements mrs y l v a m y r b e r g and m r l e i f ormegard a r e acknowledged f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e and mrs j e a n e t t e g r u n s t e i n and mrs g u n h i l d l a r s s o n f o r s k i l f u l t y p i n g o f t h e m a n u s c r i p t . t h i s s t u d y was s u p p o r t e d by g r a n t s f r o m t h e swedish m e d i c a l research c o u n c i l (3902, 7005) and t h e k a r o l i n s k a i n s t i t u t e . references 1. amundsen, p. & skalpe, 1.0.: c e r v i c a l myelography w i t h a w a t e r s o l u b l e 2. bridenbaugh, p.o. & kennedy, w.f.: s p i n a l , s u b a r a c h n o i d n e u r a l b l o c k a d e . c o n t r a s t medium ( m e t r i z a m i d e ) . n e u r o r a d i o l . 8: 209-212, 1975. i n : n e u r a l b l o c k a d e i n c l i n i c a l a n e s t h e s i a & managements o f p a i n (m.j. cousins & p.o. bridenbaugh, ed) pp. 146-175. j p l i p p i n c o t t co, p h i l a d e l p h i a , t o r o n t o , 1980. c a t h e t e r placement. a n e s t h e s i o l o g y 37: 352-353, 1972. 3. cohen, c.a. & k a l l o s , t.: f a i l u r e o f s p i n a l a n e s t h e s i a due t o s u b d u r a l 4. d i c h i r o , g.: movement o f t h e c e r e b r o s p i n a l f l u i d i n human b e i n g s . n a t u r e 5. e r i k s s o n , l . , bohm, c., wesselberg, m., b l o m q v i s t , g., l i t t o n , j., widbn,l., bergstrom, m., e r i k s s o n , k . & g r e i t z , t . : a f o u r r i n g p o s i t r o n camera system f o r e m i s s i o n tomography o f t h e b r a i n . ieee trans. n u c l e a r s c i e n c e 29: 204: 290-291, 1964. 539-543, 1982. 6. g r e i t z , t . : p o s i t r o n e m i s s i o n tomography f o r q u a n t i t a t i v e i n v i v o . s t u d i e s o f n e u r o t r a n s m i t t e r k i n e t i c s : a c r i t i c a l e v a l u a t i o n . f r o n t i e r s i n b i o c h e m i c a l and p h a r m a c o l o g i c a l research i n d e p r e s s i o n ( e d . e. usdin, m. asberg, l. b e r t i l s s o n & f. s j o q v i s t ) , pp. 69-78. raven p r e s s , new york, 1984. 7. g u s t a f s s o n , l.l., h a r t v i g , p., bergstrom, k., l i n d b e r g b., l u n d q v i s t , h., lqngstrom, b., svard, h., rane, a. & tamsen, a.: s u b m i t t e d t o b r . j. a n a e s t h e s i a . f l u i d and t h e s p i n a l d u r a m a t e r . j. n e u r o l . neurosurg. p s y c h i a t . 35: 9. o r r i s o n , w.w., e l d e v i k , o.p. & s a c h e t t , j.f.: l a t e r a l c 1 c 2 p u n c t u r e f o r 8. m a r t i n s , a.n., w i l e y , j.k. & myers, p.w.j.: dynamics o f t h e c e r e b r o s p i n a l 468-473, 1972. c e r v i c a l myelography. p a t 111: h i s t o r i c a l , anatomic and t e c h n i c a l c o n s i d e r a t i o n . r a d i o l o g y 146: 401-408, 1983. 10. r i e s e l b a c h , r.e., d i c h i r o , g., f r e i r e i c h , e.j. & r a l l , d.p.: m e t h o t r e x a t e : d i s t r i b u t i o n i n c e r e b r o s p i n a l f l u i d a f t e r i n t r a v e n o u s , v e n t r i c u l a r and lumbar i n j e c t i o n . n. e n g l . j. med. 267: 1273-1278, 1962. address f o r r e p r i n t s : ass p r o f e s s o r p e r h a r t v i g , pharm.d., ph.d. h o s p i t a l pharmacy u n i v e r s i t y h o s p i t a l s-751 85 uppsala, sweden 213 ujms110_1.pdf upsala j med sci 109:1–16, 2004 the endothelial cells in islets of langerhans review based on the doctoral thesis experimental studies on the vasculature of endogenous and transplanted islets of langerhans göran mattsson department of medical cell biology, uppsala university, uppsala, sweden abstract a free full-text copy of this article can be found at the web page of upsala j med sci: http://www.ujms.se the blood vessels of the pancreatic islets are of crucial importance for oxygen and metabolite supply, and dispersal of secreted hormones. in addition to this, endothelial cells have an important role in the revascularization process after islet transplantation. studies have reported signs of poor engraftment of transplanted islets, presumably due to impaired revascularization. the aims of this study were to investigate islet endothelial cells and the revascularization process of transplanted islets. the lectin bandeiraea simplicifolia was found to consistently stain endothelium of both endogenous and transplanted pancreatic islets. by using this marker, we investigated the vascular density of both endogenous and transplanted islets of c57bl/6 mice. one month post-transplantation, a time point when the implants are assumed to be completely revascularized, the graft vascular density was decreased at all investigated implantation sites when compared to endogenous islets. furthermore, most of the blood vessels were located in the graft connective tissue stroma. similar results were obtained six months post-transplantation and in cured diabetic animals after one month. in order to evaluate the function of intraportally transplanted islets, we developed a method to retrieve such islets. enzymatic and mechanic treatment of the liver enabled us to re-isolate the transplanted islets for further in vitro studies. these islets had decreased insulin release, insulin content and glucose oxidation rate when compared to non-transplanted control islets. to understand the role of islet endothelium in the revascularization of transplanted islets we performed angiogenesis microarray studies on islet endothelial cells, from non-cultured, cultured and transplanted islets. we found that the islet endothelium expressed mrna for both inhibitors and inducers of angiogenesis, and that this expression differed with time. in conclusion, these results provide a useful platform for further studies on the islet endothelium. 1 received 29 april 2004 accepted 7 may 2004 islet morphology the human pancreas contains approximately 1–2 million islets of langerhans, constituting 1–2% of the gland (1, 2). their size is usually in the order of 100–400 µ m, irrespective of the species studied, representing 2–3000 endocrine cells per islet (3). the islets are surrounded by a thin connective tissue capsule and consist of several different types of cells (2, 4). the islet core contains mainly beta cells, whereas the other endocrine cells, viz. alpha-, deltaand pancreatic polypeptide cells are peripherally located. the islets also contain nerves, fibroblasts, macrophages, dendritic cells and endothelial cells (ec), which are interspersed among the endocrine cells. the function of the endocrine cells is to produce the hormones, insulin and glucagon, which regulate especially carbohydrate metabolism. islet vasculature the pancreatic vasculature constitutes a complex network, which is adapted to the special needs of the exocrine and endocrine parts of the gland, respectively. the islet blood vessels are of major importance for supplying the endocrine organ with nutrients and oxygen, as well as to transport the secreted hormones into the blood stream. the islet vasculature is connected both in series and in parallel to the exocrine blood vessels, and differs morphologically and functionally from that in the exocrine parenchyma (5). the islet microvascular organization is dependent on the size of the islets (6). thus, smaller islets receive blood from one arteriole, and drain through small venules traversing the exocrine parenchyma. it has been noted that some of the islet efferent capillaries connect with acinar and/or ductular microvessels, thereby forming a so called insulo-acinar portal system (7). the larger islets, on the other hand, have 1–3 arterioles entering the islet, and the islet capillaries drain mainly into postcapillary venules at the edge of the islets, which then empty into intralobular veins. the endocrine capillaries are wider and their ec possess 10 times as many fenestrae as those in exocrine microvessels (8). these fenestrae are likely to be induced by vascular endothelial growth factor-a (vegf-a) production in the islets (9, 10), and are probably important for the high permeability of the islets capillaries. so far, no direct role of the islet vasculature in the pathogenesis of diabetes mellitus has been proven, except for the role of ec in recruitment of immune competent cells in insulitis (11, 12). furthermore, changes in the blood perfusion (13), permeability (14) and islet capillary blood pressure have been described (15). however, it is at present unknown to what extent these changes are secondary to damage to or impaired function of the beta cells per se. endothelial cells ec are lining all blood and lymph vessels and they have several unique functions, e.g. contributing to local blood flow regulation, coagulation and thrombolytic 2 processes, serving as a mechanical and immunological barrier between tissues and blood as well as participation in angiogenesis (16). the ec are adapted to the functional needs of the surrounding tissues, and thereby constitute a heterogeneous group of cells. ec differ functionally between large and small blood vessels, as well as between different organs and species (16, 17). due to this heterogeneity it is difficult to find markers which are specific for all ec. at present several techniques are used to detect ec in vitro or in histological slides, e.g. antibodies (18), lectins (19) and acetylated low density lipoprotein (dil-ac-ldl) (20). however, none of these markers consistently labels ec from all parts of the body in every application. in line with this, specific markers for the islet endothelium have been difficult to find, especially for the use in formalin-fixed and paraffin-embedded material. islet ec are highly adapted to the functional needs of the islets and previous studies have described a technique for isolation and culture of islet endothelium in rats (18) and humans (21). such in vitro studies have confirmed that there are biochemical and functional differences between endothelium from exocrine and endocrine blood vessels. diabetes mellitus diabetes mellitus is a syndrome characterized by metabolic aberrations due to an absolute or relative lack of insulin. it is commonly divided into type 1 and type 2 diabetes, respectively, both of which are likely to be disorders with a heterogeneous etiology. type 1 diabetes mellitus (insulin-dependent diabetes mellitus) is characterized by a loss of the insulin-producing beta cells, due to their destruction in an autoimmune process (22). the reasons for the specific targeting of the beta cells are at present unknown. however, the number of diabetes patients is rapidly increasing and is estimated to be 150–220 millions world wide in the year 2010, most of will be type 2 diabetes (23). type 2 diabetes is due to a combination of an impaired beta cell function and peripheral insulin resistance (24). it is more frequently seen in older people, and is often associated with obesity. however, this form of diabetes is becoming increasingly frequent, and also younger age groups are affected today (25). the number of islets in the pancreas can be normal, although it is usually decreased, albeit never to the same degree as seen in type 1 diabetes (24). transplantation to treat diabetes at present, the only known cure for type 1 diabetes is transplantation of insulin-producing cells (26, 27). type 2 diabetes patients can often achieve reversal of symptoms if increasing their physical activity in combination with weight reduction, even though glucose intolerance often remains. from now on the discussion will focus on type 1 diabetes, where replacement of beta cells has become the treatment of choice for a small group of selected patients. beta cells can be substituted either by implantation of a whole pancreas, or as isolated pancreatic islets. the former is a 3 major surgical procedure which carries significant risks for the patient (28). arguments in favor of choosing isolated islets for clinical transplantations include the technical simplicity, reduced risks for the patients and the lower medical costs associated with the procedure (26, 27). however, the results of islet transplantations have been poor until the recent application of the so called edmonton protocol. this encompasses changes in patient selection and choice of immunosuppressive drugs, and has led to a markedly improved outcome of clinical islet transplantation (29, 30). however, also when applying this protocol, transplantation of a large number of islets (>9.000 islet equivalents/kg body weight) is necessary to achieve insulinindependence. due to the limited availability of islet tissue, this severely restricts the number of patients that may be treated. methods to reduce the number of islets needed to cure a diabetic individual are therefore warranted. engraftment of transplanted islets of langerhans engraftment is the adaptation of the transplanted islets to the new environment in the implantation organ. an adequate engraftment process is of major importance for the islet graft survival and function, and constitutes a possible target for interventions to improve the outcome of islet transplantations. there are several processes involved in the engraftment, e.g. revascularization, reinnervation and reorganization of stromal and endocrine cell interactions. a rapid revascularization is crucial for islet endocrine function after transplantation, and this has been shown to occur within 7–14 days (31–34). however, the extent of the revascularization has not been thoroughly studied in detail. recent experiments on islets transplanted to the renal, splenic or hepatic subcapsular space have suggested that the angiogenic process is insufficient to achieve optimal oxygenization of the transplanted islets (35, 36). however, no measurements comparing the vascular density in endogenous islets versus islets transplanted into these implantation sites were performed. clinically, most islet transplantations are performed intraportally (27, 30), and such intrahepatic islets are difficult to visualize or retrieve, which makes post-transplantation studies difficult to perform. therefore, the development of techniques for retrieval of intraportally implanted islets would be crucial for functional studies. when islets are isolated prior to transplantation they are disconnected from the vascular network, and during the subsequent culture period they depend on diffusion of oxygen and nutrients for survival. studies have suggested that during culture, islet ec disappear or dedifferentiate (37). this means that the islet vasculature has to be rebuilt after transplantation. there are only few studies on islet endothelium (18, 21, 38, 39), and the knowledge on this cell type is therefore scarce, even though recent studies suggest that there is a vascular dysfunction in grafted islets (40). in order to investigate the islet endothelium of transplanted islets, developments of methods to provide access to these cells are mandatory. therefore, the aims of the studies presented in my thesis were to find a marker for endogenous and transplanted endothelium. furthermore, this marker should be applicable to forma4 lin-fixed, paraffin-embedded specimens and usable for comparisons of the vascular density of endogenous and transplanted islets. in order to make functional studies, we wished to retrieve transplanted islets from the liver and then finally to isolate ec from endogenous and transplanted islets. when evaluating islet allotransplantation, both the effects of rejection, immune suppressive drugs and possible recurrence of the autoimmune disease must be considered. many of the drugs used to prevent the immune system from attacking the graft are toxic for the beta cells, and may even by themselves participate in the functional impairment of grafted islets (41). in the present studies, we have consistently used syngeneically transplanted islets to circumvent these confounding factors. staining of ec in formalin-fixed samples in these initial studies we evaluated the use of several different markers (table 1) for use in formalin-fixed, paraffin-embedded specimens of different tissues from mice and rats. we found that bandeiraea simplicifolia-1 (bs-1), a lectin which binds to �-gal epitopes, stained microvascular ec in all tissues examined (figures 5 fig. 1. staining of endothelium (red) in c57bl/6 mouse brown adipose tissue (a) and small intestine (b) with bandeiraea simplicifolia. scale bars are 25 µ m. fig. 2. c57bl/6 mouse pancreatic islets stained with bandeiraea simplicifolia (a) and cd31 (b) to detect microvascular endothelial cells (red). scale bars are 25 µ m. 6 t ab le 1 . s ta in in g of e nd ot he li al c el ls i n fo rm al in -f ix ed , p ar af fi nem be dd ed t is su e sa m pl es f ro m r od en ts . –, n eg at iv e st ai ni ng ; + /– , po si ti ve s ta in in g in s om e ce ll s; + , po si ti ve s ta in in g; + + , st ro ng p os it iv e st ai ni ng ; n d , no t de te rm in ed . w f , w is ta rf ur th r at s; s d , s pr ag ue -d aw le y ra ts a nd c 57 , c 57 b l /6 m ic e. 1a and b), with the exception of the kidney where only some of the ec, especially in the medulla, were positive (42). of special interest was the finding that bs-1, in contrast to the other examined markers, consistently stained pancreatic islet endothelium (figures 2a and b). to evaluate and ensure this, we searched for unstained capillaries, i.e. blood vessels with a diameter <20 µ m and preferably containing erythrocytes, without noticing any such structures. the other investigated markers (table 1) showed no consistency in their staining of ec in sections from either mouse or rat. it should be noted in this context that many of the tested antibodies or lectins referred to above perform well in cryosectioned tissue samples (43–45). isolectin b4, an iso-form of bs-1, has previously been used as a marker for microvascular ec in mouse tissues; including the islets of langerhans (46). this lectin also binds to �-d-galactosyl residues (46), which are present on ec in many species. this may explain the ability of this lectin to stain microvascular ec in rodent tissues. identification of endothelium in endogenous and transplanted islets of particular interest was the finding that bs-1 consistently stained endothelium, not only in endogenous pancreatic islets, but also in isolated islets and syngeneically transplanted rat and mouse pancreatic islets (42) irrespective of implantation site (figures 3a and b). this means that also newly formed ec in recently revascularized islets and the connective tissue stroma can be identified with this lectin. moreover, these results imply that the staining properties of endothelium in islet grafts are independent of the origin of the newly formed microvessels, i.e. whether they derive from renal, hepatic or splenic blood vessels. recently, expression of �-gal epitopes, i.e. those binding to bs-1, was demonstrated in neonatal porcine islet cells (47), whilst no expression was seen in adult 7 fig. 3. staining of endothelial cells (red) in newly revascularized c57bl/6 mouse pancreatic islets syngeneically transplanted to kidney (a) and liver (b) with bandeiraea simplicifolia. scale bars are 25 µ m. endocrine cells (48). therefore, we further evaluated the staining specificity of bs1 by simultaneous staining with bs-1 and antibodies for insulin, glucagon or somatostatin in the same pancreatic sections (49). neither insulin-, glucagon-, nor somatostatin-positive cells in endogenous islets were stained with bs-1. thus, our results are consistent with the notion that �-gal epitopes are not expressed in adult rodent islet endocrine cells. connective tissue stroma and vasculature of transplanted islets endogenous islets of langerhans have a connective tissue capsule, which constitutes a minor part of the islet. thus, the connective tissue within the grafts, which constitutes approximately 30% of the transplant (49, 50), is likely to derive, at least partially, from a foreign body reaction. connective tissue was also formed in association with microspheres implanted into the renal subcapsular space, and constituted 57% of these grafts (49). intraportally transplanted islets were also surrounded by connective tissue. since most of these islets were entrapped in periportal areas, it was impossible to separate between connective tissue associated with graft or liver. the vascular density, i.e. the number of blood vessels per area, of the transplanted islets was decreased compared to that of endogenous islets, irrespective of the chosen implantation organ (49). furthermore, islets transplanted into the spleen had an even lower vascular density than islets transplanted beneath the renal capsule or into the liver. the capillary density was markedly higher in the connective tissue stroma of grafts implanted into kidney or spleen than in the endocrine parts of these grafts. the density of stromal capillaries appeared to be higher in the immediate vicinity of the islets at all three implantation sites (49). a markedly lower number of capillaries was found in the connective tissue surrounding microspheres implanted into the renal subcapsular space when compared to connective tissue surrounding implanted islets. thus, an angiogenic response initiated by the cells within the transplanted islets seems to be of importance to increase the vascular density in the surrounding connective tissue. this preferential distribution of graft blood vessels to the connective tissue stroma rather than among the endocrine cells has not been previously described, and its functional importance awaits further studies. however, when combining the values of vascular density for capillaries in the stromal and endocrine compartments, the value in the total graft is almost as high as in the endogenous islets. it should be noted that a lower oxygen tension in grafted islets, as well as other microvascular disturbances (35), which may be related to the location of graft capillaries, have been previously observed. this suggests that the changed capillary distribution may be of functional importance. the influence of the hormone production from intact endogenous islets on the function and revascularization of ectopically implanted islet grafts is largely unknown. to investigate this we used both normoglycemic and hyperglycemic recipients, i.e. animals with or without functioning endogenous islets (50). the 8 number of islets was chosen to either reverse (500 islets) or not reverse (200 islets) hyperglycemia. the recipients were injected with alloxan prior to transplantation and they all had blood glucose concentrations >14 mm at the time of implantation. transplantation of 500 islets fully reversed the hyperglycemia (<8 mm), while the animals transplanted with 200 islets remained slightly hyperglycemic (~11.1 mm) throughout the course of the study. the vascular density in transplanted islets was significantly decreased to the same extent, when compared to that of native islets, in both groups of recipients (50). thus, this study showed that a remaining normal number of native pancreatic islets does not affect the formation of new blood vessels in an islet graft. furthermore, the possibility to cure diabetic recipients demonstrates that the function of the islets can be adequate, even though the vascular density is decreased. we also found that the vascular density in the transplanted islets did not improve up to six months post-transplantation (50). thus, no redistribution of graft blood vessels from the connective tissue stroma into the endocrine tissue occurred with time. retrieved transplanted islets since islets implanted into the liver are injected directly into the portal vein they distribute in the portal tributaries throughout the hepatic parenchyma. this means that they are difficult to localize, and few techniques are available for their study post-transplantation. to circumvent this problem we developed a method to retrieve transplanted islets from the liver for further functional evaluations (51). on an average 25–50% of those implanted, could be retrieved from each processed mouse liver. the identity of retrieved transplanted islets was confirmed by several different methods. retrieved islets stained pink/red after intravital staining with neutral red, which allegedly selectively stains pancreatic islets (52, 53). sections from the reisolated islets also stained positive with an insulin antibody, and beta and alpha cell secretory granules were observed with transmission electron microscopy. insulin release from retrieved islets incubated with 1.67, 16.7 or 16.7 mm glucose + 5 mm theophylline was investigated either immediately after retrieval or after 3–4 days of culture and compared to non-transplanted freshly isolated or cultured islets. immediately after retrieval, the insulin release was lower compared to non-transplanted control islets during all three incubations. after culture, the insulin response to 16.7 mm glucose and 16.7 mm glucose + theophylline remained markedly impaired in retrieved islets, whereas basal insulin release was similar to that from similarly cultured control islets. retrieved transplanted islets contained less insulin, both immediately after retrieval and after culture, compared to control islets. however, whereas the insulin content of control islets decreased markedly after culture, the insulin content of retrieved islets did not change (figure 4). glucose oxidation rates of retrieved islets were markedly lower than those of control islets when exposed to 16.7 mm glucose, both immediately after retrieval and after culture. the glucose oxidation rates were not affected by culture in any of 9 the groups. there were no differences in dna content between freshly retrieved transplanted islets and freshly isolated control islets, thereby arguing against the notion that the decreased glucose-stimulated insulin release, insulin content and glucose oxidation rate of retrieved islets compared to control islets merely reflect a lower amount of islet tissue in the former preparations. another possible explanation for the poor function of the retrieved islets is that the re-isolation procedure in itself damages the islet cells. however, we used the same amount of collagenase as for isolation of islets from the pancreas (54, 55). in separate experiments we also decreased this concentration, or even omitted it completely, and nevertheless obtained similar results. however, the number of retrieved islets was markedly reduced. the mechanical dispersion of the liver used for retrieval is similar to that used during normal islet isolations and is therefore unlikely to inflict any further functional impairment, as suggested by the similar survival of the retrieved islets. an issue of importance is to what extent the observed impaired function is dependent on the implantation organ, that is the liver, or if it mainly reflects the transplantation and engraftment trauma in itself. the present functional impairment seen in intraportally implanted islets is much more pronounced than that seen after transplantation under the kidney capsule, suggesting that the implantation organ is indeed of major importance. in confirmation of this 10 fig. 4. insulin content in isolated non-transplanted c57bl/6 mouse islets (closed bars) or intraportally transplanted islets retrieved from normoglycemic (hatched bars) or cured diabetic recipients (open bars). the investigated groups were freshly isolated islets, freshly retrieved islets from normoglycemic or cured diabetic recipients, isolated and cultured islets and cultured islets retrieved from normoglycemic recipients. values are expressed as means±sem. * denotes p<0.05 when compared to corresponding isolated non-transplanted islets, whereas # denotes p<0.05 when compared to corresponding freshly isolated islets. modified from (51). notion, experimental studies in rodents have also indicated a lower long-term rate of function of intraportally transplanted islets (56). it might also be a difference if the islets are implanted as single islets or as a cluster of islets. isolation of endothelial cells from endogenous or transplanted islets when islets are isolated prior to transplantation, the efferent blood vessels are disconnected and the islet cells depend on diffusion of oxygen and nutrients. what effects this has on the islet ec are unknown, we wished to investigate this further. we were able to isolate ec from intact and dispersed freshly isolated islets; islets pre-cultured for 7 days as well as transplanted islets retrieved from both the kidney and liver. by the use of bs-1 coated dynabeads we could separate ec from contaminating cells and achieve a purity >90%. the identity of the ec was confirmed by uptake of acetylated light density lipoproteins, and staining with the lectin bs-1, as well as antibodies against angiotensin-converting enzyme and endothelial nitric oxide synthase. isolated endothelial cells have been studied with microrray technology to evaluate the presence of angiogenesis stimulators and inhibitors (57). we were consistently able to obtain expression responses in all investigated groups, i.e. ec from dispersed islets, ec outgrown from freshly isolated islets or 7 days pre-cultured islets, as well as ec outgrown from retrieved transplanted islets from both the kidney and liver, when applying the angiogenesis microarrays. a rather surprising finding is that the endothelium per se fails to produce factors able to stimulate angiogenesis, but rather produce inhibitors of this process, such as endostatin (58) and pigment epithelium-derived factor (pedf). pedf normally decreases ec proliferation (59), and induces apoptosis in ec in sprouting blood vessels, but spares those in quiescent microvessels (60). indeed, previous studies have shown that the pancreas develops substantial stromal vascularity and epithelial cell hyperplasia in the absence of pedf (61). these findings are of interest in view of our previous reports suggesting the presence of a vascular dysfunction in transplanted islets, manifested by a decreased vascular density, low oxygen pressure and low blood flow (40). it is tempting to speculate that expression of an angiogenesis inhibitor, such as pedf, may participate in this response. ongoing experiments aim to verify the accuracy of these experimental analyses. conclusions the lectin bs-1 stains endothelium in both endogenous and transplanted rodent islets of langerhans in formalin-fixed, paraffin-embedded sections. the vascular density of syngeneically transplanted islets of langerhans is decreased when compared to endogenous islets irrespective of implantation site. neither hyperglycemia nor prolonged transplantation time increased vascular density. transplanted islets 11 retrieved from the liver have decreased insulin release, insulin content and glucose oxidation compared to isolated non-transplanted islets. ec can be isolated from freshly isolated, cultured and transplanted pancreatic islets and they differ in their expression of angiogenically active substances. acknowledgements this work was carried out at the department of medical cell biology, uppsala university, sweden. i would like to thank professor leif jansson for valuable help in preparing this manuscript. own work referred to was supported by grants from the swedish medical research council (72x-109, 72xd-15043), the juvenile diabetes research foundation, the efsd/novo nordisk fund for type 2 diabetes research grant, the juvenile diabetes research foundation, the swedish diabetes association, the swedish society of medicine, the novo nordic fund, svenska barndiabetesfonden, the swedish society for medical research, the family ernfors fund, clas groschinskys minnesfond, åke wibergs stiftelse, lars hiertas minne, syskonen svenssons fond, anérs fond, jeanssons stiftelse, thurings stiftelse, ragnhild och einar lundströms minne, magnus bergvalls stiftelse and goljes minne. references 1. lacy p (1993). status of islet cell transplantation. diabetes rev 1: 76–92. 2. pipeleers d, 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25. soltesz g (2003). diabetes in the young: a paediatric and epidemiological perspective. diabetologia 46: 447–54. 26. weir gc, bonner-weir s (1997). scientific and political impediments to successful islet transplantation. diabetes 46: 1247–56. 27. ricordi c (2003). islet transplantation: a brave new world. diabetes 52: 1595–603. 28. stock pg, bluestone ja (2004). beta-cell replacement for type i diabetes. annu rev med 55: 133–56. 29. ryan ea, lakey jr, rajotte rv, korbutt gs, kin t, imes s, rabinovitch a, elliott jf, bigam d, kneteman nm, warnock gl, larsen i, shapiro am (2001). clinical outcomes and insulin secretion after islet transplantation with the edmonton protocol. diabetes 50: 710–9. 30. shapiro am, lakey jr, ryan ea, korbutt gs, toth e, warnock gl, kneteman nm, rajotte rv (2000). islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoidfree immunosuppressive regimen. n engl j med 343: 230–8. 31. menger md, vajkoczy p, leiderer r, jager s, 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secretion from hit-t15 cells and wistar rat islets. transplantation 73: 353–7. 42. mattsson g, carlsson po, olausson k, jansson l (2002). histological markers for endothelial cells in endogenous and transplanted rodent pancreatic islets. pancreatology 2: 155–62. 43. dib sa, vardi p, bonner-weir s, eisenbarth gs (1988). selective localization of factor viii antigenicity to islet endothelial cells and expression of class ii antigens by normal human pancreatic ductal epithelium. diabetes 37: 482–7. 44. mendola jf, goity c, fernandez-alvarez j, saenz a, benarroch g, fernandez-cruz l, gomis r (1994). immunocytochemical study of pancreatic islet revascularization in islet isograft. effect of hyperglycemia of the recipient and of in vitro culture of islets. transplantation 57: 725–30. 45. onrust sv, hartl pm, rosen sd, hanahan d (1996). modulation of l-selectin ligand expression during an immune response accompanying tumorigenesis in transgenic mice. j clin invest 97: 54–64. 46. laitinen l (1987). griffonia simplicifolia lectins bind specifically to endothelial cells and some epithelial cells in mouse tissues. histochem j 19: 225–34. 47. rayat gr, rajotte rv, elliott jf, korbutt gs (1998). expression of gal alpha(1,3)gal on neonatal porcine islet beta-cells and susceptibility to human antibody/complement lysis. diabetes 47: 1406–11. 48. bennet w, bjorkland a, sundberg b, davies h, liu j, holgersson j, korsgren o (2000). a comparison of fetal and adult porcine islets with regard to gal alpha (1,3)gal expression and the role of human immunoglobulins and complement in islet cell cytotoxicity. transplantation 69: 1711–7. 49. mattsson g, jansson l, carlsson po (2002). decreased vascular density in mouse pancreatic islets after transplantation. diabetes 51: 1362–6. 50. mattsson g, jansson l, nordin a, carlsson po (2003). impaired revascularization of transplanted mouse pancreatic islets is chronic and glucose-independent. transplantation 75: 736–9. 51. mattsson g, jansson l, nordin a, andersson a, carlsson p-o (2004). evidence of functional impairment of syngeneically transplanted mouse pancreatic islets retrieved from the liver. diabetes 53: 948–954. 52. bensley rr (1911). studies on the pancreas of the guinea pig. am. j. anat. 12: 297–388. 53. gray dw, gohde w, carter n, heiden t, morris pj (1989). separation of pancreatic islets by fluorescence-activated sorting. diabetes 38 suppl 1: 133–5. 54. andersson a (1978). isolated mouse pancreatic islets in culture: effects of serum and different culture media on the insulin production of the islets. diabetologia 14: 397–404. 55. carlsson po, palm f, mattsson g (2002). low revascularization of experimentally transplanted human pancreatic islets. j clin endocrinol metab 87: 5418–23. 56. hiller wf, klempnauer j, luck r, steiniger b (1991). progressive deterioration of endocrine function after intraportal but not kidney subcapsular rat islet transplantation. diabetes 40: 134–40. 57. gerritsen me, soriano r, yang s, zlot c, ingle g, toy k, williams pm (2003). branching out: a molecular fingerprint of endothelial differentiation into tube-like structures generated by affymetrix oligonucleotide arrays. microcirculation 10: 63–81. 58. liekens s, de clercq e, neyts j (2001). angiogenesis: regulators and clinical applications. biochem pharmacol 61: 253–70. 14 59. dawson dw, volpert ov, gillis p, crawford se, xu h, benedict w, bouck np (1999). pigment epithelium-derived factor: a potent inhibitor of angiogenesis. science 285: 245–8. 60. bouck n (2002). pedf: anti-angiogenic guardian of ocular function. trends mol med 8: 330–4. 61. doll ja, stellmach vm, bouck np, bergh ar, lee c, abramson lp, cornwell ml, pins mr, borensztajn j, crawford se (2003). pigment epithelium-derived factor regulates the vasculature and mass of the prostate and pancreas. nat med 9: 774–80. correspondence to: göran mattsson department of medical cell biology biomedical center, husargatan 3, box 571 uppsala university se-751 23 uppsala sweden phone number: +46 18 4714395 fax number: +46 18 4714059 e-mail: goran.mattsson@medcellbiol.uu.se 15 upsala j med sci 82: 203-208, 1977 tissue reaction to implantation of collagen film an experimental and clinical study d. bergqvist,' j. falkz and a. stahl' from the 'department of surgery, karnsjukhuset, skovde, and the 'department of toxicology, pharmacia ab, uppsala, sweden abstract a new modified collagen film was investigated to evaluate the tissue reaction and absorption time (a) after implanta tion in rabbit muscle, and (b) in a human model where collagen was implanted in the prostatic cavity during opera tion. clinical follow-up by cystoscopy and biopsy was made one week postoperatively. the animal results showed that for 6 weeks after implantation there was a cellular reaction associated with phagocytosk of the collagen fdm. then a scar was formed, similar to that in the controls. the human results showed a similar tissue reaction and no adverse effects from the collagen film. introduction collagen is a potent stimulator of platelet aggrega tion and release reaction (9, 10) and can also acti vate the intrinsic blood coagulation system (13). although it is uncertain if collagen initiates haemostatic plug formation in microvessels (4) its topical application to induce haemostasis is a theoretical possibility. patients with defective col lagen may show a slight tendency to bleed. mi crocrystalline bovine collagen preparations have been used experimentally for topical haemostasis (1,6, 8). application of collagen film to a surgically exposed surface is a new principle and is of poten tial interest due t o its promotion of haemostasis and enhancement of wound healing. this study was un dertaken to investigate the local tissue reaction to implantation of bovine collagen film. initially the tissue reaction and time of absorption were studied in animal experiments. subsequently local tissue reaction was studied in an experimental model in human beings treated surgically for benign en largement of the prostatic gland. material and methods collagen preparation the collagen film is developed from native skin collagen manufactured from the deep layers of bovine corium and sterilized by gamma-irradiation. it consists of pure col lagen fibres with an amino-acid composition very similar to that of plain catgut (arfors & holtz, personal com munication). animal experiments fifty rabbits of about 3 kg body weight, fed on a standard diet (rabbit pellets, astra ewos aj3, sodertiilje, sweden) and with free access to water, were used. the rabbits were anaesthetized intravenously with a barbiturate. the skin over the musc. longissimus dorsi was shaved, cleaned and cut through. a deep longitudinal incision was made in the muscle. in 35 rabbits a piece of about 3 x 3 cm of the collagen film was placed in the muscular incisions after having been folded twice or three times. in 15 control animals the same surgical procedure was performed but nothing was placed in the muscular incisions. the muscle was sutured superficially with plain surgical catgut 00 and the skin was sutured with stainless monofil sutures. the sites implanted with collagen film were examined after one, 2, 3 , 4 and 6 weeks and after 3 and 6 months. five rabbits were sacrificed for each examination. five control rabbits were sacrificed after respectively one, 2 and 4 weeks. sacrifice was made by an overdose of barbiturate. the implantation sites were carefully searched out, dissected free and examined macroscopi cally. human studies patients selected were those undergoing transvesical enucleation of prostatic adenomas weighing more than 50 g. a random allocation was made so that half of the patients served as controls and in half of the patients collagen film was used as a haemostatic agent. after enucleation, capsular catgut sutures were used in all pa tients. the collagen film was then placed in the prostatic cavity and fixed with a tamponade for 3 4 minutes (fig. 1). 0.5 g tranexamic acid (cyklokaprona, kabi, stock holm) was given intravenously. a three-way catheter a demeure (couvelaire, ch 22-24) was used for continuous bladder imgation. primary bladder suture was made with catgut. one week after the prostatectomy the patients were investigated with cystoscopy and biopsies were taken with the resectoscope. there were 21 control and 19 collagen-treated patients. from 15 patients (8 control and 7 collagen) further biopsies were taken 2 months after the operation. the pathologist was unaware of the group upsala j med sci82 204 d . bergqvist et al. f i g . 1 . schematic drawing of collagen inserted in the prostatic cavity and the position of the balloon of the catheter. randomization. the clinical progress of 50 patients (25 control and 25 collagen) including the patients in this study will be published in detail elsewhere ( 5 ) . histological investigation the implantation sites from the rabbits and the biopsies from the patients were fixed in 10% neutral buffered formalin, embedded in paraplast@ and sectioned for light microscopy. stainings were made with haematoxylin eosin and with van gieson’s stain. the preparations from the rabbits were scrutinized for the presence of implanted collagen, evidence of absorption and any cellular reaction involved. the biopsies were particularly examined for signs of tissue imtation such as cellular infiltration in the resected tissue. the degree of cellular infiltration was graded arbitrarily from (+) to + + +. results animal studies macroscopic examination the control incisions had healed with little scar formation and without signs of infection. the amount of scar tissue diminished with time and was hardly discernible after 4 weeks in any of the rab bits. the collagen implantation sites had also healed with scar formation and without signs of infection but these cicatrices were of a larger volume than in the controls and contained a greyish-brown flabby mass in the centre during the first 2 weeks. at 3-6 weeks, small amounts of a brown material were found in the scars. at 3 and 6 months the scar formations were very small and hardly discernible. microscopic examination one week. controls: a moderate amount of young cell-rich connective tissue with slight infiltration of polymorphs occupied almost the whole wound cavi ty. at scattered locations small amounts of fibrin were also present. adjacent to the wound margins there was necrosis of some muscle fibres. collagen film group: material from the implanted collagen film could be demonstrated in the centre of all five implantation sites by positive van gieson staining, a basophil staining with haematoxylin eosin, and a disorganized pattern of fibres not seen in normally occurring collagen. in contact with the foreign collagen were masses of fibrin and many polymorph-nucleated cells (fig. 2). peripherally there was connective tissue similar to that in the controls with a marked proliferation of fibroblasts and necrosis of some muscle fibres. two weeks. controls: in comparison with the one week controls the connective tissue was -now smaller in volume and more fibrous. the cellular in filtration was also less and n o fibrin was found. collagen film group: remains of the implanted collagen film were still present in all 5 rabbits and adjacent to this foreign material there was cellular infiltration indicating ongoing phagocytosis. be sides a decreased number of polymorphs there were many small mononucleated cells and accumulations of swollen macrophages with a brown cytoplasm and also single foreign body giant cells. the sur rounding connective tissue was fibrous as in the controls. three weeks. collagen film group: in 2 of the 5 rabbits, small remains of the implanted collagen film were found. the number of infiltrating cells was less than after 2 weeks, but in all implantation sites there were accumulations of swollen macro phages and some foreign body giant cells. in one implantation site, hypermature macrophages had formed the structure of a granuloma around remains of the collagen film. there was a small amount of fibrous connective tissue in the surroundings. four weeks. controls: a small or very small amount of fibrous connective tissue was the only finding. collagen film group: as after 3 weeks, two im plantation sites contained small remains of the col lagen film (fig. 3). in one the collagen was located in the centre of epithelioid granulomas. the other tissue reactions were essentially the same as after 3 weeks. upsala j med sci 82 tissue reaction to collagen film 205 fig. 2. one week after implantation of the collagen film into a rabbit’s skeletal muscle the implanted collagen is seen as a dark, irregular, rather coarse, fibrous material which is infiltrated by polymorphs. h-e, x250. fig. 3 . four weeks after implantation of the collagen film into a rabbit’s skeletal muscle, small amounts of the im planted collagen may be found in the centre of a granulo ma. h-e, x 100. upsala j m e d s c i 82 206 d . bergqvist et al. fig. 4 . in this sample from a patient, implanted collagen is seen a s a dark-stained curl. the infiltrating cells are pre dominantly polymorphs. v. gieson, x250. six weeks. collagen film group: minute remains of the collagen film were found in one rabbit only, located in the centre of a granuloma. swollen macrophages with brown cytoplasm were found in all rabbits but to a reduced extent in comparison with shorter implantation times. the macrophages were often situated in adipose tissue. small amounts of fibrous connective tissue were also found in the surroundings. three months. collagen film group: the implanted collagen film could not be demonstrated in any of the 5 rabbits. a few swollen macrophages could be found in adipose tissue, occupying the previous wound cavity in 3 rabbits. the amount of connec tive tissue in the surroundings was very small. six months. collagen film group: findings were similar to those after 3 months, but the histological picture was even more normalized. adverse reactions in this experimental study the collagen film caused no other tissue reaction than a reversible cellular reaction associated with phagocytosis of the col lagen film, leading to a retarded healing of the mus cle incision in comparison with the control group. upsalu j m e d sci 82 patient studies macroscopic examination at cystoscopy one week after operation a structure that was judged to be a remnant of the collagen film was seen in 9 of 19 cases. there were no other changes which could be attributed to the implanta tion of collagen and the cystoscopic findings were the same as in the control patients. microscopic examination at microscopic examination of the removed prostates all patients were found to have had a benign, often fibromuscular, hypertrophy of the prostate and there was no case of prostatitis. biopsies one week after operation in the resected tissue samples there were fo cal o r sometimes widespread slight to moderate infiltrations of inflammatory cells, mainly poly morphonuclear leukocytes. in many samples hyper emia and oedema were also evident. i n a few cases large parts of the resected tissues were necrotic and degenerated polymorphs were found. on grading the degree of cellular infiltration the collagen-group scored a mean value of 1.8k0.6 tissue reaction to collagen film 207 points and the control group scored a mean value of 2.1f0.8 (p=0.351). the grading was difficult in some cases, due to very small tissue samples. in many biopsies there were large masses of fibrin adhering to the tissue surface. some areas of the fibrin network contained numerous polymorphs and red cell debris. in a few samples small remnants of the collagen film were also found within the fibrin clots. the collagenous material was surrounded by numerous polymorphonuclear leukocytes (neu trophilic granulocytes). the cytoplasma of some polymorphs stained positively with van gieson’s stain, indicating that phagocytosis of collagen was proceeding (fig. 4). biopsies about two months after operation of the fifteen “two-month biopsies”, eleven had less cellular infiltration than the previous biopsy from the same patient. two were judged to be simi lar and in two others the degree of cellular infiltra tion was greater than before. arbitrary grading of the cellular infiltration was 1.3ko.5 in both groups. the cell populations at 2 months contained characteristically more chronic inflammatory cells such as lymphocytes and plasma cells. no remains of the collagen film were found at this time. the groups showed the same histological picture. adverse reactions n o adverse reactions were seen which could be ascribed to the use of collagen film. for an analysis of complications, see bergqvist & st%hl (5). discussion this study was undertaken to investigate the tissue reaction caused by collagen film, a new substance with an interesting potential as a haemostatic and wound healing agent. the minor tissue reactions in implantation studies in animals motivated further investigation in an experimental human model, where the results could be followed up. we chose transvesical prostatectomy as it caused some bleed ing and as follow-up can be made with cystoscopy and biopsy. this is the first study where internal application of collagen film has been investigated. n o adverse reaction could be seen ( 5 ) . further more, no untoward reactions have been reported in connection with topical application to human skin wounds (14). the histological tissue picture is very similar in both animal and human studies with much fibrin and many polymorphonuclear cells around the collagen film. polymorphonuclear leukocytes (granulocytes) contain collagenase capable of de grading collagen at normal ph (1 1 ) . after degrada tion in vivo, phagocytosis takes place, as was seen in this study. when phagocytosis is prolonged, the histological picture becomes dominated by a dif ferentiation of mononuclear phagocytes from monocytes over macrophages to epithelioid cells (3). the epithelioid cells may form structures rec ognized as granulomas. such granulomas were found in 3 rabbits 3-6 weeks after implantation of the collagen film. as the granuloma-provoking agent is destroyed, the highly differentiated mononuclear phagocytes change into less mature forms (3). in rabbits the absorption time for the collagen film is somewhere between 6 weeks and 3 months, which correlates with findings in the human biopsies where no collagen remains were seen at 2 months. after absorption of the collagen film in the rabbits, the tissues were normalized and a scar was formed similar to that in the controls. plain catgut has an amino-acid composition similar to that of the collagen film (arfors & holtz, personal communi cation) but the structure of catgut is more solid than collagen film. in rare cases, catgut sutures can re main unabsorbed for several years in humans (15). the scores after arbitrary grading of cellular reac tion and infiltration did not differ significantly be tween control and collagen patients. as collagen film is essentially a new preparation, no comparative studies can be reported. however, some investigations have been made with microcrystalline collagen. thus, hait et al. (8), ab bott & austen (1) and cochran & hait (7) carried out microscopic studies and also found a very mod erate tissue reaction, which differed minimally from control specimens. the collagen film used in this study has been shown to be non-immunogenic in rabbits (richter, 1975, personal communication). as to the haemostatic action of collagen film, no animal experiments have yet been made. to judge from the clinical part of our study there is a slight but definite haemostatic effect after prostatectomy ( 5 ) . again, microcrystalline collagen has been shown to induce topical haemostasis in different animal experiments (1, 2, 6, 8), collagen being more effective than surgicela or thrombin. vistness et al. upsulu j m e d sci 82 208 d . bergqvist et al. (16) used microcrystalline bovine collagen exter nally on human skin donor sites with good haemostatic effect. the haemostatic mechanism is undoubtedly rather complex (12, 17). on the basis of this study it can be concluded that collagen film is rapidly absorbed and it is also well tolerated by rabbit and human tissues. implantation in man would be favourable in selected situations. the haemostatic effect needs to be further evaluated but we have used it with success on raw surfaces with capillary bleeding. references 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. abbott, w. & austen, g.: microcrystalline collagen as a topical hemostatic agent for vascular surgery. surg 75: 925, 1974. the effectiveness and mechanism of collagen induced topical hemostasis. surg 78: 723, 1975. adams, d.: the structure of mononuclear phago cytes. differentiation in vivo. am j pathol 80: 101, 1975. bergqvist, d. & arfors, k.-e.: the role of red cells in haemostatic plug formation in the isolated rabbit mesentery. thromb res (in press). bergqvist, d. & st%hl, a.: evaluation of collagen film for local haemostasis after transvesical prosta tectomy. to be published. cobden, r., thrasher, e. & hams, w.: topical hemostatic agents to reduce bleeding from cancellous bone. a comparison of microcrystalline collagen, thrombin, and thrombin-soaked gelatin foam. j bone joint surg58-a: 70, 1976. cochran, g. & hait, m.: an experimental study on the healing on bone following application of a microcrystalline collagen haemostasis agent. j traum 15:494, 1975. hait. m., robb, c., baxter, c., borgmann, a. r. & tippett, l. 0.: comparative evaluation of avitene microcrystalline collagen hemostat in experimental animal wounds. ann j surg 125: 284, 1973. holmsen, h . , day, h. j. & stormorken, h.: the blood platelet release reaction. scand j haematol, suppl. 8: 3, 1%9. hovig, t.: aggregation of rabbit blood platelets pro duced in vitro by saline “extract” of tendons. thromb diath haemorrh 9: 248, 1963. lazarus, g. s., daniels, j. r., brown, r. s., bladen, h. a. & fullmer, h. m.: degradation of collagen by human granulocyte collagenolytic system. j clin in vest47: 2622, 1968. mason, r. g. & read, m. s.: some effects of a microcrystalline collagen preparation on blood. hemostasis3:31, 1974. niewiarowski, s., stuart, r. k. & thomas, d. p.: activation of intravascular coagulation by collagen. r o c soc exp biol med 123: 196, 1966. ponten, b. & nordgaard, j. 0.: the use of collagen film (cutycop) as a dressing for donor areas in split 15. 16. 17. skin grafting. scand j plastic reconstruc surg 10: 3, 1976. postlethwait, r. w., willigan, d. a. & ulin, a. w.: human tissue reaction to sutures. ann surg 181: 144, 1975. vistness, l., goodwin, d., tenery, j., ksander, g. & gruber, r.: control of capillary bleeding by topical application of microcrystalline collagen. surg 76: 291, 1974. zucker, w. h . & mason, r. g.: ultrastructural aspects on interactions of platelets with microcrystal line collagen. am j path82: 129, 1976. received december 18, 1976 address for reprints: dr david bergqvist k b s j u k h u s e t s-54001 skovde sweden upsala j med sci 82 upsala j med sci 82: 3 9 4 1 , 1977 clinical trial in patients with diabetes mellitus of an insulin-like compound obtained from plant source v. s. baldwa, c. m. bhandari,' a. pangaria and r . k . goyal from the department of medicine, s . m . s . medical college hospital, jaipur, india abstract clinical study of an insulin-like compound obtained from vegetable source (vegetable insulin) was carried out on nine patients with diabetes mellitus. the active hypoglycaemic principle, purified protein extract, was obtained from fruits as well as from tissue cultures of the plant mornordica charanlia l. this extract was homologous to insulin obtained from animal pancreas. it showed a consistent hypoglycaemic effect in patients with diabetes mellitus. the average fall in blood sugar level at the peak effect of vegeta ble insulin was found to be statistically significant. the onset of action was within 30-60 min with the peak effect six hours after the administration of the dose of plant insulin. no hypersensitivity reaction to this extract was observed in the group of patients studied. introduction a crude extract obtained from the fruit of a plant known as momordica charantia l. (bitter gourd), has been shown to possess hypoglycaemic activity when tested in rabbits ( 1 , 2 , 3). however, this ex tract was also found to have many side effects, including uterine haemorrhage in gravid female rabbits. an intraperitoneal injection of this extract invariably caused the death of the experimental animal. khanna & mohan (4) were able to extract an abortifacient factor present in this crude extract and isolated diosgenin from the fruit as well as from the in vitro tissue cultures of this plant. subse quently they were able to extract out the active principle in a pure protein from (vegetable insulin, v-insulin) which could be used as a hypoglycaemic agent i n human beings after biological standardiza tion ( 5 ) . vegetable insulin (v-insulin) is structurally and pharmacologically comparable in many re ' guest research fellow, department of pulmonary dis eases, university hospital, uppsala, sweden. spects to bovine insulin ( 5 ) . the method of extrac tion of v-insulin is similar to that of extraction of pure insulin from the pancreas of animals (6). dur ing its extraction, traces of zinc are added, resulting in the formation of colourless, needle-like crystals. the crystals of v-insulin are purified by thin-layer chromatography. the electrophoretic pattern also resembles that of bovine insulin. the infrared spectrum of p-insulin is superimposable on that of standard zinc crystalline insulin. qualitative amino acid analysis by paper chromatography and quantitative analysis by an amino acid analyser showed that p-insulin consisted of 17 amino acids. the three-dimensional structure was found to con sist of two chains of amino acids, bound together with sulphide bonds. the biological assay of the hypoglycaemic activity of v-insulin has been de termined in animal experiments (5). vegetable insulin is available as a suspension which is stable at 4°c and denatured by heat. the compound is suspended in sterile double-distilled water and ultra-violet light and potassium perman ganate fumigation is used for sterilization. the dose is so standardized that the final concentration is 40 units per ml (1.8mg per 40 units). it can be administered by the subcutaneous or the intra muscular route. material and methods nine in-patients, eight males and one female, with an age range of 16 to 52 years, from s.m.s. medical college hospital, jaipur, india, were studied after informed con sent was obtained. all had diabetes mellitus and the dura tion of their disease ranged between 3 months to 10 years. diagnosis of diabetes mellitus was confirmed by clinical examination and laboratory investigations. patients with primary or idiopathic diabetes mellitus were studied. up.rula j m e d sci 82 40 v. s. baldwa e t al. table i. clinical data of patients with diabetes mellitus case age height weight disease no. name sex (y.) (cm) (kg) (y.) type of diabetes 1 jc m 22 168 48.5 6 juvenile 2 gr m 20 168 45.0 6 juvenile 3 gs m 16 148 43.0 7 juvenile 4 pl f 20 152 39.5 8 juvenile 5 sk m 22 170 58.0 10 juvenile 6 us m 18 165 48 .o 8 juvenile 7 hr m 50 165 55.0 0.5 chemical 8 gc m 52 172 57.0 0.3 chemical 9 mr m 50 162 60.5 1 maturity onset body body duration of these patients were placed in two groups, depending on their stage of carbohydrate decompensation, as follows: (i) overt, clinical diabetes, i.e. patients with elevated fasting and random blood sugar levels (more than llomg per 100ml). they were further subdivided into juvenile and maturity onset types, depending on the age a t onset of diabetic symptoms. (ii) asymptomatic diabetes, i.e. patients with normal blood sugar levels (less than 1 lomg per 100 ml) but with an abnormal intravenous glucose tolerance test. there were 6 patients with juvenile diabetes, one with maturity onset and 2 with asymtomatic diabetes mellitus (table i). all antidiabetic medication in the form of insulin or oral hypoglycaemic agents was stopped 24 hours prior table 11. dose of vegetable insulin, according to blood sugar levels fasting blood dose of seventy of sugar level vegetable diabetes (rng%) insulin mild less than 180 10 units moderate 180-250 20 units severe 250 and above 30 units to the study. a fasting blood sugar sample was taken a t 7 a.m. before giving v-insulin. the dose of v-insulin was varied according to the severity of the disease and was de cided upon arbitrarily according to the fasting sugar level (table 11). vegetable insulin was administered subcutane ously and samples for blood sugar determination were taken at regular intervals. the first three samples were drawn at half-hourly intervals (7.30 a m . , 8.00 a.m. and 8.30 a.m.) to establish the onset of action and the subse quent samples were collected a t 11.00 am., 1.00 p m . , 3.00 p m . and 7.00 p.m. to determine the peak effect and duration of action of v-insulin. all samples were collected intravenously. blood sugar estimation was done on whole blood by the method described by king & wooton (7). all the subjects were kept fasting during the interval for col lection of samples and only plain lemon water was given if desired by the patients. a provision was kept for the administration of glucose in the event of the development of hypoglycaemic symptoms. five healthy volunteers (control group i) and 5 patients with overt diabetes mellitus (control groups 11) served as controls. a placebo injection was used. the control sub jects were kept fasting and blood samples were collected and analysed in a similar way. the administration of v insulin to control subjects was avoided due to its inherent hypoglycaemic properties, as evaluated in animal experi ments. table 111. effect of vegetable insulin and placebo on blood sugar levels results given in percentage of fall in blood sugar levels. statistical values are shown ~~~ ~~ fasting % of fall in blood sugar levels no. of values clinical sub(mean) 7.30 8.00 8.30 11.00 1 .00 3 .oo 7.00 group jects mg% a.m. a.m. a.m. a.m. p.m. p.m. p.m. healthy 5 75 5.0 5.0 5.6 5.4 5.6 5.5 5.4 controls i f 7.4 f 1 . 7 21.7 k1.4 1 1 . 8 f 1 . 6 21.4 k l . 5 diabetic 5 2 10 4.6 4.5 5.0 5.8 5.4 5.8 5.7 controls i1 k11.8 k2.0 f 2 . 6 k2.1 11.8 k1.6 f1.8 f 2 . 0 diabetes 9 295 21.5 24.8 30.2 49.2 40.3 35.9 28.8 mellitus f 1 5 . 7 k 8.9 211.0 f12.1 fy3.7 f13.4 f 1 0 . 3 f11.4 l'pscrlri j med s c i 82 results the placebo injection in the control groups did not produce any appreciable reduction in blood sugar levels at different intervals. a definite hypoglycaemic effect of v-insulin was observed in the patient group in this study. the onset of vegeta ble insulin effect was observed within +-1 hours, with the peak effect after 4 hours in 6 juvenile diabetics, after 6 hours in 2 patients with chemical diabetes mellitus, and after 12 hours in one patient with maturity onset of diabetes mellitus. all values in this study were analysed statistically by applying the paired t-test and the calculated f was 3.3 and the tabulated t was 2.3 at d.f. 8”, which was highly significant for the diabetic patient group compared with the healthy and diabetic controls during peak hours. the hypersensitivity reactions were conspicu ously absent after administration of vegetable insu lin and there was no local reaction at the site of injection. discussion the present investigation revealed that vegetable insulin has a consistent hypoglycaemic effect in pa tients with diabetes mellitus. the onset of action is similar to that of standard zinc crystalline insulin (30-60 min). however, the peak effect of vegetable insulin was seen after 4-12 hours as compared with, for 2-3 hours regular insulin. the greatest fall in blood sugar levels observed in the patient group was found to be statistically significant. there were no anaphylactic reactions to vegetable insulin how ever, as regards its long-term use, further studies are required in order to evaluate its antigenic prop erties. the availability of vegetable insulin should open new horizons in the treatment of diabetes mellitus, especially where it is taboo to use animal products. since the active principles are derived from a veg etable source, it can be obtained in abundance. further clinical trials are needed in order to establish its duration of action, assay, antigenicity and various effects on intermediary metabolism in human beings. acknowledgement the authors are grateful to dr p. khanna, department of botany, university of rajasthan, jaipur, for providing the vegetable insulin used in this study. clinicat trial of an insulin-like compound 4 1 references 1. 2. 3 . 4. 5. 6. 7. rivera, g . : preliminary chemical and pharmacological studies on “cundea mor” and “charantia l”. am j pharm i1 3: 28 1 , 194 1. rivera, g . : charantia l , 11. am j pharm 114: 72, 1942. sharma, v. n . , sogani, r. k., arora, r. b.: some observations on hypoglycaemic activity of momordica charantia. ind j med res 48: 471, 1%0. khanna, p. & mohan, s.: isolation and identification of diosgenin and sterols from fruits and in vitro cultures of momordica charantia linn. ind j exp biol 1 1 : 5 8 , 1973. khanna, p., nag, t. n. & jain, s . c.: extraction of insulin from plant cultures in vitro. third international congress of plant tissue and cell culture, held at leicester, england, july, 1974. vestling, c. s.: insulin. biochem preps6:28, 1958. king, e. j . & wooton, i.d.p.: microanalysis in medi cal biochemistry, 3rd ed. j & a churchill ltd, london, 1956. received september 20, 1976 address for reprints: dr v . s. baldwa, m.d. 12-viveka nand marg, c-scheem, jaipur, india upsula j m e d sci 82 upsala j med sci 87: 189-199, 1982 standard computer programs in statistical analysis of survival in childhood lymphoblastic leukemia bertil w. anderson' and goran gustafsson* from the department of statistics', university of uppsala and the department of pediutrics', university hospital, uppsala, sweden abstract a material comprising all children in sweden with acute lympho blastic leukemia diagnosed in the years 1973-80 was analysed sta tistically. the total number of children was 5 0 5 . studies were made of 38 different variables, using frequency tables, cross tables, life table studies ( 1 ) and linear regression analysis according to cox's method ( 2 , 4 ) . chi-square tests and log rank tests were included in the me thods. the combination of life-table studies and linear regression analysis proved to be of value in assessing the significance of different parameters and treatment programs with regard to prog nosis. the aim of this paper is to present a method for analysis of a patient material with use of standard computer programs. the re sults of the total analysis will be published elsewhere (3). introduction acute lymphoblastic leukemia (all) is a malignant disease which can occur in children of all ages. with regard to age, white blood cell count (wbc) at diagnosis and the presence or absence of cen tral nervous system (cns) involvement and/or of a mediastinal tu mor at diagnosis, the children were classified as suffereing from "high-risk leukemia" or "standard-risk leukemia" ( 3 ) . the children first received induction treatment for six weeks and if this was successful they were classified as being in com plete remission. when remission was not achieved, the children died as a result of the disease and/or the treatment. ~. after remission, prophylactic radiation of the cns was given, followed by maintenance therapy. therapy was discontinued after 13-822858 189 three years in complete continuous remission (ccr) . relapses of the disease may occur during therapy or after dis continuation of therapy, in the bone marrow, cns, testes, or other organs or a combination of these locations. following relapse, a second remission may be induced and the child may survive or new relapses may terminate life. death may also occur during a remis sion period from other causes than the disease, e.g. infection. all analysed possible outcomes of the disease are presented schematically in figure 1. diagnosis dead before remission dead in ccr dead after relapse ( s ) (drel-onther) dead after relapse (s) (drel-offther) yg o s e d a t e alive after relapse(s) alive in ccr alive after relapse(s) (arel-onther) (accr, (arel-offther) accr-ofwh er) fig.1 possible outcomes of leukemia in children. note: the notched line represents the whole group in accr, i.e. for abbreviation, see text. also those who have been treated for a shorter time than three years. material and analytical procedures in the years 1973-80, acute lymphoblastic leukemia was diagnosed in 505 children in sweden. for these children, 38 clinical varia bles, for which information was taken from the medical records, were analysed. these 38 variables were divided into four groups: 190 1. identification variables at diagnosis name, month and year of birth, age, sex, hospital, home county, municipality and parish, date of diagnosis, presence or absence of cns leukemia or mediastinal tumor, wbc, immunological classifica tion, risk group, dominating symptom at diagnosis. 2. therapy type of induction therapy, consolidation therapy, cns prophylax is therapy, and maintenance therapy, and their side effects. treat ment program. 3 . treatment results (time variables in moths) a) duration of first remission tccr = time in ccr, i.e. length of time from achieved remission to death during remission 0 ~ to first relapse 0 ~ ' to close date. every child with achieved remission had a value of one month or more €or this variable. if the child died during induction the value was 00. accr = alive in ccr, i.e. length of time from achieved remission to close date. only children who were in ccr at the close date had a value for this variable. tccr-offther = time in ccr off therapy, i.e. length of time from discontinuation of therapy to death during remission to first relapse or to close date. every child with discontinuation of therapy after 3 years in ccr had a value for this variable. accr-offther = alive in ccr off therapy, i.a. length of time from discontinuation of therapy to close date. only children who were in ccr at the close date had a value for this variable. b) patients alive at close date but after relapse arel-onther-rem = alive after relapsing on therapy, i.e. length of time from achieved remission to close date for children relaps ing during therapy. arel-onther-relapse = alive after relapsing on therapy, i.e. length of time from first relapse to close date for children relapsing during therapy. arel-offther-rem = alive after relapsing off therapy, i.e. length of time from achieved remission to close date for children re lapsing after discontinuation of therapy. arel-offther-relapse = alive after relapsing off therapy, i.e. length of time from first relapse to close date for children relapsing after discontinuation of therapy. c) dead patients dccr = died during ccr, i.e. length of time from achieved remission 191 to death during ccr. drel-onther = died after relapsing on therapy, i.e. length of time from achieved remission to death for children relapsing during therapy. time from achieved remission to death for children relapsing after discontinuation of therapy. drel-offther = died after relapsing off therapy, i.e. lengt of 4. other variables rel, = location of first relapse during therapy. rel2 location of second relapse during therapy. rel-offther = location of first relapse after discontinuation of therapy. cdccr = cause of death during ccr (e.g. infection). trel1-rel2 = length oft time in months between first and second relapse. measurements on the 38 variables for the 505 children constitut ed the data set. the data set in order to minimize the coding errors, a thorough examination of the data set comprising the following three steps was made: the data set was printed and compared with the medical records, frequence tables were used for checking missing values and out liers , cross tabulation was done to check that categorical responses were correctly classified. life tables and survival functions in the commonly used method, with for example 5-year survival, information about patients participating in the studyfora shorter time than five years would not be utilized. the proportion of pa tients surviving 5 years would in this case be: w e r of patients alive after five years in the study p5 = n-r of patients participating in the study for at least five years the life table technique, on the other hand, utilizes more in formation by computing this proportion as a cumulative proportion of surviving children. in principle this can be written as follows: where p1 is the proportion surviving one year, p2 the proportion surviving two years provided that the patients survived the first year, and so on. this technique also provides a good idea of the 192 course of the disease. the problem with different starting and follow-up times is solved by rescaling the time variables s o that all the patients start at time 0 . the end point can be one of the following: 1 ) dead (response), i.e. died during c c r or relapse. 2) withdrawn, i.e. alive in ccr at the end of the study (close date). 3) lost, i.e. patients lost at follow-up. the hazard and the density function are two ways of getting ideas of parametric models describing the survival time. the hazard function (failure rate), xi is defined as:' 2 9i a i hi ( 1 + pi) where q1 hi = the width of the i'th interval. the density function (probability of death or relapse per unit = probability of dying in interval i pi = 1 qi time), fi, is defined as: where pi = the estimate of the cumulative proportion, surviving tothe the density is sometimes called the curve of death and is in beginning of the i'th interval. fact an absolute instantaneous rate of death or relapse. the standard errors computed for the survival, hazard and den sity functions are used for computing confidence intervals andper forming tests. tables 1 and 2 and figure 2 show the computer print out of the life table and survival analysis from the program bmdp, pil, 1977 (1). 193 results table 1. example of survival analysis for female patients with achieved remission (computer print out). life table and survival rnalysis. time variable is tidiccr. grouping variable is kon. level is f: interval entered withdrawn 0.0-6.27 6.27-12.53 12.53-18.80 18.80-25.07 25.07-31.33 31.33-37.60 37.60-43.87 43.87-50.13 50.13-56.40 56.40-62.67 62.67-68.93 68.93-75.20 75.20-81.47 81.47-87.73 87.73-94.00 quantile 75th 216 24 181 10 158 10 127 1 1 96 8 80 9 67 9 53 9 40 8 31 8 22 4 18 5 13 4 9 2 7 7 estimate 19.10 median i50th) 44.22 lost 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 dead exposed 11 204.0 13 176.0 21 153.0 20 121.5 8 92.0 4 75.5 5 62.5 4 48.5 1 36.0 1 27.60 0 20.0 0 15.5 0 11.0 0 8.0 0 3.5 standard error 2.28 10.86 exdlanations of the table head: proportion proportion dead 0.0539 0.0739 0.1373 0.1646 0.0870 0.0530 0.0800 0.0825 0.0278 0.0370 0.0 0.0 0.0 0.0 0 . 0 surviving 0.9461 0.9261 0.8627 0.8354 0.9130 0.9470 0.9200 0.9175 0.9722 0.9630 1 . o o o o 1.0000 1 . o o o o 1 . o o o o 1 . o o o o cumulative hazard density survival (s.e.1 (s.e.1 1 .oooo 0.0 0.9461 0.0158 0.8762 0.0237 0.7559 0.0318 0.6315 0.0368 0.5766 0.0384 0.5460 0.0393 0.5024 0.0407 0.4609 0.0423 0.4481 0.0430 0.4315 0.0445 0.4315 0.0445 0.4315 0.0445 0.4315 0.0445 0,4315 0.0445 0.0088 0 . 0 0 8 6 0.0027 0.0000 0.0122 0.0112 0.0034 0.0000 0.0235 0.0192 0.0051 0.0000 0.0286 0.0199 0.0064 0.0000 0.0145 0.0088 0.0051 0.0000 0.0087 0.0049 0.0043 0.0000 0.0133 0.0070 0.0059 0.0000 0.0137 0.0066 0.0069 0.0000 0.0045 0.0020 0.0045 0.0000 0.0060 0.0026 0.0060 0.0000 0.0 0.0 0 . 0 0.0 0.0 0 . 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 time variable is tidiccr = time from onset to response or close date. kon = sex; level is f = sex is female. interval = time in months in ccr. entered = number of patients with a time in ccr corresponding to the interval in question. dead = number of patients responding in the interval in question, i.e. patients dying or relapsing in the interval. the important function values in table 1 are the cumulative sur vival, which forms the basis of the survival curves in figure 2. the table also gives the median estimate in the material, i.e. the time in months when half the patients have responded. table 2 gives a summary of the analyses presented in tablelfor female and male patients separately. the test statistics intable 2 represent the results of two non-parametric rank tests €or compar 194 ison of the cumulative survival functions. the low p values indi cate a difference between the two survival functions. table 2 . table summarizing the survival analyses. test statistics for comparing the proportions of females and males in ccr. summary table percent total dead censored censored f '2 1 6 88 128 0.5vzt. 2h4 146 118 0.4470 m _ _ _ _ _ _ totals 480 234 246 test statistics statistic d.f. p-value generalized wilcoxon (breslowi 18.065 1 0.0000 generalized savage (mantel-cox) 14.878 1 0.0001 dead = number of patients who have responded, i.e. died in ccr of relapsed. censored = number of patients withdrawn, i.e. the number of pa tients in ccr at close date. fig.2 is a graphical illustration of the cumulative proportions of females and males surviving in ccr as shown in table 1 . by using grouping variables, in this case sex, and comparing the times to response for different values of the grouping varia bles, good information on prognostic factors such as sex, age and wbc is obtained. a further possibility is to make the analysis below for two or more grouping variables, e.g. duration of remis sion for different risk groups of female and male patients. 195 cumulative proportion s u r v i v i n g f= female m=male ~+....+....+....+....+....+....+....+....+....+....+....+....+....+....t....+....+....+....+....+....~. 1 . 0 + m . . . . . . ........ .90 + + . m ............. . . . . . . .so + . . . . . f ...... .70 + n . . . . . . . + . f ...... .ho + m...... f ....... f . . . ... n... ... f.. .... .50 + f . . . . . . m ....... f . ..... f ..... f . . . . . f . . . .. f ...... f . 4 0 + m . . . ... m . . . ... m . . . ... m ...... m.....m.....m.. ... m ...... m .30 + .2n + + . l o + + x . 30. 4 0 . 5 0 . h 0 . 70. fig.2 plot of the cumulative proportions of females (f) and males (m) surviving in ccr versus time in ccr in months. the phglm procedure ( 2 , 4 ) the cox proportional hazard linear model to one dependent vari able can determine the "best" variable to be added to a model in a model explaining time in ccr (tccr), i.e. the variation in tccr will be explained by a set of explanatory variables. but as these variables sometimes explain the same variation (are correlated with each other), the strength of the different variables explaining tccr will be obtained, provided that the other variables are in the model. table 3 is the computer print out taken from the last step in the phglm procedure, sas supplemental library user's guide, 1 9 8 0 ( 4 ) . in the print out beta is comparable with parameters in a mul tiple linear regression model. chi-square is a measure of the 196 strength of the variable and the p value is the level of signifi cance for the variable in the model. the d value gives a measure of the contribution of the variables explaining the variation in tccr. the solution gives an answer to the question which variables are the most important of those affecting duration in ccr and is also a measure of the strength of these variables. table 3. summary of the phglm procedure (computer print out). s i j r v i v a l i n a l l stepwise proportional hazards general l i n e a r model procedure 16: 10 tuesday i december 1 i 1'381 dependent v a r i a e l e : t i d i c c r s u r v i v a l t i m e event indicator: l i c v a r i a b l e beta std. error chi-sqijare p d l p e 0,00266249 0.00041516 41.13 0.0000 0.124 eon 0.310s4665 0.13139369 5.5'? 0.0181 0.019 alder 0.00365191 0.00169567 4 . 6 4 0.0:31:3 0.016 medt 0.17321388 0.08517534 4.14 0 . ~ 4 2 0 0.014 chi-sqijahe q s t b t i s t i c s adjusted only for v a r i a b l e s i n the model u a r i u b l e chi-sqijare p d lns 0 . 0 3 0.8714 0.000 no a d d i t i o n a l v a r i a e l e s met the 0.1000 s i g n i f i c a n c e l e v e l for entry. explanations to the table: lpk = wbc, kon = sex, alder = age, medt = mediastinal tumor. the variable cns is not included in the model because it does not contribute enough to the explanation. the higher the d value of a variable, the stronger the influence of this variable on the duration of ccr. comments the aim of this communication is to demonstrate in a practical way how we have used standard computer programs in the evaluation of the influence of different clinical parameters on the outcome of a malignant disease. the most important factor in this kind of analysis is the quali ty of the selected material. this must be as complete as possible and selection should be avoided. if there is selection, its conse 197 quences must be analysed separately. selection always implies a risk of irrelevant correlations, which can lead to wrong conclu sions concerning the material. in our case there is no known selec tion, as the material includes all known cases of all in children in sweden during the period in question. no child was lost at fol low-up, which gives important strength to the material. frequency tables and cross tables analyse the material with re gard to the distribution of different variables, e.g. age, sex, risk group, location of relapse, etc. the variables can be plotted against each other in a desired way. for instance the relation be tween duration of ccr and age or sex can easily be determined, but the tables are difficult to read and the results are not easy to evaluate. life table analyses ( 1 ) offer better possibilities than frequen cy tables and cross tables of studying variables affecting the du ration of ccr versus clinical parameters and different treatment programs. the life table method gives a graphical illustration of time in ccr against parameters such as age, wbc, treatmentprograms and so on. it also permits mutual comparisons of subgroups in the material, e.g. "standard risk patients" against "high risk pa tients" with regard to sex or age. these analyses will yield vada bles explicitly describing the duration of ccr. the problemis that in one individual patient, different parameters often interact with regard to the outcome of the disease. it may thus be difficult to estimate the effect of a single parameter. we have used a line ar regression analysis as described by cox ( 2 ) to solve this prob lem. this method implies a listing of the internal order of the variables with regard to their influence on the outcome of the di sease (table 3 ) . thus we have evaluated the strength of various "high risk cri teria" in childhood lymphoblastic leukemia. acknowledgements we wish to express our most sincere thanks to a.christofferson and a.kreuger for valuable help and discussions. financial support was obtained from the swedish cancer society and selander's foun dation. patient material was obtained from the members of the swe dish child leukemia group and from the 45 departments of pediat rics in sweden who are gratefully acknowledged. 198 1. bernedette, j. & yven, k.: life tables and survival functions. in: bio-medical-computer programs, p-series (ed. m.b.braum), pp.743-770. university of california press, los anqeles, 1977. 2 . cox, d.r.: regression models and life tables. j roy statist 3 . gustafsson, g., kreuqer, a. & dohlwitz, a.: acute lymphoblastic soc b 34:187-220, 1972. leukemia in swedish children 1973-78. acta paediatr scand 70:609-614, 1981. 4 . harell, f.: the phglm procedure. in: sas supplemental library user's guide, pp.119-131. sas institute inc., cary, north caro lina, usa, 1980. accepted december 15, 1981. address for reprints: goran gustafsson, m.d. department of pediatrics university hospital s-750 14 uppsala sweden 1 99 upsala j med sci 87: 151-161, 1982 thermodynamical aspects on the determination of bicarbonate in urine morgan sohtell and bertil karlmark depart men^ of physio/ogy and medical biophysics. uppsala university, uppsalu, sweden abstract despite t h e fact that the composition o f urine varies a l o t during the day, this has essentially been neglected as a f a c t o r o f importance in determinations o f urine bicarbonate. the investigations reviewed in combination w i t h an own study shows t h a t qualitative and quantitative factors in urine composition impacts the solubility o f carbon dioxide as w e l l as the dissociation constant f o r the bicarbonate b u f f e r system. these t w o "constants" are o f outstanding importance in the determination o f bicarbonate, using the t o t a l carbonic acid method as w e l l as the carbon dioxide equilibration method. nomograms are presented t o quantify the influence o f d i f f e r e n t urine compositions on the determinations o f bicarbonate in f i n a l urine and tubular fluid. introduction since many decades it has been a well-known f a c t t h a t the ligands used i n the chemical description o f biological acid-base equilibria are influenced by d i f f e r e n t physical and chemical properties of the solution under study. f o r many biological fluids this f a c t has been made, f o r example, f o r blood (27), f o r cerebrospinal f l u i d (24, 25), f o r amniotic f l u i d (17) and also for a r t i f i c i a l fluids ( 9 , 32). a common denominator f o r these biological fluids i s the r e l a t i v e l y r e s t r i c t e d variation in composition f r o m t i m e t o t i m e and also between d i f f e r e n t individuals. as a sharp contrast, f i n a l urine varies in composition considerably even f r o m hour t o hour in t h e same individual. the circadian ph-variation (15) i s a well-known f a c t as w e l l as the postprandial "alkalaine tide". b u t the wide range in e l e c t r o l y t e composition and osmolality, makes a considerable i m p a c t on the chemical analysis o f components in the bicarbonate b u f f e r system. 151 consider t w o f i n a l urine samples f r o m the same individual , the same day; one o f them 150 mosm/kg and t h e other 600 mosm/kg. provided the osmolality r e f l e c t s the n a c l content the bicarbonate a c t i v i t i e s can be calculated using the henderson-hasselbalch equation (see table 1). thus, despite the f a c t t h a t the p h and pco2 are identical i n the same samples, the bicarbonate a c t i v i t y measured m i g h t vary around 50% depending upon chemical factors other than the bicarbonate a c t i v i t y itself. 150 mosm/kg 600 mosm/kg pka 6.117 5.919 s 0.03145 0.02915 h c o j 6 9 table 1 fitzsimons and sendroy, ( 9 ) van slyke e t al., (32) table 1.the influence o f osmolarity on the bicarbonate a c t i v i t y in urine. the calculations are based on a pcop o f 40 m m h g (5.3 kpa) and a p h o f 6.8 f o r both urines. i n biological fluids other than urines, the bicarbonate variations as a function o f thermodynamical ligands is smaller and usually well-known (for a recent review see siggaard andersen, (27)). the a i m o f the present paper i s t o present ligands f o r the bicarbonate determination in urine as a function o f d i f f e r e n t compositions o f this fluid. a review o f the l i t e r a t u r e i n this f i e l d i s presented as w e l l as a study o f primary urine (ultrafiltrate). a n evaluation o f d i f f e r e n t methods f o r the bicarbonate determination i n urine i s made. met h 0 d s the bicarbonate concentration can be analyzed i n d i f f e r e n t ways among which the determination o f t o t a l carbonic acid, tcop is the most widely used. this technique cannot, however, d i f f e r between co2, h c o j and co;-, which i s a considerable drawback when dealing w i t h fluids o f unknown p h and co2 concentrations. this is most times t h e case i n compartment studies i n or adjacent t o single cells. pcop electrodes are now made f o r in vivo studies in micropuncture research (8,30), b u t w i l l probably w a i t a couple of years f o r a more wide-spread use. the bicarbonate a c t i v i t i e s in biological fluids are determined mainly by using the equilibration technique as thoroughly used and described f o r blood and other fluids 152 (27). this technique i s also suitable f o r samples i n nano-liter size as described by k a r l m a r k and sohtell (18). for carbonic anhydrase r i c h fluids (eg blood), chemical equilibrium i s assumed and the calculation o f bicarbonate a c t i v i t y is based upon single measurements of p h and pco2. if chemical non-equilibrium (disequilibrium) i s prevailing, the henderson-hasselbalch equation cannot be used. i n such a case an immediate responding and highly specific bicarbonate electrode i s the only method. such an electrode is described (19) but i s not yet developed enough t o p e r m i t reliable and valid measurements. as long as the non-bicarbonate b u f f e r concentration i s low as compared t o the bicarbonate a c t i v i t y the equilibration technique i s rather insensitive t o differences in actual p h and pco2 and i s preferred mainly because i t s s i m p l i c i t y and also the f a c t t h a t it results i n a measure of the a c t i v i t y and not barely by the concentration. the t o t a l co2 technique f o r bicarbonate determinations gives a more unspecific t o t a l concentration o f bicarbonate buffer constituents and gives a considerable error i n acid urines. irrespective o f which o f the mentioned techniques that is t o be used f o r bicarbonate determinations i n urine, the co2 concentration must be measured. this i s easily performed w i t h a pco2 electrode as long as the sample volume i s loop1 or more. for t h e calculation of the bicarbonate a c t i v i t y t h e solubility f a c t o r ( s ) and pka are then o f v i t a l importance. i n addition, knowledge o f the actual p h i s also v i t a l i n very alkaline urines (where the amount o f co$gives a substantial contribution ( 5 10 yo ) t o totallc02) and also i n highly buffered urine. so f a r experimental investigations o f the s and pka f o r urines are not published. we used here one method described by siesjo (24) t o study s and another by siesjo (25) t o study pka. p r i m a r y urine was a r t i f i c i a l l y made as an u l t r a f i l t r a t e of r a t plasma, f i l t e r e d through a d i a f l o membrane (pm 10, amicon corp., lexington, mass., usa). a)the solubility coefficient (s): the solution investigated was acidified w i t h hci t o a p h of about 2.5 and then equilibrated w i t h the humidified 5 yo co2 in oxygen gas m i x t u r e for 1 hour a t 38oc. the carbonic acid content of an equilibrated sample was made volatile by depositing it in a h c i solution i n a conway unit, which was modified as shown i n fig.1. the co2 formed was trapped in a ba(oh)2 solution. the sealing between the plugs and t h e l i d was made by the use o f acidified carboxymethylcellulose t o avoid leakage o f co2. the diffusion time was 90 m i n and the change o f the ba(oh)2 solution due t o the reaction w i t h co2 was immediately analyzed by t i t r a t i o n w i t h h c i standard. s was measured as a relation between the t o t a l carbonic acid content (tco2) i n acidified solution and the p a r t i a l pressure o f carbon dioxide. the validity o f the method was tested by determining the s f a c t o r f o r distilled water and a 160 m m o l f l n a c l solution. 153 h c1 \ hcl + sample fig.1. the circular conway unit (made of glass), h e r e seen from t h e side. the plugs a r e s e a l e d t o t h e lid with acidified carboxymethylcellulose. b) the f i r s t a p p a r e n t dissociation c o n s t a n t (pka): the solution was f i r s t equlilibrated in a humidified g a s of 5% c o p in oxygen f o r one hour a t 38oc. the ph a t this equilibration w a s measured with a glass e l e c t r o d e and t h e p c o 2 w a s measured as described above. this value was inserted in t h e henderson-hasselbalch equation. results and discussion a) the solubility coefficient: the r e s u l t s a r e presented a t t h e bottom of table 2. van slyke et a1 (32) presented d a t a , indicating t h a t not only t h e ionic s t r e n g t h was of i m p o r t a n c e f o r t h e c o 2 solubility. of considerable i m p o r t a n c e was also t h e influence of t h e d i f f e r e n t ionic species. the solubility of c o p is depressed by ions in proportion t o t h e i r c o n c e n t r a t i o n s (table 3 ) . the t a b l e shows t h e depression of t h e solubility of co2 f o r d i f f e r e n t ions e x t r a p o l a t e d t o a c o n c e n t r a t i o n of 1 mol/l. in lower c o n c e n t r a t i o n s t h e depression in solubility is proportionally reduced (for phosphate t h i s linearity is not s t r i c t , however). i t must be pointed o u t t h a t t h e t a b l e is not useable f o r c o n c e n t r a t i o n s above 300 mmol/l of individual ions. for biological pure s a l t solutions, siggaard andersen (27), summarized t h e d i f f e r e n t d a t a of t a b l e 3 in a formula, which describes an e s t i m a t i o n of t h e ionic influence but in t e r m s of a weighed ionic s t r e n g t h . but his approximation is not valid f o r urines, due t o t h e wide range of ionic composition during d i f f e r e n t physiological d i u r e t i c conditions. 154 table 2 s 0.03304 0.03222 0.03015 0.03215 0.03007 0.0 3 2 6 2 0.03229 0.03136 0.03105 0.03223 0.03013 0.0311 0.03074 0.03065 0.03233 oc solution 38 h 2 0 38 h20 38 plasma (human) 38 h20 38 plasma (ox) 37.5 h20 38 h20 * 37.5 160 m m o l / l n a c l 38 160 m m o l / l n a c l * 38 h20 38 sera (human) 37 amnion f l u i d (human) 38 u l t r a f i l t r a t e 38 160 m m o l / l n a c l 38 h20-" bohr,c., (4) van slyke e t al., (32) -ii bartels and wrbitzky, ( 2 ) 1 8 siesjo, (24) -11 -11 -11 austin e t al., (1) -11 johnell, (17) this study (se= 0.0018,n= 24) -'i(se= 0.00004,n=30) -11(se= 0.00009,n=47) table 2. summary o f data concerning the solubility o f cop in d i f f e r e n t kinds of solutions. in case the solubility was presented as the bunsen coefficient ( l i t e r gas dissolved / l i t e r / u n i t atmosphere pressure), we have normalized it i n t o the solubility coefficients (mmol/l/mm hg). those values presented w i t h a * are a temperature correction t o 380 c f r o m the preceding value (see text). table 3 h+ h c 2 0 4 l a c t a t e c1 k+ na+ h2poi hcoj* 0.0 0 0 0 0 0.00117 0.00296 0.00130 0.0 0 2 3 8 0.00382 0.00615 0.00130 table 3. the molar depression o f carbon dioxide solubility (van slyke e t al., (32). the * denotes t h a t the value is taken f r o m harned and davies (12) as the same as t h a t f o r chloride. it lacks d i r e c t experimental support, however. 155 n o r m a l l y f i n a l u r i n e d o e s n o t c o n t a i n s i g n i f i c a n t a m o u n t s of n e i t h e r p r o t e i n s nor lipids. d u r i n g p a t h o l o g i c a l c o n d i t i o n s , h o w e v e r , t h i s could b e t h e case, a n d would t h e n m a k e a s e r i o u s i m p a c t on b i c a r b o n a t e d e t e r m i n a t i o n s . t h e solubility of c o 2 i n c r e a s e s w i t h a h i g h e r lipid c o n c e n t r a t i o n b u t d e c r e a s e s w i t h a high p r o t e i n c o n c e n t r a t i o n . r e n a l e x p e r i m e n t a l t e c h n i q u e s n o w a d a y s p e r m i t s s a m p l i n g f r o m a f f e r e n t a n d e f f e r e n t arterioles as w e l l as f r o m t h e bowman's capsule. p r i m a r y u r i n e c o n t a i n s p r o t e i n which t h u s will d e c r e a s e t h e b i c a r b o n a t e c o n c e n t r a t i o n . o n t h e o t h e r h a n d o n e c a n e x p e c t a n i n c r e a s e d b i c a r b o n a t e c o n c e n t r a t i o n in p r i m a r y u r i n e as t h e c o n c e n t r a t i o n of t h e p l a s m a p r o t e i n s i n c r e a s e s during t h e u l t r a f i l t r a t i o n . this is d u e t o a n a u g m e n t a t i o n of t h e donnan effect as w e l l as of a r e d u c e d co2 s o l u b i l i t y in p l a s m a (siggaard a n d e r s e n , pp41,(27); s o h t e l l (29)). b) t h e f i r s t a p p a r e n t d i s s o c i a t i o n c o n s t a n t : t h e r e s u l t s f r o m r a t u l t r a f i l t r a t e are shown a t t h e b o t t o m of t a b l e 4. i t m u s t b e born in m i n d t h a t o l d e r l i t e r a t u r e in t h i s f i e l d d e s c r i b e s a c i d b a s e c h e m i s t r y w i t h a n o t h e r d e f i n i t i o n of ph, t h a n t h a t of t o d a y (3). f u r t h e r m o r e , t h e a n a l y t i c a l m e t h o d s a r e c o n s i d e r a b l y i m p r o v e d . t a b l e 4 pka 6.3222 6.105 6.330 6,092 6.089 6.3089 6.09 6.112 6.3002 6.086 6.316 6.103 6.13 6.127 6.328 6.120 6.101 o c 38 38 38 38 38 38 38 37 38 37.5 38 37.5 37.5 37.5 38 37 38 solution p = o s e r u m ( h u m a n ) h = o s e r u m ( h u m a n ) s e r u m (dog) p = o p = 160 m m o l / l s e r u m (ox+dog+human) p = o p = o s e r u m (dog+human) s e r u m ( h u m a n ) s e r u m (human) p = 160 m m o l / l p = o amnion fluid u l t r a f i l t r a t e h a s t i n g s and sendroy, (13) h a s t i n g s et a1.,(14) s t a d i e a n d h a w e s , (31) robinson et al., ( 2 2 ) -1' m a c i n n e s and b e l c h e r , (20) danielson e t al., (6) dill et al., (7) h a r n e d a n d d a v i e s , (12) s e v e r i n g h a u s et al., (23) f i t z s i m o n s and s e n d r o y , (9) g a m b i n o , (10) -if(11) siesjo, (25) s i g g a a r d a n d e r s e n , (26) j o h n e l l , (17) t h i s s t u d y (se= 0.002, n = 42) t a b l e 4. s u m m a r y of d a t a , d e s c r i b i n g t h e pka in d i f f e r e n t s o l u t i o n s a t body t e m p e r a t u r e . p= 0 mol/l m e a n s a n e x t r a p o l a t i o n of d a t a f r o m salt solutions. 156 as shown in t a b l e 3, t h e p k a is s t r o n g l y i n f l u e n c e d by t h e ionic s t r e n g t h . a s p e c t r u m of f o r m u l a s i n l i t e r a t u r e are d e s c r i b e d t o s u b s t a n t i a t e t h i s r e l a t i o n . many f o r m u l a s , b a s e d on t h e debye-huckel e q u a t i o n , are r e s t r i c t e d t o t o o d i l u t e d solutions, h o w e v e r , t o b e of i m p o r t a n c e in a n a l y s i s of biological fluids. fig. 2 v i s u a l i z e s t w o of t h e s e f o r m u l a s , d e s c r i b i n g t h e p k a as a f u n c t i o n of i o n i c s t r e n g t h ( o t h e r r e l a t i o n s are d e s c r i b e d by manov et al., (21), hagg, (16), s l a t o p o l s k y e t al. (28) a n d s i g g a a r d a n d e r s e n , (27). t h e f i g u r e a l s o i n d i c a t e s r e a s o n a b l e v a l u e s o f t h e ionic s t r e n g t h in t u b u l a r fluids. i t is c l e a r l y s e e n t h a t t h e w i d e r a n g e in i o n i c s t r e n g t h in t h e s e u r i n e s m a k e s a c o n s i d e r a b l e i m p a c t on t h e p k a a n d t h u s f i n a l l y on t h e b i c a r b o n a t e d e t e r m i n a t i o n . p k h 6.3 6.1 5.9 0.2 0.4 0.6 0.8 1.0 p (mi fig. 2. p k a as a f u n c t i o n of t h e i o n i c s t r e n g t h (p). i: pka' = 6.322 0.5 c ( h a s t i n g s a n d sendroy, (13) [i: pka' = 6.316 0.512 f i ( f i t z s i m o n s a n d s e n d r o y , (9)). r e a s o n a b l e m e a n i o n i c s t r e n g t h s f o r p r o x i m a l t u b u l e (pt), e a r l y d i s t a l t u b u l e (dt) and f i n a l u r i n e are i n d i c a t e d . t h e i n f l u e n c e of ph itself on t h e f i r s t a p p a r e n t d i s s o c i a t i o n c o n s t a n t i s s p a r s e l y s t u d i e d in biological solutions. in c e r e b r o s p i n a l fluid (siesjo, (25)) a n d h u m a n a m n i o t i c fluid ( j o h n e l l , (17)) no i n f l u e n c e of p h on p k a w a s found. in s e r u m , h o w e v e r , as w e l l as in 150 m m o l / l n a c l s o l u t i o n s i g g a r d a n d e r s e n (26) f o u n d a d e c r e a s e in p k a w i t h i n c r e a s i n g ph b u t only a b o v e ph 7.0 7.5. s i m i l a r d e p e n d e n c e f o r s e r u m w a s f o u n d by s e v e r i n g h a u s et al. (23). in a l k a l i n e s o l u t i o n s c o n t a i n i n g p r o t e i n s , t h e c a r b a m i n o c 0 2 c o n c e n t r a t i o n m i g h t i n f l u e n c e on t h e b i c a r b o n a t e a c t i v i t y as i s f u r t h e r discussed by s i e s j o (25). 157 c ) u r i n a r y bicarbonate: fig. 3 shows a hypothetical urine o f p h 6.8 and pco2 o f 40 mrn h g (5.3 kpa). reasonable s and pka values are furthermore inserted i n the henderson hasselbalch equation and the bicarbonate a c t i v i t y increases w i t h increasing s f o r a certain pka. the influence o f d i f f e r e n t temperatures on the bicarbonate value i s formulated by siggaard andersen (27) and shows that 1% increase, also increases the bicarbonate a c t i v i t y w i t h about 1.6 yo. thus an analysis o f the bicarbonate concentration i n a solution f r o m a 38oc animal which is analyzed a t room temperature results i n significantly too l o w a value (more than 25%). 8.0 7.0 6.0 5.0 4.0 3.0 610 615 620 6 2 5 6 30 6 35 . . , ~ , . i . 0.0300 0.0315 0.0330 s fig. 3 the bicarbonate a c t i v i t y as a function o f pka and s in the henderson-hasseibalch equation. the example is based on equilibrium condition w i t h a p h o f 6.8 and a pco2 of 40 m m hg. 158 6.0 6.5 7.0 7.5 8.0 ph c 0 2 e q u i l i b r a t i o n c u r v e s of n o n b i c a r b o n a t e b u f f e r s . t h e r e a c t i o n e v a l u a t e d i s t h e following: 2 h p o ~ + c 0 2 + h ~ o e h~po;+ h c o ~ 2t h e c h a n g e o f h p o 4 --+ h 2 p 0 4 is e x p r e s s e d as a w h i c h also will i l l u s t r a t e t h e e q u i m o l a r i n c r e a s e in hcoj. t h e c u r v e s are c a l c u l a t e d a c c o r d i n g t o s i g g a a r d a n d e r s e n (27). t h e s o l u t i o n s used in t h e c a l c u l a t i o n s are: p h o s p h a t e b u f f e r s w i t h e q u a l c o n c e n t r a t i o n s f o r hzp0; and h p o i of 2.5 m m o l / l ( l e f t ) a n d 20 mrnol/l ( r i g h t ) a t i o n i c s t r e n g t h s of 75 rnmol/l a n d 300 m m o l / l r e s p e c t i v e l y . in t h e f i g u r e t h e d a s h e d l i n e s i n d i c a t e i s o b i c a r b o n a t e lines. for c o n c l u s i o n s , see t e x t . t h e e q u i l i b r a t i o n t e c h n i q u e f o r t h e b i c a r b o n a t e d e t e r m i n a t i o n in s o l u t i o n s c o n t a i n i n g n o n b i c a r b o n a t e b u f f e r s yields e q u i l i b r a t i o n l i n e s which are c u r v e d as shown in fig. 4. i t is w e l l visualized in t h e f i g u r e t h a t t w o d i f f e r e n t p c o 2 v a l u e s are n o t e n o u g h f o r t h e c o n s t r u c t i o n of t h e e q u i l i b r a t i o n line. acknowledgements this r e s e a r c h w a s s u p p o r t e d by t h e m e d i c a l f a c u l t y of u p p s a l a u n i v e r s i t y a n d t h e swedish s o c i e t y f o r m e d i c a l r e s e a r c h . 159 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. references austin, w.h., lacombe,e., rand, p.w. and chatterjee,m.: solubility o f carbon dioxide i n serum f r o m 15 t o 38 c. j appl physiol $3, 301-304, 1963. bartels, h. and wrbitsky,r.: bestimmung des cozabsorptionskoefficienten zwischen 15o and 38o i n wasser und plasma. pflugers a r c h 271, 162 168, 1960. bates, r.g.: e l e c t r o m e t r i c p h determination. j.wiley & sons, inc., n.y. 1954. bohr,c.: d e f i n i t i o n und methode zum bestimmung der invasionsund evasionskoefficienten bei der auflosung von gasen in flussigkeiten. werte der genennten constanten sowie der absorptionscoefficienten der kohlensaure bei auflosung i n wasser und i n chlornatriumlosung. weid ann physik chem 68, 500 525, 1899. caflish, c.r. and carter, n.w.: a m i c r o pcoz electrode. anal biochem @, 252 257, 1974. danielson, 1 . 5 , chu,h.i. and hastings, a.b.: the pk'1 o f carbonic acid i n concentrated protein solutions and muscle. j b i o l chem 131, 243 257, 1939. dill, d.b., daly, s. and forbes, i.h.: the pk' o f serum and red cells. j b i o l chem 117, 569 579, 1937. dubose, t.d., pucacco, l.r., seldin, d.w., carter, n.w. and kokko. j.p.: d i r e c t determination o f pco2 in the r a t renal cortex. j c l i n invest 62, 338 348, 1978. fitzsimons, e.j. and sendroy,j.: d i s t r i b u t i o n o f electrolytes i n human blood. j b i o l chem m, 1595 1601, 1961. garnbino, s.r.: determination o f blood p c o ~ . c l i n chem 1, 336 345, 1961. garnbino, s.r.: determination o f blood p c o ~ . c l i n chem e, 199 200, 1962. harned, h.s. and davis, r.: the ionization constant of carbonic acid in water and the solubility o f carbon dioxide in water and aqueous salt solutions f r o m 00 50°. j a m chem soc g, 2030 2031, 1943. hastings, a.b. and sendroy, j.: the e f f e c t o f variation in ionic strength on apparent f i r s t dissociation constant o f carbonic acid. j b i o l chem 65, 445, 1925. hastings, a.b, sendroy, j. and van slyke, d.d.: studies of gas and electrolyte equilibria i n blood. x11. j b i o l chem 2, 183 192, 1928. hill, a.g.: in: acid-base balance. waverly press, inc., baltimore, 1973. hagg, g.: kemisk reaktionslara. 7th ed, almqvist & wiksell, uppsala, 1964. johnell, h.e.: the f i r s t dissociation constant f o r cabonic acid and the solubility o f carbon dioxide in human amniotic fluid. scand j c l i n lab invest 27, 223 238, 1971. karlmark, b. and sohtell, m.: the determination o f bicarbonate in nanoliter samples. anal biochem 3, 1-11, 1973. khuri, r.n., agulian, s.k., bogharian,k., nassar, r. and wise, w.: intracellular bicarbonate i n single cells o f necturus kidney proximal tubules. pflugers arch 349, 295 299, 1974. 160 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. maclnnes, d.a. and belcher, d.: the thermodynamic ionization constant of carbonic acid a t 38o f r o m electromotive force measurements. j a m chem soc 37:2, 1683 -1685, 1935. manov, c.g., bates, r.g., hamer , w.j. and acree, s.f.: values of the constants i n the debye-huckel equation f o r a c t i v i t y coefficients. j a m chem soc 65, 1765, 1943. robinson, h.w., price, j.w. and cullen, g.e.: studies of the acid-base condition of blood. 111. j b i o l chem 106, 7 27, 1934. severinghaus, j.w., stupfel, m. and bradley, a.f.: variations of serum carbonic acid p k w i t h p h and temperature. 3 appl physiol 2, 197 200, 1956. siesjo, b.k.: the solubility o f carbon dioxide in the cerebral c o r t i c a l tissue o f cats. w i t h a note on the solubility o f carbon dioxide in water, 0.16 m n a c l and cerebrospinal fluid. a c t a physiol scand 2, 325 341, 1962 a. siesjo, b.k.: the bicarbonate/carbonic acid buffer system of cerebral cortex o f cats, as studied i n tissue homogenates. 11. the p k l ' o f carbonic acid a t 37.50 c, and the r e l a t i o n between carbon dioxide and ph. a c t a neurol scand e , 1 2 1 -141, 1962 b. siggaard andersen, 0.: the f i r s t dissociation exponent of carbonic acid as a function o f ph. scand j c l i n l a b invest 14, 587 597, 1962. siggaard andersen, 0.: the acid-base status of the blood. 4 t h ed., munksgaard, copenhagen, 1976. slatopolsky, e., hoffsten, r., purkerson, m. and bricker,n.s.: on the influence o f extracellular f l u i d volume expansion and o f uremia on bicarbonate reabsorption in man. j c l i n invest 9, 988, 1970. sohtell, m.: pco2 o f the proximal tubular f l u i d and the e f f e r e n t arteriolar blood i n the r a t kidney. a c t a physiol scand 105, 137 145, 1979. sohtel1,m. and karlmark, b.: i n vivo micropuncture pco2 measurements. pflugers a r c h 363, 179 180,1976. stadie, w.c. and hawes, e.r.: studies on the oxygen-, acidand base-combining properties of blood. iv. j b i o l chem 77, 265 301, 1928. van slyke, d.d:, sendroy, j., hastings , a.b. and neil, j.m.: studies o f gas and electrolyte equilibria i n blood. the s o h b i l i t y o f carbon dioxide a t 380 c i n water, salt solution, serum and blood cells. j b i o l chem e, 765 799, 1928. accepted november 6, 1981 address f o r reprints: morgan sohtell, ph.d., dept o f physiology and medical biophysics, biomedical center, box 572, 5-751 23 uppsala, sweden i 1-822851 161 upsala j med sci 83: 129-134, 1978 separation of rat-liver phosphoprotein phosphatases active on phosphorylated pyruvate kinase (type l) vincent p. k . titanji from the institute of medical and physiological chemistry, biomedical centre, university of uppsula, uppsala, sweden abstract the substrate specificity of rat liver phosphoprotein phos phatases has been investigated. the enzymes were resolved into three fractions, termed a , b and c, on elution from deae-cellulose with apparent molecular weights, as deter mined by sephadex g-200 chromatography, of approxi mately 250 000, 250000 and 140 000, respectively. all frac tions catalyzed the dephosphorylation of calf-thymus phos phohistones, salmon phosphoprotamine and rabbit skeletal muscle phosphorylase a. the major portion of the activity towards these substrates was found in fraction b. the activity towards rat liver phosphopyruvate kinase (type l) resided almost exclusively in fractions b and c. it is con cluded that rat liver contains multiple forms of phospho protein phosphatases and that phosphatases of fraction b and c are the major activities towards phosphopyruvate kinase. introduction the mechanism of hormonal regulation of carbo hydrate metabolism in the liver has been studied extensively at the level of pyruvate kinase (type l) (for recent review see ref. 1). experiments from this (1-3) and other laboratories (4-6) have estab lished that pyruvate kinase (type l) is inhibited as a result of cyclic-amp dependent phosphoryla tion. whereas the cyclic-amp dependent protein kinases which catalyse the phosphorylation of pyru vate kinase (type l) and other proteins have been investigated in great detail (for recent review and further references see 6), the phosphoprotein phos phatases which reverse the phosphorylation are still under intensive research (see 7 for further ref erences). after the discovery that a histone phosphatase preparation counterbalances the cyclic-amp de pendent phosphorylation of pyruvate kinase (type l) (8), attempts were made to purify the enzyme(s) and although an extensively purified preparation was obtained, severe losses in enzymatic activity were noted at the initial stages of the procedure (9). since this inactivation was incompatible with the known stability of the final phosphatase prep aration, it was postulated that labile enzyme forms were removed by the procedure. the aims of this investigation were to separate the phosphoprotein phosphatases of rat liver cyto sol and to compare the specificity displayed to wards phosphopyruvate kinase with that observed towards selected phosphoproteins from other organs. experimental materials. recrystallized bovine serum albumin and phe nylmethylsulfonyl fluoride were bought from sigma. sephadex g-200 was from pharmacia fine chemicals, uppsala. “buffer a” containing 10 mm imidazoi/hcl, ph 7.5, 15 mm mercaptoethanol and 2.5 mm mgci, was used as indicated. (32p)phosphoproteins. rat-liver pyruvate kinase was isolated and phosphorylated a s described earlier (8). sedimentation equilibrium experiments with pyruvate kinase were performed by the meniscus-depletion tech nique of yphantis (10, 1 i ) . a beckman model e analytical ultracentrifuge equipped with an rtic temperature control unit and an electronic control was used. all measurements were conducted in standard 12 mm double sector cells with sapphire windows. the centrifuge was run at 20°c for 18 h at 10000 rpm. pyruvate kinase was dialysed against 20 mm potassium phosphate, ph 7.0, containing 0.1 mm fructose 1,6-diphosphate and 0.1 mm dithiothreitol. assuming a partial specific volume, v , of 0.72 the molecular weight of pyruvate kinase was cal culated to be 250000. assuming e;:g=0.68 at 280 nm the maximal degree of phosphorylation was found to lie between 3.2 and 3.6 mol (32p)phosphate per mol pyru vate kinase tetrarner. analysis of cold phosphate on the unlabelled enzyme was not performed, although the dif ferent maximal levels of phosphorylation obtained with different enzyme preparations suggests the presence of 9-782853 130 v . p . k . titanji some phosphate on the unlabelled enzyme. prior to use, phosphopyruvate kinase was chromatographed on a sephadex (3-50 column equilibrated and eluted with 5 mm imidazol-hc1, ph 7.5, containing 10% glycerol, 50 mm kci, 0.1 mm fructose-l,6-diphosphate and 0.1 mm dithio threitol. (3‘)phosphohistones (sigma type iia) and (32p)phosphoprotamine were prepared and the alkali-labile phosphate content determined as described previously (9, 12). (32p)phosphorylase a was prepared using 5-10 mg of rabbit skeletal muscle phosphorylase b (boehringer) and 0.3 mg phosphorylase b kinase (sigma) essentially ac cording to the method of fischer & krebs (13). before use, (32p)phosphorylase a , which contained about 4 nmol phosphate per mg protein, was dialyzed against 5 mm imidazoi/hcl, ph 7.5, containing 1.0 mm dithiothreitol. enzyme assays and other methods. phosphoprotein phosphatase activity was assayed according to a previous method (9). the final reaction mixture contained 50 mm trislhc1, ph 7.5, i mm dithiothreitol, 0.1 mg per rnl bovine serum albumin, 2 . 5 mm mnci2 and the respective phosphoproteins: 5 p m phosphopyruvate kinase, 7 p m (32p)phosphorylase a , 20 p m (3’p)phosphohistones or 60 p m (32p)phosphoprotamine. the reaction mixture for the dephosphorylation of pyruvate kinase contained, in addi tion, 5 % glycerol and 0.05 mm fructose-1,6-diphosphate to stabilize the enzyme. the volumes of the reaction mixtures were 40 p1 for the dephosphorylation of (3’p) phosphopyruvate kinase and (3’p)phosphorylase a and 100 p1 for the other substrates. the reaction was started by addition of the protein phosphatase and allowed to continue at 30°c for 5-10 min. the release of (32p) orthophosphate was linear for at least 15 min at the en zyme concentrations used. one unit of protein phospha tase is defined as the amount of enzyme which catalyses the release of i nmol of orthophosphate per min under these conditions. the substrate is expressed as the con centration of the (3’p)phosphate moiety in the respective phosphoproteins. the specific radioactivity of the phos phoproteins ranged from 20-100 cpm.pmol-’. cyclic amp-dependent protein kinase activity was determined in “cell-sap” prepared as described earlier. one unit of pro tein kinase activity is defined as the amount of enzyme necessary to catalyze the incorporation of i pmol (”p)) phosphate into calf-thymus histones under the specified conditions (8). protein in crude fractions was assayed according to the method of lowry et al. (14) and in purified fractions from the absorbance at 280 nm assum ing e:, ;;= i . separation of protein phosphatases on deae-cellulose. freshly excised livers (60 g) from male sprague dawley rats weighing 30g-350 g were homogenized in 3 vol of 250 mm sucrose, containing 15 rnm p-mercapteoethanol, 1 mm edta (ph 7.0, naoh) and 0.1 mm phenyl methylsulfonyl fluoride. a potter-elvehjem glass homogenizer fitted with a teflon pestle was used and supernatant was applied to a deae-cellulose column (whatman de-52) (3.2x20 cm) equilibrated and eluted with buffer a containing 40 mm naci. the column was washed at a flow-rate of 2 0 0 ml/h for 5-6 h and the enzymes were eluted at a flow rate of 60 ml/h using a (500+500 ml) linear gradient of 40-350 mm nacl in buffer a. fractions of 10 ml were collected and assayed for pro tein phosphatase activity (fig. i ) sephadex g-200 chromatography. t o each of t h e pooled fractions in fig. 1 (about 50 mg protein) solid ammonium sulphate to 70% saturation was added with constant stirring. after 15 min each sample was centri fuged at 16000xg for 20 min and the precipitate dissolved in a minimal volume of buffer a and dialyzed against the same buffer containing 20% sucrose and 40 mm naci. the enzyme (about 2 ml) was then chromato graphed on a sephadex (3-200 column (2.4x57 cm) equili brated and eluted with buffer a containing 0.1 mm nacl at a flow rate of 10-12 ml/h; fractions of 2 . 6 2 . 7 ml were collected and assayed for phosphoprotein phosphatase activity. in order to estimate apparent molecular weights of the phosphatase fractions, the column was calibrated with catalase (240000), aldolase (158 ow), bovine serum albumin (67 000) and hen’s egg albumin (45 000) (results not illustrated). after the chromatography on sephadex (3-200, the enzymes were concentrated by ammonium sulphate pre cipitation (fractions a and b) or by ultrafiltration in a collodion bag (fraction c), and dialyzed at 1-2°c against buffer a containing 20% sucrose and 40 mm naci. the fractions were stored frozen in 0.1 ml portions and thawed only once prior to use. little loss of activity occurred after 1 month. ethanol precipitation at 20°c of phos phatase fractions was performed according to the method of brandt et al. (15), except that 2 4 mg protein per ml was added to 5 ml 95% ethanol. the precipitate was collected at 10000xg for 5 niin at 4”c, extracted with buffer a and dialysed against 1 i of the same buffer for 8-12 h prior to assay. results and discussion separation of rat-liver phosphoprotein phospharuses active on phosphorylated pyruvate kinase (type l ) experiments were carried out in order to compare the elution profiles of phosphoprotein phosphatases active towards (32p)phosphopyruvate kinase with those active towards other phosphoproteins. three fractions, termed a, b and c, were distinguished in order of elution from deae-cellulose (fig. 1 ) . fractions a , b and c were eluted between 0.1 0.15, 0.17-0.25 and 0.27-0.3 m nacl respectively. homogenization was performed in 30 sec with six strokes 4°c. the homogenate was centrifuged at 16000xg for 20 min and the supernatant obtained was further spun at 46000xg for 2 h. the postmicrosomal supernatant was filtered through glass wool to remove floating fat. the f l / l \ ~ i / n j m e d .s< i 83 the total recovery of (32p)phosphoprotamine phos activity (table i). almost all the activity on ( 3 2 p ) ~ h o s ~ h o r y l a t e d pyruvate kinase was recovered in fractions b and at 940 rpm. this and other steps were performed at 0phatase was usually about 5@70% of the initial , liver phosphopyruvute kinase phosphatases 13 1 n a b fraction number e \ 2 2 0 > t ? + 9 1 5 w v) 3 i $ 10 a w z i $ 05 a a m 0 a % 20 lo 60 80 f r a c t i o n number figs. i. separation of phosphoprotein phosphatase on deae-cellulose. the two panels to the same experiment. details are given in the text and in the legend to table i . the horizontal bars indicate the fractions that were used for further experiments. c (fig. 1). fraction a showed only trace activity on this substrate. (32p)phosphoprotamine, ("'p) phosphohistones and (32p)phosphorylase a of rabbit skeletal muscle were the better substrates for phos phatase fraction a. the relative ratio of (32p)phosphoprotamine/ (32p)phosphopyruvate kinase phosphatase activities was found t o be constant in different preparations of the postmicrosomal supernatant, but different for the separated fractions a , b and c (fig. 1 , table i). fraction c showed the lowest ratio of phospho protaminelphosphopyruvate kinase phosphatase activity, indicating increased preference for the latter substrate. in order to further compare the phosphatases, their physical properties were examined by chro matography on a calibrated sephadex (3-200 column. phosphatase fractions a and b had a similar apparent molecular weight, approximately 250000 (based on the mean of two determinations for each fraction). fraction c had an apparent molecular weight of 140000. analysis of samples from the different regions of the sephadex g-200 chromatograms revealed no further resolution of phosphoprotamine phosphatase from phosphopyru vate kinase phosphatase activity (results not il lustrated). the apparent molecular weights of the fractions are higher than those reported by kobayashi et al. (see footnote in ref. 16, p. 353). the reason for this difference is not known. the conditions used for the preparation and chromatography of the ex tracts in the present studies are different from those used by kobayashi et al. i t has been suggested that multiple forms of liver phosphoprotein phos phatases might be produced by the action of pro teases, released from lysosomes during vigourous homogenisation of the tissue (17). to minimize the effects of proteolysis, homogenisation was per formed under mild conditions and in the presence of phenylmethylsofonyl fluoride. although com plete inhibition of proteolysis could not be guaran teed, its effects were considered to be small, be cause fractions with apparent molecular weights lower than reported above could not be detected even after storage of the cytosol fraction for 6 h before further purification. this observation, taken together with the differences in kinetic prop erties elaborated below, supports the possibility that fraction a , b and c probably serve different functions. stability of the phosphatases under the conditions chosen for chromatography and upon storage at 2°c phosphoprotein phospha tase, fraction b seemed to be the most stable, 132 v . p . k . titanji table i. purification of phospkoprotein phosphatases f r o m rat-liver cell-sap phosphoprotamine and phosphopyruvate kinase phosphatase activities varied widely between 0.12-0.4 and 0.01 0.03 pmol (3zp)phosphate released per g wet weight of liver and min respectively (based on recoveries from cell-sap). the recovery of protein in the same fractions was about 100 mg per g of liver. in this particular experiment 14400 units of phosphoprotamine phosphatase and 1 200 units of phosphopyruvate kinase phosphatase were re covered in the cell-sap from 60 g of liver. the calculation of the recovery of enzyme activity after the sephadex column is based on the values obtained after chromatography of a portion of the activity from the deae-cellulose step. the slight activation of phosphatase b was not always obtained. the activity ratio is defined as the rate of dephosphorylation of (3zp)phosphoprotamine (60 pm) divided by the rate of the dephosphorylation of (32p)phospho pyruvate kinase (5 pm) phosphopyruvate kinase phosphatase phosphophosphatase activity ratioa protarnine yield u/mg p p r o t a m in e step (ulmg protein) (%) protein % yield 32p-pyruvate kinase cell-sap 3.0 100 0.3 100 11.6k0.6 deae-cellulose a 11.9 17.0 b 0.8 b b 25.2 44.6 0.9 19.7 16.1k2.6 c 7.4 7.1 1.7 19.2 4 . 3 5 1 . 5 a 43.5 17.8 b b sephadex g-200 b b c 114.0 59.6 3.5 22.7 31.2 13.0 2.8 2.2 7.5 5.9 activity ratio: mean+s.d. of four preparations are given for cell-sap and deae-cellulose fractions; mean values in most cases the rates of the dephosphorylation of (32p)phosphopyruvate kinase were too low for reliable estimates of two preparations are given for the sephadex g-200 fractions. to be made. followed by fraction a. phosphatase fraction c was the least stable; enzymatic activity decreased dur ing freezing and thawing in the absence of sucrose. under similar conditions, phosphatases a and b maintained full enzymatic activity. as can be seen in table i , only about 3 0 % of the phosphatase c activity applied to the sephadex (3-200 column was recovered, whereas the other two fractions were almost quantitatively recovered. earlier, a phosphoprotein phosphatase (m. w. 32 000) active towards phosphorylated pyruvate kinase was purified through an unusual ethanol precipitation step at 20°c as described by brandt et al. (9, 15). the stability of the phosphatase frac tions a , b and c , was also investigated using the ethanol treatment. we found that the enzymatic activity of fractions a and b towards (32p)phospho rylase a were increased by 50-100% after ethanol treatment (15). however, the activity of the same fraction a towards (32p)phosphoprotamine and (32p)phosphopyruvate kinase decreased to about 3 0 % of the initial values. with phosphatase frac tion b, the decrease was to about 70% of the initial activity. regardless of the substrate used, ethanol treatment either completely inactivated phos phatase c o r reduced the activity to less than 30 upsulu j m e d sci 83 50% of the initial values. whereas the precipitation of crude extracts with ethanol at 20°c facilitates the further purification of a phosphoprotein phos phatase, the same treatment reduces the activity of the deae-cellulose fractions towards all the other substrates studied except phosphorylase a. these observations might serve to explain the losses in enzymatic activity observed during our earlier attempts (9) t o purify a phosphoprotamine plus phosphopyruvate kinase phosphatase through the unusual ethanol precipitation step of brandt et al. (15). the available data do not permit us to identify the fraction yielding the low molecular weight phosphoprotein phosphatase which was purified earlier in this laboratory (9). in order to resolve this question, further purification of the enzymes would be required. the different stability properties sup port the existence of different phosphoprotein phosphatases, perhaps with overlapping specifici ties. kinetic results in view of the differences in molecular size and stability properties described above, some kinetic experiments were made to further compare the frac liver phosphopyruvate kinase phosphatases 133 table 11. effectors of phosphoprotein phosphata ses b and c the standard reaction medium containing 5 p m (32p) phosphopyruvate kinase was used. divalent cations were not included in the tests, except where specifically in dicated. phosphatase b (6.78 pg per test) and phosphatase c (4.22 p g per test) were from the deae-cellulose step. the meanfs.d. of data from four determinations are shown. other conditions were described in the experi mental section pmol releasedb min phosphos effector phatase b phatase c 1. no additions 2. 2.5 mm edta 3. 2.5 mm mgciz 4. 2.5 mm mnc1, 5 . 5 mm potassium 6. 25 mm naf 7. 1 mm atp 8. 1 mm atp+2.5 mm phosphate ph 7.5 mgcl, 5.6f0.2 2.2k0.1 48.1 f 1.1 28.15-0.08 3.02 f 0.4 2.25f0.3 2.42f1.5 46.5?2.5 29.1k 1.3 20.9k 1.9 55.5fo 46.0f2.8 13.2k0.9 10.0f0.6 8.7f0.3 46.8+0 tions b and c which showed the highest recovery of enzymatic activity towards pyruvate kinase. mn2+ and mg2+ stabilized or stimulated the activity of both fractions. the extent of stimulation ob served was greater with phosphatase b than with phosphatase c. orthophosphate, atp and n a f were inhibitory (table 11). partial inhibition of en zymatic activity by edta indicates that divalent cations are not absolutely required. the amount of phosphoprotein phosphatase ac tivity towards phosphopyruvate kinase found in the post-microsomal supernatant (table 1) was esti mated to be about 30 units per g wet weight of liver. this would be sufficient to completely de phosphorylate in about 10 secs all the pyruvate kinase present in normally fed rats (about 1 pm subunit). in other experiments, the total recovery of cyclic amp-dependent protein kinase in a post microsomal fraction was found to be 30000 units per g wet weight of liver. since histones were phos phorylated at about 50% of the rate of pyruvate kinase (ref. 8, fig. 2 ) , it was estimated that com plete phosphorylation of pyruvate kinase in the livers of normally fed rats would take about 1 sec. these findings, together with the identification of fractions b and c as the main phosphopyruvate kinase phosphatase in vitro give further support for the physiological relevance of the reversible dephosphorylation. acknowledgements this investigation was supported by grants from the swedish medical research council 13x-4485 and the medical faculty, the university of uppsala. ultracentri fugation analyses were kindly performed by dr h. per toft at this institute. references 1. 2. 3. 4. 5 . 6. 7. 8. 9. 10. 11. 12. 13. engstrom, l.: regulation of liver pyruvate kinase by phosphorylation-dephosphorylation reactions. in regulatory mechanisms of carbohydrate metabo lism (ed. v . esmann), p. 53. pergamon press, new york, 1978. ljungstrom, o., hjelmqvist, g . & engstrom, l.: phosphorylation of purified rat liver pyruvate kinase by cyclic-amp stimulated protein kinase. biochim biophys acta358: 289, 1974. engstrom, l., berglund, l., bergstrom, g., hjelm qvist, g . & ljungstrom, 0.: regulatory phosphoryla tion of purified pig liver pyruvate kinase. in lipmann symposium: energy, biosynthesis and regulation in molecular biology, p. 192. walter de gruyter, berlin, 1974. eigenbrodt, e., abdel-fattah mostafa, m. & scho ner, w . : inactivation of pyruvate kinase type m, from chicken liver by phosphorylation catalyzed by a camp-independent protein kinase. hoppe-seyler’s z physiol chem358: 1047, 1977. felicu, j. e., hue, l. & hers, h.-g.: hormonal control of pyruvate kinase activity and of gluconeo genesis in isolated hepatocytes. proc natl acad sci (usa) 73: 2762, 1976. nimmo, h. g. & cohen, p.: hormonal control of pro tein phosphorylation. in advances in cyclic nucleo tide research, vol. 8 (ed. p. greengard & g. a. robi son), p. 145. raven press, new york, 1977. titanji, v. p. k.: studies on liver phosphoprotein phosphatases. acta universitatis upsaliensis 284: 8-17, 1977. titanji, v. p. k., zetterqvist, 0. & engstrom, l.: regulation in vitro of rat liver pyruvate kinase by phosphorylation-dephosphorylation reactions cata lyzed by cyclic-amp dependent protein kinases and a histone phosphatase. biochim biophys acta 422: 98, 1976. titanji, v . p. k.: purification and properties of a phosphoprotein phosphatase from rat liver. biochim biophys acta481: 140, 1977. yphantis, d. a,: equilibrium ultracentrifugations of dilute solutions. biochem3: 297, 1964. chervenka, c. h.: long-column meniscus depletion sedimentation equilibrium technique for the analytical ultracentrifuge. anal biochem 34: 24, 1970. meisler, m. h . & langan, t. a,: characterization of a phosphatase specific for phosporylated histones and protamine. j . biol chem244:4%1, 1969. krebs, e. g. & fischer, e. h.: phosphorylase b kinase from rabbit skeletal muscle. in methods in enzymology, vol. 5 (ed. s . p. colowick & n. 0. kaplan), p. 373. academic press, new york, 1962. upsala j med sci 83 134 v . p. k . titanji 14. lowry, 0. h., rosebrough, n . j., farr, a. l. & randall, r. j . : protein measurement with the fohn phenol reagent. j biol chem 193: 265, 1951. 15. brandt, h., killilea, s. d. & lee, e. y . c.: activa tion of phosphorylase phosphatase by a novel proce dure. biochern biophys res comrn 6 1 : 598, 1974. 16. kobayashi, m., kato, k. & sato, s . : multiple molec ular forms of phosphoprotein phosphatase. biochim biophys acta377: 343, 1975. 17. lee, e. y . c., brandt, h., capulong, 2. l. & killi lea, s . d.: properties and regulation of liver phospho rylase phosphatase. enzyme regull4: 467, 1976. received m a y 30, 1978 address for reprints: vincent p. k. titanji department of medical and physiological chemistry biomedical center box 575 university of uppsala s-75123, uppsala sweden upsula j med sci 83 upsala j med sci 90: 119-125, 1985 ureteral reflux and ileal conduit pressure following diversion with a reflux-preventing technique kerstin claesson, lars frodin and lars-erik lorelius department of urology and radiology, universiv hospital, uppsala, sweden abstract i l e a l c o n d u i t u r i n a r y d i v e r s i o n was p e r f o r m e d w i t h an a n t i r e f l u x t e c h n i q u e , w i t h n i p p l i n q o f t h e u r e t e r s i n t o t h e segment, i n 63 p a t i e n t s . the p a t i e n t s were t h e n f o l l o w e d up f o r 52 2 25 months c o n c e r n i n g u r o g r a p h i c f i n d i n g s , i n f e c t i o n s and k i d n e y f u n c t i o n . u r e t e r o i l e a l s t e n o s i s d e v e l o p e d i n 3 o f 122 u r e t e r s and was s u r g i c a l l y c o r r e c t e d . r o e n t g e n o l o g i c e x a m i n a t i o n f o r u r e t e r a l r e f l u x was p e r f o r m e d a b o u t a y e a r p o s t o p e r a t i v e l y , and p r e s s u r e measurements were made i n t h e i l e a l segment. r e f l u x o f c o n t r a s t mediumwasseen i n 48 u r e t e r s a t p r e s s u r e 5 1 2 30 mm hg. when no r e f l u x was seen, t h e maximum i n f u s i o n p r e s s u r e was 62 2 34 mm hg. the b a s a l p r e s s u r e ( p r e c e d i n g c o n t r a s t i n f u s i o n ) was 24 2 29 mn, hg. r e g u l a r c o n t r a c t i o n waves w i t h p r e s s u r e r i s e i n t h e i l e a l segment were re g i s t e r e d , w i t h d u r a t i o n 10-30 seconds. the s t u d y showed no c o n n e c t i o n between u r e t e r a l r e f l u x and p r e s s u r e i n t h e i l e a l segment. c o m p l i c a t i o n s a s s o c i a t e d w i t h t h e a n t i r e f l u x o p e r a t i n g t e c h n i q u e were few. introduction i n l o n g t e r m f o l l o w u p a f t e r i l e a l c o n d u i t u r i n a r y d i v e r s i o n , h i g h i n c i d ence o f c o m p l i c a t i o n s has been found, i n c l u d i n g c h r o n i c p y e l o n e p h r i t i s , s t o n e f o r m a t i o n and d e t e r i o r a t i o n o f k i d n e y f u n c t i o n (5,8,10). these c o m p l i c a t i o n s have been t h o u g h t t o have some c o n n e c t i o n w i t h u r e t e r a l r e f l u x ( 4 ) . v a r i o u s r e f l u x p r e v e n t i n g p r o c e d u r e s have been used a t o p e r a t i o n (1). h i g h i n c i d e n c e o f u r e t e r o i l e a l s t e n o s i s has been a t t r i b u t e d t o r e f l u x p r e v e n t i n g t e c h n i q u e s f o r u r e t e r o i l e a l a n a s t o m o s i s ( 2 ) . a s u r v e y o f s i x series w i t h a n t i r e f l u x anastomosis showed s t e n o s i s i n c i d e n c e r a n g i n g from 3 t o 11 % ( 9 ) . the n i p p l e d u r e t e r o i l e a l anastomosing t e c h n i q u e d e s c r i b e d by p a t i l e t a l . ( 9 ) has been used a s a n t i r e f l u x p r o c e d u r e a t o u r c l i n i c . u r e t e r a l r e f l u x was e a r l i e r s t u d i e d t o g e t h e r w i t h r e c o r d i n g o f c o n d u i t p r e s s u r e , u s i n g s i m u l t a n e o u s l o o p o g r a p h y ( 2 ) . the aim o f t h e p r e s e n t s t u d y was 119 to register the ileal conduit pressure continuously during contrast infusion, for documentation of reflux to the ureters, and to compare the findings with the clinical features. material and methods patients sixty-three patients (45 male, 18 female, mean age 56.9 2 12.6 years) with ileal conduit were observed for 52 225 months postoperatively. the indication for urinary deviation was malignancy in 51 cases and benign, predominantly neurogenic disease in 12 cases. preoperative radiotherapy was given the 51 patients with walignant disease. three patients underwent salvage cyst ectomy after irradiation (60 gy). postoperative irradiation was given in one case. methods to 42 o f operation. the antireflux technique with nippling o f the ureters into ileal conduit (9) was used in all cases. the technique is illustrated in the fig.1. fig. 1. the nippling ureteroileal anastomosis f o r prevention of reflux intravenous uroqraphy. this exadination was performed preoperatively and 3,6 and 12 months postoperatively, and then every 1 2 t h month. infection control. clinical analysis and culture of urine from the ileal segment , collected with a single-lumen catheter, were performed routinely 3, 6 and 12 months after diversion and then at least once yearly. 120 kidney f u n c t i o n . s t u d i e s were made a t t h e above-mentioned i n t e r v a l s by measuring serum c r e a t i n i n e and cr-edta clearance. pressure s t u d i e s . retrograde c o n t r a s t i n f u s i o n o f t h e i l i a c bladder was p e r formed about one year p o s t o p e r a t i v e l y , u s i n g a m o d i f i e d foley c a t h e t e r no 1 2 ( f i g . 2), f u n c t i o n i n g as a double-lumen c a t h e t e r . p r e s s u r e registration infusion f i g . 2. f o l e y c a t h e t e r w i t h t h e b a l l o o n c u t and a s i d e h o l e made i n t h e c a n a l f o r p r e s s u r e r e g i s t r a t i o n under f l u o r o s c o p i c c o n t r o l t h e c a t h e t e r t i p was guided as c l o s e t o t h e u r e t e r a l o r i f i c e s as p o s s i b l e . through one o f t h e channels t h e i n t r a l u m i n a l p r e s s u r e was c o n t i n u o u s l y r e g i s t e r e d ( t r a n s d u c e r statham p 23 a c ) w i t h a l i n e a r w r i t e r (mingograph 800, elema-schonander, stockholm, sweden). b e f o r e i n f u s i o n o f c o n t r a s t medium t h e b a s a l l u m i n a l p r e s s u r e was r e g i s t e r e d f o r 2 minutes. t h e r e a f t e r t h e c o n t r a s t medium (isopaque 30 %, nyegaard, norway) was a d m i n i s t ered as a d r i p i n f u s i o n from a b o t t l e p o s i t i o n e d 100 cm above t h e stoma. the i n f u s i o n v e l o c i t y was 10 ml/min f o r 3 minutes, 20 ml/min f o r 5 minutes and f r e e f l o w o f c o n t r a s t u n t i l 250 m l had been i n f u s e d , w i t h f r e e o u t f l o w from t h e stoma. the f i l m s were exposed over t h e i l i a c segment, u r e t e r s and k i d n e y s a t 3 and 8 minutes and a f t e r t h e i n f u s i o n . i f a pressure r i s e was recorded i n t h e segment, a supplementary f i l m was exposed. i l e a l c o n d u i t p r e s s u r e was d e f i n e d as t h e h i g h e s t p r e s s u r e d u r i n g 30 seconds preceding t h e exposure. 121 results u r o g r a p h y . most u r e t e r s and r e n a l p e l v e s showed r e v e r s i b l e d i l a t i o n a t t h e u r o g r a p h y 3 months p o s t o p e r a t i v e l y , b u t i n o n l y 3 o f 1 2 2 u r e t e r s was t h e r e p e r s i s t e n t d i s t a l o b s t r u c t i o n . r e a q a s t o m o s i s c o r r e c t e d t h e s t e n o s i s i n t h e s e t h r e e u r e t e r s . s t o n e s . r e n a l c a l c i f i c a t i o n s were o b s e r v e d i n f o u r p a t i e n t s p r e o p e r a t i v e l y a n d d e v e l o p e d i n n i n e p o s t o p e r a t i v e l y . none o f t h e s e p a t i e n t s h a d s i g n s o f u r e t e r a l o b s t r u c t i o n . f o u r showed u r e t e r a l r e f l u x . f i v e p a t i e n t s were o p e r a t e d on f o r u r i n a r y c a l c u l i d u r i n g t h e o b s e r v a t i o n p e r i o d . r e f l u x s t u d i e s . r e f l u x was f o u n d i n 39 l o f t h e ureters. hence t h e r e ap p e a r e d t o b e no r e f l u x i n 61 %. p (mm hg) 1 n f i g . 3. maximum i n t r a l u r n i n a l p r e s s u r e ('f 1 sd) i n rnm hg ( o r d i n a t e ) a t rest and i n r e l a t i o n t o t h e t o t a l c o n t r a s t volume i n m l ( a b s c i s s a ) i n 6 3 i l e a l c o n d u i t s ( u p p e r p a n e l ) . the r e l a t e d numbers o f u r e t e r s w i t h r e f l u x ( n ) a r e shown i n t h e l o w e r p a n e l . i n t r a l u r n i n a l p r e s s u r e . a l l t h e p a t i e n t s showed a l o w e s t b a s a l p r e s s u r e < 10 rnm hg, w i t h i n t e r m i t t e n t p r e s s u r e p e a k s o f 10-30 s e c o n d s ' d u r a t i o n . the i l e a l c o n d u i t showed c o n t r a c t i o n d u r i n g p r e s s u r e p e a k s a n d was r e l a x e d a t b a s a l p r e s s ure. i n t h r e e p a t i e n t s w i t h s t e n o s i s o f t h e c u t a n e o u s s t o m a , t h e p r e s s u r e d i d 122 n o t r e t u r n t o b a s a l l e v e l between t h e c o n t r a c t i o n s . the maximum p r e s s u r e r e g i s t e r e d b e f o r e t h e s t a r t o f t h e i n f u s i o n was 24 2 29 (0-110) mm hg. d u r i n g i n f u s i o n t h e c o r r e s p o n d i n g f i g u r e s were 62 f 34 (15-150) mm hg f o r p a t i e n t s w i t h o u r r e f l u x and 5 1 ? 30 (30-110) mm hg f o r t h o s e with r e f l u x ( d i f f e r e n c e n o t s t a t i s t i c a l l y s i g n i f i c a n t ) . the maximum p r e s s u r e a t t h e o c c u r r e n c e o f r e f l u x was 28 25 (0-100) mm hg ( f i g . 3 ) . a t y p i c a l pressure r e c o r d i n g i s shown i n f i g . 4 . t -r ~ 40s t i f i g . 4. p r e s s u r e r e c o r d i n g f r o m an i l e a l c o n d u i t : f i l m exposures a r e i n d i c a t e d b y v e r t i c a l b a r s i n f e c t i o n s . o f t h o s e w i t h o u t r e f l u x t h e r e was c l i n i c a l l y m a n i f e s t p y e l o n e p h r i t i s w i t h f e v e r and f l a n k p a i n . i m p a i r m e n t o f r e n a l f u n c t i o n , measured a s e l e v a t i o n o f serum c r e a t i n i n e , o c c u r r e d i n two o f t h e s e p a t i e n t s . f u n c t i o n was l o s t i n one k i d n e y , due t o u r e t e r a l s t e n o s i s , and nephrectomy was p e r f o r m e d . another o f t h e n i n e p a t i e n t s had u r o g r a p h i c s i g n s o f p y e l o n e p h r i t i s . a l l 63 p a t i e n t s had p o s i t i v e u r i n e c u l t u r e on a t l e a s t one o c c a s i o n p o s t o p e r a t i v e l y , but o n l y t h e n i n e above-mentioned had c l i n i c a l p y e l o n e p h r i t i s . i n s i x (12.5 % ) o f t h e k i d n e y s with r e f l u x and i n t h r e e ( 4 7;) r e n a l f u n c t i o n . a t t h e end o f t h e f o l l o w u p p e r i o d , 11 p a t i e n t s showed 9-858572 123 deterioration o f renal function. six of these patients had had normal serum creatinine levels preoperatively. two o f the six had unilateral reflux and four had no reflux. three of the same six patients had had clinical pyelonephritis in the postoperative period. discussion compared with results after other techniques of operation, the complication rate in this case series was relatively low ( 5 , 8 ) , although the follow-up time is short. our investigations showed no reflux to the kidney in 61 7; of the patients, but in 39 7; the technique did not prevent reflux. the recordings in the ileal conduit showed that relatively high pressures were present locally at the site of ureteral implantation. these high pressures may be related to nonpulsatile contractions in the ileal conduit ( 3 ) . reflux could possibly entail conduction of the elevated pressures to the renal pelvis, causing intermittently high pressure to the renal parenchyma. theoretically the ureteral peristalsis might counteract this pressure r i s e , but dilation o f the ureter makes this peristalsis ineffective. further, ureteral peristalsis has been shown to lack any coordination with the contractions of the conduit (3). infections from the ileal conduit may be transferred to the renal pelvis in the same way. all o f our patients had at least one positive urinary culture from the ileal conduit during the observation time. only nine, however, had clinically manifest signs of upper urinary tract infection. a possible explan ation is that the anatomy of the renal pelvis is important for the developwent of pyelonephritis (6,7). the potential dangers of low-pressure contra high-pressure reflux are some times debated. since intermittently high pressures were registered in the con duit in almost all of our patients, this distinction cannot be made (2). a l though the incidence of pyelonephritis was slightly higher in the refluxing than in the nonrefluxing ureters, the series was too small to permit conclus ions in this respect. we found no difference in stone formation related to re flux. as, in comparison with other surgical techniques, no negative effects were found with the antireflux procedure, we are continuing to use this type of operation. references 1. ashken, m.h: urinary reservoirs. in: urinary diversion, ed. ashken, m.h.. 2. bergrnan, b., nilsson, a.e., pettersson, 5. & sundin, t.: ureteral reflux springer verlag, berlin, heidelberg, new york, 113-139,1982. from i l e a l conduit. scand j urol nephrol 12:239-242,1976. 124 3 4. 5. 6. 7. 8 . 9. 10. campbell, j.e., o l i v e r , j.a. & mckay, d.e.: dynamics o f i l e a l c o n d u i t s . r a d i o l o g y 85:338-342,1965. duggan, f.j., sanford, e . j . & rohner, t.j.: the d i s a d v a n t a g e s o f r e f l u x i n u r e t e r o i l e a l cutaneous anastomoses f o r s u p r a v e s i c a l u r i n a r y d i v e r s i o n . urol res 2:85-90,1974. harbach, l.b., h a l l , r.l., c o c k e t t , a.t.k., kaufman, j.j., m a r t i n , d.c., m i m s , m.m. & goodwin, w.e.: i l e a l l o o p cutaneous u r i n a r y d i v e r s i o n : a c r i t i c a l review. j urol 105:511-515,1971. hodson, c.j.: f o r m a t i o n o f r e n a l s c a r s w i t h s p e c i a l r e f e r e n c e t o r e f l u x n e p h r o p a t h y . c o n t r n e p h r o l 16:83-89,1979. hodson, c.j.: r e f l u x n e p h r o p a t h y : a p e r s o n a l h i s t o r i c a l r e v i e w . am j roentgenology 137:451-462,1981. k n i g h t , m.e.l. & k a n d z a r i , s.j.: u r e t e r o i l e o s t o m y : s i x t e e n y e a r s e x p e r i e n c e . west v i r g i n i a med j 70:5:119-123,1979. p a t i l , u., glassberg, m.d. & waterhouse, k . : i l e a l c o n d u i t s u r g e r y w i t h a n i p p l e d u r e t e r o i l e a l anastomosis. urology 7:594-597,1976. p h i l i p , n.h., w i l l i a m s , j.l. & e y e r s , c.e.: i l e a l c o n d u i t u r i n a r y d i v e r s i o n : long-term f o l l o w u p i n a d u l t s . br j urol 52:515-519,1980. address f o r r e p r i n t s k e r s t i n c l a e s s o n department o f urology u n i v e r s i t y h o s p i t a l 5-781 85 u p p s a l a sweden 125 upsala j med sci 81: 179-182, 1976 the effect of digitalis on regional lschaemia of the rat small intestine l. rentzhog and s. wikstrom from the department of surgery, university hospital, uppsala, sweden abstract research in recent years has shown that under certain con ditions digitalis has a strong vasoconstrictive effect in the splanchnic region. this may imply that in cases of mesenteric ischaemia, digitalization may inhibit a collateral circulation necessary for restoration of the intestinal func tion. in this investigation the effect of digitoxin on the ex change circulation of the small bowel mucosa was studied in rats with induced regional ischaemia of the intestine. on analysis 30 min after establishment of the ischaemia a statistically significant negative effect of digitoxin was observed. introduction it has long been known that digitalis has an effect on peripheral blood vessels (6, 10). until recently this has been considered of little clinical importance, but in the last few years it has received new in terest. the constrictive effect of digitalis on arterioles and veins has been studied by several authors (15, 1, 2). that digitalis has a particularly marked effect on the splanchnic vessels has been pointed out by ferrer et al. (8), harrison et al. (9) and danford (5). cohn and collaborators (4) showed that the peripheral vascular effects of digitalis may be of greater clinical significance than has been previously believed. they found that digitalization could increase the mortality of patients in shock, and assumed this to be due to its peripheral vascular effects. in acute mesenteric infarction, clearcut vascular occlusion is often not found, either angiographically or at pathology. in some clinical materials these cases comprise up to 50% of ischaemic intestinal catastrophes (14, 3). the origin of the probably causative vascular spasm may vary, but most of these patients seem t o have received high doses of digitalis. it has therefore been suspected that in many cases the vasoconstrictive effect of digitalis may be the underlying mechanism of this often fatal mesenteric vascular spasm (17, 20). the aim of the present investigation was t o de termine whether digitalization can be hazardous even in cases with more regional small bowel ischaemia. does digitalis impair the collateral circu lation necessary for restoration of the intestinal function, and thus prevent healing of the ischaemic damage? material and methods male sprague-dawley rats weighing 200-250 g were used. the animals were strictly standardized as regards iodine metabolism and iodine was added to their drinking water both before and during the experimental period. to produce standardized ischaemia of the small intestine a defined number of mesenteric end arcades (mes end arc) were ligated. the ligature material was 5-0 cardiovascular silk (ethicon). the first ligature was applied on the 6th mes end arc counted from the ileocaecal angle, and a further 7 or 11 mes end arc, in the proximal direction, were then ligated. at the same time a central loop in the devascularized intestinal segment was marked with silk threads. these were placed around the intestine without affecting the terminal blood vessels. the length of the loop always corresponded to the extent of 2 mes end arc. the mucosal circulation was assessed by a technique described by nylander & wikstrom (13), based on the fact that the passive absorption, i.e. the diffusion of a given substance from an intestinal loop of defined size, is an expression of the effective exchange circulation in the mu cosa of the intestinal segment. a radioactive iodine isotope ( n a p ) was used as the test substance. at the time of analysis the previously indicated intestinal loop (2 mes end arc) was ligated and the test dose was deposited into its lumen by transmural injection. the pylorus was tied off to prevent gastric contents from passing into the small intestine. after 30 min the rat was killed with ether. the abdomen was opened and the stomach was ligated at the cardia and resected. the isolated intestinal loop into which the test substance had been deposited was also resected. the radioactivity in the stomach, the intestinal loop and the whole body (thus excluding the stomach and test loop) was recorded. vpsala j med sci 81 180 table i. series i . characteristics of the material and percentage distribution of the radioactive dose (nai131) 30 rnin after its deposition in the isolated loop mean values with s.e. lc=laparotomy control group l . rentzhog and s . wikstrom body percentage of dose in weight experimental group n (9) isol. loop stomach body lc 20 23 1 k 3 . 8 26.1k2.2 12.0f0.8 61.9k 1.9 1 1 mes end arc, untreated 20 253f0.8 50.3 f2.4 4 . 9 f 1 . 0 44.8f2.7 1 1 mes end arc, digi toxin-treated 20 241f5.5 63.2f2.2 3 . 4 f 0 . 6 3 3 . 4 f 3 .o two series of experiments were performed. in the first series (series i) an intravenous injection of digitoxin (0.1 mg/loo g body weight) was given and 2 h later small bowel ischaemia comprising 1 1 mes end arc was produced. the exchange circulation in the ischaemic intestinal segment was analysed 30 min after establishment of the ischaemia. this series also included two control groups, one with small bowel ischaemia but not treated with digitoxin (untreated controls) and one without ischaemia, in which the exchange circulation was analysed 30 min after a lapa rotomy (laparotomy controls). in the second series (series 11) the small bowel ischaemia comprised 7 mes end arc and following establishment of the ischaemia a subcutaneous injection of digitoxin (0.1 mg/loo g body weight) was given daily for 14 days, after which the exchange circulation was analysed. the series also included an ischaemic group (7 mes end arc) treated with physiological saline instead of digitoxin (untreated controls). results in series i (table i) all animals survived. in the ischaemic animals (11 mes end arc) treated with digitoxin 50% of the given radioactive test dose remained in the isolated intestinal loop a t the end of the analytical period. the animals treated previ ously with digitoxin showed diminished absorption of radioiodide from the intestine, 63% of the dose remaining in the isolated loop. in the laparotomy controls the absorption was considerably better, and only 25% of the radioactivity was found in the intestine at the end of the analytical period. in series i1 (table 11) with the more moderate ischaemia (7 mes end arc) 4 of 22 animals in the digitoxin-treated group died. all untreated controls survived. two weeks after the ischaemia had been produced the untreated group had increased their body weight by lo%, whereas the weight of the digitoxin-treated animals was unchanged. in the un treated group 37% of the test dose remained in the isolated intestinal loop. in the group with the same degree of ischaemia but treated with digitoxin for 2 weeks the corresponding amount was 42%. discussion the observed deterioration of the exchange circula tion in the acute experiments and on analysis two weeks after establishment of the intestinal ischaemia is in agreement with previously reported findings (13, 18). the exchange circulation of the intestinal wall is a function of the mucosal circula tion and of the permeability of the mucosa and vessel walls (21). wikstrom (18) assumed that the table 11. series iz. characteristics of the material and percentage distribution of the radioactive dose (naz131) 30 rnin after its deposition in the isolated loop mean values with s.e. ~~ body weight (8) percentage of dose in mor experimental group n tality initial final isol. loop stomach body 7 mes end arc, 7 mes end arc, 37.3 f 4 . 3 8.5k 1 . 1 54.2 f 3 . 6 untreated 20 252f4.6 271f2.6 digitoxin-treated 22 4 243f3.6 243k5.0 41.6f3.7 10.7k1.0 47.4k3.2 upsala j med sci 81 effect of digitalis on bowel ischaemia 181 impaired exchange circulation in the acute stage is due mainly to a reduction of the mucosal circula tion, whereas two weeks after the ischaemic dam age has been produced secondary fibrosis in the intestinal wall also plays an important role. a deci sive factor for the degree of disturbance of the in testinal wall function following intestinal ischaemia is the availability of a collateral circulation im mediately after establishment of the ischaemia (12). in view of the fact that digitalis has been reported t o have a marked vasoconstrictive action in the splanchnic region in severe intestinal ischaemia (9, we considered that digitalization might have a de leterious effect on the so essential collateral circula tion to the ischaemic small bowel segment. the result of the acute experiment in the digitalis-treated group indicates that the mucosal circulation was, in fact, impaired. in the digitalis treated group analysed 2 weeks after establishment of the ischaemia, however, there was no such im pairment that might suggest that digitalis inhibited restoration of the intestinal circulation. the extent of the ischaemic damage was smaller, however, in the latter group than in the acute experiments, and it cannot therefore be excluded that with more ex tensive ischaemia digitalization might have this ef fect. the acute experiments indicate that digitalis can impair a collateral circulation arising from adjacent healthy intestine, by constricting the central mesenteric vessels, or that it can have a direct con strictive effect on the small vessels in the ischaemic intestinal tissue. our observations correspond t o findings in dog experiments (16) in which ouabain caused extremely high mesenteric vascular re sistance in disturbed metabolic conditions such as shock. in a smaller study the immediate effect of high doses of digitoxin o n the systemic circulation was examined. no change in the arterial blood pressure was noted 2 hours after the digitoxin injection. this speaks against the possibility that the observed im pairment of the mucosal circulation could have been caused by a toxic effect of digitoxin on the myocardium. in the long-term experiments there was some in crease in mortality among the digitalis-treated ani mals. lefer et al. (11) demonstrated in dogs that digitalis markedly reduced the arterial blood flow in the splanchnic region in haemorrhagic shock. they considered that this ischaemia could lead to a re lease of a myocardial depressant factor with a seri ous effect on the myocardial function. such an in direct depressive effect of digitalization on the myo cardium in intestinal ischaemia could explain the increased mortality in the digitalis group in our ex periments. however, it cannot be excluded that the increased mortality and the absence of a body weight increase in the digitalis group represent toxic effects of long-term treatment with high digitalis doses such as were used in these experiments. in long-term rat experiments with the same digitalis dosage as was used here, however, williams & braunwald (19) demonstrated a positive inotropic effect on the myocardium and at the same time found that this dose gave no mortality or loss of weight in intact, non-operated animals. falkenhahn (7) used the same high digitoxin doses with a good effect in tolerance experiments in rats. the present acute experiments on rats support previous observations in both clinical studies (17) and animal investigations (16) that digitalis can ag gravate an acute mesenteric ischaemia and indicate that discontinuation of digitalis therapy should be considered in the presence of both regional and more extensive intestinal ischaemia. acknowledgement this investigation has been supported by grants from the swedish medical research council (b76-17x-2809-06). skilful technical assistance was provided by mrs inger fogelberg. references 1 . braunwald, e., blodwell, r . d . , goldberg, l . j . & morrow, a. g.: studies on digitalis. j clin invest 40: 52, 1961. 2. brender, d . , strong, c. g. & shepherd, j . g.: effects of acetylstrophanthidin on isolated veins of the dog. circ res26: 647, 1970. 3. britt, l. g. & cheek, r. c.: non-occlusive mesenteric vascular disease: clinical and experimen tal observations. ann surg 169:704, 1969. 4 . cohn, j . n . , tristani, f. e. & khatri, j. m . : cardiac and peripheral vascular effects of digitalis in clinical cardiogenic shock. am heart j 78: 318, 1%9. 5. danford, r. 0.: the splanchnic vasoconstrictive ef fect of digitoxin and its reversal by glucagon. in vas cular disorders of the intestine (ed. scott j. boley). butterworth, london, 1971. 6. dock, w . & tainter, m. l . : the circulatory changes after full therapeutic doses of digitalis, with a critical discussion of views on cardiac output. j clin invest 8:467, 1930. upsala j med sd 81 182 l . rentzhog and s. wikstrom 7 . falkenhahn, a.: the influence of digitalis on the physical capacity of healthy rats in swimming experi ment. arzneim forsch 17: 551, 1967. 8. ferrer, m. i., bradley, s. e., wheeler, h. o., enson, y., preisig, r. & harvey, r. m.: the effect of di gitoxin on the splanchnic circulation in ventricular failure. circulation.32: 524, 1%7. 9. harrison, l. a,, blaschke, j., phillips, r. s . , price, w. e., cotten, m. de v. &jacobson, e. d.: effects of ouabain on splanchnic circulation. j pharmacol exp ther 169: 321, 1969. 10. katz, l. n., rodvard, s., friend, m. & rottersman, w.: the effect of digitalis on the anesthetized dog. j pharmacol exp ther 62: 1 , 1938. 11. lefer, a. m., glenn, t. m., lopez-rasi, a. m., kiechel, s. f., ferguson, w. w. & wangensteen, s. l.: mechanism of the lack of a beneficial response to inotropic drugs in hemorrhagic shock. clin pharmacol ther 12: 506, 1971. 12. lillehei, r. c., goott, b. & miller, f. a.: the physiological response of the small bowel of the dog to ischemia including prolonged in vitro preservation of the bowel with successful replacement and survi val. ann surg 150: 543, 1959. 1 3 . nylander, g. & wikstrom, s.: propulsive gastrointestinal motility in regional and graded ischemia of the small bowel. an experimental study in the rat. i. immediate results. acta chir scand, suppl. 385, 1%8. 14. ottinger, l. &austin, w. g.: a study of patients with mesenteric infarction. surg gynecol obstet 124: 251, 1%7. 15. ross, j., braunwald, e. & waldhausen, j. a.: studies on digitalis. 11. extracardiac effects of venous return on the capacity of the peripheral vascular bed. j clin invest39: 937, 1960. 16. shanbour, l.: mesenteric crisis and hemorrhagic shock enhanced by digitalis. jama 216: 1929, 1971. 17. stemmer, e. a. & connolly, j. e.: mesenteric vascu lar insufficiency. identification and management. califmedi18: 18, 1973. 18. wikstrom, s.: propulsive gastrointestinal motility in regional and graded ischemia of the small bowel. an experimental study in the rat. 11. late results. acta chir scand, suppl. 386, 1%8. 19. williams, j. f. & braunwald, e.: studies on digitalis. xi. effects of digitoxin on development of cardiac hypertrophy in the rat subjected to aortic constric tion. am j cardioll6: 534, 1965. 20. wilson, e. a.: non-occlusive mesenteric vascular disease. s afr med 547: 1470, 1973. 21. winne, d.: durchblutung und enterala resorption. z gastroenterol9: 429. 1971. received july 20, 1976 address for reprints: stig wikstrom department of surgery university hospital s-750 14 uppsala sweden upsala j med sci 81 tf-iups160014 96..112 review article the status of diabetic embryopathy ulf j. eriksson and parri wentzel department of medical cell biology, uppsala university, biomedical center, uppsala, sweden abstract diabetic embryopathy is a theoretical enigma and a clinical challenge. both type 1 and type 2 diabetic pregnancy carry a significant risk for fetal maldevelopment, and the precise reasons for the diabetesinduced teratogenicity are not clearly identified. the experimental work in this field has revealed a partial, however complex, answer to the teratological question, and we will review some of the latest suggestions. article history received 27 february 2016 revised 3 march 2016 accepted 8 march 2016 keywords anomalies; development; diabetes in pregnancy; epigenetics; gene expression; malformations introduction claes hellerstr€om (1), the senior and mentor of the authors of this text, strongly inspired the early work in the field of embryonic development (2–4) and maldevelopment (5), particularly in a diabetic environment (6–9). despite increased clinical efforts to improve glycemic control during diabetic pregnancy, the rate of congenital malformations remains increased in studies of diabetic gestation of type 1 (10–21) and type 2 diabetes (16–25). in two recent large meta-studies it was found that the malformation rate in type 1 diabetic pregnancy did not differ from that of type 2 diabetic pregnancy (26,27), and both rates were estimated to be around 5%–6%. the similar rates of malformation may relate to the higher age and concomitant adiposity in type 2 diabetic women, both of which may increase the malformation incidence in this group (28–30). the cell biological reason for the teratogenic effect of the diabetic state is not known. however, both environmental factors (maternal diabetic state and intrauterine conditions) and genetic predisposition seem to be of importance in diabetic embryopathy, i.e. this is a case of environment–gene interaction. the congenital malformations are likely to be induced in early gestation (31–33), and the risk for giving birth to a child with a malformation is enhanced by increased maternal metabolic dysregulation (34–37). alterations of maternal metabolism several teratological factors in maternal serum have been suggested, often from clinical experience, and subsequently characterized in various experimental systems. the maternal teratogenic factors most often indicated are hyperglycemia and hyperketonemia. glucose increased glucose levels are the hallmark of the diabetic state, and there is ample clinical evidence that increased glucose/ hba1c levels correlate with increased risk for congenital malformation in the offspring (32–35,37–40). in experimental studies the analogous correlation between increased serum glucose levels and increased risk for fetal malformations has been demonstrated in diabetic rodents (8,41–76). in addition, injections of glucose to pregnant non-diabetic animals have also yielded teratological effects (77,78). furthermore, in vitro culture of rodent embryos in increased glucose concentrations (48,57,64,79–95) as well as in diabetic serum (83,96–105) yields disturbed development (figure 1). the alleged teratological effect of hyperglycemia is likely to be correlated to the metabolism of glucose, since exposure to l-glucose in vitro has no negative developmental effect (79). increased embryonic uptake of glucose by existing (106), often up-regulated (107,108), glucose transporters also seems to be necessary for the teratogenic effect. the increased influx of glucose would yield increased glycolytic flux, as well as increased mitochondrial activity of the citric acid cycle and enhanced oxidative phosphorylation. several consequences of increased glucose metabolism have been suggested (109) where increased mitochondrial production of superoxide may possess the most pronounced teratogenic potential. however, also increased hexose monophosphate shunt activity, reactive oxygen species (ros)-mediated inhibition of contact ulf j. eriksson ulf.eriksson@mcb.uu.se department of medical cell biology, uppsala university, biomedical center, po box 571, se-751 23 uppsala, sweden � 2016 the author(s). published by taylor & francis. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons. org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited upsala journal of medical sciences, 2015 vol. 121, no. 2, 96–112 http://dx.doi.org/10.3109/03009734.2016.1165317 glyceraldehyde-3-phosphate dehydrogenase (gapdh), and increased formation of reactive alpha-oxoaldehydes may play roles in the diabetes-induced embryonic dysmorphogenesis. in addition, increased formation of advanced glycosylation end-products (ages) and decreased intracellular availability of arachidonic acid (and its products, the prostaglandins) are suspected players in the teratogenic drama of diabetic pregnancy (figure 2). of note, the pre-implantation embryo follows another route toward maldevelopment, since a diabetes-induced decrease of the inner cell mass (55,110) is likely to be induced by hypoglycemia, due to down-regulation of glucose transporters in the blastocyst, leading to decreased intraembryonic glucose levels (111–113) and enhanced apoptosis (110,114). ketone bodies the ketone bodies are synthesized in increased amounts in the (maternal) liver in response to the diabetic state. the two major compounds, beta-hydroxybutyrate and acetoacetate, traverse the placenta (115) and are utilized as substrate (acetoacetyl-coa) in the embryonic mitochondria. there is experimental evidence for a teratogenic role for diabetes-associated metabolites other than glucose. thus, serum from diabetic rats with normalized glucose levels is still teratogenic in in vitro cultures (103,104), and the direct exposure to increased ketone body levels in vitro yields disturbed development in cultured rodent embryos (116–123). the mechanism for ketone body teratogenesis should also include metabolism of the compounds (124). combination effect culture of rat embryos in a combination of sub-teratogenic levels of glucose and beta-hydroxybutyrate yields disturbed in vitro embryonic development, thereby supporting the notion of a synergistic relationship between diabetes-associated metabolites (90). both of these putative teratogens serve as metabolic substrates in the embryo (and mother), and the most prominent teratogen, glucose, has the most complex metabolism involving glycolysis. furthermore, the metabolic pathways of the teratogens converge at the citric acid cycle and oxidative phosphorylation in the mitochondrion, which has led to the speculation that an overactivity of the glucose ketone bodies glycolytic flux mitochondrial activity glycosylation nt stress oxidative stress apoptosis diabetes arachidonic acid / pg jnk activity pkc activity er stress hypoxic stress hexosamine stress malformation age altered (epi)genetics isoprostanes rage activation figure 2. schematic outline of the development of diabetic embryopathy. blue color marks increased activity/amount, and red color decreased or disturbed activity/ amount of compounds or processes. note that more interactions between the items are likely to be present than those denoted here, and that the putative importance of genetic predisposition is not included. figure 1. two day-9 rat embryos from a normal (left embryo) and a diabetic (right embryo) pregnancy. the latter embryo is growth-retarded (reduced crownrump length and somite number) and malformed (rotational defect, open neural tube). upsala journal of medical sciences 97 oxidative phosphorylation pathway may be at the core of the diabetic teratogenicity. alterations of fetal metabolism major teratogenic processes in embryonic tissues so far identified include alterations of signaling systems such as metabolism of arachidonic acid/prostaglandins (73,86,94,125–129). arachidonic acid/prostaglandins arachidonic acid is a polyunsaturated fatty acid present in the phospholipids of membranes of the cells. the metabolism of arachidonic acid and its products, the prostaglandins, is crucial for cellular life. in particular, arachidonic acid is involved in cellular signaling as a lipid second messenger. disturbed metabolism of arachidonic acid and prostaglandins has been found in previous studies of experimental diabetic pregnancy. intraperitoneal injections of arachidonic acid to pregnant diabetic rats diminished the rate of neural tube damage (ntd) (48) as did enriching the diet of pregnant diabetic rats with arachidonic acid (130–132). addition of arachidonic acid to the culture medium was shown to block the embryonic dysmorphogenesis elicited by high glucose concentration (48,82,94). addition of pge2 to the culture medium also blocked glucose-induced teratogenicity in vitro (86,94) as well as maldevelopment of embryos cultured in diabetic serum (101). measurements of pge2 have indicated that this prostaglandin is decreased in embryos of diabetic rodents during neural tube closure (128,133) in high-glucose-cultured embryos (128) as well as in the yolk sac of embryos of diabetic women (134). previous studies have shown that the uptake of arachidonic acid by embryonic yolk sacs is increased in a hyperglycemic environment (126). this finding would preclude an uptake deficiency of arachidonic acid in the conceptus of diabetic pregnancy, a result supported by the demonstration of unchanged concentration of arachidonic acid in membranes of high-glucose-cultured embryos in vitro (135). measurements in day-12 embryos, however, indicated a decreased arachidonic acid concentration in the offspring of diabetic rats (136). disturbances in the availability or metabolism of arachidonic acid affect the synthesis of prostaglandins. alterations in the activity of the rate-limiting enzyme cyclooxygenase (cox), which converts arachidonic acid to prostaglandin h2, may be of major importance. there are two isoforms of cyclo-oxygenase, cox-1 (constitutive) and cox-2 (inducible). a glucose-induced down-regulation of the gene expression of cox-2, as well as a gsh-dependent decrease of the conversion of the precursor pgh2 to pge2 (pge synthase), has been demonstrated (128). thus, the pge2 concentration of day-10 embryos and membranes was decreased after exposure to high glucose in vitro or diabetes in vivo. in vitro addition of n-acetylcysteine (nac) to high-glucose cultures restored the pge2 concentration (128). hyperglycemia/ diabetes-induced down-regulation of embryonic cox-2 gene expression may be an early event in diabetic embryopathy, leading to lowered pge2 levels and dysmorphogenesis, presumably because this pathway plays an important role in neural tube development. antioxidant treatment does not prevent the decrease in cox-2 mrna levels but restores pge2 concentrations, suggesting that diabetes-induced oxidative stress aggravates the loss of cox-2 activity. from these data, it may be concluded that decreased availability of arachidonic acid and the resulting decrease in several prostaglandins, in particular pge2, is likely to be involved in the teratogenicity of diabetic pregnancy (cf. figure 2) (132). other studies have shown that a diabetes-like environment decreases embryonic pge2 concentration (127,128,133) in embryonic tissues. thus, affected arachidonic acid metabolism disturbs prostaglandins and embryogenesis (137) in several ways, which emphasizes the teratological importance of prostaglandin and prostaglandin-associated pathways. diabetes-induced teratological processes several studies have suggested that diabetic embryopathy is associated with alterations of various signaling systems, which results in disturbed intracellular conditions, such as oxidative stress (62,88,90,138), nitrosative stress (139,140), endoplasmic reticulum (er) stress (140–142), and hexosamine stress (143,144). in addition, enhanced embryonic apoptosis (77,78,93,114,145–149) has been regarded as a component of diabetic embryopathy (figure 2). oxidative stress oxidative stress reflects an imbalance between production of reactive oxygen species (ros) and an ability to detoxify the reactive intermediates or to repair the resulting damage. ros can damage all components of the cell, including proteins, lipids, and dna. some ros act as cellular messengers in redox signaling, and a state of oxidative stress can therefore disturb normal cellular signaling. ros are produced through multiple mechanisms, e.g. by nadph oxidase (nox) enzymes, and in mitochondria, where about 1%–2% of electrons passing through the electron transport chain are incompletely reduced and give rise to the superoxide radical (�o�2 ). the ros with the highest capacity to cause cellular damage is the hydroxyl radical, which, once formed, will alter dna, rna, proteins, lipids, or carbohydrates without being removed by any scavenging enzyme system. the notion that diabetes is associated with oxidative stress has been suggested by several authors (150–154). for instance, increased lipid peroxidation and ros generation were found in diabetic rats, measured as increased serum 8epi-pgf2a levels (155) and increased electron spin clearance rate (156). cyclic voltammetric studies have also indicated increased levels of lipid peroxidation in diabetic rats (157), and the isoprostane 8-epi-pgf2a is increased in embryos exposed to high glucose levels in vitro (128) and diabetes in vivo (158). examination of litters of diabetic rats demonstrated lowered a-tocopherol (vitamin e) concentration in day-11 embryos and in the liver of day-20 fetuses (69). the first evidence of an involvement of oxidative stress in the pathogenesis of diabetic embryopathy was the 98 u. j. eriksson and p. wentzel demonstration that treatment of rodent embryos with antioxidative agents largely normalizes increased glucose-induced malformation rates in vitro (88, 90), observations that were repeated (91,123,159,160) and extended to in vivo studies (62,65,66,69,70,94,105,131,161–163). furthermore, antioxidative treatment was found to normalize several markers of oxidative stress, such as serum 8-epi-pgf2a levels (155), electron spin clearance rate (156), and the concentration of embryonic isoprostanes in vitro (128) and in vivo (158). evidence for diabetes-induced oxidative stress has subsequently been found in several rat models of diabetic pregnancy (164). several different compounds with antioxidative properties have been shown to diminish embryonic maldevelopment resulting from exposure to high glucose levels in vitro or to a diabetic intrauterine environment in vivo. thus, adding scavenging enzymes, e.g. superoxide dismutase (sod) (88), catalase (88), or glutathione peroxidase (88), to the culture medium protects rat embryos from dysmorphogenesis induced by high glucose concentration in vitro. the antioxidant nac blocks dysmorphogenesis in high-glucose-cultured rodent embryos (94,105,128,165–167) and neural crest cells (168,169), and addition of glutathione ester to high-glucose medium diminishes embryonic maldevelopment (91). actually, teratogenic concentrations of beta-hydroxybutyrate or the branched chain amino-acid analog alpha-ketoisocaproic acid (kic) can also be blocked by addition of sod to the culture medium (90). analogously, dietary supplementations with antioxidative compounds have been shown to diminish diabetic embryopathy in vivo. thus, administration of butylated hydroxytoulene (bht) (62), vitamin e (66,69,163), vitamin c (70,163), and folic acid (170) decreases the malformation rate in the offspring of diabetic rats and largely improves embryonic and fetal growth in vivo. alpha-lipoid acid supplementation has been found to reduce the diabetes-induced high incidence of resorptions and malformations in offspring of diabetic rodents (171–173). embryos exposed to a diabetic intrauterine milieu have demonstrated diminished malformation rates after maternal supplementation of nac (174). combined supplementation of antioxidative compounds, e.g. vitamin e and c (162), or folic acid and vitamin e (147), to pregnant diabetic rats also diminished diabetes-induced dysmorphogenesis. in a study of glucose-induced cardiac malformations in a mouse model, the administered antioxidants decreased all negative effects of hyperglycemia/oxidative stress, such as hampered migration and increased apoptosis of neural crest cells, and prevented outflow tract defects (175). in addition, pregnant diabetic mice, transgenic for the cuznsod gene (sod1) have fewer malformed offspring than the diabetic wild-type mice (138,139,142,176), illustrating the anti-teratogenic capacity of increased ros scavenging activity. embryonic neural tissue subjected to high glucose concentrations shows increased superoxide production, as measured in a cartesian diver system (177). one effect of increased intracellular ros production would be inhibition of the ratelimiting enzyme of glycolysis, glyceraldehyde-3-phosphate dehydrogenase (gapdh), since this enzyme has displayed sensitivity to ros in several different conditions of oxidative stress (178). this sensitivity resides in the thiol group of cysteine residue 149 in the active site of the enzyme (179,180). oxidation of the thiol group by no or ros leads to decreased enzyme activity (181), and blocking of this process by antioxidants protects the activity of the enzyme (182). another mechanism for gapdh inhibition also results from mitochondrial production of ros, activating poly-(adp-ribose) polymerase 1 (parp 1) by damaging dna. parp 1, in turn, induces adp-ribosylation of gapdh, leading to its inactivation and an accumulation of metabolites earlier in the metabolism pathway. in line with these considerations, decreased gapdh activity was found in rat embryos subjected to a diabetic environment both in vivo and in vitro (183). furthermore, addition of the antioxidant nac prevented the decrease in activity (183). in addition, fetuses and embryos of diabetic rodents display increased rates of dna damage (60,75,184), another indication of enhanced ros activity and damage in embryonic tissues. high-amplitude mitochondrial swelling was demonstrated in embryonic neuroectoderm of embryos exposed to a diabetic environment (185,186). this swelling decreased after antioxidative treatment of the mother (74), implicating an embryonic ros imbalance, with conceivable consequences for the rate of apoptosis in susceptible cell lineages in the embryo (167,187). the mitochondrion plays an important role in the apoptotic machinery, and previous studies have suggested that an altered apoptotic rate may affect the maldevelopment of embryos subjected to a diabetic milieu (93,145). furthermore, diabetic transgenic mice, overexpressing thioredoxin-1, have a lower incidence of malformations and decreased oxidative stress markers than diabetic wild-type mice (188), demonstrating a parallel relationship between dysmorphogenesis and degree of oxidative stress. also, it has been suggested that diabetes-induced oxidative stress in embryos may cause maldevelopment via altered tnf-alpha levels (189). in a recent study of the activity of amp-activated kinase (ampk) in embryos of hyperglycemic mice, it was demonstrated that maternal hyperglycemia stimulated ampk activity and that stimulation of ampk with 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (aicar) increased the rate of ntd in the embryos (190). in addition, stimulation of ampk by hyperglycemia, hypoxia, or antimycin a could be inhibited by antioxidants. the ampk inhibitor compound c blocked the effects of hyperglycemia or antimycin a on ntd occurrence, suggesting that diabetes/glucose-induced stimulation of embryonic ampk activity is a teratogenic consequence of oxidative stress in diabetic embryopathy (190). in support of that notion, it was reported in a subsequent study that sole stimulation of ampk disrupts embryonic gene expression and causes neural tube defects (191) the bulk of data implicates oxidative stress and ros excess as an important component in the etiology of diabetic embryopathy. the data also suggest that long-term exposure to high glucose creates embryonic ros excess either from increased ros production (177) or from diminished antioxidant defense capacity (91,159,192). the ros excess may be small, restricted to particular cell populations (193,194), and likely to vary with gestational time and nutritional status, making direct ros determinations difficult. upsala journal of medical sciences 99 increasing ros levels in embryos lead to malformations (195,196), suggesting that ros excess may also play a role in the teratogenic process(es) of phenytoin medication (197,198), ethanol abuse (193,194,199), and, possibly, thalidomide administration (200). therefore, ros excess may constitute a common element in a number of teratogenic situations, including diabetic pregnancy (cf. figure 2) (201). hypoxic stress in early organogenesis, oxygen levels are likely to be very low in the embryonic environment, and excess glucose metabolism could accelerate the rate of o2 consumption, thereby exacerbating the hypoxic state. since hypoxia can increase mitochondrial superoxide production, excessive hypoxia may contribute to oxidative stress. in a study of o2 availability in embryos of glucose-injected hyperglycemic mice, it was found that o2 availability was reduced by 30% in embryos of hyperglycemic mice (202). when pregnant hyperglycemic mice were housed in 12% o2, the ntd rate increased 8-fold in the offspring. conversely, housing pregnant hyperglycemic mice in 30% o2 significantly suppressed the effect of maternal diabetes to increase ntd (202). these observations suggest that maternal hyperglycemia depletes o2 in the embryo and that this contributes to oxidative stress and the adverse effects of maternal hyperglycemia on embryo development (figure 2). isoprostane formation isoprostanes, e.g. 8-epi-pgf2a, are prostaglandin-like compounds formed in situ from peroxidation of arachidonic acid by non-enzymatic, free radical-catalyzed reactions, and they therefore serve as indicators of lipid peroxidation (203–205). these non-classical eicosanoids possess potent biological activity as inflammatory mediators. also, the formation of isoprostanes may, in itself, consume arachidonic acid, and therefore diminish the available pool of arachidonic acid in the embryo (see above). it has been shown that a diabetes-like environment increases the isoprostane levels in embryonic tissues (128). in addition, supplementation of 8-epi-pgf2a to the culture medium caused malformations in whole-embryo culture, thereby illustrating the independent teratogenic activity of isoprostanes (206). furthermore, adding sod or nac to the culture medium with isoprostane excess normalized almost all morphological and biochemical parameters, including the elevated tissue concentration of 8-epi-pgf2a, thereby illustrating the teratogenic potential of diabetes/glucose-induced oxidative stress (cf. figure 2) (206). age formation and rage activation the biochemical process of advanced glycosylation end-product (age) formation, which is accelerated in diabetes as a result of chronic hyperglycemia and increased oxidative stress, has been postulated to play a central role in the development of diabetic complications (207). ages are known to accelerate oxidative damage to cells in a diabetic environment. examples of age-modified sites are carboxymethyllysine (cml), carboxyethyllysine (cel), and argpyrimidine. under oxidative stress due to hyperglycemia in patients with diabetes, age formation is increased beyond normal levels. rage, the receptor for age, is a transmembrane pattern recognition receptor. except for ages, rage has also other agonistic ligands: high mobility group protein b1 (hmgb1), s100/calgranulins, amyloid-beta, and mac-1. the interaction between rage and its ligands is thought to result in proinflammatory gene activation. ligand stimulation of rage initiates a signaling cascade resulting in activation of nfjb and activation of nadph-oxidase, thereby yielding increased intracellular oxidative stress. an increased accumulation of age has been found in the pathogenesis of several diabetic complications, such as cataract, retinopathy, atherosclerosis, neuropathy, and nephropathy. inhibition or knockout of rage attenuates the detrimental effects of hyperglycemia in neuropathy and nephropathy (208). it has also been suggested that age–rage activation is involved in diabetic embryopathy. thus, the embryonic formation of glycated proteins (85,209,210) has been suggested to influence the teratological events in diabetic pregnancy. it has been shown in rodent embryos cultured in high glucose that the levels of the age precursor 3-dg increase in embryonic tissues, and the addition of 3-dg to the culture medium with physiologic concentrations of glucose induces malformations, an effect that is reversible with the addition of sod (85). in a recent study of diabetic embryopathy with rage knockout mice, it was found that maternal diabetes induced more fetal resorptions, malformations (facial skeleton, neural tube), and weight retardation in the wild-type fetuses than in the rage�/� fetuses, despite similar maternal hyperglycemia. in wild-type offspring, maternal diabetes increased fetal hepatic levels of 8-iso-pgf2a and activated nfjb in the embryos, in contrast to unchanged 8-iso-pgf2a levels and nfjb activity in diabetes-exposed rage�/� offspring. these findings suggest that rage activation and oxidative stress are associated phenomena in diabetic embryopathy (figure 2). nitrosative stress nitrosamine overproduction, or ‘stress’, has been implicated in diabetic embryopathy, as a concomitant—and, in some studies, a ‘downstream’—event to oxidative stress. thus, in a study of yolk sacs of cuznsod-(sod1)-overexpressing embryos from normal and diabetic mice, it was found that the sod1-transgenic embryos were largely protected from several of the negative effects of diabetes (139). thus, diabetes-induced elevated markers of oxidative stress (4hydroxynonenal and malondialdehyde reductions) were diminished in the sod1-transgenic embryos compared to the wild-type embryos. furthermore, hyperglycemia-increased inos expression and nitrosylated protein were also diminished, and caspase-3 and caspase-8 cleavages were blocked, in the sod1-transgenic embryos. this finding suggests that oxidative stress induces inos expression, nitrosative stress, and apoptosis in diabetic embryopathy (139). in another 100 u. j. eriksson and p. wentzel study, diabetic pregnant mice were fed via gavage an inhibitor of nitric oxide (no) synthase (nos) 2,l-n6-(1-iminoethyl)lysine (l-nil; 80 mg/kg), once a day from embryonic (e) day 7.5 to 9.5 during early stages of neurulation. the treatment significantly reduced the ntd rate in the embryos, compared with that in vehicle (normal saline)-treated diabetic animals (140). in addition to alleviation of nitrosative stress, er stress was also ameliorated, as assessed by quantification of associated factors. apoptosis was reduced, indicated by caspase-8 activation. these results show that nitrosative stress is important in diabetes-induced ntds via exacerbating er stress, leading to increased apoptosis (140). the combined observations support a role for nitrosative stress in diabetic embryopathy, and suggest a ‘cross-talk’ between oxidative stress, nitrosamine stress, and er stress (see below and figure 2). er stress er stress, also named unfolded protein response (upr), is a cellular stress response related to the er, which is induced by an accumulation of misfolded proteins in the er lumen. the er stress/upr diminishes protein translation, degrades misfolded proteins, and produces chaperones involved in protein folding in order to restore normal er function. if this is not achieved, or the disruption is prolonged, the er stress/upr shifts towards promoting apoptosis. in a study of the effects of maternal diabetes on the development of oocytes and early embryos by using time-lapse live cell imaging confocal microscopy, the er displayed an increased percentage of homogeneous distribution patterns throughout the entire ooplasm during oocyte maturation and early embryo development. in addition, a higher frequency of large er aggregations was detected in oocytes and two-cell embryos from diabetic mice. these results suggest that the diabetic condition adversely affects the er distribution pattern during mouse oocyte maturation and early embryo development (211). in a study of oxidative and er stress in sod1-overexpressing mice, it was found that maternal diabetes causes increased levels of er stress markers, e.g. c/ebp-homologous protein (chop), calnexin, phosphorylated (p)-eif2alpha, pperk, and p-ire1alpha; triggered xbp1 mrna splicing; and enhanced er chaperone gene expression in wild-type embryos, whereas all these changes were blocked in the embryos of diabetic transgenic mice. this supports the notion that diminishing diabetes-induced oxidative stress, e.g. by sod1 overexpression, blocks er stress in embryos (142) a downstream effect of the er stress would be an activation of c-jun n-terminal kinase (jnk). the possible relationship between jnk1/2 activation and er stress in diabetic embryopathy was investigated in mice. maternal diabetes increased er stress markers and induced swollen/enlarged er lumens in embryonic neuroepithelial cells during neurulation. deletion of both jnk1 or jnk2 genes diminished hyperglycemia-increased er stress markers and er chaperone gene expression. in high-glucose cultured embryos, the addition of the er chaperone 4-phenylbutyric acid (4-pba) diminished er stress markers and abolished the activation of jnk1/2 and its downstream transcription factors, caspase-3 and caspase-8, as well as sox1 neural progenitor apoptosis. consequently, 4-pba blocked high-glucose-induced ntd in vivo. it was concluded, therefore, that hyperglycemia induces er stress, which yields activation of the proapoptotic jnk1/2 pathway, which yields induced neural tube apoptosis, and thereby ntd (212). autophagy is an intracellular process to degrade dysfunctional proteins and damaged cellular organelles, which, in addition, regulates embryonic cell proliferation, differentiation, and apoptosis. furthermore, blockage of this process in embryos causes ntds reminiscent of those observed in diabetic pregnancies. in a study of ntd induction in diabetic mice with or without 2%–5% trehalose water, the role of autophagy was investigated. maternal diabetes suppressed autophagy in neuroepithelial cells and altered autophagyrelated gene expression. trehalose treatment reversed autophagy impairment and prevented ntds in the embryos of diabetic pregnancies. the study demonstrates that maternal diabetes suppresses autophagy in neuroepithelial cells of the developing neural tube, leading to ntd formation (213). in a recent study of diabetes-induced cardiac malformations, it was found that the rate of atrio-ventricular septal defects (avsds) was increased concomitant with enhanced er stress in embryonic hearts. blocking of glucose-induced er stress with 4-pba in an endocardial cushion explants culture restored endocardial cell migration. the findings suggest that development of the endocardial cushions is susceptible to the insult of maternal hyperglycemia, and that diabetesinduced er stress in the developing heart mediates the negative effect on endocardial cell migration (214). the studies suggest that er stress is involved in the teratogenesis of neural and cardiac malformations in embryos exposed to diabetes or hyperglycemia, and that er stress, nitrosative stress, and oxidative stress enhance each other (figure 2). hexosamine stress increased ambient glucose concentrations yield increased uptake, phosphorylation, and metabolism of glucose, primarily by enhanced flux in the glycolytic pathway and, also, in the hexosamine biosynthetic pathway (109). this is a pathway that converts glucose to uridine diphosphate n-acetylglucosamine (udp-glcnac). under normoglycemic conditions, approximately 1%–3% of total glucose consumed by somatic cells are directed down the hexosamine pathway (215). udp-glcnac is the substrate for the majority of glycosylation in the cell, producing mucopolysaccharides (216). in this process udpglcnac is attached to serine or threonine residues of proteins, thus becoming a post-translational modification (betao-linked glycosylation), which regulates protein function in an analogous manner to phosphorylation (217). altered beta-o-linked glycosylation has been associated with a number of disease states, including cancer, inflammatory conditions, and neurodegenerative diseases (218). notably, it is also implicated as a primary mechanism behind the upsala journal of medical sciences 101 development of insulin resistance and pancreatic beta-cell destruction in type-2 diabetes (215,218) it has been suggested that hexosamine stress may play a role in diabetic teratogenesis (143,144,219). indeed, defect development has been demonstrated in pre-implantation mouse embryos, treated with glucose (27 mm) or glucosamine (0.2 mm), which was added to embryo culture media. both treatments disturbed embryo development, increased apoptosis, and decreased cell number in the resulting blastocysts (219). addition of benzyl-2-acetamido-2-deoxy-a-d-galactopyranoside (badgp), an inhibitor of o-linked beta-n-acetylglucosaminyltransferase (ogt), the enzyme which adds o-glcnac to proteins, rescued all these phenotypes in the hyperglycemia treatment group, although only mild improvement was seen in the glucosamine group (219). this may reflect the relative potencies of each hexose in their capacity to stimulate the udp-glcnac production (215). in another study, pregnant mice were injected with glucose to induce hyperglycemia, or glucosamine, to activate the hexosamine pathway directly. both treatments increased the ntd rate in the embryos, decreased gsh levels, and increased oxidative stress, as indicated by increased 2,7-dichloro-dihydrofluorescein fluorescence. glucose and glucosamine also inhibited expression of pax-3; however, all these effects were prevented by gsh ethyl ester administration (143). these findings suggest a role for hexosamine stress in diabetic embryopathy (figure 2). apoptosis the notion that apoptosis may be a component of the teratogenic process of diabetic pregnancy has been studied thoroughly. thus, there are several reports of increased rates of apoptosis in embryos exposed to a diabetic environment (77,78,146–149,167,187,220–222). in particular, there are findings indicating increased apoptotic rates already in preimplantation embryos (93,114,145). in a study of early post-implantation embryos, the expression of bcl-2 mrna was decreased and the number of deoxynucleotidyl transferase-mediated nick end labeling (tunel)-positive cells increased in embryos of diabetic rats compared to control embryos. these results suggest that a bax-regulated mitochondrial cytochrome c-mediated caspase3 activation pathway might be involved in the diabetic embryopathy (146). in another early study, it was reported that combined supplementation of folic acid and vitamin e to pregnant diabetic rats diminished diabetes-induced dysmorphogenesis and normalized apoptotic-associated protein levels (147). in another study of rodent embryos subjected to high glucose in vitro or diabetes in vivo, disturbed development was found, concomitant with increased activation of caspase-3 and other markers of apoptosis. supplementation of nac or an apoptosis inhibitor diminished both the dysmorphogenesis and apoptosis (167). exposure to a diabetic milieu during organogenesis thus increases dysmorphogenesis and apoptosis in embryos. in mice transgenic for the thioredoxin-1 gene with overproduction of the antioxidant thioredoxin-1, the incidence of diabetes-induced malformation is markedly lower in diabetic transgenic mice compared with diabetic wild-type mice. furthermore, the diabetes-induced increased markers for oxidative stress, apoptosis-promoting proteins, and cleaved caspase-3 production are all diminished in the offspring of diabetic transgenic mice compared with the offspring of diabetic wild-type mice (188). a new apoptotic pathway was recently suggested to be activated by a diabetic/hyperglycemic environment, involving the gene products of the ask1-foxo3a-tradd-caspase-8 gene (149). blocking components of this pathway diminished the ntd rate in diabetic mice. thus, hyperglycemia-induced apoptosis and the development of ntd was reduced with genetic blockage of either foxo3a or casp8, or by inhibition of ask1 by thioredoxin. in addition, examination of human neural tissues affected by neural tube defects revealed increased activation or abundance of the genes in the cascade. the conclusion was that activation of the ask1foxo3a-tradd-caspase-8 pathway participates in the development of ntds, which could be prevented by inhibiting intermediates in this cascade (149). there is, clearly, strong experimental evidence for a role of apoptosis in diabetic embryopathy (figure 2). genetics and epigenetics of diabetic dysmorphogenesis several lines of evidence support the notion that the metabolic alterations in the embryonic tissues are followed by changes in genetics (gene expression) and epigenetics (regulation of gene expression). also there should exist permissive conditions in the mother and offspring that pave the way for the diabetes-induced genetic/epigenetic changes that ultimately lead to embryonic maldevelopment, i.e. genetic predisposition in mother and child. genetic predisposition despite similar teratological exposure, the effect of any teratogen, including maternal diabetes/hyperglycemia, varies between individuals. in addition to stochastic conditions, genetic predisposition determines the effect of each teratogen on a particular individual (223,224). although predisposing genetic conditions for diabetes are clearly present in offspring of diabetic parents (225,226), as the offspring of a diabetic father has higher risk of developing the disease than the offspring of a diabetic mother (227–231), it has been established that diabetic men do not have an increased risk of fathering malformed offspring (232,233). this indicates that the genes predisposing for diabetes do not induce congenital malformations. in contrast, maternal diabetes has been suggested to be associated with down’s syndrome (234–236) and has also been suggested to predispose for optic nerve hypoplasia in female offspring (237). a genetic element may be present in the etiology of diabetic embryopathy (238), a notion supported by experimental data (58,84,138,239–242). the contribution of the fetal genome and maternal (diabetic) environment was evaluated in a rat model where the 102 u. j. eriksson and p. wentzel outcome of diabetic pregnancy in two outbred substrains of sprague-dawley rats (with low incidence, h, and high incidence, u, of skeletal malformations in the offspring), and f1 hybrids between them was studied (84). the fetuses of diabetic h mothers had no skeletal malformations, regardless of embryo type (h/h or h/u). when the diabetic mother was u or from the hybrid strain (h/u) and the offspring of the mixed h/u type, increased skeletal malformation (3%–5%) rates resulted. when the embryos contained a major u genome, either u/u or u/(h/u), further increased skeletal malformations (17%–19%) were found. these findings indicate that both the maternal and fetal genomes are involved in the etiology of diabetes-induced (skeletal) malformations in rodent diabetic pregnancy (84). when pre-implantation embryos were transferred from diabetic nod mice to non-diabetic icr mice and allowed to develop until gestation day 13, as were embryos from the reverse transfers, as well as from icr-to-icr transfers, we found 8/58 (14%), 18/45 (40%), and 0/73 (0%) malformed embryos in the nod–icr, icr–nod, and icr–icr transfers, respectively. the result thus suggests that both the embryo genotype and the maternal environment are of importance for diabetic embryopathy (58). moreover, in a recent study one-cell mouse zygotes and blastocysts were transferred from diabetic or control mice into non-diabetic pseudopregnant female recipients, and were evaluated at embryonic day 14 (243). the offspring from the diabetic rats had higher rates of malformations than the controls, and the conclusion was that exposure to maternal diabetes during oogenesis, fertilization, and the first 24 hours is enough to program permanently the fetus to develop significant morphological changes (243). in order to characterize the relative importance of the maternal and paternal genome in relation to the teratogenicity of the maternal (intrauterine) milieu, rats from two different strains were cross-mated. thus, male rats from a malformation-prone (l) and a malformation-resistant (w) strain were mated with diabetic females from the other strain to produce f1 offspring: lw (l male�w female) and wl (w male�l female), which would be genetically identical with exception of imprinted genes and mitochondrial types. however, the malformation rate was 0% in the lw and 9% in the wl offspring (244), demonstrating both a teratologic ‘dilution’ effect, as well as the importance of the maternal environment. based on metabolic data from the two types of pregnancy (indicating a more disturbed metabolic state in the diabetic l rats), the study suggested that the fetal genome controls the embryonic dysmorphogenesis in diabetic pregnancy by instigating a threshold level for the teratological insult and that the maternal genome controls the teratogenic insult by (dys)regulating the maternal metabolism (244). furthermore, when the f1 crosses were mated in a subsequent study, the malformation rate of the f2 combinations wl�wldiabetic and lw�lwdiabetic was around 5% (245), a further dilution of the teratogenic induction; however, the malformed wl�wl offspring had only agnathia/micrognathia, whereas the malformed lw�lw offspring had 60% agnathia/micrognathia and 40% cleft lip and palate. thus, despite identical autosomal genotypes, the diabetic wl and lw female rats gave birth to offspring with markedly different malformation patterns. this study suggested a teratological mechanism in diabetic pregnancy influenced by maternal metabolism and parental strain epigenetics (245). in a previous study, it could be demonstrated that a specific variant of the catalase enzyme is present in rats that are malformation-prone (cs-1a), whereas another variant of the catalase protein was present in rats that do not develop malformations in response to maternal diabetes (cs-1b) (241). thus, embryonic catalase activity was lower in embryos from normal u rats than in embryos from normal h rats, and maternal diabetes augments this difference (242). the catalase cdna and the promotor region of the catalase gene in the u and h rat were sequenced (246) and yielded one nucleotide mutation in the 5’-utr region of the u rat cdna and a heterozygocity in the u rat gene promoter. therefore, the decreased catalase mrna levels may result from different regulation of transcription (promotor), and the difference in the electrophoretic mobility in zymograms (241) may be a result of post-translational modifications of the catalase protein. using an inbred sprague-dawley strain (l) with about 20% skeletal malformations when the mother is diabetic, and inbred wistar furth rats (no diabetes-inducible skeletal malformations), a global gene linkage analysis of the skeletal malformations was performed with micro-satellites, a study which yielded strong coupling of the malformations to seven regions on chromosomes 4, 10, 14, 18, and 19, and a weaker coupling to 14 other loci in the genome. altogether we found loci on 16 chromosomes. searching for candidate genes within a distance of 10 cm from each micro-satellite yielded 18 genes that had been implicated in previous studies of diabetic embryopathy. these genes were involved in embryonic development/morphogenesis (map1b, shh, tgfb3, vegfa, dvl2, nf1, gsk3b, gap43, tgfbr3, gdf1, csf1r) (247–253), regulation of dna/rna metabolism (en2, brcc3, tp53) (166,247,254–256), regulation of apoptosis (nol3, bak1) (247), and cellular metabolism (folr1, akr1b1) (170,254,257). gene expression altered gene expression in the offspring has been demonstrated in several studies (247,250,252–254,258–262) and appears to be an integral component of the diabetic embryopathy (263). pax3 gene expression was found to be reduced in embryos of diabetic mice (77,78), and this transcription factor may regulate the gene expression of the licensing factor cdc-46 (264) and a gene, dep-1 (265), as well as p53 (255), all of which may be of importance for a correct neural tube closure. null mutation of the pax3 gene yields the splotch mouse displaying neural tube defects (77,266). it has also been shown that the decreased pax3 expression in embryos of diabetic mice could be normalized by treatment of the mother with antioxidants (202), thereby demonstrating a coupling between ros excess and a teratologically important change in gene expression. in a later study, neural crest cells (nccs) of pax3-deficient embryos displayed impaired migration and increased apoptosis. suppression of p53, either by null mutation of the p53 gene, or administration of a p53 inhibitor, pifithrin-alpha, upsala journal of medical sciences 103 prevented the defective ncc migration and apoptosis in pax3-deficient embryos, and also restored proper development of cardiac outflow tracts. pax3 thus appears required for cardiac outflow tract septation because it blocks p53 expression during ncc migration (256). the embryonic expression of genes controlling the defense against oxidative stress is sensitive to maternal diabetes. thus, in a study of pregnant diabetic rats, the embryos demonstrated decreased expression of cuznsod and mnsod (267). in addition, the expression of gpx-1 and gpx-2 was decreased compared with embryos from normal rats, enzymes that function in the detoxification of hydrogen peroxide, an important antioxidant system. the decrease in gene expression of gpx-1 was further enhanced in the malformed embryos. the immunostaining of gpx-1 displayed a general accumulation of positive cells in the cardiac tissue of all embryos. however, the non-malformed embryos had less staining than embryos from normal rats, and malformed embryos from diabetic rats had almost no staining at all (267). in a study of cardiac malformations in diabetic mouse pregnancy demonstrating dilated heart tube, smaller ventricles, conotruncal stenosis, and abnormal heart looping, ventricular septal defects were observed and actors in the tgf-b signaling that regulate heart development were down-regulated by maternal diabetes. it was concluded that the tgf-b signaling is involved in cardiac malformations in diabetic embryopathy (250). also, in a study of global gene expression in a transgenic mouse model of caudal dysgenesis, and in a pharmacological model using in situ hybridization and quantitative real-time pcr, altered expression of several molecules that control developmental processes and embryonic growth was observed. the most pronounced finding was that of altered wnt signaling, which suggests that impaired signaling in this pathway may be involved in diabetic embryopathy (252). a genome-wide investigation of gene expression in embryos from normal and diabetic mice followed by quantitative rt-pcr yielded several genes with altered expression. sequence motifs in the promoters of diabetes-affected genes suggest potential binding of transcription factors that are involved in responses to oxidative stress and/or to hypoxia, and, furthermore, around 30% of the diabetes-affected genes encoded transcription factors and chromatin-modifying proteins or components of signaling pathways that affect transcription (258). in a genomewide expression profiling in the developing heart of embryos from diabetic and control mice it was found that a total of 878 genes exhibited more than 1.5-fold changes in expression level in the hearts of experimental embryos compared with their respective controls. several genes involved in a number of molecular signaling pathways such as apoptosis, proliferation, migration, and differentiation in the developing heart were differentially expressed in embryos of diabetic pregnancy (262). several different genes and pathways have been demonstrated to be affected in diabetic pregnancy; however, there is not, as yet, any universal gene identified to be responsible for enhanced (or decreased) susceptibility to diabetic embryopathy (figure 2). epigenetics there are considerable indications that epigenetic processes play a role in diabetic embryopathy (259,268–274). in a seminal study, it was found that transient hyperglycemia induced long-lasting activating epigenetic changes in the promoter of the nfjb subunit p65 in aortic endothelial cells both in vitro and in non-diabetic mice, resulting in increased p65 gene expression. both the epigenetic changes and the gene expression changes persisted for at least 6 days of subsequent normoglycemia. furthermore, the hyperglycemiainduced epigenetic changes and increased p65 expression were prevented by reducing mitochondrial superoxide production or superoxide-induced alpha-oxaldehydes (268). analysis of gene expression data from two sets of embryos of diabetic mice suggested that maternal diabetes may increase the overall variability of gene expression levels in embryos. the suggestion was that altered gene expression and increased variability of gene expression together constitute the molecular correlates for the incomplete phenotype penetrance in diabetic pregnancy. based on this model, it was suggested that maternal diabetes reduces the precision of gene regulation in exposed individuals. loss of precision in embryonic gene regulation may include changes to the epigenome via deregulated expression of chromatin-modifying factors (259,269). in a study of maternal diabetes effect on dna methylation of imprinted genes in oocytes, it was found in sz-diabetic and nod mice that the methylation pattern of peg3 differential methylation regions (dmr) was altered, and in the sz mice demethylation was observed on day 35 after sz injection. the expression level of dna methyltransferases (dnmts) was also decreased in diabetic oocytes. these results indicate that maternal diabetes has adverse effects on dna methylation of the maternally imprinted gene peg3 in oocytes, but also that methylation in the oocytes of the offspring is normal (271). also, the expression was increased and the methylation level of h19 was decreased, whereas the expression and methylation levels of peg3 were completely opposite in placentas of diabetic mice. when embryos of normal females were transferred to normal/diabetic pseudopregnant females, the methylation and expression of peg3 in placentas were also clearly altered in the normal-to-diabetic group compared to the normal-to-normal group. however, when the embryos of diabetic females were transferred to normal pesudopregnant female mice, the methylation and expression of peg3 and h19 in placentas were similar in the two groups. the data suggest that the effects of maternal diabetes on imprinted genes may primarily be caused by the adverse uterus environment (272). in a study neural stem cells (nscs) were exposed to high glucose/hyperglycemia, and alterations were found in chromatin reorganization, global histone h3 status, and global dna methylation, as well as increased expression of dcx and pafah1b1 and decreased expression of four micrornas targeting these genes. this study suggested that hyperglycemia alters the epigenetic mechanisms in nscs, resulting in altered expression of developmental genes (273). in a genome-wide survey of histone acetylation in neurulation stage embryos 104 u. j. eriksson and p. wentzel from diabetic mouse pregnancies, it was found that exposure to maternal diabetes and, independently, exposure to high-fat diet were associated with increases and decreases of h3 and h4 histone acetylation, respectively, in the embryo. these data suggest that epigenetic changes in response to diet and metabolic conditions may contribute to increased risk for ntd in diabetic and obese pregnancies (270). in a study of embryos of pregnant hyperglycemic mice and mouse embryonic stem cells (esc), the methylation of a pax3 cpg island was decreased in embryos and esc. use of shrna in esc demonstrated that dna methyltransferase 3b (dnmt3b) was responsible for methylation and silencing of pax3 prior to differentiation and by oxidative stress. these results indicate that hyperglycemia-induced oxidative stress stimulates dnmt3b activity, thereby inhibiting chromatin modifications necessary for induction of pax3 expression, and, thus, providing a molecular mechanism for defects caused by pax3 insufficiency in diabetic pregnancy (274). epigenetic changes in the embryo caused by maternal diabetes are likely to be transferring the teratogenic input of the diabetic environment. identifying these changes is important both for the increased knowledge generated, and also for the possible anti-teratological treatment that may emerge from the identified mechanisms (figure 2). conclusions and future directions diabetic embryopathy has a complex etiology and pathogenesis. the studies of etiologic factors in the pathogenesis of congenital malformations have revealed a scenario in which the diabetic state simultaneously induces alterations in a series of teratogenically capable pathways. these pathways are intertwined, and several of them result in an imbalance of the ros metabolism, yielding ros excess in teratogenically sensitive cell populations, an imbalance ultimately causing the congenital malformations. blocking the ros excess may therefore be one valid way to diminish the disturbed development caused by the diabetic environment. upstream and downstream of the oxidative stress, however, several conditions of cellular stress are present, such as nitrosative, er, hypoxic, and hexosamine stress, as well as a possible teratogenic involvement of ages. in this area of metabolites and pathways, there may be possibilities of finding molecules to use for intervention therapy. there is also a growing understanding of the diabetesinduced alterations in genetic and epigenetic systems, which will, again, increase our knowledge, and inspire to develop new ways to block diabetic embryopathy in the future. disclosure statement the authors report no conflicts of interest. funding information the authors gratefully acknowledge the support from the swedish research council, the family ernfors fund, the novo nordisk foundation, and the swedish diabetes association. references 1. andersson a, eriksson uj, jansson l, sandler s, welsh m, welsh n. claes hellerstrom: a friendly islet explorer. diabetologia. 2007;50:4 p following 496. 2. asplund k, westman s, hellerstrom c. glucose stimulation of insulin secretion from the isolated pancreas of foetal and newborn rats. diabetologia. 1969;5:260–2. 3. aoyagi k, bergsten p, eriksson uj, 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glucose ketone bodies combination effect alterations of fetal metabolism arachidonic acid/prostaglandins diabetes-induced teratological processes oxidative stress hypoxic stress isoprostane formation age formation and rage activation nitrosative stress er stress hexosamine stress apoptosis genetics and epigenetics of diabetic dysmorphogenesis genetic predisposition gene expression epigenetics conclusions and future directions funding information references ujms109_3.pdf upsala j med sci 109: 229–238, 2004 what type of information do parents need after being discharged directly from the delivery ward? katarina johansson, elisabeth darj department of women’s and children’s health, section of obstetrics and gynaecology, uppsala university hospital, sweden abstract a free full-text copy of this article can be found at the web page of upsala j med sci: http://www.ujms.se early discharge normally means that mother and infant are discharged from the hospital between six hours and three days after delivery. early discharge with home-visits after normal delivery was introduced at uppsala university hospital in 1990. seventeen percent of the women who gave birth in 2003 in uppsala used the home-care option as an alternative to postnatal care at the hospital. the homevisiting midwives use a checklist to give and gain information about the health of the child and mother and about how breast-feeding is going. the purpose of this study was to examine the parents’ need of information after early discharge after delivery and to compare their needs with the information given according to the checklist for home-visits. forty-two couples completed the study. they were asked to formulate five questions to the midwife at the home-visit. after the questions were gathered, a content-analysis was done. three different main groups were identified: questions concerning 1) the child (68%) such as hygiene, bowel movements, burping, vomiting, eating, sleeping and sneezing 2) breast-feeding (21%) questions were asked about position while breast-feeding, nipples and amount of milk 3) the mother (11%) questions concerned afterpains, stitches, eating and drinking. the results show that the checklist worked sufficiently well as a work tool, but can be adjusted further according to the parents’ need. this study shows that they needed more information about the care of the infant, primarily concerning hygiene. introduction twenty years ago, women who had given birth to their first child stayed at a postnatal ward an average of six days in sweden (6). since then, the average stay has successively been shortened (2). early discharge after delivery in combination with a 229 received 10 march 2004 accepted 23 april 2004 home visit by a midwife was introduced in sweden 1984 as an alternative to postnatal care in the hospital ward (12). the swedish nation board of health and welfare has defined early discharge as being when both mother and infant are discharged from the hospital between six hours after birth and, at the latest, three days after birth. the concept of early discharge can also imply that both mother and infant have been discharged directly from the labour and delivery ward without spending any time on the postnatal ward. to qualify for discharge as early as six hours after birth, the following criteria must be met: the mother must be healthy and had a normal pregnancy and delivery without complications. the infant must be born during gestation week 37–42 and must be examined and determined healthy by a paediatrician before leaving the hospital. since 1990, early discharge has been used as an alternative and a compliment to postnatal care at the uppsala university hospital. this alternative type of care has been evaluated and showed that all mothers were satisfied with the care they received in their homes (1). the most common questions new mothers had concerned breast-feeding. a follow-up study showed that 95% still were breast-feeding after four months (1). a review of literature showed just one previous study concerning need of information to new parents directly discharged from the delivery ward. this study concluded that the most common areas of concerns among primiparas regarded breast-feeding, post-delivery questions and baby-caring, while multiparas had concerns about the newborn's siblings (5). grullon and grimes showed that early discharge worked well for carefully chosen families (2). medical risks for mother and child have been studied thoroughly (1, 2). an australian study showed that there was no increased risk for depression after delivery with early discharge (10). yet another study showed that early discharge is both cost-efficient and risk free if there is a well-established home visit unit. (7). seven midwives staff the outpatient care unit at the department of obstetrics and gynaecology, uppsala university hospital. this unit is called the postnatal visiting midwives group (pvmg). families who choose to be discharged early are contacted by telephone the day after they arrive home. the family will thereafter be contacted daily and offered a home visit until four days after delivery. on the fifth day, the family visits the pvmg for an examination of the infant by a paediatrician and to take a metabolic screening test on the baby. at the home visit a standardised checklist, developed by the midwives at the hospital, is used. by following it, the midwife gains information about the child’s and the mother’s health and about how breast-feeding is going. the midwife asks questions from the checklist, gets answers from the parents and then gives feedback to the parents based on the answers to the midwife’s questions. this creates a dialogue between the midwife and parents. the parents are asked if the child has urinated, had a bowel movement and about the colour and consistency of the faeces which should change from black and sticky to a less solid and lighter colour. on the third to fourth day, it should be more fluid, yellow, grainy and 230 have a sour smell. the child is undressed and the midwife examines the infant’s movements, skin colour, breathing patterns, head shape, fontanels, eyes, muscle tone and genitals. the midwife gives information about and demonstrates how the navel should be cleaned. if the child’s skin is yellow, the midwife can take a blood test for analysis of bilirubin. to get an idea about how breast-feeding is going, the midwife observes while the mother nurses. it is important that the infant has the right sucking technique. this is important so as not to develop sores or tenderness in the nipple and for the infant to get enough milk. information about the willingness of the infant to nurse and about the frequency of breast-feeding is important information. the midwife asks questions about how the mother’s breasts feel. about three days after birth, the breasts are usually swollen because the milk has begun to flow. the midwife can explain that this is normal and give ideas about steps to take should the breasts become too swollen. the midwife also informs the parents according to breast-feeding strategies for uppsala county that a pacifier should not be used in the beginning as it can affect breast-feeding in a negative manner (13). concerning the mother’s health, the midwife asks about the mother’s general health, bleeding, cramping, tears and sutures. information is given about nutrition, sleep, exercising, spouse relationships and about the care of the infant. if there is a special request, the midwife can review with the patient her chart during labour and delivery. if there isn’t a need for a home visit, the midwife asks a series of standardised questions and completes the patient’s chart. since 1997, there are laws in sweden requiring that the healthcare system systematically and continually develops to assure the quality of healthcare. among other things, this emphasises that the patient and nearest of kin should be informed and participate in decisions and that all personnel must co-operate. the aim of this study was to evaluate the quality of care by comparing the parents’ need of information with the information given by the midwife. methods a pilot test, including ten couples was conducted to evaluate the study method used. the number of informants was not decided before start. new participants were included as long as there were new questions from the parents. the midwives at the pvmg recruited parents while informing those who wanted to go home directly from the delivery ward about the home-care. an information letter was given to the parents when they were discharged, earliest six hours after the delivery. the letter held information about the purpose of the study, a request for participation and the parents were informed that participation was voluntary. ethical approval was obtained from the head of the department of obstetrics and gynaecology at the hospital and not from the ethics committee at the medical faculty of the university, as the study was designed as a quality assurance project at the clinic. the participants accepted involvement in the study at the first home visit. they 231 were asked to write down five questions they would like to ask the midwife the next day. in most cases the parents had formulated their questions before the midwife’s visit, but in some cases the parents wrote their questions during the home visit. if there were more questions written on the paper, the first five were included in the study. at the home visit, the midwife gathered the questions and answered them as a part of the home visit routine. a total of 116 questions were gathered which gave an average of 2.8 questions per couple. all information was handled confidentially. when all the material had been collected, each question was written down and the paper was cut into pieces with one question per piece. a code number was noted on each paper so we could note if the women had just got her first, second, third or fourth child. first, the material was reviewed to obtain an overview of type of questions. then, all questions were categorised into main themes (by the first author). these themes were reanalysed together with a second midwife and divided into sub-groups. this second midwife was not working at the pvmg. data were analysed by using a content analysis. as a complement to the qualitative method to analyse the questions, a quantitative method was used to calculate how many questions there were in each theme and sub-group. materials during three months, september 10 to december 15, 2001, parents were asked to participate in this study. the inclusion criteria were: 1) each family had been discharged daytime from the delivery ward without moving to the postnatal ward at the uppsala university hospital, 2) all were residents of uppsala county, 3) only swedish speaking couples were included due to practical reasons, 4) families that did not want home visits were excluded. during the study period 50 out of 53 possible couples were recruited and accepted participation. four couples were excluded, as they were not allowed to be discharged early. four couples did not return the questionnaire despite reminders. in this study, nine (21%) were first time mothers and 33 (79%) had at least two children. of these, 21 had just given birth to her second child, nine her third and three her fourth. three couples completed the study, but had no questions, when the midwife made the home-visit. questions from 39 (78 %) of the recruited couples were analysed. results three main themes were identified. questions concerned the child, breast-feeding and the mother (table 1). the main group, which contained 79 questions concerned the child and childcare, 24 questions concerned breast-feeding and 13 questions concerned the mother. the kind of questions between first time mothers and women with more than one child are presented in table 1. examples of questions are presented in italics. 232 table 1. number of question distributed in main groups according to first time mothers and mothers with more than one child. main groups first time mothers more than one child total child 23 (20%) 56 (48%) 79 (68%) breast-feeding 7 (6%) 17 (15%) 24 (21%) mother 4 (3%) 9 (8%) 13 (11%) questions about the child hyg iene in this group, 26 questions about how a child’s hygiene should be maintained are included: fifteen questions about bathing a child. ”when should one bathe the child?” seven questions about care of the navel. “how do you wash the navel?” four questions were about dry skin and redness of the bottom “can you put lotion on dry skin?” bowel movements, urination, burping and vomiting this subgroup includes questions about when and how often a child should urinate or have a bowel movement. questions about burping or vomiting are also included. there were a total of eight questions in this subgroup. “when will she have a bowel movement?” eating and sleeping parents had concerns about the activities the child devotes most of the day and night to, eating and sleeping, are included in this subgroup. they asked if the child should be allowed to steer feeding times or if he should be awaken to be nursed. ”should he be allowed to sleep as long as he wants or should i wake him after a while and try to get him to eat?” sitting, lying down questions were asked if the child should lie on his back or stomach. a few questions worried if it is dangerous if the child were to vomit while lying on his back. there were also questions about if the child can lie on his stomach and sleep. as far as questions about sitting, there were questions about if a child can sit in a soft bouncy baby chair. “how shall she lie?” nose a few questions were raised about the child’s stuffy nose and sneezing. “why does the child sneeze so often?” medical questions in this group there were 15 medically related question of which four questions were about jaundice and three about rashes. the remaining questions were about uneven breathing, eye infections, blue feet, body temperature, the risk that sick siblings 233 may infect the infant and about vaccinations. two questions were also about the bump that was seen on the chest (the sternum). miscellaneous questions not fitting into the above categories were included in this subgroup, for example, outdoor and indoor clothing, cutting the nails, crying and sibling relationships. questions about breast-feeding breast-feeding positions, nipples nine couples had questions about various breast-feeding positions, the child’s position at the breast and the correct sucking technique at the nipple. “how do you nurse lying down?” eight questions were about sores and tender nipples and breastfeeding positions. “how can i get these sores to heal?”, “which positions can i use that won’t give me sores?” time per breast-feeding, amount of milk four questions were about the amount of milk. “is there enough milk?” there were two questions about the length of time per breast-feeding. “how long should you nurse each time?” questions about mothers afterpains, stitches examples of questions about afterpains and stitches: “how long do you have afterpains?” “when will the stitches fall off?” eating, drinking, medications within this group, there were five questions about how much food and fluids to take and about drug usage during breast-feeding. some questions were about taking pain relievers. three questions were about food and fluids suited to the breast-feeding period. “can i eat as usual now?” discussion seventeen percent of the families were discharged from the university hospital in uppsala 2001, without staying at the postnatal ward. questions about childcare seem to be the most pressing questions to be answered. a great deal of all questions were about bathing the child and taking care of the navel. in the check list for home visit, information about care of the navel is included which means that there is no need for improvement because the midwife always has a dialogue with the parents on this point. when considering bathing the infant, there are still notions that the infant should not be bathed until the navel has healed. current opinions are that the 234 infant can be bathed anyway (3). in today’s segregated society, many families have lost the continuing contact with older generations who previously gave advice as to the care of an infant. in the present study only 20 % of the participating families had had their first child. eighty percent had own experience of caring for at least one new-born child before. the proportions of first time mothers and those with children before are exactly the same as in the previous study at the same clinic (1). the results show, however, that there is a great need of routinely given information about e.g. bathing the child. thus, the checklist for home visits should be redesigned to include space for this. parents also have questions about dry skin. a square for information about skin/skin colour is included in the chart/check list for home visits. it is used today to note infant jaundice. the checklist can also include a box for skin quality as well as one for jaundice. questions and answers concerning the infant’s urination and bowel movements are also important for parents. in the home visit checklist, there is a square for urination and bowel movements. by asking about the form and colour of the faeces, the midwife gains information about the child’s intake of breast milk. this information is very important for both the parents and the midwife. in this regard, the checklist for home visits works well. questions about when the infant should eat and sleep are answered by the midwife during the section about the breast-feeding interval in the checklist for home visits. the midwife’s information and the parents’ needs are met on this point. most parents know that it is now recommended that infants sleep on their backs or side to reduce the risk for sudden infant death syndrome, but it is desirable that the midwife discuss this on a routine basis. questions on why the infant sneezes or has a stuffy nose also come up. there is no special box for this but this is included in the observations the midwife makes on the general health of the baby. a small fraction of the questions was about medical aspects. this can be because only healthy infants and mothers are discharged directly from labour and delivery. it can also possibly be that parents feel secure within their own home environment with access to 24-hour advice. over 20% of all questions were about breast-feeding and these results correspond to the previous results from uppsala (1). most of them concerned breast-feeding technique and sores or tender nipples. this corresponds well with the experiences from home visits. during a home visit, the midwife observes a breast-feeding. the advantage with a home visit is that the midwife can show the mother how to find a comfortable position in the woman’s own home. problems with tender nipples or sores are less and eventually disappear completely when the mother and infant have the right technique while breast-feeding (8). the midwife’s information in this area is very thorough and is based on the home visit checklist. questions that were asked in this group were answered when the midwife observed the mother breast-feeding and, in appropriate cases, corrected the position and the infant’s position and technique. the midwife answered questions about the time per feeding during the breast-feeding interval in the checklist for the home visit. there is no box in the 235 home visit check list corresponding to questions about the amount of milk but it is common that the midwife informs the parents that there is enough raw milk (colostrums) the first few days and that the quantity of milk increases afterwards. it was not surprising and corresponds well with the midwives experience, that there were few questions concerning the mother’s health. the midwife following the checklist for the home visit answers questions about afterpains, stitches and nutrition for the breast-feeding mother. new mothers, influenced by the hormone, oxytocin, are more harmonious and better able to cope with sleeping only short periods of time (11). lundberg and öberg, found that the father’s responsibility for care was greater if the mother and infant were discharged early (4). that gives the woman time to devote herself to important tasks for both herself and the infant: breast-feeding, rest, eating and drinking. the spouse devotes more time to taking care of household chores and siblings. the results show that the checklist, developed by midwives, in co-operation with paediatricians and gynaecologists at the hospital, for home visits works well as a work tool. from the parents’ perspective, improvements can be made concerning information about childcare such as taking care of the infant’s personal hygiene, burping/vomiting and appropriate sleeping positions. documentation can also be improved concerning jaundice and the skin appearance of the infant. it could be of interest to know what kind of questions families who stay at the postnatal ward have, if it differs from those who leave the hospital early. furthermore, it could be interesting to study if the questions differed between mothers and fathers. in uppsala county there are 22% immigrants and in sweden there are totally 17 % immigrants (9). we have no information about parents who did not speak swedish. though, it would be interesting to find out if parents from other cultures have different concerns after coming home with their new-born baby. the midwives working at the pvmg will continue to develop the checklist for home visits so that it will better serve the informational needs of the parents. the demands for better documentation will be a part of changes we make in the future. references 1. darj e, stålnacke b.: very early discharge from hospital after normal deliveries, upsala journal of medical sciences 105: 57–66, 2000. 2. grullon k.e, grimes d.a.: the safety of early postpartum discharge: a review and critique. obstet gynaecol nov; 90(5):860–865, 1997. 3. gustavsson, lars h.: leva med barn, national board of health and wellfare. stockholm: gothia, 1996. 4. lundberg e, öberg s.: fathers experiences after early discharge after delivery. abstract vårdstämman. stockholm, sweden. 1999. 5. nyberg k, bernerman sternhufvud l.: mothers and fathers concerns and needs postpartum, british journal of midwifery, vol 8 (6), 387–394, 2000. 6. nilsson c.a.: goda erfarenheter av tidig hemgång efter förlossning. läkartidningen 83; 2212– 2214, 1986. 7. odelram h, nilsson b, pehrsson-lindell d, ljungkvist e.: early discharge after delivery, a safe and cost-effective form of care. läkartidningen 8; 95( 28–29 ): 3190–3194, 1998. 236 8. righard l.: are breastfeeding problems related to incorrect breastfeeding technique and the use of pacifiers and bottles? birth 25 (1): 40–44, 1998. 9. statistiska centralbyrån, internet publishing: 2001, http://www.scb.se/templates/tableorchart_26040.asp 10. thompson j. f, roberts c. l, currie m. j, ellwood d.a.: early discharge and postnatal depression: a prospective cohort study. the medical journal of australia jun 5; 172(11): 532–536, 2000. 11. uvnäs-moberg k.: antistress pattern induced by oxytocin. news physiol sci. feb;13:22–25, 1998. 12. waldenström u, sundelin c, lindmark g.: early and late discharge after hospital birth. health of mother and infant in the postpartum period. ups j med sci. 92(3): 301–314. review, 1987. 13. world health organization, w.: protecting, promoting and supporting breastfeeding: the special role of maternity services – a joint who/unicef statement. who, geneva, 1987. corresponding author: katarina johansson, rn, rm postnatal visiting midwives group department of women’s and children’s health section of obstetrics and gynaecology uppsala university hospital, se-751 85 uppsala, sweden katarina.johansson@akademiska.se 237 238 upsala j med sci 91: 17-27, 1986 determination of sialic acid residues in tkansferrin by oxidative-reductive immunoassay erik cervkn and gunnar ronquist institute of medical and physiological chemistry, biomedical center and department of clinical chemistry, university hospital, university of uppsala, sweden abstract a method i s d e s c r i b e d f o r t h e d e t e r m i n a t i o n o f s i a l i c a c i d r e s i d u e s i n g l y c o p r o t e i n s d i s p l a y i n g m i c r o h e t e r o g e n e i t y i n t h e s u g a r r e s i d u e . the new method i s based o n c o m b i n i n g an o x i d a t i v e r e d u c t i v e s t e p w i t h b i n d i n g o f t h e g l y c o p r o t e i n t o an immunoadsorbent. a f t e r a m i l d o x i d a t i o n w i t h sodium meta p e r i o d a t e t h e s u g a r i s r e d u c e d w i t h l a b e l e d s o d i u m b o r o t r i t i d e . the c h e m i c a l m o d i f i c a t i o n o f t h e s u g a r r e s i d u e s does n o t seem t o i m p a i r ' t h e b i n d i n g o f g l y c o p r o t e i n t o t h e immunoadsorbent. the p r o c e d u r e , w h i c h has been e l a b o r a t e d f o r human t r a n s f e r r i n , can be c a r r i e d o u t i n t h e p r e s e n c e o f o t h e r sub s t a n c e s i n body f l u i d s . introduction combined p h y s i o c o c h e m i c a l s e p a r a t i o n t e c h n i q u e s 1 i k e i s o e l e c t r i c f o c u s s i n g and c h r o m a t o f o c u s s i n g a r e p r e c i s e b u t time-consuming methods t h a t a r e n o t s u i t a b l e f o r use on a l a r g e m u l t i s a m p l e s c a l e b a s i s . such methods a l s o r e q u i r e e x p e n s i v e equipment and c h e m i c a l s as w e l l a s s k i l l e d manual g u i d a n c e . when complex m i x t u r e s a r e a n a l y z e d , t h e two methods g e n e r a l l y have t o be complemented w i t h some k i n d o f i d e n t i f i c a t i o n o f t h e i n v e s t i g a t e d p r o t e i n . f o r t h e s e r e a s o n s such methods a r e n o t always s u i t a b l e f o r r o u t i n e a n a l y t i c a l use on a m i c r o s c a l e b a s i s . t h e r e f o r e , new methods a r e needed t h a t a r e c o m p a t i b l e w i t h r a p i d and s i m u l t a n e o u s p r o c e s s i n g o f a l a r g e number o f samples, and w h i c h p e r m i t q u a n t i t a t i v e measurements. f o r example, t h e m i c r o h e t e r o g e n e i t y o f g l y c o p r o t e i n s m o s t l y i n v o l v e s s i a l i c a c i d and i s a s t a t i s t i c a l phenomenon, where t h e a v e r a g e c o n t e n t o f s i a l i c a c i d u s u a l l y g i v e s more i n f o r m a t i o n t h a n t h e r e l a t i v e amount of one p a r t i c u l a r i s o e l e c t r i c component o u t o f s e v e r a l . i n such cases no u s e f u l i n f o r m a t i o n i s l o s t b y r e c o r d i n g average c o n c e n t r a t i o n s o f s i a l i c a c i d . r e c e n t l y , a method has been p r e s e n t e d h a v i n g t h e d e s i r e d p r o p e r t i e s , t h e r a t i o n a l name o f w h i c h i s r a d i o l e c t i n immunoassay (1). however, a m a j o r drawback o f methods i n v o l v i n g l e c t i n s i s t h a t m o s t l e c t i n s b i n d w i t h l o w 2 868571 17 a f f i n i t y t o t h e i r c o r r e s p o n d i n g sugars. t h i s means t h a t t h e c o n d i t i o n s have t o be s t a n d a r d i z e d w i t h r e s p e c t t o s e v e r a l p a r a m e t e r s t h a t a r e d i f f i c u l t t o c o n t r o l , such as t h e t u r b u l e n c e o c c u r r i n g when r e p l a c i n g t h e b u f f e r d u r i n g w a s h i n g t h e inimunoadsorbent. hence, t h e r e i s an a c t u a l want o f r a p i d and r e l i a b l e methods f o r d e t e r m i n a t i o n o f s u g a r r e s i d u e s i n g l y c o p r n t e i n s . the p r e s e n t p a p e r d e s c r i b e s a s i m p l e and r a p i d method f o r d e t e r m i n a t i o n o f s i a l i c a c i d r e s i d u e s i n g l y c o p r o t e i n s d i s p l a y i n g m i c r o h e t e r o g e n e i t y . the method i s a t l e a s t s e m i q u a n t i t a t i v e and w i l l t h e r e f o r e r e p l a c e i n some i n s t a n c e s q u a l i t a t i v e p r o c e d u r e s l i k e i s o e l e c t r i c f o c u s s i n g . materials b u f f e r s a l t s , c i t r i c a c i d and u n l a b e l e d sodium b o r o h y d r i d e were r e a g e n t g r a d e from merck ag, darmstadt, w . germany. t r i s , t r a n s f e r r i n , n e u r a m i n i dase, a l b u m i n and n ’ n ’ d i m e t h y l f o r m a m i d e were from sigma, s t l o u i s , miss. usa. a f f i g e l 10 was f r o m b i o r a d l a b o r a t o r i e s , richmond, c a l i f . usa, a n t i t r a n s f e r r i n ( n e p h e l o m e t r i c t i t e r , 0.35 mg o f a n t i g e n h l ) f r o m k a l l e s t a d , behringwerke, f r a n k f u r t am main, w . germany, t r i t i a t e d sodium b o r o h y d r i d e (350 mci/mmol) and aquasol f r o m nen chemicals, d r e i e i c h , w . germany. b l u e d e x t r a n and sephadex 6200 were f r o m pharmacia ab, uppsala, sweden. methods p r e p a r a t i o n o f immunoadsorbent. two m l o f a f f i g e l 10 was washed 5 t i m e s w i t h an i c e c o l d s o l u t i o n c o n s i s t i n g o f 20 mm na2hp04, 100 mm nac1, ph a d j u s t e d t o 7.5 w i t h hc1 (pbs, ph 7.5). twenty mg o f t r a n s f e r r i n were d i s s o l v e d i n 1 m l o f t h e same b u f f e r , t h e g e l suspended i n a t o t a l volume o f 3 m l and t r a n s f e r r e d t o t h e t r a n s f e r r i n s o l u t i o n . a f t e r i n c u b a t i o n f o r 30 min a t room t e m p e r a t u r e w i t h g e n t l e m a g n e t i c s t i r r i n g , cac12 was added and t h e c o u p l i n g r e a c t i o n c o n t i n u e d f o r a n o t h e r 30 m i n a t room t e m p e r a t u r e . 0.2 m l of 1 m e t h a n o l a m i n e hc1, ph 8.0 were t h e n added f o l l o w e d b y i n c u b a t i o n w i t h s t i r r i n g f o r 1 h a t room t e m p e r a t u r e , t h e g e l t r a n s f e r r e d t o a p a s t e u r p i p e t t e and washed o v e r n i g h t w i t h pbs, ph 7.5 (200 ml) and t h e n w i t h 100 m l o f 150 mm nacl phosphate c i t r a t e b u f f e r p r e p a r e d b y a d d i n g s o l i d na2hp04 t o 10 mm c i t r a t e u n t i l t h e ph r e a c h e d 2.8 ( p h o s p h a t e c i t r a t e , ph 2.8). s u b s e q u e n t l y i t was washed w i t h 100 m l o f pbs, ph 7.5, c o n t a i n i n g 1 m nac1, and t h e n a g a i n w i t h p h o s p h a t e c i t r a t e . f i n a l l y i t was e q u i l i b r a t e d w i t h pbs, ph 7.5, and 20 m l o f a n t i t r a n s f e r r i n i n t h e same b u f f e r were r e c i r c u l a t e d f o r 3 h o r o v e r n i g h t a t 4oc, t h e column washed w i t h pbs, ph 7.5 c o n t a i n i n g 1 m nacl f o l l o w e d b y e l u t i o n o f t h e a n t i t r a n s f e r r i n b y t h e p h o s p h a t e c i t r a t e b u f f e r . a p p r o x i m a t e l y 10 mg o f a n t i t r a n s f e r r i n were o b t a i n e d , j u d g i n g f r o m a280 and w i t h t r a n s f e r r i n a s a s t a n d a r d . the e l u t e d a n t i t r a n s f e r r i n was n e u t r a l i z e d and 18 c o n c e n t r a t e d t o a p p r o x i m a t e l y 10 mg/ml i n pbs, ph 7.5. one m l o f a f f i g e l i n 2 ml o f pbs, ph 7.5 was t h e n added and t h e c o u p l i n g r e a c t i o n a l l o w e d t o t a k e p l a c e w i t h g e n t l e s t i r r i n g a t room t e m p e r a t u r e f o r 2h. t h e r e a f t e r , 0.2 ml o f 1m e t h a n o l a m i n e -hc1, ph 8.0 were added and t h e i n c u b a t i o n c o n t i n u e d f o r 1 h a t room t e m p e r a t u r e . f i n a l l y , t h e g e l was t r a n s f e r r e d t o pbs, ph 7.5 as a 50% packed g e l s u s p e n s i o n . neuraminidase d i g e s t i o n o f transfer& twenty mg o f human t r a n s f e r r i n c o n t a i n i n g a p p r o x i m a t e l y 0.9 pmoles o f s i a l i c a c i d were d i s s o l v e d i n 1 ml o f 0.1 m sodium phosphate b u f f e r , ph 6.5 and i n c u b a t e d w i t h 0.5 u o f n e u r a m i n i d a s e from d a c t y l i u m d e n d r o i d e s f o r 30 m i n a t 37oc. (one u n i t o f n e u r a m i n i dase i s d e f i n e d a s t h e amount n e c e s s a r y t o l i b e r a t e 1.0 umole o f s i a l i c a c i d p e r m i n a t ph 5.0 and a t 37oc). a f t e r d i g e s t i o n , b l u e d e x t r a n was added as a m a r k e r f o r t h e v o i d volume and t h e sample f r a c t i o n a t e d i n pbs, ph 7.0 (20 mm na2hp04, 150 mm nac1, ph a d j u s t e d w i t h hc1) on a sephadex 6-200 column w i t h a d i a m e t e r o f 1.2 cm and a l e n g t h o f 38 cm. the t r a n s f e r r i n was e l u t e d and l o c a t e d b y i t s a b s o r p t i o n a t 280 nm. the c o n c e n t r a t i o n o f t r a n s f e r r i n was a d j u s t e d t o 1 mg/ml w i t h n a t i v e t r a n s f e r r i n as a s t a n d a r d . m i x t u r e s o f t h e n e u r a m i n i d a s e d i g e s t e d t r a n s f e r r i n and t r a n s f e r r i n were t h e n used f o r c o m p a r a t i v e a n a l y s i s o f s i a l i c a c i d u s i n g t h e d e s c r i b e d method. a c i d h y d r o l y s i s o f t r a n s f e r r i n . ten mg o f t r a n s f e r r i n were h y d r o l y z e d i n 1 ml o f 1n h2s04 a t 8ooc f o r i h , t h e s o l u t i o n n e u t r a l i z e d w i t h k2hp04 and t h e a s i a l o t r a n s f e r r i n s e p a r a t e d on t h e sephadex 6200 column d e s c r i b e d above. comparative a n a l y s i s o f s i a l i c a c i d . the a n a l y s i s o f s i a l i c a c i d i n t r a n s f e r r i n was c a r r i e d o u t b y c o m b i n i n g an o x i d a t i v e r e d u c t i v e s t e p w i t h b i n d i n g t h e g l y c o p r o t e i n t o a n t i t r a n s f e r r i n immunoadsorbent. the volume and c o n c e n t r a t i o n s o f t h e v a r i o u s s o l u t i o n s v a r i e d a c c o r d i n g t o t h e sequence i n w h i c h t h e s e s t e p s were p e r f o r m e d and t h e p r e s e n c e o r absence o f o t h e r g l y c o p r o t e i n components t h a n t r a n s f e r r i n . the f o l l o w i n g s o l u t i o n s were used: pbs, ph 7.0: 20 mm na2hp04, 150 mm nac1, ph a d j u s t e d w i t h hc1. t r i s g l y c i n e g l y c e r o l : 0.1 m t r i s b a s e a d j u s t e d t o ph 8.0 w i t h 0.1 m g l y c i n e and g l y c e r o l added t o 0.1 m. naoh/n’n:dimethylformamide (1: 1) t o a f i n a l c o n c e n t r a t i o n o f 1.78 m c i / m l . gel p r o t e c t i n g r e a g e n t . t h i s m i x t u r e p r o t e c t e d t h e s o l i d phase g e l , b y i n h i b i t i n g t h e r e a c t i o n o f l a b e l e d compounds w i t h t h e p a r t i c u l a r t y p e o f g e l used, and was p r e p a r e d as f o l l o w s : one volume o f pbs, ph 7.0 c o n t a i n i n g 2 mm sodium m e t a p e r i o d a t e was i n c u b a t e d w i t h one volume o f t r i s g l y c i n e g l y c e r o l f o r 30 m i n a t room t e m p e r a t u r e , and t h e n w i t h one volume o f 50% 0.1 m naoh and 50% n’n’-dimethylformamide c o n t a i n i n g 2mn sodium b o r o h y d r i d e . b o r o t r i t i d e s o l u t i o n : t r i t i a t e d sodium b o r o h y d r i d e d i s s o l v e d i n 0.1 m 19 washing solution pbs, ph 7 . 5 , containing 10% nn-dimethyl-formamide. 0.1 mg of transferrin were routinely used in the assay. the oxidative step was always performed after the solutions had reached o°c in an ice-bath. sodium meta-periodate (2mm) in pbs, ph 7.0 was added for oxidation which was allowed to proceed for 10 min. the reactions were quenched with tris-gly cine-glycerol and then either reduced with borotritide or washed and equi librated with 20 ~1 of imnunoadsorbent suspension. the details are described in the figure legends. after reduction, the gel was washed free of excess reagent, transferred t o a scintillation via7 and counted by liquid scintillation using 10 ml of aquasol. fig.1 0.1 mg of transferrin was dissolved in 0.1 ml of pbs, ph 7.0 and 0.1 m l of the same buffer containing 2 mm sodium metaperiodate was mixed at ice-temperature and incubated for 10 min. a mixture from 0.1 m glycine and 0.1 m tris, ph 8.0 containing 0.1 m glycerol was then added followed by incubation with stirring at room temperature for 30 min. the mixture was kept in ic’e for 30 m.in and 0.1 m l sodium borotritide, 1.78 mci/ml, in 50% 0.1m naoh/50% n’n’-dimethylformamide added. after incubation on ice for 30 min the labeled transferrin was separated from excess reagent by passing the mixture over a sephadex 6 2 5 column. the total fractions were mixed with aquasol and counted for tritium, ( 0 ) transferrin, (0) neuraminidase-digested transferrin, (x) acid-hydrolyzed transferrin (0.1 n h2s04 for lh at 8 o o c ) . 20 results and d i s c u s s i o n one hundred ug o f t r a n s f e r r i n o r d e s i a l y l a t e d t r a n s f e r r i n were l a b e l e d w i t h sodium meta-periodate-borotritide and t h e n s e p a r a t e d f r o m excess l a b e l e d b o r o t r i t i d e b y f r a c t i o n a t i o n on sephadex 6-25 columns ( p d 1 0 , pharmacia a b ) ( f i g . 1). subsequent e s t i m a t i o n o f t h e s u r f a c e a r e a u n d e r t h e v o i d peak c o n t a i n i n g t h e l a b e l e d p r o t e i n i n d i c a t e d t h a t s i a l o t r a n s f e r r i n i n c o r p o r a t e d a p p r o x i m a t e l y 3 t i m e s more l a b e l t h a n a s i a l o t r a n s f e r r i n . the a s i a l o t r a n s f e r r i n w h i c h was p r e p a r e d b y n e u r a m i n i d a s e d i g e s t i o n o r h y d r o l y s i s i n 0.1 n h2s04 a t 8 o o c f o r 1 h gave s i m i l a r r e s u l t s . the o x i d a t i o n w i t h p e r i o d a t e i s n o t e x c l u s i v e l y s p e c i f i c f o r s i a l i c a c i d (2) and t h e r e f o r e , a c e r t a i n background a c t i v i t y i n t h e absence o f s i a l i c a c i d must be a n t i c i p a t e d . f i g . 2a e s t i m a t i o n o f t r a n s f e r r i n b i n d i n g c a p a c i t y o f t h e a n t i t r a n s f e r r i n q e l . cpi 200 t r a n s f e r r i n was l a b e l e d b y o x i d a t i o n w i t h 1 mm nai04 a t ph 7.0, o°c f o r 10 m i n f o l l o w e d b y q u e n c h i n g w i t h 0.1 m g l y c i n e t r i s c o n t a i n i n g 0.1 m g l y c e r o l and t r i t i a t i o n w i t h sodium b o r o t r i t i d e . the l a b e l e d s i a l o t r a n s f e r r i n was t h e n t r a n s f e r r e d t o pbs ph 7.5 b y g e l f i l t r a t i o n on sephadex 6-25 (pd-10 column) and d i l u t e d t o 1 mg/ml and $ s p e c i f i c a c t i v i t y o f 5x10 cpm p e r mg. samples o f 2-150 ug o f l a b e l e d t r a n s f e r r i n were t h e n d i l u t e d t o e q u a l volume and i n c u b a t e d w i t h 20 u1 o f 50% ( v / v ) a n t i t r a n s f e r r i n g e l suspension, o r c o n t r o l g e l suspension, t h e m i x t u r e d i l u t e d t o 3 m l w i t h pbs ph 7.5 and t h e g e l p e l l e t e d a f t e r w h i c h 0.1 m l o f t h e s u p e r n a t a n t was t r a n s f e r r e d t o a s c i n t i l l a t i o n v i a l and c o u n t e d w i t h aquasol u s i n g t h e t r i t i u m c h a n n e l i n a l i q u i d s c i n t i l l a t i o n coun t e r . the p o i n t s r e p r e s e n t t h e r a d i o a c t i v i t y c o n t a i n e d i n t h e s u p e r n a t a n t , i n ( x ) t e s t t u b e o n l y ; ( 0 ) : a f f i -gel -10, t r e a t e d as f o r b i n d i n g a n t i t r a n s f e r r i n ; (0): a f f i g e l c o u p l e d t o a n t i t r a n s f e r r i n . 21 labeled sialotransferrin was prepared and used to assess the antigen-binding capacity of the antitransferrin gel in relation to unspecific adsorption (fig 2a,b). it is evident from fig 2a, where a sample from the supernatant not reacting with the gel is analyzed, that virtually no background binding of labeled transferrin occurred, neither to the gel nor to the test tube. ten p1 of the antitransferrin gel bound 10-25 pg of transferrin when equilibrated with a total amount of 15-150 pg in solution, judging from the control standard curve with no transferrin antibodies (fig 2 a ) . when 100 pg of transferrin were added, approximately 22 pg bound to the gel. in fig 28 where the amount of gel was varied, figures from 18-36 pg of bound transferrin per 10 u l of antitransferrin gel ( 2 0 p i o f gel suspension) were obtained. the higher values probably represented a saturation phenomenon in the equilibrium binding of the antigen-antibody during the short (30 min) incubation time used. the results clearly show that approximately 20 ug of transferrin bound to 10 p1 of gel. cpm 150c 1000 500 i i i i 20 40 60 80 gel suspension ( p l ) :i,g. 2i’ same experiment as except that the amount of gel suspension was varied and the amount of labeled transferrin kept constant, 10.0 vg: the radioactivity remaining in 0.1 ml of supernatant after incubation of the mixture, dilution to 3 ml, and pel leting was measured. 22 fig. 3 a combination o f oxi d a t i a n r e d u c t i on w i t h binding , of t r a n s f e r r i n followed by s e p a r a t i o n of the l a b e l e d immunocomplex i n a s i n g l e step. the procedure i n f i g 2 was followed, p b s , ph 7.5 was added t o a t o t a l volume o f 0.5 ml and 20 vl of t h e a n t i t r a n s f e r r i n gel added and t h e mixture incubated w i t h vigorous shaking f o r 30 m i n a t room temperature. a f t e r i n c u b a t i o n , the gel was allowed t o sediment, t h e s u p e r n a t a n t a s p i r a t e d and t h e gel washed 5 times w i t h 5-10 m l o f washing s o l u t i o n . the f i n a l s u p e r n a t a n t was checked t o a s s u r e c l o s e t o background-levels of r a d i o a c t i v i t y : 0-0 t r a n s f e r r i n o n l y , 0-0 t r a n s f e r r i n with 25 u1 of serum. 0 20 40 60 80 100 sialotransferrln( per centof added amount of transferrin including asialotransferrin ) 23 total c pm 1o.ooc 8000 6000 4 000 2000 1 1 i i f i g . 38 c o m b i n a t i o n o f o x i d a t i o n r e d u c t i o n w i t h b i n d i n g o f t r a n s f e r r i n f o l l o w e d by s e p a r a t i o n o f t h e l a b e l e d immunocomplex'in a s i n g l e s t e p . the c o n c e n t r a t i o n o f t r i s g l y c i n e g l y c e r o l was r e d u c e d t o h a l f b y d i l u t i n g w i t h w a t e r , o t h e r w i s e t h e o x i d a t i o n q u e n c h i n g r e d u c t i on sequence f o l l o w e d . the a n t i t r a n s f e r r i n g e l was f i r s t i n c u b a t e d w i t h g & l p r o t e c t i n g r e a g e n t a t 0 c. the c o m p o s i t i o n o f t h i s r e a g e n t i s d e s c r i b e d i n t h e methods s e c t i o n . t h e r e a f t e r t h e t r a n s f e r r i n was t r a n s f e r r e d t o t h e g e l s u s p e n s i o n and t h e m i x t u r e i n c u b a t e d f o r 60 m i n a t room t e m p e r a t u r e f o l l o w e d b y washing. 0 20 40 60 80 100 sialotransferrin (per centof transferrin ) 24 i n f i g 3a-c, t h e o x i d a t i o n r e d u c t i o n o f t r a n s f e r r i n was combined w i t h b i n d i n g t h e g l y c o p r o t e i n t o t h e immunoadsorbent and s e p a r a t e d f r o m t h e m i x t u r e i n a s i n g l e s t e p . i n 3a, t h e p r o c e d u r e i n f i g 2 was f o l l o w e d and t h e g l y c o p r o t e i n i n c u b a t e d w i t h t h e g e l i n t h e p r e s e n c e o f excess r a d i o a c t i v e p r o d u c t s . the f i n a l s u p e r n a t a n t a f t e r w a s h i n g was checked t o a s s u r e c l o s e t o background l e v e l s o f r a d i o a c t i v i t y . the h i g h background a c t i v i t y w i t h a s i a l o t r a n s f e r r i n o n l y , was due t o c h e m i c a l r e a c t i o n o f t h e r a d i o a c t i v e p r o d u c t s w i t h t h e g e l m a t r i x , s i n c e i t was o b s e r v e d w i t h a s i m i l a r l y t r e a t e d g e l l a c k i n g a n t i t r a n s f e r r i n . the h i g h background a c t i v i t y was n o t r e d u c e d b y t r e a t i n g t h e g e l w i t h 1 mm sodium b o r o h y d r i d e p r i o r t o e x p o s u r e t o t h e r a d i o a c t i v e p r o d u c t s . the two c u r v e s i n f i g 3a r e p r e s e n t t r a n s f e r r i n o f v a r i o u s s i a l i c a c i d c o n t e n t w i t h o r w i t h o u t 25 u1 o f serum added b e f o r e t h e o x i d a t i v e s t e p . as e x p e c t e d t h e a d d i t i o n o f serum c o n t a i n i n g s i a l y l a t e d t r a n s f e r r i n i n c r e a s e d t h e l a b e l i n g o f t h e samples w i t h a l o w c o n t e n t o f t r a n s f e r r i n and a h i g h c o n t e n t o f a s i a l o t r a n s f e r r i n . the c o n c e n t r a t i o n o f t r a n s f e r r i n i n t h e serum sample can be e s t i m a t e d t o be 3 . 4 g / 1 f r o m t h e r a t i o o f d i l u t e d / u n d i l u t e d sample s i n c e t h e t o t a l amount o f t r a n s f e r r i n added exceeded t h e t r a n s f e r r i n b i n d i n g c a p a c i t y o f t h e immunoadsorbent. t h i s was w i t h i n t h e normal r a n g e ( 4 ) , d e m o n s t r a t i n g t h a t t h e method was r e l i a b l e q u a n t i t a t i v e l y . i n f i g 3b, t h e g e l was f i r s t i n c u b a t e d w i t h t h e g e l p r o t e c t i n g r e a g e n t b e f o r e i n c u b a t i o n w i t h t h e t r a n s f e r r i n m i x t u r e c o n t a i n i n g r a d i o a c t i v e p r o d u c t s . t h i s d e c r e a s e d t h e background r a d i o a c t i v i t y s i g n i f i c a n t l y . the o b s e r v e d background a c t i v i t y i n f i g 38, u s i n g a s i a l o t r a n s f e r r i n o n l y , was a p p r o x i m a t e l y 1/3 o f t h e i n c o r p o r a t i o n o f t r i t i u m i n t h e f u l l y s i a l y l a t e d t r a n s f e r r i n . t h i s p e r c e n t a g e was comparable t o t h e u n s p e c i f i c i n c o r p o r a t i o n i n t o a s i a l o t r a n s f e r r i n as d e m o n s t r a t e d i n f i g 1, w h i c h was most p r o b a b l y due t o i n c o r p o r a t i o n o f l a b e l i n t o o t h e r s u g a r r e s i d u e s t h a n s i a l i c a c i d . i n f i g 3c, t h e t r a n s f e r r i n was f i r s t o x i d i z e d , t h e n bound t o t h e a n t i t r a n s f e r r i n g e l , washed, and f i n a l l y i n c u b a t e d w i t h b o r o t r i t i d e and t h e a c t i v i t y i n c o r p o r a t e d i n t o t h e s o l i d phase t r a n s f e r r i n measured. the e x p e r i m e n t s i l l u s t r a t e d i n f i g 3 show t h a t t h e method p r e s e n t e d can be used t o assess s i a l i c a c i d c o n t e n t i n a g l y c o p r o t e i n and t h a t t h e sequence o f t h e v a r i o u s i n c u b a t i o n s i s n o t c r i t i c a l t o a c h i e v e a c o m p a r a t i v e a n a l y s i s o f t h e sugar. the c r i t i c a l s t e p i s t h e s e p a r a t i o n f r o m a m i x t u r e o f t h e immuno complex c o n t a i n i n g t h e l a b e l e d s u g a r . the method p r e s e n t e d may be used as an a l t e r n a t i v e t o i s o e l e c t r i c f o c u s s i n g when a l a r g e number o f samples a r e p r o c e s s e d s i m u l t a n e o u s l y t o o b t a i n i n f o r m a t i o n on t h e a v e r a g e c o n t e n t o f s i a l i c a c i d i n a g l y c o p r o t e i n . t h e method does n o t p r e s e n t l y seem t o be s e n s i t i v e enough t o measure t h e p r e v i o u s l y r e p o r t e d m i c r o h e t e r o g e n e i t y o f serum t r a n s f e r r i n i n c o n n e c t i o n w i t h 25 alcohol abuse which a t b e s t involves a d i f f e r e n c e of about 10% of t h e amount of t r a n s f e r r i n and 5% of t h e t o t a l amount of t r a n s f e r r i n b o u n d s i a l i c a c i d ( 3 ) . total cpm 20.000 15 ooc 10.000 500c i i i i 3 c combination o f oxi a ti on-reducti o n with binding of trans f e r r i n followed by s e p a r a t i o n o f t h e l a b e l e d immunocomplex i n a s i n g l e s t e p . the t r a n s f e r r i n was f i r s t o x i d i z e d , then incubated with t r i s g l y c i n e g l y c e r o l f o r 60 min a t room temperature and then with 20 ,1 of gel suspension f o r a n o t h e r 60 min. there a f t e r the gel was washed, f i r s t w i t h pbs c o n t a i n i n g 1% albumin and then with pbs ph 7.0 0.1 ml of b o r o t r i t i d e s o l u t i o n was then added and t h e volume made 0.6 ml w i t h pbs, ph 7 . 0 followed by i n c u b a t i o n f o r 5 m i n a t room t e m p e r a t u r e , washing and measuring bound r a d i o a c t i v i t y . 0 20 40 60 80 100 sialotransferrin (per cent of transferrin) 26 references 1. cervgn, e., s t i b l e r , h. & borg, s.: d e t e r m i n a t i o n o f t e r m i n a l s u g a r s i n t r a n s f e r r i n b y r a d i o l e c t i n immunoassay ( r l i a ) a new m i c r o a n a l y t i c a l p r o c e d u r e . u p s a l a j med s c i 86: 39-53, 1981. i n : the c a r b o h y d r a t e s . c h e m i s t r y and b i o c h e m i s t r y , vol i b . ( e d s . w . pigman, d h o r t o n ) p 255-280. academic p r e s s , new york and london, 1980. s t i b l e r , h., borg, s. & a l l g u l a n d e r , c . : abnormal m i c r o h e t e r o g e n e i t y o f t r a n s f e r r i n a new m a r k e r o f a l c o h o l i s m ? s u b s t a l c o h o l a c t i o n s m i s u s e 1: 247-252, 1980. 2. p e r l i n , a.s.: 3 . 4. weeke, b. & k r a s i l n i k o f f , p.a.: the c o n c e n t r a t i o n o f 2 1 serum p r o t e i n s i n normal c h i l d r e n and a d u l t s . a c t a med scand 192: 149-155, 1972. address f o r r e p r i n t s : gunnar r o n q u i s t department o f c l i n i c a l c h e m i s t r y u n i v e r s i t y h o s p i t a l s-751 85 uppsala 27 upsala j med sci 82: 1-5, 1977 cyclic 3’,5’-gmp independent protein kinase at the outer surface of intact ehrlich cells gunnar ronquist, gunnak .‘&ren, sten eklund and christer wernstedt from the institute of medical and p h y s i o l * v i c a l chemistry, u p p s a l a , s w e d e n abstract the phosphoryl group transfer from ( y ~ ~ p ) a t p into ac ceptor proteins by an endogenous protein kinase a t the surface of ehrlich cells has been further studied as regards possible stimulation by different concentrations of dibuturyl cyclic guanosine monophosphate (3’,5’-gmp). using the endogenous acceptor protein of the surface of ehrlich cells the cyclic nucleotide had no stimulatory effect on the protein kinase of the plasma membrane. the lack of stimulatory action of the cyclic nucleotide was also observed when an exogenous acceptor protein was present. instead, a slight inhibitory effect was usually seen in both types of experi ments. labeled phosphorylserine was always in exces of labeled phosphoryl threonine. both were isolated from hydrolyzed acceptor proteins. the lack of stimulation by a cyclic nucleotide on the phosphorylation of acceptor pro tein@) on the cell surface does not rule out a regulatory function by the protein kinase of the plasma membrane. instead, we propose an autoregulatory mechanism for the phosphorylation a t the cell surface. this mechanism is based upon the high sensitivity of the enzyme to ca-ions. introduction the presence of endogenous protein kinase at the surface of ehrlich cells catalyzing the phosphoryla tion of endogenous membrane protein(s) was first described 1970 (1,22) and in some following reports ( 2 , 4 , 23, 24). schlatz & marinetti described in 1971 the cyclic amp-dependent phosphorylation of iso lated rat liver plasma membrane by a partially purified protein kinase from rat liver (28). in a simi lar experiment kinzel & mueller (13) were able to demonstrate the phosphorylation of endogenous acceptor proteins at the surface of intact hela cells in the presence of a protein kinase isolated from rat skeletal muscle and ( y ~ ~ p ) a t p in the external medium. this reacting was also stimulated to a limited degree by cyclic amp. protein kinase catalyzed membrane phosphorylations, either in volving the endogenous protein kinase and an ex trinsic acceptor protein or an extrinsic enzyme and the endogenous acceptor or where both enzyme and acceptor are endogenous, have subsequently been published for many membrane systems (for refer ences, see ref. 4). few of these protein kinase mediated reactions have been cyclic amp independent ( 5 , 6, 10, 21, 31, 34) including the plasma membrane phosphorylation system for the ehrlich cell (4, 23, 24). many cyclic amp-dependent protein kinases are, however, known to be activated by more than one 3’3’-cyclic nucleotide. hence, cyclic gmp-specific kinases have in recent years been found in pig lung (16), guinea pig fetal lung (19), bovine adrenal glands (30), rat pancreas (33) and cerebellum (1 l ) , mammalian smooth muscle (7) and heart tissue (15, 17). furthermore, it has been claimed that mem brane-associated, cyclic gmp-dependent pro tein kinases might mediate the effects of cholinergic agents in a manner quite analogous to the actions of particulate and soluble cyclic amp-dependent pro tein kinases in mediating cellular responses to polypeptide hormones, adrenergic agents and neurotransmitters (26). cyclic gmp-stimulated protein kinase differs from that stimulated by cyclic amp as regards some physicochemical properties (19, 20, 32). neverthe less, enzymatic properties for both types of protein kinases are the same as regards the requirements of binding steps at 3 different sites in the molecule, namely the binding of cyclic nucleotide for activa tion, the binding of atp and mg2+ and the binding of protein substrate for the reaction (14). smooth muscle is the only tissue in which any endogenous substrate proteins for the cyclic gmp dependent protein kinases have so far been found (9), and it was of interest to extend the work on the i -772x54 upsula j m r d sci 82 2 gunnar ronquist et al. table i. effect of dibuturylcyclic g m p on the en dogenous phosphorylation of the surface mem brane of ehrlich cells the figures denote the phosphoryl group transfer in nmol/lx lo8 cells per min at 37°c into endogenous acceptor protein serp thrp peak 1 (a) incubation of intact ehrlich cells with exogenous (y”p) atp and without cyclic nucleotide in the medium 0.190 0.040 0.003 ( b ) as ( a ) but in the presence of 1 x dibuturyl cyclic gmp 0.130 0.040 0.003 endogenous protein kinase(s) as well as the en dogenous acceptor proteins at the surface of ehr lich cells so as to encompass even a study of the possible cyclic gmp dependency. the present re sults demonstrate the lack of influence of cyclic gmp in the concentration range 1 x m on the protein kinase system at the outer surface of ehrlich cells. the steric dispositions of the regu lator to the catalytic subunit have been investi gated by means of dibuturyl cyclic gmp. the reac tion was also studied with an exogenous phosphoryl group acceptor protein as well as with a purified membrane fraction. m1 x material and methods materials. all chemicals were of analytical grade, (y3’p)atp was purchased from nen chemicals, gmbh, frankfurt am main, germany. unlabeled atp (sodium form), cyclic and dibuturyl cyclic gmp as well as phosvi tin were all obtained from sigma chemical company, st. louis, mo., usa. preparation of tumor cells. the ehrlich ascites tu mour cells were grown for 8-10 days in 5-week-old male swiss albino mice obtained from the anticimex breeding farm, norrviken, stockholm. the tumour cells were separated by centrifugation of the ascitic fluid, which had been diluted without delay 10-fold with ice-cold krebs-ringer bicarbonate medium, in order to diminish the tendency of cell agglutination. the cells were then washed twice in the krebs-ringer bicarbonate medium. final washing was performed with a medium of 130 mm nacl and 25 mm kci for experiments with intact cells. for the preparation of a membrane fraction the cells were finally washed in a sucrose-(0.250 m)-tris-glycylglycine (0.016 m) buffer, ph 8.0, also containing 2 mm caci,, followed by centrifugation (25). the washings of intact cells were carried out in the international refrigerated centrifuge by acceleration for 10-15 sec up to at most upsalu j m e d sc; 82 5700 rpm followed by retardation for about 2 min. all preparatory steps were carried out at 4°c. preparation of plasma membrane fraction. the proce dure described by ronquist & christensen (25) was fol lowed exactly. the final membrane fraction was suspended in 130 mm nacl and 25 mm kci in a concen tration corresponding to about 20 mg membrane protein per ml. incubation procedure. incubations were performed di rectly after the preparation of the cells and the membrane fraction. about 1 x lo8 cells or a membrane fraction cor responding to 20 mg membrane protein were suspended in an incubation volume of 9.5 ml consisting of 130 mm nacl and 25 mm kci. it contained in addition 350 pmoles of tris-acetic acid buffer, ph 7.5, 20 pmoles of mgci,, 10 pmoles of nazhpo, (unlabeled), 5 pmoles of glutathione (reduced form) and 0.5 pmoles of ( y ~ ~ p ) a t p . in some experiments, 2.5 pmoles of phosvitin were also included. dibuturylcyclic gmp, when present, was used in final concentratims of 1 x m1 x lo-’ m. the commercial phosvitin preparation was free from protein kinase activi ty. incubation was performed at 37°c and teminated after 1 min by rapid centrifugation. the entire centrifugation procedure did not exceed 30 sec. the supernatant repre senting the external medium including the acceptor pro tein in most experiments was immediately precipitated with trichloroacetic acid (10 % final concentration). in order to completely precipitate the low molecular weight acceptor protein, 50 mg of bovine albumin were added to the precipitating agent and the cell pellets were precipi tated with trichloroacetic acid. the precipitation was al lowed to proceed for at least 24 h at 4°c. the precipitates were washed once with 10% trichloroacetic acid con taining 5 mm of unlabeled orthophosphate and 1 mm of atp. i n order to remove nucleic acid and phozpholipids, the precipitate was treated according to schneider (29). par tial hydrolysis of the schneider protein was performed using the method of lipmann (18). labeled phos phorylserine and phosphorylthreonine and phosphopep tides were isolated by ion exchange chromatography, as described previously (23). a radiometric determina tion of the different fractions from column chromato graphy was carried out in a nuclear chicago scintilla tion counter by measuring the cerenkov radiation. results during the separation process two other (32p) labelled phosphopeptides are recovered in addi tion t o the (32p)-labelied phosphorylserine and phosphorylthreonine. those peptides have been called peak i and 2 , respectively ( 2 3 ) . table i il lustrates the lack of stimulation by ix m di buturylcyclic gmp upon the endogenous mem brane-associated protein kinase on phosphoryla tion of the endogenous phosphoryl group acceptor in the surface membrane of intact ehrlich cells. instead a slight decrease of the degree of cyclic 3',5'-gmp independent protein kinase 3 table 11. effect of various concentrations of di buturylcyclic gmp on the phosphorylation of an exogenous acceptor protein by a protein kinase as sociated with the surface membrane of ehrlich cells the figures denote the phosphoryl group transfer in nmol/lx lo8 cells per min at 37°c into acceptor protein serp thrp peak 1 ( a ) phosvitin as exogenous phosphoryl group acceptor together with intact ehrlich cells 0.580 0.225 0.103 ( b ) as ( a ) plus 1 x io-'m 0.540 0.231 0.098 dibuturylcyclic gmp ( c ) as ( a ) plus 1 x m dibuturylcyclic gmp 0.473 0.197 0.076 (d) as ( a ) plus 1 x 10-4m dibuturylcyclic gmp 0.482 0.173 0.081 phosphorylation is seen in the presence of the cyclic nucleotide. this observation is in agreement with earlier findings with cyclic amp (4, 23). the ali phatic cyclic nucleotide is assumed to penetrate the plasma membrane. thus, providing the cyclic gmp-binding subunit is in trans-position to the catalytic subunit, it would nevertheless be possible for the added cyclic nucleotide t o react. however, in experiments with intact cells the possibility of binding to the receptor site by endogenous cyclic nucleotide(s) cannot be ruled out. the lack of stimulation by different concentra tions of dibuturyl cyclic gmp on the phosphoryla tion of phosvitin as exogenous phosphoryl group acceptor in the presence of intact ehrlich cells is shown in table 11. instead, a slight inhibitory ac tion is again seen, especially at the two higher con centrations. the incorporation ratio between phosphorylserine and phosphorylthreonine re mained constant and about 2 . 5 , regardless of the absznce or presence of different concentrations of dibuturylcyclic gmp in the incubation media. as mentioned before, the possibility of a reaction between cellular cyclic gmp and the receptor site of the regulatory subunit on the inner surface of the intact membrane could not be ruled out. in such a case additional cyclic gmp would have no or very little effect. therefore, experiments were also performed on a purified membrane preparation mainly consisting of vesicles (25). it is evident from table 111 that n o stimulatory effect is exerted by 1 x m dibuturylcyclic gmp in the medium also containing phosvitin and the membrane vesicles from ehrlich cells. instead, a small inhibitory effect is seen, consistent with the findings for the intact cells. the incorporation ratio between phosphoryl serine and phosphorylthreonine was increased about ~~ 3 times compared with that for intact cells. discussion we propose that the protein kinase of the plasma membrane of ehrlich cells is not dependent on either cyclic gmp in accordance with the present work or cyclic amp (4, 23). furthermore, at least some of this protein kinase is associated with the outer surface of the ehrlich cell membrane. an external protein kinase activity in rat c-6 glioma cells was claimed in a recent report to be cyclic amp-dependent (27). in the experimental system used, histone was the external phosphoryl group acceptor. n o data were presented concerning any phosphorylation of a possible endogenous ac ceptor protein of the rat glioma cells, nor was any other acceptor protein used (27). we have also studied the protein kinase at the outer surface of human glioma cells (3), as well as of ehrlich cells (24). in both types of cells we found a stimulation by cyclic amp but only when histone was the ex ogenous phosphoryl group acceptor. furthermore, in the case of ehrlich cells the maximal phosphory lation with histone as the exogenous acceptor was 8 times lower than with phosvitin as the exogenous table 111. effect of dibuturylcyclic gmp on the phosphorylation of an exogenous acceptor protein by a protein kinase associated with a plasma membrane prepparpation from ehrlich cells the figures denote the phosphoryl group transfer in nmol/mg schneider protein of membrane vesicles per min at 37°c into acceptor protein. for comparison with tables 1 and 2, 1 x lo8 ehrlich cells correspond to about i5 mg schneider protein from membrane vesicles prepared from ehrlich cells ~ serp thrp peak 1 peak 2 ( a ) phosvitin as exogenous phosphoryl group ac ceptor together with membrane vesicles from ehrlich cells 0.451 0.069 0.022 0.004 ( b ) as ( a ) plus 1 x 10-fim dibuturylcyclic g m p in incubation medium 0.440 0.046 0.022 0.004 4 gunnar ronquist et al. acceptor under otherwise same experimental condi tions (24). also, the amount of phosphorylation of histone as the exogenous acceptor did not exceed that of the endogenous acceptor of the ehrlich cells (24). the endogenous phosphorylation of ehrlich cell membranes is not stimulated by cyclic amp (23). we have therefore interpreted the effect of cyclic amp with histone as the exogenous acceptor protein as secondary, e.g. interfering with the basic histone-protein interaction with the netto-negative charges of the intact cells (24). however, the ex istence of at least two or more protein kinases at the ehrlich cell surface with different properties vis-d vis cyclic nucleotides cannot be excluded. the experiments with diffferent concentrations of cyclic gmp in the medium (table 11) showed a very constant phosphorylserine to phosphoryl threonine ratio throughout the experiments, thus favouring the view of homogeneity as regards pro tein kinase activity in the surface membrane of ehr lich cells. the labelled phosphorylserine is always in excess of labelled phosphorylthreonine ( 2 , 4, 23, 24). this observation has been confirmed to be valid also for hela cells and sv3t3-ts cells in a recent work (12). a phosphorylation and dephosphorylation cycle of membrane proteins have been suggested to oc cur, thereby changing their charge and conforma tion (8). furthermore, such changes might affect the interaction of the membrane proteins with the neighbouring lipids or lipoprotein complexes result ing in the aggregation of intramembrane particles and relative increase in the exposed free lipid bilayer phase of the membrane (8). a cyclic nucleotide dependency of the protein kinase in the surface membrane does not appear to be obligatory. instead we propose an autoregula tory mechanism based upon the actual ca’+ concentration in the microenvironment of the surface membrane. we found a strong inhibitory effect by ca2+ on the endogenous protein kinase at the membrane surface of ehrlich cells (23). maxi mal activity was obtained with mgz+, na+ and k + with no ca2+ in the system. when half the amount of mg2+ was stoichiometrically exchanged for ca2+ the protein kinase activity decreased by about 50% and when all mg2+ was replaced by ca2+, more than 80% of the activity was lost. therefore, in a maxi mally dephosphorylated state of the surface membrane of the cell, the caz+ content of the microenvironment of the cell surface might be low and the protein kinase activity high, while in a maximally phosphorylated state ca2+ ions are elec trostatically attracted and therefore the protein kinase activity is lower. acknowledgements this investigation was supported by a grant from the swedish medical research council (project no b7613x 0022812a). references i . 2. 3. 4. 5. 6. 7. 8. 9. 10. 11 agren, g . & ronquist, g . : isolation of 32p-labelled phosphorylserine from ehrlich mouse-ascites tumour cells suspended in an isotonic medium containing 32p labelled adenosine triphosphate. acta physiol scand isolation of s2p-labelled phosphorylserine and phosphorylthreonine from ehrlich mouse-ascites tumour cells, suspended in an isotonic medium con taining 32p-labelled nucleoside triphosphates or inor ganic pyrophosphates. acta chem scand 25: (32p)phosphoryl transfer by endogenous protein kinase a t the glia and glioma cell surface in culture into extrinsic acceptor proteins. acta physiol scand 92:430432, 1974. phosphorylation of endogenous membrane pro teins by endogenous protein kinase at the outer surface of ehrlich cells. upsala j med sci 81: 129 134, 1976. andrew, c., roses, a . d., almon, r. r. & appel, s. h.: phosphorylation of membranes: identification of a membrane-bound protein kinase. science 182: bacalao, j. & rieber, m . : on the properties of membrane-associated protein kinase from chinese hamster ovary cells. febs lett37:37-41, 1973. casnellie, j. e. & greengard, p.: guanosine 3’3’ monophosphate-dependent phosphorylation of en dogenous substrate proteins in membranes of mam malian smooth muscle. proc natl acad sci usa gazitt, y . , ohad, i. & loyter, a.: phosphorylation and dephosphorylation of membrane proteins as a possible mechanism for structural rearrangement of membrane components. biochim biophys acta greengard, p.: possible role for cyclic nucleotides and phosphorylated membrane proteins in postsynaptic actions of neurotransmitters. nature 260: 101-108, 1976. ho, r., russell, t., asakarva, t. & snyder, r. p.: influence on adipocyte plasma membrane bound pro tein kinase by feedback regulator. j cyc nucleo res 1:349-358, 1975. hofman, f. & sold, g . : a protein kinase activity from rat cerebellum stimulated by guanosine-3’s’-mono phosphate. biochem biophys res commun 49: 1100-1107, 1972. 79: 125-128, 1970. 2931-2934, 1971. 927-929, 1973. 71: 1891-1895, 1974. 436: 1-14, 1976. upsalu j mrd s c i 82 12. 13. 14. 15. 16. 17. 18. 19. 20. 21 22 23 24 25 kinzel, v., kiibler, d., mastro, a. m. & rosengurt, e.: phosphoproteins of the cell surface as generated by endogenous or exogenous protein kinase: stability of the 32p-labelled product. biochim biophys acta kinzel, v. & mueller, g.: phosphorylation of surface proteins of h e l a cells using an exogenous protein kinase and ( y ~ ~ p ) atp. biochim biophys acta 322: 337-351, 1973. kobayashi, r. & fang, v.: studies on cyclic gmp dependent protein kinase properties by blue dextran sepharose chromatography. biochem biophys res commun69: 1080-1087, 1976. kuo, j. f., davis, c. w. & tse, j.: depressed cardiac cyclic gmp-dependent protein kinase in spontane ously hypertensive rats and its further depression by guanethidine. nature 261: 335-336, 1976. kuo, j. f . , kuo, w-n., shoji, m., davis, c. w., seery, v. l . & donelly, t. e.: purification and gen eral properties of guanosine 3’3’-monophosphate dependent protein kinase from guinea pig fetal lung. jbiol chem251: 1759-1766, 1976. kuo, j-f., lee, t-p., reyes, p. l . , walton, k . g., donnelly, t. e. & greengard, p.: cyclic nucle otide-dependent protein kinases x . an assay method for the measurement of guanosine 3’ ,5’-monophos phate in various biological materials and a study of agents regulating its levels in heart and brain. j biol chem247: 1 6 2 2 , 1972. lipmann, f.: uber die bindung der phosphosaure in phosphoproteinen. i. mitteilung: isolierung einer phosphorhaltigen aminosaure (serinphosphosaure) aus casein. biochem z262:3, 1933. nakazava, k. & sano, m.: partial purification and properties of guanosine 3‘,5’-monophosphate-de pendent protein kinase from pig lung. j biol chem nishiyama, k., katakami, h., yamamura, h . , takar, y . , shimomura, r. & nishizuka, y.: func tional specificity of guanosine 3’,5‘-monophos phate-dependent and adenosine 3’,5’-monophos phate-dependent protein kinases from silkworm. j biol chem2.50: 1297-1300, 1975. prasad, k . n., fogleman, m., gaschler, m., sinha, p. k . &brown, l.: cyclic nucleotide-dependent protein kinase activity in malignant and cyclic amp-induced “differentiated” neuroblastoma cells in culture. bio chem biophys res commun68: 1248-1254, 1976. ronquist, g. & agren, g.: isolation of 32p-labelled phosphorylthreonine from ehrlich mouse-ascites tumour cells suspended in an isotonic medium con taining 32p-labeiled adenosine triphosphate. acta chem scand 24: 728-729, 1970. isolation of 32p-labelled phosphorylserine and phosphorylthreonine from ehrlich mouse-ascites tumour cells suspended in different isotonic media containing 32p-labelled adenosine triphosphate. acta chem scand28: 1169-1174, 1974. (32p)phosphoryl transfer by endogenous protein kinase at the ehrlich cell surface into extrinsic ac ceptor proteins. upsala j med sci 79: 138-142, 1974. ronquist, g. & christensen, h.: amino acid stimula 434: 281-285, 1976. 250:7415-7419, 1975. cyclic 3 ‘ 3 ‘-gmp independent protein kinase 5 26. 27. 28. 29. 30. 31. 32. 33. 34. tion of alkali-metal-independent atp cleavage by an ehrlich cell membrane preparation. biochim biophys acta323: 337-341, 1973. rubin, c. s. & rosen, 0. m.: protein phosphoryla tion. annu rev biochem44: 831-877, 1975. schluger, e-j. & kohler, g.: external cyclic amp dependent protein kinase activity in rat c-6 glioma cells. nature 260: 705-707, 1976. schlatz, l. & marinetti, g . v.: protein kinase mediated phosphorylation of the rat liver plasma membrane. biochem biophys res commun45: 51-56, i97 1. schneider, w. c.: phosphorous compounds in animal tissues. i . extraction and estimation of desoxy pentose nucleic acid. j biol chem 151:293-303, 1945. shima, s . , mitsunaga, m., kawashima, y., taguchi, s. & nakao, t.: studies on cyclic nucleotides in the adrenal gland. 111. properties of cyclic ampand gmp-dependent protein kinases in the adrenal gland. biochim biophys acta341: 5 6 4 4 , 1974. steiner, m.: endogenous phosphorylation of platelet membrane proteins. arch biochem biophys 171: 245-254, 1975. takai, y . , nishiyama, k., yamamura, h. & nishizuka, y .: guanosine 3’,5’-monophosphate dependent protein kinase from bovine cerebellum. purification and characterization. j biol chem 250: 4690-4695, 1975. van leemput, m., camus, j . & christophe, j.: cyclic nucleotide-dependent protein kinases of the rat pancreas. biochem biophys res comrnun 54: 182 190, 1973. weller, m., virmaux, n. & mandel, p.: light stimulated phosphorylation of rhodopsin in the retina: the presence of a protein kinase that is specific for photobleached rhodopsin. proc natl acad sci usa 72:381-385, 1975. received august 28, 1976 address for reprints: gunnar &yen, m.d. institute of physiological and medical chemistry box 575 s-75123 uppsala sweden upsulu j med sci 82 upsala j med sci 84: 228-234, 1979 thyroid function in breast cancer patients before and up to two years after mastectomy hans-olov adam?, jorgen hansenz, ake rimsten' and leif wide3 from the departments of surgery', oncologyz and clinical chemistry3, university hospital, uppsalu, sweden abstract in 41 women newly diagnosed as having breast cancer the thyroid function was assessed by determination of the serum tsh, tririodothyronine (t3), reverse tri iodothyronine (rt3), thyroxine (t4), t3-resin uptake and free t4-index. blood samples were drawn before the primary treatment and at follow-up after 1 2 0 months. there was no significant change in any of these variables during the period of observation. nor was there any difference between the values of the patients who developed recurrent disease and of those who did not. these results contradict previously proposed hypotheses of a progressive de crease in thyroid function after primary treatment and of a relation between the clinical course and the thyroid function in breast cancer patients. introduction the hypothesis that breast cancer and thyroid function are related to each other has been discussed from different viewpoints ever since beatson in 1896 (4) suggested thyroid as a treatment in advanced breast cancer. in a previous study ( 2 ) we analyzed the possible role of thyroid disease or dysfunction as an etiologic factor in breast cancer. a comparison with non-hos pitalized controls revealed that the patient group had a slightly but signifi cantly higher mean serum concentration of tsh, rt3 and t3-resin uptake and a lower t3 than the control group. these findings were considered inconsistent with a hypothyroidism, which has repeatedly been suggested as increasing the risk for breast cancer (cf 2, 1 7 ) . the same pattern of changes have, however, recently been reported to be secondary to a number of non-thyroidal diseases ( 5 ) and we concluded that the results of our study could not support the concept that a thyroid dysfunction is an etiologic factor in breast cancer. in a subsequent study (1) further support for this conclusion was obtained when the thyroid function was assessed before, during and at different times after mastectomy in breast cancer patients and compared with that of women undergoing a cholecystectomy. there were no significant differences in tsh or 228 thyroid hormone levels between the two groups. both groups showed changes after surgical trauma which were in close agreement with those previously noticed as an unspecific reaction to illness. the aim of the present study was to find out whether there is a progressive change in thyroid function and if so, whether this is in any way related to the course of the disease as repeatedly reported in earlier (6, 7, 1 0 ) and also in some recent (3, 11) studies. methods thyrotropin (tsh) in the serum was assayed by a radioimmunosorbent technique using indirectly coupled antibodies (13, 16). the results were expressed in mu/1 with the mrc preparation 68/38 used as a reference standard. the incubations were performed overnight at room temperature and the limit of sensitivity was about 0.5 mu/1 of serum. triiodothyronine (t3) in the serum was measured by solid phase radioimmuno assay with antibodies coupled directly to microcrystalline cellulosa particles activated with cyanogen bromide (15). incubations were performed at 6ooc for two hours and overnight at room temperature in 0.5% polysorbate-20 (tween-20), in a 0.05 m phosphate-buffered saline, ph 7.4. reverse triiodothyronine (rt3) was measured by a radioimmunoassay using poly ethylenglycol for separation of bound and free rt3. the reagents were obtained from biodata (rome, italy). thyroxine (t4) in the serum was determined according to the bio-rad thyroxine column test (bio-rad laboratories, richmond, california). r the t3-resin uptake test was performed with sephadex -g-25 (pharmacia, upp sala, sweden) as a resin and the results were expressed as a percentage of the mean of healthy controls. the value for free t4-index (ft4 i) was calculated by multiplying the value for t4 with the value for t3-resin uptake test divided by 100. the limits of the reference range, defined as a mean * 2 s.d. of values for healthy controls, were in this laboratory for tsh 0.5-6 mu/1, for t3 1.3-3.2 nmol/l, for rt3 0.13-0.44 nmol/l, for t4 65-165 nmol/l and for t3-resin uptake 75-115. material the study comprises 41 women without thyroid substitution therapy. only one of the patients had a history of thyroid disease. she was operated in 1942 due to a diffuse toxic goitre but euthyroid at the time of this investigation. all patients had a newly diagnosed breast cancer without known distant metastases. they were classified according to a combined clinical and histopathological staging (12). twentysix patients had no axillary metastases, two had a locally 229 table 1. staging according to a combined clinical and histopathological classification. stage definition number % 0 in situ carcinoma i no local tumour complications, no axillary metastases i1 no local tumour complications a axillary metastases b as iia but perinodal tumour growth and/or involvement of apical nodes i11 locally advanced tumour 2 5 24 59 3 7 10 24 41 100 advanced tumour but nobody had known distant metastasis at the time of primary treatment (table 1 ) . the mean age at diagnosis was 60.2 years (range 30-80 years). all patients were treated by a total mastectomy. an axillary dissection was done in all cases where a peroperative histological examination of axillary node biopsies revealed metastases. postoperative irradiation was given against the supraclavicular and parasternal region to patients with medially or centrally located cancers in stage i or iia. all 12 patients with cancers in stage iib or i11 in addition got postoperative irradiation against the axilla. the first serum sample was obtained after the initial hospital admission but before surgery. all patients were postoperatively followed regularly at the department of oncology where a second serum sample was drawn after an observa tion time varying between 7 and 27 months with a mean of 18 months. the period of observation was for one group of 12 women 7-13 months (mean 9.4) and for another of 29 women 19-27 months (mean 22.2). all sera were stored at -2ooc until they were concomitantly analyzed. results the mean values before treatment and at follow-up are shown in table 2 for the whole group and after subdivision into one group with a shorter (n = 12) and one with a longer (n = 29) observation time. the differences between the first and the second sample were very small, were not more pronounced in those fol lowed for a longer period and were all insignificant (p > 0.05, t-test). five patients had developed distant metastases during the observation period. there were no significant differences (p > 0.05, t-test) between any of the mean values for this group and for the whole group, neither before treatment nor at the follow-up (table 2). 230 t a b l e 2 . th yr oi d f u n c t i o n b ef or e tr ea tm en t ( i ) a nd a t f ol lo wup (1 1) i n th e wh ol e m a t e r i a l , i n s u b g r o u p s w it h a s ho rt er an d a l o n g e r o b s e r v a t i o n t im e an d in p at ie nt s wi th re cu rr en t di se as e. me an 2 s. d. al l p a t i e n t s p a t i e n t s wi th o b s e r v a p a t i e n t s w it h o b s e r v a p a t i e n t s w it h me ta st at ic ti on t im e 7 1 3 m o n t h s ti on t im e 19 -2 7 mo nt hs di se as e t e s t n = 41 n = 12 n = 29 n = 5 i i 1 i i 1 i i1 i i1 t s h mu /l 2. 45 t1 .3 4 3. 99 +8 .4 o1 ) 2. 75 21 . 06 2. 95 k1 .1 9 2. 32 k1 .4 4 4. 42 +1 0. 00 2) 2. 16 k0 .4 0 2. 56 20 .4 2 t 3 nm ol /l 1. 79 f0 3 5 1. 93 t0 .3 4 1. 87 ko . 3 2 1. 97 20 .2 4 1. 76 f0 .3 6 1. 92 f0 .3 8 1. 83 k0 .1 1 1. 92 f0 .2 2 rt 3 n mo l/ l 0. 34 20 .1 0 0. 32 k0 .1 1 0. 37 20 .0 8 0. 37 20 .1 2 0. 3o to .1 0 0. 34 20 .1 0 0. 34 f0 .0 6 0. 25 ko .0 4 t 4 nm ol /l 10 8f 31 1 1 15 27 12 6f 28 t 3 r e s i n 9 5 2 1 4 9 1 t 9 8 8 2 9 up ta ke % 11 1k 13 2 4 2 2 2 10 6k 29 10 82 31 10 82 14 9 2k 8 9 1 2 9 9 5 2 1 4 9 4 2 7 9 7 2 1 3 10 .4 f0 .7 11 .1 22 .1 f t 4 i 9. 62 2. 7 10 .3 f2 .4 11 .o f1 .9 11 .3 22 .0 9. 9k 2. 4 9. 62 2. 7 ’) 2 , af te r th e e x c l u s i o n o f o n e w o m a n w h o d ev el op ed o v e r t h yp ot hy ro id is m th e co rr es po nd in g va lu es w er e 2. 69 21 .3 0 an d 2. 58 f0 .3 5. o u r results were also analyzed after subdivision of the material according to the clinical and histopathological classification. this analyses did not reveal any significant differences between the stages, which is in accordance with our previous findings ( 2 ) , and no significant changes in thyroid function in any of the stages during this observational period. one 7 8 year old woman developed an overt hypothyroidism during an observation time of two years with an increase in tsh from 3.0 to 56 mu/1 with a concomi tantly subnormal t3, rt3 and t4 value. except for this woman only one had a tsh-value slightly exceeding the upper limit of the reference range. the t3 values were normal in all other women before treatment as well as at the time of follow-up . discussion a thyroid dysfunction might be involved in breast cancer disease both by in fluencing the etiologic process and the progress of the disease. it might be reasonable then to assume that such a dysfunction should take the form of a hypothyroidism or a hyperthyroidism. the hypothesis of hypothyroidism as a risk factor has at the present time been supported by the uniform finding of a higher mean tsh-concentration in breast cancer women at the time of diagnosis than in comparable controls ( 2 , 9, 1 1 , 14). values exceeding the upper limit of the reference ragne were in these studies found in 8.5% (2) 36% (3) of the pa tients. a concomitantly exaggerated tsh-response to trh-stimulation was also shown in some of the patients (3, 9, 14). women with breast cancer were there fore considered to have an increased frequency of hypothyroidism usually sub clinic (9, 11). these findings were suggested to reflect subnormal thyroid hor mone levels by mittra (9). he also proposed with support from experimental research ( 8 ) that low thyroid hormone levels render the breast epithelium more sensitive to prolactin stimulation and hypothyroidism thereby to be involved in the etiologic process. the later finding of normal plasma ti-levels ( 2 , 3, 14) and also a normal free t4-index ( 2 ) appears to invalidate this hypothesis although one recent study in fact showed a slightly decreased free t4-index ( 1 1 ) . the whole concept of hypothyroidism was questioned when thyroid function was more completely as sessed by the concomitant determination of tsh, t3, rt3, t4 and t3-resin uptake (2). the pattern of differences between patients and controls was then con sidered inconsistent with a hypothyroidism but in agreement with that found in many non-thyroidal diseases (2, 5 ) . the concept that slight changes in thyroid hormone metabolism are secondary to stress and disease in women with breast cancer might also explain why a sig nificant increase in tsh was restricted to (14) or more pronounced in (9) women with an advanced disease. 232 the present study was primarily aimed at evaluating the hypothesis of pro gressive changes in thyroid function after the primary treatment which made a control group superfluous. the study has however further confirmed our previous finding of euthyroidism in breast cancer patients. the very close agreement between the values from the first and the second sampling period is in disagreement with the findings that the thyroid function is, for one reason or another, continuously decreasing after the initial treat ment as reported by perry et al. (11). this study showed a significant decrease in free ti-index at a follow-up after 6 months. the follow-up was, however, incomplete and included only 29 of 40 patients. the concomitant increase in tsh was also insignificant. we have therefore condluded from o u r study based on a more extensive assessment of thyroid function, a longer observation time and a complete follow-up that there is no systematic change in thyroid function after the primary treatment. the thyroid function in patients who developed distant metastases and there fore ultimately will die due to the disease, has to be interpreted with caution due to the small number of patients. there is no indication from our results that recurrent disease is proceded or accompanied by a decreased thyroid func tion as previously suggested. 1. 2. 3. 4 . 5. 6. 7 . 8. 9. 10. 11. references adami, h.-o., johansson, h., thorh, l., wide, l. & akerstrom, g.: serum levels of tsh, t3, rt3, t4 and t3-resin uptake in surgical trauma. acta endocrinol 88:482-489, 1978. adami, h.-o., rimsten, a . , thorhn, l., vegelius, j. & wide, l.: thyroid dis ease and function in breast cancer patients and non-hospitalized controls evaluated by determination of tsh, t3, rt3 and t4 levels in serum. acta chir scand 144:89-97, 1978. aldinger, k., schultz, p.n., blumenschein, g.r. & samaan, n.a.: thyroid -,timulating hormone and prolactin levels in breast cancer. arch intern med 138:1638-1641, 1978. beatson, g.t.: on the treatment of inoperable cases of carcinoma of the mama: suggestions for a new method of treatment, with illustrative cases. lancet i, 104-107, 1896. bermudez, f., surks, m.i. & oppenheimer, j.h.: high incidence of decreased serum triiodothyronine concentration in patients with nonthyroidal disease. j clin endocrinol metab 41:27-40, 1975. carter, a.c.: levels of serum protein-bound iodine in patients with meta static carcinoma of the breast. j clin endocrinol metab 20:477-479, 1960. edelstyn, g.a.: thyroid function in patients with mammary cancer. lancet i, 670-671, 1958. mittra, i.: mammotropic effect of prolactin enhanced by thyroidectomy. nature 248: 525-526 r 1974. mittra, i. & hayward, j.l.: hypothalamic-pituitary-thyroid axis in breast cancer. lancet i, 885-889, 1974. myhill, j., reeve, t.s. & hales, i.b.: thyroid function in breast cancer. acta endocrinol 51:290-300, 1966. perry, m., goldie, d.j. & self, m.: thyroid function in patients with breast cancer. ann r coll surg engl 60:290-293, 1978. 233 12. 13. 1 4 . 15. 16. 17. rimsten, a . , johansson, h., stenkvist, b. & thelin, a.-m.: axillary recur rences after selective treatment of the axillary nodes in cancer of the breast. clin oncol 2: 357-364 , 1976. roos, p., jacobson, g. & wide, l.: isolation of five active tsh components from the human pituitary glands. biochim biophys acta 379:247-261, 1975. rose, d.p. & davis, t.e.: plasma thyroid-stimulating hormone and thyroxine concentrations in breast cancer. cancer 41:666-669, 1 9 7 8 . wide, l.: radioimmunoassays employing immunosorbents. in: immunoassay of gonadotropins (ed. e. diczfalusy) . acta endocrinol [suppl] 142: 207-218,1969. wide, l., nillius, s . j . , gemzell, c. & roos, p.: radioimmunosorbent assay of follicle-stimulating hormone and luteinizing hormone in serum and urine from men and women. acta endocrinol [suppl] 73:174:1-60, 1 9 7 3 . vorherr, h.: thyroid disease in relation to breast cancer. klin wochenschr 5 6 ~ 1 1 3 9 1 1 4 5 , 1 9 7 8 . accepted june 1 , 1 9 7 9 address for reprints: hans-olov adami, m.d. department of surgery university hospital 5 7 5 0 14 uppsala sweden 234 upsala j med sci 92: 19-35, 1987 some effects of metolazone on electrolyte transport bo odlind,’ bjorn beermann,’ bjorn lindstrom,3 and orjan eriksson’ ‘departments of medicine, university hospiial, u p p s a l a , ’s:t eriks hospital, stockholm and ’department of drugs, national board of health and w e y a r e , uppsala, sweden abstract metolazone a c t i o n was s t u d i e d 1) i n v i t r o on i s o l a t e d operculum of f u n d u l u s h e t e r o c l i t u s ( a c t i v e c h l o r i d e t r a n s p o r t ) using an ussinq chamber ( m e t o l a z o n e c o n c 500 pm) and i n v i v o 2 ) u s i n g t h e modified sperber t e c h n i q u e i n t h e hen (metolazone i n f u s i o n r a t e 0.75-1.2 p g / k g / m i n ) and 3 ) i n h e a l t h y v o l u n t e e r s u s i n g c l e a r a n c e t e c h n i q u e s (metolazone i n f u s i o n r a t e 10 mg/h). 1 ) metolazone reduced ( p < 0 . 0 5 ) s h o r t c i r c u i t c u r r e n t and p o t e n t i a l d i f f e r e n c e s w i t h 20% from a v e r a g e c o n t r o l v a l u e s ( p < o . o 5 ) , w h i l e d i r e c t c u r r e n t r e s i s t a n c e was unchanged. this i s comparable t o t h i a z i d e b u t much l o w e r t h a n l o o p d i u r e t i c e f f e c t s . 2 ) t r u e t u b u l a r e x c r e t i o n f r a c t i o n of metolazone before and a f t e r novobiocin ( 2 . 7 pnol/kq/rnin c o i n f u s i o n averaqed 14.1 and 4.5 %, r e s p . (o<o.ol; n = 8 ) . t h u s m e t o l a z o n e i s p a r t l y e l i m i n a t e d by r e n a l t u b u l a r s e c r e t i o n . however, t h e d i u r e t i c e f f e c t (sodium, c h l o r i d e a n d potassium e x c r e t i o n ) a n d c l e a r a n c e s of cr5’-edta a n d 1125-na-o-iodohippurate were symmetrical , i . e . independent of metolazone u r i n a r y e x c r e t i o n r a t e , a s p r e v i o u s l y shown f o r t h i z i a d e s . 3 ) renal c l e a r a n c e o f m e t o l a z o n e i n h e a l t h y v o l u n t e e r s . ( h p l c method) a v e r a g e d 173+20 m l / m i n ( n = 8 ) . p r o b e n e c i d (1 g i v . ) s i q n i f i c a n t l y r e d u c e d t h e renal c l e a r a n c e of metolazone t o 3327 m l / m i n and potassium excre t i o n w i t h maximum 30%, while d i u r e t i c a n d s a l u r e t i c e f f e c t s were s i g n i f i c a n t l y i n c r e a s e d w i t h maximum 30%. t h u s , a l s o i n humans t h e d i u r e t i c e f f e c t o f metolazone i s not coupled t o t h e u r i n a r y e x c r e t i o n r a t e of t h e drug, b u t s u o g e s t s t h a t i t s d i u r e t i c e f f e c t i s e l i c i t e d p r i m a r i l y from t h e p e r i t u b u l a r s i d e of t h e nephron. probenecid a p p a r e n t l y d i s s o c i a t e s s o d i u m from potassium excre t i o n e f f e c t s of m e t o l a z o n e . t h i s i m p 1 i e s a l u m i n a l , s o d i u m i n d e p e n d e n t k a l i u r e t i c e f f e c t of the druq. introduction. metolazone ( 7 c h l o r o 1 , 2 , 3 , 4 , t e t r a h y d r o 2 methyl 4 0 x 0 3 o t o l y l 6 q u i n a z o l inesulphonamide) i s a sulphonamide d i u r e t i c chemically r e l a t e d t o quinethazone. the d i u r e t i c a n d metabolic e f f e c t s o f m e t o l a z o n e seem 19 l a r g e l y t o be s i m i l a r t o t h o s e of t h e b e n z o t h i a d i a z i n e s . there a r e , however, a number of r e p o r t s about s t r i k i n g d i u r e t i c e f f e c t s of m e t o l a z o n e i n p a t i e n t s w i t h s e v e r e l y r e d u c e d r e n a l f u n c t i o n ( 7 , 8, 17, 19, 36) a c o n d i t i o n where " o r d i n a r y " t h i a z i d e s a r e g e n e r a l l y held t o be i n e f f e c t i v e ( 3 3 ) . some a u t h o r s h a v e t h e r e f o r e c l a s s i f i e d m e t o l a z o n e a s b e i n g more r e l a t e d t o t h e l o o p d i u r e t i c s than t o t h e t h i a z i d e s ( 4 3 ) . the aim of t h i s s t u d y was t o f u t h e r c h a r a c t e r i z e t h e e f f e c t s of m e t o l a z o n e on e l e c t r o l y t e t r a n s p o r t , i n v i t r o , using f i s h operculum and toad u r i n a r y b l a d d e r (11, 1 8 ) . s t u d i e s were a l s o per formed i n vivo,using the modified sperber t e c h n i q u e i n t h e hen (231 and u s i n g conventional c l e a r a n c e techniques i n h e a l t h y v o l u n t e e r s . the main p u r p o s e o f t h e l a t t e r s t u d i e s was t o compare m e t o l a z o n e u r i n a r y e x c r e t i o n e f f e c t r e l a t i o n s h i p w i t h t h a t o f loop d i u r e t i c s and t h i a z i d e s a s o b t a i n e d i n e a r l i e r s t u d i e s (24-30). material a n d methods. e l e c t r o o h y s i o l o g i c a l s t u d y . t h e e l e c t r i c a l t r a n s e p i t h e l i a l p o t e n t i a l d i f f e r e n c e , s h o r t c i r c u i t c u r r e n t and d i r e c t c u r r e n t r e s i s t a n c e were s t u d i e d i n o p e r c u l a r e p i t h e l i u m from fundulus h e t e r o c l i t u s . the f i s h were k e p t i n 2.5 % a r t i f i c i a l sea water f o r a t l e a s t 4 weeks a f t e r d e l i v e r y . a f t e r k i l l i n g t h e animal, t h e operculum was i m mediately removed. the e p i t h e l i u m was d i s s e c t e d f r e e from t h e o p e r c u l u m a n d was mounted a c r o s s t h e c e n t r a l hole of a g a s k e t and placed i n a n ussing cham b e r (9, 11). metolazone stock s o l u t i o n (27.3 m m ) was made by d i s s o l v i n g t h e compound i n r i n g e r s o l u t i o n a l k a l i n i z e d w i t h naoh (ph 8.9). addition of drug i n c r e a s e d the ph of t h e b a t h i n g s o l u t i o n 0.03 ph u n i t s which i t s e l f h a d no e f f e c t on t h e s t u d i e d parameters. the same t e c h n i q u e was used t o s t u d y t h e e f f e c t of m e t o l a z o n e on i o n t r a n s p o r t i n i s o l a t e d u r i n a r y bladder e p i t h e l i u m from bufo v u l g a r i s . ( 1 0 ) .the t o a d s were kept i n t a p water f o r a t l e a s t 2 weeks a f t e r d e l i v e r y . the w a t e r t e m p e r a t u r e was 8-10°c a n d t h e a n i m a l s were fed manually w i t h p i e c e s o f ox l i v e r once a week. the u r i n a r y b l a d d e r e p i t h e l i u m was d i s s e c t e d f r e e a n d placed i n a n ussing chamber a s d e s c r i b e d above f o r t h e o p e r c u l a r edithelium. t h e t r a n s p o r t of sodium a n d c h l o r i d e was determined e l e c t r o p h y s i o l o a i c a l l y by m e a s u r i n q t h e t r a n s e p i t h e l i a l p o t e n t i a l d i f f e r e n c e ( p d ; m v ) , t h e s h o r t c i r c u i t c u r r e n t ( s c c ; pa x cm-2), and t h e d c r e s i s t a n c e ( r ; o h m x cm 1. the methods used a r e d e s c r i b e d i n d e t a i l e a r l i e r ( 1 1 ) . the following spontaneous a c t i v i t y of t h e e p i t h e l i a was observed. the k i l l i f i s h o p e r c u l a r e p i t h e l i a d i s p l a y e d a t r a n s e p i t h e l i a l pd of 10.3+1.8 mv ( s e a w a t e r side n e g a t i v e ) , a scc of 49.9+4.0 pa x cm-', and a t r a n s e p i t h e l i a l d c r e s i s t a n c e o f 217+44 o h m x cm (mean + se;n=4). the corresponding values f o r 2 2 20 i s o l a t e d t o a d b l a d d e r s were 57.8+4.4 mv ( s e r o s a l s i d e p o s i t i v e ) , 82.5+8.7 pa x cm-', and 722+67 ohm x cm (mean+se;n=8). 2 the m o d i f i e d s p e r b e r t e c h n i q u e i n t h e hen. the same e x p e r i m e n t a l t e c h n i q u e was used as has been d e s c r i b e d i n d e t a i l and used i n s i m i l a r s t u d i e s e a r l i e r (23, 24, 2 6 ) . the t e c h n i q u e e n a b l e s s e p a r a t e u r i n e c o l l e c t i o n s f r o m t h e t w o i n j e c t i o n s i d e ( i s ) and c o n t r o l s i d e ( c s l u r e t e r s a n d t h e c a l c u l a t i o n o f t h e t r u e t u b u l a r e x c r e t i o n f r a c t i o n o f m e t o l a z o n e (ttefm) as is cs e x c r e t i o n o f m x 100 = ttefm ( % ) amount o f m i n f u s e d a ttef v a l u e e x c e e d i n g 5 % p r o v e s a c t i v e t u b u l a r s e c r e t i o n o f m e t o l a z o n e ( 2 3 ) . ci4 m e t o l a z o n e was i n f u s e d i n t o a l e g v e i n on t h e r i g h t s i d e o f 8 h e n s a t a r a t e o f 0.75 1.2 vg/kg/min. a f t e r a t l e a s t 4-5 c o l l e c t i o n p e r i o d s o f 10 m i n a t s t e a d y s t a t e , s o d i u m n o v o b i o c i n , an i n h i b i t o r o f o r g a n i c a n i o n t r a n s p o r t (24) w a s c o i n f u s e d a t a r a t e o f 2.7 p m o l / k g / m i n . the r e n a l c l e a r a n c e s o f cr51 edta ( c e d t a ) a n d 1 1 2 5 n a o i o d o h i p p u r a t e (chipp) w e r e d e t e r m i n e d t h o u g h o u t t h e e x p e r i m e n t s . m c l e a r a n c e s t u d i e s i n h e a l t h y v o l u n t e e r s . s e v e n h e a l t h y men, 24 t o 35 y e a r s o l d , v o l u n t e e r e d i n t h e s t u d y w h i c h was approved b y t h e l o c a l e t h i c s committee. they a r r i v e d a t t h e l a b o r a t o r y i n t h e m o r n i n g a f t e r f a s t i n g o v e r n i g h t and were s u p p l i e d w i t h i n d w e l l i n g c a n n u l a s i n t h e b r a c h i c a l v e i n s o f b o t h arms. a t z e r o t i m e , i . v . b o l u s doses o f i n u l i n ( 5 0 mg/kg) and pah ( 1 0 mg/kg) were g i v e n and f o l l o w e d b y a c o n t i n o u s i n f u s i o n o f 28 and 14 mg/min, r e s p e c t i v e l y . an e q u i l i b r i u m p e r i o d o f a t l e a s t 1 h o u r was a l l o w e d b e f o r e s t a r t i n g t h e c l e a r a n c e d e t e r m i n a t i o n s . m e t o l a z o n e was g i v e n as an i . v . b o l u s o f 1 0 mg f o l l o w e d b y a c o n t i n u o u s i . v . i n f u s i o n o f 10 mg/h 1.5 t o 2 . 5 h r s a f t e r t h e s t a r t o f t h e i n u l i n p a h i n f u s i o n . p r o b e n e c i d was a d m i n i s t e r e d as an i . v . b o l u s o f 1 g i n 50 m l s a l i n e a t 2.5 h r s a f t e r s t a r t o f t h e i n f u s i o n o f metolazone, w h i c h was c o n t i n u e d f o r a n o t h e r 3 h r s . the s u b j e c t s e m p t i e d t h e i r u r i n a r y b l a d d e r s b e f o r e t h e e x p e r i m e n t . u r i n e was c o l l e c t e d i n p e r i o d s o f 30 m i n and b l o o d samples were drawn w i t h i n 3 m i n o f t h e b e g i n n i n q and t h e end o f each c o l l e c t i o n p e r i o d . f r o m t h e s t a r t o f t h e e x p e r i m e n t t o t h e i n j e c t i o n o f m e t o l a z o n e t h e s u b j e c t s d r a n k 200 m l t a p w a t e r / h . u r i n e l o s s e s o f e a c h p e r i o d a f t e r t h e i n j e c t i o n o f t h e d i u r e t i c i n excess o f t h e o r a l w a t e r r e p l a c e m e n t was r e p l a c e d volume f o r volume by c o n t i n u o u s i . v . i n f u s i o n o f s a l i n e d u r i n g t h e s u b s e q u e n t 21 p e r i o d . food i n t a k e a n d smoking were not allowed d u r i n g t h e experiment. except when voiding, t h e s u b j e c t s were supine d u r i n q t h e experiment. determination of metolazone. a l i q u i d c h r o m a t o g r a p h i c method was s e t u p f o r t h e d e t e r m i n a t i o n o f m e t o l a z o n e i n plasma and u r i n e . t h i s u t i l i z e d a 6000a p u m p , a u6k i n j e c t o r (waters assoc, m i l f o r d , m a , u s a ) and a 970 f l u o r e m e t r i c d e t e c t o r ( s p e c t r a p h y s i c s , s a n t a c l a r a , c a y usa) o p e r a t e d a t an e x i t a t i o n wavelength o f 230 nm and equipped w i t h a 420 nm emission f i l t e r . the column ( s t a i n l e s s s t e e l , 1 5 0 m m x 4 , 6 m m i.d.) w a s s l u r r y packed w i t h lichrosorb rp-18.5 pm p a r t i c l e s i z e (merck, darmstadt, g r f ) a n d e l u t e d w i t h 40% a c e t o n i t r i l e i n 0,ol m ph 7 phos phate b u f f e r a t a r a t e of 1 . 2 r n l / m i n . the sample ( 0 . 5 and 0.1 ml were used f o r plasma and u r i n e r e s p e c t i v e l y ) was added t o 0 . 5 ml of s a t u r a t e d sodium hydrogen-carbonate s o l u t i o n and e x t r a c t e d w i t h 5 ml e t h y l a c e t a t e i n a s c r e w c a p p e d t u b e ( s h a k e b o a r d 10 m i n ) . a f t e r c e n t r i f u g a t i o n ( 5 0 0 g ) 4 ml o f t h e o r g a n i c phase was removed and evaporated w i t h n i t r o g e n g a s . the r e s i d u e was r e d i s s o l v e d in 500 pl of t h e e l u e n t s y s t e m and u s u a l l y a 20 p1 a l i q u o t was i n j e c t e d i n t o t h e l i q u i d chromatograph. these volumes could be a d j u s t e d t o t h e c o n c e n t r a t i o n range analysed. t h e c a l i b r a t i o n graph was c o n s t r u c t e d by adding known amounts of metolazone t o plasma o r u r i n e and a n a l y z i n g them according t o t h e method d e s c r i b e d . the r e s u l t i n g peak h i g h t s were p l o t t e d v e r s u s t h e c o n c e n t r a t i o n . the t o t a l recovery when e x t r a c t i n g metolazone from plasma ( u r i n e ) a v e r a g e d 88%. the p r e c i s i o n o f t h e method was 4.5 and 7% (n=10) a t 400 and 500 n g h l plasma, r e s p e c t i v e l y . chemical d e t e r m i n a t i o n s . concentrations of i n u l i n and pah were d e t e r m i n e d a c c o r d i n g t o s c h r e i n e r ( 3 7 ) and b r u n ( 6 1 , r e s p e c t i v e l y . i n t h e v o l u n t e e r study c o n c e n t r a t i o n s of sodi urn, p o t a s s i urn and chl o r i de were determined according t o r o u t i n e methods a t t h e h o s p i t a l and i n t h e experimental study a s p r e v i o u s l y d e s c r i b e d ( 2 4 ) . i s o t o p e d e t e r m i n a t i o n s . spectrometer and carbon-14 a c t i v i t i e s by l i q u i d s c i n t i l l a t i o n counting. and cr5' a c t i v i t i e s were d e t e r m i n e d i n a two-channel s c i n t i l l a t i o n m a t e r i a l s . metolazone and ci4 metolazone ( s p e c . a c t . 1.45 mci/mmol) was o b t a i n e d from p h a r m a c i a a b , uppsal a sweden. probenecid ( i n j e c t i o n "benemid") 0 , l q/ml was 22 d e l i v e r e d by merck f r o s t research l a b o r a t o r i e s , m o n t r e a l , canada. p a h ( p a r a ami n o h i p p u r a t e s o d i u m ) and i n u l i n ( i n u t e s t ) were s u p p l i e d by merck sharp and dohme, rahway, nj, and laevosan g e s e l l s c h a f t , l i n z , a u s t r i a , r e s p e c t i v e l y . s o d i u m n o v o b i o c i n was o b t a i n e d f r o m m e r c k s h a r p a n d dohme, rahway, nj. c r edta was b o u g h t f r o m b e h r i n g w e r k e a g , v a r b u r g , germany a n d 1125-na-0 i o d o h i p p u r a t e f r o m k a b i d i a g n o s t i k a ab, s t u d v i k , sweden. 5 1 s t a t i s t i c a l a n a l y s i s . r e s u l t s a r e g i v e n a s a r i t h m e t i c mean + sd. d i f f e r e n c e s i n c l e a r a n c e o r e x c r e t i o n v a l u e s w e r e a n a l y s e d a c c o r d i n g t o t h e c o n c e p t o f c o n f i d e n c e i n t e r v a l s . p v a l u e s < 0.05 were c o n s i d e r e d as s i g n i f i c a n t . the d i f f e r e n c e be tween p r e d r u g and p o s t d r u g v a l u e s f o r f i s h operculum a n d t o a d b l a d d e r w e r e e v a l u a t e d b y s t u d e n t s t t e s t ( t w o s i d e d t e s t ) f o r p a i r e d v a r i a t e s and con s i d e r e d s i g n i f i c a n t a t p < 0.05 ( * ) . results. e l e c t r o p h y s i o l o g i c a l s t u d i e s . m e t o l a z o n e ( 5 0 0 pm) r e d u c e d t h e s h o r t c i r c u i t c u r r e n t ( s c c ) and t h e p o t e n t i a l d i f f e r e n c e (pd) i n t h e i s o l a t e d e p i t h e l i u m o f f u n d u l u s h e t e r o c l i t u s w i t h a b o u t 2 0 % ( p < 0.051, w h i l e t h e d i r e c t c u r r e n t r e s i s t a n c e ( r ) was unchanged ( t a b l e i ) . m e t o l a z o n e ( 5 0 0 pm) e x c e r t e d o n l y s m a l l e f f e c t s on pd, scc and r ( 1 4 t o + 1 8 % ; p > 0 . 0 5 ) when a d m i n i s t e r e d t o t h e s e r o s a l a n d mucosal s i d e , r e s p e c t i v e l y , o f t o a d u r i n a r y b l a d d e r ( t a b l e i ) . t a b l e i. e f f e c t s o f m e t o l a z o n e on t h e p o t e n t i a l d i f f e r e n c e (pd), s h o r t c i r c u i t c u r r e n t (scc) and d i r e c t c u r r e n t r e s i s t a n c e ( r ) i n i s o l a t e d o p e r c u l a r e p i t h e 1 i a o f fundul u s h e t e r o c l i t u s and i n i s o l a t e d t o a d b l a d d e r .the d r u g ( 5 0 0 pm) was added t o t h e s e r o s a l ( s ) o r mucosal (m) s i d e o f t h e e p i t h e l i u m . the e l e c t r i c a l p a r a m e t e r s w e r e r e c o r d e d b e f o r e a n d o n e h o u r a f t e r m e t o l a z o n e exposure. r e s u l t s a r e g i v e n a s p e r c e n t change from c o n t r o l v a l u e s . the number o f e p i t h e l i a t e s t e d a r e g i v e n w i t h i n p a r e n t h e s e s ( x d e n o t e s p<0.05). pd scc r % change f r o m c o n t r o l . o p e r c u l a r e p i t h e l i u m s ( 4 ) -20.6 + 2.8' -19.5 + 4.5' -0.1 + 8.7 s ( 4 ) -0.4 + 6 . 2 -14.2 + 7.1 +18.0 + 11.0 u r i n a r y b l adder m ( 4 ) + 6 . 4 + 4.9 +15.8 + 5.5 -5.8 + 8.2 u r i n a r y b l a d d e r , 23 the modified sperber technique. a t s t e a d y s t a t e t h e ttefm value of ci4 metolazone averaqed 14.1 + 4.2 % ( c a l c u l a t e d a s means of 4-5 c o l l e c t i o n p e r i o d s of 1 0 min from e a c h of 8 a n i m a l s ) . a f t e r a new steady s t a t e had been reached during novobiocin c o i n f u s i o n , t h i s v a l u e was s i q n i f i c a n t l y ( p < 0 . 0 0 1 ) r e d u c e d t o 4 . 5 2 2 . 0 % ( c a l c u l a t e d a s means from 3 ( 4 ) c o l l e c t i o n p e r i o d s of 10 min from each of 8 a n i m a l s ) . corresponding r e s u l t s f o r t o t a l ( i n j e c t i o n s i d e p l u s c o n t r o l s i d e ) u r i n a r y e x c r e t i o n was 58.8 + 6.0% and 37.7 + 2.9% ( p < 0.001), o f t h e i n f u s e d amount, r e s p e c t i v e l y . the u r i n a r y e x c r e t i o n r a t e of c h l o r i d e , sodium and w e r e s y m m e t r i c a l t h o u q h o u t a l l p o t a s s i u m a s w e l l a s c e d t a a n d c experiments. ( c f . fig 1). hi p p f:g 1. typical r e s u l t s from one experiment with t h e modified sperber technique. i n f u s i o n r a t e o f ~ 1 4 metolazone was 0 . 9 pg/kg/min. the t r u e t u b u l a r ex c r e t i o n f r a c t i o n o f metolazone ( t t e f m ) , sodium, c h l o r i d e and p o t a s s i u excretion i"5-na-0-iodohippurate from i n j e c t ion and c o n t r o l kidneys, r e s p e c t i v e l y , a r e shown. a t h e renal c l e a r a n c e s of cr y iedta a n d .. c 0 3 0 2 o l o 0-10 20-30 lo-so collection period (min) clearance s t u d i e s i n heal thy v o l u n t e e r s . the plasma l e v e l s of m e t o l a z o n e were s l o w l y i n c r e a s i n g a t t h e t i m e o f i n j e c t i o n of p r o b e n e c i d when they averaged 402 2 64 n q / m l ( f i g 21. the renal c l e a r a n c e of metolazone ( f i g 2 ) was almost i d e n t i c a l d u r i n q t h e two p e r i o d s b e f o r e probenecid a d m i n i s t r a t i o n ( p e r i o d s 2 a n d -1) when i t averaged 184 + 25 a n d 173 + 20 m l / m i n , r e s p e c t i v e l y . accordingly, period -1 was c h o s e n as con t r o l p e r i o d . the i n j e c t i o n o f probenecid caused a decrease i p renal c l e a r a n c e o f metolazone t o 33 + 7 ml/min, which corresponds t o 18 + 2.8 per c e n t o f t h e 24 c l e a r a n c e d u r i n g p e r i o d -1. the d e c l i n e i n c l e a r a n c e was followed by a con t i n u o u s r i s e i n plasma l e v e l s of metolazone u p t o 930 2 204 ng/ml a t 3 h a f t e r t h e a d m i n i s t r a t i o n o f probenecid ( f i g 2 ) . 1000 e 2 500 1 * * * * * * * * 1 ij' f ' j ' 2 ' 1 ' 1 2 ' 3 ' 4 ' 5 ' 6 ' perlod p fig 2 . .' 150e i e 100 50 c m 2501 200 u m 100, * * * i * * t * * * * * * t t plasma l e v e l s ( s i ~ ) , renal c l e a r ance (cm) a n d u r i n a r y e x c r e t i o n ( u m ) of metolazone during i n t r a venous i n f u s i o n of metolazone b e f o r e and a f t e r i n j e c t i o n of probenecid ( p ) . * p<0.05 in com parison t o c o n t r o l period (hatched a r e a ) . 25 the u r i n a r y f l o w d u r i n g t h e p e r i o d p r e c e e d i n q t h e i n j e c t i o n o f p r o b e n e c i d was 4 . 1 + 0.6 m l / m i n i n mean ( f i g 3 ) . the i n j e c t i o n o f p r o b e n e c i d c a u s e d a s l i g h t b u t i n s i g n i f i c a n t decrease i n d i u r e s i s w h i c h was f o l l o w e d b y a s i g n i f i c a n t mean i n c r e a s e ( p < 0.05) i n e x c r e t i o n o f u r i n e , d u r i n g p e r i o d 3 and 4 when t h e d i u r e s i s averaged 5.3 1.3 (131.4 2 32.4 p e r c e n t o f c o n t r o l ) a n d 4.9 + 1.6 ( 1 2 0 . 3 + 1 1 . 0 p e r c e n t ) m l / m i n , r e s p e c t i v e l y . the response t o t h e i n j e c t i o n o f p r o b e n e c i d w i t h r e g a r d s t o t i m e f o r maximal c h a n g e d i f f e r e d be t w e e n i n d i v i d u a l s . thus, t h e maximal i n d i v i d u a l i n c r e a s e i n d i u r e s i s averaged 155.9 + 15.4 der c e n t o f c o n t r o l d i u r e s i s ( p < 0.001). * * m 1 1 2 3 4 5 p * * 1 6 period f i g 3 a,b. u r i n a r y o u t p u t o f w a t e r ( u h ~ o ) c h l o r i d e ( u c i ) , sodium (uva+) and p o t a s s i u m ( u k + ) d u r i n g i n t r a v e n o u s i n f u s i o n o f m e t o l a z o n e b e f o r e and a f t e r i n j e c t i o n o f p r o b e n e c i d ( p ) . * p < 0 . 0 5 i n comparison t o c o n t r o l p e r i o d ( h a t c h e d a r e a ) . * * * m 1 1 2 3 4 5 8 ke 1 1 2 3 m + 1 4 5 6 period 1 6 period 1 6 period 26 gfr was 1 1 2 + 1 4 ml/min d u r i n g t h e period b e f o r e t h e i n j e c t i o n of metolazone. i t did not d i f f e r from t h a t during t h e c o n t r o l p e r i o d when i t averaged 1 0 9 + 13 rnl/min. probenecid caused a s i g n i f i c a n t increase in gfr during periods 3 ( 1 2 3 5 1 6 ml/min) and 4 (114 + 9 ml/min). this increase could a t l e a s t i n part explain the increased d i u r e s i s during those periods, as cor responding mean fractional excretion of water ( e f ) ( 4 . 5 2 1.4 a n d 4.3 + 1.3 per cent, respectively) did n o t d i f f e r s i g n i f i c a n t l y from t h a t during the con t r o l period (3.8 + 0.5 per c e n t ) . however, the maximal individual ef ( 6 . 1 + 0.6 per cent; period 4.7 + 1) corresponded t o 163.3 + 25.7 per cent of control ( p < 0,001). ( f i g 4 ) . "2o h2° ef per cent h20 k ' na' cii" * * * * * i * na+ ci h p fig 4. maximal change ( u n f i l l e d a r e a ) in fractional ex c r e t i o n ( e f ) o f water, sodium, chloride and potas sium during intravenous infusion of metolazone before and a f t e r injection of probenecid. ** p<o.ol *** p<o.ooi in comparison t o control period (hatched a r e a ) . k' n a t r i u r e s i s averaged 546 + 7 3 pmol/min during the control period ( f i g 3 ) . after probenecid i t was s i g n i f i c a n t l y increased ( p < 0.05) during p e r i o d s 3 5 with maximal n a t r i u r e s i s averaging 696 + 117 pmolhin ( 1 2 7 . 8 + 13.2 per c e n t ) . again, increased g f r could in p a r t explain the change during p e r i o d s 3 a n d 4 a s t h e f r a c t i o n a l e x c r e t i o n of sodium ( e f n a ) d i d n o t d i f f e r from control value, which amounted t o 3 . 6 + 0 . 5 per c e n t . however, mean e f n a was s i g n i f i c a n t l y g r e a t e r ( p < 0 . 0 5 ) t h a n control value during period 5 ( 5 . 0 + 1.3 per c e n t ) . also, maximal individual e f n a ; 5.4 1.1 per c e n t was seen d u r i n g period 5.0 + 1.1 (150.3 + 23.4 per cent of control value; p < 0.01; fig 4 ) . chloride excretion was s i g n i f i c a n t l y higher during p e r i o d s 3 5 t h a n d u r i n g t h e c o n t r o l p e r i o d when i t averaged 5 6 0 + 77 pmol/min. ( f i g 3 ) . maximal chloruresis was 719 2 103 pmol/min ( 1 2 9 . 4 + 16.7 per cent of control) in mean. the fractional excretion of chloride was 5.0 + 0.4 per cent during the control period. i t increased t o 5.4 + 0.5 ( p < 0.05) a n d 5.9 + 0.6 ( p < 0.01) per cent 21 d u r i n g p e r i o d s 4 a n d 5 , r e s p e c t i v e l y . mean value f o r h i g h e s t i n d i v i d u a l r i s e was 6.9 0.6 per c e n t ( p e r i o d 4.9 + 1 . 2 ) ; ( f i g 5). fig 5. d i s s o c i a t i o n of t u b u l a r e f f e c t s of metolazone on sodium and potassium t r a n s p o r t . f r a c t i o n a l e x c r e t i o n of sodium ( e f ) and potassium ( e f ) b e f o r e ( 0 ) n g f t e r ( 0 ) probenecid &e shown. efk+ per cent 10 e f ~ ~ . per cent t h e e x c r e t i o n o f p o t a s s i u m a v e r a g e d 99 + 36 pmol/min during t h e c o n t r o l p e r i o d . ( f i g 3 ) . the i n j e c t i o n of probenecid caused a f a l l i n k a l i u r e s i s which was s i g n i f i c a n t ( p < 0 . 0 5 ) d u r i n g p e r i o d s 2 and 6 , when i t averaqed 71 + 22 ( 7 6 . 1 + 1 4 . 3 p e r c e n t ) a n d 77 + 3 3 ( 7 0 . 3 + 2 8 . 4 p e r c e n t ) p m o l / m i n , r e s p e c t i v e l y . the f r a c t i o n a l e x c r e t i o n of potassium was 23.8 + 9.0 per c e n t during t h e c o n t r o l p e r i o d . i t decreased t o 19.6 + 7.0 ( p < 0 . 0 5 ) , 1 7 . 4 + 5.1 ( p < 0.01) a n d 19.0 + 6.0 ( p < 0.05) during p e r i o d s 1, 2 and 3 , r e s p e c t i v e l y . the most pronounced i n d i v i d u a l decrease was down t o 14.6 + 6 . 8 p e r c e n t ( p < 0.001) d u r i n g period 4.4 + 1.7. ( f i g 4 ) . renal c l e a r a n c e of p a h decreased from c o n t r o l value of 509 + 89 t o an a v e r a g e o f 357 + 86 m l / m i n d u r i n g t h e s i x p e r i o d s f o l l o w i n g probenecid which cor responds t o 70.0 + 14.8 per c e n t of c o n t r o l value ( p < 0.01). discussion. metolazone (500ph1) caused a 20% r e d u c t i o n o f t h e t r a n s e p i t h e l i a l p o t e n t i a l d i f f e r e n c e and s h o r t c i r c u i t c u r r e n t without a f f e c t i n g t h e d c r e s i s t a n c e i n i s o l a t e d o p e r c u l a r e p i t h e l i a o f f u n d u l u s h e t e r o c l i t u s ( t a b l e i ) , a n e p i t h e l i u m t h a t t r a n s p o r t s c h l o r i d e e l e c t r o g e n i c a l l y (9). t h i s m o d e r a t e d e c r e a s e p r o b a b l y r e f l e c t s u n s p e c i f i c a c t i o n s o f m e t o l a z o n e . " c l a s s i c a l " t h i a z i d e s e.g. h y d r o c h l o r o t h i a z i d e have no e f f e c t on the o p e r c u l a r e p i t h e l i u m while loop d i u r e t i c s cause a profound i n h i b i t i o n of c h l o r i d e t r a n s p o r t i n t h i s e p i t h e l i u m (11). moreover, we f o u n d no e f f e c t s of m e t o l a z o n e ( 5 0 0 p m ) on e l e c t r i c a l p a r a m e t e r s i n t h e i s o l a t e d b l a d d e r of bufo v u l g a r i s ( t a b l e i ) . t h i s i s con t r a r y t o t h e i n h i b i t i o n o f scc and p o r e p o r t e d by r i v e c c a e t a 1 ( 3 4 ) 28 s u g g e s t i n g an i n h i b i t i o n b y m e t o l a z o n e o f a c t i v e sodium t r a n s p o r t . the d i f f e r e n c e c o u l d r e f l e c t a s p e c i e s d i f f e r e n c e . r i v e c c a e t a1 (34) t h u s u s e d b l a d d e r s f r o m b u f o m a r i n u s , a s a l t w a t e r t o a d w h i l e o u r o b s e r v a t i o n s were made on b u f o v u l g a r i s , a f r e s h w a t e r t o a d . r e s u l t s f r o m s i m i l a r s t u d i e s w i t h o t h e r b e n z o t h i a z i d e s a d d s t o t h e c o m p l e x i t y . f o r i n s t a n c e , p e n d l e t o n e t a1 (311 f o u n d b e n z o f l u m e t h i a z i d e t o decrease scc when added t o s e r o s a l s o l u t i o n and t o s l i g h t l y i n c r e a s e s c c when a d d e d t o t h e mucosal s i d e o f t h e b l a d d e r o f bufo m a r i n u s . marumo e t a1 ( 2 0 ) o n t h e o t h e r h a n d f o u n d h y d r o c h l o r o t h i a z i d e t o cause a p a r t i a l i n h i b i t i o n o f scc a f t e r mucosal a d d i t i o n and no e f f e c t s on t h e s e r o s a l s i d e o f t h e f r o g rana c a t e s b e i a n a b l a d d e r . s c h n i e d e r s and l u d e n s ( 3 5 ) o b s e r v e d a 50% d e c r e a s e o f t h e scc by 640pm h y d r o c h l o r o t h i a z i d e , added t o b o t h s i d e s o f t h e chamber. thus, i t a p p e a r s t h a t t h e e p i t h e l i a s t u d i e d d i f f e r much i n t h e i r s e n s i t i v i t y t o t h i a z i d e s . a l s o , i t seems t h a t i f t h e e f f e c t s o f d i f f e r e n t t h i a z i d e s a r e t o b e c o m p a r e d t h e same e p i t h e l i u m s h o u l d b e u s e d . i n t e r e s t i n g l y , i n a r e c e n t s t u d y b y s t o k e s e t a1 ( 4 0 ) a t h i a z i d e s e n s i t i v e e l e c t r i c a l l y n e u t r a l t r a n s p o r t svstem has been d e s c r i b e d i n t h e b l a d d e r o f t h e w i n t e r f l o u n d e r . i n t h i s system, h y d r o c h l o r o t h i a z i d e and m e t o l a z o n e ( b u t n o t f u r o s e m i d e ) had q u a l i t a t i v e l y s i m i l a r a c t i o n s . u s i n g t h e m o d i f i e d sperher t e c h n i q u e i t was found t h a t c14 m e t o l a z o n e was s u b j e c t t o a c t i v e t u b u l a r s e c r e t i o n i n t h e k i d n e y ( f i g 1 ) . the ttef v a l u e o f 14% shows a t u b u l a r s e c r e t i o n t h a t i s s m a l l e r t h a n f o r h y d r o c h l o r o t h i a z i d e (ttef 59%) i n t h i s s p e c i e s ( 2 8 ) . n o v o b i o c i n c o i n f u s i o n r e d u c e d t h e s t e a d y s t a t e ttef v a l u e o f m e t o l a z o n e t o 1/3 o f p r e i n h i b i t i o n v a l u e s w h i c h i s e v i d e n c e f o r a c t i v e s e c r e t i o n o f m e t o l a z o n e by an o r g a n i c a n i o n t r a n s p o r t sys t e m ( 2 4 ) . t h r o u g h o u t a l l e x p e r i m e n t s , t h e u r i n a r y sodium c h l o r i d e e x c r e t i o n was s y m m e t r i c a l ( f i g 1). thus, t h e s a l u r e t i c e f f e c t was n o t c o u p l e d t o t h e t u b u l a r s e c r e t i o n o f t h e d i u r e t i c . t h i s s u g g e s t s t h a t t h e s a l u r e t i c e f f e c t o f m e t o l a z o n e i s n o t p r i m a r e l y l i n k e d t o t h e u r i n a r y e x c r e t i o n o f t h e d r u g ( l u m i n a l e f f e c t ) . a p r e d o m i n a n t a c t i o n f r o m t h e b l o o d ( p e r i t u b u l a r ) s i d e o f t h e nephron i s t h e r e f o r e proposed, i n a n a l o g y w i t h p r e v i o u s s u g g e s t i o n s f o r h y d r o c h l o r o t h i a z i d e and c h l o r o t h i a z i d e based on s i m i l a r e x p e r i m e n t s (28). t h i s t h e n i s d i f f e r e n t f r o m t h e p r e d o m i n a n t e l y l u m i n a l e f f e c t s o f l o o p d i u r e t i c s , s u c h a s f u r o s e m i d e , p i r e t a n i d e o r e t h a c r y n i c a c i d ( 2 4 ) . a l s o , t h e p o t a s s i u m e x c r e t i o n was s y m m e t r i c a l d u r i n g m e t o l a z o n e i n f u s i o n ( f i g 1 ) a s p r e v i o u s l y s h o w n f o r h y d r o c h l o r o t h i a z i d e w h i l e c h l o r o t h i a z i d e showed a s e c r e t i o n d e p e n d e n t k a l i u r e s i s ( 2 8 ) . it h a s b e e n p r o p o s e d t h a t t h i s r e f l e c t s a n i n h i b i t i o n o f c a r b o n i c anhydrase b y c h l o r o t h i a z i d e i n t h e a v i a n nephron. the l a c k o f a s i m i l a r e f f e c t o f m e t o l a z o n e s u p p o r t s t h i s i n t e r p r e t a t i o n s i n c e m e t o l a z o n e i s a p o o r i n h i b i t o r o f c a r b o n i c anhydrase ( 1 2 ) 29 i n n o n h y d r a t e d b u t u r i n e l o s s s u b s t i t u t e d h e a l t h y v o l u n t e e r s , m e t o l a z o n e i n f u s i o n ( 1 0 mg b o l u s a n d 1 0 m g / h r ) g a v e c l e a r d i u r e t i c b u t e s p e c i a l l y s a l u r e t i c e f f e c t s w i t h f r a c t i o n a l e x c r e t i o n o f w a t e r a n d s o d i u m c h l o r i d e i n s t e a d y s t a t e o f 3.5 5% ( f i g 2 and 3 ) . i n p r e v i o u s s i n g l e dose e x p e r i m e n t s , maximal f r a c t i o n a l sodium e x c r e t i o n o f 4 7% has been r e p o r t e d f o l l o w i n g 5 1 0 mg metolazone i n t r a v e n o u s l y ( 8 , 32, 39, 4 1 ) . thus, t h e maximal d i u r e t i c and s a l u r e t i c e f f i c i e n c y o f m e t o l a z o n e i s much s m a l l e r t h a n t h o s e o f l o o p d i u r e t i c s as r e v i e l e d i n s i m i l a r s t u d i e s ( 2 5 , 29, 3 0 ) . it i s o f i n t e r e s t t h a t i n h y d r a t e d s u b j e c t s metolazone had no ( a d d i t i v e ) d i u r e t i c e f f e c t (21, 32, 38, 3 9 ) . use o f a s t e a d y s t a t e e x p e r i m e n t a l t e c h n i q u e w i t h c o n t i n u o u s i n f u s i o n o f t h e d r u g a n d f u l l s u b s t i t u t i o n o f d i u r e t i c i n d u c e d volume l o s s e s has p r o v e n s u c c e s s f u l i n p r e v i o u s s t u d i e s o f d i u r e t i c e x c r e t i o n e f f e c t r e l a t i o n s h i p i n h e a l t y v o l u n t e e r s ( 2 5 , 29, 3 0 ) . t h i s t e c h n i q u e r e d u c e s i n t e r p r e t a t i o n d i f f i c u l t i e s t h a t may a r i s e i n s i n g l e dose e x p e r i m e n t s due t o t h e t i m e c o u r s e o f d i u r e t i c e x c r e t i o n ( c f . 1 6 ) o r due t o a c u t e t o l e r a n c e d e v e l o p e m e n t . ( l 5 ) we f o u n d t h e r e n a l c l e a r a n c e o f m e t o l a z o n e i n h e a l t h y v o l u n t e e r s t o a v e r a g e 1 7 3 t o 1 8 4 m l / m i n i n s t e a d y s t a t e ( f i g 4 ) . i n v i e w o f t h i s a n d p r e v i o u s r e p o r t s o f a plasma p r o t e i n b i n d i n g o f m e t o l a z o n e o f 95% ( 4 2 ) i t i s c l e a r t h a t t u b u l a r s e c r e t i o n c o n t r i b u t e s s u b s t a n t i a l l y t o t h e r e n a l e l i m i n a t i o n o f t h e d r u g i n humans. i n c o n f i r m a t i o n o f t h i s , p r o b e n e c i d r e d u c e d t h e u r i n a r y e x c r e t i o n r a t e o f m e t o l a z o n e a n d i t s c l e a r a n c e a s much a s down t o 18% o f p r e i n h i b i t i o n v a l u e s . ( f i g 4 ) . i n s p i t e o f t h i s , p r o b e n e c i d d i d n o t r e d u c e t h e d i u r e t i c o r s a l u r e t i c e f f e c t s o f m e t o l a z o n e ; on t h e c o n t r a r y t h e r e was an i n c r e a s e i n t h e s e e f f e c t s ( f i g 2 a n d 3 ) . t h e r e f o r e , i t a p p e a r s t h a t a l s o i n humans m e t o l a z o n e e x c e r t s i t s d i u r e t i c / s a l u r e t i c e f f e c t s p r i m a r i l y f r o m t h e p e r i t u b u l a r ( b l o o d ) s i d e o f t h e nephron and n o t f r o m t h e l u m i n a l ( u r i n e ) s i d e . t h i s i s c o n t r a r y t o t h e c a s e f o r s e v e r a l l o o p d i u r e t i c s ( 2 5 , 29, 3 0 ) . a p r o b e n e c i d i n d u c e d i n c r e a s e i n t h e d i u r e s i s / s a l u r e s i s , s i m i l a r t o o u r f i n d i n q f o r metolazone, has, however, been d e s c r i b e d by b r a t e r ( 5 ) f o r c h l o r o t h i a z i d e , t h u s a d d i n g a n o t h e r t h i a z i d e l i k e p r o p e r t y t o metolazone. i n t e r e s t i n g l y , p r o b e n e c i d d e c r e a s e d m e t o l a z o n e i n d u c e d a b s o l u t e and f r a c t i o n a l p o t a s s i u m e x c r e t i o n ( f i g 3 and 4 ) . the i n h i b i t o r t h e r e f o r e d i s s o c i a t e d t h e e f f e c t s o f m e t o l a z o n e on t u b u l a r sodium t r a n s p o r t f r o m t h a t on p o t a s s i u m t r a n s p o r t ( c f . f i g 5 ) , w h i c h i s e s p e c i a l l y n o t e w o r t h y s i n c e i t i s g e n e r a l l y h e l d t h a t t h e g r e a t e r sodium l o a d t h a t appears a t t h e d i s t a l t u b u l a r s i t e s o f sodium-potassium exchange, t h e g r e a t e r t h e o u t p u t o f p o t a s s i u m ( c f . 14). thus, i t appears t h a t m e t o l a z o n e c o u l d have s e p a r a t e e f f e c t s on sodium and p o t a s s i u m e x c r e t i o n i n h e a l t y s u b j e c t s ( f i g 5 ) . moreover, t h e s e r e s u l t s a r e c o m p a t i b l e w i t h a d i r e c t l u m i n a l k a l i u r e t i c e f f e c t o f m e t o l a z o n e . m e t o l a z o n e has a 30 " p o t a s s i u m s p a r i n g " e f f e c t , w h i c h i s d i f f e r e n t f r o m t h e w e l l d e f i n e d p o t a s s i u m s p a r i n g e f f e c t s o f b o t h a m i l o r i d e and a1 d o s t e r o n e a n t a g o n i s t s . ( 3 2 however, a1 t h o u g h an a n t i k a l i u r e t i c e f f e c t o f metolazone h a s b e e n d o c u m e n t e d ( 2 1 , o t h e r a c u t e c l e a r a n c e s t u d i e s i n h e a l t h y s u b j e c t s have shown i n c o n s i s t a n t e f f e c t s on p o t a s s i u m e x c r e t i o n ( 2 1 , 3 9 ) o r i n c r e a s e d p o t a s s i u m e x c r e t i o n ( 2 2 , 38, 4 1 ) . i n p a t i e n t s w i t h o e d e m a t o u s d i s o r d e r s , m e t o l a z o n e i n c o n s i s t e n t l y cause h y p e r k a l i u r i a a s s o c i a t e d w i t h h y p o k a l e m i a . ( 1 9 ) . t h i s m i q h t be r e l a t e d t o t h e p r e s e n c e o f h y p e r a l d o s t e r o n i s m , s i n c e d o c a p r e t r e a t m e n t r e v e r s e d m e t o l a z o n e a n t i k a l i u r e s i s i n h e a l t h y s u b j e c t s . m o r e o v e r , i n c o m b i n a t i o n t h e r a p y o f s e v e r e oedematous d i s o d e r s w i t h l o o p d i u r e t i c s and metolazone, ex c e s s i v e p o t a s s i u m e x c r e t i o n and s e v e r e h y d o k a l e m i a c o u p l e d t o t h e d i u r e t i c e f f e c t h a s b e e n d e s c r i b e d ( 4 ) . t h e r e f o r e , f r o m a c l i n i c a l p o i n t o f v i e w m e t o l a z o n e does n o t seem t o o f f e r any advantage o v e r " o r d i n a r y " t h i a z i d e s w i t h r e g a r d t o p o t a s s i u m l o s s e s . m e t o l a z o n e i n f u s i o n c a u s e d n o c h a n g e s i n gfr a s m e a s u r e d b y t h e r e n a l c l e a r a n c e o f i n u l i n . t h i s i s p e r h a p s n o t s u r p r i s i n g c o n s i d e r i n g t h a t we sub s t i t u t e d f o r u r i n e volume l o s s e s f o r each p e r i o d and i t i s i n a q r e e m e n t w i t h o t h e r r e p o r t s i n h y d r a t e d s u b j e c t s ( 2 2 , 32, 38-40). b e n n e t t and p o r t e r ( 2 ) even f o u n d an i n c r e a s e d gfr a f t e r m e t o l a z o n e w h i l e gfr was u n c h a n q e d a f t e r c h l o r o t h i a z i d e . t h i s c o u l d perhaps be due t o d i f f e r e n t a d m i n i s t r a t i o n s o f t h e t w o d r u g s m e t o l a z o n e was g i v e n as a b o l u s w h i l e c h l o r o t h i a z i d e was g i v e n a s a b o l u s a n d a s u s t a i n i n g i n f u s i o n . we f o u n d t h a t t h e gfr i n c r e a s e d ( s i g n i f i c a n t l y d u r i n g p e r i o d s 3 a n d 4 ) a f t e r t h e p r o b e n e c i d i n j e c t i o n . the r e a s o n f o r t h i s i s u n c l e a r . i n o u r e x p e r i e n c e f r o m s i m i l a r s t u d i e s w i t h o t h e r d i u r e t i c , p r o b e n e c i d i n j e c t i o n has e i t h e r l e f t gfr unchanged ( 2 5 ) o r decreased ( 2 9 , 3 0 ) . renal plasma f l o w , as d e t e r m i n e d by t h e r e n a l c l e a r a n c e o f pah, was as e x p e c t e d s i g n i f i c a n t l y r e d u c e d by probenec d; an e f f e c t o f i n h i b i t i o n o f pah s e c r e t i o n . on t h e w h o l e , m e t o l a z o n e e f f e c t s o n e l e c t r o l y t e t r a n s p o r t seem t o b e q u a 1 i t a t i v e l y s i m i l a r t o t h o s e o f " c l a s s i c a l " t h i a z i d e s . the f a c t t h a t m e t o l a z o n e ( a l o n e o r i n c o m b i n a t i o n w i t h a l o o p d i u r e t i c ) can be an e f f e c t i v e d i u r e t i c a g e n t i n p a t i e n t s w i t h a p p a r e n t r e s i s t a n c e t o d i u r e t i c s e.g. i n r e n a l f a i l u r e , has been t a k e n as a c l a i m f o r some u n i q u e p r o p e r t i e s o f t h i s d r u g a s c o m p a i r e d t o t h e t h i a z i d e s . however, some o b s e r v a t i o n s w i t h b e n z o t h i a z i d e s (1, 3 3 ) i n d i c a t e t h a t i f t h e dose i s i n c r e a s e d , t h e s e d i u r e t i c s may a l s o be e f f e c t i v e i n s i m i l a r s i t u a t i o n s . a c l e a r answer t o t h i s q u e s t i o n a w a i t s p r o p e r l y d e s i g n e d c l i n i c a l t r i a l s . 31 acknowledgments. t h e a u t h o r s a r e i n d e p t e d t o mrs s i g r i d s a n d b e r g , f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e . we want t o thank pharmacia a b , uppsala, sweden f o r providing t h e metolazone s o l u t i o n s . 1. 2. 3. 4 . 5. 6 . 7 . 8. 9. references beermann, b . , o d l i n d , b . and w i b e l l , l . : d i u r e t i c e f f e c t a n d p h a r macoki n e t i c s o f t i z o l emi de i n s u b j e c t s w i t h normal and decreased renal f u n c t i o n . c l i n . nephrol. 1 9 ( 3 ) :124-131, 1983. bennett, w . m. and p o r t e r , g . : comparison o f i n t r a v e n o u s c h l o r o t h i a z i d e and metolazone i n normal man. curr. ther. res. 22:326-334, 1977. b e n t l e y , p.j.: a m i l o r i d e : a p o t e n t i n h i b i t o r of sodium t r a n s p o r t a c r o s s t h e toad bladder. j . p h y s i o l . 195:317-330, 1968. b l a c k , w.d., s h i n e r , p . t and roman, j . : severe e l e c t r o l y t e d i s t u r b a n c e s a s s o c i a t e d w i t h metolazone and furosemide. s o u t h e r n med. j . 71:380-381, 1978. brater, c . d . : i n c r e a s e i n d i u r e t i c e f f e c t of c h l o r o t h i a z i d e by probenecid. c l i n . pharmacol ther. 23 (3):259-265, 1978. b r u n , c.: a r a p i d method f o r t h e d e t e r m i n a t i o n o f para-aminohippuric a c i d i n kidney f u n c t i o n t e s t . j . lab. c l i n . med. 37:955-958, 1951. craswell, p.w., ezzat, e . , kopstein, j . , varghese, z . and moorhead, j . f . : use o f m e t o l a z o n e , a d i u r e t i c , i n p a t i e n t s w i t h renal d i s e a s e . nephron d a r g i e , h . j . , a l l i s o n , m . e . m . , kennedy, a.c. a n d gray, m.j.: e f f i c a c y of metolazone i n p a t i e n t s w i t h r e n a l edema. c l i n . nephrol 2 ( 4 ) :157-160, 1974. degnan, k.j., k a r n a k y , j r . k . j . and z a d u n a i s k y , j . a . a c t i v e c h l o r i d e t r a n s p o r t i n t h e i n v i t r o o p e r c u l a r s k i n o f a t e l e o s t ( f u n d u l u s h e t e r o c l i t u s ) , a g i l l l i k e e p i t h e l i u m r i c h i n c h l o r i d e c e l l s . j . p h y s i o l . 1 2 ~ 6 3 7 3 , 1973. 271 : 155-191, 1977. 10. e r i k s s o n , 0.: e f f e c t s o f s t a n d a r d d i u r e t i c s a n d orthovanadate on sodium t r a n s p o r t a c r o s s i s o l a t e d f r o g s k i n . acta p h y s i o l . scand. 1 2 2 : 2 4 9 2 6 0 , 1984. the u s e of i s o l a t e d f i s h o p e r c u l a r e p i t h e l i u m a s a t i s s u e f o r studying i n t r i n s i c a c t i v i t i e s of loop d i u r e t i c s . acta physiol . scand. 125:55-66,1985. 12. fernandez, p . and p u s c h e t t , j . : proximal t u b u l a r a c t i o n s of metolazone and c h l o r o t h i a z i d e . amer. j . p h y s i o l . 225:4, 954-961, 1973. 13. f r i z z e l l , r . a . , smith, p . l . , vosburgh, e . and f i e l d , m . : coupled sodium c h l o r i d e i n f l u x a c r o s s b r u s h border of f l o u n d e r i n t e s t i n e . j membr. b i o l . 11. eriksson, o., yayer-gostan, n. and w i s t r a n d , p.j.: 46:27-39, 1979. 32 14. g i e b i s c h , g.: e f f e c t s o f d i u r e t i c s on r e n a l t r a n s p o r t o f p o t a s s i u m . i n : m a r t i n e z m a l d o n a d o m . e d i t o r m e t h o d s i n p h a r m a c o l o g y . r e n a l pharmacology. new york, london, pleunum p r e s s 4:121-166, 1976. 15. h a m m a r l u n d , m . m . , o d l i n d , b . a n d p a a l z o w , l.k.: a c u t e t o l e r a n c e t o f u r o s e m i d e d i u r e s i s i n humans. pharmacokinetic-pharmacodynamic model i n g . j .pharmacol exp. ther. 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medicine u n i v e r s i t y hospital 751 85 uppsala sweden 35 upsala j med sci 92: 215, 1987 addendum i n t h e l a s t i s s u e o f t h e j o u r n a l (ujms v o l 92, no 1 ) 2 r e f e r e n c e s i n t h e paper o f lundberg, p.o. e t a l . (pages 59-64) were n o t i n c l u d e d , namely r e f e r e n c e s 7 and 8. they a r e g i v e n below: 7. lundberg, p.o., muhr, c., bergstrom, k., thuomas, k.-a., h a r t v i g , p., l u n d q v i s t , h., a n t o n i , g. & ldngstrom, 6.: pet and nmr i n d i a g n o s i s o f p i t u i t a r y adenomas. i n : pet and nmr: new p e r s p e c t i v e s i n n e u r o i m a g i n g and i n c l i n i c a l n e u r o c h e m i s t r y . l i s s s c i e n t i f i c p u b l i c a t i o n s , new york, 1986 ( i n p r e s s ) . 8. m o l i t c h , m.e., e l t o n , r.l., b l a c k w e l l , r.e., c a l d w e l l , b., chang, r.j., j a f f e , r., j o p l i n , g., robbins, r.j., tyson, j. & t h o r n e r , m.o.: bromo c r i p t i n e as p r i m a r y t h e r a p y f o r p r o l a c t i n s e c r e t i n g macroadenomas: r e s u l t s o f p r o s p e c t i v e m u l t i c e n t e r s t u d y . j c l i n e n d o c r i n o l metab 60:698-705, 1985. 215 upsala j med sci 93: 201-214, 1988 storage of human red blood cells and platelets some aspects concerning the factors leading t o storage lesion characterized as morphological changes and vesiculation. minireview based on a doctoral thesis christel solberg department of biochemistry, institute of medical biology, university of tromsm, troms#, norway abstract y 1. storage renders erythrocytes more responsive to thermally induced morphological changes, especially the shedding of microvesicles. 4-8 week old cells can be morphologically "rejuvenated" by heating. 2. if ph increases during storage of platelets an extensive loss of small particles occurs. the platelet disintegration is associated with a loss in the metabolic activity, discharge of ldh, increased suseptibility to phospholipid hydrolysis by phospholipase c and is found to be initiated during the actual preparation of platelet concentrates. 3. activation of platelets during preparation can be decreased by shortening the first centrifugation time or by using adenine in the anticoagulant. 4. a 4 hour prestorage of the whole blood unit prior to centrifugation strongly decreases the activation of platelets upon stimuli and results in platelet concentates much more stable to storage. introduction biochemical and morphological changes in erythrocytes during storage. upon storage, b series of metabolic and morphological changes occur in the erythrocyte producing eventually a non-viable cell form that is rapidly removed from the body upon transfusion. to understand these changes, it is necessary to understand the structure and properties of the fresh erythrocyte. the red cell membrane consists of a lipid bilayer of several differenl classes of lipids (30) with an ssyinetric distribution of the individual lipid types (64). the cholinc-containing phospholipids ( pc, sm) are located mainly in the outer layer and the amino phospholipids (ps, pe) preferentially in the inner layer. 13-888572 20 1 phosuliolipid composition in fresh human erythrocytes (30). phosoholipid class molar uercentaee sm sphingomyelin 24.5f2.0 pc pliosphatidylcholine 25.4fl.l ps phosphatidylserine 5.8f1.7 pe phosphatidylethanolamine 27.0f1.7 pip phosphatidylinositol-4,5-bisphosphate 1 .4m0.3 pi phosphatidyl inositol 1.2m.1 pa phosphatidic acid 2.2m.2 lysophosphatidylethanolamine 1.3io.3 lysophosphatidyl choline 1.om.3 integral membrane proteins penetrate the lipid bilayer and interact with the hydrophobic lipid core. these include receptor and antigen-bearing proteins, such as glycophorin a and the anion transport channel band 3. the latter is most important in that the protein cytoskeleton is anchored to band 3. the red cell cytoskeleton consists of a highly, organized filamentous network mainly consisting of spectrin (band 1, 2), ankyrin (band 2 4 , band 4.1 and actin (band 5) (13,49,50,78,79). the metabolic changes occuring during storage result in membrane reorganisation and change in morphology. during storage the red cell undergoes a defined series of morphological changes (fig.1) fig.1. scanning electron micrograph of stored red cells. it passes from the smooth discocyre (1) to a crenated disc (2, echinocyte i; 3, echinocyte 11) thereafter it swells to a crenated sphere (4, echinocyte 111) and finally to a sphere with multiple short, thin spicules (5, sphero-echinocyte) that will gradually shed off (6, spherocyte) (16,45,47). during storage intracellular ca ’+ increases and atp decreases (25,33,37,73,96). the echinocyte formation and vesiculation (budding of the thin spicules) can be induced by treating fresh cells with 202 ca2+ and ionophore a 23187 (1,z). echinocyte transformation can also be induced by metabolic depletion simply by incubating the red cells at 37°c for 24 h and results in two different reactions (69). one is the increase in lysolecithiri in the outer phospholipid bilayer resulting in an expansion in the outer leaflet and echinocytosis (42), the other slower change is the suggested rearrangement of the membrane proteins (29). atp does not directly control either the form or deformability of the membrane, but events secondary to atp depletion are responsible for the induced changes (28). the degree of irreversible change upon metabolic depletion depends of many external factors such as: rate, length of time of at€' depletion, temperature, ph, ca 2 + , mg 2 + , plasma, oxygen availability. after about 30 h atp depletion major lesions occur in the form of surface components loss (vesiculation) increase in cytoplasmic viscosity and osmotic fragility (93). the earliest change in the membrane structure upon atp depletion is the increase in the amount of spectrin-actin-4.1 complex that can be extracted from the membrane by exposure to low-ionic strength buffers (48). band 4.1 is associated with the lipid membrane by a polyphospho-inositide (pip,,pip)-regulated binding to glycophorin ( 5 ) . the binding sites for actin and band 4.1 are localized to the tail of the spectrin tetramer. near to the head region of spectrin is the binding site for ankyrin (89). the spectrin 8-chain has four phosphorylation sites in the end of the molecule where the end-to-end association with the a-chain occurs and close to the ankyrin binding sites. a confoimationally intact cytoskeleton is essential for maintaining the cell shape and deformability. upon heating a decrease in spectrin extractability can be measured prior to onset of red cell fragmentation and shape change (56). conformationally intact spectrin is necessary for preserving the membrane phospholipid asymmetry. when intact red cells are oxidised with agents that selectively crosslink spectrin, 30-50% of the inner membrane phospholipids, phosphatidylserine (ps) and phosphatidylethanolamine (pe), become accessible at the outer membrane surface (32). if these amino-phospholipids become exposed on the outer surface of the red cell or platelet, they will activate the conversion of prothrombin to thrombin and promote intravascular clotting (65,94). exposure of ps and pe on the exterior of blood cells also makes them more adherent to endothelial cells (75) and increases the trapping into the reticuloendothelial cells (76). while the relationship between spectrin phosphorylation and shape change is only indirect (4,68), it is clearly evident that the phospholipid asymmetry and shape change are atp-dependent (77). the maintenance of the membrane phospholipid asymmetry is due to two different processes: ankyrin has also a phosphorylation site localized in the spectrin-binding domain (12). i ) ii) binding of aminophospholipids to spectrin (32,so). "pumping" of lipids from the outside back to the inside, by the bidirectional atp-dependent "flip-flop" enzyme (flippase) (92). although there is only 1.4% phosphatidylinositol-4,5.bisphosphate (pip , ) in the membrane it is evident that the conversion of pip, to p i is directly correlated to atp depletion (30). concomit tant with this conversion phosphatidic acid will dephosphorylate to diacylglycerol. taken together 203 the changes in lipid population will shrink the inner membrane monolayer by 0.6%, which is enough to convert discocytes to echinocytes iii (10). high levels of intracellular calcium activate the ca '+ -dependent phospholipase c resulting in the ' degradation of pip and pip to the fusogenic diacylglycerol (3). in conclusion, to survive in the circulation the erythrocyte must be both strong and flexible (49,50,57). the "cellular deformability" is necessary for passage through the microcapillaries and especially through the narrow portals of the spleen. the biconcave disc-shape of normal eryth rocytes creates a surface area-to-volume relationship that is advantageous for this purpose. the sphere has in contrast to the discoid form minimum area-to-volume ratio and is therefore far less flexible and deformable. stored cells not only lose in vivo viability because of increased trapping in the spleen but also disappear from the circulation because of increased adhesion to endothelial cells. preparation and storage of platelets whereas the human red cell is a rather simple non-nucleated cell consisting of a flexible and very stable, uniformly built up membrane enclosing a near-saturated solution of hemoglobin with the only function to transport 0 and co , , the platelet is a complex, non-nucleated, very active cell. platelets have, in addition, mitochondria performing oxidative phosphorylation and resulting in 36 atp/glucose molecule instead of only 2 atp/glucose in the glycolytic pathway. the adenine nucleotides in the platelets are enclosed in three physically distinct compartments, 64% in the dense granule pool (human atp/adp ratio = 0.7), 32% in the metabolic pool (ate'/adp ratio = 4-5) and 4% in the f-actin bound pool (adp only) (36). the platelet plasma membrane is very similar to the erythrocyte membrane, the phospholipids are asymmetrically arranged in the same pattern (20.95). spectrin-like molecules underlying the membrane have been found (23) with concomittant changes in the organization of cytoskeletal elements and the exposure of internal phospholipids upon activation of platelets (9,21). the platelet membrane has, in addition, several different receptors and is easily activated by many different agents (22,39,55). many of these substances can also be released during the activation process itself such as adp and serotonin (released from the platelet dense granulae), arachidonic acid and thromboxane a, (from the phospholipid breakdown in the platelet membrane), thrombin (from the activation of prothrombin by the coagulation cascade) and collagen (released from injured epithelial cells). activation can also be induced by physical stimuli such as cell-cell contact which occurs, for example, during mixing (6.7). centrifugation (53,74) and gel filtration (74). activation of the platelet membrane results in the breakdown of phospholipids and especially in changes in the small phosphatidylinositol pool. the polyphosphatidylinositol (pjp ) hydrolysis to the second messengers inositoltriphosphate (jp ) and diacylglycerol (dg) is the initial agonist (e.g., thrombin, collagen, platelet activating factor, thromboxane a , )-stimulated event in the phosphatidylinositol turnover (14,51,63). jp, mobilizes ca2+ from intracellular stores and initiates pi hydrolysis. dg 204 and ca2' in concert serve as a direct activator of protein kinase c resulting in the phosphorylation of the 40 kda protein and the ca2'-enhanced serotonin release from dense granulae (39). dg, once produced, disappears very rapidly; it is converted either to phosphatidic acid or by the action of diglyceride lipase degraded to glycerol and arachidonic acid (1 1,39). the ca 2+ mobilization also activates phospholipase a further facilitated by dg (40) and results in arachidonic acid release from the major phospholipid pools (pc, pe, ps) (17,70). liberated arachidonic acid is rapidly converted to prostaglandins and thromboxanes which in turn are capable of activating other phospholipids in the membrane resulting in a positive feedback loop (35). the activation of plc and pla, working together leads to an amplified response (8,17). both enzymes lead to arachidonic acid liberation different ways (91). plc has an ph optimum of 7.5, is specific for phosphatidylinositol and liberates diglyceride. pla, activity is enhanced by increasing the ph (7.5 to 9.5), hydrolyses pc, pe, ps and liberates free fatty-acid and lysophospholipids (17). there is also a synergistic amplifiction in the platelet response as a result of simultaneously stimulation by different agonists (8,26,66). in morphological terms the activation of platelets is seen as a change from a smooth disc to a sphere with pseudopods. the first phase in platelet activ-ation is a reversible shape change. if the cell is more strongly activated a second phase will follow which is associated with release of the granulae content and irreversible changes occur (44). a na'/h+ antiporter is involved in the pathway leading to arachidonic acid mobilization by epineph rine, adp and low concentrations of thrombin (8). removal of extraplatelet na' or increasing extraplatelet h t (i.e. decreasing extraplatelet ph from 7.35 to 6.8) causes a blockage in the na'lh' exchange system and inhibits platelet aggregation (88). the morphological change in stored platelets is found to be well correlated with the posttransfusion viability and those concentrates having the highest proportion of discoid platelets also have the best survival (41). there is also a close correlation between the ph in the platelet concentrate (pc) after storage and the morphology (83). at ph 7.2-6.8 platelets maintain a normal discoid shape. with decreasing ph, platelets swell and at ph less than 6.0 all platelets are spherical (59). this fall in ph is due to lactate production from glycolysis (58). if the platelets are supplied with enough 0 2 , lactate production and thereby ph decrease is prevented. a new generation of storage bags with increased gas permeability has therefore been developed (61). the high gas permeability very effectively prevents the ph fall below 6.0. in the first generation of platelet storage bags the shelf life for pc was limited to 3 days because the rapid decrease in ph. in the second generation of platelet storage bags the shelf life has been extended to 5-7 days. loss of respiratory metabolism as observed by rising po, and decreasing p c 0 , results in decreasing atp levels. the platelet storage lesion, measured as the decrease in platelet count, increasing discharage of intracellular lactate dehydrogenase (ldh), decrease in the extent of photometrically measured shape change induced by adp and the ability of platelets to recover from hypotonic shock rue all found to be highly correlated to platelet atp (34). for maintaining the phophoinositide cycle, atp is necessary. it has recently been shown that the (pla , ), a stimulation that is even though they hydrolyse phospholipids in 205 generation of pip 2 , and not the second messengers ip and dg, controls the polymerization of actin and the mechanism of platelet shape change mechanism (43). the inability to produce pip, could therefore be a limiting factor for in vivo viability and function. during storage the cells can start to bud off membrane (vesiculation) resulting in a irreversibly changed cell membrane. in the erythrocyte this is a relatively slow process and the uniformly built up cell membrane renders the erythrocyte a good model for studying the vesiculation process. in the platelet, the cell interior consists of several different organelles and an internal surface connecting system (52). the main function of the platelet is the activation and aggregation process. the storage lesion of platelets in the form of buddindfragmentation, could, because of rhe risk for an accelerated "self-activation", be explosive. from an deeper understanding of the circumstances responsible for and the mechanism of vesiculation/fragmentation it will be possible to change the preparation and storage properties and in that way make more storage-stable, viable blood cells. results and discussion a. red cell storage and vesiculation fresh red cells are essentially unaffected by heating below the thermal transition temperature of spectrin (49"c), but above rhis temperature are very rapidly changed to large smooth spheres and subsequently to smaller smooth spheres (81). during in vitro storage of red cells metabolic and morphological changes gradually occur. some of the morphological changes are easily reversible, sometimes simply needing increases in intracellular atp for reversibility, whereas some changes are irreversible e.g. the transformatin to speroechino cytes or sperocytes. during storage of red cells to the time of outdaling (4-8 weeks) the proportion of echinocytes(e)ii and eiii is increased. a 1 minute heat treatment at 46°c of 4-8 weeks stored blood, readily increased the proportion of the discoid (d+ei) (i.e., morphologically "younger") cell forms (from 40 to 55%) concornitlatit with a decrease in the proportion of eiii. recent findings of others also show that red cells, stored for 42 days in sagm-solution, show a significantly increased (39% to 51%) precentage of discoid formed cells (d+ei) upon a one hour incubation at 37'c. this latter improvement in morphology does not, however, result in any change in the in vivo posttransfusion survival, potassium leak, glucose consumption or atp concentration (38). it is thus highly questionable whether this morphological improvement we observe using the 46'c treatment represents a true "rejuvenation" of the aged erythrocytes. if the stored cells instead are rejuvenated (by incubation with adenosine or adenine to raise the intracellular atp level), the improvment in morphology is followed by an increased in vitro viability (37,47,90). prolonged heating of stored cells at 46'c resulted in a fall in the precentage discoid formed cells and a marked increase in the spheroechinocytic cells (i.e., "accelerated ageing"). thus indicating thal lhere are still unrepaired changes in the stored cell membrane, making the cells more 206 susceptible to heating than are fresh cells. at 48 c, extensive shedding of microvesicles and a gradual increase in the number of spherocytes occurs. at 5ooc a rapid and extensive microvesiculation associated with a rapid conversion to spherocytes of different sizes is observed. during atp-depletion, spectrin will be dephosphorylated and result in echinocytosis. the loss of the interaction between the spectrin-cytoskeleton and the phospholipid-bilayer could explain the facilitated vesiculation occuring upon heating around the spectrin-transition temperature (67,78,81). the spherocyte was found not to respond morphologically to heating other than by lysing. longster (47) has also shown, that stored cells which already have reached the "smooth sphere state, were similar to the type of cells which were rapidly removed from the circulation upon transfusion. he also found that these cell types cannot revert to disc form upon incubation at 37°c with or without added adenine. the speroechinocyte, but not the earlier morphological forms, is also readily lysed upon treatment with phospholipase c (plc, bacillus cereus) and this enzyme can be used as a tool for assessing the degree of in vitro ageing of stored red cells (45). this would suggest some marked differences in the nature of the cell membrane in spheroechinocytes. most probably a lowering of the lateral packing pressure in the membrane (24), although other membrane rearrangements cannot be excluded. little (46) showed that glass-bottle stored rbc were more susceptible to plc lysis than plastic bag ( d e w containing) stored cells. b . storage of platelet concentrate in the first generation containers (pl-146). the phospholipids of fresh human platelets are extremely resistant to hydrolysis by externally added plc (82). this was also the case with activated fresh platelets. during storage, presumably as a result of decreases in the lateral packing of phospholipids in the external leaflet, the suceptibility to plc degradation increases. these suggested ch'anges in membrane packing pressure occumng both in stored red cells and stored platelets could arise from a build up in the levels of components such as diglyceride, lysophospholipids or free fatty acids in the membrane (20). the extent of phospholipid hydrolysis upon incubation of platelets with plc is dependent on the plasma ph after storage (82). pc with a ph 6.6-6.8 resulted in only 1045% phospholipid hydrolysis. lower ph resulted in higher degradation. increasing ph resulted in a more marked increase in the susceptibility to hydrolysis by plc and this was found to be very extensive (50-60%) at ph>7.4. note that the ph values refer to the plasma ph in the concentrates after storage. all platelets were incubated with plc at the same ph. in pc with ph>7.3 after storage, there was also a prominent change in morphology. the size distribution of pc shows that the platelets have become smaller and the pc contaiii an increased number of microvesicles/platelet fragments (83). a quarter of the platelets were balloon-formed or lysed and the discharge of ldh was elevated. the storage lesion was also found to be more marked for pc of lower ph after storage. the progressive disc-to-sphere swelling was reversible as long as the ph was >6.1. 207 c. storage of platelet concentrate in second veneration containers (pl 1240. pl 7321. i) influence of storage containers. a limiting factor for platelet storage in the first generation of bags, was rate of diffusion of gases, especially 0, through the bag walls. types of plastics (polyolefin, fenwal type pl 732) (61) or by using other types of plasticizer in pvc bags (fenwal 1240, cuttler clx) (31) the gas diffusion could be increased. when pc stored in the first generation of bags (pl 146) were compared to pc in the second generation of bags (pl 1240), at storage expiry the latter were, in our hands, found on average to have become much more alkaline (84,85). in view of the well established connection between changes in ph during storage and loss of vkblility (41,58,60,61) the above observation would lead one to expect an enhanced loss of platelet quality in the second generation of bags. indeed, this rise in ph during storage was associated with extensive evidence of platelet degradation in the form of decreased platelet count, high levels of extracellular ldh activity and very abnormal platelet size distribution profiles as shown in the coulter counter. this latter feature arose, from platelet disintegration as seen in electron microscopy ( 8 5 ) and with the subsequent appearance of large numbers of small particles (84). there was a strong correlation between the extent of ph increase during storage and the increase in relative number of small particles (e0.85). isolation of these small particles showed that they g had a very considerable procoagdant activity, measured as platelet factor 3 activity (pf3) (84). similar findings have recently been found by miller and bode (54). using a flow cytometer these workers have shown the occurrence of small particles in stored pc and that these particles have pf3 activity. they have also found that thrombin inhibitors in the anticoagulant inhibit thrombin production and the release of pf3 (18,19). the discharge of ldh was also strongly correlated with the ph changes. in pl 1240 the ldh discharge was several-fold higher than that observed in pl 146 bags. the average ldh discharge in 3 pc after 4 days storage in pl 146 (i.e. storage life expiry) was 11% and in pl 1240 30% which rose to 45% after 6 days storage (storage life expiry for pl 1240 stored pc). a major difference between pl 146 and pl 1240 is the nature of the plasticizer used in the manufacture of the plastic from which the storage bag is constructed. in pl 146 dehp (di-ethyl-hexyl-phthalate) plasticizer is used and this is easily extracted from the plastic, is fat-soluble and is known to be accumulated in the cell membrane (71). dehp has been found to inhibit phospholipase a , activity and can thereby decrease the platelet aggregation ability (72). this chemical has also been shown to inhibit the vesiculation from erythrocytes (27). the storage life for red cell decreases from 35 to 21 days if types of storage bag, other than dehp containing ones, are used (62,71). pl 1240 and clx bags contain tri(2-ethylhexyl) trimellitate (tehtm) as plasticizer. tehtm is less fat soluble and results in a 30-fold reduction of plasticizer accumulated in platelet concentrates during 5 day storage at 22'c (i.e. 15 pg/ml of tehtm compared with 450 pg/ml of d e w ) (31). the higher gas diffusion, in the second generation of bags, not only hinders the development of it was found that by using very thin-walled bags (60), other anaerobic conditions and the resulting lactate production but concomittantly accelerates co diffusion out of the bags. an early rise in ph can sensitize the platelet to further damage resulting in less co, production leading to further ph rise. a vicious circle resulting in high ph, loss of metabolic activity and platelet disintegration can in this way be established. indeed, murphy (61) has shown that deleterious changes and decrease in in vivo viability associated with high ph, could be largely prevented by storing pc in an atmosphere containing 10% co, . -100ae z 0 0 w u 8 50 6 w u $ 75 25 5 u a: ii. influence of memuation method . platelets are isolated from whole blood by centrifugation. by using different recommended centrifugation methods we found that the metabolic activity during and after storage in the pc was very different depending on the precise centrifugation method used (85,86). already the day after preparation there was a highly significant difference between the po, values in the bags (86). depending on the platelet concentration, ph and the metabolic activity in the pc, the ldh discharge and the extent of build up of small paraticles (s.p.) after storage could be predicted, to 90% (ldh) and 86% (s.p.) respectively by the use of multivariate data analysis (86). this close correlation between the aspects of the storage lesion and loss of metabolic activity was confirmed in the recently published results of holme (34) showing the correlation between metabolic activity, atp concentration and storage lesion. our findings concerning the strong influence of the centrifugation method used and the subsequent metabolic activity in stored pc was, however, in great contrast with experience reported widely in the u.s.a. the major difference between the blood collection and handling procedures in u.s.a. and our own is the time between blood withdrawal and subsequent preparation. in the u.s.a. about 80% of the blood is collected in blood-mobiles, resulting in a gap of several hours between donation and the separation of blood components. in sweden and norway the preparation of components is supposed to be carried out within two hours after phlebotomy (15). 0 i 1 l h dh 3 h b h time of storage of whole blood fig. 2. changes in aggregation response in whole blood upon storage from four different donators. 209 with the use of a whole-blood aggrometer, changes in the aggregation response after phlebotomy could be followed in whole blood, i.e., without any manipulations of the blood (fig. 2). the aggregation response measured at 37°c starts to decrease after 2 hours. after 3 hour the aggrega tion was less than half the maximum response (87). preparation of prp after a 4 hour prehold of whole blood results in a significantly decreased aggregation response upon stimulation with collagen. using lower (cyclooxygenase-dependent) doses resulted in no aggregation (measured as change in light absorption), whereas prp prepared exactly 60 minutes after blood withdrawal yielded a high aggregation response. ' using cpd-adenine instead of cpd alone as 'anticoagulant was also found to decrease significantly the aggregation response to physiological doses of collagen. the superior morphological state of 6 day-stored platelet concentrates from platelets prepared after 4h-rested blood compared with immediately processed blood is clearly apparent in electron microsc opy (fig.3.) fig.3. transmission electron micrographs of 6 day-stored pc. a= pc prepared close to phlebotomy showing a high number of lysed or fragmented platelets, which correpond to the high ldh discharge. b= pc prepared after 4 h prestorage of whole blood showing discoid platelets with well preserved internal structure and almost no lysed cells. a 4 hours prehold of cpd-whole blood prior to preparation in the secondgeneration of bags results in less activation during preparation and less storage lesion. the combination of storage a1 higher ph and no dehp in cpd-plasma renders the time delay before preparation as a new important parameter to be considered in platelet storage. in norway and sweden and conceivably in other regions of the world as well, present blood bank practise is to process blood as soon as possible after donation. in such a situation, the combina tion of the second generation storage containers and adenine-free anticoagulant can very easily yield substandard, potentially dangerous platelet preparations after storage. the uncovering of this fact together with what appears to be a very simple way of avoiding this problem is undoubtedly the most import'anl aspect of the work described in this thesis. 210 references 1. 2. 3 4. 5 . 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. allan, d., michell, r.h. 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(1987) stimulation of ca*+-activated human platelet phospholipase a by diacylglycerol. biochem. j. m , 779. 41. kunicki, t.j., tucceli, m., becker, g.a., aster, r.h. (1975) a study of variables affecting the quality of platelets stored at "room temperature". transfusion u, 414. 42. lange, y., slayton, j.m. (1982) interaction of cholesterol and lysophosphatidylcholine in determing red cell shape. j. lipid res. 2, 1121. 43. lassing, i., lindberg, u. (1988) evidence that the phosphatidylinositol cycle is linked to cell motility. exp. cell res. 174, 1. 44. leung, l., nachman, r. (1986) molecular mechanisms of platelet aggregation. ann. rev. med. 37, 179. 45. little, c., rumsby, m.g. (1980) lysis of erythrocytes from stored human blood by phospho lipase c (bacillus cereus). biochem. j. m, 39. 46. little, c., rumsby, m.g., johansen, s. 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(1983) membrane skeletal dynamics: role in modulation of red cell deformability, mobility of transmembrane proteins and shape. sem. in hematol. 20, 175. 80. sikorski, a.f., michalak, k., babrowska, m. (1987) interaction of spectrin with phospholipids. quenching of spectrin intrinsic fluorescence by phospholipid suspensions. biochim. biophys. acta 904, 5 5 . 81. solberg, c., litt1e.c. (1984) morphological changes during heating of erythrocytes from stored human blood. j.thenn biol. e, 221. v o x sang. b, 199. 213 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. solberg, c., little, c., holme, s., aakre, s.-e. (1986) effect of phospholipase c (bacillus cereus) on freshly isolated and 4-day stored human platelets. biochem. 5.222, 389. solberg, c., holme, s., little, c.(1986) morphological changes during storage of platelet concentrates in first-generation 3-day container. vox sang.50,70. solberg, c., 0sterrud, b., little, c. 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(1979) purification of two spectrin-binding proteins: biochemical and electron microscopic evidence for site-specific reassociation between spectrin and bimd 2.1 and 4.1. proc. natl. acad. sci. u.s.a. 76, 5192. usry, r.t., moore, g.l., manalo, f.w. (1975) morphology of stored rejuvenated human erythrocytes. vox sang. 3, 176. van den bosch, h. (1983) phospholipases: link between membrane phospholipids and arachino date metabolites. life sci. 54, 1. williamson, p., antia, r., schlegel, r. (1987) maintenance of membrane phospholipid asymmetry. lipid-cytoskeletal interaction or lipid pump? febs m, 316. wolfe, l.c. (1985) the membrane and the lesions of storage in preserved red cells. tr'ansfusion 2, 185. zwaal, r.f.a., comfurius, p., van deenen, l.l.m. (1977) membrane asymmetry and blood coagulation. nature 2$h, 358. zwaal, r.f.a. (1978) membrane and lipid involvement in blood coagulation. biochim. biophys. acta s, 163. akerblom, o., de verdier, c.-h., finnson, m., garby, l., hogman, c.f., johansson, s.g.o. (1967) further studies on the effect of adenine in blood preservation. transfusion 7, 1. adress for correspondence: christel solberg institute of medical biology university of tromsg 9001 tromsg, norway 214 upsala j med sci 81: 65-69, 1976 a solid phase radioimmunoassay for pancreatic glucagon gudmar lundqvis’t, john edwards and l e i f wide from the department of clinical chemistry and department of histology, university of uppsala, uppsala, sweden abstract a solid phase radioimmunoassay procedure has heen ap plied for the determination of glucagon and some variables of the assay procedure were studied. the sensitivity of the assay was 10 pg of glucagon for samples assayed in tripli cate. this assay method which has a high precision and is technically simple as compared with other radioimmuno assays, was found to he suitable for studies on glucagon release in vitro. introduction since the presentation of the first radioimmuno assay method for glucagon (19, 20), several reports have been published on the measurement of this hormone using different techniques for the separa tion of antibody-bound from unbound hormone in the assay procedure (1, 8, 12, 18, 15, 13, 4, 9, 2). a particular complication in all glucagon immuno assays is the susceptibility of glucagon, and espe cially iodinated glucagon, to enzymatic degrada tion. the enzymatic damage can be reduced by the addition of trasylol, a proteinase inhibitor, to the incubation mixture ( 5 ) . however, there is still a demand for shorter incubation times in the assay procedure to reduce the exposure of glucagon to the degrading enzymes. a solid phase radioimmunoassay procedure, in which antibodies are chemically coupled t o an in soluble polysaccharide, has been introduced for the assay of protein and polypeptide hormones ( 2 2 , 24). in the present investigation this radioimmuno sorbent technique was applied for the determination of glucagon and some variables of the assay pro cedure were studied. this assay method which produces a simple and efficient separation of antibody-bound from unbound glucagon was found . to be suitable for studies on glucagon release in vitro. material and methods chemicals crystalline pork glucagon (kindly supplied by dr l. hed ing, novo industri, denmark, lot. nr. b66) was used for the induction of antibodies, and beef-pork glucagon (sigma chemical co., st. louis, usa) was used for the preparation of 1125-glucagon and for the standards in the assay procedure. synthesized human gastrin i (imperial chemical in dustries ltd., england), crystalline pork insulin (novo industri, denmark, lot. nr. s21166), pork secretin and cholecystokinin-pancreozymin (kindly supplied by prof. v. mutt) were used in the cross-reactivity studies. was obtained as n a p 5 in naoh solution from the radiochemical centre, amersham, england. rabbit albumin (fraction v) was obtained from mann research lab., new york, usa, bovine albumin (fraction v) from armour pharmaceuticals ltd., eastbourne, england and carbodiimide (morpho-cdi) was from aldrich chem. co., milwaukee, usa. “trasylol” was supplied by bayer, leverkusen, west germany. freunds adjuvant was obtained from difco labs., detroit, usa, and tween 20 was obtained from sigma chemical co., st. louis, usa. microcrystalline cellulose and other reagents of analytical grade were from e. merck ag, darmstadt, w. germany. preparation of antisera pork glucagon coupled to rabbit albumin by carbodiimide was administered to rabbits in the antibody production process (7, 6). the animals were immunized by series of 3 weekly intramuscular injections of antigen containing 5 mg glucagon. this procedure was repeated every 2 months for a period of 6-12 months. antisera were stored at -20°c until the preparation of the immunosorbent. preparation of i’2s-glucagon beef-pork glucagon was labelled with iiz5 using the chloramine-t method of hunter & greenwood (10) as modified by edwards et al. (4). separation of the iodinated hormone from free was performed on a sephadex g25 column (25x 1 cm). it has been shown that glucagon and labelled glucagon normally remain at the point of application after paper chromatography on whatman 3mm paper with veronal buffer, ph 8 . 6 (20). this method was used in order to 5-162852 upsala j med sci 81 66 g. lundqvist et al. estimate the immunoreactivity of the labelled glucagon. normal serum and glucagon antiserum (1 : 5 dilution) were incubated for 24 hours with the labelled glucagon (100-200 pg) and trasylol(l000 kiu/ml). after chromatography of the incubation mixtures, the papers were cut into 1 cm strips and the distribution of radioactivity was then de termined. detection of antibodies a solid phase “sandwich-technique” (25) was used as a sensitive test of the antisera. five mg of glucagon was coupled to 100 mg of cyanogen bromide-activated cel lulose (using the same method as for the coupling of anti bodies, see below), and 0.2 ml of this cellulose-glucagon suspension ( 5 mglml) was incubated for 24 hours with 50 pl of a 1 : 10 dilution of the test antisera. after centrifuga tion and washing, the cellulose-glucagon-antibody com plex was incubated with 10cl200 pg of 1125-glucagon in a second incubation of 24 hours. the cellulose-glucagon antib~dy-i~*~-glucagon complex was then separated from the free 1125-glucagon by centrifugation and washing be fore determination of the radioactivity of the cellulose bound 1125-glucagon. antisera which resulted in more than 50 per cent binding of 1125-glucagon to the cellulose parti cles were selected for further studies. to decide which one of the antisera would give the highest sensitivity in the assay system, titration curves were made with and without the addition of a constant amount of the unlabelled antigen as described by hurn & landon (1 1). when 20% of the 1125-glucagon was bound by each antiserum, the antiserum which showed the largest reduction in antibody-bound i’25-glucagon in the presence of the added unlabelled glucagon was subsequently used in the assay. activation of cellulose and coupling of antibodies antibodies were coupled to cyanogen bromide-activated microcrystalline cellulose as described in detail previously (23,24). the sodium sulphate precipitated gamma globulin fraction from 0.1 ml of antiserum was incubated with 300 mg of the activated cellulose in the coupling procedure. a suspension of 5 mg/ml of the cellulose-bound antibody could then be used in the assay system. the amount of immunosorbent was chosen so that in the absence of un labelled glucagon, about 20 per cent of the 1125-glucagon was bound to the immunosorbent during an incubation time of 16-18 hours. immunoassay procedure standards and samples were assayed in triplicate. each assay tube contained 200 p1 of a suspension of the immunosorbent ( 5 mg/ml) in 0.1 m phosphate buffer, ph 7.5 containing 0.5 mg/ml bovine plasma albumin and 0.5 % tween 20, approx, 100 pg of 1125-glucagon in 100 pi of the same phosphate buffer, and 200 p1 of sample or standard. trasylol (100 p1 of a solution containing 5 0 0 0 kiu/ml saline) was added to each tube to prevent degradation of glucagon during the assay. the tubes were then incubated for 16-18 hours at room temperature with constant rota tion in a “rotamix” (heto, birkerod, denmark) to keep the cellulose particles in suspension. separation of the glucagon-antibody-cellulose complex from unbound glucagon was performed by centrifugation at 3 000 y m for one minute in an mse super medium centrifuge (mse, london, england). the cellulose particles were then washed three times with 1.5 ml volumes of saline before determination of their radioactivity in a gamma counter. in order t o test the non-specific binding of i’2s-glucagon to the cellulose-globulin complex, the sodium sulphate pre cipitated gamma globulin fraction of normal rabbit serum was coupled to cellulose as described for the preparation of the immunosorbent, and used in the assay conditions. isolation and incubation of guinea-pig islets of langerhans islets of langerhans were isolated from guinea-pig pancreas using collagenase, and incubated in groups of ten using methods described in detail elsewhere (4). the islets were incubated in the presence of 5 . 5 mm glucose and, in order to inhibit the glucagon release from the islets, with the addition of 5 mm octanoic acid to the incubation medium. samples of the incubation medium were stored at -20°c until the determination of their glucagon content. medium taken before the incubations served as controls in the assay. results iodination of glucagon the specific activity of iodinated glucagon was 250-300 pcilpg, while the iodination damage was about 15 % shown by tca precipitation and paper chromatography. comparisons between the paper chromatography of 1125-glucagon incubated with normal serum and antiglucagon serum showed that 80% of the radioactivity, which remained at the origin with normal serum, migrated in the presence of antiglucagon serum. the results indicated that at least 80 % of the 1125-giucagon was immunoreactive. the iodinated hormone was stored at -20°c and could be used for 4 to 6 weeks. antibody production and selection of antiserum samples of antisera from 3 of the 6 animals used in the immunization procedure showed sufficient bind ing capacities of labelled glucagon to be used in the assay. 1 ml of the antiserum with the highest bind ing capacity could be used for about 3 000 analyses. from the titration curves with and without the addi tion of unlabelled glucagon it was found that with different antisera reduction of 10-24 % of the antibody-bound radioactivity were caused by addi tion of 200 pg unlabelled glucagon. upsala j med sci 81 a solid phase radioimmunoassay f o r glucagon 67 hours of incubation with little further increase after this time (fig. 1). an incubation time of 16-18 hours was subsequently used in the assay system. standard curve a standard curve for determinations in triplicate over the range 0-500 pg glucagon per sample is shown in fig. 2 . the standard curve is presented both as radioactivity bound to the particles vs. dose and after logit transformation according to rodbard et al. (16). the average of the standard errors of the z f2000. 6 12 24 18 hours of lncu8atfon 2 ~ i ~ . 1 . the uptake of 1125-glucagon by cellulose-bound antibody. the immunosorbent was incubated with labelled glucagon under the conditions of the assay and the radio activity of the cellulose-bound 1125-glucagon determined at the times shown. mean for the triplicate samples was 0.5 % of the radioactivity bound at each level of added giucagon. the sensitivity of the assay expressed as the lowest assay incubation temperature in order to study the influence of incubation tem perature upon the antigen-antibody reaction, incuba tions were performed at room temperature and at 4°c. although the standard curves were parallel, the uptake of labelled hormone was increased when incubated at room temperature and assays were therefore performed at room temperature. assay incubation time in the glucagon assay at room temperature it was shown that the uptake of 1125-glucagon on the immunosorbent was rapid during the first twelve 23001 i s.e.m. of triplicate samples 2 i1 1900 0 u, 1800 2 a 0' i 1700 t 1600 t 15001 amount of glucagon which could be detected from a sample containing zero glucagon with 95% confi dence when the samples were assayed in triplicate, was calculated to be approx. 10 pg of glucagon. twenty pg of glucagon could be detected for sam ples assayed in duplicate. the coefficient of varia tion for means of triplicate within an assay was calculated to be about 3 % at a glucagon level of 29!2-400 pg. specificity of the assay samples containing pork insulin, synthetic human gastrin i, pork secretin or pork cholesystokinin l 0 05 1.0 1.5 20 2.5 0.25 05 lo 1.5 2.02.5 glucagon l n g / m l i glucagoh(ng/mt i -1 fig. 2. glucagon assay standard curve. a single assay was ratio bit is expressed as a percentage, where b equals performed as described in the text and the radioactivity of activity bound at each level of added glucagon and t* the cellulose-bound 1125-glucagon was determined for addiequals activity bound in the absence of unlabelled tions of unlabelled glucagon over the range 0-500 pg. the glucagon. upsala j med sci 81 68 g. lundqvist et ul. pancreozymin at concentrations from 1 to 1000 ng/ml were incubated with the immunosorbent and 1125-glucagon under the conditions of the assay. it was found that there was no inhibition of the bind ing of labelled glucagon to antibody by these hormones. samples containing guinea-pig glucagon and beef-pork glucagon gave parallel dilution curves in the assay procedure. glucagon release from isolated guinea-pig islets the rate of glucagon release from guinea-pig islets incubated in vitro in medium containing 5.5 mm glucose was found to be 1.50fo. 12 ng/islets/30 min ( 1 1 observations). when 5 mm octanoic acid was added to the incu bation medium, glucagon release from the isolated islets was markedly inhibited. the rate of glucagon release in the presence of 5.5 mm glucose and 5 mm octanoic acid was 0.44f0.18 ng/lo islets/30 min (9 observations). possible interference by the incuba tion media was excluded by the use of control samples. discussion several methods have previously been reported for the separation of antibody-bound glucagon from unbound glucagon in radioimmunological assay methods. when comparing their relative merits, the efficiency in separation of free from bound hormone as well as the technical simplicity and speed of the analyses have to be taken into consideration. for glucagon measurements the ability of the method to avoid interference from damaged hormone is also important, since glucagon is very unstable to en zymatic degradation. in this respect, the radioimmunosorbent tech niques offer some advantages over other methods of radioimmunoassay (25). an almost complete sep aration of free hormone from bound is obtained and the reported rapid dissociation of the antibody antigen complex (15) was not observed during the separation procedure of this system. proteolytic enzymes in the antisera are removed before the immunosorbent is used in the assay and the amount of proteolytic enzymes in the assay incubation mix ture are therefore very small. the binding of glucagon to antibody could be temperature dependent for some antisera (9). when this was investigated in our assay system only small differences were found between the ratio of antibody-bound glucagon to unbound in incubations at room temperature and at 4°c with the particular antisera used. the non-specific adsorption of radioactive mater ial to the cellulose particles in the assay was very low, below 0.5% of added radioactivity. the back ground radioactive contamination of the antibody bound hormone was therefore small, contributing to the high precision of the assay. interference by damaged i'*s-glucagon which has been shown to cause errors in other assay methods (2), was negli gible. in this radioimmunosorbent glucagon assay the sensitivity obtained was 10 pg of glucagon per sam ple, comparable to the sensitivity of the ethanol precipitation assay described by heding (9). in other assay systems 20-200 pg could be detected with the same confidence (3, 8, 15, 18, 21). the uptake of the labelled glucagon by the immuno sorbent was rapid during the first 12 hours of incu bation, and longer assay incubation times were un necessary. the high sensitivity could therefore be obtained in spite of the relatively short incubation time of 16-18 hours as compared with other analyse methods where incubation times of up t o 72 hours are used. the cross-reaction of the glucagon antisera with other pancreatic hormones or hormones with simi lar structure to glucagon was studied. since purified hormones of guinea-pig were not available, samples of hormones from other species were used. no cross-reaction was observed with pork insulin, hu man gastrin i , pork secretin or pork cholecystoki nin-pancreozymin. the separation of antibody-bound hormone from free is technically simple and rapid, and using automatic techniques (24), it is possible to perform the separation procedure for 200 tubes in 45 min. when glucagon release from isolated guinea-pig islets was measured, an inhibition of glucagon re lease was obtained by octanoic acid. it has been found previously that fatty acids inhibit glucagon release both from isolated islets incubated in vitro (3) and from the pancreas in vivo (14). the levels of glucagon release from the isolated guinea-pig islets incubated in the presence of glucose and of octanoic acid compare favourably with those found using ethanol precipitation assay method (4). the basal glucagon secretion in the presence of glucose also upsala j med sci81 a solid phase radioimmunoassay f o r glucagon 69 12. lawrence, a. m.: radioimmunoassayable glucagon levels in man: effects of starvation, hypoglycemia, and glucose administration. proc nat acad sci 55: 316, 1966. 13. leclercq-meyer, v., mialhe, p. & m a l a i s e , w. j.: assay of glucagon using dextran-charcoal. dia betologia 6: 121, 1970. 14. luyckx, a. s. & lefebvre, p. j . : arguments for a regulation of pancreatic glucagon secretion by circulating plasma free fatty acids. proc soc exp biol med usa 133: 524, 1970. 15. nonaka, k. & fob, p. p.: a simplified glucagon im munoassay and its use in a study of incubated pancreatic islets. proc socc exp biol med 130: 330, 1969. 16. rodbard, d., rayford, p. l., cooper, j. a. & ross, g. t.: statistical quality control of radioimmuno assays. j clin endocr28: 1412, 1968. 17. samols, e., tyler, j., megyesi, c. & marks, v.: immunochemical glucagon in human pancreas, gut and plasma. lancet 2: 727, 1966. 18. shima, k. & fob, p. p.: a double antibody assay for glucagon. clin chim acta22: 511, 1968. 19. unger, r. h., eisentraut, a. m., mccall, m. s . , keller, s . , lanz, h . c. & madison, l. l.: glucagon antibodies and their use for immunoassay for glucagon. proc exp biol med 102: 621, 1959. 20. unger, r. h . , eisentraut, a. m., mccall, m. s . & madison, l . l.: glucagon antibodies and an im munoassay for glucagon. j clin invest 40: 1280, 1961. 21. vance, j.e., buchanan, k. d., challoner, d. r. & williams, r. h.: effects of glucose concentration on insulin and glucagon release from isolated islets of langerhans of the rat. diabetes 17: 187, 1968. 22. wide, l. & porath, j.: radioimmunoassay ofproteins with the use of sephadex-coupled antibodies. bio chim biophys acta 130: 257, 1966. 23. wide, l., axen, r. & porath, j.: radioimmuno sorbent assay for proteins. chemical couplings of antibodies to insoluble dextran. immunochemistry 4:381, 1967. 24. wide, l.: radioimmunoassays employing immuno sorbents. in immunoassays of gonadotrophins (ed. e. diczfalusy), acta endocr 6 3 , suppl. 142: 207, 1969. 25. solid phase antigen-antibody systems. in radio immunoassay methods (ed. k. e. kirkham and w . m. hunter), p. 405. churchill livingstone, edin burgh &london, 1971. corresponds to that from isolated rat islets assayed using a double-antibody assay (21). this assay has primarily been developed for studies on glucagon release in vitro. the specific problems with plasma measurements of pancreatic glucagon (reviewed by heding 1971) and the addi tional difficulties caused by the presence of gut glucagon-like immunoreactivity (17) have not yet been studied in this system. acknowledgements financial support from the swedish medical research council, the medical faculty of uppsala, the swedish diabetes association and the ahlen foundation is grate fully acknowledged. 1 . 2. 3 . 4. 5 . 6. 7. 8 . 9. 10. 1 1 . references , assan, r., rosselin, g., drouet, j., delais, j., tchobroutsky, g. & derot, m.: dosage radio immunologique du glucagon plasmatique chez i’homme. ann endocr27: 690, 1966. buchanan, k . d. & mccarroll, a. m.: comparison of methods of separation of free from bound hormone in the radioimmunoassay of insulin and glucagon. in radioimmunoassay methods (ed. k. e . kirkham and w. m. hunter), p. 266. churchill livingstone, edinburgh and london, 1971. edwards, j. c., howell, s. l . & taylor, k. w.: fatty acids as regulators of glucagon secretion. nature 224: 808, 1969. radioimmunoassay of glucagon released from iso lated guinea-pig islets of langerhans incubated in vitro. biochim biophys acta 215: 297, 1970. eisentraut, a. m., whissen, n. & unger, r. h.: incubation damage in the radioimmunoassay for hu man plasma glucagon and its prevention with “trasylol”. amer j med sci225: 137, 1968. goldfine, i. d. & ryan, w. g.: rapid production of glucagon antibodies. horm metab res 2: 47, 1970. goodfriend, t . l., levine, l . & fasman, g. d.: antibodies to bradykinin and angiotensin: a use of carbodiimides in immunology. science 144: 1344, 1964. hazzard, w. r., crockford, p. m., buchanan, k . d., vance, j. e., chen, r. & williams, r. h.: a double antibody immunoassay for glucagon. diabetes 17: 179, 1966. heding, l . g.: radioimmunological determination of pancreatic and gut glucagon. diabetologia 7: 10, 1971. hunter, w. m. & greenwood, f. c.: preparation of i ~ d i n e ’ ~ ~ l a b e l e d growth hormone of high specific activity. nature, london 194: 495, 1962. h u m , b. a. l . & landon, j . : antisera for radioimmunoassay. in radioimmunoassay methods (ed. k . e . kirkham and w. m. hunter), p. 121. churchill livingstone, edinburgh and london, 1971. received september 9, 1975 address for reprints: gudmar lundqvist, m.d. department of clinical chemistry university hospital sweden s-750 14 uppsala upsalu j m e d s c i 81 upsala j med sci 81: 97-102, 1976 gastric evacuation and propulsive intestinal motility in acute afferent loop syndrome in the rat sven dahlgren from the department of surgery, university hospital, u p p s a l a , sweden abstract the acute afferent loop syndrome, i.e. occlusion of the afferent loop after partial gastrectomy by the billroth i1 method, was produced in the rat. in a primary session a gastrojejunostomy with division of the pylorus was performed. 2-3 months later the afferent loop was ligated.. the gastric evacuation and the propulsive motility of the intestine were studied quantitatively, using an inert radio isotope. both the gastric evacuation and the propulsive in testinal motility were considerably delayed in als, both in relation to the laparotomized controls and in relation to previous findings in mechanical intestinal obstruction and paralytic ileus due to retroperitoneal irritation or bacterial peritonitis. introduction the gastric evacuation and propulsive intestinal motility in different pathological conditions of the abdomen and after different intra-abdominal oper ations have been described by several authors (7-1 1, 13, 14). both gastric evacuation and intestinal propulsion thus appear to be easily affected by different forms of trauma to the abdomen and abdominal organs. the stomach evacuates, however, even in the pres ence of a low small bowel obstruction ( i i ) . similar ly, according to t h e same authors, a slow propulsion of the intestinal contents takes place above the obstruction. it was considered of interest to find out how the gastrointestinal propulsive motility is af fected by an even more violent acute abdominal catastrophe than obstructive ileus. for this purpose the acute afferent loop syndrome (als) was used. this condition comprises a total obstruction of the pressure in the occluded loop, and in dog experi ments the pressure increased in some cases up to 100 mmhg. within 24 hours the occluded loop shows signs of necrosis, there is evidence of hepatocellular degeneration in the liver, and a fully developed pancreatitis is observed (3). death may follow within a few days, both in man and in ex perimental animals ( 2 ) . the syndrome thus ful fils the criteria of an acute severe abdominal catastrophe. the present study was considered of importance also for understanding of the acute af ferent loop syndrome. material 112 male rats of the same strain (sprague-dawley) were used. the animals were part of two series of a study of the pathophysiology in the acute afferent loop syndrome (1). the distribution of the animals into groups, and their body weights, are given in table i. methods operative technique. in a primary session a gastroenteros tomy with division of the pylorus was performed. two months later the main experiment was carried out, whereby the afferent loop was ligated with a 3-0 silk liga ture close to the gastroenterostomy (als series) (fig. 1). the abdomen was closed with single silk sutures in two layers. further details of the operation have been given in (1). the control rats (laparotomized c o n t r o l s l c ) also underwent gastroenterostomy 2 months prior to the main experiment; this comprised the same dissection in the abdomen, the afferent loop being brought forward and a silk ligature being placed around it close to the gastro enterostomy, but without tying of the ligature. afferent loop a partial gastrectomy by the the gastric evacuarion and propu~sive intestinal motility 'i method. in als, which may appear in were analysed by a method previously described in detail the immediately postoperative stage after a ( 5 ) . a standardized test meal containing a radioactive gastrectomy or much later ( 2 ) , there is a rise in substance (nals1cr04) was deposited in the stomach im upsala j m e d sci 81 7 -162852 98 s . dahlgren fig. 1 . acute afferent loop syndrome in the rat. mediately after the afferent loop had been ligated or, in the control experiments, after the dissection of the afferent loop. after 0 . 5 , 4 , 8 and 12 hours (als series) and 0.5 and 4 hours (control series) the animals were anaesthetized with ether on an open mask and killed by cardiac puncture and exsanguination. the abdomen was opened by a large incision and ligatures were placxd around the cardia and around the efferent loop at the gastroenterostomy. prior to this the entire afferent loop had been excised ( 1 ) . the whole gastrointestinal tract from the stomach to the anus was then taken out by careful dissection. the organs were then placed on a plexiglas plate which was moved under a scintillation detector synchronously with an automatic linear recorder, connected to the re cording unit of the scintillation detector. a quantitative study of the distribution of the radioactive test dose was then made, a planimetric analysis being performed on the area corresponding to the stomach and on each of 10 equal fractions into which the area of the small intestine had been divided. the caecum and colon were anlysed plani metrically as one combined area. each planimetrically determined area was expressed in percent of the total area of the curve, i.e. in per cent of the total amount of radioactivity in the gastrointestinal tract, corresponding to the radioactive dose. further information was obtained by analysing the re sults by the so-called quotient method described by grev sten, johansson & nylander ( 6 ) : x1 a=? v + z x, j = i where &=the propulsive quotient for intestinal fraction i xi=the radioactive content of fraction i v=the radioactive content of the stomach 2x,=the radioactive content of all fractions proximal to j = 1 fraction i ii r e s u l t s t h e increase in weight in t h e different groups be tween t h e primary operation and t h e main experi ment is given in table i . t h e s a m e table gives the haematocrit value a t t h e primary operation and at the main experiment and the number of animals in each group. only a few animals in t h e group with 12 hours’ occlusion of t h e afferent loop died. t h e ani mals were in surprisingly good condition a t the time of the main experiment, with t h e exception of the 12-hour group, which appeared extremely lethargic a n d ruffled and showed a very pale oral mucosa and a p o o r tendency t o bleeding from t h e tail, as signs of peripheral vascular contraction. gastric evacuation t h e gastric evacuation is given as t h e proportion of t h e radioactive d o s e remaining in t h e stomach a t a given time point, expressed in p e r cent of t h e total d o s e administered. t h e mean values and the errors of the means for t h e percentage of t h e test d o s e recorded in t h e stomach and analysed plani metrically are given f o r the different groups in table 11. figure 2 shows the corresponding values plotted in a coordinate system, where the radioac tive gastric content is given o n t h e ordinate a n d t h e duration of occlusion of the loop o n the abscissa. it is seen that t h e gastric evacuation is exponential, a n d is markedly delayed in t h e afferent loop syndrome compared with t h e laparotomized con trols. since t h e comparison of importance was that be tween laparotomized controls and a l s animals, t h e motility in intact controls with a gastroenterostomy alone w a s not studied in these experiments. t h e gastric evacuation in such animals h a s been shown b y nylander & wikstrom (lo), a n d their results a r e also included in fig. 2 (ic). t h e difference be tween t h e gastric evacuation in t h e control and a l s groups i s significant, both at experimental times of 0.5 and 4 hours (p<0.0005). jjpsala j m e d sci 81 gastro-intestinal motility in acute afferent loop syndrome 99 table i . data concerning the two series no. of initial final group animals initial wt. final wt. haematocrit haematocri t control series 0.5 hr 16 233210.7 385k10.3 48f0.7 33k1.1 4 hrs 19 244f 7.9 400f 8.0 49f0.4 32+ 1.2 als series 0.5 hr 17 230f 4.8 394f 12.6 49f0.5 3651.6 4 hrs 24 244f 6.2 231f 7.5 5 0 f 0 . 7 35f1.5 8 hrs 18 238f 5.3 318k10.5 4 7 f 0 . 7 36f1.2 12 hrs 18 238f 5.7 406+ 7.8 4 9 f 0 . 4 31f1.0 table 11. planimetric analysis of the percentage distribution of the radioactive dose in the stomach, in fractions of the small intestine and in the colon laparotomy controls afferent loop syndrome 0.5 hr 4 hrs 0.5 hr 4 hrs 8 hrs 12 hrs stomach fraction 1 2 3 4 5 6 7 8 9 10 caecumi -colon 67.8 k4.20 19.5f2.10 8 . 4 k 1.66 3.4f1.12 1.0k0.70 0.1 k0.14 0.4f0.38 0.1 f0.09 30.8f7.75 5 . 9 f 1 . 3 8 5.2 f0.98 1.820.63 2.6 f 0.94 2 . 9 f 1.05 6.2f 1.67 12.5 f 3.70 1 l.of3.60 6.823.30 8 .of3.57 8.8k4.89 89.9f2.36 65.5 f 6 . 0 5 8.8f1.82 14.1k1.97 1.4f0.69 5.3f 1.10 0.2f0.20 3.3f0.97 3.1 f 1.08 3.0k1.20 3.4f1.50 i .4f0.74 1.3f0.24 1.1 k0.22 0.8f0.19 63.426.69 42.7k7.32 18.2 k2.49 15.3 f 2 . 4 3 10.823.03 9.122.36 4 . 3 f 1 . 4 8 6. i f 1.34 2 . 9 f 1.29 6 . 4 k 1.75 1.0f0.71 6.4k2.12 0.2f0.24 4.1f1.30 2.8+ 1.03 2.7+ i .95 1.7 + 1.09 0.7f0.48 2.622.15 propulsive intestinal motility the percentage distribution of the test dose in the 10 equal, consecutive fractions of the small in testine and the colonic fraction, as obtained by planimetry, is given in table 11. the same values are illustrated graphically in fig. 3 for control and als animals a t experimental times of 0.5 and 4 hours, and i n fig. 4 for 4-hour control animals and for als animals a t 8 and 12 hours. fractions con taining less than 5 % of the total test dose were not included in the subsequent analysis. it was found that the propulsion in the distal direction after 0.5 hours had reached the second fraction in the control cases and the first fraction in the als group. after 4 hours the colonic fraction was reached i n the controls and the second fraction in the als group. after 8 and 12 hours the second and fifth fractions, respectively, had been reached in the als group. thus the propulsion was considerably delayed in the acute afferent loop syndrome. the relationship between gastric evacuation and propulsive intesti nal motility is evident from the propulsive quotients given numerically in table 111 and diagrammatically in fig. 5. in the acute afferent loop syndrome there is thus a strong inhibition of the intestinal propul sion. % x-x l c 0-oals a-a ic { n y l a n d e r b w i k s t r o m 1967) 2o 1 2 4 6 8 10 12 hrs fig. 2 . diagram illustrating gastric emptying. upsala j med sci 81 100 s . dahlgren table 111. the propulsive quotient fraction groups 1 2 3 4 5 6 7 8 9 10 colon lc 0.5 h r 0.29 0.10 lc 4 h r s 0.19 0.14 0.04 0.06 0.06 0.13 0.23 0.16 0.09 0.09 0.09 als 0.5 hr 0.10 als 4 hrs 0.22 0.08 als 8 hrs 0.29 0.13 als 12 hrs 0.36 0.16 0.09 0.09 0.08 discussion the inhibitory effect of laparotomy on the gastrointestinal motility has been pointed out previ ously by nylander & wikstrom ( l o ) , nylander & svensson ( 1 i ) and kylberg (8), among others. in the present control rats the laparotomy was combined with a dissection of the afferent loop corresponding to that required in order to place a ligature around the afferent loop and thereby produce an afferent loop syndrome. in this way the inhibitory effect was even more pronounced than in the previously de scribed laparotomies. despite the relatively strong inhibition of the intestinal motility after the laparo tomy, there was a significantly more delayed gastric evacuation in the als groups and a delay of the propulsive motility. in als the entire small in $ 100 1 n=17 0 . 5 hr als % 80 i testine is intact, since the pathological process lies in the blind portion which is occluded from the efferent intestinal segment leading from the stomach. even though this efferent part is com pletely intact, as well as the communication be tween the stomach and the efferent loop, als leads to pronounced gastric and intestinal paralysis, which was found in these studies to be more marked than has been described in experimental intestinal obstruction in the rat ( 1 1). there are certainly sev eral reasons for this. in als the pathological pro cess is localized to the upper part of the abdomen in the vicinity of the stomach, while that in a low small bowel obstruction lies i n the lower part of the abdomen. als has, in addition, a direct effect on three organs, the duodenum, liver and pancreas. of n=22 4 hrs a i s 1 2 3 4 5 6 i 8 9 10 z 1 0 0 1 as16 0.5 hr c o n t r o l s % 100 80 60 40 20 0 1 2 3 4 5 6 7 8 9 1 0 1 n=19 4 hrs controls h,, 1 2 3 4 5 6 7 8 9 1 0 colon f i g . 3. histograms representing the quantitative distribu tion of the radioactive substance in the gastrointestinal upsala j med sci 81 sample. control groups 0.5 and 4 hours and als groups 0.5 and 4 hours. gastro-intestinal motility in acute afferent loop syndrome 101 100 80 60 . ic loo1 80 a. n=18 8 hrs als 0 h ::l 20 1 2 3 4 5 6 7 8 9 10 n=19 4 hrs controls 1 2 3 4 5 6 7 8 9 1 0 e o l o n % 100 60 n=18 12 h r e als 4: 20 l 1 2 3 4 5 6 7 8 9 10 colon -19 4 hrs c o n t r o l s ::k 0 , , rc-h , , , 1 2 3 4 5 6 7 8 9loealon f i g . 4 . histograms representing the quantitative distribu tion of the radioactive substance in the gastrointestinal sample. control group 4 hours and als groups 8 and 12 hours. probable importance for the development is the di lated, tense duodenal loop, i n which rnucosal necrosis gradually occurs (3). this necrosis gives a possibility of leakage into the peritoneum of both bacteria from the bacteriarich duodenal fluid (1) and toxins. supporting this assumption are the signs of peritonitis with increased fluid in the peritoneum which were observed i n all cases. in als, pancreatitis occurs regularly (3, 4, 13). this also contributes both to peritonitis and t o paralytic ileus, which is a regular component in all cases of pancreatitis. added to this is the fact that als also gives rise to biliary stasis and hepato cellular degeneration (3), which can contribute to deterioration of the liver function and thereby re duction of the capacity of general detoxification. paralytic ileus has been produced previously in a-a lc l h r s 0 4 lc 0 5 " ._. a l s 1 2 " 0-0 als 8 " 04 a l s l '' a a l s 0 5 " i . . . . . . . . . . , 1 2 3 l 5 6 7 8 9 10 colon froction fig. 5. diagram illustrating propulsive quotient values. the rat both by means of bacterial peritonitis (8) and through retroperitoneal irritation (9). the paralysis of the gastrointestinal tract occurring in the acute afferent loop syndromes seems to be more pro nounced, however. contributing to this is thus the combination of pancreatitis, intestinal necrosis, hepatocellular degeneration and peritonitis, and in the final phase the irreversible shock which de velops. references 1. 2 . 3. 4 . 5. 6. bengtsson, s . , dahlgren, s . & hellsing, k.: the path ophysiology of the acute afferent loop syndrome. acta chir scand 1976. (in press.) dahlgren, s . : the afferent loop syndrome. acta chir scand, suppl. 327, 1964. dahlgren, s . & thorell, j.: histological changes in the duodenum, pancreas and liver in the acute affer ent loop syndrome. in the afferent loop syndrome (ed. s . dahlgren). acta chir scand, suppl. 327: 65, 1964. dahlgren, s . & stenram, u . : acute pancreatitis and hepatic necrosis in the acute afferent loop syndrome-a histopathological study in the rat. up sala j med sci 1976. (in press.) derblom, h . , johansson, h . & nylander, g . : a sim ple method of recording quantitatively certain gastro intestinal motility functions in the rat. acta chir scand 132: 154, 1966. grevsten, s . , johansson, h. & nylander, g.: analysis of a quantitative method of evaluation a s applied to the propulsive gastrointestinal motility in the rat. acta chir scand 133: 563, 1967. upsala j med sci 81 102 s. dahlgren 7. johansson, h. & nylander, g.: ileo-colic transit in rats subjected to ileocaecal resection. acta chir scand 135: 455, 1969. 8. kylberg, f.: gastric evacuation and propulsive in testinal motility in experimental perforation peri tonitis. scand j gastroent5: 593, 1970. 9. lindquist, b.: propulsive gastrointestinal motility re lated to retroperitoneal irritation. acta chir scand, suppl. 384, 1968. 10. nylander, g. & wikstrom, s.: gastric emptying and propulsive intestinal motility following partial gastric resection, gastroenteroanastomosis, and abdominal “trunk” vagotomy in the rat. acta chir scand 133: 41, 1967. 11. nylander, g. & svensson, a.: gastric emptying and propulsive intestinal motility in experimental intestinal obstruction. acta chir scand 134: 135, 1968. 12. parkkulainen, k. v.: simple low small bowel obstruc tion. acta chir scand, suppl. 290, 1962. 13. port, m . & gelb, a.: acute afferent loop syndrome simulating acute pancreatitis. amer j gastroent 53:36, 1970. 14. wikstrom, s.: propulsive gastrointestinal motility in regional and graded ischemia of the small bowel. acta chir scand, suppl. 386, 1%8. received november i , 1975 address for reprints: sven dahlgren karnsjukhuset skovde fack s-54101 skovde sweden upsulu j med sci 81 upsala j med sci 92: 253-257, 1987 vitamin a and pcarotene concentrations at different depths of the epidermis: a preliminary study in the cow snout anders vahlquist, eva stenstrom and hans torma department of dermatology, uppsala uniuersity, uppsala, sweden abstract vitamin a (retinol) is an anti-keratinizing agent essential for normal epithelial differentiation. in order to examine the epidermal distribution of vitamin a and provitamin a (i3-carotene), we took advantage of the extraordinarily thick snout epidermis of the cow which can be cut horizontally into at least 6 layers, representing keratinocytes at different stages of maturation. extracts of saponified samples were analyzed for retinol and @-carotene by reversed phase high-performance liquid chromatography. the highest retinol concentration (0.8 pg/g protein; n=3) was recorded closest to the dermis; progressively decreasing amounts of retinol were found in the upper parts of epidermis. maximum values of @-carotene (1.0 kgg/g; n=7) were found in the lower parts of epidermis; substantially lower levels were seen at the dermal transition zone and in the upper parts of epidermis. the results suggest that the endogenous concentration of vitamin a in snout epidermis is inversely related to the degree of cellular differentiation. introduction epidermal keratinocytes, while undergoing terminal differentiation, continuously move outwards from the basal layer to the skin surface .the mechanisms controlling these events are still not clear. vitamin a is a potent inhibitor of keratinization (4) and, in experimental animals, deficiency of the vitamin causes epithelial hyperkeratosis (13). it may thus be proposed that normal keratinocytes become progressively deficient in vitamin a as they move towards the surface, thereby facilitating their transformation into corneocytes. this 'programmed vitamin a-deficiency' could operate through a restricted supply of vitamin a to the upper parts of epidermis or through an inherent decline in the retinoid-responsiveness of keratinocytes as they move away from the basal layer. whereas the former mechanism does not seem to have been investigated, the latter is supported by the work of green and watt (5), who showed that cultured keratinocytes from various sources (conjunctiva, esophagus, vagina and epidermis) differ in their responsiveness to retinoids in vitro, with epidermal keratinocytes being most resistant to the anti-keratinizing action of the compounds. in vivo, vitamin a is transported to the target cells by serum retinol-binding protein (rbp) which delivers the vitamin by binding to cell-surface receptors (9,lo). subsequently, retinol enters 253 a series of metabolic steps leading to the formation of e.g. retinyl esters, retinaldehyde and retinoic acid. whether i3-carotene (provitamin a) contributes to the supply of retinol to the epidermis is uncertain. however, an additional function of the carotenoid pigments in epidermis may be related to their capacity to quench free radicals. in previous analyses of epidermal vitamin a and carotenoids, we used specimens of whole epidermis, i.e. mixtures of cells at different stages of keratinization (12). in the present study, we have focused on the distribution of retinol and &carotene in cow snout epidermis which can be easily cut into 5 or 6 horizontal layers of sufficient s size to permit chemical characterization (7). material and methods cow snouts were removed immediately after slaughter and were kept cool in the dark until cut into cubes (20x20~5 mm) within 30 min. the samples were then firmly squeezed between two glass plates and frozen by flushing with liquid c 0 2 . slices of 0.1 mm thickness were produced with the sample mounted horizontally in a hand driven cryostat (leitz wetzler, frg). a punch biopsy from the center of each slice was preserved for routine histology. the rest of the slices were kept frozen (-70°c) until hydrolyzed in ethanolic koh (80°c; 20 min) and extracted with n-hexane as previously described for human skin (1 1). the evaporated extracts were redissolved in methanol and analyzed for retinol by high-performance liquid chromatography (hplc) as described elsewhere (11). a separate series of samples were analyzed for carotenoids by a modification of the hplc procedure of driskell et a1 (3). thus, the sample extract (prepared as before) was dissolved in 100 ~1 of acetonitrile and applied to a zorbax ods column (4.6 x 150 mm) coupled to a dual pump system (altex 110, altex scientific inc., berkeley, ca.) initially delivering acetonitri1e:dichloromethane: methanol (83:2:15) at a flow rate of 1.5 ml/min for 6 min. the composition of the mobile phase was subsequently shifted (concave gradient) to 70:18:12, reaching a plateau at 12 min. the eluate was monitored by uv-absorption at 436 nm ( waters 440, waters assoc. inc, milford, ma.). standard curves (peak height vs. mass) were derived by injecting variable amounts of a solution containing eight different authentic caroteinoids. in a typical experiment, &carotene eluted at 27 min. the &carotene and retinol concentrations were related to the protein content of the sample as determined by the biuret assay (12). results histological preparations of the tissue sections produced by cutting a cow snout specimen parallel to the surface are shown in fig. 1. as indicated, the first section comprises the stratum corneum and the upper portion of stratum granulosum. the second and third sections comprise the upper and mid portions of the stratum spinosum; sweat ducts penetrating the epidermis can be seen. the three last sections contain the basal layers and variable amounts of papillary dermis. 254 figure 1. sections of epidermis stained with hematoxylin and eosin. six epidermal slices of 0.1 m m thickness taken through the epidermis from the stratum corneum (1) to the papillary dermis (6). fig.:! shows the mean retinol and s-carotene concentrations in the sections produced from 10 samples of epidermis. the highest retinol concentration is seen at the dermal transition zone (section no. 6) and the lowest concentration is in the upper part of stratum spinosum (section no.2). occasionally, 3-dehydroretinol (vitamin a2) was detected in the samples (data not shown), but not to the same extent as in human epidermis (12). the concentration profile of s-carotene is different from that of retinol; high values are seen in mid to lower parts of epidermis and low values are seen both in the stratum corneum and in the dermal transition zone. small amounts (<0.1 pg/g protein) of lutein were also present in the samples. this carotenoid appeared to be evenly distributed in the epidermis (data not shown). figure 2. changes in retinol and &carotene contents of epidermis with depth in the epidermis. mean ( s e ) values for three (retinol) and seven (@-carotene) samples, respectively. factors to convert the values to nmol/g: x3.497 ( v i t a m i n a ) a n d x1.863 (&carotene). . . , , . . 1 2 3 4 5 6 stratum basal corneum layer no. 255 discussion the low retinol concentration found in the upper layers of epidermis (saponified samples) ,is consistent with the proposed 'programmed vitamin a-deficiency' in the keratinizing zone. however, it should be emphasized that the number of samples investigated was small and that it is not known whether the observed retinol concentration is sufficiently low to precipitate terminal differentiation of keratinocytes. also, the vitamin a status of a tissue depends not oniy on its total content of retinol; a number of other factors, including variable formation of inactive retinyl esters or highly active retinoic acid (not detected by our assay) may markedly influence the expression of retinoid activity. the origin of the retinol gradient in snout epidermis should be sought among one or several of the following explanations: (a) restricted passage of serum retinol-rbp to the outer parts of epidermis (l), (b) decreased number of rbp receptors on differentiated keratinocytes (8), (c) enhanced metabolic degradation of retinol in upper epidermis, and (d) destruction of retinol by solar radiation penetrating the superficial layers of the integument ( 2 ) . the possibility of a decreased uptake of retinol in the upper epidermis is supported by a recent in vitro study on cow snout. autoradiography showed that the uptake of tritiated retinol from rbp is highest in the basal layers of epidermis (torma, gillberg & vahlquist, to be published). although one should excercise care when extrapolating results from cow snout to other types of epithelia, there are indications that a similar concentration gradient of vitamin a exists in human epidermis. for example, the stratum corneum of the human foot sole contains much less vitamin a than the underlying epidermis (12). unfortunately, the thickness of human epidermis (about 0.1 mm) precludes sectioning of the samples by the technique used for snout epidermis. clearly, further studies on the occurrence, metabolism and function of vitamin a in different types of epithelia are required before the observed vitamin a gradient in epidermis can be implicated in the process of keratinization. it is noteworthy that the distributions of retinol and 8-carotene in epidermis differ. using less sophisticated methods, we (12) and others ( 6 ) reported previously that human epidermis has a higher affinity for carotenoids than dermis, but it is not known to which epidermal component(s) the compounds bind. as yet, there is no proof that epidermal &carotene is converted to retinol but other processes, such as quenching of free radicals, may be equally important functions of the carotenoids in the epidermis. tentatively, the accumulation of carotenoids in the lower parts of epidermis could help to protect the proliferating basal cells from solar damage. acknowledgements the expert technical assistance of ms i. pihl-lundin is gratefully acknowledged. financial support was recieved from the swedish medical research council (proj. no. 03x-07133), the welander foundation and the finsen foundation. 256 references 1. benoldi, d., manfredi, g . , pezzarossa, e. & allegra, f.: retinol-binding protein in normal human skin and in cutaneous disorders. br j dermatol 105:659-665,1981. 2. berne, b., nilsson, m. & vahlquist, a.: uv-irradiation a n d cutaneous vitamin a:an experimental study in rabbit and human skin. j invest dermatol 83401-404, 1984. 3. driskell, w.j., bashor, m.m. & neese, j.w.: beta-carotene determined in serum by liquid chromatography with an internal standard. clin chem 29:1042-1044, 1983. 4. fuchs, e. & green, h.: regulation of terminal differentiation of cultured human keratinocytes by vitamin a. cell 25:617-625, 1981. 5. green, h . & watt, f.m.: regulation by vitamin a of envelope cross-linking in cultured keratinocytes derived from different human epithelia. mol cell biol 21115-1117, 1982. 6 lee,r., mathews-roth, m.m., pathak, m.a. & parrish, j.a.: the detection of carotenoid pigments in human skin. j invest dermatol 64:175-177, 1977 7. long, v.j.w.: variation in lipid composition a t different depths in the cow snout epidermis. j invest dermatol55269-273,1970. 8. rask, l., anundi, h., bohme, j. e t al.: structural and functional studies of vitamin a-binding proteins. ann ny acad sci 359:79-90, 1981. 9. rask, l. & peterson, p.a.: in vitro uptake of vitamin a from retinol-binding plasma protein to mucosal epithelial cells from the monkey's small intestine. j biol chem 251:6360-6366, 1976. 10. torma, h. & vahlquist, a.: vitamin a uptake by human skin in vitro. arch dermatol res 11. vahlquist, a.: vitamin a in human skin. i. detection and identification of retinoids in normal epidermis. j invest dermatol 79239-93, 1982. 12. vahlquist, a., lee, j.b., michaelsson, g . & rollman, 0.: vitamin a in h u m a n skin. 11. concentrations of carotene, retinol and 3-dehydroretinol in various components of normal skin. j invest dermatol 7994-97, 1982. 13. wolbach, s.b. & howe p.r.: tissue changes following deprivation of fat-soluble a vitamin. j exp med 43:753-777,1925. 276:390-395,1984. adress reprint requests to: dr anders vahlquist, dept of dermatology, university of uppsala, akademiska hospital, s751 85 uppsala, sweden. 257 upsala j med sci 87: 243-250, 1982 effect of chemical modification of a histidine and a lysine residue of pea seed nucleoside diphosphate kinase bror edlund*, carl henrik heldin and lorentz engstrom from the institute of medical and physiological chemistry, biomedical centre, university of uppsala, sweden *present address: department of clinical chemistry, university hospital, university of uppsala, sweden abstract chemical m o d i f i c a t i o n o f a h i s t i d i n e and l y s i n e r e s i d u e i n a c t i v a t e s pea seed n u c l e o s i d e d i p h o s p h a t e k i n a s e (ndp k i n a s e ) . r e a c t i v e l y s i n e r e s i d u e , a t t h e a c t i v e s i t e o f pea seed ndp k i n a s e , i n a d d i t i o n t o t h e h i s t i d i n e r e s i d u e p h o s p h o r y l a t e d b y t h e s u b s t r a t e atp as a con sequence o f t h e enzyme r e a c t i o n . the presence o f a r e a c t i v e l y s i n e a t t h e a c t i v e s i t e o f t h e enzyme c o u l d e x p l a i n why a s m a l l amount o f n-e-phospho l y s i n e , as w e l l a s 1 p h o s p h o h i s t i d i n e and 3 p h o s p h o h i s t i d i n e y i s formed on a l k a l i n e h y d r o l y s i s o f t h e enzyme. thus t h e r e seems t o be a introduction e a r l i e r s t u d i e s have i n d i c a t e d t h a t ndp k i n a s e ( n u c l e o s i d e d i p h o s p h a t e : atp t r a n s p h o s p o r y l a s e ec 2.7.4.6) f r o m pea seed i s a t e t r a m e r i c p r o t e i n w i t h a m o l e c u l a r w e i g h t o f 70 000, w i t h f o u r i d e n t i c a l s u b u n i t s , each c o n t a i n i n g an a c t i v e s i t e (1,2). i n t e r m e d i a t e l y p h o s p h o r y l a t e d b y i t s s u b s t r a t e atp on a h i s t i d i n e r e s i d u e a t t h e a c t i v e s i t e , presumably as 1 p h o s p h o h i s t i d i n e , t h e phosphoamino a c i d d o m i n a t i n g i n an a l k a l i n e h y d r o l y s a t e o f p h o s p h o r y l a t e d ndp k i n a s e f r o m baker’s y e a s t (3,4). the amino a c i d sequences o f t h e d o m i n a t i n g p h o s p h o p e p t i d e s f r o m t h e a c t i v e s i t e o f t h e pea seed enzyme i n a c t i v a t e d i n t w o d i f f e r e n t ways, 3. w i t h a l k a l i and a c i d , and degraded w i t h two d i f f e r e n t p r o t e a s e s , t r y p s i n and p e p s i n r e s p e c t i v e l y , a r e o v e r l a p p i n g (5,6) i n d i c a t i n g t h a t t h e p h o s p h o r y l g r o u p i s bound t o t h e same h i s t i d i n e r e s i d u e i n t h e enzyme i r r e s p e c t i v e of t h e i n a c t i v a t i o n method used. two t r y p t i c p h o s p h o p e p t i d e s w i t h t h e same amino a c i d sequence b u t d i f f e r evidence has a l s o been o b t a i n e d , t h a t t h e enzyme i s i n g i n s t a b i l i t y t o a c i d o f t h e p h o s p h o r y l bond a r e o b t a i n e d , p r o b a b l y b y a p h o s p h o r y l g r o u p m i g r a t i o n f r o m 1 p h o s p h o h i s t i d i n e t o 3 p h o s p h o h i s t i d i n e ( 7 ) . a l k a l i n e h y d r o l y s i s o f p h o s p h o r y l a t e d pea seed ndp k i n a s e g i v e s m a i n l y p h o s p h o p e p t i d e s , and i n a d d i t i o n s m a l l amounts o f p h o s p h o h i s t i d i n e and n-e 243 phospholysine (8,9). the present investigation was therefore focused on the possibility that there might exist, in addition to the reactive histidine residue, a lysine residue, essential for enzyme activity, in the active site of pea seed ndp kinase. the active site and more chemically reactive than the rest of the amino acid residues of the enzyme. modification of one lysine and one histidine residue were to inactivate the enzyme and if substrate and product protected the enzyme from inactivation. the enzyme was therefore treated with 1 -fluoro-2,4-dinitrobenzene (fdnb) and 2,4,6-trinitrobenzene sulfonic acid (tnbs) which are known to react preferenti ally with lysine residues (10,11,12) and diethylpyrocarbonate (dpc) which reacts with histidine residues (13,14). was studied in relation to the degree of chemical modification obtained, also in the presence of the substrate atp and product adp. if so, these amino acid residues should be exposed in it was considered that support for this view would be obtained if chemical the activity of the treated enzyme materials and methods the enzyme was prepared as previously described (8). an absorbance value of e:io = 14.1 was used for the purified enzyme (8). the enzyme activity was determined by a coupled assay according to mourad and parks, using dgdp as nucleoside diphosphate (15). fdnb was obtained from pierce, tnbs from sigma, dpc from eastman and sodium dodecylsulfate (sds (hydroxymethyl )-methyl -2-amino-ethanol sulfonic british drughouse ltd. all chemicals were of h spectroscopic measurements were made with a pmq using a recorder for registration. reaction of ndp kinase with fdnb. to 20 m from pierce. n-tris acid (tes) was purchased from ghest quality available. i1 zeiss spectrophotometer, of ndp kinase (15 pmoles/l) in tes buffer (0.2 moles/l, ph 8.5) 1.6 ml of a solution of fdnb in ethanol (0.1 moles/l) were added. the solution was kept at room temperature (23oc). aliquots (1 ml) were taken at fixed intervals and chromatographed at 23oc on a sephadex 6-50 column (1.4~16 cm) equilibrated and eluted with sodium phosphate buffer (0.05 moles/l, ph 6.5). fractions eluted with the void volume were measured and the molar amount of incorporated dinitrophenyl groups was calculated. a molar absorbance of 16 000 cm-l at 365 nm was used for the dinitrophenyl groups (10) and the number of modified lysine residues per subunit was calculated. activities were determined and compared with that of a sample chromatographed without previous addition of fdnb. reacting with dpc were determined by difference spectroscopy at 240 nm, using a molar absorbance of 3200 cm-' (13). the absorbance at 280 nm of the the enzyme treatment of ndp kinase with dpc. the number of histidyl residues the carbetoxylation of the enzyme was 244 carried out in sodium phosphate buffer (0.05 moles/l, ph 6.5) or in triethanol amine-acetic acid buffer (0.05 moles/l, ph 7.4). were prepared ex tempore. the final concentration of dpc was 1 mmole/l and that of the enzyme 15 umoles/l. calculated in relation to that of the enzyme treated in the same way but without addition of dpc. in sodium hydrogen carbonate (0.25 moles/l), 1 ml of tnbs (1.7 mmoles/l) were added at zero time. a blank solution without ndp kinase was also prepared. the reaction mixtures were kept at 4ooc. aliquots were taken for enzyme assay at fixed intervals. at the same time 100 u1 of sample and blank solutions were diluted with 100 u1 of 10% (w/v) sds in water followed immediately by 50 111 of hc1 (1 mole/l) and 3 ml of hc1 (0.01 moles/l) (12). the difference in absorb ance between sample and blank solutions at 344 nm was determined and the number of trinitrophenyl groups incorporated was calculated from the molar absorbance of 11 000 cm-l at 344 nm given for a trinitrophenyl group in a protein (12). activity remaining after the experimental period. experiments the final ndp kinase concentration was about 0.2 umoles/l. enzyme solutions contained magnesium acetate (1.5 mmoles/l) in order to obtain the nucleotides in the magnesium form. or adp (0.8 mmoles/l) were incubated at 3ooc with dpc (1 mmole/l) in tri ethanolamineacetic acid buffer (40 mmoles/l, ph 7.4) for 10 min, tnbs (2 mmoles/ 1) in sodium hydrogen carbonate (10 mmoles/l) for 30 min or fdnb (20 mmoles/l) in sodium dihydrogen carbonate (40 mmoles/l) for 30 min. activity was compared with that of a control solution in which the modifying agent was omitted. was dissolved in ethanol, the same amount of ethanol was included in the control experiment (5% to 10% (v/v)). these ethanol concentrations. solutions of dpc in ethanol the extent of inactivation of the enzyme was trinitrophenylation of lysine residues. to 1 ml of ndp kinase (30 pmoles/) the extent of inhibition was calculated as per cent o f enzyme inactivation of ndp kinase in the presence of atp and adp. in all the samples containing atp (0.8 moles/l) the residual enzyme in the experiments with dpc and fdnb, where the reagent the enzyme activity was not affected by results and discussion the reaction of pea seed ndp kinase with fdnb indicates that there was one lysine residue per subunit which reacted faster than the other nine (2). all enzyme activity was lost when this residue had reacted, but the enzyme activity seemed to decrease more rapidly than the dinitrophenylation of the lysine residue, indicating that another kind of amino acid residue was also reacting. at 365 nm and would therefore not have been detected by the method used for measuring the degree of dinitrophenylation of the enzyme in the present work imidazole-dinitrophenyl -histidine does not show an absorbance peak 245 (16). it is therefore possible that a histidine residue was also blocked by fdnb, leading to inactivation of the enzyme (fig. 1 ) . i i 1.0 0.5 time i min fig. 1. filled circles represent enzyme activity remaining and open circles represent dnp groups incorporated per mole of subunit. inhibition of pea seed ndp kinase by fdnb at ph 8.5. the carbetoxylation at ph 7.4 of one histidine residue per subunit o f the enzyme out o f three (2) leads to inactivation 100 s c 2 z # 5 0 t l 6 x > i v 6 y > 8 ph 7.4 h ph 6.5 d--o n 1 2 l w moles of histidine residues modified per mole of subunit of enzyme (fig. 2). 1 fig. 2. enzyme activity remaining at ph 7.4 and open squares represent enzyme activity remaining at ph 6.5 after modification of the indicated number of histidine residues per subunit. modification of histidine residues of pea seed ndp kinase by carbet 'oxylation with dpc and its effect on enzyme activity. filled squares represent 246 at ph 6.5 two histidine residues reacted before the enzyme was completely inactivated. the enzyme from beef heart cytosol has also been inactivated by carbetoxylation (17). tion of the enzyme, supporting the view that the enzyme contains a lysine residue which is essential for its activity (fig. 3 ) . incubation, all ten lysine residues in each subunit were modified, showing that all of them became accessible to tnbs. the trinitrophenylation of pea seed ndp kinase leads to rapid inactiva however, on prolonged 1001 1 s i c3 z 5 i w cr t t 1 a l 0 w i t n z w 50 100 time ( min 1 v) w fig. 3 . inhibition and modification of pea seed ndp kinase by tnbs. filled circles represent enzyme activity remaining and open circles trinitrophenyl groups incorporated per subunit after the indicated incubation period. the fact that the substrate atp and the product adp protect, at least partially, the enzyme from inactivation by the reagents used here, indicates that the reactions involve the active site of the enzyme (table 1). nucleo tides have also been shown to protect some ndp kinases from inactivation by p-chloromercuribenzoat (pcmb) (18,19,20,21). it has been discussed that such a sulfhydryl group may be essential for the qurtenary structure of the enzyme and not a part of the active site of the enzyme (18,211. pea seed ndp kinase does not contain any sh-group at all (2). lysine residue in addition to a reactive histidine residue in the active site of pea seed nucleoside diphosphate kinase. thus there seems to be a reactive 247 table 1. fdnb, dpc and tnbs. addition of nucleotides. same specific activity (1 200 units/mg) as when the enzyme was kept in tri ethanolamine-acetic acid buffer (0.01 moles/l , ph 7.4). obtained in duplicate experiments are given below. effect of atp and adp on the inactivation of pea seed ndp kinase by in each series a control experiment was made with no in the control experiments the enzyme showed the the mean values for details see text. additions atp (control) fdnb fdnb + atp adp (control ) fdnb + adp atp (control ) dpc dpc + atp adp (control) dpc + adp atp (control tnbs tnbs + atp adp (control tnbs + adp residual enzyme activity (per cent) 100 47 80 100 84 100 4 39 100 75 100 58 81 100 75 acknowledgements this work was supported by the swedish medical research council (project no. 13x-50). the skilful technical assistance by mrs. jill ekstrom i s gratefully acknowledged. references 1. edlund, b.: tetrameric structure of nucleoside diphosphate kinase from 2. edlund, b.: evidence for identical subunits in pea seed nucleoside 3. edlund, b. & wdlinder, 0.: evidence for an intermediary phosphorylation pea seed. febs letters 13:56-58, 1971. diphosphate kinase. of nucleoside diphosphate kinase from pea seed. 1974. febs letters 38:222-224, 1974. febs letters 38:225-228, 248 4. edlund, b., rask, l., olsson, p., wblinder, o., zetterqvist, u. & engstrom, l.: from baker’s yeast and purification of 1-phosphohistidine as the main phosphorylated product of an a1 kal ine hydrolysate of enzyme incubated with adenosine ( pi-triphosphate. eur j biochem 9:451-455, 1969. 5. edlund, b.: active site phosphopeptides from pea seed nucleoside diphosphate kinase. uppsala j med sci 79:143-147, 1974. 6. edlund, b. & engstrom, l.: a peptic phosphopeptide from the active site of pea seed nucleoside diphosphate kinase. febs lett 47:279-283, 1974. 7. hultquist, d.e.: the preparation and characterization of phosphorylated derivatives of histidine. biochim biophys acta 153:329-340, 1968. 8. edlund, b.: purification o f nucleoside diphosphate kinase from pea seed and phosphorylation o f the enzyme with adenosine ( acta chem scand 25:1370-1376, 1971. liver protein rapidly phosphorylated by adenosine triphosphate. o f 1 32 p-phosphohistidine, 3-32p-phosphohistidine and n-~~~~p-phosphohis tidine from 32p-labelled protein. enzymology (ed. c.h.w. hirs), 11: p. 551. new york, academic press, inc., 1967. preparation of crystalline nucleoside diphosphate kinase 32 32 p)triphosphate. 9. wblinder, o., zetterqvist, u. & engstrom, l.: purification of a bovine isolation j biol chem 243:2793-2798, 1968. 10. hirs, c.h.w.: reaction with reactive aryl halides. in: methods in 11. habeeb, a.f.s.a.: determination of free amino groups in proteins by trinitrobenzenesulfonic acid. anal biochem 14:328-336, 1966. 12. haynes, r., osuga, d.t. & feeney, r.e.: modification of amino groups in inhibitors of proteolytic enzymes. biochemistry 6:541-547, 1967. 13. pradef, l.-a. & kassab, r.: site actif des atp:guanidine phosphotrans ferases. 11. mise an evidence de residue histidine essentiels au moyen du pyrocarbonate d’ethyle. biochim biophys acta 167:317-325, 1968. histidyl residues in arginine oxygenase (decarboxylating). with amino acid oxidases. eur j biochem 19:270-275, 1971. 11. isolation and kinetics. j biol chem 241:271-278, 1966. 14. thome-beau, f., l5-thi-lan, olomucki, a. & van thoai, n.: essential comparison 15. mourad, n. & parks j:r, r.e.: erythrocytic nucleoside diphosphokinase. 16. henkart, p. : 17. colomb, m.g., cheruy, a. & vignais, p.v.: the chemistry and identification o f im-dinitrophenyl histidine. j biol chem 246:2717-2713, 1971. beef heart cytosol. 11. characterization of the phosphorylated inter mediate. biochemistry 11:3378-3389, 1972. nucleoside diphosphokinase from 18. agarwal , r.p. & parks, j:r, r.e.: erythrocytic nucleoside diphosphokinase. v. some properties and behavior of the pi 7,3 isoenzyme. j biol chem 246:2258-2264, 1971. 249 19. palmieri, r., yue, r.h., jacobs, h.k.,maland, l., wu, l. & kuby, s.a.: nucleoside triphosphate-nucleoside diphosphate transphosphorylase (nucleoside diphosphokinase). 111. subunit structure of the crystalline enzyme from brewer’s yeast. j biol chem 248:4486-4499, 1973. 20. colomb, m.g., chernuy, a. & vignais, p.v.: adenosine diphosphate as a regulatory ligand in beef heart cytosol nucleoside. biochemistry 13:2269-2277, 1974. 21. minh duc, d., lascu, i . , porumb, h., gozia, o., schell, h.d. & barzu, 0.: differential sensitivity to p-chloromercuribenzoate and urea o f soluble and sepharose-bound pig heart nucleoside diphosphate kinase. febs lett 127: 281 -284, 1981. address for reprints: docent bror ed1 und department o f c1 inical chemistry university hospital s-750 14 uppsala 14 sweden 250 upsala j med sci 91: 257-262, 1986 image analysis in whole-body autoradiography (wba) roland d'argy department of toxicology, uppsala university, uppsala biomedical center, box 594, s-75124 uppsala, sweden i n t r o d u c t i o n i m a g e p r o c e s s i n g by c o m p u t e r s h a s b e c o m e i n c r e a s i n g l y i m p o r t a n t f o r a wide v a r i e t y of f i e l d s , r a n g i n g f r o m a e r i a l a n d s p a c e p h o t o g r a p h y t o m e d i c a l diagnosis e.g. i m a g e s a s s e m b l e d f r o m x-ray d a t a o r f r o m positron emission t o m o g r a p h y (pet). during t h e l a s t f i v e y e a r s , c o m p u t e r a s s i s t e d i m a g e a n a l y s i s h a s a l s o b e e n u s e d f o r d i f f e r e n t k i n d s of a u t o r a d i o g r a p h i c a p p l i c a t i o n s . most of t h e s e s t u d i e s , h o w e v e r , d e a l with v a r i o u s a s p e c t s of t h e brain. thus, q u a n t i t a t i v e r e c e p t o r a u t o r a d i o g r a p h y h a s b e e n used in in v i t r o s t u d i e s on t h e d i s t r i b u t i o n of t r a n s m i t t o r r e c e p t o r s in t h e c e n t r a l n e r v o u s s y s t e m ( 5 7 , 12, 13). also, q u a n t i t a t i v e a u t o r a d i o g r a p h i c in vivo t e c h n i q u e s , b a s e d on s t u d i e s of b r a i n f u n c t i o n s a n d using c o m p u t e r i z e d d e n s i t o m e t r y , h a v e b e e n d e s c r i b e d (3, 4, 11). wba (9, 10) is a w e l l e s t a b l i s h e d r a d i o t r a c e r t e c h n i q u e f o r s t u d y i n g t h e d i s t r i b u t i o n of l a b e l l e d c o m p o u n d s in t h e e n t i r e a n i m a l . p r a c t i c a l l y a l l t i s s u e s , e v e n v e r y s m a l l o n e s , c a n b e s t u d i e d . in p r e g n a n t a n i m a l s , t h e c o n c e n t r a t i o n of r a d i o a c t i v i t y in t h e f e t a l t i s s u e s m a y b e c o m p a r e d w i t h t h a t of t h e m o t h e r . i n f o r m a t i o n a b o u t m e t a b o l i s m a n d t i s s u e binding m a y b e o b t a i n e d by d i f f e r e n t a p p l i c a t i o n s of wba ( f o r r e v i e w , see r e f . 1). t h e e x t r a c t i o n of a l l i n f o r m a t i o n f r o m such s t u d i e s is v e r y t e d i o u s a n d t i m e consuming. e a r l i e r , d e n s i t o m e t r i c d a t a w e r e o b t a i n e d by a m a n u a l d e n s i t o m e t e r a n d a s e m i q u a n t i t a t i v e s t a i r c a s e of r a d i o a c t i v e s t a n d a r d s , o r by d i r e c t i m p u l s e c o u n t i n g of punched t i s s u e p i e c e s (2). r e c e n t d e v e l o p m e n t of c o m p u t e r a s s i s t e d i m a g e a n a l y s i s h a s now g r e a t l y i m p r o v e d t h e possibilities t o m e a s u r e a n d d e s c r i b e visual i n f o r m a t i o n o b t a i n e d f r o m t h e v a r i o u s wba t e c h n i q u e s . in t h e following, s o m e e s s e n t i a l s t e p s involving c o m p u t e r a s s i s t e d i m a g e a n a l y s i s will b e b r i e f l y discussed. f i r s t , a s h o r t d e s c r i p t i o n will b e given of t h e e n t i r e i m a g e a n a l y s i s s y s t e m , a n d t h e i m a g e pick-up p r o c e d u r e s , a n d b r i e f l y s o m e main p r o c e s s i n g commands"' u s e d in a n a l y z i n g whole-body a u t o r a d i o g r a m s . finally, s o m e of t h e s e p r o c e d u r e s will b e e x e m p l i f i e d . 1 i1 ... 257 video camera disc memory digitalizato light box fig. i---]-l digitizer pad fl . block d i a g r a m of t h e i m a g e a n a l y s i s s y s t e m . image analysis system t h e s t r u c t u r e of t h e i m a g e a n a l y s i s s y s t e m is shown in f i g u r e 1. t h e a u t o r a d i o g r a p h i c f i l m is t r a n s i l l u m i n a t e d by a r e o s t a t c o n t r o l l e d light-box. by p r o p e r c h o i c e of t h e c a m e r a l e n s a n d i t s d i s t a n c e t o t h e v i d e o c a m e r a , a l m o s t a n y s i z e of a u t o r a d i o g r a m c a n h e a n a l y s e d . a m i c r o c o m p u t e r w i t h a d i s c m e m o r y for t h e s t o r a g e of p i c t u r e s (up t o 120 p i c t u r e s c a n b e s t o r e d in t h e m e m o r y ) r e c e i v e s i n p u t d a t a f r o m t h e v i d e o c a m e r a v i a a n i m a g e d i g i t i z e r . t h e o u t p u t d a t a in t h e f o r m of p r o c e s s e d i m a g e s a r e s t o r e d in t h e i m a g e m e m o r y , w h e r e up t o f o u r p r o c e s s e d p i c t u r e s c a n b e a l t e r n a t i v e l y displayed o n t h e s c r e e n ( o u t p u t i m a g e ) . to t h e c o m p u t e r , a d i g i t i z e r p a d a l l o w s t h e o p e r a t o r to i n d i c a t e o b j e c t s or r e g i o n s of i n t e r e s t in t h e i m a g e , a n d t h e position, t h e m e a n o p t i c a l d e n s i t y , t h e s t a n d a r d d e v i a t i o n , a n d t h e a r e a s i z e a r e displayed on t h e t e r m i n a l , or could b e p r i n t e d on paper. in a d d i t i o n t o t h e d i s c m e m o r y , t h e s y s t e m i s a l s o c o m p l e m e n t e d w i t h a t a p e m e m o r y f o r s t o r a g e a n d back-up of t h e p r o g r a m s . image pick-up system w e h a v e c h o s e n a v i d e o c a m e r a f o r t h e i m a g e pick-up s y s t e m , b e c a u s e i t h a s t h e a d v a n t a g e of b e i n g fast, s e n s i t i v e , a n d r a t h e r c h e a p . t h e i m a g e in t h e c a m e r a t u b e i s d i v i d e d by t h e v i d e o d i g i t i z e r i n t o a n a r r a y of 384 h o r i z o n t a l c o l u m n s a n d 2 4 1 v e r t i c a l rows, t h u s producing 92 5 4 4 e q u a l a n d d i s c r e t e s q u a r e s (pixels). t h e p i x e l i s t h e b a s i c e l e m e n t of t h e p i c t u r e a n d i t s s i z e a l s o d e f i n e s t h e o p t i c a l r e s o l u t i o n of t h e s y s t e m . e a c h p i x e l is t h e n a s s i g n e d a v a l u e b a s e d on t h e g r a y l e v e l o n t h a t m i n u t e p o r t i o n of t h e p i c t u r e which i t e n c o m p a s s e s . t h e d i g i t i z e r p r o v i d e s 256 d i f f e r e n t g r a y levels. a s e r i e s of g r a y f i l t e r s f r o m known o p t i c a l d e n s i t i e s c a l i b r a t e t h e g r a y l e v e l s to o p t i c a l d e n s i t y (od), a n d t h e n a s e c o n d c a l i b r a t i o n m a y h e p e r f o r m e d , using s t a n d a r d s w i t h known a m o u n t s of r a d i o a c t i v i t y , t h u s c o n v e r t i n g t h e o p t i c a l d e n s i t i e s t o r e a l c o n c e n t r a t i o n values. 258 image processing p r o c e d u r e s i n wba in o r d e r t o e x t r a c t as m u c h i n f o r m a t i o n as possible f r o m d i f f e r e n t wba t e c h n i q u e s , a n u m b e r of i m a g e e n h a n c e m e n t f u n c t i o n s c a n b e g e n e r a t e d . t h e s e h a v e b e e n e s p e c i a l l y a d a p t e d f o r t h e a n a l y s i s of whole-body a u t o r a d i o g r a m s . a d e t a i l e d d e s c r i p t i o n of b a s i c i m a g e e n h a n c e m e n t p r o c e d u r e s (8) a n d pseudo-colour c o d i n g (3) h a s b e e n r e p o r t e d e a r l i e r . an a u t o r a d i o g r a d h i c i n v e s t i g a t i o n includes v a r i o u s l e v e l s of t h e body, a n d t h u s m o s t t i s s u e s a n d fluids c a n b e s c a n n e d . i t is possible t o o b s e r v e w h e t h e r t h e r a d i o a c t i v e [eve1 is p r e s e n t in s m a l l s t r u c t u r e s as t h e m u s c l e l a y e r of t h e i n t e s t i n a l t r a c t , or in t h e mucosa. in t h e e y e , t h e r a d i o a c t i v i t y c a n b e t r a c e d t o e.g. t h e c o r n e a , r e t i n a , p i g m e n t l a y e r s , or a q u e o u s humour. small e n d o c r i n e o r g a n s will show up, s u c h as t h e p i t u i t a r y , t h y r o i d a n d d i f f e r e n t l a y e r s of t h e a d r e n a l c o r t e x . when i n v e s t i g a t i n g p r e g n a n t a n i m a l s , t h e a c t i v i t y c a n b e c o m p a r e d in b o t h t h e m a t e r n a l a n d t h e f e t a l tissues. [jp t o now, t h e q u a n t i t a t i o n of s u c h s m a l l s t r u c t u r e s h a s b e e n v e r y h a r d a n d time-consuming. however, by s u i t a b l e i m a g e p r o c e s s i n g p r o c e d u r e s as m a g n i f c g t i o n , h i s t o g r a m geneyation, a n d oblect-c~assiffication e v e n t h e q u a n t i t a t i o n of s u c h s m a l l s t r u c t u r e s c a n b e p e r f o r m e d v e r y r a p i d l y a n d w i t h high a c c u r a c y . magnified p i c t u r e . t h e r e a r e t w o m e t h o d s of p e r f o r m i n g m a g n i f i c a t i o n w i t h t h i s s y s t e m . a z o o m l e n s on t h e v i d e o c a m e r a c a n b e used t o m a g n i f y o p t i c a l l y t h e i m a g e (fig. 2 b,c,d), or t h e i m a g e c a n b e m a g n i f i e d in t h e c o m p u t e r (fig. 2 h) by s o f t w a r e o p e r a t i o n . when d o u b l e m a g n i f i c a t i o n i s p e r f o r m e d , e a c h p i x e l of a c e r t a i n g r a y l e v e l i s r e p l a c e d by 2 x 2 p i x e l s of e q u a l v a l u e . g r a y l e v e l h i s t o g r a m plot. t h e h i s t o g r a m is a g r a p h i c a l p l o t of t h e g r a y s c a l e d i s t r i b u t i o n in t h e p i c t u r e . t h e h o r i z o n t a l a x i s r e p r e s e n t s t h e g r a y s c a l e , s t a r t i n g w i t h 0 ( w h i t e ) a n d r a n g i n g t h r o u g h 255 (black), a n d t h e v e r t i c a l a x i s r e p r e s e n t s t h e n u m b e r of p i x e l s (fig. 2 e,f,g). g r a y l e v e l window m e a s u r e m e n t . the h i s t o g r a m p r o d u c e d as a b o v e c a n b e a n a l y z e d by s p e c i f y i n g , w i t h t h e d i g i t i z e r pad, a n y t w o g r a y levels, which r e p r e s e n t a window ( r e g i o n ) of i n t e r e s t . this p r o g r a m will c o m p r i s e t h e n u m b e r of p i x e l s ( t h e s e l e c t e d a r e a within t h e p i c t u r e ) f a l l i n g within t h i s g r a y l e v e l window, a n d i t will p o i n t o u t t h e m e a n d e n s i t y , s t a n d a r d d e v i a t i o n , t h e s e l e c t e d g r a y l e v e l s in t h e window, a n d t h e t o t a l n u m b e r of pixels ( t h e a r e a ) within t h e window. t h e s e l e c t e d p i x e l s will t h e n i m m e d i a t e l y s h o w u p on t h e i m a g e display (fig. 2 b,c,d). this p r o c e d u r e c a n b e u s e d i n t e r a c t i v e l y by t h e o p e r a t o r . o b j e c t c l a s s i f i c a t i o n by g r a y l e v e l windows. by i n s p e c t i o n of t h e h i s t o g r a m of a p i c t u r e , o n e m a y g u e s s w h e r e t h e t h r e s h o l d s should b e l o c a t e d f o r a c l a s s i f i c a t i o n of t h e i m a g e i n t o i t s d i f f e r e n t p a r t s . t o d o this, o n e a s s u m e s t h a t e a c h p e a k of t h e h i s t o g r a m c o r r e s p o n d s t o a c e r t a i n a n a t o m i c a l s t r u c t u r e of t h e a u t o r a d i o g r a m . c l a s s i f i c a t i o n of a n i m a g e on t h e basis of i t s g r a y l e v e l h i s t o g r a m h a s b e e n shown t o b e e s p e c i a l l y u s e f u l 259 h i fig. 2 a-i. a s e q u e n c e of p i c t u r e s f r o m t h e i m a g e display, i l l u s t r a t i n g s o m e main i m a g e p r o c e s s i n g p r o c e d u r e s in w r a . in (a), t h e d i s t r i b u t i o n of a b e n z o d i a z e p i n a n t a g o n i s t ( r o 15-1788) i s shown in t h e o r i g i n a l a u t o r a d i o g r a m . note t h e high a c t i v i t y in t h e b r a i n ( w h i t e a r e a ) . in (b-d), o p t i c a l l y m a g n i f i e d a n d d i g i t i z e d p i c t u r e s of t h e b r a i n , s e l e c t e d f r o m t h e original, a n d t h e i r c o r r e s p o n d i n g g r a y l e v e l h i s t o g r a m s , a r e shown just b e l o w (el, ( f ) , a n d (g). by s e l e c t i o n of d i f f e r e n t g r a y l e v e l windows in (e), (f), a n d (g) d r o p s in t h e h i s t o g r a m -, a c l a s s i f i c a t i o n of v a r i o u s b r a i n a r e a s is displayed as a c h e s s p a t t e r n in (b), (c), a n d (d). fig. 2 h i l l u s t r a t e s a c o m p u t e r m a g n i f i c a t i o n r o u t i n e , a n d e x c l u s i v e l y t h e s e l e c t e d g r a y l e v e l s o b t a i n e d f r o m t h e brain c l a s s i f i c a t i o n in (d). fig. 2 i i l l u s t r a t e s s o m e c o m p u t e r r o u t i n e s , s u c h as "retouch", "text-writing", plus "arrows" t o m a r k tissues. 260 a b c d fig. 3 a f . e f s e q u e n c e of p i c t u r e s i l l u s t r a t i n g o r d i n a r y c o n t r a s t e n h a n c e r n e n t (a. b, c). a n d a n o t h e r c o m p u t e r r o u t i n e ( d , e, f ) t h a t b r i n g s o u t d e t a i l s a n d c o n t o u r s , w h i c h a p p e a r d i f f u s e in t h e o r i g i n a l p i c t u r e . t h e s e q u e n c e i l l u s t r a t e s t h e d i s t r i b u t i o n of c l a b e l l e d d e p r i n y l in a r a t b r a i n . fig. 3 c s h o w s t h e r e s u l t of c o n t r a s t e n h a n c e m e n t , w h e n t h e g r a y l e v e l s o b t a i n e d f r o m t h e n a r r o w h i s t o g r a m ( b ) of t h e o r i g i n a l a u t o r a d i o g r a m (a) a r e s p r e a d o v e r a w i d e r r a n g e . in ( f ) , t h e c o n t o u r s of t h e n u c l e u s c a u d a t u s / p u t a m e n a r e m a d e c l e a r by t h e u s e of t h e h i g h e s t g r a y l e v e l p i x e l s (cf. b). t h e s e a r e m a r k e d in b l a c k (e) a n d s e l e c t e d f r o m t h e r e g i o n of i n t e r e s t (d). in m e a s u r i n g c o m p l i c a t e d s t r u c t u r e s as, e.g., t h e i n t e s t i n a l m u c o s a , t h e v a r i o u s p o r t i o n s of t h e b r a i n ( f i g . 2 h), a n d d i f f e r e n t p a r t s of t h e s p l e e n ( r e d a n d w h i t e pulp). t h i s p r o c e d u r e m i g h t a l s o b e u s e d t o e x c l u d e a c t i v i t y in blood v e s s e l s o r b i l e d u c t s , t h u s m e a s u r i n g t h e a c t i v i t y of t h e l i v e r p a r e n c h y m e e x c l u s i v e l y . e a c h c l a s s of g r a y l e v e l s (a c e r t a i n a n a t o m i c a l s t r u c t u r e ) c a n t h e n a l s o b e g i v e n a c e r t a i n c o l o u r . c o n c l u s i o n s a s i n g l e l o w c o s t i m a g e a n a l y s i s s y s t e m h a s b e e n d e s c r i b e d . t h e i m a g e p r o c e s s i n g s y s t e m e x p l a i n e d in t h i s r e p o r t h a s b e e n e s p e c i a l l y a d a p t e d t o w b a . i t h a s l a r g e l y i n c r e a s e d t h e p o s s i b i l i t i e s t o m e a s u r e a n d d e s c r i b e v e r y s m a l l a n d h e t e r o g e n o u s s t r u c t u r e s . c o n t r a s t e n h a n c e m e n t r o u t i n e s h a v e a l s o b e e n i l l u s t r a t e d , t h u s g i v i n g t h e o b s e r v e r f u r t h e r i n f o r m a t i o n . 17-868573 26 1 a c k n o w l e d g e m e n t s w e a r e m u c h i n d e b t e d t o dr. g o r a n s p e r b e r , d e p t . of p h y s i o l o g y a n d m e d i c a l p h y s i c s , u p p s a l a u n i v e r s i t y , w h o d i d t h e c o m p l e t e p r o g r a m m i n g of o u r i m a g e a n a l y s i s s y s t e m . 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. r e f e r e n c e s d ' a r g y , r. & u l l b e r g , s.: whole b o d y a u t o r a d i o g r a p h y . s c i e n c e t o o l s 32: 23-26, 1985. b e r l i n , m. & u l l b e r g , s.: a c c u m u l a t i o n a n d r e t e n t i o n of m e r c u r y in t h e m o u s e . i 111. a r c h e n v i r o n h e a l t h 6: 589-616, 1963. g o o c h e e , c., r a s b a n d w. & s o k o l o f f , l.: c o m p u t e r i z e d d e n s i t o m e t r y a n d c o l o r c o d i n g of / "c/ d e o x y g l u c o s e a u t o r a d i o g r a p h s . ann n e u r o l 7: 359-370, 1980. g o o c h e e , c., r a s b a n d , w. & s o k o l o f f , l.: a p p l i c a t i o n of c o m p u t e r a s s i s t e d i m a g e p r o c e s s i n g t o a u t o r a d i o g r a p h i c m e t h o d s f o r s t u d y i n g b r a i n f u n c t i o n s . t i n s 6: 256 260, 1983. p a l a c i o s , j.m., n i e h o f f , d.h. & k u h a r , m.j.: r e c e p t o r a u t o r a d i o g r a p h y w i t h t r i t i u m s e n s i t i v e f i l m : p o t e n t i a l f o r c o m p u t e r i z e d d e n s i t o m e t r y . n e u r o s c i l e t t p e n n e y , j.b., p a n , h.s., young, a.b., f r e y , k.a. & d a u t h , g.w.: q u a n t i t a t i v e a u t o r a d i o g r a p h y of ( 3 h) m u s c i m o l b i n d i n g in r a t b r a i n . s c i e n c e 214: 1036-1038, 1981. r a i n b o w , t.c., b l e i s c h , w.v., riegon, a. & m c e w e n , b.s.: q u a n t i t a t i v e d e n s i t o m e t r y of n e u r o t r a n s m i t t e r r e c e p t o r s . j n e u r o s c i m e t h o d s 5: 127-138, 1982. r o s e n f e l d , a. & k a k , a.c.: in: d i g i t a l p i c t u r e p r o c e s s i n g , vol. i , pp. 209-265, a c a d e m i c p r e s s , n e w york a n d l o n d o n , 1982. u l l b e r g , s.: s t u d i e s o n t h e d i s t r i b u t i o n a n d fate of s 3 l a b e l l e d b e n z y l p e n i c i l l i n in t h e body. a c t a r a d i o 1 (suppl) 118: 1-1 10, 1954. u l l b e r g , 5.: t h e t e c h n i q u e of w h o l e b o d y a u t o r a d i o g r a p h y . c r y o s e c t i o n i n g o f l a r g e s p e c i m e n s . s p e c i a l i s s u e o n whole-body a u t o r a d i o g r a p h y , pp. 2-29, s c i e n c e tools, l k b i n s t r u m e n t j o u r n a l , b r o m m a , s w e d e n , 1977. s o k o l o f f , l.: m o d e l i n g m e t a b o l i c p r o c e s s e s in t h e b r a i n in vivo. ann n e u r o l (suppl) u n n e r s t a l l , j. r., k u h a r , m.j., n i e h o f f , d.j. & p a l a c i o s , j.m.: b e n z o d i a z e p i n e r e c e p t o r s a r e c o u p l e d t o a s u b p o p u l a t i o n of y a m i n o b u t y r i c a c i d ( g a b a ) r e c e p t o r s : e v i d e n c e f r o m a q u a n t i t a t i v e a u t o r a d i o g r a p h i c s t u d y . 3 p h a r m a c o l e x p t h e r u n n e r s t a l l . j.r., n i e h o f f , d.l., k u h a r , m.j. & p a l a c i o s , j.m.: q u a n t i t a t i v e r e c e p t o r a u t o r a d i o g r a p h y using ( h) u l t r o f i l m : a p p l i c a t i o n t o m u l t i p l e b e n z o d i a z e p i n e r e c e p t o r s . j n e u r o s c i m e t h o d s 6: 59-73, 1982. 25: 101-105, 1981. 15; s1-s11, 1984. 218: 797-804, 1981. a d d r e s s f o r r e p r i n t s : r o l a n d d ' a r q y u p p s a l a u n i v e r s i t y d e p a r t m e n t of t o x i c o l o g y u p p s a l a b i o m e d i c a l c e n t e r box 594 5-751 24 u p p s a l a , s w e d e n 262 upsala j med sci 80: 113-117, 1975 mode of spontaneous onset and termination of supra ventricular tachyarrhythmias b e n g t furberg and l a r s n o r d g r e n from the d e p a r t m e n t of clinical physiology, university h o s p i t a l , u p p s a l a , s w e d e n abstract the present study, a recording technique which ecg tracings from 16 patients with spontaneous onset gives a high r e s o h t i o n of t h e atrial waves has been and/or termination of supraventricular tachyarrhythmias used, thereby offering better possibilities for pre (svta) were studied. of these recordings, 13 were made cise assessment of p wave aberration and p-p in with a special technique which gives a high resolution of the t e r v a l s . atrial waves. at the onset of svta, the first atrial wave invariably had an aberrant configuration. the coupling index (coupling interval (p-'p) preceding cycle length) was 0.50 or less in 9 of 15 cases but more than 0.60 in 4 cases. in the 5 cases of onset of atrial fibrillation, the intervals m a t e r i a l a n d m e t h o d s the study was performed on ecg tracings from 16 pa between the first few atrial waves corresponded to a fretients (9 men and 7 women, with ages between 19 and 7 2 ) quency of 300-350 per minute. acceleration of the atrial activity occurred within the first 30 seconds. at the ter mination of svta, no successive modification of the atrial activity was found. the termination often did not occur a t or shortly after a qrs complex. it is concluded that a premature atrial beat-even a single one-with a short coupling interval may well initiate a circus-movement svta, while an ectopic atrial beat with a long coupling interval apparently must be followed by repeated rapid discharges from the ectopic focus in order for svta to ensue. the functional conditions of the atria may then determine which kind of svta eventually results. i n t r o d u c t i o n t h e mechanisms responsible for the initiation, maintenance and termination of supraventricular tachyarrhythmias (svta), have been the subject of continuing controversy and investigation. experi ments with electrical o r chemical stimulation of t h e with spontaneous onset and/or termination of svta. i n tracings with more than one bout of svta a representa tive one was selected for the s t u d y . the change from sinus rhythm to atrial fibrillation was studied in 5 tracings, to atrial flutter in 8 and to atrial tachycardia in 2. the rever sion to sinus rhythm from atrial fibrillation was studied in 2 tracings, from atrial flutter in 6 and from atrial tachy cardia in 2 . i n 3 patients the ecg was recorded with conventional amplification ( 1 mv=lo mm), lead v , and at least 3 other leads being recorded. i n 13 patients a special recording technique was used (7), using three bipolar leads. the common reference electrode was placed at the midline of the angle of sternum and the three different electrodes were placed at the highest attainable point of the armpit in the left mid-axillary line (s 1 ) . at the caudal end of the sternal body (s2) and at a point over the vertebral column at the transthoracic level of the sternal angle (s3). the three bipolar leads were recorded with conventional amplification, as well as with a tenfold grea ter amplification (0.1 m v = l o mrn), using a 3-channel dif ferential preamplifier and a mingograph 81 (siemens atrium, while yielding important information con cerning the electro-physiological characteristics of s v t a , have not solved t h e problems of onset and elema l t d . , for measurements, the system illustrated in fig. i was used, the svta type was defined according to t h e main atrial wave frequency. an atrial wave frequency of termination of spontaneous s v t a . since the first electrocardiogram showing t h e onset of atrial fibrillation was presented in 1918 150-210/min was judged as supraventricular tachycardia (svt), of 220-340 as atrial flutter (afu) and of 350 or more as atrial (afi), (lo), numerous tracings of s v t a onset and termi nation have been published. most of these tracings were made with a conventional recording techni que, which is not sufficiently sensitive to clearly (exhibit changes in the atrial wave configuration. in r e s u l t s t h e tracings of s v t a with the special recording technique permitted analysis of the atrial wave con 8-752858 upsala j med sci 80 114 b . furberg cind l . nordgrrn f i g . i . onset and termination of atrial flutter (patient no. 9). the nomenclature of the atrial waves is shown. the three special leads s i-s 3 are recorded with conventional and tenfold amplification. f i g . 2. short episode of atrial flutter (patient no. 12). note the long coupling interval. upsala j med sci 80 onset rind terminrrtion of tcichyrirrhythmiris 1 15 fig. 3. onset of atrial fibrillation (patient no. 4). the premature atrial beat is followed by a rapid atrial tachy cardia and within 2 s by atrial fibrillation. figuration and intervals; this had been difficult t o obtain with the conventional recording technique. examples of these new recordings a r e shown in figs. 1-3. at the onset of s v t a , the first atrial wave ('p) invariably had an aberrant configuration in com parison t o the ordinary p wave. t h e following atrial waves ( w " p ) of the s v t a had the same configura tion a s 'p in the 2 s v t cases and in 6 of t h e 8 afu cases. in the 5 afi cases the configuration changed. in 9 of the i5 cases, t h e 'coupling index' (the ratio of coupling interval (p-'p) t o preceding cycle length (p-p)) was 0.50 o r less, while it was more than 0.60 in 1 afi case, in 2 a f u cases and in 1 s v t case. acceleration of the atrial activity after *p was seen in t h e 5 afi cases, in 3 of the afu cases and in none of t h e s v t cases. t h e final atrial wave frequency was always obtained within 30 s. t h e termination of s v t a occurred in 5 of t h e 10 cases studied within 30 cs from the beginning of a q r s complex. t h e point of termination was de ' fined as the start of the p' wave plus t h e pz-p1 interval. there was no deceleration of the last few atrial impulses before t h e termination and t h e form of t h e atrial waves did not change. the first atrial wave after termination of s v t a exhibited a n ordi nary configuration in all but o n e case. discussion when dealing with the mechanism of s v t a onset and maintenance, some authors have concluded that a t least s v t and a f u , and possibly afi, are evoked b y a n ectopic atrial focus with a high impulse-formation rate. other authors have con cluded that s v t a depends o n the establishment of a n atrial circus movement with o r without engage ment of the atrio-ventricular node. from experi mental studies there is evidence that s v t a can result from either of these mechanisms. t h e argu ments in favour of both hypotheses have been re viewed by hecht e t al. 1953 (4), scherf, schaffer & blumenfeld 1953 ( 1 i ) , katz & pick 1960 (9, rytand 1966 (8) and guiney & lown 1972 (3). studies of the upsala j med sci 80 116 b . furherg find l . n o r d g r e n table i . o n s e t of suprciventricular t ~ i ~ i i y ~ i r r i i y t i i m i ~ i final atrial constant patient ordinary p-'p 'p-*p zp-3p 3p-4p frequency p-'p configuration no. p-p, cs (cs) (cs) (cs) (cs) (waveslmin) p-p of lp-"p sinus rhythm to atrial fibrillation, n = 5 1 186 38 20 2 83 40 17 3 65 43 19 4 110 26 26 5 80 28 19 sinus rhythm to citrialflutter, n =8 6 126 38 22 7 84 46 30 8 108 54 27 9 80 35 26 10 80 40 28 1 1 81 52 28 12 84 1 9 29 13 216 112 28 sinus rhythm t o atrial tachycardia, n =2 14 i 0 49 38 15 98 40 39 17 18 17 26 18 20 20 25 26 19 21 26 22 36 40 16 17 16 19 19 20 20 25 26 20 19 27 21 3 1 41 400 390 410 430 360 340 330 230 280 280 330 23 0 270 160 150 0.20 0.48 0.66 0.24 0.35 0.30 0.55 0.50 0.44 0.50 0.64 0.94 0.52 0.70 0.41 + + spontaneous onset and termination of s v t a may have some bearing on this problem. killip & gault (6) studied changes from sinus rhythm to afi in 14 patients. an atrial premature discharge preceded the onset of afi in all 14 episodes. i n 10 of these t h e premature p wave was immediately followed by afi. i n the o t h e r 4 cases t h e premature p wave appeared t o initiate a brief run of s v t (less than 6 beats) which accelerated to afi. t h e relative pre maturity of ectopic atrial beats was expressed by calculating the coupling index. t h e coupling index was less than 0.50 in 9 of t h e 14 patients. killip & gault t h u s concluded that a spontaneously occur ring atrial premature impulse may initiate afi. when the coupling index is less than 0.50, the chance of ensuing afi is high; when it i s greater than 0.60, t h e chance of ensuing afi is small. bennett & pentecost ( 1 ) studied t h e onset of afi in 8 patients with acute myocardial infarction. one intra-atrial and o n e surface lead were registered. a table 11. t e r m i n a t i o n of s u p r a v e n t r i c u l a r tachyarrizythmia termination within patient ordinary p4-p3 p3-pz pz-pl p '-p p'-p 30 cs from no. p-p, cs (cs) (cs) (cs) (cs) p-p beginning of qrs atrial fibrillation to sinus rhythm, n =2 5 80 19 20 22 83 1.04 + 16 69 17 16 16 52 0.75 + atrialgutter to sinus rhythm, n = 6 6 126 18 18 16 27 0.21 8 1 08 27 25 25 86 0.80 + 9 80 21 22 20 104 1.30 1 1 81 20 20 22 96 1.19 12 84 29 26 27 96 1.14 + 13 216 22 22 22 132 0.61 + attriaf tachycardia to sinus rhythm, n = 2 1.09 14 70 36 37 36 76 15 98 39 40 41 1 02 1.04 vpsala j med sci 80 onset crnd termincition of tcrchycirrliytlimitrs 1 17 total of 32 episodes were studied, and o n each occa sion the arrhythmia was preceded by a premature atrial beat. t h e premature atrial beat was always followed by a rapid regular atrial tachycardia of variable duration with a rate of approximately 340 beats/min; on some occasions the tachycardia lasted as little as 1 o r 2 s, but at other times the duration was up t o 30 s. bigger & goldreyer ( 2 ) studied the repeated onset of paroxysmal s v t in 5 patients. t h e s v t always began with a n atrial premature depolarization (apd). spontaneous episodes of s v t were in itiated only by apds occurring in the relative a-v refractory period, while electrical stimuli during the atrial vulnerable period did not elicit s v t . from these and other findings bigger & goldryer con cluded that paroxysmal s v t is most often d u e to reentry utilizing t h e a-v conduction system. t h e results of the present study in general cor respond well with t h e results of the earlier reports. t h e first premature atrial wave was shown to b e aberrant in all cases studied and the coupling inter val was in most cases short. the coupling index, however, was more than 0.60 in 4 of our cases (fig. 2 shows o n e of these cases). in these cases t h e first premature atrial wave could scarcely have occurred during the vulnerable period of t h e atria o r t h e rela tive refractory period of the a-v conduction system. in these cases, therefore, the onset of s v t a probably depended o n a rapidly firing ectopic focus rather than o n circus movement. o u r study of t h e termination phase indicates that there is not a successive modification of atrial activ ity during the last seconds of a svta: the s v t a always ended abruptly in o u r cases. rytand (9) re ported similarly for 3 cases of a f u , while bennett & pentecost ( l ) , in their study of afi in patients with acute myocardial infarction, found that a change of atrial wave form in the intra-atrial lead preceded t h e cessation of afi o n all 28 occasions observed. several explanations f o r the interruption of s v t a have been offered, for example, exit block from a rapidly firing ectopic focus o r interruption of a circus movement with o r without engagement of the atrio-ventricular node. one possible cause of such interruption could b e retrograde depolariza tion of the atrio-ventricular node. in t h e latter case, the s v t a should b e expected to terminate a t o r shortly after a q r s complex. this was observed in only 5 of our cases of afi or a f u , so that this explanation is probably not t h e only one. our results t h u s indicate that the theory of circus movement does not explain the mode of spontane ous onset o r termination of s v t a in all cases. onset and maintenance of s v t a in some cases may perhaps be governed b y different mechanisms, the onset being initiated from an atrial ectopic focus and a circus movement being responsible for the maintenance. the importance of the vulnerable period of the atria could b e that o n e single ectopic and premature atrial impulse occurring during the vulnerable period of t h e atria may initiate a circus movement s v t a , while an ectopic atrial beat with a long coupling interval must be followed by re peated rapid discharges from the ectopic focus in order for s v t a to ensue. the functional conditions of the atria may then determine which kind of s v t a eventually results. references 1. 2. 3. 4. 5 . 6. 7. 8. 9. 10. 11. bennett, m. & pentecost, b.: the pattern of onset and spontaneous cessation of atrial fibrillation in man. circulation4/:981, 1970. bigger, t. & goldreyer, b.: the mechanism of supraventricular tachycardia. circulation 42: 673, i 970. guiney, t. & lown, b.: electrical conversion of atrial flutter to atrial fibrillation. brit heart 5 3 4 : 1215, 1972. hecht, h . , katz, l., pick, a . , prinzmetaf, m. & rosenblueth, a.: the nature of auricular fibrillation and flutter. a symposium. circulation 7: 591, 1953. katz, l. & pick, a.: current status of theories of mechanisms of atrial tachycardias, flutter and fibrilla tion. progr cardiov dis 2: 650, 1960. killip, t. & gault, j . : mode of onset of atrial fibrilla tion in man. am heart j 70: 172, 1965. nordgren, l.: a new method to study atrial activi ty-intended for clinical use. acta soc med upsal 74: 186, 1969. rytand, d.: the circus movement (entrapped circuit wave) hypothesis and atrial flutter. ann int med 65: 125, 1966. rytand, d.: electrocardiographic patterns at the termination ofatrjal flutter. am heart 574: 741, 1967. semerau, m . : uber riickbildung der arrhythmia perpetua. arch klin med 126: 161, 1918. scherf, d., schafter, a . i . & blumenfeld, s . : mechanism of flutter and fibrillation. arch intern med 91: 333, 1953. received november 28, 1974 address for reprints: bengt furberg, m.d. department of clinical physiology central hospital s-800 07 gavle sweden upsola j med sci 80 upsala j med sci 84: 67-74, 1979 the effect of short-term high-dose treatment with methenamine hippurate of urinary infection in geriatric patients with indwelling catheters i. the preparation and morphology of a quantified urine sediment bo norberg, astrid norberg, u l f parkhede, hans gippert and mbns akerman departments of internal medicine, pathology and education, univer.tity of lund and the school of nursing, lund, sweden. 3 a4 riker laboratories, skurholmen, swyden abstract a quantified sediment of the urine from patients with indwelling catheters was prepared by fixation of 0.1 ml urine in 0.9 ml 2% glutaraldehyde immediately after sampling. slide preparations were then made from 0.2 ml of the glutaral dehyde suspension by means of a cytocentrifuge. were properly contrasted by the may-grunwald-giemsa stain but haematoxylin eosin and the papanicolaou stain were superior as regards leukocyte morphology. it is suggested that g l u t a r a l d e h y d e c y t o c e n t r i f u g e preparations of the urine cytology may be useful in the evaluation of urinary infection and in the evaluation of the therapy of urinary infection. bacteria and epithelial cells introduction the microscopic examination of urine sediments is a rapid and simple proce dure which provides essential information in many cases of kidney disease or infections in the urinary tract. the conventional urinary sediment has, how ever, serious pitfalls, e.g. low reproducibility, low precision and high vul nerability to delay in transport and preparation (1-10). it nevertheless seemed desirable to make a quantified urine sediment from patients with indwelling catheters in order to evaluate urinary infection and the effects of therapy. since delay in transport and preparation could not be avoided under the conditions prevailing, we chose to "freeze" the cell picture at the moment of sampling by fixation. the aim of the present study was to identify a simple and rapid method of sampling, fixing and quantifying the cytology of the urine from patients with indwelling catheters. different staining procedures were also compared as re gards the contrast and detail revealed in the morphology of bacteria and leu kocytes. 67 material and methods the urine was sampled from 14 inpatients at the somatogeriatric wards at saint lars hospital, lund, twice a week during the pre-treatment control period, days 10-17 , during treatment with methenamine hippurate (mh, hiprexr, riker laboratories, loughborough, leicestershire, england), 2 g x 3 , days 18-52 and during the post-treatment control period, days 6 6 7 3 . during days 1-9 different cytocentrifuge preparations of leukocytes and bacteria were evaluated. all patients had an indwelling catheter with continuous flow of the urine in to a bag with a valve preventing back flow (closed system) for months or years prior to the present study. the catheter was plugged 20-30 minutes before sam pling. the urine, approximately 15 ml, was collected in a sterile plastic tube and thoroughly shaken in order to disperse the solid matter. the specimen (0.1 ml) was then immediately transferred to 0.9 ml 2% glutaraldehyde in phosphate buffer, 0.135 m, ph 7 . 4 . cells and bacteria were spun down on slides 2-4 hrs later by means of a cytocentrifuge (shandon-elliot cytospin), 1,000 r.p.m., 10 minutes, 0 . 2 ml of the cell suspension. during the present study 197 double preparations of urine sediments were ob tained. one preparation was always stained according to may-griinwald-giemsa (mgg), which was chosen as reference stain to confirm with the haematological traditions of the laboratory. other routine stains available in the laboratory were used for the remaining preparation and compared with mgg: haematoxylin eosin (mayer's h a e m a t o x y l i n e r y t r o s i n ) , the papanicolaou stain, periodic acid schiff (pas), methyl green-eosin or haematoxylin-eosin-methylene blue. in ad dition, staining with acridine orange at low ph ( 4 ) was tried. the evaluation of the staining procedures was performed by means of a zeiss photomicroscope equipped with a planachromatic x 100 objective (cf. figs 1-3). results the cytological picture of urine sediments occasionally shed light on the associated disease; the renal lesions in a patient with hyperparathyroidism and urinary tract infection were confirmed by the finding of numerous hyaline casts with embedded leukocytes, sugcestive of protein loss in the urine and re nal inflammation (fig. 1). the glutaraldehyde fixation was so rapid and effective that leukocytes were caught in different stages of the locomotory cycle (fig. zb), in different sta ges of phagocytosis (fig. 2b) and in different stages of disintegration (figs. l b , 3). the staining of g l u t a r a l d e h y d e c y t o c e n t r i f u g e preparations entailed several problems. the basic haematological stain, may-grunwald-giemsa (mgg), produced 68 f i g . 1. from p a t i e n t no. 8, an 80-year-old man w i t h c e r e b r a l a r t e r i o s c l e r o s i s , a m o d e r a t e d e g r e e of r e n a l f a i l u r e ( s c r e a t . 130-150-140 m m o l / l ) , and hyper p a r a t h y r o i d i s m (s-ca 3.7-2.5-2.8 mmol/l, s-p 0.8-0.8 m o l / l , s-parathormone 690-510 p m o l / l ) . the u r i n e s e d i m e n t c o n t a i n e d numerous h y a l i n e c a s t s w i t h em bedded l e u k o c y t e s . the l e u k o c y t e c a s t s s u g g e s t e d r e n a l l e s i o n s i n d u c e d by h y p e r c a l c e m i a o r u r i n a r y t r a c t i n f e c t i o n , o r b o t h . s t a i n i n g a c c o r d i n g t o may griinwald-giemsa. f i g . 1 a . low-power f i e l d , m a g n i f i c a t i o n x 1 3 4 . two h y a l i n e c a s t s (cy) w i t h em bedded l e u k o c y t e s . e x c e p t f o r t h e c a s t s , t h e p i c t u r e i s t y p i c a l of t h e u r i n e s e d i m e n t from a p a t i e n t w i t h i n d w e l l i n g c a t h e t e r and h e a v i l y i n f e c t e d u r i n e , i . e . numerous b a c t e r i a and l e u k o c y t e s , some i n l y s i s . t h i s specimen w a s ob t a i n e d a f t e r t r e a t m e n t w i t h methenamine h i p p u r a t e , 2 g x 3 , f o r 2 2 d a y s . f i g . 1 b . d e t a i l from f i g . l a , m a g n i f i c a t i o n x l , 3 3 6 of a p a r t of a h y a l i n e c a s t ( e n c l o s e d a r e a ) . two l e u k o c y t e s ( l ) , s u r r o u n d e d by b r i g h t h a l o s , a r e embedded i n t h e c a s t . two c e l l s i n l y s i s (c) are a l s o s e e n , one embedded i n t h e c a s t . b: b a c t e r i a , p r o b a b l y p r o t e u s m i r a b i l i s , which were r e p e a t e d l y c u l t u r e d from t h e u r i n e of t h i s p a t i e n t . a b r i l l i a n t c o n t r a s t i n e p i t h e l i a l c e l l s and b a c t e r i a ( f i g . 2 a ) . the l e u k o c y t e s were, however, o v e r l o a d e d w i t h s t a i n which v e i l e d i n t r a c e l l u l a r d e t a i l s ( f i g s . 69 f i g . 2a. the u r i n e s e d i m e n t from an 82-year-old f e m a l e , p a t i e n t no. 5 , p r i o r t o t r e a t m e n t w i t h methenamine h i p p u r a t e . the c y t o l o g i c a l p i c t u r e w a s dominated by b a c t e r i a of which two s t r a i n s a r e shown, b a p p a r e n t l y s t r e p t o c o c c u s f a e c a l i s , b a p p a r e n t l y e c o l i , b o t h of which were found i n h e r u r i n e c u l t u r e s . the u p p e r p a r t of t h e m i c r o p h o t o g r a p h i s dominated by an e p i t h e l i a l c e l l , t h e b o r d e r s of which a r e i n d i c a t e d by a r r o w s . n : l: l e u k o c y t e s , one p r o b a b l y l o c a t e d w i t h i n thoc y t o p l a s m of t h e e p i t h e l i a l c e l l . s t a i n i n g a c c o r d i n g t o may-grunwald-giemsa. m a g n i f i c a t i o n ~ 1 , 3 3 6 . f i g . 2 b . u r i n e s e d i m e n t from an 82-year-old f e m a l e , p a t . no. 7 , p r i o r t o t r e a t ment w i t h methenamine h i p p u r a t e . leukocytes c a u g h t i n t h e h u n t and phagocy t o s i s of b a c t e r i a . many of t h e l e u k o c y t e s h a v e t h e e l o n g a t e d s h a p e t y p i c a l of c e l l s i n l o c o m o t i o n , amoeboid movement c o n f i g u r a t i o n . p h a g o c y t o s i s i s p e r f o r med by e x t e n s i o n of t h e t h i n g r a n e l a f r e e c y t o p l a s m i c v e i l i n t h e f r o n t p a r t of t h e l e u k o c y t e s , t h e l a m e l l i p o d i u m ( i p ) towards and around t h e b a c t e r i a (b). s e v e r a l b a c t e r i a are found w i t h i n l e u k o c y t e s and s u r r o u n d e d by p h a g o c y t i c v a c u o l e s ( s e e e . g . a r r o w ) . s t a i n i n g a c c o r d i n g t o may-grunwald-giemsa. mag n i f i c a t i o n ~ 1 , 3 3 6 . l 2 n u c l e u s of t h e e p i t h e l i a l c e l l . 1, 2a) w i t h r a r e e x c e p t i o n s ( f i g . 2 b ) . mgg s t a i n a s r e g a r d s l e u k o c y t e morphology and c l e a r l y i n f e r i o r t o vgc i v t h e s t a i n i n g of h a c t e r i a . the pas s t a i n v7as n e a r l y as bac! as t h e 70 3a 3b f i g . 3 . the h e m a t o x y l i n e o s i n s t a i n p r o v i d e d b e t t e r t r a n s p a r e n c y of c y t o p l a s m and n u c l e u s t h a n t h e may-griinwald-giemsa s t a i n b u t b a c t e r i a was h a r d l y v i s i b l e ( b ) . it i s e v i d e n t t h a t t h e v a s t m a j o r i t y of l e u k o c y t e s i n t h e u r i n e of p a t i e n t s w i t h i n d w e l l i n g c a t h e t e r s are p o l y m o r p h o n u c l e a r s . amc: amoeboid movement c o n f i g u r a t i o n , t h e e l o n g a t e d c e l l s h a p e s u g g e s t i v e of l o c o m o t i o n a t t h e moment of f i x a t i o n . some l e u k o c y t e s were f i x e d i n t h e s t a t e of p y c n o s i s ( p ) , k a r y o r r h e x i s ( k ) o r c y t o l y s i s ( c ) . u r i n e s e d i m e n t from an 81-year-old m a l e , p a t i e n t n o . 2 , w i t h c e r e b r a l a r t e r i o s c l e r o s i s and a m o d e r a t e d e g r e e of c a r d i a c f a i l u r e , a f t e r t r e a t m e n t w i t h methenamine h i p p u r a t e f o r 1 7 d a y s . m a g n i f i c a t i o n 1 , 3 3 6 . haematoxylin-eosin and t h e p a p a n i c o l a o u s t a i n p r o v i d e d i n t e r e s t i n g a l t e r n a t i v e s t o t h e mgg s t a i n . the n u c l e a r and c y t o p l a s m i c d e t a i l s o f l e u k o c y t e s w e r e b e t t e r v i s u a l i z e d t h a n i n mgg-stained p r e p a r a t i o n s ( f i g . 3) w i t h o u t l i t t l e d i f f e r e n c e between t h e he s t a i n and t h e p a p a n i c o l a o u s t a i n . a t l e a s t 90-99% of t h e l e u k o c y t e s i n t h e u r i n e of t h e s e c a t h e t e r i z e d p a t i e n t s were g r a n u l o c y t e s . t h e l i a l c e l l s were s t a i n e d p r o p e r l y by b o t h he and t h e p a p a n i c o l a o u s t a i n . t e r i a were o f t e n h a r d l y v i s i b l e i n p r e p a r a t i o n s s t a i n e d by he and o n l y s l i g h t l y e a s i e r t o s e e i n p r e p a r a t i o n s s t a i n e d by t h e p a p a n i c o l a o u s t a i n . epi bac o t h e r s t a i n i n g 71 p r o c e d u r e s w i t h m e t h y l g r e e n and e o s i n , he combined w i t h m e t h y l e n e b l u e and ac r i d i n e o r a n g e a t low ph d i d n o t improve t h e c y t o l o g i c a l p i c t u r e . t r e a t m e n t of t h e p a t i e n t w i t h mh, 2 g x 3 d a i l y , d i d n o t i n h i b i t l e u k o c y t e a c t i v i t y , a s r e f l e c t e d by m o r p h o l o g i c a l s i g n s of locomotion and p h a g o c y t o s i s . both c l i n i c a l and c y t o l o g i c a l e f f e c t s of mh t r e a t m e n t w e r e , however, ambiguous. i n some p a t i e n t s t h e u r i n e c l a r i f i e d , i n o t h e r p a t i e n t s i t d i d n o t . i n some pa t i e n t s , t h e p r e s e n c e of e p i t h e l i a l c e l l s w i t h o u t l e u k o c y t e s and b a c t e r i a sug g e s t e d a b e n e f i c i a l e f f e c t of mh t r e a t m e n t , i n o t h e r p a t i e n t s p y u r i a and bac t e r i u r i a p e r s i s t e d . i t w a s e v i d e n t t h a t a p o s s i b l e r e d u c t i o n i n p y u r i a , bac t e r i u r i a and o t h e r c o m p l i c a t i o n s of c a t h e t e r i z a t i o n had t o b e e v a l u a t e d by more q u a n t i t a t i v e methods amenable t o s t a t i s t i c a l t e s t s . discussion the u r i n e from p a t i e n t s w i t h i n d w e l l i n g c a t h e t e r s i s u s u a l l y h e a v i l y con t a m i n a t e d by b a c t e r i a w i t h s e c o n d a r y i n v a s i o n o f l e u k o c y t e s . the c o n t a m i n a t i o n o f t h e u r i n e cannot b e e v a l u a t e d by c o n v e n t i o n a l microscopy of t h e s e d i m e n t un l e s s m i c r o s c o p e , c e n t r i f u g e and m i c r o s c o p i s t are v i r t u a l l y a t t h e p a t i e n t ’ s bed s i d e . leukocytes d i s i n t e g r a t e ( c f . 2 , 5 , 1 0 ) and b a c t e r i a m u l t i p l y e v e r y 20 60 m i n u t e s . the c l o u d y u r i n e of c a t h e t e r i z e d p a t i e n t s due t o t h e p r e s e n c e of mucus, s a l t p r e c i p i t a t e s and a g g r e g a t e s of b a c t e r i a , l e u k o c y t e s and e p i t h e l i a l c e l l s w a s a m a j o r s o u r c e of v a r i a t i o n between d o u b l e p r e p a r a t i o n s from t h e same sam p l e . o t h e r p i t f a l l s were l o s s of c e l l s d u r i n g c e n t r i f u g a t i o n due t o a d h e r e n c e t o t h e w a l l s of t h e p l a s t i c chamber of t h e c e n t r i f u g e , l o s s of c e l l s i n t o t h e f i l t e r p a p e r d u r i n g c e n t r i f u g a t i o n , and l o s s of c e l l s from t h e s l i d e s d u r i n g t h e d r y i n g and s t a i n i n g . the l e u k o c y t e s p r e s e n t i n t h e glutaraldehyde-cytocentrifuge s e d i m e n t s of ca t h e t e r u r i n e c o n s i s t e d t o 90-99% of g r a n u l o c y t e s . ( f i g s . 1-3.) t h i s f i n d i n g i s i n agreement w i t h o b s e r v a t i o n s of p r e v i o u s a u t h o r s on p a t i e n t s w i t h bac t e r i u r i a ( 5 ) . the morphology of t h e b a c t e r i a w a s sometimes s u f f i c i e n t l y d i s t i n c t t o p e r m i t a t e n t a t i v e i d e n t i f i c a t i o n of b a c t e r i a l s p e c i e s ( f i g . 2 a ) . i n c o n v e n t i o n a l c o v e r s l i p p r e p a r a t i o n s o f u r i n e s e d i m e n t , v i t a l l e u k o c y t e s are sometimes s e e n h u n t i n g and p h a g o c y t i s i n g b a c t e r i a . s e e n i n a i r d r i e d p r e p a r a t i o n s of l e u k o c y t e s u s p e n s i o n s , a p p a r e n t l y due t o t h e s l o w n e s s of t h e method; t h e l e u k o c y t e s s t o p h u n t i n g and t a k e up a s t a t i o n a r y s p h e r i c a l form d u r i n g p r e p a r a t i o n . the g l u t a r a l d e h y d e f i x a t i o n was r a p i d and e f f e c t i v e , as e v i d e n c e d by t h e f i n t h i s phenomen i s n o t d i n g of l e u k o c y t e s w i t h t h e e l o n g a t e d s h a p e of moving c e l l s and l e u k o c y t e s i n d i f f e r e n t s t a g e s of d i s i n t e g r a t i o n . the l e u k o c y t e s were f i x e d i n t h e rounded s h a p e of suspended c e l l s , n o t i n t h e 72 f l a t t e n e d s h a p e of smeared and a i r d r i e d c e l l s . t h u s became l o c a t e d i n d i f f e r e n t o p t i c a l p l a n e s on t h e s l i d e d u e t o t h e r e s t r i c t e d d e p t h of t h e v i s u a l f i e l d ( 0 . 2 0 . 4 ~ m ) . of b a c t e r i a more d i f f i c u l t . the l e u k o c y t e s and t h e b a c t e r i a t h i s f a c t r e n d e r e d t h e a s s e s s m e n t the glutaraldehyde-cytocentrifuge method had some o b v i o u s a d v a n t a g e s o v e r t h e c o n v e n t i o n a l c o v e r s l i p p r e p a r a t i o n of u r i n e s e d i m e n t and t h e c o n t r a s t i n g p r o c e d u r e s d e s c r i b e d by p r e v i o u s a u t h o r s (1-10). the c y t o l o g i c a l and b a c t e r i o l o g i c a l p i c t u r e of t h e u r i n e w a s f r o z e n i n t h e s t a t e e x i s t i n g a t t h e moment of s a m p l i n g . cells and b a c t e r i a on t h e s l i d e were l i n e a r l y c o r r e l a t e d t o t h e c e l l u l a r i t y of t h e o r i g i n a l sample and were p r o p e r l y c o n t r a s t e d by r o u t i n e c y t o l o g i c a l s t a i n s . the p r e p a r a t i o n of t h e u r i n e d u r i n g i t s t r a n s f e r from t h e c a t h e t e r t o micro s c o p e w a s r e l a t i v e l y s i m p l e and r a p i d . i n s p i t e o f t h e p i t f a l l s a s s o c i a t e d w i t h s a m p l i n g , p r e p a r a t i o n and a s s e s s m e n t of t h e i n f l a m m a t o r y c y t o l o g y of t h e u r i n e from p a t i e n t s w i t h i n d w e l l i n g c a t h e t e r s , t h e glutaraldehyde-cytocentrifuge s e d i m e n t p r o v i d e d a rough q u a n t i t a t i v e measure of u r i n e c o n t a m i n a t i o n . b a c t e r i a were b e s t v i s u a l i z e d by t h e mgg s t a i n , l e u k o c y t e s by he o r by t h e p a p a n i c o l a o u s t a i n . acknowledgements no * 1. 2. 3 . 4 . 5. 6 . 7 . 8 . 9. 1 0 . t h i s work w a s s u p p o r t e d by t h e swedish medical r e s e a r c h c o u n c i l p r o j e c t 2296 and no. 5 3 6 2 and k r s n t s frorii t h e h e d i c a l f a c i l l t y of lund. references b r a d l e y , j . m . & l i t t l e , m . b . : q u a n t i t a t i v e u r i n e c u l t u r e s . b r i t med j 11:361-363, 1963. g a d e h o l t , h . : p e r s i s t e n c e o f b l o o d c e l l s i n u r i n e . a c t a med scand 183:49 54, 1968. h e l g a s o n , s . & l i n d q v i s t , b.: e o s i n o p h i l u r i a . scand j u r o l nephrol 6:257 259, 1972. k r o n v a l l , g . & myhre, e . : d i f f e r e n t i a l s t a i n i n g of b a c t e r i a i n c l i n i c a l specimens u s i n g a c r i d i n e o r a n g e b u f f e r e d a t low ph. acta p a t h m i c r o b i o l scand s e c t b 85:249-254, 1977. l i n d q v i s t , b. & wahlin, a . : d i f f e r e n t i a l c o u n t of u r i n a r y l e u c o c y t e s and r e n a l e p i t h e l i a l c e l l s by p h a s e c o n t r a s t microscopy. acta med scand 198:505-509, 1975. l i t t l e , p . j . : u r i n a r y w h i t e c e l l e x c r e t i o n . l a n c e t i:1149-1151, 1962. l i t t l e , p . j . : t h e w h i t e c e l l e x c r e t i o n r a t e . b r j u r o l 36:360-363, 1964. p r e s c o t t , l.f. & b r o d i e , d . e . : a s i m p l e d i f f e r e n t i a l s t a i n f o r u r i n a r y s e d i m e n t . l a n c e t i i : 9 4 0 , 1964. s t e r n h e i m e r , r . & malbin, b.: c l i n i c a l r e c o g n i t i o n of p y e l o n e p h r i t i s , w i t h a new s t a i n f o r u r i n a r y s e d i m e n t s . am j med 11:312-323, 1951. wahlin, a . : d i f f e r e n t i a l c o u n t of u r i n a r y l e u k o c y t e s and r e n a l e p i t h e l i a l c e l l s . a comparison between p h a s e c o n t r a s t m i c r o s c o p y of u n s t a i n e d s e d i ments and l i g h t microscopy of f i x e d and s t a i n e d s p e c i m e n s . uppsala j med s c i 82:43-47, 1977. a comparison of t h e u r i n a r y w h i t e c e l l c o n c e n t r a t i o n w i t h 73 received july 1978. accepted october 1978. address for reprints: bo norberg, m . d . department of internal medicine university hospital of lund s-221 85 lund, sweden 74 upsala j med sci 79: 106-108, 1974 improved polymeric contrast agents for roentgenologic examination of the gastrointestinal tract ii. preliminary report on animal investigations lars b j o r k , u n o e r i k s o n , j o h n falk, bjorn i n g e l m a n a n d g e r t l i n d b l a d from t h e departments of diagnostic radiology a n d clinical c h e m i s t r y , university h o s p i t a l , u p p s a l a , a n d pharmacia a b , u p p s u l a , s w e d e n abstract a new improved water-soluble polymeric contrast agent has been studied in animal experiments. on oral and intraperitoneal administrations in mice, this polymer was found to have a very low toxicity. the contrast agent was used in connection with the roentgenologic examination of the gastrointestinal tract of dogs with good results. further improvements are possible. introduction the polymeric contrast agents described in our earlier publication in investigative radiology were found t o have a very low toxicity in mice after oral and intraperitoneal administration.' the im proved polymeric contrast agents described in the preceding part i of the present report also seem to have a very low toxicity and in addition possess favourable properties for roentgenologic examina tion of the gastrointestinal tract. in this report, some preliminary studies of toxicity i n mice as well as some preliminary roentgenologic studies in dogs are described. studies of toxicity in mice a brief report is given below on som experiments on mice with the polymeric substance 730e de scribed in part i. (the weight average molecular weight h?, was about 5 000.) for the studies a neutral aqueous solution of the sodium salt of the substance 730e was used. the solution contained 50 gram of substance per 100 ml of solution corresponding to an iodine content of about 180 mg per ml of solution. the solution was sterilized by autoclaving at ii0"c for 30 minutes. in these preliminary experiments very large doses were given orally and intraperitoneally i n order to gain a general impression of the doses that should be used in future more detailed toxi city studies. ten mice were given a single oral dose of the solution, corresponding to 8 g of the substance per kg body weight. 5 mice were sacrificed after some hours and the other 5 mice were sacrificed after a week. none of the mice showed any toxic symptoms during the observation periods. at autopsy, no change were observed macroscopically. histologic examinations performed on kidneys, liver, stomach and different parts of the intestines revealed n o changes. ten mice were given a single intraperitoneal dose of the solution, corresponding to 8 g of the sub stance per kg body weight. 5 mice were sacrificed after some hours and the other 5 mice were sacrificed after a week. none of the mice showed any toxic symptoms during the observations periods at autopsy, no changes were observed macroscopi cally. histologic examinations performed on kidneys, liver, stomach and different parts of the intestines revealed no changes. six mice were given an oral dose of the solution daily for 5 days, corresponding to 8 g of the substance per kg body weight per day. 3 mice were sacrificed after some hours and the other 3 mice were sacrificed after a week. none of the mice showed any toxic symptoms during the observation periods. at autopsy, no changes were observed macroscopically. histologic examinations performed on kidneys, liver, stomach and different parts of the intestines revealed no changes. six mice were given an intraperitoneal dose of upsala j med sci 79 improved polymeric contrust agents. i 1 107 vestigation. in the morning, 40 ml of the contrast solution w a s given orally to each dog through a gastric tube. roentgenograms of the stomach and intestine were taken at 15-30 minute intervals. one week later t h e same dogs w e r e given 40 ml of gastrografin (methyl-glucamine diatrizoate + sodium diatrizoate; schering ag, berlin), which had been diluted with distilled water t o the same iodine content as the solution of 7 3 0 e , i.e. to i80 mg/ml. t h e s a m e roentgenologic procedure was followed a s with 730 e. with 730 e good visualization of t h e stomach and the intestines was obtained. n o precipitation of the contrast medium was observed. t h e contrast medium was observed to mix well with t h e con tents of the gastrointestinal tract. t h e contrast medium also coated the surface of the mucosa of the intestinal tract and a good double-contrast effect was observed in gas-filled parts of the intestines. t h e contrast filling of colon was good in all 3 dogs. fig. 1. shows a roentgenogram from a dog 1 hour after oral administration of 730 e. good contrast filling of the stomach and the intestines was generally obtained with gastro grafin. however, in 2 of t h e dogs a considerable flocculation of t h e contrast medium was observed in the stomach and later precipitated contrast medium was seen as granular matter in t h e small intestine. t h e contrast filling of colon was some what poorer with gastrografin than with 7 3 0 e , particularly in t h e two cases where flocculation of gastrografin occurred. fig. 2 shows a roentgeno gram from a dog 2 hours after oral administration of gastrografin. there were no marked differences in the passage time through the small intestine between t h e two contrast media. n o excretion of contrast medium in t h e renal pelvis o r in the urinary bladder was observed roentgenologically on a n y occasion with either contrast media in these 3 dogs. n o clinically mani fest side effects were observed in any dog in these experiments. f i g . 1 . roentgenogram from a d o g 1 h o u r after o r a l administration of 730 e. the solution daily for 5 days, corresponding to 8 g of the substance per kg body weight per day. 3 mice were sacrificed after some hours and the other 3 mice were sacrificed after a week. n o n e of the mice showed any toxic symptoms during the observation periods. at autopsy, n o changes were observed macroscopically. histologic exami nations performed on kidneys, liver, stomach and different parts of t h e intestines revealed n o changes. r o e n t g e n o l o g i c s t u d i e s i n dogs t h e solution of substance 7 3 0 e which was used for studies of toxicity in mice was also used for roentgenologic studies of the gastrointestinal tract in dogs. t h u s , the solution contained 50 g sub stance per 100 ml solution, corresponding t o an iodine content of about 180 mg per ml. three dogs (beagles) weighing from 7-11 kg were fasted from the evening prior to the in discussion the polymeric contrast agent used in the animal studies reported here has very high solubility in water even a t low ph values. this contrast agent is not precipitated in acid gastric juices, whereas water-soluble iodine-containing contrast media in upsala j med sci 79 108 l . b j o r k et al. references 1. bjork, l., erikson, u . & ingelman, b.: polymeric contrast media for roentgenologic examinfitions of gastrointestinal tract. investigative radiology 5: 142, 1970. received december 22. 1973 address for reprints: u. erikson, m.d. department of diagnostic radiology university hospital s-750 14 uppsala sweden fig. 2. roentgenogram from a dog 2 hours after oral administration of gastrografin. current use are precipitated at low ph values and therefore can flocculate and precipitate in the stomach. (for example, diatrizoate preparations precipitate at about ph 3 . ) as this new contrast agent is a polymeric substance, it also has more favourable physical-chemical properties than those of the water-soluble contrast agents of com paratively low molecular weight used at present for this purpose. the preliminary toxicity studies as well as the preliminary roentgenologic studies in animals have shown that further investigations of this type of polymeric contrast agents are justified. the polymers can be further improved. upsala j med sci 79 upsala j med sci 82: 55-59, 1977 corticosteroid therapy in regional small bowel lschaemia an experimental study in rats l. rentzhog and s. wikstrom from the department of surgery, university hospital, uppsala sweden abstract corticosteroid therapy in pharmacological doses has a well documented positive effect in shock caused by severe in testinal ischaemia. i n this study the effect of high doses of corticosteroids on the exchange circulation of the mucosa was analysed in varied, regional small howel ischaemia. thirty minutes after establishment of moderate ischaemia the exchange circulation in the mucosa of the ischaemic intestinal segment in animals treated with 100 mg/kg hydrocortisone showed no improvement compared with un treated animals. higher corticosteroid doses impaired the exchange circulation. on analysis 7 days after establishment of the ischaemia, treatment with 100 mg/kg hydrocortisone during the first 3 days was found to have impaired the exchange circulation. the same treatment in rats with more severe intestinal ischaemia gave a greatly increased mortali ty. possible reasons for the impaired mucosal circulation following corticosteroid therapy in pharmaeo-logical doses in regional small bowel ischaemia are discussed. one possi bility is that corticosteroids induce a “steal syndrome” due to the better vasodilative effect on healthy than on ischae mic intestinal tissue. introduction in most experimental studies concerning ischaemia of the small intestine the ischaemia has been pro duced by central, intermittent interruptions of the blood flow. the significance of bacterial endotoxin in this connection has been pointed out by several authors (13, 19). selkurt et al. (16) discussed the importance of vasoactive substances in intestinal ischaemia. kobold & thal (8) identified a vasoac tive peptide which was liberated in different types of experimental and clinical ischaemia of the small intestine. other authors (2, 6, 7) have shown that lysosomal enzymes are released in association with ischaemia in the splanchnic region, and this may well lead to a serious impairment of myocardial function (10, 18). it seems likely that abnormal bacteria-dependent endotoxin production, ischaemic destruction of lysosomes, release of vascoactive substances and activation of vasoconstrictive reflexes can also be induced in connection with regional small bowel ischaemia. these local factors can affect the collat eral circulation and can also probably cause a re lease of a myocardial depressant factor (10). lil lehei et al. (1 1 ) pointed out that the ability of the intestine to survive a regional ischaemia is highly dependent upon the collateral flow from adjacent intestinal segments. norlcn, rentzhog & wikstrom (14) have demon strated that antibacterial and antithrombotic therapy improve t h e exchange circulation and chances of survival of the mucosa in regional small bowel ischaemia. corticosteroids in gram doses increase the survi val in intestinal ischaemic shock (1, 10). the effect of cortisone in pharmacological doses in severe in testinal ischaemia has been discussed. altura & altura ( 1 ) claim that in these doses corticosteroids unspecifically inhibit the effect of vasoconstriction that may be released in intestinal ischaemia. lil lehei et al. (12) assumed that corticosteroids act as alpha receptor blocking agents but provided no de finite evidence for this assumption. bruns & con nolly (3), on the other hand, considered that the effect was a potentiation of the vasoconstrictive action of the catecholamines. weissman & thomas (17) and glenn & lefer (6) were of the opinion that the main effect of corticosteroids was t o prevent lysosomal destruction and thereby the release of de leterious lysosomal enzymes. the effect of corticosteroids in gram doses in re gional small bowel ischaemia does not appear to have been studied previously. we therefore consid upsaln j m e d sci 82 56 l . rentzhog and s. wikstrom ered it of great interest t o examine t h e influence of nal ischaemia-two comprising 1 1 mes end arc and two i5 pharmacological doses of corticosteroids o n the ex change circulation of the mucosa in standardized, regional small bowel ischaemia both immediately and o n e week after establishment of t h e ischaemia. m a t e r i a l a n d m e t h o d s male sprague-dawley rats weighing 200-300 g were used. the animals were strictly standardized as regards iodine metabolism and were given iodine in their drinking water both before and during the experimental period. standardized small bowel ischaemia was produced by ligation of a defined number of mesenteric end arcades (mes end arc). the ligature material was 5 : o cardiovascu lar silk (ethicon). the first ligature was applied on the 6th mes end arc counted from the ileocaecal angle, and a further 1 1 or 15 mes end arc, in the proximal direction, were then ligated. at the same time a central loop in the devascularized intestinal segment was marked with silk threads. these threads were placed around the intestine without affecting the terminal vessels. the length of the loop thus marked always corresponded to the extent of 2 mes end arc. the mucosal circulation was assessed by a technique described by nylander & wikstrom (15), based on the fact that the passive absorption, i.e. the diffusion of a given substance from an intestinal loop of defined size, is an expression of the effective exchange circulation in the mucosa of the intestinal segment. a radioactive iodine isotope ( n a p ) was used as the test substances. at the time of analysis the previously marked intestinal loop (2 mes end arc) was ligated and the test dose was deposited into its lumen b y transmural injection. the pylorus was tied off to prevent gastric contents from passing into the small intestine. after 30 min the animal was killed with ether. the abdomen was opened and the stomach was ligated a t the cardia and resected. the isolated intestinal loop into which the test substance had been deposited was also resected. the radioactivity in the stomach, the in testinal loop and the whole body (thus excluding the stomach and the test loop) was recorded. two series of experiments were performed. in all ani mals a catheter was inserted into the vena cava viajugular vein by technique described by engberg (4), and left in sitii. in series i (table i) small bowel ischaemia comprising 1 i mes end arc was produced in four groups of animals. in two groups an intravenous injection of hydrocortisone (soh-cortep, upjohn) in a dose of 100 mg and 300 mg, respectively, per kg body weight, dissolved in 0.2 ml physiological saline was given immediately before the ischaemia was established. the third group was simultaneously given methylprednisolone (medrone@, up john), 30 mg per kg dissolved in 0.2 ml physiological saline. the fourth group, as well as a laparotomy control group (no ischemia) was given 0.2 ml physiological saline alone. the exchange circulation in the ischaemic intestinal segment was analysed 30 min after establishment of the ischaemia. series i1 (table 11) consisted of four groups with intesti upsala j med sci 82 mes end arc. in all groups the exchange circulation was analysed 7 days after establishment of the ischaemia. two of the groups (one 1 1 and one 15 mes end arc) were treated with hydrocortisone. the first dose (100 mg/kg) was given immediately before the ischaemia was produced, and the next dose (100 mg/kg) 2 h later. a further two doses of 50 mg/kg were given during the rest of the first 24 hours. in the following 2 days four doses of 50 mg/kg were given per day. the other two groups-ischaemic controls-were given physiological saline of the same volume as the hydrocortisone doses in the treated groups. in series i1 a morphological evaluation of the ischaemic intestinal segment was made (table 11). the material was divided into three different types according to the macroscopical appearance of the small intestine, as fol lows: t y p e 1 . the intestinal wall appeared intact, apart from moderate thickening. t y p e 2 . varying length of the intestinal wall were con siderably thickened. the width of the lumen was normal both above and below the thickened area of the intestinal wall. t y p e 3. the intestinal wall was thickened as in type 2, but proximal to the thickened area the lumen was dilated to the extent of an ileus state. the results of the exchange circulation analyses in the ischaemic segment have been recorded only for type 1 in the respective experimental groups. r e s u l t s i n series i , in which the exchange circulation of the intestinal mucosa was analysed 30 min after laparotomy or establishment of regional small bowel ischaemia ( 1 1 mes end arc), all animals sur vived. in table i it is seen that in the laparotomy control group 26% of the radioactive dose remained in the isolated intestinal loop and a t the end of the analytical period. in the untreated ischaemic group the absorption from the isolated loop was consider ably impaired 45% of the radioactive dose remain ing in t h e loop. treatment with 100 mg/kg hydro cortisone did not improve the exchange circula tion significantly in comparison with the untreated ischaemic group; in this case 48% of the dose re mained in the loop. following treatment with the higher dose of hydrocortisone (300 mg/kg) and with methylprednisolone (30 mg/kg) the absorption of the radioactive iodine was reduced, 67% and 66% of the dose, respectively, remaining in the loop a t the end of the analytical period. half of the animals treated with the higher dose of hydrocortisone (300 mg/kg) also received 2 ml physiological saline in a continuous intravenous infusion during the experi mental period, but these animals showed no differ corticosteroid therapy in bowel ischaernia 57 table i. series z. characteristics of the material and percentage distribution of the radioactive dose 30 rnin after its deposition in the isolated loop ( m e a n values with s . e . ) body percentage of dose i n weight exp. group n fg) isol. loop stomach body lc 20 23 1 k 3 . 8 26.1 k 2 . 2 12.0k0.8 61.9k1.9 11 mes end arc, untreated 18 242 2 6.8 45.2k 3.2 6 . 7 k 0 . 8 48.1 k 2 . 7 11 mes end arc, hydrocortisone treated (100 mg/kg) 20 2 5 2 k l . l 49.1 k 4 . 0 10.1f 1.3 40.8k3.1 11 mes end arc, hydrocortisone treated (300 mg/kg) 20 240 2 5.2 66.8 k 3 . 2 2.2k0.3 31.0k3.2 11 mes end arc, methylpredni solone treated 10 270f 1 .o 65.8 k5.1 1.9k0.4 3 2 . 3 f 4 . 8 ence in absorption of the test dose from the animals given hydrocortisone alone (65% and 68%, respec tively, of the test dose remaining in the loop). all animals that were given the higher hydrocortisone dose (300 mg/kg) are therefore placed in one group in table i. in series i1 the exchange circulation of the small bowel mucosa was analysed 7 days after establish ment of the ischaemia. in table i1 it is seen that in the untreated ischaemic group comprising 1 1 mes end arc 3 of 21 animals died. morphologically, one of these 3 animals was assigned t o type 2 . the other 2 animals that died were of type 3 , exhibiting acute perforation and peritonitis. of the surviving ani mals in this group 2 were assigned t o type 3 , with severe ileus, and one of them also had perforation. the other 16 animals in this group were assigned to type 1. in the group with ischaemia comprising 11 mes end arc and treated with hydrocortisone, one of 18 animals died. this animal was of type 3. one of the surviving animals of this group was also as signed to type 3 , with perforation. it is also seen in table i1 that in the hydrocortisone-treated ischaemic group the exchange circulation in the ischaemic intestinal segment was sinificantly im paired compared with the untreated group. thus 50% of the radioactive dose remained in the iso lated loop in the former group, and 3 5 % in the untreated group. among the animals with severe bowel ischaemia (15 mes end arc) (table ii), only one could be assigned to type 1 . the others were of type 3, and four of these died. hydrocortisone treatment com bined with this severe degree of ischaemia resulted in death in all cases. morphologically all animals in this group were assigned t o type 3. discussion nylander & wikstrom (15) showed that the ex change circulation of the mucosa was greatly im paired immediately following establishment of small table 11. series zz. characteristics of the material, morphological types and percentage distribution of the radioactive dose 30 min after its deposition in the isolated loop ( m e a n values with s . e . ) morphological types body weight (g) percentage of dose in mor exp. group n tality i i1 i11 initial final isol. loop stomach body 11 mes end arc, untreated 21 3 16 1 4 284k7.6 285k9.3 34.9k4.2 4 . 7 k 0 . 3 60.4f4.1 11 mes end arc, hydrocortisone treated 18 1 16 2 268k1.1 2 4 3 f 4 . 5 4 9 . 8 f 4 . 5 4 . 1 k 0 . 6 46.1k5.5 15 mes end arc, untreated 12 4 1 11 234k3.4 15 mes end arc, hydrocortisone treated 12 12 12 270f2.9 upsulu j m r d sci 82 58 l . rentzhog and s. wikstrom bowel ischaemia. this finding was confirmed in t h e present investigation. a release of lysosomal en zymes and of vasoactive substances, as well as the influence of bacterial endotoxins, may all presuma bly influence the collateral circulation and thereby the exchange of the mucosa in the ischaemic in testinal segment. theoretically it would therefore seem probable that hydrocortisone would improve the exchange circulation, but no effect of this corti costeroid in a dose of 100 mg/kg was found in the acute experiments. altura & altura (1) reported, however, from acute experiments in the rat, that the survival in intestinal ischaemic shock was only im proved at a dose of 300 mglkg hydrocortisone or 30 mglkg methylprednisolone. in our experiments this high dose of hydrocortisone and of methylpredni solone impaired the exchange circulation in the ischaemic intestinal segment, indicating that these steroid doses have a negative effect on the collateral circulation. one possible reason for this surprising effect of high glucocorticoid dose may be a negative in fluence on the central circulation. in a pre-experi mental study, however, no such influence was ob served. in rats with catheters inserted into the fe moral artery for continuous pressure recording, a transient reduction of the systemic blood pressure was noted immediately after administration of the glucocorticoid, followed by normalization or a slight pressure increase. another possibility is that a strong general vaso dilative effect of high doses of glucocorticoids might unmask a hypovolaemia caused by loss of fluid through oedema, for instance, in the ischaemic tissue in these animals. infusion of physiological saline after administration of the corticosteroid, however, did not affect the results. a further conceivable explanation is that corticosteroids impair the exchange circulation of the mucosa by inducing a “steal syndrome” due to more effective vasodilatation in healthy than in ischaemic intestinal tissue. it is possible that metabolic factors in ischaemic tissue and any re lease of certain vasoactive substances may counteract a favourable effect of the corticosteroids on the circulation in an ischaemic intestinal seg ment. the net effect may then be an impaired circu lation in the ischaemic segment. folkow ( 5 ) has demonstrated that the circulation of the intestinal wall is built up of several parallel vascular systems. our method measure3 indirectly l / l , c o / ( i j m r d s c i x 2 the circulation in the mucosal vascular system. it is possible that high corticosteroid doses do not im pair the total circulation of the ischaemic intestine despite a reduction of the exchange circulation of the mucosa. a redistribution of blood to deeper layers of the intestinal wall is conceivable, especial ly to the vascular system in the muscular layer. the presence of an adequate circulation during the first days after the development of intestinal ischaemia is of decisive importance for survival and restoration of the intestine. during this initial ischaemic period the available intestinal circulation would seem to be most susceptible to a negative influence of a high sympathetic tone and local re lease of vasoactive substances, endotoxins and lysoenzymes. high corticosteroid doses during the first 2-3 days might have a positive effect on the local damage and improve the chance of survival. in our second series of experiments, however, there was no definite positive effect of intensive cor ticosteroid therapy during the first three days following establishment of the ischaemia. in rats with severe ischaemia (15 mes end arc) the high dose of corticosteroid had a distinctly negative ef fect on the survival. in untreated animals with ischaemia comprising 11 mes end arc the exchange circulation was consid erably better one week after than immediately after establishment of the ischaemia. this is in full agreement with previous results of nylander & wikstrom (15). on the other hand, treatment with a corticosteroid in gram doses during the first 3 days after ischaemia had been produced resulted in no improvement of the mucosal circulation 7 days la ter. a probable explanation, as in the acute experi ments, is that gram doses of corticosteroids impair the collateral circulation in regional ischaemia due to a “steal syndrome”. another possibility is that corticosteroid therapy favours bacterial invasion in this ischaemic intestinal wall, with consequent oedema and fibrosis, leading to an impairment of the mucosal circulation. acknowledgement this investigation has been supported by grants from the swcdish medical research council (b76-17x-2809-06). excellent technical assistance was provided by mrs inger fogelberg. corticosteroid therapy in bowel ischaemia 59 references 1 . altura, b. m. & altura, b. t.: peripheral vascular actions of glucocorticoids and their relationship to protection in circulatory shock. j pharmacol exp ther 190: 300, 1974. 2. bitensky, l., chayen, j., cunningham, j. g . & fine, j . : behaviour of lysosomes in hemorrhagic shock. nature, london, 199: 493, 1963. 3 . bruns, d . l. & connolly, j . e.: comparative study of the effectiveness of adrenal cortical compounds in hemorrhagic shock. surg forum 10: 382, 1960. 4. engberg, a , : a technique for repeated renal clearance measurements in undisturbed rats. acta physiol scand 75: 170, 1969. 5 . folkow, b.: role of nervous system in the control of muscular tone. circulation 21: 760, 1960. 6. glenn, t . m . & lefer, a . m.: protective effect of thoracic lymph diversion in hemorrhagic shock. amer j physiol219: 1305, 1970. 7. janoff, a , , weissmann, g . , zweifach, b. & thomas, l.: pathogenesis of experimental chock: 1v. studies on lysosomes in normal and tolerant animals sub jected to lethal trauma and endotoxemia. j exp med 116:451, 1962. 8. kobold, e . e. & thal, a. p.: quantitation and identification of vasoactive substances liberated dur ing various types of experimental and clinical intesti nal ischemia. surg gyn obst 117: 315, 1963. 9. lefer, a . m.: role of myocardial depressant factor in the pathogenesis of circulatory shock. fed proc 29: 1836, 1970. 10. lefer, a. m. & vernier, r. l.: role of corticosteroids in the treatment of circulatory collapse states. clin pharm ther 11:630, 1970. 1 1 . lillehei, r. c., goott, b. & miller, f. a,: the physiological response of the small bowel of the dog to ischemia including prolonged in vitro preservation of the bowel with successful replacement and survi val. ann surg 150: 543, 1959. 12. lillehei, r. c., longerbeam, j . k . , block, j. h. & manax, w. g . : the modern treatment of shock based on physiological principles. clin pharm ther 5: 63, 1964. 1 3 . milliken, j . , nahor, a . & fine, j.: study of the factors involved in the development of peripheral vascular collapse following release of the occluded superior mesenteric artery. brit j surg52:699, 1965. 14. norlen, k . , rentzhog, l. wikstrom, s . : experimen tal ischemia of the small intestine. effect of antibotic and antithrombotic drugs on the mucosal exchange circulation. acta chir scand 141: 780, 1975. 15. nylander, g . & wikstrom, s.: propulsive gastrointestinal motility in regional and graded ischemia of the small bowel. an experimental study in the rat. i. immediate results. acata chir scand, sup pl. 385, 1968. 16. selkurt, e. e., scibetta, m. p. & cull, t. e.: hemodynamies of intestinal circulation. circ res 6:92, 1958. 17. weissman, g . & thomas, l.: studies on lysosomes: i. effects of endotoxin tolerance and cortisone on the release of acid hydrolases from a granular fraction of rabbit liver. j exp med 116:433, 1962. 18. williams, l . f., jr., goldberg, a. h., polansky, b. j. & byrne, j . j . : myocardial effects of acute intestinal ischemia. surgery 66: 138, 1969. 19. witznitzer, t. & rozin, r.: bacterial factor in shock following superior mesenteric artery occlusion. israel med j 22: 4 , 1963. received july 20, 1976 address for reprints: stig wikstrom, m.d. department of surgery university hospital s-750 14 uppsala sweden upsulu j med sci 82 upsala j med sci 83: 135-139, 1978 effect of the endocrine state of blastocyst donors on the time required for initiation of tropholalast outgrowth grels n r s l u n d and orjan lundkvist from the reproduction research u n i t , biomedicul centre, uppsala, sweden abstract blastocysts from mice in a state of delayed implantation af ter ovariectomy were recovered on day 5 , 7 or 9 (day 1 was the day a vaginal plug was found). blastocysts were also re covered on day 7 from animals that had received an injection of oestradioll7p 8, 16 or 24 hours earlier. the blastocysts were incubated in a modified brinster’s medium to which serum had been added and the time of initiation of blasto cyst outgrowth was recorded. blastcysts from early delay similar to those occurring on blastocyst activation during implantation. since the endocrine state of the host animal determines the activity of the blastocysts in vivo, it is also possible that the same state of the blastocyst donors determines the activ ity of blastocysts in vitro. knowledge of this matter is important for culture experiments. where the level of blastocyst activity is crucial. the aim of this study was therefore to test grew out before those from late delay. a steady state of outgrowth time was achieved on day 7. it is therefore sug gested that blastocysts for culture experiments in which the metabolic activity level is crucial should not be recovered before the steady state is attained. oestrogen injections caused earlier outgrowths, at least after 16 h, which indi cates that the earlier outgrowth soon after ovariectomy might also he caused by a residual effect of the oestrogen. whether the interval between the start of the culture and the beginning of outgrowth of the blastocysts is to the endocrine state of the blastocyst donors. mouse blastocysts were recovered at dif ferent times both during experimental delay of im plantation and during oestrogen-induced activation for implantation. a state of low metabolic activity (8, 1 1 , 19), and given subcutaneously daily, the day of flushing excluded show a characteristic ultrastructure ( 1 , 13). the depression of the blastocyst activity during delay probably depends both on the exogenous adminis tration of progesterone (9) and on the declining in fluence of ovarian oestrogen, since if oestrogen is given, the blastocysts will become activated and start to implant (7, 20). mouse blastocysts in vitro will grow out on the bottom of the culture dish within a few days. this ( 1 ) . blastocysts were recovered on day 5 , 7 or 9 during delay of implantation and on day 7-8, 16 or 24 h after a s. c. injection of 0.1 pg oestradiol-17p (ae leo, helsingborg, sweden) dissolved in propylene glycol ( 1 pglml). the uteri were flushed with culture medium, that had previ ously been equilibrated with 5 % co, in air overnight. during the flushing procedure the medium was collected in petri dishes containing liquid paraffin in order to main tain the p h during the subsequent search for blastocysts. brinster’s medium for ovum culture was used (2), in which process has been regarded as an analogue ofimplan ultrastmctural (naeslund, g. 8~ nilsson, 0.: to be published) and metabolic (9, 15, 16, 18) changes the lactate had been replaced with 1.0 mg/ml of glucose, which is appropriate from the &cell stage on in the mouse centration of 1 % was added to permit outgrowths (5). tation (6) and has been found to be associated with (3). fetal calf serum (flow, ayrshire, scotland) in a con immediately after flushing the blastocysts were trans unsalu j m r d sc.i 83 136 g . n z s h n d and 0. lundkvist ferred to 0.1-ml droplets of the medium under liquid paraf fin by means of glass capillaries connected to an agla micrometer syringe (burroughs wellcome & co, london, england). the droplets were made the day before the culture started and were kept in plastic petri dishes (falcon, oxnard, california, usa) at 37°c in an atmosphere of 5 % cop in air. four to five blastocysts were placed in each droplet. observations of the blastocysts were made with an in verted microscope (biovert, reichert, austria), bright ground microscopy being used for screening purposes and phase contrast (magnification x160) to detect the first signs of outgrowth (fig. ! and 2 ) . the initiation of the outgrowth was defined as the moment when growing cells were first seen outside the blastocyst contour. the ob servations were made every 8 h and always by the same person. the first observation of an outgrowth was re corded as occumng in the middle of the previous 8-hour interval. analysis of variance was used to detect signifi cant differences between the experimental groups. fig. 1 . blastocysts in culture drop lets 24 h after explantation; day-7 group. the blastocysts are expanded without signs of trophoblast outgrowth. bright field. x250. results the frequency distributions for the moment of initi ation of blastocyst outgrowth are summarized in fig. 3. the means for the groups, and the number of animals, blastocysts, and droplets are given in table 1. the means for every droplet were used as observations in the subsequent tests of significance (table 11). the duration of the delay influenced the moment of initiation of outgrowth, as the blastocysts from day 5 started to grow out 13 h earlier, on the, aver age, than the blastocysts from day 7, a statistically significant difference, whereas the difference be tween day-7 blastocysts and those from day 9 was non-significant. of the blastocysts from day 5 , those encased in zona pellucida grew out earlier f i g . 2 . outgrowing trophoblast cells around two blastocysts 64 h after explantation; day-7 group. phase contras. ~ 2 5 0 . upsalu j med sci 83 effect of blastocyst donors on trophoblast outgrowth 137 table i . time required f o r initiation of outgrowth in vitro of blastocysts f r o m donors in various endocrine states hours after initiation no. of of culture no. of blastono. of group ( m + s .d.) animals cysts droplets delay of implantation day 5 day 7 day 9 4 4 f 9 19 33 8 57+ 10 40 97 21 61f 7 40 i18 24 oestrogen activation oestrogen 8 h 6 0 f 4 6 32 6 oestrogen 24 h 9 f 4 8 29 6 oestrogen 16 h 4 7 f 8 14 22 5 (mean 39 h) than the zona-free ones (mean 47 h). no tests of significance were performed, because of the small number of zona-encased blastocysts. oestrogen injected into the donor mice had a d a y 5 1 0 7 2 96 l2oh 27 33 3 2 day 7 0 2 4 48 * 96 120 h 6 2 7 32 34 0 24 4 8 * 7 2 9’6 120 h d a y 9 d a y 9 2 7 32 34 2 9’6 120 h 1 oestr 8 h , i 0 2 4 4 8 * 7 2 9 6 120 h oestr 16 h &* 4 9 6 120 h 0 2 4 7 2 oestr 2 4 h 4 8 72 96 120 h fig. 3. histograms representing the frequency distnbu tions for the appearance of the first outgrowth. the figures within or above rectangles represent the number of blastocysts that started to grow out during each 8-hour interval. arrows indicate means. day 5: blastocysts reco vered on day 5 during delay of implantation.-day 7: blastocysts recovered on day 7 during delay of implanta tion. day 9: blastocysts recovered on day 9 during delay of implantation. oestrogen 8 h: blastocysts recovered on day 7, 8 h after an oestrogen injection. oestrogen 16 h: blastocysts recovered on day 7, 16 h after an ostrogen in jection. oestrogen 24 h: blastocysts recovered o n day 7 , 24 h after an oestrogen injection. marked effect on the blastocysts in vitro compared with those from day 7 of delay. no difference in the appearance of outgrowths was seen 8 h after the injection, whereas after 16 h there was a statistically significant difference. the effect was considerable 24 h after the injection, as the blastocysts grew out on the average 48 h earlier than the day-7 blasto cysts from animals in a state of delay. discussion blastocyst activity has often been determined by measuring specific metabolic steps, such as the in corporation of rna precursors (4, 12, 18) and of amino acids (16, 19) and the production of carbon dioxide (9, 14, 15). in the present experiment a new method was tried-determination of the time re quired for initiation of trophoblastic outgrowth in vitro. this method supplements others by taking into account the proliferating capacity of tropho blast cells. the technique is simple and easy to handle, which is an advantage when several ex perimental groups are to be tested. the moment of initiation of blastocyst outgrowth was estimated by microscopic observations of the blastocyst cultures. however, the very first cells table 11. significance levels f o r the differences be tween groups group significance level day 5 vs. day 7 day 7 vs. day 9 day 7 vs. oestrogen 8 h day 7 vs. oestrogen 16 h day 7 vs. oestrogen 24 h pc0.005 non-significant no apparent difference pc0.05 a manifest difference upsala j med sci 83 138 g . n a l u n d and 0. lundkvist growing out are difficult to distinguish, but as trophoblast cells proliferate rapidly, judging from the changes that occurred in the same outgrowth area between two observations, the influence of this error is probably of minor importance. the sensitiv ity of the method depends upon the length of the in terval between two observations. too close obser vations might disturb the growth of the blastocysts and in the present experiment an interval of 8 h was found suitable, which resulted in an accuracy of k 4 h for every single observation. the reliability of this method was judged to be adequate for the present purpose, as no statistically significant differences were observed between vari ous batches of day-7 blastocysts cultured during different periods of the experiment. it is therefore concluded that the present technique of measuring the time required for blastocysts to start their out growth in vitro can be useful for evaluating factors that influence the blastocyst activity. the results demonstrate that the activity of the blastocyst in vitro is, in fact, influenced by the endocrine state of the blastocyst donor. thus, delayed blastocysts flushed soon after the ovariectomy grew out faster than those flushed a few days later. however, from day 7 onwards (i.e. after 4 days diapause) no difference were ob served between the groups of delayed blastocysts (table i). the conclusion may therefore be drawn that the slow attainment of a steady state of blasto cyst ultrastructure (1, 13) and metabolic activity ( 1 1) during delay corresponds to a similar attain ment of activity in vitro. when oestrogen was injected into the donor ani mals, their blastocysts grew out faster in vitro. this effect was more marked the longer after the oestrogen injection the blastocysts were recovered. further, blastocysts from day 5 of normal, non delayed pregnancy will grow out faster than those from animals during delay of implantation (naeslund, g . : unpublished observation). thus, if blastocysts are influenced by oestrogen before be ing cultured the time required for initiation of out growth in vitro will change. therefore, in culture experiments where the level of blastocyst activity is crucial, it is important to define strictly the en docrine state of the blastocyst donors and the time of recovery. furthermore, if a basic level is desired, the present experiment suggests that delayed blastocysts should not be recovered earlier than day 7. u p s u h j m e d s c i 83 the outgrowth of trophoblast cells i n vitro'has been regarded as an analogue of the attachment and invasion of trophoblast cells in vivo (6). however, the blastocyst in vitro lives in a totally different environment and faces quite a different material for growth than the blastocyst in the uterine cavity. one possible way of determining whether the cells of the blastocyst respond differently to these two conditions is to compare the intracellular changes in vitro with those in v i v o . this kind of study requires well defined culture conditions to avoid irrelevant influences on the morphology. the present design for blastocyst culture seems suitable for this purpose and will be used in future experiments for ultrastructural comparisons of blastocyst develop ments in vivo and in vitro (naeslund, g. & nilsson, 0.: to be published) and for testing some hypo theses on the control of blastocyst growth. acknowledgement§ we thank mrs barbro einarsson for skilful technical as sistance and dr gunnar ekbohm for statistic consultation. the study was financially supported by the swedish med ical research council (project no. 12x-70 to professor ove nilsson) and the swedish foundation for prenatal research. references 1. bergstrom, s . : delay of blastocyst implantation in the mouse by ovariectomy or lactation. a scanning electron microscope study. fertil steril23: 548, 1972. 2. brinster, r. l.: a method for in vitro cultivation of mouse ova from two-cell to blastocyst. exp cell res 32: 205, 1963. 3. brinster, r. l . & thomson, j. l.: development of eight-cell mouse embryos in vitro. exp cell res 42: 308, 1966. 4. gulyas, b. j. & daniel, j. c., jr: incorporation of labeled nucleic acid and protein precursors by diapausing and nondiapausing blastocysts. b i d re prod2: 11, 1969. 5 . gwatkin, r. b. l.: defined media and development of mammalian eggs in vitro. ann ny acad sci 239: 79, 1966. 6. amino acid requirements for attachment and out growth of the mouse blastocyst in vitro. j cell physiol 68: 335, 1966. humphrey, k . w.: the induction of implantation in the mouse after ovariectomy. steroids 19: 591, 1967. maclaren, a.: blastocyst activation. in the regula tion of mammalian reproduction (ed. s. j. segal, r. crozier, p. a. corfman & p. g . condliffe), pp. 321-328. charles c. thomas, springfield, ill., 1973. maclaren, a. & menke, t. m.: co, output of mouse blastocysts in vitro, in normal pregnancy and in delay. j reprod fertil (suppl.) 14: 23, 1971. effect of blastocyst donors on trophoblast outgrowth 139 10. mayer, g . : delayed nidation in rats: a method of exploring the mechanisms of ovo-implantation. i n de layed implantation (ed. a. c. enders), pp. 213-231. univ. chicago press, chicago, 1963. 1 i . menke, t. m.: changes in mouse blastocyst carbon dioxide production as a function of time postcoitum in delay of implantation during lactation or following ovariectomy. biol reprod 7: 414, 1972. 12. mohla, s . & prasad, m. r. n . : early action of oestrogen on the incorporation of [3h]uridine in the blastocyst and uterus of rat during delayed implanta tion. j endocnnol49:87, 1971. 13. nilsson, 0.: the morphology of blastocyst implanta tion. j reprod fertil39: 187, 1974. 14. torbit, c . a. & weitlauf, h.m.: the effect of oestrogen and progesterone on co, production by ‘delayed implanting’ mouse embryos. j reprod fertil 39: 379, 1974. production of carbon dioxide in vitro by blasto cysts from intact and ovariectomized mice. j reprod ferti142: 45, 1975. 16. weitlauf, h.m.: i n vitro uptake and incorporation of amico acids by blastocysts from intact and ovari ectumized mice. j exp zoo1 183: 303, 1973. 17. metabolic changes in the blastocysts of mice and rats during delayed implantation. j reprod fertil 39: 213, 1974. 18. effect of uterine flushings on rna synthesis by ‘implanting’ and ‘delayed implanting’ mouse blasto cysts in virro. biol reprod 14: 566, 1976. 19. weitlauf, h. m . & greenwald, g. s . : a comparison of 35s methionine incorporation by the blastocysts of normal and delayed implanting mice. j reprod fertil zu: 203, 1965. 20. yoshinaga, k. & adams, c. e.: delayed implantation in the spayed, progesterone treated adult mouse. j reprod fertill2: 593, 1966. 15. received january 18, 1978 address for reprints: g . nsslund department of anatomy biomedical centre box 571 s-751 23 uppsala sweden upsala j med sci 83 upsala j med sci 81: 183-187, 1976 preliminary report on angiography with polymeric contrast agents in rabbits and dogs lars bjork, uno erikson and bjorn ingelman from the departments of diagnostic radiology and clinical chemistry, university hospital, uppsala, and pharmacia ab, uppsala, sweden abstract polymeric, water-soluble, iodine-containing contrast agents have been synthesized and tested in renal angiography and thoracic aortography in rabbits and in femoral arterio graphy in dogs. good filling of the arteries and also of the veins was obtained. polymeric, water-soluble, iodine-containing con trast agents of different types intended for use in, for example, angiography, lymphography, uro graphy and hysterosalpingography have been pre pared (1). special polymeric contrast agents for the gastrointestinal tract which have a high solubility in water even at relatively low ph values, and there fore d o not precipitate in the stomach, were also prepared. in earlier publications we have reported on animal investigations with such polymeric con trast agents in the radiological examinations of the gastrointestinal tract ( 2 , 3 , 4 ) . the polymers are of the type presented in the schematic fig. 1 a . in this figure a denotes iodine substituted benzene derivatives (preferably 2,4,6 triiodobenzoic acid derivatives) and b denotes intermediate hydroxyl-bearing aliphatic bridges. group a , for example, has the structure shown in fig. 16 or in 1 c . for contrast agents for oral use, long bridges ( b ) with several hydroxyl groups of the type shown in fig. i d were chosen (2), or such bridges in which the hydroxyl groups were partly replaced by glycerol ether groups (3, 4). for other uses (for instance for angiography and for uro graphy) shorter bridges, for example of the type shown in fig. l e , were chosen. as iodine-substi tuted benzene derivative a , we have in most poly mers preferred the structure shown in fig. 1 c . some of the angiographies performed in rabbits and dogs with the aid of some of the water-soluble, iodine-containing polymers synthesized will be de scribed in this report. materials and methods polymeric contrast agents the iodine-containing polymeric agents used in the animal investigations described in this report were given the code numbers 483,549,566,599,602,616 and 727 e. they had been prepared by reacting 5-acetylamino-2,4,6-tri iodo-n-methyl-isophtalic acid monoamide or 3-acetyl amino-5-acetylamino-2,4,6-triiodo-benzoic acid in alkaline aqueous solution with 1,4-butandioldiglycide ether in molar ratios close t o one to one according to the methods described in reference (1). in all these polymers the bridgeb was of the type shown in fig. 1 e. the iodine-substituted benzene derivative group a was in all these polymers of the type shown in fig. 1 c , with the exception of polymer 727e in which a was of the type shown in fig. 1 b. polymers in which a has the structure shown in fig. 1 b are easily discoloured and the structure shownin fig. i c was therefore preferred. some data regarding these polymers are shown in table i. the code numbers are given in the first column. in the second column, the weight average molecular weights &fw (determined by light scattering by dr k. gra nath) are shown. the f i w value of polymer 483 was never determined. however, polymer 483 was rather similar to polymer 566, to judge from some preliminary gel chro matography experiments. the iodine contents of the dry polymers are given in the third column. (all the polymers had been reprecipitated as polyacids after the synthesis, with the exception of 727 e which had been isolated as the sodium salt after several reprecipitations with acetone. the precipitated polymers had been dried a t about 50°c under vacuum.) the type of the iodine-containing unit a and the type of the bridge b (cf. fig. 1) are also shown in the table. the type of salts in the solutions used in the animal investigations described in this report are shown in the final column. these solutions contained 200-220 mgi/ ml. solutions of conray meglumine which had been di luted to the same iodine content were used for comparison. the molecular weight distributions of these polymers are rather broad as, for the introductory animal tests reported here, the polymers were not fractionated in order to obtain products with narrow molecular weight distribu tions. for several of the polymers a low average degree of polymerization (and thus a relatively low average mo lecular weight) was chosen in order that the molecular weight distribution would be such that all of the material upsala j med sci 81 184 l . bjork et al. cooh cooh oh oh oh oh i i i i d ) ~h,chch,och,chch,o(ch1), och,chch,och,chchr el -ch~ch(oh)ch~olch~~,och~chlohlch~ fig. 1. ( a ) basic structure of the polymeric contrast sub stances. a indicates iodine-substituted benzene deriva tives, mainly 2,4,6-triiodobenzonic acid derivatives. b in dicates hydroxyl-bearing aliphatic bridges. ( b ) example of group a . ( c ) example of group a . ( d ) example of the bridge b . ( e ) example of the bridge b . in the polymers described in this report the bridge b was of the type shown in fig. 1 e and the iodine-substituted benzene de rivative group a was of the type shown in fig. 1 c with one exception. would be well below the limit of permeability of the renal glomeruli. analytical gel chromatography with polymer 599 (which in the animal tests has shown interesting prop erties) showed that about 15% by weight of this polymer product had molecular weights above 20000 and that about 20% by weight had molecular weights below 3 000. renal angiography and thoracic aortography in rabbits female white new zealand rabbits weighing approxi mately 2.5 kg were used as experimental animals. the rabbits were anaesthetized with intravenous injec tion of phenobarbital, the iliac artery on one side was ex posed and a fine polyethylene catheter was introduced into the artery and advanced with its tip immediately above the renal arteries. one of the kidneys, usually the left, was selected for the angiographic studies. an x-ray tube with a 0.6 mm focal spot and a seven .table i m = methylglucamine, na= sodium inch cesium-iodine image intensifier and a 70 mm camera operated at 1 frame/s were used to record the angio grams. the following polymeric contrast agents were tested and compared with conray meglumine: code numbers 483,549,566,599,602,616 and 727 e. the iodine content of the solutions used was adjusted to about the same level (200-220 mgi/ml). each of the new compounds was tested in four rabbits in renal arteriography. in each rabbit four injections of the new compound were alternated with injections of similar amounts of diluted conray meglumine, 0.5 ml per kg body weight being injected on each occasion. in the ab sence of a suitable automatic injector for small volumes the injections were done manually. the injections were timed by a stop-watch and the average rate found to be 0.5 ml/s with only minor variations. the electrocardio gram was recorded continuously before, during and after the injections. the blood pressure was measured inter mittently using the injection catheter. in four rabbits the catheter was advanced to the ascend ing aorta and thoracic aortograms were performed in each animal with four polymeric contrast agents (549,566, 602 and 616). one millilitre per kg body weight was in jected on each occasion. the iodine content of the solu tions was again 200-220 mg/ml. femoral arteriography in dogs in four mongrel dogs (weighing approximately 25 kg each) the femoral artery on one side was exposed under general anaesthesia, and a catheter introduced and manipulated into the contralateral iliac artery and advanced until its tip was in the femoral artery. femoral arteriography in the dogs was performed with the same technique as described by bjork ( 5 ) . comparative injections of polymeric contrast agents (483, 549, 566 and 616) and conray meglumine were made. the contrast solutions used had been diluted to about the same iodine content (200-220 mgi/ml). the weight of the leg of each dog was estimated and the dose of contrast medium used was 1 ml per kg leg weight. full-size angiograms of the leg were obtained using a frankling roll film changer. iodine content type of of dry polymer iodine polymer code (1) as polyacid containing type of type of salt number m w (2) as sodium salt unita bridgeb in test solution 483 46.1 (1) c e m 549 4 0 0 0 45.5 (1) c e m 566 5000 46.1 (1) c e m 599 14 ooo 44.7 (1) c e m 602 41 000 44.7 (1) c e na 616 7000 45.5 (1) c e m 727 e 38 000 41.6 (2) b e na upsula j med s c i 81 angiography with polymeric contrast agents 185 fig. 2. renal angiography in rabbit. polymer 727 e. (a) arterial phase. (b) 12 seconds after injection. very good filling of intra renal veins. (c) 3 min after injection. dense filling of collecting tubes in the papillae. results renal angiography and thoracic aortography in rabbits judging by experience at the injections, the viscos ity of the solutions of code numbers 483, 549, 566, 599 and 616 was only moderately higher than that of the diluted conray meglumine solution used for comparison. the viscosity of the solutions of com pounds 602 and 727 e was definitely higher than that of conray meglumine. however, with the small amounts used in the experiments no difficulty in injecting the contrast media was encountered. the arterial filling was, as expected, similar with all contrast media used. the flow of the solutions of compounds 602 and 727 e in the vessels was slower than with the other compounds. the venous filling was in all instances better with the new polymeric compounds than with similar in jections of conray meglumine. this was partic ularly marked with the compounds 599, 602 and 727 e (fig. 2. polymer 727 e), with which a very marked filling of even small veins in the kidneys was seen. the appearance time (i.e. the time from the start of the injection until contrast was visible in the veins) was on average 30% longer with the com pounds 483, 549, 566 and 616 than with conray meglumine, and about 50% longer with compound 599. with the compounds 602 and 727 e the appear ance time was on average twice as long as with con ray meglumine. fig. 3. thoracic aortogmphy in rabbit. polymer 602. upsala j med sci 81 186 l . bjork et al. f i g . 4. femoral arteriography in dog. polymer 566. (a) early arterial phase. ($3) very good filling of the veins in the leg. the excretion of contrast medium into renal pelvis started at almost the same time with the com pounds 483,549, 566 and 616 as with conray meg lumine. with the compound 599 there was some de lay in the appearance in the renal pelvis and with the compounds 602 and 727 e there was a marked delay in appearance. with the latter three com pounds there was also very dense filling, not only of the renal pelvis but also of the collecting tubes in the intrarenal papillae (fig. 2. polymer 727 e). the changes in heart rate and blood pressure after injection of conray meglumine and the test compounds were usually slight. from these experi ments it is difficult to draw any definite conclusions but there was a tendency towards less effect on the heart rate and blood pressure with the new com pounds than with conray meglumine. the four contrast agents (549, 566, 602 and 616) tested in thoracic aortography in four rabbits all gave satisfactory arteriograms with good filling of the arteries and also of the veins. the high viscosity of the solution of compound 602 proved to be n o hindrance to rapid aortic injection for good filling of the thoracic aorta and its branches in these small animals (fig. 3 . polymer 602). upsala j med sci 81 femoral arteriography in dogs with conray meglumine there was good arterial filling, as expected. however, the filling of major veins was poor with all 16 injections. with the four polymeric agents tested (483, 549, 566 and 616), however, good filling of the veins was seen 12 times out of 16 (fig. 4. polymer 566). the arterial filling was good in all animals. the appearance time in the veins was always longer (on average 40% longer) with the polymeric contrast agents than with conray meglumine. discussion these preliminary experiments, as well as other experiments with similar polymers, have shown that polymeric contrast agents may be useful for, e.g. angiography and urography . the experiments also indicate that some of the expected advantages are present, particularly with agents of high molec ular weight. these include better visualization of veins and also denser contrast excretion in the kid neys, provided that the sizes of the polymer mole cules are not so great that they are unable to pass the renal glomeruli and easily be excreted with the angiography with polymeric contrast agents 187 urine. the circulation time, as reflected by the appearance time in the veins following intra-arterial injection, was also longer with all polymeric con trast agents tested. the differences observed between conventional contrast agents and polymeric contrast agents might be due t o several factors, including larger molecular size (resulting in lower diffusion rate, higher viscosity, less osmotic activity etc.), less pronounced effects on the local vascular bed and less pronouned effects on the circulation. the results of the studies performed give guid ance in the choice of suitable molecular weights for these polymers. however, further experiments are needed to compare polymeric contrast agents with different and well-defined molecular weight distributions and to establish suitable molecular weight distributions of such polymers for various uses. references 1 . bjork, l., erikson, u . , granath, k . , ingelman, b. & lindberg, b.: swedish patent no. 348 110, filed 1966, united states patent no. 3852341 and german pat ent no. 1617743, filed 1%7. 2. bjork, l., erikson, u. & ingelman, b.: polymeric con trast media for roentgenologic examination of gastro intestinal tract. investigative radiology5: 142, 1970. 3. bjork, l., erikson, u., ingelman, b. & zaar, b.: im proved polymeric contrast agents for roentgenologic examination of the gastrointestinal tract. i. upsala j med sci 79: 103, 1974. 4 . bjork, l . , erikson, u., falk, j., ingelman, b. & lind blad, g.: improved polymeric contrast agents for roentgenologic examination of the gastrointestinal tract. 11. upsala j med sci 79: 106, 1974. 5 . bjork, l.: arterial venography of the leg. experimental studies in dogs. upsala j med sci8j: 119, 1976. received j u n e 8, 1976 address for reprints: uno erikson, m.d. department of diagnostic radiology university hospital s-750 14 uppsala 14 sweden upsala j med sci 81 upsala j med sci 84: 9-20, 1976 the effect of glucose-, arginineand leucine-deprivation on mouse blastocyst outgrowth in vitro' grels naeslund from t h e reprciductiori resecirclr u n i t , biotnedic~il c e n t r e , u p p s u l o , s w e d e n abstract to estimate the degree of trophoblast outgrowth in vitro, mouse blasto cysts obtained after delay of implantation were cultured either in a modified brinster medium or in the same medium with exclusion of various combinations of glucose, arginine and leucine. trophoblast outgrowth was prevented only in a medium from which all three substances were excluded. in this medium the blastocysts remained expanded for 5 days without signs of trophoblast outgrowth a growth arrest in vitro. after transfer of blastocysts growth arrested in vitro for 5 days to a complete medium including both glucose and the two amino acids, normal out growths occurred within two or three days. the growth-arrested blastocysts also developed normally for at least one week when transplanted into salping ectomized foster mothers. it is concluded that blastocyst activation in vitro can be controlled by a few nutrients in a way reminiscent of the activation prior to implantation in utero. blastocysts activated in utero by systemic administration of oestrogen for various lengths of time before the start of culture, or in vitro by preincubation in a medium containing glucose and all amino acids, also grew out in the growth arrest medium if they had been activated for a sufficiently long time, 18 h in utero and 1 h in vitro, thus indicating that when a blastocyst has reached a certain degree of activation its growth can not be arrested by exclusion of glucose, arginine and leucine. introduction activation of a uterine mouse blastocyst by systemic administration of oestrogen during experimental delay of implantation might be due, for instance, either to disappearance of a uterine inhibitor ( z o ) , or to an increase of 'part of this study was presented at the society for the study of fertility, sheffield, england, 1976. 9 nutrients in the uterine secretion ( 9 , 1 3 ) . similar ideas can be applied to conditions in vitro. thus, when blastocysts in delay are transferred from the uterine cavity to a medium containing serum, glucose and amino acids, the trophoblast cells become activated (15) and grow out ( 8 ) , indicating either that the blastocyst gets released from the uterine inhibitor o r that on transfer to the culture medium it is supplied with nutrients that are lacking in the uterus (25). the uterine secretion, which is assumed to harbour the factors controlling mouse blastocyst activity during implantation, is increased a few hours after an oestrogen injection (17). further, its composition is changed around the time of implantation according to various assays of proteins and carbohydrates. the total protein content in rat and mouse uterine secretion has thus been found to increase and the presence of new serum and non-serum proteins has been observed ( 2 , 2 4 ) . it is evident that in vitro, as in utero, serum or a few other macromolecular components are necessary for outgrowth (7). fructose is present in increasing amounts when the roe deer blastocyst in diapause starts implanting (l), whereas in the mouse, studies suggest that glucose is the more important carbohydrate for implantation ( 1 9 ) . in vitro, glucose has also been found necessary for hatching of zona-encased mouse blastocysts from superovulated mice ( 2 8 ) . however, experiments on mice during delay of implantation have shown that even when glucose is excluded from the culture medium blastocyst outgrowths are obtained (15). in vitro, amino acids have also been found important, as they affect blastocyst hatching, attachment and outgrowth and thus may be of significance for implantation. in one study blastocysts could be kept expanded without trophoblast outgrowth when arginine or leucine was excluded from the medium ( 8 ) , but with another experimental set-up these amino acids only inhibited outgrowth to some extent ( 2 3 ) . in utero the concentrations of various amino acids, including arginine and leucine, were the same at implantation and during delay (9). since there are indications that glucose, argjnine and leucine are critical nutrients, the present experiments were undertaken to test the influence of exclusion of glucose and the two amino acids from the culture medium on the outgrowth of delayed mouse blastocysts, in order to find out whether growth arrest reminiscent of delayed implantation could also be obtained in vitro. since this purpose was achieved, blastocysts in various degree of activation were also evaluated to examine whether a critical stage of blastocyst acti vation exists beyond which the blastocyst loses its capability of being growth-arrested in vitro. 10 material and methods brinster's medium for ovum culture (3) with replacement of lactate by 1.0 mg/ml of glucose, which is regarded appropriate from the 8-cell stage onwards ( 4 ) , was used as control medium. the amino acids of eagle's basal medium (5) stored in two stock solutions with 100 times the final concentration, one with cystine and tyrosine dissolved in 0.1 n hc1 and one with the other amino acids, dissolved in water. glutamine was added as dry powder. foetal calf serum (flow laboratories, irvine, scotland), dialysed against 100 times its volume of isotonic nacl at 4oc with two changes daily for three days to remove glucose and amino acids, was used within a week after the dialysis (8). reagent grade water from a milli-q2 system (millipore corp., bedford, mass., u.s.a.) was used. this water had a specific resistance of about 18 mrfcm. six types of media, all differing with regard to the presence of the three components glucose, arginine and leucine (fig. l), were employed. these were: 1 . a control medium containing glucose and all amino acids. 2. an experimental medium without addition of glucose. 3. an experimental medium without addition of arginine and leucine. 4 . an experimental medium without addition of glucose and arginine. 5. an experimental medium without addition of glucose and leucine. 6 . an experimental medium without addition of glucose, arginine or leucine. all media contained 1 % dialysed foetal calf serum, except in experiments where the effect of various serum concentrations were tested. virgin, albino mice of the nmri strain (anticimex, stockholm, sweden) were caged with males overnight and the day on which a vaginal plug was found was called day 1 of pregnancy. ovariectomy was performed on day 3 to induce delay of implantation and 1 mg of the depot preparation medroxiprogesterone (depo provera, upjohn co, u.s.a.) was given subcutaneously at ovariectomy and again 5 days later (30). the normal animals were taken for experimentation on the afternoon of day 4, while the animals in delay of implantation were used on day 8-12, as a steady state of outgrowth activity is attained on day 7 (16). the uterine horns were flushed with phosphate-buffered saline (pbs) containing magnesium and calcium (sbl, stockholm, sweden) with addition of 1% dialysed foetal calf serum to prevent the blastocysts from becoming adhesive. the culture medium was equlibrated against an atmosphere of 5% c 0 2 in air for a t least one hour and droplets of the medium with a volume of 0.1 ml were then covered with liquid paraffin (3) and kept in plastic tissue culture dishes (type 3001f, falcon, oxnard, u.s.a.) at 3 7 o c in an atmosphere of 5% c 0 2 in air. as a low oxygen tension has been observed in the rat uterus around the time of implantation, and as changes in oxygen tension might influence carbohydrate metabolism ( 2 9 ) , tests on the effect of a low-oxygen milieu were also carried out by using airtight jars containing the culture dishes and flushed with a mixture of 5% c o and 5% o2 in n 2 2 slight residual gas pressure. the jars were placed in a culture box at a temperature of 3 7 o c and the dishes were checked daily. if some pressure was still left the gas atmosphere was considered unchanged. and then closed with a ' the blastocysts were transferred to the droplets, about 5 in each, by means of glass capillaries connected to an agla micrometer syringe (burroughs wellcome and co., london) and were then observed daily for 5 days in an inverted microscope (biovert, reichert, austria). phase-contrast microscopy (magnification x 160) was used to detect the first signs of outgrowth (16). to examine the influence of various media on the outgrowth, blastocysts from mice in delay of implantation were cultured in media from which glucose and/or amino acids had been excluded. in order to check the capacity for subsequent outgrowth in blastocysts whose growth had been arrested & vitro, the medium without glucose, arginine or leucine was exchanged for the control medium. to check their ability to implant, blastocysts from delay of implantation that had been growth-arrested in vitro for 6 days were transferred to the control medium for 24 h to activate them, and were then transplanted in pbs and 5 % dialysed serum to the left uterine horns of nmri mice on day 4 of pregnancy. the mice had been bilaterally salpingectomized on day 2. uterine swellings observed after a week were embedded in paraffine and sectioned for light microscopy. to examine whether a critical stage of blastocyst activation exists beyond which the blastocyst loses its ability to be growth-arrested in vitro, experiments were performed in which blastocysts from delay of implantation were activated either in vitro or in utero before culture in the growth arrest medium. for activation in vitro, blastocysts were pre-incubated for 1, 3 or 6 h in the control medium, whereas for activation in utero blasto cysts were taken from animals that had received 0.1 ug of 1 7 8-oestradiol 12, 1 8 or 24 h before the start of culture (fig.3 1. results the blastocysts from animals in delay of implantation generally became contracted during the process of recovery and transfer to the droplets, probably because they were zona-free, but in the control medium they expanded within 2 4 h and within three days the trophoblast cells of the blastocysts had grown out producing a pile of cells (fig. 1). in media without glucose andlor amino acids the outgrowth was affected. _-___-_--___-__ with exclusion of glucose $line the blastocysts became expanded within 24 h 12 but the first outgrowths did not appear until days 3 to 5. after 5 days all blastocysts had grown out, the outgrowths being smaller, however, than in the control medium. with exclusion of grgin_ine and leucine, exclusion of -g_l_u_c_o_s_e_ and arginine and exclusion of glucose and leucine, some of the blastocysts grew out on day 4 to 5 , while the others remained expanded, but showed no trophoblast outgrowth (fig. 1). when all three nutrients glu_cose, -------arginine --_-_ and _____-----leucine were excluded from the medium, all blastocysts still remained expanded without any signs of trophoblast outgrowth after 5 days in culture a growth arrest in vitro. _____---- 0 0 in m . 50 40 30 20 10 c g al ga gl gal fig. 1. no. of outgrowing blastocysts to total number of blastocysts after 5 days in culture. two to four replicate experiments were performed in each group. the height of the filled bars indicates the number of blastocysts, that had grown out after 5 days’ culture. the remaining blastocysts were expanded. “c“ indicates the con&rol medium; the other bars represent experimental media with exclusion of different components. (g = glucose, a l = arginine and leucine g a = glucose and arginine,g l = glucose and leucine, g a l = glucose, arginine and leucine). fig. 2. a blastocyst growth-arrested for 5 days in amedium from which glucose, arginine and leucine were excluded. the blastocyst is expanded and somewhat rugged in outline. the embryoblast has partly loosened from the inner surface of the trophoblast. bright field microscopy. 13 most of the blastocysts in all types of media the control medium as well as the media from which nutrients were excluded had become attached to the plastic surface within a day and remained so throughout the time of culture.' in a medium containing neither glucose, arginine nor leucine the longest diameter of the blastocysts slowly increased from about 100 um after 2 4 h to about 150-200 pm after 5 days and the blastocyst contour became somewhat rugged (fig. 2 ) . the embryoblast of most blastocysts showed a peculiar change after 2 to 3 days, in the form of loosening of most of the contact with the inner surface of the trophoblast, leaving only attachment with thin connections from the embryoblast edges (fig. 2). this change was observed in all five types of experimental media with exclusion of nutrients. the viability of the trophoblasts of the blastocysts growth-arrested & vitro in the medium with glucose, arginine and leucine excluded was tested by changing the experimental medium for the control medium after 5 days. it was then observed that outgrowths with a normal appearance and size occurred 2 to 3 days after the change of medium (30 blastocysts from three replicate exper iments). the viability of the embryoblasts was tested by transplantation experiments. out of 23 blastocysts growth arrested in vitro for 6 days in the medium with glucose, arginine and leucine excluded and transplanted to the left uterine horns of ten bilaterally salpingectomized recipients in two replicate exper iments, 5 animals had uterine swellings after one week. three of these showed only decidual tissue, while the other 2 contained embryos with a normal appearance (neural tube and primitive heart). blastocysts from day 4 of normal pregnancies, in one experiment, sometimes hatched (2/10 blastocysts), and zona-free blastocysts always grew out when glucose alone was excluded and 1% dialysed serum was added. also with 10% dialysed serum, as in wordinger's and brinster's study (28), 4 / 1 0 blastocysts hatched and all those that hatched grew out on day 3 to 5. in a medium from which glucose, arginine and leucine were all excluded, however, all 15 blastocysts had hatched after 3 to 4 days and subsequently remained expanded and relatively large without any signs of trophoblast outgrowth (two replicate experiments). a few variants of the culture system were tried. an atmosphere containing only 5 % o 2 with 5 % c 0 2 in n change the effect of the various media on the outgrowth (5 to 8 blastocysts in each group, two replicate experiments). the expanded blastocysts cultured in a medium deprived of glucose, arginine and leucine in a low oxygen milieu appeared similar to those cultured in 20% oxygen. when serum was excluded from the control medium the blastocysts contracted after 2 to 3 days (10 instead of the usual 2 0 % 0 2 2 (air) did not 14 blastocysts from two replicate experiments), whereas exclusion of serum from the medium containing no glucose, arginine of leucine did not cause contraction; the blastocysts were still expanded for one to 2 weeks and the embryoblast loosened just a s in the presence of serum ( 2 0 blastocysts from two replicate experiments). when the concentration of dialysed serum was increased to 2 0 %, 3 of 10 blastocysts grew out even when all three nutrients were excluded (two replicate experiments). experiments with blastocysts at various degrees of activation showed that blastocysts that had been preincubated in the control medium for 1 h or longer or taken from animals that had received oestradiol 18 h earlier were able to grow out in the medium without glucose, arginine or leucine (figs. 3 and 4 ) . the outgrowths after 1 and 3 h preincubation in the control medium were small and did not appear until after 5 days in the growth-arrest medium. 0 0 0 “ n \ \ \ i 0 0 0 0 0 0 “ c i 12h 18h 2 4 h in utero lh 3h 6h in vitro fig. 3. capability of the growth arrest medium (with glucose and arginine-leucine excluded) to impede outgrowth of blasto cysts activated in utero o r in vitro. they were activated in utero by giving 0.1 pg 17-8-oestradiol to the hnimal 12, 18 or 2 4 h before start of culture and & vitro by incubating them in the control medium 1, 3 or 6 h before change of culture medium. two replicate experiments in each group. concerning filled and un filled bars see fig. 1. fig. 4 . trophoblast outgrowth from a blastocyst activated by 0.1 pg 17-8 oestradiol 2 4 h before start of culture and then incubated for 5 days in a medium with glucose, arginine and leucine excluded. phase contrast microscopy. 15 discussion when using trophoblast outgrowth as a measure of the growth-promoting potential of various culture media, it is an advantage to use blastocysts from animals in delay of implantation, as they are zona-free (12) and have a defined capacity for outgrowth ( 1 6 ) . in the present experiments the blasto cysts were observed for 5 days, because in a medium permitting normal out growth most delayed blastocysts grow out within 3 days and only few later ( 1 6 ) . since in earlier assays of the trophoblast outgrowth no such standardized systems have been used, comparisons of the various results should be made with caution. the proliferation of animal cells in culture quite generally can be stimulated by a variety of substances. for some such substances, like serum and specific growth-stimulating factors, the rate of proliferation is a function of the concentration (26,27), and also in the case of others, like amino acids, proliferation is now thought to depend on the concentration (11). the present experiments focus on the influence of this type of substance on blastocyst growth and viability. the exclusion of arginine and leucine from a blastocyst culture medium has been reported by some authors to result in a total inhibition of trophoblast outgrowth (8). others, however, have found less complete inhibition when the two amino acids are omitted (23). these discrepancies might be due to the use of different materials for cell support (glass versus collagen) or to the culture of different types of blastocysts (cultured from 2-cell stage versus flushed on day 4 ) . however, since it was also found in the present experiments with blastocysts delayed in utero that exclusion of arginine and leucine only inhibited outgrowth to some extent, these amino acids are probably not the only growth controlling factors. glucose exclusion from a medium has been reported to almost totally inhibit hatching of zona-encased blastocysts (28). the degree of outgrowth, however, might have been difficult to test in the model used, as the blasto cysts, which derived from superovulated animals, rarely hatched and thus very few had the opportunity to grow out. in these experiments the blastocysts were further cultured on collagen and the observation time was only 48 h. consequently the small, late appearing outgrowths that were observed in the present study might have been missed with this experimental set-up. in the present experiments, the growth of normal day-4 blastocysts was tested in a medium from which only glucose was excluded, those that hatched always grew out. neither were blastocysts obtained during diapause completely inhibited in media without glucose, and therefore lack of glucose in the uterine secretion is not likely alone to cause a delay in utero. 16 media from which all three nutrients glucose, arginine and leucine were excluded allowed the blastocysts to remain expanded, i.e. their growth was arrested, reminiscent of blastocysts delayed in utero. the blastocysts were observed regularly for 5 days, but some blastocysts kept in vitro for a longer time remained expanded for about two weeks, though they became more irregular in outline. when the excluded components were added, the blastocysts were activated and outgrowths occurred after two to three days corresponding to the response by blastocysts in uterine days corresponding to the response by blastocysts in uterine diapause when transferred to a complete medium (16). since the shape and size of the outgrowths were similar in both cases, the trophoblast cells were probably undamaged by the exclusion of glucose and the two amino acids. ultrastructural studies are in progress with the aim of shedding further light on growth-arrest of blastocysts in vitro. blastocysts whose growth was arrested in vitro increased their diameter slowly. this expansion could explain why day-4 blastocysts, only some of which hatched in a medium deprived only of glucose, always hatched when both glucose and the two amino acids were excluded. the observed loosening of the embryoblast could be due to the loose connection between trophoblast and embryoblast ( 1 4 ) , in combination with the enlargement of the blastocyst in the growth-arrest medium. it might also indicate an adverse effect of the growth-arrest medium on this part of the blastocyst, since the embryoblast is more dependent on the amino acid concen tration than the trophoblast (23). however, the blastocysts that were growth arrested in vitro developed normally for at least one week when transplanted to salpingectomized pregnant mice. sherman and barlow (21) reported that blastocysts in media with exclusion of only arginine and leucine survived a transplantation for about a week. in the present experiments no attempt was made to standardize the transplantation technique which would have been necessary in order to correlate the success rate with that of blastocysts not cultured. the transplantation results indicate, however, that both embryoblast and trophoblast could survive in the growth arrest medium. experiments with activated blastocysts showed that the growth-arrest medium did in fact allow trophoblast outgrowth from blastocysts activated by systemic oestrogen or by previous incubation in a complete medium, where blastocysts become metabolically activated (15). this suggests that the growth-arrest is due to an absence of sufficient activation in vitro and not to a hostile environment making outgrowth impossible. when the blastocyst has reached a certain degree of activation its growth could no longer be arrested in vitro. the necessary activation time was 18 h in utero and only 1 h & vitro perhaps reflecting the time lag between oestrogen administration and 2-792854 17 production of uterine secretion. as blastocysts are able to grow out in a growth-arrest medium provided they are sufficiently activated, use of this experimental system might be one simple way to determinate the degree of , blastocyst activation. attachment of the blastocysts to the surface has been considered a stage indicating a subsequent outgrowth (8). blastocysts from delay of implantation cultured in a complete medium have been observed first to attach and then to loosen from the substratum before the start of outgrowth, indicating changes in the characteristics of the blastocyst surface (22). also with the present experimental set up this phenomenon was observed in the control medium. the type of surface probably has a profound effect on attachment. moreover, the definition of attachment as the capacity of the blastocysts to adhere to the solid surface even also when the culture dish is moved (23) is rather unprecise in the present experiments blastocysts attached even in a growth-arrest medium, indicating a poor correlation between attachment and outgrowth. in utero the blastocyst coat in delay has a negative electrical surface charge which, probably because of changes in the surface coat, decreases before implantation, thus facilitating attachment (18). the surface coat of a blastocyst in vitro, however, might behave differently, thus explaining why growth-arrested blastocysts are as firmly attached to the substratum as outgrowing ones. experiments with surface indicators such as alcian blue and con-a on blastocysts in vitro are under way. the serum used in the medium represents an undefined factor. normal blasto cyst outgrowth has, however, been observed in serum-free media when the blastocysts have grown on a collagen surface, which should indicate that protein from serum is necessary only to make the plastic surface physically suitable (10). however, a special cell spreading factor for different types of cells have also been found in fetal calf serum (6) and might also be required for blastocysts in vitro (7). when, in the present experiments, the serum concentration was increased, some blastocysts grew out even in the growth-arrest medium, suggesting that components present in dialysed serum might activate blastocysts when present in a high concentration but not in a lower concentration. alternatively, a high serum concentration modifies the plastic surface so that even a partly growth-arrested blastocyst can pro liferate. the present observation that blastocysts whose growth had been arrested in vitro were able to expand without serum, whereas blastocysts in a complete medium became contracted in a serum-free milieu, indicates that serum is important mainly for the outgrowing blastocyst. the implication from the present experiments for conditions in utero is that absence or a low concentration of both glucose and amino acids could keep a blastocyst in a state of delay until the missing nutrients are produced 18 by the endometrium. there might, however, be different ways in which growth of a blastocyst, is arrested. it is possible that in utero an arrest may occur due to absence of macromolecular components, and in vitro due to other factors. for example blastocysts delayed in utero are arrested in another phase of the cell cycle than blastocysts in media from which only arginine and leucine are excluded (21). acknowledgements i wish to thank mrs barbro einarsson for skilful technical assistance. this study was financially supported by the swedish medical research council (grant no. 12x-70 to professor ove nilsson). references 1. aitken, r.j.: uterine secretion of fructose in the roe dear. j reprod fertil 46: 439-440, 1976, 2. aitken, r.j.: changes in the protein content of mouse uterine flushings during normal pregnancy and delayed implantation, and after ovariectomy and oestradiol administration. j reprod fertil 50: 29-36, 1977. 3. brinster, r.l.: a method for in vitro cultivation of mouse ova from two cell to blastocyst. exp cell fes 32: 205-208, 1963. 4. brinster, r.l. & thomson, j.l.: development of eight-cell mouse embryos in vitro. exp cell res 42: 308-315, 1966. 5. eagle, h.: nutrition needs of mammalian cells in tissue culture. science 122: 501-504, 1955. 6. grinnell, f.: biochemical analysis of cell adhesion to a substratum and its possible relevance to cell metastasis. in: membranes and neoplasia: new approaches and strategies, record of a workshop in keystone, col., 1976; ed. v.t. marchesi, pp. 227-236. alan r. liss, new york, 1976. ann ny acad sci 139: 79-90, 1966. the mouse blastocyst in vitro. j cell physiol 68: 335-344, 1966. in the mouse. int j fertil 14: 101-105, 1969. blast outgrowth in the mouse. j embryol exp morphol 30: 21-30, 1973. mark, 1978. 7. gwatkin, r.b.l.: defined media and development of mammalian eggs in vitro. 8. gwatkin, r.b.l.: amino acid requirements for attachment and outgrowth of 9. gwatkin, r.b.l.: nutritional requirements for post-blastocyst development 10. jenkinson, e.j. & wilson, i.b.: in vitro studies on the control of tropho 11. mckeehan, w.l. & ham, r.g.: cited in personal communication with b. wester 12. mclaren, a.: the fate of the zona pellucida in mice. j embryol exp morph 23: 1-19, 1970. 13. mclaren, a.: blastocyst activation. in: the regulation of mammalian re production (ed. s.j. segal, r. crozier, p.a. corfman & p.g. condliffe), pp. 321-328. charles c. thomas, springfield, ill., 1973. cyst substages. j embryol exp morphol 32: 675-695, 1974. at the society for the study of fertility (annual conference, sheffield, england, 1976). donors on the time required for initiation of trophoblast outgrowth. ups j med sci 83: 135-139, 1978. 39: 187-194, 1974. implantation in the mouse. contraception 11: 441-450, 1975. 14. nadijcka, m. & hillman, n.: ultrastructural studies of the mouse blasto 15. naeslund, g.: activation of the mouse blastocyst in vitro. paper presented 16. naeslund, g. & lundkvist, 0 . : effect of the endocrine state of blastocyst 17. nilsson, 0.: the morphology of blastocyst implantation. j reprod fertil 18. nilsson, o., lindqvist, i. & ronquist, g.: blastocyst surface charge and 19 1 9 . 20. 2 1 . 22. 23. 24. 25. 26. 2 7 . 2 8 . 29. 3 0 . nilsson, b.o., ostensson, c., eide, s. & hellerstrom, c.: role of glucose in the mouse uterine secretion for the activation of the implanting mouse blastocyst. submitted to cell tissue res, 1978. psychoyos, a., bitton-casimiri, v. & brun, j.l.: repression and activation of the mammalian blastocyst. i n : regulation of growth and differentiated function in eucaryote cells (ed. g.p. talwar), pp. 509-514. raven press, new york, 1975. sherman, m.i. & barlow, p.w.: deoxyribonucleic acid content in delayed mouse blastocysts. j reprod fertil 29: 123-126, 1 9 7 2 , sherman, m.i. & wudl, l.r.: the implanting mouse blastocyst. in: the cell surface in animal embryogenesis & development (ed. g, poste & g.l. nicholson). north holland, amsterdam, 1977. spindle, a.i. & pedersen, r.a.: hatching, attachment and outgrowth of mouse blastocysts in vitro: fixed nitrogen requirements. j exp zoo1 186: 305-318, 1973. surani, m.a.h.: uterine luminal proteins at the time of implantation i n rats. j reprod fertil 48: 141-145, 1976. weitlauf, h.m.: metabolic changes in the blastocysts of mice and rats during delayed implantation. j reprod fertil 39: 213-224, 1974. westermark, b . : proliferation control of cultivated human glia-like cells under "steady state" conditions. exp cell res 69: 259, 1971. westermark, b . & wasteson, a . : a platelet factor stimulating human normal glial cells. exp cell res 98: 170-174, 1976. wordinger, r.j. & brinster, r.l.: influence of reduced glucose levels on the in vitro hatching, attachment, and trophoblast outgrowth of the mouse blastocyst. dev biol 53: 294-296, 1976. yochim, j.m. & mitchell, j.a. intrauterine oxygen tension in the rat during progestation: its possible relation to carbohydrate metabolism and the regulation of nidation. endocrinology 83: 706-713, 1968. yoshinaga, k . & adams, c.e.: delayed implantation in the spayed, pro gesterone treated adult mouse. j reprod fertil 1 2 : 593-595, 1966. accepted november 2 8 , 1978 address for reprints: g. naeslund department of anatomy biomedical centre box 5 7 1 s-751 23 uppsala sweden 20 upsala j med sci 82: 11-14, 1977 adenylate kinase activity in cerebrospinal fluid in connection with transitory lschaemic attacks goran frithz,' per ericsson' and gunnar ronqutst* from the departments of 'znternaf medicine and zclinical chemistry central hospital, s-63188 eskilstuna, sweden abstract adenylate kinase activity was measured in cerebrospinal fluid of healthy normal individuals and those having suf fered from transitory ischaemic attacks (tia). normally, no adenylate kinase was present in cerebrospinal fluid. a slight but distinct activity was always registered in the 11 cases studied in connection with tl4. cerebrospinal fluid of 2 patients was also analysed in a symptom-free interval (at least 2 weeks after the stroke) and no adenylate kinase activity was found. introduction in transitoric ischaemic attack (tia) the clinical manifestations may be pronounced while at the same time the underlying cerebral disorder is subtle in its changes. it has consequently proved difficult to substantiate the pathologic conditions with ordi nary laboratory methods. thus, cerebral angio graphy and scintigraphy generally d o not give any further information about the localized area involved ( 2 ) . nevertheless, there are reasons t o be lieve that cell damage-reversible and possibly ir reversible to some extent-occurs. consequently, a leakage takes place of intracellular compounds into the extracellular medium. among these, enzymes are of special interest as indicators of cell damage and could be expected to appear primarily in the cerebrospinal fluid (csf). the normal blood csf barrier results in csf-enzyme concentra tions that are relatively independent of their serum levels (1, 7). enzyme determinations, especially asat and ld, have been performed to a limited extent in the csf in various pathological conditions, such as cerebral infarction, tumour, and multiple sclerosis (4, 8, 9, 19). it was recently shown (3) that adeny late kinase is a more sensitive indicator of slight cell damage, at least for the myocardium, than is asat and ld. to our knowledge, no studies have so far been published concerning adenylate kinase activity in cerebrospinal fluid, neither under normal condi tions nor in connection with tia. nor have any other enzymes been determined in csf in connec tion with tia. the aim of the present study was to investigate whether the adenylate kinase in csf could be used as a sensitive marker of impaired cells of the brain tissue in connection with tia. patients and methods according to who criteria (16) transitory ischaemic at tack (tia) is defined as a focal neurological deficit on a vascular basis and commonly lasts some minutes (though never exceeding 24 h), leaving no residual deficit. patients no. 1. female, aged 64. known mitral stenosis. aphasia and weakness of right arm twice during 4 h. duration of each spell, 15-30 min. no. 2. male, aged 60. sudden vertigo, dysarthria and numbness in the face for 2 h . three months later admitted comatose and died within a few hours. autopsy revealed complete thrombosis of the left carotis interna artery and a large cerebral infarction. no. 3. male, aged 56. hypertension and hyperlipo proteinemia for a t least 3 years. sudden dysarthria and right hemiparesis; was unable to walk. no. 4. male, aged 60. weakness and paresthesia in the left arm and to a slight degree in left leg. unsteadiness on attempt to walk. transitory babinski sign on left side. attack lasting for about 3 h. upsala j m e d sci 82 12 g . frithz et al. table i. adenylate kinase activity in csf from i 1 patients with tza analysed for adenylate kinase activity. enzyme activity was expressed in milliunits ( m u ) per ml as has been de scribed previously (3). adenylate patient kinase activity no. (mulml) r e s u l t s 1 2 3 4 5 6 7 8 9 10 1 1 0.95 1.15 0.85 0.70 1.20 0.65 0.80 0.50 0.60 1.05 0.75 no. 5 . female, aged 70. woke up with a left-sided hemiparesis, which disappeared within 2 h. no. 6. male, aged 61. hypertension for a t least 10 years. diabetes mellitus for one year. paresis of the right arm and a short attack of blurred vision. no. 7. male, aged 65. headache, rightsided hemiparesis and aphasia. turned out to have a polycytemia vera. no. 8 . male, aged 47. hypertension known for 3 years. short attack of dysarthria and weakness of the right arm for 3 h. no. 9 . male, aged 59. headache, dysarthria and right sided facial palsy for 4 h. no. 10. male, aged 80. aphasia and right-sided hemiparesis for one h. no. 1 1 . female, aged 73. hypertension and diabetes mellitus for 5 years. left-sided hemiparesis and babinski’s sign for 12 h. was re-admitted 2 months later with com plete left-sided hemiplegia. lumbar puncture was performed well within 24 h after onset of symptoms. in cases 3 and 7 another spinal fluid examination was made after at least 2 weeks, in a symptom-free interval. simultaneously a blood sample was drawn for analysis of serum adenylate kinase activity. normals: cerebrospinal fluid from 18 patients without any sign of neurological disorder was obtained in con nection with lumbar tap for spinal anesthesia. to rule out any presence of blood in the spinal fluid, sample controls were routinely run for hemoglobin analysis as has been described for serum in an earlier paper (3). 0.5 ml of spinal fluid was routinely used for analysis. the samples were immediately chilled with ice and brought to the laboratory for analysis. the method de scribed by frithz et al. (3) was followed exactly, with the exception that the spectro-photometric analysis was performed in a zeiss spectro-photometer connected to an oltronic stabilizer to eliminate background fluctuations. duplicate controls containing all compounds except the sample, were always run concomitantly, thereby correct ing for the small background activity due to slow physicochemical decay of atp and possible adp con tamination of the atp batch. serum was concomitantly n o adenylate kinase activity was detected in any of the control spinal fluid samples, provided that the samples were not contaminated with blood. there fore, we have reason to believe that normally n o adenylate kinase is present in spinal fluid. t h e 11 patients with t i a all displayed a clear adenylate kinase activity (table i). t h e highest activity observed was 12.0 and the lowest 5.0 mu/ml. n o o n e of the patients had any elevation of the adenylate kinase in serum according to the standard levels determined earlier (3). in t h e two cases, numbered 3 and 7, with activities around 8 mu/ml during the t i a , new analyses were performed after recovery (at least 14 days after the tia). n o adenylate kinase activities were detected in t h e csf on these symptom-free occasions. discussion although t h e clinical picture of t i a is alarming, the underlying conditions are not easily demonstrated by laboratory methods. also, the patho physiological process in the brain parenchyma is subtle, and the changes a r e probably reversible. therefore, ordinary angiograms and brain scinti grams are of no specific aid in visualizing the pathologic condition (2). however, studies on re gional blood flow in patients with t i a have re vealed an impaired circulation (6, 10, 11, 12). thus, there is experimental evidence of reduced blood supply t o distinct areas of the brain during and some time after tia. since brain tissue and especially the neuron a r e highly dependent o n the oxygen sup ply f o r its metabolism and normal function, there is good reason to assume changes on a cellular level in a n y case of reduced oxygen supply. the aerobic metabolism including the tricarboxylic acid cycle plays a central role in brain tissue f o r the mainte nance of normal levels of a t p (13). furthermore, it has been claimed recently that t h e integrity of the plasma membrane, as assessed by its ability to prevent leakage of intracellular en zymes, is dependent upon the energy content of the cell ( 5 , 15, 17). since it was demonstrated in an earlier paper (3) that adenylate kinase was more sensitive a s a marker of ischemic injury of the u p s a h j med sci 82 adenylutr kinase und tia 13 myocardium than are the transferases (asat, alat) and lactate dehydrogenase (ld) it was tempting to study the possible presence of adeny late kinase in spinal fluid in connection with tia. the question then arises whether adenylate kinase is normally present in spinal fluid, e.g. in enlarged quantities with increased age as reported by spolter & thomson (14) who found such an increase for asat and ld. the normals in the present study ranged in age from 24 to 80 years and it was not possible to detect adenylate kinase activ ity in their spinal fluids in any single case. therefore the normals studied were comparable on a n age basis to the persons suffering from tia. further more, the enzyme seems to disappear completely after a certain time after the tia. the disappear ance of the enzyme most probably indicates a re stitution of the intracellular metabolism also com prising the integrity of the plasma membrane. the disappearance of the enzyme may also be due to a total loss of the functions of some cells concerned. the levels of adenylate kinase activity in csf were not high compared with the activities found in serum in connection with myocardial infarction (3) though clearly demonstrable, especially since nor mally no adenylate kinase is present in csf. this finding is however not surprising considering the subtle changes of the brain parenchyma that proba bly take place. the patients differed from each other as regards the level of adenylate kinase activ ity in spinal fluid. all patients with tia so far examined displayed a clear-cut adenylate kinase activity in their csf. due to the limited number of patients investigated, it is not possible to state whether the occurrence of adenylate kinase in spi nal fluid in connection with tia is obligatory or not. the variability of the level of enzyme activity may reflect variation of the amount of brain tissue involved, although the topographic location of the amount of the tia also might influence the amount of enzyme released into the spinal fluid. a method has been described in which the extent of myocardial infarction in man is assessed by mathematical analysis of the rise in plasma enzyme levels, mainly involving asat, alat and ld (18). furthermore the amount of adenylate kinase in serum after infarction parallels that of the trans ferases and ld (3). therefore, a higher level of adenylate kinase activity in spinal fluid during and after tia might indicate a more extensive involve ment of also the brain parenchyma. acknowledgements our thanks are due to mrs maryanne hedstrom. b.a., for excellent technical assistance. we also thank miss anna eckerdahl, head librarian, for kind cooperation. this investigation was supperted by grants from the sodermanland county council, sweden. references 1 . chutorian, a . , gold, a. & carter, s.: cerebrospinal 2 3 4 . 5 . 6. 7. 8 9 10. 11. 12. 13. 14. 15. 16. fluid and serum enzymes in neurological disorders of childhood. trans amer neurol ass 91: 206, 1966. cronquist, s. & muller, r.: brain scanning i n cere brovascular lesions. acta radio1 [diagn] 13: 659, 1972. frithz, g., ericsson, p. & ronquist, g.: serum adenylate kinase activity in the early phase of acute myocardial infarction. upsala j med sci. in press, 1976. green, j. b., oldewurtel, h., o’boherty, d., forster, f. & sanchez-longo, l.: cerebrospinal fluid glutamic oxalacetic transaminase activity in neuro logic disease. neurology 7: 3 13, 1957. hallak, g. & wilkinson, h . : action of metabolic in hibitors on the release of intracellular enzymes from human and rat lymphocytes and human erythrocytes. clin chim acta66:251, 1976. heiss, w.-d., reisner, th., herless, h.-j. & bruck, j.: storungen der regionalen hirndurchblutung bei vaskular bedingten passageren neurologischen aus fallen. wien klin wschr86: 614, 1974. jefferson, m.: the cholinesterase activity of cere brospinal fluid. clin sci 13: 599, 1954. katzman. r., fishman, r. & goldensohm, e.: glutamic oxalacetic transaminase activity in spinal fluid. neurology 7: 853, 1957. lowenthal, a., van sande, m. & karacher, d.: heterogeneity of lactic and malic dehydrogenase in cerebrospinal fluid. j neurochem 7: 135, 1961. paulson, 0 b.: regional cerebral blood flow in cere bral infarction and transient ischemic attacks. rev electroencephalogr et clin neurophysioll: 210, 1974. paulson, 0. b., lassen, n . & skinhnj, e.: regional cerebral blood flow in apoplexy without arterial oc clusion. neurology 20: 125, 1970. rees, j., du boulay, g., bull, j., marshall, j., russel, r. & symon, l . : regional cerebral blood flow in transient ischemic attacks. lancet zz: 1210, 1970. somjen, g., rosentahl, m., cordingeby, g., l a man na, j. & lothman, e.: potassium, neuroglia and oxi dative metabolism in central gray matter. fed proc 35: 266, 1976. spolter, h. & thomson, h.: factors affecting lactic dehydrogenase and glutamic oxalacetic transaminase activities in cerebrospinal fluid. neurology 12: 53, 1962. weed, r. l . , l a salle, p. l . & mevitt, e. w.: metabolic dependence of red cell deformability. j clin invest48: 1794, 1%9. who: cerebrovascular diseases: prevention, treat upsalo j med sci 82 14 g . frithz et at. ment and rehabilitation. wld hlth org, techn rep ser no. 469, 1971. 17. wilkinson, j. h. & robinson, j. m.: effect of energy-rich compounds on release of intracellular en zymes from human leucocytes and rat lymphocytes. clin chem20: 1331, 1974. 18. witteveen, s . , hemker, h., hollaar, l. & haemens, w.: quantitation of infarct size in man by means of plasma enzyme levels. british heart 537: 795, 1975. 19. wolintz, a,, jacobs, l., christoff, n . , solomon, m. & chernik, n.: serum and cerebrospinal fluid en zymes in cerebrovascular disease. arch neurol chicago20: 54, 1969. received september 5,1976 address for reprints: goran frithz, m.d. department of internal medicine central hospital s-63 1 88 eskilstuna sweden upsala j med sci 90: 163-168, 1985 fine-needle biopsies of renal tkansplants in clinical rejection monitoring jan wahlberg,' ulla backman,2 lars frodin,' bjorn stenkvist3 and gunnar tufveson' departments of 'urology, 2nephrology and 3pathology, university hospital, uppsala, sweden abstract f i n e n e e d l e a s p i r a t i o n b i o p s y (fnab) o f r e n a l a l l o g r a f t t r a n s p l a n t s has been used a t uppsala u n i v e r s i t y h o s p i t a l f o r 3 y e a r s . e x p e r i e n c e from 5 1 c o n s e c u t i v e p a t i e n t s ( f r o m 1 4 y e a r s ) w i t h 333 fnab was r e v i e w e d . r e p r e s e n t a t i v e m a t e r i a l was o b t a i n e d i n more t h a n 70 5 o f t h e b i o p s i e s . e l e v e n r e j e c t i o n e p i s o d e s i n 1 2 p a t i e n t s were c o n f i r m e d w i t h t h i s method. one was n o t r e c o g n i z e d . s i g n i f i c a n t i n f l a m m a t i o n i n t h e k i d n e y w i t h o u t c l i n i c a l r e j e c t i o n was found i n 22 p a t i e n t s . the p o s s i b l e causes o f such i n f l a m m a t i o n a r e d i s c u s s e d . repeatedly r e c o r d e d i n f l a m m a t i o n i n t h e k i d n e y w i t h m i n o r or no e f f e c t on g r a f t f u n c t i o n may some t i m e s be caused b y v i r a l i n f e c t i o n . the c l i n i c a l v a l u e o f fnab i n v a r i o u s i m munosuppressive regimens i s d i s c u s s e d . introduction f i n e n e e d l e a s p i r a t i o n b i o p s y (fnab) as a d i a g n o s t i c t o o l i n c l i n i c a l r e n a l t r a n s p l a n t a t i o n was f i r s t r e p o r t e d b y p a s t e r n a c k e t a l . i n 1973 ( 9 ) , and t h e method was r e f i n e d by hayry & von w i l l e b r a n d ( 4 ) . i n 1 9 8 1 uppsala became one o f t h e f i r s t f i v e t r a n s p l a n t c e n t r e s t o use t h i s method, w h i c h has g a i n e d c l i n i c a l acceptance and c u r r e n t l y i s used i n a t l e a s t 50 t r a n s p l a n t c e n t r e s t h r o u g h o u t t h e w o r l d . two i n t e r n a t i o n a l workshops have been c o n d u c t e d on t h e i s s u e ( 7 ) . much e f f o r t has been d e v o t e d t o i n v e s t i g a t i n g t h e r e l i a b i l i t y o f fnab i n r e n a l t r a n s p l a n t s and t o r e f i n e m e n t o f t h e c y t o l o g i c d i a g n o s i s , e.g. u s i n g immune h i s t o c h e m i c a l t e c h n i q u e s ( 3 ) . l e s s i n f o r m a t i o n i s a v a i l a b l e c o n c e r n i n g t h e i m p a c t o f d i f f e r e n t immunosuppressive regimens on t h e method's c l i n i c a l u s e f u l ness. i n s t e a d o f comparing fnab r e s u l t s w i t h t h e h i s t o l o g i c r e p o r t s , we have re l a t e d them t o t h e c l i n i c a l s t a t u s o f t h e p a t i e n t s . a r e t r o s p e c t i v e r e v i e w o f o u r own e x p e r i e n c e i s now p r e s e n t e d . the i m p a c t o f immunosuppression w i t h l o w dose s t e r o i d s and c y c l o s p o r i n or a z a t h i o p r i n e on fnab was s t u d i e d . 163 material and methods p a t i e n t s the s t u d y c o m p r i s e d 5 1 c o n s e c u t i v e p a t i e n t s who had r e c e i v e d k i d n e y a l o , g r a f t and were i n v e s t i g a t e d w i t h fnab d u r i n g t h e i r i n i t i a l h o s p i t a l s t a y r immunosuppressive r e g i m e n c o n s i s t e d i n 1 8 cases o f a z a t h i o p r i n e ( i m u r a n , wellcome, london, uk) and low-dose s t e r o i d s (aza) as o u t l i n e d by mcgeown e t a l . ( 8 ) and 30 p a t i e n t s r e c e i v e d c y c l o s p o r i n r (sandoz, b a s l e , s w i t z e r l a n d ) and low-dose s t e r o i d s (cya) a c c o r d i n g t o t h e p r o t o c o l o f t h e s c a n d i n a v i a n m u l t i c e n t e r t r a n s p l a n t study ( 6 ) . three p a t i e n t s were s w i t c h e d f r o m one r e g i m e n t o t h e o t h e r (aza/cya). c l i n i c a l r e e v a l u a t i o n the the c l i n i c a l c o u r s e was r e t r o s p e c t i v e l y r e e v a l u a t e d . a d i a g n o s i s o f r e j e c t i o n was based on c l i n i c a l o b s e r v a t i o n s o f w e i g h t g a i n , f e v e r and p a i n o v e r t h e g r a f t c o n c o m i t a n t w i t h r i s i n g serum c r e a t i n i n e . i n a d d i t i o n , most o f t h e p a t i e n t s responded w i t h f a l l o f serum c r e a t i n i n e t o supplementary s t e r o i d t r e a t ment. f i n e n e e d l e a s p i r a t i o n fnab was p e r f o r m e d as d e s c r i b e d by hayry & von w i l l e b r a n d ( 5 ) . b r i e f l y , a s p i n a l n e e d l e was i n t r o d u c e d under s t e r i l e c o n d i t i o n s i n t o t h e g r a f t and cells f r o m t h i s t i s s u e were a s p i r a t e d i n t o a s y r i n g e c o n t a ~ n ~ n g t i s s u e c u l t u r e medium. a c a p i l l a r y b l o o d sample was c o l l e c t e d a t t h e same t i m e t o p e r m i t c a l c u l a t i o n o f a c o r r e c t e d i n c r e m e n t . these b i o p s i e s u s u a l l y were p e r f o r m e d a t r e g u l a r i n t e r v a l s ( t h r i c e w e e k l y ) d u r i n g t h e i n i t i a l p m t o p e r a t i v e s t a y . some b i o p s i e s were p e r f o r m e d l a t e r , a s need arose. t a b l e 1. number o f b i o p s i e s a c c o r d i n g t o iwmunosuppressive r e g i m e n no o f t o t a l n o no o f n o n r e p r e s e n t treatment * p a t i e n t s o f fnab a t i v e fnab c y a aza aza/cya 30 190 56 18 106 31 3 37 5 t o t a l 5 1 333 92 * d e f i n i t i o n s i n m a t e r i a l and methods 164 evaluation of fnab cytologic evaluation of the biopsy specimens was performed as previously de scribed (11). essentially this method transposes the observed occurrence of in flammatory cells within the graft to a numerical value called the corrected increment. a corrected increment higher than 2.9 was classified as significant inflammation. only biopsies containing 10 o r more kidney tissue cells per 100 inflammatory cells were regarded as representative. results table 1 summarizes the total number of biopsies and the number of nonrepre sentative specimens in the three treatment groups. more than 70 per cent of the biopsies were representative. in this respect there was no clear difference ac cording to immunosuppressive regimen. the number of biopsies per patient was highest in the aza/cya group, the reason being that these three patients had a more difficult and protracted postoperative course than the average. table 2 . number of patients with ( > 2 . 9 ) o r without ( < 2 . 9 ) inflamnation in fnab at the start of clinical rejection corrected increment treatment > 2 . 9 < 2.9 cya 0 0 aza/cya 2 1 ** total with rejection 8 4 aza 6 3 * * next biopsy positive in 2 of these patients ** next biopsy positive table 2 presents the corrected increment in the cases with clinical signs of rejection. in 11 of the 12 episodes of rejection there was significant rise in corrected increnent. in three patients, however, the increment was below the significance level ( > 2 . 9 ) in the morning of the day when rejection started, but had risen when the next fnab was taken. f o r the fourth patient without significant corrected increment no further fnab information was available. not ably, there was no rejection episode in the cya group. the three patients on aza/cya had rejection only when on the aza regimen. 165 t a b l e 3. c o r r e c t e d i n c r e m e n t b u t w i t h o u t c l i n i c a l r e j e c t i o n s i g n s number o f p a t i e n t s and number o f fnabs w i t h h i g h no o f h i g h i n c r e m e n t s / p a t i e n t no o f treatment 1 2 3 4 5 >6 p a t i e n t s aza 4 1 c y a 5 6 aza/cya 1 2 a 1 12 1 2 the number o f h i g h c o r r e c t e d i n c r e m e n t s ( > 2.9) i n t h e absence o f r e j e c t i o n s i g n s i s shown i n t a b l e 3. a l t o g e t h e r 22 o f t h e 51 p a t i e n t s t h u s had a t l e a s t one such fnab b u t no c l i n i c a l s i g n s o f g r a f t r e j e c t i o n . r e t r o s p e c t i v e a n a l y s i s o f a l l t h e s e o c c a s i o n s was n o t w i t h i n t h e scope o f t h e p r e s e n t s t u d y . however, c o n c o m i t a n t c y t o m e g a l o v i r u s i n f e c t i o n was known f r o m v i r a l i s o l a t i o n or con v e n t i o n a l s e r o l o g y t o have been p r e s e n t i n t w o o f t h e f i v e p a t i e n t s w i t h a t l e a s t f o u r h i g h i n c r e m e n t s . f o r t h e o t h e r t h r e e p a t i e n t s no such i n f o r m a t i o n was a v a i l a b l e . discussion fnab was e q u a l l y r e l i a b l e i n p a t i e n t s w i t h cya and i n t h o s e w i t h aza i m munosuppression. more t h a n 70 p e r c e n t o f t h e r o u t i n e l y p e r f o r m e d b i o p s i e s y i e l d e d r e p r e s e n t a t i v e specimens. t h i s p e r c e n t a g e s h o u l d be i m p r o v a b l e w i t h use o f u l t r a s o n i c g u i d i n g , e s p e c i a l l y i f t h e k i d n e y i s d i f f i c u l t t o p s l p a t e . i f a r e p r e s e n t a t i v e specimen i s p a r t i c u l a r l y d e s i r a b l e , t h e p r o b a b i l i t y o f success can be i n c r e a s e d by making s e v e r a l b i o p s i e s . the p r o c e d u r e is i n no way h a r m f u l t o t h e p a t i e n t , i n o u r e x p e r i e n c e o r i n t h a t f r o m o t h e r c e n t r e s ( 7 ) . we have p e r f o r m e d more t h a n 700 b i o p s i e s w i t h o u t c o m p l i c a t i o n s a p a r t from b r i e f , m i l d h a e m a t u r i a . the main advantage o f a s p i r a t i o n o v e r " t r u c u t " b i o p s y i s , i n f a c t , t h a t fnab can be p e r f o r m e d as o f t e n as n e c e s s a r y , thus p e r m i t t i n g immunologic m o n i t o r i n g o f t h e p a t i e n t . the c l i n i c a l r e l e v a n c e o f t h i s m o n i t o r i n g can p e r h a p s be debated. when t h e aza im-nunosuppressive r e g i m e n was used, 11 o f 12 c l i n i c a l re j e c t i o n e p i s o d e s were diagnosed. s i g n s o f i n f l a m m a t i o n i n t h e k i d n e y a r e n o t pathognomonic f o r r e j e c t i o n . thus c o r r e c t e d i n c r e m e n t s o f > 2.9 were o f t e n seen w i t h o u t c l i n i c a l s i g n s o f r e j e c t i o n . the r e a s o n was n o t c l e a r , t h o u g h s e v e r a l e x p l a n a t i o n s may be proposed. f i r s t , n o t a l l immune r e a c t i o n s t o t h e g r a f t may damage g r a f t f u n c t i o n . i n a d d i t i o n , subc i n i c a l r e j e c t i o n e p i s o d e s may o c c u r . 166 t h i r d l y , v i r u s i n f e c t i o n s w i t h i n t h e g r a f t may cause immune r e a c t i o n s t o v i r a l a n t i g e n s w i t h l i t t l e e f f e c t on k i d n e y f u n c t i o n b u t e v o k i n g an i n f l a m m a t o r y re sponse. f o u r t h l y , t r a n s p l a n t a t i o n o f organs always i n d u c e s i s c h a e m i c damage t o t h e g r a f t t h a t may l e a d t o some i n t r a g r a f t i n f l a m m a t i o n . thus, s y n g e n e i c r a t a l l o g r a f t s a r e i n f i l t r a t e d w i t h l y m p h o c y t e s , b u t t h e g r a f t s a r e n o t r e j e c t e d (1). i r r e s p e c t i v e o f t h e cause o f i n f l a m m a t i o n o b s e r v e d i n fnab from n o n r e j e c t i n g p a t i e n t s , i t s f r e q u e n t o c c u r r e n c e must l e a d t o c a u t i o n i n a d m i n i s t e r i n g a n t i r e j e c t i o n t h e r a p y t o p a t i e n t s w i t h h i g h c o r r e c t e d i n c r e m e n t s u n l e s s t h e r e a r e c l i n i c a l s i g n s o f r e j e c t i o n . i f h i g h i n c r e m e n t s a r e r e p e a t e d l y found i n a p a t i e n t w i t h o u t c l i n i c a l s i g n s o f r e j e c t i o n , a s e a r c h f o r v i r u s i n f e c t i o n is ad v i s a b l e ( u n p u b l i s h e d r e s u l t s ) . from o u r t o t a l e x p e r i e n c e o f fnab i n a z a t r e a t e d p a t i e n t s , i t seems w a r r a n t a b l e t o i n c l u d e t h e s e b i o p s i e s i n t h e p o s t o p e r a t i v e ( f i r s t 3 weeks) m o n i t o r i n g o f r e n a l t r a n s p l a n t p a t i e n t s . i f r e j e c t i o n i s sus p e c t e d , a t l e a s t two b i o p s i e s s h o u l d be p e r f o r m e d , i f necessary g u i d e d by u l t r a s o u n d s c a n n i n g , t o i n c r e a s e t h e p o s s i b i l i t y o f a w e l l r e p r e s e n t a t i v e b i o p s y . t h i s s t u d y d e m o n s t r a t e s t h e r a r i t y o f c l i n i c a l r e j e c t i o n e p i s o d e s w i t h o u r p r e s e n t cya regimen d u r i n g t h e p o s t o p e r a t i v e p e r i o d . i n c l u s i o n o f fnab i n t h e r o u t i n e m o n i t o r i n g o f c y a t r e a t e d p a t i e n t s is t h e r e f o r e o f q u e s t i o n a b l e j u s t i f i c a t i o n . we s h a l l c o n t i n u e t o use t h e p r o c e d u r e f o r s e v e r a l reasons, however. c y c l o s p o r i n i s a n e p h r o t o x i c d r u g and t h e serum l e v e l o n l y p a r t l y r e f l e c t s i t s t o x i c i t y . some i n f o r m a t i o n o f t h e n e p h r o t o x i c a c t i o n can be g a i n e d f r o m s t u d y i n g t h e morphology o f t h e t u b u l a r c e l l s i n t h e a s p i r a t e s ( 1 2 ) . as e a r l i e r s t a t e d , r e p e a t e d l y h i g h i n c r e m z n t s have l e d us t o i n t e n s i f y s e a r c h f o r v i r a l i n f e c t i o n s . use o f fnab s h o u l d f a c i l i t a t e comparisons w i t h some o t h e r , r e c e n t l y suggested m o n i t o r i n g a l t e r n a t i v e s ( 2 , l o ) . acknowledgements we w i s h t o t h a n k mrs i n g e g e r d h a k i n g and mrs norma jansson f o r e x p e r t t e c h n i c a l a s s i s t a n c e . the c l i n i c a l r e s e a r c h was s u p p o r t e d by t h e tore n i l s s o n and maud & birger gustavsson f o u n d a t i o n s and by t h e swedish m e d i c a l research c o u n c i l . references 1. claesson, k., forsum, u . , k l a r e s k o g , l., l a r s s o n , e., f r o d i n , l . & tufveson, g.: t c e l l s u b s e t s and i a e x p r e s s i n g c e l l s i n t r a n s p l a n t e d r a t k i d n e y s . t r a n s p l a n t proc. i n p r e s s . 167 2. claesson, k . , ronnblom, a., alm, g.v. & tufveson, g.: antiviral activity appearing in serum of renal transplant recipients. transplantation 39:32, 1984. odling, b., stenkvist, b. & tufveson, g.: methods for monitoring t lympho cyte subsets in renal allografts. transplant proc 15:1178,1983. 4. hayry, p. & von willebrand, e.: monitoring of human renal allograft reject ion with fine needle aspiration cytology. scand j immunol 13:87,1981. 3. forsurn, u . , claesson, k., frodin, l., karsson-para, a., klareskog, l., 5. 6 . 7. 8. 9. 10. 11. 12. hayry, p. & von willebrand, e.: practical guide lines f o r fine needle as piration biopsy of human renal allografts. ann clin res 13:288, 1981. klintmalm, g., brynger, h., flatmark, a., frodin, l . , husberg, b., thorsby, e. & groth, c.g.: the blood transfusion and dr-matching effect on cadaveric renal transplantation has been abolished by the use of cyclosporin. transplant proc. in press. kreis, h. & droz, d. (eds.) proc of 2nd int workshop on renal transplant cytology, 1984. in press. mcgeown, m.g., loughridqe, w.g.g., alexander, j.a., mcevoy, j., kennedy, j.a., douglas, j., clarke, s.d. & hewitt, j.c.: one hundred kidney trans plants in t h e belfast city hospital. the lancet 24:648,1977. pasternack, a., virolainen, m. & hayry, p.: fine needle aspiration biopsy in the diagnosis of human renal allograft rejection. j urol 109:167,1973. tufveson, g., gronowitz, j.s., larsson, a., kallander, c.f.r., claesson, k., sjoberg, 0 . wahlberg, j. & frodin, l.: the possible relation between serum levels of deoxythymidine kinase and rejection o r infection in renal transplant patients. in proc of 16th clin immunol. lyon, france, 1984. in press. von willebrand, e.: fine needle aspiration cytology of human renal trans plants. clin imnunol immunopathol 17:309, 1980. von willebrand, e. & hayry, p.: cyclosporine a deposits in renal allo qrafts. the lancet ii:189, 1983. int course on transplantation & address for reprints jan wahlberg department o f urology university hospital s-751 85 uppsala sweden 168 upsala j med sci 92: 115-146, 1987 the complete amino acid sequence of human serum retinol-binding protein lars rask', helena anundi', jan fohlman3 and per a. peterson4 department of cell research, university of uppsala and swedish university of agricultural sciences, uppsala, sweden abstract the complete amino acid sequence of human serum retinol-binding protein (rbp) including the distribution of its three disulfide bridges, has been determined. the protein consists of 1 8 2 amino acid residues, the order of which was determined following the isolation of five cnbr-fragments. direct amino acid sequence analysis in an automatic liquid phase sequencer provided almost the entire sequences of the five cnbr-fragments. several sets of enzymatically derived peptides of rbp were also used to elucidate the.primary structure. rbp displays significant homology to bovine p-lactoglobulin, human al-microglobulin and rat al-microglobulin. rbp contains an internal homology. thus, residues 36 to 83 display statistically significant homology with residues 9 6 to 1 4 1 . introduction from its site of synthesis in the liver ( 2 8 , 3 6 , 4 8 ) the retinol-binding protein (rbp)' carries one molecule of retinol ( 2 0 , 3 3 , 3 4 ) to vitamin a requiring cells. while transporting retinol in plasma, rbp forms a stable complex with thyroxine-binding prealbumin ( 2 0 , 3 3 ) . this complex formation prevents rbp, which has a molecular weight of 21 000, to pass the kidney glomeruli ( 3 4 ) . cells requiring vitamin a express a receptor for rbp on their cell membranes ( 1 9 , 4 1 1 . on recognizing rbp the receptor takes up the vitamin. simultaneously, rbp undergoes a conformational change, the nature of which is presently unknown. this conformational change does not allow a sustained binding between rbp and prealbumin ( 1 9 , 4 3 1 . due to the abolished protein-protein interaction the free rbp molecule becomes degraded in the 'abbreviations used are: rbp retinol-binding protein dansyl 1-dimethyl-aminoaphthalene-5-sulphonyl chloride edta ethylenediaminotetraacetate cm-cysteine carboxymethylcysteine cys-a cysteic acid 8-878572 115 kidney following glomerular filtration and reabsorption in the tubuli cells (34). to understand how rbp interacts with retinol, prealbumin and the cell surface receptor and how these interactions may become modulated, it appeared of importance to establish the amino acid sequence of rbp. the primary struc ture of rbp was also a prerequisite for the interpretation of high-resolu tion x-ray crystallographic data. in this communication we describe the complete amino acid sequence of human rbp. part of this information has appeared in preliminary form (40). a partial primary structure of human rbp has also been reported by kanda and goodman ( 2 1 ) . recently, a cdna clone encoding human rbp has been analysed ( 5 ) . materials and methods isolation of rbp the rbp used in the sequence studies was isolated from human serum (34) and urine (35). the purity of the rbp preparations was assessed as described (34,351. peptide nomenclature the peptides obtained after cyanogen bromide cleavage are designated a . b, and c followed in some instances of a number and a letter, indicating the order of emergence of a particular peptide during fractionation. h denotes a peptide obtained after acid cleavage. peptides isolated after digestion of rbp by trypsin, chymotrypsin, thermolysin, and clostripain are symbolized by r, rc, rt and c1, respectively. peptides obtained from cnbr-fragment a3b after digestion with clostripain are designated a3b and those from tryptic and chymotryptic digestions of cnbr fragment c, c and ct, respectively. peptides isolated from cnbr fragment a1 after digestion with staphylococcus aureus protease v 8 are called sa sb, with chymotrypsin ac, with thermolysin at, with pepsin ap, with subtilisin as and with clostripain ac1, respectively. peptides obtained after cleavage of unreduced rbp with acid, trypsin and pepsin are denoted s, t and p, respectively. the numbers that follow the symbols indicate the order of emergence of a particular peptide during fractionation. reduction, alkylation, cnbr-fragmention and acid cleavage these procedures were carried out as described (51). enzymatic digestion of rbp and rbp fragments trypsin digestions were performed on samples (0.1 to 3 pmoles) in 0.2 m nh4hc03, ph 8.0, at protein to enzyme ratios of 1oo:l to 5 0 : l . the protein or peptide concentration was usually between 5 and 10 mgfml. digestions were carried out at 37o for 3 hours and were terminated by lyophilization. a-chymotrypsin and subtilisin digestions were performed similarly. also thermolysin digestions were and 116 conducted in the same fashion but the buffer was 0 . 2 m nh4hco3, ph 8.0, containing 5 mm cac12 and the reaction was terminated after 2 pepsin digestions were carried out at an enzyme to substrate ratio of 1:50 (w/w) at 37o for 3 hours in 5% (v/v) formic acid. digestions with clostripain were performed at ph 7.8 in 0.1 m nh4hc03. containing 2 mm dtt and 1 mm cac12. after 2 to 3 hours at 37o the reaction mixture was lyophilized. the substrate to enzyme ratio was 5o:l. staphylococcus aureus protease v8 was used at a protein to enzyme ratio of 50:l. the substrate was dissolved in 0.2 m nh4hc03, ph 8.0, containing 1 mm edta and after 2 hours at 37o the digestion mixture was lyophilized. hours. to investigate the distribution of the disulfide bridges of rbp, the cnbr fragments al, a2, a3a and a3b, which were held together by disulfide bonds (fraction a of fig. la), were digested with trypsin and pepsin. the cnbr fragment mixture, at a concentration of about 10 mg/ml in 0.2 m tris-acetate buffer, ph 6 . 0 , was digested for 8 hours at 37o with trypsin at an enzyme to substrate ratio of 1:50. the same amount of cnbr-fragments in 0.2 m sodium acetate buffer, ph 5.0, was digested with pepsin (final concentration 0.2 mg/ml) for 8 hours at 37o. carboxypeptidase a and b digestions were carried out as described (51). peptide fractionation large peptides of rbp were usually separated by gel chromatography on columns of sephadex g 1 0 0 and g 5 0 equilibrated with 0.05 m sodium acetate buffer, ph 5.0, containing 6m guanidine-hc1 or with 10% propanol 0.025% ammonia in water. smaller peptides were purified by high voltage electrophoresis in pyridine-acetate buffer, ph 6.5 (pyridine:acetic acid:water, 100:3:897 v/v) and ph 3.5 (pyridine:acetic acid:water, 1:10:189 v/v). the electrophoreses were carried out on 60 to 100 cm long whatman no 3mm papers at 40 v/cm for 60 to 100 min. further purification of impure peptide fractions was accomplished by descending paper chromatography developed with butano1:acetic acid:water:pyridine (15:3:12:10 v/v). localization of peptides was accomplished by staining the papers with fluorescamine. purified peptides were eluted from papers with 0.1% ammonia. some peptides were purified by ion exchange chromotography on deae sepharose columns equilibrated with 0.02 m nh4hc03. the applied sample was usually eluted with a 250 ml linear gradient of nh4hc03 from 0.02 to 0.2 m. the occurrence of peptides in the effluent was monitored by measuring the absorbance at 2 2 0 nm or at 280 nm. occasionally aliquots were withdrawn for ninhydrin analysis (see below). peptide digests were also separated on a modified jeol-5 ah amino acid analyzer (18). the column (11~0.5 cm), maintained at a temperature of 50°, 117 contained the jeol type ar-15 sulfonated resin. the applied material, usually between 15 and 30 mg of peptide mixture, was eluted as described (18). the flow rate was 1.85 ml/min and fractions of 3.0 ml were collected. for the separation of some peptides advantage was taken of column zone electrophoresis (37). the column (86x1 cm), packed with water-pyridine-extracted cellulose and cooled by running water, was equilibrated with a ph 1.9 buffer composed of acetic acid:formic acid:water (78:25:897 v/v). after application the samples were usually displaced downward to an appropriate starting point and runs were conducted at 1000 v for 8 to 12 hours. after electrophoresis the column, which had a total free liquid volume of about 60 ml, was eluted at a flow rate of 12 ml/hour (13). high pressure liquid chromatography was also used to purify some peptides. two model lloa pumps (altex, berkley, california), an altex model 400 solvent programmer and a micro-bondapak c18 column (300x3.9mm, waters associates inc., milford, mass.) were used. the column was equilibrated with 2 mm ammonia adjusted to ph 2 . 4 with trifluoroacetic acid and 5% methanol. elution was accomplished with a linear gradient of methanol from 5% to 55% followed by 30 ml of 55% methanol in the ammonia trifluroacetic acid buffer. peptides in the effluent were detected by measuring the absorbance at 206 nm. the flow rate was 24 ml/h and fractions of 0 . 4 ml were collected. alkaline hydrolysis and ninhydrin analysis for alkaline hydrolysis appropriate aliquots were evaporated to dryness at 110'. 0.5 ml of 2.5 m naoh was added. the hydrolysis was carried out at lloo for 3 hours. following neutralization with 1.0 ml of 1.5 m acetic acid, 1.0 ml of the ninhydrin reagent was added. after 15 min in a boiling water bath each fraction was diluted with 2 ml of 50% ethanol and the absorbance at 570 nm was estimated.. amino acid analyses amino acid analyses were carried out as described (51). amino acid sequence determinations automatic amino acid sequence determinations were carried out as described (51). amino acid sequence determinations were also accomplished with the dansyl end group method in conjunction with the edman technique (17). dansyl amino acids were identified by two-dimensional chromatography on 5x5 cm polyamide thin layer sheets. the solvent systems used were those of woods and wang (56). statistical analyses for relatedness of rbp sequence to other proteins -the rbp sequence was compared to a data file containing sequences of other proteins ( 6 9 ) , with the search program (9). the program align ( 2 9 ) was used to analyse the alignment of homologous sequences. the matrix bias parameter and the break penalty parameter were set to 2 and 6, respectively. these values are appropriate when comparisons are made between distantly related sequences. 118 results isolation and nh2-terminal amino acid sequence determination of human rbp cnbr-fragments -rbp was cleaved with cnbr and the resulting fragments purified by repeated gel chromatographies (fig. 1 and 2). the amino acid compositions and the yields of the fragments are summarized in table 1. since human rbp contains four methionines (43) five cnbr-fragments were expected. however, six fragments were isolated (table 1). this result together with the observations that the amino acid composition of fragment a2 is almost identical to the combined composition of fragments b and a3b, that a2 contains more homoserine residues than anyone of the other fragments and that the yields of fragments a2, b and a3b vary somewhat from preparation to preparation, strongly suggested that fragment a2 was a product of incomplete cnbr-cleavage. fragment a1 was the only one lacking homoserine (table 1). fig. 1. gel chromatography on a column (142~1.5 cm) 0 w ;'ol of sephadex-g-io0 equilibrated with 0.05 m sodium acetate buffer, ph 5.0, containing 6m guanidine-hc1. a . the sample, 80 mg of cnbr-cleaved rbp, was eluted at a flow rate of 4 ml/h and fractions of 1.5 ml were collected. the bars denote materials which were pooled, desalted and lyophilized. b. fraction a fig. 1. a was dissolved in 3 ml of 1 m tris-c1 buffer,ph 8.0, containing 6 m guanidine-hc1 and 50 mm edta. after the addition of dithiothreitol to a final concentration of 10 mm the sample was incubated for 30 min at room temperature. iodoacetic acid to a final concentration of 25mm was then added and after another 15 min in the dark the sample was exhaustively dialyzed against the equilibrating buffer of the column. the sample was then chromatographed on the sephadex g-100 column under conditions identical to those described above. 'the bars denote materials which were use6 in subsequent analyses. fig. 2. gel chromatography on a column (125 x 1.5 cm) of sephadex g-50 equilibrated with 0.05 m sodium acetate buffer, ph 5.0, containing 6 m guanidine-hc1. the material subjected to fractionation was fraction a3 of fig. 1b. the column was operated with a flow rate of 6 ml/h and 1.5 ml fractions were collected. material denoted by bars were pooled, desalted and lyophilized. 119 table 1. amino acid compositions of cyanogen bromide fragments of human rbpa the integral values in parantheses are based on the sequence. cnbr-a1 cnbr-a2 cnbr-a3a cnbr-a3b cnbr-b cnbr-c rbp" residue 89-182 28-73 1-27 54-73 28-53 74-88 1-182 lysine 3.41(3) 2.75(3) 2.00(2) l.oo(1) 1.86(2) 1.82(2) 10.19(10) histidine 1.88(2) 0.26 2.00(2) ar g in ine 7.88(8) 2.36(2) 3.68(4) 2.09(2) 0.30 14.02(14) cm-cysteine 4.12(4) 1.33(1) 1.21(1) 0.72(1) 4.91(6) aspartic acid 14.01(14) 6.90(8) 3.36(3) 3.54(4) 4.00(4) 2.00(2) 26.03(27) threonineb 3.45(3) 1.90(2) 1.13(1) 0.95(1) 1.25(1) 2.60(3) 9.35(9) ser ineb 5.67(6) 2.65(2) 3.15(3) 1.23(1) 1.35(1) 0.38 10.89(11) homoserine 0.05 0.56(2) 0.39(1) 0.42(1) 0.40(1) 0.48(1) glutamic acid 10.30(10) 5.94(5) 2.30(2) 0.43 4.38(5) 1.41(1) 18.28(18) proline 3.36(3) 1.36(1) 1.10(1) 1.18(1) 6.84(5) glycine 6.23(6) 2.87(3) 1.17(1) 1.20(1) 2.00(1) 1.41(1) 11.21(11) alanine 5.35(5) 4.08(5) 2.36(2) 2.57(3) 2.01(2) 1.12(1) 13.90(13) valinec 5.95(6) 3.69(4) 2.05(2) 1.97(2) 1.89(2) l.oo(1) 12.20(13) methionine 3.84(4) isoleucinec 3.07( 3) 0.78( 1) 0.68(1) 3.96(4) leucine 9.00(9) 3.89(4) 1.65(2) 1.95(2) 0.36 12.78)13) tyrosine 6.75(7) 0.41 0.93(1) 0.37 8.06(8) phenylalanine 3.29(3) 2.24(2) 2.64(3) 0.34 1.81(2) 1.80(2) 10.14(10) tryptophan 1.48(2) 0.61(1) 0.67(1) 0.71(1) 5.22(4) yieldf ( x ) 80 40 82 45 40 85 aexcept where noted all figures are average values of one 24 h and one 72 bvalues obtained by extrapolation to 0 h hydrolysis. '72 h hydrolysis value. ddata taken from ref. 40. edetermined spectrophotometrically. fthese data are based on one preparation from 6pmoles of rbp. other prepara hydrolysis tions gave similar values although the yields of fragments a2, a3b and b somewhat v a r i e d . intact, reduced and carboxymethylated rbp and the six cnbr-fragments were separately subjected to nh2-terminal amino acid sequence determination in an automatic liquid phase sequencer. by this procedure almost the entire primary structure of fragments a3a, b, a3b and c could be elucidated (table 2 and fig. 3). the nh2-terminal amino acid sequences of fragments a2 and b were identical confirming that fragment a2 is the result of incomplete cnbr cleavage of rbp. the nh2-terminal amino acid sequence of intact rbp provided unambiguous information for 40 residues (table 2 and fig. 3). this information was sufficient to establish that fragment a3a is the nh2-terminal cnbr-fragment and that it is followed by fragment b(a2) in the sequence. alignment of the cnbr-fragments of human rbp in order to establish the order of the cnbr-fragments of rbp the intact protein was digested separately with several enzymes and during fractionation methionine-containing peptides were particularly looked for. during the course of this work a number of other 120 peptides were also obtained, some of which were important for establishing the primary structure of rbp. thus, in this section such peptides will also be described. reduced and carboxymethylated rbp was digested with trypsin, chymotrypsin and thermolysin, respectively. after lyophilization each digest was separately subjected to gel chromatography on a column of sephadex g-25 (fig. 4 ) . nh2 terminal amino acid sequence determinations demonstrated that all peptide fractions obtained were impure. further purification was accomplished by combining high-voltage paper electrophoresis with paper chromatography. the amino acid compositions, the purification procedure and the amino acid sequence of each peptide are presented in table 3a. peptides r5, r10, rc2, rc3, rc8 and rt1 contained methionine residues. the amino acid sequences of peptides r5, r10 and rc2 (table 3b) established that cnbr-fragments a3a and b were juxtaposed (see above). peptide rt1 connected cnbr-fragments b and a3b. since cnbr-fragment a 1 lacked homoserine it had to be the cooh-terminal fragment. consequently, the only remaining fragment, c, had to be positioned in between fragments a3b and al. this notion was supported by the observation that peptide rc8 connected c with a1 (table 3). further support for the order of the cnbr-fragments was obtained from analyses of two other peptides. after cleavage of intact, reduced and carboxymethylated rbp with clostripain, two peptides were isolated following gel chromatography on a column of sephadex g-100 (fig.5). after rechromatography on the same column of the peaks denoted c1 i and c1 i1 (fig. 5b and c) amino acid analyses demonstrated that c1 i comprised 59 and c1 i1 41 amino acid residues (table 4 ) . nh2-terminal amino acid sequence determination in the automatic sequencer of fragment c1 i provided almost its entire structure (table 4). this result definitely showed that cnbr-fragment a3a was followed by b and a3b. the nh2-terminal 15 residues of fragment c1 i were determined (table 4 ) which connected fragment a3b with c. thus, the order of the cnbr-fragments of rbp is a3a-b-a3b-c-a1. cnbr-fragment a2 occurs as a consequence of the incomplete cleavage of the met-ser bond joining cnbr fragment b with a3b (see fig. 6). fig. 3. (next page) the yields of pth-amino acids in each degradation cycle. the materials subjected to amino acid sequence determination contained a) intact, reduced and alkylated rbp; 120 nmoles. b ) cnbr-fragment a3a; 90 nmoles. c) cnbr fragment a2; 210 nmoles. d ) cnbr-fragment b; 110 nmoles. e ) cnbr-fragment a3b; 80 nmoles. f) cnbr-fragment c; 120 nmo1es.g) cnbr-fragment al; 240 nmoles and h ) acid cleavage fragment h-2; 220 nmoles. the initial yields, which in the figure are normalized to 100% were a) 7 4 nmoles. b) 52 nmoles. c ) 130 nmoles. d ) 61 nmoles. e) 49 nmoles. f) 67 nmoles. g ) 138 nmoles and h) 113 nmoles, respectively. 121 -1 * w 1 i i i (3 e -0 0 0 bod.obb,, t o o ' 0 % o c a o 0 a0 o o o o o 0 : o 08, 50 0 b o o o o o a 00 0 -_ 0 0 8 0 10 0 00,". oo 0 o 0 0 qj 0 0 2 i 8 1 d i 1 i i .h 0 0 0 0 0 0 %o 0 00 0 0 0 0 soo 0 _ _ 0 oo ,-o" 0 *o% i i table 2. nh2-terminal amino acid sequence determination of cnbr-fragments of rbp (a) and of intact rbp (b). material amino acid sequence a . a1 lys-tyr-trp-gly-val-ala-ser-phe-leu-gln-lys-gly-asn -asp-asp-his-trp-ile-val-asp-thr-asp-tyr-asp-thr-tyr -ala-val-gln-tyr-ser-cmcys-arg-leu-leu-asn-leu-asp -gly-thr-cmcys-ala-asp-ser-tyr-ser-phe-val -1le-val-ala-glu-phe-ser-val-asp-glu-thr-gly-gln a2 ala-lys-lys-asp-pro-glu-gly-leu-phe-leu-gln-asp-asn a3a glu-arg-asp-cmcys-arg-val-ser-ser-phe-arg-val-lys-glu -am-phe-asp-lys-ala-arg-phe-ser-gly-thr a3b ser-ala-thr-ala-lys-gly-arg-val-arg-leu-leu-asn-asn -trp-asp-val-cmcys b ala-lys-lys-asp-pro-glu-gly-leu-phe-leu-gln-asp-asn -1le-val-ala-glu-phe-ser-val-asp-glu-thr-gly c val-gly-thr-phe-thr-asp-thr-glu-asp-pro-ala-lys-phe b. rbp glu-arg-asp-cmcys-arg-val-ser-ser-phe-arg-val-lys-glu -asn-phe-asp-lys-ala-arg-phe-ser-gly-thr-trp-tyr-ala -met-ala-lys-lys-asp-pro-glu-gly-leu-phe-leu-gln-asp 122 20 10 20 s 0 n 2 10 w z p 2, 10 c n 100 200 effluent volume (mi) effluent volume ( m l ) fig. 4 . gel chromatography of tryptic (a), chymotryptic (b) and thermolytic (c) digests of rbp. in each case 2 p m o l e s of reduced and carboxy methylated rbp were digested. the columns (152~1.5~111) of sephadex g-25 were equilibrated with 0.025% ammonia and 10% n-propanol in water. fractions of 2.0 ml were collected at 10-min intervals. the bars indicate fractions pooled and lyophilized. the peptides have the same designations as in table 3. fig. 5. gel chromatography on a column (175~1.2 cm) of sephadex g-100 equilibrated with 0.05 m sodium acetate buffer, ph 5.0, containing 6 m guanidine-hc1 of a clostripain digest of 3 . 1 p m o l e s of carboxy methylated rbp (a). fractions denoted by the bars (c1 i and c1 11) in a were pooled, desalted, lyophilized and resubjected to the same sephadex g-100 column (b and .c). the flow rate of the column was 2.8 ml/h and fractions were collected every 25 min. fractions denoted by the bars in b and c were pooled, desalted and subjected to amino acid analysis and sequential degradation. the complete amino acid sequence of cnbr-fragment a3a -the complete amino acid sequence of cnbr-fragment a3a was obtained by automatic amino acid sequence determination of intact rbp (table 2b). this sequence was corroborated by direct automatic sequencing of fragment a3a (table 2a), which 123 provided information for 23 out o f the 27 residues. additional information was obtained from the amino acid sequences of peptides r3, r4, r6, r10, rc2, rc4, rc6, rc7, rt5 (table 3) and clostripain fragment c1 i1 (table 4) as summarized in fig. 6. table 3. amino acid compositiona and amino acid sequenceb of some tryptic, chymotryptic and thermolytic peptides of carboxy methylated rbp peppur i tide f ica de amino acid composition yield' t ion' sigpro nace t ion dure a _ trypt ic r1 r2 r3 r4 r5 r6 r7 r8 r9 r10 k0.8d2.2e1. 8p3.0g1.0a1. ll1 .2 c0.9d3.0t0.9s0.8g1 .oa1. ll3.oy0.9 k2. od2. le1. 1'0. 8f1. 0 r2. oco. 8d1. oeo. 7 k2. 4a2. omo. 9 r 1 . 3% .9'0. gf1. 0 r1. ogo. 7 k1 .4t0.9s1.0g1 .0a1.8m0.9y0. 8f1 .o r1. ovo. 9 kl.of1.0 chymotryptjc rc 1 kl. 7d0.9e1. lp1 .0g1.0a0.9l0.9f0.9 rc2 k2,3d1. 1e1.0p1.3g1.4a2. lm1 .of1.o rc 3 d1.3t1 .2s0. ge2.0g1 . o v l .2m0.8 rc4 r2.0c0. 7d0.9s1. 7e1.4v1 .0f0.9 rc5 r0.7k1.2t1 .oso.gg1. la1 .gv1. 1 rc6 k1. 2r1 .2d0. 7a0. 8f0. 8 rc8 k2. omo. gy1* thermolytic rt 1 rt2 rt3 d1. 8e1. olo. 8 rt4 oao. 8'0. 4'0.4 rt5 k o . 8d1. o e 1 . 2'0.8 rc 7 k i . 3r1.3do. 9%. 9'1. a f i .i d1. 2t1 . o s l .0e2. lg1. lvl.om0.8 k0.9d1.9t1. 7e1. lp1 .oa1 .0f0.8 moles 184 385 231 180 92 274 233 319 57 75 233 114 435 87 55 473 194 35 48 356 215 200 293 z 9.2 19.5 11.7 9.1 4.6 13.7 11.7 16.0 2.9 3.7 11.7 3.8 21.7 4.3 2.8 23.7 9.7 1.8 2.4 17.8 10.8 10.0 14.7 residue ab 140-150 ab 122-133 abd 11-17 abc 1-5 ab 26-30 ab 6-10 abd 61-62 ab 59-60 ab 86-87 ab 20-29 abcd 28-36 abcd 26-36 abc 46-53 abd 1-9 ab 54-61 abdc 16-20 abdc 10-15 ab 87-90 abd 47-54 abd 77-85 abc 37-40 abc 41-44 abd 11-14 amino acid sequence e tryptic r1 asx-pro-asx-gly-leu-pro-pro-glx-ala-glx r2 leu-leu-asx-leu-asx-gly-thr-cmcys-ala r3 val-lys-glx-asx-phe-asx r4 glx-arg-asx-cmcys-arg r5 ala-met-ala-lys r6 val-ser-ser-phe-arg r7 val-arg r8 gly-arg r9 phe-lys r10 phe-ser-gly-thr-(-)-tyr-ala-met 124 chymotryptic rc1 ala-lys-lys-asx-pro-glx-gly-leu-phe rc2 ala-met-ala-lys-lys-asx-pro-glx-gly-leu-phe rc3 ser-val-asx-glx-thr-gly-glx-met rc4 glx-arg-asx-cmcys-arg-val-ser-ser-phe rc5 ser-ala-thr-ala-lys-gly rc6 asx-lys-ala-arg-phe rc7 arg-val-lys-glx-asx-phe rc8 lys-met-lys-tyr thermolytic rt1 val-asx-glx-thr-glv-glx-met-ser rt2 phe-thr-asx-thr-glk-asx-pro-ala rt3 leu-glx-asx-asx rt4 ile-val-ala-glx rt5 val-lys-glx-asx a) b) c) d) the letters have the following meaning: a, gel chromatography all analyses are 24h hydrolysis values. all sequence analyses were carried out by the dansyl-edman technique. yields are not corrected for material taken for analyses during the course of the purification procedure. on sephadex g-25; b, high voltage paper electrophoresis at ph6.5; c, high voltage paper electrophoresis at ph 3 . 5 ; d, paper chroma table 4a. amino acid composition of two clostripain peptides obtained from carboxymethylated rbpa amino c1 i residue 63-121 c1 i1 residue 20-60 acid found to sequencea found to sequenced nearest nearest integer integer molelmo le mole/mole lysine 3.8 4 4 2.7 3 3 histidine 1.0 1 1 0.3 arginine 1.3 1 1 1.2 1 1 cm-cysteine 1.7 2 2 0.1 aspart ic acid 10.7 11 12 4.1 4 4 threonineb 4.4 4 5 2.7 3 3 serineb 2.0 2 2 3.3 3 3 glutamic acid 3.1 3 3 5.1 5 5 proline 1.4 1 1 1.0 1 1 glycine 3.4 3 3 3.7 4 4 alanine 4.0 4 4 4.8 5 5 valine' 4.6 5 5 2.1 2 2 methionine 1.6 2 2 1.6 2 2 isoleucinec 1.3 1 1 1.0 1 1 leucine 3.4 3 3 2.0 2 2 tyrosine 3.4 3 4 1.0 1 1 phenyl alanine 2.8 3 3 2.8 3 3 tryptophan 2.6 3 3 0.6 1 1 yield ( x ) 45 68 aexcept where noted all figures are average values of one 24 h and one bvalues obtained by extrapolation to 0 h hydrolysis. c72 h hydrolysis value. dcalculated from fig. 6. 72 h hydrolysis. 125 table 4b. amino acid sequence of clostripain fragments c1i and clii. peptide designation amino acid sequence c1 i leu-leu-asn-asn-trp-asp-val-cmcys-ala-asp-met-val-gly thr -phe c1 i1 phe-ser-gly-thr-trp-tyr-ala-met-ala-lys-lys-asp-pro-glu gly-leu-phe-leu-gln-asp-asn-ile-val-ala-glu-phe-ser-val asp-glu-thr-gly-gln-met-ser-ala-thr-ala afor both peptides 120 nanomoles were subjected to automatic sequence determination in the liquid phase sequencer. the repetetive yield for c1 i1 was 92% and for c1 184%. fig. 6. amino acid sequences of peptides used to estab lish the primary struc ture of rbp. for desig nations of peptides, see text. j denotes nhz-terminal amino acid sequencing by means of liquid phase sequencer or by the manual dansyl-edman procedure. -denotes amino acid residues released after carboxypeptidase diges tion as quantitated by amino acid analysis. the complete amino acid sequence of cnbr-fragment b thirteen out of the 26 amino acid residues of cnbr-fragment b were obtained by automatic amino acid sequence analysis of intact rbp (table 2b). by the same procedure fragments b and a2 provided 24 and 25 residues, respectively. corroborative information was obtained from peptides r10, rc1, rc2, rc3, rt1, rt3, rt4 (table 3) and ci i1 (table 4). the complete amino acid sequence of cnbr-fragment a3b automatic amino acid sequence analysis of cnbr-fragment a2b provided information for 17 out of the 20 positions (table 2a). clostripain .eptide c1 i1 (table 4) corroborated the nh2-terminal sequence of the fragment and peptide c1 i (table 4) established other peptides like r7, r8 and rc5 (table 3) supported the established sequence. however, since the amino acid sequence of the cooh-terminal half of fragment a3b relied only on the cooh-terminal region of a3b (fig.6). 126 analyses performed on rather large peptides, a3b was digested with clostripain. the peptide mixture was fractionated by deae-sepharose ion exchange chromatography. three peptides were obtained (fig. 7). the combined amino acid composition of peptides a3b1, a3b2 and a3b3 was identical to that of fragment a3b (table 5) and with the amino acid sequence of the three peptides (table 5) the sequence of cnbr-fragment a3b (fig.6) was ascertained. the complete amino acid sequence of cnbr-frament c the amino acid sequence of the cnbr-fragment c, comprised of 15 amino acid residues (table l), was elucidated by automatic sequencing of the intact fragment, which yielded information in 13 positions (table 21, and by sequence analysis of peptides r9and rc8 (table 3). corroborative information was obtained from peptide c1 i (table 4) and from tryptic and chymotryptic peptides of fragment c. these peptides, c1, c2, ct1 and ct2 were isolated by high voltage paper electrophoresis and paper chromatography (table 4) and their sequences established the primary structure of cnbr-fragment c (table 6 and fig. 6). table 5. amino acid composition and amino acid sequence of clostripain peptides derived from 0.8 pole of cnbr-fragment a3b peptide designation amino acid compositiona yield residue nmoles 1 40 54-60 36 61-62 a3b2 r1.o vo.9 290 a3b3 c0.9 d3.6 a1.l vl.o m0.9bl1.6 380 48 63-73 a. a3bl k1.o r0.9 t0.9 so.9 g1.2 a1.9 320 amino acid sequence' b. a3bl ser-ala-thr-ala-lys-gly-arg a3b2 val-arg a3b3 leu-leu-asn-asn-trp-asp-val-cmcys-ala athe amino compositions are based on 24 hydrolysis values only. bdetermined as homoserine . cfor a3bl (68 nanomoles) and a3b3 (82 nanomoles) sequence determination was accomplished in an automatic liquid sequencer. peptide a3b2 (32 nanomoles) was analyzed by the manual dansyl edman technique. the complete amino acid sequence of cnbr-fragment a1 cnbr-fragment a1 repre sents more than half of rbp. automatic sequencing of the entire alfragment provided information for 48 out of its 94 residues (table 2). since 127 100 200 effluent volume tml) fig. 7. deae-sepharose ion exchange chromatography of a clostri pain digest of 0.8 pmoles of cnbr-fragment a3b. the column (12x1 cm) was equilibrated with 0.02 m nh4hc03. the applied sample was eluted with a 250 ml linear gradient of nh4hc03 from 0.02 to 0.2 m. the flow rate was 18ml/h and fractions of 2 ml were collected. the bars indicate material pooled, lyophilized and used in further analyses. preliminary studies had shown that cnbr-fragment a1 contained a single aspartyl-prolyl bond, which is sensitive to acid proteolysis (23), fragment a1 was cleaved with formic acid. two fragments of similar size, table 6 . amino acid composition and amino acid sequence of tryptic and chymotryptic peptides obtained from cnbr fragment ca peptide puri designaamino acid compositionb yield fication residue t ion proce a nmoles 5 74-85 i -&.? e1.2 p1.2 g1.o 190 9-5 tryptic c1 k0.9 d1.e t? . b d c2 k1.o m0.6dfl.l 280 14 b 86-88 b 74-82 ct2 k1.8 m0.5dp0.9 a1.2 f1.o 150 7.5 b 83-88 ct1 d2.1 t2.8 e1.o g1.l vl.o f0.9 lg0 a1.o vo.8 ro.9 chymotryptic amino acid sequencee b. tryptic c1 val-gly-thr-phe-thr-asx-thr-glx-asx c2 phe-lys ct1 val-gly-thr-phe-thr-asx-thr-glx-asx ct2 pro-ala-lys-phe-lys chymotryptic =two pmoles of fragment c were subjected to each enzymatic digestion. bthe amino acid compositions are based on 24 h hydrolysis values only. cb denotes high voltage paper electrophoresis at ph 6.5 and d denotes ddeterminated as homoserine eall sequence determinations were carried out with the manual dansyl-edman paper chromatography. technique. except for peptide c2 ( 5 0 nanornoles) 120 nanomoles of each pep tide were subjected to manual derradation. 128 as shown by sds-polyacrylamide gel electrophoresis, and charge, as evidenced by ion-exchange chromatography, were obtained in excellent yield. since the properties of the two fragments precluded their separation, intact fragment a1 was succinylated prior to the acid cleavage. the cleavage mixture was added to the automatic sequencer without prior peptide separation. as expected, the only amino acid sequence obtained, was that of the cooh-terminal acid cleavage fragment h-2 (fig.3,6). this information together with the nh2-terminal auto matic amino acid sequence analysis of intact fragment a1 gave almost all of the primary structure of a1 (see fig.6). fragment a1 was digested with staphylococcus aureus protease v8 and the resulting peptide mixture was resolved by gel chromatography on a column of sephadex g-50 (fig.8). two peptides, denoted sa and sb in fig. 8a were further purified by column electrophoresis (fig. 8b and c). amino acid analysis and automatic sequencing o f the two peptides demonstrated that sa represented the nh2-terminal part of a1 (table 7). the cooh-terminal fragment sb gave clear sequence information in 23 out of its 24 positions (table 7). however, this information did not establish a connection between the nh2-terminal region of fragment a1 and the cooh-terminal acid cleavage fragment h-" (see fig. 6 and fig. 3.) to obtain further amino acid sequence information about cnbr-fragment al, this fragment was separately digested with chymotrypsin, thermolysin, pepsin and subtilisin. all digests were subjected to ion-exchange chromatography (fig. 9). table 8 summarizes the amino acid compositions and sequences of the isolated peptides. fragment a1 was also digested with clostripain and the digest was fractionated on a sephadex g 5 0 column (fig. lo). fraction i con tained aggregated material and fraction vi contained a single peptide. all other fractions were further purified by due-sepharose ion-exchange chromato graphy (fig. 10). a total of twelve clostripain peptides were recovered and they made up the entire a 1 fragment (table 9). it should be noted that pep tides acl i1 1 and acl i11 1 were identical and that peptides ac1 i1 4, ac1 iv 1, ac1 iv 2 and ac1 iv 3 probably arose by thermolysin-like activity present in the clostripain preparation (see table 9). the peptides obtained from the various digests (table 8 and 9) together with the tryptic peptides r1 and r2 (table 3) gave the entire sequence of cnbr-fragment al. the gap between the nh2-terminal amino acid sequence (residues 89-136) and the sequence of the acid cleavage fragment h-2 (residues 141-175) was bridged by clostripain peptide ac1 i1 2 (table 91, the chymotryptic peptide ac4 (table 8) and the thermolysin peptide at2 (table 8). to firmly establish the sequence of peptide 129 ac1 i1 2 it was subjected to carboxpeptidase digestion (fig. 6) in addition to nh2-terminal sequencing. thus, the information obtained was sufficient .to establish the primary structure of cnbr-fragment al. table 7a. amino acid composition of two staphylococcus aureus protease v8 peptides derived from cnbr-fragment ala*b sa residue 89-158 sb residue 159-182 amino acid found to neasefound to nease rest quenrest quen intecec intecec aer ger mole/ mole/ lysine 2.8 3 histidine 1.0 1 arginine 3.6 4 cm-cysteine 2.4 2 threonined 3 . 3 3 serined 4.7 5 glumatic acid 8.1 8 proline 2.9 3 glycine 4.0 4 alanine 3.9 4 vaiinee 4.6 5 methionine isoleucinee 1.6 2 leucine 5.2 5 tyrosine 4.8 5 phenylalanine 3.0 3 tryptophan 1.7 2 aspartic acid 10.6 11 3 1 4 2 11 3 5 8 3 4 4 2 5 5 3 2 0 . 3 0.9 1 3.6 4 2.0 2 2.8 3 1.1 1 2.3 2 1.9 2 1.2 1 5 0.9 0.7 1 3.8 4 1.7 2 1 4 2 3 1 2 2 1 1 1 4 2 yield 76% 48% atwo pmoles of cnbr-fragment a1 were digested with the enzyme. bexcept where noted all values are average values of one 24 h and one 72 h cobtained from the sequence shown in fig. 6. dvalues obtained by extrapolation to 0 h hydrolysis. e72 h hydrolysis value hydrolysis. table 7b. amino acid sequence analyses of staphylococcus aureus protease v8 peptides obtained from cnbr-fragment ala peptide designation amino acid sequence sa lys-tyr-trp-gly-val-ala-ser-phe-leu-gln-lys-gly -am-asp-asp-his-trp-ile-val-asp-thr-asp-tyr sb -leu-cmcys-leu-ala-arg-gln-tyr-arg-leu-ile-val -his-asn-gly-tyr-cmcys-asp-gly-arg-ser-glu-arg a m 'both peptides were degraded in the automatic liquid phase sequencer. the overall repetitive yield was 89% for peptide sa and 91% for peptide sb. in each case 210 nanomoles were subjected to analysis. 130 s a a 100 200 effluent volume i ml ) effluent volume (ml) fig.8. purification of staphylococcus aureus, protease v8 peptides obtained from digestion of 2 pmoles -of cnbr-fragment al. the digest was applied onto a sephadex g-50 column (110x2 cm) equilibrated with 0.025% ammonia 10% propa no1 ( a ) . fractions of 2 . 0 ml were collected at 9-min intervals. the denote materials (sa and sb) which were further purified by column electro phoresis at ph 1 . 9 (b and c). after completed electrophoretic separation the columns were eluted at a flow rate of 1 2 ml/hour. fractions of 1 . 0 ml were collected. the occurrence of peptides in the effluent was monitored by measur ing the absorbance at 280 nm. in addition 25 pl-aliquots from each fraction were subjected to alkaline hydrolysis and the ninhydrin reaction. the color developed was measured at 570 nm. fractions indicated by the bars were pooled and lyophilized. fig. 9. ion exchange chromatography on a a 1 polystyrene resin (jeol ar-15) of peptides derived from cnbr fragment a1 after digestion with with chymotrypsin ( a ) , thermoly sin (d). the digests represent ing 0.8 pmoles ( a and 2) and 1 . 3 pmoles (g and c ) of fragment a1 column. fractions of 3.0 ml of 3 . 0 ml were collected at a flow rate of 110 ml/h. the bars ( 2 denote highly purified peptides used in subsequent analyses (see table viii). further experimen1 tal details are given under fraction number methods. the ninhydrin color at 570 nm is expressed in arbitrary units (a.u. ). i a c 5 sin (b), pepsin ( c ) and subtili were separately applied onto the i _ d 9-878572 1 3 1 t e c 2 2.0 2 hl lu 0 z < 1.0 m a 0 4 % 132 i i i i c m 100 200 table 8. amino acid compositiona and amino acid sequenceb of some chymo tryptic, thermolytic, peptic and subtilisin peptides derived from cnbr-fragment a1 peptide des igna t ion amino acid composition yield' residue a_. chymo trypt icd ac 1 ac2 ac3 ac4 ac5 thermolytic at 1 at2 at3 at4 at5 at6 at7 peptice ap1 ap2 ap3 ap4 ap5 ap6 subtilisind as 1 as 2 as 3 moles % cl.o d2.7 t0.8 s0.9 g1.o a0.6 l0.9 '0.8 190 24 d1.7 t1.l '0.3 '0.3 '1.0 400 50 90 11 210 26 d0.3 sl.o e0.3 g1.l a1.l '0.9 f 1 . o r0.9 d2.2 s0.9 e1.o p2.9 g1.l a0.8 l1.o r1.o e1.l a0.9 y1.0 220 28 660 51 l4 150 12 600 46 530 41 320 25 330 25 d3.0 t2.1 s0.3 a0.8 v0.6 i0.4 lo.l '1.8 k 1 . o r1.l d2.4 sl.l e2.2 '2.8 g1.3 a1.l l1.l 180 r1.o c0.9 sl.l e1.3 v0.9 l1.o '0.9 r2.0 e4.2 v0.4 i0.4 k0.8 g1.o yl.o r1.l e1.2 a0.9 l1.0 r1.0 l1 .o y o . 9 cl.l d2.2 t1.2 s0.9 g1.o a1.o ' 0 . 3 d0.3 e1.2 a0.9 vl.o '2.0 k 1 . o h0.9 d3.0 e1.l g1.o l1.l k1.o s0.9 e0.3 g1.o a0.9 "1.1 '1.0 r1.o e1.2 a0.9 r1.l l1.0 '0.9 d2.0 t0.9 g0.9 lo.9 co.9 a1.1 l2.0 v1.0 f1.o 680 52 220 17 420 32 710 55 340 26 730 56 370 46 80 10 90 11 124-133 106-111 92-96 138-148 162-165 106-115 138 150 116-122 161-164 151-158 165-167 89-92 126-133 114-118 97-104 162-164 89-95 165-167 124-128 159-162 136-137 amino acid sequence b. chymotryptic ac 1 asx-leu-asx-gly-thr-cmcys-ala-asx-ser-tyr ac2 ac 3 gly-val-ala-ser-phe ac4 ser-arg-asx-pro-asx-gly-leu-pro-pro ac5 ala-arg-glx-tyr thermolytic at1 ile-val-asx-thr-asx-tyr-asx-thr-tyr at2 ser-arg-asx-pro-asx-gly-leu-pro-pro-glx-ala at3 val-glx-tyr-ser-cmcys-arg-leu at4 leu-ala-arg-glx at5 ile-val-arg-glx-arg-glx-glx-glx at6 tyr-arg-leu at7 lys-tyr-(-)-gly i le -val asx-thr -asx-tyr peptic ap1 asx-gly-thr-cmcys-ala-asx-ser ap2 tyr-ala-val-glx-tyr ap3 leu-glx-lys-gly-asx-asx-asx-his ap4 ala-arg-glx ap5 lys-tyr-(-)-gly-val-ala-ser ap6 tyr-arg-leu subtilisin as 1 asx-leu-asx-gly-thr as2 leu-cmcys-leu-ala as3 val-phe 133 aall analyses are 24 h hydrolysis values. ball sequence analyses were carried out by the manual dansyl-edrnan technique. cyields are not corrected for material taken for analyses during the course of dthe enzymatic digestions were carried out on 0.8 gmoles of fragment al. ethe enzymatic digestion was carried out on 1.3 moles of fragment al. the purification procedure. table 9. amino acid compositiona and amino acid sequenceb of clostripain peptides obtained after digestion of 0.75 gmole of [ i4c] carboxy methylated cnbr-fragment a1 peptide yield residue desigamino acid composition nmoles % nation a. 250 33 140-153 a 'i1 a c1 112 r1.3 c1.2 d3.1 t1.o s2.9 g1.l a1.4 '1.0 l2.2 '1.0 f2.0 240 150 2o 32 122-139 122-134 80 11 106-115 a c1 113 a c1 114 a c1 1111 k1.3 r1.o d2.1 '0.4 e2.2 p3.0 g1.4 a1.3 '0.9 i0.7 l1.2 230 8o 31 l1 154-163 140-153 a c1 1112 r2.0 cl.l d0.4 e4.0 g0.3 a1.2 '0.3 l2.3 280 37 167-177 a c1 1113 h0.8 r1.o c1.2 d2.0 e0.4 g1.7 '0.7 i0.5 l1.l '0.8 70 9 178-180 60 8 181-182 a c1 iv1 a c1 iv2 d1.0 l0.g 300 40 116-121 a c1 iv3 340 45 164-166 a c1 v1 a c1 vll 530 71 89-105 k1.l r1.o d2.2 e2.1 p3.1 g1.2 a1.l v0.7 '0.5 l1.o c1.3 d3.0 t1.o '2.0 g1.l a1.o l3.0 '1.0 d3.0 t1.6 a1.o '0.8 i0.5 l0.2 '1.7 r 1 . 0 sl.1 e1.2 r1.o e1.2 '1.0 r1.o c1.2 s1.0 e1.2 v1.0 y1.0 k1.9 h0.8 d3.0 '0.9 e1.2 g2.1 a1.o v0.9 l1.o y0.9 f 1 . o amino acid sequence b. ii c1 i11 a c1 i12 a c1 i13 a c1 i14 a c1 1111 a c1 1112 a c1 1113 a c1 iv1 a c1 iv2 a c1 iv3 a c1 v1 asp-pro-asn-gly-leu-pro-pro-glu-ala-gln-lys-ile leu-leu-asn-leu-asp-gly-thr-cmcys-ala-asp-ser-tyr-ser-phe-val-phe-ser leu-leu-asn-leu-asp-gly-thr-cmcys-ala-asp-ser-tyr ile-val-asp-thr-asp-tyr-asp-thr-tyr-ala asp-pro-asn-gly-leu-pro-pro-gln-ala-gln-lys-ile-val gln-arg-gln-glu-glu-leu-cmcys-leu-ala-arg leu-ile-val-his-asn-gly-tyr-cmcys-asp ser-glx-arg asx-leu val-glx-tyr-ser-cmcys-arg glx-tvr-ara -arg a c1 vi1 lys-t;r-trp-gly-val-ala-ser-phe-leu-gln-lys-gl~-asn-asp-as~ aall analyses are 24 h hydrolysis values. bthe peptides designated a c1 11, a c1 i11 and a c1 vi were all subjected to automatic sequence analysis in the liquid phase sequencer. between 50 to 110 nanomoles of peptide were used in these analyses. the repetitive yield varied between 91 and 94%. peptides designated a c1 iv and a c1 v were degraded manu ally with use of the dansyl-edman technique. between 30 and 70 nanomoles of peptide were used. fication procedure. cyields have not been corrected for material taken for analyses during the puri the cooh-terminal amino acid sequence of rbp we have previously suggested that the cooh-terminus of rbp is involved in the regulation of the catabolism of the protein and we obtained data that the cooh-terminal residue of is rbp 134 arginine (43). other authors have obtained other cooh-terminal sequences for rbp (12,55). it was, therefore, of importance to clarify this discrepancy. fig. 6 shows that amino acid sequence determinations of peptides sb (table 7) and ac1 iv 1 (table 9) provided the sequence arg-asn-leu. to corroborate this information, intact, reduced and carboxmethylated rbp as well as cnbr-fragment a1 and peptide sb were separately subjected to carboxypeptidase a and b diges tions. fig. 11 summarizes the results obtained with fragments a1 and peptide sb. both types of materials clearly showed that the cooh-terminal sequence is asn-leu. the digestions of peptide sb also provided strong support for the established sequence (fig.61, k. that arginine preceeds the asparagine. the arginine was not as evident when carboxypeptidase b digestions of fragment a1 (fig.lla) and intact rbp (not shown) were carried out. the reason for this was that several amino acid residues were released almost simultaneously on the addition of carboxypeptidase b. however, the carboxypeptidase digestions together with the amino acid sequence information summarized in fig.6 establish the cooh-terminal sequence of rbp. 20 lo 60 incubation time ( m i n ) fig.11. carboxypeptidase digestions of cnbr-frag ment a1 ( a ) and staphylococcus aureus protease v8 peptide sb (g) . the samples, each comprising 50 nanomoles of peptide were mixed with carboxypeptidase a (40ug).after 30 min of incubation carboxypeptidase b (25pg) was added (arrow). samples were withdrawn at the indicated times and amino acids released were identified and quantitated by amino acid analysis. the values given in the figure have been corrected for the presence of free amino acids in the carboxypeptidase preparations and in the peptide fractions. the symbols in the figure are : 0---0, leucine;-, asparagine;c--a, arginine; o o , alanine w, tyrosine. localization of disulfide bridges in rbp intact rbp was subjected to acid cleavage and gel chromatography on a column of sephadex g-100. fig. 12 depicts the chromatogram. fraction si was subjected to amino acid analysis and nh2 terminal amino acid sequence determination (table 10) which clearly showed that si corresponds to residues 8 3 to 140 (fig.6.). thus, half-cystines 120 and 129 must form a disulfide bridge as rbp does not contain any free sulfhyd ryl groups. 135 e w2.0 e 0 effluent volume ( m l fig. 12 gel chromatography of 3 pmoles of acid-cleavaged rbp on a column (16ox2cm) of sephadex g-100 equilibrated with 0.05 m sodium acetate, ph 5.0, containing 6m guanidine-hc1. the acid cleavage was obtained by incubating the protein in 70% (v/v) formic acid at 37' for 24 h. after this period of time the formic acid was diluted with h20 and the protein was lyophilized. the column had a flow rate of 3.4 ml/h and fractions of 2.0 ml were collected. material denoted by the bar was pooled, dialyzed and lyophilized. table 10 a . amino acid composition of acid cleavage fragment si derived from intact r b p ~ . ~ amino acid si residue 83-140 found to nearest integer sequence' lysine histidine arginine cm-cysteined aspartic acid threoninee serinee glutamic acid proline glycine alanine valinef methionine isoleuc inef leucine tyrosine phenylalanine tryptophan 3 . 7 1.0 2.0 2.1 9.6 3 . 1 4.7 2 . 3 1 . 3 3 . 2 4 . 0 3.8 1.0 1.1 4 . 3 5.1 4.0 1.9 moleimole 4 1 2 2 10 3 5 2 1 3 4 4 1 1 4 5 4 2 4 1 2 2 10 3 5 2 1 3 4 4 1 1 4 5 4 2 yield 27% athree vmoles of rbp were subjected to acid cleavage. bexcept where noted all values are average values of one 24 h and one 72 h 'calculated from the sequence shown in fig.6 (residues 8 3 1 4 0 ) . dthe acid cleavage fragment was reduced and carboxymethylated after the gel '=values obtained by extrapolation to 0 h hydrolysis. f72 h hydrolysis value. table 10 b. amino acid sequence analysis of acid cleavage fragment sia pro-ala-lys-phe-lys-met-lys-tyr-trp-gly-val-ala-ser-phe-leu-gln lys-gly-asn-asp-asp-his-trp-ile-val-asp-thr-asp-tyr-asp-thr-tyr ala-val-gln-tyr-ser-cmcys-arg-leu-leu-asn adegradation was accomplished on 190 nanomoles of peptide in the automatic hydrolysis. chromatography separation (see fig.12). sequencer. the repetitive yield was 93%. 136 material corresponding to fraction a of fig. 1a which comprised the half cystine-containing cnbr-fragments of rbp, was separately subjected to trypsin digestion at ph 6.0 and pepsin digestion at ph 5.0. the digests were separate ly subjected to sephadex g-50 gel chromatography in dilute acetic acid (fig.13). fractions denoted t and p in fig. 13 contained half-cystine as monitored by amino acid analysis. these two fractions were pooled and lyophi lized. fraction t was further purified by column electrophoresis (fig.14) and the pooled fraction t1 was subjected to performic acid oxidation and re electrophoresed (fig. 14) to yield peptides t1a and t1b. both the amino acid composition and the sequence establish that peptide t1a corresponds to residues 167 to 177 and t1b to residues 63 to 73 of rbp (table 11 and fig. 6).thus, the half-cystines in positions 70 and 174 of rbp form a disulfide bridge. fraction p fig. 13 was further purified by column electrophoresis (fig. 14). both peptide p1 and p2 contained half-cystine. after performic acid oxidation and re-electrophoresis peptide p1 appeared as a single homogenous peak (not shown). amino acid analysis and sequence determination (table 11) demonstrated that peptide p1 corresponded to residues 118 to 135, which corroborates the previously established disulfide bond between half cystines 120 and 129 ( s e e above). peptide p2 was further purified by high pressure liquid chromtography (fig. 15) to yield fraction p2a. after performic acid oxidation this material was re-subjected to high pressure liquid chromatography. fig. 15b demonstrates that three peptides, p2a1, p2a2 and p2a3, could be isolated. table 11 ascer tains that p2a1 and p2a2 represent residues 1 to 9 of rbp and that p2a3 corre sponds to residues 159 to 161. thus, the third disulfide bond engages the half-cystines in positions 4 and 160. the complete amino acid sequence of human rbp, including the disulfide bridges, is depicted in fig. 16. discussion prior t o the analysis of the rbp sequence reported here, two laboratories presented partial nh2-terminal sequence information (27,38). their information is in full agreement with the sequence elucidated here. after completion of this study kanda and goodman (21) reported the primary structure for the nh2 terminal 121 positions of human rbp. although their data are generally in good agreement with those described here, some noteable differences occur in posi tions 50 to 53 (our numbering), 58 to 60, 111 to 114 and 119 to 120. in most of these positions kanda and goodman assigned amino acid residues from data obtained by cooh-terminal digestions or from the amino acid composition of 137 fig.13. gel chromatography of trypsin (4) and pepsin ( g ) digested disulfide-linked cnbr-fragments al,a2, a3a and a3b (cf. fraction a of fig.la) on a column (100x2 cm) of sephadex g 5 0 equili brated with 10% (v/v) acetic acid. five pmoles of rbp cleaved with cnbr were subjected to gel chromatography as described in the legend of fig.la. material corresponding to fraction a of fig.la was pooled, desalted, lyophilized and divided into two equal parts. one part was dissolved in 4 ml of tris-acetate buffer, ph 6.0, and the other part was dissolved in 4 ml of 0 . 2 m sodium acetate buffer, ph 5 . 0 . each enzyme (0.8 mg) was added to one aliquot and the digestions were allowed to proceed for 8 hours at 3 7 o . the samples were then immediately applied onto the columns, which were eluted at a flow rate of 6.0 ml/hour. fractions of 2 . 0 ml were collected. aliquots (50 p1) from every third fraction were subjected to amino acid analysis. fractions denoted by the bars contained cysteine and accordingly were pooled and lyophilized. fig. 1 4 . fraction t of fig. 13a was subjected to elec trophoresis on a column of cellulose at ph 1.9 (&).fractions denoted t1 in &, which contained cysteine as monitored by amino acid analyses on aliquots from alternate fractions, were pooled, lyophilized, subjected to performic acid oxida tion and re-electrophoresed under identical conditions ( g ) . fractions denoted by the bars were pooled and lyophilized. material in frac tion p of fig. 12b was also subjected to elec trophoresis at ph 1.9 ( c ) . material denoted by the bars contained cysteine as revealed by amino acid analysis performed on aliquots withdrawn from every second fraction. conse quently, those fractions were pooled and lyo philized. the experimental details were the same as in fig. 8b and c. i ‘2 fig.15. (next page) high pressure liquid chromatography of the material designated p2 in fig.14c ( a ) . the c18 reversed phase column was equilibrated with 2 mm ammonia, adjusted to ph 2.4 with trifluoroacetic acid, and 5% methanol. the applied material was eluted with an 80-ml linear gradient of methanol from 5 to 55% followed by 30 ml of 55% methanol. the flow rate of the column was 24 ml/h and fractions were collected at 1 min intervals. all absorbance peaks were separately pooled and aliquots from each were subjected to amino acid analysis. only the fraction denoted p2a contained significant amounts of cysteine. therefore, this fraction was subjected to performic acid oxidation and re-chromatographed on the c 18 column (g) under conditions identical to those described above. fractions denoted by the bars were pooled and lyophilized. 138 a3 0.2 e 2 w 0.1 w 0 z 4 m m m si a of .l 0.04 fig.ls. a _ p2 a b p2aa p2a3 p2 x 2 i 1 100 2 00 retention time (rnin) 0 o w ul < r o n u a s z 4 0 0 3 i i fig.16. the complete amino acid sequence of human rbp depicting the distribution of the three disulfide bonds. 139 table 11. amino acid compositiona and amino acid sequenceb of tryptic and peptide desigamino acid composition nation yield' residue peptic cysteine-containing rbp peptides a. tr yp t ic nmoles % h1.o r1.3 d5.6 m0.6dg2.1 a1.2 '1.1 '1.8 i 0 . 8 l2.3 yl.o 520 h1.2 r1.2 '1.1 d2.3 g2.2 '0.9 '0.9 l0.8 ' 0 . 8 250 10 167-177 '1.0fd4.0 m0.7dal.2 '1.0 l2.2 350 14 6373 peptic p1 r1.o c1.8fd2.9 t1.o '2.9 g1.l a1.o l3.0 y1.5 f0.7 390 16 118-135 p2 r2.1 c1.zed1.2 s1.9 e1.0 v1.0 l2.2 f1.l 440 18 p2a1 r1.9 cl.ofd1.o s1.8 e0.9 vl.l f0.7 150 6 1-9 p2a2 r2.1 c0.9fdl.l s2.0 .o '1. 1 f0.6 130 5 1-9 p2a3 '1.0 l1.9 200 8 159-161 21 amino acid sequence b. f1a leu-ile-val-his-asn-gly-tyr-tyr-cysa-asp-gly t1b leu-leu-asn-asn-trp-asp-val-cysa-ala p1 tyr-ser-cysa-arg-leu-leu-asn-leu-asp-gly-thr-cysa-ala-asp-ser p2a1 glu-arg-asp-cysa-arg-val-ser p2a2 glu-arg-asp-cysa-arg-val p2a3 leu-cysa-leu aall analyses are 24 h hydrolysis values. tryptophan was not determined. ball peptides except p2a3 were degraded in the automatic sequencer. peptide p2a3 was analyzed by the manual dansyl-edman method. between 70 and 160 nano moles of each peptide were subjected to the automatic sequencer. the repetitive yield varied between 87 and 94% for the different peptides. of peptide p2a3 50 nanomoles were used for the amino acid sequence determination. 'yields were not corrected for material taken for analyses during the isolation procedure. ddetermined as homoser ine edetermined as cysteine fdetermined as cysteic acid after performic acid oxidation peptides. in contrast, these positions are in our sequence analyzed by nh2 terminal degradation of several peptides. in all other positions identical residues were found although kanda and goodman could not unequivocally assign amides and acids in few positions. the sequence of human rbp predicted from a cdna clone (5) agrees completely with the one described here, except in the cooh-terminus (see below). the cooh-terminus of rbp has received particular attention in as much as two forms of rbp exist physiologically (34). they differ in their ability to interact with prealbumin. the non-bound form contains very little retinol, has a changed conformation (42) and is more acidic than the prealbumin-binding species (43). we previously reported that the more acidic form lacked arginine in its cooh-terminus (43). this erroneous information, which was obtained by carboxypeptidase b digestion, probably arose from the occurrence of trypsin like activity present in the carboxypeptidase preparation. two other laborato 140 ries have also attempted to establish the cooh-terminal sequence of human rbp (12,55). in both cases data were obtained which do not agree with the present results. however, in the present study the cooh-terminal sequence was estab lished not only by carboxypeptidase digestions but also by nh2-terminal se quencing of peptidose whose amino acid compositions corroborated the results obtained. nevertheless, the amino acid sequence predicted from a cdna clone encoding human rbp is one residue longer in the cooh-terminus than the determined protein sequence (5). analysis of a cdna clone for rat rbp also predicted an additional amino acid residue compared to the determined protein sequence of human and rabbit rbp (51). neither the data on the rabbit nor on the human sequence support the presence of an additional residue of leucine as the cooh terminus, although admittedly it is difficult with available sequencing tech niques to distinguish the sequence -am-leu-cooh from asn-leu-leu. however, since the cooh-terminus of rbp is located at the surface and seems to be quite flexible (30), it is possible that the additional cooh-terminal leucine resi due encoded by the rbp gene might be removed in a post-translation event. the amino acid sequence of rbp was subjected to a computer search to inves tigate whether any of the previously sequenced proteins would display any structural homology to rbp (6,9). three proteins, p-lactoglobulin (3), human al-microglobulin (11,25) and rat a2-microglobulin ( 5 4 ) were found. the se quences of these proteins, which are of similar sizes, were aligned to that of human rbp by the computer program align (29). the alignment scores are shown in table 12. as all values above 3 are regarded as significant this analysis clearly shows that all four protein sequences are related to each the same conclusion has been reached by two other laboratories (15,32). a closer l o o k at the aligned sequences (fig.17) shows that in eight instances only one type of amino acid residue occupies the same position in all four sequences. in another 20 positions only two alternative amino acids exist. it can accord ingly be inferred that the four proteins belong to the same protein super family. other. three of the four proteins, rbp (28,36), the rodent a2-microglobulin ( 4 4 1 , and most probably human al-microglobulin (1) are produced in liver cells. rodent a2-microglobulin and, to a certain extent, rbp are under androgen control (10,45,49). the same might also hold true for human al-microglobulin (53). the synthesis of rbp and of rodent a2-microglobulin is also influenced by glucocorticoids (2,4). whether any physiological similarities exist between these three liver-produced proteins remains to be established as the molecular functions of rodent a2-microglobulin and of human al-microglobulin are still unknown. 141 table 12. alignment scores for comparisons of human rbp with bovine p lactoglobulin, human al-microglobulin and rat a2-microglobulin bovine human a1 rat a2 0-lactomicromicro globulin globulin globulin human rbp (1-182) 5.97 8.21 5.18 bovine 0-lactoglobulin (1-162) 7.53 10.69 human al-microglobulin the alignment scores, which were obtained with the use of the program align, represent the number of standard deviations of the real score above the ran dom score. numbers in parentheses: residue numbers compared. the sequnces of bovine b-lactoglobulin, human were taken from ref. 3. 25 and 54, respectively. (1-167) 9.35 al-microglobulin, and rat a2-microglobulin 0 0 . 0 b ! 1 0 r6p 0 : . .. !.!* t t o 0 1 2 0 1 4 " fig.17. comparison of the amino acid sequence of human rbp with the sequence of bovine 0-lactoglobulin (3), human al-microglobulin (1,441, and rat a2-microglobulin (45). the alignment was obtained by maximizing the homology using the computer program align. boxes: residues shared by the rbp sequence and any of the other sequences. arrows: positions with a single amino acid residue shared by all the four proteins. stars: positions with two amino acidresidue alternatives for the four sequences. bovine p-lactoglobulin has been reported to bind retinol in a similar way to rbp (14). indeed, b-lactoglobulin has been suggested to function in binding, protecting and facilitating the uptake of retinol in the intestine of suckling young animals (16,32). the four proteins of the rbp superfamily might also have similar tertiary structures. disulfide bonds homologous to that between 142 residues 7 5 and 1 8 0 in rbp are also found in 8-lactoglobulin ( 2 6 ) and in human a-microglobulin ( 4 4 ) and a disulfide bound homologous to that between residues 125 and 1 3 4 in rbp is present in 0-lactoglobulin. moreover, recently the three-dimensional structures of both rbp ( 3 0 ) and 0-lactoglobulin ( 4 6 ) were reported. the polypeptides folds of the two proteins are remarkably similar. the computer analyses suggested that rbp might have arisen by an internal duplication of its primordial gene. residues 3 6 8 3 and 9 6 1 4 1 of human rbp display statistically significant homology ( 6 ) . a similar internal homology has been noted in p-lactoglobulin ( 2 2 ) . this internal homology would suggest that the primordial gene for rbp once coded for a protein with a molecular weight of about 1 4 , 0 0 0 . this is the i molecular weight of the intracellular retinol-binding protein (31) but the amino acid sequence of that protein is not homologous t o that of serum rbp (39,501. however, piscine serum rbp which does not bind to prealbumin has a molecular weight of about 16,000 ( 4 7 ) and therefore the possibility was raised that the gene for serum rbp underwent a partial duplication after the divergence of fish and mammals. the three-dimen sional structure of rbp is also consistent with a partial duplication event. however, the retinol binding site is formed by side-chains from both putative duplicated portions (30). it is therefore probable that the two 'homologous portions found in mammalian rbp also are present in piscine rbp, assuming that the site for retinol has been conserved. moreover, the exon-intron organization and the nucleotide sequence of the rat rbp gene ( 2 4 ) did not show any obvious similarities between the portions of the gene encoding residues 3 6 8 3 and 9 6 1 4 1 . these data together with the similarities in three-dimensional structure between rbp and p-lactoglobulin suggest that if a partial duplication has been involved in the evolution of rbp, it occurred before the divergence of rbp from p-lactoglobulin and the other proteins in the rbp superfamily. acknowledgements expert technical assistance was provided by mr kjell andersson, mr jorgen ericson, ms inga sjoquist and ms yvonne tillman. the computer analyses were kindly carried out by ms elisabetta rossi. some of the urinary rbp used in these analyses was a gift from the late dr ingemar berggird and his col leagues. the constant support and advice provided by dr hans bennich proved invaluable. the patient typing of this manuscript could only have been accom plished by ms anna-greta lundquist and ms margareta moliteus. this work was supported by grants from the swedish medical research council. 143 references 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. berggird,b., ekstrom,b., & bkerstrom,b. :al-microglobulin. scand.j.clfn. lab.invest.40:suppl.154,63-71,1980. borek, c., smith,j.e., sopran0,d.r. & goodman, d.s.: regulation of retinol-binding protein metabolism by glucocorticoid hormones in cultured hhiiec3 liver cells. endocrinology 109:386-391, 1981. braunitzer,g., chen,r., schrank,b. & stang1,a.: die sequenzanalyse des 0 lactoglobulins. hoppe-seyler's z. physiol.chem. 354:867-878, 1973. chen,c.-l.c. & feigelson,p.: hormonal induction of a2p-globulin synthesis in isolated rat hepatocytes. biochemistry 17: 5308-5313, 1978. colantuoni,v., romano,v., santoro,c., costanzo,f., raugei,g., and cortese,r.: cloning and sequencing of a full length cdna for human retinol-binding protein . nucleic acids res.l1:7769-7776,1983. dayhoff, m.o.: atlas of protein sequence and structure. 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78:3478-3482,1981. white,g.h., western,s.m., & glover,j.: carboxy-terminal sequence of retinol-binding protein from human plasma. febs lett. 27:107-110,1972. woods,k.r., & wang,k.t.: separation of dansyl-amino acids by polyamide layer chromatography. biochim.biophys.acta 133:369-370,1967. biochem.biophys.acta 127:82-87,1966. address for reprints: lars rask department of cell research uppsala biomedical center box 596 5-751 24 uppsala, sweden 3present address : department of infectious diseases university hospital s-751 85 uppsala 2present address : national board of occupational safety and health ekelundsvagen 16 5-171 84 solna 4present address : department of immunology research institute of scripps clinic 10666 north torrey pines road la jolla, ca 92037, usa 146 upsala j med sci 87: i 1 1 1 17. 1982 fatty acid composition of serum lipids in diabetic children and their matched healthy controls u . ewald', i.-b. gustafssoni, t. tuverno' a n d b. vessby' dapurtment of purdiatric,s' utzd grrirrtric..y', uniivr.<ity hospittrl, uppsttltr , s\twieti abstract t h e f a t t y a c i d c o m p o s i t i o n s of t h e s e r u m c h o l e s t e r o l esters, phospholipids a n d t r i g l y c e r i d e s w e r e d e t e r m i n e d in 28 insulin-dependent d i a b e t i c c h i l d r e n a n d 1 3 h e a l t h y c o n t r o l s . t h e d i a b e t i c c h i l d r e n w e r e o n a r e g u l a t e d d i e t a n d t h e d i s e a s e w a s under g o o d c o n t r o l . t h e r e l a t i v e c o n t e n t s of l i n o l e i c a n d a r a c h i d o n i c a c i d s w e r e h i g h e r in t h e s e r u m lipids of t h e d i a b e t i c s t h a n in t h e c o n t r o l s as could be e x p e c t e d f r o m t h e d i e t a r y a d v i c e given. h o w e v e r , t h e d e g r e e of d i a b e t i c c o n t r o l w a s n o t s i g n i f i c a n t l y c o r r e l a t e d to t h e f a t t y a c i d c o n t e n t of a n y lipid f r a c t i o n . t h e s e r u m c o n c e n t r a t i o n of a p o l i p o p r o t e i n f a p o ) a-i w a s d i r e c t l y c o r r e l a t e d to t h e c o n t e n t of p o l y u n s a t u r a t e d fat a n d t h e r a t i o b e t w e e n p o l y u n s a t u r a t e d a n d s a t u r a t e d f a t t y a c i d s in t h e t r i g l y c e r i d e s . t h e a p o a-i1 c o n c e n t r a t i o n w a s s i g n i f i c a n t l y c o r r e l a t e d t o t h e r a t i o b e t w e e n homo-gamma-linolenic a n d a r a c h i d o n i c a c i d in a l l s e r u m lipid e s t e r s . no s i m i l a r r e l a t i o n s h i p s w e r e s e e n a m o n g t h e h e a l t h y c o n t r o l s . t h e d i f f e r e n t r e l a t i o n s h i p s b e t w e e n s e r u m a p o l i p o p r o t e i n c o n c e n t r a t i o n s a n d t h e f a t t y a c i d c o m p o s i t i o n in s e r u m in d i a b e t i c s a n d c o n t r o l s i s c o m p a t i b l e w i t h t h e h y p o t h e s i s t h a t n o t only t h e q u a n t i t y b u t a l s o t h e q u a l i t y of t h e s e r u m l i p o p r o t e i n s a r e d i f f e r e n t in t h e s e t w o c a t e g o r i e s of c h i l d r e n . introduction j u v e n i l e o n s e t , insulin-dependent d i a b e t i c s show a n i n c r e a s e d i n c i d e n c e of e a r l y a t h e r o s c l e r o t i c c a r d i o v a s c u l a r d i s e a s e . an i n c r e a s e d a m o u n t of p o l y u n s a t u r a t e d f a t t y a c i d s ( p u f a ) in t h e d i e t c o n t r i b u t e s to a r e d u c t i o n of a t h e r o s c l e r o t i c c a r d i o v a s c u l a r d i s e a s e , at least in n o n d i a b e t i c s (3, 14, 16). a r e c e n t s t u d y s u g g e s t s t h a t a n i n c r e a s e d i n t a k e of p u f a d e c r e a s e s t h e i n c i d e n c e of v a s c u l a r c o m p l i c a t i o n s i n a d u l t o n s e t d i a b e t i c s (10). t h e m e c h a n i s m f o r t h i s b e n e f i c i a l effect i s n o t c l e a r . t h e p o l y u n s a t u r a t e d f a t t y a c i d a r a c h i d o n i c a c i d (20:4) is t h e p r e c u r s o r of t h r o m b o x a n e a 2 (txa2) a n d p r o s t a c y c l i n (pgiz), a n d e i c o s a p e n t a e n o i c a c i d (20:5) i s t h e p r e c u r s o r of t h e c o r r e s p o n d i n g t x a 3 a n d pgi3. in e s c i m o e s a v e r y low i n c i d e n c e of a t h e r o s c l e r o s i s w a s a s s o c i a t e d w i t h a c h a n g e in s e r u m f a t t y a c i d p a t t e r n i n f a v o u r of 20:5 a n d a s u b s e q u e n t c h a n g e i n p c / t x f o r m a t i o n i n f a v o u r of t x a 3 / p g i 3 ( 5 ) . increased platelet aggregation associated with a disturbed prostaglandin/thromboxane formation has been shown i n diabetics with vascular disorder (8). we have recently shown that diabetic children have a diminished vascular response to post-ischaemic hyperaemia (6). this response i s t o a major part dependent on pgi2 formation (6). a f i r s t step i n studying these interactions i s a description of the serum fatty acid composition i n a group of diabetic children without atherosclerosis or diabetic vascular disease compared with the pattern i n healthy matched controls, which i s the aim of this work. subjects the study comprised 28 children aged 4 to 17 years (17 girls and i 1 boys) with insulin dependent diabetes mellitus (iddm). the height, body weight and growth velocity were i n a l l cases within 22 sd for swedish children (12). none of the children had albuminuria, hypertension or ophthalmological or other clinical signs of vascular disease. they were being treated with two doses of intermediate monocomponent porcine insulin daily. some of them were also receiving a rapid monocomponent insulin i n the morning and/or afternoon. their total daily insulin dosage was 0.5 1.2 iu/kg body weight. they were a l l treated according t o the same general principles, including prescriptions of regulated diet, aiming at a fat consumption of 30-33% of the energy intake and a p/s (polyunsaturated/saturated) ratio of about 0.5. judging from clinical and laboratory findings the diabetic disease was under good control (haemoglobin a1 10.8+1.8%, fasting serum glucose 9.6l0.5 mmol/l, serum triglyceride concentations (tg) 0.7220.23 mmol/l, and cholesterol concentrations (chol) 4.59'0.78 mmolf i. thirteen of the diabetic children had peers of the same age and sex, living i n the same area and most often attending the same class a t school, participating as matched healthy controls. this study i s part of an investigation approved by the ethical committee of the medical faculty of the university of uppsala. methods a l l blood specimens were taken fasting at 08.00-08.30 in the morning before breakfast and insulin injection. serum glucose and h b a l were determined by routine methods (yellow spring instruments 23 am glucose analyzer and h b a l microcolumns, bio-rad laboratories). tg and chol in serum were determined by semiautomated methods in an lkb system (2071, 2074, 20861, using commercial test kits (test combination from boehringer-mannheim and nyco-test cholesterol from nyegaard). the concentrations of serum apolipoproteins (apo) b, a-i and a-i1 were determined by electroirnmunoassay using monospecific antibodies as earlier described i n detail (21). the fatty acid compositions of serum triglycerides (tg) cholesterol esters (ce) and phospholipids (pl) were determined in blood serum. the blood samples were allowed to coagulate for two hours and serum was collected by low speed centrifugation at +4oc. the 112 s e r u m lipids w e r e e x t r a c t e d w i t h c h l o r o f o r m m e t h a n o l a n d s e p a r a t e d by t h i n l a y e r c h r o m a t o g r a p h y a c c o r d i n g to a m e t h o d previously d e s c r i b e d (i). t h e fatty a c i d s of t h e i s o l a t e d lipid f r a c t i o n s , t r i g l y c e r i d e s , c h o l e s t e r o l e s t e r s a n d phospholipids, w e r e t r a n s e s t e r i f i e d to m e t h y l e s t e r s (17) a n d s e p a r a t e d i s o t h e r m a l l y at 185oc o n a 10% sp 2340 c o l u m n in a p y e u n i c a m 104 gas-liquid c h r o m a t o g r a p h e q u i p p e d w i t h a n a u t o m a t i c s a m p l e i n j e c t o r ( h e w l e t t p a c k a r d co.) r e t e n t i o n t i m e s of known f a t t y a c i d s t a n d a r d s (supelco inc.,) w e r e used f o r i d e n t i f y i n g t h e p e a k s of t h e c h r o m a t o g r a m (9). t h e r e l a t i v e c o m p o s i t i o n s of t h e d i f f e r e n t i d e n t i f i e d f a t t y a c i d s ( e x p r e s s e d in %) a n d t h e i r r e t e n t i o n t i m e s w e r e c a l c u l a t e d by a h p 3380a d i g i t a l i n t e g r a t o r . t h e s t a t i s t i c a l c a l c u l a t i o n s w e r e p e r f o r m e d by t h e sas statistical p r o g r a m p a c k a g e on i b m 155-158 a t u p p s a l a university. in c o m p a r i s o n s b e t w e e n d i a b e t i c s a n d n o n d i a b e t i c s only p a t i e n t s w i t h m a t c h e d c o n t r o l s w e r e included a n d t e s t e d a g a i n s t t h e i r c o n t r o l s in t w o t a i l e d p a i r e d t-tests. when c o r r e l a t i o n s within t h e d i a b e t i c g r o u p w e r e p e r f o r m e d , a l l t h e 28 d i a b e t i c s u b j e c t s w e r e included. results table 1: r e l a t i v e c o n c e n t r a t i o n of f a t t y a c i d s a n d p i s r a t i o s in s e r u m lipid e s t e r s of 1 3 d i a b e t i c c h i l d r e n c o m p a r e d w i t h t h e i r m a t c h e d h e a l t h y c o n t r o l s ( m e a n 5 sd), (* = ~ ~ 0 . 0 5 , * * = p<o.oi). 14:o 16:o 16:l i8:o 18:l 18:2 18.3 20:3 20:4 20:5 p i s c e c o n t r o l s d i a b e t i c s 0.850.2 0.720.2 11.951.9 11.451.0 3.551.02 2.950.4* 1.050.2 1.050.7 20.623.0 18.051.6 ** 56.455.9 59.953.0 * 1.151.1 0.720.8 0.850.7 0.720.7 3.651.4 4.22 i , 3 * 0.520.5 0.721.1 c o n t r o l s 2.450.8 26.555.3 5.151.4 4.651.5 43.022.7 15.524.9 1.750.7 0.450.7 0.820.5 0.050. i t g d i a b e t i c s 1.750.7 25.321.9 4.620.9 4.650.9 41.252.4 19.352.9 * 1.850.5 0.220.3 1.2+0.6** 0 . 050 . 0 4.551.0 5.050.6* 0.550.2 0.720.1 p l c o n t r o l s d i a b e t i c s 0.450.1 0.350.1 32.854.4 32.754 .o 0.450.4 0.520.3 16.352.2 15.552.3 * 13.751.8 12.321.4 * * 24.154.1 25.623.5 * 0.820.5 0.520.3 * * 4.351.5 4.620.6 6.421.7 7.35 1.8 * * 0.951.4 0.720.7 0.651.1 0.720.1 * * t h e f a t t y a c i d c o m p o s i t i o n of c e , t g a n d p l of t h e d i a b e t i c c h i l d r e n a n d t h e i r c o n t r o l s a r e given i n t a b l e 1 . t h e m o s t s t r i k i n g d i f f e r e n c e s b e t w e e n d i a b e t i c s a n d c o n t r o l s w e r e f o u n d w i t h i n t h e l o n g c h a i n e d u n s a t u r a t e d f a t t y acids. t h e l i n o l e i c a c i d (18:2) a n d 20:4 c o n t e n t in all lipid f r a c t i o n s w e r e s i g n i f i c a n t l y higher in t h e d i a b e t i c s t h a n i n t h e c o n t r o l s . 18:2 in c e w a s t h u s a b o u t 60% in d i a b e t i c s c o m p a r e d w i t h 56% in t h e controls. o n t h e o t h e r hand, o l e i c a c i d (18:l) a n d linolenic a c i d (18:3) w e r e s i g n i f i c a n t l y l o w e r in d i a b e t i c s in c e a n d pl. t h e 18:2/18:1 r a t i o was significantly higher in d i a b e t i c s in a l l s e r u m lipid e s t e r s . t h e p / s r a t i o w a s higher in p l a n d ce. t h e d e g r e e of d i a b e t i c c o n t r o l , m e a s u r e d as h b a l , w a s n o t significantly c o r r e l a t e d to t h e f a t t y a c i d c o n t e n t of a n y lipid f r a c t i o n . t h e c o n c e n t r a t i o n of a p o a-i in s e r u m of t h e d i a b e t i c children was significantly positively c o r r e l a t e d to t h e c o n t e n t of homo-gamma-linolenic a c i d (20:3), 20:4 and t h e r a t i o 18:2/18:1 and n e g a t i v e l y c o r r e l a t e d to t h e c o n t e n t of 18:l in t h e t r i g l y c e r i d e f r a c t i o n while no s u c h c o r r e l a t i o n w a s s e e n in c e o r pl. t h e c o n c e n t r a t i o n of s e r u m a p o a-i1 w a s d i r e c t l y c o r r e l a t e d to t h e c o n t e n t of in 20:3 tg a n d t o t h e r a t i o 20:3/20:4 in c e and tg. t h e c o n c e n t r a t i o n s of s e r u m t r i g l y c e r i d e s w e r e inversely c o r r e l a t e d t o t h e c o n t e n t of 20:4 in p l a n d d i r e c t l y c o r r e l a t e d to t h e 20:3/20:4 r a t i o in t h e s a m e f r a c t i o n . t h e s e r u m c h o l e s t e r o l was n e g a t i v e l y c o r r e l a t e d with t h e c o n t e n t of 20:5 in pl. t a b l e 2. within t h e group of h e a l t h y c o n t r o l s t h e r e w e r e no s i g n i f i c a n t c o r r e l a t i o n s b e t w e e n t h e a p o a-i c o n c e n t r a t i o n a n d any f a t t y a c i d c o n c e n t r a t i o n . t h e s e r u m c o n c e n t r a t i o n of a p o a-i1 a n d a p o r w e r e significantly n e g a t i v e l y c o r r e l a t e d t o t h e c o n t e n t of 14:o in tg. t a b l e 2: c o r r e l a t i o n s b e t w e e n p l a s m a lipid a n d apolipoprotein c o n c e n t r a t i o n s a n d t h e r e l a t i v e c o n c e n t r a t i o n of s o m e f a t t y a c i d s in s e r u m lipid e s t e r s of 28 d i a b e t i c children.only t h e s i g n i f i c a n t c o r r e l a t i o n s a r e presented.. tg vs pl 20:4 p l 20:3/20:4 chol vs p l 20:5 apo a-i vs tg 18:l 20:3 20:4 18:2/18:1 a p o a-i1 v s c e 20:3/20:4 tg 20:3 t g 20:3/20:4 r = -0.52 r = +0.56 r = -0.53 r = -0.55 r = +0.54 r = +0.50 r = +0.51 r = +0.60 r = +0.62 r = +0.57 p co.01 p c o . 0 1 p <0.01 p co.01 p co.01 p <0.01 p co.01 p <0.001 p <0.001 p co.01 discussion a r e g u l a t e d d i e t is t h e basis f o r a l l t r e a t m e n t p r o g r a m m e s f o r d i a b e t e s mellitus. during r e c e n t y e a r s t h e high fat, low c a r b o h y d r a t e d i e t h a s been abandoned in f a v o u r of a d i e t c o n t a i n i n g a high a m o u n t of c o m p l e x c a r b o h y d r a t e s w i t h a r e l a t i v e l y low fat c o n t e n t . with r e g a r d t o t h e d i e t a r y f a t t y a c i d s t h e r e l a t i v e c o n t e n t of p o l y u n s a t u r a t e d fats should b e m o d e r a t e l y i n c r e a s e d (1 5). t h e h e a l t h y c o n t r o l s showed a proportion of linoleic a c i d which w a s similar to t h a t e a r l i e r r e p o r t e d i n a n u r b a n p o p u l a t i o n of h e a l t h y swedish c h i l d r e n (13). t h e h i g h e r c o n t e n t of p u f a in t h e s e r u m lipid e s t e r s in t h e d i a b e t i c c h i l d r e n r e f l e c t s t h e d i e t a r y a d v i c e given. t h e f a t t y a c i d c o n t e n t of t h e t r i g l y c e r i d e s m e r e l y m i r r o r s t h e fat composition of t h e d i e t d u r i n g t h e d a y s p r e c e d i n g t h e test, while t h e c h a n g e of t h e f a t t y a c i d p a t t e r n in t h e c h o l e s t e r o l e s t e r s a n d phospholipids i s m o r e g r a d u a l o v e r a period of s e v e r a l w e e k s (2). in t h e p r e s e n t s t u d y t h e r e w a s n o s i g n i f i c a n t c o r r e l a t i o n b e t w e e n t h e d e g r e e of d i a b e t i c c o n t r o l a n d t h e f a t t y a c i d p a t t e r n in t h e s e r u m lipid e s t e r s . assuming t h a t t h e p / s r a t i o i n t h e s e r u m lipids is c o r r e l a t e d to t h e d i e t a r y a d h e r e n c e , t h i s i n d i c a t e s t h a t t h e r e w a s no significant r e l a t i o n s h i p b e t w e e n t h e d e g r e e of d i e t a r y a d h e r e n c e a n d t h e d e g r e e of d i a b e t i c c o n t r o l . h o w e v e r , t h i s d o e s c e r t a i n l y n o t m e a n t h a t t h e d i e t a r y a d h e r e n c e i s of no i m p o r t a n c e f o r t h e d i a b e t i c c o n t r o l . r a t h e r , t h e l a c k of c o r r e l a t i o n m a y b e d u e to t h e fact t h a t t h e d i a b e t i c c o n t r o l i s i n f l u e n c e d n o t only by t h e fat q u a l i t y of t h e d i e t b u t a l s o by o t h e r p r o p e r t i e s of t h e d i e t as w e l l as by t h e a d m i n i s t r a t i o n of e x o g e n o u s insulin. a new f i n d i n g w a s t h e p o s i t i v e r e l a t i o n s h i p b e t w e e n t h e s e r u m c o n c e n t r a t i o n of a p o a-i and t h e r e l a t i v e c o n t e n t of p o l y u n s a t u r a t e d fat a n d t h e p / s r a t i o in t h e s e r u m triglycerides. a p o a-i i s t h e m a j o r p r o t e i n c o m p o n e n t of t h e high d e n s i t y l i p o p r o t e i n s (11). insulin d e p e n d e n t d i a b e t i c c h i l d r e n h a v e h i g h e r a-i c o n c e n t r a t i o n s t h a n r n a t c h e d h e a l t h y controls (7). in s p i t e of high hdl l e v e l s t h e i n c i d e n c e of a t h e r o s c l e r o t i c v a s c u l a r d i s e a s e in d i a b e t i c s is high. i t h a s b e e n p o s t u l a t e d t h a t t h e h d l c o m p o s i t i o n m a y b e q u a l i t a t i v e l y d i f f e r e n t in d i a b e t i c s c o m p a r e d w i t h t h a t of h e a l t h y c o n t r o l s (4). t h e p o s i t i v e c o r r e l a t i o n between t h e a p o a-i c o n c e n t r a t i o n a n d t h e c o n t e n t of p o l y u n s a t u r a t e d fat i n t g m a y indicate s u c h a d i f f e r e n c e . in n o n d i a b e t i c s a high i n t a k e of p o l y u n s a t u r a t e d fats h a s b e e n shown to d e c r e a s e t h e s e r u m a p o a-i l e v e l s , a t l e a s t d u r i n g s h o r t t e r m d i e t a r y t r e a t m e n t (19, 20). t h e l a c k of c o r r e l a t i o n b e t w e e n t h e s e r u m a p o a-i v a l u e s a n d t h e p / s r a t i o in t h e other s e r u m lipid e s t e r s in t h i s s t u d y i n d i c a t e s t h a t t h e a-i c o n c e n t r a t i o n w a s n o t r e l a t e d t o t h e i n t a k e of p o l y u n s a t u r a t e d fats d u r i n g longer t i m e periods. t h e p o s i t i v e c o r r e l a t i o n between a-i a n d t h e c o n t e n t of p u f a in t h e s e r u m t r i g l y c e r i d e s i n d i c a t e s a p o s i t i v e relationship b e t w e e n t h e p o l y u n s a t u r a t e d d i e t a r y f a t i n t a k e d u r i n g t h e l a s t d a y s b e f o r e t h e s a m p l e w a s t a k e n a n d t h e s e r u m a p o a-i levels. possibly a high i n t a k e of p u f a mirrors a high total fat i n t a k e . i n t r o d u c t i o n of n e u t r a l fat in t h e i n t e s t i n e i n d u c e s synthesis of a p o a-i i n t h e i n t e s t i n a l c e l l s (18). we h a v e e a r l i e r d e s c r i b e d a p o s i t i v e r e l a t i o n s h i p b e t w e e n t h e s e r u m a p o a-i1 c o n c e n t r a t i o n a n d t h e s e r u m t r i g l y c e r i d e c o n c e n t r a t i o n in d i a b e t i c c h i l d r e n (7). t h e present s t u d y s h o w s t h a t b o t h a-i1 a n d t g a r e d i r e c t l y c o r r e l a t e d to t h e r a t i o 20:3/20:4 in t h e s e r u m lipid e s t e r s . t h e t g c o n c e n t r a t i o n w a s i n v e r s e l y c o r r e l a t e d w i t h 20:4 i n tg. w e have no e x p l a n a t i o n f o r t h e s e f i n d i n g s o r f o r t h e n e g a t i v e c o r r e l a t i o n b e t w e e n t h e s e r u m cholesterol c o n c e n t r a t i o n a n d t h e 20:5 c o n t e n t i n pl. o n e m a i n f i n d i n g in t h e p r e s e n t s t u d y w a s t h e c o n f i r m a t i o n of t h e d i f f e r e n c e b e t w e e n d i a b e t i c c h i l d r e n a n d h e a l t h y c o n t r o l s w i t h r e g a r d to t h e r e l a t i o n s h i p s b e t w e e n s e r u m apolipoprotein c o n c e n t r a t i o n s a n d s e r u m lipid c o m p o s i t i o n . w e h a v e e a r l i e r shown i i5 diverging relationships in insulin t r e a t e d d i a b e t i c children and h e a l t h y c o n t r o l s b e t w e e n s e r u m apolipoprotein c o n c e n t r a t i o n s a n d s e r u m lipid c o n c e n t r a t i o n s (7). t h i s s t u d y shows t h a t t h e t w o groups d i f f e r a l s o with r e g a r d to t h e relationships b e t w e e n s e r u m apolipoprotein c o n c e n t r a t i o n s a n d t h e f a t t y a c i d composition of t h e s e r u m lipid e s t e r s . this is c o m p a t i b l e with t h e view t h a t n o t only t h e q u a n t i t y b u t a l s o t h e q u a l i t y of t h e s e r u m lipoproteins a r e d i f f e r e n t in t h e s e t w o c a t e g o r i e s of children (4). acknowledgement this s t u d y was s u p p o r t e d by g r a n t s f r o m ab novo, malmo, sweden a n d by t h e swedish m a r g a r i n e industrial association f o r nutritional-physiological r e s e a r c h . 1. 2. 3. 4. 5. 6. 7. 8. 9. 10 i 1 12 1 3 14 i16 references boberg, j.: s e p a r a t i o n of labelled p l a s m a a n d t i s s u e lipids by thin l a y e r chromatography. a q u a n t i t a t i v e m e t h o d o l o g i c a l study. c l i n c h i m a c t a 14:325-334, 1966. boberg, j., g u s t a f s s o n , i-!3., k a r l s t r o m , b., 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validity of hill's equation in an artificial actomyosin streaming system erik c e r v e n the faculty of pharmaceutical sciences, university of tokyo, bunkyo-ku, tokyo 113, japan a b s t r a c t t h e r o t a t i o n o f a n a c t o m y o s i n m o t o r ( 9 , 1 1 ) , a s s e m b l e d f r o m b l a d e s , o n e s i d e o n t o w h i c h f a c t i n o f u n i f o r m p o l a r i t y w a s a t t a c h e d , s u s p e n d e d i n a s o l u t i o n o f h e a v y m e r o m y o s i n (hmm), w a s m o d e l l e d a s d u e t o s l i d i n g o f h m m o v e r t h e m a r g i n s o f t h e b l a d e s , w h e r e b y t h e w o r k r e s u l t i n g f r o m a t p a s e a c t i v i t y i s u s e d f o r p u s h i n g b u l k f l u i d c o n t a i n i n g h m m f r o m t h e l e a d i n g s u r f a c e o f t h e b l a d e o v e r t h e f o r c e g e n e r a t i n g f i l a m e n t s t o t h e b a c k s u r f a c e , w h i c h l e a d s t o i n c r e a s e d s l i d i n g v e l o c i t y . t h e a m o u n t o f h m m c o n t r i b u t i n g t o f o r c e g e n e r a t i o n i s d i v i d e d i n t o o n e c o m p o n e n t p e r p e n d i c u l a r t o t h e f i l a m e n t s , w h i c h i s d i f f u s i o n l i m i t e d a n d r e g u l a t e d b y a c o m p o n e n t p a r a l l e l t o t h e f i l a m e n t s , r e p r e s e n t e d b y t h e m o v e m e n t o f t h e b u l k f l u i d , t h e s u p p l y o f n e w h m m a n d t h e o b s e r v a b l e v e l o c i t y o f r o t a t i o n o f t h e b l a d e . u s i n g h i l l ' s e q u a t i o n ( 6 ) w h i c h e s s e n t i a l l y s t a t e s t h a t t h e p r o d u c t o f a v i r t u a l f o r c e a n d a v i r t u a l v e l o c i t y i s c o n s t a n t w i t h i n t h e r a n g e of o b s e r v a b l e f o r c e s a n d v e l o c i t i e s , t h e f o r c e c a n b e e x p r e s s e d a s v e l o c i t y , g i v i n g a s i m p l e f i r s t o r d e r d i f f e r e n t i a l e q u a t i o n . a s o l u t i o n t o t h i s e q u a t i o n c a n b e f i t t e d t o t h e o b s e r v e d d a t a w i t h p h y s i c a l l y m e a n i n g f u l c o n s t a n t s i n terms o f m a s s , v i s c o s i t y , t h e f o r c e v e l o c i t y c o n s t a n t a n d a c o n s t a n t e x p r e s s i n g t h e q u a l i t y o f t h e p r e p a r a t i o n . c o n t r o l e x p e r i m e n t s were p e r f o r m e d , u s i n g f i x e d b l a d e s . i n t h e s e e x p e r i m e n t s , b u l k f l o w a r o u n d t h e m a r g i n s d i d n o t i n c r e a s e a s a r e s u l t o f a t p a s e a c t i v i t y a n d n o m o v e m e n t o r s t r e a m i n g w a s o b s e r v e d . t h e r e s u l t s s h o w t h a t h i l l ' s e q u a t i o n may b e a p p l i c a b l e t o s t r e a m i n g c a u s e d b y a c t o m y o s i n i r r e s p e c t i v e o f i t s c o n f i n e m e n t t o s k e l e t a l m u s c l e a n d i n d e p e n d e n t l y o f p r e f e r r e d m o l e c u l a r m o d e l , a n d l e a d s t o s e v e r a l v e r i f y a b l e p r e d i c t i o n s . 89 i n t r o d u c t i o n h i l l ' s e q u a t i o n i s a n e m p i r i c a l e q u a t i o n w h i c h d e s c r i b e s t h e f o r c e v e l o c i t y r e l a t i o n s h i p o f c o n t r a c t i n g s k e l e t a l m u s c l e ( 6 ) . i t c a n b e d e r i v e d f r o m c o n s i d e r a t i o n s o f t h e m o l e c u l a r m e c h a n i s m o f a c t o m y o s i n i n t e r a c t i o n , p r o v i d i n g a m a t h e m a t i c a l f o u n d a t i o n f o r t h e s l i d i n g f i l a m e n t m o d e l o f m u s c u l a r c o n t r a c t i o n ( 7 ) . t h i s s h o w s t h a t t h e m a c r o s c o p i c b e h a v i o u r o f w h o l e m u s c l e may f o l l o w f r o m p o s t u l a t e d m o l e c u l a r e v e n t s ( 7 , 8 ) a n d r a i s e s t h e q u e s t i o n how c l o s e t o t h e e x p e r i m e n t a l m o l e c u l a r l e v e l h i l l ' s e q u a t i o n may a c t u a l l y b e v a l i d . i n t h e p r e s e n t r e p o r t , it i s s h o w n t h a t t h e m o v e m e n t o f a n a r t i f i c i a l a c t o m y o s i n s t r e a m i n g s y s t e m , t h e " a c t o m y o s i n m o t o r " ( y , l l ) , i s c o m p a t i b l e w i t h h i l l ' s e q u a t i o n . t h i s t a k e s h i l l ' s e q u a t i o n f r o m w h o l e m u s c l e , b y p a s s i n g t h e c e l l u l a r a n d s a r c o m e r i c l e v e l s , d o w n t o t h e s u p r a m o l e c u l a r l e v e l a s r e p r e s e n t e d b y t h e j o i n t a c t i n s t r a n d s o n t h e b l a d e s o f t h e a c t o m y o s i n m o t o r . t h e r e f o r e , t h e p r e s e n t a n a l y s i s j u s t i f i e s m o d e l s w h e r e t h e a c t o m y o s i n i n t e r a c t i o n i n s u p r a m o l e c u l a r s t r u c t u r e s i t s e l f , r e g a r d l e s s of e l a s t i c i t y o r o t h e r m a c r o s c o p i c p a r a m e t e r s o f w h o l e s k e l e t a l m u s c l e o r s a r c o m e r e s , p r o v i d e s t h e b a s i s of h i l l ' s e q u a t i o n . methods t h e m e t h o d s h a v e b e e n d e s c r i b e d b e f o r e ( 9 , 1 1 1 . b r i e f l y , a c t i n w a s p r e p a r e d f r o m c o n t r a c t e d a c e t o n e d r y p o w d e r ( 3 , l o ) a n d h e a v y m e r o m y o s i n b y t r y p t i c d i g e s t i o n o f m y o s i n f o r 5 m i n a t 2 0 c a t a w e i g h t r a t i o ( t r y p s i n l m y o s i n ) of 1 1 2 0 0 . t h e m y o s i n h a d b e e n e x t r a c t e d f r o m m i n c e d m u s c l e i n 0.2 m p h o s p h a t e b u f f e r , ph 6 . 4 6 . 5 , c o n t a i n i n g 0.5 m p o t a s s i u m c h l o r i d e a n d i n a d d i t i o n p h o s p h o c r e a t i n e t o a v o i d a c c u m u l a t i o n o f adp a s p r e v i o u s l y c a u t i o n e d ( 5 ) . t h e a c t i n was p o l y m e r i z e d o n t o p o l y l y s i n e c o a t e d t e f l o n a t t a c h e d t o mica o n a n " a c t o m y o s i n m o t o r " a s d e s c r i b e d p r e v i o u s l y ( 9 , l l ) . t h e a c t o m y o s i n m o t o r h a s s i x b l a d e s w i t h a l l t o g e t h e r 2 4 m a r g i n s . i n a c o n t r o l e x p e r i m e n t , 2 4 b l a d e s were a t t a c h e d t o t h e i n n e r s u r f a c e o f a c y l i n d r i c a l c o n t a i n e r , w i t h f r e e m a r g i n s f a c i n g a 2 m m s l i t a n d t h e b o t t o m o f t h e c o n t a i n e r . t h e a c t o m y o s i n m o t o r a n d t h e c o n t r o l d e v i s e a r e s c h e m a t i c a l l y i l l u s t r a t e d i n f i g . 1 a a n d b , r e s p e c t i v e l y . a f t e r p o l y m e r i z a t i o n , n a t i v e t r o p o m y o s i n ( 4 ) , c o n t a i n i n g b o t h t r o p o n i n a n d t r o p o m y o s i n was e q u i l i b r a t e d w i t h t h e a c t i n , t h e b l a d e s w a s h e d b r i e f l y i n b u f f e r e d k c 1 a n d s t o r e d i n t h e c o l d room o v e r n i g h t . b e f o r e t h e e x p e r i m e n t , t h e d e v i s e was t r a n s 0 f e r r e d t o a s o l u t i o n c o n t a i n i n g 1 g / 1 'of h m m i n 9 0 m m k c 1 , 4 m m mgc1, 1 0 m m m o p s , p h 7.0 f o l l o w e d b y t ' h e a d d i t i o n o f a t p t o 2 m m a n d ca i o n s t o 3 0 um. t h e m o v e m e n t w a s o b s e r v e d u s i n g a l o w m a g n i f y i n g m i c r o s c o p e a n d v i d e o e q u i p m e n t a n d s u b s e q u e n t l y q u a n t i f i e d b y m a n u a l l y m e a s u r i n g t h e a n g l e o f m o v e m e n t on t h e tv s c r e e n . f i g . 1. a . i l l u s t r a t i o n o f t h e f l o w o f h m m c o n t a i n i n g b u f f e r a r o u n d t h e m a r g i n s o f t h e " a c t o m y o s i n m o t o r " a s i t r o t a t e s i n t h e s o l u t i o n w h e r e i t i s s u s p e n d e d b y a f l o a t i n g c o r k t h e a c t o m y o s i n mo t o r c o n s i s t s o f s i x b l a d e s , o n e s i d e o n t o w h i c h a c t i n o f u n i f o r m p o l a r i t y h a s b e e n a t t a c h e d , t h e a r r o w h e a d d i r e c t i o n p o i n t i n g t o w a r d s t h e b l a d e . a s a r e s u l t o f f o r c e g e n e r a t i o n d u e t o t h e i n t e r a c t i o n o f h m m w i t h a c t i n , t h e b l a d e s a r e p u s h e d f o r w a r d a n d s o l u t i o n s t a r t s t o f l o w a r o u n d t h e m a r g i n s ( i n d i c a t e d b y b l a c k a r r o w s f b. i l l u s t r a t i o n o f t h e f l o w o f s o l u t i o n a t t h e m a r g i n s o f t h e c o n t r o l d e v i s e i n w h i c h t h e b l a d e s a r e f i x e d . i n t h i s c a s e , t h e f o r c e c a n n o t b e u t i l i z e d f o r p u s h i n g m o r e h m m f r o m t h e a n t e r i o r s i d e o f t h e b l a d e s o n t o t h e m a r g i n a l f i l a m e n t s . r e s u l t s a n d discussion t h e m o v e m e n t o f f l u i d a r o u n d t h e m a r g i n s o f t h e s i x s u s p e n d e d b l a d e s o f t h e a c t o m y o s i n m o t o r a n d t h e c o n t r o l d e v i s e a r e i l l u s t r a t e d i n f i g . 1 a a n d b , r e s p e c t i v e l y . i n t h e a c t o m y o s i n m o t o r , t h e f o r c e g e n e r a t e d i s p r e s u m e d t o b e p r o p o r t i o n a l t o t h e a m o u n t o f h m m w h i c h c a n d i f f u s e o n t o t h e a c t i n f i l a m e n t s a t t h e i n i t i a l v e l o c i t y t i m e s t h e a m o u n t o f h m m n e w l y s u p p l i e d b y t h e b u l k f l o w a r o u n d t h e m a r g i n s , w h i c h i s p r o p o r t i o n a l t o t h e o b s e r v e d v e l o c i t y 91 o f r o t a t i o n , v . i n t h e c o n t r o l d e v i s e , t h e b l a d e s a r e f i x e d t o t h e w a l l o f t h e c y l i n d e r a n d , a s a c o n s e q u e n c e , n o b u l k f l u i d c a n b e p u s h e d a r o u n d t h e m a r g i n s a n d t h e s t r e a m i n g w i l l r e m a i n a t t h e i n i t i a l d i f f u s i o n l i m i t e d v e l o c i t y . t h e a v e r a g e i n t e r m o l e c u l a r d i s t a n c e i n a s o l u t i o n c o n t a i n i n g 1 g / 1 o f h m m ( w h i c h i s s u f f i c i e n t t o o b s e r v e m o v e m e n t ) i s a p p r o x i m a t e l y 1 0 0 0 a , a n d t h e d i a m e t e r o f t h e a c t i v e g l o b u l a r p o r t i o n of o n e s u b u n i t o f h m m i s i n t h e r a n g e o f 1 0 0 a , a p p r o x i m a t e l y 1 0 t i mes l e s s . if i t i s a s s u m e d t h a t t h e h m m w h i c h i n t e r a c t s w i t h a c t i n i s t r a n s l o c a t e d u n d e r n o l o a d a l o n g t h e f i l a m e n t w i t h a v e l o c i t y o f a t l e a s t 1 2 u m / s ( f i g . 2 ) i t w o u l d h a v e t o b e r e p l a c e d w i t h i n l e s s t h a n 0 . 8 m s w h i c h i s t h e t i m e i t t a k e s f o r i t t o l e a v e a n e l e m e n t a r y s p a c e o f 1 0 0 a o n t h e f i l a m e n t . s i n c e t h e i n t e r m o l e c u l a r d i s t a n c e i n s o l u t i o n i s , o n a v e r a g e , 1 0 t i m e s l o n g e r t h a n t h a t , a n d t h e o r i e n t a t i o n o f h m m i s r a n d o m , t h e p r o b a b i l i t y o f s u c h i n s t a n t a n e o u s r e p l a c e m e n t f r o m t h e s o l u t i o n o f h m m m o l e c u l e s m o v i n g o n t h e f i l a m e n t s i s e v i d e n t l y l o w . t h e c o n t r i b u t i o n t o t h e p r o b a b i l i t y g i v e n b y e a c h m o l e c u l e i s p r o v i d e d b y e q u a t i o n ( 1 1 , w h i c h d e s c r i b e s t h e t h r e e d i m e n s i o n a l d i f f u s i o n c a s e : i n t h i s e q u a t i o n , d x i s o f t h e o r d e r o f m a g n i t u d e o f t h e m o l e c u l a r d i m e n s i o n s o f t h e h m m m o l e c u l e w h i c h i s t o b e r e p l a c e d , d t h e d i f f u s i o n c o e f f i c i e n t o f h m m a n d x a m u l t i p l e o f t h e a v e r a g e i n t e r m o l e c u l a r d i s t a n c e b e t w e e n h m m m o l e c u l e s i n t h e s o l u t i o n . t h e r e l e v a n t t i m e i s s h o r t e r t h a n 1 m s w h i c h c o r r e s p o n d s t o t h e d i s t a n c e o f " e l e m e n t a r y " d i s p l a c e m e n t o f a n h m m m o l e c u l e w i t h t w o h e a d s a s s u m i n g t h a t i t s m a x i m a l v e l o c i t y i s h i g h e r t h a n 1 2 u m / s , w h i c h i s t h e o b s e r v e d v e l o c i t y o f r o t a t i o n i n t h e e x p e r i m e n t a n a l y s e d ( f i g . 2 ) . e q u a t i o n ( 1 ) d o e s n o t a l l o w f o r s t e r i c f a c t o r s i n t h a t o n l y c e r t a i n o r i e n t a t i o n s o f o n d i f f u s i n g h m m c a n c o n t r i b u t e t o f o r c e g e n e r a t i o n . f r o m t h e c o n s i d e r a t i o n s a b o v e , i t seems r e a s o n a b l e t o a s s u m e t h a t t h e b u l k f l o w a r o u n d t h e m a r g i n s s u p p l i e s h m m t o s u s t a i n move m e n t , a n d t h a t a s more h m m i s a d d e d w h e n t h e v e l o c i t y of s t r e a m i n g i n c r e a s e s , t h e f o r c e d e v e l o p m e n t w o u l d a l s o i n c r e a s e a s a f u n c t i o n o f t h e a m o u n t o f h m m s u p p l i e d . t h i s s i t u a t i o n r e s e m b l e s t h a t w h e n t h e d e g r e e of o v e r l a p i n t h e s a r c o m e r e of s k e l e t a l m u s c l e d e t e r 92 m i n e s how m u c h m y o s i n i s s u p p l i e d t o t h e f o r c e g e n e r a t i n g s y s tem a n d i s i n d e p e n d e n t o n w h e t h e r t h e h m m i s s u p p l i e d i n d i v i d u a l l y o r i n t h e f o r m o f s m a l l a g g r e g a t e s . t h e p r e s e n c e o f a g g r e g a t e s o f h m m i n t h e p r e s e n t s y s t e m w o u l d s t r o n g l y f a v o u r t h e a r g u m e n t s p r e s e n t e d b e c a u s e of t h e l o n g e r i n t e r m o l e c u l a r d i s t a n c e b e t w e e n t h e o l i g o m e r s a n d t h e i r s l o w e r d i f f u s i o n . f i n a l l y , t h e i d e a t h a t t h e f l o w a r o u n d t h e m a r g i n s s u p p l i e s h m m t o t h e p r e s e n t f o r c e g e n e r a t i n g s y s t e m i s c o m p a t i b l e w i t h t h e r e c e n t o b s e r v a t i o n o f a c o m p a r a t i v e l y s l o w v e l o c i t y o f s t r e a m i n g ( 1 u m / s ) i n a s y s t e m w i t h e i t h e r o n e o r s e v e r a l h u n d r e d c l o s e l y a p p o s e d b u t i m m o b i l e m a r g i n s o f p o l a r i z e d a c t i n ( 1 2 ) r e l a t i v e t o t h e m a x i m a l v e l o c i t y o f m o v e m e n t o b s e r v e d i n t h e p r e s e n t s y s t e m , w h i c h i s 40 u m / s ( 1 1 ) . a s s u m i n g l a m i n a r f l o w , t h e s u p p l y o f h m m t o t h e f o r c e g e n e r a t i n g s i t e i s a l i n e a r f u n c t i o n o f v e l o c i t y , w h i c h i s d e s c r i b e d b y : = k v v f u f t o t t h i s e q u a t i o n e x p r e s s e s t h a t t h e t o t a l f o r c e d e v e l o p m e n t , f t o t , i s r e g a r d e d a s p r o p o r t i o n a l t o a p r o d u c t o f t w o c o m p o n e n t s , o n e o f w h i c h i s t h e e x t r a s u p p l y of h m m i n t h e l a m i n a r f l o w , t h e " o v e r l a p " , r e p r e s e n t e d b y v , w h i c h i s t h e o b s e r v e d v e l o c i t y o f s t r e a m i n g , a n d t h e o t h e r o f w h i c h i s t h e s p e c i f i c f o r c e d e v e l o p m e n t o f h m m , r e l a t e d t o a u n i t a r y a m o u n t o f t h a t p r o t e i n a n d d e n o t e d b y s t a n d s f o r " u n i t a r y " . k i s a c o n s t a n t w h i c h r e f u . t h e i n d e x " u " l a t e s t h e v e l o c i t y o f s t r e a m i n g t o t h e a d d i t i o n a l f o r c e p r o v i d e d b y some o f t h e h m m m o l e c u l e s s u p p l i e d i n t h e f l o w w h e n t h e y i n t e r a c t w i t h t h e a c t i n s t r a n d s . t h e u n i t a r y f o r c e , f u , w h i c h c a n n o t b e r e l a t e d t o a n y m o l e c u l a r m e c h a n i s m i n t h e p r e s e n t t r e a t m e n t wo u l d b e r e l e v a n t t o i t a n d t o t h e o b s e r v e d m o v e m e n t a n d a c c e l e r a t i o n o f t h e a c t o m y o s i n m o t o r . t h e f o r c e p r o d u c e d b y t h e e l e m e n t a r y c y c l e s o f h m m l e ' s d s t o a c c e l e r a t i o n o f t h e d e v i s e u n t i l i t s r o t a t i o n a l v e l o c i t y i s b a l a n c e d b y t h e f l o w f r i c t i o n . t h i s c a n b e d e s c r i b e d b y : v c v e f t o t m a = k ( 3 ) w h e r e m i s t h e m a s s o f t h e a c t o m y o s i n m o t o r , k e a n e x p e r i m e n t a l c o n s t a n t r e l a t i n g t o t h e l e n g t h o f t h e m a r g i n s , t h e q u a l i t y o f t h e p r o t e i n s a n d o f t h e p r e p a r a t i o n a n d p r e s u m a b l y t h e l e n g t h o f t h e a c t i n f i l a m e n t s a n d t h e i r c o h e r e n c e ( c f . 2 ) . c i s t h e f r i c 93 t j o n a l c o n s t a n t . i f ( 2 ) i s i n s e r t e d i n t o ( 3 ) , we g e t : m a = k v f c v ( 4 ) u w h e r e f i s t h e u n i t a r y f o r c e . k v a n d k a r e i n c o r p o r a t e d i n t o t h i s n e w c o n s t a n t k. t h i s e x p r e s s i o n p r o v i d e s t h e o b s e r v e d a c c e l e r a t i o n d v / d t , w h i c h i s g j v e n b y : u d v k f u v cv_ d t m m ( 5 h i l l ' s e q u a t i o n , u s u a l l y g i v e n i n t h e f o r m ( p + a ) * ( v i b ) = c , w h e r e p i s t h e t e n s i o n o f c o n t r a c t i n g m u s c l e , v t h e v e l o c i t y o f c o n t r a c t i o n , a n d a a n d b c o n s t a n t s o f d i m e n s i o n f o r c e a n d v e l o c i t y , r e s p e c t i v e l y ( c i s t h e f o r c e v e l o c i t y c o n s t a n t ) , e s s e n t i a l l y s t a t e s t h a t t h e p r o d u c t o f a v i r t u a l f o r c e a n d a v i r t u a l v e l o c i t y o f c o n t r a c t i o n i s c o n s t a n t w i t h i n t h e r a n g e o f o b s e r v a b l e f o r c e s a n d v e l o c i t i e s a n d t h u s , t h a t : * * f v = k * * w h e r e k i s t h e f o r c e v e l o c i t y c o n s t a n t a n d f a n d v t h e v i r t u a l f o r c e a n d v e l o c i t y , r e s p e c t i v e l y . i n t h e c a s e o f t h e a c t o m y o s i n m o t o r , t h e t o t a l f o r c e i n c r e a s e s l i n e a r l y w i t h v e l o c i t y d u e t o t h e i n c r e a s e d o v e r l a p r e s u l t i n g f r o m t h e s u p p l y o f h m m o v e r t h e e d g e s w h i l e t h e e l e m e n t a r y f o r c e fu c o n t r i b u t e s t o t h e t o t a l f o r c e a c c o p d i n g t o e q ( 2 ) . as t h e v e l o c i t y i n c r e a s e s , t h e f l o w f r i c t i o n , a n d c o n s e q u e n t l y t h e t e n s i o n i n c r e a s e s , a n d t h e r e f o r e , t h e s i t u a t i o n i s a n a l o g o u s t o t h a t o f c o n t r a c t i n g m u s c l e , t h e o n l y d i f f e r e n c e b e i n g t h a t t h e o v e r l a p a l s o i n c r e a s e s w h e n t h e v e l o c i t y i n c r e a s e s . s i n c e h i l l ' s e q u a t i o n , r e l a t i n g t e n s i o n t o v e l o c i t y , h o l d s i r r e s p e c t i v e o f t h e d e g r e e o f o v e r l a p i n s k e l e t a l m u s c l e , i t i s a p p r o p r i a t e t o u s e i t a l s o f o r t h e " u n i t a r y " f o r c e i n t h e p r e s e n t t r e a t m e n t . u s i n g ( 6 ) , t h e f o r c e c a n b e e l i m i n a t e d f r o m e q ( 5 ) s o t h a t i t t a k e s t h e f o r m : d t m m (7) x d v * + a v = b d t w h e r e a a n d b a r e c o n s t a n t s g i v e n b y c/m a n d k k f m , r e s p e c t i v e l y . t o m a k e t h e s u b s t i t u t i o n ( 7 ) i s e s s e n t i a l l y t o s t a t e t h a t h i l l ' s e q u a t i o n i s v a l i d a t t h e u n i t a r y l e v e l d e f i n e d i n ( 2 ) . i n t h e f o l l o w i n g , some c o n s e q u e n c e s o f t h i s p o s t u l a t e a r e e x a m i n e d . a s o l u t i o n t o e q u a t i o n ( 8 ) i.s: b a t 0 a v = ( b -) e ( b a) w h e r e b i s h i l l ' s c o n s t a n t , b , o r , b b a t b b b a2 a a a a x = (-) e + (-h ) t (-2-) w h i c h i s e q u i v a l e n t t o : kkm bm kk kkm bm c c c c 2 c x = (-7 + ( b ) t (-) ( 9 ) ( 1 1 ) w h e r e x i s t h e o b s e r v e d a n g l e o f r o t a t i o n , m e a s u r i n g d i s t a n c e , a n d b i s h i l l ' s c o n s t a n t , b . w i t h e x p e r i m e n t a l l y d e t e r m i n e d c o n s t a n t s t h i s e q u a t i o n f i t s q u a l i t a t i v e l y t h e o b s e r v e d a n g l e o f r o t a t i o n i n p r e p a r a t i o n s o f h i g h e n o u g h q u a l i t y t o a l l o w s u s t a i n e d r o t a t i o n a n d d e t e r m i n a t i o n o f t h e r a t i o b / a ( f i g . 2 ) . t h e d e c r e a s e d v e l o c i t y o f r o t a t i o n o b s e r v e d a f t e r 3 0 5 0 m i n i n t h e f i g u r e i s m o s t l i k e l y d u e t o a t p d e p l e t i o n , a n d c o r r e s p o n d s t o t h e t i m e r e q u i r e d t o o b s e r v e a s h i f t o f t h e a b s o r p t i o n a t 2 8 4 nm o f h m m , w h i c h i s d u e t o t h e a c c u m u l a t i o n o f a d p ( c e r v e n , 1 9 8 5 , u n p u b l i s h e d o b s e r v a t i o n s ; c f . r e f . 5 ) . i n e x p e r i m e n t s w h e r e t h e r o t a t i o n slows d o w n e a r l i e r , p e r t u r b a t i o n o f t h e a c t i n c a n n o t b e e x c l u d e d t o b e a n a d d i t i o n a l f a c t o r . i n t h e p r e s e n t s t u d y , s u c h r a p i d c e s s a t i o n o f r o t a t i o n a f t e r a n i n i t i a l b u r s t w a s o b s e r v e d w h e n t h e a c t o m y o s i n m o t o r was u s e d i m m e d i a t e l y a f t e r p r e p a r a t i o n . t h e m a x i m a l v e l o c i t y i s g i v e n b y t h e s l o p e o f t h e c u r v e , o r : kk v = (b ) = ( k k b c ) 1 max c c 95 f i g . 2. e x p e r i m e n t a l r e s u l t s u s i n g a n " a c t o m y o s i n m o t o r " ( 9 , 11, f i g . 1 a ) . t h e a n g u l a r r n o v e r n e n t i s p l o t t e d a g a i n s t time. t h e e x p e r i m e n t a l c o n s t a n t s were d e t e r m i n e d b y c u r v e f i t t i n g a n d t h e c u r v e i s d e s c r i b e d b y t h e e q u a t i o n : x = 2 0 . 7 5 e " l t + 2 . l t 2 0 . 7 5 a n d , a s e x p e c t e d , i s p r o p o r t i o n a l t o t h e f o r c e c o n s t a n t r e p r e s e n t i n g t h e q u a l i t y of t h e p r e p a r a t i o n a n d t h e f l o w s u p p l y c o u p l i n g o f hmm, k , t h e f o r c e v e l o c i t y c o n s t a n t , k , a n d i n v e r s e l y p r o p o r t i o n a l t o t h e f r i c t i o n a l c o n s t a n t , c , o r t h e v i s c o s i t y . t h e o b s e r v e d v e l o c i t y a t t i m e t i s g i v e n b y : = ( b c k k ) l c e-mt ( b c k k ) 1 c c a n d t h e a c c e l e r a t i o n b y : as e x p e c t e d , t h e m a s s o f t h e r o t o r d o e s n o t a f f e c t t h e m a x i m a l v e l o c i t y b u t , p r o v i d i n g i n e r t i a , a p p e a r s i n t h e e x p o n e n t i a l term d e l a y i n g t h e a c c e l e r a t i o n o f t h e r o t o r . a l s o t h e f r i c t i o n c o n s t a n t a n d c o n s e q u e n t l y t h e v i s c o s i t y d e c r e a s e s t h e a c c e l e r a t i o n t e r m , b u t t h i s d e p e n d e n c e may n o t b e s t r a i g h t f o r w a r d s i n c e a m e d i u m o f h i g h e r v i s c o s i t y c o u l d p r o v i d e a h i g h e r c o u n t e r f o r c e r e l a t i v e t o w h i c h t h e o b s e r v a b l e a c c e l e r a t i o n t a k e s p l a c e . t h e p r e s e n t t r e a t m e n t d o e s n o t e l a b o r a t e o n h i l l ' s c o n s t a n t b , t h e n a t u r e of w h i c h i s p o o r l y k n o w n , b u t m a y b e r e l a t e d t o t h e c h e m i c a l r e a c t i o n r a t e o r t h e " i n c r e a s e o f e n e r g y r a t e p e r g w e i g h t d e c r e a s e o f l o a d " ( 6 ) . t h e c h e m i c a l r e a c t i o n r a t e s i n u n i d i r e c t i o n a l s t r e a m i n g s y s t e m s may i n v o l v e c o n s i d e r a b l e u t i l i z a t i o n o f atp a n d may b e q u a l i t a t i v e l y d i f f e r e n t f r o m t h o s e d u r i n g c o n t r a c t i o n o f m u s c l e w h e r e t h e s y m m e t r y o f t h e s a r c o m e r e p r o v i d e s a n i d e a l f r a m e w o r k f o r f u t i l e c y c l e s o r a t p r e g e n e r a t i n g c i r c u i t s . t h e p r e s e n t a n a l y s i s i n d i c a t e s t h a t h i l l ' s e q u a t i o n e x p r e s s e s a n i n h e r e n t p r o p e r t y i n v e c t o r i a l l y c o n t r a c t i n q a c t o m y o s i n a s s e m b l i e s , i r r e s p e c t i v e o f m u s c l e e l a s t i c i t y o r f o r c e t r a n s m i s s i o n i n w h o l e m u s c l e a n d e v e n i r r e s p e c t i v e o f t h e p r e s e n c e o r a b s e n c e o f a m y o s i n f i l a m e n t , a n d l e a d s t o s e v e r a l v e r i f y a b l e p r e d i c t i o n s . t h e s e p r e d i c t i o n s , f o r e x a m p l e t h o s e r e l a t i n g t o t h e i n f l u e n c e o f mass a n d v i s c o s i t y o n t h e c o o r d i n a t e a n d v e l o c i t y o f m o v e m e n t ? a r e i n t u i t i v e l y a c c e p t a b l e , a n d c o u l d b e c h e c k e d f o r e x a m p l e b y c h a n g i n q t h e mass o f t h e r o t o r o r t h e v i s c o s i t y o f t h e m e d i u m b y a l t e r i n g f o r e x a m p l e t h e c o n c e n t r a t i o n o f h m m o r t h e t e m p e r a t u r e . b e c a u s e o f t h e d i f f i c u l t y i n o b t a i n i n g e x a c t l y e q u i v a l e n t p r e p a r a t i o n s of a c t o m y o s i n m o t o r s , w h i c h w o u l d r e q u i r e a c c u r a c y a t t h e n a n o m e t e r l e v e l , a n d s i n c e o n e p r e p a r a t i o n c a n o n l y b e u s e d o n c e , i t i s a t p r e s e n t i m p o s s i b l e t o p e r f o r m s u c h c o m p a r a t i v e e x p e r i m e n t s . t h e m a i n c o n c l u s i o n o f t h e p r e s e n t t h e o r e t i c a l a n d s e m i e m p i r i c a l r e s u l t s r e l a t e t o t h e v a l i d i t y o f h i l l ' s e q u a t i o n a n d p a r t j c u l a r l y i t s i n v e r s e f o r c e v e l o c i t y r e l a t i o n s h i p . a s s u c h , t h e p r e s e n t r e s u l t s b a s e d o n t h e a s s u m p t i o n o f a u n i t a r y f o r c e e x e r t e d b y h m m a r e t h e f i r s t i n d i c a t i o n t h a t t h e f o r c e v e l o c i t y r e l a t i o n s h i p m a y h o l d o u t s i d e t h e s a r c o m e r e . t h i s i n f o r m a t i o n w o u l d b e i m p o r t a n t t o u n d e r s t a n d t h e m o l e c u l a r m e c h a n i s m o f a c t o m y o s i n c o n t r a c t i o n , w h i c h i s n o t y e t k n o w n , b u t f o r w h i c h s e v e r a l more o r l e s s e x a c t t h e o r i e s h a v e b e e n p r o p o s e d . acknowledgements t h i s w o r k was s u p p o r t e d b y t h e j a p a n r e s e a r c h d e v e l o p m e n t c o r p o r a t i o n ? t h e s w e d i s h b o a r d o f t e c h n i c a l d e v e l o p m e n t t h r o u g h t h e s w e d e n j a p a n f o u n d a t i o n f o r r e s e a r c h a n d d e v e l o p m e n t ? t h e f a c u l t y o f p h a r m a c e u t i c a l s c i e n c e s , u n i v e r s i t y o f t o k y o a n d a g r a n t f r o m f o r t i a a b a n d t h e u n i v e r s i t y o f u p p s a l a . t h e a u t h o r i s g r a t e f u l t o p r o f . h. s h i m i z u f o r v a l u a b l e c r i t i c i z m a n d t o m e m b e r s o f h i s l a b o r a t o r y f o r e x p e r i m e n t a l a d v i s e a n d f o r r e a d i n g t h e m a n u s c r i p t . t h a n k s a r e a l s o e x t e n d e d t o m e m b e r s o f t h e i n t e r n a t i o n a l m u s c l e r e s e a r c h c o m m u n i t y , w h o s e c o m m e n t s a s r e v i e w e r s h a v e l e a d t o i m p r e v e m e n t s of t h e p r e s e n t i n v e s t i g a t i o n . 97 references 1. 2 . 3 . 4. 5. 6 . 7 . 6 . 9 . 1 0 . 11. 1 2 . b r o w n , s. s . : i n l. w i l s o n ( e d ) m e t h o d s i n c e l l b i o l o g y v o l 2 4 p t a . d i r e c t i o n a l g r o w t h o f a c t i n o f f p o l y l y s i n e c o a t e d b e a d s . a c a d e m i c p r e s s , n e w y o r k , l o n d o n , p a r i s , s a n d i e g o , s a n f r a n s i s c o , s a o p a u l o , s y d n e y , t o k y o , t o r o n t o 1 9 6 2 p p 2 9 1 3 0 0 c e r v e n , e : e v i d e n c e o f " i n t e r f i l a m e n t c o o p e r a t i v i t y " i n t h e p o l y m e r i z a t i o n o f a c t i n . 3 : r d i n t l c o n g r c e l l b i o l a b s t r n:o 4 1 9 1 , 1 9 8 4 d r a b i k o w s k i , w . & g e r g e l y , 3 : t h e e f f e c t o f t h e t e m p e r a t u r e o f e x t r a c t i o n o n t h e t r o p o m y o s i n c o n t e n t i n a c t i n . 3 b i o l chem 2 3 7 : 3 4 1 2 3 4 1 7 , 1 9 6 2 e b a s h i , s. & e b a s h i , f : a new p r o t e i n c o m p o n e n t p a r t i c i p a t i n g i n t h e s u p e r p r e c i p i t a t i o n o f m y o s i n 8. 3 b i o c h e m 5 5 : 6 0 4 6 1 3 , 1 9 6 4 g a r r i g o s , m . , m o r e l , 3 . e . & d e l a t o r r e , 3 . g : r e i n v e s t i g a t i o n o f t h e s h a p e a n d s t a t e o f h y d r a t i o n o f t h e s k e l e t a l myo s i n s u b f r a g m e n t 1 monomer i n s o l u t i o n . b i o c h e m i s t r y 2 2 , 4 9 6 1 h i l l , a . v : t h e heat o f s h o r t e n i n g a n d t h e d y n a m i c c o n s t a n t s o f muscle. p r o c roy s o c b 1 2 6 : 1 3 6 1 9 5 , 1 9 3 6 h u x l e y , a . f : muscle s t r u c t u r e a n d t h e o r i e s o f c o n t r a c t i o n . p r o g b i o p h y s mol b i o l 7 : 2 5 7 3 1 7 , 1 9 5 7 s h i m i z u , h : i n s t a b i l i t y t h e o r y o f b i o l o g i c a l m o t i l i t y . i . on t h e s t a t i s t i c a l m e c h a n i c a l n a t u r e o f b i o l o g i c a l m o t i l i t y . 3 p h y s s o c j a p a n 3 2 : 1 3 2 3 1 3 3 0 , 1 9 7 2 s h i m i z u , h: i n g . h . p o l l a c k & h. s u g i ( e d s ) c o n t r a c t i l e mec h a n i s m s i n m u s c l e . an a c t o m y o s i n m o t o r p l e n u m p u b l i s h i n g c o r p u l b r e c h t , m . , g r u b h o f e r , n . , 3 a i s l e , f. & w a l t e r , s : die e r s h o p f e n d e r e i n i g u n g v o n a k t i n p r e p a r a t e n u n d art d e r p h o s p h a t h a l t i g e n p r o s t h e t i s c h e n g r u p p e n v o n g u n d f a c t i n . b i o c h i m b i o p h y s a c t a 4 5 : 4 4 3 4 5 9 , 1 9 6 0 y a n o , m., y a m a m o t o , y. & s h i m i z u , h : an a c t o m y o s i n m o t o r . n a t u r e 2 9 9 : 5 5 7 5 5 6 , 1 9 6 2 y a n o , m., m i o h , h . & s h i m i z u , h : i n h . i s h i k a w a , s. h a t a n o & h . s a t o ( e d s ) c e l l m o t i l i t y : m e c h a n i s m a n d r e g u l a t i o n . p r o c yamada c o n f e r e n c e x on c e l l m o t i l i t y 11: m e c h a n i s m a n d r e g u l a t i o n . t h e s l a v i n g p r i n c i p l e i n c h e m o m e c h a n i c a l e n e r g y c o n v e r s i o n m o t i o n d e p e n d e n t a c t o m y o s i n a t p a s e a c t i v i t y d u r i n g r e c o n s t i t u t e d s t r e a m i n g . u n i v e r s i t y o f t o k y o p r e s s 1 9 6 5 , p p 4 9 6 9 , 1 9 6 3 1 9 8 4 , p p 4 2 9 4 3 6 6 9 9 6 98 upsala j m e d sci 91: 53-66, 1986 perception of efforts in working postures m . wangenheim,' s. carlsoo,' b. nordgren,' and k. l i n r o t h 4 ' r e s e a r c h foundation f o r occupational safety and health in the swedish construction industry and department of rehabilitation medicine, university hospital, uppsala, 'departnirnt of a n a t o m y , karolinska institute, stockholm, 'department of rehabilitation medicine, university hospital, uppsala, 4research foundation f o r occupational safety and health in the swedish construction industry, sweden abstract the aim o f t h i s s t u d y i s t o o b t a i n an u n d e r s t a n d i n g o f t h e s u b j e c t i v e d i s c o m f o r t o f w o r k i n g p o s t u r e s and t h e degree o f p h y s i c a l e f f o r t t h e s e p o s t u r e s g i v e r i s e t o . 31 s t u d e n t s have r a t e d t h e i r p e r c e i v e d e x e r t i o n on borg's s c a l e (1982) based on a c o l l e c t i o n o f photographs o f d i f f e r e n t work p o s t u r e s o f v a r y i n g c o m p l e x i t y , i n c l u d i n g a k i n a e s t h e t i c d e s c r i p t i o n ( 2 ) . each one o f t h e 78 p o s t u r e s was h e l d f o r 45 seconds, w i t h r a t e d p e r c e i v e d e x e r t i o n (rpe) g i v e n a f t e r 15, 30 and 45 seconds. the whole t e s t s e r i e s was r e p e a t e d t w i c e . the r e s u l t s show t h a t i t i s p o s s i b l e t o e v a l u a t e d i f f e r e n t body p o s t u r e s b y rpe a c h i e v i n g a t o t a l r e l i a b i l i t y a v a l u e o v e r 0.96. d e s p i t e t h e f a c t t h a t t h e r a t i n g p a t t e r n between t h e d i f f e r e n t p o s t u r e s was t h e same f o r a l l s u b j e c t s , i t i s shown t h a t each person assessed a c c o r d i n g t o a c e r t a i n i n d i v i d u a l l y i n f l u e n c e d p a t t e r n . a c e r t a i n p e r s o n can f o r example, d e v i a t e c o n s i s t e n t l y t o w a r d s h i g h e r v a l u e s on borg's s c a l e , a s o c a l l e d " h i g h r a t e r " . f i n a l l y , t h e d i f f e r e n t p o s t u r e s were grouped a c c o r d i n g t o how t h e r a t i n g s changed i n t i m e ( r e g r e s s i o n s l o p e between t h e rpe's a f t e r 15, 30 and 45 seconds). the t h i r d c l u s t e r , w h i c h showed a h i g h b a s i c r a t i n g l e v e l and t h e g r e a t e s t r e g r e s s i o n s l o p e , i n c l u d e d o n l y extreme o u t e r l i m i t s o f movement. t h i s i n d i c a t e s t h a t t h e o u t e r l i m i t p o s t u r e s have a tendency t o s t a t i c l o a d and need t o be s t u d i e d b y more a d d i t i o n a l methods when r e m a i n i n g unchanged even f o r s h o r t p e r i o d s . 53 i n t r o d u c t i o n the a b i l i t y t o adapt and f r e e l y m a i n t a i n a body p o s t u r e , e.g. a p o s t u r e w i t h o u t e x t e r n a l s u p p o r t always i n v o l v e s a c e r t a i n degree o f p h y s i c a l s t r e s s . the degree o f s t r e s s d i f f e r s o f course from one p o s t u r e t o another. it i s t h e w e i g h t o f t h e d i f f e r e n t p a r t s o f t h e body, t h e s o c a l l e d f u n c t i o n a l u n i t s (fu), and t h e energy r e q u i r e d t o make them move t h a t causes t h e s t r e s s . t h i s s t r e s s i n v o l v e s l o a d on j o i n t s and muscles. a s t h e t o r q u e around t h e j o i n t s changes, so does t h e p e r c e p t i o n o f e f f o r t o r d i s c o m f o r t . when making an ergonomical e v a l u a t i o n o f body p o s t u r e s o c c u r r i n g i n p h y s i c a l work, i n o t h e r words work postures, an anatomical / p h y s i o l o g i c a l a n a l y s i s should a1 so be performed p a r a l l e l t o a p s y c h o l o g i c a l s u b j e c t i v e assessment, t o come as c l o s e as p o s s i b l e t o a r e l i a b l e n d i a g n o s i s n (8). the aim o f t h i s study i s t o o b t a i n an u n d e r s t a n d i n g o f t h e s u b j e c t i v e d i s c o m f o r t o f working p o s t u r e s and t h e degree o f p h y s i c a l e f f o r t these p o s t u r e s g i v e r i s e t o . carlsoo e t a1 ( 2 ) have d e s c r i b e d d i f f e r e n t work p o s t u r e s from a k i n a e s t h e t i c , t h a t i s anatomical p h y s i o l o g i c a l b a s i s . they d e s c r i b e t h e c h a r a c t e r i s t i c f e a t u r e s o f t h e muscular a c t i v i t y around t h e j o i n t s . each p o s t u r e i s i l l u s t r a t e d by a s p l i t image p i c t u r e . i n o r d e r t o make t h e d e s c r i p t i o n s u f f i c i e n t l y d e t a i l e d w h i l e r e m a i n i n g a t t h e same t i m e easy t o f o l l o w , t h e body was d i v i d e d up on t h e b a s i s o f t h e g r e a t j o i n t s and j o i n t s y s t e m s i n t o 14 " f u n c t i o n a l u n i t s " . the 14 fus are: head/neck, b a c k / t r u n k and 12 e x t r e m i t y fus. each e x t r e m i t y has been broken down i n t o t h r e e p a r t s . each p a r t covers one o f t h e t h r e e m a j o r j o i n t s as w e l l as t h e d i s t a l p a r t , shoulder/upper arm, elbow/forearm and so on. it i s these p i c t u r e s o f d i f f e r e n t p o s t u r e s f o r each fu which form t h e m a t e r i a l f o r s u b j e c t i v e e v a l u a t i o n o f e x e r t i o n i n t h e p r e s e n t study, whatever ergonomic e v a l u a t i o n technique i s adopted, t h e purpose i s t o o b t a i n a q u a n t i t a t i v e and/or q u a l i t a t i v e e v a l u a t i o n o f even a complex 54 p o s t u r e . q u e s t i o n n a i r e s a r e commonly-used f o r w o r k p o s t u r e s e.g. i n o f f i c e s o r m o t o r v e h i c l e s . l a b o r a t o r y c o n s t r u c t i o n o f a r e p l i c a o f a work s i t u a t i o n and b i o m e c h a n i c a l a n a l y s i s i s a n o t h e r , more o b j e c t i v e method ( 3 ) . man’s a b i l i t y t o grade and g i v e v e r b a l e x p r e s s i o n t o t h e d i s c o m f o r t and s t r e s s e x p e r i e n c e d i n d i f f e r e n t work p o s t u r e s has a l s o been employed as an i n v e s t i g a t i v e method ( 1 0 ) . van wely ( 1 0 ) has shown t h a t a h i g h degree o f c o r r e l a t i o n e x i s t s between s u b j e c t i v e p e r c e p t i o n o f a w o r k p o s t u r e and t h e c o m p l a i n t s t o w h i c h i t g i v e s r i s e . work p s y c h o l o g i s t gunnar borg (1) showed t h a t s u b j e c t i v e p e r c e p t i o n can be q u a n t i f i e d u s i n g a c a t e g o r y s c a l e w i t h r a t i o p r o p e r t i e s . i t i s t h i s method w h i c h has been used i n t h e p r e s e n t s t u d y . the s c a l e used i n t h e f o l l o w i n g t e s t s c o n s i s t s o f a l i m i t e d r a n g e o f numbers, 0-10. d i f f e r e n t v e r b a l e x p r e s s i o n s a r e c o r r e l a t e d t o t h e numbers. 0 r e p r e s e n t s n o t h i n g a t a l l , 0.5 e x t r e m e l y weak, 1 v e r y weak, 2 weak, 3 moderate, 4 somewhat s t r o n g , 5 s t r o n g , 7 v e r y s t r o n g and 1 0 e x t r e m e l y s t r o n g . the p e r c e i v e d e x e r t i o n t o w h i c h a p o s t u r e g i v e s r i s e , depends o f c o u r s e on whether t h e p o s t u r e i s momentary, f o r m i n g p a r t , f o r example, o f a sequence o f movements, o r o f l o n g e r d u r a t i o n ( 4 ) . i n t h e l a t t e r case, t h e i s o m e t r i c m u s c u l a r l o a d p l a y s such a d e c i s i v e and s p e c i a l r o l e i n t h e e v a l u a t i o n o f t h e work p o s t u r e t h a t a s p e c i a l s t u d y such as rohmert (9) suggests, w o u l d be necessary. the s t u d y d e s c r i b e d be1 ow r e 1 a t e d o n l y t o m o m e n t a r i l y a d o p t e d p o s t u r e s o f s h o r t d u r a t i o n . i n a p i l o t s t u d y t h e p o s t u r e s were e v a l u a t e d a f t e r 6 seconds. whatever t h e r e s u l t s showed t h a t t h e t h r e s h o l d o f p e r c e p t i o n o c c u r s l a t e r . t h e r e f o r e e v a l u a t i o n was made a f t e r 15, 30 and 45 seconds. 55 material and methods the s u b j e c t s were 8 male students, aged 23-35 y e a r s and 20 female s t u d e n t s aged 20-38 y e a r s from t h e physiotherapy department, u n i v e r s i t y h o s p i t a l , uppsala. none had any p r e v i o u s r e c o r d o f c o m p l a i n t s o f t h e muscul a r s k e l e t a l system. the s u b j e c t s were asked t o t a k e up and h o l d t h e v a r i o u s p o s t u r e s d e s c r i b e d i n t h e p r e v i o u s l y mentioned c o l l e c t i o n o f p o s t u r e photographs. each p o s t u r e was adopted from a symmetrical u p r i g h t stance a t r e s t , and compared w i t h t h i s stance. though t h e l o w e r e x t r e m i t i e s as w e l l as t h e back a r e s u b j e c t t o s t r e s s due t o t h e f a c t t h a t t h e p o s t u r a l muscles a r e t o some e x t e n t a c t i v e i n t h e u p r i g h t stance, t h e h e a l t h y s u b j e c t does n o t p e r c e i v e t h i s as s t r e s s f u l as l o n g as t h e d u r a t i o n o f t h e p o s t u r e i s l i m i t e d t o 4 5 seconds. the degree o f e x e r t i o n o f t h e p o s t u r e has t h e r e f o r e been graded 0 i n t h e e v a l u a t i o n s c a l e g i v e n above. the p o s t u r e d i f f e r s from t h e i n t e r n a t i o n a l l y a c c e p t e d anatomic p o s t u r e o n l y i n s o f a r as t h e arms hang f r e e by t h e s i d e s and are n o t , as i n t h e anatomic p o s t u r e , supined. the p o s t u r e s t h e s u b j e c t s were r e q u i r e d t o adopt a r e shown i n f i g s . 2-9. these a r e 10 d i f f e r e n t head postures, 8 shoulder-arm postures, 12 elbow postures, 9 h a n d w r i s t postures, 10 back-trunk p o s t u r e s , 12 h i p p o s t u r e s , 13 knee p o s t u r e s and 5 f o o t postures. a l l 78 postures, were h e l d unchanged f o r 45 seconds. s o c a l l e d normal p o s t u r e s ( f o r each fu) as w e l l as m i r r o r i m a g e p o s t u r e s ( c r o s s e d o u t i n f i g u r e s 2, 3 and 6) were n o t r a t e d . the s u b j e c t e v a l u a t e d t h e degree o f e x e r t i o n a s s o c i a t e d w i t h each p o s t u r e a f t e r 15, 30 and 45 seconds r e s p e c t i v e l y . the rpe was r a t e d a c c o r d i n g t o t h e p r e v i o u s l y mentioned s c a l e ad modum borg ( 1 ) . b e f o r e t h e t e s t s began, each s u b j e c t was c a r e f u l l y i n s t r u c t e d i n t h e v a r i o u s p o s t u r e s and had an o p p o r t u n i t y t o p r a c t i s e u s i n g t h e s c a l e i n d i f f e r e n t t e s t s . i n t h e f i r s t t e s t an e v a l u a t i o n o f t h e area o f d i f f e r e n t g e o m e t r i c a l s u r f a c e s was made. i n t h e second t e s t d i f f e r e n t shades o f w h i t e and grey were evaluated. taste t e s t s were a l s o performed u s i n g s o l u t i o n s o f v a r y i n g a c i d i t y . f i n a l l y t h e s u b j e c t s were asked t o demonstrate g i v e n percentage p a r t s o f t h e i r maximal s t r e n g t h o f g r i p and back e x t e n s i o n . 56 to p r e v e n t p e r c e p t i o n o f one p o s t u r e i n f l u e n c i n g e v a l u a t i o n o f t h e n e x t , t h e r e was c o n s t a n t v a r i a t i o n between t h e f u n c t i o n a l u n i t s employed and o f more and l e s s s t r e s s f u l p o s t u r e s . the whole s e r i e s was r e p e a t e d t w i c e . results an a n a l y s i s o f v a r i a n c e * was p e r f o r m e d on t h e e n t i r e m a t e r i a l , 78 body p o s t u r e s , 3 r a t i n g s ( 1 5 , 30 and 45 sec.) and two r e p e t i t i o n s o f t h e e n t i r e t e s t s e r i e s f o r 31 s u b j e c t s . the a n a l y s i s showed t h a t as expected, t h e r a t i n g s o f t h e d i f f e r e n t p o s t u r e s v a r i e d s i g n i f i c a n t ' l y (p<o.ool). d i f f e r e n t o b s e r v a t i o n a l t i m e s ( 1 5 , 30 and 45 sec . i produced s i g n i f i c a n t l y v a r y i n g r e s u l t s . t h a t i s : a l l r a t i n g s a t 15 sec. had a t r e n d d i s s i m i l a r t o t h e r a t i n g s a t 30 and 45 sec. a n a l y s i s o f t h e r e p e t i t i o n o f t h e e n t i r e t e s t gave no s i g n i f i c a n t d i f f e r e n c e between t h e two t e s t s e r i e s . t a b l e 1 g i v e s t h e c o r r e l a t i o n c o e f f i c i e n t s , mean v a l u e s , s t a n d a r d d e v i a t i o n s and r a n g e ( d i f f e r e n c e between h i g h e s t and l o w e s t v a l u e s used on borg's s c a l e ) f o r each s u b j e c t on t h e e n t i r e m a t e r i a l . d e s p i t e t h e f a c t t h a t t h e r a t i n g p a t t e r n between t h e d i f f e r e n t p o s t u r e s was t h e same f o r a l l s u b j e c t s , c e r t a i n p o s t u r e s were r a t e d h i g h i n r e l a t i o n t o t h e o t h e r s , and, c o n v e r s e l y , i t i s shown t h a t e v e r y p e r s o n tended t o assess a c c o r d i n g t o a c e r t a i n i n d i v i d u a l l y i n f l u e n c e d p a t t e r n . it i s p r i m a r i l y t r u e t h a t c e r t a i n p e r s o n s t e n d e i t h e r t o d e v i a t e c o n s i s t e n t l y t o w a r d l o w e r o r h i g h e r values, t h e s o c a l l e d " h i g h r a t e r s " and "1 ow r a t e r s " . f o r example, i n d i v i d u a l 4 had a mean (rpe) v a l u e o f 0.52 and i n d i v i d u a l 26 a mean (rpe) v a l u e o f 5.72 f o r t h e 45 second r a t i n g s ( t a b l e 1). a n o t h e r t y p e o f p e r s o n a l d e v i a t i o n i s t h e r a n g e c o v e r e d by t h e e s t i m a t e r , t h a t i s , t h e d i f f e r e n c e between t h e h i g h e s t and l o w e s t r a t i n g s used. here we f i n d i n d i v i d u a l s who use t h e whole o f t h e 1 0 p o i n t r a n g e and o t h e r s *data program bmdpbv: bmdp b i o m e d i c a l computer programs. p s e r i e s , 1979. u n i v e r s i t y o f c a l i f o n i a press. 57 who t r y t o keep w i t h i n a l i m i t e d p a r t o f t h e s c a l e . f o r example, i n d i v i d u a l 24’s range was 1 0 and i n d i v i d u a l s 3 and 29 had a r a n g e 4 f o r t h e 45-second r a t i n g s ( t a b l e 1 ) . the a b i l i t y o f t h e i n d i v i d u a l s t o r e p e a t t h e i r r a t i n g s a t e x a c t l y t h e same e x p o s i t i o n was good. a c o l l a t i o n o f t h e c o r r e l a t i o n c o e f f i c i e n t s a t t h e t h r e e t i m e i n t e r v a l s i s p r e s e n t e d i n t a b l e 1. t h i s i s complemented by an a l f a m o d e l r e l i a b i l i t y t e s t ( 5 , 6 ) . 28 s u b j e c t s w i t h an a v a l u e 0.96 o r more were s e l e c t e d f o r f u r t h e r p r o c e s s i n g : 3 were dropped. the 3 who were dropped, i n d i v i d u a l s 2, 3 and 28, a v a l u e s 0.94 0951, a l s o had t h e l o w e s t c o r r e l a t i o n c o e f f i c i e n t s ( t a b l e 1 ) . these t h r e e d i f f e r e d i n t h e i r r a t i n g s o f some s i t u a t i o n s , t h e r e b y d e m o n s t r a t i n g i n a b i l i t y t o p e r c e i v e body p o s t u r e s . an a n a l y s i s was p e r f o r m e d o f 28 r e 1 i a b l e i n d i v i d u a l s ’ r a t i n g p a t t e r n s , based on a c l a s s i f i c a t i o n o f t h e i r t o t a l mean v a l u e s o v e r a l l t h e r a t i n g s ( 7 8 p o s t u r e s , 30 second r a t i n g s , r e p e a t e d t w i c e ) . a f t e r d i v i d i n g t h e i n d i v i d u a l s ( i n t o t h r e e groups o f equal s i z e ) a c c o r d i n g t o l o w mean r a t i n g combined w i t h l o w s t a n d a r d d e v i a t i o n ( s . d . ) , i n t e r m e d i a t e mean rating/s.d. and h i g h mean rating/s.d., an a n a l y s i s o f v a r i a n c e was p e r f o r m e d on 6 persons i n each o f t h e t h r e e groups. the most i m p o r t a n t r e s u l t t o emerge f r o m t h i s a n a l y s i s was t h a t t h e t h r e e groups showed a n o n s i g n i f i c a n t d i f f e r e n c e i n t h e t r e n d o f t h e i r r a t i n g p a t t e r n s . the d i f f e r e n t p o s t u r e s were a l s o grouped a c c o r d i n g t o t h e r a t i n g change i n t i m e . a c l u s t e r a n a l y s i s c a l l e d m i c k a u s i n g 3 g r o u p s ( 7 ) produced t h e b e s t v a l u e s and i s p r e s e n t e d i n f i g . 1. c l u s t e r 3, w h i c h showed a h i g h b a s i c r a t i n g l e v e l and as f i g u r e 1 i n d i c a t e s a g r e a t r e g r e s s i o n s l o p e between 15, 30 and 45 seconds, i n c l u d e d o n l y extreme o u t e r l i m i t s o f movement, a p a r t f r o m two upper-arm p o s t u r e s a t h e a d l e v e l . c l u s t e r s 1 and 2 i n c l u d e d l e s s s t r e s s f u l p o s t u r e s . the above time-development t e n d e n c i e s i n d i c a t i n g t h a t t h e o u t e r l i m i t p o s t u r e s i n f i g u r e s 2-9 have a tendency t o s t a t i c l o a d and need t o be s t u d i e d by o t h e r means when r e m a i n i n g unchanged even f o r s h o r t p e r i o d s . table 1 . c o r r e l a t i o n c o e f f i c i e n t s between t h e two t e s t s e r i e s , mean val ues , s t a n d a r d d e v i a t i o n and range ( d i f f e r e n c e between z e r o and h i g h e s t v a l u e s ; a1 1 persons had 0 a s lowest r a t i n g ) f o r a l l 78 p o s t u r e s , 2 t e s t s e r i e s and 3 o b s e r v a t i o n times ( 1 5 , 30, 45 s e c . ) . rpe-rated perceived e x e r t i o n on borg’s s c a l e . s . d . standard d e v i a t i o n . subjcci 1 2 3 4 5 6 7 6 9 1c 1 1 1 2 1 3 1 4 1 5 1 6 17 1 8 1 9 2u 2 1 22 23 2 4 2 5 2 6 2 7 2 8 2 9 30 3 1 c o r r l l a l l o n c o e r r l c l c n l 1 5 3 0 4 5 0 . 7 1 0 . 8 9 0 . 9 1 0 . 6 1 0 . 7 3 0 . 8 1 0 . 7 3 0 . 7 0 0 . 6 5 0 . 7 9 0.88 0 . 9 2 0 . 7 8 0 . 7 6 0 . 8 5 0 . 8 0 0 . 7 7 0 . 7 9 0 . 8 1 0 . 8 4 0 . 8 6 0 . 8 6 0 . 8 2 0 . 7 7 0 . 8 7 0 . 9 1 0 . 9 0 0 . 7 0 0 . 8 1 0 . 6 2 0 . 8 0 0 . 9 1 0 . 9 4 0 . 8 4 0 . 9 1 0 . 9 1 0 . 6 4 0 . 8 6 0 . 9 0 0 . 7 6 0 . 8 7 0 . 9 7 0 . 8 6 0 . 6 9 0 . 9 1 0 . 8 3 0 . 8 7 0 . 9 4 0 . 8 5 0 . 8 9 0 . 9 3 0 . 8 2 0 . 9 0 0 . 9 1 0 . 8 2 0 . 8 6 0 . 8 3 0 . 8 3 0 . 8 8 0 . 8 9 0 . 7 1 0.78 0.84 0 . 8 2 0 . 8 7 0 . 9 1 0 . 8 9 0 . 8 4 0 . 6 9 0.83 0 . 8 3 0 . 6 8 0 . 7 1 0 . 7 7 0 . 8 2 0 . 7 2 0 . 8 0 0 . 7 6 0 . 6 3 0 . 9 0 0 . 9 2 0 . 4 6 0.80 0 . 8 3 0 . 7 7 0.82 0 . 6 3 0.88 0 . 8 9 0 . 9 1 0 . 8 7 0 . 9 2 0 . 9 2 w l a n rf’l 1 5 3 0 4 5 0 . 5 8 0 . 9 9 1 . 4 3 1 . 8 2 2 . 2 4 2 . 6 7 0 . 5 8 1 . 1 2 1 . 6 6 0 . 5 0 0 . 9 2 0 . 5 2 1 . 1 0 1 . 6 3 2 . 1 8 2 . 9 4 3 . 4 5 3 . 9 5 2 . 1 7 2 . 7 3 3 . 1 1 1 . 1 1 1 . 4 7 1 . 9 8 0 . 7 9 1 . 2 3 1 . 7 1 2 . 3 3 2 . 5 6 2 . 7 9 1 . 3 1 1 . 6 9 2 . 0 4 0 . 8 0 1 . 1 7 1 . 4 9 2 . 3 0 2 . 9 7 3 . 4 2 2 . 5 6 1 . 5 2 4 . 1 4 1 . 3 8 1 . 6 9 2 . 0 0 2 . 1 1 2 . 9 2 3 . 7 8 1 . 4 1 1 . 8 9 2 . 6 1 2 . 6 7 3 . 4 7 4 . 2 6 3.87 4 . 1 7 4 . 5 5 1 . 7 0 2 . 2 5 2 . 8 6 1 . 6 0 3 . 1 2 4 . 5 9 2 . 5 9 2 . 9 9 3 . 7 0 2 . 0 2 2 . 8 3 3 . 5 1 2 . 9 6 3 . 5 7 4 . 0 0 1 . 6 8 2 . 1 4 2 . 7 0 4 . 2 3 5 . 0 5 5 . 7 2 2 . 9 2 3 . 5 5 4 . 0 1 0 . 4 3 0 . 7 6 1 . 2 0 0 . 7 5 1 . 1 4 1 . 5 5 1 . 5 4 1 . 7 0 1 . 6 5 2 . 5 7 3 . 1 8 3 . 5 4 5d 1 5 3 0 4 5 0 . 5 9 1 . 0 1 1 . 2 8 0 . 7 4 0 . 9 1 1 . 2 1 0 . 4 7 0 . 6 2 0.’77 0 . 7 2 1 . 1 6 1 . 7 2 0 . 6 9 1 . 0 1 1 . 3 3 0 . 8 4 1 . 0 8 1 . 2 6 1 . 1 6 1 . 3 0 1 . 4 7 1 . 0 9 1 . 1 7 1 . 3 6 0 . 8 6 1.oh 1 . 2 4 0 . 9 3 0 . 9 9 1 . 1 2 1 . 2 2 1 . 5 3 1 . 8 2 0 . 9 0 1 . 1 8 1 . 4 0 0 . 9 3 1 . 1 7 1 . 3 6 1 . 0 0 1 . 2 5 1 . 3 7 0 . 7 6 0 . 8 9 0 . 9 7 0 . 9 9 1 . 2 9 1 . 6 1 0 . 9 8 1 . 2 3 1 . 5 6 0 . 9 r 1 . 4 7 1 . 8 4 1 . 0 4 1 . 2 2 1 . 2 2 1 . 1 2 1.411 1 . 5 9 n . 9 0 1 . 4 3 1 . 9 2 1 . 0 3 1 . 3 2 1 . 5 2 1 . 2 6 1 . 6 4 1 . 9 0 1 . 2 8 1 . 5 7 1 . 7 6 1 . 1 0 1 . 2 0 1 . 5 0 1 . 6 0 1 . 8 4 2 . 0 4 1 . 1 2 1 . 3 4 1 . 5 4 0 . 6 6 11.77 1.ou 0 . 6 2 0 . 6 2 0 . 9 8 1 . 1 5 1 . 2 1 1 . 2 6 1 . 4 1 1 . 7 1 1 . 9 1 r a n g e 1 5 30 4 5 3 . 5 5 . 0 6 . 0 3 . 5 5 . 0 6 . 0 2 . 5 3 . 5 4 . 0 3 . 0 4 . 5 7 . 5 3 . 1 8 . 3 5 . 3 4 . 8 6 . 0 6 . 8 4 . 5 5 . 0 6 . 0 4 . 0 4 . 0 5 . 5 3 . 5 5 . 0 5 . 5 8 . 0 4 . 5 4 . 6 4 . 5 6 . 0 7 . 0 3 . 5 4 . 5 5 . 5 4 . 3 5 . 0 5 . 8 4 . 0 6 . 0 7 . 0 3 . 0 3 . 6 3 . 7 3 . 5 5 . 0 6:6 4 . 5 6 . 5 7 . 6 5 . 0 7 . 1 8 . 3 7 . 5 8 . 0 8 . 5 4 . 0 5 . 0 5 . 8 1.0 6 . 0 9 . 5 5 . 0 6 . 0 7 . u 6 . 0 8 . 0 9 . 5 6 . 5 6 . 5 1 0 . 0 4 . 0 5 . 5 7 . 0 7 . 5 7 . 0 8.u 6 . 0 6 . 8 7 . 8 7 . 0 4 . 0 5 . 0 2 . 5 3 . 5 4 . 0 5 . 0 5 . 0 4 . 3 6 . 5 7 . 5 9 . 0 59 r a t e d p e r c e i v e d e x e r t i o n i d5 c if 30 o b s e r v a t i o n a l t i m e s ( s e c . ) p o s t u r e s i n c l u d e d i n : c l u s t e r 1 1.1.12 1.1.3 1.1.430 1.1.6 3.2.1 3.1.1 3.0.1 8.1.3 9.1.2 9.0.1 9.0.2 9.1.3 9.3.1 9.3.2 9.3.3 10.1.3 c l u s t e r 2 1.2.6 2.1.43' 2.1.6 3.3.2 3.3.1 3.3.3 3.2.2 3.2.3. 3.1.2 3.1.3 3.0.2 3.0.3 4.1.6 whole hano s u p p o r t h a l f hand s u p p o r t k n u c k l e s u p p o r t f i n g e r t i p support 8.2.6 8.3.6 1.1.i30 8 . 1 . 1 ~ ~ 8.1.430 8.1.6 9.1.1 9.1.4 9.2.2 9.2.3 10.0.1 10.0.2 10.1.1 10.1.2 10.2.2 10.3.1 10.3.2 10.4.1 10.4.2 10.4.3 11.1.12 11.1.3 11.1.6 11.0.1 c l u s t e r 3 1.2.12 1 2 . 1 ~ ~ 1.2.3 1.2.4 2.2.12 2.2.1 30 30 2.2.3 2 .2.430 2.1.12 2.1.1 30 4.1.12 . 4.1.3 4.1.9 f i n g e r support 8.2.12 8.2.3 8 . 2 . 1 ~ ~ 8.2.430 9.2.1 10.2.1 11.1.9 c l u s t e r a n a l y s i s u s i n g 3 groups. the d i f f e r e n t p o s t u r e s a r e f o l l o w i n g t h e i r time-development tendencies ( r p e a t 3 observa t i o n p o i n t s , 15, 30 and 45 sec.). the p o s t u r e s i n c l u d e d i n each c l u s t e r a r e g i v e n a f t e r t h e i r code. the f i r s t number i n each code r e l a t e s t o t h e f u n c t i o n a l u n i t , t h e second number t o t h e s t r e s s l e v e l and t h e t h i r d t o t h e p o s i t i o n on a normal c l o c k . l a s t l y , t h e mean v a l u e s f o r a l l p o s t u r e s a f t e r 30 seconds were f i l l e d i n on t h e 8 p o s i t i o n c h a r t s ( f i g . 2-9). s i n c e t h e p u r p o s e was t o assess "momentari l y a d o p t e d " p o s t u r e s , t h e 30 second r a t i n g was s e l e c t e d . the 15 second r a t i n g i s r e g a r d e d as b e i n g on t h e t h r e s h o l d o f p e r c e p t i o n f o r t h e " l i g h t " p o s t u r e s , t h e t h r e s h o l d i s p r o b a b l y passed a t 30 seconds, and a f t e r 45 seconds t h e r e i s a r i s k o f s t a t i c l o a d , p a r t i c u l a r l y i n t h e case o f e x t r e m e o u t e r l i m i t p o s t u r e s . discussion the p r e s e n t m a t e r i a l shows t h a t i t i s p o s s i b l e t o e v a l u a t e d i f f e r e n t body p o s t u r e s b y r a t e d p e r c e i v e d e x e r t i o n . new s u b j e c t i v e r a t i n g s s h o u l d be o b t a i n e d when u s i n g c r i t e r i a o f t h i s n a t u r e on o t h e r homogeneous work-groups. the r a t i n g c r i t e r i a w h i c h t h e a u t h o r s f e e l i s o f g e n e r a l a p p l i c a b i l i t y i s p r e s e n t e d i n a guide f o r e v a l u a t i o n o f body p o s t u r e s c a l l e d p o s t u r e s and l o a d s (2). the advantages a r e : s t a n d a r d i s e d , qua1 i f i e d r a t i n g s o f d i f f e r e n t w o r k s i t u a t i o n s f r o m t h e ergonomic p o i n t o f view, w i t h o u t r e q u i r i n g t h e a n a l y s t t o possess a q u a l i f i e d knowledge o f anatomy, p h y s i o l o g y and b i o m e c h a n i c s . the d i s a d v a n t a g e s a r e : a p o s s i b l e degree o f i n a c c u r a c y i n t h e r a t i n g s . f o r example, i t i s presumed, i n a n a l y s i n g t h e p o s t u r e o f one e x t r e m i t y , t h a t t h e o t h e r e x t r e m i t y i s a t r e s t : i f t h i s i s n o t s o , t h e s t r e s s i s n o r m a l l y g r e a t e r . it i s f u r t h e r assumed t h a t t h e body i s s t a n d i n g u p r i g h t . i n o t h e r c i r c u m s t a n c e s , b e n d i n g f o r w a r d f o r example, t h e s t r e s s on t h e head o r t h e u p p e r e x t r e m i t i e s i s d i f f e r e n t . i f t h e s u b j e c t ' s movement p a t t e r n i s abnormal, f o r example a s a r e s u l t o f d i m i n i s h e d m o b i l i t y i n some p a r t o f t h e body o r because t h e s u b j e c t a v o i d s c e r t a i n p o s t u r e s as a r e s u l t o f i n j u r y o r p a i n , e r r o r s may o c c u r . o b v i o u s l y t h e r e i s a l s o a p o s s i b i l i t y t h a t a s i t u a t i o n may be assessed i n c o r r e c t l y because t h e p o s t u r e s i n c l u d e d c a n n o t f o r e s e e e v e r y i m a g i n a b l e s i t u a t i o n and t h e a n a l y s t has t o s e l e c t t h e p i c t u r e t h a t m o s t c l o s e l y resembles t h e p o s t u r e . 61 fig. 2. p o s i t i o n c h a r t showing t h e d i s t r i b u t i o n o f mean rpe o n t h e m e c k . each p o s i t i o n i s r e p r e s e n t e d b y a s p l i t p i c t u r e showing t h e head f r o m t h e f r o n t and f r o m t h e s i d e . each p i c t u r e has a code number, as w e l l as a l a r g e number d e s c r i b i n g t h e mean rpe. the p o s t u r e s c r o s s e d o u t on t h e l e f t , r i g h t were n o t assessed. n e i t h e r was t h e normal p o s i t i o n o f r e s t . w h i c h a r e m i r r o r i m a g e s o f t h e ones on t h e f i g . 3 . p o s i t i o n c h a r t showing t h e d i s t r i b u t i o n o f mean rpe on t h e s h o u l d e r / u p p e r arm. the p r o j e c t i o n s i n each s p l i t p i c t u r e a r e 45 t h e f r o n t a l p l a n e . the p o s t u r e s c r o s s e d o u t were n o t assessed f o r t h e same r e a s o n s as i n f i g u r e 2. o f 62 p o s i t i o n c h a r t showing t h e d i s t r i b u t i o n o f mean rpe on t h e the s t r u c t u r e i s t h e same as i n f i g u r e 2, e x c e p t f o r t h e movement p a t t e r n . the p i c t u r e s on t h e l e f t show p r o n a t i o n and on t h e r i g h t , s u p i n a t i o n . i n each h o r i z o n t a l s e r i e s o f p i c t u r e s t h e a n g l e between t h e u p p e r arm and f o r e a r m i s t h e same. i n t h e t o p s e r i e s t h e arm i s f l e x e d m a x i m a l l y , w h i l e i n t h e b o t t o m s e r i e s i t i s f u l l y extended. f i g . 5. p o s i t i o n c h a r t showing t h e d i s t r i b u t i o n o f mean rpe on t h e =/hand. on t h e l e f t t h e hand i n a f o r c e g r i p i s r o t a t e d showing d i f f e r e n t p o s s i b l e e x t r e m e p o s i t i o n . on t h e r i g h t , f i v e s u p p o r t p o s t u r e s , s t a n d i n g w h i l s t s u p p o r t i n g t h e body a t w a i s t l e v e l , a r e shown. 63 f i g . 6. p o s i t i o n c h a r t showing t h e d i s t r i b u t i o n o f mean r p e on t h e b a c k / t r u n k . the back i s r o t a t e d around t h e normal r e s t p o s i t i o n i n t h e c e n t r e , s t a r t i n g from maximum f l e x i o n 8.2.12, around t h e c l o c k t o f o r w a r d f l e x i o n d e m o n s t r a t i n g two s p e c i a l cases: 8.2.6 b e n t knees and 8.3.6 s t r a i g h t knees, and f u r t h e r back t o 8.2.12. the p o s t u r e s c r o s s e d o u t were n o t assessed f o r t h e same reasons as i n f i g . 6. 7 . p o s i t i o n c h a r t showing t h e d i s t r i b u t i o n o f mean rpe on t h e 9h i p t h i g h . l e f t : t h e h i p i s r o t a t e d t o l e f t and r i g h t . c e n t r e l e f t : a few s p e c i a l cases. top r i g h t : d i f f e r e n t f l e x i o n s . bottom r i g h t : t h r e e s p e c i a l cases. 64 8. p o s i t i o n c h a r t showing t h e d i s t r i b u t i o n o f mean rpe on t h e -7t knee eg. l e f t : t h e h i p i s r o t a t e d l e f t and r i g h t e x c e p t f o r 10.0.2 . demonstrating a person s t a n d i n g on one f o o t . top r i g h t : two d i f f e r e n t f l e x i o n s . the remaining f i v e p i c t u r e s demonstrate s p e c i a l cases. 9 . p o s i t i o n c h a r t showing t h e d i s t r i b u t i o n o f mean rpe on t h e %%7foot. 11.1.12, d o r s i f l e x i o n 11 . i .3, i n v e r s i o n 11 .i .6, p l a n t a r f l e x i o n 11.1.9, e v e r s i o n 5-868571 65 references 1. borg, g.a.v.: a c a t e g o r y s c a l e w i t h r a t i o p r o p e r t i e s f o r i n t e r m o d a l and i n t e r i n d i v i d u a l comparisons. i n : h.-g. g e i s s l e r & p e t z o l d (eds.) psychophysical judgment and t h e process o f p e r c e p t i o n . b e r l i n : veb deutscher v e r l a g d e r wissenschaften, 1982. 2. carlsoo, s . & hammarskjold, e.: postures and l o a d s . the research foundation f o r occupational s a f e t y and h e a l t h i n t h e swedish c o n s t r u c t i o n i n d u s t r y , box 94, s-182 11 sweden, 1985. 3. c h a f f i n , d.b., h e r r i n , g.d., k e y s e r l i n g , m. & garg, a , : a method f o r e v a l u a t i n g t h e biomechanical s t r e s s e s r e s u l t i n g from manual m a t e r i a l s h a n d l i n g j o b s . am i n d hyg assoc j 38: 662-675, 1977. 4. c o r l e t t , e.n. & manenica, i.: the e f f e c t s and measurement o f w o r k i n g postures. a p p l i e d ergonomics 11: 7-16, 1980. 5. cronbach, l.j.: c o e f f i c i e n t alpha and t h e i n t e r n a l s t r u c t u r e o f t e s t s . psychometrika 16:297-334, 1951. 6. k r i s t o f , w.: the s t a t i s t i c a l t h e o r y o f speed-up r e l i a b i l i t y c o e f f i c i e n t s when a t e s t has been d i v i d e d i n t o several equal p a r t s . psychometrika 28: 221-38, 1963. 7. mcrae, d.j.: micka, a fortran i v i t e r a t i v e k-means c l u s t e r a n a l y s i s program. behavl s c i 16: 423-424, 1971. 8. m u r r e l l , k.f.h.: ergonomics. chapman och h a l l , 1965. 9. rohmert, w.: untersuchungen i i b e r muskelermiidung und a r b e i t s g e s t a l tung. b e r l i n , koln, f r a n k f u r t : b e u t h v e r t r i e b , 1962. 10. stevens, s . s . & galanter, e.h.: r a t i o scales and category scales f o r a dozen perceptual continua. j o u r n a l o f experimental psychology 54: 377-411, 1957. 11. van wely, p.: design and disease. a p p l i e d ergonomics 1: 262-269, 1969. address f o r r e p r i n t s : michael wangen h e i m research foundation f o r occupational s a f e t y and h e a l t h i n t h e swedish cons t r u c ti on i n du s t ry box 94 s-182 11 danderyd sweden 66 upsala j med sci 84: 259-274, 1979 serum concentrations of estrone, androstenedione, testosterone and sex-hormonebinding globulin in postmenopausal women with breast cancer and in age-matched controls hans-olov adami, m.d.', elof d. b. johansson, m.d.', jan vegelius, ph.d.3 and arne victor, m.d.' from the department3 of surgery, obstetrics & gynecology, univerjity hospital, uppsala, sweden and department ttf statistics, uppsalu university, uppsala, sweden abstract the c o n c e n t r a t i o n s o f e s t r o n e ( e l ) , a n d r o s t e n e d i o n e ( a ) , t e s t o s t e r o n e ( t ) a n d s e x h o r m o n e b i n d i n g g l o b u l i n (shbg) i n t h e s e r u m were d e t e r m i n e d i n 1 2 2 p o s t m e n o p a u s a l women, u n s e l e c t e d w i t h r e s p e c t t o a g e a n d s t a g e o f d i s e a s e a n d w i t h a n e w l y d i a g n o s e d b r e a s t c a n c e r . the r e s u l t s were c o m p a r e d w i t h t h o s e i n 1 2 2 a g e m a t c h e d women w i t h o u t b r e a s t c a n c e r , s e l e c t e d f r o m t h e p o p u l a t i o n r e g i s t e r . the p a t i e n t s were f o u n d t o h a v e a s i g n i f i c a n t l y h i g h e r mean l e v e l t h a n t h e c o n t r o l s o f e l ( 1 3 2 a n d 108 p m o l / l ) , a ( 2 . 5 a n d 1.6 n m o l / l ) a n d ' t ( 1 . 5 4 a n d 1.38 n m o l / l ) a n d a lower l e v e l of shbg ( 4 0 . 2 a n d 4 7 . 3 n m o l / l ) i n t h e s e r u m . a mul t i p l e r e g r e s s i o n a n a l y s i s r e v e a l e d i n t h e c o n t r o l g r o u p t h a t t h e s e r u m l e v e l o f el w a s s i g n i f i c a n t l y c o r r e l a t e d t o a (r=0.48, p < 0.001) a n d t ( r = 0 . 4 5 , p < 0 . 0 0 1 ) . i n t h e p a t i e n t g r o u p e l w a s o n l y s l i g h t l y c o r r e l a t e d t o t ( r = 0 . 2 5 , p < 0 . 0 1 ) a n d n o t t o a ( r = 0 . 1 0 , p > 0 . 0 5 ) . a s i g n i f i c a n t n e g a t i v e c o r r e l a t i o n was f o u n d b e t w e e n shbg a n d w e i g h t i n b o t h g r o u p s . o t h e r w i s e n o s i g n i f i c a n t c o r r e l a t i o n s were f o u n d b e t w e e n a n y o f t h e hormone l e v e l s a n d a g e , s t a g e o f d i s e a s e o r w e i g h t . i t w a s c o n c l u d e d t h a t a n i n c r e a s e d a v a i l a b i l i t y o f a a n d t i l e a d i n g t o a n i n c r e a s e d a n d r o g e n i c s t i m u l a t i o n a n d t h e r e f o r e d e c r e a s e d shbg a n d a n i n c r e a s e d e l l e v e l , i s t h e most r e a s o n a b l e e x p l a n a t i o n f o r t h e f i n d i n g s . the l a c k of c o r r e l a t i o n b e t w e e n e l a n d a i n t h e p a t i e n t g r o u p i s however d i f f i c u l t to e x p l a i n a n d t h e r e s u l t s d o n o t seem t o f i t i n t o a d e f i n i t e h y p o t h e s i s . introduction e p i d e m i o l o g i c a l a n d c l i n i c a l e v i d e n c e t h a t t h e r i s k f o r b r e a s t c a n c e r is i n f l u e n c e d by e n d o c r i n e f a c t o r s ( 4 0 ) h a s i n i t i a t e d numerous s t u d i e s d e s i g n e d t o d e f i n e a s p e c i f i c e n d o c r i n e e n v i r o n m e n t t h a t p r e d i s p o s e s t o b r e a s t c a n c e r . major i n t e r e s t h a s b e e n p a i d t o t h e u r i n a r y e x c r e t i o n o f a n d r o g e n a n d e s t r o g e n m e t a b o l i t e s . a l t h o u t h c o n t r a d i c t o r y i n many i n s t a n c e s , t h e r e s u l t s from t h e s e s t u d i e s h a v e i n i t i a t e d s e v e r a l h y p o t h e s e s a n d t w o o f them h a v e a t t r a c t e d s p e c i a l i n t e r e s t . 8-792856 259 according to the first of them a decreased urinary excretion of etiochola bolone and androsterone was associated with an increased risk of breast cancer and in the manifest disease with a poor prognosis and a poor response to endo crine ablative surgery (cf. 19). contradictory results have however been re ported (cf. 62) and the androgen excretion has also been shown to be decreased as a nonspecific consequence of illness (61). secondly, the "estriol hypothesis" was derived from the fact that carcino genic potential has been demonstrated for estrone (el) and estradial ( e 2 ) in experimental research but not for estriol (e3) and that e3 has the capacity to impede certain estrogenic activities of el and e 2 (33). the relative amount of e3 in relation to el and e 2 was therefore suggested as an important determinant for breast cancer risk. this hypothesis was supported when population studies revealed a parallelism between the urinary estriol ratio and a high, intermedi ate and low breast cancer risk, respectively (13, 41). the difference was most marked in young women, a finding combined with the previous finding of a reduced breast cancer risk due to an early age at first birth (40). recent research has revealed several lines of data which has seriously brought into challenge the concepts that the pattern of excretion of the urinary androgens and estrogens is causally related to the etiology of breast cancer (cf. 32, 6 2 ) . results derived from determinations of the steroid hormones in serum are very few and have not resulted in any convincing alternative hypo thesis (32). the design of the present investigation was not primarily aimed at analysis of steroid hormones and their binding globuline. the availability of serum within the frame of an ongoing epidemiologic breast cancer study which included an assessment of thyroid function and an access to recently developed radio immunoassays did, however, enable the study. the aim was therefore to compare the serum concentrations of el, androstenedione (a), testosterone (t) and sex hormone-binding globulin (shbg) in an unselected series of postmenopausal women with a newly diagnosed breast cancer with those in a group of age-matched, non-hospitalized, postmenopausal women without breast cancer. subjects and methods patients this study was based on 149 postmenopausal women included in a series of 179 women with breast cancer registered consecutively during five months in four swedish counties. the population within this area was uniform as to race and nationality. only two of the breast cancer patients who were diagnosed during the observation period (2/181) refused therapy and participation in the study. hospital records were written on a special form and all patients answered a comprehensive questionnaire concerning their reproductive history, gynecologic 260 d i s e a s e s , h e i g h t , w e i g h t , d r u g consumption and o t h e r f a c t o r s of e p i d e m i o l o g i c i n t e r e s t . a l l p a t i e n t s were c l a s s i f i e d a c c o r d i n g t o t h e t n m c l a s s i f i c a t i o n ( 2 3 ) ( t a b l e 1 ) . two blood s a m p l e s were drawn f o r a n a l y s i s of shbg and hormones i n t h e serum. the s e r a were d i r e c t l y s e n t t o uppsala and a r r i v e d t h e same e v e n i n g or t h e n e x t morning and were f r o z e n and s t o r e d a t -9ooc u n t i l a n a l y z e d . the f i r s t s e r u m sample w a s t a k e n a f t e r a d m i s s i o n t c t h e h o s p i t a l b u t b e f o r e o p e r a t i o n and was used for t h e d e t e r m i n a t i o n o f f o l l i c l e s t i m u l a t . i n g hormone (fsh) and f o r t h e a s s e s s m e n t of t h y r o i d f u n c t i o n . the second serum sample was drawn randomly be tween 0 9 . 0 0 a m and 04.00 pm more t h a n o n e w e e k p o s t o p e r a t i v e l y , t h e median t i m e b e i n g t h r e e w e e k s and t h r e e q u a r t e r s o f t h e s a m p l e s t a k e n w i t h i n s i x w e e k s . the s e c o n d serum sample u a s u s e d f o r a n a l y s e s of t h e s t e r o i d hormones and shbg. t a b l e 1 . s t a g i n g a c c o r d i n g t o t h e number tnm c l a s s i f i c a t i o n of women t n m c l a s s i f i c a t i o n 301 c a t e g o r y number % i 52 37 20 i1 6 8 48 i11 13 9 i v 8 6 1 4 1 100 45 55 65 1 1, 85 95 age f i g . 1 . age d i s t r i b u t i o n o f 1 4 1 pa t i e n t s and c o r r e s p o n d i n g c o n t r o l s c o n t r o l s the c o n t r o l g r o u p c o n s i s t e d o f age-matched women w i t h o u t b r e a s t c a n c e r t h i s s t u d y t h o s e 151 w i t h i n t h e t o t a l c o n t r o l g r o u p o f 1 7 9 women who were menopausal were i n c l u d e d . they were s e l e c t e d from t h e c o m p u t e r i z e d p o p u l a i n p o s t i o n r e g i s t e r w i t h i n e a c h c o u n t y where t h e women closest i n a g e t o e a c h r e s p e c t i v e b r e a s t c a n c e r p a t i e n t was c h o s e n . c o n t r o l women who r e f u s e d t o p a r t i c i p a t e (25=14 % ) were r e p l a c e d by a n a l t e r n a t i v e c o n t r o l s e l e c t e d i n t h e same way a s t h e f i r s t o n e . a s a r e s u l t e a c h b r e a s t c a n c e r p a t i e n t had o n e age-matched con t r o l w i t h n o t more t h a n 3 d a y s a g e d i f f e r e n c e t o t h e c o r r e s p o n d i n g p a t i e n t . a l l women i n t h e c o n t r o l g r o u p answered a q u e s t i o n n a i r e i d e n t i c a l to t h a t i n t h e p a t i e n t g r o u p . i n a d d i t i o n t h e y were a l l examined a t t h e o f f i c e o f t h e i r d i s t r i c t m e d i c a l n u r s e by o n e o f u s (hoa). a t t h e e x a m i n a t i o n blood s a m p l e s f o r a n a l y s i s o f hormones i n s e r u m were drawn and c e n t r i f u g e d . t h i s o c c u r r e d , a s i n 26 1 t h e p a t i e n t g r o u p , randomly between 0 9 . 0 0 am and 04.00 pm. the sera were d i r e c t l y s e n t t o uppsala i n t h e same way a s i n t h e p a t i e n t g r o u p and were f r o z e n and s t o r e d a t -90 c . the s u b s e q u e n t a n a l y s i s i n c l u d e d , a s i n t h e p a t i e n t g r o u p , fsh and shbg i n a d d i t i o n t o t h e s t e r o i d hormones. 0 s e l e c t i o n of t h e p o s t m e n o p a u s a l g r o u p when comparing t h e age-matched p a i r s b o t h t h e p a t i e n t and t h e c o r r e s p o n d i n g c o n t r o l were p o s t m e n o p a u s a l a c c o r d i n g t o h i s t o r y and a n i n c r e a s e d c o n c e n t r a t i o n of fsh i n t h e s e r u m ( 6 0 ) i n 141 i n s t a n c e s . the a g e d i s t r i b u t i o n of t h i s g r o u p i s shown i n f i g . l . , t h e a v e r a g e and t h e median a g e b e i n g 68 y e a r s . i n t h e s e g r o u p s two p a t i e n t s and o n e c o n t r o l had t o be e x c l u d e d due t o e s t r o g e n t r e a t ment. i n t h e r e m a i n i n g g r o u p p o s t o p e r a t i v e l y t a k e n s e r u m s a m p l e s were a v a i l a b l e i n o n l y 1 2 2 p a t i e n t s . with r e s p e c t t o t h e f a c t o r s r e l e v a n t f o r t h e i n v e s t i g a t i o n , t h e loss c a n be c o n s i d e r e d random and t h e 1 2 2 p a i r s a n a l y z e d t h u s r e p r e s e n t a t i v e f o r t h e whole p o s t m e n o p a u s a l g r o u p s . radioimmunoa s s a y s t e s t o s t e r o n e : an a n t i b o d y r a i s e d i n s h e e p a g a i n s t testosterone-3-0-carboxy m e t h y l b o v i n e serum a l b u m i n was used ( g i f t from d r . l a r s e r i k e d q v i s t , royal v e t e r i n a r y high s c h o o l , u p p s a l a , sweden). the a n t i b o d y c r o s s r e a c t s 56 % w i t h d e h y d r o t e s t o s t e r o n e and 7 % w i t h a n d r o s t e n e d i o n e b u t l e s s t h a n 1 % w i t h dehydro e p i a n d r o s t e r o n e and e s t r a d i o l . i t was used i n a 1:15 000 d i l u t i o n w i t h e t h a n o l . a s t r a c e r 1 , 2 , 6 , 7 h t e s t o s t e r o n e w i t h a s p e c i f i c a c t i v i t y of 8 5 c i / m m o l p u r c h a s e d from t h e n e w england n u c l e a r , boston m a s s a c h u s e t t s , u s a w a s u s e d . i t was d i l u t e d t o g i v e a p p r o x i m a t e l y 85 pg p e r a s s a y t u b e . the p r o c e d u r e and r e a g e n t s of t h e a s s a y were t h e same a s d e s c r i b e d p r e v i o u s l y ( 1 4 ) . 0.4 m l a l i q u o t s of plasma were e x t r a c t e d w i t h 4 m l of d i e t h y l e t h e r f o r t h e a s s a y . a b l a n k of 55 pg (cv 2 1 % ) was found i n c h a r c o a l t r e a t e d plasma a t t h i s e x t r a c t i o n volume and r e s u l t s were c o r r e c t e d f o r t h i s plasma b l a n k . the w i t h i n and between a s s a y co e f f i c i e n t s o f v a r i a t i o n were found t o be 1 4 and 18 % , r e s p e c t i v e l y . 3 a n d r o s t e n e d i o n e : a p r e v i o u s l y d e s c r i b e d a n t i b o d y r a i s e d i n a d u l t ewes a g a i n s t androstenedione-3-oxime human s e r u m albumin ( g i f t from dr. guy e . abra ham) was u s e d . the a n t i b o d y c r o s s r e a c t s 25 % w i t h d e h y d r o e p i a n d r o s t e r o n e ( 2 ) . -3 i t w a s u s e d i n a 1:500 d i l u t i o n . 1 , 2 , h a n d r o s t e n e d i o n e w i t h a s p e c i f i c a c t i v i t y of 4 0 c i / m m o l p u r c h a s e d from n e w england n u c l e a r was used a s t r a c e r . i t was d i l u t e d t o g i v e a p p r o x i m a t e l y 7 2 pg p e r a s s a y t u b e . the p r o c e d u r e and r e a g e n t s of t h e a s s a y were t h e same a s d e s c r i b e d p r e v i o u s l y ( 1 4 ) . 0 . 1 m l a l i q u o t s of s e r u m were e x t r a c t e d w i t h 1 . 5 m l of d i e t h y l e t h e r f o r t h e a s s a y . a plasma blank of 1.03 pg (cv 20 % ) was found i n c h a r c o a l t r e a t e d plasma a t t h i s e x t r a c t i o n volume and t h e r e s u l t s were c o r r e c t e d f o r t h i s b l a n k . the w i t h i n and between c o e f f i c i e n t s of v a r i a t i o n f o r t h e a s s a y were found t o be 10 and 20 % , r e s p e c t i v e l y . 262 e s t r o n e was d e t e r m i n e d by radioimmunoassay a s d e s c r i b e d by axelsson e t a l . (7). sex-hormone-binding g l o b u l i n w a s a s s a y e d by t h e ammoniumsulphate p r e c i p i t a t i o n t e c h n i q u e d e s c r i b e d by rosner ( 5 0 ) and t h e r e s u l t s are e x p r e s s e d a s t h e dihydrotestosterone-binding c a p a c i t y of plasma i n nmol/l. c a l c u l a t i o n s and s t a t i s t i c a l methods a s a n i n d e x o f o b e s i t y i n d e p e n d e n t of h e i g h t w e u s e d t h e i n d e x o f q u e t e l e t ( w e i g h t / h e i g h t ) which was shown t o f u l f i l l i m p o r t a n t c r i t e r i a f o r s u c h a n i n d e x ( 2 9 ) . a s t h e shbg, el and t showed a skewed d i s t r i b u t i o n t h e i r l o g a r i t h m i c v a l u e s were c a l c u l a t e d . t h i s made t h e normal d i s t r i b u t i o n a s s u m p t i o n of t h e t t e s t f o r matched g r o u p s more p l a u s i b l e . 2 the c o r r e c t i o n f o r t h e r e l a t i v e l y h i g h plasma b l a n k o f a by s u b t r a c t i o n o f i t s mean v a l u e g a v e some n e g a t i v e r e s u l t s . i f t h e s e v a l u e s had been d i s c a r d e d t h e mean e s t i m a t e o f a would have become p o s i t i v e l y b i a s e d . they were t h e r e f o r e i n c l u d e d i n t h e c a l c u l a t i o n which, however, made t h e u s e o f l o g a r i t h m e d v a l u e s i m p o s s i b l e . due t o t h e s k e w e d d i s t r i b u t i o n of a wilcoxon's n o n p a r a m e t r i c m a t c h e d p a i r s s i g n e d r a n k s test w a s u s e d a s a complement t o t h e t-test. i n t h e c a l c u l a t i o n of t h e c o n f i d e n c e i n t e r v a l s of t h e p o p u l a t i o n means of a t h e s t a n d a r d d e v i a t i o n o f t h e b l a n k had t o be i n c l u d e d . the p r o d u c t moment c o r r e l a t i o n c o e f f i c i e n t w i t h t h e c o r r e s p o n d i n g p-value o f t h e i n d e p e n d e n c e h y p o t h e s i s was u s e d a s a n measure o f c o r r e l a t i o n . a m u l t i p l e r e g r e s s i o n a n a l y s i s was a l s o a p p l i e d t o e v a l u a t e how much of t h e v a r i a n c e i n e l c o u l d be e x p l a i n e d by v a r i a n c e i n o t h e r " i n d e p e n d e n t " v a r i a b l e s . a l l v a l u e s r e f e r t o t w o t a i l e d tests. when v a l u e s were m i s s i n g i n t h e p a t i e n t or t h e c o r r e s p o n d i n g c o n t r o l , b o t h were e x c l u d e d from t h e c a l c u l a t i o n s v a r i a b l e . e s t r o n e ( e l ) . c a n t l y h i g h e r (p ( t a b l e 2 ) . t h e r e v a l u e s e x c e e d i n g results the mean v a l u e i n t h e p a t i e n t g r o u p (132 p m o l / l ) w a s < 0.01) t h a n t h e mean v a l u e i n t h e c o n t r o l g r o u p ( 1 0 8 was a wide r a n g e o f v a r i a t i o n i n b o t h g r o u p s ( f i g . 2) 200 pmol/l were found i n o n l y 15 c o n t r o l s compared t o f o r t h a t s i g n i f i pmol/l) b u t 29 pa t i e n t s . the means c a l c u l a t e d on a r i t h m e t i c v a l u e s were 152 pmol/l ( 4 1 . 2 pg/ml) i n t h e p a t i e n t g r o u p and 1 2 4 pmol/1 (33.5 pg/ml) i n t h e c o n t r o l g r o u p . a n d r o s t e n e d i o n e ( a ) . the d i s t r i b u t i o n was s l i g h t l y s h i f t e d t o h i g h e r v a l u e s i n t h e p a t i e n t g r o u p ( f i g . 3 ) whose mean v a l u e a f t e r c o r r e c t i o n f o r t h e b l a n k was 2 . 5 nmol/l (0.71 ng/ml) which i s s i g n i f i c a n t l y h i g h e r (p=0.009, t t e s t ; p0.006, wilcoxon's t e s t ) t h a n t h e mean v a l u e i n t h e c o n t r o l g r o u p of 1.6 n m o l / l (0.46 ng/ml) ( t a b l e 2 ) . v a l u e s lower t h a n t h e mean v a l u e of t h e blank (3.7 nmol/l) were found i n 16 p a t i e n t s and 2 2 c o n t r o l s . t e s t o s t e r o n e ( t ) . even t h e d i s t r i b u t i o n of testosterone was s h i f t e d t o 263 a j 4 u m u m m c a o u ' 4 a m u c z m u m ar -4 0 m 4 w 3 [ r q u c 0 -4 -4 4 e c cnc aj 3 d m i a c m .rl 5 aj z aj 0 aj 9 w c c r n o a j u e oh0 a, s m u m w a 0 m m u c -4 m e aj .a e d -4 ci (i) aj u i u ffl d z ci c u (i) u c aj .rl u m a m rl z u c 0 m u c ar .4 u m a tr 0 0 0 c c c 0 m c m c c i 00 m w 0 c lo -3 ? i n c ? n m c d d \ 2 ci w c m 0 0 0 m c 0 n c n i c c w c c m i m c m n ? 5 4 0 4 n v 0 0 m m c 0 w c 0 m c i 00 n c w m c m w c i m -r c q m c d \ 4 0 5 €+ w 0 0 0 m 0 m c 0 0 m 0 in i 0 q -3 w p -j m m q i w w w n 0 xr d \ rl 0 5 u z m m 264 number of women 7 / / / / l patients controls 30 20 i a r 121 number of women 30. 201 estrone pmolll patients 0 controls 2 3 anorostenedione nrnolll fig. 2. distribution of serum levels fig. 3. distribution of serum levels of estrone in patient and control of androstenedione in patient and group control group number of women 0 patients 0 controls l,25 150 175 2po 2.25 2,50 2.75 "3,oo testosterone nmolll number of women patients controls n 50 60 70 80 290 shbg nmolll fig. 4. distribution of serum levels fig. 5. distribution of serum levels of testosterone in patient and control group patient and control group of sex-hormone-binding globulin in 265 higher values in the patient group (fig. 4 ) but the difference in mean values between the groups was very small albeit significant (p < 0.05)(table 2). the skewness of the distributions (fig. 4 ) was less pronounced than for el (fig. 2) and a (fig. 3 ) and the arithmetic means 1.65 nmol/l ( 0 . 4 8 ng/ml) and 1.52 nmol/l ( 0 . 4 4 ng/ml) close to the logarithmic. sex-hormone-binding globulin (shbg). in contrast to e l , a and t this dis tribution was shifted towards lower values in the patient group (fig, 5 ) . the difference is highly significant (p < 0 . 0 1 ) . due to some very high values in the patient group (fig. 5 ) the mean values 4 0 . 2 nmol/l and 4 7 . 3 nmol/l differ less than the median values 4 0 . 1 and 5 0 . 9 , respectively (table 2). the arith metic means were 4 5 . 2 and 5 0 . 8 nmol in the patient and control groups. relations to age, weight and stage of disease. subgrouping of the material according to age revealed no obvious trend in the differences between patients and controls for any of the variables. thus in all decades from 4 5 to 7 5 years or more the patients, on the average, had a slightly higher a and t concentra tion in the serum. e l concentration did not differ between patients and controls aged 4 5 5 4 years but the patients had higher mean values in all subsequent age groups. the patients had a lower mean shbg value in all groups except in that including women over 7 5 years of age. not any of the variables was significantly correlated to age, either in the control or in the patient group (table 3 ) . table 3 . correlation coefficients to age, weight, quetelet’s index and stage of disease according to the tnm-classification correlated to test group age quetelet’s tnm weight index classification e l patient 0;ol 0.05 0 . 0 7 0 . 0 4 control 0.07 0 . 1 4 0. 22l’ a patient 0.00 0.07 0.09 0 . 0 8 control 0 . 0 1 0.03 0.00 t patient -0.11 -0.04 -0.03 -0.01 control -0.09 0.01 0 . 0 5 0 . 0 1 4 / 4 / shbg patient 0.13 0 . 2 8 3 / 0 . 3 1 control 0.12 -0.29 3 / 0.34 2 i / quetelet’s index = weight/height 2 / p < 0 . 0 5 3 / p < 0.01 4 / p < 0 . 0 0 1 266 350 300 250 _i . . . . . . . . . . . . . . . . .. . . . . . . . -. . ? ... .. i . . . . . . . . . . . . . . . . . . . . 250 2w 150 i00 50. i 0 2 l 6 8 1 0 350. 300. 2507 a n d l l 200. 150 100. 5 0 f i g . 6 . c o r r e l a t i o n b e t w e e n s e r u m l e v e l s of e s t r o n e a n d a n d r o s t e n e d i o n e i n t h e c o n t r o l g r o u p 150. loo. 0 p w l loo! .. . . . . . . . . . . . . .. . . . . . . . . " . . . . . . .i .. . . . . . . . . . . . . . " . ... i = m o pzod5 50. . . . . . . . . . . . . . . . " . . . . . . r = oh5 pcmol f i g . 7 . c o r r e l a t i o n b e t w e e n s e r u m l e v e l s of e s t r o n e a n d t e s t o s t e r o n e i n t h e c o n t r o l g r o u p e l pmdll mi r 5 0.25 p 4 0 1 . . . . . / . ." . . . . . . . .... . . .. . . . . . . . . . . . . . . " * u : .: _. . . . . . . i 015 (0 1:5 2b 25 3'0 15 t nrnolll f i g . 9. c o r r e l a t i o n b e t w e e n s e r u m l e v e l s of e s t r o n e a n d t e s t o s t e r o n e i n t h e p a t i e n t g r o u p 267 weight varied from 41 to 110 kg with a close accordance between the distribu tions and a mean weight of 65.55 kg in the patient group and 65.75 kg in the control group. the same accordance was found concerning height with a range from 140 to 178 and mean values of 161.7 and 161.4 cm, respectively. the differences were, as reported elsewhere (4), not statistically significant in either weight, height or the quetelet’s index. when weight and the index for overweight (quetelet’s index) were correlated to the steroid hormone levels the correlation coefficients were very low (table 3). except for a low degree of significance (p < 0.05) for the correlation be tween el and quetelet’s index in the control group (table 3) they were all in significant (p > 0.05). the same was true in the patient group in the correla tion to the stage of the disease according to the tnm classification (table 3). a slight but significant negative correlation was found between quetelet’s index and the shbg, in both groups (table 3). interrelations between el, a and t were finally analyzed with calculation of correlation coefficients and with a multiple regression analysis using el as a dependent variable and a and t as independent variables in addition to age and the weight index. in the control group there was a highly significant correlation between el and a (r=0.48) (fig. 6) and el and t (r=0.45) (fig. 7). the situation was quite different in the patient group where no significant correlation (r=0.10) was found between el and a (fig. 8) and a low correlation (~0.25) between el and t (fig. 9). according to the multiple regression analysis the variances in the serum level of el could be explained to 14 % and 58 % in the patient and control group, respectively, by variations primarily in a and t with very little contri bution to the determination coefficient from age and the weight index. shbg was not significantly correlated to el, a or t in any of the groups (r=0.17). discussion the estrogen and androgen metabolism and the serum concentration after the menopaus differ in many aspects from the premenopausal state. the serum concen trations of estradiol and estriol were thus generally found to be low (8, 15, 16, 17, 27, 35, 46) while estrone is the dominant estrogen in the postmenopause with normal values in most reports which were in agreement with the mean value in our control group (17, 25, 27, 35, 42, 46) and in some reports even higher (3, 9, 58). a l s o concerning testosterone our study revealed a mean value in accordance with previous authors (3, 10, 24, 30, 58) although lower (25, 42) as well as higher (16) normal values have been reported. our mean value for andro stenedione in the control group when corrected for the blank, is in the range found by some other investigators using the same antibody for the radioimmuno a s s a y ( 2 , 3 , 4 2 ) b u t somewhat lower t h a n t h a t r e p o r t e d by o t h e r s who u s u a l l y found v a l u e s i n t h e i n t e r v a l 0.75-1.09 ng/ml ( 1 6 , 1 7 , 2 5 , 5 8 ) . the u s e of n o n h o s p i t a l i z e d c o n t r o l s made a c o m p a r a b i l i t y w i t h t h e p a t i e n t g r o u p w i t h r e g a r d t o a r e c e n t o p e r a t i o n i m p o s s i b l e . s u r g i c a l trauma h a s been shown t o i n d u c e a s m a l l and t r a n s i t o r y f a l l of a and t b u t no change o f e l i n normal women ( 2 8 ) . the f a c t t h a t t h e s e c h a n g e s were n o r m a l i z e d w i t h i n one w e e k ( 2 8 ) and t h a t o u r p a t i e n t s had h i g h e r mean v a l u e s t h a n t h e c o n t r o l s seems t o e x c l u d e p o s t o p e r a t i v e c h a n g e s a s a cause of t h e d i f f e r e n c e s found i n t h i s s t u d y . d i u r n a l v a r i a t i o n s o f a and t i n women were shown by vermeulen ( 5 8 ) whereas r e s u l t s c o n c e r n i n g e l have been c o n t r a d i c t o r y ( 8 , 5 8 ) . a s i g n i f i c a n t d i f f e r e n c e i n t h e a v e r a g e s a m p l i n g t i m e between p a t i e n t s and c o n t r o l s was, however, con s i d e r e d u n l i k e l y and t h e c h a n g e s d u r i n g t h e day too s m a l l ( 5 8 ) t o a c c o u n t f o r t h e d i f f e r e n c e s f o u n d between t h e g r o u p s c o n c e r n i n g a and t. accumulated e v i d e n c e now s u p p o r t t h e view t h a t n e g l i g i b l e amounts of estro g e n s a r e s e c r e t e d by t h e p o s t m e n o p a u s a l o v a r i e s or a d r e n a l s ( 1 2 , 1 6 , 2 5 , 3 6 3 8 , 4 3 ) . i n s t e a d most e s t r o g e n s a f t e r t h e menopause a r e d e r i v e d from t h e p e r i p h e r a l c o n v e r s i o n of t h e a n d r o g e n s ( 1 7 , 3 4 , 3 7 ) s e c r e t e d by t h e o v a r i e s and a d r e n a l s ( 1 6 , 1 7 , 2 5 , 2 6 , 3 6 ) . thus e l c a n be a c c o u n t e d f o r n e a r l y e x c l u s i v e l y by t h e p e r i p h e r a l c o n v e r s i o n o f a w h e r e a s t h e p r o d u c t i o n of e 2 i n t h e blood is m a i n l y due t o t h e c o n v e r s i o n of t b o t h d i r e c t l y and i n d i r e c t l y v i a a and e l ( 3 4 , 3 8 ) . t h e r e f o r e t h e h i g h e r mean c o n c e n t r a t i o n of e l found i n women w i t h b r e a s t c a n c e r i n t h i s s t u d y h a s t o be d i s c u s s e d i n terms of a lowered m e t a b o l i c c l e a r a n c e r a t e or a n i n c r e a s e d p r o d u c t i o n a s a consequence of e i t h e r i n c r e a s e d a v a i l a b i l i t y o f plasma p r e c u r s o r s or o f a n i n c r e a s e d e x t e n t of c o n v e r s i o n of t h e s e p r e c u r s o r s t o t h e p r o d u c t hormone ( 3 9 ) . the m e t a b o l i c c l e a r a n c e r a t e o f e s t r o g e n s ( e l , e 2 , e 3 ) h a s been shown t o be d e c r e a s e d i n p o s t m e n o p a u s a l women compared t o premenopausal ( 1 5 , 3 5 ) b u t i n b r e a s t c a n c e r p a t i e n t s t h e r e h a s r a t h e r been a t e n d e n c y t o h i g h e r m e t a b o l i c c l e a r a n c e o f e l ( 3 1 ) and even a ( 3 1 , 4 5 ) t h a n i n n o r m a l s . the c o n v e r s i o n r a t e o f a n d r o s t e n e d i o n e t o e s t r o n e i n b r e a s t c a n c e r p a t i e n t s d i d n o t d i f f e r from t h a t i n normal c o n t r o l s ( 3 1 , 4 5 ) . b u t i n women w i t h endo m e t r i a l c a r c i n o m a ( 1 8 , 5 3 ) and e n d o m e t r i a l h y p e r p l a s i a ( 5 3 ) a n i n c r e a s e d con v e r s i o n was found and s u g g e s t e d an i m p o r t a n t e t i o l o g i c f a c t o r ( 3 9 ) . these f i n d i n g s m i g h t imply t h a t t h e p r i n c i p a l r e a s o n f o r t h e h i g h e r e s t r o n e l e v e l i n t h e b r e a s t c a n c e r g r o u p i s a n i n c r e a s e d a v a i l a b i l i t y of t h e p r e c u r s o r s , m a i n l y a and t . t h i s c o n c e p t was s u p p o r t e d by t h e s i g n i f i c a n t l y h i g h e r v a l u e s found i n t h e p a t i e n t g r o u p i n o u r s t u d y ( t a b l e 2 ) . a s l i g h t d i f f e r e n c e i n t h e same d i r e c t i o n w a s a l s o s u g g e s t e d f o r a n d r o s t e n e d i o n e i n a r e c e n t r e p o r t by rose e t a l . ( 4 8 ) . i n c r e a s e d t e s t o s t e r o n e l e v e l s were a l s o r e c e n t l y f o u n d i n b r e a s t c a n c e r p a t i e n t s by mcfadyen e t a l . ( 4 4 ) and s a r f a t y e t a l . ( 5 1 ) a l t h o u g h t w o p r e v i o u s r e p o r t s a r e i n d i s a g r e e m e n t w i t h t h i s f i n d i n g ( 2 2 , 5 6 ) . whereas i n t h e c o n t r o l g r o u p a c o n s i d e r a b l e p a r t o f t h e e s t r o n e v a r i a t i o n s 269 were r e l a t e d t o t h e a v a i l a b i l i t y o f p r e c u r s o r s ( f i g s 6 and 7 ) , t h e l a c k o f c o r r e l a t i o n between e l and a and t h e l o w c o r r e l a t i o n between e l and t i n t h e p a t i e n t g r o u p a r e c o n f u s i n g and s u g g e s t t h a t o t h e r f a c t o r s have a n i n f l u e n c e on t h e s e r u m e s t r o n e l e v e l . the r e s u l t s of poortman e t a l . ( 4 5 ) and k i r s c h n e r e t a l . (31) a r e a l s o i n c o n s i s t e n t w i t h t h e c o n c e p t o f a n i n c r e a s e d a v a i l a b i l i t y of p r e cursor hormones i n b r e a s t c a n c e r p a t i e n t s a s t h e y found no d i f f e r e n c e i n t h e a n d r o s t e n e d i o n e p r o d u c t i o n r a t e compared t o age-matched c o n t r o l s . although t h e p r o d u c t i o n of a and t c a n be a c c o u n t e d f o r e x c l u s i v e l y by a d r e n a l and o v a r i a n s e c r e t i o n and i n t e r c o n v e r s i o n between t h e s e hormones ( 5 8 ) a n a d d i t i o n a l source of e s t r o g e n was s u g g e s t e d by h i l l e t a l . ( 2 1 ) . they demon s t r a t e d t h e p r o d u c t i o n of e s t r o g e n s by b a c t e r i a from t h e b i l i a r y s t e r o i d s re l a t e d t o t h e f a t c o n t e n t i n t h e d i e t . t h i s c o u l d t h e n l i n k t h e o b s e r v e d g r e a t i n t e r n a t i o n a l v a r i a t i o n s i n b r e a s t c a n c e r i n c i d e n c e o b v i o u s l y r e l a t e d t o e n v i r o n m e n t a l f a c t o r s ( 4 ) t o an e n d o c r i n e h y p o t h e s i s . the d i s t r i b u t i o n and mean v a l u e of shbg i n t h i s m a t e r i a l is i n t h e r a n g e p r e v i o u s l y r e p o r t e d i n normal women ( 4 9 ) . v a l u e s i n b r e a s t c a n c e r p a t i e n t s a r e n o t a v a i l a b l e f o r c o m p a r i s o n . serum shbg l e v e l s a r e known to be r e g u l a t e d by t h e b i o l o g i c a l a c t i v e , unbound hormone f r a c t i o n w i t h t h e a n d r o g e n s h a v i n g a n i n h i b i t o r y and e s t r o g e n s a s t i m u l a t o r y i n f l u e n c e ( 6 , 5 7 ) . t h y r o i d f u n c t i o n , a s s e s s e d i n t h i s m a t e r i a l by d e t e r m i n a t i o n of tsh, t3, rt3, t4 and t 3 r e s i n u p t a k e and r e p o r t e d e l s e w h e r e ( 5 ) , is a l s o known t o i n f l u e n c e t h e shbg l e v e l ( 6 ) and t h e s t e r o i d hormone m e t a b o l i s m ( 5 4 ) . the r e s u l t s were, however, m a i n l y w i t h i n t h e l i m i t s o f t h e r e f e r e n c e r a n g e and no s i g n i f i c a n t c o r r e l a t i o n s t o shbg, e l , a or t were f o u n d . the lower c o n c e n t r a t i o n of shbg i n t h e p a t i e n t g r o u p s p e a k s a g a i n s t i n c r e a s e d e s t r o g e n i c i t y i n b r e a s t c a n c e r p a t i e n t s and might r a t h e r i n d i c a t e a predominance of a n d r o g e n i c i n f l u e n c e ( 6 ) c o n s i s t e n t w i t h t h e c o n c e p t o f i n c r e a s e d p r o d u c t i o n of a and t. t h e r e i s a well-known r e l a t i o n between t h e b i o l o g i c a l a c t i v i t y o f s t e r o i d h o r mones and t h e i r b i n d i n g t o shbg. c o n s e q u e n t l y t h e b i n d i n g o f t e s t o s t e r o n e is h i g h and t h a t o f a n d r o s t e n e d i o n e and e s t r o n e v e r y l o w or n e g l i g i b l e ( 6 , 4 9 ) . a lower shbg l e v e l i n t h e p a t i e n t g r o u p w i l l t h e r e f o r e p r i m a r i l y a f f e c t testo s t e r o n e and cause a n i n c r e a s e d f r a c t i o n o f t h e unbound hormone. an a d d i t i o n a l consequence would b e a n i n c r e a s e d m e t a b o l i c c l e a r a n c e r a t e o f t e s t o s t e r o n e ( 1 1 , 57) which c o u l d d i m i n i s h t h e i n f l u e n c e of a n i n c r e a s e d p r o d u c t i o n r a t e on t h e p e r i p h e r a l c o n c e n t r a t i o n ( 1 ) . the a b i l i t y o f a d c p o s e t i s s u e t o m e t a b o l i z e c 1 9 s t e r o i d s t o e s t r o g e n s h a s been d e m o n s t r a t e d i n v i t r o ( 5 2 ) . a p o s i t i v e c o r r e l a t i o n was a l s o found between w e i g h t and c o n v e r s i o n r a t e of a t o e l i n normal p o s t m e n o p a u s a l women ( 5 3 ) and i n women w i t h e n d o m e t r i a l c a n c e r ( 4 7 ) . the l a c k of s i g n i f i c a n t c o r r e l a t i o n between w e i g h t and s e r u m e s t r o n e l e v e l s i n normal p o s t m e n o p a u s a l women found i n t h e p r e s e n t s t u d y , i s i n a c c o r d a n c e w i t h t h e f i n d i n g s o f j u d d e t a l . ( 2 5 , 2 7 ) b u t a 270 positive correlation was found in women with endometrial carcinoma ( 2 7 ) . our study did not reveal any difference in weight or in different weight indices between patients and controls ( 4 ) and no correlations to e l , a or t except for a low correlation between e l and quetelet's index in the control group. if the amount of fat tissue is positively correlated to the conversion rate of a to e l , an influence on the serum levels might be obscured if the body weight is nega tively correlated to the percentage of e l derived from plasma a which enters the circulation, as noticed by grodin et al. ( 1 7 ) . but if obesity was a risk factor for breast cancer and the amount of fat tissue quantitatively influenced the aromatization of estrogen precursors, then the conversion rate of a to e l ought to be increased in breast cancer patients. this was, as pointed out above, not confirmed by poortman et al. ( 4 5 ) or by kirschner et al. ( 3 1 ) who found a con version rate in close agreement with other studies in normal postmenopausal women ( 3 7 , 5 3 ) . a negative correlation between shbg and obesity, confirmed in this study, was previously noticed by vermeulen et al. ( 5 7 ) . the conversion rate of a to e l has repeatedly been found to be 1 . 2 1 . 3 % in premenopausal women ( 3 7 , 5 3 ) and at least two times higher in postmenopausal women ( 1 7 , 3 7 , 3 8 , 4 5 , 5 3 ) . the concept of a progressive increase with advancing age, as suggested by hemsell et al. ( 2 0 ) has less support and was not confirmed in women with endometrial carcinoma ( 4 7 ) . it is also in disagreement with the lack of correlation between age and excretion of urinary estrogens found by thijssen et al. ( 5 5 ) in normal postmenopausal women. neither was there any cor relation between age and serum levels of e l or a in the present study nor in those by judd et al. ( 2 5 , 2 7 ) . in conclusion, serum concentrations of e l and shbg have, to our knowledge, not been studied before in breast cancer patients and thus the increased e l and decreased shbg never observed, whereas the increased a and t levels have some support in a few recent studies. although an increased production of estrogen precursors seems to be the most reasonable explanation for these findings, they are difficult to fit into a definite hypothesis and uniform support for this view was not perceivable in earlier studies. the significance of these findings needs therefore further conformation, primarily by studies of steroid hormone kinetics including production, conversion and metabolic clearance in breast cancer patients and comparable controls. ack€?owledgment this study was supported by contract no. 1-cb 5 3 9 6 8 from the national cancer institute, usa and by grants from the swedish medical research council no. 0 3 4 9 5 and the ford foundation. 27 1 references 1 . abraham, g . 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, van d e r s t r a e t e n , m. & o r i e , n . : c a p a c i t y o f 58. v e r m e u l e n , a.: the hormonal a c t i v i t y of t h e p o s t m e n o p a u s a l o v a r y . j c l i n 59. v i c t o r , a , , weiner, e . & j o h a n s s o n , e.d.b.: r e l a t i o n between s e x hormone 61. zumoff, b . , bradlow, h . l . , g a l l a g h e r , t.f. & hellman, l.: d e c r e a s e d c o n v e r 62. zumoff, b., fishman, j., bradlow, h.l. & hellman, l.: hormone p r o f i l e s i n accepted j a n u a r y 25, 1979 address f o r r e p r i n t s : hans-olov adami, m.d. department o f s u r g e r y u n i v e r s i t y h o s p i t a l 5-750 14 uppsala sweden 274 u p s a l a j m e d sci 79: 84-89, 1974 metabolic aspects of physical training in male patients after myocardial infarction a r n e b j e r n u l f a n d j o n a s b o b e r g from the departments of internal medicine and clinical physiology, university hospital, uppsala, and the depart ment of geriatrics, university of uppsala, sweden a b s t r a c t twenty-six post infarct patients were selected and ad mitted to hospital 3 months after the onset of the myocardial infarction for an investigation including lipid studies and glucose and fat tolerance tests. after this initial hospital stay the patients were randomly divided into a training (13 patients) and a reference (13 patients) group. during the following 3 months the first group of patients were physically trained (entirely in doors, three sessions a week individually). thereafter the patients of both groups were readmitted to hospital for a second investigation, identical with the initial one. the oral glucose tolerance (ogt)' was improved in the younger (less than or equal to 55 years) patients of the trained group. there were no significant changes of cholesterol or triglyceride levels after the training or reference periods measured 5 days after the last train ing session. i n t r o d u c t i o n t h e risk f a c t o r s for ischaemic h e a r t disease ( i h d ) , n o w a d a y s well k n o w n , include c i g a r e t t e smoking, hyperlipidaemia, r e d u c e d g l u c o s e t o l e r a n c e a n d raised blood pressure ( i , 13, 17, 26, 39). a t t e m p t s at c o m b a t i n g t h e s e f a c t o r s h a v e included re c o m m e n d a t i o n s for c h a n g e s i n diet, s m o k i n g with d r a w a l , a n d different d r u g t h e r a p i e s . during r e c e n t y e a r s the interest in physical training as an addi tional f o r m of t h e r a p y in primary prevention of ihd h a s increased. t h e e x p e c t e d benefits are a d e c r e a s e of s e r u m triglycerides ( t g ) a n d serum c h o l e s t e r o l (chol) c o n c e n t r a t i o n s (24, 27, 29, 34). p h y s i c a l training as secondary p r e v e n t i o n o f ihd has a l s o gained i n t e r e s t b e c a u s e of o t h e r benefits such as a c h i e v e m e n t of a higher physical w o r k c a p a c i t y , and i n c r e a s e d self-confidence of b o t h patient a n d his relatives ( 2 , 5, 33). the following abbreviations were used: ogt oral glu cose tolerance, ivgt intravenous glucose tolerance. ?'his r e p o r t d e s c r i b e s e x p e r i e n c e s o f t h e effect o f physical training o n p l a s m a lipids a n d blood g l u c o s e metabolism in male p a t i e n t s r e c o v e r e d f r o m t h e i r first myocardial infarction. m a t e r i a l selection of patients the material was selected from all male patients treated at the university hospital, uppsala during the period december 15, 1970-march 15, 1972 for their first myocar dial infarction. patients 70 years old or older were excluded, as well as all patients with the following conditions: treated dia betes mellitus, diastolic blood pressure exceeding l 10 mmhg a t rest (indirect method), heart volume exceeding 600 ml/m2 body surface area (supine posture), cardiac arrhythmias (e.g. regularly recurring ves during exer cise, or atrial fibrillation), a history of pain or aching in the back or the larger joints or pulmonary disease. no patients treated with digitalis, beta receptor blocking agents, or lipid lowering drugs were included in the study. the material is a subsample of the series previously described and compared with the whole infarct popula tion treated at the hospital during a two-year period march 15, 1969-march is, 1971 (2). after the above selections there remained 26 patients. who were assigned randomly to two groups, a so-called training group (13 patients) and a reference group (13 patients). the former group underwent a 3-month period of physical training (see below) while the reference group served as a control. clinical characteristics of the material have been presented elsewhere (2, 3, 4). drop-out of patients three patients from the trained group and 5 patients from the reference group dropped out from the physical train ing or were excluded for the following reasons: aggrava tion of angina pectoris during training ( 1 patient), rein, farction ( 1 patient) (not in connection with any training session), ulcerative proctitis ( 1 patient). one patient did not wish to take part in any follow-up investigations. four patients dropped out for technical reasons: vaso vagal reactions (2 patients), incomplete blood sampling (2 patients). upsala j med sci 79 infarction, physical training and metabolic changes 85 table i. a g e , body weight, and blood plasma concentrations of triglycerides and cholesterol before and after the training and reference periods plasma trielvcerides” i < (mmole/l) body weight plasma cholesterol (kg)i i1 (mgil00 ml) age (years) before after before after before after before after trainedgroup mean . 56 75.9 75.5 2.83 2.53 2.64 2.39 266 237 (0.421) (0.379) ( n = 10) s.e.m. 2.3 2.5 2.8 0.36 0.30 (-0.945) (-0.953) 13 9 h 2s.e.m. -0.4 k 0 . 7 -0.30 k0.25 (-0.042 k0.044) -29 + i 7 ns ns ns ns reference group mean 52 76.2 76.2 2.35 2.24 2.29 2.19 289 273 ( n =8) s.e.m. 3.0 3.3 3.5 0.21 0.17 (-0.962) (-0.965) 13 13 (0.359) (0.340) a i s . e . m . 0 -0.1 lk0.13 (-0.019k0.024) -16 *7 ns ns ns ns a calculations made without (i) and with (11) logarithmic transfer of the values. the 10 logarithmic values are given within brackets. general procedure three and six months after the onset of the acute my ocardial infarction the patients were readmitted to hos pital for investigations, which have been described pre viously (2, 3). in addition during the last year of the study oral and intravenous glucose tolerance tests and also an intravenous fat tolerance test were performed on different days before and after the training or reference periods. furthermore, adipose tissue biopsies were taken for determination of fat cell size. the patients of the trained group were then trained 3 times weekly as pre viously described (3). each session started with 10 min rest, and this was followed by 10 min of calisthenics under the leadership of a qualified physiotherapist. this was followed by three 10-min sessions of cycling on an ergometer bicycle with varying work loads. the training session ended with 10 min rest on a couch. it was con sidered desirable that the work load should reach but not exceed the pain threshold for the patients with angina pectoris. n o instructions were given to the patients concerning dietary modifications. methods the patients came to the laboratory in the morning after fasting and refraining from smoking since the night be fore. teflon catheters (stille infart i . 15 mm, stockholm) were inserted percutaneously in a cubital vein for blood sampling. determinations of the plasma concentrations of cho lesterol and triglycerides were made by an autoanalytical technique (32). the blood glucose concentrations were determined on venous blood plasma by the glucose oxidase method (2 1). the serum insulin concentration was determined by a “solid phase” radioimmunological technique (phadebas insulin test, pharmacia) based upon the method described by wide & porath (40). the serum uric acid was analysed by a method described by sobel & kim (36). the oral glucose tolerance (ogt) test was performed as follows: the patient drank about 200 ml water containing 100 g glucose. blood samples for analysis of glucose and insulin concentrations were taken immediately before (o), 30, 60, and 120 min after inges tion of the glucose. for the intravenous glucose tolerance (ivgt) test, 0.5 g glucose per kg body weight was given intrave nously during 2 min in the form of a 50% solution. blood samples were taken for determination of blood glucose before and 20, 30, 40, 50 and 60 rnin after, and of serum insulin before and 4, 6, 8, and 60 min after the start of the glucose injection. early insulin response was cal culated according to the method of thorell et al. (38). the ivgt was expressed as the k value calculated from the formula 0.693 x loo/ glucose half time (25). the intravenous fat tolerance test (ivftt) was ,per formed in accordance with the method of carlson & rossner (16). the fat cell diameter was measured on cells obtained by adipose tissue biopsy according to the method of sjo strom et al. (35). statistical concepts and methods as described by hoe1 were used (22). results n o significant differences were found between the trained and the reference group with respect to age, height and body weight (table i). in the 10 patients of the trained group the mean age was 56 years, the initial weight 75.9 and the final weight 75.5 kg, and the mean height 176 cm. in upsala j med sci 79 86 a . bjernulfand j . boberg table 11. a g e , body weight, and intravenous f a t tolerance and f a t cell diameter before and after the training and reference periods intravenous fat body weight tolerance, k fat cell diameter (kg) (%/rnin) ( c l ) age (years) before after before after before after trained group mean 53.6 76.4 75.6 2.64 3.11 89.9 85.4 s.e.m. 2.1 3.2 3.8 0.66 0.83 6.58 4.00 range 44-59 65.1-89.8 63.1-94.0 0.7-5.1 i . 1-7.7 61.5-108.0 67.8-93.6 n 7 7 7 6 reference group mean 58.7 75.4 75.2 3.53 4.05 89.4 89.0 s.e.m. 6.6 4.0 4.1 0.53 0.5 i 3.44 5.13 n 7 7 7 6 range 49-66 63.4-94.6 61.6-95.2 2.2-6.0 2.6-6.0 72.8-99.0 66.0100.7 the 8 patients of the reference group the correference period. in the training group there was responding figures were 52 years, 76.2 and 7 6 . 2 a reduction of 0.30-+0.25 mmoles/l ( 1 i % ) com kg, and 174 cm. pared with 0.11ko.13 mmoles/l in the reference group (table i ) . in the trained group 6 of the effect o f training o n lipid metabolism patients achieved a decrease i n plasma tg. ail there were no significant changes of the mean these patients had initial tg values more than 1.90 plasma tg concentration after the training or mrnoles/l. table 111. the effect of physical training on fasting blood glucose, and serum insulin, oral glucose tolerance, intravenous glucose tolerance and the serum insulin response after glucose given orally and intravenously mean values 2 standard errors of the means (s.e.m.) are given oral glucose test o g t (mg/100 ml) insulin (microunits/ml) age body weight n (years) 0%) s u m n s u m 7rained group total before after before after 6 5 years reference group total before after before after 6 5 5 years 80.8 23.7 81.0 24.2 8 52.4 23.1 81.1 2 5 . 4 82.1 2 6 . 0 5 4 7 . 0 2 2 . 3 77.3 2 2 . 6 9 5 4 . 4 2 3 . 0 76.1 5 3 . 1 78.2 2 4 . 4 76.8 25.6 5 4 8 . 4 2 3 . 3 0‘ 81 ?3 81 2 2 82 2 4 81 2 4 85 2 4 83 2 5 86 5 8 83 2 8 (0-60-120’) 328 2 2 2 298 2 1 5 350 +20 ** 5 294 2 16 8 349 2 3 3 374 2 2 5 3 14 2 3 2 354 2 4 2 7 5 0‘ 12 2 2 14 2 2 1 1 221 14 2 2 19 2 5 14 + 3 17 2 3 11 2 2 (mo-120’) 175 2 3 9 151 2 2 8 146k32 134 2 3 3 228 2 9 3 178 2 5 7 131 2 2 7 108 2 1 7 * * p < 0.01 (significance for paired differences), n = n u m b e r of patients. the o g t test and ivgt test were performed a s described under methods. o g t is expressed a s the fasting blood glucose (0 ) and the sum of the values for blood glucose concentration at 0, 60 and 120 min after the glucose intake. serum insulin values during the o g t test are presented in the same way. ivgt is presented as the k value (25) and the serum insulin response during the ivgt test is expressed a s early response (38) (see under methods) and the concentration 60 min after glucose injection. uvsala j med sci 79 infar neither were there any significant changes of the mean plasma cholesterol concentration. in the trained group there was a reduction of i i % of the pretraining value, on the average, from 266 t o 237 mg/l00 ml. in the reference group the cholesterol value decreased from 289 to 273 mg/loo ml (ta ble i ) . there were no significant changes in body weight after the training or reference period (ta ble i). as shown in table i1 there were no significant changes of fractional removal rate of exogenous triglycerides (intravenous fat tolerance) after the training or reference periods. neither were there any significant changes of the fat-cell diameter. effects of training on carbohydrate metabolism in a sample of patients investigated during the last year of the study, oral and intravenous glucose tolerance and serum insulin were investigated be fore and after the training and reference periods. results of these investigations are presented i n table 111. no changes in fasting blood glucose concentrations were obtained in any group, which is in agreement with results for the whole material presented elsewhere (4). a significant improve intravenous gliucose test [vgt insulin (microunitslml) y k-value n early response 1.320.2 63213 34210 1.120. i 43 2 i7 3 2 2 10 7 4 1.420.3 1.220.2 4 1 .020. 1 103 248 392 10 1.120.1 97 248 3 3 4 5 1.120.2 0.920.1 8 4 vction, physical training and metabolic changes 87 ment in glucose tolerance (lower sum of glucose values 0-60-120 min (6) after the ingestion of glu cose) was obtained in the younger patients (less than or equal to 5 5 years) of the trained group as measured by ogt. the sum decreased from 3 5 0 t 2 0 to 294216 mg/100 ml (p<o.oi). there were no significant changes of the insulin con centrations. ivgt did not change significantly in any group and neither did the early insulin re sponse. effect of training on serum uric acid concentration the serum uric acid was measured before and after the training and the reference period. the changes were not significant. in the trained group the value before training was 6.4 t 0 . 2 and after 5.8 t 0 . 3 mgl100 ml. in the reference group the before-value was 6.0r0.4 and the after-value 5.6 k0.4 mg/100 ml. discussion it has been known for some years that physical effort of long duration reduces the tg level in the blood of healthy men (14, 15). holloszy et al. (24) studied i5 healthy middle-aged men and noted a tg reduction of 40% towards the end of a 6-month period of physical training. keul et al. (27) found that even a 4-week period of training reduced the fasting level of tg by 35% in men aged 20-30 years and by 15% in men 50-60 years old. siege1 et al. (34), however, found no significant change in the tg level on training of blind patients, while mann et al. (29) obtained increased levels after a period of physical training of healthy middle-aged men. this elevation, however, was considered to be caused by an increased intake of carbohydrates during the training period. the body weight was unchanged. most authors now agree that physical training gives a sustained reduction of the plasma tg in healthy persons, at least with a training session two or three times weekly. this would mean that one of the risk factors for the occurrence of ischaemic heart disease would be reduced con siderably (17). after training of a group of middle-aged patients with previous myocardial infarction, and excluding those who did not show any circulatory training effect, bjorntorp et al. (6) noted a significant reduc tion of the tg levels after training. however, upsala j med sci 79 88 a . bjernulfand j . boberg these authors also observed a significant decrease in body fat during training. in the present study, n o significant correlation could be established between training effect and t g reduction. neither was there any decrease in body weight during the training period. further more, in this series a limited number of 10 post myocardial infarct patients were trained and the blood samples for plasma lipid determination were taken only once 5 days after the last training ses sion. it is possible that the non-significant reduc tion of 1 1 % of the pretraining value might have been more pronounced if blood samples had been taken 2 o r 3 days after the last training session (24). an elevation of the plasma cholesterol con centration is believed t o comprise a n important risk factor for ischaemic heart disease (26). several authors have reported a reduction of t h e choles terol level after increased physical activity or train ing in healthy persons (12, 19, 29, 30), while others have found n o change in the value after a training period (20, 37). t h e plasma cholesterol concentra tion h a s been reported to b e lower after physical training even in the absence of simultaneous body weight reduction (18, 28, 34). o n training of pa tients with earlier cardiac infarction bjorntorp e t al. (6) found n o significant decrease of the plasma cholesterol, and neither w a s a n y significant reduc tion noted in the present series. bjorntorp e t al. (7) have recently made a com parison of glucose tolerance (ogt) in trained and untrained healthy middle-aged men. they found that the trained men had a significantly higher glucose tolerance and lower plasma insulin values o n oral glucose loading. furthermore these sub j e c t s achieved a lower fat cell weight and a higher removal rate of exogenous triglycerides. in the present series of patients a n improved o g t was achieved in the younger trained patients (less than or equal t o 55 years). the corresponding serum in sulin values were non-significantly lower after training. in a study of the effect of training after myocardial infarction, bjorntorp e t al. (6) have recently also found a slightly increased glucose tolerance after training (significant decrease of the 90 min value o n oral glucose loading), but especi ally noted lower plasma insulin values after ogt. improvement of i v g t after training of patients with asymptomatic glucose intolerance h a s also been achieved by others (9). i t has been postulated upsola j med sci 79 that the physical training, which among other ef fects increases the aerobic capacity of t h e skeletal muscles (8, 23), makes the trained muscle better equipped to oxidate energy-rich substrates of dif ferent kinds (both carbohydrates and lipids), elevated serum uric acid concentrations have been related t o an increased risk for ischaemic heart disease (3 i ) . after a period of physical train ing in healthy men, both reduced and unchanged serum uric acid levels have been reported (10, 1 i ) . t h e reduced levels have been explained by a n increase of the plasma volume caused by the train ing, and possibly also by a n increased excretion of uric acid. in the present series a significant in crease of the blood volume was noted (2), but there were no significant changes of the serum uric acid and thus no training effect achieved. a c k n o w l e d g e m e n t s the authors are indebted to associate professor leif wide and dr goran walldius, m.d. for advice and ana lytical help r e f e r e n c e s i . 2. 3 . 4. 5: 6. 7. 8 . berchthold, p., bjorntorp, p., gustafson, a . , lind holm, b., tibblin, g. & wilhelmsen, l.: glucose tolerance, plasma insulin and lipids in relation to adipose tissue cellularity in men after myocardial in farction. acta med scand 191:35, 1972. bergstrom, k . , bjernulf, a . & erikson, u . : work capacity and heart and blood volumes before and after physical training in male patients after myocar dial infarction. scand j rehab med (in press). bjernulf, a . : haemodynamic aspects of physical training after myocardial infarction. acta med scand, suppl. 548, 1973. bjernulf, a . , boberg, j . & froberg, s . 0.: the effect of short and prolonged exercise before and after physical training in male patients after myocardial infarction. admitted to scand j clin lab invest. bjernulf, a . & johannesson, k.: psychological as pects of physical training after myocardial infarc tion. (to be published.) bjorntorp, p., berchthold, p., grimby, g., lindholm, b., sanne, h . , tibblin, g. & wilhelmsen, l.: effects of physical training on glucose tolerance, plasma in sulin and lipids and on body composition in men after myocardial infarction. acta med scand 192: 439, 1972. bjorntorp, p., fahlen, m., grimby, g., gustafson, a , , holm, j., renstrom, p. & schersttn, t.: carbo hydrate and lipid metabolism in middle-aged, phys ically welltrained men. metabolism 21: 1037, 1972. bjorntorp, p., fahlen, m., holm, h., schersten, t. & szostak, v.: determination of succinic oxidase activity in human skeletal muscle. scand j clin lab invest 26: 145, 1970. znfar 9. boberg, j . , furberg, b. & hedstrand, h.: indices of lipid transport and serum insulin in patients with asymptomatic glucose tolerance. proc. of idf 8th congress, brussels 1973 (in press). 10. bosco, j. s . , greenleaf, j. e., kaye, r. l. & aver kin, e. g.: reduction of serum uric acid in young men during physical training. amer j cardiol 25: 46, 1970. 11. calvy, g. l . , cady, l. d., mufson, m. a,, nierman, j. & gertler, m. m.: serum lipids and enzymes. their levels after high-calori-high-fat intake and vigorous exercise regiment in marine corps recruit personnel. jama 183.87. 1963. 12. campbell, d. e.: influence of several physical activi ties on serum cholesterol concentrations in young men. j lipid res6:478, 1965. 13. carlson, l. a , : serum lipids in men after myocar dial infarction. acta med scand 167: 399, 1960. 14. carlson, l. a. & froberg, s . 0.: blood lipids and glucose levels during a ten-day period of lowcalorie intake and exercise in man. metabolism 16:624, 1967. 15. carlson, l. a. & mossfeldt, f.: acute effects of prolonged heavy exercise on the concentration of plasma lipids and lipoproteins in man. acta physiol scand 62: 5 i , 1964. 16. carlson, l. a. & rossner, s . : a methodological study of an intravenous fat tolerance test with intra lipida emulsion. scand j clin lab invest 29:271, 1972. 17. carlson, l. a. & bottiger, l . e.: ischaemic heart disease in relation to fasting value of plasma tri glycerides and cholesterol. lancet i : 865, 1972. 18. dalderup, l. m., de voogd, n., meyknecht, e. a. m. & den hartog, c . : the effects of increas ing the daily physical activity on the serum choles terol levels. nutr dieta 9: 112, 1967. 19. golding, l. a.: effects of physical training upon total serum cholesterol levels. res quarterly 32: 499, 1961. 20. goode, r. c., firstbrook, j. b . & shephard, r. j.: effects of exercise and a cholesterol-free diet on human serum lipids. canad j physiol pharmacol44: 575, 1966. 21. hjelm, m. & de verdier, c. h.: a methodological study of the enzymatic determination of glucose in blood. scand j clin lab invest /5:415, 1963. 22. hoel, p. g.: elementary statistics, 2nd ed. wiley, new york, 1967. 23. holloszy, j. 0.: biochemical adaptations in muscle. effects of exercise on mitochondria1 oxygen uptake and respiratory enzyme activity in skeletal muscle. j biol chem 242: 2278, 1967. 24. holloszy, j. o., skinner, j . s . , gelson, t. & cure ton, t. k . : effects of a six month program of endur ance exercise on the serum lipids of middle-aged men. amer j cardiol 14:753, 1964. 25. ikkos, d. & luft, r.: on the intravenous glucose tolerance test. acta endocrinol (kbn) 25:312, 1957. 26. kannel, w. b., dawber, t. r., friedman, g . d., glennon, w. e . & mcnamara, p. m.: risk factors on coronary heart disease. an evaluation of several vction, physical training and metabolic changes 89 serum lipids as predictors of coronary heart disease. the framingham study. ann intern med 61:888, 1 964. 27. keul, j . , doll, e. & haralambie, g.: freie fett sauren, glycerin, und triglyceride im arteriellen und femoralvenosen blut vor und nach einem vier wochigen korperlichen training. f'flugers arch 316: 194, 1970. 28. kilbom, a., hartley, l . h. h., saltin, b., bjure, j . , grimby, g . & astrand, i.: physical training in seden tary middle-aged and older men. i. medical evalua tion. scand j clin lab invest 24: 315, 1969. 29. mann, g. v., garrett, h. l., farhi, a., murray, h. & billings, f. t.: exercise to prevent coronary heart disease. amer j med 46: 12, 1969. 30. montoye, h. j . , van ;-fuss, w. d., brewer, w. d . , jones, e. m., ohlson, m. a,, mahoney, e. & olson, h.: the effects of exercise on blood cholesterol in middle-aged men. amer j clin nutrition 7: 139. 1959. 31. myers, a. r., epstein, f. h., dodge, h. j. & mik kelsen, w. m . : the relationship of serum uric acid to risk factors in coronary heart disease. amer j med 45:520, 1968. 32. rush, r. l., leon, l . & turrell, j.: automated simultaneous cholesterol and triglyceride determina tion on the autoanalyzer i1 instrument. in advances in automated analysis. technicon international congress 1970, vol. 1, p. 503. 33. sanne, h . , grimby, g. & wilhelmsen. l.: physical training during convalescence after myocardial in farction. in physical fitness and coronary heart disease. munksgaard, copenhagen, 1971. 34. siegel, w., blomquist, g. & mitchell, j. h.: effects of a quantitated physical training program on middle aged sedentary men. circulation 41: 19, 1970. 35. sjostrom, l., bjorntorp, p. & vrana, j . : microscopic fat cell size measurement on frozen-cut adipose tissue in comparison with automatic determination of osmiumfixed fat cells. j lipid res 12-521. 1971. 36. sobel, r . c. & kim, j.: a modified carbonate-phos photungstate method for the determination of uric acid and comparison with the spectrophotometric uricase method. amer j clin pathol 28: 152. 1957. 37. taylor, h. l., anderson, j . t. & keys, a.: studies on diet, physical activity and serum cholesterol con centration. circulation 24: 1055, 1961. 38. thorell, j. i., nosslin, b. & sterky, g.: estimation of the early insulin response to intravenous glucose injection. j lab clin med 1973 (in press). 39. wahlberg, f.: intravenous glucose tolerance in myocardial infarction, angina pectoris and inter mittent claudication. acta med scand, suppl. 453, 1966. 40. wide, l. & porath, j.: radioimmunosorbent assay of proteins with the use of sephadex coupled anti bodies. biochem biophys acta 130: 257, 1966. received october 20, 1973 address for reprints: a . bjernulf, m . d. ovansiljans halsov%rdscentral, s-792 00 mora, sweden. upsala j med sci 79 upsala j med sci 87: 201-213, 1982 serum levels of cortisol, dehydroepiandrosterone, dehydroepiandrosterone sulphate, estrone and prolactin after surgical trauma in postmenopausal women h. 0. adami’, 0. axelsson’, k. carlstrom4, j . vegelius3 and g. akerstrom’ from the departments of surgery’, obstetrics and gynaecology* and statistics3, university of uppsala and the hormone laboratory, department of obstetrics and gynaecology4, huddinge university hospitul, huddinge, sweden abstract changes in serum hormone concentrations induced by surgical trauma were studied by determination of cortisol, dehydroepiandrosterone (dha) , dehydroepiandrosterone sulphate (dhas) , estrone ( e l ) and prolactin in 25 postmenopausal women. blood samples were collected before, during and after mastectomy (14 women) and cholecystectomy (11 women). a slight peroperative increase in dha preceded a marked postoperative decrease whereas no sig nificant changes were seen concerning dhas . the posttraumatic increase in cortisol values was delayed in relation to that of dha, reaching its maximum on the f i r s t postoperative day. there was a pronounced postoperative in crease in estrone which was only slightly (r = 0.3) correlated to the con comitant changes in the serum levels of dha and cortisol indicating that other factors than increased availability of precursor steroids might influence this change. prolactin levels showed an about fourfold peroperative increase and were normalized on the f i r s t day after surgery. n o significant differences in preoperative values were seen between the groups although generally more pronounced and retarded changes were seen after cholecystectomy than after mastectomy. introduction the s t r e s s of a surgical trauma will cause changes in the serum levels of pituitary (10.18) a s well as adrenal hormones (10). in postmenopausal women the bulk of plasma estrogens originates from peripheral cotversion of adrenal steroids (13,15). in the present study the serum concentiations of cortisol, 20 1 dehydroepiandrosterone (dha) , dehydroepiandrosterone sulphate (dhas) , estrone and prolactin were measured in postmenopausal women before, during and after a surgical trauma. the purpose of the study was to estimate stress induced changes in serum concentrations of these hormones in postmenopausal women. material and methods patients this study was performed on 25 female patients. fourteen were undergoing simple o r modified radical mastectomy for primary breast carcinoma and 11 were undergoing cholecystectomy for gallstone disease. the mean age of the mastectomy group was 66 years (range 48-85) and of the cholecystectomy group 62 years (range 52-71). all patients were postmenopausal according to history and to an fsh concentration in the serum exceeding 3 ug/l (26). no women had undergone a surgical menopause. two patients in the breast cancer group were treated with glucocorticoids a t the time of s u r g e r y and were not included in the statistical calculations. they a r e reported on separately. the other patients were free from medications. / the d r u g s used for premedication and the methods for anesthesia were identical in the groups. the operations were all uncomplicated. on the day of s u r g e r y all patients were fasting with intravenous fluid therapy 2000-2500 ml glucose electrolyte solutions. all mastectomy patients had peroral feeding on the f i r s t postoperative day while 7 of the cholecystectomy patients were fasting until the second postoperative day. venous blood samples were drawn a t different times in relation to s u r g e r y as shown in table i. sample i was collected on the day of admission a t about 10 a . m . , sample i1 on the day of s u r g e r y preoperatively a t about 8 a.m. and sample i11 half an hour after the s t a r t of s u r g e r y on the average a t 1 2 a.m. (range 9 a.m.-4p.m.). samples iv-vi were collected on the f i r s t , third and fifth postoperative day, respectively, a t about 8 a.m. sample vi was not originally included in the study and was later taken only in those 7 patients with breast cancer and 9 with gallstone disease, who were still hospitalized on t h e fifth postoperative day. the last sample (vii) was taken randomly during t h e day a t the f i r s t postoperative visit to the out-patient clinic. this occurred in 2/3 of the cases 2-4 weeks after the operation and in all in stances within 12 weeks. the serum was stored a t -20' c until all samples for each patient were concomitantly analysed . 202 hormone analyses cortisol in serum (s-cortisol) was determined by a radioimmunoassay without prior extraction using a commercial kit from diagnostic products corp., los angeles, calif., u . s . a . the method uses an (1251)cortisol tracer and free and bound antigen are separated by a double antibody-polyethylene glycol precipitation technique. s-dehydroepiandrosterone (dha) was determined after ether extraction by radioimmunoassay using anti-dehydroepiandrosterone-l7-(carboxymethyl oxime) bovine serum albumin (hypolab, s . a . , coinsins, switzerland). this antibody crossreacts to 7.3% with 5-androstene-3pi 1 7 ~ d i o l and to 4.1% with 5-pregnenolone . these steroids together will thus account for approximately 3.5% of the dha values obtained ((calculated from serum steroid levels given in (1,3,4,22)). s-dehydroepiandrosterone sulphate (dhas) was measured by a modification (9) of the procedure of metcalf (16). the method includes hydrolysis of dhas by autoclaving diluted serum a t ph 4.5 a t 120' c , extraction with diethyl ether and radioimmunoassay as described for unconjugated dha . unconjugated dha will account for approximately 1% and the sulphates of 5-androstene-3pi 17b-diol and 5-pregnenolone for less than 1% of the values (calculated from 1,5,22,23)). s-estrone was measured after ether extraction by a radioimmunoassay (6) using anti-estrone-6(carboxymethyl oxime) bovine serum albumin (generously supplied by d r . gordon niswender , colorado state university, fort collins, colorado, u . s. a . ) . s-prolactin was determined using a commercial kit from kabi ab , stockholm, sweden. bound and free (1251) prolactin were separated by precipitation with polyethylene glycol. the values a r e expressed a s /ug/l of human prolactin nih v . l . s . 1. within and between assay variations were f o r cortisol 4.5 and 7.0%, for dha 5.4 and 7.1%, for dhas 8.1 and 12.1%, for estrone 4.5 and 6.2%, and f o r prolactin 7.2 and 14.6%, respectively. statistical methods all p-values refer to comparison with the f i r s t ( i ) serum sample using a two-tailed wilcoxon matched-pairs signed-ranks t e s t . the product moment correlation coefficient was used a s a measure of correlation. 203 results 1500 surgery 1500 i i 1°00 * 1000; i i i 500 500 (9) (11) cp, (11) (11) (7) (11) i 0i i i i i 1 1 time-course of serum levels the mean steroid and prolactin levels for the 2 groups a t different sampling times are given in tables 1 and 2 in figs. la-e. n o significant differences were found between the 2 groups concerning the initial levels. cortisol. (fig. l a ) . the highest mean value for cortisol was noted on the f i r s t postoperative day when it was significantly ( p < 0.05) higher than the initial values (sample i ) in both groups of patients. surg? i 4 i (10) (11) 00) (9) (11) (9) (11) i 0 1 i i i i i 1 fig. 1 a . concentration of cortisol in the serum before, during and after surgery in the mastectomy and cholecystectomy group. the mean value, sem and number of observations (within brackets) is shown for each measurement period. significant differences related t o the f i r s t value (i) a r e indicated a s *(p < 0.05). , dha. (fig. l b ) . the highest mean dha value was noted on the day of s u r g e r y , half an hour after the s t a r t of s u r g e r y , in both groups. this value was significantly (p < 0.05) higher than the initiai value in the gallstone group only, while no significant increase was noted for the breast cancer group. in both groups the mean dha values decreased postoperatively, being significantly lower than the initial values on day 3 after surgery in the breast cancer group ( p < 0.01) and on day 5 in the gallstone group (p < 0.01). 204 t ab le 1 . b re as t ca nc er g ro up . m ea n va lu es , st an d ar d d ev ia ti on s an d n um be r of ob se rv at io ns ( w it hi n br ac ke ts ) fo r co rt is ol , d h a , d h a s , e l an d pr ol ac ti n a t di ff er en t m ea su re m en t pe ri od s in r el at io n to m as te ct om y. pr eo pe ra ti ve pe ro pe ra ti ve po st op er at iv e a t ad m is si on da y of op er at io n + h ou r af te r da y 1 da y 3 da y 5 m or e th an 2 st ar t of su r w ee ks p os to p g er y ra nd om ly d ur in g h or m on e 1 0 .3 0 a .m . 0 8 .0 0 a .m . 9 a. m .4 p .m . 0 8 .0 0 am . 08 .0 0 a. m . 08 .0 0 a. m . th e da y i i1 i1 1 iv v v i v ii c or ti so l nm ol /l 59 32 22 0 d h a n m ol /l 20 21 3 (9 ) (1 2) (5 ) (1 2) (5 ) d h a s nm ol /l 29 37 k1 64 4 e i pm ol /l 15 9k 85 p ro la ct in ug /l 4. 42 22 .8 2 / 64 8k 15 6 (1 1 ) (1 1 ) 26 60 k1 46 0 (8 ) 17 0k 78 (1 1 ) 6. 44 23 .4 3 (8 1 19 21 3 44 62 26 6 (9 ) 22 21 5 (1 0) 20 83 21 16 3 (8 ) 13 0k 67 (1 0) (9 ) 25 .4 42 11 .5 4 87 02 33 5 (1 1 ) 21 +1 0 (1 2) (7 ) (1 2) 6. 78 k5 .8 6 (8 ) 20 45 21 26 0 22 6+ 98 55 0k 16 7 (1 1 ) (1 1 ) (6 ) (1 2) 6. 67 23 .3 0 (6 ) 11 25 17 93 21 11 3 14 1k 78 77 02 24 4 (7 ) 16 k1 3 (7 ) 15 73 21 28 3 (6 ) (7 ) (7 ) 14 1+ 10 4 8. 03 k3 .6 0 5 0 12 1 4 2 (1 1 ) 16 29 (1 2) (6 ) (1 2) (5 ) 27 47 21 00 7 10 72 52 7. 54 k5 .1 4 5.3 a s c '5 tco rl +i in in m in n +i -3 0 t m n rl rl w n cn w m rl +i v n co +i n 4 n m w p (d cn +i m n rl n co 0 0) +i rl in v rl n ln +i w r l n m c n m n 0, w 0 p +i w q rl n rl rl w co ln r +i lo (d co n rl rl w co n r(d rl n c o rl h l n +i 0 +i rl +i m n r l n m +i a rl c-n +i o o n o o o n o c n r l r l v c o o r l * p r l w m w ~ w c r ) ~ ~ ~ 09 n 5: w n 0 rl w m cn co +i n t u) rl n cn w co p +i co v rl n 0 rl w cn cn n rl +i fu in ln m n co w ppco rl ln n v r l m m n v +i co +i + i n r l n v +i n rt to +i d m n n rl n n c o r l ( d d n r n r l * p ~ w n w n w r l w ~ w c h z a rl w \ tn f\ c 4 4 v m 0 5.3 a .i 3 206 dha nmol/ i surgery i 4 0 1 i mastectomy i i 10 (12) (11) \lo) (12) (11) (7) (12) 0 i i i1 iii iy y yi yii dha nmol/ i cholecystectomy 40 30 20 10 0 i i1 if1 iy y yi yii fig. 1 b . concentration of dha in the serum before, during and after s u r g e r y in the mastectomy and cholecystectomy group. the mean value, sem and number of observations (within brackets) is shown for each measurement period. significant differences related to the f i r s t value (i) a r e indicated as *(p < 0.05) and **(p < 0.01). dhas. (fig. l c ) . due to lack of serum, determination of dhas was performed only on a limited number of patients. there were no significant changes in the serum levels of di-ias in any of the groups. e l . (fig. i d ) . the highest mean estrone level was noted on the f i r s t post operative day in the gallstone group ( p < 0.01) a s well a s in the breast cancer group ( p < 0.05). in the gallstone group the mean estrone level was still elevated 3 days after s u r g e r y ( p < 0.01) but after t h a t they returned to initial values. in the breast cancer group the estrone values decreased rapid ly after the f i r s t postoperative day and became lower than the initial values in the last sample. 207 4000 surgery 4000 i t i i i 3000 2000 1000g1(7) fig. 1 c. concentration of dhas in the serum before, during and after surgery in the mastectomy and cholecystectomy group. the mean value, sem and number of observations (within brackets) is shown for each measurement period. e l e l pmol / i mastectomy pmol / i cholecystectomy surgery i i i i i i i i & 191 (9) (8) (10) (9) (9) 0 , 300 200 100 0 208 surgery 300 i i 200 100 i * i i i (12) (11) (lo) (12) (12) (7) (12) (11) (11) (10) (10) (11) (9) (11) i i i i i i i 1 0’ 1 1 i i i i 1 i prolactin. (fig. l e ) . the concentration of serum prolactin was measured only on a limited number of patients due to the lack of serum, which in 5 patients gave cause for the use of sample i1 instead of sample i a s a preoperative reference. the serum levels were markedly increased during surgery (sample 111) in both groups (p < 0.01). prolac tin prolac t in u g / l mastectomy )jg / i c h olecyste ct om y 40 30 20 10 0 surgery i i i \ i \ i i1 ill iv v vi vii 40 3 0 20 10 0 surgery it a if *i fig. 1 e. concentration of prolactin in the serum before, during and after surgery in the mastectomy and cholecystectomy group. the mean value, s e m and number of observations (within brackets) is shown for each measurement period. significant differences related to the f i r s t value ( i ) a r e indicated a s * ( p < 0.05) and ** ( p < 0.01). dha and estrone values from 2 women on glucocorticoid treatment were sepa rately analysed. one of them received hydrocortisone 100 mg x 4 intra venously on the day of surgery and the f i r s t postoperative day and after that prednisolone orally in doses declining from 5 mg x 4 (day 2 ) to 5 mg x 1 (day 5). she had uniformly low dha values ( 2 . 5 3 . 8 nmol/l). the estrone levels were below the sensitivity of the method a t all measurement periods. the other woman was given hydrocortisone 100 mg x 2 intravenously on the day of surgery only and was the only one showing a definitely decreased e l value from sampling period i to iv (240.5 to 111.0 pmol/l). her dha values decreased already in sample iv. 209 correlations when values from the two groups during the postoperative period (samples iv-vii) were combined and the changes from the first to the fourth sampling period were studied by a regression analysis, a signficant correlation was found between the changes in cortisol and dha ( r = 0.83). poor correlation were however found between the changes in cortisol and estrone ( r = 0.38) and in dha and estrone (r = 0.33). uniformly lower correlations were found when the same calculations were repeated using the perioperative (sample 111) dha value instead of the f i r s t postoperative value (sample iv) or when the lowest value instead of the first one was used a s a reference. discussion i t is well known that surgical trauma, a s in the present s t u d y , causes in creased cortisol levels, secondary to an increased pituitary secretion of acth. no changes in serum cortisol were found after 30 minutes of surgery neither in the present study nor in the investigation carried out b y charters e t al. (10) but would probably have been shown with another sampling sche dule including samples taken later than 30 minutes during surgery. in view of the pattern observed for cortisol, the time-courses of another adrenocortical steroid, dha, and its sulphate dhas are interesting. maximum values for dha occurred earlier than the maximum in cortisol, i.e. 30 minutes after the s t a r t of surgery (sample 111), and this increase, although not statistically significant was more pronounced in the gallstone group. following the first postoperative day (sample iv), however, the time course of dha in serum was similar to that of cortisol in both groups. dhas was not signi ficantly changed in any of the groups. the initial discrepancies between the time courses of cortisol and dha might be due to changes in the balance between unconjugated and conjugated dha or indicate differences in the regulation of dha and cortisol. a lot of evi dence have been accumulated f o r the existence of an "adrenal androgen sti mulating hormone" o r 9-eticulotrophin" differing from acth, which selectively stimulates the adrenal dha synthesis (14). the nature of this hormone ( s ) is not known. its action seems to require a sufficient acth stimulation of the adrenal cortex. prolactin, being increased during surgery (18) has been suggested as the adrenal androgen stimulating hormone ( 1 6 ~ 1 ) . we found highly increased prolactin levels during surgery and this increase was more pronounced in the gallstone group. the role of prolactin has, however, 210 been seriously questioned in this respect (14). other protein hormones such a s hgh, fsh, lh and tsh have also been proposed a s possible adrenal androgen stimulating hormones. increased levels of these hormones during surgery have been reported (2,10), but they seem t o be even less possible than prolactin in an adrenal stimulating role (14). charters e t al. (10) found maximum cortisol values after 2 3 hours of surgery. i t is very likely that maximum dha values also occur a t this time, since dha follows closely t o cortisol in acth stimulation tests (7,21). a t the first postoperative day charters e t al. (10) noticed still significantly elevated cortisol values. in the present study cortisol but not dha, was significantly elevated on this day. one explanation f o r this discrepancy may be the shorter biological half-life for dha, i.e. 1 2 hours, compared' with 3 hours for cortisol (7,12,19). from the f i r s t postoperative day the time courses for cortisol and dha were rather similar indicating a common regulator of the 2 steroids a t this time, i . e . acth. significantly decreased dha values were noted on the third day after s u r g e r y (sample v) in the breast cancer group and on the fifth day (sample vi) in the gallstone group. a tendency towards lower dhas values was also noted in these samples. one may speculate over a negative rebound effect of the previously increased cortisol levels upon the adrenal androgen biosynthesis . decreased values for dhas a s well a s for another adrenocortical androgen, androstenedione , following mastectomy have previously been reported by wang e t al. (24,25). maximum estrone levels were observed on the f i r s t postoperative day con comitantly with the maximum cortisol values. already in 1959 brown (8) showed that administration of acth caused an increase in urinary estrogens in oophorectomized women. i t is well known (17,20) that acth stimulation increases estrone production in postmenopausal women. thus, the maximum estrone levels on the first postoperative day may be due to an acth induced increase in the production of adrenal estrone precursors, notably andro stenedione. however, i t should be pointed out that acth has been reported to cause changes in the balance between unconjugated and sulphoconjugated steroids (11) , notably by increasing sulphatase activity. the low degree of correlation between the increase of the presumed precursor dha and estrone might be due to a change in the conversion rate a s a function of the precursor concentration as suggested by vermeulen and verdonck ( 2 2 ) . 21 1 one woman on continuous glucocorticoid treatment showed no changes a t all in her serum levels of dha and estrone. another woman who got glucocorticoid injections only on the day of surgery showed a definite decrease of the serum levels of dha, and estrone on her f i r s t postoperative day; a result strongly pointing to the important role the adrenals a r e playing for the formation of estrone in postmenopausal women. the more pronounced changes in the serum hormone levels observed in the gallstone group (tables 1 + 2, figs. 1) could be due to the greater surgical trauma experienced by these patients. the reason, however, for the observed rise of estrone already in sample i1 in the gallstone group remains obscure. the presence of a preoperative s t r e s s a s an explanation is not supported by any other hormonal changes. besides the previously known influence of s t r e s s and surgical trauma on the pituitary adrenal axis, the present results also indicate effects upon the steroid balance which a r e not related to the action of acth upon the adrenocortical de novo steroid biosynthesis. acknowledgement this study was supported by the swedish medical research council (3495). references 1. 2 . 3. 4. 5. 6. 7. 212 abraham, g. & marculis, g . b . : effect of exogenous estrogen on serum pregnenolone , cortisol and androgens in postmenopausal women. obstet gynecol 5: 271, 1975. adami, h . 0 . johansson, h . thorcn, l . wide, l. & akerstrom, g.: serum levels of tsh, t3, rt3 and t3-recine uptake in surgical trauma. acta endocrinol (kbh) 88: 482, 1978. anderson, d.c. & yen, s.s.c.: effects of estrogens on adrenal 3 p h y d r o x y steroid dehydrogenase in ovariectomized women. j clin endocrinol metab 43: 561, 1976. anderson, d.c. child, d.f. & bu'lock, d.e.: adrenal steroids in hirsutism and related conditions. in: (ed: james, v.h.t. serio, m. giusti, g. & martini, l . (eds). the endocrine function of the human adrenal cortex. proc serono symp, vol. 18: pp. 301 324. academic press, london, new york, san francisco, 1978. aso, t . aedo, a . r . & cekan, s . z . : simultaneous determination of the sulphates of dehydroepiandrosterone and pregnenolone in plasma by radioimmunoassay following a rapid solvolysis . j steroid biochem 8: 1105, 1977. axelsson, 0. englund, d.e. luukkainen, t . & johansson, e.d.b.: a radioimmunoassay of oestrone in plasma. plasma levels of oestrone and oestradiol in oophorectomized rhesus monkeys during treatment with subcutaneous implants containing oestrone. acta endocrinol (kbh) 87 : 609, 1978. brody, s . carlstrom, k. lagrelius, a . lunell, n . 0 . & rosenborg, l . : adrenal steroid synthesis and bone mineral density in postmenopausal women. submitted for publication 1981. 8. 9. 10. 11. 1 2 . 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. brown, j . b . falconer, c.w.a. & strong, j . a . : urinary oestrogens of adrenal origin in women with breast cancer. j endocrinol 19: 52, 1959. carlstrom, k . damber, m.-g. furuhjelm, m . joelsson, i . lunell, n . 0 . & von schoultz, b. : serum levels of total dehydroepiandrosterone and total estrone in postmenopausal women with special regard to carcinoma of the uterine corpus. acta obstet gynecol scand 58: 179, 1979. charters, a.c. odell, w.d. & thompson, j.c. : anterior pituitary function during surgical s t r e s s and convalescence. radioimmunoassay measurements of blood, tsh, lh, fsh and growth hormone. j clin endocrinol metab 29: 63, 1969. dominguez, o.v. loza, c . a . moran, l . z . & valencia, a.s. : acth and sulfatase activity. j steroid biochem 5: 867, 1975. gallagher, t . f . : steroid hormone metabolism and the control of adrenal secretion. harvey lectures 52: 1 , 1958. grodin, j . m . siiteri, p.k. & mcdonald, p.c. : source of estrogen pro duction in postmenopausal women. j clin endocrinol metab 36: 207, 1973. grumbach, m . m . richard, g . e . conte, f.a. & kaplan, s . l . : clinical disorders of adrenal function in puberty: an assessment of the role of the adrenal cortex in normal and abnormal puberty in man and evidence for an acth-like pituitary adrenal androgen stimulating hormone. (ed: james, v.h.t. serio, m . giusti, g. & martini, l . ) in the endocrine function of the human adrenal cortex. proc serono symp, vol 18: 583 612. academic press, london, new york, san francisco, 1978, p p . longcope, c. : metabolic clearance and blood production rates of estro gens in postmenopausal women. am j obstet gynecol 111: 778, 1971. metcalf, m . g. : dehydroepiandrosterone sulfate in plasma: hydrolysis, extraction and radioimmunoassay . steroids 28: 311, 1976. murakami, t . yamaji, t . & ohsawa, n . : the effect of acth adminis tration on serum estrogens, lh and fsh in the aged. j clin endocrinol metab 42: 88, 1976. noel, g.l. s u h , h.k. stone, g. j . & frantz, a.g. : human prolactin and growth hormone release during s u r g e r y and other conditions of s t r e s s . j clin endocrinol metab 35: 840, 1972. rosenfeld, r.s. rosenberg, b . j . fukushima, d . k . & hellman, l . : 24-hour secretory pattern of dehydroisoandrosterone and dehydro iso androsterone sulfate. j clin endocrinol metab 40: 850, 1975. vermeulen, a . : the hormonal activity of the postmenopausal ovary. j clin endocrinol metab 42: 247, 1976. vermeulen, a , & ando, s. : prolactin and adrenal androgen secretion. clin endocrinol 8: 295, 1978. vermeulen, a . & verdonck, l . : factors affecting s e x hormone levels in postmenopausal women. j steroid biochem 11 : 899, 1979. vihko, r . : gas chromatographic-mass spectrometric studies on solvo lyzable steroids in human plasma. acta endocrinol (kbh), suppl 109, 1966. wang, d . y . bulbrook, r.d. herian, m . & hayward, j . l . : studies on the sulphate esters of dehydroepiandrosterone and androsterone in the blood of women with breast cancer. europ j cancer 10: 447, 1974. wang, d . y . bulbrook, r . d .& hayward, j . l . : plasma androstenedione levels in women with breast cancer. europ j cancer 13: 187, 1977. wide, l. nillius, s.j. gemzell, c.a. & roos, p.: radioimmunosorbent assay of follicle-stimulating hormone and luteinizing hormone in serum and urine from man and women. acta endocrinol (kbh) suppl 174, 1973. 583-612. address for reprints: d r . ove axelsson department of obstetrics and gynaecology university hospital s-750 14 uppsala sweden 213 upsala j med sci 92: 37-45, 1987 endorphin activity in cerebrospinal fluid prior to elective cesarean section and in early puerperium sven lyrenas,' fred n ~ b e r g , ~ gunilla willdeck-lund,' leif l i n d ~ t r o m , ~ bo lindberg' and lars terenius4 'departments o obstetrics and gynecology and 'anesthesiology, university hospital, uppsala, fpsychiatjic research center, and 4department of pharmacology, university of uppsala, uppsala, sweden abstract opioid activity in cerebrospinal fluid (csf) was estimated by radioreceptor assay (rra) in samples obtained from ten women a t term pregnancy and in the early puerperal period. the samples were fractioned on sephadex g-10 columns and two opioid receptor active fractions, fi and fii, were recovered. two pools of fii materials from pregnant and puerperal women, respectively, were further analyzed by electrophoresis and the concentrations of opioid activity were measured by radioreceptorassay. there was a significant rise in receptor-assayed fii opioid activity in late pregnancy a s well a s in the early puerperal period compared to that of healthy, nonpregnant, nonpuerperal females. pooled fii material obtained before delivery could be separated into two major components tentatively assigned the hexapep tide [metlenkephalin-lys6 and the heptapeptide [metlenkephalin-arg6-phe7. these two opioid peptides both have their origin in the [metlenkephalin precur s o r , proenkephalin a . in the puerperal period there was predominance of only one of these components. @ introduction the known number of endogenous opioid peptides (endorphins) which may act as ligands to opiate receptors has increased rapidly in the past few years. by the use of molecular genetics, the amino acid sequences of three different pep tide precursors to these peptides have been predicted. the f i r s t , proopio melanocortin, is the precursor of p-endorphin ( 1 3 ) . the other two a r e proenkephalin a (1, 6 , 1 2 ) and proenkephalin b ( l o ) , precursors to enkephalins and dynorphins , respectively. thus, there a r e three families of endorphins 37 which show affinity in varying degree t o different types of opioid receptors. endogenous opioids have been reported t o provide pain relief in labour. thus oyama et al. in 1980 (18) described complete analgesia in 1 4 women who had received 1 mg of p-endorphin by intrathecal injection in the early stage of labour. a gradual rise in the pain threshold for electric stimuli during the final week prior to parturition has also been registered in r a t experiments ( 4 ) . the pain threshold was rapidly normalized following delivery and it was also abolished in naltrexone treated pregnant animals. prenatal naloxone also affected morphine sensitivity of the off-spring (7). in the present investigation a screening of cerebrospinal fluid (csf) sub stances with opioid receptor affinity was conducted in late pregnancy and in the puerperal period. csf samples were obtained near the end of the third trimester before onset of labour and also during the early puerperal period in the same women. the aim of the study was to establish whether the amounts of endorphins in csf a r e increased in the final stage of pregnancy and if such an increase could be related to any of the three opioid peptide precursors. material and methods the study included two groups of women. 1) one group was composed of 10 healthy pregnant women (mean age 30 years, range 22-37) who underwent elective cesarean section a t term due t o pelvic disproportion. the women had all volunteered for the study. there were no drugs given prior to delivery. the operations were per formed during spinal anesthesia and prior to administration of the anesthetic d r u g , a 5 ml sample of csf was removed through a 25 gauge needle. the cesarean sections were all performed between 10 and 1 2 am and the anesthetic used was 50-75 mg of xylocain@. a second lumbar puncture was done one week post partum when another 5 ml of csf was sampled. 2) the control group was composed of 16 nonpregnant, nonpuerperal healthy females who volunteered for the s t u d y . the mean age of this group was 26.1 years (range 18-35). samples of 5 ml csf were obtained from each woman with the same technique as for the women in group 1 . the investigation was approved by the ethics committee of the medical the lumbar punctures were done at the level of l:3-l:4 with the woman in a faculty. 38 sitting position. the average collection time was 15 minutes. csf samples were taken in plastic tubes, frozen and kept a t -7ooc until analyzed. biochemical methods the standard peptides [metlenkephalin and [metlenkephalin-arg6-phe7 were obtained from bachem, bubendorf , switzerland, whereas dynorphinl-8 and [met] enkephalin-lys6 were supplied by peninsula laboratories, san carlos, usa. equipment for gel filtration (sephadex@ g-10) and the agarose (agarose-c) used in the electrophoretic separation were obtained from pharmacia fine chemicals, uppsala , sweden. the opioid activity in each sample was analyzed according to terenius and wahlstrom (20). in this method the opioid active material is separated into two major fractions called fi and fii, by chromatography on sephadex g-10 columns. these fractions a r e further tested for receptor-binding affinity in a radioreceptorassay (rra) , which uses synaptic plasma membranes from r a t brain excluding cerebellum with 3h-labelled dihydromorphine as a competing radio ligand. each assay included a calibration curve with [metlenkephalin, and the binding activity of the tested fractions was expressed in [met] enkephalin equivalents. the biochemical characteristics of these fractions have been studied extensively in this laboratory (15, 16, 17). the contents of fraction i include hydrophilic peptides with eight or more amino acids such a s dynorphin a ( d y n ~ r p h i n ~ ~ ~ ) and its shorter fragments (i. e . dynorphinl-8) while fraction ji contains enkephalin peptides with six to eight amino acids including [met] enkephalin-lys6 and [met] enkephalin-arg6-phe7. column electrophoresis in agarose suspension of pooled fii material was performed according to nyberg and terenius (16). for this procedure, the freeze-dried fii materials from the ten pregnant women were combined into two pools, one consisting of material collected in term pregnancy before cesarean section and one consisting of fii material from samples collected in the early puerperal period. the duration of each electrophoresis was five hours a t a current of 10 ma and a voltage of 1,000 v. fractions of 0 . 4 ml were then suctioned from the column and separated from agarose by the use of a centri fuge. the distribution of the endorphin activity was monitored by radioreceptor assay. calibration of the column had been performed earlier with samples of known enkephalinand dynorphin-related peptides . 39 results the csf levels of fi and fii in the cesarean section group and in the control group a r e shown in figs. 1 and 2 . in these patients, no general in crease was found in fi either a t the time of cesarean section or in the early puerperium in comparison with the control group. f i i , on the other hand, was significantly increased both before cesarean section and in the early puerperium compared to the control group (pc0.01 and p<o.ol, respectively, student's t-test on independent samples; p<o.ol and p<o.ol, mood's median t e s t ) . there was no significant change in fii levels in the early puerperium compared t o those in late pregnancy (t-test on paired samples; mood's median t e s t ) . twg women (no. 1 and no. 7) had very high fii levels a t the time of cesarean section. following delivery this fraction decreased. in one of the women (no. 1) a similar pattern was found for fi while for the other woman fi was not elevated. 9.\ \ i controls pregnant after fraction i mivery fig. 1 s e c t i o n ( o ) , i n e a r l y puerperium ( o ) , and i n 16 h e a l t h y female c o n t r o l s (x). csf l e v e l s of f r a c t i o n i o p i o i d p e p t i d e s i n 10 women p r i o r t o cesarean 40 after column electrophoresis of the pooled fii material from samples in late pregnancy, the opioid activity could be separated mainly into two compo nents. fig. 3a shows the levels of these components in the group of ten women prior to cesarean section. in the puerperal period there was a predominance of only one of these components (fig. 3b). 20 i 4 10 m x x x x p i i 0 ;& 3 controls pregnant after delivery fractlon i 1 fig. 2 csf l e v e l s of f r a c t i o n i1 o p i o i d p e p t i d e s i n 10 women p r i o r t o cesarean s e c t i o n ( o ) , i n e a r l y puerperiwn ( o ) , and i n 1 6 h e a l t h y female c o n t r o l s (x). discussion there a r e two principal methods to measure opioid peptides in body fluids: by radioreceptorassay ( r r a ) and radioimmunoassay (ria). ria is the most commonly used method when analyzing endorphins in body fluids. the major advantage of the ria is that i t will give information about individual opioid peptides while r r a determinations a r e based on functional opioid activity. though r r a does not distinguish between individual endorphins i t will, however, give a more complete information about the total amount of endorphin 41 1. [med enkephalin i 2. [med enkephalin-arg6-phe7 l . 2 0 30 40 l o 10 '1 1 20 30 40 fraction number fig. 3 column electrophoresis (ph 2 . 7 ) in agarose suspension of pooled csf fraction 11 opioid peptides from 10 women: a) in term pregnancy prior to elec tive cesarean section and b) one week following delivery. fractions were isolated and subjected to radioreceptorassay. for comparison, runs of some known opioid peptides are also shown in the figure. activity. i t has earlier been shown in this laboratory by nyberg and terenius (15) that classical endorphins such a s enkephalin , p-endorphin and dynorphin contribute less than 10 per cent of the total receptor activity in csf. the activity of the endocrine system during pregnancy and in labour has been studied by several investigators. the results reported on @-endorphin and @-endorphin-like peptides in maternal plasma during pregnancy and delivery are 42 rather conflicting. goland et al. (5) found no difference in p-endorphin plasma concentrations in either the f i r s t , second or third trimester in comparison with values in nonpregnant women. genazzani e t al. ( 3 ) found progressively in creased p-endorphin plasma levels from the 27th week of gestation and the highest concentration a t term. newnhamn e t al. (14) reported a pattern similar to that of genazzani but also a progressive rise even for p-lipotrophin. newn ham's group also reported unchanged levels of maternal plasma [met] enkephalin during pregnancy. on the other hand, hoffman et al. (8) found significantly lower plasma levels of p-endorphin in each trimester of gestation than in the levels of nonpregnant control subjects. much less is known about opioid peptide secretion within the cns and reports on endorphins in csf during pregnancy a r e r a r e . datta e t al. ( 2 ) found no differences in csf levels of immunoreactive p-endorphin between nonpregnant women and women a t term prior t o elective cesarean section. in fact, the csf levels showed no elevation a t term after active labour f o r 6 to 8 hours. p-endorphin is synthesized both from the anterior lobe of the pituitary gland and from the brain. schlachter e t al. (19) found significant amounts of this peptide in csf, b u t not in plasma, five days after complete hypophysectomy in 13 patients. no correlation between the concentrations of p-endorphin in plasma and in csf has been found (2, 19). the present study provides evidence that there is an increased amount of substances in csf with opioid receptor affinity in late pregnancy as well a s in early puerperium compared to those found in nonpregnant, nonpuerperal women. a t the lumbar level we found that most of opioid receptor-active material in fii could be traced to proenkephalin a which is the [metlenkephalin precursor. since enkephalins seem to be the main opioid peptides in the spinal cord (9) this is not unexpected. there were, however, no signs of increased levels of the pentapeptides [metlenkephalin or [leulenkephalin in the csf samples. instead, electrophoresis gave evidence of other forms of enkephalin-containing polypeptides (ecps) , tentatively assigned the hexapeptide [metlenkephalin-lys6 and the heptapeptide [metlenkephalin-arg6-phe7. kofinas e t al. (11) found that vaginally delivered women had significantly higher plasma p-endorphin levels than those women delivered by cesarean section. thus, s t r e s s caused by cesarean section does not seem to elevate circulating amounts of this opioid peptide. our finding that there is a moderate fii increase in late pregnancy before elective cesarean section is most likely not only an effect of s t r e s s in connection with the operation. the role of endorphins in the physiology of pregnancy is a t present unclear. the differences in csf levels of some endorphins before and after delivery 43 found in this study indicate that endogenous opioid peptides may play a role in these events also in humans. further studies a r e needed in order to elucidate the importance of the different opioid peptides for the delivery and the adaptation of labour pains in humans. such studies are under progress. acknowledgements the authors are grateful to m s inga hansson for skillful technical assistance. this work was supported by syskonen svensson's fond and the swedish medical research council, grant no. 5095 ( l . lindstrom) . references 1. 2. 3. 4. 5 . 6. 7. 8. 9. 10. 11 12. 13. 14. 15. 44 comb, m., seeburg, p.h., adelman, j., eiden, l. & herbert, e.: primary structure of the human metand leu-enkephalin precursor and its mrna. nature 295: 663-666, 1982. datta, s . , steinbrook, r.a., carr, d.b., naulty, j.s. & lee, c.: plasma and cerebrospinal fluid endorphin levels during pregnancy. anaesthesiology genazzani, a.r., facchinetti, f., & parrini, d.: $-lipotrophin and $-endorphin plasma levels during pregnancy. clin endocrinol 14: 409-418, 1981. gintzler, a.r.: endorphin-mediated increases in pain threshold during pregnancy. science 210: 193-195, 1980. goland, r.s., wardlaw, s.l., stark, r.i. & frantz, a.g. : human plasma $-endorphin during pregnancy, labor and delivery. j clin endocrinol metab gubler, u., seeburg, p., hoffman, b.j., gage, p.l. & udenfriend, s.: molecular cloning establishes proenkephalin as precursor of enkephalin containing peptides. nature 295: 206-208, 1982. hetta, j. & terenius, l.: prenatal naloxone affects survival and morphine sensitivity of rat offspring. neuroscience letters 16: 323-327, 1980. hoffman, d.i., abboud, t.k., haase, h., hung, t. & goebelsmann, u.: plasma $-endorphin concentrations prior to and during pregnancy, in labor and after delivery. am j obstet gynecol 150: 492-496, 1984. hokfelt, t., johansson, o., ljungdahl, i . , lundberg, j.m. and schultz berg, m.: peptidergic neurons. nature 284: 515-521, 1980. kakidani, h., furutani, y., takahashi, h., noda, m., morboto, y., hirose, t., asai, m., inayama, s., nakanishi, s. & numa, s . : cloning and sequence analysis of cdna for porcine $-neo-endorphin/dynorphin precursor. nature 298: 245-249, 1982. kofinas, g.d., kofinas, a.d. & tavakoli, f.m.: maternal and fetal f3-endorphin release in response to the stress of labor and delivery. am j obstet gynecol 152: 56-59, 1985. noda, m., furutani, y., takahashi, h., toyosato, m., hirose, t., inayama, s., nakanishi, s. & numa, s. : cloning and sequence analysis of cdna for bovine adrenal preproenkephalin. nature 295: 202-206, 1982. nakanishi, s., inoue, a., kita, t., nakamura, m., chang, a.y.c., cohen, s.n. & numa, s.: nucleoti.de sequence of cloned cdna for bovine c o r t i c o t r o p i n f 3 l i p o t r o p i n precursor. nature 278: 423-427, 1979. newnhamn, j.p., tomlin, s., ratter, s.j., bourne, g.l. & rees, l.h.: endogenous opioid peptides in pregnancy. br j obstet gynaecol 90: 535-538, 1983. nyberg, f. & terenius, l.: endorphins in human cerebrospinal fluid. life sci 31: 1737-1740, 1982. 57: a380, 1982. 52: 74-78, 1981. 16. nyberg, f. & terenius, l.: electrophoresis of opioid peptides on columns with agarose suspension. analyt biochem 134: 245-253, 1983. 17. nyberg, f., wahlstrom, a., sjolund, b. & terenius, l.: characterization of electrophoretically separable endorphins in human csf. brain res 259: 18. oyama, t., matsuki, a., taneichi, t., ling, n., & guillemin, r.: $-endorphin in obstetric analgesia. am j obstet gynecol 137: 613-616, 1980. 19. schlachter, l.b., wardlaw, s.l., tindall, g. & frantz, a.g.: persistence of $-endorphin in human cerebrospinal fluid after hypophysectomy. j clin endocrinol metab 57: 221-224, 1983. 20. terenius, l. & wahlstrom, a.: morphine-like ligand for opiate receptors in human csf. life sci 16: 1759-1764, 1975. 267-274, 1983. address f o r reprints sven lyrenas, m . d . department of obstetrics & gynecology akademiska sjukhuset s-751 85 uppsala, sweden 45 upsala j med sci 87: 163-174, 1982 oxygen tension alterations in the intervertebral disc as a response to changes in the arterial blood sten holm’ and gunnar selstam’ from the department of orthopaedic surgery i , sahlgren hospital’, giiteborg and the department of physiology, university of urneii2, urneb, sweden abstract measurements of oxygen tension in the canine lumbar intervertebral disc, by the use of a polarographic oxygen electrode, were performed. the oxygen tension in the arterial blood was changed by regulating the oxygen concen tration in the inspired air. the alterations in oxygen tension in the nucleus pulposus, as a function of distance to the vertebral endplate were determined. the response times registered in the disc matrix were relatively short (with in five min.), which implies an efficient solute transport from the vertebral blood pool underneath the hyaline cartilage into the avascular intervertebral disc. introduction the largest avascular tissue in the body is the disc between the vertebrae. the intervertebral disc is a specialized connective structure designed to give strength and mobility. for its normal function it must possess visco-elastic properties. metabolic and/or mechanical changes are liable to interfere with properties that are needed for equalization and absorption of the various stresses put upon the vertebral column ( 1 3 , 16). as the intervertebral discs are large and avascular the question has therefore been asked whether or not diffusion alone could adequately supply nutrients to the cells in various parts of the disc ( 1 1 , 12). solute gradients (sulphate, oxygen, and lactate) in various places in the i63 disc have recently been measured and shown to be steep and very sensitive to the distance from the endplates and partially, but to a lesser extent (in the central disc), dependent on the distance from the periphery of the annulus fibrosus (5, 1 7 ) . despite the relatively low partial pressures of oxygen in the central area of the disc, an aerobic metabolism was found and thus a continuous supply of oxygen is needed ( 4 , 5 ) . vascular blood pools are present both underneath the cartilaginous endplate as well as in the area adjacent to the annulus fibrosus ( 1 7 , 18). nourishing solutes can be principally supplied to the disc via two main routes, namely through the periphery of the surrounding annulus fibrosus and through the central portion of the vertebral endplate (2, 12, 1 6 , 1 8 ) . it must, however, be taken into consideration that there may be variations in the effectiveness of the important diffusion routes due to the characteristics of the solute itself, as well as the transport properties due to the degree of degeneration of the disc system ( 1 3 ) . in the peripheral parts of annulus fibrosus it is reasonable to believe that the disc cells are supplied with oxygen from the capillaries outside the annulus periphery with perhaps a small, additional contribution from the vertebral endplate, although the latter pathway has been shown to give almost zero diffusion in experiments performed using the sulphate ion as a tracer ( 1 7 ) . the nucleus pulposus as well as the inner part of the annulus are, however, supplied with solutes by transport mainly through the vertebral endplate. in an avascular structure as the disc, the distances from nourishing fluids to the central parts of the tissue are very long ( 5 ) . theoretical calculations based upon adequate oxygen consumption rates show that the oxygen needed for cell metabolism available in the central canine nucleus pulposus was predominately diffusing through the vertebral endplates (5, 16). these calculations also give indications that oxygen, an uncharged little molecule in contrast to large and especially negatively charged complexes, can 164 diffuse into the central parts of the disc within minutes. the purpose of the experiment reported here was to study, for the first time, changes under in vivo conditions intradiscal oxygen partial pressure when the arterial oxygen partial pressure was altered. materials and methods measurements were taken in lumbar discs ( l 3 l6) from seven adult labrador dogs ( 3 5 years old). anaesthesia with pentothal sodium (abbott, italy) was injected intravenously (30 mg/kg body weight) and maintained during the operation. all animals were intubated and ventilated with an engstroem respirator. the ventilation rate was kept constant throughout the experiment and was 16 strokes per min. the stroke volume (ranging from 4 6 litres) was chosen to give normal values for the arterial oxygen tension and the partial pressure of co arterial oxygen tensions were obtained by changing the partial pressure of oxygen in the inspired gas, keeping the stroke volume and the respiratory rate constant. when the animals were ventilated with room air. the different 2 the femoral artery was cannulated with a pe 120 catheter, which was used for blood sampling. blood pressure was controlled regularly, using a pressure transducer (elema, emt-35 solna, sweden). the blood samples were taken and analysed immediately in an automatic bloodgas analysator (abl-1, radiometer, copenhagen). the oxygen probe used consisted of a modified polarographic electrode protected by a stainless steel cannula (1, 4 ) . the electrode had an overall diameter of 1.0 millimeter and a thin rubber modified polystyrene membrane was dipcoated onto it ( 4 , 15). the electrode was bathed just prior to each run in warmed ( 3 7 o c ) oxygen-saturated saline, as well as in solutions equili brated with different oxygen concentrations. these solutions served as cali bration tensions for the electrode (14). the arterial oxygen tension was also checked with the electrode used for intradiscal measurements ( 8 ) . for zero oxygen tension calibration, pure 165 nitrogen in saline, contained in specially designed test-tubes to prevent oxygen interface, was used. calibration checks were immediately done ere ceeding and following each introduction into either the blood or the inter vertebral disc. an abdominal midline incision was made and by carefully performed surgery, in order to prevent bleeding, the lumbar region of the spine was approached. the ventral aspects of the lumbar ( l 3 l6) discs were freed by blunt dis section and under careful haemostasis. the probe attached to the specially designed electrode holder of the manipulator was introduced into the nucleus pulposus of the disc. after allowing a few minutes for equilibration to steady state, oxygen tensions were registered. only one introduction per disc was made in order not to change the matrix properties and disturb the cells. by means of a modified horsley-clark micromanipulator, which was attached to a mechanical support, it was possible to reach any position with the probe in the disc via the exposed annulus periphery of the vertebral column. the position and angle of the electrode could, at any time, be registered on the scales of the instrument. verification of the measuring point was also made by means of x-ray pictures. the position of the electrode could with these methods be determined with an accuracy of 2 0 . 4 millimeters. results the effect of changes in the arterial oxygen tension of the disc tissue is shown in fig. 1. changes in the arterial oxygen tension, after each change of oxygen concentration in the inspired gas, were noted almost immediately (within 10 seconds). as can be seen from this figure, changes in the arterial oxygen tension caused changes in the intradiscal oxygen tension. these changes occurred within minutes (response time) (fig. 2). in order to compare the different experiments, the first oxygen tension response "the response time" is defined as the time when the first significant increase in intra discal oxygen tension was noted. the time for reaching equilibrium "equilibrium time" was determined when no changes in oxygen tension were 166 noticed over a period of at least 5 minutes. 10.0 i 2 6 1 2 3 l 5 7 8 time i hours i fig. 1 . schematic presentation of results from a typical experiment showing oxygen tension changes in the arterial blood and in the intervertebral disc (nucleus pulposus) . seven experiments, where the distance from the electrode to the vertebral endplate was varied, but otherwise carried out in an identical fashion, are summarized in table 1 . the distance to the vertebral endplate from the measuring point ranged from 0.4 to 2 . 1 millimeters. the response time never exceeded 5 min. the response time, plotted as a function of the distance from the hyaline cartilage to the tip of the electrode is shown in fig. 3. the response times were longer the longer the distance from the endplate. the time for reaching oxygen equilibrium varied from approximately 15 to 145 min. (table 1 ) . there were longer equilibrium times when the electrode was situated further away from the endplate. the longest equilibrium times were seen when maximal alterations were performed, i.e. when altering ventilation from the highest oxygen concentration level to a normal state (air). 167 table 1 . oxygen tension measured in the arterial blood and in the interverte bra1 disc both initially and after alterations of the oxygen tension in the respirating air. response time and approximate equilibrium time at various distances from the endplate are also included. 0 . 8 1 . 1 1 . 3 1 . 5 1 . 6 2.1 distance oxygen tension to changes in endplate respirating air (mm) (kpa) basal levels alteration i i1 i11 iv 1 ) 1 1 1 1 0 . 4 basal levels alteration i i1 111 iv 1 1 11 1 1 basal levels alteration i i1 i11 iv $ 1 1 1 1 1 basal levels alteration i i1 i11 iv ( 1 $ 1 11 basal levels alteration i i1 111 iv ' 1 1' 1' basal levels alteration i i1 111 iv ' 1 1' 1' basal levels alteration i i1 i11 iv 11 1' oxygen tension (kpa) blood 12.7 30.0 51.7 70.3 13.1 13.9 35.0 5 5 . 4 75.6 14.1 12.9 28.7 5 0 . i 68.9 13.0 11.9 32.7 5 0 . 9 72.4 12.1 12.8 3 2 . 3 5 2 . 8 72.1 13.2 12.4 33.4 53.1 74.8 12.6 13.6 30.9 51.2 7 2 . 0 13.7 disc response time in disc (mid approx. eq time in disc (min) 1.9 3 . 9 5.8 7 . 3 2 . o 1 . 5 3 . 6 5 . 8 7 . 8 1.6 1 .o 3 . 5 4 . 8 6 . 2 1 .o 0 . 9 3 . 3 5 . 2 6 . 8 0 . 9 0 . 8 3 . 7 4 . 9 6 . 3 0 . 9 0 . 9 3.1 4.9 6 . 4 1 . o 0 . 6 3.1 4 . 4 5 . 9 0 . 7 1.6 2.0 2.4 3.1 2 . 1 2 . 3 2.8 3.6 2 . 3 2 . 7 3 . 1 4.1 2 . 6 3 . 0 3 . 3 4 . 3 3.1 3.4 3.6 4 . 3 3 . 3 3 . 4 3 . 8 4.5 3 . 8 3.7 4.1 4 . 7 15 2 0 15 70 30 30 40 100 40 35 35 95 50 45 45 90 85 60 65 110 95 105 95 100 135 85 105 145 168 5 0 l o . . . . . . . . 5 5 5'u*. 4 8 4 2 0 2 4 6 b 10 l 2 0 2 . . . . l 6 b 10 time i rnl" 1 fig. 2. enlarged views of areas marked in fig. l . , showing intradiscal oxygen tension before and after the alterations of the oxygen tension in the respiring air. from this and similar plotted graphs the corresponding response times have been obtained. fig. 3. response time as a function of distance from the hyaline cartilage of the vertebral endplate in the region of the nucleus pulposus. the roman numerals correspond to the altera tions in the respirating air. discussion the results clearly indicated that within minutes there was an obvious change in the intradiscal oxygen tension. the response time (2 4 min.) must be considered to be short in this avascular system, taking into account the distance from the nearest blood pool to be between 0.5 and 2.2 millimeters. an explanation for this very quick response could be that oxygen transport 111-822857 169 is more efficient through a cartilaginous tissue such as the intervertebral disc, under in vivo conditions, than when incubating excised specimens in vitro. a very important factor in the present situation is that there will be no blockage of blood capillaries underneath the hyaline cartilage, and therefore, the exchange of solutes between tissue and body fluids will not, from this point of view, be specially restricted. other factors, which may affect the oxygen response (and probably the solute gradients) in an avascular structure could be cell distribution (disturbed cell function i.e. shock effects) and derangements of the tissue constituents when introducing the electrode. previous studies indicate, however, that the latter factor is not of significant importance ( 5 ) . the results of the intradiscal oxygen tension measurements obtained in this investigation show low values from the start at a norma1,resting respira tion rate, thus increasing when the arterial oxygen tension was increased. that the changes in intradiscal oxygen tensions were due to a leakage of oxygen along the electrode needle is most unlikely, since very small arterial oxygen tension increases immediately gave intraaiscal responses. it cannot be fully excluded that such a leakage could, however, cause a constant but very small error when measuring during resting conditions for longer periods of time. the equilibrium times found in this investigation are in agreement with those found for cartilage, consideringrdihusion from zme side (one endplate in the disc system) ( 1 1 ) . for the disc we find that the longer the distance to the endplate or the greater the change in arterial oxygen tension, the longer the equilibrium time. there is, however, a possible source of error in assessment on the recorder chart, when equilibrium is obtained. we standardized this procedure by taking the reading when an absolute constant value (as could be seen) was obtained for at least 5 min. but of course it is not possible to detect extremely small variations in the oxygen tension when gradually approaching the equilibrium value in our-experimental set-up. thus, it is possible, from 170 this point of view, that our values represent a slight underestimation of the real equilibrium time in the disc tissue. the oxygen equilibrium obtained is, however, affected by several other factors such as: variations in the diffusion constant, diffusion capacity of the system, blood capacity, and variations in cell respiration. it is reasonable to believe that these factors act together and perhaps it is impossible to distinguish between them. while some factors probably remain constant, variations in cell respiration could be very important in this avascular system, at least at low oxygen concentrations where steep solute gradients exist (5, 17). ‘ 7 other factors, which might affect the oxygen situation and create differen ces in ,the intradiscal oxygen tension level when comparing different discs, are the size and thus the dimensions of the system. theoretically performed calculations and in vivo studies of 35s labelled sulphate transport show that small variations, for example in the diffusion area of the endplate, will greatly affect the situation for the centrally located nucleus tissue (16). transport of oxygen through the tissue, predominantely by diffusion, seems to be sufficient for cell metabolism since the oxygen tension in the tissue ranges from 0.6 to 1.9 kpa when the arterial oxygen tension is 1 1 . 9 to 13.9 kpa. since response time is a few min., the transport of oxygen may be even sufficient to meet a slightly increased demand. it should, however, be pointed out that the measurements of oxygen tension and oxygen consumption are done in vivo under resting conditions. whether metabolic processes such as oxygen consumption change with dynamic movements of the spine or with increased vertical pressure is, at present, not fully known, although experiments in progress indicate an increased solute transport and metabolic rate with in creasing spinal movements. the first increase of arterial oxygen tension always caused a greater in crease in intradiscal oxygen tension than the later alterations i1 and 111. this is probably due to the fact that before alteration i, when the arterial oxygen tensions were in the range of 1 1 . 9 to 1 3 . 9 kpa, the haemoglobin is only partially saturated. after alteration i, much more oxygen is transported 171 per unit time. after alterations i1 and 111, only the amount of physically dissolved oxygen could be increased, which still constitutes the smaller por tion of the total oxygen content and this amount is directly proportional to the oxygen tension. the possibility that high oxygen concentration in the arterial blood reduces the blood flow by vasoconstriction (3, 6 , 7 ) must also be taken into consideration when dealing with diffusion from small vessels both outside the periphery of the annulus fibrosus and in the vertebral end plate. on the other hand, low oxygen concentrations have been reported to affect cell metabolism in several ways (9, 10). there are obvious difficulties in measuring oxygen tension in the super ficial layer of the vertebral body. it is at present impossible to measure oxygen tension in the capillaries close to the hyaline cartilage and how much lower the tension is in relation to the capillary network, which is present 1 2 millimeters underneath the cartilaginous vertebral endplate. intradiscal measurements very close to the endplate show oxygen tensions of about 8 kpa (5). the hyaline cartilage of the endplate therefore seems to be a relatively small barrier. furthermore, the capillary network just below the cartilage under the nucleus seems to be adequately supplied with vessels ( 1 8 ) . according to earlier studies, the central parts of the disc are very close to being deprived of solutes and, due to the long distances to the nearest blood supply, an anaerobic metabolism is predominant, with high lactic acid production on low oxygen tensions (5, 1 8 ) . small reductions in the arterial oxygen tension close to the vertebral endplate, or a decreasing diffusion capacity, can, from a theoretical point of view, easily affect the nutritional situation. how physiological changes such as aging or pathological conditions may affect the supply of nutritional solutes, including oxygen, and the disposal of waste products for the cells in the disc is an area where we have only scattered data but which, nevertheless, may prove important for the understanding of many abnormal stages of the back. 172 acknowledgements this study was supported by grants from the swedish work environment fund, carin trygger’s foundation, greta and einar asker’s foundation, riksfgrbundet mot reumatism, and gijteborgs lzkareszllskap. 1 . 2 . 3. 4. 5. 6. 7. 8. 9. 10. 1 1 . 12. 13. 14. 15. 16. 17. 18. references alm, a. & bill, a: the oxygen supply to the retina, 1. effects of changes in intraocular and arterial blood pressures, and in arterial po2 and pco2 on the oxygen tension in the vitreous body of the cat. acta physiol scand brodin, h: paths of nutrition in articular cartilage and the intervertebral disc. acta orthop scand 24:177-180, 1955. , dollery, c.t., bullpitt, c.j. & kohmer, e.m: oxygen supply to the retina from the retinal and choroidal circulations at normal and increased arterial oxygen tensions. invest ophth 8:588-596, 1969. ejeskzr, a. & holm, s: oxygen tension measurements in the intervertebral disc. upsala j med sci 84:83-93, 1979. holm, s., maroudas, a., urban, j., selstam, g. & nachemson, a: nutrition of the intervertebral disc: solute transport and metabolism. connect tissue res 8:lol-119, 1981. kety, s.s. & schmidt, c.f: the effects of altered arterial tensions of carbon dioxide and oxygen on cerebral blood flow and cerebral oxygen con sumption of normal young men. j clin invest 27:484-492, 1948. leninge-follert, e., lubbers, d.w. & wrabetz, w: regulation of local tissue p 0 2 on the brain cortex at different arterial oxygen pressures. pfluegers arch 359:81-95, 1975. lubbers, d.w: methods of measuring oxygen tensions of blood and organ surfaces. oxygen measurements of blood and tissues. (ed. j.p. payne & d.w. hill) pp. 103-128, churchill, 1966. marcus, r.e: the effect of low oxygen concentration on growth, glycolysis and sulphate incorporation by articular chondrocytes in monolayer culture. arthritis rheum 16:646-656, 1973. marcus, r.e. & srivastava, v.m.l: effect of low oxygen tensions on gluco semetabolizing enzymes in cultured articular chondrocytes. proc soc exp biol med 143:488-491, 1973. maroudas, a., stockwell, r.a., nachemson, a. & urban, j: factors involved in the nutrition of the human lumbar intervertebral disc: cellularity and diffusion of glucose in vitro. j anat 120:113-130, 1975. nachemson, a., lewin, t., maroudas, a. & freeman, m.a.r: in vitro diffusion of dye through the endplates and the annulus fibrosus of human lumbar inter vertebral discs. acta orthop scand 41:589-607, 1970. naylor, a: the biophysical and biomechanical aspects of intervertebral disc herniation and degeneration. ann r coll surg engl 31:91-114, 1962. silver, i: the measurements of oxygen tension in tissues. in: oxygen measurements of blood and tissues. (ed. j.p. payne & d.w. hill) pp. 133-154, churchill, 1966. soutter, l.p., conway, m.j. & parker, d: a system for monitoring arterial oxygen tension in sick new born babies. biomed eng 10 (7), 257-260, 1975. urban, j.p.g: fluid and solute transport in the intervertebral disc. thesis, london, 1977. urban, j., holm, s., maroudas, a. & nachemson, a : nutrition of the inter vertebral disc: an in vivo study of solute transport. j clin orthop 129: 101-114, 1977. urban, j., holm, s. & maroudas, a: diffusion of small solutes into the intervertebral disc: an in vivo study. biorheology 15:203-223, 1978. 84:261-274, 1972. 12-822857 173 received october 1, 1 9 8 1 . address for reprints: sten holm, ph.d. department of orthopaedic surgery i sahlgren hospital university of gijteborg s-413 45 gijteborg sweden 174 upsala j med sci 95: 1-29 hyperprolactinaemia-a clinical study with special reference to long-term follow-up, tkeatment with dopamine agonists, and pregnancy carsten rasmussen department of obstetrics and gynaecology, university hospital, uppsala, sweden although our present knowledge of hyperprolactinaemia as a cause of galactorrhoea and menstrual irregularities has its origin in the early 1970s, observations dating back to the mid-1 9th century pointed to these associations, as mentioned by blackwell (8). classically, three clinical entities involving galactorrhoea and amenorrhoea have been identified: the chiari-frommel syndrome, with persistence of postpartum amenorrhoea and galactorrhoea for more than one year after delivery without any evidence of a pituitary tumour; the ahumada del castillo syndrome, the spontaneous onset of amenorrhoea and galactorrhoea without any obvious pituitary tumour; and the forbes-albright syndrome, which is comparable to the above but with roentgenological evidence of a pituitary adenoma. since the development of a specific radioimmunoassay for measurement of human prolactin (prl) (36), it has been shown that in all of these three syndroms the serum prl concentration is elevated. the hormone prl was discovered in 1928 by stricker and grueter, who first described its lactogenic actions in mammals (cited by rillema et al. (68)). it is only in the last two decades that the importance of this hormone in human reproductive disorders has been recognized. the discovery of lactogenic activity in highly purified human growth hormone (47) initiated a ten-year controversy concerning the existence of a separate human prl. it was long assumed that pituitary prl activity was an intrinsic property of growth hormone. however, in 1970 a human prl molecule was identified (29) and was soon also isolated from the pituitary gland (37, 46). the prl molecule is a single polypeptide containing 198 amino acids (74). the prl molecule in man has been shown to be heterogeneous (78) and to have different distinct forms, little, big and big-big, with decreasing bioactivity and increasing 1-908571 1 molecular weight, in that order (91). the most common molecular weight is that of, little prl (22000), which is believed to be the bioactive form of the hormone. these heterogeneous forms of prl have been found in both normal and hyperprolactinaemic states. this matter is further complicated by the finding of functional heterogeneity within the population of the lactotrophs in the pituitary gland as illustrated by major differences in both synthetic rates and thyreotropin releasing hormone (trh) responsiveness (86). although the role of prolactin-inhibiting factors in the control of prl secretion is now well established, there is substantial evidence to suggest that stimulatory factors (prolactin-releasing factor) may also play an important part. hypothalamic control of prl secretion seems to be mediated by at least two different prolactin-inhibiting factors, which are synthesized by tubero-hypophyseal neurons (91). the role of hypothalamic dopamine as a major prolactin-inhibitory factor is clearly documented. dopamine is secreted directly into the hypophyseal portal blood system and binds, within the pituitary gland, to receptors on the cell membrane of the lactotrophs through which a direct inhibitory effect is exerted (17, 44). evidence suggest that gamma-aminobutyric acid (gaba), in addition to dopamine, functions as a prolactin inhibitor. however, it seems that the prl inhibitory activity of dopamine is far greater than that of gaba (91). one special feature of the synthesis and release of dopamine is the inhibitory action of prl via a short-loop feedback mechanism (34). production of dopamine by the posterior lobe of the pituitary gland has also been demonst rated (65). for many years it was believed that the action of inhibitory factors was the only mechanism for regulating prl secretion, but there is now considerable evidence to suggest that stimulatory factors also play an important role. experimental observations in several species, including humans, indicate that trh, vasoactive intestinal peptide, cholecystokinin and angiotensin ii may be involved in the control of prl secretion (91). thus, prl secretion seems to be under the control of a complex dual regulatory system which involves both inhibitory and stimulatory factors. in 1676 the physician dodart (22) described ergot-related cessation of lactation. this was the first medical correlation between an ergot alkaloid and endocrine function. a search for an ergot derivative that could suppress lactation in humans with minimal or no ergotoxic effects was initiated in the 1960s. several ergot alkaloids were 2 shown to inhibit prl secretion and suppress lactation in animals, but could not be used in humans because of their severe side effects. bromocriptine (brcr) (2-bromo-alpha-ergocryptine mesylate), a semi-synthetic ergot alkaloid which stimulates dopamine receptors and inhibits prl secretion, was introduced in the early 'seventies (25) and proved to be a highly effective and well tolerated drug in reducing serum prl levels in patients with hyperprolactinaemia. it is generally accepted today that brcr therapy is the treatment of choice for most infertile women with hyperprolactinaemia and amenorrhoea (63). the treatment results in rapid normalization of serum prl levels and restoration of ovulatory function and fertility in more than 90% of hyperprolactinaemic women (5). it is still unclear, however, whether the suppressive effect of brcr on prolactin hypersecretion is permanent (28), or whether the hyperprolactinaemia may return after discontinuation of the drug as has been found in some studies (7, 51, 77). the major risk associated with pregnancy in women harbouring a pituitary adenoma is expansion of the tumour, resulting in compression of the surrounding neural structures. bergh et al. (3), however, found that the risk of pregnancy-related tumour growth in hyperprolactinaemic women treated with bromocriptine was low. in a review nillius et al. (61) noted that clinical complications due to growth of a prolactinoma occurred in only 2% of 488 pregnancies i n women with hyperprolactinaemia who were given primary brcr therapy. however, there are only few reports on the long-term effects of pregnancy on prl hypersecretion or on the effect of multiple pregnancies in hyperprolactinaemic women treated with brcr. the aetiology and natural history of prolactinomas are largely unknown. it is not clear which or how many of the small prolactinomas which enlarge and become macroprolactinomas or invasive lesions (8). long-term studies have indicated that the frequency of further enlargement of the tumour among patients with microprolactinomas is low (48, 59, 76, 89). it has been reported that brcr effectively reduces the size of prolactinomas (53, 80). however, re-expansion of large pituitary adenomas has occurred soon after withdrawal of brcr treatment (82). in such cases sellar changes indicating intra-pituitary adenomas have been observed by plain radiography and multidirectional tomography. a disadvantage of these examinations is that the change in the size of an adenoma is assessed indirectly. modern radiological technology, such as computed tomography, is superior, as it provides information not only about the sella turcica but also about the pituitary 3 gland itself. during evaluation of the radiographs of the sella turcica calcific deposits in pituitary adenomas have, although rarely, been observed (43). from radiological studies an incidence of granular calcification within pituitary adenomas of 0.3 to 14% has been reported (1 3). however, it is not known whether these calcifications represent regression or progression of a pituitary tumour. the dopamine agonist brcr is the most commonly used drug in the treatment of hyperprolactinaemia (84). adverse reactions are often a problem at the beginning of the treatment and sometimes persist during chronic treatment. moreover, the drug usually has to be taken several times a day to maintain the therapeutic effect, although some patients can be effectively treated with a once-daily dose (14). for this reason efforts have been made t o find dopamine agonists with a more favourable profile of adverse effects and a longer duration of action. other ergot derivates such as lisuride, metergoline, and pergolide mesylate have been used, but these have essentially the same action as brcr and cause similar adverse reactions (75, 58). cv 205-502 is a new long-acting, non-ergot dopamine agonist which effectively inhibits prl secretion in healthy volunteers (30). we have performed the first clinical trials with this new drug in hyperprolactinaemic women. evaluations of hyperprolactinaemic women have revealed a wide range of variations in clinical symptoms. further knowledge of such variations is of importance when decisions have t o be taken as t o how to treat and follow up patients with hyperprolactinaemia. aims of the investigation this investigation was undertaken 1. t o determine whether long-term treatment with brcr in hyperprolactinaemic women could result in permanent suppression of the prl hypersecretion after discontinuation of the therapy; 2. effects of multiple pregnancies on hyperprolactinaemic women; to study the long-term effects of pregnancy on prl hypersecretion and the 4 3. to examine the changes (regression/progression) of the sella turcica during long-term follow-up in hyperprolactinaemic women and to evaluate the risk of serious tumour enlargement in women with a prl-secreting adenoma; 4. follow-up of women with hyperprolactinaemia; and to investigate how often clinical investigations are necessary for a safe 5. to investigate the duration of action, efficacy and side effects of a new non ergot doparnine agonist, cv 205-502, in the treatment of women with hyperprolactinaemia. results a: the effects of long-term bromocriptine treatment and pregnancy in women with hype rprolactinaemia the principal aim of these studies was to determine whether long-term treatment with brcr can cause permanent suppression of prl levels in serum even after withdrawal of the drug. further aims were to evaluate the long-term effect of pregnancy on prl hypersecretion and to examine the radiological changes of the sella turcica during long-term follow-up in hyperprolactinaemic women prolactin secretion and menstrual function after long-term bromocriptine treatment seventy-five women, 16 to 40 years of age, with long-standing amenorrhoea and hyperprolactinaemia were followed up after discontinuation of long-term brcr treatment. the prl concentration before treatment ranged between 25 and 1470 ug/l (mean 135 ug/l). forty-nine of the women had pretreatment serum prl concentrations below 100 ug/l, and 26 women had levels above 100 ug/l. brcr had been given in daily doses of between 2.5 and 50 mg for up to 65 months (median 24 months). twenty-two women had previously had a brcr-induced term pregnancy and six women had had two term pregnancies. treatment with brcr was not restarted in 33 women (44%). the prl levels in the serum in this group had decreased by 55 % after 6 months' follow-up as compared 5 with the pretreatment values. three of these 33 women had become normoprolactinaemic. regular menstruation occurred for at least 12 months in 19 of the 33 women after discontinuation of the treatment. most of the women with menstruation had prl levels of less than 100 ug/l before treatment. when the group of 33 patients was subdivided into those with prl concentrations above and below 100 ug/l, it was found that the women with prl levels above 100 ug/l showed a more marked decrease in their prl secretion than those with only moderate hyperprolactinaemia. there was no correlation in the 33 women between the pretreatment prl levels and the levels recorded after brcr withdrawal. ten women in the group of 33 were followed up for a number of years after discontinuation of treatment. after 5 years the mean prl value had decreased by 54 % compared with those before therapy. seven women had regular menstrual bleeding, and two patients we re no rmop ro lacti n ae mic. treatment was reinstituted in 42 of the 75 women (56%) within 3 months after discontinuation of therapy, mainly because of increasing prl levels in the serum. in 18 of these 42 women, treatment was discontinued a second time. six of these patients had regular menstrual bleeding after 6 months' follow-up and three of them were normoprolactinaemic. return of menstruation and normalization of prolactin in hyperprolactinaemic women with bromocriptine-induced pregnancy fifty-eight hyperprolactinaemic women, 22 to 36 years of age, were followed up for 13 to 108 months after a total of 73 bromocriptine-induced term pregnancies to investigate whether pregnancy had any adverse long-term effects on the hyperprolactinaemic state. fifteen of these women had had two term pregnancies, 13 of which were induced by brcr. the pretreatment prl concentrations ranged from 24 to 1470 ug/l (mean 92 ug/l). the treatment had been given in daily doses of between 2.5 and 30 mg for 1 to 48 months before conception occurred. none of the women showed evidence of extrasellar extension of a presumptive prolactinoma or of an empty sella at the radiological examination. pituitary tumour complications during pregnancy occurred in two women, who developed visual field defects in the third trimester. these defects improved when brcr was reinstituted a marked decrease in serum prl concentrations, i.e. a decrease of 50% or more of 6 the pretreatment value, occurred in 20 of the women (34%) after pregnancy, lactation and weaning. seven of them regained regular uterine bleeding and four had normal prl levels in serum after completion of breast-feeding. no decrease in serum prl was found in 36 women (62%), of whom five regained regular menstrual function. higher serum prl levels after pregnancy were found in two women, neither of whom displayed any change in the radiological appearance of the sella turcica or any symptoms of tumour growth. spontaneous uterine bleedings returned in 16 of the 58 women after pregnancy (27%). twelve of the women regained regular uterine bleedings and four had oligomenorrhoea. with only two exceptions, the women in whom spontaneous uterine bleedings returned had prl levels in their serum below 100 ug/l before treatment and pregnancy. this group of women was followed up for 6 years without any evidence of a further increase in the prl levels being found. fifteen women had two term pregnancies of which 1 3 were induced by bromocriptine. there was no difference between the mean serum prl level before treatment and that after the first pregnancy, but after the second pregnancy the mean prl level in serum had decreased compared with the level after the first pregnancy. long-term radiographic follow-up of the sella turcica in hyperprolactinaemic women and microcalcification in prolactin-secreting pituitary adenomas in this study 73 hyperprolactinaemic women, 17 to 56 years of age, were followed up for 5 to 13 years after the initial radiological examinations of the sella turcica. at the first examination standard lateral and postero-anterior coned down views of the sella turcica showed a normal sella in 16 women. minor radiological changes classed as b l b 3 were observed in 38 women, while 19 women displayed pronounced asymmetry that was assigned to class 84-85. in four patients there was suprasellar extension of the tumour. one patient had an empty sella. the prl concentration in the serum before treatment ranged between 21 and 1500 ug/l (mean 108 ug/l). the mean serum prl level was significantly higher in the 84-85 group than in groups bo and bl-b3. brcr had been given in daily doses of between 2.5 mg and 50 mg for 2 to 132 months in all patients except one, who received no treatment. sixty-seven women were normoprolactinaemic during treatment and had regular menstrual bleeding. forty-eight brcr-induced pregnancies occurred in 35 of the women 7 during the follow-up. thirteen of them had two term pregnancies, among which ten were induced by brcr. enlargement of the pituitary fossa was found in 25 (34%) of the 73 women. the serum prl concentration in this group ranged between 21 and 1470 ug/l (mean 107 ug/l). seven of these 25 women showed progression of the sellar asymmetry during pregnancy, with alteration in the classification of the sella turcica. all of them had been treated with 5 mg of bromocriptine daily for 2 to 20 months (median 4 months). in all 7 patients the brcr treatment was stopped as soon as pregnancy was confirmed. alt of them were treated with brcr postpartum. during this treatment three of them showed regression of the sellar asymmetry but without normalization of the sella. two women had two term pregnancies, and one of them showed further progression during the second pregnancy. marked progression during pregnancy with development of suprasellar extension of the tumour was seen in one woman. brcr was reinstituted during the pregnancy and resulted in tumour regression. brcr treatment was restarted after weaning and the patient conceived soon again and this time had a normal pregnancy with no further progression of the sellar asymmetry. non-pregnancy-related progression occurred in 18 of the 25 women. minor progression with erosion and thinning of the laminae durae was observed in eight women and major progression with a change in the classification occurred in ten women. brcr had been given in a mean dose of 7.3 mg daily (range 2.5 to 50 mg daily) for 18 to 132 months (median 60 months). there was no difference in the mean prl level in the serum before treatment between the group with minor and that with major progression. during the follow-up period six of these women had one brcr-induced term pregnancy and three of them had two such pregnancies without any progression of the asymmetry. radiological regression occurred in 14 (20%) of the 73 women. three of these 14 women showed suprasellar extension of the tumour. minor regression was noted during treatment in six women, while marked regression with a change in classification was found in five women. brcr had been given for 38 to 11 2 months (median 78 months) with a mean dose of 6.9 mg. in three women with suprasellar extension of the tumour, repeated computed tomography showed regression of the extension after brcr treatment for 4, 6 and 10 months, respectively, but there was 8 no further regression in the sellar asymmetry. pregnancy had occurred during treatment with brcr in four patients, resulting in four normal children. two patients had had two pregnancies. there was no progression of the radiological sellar changes during or after the pregnancies. no difference in the mean p r l level in serum before treatment or in the duration of treatment was found between the group with minor and the group with major regression. there was no difference in the mean duration of treatment between the group with regression and the group with non-pregnancy-related progression. thirty-four women (46%) did not show any change in their sellar configuration during the follow-up. brcr treatment had been given for 4 to 126 months (median 58 months) in a mean dose of 7.0 mg. one brcr-induced term pregnancy was experienced by 18 of these women, while six of them had two term pregnancies. within the whole group of 73 women, the mean prl level in the serum before treatment was lower in the group with no radiological changes than in the group with regression. lntrasellar calcifications were seen in eight of the 73 women. one woman had a curvelinear calcification in the region of the anterior superior aspect of the pituitary capsule. one woman had developed a solitary calcification after a diagnostic fine needle aspiration of the pituitary tumour. the remaining six women displayed granular calcifications in the anterior portion of the sella turcica. progression of these calcifications was observed in three of the women and in two of these the calcification increased in association with radiographical regression of the sellar changes. we were not able to find any factor which could predict the occurrence of the calcific deposit. b: treatment with a new long-acting dopamine agonist (cv 205-502) in women with hyperprolactinaemia short-term treatment with cv 205-502 in a dose-ranging study cv 205-502 was given to 24 hyperprolactinaemic women of ages 18 to 47 years for 7 days. twenty-one had had brcr treatment for 2 to 69 months before they entered the study. three had never received any treatment with a dopamine agonist. the pretreatment prl levels in the serum ranged between 22 9 and 320 ug/l (mean 85 ug/l). the women were randomly allocated double-blind to one of three treatment groups, which received cv 205-502 once daily for 7 days in a ' dose of 0.01 mg (group l ) , 0.03 mg (group 2) or 0.06mg (group 3). two patients in each group were given placebo. a prl day curve was obtained during the 1st and 7th days. on day 8 blood samples were obtained 23 and 24 hours after the last treatment dose. the serum prl concentrations decreased dose-dependently in all patients given active substance. group 3 (0.06 mg) showed the most pronounced effects of the drug. four patients became nonoprolactinaemic, with serum prl levels below 10 ug/l, and in these patients the prl secretion was suppressed for at least 24 hours. in the remaining two women the prolactin level decreased by more than 50%. the side effects were mild and transient. no patients discontinued the study. long-term treatment with a new non-ergot long-acting dopamine agonist, cv 205 502 in women with hyperprolactinaemia twenty-four hyperprolactinaemic women, aged 22 to 48 years, were treated with cv 205-502 for 6 months. all except one had been treated with brcr for 1 to 108 months. five of the women had discontinued the brcr treatment because of undesirable side effects. the serum prl concentrations ranged between 31 and 11 00 ug/l (mean 84 ug/l). a double-blind placebo-controlled design was used for the first 4 weeks, in which the patients were randomly assigned to receive either placebo or 0.05 mg of cv 205 -502 once daily. the patients who had had placebo started with 0.05 mg of cv 205 -502 at the end of week 4, while in the cv 205-502 treated patients who had not become normoprolactinaemic the daily dose was increased by 0.025 mg. thereafter the study was open-label and the dose was increased by 0.025 mg daily every 4 weeks until the plasma prl levels were within the normal range or until the study period ended at 6 months. all the patients, including those who started with placebo, received 6 months' treatment with cv 205-502. the maximum dose was 0.175 mg once daily. cv 205-502 therapy decreased the serum prl levels in all the patients. the mean prl concentration had decreased from baseline by an average of 77% after 4 weeks of treatment and by 90% at week 24. the treatment resulted in normalization 10 of prl secretion in 17 of the 24 women receiving daily doses of 0.05 to 0.15 mg (mean 0.075 mg) of the drug. the prl level was still elevated in seven of the 24 women at the end of the study. in three of these women a marked decrease in the prl concentration occurred, compared with the pretreatment value while four of the seven women responded to c v 205-502 with only a minor decrease within the 6 months. one of these patients had previously received brcr with normalization of prl secretion and restoration of menstrual function. regular menstrual bleeding occurred in 16 of the 17 women whose prl levels were normalized. ovulation was confirmed by progesterone determinations in 15 women. furthermore, regular menstrual function occurred in four of the seven women despite persistently elevated prl levels. ovulation was confirmed in all four women. mild to moderate galactorrhoea was found at the baseline examination in 19 of the 24 patients. after 6 months of treatment some mild galactorrhoea was still present in six women. c v 205-502 was well tolerated and the side effects, which mainly consisted of nausea and tiredness, were mild and transient and occurred mostly at the beginning of the drug therapy. no patients discontinued the study. discuss ion in these studies we have investigated the long-term effects of treatment with dopamine-agonists and of pregnancy on hypersecretion of prolactin. in some patients significant changes in the prl secretion had occurred during the study while in others the changes in serum prolactin concentrations were regarded to be within the normal variation of prl secretion. prolactin i s synthesized and secreted from the lactotropic cells of the adenohypophysis. the hormone is released in pulses of varying magnitude superimposed on a continuous basal secretion (45). in normal subjects the highest plasma prl concentrations occur during sleep (72). moreover, prl secretion is increased by a number of physical, emotional, or combined stress stimuli. at the time of puberty in girls, the mean serum prl level increases significantly to reach the adult female range (23) and remains unchanged until menopause. during pregnancy the serum prl level begin to rise in the first trimester and increases 1 1 progressively to ten times or more the concentration of non-pregnant women at term (83). hyperprolactinaemia is often associated with hypogonadism with or without galactorrhoea. high prl concentrations have an inhibitory effect on follicular growth and steroidogenesis (52), and in addition there is a loss of normal surge of lh secretion in response to the administration of oestrogen (32), and spontaneous pulses of lh secretion are infrequent or absent (9). reduction of circulating prl, by treatment with brcr, is associated with a rise in the serum oestradiol concentration (73), a return of lh positive feedback responses (l), and restoration of lh pulsatility (9). moult et a1 (56) proposed that brcr acts directly on the pituitary to reduce the serum prl level, and that this in turn allows hypothalamic dopamine production t o fall, restoring normal lh pulsatility. the primary reason for treatment of hyperprolactinaemia in women is infertility, but in some cases treatment may be warranted because of troublesome galactorrhoea or because of symptoms due to oestrogen deficiency. not all patients with hyperprolactinaemia require treatment and those who do not should be kept under observation. however, there are data suggesting that patients with untreated hyperprolactinaemic amenorrhoea may be at risk of developing osteoporosis (41, 42). this possibility should be kept in mind when making the decision as to whether women with hyperprolactinaemia should be treated or not. only limited data are available concerning the natural history of hyperprolactinaemia in patients with and without a pituitary tumour and the long-term effects of brcr treatment and of pregnancy on these conditions. hyperprolactinaemia and long-term bromocriptine treatment hyperprolactinaemia is a common clinical disorder and has been found to be present in a significant proportion of women with amenorrhoea, with an incidence varying between 13 and 20% (2, 63). several studies have documented that brcr therapy effectively lowers the prl level to normal and restores ovulatory function in more than 90% of women with hyperprolactinaemia (5, 28, 55). in addition, this drug has been found to reduce the size of a prolactinoma in a large percentage of patients (53, 80). it is still unclear, however, whether the suppressive effect of the drug on prl hypersecretion is permanent (28). 12 in the present investigation we followed up 75 hyperprolactinaemic women after discontinuation of long-term brcr treatment. hyperprolactinaemia returned rapidly in most of the patients (56%) after withdrawal of the drug, which is in agreement with other reports (7, 50, 51, 87). however, as many as 44% of the women, who had been treated with brcr for up to 65 months, had no need for reinstitution of therapy within 6 months. the prl levels in this group had decreased by 55% compared with the pretreatment values. in the group of women who had discontinued brcr treatment after another treatment period of 5 to 81 months, eight were still without need of treatment at the 6-month follow-up. persistent serum prl normalization occurred in three of the women in each of the two groups. moriondo et al (55) reported persistent normalization of serum prl levels after brcr withdrawal in 11 % and 22% of 36 women treated for 1 and 2 years, respectively. the observation time was only 45 days, which might explain the higher success rate. maxon et al. (51) found that prl concentrations did not usually plateau until at least 40 days after cessation of therapy and concluded that decisions regarding tumour activity based on the prl concentration should not be made until at least 6 weeks after withdrawal of bromocriptine treatment. this is in accordance with the results in ten women followed up in our study for 5 years after discontinuation of treatment. it was found that the serum prl levels rapidly returned within 3 months to a stable plateau and remained at this level for the rest of the follow-up period. when the group of patients who were still without treatment were subdivided into those with serum prl levels above and below 100 ug/l, it was found that the women with prl levels above 100 ug/l showed a more marked decrease in their prl secretion than those with only moderate hyperprolactinaemia. these results are in agreement with those of eversmann et al (24), who concluded that the degree of persistent prl suppression correlated to the height of the prl level before treatment and to the duration of brcr treatment. this is partially in contrast with the results of moriondo et al (55), who found a positive correlation between basal prl levels and those recorded after brcr withdrawal. one explanation for the difference from our results may be that most of our patients had had treatment for a longer period than in the study by moriondo et al (55). in a previous study bergh et al. (2) found that nine women with slightly elevated prl levels had become normoprolactinaemic at follow-up after 1 to 4 years without treatment. this is in accordance with the report by pepperell (64) who concluded 13 that one third of patients with mild hyperprolactinaemia can have spontaneous resolution of their hyperprolactinaemia. in a group of women with idiopathic ' hyperprolactinaemia, martin et al (49) found that the p r l levels returned to normal spontaneously in 14 of 41 of the patients after a follow-up of 5 years, but that there was no spontaneous regression in patients with prl levels above 60 ug/l. however, it is unlikely that p r l secretion would become normal spontaneously in a large group of women with marked hyperprolactinaemia. rjosk et al (69) found no evidence of p r l normalization in 34 untreated patients with microprolactinomas who were followed up for 6 years and in another study spontaneous regression occurred in only 7% of 43 untreated patients with hyperprolactinaemia (48). none of the ten women in this study who were followed up for up to 5 years after brcr therapy showed any signs of spontaneous regression. it is much more probable that in most of the patients the p r l decrease is related to the long-term brcr treatment. re-expansion of prolactinomas occurred in a large proportion of patients in a recent study when brcr therapy was discontinued (53). in this study, brcr had been given for only one year. possibly a more prolonged treatment period is necessary to achieve more permanent tumour regression. one of our patients, with a large pituitary tumour with suprasellar extension and who was given brcr treatment for 2 years, still has a nearly normal serum prl level and normal menstruation after 8 years without therapy. radiological follow-up has shown no evidence of tumour regrowth. this finding is in agreement with the report by johnston et al (39), who followed up 15 hyperprolactinaemic patients after withdrawal of bromocriptine treatment which had been given for 1.5 to 7 years and concluded that rapid tumour regrowth is uncommon and of small extent. hyperprolactinaemia and pregnancy restoration of ovarian function and induction of pregnancy are now easily achieved in hyperprolactinaemic women with amenorrhoea and infertility by means of prl lowering treatment with brcr (3, 63, 79). brcr is generally found to enable about 80% or more of women with hyperprolactinaemic infertility to become pregnant (88). the major risk associated with pregnancy in women harbouring a prolactinoma is expansion of the tumour during pregnancy. however, several studies have shown that the risk of developing serious pituitary tumour complications during pregnancy 14 is low (3, 21, 38). nillius et al (61) reviewed 488 pregnancies and found that only 2% of the hyperprolactinaemic women developed visual complications due to rapid prolactinoma enlargement. the risk is also small i n patients with larger prolactinomas (6). in a recent review, molitch (54) summarized 16 series reported in the literature encompassing 246 microprolactinomas and 45 macroprolactinomas left untreated before pregnancy was achieved.. only 1.6% of the women with microprolactinomas had symptoms of tumour enlargement. of the 45 women with macroprolactinomas, 15.5% had symptomatic, and 8.9% asymptomatic tumour enlargement. studies on the long-term effect of pregnancy on prl hypersecretion have been sparse. in 1979 cowden and thomsen (16) reported on a hyperprolactinaemic woman in whom a brcr-induced pregnancy was followed after breast-feeding by the return of spontaneous menstruations and normoprolactinaemia. resolution of hyperprolactinaemia after brcr-induced pregnancy has now been decribed in several papers (4, 12, 18, 71). however, there are only few reports on the long-term effects of pregnancy in women with hyperprolactinaemia (38). in the present study 58 hyperprolactinaemic women were followed up for 1 to 9 years after at least one brcr-induced pregnancy. the serum prl level decreased markedly in 20 of the women (34%) after pregnancy, lactation and weaning. four had become normoprolactinaemic. only two women showed a significant increase in prl secretion after pregnancy, but they had no symptoms or signs of tumour complications. spontaneous uterine bleedings returned in 16 of the 58 women after pregnancy (27%). all except two of these 16 women had prl levels of c 100 ug/l before treatment. this group of women was followed up for 6 years without any evidence being found that the prl levels increased again. the 15 women who experienced two term pregnancies had lower prl levels in their serum after the second pregnancy than after the first, which confirms the observation that prl hypersecretion seems to decrease after repeted pregnancy (1 9). prolactinomas and long-term bromocriptine treatment the prolactinomas represent a common and complex clinical entity. autopsy series have yielded evidence that pituitary microadenomas occur in 20 to 30 yo of pituitary 15 glands examined during routine postmortem examinations. approximately 40 yo of these tumours stain positively for prl (1 1, 15). it is likely that microand macroprolactinomas are two distinct entities, one destined to enlarge, possibly rapidly (82), the other remaining inconsequential throughout most of the patient's life, although gradual tumour growth over decades is possible. sisam et al. (76) found no evidence of even subtle tumour growth in 38 untreated hyperprolactinaemic women with microprolactinomas, who were followed up for an average of 31.7 months. in another study, 43 untreated hyperprolactinaemic patients were followed up for 3 to 20 years and signs of tumour enlargement were observed in only two patients (48). on the other hand, pontiroli and falsetti (66) reported the sudden development of radiologically verified prolactinomas i n 14 of 65 hyperprolactinaemic patients followed up for 3 years. in this present study 73 hyperprolactinaemic women were followed up for 5 to 13 years after the initial radiological examination of the sella turcica. radiological signs of progression were noted in 25 of the 73 patients (34%). in seven women this progression occurred during pregnancy. evidence of minor or major non-pregnancy related progression during the follow-up period was found in 18 of the 73 patients. we were not able to find any factor such as prl levels or grade of sellar asymmetry which could predict this progression. it is well known that there is a risk of pituitary tumour expansion during pregnancy in hyperprolactinaemic women. however, only one woman had serious clinical complications which demanded reinstitution of brcr therapy. twenty-two of the women had a term pregnancy without any radiological signs of progression. these results, like those of previous studies (3, 21), show that the risk of clinically significant tumour expansion during pregnancy is low. radiological regression occurred in '1 4 women (20%). there was no difference in the mean serum prl level before treatment between the group with progression and the group which showed regression. the normal sella turcica varies markedly in shape (60). the pituitary gland itself occupies only 50-85% of the pituitary fossa (20). the gland is surrounded by venous plexus communicating with the sinus cavernosus. therefore, the pituitary gland can increase considerably in size at the expense of venous filling and can even double in size during pregnancy without altering the bony margins of the sella (20). it is unlikely that minor changes in the shape or the bony margins of the sella turcica constitute evidence of an intrasellar lesion. in a study of 205 autopsy cases muhr et 16 al. (57) found that asymmetries of the sellar floor with a slope of 2 to 140 and minor cortical changes were frequent, and correlated poorly to the presence of microadenomas. furthermore, a normal-sized sella turcica can well harbour an adenoma in the pituitary gland. still, it is probable that radiographical regressive or progressive changes of the sella turcica over time in individual patients are an expression of an increased or decreased tumour size. annual radiological examinations of the sella turcica have previously been recommended in the follow-up of the hyperprolactinaemic patients (2). our present study shows, however, that from a clinical point of view such short intervals are unnecessary. in none of our patients did repeat sellar x-rays give any information which altered the clinical management. furthermore, a disadvantage of standard coned down views and multidirectional tomography is that the change in size of an adenoma is only recorded indirectly. modern radiological technology, such as computed tomography is superior to plain sellar x-ray as it provides information not only about the sella turcica but also about the pituitary gland itself. with this technique it is possible to detect and follow the changes in a microadenoma before a change in the configuration of the sella turcica appears (10). weiss et a1 (89) concluded that computed tomography rather than endocrine or ophthalmological evaluation provides the best index of early tumour growth. however, standard radiological examination of the sella turcica is an easily performed low-cost examination, involving low radiation doses. it permits a preliminary evaluation of the sella turcica, if necessary before the use of more detailed procedures. granular calcifications, a finding of unknown clinical meaning, were found in the anterior portion of the sella turcica, in six women (8%). this incidence is in agreement with the results of a compilation of 13 radiological series (13), where the incidence of granular calcifications within a pituitary adenoma varied between 0.3 and 14%. the aetiology of the calcifications is not known. landolt and rothenbuhler (43) reported that calcification occurred in necrotic cells and classified this as dystrophic calcification. in contrast, rilliet et al. (68) found histologically that calcifications were most frequently located extracellularly. they concluded, like guay et al. (33), that the presence of calcification suggests a slowly growing neoplasm. in our study two patients showed progression of the calcific deposit accompanied by radiographical signs of regression of the sellar asymmetry. in one woman progression of the sellar asymmetry occurred without any change in calcification. thus it is still unclear why 2-908571 17 these calcifications develop and if they are of any clinical importance. cv 205-502, a new dopamine agonist bromocriptine is the most widely used drug in the treatment of hyperprolactinaemic states. despite a relatively long duration of action, prolonged suppression of serum prl in human subjects usually requires oral administration 2 or 3 times a day. in addition, adverse reactions including nausea, emesis and hypotension are often seen at the beginning of the treatment, and sometimes such problem persist during therapy. these adverse reactions together with the need for multiple doses during the day, have prompted a search for a new compound with greater potency for inhibiting prl secretion without a concomitant increase in undesirable effects. a short-term study with cv 205-502, a new long-acting dopamine agonist, showed that this drug effectively inhibited prl secretion for at least 24 hours. it was found that the highest used dose of 0.06 mg was most effective but probably not optimal, as two of the women did not become normoprolactinaemic. in most of the patients in whom the prl level was restored to normal, this happened within 6 hours after the first dose of cv 205-502. the side effects were all transient and mild. several of the patients who had experienced severe side effects during bromocriptine treatment had no problem in tolerating cv 205-502. the prolonged study confirmed the efficacy of single daily doses of cv 205-502 in suppressing prl secretion and in restoring menstrual function. seventy per cent of the women became normoprolactinaemic. in a previous study (5) it was found that brcr restored ovulatory function in about 90% of patients with hyperprolactinaemia. one explanation for the difference between cv 205-502 and brcr in the effect on the serum prl levels may have been that the patients in the cv 205-502 trial included a high proportion of women who had been hyporesponders to previous dopamine agonist treatment. secondly, the treatment in this study was stopped at a dose of 0.175 mg daily. the treatment continued in 21 of the 24 women for further 6 months (max dose 0,325 ug/l). at 12 months' follow-up all except two of the women now were normoprolactinaemic. both had been hyporesponders to previous treatment with dopamine agonists. brcr treatment has no uniform effect on prl secretion, i.e. some patients need higher doses than others, even if their serum prl levels are similar. according to thorner et al. (81), it is not possible on the basis of 18 the pretreatment prl level, the tumour size or the response to a single dose of bromocriptine, to determine in which patient the prl level will fall to normal during chronic therapy. these authors speculated that the wide spectrum of results is most likely due to the differential sensitivity of the lactotrophs to dopamine stimulation. the same mechanism could explain why not all of our patients responded satisfactorily to cv 205-502 therapy. one patient in our study achieved no therapeutic effect of cv 205-502 even though she had previously responded to brcr. this example illustrates the fact that prl suppression may vary significantly in the same patients in response to different dopamine agonists. franks et al (27) showed that higher doses of pergolide mesylate did not markedly affect prl secretion in brcr-resistent women with hyperprolactinaemia. however, kleinberg et al (40) described a patient in whom 30 mg of brcr daily had no effect while 0.3 mg of pergolide mesylate per day normalized the serum prl level. furthermore, they found one woman who was unable to tolerate brcr because of gastrointestinal distress but responded well to pergolide and one other woman who was unable to tolerate pergolide for the same reason but took brcr without side effects. thus, there seem to be marked individual variations in the response to different dopamine agonists. four of our hyporesponders had normal ovulatory function at the end of the study, according to the serum progesterone levels in the luteal phase of the menstrual cycle. similar observations of restoration of gonadal function in women whose prl levels were not suppressed to the normal range have been made previously (81). thus the significance of the need to suppress prl levels completely within normal limits remains to be established. the adverse effects of cv 205-502 in this study were all mild and transient which is in agreement with another report (35). most of the women who had experienced adverse effects during brcr therapy had no tolerance problems while taking cv 205-502. thus cv205-502 seems to be a valuable alternative to the dopamine agonists which are used today in the treatment of patients with prl disorders. 19 1. 2 . 3 . 4. 5. 6 . 7. 8. 9 . aono t., m i y a k e a , , y a s u d a t.s.m., koike k . & k u r a c h i k . ( 1 9 7 9 ) r e s t o r a t i o n of o e s t r o g e n positive feedback effect on l h r e l e a s e by b r o m o c r i p t i n e in h y p e r p r o l a c t i n a e m i c p a t i e n t s w i t h g a l a c t o r r h o e a a m e n o r r h o e a . acta e n d o c r i n o l ( c o p e n h ) . 9 1: 5 9 1 . b e r g h t., nillius s.j. & w i d e l . ( 1 9 7 7 ) h y p e r p r o l a c t i n a e m i a in a m e n o r r h o e a i n c i d e n c e a n d clinical s i g n i f i c a n c e . acta e n d o c r i n o l ( c o p e n h ) . 8 6 : 6 8 3 . b e r g h t., nillius s.j. & w i d e l. ( 1 9 7 8 ) clinical c o u r s e a n d o u t c o m e of p r e g n a n c i e s in a m e n o r r h o e i c w o m e n w i t h h y p e r p r o l a c t i n a e m i a a n d p i t u i t a r y t u m o u r s . b r med j. 1 : 875. b e r g h t., nillius s.j., l a r s s o n s.-g. & w i d e l . ( 1 9 8 1 ) e f f e c t s of b r o m o c r i p t i n e i n d u c e d p r e g n a n c y on p r o l a c t i n s e c r e t i n g p i t u i t a r y t u m o u r s . acta e n d o c r i n o l ( c o p e n h ) . 9 8 : 333. b e r g h t. & n i l l i u s s j . ( 1 9 8 2 ) p r o l a c t i n o m a s : f o l l o w u p of m e d i c a l t r e a t m e n t . i n a clinical p r o b l e m : m i c r o p r o l a c t i n o m a ( e d s g.m. molinatti) p 1 1 5 . oxford, e x c e r p t a medica. b e r g h t., n i l l i u s s.j., e n o k s s o n p., l a r s s o n s.-g. & w i d e l . ( 1 9 8 2 a ) b r o m o c r i p t i n e i n d u c e d p r e g n a n c i e s in w o m e n w i t h l a r g e p r o l a c t i n o m a s . clin endocrinol. 17: 6 2 5 . b e r g h t., nillius s.j. & w i d e l. ( 1 9 8 2 b ) m e n s t r u a l f u n c t i o n a n d s e r u m p r o l a c t i n levels a f t e r l o n g t e r m b r o m o c r i p t i n e t r e a t m e n t of h y p e r p r o l a c t i n a e m i c a m e n o r r h o e a . clin endocrinol. 1 6 : 5 8 7 . b l a c k w e l l r.e. ( 1 9 8 5 ) diagnosis a n d m a n a g e m e n t of p r o l a c t i n o m a s . f e r t i l steril. 4 3 : 5. b o h n e t h.g., d a h l e n h.g., w u t t k e w . & s c h n e i d e r h . p . g . ( 1 9 7 6 ) h y p e r p r o l a c t i n e m i c a n o v u l a t o r y s y n d r o m e . j clin e n d o c r i n o l metab 4 2 : 1 3 2 . 10. b o n n e v i l l e j . f . , p o u l i g n o t d., c a t t i n f., c o u t u r i e r m., mollet e. & d i e t e m a n t j.l. ( 1 9 8 2 ) c o m p u t e d t o m o g r a p h i c d e m o n s t r a t i o n of t h e e f f e c t s of b r o m o c r i p t i n e on p i t u i t a r y m i c r o a d e n o m a size. radiology. 1 4 3 : 45 1 . 20 1 1 . b u r r o w g.n., w o r t z m a n g., r e w c a s t l e n.b., h o l g a t e r.c. & kovacs k . ( 1 9 8 1 ) m i c r o a d e n o m a s of t h e p i t u i t a r y a n d a b n o r m a l s e l l a r t o m o g r a m s in a n u n s e l e c t e d a u t o p s y s e r i e s . n e n g l j med. 3 0 4 : 1 5 6 . 1 2 . c a m p a g n o l i c., b e l f o r t e l., m a s s a r a f.. p e r i s c. & m o l i n a t t i g.m. ( 1 9 8 1 ) p a r t i a l r e m i s s i o n of h y p e r p r o l a c t i n e m i c a m e n o r r h e a a f t e r b r o m o c r i p t i n e i n d u c e d p r e g n a n c y . j e n d o c r i n o l i n v e s t . 4: 8 5 . 13. c e c c h i n i a . ( 1 9 6 8 ) l e calcificazioni negli a d e n o m i ipofisari. m i n e r v a radiol. 1 3 : 4 9 0 . 1 4 . ciccarelli e., mazza e., g h i g o e., guidoni f., b a r b e r i s a . , m a s s a r a f. & c a m a n n i f. ( 1 9 8 7 ) l o n g t e r m t r e a t m e n t w i t h o r a l s i n g l e a d m i n i s t r a t i o n of b r o m o c r i p t i n e in p a t i e n t s w i t h h y p e r p r o l a c t i n e m i a . j e n d o c r i n o l i n v e s t . 1 0 : 5 1 . 1 5 . costello r . t . ( 1 9 3 6 ) subclinical a d e n o m a of t h e p i t u i t a r y g l a n d . am j p a t h o l . 12: 2 0 5 . 1 6 . c o w d e n e . a . & t h o m s e n j . a . ( 1 9 7 9 ) r e s o l u t i o n of h y p e r p r o l a c t i n a e m i a a f t e r b r o m o c r i p t i n e i n d u c e d p r e g n a n c y . l a n c e t . 1: 6 1 3 . 17. c r o n i n m.j., c h e u n g c.y., wilson c.b., j a f f e r.b. & weinerr.1. ( 1 9 8 0 ) ( 3 h ) s p i p e r o n e b i n d i n g to h u m a n a n t e r i o r p i t u i t a r i e s a n d p i t u i t a r y a d e n o m a s s e c r e t i n g p r o l a c t i n , g r o w t h h o r m o n e , a n d a d r e n o c o r t i c o t r o p i c h o r m o n e . j clin e n d o c r i n o l m e t a b . 5 0 : 387. 1 8 . c r o s i g n a n i p.g., f e r r a r i c., s c a r d u e l l i c., picciotti m.c., c a l d a r a r . & m a l i n v e r n i a . ( 1 9 8 1 ) s p o n t a n e o u s a n d i n d u c e d p r e g n a n c i e s i n h y p e r p r o l a c t i n e m i c w o m e n . o b s t e t g y n e c o l . 5 8 : 7 0 8 . 1 9 . d a y a s., s h e w c h u k a.b. & b r y c e l a n d n . ( 1 9 8 4 ) t h e e f f e c t of m u l t i p a r i t y on i n t r a s e l l a r p r o l a c t i n o m a s . am j o b s t e t g y n e c o l . 1 4 8 : 5 1 2 . 2 0 . dichiro g . & nelson k.b. ( 1 9 6 2 ) t h e v o l u m e of t h e sella t u r c i c a . am j r o e n t g e n o l r a d i u m t h e r n u c l med. 87: 9 8 9 . 21 2 1. d i v e r s w.a. & yen s.s.c. ( 1 9 8 3 ) p r o l a c t i n p r o d u c i n g m i c r o a d e n o m a s in p r e g n a n c y . o b s t e t gynecol. 6 1: 4 2 5 . 2 2 . d o d a r t ( 1 6 7 6 ) c i t e d in: editorial " b r o m o c r i p t i n e a c h a n g i n g s c e n e " ( 1 9 7 5 ) . b r med j 4: 6 6 7 2 3 . e h a r a y . , yen s.s.c. & s i l e r t . m . ( 1 9 7 5 ) s e r u m p r o l a c t i n l e v e l s d u r i n g p u b e r t y . a m j o b s t e t g y n e c o l . 1 2 1: 9 9 5 . 2 4 . e v e r s m a n n t., f a h l b u s c h r . , rjosk h.k. & von w e r d e r k . ( 1 9 7 9 ) p e r s i s t i n g s u p p r e s s i o n of p r o l a c t i n s e c r e t i o n a f t e r l o n g t e r m t r e a t m e n t w i t h b r o m o c r i p t i n e in p a t i e n t s w i t h p r o l a c t i n o m a s . acta e n d o c r i n o l ( c o p e n h ) . 92: 4 1 3 . 2 5 . f l u c k i g e r e. ( 1 9 7 6 ) t h e p h a r m a c o l o g y of b r o m o c r i p t i n e . i n p h a r m a c o l o g i c a l a n d clinical a s p e c t s of b r o m o c r i p t i n e ( p a r l o d e l ) ( e d s . r.i.s. bayliss, p. t u r n e r & w.p. m a c l a y ) p 12. mcs c o n s u l t a n t s , t u n b r i d g e wells, kent. 2 6 . f l u c k i g e r e . ( 1 9 8 6 ) p h a r m a c o l o g i c a l p r o f i l e of d o p a m i n o m i m e t i c d r u g s . i n e n d o c r i n o l o g y ' 8 5 ( e d s g.m. molinatti & l. m a r t i n i ) p 2 6 1 . e l s e v i e r s c i e n c e p u b l i s h e r s . a m s t e r d a m . 2 7 . f r a n k s s., h o r r o c k s p.m., l y n c h s.s., b u t t w.r. & l o n d o n d . r . ( 1 9 8 3 ) e f f e c t i v e n e s s of p e r g o l i d e m e s y l a t e in long t e r m t r e a t m e n t of h y p e r p r o l a c t i n a e m i a . b r med j . 2 8 6 : 1 177. 2 8 . f r a n k s s. & j a c o b s h.s. ( 1 9 8 3 ) h y p e r p r o l a c t i n a e m i a . clin endocrinol m e t a b . 12: 6 4 1 . 2 9 . f r a n t z a.g. & k l e i n b e r g d.l. ( 1 9 7 0 ) p r o l a c t i n : e v i d e n c e t h a t it is s e p a r a t e f r o m g r o w t h h o r m o n e in h u m a n blood. science. 1 7 0 : 7 4 5 . 3 0 . gaillard r . , n o r d m a n n r., p e t c h e r t.j. & b r o w n e l l j . ( 1 9 8 6 ) a novel octahydrobenzo(g)quinoline, c v 2 0 5 5 0 2 , w i t h p o t e n t d o p a m i n e a g o n i s t p r o p e r t i e s . i n e n d o c r i n o l o g y '85 ( e d s g.m. molinatti & l . m a r t i n i ) , p 3 0 5 . e l s e v i e r s c i e n c e p u b l i s h e r s , a m s t e r d a m . 22 3 1. 3 2 . 3 3 . 34. 35. 3 6 . 3 7. 38. 3 9 . gaillard r.c., b r o w n e l l j., a b e y w i c k r a m a k . & m u l l e r a.f. ( 1 9 8 8 ) cv 2 0 5 5 0 2 , a novel octahydrobenzo(g)quinoline w i t h p o t e n t , specific a n d l o n g l a s t i n g d o p a m i n e a g o n i s t p r o p e r t i e s . in new activities in t h e d o p a m i n e a g o n i s t field ( e d s g.m. b e s s e r ) p 13. p a r t h e n o n p u b l i s h i n g g r o u p , c a m f o r t h uk. g l a s s m.r., s h a w r.w., b u t t w . r . , e d w a r d s r . l . & l o n d o n d.r. ( 1 9 7 5 ) an a b n o r m a l i t y of o e s t r o g e n f e e d b a c k in a m e n o r r h o e a g a l a c t o r r h o e a . b r med j. 3: 2 7 4 . g u a y a.t., f r e e m a n r . , rish b.l., s p e r b e r e. & woolf p.d. ( 1 9 7 8 ) calcified p i t u i t a r y t u m o r w i t h h y p e r p r o l a c t i n e m i a : s e l e c t i v e r e m o v a l by t r a n s s p h e n o i d a l a d e n e c t o m y . f e r t i l s t e r i l . 2 9 : 5 8 5 . g u d e l s k y g.a. & p o r t e r j.c. ( 1 9 8 0 ) r e l e a s e of d o p a m i n e f r o m t u b e r o i n f u n d i b u l a r n e u r o n s i n t o p i t u i t a r y stalk blood a f t e r prolactin or haloperidol a d m i n i s t r a t i o n . e n d o c r i n o l o g y . 106: 5 2 6 . v a n d e r h e i j d e n p.f.m.. rolland r., l a p p o h n r.e., c o r b e i j r.s., d e g o e i j w.b.k.m.v. & b r o w n e l l j . ( 1 9 8 8 ) cv 2 0 5 5 0 2 , a n e w l o n g a c t i n g d o p a m i n e a g o n i s t f o r t h e t r e a t m e n t of h y p e r p r o l a c t i n e m i a : clinical e x p e r i e n c e a n d h o r m o n a l f i n d i n g s i n a 3 m o n t h s t u d y in h y p e r p r o l a c t i n e m i c w o m e n . in new activities in t h e d o p a m i n e a g o n i s t field ( e d s g.m. b e s s e r ) p 2 7 . p a r t h e n o n p u b l i s h i n g g r o u p , c a m f o r t h uk. h w a n g p., g u y d a h . & f r i e s e n h . ( 1 9 7 1 ) a r a d i o i m m u n o a s s a y for h u m a n prolactin. p r o c nat acad sci usa.. 68: 1 9 0 2 . h w a n g p., g u y d a h . & f r i e s e n h . ( 1 9 7 2 ) p u r i f i c a t i o n of h u m a n p r o l a c t i n . j biol c h e m . 2 4 7 : 1 9 5 5 . j e w e l e w i c z r . & v a n d e w i e l e r.l. ( 1 9 8 0 ) c l i n i c a l c o u r s e a n d o u t c o m e of p r e g n a n c y i n t w e n t y f i v e p a t i e n t s w i t h p i t u i t a r y m i c r o a d e n o m a s . am j o b s t e t gynecol. 1 3 6 : 3 3 9 . j o h n s t o n d.g., k e n d a l l t a y l o r p., w a t s o n m . , hall k . , p a t r i c k d. & cook d.b. ( 1 9 8 4 ) e f f e c t of d o p a m i n e a g o n i s t w i t h d r a w a l a f t e r l o n g t e r m t h e r a p y in prolactinomas. t h e l a n c e t . 28: 187. 23 4 0 . kleinberg d.l., boyd a.e., w a r d l a w s., f r a n t z a.g., g e o r g e a , , b r y a n n., hilal s., g r e i s i n g j., h a m i l t o n d. & s e l t z e r t. ( 1 9 8 3 ) p e r g o l i d e for t h e t r e a t m e n t of p i t u i t a r y t u m o r s s e c r e t i n g p r o l a c t i n o r g r o w t h h o r m o n e . n e n g l j med. 3 0 9 : 7 0 4 . 41. klibanski a., n e e r r., b e i t i n s i., r i d g w a y e . & m c a r t h u r j. ( 1 9 8 0 ) d e c r e a s e d b o n e d e n s i t y in h y p e r p r o l a c t i n a e m i c w o m e n . n e n g l j med. 3 0 3 : 1 5 1 1 . 4 2 . l a m s.y., b a k e r h.w.g., s e e m a n e . & p e p p e r e l l r.j. ( 1 9 8 8 ) g y n a e c o l o g i c a l d i s o r d e r s a n d risk f a c t o r s in p r e m e n o p a u s a l w o m e n p r e d i s p o s i n g t o osteoporosis. b r j o b s t e t g y n a e c o l . 9 5 : 9 6 3 . 4 3 . l a n d o l t a . m . & r o t h e n b u l e r v . ( 1 9 7 7 ) p i t u i t a r y a d e n o m a calcification. arch p a t h o l l a b med. 1 0 1: 2 2 . 44. l e b l a n c h., l a c h e l i n g.c.l., abu-fadil s. & yen s.s.c. ( 1 9 7 6 ) e f f e c t s of d o p a m i n e infusion on p i t u i t a r y h o r m o n e s e c r e t i o n s in h u m a n s . .i clin e n d o c r i n o l m e t a b . 4 3 : 6 6 8 . 45. l e i g h t o n p.c., mcneilly a.s. & c h a r d t. ( 1 9 7 6 ) s h o r t t e r m v a r i a t i o n in blood l e v e l s of prolactin in w o m e n . j e n d o c r i n o l . 6 8 : 177. 4 6 . l e w i s u.j., s i n g h n.p. & s e a v e y b.k. ( 1 9 7 1 ) h u m a n p r o l a c t i n : isolation a n d s o m e p r o p e r t i e s . b i o c h e m b i o p h y s r e s c o m m u n . 4 4 : 1 1 6 9 . 4 7 . l y o n s w.r., li c.h. & j o h n s s o n r.e. ( 1 9 5 8 ) t h e h o r m o n a l c o n t r o l of m a m m a r y g r o w t h a n d l a c t a t i o n . i n r e c e n t p r o g r e s s in h o r m o n e r e s e a r c h ( e d s g p i n c u s ) p 2 1 9 . a c a d e m i c p r e s s , new york. 48. m a r c h c.m., kletzky o.a., d a v a j a n v., t e a l j . , w e i s s m., apuzzo m.l.j., m a r r s r.p. & mishell d.r. ( 1 9 8 1 l o n g i t u d i n a l e v a l u a t i o n of p a t i e n t s w i t h u n t r e a t e d p r o l a c t i n s e c r e t i n g p i t u i t a r y a d e n o m a s . am j o b s t e t g y n e c o l . 1 3 9 : 8 3 5 . 4 9 . m a r t i n t.l., kim m . & m a l a r k e y w.b. ( 1 9 8 5 ) t h e n a t u r a l h i s t o r y of idiopathic h y p e r p r o l a c t i n e m i a . j clin e n d o c r i n o l metab. 6 0 : 855. 24 50. 5 1 . 5 2 . 53. 54. 55. 5 6 . 5 7. mattei a.m., f e r r a r i c., r a g n i g., b e n c o r., picciotti m.c., r a m p i n i p., c a l d a r a r. & c r o s i g n a n i p.g. ( 1 9 8 4 ) s e r u m p r o l a c t i n a n d o v a r i a n f u n c t i o n a f t e r d i s c o n t i n u a t i o n of d r u g t r e a t m e n t f o r h y p e r p r o l a c t i n a e m i a : a s t u d y w i t h b r o m o c r i p t i n e a n d m e t e r g o l i n e . b r j o b s t e t g y n a e c o l . 9 1: 2 4 4 . m a x s o n w.s., d u d z i n s k i m . , h a n d w e r g e r s.h. & h a m m o n d c.b. ( 1 9 8 4 ) h y p e r p r o l a c t i n e m i c r e s p o n s e a f t e r b r o m o c r i p t i n e w i t h d r a w a l i n w o m e n w i t h p r o l a c t i n s e c r e t i n g p i t u i t a r y t u m o r s . f e r t i l steril. 41: 2 1 8 . m c n a t t y k.p. ( 1 9 7 9 ) r e l a t i o n s h i p b e t w e e n p l a s m a p r o l a c t i n a n d t h e e n d o c r i n e m i c r o e n v i r o n m e n t of t h e d e v e l o p i n g h u m a n a n t r a l follicle. f e r t i l steril. 3 2 : 433. molitch m.e., e l t o n r.l., b l a c k w e l l r.e., c a l d w e l l b., c h a n g r.f., j a f f e r., joplin g., r o b b i n s r.j., t y s o n j. & t h o r n e r m.o. ( 1 9 8 5 a ) b r o m o c r i p t i n e a s p r i m a r y t h e r a p y f o r p r o l a c t i n s e c r e t i n g m a c r o a d e n o m a s : r e s u l t s of a p r o s p e c t i v e m u l t i c e n t e r s t u d y . j clin e n d o c r i n o l m e t a b . 6 0 : 6 9 8 . m o l i t c h m.e. ( 1 9 8 5 b ) p r e g n a n c y a n d t h e h y p e r p r o l a c t i n e m i c w o m a n . n e n g l j m e d . 3 12: 1 3 6 4 . m o r i o n d o p., t r a v a g l i n i p., nissim m., c o n t i a . & f a g l i a g. ( 1 9 8 5 ) b r o m o c r i p t i n e t r e a t m e n t of m i c r o p r o l a c t i n o m a s : e v i d e n c e of s t a b l e p r o l a c t i n d e c r e a s e a f t e r d r u g w i t h d r a w a l . j clin e n d o c r i n o l m e t a b . 60: 7 6 4 . moult p.j.a., r e e s l.h. & b e s s e r g.m..( 1 9 8 2 ) p u l s a t i l e g o n a d o t r o p h i n s e c r e t i o n in h y p e r p r o l a c t i n a e m i c a m e n o r r h o e a a n d t h e r e s p o n s e to b r o m o c r i p t i n e t h e r a p y . clin endocrinol. 16: 1 5 3 . m u h r c., b e r g s t r o m k., g r i m e l i u s l. & l a r s s o n s.-g. ( 1 98 1 ) a parallel s t u d y of t h e r o e n t g e n a n a t o m y of t h e s e l l a t u r c i c a a n d t h e h i s t o p a t h o l o g y of t h e p i t u i t a r y g l a n d in 2 0 5 a u t o p s y s p e c i m e n s . neuroradiology. 2 1: 55. 25 58. m u l l e r e.e., c o c c h i d., p a m e r a i a.e., gil-ad i . , l o c a t e l l i v . & m a n t e g a s s a p . ( 1 9 7 8 ) p i t u i t a r y h o r m o n e s a n d e r g o t a l k a l o i d s . pharmacology. suppl. 1: 63. 5 9 . n a b a r r o j.d.n. ( 1 9 8 2 ) p i t u i t a r y prolactinomas. clin endocrinol. 17: 1 2 9 . 6 0 . n a c h t i g a l l r.d., monroe s.e., wilson c.b. & j a f f e r.b. ( 1 9 8 1 ) p r o l a c t i n s e c r e t i n g p i t u i t a r y a d e n o m a s in w o m e n . am j o b s t e t gynecol. 140: 3 0 3 . 6 1 . n i l l i u s s.j., r o j a n a s a k u l a . & b e r g h t . ( 1 9 8 5 ) m a n a g e m e n t of prolactinomas in p r e g n a n c y . i n p r o l a c t i n o m a s ( e d s lm auer, g leb, g t s c h e r n e , w u r d l , g f w a l t e r ) p 3 7 3 . w a l t e r d e g r u y t e r , berlin. 6 2 . n o r d m a n n r., f l u c k i g e r e.w., p e t c h e r t.j. & b r o w n e l l j. ( 1 9 8 8 ) e n d o c r i n e a c t i o n s of t h e p o t e n t d o p a m i n e d2-agonist c v 2 0 5 5 0 2 a n d r e l a t e d octahydrobenzo(g)quinolines. d r u g s of t h e f u t u r e . 1 3 , 9 5 1 . 6 3 . p e p p e r e l l r.j. ( 1 9 8 1 ) prolactin a n d r e p r o d u c t i o n . f e r t i l steril. 35: 2 6 7 . 6 4 . p e p p e r e l l r.j., martinez c., dickinson a . ( 1 9 8 3 ) n a t u r a l history of p a t i e n t s w i t h hyperprolactinaemia. ciin reprod a n d fertil. 2: 237. 6 5 . p e t e r s l.l., h o e f e r m.t. & b e n j o n a t h a n n . ( 1 9 8 1 ) t h e posterior pituitary: regulation of a n t e r i o r pituitary prolactin secretion. science. 2 13: 6 5 9 . 6 6 . pontiroli a.e. & falsetti l. ( 1 9 8 4 ) development of pituitary a d e n o m a i n w o m e n w i t h h y p e r p r o l a c t i n a e m i a : c l i n i c a l , e n d o c r i n e , a n d radiological characteristics. br med j. 2 8 8 : 5 15. 6 7 . r i l l e m a j.a., e t i n d i r . n . , o f e n s t e i n j . p . , w a t e r s s.b. ( 1 9 8 8 ) m e c h a n i s m s of prolactin action. i n t h e physiology of reproduction (eds. e knobil & j neil11 p 2 2 1 7 . raven p r e s s , new york. 6 8 . rilliet b., mohr g., r o b e r t f. & h a r d y j . ( 1 9 8 1 ) calcifications i n pituitary a d e n o m a s . surgical neurology. 15: 2 4 9 . 26 6 9 . r j o s k h.k., f a h l b u s c h r. & v o n w e r d e r k. ( 1 9 8 2 ) s p o n t a n e o u s d e v e l o p m e n t of h y p e r p r o l a c t i n a e m i a . acta e n d o c r i n o l ( c o p e n h ) . 1 0 0 : 3 3 3 . 7 0 . roos p., n y b e r g f. & w i d e l. ( 1 9 7 9 ) isolation of h u m a n p i t u i t a r y p r o l a c t i n . biochim b i o p h y s acta. 5 8 8 : 3 6 8 . 7 1. ruiz-velasco v . & tolis g. ( 1 9 8 4 ) p r e g n a n c y in h y p e r p r o l a c t i n e m i c w o m e n . f e r t i l steril. 4 1 : 7 9 3 . 7 2 . s a s s i n j.f., f r a n t z a.g., w e i t z m a n e.d. & k a p e n s. ( 1 9 7 2 ) h u m a n p r o l a c t i n : 2 4 h o u r p a t t e r n w i t h i n c r e a s e d release d u r i n g s l e e p . s c i e n c e . 1 7 7 : 1 2 0 5 . 73. s e p p a l a m . , u n n e r u s h.a., h i r v o n e n e . & r a n t a t . ( 1 9 7 6 ) b r o m o c r i p t i n e i n c r e a s e s p l a s m a e s t r a d i o l 1 7 c o n c e n t r a t i o n i n a m e n o r r h e a p a t i e n t s w i t h n o r m a l s e r u m p r o l a c t i n . j clin e n d o c r i n o l m e t a b . 4 3 : 4 7 4 . 7 4 . s h o m e b. & p a r l o w a.f. ( 1 9 7 7 ) h u m a n p i t u i t a r y p r o l a c t i n ( h p r l ) : t h e e n t i r e l i n e a r a m i n o a c i d s e q u e n c e . j clin e n d o c r i n o l m e t a b . 4 5 : 1 1 1 2 . 7 5 . s i l v e s t r i n i f . , liuzzi a . & c h i o d i n i p . g . ( 1 9 7 8 ) e f f e c t of e r g o t a l k a l o i d s on g r o w t h h o r m o n e a n d p r o l a c t i n s e c r e t i o n in h u m a n s . p h a r m a c o l o g y . s u p p l . 1: 7 8 . 7 6 . s i s a m d.a., s h e e h a n j.p. & s h e e l e r l.r. ( 1 9 8 7 ) t h e n a t u r a l h i s t o r y of u n t r e a t e d m i c r o p r o l a c t i n o m a s . f e r t i l s t e r i l . 4 8 : 6 7 . 77. s o b r i n h o l.g., n u n e s m.c., c a l h a z j o r g e c., m a u r i c i o j.c. & s a n t o s m.a. ( 1 9 8 1 ) e f f e c t of t r e a t m e n t w i t h b r o m o c r i p t i n e on t h e size a n d a c t i v i t y of p r o l a c t i n p r o d u c i n g p i t u i t a r y t u m o u r s . acta e n d o c r i n o l ( c o p e n h ) 9 6 : 2 4 . 78. s u h h.k. & f r a n t z a.g. ( 1 9 7 4 ) size h e t e r o g e n e i t y of h u m a n prolactin in p l a s m a a n d p i t u i t a r y e x t r a c t s . j clin e n d o c r i n o l m e t a b . 3 9 : 9 2 8 . 27 7 9 . t h o r n e r m.o., e d w a r d s c.r.w., c h a r l e s w o r t h m . , d a c i e j.e., moult p.j.a., r e e s l . h . , j o n e s a.e. & b e s s e r g.m. ( 1 9 7 9 ) p r e g n a n c y i n p a t i e n t s p r e s e n t i n g w i t h h y p e r p r o l a c t i n a e m i a . b r med j . 2: 77 1 . 80 8 1 8 2 8 3 84. 85. 8 6 . t h o r n e r m.o., m a r t i n w.h., rogol a.d., m o r r i s j.l., p e r r y m a n r.l., c o n w a y b.p., h o w a r d s s.s., w o l f m a n m.g. & m a c l e o d r.m. ( 1 9 8 0 a ) r a p i d r e g r e s s i o n of p i t u i t a r y p r o l a c t i n o m a s d u r i n g b r o m o c r i p t i n e t r e a t m e n t . j clin e n d o c r i n o l metab. 5 1: 4 3 8 . t h o r n e r m.o., s c h r a n h . f . , e v a n s w.s., rogol a.d., m o r r i s j.l. & macleod r.m. ( 1 9 8 0 b ) a broad s p e c t r u m of prolactin s u p p r e s s i o n by b r o m o c r i p t i n e in h y p e r p r o l a c t i n e m i c w o m e n : a s t u d y of s e r u m p r o l a c t i n a n d b r o m o c r i p t i n e levels a f t e r a c u t e a n d c h r o n i c a d m i n i s t r a t i o n of b r o m o c r i p t i n e . j clin e n d o c r i n o l m e t a b . 50: 1 0 2 6 . t h o r n e r m.o., p e r r y m a n r.l., rogol a.d., c o n w a y b.p., macleod r.m., login i.s. & m o r r i s j.l. ( 1 9 8 1 ) r a p i d c h a n g e s of p r o l a c t i n o m a v o l u m e a f t e r w i t h d r a w a l a n d r e i n s t i t u t i o n of b r o m o c r i p t i n e . j clin e n d o c r i n o l m e t a b . 5 3 : 480. t y s o n j.e., h w a n g p., g u y d a h. & f r i e s e n h.g. ( 1 9 7 2 ) s t u d i e s of p r o l a c t i n s e c r e t i o n in h u m a n p r e g n a n c y . am j o b s t e t g y n e c o l . 113: 1 4 . v a n c e m.l., e v a n s w.s. & t h o r n e r m.o. ( 1 9 8 4 ) b r o m o c r i p t i n e . ann i n t e r n med. 1 0 0 : 7 8 . v e n e t i k o u m . s . , b u r r i n j.m., w o o d s c.a., yeo t . h . , b r o w n e l l j . & a d a m s e . f . ( 1 9 8 7 ) e f f e c t s of t w o n o v e l d o p a m i n e r g i c d r u g s , cv 2 0 5 5 0 2 a n d c q p 2 0 1 4 0 3 , o n p r o l a c t i n a n d g r o w t h h o r m o n e s e c r e t i o n by h u m a n p i t u i t a r y t u m o u r s in v i t r o . a c t a e n d o c r i n o l ( c o p e n h ) . 1 1 6 : 2 8 7 . w a l k e r a.m. & f a r q u h a r m.g. ( 1 9 8 0 ) p r e f e r e n t i a l r e l e a s e of n e w l y s y n t h e s i z e d p r o l a c t i n g r a n u l e s is t h e r e s u l t of f u n c t i o n a l h e t e r o g e n e i t y a m o n g m a m m o t r o p h s . e n d o c r i n o l o g y . 1 0 7 : 1 0 9 5 . 28 87. w a n g c., l a m k.s.l., ma j.t.c., c h a n t., liu m.y. & y e u n g r . t . t . ( 1 9 8 7 ) l o n g t e r m t r e a t m e n t of h y p e r p r o l a c t i n a e m i a w i t h b r o m o c r i p t i n e : e f f e c t of d r u g w i t h d r a w a l . clin e n d o c r i n o l (oxf). 27: 3 6 3 . 88. w e i l c. ( 1 9 8 6 ) t h e s a f e t y of b r o m o c r i p t i n e in h y p e r p r o l a c t i n a e m i c f e m a l e infertility: a l i t e r a t u r e r e v i e w . c u r r med r e s o p i n . 10: 1 7 2 . 8 9 . w e i s s m.h., teal j . , g o t t p., w y c o f f r., yadley r., apuzzo m.l.j., g i a n n o t t a s.l., k l e t z k y 0. & m a r c h c . ( 1 9 8 3 ) n a t u r a l h i s t o r y of m i c r o p r o l a c t i n o m a s : s i x y e a r follow-up. n e u r o s u r g e r y . 1 2 : 1 8 0 . 9 0 . w i d e l. ( 1 9 6 9 ) r a d i o i m m u n o a s s y s e m p l o y i n g i m m u n o s o r b e n t s . acta e n d o c r i n o l ( s u p p l ) ( c o p e n h ) . 1 4 2 : 2 0 7 . 9 1. yen s.s.c. ( 1 9 8 6 ) p r o l a c t i n in h u m a n r e p r o d u c t i o n . in r e p r o d u c t i v e e n d o c r i n o l o g y ( e d s s.s.c. yen a n d r.b. j a f f e ) p 237. s a u n d e r s , p h i l a d e l p h i a . 29 upsala j med sci 79: 63-64, 1974 the amount of zinc detected in washed human spermatozoa ,jan friberg and ove nilsson department of obstetrics and gynecology, and znstitute of human anatomy, university of uppsala, uppsala, sweden abstract samples of human semen, the supernatants from washings of these samples, and the suspensions of washed sperma tozoa, were examined for zinc in a perkin-elmer spectro photometer. almost all the zinc was found in the original semen samples and in the first washings. zinc could not be detected in the heads of fourfold-washed spermatozoa on analysis in a wavelength dispersive x-ray spectrophoto meter attached to a scanning electron microscope. introduction high concentrations of zinc have been demon strated in seminal fluid (1, 8, 9, 11) and in prostatic fluid (2, 6, 7). mawson & fischer (8) performed zinc analysis of the ashed infranatant after centrifugation of human ejaculates and ob served about 2 mg zinc/g dry weight. hall (3) used an x-ray microprobe to examine the zinc content of individual spermatozoa and found a high concentration in the heads of the sperm cells. since unwashed spermatozoa were used, an unintentional contamination of the samples with zinc from the seminal fluid cannot be excluded. this possibility is strengthened by the finding that spermatozoa which were obtained from pa tients with infertility problems and/or suspected prostatitis and then separated by density gradient centrifugation and analysed by atomic absorption spectrophotometry showed a mean zinc concentra tion of 5.5 pg/los spermatozoa (5). t o obtain information about the content of zinc in carefully washed human spermatozoa from normal donors, analyses were performed in the present study both by atomic absorption spectrophotometry and by a wavelength dispersive x-ray spectrophotometer attached to a scanning electron microscope. was previously demonstrated by artificial insemination. two ml of each semen sample (average 40-60 million sperm cells/ml) were spun at 1500 rpm, washed 4 times in 0.01 m phosphate-buffered saline (pbs), p h 7.4, and finally suspended in the same volume of pbs as the original sperm sample. the motility of the spermatozoa was not markedly affected by this treatment. the semen samples, the supernatants from each washing, and one lot of the fourfold-washed sperm suspension, were subjected to zinc analysis in a perkin-elmer spectro photometer using the niethod described by parker (10) and kahnke (4). small drops of suspended, washed spermatozoa were placed o n a stage of pure aluminium adapted for a jeol smu-3 scanning electron microscope. the heads of single spermatozoa were then examined for their zinc content by means of a wavelength dispersive x-ray spectrophoto meter. the microscope was operated at 25 k v with 0.01 pa and 10 cps f o r full scale o n the recorder. the minimum detection level of zinc under the conditions used was calculated to be about 1 x gram zinc. results zinc analyses of the sperm samples, the super natants from each washing, and the final sperm suspensions showed that almost all the zinc was found in the original semen samples and the supernatants from the first washings. the con centration of zinc in the final solution of four fold-washed spermatozoa was about 9 x 10-8 mol/ ml or about 12 pg/108 spermatozoa. no zinc couid be demonstrated in the heads of individual spermatozoa when investigated by means of the dispersive x-ray spectrophotometer attached to the scanning electron microscope. this is only to be expected since the amount of zinc in a single washed spermatozoon was about 1 >: 10-13 gram. discussion the in the present study indicate that only minute amounts of zinc can be present in upsala j med sci 79 material and methods two masturbated ejaculates from each of 4 fertile donors were used for the present study, the fertility of the donors 64 j . friberg and 0. nilsson the sperm cells. the high zinc concentrations previously ascribed to spermatozoa were probably due t o contamination with zinc from the seminal fluid. the minute amounts of zinc present in human spermatozoa, according t o the results in this study, may derive from contamination by epithelial cells and cellular debris or from the carbonic anhydrase contained in the spermatozoa. the experiments do not exclude the possibility that some zinc may have leaked out from the spermatozoa during the washing procedures. the healthy donors were found to have a zinc concentration of about 12 p g / 10s spermatozoa. this figure lies in the upper range of values ob tained from patients with infertility problems a n d / o r suspected prostatitis (mean 5.5 p g / los spermatozoa, range 0.20-12.63) (5). however, the results are sufficiently similar to permit the con clusion that the zinc concentration of human spermatozoa is low, lying around some pg/los spermatozoa. acknowledgement d r h. ulfendahl is gratefully acknowledged for the analyses by the atomic absorption spectrophotometer and mr k. ayaghi, jeol europe, for the analysis by the dis persive x-ray spectrophotometer. this study was supported by the swedish medical research council (project no. 12x-70) and by the ford foundation (grant no. 66-405 to professor carl gemzell). references 1. eliasson, r. 8; lindholmer, c.: zinc in human seminal plasma. andrologie 3: 147, 1971. 2. gyorkey, f., min, k.-k'., huff, j. a. & gyorkey, p.: zinc and magnesium in human prostate gland: normal, hyperplastic and neoplastic. cancer res 27: 1348, 1967. 3. hall, t. a.: the microprobe analyses of zinc in mammalian sperm cells. in optique des rayons x et microanalyse (ed. r. castaing & p. deschamps), pp. 679-686. j. philibert hermann, paris, 1966. 4. kahnke, m. j.: atomic absorption spectorphotometry applied to the determination of zinc in formalinized human tissue. atomic absorption newsletter 5: 7, 1966. 5. lindholmer, c. & eliasson, r.: zinc and magnesium in human spermatozoa. in1 j fert 17: 153, 1972. 6. mackenzie, a. r., hall, t. & whitmore, w. f.: zinc content of expressed human prostatic fluid. nature 193: 72, 1962. 7. mawson, c. a. & fischer, m. i.: the occurrence of zinc in the human prostate gland. can j med sci 30: 336, 1952. upsala j m e d sci 79 8 . 9. 10. 11. zinc and carbonic anhydrase in human semen. biochem j 55:696, 1953. zinc in aspermic human semen. nature, 177: 190, 1956. parker, h. e.: magnesium, calcium and zinc in animal nutrition. atomic absorption newsletter 2: 23, 1963. schirren, c., beltermann, r., haensch, m., kohn, d. & lossin, j.: biochemische untersuchungen am menschlichen spermaplasma: zincund phosphohexose isomerase-aktivitat. arch klin exp dermatol 218: 323, 1964. received m a y 4, 1973 address for reprints: jan friberg department of obstetrics and gynecology university hospital s-750 14 uppsala 14 sweden upsala j med sci 91: 269-272, 1986 application of whole-body autoradiography to distribution studies of volatile substances kerstin bergman toxicology laboratory, national food administration, box 622, s-75126 uppsala, sweden introduction exposure to volatile substances of toxicological significance takes place most frequently in occupational settings (e.g. organic solvents, plastics monomers), but volatile substances may be encountered also in e.g. clinical practice (anesthetic agents) or as constituents of tobacco smoke or food. the volatility of a chemical substance at a certain temperature is expressed by its vapour pressure. conventional whole-body autoradiographic techniques cannot be used for distribution studies of compounds exerting significant vapour pressures at or above -20 c. the autoradiographic registration of volatile substances can only be accomplished by working at low temperatures. theoretically, evaporation occurs at all temperatures above absolute zero, but a negligible evaporation can be said to occur when the vapour pressure is less than 0.5 mm hg (3). today, most low-temperature auto radiography is performed at temperatures around -8ooc, which are sufficient to lower the vapour pressure of e.g. most organic solvents to 0.5 mm hg or less. 0 low-temperature autoradiography of volatile substances the preparation of a whole-body autoradiogram requires a flat surface to be pressed against x-ray film and, since thin sections warm up more easily than thick ones, all low-temperature autoradiographic methods found in the literature describe techniques to flatten the frozen animal resulting in thick slabs or slices ( 3 ) . after rapid freezing, usually in liquid nitrogen, the animal is milled down or sawn sagittally to a body level suitable for auto radiography. improved sawing equipment has allowed the preparation of 5-6 sections ( 2 3 mm thick) from a mouse, both sides of which can be pressed against x-ray film ( 3 ) . film exposure must take place at a low temperature, e.9. over solid carbon dioxide or in a low-temperature freezer. 269 conventional autoradiography of volatile substances whole-body autoradiography, like any other tracer technique, does not supply any information about the nature of the registered radioactivity, which may belong both to the administered substance and/or to its metabolites. however, when doing autoradiography of volatile substances it is possible to take advantage of their volatility to distinguish between the volatile substance itself and its usually non-volatile metabolites. low-temperature autoradiography registers the total radioactivity, i.e. both volatile and non-volatile radioactivity (autoradiogram a). if conventional autoradiography with tape-fastened and freeze-dried 20 sections is used, the volatile parent substance will evaporate and only the non-volatile metabolites are registered (autoradiogram b). the tape-fastened sections may be taken from the thick sections, after their film exposure at low temperature, or from a separate series of animals. to ensure a complete evaporation of volatile radioactivity the thin sections may be warmed carefully prior to film exposure (3) . low-temperature autoradiography in combination with conventional auto radiography has been successfully applied to distribution studies of several organic solvents (2,3,9,11,12), anesthetic agents (6,7,8,10), ethanol (l), dimethyl mercury (14) and volatile n-nitroso compounds (5,131. whole-body autoradiography of non-extractable metabolites some low-temperature autoradiographic investigations have included the study of covalently bound metabolites, which are associated with tissue injury (2,3,4,9,10,11,12,13). an extraction procedure for tape-fastened sections, which removes non-bound radioactivity, has been developed (3). usually, pairs of one extracted and one non-extracted section are exposed and developed together in order to enable an estimate of the amount of bound radioactivity in relation to total non-volatile radioactivity (autoradio gram c). conclusions the technique of low-temperature autoradiography has been successfully applied to distribution studies of several important groups of volatile substances, e.g. organic solvents, anesthetic agents and n-nitroso compounds. it has been possible to take advantage of the volatility of these substances in order to separate between the substances themselves and their non-volatile metabolites, which can be registered with conventional whole cerebellum spinal nerves lung bone marrow meninges liver €at epididymis testis nasal mucosa lung kidney pancreas salivary gland liver intestinal contents n a s a l mucosa bronchi kidney liver whole-body autoradiography of 14c-chloroform (survival time 2 hours) a . low-temperature (-8ooc) autoradiography: registration of total radioactivity b. c o n v ~ n t ~ o n a l autoradiography: registration of non-volatile metabolites. c. a ~ ~ o r a d ~ o g r a f ~ y of an extracted section: registration of covalently bound (reprinted from acta pharmacol toxicol,4l(suppl.l):22-23,1977. with permission) (chloroform and non-volatile metabolites) metabolites. 271 body autoradioqraphy. references 1. akesson, c. : autoradiographic studies on the distribution of 4c-2-ethanol and its non-volatile metabolites in the pregnant mouse. arch int pharmaco dyn th6r 209: 296-304, 1974. distribution and elimination studies of some organic solvents. scand j work environ health 5 (suppl.1): 1-263, 1979. distribution studies of organic solvents. crc crit rev toxicol 12: 59-118, 1983. 2. berqman, k.: whole-body autoradiography and allied tracer techniques in 3. berqman, k.: application and results of whole-body autoradioqraphy in 4. 5. 6. 7. 8. 9. 10. 11. 12. berqman, k., danielsson, b.r.g. & d'arqy, r.: tissue disposition of carbon disulf ide. i. whole-body autoradiography of 5sand c-labelled carbon disulfide in adult male mice. acta pharmacol toxicol 54: 141-150, 1984. brittebo, e.: distribution and metabolism of some n-nitroso compounds. acta universitatis upsaliensis, abstracts of uppsala dissertations from the faculty of pharmacy, 44, 1979. cohen, e.n. & hood, n.: application of low-temperature autoradioqraphy to studies of the uptake and metabolism of volatile anesthetics in the mouse. i. chloroform. anesthesiology 30: 306-314, 1969. cohen, e.n. & hood, n.: application of low-temperature autoradioqraphy to studies of the uptake and metabolism of volatile anesthetics in the mouse. 11. diethyl ether. anesthesiology 31: 61-68, 1969. cohen, e.n. & hood, n.: application of low-temperature autoradiography to studies of the uptake and metabolism of volatile anesthetics in the mouse. 111. halothane. anesthesiology 31: 553-559, 1969. danielsson, b.r.g., bergman, k. & d'arqy, r.: tissue disposition of carbon disulfide. 11. whole-body autoradioqraphy of disulfide in pregnant mice. acta pharmacol toxicol 55: 410-417, 1984. danielsson, b.r.g., ghantous, h. & dencker, l.: accumulation in murine amniotic fluid of halothane and its metabolites. acta pharmacol toxicol 55: 410-417, 1984. ghantous, h., danielsson, b.r.g., dencker, l., gorczak, j. & vesterberg, 0.: trichloroacetic acid accumulation in murine amniotic fluid after tri and tetrachloroethylene inhalation. acta pharmacol toxicol 58: 104-114, 1986. inqebrigtsen, k. & walde, a. : the distribution of (tbt) following inhalation by the rat: a whole-body autoradioqraphic study. acta pharmacol toxicol 51: 203-208, 1982. sand "c-labelled carbon 'c-p-tert. -butyltoluene 13. lofberq, b.: tissue specificity of n-nitrosoamine metabolism: studies in pregnant and non-pregnant rodents. acta universitatis upsaliensis, comprehensive summaries of uppsala dissertations from the faculty of pharmacy 4, 1985. 14. gstlund, k.: studies on the metabolism of methyl mercury and dimethyl mercury in mice. acta pharmacol toxicol 27 (suppl.1): 47-50, 1969. address for reprints: kerstin berqman toxicology laboratory national food administration box 622 s-751 26 uppsala, sweden 272 upsala j med sci 90: 127-132, 1985 intravesical ethoglucid (epodylr) for weatment of noninvasive bladder cancer (stage ta) anders larson and ake fritjofsson department of urology, university hospital, uppsala, sweden abstract widespread, we 1 d i f f e r e n t i a t e d ( g r a d e i) b l a d d e r turnours c o n f i n e d t o t h e rnucosa ( s t a g e ta) were t r e a t e d w i t h r e g u l a r i n t r a v e s i c a l i n s t i l l a t i o n s o f e t h o g l u c i d ( e p o d y l ) n 24 p a t i e n t s . the t h e r a p e u t i c s c h e d u l e c o u l d b e f o l l o w e d i n a l l b u t one p a t i e n t , i n whom s i d e e f f e c t s n e c e s s i t a t e d c e s s a t i o n o f t r e a t m e n t . complete r e s p o n s e was o b t a i n e d i n 7 5 76 o f t h e p a t i e n t s , and d u r i n g c o n t i n u e d p r o p h y l a c t i c t h e r a p y 90 5 remained t u m o u r f r e e . a f t e r t e r m i n a t i o n o f t h e t r e a t ment,however, new tumours appeared i n 60-80 % o f t h e p a t i e n t s . r i n t r o d u c t i o n s u p e r f i c i a l b l a d d e r tumours ( s t a g e ta and t l ) have a l w a y s been r e g a r d e d w i t h some d i s t r u s t because o f t h e h i g h r e c u r r e n c e r a t e a f t e r e n d o s c o p i c r e s e c t i o n o r f u l g u r a t i o n . moreover, tumours d e v e l o p i n g a f t e r r e s e c t i o n have sometimes been o f h i g h e r s t a g e o r grade. c o n s e q u e n t l y , t h e e v o l u t i o n o f a n t i t u m o u r chemother a p e u t i c d r u g s f o r i n t r a v e s i c a l use a r o u s e d e x p e c t a t i o n s o f more permanent c u r e o f s u p e r f i c i a l b l a d d e r tumours a s an a l t e r n a t i v e t o c o n v e n t i o n a l e n d o s c o p i c r e s e c t i o n or as p r o p h y l a c t i c a d j u v a n t t o t r a n s u r e t h r a l t r e a t m e n t . d u r i n g t h e p a s t 25 y e a r s s e v e r a l c y t o t o x i c a g e n t s have come i n t o use. numerous s t u d i e s have been done t o d e t e r m i n e w h i c h p a t i e n t s s h o u l d r e c e i v e chemotherapy, when t o i n i t i a t e t r e a t m e n t , and what a g e n t , dose, t r e a t m e n t s c h e d u l e and d u r a t i o n o f t r e a t m e n t a r e most a p p r o p r i a t e f o r s u p e r f i c i a l b l a d d e r tumours. d e f i n i t i v e an swers t o t h e s e q u e s t i o n s a r e s t i l l a w a i t e d . t h i o t e p a seems t o have been t h e f i r s t a g e n t u t i l i z e d f o r t h i s purpose. a l though h i g h l y e f f e c t i v e i n some cases o f h i g h l y d i f f e r e n t i a t e d b l a d d e r tumour, t h i s d r u g has n e v e r a c h i e v e d p o p u l a r i t y , m a i n l y because o f i t s s e r i o u s s i d e e f f e c t s , t h e c h i e f o f w h i c h i s rnyelosuppression due t o a b s o r p t i o n t h r o u g h t h e b l a d d e r rnucosa. r e t h o g l u c i d ( e p o d y l ) , i n t r o d u c e d i n 1971 b y r i d d l e & w a l l a c e ( 9 ) , i s an o t h e r d r u g used f o r t o p i c a l chemotherapy. i t i s an a l k y l a t i n g a n t i n e o p l a s t i c 127 a g e n t with h i g h e r m o l e c u l a r w e i g h t t h a n t h i o t e p a . systemic s i d e e f f e c t s a r e un common, presumably because none or v e r y l i t t l e o f t h e d r u g i s absorbed from t h e b l a d d e r . a l t h o u g h new d r u g s such as a d r i a m y c i n ( d o x o r u b i c i n h y d r o c h l o r i d e ) and m i t o m y c i n c have w e l l e s t a b l i s h e d e f f i c a c y a g a i n s t s u p e r f i c i a 1 b l a d d e r c a n c e r , e p o d y l i s s t i l l e x t e n s i v e l y used i n many c e n t r e s , e s p e c i a l l y i n europe, a s s t a n d a r d t r e a t m e n t o f s u p e r f i c i a l tumours o f t h e b l a d d e r , the aim o f t h i s s t u d y was t o d e t e r m i n e t h e t h e r a p e u t i c e f f e c t o f i n t r a v e s i c a 1 e p o d y l on s u p e r f i c i a l p a p i l l a r y c a n c e r o f t h e b l a d d e r , w i t h o u t i n v a s i o n i n t o t h e l a m i n a p r o p r i a ( s t a g e ta) and o f l o w m a l i g n a n c y g r a d e ( g r a d e 1). material and methods the s e r i e s c o m p r i s e d 24 p a t i e n t s , 2 1 men (mean age 66, r a n g e 33-85 y e a r s ) and t h r e e women (aged 68, 76 and 8 1 ) . a l l p r e s e n t e d w i t h e x t e n s i v e b l a d d e r tu mours. i n 1 2 cases t h e s e tumours were r e c u r r e n c e s a f t e r f r e q u e n t t r a n s u r e t h r a l r e s e c t i o n and/or f u l g u r a t i o n f o r p a p i l l o m a . the o t h e r 1 2 p a t i e n t s had n o t p r e v i o u s l y r e c e i v e d t r e a t w e n t . i n a l l t h e p a t i e n t s s u r g i c a l b i o p s y was t a k e n f o r p a t h o l o g i c c l a s s i f i c a t i o n o f t h e tumour, w h i c h o t h e r w i s e was l e f t i n t a c t . to make t h e s e r i e s as homogeneous a s p o s s i b l e , o n l y p a t i e n t s with tumours c l a s s i f i e d as s t a g e ta (uicc), g r a d e i (who) were i n c l u d e d i n t h e s t u d y . f o r t r e a t m e n t w i t h e p o d y l ( i c i , u k ) , t h e r i d d l e w a l l a c e ( 9 ) s c h e d u l e was used. a s o l u t i o n o f 1 m l e p o d y l i n 100 m l d i s t i l l e d w a t e r was i n s t i l l e d with a g l a s s s y r i n g e i n t o t h e empty b l a d d e r , and t h e s o l u t i o n was r e t a i n e d for one h o u r . the s c h e d u l e f o r i n s t i l l a t i o n s was a s f o l l o w s : once weekly for 3 months, t h e n f o r t n i g h t l y f o r 3 months, t h e n m o n t h l y f o r 1 year, t h e n m o n t h l y t o e v e r y t h i r d month. the t r e a t m e n t was g i v e n on an o u t p a t i e n t b a s i s . c y s t o s c o p i c checks were made e v e r y t h i r d month. results the r e s u l t s a r e s u r v e y e d i n f i g . 1. complete r e g r e s s i o n (cr) was o b t a i n e d 128 n c .rl a, u a, ci ci 3 a, ci 0 z 4 m c 4.j c e n m 3 m ci e r n .d a, x a, o v ) a , i ci c i m 3r oc, a c c i o e 0 z m 0, .ri ci 3 d a, o u c c m a , c i e cic, 3 m 4 129 i n 1 8 o f t h e 24 p a t i e n t s a f t e r 3 t o 6 months o f t h e r a p y . e l e v e n o f t h e 1 8 re c e i v e d f u r t h e r p r o p h y l a c t i c t r e a t m e n t f o r p e r i o d s r a n g i n g f r o m 1 t o 5 y e a r s , and d u r i n g t h e s e p e r i o d s 10 o f t h e p a t i e n t s remained f r e e f r o m turnour. a f t e r t h e t r e a t m e n t was stopped, however, s i x had r e c u r r e n c e , a l l w i t h i n 1 8 m o n t h s . four p a t i e n t s have remained t u m o u r f r e e d u r i n g f o l l o w u p f o r p e r i o d s o f 3 months t o 6 y e a r s . one p a t i e n t showed r e c u r r e n c e o f tumour w h i l e u n d e r g o i n g p r o p h y l a c t i c t r e a t m e n t . f i v e o f t h e 18 p a t i e n t s w i t h c r were n o t s u b s e q u e n t l y g i v e n p r o p h y l a c t i c t r e a t m e n t . i n f o u r o f them t h e r e was r e c u r r e n c e o f tumour a f t e r 3 t o 9 months, w h i l e one has remained t u m o u r f r e e d u r i n g two y e a r s o f o b s e r v a t i o n . i n 6 o f t h e 24 p a t i e n t s t h e e p o d y l i n s t i l l a t i o n s d i d n o t r e s u l t i n cr. two o f t h e 18 p a t i e n t s w i t h cr f a i l e d t o a t t e n d for f o l l o w u p i n v e s t i g a t i o n . l o c a l s i d e e f f e c t s i n t h e form o f f r e q u e n c y o f m i c t u r i t i o n and d y s u r i a were e x p e r i e n c e d by 1 0 ( 4 0 % ) o f t h e p a t i e n t s , b u t n e c e s s i t a t e d d i s c o n t i n u a t i o n o f t r e a t m e n t o n l y i n one. there were no s y s t e m i c s i d e e f f e c t s . a l l t h e p a t i e n t s w i t h r e c u r r e n c e o f tumour have r e c e i v e d f u r t h e r t r e a t m e n t , w i t h i n t e n s i f i e d t r a n s u r e t h r a l r e s e c t i o n and/or a d d i t i o n a l chemotherapy. discussion i n t h i s series o f p a t i e n t s , i n t r a v e s i c a l i n s t i l l a t i o n s o f e p o d y l f o r wide spread, n o n i n v a s i v e ( s t a g e ta), h i g h l y d i f f e r e n t i a t e d ( g r a d e i ) b l a d d e r c a n c e r i n i t i a l l y gave c o m p l e t e e r a d i c a t i o n o f tumour i n 75 % o f t h e cases. when t h e c u r a t i v e t r e a t m e n t was n o t superseded b y p r o p h y l a c t i c chemotherapy, however, new tumours appeared i n 80 % o f t h e p a t i e n t s w i t h i n 3 t o 9 months. when p r o p h y l a x i s was g i v e n , 90 x o f t h e p a t i e n t s remained t u m o u r f r e e d u r i n g t h e t r e a t m e n t p e r i o d . a f t e r t e r m i n a t i o n o f t h e p r o p h y l a x i s , 60 l o f t h e s e p a t i e n t s a l s o sooner or l a t e r showed new tumours. comparison o f o u r r e s u l t s w i t h t h o s e o f o t h e r i n v e s t i g a t o r s i s n o t easy, s i n c e such s t u d i e s commonly do n o t d e s c r i b e i n d e t a i l how many o f t h e s o c a l l e d s u p e r f i c i a l tumours were, i n f a c t , i n s t a g e ta or t 1 , how many were p a p i l l a r y tumours o r p r i m a r y c a r c i n o m a i n s i t u and how many p a t e n t s had been g i v e n epo d y l as t r e a t m e n t w i t h c u r a t i v e i n t e n t or a s p r o p h y l a x s f o l l o w i n g t r a n s u r e t h r a l tumour r e s e c t i o n . moreover, f i g u r e s f o r r e s p o n s e r a t e have sometimes i n c l u d e d b o t h c o m p l e t e and p a r t i a l r e g r e s s i o n o f tumour. i n some s e r i e s r e p o r t e d i n t h e l i t e r a t u r e , however, s u p e r f i c i a l b l a d d e r tumours were managed e s s e n t i a l l y as i n o u r p a t i e n t s , w i t h i n t r a v e s i c a l i n s t i l l a t i o n s o f e p o d y l g i v e n a s t h e r a p y and n o t s i m p l y as complement t o endo s c o p i c p r o c e d u r e s . i n comparison w i t h such series, o u r f i g u r e s f o r c o m p l e t e 130 tumour r e g r e s s i o n l l , l 2 ) , e q u a l t h o s e f o r m i t o m y c i n c (41, and a r e c l e a r l y s u p e r i o r t o r e s u l t s w i t h t h i o t e p a (3,7,14) and a d r i a m y c i n ( 2 , 5 ) . e q u a l or exceed t h o s e s t a t e d f o r e p o d y l t r e a t m e n t (1,9,10, our series comprised o n l y p a t i e n t s w i t h p r e c i s e l y d e f i n e d p a t h o l o g i c s t a g e o f tumour, ta. this may e x p l a i n t h e more f a v o u r a b l e response r a t e among o u r p a t i e n t s t h a n i n o t h e r s e r i e s , w h i c h i n c l u d e d t i s , a tumour w i t h p r o g n o s i s o f t e n much worse t h a n f o r o t h e r s u p e r f i c i a l tumours. the o b s e r v a t i o n t h a t new tumours so o f t e n arise a f t e r t e r m i n a t i o n o f i n i t i a l t r a n s u r e t h r a l r e s e c t i o n and/or i n t r a v e s i c a l chemotherapy t h a t has e l i m i n a t e d e x i s t i n g tumours s u p p o r t s t h e t h e o r y p o s t u l a t e d by soloway ( 1 3 ) and o t h e r s , v i z . t h a t t h e l a t e r l e s i o n s a r e n o t " r e c u r r e n c e s " b u t a r e i n d e e d new, p o s s i b l y produced by c a r c i n o g e n s or o t h e r , s t i l l obscure a e t i o l o g i c f a c t o r s . l o c a l s i d e e f f e c t s o c c u r r e d i n 40 5 o f o u r p a t i e n t s , e s p e c i a l l y d u r i n g t h e f i r s t phase o f t r e a t m e n t , when t h e i n s t i l l a t i o n s had t o be f a i r l y f r e q u e n t . b u t o n l y i n one case were t h e s i d e e f f e c t s so s e v e r e a s t o n e c e s s i t a t e d i s c o n t i n u a t i o n o f t h e t r e a t m e n t . i n t h e o t h e r p a t i e n t s f r e q u e n c y and d y s u r i a were t r a n s i e n t , and a f t e r some a d j u s t m e n t o f dosage and t r e a t m e n t s c h e d u l e t h e i n s t i l l a t i o n s c o u l d be c o n t i n u e d . our r e s u l t s i n t h i s r e s p e c t c o n t r a s t f a v o u r a b l y with e a r l i e r r e p o r t e d o b s e r v a t i o n s t h a t e p o d y l t r e a t m e n t had t o b e s t o p p e d be cause o f s e r i o u s s i d e e f f e c t s i n 5 t o 25 % o f cases ( 8 ) . much a t t e n t i o n has been g i v e n t o t h e r i s k t h a t tumours may d e v e l o p as a com p l i c a t i o n o f chemotherapy ( 6 ) . i n o u r s t u d y , however, t h e h i s t o l o g i c appearance o f new tumours a p p e a r i n g d u r i n g or a f t e r t h e p r o p h y l a c t i c t r e a t m e n t d i d n o t d i f f e r f r o m t h a t i n t h e i n i t i a l turnours, a l t h o u g h i n some cases t r e a t m e n t h a d been g i v e n f o r up t o 5 f y e a r s . i n c o n c l u s i o n , o u r s t u d y showed t h a t p a t i e n t s w i t h s u p e r f i c i a l b l a d d e r t u mours o f s t a g e ta, g r a d e i a r e s u i t a b l e f o r i n t r a v e s i c a l chemotherapy. compared w i t h o t h e r c h e m o t h e r a p e u t i c a g e n t s , e p o d y l has p r o v e d f a v o u r a b l e f o r t h i s p u r pose. the advantages o f e p o d y l a r e i t s documented e f f e c t i v e n e s s , r e l a t i v e l a c k o f s i d e e f f e c t s and f a i r l y l o w c o s t . references 1. c o l l e e n , s., ek, a . , h e l l s t e n , s. & lindholm, c.-e.: i n t r a c a v i t a r y e p o d y l f o r m u l t i p l e , n o n i n v a s i v e , h i g h l y d i f f e r e n t i a t e d b l a d d e r tumours. scand j urol n e p h r o l 14:43-45,1980. 2. edsmyr, f., b e r l i n , t . , boman, j . , duchek, m . , e s p o s t i , p.l., g u s t a f s s o n , h. w i j k s t r o m , h. & c o l l s t e , l.c.: i n t r a v e s i c a l t h e r a p y w i t h s u p e r f i c i a l b l a d d e r tumours. e u r u r o l 6: 132-136,1980. tumours b y i n s t i l l a t i o n o f t h i o t e p a and 5 f l u o r a c i l i n v e s t u r o l 2: 381, 1966. 3 . e s q u i v a l , e.l. mackenzie, a.r. & whitmore, w.f.: treatment o f b l a d d e r 131 4. 5. 6 . 7. 8. 9. 10. 11. 12. 13. 14. harrison, g.s.m., green, d.f., newling, d.w.w., richar'ds, b., robinson, m.r.g. & smith, p.h.: a phase i1 study of intravesical mitomycin c in the treatment of superficial bladder cancer. br j urol 55: 676-679,1983. jauhiainen,k. & alfthan,o.: die behandlung von carcinoma in situ der , harnblase mit lokaler adriamycininstillation. akt urol 15: 129-133, 1984. lunglmayr, g.: zur frage der cytostatischen recifivprofylaxe von ober flachlichen blasentumoren. der urologe: a 11, 94, 1972. mitchell, r.j.: intravesical thiotepa in the treatment of transitional cell bladder carcinoma. br j urolr43: 185, 1971. nielsen, h.v. & thybo, e.: epodyl treatment o f bladder tumours. scand j urol nephrol 13: 59, 1979. riddle, p.r. & wallace, d.m.: intracavitary chemotherapy f o r multiple non invasive bladder tumours, br j urol 43: 181-184, 1971. riddle, p.r.: the management o f superficial bladder tumours with intra vesical epodyl. br j urol 45: 84-87, 1973. robinson, m.r.g., sbetty, m.b, bastable, j., glashan, r.w. & smith, p.h.: intravesical epodyl in the management of bladder turnours: combined ex perience of the yorkshire urological cancer research group. j. urol 118: 972,1977. smith, j.m., lane, v. & o'flynn, j.d.: epodyl in management of non invasive vesical neoplasms. urology 11: 474, 1978. soloway, m.s.: intravesical and systemic chemotherapy in the management of superficial bladder cancer. urol clin north am ll,no 4,1984. veeneman,r.j., dean, a.l., uson, a.c., roberts, m. & longo, f: thiotepa bladder instillations; therapy and prophylaxis o f superficial bladder tumours. j urol 101: 711, 1969. r . address for reprints: ake fritjofssan department of urology university hospital 5-751 85 uppsala sweden 132 upsala j med sci 82: 221-230, 1977 the motherkhiid dyad-nutritional aspects symposium organized by the swedish nutrition foundation and uppsala university, uppsala, june 20-22, 1977 abstracts i. introductory lectures chairman: b . vahtquist 1. the physiology of pregnancy and lactation by a. m. thomson and f. e. hytten the history of the development of this subject will be reviewed briefly, with special reference to the personal experience of the authors. reliable infor mation on nutritional physiology is mostly rather recent, and many of the earlier ideas, based on misleading ideas and scanty data, have proved to be erroneous. comments will be made on practical aspects of physiological research on pregnant and lactating women. 2. psycho-social aspects of the mother/ father/child unit by n. newton attachment, bonding, engrossment are terms used to describe the behavior of the father-mother infant unit in the early weeks of life. all three members of the triad may have a basic falling in love experience, but biological differences modify the way such interest is shown by each member of the triad. often overlooked is the fact that primary family bonding is reinforced through the ongoing pleasures of skin contact, breast feeding and coitus. these primary love relationships may flourish best when protected from undue stress by an affection ate cooperative wider social network. 11. nutritional needs of t h e mother and the child chairman: r . g . whitehead 1. nutritional needs of the pregnant and lactating mother by g. h. beaton the definition of nutritional needs during preg nancy and lactation remains as much an art as a science. in many areas, such as trace element re quirements, our knowledge is very fragmentary and based largely on animal studies of the effects of deficiency rather than the prediction of need. even in the well-studied areas of human nutrition, opin ions remain divided about requirements in this period. three basic approaches have been fol lowed, often with discordant results: (i) a factorial approach based upon a knowledge of the physio logy of pregnancy and lactation coupled with esti mates of the nutrients deposited in or utilized by the foetus and infant, (ii) an experimental approach based upon feeding trials with a variety of ‘end points’ used t o assess adequacy of intake, and (iii) an epidemiologic approach in which the end point is usually birth weight and infant growth rate. by the first approach, only modest increases in protein requirements are estimated in pregnancy, nitrogen balance studies suggest that appreciably higher amounts of nitrogen can be deposited during preg nancy than can be accounted for in the factorial models; feeding trials suggest that energy intake may be the important determinant of birth weight if protein intakes are at all reasonable. if one accepts that haemodilution and fall in circulating haemoglo bin levels are a normal part of pregnancy, then the factorial approach suggests that iron requirements in the previously well nourished woman undergo relatively small changes during pregnancy. con versely, administration of large amounts of iron can maintain a higher level of haemoglobin in the ma ternal circulation. which approach is correct? it is the position of the present author that nutrient in takes during pregnancy and lactation should be adequate to ( a ) permit adequate growth and “com position” of the foetus and infant and ( b ) ensure that the mother entering pregnancy in a well nourished state will end lactation in an equally well nourished state. criteria of assessment of requirement should be based upon these objectives; criteria applied dur ing pregnancy and lactation must take into account upsala f med sri82 222 abstracts the normal physiological adjustments of the mater nal organism. in practice it is to be recognized that in addition to the normal requirements of pregnancy as defined above, there may also be a therapeutic requirement for rehabilitation of the previously un dernourished mother. it is well documented that a number of physiolog ical adjustments take place during pregnancy and lactation. usually these have the effect of minimiz ing maternal needs/improving the efficiency of the maternal organism and further assuring the ade quacy of nutrient supply for the foetus (even at the expense of maternal tissues if total intake is inade quate). seemingly the foetus functions as an effec tive “parasite”. energy intake and utilization seems to be an important exception. in the normal cycle of pregnancy and lactation considerable energy stores are deposited during pregnancy in anticipation of the needs of lactation. if energy intakes are inade quate the stores are smaller, but so too is the foetus. the “parasitic” theory does not hold. 2. nutritional needs of the foetus and the young child by e. m. widdowson one of the most economical processes from the energy point of view is the making of a baby. the biggest component in the amount of energy required is the gain in maternal tissues; the energy value of the protein and fat in the foetal body at term ac counts for less than 10% of the total. the require ments of energy for new body tissue expressed as kcal/kg day are fractionally higher ‘during the two months before birth than they will ever be again. the organism is becoming larger all the time, how ever, and the total energy requirements for new body tissue are higher for the 2 months after birth than for the two months before. there is also an in crease in energy requirement for maintenance after birth, partly due to losses of nutrients by the bowel but, more important, because energy is needed to maintain the body temperature in an environment that is generally cooler than the uterus. the amount of protein laid down in the body tissues per day is higher during the last two months of gestation than after birth. calcium and phos phorus are also retained by the body at a greater rate before birth than after if the infant is fed on human milk. balance studies suggest that this is not so if the food is whole cow’s milk, but it is difficult to be sure about the observations. the small preterm infant lacks body fat and its dominating requirement is for energy and protein. problems about supplies of calcium and phosphorus for these infants will be discussed. 111. non-nutritional diseases influencing the health o f t h e pregnant mothers and their offspring chairman: r . g. hendrickse 1. antenatal bacterial infection-the role of nutrition in a leading cause of death by n. tafari and r. l. naeye a recent study of excessive perinatal mortality of 66/ 1 000 live births among 25 000 pregnancies in a preindustrial community (ethiopia) identified causes of death in over95% examined by necropsy. the fetal : neonatal death ratio was 2.7 : 1 indicat ing that most of the diseases had maternal origin. amniotic fluid infection syndrome was the leading cause of death with a rate of 21.8 per 1000 live births, a rate three times higher than that seen in an industrial community (usa). the infection was highest in the most poor of gravida, in those en gaged in hard physical work during pregnancy and in twins. the incidence of the disorder was similar in the two communities at mid-gestation. between 34 and 38 weeks gestation, the frequency drops in the us population while the same rate is maintained in ethiopians. the drop in the frequency of the infection among the us population coincides with the normal appearance in amniotic fluid of a polypeptide that requires zinc for full antimicrobial activity. examination of amniotic fluid at term among ethiopian gravida showed that 90% lacked antimicrobial activity. the lack in antimicrobial activity was correlated with energy-protein under nutrition and low levels of zinc concentration in amniotic fluid. while bacteria gain easy access to invade fetal membranes as evidenced by the fre quency of chorioamnionitis of 151.8 per 1000 live births in the present study, it is only in 14.4% (21.8/1000 live births) that the infection is fatal. it is, therefore, reasonable to postulate that in the deficiency in the defense against bacterial infection during late gestation, the antimicrobial properties of amniotic fluid play a crucial role. deficiencies in the upsula j med sci 82 abstracts 223 polypeptide may require expensive supplementafetal hypoxia, but also in intrauterine malnutrition tion, but if the deficiency can be corrected in at and delivery of a small-for-date fetus. consequent least a proportion of gravida with administration of ly, there is no sharp borderline between nutritive zinc during late gestation, a major break through in and non-nutritive complications influencing the the solution of a leading perinatal health problem health of the offspring. can be anticipated. 2. pregnancy and delivery complications. mothers at risk by 2. stembera the results of research and of longstanding clinical experience in countries with advanced perinatal medicine have proven that for different non nutritive complications of pregnancy and labour in fluencing the health of the mother and her offspring several common principles for elaboration of an effective prevention are of importance. 1 . the weight of individual risk factors is fie quently different for maternal and fetal health (e.g. heart disease is more consequential for the mother iv. factors affecting the nutrition o f mother and child in pre-industrial countries chairman: m . behar 1. cultural traditions and nutritional taboos related to pregnancy and lactation by d* b* (abstract not received.) 2. quantity and composition of breast milk in ma’nourished by b. belavady than for the fetus. post-term pregnancy, on the other hand, is relevant for the fetus but not for the mother .) 2 . the final effect of a risk factor upon the fetus is destined not only by its kind and weight but also by the week of pregnancy at which it started to act and by the length of the action. according to this a single risk factor can cause congenital malforma tion, premature delivery, stillbirth or some kind of perinatal morbidity. 3 . the improving mother-child care does not de crease the weight of any risk factor, but changes the frequency of its occurrence. as a result, the succes sion of their occurrence in the population of preg nant women is changing. 4 . a successful prevention of one risk factor can call forth a risk on another area. e.g. introduction of legal abortions decreased maternal mortality due to sepsis as a consequence of criminal abortions. the risk of premature delivery, however, in creased. an intensive care for pregnant women suf fering from diabetes caused a manifold decrease of perinatal mortality. but the infants share increas ingly in perinatal morbidity. 5. the most serious complications for the fetus are those resulting in delivery of a very premature fetus or inception of a fetoplacental dysfunction. even if the latter derangement is caused by non nutritive complications (e.g. gestosis, postmaturi ty), it frequently results not only in acute or chronic lactation imposes stress on women and hence, nutritional status and dietary intake may be ex pected t o play an important role in breast milk se cretion. breast feeding for long periods is com mon among women belonging to the poor socio economic groups in developing countries as com pared to women in the affluent countries. among the women of the former group, the dietary intake of all nutrients is low and their nutritional status is not satisfactory. however, clinical deficiency dis eases are not common among nursing women. these women are considered to be “apparently” normal by us and only those who show specific signs of deficiency are referred to as “malnour ished”. studies available indicate a daily secretion of milk of the order of 400-700 ml for over a one year period in undernourished women. reported yields in well-nourished mothers were 850 ml or less over a duration of six months or less. in a report from egypt, malnourished mothers had lower yields than normal mothers. the total protein concentration and fractionation in milk appears to be normal in the “apparently” normal mothers. in severely under-nourished, the ratio of casein to whey proteins showed an in crease. special proteins like lysozyme, lactoferrin and immuno-reactive proteins are not low. the fat content is either low or in the low normal ranges of values observed in well-nourished women. lactose upsala j med sci82 224 abstracts is slightly higher and the calorie content per 100 ml milk is not very different. calcium, iron and phosphorus content are in the normal ranges though early studies had indicated low calcium content in milk of chinese women. the unsatisfactory dietary intake of nutrients by the mothers is reflected in vitamin concentration in milk. all the vitamins studied are low in milk of under-nourished mothers. supplementation with different nutrients indi cated that there was a direct relation between the intake of vitamins by the mother and their concen tration in milk. such a relationship was not ob served with other nutrients. one unusual feature in the composition of milk of under-nourished mother was the high concentration of creatine and creatinine in milk. the protein in take of the mother affected the creatine concentra tion in milk. 3. patterns of breast feeding and weaning by y. hofvander in the who collaborative study on breast feeding three widely different socio-economic groups are being investigated as regards length of breast feed ing and weaning: the urban elite, the urban poor and the traditional rural population. although con ditions vary greatly area-wise some common pat terns may be identified. in the well educated elite group comprising pro fessionally active mothers the period for whole breast feeding is usually very short, 6-8 weeks or less, partly because the paid maternity leave period seldom is longer, but also because artificial feeding is considered fashionable. the pattern in the other extreme, the traditional rural population, is more varying. in general, breast feeding continues for a long time although the period for whole breast feeding may vary from half a year to more than a year. in certain cultures abrupt weaning takes place after half a year or sometimes less to ensure a new pregnancy. in most cultures children are abruptly weaned when the mother becomes pregnant. exceptions are certain eskimo tribes who breast feed all through the next pregnancy. the urban poor sector-from public health point of view an increasingly important g r o u p t e n d s to have a brief period of whole breast feeding while partial breast feeding may continue for a long time, a pattern which in the given environment is un satisfactory from immunological and nutritional points of view and predisposes t o gastroenteritis and malnutrition. the pattern of breast feeding and weaning is de cisive to a great extent for the state of nutrition, health and development in the young child. pro vided milk produce is ample whole breast feeding safeguards the child nutritionally and immunologi cally for half a year or even longer. inadequate milk production may explain faltering growth from 3 4 months or even earlier. gradual weaning on realis tic and nutritionally adequate foods from 4-6 months is generally advocated. 4. effect of maternal nutrition on the mother/child dyad by a . lechtig, h. delgado, r. martorell, ch. yarbrough and r. e. klein data from the incap longitudinal study suggest that maternal nutrition, both before and during pregnancy, has an effect on birth weight. the rela tive contribution of calories and proteins to an in crease in birth weight depends on the balance of these nutrients in the home diet of the population under study, type of physical activity, prevalence of disease and magnitude of the maternal nutritional stores before pregnancy. the anticipated impact of a nutritional intervention on birth weight ranges between 25 and 84 g of birth weight per 10000 calories ingested during pregnancy. the expected reduction in the proportion of low birth weight babies following a nutritional intervention will de pend on the estimated fetal weight increase and on the proportion of lbw babies existing prior to the intervention. in addition, improved maternal nutrition contri butes to decrease infant mortality rates and im proves breast milk output and nutritional status of the breast-fed infant at least during the first six months of age. caloric supplementation during pregnancy also reduces the risk of low weight gain during pregnancy, decreases the duration of post partum amenorrhea and may increase the probabil ity of a shorter birth interval. maternal height, head and arm circumference may be used as simple indi cators to select target populations for health and nutritional interventions. food supplementation during pregnancy also decreased diastolic blood pressure and increased the prevalence of lower upsala j med sci82 abstracts 225 limbs edema. the public health relevance of these findings is discussed and recommendations made to decrease the high world wide incidence of fetal growth retardation. 5. impact of fertility regulation on the health and nutrition of mother and child by c. c. standley fertility regulation is defined as control over the timing, spacing and number of pregnancies. its impact on the health and nutrition of mothers and children is discussed from several points of view: ( a ) in the absence of the practice of family plan ning, how d o the timing, spacing and number of pregnancies affect these parameters? the data from developed countries are briefly summarized and re sults from who studies in developing countries are presented in greater detail. ( b ) what direct evidence is there for the benefits of family planning? (c) what adverse effects do specific methods of fertility regulation have on health and nutritional status on mothers and children? results of recent who studies are discussed. v. factors affecting t h e nutrition o f mother and child in industrialized countries chairman: g . h . beaton 1. the mother’s and father’s role and function in an amuent society by r. liljestrom (abstract not received.) 2. the mother’s emotional contact with the newborn infant by e. lagercrantz the emotional relation between mother and infant is of the utmost importance for the infant’s physical health and its harmonious mental development. the mother’s emotional attitude to the child is far more important than the routines with which she takes care of the infant. these attitudes are the result of her long individual development from infant to woman. but the mental state during pregnancy is al so very important as motherly feelings for the ex pected child develop which later take concrete ex pressions in the mother and child interaction. the motherhood and the mother-child relationship will be discussed from several points of view: mothers’ interaction with their newborn infants. mothers’ conflicts in pregnancy and postpartum. children’s development at 6 and 18 months re lated to the emotional contact with the mother. 3. the mother’s choice of food for herself and her baby by l. aldrahamsson the possibilities for mothers to make a real choice of foods are often limited by several factors. among the limiting or decisive factors are, e.g., food availability, economic situation, tradition, food and nutrition education, especially from medi cal personnel, and food legislation that controls advertising and product quality. these factors in fluence the nutritional value of the diet and are, therefore, all of importance for the development of the child, either indirectly during pregnancy and lactation or directly such as during the weaning period. the pregnant mother’s nutritional status affects the development of the fetus. the availability of foods is generally sufficient in industrialized countries. however, in some countries there are pockets of poverty. lack of education, often com bined with poverty, has a negative effect on the food habits even in industrialized countries. the need for food during pregnancy varies with the manner and standard of living. in affluent groups, physical activity is often reduced during pregnancy. as in this group the mothers also can choose among an abundant variety of foods, there is a risk of overfeeding the child already during uterine life. furthermore, health and nutrition information has perhaps gone beyond its objectives: compare the saying “a pregnant mother must eat for two” and “one tooth for each child”. this, in combination with a short period of breast-feeding, makes it dif ficult for mothers to reduce their body weight to what it was before pregnancy. during early infancy breast-feeding ought to be the chosen feeding practice. however, in in dustrialized countries different sociological and cultural factors make the mothers choose bottle upsula j m e d sci82 226 abstracts feeding instead. in some countries weaning may start very early, as for example in usa, where solid foods are introduced by the mothers even earlier than the already too early time suggested. the choice of food for the weaning child varies greatly from one socio-economic group to another. one debated subject has been the differences be tween industrially produced and home-prepared food. in sweden the frequency of iron deficiency anaemia has almost been eliminated completely among small children. this is due to the ready-to eat, enriched milk cereal based products commonly used from about 4-6 months of age. very few fami lies prepare this type of food at home. fruit and vegetable purees or mixed baby-foods in jars were served daily to 78% of swedish infants (0-11 months) and to 5 % of children between 20-24 months in 1972. since then baby foods have been much discussed, including in the news media. if the study were repeated today the figures would prob ably be lower. today many mothers consider baby foods in jars t o be too expensive compared to home prepared food. on the other hand, there are moth ers who serve their children industrially produced food because they do not have enough confidence to choose and prepare adequate meals. 4. causes and consequences of early weaning by s. sjolin around 1970 breast feeding in most industrialized countries was in general limited to a very brief period. even people, who since long had had a thorough insight in these matters did not seem to worry, and only a few studies had been undertaken to reveal causes and consequences of this remark able manifestation of civilization. an explanation to this relaxed attitude may have been that infant morbidity and mortality continued t o drop in these countries despite a successively shortened breast feeding time. seemingly, infants could manage quite well without breast milk. most attempts to reveal the causes of the decreas ing breast feeding rate in industrialized countries have failed to disclose the causes, but have un equivocally demonstrated a number of background conditions related to the duration of breast feeding. determining factors are e.g. the mother’s age, civil status, education, social class and experience of breast feeding. young, single mothers, belonging to the lowest social class and with few school years tend to breast feed the shortest time. these factors, however, are not in themselves to be regarded as immediate causes of early weaning. by means of a prospective in-depth study of 71 mother+hild pairs we have been able to show that the direct reasons for early weaning could be found and could be divided into those referable to the mother herself (51.4%), to the environment (6.6%) and to the interplay between the mother and the environment (41.3%). during the course of lacta tion each mother ran into breast feeding difficulties on one or more occasions (“lactation crises”), trig gered by many different types of physical and/or environmental stress factors. the consequences of early weaning, which were so obvious and serious even in industrialized countries before basic hygienic measures were commonly adopted, have recently again attracted an increasing interest of research workers. it now seems evident that the consequences of early wean ing even under excellent hygienic conditions are more important to the health of infants than was believed some 10 to 20 years ago. above all the increased risk of alimentary tract infections, of al lergic reactions and of malabsorption have recently been convincingly established. since long it has further been known that artificial feeding also re sults in a higher incidence of constipation and obesi ty. there are, however, no indications that the early introduction of cow’s milk formulas should lead t o an increased infant mortality. 5. overfeeding during infancy by 0. wolff it is possible that overnutrition may occur already in intrauterine life. measurements of skinfold thick ness in newborn infants show a correlation with the mothers’ skinfold thickness. this correlation may be due t o genetic factors and/or t o excessive placen tal transfer of fatty acids from the obese mother t o the foetus. overfeeding is more common in infants who are artificially fed than in those who are breastfed. in accurate reconstitution of the feed may be one reason for overconsumption of nutrients by the arti ficially fed infant. there is a tendency to give non milk solids, normally as cereals, already at an early age, to both bottle and breastfed infants, and the introduction of such foods during the first month of life is associated with accelerated weight gain. upsala j med sci 82 abstracts 227 disturbances in the emotional relationship be tween the mother and her infant may lead to over feeding. overnutrition in infancy has certain effects: i . in adipose tissue it leads to an increase in mean fat cell size and in fat cell number. whether this increased cellularity persists indefinitely and whether it influences prognosis is not known. 2. it accelerates linear growth. 3. it predisposes to respiratory infections. as far as the prognosis of obesity in infancy is concerned, there is agreement in the literature that though the majority of obese infants will be of nor mal weight in childhood, obese infants run a greater risk of remaining obese than d o infants of normal weight. estimates suggest that about 20% may re main obese. retrospective studies of obese children suggest that in 40-50% of the children the obesity had its onset in infancy. the incidence and prognosis of obesity in infancy differ between different social classes and com munities and may be influenced by health educa tion. 6. the responsibility of the food industry and the importance of its cooperation with nutrition research institutes by l. hambraeus scientific and technological advances in the food industry have made it possible to produce a variety of new foods that combine acceptability and good nutritional values from edible raw material. how ever, the food industry has a great responsibility in the use of their innovative technology and espe cially in the marketing of new food products. food analogues, i.e. foods that duplicate the composition of a conventional food as well as its role in the diet, can, however, influence the pattern of food con sumption and secondarily give rise to unwanted nutritional effects. this can be especially harmful when the product it replaces contributes signifi cantly to the daily allowance of any essential nutrient. collaboration between the food industry and nutrition research institutes of the university is advantageous for several reasons. first it is essen tial that the producers give appropriate considera tion to the nutritional quality of their products and that new processes do not harm this quality. sec ondly it is essential that nutritionists in their nutri tion education activities stimulate the food industry to produce suitable products for the market, e.g. low fat milk, low fat margarine. the role of nutri tion researchers in informing the food industry of the expected development in consumers’ needs and nutrition recommendations, to adapt their product development to the coming market, is another ex ample of fruitful collaboration. a more complex practical problem is to find how the rights of the research fellow and inventor should be defended, since he seldom has the economic possibilities of marketing his knowledge and inventions and easily could be overrun by industry. it is essential that the use of nutrition facts in marketing of food products is well balanced. there is a large risk of using fortification as a means for marketing if fortification is used without legal restrictions. there is a need for the establishment of a policy of the use of fortifi cation and enrichment. it also seems essential to establish internationally applicable guidelines for the proper development and adaptation of food technology innovations. one area which has been most discussed during recent years is the marketing ethics of infant food industry on a national as well as an international basis. in 1975 the international pediatric associa tion adopted recommendations for action programs for encouraging breast feeding and stressed a need for curtailing promotion of artificial feeding. it is in this respect of interest to note that already in 1964 a group of swedish pediatricians worked out “medi cal standards for marketing of infant foods”. the fact that the swedish baby food industry has re spected the adopted rules and shown a clear ambi tion to follow them is a good example of fruitful collaboration and responsibility. the various nutrition foundations seem to offer a suitable forum for nutrition and food scientists of the universities, those engaged in nutrition educa tion of the public and representatives of food in dustry to discuss mutual problems. in sweden we have also had fruitful collaboration between the national board of health and welfare and the food industry in launching the campaign “diet and exer cise” throughout the country. upsala j med sci 82 228 abstracts v l . public health programmes intended to support t h e mother/child dyad. t h e role of primary health services chairman: 0. mellander 1. an integrated mch package delivery as part of primary health care by a. petros-barvazian (abstract not received.) 2. nutrition programmes as integrated part of general health programmes for mother and child by m. behar children from conception until they are fully in corporated in their family diet-at about the age of three-are considered the most vulnerable to malnutrition, and in whom the consequences of malnutrition are the most serious. pregnant and lactating mothers and children up to about 3 years of age should therefore have priority in the public health activities to correct malnutrition. there is a further need to identify and concentrate attention on the population groups and families at greater risk. among the limitations and pitfalls which have prevented a more effective action of the health services t o correct malnutrition in mothers and children, the following ones are discussed: insuffi cient coverage of the services; their disease rather than health orientation; the isolation of nutrition oriented activities from other health services; the concentration of attention on the food components of the problem without enough recognition of other responsible factors which the health workers con tribute to reduce; the inadequate planning and im plementation of “nutrition interventions”, e.g. food supplementation and nutrition intervention. activities, specifically oriented to prevent and correct malnutrition in mothers and children, that could be carried out at the primary level of health care as regular integrated components of the pro gramme are suggested. they are classified into three categories: monitoring of the nutritional situa tion, nutrition promotion and care of the malnour ished. areas in which further experience is needed for the effective application of available general princi ples into health programmes at the primary level of health care, in order to improve the nutritional situ ation of mothers and children, are suggested. 3. family planning programme as integrated part of general health programmes for mother and child by f. sai the importance of postponing the first pregnancy until the early twenties, spacing children at inter vals of about three years and stopping childbearing after the age of 35 for the health of women and their children, is analysed. evidence is adduced from different parts of the world t o show the age and parity related morbidities and mortalities. the con tribution made by family planning programs essen tially consisting of (i) efforts to delay the first pregnancy until the early twenties; (ii) spacing children at intervals of not less than three years; and (iii) stopping childbearing when the mother is 35 years of age, is illustrated. with this as a background, strategies are put for ward for the integration of family planning pro grams into mch and general health programs for mother and child. these strategies lean heavily on overall community involvement, the use of non health personnel in delivery systems and education programs, and an advocacy for the liberalisation of abortion and sterilisation laws together with the removal of restrictive legislation which does not take cognizance of the scientific facts on the rela tive safety of contraceptives. 4. socioeconomic planning. legislative measures to improve the nutritional status of the motherlchild dyad by m. manciaux and m. pechevis for many years the problem of improving the socioeconomic conditions relating to pregnancy and early infancy has been attacked by the passage of legislation to protect working women in developing countries. the nutritional benefits of such legisla tion were dependent on preand post-natal leaves and on the provision of nursing “breaks”. later, with the decline in breast feeding, some specific steps were taken to improve maternal and child uvsuk~ j med sci82 abstracts 229 nutrition by providing maternity and family allow ances, clean milk and supplementary foods at little or no cost. ilo, who, and paho have reviewed such legislation. however, the inadequacies of these measures, and the difficulties in implementing them, are quite evident: some were limited to working women or to a special social o r professional group. also, such legislation, even when it exists in a develop ing country-based on a model adapted from an industrialized country-affects relatively small numbers of the population, and does not protect those who have the greatest needs, namely preg nant and lactating mothers and infants in rural and periurban areas. within this context, the diminution of breast feeding, the misuse of western com mercialized dietetic products and uncontrolled fertility have had disastrous effects. newer knowledge concerning nutritional needs and the qualities of breast milk and changes in the processing of commercialized infant milks and foods have resulted in newer legislative approaches which require that the labels include details about the various components, methods of use, etc. certain legislative measures are designed to de fine the import requirements, regulation of free dis tribution to consumers, and advertising to the public. recent legal suits have resulted in expand ing the public awareness of these problems and will speed further legislative changes. because of such events, each country will be obliged to define its own nutritional policy, depend ing on internal conditions and its priorities: better application of existing measures, extending benefits to the entire population, adoption of new criteria for mother and infant foods, making them safer and better adapted to the nutritional needs of the mother-child dyad. vii. general discussion chairman: d. b . jellgfe recommendations for action at country level. role of national governments, international agencies and other bodies upsala j med sci 82 upsala j med sci 80: 118-121, 1975 hig h-resolution arteriography of the major peripheral vessels lars b j o r k and tamas sandor from the d e p a r t m e n t of r a d i o l o g y , h a r v a r d medicul school and p e t e r bent brigham hospitul, b o s t o n , m a s s a c h u s e t t s , u s a abstract in experimental studies using a 0.1 mm focal spot tube, rare-earth screens, cronex 4 film and diluted contrast agent (120 mgi/ml), 6xmagnification showed detail in the walls of major arteries that could not be identified with standard radiography or 3 xmagnification. the method is applicable to studies of arteriosclerotic changes in the wall of the femoral artery in patients. introduction high-degree magnification radiography can en hance the visualization of small image details and can even delineate small vessels not visible on the original radiograph (1). although magnification has little effect on larger images, such as those of major arteries, it can significantly improve delineation of the walls of these arteries. we are currently de veloping an angiographic method for the study of early small arteriosclerotic changes in large vessels. such a technique will be useful in man for early diagnosis and objective assessment of the effects of dietary and/or drug treatment for arteriosclerosis. the experiments described here dealt with magnification of the femoral artery in dogs and au topsy specimens of human superficial femoral arteries. theoretical considerations and experimental procedures the efficiency of early diagnosis of arteriosclerosis depends on how well the walls of the artery can be visualized. in this context, improved image quality means an image that has better contrast and higher resolution than can be obtained with conventional techniques. such improvement was recently asses sed with regard t o the contrast of small blood-vessel images. image contrast was calculated i n terms of magnification by using computer simulation techni que ( i ) ; the results are shown i n fig. 1 . the curves indicate that a significant increase i n contrast oc curs when the effective focal-spot size is smaller than 0.2 mm and magnification is about 6 x (pro vided that a par-speed film-screen combination or an equivalent line spread function is used). these results have been experimentally verified ( i ) and were applied i n the present study. experiments in dogs material and methods six mongrel dogs weighing 20 to 25 kg were anesthetized with an intravenous injection of sodium barbiturate. they were intubated but no artificial respiration was used. a catheter was introduced into the femoral artery on one side and manipulated via the aortic bifurcation into the distal femoral artery on the other side. this artery was exposed at the groin to permit subsequent occlusion of the artery around the catheter. an x-ray tube with a 0.1-mm focal spot was used for taking all magnification arteriograms. the design of the tube was such that it had an inherent current flow of 30 ma; the kilovoltage had a range of 50 t o 100 kv with 10 kv increments. single exposures were obtained of the femoral artery at the mid-thigh. exposure factors were 60 to 80 kv and 30 ma; exposure time varied from 0.2 to 1.2 sec. magnifica tion of 3 t o i o x was used. special rare-earth screens (a mixture of terbium-activated yttrium and gadolinium oxysulfde) with high definition were used in combina tion with cronex 4 film. these screens had an 8 to 10 line pairslmm resolution measured with a line test pattern at 40 kv. the injection of contrast medium was prolonged to approach homogeneous filling of the arterial system dur ing the long exposure times used in this experiment. injec tions were made either by hand o r by an automatic in jector. in both instances the rate of injection was adjusted t o permit complete replacement of blood flow with con trast medium. the contrast media were renografin 60 with an iodine content of 290 mg/ml and renografin 60 upsala j med sci 80 fast film-screen combination 7 ’ $ 6 iin 7 200p // 0 2 4 6 8 10 magnification f i g . i . contrast calculated in terms of magnification for 50, 75, 100, 150 and 200 p n blood vessel images. the dimensions of the focal spots are 0.05, 0.1, 0.2 and 0.3 mm. the line spread function of kodak blue brand film and radelin tf-2 screens were used in the computa tions. diluted with distilled water to 70, 100, 120 and 145 mgi/ml. in addition, 20 t o 30 ml of air using high injection rate was injected after filling the catheter with reno g r a f n 60. this created a coating of contrast medium on the surfaces of the walls of the arteries distended by the air. before the injection of air either alone o r for the double contrast effect, the femoral artery was occluded by tying a cotton band around the catheter in the groin. results the best results with regard to definition and con trast were obtained using 70 kv for the exposures. images of high contrast could be obtained regularly at this kv value. at the same time the transparency of the images was satisfactory to permit evaluation of wall definition in larger vessels and even evalua tion of vessels crossing each other. exposures were also taken at 6 x with high plus screens and cronex 4 film. it was found that the image quality with rare-earth screens was significantly better than that obtained by high plus and cronex 4 film-screen combination. renografin 60 with an iodine content of 290 1 mg/ml was too concentrated to delineate the fine arteriography of major peripheral vessels i 19 details of larger vessel walls. renografin diluted t o 120 mg/ml gave the best results (fig. 2). the prob lem of oversaturation of the blood-vessel image was minimized with this concentration. air alone as contrast medium gave satisfactory definition of the major arteries. the double-contrast method proved very satisfactory, and the images obtained were of very high quality with high resolution and contrast. because the arteries were all normal, it was dif ficult to evaluate whether the information content vaned with different degrees of magnification. experiments o n specimens material and methods two 7 cm long specimens of the superficial femoral artery were obtained from two cadavers. one was from a 90 year-old woman with numerous partially calcified arteriosclerotic changes in the wall. the other was from a 25-year-old woman with a normal artery. these t w o artery specimens were filled with various types of contrast ma terial and placed centrally in an l l cm thick plastic con tainer filled with water. the 1 1 cm thickness reproduces the scatter and absorption generated by the thigh of an ordinary supine patient. the same radiographic technique was used a s in the canine experiments. subjective comparison between the amount of information in images obtained at various magnification was performed. images were obtained of the arteries filled with air only. double contrast was then created by coating the inner surface of the artery with dionosil, leaving the arteries otherwise filled with air. finally, the arteries were filled with dionosil (iodine content 270 mglml) and dionosil diluted to 135 mgi/ml. dionosil was chosen because i t is almost non-diffusible and permits repeated experiments on the same specimen. results as in the dog experiments, 70 kv proved to offer maximum-definition angiograms. angiograms were taken at 1 . 2 , 3 , 6 and 10 magnifications. with the 6x magnification, minor detail, particularly the serrate appearance of the slightly contracted normal femoral artery, could be clearly demonstrated, whereas this was’not possible with the 3 x magnifi cation when other factors were equal (fig. 3 ) . magnification higher than 6 x gave no additional information. air contrast, double contrast and complete filling of the lumen of the arteries with dionosil were all satisfactory for achieving gooddetail images of the arteries and their walls. however, the density of undiluted dionosil was too high to permit penetra upsala j med sci 80 120 l . bjork and t . sandor tion in the center of these large arteries. the best definition was obtained with double contrast. the rare-earth screens combined with cronex 4 film proved t o be superior t o the high-plus cronex 4 film-screen combination. discussion these experiments show that 6 x magnification with a 0.1 mm focal spot can show detail in the walls of major arteries that cannot be identified with standard radiography or even 3 x magnification. it is desirable to be able to achieve this degree of enlargement when studying the femoral artery in patients. in many angiographic rooms this degree of magnification can be obtained by raising the tabletop to a high position and placing the cassette on the floor. this would permit the patient t o re main supine during the study and thus greatly facili tate the reproducibility of the imaging from one occasion to another. the rareearth screen in combination with cronex 4 film seems to be the most suitable combi nation for this type of study. they represent high resolution and a speed increase by a factor of about 1.8 greater than the high-plus cronex 4 film-screen combination. substitution of cronex 4 with green sensitive agfa gevaert curix ortho film can pro f i g . 2. effect of variation of iodine concentration of con trast medium in femoral arteriography in the dog. (a) 145 mgi/ml. @) 120 mgi/ml. (c) 100 mgi/ml. 6 x magnifica tion, 70 kv, 30 ma, 0.6 sec 0.1 mm focal spot. injection rate and amount of contrast agent were the same in three angiograms. upsala j med sci 80 arteriography of major peripheral vessels 121 fig. 3 . normal human femoral artery specimen partially filled with dionosil. (a) x 6 . (b) x 3 . other factors being equal: 0.1 mm focal spot, 7 0 kv, 30 ma. better detail visualization with 6 x magnification, particularly of ser rated appearance of the wall of the artery. vide a further speed increase by about 50%. this type of angiography requires single exposures only and involves no risk of overheating t h e tube with a microfocal spot. diluted contrast medium with a n iodine content of approximately 120 mgi/ml is probably best f o r demonstrating both small vessels and details in the walls of large arteries. this is in agreement with previous determinations of t h e iodine concentration required t o achieve good angiographic results ( 2 ) . this could be achieved by injecting a fairly large amount of diluted contrast medium during a period ' long enough t o cover the relatively long exposure necessary. it is expected that these injections of diluted contrast media would be well tolerated by patients. other modifications of the angiographic techni ques such a s use of carbon dioxide or oxygen alone o r in combination with iodine-containing contrast agents t o create a double-contrast effect, a r e more difficult to use in patients because these techniques probably will require the occlusion of the artery with a balloon or a similar device. while air alone as contrast gave satisfactory definition of the major arteries it is questionable whether small changes in the walls were in fact detected with this method. it may require the use of x-rays generated a t much higher kv to take full advantage of air as contrast for large vessel angiography. from t h e a b o v e it seems that 6x magnification arteriography of major arteries using a 0.1 mm focal spot would b e a feasible and reproducible method in most patients. t h e risks involved would most likely be n o greater than with other angiographic studies. the additional injection of 20-30 ml of diluted water-soluble contrast medium in the iliac o r femoral artery will hopefully be well tolerated. a c k n o w l e d g e m e n t this work was supported by usphs grants gml8674 and hl11668. r e f e r e n c e s 1. sandor, t., adams, d. f., herman, p. g., eisenberg, h. & abrams, h . l . : the potential of magnification angiography. am j roentgenoll20: 916921, 1974. 2. swart, b. & dingendorf, w.: experimenteller beitrag zur optimalen gefassdarstellung. fortschr rontgenstr 97: 6 3 7 4 4 8 , 1962. received june 15, 1974 address for reprints: larsbjork, m . d . department of radiology harvard medical school 25 shattuck street boston, massachusetts 021 15 usa upsala j m e d sci 80 upsala j m e d sci 87: 251-258, 1982 effects of chemical modification of lysine, tyrosine and tryptophan residues in pea seed nucleoside diphosphate kinase and inhibition of the enzyme with antibodies bror edlund department of clinical chemistry, university hospital, and institute of medical and physiological chemistry, university qf uppsala, uppsala, sweden abstract pea seed n u c l e o s i d e d i p h o s p h a t e k i n a s e (ndp k i n a s e ) i s an o l i g o m e r i c , t e t r a m e r i c enzyme w h i c h has been shown t o be p h o s p h o r y l a t e d b y i t s s u b s t r a t e atp, presumably f o r m i n g an 1 p h o s p h o h i s t i d i n e a t i t s a c t i v e s i t e . has a l s o a r e a c t i v e l y s i n e r e s i d u e been d e m o n s t r a t e d a t t h e a c t i v e s i t e . i n t h e p r e s e n t i n v e s t i g a t i o n n i t r a t i o n of a t y r o s i n e r e s i d u e i s shown t o i n a c t i v a t e t h e enzyme. enzyme on m o d i f i c a t i o n o f t h e s e e s s e n t i a l l y s i n e and t y r o s i n e r e s i d u e s , s i n c e d o u b l e d i f f u s i o n e x p e r i m e n t s w i t h an i n h i b i t o r y a n t i s e r u m shows no d i f f e r e n c e i n r e a c t i v i t y between t h e n a t i v e enzyme and t h e m o d i f i e d enzyme. i t i s a l s o f o u n d t h a t m o d i f i c a t i o n o f a l l t r y p t o p h a n r e s i d u e s i n t h e enzyme r e d u c e s t h e enzyme a c t i v i t y o n l y t o a s m a l l degree, i n d i c a t i o n t h a t t h e s e h y d r o p h o b i c amino a c i d r e s i d u e s a r e n o t d i r e c t l y i n v o l v e d i n t h e c a t a l y t i c p r o c e s s . r e c e n t l y there seems o n l y t o be a s l i g h t s t r u c t u r a l change i n t h e introduction atp: n u c l e o s i d e d i p h o s p h a t e k i n a s e t r a n s p h o s p h o r y l a s e ( e c 2.7.4.6) (ndp k i n a s e ) f r o m pea seed i s an o l i g o m e r i c , t e t r a m e r i c enzyme w i t h a m o l e c u l a r w e i g h t o f 70 000 d a l t o n s ( 1 ) . s u b s t r a t e atp, presumably f o r m i n g an 1 p h o s p h o h i s t i d i n e r e s i d u e a t i t s a c t i v e s i t e ( 2 , 3 ) . the amino a c i d sequence a r o u n d t h i s p h o s p h o h i s t i d i n e r e s i d u e has been i n v e s t i g a t e d and f o u n d t o c o n t a i n a l y s i n e r e s i d u e ( 4 , 5 ) . s p e c i f i c f o r c e r t a i n amino a c i d r e s i d u e s , and i n a d d i t i o n t o a r e a c t i v e h i s t i d i n e r e s i d u e , has a r e a c t i v e l y s i n e r e s i d u e o f i m p o r t a n c e f o r enzyme a c t i v i t y been f o u n d ( 6 ) . sequence o f t w e n t y amino a c i d r e s i d u e s f r o m t h e a c t i v e s i t e o f pea seed ndp k j n a s e (51, and t h e r e i s n o e v i d e n c e f o r more t h a n one p o l y p e p t i d e c h a i n i n t h e enzyme ( 1 ) . s t i l l be a t y r o s i n e o r t r y p t o p h a n r e s i d u e i n t h e a c t i v e s i t e o f t h e enzyme i t i s i n t e r m e d i a t e l y p h o s p h o r y l a t e d b y i t s i n a r e c e n t r e p o r t t h e n a t i v e enzyme has been t r e a t e d , w i t h r e a g e n t s no a r o m a t i c amino a c i d r e s i d u e a p a r t f r o m h i s t i d i n e has been f o u n d i n t h e however, t h e p r e s e n t i n v e s t i g a t i o n was made s i n c e t h e r e m i g h t 25 1 at some distance in the primary structure from the histidine residue that is phosphorylated. benzyl bromide (hnb-bromide) and tetrani tromethane (tnm) to react with trypto phan and tyrosine residues respectively (7,8). it also seemed to be of interest to study if antibodies against the enzyme were inhibitory to enzyme action and if chemical modification altered its antigenicity, as has been found with other enzymes, for a review see ( 9 ) . in the present investigation the effect of antibodies on enzyme activity has been studied as well a s the reactivity in an immunodiffusion test of native ndp kinase and enzyme modified by fluoro-2,4-dinitrobenzene (fdnb) and tnm. thus the native enzyme has been treated with 2-oh-5-no2 the effect of chemical modification and antibodies on enzyme activity are discussed. materials and methods pea seed ndp kinase was prepared as previously described ( 3 ) . enzyme activity was measured according to monrad and parks, using dgdp for nucleoside diphosphate (10). protein was measured by a folin method, using human serum albumin as standard (11). photometric measurements were made, using a zeiss pmqii spectrophotometer, equipped with an automatic cell unit recorder. 2-oh-5-n02-benzyl bromide (hnb-bromide) and tetranitromethane (tnm) were from sigma. innnunodiffusion experiments were performed according to ouchterlony (12). agarose 1'industrie biologique francaise (0,8% w/v) in a sodium diemal buffer (75 mmoles/l , ph 8.6) containing calcium lactate (2 mmoles/l) was used. after visible precipitation lines had formed, usually within 24 hours at room temperature, the agarose layers were extracted with nacl (0,9% w/v) dried and stained with coomassie brilliant blue r-250 essentially according to laurel1 (13). 1 ml aliquots of a ndp kinase solution, 1 mg per ml of a phosphate buffer (0.1 moles/l, ph 7,4) were incubated with 25-50 pl-of hnb-bromide (0,2-0,4 moles/l) dissolved in acetone. the hnb-bromide solutions were made ex tempore using redistilled acetone. the incubation mixtures were then cooled in an ice-water bath and the hnb-hydroxide formed was separated by centrifugation at 4000 rpm for 5 min. the supernatant solutions were chromatographed at room temperature on 0,9x60 cm sephadex 6-50 columns, equilibrated and eluted with sodium borate buffer (0,ol moles/l, ph 8,5). absorbancy at 410 nm of the void volume fractions were determined. a control experiment was run in parallel with no hnb-bromide added. it was found that the enzyme activity was unaffected by the acetone concentrations used (up to 5% v/v). reaction of tryptophan residues with hnb-bromide. the enzyme activity, protein concentration and the specific activity of each test sample was then compared to that 252 of the control experiment and the number of tryptophan groups modified per subunit was calculated using a molar absorbance value of 18 000 cm-l for the hnb-groups in the protein (7). moles/l) in sodium borate buffer (0,l moles/l, ph 8.5) were added 50 p1 o f a solution of tnm in ethanol (1 mole/l). mixture kept at 3ooc. ed in fig. 2, and chromatographed on 0,9x60 cm sephadex 6-50 columns, equili brated and eluted at room temperature with sodium borate buffer (10 mmoles/l, ph 8.5). the absorbancy at 428 nm of the void volumes was determined, as well as protein concentration. subunit was calculated from the molar absorbance of 4 300 cm-l for a nitro tyrosine group in a protein (14) and the protein content. activity was determined for each sample and compared to that of a control sample treated in the same way but without tnm added. rabbits by injecting subcutaneously 1 ml of a 1:l suspension of freund's complete adjuvant (difco, usa) and pea seed ndp kinase ( 2 mg/l) in a sodium phosphate buffer ( 0 , l moles/l, ph 7.4) containing nacl (0,15 moles/l). one week later an intramuscular injection of the same amount of enzyme was made, but now using freund's incomplete adjuvant (difco, usa), and a booster dose was given in the same way after another week. the rabbits were starved over night and bled through the ear vein to harvest serum. of the antiserum was 85 g/l. antiserum were added to 20 pg of pea seed ndp kinase in 100 111 o f a triethanol amine-acetic acid buffer (10 mmoles/l, ph 7,4). kept at 37' for 30 min, followed by dilution in an ice-cold tris-acetic acid buffer (0,15 moles/l, ph 7,4). of a sample incubated at 37oc for 30 min without antiserum added. activity of the enzyme (1500 units/mg) was unaffected by this treatment. enzyme tests were performed in duplicate. treated with 14c-fdnb and tnm. ly as described before (5), except that only a twofold molar excess of 14c fdnb (17 mci/mmole) over ndp kinase was used. samples were taken after a reaction time of 12 hours, when 0,8 moles of 14c-dnp-lysine residues had been formed per mole of subunit, and when 25% of the enzyme activity remained. sample was also taken before the addition of fdnb (control sample). immunological reactivity of the enzyme was tested as described in fig. 4 a. ndp kinase was also inactivated by nitration with tnm as described above. nitration of tyrosine residues with tnm. to 3,5 ml o f ndp kinase (31 after mixing was the incubation 0,5 ml samples were taken at time invervals, as indicat the number of nitrotyrosine residues formed per the specific enzyme production of antiserum. antibodies against ndp kinase were rised in protein concentration 25-200 ul of rabbit inactivation of pea seed ndp kinase by antibodies. the incubation mixtures were the specific activities were compared to that all the specific reactivity of inhibitory antibodies against the native enzyme and enzyme the enzyme was treated with i4c-fdnb essential a the 17-822858 253 a control sample was taken before addition of tnm and treated in the same way. samples were taken 95 min after the addition of tnm, when 1,o mole of tyrosine residues had reacted per mole of subunit o f the enzyme (21, 5% of the enzyme activity remaining. the samples were tested for immunological reactivity as described in fig. 4 6. results and discussion there seems to be a tyrosine residue more reactive to tetranitromethane than the other tyrosine residues in each subunit of the enzyme, and nitration of this tyrosine residue leads to inactivation of the enzyme, indicating that this tyrosine residue is of importance for enzyme activity as seen in fig. 1. q 50 100 time i m i n l fig. 1. inactivation o f pea seed ndp kinase by nitration of tyrosine. open circles indicate enzyme activity remaining, filled circles indicate number of nitrotyrosine residues formed per subunit. for details see text. the product adp (4 moles/l) did not protect the enzyme from inactivation. this is in contrast to its protection of the enzyme from inactivation by 2,4 dinitrofluorobenzene (6) , zy4,6-trinitrobenzene sulfonic acid (6) and diethyl pyrocarbonate (6). enzyme on chromatography on sephadex 6-200, indicating that the enzyme was not dissociated into its subunits on nitration. fifty percent of the enzyme activity remained after reaction of all three tryptophan residues in each subunit by hnb-bromide (fig. 2). neither did the nitroenzyme separate from the native 254 ’c % 2 25 t \ hl a 1 i i fig. 2. filled number modification of pea seed ndp-kinase with 2-oh-5-no -benzyl bromide. triangel s indicate enzyme activity remaining after midification of the of tryptophan residues per subunit given on the abscissa. for details see text. therefore, modification of the tryptophan residues does not seem to directly involve the active site. as seen in fig. 3 , the native enzyme was inactivated in direct proportion to the relative amount of antiserum added, strongly indicating antigenic sites involving the active site of the enzyme. diffusion or trapping of active enzyme in antigen-antibody comp’lexes seem to have occurred since some enzyme activity was regained by using a large excess of anti serum. thus, 30% of enzyme activity remained using 850 pg of antiserum per pg o f a certain restriction o f substrate antiserum per ug o f ndp kinase. i i i i i 100 200 300 400 500 pg antiserum / g ndp k i n a s e t.’ fig. 3 . filled circles indicate enzyme activity after incubation of native enzyme with antiserum. for details see text. inactivation of pea seed ndp kinase by specific rabbit antiserum. 255 9sz thus apart from the histidine residue phosphorylated during enzyme action (2,3) there seem to be a lysine (5) and a tyrosine residue essential for enzyme activity in pea seed ndp kinase. acknowledgements this work was supported by the swedish medical research council (project no. 13x-50). discussions and kind support of this work. i wish to thank professor lorentz engstrom for valuable the skilful technical assistance of 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 72. mrs. jill ekstrom is gratefully acknowledged. references edlund, b.: evidence for identical subunits in pea seed nucleoside di phosphate kinase. febs lett 38:222-224, 1973. edlund, b., rask, l., olsson, p., wblinder, o., zetterqvist, u. & engstrom, l.: yeast and purification of 1-phosphohistidine as the main phosphorylated product of an alkaline hydrolysate of enzyme incubated with adenosine ( triphosphate. edlund, b.: and phosphorylation of the enzyme with adenosine ( acta chem scand 25:1370-1376, 1971. edlund, b.: phate kinase. upsala j med sci 79:143-147, 1974. edlund, b. & engstrom, l.: pea seed nucleoside diphosphate kinase. febs lett 47:279-283, 1974. edlund, b., heldin, c.h. & engstrom, l.: effect of chemical modification of a histidine and a lysine residue of pea seed nucleoside diphosphate kinase. upsala j med sci (in press). barman, t.e. & koshland, d.e. j:r: a colorimetric procedure for the quantitative determination of tryptophan residues in proteins. j biol chem riordan, j.f. , sokolovsky, m. & valler, b.l.: tetranitromethane. a reagent for the nitration of tyrosine and tyrosyl residues of proteins. j am chem soc 88:4104-4105, 1966. arnon, r. in current topics in microbiology and immunology 54:47-61, 1971. mourad, n. & parks, j:r, r.e.: erytrocytic nucleoside diphosphate kinase. 11. isolation and kinetics. j biol chem 241:271-278, 1966. lowry, o.h., rosebrough, n.j., farr, a.l. & randall, r.j.: protein measure ments with the phenol reagent. j biol chem 193:265-275, 1951. ouchterlony, u. in handbook of experimental immunology fed. d.m. weir) p. 655-706. oxford and edinburgh, blackwell sci publ, 1967. preparation of crystalline nucleoside diphosphate kinase from baker’s 32 p) eur j biochem 9:451-455, 1969. purification of nucleoside diphosphate kinase from pea seed 32 p ) triphosphate. active site phosphopeptides from pea seed nucleoside diphos a peptic phosphopeptide from the active site of 242:5771-5776, 1968. 257 13. laurell, c.-b.: electroimmunoassay. scand j clin lab invest 29, suppl 124 14. ness, s . , schmidt, w . & schneider, f.: chemische modifizierung und kata 21-37, 1972. lytische aktivitat der carbonate-hydro-lyase b aus rindererythrozyten. modifizierung von arginin, histidin, lysin, tyrosin und tryptophan. hoppe-seyler’s z physiol chem 352:355-368, 1971. address for reprints: docent bror edlund department o f c1 inical chemistry university hospital s-750 14 uppsala sweden 258 upsala j med sci 91: 29-36, 1986 induction of skeletal malformations in the offspring of rats fed a zinc deficient diet johan styrud, v. elisabeth dahlstrom and ulf j. eriksson department of medical cell biology, university of uppsala, uppsala, sweden abstract p r e g n a n t rats w e r e subjected t o a t r a c e m e t a l poor d i e t (1.2 ppm zinc, 5.9 ppm copper, 40 ppm manganese) during t h e e n t i r e gestation. the r a t mothers did not gain weight during pregnancy and showed d e c r e a s e d liver weight and lowered s e r u m glucose levels on g e s t a t i o n a l day 20. the offspring exhibited d e c r e a s e d body and p l a c e n t a l weights, delayed ossification of t h e skeleton, and a n increased resorption rate. we also found 4 % s k e l e t a l malformations in t h e offspring (0 % in t h e controls), which closely resembled a t y p e of malformation previously encountered in r a t s when t h e m o t h e r was manifest d i a b e t i c (i..~. sacral dysgenesis). the z i n c l e v e l s w e r e d e c r e a s e d and manganese levels increased t o t h e s a m e e x t e n t in offspring of trace m e t a l r e s t r i c t e d ( t h i s study) and manifest d i a b e t i c r a t s (previous studies). f u r t h e r m o r e , when pregnant rats on t h e trace m e t a l r e s t r i c t e d d i e t w e r e resupplemented with 75 ppm z i n c in t h e drinking w a t e r t h e offspring largely normalized t h e i r s o m a t i c and p l a c e n t a l growth, s k e l e t a l maturation, as well as t h e i r z i n c and manganese levels. in addition, t h e f e t u s e s of t h e z i n c resupplemented rats did not show a n y malformations. the possibility of common t e r a t o l o g i c a l mechanisms in m a t e r n a l d i a b e t e s and t r a c e m e t a l deficiency may t h e r e f o r e be considered. introduction the etiology behind disturbed f e t a l growth and congenital malformations in t h e offspring of d i a b e t i c m o t h e r s i s at p r e s e n t n o t clear (10,13,18,20,21). in previous exper i m e n t a l a t t e m p t s t o study t h i s problem w e have described a rat model f o r d i a b e t i c pregnancy in which d e c r e a s e d f e t a l weight, delayed s k e l e t a l ossification and a n increased number of f e t a l resorptions and s k e l e t a l malformations w e r e found (6,8). f u r t h e r m o r e , t h e t w o types of skeletal malformations found in this r a t model (micrognathia and sacral dysgenesis, cf. refs. no. 7-10) show a s t r o n g g e n e t i c dependence to t h e rat s t r a i n used, s i n c e w e have been unable to induce t h e s e malformations in t h e offspring of manifest d i a b e t i c rats f r o m o t h e r s t r a i n s (11,121. in addition, w e have shown t h a t t h e offspring of t h e d i a b e t i c r a t s in our model are zinc-deficient but have normal levels of copper and increased levels of manganese, thus establishing a link b e t w e e n m a t e r n a l 29 d i a b e t e s and f e t a l t r a c e m e t a l disturbances (9). in t h e p r e s e n t s t u d y w e have used t h i s malformation-prone rat s t r a i n , which is presently k e p t in a colony in uppsala (denoted tj rats"), in a n a t t e m p t t o e l u c i d a t e t h e t e r a t o g e n i c i t y of trace m e t a l disturbances in t h e absence of m a t e r n a l diabetes. materials and methods r a t s f r o m our l o c a l colony in uppsala ("u rats") w e r e used in t h e p r e s e n t investigation. this colony consists of a sprague-dawley derived s t r a i n of albino rats in which t h e offspring consistently exhibit a 1 5 2 0 % incidence of s k e l e t a l malformations when t h e m a t e r n a l rat has been m a d e d i a b e t i c with s t r e p t o z o t o c i n before pregnancy (12). the total incidence of all t y p e s of f e t a l malformations is less t h a n 1 % in t h e c o n t r o l preg nancies of this s t r a i n (12). the (u) rats in t h e p r e s e n t study w e r e all non-diabetic and kept in a light and t e m p e r a t u r e controlled environment with a 1 2 + 1 2 h light-dark cycle. two to t h r e e f e m a l e rats w e r e housed per c a g e and w e r e f e d s t a n d a r d rat p e l l e t s contain ing 1 9 2 ppm zinc, 33 ppm copper and 1 3 2 ppm manganese (r 3, ewos ab, sodertalje, sweden) and t a p w a t e r a d libitum before t h e o n s e t of pregnancy. they w e r e m a t e d overnight with male r a t s from t h e s a m e colony and conception w a s confirmed by examin a t i o n of vaginal s m e a r s f o r t h e presence of sperm in t h e morning. the d a y when a positive vaginal s m e a r w a s obtained was denoted g e s t a t i o n a l d a y 0 and f r o m t h a t day e a c h pregnant animal was k e p t in a stainless steel c a g e (stiilstandard as, norway) with a m e t a l grid floor and without wood c u t t i n g s in o r d e r t o p r e v e n t t h e rats from ingesting t r a c e m e t a l s from o t h e r sources t h a n t h e food and drinking water. on gesta tional day 0 t h e s t a n d a r d food was changed t o a s p e c i f i c trace m e t a l poor diet: 1.2 ppm zinc, 5.9 ppm copper, 40 ppm manganese (ewos ab, sodertalje, sweden). about half t h e pregnant animals received zinc supplemented drinking w a t e r with a z i n c concen t r a t i o n of 1 5 ppm (solvezinc, tika ab, lund, sweden), which w a s markedly increased in comparison with ordinary t a p w a t e r (0.1 ppm zinc). t h e amount of food and w a t e r consumed by t h e pregnant rats was assessed t w i c e a week. on g e s t a t i o n a l days 0 and 20 t h e m a t e r n a l s e r u m glucose levels w e r e e s t i m a t e d (glucose analyzer 2 , beckman inc., fullerton, ca, usa). t h e c o n t r o l pregnant rats on s t a n d a r d d i e t are d e n o t e d n , t h e zinc deficient zd, and t h e z i n c resupplemented rats are denoted zdz in t h e following. on g e s t a t i o n a l day 20, t h e pregnant rats w e r e weighed and killed by a blow to t h e neck. the m a t e r n a l liver w a s dissected o u t a n d weighed, t h e f e t u s e s w e r e e x t e r i o r i z e d and examined with r e s p e c t to weight, viability and o c c u r r e n c e of f e t a l malformations. from e a c h u t e r i n e horn o n e f e t u s was randomly chosen and processed f o r whole body trace m e t a l determination. these f e t u s e s and t h e m a t e r n a l livers w e r e placed in pre-weighed, acid-washed plastic beakers and c u t u p thoroughly with a p a i r of s t a i n l e s s steel scissors and freeze-dried to c o n s t a n t weight f o r a b o u t 2 4 h (gt 2 f r e e z e drier, leybold-heraeus ag, west germany), in o r d e r to r e c o r d t h e specimens dry weights. they w e r e t h e n t r a n s f e r r e d to a platinum crucible, ashed overnight in a t e m p e r a t u r e programmed f u r n a c e (carbolite, sheffield, u.k.) and analyzed by a t o m i c absorption 30 spectrophotometry. zinc, copper and manganese w e r e d e t e r m i n e d on a varian aa-6 i n s t r u m e n t in a n oxidizing air-acetylene f l a m e at wavelengths of 213.9 nm, 324.7 nm and 279.5 nm, respectively. one non-malformed (chosen a t random) and all t h e malformed f e t u s e s per individual horn w e r e processed f o r skeletal staining (24). these f e t u s e s w e r e e v i s c e r a t e d , fixed in 7 0 % e t h a n o l f o r 10-30 days, t r e a t e d with 1 % k o h and s t a i n e d with alizarin red as described previously (7). subsequent evaluation of s k e l e t a l development was m a d e by counting t h e visualized ossification c e n t e r s in six d i f f e r e n t locations in a c c o r d a n c e with aliverti and coworkers (1). the probability (p) of a c h a n c e d i f f e r e n c e b e t w e e n means was e s t i m a t e d by student's t w o tailed t-test. d a t a are given as means k s.e.m. table i. body weight, body weight gain during pregnancy, liver weight and serum glucose c o n c e n t r a t i o n of t h e normal (n), zinc-deficient (zd) and zinc-resupplemented (zdz) pregnant rats on g e s t a t i o n a l day 20. means * s.e.m. group no.of maternal maternal maternal maternal (g) day 0-day 20 (g) (m m o w r a t s body weight weight gain liver weight serum glucose (g) n 3 371 * 26 137 k 1 5 11.7 2 0.5 5.6 k 1.3 zd 11 242 k 8'" -1 k 5'" 7 . 7 ? 0.4"' 2.3 ? 0.3'" zdz 6 356k11 112+15++ 11.0+ 0.3 3.2 f 0.3" ~ significances: ++ p<0.01 vs. n rats; +++ ~ < o . o o i vs. n rats. results during g e s t a t i o n a l days 0-20 t h e normal (n), zinc-deficient (zd) and zinc-deficient and resupplemented r a t s (zdz) consumed t h e s a m e a m o u n t of food and w a t e r ( d a t a n o t shown). the body weight and body weight i n c r e a s e during pregnancy d i f f e r e d significantly between t h e groups, t h e zd group did not gain any weight at all, whereas t h e zdz rats showed normal body weights on g e s t a t i o n a l day 20 (table i). t h e zd m a t e r n a l r a t s exhi bited d e c r e a s e d liver weights on d a y 20 in c o n t r a s t t o t h e zdz r a t s whose m a t e r n a l liver weight did n o t d i f f e r f r o m t h e normals (table i). on g e s t a t i o n a l day 0 t h e r e w e r e no dif f e r e n c e s in serum glucose levels b e t w e e n t h e groups ( d a t a n o t shown), but on pregnancy day 20, t h e s e r u m glucose levels of t h e zd and zdz r a t s w e r e both significantly d e c r e a s e d in comparison with t h e n rats (table i). the number of viable offspring was slightly lowered in t h e zdz group (table 11). the number of non-viable f e t u s e s was increased in both t h e zd and zdz groups, t h i s i n c r e a s e was most pronounced in t h e zd group. f e t a l body and p l a c e n t a l weights w e r e slightly low er t h a n normal in t h e zdz group, whereas t h e y w e r e f u r t h e r d e c r e a s e d in t h e zd group (table 11). in four of t h e eleven zd l i t t e r s a t o t a l of f i v e malformed f e t u s e s w e r e found. 31 table 11. number of viable and non-viable offspring (per u t e r i n e horn), f e t a l body weight and p l a c e n t a l weight of t h e offspring of normal (n), zinc-deficient (zd) and zinc-resupplemented (zdz) rat m o t h e r s on g e s t a t i o n a l d a y 20. means ? s.e.m., n d e n o t e t h e number of u t e r i n e horns examined. group n no. of no. of f e t a l p l a c e n t a l viable non-viable body-weight weight offspring offspring (g) (g) n 6 6.5 * 0.6 0.2 * 0.2 4.6 2 0.3 0.50 ? 0.01 zd 2 2 5.7 ? 0.5 1.3 i 0.3"' 2.7 ? 0.1'" 0.39 ? 0.01+++ zdz 1 2 5.3 2 0.4' 0.6 t 0.1"' 3.8 ? 0.2"' 0.49 ? 0.01' significances: + p<0.05 vs. n fetuses; ++ pc0.01 vs. n f e t u s e s ; +++ p<o.ool vs. n fetuses. all f i v e f e t u s e s had malformation of t h e s a c r a l dysgenesis t y p e (figure i, cf. refs. no. 6, 9 , l l ) . the rate of malformation was 3.9% (5/128) in t h e zd group, whereas i t was z e r o in t h e n (0/39) and zdz (0/63) groups. the f i v e malformed f e t u s e s in t h e zd group weighed t h e s a m e (2.7 2 0.1 g) as t h e non malformed ones, whereas t h e p l a c e n t a e of t h e malformed f e t u s e s w e r e slightly lighter (0.34 k 0.02 g) t h a n t h e i r non-malformed l i t t e r m a t e s (p < 0.001, table 11). figure i. alizarin r e d s t a i n e d day-20 f e t u s e s f r o m normal (n, l e f t ) , zinc-deficient (zd, middle), and zinc-resupplemented (zdz, right) rats. the zd f e t u s shows s a c r a l dysgenesis, (cf. refs. no. 7-10,121 t h e o t h e r t w o f e t u s e s show normal morphology. 32 table 111. maternal liver c o n c e n t r a t i o n s of trace m e t a l s in n o r m a l (n), zinc-deficient (zd) and zinc-resupplemented (zdz) rats on g e s t a t i o n a l day 20. means f s.e.m. group no. of concentration (mg/kg dry liver weight) rats zinc copper manganese n zd zdz 3 11 6 104 ? 3 16.0 ? 0.4 8.1 2 0.3 81 5 2'" 13.8 f 0.3'" 7.9 2 0.4 104 f 2 16.0 2 0.2 7.2 ? 0.1"' significances: + p<0.05 vs. n rats; ++ pc0.01 vs. n r a t s ; +++ p<o.ool vs. n r a t s . the m a t e r n a l livers of t h e zd rats a c c u m u l a t e d less zinc and copper during g e s t a t i o n (table 111). the zdz rat showed normal z i n c and copper levels, whereas t h e accumulation of manganese w a s lowered (table 111). t h e zd f e t u s e s exhibited g r e a t l y diminished ac cumulation of z i n c and slightly increased l e v e l s of manganese (table iv).the zdz f e t u s e s showed less pronounced a l t e r a t i o n s in t h e i r accumulation of trace m e t a l s t h a n t h e zd offspring, but nevertheless had lowered zinc and increased manganese levels compared t o t h e controls (table iv). the copper l e v e l s w e r e similar in all groups of f e t u s e s (table iv). the sum of ossification c e n t e r s in s i x d i f f e r e n t locations (sternum, m e t a c a r p u s , m e t a t a r sus, a n t e r i o r and posterior proximal phalanges, caudal v e r t e b r a e , cf. ref. no. 1 ) was de c r e a s e d in t h e zd f e t u s e s and normal in t h e zdz offspring (figure 11). table iv. t o t a l body concentrations of trace m e t a l s in f e t u s e s of normal (n), zinc-deficient (zd) and zinc-resupplemented (zdz) rats on g e s t a t i o n a l day 20. means f s.e.m. group no. of concentration (mg/kp d r y body weight) zinc copper manganese f e t u s e s n zd zdz 6 1 3 1 2 1 10.920.4 2 2 1 0 5 * 2"' 10.6 ? 0.4 1 2 122 f 2+++ 10.7 f 0.3 0.920 .o 2 1.1 2 o.l+++ 1.0 f 0.1+++ significances: + p<0.05 vs. n fetuses; ++ pc0.01 vs. n fetuses; +++ p<o.ool vs. n fetuses. 3 -868571 33 discussion the major finding in t h i s s t u d y was t h e demonstration of fetal malformations in t h e t r a c e metal-restricted pregnancies, of a t y p e similar to those appearing in t h e offspring of manifest diabetic rats (7,8). in addition, t h e pregnant ( u ) r a t s on t h e t r a c e m e t a l poor d i e t produced f e t u s e s with s e v e r a l c h a r a c t e r i s t i c s i n common with offspring of manifest d i a b e t i c (u) rats (on s t a n d a r d diet): decreased f e t a l weight ( 6 , 7 ) , increased resorption rate ( 6 , 7 ) , delayed s k e l e t a l ossification (7,8), as well as d e c r e a s e d z i n c and increased manganese accumulation in t h e f e t u s e s (9). this suggests, t h a t t h e t e r a t o g e n i c processes i n t h e t w o d i f f e r e n t t y p e s of (u) rat pregnancy -&g. m a t e r n a l t r a c e m e t a l deficiency and m a t e r n a l d i a b e t e s may s h a r e s o m e etiological mechanisms. the i m p o r t a n c e of zinc in t h i s c o n t e x t is i l l u s t r a t e d by t h e e f f e c t s of i t s administration to t h e trace m e t a l r e s t r i c t e d rat mothers. in t h e zdz l i t t e r s t h e r e w e r e no malformations, t h e s k e l e t a l ossification was normal and t h e f e t a l resorption r a t e , body and placental weights w e r e also largely normalized. o t h e r a u t h o r s have suggested t h a t changes in m a t e r n a l l e v e l s of trace m e t a l s may b e involved in t h e induction of congenital malformations both in humans (2,3,14,17,23) and animals (15,16). diabetic individuals a r e f u r t h e r m o r e known to lose t r a c e metals, via increased uri nary output in relation to t h e i r d e g r e e of m e t a b o l i c c o n t r o l (19). a l t e r e d levels of trace metals, in particular zinc, may t h e r e f o r e b e of significance f o r t h e induction of malforma t i o n s i n d i a b e t i c pregnancy. t h e possible t e r a t o g e n i c a c t i o n s of a n embryo-fetal zinc de z a, t ar 0 c 0 a 0 = v) v) 0 0 z 4 . c . .c 0 30 20 10 0 i 6 n zd zd malf l a zdz figure 11. t o t a l number of ossification c e n t e r s in s i x a n a t o m i c a l l o c a t i o n s (cf. ref. no. 1) in t h e non-malformed and malformed (malf) f e t u s e s of n o r m a l (n), zinc-deficient (zd) and zinc-resupplemented (zdz) rats on g e s t a t i o n a l day 20. means i s.e.m. significance: +++ p<o.ool vs. n fetuses. 34 ficiency are several. this trace m e t a l s e r v e s as a co-factor of a number of enzymes, g.g. thymidine kinase, dna-polymerase and superoxide dismutase, and a deficiency may t h e r e f o r e inhibit t h e biosynthesis of dna or affect t h e defense a g a i n s t f r e e oxygen radicals in t h e conceptus ( 4 , 5 , 2 2 ) . the significance of t h e concomitantly increased f e t a l manganese levels in t h i s s t u d y as well as in f e t u s e s of manifest d i a b e t i c rats ( 9 ) remains t o b e eluci dated. explain all t h e malformations s e e n in t h e d i a b e t i c r a t pregnancy, s i n c e t h e frequency of s k e l e t a l malformations was markedly lower in t h i s investigation t h a n in t h e offspring of manifest d i a b e t i c ( u ) r a t s (4% versus 15-20%, cf. refs. no. 7 8 ) . f u r t h e r m o r e , a l l t h e mal f o r m a t i o n s w e r e of t h e s a c r a l dysgenesis t y p e in t h e offspring of t r a c e m e t a l r e s t r i c t e d r a t s , whereas in d i a b e t i c rat pregnancy both micrognathia and sacral dysgenesis are en c o u n t e r e d (7-10). in addition, manifest d i a b e t i c r a t s of t h i s malformation-prone s t r a i n a p p e a r to give birth t o a g r e a t e r proportion of micrognathias, when t h e m o t h e r is zinc supplemented (eriksson, unpublished). a l t e r e d f e t a l zinc levels may t h e r e f o r e play a role in t h e induction of congenital malformations, and may b e of s p e c i f i c significance f o r t h e etiology of t h e s a c r a l dysgenesis syndrome. this idea a w a i t s f u r t h e r investigation. a trace m e t a l disturbance of t h e t y p e produced in t h e p r e s e n t s t u d y may not, however, acknowledgements the a u t h o r s a r e g r a t e f u l to a s s o c i a t e professor p e r mattsson, the swedish national food administration f o r help with t h e analyses of trace metals, and to lars j o r h e m , arne p e t t e r s s o n and birgitta sundstrom f o r e x p e r t t e c h n i c a l a s s i s t a n c e with a t o m i c absorption spectrophotometry. astrid nordin, olov rosendal and p a r r i wentzel are acknowledged f o r t h e i r help in care of t h e animals a n d tissue sampling. this investigation w a s supported by t h e swedish medical r e s e a r c h council ( 1 2 x 1 0 9 , post-doctoral fellowship 12p-6346 t o uje), t h e "expressen" p r e n a t a l r e s e a r c h foundation, the medical faculty of t h e uni v e r s i t y of uppsala, the swedish d i a b e t e s association and the nordic insulin fund. % references 1. aliverti, v., b o n a h m i , l., giavini, e., leone, v.g., and mariani, l.: the e x t e n t of f e t a l ossification as a n index of delayed development in t e r a t o g e n i c s t u d i e s on t h e rat. teratology 20:2372 4 2 , 1 9 7 9 . 2. breskin, m.w, worthington-roberts, b.s., knopp, r.h., brown, z., plovie, b., mottet, n.k., and mills, j.l.: f i r s t t r i m e s t e r s e r u m zinc c o n c e n t r a t i o n s in human pregnancy. am. j. clin. nutr. 38:943-953, 1 9 8 3 . 3. cavdar, a.o., arcasoy, a., cin, s., and giimiis, h.: zinc deficiency in geophagia in turkish children and response to t r e a t m e n t with zinc sulphate. haematologia 65:403 4 0 8 , 1 9 8 0 . 4. dreosti, i.e., grey, p.c. and wilkins, p.j.: deoxyribonucleic a c i d synthesis, protein synthesis and t e r a t o g e n e s i s in zinc-deficient rats. s. afr. med. j. 46:1585-1588, 1 9 7 2 . 5 . duncan, j.r., and hurley, l.s.: thymidine kinase and dna polymerase a c t i v i t y in n o r m a l and z i n c d e f i c i e n t developing rat embryos. proc. soc. exp. biol. med. 159:39 4 3 , 1 9 7 8 . 6. eriksson, u.j., andersson, a., efendic, s., elde, r., and hellerstrom, c.: diabetes in pregnancy: e f f e c t s on t h e f e t a l and newborn r a t with p a r t i c u l a r regards to body weight, s e r u m insulin c o n c e n t r a t i o n and p a n c r e a t i c c o n t e n t s of insulin, glucagon a n d s o m a t o s t a t i n . a c t a endocrinol. (copenh.) 94:354-364, 1 9 8 0 . 7. eriksson, u.j., dahlstrom, e., larsson, k.s., and hellerstrom, c.: increased incidence of congenital malformations in t h e offspring of d i a b e t i c rats and t h e i r prevention by m a t e r n a l insulin therapy. d i a b e t e s 31:l-6, 1982. 35 8. eriksson, u.j., dahlstrom, e., and hellerstrom, c.: diabetes in pregnancy: s k e l e t a l malformations in t h e offspring of diabetic r a t s a f t e r i n t e r m i t t e n t withdrawal of insulin in e a r l y gestation. diabetes 32:1141-1145, 1983. 9. eriksson, u.j.: diabetes in pregnancy: r e t a r d e d f e t a l growth, congenital malformations and f e t o m a t e r n a l c o n c e n t r a t i o n s of zinc, copper and manganese in t h e rat. j. nutr. ' 10. eriksson, u.j.: congenital malformations in d i a b e t i c animal models a review. diab. 11. eriksson, u.j., lewis, n.j., and freinkel, n.: growth r e t a r d a t i o n during e a r l y organo 1 2 . eriksson, u.j., dahlstrom, v.e., and styrud, j.: metabolically d e t e r m i n e d teratogenesis: 13. freinkel, n.: of pregnancy and progeny. banting l e c t u r e 1980. d i a b e t e s 29:385-394, 14. hambridge, k.m., neldner, k.h., and walravens, p.a.: zinc, a c r o d e r m a t i t i s e n t e r 15. hurley, l.s.: t e r a t o g e n i c a s p e c t s of manganese, zinc, and copper nutrition. physiol. 16. hurley, l.s., and swenerton, h.: congenital malformations resulting f r o m zinc de 17. jameson, s . : e f f e c t s of z i n c deficiency in human reproduction. thesis. a c t a med. scand. 18. kucera, j.: r a t e and t y p e of congenital anomalies among offspring of d i a b e t i c women. j. reprod. med. 7:61-70, 1971. 19. mcnair, p., kiilerich, s., christiansen, c., christiansen, m.s., madsbad, s . , and transbol, i.: hyperzincuria in insulin t r e a t e d d i a b e t e s mellitus i t s relation to glucose homeo s t a s i s and insulin administration. clin. chim. a c t a 112:343-248, 1981. 20. mills, j.l.: malformations in i n f a n t s of d i a b e t i c mothers. teratology 25:385394, 1982. 21. pedersen, j.: the pregnant d i a b e t i c and her newborn. 2nd edit., copenhagen, munks 22. prasad, a s . , and oberleas, d:: thymidine kinase a c t i v i t y and incorporation of thy 23. sever, l.e., and emanuel, i.: is t h e r e a connection b e t w e e n m a t e r n a l zinc deficiency 114:477-486, 1984. res. 1:57-66, 1984. genesis in embryos of experimentally d i a b e t i c rats. diabetes 33:281-284, 1984. malformations and m a t e r n a l diabetes. biochem. soc. trans. 13:79-82, 1985. 1980. opathica and congenital malformations. l a n c e t 1:577-578, 1975. rev. 61:249-295, 1981. ficiency in rats. proc. soc. exp. biol. med. 123:692-697, 1966. suppl 593:l-89, 1976. g a a r d , 1977, pp. 1-280. midine i n t o dna in zinc-deficient tissue. j. lab. clin. med. 83:634-639, 1974. and congenital malformations of t h e c e n t r a l nervous s y s t e m in man? teratology 7:117 118, 1973. alizarin red s method f o r f e t a l bone. stain technol. 39:61-63, 1964. 24. staples, r.e., and schnell, v.l.: r e f i n e m e n t s in rapid clearing technique in t h e koh adress r e p r i n t r e q u e s t s to: dr. ulf j. eriksson d e p a r t m e n t of medical c e l l biology university of uppsala biomedicum p.o. box 571 s-751 23 uppsala sweden 36 upsala j med sci 84: 235-246, 1979 a prospective epidemiological survey of cerebrovascular disease in a swedish community andreas terent from t h e department of internal medicine, university hospital, u p p s a l a , s w e d e n abstract a prospective epidemiological study of cerebrovascular diseases and tran sient ischemic attacks (tia) is presented. during a three-year period the an nual incidence of strokes was 2.90 and of tia 0.45 per thousand population. this difference in incidence and the disparities in age characteristics favour the hypothesis that tia precedes only a minority of the strokes. the short-term mortality is high among the stroke patients. introduction chronic illness, such as cardiovascular disorders and malignant neo plasms, now dominate the panorama of diseases in middle-aged and old people in the industrialized countries (15). in sweden, as in many other countries, cere brovascular disease(stroke) is one of the leading causes of death ( 8 , 17). cerebrovascular disease (0) ranks as the number-one diagnosis on the list of days spent in hospital in the uppsala region (20). numerous community-based ( 3 , 4, 5, 10, 11, 12, 16, 18, 23, 24, 27, 30, 35, 41) as well as hospital-based (1, 2, 7, 28, 40) studies of cvd have been pub lished, but s o far very few have dealt with all kinds of stroke at all ages (27, 41). the aim of the present investigation was therefore to register all strokes in a well-defined area. the prognosis in cvd and factors influencing it were also studied, as well as socio-medical aspects o f the disease, and these results will be reported elsewhere. the information collected should provide a basis for the planning of care of patients with diseases of this group ( studied area and population the municipality of sijderhamn consists of 7, 32). a small town with rural surroun dings. in this self-administered area, of 1,062 sq.km, the population was 32,250 in 1975 at the beginning of the study and 32,000 in 1978 at the end 235 (25). the age distribution was similar to that of the general swedish popula tion (fig. 1 ) . there is only one hospital in the community. the general prac titioners are usually eight in number and they work in close co-operation with the staffs o f the medical and surgical departments of the hospital. methods i n may 1975 a stroke register, for recording of all cases of stroke, both those with long-standing symptoms and those with transitory ischemic attacks (tia), was set up at the sijderhamn hospital, for the purpose of the present 8 y e a r s 90-94 80-84 70-74 60-64 5054 404l 30-34 20-24 10-14 04 i 1 i i 1 1500 1000 500 500 1000 1500 no.of i n d i v i d u a l s d p a g e , y e a r s 90-94 8 0-84 7 0-74 6c-64 50-54 40-4l 30-36 20-24 10-14 0 l i i 1 i i 3bo 200 100 100 200 300 no.of i n d i v i d u a l s . l o 0 0 fig. 1 a. fig. 1 b. fig. 1 . the population by age in sijderhamn ( 1 a ) compared with the swedish population ( 1 b ) on 31 december, 1 9 7 4 . 236 study. before the registration started the diagnostic criteria and the therapy t o be given were defined. information concerning the stroke registration, including the diagnostic criteria and therapy, was given continuously to all doctors at the hospital, and to the general practitioners in the district. all nursing institutions were repeatedly asked to report definite and suspected cases of stroke. the hospital records were checked daily by the registered nurse. all death certificates for permanent residents of sijderhamn, issued in the district during the period or obtained from the institute of forensic medicine in uppsala, were examined. one important function in the study was carried out by a special registered nurse, who was responsible for the tracing and primary registration of new cases and for calling patients for follow-up. the initial physical examination, either in hospital or elsewhere, was performed in all cases by members of the medical staff, with use of a special form. interviews were conducted and physical examinations performed on admis sion to hospital, after three months and then once a year. in the event of a new stroke the procedure was started from the beginning again. after exactly three years the registration was stopped but the follow-up, including the examinations mentioned above, is still continuing. the same doctor (the author) was responsible for the final registration, throughout the study period. de f in i t ions the definitions of stroke and tia are in accordance with the recommenda tions of who. thus, stroke was defined as "rapidly developed clinical signs of focal (and/or global) disturbance of cerebral function, lasting longer than 24 hours or leading to death with no apparent cause other than vascular". the term "global" mainly applies to the cases of subarachnoid hemorrhage. tia was defined as "rapidly developed clinical signs of focal cerebral dysfunction of presumed vascular origin not lasting more than 2 4 hours". the term recurrent stroke was used for a new stroke taking place more than three weeks after an initial stroke. i n the case of tia recurrence meant a new attack after a symptom-free interval of more than 2 4 hours. comments on diagnoses the diagnosis was based mainly on clinical observations. a diagnosis of hemispheric lesion was given if one o r more of the follow ing were observed: hemiparesis, grasset's phenomenon, hemisensory l o s s , apha sia, facial nerve paresis of the central type, eye deviation, homonymous anop sia, abnormal finger-to-nose test of the non-atactic type, or unilateral posi tive plantar reflex. 237 a brainstem lesion was indicated by cranial nerve deficits of the lower motor neuron type. doll's eye (loss of eye movements when the head is quickly rotated) or unilateral loss of the pupillary reflex was also taken as a sign of a brainstem lesion. cerebellar lesions are known to present with a large variety of symptoms, but a combination of typical tremor in the finger-to-nose test, dysdiadochoki nesis and nystagmus were primarily regarded as signs of cerebellar dysfunction. a macroscopically hemorrhagic cerebrospinal fluid (csf) indicated intracerebral breakthrough-bleeding or subarachnoid hemorrhage, depending on the presence or absence of localizing symptoms. an embolic infarction was suspected in patients with mitral valve disease with atrial fibrillation, patients with paroxysmal atrial fibrillation of other causes and in patients with infarction. exclusions patients who were unconscious at the onset of other recent myocardial neurological symptoms were excluded from the tia group because of the possibility of the attack being an epileptic seizure. migraine patients, who sometimes fit the definition of tia, were naturally not included. drop attacks or vertigo without any other symptoms were not sufficient for a diagnosis of tia because of difficulties in the differential diagnosis. treatment design the patients were treated according to the relevant principles of the medical department, which meant that no intensive care was available except for a few cases. neither was it the rule to use vasodilators, edema-reducing agents or low-molecular weight dextran as a regular procedure. early mobilization was prescribed, except in the case of subarachnoid hemorrhage. anticoagulant treatment with warfarin sodium was given to patients with tia or stroke due to embolism. the tia patients received this treatment for two years. no time limits were set for the embolism patients. the tia patients with unilateral symptoms who showed corresponding carotid lesions at angiography were consi dered for surgery. stat is t ics the incidence density (id, in the text simply called incidence) is expres sed as the number of new cases per 1,000 population and year. the five-yearly increase of the incidence was estimated by adjusting the formula id = a . k to the stroke and the tia groups, respectively. "t" stands for the number given to each consecutive five-year age group in which stroke cases were registered; the lowest age group in which stroke appeared being no. 1. thus, when t increa t 238 sed one unit, there was a simultaneous increase in id by (k 1 ) * 100 %. the chi-square test was used when testing variables within the stroke and the tia groups, respectively. when testing for differences between these two groups the variables studied, e.g. mean age, were thought to have a normal distribution due to the large number of observations (the central limit theorem). the cri tical value for a at the 95 % significance level was 1.645 in one-sided tests. i i 15 10 5 a g e . years 90-9d 80-8l 70-76 6 0 6 4 50-5c 40-44 30-34 i i i 5 10 15 a g e , i 30 20 10 10 20 30 i i 1 1 i no. of p a t i e n t s fig. 2. a fig. 2 b. ?ig. 2. the sex and age distribution of cases of stroke (2 a) and tia (2 b ) in jijderhamn 1975 1978. 239 results between 1 may 1975 and 30 april 1978, a total of 338 strokes and 51 tias were registered, of which 57 strokes and 7 tias were recurrences (table 1). the number of strokes and tias were almost identical in each year of the study. the sex and age distribution are shown in fig. 2. the mean age in the stroke group was 71.1 + 1.7 years for men, 75.4 + 1.7 years for women for both sexes combined. up to the age of 80 years there tely large number of men (x2 = 10.85 to be compared to obs and 73.1 z 1.2 years was an unproportiona l x o.95(4) = 9.488). table 1. number of cases of stroke and tia, soderhamn, 1975-78. new stroke recurrence new tia recurrence first year 92 6 14 2 second year 95 23 16 3 third year 94 28 14 2 28 1 57 44 7 in the tia group the mean age for men was 67.7 z 5.2, for women 66.7 z 3.7 and for the whole group 67.2 3.6. there was no significant sex difference in this group with respect to age. on the other hand, there was a significant age difference (a 0.95 group. = -11.20) between the total stroke group and the total tia the relative risks of stroke and tia were found to increase exponentially with age (fig. 3), but some differences were found between them. firstly, there were no tia patients below the age of 40 years, whereas there were three stro kes in this age group (one cerebellar hemorrhage and two subarachnoid hemorr hages). secondly, the incidence of stroke increased by 55 % per 5-year interval compared with 36 % for tia. the overall incidence of stroke was 2.90/1000/year and that of tia 0.451 1000/year. after correction for differences in age and sex, the corresponding incidences in the total swedish population are calculated to be 2.50/1000/year and 0.39/1000/year, respectively. up to 30 april, 1978, 130 (46 % ) of the stroke patients and 3 (7 x ) of the tia patients had died. during the same period of time 4 tia patients developed completed stroke, including one of those who died. a history of tia was found in 39 (14 %) of the patients with completed stroke. lumbar puncture was performed in 60 'z of all patients. the other diagnos tic procedures are listed in table 2 and the different types of stroke in table 3. 240 table 2 . diagnostic procedures besides clinical examination and lumbar punc ture. no. of patients stroke tia computed tomography 3 0 cerebral arteriography 56 10 isotope-encephalography and eeg 22 2 isotope-encephalography 10 4 eeg 32 4 x-ray of skull or echoencephalo76 4 graphy 50 10 5 table 3. types of stroke. csf examitotal nation autopsy no. z cerebral infarction 1 2 8 4 132 4 7 . 0 intracerebral hemorrhage 4 0 9 49 1 7 . 4 subarachnoid hemorrhage 12 3 15 5 . 3 unclassified 85 3 0 . 3 281 100.00 iolog incidence a a a h a a a 0 a 0 a 0 a a 0 a a 00 0.1 %o 40 50 60 70 80 90 n i 1 1 i i i i fig. 3 . the age-specific incidence of stroke (triangles) and tia (circles) in sijderhamn in the pe riod 1975 1 9 7 8 . age, years 241 it is expected that some, at least, of the patients in the 'unclassified' group in table 3 will be assigned to one of the other three groups when the results of all diagnostic procedures have been analysed. this will be reported in a future communication. during the study 31 cases of suspected stroke were excluded. the most common reason was the demonstration of a cerebral tumor or an extradural hemorr hage. other diagnoses giving reason for exclusion were bell's palsy, parkinson's disease and peripheral neuropathy. of the patients registered, 90.7 z were examined and treated at the medi cal department from the time of onset of symptoms. thus, less than 10 z of the patients were primarily treated in their own home or an old people's home, or were found dead from a stroke. except in the last case they were all examined, mostly several times, by the same doctor who was responsible for the final registration. discussion the choice of a small and very well-defined geographical area has the advantage that it diminishes the risk of patient l o s s . a final check-up showed that during the three-year period less than 10 per cent of the patients with stroke had been treated or had been found dead before admission to hospital. this figure is well at a level with those reported by other investigators ( 4 , 16, 36). by using the who definitions for stroke and tia, a comparison with other results is possible. on the other hand, there i s still a lack of simple and safe methods for diagnosing different types of strokes. i n some comparable studies (3, 35) the diagnosis of a hemorrhagic lesion was based on the same criteria as were used here, and a similar definition was also used by kannel (22) in the framingham study. the diagnosis of thrombotic or embolic lesions has been made without any uniformity. the only factor in common in previously presented epidemiological studies has been an exclusion diagnosis with absence of evidence of intracrsnial bleeding (9). i n the present study the lesion was considered of thrombo-embolic origin if the csf was non-hemorrhagic or if autopsy showed cerebral infarction. however, 30 z of the cases still remain unclassified. the tia diagnosis is solely clinical (16). although the great majority of the tia population have arteriographically visible lesions in the neck vessels (31, 36, 38, 39), tia patients lacking these have the same prognosis (39). i n spite o f exclusion of some cases with a difficult differential diagnosis, tia as a whole i s more unreliable as a diagnostic entity than stroke, as the fin dings may have disappeared at the clinical examination. 242 the frequency of autopsy was low in this material, owing to the traditions in this area. however, even in the case of a high autopsy rate it may be diffi cult to draw firm conclusions from the autopsy in every case (21, 24, 27). thus, jorgensen and torvik (21) found evidence of cvd in 320 patients, of whom only 196 had had previous clinical signs. recently the combined use of computed tomography and csf spectrophotometry has been reported to increase the diagnos tic reliability (37). the precision of the measurement must be high to permit an extrapolation of the incidence rates from sijderhamn to other communities (29). although the population of sijderhamn is rather small, this precision is certainly increased by the fact that a three-year period was used for the study. the incidence of new strokes in the present material, 2.9/1000/year, may by compared with that found previously in other countries. in finland (4) an incidence of 1.42/1000/ year has been reported, while in denmark (7) it has been estimated to be 2.2/1000/year for males, and 2,5/1000/year for females. these seemingly large differences between the finnish figure, on the one hand, and the figures from the danish and the present study, on the other, are reversed if the annual age -specific incidences are calculated (table 4). in this case the incidence is highest in finland and lowest in denmark, with the sijderhamn figures in the intermediate position. this again illustrates the great influence of the age composition of the population on the incidence (25). the proportion of the population above 65 years in the finnish study was 5.2 per cent, while the corresponding figures for the danish and the present study were 21 and 18.2, respectively. in gothenburg, one study with stroke registration in people up to 66 years of age has been published (16). the incidence of new strokes in that city was 0.52/1000/year, compared with 0.53/1000/year in the same age group in sijderhamn. thus, there seems to be no difference in this respect between a big city and a small town. one retrospective investigation of hospital records in table 4. annual incidence of stroke in some scandinavia communities, per 1,000 population. commun it y study males females years age (years) age (years) 55-64 65-74 75-84 55-64 65-74 75-84 fredriksberg 1971-73 4.5 8.6 14.9 1.9 5.1 14.2 denmark (35) espoo-kauniainen 1972-73 4.9 12.2 26.8 2.5 9.5 21.5 finland (3) sijderhamn 1975-78 3.4 10.6 22.7 1.0 7.3 17.2 sweden 17-792856 243 t h e uppsala r e g i o n i n 1964 r e v e a l e d an i n c i d e n c e of 2 . 3 / 1 0 0 0 / y e a r , i n c l u d i n g some c a s e s (18 x ) n o t s a t i s f y i n g t h e s t r o k e c r i t e r i a and some w i t h t i a ( 3 3 ) . i n a n o t h e r r e t r o s p e c t i v e s t u d y of h o s p i t a l i z e d p a t i e n t s , f r i t h z (13) c a l c u l a t e d t h e i n c i d e n c e of s t r o k e below 70 y e a r s of a g e t o b e 0 . 3 6 / 1 0 0 0 / y e a r . t h i s i n c i d e n c e i s based on f i g u r e s from 1967 t o 1971. the p r e s e n t c o r r e s p o n d i n g f i g u r e i n sijderhamn i s 1 . 0 / 1 0 0 0 / y e a r . a d e c l i n e w a s r e p o r t e d f o r t h e two d e c a d e s up t o 1971 ( 6 , 1 4 ) , a f t e r which t h e l e v e l remained unchanged up t o 1976 ( 1 9 ) . though t h e r e h a s been a s h i f t towards a l a r g e r p r o p o r t i o n of o l d p e o p l e between t h e two p e r i o d s 1967 1971 and 1975 1978, t h i s c a n n o t p o s s i b l y e x p l a i n t h e d i f f e r e n c e between 0.36 and 1.0. t h i s d i s c r e p a n c y p o s s i b l y r e f l e c t s t h e d i f f i c u l t i e s of c a s e f i n d i n g i n r e t r o s p e c t i v e s t u d i e s . the t i a i n c i d e n c e i n sweden i s s o f a r unknown. the i n c i d e n c e i n sijderhamn, 0 . 4 5 / 1 0 0 0 / y e a r , c a n o n l y be compared w i t h f i n d i n g s i n t h e usa ( t a b l e 5 ) . i n t a b l e 5 . annual i n c i d e n c e o f t i a and s t r o k e i n v a r i o u s communities, p e r 1,000 p o p u l a t i o n . community age t i a s t r o k e y e a r s sijderhamn a 1 1 0.45 2.90 r o c h e s t e r ( 2 7 , 41) a l l 0.31 1.54 s e a l beach (12) > 52 1 . 1 7 . 1 r o c h e s t e r ( 4 1 ) t h e i n c i d e n c e of t i a a t a l l a g e s w a s 0.31/1000/year. i n s e a l beach ( 1 2 ) , w i t h a p o p u l a t i o n above t h e age of 55 y e a r s i t was 1 . 1 / 1 0 0 0 / y e ~ r , compared w i t h 1 . 0 / 1 0 0 0 / y e a r i n t h e same a g e c l a s s e s i n sijderhamn. thus, s t r o k e seems t o be a b o u t s i x t i m e s more f r e q u e n t t h a n t i a . our f i n d i n g s , as w e l l a s r e p o r t s of o t h e r a u t h o r s ( t a b l e 6 ) , show t h a t a h i s t o r y of t i a i s known i n o n l y 15 20 p e r c e n t of t h e s t r o k e p a t i e n t s ( 3 , 1 6 ) . there i s one e x c e p t i o n , namely t h e s t u d y from r o c h e s t e r ( 2 7 ) . here 73 p a t i e n t s o u t of 7 7 7 w i t h presumed c e r e b r a l i n f a r c t i o n had e x p e r i e n c e d t i a , c o n s t i t u t i n g o n l y 5.9 p e r c e n t of t h e t a b l e 6 . h i s t o r y of t i a i n s t r o k e p a t i e n t s i n v a r i o u s s t u d i e s . no. of age no. w i t h p e r c e n t a g e community p a t i e n t s y e a r s t i a w i t h t i a sijderhamn 2 8 1 a 1 1 39 13.9 espoo-kauniainen ( 9 ) 286 a 1 1 44 15.4 harlem (23) 328 a 1 1 41 12.5 r o c h e s t e r ( 3 , 41) 1245 a l l 73 5.4 244 total number of 1,245 stroke patients. this latter low figure supposedly ref lects the difficulties in identifying this diagnosis retrospectively. in saderhamn the youngest cases with stroke come at a lower age than those with tia. in contrast, however, the mean age is lnwer in the tia group. a l s o , the incidence of tia increases more slowly than that of stroke. all these obser vations support the hypothesis that tia precedes only a minority of strokes. of course some tia may be ignored by the patients, but probably not to such a degree that these differences can be explained simply as missing registrations. if this hypothesis is true, only a minority of the strokes may be prevented by good care of tia patients. on the other hand, the poor prognosis for tia pa tients (26, 41) justifies active investigation and treatment. 1 . 2. 3. 4 . 5. 6. 7. 8. 9 . 10. 1 1 . 12. 13. 14. 15. 16. 1 7 . 18. references acheson, j., acheson, h.w.k. & tellwright, j.m.: the incidence and pattern of cerebrovascular disease in general practice. j r coll gen pract 16: 428, 1968. acheson, r.m. & fairbairn, a.s.: burden of cerebrovascular disease in the oxford area in 1963 and 1964. br med j 2: 621, 1970. aho, k.: incidence, profile and early prognosis of stroke, epidemiological and clinical study of the 286 persons with onset of stroke. academic dissertation, helsinki, 1975. aho, k. & fogelholm, r.: incidence and early prognosis of stroke i n espoo-kauniainen area, finland in 1972. stroke 5: 658, 1974. alter, m., christofferson, l., resch, j., myers, g. & ford, j.: cerebro vascular disease, frequency and population selectivity in an upper mid western community. stroke 1: 454, 1970. aurell, m. & hood, b.: cerebral hemorrhage in a population after a decade of active antihypertensive treatment. acta med scand 176: 377, 1964. bruun, b. & richter, r.w.: the epidemiology of stroke in central harlem. stroke 4: 406, 1973. causes of death 1974. statistical abstract of sweden national central bureau of statistics, stockholm, 1976. cervantes, f. & schneiderman, l.: anticoagulants in cerebrovascular disea se a critical review of studies. arch int med 135: 875, 1975. eckstrom, p., brand, f., edlavitch, s. & parrish, h.: epidemiology of stroke in a rural area. public health report 84: 876, 1969. eisenberg, h., morrison, j., sullivan, p. & foote, f.: cerebrovascular accidents, incidence and survival rates in a defined population, middlesex county, connecticut. jama 189: 883, 1964. friedman, g.d., wilson, w.s., mosier, j.m., colandrea, m.a. & nichaman, m.z.: transient ischemic attacks in a community. jama 210: 1428, 1969. frithz, g.: studies on cerebrovascular disease. acta universitatis upsa liensis, abstracts of uppsala dissertations from the faculty of medicine 209, 1974. frithz, g., hood, b., hansson, l. & bjgrk, s.: cerebrovascular lesions. acta med scand 196: 35, 1974. glazier, w.h.: the task of medicine. scientific american 228: 13, 1973. harmsen, p. & tibblin, g.: a stroke register in ggteborg, sweden. acta med scand 191: 436, 1972. hatano, s.: who stroke controle programme. who chronical 26: 456, 1972. heyman, a., karp, h.r., heyden, s., bartel, a., cassel, j.c., tyroler, h.a., coroni, j., hames, c.g. & stuart, w.: cerebrovascular disease in the bi-racial population of evans county, georgia. stroke 2: 509, 1971. 245 1 9 . 2 0 . 2 1 . 2 2 . 2 3 . 2 4 . 2 5 . 2 6 . 2 7 . 2 8 . 2 9 . 3 0 . 3 1 . 3 2 . 3 3 * 3 4 . 3 5 . 3 6 . 3 7 . 3 8 . 3 9 . 4 0 . 4 1 . hillerdal, g. & aberg, h.: acute cerebrovascular lesions in uppsala in 1 9 7 6 (summary in english). lakartidningen 75: 2 2 3 1 , 1 9 7 8 . in-patient statistics from hospitals in the uppsala region 1 9 6 8 . national board of health and welfare, sweden, 1 9 7 2 . jorgensen, l. & torvik, a.: ischemic cerebrovascular disease in an autopsy series. j neurol sci 9: 2 8 5 , 1969. kannel, w.b., dawber, t.r., cohen, m.e. & mcnamara, p.m.: vascular disease of the brain epidemiologic aspects the framingham study. am j public health 5 5 : 1355, 1 9 6 5 . karp, h., heyman, a., heyden, s., barel, a., tyroler, h. & hames, c.: transient cerebral ischemia, prevalence and prognosis in a bi-racial community. jama 2 2 5 : 125, 1 9 7 3 . katsuki, s . , omae, t. & hirota, y.: epidemiological and clinicopathologi cal studies on cerebrovascular disease. kyoshu j med sci 1 5 : 1 2 7 , 1 9 6 4 . marquardsen, j.: the natural history of acute cerebrovascular disease. acta neurol scand suppl 38: 45, 1969. marshall, j.: the natural history of tia. quart j med 3 3 : 309, 1 9 6 4 . matsumoto, n., whisnant, j., kurland, l. & okazaki, h.: natural history of stroke in rochester, minnesota. stroke 4 : 2 0 , 1 9 7 3 . melamed, e., cahane, e., carmon, a. & lavy, s.: stroke in jerusalem district 1 9 6 0 through 1 9 6 7 . stroke 4 : 4 6 5 , 1 9 7 3 . organi: iix control of stroke in the community (ed hatano). pp. 9 11, geneve, 1 9 7 3 . ostfeld, a.m., shekelle, r.b. & klavans, h.l.: transient ischemic attacks and risk of stroke in an elderly poor population. stroke 4: 9 8 0 , 1 9 7 3 . pessin, m., duncan, g., mohr, 3. & poskanzer, d.: clinical and angiogra phic features of carotid transient ischemic attacks. n eng j med 296: 3 5 8 , 1 9 7 6 . report of the joint committe for stroke facilities. stroke 3 : 3 6 0 , 1 9 7 2 . sjgstrom, a.: hospitalized cases of stroke in a swedish hospital region. iqstroke (eds engel, a. & larsson, t.) pp 4 1 5 0 , stockholm nordiska bokhandelns forlag, 1 9 6 7 . statistical abstracts of sweden, 1 9 7 0 and 1 9 7 5 . stensgaard-hansen, b. & marquardsen, j.: incidence of stroke in fredriks berg, denmark. stroke 8: 6 6 3 , 1 9 7 7 . swanson, p., calcchini, p., dyken, m., gotshall, r., haerer, a., poskan zer, d., price, t. & coneally, m.: a cooperative study of hospital fre quency and character of transient ischemic attacks. jama 237: 2 2 0 2 , 1 9 7 7 . soderstrsm, c.e.: diagnostic significance of csf spectrophotometry and computed tomography in cerebrovascular disease. stroke 8 : 6 0 6 , 1 9 7 7 . toole, j., janeway, r., choi, k., cordell, r., cavies, c., johnston, f. & mille, h.: transient ischemic attacks due to atherosclerosis. arch neurol 32: 5 , 1975. toole, j. & yuson, c.: transient ischemic attacks with normal arterio grams. ann neurol 1: 1 0 0 , 1977. wallace, d.: cerebrovascular disease occurrence in australia. med j aust 2 : 1 0 9 3 , 1 9 7 1 . whisnant, j.p., matsumoto, n. & elveback, l.r.: transient cerebral ische mic attacks in a community, rochester, minnesota. mayo clin proc 48: 1 9 4 , 1 9 7 3 . received auril 3 . 1 9 7 9 adress for reprints: dr andreas terent department of internal medicine university hospital s-750 1 4 uppsala 1 4 sweden 246 upsala j med sci 79: 28-38, 1974 haemostatic plug formation in the rabbit mesentery a methodological study d. bergqvist department of experimental medicine, pharmacia ab, and department of surgery, university hospital, uppsala, sweden abstract a methodological study has been carried out using the rabbit mesentery for the investigation of the initial haemostatic mechanism in the microvasculature. heaemo static plug formation in proximal arteriolar ends differ from that in venules in many respects. the arteriolar haemostatic plug formation time is shorter than the venular and it is vessel size dependent in arterioles but not in venules. the frequency of rebleeding is higher and the rebleeding time is longer in venules than in arterioles. it is stressed that. the same vessel type and size should be used in comparative studies if comparable results are to be obtained. it is also shown that most of the rebleedings appear within 10 minutes of the cessa tion of the primary bleeding and that the haemostatic plug formation time shows a skew distribution, which should be taken into account in statistical calcula tions. the importance of using an adequate experi mental technique including a sharp and swift transec tion of the vessel is also stressed. introduction the mesenteric preparation of different animal species has been used for various microcirculatory investigations, particularly since the report of the method by chambers & zweifach (6). several authors have used this method when studying the initial haemostatic process (1, 7 , 12, 16, 20, 21, 22, 23, 27, 29, 30, 36). among these authors hugues (22) made the most careful methodological study, although all have used the model to study different aspects of the platelet reaction in the early haemo static mechanism. with a few exceptions (8, 9, 28) most investigators agree on the fundamental role played by the platelets in sealing a bleeding microvessel. apitz ( 1 ) called the aggregate of platelets >>die blutstillende thrombose. and zucker (36) called it >>the platelet plug,,. their histo logical findings were verified by the ultrastructural upsala j m e d sci 79 studies on the shaemostatic platelet plugx made by kjaerheim & hovig (27), who for the first time showed that the plug was composed of densely packed platelets. thus many investigations, dealing with different aspects of the haemostatic mechanism have been carried out in transected and punctured mesenteric microvessels. however, there are many discrepancies in the results, especially in the eva luation of arteriolar versus venular haemostasis and the occurrence and nature of rebleedings (1, 7 , 12, 16, 22, 30, 36). because of these contra dictory findings it is not possible to use the mesenteric model for haemostatic studies without further methodological investigations. the aim of this investigation therefore was to: 1. standardise the mesenteric model for studies of the initial haemostatic mechanism 2. study the influence of vessel type and size on the haemostatic plug formation time 3. study the rebleeding pattern 4. establish the importance of these factors to form a basis for further physiological, patho physiological and pharmacological studies. materials and methods 112 new zealand white rabbits of both sexes ( 2 . 7 + 0.5 kg), fed on a standard diet (ewos pellets, astra ewos ab, sodertalje or teknosan pellets, ferrosan ab, malmo, sweden), were used. the animals were starved at least 12 hours before the experiment. prior to anaesthesia bleeding time, whole blood coagulation time and haematocrit were performed in all rabbits as a screening method to ex clude abnormal animals. the. bleeding time was measured in the following way: the rabbit ear was warmed for 2 minutes with a heating lamp and veins of 0.2-0.5 mm diameter haemostatic plug formation in the rabbit mesentery 29 were transected. the mean of four bleeding times was recorded in every rabbit. whole blood coagulation time was determined by breaking off a short length of blood-filled capillary tube every 30 second until a fibrin strand had formed. two measurements were performed in every rabbit. haematocrit was measured in triplicate with a micro haematocrit centrifuge (10 000 g for 5 min; international equipment co., boston, usa). the animals were anaesthetised with 20% urethane in 0.9% saline (kebo, stockholm, sweden) by intravenous injection (4). through a small midline incision the distal part of ileum was exteriorised over a siliconised glass plate in a movable, electrically heated microscope table. during the experiment the preparation was continuously super fused with thermostated tyrode's solution of 37.5-39.o"c and p h 7.4. the bowels were covered with moistened swabs covered with plastic foil to minimise heat loss by evaporation. the excess fluid was removed by suction. with the electrically heated table and a heating lamp the rectal temperature of the animals was kept at 37.5-39.0"c during the experiment. the temperature was continuously monitored with a thermistor probe (laboratorio richerche elettroniche, milan, italy). for observations on the mesenteric microcirculation a leitz biomed intravital microscope with a leitz ultrapak 6 . 5 x objective at a total magnification of 81 x was used. the diameter of the vessels was measured with a calibrated ocular graticule. the diameter was measured before transection and the vessel type was identified, i.e. arteriole or venule. in the first part of the study (35 animals) a small opthalmic knife was used for transection of the micro vessels and in the second part (77 animals) this was performed with a disposable gillette scalpel blade, roo, 80 "i or 5 0 y 4 0 shape e/11. vessels used were in the transparent part of the mesentery giving the upper limit of their sizes as about 110 pm. the time from transection until the bleeding stopped was recorded as well as the frequency and time for rebleedings. every transected vessel was observed for about 20-30 minutes. statistical methods the mean value, the standard deviation and the regression coefficient was calculated according to standard methods (34). the haemostatic plug formation time had a skew distribution (vide infru). for statistical purposes the original values are rendered more normally distributed by logarithmic transformation. in testing differences in haemostatic plug formation times between the different vessel ends the sign test was used (lo), and significance was considered when p<0.05. in testing differences in frequencies of rebleeding the rank-sum test was used (10). both the sign test and the rank-sum test are distribution free tests, i.e. avoid assumptions about the nature of the distribution function of the data. dejkitions haemostatic plug: the platelet mass formed at the ends of a transected bleeding vessel. primary bleeding time or primary haemostatic plug formation time: the time from transection until the bleeding f r s t stops (pht). rebleeding: bleedings occurring at intervals through the initially efficient haemostatic plug. total rebleeding time: the total of all the rebleeding times recorded in one vessel. total bleeding time or total haemostatic plug formation time: the sum of the primary bleeding time and the total rebleeding time (tht). 0 a r r e r d e s venules 0 fig. 1 . the frequency (in per cent) of non-bleeding vessels in relation to the vessel diameter when an 10 20 m lo 9 60 ;o % gb l i b 1io 120 ophthalmic knife is used for the vessel dameter i,u) transection. i upsalu j med sci 79 30 d . bergqvist 0 arreraks venules 0 0 0 f i g . 2. the ratio of proximal to distal bleeding vessel ends after tion to the vessel diameter. 1 z e 9 t 10 n, 30 lo 50 60 70 80 90 too 110 120 transection with a scalpel in rela vessel dtamerer (pl results the bleeding time from transected ear veins is 86?31 seconds, the coagulation time 136?78 seconds and the haematocrit 3 8 . 7 c 3 . 6 . non of the animals was rejected from the study because of an extreme value. after transecting a microvessel in the mesentery cleanly and swiftly with a sharp knife, bleeding will immediately start from the transected vessel. within a few seconds a haemostatic plug starts to grow because of platelets adhering to the injured vessel and aggregating to each other. the plug continues to grow until the bleeding stops after a variable length of time. when a haemostatic plug, sufficient to stop bleeding, was formed at a proximal arteriolar end, blood will continue fo flow into the nearest vessel branch. in both venular ends and in the distal arteriolar end the blood flow will either be re versed, or stopped if anastomoses are absent. this primary haemostatic plug is not always stable and one o r more rebleeding may occur. in such a case new channels are opened up through the plug and no fragmentation of the plug occurs. with a blunt blade a certain number of vessels fail to bleed at all. fig. 1 shows the percentage of non-bleeding vessels in the different vessel size groups based on calculations from the experiments where the oph thalmic knife was used (35 animals). at least for arterioles there is a greater number of non-bleeding vessels when the diameter is small ( r = 0 . 8 5 for arterioles; p<o.ol. r=0.67 for venules; p > o . o s ) . the rest of the results are based on calculations from the animals in which sharp scalpel blades were used for transection. after transection of a venule bleeding usually occurs from both ends of the transected vessel, but it seldom occurs from the distal end of a transected arteriole as can be seen from fig. 2. n o correlation could be demonstrated between the ratio (number of proximal bleeding vessel ends per number of distal bleeding vessel ends) and the vessel size (r for arterioles is 0.37 and r for venules is 0 . 6 4 ; p > 0 . 0 5 ) . the total and primary haemostatic plug formation times for different vessel sizes are given in table i and this is demonstrated graphically in figs. 3 and 4. it can be seen, that the haemostatic plug formation time, both the total and the primary, increases with increasing arteriolar size. there is a good correlation between arteriolar diameter and the haemostatic plug formation time (table 11). on the other hand there is no correlation between venular size and haemostatic plug formation time (table 11). from figs. 3 and 4 and table i it can also be seen that the total venular rebleeding time is much greater than the total rebleeding time for the proximal arteriolar end, and that there is a great variation as far as the distal arteriolar end is concerned. the haemostatic plug formation time for distal upsala j med sci 79 haemostatic plug,formation in the rabbit mesentery 3 1 table i. total ( t h t ) and primary ( p h t ) haemostatic plug formation times f o r arterioles and venules the mean value and s.d. are given arterioles venules vessel proximal end distal end proximal end distal end size olm) tht pht tht pht tht pht tht pht 7 3 7 2 4 9 2 7 2 2 9 62$.73 5 6 1 6 7 13 3 5 2 3 6 3 1 2 3 5 102+ 127 861-88 200f256 166f238 277f281 2322266 20 41 2 4 6 32_+31 138k216 1172209 2702300 2012273 2782268 2241246 26 4 5 k 4 3 3 7 2 3 5 113f203 8 8 2 1 9 7 290+304 221f279 2322255 195i-245 33 6 6 k 5 4 4 9 2 4 2 1782259 1772260 2622218 1762187 2932287 245+278 40 7 5 f 7 3 6 2 2 6 5 2572318 252+317 1892381 1452128 353f322 283f306 46 8 5 2 7 5 6 7 f 6 1 3852396 2142324 2502239 126+134 391f302 2952308 53 99+86 7 7 2 6 2 2622332 1582276 2211-204 1492134 386+314 3102307 59 9 9 2 7 1 8 8 f 6 8 206f281 1881-280 2542273 1952269 3182287 2612277 66 1231103 101f98 363_+364 314_+354 2522287 2291296 3472312 260_+279 79 139k91 121+89 2342270 2342270 3832310 3142287 3532293 283f275 92 1 6 4 2 9 9 1471-92 366f365 3652377 2641-271 2292261 302k266 230k243 106 1992109 1842106 4 3 6 t 3 3 0 4092350 arteriolar ends is significantly longer than that for the proximal ends ( p < o . o o l ) , but the corresponding venular haemostatic plug formation times d o not differ significantly ( p > o . o s ) . the haemostatic plug formation time for proximal arteriolar ends is statistically shorter than that for venules ( p < o . o o l ) . the difference between venular and distal arteriolar haemostatic plug formation time is insignificant for the primary time (p>o.os) and of borderline significance for the total time (o.ol<p<o.os). the o a total bleedmg ome prmary bleedrnq t m e arterroles 0 . 4501 4001 300 i a difference in haemostatic plug formation times between arterioles and venules diminishes with increasing vessel size (figs. 3 and 4). if the frequency of rebleeding is plotted against the total rebleeding time the origo represents an absolutely stable haemostatic plug. in proximal arteriolar and venular ends there is a correlation between the frequency of rebleeding and the total rebleeding time ( r 0.77 and o.86;p<o.0li see fig. 5 ) . there is no correlation between the frequency of p /’ / p f 0 dsral end ,/’ proxtmal end q 1 1 i i i i i i i 1 i 1 1 10 20 30 lo 50 60 70 80 90 100 110 120 vessel s u e (,urn) fig. 3. the arteriolar haemostatk plug formation time in relation to the vessel diameter. upsala j med sci 79 32 d . bergqvist 255 c e 200 e 150 & 100 400 3501 300 o d total bleedmg f m e pr,mary bleedtng lime venuies @ a a a i%fa/ end 0 proxrmal end f i g . 4. the venular haemostatic 10 20 30 40 50 60 70 80 90 100 110 120 plug formation time in relation to the vessel diameter. i o l i _ i i i i 1 1 vessel s,ze (,urn) 1 6 154 i ’-1 j 3 1 t ee 0 3 0 a . a a i a a a a a a a a a n ::lo o 0 o 0 0 so 100 150 200 rebleeding and the vessel size, nor is there a n y correlation between the total rebleeding time and vessel size (table 11). the frequency of rebleeding i n venules is significantly higher than i n arterioles @ < o . o l ) . figs. 6 and 7 show the number of rebleeding plugs expressed as a percentage of the total in relation to the time between the cessation of primary bleeding and the start of the first rebleeding. within 10 minutes after the formation of the primary haemostatic plug almost all (>97%) rebleedings have occurered, and within 5 minutes more than 70% of the rebleedings have occurred. every line represents one vessel size and the time for re bleeding t o occur is independent of the vessel size. the distribution of the haemostatic plug formation times is skew, that is, even in normal rabbits some vessels bleed for a very long time. the longest haemostatic plug formation times are seen in distal arteriolar ends and in venules. in figs. 8 and 9 the distribution curves are given for vessels of the sizes 26 and 53 p m . the skew distribution is rendered more normal after logarithmic trans formation, discussion rorai reb/eed(ng r m e isec / since urethane is known to induce haemolysis ( 5 ) with the release of adenosine diphosphate, adp, the use of anaesthesia in haemostatic studies may be theoretically unacceptable. adp is, f i g . 5 . the stability of the haemostatic plug is shown by plotting the frequency of rebleeding against the total rebleeding time. origo represents an absolutely stable plug. upsala j med sci 79 huemostutic p l u g formation in the ruhbit mesentery 3 3 table 11. correlation coefficients between the vessel diameter in pm and different parameters f o r the four vessel segments studied ns: not significant. pco.001 correlation ( r value) between vessel size and ~ proximal distal proximal distal arteriolar end arteriolar end venular end venular end primary haemostatic plug formation time 0.99*** 0.92*** 0.51 ns 0.23 ns total haemostatic plug formation time 0.99 * * * 0.84*** 0.42 ns 0.31 n s 0.48 ns frequency of rebleeding 0.29 ns 0.06 ns -0.34 ns total rebleeding time 0.60 ns 0.01 ns -0.19 ns 0.57 ns 100 3 c u k w m e w 5 9 y 50 u p f d 9 % 0 $ 2 2 60 in) i80 240 300 360 120 a80 540 600 i m e fsec i ilme ( s k i given above the curve). 1 120 f i g . 6 . the number of re bleeding arteriolar haemo static plugs expressed as per cent of total number of re bleeding plugs plotted against the time between the first arrest of bleeding and the start of the first rebleed ing. every line represents l&j 5$ 6& one vessel size (diameter 3-742854 upsala j m e d sci 7 9 34 d. bergqvist h 2 0 a e c 0 i promma/ venules i i r m fsec) rime fsec) however, rapidly metabolized and removed from plasma (17, 18, 24). we have evaluated the effect of different anaesthetic agents on the formation and stability of t h e haemostatic plug using the mesenteric model and found no significant differences between urethane, chloralose and mebumal sodium anae sthesia or neurolept analgesia (4). as rabbits anaesthetised with urethane were stable in acid base balance and blood pressure, and as urethane was the agent most easy to handle in our hands, we decided to use it in our further studies. it must be borne in mind, however, that our haemostatic studies were made while the mesenteric fig. 7. the number of re bleeding venular haemostatic plugs expressed as per cent of total number of rebleed ing plugs plotted against the time between the first arrest of bleeding and the start of the first rebleeding. every line represents one vessel size (diameter given above the curve). preparation was continuously superfused with tyrode’s solution. the superfusion milieu is standardised as far as possible, for ions, ph, temperature and flow. because of the superfusate it must be stressed, that the haemostatic plug formation time obtained with the present method may not be comparable to bleeding times obtained with other techniques, for instance described by duke ( 1 1 ) or i v y (25). comparisons between these two types of techniques should thus be very care ful. using a continuous superfusate, blood and various tissue factors are washed away. there are also differences between the tissues themselves, ffpsulu j med sci 79 haemostatic plug formation in the rabbit mesentery 35 fig. 8. the distribution of the arteriolar haemostatic plug formation time shown l 200 3w 100 500 i.e. anatomical localisation, thickness, forces of pressure and pulling and numbers of transected vessels. another factor of importance is the transection technique. the phenomenon of nonbleeding vessels has been described before but not the difference between vessel types ( 1 , 22, 3 3 , 36). endothelial adhesion was previously considered to be the main haemostatic mechanism in capillaries (15, 33), but jsrgensen & borchgrevink ( 2 6 ) showed histo logically that platelet plugs were formed in capillaries too. from our study it is evident (fig. i), that the frequency of non-bleeding vessels, when using a blunt knife, is somewhat higher in arterioles than in venules, and that the frequency is correlated to the diameter of the arteriole. the theory of endo thelial adhesion and the frequent occurrence of non-bleeding vessels are probably due to the fact that the importance of sharp knives and swift 6w 760 /set/ for two vessel sizes. transections in previous haemostatic experiments wasnot considered. inconclusion, theseresultsmade us use disposable knife blades in the main study. the frequency of non-bleeding vessels could then be neglected and could well be compared with hugues' (22) results. some authors studying the initial haemostatic mechanism have used puncture of microvessels instead of transection (7, 14, 19, 36). in those studies the size of the puncture hole, the thickness and elasticity of the vessel wall and the adjacent tissue will probably influence the bleeding time. when a vessel is punctured there is a combination of a haemostatic plug outside the vessel and a microthrombus inside it. after transection of a venule bleeding occurred from both ends of the transected vessel, while bleeding was less common from the distal than from the proximal arteriolar end, which almost ,urn proxmal venuk 26,um fig. 9. the distribution of formation time shown for roxrmal 5 3 p m dsra! 2 6 p m the venular haemostatic plug i i i i i i i na 200 3aj 403 mo 600 700 .do & fsccl two vessel sizes. vpsala j med sci 79 36 d. bergqvist always bled (fig. 2). although this phenomenon has been briefly mentioned by various authors (1, 13, 22, 33, 36), no systematic study of this problem has been carried out. fig. 2 shows that the number of observations from distal arteriolar ends is less than from the other vessel ends, a fact that must be remembered in the further discussion. the pressure differences, that exist between proximal and distal arteriolar ends, could in part explain this observation. a pressure difference should also exist between the both venular ends, but this will not be so pronounced as in arterioles and probably is of no importance since the venular ratio is near 1 . there are contradictory results in the literature as far as the relationship between the haemostatic plug formation time and vessel type and diameter is concerened ( 1 , 22, 33, 36). hugues (22) observed the importance of the vessel type, but apart from his study and as far as the vessel size is con cerned, no systematic studies on these problems have been carried out. hugues (22) found the longest bleeding times in proximal arteriolar ends, whereas bleeding times from the other vessel seg ments were of the same magnitude and about half that seen in the proximal arteriolar ends. in his calculations hugues did not use the 11% of the transected vessels, where the bleeding time exceeded 10 minutes, which was seen in venules and distal arteriolar ends-a finding that fits well with our experience (vide infru). this is probably the explanation why hugues found the primary haemostatic plug formation time referred to above, where the proximal arteriolar end showed the longest bleeding time. he pointed out that the most reproducible results were obtained from the proximal arteriolar ends, but we suggest that valuable in formation also can be obtained when the venules are taken into consideration. thus we found aspirin to have no effect on arteriolar haemostatic plug formation time, whereas venular haemostatic plug formation time was significantly shortened (2). in his study hugues (22) also studied the vessel contraction in connection with haemostatic plug formation. he found no correlation between the degree of contraction and the bleeding time. we found that the haemostatic plug formation time is longer and the rebleeding tendency greater in venules than in arterioles, and that the time for haemostatic plug formation is significantly longer in the distal than in the proximal arteriolar end. upsala j med sci 79 these investigations clearly show, that in studies of haemostatic plug formation one must be careful to differentiate between arterioles and ven.ules as well as taking into account the vessel diameter at least in arterioles. it can be concluded, that the same vessel size and type should be used in com parative studies, if comparable results are to be obtained. this fact has not always been taken into consideration (12, 16, 30). the literature concerning the nature and occurrence of rebleedings is contradictory ( 1 , 7, 12, 16, 20, 22, 30, 36), and no systematic study on this phenomenon has been made. the rebleeding always started with the opening up of new channels through the formed plug as pointed out by zucker (36). however, some authors have remarked on the frequency of fragmentation both in rebleedings (35) and during the growth of primary plugs (3 1, 32). we found the lowest frequency of rebleeding in the distal arteriolar ends, but the length of the total rebleeding time varied. this time and the frequency of rebleeding was fairly constant for the proximal arteriolar end, while both parameters were longer for the two venular segments. both proximal vessel ends showed some correlation between the frequency of rebleeding and the total rebleeding time. on the other hand the rebleeding pattern was independent of the vessel size in the range investigated. our results, showing that re bleedings are more frequent in venules, speak against the hypothesis claimed by aptiz ( l ) , that pressure is of prime importance for the rebleedings to occur. thus it is also of considerable importance t o define the vessel type when studying the re bleeding pattern, whereas the vessel size seems to be of less importance within the range under in vestigation. we have found, that the rebleeding pattern is a useful parameter, for instance in pharmacological studies. thus, the rebleeding fre quency was significantly decreased in rabbits treated with aspirin (2), ether ( 4 ) and sulfinpyrazone or phenylbutazone (unpublished results), but the haemostatic plug formation time was normal in all groups except the aspirin group in which the venular haemostatic plug formation time was shortened. as can be seen from figs. 6 and 7 the majority of rebleedings (>97%) occurred within 10 minutes of the formation of the primary haemostatic plug. using this experimental model system it would thus haemostatic plug formation in the rabbit mesentery 37 be sufficient in normal, untreated rabbits to observe for a period of 10 minutes after primary haemo statis. the distribution of haemostatic plug formation time has not been studied in detail before, although hugues (22) found that in i i % of transected venules and distal arteriolar ends the bleeding continues for more than 10 minutes. in these cases he found fragmentation from already formed plugs or no plugs at all. in our studies it was extremely rare to find a total absence of plugs. on the other hand the growth rate of the plugs in venules and distal arteriolar ends was very slow, when bleeding was prolonged (3). as can be seen from figs. 8 and 9 the haemostatic plug formation time shows a skew distribution in our material, the skewness being more pronounced for venules and distal arteriolar ends. since prolonged bleeding times are a part of the normal bleeding time distribu tion it is our opinion, that they must be included in calculations on haemostatic plug formation time. in our material logarithmic transformation of the haemostatic plug formation time render it more normally distributed and in our opinion this method should be used, when statistical comparisons are made. conclusions i . a swift and clean transection with a sharp knife should be used in experiments on haemostatic plug formation. 2. the ratio of bleeding proximal ends to bleeding distal ends is between 2 and 3 for arterioles and about 1 for venules. this ratio is not correlated to the vessel size. 3. total and primary haemostatic plug formation time in proximal arteriolar ends are statistically shorter than those in venules and distal arteriolar ends. 4. total and primary haemostatic plug formation time in arterioles but not in venules are correlated to the vessel diameter. 5. the difference in haemostatic plug formation time between arterioles and venules decreases with increasing vessel size. 6. the total rebleeding time is longer in venules than in proximal arteriolar ends. distal arteriolar ends show a variable pattern. 7. the frequency of rebleeding in proximal arteriolar and venular ends is correlated with total , rebleeding time. 8. there is no correlation between vessel diameter and total rebleeding time or frequency of re bleeding. 9. the frequency of rebleeding is lowest for distal arteriolar ends, followed by the proximal arteriolar ends and both venular ends. 10. most rebleedings (>97%) have occurred within 10 minutes after the formation of the primary haemostatic plug. 1 i . the haemostatic plug formation time shows a skew distribution which can be rendered more normal by logarithmic transformation. references 1 . apitz, k.: die bedeutung der gerinnung und thrombose fur die blutstillung. virchows arch path anat 308: 540, 1942. 2. arfors, k.-e.,bergqvist,d.,bygdeman,s., mckenzie, f. n. & svensjo, e.: the effect of inhibition of the platelet release reaction on platelet behaviour in vitro and in vivo. scand j haematol 9: 322, 1972. 3. bergqvist, d. & arfors, k.-e.: the growth rate of haemostatic plugs in the rabbit mesentery. proc. vii conf. microcirculation, aberdeen, 1972. karger, basel, 1973. p. 186. 4. bergqvist, d., mckenzie, f. n. & arfors, k.-e.: influence of various anaesthetic agents on the formation and stability of haemostatic plug in the rabbit mesentery. upsala j med sci, in press. 5 . bree, m. & cohen, b. effects of urethane anaesthesia on blood and blood vessels in rabbits. lab animal care 15: 254, 1965. 6 . chambers, r. & zweifach, b. w.: topography and function of the mesenteric capillary circulation. am j anat 75: 173, 1944. 7. chen, t . i . & tsai, c . : the mechanism of haemo stasis in peripheral vessels. j physiol 107: 280, 1948. 8 . cruz, w. 0.: studies on hemostasis in vivo. henry ford hospital symp. blood platelets. little, brown & co., boston, 1961. 9. cruz, w. 0.: the significance ofa smooth muscle com ponent in hemostatis. proc soc exptl biol med 119: 876, 1965. 10. dixon, w. j . & massey, f . j., jr: introduction to statistical analysis, mcgraw-hill, new york, 1957. 1. duke, w . w.: the relation of blood platelets to hemorrhagic disease; description of a method for determining the bleeding time and coagulation time and report of three cases of hemorrhagic disease relieved by transfusion. j am med assoc 55: 1185, 1910. 2 . evans, g . , packham, m. a., nishisawa, e. t., mustard, j . f. & murphy, e. a . : the effect of acetylsalicylic acid on platelet function. j exptl med 128: 877, 1968. 13. french, j. e., macfarlane, r. g. & sanders, a. g . : the structure of haernostatic plugs and experimental thrombi in small arteries. brit j exptl path45: 467, 1964. upsala j m e d sci 79 38 d . bergqvist 14. herrmann, r . , frank, j . d. & marlett, d. l.: an in vivo technique for assessing the formation of a hemostatic platelet plug. proc soc exp!l biol med 128: %0, 1968. 15. herzog, f.: die rolle der capillaren bei der blut stillung. pfliigers arch gesamt physiol207: 476, 1925. 16. hirsh, j . , buchanan, m., glynn, m. & mustard, j . f.: effect of streptokinase on hemostatis. blood 32: 726, 1968. 17. holmsen, h. & stormorken, h.: kinetic studies on the breakdown of adenosine diphosphate in human plasma. scand j clin lab invest suppl. 84, p. 138. 1965. 18. holmsen, i . & holmsen, h.: breakdown of adenosine nucleotides in whole blood. 111 congr int soc thromb haemost. washington 1972. abstract volume p. 206. 19. honour, a. j . & mitchell, j . r. a,: platelet clumping in injured vessels. brit j exptl path 45: 75, 1964. 20. hovig, t., rowsel!, h. c., dodds, w. j., jsrgensen, l. & mustard, j. f.: experimental hemostatis in normal dogs and dogs with congenital disorders of blood coagulation. blood30: 636, 1967. 21. hovig, t., dodds, w. j., rowsell, h. c. & mustard, j. f.: the transformation of hemostatic platelet plugs in normal and factor ix deficient dogs. amer j path5.3: 355, 1968. 22. hugues, j . : contribution a i’etude des facteurs vasculaires et sanguins dans i’hemostase spontanee. arch internat physiolll: 565, 1953. 23. hugues, j . : metamorphose visqueuse des plaquettes e t formation du clou hemostatique. thromb diath haemorrh 3: 34, 1959. 24. ireland, d. m. & mills, d. c. b.: degradation of adenosine diphosphate in plasma. biochem j 92: 30p, 1%4. 25. ivy, a. c., shapiro, p. f. & melnick, p.: the bleeding tendency injaundice. surg gynec obstet60: 781,1935. 26. jsrgensen, l. & borchgrevink, c.: the platelet plug in normal persons. i. the histological appearance of the plug 15 to 20 minutes and 24 hours after the bleeding and its role in the capillary hemo stasis. acta path microbiol scand57: 40, 1963. 27. kjaerheim, a. & hovig, t.: the ultrastructure of haemostatic blood platelet plugs in rabbit mesenterium. thromb diath haemorrh 7: i , 1962. 28. macfarlane, r. g.: critical review: the mechanism of haemostasis. quart j med n s 10: 1 , 1941. 29. marr, j . , barboriak, j . j . & johnson, s. a.: relation ship of appearance of adenosine diphosphate, fibrin formation and platelet aggregation in the haemostatic plug in vivo. nature 205: 259, 1965. 30. packharn, m. a,, warrior, e. s., glynn, m. f., senyi, a. s. & mustard, j . f.: alteration of the response of platelets t o surface stimuli by pyra zole compounds. j exptl med 126: 171, 1967. 31. roskam, j . : role of platelets in the formation of a hemostatic plug. blood platelets. little, brown & co., boston, mass., 1961. 32. roskam, j . , hugues, j., bounameaux, y. & salmon, j.: the part played by platelets in the formation of an efficient hemostatic plug. thromb diath haemorrh 3: 510, 1959. 33. sanders, a . g.: in vivo observations on haemostasis in the hamster. symp. zoo1 soc lond 27: 109, 1970. 34. snedecor, g. w. & cochran, w . g.: statistical methods. the iowa state univ. press. ames, iowa, usa. 6th ed. 1967. 35. stormorken, h. & owren, p. a.: physiopathology ofhemostatis. sem hemat8: 3, 1971. 36. zucker, m. b.: platelet agglutination and vaso constriction as factors in spontaneous hernostasis in normal, thrombocytopenic, heparinized and hypo prothrombinemic rats. amer j physiol148: 275, 1947. received m a y 16, 1973 address for reprints: david bergqvist department of experimental medicine pharmacia ab, box 604 s-751 25 uppsala 1 sweden upsala j med sci 79 upsala j med sci: 81: 103-108, 1976 rehabilitation of patients with low back disorders in the county of uppsala soren hilding, herman hedqvist, brita arbman and anders wigren from the department of orthopaedic surgery, university hospital, uppsala and the rehabilitation centre of uppsala county council, sweden abstract low back pain is a common cause of inability to work. at the rehabilitation centre of the uppsala county council an annual number of more than 100 patients with this condition undergo an investigation aimed at evaluating their capacity for work and their need for rehabilitation. by compiling and analysing the informa tion recorded in the case journals of the patients with low back disorders during the years 1969 to 1971, it was hoped that more knowledge would be gained about the factors constituting a hindrance to rehabilitation. the present paper is a preliminary report on an analysis of 47 case journals unsystematically selected from the entire ma terial. the study verifies a number of previously reported observations, e.g. the low frequency of roentgenological changes. it is also noted that in a remarkably large number of patients no or only slight abnormalities were found on physical examination. compared with the results of other investigations, there was a very low frequency of concurrent psychiatric disorders and abuse of alcohol. it was considered from this analysis that the information in the case journal material in question is adequate for elucidating the relevant problems in the planned larger investigation; further, it provides motivation for a future follow-up study. introduction and background a large number of people of occupational age suffer from low back pain, which in many cases causes inability to work. this problem has long been a subject of research efforts, and numerous investi gations have been made, especially in the indus trialized countries, with the aim of shedding light not only on its orthopaedic and general medical aspects but also on its psycho-social effects. in scandinavia, studies have been reported by hult (1954), gogstad (1962), magnusson (1968), sparup (1969), horal (1969), natvig (1970), tufvesson and co-workers (1970) and westrin (1970), among others. in sweden those people who because of illness or disablement are unable to carry out their ordi nary work generally undergo both medical and occupational rehabilitation. with respect to the latter, in order t o make a more accurate evalua tion of the patient’s capacity for work and need for rehabilitation, a relatively extensive investiga tion is undertaken. at the rehabilitation centre of the uppsala county council, about 350 patients are investi gated each year. about 35% of these are cate gorized as patients with low back disorders. despite considerable efforts at medical and oc cupational rehabilitation, the impression is ob tained that many of these patients, because of their low back pain, have great difficulty in readjusting to working life. by compiling and analysing the in formation recorded in the case journals of the rehabilitation centre concerning patients pre dominantly disabled by a low back disorder, and by examining the patients 4 to 5 years later, it was hoped that more knowledge would be gained about the factors constituting a hindrance to rehabilita tion. the present paper concerns the first part of the investigation, i.e. an analysis of case journals from the rehabilitation centre. an attempt was made to find out whether any correlations existed between particular symptoms, in combination with objective findings, and the outcome of the rehabilitation. the patients’ physi cal and intellectual capacities and their social situa tion also seemed worthy of investigation in this context. it was considered that some type or types of patients with low back pain in whom the rehabili tational measures had a particularly good or poor effect might be distinguishable. further, it was hoped that the results of this investigation would upsala j med sci 81 \ 104 s . ffilding et al. have practical benefits such as: ( 1 ) improvement of the routine rehabilitational investigation; (2) es tablishment of appropriate methods of medical treatment; ( 3 ) improvement of prognostic evalua tion; (4) establishment of measures to prevent re currence. material, methods and definitions during the years 1969 to 1971, 977 patients were investi gated at the rehabilitation centre in uppsala. of these, about 35 9% (340 patients) were considered to have a low back disorder as their main diagnosis. 1971 was chosen as the last year in order to have at least 4 years between the investigation and the planned follow-up examination or, to use a mare modern term, the product control. from the relevant case journal material, 47 were se lected unsystematically. the patients were divided into age groups of 5 years, with 15 years as the lower limit and 49 years as the upper. patients with low back disorders get in contact with rehabilitation centre mainly in two ways, either through the county employment board, or by referral from the department of orthopaedics a t the university hospital of uppsala. orthopaedic examination the examination was performed at the dept. of ortho paedics and with a few exceptions, by the same doctor. this latter was considered an advantage and is a reason able guarantee of relatively good uniformity in the evalu ation of the signs and symptoms. the patients were clas sified essentially according to the same principles as were used by lennart hult in the munkfors investigation in 1954. lumbar spine insufficiency by lumbar spine insufficiency is meant a condition with a more or less intermittent sensation of fatigue or pain located in the form of a band over the lumbar spine. the symptoms are often provoked by physical work in the forward bending position. recurrent lumbago this is defined as repeated attacks of aching, pain on movement, and stiffness in the lumbar spine. between attacks the patient is relatively free from symptoms. chronic lumbago these patients have the same symptoms as the fore going, but the symptoms are more persistent and there are no completely free intervals. sciatica this group have not only symptoms from the lumbar spine but also radiating pain in the lower extremities, usually unilateral. the patient often has a concomitant sensation of numbness or paresthesia in the foot and toes. by concurrent symptoms from the cervical and thoracic spine is meant aching, pain and stiffness in these spinal areas, in some cases combined with radiating pain in the upper extremities. the objective findings in the orthopaedic examina tion were also classified in accordance with the defini tions in the above-mentioned investigation by hult: static spinal deformity by this is meant deviations from the normal configuration of the spinal column, e.g. kyphosis, lordosis or scolio sis. restricted mobiliiy extension, forward flexion and lateral flexion of the spine were evaluated. from the findings at the ortho paedic examination the patients were divided into two groups, one with restricted mobility and one with pro nounced restricted mobility. this was only a rough classification but seemed to be the only one practicable at this stage of the investigation. lasegue's sign this sign was tested in all patients, and for the purpose of this investigation it was considered sufficient to note only a positive finding. neurological deficit by this is meant here the occurrence of sensory impair ments, paresthesia, paresis, muscular atrophy and ab sence of the patellar or achilles reflexes. the patients with lumbo-sacral rhizopathy were also divided into two groups according to whether or not the level of the lesion could be determined. general medical evaluation a detailed history was first taken, including both previous illnesses and the present condition. any psychological disorders or abuse of alcohol were noted, and patients with such symptoms were always examined by a psychi atrist. a thorough physical examination was carried out and laboratory tests included blood analyses for haemo globin concentration (expressed in grams per cent), red and white blood counts and sedimentation rate, and uri nary analysis for protein and glucose. it was considered sufficient to record only the occurrence of disease in the respective organic systems. additional examinations roentgenological examination of the lumbar spine was performed in most patients, with special regard to disc degeneration, spondylosis and spondylolisthesis. if lum bar myelography with contrast medium was carried out, this was also noted. examination of isometric muscle strength was per formed in all patients at the department of clinical physiology of the university hospital. this included tests of the muscle power and function in the upper and lower extremities and lumbar spine. any pain in connec tion with the examination was noted. for the present upsala j med sci 81 rehabilitation of patients with low back disorders 105 table i. a g e distribution of the patient material age patients no. of 15-19 4 20-24 7 30-34 4 35-39 10 40-44 6 45-49 2 total 47 25-29 14 study a recording was made only of whether the muscle strength was considered normal or reduced. a physicul work test was also usually included in the investigation. this also was performed at the dept. of clinical physiology, using a bicycle ergometer with in creasing work loads. the heart rate, respiratory fre quency and blood pressure were recorded continuously. the patients were classified according to whether their physical work capacity was good, ordinary, somewhat low, low or very low. a psychological examination was carried out in most cases. this was undertaken by a psychologist attached to the rehabilitation centre, with the aim of assessing the patient’s intellectual and practical abilities. the patients were graded according to the scale: sub-normal, normal and above normal. an extensive social investigation was performed, comprising school and vocational education, the occupa tional situation, absence from work, periods on the sick list, family conditions, housing and financial situation, hobbies and any criminal record. it was considered im portant to get an idea, if possible, of the patients’ annual incomes prior to the rehabilitational investiga tion and to compare them with those of the rest of the population in the years in question. useful information on incomes was obtained from about half of the patients, but it should be pointed out that even these are only rough estimates and that the figures are therefore uncer tain. the definitions of low and high incomes used by holmberg & strom (3) were applied in the present anal yses. the income data of the low-income investigation are based on an analysis of swedish people in 1966. in the investigation of low-income groups the income limits were listed in relation to the wages development from 1966 to 1970. the following income groups were thus defined: a low income meant an annual income of less than 20000 sw. kr, an average income an annual income of 20000 to 40000 kr, and a high income an annual income of more than 40000 kr. a rehabilitational investigation always results in a rec ommendation, and a t present there are more than 50 different alternatives. the alternatives most relevant for patients with low back disorders may be summarized as follows: return to previous work; change to different occupation; vocational training (patients who have not previously had such training); re-training (patients with previous vocational training who are trained for another occupation); higher education (junior secondary school, upper secondary school, university or equivalent); con tribution towards a business; and retirement pension. results of the 47 patients, 34 were men and 13 women. four of them were of foreign nationality. thirty-three of the patients were referred for the investigation by the county employment board, and the rest by the department of orthopaedics at the university hospital. the age distribution is presented in table i. all age groups are represented, though somewhat unevenly. the general medical examination revealed that one patient had concurrent cardiopulmonary dis ease, 3 patients gastrointestinal diseases, 3 patients urogenital diseases and one patient contact allergy. four of the patients were suffering from psychiatric disorders and 3 were heavy drinkers. the results of the orthopaedic examination are given in tables i1 and 111. in 31 patients, i.e. the majority, lumbar spine insufficiency was the domi nant symptom. sixteen suffered from lumbago or sciatica. a few patients also had concurrent symp toms from the cervico-thoracic spine. in 19 of the patients no abnormal findings were made at the orthopaedic examination, and in the remaining 28 the main finding was restricted mobility of the lumbar spine, but in no case was this restriction pronounced. thirty-one patients had a roentgenological ex amination of the lumbar spine. in 8 patients, one or several discs in the lumbar spine were lower than normal, indicating degeneration. 10 patients also exhibited osteophytes on the margins of the vertebrae (spondylosis deformans). in one patient table 11. s y m p t o m s a t the orthopaedic examina tion lumbar spine insufficiency 31 lumbago recurrent 2 lumbago chronic 4 sciatica, back symptoms predominating 4 sciatica, leg pain 0 sciatica, equally troubled by both concurrent symptoms from cervico thoracic spine 4 concurrent lesion or disease of lower extremities 3 6 upsala j med sci81 106 s . hilding et al. table 111. findings at orthopaedic examination no abnormalities 19 restricted mobility 20 unqualified gainful employment 32 greatly restricted mobility 1 qualified gainful employment 14 laskgue's sign positive 12 light gainful employment 16 table v. occupational situation prior to the re habilitational investigation static spinal deformity 10 housewife 1 neurological deficit with established heavy gainful employment 30 neurological deficit with level uncertain level of lesion 2 3 with sciatica and with neurological deficit, lumbar myelography with contrast medium was per formed, with no evidence of herniation of the discs. examination of the isometric muscle strength revealed reduced muscle strength in 15 of the pa tients and normal strength in the remainder. a physical work test was performed in 38 patients. in 9 of them the physical work capacity was above normal, in 24 normal and in 5 subnormal. the results of the psychological examination, which was carried out on 40 of the patients, can be seen in table iv. the social investigation showed that 32 of the patients had only completed the compulsory school education, one patient higher school education and 15 patients vocational training. table v shows the occupational situation of the patients prior to the rehabilitational investigation. it is seen that the majority-about 30 of the pa tients-had heavy, unqualified work. fairly reliable information concerning annual incomes was obtained for 23 patients and are di vided into low, average, and high incomes in table vi. about half the patients whose incomes could be ascertained lay within the average income group, a few had a high income and the rest, mostly wom en, were in the low income group. during the 3 years immediately preceding the in vestigation, 25 of the patients had been o n the sick list for more than 6 consecutive months or for short, frequent periods. four patients were unemployed at the time of table iv. psychological examination the investigation. the family situation can be seen in table vii. the majority of the patients (36) were married or cohabiting, and 20 of these had a gain fully employed husband/partner. the housing situa tion could be regarded as unsatisfactory in 6 cases. twenty-two of the patients stated that they had ac tive hobbies, and in 10 of them these involved physical activity. two of the patients had criminal records. the recommendations resulting from the rehabil itational measures are listed in table viii. the two predominant recommendations were change of occupation and vocational training. discussion as it was the intention to analyse the entire case journal material, it was considered necessary when selecting patients for the present pilot study only to take into account the completeness of the jour nals, and not the exact distribution of the total ma terial with regard to sex, etc. for the same reason it was not considered to satisfy statistical demands of randomness. the results reported here are derived from a small portion of a series of patients who underwent rehabilitational investigation during 3 consecutive years. as mentioned previously, the present study is to be regarded as a preliminary report for the planning of a broader investigation including the whole series of patients. n o definite conclusions can therefore be drawn concerning the patients t o h l p wt distribution of incomes prior to rehabi subnormal intellectual ability 4 normal intellectual ability 26 above normal intellectual ability 10 l a " l c "i. litation subnormal practical ability 3 low income 7 ( 1 man, 6 women) normal practical ability 24 average income 13 (12 men, 1 woman) above normal practical ability 13 high income 3 (men) examination not performed 7 no evaluable information 24 wpsala j med sci 81 rehabilitation of patients with low back disorders 107 table vii. family conditions married or cohabiting 36 20 2 3 husband or wife gainfully employed sole family supporter 3 single without duty to support family separated or divorced at time of investigation family problems 3 with low back disorders undergoing rehabilitational measures. in choosing the lower age limit consideration was taken of the fact that an increasing number of people undergo rehabilitational measures at a very young age and often at the end of the compulsory school period. the upper limit of 49 years may seem rather low, but this limit was chosen for practical reasons, so that all patients in the ma terial should have a true possibility of utilizing the recommendations following the rehabilitational investigation. the total rehabilitation of a patient is often a long procedure and in practice covers several years. this material was somewhat un evenly distributed between the age groups. thus there were two peaks, the 25-29 year group and the group of 35-39 years. this age distribution will cer tainly not reflect that of the total material, which could only be roughly estimated but which probably has a peak at 3 5 4 9 years. for this pilot study it was considered most important, however, t o in clude patients from every age group. most of the patients had symptoms of lumbar spine insufficiency and only a few had more severe conditions such as lumbago and sciatica. in 19 of the patients, normal conditions were found on ex amination, and in the rest only mild disorders were observed. other investigations on similar materials (e.g. natvig, 1970) have revealed a considerably higher frequency of both mild and severe symp toms and signs. this may be explained by the fact that the age distribution of the present small se ries of patients probably deviates somewhat from that of the total material, in which it is expected that higher ages will predominate. among the 31 patients who underwent roent genological examination, pathological changes were found to only a very small extent. l a rocca & macnab (1970), and previously other authors, have demonstrated the limited value of roentgenological examination of patients with low back pain. even from a prognostic aspect the roentgenological ex amination is not reliable, especially in young work ing people. in the younger age groups it is prob ably realistic, therefore, to assume that a correct evaluation can be made without roentgenological examination. other somatic diseases were present t o a strik ingly small extent, and it is of even greater interest that only 4 patients had a history of psychiatric disorder and only 3 were heavy drinkers. other investigations of this category of patients have revealed psychiatric disorders in about 40% and alcoholism or abuse of alcohol in 18% (natvig, 1970). this may be explained by a non-representa tive age distribution, but the low figures are inter esting. the majority of the patients were tested for muscle strength, physical work capacity and intel lectual capability. on the whole it may be said that the patients appeared to be normal in these respects. it is evident from the social investigation that the education of most of the patients ended with the compulsory school period, after which they had unqualified, heavy work. as mentioned previously, it was difficult to ob tain reliable information from the case journals concerning the patients’ incomes prior to the re habilitational investigation. information on income was available for only about half of the patients. in a few cases this information concerned hour ly wages, in others weekly or monthly wages and in a few, the annual income. thus it must be pointed out again that the income figures are un certain. nevertheless the patients were placed in their respective income groups with respect to annual income. it may also be mentioned that the income distribution showed similarities to the statistics for all gainfully employed persons re ported in the 1966 state investigation of low-income groups. the well known sex+difference with re table viii. recommendations of rehabilitation centre following investigation return to previous work 2 retraining 4 higher education 7 contribution towards a business 2 pension 1 change of occupation 16 vocational training 15 upsala j med sci 81 108 s . hilding et al. spect to income is also evident in this small ma terial. it was found that half of the patients had been on the sick-list for long periods prior to the reha bilitational investigation. this is an unexpectedly low figure in view of the fact that low back pain is one of the main causes of absence from work on account of illness, and that there was a waiting period of several months €or investigation at the rehabilitation centre. the housing situation could be regarded as un satisfactory for only 6 of the patients. as this is a very important factor socially, which is certainly of considerable importance for adjustment to em ployment, it should be of interest to find out whether this low figure is representative for the total material. the recommendations of the rehabilitation centre for this group of patients comprise seven alternatives. the two predominant recommenda tions were change of occupation and vocational training. in only one case was an early retirement pension suggested. since there is reason to expect that in the total material from the years in question there will be a greater number of patients in the age group 4 0 4 9 years, the latter alternative, an early pension, will probably have a higher frequency in the planned, broader investigation. the recom mendations will probably have their greatest inte rest, however, when related to the occupational situation of the patients 4-5 years after the reha bilitational investigation. it is planned to elucidate this question in a future follow-up study. references 1 . 2. 3 . 4. 5 . gogstad, a. c.: psykiatriske klienter i attforings institutt. nord psykiatr tidskr 1: 1962. gogstad, a. c.: etterundersokelse av rehabilite ringsforanstaltninger. nord med 8, xi, 1962,68: no. 45, 1962. holmberg, p. & strom, h.: lgginkomstutredningens forsta betiinkande i sammandrag. allmanna forlaget, stockholm, 1970. horal, 1.: the clinical appearance of low back dis orders in the city of gothenburg. acta orthop scand, suppl. 118, 1969. hult, l.: the munkfors investigation. a study of the frequency and causes of the stiff neck brachialgia and lumbago-sciatica syndromes, as well as observations on certain signs and symptoms from the dorsal spine and the joint of the extremities in industrial and forest workers. acta orthop scand, suppl. 16, 1954. 6. 7. 8. 9. 10. 11. hult, l.: cervical, dorsal and lumbar-spinal syn dromes. a field investigation of a non-selected ma terial of 1200 workers in different occupations with special reference to disc degeneration and so-called muscular rheumatism. acta orthop scand, suppl. x v i i , 17, 1954. l a rocca, h . & macnab, i.: value of pre-employ ment radiographic assessment of the lumbar spine. canad med ass j 4: 49, 1969. magnusson, r.: rehabilitering av skogsarbetare med ryggbesvar. lakartidn no. 5 , 1968. natvig, h.: sociomedical aspects of low back pain causing prolonged sick leave. acta socio-med scand sparup, k . h.: a sociomedical evaluation of back insufficiency. scand j rehab med i : 74, 1969. tufvesson, b . , cardell, h. & martelius, e.: reha bilitering av 240 ryggpatienter. lakartidningen, no. 2-3: 117, 1970. . __. 46, 1970 12. westrin, c. g.: low back sick-listing. a nosological and medical insurance investigation. acta socio med scand2-3: 127, 1970. received october 29, 1975 address for reprints: soren hilding, m.d. dept. of orthopaedic surgery university hospital s-750 14 uppsala sweden upsala j med sci 81 upsala j med sci 85: 265-282, 1980 the teorell membrane oscillator--a complete nerve model ulrich f. franck technical university, aachen, germany abstract the membrane oscillator discovered by t. teorell in1954 (10) is one of the most remarkable kinetic systems exhibiting spontaneous periodic behaviour under constant environmental conditions. it can be shown and demonstrated experimentally that the intrin sic reason for the occurrence of oscillations in the teorell oscillator, like in other physicochemical and biological oscil latory systems, is an appropriate antagonistic actign of a la bilizing positive and a recovering stabilizing negative feedback. each kind of feedback causes a group of characteristic temporal phenomena, which are observed in all oscillatory systems inclu ding the living excitable nerve such as: instability, bistability, excitability, propagation of excitation and recovery, refractori ness, abolition, accommodation etc. . introduction: in gottingen in 7951 torsten teorell presented at the 50th hauptversammlung of the deutsche bunsen-gesellschaft a most memo rable lecture: "on the quantitative treatment of membrane per meability". this lecture and its publication(9) i n i t i a t e d a s t r o n g new development in membrane science, and it was quite a convin cing confirmation of the rightness and fruitfulness of these new conceptions when in 1954 his discovery of spontaneous membrane flux oscillations in a ion-exchange membrane system became known. though 2 6 years have passed since that event, the teorell membrane oscillator has thoroughly retained its importance and actuality not only as an exceptional ion-exchange membrane system but also as a general kinetic model for all physicochemical and biological oscillatory systems due to its remarkable transparent kinetics. 265 1 ) n o n l i n e a r p r o p e r t i e s of ion-exchange membranes a s i s well-known, i n t h e t e o r e l l membrane o s c i l l a t o r t h r e e f o r c e s a r e e f f e c t i v e s i m u l t a n e o u s l y ( d i f f e r i n g p o t e n t i a l , p r e s s u r e and' s a l t c o n c e n t r a t i o n on b o t h s i d e s o f t h e membrane) c a u s i n g t h r e e k i n d s of f l u x e s a c r o s s t h e membrane ( e l e c t r i c c u r r e n t , volume f l u x o f f l u i d and s a l t f l u x ) , which a r e m u t u a l l y c o u p l e d t o a h i g h d e g r e e . the e s s e n t i a l k i n e t i c f e a t u r e of t h e t e o r e l l o s c i l l a t o r i s i t s p r e s s u r e d e p e n d e n t non monotonic c u r r e n t v o l t a g e c h a r a c t e r i s t i c (12) ( f i g . i ) , g i v i n g r i s e t o i t s i n s t a b i l i t y p r o p e r t y , which i s one of t h e n e c e s s a r y p r e r e q u i s i t e s f o r t h e occurrence of o s c i l l a t i o n s . with r e s p e c t t o t h e d i r e c t i o n s of t h e d r i v i n g f o r c e s and t h e s i g n of t h e membrane c h a r g e f o r m a l l y e i g h t cases of c o m b i n a t i o n are p o s s i b l e . because, however, n o n l i n e a r c u r r e n t v o l t a g e c h a r a c t e r i s t i c s o n l y o c c u r i n t h e t e o r e l l system under c o n d i t i o n s of opposing e f f e c t s of v o l t a g e and h y d r o s t a t i c p r e s s u r e , o n l y t h e f o u r cases shown i n f i g . 2 remain t o be c o n s i d e r e d h e r e . the pronounced v o l t a g e dependence of t h e membrane r e s i s t a n c e i s c a u s e d by t h e e l e c t r o o s m o t i c volume f l u x , which c a r r i e s e i t h e r c o n d u c t i n g s a l t i n t o o r o u t of t h e p o r o u s ion-exchange membrane. i n t h e f o u r c a s e s i n q u e s t i o n t y p i c a l n o n l i n e a r c u r r e n t v o l t a g e c h a r a c t e r i s t i c s a r i s e a s a consequence of r e s i s t a n c e t r a n s i t i o n s o c c u r r i n g i n t h e v o l t a g e r a n g e where a r e v e r s a l o f t h e volume f l u x t a k e s p l a c e e x h i b i t i n g e i t h e r non-monotonic n-shaped o r mo n o t o n i c f -shaped c u r v e s . f i g . 3 g i v e s t y p i c a l e x p e r i m e n t a l m e a s u r e m e n t s of s u c h n o n l i n e a r c h a r a c t e r i s t i c s c o n c e r n i n g c o a r s e g r a i n e d c a t i o n and anion-exchange membranes whose h i g h p o r o s i t y a l l o w s s t r o n g c o n v e c t i o n a l f l u x e s . i t may be mentioned h e r e , t h a t a l s o i n n o n c o n v e c t i o n a l membranes n o n l i n e a r c h a r a c t e r i s t i c s of nand / t y p e a r e t o be e x p e c t e d namely under c o n d i t i o n s where v o l t a g e d e t e r m i n i n g p r o p e r t i e s of t h e membrane depend upon t h e v o l t a g e i t s e l f . t h i s i s t h e case f o r i n s t a n c e when t h e membrane c h a r g e ( ? ) o r i t s s i g n ( 0 ) o r t h e se l e c t i v i t y change s t r o n g l y w i t h i n a s u f f i c i e n t l y narrow r a n g e of t h e membrane p o t e n t i a l ( f i g . 4 ) . 266 a b c f i g . 1 the t e o r e l l membrane o s c i l l a t o r a ) s e t u p ; b) c u r r e n t v o l t a g e c h a r a c t e r i s t i c s ( p a r a m e t e r : p); c ) o s c i l l a t i o n s of membrane p o t e n t i a l and h y d r o s t a t i c p r e s s u r e d i f f e r e n c e p ( 1 2 , 1, 5 ) . electroosmotic s y m po~ow cat$--eschange membrane , pdtdus anmn-erchange membrane f i g . 2 a ) s e t u p ; b) membrane r e s i s t a n c e r a s a f u n c t i o n of t h e membrane n o n l i n e a r e l e c t r o o s m o t i c ' membrane s y s t e m s p o t e n t i a i e ; c ) t t c u r r e n t v o l t a g e c h a r a c t e r i s t i c s ; glass-sinter 0 : -1 anion-exchanger grains w: +i f i g . 3 examples of n o n l i n e a r c u r r e n t v o l t a g e c h a r a c t e r i s t i c s of p o r o u s ion-exchange membranes ( p a r a m e t e r : p h y d r o s t a t i c p r e s s u r e mm column of w a t e r ) ( l ) . 267 -qf -qj i ie i i ' i i f i g . 4 non-convectional membrane s y s t e m s (x: membrane c h a r g e ; w : s i g n of membrane c h a r g e ; g : conductivity) !raw -5a7 0 ,500 +?om f i g . 5 membrane c o n d u c t a n c e a s a f u n c t i o n of membrane c h a r g e ( t . t e o r e l l 1951 ) (9). i n 1951 t . t e o r e l l has shown t h a t t h e c o n d u c t i v i t y o f a n i o n exchange membrane depends on t h e c o n c e n t r a t i o n of i t s f i x e d i o n c h a r g e ? ( f i g . 5 ) . hence t h e c o n d u c t i v i t y of a membrane e x h i b i t i n g v o l t a g e dependence of ? i s a f u n c t i o n of t h e membrane v o l t a g e t o o . the t h i r d c a s e of f i g . 4 ( v o l t a g e d e p e n d i n g s e l e c t i v i t y ) c o r r e s ponds t o t h e well-known c o n c e p t of t h e hodgkin-huxley t h e o r y of e x c i t a b l e b i o l o g i c a l t i s s u e s . 2) the feedback concept of n o n l i n e a r p h y s i c o c h e m i c a l systems the main c o n c e r n of t h i s t e x t i s t o show t h a t a l l k i n e t i c a l pheno mena r e s u l t i n g from n o n l i n e a r f l u x f o r c e c h a r a c t e r i s t i c s a r e b r o u g h t f o r t h i n a c t u a l f a c t by f e e d b a c k mechanisms which a l l p h y s i c o c h e m i c a l o s c i l l a t o r y s y s t e m s i n c l u d i n g t h e t e o r e l l o s c i l l a t o r and t h e e x c i t a b l e n e r v e have i n common ( 3 , 6 ) . 268 b e f o r e d o i n g s o , w e have t o l o o k a l i t t l e c l o s e r i n t o f e e d b a c k p r o c e s s e s o c c u r r i n g i n p h y s i c o c h e m i c a l s y s t e m s . a s w e l l known f e e d b a c k a r i s e s when a p r o c e s s a c t s k i n e t i c a l l y upon i t s e l f . i t c o n s i s t s t h e r e f o r e b a s i c a l l y i n a c l o s e d c h a i n of a c t i o n c a u s i n g t h e known e f f e c t s of " s e l f e n h a n c e m e n t " i n case of p o s i t i v e f e e d b a c k o r " s e l f i n h i b i t i o n " i n c a s e of n e g a t i v e f e e d b a c k . such p r o c e s s e s of s e l f i n f l u e n c e may a r i s e i n two ways ( f i g . 6 ) : n 1 +tk+ i d + x l p systemic leedbach f i g . 6 "non-systemic" and " s y s t e m i c " f e e d b a c k i n physicochemi c a l s y s t e m s . i f t h e out.put of a t r a n s m i s s i o n system ( t h a t may be an e l e c t r o n i c a m p l i f i e r , a c h e m i c a l r e a c t i o n , a n e l e c t r o d e o r a membrane s y s t e m e t c . ) a c t s k i n e t i c a l l y upon i t s cwn i n p u t , t h e n a f e e d b a c k s i t u a t i o n a r i s e s i n which t h e o u t p u t " e f f e c t " i n f l u e n c e s i t s own " c a u s e " . t h i s mode of f e e d b a c k h a s no e f f e c t upon t h e p r o p e r t i e s of t h e t r a n s m i s s i o n system. f o r t h i s r e a s o n it s h a l l b e d e s i g n a t e d h e r e a s "non-systemic " f e e d b a c k ( 4 ) . i n p h y s i c o c h e m i c a l and b i o l o g i c a l s y s t e m s , however, most f e e d b a c k mechanisms a c t n o t upon t h e i n p u t b u t i n s t e a d upon t h e p r o p e r t i e s of t h e t r a n s m i s s i o n s y s t e m . t h i s k i n d of f e e d b a c k c o r r e s p o n d i n g l y s h a l l be d e s i g n a t e d a s " s y s t e m i c " f eedback . feedback l o o p s i n open s y s t e m s may c o n c e r n t h e f o r m a t i o n o r t h e consumption of t h e k i n e t i c s p e c i e s x and may be a s a l r e a d y mentioned of p o s i t i v e o r n e g a t i v e n a t u r e . i n t h i s way f o u r d i f f e r e n t f e e d b a c k s i t u a t i o n s are p o s s i b l e b e i n g c a l l e d : backward a c t i v a t i o n , forward i n h i b i t i o n , backward i n h i b i t i o n and f o r w a r d a c t i v a t i o n ( f i g . 7 ) ( 7 ) . 269 backward activation forward inhibition backward lnhibltlon forward activation positive feedback negative feedback f i g . 7 p o s i t i v e and n e g a t i v e f e e d b a c k i n open s y s t e m s . o s c i l l a t o r y s y s t e m s c o n t a i n a t l e a s t two s i m u l t a n e o u s p r o c e s s e s . by mutual ( c r o s s ) c o u p l i n g between t h e s e p r o c e s s e s 1 6 d i f f e r e n t p o s s i b i l i t i e s have t o be t a k e n i n t o c o n s i d e r a t i o n ( f i g . 8 ) . i n t h e i r o v e r a l l e f f e c t s t h e y l e a d t o f o u r c a s e s e a c h f o r backward a c t i v a t i o n , f o r w a r d i n h i b i t i o n , backward i n h i b i t i o n and f o r w a r d a c t i v a t i o n ( 6 ) f i g . 8 feedback by c o u p l i n g of two s i m u l t a n e o u s p r o c e s s e s ( 6 ) . i n m o s t r e a l i s t i c f e e d b a c k s y s t e m s t h e t y p e s of e f f e c t i v e f e e d back p r o c e s s e s c a n be found o u t d i r e c t l y by e x p e r i m e n t a l methods. because a l l c o u p l i n g o r f e e d b a c k mechanisms a r e r e p r e s e n t e d by r e a l r e a c t i o n s o r t r a n s p o r t a t i o n p r o c e s s e s of d e f i n e d r a t e s , a l l f e e d b a c k p r o c e s s e s n e c e s s a r i l y need t i m e f o r t h e i r p r o c e e d i n g . the t i m e d e l a y i n p h y s i c o c h e m i c a l f e e d b a c k l o o p s v a r i e s i n a l a r g e scale of magnitude. i n t h e case of t h e t e o r e l l s y s t e m i t r a n g e s i n m i n u t e s , i n t h e case of t h e n e r v e membrane i n m i l l i s e c o n d e s . 3 ) the temporal b e h a v i o u r o f feedback s y s t e m s the t e m p o r a l b e h a v i o u r of p o s i t i v e and n e g a t i v e f e e d b a c k s y s t e m s i s of e s s e n t i a l i m p o r t a n c e f o r t h e o c c u r r e n c e and t h e t e m p o r a l p a t t e r n of p h y s i c o c h e m i c a l o s c i l l a t i o n s . 270 .0 63 0 0 f i g . 9 n o n l i n e a r f l u x f o r c e c h a r a c t e r i s t i c s a s a r e s u l t of f e e d b a c k a c t i o n ( 4 ) . f i g . 9 i l l u s t r a t e s t h e g e n e r a l t e m p o r a l b e h a v i o u r of t h e f o u r modes of c o u p l i n g l e a d i n g t o p o s i t i v e o r n e g a t i v e f e e d b a c k . assuming f o r t h e s a k e of s i m p l i c i t y t h a t t h e f o r m a t i o n and con sumption o f t h e k i n e t i c a l s p e c i e s x are p r o c e s s e s of f i r s t o r d e r , a p l o t o f v e r s u s x g i v e s t w o s t r a i g h t l i n e s which i n t e r s e c t a t t h e s t e a d y s t a t e v a l u e x s t . t h e i r s l o p e s c o r r e s p o n d d i r e c t l y t o t h e r e l e v a n t r a t e c o n s t a n t k o r t h e c o n d u c t a n c e g . s e l f c o u p l i n g a l t e r s t h e s l o p e of t h e k i n e t i c c h a r a c t e r i s t i c be l o n g i n g t o t h e f e e d b a c k p r o c e s s i n q u e s t i o n . i n case of f o r w a r d p o s i t i v e f e e d b a c k it i s g e t t i n g f l a t t e r w i t h i n c r e a s i n g x and it i s g e t t i n g s t e e p e r i n c a s e of n e g a t i v e f e e d b a c k . f i g . 9 shows i n p a r t i c u l a r how non-monotonic n-shaped c u r v e s re s u l t from p o s i t i v e f e e d b a c k and monotonic i s h a p e d c u r v e s from n e g a t i v e f e e d b a c k . p r o v i d e d t h s t t h e p o s i t i v e f e e d b a c k i s s u f f i c i e n t l y s t r o n g t h r e e i n t e r s e c t i o n r e p r e s e n t i n g s t e a d y s t a t e s may o c c u r between t h e c u r v e s of consumption of x e x h i b i t i n g b i s t a b i l i t y and i n s t a b i l i t y r e s p . . p o s i t i v e f e e d b a c k l a b i l i z e s t h e system, and it i s t h e i n t r i n s i c r e a s o n f o r a l l i n s t a b i l i t y phenomena of a u t o c a t a l y t i c and o t h e r s e l f e n h a n c i n g s y s t e m s . 27 1 i n c o n t r a s t n e g a t i v e f e e d b a c k h a s a s t a b i l i z i n g e f f e c t . i t g e n e r a t e s a l w a y s one s t a b l e s t a t e o n l y . the bottom row o f g r a p h s shows t h e "dynamic d i a g r a m s " which d e s c r i b e t h e o v e r a l l t e m p o r a l v a r i a t i o n of x a s a f u n c t i o n of x it... s e l f . the i n s e r t e d a r r o w s i n d i c a t e t h e d i r e c t i o n s o f t h e r e s u l t i n g t e m p o r a l v a r i a t i o n s , and which i l l u s t r a t e how s t a b i l i t y and i n s t a b i l i t y a r i s e from t h e v a r i a t i o n s i n t h e immediate n e i g h b o u r hood of a p a r t i c u l a r s t e a d y s t a t e . f o r p o s i t i v e a s w e l l as f o r n e g a t i v e f e e d b a c k c h a r a c t e r i s t i c msdes of t e m p o r a l b e h a v i o u r r e s u l t a s a d i r e c t consequence of t h e f e e d b a c k d e l a y . concerning i o n i c membranes, a l r e a d y i n 1951 t . t e o r e l l p o i n t e d o u t i n t h e p a p e r c a p a c i t a n c e " a l r e a d y mentioned t h a t i n s u c h membranes "pseudo and " p s e u d o i n d u c t a n c e ' ' b e h a v i o u r are o b s e r v e d (fig.10). kupuztfw ,, ku,ztbw -indukhu " sponnung (zstmp _ _ _ _ . indukfiv " zeitlicher spannungsverlauf verschiedener s ysteme elektroly ten-membran (nach einschalten eines konstanten stromes). das system zeigt scheinbnre kapazitat. induktivitat bzw. kapazitjt + lnduktivitat f i g . 1 0 p s e u d o c a p a c i t i v e and p s e u d o i n d u c t a n c e b e h a v i o u r o f ion-exchange membranes ( t . t e o r e l l 1 9 5 1 ) ( 9 ) . f i g . 1 1 temporal b e h a v i o u r o f f e e d b a c k s y s t e m s compared w i t h e l e c t r i c c a p a c i t a n c e and i n d u c t a n c e c i r c u i t s ( 6 ) . 272 fig. 11 i l l u s t r a t e s pattern r e s e m b l i n g t h a t of a c a p a c i t y . systems c o n t a i n i n g n e g a t i v e f e e d b a c k behave c o r r e s p o n d i n g l y l i k e i n d u c t a n c e s e x h i b i t i n g o v e r s h o o t phenomena which i n f a c t are r e c o v e r y phenomena as a c o n s e quence o f t h e s t a b i l i z i n g a c t i v i t y of t h e n e g a t i v e f e e d b a c k . now w e a r e i n a p o s i t i o n t o i d e n t i f y t h e n a t u r e of f e e d b a c k l o o p s e x i s t i n g i n a p a r t i c u l a r s y s t e m by s t u d y i n g i t s t e m p o r a l b e h a v i o u r . t h a t p o s i t i v e feedback a c t u a l l y l e a d s t o t e m p o r a l i n t h i s c o n t e x t it might b e s t a t e d t h a t from t h e f e e d b a c k p o i n t of view it i s e v i d e n t t h a t t h e t e m p o r a l b e h a v i o u r of p h y s i c o c h e m i c a l s y s t e m s i s q u i t e t h e same a s it i s well-known from f e e d b a c k i n e l e c t r i c c i r c u i t s o r t e c h n i c a l c o n t r o l s y s t e m s , and i n a c t u a l f a c t t h e o c c u r r e n c e of o s c i l l a t i o n s i n p h y s i c o c h e m i c a l and b i o l o g i c a l s y s t e m s o b v i o u s l y i s b a s e d on t h e same dynamical p r i n c i p l e s a s it i s t h e case i n e l e c t r i c a l s y s t e m s whose o s c i l l a t o r y b e h a v i o u r a s a r e s u l t of f e e d b a c k a c t i o n i s q u i t e s e l f e v i d e n t f o r u s . 4 ) the p r i n c i p l e of a n t a g o n i s t i c feedback. i t i s a h i g h l y i n t e r e s t i n g f a c t t h a t a l l p h y s i c o c h e m i c a l o s c i l l a t o r y s y s t e m s found so f a r c l e a r l y e x h i b i t p o s i t i v e and n e g a t i v e f e e d b a c k s i m u l t a n e o u s l y . i t i s most l i k e l y t h a t o s c i l l a t i o n s i n such s y s t e m s a r e t h e r e s u l t of a g e n e r a l k i n e t i c p r i n c i p l e which i s r e p r e s e n t e d i n f i g . 1 2 i n form of a g e n e r a l f e e d b a c k p a t t e r n . i t may b e d e s i g n a t e d h e r e a s t h e " p r i n c i p l e o f a n t a g o n i s t i c f e e d b a c k of p h y s i c o c h e m i c a l o s c i l 1 a t o r s l 1 ( 3 ) . f i g . 1 2 a n t a g o n i s t i c f e e d b a c k i n o s c i l l a t o r y open s y s t e m s c o n t a i n i n g two s i m u l t a n e o u s p r o c e s s e s (3). according t o t h i s p r i n c i p l e t h e o s c i l l a t i o n s a r e u n d e r s t o o d a s a consequence o f an a n t a g o n i s t i c i n t e r a c t i o n o f a r e l a t i v e l y f a s t a c t i n g p o s i t i v e f e e d b a c k o f l a b i l i z i n g t e n d e n c y and a s l o w e r ac t i n g n e g a t i v e f e e d b a c k of s t a b i l i z i n g r e c o v e r i n g t e n d e n c y . t h i s c o n c e p t of o s c i l l a t o r y s y s t e m s c o n s i s t i n g of two d i s t i n c t l o o p s of p o s i t i v e and n e g a t i v e f e e d b a c k r e q u i r e s a t l e a s t two k i n e t i c a l v a r i a b l e s , one f o r e a c h f e e d b a c k mechanism. each l o o p , however, may c o n t a i n s e v e r a l v a r i a b l e s i n series. a l l t h e s e va r i a b l e s p a r t i c i p a t e i n t h e o v e r a l l o s c i l l a t o r y p r o c e s s . i n most r e a l cases of o s c i l l a t i o n s it i s p o s s i b l e t o f i n d o u t from t h e r e c o r d i n g s t o which c l a s s of f e e d b a c k l o o p a p a r t i c u l a r o s c i l l a t i n g v a r i a b l e b e l o n g s ( f i g . 1 3 ) . a l s o a g e n e r a l c l a s s i f i c a t i o n of t h e t e m p o r a l phenomena of f e e d b a c k s y s t e m s can b e g i v e n now w i t h r e s p e c t t o t h e n a t u r e of t h e f e e d b a c k b e i n g r e s p o n s i b l e f o r a p a r t i c u l a r phenomenon ( f i g . 1 4 ) . teorell membrane b 2 reaction bray reaction i r o n in hno, variable 01 br' ~ l ~ l l ) ) j $ ! ~ poslttve feedback tt t t negative variableof cl?+w 'w,,,!ozw iw leedback t t 1t f i g . 1 3 s i m u l t a n e o u s o s c i l l o g r a m s of v a r i a b l e s b e l o n g i n g t o d i f f e r e n t f e e d b a c k l o o p s . v a r i a b l e s i n v o l v e d i n p o s i t i v e f e e d back l o o p s e x h i b i t f l i p f l o p t y p e o s c i l l o g r a m s showing i n s t a b i l i t y f e a t u r e s ( ) . o s c i l l o g r a m s o f v a r i a b l e s b e l o n g i n g t o n e g a t i v e f e e d b a c k l o o p s u s u a l l y have s i m p l e saw-tooth s h a p e w i t h o u t marks of i n s t a b i l i t y (b.z.: belousov z h a b o t i n s k y r e a c t i o n ) ( 6 ) . mi. f i g . 1 4 c l a s s i f i c a t i o n of t e m p o r a l phenomena of f e e d b a c k s y s t e m s (4). 274 positive feedback -xrn -ef3 lo l m ( 9 m ) forward inhibition t , i f 9, i\-.+ l a b i l i z a t i o n : -:: , e * tdlb $-bj pseudo capactlance dt c' ._ + v < increasing voltage inhlbits the membraneconductance by concentratmn profile deformation negative feedback j : i t tforward activation -e e ---?s t a b i l i z a t i o n t pseudo-inductance e lncrea~ing voltage actovates the membrane conductance by increaslng hydrostatic pressure f i g . 1 5 p o s i t i v e and n e g a t i v e feedback a r i s i n g i n t h e teorel.1 system. 275 5 ) the feedback s i t u a t i o n i n t h e t e o r e l l membrane o s c i l l a t o r l e t u s now l o o k a t t h e t e o r e l l membrane system from t h e f e e d b a c k p o i n t of view. t h e r e i s c l e a r l y a p o s i t i v e f e e d b a c k l o o p p r o v a b l e w i t h r e s p e c t t o t h e transmembrane p o t e n t i a l , and which m a n i f e s t s i t s e l f i n t h e v o l t a g e dependence of t h e membrane r e s i s t a n c e ( f i g . 1 5 ) . the " f o r m a t i o n " and "consumption" of t h e k i n e t i c a l s p e c i e s x c o n c e r n s p r i m a r i l y t h e s a l t c o n t e n t i n s i d e t h e membrane a s a r e s u l t of t h e volume f l u x d r i v e n by t h e e l e c t r o o s m o t i c e f f e c t of t h e membrane p o t e n t i a l i n s i d e t h e ion-exchange membrane. because t h e s a l t c o n t e n t of t h e membrane d e t e r m i n s t h e membrane r e s i s t a n c e , t h e r e e x i s t under c u r r e n t clamp c o n d i t i o n s a d i r e c t r e l a t i o n s h i p between t h e s a l t c o n t e n t and t h e membrane p o t e n t i a l . t h e r e f o r e w e may r e p l a c e x d i r e c t l y by t h e v o l t a g e e a s t h e cha r a c t e r i s t i c k i n e t i c a l v a r i a b l e o f t h e p o s i t i v e f e e d b a c k l o o p . i t i s e a s y t o show t h a t i n t h e t e o r e l l membrane s y s t e m t h i s l o o p i s r e a l i z e d by a f o r w a r d i n h i b i t i o n c o n s t e l l a t i o n . here, a n i n c r e a s i n g v o l t a g e i n h i b i t s t h e membrane c o n d u c t a n c e by a c o r r e s p o n d i n g d e f o r m a t i o n of t h e c o n c e n t r a t i o n p r o f i l e . the p o s i t i v e f e e d b a c k l e a d s t o p s e u d o c a p a c i t i v e b e h a v i o u r , t o a non-monotonic c u r r e n t v o l t a g e c h a r a c t e r i s t i c and t o a c o r r e s p o n d i n g dynamic r e l a t i o n s h i p between and e . de d t the n e g a t i v e f e e d b a c k of t h e t e o r e l l o s c i l l a t o r ( f i g . 1 5 ) comes a b o u t as a r e s u l t o f t h e volume f l u x dependence o f t h e h y d r o s t a t i c p r e s s u r e , which i s b e s i d e s t h e membrane p o t e n t i a l t h e o t h e r d r i v i n g f o r c e o f t h e volume f l u x . the n e g a t i v e f e e d b a c k l o o p r e p r e s e n t s h e r e a t y p i c a l example of mutual c o u p l i n g o f two s i m u l t a n e o u s p r o c e s s e s whose o v e r a l l e f f e c t c o n c e r n s a f o r w a r d a c t i v a t i o n . i n c r e a s i n g v o l t a g e a c t i v a t e s h e r e t h e membrane c o n d u c t a n c e by i n c r e a s i n g h y d r o s t a t i c p r e s s u r e . 6 ) the t e o r e l l membrane o s c i l l a t o r a s a "complete nerve analogue". s u r v e y i n g t h e l i s t of f e e d b a c k phenomena of f i g . 1 4 w e a r e s t r o n g l y reminded of t h e phenomenology of t h e e x c i t a b l e n e r v e and o t h e r b i o l o g i c a l e x c i t a b l e t i s s u e s . tab. 1 sums up t h e p r o p e r t i e s which are c o n s i d e r e d a s f u n d a m e n t a l phenomena of t h e n e r v e membrane. 276 p h y s i c o c h e m i c a l s y s t e m s e x h i b i t i n g t h e s e p r o p e r t i e s a r e d e s i g n a t e d as"comp1ete n e r v e models" o r " n e r v e a n a l o g u e s " . table 1. f u n d a m e n t a l e l e c t r o p h y s i o l o g i c a l p r o p e r t i e s o f e x c i t a b l e tissues i n s t a b i l i t y : i m p u l s e response: p r o p a g a t i o n : r h y t h m i c a l a c t i v i t y : b i s t a b i l i t y e x c i t a b i l i t y ( t r i g g e r a b i l i t y ) t h r e s h o l d phenomena a b o l i t i o n e x c i t a b l e a c t i o n p o t e n t i a l s p o n t a n e o u s r e c o v e r y o f e x c i t a t i o n r e f rac t o r i n e s s accommodation o v e r s h o o t phenomena two-way p r o p a g a t i o n of e x c i t a t i o n p r o p a g a t i o n o f a c t i o n p o t e n t i a l t r i g g e r a b i l i t y o f p r o p a g a t i o n phenomena s a l t a t o r i c p r o p a g a t i o n o s c i l l a t o r y phenomena obviously t h e n e r v e r e p r e s e n t s k i n e t i c a l l y a s y s t e m of a n t a g o n i s t i c f e e d b a c k t o o . hence w e may d e f i n e a c o m p l e t e n e r v e a n a l o g u e b r i e f l y as a system e x h i b i t i n g a n t a g o n i s t i c f e e d b a c k a s o u t l i n e d h e r e . there a r e s e v e r a l a r t i c l e s p u b l i s h e d by t . t e o r e l l t w e n t y y e a r s ago which c l e a r l y show t h a t a l r e a d y i n t h e f i f t i e s he w a s q u i t e aware and c o n v i n c e d t h a t h i s membrane o s c i l l a t o r i n a c t u a l f a c t r e p r e s e n t s a c o m p l e t e n e r v e a n a l o g u e (11 i s ) i n p a r t i c u l a r by a i d of a n a n a l o g u e c o m p u t a t i o n program d e r i v e d from t h e s e t u p and t h e d a t a of t h e membrane o s c i l l a t o r t e o r e l l d e m o n s t r a t e d c o n v i n c i n g l y a g r e a t d e a l o f t h e s e e x c i t a b i l i t y phe nomena. but a l s o under s u i t a b l e c o n d i t i o n s t h e s e phenomena c a n b e d e m o n s t r a t e d i n t h e r e a l t e o r e l l membrane s y s t e m . because t h e a u t h o r d o e s n o t know e x a c t l y which o f t h e s e e x p e r i ments have a l r e a d y been c a r r i e d o u t by t . t e o r e l l h i m s e l f , some of t h e a u t h o r ' s r e s u l t s may be g i v e n h e r e i n o r d e r t o show t h e e f f i c i e n c y and t h e c o m p l e t e n e s s of t h e n e r v e a n a l o g u e p r o p e r t i e s of t e o r e l l ' s membrane system. tab. 2 g i v e s a l i s t of m a t e r i a l s s u i t a b l e f o r membranes i n t h e t e o r e l l o s c i l l a t o r . most of t h e e x p e r i m e n t s shown i n t h e f o l l o wing p i c t u r e s have been c a r r i e d o u t by means of d u r a n s i n t e r g l a s s 277 2 membranes of a t h i c k n e s s of . 7 mm and a n a r e a of .3 c m ( x : m o l / l , w i d t h of p o r e s : ~9 p). l o * table 2 . membrane m a t e r i a l s s u i t a b l e f o r t e o r e l l o s c i l l a t i o n s p o r c e l a i n ( t e o r e l l ) 7 r p o r o u s g l a s s s i n t e r ( t e o r e l l ) 11.i p o r o u s d u r a n g l a s s s i n t e r 9 r p o r o u s p o l y v i n y l c h l o r i d e ( ' p o r v i c ' ) 4 p 2 . 5 p n u c l e p o r f i l t e r (meares & p a g e ) i p q u a r t z powder 5 v p o r o u s p o l y s t y r o l ( f l e x o l i t h ' ) a120 3-po wde r 21.1 c a t i o n e x c h a n g e r g r a i n s ( t e o r e l l ) anion-exchanger g r a i n s e cation-exchange anion-xchsnge membrane membrane f i g . 1 6 t h r e s h o l d b e h a v i o u r of f i g . 1 7 a c t i o n p o t e n t i a l re t h e tna ( t e o r e l l nerve analogue). s p o n s e of t h e tpja. f i g . 1 6 shows t h e well-known t e m p o r a l p a t t e r n of t h e t h r e s h o l d b e h a v i o u r of s u c h a n e x c i t a b l e d u r a n g l a s s membrane. i n t h i s case t h e compartments are v i r t u a l l y u n l i m i t e d so t h a t no o s c i l l a t i o n s can o c c u r . 278 e x c i t a b i l i t y i n s u c h a b i s t a b l e s y s t e m i s , a s well-known, a two s i d e d phenomenon w i t h r e s p e c t t o b o t h s t a b l e s t a t e s , which are c o m p l e t e l y e q u i v a l e n t i n t h e k i n e t i c a l s e n s e . s u p e r t h r e s h o l d s t i m u l i s h i f t t h e membrane r e s i s t a n c e beyond t h e v a l u e o f t h e un s t a b l e c o n c e n t r a t i o n p r o f i l e , s u b t h r e s h o l d s t i m u l i don't reach it. simply by l i m i t a t i o n of one compartment f i t t e d w i t h a n o v e r f l o w t h e t e o r e l l system e x h i b i t s t r i g g e r a b l e a c t i o n p o t e n t i a l s ( f i g . 1 7 ) . i t depends upon on which s i d e of t h e s e t u p t h i s compartment l i m i t a t i o n i s made whether a n upward o r a downward a c t i o n p o t e n t i a l o c c u r s . the r e c o v e r y i s t h e r e s u l t of t h e v a r i a t i o n of t h e hydro s t a t i c p r e s s u r e c a u s e d by t h e volume f l u x d u r i n g t h e p e r i o d of e x c i t a t i o n . the o v e r f l o w p r e v e n t s r e p e t i t i v e e x c i t a t i o n . f i g . 18 r e f r a c t o r i n e s s of :ti.-+ t h e tna. f i g . 1 9 a b o l i t i o n o f ac t i o n p o t e n t i a l of t h e tna. f i g . 18 d e m o n s t r a t e s a t t h e same membrane s y s t e m t h e phenomenon of r e f r a c t o r i n e s s by a p p l y i n g o f two s t i m u l i of t h e same s t r e n g t h w i t h i n c r e a s i n g t e m p o r a l d i s t a n c e s . with d e c a y i n g r e f r a c t o r i n e s s r e a l i z e d by d e c a y i n g h y d r o s t a t i c p r e s s u r e , t h e s y s t e m r e g a i n s i t s e x c i t a b i l i t y . f i g . 1 9 d e m o n s t r a t e s t h e phenomenon of a b o l i t i o n of t h e a c t i o n p o t e n t i a l by c o u n t e r a c t i n g s t i m u l i . i n c a s e of t h e e x c i t a b l e n e r v e i. t a s a k i h a s d e m o n s t r a t e d a b o l i t i o n i n 1 9 5 6 (8). a l s o t h e phenomenon of e x c i t a t i o n p r o p a g a t i o n c a n be d e m o n s t r a t e d by means of t e o r e l l ' s membrane system ( f i g . 2 0 ) . p r o p a g a t i o n i s a 279 d i r e c t consequence o f t h e b i s t a b i l i t y of t h e system c o n t a i n i n g s t r o n g p o s i t i v e f e e d b a c k . the p r o p a g a t i o n may c o n c e r n t h e c o n v e r s i o n of t h e u p p e r s t a t e i n t o t h e lower s t a t e o r t h e r e v e r s e d p r o cess. a l s o s a l t a t o n i c p r o p a g a t i o n c a n b e r e a l i z e d i n t e o r e l l ' s n e r v e model ( 2 ) . @ membrane f i g . 2 0 p r o p a g a t i o n of f i g . 2 1 a p r o j e c t of a t n k s t a t e t r a n s i t i o n s ( 2 ) e x h i b i t i n g a c t i o n p o t e n t i a l a ) from lower t o u p p e r s t a t e ; p r o p a g a t i o n . b ) from upper t o l o w e r s t a t e ; p r o p a g a t i o n of a c t i o n p o t e n t i a l s s h o u l d be d e m o n s t r a t a b l e t o o by means of t h e t e o r e l l system. f i g . 2 1 shows a p r o j e c t of a s e t u p which s h o u l d e x h i b i t a c t i o n p o t e n t i a l p r o p a g a t i o n . it c o n s i s t s of a n a r r a y of compartments bounded by i o n i c c o n d u c t i n g w a l l s on b o t h s i d e s of t h e membrane a l l o w i n g t h a t l o c a l i z e d h y d r o s t a t i c p r e s s u r e c a n be m a i n t a i n e d . t h e r e i s no d o u b t t h a t t h i s model w i l l work and it c a n b e p r e d i c t e d on t h e b a s i s of t h e e x p e r i e n c e s w i t h t h e t e o r e l l s y s t e m t h a t i n t h i s n e r v e a n a l o g u e a wave of a c t i o n p o t e n t i a l accompanied by a wave of h y d r o s t a t i c p r e s s u r e and a d o u b l e wave o f eddy c u r r e n t s would t r a v e l a l o n g t h e membrane a f t e r a l o c a l s t i m u l a t e d e x c i t a t i o n . t h i s p r o j e c t , whose r e a l i z a t i o n would b e v e r y l a b o r i o u s , i s men t i o n e d h e r e i n o r d e r t o show t h a t t h e m i s s i n g of a c t i o n p o t e n t i a l 280 propagation experiments is no disproof of the completeness of the nerve analogue property of teorell's membrane system. fig. 22 synopsis of the general phenomenology 0 s systems con taining antagonistic feedback. concluding a synopsis of physicochemical oscillations are shown in fig. 22. these systems which are regarded as nerve models are of extremely different substantial nature, but they have obvious ly one property in common: the antagonistic feedback. from this point of view it is no more surprising that they exhibit neverthe less an identic phenomenology. among these systems teorell's mem brane oscillator certainly is the system being best understood now due its simplicity and transparent kinetics. references 1. franck, u.f.: uber das elektrochemische verhalten von porosen ionenaustauschermembranen. ber. bunsenges. phys.chem.67: 657-671, 1963 nen. ber. bunsenges. phys.chem. 71: 789-799, 1967 cillatory systems faraday symp. chem.soc. 9: 137-149, 1974 angew.chem. int.ed.eng1. 17: 1-15, 1978 in coarse-grained ion-exchanger membranes. electrochimica acta 23: 1081-1091, 1978 2. franck, u.f.: phanomene an biologischen und kunstlichen membra 3. franck, u.f.: kinetic feedback processes in physicochemical os 4. franck, u.f.: chemical oscillations 5. franck, u.f.: a quantitative treatment of oscillatory phenomena 28 1 6. franck, u.f.: feedback kinetics in physicochemical oscillators. 7. higgins, j.: oscillating reactions 8. tasaki i.: j.g.physio1. 39: 377, 1956 9. teorell, t.: zur quantitativen behandlung der membranpermeabi 10.teore11, t: expt.cel1 res., 1955, suppl. 3, 339 11.teorel1, t: biophysical aspects on mechanical stimulation of 12.teorel1, t: oscillatory electrophoresis in ion exchange mem 13.teorel1, t: excitability phenomena in artificial membranes. bio ber.bunsenges.phys.chem.84: 334-341, 1980 1nd.eng.chem. 59: 19-62, 1967 litat. 2.f.elektrochem. 55: 460-469, 1951 excitable tissues, acta soc.med.upsa1iensi.s 64: 341-352, 1959 branes. arkiv for kemi 18(22): 401-408, 1961 physical j. 2: 27-52, 1962 received 80 10 17 address for reprint requests: ullrich franck institut fur physikalische chemie, tempelgraben 59, aac hen , germany 282 upsala j med sci 81: 71-78, 1976 lipoprotein composition and lipoprotein interrelations in 50-year-old men with hyperlipoproteinaemia bengt vessby, hans hedstrand and u l f olsson from the departments of geriatrics, lnternal medicine, and statistics, university of uppsala, sweden al3 strac t the serum lipoprotein (lp) composition and lp lipid inter relations were studied in 50-year-old men with different types of hyperlipoproteinaemia (hlp) and in randomly sampled healthy controls from the same population. the ratio cholesterol/triglycerides in very low density lipoproteins (vldl) was high in hlp type 111. the other types of hlp showed ratios not significantly different from the controls. the low density lipoprotein (ldl) cholesterol concentration was similar in controls, type 111 and type iv while, by definition, higher values were seen in type i i a and iib. all types of hlp showed statistically significantly higher ldl triglycerides than the controls. hlp type i1 a and i1 b showed cholesterol/triglyceride ratios in ldl similar to the controls. the corresponding ratio in type i v was lower than in the control subjects but the lowest ratio was seen in type 111 with a mean value below the 5th per centile of healthy controls. the high density lipoprotein (hdl) cholesterol con centration was decreased in hlp type iv. apparently elevated hdl triglyceride levels were seen in all types of hlp with the highest mean value in type 111. the lp lipid interclass relationships were analysed in the random sample of healthy men and compared to cor responding relationships in the different types of hlp. apart from hlp type iii and hlp type i v with low ldl cholesterol levels all other types of lp patterns seemed to conform to a common model of lp interconversions. in type iv a significant negative correlation between vldl concentration and ldl cholesterol levels was demonstrated in subjects with low ldl cholesterol as well as a direct relationship between ldl cholesterol concentration and the cholesterol/triglyceride ratio in vldl. there were no significant correlations between ldl cholesterol concentra tion and vldl lipid variables in other types of hlp and normolipidaemia. introduction hyperlipoproteinaemia (hlp) is one of the major risk factors for development of premature athero sclerotic cardiovascular disease (14, 3). according to suggestions by fredrickson et al. (7, 8) h l p are divided into separate ‘types’. the classification of h l p is based on the definition of upper ‘normal’ limits for the concentration of very low density lipoproteins (vldl, d< 1.006) and low density lipoproteins (ldl, d= 1.006-1.063) (1). the pre valence of h l p in a population, and also the rela tive frequency of the different types of h l p (13), thus depends on which cutting points are chosen. lipid levels vary with sex and age as well as ethnic and geographic factors. this study was undertaken to characterize lipoprotein (lp) com position and l p lipid interrelations in different types of h l p in 50-year-old men. the lipid levels in the isolated l p density fractions and the statisti cal interrelationships between the lp fractions were studied and compared to corresponding rela tions in randomly sampled healthy controls. material and methods subjects during 1971-74 all men aged so (born 1921-24) living in and around the town of uppsala in eastern sweden were invited to a health screening for risk factors for coronary heart disease. the adherance rate was 84%. all men with triglyceride and/or cholesterol concentration in serum in the two top decentiles of the population at the initial screening were referred to the department of geriatrics for a complete lp analysis. the lp patterns were classi fied according to fredrickson, levy & lees (8). cutting points for hlp were for vldl triglycerides and ldl cholesterol 1.5 mmol/l and 190 mg/loo ml respectively corresponding to the 85th percentile in a randomly sampled population of healthy 50-year-old men (13). i n the present study all samples showing a characteristic lp pattern diagnostic for hlp according to who ( 1 ) ana lysed from january 2,1972 through january 31,1973 were included ( n = l l l ) . diagnosis of hlp type i11 was based on the demonstration of a typical ‘floating @’ pattern in vldl.’ no samples from subjects suffering from insulin * @-migrating lipoproteins demonstrated at agarose electrophoresis in the supernatant fraction of plasma after ultracentrifugation at d = 1.006. upsala j med sci 81 7 2 b . vessby et al. table i. triglyceride and cholesterol concentration in serum lipoprotein density fractions in 50-year-old men with normal lipoprotein pattern and hyperlipoproteinaemia (mean fs.e.) vldl" ldl hdl lipoprotein triglyceride cholesterol triglyceride cholesterol triglyceride cholesterol pattern (n) ( m m o ~ / ~ ) (mg/100 ml) (mmol/l) (mg/iw ml) (mmol/l) (mg1100 mi) controls (92) 1.03f0.06 i1 a (38) 1.02f0.05 *** i1 b (28) 2.12+0.11 * * * 111 (8) 2.18f0.26 *** iv (37) 2.85f0.38 21f 1 23+ 2 *** 41+ 2 *** 9 9 f 15 *** 532 6 0.49f0.01 155f 3 *** *** 0.68f0.03 224f 8 *** *** 0.70f0.02 219f 4 *** 0.71fo. 10 143f20 *** 0.61f0.03 145f 5 0.24f0.01 48f 1 * * 0.27f0.01 4 6 f 2 *** 0.29f0.01 4 3 f l *** 0.34f0.04 4453 *** *** 0.30f0.01 36f i a the tests involving vldl lipid concentrations were performed also on logarithmic transformed values with identical results regarding the demonstrated group differences. * 9 , ** ***=significant on the 2, 1, and 0.1% level respectively compared with normals when tested with student's t-test. deficient diabetes, hypothyreosis or renal disease were in cluded. one man had suffered from a myocardial infarc tion. one had angina pectoris and was treated with a /3 receptor blocking drug and one suffered from intermittent claudication. n o other subjects were on regular treat ment. seventeen men had a reduced glucose tolerance at intravenous glucose tolerance test. three of those showed a moderately increased fasting blood glucose concentra tion without glucosuria. controls the control subjects (n=92) were apparently healthy men randomly sampled from the same population of 50-year old men. excluded from this material were obese persons (subjects with weightlheight index above 1.10) and per sons with clinical or laboratory signs of disease. the con trol population has earlier been described in detail (13). lp analysis blood samples from subjects fasted over night were al lowed to clot at room temperature and edta was added as a 5 % solution to a final concentration of 0.05%. vldl, ldl and high density lp (hdl, d>1.063) were isolated by consecutive spins at 15°c in a l2 65b beckman preparative ultracentrifuge according to have1 et al. (1 1) using a 40.3 rotor. vldl was isolated as the top frac tion after centrifugation of serum at d=1.006 for 16 h at 40000 rpm. the bottom fraction was then centrifuged at d=1.063 at 40000 rpm for 20 h. the top and bottom fraction after the second centrifugation contained ldl and hdl respectively. a detailed description of the isola tion procedure has been given (2). whole serum as well as the isolated l p classes were extracted manually with isopropanol. triglyceride and cholesterol concentrations in the l p feactions were determined in a technicon auto analyzer type i1 (23). the sum of cholesterol and tg concentrations in vldl, ldl and hdl, was always within 100fio% of whole serum cholesterol and tri glyceride concentrations respectively. immediately after the centrifugation whole serum and the top and bottom fraction at d= 1.006 were subjected to agarose electrophoresis according to noble (19). a 1 % agarose gel containing 0.25% albumin was used. the electrophoresis was run in a barbital buffer, ph 8.6, at 16 v/cm for 1 hour and the gel was stained in sudan black. statistics significant differences between mean values were estimat ed with student's t-test (two-tailed tests). vldl tri glyceride and cholesterol concentrations as well as ldl triglyceride concentrations were tested also after loga rithmic transformation because of a skewed distribution of these variables (13). correlation analyses ( 5 ) were per formed at the uppsala university data center on an ibm 370/155 computer using program bmd02r (4). the accepted level for statistical significance was p<0.02. a significance at the 5 % level would be expected to occur by chance alone in one test of 20. because of the number of significance tests performed (e.g. table i and iv-vi) the minimum requirement for statistical significance was set at 2% to reduce the risk of chance significances. since the different groups of h l p were obtained by selection, i.e. by assigning subjects with values above and below certain limits for some variables to different groups, there is a certain accordance between the group compari sons of mean values and the reported correlation coeffi cients. by definition, significant group differences were obtained for the group assignment variables. similarly, group differences should be expected for variables highly correlated with the group assignment variables. upsala j med s c i s l lipoprotein composition in 50-year-old men with hyperlipoproteinaemia 73 table 11. cholesterolltriglyceride ratio (mean ks.e.1 in serum lipoprotein density fractions in 50-year-old men with normal lipoprotein pattern and hyperlipoproteinaemia vldl" ldl hdl lipo-, mgll00 ml mglloo ml mglloo ml protein pattern ( n ) mmol/l mmol/l mrnol/l controls (92) 20.4f0.6 325f 7 2 2 1 f 9 *** i i a (38) 22.2k1.2 340211 172? 8 *** iib (28) 19.9k0.8 317f 8 154k 7 *** *** 111 (8) 44.8k3.6 2 0 2 t 1 0 1 5 5 f 3 9 *** * * * iv (37) 19.3k0.7 2 5 1 f l l 1 2 8 5 7 the tests involving vldl lipid concentrations were performed also on logarithmic transformed values with identical results regarding the demonstrated group dif ferences. *, **, ***=significant on the 2, 1, and 0 . 1 % level rspectively compard with normals when tested with student's t-test. results by definition h l p type iv and i i b had increased vldl concentrations compared with the control subjects (table i). the criteria for diagnosis of type 111 h l p do not include a requirement for increased lipid levels in vldl o r ldl (1). however, on the average h l p type 111 showed increased vldl tri glyceride levels. the vldl cholesterol concentra tion was by far highest in type 111. the ldl cholesterol concentration was on the average similar in normals, type 111 and type iv. significantly higher values were, by definition, seen in type i i a and iib. all types of h l p showed sig nificantly higher ldl triglycerides than the normal controls. type 111, i i b and i i a showed the most pronounced rise while type iv showed ldl tri glyceride levels intermediate between normals and the other types of h l p with a significantly lower mean value than in type i i b ( p < o . o l ) . also in hdl the lipid levels differed in the various types of hlp. type i i a showed hdl cholesterol concentrations similar to the normals. types i i b and 111 tended to show lower mean values than the controls but the differences were not statistically significant. in type iv there was a clearly lower hdl cholesterol concentration than in the control group. apparently elevated hdl tri glyceride concentrations were seen in all types of hlp. the highest mean value was seen in type 111. in an effort to gain further information regard ing the lipid composition in different hyperlipo proteinaemic states the ratios between cholesterol and triglyceride concentrations in vldl, ldl and hdl were computed and compared with normals (table 11). although not significantly different there was a tendency to a higher vldl ratio in i1 a and lower in type iv than in normals. type 111 was characterized by a very high ratio in vldl. h l p types i1 a and i i b showed cholesterol/tri glyceride ratios in ldl similar to the controls. type i i a showed the highest mean value although not significantly different from the two other groups. the ratio in type iv was on the average table 111. relationships between triglyceride ( x ) and cholesterol ( y ) concentration in serum lipoprotein in 50-year-old men with different lipoprotein patterns linear correlation coefficients ( r ) , slope ( b ) and intercept ( a ) of regression equations (y=a + b x ) lipoprotein pattern lipocontrols (n=92) 11 a (n=38) i i b (n=28) 111 ( n = 8 ) iv (n=37) protein fraction r b a r b a r b a r b a r b a *** *** *** * * *** * * vldl 0.89 18.9 1.2 0.70 22.8 -0.3 0.68 12.6 14.6 0.84 50.6 -11.5 0.95 14.9 10.3 * * * *** ** * * * ** * * * *** ** *** ldl 0.60 169 72 0.49 140 129 0.52 98 150 0.94 193 7 0.44 72 101 *** *** *** ** *** hdl 0.10 21 44 0.23 46 33 -0.05 -8 46 0.04 4 43 -0.06 -7 38 * > , ** ***=significantly different from zero on the 2, 1, and 0.1 % level respectively. upsula j m e d sci 81 74 b . vessby et al. table iv. relationships between lipoprotein lipid variables in the very low and low density lipoproteins in 50-year-old men with different types of serum lipoprotein patterns linear correlation coefficients ( r ) ldl controls (n=92) iia (n=38) i1 b (n = 28) iv (n=37) vldl" tg chol chol/tg tg chol chol/tg tg chol chol/tg tg chol chol/tg * ** *** *** tg 0.26 0.06 -0.27 0.22 0.03 -0.21 -0.08 -0.08 0.06 0.05 -0.62 0.55 ** ** *** * * * * *** chol 0.35 0.11 -0.30 0.52 0.08 -0.55 0.27 0.00 -0.28 0.33 -0.40 -0.68 *** *** * * * * ** chol/tg 0.21 0.11 -0.10 0.59 0.06 -0.66 0.44 0.13 -0.41 0.62 0.43 -0.32 vldl triglyceride and cholesterol concentrations were logarithmic transformed before calculations. * , 1 ** ***=significant on the 2, 1, and 0.1 % level. clearly lower than the controls. the lowest ratios, however, were seen in type i11 where the mean ratio was well below the 5th percentile of randomly sampled healthy controls. i n h d l all types of h l p exhibited a mean value for the cholesterol/triglyceride ratio below the con trols. type i i a showed a moderately decreased ratio, in type iv the ratio was reduced with nearly 50%. types i i b and i11 showed similar average ratios. the relation between cholesterol and triglycer ides in the l p fractions were studied by correla tion analysis in the different types of h l p and com pared to those found in the population of randomly sampled healthy controls (table 111). in the controls a strong positive correlation was seen between vldl triglycerides and cholesterol (r=0.89). a less pronounced but clearly significant correlation was also seen in ldl (r=0.60). in hdl, however, there was no significant correlation between triglyc erides and cholesterol (r=0.10). the same pattern was seen in all types of hlp: a significant positive correlation between cholesterol and triglycerides in vldl and ldl, no correlation in hdl (table 111). when lp lipid interclass relationships were analyzed in the control sample certain significant lipid relationships were revealed. these were basi cally similar to those distinguished also in h l p types i i a , i i b and iv (tables iv-vi). these cor relations were not studied in h l p type 111 because of the low number of subjects in this group. the ldl triglyceride concentration tended to table v. relationships between lipoprotein lipid variables in the very low and high density lipoproteins in 50-year-old m e n with different types of serum lipoprotein patterns linear correlation coefficients ( r ) hdl controls (n =92) i i a (n=38) i1 b (n =28) iv (n=37) vldl" tg chol chol/tg tg chol chol/tg tg chol chol/tg tg chol chol/tg *** *** *** ** * * *** *** *** tg 0.36 -0.41 -0.51 0.07 -0.43 -0.42 0.23 -0.01 -0.12 0.47 -0.52 -0.54 *** *** *** *** *** *** * * *** chol 0.37 -0.46 -0.54 0.26 -0.54 -0.60 0.27 -0.04 -0.20 0.52 -0.42 -0.57 * * * chol/tg 0.07 -0.14 -0.13 0.28 -0.41 -0.50 0.03 -0.03 -0.08 0.15 0.18 0.09 vldl triglyceride and cholesterol concentrations were logarithmic transformed before calculations. * , , ** ***=significant on the 2, 1, and 0.1 % level. upsulu j med sci 81 lipoprotein composition in 50-year-old men with hyperlipoproteinaemia 75 table vi. relationships between lipoprotein lipid variables in the low and high density lipoproteins in 50-year-old-men with different types of serum lipoprotein patterns linear correlation coefficients ( r ) hdl controls (n=92) i i a (n=38) iib (n=28) iv (n=37) ldl tg chol chol/tg tg chol chol/tg tg chol chol/tg tg chol chol/tg ** ** * * * tg 0.28 -0.19 -0.30 0.28 -0.37 0.43 0.30 -0.10 -0.27 0.29 -0.09 -0.39 chol 0.04 -0.04 -0.11 0.09 0.01 0.12 0.03 -0.11 -0.11 -0.22 0.30 0.17 * * *** * * * *** ** chol/tg -0.28 0.20 0.22 -0.31 0.43 0.59 -0.34 0.01 0.22 -0.54 0.31 0.59 * * * ***=s. ignificant on the 2, 1, and 0.1 % level. , > vary positively with vldl lipid levels (significant in controls and type i1 a) (table iv) and was signi ficantly positively correlated to the cholesterol/ triglyceride ratio in vldl in both h l p types i1 a , iib and iv. the ldl cholesterol/triglyceride ratio tended to vary inversely with vldl lipid con centration (significant in controls, types i1 a and iv) and with the vldl ratio (significant in type iia). the ldl cholesterol concentration on the other hand did not show any significant relation ships with lipids in other lp classes in either controls, i1 a or i1 b with remarkably low cor relation coefficients when related t o all other lipid variables. in type i v , however, a negative correla tion to vldl lipid concentrations as well as a positive correlation to the vldl cholesterol/tri glyceride ratio was demonstrated. the negative correlation to vldl as well as the positive correla tion to the vldl ratio was due to the presence of a number of patients with h l p type iv with high vldl triglyceride levels and low ldl cholesterol levels. hdl triglyceride and cholesterol concentrations generally tended to show a positive and negative relation to vldl lipid concentration respectively (table v). the hdl ratio was negatively correlat ed to the vldl lipid concentration. the h d l triglyceride concentration was positively respec tively negatively correlated to ldl triglyceride levels and the ldl cholesterol/triglyceride ratio in the control groups (table vi). the hdl choles terol concentration in type i i a and the h d l tri glyceride concentration in type iv were positively respectively negatively correlated to the ldl ratio. in contrast to the other variables which showed apparently linear interrelationships the h d l ratio gave curved relations to other variables when plotted. discussion l p lipid composition and l p lipid interrelations were studied in h l p and in an apparently healthy control population of 50-year-old men. it was shown (table i) that the l p patterns in h l p differ ed from the normal l p pattern not only in regard of vldl triglyceride and ldl cholesterol concentra tion. in spite of great variations in vldl con centration the lipid composition, as mirrored by the cholesterol/triglyceride ratio (table 11) did not differ from normal in types i1 a , i i b and iv. h l p type i11 was characterized by a high cholesterol/ triglyceride ratio (table 11) caused by a dramati caliy increased cholesterol level in vldl (table 1). the h l p discovered in the investigated popula tion were moderate as can be expected when a screening of a general population is performed. the mean values for ldl cholesterol in type i1 were about 220 mgl100 ml. concomitant with the in creased cholesterol levels, type i1 a and i1 b show ed increased ldl triglyceride concentrations of a similar relative magnitude resulting in a 'normal' cholesterol/triglyceride ratio (table 11). slack and mills (26) reported a low proportion of triglycerides in the total ldl lipid in patients with familial hyper-p-lipoproteinaemia and suggested the pre sence of an abnormal l p in this disease. in a recent report (20) type i1 a with tendon xanthomata upsala .i med sci 81 76 b . vessby et al. were found to have particularly high cholesterol content in relation to triglyceride content. in the mild cases of type i1 without xanthoma tendinosum, most of which probably were of non-familial origin, in the present population, no abnormality was found in regard of the cholesterol/triglyceride ratio. the linear relation between the cholesterol and triglyceride content in ldl in healthy 50-year-old men does not pass through the origin, i.e. the inter cept ( a ) is significantly different from zero (table 111). thus, the ratio cholesterol/triglycerides is a somewhat artificial concept which will increase with decreasing lipid content of ldl. a change of the ratio may not necessarily mean a change in l p composition, especially at low lipid concentrations in ldl. as h l p types i11 and iv (table 11) showed a low mean ratio in spite of normal ldl cholesterol levels this, however, seems to imply a real change in l p composition in these types of h l p compared to normal l p composition. the density range 1.006-1.063 comprises two lp fractions: ldl, (d= 1.006-1.019 corresponding to sf 12-20) and ldl, (d=1.01!l1.063, sf 0-12). the ldl, particles are bigger and relatively more tri glyceride rich than ldl, (25). normally ldlz is the quantitatively totally dominating fraction, as is also seen in h l p type i1 (4). the ldl cholesterol/ triglyceride ratio in normals, types i1 a and i1 b is thus mainly reflecting the composition of the ldl, particles. the heterogeneity of the ldl density fraction may explain the lower correlation recorded between cholesterol and triglyceride concentration in ldl than in vldl (table 111). both h l p types 111 and iv were characterized by considerably increased ldl triglyceride concentra tions when compared with normals (table i). as the ldl cholesterol levels were normal, the ldl cholesterol/triglyceride ratios in type 111 and iv were low. the lower ratio in type 111 than in type iv was caused by a higher mean value for ldl tn glycerides (tables 1-11). the very low ldl ratio in type 111 mirrors a change in the relation between ldl, and ldl, first reported by gofman et al. (16) in patients with xanthoma tuberosum (e.g. probably type 111). an increased concentration of ldl, in relation to ldl, probably reflects the accumulation of par ticles metabolically intermediate between vldl and ldl, because of a block in the normal ca tabolism of vldl (21). the group of lipid patterns classified as h l p type iv is probably heterogeneous representing pathogenetically different disorders with an in crased vldl concentration in common but with diverging ldl concentration and composition. some type i v may be genetically related to com bined hyperlipidaernia (22). other l p patterns classified as type iv have been suggested to be associated with a retarded conversion of vldl to l d l because of a relatively decreased vldl tri glyceride clearance (28). also the density fraction > 1.063 contains two or more populations of l p particles with different lipid composition (24). hdl, (d= 1.063-1.125) contains somewhat less triglycerides and more cholesterol than hdl, (d=1.125-1.21) which is also more rich in protein content. nichols reported (17) that hdl, and hdl, may vary in concentration relatively in dependent of each other. he did not find any rela tionship between the cholesterol and triglycerides in hdl in either normals or hlp. in this study low cholesterol levels in h d l in combination with an increased vldl lipid concentration was seen i type iv which may reflect the redistribution of some l p components (mainly hdl,) from hdl to vldl after the introduction of triglyceride-rich vldl into the circulation (12). a reciprocal rela tionship between hdl and vldl concentration is present during conditions of varying vldl con centration ( i 5 ) . h d l triglyceride concentration was significantly increased in all types of h l p compared with the normal controls (table i). these ‘hdl triglyceride’ determinations probably measure not only hdl l p triglycerides but also some diglycerides and mono glycerides produced during the hydrolysis of tri glyceriderich l p and recovered in the ultracentri fuge fraction with d> 1.063. the hdl ‘triglyceride’ determinations is certainly both less specific and less accurate ( 2 ) than the triglyceride determination in the other l p classes. one explanation for actually incrased hdl tri glyceride levels in h l p may be a suggested non enzymic exchange of hdl esterified cholesterol for vldl triglycerides secondary to lecithin choles terol acyl transferase (lcat) dependent esterifica tion of free cholesterol in hdl (18). high hdl triglyceride concentrations may possibly be re garded as reflecting an accelerated break down of triglyceride-rich l p particles with a requirement of disposal of much vldl surface material. n o signif icant correlation was found between hdl triglyc upsala j med sci 81 lipoprotein composition in 50-year-old men with hyperlipoproteinaemia 77 eride and cholesterol concentrations (table 111). thus no conclusion can be drawn from changes in the hdl cholesterol/triglyceride ratio regarding changes in hdl lipid composition. when lipid variables show either a strongly positive or a strongly negative interrelationship, some common determinant of the serum concentra tions of these lipids seems probable. we have studied linear correlations between l p lipid variables in the random sample of healthy men and in different types of h l p (table iv-vi). although the conclusions drawn from this study are valid only for middle-aged men the results were in good agreement with those found in investigations of other populations. the positive relationship between vldl con centration and the triglyceride concentration in ldl and hdl as well as the negative relationship between vldl concentration and hdl choles terol content is in agreement with earlier studies by lindgren, freeman & nichols (16) and ewing, freeman & lindgren (6). in the present study ldl cholesterol concentra tion varied remarkably independent of all other l p lipid variables in the controls, types i i a and i i b . in type i v , however, the ldl cholesterol con centration showed a significant negative relation ship to vldl lipid levels and a positive correlation to the cholesterol/triglyceride ratio in vldl due to the presence of a group of patients with high vldl triglyceride levels and low ldl cholesterol levels. gofman & tandy reported (10) that a regression curve of ldl on vldl levels studied in two large samples of human males showed a steady rise in ldl levels with increasing levels of vldl. at very high levels of vldl, however, the curve sloped downwards and the relationship between those two classes became inverse rather than di rect. the reason for this may be a retarded inter conversion of vldl to ldl because of a relatively deficient triglyceride removal capacity (28). the negative correlation between ldl cholesterol and the vldl cholesterol/triglyceride ratio is also compatible with this hypothesis. the tendency to a negative relationship between ldl and vldl ratios seen in h l p type i i a as well as the positive correlation between ldl tri glycerides and the vldl ratio in types iia, iib and iv may be due to a certain accumulation of particles with a density and composition inter mediate between vldl and ldl in some patients associated with a ‘late pre-p’ pattern in d < i .006 on agarose electrophoresis (27). in conclusion all the moderate h l p studied in this investigation showed similar l p lipid interclass interrelationships although they are conventionally divided into different types of h l p because of diverging mean values for l p lipid concentrations. apart from h l p type i11 and h l p type iv with low ldl cholesterol levels all other types of l p patterns seemed to conform to a common model of l p interconversions where the different h l p types represented different tails of the same spectrum. no clear qualitative differences in l p interrela tionships between the h l p types were observed. the basically quantitative differences in the statisti cal correlations between the l p lipids in the healthy controls and types i i a , i i b and some of the type iv may be due to the ‘truncation’ of the spectrum caused by the classification. by simple measures such as dietary modifications, many of these moderately expressed h l p may be transformed to other types of h l p patterns or to a normal l p pattern. h l p type i11 is probably due to a block in the catabolism between vldl and ldl with ac cumulation of ‘intermediary’ lp. in type iv a negative correlation exists between vldl con centration and ldl cholesterol levels which also at least in some cases, may be caused by a block in the conversion of vldl to ldl although at an earlier step in the catabolism of triglyceride-rich lp. acknowledgements this work was supported by grants from the swedish medical research council (19x-204 and 876-4679-01). references 1. 2. 3. 4 . 5. beaumont, j. c., carlson, l. a . , cooper, g . r., fejfar, z . , fredrickson, d. s . & strasser, t . : classi fication of hyperlipidemias and hyperlipoprotein emias. bull wld hlth org43.891, 1970. carlson, k. : lipoprotein fractionation. j clin pathol, suppl. 5: 32, 1973. carlson, l. a. & bottiger, l. e.: ischaemic heart disease in relation to fasting value of plasma tri glycerides and cholesterol. lancet i : 865, 1972. dixon, w. j . (ed.): bmd biomedical computer programs. university of california press, berkeley, 1973. draper, n. r. & smith, h.: applied regression anal ysis. john wiley & sons inc., new york, 1966. upsala j med sci 81 78 b. vessby et al. 6. ewing, e. m., freeman, n. k. & lindgren, f. t.: the analysis of human serum lipoprotein distribu tion. advan lipid res3: 25, 1965. 7. fredrickson, d. s. & lees, r. s . : a system for phenotyping hyperlipoproteinemia. circulation 32: 321, 1965. 8. fredrickson, d. s., levy, r. i. & lees, r . s . : fat transport in lipoproteins: an integrated approach to mechanisms and disorders. new engl j med 276: 34, 1967. 9. gofman, j. w., de lalla, o., glazier, f., freeman, n . k., lindgren, f. t., nichols, a. v., strisower, b. & tamplin, a. r.: the serum lipoprotein transport system in health, metabolic disorders, atherosclerosis and coronary heart disease. plasma (milan) 2: 4 13, 1954. 10. gofman, j. w. & tandy, r.: in atherosclerotic vascular disease, p. 162. appleton-century-crofts, new york, 1967. 11. havel, r. j., eder, h. a. & bragdon, j. h.: the determination and chemical composition of ultra centrifugally separated lipoproteins in human serum. j clin invest34: 1345, 1955. 12. havel, r. j., kane, j. p. & kashyap, m. l.: inter change of apolipoproteins between chylomicrons and high density lipoproteins during alimentary lipernia in man. j clin invest 52: 32, 1973. 13. hedstrand, h. & vessby, b.: serum lipid levels and lipoprotein composition in healthy 50-year-old men in sweden. upsala j med sci 81: 37, 1976. 14. kannel, w. b., castelli, w. p., gordon, t. & mcnamara, p. m.: serum cholesterol, lipoproteins, and the risk of coronary heart disease. ann intern med74: i , 1971. 15. levy, r. i . , lees, r. s . & fredrickson, d. s . : the nature of prebeta (very low density) lipoproteins. j clin invest 45: 63, 1966. 16. lindgren, f. t., freeman, n. k. & nichols, a. v.: in metabolism of lipids as related to athero sclerosis, p. 62. ch. c. thomas, springfield, illinois, 1965. 17. nichols, a . v.: human lipoproteins and their inter relationships. adv biol med phys 1 1 : 109, 1967. 18. nichols, a. v. & smith, l.: effect of very low density lipoproteins on lipid transfer in incubated serum. j lipid res 6: 206, 1965. 19. noble, r. p.: electrophoretic separation of plasma lipoproteins in agarose gel. j lipid res 9: 693, 1968. 20. olsson, a. g . & carlson, l. a,: studies in asympto matic primary hyperlipidaemia. i. types of hyperlipo proteinaemias and serum lipoprotein concentrations, compositions and interrelations. acta med scand, suppl. 580, 1975. 21. quarfordt, s. h., levy, r. i. & fredrickson, d. s.: the kinetic properties of very low density lipopro tein triglyceride in type 111 hyperlipoproteinemia. biochim biophys acta296: 572, 1973. 22. rose, h . g., kranz, p., weinstock, m., juliano, j. & haft, j. i . : combined hyperlipoproteinemia. evi dence for a new lipoprotein phenotype. athero sclerosis20: 51, 1974. 23. rush, r. l., leon, l. & turrel, j.: automated simultaneous cholesterol and triglyceride determina tion on the auto analyzer@ i1 instrument. advances in automated analysis 1 : 503, 1971. 24. scanu, a. & granda, j. l.: effects of ultracentrifuga tion on the human serum high-density (1.063 < p < 1.21 g/ml) lipoprotein. biochemistry5: 446, 1966. 25. shore, b. & shore, v.: some physical and chemical properties of the lipoproteins produced by lipolysis of human serum sf 2 w o o lipoproteins by postheparin plasma. j atherosclerosis res2: 104, 1962. 26. slack, j. & mills, g. l.: anomalous low density lipoproteins in familial hyperbetalipoproteinaemia. clin chim acta29: 15, 1970. 27. vessby, b . , carlson, l. a. & hedstrand, h.: cholesterol and triglyceride composition of very low and low density lipoproteins in hyperlipo proteinaemias, p. 882. springer verlag, berlin, hei delberg, new york, 1974. 28. vessby, b. & lithell, h.: dietary effects on lipo protein levels in hyperlipoproteinemia. delineation of two subgroups of endogenous hypertriglycerid emia. artery i : 63, 1974. received january 20, 1976 address for reprints: b. vessby, m.d. department of geriatrics box 641 s-75 1 27 uppsala sweden upsala j med sci 81 upsala j med sci 81: 189-191, 1976 fa mi lia i ovarian carcinoma l. thor, b. h . persson and b. kjessler from the department of obstetrics and gynaecology, university hospital, uppsala, sweden abstract familial aggregation of patients with ovarian carcinoma is unusual. a family with four affected members in three con secutive generations is described. the tumors were all of the serous papillary adenocarcinoma type. the pattern of ap pearance of the malignant disorder in the present family may he explained as the result of transmission of a dominant mutant autosomal gene. the future long term management of such a family might include prophylactic oophorectomy in certain family members, and possibly selective termina tions of pregnancies with female fetuses in high-risk women. introduction familial aggregation of patients with ovarian adeno carcinoma is unusual. thus, only three cases with affected near relatives were observed in a study of 110 patients with ovarian cancer ( 5 ) . so far only seven families have been described, where ovarian adenocarcinoma has been present in two consecutive generations (1, 2, 3 , 4 , 6). we here describe a family with serous papillary adenocarcinoma of the ovary present in four family members belonging to three consecutive genera tions, suggesting the transmission of a mutant domi nant gene as a possible cause of this site-specific cancer. methods all affected members of the present family have been investigated and treated at the department of obstetrics & gynaecology, university hospital, uppsala, where the first case appeared in 1925. histopathological diagnosis were obtained by ana lyses of pathology specimens derived from opera tions and post mortem autopsies. family history relevant family data and medical histories appear from the pedigree (fig. 1) and table i. case reports i: 1. the patient was admitted after six months of symptoms including abdominal distension. a large pelvic mass was present. at operation, bilateral ovarian tumors with papillary projections were ob served together with general engagement of adja cent tissues. she died at the age of 47. 11: 2. this patient was originally seen because of a large pelvic mass, subsequently found to consist of large bilateral cysts which were radically removed. three years later and despite cytostatic treatment, the patient died from a recurrent tumor with ascites after a short course. she died at the age of 55. 111: 3. this patient was admitted after two weeks of abdominal distension with a large pelvic mass present. laparotomy revealed wide-spread ab dominal carcinosis of ovarian origin. cytostatic therapy did not prevent her death six months later at the age of 45. 111: 6. chest symptoms of nearly one year’s dura tion brought this patient to attention, and she was found to have pleural effusion and metastatic lung tumors. by means of fineneedle biopsy a large pel ? i m 0 0 n o r m a l male or female 0 d i e d of o v a r i a n c a n c e r @ p r o p h y l a c t i c o o p h o r e c t o m y i n o o f f s p r i n g 7 d e c e a s e d fig. 1. pedigree of a family with ovarian cancer. upsulu j med sci 81 190 l . thor et al. table i. clinical and histopathological data on six members of a family with ovarian adenocarcinoma age at histopathologic first addiagnosis duration until mission of the tumor type of therapy fatal outcome i: 1 41 papillary serous 11: 2 51 papillary serous adenocarcinoma adenocarcinorna 111: 3 45 papillary serous 111: 6 38 papillary serous 111: 2 46 normal ovarian 111: 4 41 normal ovarian adenocarcinoma adenocarcinoma tissue tissue operations and radia (1) bilateral salpingo tion therapy oophorectorny +radia tion therapy; (2) cyto static therapy therapy operation+cytostatic cytostatic therapy prophylactic bilateral prophylactic bilateral oophorectomy oophorectorny vie mass also present was found to consist of ovarian papillary adenocarcinoma. she died seven months later after several courses of cytostatic treatment at the age of 38. iii:2&4. due to the family history these two sisters demanded prophylactic oophorectomy to be performed, and their ovaries were found to be histo logically normal. 11: 3. this 66 year old sister of 11: 2 is still healthy, and no pathological findings have been demon strated at repeated gynecological examinations. 111: 7&8. these two daughters of 11: 3 are 45 and 41 years respectively. they are both in goodgeneral condition. additionally iv: 8 has recently been found to be normal at gynecological examination. discussion in the present family all affected members suffered from papillary serous adenocarcinoma. this type of ovarian malignancy also seems to have been pre vailing in the previously reported families ( 1 , 2, 3 7 4 ) . the probability of familial association of this site -specific malignancy by chance, has been re garded as extremely remote, and other explanations must therefore be considered. the appearance of the same type of rare disorder in consecutive gene rations suggests the possibility of transmission of a dominant autosomal gene according to the men delian laws of inheritance. the family patterns reported by liber (4), lewis & davison (2), li et al. (3), lynch & krush (6) < 1 year 31 year 6 months 7 months and fraumein et al. (1) as well as in the present family are consistent with such an explanation. however, a polygenic mode of inheritance must also be considered. in other families with aggregation of affected sibs as described by mccrann et al. (7) and fraumein et al. (i), transmission of rare recessive genes might explain the findings, though genetic transmission of the disease could not be proven. if mutant genes sometimes may be of importance in the etiology of malignant ovarian disease, they may of course differ with regard to mode of expres sion and transmission. li et al. (3) reported a large kindred, where a phenotypically normal male might possibly have transmitted a mutant gene, to his two daughters affected by abdominal carcinomatosis with un known, primary site. so far there are no other re ports on suggested male transmission of mutant genes for ovarian malignancy. neither are there any reports on males affected with gonadal carcinoma as a result of suggested inheritance of mutant genes. the clinical management and counselling of families with several affected members must there fore be based on thorough considerations of all family data available for each specific family. in the present kindred, the malignant ovarian dis order has been restricted in its appearance to descendants within one of the two “female” branches of the family, i.e. in 11: 2 and her offspring, while ii:3 and her progeny have so far escaped the disease. this finding is consistent with an in heritance pattern of a dominant mutant gene, where upsala j med sci 81 familial ovarian carcinoma 191 ii:3 may be a non-carrier of the mutant gene. evidences in favor of such an interpretation are her present health condition together with her present age, which seems to be beyond the actual risk pe riod within which the affected members of the family were taken ill. if ii:3 is a non-carrier, her two healthy daughters and their progeny may be considered not to be a t risk. regular clinical con trols have been recommended for 111: 7 and 111: 8. surgical intervention has for the moment not been considered necessary. in the affected family branch, 111: 2 and 111: 4 had their ovaries removed prophylactically without histological signs of malignancy. there are two female descendants, iv: 3 and iv: 5 , 8 and 17 years old respectively, who may be considered to be at considerable risk. these two females will require regular and thorough clinical controls until they have the number of children they want, when prophylactic oophorectomy may be indicated. for the long term management of the present family it must be taken into consideration that only female members have been affected, and are likely transmittors, and that, so far, there is no evidence of male transmission of the alleged mutant gene. prenatal sex determination and selective abortion of female fetuses in future pregnancies of iv: 3 and iv:5 could therefore possibly reduce the rate of long term anguish for having in fact “affected” fe male offspring. should it be, that male members of the present family d o not transmit the genes for this disorder, then selective termination of pregnancies with female fetuses may in addition help to even tually eliminate the adverse gene from this family. references 1 . fraumein, j. f., jr, grundby, g . w . , creagan, e. t. & everson, r. b.: six families prone to ovarian cancer. cancer 36: 364-369, 1975. 2 . lewis, a. c. w. & davison, b. c. c.: familial ovarian cancer. lancet ii: 235-237, 1%9. 3 . li, f. p., rapaport, a. h . , fraumein, j. f., jr & jen sen, r. d.: familial ovarian carcinoma, jama 214: 1559-1561, 1970. 4. liber, a. f.: ovarian cancer in mother and five daughters. arch parhol49: 280-290, 1950. 5 . lynch, f. w . : a clinical review of 110 cases of ovarian carcinoma. am j obstet gynec 32: 753, 1936. 6. lynch, h. t. & krush, a . j.: carcinoma in the breast and ovary in three families. surgery, gynecology & obstetrics 133: 644-648, 1971. 7. maccrann, d. j . , marchant, d. j . & bardawil, w. a.: ovarian carcinoma in the three teenage siblings. ob stet gynecol43: 132-137, 1974. received march 28, 1976 address for reprints: berndt kjessler, m.d. department of obstetrics & gynecology university hospital s-750 14 uppsala sweden upsala j med sci 81 upsala j med sci 83: 141-144, 1978 prolonged serum insulin decreasing effects of two synthetic somatostatin analogues studied in vivo by a new animal method preliminary communication georg herbai,' sighild westman-naeser,' l h r s carlsson3 and klas nilsson3 from the department of 'internal medicine (endocrinological section) and department of 2histology and clinical cytology, university hospital, uppsala, and 3department of peptide chemistry, ferring ab, malmo, sweden abstract a newly developed in vivo method, using the ob-ob strain of obese-hyperglycaemic mice with permanently very high serum insulin values, makes it possible to detect more pro longed serum insulin lowering properties than in normal animals. two newly synthesized analogues of somatostatin, o-alanine-somatostatin and des-alanine-des glycine-des amino-somatostatin produced a more prolonged and greater decrease in the serum insulin values of ob-ob mice than did somatostatin. our new in vivo method makes it possible to investigate the duration of insulin suppression of new derivatives. introduction somatostatin research has undergone an explosive development in the last few years and an abundant literature has resulted. several extensive and de tailed reviews have been published ( 6 , 8 , 23). a n i m a l e x p e r i m e n t s . insulin release inhibiting ef fects of somatostatin have been studied in isolated rat pancreas (3, 5, 12) and in perfused canine pancreas (1 1). glucose, triglycerides and casein administered intraduodenally in conscious dogs cause an increasing glycagon-like immunoreactivity (gli) in serum. somatostatin infusion inhibits such gli increases (19). somatostatin has been detected in the hypothalamus, posterior pituitary, pancreatic islets (a, or d-cells), stomach and intestine in rats with an irnmunofluorescence technique (10). all these experiments indicate the complex biological effects of somatostatin and several interactions with other hormone systems. h u m a n e x p e r i m e n t s . somatostatin inhibits the arginine-dependent growth hormone release and its basal secretion in the normal male (21). moreover, somatostatin suppresses the release of glucagon, insulin and growth hormone in normal, diabetic, acromegalic and in hypopituitary patients (14). in normal and acromegalic patients somatostatin infu sion inhibits the sleep-related peak of growth hormone release (15). trh-elicited tsh release is also inhibited by somatostatin (16, 2 2 , 2 5 ) . serum glucose and insulin values are depressed by somatostatin injections (1) and somatostatin also lowers the serum insulin values stimulated by glucagon and tolbutamide injections (4). the basal and foot-stimulated secretions of gastrin and gastric acid are also decreased by somatostatin ( 2 ) . the aim of our studies was to develop a suitable in vivo method for following the time course after a single injection of somatostatin into the animal. similar experiments with insulin lowering effects were made in humans given somatostatin infusions (1, 13, 14, 17). in all these studies somatostatin elicited a short insulin inhibition followed by a re bound effect. the biological half-life of somatosta tin is very short: 4-6 min (8). our in vivo animal method, using ob-ob mice with very high serum insulin values demonstrated a dose-dependent somatostatin effect with a decrease in the serum insulin values which lasted for only a short time (9). in this preliminary communication two newly synthesized somatostatin derivatives are described which exhibit markedly prolonged serum insulin depressing effects. material and methods male 5-7 months old obese-hyperglycaemic mice, with high serum insulin values (150-250 ngiml), were selected, unsalo j mrd .5( i 83 142 g. herbai et al. table i . the amino acid sequence of the original somatostatin (1) and of two recently synthesised ( 2 , 3 ) somatostatin analogues 1. original gh-rih: h-ala-gly-cys-ly s-asn-phe-phe-trp-lys-thr-phe-thr-ser-cys-oh 2. somatostatin analogue synthesised by changing the first amino acid to d-alanin: h-d-ala-gly-cys-lys-asn-phe-phe-trp-ly s-thr-phe-thr-ser-cys-oh 3 . somatostatin analogue produced newly by omission of the two first amino acids and the amino group of the third (des-ala-des-gly-desamino polypeptide): schzchzco-ly s-asn-phec-phe-trp-ly s-thr-phe-thr-ser-cy s-oh 6 animals per group. all animals had body weights be tween 48 and 55 g. somatostatin was synthesized by a step-wise frag mentary condensation technique (9). the two ana logues were synthesized by a classical step-wise frag mentary condensation technique at the department of peptide chemistry, ferring ab, malmo, sweden. they were purified by ion-exchange chromatography on carboxymethyl cellulose (whatman cm 2 3 ) . analytical data d-ala l-somatostatin: homogeneous in three different tlc-systems. [a]i4: -41", c 0.48 in 1 % acetic acid. amino acid analysis: ala 0.99, gly l.&, cys 2.0, lys 2.11, asp 1.00,phe2.85, thr 1.92, ser0.94. des-ala1-des-gly2-desarnino-somatostatin: homogene ous in three different tlc-systems. [a]$: -47", c 0.42 in 1 % acetic acid. amino acid analysis: lys 2 . 0 4 , asp i .oo, phe 2.88, thr 1.94, ser 0.93. immuno-reactive serum insulin was determined by double-antibody radinimmuno-assay (7), using a kit ob tained from the radiochemical centre, amersham, eng land. crystalline mouse insulin (24 iu/mg) was used as standard. the animals were conscious and unanesthetised during the experiments. blood samples were obtained by puncture of the orbital venous plexus with a thin-walled pasteur pipette before and after the injection of control saline, somatostatin, or synthesized analogues. in this preliminary report the lowest effective dosage of the drugs, found in an earlier study (9), was used: 100 pg/kg. the solutions (0.1 ml) were injected rapidly into a tail vein. blood samples were taken from all animals at zero, 8, 16, 32, 64 and 128 min after the start. the small blood samples were collected and stored as described previ ously (9). results the chemical structure of somatostatin and the synthetic analogues are given in table i. the first analogue ( 2 ) has d-alanine in the first position of the cjpjulo j m p ~ s c i 83 chain in contrast to l-alanine as in somatostatin itself. the second analogue (3) lacks the first two amino acids, on the n-terminal end of the chain alanine and glycine, together with the amino group of the third, with an unchanged cys-cys bridge. the serum insulin values, initially and at several times up to 128 min after drug administration, are given in ng/ml in fig. 1 . control saline injections did not alter the insulin values. somatostatin injection resulted in rapidly decreasing serum insulin value, which returned to the normal initial value of about 300 ng/ml after 30 min. both synthetic analogues also caused and markedly and rapidly reduced the 300 e e \ 5 2 0 0 0 0 z _i 3 v, z r 3 100 0 0 8 16 32 6l 128 t i m e minutes fig. i . serum insulin values at different times after injec tion of the drugs in vivo. six animals per group. the controls received physiological saline. all drugs were in jected at zero time i.v. in single doses of 100 pg/kg. effects of somatostatin analogues on serum insulin 143 insulin values. in contrast to the fairly rapid recov ery after somatostatin, both the synthetic deriva tives produced a prolonged depression of the insulin value, which lasted throughout the period of obser vation, i.e. 128 minutes. discussion the great difficulty in the therapeutic use of somatostatin in endocrine disorders has been the lack of a long-acting derivative. several research groups have synthesized and tested a large number of chemically different derivatives (18, 20, 23). some attempts were made to shorten the tetra decapeptide chain by excluding one or several amino acids. another structure-activity relation in vestigated, was whether cleavage of the disulphide link in the polypeptide resulted in alterations of somatostatin activities. another possibility was to analyse the separation of the activities of so matostatin derivatives inhibiting growth hormone and influencing insulin and glucagon. the evalua tion of the different activities was made by studying the hormone effects in vitro and in some experi ments in vivo. in these studies it was found that many shorter amino acid analogues had lower activities than somatostatin in several types of tests. as far as we know, no new method has been described, which permits tests of somatostatin and different structural analogues, by single injections of the agent in vivo with subsequent monitoring of the serum hormone values at different times after injection. our previous study (9) showed a statisti cally significant dose-response effect in vivo for serum insulin-inhibiting effects with different doses of somatostatin injected into obese-hyperglycaemic mice. in our system, somatostatin had a very short lived serum insulin diminishing effect in the dosage used. in the present investigation of the two somatostatin analogues, both were injected in rela tively low doses into each group of animals. it was of great interest to note that both d-ala-somatostatin and des-ala-des-gly-desamino-somatostatin had markedly prolonged serum insulin depressant effects. these prolonged insulin inhibiting effects in vivo were obtained in a complex hormonal environ ment. some hours after the experiment the mice seemed to have recovered completely. experiments with lean litter-mates of the ob-ob mice, which have very low serum insulin values, showed that they were completely uninfluenced by somato statin. the synthesis of the two new derivatives tested was modelled on earlier findings by vavra et al. (24). they modified vasopressin by replacing l-arginine in position 8 by d-arginine and replacing the terminal amino group by p-mercaptopropionic acid, the antidiuretic effect. this resulted in a marked prolongation of the anti-diuretic effect, which was tested in both animals and in patients with diabetes insipidus. the results of this study also demonstrate the usefulness of the “ob-ob in vivo”-method for test ing the duration of the insulin suppressive effect of somatostatin derivatives, which can perhaps give a possibility of finding a new therapeutic agent. references 1 . 2. 3. 4 . 5 . 6 . 7. 8. 9. alberti, k . g. m. m., juel christensen, n . , engkjaer christensen, s . , prange hansen, aa., iversen, j . , lundbaek, k., sayer-hansen, k. & @rskov, h.: in hibition of insulin secretion by somatostatin. lancet ii: 1299, 1973. bloom, s . r., mortimer, c. h., thorner, m. o., besser, g. m., hall, r . , gomez-pan, a., roy, v. m., russel, r. c. g., coy, d. h., kastin, a. j. & schally, a. v.: inhibition of gastrin and gastric-acid secretion by growth-hbrmone release inhibiting hormone. lancet ii: 1106, 1974. efendic, s . & luft, r.: studies on the inhibitory ef fect of somatostatin on glucose induced insulin re lease in the isolated perfused rat pancreas. acta en docr (kbh.), 78: 510, 1975. gerich, j . e . , lorenzi, m., schneider, v . & forsharn, p. h.: effect of somatostatin on plasma glucose and insulin response to glucagon and tolbutamide in man. j clin endocr metab39 1057, 1974. gerich, j. e., lovinger, r. & grodsky, g. m . : inhibi tion by somatostatin of glucagon and insulin release from the perfused rat pancreas in response to ar ginine, isoproterenol and theophylline. evidence for a preferential effect of glucagon secretion. endocrinol guillemin, r. & gerich, j. e.: somatostatin: physiological and clinical significance. ann rev med 27: 379, 1976. hales, c. n . & randle, b. j.: immuno-assay of insu lin with anti-body precipitate. biochem j 88: 137, 1963. hansen, a. p. & lundbaek, k.: somatostatin: a re view of its effects, especially in human beings. diab ete metabolisrne (paris) 2: 203, 1976. herbai, g., westman-naeser, s., carlsson l., melin, p. mulder t, j.: the dose dependent serum insulin lowering effect of somatostatin (gh-rif) in vivo in the obese-hyperglycaemic (ob-ob) syndrome. ups j med sci 82: 255. 1977. ogy 96: 749, 1975. 144 g . herbai et al. i unscrlu j m e d s c i 83 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. hokfelt, t., efendic, s . , hellerstrom, c., johansson, o., luft, r. & arimura, a,: cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special references to the a,-cells of the pancreatic islets and to the hypothalamus. acta en docr (kbh) suppl. 200,80: 5 , 1975. iversen, j.: inhibition of pancreatic glucagon release by somatostatin: in vitro. scand j clin lab invest 33: 125, 1974. johnson, d. g . , ensinck, j. w., koerker, d., palmer, j. & goodner, c. j.: inhibition of glucagon and insulin secretion by somatostatin in the rat pancreas perfused in situ. endocrinology 96: 370, 1975. leblanc, j . r., anderson, m. b., siegel, m. b. & yen, s. s. c.: inhibitory action of somatostatin on pancreatic a and p cell function. j clin endocrinol metab40: 568, 1975. mortimer, c. h., turnbridge, w . m. g., carr, d., yeomans, l . , lind, t., coy, d. h., bloom, s . r., kastin, a . , mallinson, c. n . , besser, g. m., schally, a. v. & hall, r.: effects of growth-hormone release inhibiting hormone on circulating glucagon, insulin, and growth hormone in normal, diabetic, ac romegalic, and hypopituitary patients. lancet i : 697, 1974. parker, d. c., rossman, l. g., siler, t. m., rivier, j., yen, s. s. c. & guillemin, r.: inhibition of the sleeprelated peak in physiologic human growth hormone release by somatostatin. j clin endocr metab 38: 496, 1974. patel, y. c., alford, f. p. & burger, h . g.: the 25-hour plasma thyrotrophin profile. clin sci 43: 71, 1972. peracchi, m., reschini, e., cantalamessa, l., giusti na, g., cavagnini, f., pinto, m. & bulgheroni, p.: effect of somatostatin on blood glucose, plasma growth hormone, insulin, and free fatty acids in nor mal subjects and acromegalic patients. metabolism 23: 1009, 1974. rivier, j., brazeau, p., vale, w. & guillemin, r.: somatostatin analogs. relative importance of the di sulfide bridge and of the ala-gly side chain for bio logical activity. j med chemistry 18: 123, 1975. sakurai, h., dobbs, r. e. & unger, r. h.: the effect of somatostatin on the response of gli to the in traduodenal administration of glucose, protein, and fat. diabetologia 11: 427, 1975. sarantakis, d., mckinley, w. a., jaunakais, i., clark, d. & grant, n. h.: structure activity studies on somatostatin. clin endocrin 5 , suppl.: 275% 1976. siler, t. m., vandenberg, g., yen, s . s. c., brazeau, p., vale, w. & guillemin, r.: inhibition of growth hormone release in humans by somatostatin. j clin endocr metab37: 632, 1973. siler, t. m., yen, s . s . c., vale, w. & guillemin, r.: inhibition by somatostatin on the release of tsh in duced in man by thyrotropin-releasing factor. j clin endocrinol metab38: 742, 1974. vale, w., brazeau, p., rivier, c., brown, m., boss, b., rivier, j., burgus, r., ling, n. & guillemin, r.: somatostatin, recent progr hormone res 31: 365, 1975. 24. vavra, i., machova, a., holeeek, v . , cort, j . h., zaoral, m. & sorm, f.: effect of a synthetic analogue of vasopressin in animals and in patients’with dia betes insipidus. lancet i: 948, 1968. 25. weeke, j., hansen, a . p. & lundbaek, k.: the in hibition by somatostatin of the thyrotropin response to thyrotropin-releasing hormone in normal subjects. scand j clin lab invest 33: 101, 1974. received november 18, 1977 address for reprints: georg herbai, m.d. department of internal medicine university hospital s-750 14 uppsala sweden upsala j med sci 82: 15-19, 1977 a solid phase radioimmunoassay method for ferritin in serum using 1251-labelled ferritin l e i f wide and gunnar birgegard from the departments of clinical chemistry and internal medicine, university hospital, uppsala, sweden abstract a solid phase radioimmunoassay method for ferritin in serum was developed using ferritin conjugated with an 1251-labelled ester. the method was based upon competitive inhibition utilizing the free antigen-binding sites of anti bodies to ferritin bound to a ferritin which had been chemi cally coupled to insoluble polysaccharide particles. the method was evaluated with regard to specificity, sensitivity, precision and practicability and was found to be a robust method suitable for the assay of ferritin in human serum in clinical studies. there was a significant (p<o.ool) differ ence between the ferritin levels in healthy men and women, mean values 100 and 66 arb u/i respectively. male blood donors had significantly (p<0.002) lower ferritin levels in serum (mean 68.5 arb u/l) than other healthy men. patients with iron overload or hepatic damage had high values, as expected, and the serum levels of ferritin reflected changes in iron deposits during phlebotomy and iron treatment in 3 healthy men. introduction in 1972 an immunoradiometric assay for ferritin in serum was described by addison et al. (1). since then several laboratories have used this method (8, 9, 1 1, 14) for which a production of an immuno sorbent for ferritin antibody, labelling of the anti body and subsequent elution of the labelled anti body is necessary. even if not all workers find the immunoradiometnc assay for ferntin as “exasperat ingly difficult” t o handle as crosby (3, there is a need for a simpler method. radioimmunoassays employing labelled antigens instead of labelled anti bodies are commonly used in many laboratories for the assay of different antigens. ferritin has been labelled with lz5i by the chloramine-t technique but only to a very low specific activity (12). attempts to label ferritin by that method to high specific ac tivity were unsuccessful in our laboratory. the present work presents a method for measuring fer ritin in serum by using ferntin labelled with a p hydroxyphenylpropionic acid, n-hydroxysuccin imid ester, which had been iodinated according to bolton & hunter (3), in a radioimmunoassay sys tem with antibodies coupled to a solid phase. materials and methods ferritin preparation. ferritin was purified from human spleen by a modification of the method described by drys dale & munro (7), including a final repeated chromato graphy on sepharose 6b. the preparation was kindly supplied by dr tor olofsson, university hospital, lund, sweden. this preparation was used for immunization, for labelling with lz5i and as a provisional reference stand ard preparation. antisera to ferritin. three different antisera were com pared. two antisera were raised in two rabbits by weekly injections for 4 weeks with 0.25 mg of the ferritin prepara tion in freund‘s adjuvant. the animals were bled 3 weeks after the last injection. the third antiserum was a com mercial product from behringwerke, west germany. labelling of ferritin. to 400 mg of p-hydroxyphenyl propionic acid n-hydroxysuccinimide ester was added 2 mci n a t for labelling of the ester according to the technique of bolton & hunter (3). the iodinated ester was conjugated to 15 p g of the ferritin preparation by in cubation at 0°c for 40 min. the labelled product was puri fied by chromatography on a sephadex g 150 column. two peaks with labelled material (apart from the free iodine) were obtained. the first peak was eluated with the void volume and contained almost all immunoreactive material. the second peak followed immediately after the first and had a very low immunoreactivity with the anti ferritin antibodies. the radioactivity bound to immuno reactive material comprised less than 30% of the total proteinbound radioactivity. solidphase coupling of anti-ferrifin. for direct coupling of antibodies 5 to 10 p1 of the antisera were coupled to 100 mg of cnbr-activated ultrafine sephadex@ or micro crystalline cellulose as previously described (17). anti bodies were also indirectly coupled (19), i.e. ferritin (200 pg) was first coupled to the cnbr-activated particles (100 mg) and antifemtin-antibodies were then bound to the coupled ferritin. the particles were suspended in assay buffer solution. upsulri j m e d sci 82 l . wide and g. birgegdrd 16 95 90 80 * 70 s .$ 60 c' 50 2 p lo z 30 20 10 5 ferritin o r b u i i s 10 20 t o i a o 200 500 fig. 1 . logit representation of the dilution curve for the reference standard and for serum sample with high ferritin content. assay buffer solution. 0.05 m phosphate buffer, ph 7.4, containing 0.01 m edta, 0.2% bovine serum albumin, 0.02 % nan,, 0.08 m nacl and 0.5 % tweena 20. the ph of the final solution was adjusted to 7.4. performance o f a s s a y . the assays were technically per formed a s previously described for the assay of gonado tropins (19). all samples were run in duplicate. 0.1 ml of test serum was incubated with 1 ml of the solid phase antibody suspension. the amount of polymer-coupled antibodies was chosen so that about 10% of added la belled ferritin was bound after incubation for about 20 hours. after 3 4 h of incubation at room temperature the labelled ferritin (40 000 cpm) was added in 0. i ml of assay buffer solution and the mixture was further incubated for 20 hours with a slow vertical rotation of the test tubes. after washing three times with saline with o . s % tween 20 the remaining radioactivity was measured in a gamma counter. the assays were calibrated using the ferritin preparation diluted in 25% serum containing a small amount of ferritin as a provisional reference standard. the values given are expressed in arbitrary units per litre, obtained by giving the geometric mean for a population of 25 normal young men (no blood donors), age 26-30 years, the value of 100. the results of the assay and dif ferent quality control parameters were calculated using a compucorp 445 desk computer with a logit-log program gel chromatography. fractionation of 3 ml of a serum sample from a healthy young man was performed by gel chromatography on a 2 6 x 9 3 0 mm sephadex g 200 column at +4"c in a buffer solution of ph 7.5 containing 0.1 m upsciln j m r d s o 82 (19). tris-hci, 0.2 m naci, 2 mm edta, 0.02% nan,. flow rate was 9 ml/h and 3 ml fractions were collected.' subjects. serum samples were obtained from the fol lowing subjects: 25 male and 33 female medical students 26-30 years old. blood donors were excluded as well a s pregnant women. thirty-seven male blood donors, ran domly selected, were investigated. two patients with ex cess iron due to repeated transfusions, one with pure red cell aplasia and one with anaemia of chronic renal failure, as well a s 4 patients with well established alcoholic liver cirrhosis were selected. three healthy young males were phlebotomized once weekly until they were anaemic and iron depleted, after which parenteral iron treatment was given in a dose not quite sufficient to restore the haemo globin level to the initial values. from this experiment samples were selected before phlebotomy, at the time of lowest haemoglobin level and after iron treatment. all serum samples were kept at -20°c until analysed. r e s u l t s using the conjugation method with iodinated ester, ferritin was labelled with '"1 to a specific activity of about 10 ci/g. t h e labelled ferritin was found to be sufficiently stable, when stored a t + 4 t , to be used in the assays for at least 4 months. t h e anti sera were tested for ferritin-binding capacity both after direct and indirect coupling. the three anti sera could b e used for 250 0001100 000 assays per ml with both variants of the coupling method. t h e esti mated ferritin concentration in serum was not sig nificantly different (p>0.05, paired t-test) when the three antisera were compared, testing serum samples from 10 different individuals. t h e sepha dex@ particles gave a slightly better precision than microcrystalline cellulose. t h e slope of the stand ard line was steeper in assays with indirectly than with directly coupled antibodies (mean values: 1.22 versus 0.97). t h e ferritin concentration that gave a 50% inhibition to the assay was 50 arb. u/i with the indirect and 110 arb. u/l with the direct coupling. one of the antisera raised in our laboratory and in directly coupled to sephadex@ particles was chosen for further experimental and clinical studies. quality control data were obtained from nine as says over a period of 4 months which included duplicate tests o n a total of 793 serum samples. t h e average percentage of labelled ferritin bound to b" w a s 10.1k1.5 (s.d.). t h e mean +_s.d. v a l u e s for the slope was 1.22k0.07 and for the 50% inhibition 50.2k5.8 arb. u/l. the intra-assay variation calcu lated as the average percentage standard error of the mean of duplicates giving an inhibition between 20 and 80% of total bound was 4.7. t h e i n t e r f i n t r a radioimmunoassay for ferritin in serum 17 e280 nm 2.0 1.5 1.c 0.5 ferritin arb u / i i0 5 , 1 fig. 2. ferritin concentra tion in fractions from a se phadex g-200 gel chromato graphy of a serum sample from a healthy young man. 30 lo 50 so m 80 90 ioo 110 120 assay variation calculated as the coefficient of var iation from nine repeated assays of control samples on three levels of ferritin concentration was: 8% for 19 arb. u/l, 6.5% for 73 arb. u/l and 4 . 4 % for 180 arb. u/l. the average detection limit (p=0.0.5) of the nine assays was 4.6 arb. u/i of serum. the dose-response curve for serial dilutions of the reference standard, diluted in 2.5 % serum, par alleled those for dilutions of sera with high ferritin content (fig. 1). a fresh serum sample from a healthy young male (80 arb. u of ferritin per litre) was chromatographed on a sephadex g 200 column and all fractions were assayed for ferritin content. the sensitivity was 1 ..5 arb. u/l. two immunoreac tive peaks eluted soon after the void volume were detected (fig. 2). this elution pattern for immuno reactive ferritin was almost identical with that ob tained for the radioactivity when '251-labelled ferri tin added to normal serum was gel chromato graphed on the same sephadex g-200 column. the levels of ferritin in serum in normal males and females and male blood donors is shown in fig. 3. the geometric mean for males was given the value of 100 arb. u/l (range expressed as 9.5 7% lim its: 40.3-246), which was significantly (p<o.001) higher than that for women, 66 arb. u/l (range 26 173). male blood donors had significantly (p<0.002) lower levels than other normal men; geometric mean 68.5 arb. u/l. the 4 patients with liver cirrhosis had values of 2 -771x54 i j o i i o is0 160 170 fraction number 261, 292, 983 and 2496 arb. u/l. the highest value recorded, 3640 arb.u/1, was in a patient with chronic renal failure and iron overload due to re peated transfusions and oral iron therapy. her bone marrow was stained for hemosiderin and showed a strongly positive reaction. one patient with pure red cell aplasia, who had had several blood trans fusions, rose from 433 arb.u/l to 1404 arb.u/l during a 3 month period of repeated transfusions. the ferritin levels in serum during induction of anaemia and after iron treatment of 3 healthy young men is shown in fig. 4. ferritin fell to a subnormal mean value when iron stores were depleted (no marrow hemosiderin could be detected). when iron ferritin i males femoles male blood donors f i g . 3. serum ferritin levels in normal males, females and male blood donors. (jp.su1u j med sci 82 18 l . wide and g. birgegdrd 140’ im 120. 110. 100. 90i f e d i n so 13 i& 15 16 19 w k s fig. 4. mean hb concentrations and serum ferritin levels in 3 healthy young men during phlebotomy and iron treat ment. was injected, ferritin levels rose, in one case almost 10-fold. the iron dose was chosen so that iron stores would again be depleted before the initial haemoglobin level was reached, as also occurred: there was no stainable iron in the bone marrow and serum ferritin levels fell again to low levels. discussion one prerequisite for achieving high sensitivity in a competitive inhibition radioimmunoassay technique for ferritin using labelled ferritin is the labelling of this protein to a sufficiently high specific activity. using the conjugation technique with ‘251-labelled ester, a specific activity of about 10 cilg was ob tained in the present study, which is about 1000 times higher than that obtained by the chloramine-t technique (12). the labelled ferritin had to be further purified by gel chromatography, as less than 30 % of the 1251-labelled protein was immunoreac tive. it seems likely that this was due mainly to heterogeneity of the ferritin preparation used for labelling, and, to a lesser degree, a result of the labelling procedure, since this method is expected to damage the protein very little (3). two variants were used for coupling of antibodies. the observa tion of a higher value for the slope and a lower ferritin concentration at 50 76 inhibition using the indirect vs. the direct coupling method is in agree ment with results from assay of gonadotropins (19). ilprtrirt .i mc.d y( 182 support for a high specificity of the ferritin assay was obtained from several experiments with the method. on gel chromatography of serum, the im munoreactivity was eluted in the same position as labelled ferritin. there was no difference in relative ferritin activity in serum when the three different antisera were compared in the assay. the relation between mean ferritin concentration in serum from healthy men and women was similar to that re ported by other investigators ( 1 , 4 , 8, 9, 16). male blood donors had significantly lower mean ferritin concentrations than other healthy males of the same age. the results of the clinical material and the variation in immunoreactive ferritin concentra tion in serum for the three men participating in the phlebotomy study with iron therapy give further evidence for the high specificity of the ferritin assay. several types of ferritin have been isolated from various tissues in man (6, 10) and some of them have been shown to cross-react immunologi cally (2, 13). however, it is not known to what degree they cross-react in our radioimmunoassay system. the values for precision and sensitivity of the method were acceptable for the application of the method for the assay of ferritin in serum clini cal studies. the method, which is technically simple to per form in a clinical chemical laboratory, was robust and suffered from only a small intraand inter-assay variation. there was no detectable change in the activity of control samples when different batches of labelled ferritin were compared. the labelled ferritin could be used for at least four months. all three antisera could be used for a large number of tests and with such high “titres” it is unlikely that this solid phase technique will be found wasteful of antibodies when compared with other radioim munoassay systems ( i 8). for several reasons, we have chosen to express the activity in relation to the mean activity obtained in healthy men, no blood donors, 20-30 years of age. there is no international standard preparation of ferritin. we had no evidence for the homogeneity of the ferritin preparation which was available. furthermore, stauffer & greenham (15) have re cently shown that variations in iron content of the ferritin molecule constitute a serious source of error i n estimations of ferritin protein by the method of lowry et al. expressing results in relation to a normal popula tion simplifies calibration of new reference standard rudioimmunoassuy f o r ferritin in serum 19 solutions in our own laboratory and facilitates com parisons of our results with those from other labora tories. in conclusion: a method is described for the as say of ferritin in serum, which is based o n the use of labelled ferritin in a solid phase system and which has been found to be a robust method suitable for clinical investigations. acknowledgements the study was supported by a grant from the swedish medical research council (637). we are indebted to dr tor olofsson, lund, sweden, for supply of the femtin preparation, and to mrs anna-lena barmark and mr christer bengtsson for valuable technical assistance. references i . 2. 3 . 4 5 . 6. 7. 8. 9. 10. 11. addison, g. m., beamish, m. r., hales, c. n., hodgkins, m., jacobs, a. & llewellin, p.: an im munoradiometric assay for ferritin in the serum of normal subjects and patients with iron deficiency and iron overload. j clin path 25: 326, 1972. alfrey, c. p., lynch, e. c. & whiteley, c. e.: characteristics of ferritin isolated from human mar row, spleen, liver and reticulocytes. j lab clin med 70:419, 1967. bolton, a. e . & hunter, w. m.: the labelling of pro teins to high specific radioactivities by conjugation to a lz5i-containing acylating agent. biochem journal 133: 529, 1973. cook, j. d., lipschitz, d. a , , laughton, ch., miles, e. m. & finch, c. a.: serum ferritin as a measure of iron stores in normal subjects. am j clin nutr 27: 68 i , 1974. crosby, w. h.: editorial new eng j med 294: 302, 1976. drysdale, j. w., arosio, p., adelman, t., hazard, j . t. & brooks, d.: isoferritins in normal and dis eased states. in proteins of iron storage and trans port in biochemistry and medicine (ed. r. chrighton), p . 359. north-holland publishing company, amster dam, 1975. drysdale, j. w. & munro, h. n.: small-scale isola tion of ferritin for the assay of the incorporation of ''c-labelled amino acids. biochem j 95: 851, 1965. halliday, j. w., gera, k . l . & powell, l. w.: solid phase radioimmunoassay for serum ferritin. clin chim acta58: 207, 1975. leyland, m. j . , ganguli, p. c., blower, d. & dela more, i. n.: immunoradiometric assay for ferritin in human serum. scand j haematol14: 385, 1975. linder, m. c. & munro, h. n.: metabolic and chemi cal features of ferritins, a series of iron-inducible tis sue proteins. am j path 72: 263, 1973. miles, l. e. m., lipschitz, d. a. & bieber, c. p.: measurement of serum ferritin by a 2-site immuno radiometric assay. anal biochem61: 209, 1974. 12. porter, f. s.: the effect of iron content on the be havior of human ferritin in an inhibition-type radio immunoassay. j lab clin med 83: 147, 1976. 13. powell, l . , albert, e., isselbacher, k. j. & drysdale, j. w.: human isoferritins: organ specific iron and apoferritin distribution. brit j haernat 30: 47, 1975. 14. siimes, m. a., addiego, j. e. & dallman, p. r.: ferritin in serum: diagnosis of iron deficiency and iron overload in infants and children. blood43: 581, 1974. 15. stauffer, c. e. & greenham, c. c.: sources of error in measuring ferritin. clin chem22: 1755, 1976. 16. walters, g. o., jacobs, a., worwood, m., trevett, d. & thomson, w.: iron absorption in normal sub jects and patients with idiopathic haemochromatosis: relationship with serum femtin concentration. gut 16: 188, 1975. 17. wide, l.: radioimmunoassays employing immuno sorbents. acta endocrinologica, suppl. 142: 207, 1969. 18. wide, l.: radioimmunosorbent assay of steroids. discussion in: steroid immunoassay (ed. e. h. d. cameron et al.), pp. 267-269. alpha omega h b l . ltd., cardiff, 1975. 19. wide, l . , nillius, s. j., gemzell, c. & roos, p.: radioimmunosorbent assay of follicle-stimulating hormone and luteinizing hormone in serum and urine from men and women. acta endocrinologica, suppl. 174: i , 1973. received january 24,1977 address for reprints: leif wide, m.d. departments of clinical chemistry university hospital s-750 14 uppsala upsflln j m e d sci 82 plenary lectures 11 1 the basal lamina i n epithelial-mesenchymal morphogenetic interactions, m. b e r n f i e l d ( s t a n f o r d , c a l i f . , u s a ) . the g e n e r a t i o n of s t r u c t u r a l form of s e v e r a l embryonic e p i t h e l i a l o r g a n s , s u c h as t h e l u n g , k i d n e y , mammary and s a l i v a r y g l a n d s , o c c u r s by a common s e q u e n c e . t h e l i a l c e l l s , and d u r i n g d e v e l o p m e n t , t h e bud u n d e r g o e s a d i s t i n c t i v e s e q u e n c e of f o l d i n g and b r a n c h i n g which r e s u l t s i n a morphology t h a t i s c h a r a c t e r i s t i c of t h e o r g a n . l u l a r l o o s e c o n n e c t i v e t i s s u e , o r mesenchyme, which i s a b s o l u t e l y r e q u i r e d f o r t h e morphogenesis. m a t e r i a l s , f i b r i l l a r c o l l a g e n and a w e l l d e f i n e d b a s a l l a m i n a . each o r g a n arises a s a rounded bud c o n s i s t i n g of a l a y e r of e p i the e p i t h e l i a a r e s u r r o u n d e d by a h i g h l y c e l between t h e e p i t h e l i u m and mesenchyme l i e amorphous with mouse embryonic s a l i v a r y g l a n d s , t h e mesenchyme, amorphous m a t e r i a l s a n d f i b r i l l a r c o l l a g e n c a n b e removed by m i c r o d i s s e c t i o n i n c o l l a g e n a s e , y i e l d i n g e p i t h e l i a which r e t a i n t h e b a s a l l a m i n a . such e p i t h e l i a m a i n t a i n normal m u l t i l o b u l a r morphology, b u t r e q u i r e r e c o m b i n a t i o n w i t h mesenchyme f o r c o n t i n u e d b r a n c h i n g m o r p h o g e n e s i s . by b r i e f t r e a t m e n t w i t h nanogram amounts of t e s t i c u l a r h y a l u r o n i d a s e . t r e a t m e n t c a u s e s t h e c e l l s t o round up and r e s u l t s i n d i s r u p t i o n of c e l l u l a r a d h e s i o n s , d i s o r g a n i z a t i o n of c y t o s k e l e t a l s t r u c t u r e s a n d , when t h e e p i t h e l i u m is recombined w i t h mesenchyme, t h e l o s s of m u l t i l o b u l a r morphology. these o b s e r v a t i o n s s u g g e s t t h a t t h e b a s a l l a m i n a i s r e q u i r e d f o r m a i n t e n a n c e of normal morphology. however, d e l a y i n g r e c o m b i n a t i o n w i t h mesenchyme f o r 2 h r s , d u r i n g which t h e e p i t h e l i u m r e p l a c e s t h e l a m i n a , r e v e r s e s t h e a l t e r a t i o n s and p r e v e n t s t h e l o s s of morphology. thus t h e mesenchyme may h a v e a p r o p e r t y which i s d e l e t e r i o u s t o e p i t h e l i a l r e c o v e r y from h y a l u r o n i d a s e , b u t i s n o r m a l l y i n v o l v e d i n morphogenesis. the l a m i n a c a n b e c o m p l e t e l y removed such the l a m i n a c o n t a i n s g l y c o s a m i n o g l y c a n (gag) and l a b e l i n g s t u d i e s show t h a t c a . h a l f of t h e l a b e l e d gag i s h y a l u r o n i c a c i d , c h o n d r o i t i n 4 i s t w i c e c h o n d r o i t i n & s u l f a t e and c h o n d r o i t i n i s i n t r a c e amounts. u l t r a s t r u c t u r a l s t u d i e s u s i n g t a n n i c a c i d f i x a t i o n and r u t h e n i u m r e d s t a i n i n g reveal t h e l a m i n a t o b e composed of components i n o r d e r e d p e r i o d i c a r r a y s i n t i m a t e l y a s s o c i a t e d w i t h t h e plasmalemma. the b a s a l l a m i n a may c o n s i s t , i n p a r t , of s u p r a m o l e c u l a r complexes of h y a l u r o n i c a c i d and p r o t e o g l y c a n which a r e upsala j med sci 82 112 plenary lectures organized into an extracellular scaffolding that imposes structural form on the epithelium. in intact glands, the gag in the basal lamina is rapidly turning over. laminar gag is lost more rapidly at the distal ends of the lobules, the sites of greatest cell proliferation and change in cell shape, and less rapidly within the interlobular clefts. this differential rate of loss results in more gag accumulating within the clefts and less on the lobules. the pattern of gag metabolism is not secondary to lobular growth, nor is it altered by inhibition of cell proliferation or by inhibitors of collagen secretion or cross-linking. a similar pattern is seen during the branching morphogenesis of several embryonic epithelia, consistently reflecting the morphologic changes characteristic of each organ, but is not observed on unbranched epithelia. thus, differential laminar gag turnover is associated with branching morphogenesis. the pattern of laminar gag metabolism occurs only in the presence of mesen chyme. the mesenchyme is not involved in the synthesis, deposition or organization of the lamina, but culture in combination with mesenchyme causes a loss of gag from the epithelium. the mesenchyme possesses activity which removes laminar gag, producing a heterogeneous mixture of small molecular weight components. the activity requires living mesenchymal cells and is not duplicated by medium conditioned by mesenchyme. mesenchyme, therefore, is responsible for the degradation involved in the differential turnover of laminar gag. the since embryonic salivary epithelia require a gag-rich basal lamina to maintain morphology, and require mesenchyme for continued branching morpho genesis, it is likely that the effect of the mesenchyme on gag within the lamina is involved in the changes in epithelial morphology which occur during development. upsulu j med sci 82 upsala j med sci 79: 90-93, 1974 parathyroid hormone and gastric mucosal surface ultrastructure b. frenning, h. johansson and p. g . ohrn from the departments of medicine, surgery and clinical research ii, university hospital, u p p s a l a , sweden abstract examination in the sem of gastric mucosa from rats given parathyroid hormone appeared to reveal an in creased incidence of unspecific damage on the luminal surfaces of the gastric mucosal surface epithelial cells. the finding may he related to the suggested ulcer ogenic effect of the hormone. introduction a relationship between hyperfunction parathyroid glands and gastrointestinal of the disturb ances and gastric ulcers is often suggested both in man and in experimental animals (8, 9, 10). there appears t o be no difference between man and the rat in this respect, though the basal gastric secretion is increased in at least some of the patients with hyperparathyroidism (3, 4, 15, cf. 2 ) , whereas it is decreased by treatment with parathyroid hormone (pth) i n the rat (1, 13). in a previous investigation (13) an increased serum calcium level, a reduced secretion rate and reduced acidity in the basal gastric secretion were noted in rats treated with pth for 14 days. this result might have been due to an inhibitory effect of this hormone on the gastric secretion, but an increased permeability of the gastric mucosa caus ing an increased back-diffusion of hydrogen ions would have given the same result (5, 1 1 , 12, 14). to examine the effects of pth further gastric mucosa from rats treated with the hormone and from untreated controls were examined in the scanning electron microscope (sem). a prelimin ary report of the findings is given in this paper. material and methods the experiments were performed on male albino rats weighing about 200 g. five rats were given parathyroid hormone (para-thor-mone, ely, lilly) s.c. twice a day for 14 days in a dose of 40 usp units/100 g body weight/ day. three control animals were given physiological saline s.c. twice a day. under general anaesthesia 2.5 55 glutaraldehyde in phosphate buffer was instilled intragastrically via a rub ber tube. five minutes later the stomachs were removed and specimens from the glandular portions were taken in duplicate. after the initial glutaraldehyde fixation post fixation was performed in 1% osmium tetroxide. the specimens were dehydrated in a graded acetone series. after drying they were mounted on brass stubs with an adhesive and electrical continuity was ensured by paint ing with colloidal silver. they were covered by cold palladium in a vacuum-evaporator (jeol, j e e 48) and exa mined in a jeol sem (jsm-u3). for details regarding the preparation procedure see (5) or (7). results the appearance of gastric mucosa from a rat given pth is shown in fig. 1 . the surfaces of quite a few cells were irregular and appeared unspecific ally damaged. no changes were observed along the intercellular borders. fig. 2 shows a similar area from an untreated control animal. in figs. 3 and 4 damaged and normal surface epithelial cells are seen at a higher magnification. in both these micrographs some microvilli are observed. most of these structures appeared t o be covered by mucus, however. it must be pointed out that signs of unspecific damage similar to those shown in figs, 1 and 3 were occasionally seen on the upsala j m e d sci 79 parathyroid hormone and gastric mucosa 91 f i g . i . micrograph of gastric mucosa from a rat treated with parathyroid hormone for 14 days. the openings of control specimens. the changes observed on specimens from pth treated rats seemed to be more pronounced, however, and the damaged cells more numerous. discussion the type of damage noted on gastric mucosa from pth treated rats was not observed on cat gastric mucosa exposed to aspirin, acetic acid or hyperos motic sodium chloride solutions (5, 6, 7). sub sequent to exposure of gastric mucosa to these weak acids (7) or to large osmolaiity variations (6) the appearance of the gastric mucosal surface epithelium was also changed from normal. in these cases the changes were of different natures, how ever. as to the origin of the observed changes it is only possible t o speculate. one suggestion is that pth treatment may make the cell surfaces more susceptible for peptic digestion. the changes appear to be sufficient to explain the decreased gastric secretion observed after pth treatment of ~ three crypts are seen. some cells appear unspecifically damaged. ( ~ 3 0 0 0 . ) rats as an effect of an increased back-diffusion of hydrogen ions (5, 11, 12, 14). they may be related to the suggested ulcerogenic effect of the hor mone. acknowledgements we are very grateful to mrs margareta einarsson and mr b. wieselblad for technical assistance. the work was supported by the swedish medical research council, project no. 17x-3498. references arnthorsson, g . , o h m , p. g . & segerstrom, a.: gastric secretion in relation t o parathyroid dysfunc tion in rats with chronic gastric fistulae. upsala j med sci 78:55-59, 1973. barreras, r. f . : calcium and gastric secretion. gastroenterology64: 1168-1 184, 1973. barreras, r . f. & donaldson, r . m., jr: role of calcium in gastric hypersecretion, parathyroid ade noma and peptic ulcer. n engl j med 276: 1122 1124, 1967. christiansen, j . & aagaard, p.: parathyroid adenoma and gastric acid secretion. scand j gastroent 7:445 449, 1972. upsala j med sci 79 92 b . frenning et al. f i g . 2 . micrograph of gastric mucosa from a control animal. here also the openings of three crypts are ob served. ( ~ 3 0 0 0 . ) f i g . 3. damaged surface epithelial cells on gastric mucosa from a pth treated rat. along the intercellular borders some microvilli are seen. ( ~ 6 0 0 0 . ) upsala j med s c i 79 parathyroid hormone and gastric mucosa 93 f i g . 4. normal surface epithelial cells. some microvilli can be observed in this micrograph also. most of these 5. 6 7. 8. 9. 10. 11. 12. 13. frenning, b.: ion transport and surface ultrastructure in the non-secreting stomach. upsala j med sci, suppl. 13, 1972. the effects of large osmolality variations on the gastric mucosal surface ultrastructure. scand j gastroent 8: 185-192, 1973. frenning, b. & obrink, k. j.: the effects of acetic and acetylsalicylic acids on the appearance of the gastric mucosal surface epithelium in the scanning electron microscope. scand j gastroent 6: 605-612, 1971. hellstrom, j.: hyperparathyroidism and gastroduo denal ulcer. acta chir scand 116:207-221, 1959. johansson, h . , thoren, l. &werner, i . : hyperpara thyroidism. clinical experiences from 208 cases. upsal j med sci 77:41-46, 1972. kelly, t. r. : relationship of hyperparathyroidism to peptic ulcer. arch surg (chicago) 101: 193-199, 1970. nordgren, b.: the rate of secretion and electrolyte content of normal gastric juice. acta physiol scand, suppl. 202, 1963. obrink, k. j.: studies on the kinetics of the parietal secretion of the stomach. acta physiol scand, suppl. 51, 1948. ohm, p. g., frenning, b., hessman, y . , johansson, h . & segerstrom, a,: influence of parathyroid hor mone and vitamin d on basal gastric secretion in rats. scand j gastroent (in press). structures appear to be covered by mucus, however. ( x6000.) 14. teorell, t.: electrolyte diffusion in relation to the acidity regulation of the gastric juice. gastroentero logy 9: 425-443, 1947. 15. ward, j. t . , adesola, a. 0. & welbourn, r. b.: the parathyroids, calcium and gastric secretion in man and the dog. gut 5: 173-183, 1964. received october 28, 1973 address for reprints: bertil frenning, m.d. department of medicine university hospital s-750 14 uppsala sweden upsala j med sci 79 upsala j med sci 87: 175-177, 1982 peripheral embolization of separated angiographic catheter b. almgren’ and a. hemmingsson’ departments of surgery’ and diagnostic radiology‘, university hospiial, uppscrlcr, swcjjen abstract a c o m p l i c a t i o n o f s e p a r a t i o n o f an a n g i o g r a p h i c c a t h e t e r i s described. con t r i b u t i n g f a c t o r s and p r e v e n t i v e measures a r e discussed. introduction c a t h e t e r s e p a r a t i o n i s an e x t r e m e l y r a r e compl c a t i o n o f angiography. an i n c i d e n c e o f 0.0008 p e r c e n t has been r e p o r t e d ( 3 ) , b u t a l s o a h i g h e r o f 2 p e r c e n t , when angiography has been performed t h r o u g h s y n t h e t i c v a s c u l a r g r a f t s ( 2 ) . i n a l l p r e v i o u s l y r e p o r t e d cases t h e broken end o f t h e c a t h e t e r has r e m a i n e d , a t t a c h e d t o t h e v e s s e l o r g r a f t w a l l a t t h e c a t h e t e r e n t r y s i t e and i n no case p e r i p h e r a l e m b o l i z a t i o n has occurred. we h e r e r e p o r t a case u n i q u e i n t h a t t h e separated fragment embolized t o t h e p e r o n e a l a r t e r y v i a bypass g r a f t . case report a 35-year o l d man had undergone a f e m o r o t i b i a l bypass w i t h autogenous sa phenous v e i n g r a f t o f t h e r i g h t l e g because o f a r t e r i o s c l e r o s i s 21 months be f o r e t h i s admission. angiography was now requested because o f r e c u r r e n t r e s t p a i n and u l c e r a t i o n o f t h e g r e a t toe. angiography was performed u s i n g a p o l y e t h e n e c a t h e t e r , i.d. 1.58 mm, 0.d. 2.08 mm ( r a d i c a t h ) w i t h 4 s i d e h o l e s a f t e r p u n c t u r e o f t h e r i g h t common femoral a r t e r y . no d i f f i c u l t i e s were encountered d u r i n g t h e i n s e r t i o n o r t h e study. d u r i n g w i t h d r a w a l f o l l o w i n g c o m p l e t i o n o f a r t e r i o g r a p h y t h e c a t h e t e r became f i x e d a t t h e p u n c t u r e s i t e and r e s i s t e d f u r t h e r removal. a g u i d e w i r e was r e i n s e r t e d t o r e l e a s e t h e f i x a t i o n and t h e c a t h e t e r was g e n t l y p u l l e d . d u r i n g t h i s m a n i p u l a t i o n t h e c a t h e t e r separated a t t h e l e v e l o f t h e f i r s t s i d e h o l e , l e a v i n g a p p r o x i m a t e l y 2 cm o f t h e c a t h e t e r i n t h e common femoral a r t e r y . this was c o n f i r m e d by f l u o r o s c o p y . the fragment was t h e n c a r r i e d by t h e b l o o d stream a l o n g t h e bypass g r a f t down i n t o t h e p e r o n e a l a r t e r y , which was t h e o n l y p a t e n t a r t e r y o f t h e l o w e r l e g . a r a d i o g r a p h demonstrated t h e fragment i n t h e m i d d l e p a r t o f t h e a r t e r y j u s t p r o x i m a l t o an o c c l u s i o n ( f i g 1 ) . the a r t e r y was s u r g i c r i75 a l l y e x p l o r e d and t h e c a t h e t e r t i p removed. the p a t i e n t r e c o v e r e d w i t h o u t com p l i c a t i o n . discussion our case demonstrates t h a t p e r i p h e r a l e m b o l i z a t i o n o f a c a t h e t e r fragment i s a p o s s i b l e c o m p l i c a t i o n o f c a t h e t e r s e p a r a t i o n d u r i n g angiography. s i n c e em b o l i z a t i o n d i d n o t occur i n any o f t h e s i x d e s c r i b e d cases o f c a t h e t e r separa t i o n (1,2,4,5,6), some p r e v e n t i v e f a c t o r s must e x i s t . i n t h o s e cases t h e broken segments were 5 cm up t o 28 cm i n l e n g t h , compared t o 2 cm i n our case, and r e mained f i x e d a t t h e c a t h e t e r e n t r y s i t e , f i g . 1. p e r o n e a l a r t e r y j u s t p r o x i m a l t o an o c c l u s i o n ( b ) . a r t e r i o g r a p h y o f r i g h t l o w e r l e g ( a ) . separated c a t h e t e r t i p i n t h e i76 s u r g i c a l removal o f t h e separated fragment was necessary i n our case, as i n a l l o t h e r cases, b u t one ( 4 ) , which i n d i c a t e s t h a t c a t h e t e r s e p a r a t i o n i s a r a t h e r s e r i o u s c o m p l i c a t i o n o f angiography. we w i l l n o t r e p e a t t h e adequate d i s c u s s i o n s o f t h e p r e v i o u s r e p o r t s o f ca t h e t e r s e p a r a t i o n , b u t o n l y p o i n t o u t some s a l i e n t f e a t u r e s , common t o a l l cases. c a t h e t e r s e p a r a t i o n has o c c u r r e d e x c l u s i v e l y d u r i n g w i t h d r a w a l , b o t h f r o m s y n t h e t i c v a s c u l a r g r a f t s and t h e p a t i e n t ' s n a t i v e v e s s e l . regardless o f g r a f t o r v e s s e l , i n d u r a t i o n and f i b r o s i s have e x i s t e d a t t h e c a t h e t e r e n t r y s i t e s . i n most cases p o l y e t h y l e n e c a t h e t e r s , which a r e s o f t , p l i a b l e and e a s i l y s t r e t c h e d , have been used. the r i s k o f f i x a t i o n t o a rough edge i s g r e a t e r w i t h a c a t h e t e r w i t h s i d e h o l e s t h a n w i t h an endhole. the awareness o f these p o t e n t i a l l y p r o v o k i n g , b u t n o t i n e v i t a b l e f a c t o r s i n a d d i t i o n t o recommended p r e c a u t i o n s d u r i n g c a t h e t e r w i t h d r a w a l s h o u l d m i n i m i z e t h e r i s k o f c a t h e t e r s e p a r a t i o n and consequently p e r i p h e r a l e m b o l i z a t i o n o f broken fragments. 1. 2. 3. 4. 5. 6. references feigenbaum, l. & grollman, j.h.: c a t h e t e r s e p a r a t i o n i n a o r t o f e m o r a l g r a f t s d e s p i t e w i r e p r o t e c t i o n . am j roentgenol 134:581-582, 1980. mani, r.l. & c o s t i n , b.s.: c a t h e t e r angiography t h r o u g h a o r t o f e m o r a l g r a f t s : p r e v e n t i o n o f c a t h e t e r s e p a r a t i o n d u r i n g withdrawal. am j roentgenol 128: ravin, c.e. & koehler, p.r.: reuse o f d i s p o s a b l e c a t h e t e r s and g u i d e w i r e s . radiology 122:577-579, 1977. soo, c.s., chuang, v.p. & wallace, s.: n o n s u r g i c a l r e t r i e v a l o f a severed c a t h e t e r from femoral a r t e r y u s i n g a m y l a r sheath. am j roentgenol 135: weinshelbaum, a . & carson, s.n.: s e p a r a t i o n o f a n q i o g r a p h i c c a t h e t e r d u r i n g a r t e r i o g r a p h y t h r o u g h v a s c u l a r g r a f t . am j roentgenol 134:583-584, 1980: zablow, b.c., gordon, d.h. & m a r t i n , e.c.: s e p a r a t i o n t o a femoral a r t e r y c a t h e t e r d u r i n g withdrawal. radioloqy 133:244-245, 1979, 328-329, 1977. 400-401, 1980, received y e b r u a r y 20, 1 yu2 address f o r r e p r i n t s : bo almgren, m.d. department o f surgery u n i v e r s i t y h o s p i t a l s-750 14 uppsala sweden 177 upsala j med sci 84: 275-280, 1979 clinical and surgical staging of hodgkin's disease gunnar gustafsson,' henry johansson,' bjorn molin,' christer sundstrom3 and anne-marie thelin2 from the departments of surgery', oncologyz and pathology3, university hospital, u p p s a l a , sweden abstract staging laparotomy was performed in 36 patients with hodgkin's disease. the surgical procedure changed the stage in 1 4 cases or 36 % and also revealed sub diaphragmatic disease in 1 4 patients. mixed cellularity and nodular sclerosis were the main histopathological types, contributing 4 4 % and 4 2 % , respectively, of the whole material. postoperative complications occurred in 8 cases (22 % ) none was fatal. the average hospital stay was 9 . 5 days, the longest being 22 days. introduction since thomas hodgkin in 1832 described the disease that now bears his name ( 7 ) , the condition has had a very dismal prognosis for decades. there was little or no improvement in the treatment of the disease until the development of high energy irradiation, which allowed the delivery of tumoricidal doses to restrict ed areas and to large volumes of the tumor. in addition there was progress in chemotherapy. soon, however, it was apparent that a high proportion of the treated patients had prior undetected abdominal lesions. in order to detect and further treat occult intra-abdominal involvement the staging operation was grad ually developed, starting with kaplan's group (9). at the university hospital of uppsala, the first staging operation was per formed in june 1 9 7 2 and now, six years later, we want to present our experience from 36 patients with hodgkin's disease, who have undergone such an operation. material and methods twenty-two males and fourteen females were included in the series. mean age for onset of the disease: males 3 5 . 1 years (range 21 7 0 years) females 26.3 " ( " 1 4 4 1 'i ) seventeen of the men and all the women (thirty-one patients) were younger than 50 years. 19-792856 275 the diagnosis of hodgkin's disease was made from lymph node biopsies. the histological subtyping was made according to the rye classification (12). ' the patients were preoperatively examined and clinically staged by means of non-operative methods, i.e. physical examination, routine hematological analy ses, which were performed by an automatic cell counter (coulter counter s ) , serum electrophoresis, liver function tests such as bilirubin ( 1 4 ) , transphera ses and alkaline phosphatase ( 1 5 ) , chest x-ray, bone marrow biopsy, lymphangio graphy, scintigraphy of the liver and spleen using ''tern tin or sulphur colloid. all the patients underwent staging laparotomy. the aim of that operation was to examine whether the disease had any manifestations below the diaphragm. the operative procedure included splenectomy (also removal of any existing additional spleen), wedge biopsy of the liver, and lymph node sampling in the areas recommended by the stanford group ( 4 , 5 , 6 ) , i.e. the splenic hilar region and the coeliac region but also along the aorta and the major vessels or any obviously enlarged node. the lymph node sampling sites and the splenic pedicle were marked with silver clips. to afford protection from radiation the ovaries were moved to the anterior superior aspect of the uterus. the position of the ovaries after transfer was marked with silver clips. results symptoms at onset are shown in table 1 . the most common initial symptom was enlarged cervical lymph nodes or palpable nodes in the supraclavicular region, altogether in 7 0 %. one patient showed a palpable mass in the axilla, one had a tumor in the biceps region of his left upper extremity (6 % ) . other symptoms (including fever, tiredness, coughing and an increased blood sedimentation rate) were noted in 6 patients ( 1 6 % ) . "b-symptoms", like night sweating, itching or weight loss was at onset present in 3 cases (8 % ) . table 1. symptoms at onset in hodgkin's disease symptoms number % enlarged cervical or supraclavicular lymph nodes right side 16 left " 8 bilateral 1 70 palpable mass in the axilla or in the biceps region of the arm 2 6 other symptoms 6 1 6 "b-symptoms" 3 8 276 besides palpation, pulmonary x-ray was the most valuable clinical staging tool, often exposing mediastinal masses. the results given as positive findings of the different investigations are shown in table 2. table 2. patients with positive results in clinical staging of hodgkin’s disease method of examination number of positive findings investigated patients number % pulmonary x-ray 36 1 6 44 scintigraphy of the liver and spleen 3 3 2 6 liver function tests 36 1 5 42 lymphang iog raphy 36 9 25 bone marrow smears and histological examination of aspirated particles showed in 8 cases slight, non-specific reactions. no signs of hodgkin’s disease were found in the examined bone marrow specimens. scintigraphy of the liver showed i n two cases such changes that hodgkin’s disease of the liver was suspected. none of them had, however, histological signs of the disease in their liver biopsies at laparotomy. pathological liver function tests were observed in 15 patients and almost exclusively involved the transpherases without any pathological change of the alkaline phosphatase values. usually there was only a slight rise in the values of one or the other of the transpherases, but in none of these cases was the liver biopsy positive. one patient with histologically verified liver involvement also had pathological values of alkaline phosphatase and transpherases simultaneously. lymphangiography was performed in 30 cases. i n another 3 patients such an attempt technically failed. 9 lymphangiographies were considered positive, whereas 21 were negative. among the positive cases 5 showed histological signs of hodgkin’s disease in the lymph nodes. i n 4 cases histological examination of the collected lymph nodes could not confirm the lymphangiographic finding. i n 2 cases there were positive paraaortic lymph node biopsies in spite of negative lymphangiographies, and in another 5 cases positive lymph nodes were collected from the splenic hilar region. changes in the clinical staging as a result of the operative findings are illustrated in table 3. a tendency to a further concentration of the patients in stage i1 and i11 was seen following the operative procedure, although the chang ings were not statistically significant. 14 patients (39 % ) were registered in a different stage after laparotomy than initially, 8 of them in a more advanced group. 277 table 3 . distribution of different stages before and after surgical staging clinical stage before laparotomy after laparotomy i 10 7 i1 14 15 i11 10 12 iv 2 2 the histopathologic type (table 4) was distributed among mixed cellularity (mc) 16 cases (44 % ) , nodular dominance (lp) 5 ( 1 4 % ) . none cytic depletion (ld). further types in 7 cases ( 1 9 % ) which 4 cases, mc and lp in 2 cases sclerosis (ns) 1 5 (42 % ) and lymphocytic pre of the cases was initially classified as lympho control of the material has revealed mixing of means that there was both the mc and ns type in and mc and ld in 1 case. table 4. distribution of different types in hodgkin's disease % histopathologic type number lymphocytic predominance 5 14 nodular sclerosis mixed cellularity 1 5 1 6 42 44 lymphocytic depletion postoperative complications occurred in 8 patients (22 % ) . the complications are outlined in table 5. no postoperative death has occurred. table 5. postoperative complications after staging laparotomy in cases of hodgkin's disease complication number of patients pneumonia 3 fever without signs of infection subcutaneous wound rupture 2 1 pancreatitis i abdominal pain without signs of ileus, pancreatitis or peritonitis 1 8 the postsurgical stay averaged 9.5 days. the shortest stay was 5 days and the longest 2 2 days. 278 discussion the spleen often seems to be involved in hodgkin’s disease. splenectomy has also been stressed as being important in assessing the extent of the disease ( 1 , 4 , 5 ) . in our study involvement of the spleen was demonstrated in 1 / 3 of the patients but in nearly all of these cases there were simultaneously histological signs of perihilar lymph node engagement. positive lymph nodes were found in various localizations most often in the vicinity of the spleen or the splenic vessels but also along the aorta. splenic involvement seems to increase the risk of liver engagement ( 3 ) . ~n our material only two patients showed histopathologic signs of hodgkin’s dis ease in the liver in spite of nearly 50 % demonstrated positive liver function tests. often an isolated transpherase value was abnormal. one of the patients with liver engagement had multiple abnormal values including that of alkaline phosphatase. this is in agreement with the assumption that multiple abnormal test reactions in the liver battery are associated with an increased likelihood of liver involvement in hodgkin’s disease ( 7 ) . a positive liver scintiyraphy may be an indication in the same direction. in 2 cases there were, however, suspected changes of the liver scintigraphy, but none of these patients had positive liver biopsies. unfortunately, the two pa tients with liver involvement were not examined in that way preoperatively. lymphangiography showed both false positive and false negative findings. in 4 cases the pathologist could find only benign changes in spite of positive lymphangiography and apparently representative biopsies. in 2 cases there were positive findings histologically in the paraaortic lymph nodes without any indi cations preoperatively of such a spreading of the disease. another 5 cases showed a positive histology in spite of negative lymphangiography, but these nodes were all collected from the splenic hilar region. however, it must be stressed that the radiographs should be available at the operating theatre. an immediate examination by the pathologist of collected specimens is further recommended in cases where there are doubts about the lymphangiographic find ings vis-a-vis the macroscopic findings during the laparotomy. histologically mixed cellularity constituted the major type and together with nodular sclerosis represented more than 8 5 % of the whole material. other find ings of interest by the pathologist were an abundance of eosinophilic cells in the spleen in many cases, although there was no proof of hodgkin’s disease. if this is of any importance can perhaps be revealed by future controls. the role of splenectomy in the treatment of patients with hodgkin’s disease is controversial. whether it adds to the survival as a whole is still unknown. improved tolerance to subsequent cytostatic therapy may be achieved by splen ectomy at least in patients with advanced disease. conflicting data exist whether or not there is a definite risk of postsplenectomy infections in 279 individuals with hodgkin‘s disease (2,6). in our series a 30 years old man de veloped a septicemia 4 years after splenectomy. although intensive treatment including antibiotics and steroids was given his severe infection had a fatal outcome. this case of splenectomy undoubtly points to the risk but the possi bilities of positive findings in the spleen strongly advocate splenectomy as a part in staging procedure of hodgkin‘s disease. 1. 2. 3. 4. 5. 6 . 7. 8 . 9. 10. 11. 12. 13. 14. 15. references cannon, w.b. & nelsen, t.s.: staging of hodqkin‘s disease. a surgical per spective. am j surg 132:224-230, 1976. cooper, a., ironside, p.n.j., madigan, j . p . , morris, p.j. & ewing, m.r.: the role of splenectomy in the management of advanced hodgkin’s disease. cancer 34:408-417, 1974. chilcote, r.r., baehner, r.l. & hammond, d.: septicemia and meningitis in children splenectomized for hodqkin’s disease. new engl j med 295:798-800, 1976. enright, l.p., trueblood, w.w. & nelsen, t.s.: the surgical diagnosis of abdominal hodgkin’s disease. surg gynecol obstet 130:853-858, 1970. glatstein, e., guernsey, j.m., rosenberg, s.a. & kaplan, h.l.: the value of laparotomy and splenectomy in the staging of hodgkin’s disease. cancer glatstein, e., trueblood, h.w., enright, l.p., rosenberg, s.a. & kaplan, h.s.: surgical staging of abdominal involvement in unselected patients with hodqkin’s disease. radiology 97:425-432, 1970. hodgkin, t.: on some morbid appearances of the absorbent glands and spleen. medico-chirurgical transactions 17:68-114, 1832. irving, m.: the role of surgery in the management of hodgkin’s disease. br j surg 62:853-862, 1975. kaplan, h.s., dorfman, f.f., nelsen, t.s. & rosenberg, s.a.: staging laparo tomy and splenectomy in hodgkin‘s disease: analysis of indications and patterns of involvement in 285 consecutive, unselected patients. nci monogr 36:291-301, 1973. keinert, k., kober, b. et al.: die splenektomie bei der lymphogranulamatose in indikation und ergebnisse. arch geschwulstforsch 46:311-315, 1976. kolygin, b.a. & fedoreev, g.a.: exploratory laparotomy and splenectomy in the diagnosis of hodgkin’s disease in children. j surg oncol 8:345-349,1976. lukes, r.j., craver, l.f., hall, th.c., rappaport, h. & ruben, f.: report of the nomenclature committee. cancer res 26:1311, 1966. meeker jr., w.r., richardson, j.d., west, w.o. & parker, j.c.: critical evaluation of laparotomy and splenectomy in hodgkin‘s disease. arch surg 105:222-229, 1972. michaelson, m.: evaluation of a method for determination of bilirubin in 24:709-718, 1969. serum using direct spectrophotometry. scand j clin lab invest 30:387-390, 1972. the scandinavian society for clinical chemistry and clinical physiology: recommended methods for the determination of four enzymes in blood. scand j clin lab invest 33:291-306, 1974. received april 20, 1979 address for reprints: gunnar gustafsson, m.d. department of surgery university hospital s-750 14 uppsala, sweden 280 upsala j med sci 98: 335-338, 1993 6.1.2.2 quality specifications for haemoglobin a l c assays in the monitoring of diabetes mogens lytken larsen,* per hyltoft petersen* and callum g . fraser** *department of clinical chemistry, odense university hospital, dk-5000 odense c, denmark, **department of biochemical medicine, ninewells hospital and medical school, dundee d d l 9sy, scotland analytical goals for the performance characteristics of assays of haemoglobin a l c (hba,,) have been investigated based on within-subject biological variation and on the clinical significance of a certain change in hba,, concentrations in the individual during monitoring of metabolic control. the derived goals are highly dependent on the assumptions made but, when several goals are to be considered, the most demanding should be used. clinical situation measurements hba,, are used to monitor glycemic control in diabetes mellitus (1). a strong and curvilinear relation between hba,, and mean blood glucose concentration over the preceding 6-8 weeks has been demonstrated (2). moreover, regular measurements of hba,, has been shown to lead to changes in diabetes treatment and improvement of metabolic control, indicated by a lowering of hba,, values (3). the objectives of monitoring glycated haemoglobin are to reach near-normal and steady concentrations. consequently, clinical strategies have to be analyzed to generate goals for analytical quality. characteristics of the method the present evaluation of quality specifications has been based on both clinical goals setting (4), biological variation data ( 5 , 6) and theoretical goal setting (7). data on biological variation are obtained from studies using an isoelectric focusing method for measurements of hba,, (4, 5 ) . 335 models for evaluation of quality specifications to define the clinical goals for analytical quality, it was important to understand the relationship between quality of performed analyses and medical needs (4). clinicians have proposed that at a change of 1% hba,, in an individual (e.g. from 9.5% to 10.5%) should be considered a significant change in mstabolic control and cause clinical attention. a change of 2% hba,, should make the physician intervene actively (5, 8, 9). concerning the significance of a change in hba,, in the individual, the within-subject biological variation (cv,,) is determining and analytical imprecision become most demanding. the two concepts that provide the basis for the analytical goal-setting for tests used in monitoring such changes are: a. the postulate of harris (10) that analytical imprecision (cv,) should be less than, or equal to, one-half the within-subject biological variation (cv,,), that is: cva 50.5 . (cv,,) b. the proposal that analytical imprecision depends in the change (d) that it is desirable to detect clinically, the probability that such a change should exist, and the within-subject biological variation, according to the formula (1 1): cv, <[(d2/2 z2) -cv,, 2 ] 112 where z the z-statistic, which depend solely on the probability selected for significance. evaluation of quality specifications a. we have previously estimated within-subject variation in insulin-dependent diabetics (5) and found cv,, = 4.1%. using the postulate of harris, the estimated goal for imprecision in the hbl4,, assay is thus: cv, 50.5 * 4.1 = 2.1%. attention to significant changes in hba,, concentrations should be drawn early, consequently the probability that a change in results of 1% hba,, (which is change of 10%) was set moderately low, at the 0.80 confidence level. from the above formula: b. cv, <[(102/2 * 1.2g2) -4.l2l1l2 i.e. cva <3.7% a change of 2% hba,, in an individual (which is about a 20% change) would cause therapeutical intervention and the physician should be very confident that this change is 336 significant; the probability is therefore set at 0.99 and the analytical goal becomes cv, < 3.6%. discussion goal setting cannot be static and must evolve with new knowledge and concepts. clinical strategies should be analyzed in detail as the needs for analytical quality are different i n various situations. we have previously evaluated analytical goals based on the criterion that hba,, concentrations should be below a certain level in patients with diabetes mellitus (12). accepting this strategy makes analytical bias important. the present evaluation of analytical goals is based on clinical significance of a certain change within the individual during monitoring of hba,,. since monitoring is usually performed in a single laboratory, using one analytical method, the problem, of bias is somewhat irrelevant, whereas the within-subject biological variation is determining and analytical imprecision most demanding. we find it interesting that the goals in the two clinical monitoring situations were almost identical. moreover, the goals derived by using this model were more stringent than those empirically proposed by an expert committee (13), namely cv, less than five percent. conclusions 1. 2. 3. 4. 1. a number of different goals for analytical quality can be derived when different clinical situations using the same quantity are analyzed. when the clinical situation is well defined, the use made of the result should be the basis for goal-setting; if this is unknown, data on biological variation should be the basis for the goal. in monitoring of patients using results from the same laboratory the analytical imprecision is the most important of the performance characteristics. if several goals are considered to be applicable, the most demanding should be used. references singer, d.e., coley, c.m., samet, j.h. & nathan, d.m. tests of glycemia in diabetes mellitus. ann int med 1989;110:125-37. 337 2. 3. 4. 5 . 6. 7. 8. 9. 10. 11. 12. 13. svendsen, p.aa., lauritzen, t., soegaard, u. & nerup, j. glycosylated haemoglobin and steady-state mean blood glucose concentration in type i (insulin-dependent). diabetes. diabetologia 1982;23:403-5. larsen, m.l., horder, m. & mogensen, e.f. effect of long-term monitoring of glycosolated hemoglobin levels in insulin-dependent diabetes mellitus. n eng j med 1990;323: 1021-5. larsen, m.l., blaabjerg, o., petersen, p.h., hansen, h. & hoorder, m. analytical goal setting prior to selection of a method for glycated haemoglobin. scand j clin lab invest 1990;50:715-21. petersen, p.h., larsen, m.l. & horder, m. prerequisites for the maintenance of a certain state of health by biochemical monitoring. in: harris, e.k., yasaka, t., eds. maintaining a healthy state within the individual. amsterdam; elsevier 1987; 147-58. fraser, c.g. the application of theoretical goals based on biological variation of proficiency testing. arch pathol lab med 1988;112:404-15. fraser, c.g. & harris, e.k. generation and application of data on biological variation in clinical chemistry. crit rev clin lab sci 1989;27:404-37. walinder, o., wibell, l. & bostrom, h. the clinical value of hba,, determinations. acta med scand 1980; suppl. 639:17-22. dunn, p.j., cole, r.a., soeldner, j.s. et al. stability of hemoglobin a l c levels on repetitive determinations in diabetic outpatients. j clin endocrin metab 1981;52: 1019-22. harris, e.k. statistical principles underlying analytical goalsetting in clinical chemistry. am j clin pathol 1979;72:374-82. fraser, c.g., petersen, p.h. & larsen, m.l. setting analytical goals for random analytical error in specific monitoring situations. clin chem 1990;36: 1625-28. petersen, p.h., larsen, m.l. & fraser, c.g. the quality needed for measuring glycated haemoglobin. an application. upsala j med sci 1990;95: 195-90. national diabetes data group: report of the expert committee of glycosylated hemoglobin. diabetes care 1984;7:602-6. correspondence: mogens lytken larsen, department of clinical chemistry, odense university hospital, dk-5000 odense c, denmark 338 upsala j med sci 81: 159-166, 1976 detection and characterization of hyperlipoproteinaemia in middle-aged men hans hedstrand and bengt vessby from the departments of internal medicine and geriatrics, university hospital, uppsala, sweden abstract serum lipoprotein (lp) concentrations were determined and lp patterns were classified in 261 middle-aged men, re cruited from a health examination survey, with serum lipid values above the 80th percentile of the same population. individuals with hyperlipoproteinaemia (hlp) and normolipidaemic controls were characterized also regarding family history of cardiovascular disease, do-economic factors and clinical and laboratory variables. subjects with hlp type iv-v and i i b were overweight and showed hyperuricaemia and hyperinsnlinaemia compared with normolipidaemic controls and subjects with hlp type i i a . the latter showed elevated erythrocyte sedimentation rate. in spite of being overweight, subjects with hlp type iii showed normal fasting values of insulin and uric acid in serum and normal early insulin response to intravenous glucose. the glucose tolerance did not differ significantly between the groups. men with hlp types iv-v had predo minantly sedentary occupations, in contrast to those with type iia. there were significantly more smokers in the groups with hlp type iib and iv-v than in the control group. thus, individuals with different types of hlp tend to show different metabolic profiles hut also different socio economic and clinical patterns, suggesting that exogenous factors are of importance in the expression of the lp abnormalities. introduction in prospective studies, several risk factors for the development of atherosclerotic coronary heart dis ease (chd) have been identified. these factors include elevated concentrations of cholester ol (21) and triglycerides (11) in serum. the hyperlipoproteinaemias (hlp) are, according to fredrickson et al., divided into separate types with different lipoprotein (lp) patterns (16). etiological ly, hlp may be primary, due to genetically de termined metabolic defects, or secondary to dis orders such as diabetes mellitus and hypo thyroidism. however, environmental factors, e.g. dietary habits, stress, physical activity, may also influence the serum lipid levels. this report concerns a study of the serum lp composition in middle-aged men with hyper lipidaemia recruited from a health examina tion survey. subjects with different types of lp pattern were characterized regarding family history of cardiovascular disease, smoking habits, physical activity and socio-economic factors. the associa tion between different types of hlp and some clini cal and laboratory variables were also studied. materials a health examination was offered to all men living in the city of uppsala and born in 1920-24 (19). a total of 2322 men were examined, giving a participation rate of 83.9%. the present study comprised all men analysed between sept. 1971 and sept. 1973 with hyperlipidaemia defined as the mean of two values of serum cholesterol and/or triglycerides (tg) above the 80th percentile in this popula tion (291 mgll00 ml and 2.63 mmol, respectively. the hyperlipidaemic subjects were invited to a special lipid clinic for lipoprotein analysis. the following individuals were excluded: 23 men with hypertension, supine diastolic blood pressure (dbp)slos mmhg, 3 men with treated diabetes mellitus and one man with untreated hypothy roidism. a total of 270 men were invited to the lipid clinic but 9 of them did not show up. ten of the 261 subjects with hyperlipidaemia suffered from medical disorders. two of them had angina pectoris and one had suffered from a myocardial infarction. one case each of intermittent claudication, operated aortic coarctation, morbus crohn, and morbus parkinson were included. three men were treated with drugs: one hyperlipidaemic, one man with corticosteroid treated bronchial asthma and one with epilepsy. a control group consisting of 100 men, hypertensives excluded (supine dbp2105 mmhg), with serum lipid val ues below the 80th percentile at the initial examination were randomly selected: 5 men consecutively examined on the 15th of each month throughout the investigation were chosen. the laboratory and clinical variables as well as socio-economic data of this group were compared with those of the subjects with different types of hlp. the lp values in the different types of hlp were also upsala j med sci 81 160 h . hedstrand and b . vessby compared with the corresponding values in a randomly selected group of 92 apparently healthy men from the same population study (12). methods lipid and lipoprotein analyses all serum lipid and lp analyses were performed at the department of geriatrics, university of uppsala. blood samples were drawn after an overnight fast and serum was collected by low speed centrifugation. edta was added to a final concentration of 0.05%. separation of lp den sity fractions by preparative ultracentrifugation accord ing to have1 et al. (18) was begun on the same day. after centrifugation at 15°c for 16 hours at 40000 rpm using a 40.3 rotor the top fraction corresponding to the very low density lipoprotein fraction (vldl, dc1.006) was col lected. from the bottom fraction at d = 1.006 the low den sity lipoprotein (ldl, d=1.006-1.063) and high density lipoprotein (hdl, d>1.063) fractions were isolated either by a second spin at d = 1.063 in the preparative ultra centrifuge or by precipitation of ldl by a heparin manganese chloride solution (6). cholesterol and tg analyses in serum and in the isolated density fractions were performed by semi-automatic techniques in a tech nicon auto-analyzer type i1 according to rush et al. (27). the recovery of the lp analysis, i.e. the sum of cholesterol and tg in vldl, ldl and hdl, was always within 100+10% of total serum cholesterol and tg, re spectively. immediately after the ultracentrifugation the top and bottom fractions recovered at d=1.006 as well as whole serum were subjected to agarose electrophoresis accord ing to noble (25). a 1 % agarose gel containing 0.25 % albumin was used and the chromatogram was stained in sudan black. the lp patterns were classified according to fredrikson et al. using the recommendations by beaumont et al. (2). limits for hlp corresponding to the 85th percentile in a random sample of healthy men from the same population study were 190 mgll00 ml and 1.50 mmoln for ldl cholesterol and vldl respectively (12). the definition of type i11 hlp was based on the presence of a slow-moving band in vldl on agarose gel electro phoresis, with beta or close to beta mobility, in combina tion with a high ratio cholesterol/tg in vldl and a high “111-index” (29). in 6 individuals no complete ultracentrifuge analysis was obtained because of a low recovery of tg in the lp density fractions. however, the information gained from the analyses performed including serum tg and chol esterol concentrations, cholesterol concentrations in the lp classes and the results of the agarose electrophore sis was sufficient for an adequate classification of the lp pattern. other laboratory investigations the analyses of serum uric acid (sua), erythrocyte sedimentation rate (esr) and haematocrit were performed with the methods used routinely at the de partment of clinical chemistry, university hospital, de scribed elsewhere (19). the intravenous glucose tolerance test was performed as described before (19). the subjects were asked to be fasting after midnight the day before the examination. no other dietary prescriptions were given. serum insulin was determined in duplicate with the phadebas insulin test (pharmacia ab, uppsala, sweden). this method is based upon the radioimmunosorbent tech nique, described by wide et al. (30). the early serum insulin response to intravenous glucose was expressed as the mean value of the serum insulin concentrations de termined at 4 , 6 and 8 minutes after the start of the glucose injection. the late serum insulin response was expressed as the value at 60 minutes. the serum insulin index was defined as the ratio between early insulin response and basal serum insulin concentration (5,31). clinical investigations the supine bp was measured with a mercury manometer after 10 min rest and read to the nearest 5 mmhg. the dbp was recorded when the sound disappeared entirely (korotkoff phase 5 ) . the tables of lindeberg et al. were used for calculation of relative body weight (22). subscapular skinfold thick ness was measured with a harpenden caliper. a 12-lead electrocardiogram was recorded and clas sified according to the minnesota code. questionnaire the family history, as well as the information regarding physical activity, was obtained by a self-administered questionnaire a d rnodum collen (13). the question and the classification of physical activity used have been pre sented elsewhere (19). the coding of marital status and social group was based on interview reports. the three conventional social classes were used. the information of smoking habits was also recorded by interview. statistical calculations conventional methods were used for calculation of mean value and standard deviation (s.d.). significances of dif ferences between mean values were estimated with stu dent’s t-test (two-tailed test). logarithm transformed val ues were used when testing the means of serum insulin concentrations, k-values and esr because these parameters had a skew distribution (19). for the same reason serum tg concentrations, vldl and ldl tg and vldl cholesterol concentrations were also tested after logarithmic transformation (12). the x2-test with yates’ correction was used for comparison of frequencies. the accepted level of significance was p<o.o5. results lipid and lipoprotein analyses the mean values for serum cholesterol of the 261 men at the initial screening, at the second examina tion and at the lipid clinic were 292, 299 and 283 mgl100 ml, respectively. the difference between upsala j med sci 81 hyperlipoproteinaemia in middle-aged men 161 table i. classijication of lipoprotein pattern in 50 year-old men with hyperlipidaemia lipoprotein number of frequency pattern subjects (%) normal 42 16.1 i1 a 77 29.5 i1 b 55 21.1 i11 17 6.5 iv" 68 26.1 v 2 0.7 total 26 1 100.0 a complete ultracentrifuge data missing in 6 subjects. the first two values was not significant. the analyses at the lipid clinic showed significantly lower values than at the initial (p<0.05) as well as at the second examination (p<o.ool). the cor responding values for serum tg were 3.32,3.21 and 3.04 mmol/l, respectively. these values did not dif fer significantly. the mean serum cholesterol and tg concentrations of the 23 men who were ex cluded because of hypertension did not differ significantly from those of the remaining group of hyperlipidaemics . the classification of the lp patterns are shown in table i and the lipid concentrations in the serum l p density fractions of the different types of h l p are shown in table 11. the type i i a and iib ldl cholesterol is high by definition. correspondingly, type iib and iv showed high vldl tg levels. the vldl cholesterol concentration in type i11 was considerably higher than in both types i i a and iib (p<o.ool) and also higher than in type iv @<0.01). the ldl cholesterol was lower in type i11 than in types i i a and iib (p<o.ool), but on the average higher than in type iv (p<o.oi). other laboratory investigations the mean of esr was significantly higher in type i i a than in the control group (fig. 1). the same tendency was seen in type iib ( p = o . o 6 ) . the differences in mean haematocrit values were numerically small. however, the values in all h l p groups were significantly higher than in the control group. the highest value, 44.1k2.5%, was obtained in type iib @<0.001). the haematocrit in type i i a was 43.5k2.5% ( p < o . o l ) and in type iv v 43.3+2.4% (p<0.05), while the controls had a value of 42.5k2.4%. the mean s u a concentrations were signifi cantly increased in types iib and iv-v compared with the control group as well as with the other two types of h l p (fig. 1 ) . the results of the serum insulin determinations are summarized in fig. 2. there were no significant differences between the mean k-values of the five groups. no significant differences were observed be tween the group of 42 men with normal l p pattern (table i) and the control group with regard to laboratory investigations. clinical investigations the mean systolic blood pressure was 137.1k 16.3 mmhg in h l p type iib which was significantly higher (p<0.05) than in the control group, 131.5k 16.3 mmhg. the mean dbp was significant ly higher (p<0.05) in types iv-v than in the control group. the values were 85.1k9.3 mmhg and 81.7+ 10.4 mmhg, respectively. no other signifi cant differences were found. the average pulse rate were the same in all groups. the weight index was significantly higher in types iib, i11 and iv-v than in the control group as well as in type i i a (fig. 1). subjects with hlp types iib and iv-v had an increased skinfold thickness compared with the controls. a history of weight gain of more than 10 kg after the age of 30 was reported by 57% and 44% of the subjects with h l p types iv-v and iib, respectively. this was significantly ( p < o . o l ) more frequent than among men with type i1 a (24 %) and in the control group (23 %). electrocardiogram the resting ecg was classified according to the minnesota code. there were no reportable items in 54.7 % of the ecgs of the subjects with h l p type i i a compared with 72.3% in types iv-v. this difference was significant (p<0.05). in the control group, 61.0% of the ecgs were without reportable items. however, this seems a somewhat low fre quency since in the total population of more than 2000 men of the same age the corresponding fre quency was 69.7%. the frequencies of signs of chd were too low to allow any conclusions. aspects on the family history of cardiovascular deaths the frequencies of parents dead from myocardial infarctions or cerebrovascular lesion did not differ 11 -762853 upsala j med sci 81 162 h . hedstrand and b . vessby table 11. lipoprotein lipid concentrations in lp density fractions in 50-year old men with different types of hyperlipoproteinaemia and in a sample of healthy men from the same study serum vldl ldl chol chol hlp (mg/ tg chol tg (mgl tg type n 100 ml) (mmolh) (rng1100 ml) (mmol/l) 100 ml) (mmol/l) i1 a 77 305f40 2.00 23 0.98 226f37 0.69 i1 b 55 315f25 3.24 43 2.12 218f21 0.72 i11 17 321f67 3.74 92 2.42 173f56 0.80 iv" 68 254f39 4.05 58 2.95 145f30 0.63 popul. 92 238k38 1.80 21 1.03 155f34 0.49 (1.832k0.083) (2.293 f0.087) (1.330f0.189) ( 1 .v4+0.140) (2.505 f 0 . 0 7 1) (1.615f0.117) (2.318k0.097) (1.852k0.095) (2.532k0.176) ( 1 379f0.267) (2.305 f0.242) (1.881 f 0.151) (2.561fo.177) (1.697f0.223) (2.406f0.209) (1.780f0.132) sample (2.233fo. 142) (1 252 f 0.246) (1.959 f 0.227) (1.677f0.109) log (mean valuex 100)ks.d. in parentheses. ultracentrifuge data missing in 6 subjects. between the four groups of hlp. nor were there any differences when compared to the controls. the frequency of a history of diabetes mellitus among parents of men with different types of hlp was similar, around 6%, and did not differ from that of the controls. marital status and social class when .comparing marital status no differences were found between the groups of men with different types of hlp and the control group. there were significantly fewer men with hlp type i i a than in the control group who were clas fasting serum early serum insulin insulin response e r y t hrocy te serum urlcacld sedimentation rate mg,,oom, weight index subscapular index mm skinfold **+ n = 72 52 17 68 94 n = 69 52 17 66 92 fig. 1. erythrocyte sedimentation rate, serum uric acid, weight index and subscapular skinfold thickness in 50 year-old men with different types of hyperlipoprotein aemia (hlp) compared with controls (**p<o.oi, ***p<0.001). late serum serum insulin insulin response index index pu/rnl y y * 1 *** i 20 40 81) 10 20 l o 0 0 0 n= 71 52 17 68 93 n = 69 52 17 66 92 fig. 2. serum insulin concentrations in 50-year-old men with different types of hyperlipoproteinaemia (hlp) com pared with controls (**p<o.oi, ***p<o.ool). upsala j med sci 81 hyperlipoproteinaemia in middle-aged men 163 physical activity the degree of physical activity at work and during leisure reported by the men is shown in table v . subjects with h l p type i i a had sedentary work less often than the controls and also in comparison with the subjects with types iib and iv-v @<0.01). the frequency of subjects with types iv-v who had predominantly sedentary work was significantly increased compared with the controls. correspondingly, men with type i i a showed the highest frequency of heavy manual work, the differ ence being significant compared with men with types iv-v @<0.01). in the group with types iv-v, the frequency of men with heavy manual work was significantly lower than in the control group. no significant differences were found when comparing activity during leisure time in the differ ent groups. hdl chol tg (mmol/l) 42fg 0.28k0.05 41k8 0.34k0.09 37+9 0.31k0.13 a+13 0.24f0.06 sified in social class i (table 111). there was an overrepresentation of men in' type i1 b classified in social class 2. in type i i a more men belonged to social class 3 than in type iv @<0.05). smoking habits there were 46% smokers in the control group (ta ble iv). this number corresponded well t o the 51 % smokers found in the total material of more than 2000 men. the smoking group comprised both cigarette, cigar and pipe smokers. in the groups with types iib and iv-v there were significantly more smokers than among the controls. this differ ence was caused by a higher frequency of cigarette smokers (40%) among the men with types iib and iv-v than in the control group (26%). table 111. social class classification (%) of 50 year-old men with hyperlipoproteinaemia com pared with normolipidaemic controls lipoprotein pattern social iia i i b iv-v controls class [n=77) (n=55) (n=70) (n=loo) 1 6.5* 9.1 20.0 19.0 2 35.1 49.1* 42.9 31.0 3 58.4 41.8 37.1 50.0 * p<0.05 compared with controls. discussion the purpose of this investigation was to study the association between different types of hlp and case history, clinical and laboratory data. the population was homogeneous with regard t o age, sex and geographical factors. the hyperlipidaemic group was unselected with the exception that sub jects with hypertension and secondary hyper lipidaemia were excluded. serum lipid levels were moderately elevated in these hyperlipidaemics. only one subject had a serum cholesterol value above 400 mg/100 ml. most other studies of hyperlipidaemics as well as of lp composition in different types of h l p have been concerned with selected materials of patients with extreme hyperlipidaemia. these studies may not be representative of the moderate h l p found in the general population. table iv. smoking habits (%) among 50-year-old men with hyperlipoproteinaemia compared with normolipidaemic controls lipoprotein pattern smoking iia iib iv-v controls habits (n=77) (n=55) (n=70) (n=loo) never smoked 18.2 14.5 17.1 26.0 ex-smokers 26.0 18.2 20.0 28.0 smokers 55.8 67.3* 62.9* 46.0 * p<0.05 compared with controls. upsala j med sci 81 164 h . hedstrand and b . vessby table v. prevalence of different categories of physical activiv at work and during leisure in 50-year-old men with hyperlipoproteinaemia compared with normolipidaemic controls physical activity i1 a iib iv-v controls (fl=77)“ (fl=55)” (n=70)* (n=loo)‘ at work predominantly standing 25.7 36.5 30.3 30.6 the same+fifting and 31.1 11.5 13.6 17.3 heavy manual work 25.6 19.3 4.5* 16.3 leisure time inactive 18.9 21.6 15.4 17.5 some activity 32.4 39.2 44.6 37.1 regular activity 40.5 39.2 36.9 39.2 regular hard physical 8.2 0.0 3.1 6.2 training * p < 0 . 0 5 , ** p<o.ol compared with controls predominantly sedentary 17.6** 32.7 51.5* 35.7 and walking handling heavy objects data incomplete in 3 . 4 and 2 subjects respectively. seventeen subjects (6.5 %) of the hyper lipidaemics were classified as type 111, cor responding to a prevalence of around 1 % in the total population. this seems to be a fairly high prevalence, although comparisons with other studies are uncertain because of dissimilarities in the materials and the criteria used for classification, which in the present study included a qualitative aspect, the requirement of a “floating beta” band in vldl, a high vldl cholesterol/tg ratio and a high “‘111-index” (29). a high haemoglobin level in men has been re ported to be a risk factor for the development of chd (9, 20, 32). the haematocrit has also been demonstrated to be related to both the later de velopment of myocardial infarction and to death from other causes (28). however, using a multiple logistic model, a high haematocrit reading did not signifkantly correlate to an increase in events re lated to chd (32). a positive correlation between haemoglobin levels and serum lipid levels has been reported and suggested to be dependent on changes in plasma volume (7). cigarette smokers have higher haematocrit values and haemoglobin levels than non-smokers, presumably because of an im paired respiratory function as well as increased blood levels of carbon monoxide. in our study there was an increased frequency of smokers among men with h l p who were also characterized by higher haematocrit values, compared with the controls. an elevated esr in subjects with hlp has earlier been reported in a study of asymptomatic subject with marked hyperlipidaemia (10). an elevated esr has also been found to be a “risk factor” of chd (9). the mechanism is unclear. a high l p level has been suggested to influence the esr through increased binding of lysolecithin (8). silent vascular disease caused by the hyperlipidaemia has also been discussed as a possible reason for the raised esr (10). the sua concentration was higher in subjects with h l p types i i b and iv-v than in controls. an as sociation between sua and obesity (14, 23) and hypertriglyceridaemia (15) has been found. fur thermore, in a recent study the sua level has been demonstrated to be correlated to both the vldl tg concentration and body weight independently (26). the average weight index and skinfold thickness in h l p types i i b and iv-v were significantly greater than in the control group and in type i i a , indicating an increased degree of obesity. support ing this was the finding of a higher frequency of a history of weight gain after the age of 30 in subjects with types i i b and iv-v. some marginal differ ences in supine blood pressure could probably be explained by differences in body weight between the h l p types. i t has recently been suggested that there are two forms of obesity (4). one form, acquired in adult upsala j med sci 81 age, is characterized by hypertrophy of fat cells. this type is related t o caloric balance and associat ed with metabolic disturbances. the other form, with onset in childhood, is characterized by an in creased number of fat cells. it has been shown that the adipose tissue fat cell size is a more important determinant than the adipose tissue size for the in sulin response to glucose tolerance test (3). in this study, significantly more men with hlp types i i b and iv-v reported a history of weight gain after the age of 30 than the men in type i i a and in the con trol group. this indicates that there might be more subjects with the hypertrophic type of obesity in types i i b and iv-v, which may be an explanation for the hyperinsulinaemia found in these types of hlp. in contrast to the findings in types i i b and iv-v, the h l p type i11 subjects did not, on average, show elevated fasting serum insulin concentration and early serum insulin response, in spite of a high weight index. only the late serum insulin response was increased, compared with the controls. the sua value too was normal in type 111. with reser vation for the limited number of type i11 subjects these findings may indicate that the metabolic pat tern presumably characteristic of types i i b and iv-v is not seen in the type 111 disorder, which might constitute a pathogenetically different entity. in contrast to other reports, no significant differ ences in glucose tolerance were found between the different hlp groups and the control group. considering socio-economic data, there seemed to be an association between social class and degree of physical activity a t work which might be re flected in the distribution of hlp types. the socio-economic status may also influence the smoking habits. there were significantly more smokers in h l p types i i b and iv-v than in the control group. this is in accordance with the find ings in a study in asymptomatic primary hyper lipidaemia (26). the explanation for this association is unclear. the results of this study indicate that individuals with different types of h l p not only show typical lp pattern but even different socio-economic and clinical patterns, thus supporting the view that ex ogenous factors are important for the expression of l p pattern abnormalities. the type i1 a subject is a lean, physically hard working man in social class 3 with elevated esr but with most of the metabolic parameters normal. the type iv-v subject is an hyperlipoproteinaemia in middle-aged men 165 overweight, predominantly sedentary smoker in social class 1 or 2 with elevated sua and hy perinsulinaemia. acknowledgements this study was supported by grants from the swedish medical research council (19x-204 and 19x-3116), pharmacia ab and uppsala lans landsting. references 1 . albrink, m. j., meigs, j. w. & gramott, m. a.: weight gain and serum tnglycendes in normal men. n engl jmed266:484, 1962. 2. beaumont, j. l., carlson, l. a., cooper, g. r., 3. 4. 5. 6. 7. 8. 9. 10. 1 1 . 12. fejfar, z., fredrickson, d. s. & strassir, t.: clas sifications of hyperlipidaemias and hyperlipo proteinaemias. bull wld hlth org43: 891, 1970. bjomtorp, p., berchtold, p. & tibblin, g.: insulin secretion in relation to adipose tissue in men. dia betes 20: 65, 1971. bjorntorp, p. & sjostrom, l.: number and size of adipose tissue fat cells in relation to metabolism in human obesity. metabolism20:703, 1971. boberg, j . , hedstrand, h. & wide, l.: the early serum insulin response to intravenous glucose in pa tients with decreased glucose tolerance and in sub jects with heredity for diabetes mellitus. scand j clin lab invest36: 145, 1976. burstein, m. & samaille, j.: sur un dosage rapide du cholesterol lie aux aet aux p-lipoprottines du serum. clin chim acta5:609, 1%0. bottiger, l. e. & carlson, l. a.: relation between serum cholesterol and triglyceride concentration and haemoglobin values in non-anaemic healthy persons. brit med jzzz:731, 1972. bottiger, l. e.: erythrocyte sedimentation rate and plasma lipids. acta med scand 193: 53, 1973. bottiger, l. e. & carlson, l. a.: the stockholm prospective study. 2. new events of coronary heart disease in men in relation to findings at initial exami nation, 9-year follow-up. in: early phases of coro nary heart disease. the possibility of prediction. skandia international symposia. nordiska bok handeln ab, stockholm 1973. bottiger, l. e., carlson, l. a., ekelund, l. g. & olsson, a. g.: raised erythrocyte sedimentation rate in asymptomatic hyperlipidaemia. brit med j zz: 681, 1973. carlson, l. a. & bottiger, l. e.: ischaemic heart disease in relation to fasting value of plasma triglycerides and cholesterol. lancet i : 865, 1972. hedstrand, h. & vessby, b.: serum lipoprotein con centration and composition in healthy 50-year-old men. uusala j med sci81: 37. 1976. 13. collen,m. f., cutler, j. l., siegelaub, a. b. & cella, r. l.: reliability of a self-administered medical questionnaire. arch int med 123:664, 1%9. 14. evans, j. g. & morrison, r. b. i.: the carterton upsala j med sci 81 166 h . hedstrand and b . vessby study: 5 . serum uric acid levels of a sample of new zealand european adults. n z med j 70: 306, 1969. 15. feldman, e. b. & wallace, s. l.: hyper triglyceridaemia in gout. circulation 29: 508, 1964. 16. fredrickson, d. s., levy, r. i. & lees, r. s.: fat transport in lipoproteins: an integrated approach to mechanisms and disorders. n engl j med 276:34, 1967. 17. glueck, c. j., levy, r. i. & fredrickson, d. s.: immunoreactive insulin, glucose tolerance and carbohydrate inducibility in types 11, 111, iv and v hyperlipoproteinaemia. diabetes 18: 739, 1969. 18. havel, r. j., eder, h. a. & bragdon, j. h.: the determination and chemical composition of ultracentrifugally serarated lipoproteins in human serum. j clin invest34: 1345, 1955. 19. hedstrand, h.: a study of middle-aged men with particular reference to risk factors for cardiovascular disease. upsala j med sci, suppl. 19, 1975. 20. kannel, w. b., castelli, w. p. & mcnamara, p. m.: the coronary profile: 12-year follow-up in the framingham study. j occup med 9: 61 1, 1967. 21. kannel, w. b., castelli, w. p., gordon, t. & mcnamara, p. m.: serum cholesterol, lipoproteins, and the risk of coronary heart disease. ann intern med74: 1, 1971. 22. lindeberg, w., natvig, h., rygh, a. & svendsen, k.: hoyde og vektundersokelser hos voksne menn og kvinner. tidskr n laegeforen 76: 361, 1956. 23. myers, a. r., epstein, f. h., dodge, h. j. & mik kelsen, w. m.: the relationship of serum uric acid to risk factors in coronary heart disease. amer j med 45:520, 1968. 24. nikkila, e. a.: control of plasma and liver triglyceride kinetics by carbohydrate metabolism and insulin. adv lipid res 7: 63, 1969. 25. noble, r. p.: electrophoretic separation of plasma lipoproteins in agarose gel. j lipid res 9: 693, 1968. 26. olsson, a. g.: studies in asymptomatic primary hyperlipidaemia. 11. clinical findings. acta med scand 197:477, 1975. 27. rush, r. l., leon, l. & turrell, j.: automated simultaneous cholesterol and triglyceride determina tion on the auto analyzer i1 instrument. advances in automated analysis. thurman associates. vol 1:503, 1971. 28. tibblin, g.: risk factors for developing myocardial infarction and other diseases. the “men born in 1913” study. i n preventive cardiology (ed. g. tib blin, a. keys & l. werko), almqvist & wiksell, stockholm, 1972. 29. vessby, b.: studies on the serum lipoprotein compo sition in 50-year-old men. a suggestion of chemical criteria for diagnosis of hyperlipoproteinaemia type 111 (broad-p-disease). clin chim acta 69: 29, 1976. 30. wide, l., axen, r. & porath, j.: radioimmunosor bent assay of proteins. chemical couplings of antibodies in insoluble dextran. immunochemistry 4:381, 1967. 31. wide, l. & ohlsson, e.: impaired early insulin re sponse to glucose in non-diabetic patients with necrobiosis lipoidica. i n radioimmunoassay. meth odology and application in physiology and in clinical studies (ed. r. levine & pfeiffar, e. f.). horm metab res, suppl.5: 130, 1975. 32. wilhelmsen, l., wedel, h. & tibblin, g.: multivariate analysis of risk factors for coronary heart disease. circulation48: 950, 1973. received june 27, 1976 address for reprints: bengt vessby, m.d. institute of geriatrics uppsala 1 sweden box 641, s-75127 upsala j med sci 81 upsala j med sci 87: 119-125, 1982 alcohol intake, serum p2-microglobulin and ventricular extrasystoles factors related to death in five-year follow u p of middle-aged men a . urban waern', c. lidell' and k. hellsing' from the departments of internal medicine' clinical chemistry2, university hospital, u p p s t i l t i . sttvden abstract common causes of death in middle-aged men in sweden are ischemic heart disease and malignant tumours. in this work the authors relate some findings in a healthy survey of sixty-year old men to mortality in the ensuing five-year follow-up period. of the original population of 331, 60-year old men 15 sub jects (4.5%) died. in the follow-up period. the factors tested for relation to death in the follow-up period were: indices of alcohol abuse, serum con centration of b2-microglobulin and occurrence of ventricular extrasystoles in a long term (6 hrs) ambulatory electrocardiogram. it was found that in seven of the fifteen deceased subjects at least episodical heavy abuse of alcohol had prevailed. this was not evident at the health examination by use of serum gammaglutamyltransferase analysis, nor through the death certificates, but through hospital records and registration at the temperance board. four subjects died (1.2%) of malignant tumours, and three of these subjects had serum-b2-microglobulin values above the 95th percentile in the study popula tion. an increased risk of death, though statistically not significant, was noted in men with ventricular extrasystoles compared to those without ventri cular extrasystoles in long term electrocardiogram. however, all men with ventricular extrasystoles ? 30/hour and those with ventricular tachycardia were alive five years later. among the men w?o died due to ischemic heart disease, ventricular extrasystoles in the long-term electrocardiogram prevailed only in subjects with indices of alcohol intake. alcohol induced fatal arr hythmias might thus be the terminating event in middle-aged men with tradition al risk-factors for ischemic heart disease. material and methods all men born in 1915, living in uppsala were invited to a health examina tion at their age of sixty years in 1975. the procedures of this health in vestigation have been described in detail elsewhere (15). a total of 331 60 year old men took part, which corresponded to a participation rate of 78.4%. i i9 l a b o r a t o r y i n v e s t i g a t i o n s c l u d e d a n a l y s i s o f s p 2 m i c r o g l o b u l i n ( 6 ) and s-gammaglutamyltransferase (s-gt) ( 1 1 ) . f u r t h e r m o r e each s u b j e c t was i n s t r u c t e d t o c o l l e c t a l l u r i n e d u r i n g t h e 24 h o u r s f o l l o w i n g t h e h e a l t h e x a m i n a t i o n . a n a l y s i s f o r any con t e n t s o f e t h y l a l c o h o l i n u r i n e was c a r r i e d o u t a c c o r d i n g t o t h e method b y bonnichsen e t a1 ( 1 ) . the serum samples were t a k e n i n t h e f a s t i n g s t a t e i n t h e m o r n i n g and i n m e d i c a l h i s t o r y m o d i f i e d a f t e r c o l l e n ( 2 ) i f he had i n g e s t e d any a l c o h o l i c beverages t h e day p r i o r t o t h e h e a l t h s c r e e n i n g . the r e g i s t e r s o f h o s p i t a l r e c o r d s f o r i n h o s p i t a l s t a y a t t h e u n i v e r s i t y h o s p i t a l o f uppsala were searched. f u r t h e r m o r e t h e r e g i s t e r o f t h e temperance board was e v a l u a t e d , f o r s u b j e c t s b e l o n g i n g t o t h e a c t u a l s t u d y p o p u l a t i o n , r e g i s t e r e d , due t o a l c o h o l abuse. each p a r t i c i p a n t was asked by means o f a s e l f a d m i n i s t e r e d q u e s t i o n n a i r e death c e r t i f i c a t e s the d e a t h c e r t i f i c a t e s f o r a l l s u b j e c t s i n t h e s t u d y g r o u p who d i e d i n t h e f i v e y e a r p e r i o d (1975-1980) f o l l o w i n g t h e h e a l t h e x a m i n a t i o n , were o b t a i n e d i n c o p i e s f r o m t h e n a t i o n a l bureau o f s t a t i s t i c s i n stockholm. long-term ecg d a i l y a c t i v i t i e s . a p o r t a b l e one-channel t a p e r e c o r d e r (hrb2 s r a h e l l i g e , sweden) was used. the e l e c t r o d e s were a t t a c h e d i n b i p o l a r l e a d v e r t i c a l l y o v e r s t e r n u m . . a n a l y s i s was c a r r i e d o u t t h r o u g h a scanner w i t h 2 0 f o l d t i m e c o m p r e s s i o n (memoport h e l l i g e , sweden). g r a p h i c p a p e r w i t h paper-speed 125 mm/sec f o r subsequent manual e v a l u a t i o n o f a r r h y t h m i a s . a 6 h o u r a m b u l a t o r y ecg r e c o r d i n g was c a r r i e d o u t d u r i n g u n r e s t r i c t e d the e l e c t r o c a r d i o g r a m s were f u l l y p r i n t e d o u t on s t a n d a r d e l e c t r o c a r d i o a r r h y t h m i a c l a s s i f i c a t i o n i n t h e l o n g t e r m ecg complex (> 0.12 s e c ) , w i t h o u t a p r e c e d i n g p-wave. f r e q u e n t ves were d e f i n e d as a mean h o u r l y c o u n t 530. complex v e s c o m p r i s e d bigeminy, c o u p l e t s , m u l t i f o r m i t y , v e n t r i c u l a r t a c h y c a r d i a ( v t ) (> 3 ves c o n s e c u t i v e l y w i t h r a t e > l o 0 p e r m i n ) , and t h e r on t phenomenon (rr-/qt 2 0 . 8 5 ) . a v e n t r i c u l a r e x t r a s y s t o l e (ves) was d e f i n e d as a p r e m a t u r e w i d e qrs results t a b l e s 1-3 show a l l 15 s u b j e c t s ( 4 . 5 % ) who d i e d i n t h e f i v e y e a r s f o l l o w i n g t h e h e a l t h e x a m i n a t i o n . a l l s u b j e c t s underwent a u t o p s y . seven 120 s u b j e c t s d i e d o u t s i d e h o s p i t a l . the s u b j e c t s who d i e d a r e grouped i n t h e t h r e e t a b l e s a c c o r d i n g t o causes o f d e a t h , nemely i s c h e m i c h e a r t d i s e a s e ( i h d ) , m a l i g n a n t tumours and o t h e r causes r e s p e c t i v e l y . e i g h t s u b j e c t s d i e d due t o i h d , f o u r s u b j e c t s due t o a m a l i g n a n t tumour and t h e r e m a i n i n g t h r e e o f o t h e r causes. t a b l e 1. s u b j e c t s ( n o 1-8) who d i e d due t o i s c h e m i c h e a r t d i s e a s e ( i h d ) . s u b j e c t s-gt age a t d e a t h i n d i c e s o f a l c o r i s k f a c t o r s f o r i h d ves i n no u k a t / l y e a r s h o l i n t a k e ( a b b r e v i a t i o n s se l o n g t e r m be1 ow) ecg 1 0.70 61 r e g i s t e r e d a t s + temperance b o a r d 2 0.50 62 3 0.17 62 s hl,ht 0 0 4 0.22 61 0 5 0.68 61 a l c o h o l i s m (dept o f s p s y c h i a t r y ) . a c u t e a l c o h o l p a n c r e a t i t i s p s y c h i a t r y ) . pos q u e s t i o n n a r y r e p l y 6 0.38 61 a l c o h o l i s m (dept o f hl, s 7 0.18 60 a l c o h o l i n u r i n e hl, s 8 0.51 61 pos q u e s t i o n n a r y r e p l y + a b b r e v i a t i o n s : hl = h y p e r l i p i d e m i a , ht = h y p e r t e n s i o n , s = tobacco smoking, + = o c c u r r e n c e o f ves, 0 = no o c c u r r e n c e o f ves, p e r f o r m e d = no l o n g t e r m ecg i n d i c e s o f a l c o h o l i n t a k e as can be seen f r o m t a b l e 1-3 i n d i c e s o f a l c o h o l i n t a k e were f o u n d i n 9 o f t h e 15 deceased s u b j e c t s . however, i n t w o o f t h e cases ( n o 8 and no 1 1 ) t h e i n d e x r e g i s t e r e d was a p o s i t i v e r e p l y o f t h e q u e s t i o n n a i r e . these cases c e r t a i n l y r e p r e s e n t e d o c c a s i o n a l i n t a k e . i n t h e r e m a i n i n g 7 s u b j e c t s however, t h e r e were s i g n s o f a t l e a s t e p i s o d i c a l heavy a l c o h o l abuse. f o u r o f t h e f i f t e e n s u b j e c t s were r e g i s t e r e d a t t h e temperance board ( 2 7 % ) w h i c h s h o u l d be compared t o 7% i n t h e r e m a i n i n g p o p u l a t i o n a l i v e . serum b 2 m i c r o g l o b u l i n s u b j e c t ( n o 1 1 ) t h e tumour was known a t t i m e o f t h e h e a l t h e x a m i n a t i o n . the v a l u e s o f b 2 m i c r o g l o b u l i n s h o u l d be j u d g e d a g a i n s t t h e mean v a l u e i n t h e r e m a i n i n g p o p u l a t i o n a l i v e w h i c h was 2.05 ? 0.48 m g / l . t a b l e 2 shows t h e s u b j e c t s who d i e d due t o a m a l i g n a n t tumour. i n one 121 table 2. subjects (no 9-12) who died due to a malignant tumor. subject s-p2 s-gt age at death origin of indices of alcohol intake no mg/l ukat/l years ma1 .tumor 9 2.40 2.34 61 kidney 10 3.82 0.23 63 lungs registered at temperance board alcoholism (dept of psy chi atry) 1 1 2.92 0.20 61 prostata pos questionnary reply 12 4.28 0.70 64 gal 1 bladder table 3. subjects no subjects (no 13-15) who died of other causes than ihd and malignant tumor. s-gt age at death cause of death indices of alcohol intake ukat/l years 13 0.24 62 amyotrofic lateral scle rosis 14 0.64 62 perforating acute alcohol pancreatitis ulcus ventri cul i pos questionnary reply aortic aneurysm reg at temperance board 15 0.33 64 ruptured reg at temperance board a1 coho1 i sm (dept of psychiatry) long-term ecg subjects in the entire screened population. ves were noted in 121 of these recordings (41.4%). among the eight subjects who died due to ihd seven under went long-term ecg, four of these had ves on the long-term ecg, but none however had vt, frequent ves or r/t as defined above. the risk of death due to ihd was 3 times higher in men with ves compared to those without ves. this difference was however not statistically significant (p = 0.2, fischers exact test). technically satisfactory long-term ecg recording were obtained in 292 discussion increased alcohol intake has been shown by several authors (3, 8, 10) to be related to higher mortality and morbidity, of all causes compared to that among low consumers and abstainers. in our study nearly half of the deceased men had indicators of at least epidosical alcohol abuse. this was not dis closed by the s-gt values which previously (13, 14, 16, 17) has been related 122 to increased alcohol intake, and in only one of the subjects alcoholismus chronicus was mentioned in the death certificate as contributory cause o f death. increased alcohol intake as contributory, or main cause of death, is certainly underreported in the national statistics on mortality. further one might speculate if those subjects whose findings suggested at least episodical heavy abuse of alcohol, voluntarily had abstained from alcohol intake the weeks preceding the health examination. the reported rapid decrease of s-gt activity ( t 1 7-10 days) at alcohol abstention might thus explain the un 2 foreseen low s-gt values. serum microglobulin is a small protein (m.w. 11.800), reported by some authors (9, 12, 13) to show elevated serum concentrations in patients suffer ing from malignant tumours. in our study only four (1.2%) of the originally investigated population had died due to a malignant tumour in the five-year follow-up period. however, in three of these four subjects the serum d2-microglobulin values were found above the 95th percentile in the entire population. the erytrocyte sedimenta tion rate (esr) was not elevated in any of these cases, nor were there any other signs of inflammatory disorders or decreased renal function, the latters (5, 12) have been reported as important causes of elevated serum p2-micro globulin. further studies are needed to clarify the possible role of serum d 2 microglobulin as a possible early circulating marker of a malignant tumour. some studies ( 4 , 7 ) performed, have not found a correlation between occur rence of ves and fatal outcome of ihd. hinkle et a1 (7) investigated 283 actively employed men with a median age of 55 years, with continous 6 hours ambulatory ecg. they found that frequently occuring ves (>lo/looo) was associated with a ten fold increase in the risk of developing death due to ihd, in comparison to men without ves. as in our study deaths due to ihd, was not predicted by occurrence of vt or coupled ves. increased 3 times if ves were noted, during a 6 hour continous recording. however, all men with ves > 29/hour and those with vt were alive five years after the initial investigation. the findings of ves in the group who died due to ihd was confined to men with indices of alcohol intake. the wellknown arrhytmogenic action of ethyl alcohol and it’s metabolites certainly worsens the long-time prognosis concerning ihd in subjects with traditional risk factors for ihd. in our follow-up study during five years the risk of death due to ihd was 123 references bonnichsen, r.k. & theorell, h.: an enzymatic method for the microdeter mination of ethanol. scand j clin lab invest 3:58, 1951. collen, m.f., cutler, j.l., siegelaub, a.b. & cella, r.l.: reliability of a self-administered medical questionnaire. arch intern med 123:664, 1969. dyer, a., stamler, j., paul, o., berkson, d., lepper, m., mckean, h., shelle, r., lindberg, h. & garside, d . : alcohol consumption, cardio vascular risk factors and mortality in two chicago epidemiologic studies circulation 56:1067, 1977. desai, d.c., hershberg, p.i. & alexander, s.: clinical significance of ventricular premature beats in an out-patient population. chest 64:564, 1973. evrin, p.-e. & wibell, l.: serum b2-microglobulin in various disorders. clin chem acta 43:183, 1973. hellsing, k. & enstrom, h.: pre-treatment of serum samples for immuno nephelometric analysis by precipitation with polyethylene glycol. scand j clin lab invest 37:529, 1977. hinkle, l.e., carver, s.t. & stevens, m.: the frequency of asymptomatic disturbances o f cardiac rhythm and conduction in middle-aged men. am j cardiol 24:629, 1969. schmidt, w. & delint, j.: causes of death o f alcoholics. q j stud alc 33:171 , 1972. shuster, j., gold, p. & poulik, m.d.: b2-microglobulin levels in cancerous and other disease states. clin chem acta 67:307, 1976. sundby, p.: alcoholism and mortality. universitetsforlaget, blindern, oslo, norway 1967. szasz, g.: a kinetic photometric method for serum gamma glutamyltrans peptidase. clin chem 15:124, 1969. talal, n., grey, h., zvaifler, n., michalski, j. & daniels, t.: elevated salivary and synovial fluid be-microglobulin in sjogrens syndrome and rheumatoid arthritis. science 188:1196, 1975. teasdale, c., mander, a.m., fifield, r., keyser, j.n., newcombe, r.g. & hughes, l. e. : ser~m-8~-microglobulin in controls and cancer patients. clin chem acta 78:135, 1977. trell, e., sternby, n. & peterson, b.: alcohol abuse, s-gt values and premature death in middle-aged men. lakartidningen 77:2904, 1980. waern, u.: health and disease at the age o f sixty. upsal j med sci 83: 153, 1978. 1. 2. 3 . 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 124 1 6 . w h i t f i e l d , j . b . , hensley, w.j., bryden, d . & g a l l a g h e r , h . : some l a b o r a t o r y c o r r e l a t e s o f d r i n k i n g h a b i t s . a n n clin biochem 15:297, 1978. 1 7 . whiteland, t . p . , clarke, c . a . & w h i t f i e l d , a . g . w . : biochemical and hematological markers of alcohol i n t a k e . lancet i : 9 7 8 , 1978. 18. wu, a , , s l a v i n , g . & levi, a.j.: a s e , and h i s t o l o g i c a l l i v e r damage i n alcoholism. am j g a s t r o e n t e r o l 65:318, 1976. elevated serum gamma-glutarnyltransfer received june 1 , 1981 address f o r r e p r i n t s : a. urban waern, m . d . department of i n t e r n a l medicine u n i v e r s i t y hospital sweden s-750 1 4 uppsala 125 upsala j med sci 78: 145-149, 1973 an in-vitro method of investigating the rates of transcapillary exchange and net filtration by single-injection technique bo aberg and sven-olof lindahl from the institute of physiology and medical biophysics, biomedical centre, university of uppsala, uppsala, sweden abstract a method of preparing a cat's hind leg that is suitable for studying the rates of transcapillary solute and solvent movements is described. the preparation may be perfused at any desired venous pressure. a bolus may be injected into the capillary network without altering either the perfusion pressure or the flow rate (while continuously recording the weight of the isolated leg). a new method of calculating extractions is introduced. this method uses the estimate of the correlation between the relative eon centrations of reference and test substances in the venous outflow. introduction the starling concept of paracapillary fluid circula tion is well known. however, in studies of the movement of uncharged molecules from within the capillary to the extravascular space of the tissue, the effect of a transcapillary fluid flow on diffusion has all too frequently been ignored. one result of this omission has been that in ex planations of the transcapillary separation between uncharged molecules of different sizes the pos sibility of the diffusion being modified by bulk flow has often been overlooked. this report describes an experimental approach suitable for examining whether the effects of con vection on diffusion may be disregarded in ac counting for the transport of uncharged solutes in the capillary bed of a hind leg a cat. methods preparation cats were anaesthetized with chloroform (30 mg/kg body weight intravenously). isolated from and nembutalb an amount of heparin just sufficient t o prevent coagulation (about 10 000 iu) was administered. after the paw was removed, a circular incision with a cautery was made in the skin ' j u s t below the inguinal ligament and just above the greater 10 732853 trochanter. by blunt dissection, all muscular bundles were freed and cut down lo thz hip joint. ligatures were care fully tied around each muscle to prevent bleeding. with the excep:ion of the femoral artery and veins, all vessels were ligated separately. after the leg was exarticulated, the animal was bled. mean-while the femoral artery and vein of the leg were cannulated, the pareparation was laid o n a balance (with an accuracy of 25 mg), and attached to the perfusion equipment, as shown in fig. 1 a and b. from a reservoir the thermostated-controlled and oxygenated blood of the cat was circulated by a variable-speed peristaltic pump through the arterial and venous vessels, by-passing an injection loop, and returned to rhz reservoir, by-passing a sampling loop. the mean blood-perfusion flow was about 15 ml/min. papaverin (usually around 60 mg) was introduced into the blood to produce a maximal dilatation of thz blood vessels of the preparation, which was reached when an additional dose of papaverin did not furiher decrease the perfusion pressure. the venous pressure depended on thz lengths and diam eters of the outflow catheters. apparatus the experiment was started with the venous blood flow ing thrsugh c (fig. 1 b). when th: leg weight was con stant and th: venous pressure stable, an injection was given by switching the arterial blood flow through th: injecticn loop, which contained the bolus (tube a, fig. 1 b). as soon as possible after the injection, the outflow was switched to route c-d-e for sampling. about 30 samples were taken during thvenous passage of cne bo lus, which on the average lasted for 20-60 sec. by changing the venous outflow route from c t o f-d with e closed, the venous pressure increased. this in crease was controlled by varying the length a n d / o r ths diameter of the catheter. this procedure caused the tissue to become oedematous. after a few seconds, when the leg weight increased at a constant speed and the venous pressure was again stable, a new bolus was injected and collected by opening e. the bolus in the injection loop comprised a mixture of five parts of homologous blood, one part of glucose and raffinose (about 930 m m of each) and on: part of 3 mg/ml evans blue in physiological saline, which served to label the albumin. the injection loop contained about 1.3 ml. upsala j m e d sci 78 146 b . dberg and s . 0 . lindahl w d recorder force transducer fig. i a. a schematic drawing of the ex perimental set-up. the hind leg of a cat is placed o n the balance and the arterial and venous cannulae are connected to the femoral artery and vein. leg weight (oedema formation) and arterial and venous pressures (pa and pv) are measured, and the frac tion collector is connected to the event marker on the recorder for measuring sam pling time. the thermostate-controlled and oxygenated blood is circulated through the leg via the injection and sampling device (id) shown in fig. 1 b. analyses 1. venous-outflow concentration curves, as shown in serial samples of the outflow, each about 250 in fig. 2, in which the concentrations were given as relative volume, were collected in small, weighed glass which to the concentrations in the injection solution (relative were then weighed again and immersed i n 2.00 ml of concentrations). physiological saline in centrifuge tubes. after the mixing, 2. the relative concentrations of raffinose and glucose aliquots were analysed for glucose by a g~ucose-oxidase plotted against the relative concentration of albumin la method (1, 6 ) and for raffinose by determining fmctose belled with evans blue for each sample, as shown in fig. after a seliwanoff reaction (8). the remainder was centri3. the plots appeared to lie o n straight lines, and the fuged and the analysed for e~~~~ blue at 610 regression coefficients were calculated according to bart nm in a zeiss pmq spectrophotometer. lett ( 2 ) . the lines pass close to the origin, which means the three measurements mentioned above were made that the regression coefficient equals c a / c , , where cd is on ( a ) blood s a m p ~ e s drawn prior to the bolus injection the concentration of the diffusible and c, that of the (“blanks”), ( b ) the bolus itself, and (c) the serial samples reference substance. from the venous outflow. 3. extractions calculated according to the formula e = (c,-ccd)/c, o r l c d / ’ c , ( 3 , 4 , 5). thus, extraction can be calculated from the regression coefficient in fig. 3. ex traction expresses the fraction of the total amount lost calculations the were carried out with the help of a from the bolus or any part thereof, which was eliminated digital computer (cd 3600 or ibm 370) and were pref r o m the blood through the capillary wall. sented in the following forms: to pressure transducer from pump -+ to artery 4 to sampling from vein fig. i b. the injection and sampling device. upper catheter is “arterial”. blood goes via either route a (injection) o r route b. below the “venous” catheter is shown; route c, free flow; route c-d-e, sampling in free flow. route f includes a thin ex changeable catheter producing venous con gestion with or without sampling. upsala j m e d sci 78 investigating rates of transcapillary exchange 147 25 20 15 10 5 n yo of injection conc. x a glucose 0 raffinose x evans blue 0 5 10 15 20 rn\ fig. 2. the relative venous concentrations of albumin (tagged with evans blue), raffinose and glucose in bal anced-weight conditions (no net filtration), presented ac cording to chinard and crone (4, 5, 6 ) . discussion methods the preparation combines some of the qualities of those previously described (5, 7). one important problem is whether or not the perfused organ has suffered from the operation technique. fortu nately, most preparations were successful, in the sense that they were in weight balance (no oedema formation). this balance could be maintained for hours, provided the blood was well oxygenated. occasionally, however, some preparations showed a slow but steady oedema formation, even when the venous pressure was negative. these prepara tions were discarded. i n order to obtain a homogeneous distribution of the bolus within the arterial vessel leading to an organ, the injection has to be fairly rapid. when this is given by a syringe, such a rapid in jection is accompanied by a transient change in both blood pressure and flow. it cannot be safely assumed that this has any effect on the exchange between capillary and tissue. when the injection is given by a separate injection loop, such transi ents are avoided or at least made small enough not to be detected by the arterial-pressure trans ducer. although the bolus concentration is 540 mm and might therefore be expected to cause a loss of water from the tissue, only in one instance was there even a slight leg-weight decrease (about 3.4 x g/sec, 100 g tissue). the explanation of this might be that the reflection coefficient for raffinose and glucose are so low as to have little osmotic effect across a capillary wall. these sugars might rapidly disappear from the blood stream, and the bolus thus behaves as if it were virtually isotonic in the exchange capillaries. c o n c . diff. , identity l i n e 25 20 15 10 5 / / raffi nose glucose raffinose & glucose (congested) a raffinose 4 glucose conc. ref. 5 10 15 20 25 fig. 3. the relative concentration of the diffusible molecule (conc. diff.) plotted against the relative con centration of the reference (conc. ref.). the continuous lines are the best fit of straight lines calculated according t o bartlett (3). the angles of inclination of these lines are used to calculate the extractions. note that during rapid oedema formation (raffinose and glucose (congested)) the extrations of raffinose and glucose are of about the same magnitude. upsala j m e d sci 7% 148 b . aberg and s . 0 . lindahl calculations the slop: of the regression line between reference and test-solute concentrations in the venous out flow is acd/ac,.. this ratio differs from the ratio c,/c, if the line does not pass through the origin. there are three possibilities for cd not t o be zero when c, is zero: 1. the diffusible substance passes back from the tissue to the blood (back diffusion or back transport). if this blood-tissue-blood transport were slower o r faster than the intravascular passage of the bolus through the exchange vessels, there would be a time-lag between the venous appear ance of the reference and test substance (compare the results in ref. (9)). in this case, the cd/c, ratios would not fall on a straight line but would form a loop. fig. 3, however, showed no sign of such a loop. 2. the reference substance follows an axial stream in the blood, whereas the test solute does not. in that case, there would also be a different appearance time between the “faster” reference solute and the test solute. for the substances used, this possibility seems excluded. 3. the base-line in fig. 2 has not been deter mined correctly. this would appear as a constant addition to the regression line (a parallel displace ment of the regression line), if the concentration ratios fall on a straight line. in that case, the cal culated ratio a c d / a c r would probably be a more reliable estimate of the extraction than the ratio the advantage of this extraction-estimate meth od (regression method) is its simplicity. i t further gives information about the constancy of the ex traction, it rules out the errors in concentration measurements a t the beginning and the end of the venous-outflow-concentration curves, as these points bear little weight in calculating the regres sion line, and lastly it involves the possibility of excluding errors in base-line determinations. whatever method is used for calculating ex tractions, the values obtained will be erroneously low if the reference substance leaks out from the vessels. in that case, the concentration of the ref erence solute in the venous outflow must be cor rected by multiplying by a correction factor (1 + the lost fraction of the reference substance). whether or not such a leak of the reference sub stance used in the work described in this paper occurs will be further investigated. upsala j m e d sci 78 c d / cr. preliminary results in the balanced-weight condition (when the per fused leg suffers no net change in weight during the bolus passage), the relative amounts of glucose and raffinose lost to the tissue seem proportional to their diffusion coefficients in free solution and the results seem to confirm those of crone (5) (figs. 2 and 3). i n the experiment cited here, the (raffinose/glucose) extraction ratio amounted to 0.65, which equals the free diffusion coefficient ratio (0.434/0.673 = 0.645). on the other hand, when there is venous con gestion, the loss of both raffinose and glucose is increased. with increasing speed of oedema forma tion, raffinose is lost relatively more quickly than glucose, until the differences in their relative concentrations in the venous outflow finally vanish (fig. 3). the preparation described here and the regres sion method of calculate extractions is to be used in further studies of the transcapillary transport of raffinose and glucose, both in stable weight conditions and during oedema formation. acknowledgements this work was supported by grants from the medical faculty, uppsala university, the swedish medical research council (project no. 14x-629) and the national institutes of health (grant no. 5 r 0 1 h e 12960-08). the authors also wish to express their gratitude to miss carin ferner for valuable technical assistance. this paper was translated by neil tomkinson. references 1. aberg, b.: interference of light o n the determination of low glucose concentrations with glucose exidase. acta physiol scand 71: 186-193, 1967. 2. bartlett, m. s.: fitting a straight line when both vari ables are subject to error. biometrics 5: 207-212, 1949. 3. chinard, f. p. & enns, t.: transcapillary pulmonary exchange of water in the dog. am j physiol 178: 197 202, 1954. 4. crone, c.: o m diffusionen af nogle organiske non elektrolyter fra blod ti1 hjernevaev. (thesis.) munks gaard, copenhagen, 1961. 5. crone, c.: the permeability of capillaries in various organs as determined by use of the “indicator diffusion” 6. 7. method. acta physiol scand 58: 292-305, 1963. keilin, d. & hartree, e. f.: specificity of glucose oxi dase (notatin). biochem j 50: 331-334, 1952. pappenheimer, j. r. & soto-rivera, a,: effective os motic pressure of the plasma proteins and other quantities associated with the capillary circulation in the hindlimbs of cats and dogs. a m j physiol 152: 471 491, 1948. investigating rates of transcapillary exchange 149 8. roe, j. h., epstein, j. h. & goldstein, n. p.: a photometric meth3d for the determination of inulin in plasma and urine. j biol chem 78: 839-845, 1949. 9. martin, p. & yudilevich, d.: a thzory for the quantifi cation of transcapillary exchange by tracer dilution curves. am j physiol 207: 162-168, 1964. received february 17, 1972 revised mars is, 1973 address for reprints: bo aberg institute of physiology and medical biophysics uppsala university biomedical center box 512 s-751 23 uppsala 1 sweden upsala j m e d sci 78 upsala j med sci 90: 139-145, 1985 biochemical and morphologic studies of the prostate gland in men subjected to radical cystectomy bernd stegmayr,’ christer busch,’ b;ke fritjofsson3 and gunnar ronquist4 ‘department of internal medicine, central hospital, eskilstuna and the ’departments of pathoiogy, 3urology, and 4clinical chemistry, university hospital, uppsala, sweden abstract whole human p r o s t a t e g l a n d s o b t a i n e d from p a t i e n t s u n d e r g o i n g r a d i c a l c y s t ectomy were d i s s e c t e d i n t o t h r e e p a i r e d l o b e s and t h e v a r i o u s p a r t s o f t h e g l a n d were s u b j e c t e d t o b i o c h e m i c a l and m o r p h o l o g i c e x a m i n a t i o n . no d i s t i n c t d i f f e r ences were f o u n d between t h e p r o s t a t e l o b e s i n r e g a r d to c o n t e n t o f d i v a l e n t c a t i o n s , a c i d phosphatase and atpase. these f i n d i n g s were c o n c o r d a n t w i t h ob s e r v a t i o n s a t l i g h t microscopy. hence, d e s p i t e d i s c e r n i b l e change i n c e l l u l a r appearance, no d i s t i n c t b o r d e r was o b s e r v e d between l o b e s i n d i c a t i n g s e p a r a t e “compar tmen t s “ introduction a c i d phosphatase, zn2+ and mg2+ a r e w e l l known s e c r e t o r y p r o d u c t s i n human s e m i n a l plasma (4,5,6,9 ).an mg2+ and ca2+-dependent atpase system was f o u n d t o be a n o t h e r i m p o r t a n t s e m i n a l component ( 1 0 ) . s t u d i e s o f s p l i t e j a c u l a t e r e v e a l e d a l l t h e s e p a r a m e t e r s t o be p a r t o f t h e human p r o s t a t i c s e c r e t i o n (1,lo). i n f u r t h e r a n a l y s e s t h i s atpase a c t i v i t y was f o u n d t o be a s s o c i a t e d w i t h t h e p r e s e n c e o f o r g a n e l l e s i n t h e s e m i n a l plasma ( 1 0 ) . the aim o f t h i s s t u d y was t o c l a r i f y more t h o r o u g h l y t h e e x t e n t t o w h i c h t h e s e o r g a n e l l e s , l a t e r d e n o t e d prostasomes ( 2 , 1 2 ) , and a l s o t h e o t h e r sub s t a n c e s , a r e p r e s e n t i n and e x c r e t e d f r o m t h e human p r o s t a t i c g l a n d , and wheth er l o c a l d i f f e r e n c e s w i t h r e g a r d t o c o n t e n t o f t h e s e s u b s t a n c e s e x i s t i n d i f f e r e n t p a r t s o f t h e p r o s t a t e . material and methods p r o s t a t i c t i s s u e was o b t a i n e d f r o m seven men aged 5 9 68 y e a r s who were un d e r g o i n g r a d i c a l c y s t e c t o m y , i n c l u d i n g t o t a l p r o s t a t e c t o m y , because o f b l a d d e r c a n c e r . none o f t h e men had had symptoms or s i g n s o f p r o s t a t i c d i s e a s e b e f o r e 10-858572 139 the operation. all had received preoperative irradiation to a dosage of 40 gy over 3 weeks, and cystectomy and urinary diversion were performed 3 weeks after termination of radiotherapy. immediately after the operation the prostatic gland was dissected free from the bladder, the seminal vesicles and adhering tissues. the prostate was then further dissected into three paired lobes as described by tissell & salander in 1975 (13). in three of the seven cases the prostatic tissue was immediately prefixed in isotonic glutaraldehyde (3 % ) buffer solut ion (280 mosmol/l), kept at 4oc until fixation for electron microscopy with osmium tetroxide,and embedded in epon according to ronquist et al. (10). in the other four cases all the material was immediately stored in a moist chamber at o0c and kept in a solution of ice and water to maintain temperature stability. after 4 to 7 hours representative specimens of prostatic tissue were carefully cut from this material and prefixed in glutaraldehyde for electron microscopy as outlined above. from the remaining prostatic cut sections,secretory fluid was gently squeezed from the ductules and collected in small plastic tubes. the fluid was diluted 1:3 with 0.15 m nacl solution and centrifuged at 2 000 x g for 10 minutes. the residual pellet, containing cells and cell debris, was dis carded and the supernatant was used for biochemical analyses. magnesium, calcium and zinc were determined in an atomic absorption emission spectrophotometer. acid phosphatase was measured colorimetrically according to a tartrate-inhibition method (7). mg2+and ca2+-dependent atpase activity was measured according to ronquist et al. (10). the protein concentration was meas ured with the method of lowry et al. (8). to avoid influence of possible ex ternal fluid on concentration, the parameters were related to the protein con centration of the fluid. in three patients the prostatic dissection disclosed well-defined periureth r a l hyperplastic nodules, the largest with a diameter of 20 mm. the nodules were removed from the prostatic tissue without rupture of the surrounding cap sule. the solitary nodules likewise were sectioned for electron microscopy. moreover, secretion was obtained by gentle squeezing for the aforementioned biochemical investigations. no contamination with prostatic tissue material was possible, as the nodules were separately prepared. results biochemical investigations the values for acid phosphatase, calcium, magnesium and zinc were as expect ed for prostatic fluid, although the analyzed specimens were obtained from squeezing of the dissected glandular material (table 1). in all cases the mg 2+ 140 2 and ca d e p e n d e n t atpase a c t i v i t y c o u l d r e a d i l y b e r e c o r d e d and was p r e s e n t a t a l e v e l h i g h e r t h a n t h a t e x p e c t e d f o r mixed s e m i n a l plasma ( c o n t a i n i n g s e c r e t i o n a l s o from s e m i n a l v e s i c l e s , t e s t e s and e p i d i d y m e s ) . the h y p e r p l a s t i c n o d u l e s d i s p l a y e d e s s e n t i a l l y t h e same c o n t e n t o f t h e s e s u b s t a n c e s a s t h e p r o s t a t i c t i s s u e . t a b l e 1. o f f l u i d from t h e p r o s t a t e g l a n d i n 4 men and i n adenornatous t i s s u e from 3 o f t h e same g l a n d s atpase, a c i d p h o s p h a t a s e (ap), c a l c i u m ( c a ) , magnesium (mg) and z i n c ca mg zn atpase -1 ap (nmol x min ( m k a t x g x g p r o t e i n ) p r o t e i n 1 ) -1 ( a m 0 1 x g p r o t e i n ’ ) p r o s t a t i c a l a n d mean 54.5 235 1 7 1 44.4 21.0 sdn-l 41.3 197 111 1 7 . 1 1 2 . 0 r a n g e 8 2 1 7 0 59.0-684 72.2-396 24.8-68.0 7.9-47.0 h y p e r p l a s t i c n o d u l e s m a n 50.1 1 2 9 . 3 1 3 5 54.2 21.6 son-’ 1 6 . 5 5 3 . 5 48.7 29.6 5 . 3 r a n g e 38-6-69. o 9 1 .o-190 98-6-190 34.8-88.3 16.3-26.9 the c o n c e n t r a t i o n o f t h e s e s u b s t a n c e s v a r i e d t o some e x t e n t w i t h i n d i f f e r e n t p r o s t a t i c l o b e s , b u t w i t h o u t s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s ( t a b l e 2 ) . nor was any mutual c o r r e l a t i o n found between t h e p a r a m e t e r s w i t h d i f f e r e n t l o c a t i o n s i n t h e g l a n d . t a b l e 2. a s i n t a b l e 1. means ( s o n 1 ) ] v a l u e s f o r p r o s t a t i c f l u i d a c c o r d i n g t o l o b e o f o r i g i n [ a b b r e v i a t i o n s atpase ap ca mg zn lobes from 4 g l a n d s d o r s a l l a t e r a l 4 0 . 9 258.9 1 7 8 . 0 4 4 . 1 23.2 ( 2 4 . 3 ) ( 2 1 2 . 9 ) ( 1 0 1 . 8 ) (20.8) ( 1 2 . 6 ) 75.7 298.0 144.0 4 0 . 1 21.9 ( 6 4 . 1 ) ( 2 6 9 . 0 ) ( 1 1 4 . 0 ) (11.1) (17.5) medial 42.0 149.0 192.0 49.1 1 7 . 8 ( 1 5 . 1 ) ( 9 5 . 2 ) ( 1 4 1 . 0 ) ( 2 1 . 4 ) ( 6 . 2 ) 141 f r u c t o s e was n o t d e t e c t e d i n any o f t h e s e f l u i d sl g l a n d . jecimens f r o m t h e p r o s t a t e f i g . 1 a. s e c t i o n showing t h e d o r s a l l o b e p a t t e r n . a c i n i a r r a n g e d i n a p a r a l l e l p a t t e r n w i t h s i m p l e p a p i l l a r y p r o j e c t i o n s ( o r i g i n a l m a g n i f i c a t i o n x 4 1 ) . b. l a t e r a l l o b e p a t t e r n w i t h i r r e g u l a r a c i n i and c o a r s e stroma. n o t e t h e mild i n f l a m m a t o r y changes and s l o u g h i n g o f t h e g l a n d u l a r e p i t h e l i u m , p r o b a b l y due t o t h e p r e o p e r a t i v e i r r a d i a t i o n ( x 4 1 ) . c . m e d i a l l o b e p a t t e r n w i t h p r o m i n e n t p a p i l l a r y f o r m a t i o n s ( x 41). 142 liaht microscow the morphologic pattern within each isolated lobe was generally variable (fig. l), 1.e. did not show purely medial, lateral o r dorsal lobe chanacterist ics as described by earlier authors (11). in two cases, however, pure patterns were observed, one with a typical dorsal lobe pattern and another with a typical lateral one. transmission electron microscopy fig. 2 shows the intracellular location of prostasomes within other, bigger organelles, the storage vesicles. very few, if any, prostasomes are free in the cytoplasm (2). the size and general appearance of the intracellular prostasomes are the same as in prostasomes isolated from prostatic fluid and seminal plasma (2). discussion the patients from whom the tissue material was obtained had been irradiated preoperatively because of bladder cancer. as the radiotherapy was directed not only towards the bladder, but also to the pelvic lymph nodes, shedding irradi ation could have involved the prostatic tissue used in our investigations. the -registered biochemical values thus may have been lower than in nonirradiated men, but this did not seem to be the case. ultrastructural, atrophic changes may also be expected after radiotherapy. in the present study there were no distinct differences between the prostate lobes in regard to their content of divalent cations, acid phosphatase and atp ase. convincing evidence has accumulated that this latter enzyme system is in timately linked to the enveloping membrane of organelles (prostasomes) occurr ing free in prostatic fluid and seminal plasma (10) as well as in prostatic tissue (secretory cells and acinar ducts, cf reference 2 and fig. 2). the atpase activity may function as a quantitative measure of the presence of prostasomes. these findings were concordant with the observations at light microscopy. hence, although a change in cellular appearance was discernible, no distinct boundaries were observed between lobes that could indicate "com partments". our study also indicated similarities between hyperplastic and normal prost atic tissue, although the hyperplastic tissue did not macroscopically present ducts connecting with other prostatic ducts. earlier authors (14) showed that the zinc content/g tissue was at least equal in hyperplastic and normal prost atic tissue. in o u r cases, moreover, the hyperplastic nodules were enveloped in a fibrous capsule. this investigation supports the view that hyperplastic tissue may develop from obstructed parts o f the true prostatic gland, because of the demonstrated similarities between the two tissue t y p e s . 143 144 fig. 2 a. storage vesicles ( s ) in epithelial prostatic cells. the storage vesicles containing prostasomes are encased in a trilaminar membrane (x 2 7 000) b. higher magnification of membraneenveloped prostasomes (p) in storage vesicles ( x 6 7 0 0 0 ) references 1. 2. 3 . 4. 5. 6. 7. 8. 9. 10. 11. 1 2 . 1 3 . 1 4 . brody, i., ronquist, g., gottfries, a. & stegmayr, b.: abnormal deficiency of both mgand ca-dependent adenosine triphosphatase and secretory granules and vesicles in human seminal plasma. scand j urol nephrol 1 5 : 8 5 9 0 , 1 9 8 1 . brody, i., ronquist, g. & gottfries, a,: ultrastructural localization o f the prostasome an organelle in human seminal plasma. upsala j med sci 8 8 : 6 3 8 0 , 1 9 8 3 . clark, m.a., o'connell, k.j. & edson, m.: scanning elect ron microscopic studies of control and hypertrophic human prostate glands. j urol 1 1 0 : 5 5 8 5 6 0 , 1 9 7 3 . eliasson, r. & lindholmer, c.: zinc in human seminal plasma. andrologie 3 : 1 4 7 1 5 3 , 1 9 7 1 . eliassan, r. & lindholmer, c.: magnesium in human seminal plasma. invest urol 9 : 2 8 6 2 8 9 , 1 9 7 2 . fisher, e.r. & jeffrey, w.: ultrastructure of human normal and neoplastic prostate, with comments relative to prosta tic effects of hormonal stimulation in the rabbit. am j clin path 4 4 : 1 1 9 1 3 4 , 1 9 6 5 . jacobsson, k.: the determination of tartrate-inhibited phosphatase in serum. scand j clin lab invest 1 2 : 3 6 7 3 8 0 , 1 9 6 0 . lowry, d.h., rosebrough, n.j., farr, a.l. & randall, r.j.: protein measurement with the folin phenol reagent. j biol chem 1 9 3 : 2 6 5 2 7 5 , 1 9 5 1 . mawson, c.a. & fisher, m.i.: the occurrence of zinc in the human prostate gland. canad j med sci 3 0 : 3 3 6 3 3 9 , 1 9 5 2 . ro mg" and ca prostatic fluid. parts i & 11. andrologia 1 0 : 2 6 1 2 7 2 , 4 2 7 4 3 3 , 1 9 7 8 . salander, h., johansson, s. & tisell, l.e.: the histology of the dorsal, lateral and medial prostatic lobes in man. invest urol 1 8 : 4 7 9 4 8 3 , 1 9 8 1 . stegmayr, b. & ronquist, g.: promotive effect on human sperm progressive mortality by prostasomes. urol res 1 0 : 2 5 3 2 5 7 , 1 9 8 2 . tisell, l.e. & salander, h.: the lobes of the human prostate. scand j urol nephrol 9 : 1 8 5 1 9 1 , 1 9 7 5 . tisell, l.e., fjelkegard, b. & leissner, k.h.: zinc concentration and content of the dorsal, laterla and medial prostatic lobes and of periurethral adenomas in man. j urol 1 2 8 : 4 0 3 4 0 5 , 1 9 8 2 . uist, g2+ gottfries, a., brody, i. & stegmayr, €3.: an -stimulated adenosine triphosphate in human address for reprints: ake fritjof sson department of urology university hospital s 7 5 1 8 5 uppsala sweden 145 upsala j med sci 90: 173-179, 1985 arterial hypertension-a disease of the juxtaglomerular apparatus? a. erik g . person and uk boberg department of urology, university hospital, and the department of physiology and medical biophysics, university of uppsala, sweden abstracl from a s p e c i a l s t r a i n o f g e n e t i c a l l y h y p e r t e n s i v e r a t s , t h e m i l a n h y p e r t e n s i v e s t r a i n (mhs), a r t e r i a l h y p e r t e n s i o n can be t r a n s p l a n t e d w i t h t h e k i d n e y t o t h e m i l a n n o r m o t e n s i v e s t r a i n (mns). d u r i n g development o f h y p e r t e n s i o n i n mhs r a t s t h e r e was an a c t i v a t i o n o f t h e t u b u l o g l o m e r u l a r feedback c o n t r o l t h a t re duced g l o m e r u l a r f i l t r a t i o n r a t e , l e a d i n g t o r e t e n t i o n o f e l e c t r o l y t e s and f l u i d . t h i s i n c r e a s e d e x t r a c e l l u l a r f l u i d volume r e d u c e s feedback s e n s i t i v i t y , b u t i n a f a s h i o n t h a t g i v e s r i s e t o c h r o n i c e x t r a c e l l u l a r f l u i d e x p a n s i o n and can t h e r e b y r a i s e t h e b l o o d p r e s s u r e . i n a l i m i t e d sense, a r t e r i a l h y p e r t e n s i o n i n t h e s e a n i m a l s e x i s t s t o p r e v e n t t h e k i d n e y f r o m r e t a i n i n g more e x t r a c e l l u l a r f l u i d volume. the a l t e r e d f u n c t i o n i n t h e j u x t a g l o r n e r u l a r a p p a r a t u s o f t h e mhs r a t s t h u s may e x p l a i n t h e r i s e i n a r t e r i a l b l o o d p r e s s u r e . introduction the t u b u l o g l o m e r u l a r feedback c o n t r o l (tgf) i s i m p o r t a n t f o r t h e c o n t r o l o f e x t r a c e l l u l a r f l u i d volume and a r t e r i a l b l o o d p r e s s u r e ( 1 1 , 1 9 ) , recent e x p e r i ments i n o u r l a b o r a t o r y (5,12,13) have i n d i c a t e d t h a t m a l f u n c t i o n i n g o f t h i s mechanism may be t h e cause o f development o f a r t e r i a l h y p e r t e n s i o n i n r a t s o f t h e m i l a n h y p e r t e n s i v e s t r a i n (mhs). some e v i d e n c e i n s u p p o r t o f t h i s c o n c e p t i s d i s c u s s e d i n t h e p r e s e n t paper. i n r e c e n t y e a r s t h e g r e a t i m p o r t a n c e o f t h e i n t e r s t i t i a l space and i t s p r e s s u r e s f o r t h e f u n c t i o n o f t h e k i d n e y has become a p p a r e n t . i n t h e r a t , t h e r e n a l i n t e r s t i t i a l h y d r o s t a t i c p r e s s u r e (pint) i s a b o u t 1-2 mm hg under c o n t r o l c o n d i t i o n s , and i n c r e a s e d by a b o u t 2 mm hg d u r i n g s a l i n e volume e x p a n s i o n b y 5 5 o f body w e i g h t (16,zo). the i n t e r s t i t i a l o n c o t i c p r e s s u r e ( 3 . ) was f o u n d t o b e a b o u t 4-5 mm hg and t o d e c r e a s e b y about 2 vm hg d u r i n g s a l i n e volume ex p a n s i o n (16). c o n s i d e r a b l e change e v i d e n t l y o c c u r r e d i n t h e n e t i n t e r s t i t i a l p r e s s u r e ( p . -$. i n t ) d u r i n g s a l i n e volume expansion, a s i t u a t i o n which we have i n t i n t 173 d e s c r i b e d a s " i n t e r s t i t i a l oedema". under c o n d i t i o n s o f d e h y d r a t i o n , o n t h e o t h e r hand, t h e r a t s showed v e r y l o w h y d r o s t a t i c and h i g h i n t e r s t i t i a l o n c o t i c p r e s s u r e , c o l l e c t i v e l y d e s i g n a t e d " i n t e r s t i t i a l d e h y d r a t i o n " i n o u r s t u d i e s . i n t h e d i s c u s s i o n o f volume r e g u l a t i o n , t h e nephron may be c o n s i d e r e d as two u n i t s . one i s v o l u m e r e g u l a t i n g , c o n s i s t i n g o f t h e g l o m e r u l u s , p r o x i m a l t u b u l e , and l o o p o f h e n l e t o t h e macula densa, a t w h i c h p o i n t t h e p o s s i b i l i t y o f a f e e d back l o o p e x i s t s . the o t h e r u n i t may be c a l l e d t h e f i n e a d j u s t i n g e l e c t r o l y t e and w a t e r e x c r e t o r y u n i t , c o n s i s t i n g o f t h e d i s t a l t u b u l e and c o l l e c t i n g d u c t s . c o n c e r n i n g t h e v o l u m e r e g u l a t i n g u n i t , w h i c h we s h a l l d i s c u s s i n t h i s paper, a l a r g e f l u i d volume i s f i l t e r e d i n t o bowman's space and from t h e r e f l o w s i n t o t h e p r o x i m a l t u b u l e . f l u i d r e a b s o r p t i o n i n t h i s segment u s u a l l y i s a c o n s t a n t f r a c t i o n o f t h e t o t a l f i l t r a t e , a b o u t 2 / 3 . i t would seem t h a t t h e mechanism o f t h i s f l u i d r e a b s o r p t i o n i s m u l t i f a c t o r i a l , and a l s o t h a t a change i n i n t e r s t i t i a l p r e s s u r e i n d u c e d b y a l t e r e d c a p i l l a r y p r e s s u r e can modulate f l u i d t r a n s f e r ( 6 , 7 ) . i n a s i t u a t i o n t h a t l e a d s t o " i n t e r s t i t i a l oedema", t h e p r o x i m a l t u b u l a r f l u i d r e a b s o r p t i o n i s reduced, whereas d u r i n g " i n t e r s t i t i a l d e h y d r a t i o n " i t i s i n c r e a s e d . t h i s a l t e r a t i o n i n f l u i d a b s o r p t i o n r a t e may be due t o an e f f e c t on a d i r e c t pressure-dependent f l u i d t r a n s f e r a c r o s s t h e p r o x i m a l t u b u l a r w a l l (1, 10). the n e x t segment down t h e nephron t h r o u g h w h i c h t h e f l u i d passes i s t h e l o o p o f henle. d u r i n g t h i s passage e l e c t r o l y t e s a r e p r e f e r e n t i a l l y r e a b s o r b e d , w h i l e w a t e r i s t r a n s p o r t e d t o a l e s s e r e x t e n t . t h i s e x p l a i n s t h e l o w sodium c o n c e n t r a t i o n i n t h e e a r l y d i s t a l t u b u l e . i f t h e f l u i d f l o w t h r o u g h t h i s seg ment i s i n c r e a s e d , e l e c t r o l y t e r e a b s o r p t i o n w i l l a l s o be i n c r e a s e d , b u t n o t f u l l y p r o p o r t i o n a t e t o t h e i n c r e a s e i n f l o w , w i t h t h e r e s u l t t h a t t h e e a r l y d i s t a l sodium c h l o r i d e c o n c e n t r a t i o n , w h i c h n o r m a l l y i s l o w , w i l l r i s e ( 8 , 1 4 ) . the s i t e o f c o n t a c t between t h e a l t e r e d c e l l s i n t h e d i s t a l t u b u l e , t h e macula densa c e l l s and a r t e r i o l e s i n t h e v a s c u l a r p o l e o f t h e k i d n e y , and t h e renin-granule-containing c e l l s c o n s t i t u t e t h e j u x t a g l o m e r u l a r a p p a r a t u s . t h i s i s an i m p o r t a n t r e g u l a t o r y u n i t , t h a t can sense t h e c h l o r i d e c o n c e n t r a t i o n i n t h e d i s t a l t u b u l a r f l u i d . the system is a c t i v a t e d when t h e e l e c t r o l y t e concen t r a t i o n i n c r e a s e s , l e a d i n g t o r e d u c t i o n i n g l o m e r u l a r c a p i l l a r y p r e s s u r e and g l o m e r u l a r f i l t r a t i o n r a t e (gfr) v i a a c t i v a t i o n o f t h e tgf mechanism, and t o i n c r e a s e d r e n i n r e l e a s e . the b a s i c f u n c t i o n o f a v o l u m e r e g u l a t i n g u n i t i n t h e nephron can now be o u t l i n e d . i f gfr f o r some r e a s o n t e n d s t o i n c r e a s e a s a r e s u l t o f c o n s t a n t f r a c t i o n a l p r o x i m a 1 r e a b s o r p t i o n , t h e f l u i d d e l i v e r y t o t h e l o o p o f h e n l e w i l l be en hanced. an i n c r e a s e d f l o w t h r o u g h t h i s nephron segment w i l l i n c r e a s e t h e e l e c t r o l y t e c o n c e n t r a t i o n i n t h e e a r l y d i s t a l t u b u l e and t h e r e b y a c t i v a t e t h e j u x t a g l o m e r u l a r a p p a r a t u s t o l o w e r t h e g f r and p r o d u c e r e n i n . i t has a l s o been found, however, t h a t t h e tgf mechanism i s o n l y s l i g h t l y a c t i v a t e d under c o n t r o l 174 conditions (11,151, i.e. the operational point is close to no activation at all (fig. 1). this means that under control conditions there is very little activ ation of the tgf system, just as there is only very low renin production. the question therefore arises: when is the feedback system activated to e f fect appreciable reduction of gfr '? from several lines of evidence we have de duced that changes in the extracellular fluid volume o r the blood pressure will alter the sensitivity of the tgf via changes in renal interstitial hydrostatic and oncotic pressures (11). under conditions of saline volume expansion o r post unilateral nephrectorny, o r in the initial period of ureteral occlusionall situations with "interstitial oedema"a low o r abolished tgf response was found, as shown by a shift of the response curve to the right in fig. 1. this of course constitutes an important resetting of the sensitivity to avoid reduc tion of gfr in a situation with demand f o r increased fluid excretion. gfr con sequently can remain high despite increased distal delivery o f fluid. i n situat ions such as saline volume expansion, renin release also is low. (mmhg) glomerular c a p i l l a r y p r e s s u r e 0 operation point 60 e r s t i t i a i o edema " 5 5 50 c o n t r o l 4 5 i n t e r s t i t i a i de h y dr a t ion " 4g 20 30 40 10 loop of henle f l o w r a t e (nllmin) fig. 1. glomerular capillary pressure estimated for stop-flow pressure in re lation to the f l o w rate in the loop o f henlc. comparisons under con trol conditions or with "interstitial oedema" o r "interstitial dehyd ration". the operation point for the mechanism is shown. 175 in states of dehydration, hypovolaemia o r hypotension, or following unilater al ureteral occlusion all situations with "interstitial dehydration"we found high tgf sensitivity with a low flow threshold and a large reduction o f maximal glomerular capillary pressure in response to increased flow. this im plies a shift of the response curve to the left compared with the control curve in fig. 1. further, in all of these situations with "interstitial dehydration", although the distal delivery of fluid was normal o r even subnormal, the increase in sensitivity was so large that the feedback was greatly activated to reduce glomerular capillary pressure and gfr. in dehydration, hypovolaemia and hypo tension the renin releaseincreased, indicating that activation of the t g f mech anism also proceeds parallel with renin release, a concept which accords with findings in earlier direct micropuncture studies ( 9 ) . the tgf thus is activated as a consequence of the extracellular fluid needs and the blood pressure level, and not solely as a result of increased delivery of fluid. this interrelationship, representing the influence of renal interstitial pressure on renal function, is illustrated in fig. 2. g f r and tubular fluid reabsorption, the latter being influenced by interstitial pressure, determine the excretion rate. furthermore, gfr and fluid reabsorption up to the early distal tubule determine the fluid load tothe juxtaglomerular apparatus. the response of this apparatus then depends on the degree of stimulation of the renal interstitial pressure. in situations with high-degree stimulation of the interstitial pressure receptor mechanism, such as hypotension, hypovolaemia and dehydration, the sensitivity of the tgf mechanism will be so high that even nor mal o r reduced distal delivery of fluid will be sufficient to activate the jux taglomerular apparatus that reduces gfr and increases renin production. these alterations will in turn change the urine excretion rate, which will lead to restoration of the fluid balance towards the control level. this feedback con trol loop will then act to restore the disturbed extracellular fluid volume balance to the control level and to the control operation point. such resetting of the tgf sensitivity is therefore an important determinant of body fluid vol ume and an important factor in normalisation of different types of volume o r blood pressure disturbance. the tgf mechanism will strive to restore the fluid volume of the body so that it attains the operation point on the control curve. in this fashion the t g f can determine the total volume of body fluid. the link between extracellular fluid volume and blood pressure is not yet clear, though theories concerning the mode of control have been proposed (17). it is clear, however, that in patients without kidneys the extracellular volume influences blood pressure. since the t g f mechanism and its resetting are im portant for the actual volume of the extracellular fluid, we made comparisons of the t g f and its resetting between rats of the milan hypertensive strain 176 c ns i i w tubular apparatus v i angiotensinl tubular . l i i rurine excretion] v renal lntersti t ial extra cell. vdume pressure 4 fluid f i g . 2. b l o c k diagram i l l u s t r a t i n g t h e i n f l u e n c e o f r e n a l i n t e r s t i t i a l p r e s s u r e i n r e n a l volume r e g u l a t i o n . reproduced by p e r m i s s i o n from t h e e d i t o r o f kidney i n t e r n a t i o n a l ( f r o m r e f e r e n c e 11). (mhs) and t h e m i l a n normotensive s t r a i n (mns). these s t u d i e s were o f p a r t i c u l a r i n t e r e s t because t h e h y p e r t e n s i v e disease i n mhs r a t s can be t r a n s p l a n t e d w i t h t h e kidney ( 2 ) . because o f d i f f e r e n c e s i n r e n a l f u n c t i o n , we s t u d i e d mhs r a t s d u r i n g t h r e e phases o f l i f e , and mns r a t s a t corresponding ages. the r a t s were f i r s t s t u d i e d i n t h e p r e h y p e r t e n s i v e phase, a f t e r weaning a t t h e age o f 4-5 weeks, when t h e i r weight was 60-100 kg. the second phase was d u r i n g development o f h y p e r t e n s i o n , which occurs a t 5-7 weeks when t h e body weight i s 90-150 g, and t h e t h i r d was an a d u l t phase, a t age about 3 months w i t h weight 250-350 9. e a r l i e r s t u d i e s by o t h e r a u t h o r s ( 3 ) showed t h a t mhs r a t s i n t h e p r e h y p e r t e n s i v e phase had h i g h gfr and low r e n i n p r o d u c t i o n compared w i t h mns r a t s . i n r e c e n t i n v e s t i g a t i o n s ( 5 , 1 2 ) we found t h a t i n t h e p r e h y p e r t e n s i v e phase t h e mhs r a t s had no tgf re sponse a t a l l , w h i l e mns r a t s showed normal response. t h i s absence o f a feed back response can e x p l a i n t h e h i g h gfr, s i n c e t h e o p e r a t i o n p o i n t u s u a l l y shows some s l i g h t a c t i v a t i o n o f t h e tgf mechanism i n response t o reduced gfr. these f i n d i n g s a r e a l s o i n l i n e w i t h t h e low r e n i n r e l e a s e t h a t was observed. d u r i n g development o f h y p e r t e n s i o n , by c o n t r a s t , t h e mhs r a t s d i s p l a y e d a tgf mechanism w i t h very h i g h s e n s i t i v i t y , w i t h a low f l o w t h r e s h o l d l e a d i n g t o 177 activation o f the tgf to reduce gfr. in this phase there is relative reduction in gfr and relative increase in renin production and sodium retention. these observations can readily be explained by the high feedback sensitivity, which will reduce gfr and retain electrolytes. the process thus will lead to fluid retention in the mhs animals that will tend to normalize tgf sensitivity but also increase arterial blood pressure. indeed, in the adult mhs rats we found a normal tgf response. however, both in the adult mhs rats and during the de velopment of hypertension, the resetting of tgf sensitivity was impaired. dur ing volume expansion the high sensitivity was only slightly reduced, and was not reset to a lower level as it was in the mns rats. we also found that the inter stitial pressure was normal both under control conditions and during volume ex pansion. a change in interstitial pressure could therefore not explain the im paired tgf resetting. the mechanism of action, speculatively, seems to be as follows. there is an increased transmembrane electrolyte transport in red cells and proximal tubular cells of mhs rats. many cells in the mhs rat appear to have this transport ab normality (4). if the abnormal transport occurs also in the macula densa cells, an increase in chloride o r sodium chloride transport may activate the feedback and explain the high sensitivity that develops in this situation. the impaired resetting during volume expansion will then keep gfr low and retain fluids and electrolytes even when there is a volume overload. the result will be a rise in arterial blood pressure. both the volume expansion and the increase in blood pressure can then restore the tgf sensitivity to a normal level. thus, in a limited sense it appears that the high arterial pressure in the mhs rats exists in order to prevent the kidney from retaining an even greater extracellular volume of fluid. these studies have convinced us that the tgf mechanism plays an in 1. 2 . 3. 4. 178 important r o l e in the development and maintenance of arterial hypertension the mhs rats. references agerup, b . & persson, a.e.g.: modulation of proximal tubular hydraulic conductivity by peritubular capillary oncotic pressure. acta physiol scand 115:355-359,1982. bianchi, g., fox, u., difrancesco, c.f., giovanetti, a.m. & pagetti, d.: blood pressure changes produced by kidney crosstransplantation between spontaneously hypertensive rats and normotensive rats. clin sci mol med 47:435-448,1974. bianchi, g., baer, p.g., fox, u., duzzi, l . , pagetti, d. & giovanetti, d.m.: changes in renin, water balance and sodium balance during develop ment of high blood pressure in genetically hypertensive rats. circ res 37:153-161,1975. bianchi, g., ferrari, p. & barber, b.r.: the milan hypertensive strain. in handbook of hypertension, vol. 4. experimental and genetic models o f hypertension. ed. de jons. elsevier scient pub1 1984. boberg, u. & persson, a.e.g.: increased tubuloglomerular feedback activity in milan hypertensive rats. am j physiol: submitted. brenner, b.m., falchuk, k.h., keimowitz, r.l. & berliner, r.w.: the relat ionship between capillary protein concentration and fluid reabsorption by the renal proximal tubule. j clin invest 48:1519-1531,1969. ichikawa, i., hoyer, j.r., seiler, m.w. & brenner,b.m.: mechanism of glomerulotubular balance in the setting of heterogeneous glomerular in jury. j clin invest 69:185-198,1982. morgan,t. & berliner, r.w.: a study by continuous microperfusion of water and electrolyte movements in the loop of henle and distal tubule o f the rat. nephron 6:388-405,1969. morgan, t. & davis, j.m.: renin secretion at the individual nephron level. pflugers arch 359:23-31,1975. persson, a.e.g., schnermann,j., agerup, b. & eriksson, n-e.: the hydraulic conductivity of the rat proximal tubular wall. determinations with coll oidal solution. pflugers arch 36095-44,1975. persson, a.e.g., boberg, u., hahne,b., muller-suur, r., norlhn, b-j. & selen, g. interstitial pressure as a modulator of the tubuloglomerular feedback control. kidney int 22:122-128,1982. persson, a.e.g., bianchi, g. & boberg, u.: evidence of defective tubulo glornerular feedback control in rats of the milan hypertensive strain (mhs). acta physiol scand 122:217-219,1984. persson, a.e.g., bianchi, g. & boberg, u.: tubuloglomerular feedback in hypertensive rats of the milan strain. acta physiol scand 123:135-146,1985. schnermann, j.: microperfusion study of single short loop o f henle in rat kidney. pflugers arch 300:255-282,1968. schnermann, j., persson, a.e.g. & agerup, 6 . : tubuloglomerular feedback: nonlinear relation between glomerular hydrostatic pressure and loop of henle perfusion. j clin invest 58:862-869,1973. selen,g. & perssor1,a.e.g.: hydrostatic and oncotic pressures in the inter stitium of dehydrated and volume-expanded rats. acta physiol scand 117: de wardener, h.e. & macgregor,g.a.: dahl's hypothesis that a saluretic substance may be responsible for a sustained rise in arterial pressure: its possible role in essential hypertension. kidney int 18:l-9,1980. wolgast,m., persson,e., schnermann,j., ulfendah1,h. & wunderlich,p.: the colloid osmotic pressure of the subcapsular interstitial fluid of rat kid neys during hydropenia and volume expansion. pflugers arch 340:123-131, 1973. wright, f.s. & briggs, j.p.: feedback control o f glomerular blood flow, pressure and filtration rate. physiol rev 59:958-1006,1979. wunderlich,p., persson,e., schnermann,j., ulfendah1,h. & wolgast,m.: hy drostatic pressure in the subcapsular interstitial space o f rat and dog kidneys. pflugers arch 328:307-319,1971. 75-81,1983. 5. 6. 7. 8 . 9. 10. 11. 1 2 . 13. 14. 15. 16. 1 7 . 18. 19. 20. address for reprints a. erik g. persson department of urology university hospital s-751 85 uppsala sweden 179 upsala j med sci 85: 97-102, 1980 significance, importance and equality three basic concepts in the analysis of a difference adam taube department of statistics, university of uppsala, uppsala, sweden abstfact by means of data from fictitious cross over trials, it is first demonstrated that a statistically significant difference is not necessarily of a practically important order of magnitude. this fact is of special interest when the number of observations is large. second, a statistically non significant difference does not prove the hypothesis about equality between, say, treatment effects. this fact is of special interest when the number of observations is small. for investigating whether equality is plausible, confidence intervals are more use ful than non significant results from tests of significance. introduction the purpose with this little note is to give an illustration of how elemen tary statistical methods can be applied in order to answer two principally dif ferent questions about a comparison between means or between proportions. the first question is whether a difference is statistically significant and the second whether it is of an important order of magnitude. if both these questions lead to negative answers, it is natural to ask whether it can be said that the two groups (treatments etc) being compared, are equal. the concepts "signifi cance", "importance" and "equality" relate to three different aspects of a com parison. these aspects are all of interest both in observational studies and in experimental situations. material and methods as an example, we consider a cross over trial, where two drugs, a and b, are being compared. for simplicity, we assume that the results concerning each of them can be given in the form tlimprovementtt or "no improvement". thus, the data are of the kind illustrated in table 1. 7-802858 97 table 1. frequencies in a cross over trial. drug a no improvement improvement total drug b no improvement a b a+b improvement c d c+d total a+c b+d n the standard analysis concerning the statistical significance of the effect difference, is the well known mcnemar test (2) 1 d. fr 2 x2 = (b c) /(b + c) which is based only on those individuals who give different judgements of the two drugs. if this test statistic gives a sufficiently large value, the conclu sion is that the two treatments have different effects. the result of this test is unaffected by the magnitude of the two frequencies a and d. it is obvious that if the frequencies b and c constitute a very minor frac tion of the total number of observations n, the result of the above test might r be of little or no interest. this can be so, even if the xd-value i s very large, indicating a strong statistical significance. instead, one ought to study the rates of improvement ,. a pa = (b+ d)/n and pb = (c + d)/n a and we notice that their difference, d, can be written as “ . a a d = pa pb = (b c)/n this is the appropriate measure let us now define a variable of whether the difference is important o r not. xa such that fl if improvement with drug a x = j a l 0 if no improvement with drug a and similarly for ence z. = x. xy where i = 1, 2, ..., n. if, for a certain individua1,there is no difference in effect between drug a and drug b we get better than b we get obviously, the variable z will have the values -1, 0 and + 1 with the frequen xb . for each of the n individuals, we can form the differ a 1 1 z = 0. if a is z = 1 and if drug b is better than drug a we get z = -1. 98 ties c, (a + d) and b respectively, as illustrated in fig. 1 fig. 1. b distribution of z = xa x 1 0 + 1 z we notice that the average of the variable z is and it is easily verified that the standard deviation s is obtained from 2 s 2 = [n(b + c ) (b c) ]/(n 1) n if n is large, z can be assumed to be a normally distributed variable irre spective of the shape of the distribution of the variable z. therefore, we can give a confidence interval for the true difference d by means of z 2 k se(z) where se(z) = s z / a and the value of k is obtained from a table of the standard normal distribution. we denote the upper and lower confidence limits du and dl and apply 95% confi dence level, when calculating them for the six different data sets preserted in table 2. h h instead of using mcnemar's test, it is possible to form the critical ratio c . r . & (i 0 ) / s z this will give a very similar result, since the two test statistics are strong ly related. in fact, it can easily be shown that 2 2 2 ( c . r . ) = x (n l)/(n x ) 99 table 2 . data from six fictitious trials. frequendata set cies i i1 i11 iv v vi a 10 1 2 0 3 2 0 1 0 120 32u b 1 5 1 5 1 5 12 1 2 1 2 c 5 5 5 b 8 8 d 20 260 660 20 260 660 n 50 400 1000 50 400 1000 5.0* 5.0* 5.u* .8 .8 .s 2 x c . r . 2.33* 2.25* 2.24* -89 .89 .89 a y o 0 3.2% .3% .1% -9.6% -1.2% -.5% duioo 36.8% 4.7% 1.9% 25.6% 3.2% 1.3% * ) significant. p < .05 discussion wnen comparing data sets i, i1 and i11 we notice that the x -values are ex2 actly the same, indicating a statistically significant difference. however, the confidence interval for this difference is very wide in data set i, where n=50, while it is quite narrow and not far from the point zero in data set 111, where n = 1000, for this latter material, it can be argued that, in spite of the fact that the result is statistically significant, the difference might be without any practical importance. indeed, there is no contradiction in this: the results from the statistical test procedure just tells us whether a difference is larger than what could be due to chance, if the so called null hypothesis is true. when the number of observations is large, even a very unimportant difference can be statistically significant n fig. 2a. data sets i, i1 and 111. 95 % confidence intervals for the x2 =5.0 true difference d.loo%. ( 1 ) i i i i a ' 5 0 1 . 0 10 20 30 d . l o o e % it is essential to stress that the judgement whether a difference should be considered as being important or not, is not possible to do merely by means of statistical techniques. the statistician can present a confidence interval, but it must be up to the subject matter specialist to decide the magnitude of what should be considered as an important difference. 2 the data sets iv, v and vi all give small x -values. this means that no sta tistically significant differences have been found. the corresponding confidence 100 intervals always contain the point zero, but this is certainly not enough in order to demonstrate that the two drugs have equal effects. we notice that in data set ivy where n = 50, the confidence interval is quite wide. n fig. 2b. data sets iv, v and vi. 95% confidence intervals for the true difference d*loo%. x 2 = 0 . 8 an interesting interpretation of a confidence interval in a situation like this is that all possible values of the true difference d within the calculated limits are not in contradiction with the data. this means that even if data set iv gives quite a low y, -value, there is still a possibility that the true dif ference could be of the magnitude 2 5 % . however, when we consider data set vi, we find a very narrow confidence interval, indicating that even as small values of the difference d as, say 1.5%, would be in contradiction with the data. 2 until now, we have just used the confidence level 95%. for data set vi, the h h 99% confidence limits for d would be dl 100 = -.8% respectively, thus giving an interval which includes the above mentioned value of 1.5%. apparently, the conclusion about which values of u that are possible or not, is not only depending upon the number of observations, n, but also upon the confidence level chosen. and du 100 = +1.6% if it is desired to establish whether there is equality between the two treatments, it must be recognized that it is not possible to prove that for a finite number of observations, the confidence interval f o r d will always have a certain length, unless b = c = 0 , which is the uninteresting case, when there is no random variation involved. therefore, the equality statement must be substituted with the condition that the true difference is smaller than a certain value, i.e. [di < d,, where d, is tile smallest non-zero value of the difference which is of any interest to discover. if d' is specified by the sub ject matter specialist and also the confidence level and the desired power of the test is decided upon, it is possible to calculate the necessary number of observations in order to establish "equality" in accordance with this modified meaning of the word. one gets the impression that this procedure has seldombeen applied, when it is stated in medical articles that "there is no difference" or that "the treatments are equal". d = 0. indeed, everything in this discussion is of a very basic, elementary charac ter. as the references (1, 3, 4, 5, 6) to this little note demonstrate, the problems touched upon are mentioned now and then in various journals. it is the 101 the author's impression that they are not sufficiently stressed in statistical text books, and it is easy to find applied scientific articles where the results from tests of significance have been interpreted incorrectly. 1. 2. 3. 4. 5. 6. references lutz, w. & nimmo, i.a.: the inadequacy of statistical significance. europ j of clin investigation 7:77-78, 1977. mcnemar, i.: note on the sampling error of the difference between correlated proportions or percentages. psychometrika 12:153-157, 1947. rennie, d.: vive la diffgrence (p < 0.05). new eng j med 299:828-829, 1978. spriet, a. & bieler, d.: when can "non significantly different" treatments be considered as "equivalent"? b r j clin pharmacol 7:623-624, 1979. taube, a.: blev det inte signifikant? slutsatser vid statistisk hypotes pr6vning. lakartidningen 69:65-68, 1972. (swe) wade, c.l. & waterhouse, j.a.n.: significant or important? br j clin phar macol 4 : 411-412 , 1977. accepted january 15, 1980 a d d r e s s for r e n r i n t e : adam taube university of uppsala department of statistics p.o. box 513 s-751 20 uppsala sweden 102 upsala j med sci 80: 122-126, 1975 results of partial thyroidectomy for thyrotoxicosis henry johansson,' folke nilsson,' a k e rimsten,' anders parrow,2* gudrun jonsell,3 magnus michaelsson" from the ' d e p a r t m e n t of surgery, the =department of internal medicine, a n d the qepartment of pediatrics, university h o s p i t a l , u p p s a l a , s w e d e n abstract in a retrospective study, two series of surgically treated patients with thyrotoxicosis were compared. the series dif fered with respect to preoperative treatment and operative technique. in one series a combined preoperative treatment with an antithyroid drug and i-thyroxine was given and the recurrent nerves and parathyroid glands were routinely identified. in the other series no such operative routine was followed and iodine was given preoperatively. the compli cation rate was low in both series. there was no postopera tive mortality. the existing differences, although subtle, in the frequency of recurrent nerve paralysis, postoperative hypoparathyroidism or hypothyroidism, as well as recur rent toxicosis, favoured the surgical approach with the combined preoperative treatment and a meticulous techni que with identification of recurrent nerves and parathyroid glands. introduction subtotal thyroidectomy is widely employed as therapy for thyrotoxicosis. clear indications exist for such a treatment and surgery often is thought to offer prompt and permanent relief. in addition to surgery, antithyroid drugs and radio-iodine must also be considered as sometimes superior modes of therapy. the choice of therapy is debated, however, owing to the simple fact that none is ideal, each having its own advantages and disadvantages. immediate surgical complications are mortality and recurrent nerve injuries and as remote se quelae, toxicosis, hypofunction of the thyroid andlor the parathyroids are considered. the com plication rate is usually low (2, 6) though the major complication of surgery, hypothyroidism, has been observed in certain studies to have an overall inci dence of as high as 49 % (7). variations in complication rate certainly reflect differences in preand postoperative care, as well as the surgical procedure itself. wide experience in * present address: county hospital, enkoping, sweden. upsala j med sci 80 thyroid surgery and careful attention to details in operative techniques seem to lower the complica tion rate (5, 8, 10, 1 i ) . at the university hospital of uppsala we have, since 1965, used standardized methods for the treatment of thyrotoxicosis, employing preopera tive antithyroid drugs and /-thyroxine, an operative technique where meticulous care is taken in the identification of the parathyroid glands and the re current nerves and careful follow-up examinations. i n this retrospective study, the results of the above outlined regimen are compared with those of earlier series without such an established program. the findings favour the principles for surgical treatment of thyrotoxicosis including the preopera tive schedule, and operative techniques with routine identification of the parathyroid glands and recurrent nerves. material series i , o p e r a t e d b e t w e e n 1968 a n d i972 the series consisted of 108 consecutive patients who un derwent surgery for thyrotoxicosis. the diagnosis, sex and age distribution are seen in tables i and 11. the yearly incidence from 1968 to 1972 is presented i n table 111. surgery was the primary treatment in 99 cases and 9 patients were referred to surgery when medical treatment led to recurrent toxicosis. f'reoperatively the patients were treated with an anti thyroid drug, as a rule carbimazole (neomercazol", british sheerings ltd.) supplemented with i-thyroxine (levaxin", nyegaard a/s). this treatment was given on an ambulatory basis and usually continued for 3 months. on the day of operation the antithyroid treatment was discontinued. thyroxine was given postoperatively in most cases with nodular, and in some cases with diffuse, goitre, in an initial daily dose of 0.1 mg. bilateral thyroid resection was performed for diffuse and nodular goitre and unilateral thyroid resection for toxic adenomas. in the bilateral resection, thyroid rem nants of 4-6 g were left. important stages in the surgical procedure were as follows: partial thyroidectomy f u r thyrutuxicosis i23 table i . dirrgrzosis ctnd sex distribution of patients operrrted f b r thyrotoxicosis betkrven i968 and 1972 table 111. dicigno5i.y and yearly incidence of pu tients operrrted f o r thyrotoxicosis between 1968 and i972 m=male, f=female diffuse nodular adenoma diffuse nodular adenoma total m f m f m f m f i968 i5 16 3 1969 16 14 2 1970 1 1 8 i 1971 4 10 0 50 49 9 i08 1972 4 1 3 10 40 9 40 2 7 21 87 1 . transversal skin incision approximately 2 cm above the sterno-clavicular joints. 2. separation and retraction of the sternothyroid and sternohyoid muscles in the mid-line. 3. separate ligation of the superior thyroid artery t o avoid injury of the external laryngeal nerve. 4 . exposure of inferior thyroid artery with ligation in continuity. 5 . identification of recurrent nerves. 6. identification, if possible, of parathyroid glands. before a bilateral resection, a thorough search was made for at least one parathyroid gland. usually at least two para thyroid glands were identified and left at every operation. 7. meticulous hemostasis before closing the wound, and drainage catheters (suction drainage according to redon jost) for 48 hours. the vocal cords were inspected prior to the operation, at the extubation and 3 4 days after surgery. serum calcium and phosphorous levels were determined before and after the operation. the thyroid status was followed by determinations of protein-bound iodine (pbj) or serum-thyroxine (t4) and triiodothyronine (t,-resine test) as well a s by measuring thyroid stimulating hormone (tsh). the patients were examined 2 weeks, and 2, 6 , 18 months postoperatively and thereafter annually. the re sults of the present study were calculated on a mean follow-up time of 30+2 months. series 11, operated upon between 1950 and 1964 the series consisted of 114 women and is presented in table iv. the mean age of the patients at the time of operation was 34 years ( 1 6 4 9 ) . for 2-3 weeks preopera tively the patients were hospitalized and prepared for sur gery with oral iodine. this treatment was started with low doses and the dosage was increased until symptoms of thyrotoxicosis disappeared or were significantly reduced. postoperatively the dosage was successively diminished and the treatment was usually terminated on the fifth day. the operation consisted, with a few exceptions, of bilateral resection of the thyroid. parathyroid glands and recurrent nerves were not regularly identified. in other respects the operations followed in principle the proce dures described for series i . at the follow-up examinations all patients were investi gated with serum determinations of cholesterol, triglycerides and tsh. pbi and basal metabolic rate (bmr) were also measured when hypothyroidism was suspected. the mean follow-up time was 12 years (6-20) after operation. only women fertile at the time of operation were in cluded in this material as the primary aim of the follow-up examination was to study the influence of the surgical treatment of thyrotoxicosis on fertility, pregnancies and children born thereafter. these aspects of the follow-up will be published elsewhere. in the following text the cases in series i will be referred to a s the recent series and those in series i1 as the early series. the two series were worked up with regard to postoperative bleedings (series i), vocal cord paralysis, hypoparathyroidism, hypothyroidism, and recurrent thyrotoxicosis (series i, 11). the diagnosis of hypothyroidism is based on evaluation of clinical symptoms, and lowered values of pbi, t4 or t,, sometimes in combination with an increase in tsh. results murtalitv table 11. diagnosis and a g e distribution of patients operated f o r thyrotoxicusis between 1968 and 1972 diffuse nodular adenoma total age there was no peror postoperative mortality in either series i or series 11. -20 1 1 0 2 20-29 12 2 1 15 30-39 14 3 2 19 40-49 11 14 2 27 50-59 7 14 1 22 postoperative bleeding 6 0 6 9 4 12 2 18 in series i, 6 patients (6 %) had postoperative bleed 70-79 1 3 1 5 ing which required reoperation with hemostasis. in total 50 49 9 108 no case tracheostomy was necessary. upsaia 3 med sci 80 124 h . johansson el al. table i v . diagnosis of female patients operated for thyrotoxicosis between 1950 and 1964 diffuse nodular ‘‘undetermined’’ 82 25 7 paralysis of recurrent nerve in series i , none of the patients (0%) had persistent paralysis. unilateral and, within 3 months, transient paralysis occurred in 4 patients (4 %). in series 11, 3 patients (2%) suffered persistent unilateral vocal cord paralysis. postoperative hypoparathyroidism in series i , none of the patients (0 %) had persistent hypoparathyroidism. overt but transient hypopara thyroidism arose in 8 patients (7%). two of these patients required extra calcium for a few days. in series 11, one patient required vitamin d sup plementation (dygratyp, ferrosan, sweden) imply ing a persistent postoperative hypoparathyroidism of less than 1 %. postoperative hypothyroidism in series i , most (84%) of the patients with nodular goitre and many (38 %) of those with diffuse goitre had regular administration of thyroxine post operatively (table v). 12 patients (11%) were characterized as hypothyroid, with one exception all having been operated for a diffuse goitre. in series 11, 50% of the patients with nodular goitre and 14% of those with diffuse goitre had regular administration of thyroxine postoperatively. 8 women (7%) were classified hypothyroid, 6 of whom were already treated with thyroxine. the values of tsh, cholesterol and triglycerides in series i1 found at the follow up are shown i n table vi. the patients submitted to partial thyroidectomy had on the average a higher tsh -value than the corresponding controls (normals). the values of cholesterol and triglycerides did not differ from the normals. postoperative recurrent toxicosis in series i , 2 patients (2%) had a relapse within 12 months of surgery. both (women, age at time of operation 18 and 27, respectively) were given anti vpsala j med sci 80 thyroid drugs for 2 years and still remain euthyroid 6 and 3 months after discontinued therapy. in series 11, 7 women ( 6 % ) had a relapse, 4 of them within 12 months. only one was treated and cured with a thyrostatic drug, all the others were treated with radioiodine, hitherto remaining eu thyroid. discussion the frequency of thyrotoxicosis in this region has been relatively constant for the last 20-25 years. the calculated incidence is around 30/100000, for women, 40/100000. during 1970-72 there was a di minishing number of operations for thyrotoxicosis mainly depending o n an increased use of antithyroid drugs for the treatment of the toxic diffuse goitre. however, it has been shown that also after long term antithyroid drug therapy only 20-35 % of the patients experienced prolonged remission ( 3 , 9 ) and an extension of the antithyroid dose did not obvi ously reduce the number of recurrences. a still lower frequency of prolonged remission after anti thyroid drug therapy is demonstrated in patients with grave’s disease and enlarged goitre (9). ap parently long time antithyroid drug therapy can above all be expected to be successful in cases with small goitre, short duration of symptoms and with a simultaneous reduction in the size of the goitre dur ing the treatment. these facts have in our clinic been reflected in an increased number of operations for toxic goitre during the last 2 years. in spite of certain differences in the indication, the types of operations have been identical and in both series, with very few exceptions (unilateral resection in toxic adenomas), bilateral, partial re section was performed. however, the modes of table v. hypothyroidism afrer operation for thyro toxicosis, between 1968-1972, with regard to dia gnosis and postoperative thyroxine supplementa tion (+) or not (-) diffuse nodular adenoma n =50 n =49 n =9 + f + postoperative 3 8 0 1 0 0 hypothyroidism thyroxine supplementation postoperative 19 31 41 8 8 1 partial thyroidectomy f o r thyrotoxicosis 125 a routine identification of parathyroid glands is, however, no guarantee for the avoidance of post operative hypoparathyroidism. it is not always pos sible to find the parathyroids (12) and in attempting to identify the glands there is always the risk of damaging their vascular supply ( 1 ) . such an injury may explain the overt but tran sient hypoparathyroidism in the 8 patients in the recent series. we would nevertheless emphasize the usefulness of the routine identification of the parathyroids, as this technique gives a familiarity with the anatomy leading to an increased skill in the handling of more radical thyroid procedures and parathyroid surgery. the problem of the exact estimation of the fre quency of postoperative hypothyroidism is well known and, in our two series, the liberal postopera tive administration of thyroxine, contributes to the difficulty. the reason for our thyroid hormone treatment in nodular goitre is to avoid a recurrency of nodules and, i n diffuse goitre, to decrease the risk of the postoperative exophthalmus syndrome. however, the small daily dose, 0.1 mg i-thyroxine, cannot conceal the appearance of a real thyroid hypofunction. postoperative hypothyroidism usually appears early, and more than 90% are revealed within 12 months (7). in the early series only 2 new hypothyroid sub jects were discovered 12 years after thyroid sur gery, and i n the recent series all patients characterized as hypothyrotic were observed within one year. however, our studies showing elevated tsh levels in patients followed for an average of 12 years indicate not only an early subtle thyroid hormone deficiency ( 2 ) ; for a considerable time af ter the operation the thyroid remnants are unable to maintain a state of euthyroidism without an exces sive stimulation. thus there is always the risk of a late postoperative hypothyroidism underlining the importance of life-long follow-up. our frequencies of recurrent toxicosis are within those reported in the literature. an interesting observation is that, taken together, the percentage of hyperand hypothyroidism postoperatively is identical-13-in our two series. these complica tions reflect our inability to individually and exactly decide the optimal size of the thyroid remnant. the size of the remnant and the degree of lymphoid infiltration are crucial factors for a development of table vi. values of t s h , cholesterol and triglyce rides in female patients operated between 1950-1964. normal values of t s h in f e m a l e s of corresponding age are given in parentheses mean ksd tsh (units) 12.9 3.4-49.0 cholesterol (mg %) 252 164-384 triglycerides (mg %) 106 38-290 (5.5) (1.2-25.0) therapy are definitely different with respect to operative techniques with routine identification of the parathyroid glands and the recurrent nerves in series i, and t o the preoperative treat ment which is completely changed between the two series. the complication rate must be considered ac ceptable in both series, and the differences subtle. such a similar and low incidence of complications might be explained more by the fact that in both series our preoperative evaluations, as well as the operations, have been performed by experienced surgeons. some observations are, however, worthy of further elucidation. none of the series suffered from postoperative mortality due to crisis, haemorrhage or tetany. this can be attributed to the adequate preoperative treatment, either with iodides or antithyroid drugs, and the advantages of modern surgery and anesthesiology. without a doubt the preoperative treatment of today, antithyroid drugs in combina tion with thyroxine, allows of an uncomplicated postoperative course-no abnormal increase in pulse rate o r elevation of body temperature was found. not one crisis was observed i n the so treated patients, while in the early series one severe thyroid storm appeared, requiring intensive care. the absence of recurrent nerve paralysis in the series with routine identification of the nerves indi cates the importance of such a procedure. persis tent vocal cord paralysis is reported to be rare with such an operative technique 0-0.3% (4, 12). this observation is also well confirmed in the compari son between our series with and without routine nerve identification, 0 and 2 %, respectively. our frequency of permanent postoperative hypoparathyroidism is extremely low and consist ent with most recent series, in which, in the hands of experienced surgeons, the percentage ranges from 0 t o 1.4 ( 2 ) . upsala j med sci 80 126 h . johansson et al. p o s t o p e r a t i v e hypofunction. in t h e c h o i c e b e t w e e n a r e c u r r e n t toxicosis or a hypothyroid s t a t e , t h e l a t t e r is definitely p r e f e r r e d , since it is a m e n a b l e t o p r o m p t t r e a t m e n t . t h e signs of a h i t h e r t o l o w e r f r e q u e n c y of r e c u r rent toxicosis in t h e r e c e n t series m a y b e an a d v o cate f o r a m o r e radical r e s e c t i o n , w h i c h is certainly t h e c o n s e q u e n c e of t h e m o r e e x t e n s i v e dissection t e c h n i q u e n e e d e d for t h e routine identification of r e c u r r e n t nerves a n d parathyroid glands. references 1 . alveryd, a.: parathyroid glands in thyroid surgery. acta chir scand, suppl. 389, 1968. 2. barnes, h . v . & gann, d. s.: choosing thyroidectomy in hyperthyroidism. surg clin n amer 54: 289, 1974. 3 . gillquist, j., karlberg, b., sjodahl, r. & tegler, l.: preoperative treatment of hyperthyroidism. acta chir scand 140: 23, 1974. 4 . hawe, p. & lothian, k. r.: recurrent laryngeal nerve injury during thyroidectomy. surg gynec ob stet 110: 488, 1960. 5. heyman, p.: atoxic and toxic goiter. endemiology, symptomatology and surgical treatment. acta chir scand, suppl. 289, 1962. 6 . lamberg, b.: skoldkortelns sjukdomar. remedia fennica, helsingfors, 1969. 7 . michie, w., pegg, c. & bewsher, p.: prediction of hypothyroidism after partial thyroidectomy for thyrotoxicosis. brit med j i: 13, 1972. 8. riddell, v . : thyroidectomy. prevention of bilateral recurrent nerve palsy. brit j surg57: 1 , 1970. 9 . shizume, k., irie, m., nagataki, s., matsuzaki, f., shishiba, y . , suematzu, h. & tsushima, t.: long term result of antithyroid drug therapy for grave’s disease: follow up after more than 5 years. en docrinol japon 17: 327, 1970. 10. thompson, n . w., olsen, w. r. & hoffman, g . l.: the continuing development of the technique of thyroidectomy. surgery 73: 913, 1973. 1 1 . thoren, a. & wijnbladh, h.: operative treatment of thyrotoxicosis. acta endocrin 22: 224, 1956. 12. wade, j . s . h.: the morbidity of subtotal thyroidectomy. brit j surg48: 25, 1960. received february 13, 1975 address for reprints: h . johansson, m.d. department of surgery university hospital s-750 14 uppsala 14 sweden upsala j med sci 80 upsala j med sci 95: 3 1 4 4 metabolism in vitro of cartilage proteoglycans in rat (pre)chondrocytes from different embryonic regions johan styrud, erik unger* and ulf 3. eriksson department of medical cell biology, university of uppsala, biomedicum, uppsala, sweden and *department of veterinary medical chemistry, swedish university ofagricultura1 sciences, biomedicum, uppsala, sweden abstract diabetes in the mother may cause disturbances in the chondrocyte development in the embryo. a rat model was used to investigate whether this was reflected in the production of proteoglycans by cells from two embryonic regions. one of these re gions is resistant (limb bud) and the other susceptible (mandibular arch) to malfor mation in diabetic pregnancy. chondroitin sulphate proteoglycans from cultures of day-12 rat embryo limb bud and mandibular arch chondrocytes were extracted with guanidine-hcl and anal yzed by gel chromatography after in vifro 35s-sulphate-labeling. two sizes of pro teoglycans (kav 0.26 and 0.66 on cl-2b sepharose) were found in both types of chondrocytes and in all media. the polysaccharide chain length was the same (kav 0.36 on cl-6b sepharose) for both proteoglycans. elevated levels of d-glucose or p-hydroxybutync acid had no effect on either pro teoglycan size or proportion, nor on polysaccharide chain length. however, there were differences (in all culture conditions) between limb bud and mandibular arch cultures in that the larger proteoglycan accounted for 80 yo of total radioactivity in the limb bud cultures, 53 % in the mandibular arch cultures, and only 25-29 % in the media from both types of cultures. furthermore, different ratios between radio active proteoglycans in medium and matrix suggested markedly different effic iencies for matrix formation in the two cell types. these findings indicate differences in the metabolism of the proteoglycans in these two cell types which may be related to the induction of mandibular malformation in diabetic pregnancy. 31 introduction maternal diabetes causes an increased incidence of malformations in the off spring, a great proportion of which involve disturbances in embryonic chondro genesis. several experimental studies have indicated that diabetes per se affects chondrocyte development and function (1). rib cartilage from fetuses made hyper glycemic by chronic d-glucose infusion into the mother showed decreased thym idine incorporation in vivo and in vitro (2, 3). in streptozotocin-diabetic rats the in corporation of sulphate in demineralized bone matrix was decreased and the pro teoglycan produced was smaller than that in normal cartilage (4). the synthesis of growth plate proteoglycans was almost abolished in untreated diabetic rats, whereas insulin treatment partly normalized the proteoglycan production (5). two populations of proteoglycans have been found in costal cartilage from mice (6), and rats (7), and whilst experimental diabetes in rats decreased production of both populations of proteoglycans the larger form was suppressed to a greater extent (7). these alterations in proteoglycan production were more pronounced in diabetic rats from a strain prone to skeletal malformations than in rats from a non-malform ation-prone strain (7). insulin treatment of the diabetic rats normalized proteoglycan production. these results indicate that a diabetic environment inhibits the synthesis of cartilage-associated proteoglycans. (pre)chondrocyte cells from two different regions of rat embryos showed mark edly decreased thymidine incorporation when exposed to increased levels of p-hy droxybutyric acid in culture (8). raised d-glucose concentration caused a marginal decrease in thymidine incorporation in mandibular arch chondrocytes but this effect was only found in cells isolated from the malformation-prone rat strain (8). the aim of the present work was to study the effects of a diabetes-like in vitro en vironment on the production of proteoglycans by the chondrocytes. for this, a "micromass" technique was utilized, in which undifferentiated (pre)chondrocyte cells matured into alcian blue stainable chondrocytes over six days of culture (9, 10, 1 1, 12). the (pre)chondrocytes were isolated from day-i2 embryos of rats of the 32 malformation-prone strain (1 3) and cultured in different d-glucose and p-hydroxy butyric acid concentrations. materials and methods sprague-dawley-derived rats from a colony in uppsala were used in this study. the fetuses of these rats have an elevated rate of congenital malformations when the mother is diabetic (1 3, 14, 15). female rats were caged overnight with male rats from the same strain and the day of conception, i.e. presence of sperm in a vaginal smear, was designated day zero of pregnancy. on gestational day 12 the rats were decapitated and the embryos were dissected out. isolated (pre)chondrocyte cells were prepared and cultured in a micromass in vitro system, as described in detail elsewhere (8). in previous studies isolation and culture of (pre)chondrocytes from gestational days 11 and 13 were also attempted, but led to almost no growth of the cells in v i m (8). briefly, the limb bud and the mandibular arches were dissected free, pooled sep arately and trypsinized for 8 to 9 minutes at 37°c until single cell suspensions were obtained. non-viable embryos or embryos with less than 30 somites were dis carded. the cells were carefully suspended in the medium (rpmi 1640, supple mented with 10 yo fetal calf serum) (flow laboratories, irvine, uk) by vortex mixing for 5 minutes and filtered through a nitex monofilament screen (mesh diameter 20 pm). the cell concentration was determined using a haemocytometer and ad justed to 1.8-2.2.1 07 cells/ml for limb bud and 1.5-2.0.1 07 cells/ml for mandibular arch (pre)chondrocyte cells. in each experiment 20 pi of a cell suspension was placed as a single droplet on the dry base of a 35 mm tissue culture dish (cluster 3406, costar, cambridge, ma, usa).the dishes were then maintained in an incu bator (95 % air and 5 yo c02; assab, sundbyberg, sweden) at 37°c for 90 minutes, to allow the cells to attach to the substratum, before each dish was carefully flooded with 2 ml of the medium (1 1.1 mmol/l d-glucose). after 24 hours culture, the medium was changed and the (pre)chondrocytes were subsequently 3-908571 33 cultured for five days in either 11.1 mmola d-glucose, 44.4 mmol/l d-glucose or , with 32.0 mmol/l p-hydroxybutyric acid (in the presence of 11.1 mmol/l d-glucose). on day 3 of culture the medium was changed and 100 pci nan 35s-so4 (carrier free) (amersham international plc, amersham, buckinghamshire, uk) was added, and the culture was continued for a further three days. the medium was then saved and pooled for each culture condition and 200 pi guanidine buffer (4 m guanidine hci in 0,05 m acetate buffer, ph 5.8, containing 0.2 % triton x-100, 1 mm n-ethyl maleimide, 0.1 m e-amino-n-caproic acid, 5 mm benzamidine and 10 mm edta, all from sigma chemical co., st. louis, mo, usa) (16) was added to each dish and the cultures were scraped off with a "rubber policeman" into plastic tubes. the cul tures were extracted for 72 hours at 4"c, centrifuged at 15 000 x g for 15 minutes and dialyzed against 2 x 50 volumes (approximately 50 ml) of guani dine buffer for 24 hours. cartilage extracts were chromatographed in guanidine buffer on a sepharose cl-26 (pharmacia fine chemicals, uppsala, sweden) column (88 x 1 cm). dextran blue (pharmacia fine chemicals) and dnp-alanine (sigma chemical co.) were used to indicate the void (vo) and the total (vt) volumes of the column. to each 1 ml fraction 4 ml emulsifier scint 299 (packard ab, stockholm, sweden) was added and the radioactivity was estimated in a liquid scintillation counter (no. 300-c, packard ab). disintegrations per minute (d.p.m.) were calculated by external standardization. the pooled medium samples were diluted with a 0.14 m nacl 10 mm tris buffer, ph 8, applied to a 0.5 ml deae-sepharose (pharmacia fine chemicals) column, washed with the tris-buffer, then with a 0.14 m nacl 20 mm na-acetate buffer, ph 4, and subsequently eluted with 1.5 m nacl 20 mm na-acetate buffer, ph 4. the medium samples were then chromatographed in the same manner as the extracts. chondroitinase abc digestion followed by gel chromatography was performed to determine chondroitin sulphate content. proteoglycans were degraded by incu bation for 15 hours at 4°c with 0.05 units of chondroitinase abc (sigma chemical co.) in 0.05 m trisihci, ph 8.0, containing 0.03 m sodium acetate and 0.1 mg/rnl 34 bovine serum albumin. the samples were chromatographed on a sepharose cl 6b column (pharmacia fine chemicals) with blue dextran and dnp-alanine as markers for void and total volumes (1 7). for polysaccharide chain length determination the samples were incubated overnight in 0.5 m naoh at 4°c. after neutralization with tris-hci, ph 8.0, and hci the samples were chromatographed on a sepharose cl-6b column, eluted with 0.01 m tris buffer, ph 8.0, containing 0.14 m nacl and 0.02 % nan3. blue dex tran and dnp-alanine were used as markers for void and total volumes, respect ively. 15 10 h c? a el a, p ' 5 m 5 a .h 3 1.5 4 cd 1.0 in m 0.5 -peak' ipeak ii * i 1 i i 10 20 30 40 v fraction number 1 50 figure 1. cl-2b sepharose chromatogram of extracted 3%-labeled proteo glycans from cultured limb bud chondrocytes (top panel) and corresponding culture medium (lower panel). 35 results after digestion with chondroitinase abc, more than 95% of the 3%-macro molecules present in the dialyzed extracts of limb bud and mandibular arch chondrocyte cultures, eluted as low molecular weight material following chromato graphy on sepharose cl-66, demonstrating that the label was mainly present in chondroitin sulphate (data not shown). 1.5 1 .o h 9 a e a, ‘5 2 0.5 a h v 1.5 u -fl u 3 $ 1.0 lo m 0.5 0 1 10 20 30 40 v fraction number v o 50 figure 3. cl-26 sepharose chromatogram of extracted 35s-labeled proteo glycans from cultured mandibular arch chondrocytes (top panel) and corresponding culture medium (lower panel).the large peak in the vt position consists of free sulphate. 36 when dialyzed chondrocyte extracts were analyzed by gel chromatography on sepharose cl-2b, the 35s-labeled material was separated into two peaks ( i and ii; kav about 0.3 and 0.7) in which peak i was dominant in the cell culture and peak ii was dominant in the medium (figures 1 and 2). due to the low total incorporation in mandibular arch cultures, the free sulphate peak is relative large (figure 2). elevated ambient concentrations of d-glucose (44.4 mmol/l) or 0-hydroxybutyric acid (32.0 mmol/l) had no effect on either the kav values (figures 3 and 4) or the relative proportions of the two peaks (figure 5) in the limb bud or mandibular arch cell cultures. in particular, there were no significant differences between the amount of 35s incorporation in peak i in different culture conditions in mandibular arch medium (figure 5). table 1. combined kav values on sepharose cl-6b of proteoglycans isolated from cell and medium fractions of limb bud (lb, n = 10) and mandibular arch (ma, n = 9) chondrocytes. mean +. sem of values from all types of culture conditions (see text). lb 0.26 l 0.01 0.62 f 0.01 0.28 f 0.01 0.67 f 0.01 ma 0.29 l 0.01 0.66 f 0.02 0.27 ~ 0 . 0 1 0.69 i 0.02 combined data for limb bud and mandibular arch chondrocytes from all three types of culture conditions yielded kav values of approximately 0.28 and 0.66, re spectively, for the two different proteoglycans (table 1). incorporation of 35s into the larger proteoglycan (peak i) was significantly greater in the limb bud cells.than 37 in the mandibular arch cells (table 2). in contrast, there was no statistically signifi cant difference between the distribution of the label between the two proteoglycans in the medium from limb bud and mandibular arch chondrocyte cultures (table 2). the total sulphate incorporation of limb bud chondrocytes was more than tenfold greater than that of mandibular arch cells. furthermore, the ratio of medium-to chondrocyte radioactivity was markedly different in the two types of culture. in limb bud cultures the ratio was 1 :6, whereas in mandibular arch culture the ratio was approximately 1 :1 (table 2). table 2. the amount of 3%-radioactivity in peak i (expressed as a percentage of the total 3%-incorporation in eluted macromolecules) and total 3%-radioactivity (d.p.m.per 6 culture dishes) from cultured chondrocytes and media. in the last column the ratio between total 3%-incorporation in the culture and in the media is shown. mean & sem of pooled values from all types of conditions per lb (n = 10) or ma (n = 9). ---i--------------------------------------- culture medium ratio peak i% 358.103 peak i% 35s.103 m:c lb 8 0 ~ 2 125.629.6 2 9 f 2 21.9 f 9.6 1 :5.7 ma 53+3*** 9.6 & 0.4*** 2 5 + 5 11.2 f 0.6** 1 :0.9 ** = p < 0.01 *** = p< 0.001 gel chromatography on sepharose cl-6b of alkali-released polysaccharide chains from proteoglycans in limb bud cell extracts and media showed a single peak. this suggest that there was no significant difference in polysaccharide chain length between the proteoglycans in the cells and media (data not shown). the kav value was about 0.36 in all peaks, indicating a molecular weight of about 35000 according to the calibration of wasteson (18). 38 0.8 0.7 0.6 0,5 0,4 03 02 figure 3. kav values for the small (peak i t , upper part of diagram) and large (peak i, lower part of diagram) proteoglycan isolated from limb bud (lb) or mandibular arch (ma) chondrocytes, cultured in 11.1 mmolll d-glucose (lb 11, ma 1 l ) , or 32 mmolll p-hydroxybutyric acid (lb 32, ma 32), or 44 mmolll d-glucose (lb 44, ma44). mean sem, 3 s n i 4 . kav culture medium i i i m f i 0 8 0 11 p i i i i i i 03 0.7 0.6 0 3 0.4 03 02 figure 4. kav values for the small (peak ii, upper part of diagram) and large (peak i, lower part of diagram) proteoglycan in medium from limb bud (lb) or mandibular arch (ma) chondrocytes, cultured in 11.1 mmola d-glucose (lb 11, ma 1 l ) , or 32 mmol/l p-hydroxybutyric acid (lb 32, ma 32), or 44 mmolll d-glucose (lb 44, ma 44). mean f sem, 3 i n 1 4 . € m i a 0 0 p p 0 i i i i i i 39 discussion the present results show, that chondrocytes from limb buds and mandibular arches both produced two major types of proteoglycans. the larger of these (peak i , kav 0.28) was predominant in the limb bud extracts, whereas in the mandibular arch cells the peaks were evenly distributed. in the culture medium, the smaller proteo glycan (peak ii, kav 0.66) was predominant, and this pattern was similar in media from both types of chondrocyte cultures. the production of proteoglycans, as reflected in 35s-sulphate incorporation into extracted macromolecules, was markedly lower in mandibular arch cultures compared to limb bud cultures. further more, the different ratios between radioactive proteoglycans in medium and matrix showed that 85 % of the proteoglycans in the limb bud cultures remained in the matrix, in contrast to only about 50 yo of the mandibular arch proteoglycans. 60 yo 40 20 0 0 culture extract 0 culture medium 8 p 3 € 4 f f i i i i i i, figure 5. amount of 3%-radioactivity in peak i expressed as a percentage of to tal 35s-incorporation in eluted macromolecules from cultured limb bud (lb) and mandibular arch (ma) chondrocytes (top row of values), and the corresponding cul ture media (lower row of values). culture conditions, cf. legends to figures 3 and 4. mean l sem, 3 i n i 4. 40 this suggests differences in the metabolism of the proteoglycans in the two cell types. however, there were no detectable differences in kav values, or relative pro portions of proteoglycans of each cell type when cultured in different concentrations of d-glucose or p-hydroxybutyric acid. likewise, the kav and relative proportions of the proteoglycans did not change in the corresponding media. the polysaccharide chain length was also similar in the proteoglycans of all samples. in previous in v i m studies, we found that the total dna content of mandibular arch cultures was about 50 % of that of limb bud cultures, implicating a greater rate of sulphate incorporation per cell in the latter chondrocytes. mandibular arch chondrocytes from embryos of rats of the local colony are more sensitive to increased ambient d-glucose and p-hydroxybutyric acid levels than limb bud chondrocytes (8). this corresponds to the micrognathia and normal limb development in the offspring of diabetic rats of this strain (13, 14). the critical period for induction of diabetes-related skeletal malformations in this rat strain has recently been determined to be gestational days 6-10 (15), which suggests that the chon drocytes isolated on day 12 in the present study may already have passed their pe riod of maximal sensitivity to a diabetic environment. human endothelial cells exposed to high concentrations of d-glucose display signs of dna damage (1 9). connective tissues in diabetic rats has an increased rate of synthesis of fibronectin (20). increased d-glucose levels have been shown to exert effects on the gene expression of fetal cells. culture of umbilical cord vessel endothelium in high d-glucose concentration caused marked changes in mrna levels of several matrix proteins, including collagen and fibronectin (21). in fetal rat brain, increased p-hydroxybutyric acid levels yield decreased production of purine and pyrimidine moities, indicating a direct inhibitory effect on the biosynthesis of rna and dna (22, 23). in contrast, the results of the present study suggest that embryonic production of cartilage-associated proteoglycans is not markedly af fected by elevated ambient d-glucose and (3-hydroxybutyric acid levels. in the present study, we have demonstrated that both limb bud and mandibular arch chondrocytes produce two size populations of proteoglycans. the kav values 41 of these proteoglycans are similar in the two types of chondrocyte cultures and are not influenced by the culture conditions. the length of the chondroitin sulphate chains of the proteoglycans is similar in all chondrocyte cultures and media, regard less of origin of the cells and culture conditions. in contrast, the limb bud and mandibular arch chondrocytes differ markedly in the total amount of proteoglycan produced and secreted to the culture medium. furthermore, different ratios between radioactive proteoglycans in medium and matrix suggest markedly different efficiencies for matrix formation in the two types of cultures. acknowledgements this investigation was supported by the swedish medical research council (grant no. 12x-74751, the "expressen" prenatal research foundation, the swe dish diabetes association, the swedish "forenade liv" mutual group life in surance company, the nordic insulin fund, the swedish hoechst diabetes fund and the juvenile diabetes foundation. references 1. sternberg, m., cohen-forterre, l.& peyroux, j.: connective tissue in diabetes mellitus: biochemical alterations of the intercellular matrix with special refer ence to proteoglycans, collagens and basement membranes. diab metabol (paris) 1 1 :27-50, 1985. heinze, e., brenner, r., nguyen-thi, c. h., vetter, u., leupold, d.& pohlandt, f. : skeletal growth in fetal rats, effects of glucose and amino acid. diabetes heinze, e.& vetter, u.: skeletal growth of fetuses from streptozotocin diabetic rat mothers: in vivo and in vitro studies. diabetologia 30: 100-1 03, 1987. weiss, r., gorn, a.&nimni, m.: abnormalities in the biosynthesis of cartilage and bone proteoglycans in experimental diabetes. diabetes 30: 670-677, 1981. 2. 35: 222-227, 1986. 3. 4. 42 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. axelsson, i., lorentzon, r.& pita, j.: biosynthesis of rat growth plate proteo glycans in diabetes and malnutrition. calcif tissue int 35: 237-242, 1983. venn, g.& mason, r : absence of keratan sulphate tissues of mouse and rat. biochem j 228:443-450, 1985. unger, e., kjellen, l.& eriksson, u. j.: effect of insulin on the altered produc tion of proteoglycans in rib cartilage of experimentally diabetic rats. submitted for publication, 1989. styrud, j. & eriksson, u. j. effects of d-glucose and p-hydroxybutyric acid on the in vitro development of (pre)chondrocytes from embryos of normal and diabetic rats. accepted for publication in acta endocrinol (copenh), 1990. hassel, j. r., pennypacker, j. p a lewis, c. a.: chondrogenesis and cell pro liferation in limb bud cell cultures treated with cytosine arabinoside and vitamin a. exp cell res 1 12:409-417, 1978. hassel, j. r.& horigan, e. a.: chondrogenesis: a model development system for measuring teratogenic of compounds. teratogenesis, carcinogenesis, and mutagenesis 2:325-331, 1982. osdoby, p.& caplan, a. 1.: osteogenesis in cultures of limb bud mesenchymal cells. develop biol 73:84-102, 1979. paulsen, d. f.& solursh, m.: microtiter micromass cultures of limb-bud mesen chymal cells. in vitro 24:138-149, 1988. eriksson, u. j., dahlstrom, e., larsson, k. s.& hellerstrom, c.: increased incidence of congenital malformations in the offspring of diabetic rats and their prevention by maternal insulin therapy. diabetes 31 : 1-6, 1982. eriksson, u. j., dahlstrom, e.& hellerstriim, c.: diabetes in pregnancy: skeletal malformations in the offspring of diabetic rats after intermittent with drawal of insulin in early gestation. diabetes 32: 1 141 -1 145, 1983. eriksson, r. s. m., thunberg, l.& eriksson, u. j.: effects of interrupted insulin treatment on fetal outcome of pregnant diabetic rats. diabetes 38: 764-772, 1989. sajdera, s. w.& hascall, v. c.: protein polysaccharide complex from bovine nasal cartilage: a comparison of low and high shear extraction procedures. j biol chem 244:77-87, 1969. kolset, s. o., kjellen, l., seljelid, r.& lindahl, u.: changes in glycosamino glycan biosynthesis during differentiation in vitro of human monocytes. biochem j 210: 661-667, 1983. wasteson, a.: properties of fractionated chondroitin sulphate from ox nasal septa. biochem j 122: 477-485, 1971. lorenzi, m., montisano, d. f., toledo, s.& barrieux, a.: high glucose induces dna damage in cultured human endothelial cells. j clin invest 77: 322-325, 1986. 43 20. 21. 22. 23. phan-thanh, l., robert, l., deroette, j. c.& labat-robert, j.: increased biosynthesis and processing of fibronectin in fibroblast from diabetic mice. proc natl acad sci 84: 191 1-1915, 1987. cagliero, e., maiello, m., boeri, d., roy, s.& lorenzi, m.: increased expression of basement membrane components in human endothelial cells cultured in high glucose. j clin invest 82:735-738, 1988. bhasin, s.& shambaugh, g. e.: fetal fuels. v. ketone bodies inhibit pyrimidine biosynthesis in fetal rat brain. am j physiol 243:e234-e239, 1982. shambaugh, g. h., angulo, m. c.& koehler, r. r.: fetal fuels: vii ketone bodies inhibit synthesis of purines in fetal rat brain. am j physiol 247:elll el 17, 1984. correspondence to: dr. johan styrud department of medical cell biology biomedicum p.o. box 571 s-751 23 uppsala sweden 44 upsala j med sci 93: 233-244, 1988 sequential changes of the plasma-protein pattern in cases of hepatitis a claes-otto kindmark,' carl-bertil laurell,2 sten iwarson3 and kennet p e r s o n 4 'department of infectious diseases, university hospital, upqsala, 'department of clinical chemistry, university of lund, malmo general hospital, mulmo, department of infectious diseases, university of gothenburg, gothenburg, 4department of virology, university of lund, malmo general hospital, malmo, sweden abstract t h e l e v e l o f 21 plasma p r o t e i n s was f o l l o w e d in h e p a t i t i s a f o r t w o m o n t h s a f t e r o n s e t o f i c t e r u s . t h e mean c o n c e n t r a t i o n o f a a n t i t r y p s i n , orosomucoid, h a p t o g l o b i n , c r e a c t i v e p r o t e i n ( c r p ) a n d a a n t i c h y m o t r y p s i n i n c r e a s e d u n i f o r m e l y during t h e f i r s t week o f h e p a t i t i s a. t h u s , t h e y d i f f e r f r o m t h a t o f i n o c u l a t i o n h e p a t i t i s e a r l i e r d e s c r i b e d . t h e mean c u r v e f o r i g m was h i g h e r in h e p a t i t i s a t h a n t h e c o r r e s p o n d i n g r e s u l t s f o r i n o c u l a t i o n h e p a t i t i s during t h e f i r s t week o f illness, but because o f g r e a t i n t e r i n d i v i d u a l d i f f e r e n c e s in c o n c e n t r a t i o n s i g m d e t e r m i n a t i o n s can n o t b e u s e d t o d i s c r i m i n a t e b e t w e e n t h e t w o diseases in a g i v e n case. i g a l e v e l s w e r e s l i g h t l y i n c r e a s e d e a r l y in h e p a t i t i s a but n o c h a n g e in i g c l e v e l s was o b s e r v e d . p r e a l b u m i n was t h e b e s t m i r r o r o f t h e p a t i e n t s ' r e c o v e r y o r d e t e r i o r a t i o n . 1 1 introduction t h e d e t e c t i o n o f plasma p r o t e i n a b n o r m a l i t i e s i s o f v a l u e in t h e d i a g n o s i s a n d f o l l o w u p of h e p a t i c d i s o r d e r s i n c l u d i n g h e p a t i t i s . a s e a r l y as t h e n i n e t e e n f o r t i e s , g r a y a n d b a r r o n ( 8 ) showed t h a t h e p a t i t i s i s accompanied by a p o l y clonal t y p e o f hypergammaglobulininemia a n d t h i s h a s s i n c e b e e n c o n f i r m e d by o t h e r s (3,4,7). wollheim ( 2 2 ) was t h e f i r s t t o r e c o g n i z e a d i f f e r e n c e b e t w e e n h e p a t i t i s a a n d h e p a t i t i s b, namely, a s u b s t a n t i a l i n c r e a s e o f i g m in most cases of h e p a t i t i s a but in o n l y h a l f o f t h o s e o f h e p a t i t i s b. this d i f f e r e n c e h a s b e e n w i d e l y d i s p u t e d (6,12,14,19). s c a t t e r e d o b s e r v a t i o n s h a v e b e e n r e p o r t e d o n t h e v a r i a t i o n o f some p r o t e i n s during t h e c o u r s e o f h e p a t i t i s . b e l f r a g e ( 3 ) o b s e r v e d a low h a p t o g l o b i n l e v e l in s e r a f r o m p a t i e n t s w i t h h e p a t i t i s b, but n o t in s e r a f r o m t h o s e w i t h hepa t i t i s a. m u l l e r a n d m u l l e r v o n v o i g t ( 1 6 ) s t u d i e d t h e v a r i a t i o n s in 16 individ u a l plasma p r o t e i n s in cases o f l i v e r disease a n d f o u n d a s e r i e s o f s i g n i f i c a n t changes in p a t i e n t s w i t h a c u t e h e p a t i t i s . t h e i r findings r e f e r t o s i n g l e o b s e r v a t i o n s during t h e e a r l y p h a s e of t h e disease. 15-888572 233 we h a v e p r e v i o u s l y r e p o r t e d o n t h e s e q u e n t i a l changes in t h e p a t t e r n o f plasma p r o t e i n s in h e p a t i t i s b 111). o u r findings h a v e b e e n c o n f i r m e d t o a, major e x t e n t (5,15,20.21) t h o u g h r e s u l t s h a v e b e e n i n t e r p r e t e d as a p p l y i n g t o a c u t e v i r a l h e p a t i t i s in g e n e r a l r a t h e r t h a n t o h e p a t i t i s b alone. t h e aim o f t h e p r e s e n t i n v e s t i g a t i o n was t o study t h e s e q u e n t i a l c h a n g e s in plasma p r o t e i n s in p a t i e n t s w i t h h e p a t i t i s a during a n a c u t e epidemic e n a b l i n g t h e c h a n g e s t o b e compared t o t h o s e r e p o r t e d in h e p a t i t i s b ( 1 1 ) . m a t e r i a l a n d methods p a t i e n t s : thirty people a g e d 18-54 (mean 34) y e a r s w e r e i n f e c t e d w i t h h e p a t i t i s a in a f a c t o r y w h e r e a p o i n t s o u r c e o u t b r e a k o c c u r r e d . accumulated e v i d e n c e r e p o r t e d e a r l i e r ( 1 8 ) r e v e a l e d t h a t t h e y w e r e i n f e c t e d in t h e f a c t o r y canteen. t h e epidemic was o f s h o r t d u r a t i o n . t h e d i a g n o s i s was based o n epidemiological e v i d e n c e a n d a n t i h a v d e t e r m i n a t i o n s . none o f t h e p a t i e n t s showed a n y serological e v i d e n c e o f h e p a t i t i s b. t h e c l i n i c a l c o u r s e o f t h e h e p a t i t i s a i n f e c t i o n was u n e v e n t f u l a n d a l l p a t i e n t s h a d normal l e v e l s o f s e r u m bilirubin a n d s e r u m alanine amino t r a n s f e r a s e ( s a l a t 1 w i t h i n t h r e e m o n t h s o f o n s e t of t h e i r i l l n e s s . blood sampling: t h e f i r s t b l o o d samples w e r e o b t a i n e d w i t h i n one week of o n s e t o f symptoms. t h e m a j o r i t y o f samples w e r e o f s e r u m w i t h o n l y a f e w plasma samples f r o m t h e f i r s t week o f t h e i l l n e s s . plasma samples w e r e t h e n t a k e n a t w e e k l y i n t e r v a l s f o r a t least t e n weeks. t h e f o l l o w i n q 21 plasma p r o t e i n s w e r e d e t e r m i n e d : prealbumin, albumin, orosomucoid, a l i p o p r o t e i n . a a n t i t r y p s i n , a a n t i c h y m o t r y p s i n , ceruloplasrnin, a -macroglobulin, h a p t o g l o b i n , hemopexin, f i b r o n e c t i n ( e a r l i e r w r o n g l y d e s i g n a t e d ahf), t r a n s f e r r i n , c3, c4, plasminogen. p r o t h r o m b i n , p r o p e r d i n . i m m u n o g l o b u l i n c, i m m u n o g l o b u l i n a, i m m u n o g l o b u l i n m a n d c r e a c t i v e p r o t e i n ( c r p ) . a l l t h e p l a s m a p r o t e i n analyses w e r e p e r f o r m e d by electroimmunoassay ( 1 3 ) w i t h t h e m o d i f i c a t i o n s d e s c r i b e d e a r l i e r (10,171. 1 2 r esu lts t h e p r o t e i n p a t t e r n u s u a l l y f o l l o w e d t h e same c o u r s e in a l l p a t i e n t s , t h o u g h it v a r i e d in i n t e n s i t y a n d in t h e d u r a t i o n of abnormal values. t h e r e s u l t s o f t h e d e t e r m i n a t i o n s o f al a n t i t r y p s i n , orosomucoid a n d h a p t o g l o b i n a r e summarized in fig. 1. t h e y a r e p l o t t e d a g a i n s t t h e t i m e a f t e r i c t e r u s was o b s e r v e d . c u r v e s f o r t h e mean v a l u e s a r e g i v e n in t h e f i g u r e s . 234 2 6 0 2 0 0 1 6 0 1 2 0 8 0 lo % 2 00 160 1 2 0 80 lo g/l 2 4 2 0 1 6 1 2 0 8 0 4 fig. u, a t r > 1 2 3 4 5 6 7 8 9 1 0 11 weeks o r o s o h p *i . + 1 2 3 4 5 6 7 8 9 1 0 1 1 w e e k s 1 acute phase reactants (al-antitrypsin, orosomucoid, hapto globin) during the course of hepatitis a . the normal (95 per cent) ranges a r e shaded and the mean value lines connected. 235 d u r i n g t h e f i r s t 2-3 weeks a u n i f o r m r i s e o f t h e s e t h r e e acute-phase r e a c t a n t s i s noted. t h e n a g r a d u a l decrease o f t h e mean c o n c e n t r a t i o n s o f these, t h r e e acute-phase r e a c t a n t s o c c u r s o v e r a p e r i o d o f 5 weeks. h a v i n g r e t u r n e d t o t h e normal r a n g e t h e mean f o r a l a n t i t r y p s i n , orosomucoid a n d h a p t o g l o b i n remained normal during t h e r e s t o f t h e p e r i o d o f o b s e r v a t i o n . a a n t i c h y m o t r y p s i n n o t s h o w n in a n y f i g u r e followed t h e p a t t e r n o f orosomucoid v e r y closely. d e t e r m i n a t i o n s o f c r e a c t i v e p r o t e i n (fig. 2 w e r e i n i t i a l l y s l i g h t l y t o m o d e r a t e l y i n c r e a s e d but decreased c o n s i d e r a b l y a f t e r t h e f i r s t t w o weeks. 1 c r p 1 2 3 4 5 6 7 8 9 1 0 11 w e e k s f i g . 2 c r e a c t i v e protein ( c r p ) d u r i n g t h e course of tiepatitis a . s e v e r a l p r o t e i n s showed a n i n i t i a l f a l l in c o n c e n t r a t i o n w i t h t h e lowest v a l u e a r o u n d t h e 10-14 d a y o f i l l n e s s f o l l o w e d by a g r a d u a l r e t u r n t o w a r d s normal values. p r e a l b u m i n ( f i g . 31, albumin, hemopexin a n d a l i p o p r o t e i n demon s t r a t e t h i s p a t t e r n . s t a r t i n g f r o m t h e normal r a n g e i n i t i a l l y t h e mean s e r u m a l b u m i n v a l u e r e a c h e d i t s lowest p o i n t ( 3 6 g / l ) during t h e second week a n d r e t u r n e d t o t h e normal r a n g e a f t e r one m o n t h . t h e mean c u r v e f o r hemopexin i n d i c a t e s v a l u e s well below t h e normal r a n g e by t h e time t h e p a t i e n t was h o s p i t a l i z e d ( 7 8 % o f t h e normal mean). t h e mean c u r v e r e a c h e d i t s lowest p o i n t (63% o f t h e normal mean) t e n d a y s a f t e r o n s e t o f t h e i l l n e s s a n d r e t u r n e d t o i t s s t a r t i n g l e v e l in t h e third week. t h e mean c u r v e c o n t i n u e d t o i n c r e a s e g r a d u a l l y a n d r e a c h e d i t s h i g h e s t v a l u e s ( 8 7 % o f normal mean) a f t e r 7 weeks a n d remained a t t h a t l e v e l during t h e r e s t o f t h e p e r i o d o f o b s e r v a t i o n . 236 p r e a l b 140 % i 1 2 0 1 0 0 80 60 2 o t i 9 1 2 3 4 5 6 7 8 9 10 11 w e e k s fig. 3 prealbumin during the course of hepatitis a . too f e w plasma samples w e r e a v a i l a b l e f r o m t h e f i r s t week of i l l n e s s t o allow a n y d e f i n i t e c o n c l u s i o n s t o b e d r a w n a b o u t changes in p r o t h r o m b i n l e v e l s during t h i s p e r i o d . t h e mean c o n c e n t r a t i o n o f p r o t h r o m b i n in plasma samples a r o u n d d a y 10 was 80% of t h e normal mean. t h e r e a f t e r t h e mean p r o t h r o m b i n l e v e l r e m a i n e d normal. a third g r o u p o f p r o t e i n s i n c l u d i n g t r a n s f e r r i n (fig. 4 ) . a2-macroglobulin ( f i g . 4 ) , ceruloplasmin a n d p r o p e r d i n showed o n l y m i n o r changes a n d t h e mean c u r v e remained e s s e n t i a l y c o n s t a n t w i t h i n t h e normal r a n g e during t h e whole t i m e of o b s e r v a t i o n . t h e same appears t r u e f o r plasminogen, f i b r o nectin, c 3 a n d c4, p r o v i d e d allowances a r e made f o r t h e p a u c i t y o f plasma samples c o l l e c t e d during t h e f i r s t week of t h e i l l n e s s . 237 t 140 1 2 0 100 a o 60 40 2 0 yo 2 40 2 00 1 6 0 1 2 0 80 40 fig. t 8 i 8 t f 1 2 3 4 5 6 7 8 9 10 11 w e e k s a 2 m a k r o 8 4 transferrin and a2macroglobulin during the course of hepatitis a . t h e i g m v a l u e s v a r i e d g r e a t l y between t h e d i f f e r e n t p a t i e n t s a l t h o u g h t h e i n d i v i d u a l v a l u e s f o l l o w e d t h e mean c u r v e (fig. 5 ) . most p a t i e n t s a l r e a d y h a d peak l e v e l s of i g m o n admission, c o i n c i d i n g w i t h t h e appearance o f jaundice. t h e v a l u e s g r a d u a l l y r e t u r n e d t o normal during t h e f o l l o w i n g months. t h e mean c u r v e s o f l e v e l s o f i g c a n d i g a w e r e b o t h w i t h i n t h e normal r a n g e during t h e whole p e r i o d of o b s e r v a t i o n . a s l i g h t i n c r e a s e in i g a l e v e l s ( f i g . 5 ) was, however, o b s e r v e d during t h e f i r s t t w o weeks o f illness. 238 . . 4 8 1 2 3 4 5 6 7 8 9 1 0 1 1 weeks . 7 1 2 3 4 5 6 7 8 9 1 0 1 1 w e e k s fig. 5 immunoglobulin m and a during hepatitis a . discussion t h e d a t a p r e s e n t e d a r e based o n samples d r a w n f r o m p a t i e n t s s u f f e r i n g f r o m h e p a t i t i s a . none o f t h e p a t i e n t s showed a n y serological e v i d e n c e o f h e p a t i t i s b. thus, t h e d a t a o b t a i n e d m e r i t a comparison o f t h e s e q u e n t i a l c h a n g e s in p a t t e r n of plasma p r o t e i n s in h e p a t i t i s a w i t h t h o s e r e p o r t e d e a r l i e r f o r h e p a t i t i s b ( 1 1 1 . since t h e p r e s e n t a t i o n o f t h e h e p a t i t i s b s e r i e s t h o s e s e r a h a v e b e e n c h e c k e d f o r a n t i b o d i e s t o h e p a t i t i s a as t h i s t e s t became available. no e v i d e n c e o f h e p a t i t i s a was f o u n d in t h o s e sera. 239 m u c h a t t e n t i o n h a s b e e n p a i d t o t h e changes in t h e s e r u m i m m u n o g l o b u l i n c o n c e n t e r a t i o n in cases o f h e p a t i t i s a a n d b . t h e i g m l e v e l s in h e p a t i t i s a r e p o r t e d h e r e showed t h a t t h e mean c u r v e f o r i g m r e a c h e d m u c h h i g h e r ’ v a l u e s t h a n t h e c o r r e s p o n d i n g c u r v e f o r h e p a t i t i s b. this is e v i d e n t f r o m t h e o n s e t o f t h e i l l n e s s w h e n t h e mean c u r v e f o r i g m s t a r t s a t 3.2 g / l compared t o 1 g / l f o r h e p a t i t i s b ( 1 1 ) . s u b s t a n t i a l i n t e r i n d i v i d u a l c h a n g e s w e r e seen in h e p a t i t i s a, i.e. t h e r e w e r e just as many p a t i e n t s s u f f e r i n g f r o m h e p a t i t i s a w i t h a n i g m v a l u e well a b o v e 4 g / l during t h e f i r s t week o f i l l n e s s as t h e r e w e r e p a t i e n t s w i t h a n i g m v a l u e below 2 g/l. thus, i g m v a l u e s a f f o r d t h e c l i n i c i a n l i t t l e , if any, d i a g n o s t i c g u i d a n c e in a n y g i v e n case. in t h e c o u r s e o f h e p a t i t i s a we did n o t o b s e r v e a n y c h a n g e in i g c c o n c e n t r a t i o n s . t h u s , o u r r e s u l t s c o n f i r m e d t h o s e r e p o r t e d by k r u g m a n , giles a n d hammond ( 1 2 ) a n d by c i l e s a n d k r u g m a n (6) but n o t t h o s e of lo grippo, h a y a s h i a n d s h a r p l e s s ( 1 4 ) . bevan, taswell a n d c l e i c h ( 4 ) o r wollheim ( 2 2 ) . t h e s l i g h t i n c r e a s e in t h e i g a l e v e l s during t h e f i r s t t w o weeks o f h e p a t i t i s a r e p o r t e d h e r e was n o t o b s e r v e d f o r h e p a t i t i s b ( 1 1 ) . a f t e r t h e i n i t i a l m i n o r i n c r e a s e t h e mean c u r v e f o r i g a was almost i d e n t i c a l f o r h e p a t i t i s a a n d b . t h i s a g r e e s w i t h d a t a r e p o r t e d by wollheim ( 2 2 ) . no e x p l a n a t i o n c a n b e o f f e r e d f o r t h e m a r k e d r e a c t i o n o f i g m in m a n y p a t i e n t s in t h e e a r l y phase o f h e p a t i t i s a a t t h e t i m e w h e n o n l y a m i n o r r e a c t i o n is f o u n d in h e p a t i t i s b . p e r h a p s t h e peak in i g m l e v e l s in h e p a t i t i s a may coin c i d e w i t h t h e peak o f v i r u s s h e d d i n g in this disease t h o u g h it remains t o b e s h o w n t h a t t h e p a t i e n t s w i t h t h e h i g h e s t i g m v a l u e s a r e t h o s e t h a t s h e d t h e g r e a t e s t n u m b e r o f v i r u s p a r t i c l e s . t h e s l i g h t i n c r e a s e in plasma i g a l e v e l s during t h e f i r s t t w o weeks o f i l l n e s s in h e p a t i t i s a but n o t in h e p a t i t i s b remains u n c l e a r . h e p a t i c oedema w i t h c o n s e q u e n t l y i n c r e a s e d p o r t a l venous p r e s s u r e e a r l y in h e p a t i t i s a m i g h t b e a n e x p l a n a t i o n . t h e i n c r e a s e d p r e s s u r e in t h e p o r t a l v e i n a f f e c t s t h e b l o o d f l o w in t h e i n t e s t i n e . t h e i g a p r o d u c e d t h e r e m i g h t t h e r e b y b e f o r c e d b a c k i n t o t h e s y s t e m i c c i r c u l a t i o n . t h e c o n s t a n t r i s e in t h e v a l u e s o f t h e acute-phase r e a c t a n t s (a a n t i t r y p s i n , orosomucoid, haptoglobin, crp, a n d al a n t i c h y m o t r y p s i n 1 w h i c h is c h a r a c t e r i s t i c o f b a c t e r i a l i n f e c t i o n s , t r a u m a t i c lesions ( 2 ) a n d a s e p t i c n e c r o s i s ( 10) i s also d e m o n s t r a t e d in t h e e a r l y phase o f h e p a t i t i s a, as r e p o r t e d h e r e . this c o n t r a s t s t o t h e f i n d i n g s in h e p a t i t i s b ( 1 1 ) . f u r t h e r m o r e , in h e p a t i t i s a t h e h a r m o n i o u s r i s e o f a l a n t i t r y p s i n , orosomucoid a n d h a p t o g l o b i n during t h e f i r s t week a f t e r t h e appearance of j a u n d i c e is a r e a c t i o n o f g r e a t r e g u l a r i t y , 1 240 as s h o w n in f i g . 6. in t h i s f i g u r e t h e mean c u r v e s a r e g i v e n f o r t h e s e t h r e e acute-phase r e a c t a n t s during t h e f i r s t week o f i l l n e s s . t h e scales u s e d a r e s u c h t h a t t h e v a l u e s f o r t h e s e t h r e e p r o t e i n s in h e a l t h y i n d i v i d u a l s w i l l b e a r o u n d 100% f o r a a n t i t r y p s i n a n d orosomucoid a n d 1 g / l f o r h a p t o g l o b i n . t h i s p r o f i l e i s q u i t e d i f f e r e n t f r o m t h e one we h a v e r e p o r t e d f o r h e p a t i t i s b ( 1 1 ) . w h i c h i s i n c l u d e d in f i g u r e 6 f o r r e f e r e n c e . t h u s , t h e r e s p o n s e o f t h e s e a c u t e p h a s e p r o t e i n s may b e u s e d a s a d i a g n o s t i c t o o l f o r d i f f e r e n t i a t i o n b e t w e e n h e p a t i t i s a a n d b, p a r t i c u l a r l y in t h o s e cases w h e n serological t e s t s in t h e s e diseases h a v e n o t g i v e n u n e q u i v o c a l r e s u l t s . t h e s e f i n d i n g s also s t r e s s t h e f a c t t h a t t h e d e t e r m i n a t i o n o f one acute-phase r e a c t a n t may n o t always s u f f i c e a n d m u c h b e t t e r i n f o r m a t i o n can b e o b t a i n e d if 2 o r 3 acute phase p r o t e i n s a r e d e t e r m i n e d simultaneously. 1 100 200 % d, atr oroso 1.0 2.0 g / l f i g . 6 relation b e t w e e n individual values of a ; l n t i t r y p s i r , orosomucoid and haptoglobin d u r i n g t h e f i r s t week o f hepatitis a . t h e normal ( 9 5 p e r c e n t ) r a n g e s a r e shaded and t h e mean value l i n e s ( x x) c o n n e c t e d . t h e corresponding mean value lines f o r h e p a t i t i s b ( 0 0 ) i s g i v e n f o r comparison. ( t h e individual v a l u e obtained f o r h e p a t i t i s b h a v e b e e n r e p o r t e d earlier ( 1 1 ) ) . 1 24 1 t h e c o n c e n t r a t i o n o f c r e a c t i v e p r o t e i n may also a s s i s t in d i f f e r e n t i a t i o n b e t w e e n h e p a t i t i s a a n d b e a r l y in t h e illness. c o m p a r i n g t h e mean c u r v e o b t a i n e d f o r crp in h e p a t i t i s a w i t h t h a t of h e p a t i t i s b a r r i v e d a t e a r l i e r ( 1 1 ) s i g n i f i c a n t l y h i g h e r l e v e l s w e r e seen in h e p a t i t i s a t h a n in h e p a t i t i s b, c o n f i r m i n g t h e r e s u l t s o b t a i n e d by h e d l u n d ( 9 ) . t h e d i f f e r e n t r e s u l t s f o r t h e acute-phase r e a c t a n t s r e p o r t e d h e r e compared t o t h o s e r e p o r t e d f o r h e p a t i t i s b e a r l i e r ( 1 1 ) s u g g e s t s t h a t t h e p a t h o l o g y o f t h e 2 t y p e s o f h e p a t i t i s is q u i t e d i f f e r e n t . t h e f i n d i n g s w o u l d b e c o n s i s t e n t w i t h a p r o n o u n c e d c e l l u l a r r e a c t i o n w i t h an i n c r e a s e d a c t i v i t y of g r a n u l o c y t e s o r monocytes in h e p a t i t i s a but n o t in h e p a t i t i s b. when m o n i t o r i n g t h e p a t i e n t s s u f f e r i n g f r o m h e p a t i t i s a it i s h e l p f u l t o h a v e a v z r i a b l e t h a t r e f l e c t s t h e c l i n i c a l c o u r s e of t h e disease. t h e s a l a t , v e r y o f t e n u s e d in t h i s r e s p e c t , c o u l d p r o v i d e e r r o n e o u s i n f o r m a t i o n as l e v e l s may decrease i n s t e a d o f i n c r e a s e w h e n t h e p a t i e n t i s g r a d u a l l y g e t t i n g c r i t i c a l l y ill. t h e r e s u l t s o f t h i s s t u d y w o u l d seem t o i n d i c a t e t h a t t h e p r e a l b u m i n v a l u e s w e r e t h e b e s t m i r r o r o f t h e p a t i e n t s ' r e c o v e r y o r d e t e r i o r a t i o n during t h e c o u r s e o f h e p a t i t i s a . t h i s i s in agreement w i t h e a r l i e r r e p o r t s (1,11,15,16,20,21). refer en c es 1. a l y , f.w. e schaup, h.: eine q u a n t i t a t i v e bestimmung d e s t r y p t o p h a n r e i c h e n serumpraealbumin im a g a r a g a r . c l i n chim a c t a 4:88-95, 1959. 2. aronsen, k . f . , ekelund, c., k i n d m a r k , c.-0. e l a u r e l l , c . b . : s e q u e n t i a l changes of plasma p r o t e i n s a f t e r s u r g i c a l t r a u m a . scand j c l i n l a b i n v e s t 29, s u p p l . 124:127-136, 1972. 3. b e l f r a g e , s . : plasma p r o t e i n p a t t e r n in c o u r s e o f a c u t e i n f e c t i o u s disease. a c t a med scand, s u p p l . 395, 1963. 4. bevan, c., taswell, h . f . e cleich, c . j . : serum immunoglobulin l e v e l s in b l o o d d o n o r s i m p l i c a t e d in t r a n s m i s s i o n n o f h e p a t i t i s . jama 203:38-40, 1968. 5 . carlson, j. e e r i k s s o n , s.: a a n t i t r y p s i n a n d o t h e r a c u t e phase r e a c t 1 a n t s in l i v e r disease. a c t a med scand 207:79-83, 1980. 242 6 . giles. j.p. & krugman, s.: v i r a l h e p a t i t i s . jama 208:497-503, 1969. 7 . 8 . 9. 10. 11. 12. 13. 14. 15. 16. cleichmann, e . & deicher, h. : q u a n t i t a t i v e immunoglobulin-bestim m u n g e n im serum b e i e n t z u n d l i c h e n l e b e r k r a n k h e i t e i n . k l i n wochenschr 46:171-176, 1968. g r a y , s.j. & cuzman b a r r o n , e.s.: t h e e l e c t r o p h o r e t i c analyses o f t h e s e r u m p r o t e i n s in diseases o f t h e l i v e r . j c l i n i n v e s t 22:191-200, 1943. h e d l u n d , p. : c l i n i c a l a n d e x p e r i m e n t a l s t u d i e s o n c r e a c t i v e p r o t e i n ( a c u t e phase p r o t e i n ) , t h e s i s . a c t a med scand, s u p p l . 361, 1961. johansson, b . g . , k i n d m a r k , c . 0 . . t r e l l , e.y. & wollheim, f . a . : s e q u e n t i a l changes o f plasma p r o t e i n s a f t e r m y o c a r d i a l i n f a r c t i o n . scand j c l i n l a b i n v e s t 29, s u p p l . 124:117-126, 1972. k i n d m a r k , c.-0. & l a u r e l l . c . b . : s e q u e n t i a l changes o f t h e plasma p r o t e i n p a t t e r n in i n o c u l a t i o n h e p a t i t i s . s c a n d j c l i n l a b i n v e s t 29, s u p p l . 124:105-115, 1972. k r u g m a n , s . , giles, j.p. e hammond, j . : i n f e c t i o u s h e p a t i t i s . jama 200:365-373, 1967. l a u r e l l , c . b . : electroimmuno assay. scand j c l i n l a b i n v e s t 29, s u p p l . 124:21-37, 1972. logrippo, g . a ' , hayashi, h. & sharpless, n. : immunoglobulins a n d i n t e r f e r o n responses in i n f e c t i o u s a n d t r a n s f u s i o n associated h e p a t i t i s . h e n r y f o r d h o s p i t a l medical b u l l e t i n 15:57-63, 1967. m u r r a y l y o n , i .m. & williams, r . : q u a n t i t a t i v e immunoelectrophoresis o f plasma p r o t e i n s in a c u t e v i r a l h e p a t i t i s , e x t r a h e p a t i c b i l i a r y o b s t r u c t i o n , p r i m a r y b i l i a r y c i r r h o s i s a n d i d i o p a t h i c haemochromatosis. c l i n chim a c t a 51:303-308, 1974. m u l l e r , h. e. & m u l l e r v o n voigt, i . : q u a n t i t a t i v e immunologische s e r u m p r o t e i n u n t e r s u c h u n g e n b e i l e b e r e r k r a n k u n g e n . z i m m u n i t a t s f o r s c h 133: 366-375, 1967. 243 17. persson, k . , k i n d m a r k . c.-o., p e n s k y , j. e naff, c . : q u a n t i t a t i v e measurement o f p r o p e r d i n in normal human s e r u m by electroimmunoassay a n d s i n g l e r a d i a l immunodiffusion. c l i n e x p lmmunol 29:84-88, 1977. 18. persson, k . , hansson. b.c., iwarson, s., johansson, t., k i n d m a r k , r c . 0 . & n o r d e r , h.: serologisk d i a g n o s t i k a v h e p a t i t a med h a v a b o c h s p e c i f i k i g m med e l l s a ( a b s t r a c t in s w e d i s h ) . hygiea, a c t a soc med suec 88:333. 1979. 19. peters, r . l . e ashcavai, m.: i m m u n o g l o b u l i n l e v e l s in d e t e c t i o n o f v i r a l h e p a t i t i s . am j c l i n p a t h 54:102-109, 1970. 20. skrede, s . , blomhoff, j.p., elgjo, k . e cjone, e . : s e r u m p r o t e i n s in diseases o f t h e l i v e r . scand j c l i n l a b i n v e s t 35:399-406, 1975. 21. skrede, s., blomhoff. j.p. e cjone, e . : biochemical f e a t u r e s o f a c u t e a n d c h r o n i c h e p a t i t i s . a n n c l i n res 8:182-199, 1976. 22. wollheim, f . a . : i m m u n o g l o b u l i n s in t h e c o u r s e o f v i r a l h e p a t i t i s a n d in c h o l e s t a t i c a n d o b s t r u c t i v e jaundice. a c t a med scand 183:473-479, 1968. a d d r e s s f o r r e p r i n t s : c . 0 . k i n d m a r k , m.d., d e p a r t m e n t o f i n f e c t i o u s diseases, u n i v e r s i t y hospital, s-751 85 uppsala, sweden. 244 upsala j med sci 91: 233-238, 1986 the technique of whole body autoradiography-some examples of applications lars-erik appelgren department of pharmacology and toxicology, faculty of veterinary medicine, the swedish university of agricultural sciences, biomedicum, uppsala, sweden introduction when sven u l l b e r g i n t h e b e g i n n i n g o f t h e 1950’s s t a r t e d h i s ph.d. t h e s i s work a l l d e t e r m i n a t i o n s o f t h e p e n i c i l l i n c o n c e n t r a t i o n i n organs o f t h e li v i n g organism were done by m i c r o b i o l o g i c a l i n h i s a t t e m p t t o i n v e s t i g a t e t h e f a t e o f p e n i c i l l i n i n v i v o u l l b e r g t r i e d d i f f e r e n t , by t h e n r e l a t i v e l y new methods. he produced r a d i o l a b e l l e d p e n i c i l l i n by growing t h e p e n i c i l l i n p r o d u c i n g f u n g i i n a r a d i o a c t i v e ( 3 5 s ) g r o w t h medium. a f t e r a h i n i s t r a i o n o f t h i s l a b e l l e d p e n i c i l l i n t o e x p e r i m e n t a l animals measurements o f p e n i c i l l i n c o n c e n t r a t i o n s i n d i f f e r e n t organs were t h e n performed w i t h geiger m h l e r t e c h n i q u e and a u t o r a d i o g r a p h y o f s e c t i o n s from e x c i s e d organs. a prob lem with which i s a water s o l u b l e substance, may be d i s s o l v e d when p r e p a r i n g t h e h i s t o l o g i c a l sec t i o n s used. to be a b l e t o make a d i r e c t comparison o f t h e r a d i o a c t i v i t y i n d i f f e r e n t organs sven u l l b e r g e l a b o r a t e d a method (31 which makes i t p o s s i b l e t o s i m u l t a n e o u s l y gans i n t h e body w i t h o u t r i s k i n g leakage o f water s o l u b l e substances. the d i f f i c u l t p r e s e l e c t i o n o f organs which f o r p r a c t i c a l reasons u s u a l l y has t o be done i s t h u s needless. techniques. t r a d i t i o n a l a u t o r a d i o g r a p h y i s t h e f a c t t h a t p e n i c i l l i n , l o c a l i z e a r a d i o a c t i v e substance i n p r a c t i c a l l y a l l t h e o r description of the technique the technique can b r i e f l y be d e s c r i b e d as f o l l o w s (4): a f t e r a d m i n i s t r a t i on o f t h e r a d i o a c t i v e substance t h e animals a r e s a c r i f i c e d a f t e r d i f f e r e n t s u r v i v a l t i m e s by an a n a e s t h e t i z i n g agent. t h e i r bodies a r e immediately f r o z e n i n a m i x t u r e o f hexane and s o l i d carbon d i o x i d e and t h e n s e c t i o n e d i n a s p e c i a l microtome a t =: -2ooc. to be a b l e t o h a n d l e t h e l a r g e t i s s u e s e c t i o n s a p i e ce o f scotch t a p e i s pressed o n t o t h e b l o c k b e f o r e s e c t i o n i n g . it i s p o s s i b l e t o t a k e s e c t i o n s from animals up t o t h e s i z e o f e.g. a newborn p i g by t h i s technique. a f t e r f r e e z e d r y i n g t h e s e c t i o n s they a r e p r e s s e d a g a i n s t a photo g r a p h i c emulsion. a f t e r exposure t h e s e c t i o n s a r e separated from t h e f i l m s and t h e f i l m s a r e developed t o v i s u a l i z e t h e b l a c k e n i n g caused by t h e r a d i o a c t i v e 233 s u b s t a n c e i n t h e d i f f e r e n t o r g a n s . a u t o r a d i o g r a m s are shown i n f i g . 1. s o l u t i o n t o t h e p r o b l e m i s a s i n g e n i o u s a s s i m p l e . the who: l e body a u t o r a d i o g r a p h y as i n t r o d u c e d by u l l b e r g h a s b e e n i n c r e a s i n g l y u s e d i n o u r d e p a r t m e n t s b u t a l s o i n many l a b o r a t o r i e s i n p r a c t i c a l l y a l l p a r t s o f t h e w o r l d . the p i o n e e r work i n c l u d e d s e c t i o n i n g t h e s p e c i m e n s i n a f r e e z e room ( 2 o o c ) w h i c h m e a n t t h a t f u r coats and g l o v e s were n e c e s s a r y e q u i p m e n t . some i m p r o v e m e n t s o f t h e t e c h n i c a l d e t a i l s when h a n d l i n g t h e embedding o f t h e s p e c i m e n s and t h e s e c t i o n i n g h a s b e e n i n t r o d u c e d d u r i n g t h e y e a r s . the i n t r o d u c t i o n o f a c r y o s t a t e w i t h a s p e c i a l l y d e s i g n e d , heavy m i c r o t o m e h a s f a c i l i t a t e d t h e work c o n s i d e r a b l y . the d e v e l o p m e n t a l work f o r an a u t o m a t i c sec t i o n i n g m a c h i n e h a s l a t e l y been s u c c e s s f u l and a p r o t o t y p e f o r t h i s p u r p o s e is now t e s t e d a t t h e d e p a r t m e n t o f t o x i c o l o g y , u p p s a l a u n i v e r s i t y . examples o f w h o l e body s v e n u l l b e r g ’ s o t h e r f i . 1 whole body a u t o r a d i o g r a m s from mice i n j e c t e d w i t h 3 s s p r o m e t h a z i n e ( a ) +s-hydroxyethyl p r o m e t h a z i n e ( 5 ) . w h i t e areas c o r r e s p o n d t o h i g h a m o u n t s of r a d i o a c t i v i t y . note t h e d i f f e r e n t u p t a k e of t h e compounds i n t h e b r a i n , w h i c h e x p l a i n s why s u b s t a n c e (b) d i d n o t c a u s e d r o w s i n e s s when u s e d f o r e . g . a l l e r g i c r h i n i t i s ( 2 ) . use of the technique f o r r e f e r e n c e s e x e m p l i f y i n g t h e u s e o f t h e t e c h n i q u e t h e r e a d e r is r e f e r r e d t o (4), w h e r e an e x t e n s i v e b i b l i o g r a p h y of a p p l i c a t i o n s is p u b l i s h e d . the most a p p a r e n t a d v a n t a g e o f t h e w h o l e body a u t o r a d i o g r a p h y t e c h n i q u e as a s c i e n t i f i c t o o l is t h a t it o f f e r s a d e t a i l e d , b u t a l s o c o m p l e t e , view o f t h e d i s t r i b u t i o n o f a n i n j e c t e d compound i n a l l t h e t i s s u e s of t h e body. t h i s f a c t f a v o u r s t h e 234 p o s s i b i l i t y t o make unexpected d i s c o v e r i e s and a l s o c o n t r i b u t e s t o s u g g e s t i o n s f o r f u r t h e r research. the method is a l s o e x t e n s i v e l y used f o r r o u t i n e scree n i n g work i n t h e pharmaceutical i n d u s t r y . the c o m b i n a t i o n of u l l b e r g ’ s method w i t h o t h e r t e c h n i q u e s has t o l a r g e e x t e n t c o n t r i b u t e d t o e l u c i d a t i n g t h e mech a n i s m s o f a c t i o n s f o r many endogenous s u b s t a n c e s as well a s the p r i n c i p l e f o r c e r t a i n p h a r m a c o l o g i c a l and t o x i c o l o g i c a l e f f e c t s . as an example can be men t i o n e d t h e s t u d i e s w h i c h g a v e t h e e x p l a n a t i o n why two a n t i h i s t a m i n e s ( p r o m e t h a z i n e and h y d r o x y e t h y l p r o m e t h a z i n e ) i n f l u e n c e d t h e slee p i n g p e r i o d a f t e r b a r b i t u r a t e a d m i n i s t r a t i o n i n d i f f e r e n t ways ( f i g . 1). an i m p o r t a n t q u e s t i o n when s t u d y i n g the d i s t r i b u t i o n o f a n t i b i o t i c s is the con c e n t r a t i o n i n t h e s i t e o f t h e i n f e c t i o n , e.g. t h e a b i l i t y t o p e n e t r a t e the membrane o f an e n c a p s u l a t e d a b s c e s s . the c o n d i t i o n f o r 3 5 s p e n i c i l l i n i n t h i s c o n t e x t is shown i n f i g . 2. a u t o r a d i o g r a p h i c abscess membrane abscess c o n t e n t f i g . 2 autoradiogram showing the d i s t r i b u t i o n of r a d i o a c t i v i t y i n two 30 day o l d ab scesses a f t e r i n t r a v e n o u s i n j e c t i o n o f 3 5 ~ p e n i c i l l i n i n t o a mouse. white areas re p r e s e n t h i g h amounts of r a d i o a c t i v i t y . p e n i c i l l i n p e n e t r a t e s the abscess membrane and a r e l a t i v e l y h i g h c o n c e n t r a t i o n is s e e n immediately i n s i d e t h e membrane. the con c e n t r a t i o n is t h e n d i m i n i s h e d towards t h e c e n t e r of t h e a b s c e s s ( 3 ) . from t h e t o x i c o l o g i c a l p o i n t o f view r e t e n t i o n of a s u b s t a n c e i n a c e r t a i n tissue may be o f i n t e r e s t . the p r e v i o u s l y n o t known, s t r o n g a c c u m u l a t i o n o f t e t r a c y c l i n e s i n bone and t e e t h , w h i c h was o b s e r v e d by whole body a u t o r a d i o g r a p h i c s t u d i e s i n 1956 h a s c o n t r i b u t e d t o t h e u n d e r s t a n d i n g of t h e s i d e e f f e c t s r e p o r t e d r e g a r d i n g f o e t a l growth and m a l f o r m a t i o n s of the enamel i n the d e c i d u o u s t e e t h when u s i n g these a n t i b i o t i c s i n p r e g n a n t i n d i v i d u a l s . to be a b l e t o e v a l u a t e the a u t o r a d i o g r a p h i c image i n a q u a n t i t a t i v e way d i f f e r e n t methods have been t r i e d . simple s y s t e m s u s i n g ’ i s o t o p e staircases’, t i n y p h o t o c e l l s and advanced systems w i t h c o m p u t e r i z e d image a n a l y s i s have 235 been used. whole body s e c t i o n s have a l s o been used f o r p a r a l l e l i n v e s t i g a t i o n s such as q u a n t i t a t i o n and r a d i o c h e m i c a l a n a l y s i s o f p i e c e s punched o u t from t h e d i f f e r e n t t i s s u e s i n t h e s e c t i o n s . h i s t o c h e m i c a l methods have been a d j u s t e d t o whole body s e c t i o n s i n a t t e m p t s t o f u r t h e r e x p l a i n t h e mechanism o f a c t i o n i n r e l a t i o n t o an a u t o r a d i o g r a p h i c a l l y found l o c a l i z a t i o n , e.g. enzyme i n h i b i t i o n . substances with f l u o r e s c e n c e o f t h e i r own (e.9. t e t r a c y c l i n e s and c e r t a i n m y c o t o x i n s ) have been i d e n t i f i e d d i r e c t l y i n whole body s e c t i o n s by u l t r a v i o l e t r a d i a t i o n . by u s i n g a s p e c i a l d r y mounting technique whole body sec t i o n s and p h o t o g r a p h i c emulsions can be permanently mounted t o enable a more p r e c i s e l o c a l i z a t i o n o f t h e r a d i o a c t i v i t y t o s p e c i a l t y p e s o f c e l l s (’semi m i c r o t e c h n i q u e ’ ) (fig. 3 ) . when i n t e r p r e t i n g t h e autoradiograms i t i s impor t a n t r e p r e s e n t t h e o r i g i n a l substance as w e l l as r a d i o l a b e l l e d m e t a b o l i t e s . as an example can be mentioned t h a t t h e r a d i o a c t i v i t y i n f i g . 3 i n t h e a d r e n a l c o r t e x r e p r e s e n t s o n l y 10 % o f un changed c h o l e s t e r o l a c c o r d i n g t o p a r a l l e l radiochromatographic s t u d i e s . the m a j o r i t y o f 1 4 c r e p r e s e n t s d i f f e r e n t c h o l e s t e r o l e s t e r s which a r e t h e s t o r a g e forms f o r t h e s t a r t i n g m a t e r i a l f o r s y n t h e s i s o f c o r t i c a l s t e r o i d hormones. t o remember t h a t t h e r a d i o a c t i v i t y can g l o m e r u l a r zone f a s c i c u l a r zone r e t i c u l a r zone m e d u i la f i g . 3 d f t a i l o f ’semi microauto radiogram (whole body s e c t i o n and p h o t o g r a p h i c emulsion permanently mounted t o g e t h e r ) from a mouse in j e c t e d i n t r a v e n o u s l y with “c-cho l e s t e r o l and s a c r i f i c e d a f t e r f o u r days. b l a c k areas correspond t o h i g h amounts o f r a d i o a c t i v i t y . the r a d i o a c t i v i v t y i s c o n f i n e d t o o n l y one c e l l t y p e l a y e r o f t h e c o r t e x (zona f a s c i c u l a t a ) (1). whole body autoradiography i m p l i e s t h a t r a d i o l a b e l l e d substances a r e a t hand. i n t h e l a t e 1950’ t h e r e were n o t t o o many l a b e l l e d p h a r m a c o l g i c a l or t o x i c o l o g i c a l substances a v a i l a b l e . beside t h e b i o s y n t h e s i s o f l a b e l l e d sub 236 s t a n c e s u l l b e r g and h i s coworkers s p e n t much time f o r ' c h e m i c a l ' l a b e l l i n g of i n t e r e s t i n g compounds b y e x c h a n g e r e a c t i o n s ( t r i t i u m , i o d i n e exchange etc.). to p o s s i b l y improve the a u t o r a d i o g r a p h i c r e s o l u t i o n , an i n v e n t o r y o f r a d i o n u c l i d e s w i t h s u i t a b l e low e n e r g y r a d i a t i o n was made i n c o l l a b o r a t i o n w i t h t h e royal t e c h n i c a l c o l l e g e i n stockholm. s e v e r a l o f these r a d i o n u c l i d e s were s u c c e s s f u l l y a p p l i e d i n b i o l o g i c a l e x p e r i m e n t s . a l s o v e r y s h o r t l i v e d r a d i o n u c l i d e s s u c h a s "f ( h a l f l i f e 110 min.) h a v e been used f o r b o t h whole body a u t o r a d i o g r a p h y a n d t h e a b o v e mentioned 'semi m i c r o t e c h n i q u e ' . i n t e r e s t i n g a r e a l s o t h e t o use t h e v e r y same s e c t i o n f o r l o c a l i z a t i o n of ''ca as well as "f by u s i n g t h e i r d i f f e r e n c e s i n h a l f l i v e s . s e v e r a l v o l a t i l e s u b s t a n c e s have been s t u d i e d by a s o f i s t i c a t e d method. af ter f r e e z i n g t h e whole a n i m a l , t h e s p e c i m e n s are k e p t a t 8 o o c and after saw i n g t h e b l o c k i n t o two p a r t s a t d r y ice t e m p e r a t u r e the ' h a l v e s ' are p l a c e d a g a i n s t x-ray films and exposed w h i l e k e p t a t the same low t e m p e r a t u r e . by t h i s method it is p o s s i b l e t o l o c a t e q u i t e many v o l a t i l e s u b s t a n c e s as well a s t h e i r l a b e l l e d m e t a b o l i t e s . when s e c t i o n s p r e p a r e d i n t h e t r a d i t i o n a l way are t h e n e x p o s e d , o n l y t h e n o n v o l a t i l e l a b e l l e d material is v i s i b l e i n the a u t o r a d i o g r a m s . after e x t r a c t i o n w i t h d i f f e r e n t s o l v e n t s r e e x p o s u r e o f t h e secti o n s can r e v e a l i f f i r m l y ( c o v a l e n t l y ? ) bound m e t a b o l i t e s a r e p r e s e n t . d u r i n g pregnancy and t o x i c o l o g y e v a l u a t i o n of compounds p o s s i b l y e f f e c t i n g p r e g n a n t women many c o n t r i b u t i o n s have been a c h i e v e d t h r o u g h t h e good r e s o l u t i o n of whole body a u t o r a d i o g r a p h y making it p o s s i b l e t o trace s u b s t a n c e s i n o r g a n s o f the growing f o e t u s . the whole body a u t o r a d i o g r a p h y t e c h n i q u e h a s a l s o been v e r y u s e f u l f o r f i n d i n g s u i t a b l e s u b s t a n c e s w i t h s e l e c t i v e l o c a l i z a t i o n f o r d i a g n o s t i c and/or t h e r a p e u t i c u s e s i n c o n n e c t i o n w i t h n e o p l a s t i c diseases. s u b s t a n c e s w i t h s p e c i f i c l o c a l i z a t i o n t o c e r t a i n may be l a b e l l e d w i t h y e m i t t i n g i s o t o pes f o r s u c h p u r p o s e s . lately t h e u s e of s h o r t l i v e d p o s i t r o n e m i t t i n g n u c l i des i n c o n n e c t i o n w i t h p o s i t r o n e m i s s i o n tomography (pet) h a s a l s o i n c r e a s e d t h e need f o r f i n d i n g s u b s t a n c e s w i t h s e l e c t i v e l o c a l i z a t i o n s . p o s s i b i l i t i e s i n c o n n e c t i o n w i t h d r u g use t i s s u e s conclusion t h a t t h e whole body a u t o r a d i o g r a p h y t e c h n i q u e is s t i l l r e l e v a n t and up t o date w i t h i n b i o m e d i c a l research is shown by its e x t e n s i v e use ( a l o n e and i n c o m b i n a t i o n w i t h o t h e r methods) i n f i e l d of pharmacology and t o x i c o l o g y . t h e i n t r o d u c t i o n o f pet h a s a l s o g i v e n c u r r e n t i n t e r e s t t o the p o s s i b i l i t i e s t o f i n d d i s t i n c t , s p e c i f i c l o c a l i z a t i o n s f o r which t h e whole body a u t o r a d i o graphy i s s t i l l t h e u n r i v a l l e d method. t h e 231 references 1. appelgren, l.-e.: s i t e s o f s t e r o i d hormone formation. a u t o r a d i o g r a p h i c s t u d i e s u s i n g l a b e l l e d p r e c u r s o r s . acta p h y s i o l scand (supel 301) 1-108, 1967. 2. hansson, e. schmiterlow, c.g.: a comparison o f t h e d i s t r i b u t i o n , e x c r e t i o n and metabolism o f a t e r t i a r y (promethazine) and a q u a r t e n a r y ( a p r o b i t ) p h e n o t h i a z i n e com pound l a b e l l e d w i t h s 3 ' . archs i n t pharmacodyn thcr 131:309-324, 1961. 3. u l l b e r g , s.: s t u d i e s on t h e d i s t r i b u t i o n and f a t e o f s3 ' l a b e l l e d b e n z y l p e n i c i l l i n i n t h e body. acta radio1 (suppl 118) 1-110, 1954. 4. u l l b e r g , s.: the technique o f whole body autoradiography. c r y o s e c t i o n i n g o f l a r g e specimens. science tools. the lkb i n s t r u m e n t j o u r n a l (sweden). s p e c i a l i s s u e on whole body autoradiography 1-29, 1977. address f o r r e p r i n t s : l a r s e r i k appelgren dept o f pharmacology toxicology biomedicum, box 573 s-751 23 uppsala, sweden 238 upsala j med sci 87: 259-267, 1982 rapid regression of pituitary tumours during bromocriptine treatment of women with h yperprolactinaemia torbjorn bergh, sven johan nillius, per olof lundberg, ulf mostrom, paul enoksson, lena ohman and leif wide departments of obstetrics and gynaecology, neurology, neuroradiology, ophthalmology and clinical chemistry, university hospital, uppsala, sweden abstract four hyperprolactinaemic women with large pituitary adenomas with suprasellar extension were given primary tumour therapy with bromocriptine. the treatment resulted in rapid tumour regression in all the women, a s verified by repeated computerized tomography (ct) scans. pronounced visual field defects were present in three of t h e four women before treatment. all of them had marked improvement of vision within a few days after t h e initiation of bromocriptine therapy and they regained normal or nearly normal visual fields during t h e treatment. the raised serum prolactin concentrations decreased to normal levels in all t h e women. t h u s , medical treatment with bromocriptine can induce rapid tumour regression in patients with hyper prolactinaemia and large pituitary tumours. introduction the management of patients with prolactin-secreting pituitary tumours is still controversial (4). three main treatment alternatives a r e available, namely s u r g e r y , radiotherapy and medical treatment with dopamine agonists , like bromocriptine . transsphenoidal microsurgery with selective adenomectomy has been widely used during t h e last few years. in patients with prolactin secreting pituitary microadenomas, experienced neurosurgeons can produce excellent results b u t the cure r a t e decreases markedly in patients with large prolactinomas (5, 7 ) . radiotherapy can be given a s external or internal pituitary irradiation with few immediate complications (8, 1 3 ) . however, radiotherapy alone seldom normalizes hyperprolactinaemia and gonadal function within a reasonable period of time (6) and the long-term effects on endocrine function a r e unknown. here we report on results of primary bromocriptine treatment of four hyperprolactinaemic women who had large pituitary tumours with suprasellar extension. bromocriptine induced a dramatic reduction in tumour size with rapid improvement of visual field defects. 259 patients and methods four hyperprolactinaemic women with large pituitary tumours were treated with bromocriptine. none of the women had received prior pituitary tumour therapy with irradiation or surgery. clinical details are given in the case reports and table 1. prolactin in serum was measured radioimmunologically by the use of 1251-labelled human prolactin and rabbit anti-human prolactin antibodies coupled to cnbr-activated ultrafine sephadex particles (17). a prolactin preparation (vls 3) supplied by the national institutes of health, bethesda was used as a reference standard. in our laboratory the normal range for healthy women of fertile age is 2-15 pg/l (mean 6.5 pg/l) (1). the visual fields were tested kinetically with the goldmann perimeter and the following stimuli were used: v/4, 0 / 4 , 0 / 3 and 0 / 2 . radiological examination of the sella turcica included hypocycloidal poly tomography and computerized tomography (ct-scan , em1 ct 1010) with intravenous administration of iodinated contrast medium. the pituitary fossa was classified from the skull x-rays according to thorner e t al. (14), (e.g. b4: asymmetry > 3 mm with double contour throughout, b5: ballooned fossa, e: erosion). bromocriptine was given in doses of between 5 and 15 mg/d. during treatment the women were followed-up with repeated visual field examinations and ct-scans. table 1. pituitary fossa classification and serum prolactin levels in four bromocriptine-treated women with large pituitary tumours. prolactin, pg/l before during bromocriptine treatment age sella therapy 1 month 2 months 6 months classifi cation* patient 1 36 b4e 1340 67 1 2 14 patient 2 56 b4e 1385 70 26 13 patient 3 71 b5e 46 <0.5 <0.9 <0.1 patient 4 74 b5e 5500 210 88 66 "according to thorner et al. (14). 260 case reports patient 1 was a 36-year-old woman with secondary amenorrhoea since 1965. she was treated with human gonadotrophins in 1968 and had an uneventful term pregnancy. sellar x-ray was not performed. after the pregnancy she continued to have amenorrhoea. in 1971 sellar x-ray showed a slight asymmetry of the sellar floor ( b 2 ) . she was then lost to follow-up. in 1980 she consulted a gynaecologist for vaginal discomfort and the long-standing amenorrhoea and galactorrhoea was noticed. her pretreatment serum prolactin level was 1340 pg/1. sellar x-ray showed evidence of tumour progression since 1971 with pronounced asymmetry of the pituitary fossa and local erosion of the sellar floor (b4e). a t ct-scan, there was a small suprasellar extension of a pituitary tumour but the visual acuity and the visual fields were normal. bromocriptine treatment (7.5 mg daily) resulted in normal prolactin levels within 2 months. a t a repeat ct-scan after 4% months of bromocriptine therapy, the suprasellar portion of the tumour was no longer visible. regular ovulatory menstruations started after 3 months of treatment during which the patient experienced markedly increased general well-being with return of libido. patient 2 was a 56-year-old nullipara with 21 years of amenorrhoea galactorrhoea. in the beginning of the amenorrhoea period she consulted several doctors but no x-ray examination of the pituitary fossa was performed. in 1980 she saw a doctor because of fear of breast cancer. a serum prolactin level of 1385 pg/l was found. sellar x-ray showed a large asymmetrical pituitary fossa (b4e) and ct-scan visualized a pituitary tumour with large suprasellar extension. the visual field examination showed unrestricted periphery, an absolute temporal scotoma in the right field and a relative temporal hemianopia in the left field. the visual acuity was 0.9 in the right eye and 1.0 in the left eye. bromocriptine was given in increasing doses up to 15 mg daily after 5 days of treatment. the visual fields improved within 6 days and were normal within 2 months. repegt ct-scan after 2 weeks of bromocriptine treatment showed marked tumour regression but a small suprasellar portion still remained. after 2 months of treatment there were no longer any signs of suprasellar extension of the tumour (fig. 1). the prolactin levels had decreased but were still slightly raised (table 1). patient 3 was a 71-year-old nullipara with menopause a t the age of 48. in 1980 she was operated with cholecystectomy because of chronic cholecystitis . postoperatively, the patient had a period of confusion and fatigue. a 6-month history of headache and decreased visual acuity was detected. the patient was found to have hyperprolactinaemia and visual field defects and was referred for evaluation of a suspected pituitary tumour. 26 1 fig. 1. head ct-scans of a 56-year-old woman before (top) and after 2 weeks (middle) and 2 months (below) of bromocriptine treatment. after 2 months of therapy the suprasellar portion of the tumour is no longer visible. 262 a t admittance the visual acuity was 0.7 in the right eye and 0.8 in the left eye. visual field examination showed relative bitemporal hemianopia. sellar x-ray showed a ballooned pituitary fossa and ct-scan revealed a pituitary tumour with large suprasellar extension. the prolactin level in serum was 46 pg/l (table 1). bromocriptine therapy (7.5 mg daily) normalized the raised serum prolactin concentration within one week. some improvement of the visual fields was recorded after one week and after 6 weeks of treatment the improvement was marked with visual acuity of 0.9 in the right and 1.0 in the left' eye. after four and a half months the left visual field appeared normal and the right field had further improved. evidence of rapid tumour regression was also found a t the radiological examination. a ct-scan after 6 weeks of treatment showed regression of the suprasellar extension and after 8 months of therapy only a small suprasellar tumour portion was visible. during the bromocriptine treatment the patient reported loss of headache and a pronounced increase of general well-being. patient 4 was a 74-year-old woman with 2 term pregnancies and regular menstruations until the age of 48. she had been treated with prednisolone (5 m g daily) for one year because of polymyalgia rheumatica. the patient was admitted to the hospital with a 6-month history of visual deterioration. the visual acuity was 0.8 in the right and 0 . 1 in the left eye. she had almost complete temporal hemianopia in the right field. left visual field examination showed complete temporal hemianopia and poor vision in the nasal field. x-ray showed enlargement of the pituitary fossa w i t h destruction of the sellar floor and the dorsum sellae and ct-scan revealed a large suprasellar extension of the pituitary tumour. the prolactin concentration in serum was 5700 pg/l. transnasal aspiration biopsy confirmed the diagnosis of a pituitary adenoma. the tumour was classified a s an invasive pituitary adenoma ( 9 ) . bromocriptine therapy (5 mg daily) resulted in a rapid decrease of the high serum prolactin concentration but the prolactin level was still slightly raised after 6 months of treatment (table 1). during the bromocriptine therapy, a rapid improvement of the visual acuity and visual field defects was recorded. after two weeks of treatment the visual acuity was 1 . 0 in both eyes and only small bitemporal defects remained. three and a half months later the left visual field appeared normal (fig. 2 ) . repeat ct-scan after 5 weeks of therapy showed regression of the suprasellar tumour. after 7 months of bromocriptine treatment only a minor suprasellar portion remained. during the treatment the patient experienced a marked increase in well-being. 263 fig. 2 . visual fields of a 74-year-old woman before and after 1 , 2 and 16 weeks of bromocriptine therapy. the bitemporal hemianopsia rapidly improved during the treatment. discussion medical treatment with brornocriptine resulted in tumour regression in our four hyperprolactinaemic patients who had large pituitary tumours with suprasellar extension. three of the women had visual field defects which rapidly disappeared or markedly improved during the treatment. repeated ct-scans showed regression of the suprasellar extension of the tumours in all four women. the rapidity of the tumour shrinkage was remarkable in three of the patients. radiologically demonstrable tumour regression was evident after only 2-6 weeks of bromocriptine treatment. the daily dose of bromocriptine 264 was only 5-7.5 mg in three of the four women. the improvement of the visual fields was observed even earlier, being obvious after only a few days of treatment. the results agree well with those of other recent studies in which bromocriptine-induced regression of pituitary tumours has been objectively verified by repeated ct-scans or contrast radiology (2,3,10,11,12,15,16,18). the prolactin levels before treatment was very high in three of our patients. one woman (patient 3) had moderate hyperprolactinaemia (46 pg/l) before treatment despite evidence of a large pituitary tumour with suprasellar extension. i t can be questioned if she had a prolactinoma. the prolactin hypersecretion may be caused b y disturbed production or transportation of the prolactin-inhibiting factor (pif) because of pressure from a large non functional adenoma, i t is interesting t h a t bromocriptine had a regressing effect also on this tumour. wollesen e t al. (18) recently showed t h a t bromocriptine was effective in reducing the size of not only prolactin secreting b u t also nonsecreting extrasellar pituitary tumours. transsphenoidal adenomectomy has frequently been recommended for treatment of hyperprolactinaemic patients with evidence of pituitary tumours. however, surgical treatment of large prolactinomas is associated with poor results (5, 7). medical treatment with dopamine agonists like bromocriptine is well tolerated and has no serious side-effects. this and other recent clinical studies have demonstrated t h a t bromocriptine can have a remarkably rapid tumour-regressing effect on large pituitary adenomas. i t is therefore worth while to s t a r t with bromocriptine treatment even if s u r g e r y is planned. the likelihood is great that the tumour will decrease in size and become more easy to remove surgically later. our results like those of other recent studies strongly suggest t h a t bromocriptine i s the primary treatment alternative in patients with large prolactinomas. acknowledgments the study was supported by the swedish medical research council (grant no 13x-3145). we thank mrs birgitta bohman for excellent secreterial assistance. references 1. bergh, t . , nillius, s. j . and wide, l . : bromocriptine treatment of 42 hyperprolactinaemic women with secondary amenorrhoea. acta endocrinol 2. chiodini, p . , luizzi, a . , cozzi, r . , verde, g . , oppizzi, g . , dallabonzana, d . , spelta, b . , silvestrini, f . , borghi, g . , luccarelli, g. , rainier, e . and horowski, r . : size reduction of macroprolactinomas 88: 435-451, 1978. 265 3. 4. 5. 6. 7. 8. 9. 10. 11. 1 2 13, 14 15. by bromocriptine or lisuride treatment. j clin endocrinol metab corenblum, b . and hanley , d . a . : bromocriptine reduction of prolactinoma size. fertil steril 36 : 716-719, 1981. edwards, c. r . w . and feek, c.m. : prolactinoma: a question of rational treatment. b r med j 283:1561-1562, 1981. fahlbusch, r . : surgical failures in prolactinomas. in: pituitary adenomas. biology, physiopathology and treatment (eds . p . j . derome , c. p. jedynak & f . peillon) , pp . 273-284. asclepios publishers, france, 1980. gomez, f. , reyes, f . i. and faiman, c. : nonpuerperal galactorrhea and hyperprolactinemia . clinical findings, endocrine features and therapeutic responses in 56 cases. am j med 62:648-660, 1977. hardy, j . : transsphenoidal microsurgical treatment of hypersecreting pituitary adenomas. in : endocrinology 1980 (eds . i. a . cumming , j . w . funder & f . a . 0. mendelsohn) , pp . 715-722. australian academy of science, canberra, 1980. kelly, w.f., doyle, f.h., mashiter, k., banks, l.m., gordon and h . , joplin , g . f . : pregnancies in women with hyperprolactinaemia : clinical course and obstetric complications of 41 pregnancies in 27 women. b r j obstet gynaecol 86: 698-705, 1979. lundberg, p . o . , drettner, b . , hemminsson, a . , stenkvist, b . and wide, l. : the invasive pituitary adenoma. a prolactin-producing tumor. arch neurol 34:742-749, 1977. 53:737-743, 1981. mcgregor, a.m., scanlon, m.f., hall, r. and hall, k . : effects of bromocriptine on pituitary tumour size. b r med j 2:700-703, 1979. prescott, r . w .g. , johnston, d.g. , kendall-taylor, p . , crombie, a . , hall, k . , mcgregor, a . and hall, r . : hyperprolactinaemia in men response to bromocriptine therapy. lancet i : 245-248, 1982. spark, r . f . , baker, r . , bienfang, d.c. and bergland, r . : bromocriptine reduces pituitary tumor size and hypersecretion. requiem for pituitary surgery? jama 247: 311-316, 1982. thorner, m.o., besser, g . m . , jones, a . , dacie, j. and jones, a . e . : bromocriptine treatment of female infertility : report of 13 pregnancies. b r med j 4: 694-697, 1975. thorner, m.o., edwards, c.r.w., charlesworth, m., dacie, j . e . , moult, p.j.a., rees, l.h., jones, a . e . and besser, g.m.: pregnancy in patients presenting with hyperprolactinaemia. b r med j 2: thorner, m.o., martin, w.h., rogol, a.d., morris, j . l . , perryman, r.l., conway, b . p . , howards, s . s . , wolfman, m.g. and macleod, 771-774,1979. 266 r .m. : rapid regression of pituitary prolactinomas during bromocriptine treatment. j clin endocrinol metab 51: 438-445, 1980. 16. wass, j.a.h., williams, j . , charlesworth, m., kingsley, d.p.e., halliday, a.m., doniach, i . , rees, l . h . , mcdonald, w.i. and besser, g.m.: bromocriptine in management of large pituitary tumours. b r med j 284: 1908-1911, 1982. 17. wide, l. : use of particulate immunosorbents in radioimmunoassay . in: immunochemical techniques, vol. b. (eds. h. van nunakis & j . j . langone) vol. 73 of methods in enzymology (eds. s.p. colowick & s.p. kaplan) p p . 204-224. academic press, new york, 1981. 18. wollesen, f . , andersen, t . and karle, a . : size reduction of extrasellar pituitary tumors during bromocriptine treatment. ann i n t med 96: 281-286, 1982. address for reprints : dr. torbjorn bergh department of obstetrics and gynaecology university hospital s-750 14 uppsala 14 sweden 267 upsala j med sci 81: 109-1 1 1 , 1976 influence of sympathetic nerve activity on renal haemodynamics and release of renin a preliminary communication l.-e. lorelius, p.-0. lofroth, l. wiklund, c. morlin and h. &erg from the departments of diagnostic radiology, radiophysics, anaesthesiology, and internal medicine, university hospital, uppsala, sweden al3 s tract the role of the sympathetic nerve system is still unknown in different types of hypertension. the procedure used in the evaluation of suspect renovascular hypertension has been revised in such a way that renal blood flow, renal arterial blood pressure and plasma renin activity in renal vein could be determined prior to and after splanchnic blockade. a new devic-videovolumeter-has been used to investigate the blood flow. some preliminary data are given. introduction the significance of the sympathetic nerve system in different types of hypertension is still obscure in many respects (5). it is known, however, that this nerve system is one of several mechanisms in volved in the regulation of renin release (4, 9). renin has attracted considerable interest in recent years in association with hypertension, not only the secondary but also the primary form (2), and con sequently increasing research has been devoted to the role of the sympathetic nerve system in these conditions. as the kidney plays a central part not only with regard to renin but also concerning other, partly unknown, circulation-regulating substances, it would be of value to gain more knowledge on the effect of sympathetic impulses on the haemodyna mics of the kidney and on renin release. in the autumn of 1975 an investigation on these problems was begun, with the use of partly new methods. a preliminary report of the initial results is pre sented in this paper. material six p a t i e n t s 4 men and 2 women-in the age range 36 to 63 years, with hypertension of suspected renal origin, have been investigated hitherto. the patients took no medication for at least 4 weeks prior to the investiga tion, and for the last week of these 4 they stayed in hospital. for 5 days prior to the investigation a low sodium diet was given ( c . 20 mmol na+ per day) and also 40 mg furosemide (lasixa) daily. this is the routine pro cedure in our department in all investigations for suspect ed renovascular hypertension (7). method for investigating the effect of splanchnic blockade on the renal haemodynamics and renin release, we chose to make an extra study of the right kidney in addition to the routine investigation of both kidneys aimed a t revealing the presence of any significant renal arterial stenosis. the reason for this choice was that on the right side there is a greater distance between the kidney and the lung, which diminishes any possible influence of the lung on the '33xenon measurement of the renal blood flow. a polyethylene catheter with an outer diameter of 18 gauche was inserted so that its tip lay at the level of the right anterior margin of the vertebral body l 1. this was done on the day before the investigation, so that the procedure should not affect the conduction capacity of the autonomous nerves and thereby the sympathetic flow to the kidney at the time of the investigation. the catheter was inserted under general anaesthesia, which was in duced by 200-500 mg propanidid (epontol"). on the morn ing of the investigation day a catheter was inserted into the femoral vein by the seldinger technique and advanced further. blood samples were taken from the inferior vena cava and the left and right renal veins for renin deter mination. this catheter was left in situ with its tip in the right renal vein for further sampling. a red odman catheter with end-holes and no side-holes was then in troduced through the contralateral femoral artery, also by the seldinger technique. the pressures in the aorta and right renal artery were then recorded via the latter cathe ter, using an e m t 34 pressure transducer (siemens elema, sweden). the renal blood flow was then meas ured by videodensitometry, with selective injection of upsala j meed sci 81 110 l.-e. lorelius et ai. 8 ml angiografv into the right renal artery under a pressure of 295 kpa. the results were analysed later by a videovolumeter (1). selective arteriography was sub sequently performed, with 12 ml angiografin@, under a pressure of 295 kpa, and with an exposure frequency of three frames per s for 3 s, two frames per s for 2 s , one frame every other s for 6 s, or (in 2 cases) with cine-re cording at 50 frames per s. the renal blood flow was then determined by selective injection 0.3-0.4 mci lsxe into the right rend artery via the same catheter as mentioned above, which had been left in an unchanged position. recordings were made over the kidney with a nai scintillation spectrometer. the detector, which had a crystal diameter of 5 cm, was fixed in a cylindrical collimator with the anterior surface of the crystal lying about 8 cm from the margin of the collimator. the collimator was placed as close to the patient's skin as possible. the detector was placed on the ventral side of the patient, and was centred over the kidney under fluo rescent control. the number of impulses that passed through the analyser was recorded in both analogue and digital form for subsequent analysis in a 2-compartment model. further recordings of the blood pressure in the right renal artery were made, after which blood samples were again taken from the right renal vein for renin determina tion. with the patient in the same position, splanchnic blockade was induced with 40 ml of 0.25% plain bupi vacaine (marcaine3 injected into the polyethylene catheter inserted on the previous day. the arterial blood pressure was recorded 10 and 20 min after induction of the blockade, and the renal blood flow was again determined by the xenon method. blood samples were also taken for renin determination. the patient, the collimator and the polyethylene catheter were kept in the same position throughout the examination. with the patient still in the same position, densitometry and angiography were repeated, after which the arterial blood pressure was recorded and blood samples were again taken from the right and left renal veins for renin determination. the investigation was completed with a left-sided renal angiography. the plasma renin activity was determined by a modified radioimmunoassay method (8). results after the induction of splanchnic blockade the mean blood pressure in the renal artery decreased by 15 to 20% in all assessable patients. this blood pressure reduction is of the same order of magni tude as has been reported previously after splanch nic blockade in patients without cardiovascular dis orders (9). the total blood flow through the kidney, meas ured with 133xe, decreased in all patients. our preliminary results indicate that there is good correspondence between blood flows measured densitometrically and those calculated from the 133xe washout curve. moreover, from the 3 patients for whom technically satisfactory 'curves were obtained by both methods of flow measure ment, it seemed that flows measured from video densitometric recordings over the areas in which the renal cortex dominates corresponded relatively well with the flows calculated from the most rapid component of the 133xe curve. in 4 patients the blood flow through the renal cortex decreased after the blockade, while in one patient it increased. one patient was excluded from these results for technical reasons. after the blockade the vascular resistance in the investigated kidney was reduced in 3 patients, un changed in 2 and somewhat increased in one (this latter patient had a vasovagal reaction). in the pa tients with the highest mean arterial blood pressures the vascular resistance was reduced, while in those with more normal pressures it was less affected. despite the decrease in blood pressure and in the total renal blood flow, an increased release of renin was not noted in any of the patients. discussion renovascular hypertension is still, more than 40 years after goldblatt's important discoveries, diffi cult to understand in certain respects. by deter mination of the plasma renin activity (pra) in the renal veins, significant stenosis of the renal arteries can be revealed. more problematical, however, is the question of how, with a normal peripheral pra, stenosis can give rise to hypertension ( 6 ) . the mechanisms of renin release have been part ly established, but we still have relatively little knowledge about the interactions between them and their individual contributions to the total events. how great, for example, is the role of the sympa thetic nerve system in comparison with that of the baroreceptor mechanism? further, it is possible that the answer to this question might not be the same for individuals with healthy kidneys as for those with disorders of the kidneys and renal arteries. one of the aims of the present investiga tion is t o elucidate the role of the sympathetic nerve system in renal arterial stenosis. this is only a preliminary report and is intended mainly as a methodological description. some observed ten dencies are of interest, however, and deserve some discussion. despite the fact that the autonomous upsala j med sci 81 influence of sympathetic nerve on renal haemodynamics 1 1 1 nerve blockade resulted in a reduced perfusion pressure in the kidney, pra did not increase. the explanation for this must be that this blockade, which acts mainly upon the sympathetic nerves, eliminates the effect of a reduced renal arterial pressure. it is too soon to draw definite conclu sions, as our investigation is only in its early stages. it would seem reasonable to propose, however, that the sympathetic nerve system plays a predominant role in the release of renin and perhaps over shadows the effect mediated by baroreceptors in afferent renal arterioles. no absolute conclusions can yet be drawn as to differences in the behaviour of the sympathetic nerve system in kidneys with normal arteries and those with arterial stenosis. another interesting finding is the relative reduc tion in the cortical blood flow after the splanchnic blockade. corresponding shunting, but in the other direction-from medulla to cortex-has been ob served in dogs after stimulation of the renal nerves (3). finally, it must be mentioned that none of the patients suffered any noteworthy discomfort from the investigation, even though it was somewhat longer than the selective renal vein catheterization, including nephroangiography, usually undertaken in these patients with renal arterial stenosis. no complications occurred in connection with the investigation, which was approved by the ethical committee of the faculty of medicine. references 1 . bjork, l., erikson, u. & hallstrom, a,: the video volumeter. upsala j med sci 79: 148, 1974. 2. brunner, h. r., et al.: essential hypertension: renin and aldosterone, heart attack and stroke. n engl j med286:441, 1972. 3. l a grange, r. g., sloop, c. h. & schmid, h. e.: selective stimulation of renal nerves in the anesthe tized dog. circ res33: 704, 1973. 4. michelakis, a. m. & mcallister, r. g.: the effect of chronic adrenergic receptor blockade on plasma renin activity in man. j clin endocrinol metab 34:386, 1972. 5 . dequattro, v . & miura, y.: neurogenic factors in human hypertension: mechanism or myth? am j med 55: 362, 1973. 6. schambelan, m., glickman, m., stockigt, j. r. & biglieri, e . g.: selective renal-vein renin sampling in hypertensive patients with segmental renal stenosis. n engl j med 290: 1153, 1974. 7. strong, c. g., hunt, j. c., sheps, s. g., tucker, r. m. & bernatz, p. e.: renal venous renin activity. enhancement of sensitivity of lateralization by so dium depletion. am j cardiol27: 602, 1971. 8. wide, l.: personal communication. 9. wiklund, l.: postoperative hepatic blood flow and its relation to systemic circulation and blood gases during splanchnic blockade and fentanyl analgesia. acta anaesthesiol scand (suppl.) 58: 5 , 1975. 10. winer, n., chokshi, d. s., yoon, m. s. & freedman, a. d.: adrenergic receptor mediation of renin secre tion. j clin endocrinol metab 29: 1168, 1969. received j a n u a r y 29, 1976 address for reprints: hans aberg, m.d. department of internal medicine university hospital s-750 14 uppsala sweden upsala j med sci 81 114 plenary lectures structural and physiological aspeci's of & l ? k x m l ~ heparin.. alan a. h o r n e r (department of physiology, university of toronto, toronto, ontario canada). evidence w i l l be presented that the s t r u c t u r a l i n t e g r i t y of macro molecular heparin, the multi-chain fonn of heparin isolated fram pronase digested r a t skin (horner, j. biol. chem. 246, 231, 1971) depends on a unique polypeptide core containing only s e r i n e and glycine i n equim0lar p r q r t i o n s . degradation studies using alkali i n t h e presence of h-borohydride, n i t r o u s acid and an endqlycosidase present i n muse mastocytml t i s s u e (robinson, horner, h z k , &ren and lindahl, manuscript in preparation) . the data supporting the proposed structure is derived f r a n 3 it follows f r a n this nodel that t h e individual heparin chains linked t o the polypeptide core are considerably longer than those of ccormercial heparin. (young and homer, manuscripts i n preparation), of the sequential break dawn of m a c r m l e c u l a r heparin by several depolymrases present i n normal rat tissues, yielding chains i n t h e same molecular s i z e range as c o m r c i a l heparins. endoqlycosidase in t h a t products of d i f f e r e n t s i z e s are produced. small i n t e s t i n e contains two depolymrase a c t i v i t i e s with ph o p t h a of 6.0 aryj 7.4, w h i l s t plasma contains a c t i v i t y w i t h a ph o p t i m u m of 6.0 only. the plasma e n z p p r d u c t fonned a t ph 6.0 is l a r g e r than t h e i n t e s t i n a l enzyme product formed a t ph 6.0. these depolymerizing enzymes are not necessarily a l l endqlycosidases, one may cleave t h e proposed polypeptide core structure. examples w i l l be given, fram work i n progress i n t h i s laboratory these enzyms appear t o act d i f f e r e n t l y f r m the mastocytomal the the possible physiological significance of t h e breakdmm of macro molecular heparin i n vivo w i l l be discussed, with p a r t i c u l a r reference t o the r e l a t i o n s h i p betwen endogenous heparin arid lipoprotein lipase. the pathological jmplications of impaired depolymerization of macranolecular heparin have been studied i n an i n d i r e c t manner by feeding an atherogenic d i e t t o squirrel mnkeys. m e a n s was a c c q a n i e d by t h e accumulation of macromolecular heparin i n the small i n t e s t i n e , a t i s s u e in which only low m l e c u l a r weight heparin was found i n healthy m n t r o l s . macromolecular heparin inhibits lipoprotein l i p a s e a c t i v i t y i n v i t r o (homer, prcc. n a t . acad. sci. u.s.a. 6 9 , 3469, 1972). support the hypothesis of z i l v e r s n i t (circulation res. 2, 633, 1973) which contends t h a t lipoprotein l i p a s e and endogenous heparin are s i g n i f icant factors in the a e t i o l q y of atherosclerosis. demonstrates that the enzymic depolymerization of m a c r m l e c u l a r heparin is an additional f a c t o r which must be considered. the induction of atherosclerosis by d i e t a r y therefore the r e s u l t s of the s q u i r r e l monkey feeding experiment the present work upsala j med sci 82 upsala j med sci 98: 415-416, 1993 6.3.2 databases for facilitating work on setting quality specifications: biological variation c. g. fraser department of biochemical medicine, ninewells hospital and medical school, dundee, scotland data on within-subject and between-subject biological variation are essential prerequisites to many strategies used to set quality specifications. the data in the literature have been collated in three reviews. these and data published to date in 1992 are listed here to facilitate future work. data to 1980 ross jw. evaluation of precision. in, werner m, ed. crc handbook of clinical chemistry. boca raton, fla, crc press, vol 1, pp 391-422. data, 1981-1987 fraser cg. the application of theoretical goals based on biological variation data in clinical chemistry. arch pathol lab med 1988; 112: 404-415. data, 1988-1991 fraser cg. biological variation in clinical chemistry. an update: collated data, 1988-1991. arch pathol lab med 1992; 116: 916-923. data, 1992 to date ortola j, castineiras mj, fuentes-arderiu x. biological variation data applied to the selection of serum lipid ratios used as risk markers of coronary heart disease. clin chem 1992; 38: 56-59. kafonek sd, derby ca, bachorik ps. apolipoproteins in patients referred to a lipid clinic. clin chem 1992; 38: 864-872. biological variability of lipoproteins and 41 5 fuentes-arderiu x, albert p. within-subject biological variation of pituitary ovarian axis hormones and desirable imprecision. clin chim acta 1992; 207: 257-259. dot d, miro j, fuentes-arderiu x. within-subject biological variation of hematological quantities and analytical goals. arch pathol lab med 1992; 116: 825-826. pagani f, panteghini n. biological variability of lipoprotein (a) [lp(a)] in serum. clin chem 1992; 38: 1058-1059 gallacher sk, johnston lk, milne db. short-term and long-term variability of selected indices related to nutritional status. xi. vitamins, lipids, and protein indices. clin chem 1992; 38: 1149-1153. jimenez cv. variabilidad bilogica intraindividual de las magnitudes citohematologicas como objectivo de calidad analitica (within-subject biological variation of haematological quantities as analytical quality goals). quim clin 1992; 11: 147-150. simon m, macia m, ribera c, et al. objectivos analyticas de calidad en la determinacion de diversos constuyentes en orina (analytical quality goals for various quantities in urine). quim clin 1992; 11: 156-160. alvarez v, hernandez a, macia m, et al. election del major especimen para la determinacion de constituyentes en orina. us0 de 10s datas de variabilidad biologica (selection of the best specimen for analysis of urine quantities: use of data on biological variation). quim clin 1992; 11: 161-165. correspondence: callum g fraser, department of biochemical medicine, ninewells hospital and medical school, dundee dd1 9sy, scotland. 416 upsala j med sci 79: 39-44, 1974 influence of various anaesthetic agents on the formation and stability of haemostatic plugs in the rabbit mesentery d. bergqvist, f. n. mckenzie and k.-e. arfors department of experimental medicine, pharmacia ab, uppsala, sweden, departmeni of surgery, university hospital, uppsala, sweden, and department of surgery, university of aberdeen, aberdeen, scotland abstract the effect of five different anaesthetic agents (urethane, chloralose, pentobarbital, fentanyl-fluanisone and ether) on the formation and stability of haemostatic plugs in transected microvessels of the rabbit mesentery was studied. ether significantly decreased the frequency of rebleeding through previously stable haemostatic plugs. there were no signifcant differences between the other four agents although there was a tendency to de creased plug stability during pentobarbital anaesthesia. it is concluded that, despite some theoretical disad vantages, urethane offers the satisfactory form of anaesthesia static plug formation using the paration. most convenient and for studies on haemo rabbit mesenteric pre introduction observations o n the response to microvascular transection or puncture in the mesentery of ana esthetised animals has been extensively used as a means of studying haemostatic plug formation. different workers using the mesenteric technique have employed a wide variety of anaesthetic agents. thus barbiturates (11, 12, 14, 15, 19, 24, 25), chloralose (16, 17), urethane (1, 8, 14, 17), mor phine (16) and ether ( 1 ) are among the different anaesthetic agents which have been used. the milieu in which platelets aggregate to form a haemostatic plug should appropriately be con sidered prior to interpretation of results obtained using the method. this view is strengthened by recent reports of anaesthetic-induced changes in platelet reactivity in vivo (6, 20). a study of the effect of five commonly used anaesthetic agents on the formation and stability of haemostatic plugs was therefore undertaken to define the suitability of different forms of anaesthesia in studies of the initial haemostatic mechanism, using the rabbit 1 mesenteric preparation. materials and methods m a t e r i a l new zealand white rabbits (weight 2.5h0.5 kg), fed on a standard diet (teknosan pellets, ab ferrosan, malmo, sweden) were used. five groups, each of 5 aminals, were studied. 1. ethylcarbamate (urethane, kebo, stockholm, sweden) 2. alpha-d-glucochloralose (chloralose@, merck, darm stadt, west germany) 3 . sodium pentobarbital (nembutap, abbott labora tories ltd., north chicago, usa) 4. fentanyl+fluanisone (hypnorm@ a d us. vet., ab leo, halsingborg, sweden) 5. diethylether (aether ad narcosin, skinska bomulls krutsfabriks ab, dosjebro, sweden). in a further 3 groups, each of 5 animals, the effects of anaesthesia with urethane, pentobarbital or ether on arterial blood p h , pc0, and po, were studied. a n a e s t h e t i c t e c h n i q u e 1 . urethane was administered i.v. as a 20% solution in 0.9% saline. supplementary doses were given inter mittently as required t o a total amount of 1.4-r-0.1 g/kg. 2. chloralose anaesthesia was induced using 5% chlora lose in 5% sodium borate given i.v. (2) and maintained by intermittent i . v . infusion of 0.8% chloralose in saline. this solution was kept at 30 to 40°c and continuously stirred and before injection it was filtered. local ana esthesia with 1% xylocaine (astra lakemedel ab, soder talje, sweden) was needed for skin incision t o suppress the hyperexcitability on tactile stimulation after clora lose infusion. the amount of chloralose used for in duction was 68.0?16.4 mg/kg and for maintenance 3 5 . 3 2 3 0 . 5 mg/kg. 3 . sodium pentobarbital was administered slowly i.v. a s a 12.5 mg per ml solution in physiological saline. small fortification doses were given a s required. the total amount used was 36.226.9 mg/kg. 4. neurolept analgesia was achieved by intermittent i.m. injection of hypnorm (fentanyl 10 mglml and fluanisone 0.2 rngiml). the ,mount used was 0 . 8 2 t 0.20 mltkg. 5 . ether anaesthesia was achieved by drip on open mask. upsula j med sci 79 40 d . bergqvisf et (11. the injection were given into the marginal ear vein. with each agent, anaesthesia was kept at a depth suffi cient to inhibit the blink and paw reflex while main taining regular respiration. the depth of anaesthesia was assessed every 5 minutes. operative technique the rabbits were starved for about 12 hours before the experiment. before anaesthesia, a central ear artery was cannulated (silasticm, dow coming, midland, usa) for sampling and blood pressure measurement before and during induction. after induction, blood pressure was measured through a teflon cannula (ab stille-werner, stockholm) placed in the femoral artery, using a strain gauge pressure transducer (statham) and an ultralette u v recorder (abem, stockholm). pressure measure ments were not obtained during induction with ether. the heart rate was obtained from the pressure curves. the procedure for haemostatic experiments is de scribed in detail elsewhere (3, 4). briefly, mesenteric arterioles and venules, each divided according to size into two groups, 20-<40 pm and 40-<60 pm, were cleanly transected, using a fresh gillette scalpel blade, shape e/i 1 . the primary haemostatic plug formation time, i.e. from transection to the first cessation of bleeding, and the time and frequency of rebleeding through previously stable haemostatic plugs were re corded. the sum of the primary haemostatic plug formation time and the rebleeding times in one vessel gives the total haemostatic plug formation time. in each animal 4 transections in each of the two arteriolar subgroups and 5 transections in each of the two venular subgroups were made. each transected vessel was ob served for 15-20 minutes. laboratory technique blood from the central ear artery was taken for de termination of haematocrit and coagulation time before and 30 minutes after the start of the experiment and at the end of the experiment. haematocrit was measured in triplicate using a micro-haematocrit centrifuge (10000 g for 5 min; international equipment co., boston, usa). coagulation time on 1 ml whole blood was determined in duplicate in unsiliconised glass tubes. at the same time intervals the bleeding time after cutting small peripheral ear veins, 0.2-0.5 mm, was measured. the mean value of four bleeding times was calculated on each occasion. in the 15 animals used for acid-base studies blood samples were obtained through a cannula inserted into the central ear artery before and at 15, 30, 60, 90 and 120 minutes after the induction of anaesthesia. the animals were other wise prepared in the same way as the rabbits used for the haemostatic plug experiments. ph, pc0, and po, were measured with an acid-base analyzer (model 213, instrumentation laboratory inc., lexington, usa). statistical methods the haemostatic plug formation times from transected mesenteric vessels have a skew distribution (4). for statistical purposes the original values were rendered upsala j med sci 79 more normally distributed by logarithmic transformation. the mean logarithmic haemostatic plug formation time for each vessel type in each group was calculated and the group means compared by one-way-layout analysis of variance (23). when this test showed significant differences between the groups, simultaneous 95% con fidence limits were calculated for differences between the groups according to the tand s-methods of scheffe (22). the frequency of rebleeding was calculated for both vessel types and subgroups together for each animal and the differences in the frequencies com pared using the rank sum test (9). this test avoids assumptions on the nature of the frequency distribu tion of the data. the significance of changes in haema tocrit, coagulation time, bleeding time, ph, pco,, po, and blood pressure was examined by the student [-test. results the depth of anaesthesia was kept at an even level by frequent reassessment of the blink reflex, paw reflex and the rate and depth of respiration. despite this, two animals in the chloralose group died of respiratory failure. they were excluded from the study. under cloralose anaesthesia the rabbits tended to be hyperexcitable with excessive reaction to noise and exaggerated tendon reflexes. with neurolept analgesia, there is no real ana esthesia, only profound sedation and analgesia. the values of ph, pcoz and poz in three groups studied are shown in table i . there was a significant decrease of ph at 15 minutes in the ether group, a slight but significant increase of poz at 90 minutes in the urethane group and a significant decrease of poz at 15 minutes in the pentobarbital group. during induction the mean arterial pressure increased between 20 and 30 mm hg in animals anaesthetised with urethane and chloralose, some what less after sodium pentobarbital and not at all after neurolept analgesia (fig. 1). thereafter, blood pressure in the urethane group decreased and remained constant at the control value while after chloralose, arterial pressure remained approxi matively 10 mmhg above the control value. the immediate increase in pressure after sodium pento barbital injection was followed by a progressive decline throughout the period of observation. after both neurolept analgesia and ether anaesthesia a gradual fall below control values was observed. the changes in arterial blood pressure within and between the different groups were not statisti cally significant. the heart rate increased significantly after in influence of unaesthetic ugents on huemostatic plugs 41 table 1 . effect of urethane, sodium pentobarbital and mean value and s.d. of 5 experiments in each group on arterial p h , p c 0 , and po, s,p<o.ol urethane control 7.43 f 0.03 i5 min 7.442 0.05 30 min 7.4420.06 60 min 7.46k0.06 90 min 7.45k0.08 120 min 7.45 k 0.05 sodium pentobarbital control 7.49 k 0.03 15 min 7.47+ 0.05 30 min 7.47 1 0.04 60 rnin 7.48 1 0.04 90 min 7.51 10.05 120 min 7.48 k 0.05 ether control 7.43 f 0.03 15 min 7.31f0.07. 30 rnin 7.37 f 0.08 60 min 7.39f0.06 90 min 7.38 f 0.07 120 min 7.3610.08 34.1 k 0 . 9 35.6f 1.4 35.9+ i . i 35.4f0.6 34.6+ 1.4 35.1 f 1.4 34.4f3.4 36.2+ 1.8 36.8k3.1 36.9k 3.5 35.9k2.8 36.912.1 33.1 1 3.1 31.3 1 2 . 4 29.8k2.9 29.5k6.2 30.0k5.0 32.2k4.4 77.9f 1.5 79.1f8.0 77.6k4.7 80.9 if6.7 85.0+4.6* 81.0k4.1 80.315.7 70.2+ 1.9. 71.3 f 4 . 6 72.0f7.1 78.9f7.6 76.0 i: 4.4 87.2 1 4.7 91.9 1 9.3 90.718.8 8 9 . 8 1 13.2 86.6f9.8 85.9 f 12.0 duction and remained elevated in all groups @ <0.005) except neurolept analgesia where no change occurered. changes in the bleeding time from small ear veins and in coagulation time and haematocrit are shown in table 11. the haematocrit remained about the same level throughout the experiment in all the groups. the coagulation time showed a tendency to increase, but this change was not statistically significant in any group. variable fluctuations in bleeding time were seen in the different groups. the only value of statistical significance was the decrease in bleeding time at 30 minutes after sodium pentobarbital anaesthesia @<0.01) and this was followed by a return to near control values by the end of the experiment. the total and primary haemostatic plug forma tion times from transected mesenteric vessels is shown in table 111. analysis of variance revealed no significant differences between the groups. the frequency of rebleeding (table iv) was signi ficantly less for arterioles than for venules in the different groups @<0.005) except for ether. table , iv also shows that the venules in the ether 110 100 3 v) yi g 90 2 b o 2 70 i0 3 60 # i a urefbane cbioraiose knfobarbrfal neuroiepr efher +i i , i 1 i i ? 10 20 30 lo 50 60 70 mtn afrer induction c . 2 $ 2 f i g . i . the effect of different anaesthetic agents on mean arterial blood pressure before and during induction of anaesthesia and at intervals after start of experiment. each point represents the mean of 5 observations. table 11. effect of different anaesthetic agents on bleeding time (bt, seconds), coagulation time ( c t , seconds) and haematocrit (hct, %) before induction of anaesthesia and at 30 and 70 minutes after start of experiment mean values and s.d. of 5 experiments in each group * = p < 0.01 control 30 min. 70 min. urethane bt 76+11 53+14 5 1 1 1 4 ct 259 k 87 288 f 47 3 1 5 f 4 2 hct 38.8k2.6 40.2k3.5 3 7 . 8 1 1.9 chloralose bt 5 8 k 9 75+ 10 4 3 1 9 ct 291 k 2 0 303 k 34 330+ 32 hct 37.8k1.9 36.4f4.2 37.0f4.1 sodium pentobarbital bt 62+11 4155. 5 5 1 6 ct 228 t 34 249158 2 6 7 1 3 7 hct 3 9 . 0 t 3 . 4 39.412.8 39.613.0 neurolept ct 225 & 30 270k28 263 1 29 hct 3 6 . 4 t 1.1 34.4k1.5 35.8k2.2 ether 3t 68 2 24 6 4 1 13 6 2 1 18 ct 243 & 63 273 + 34 270 k 28 hct 34.2& 4.1 35.253.6 33.0k5.1 bt 6 6 + 7 7 2 1 2 1 5 1 1 1 1 upsala j med sci 79 42 d . bergqvist et al. table 111. effect of different anaesthetic agents on total ( t h t ) and primary ( p h t ) haemostatic plug formation times (seconds) in transected mesenteric arterioles and venules each value represents the mean and s.d. of 20 observations o n transected arterioles and 25 observations on transected venules in each vessel subgroup arteriole venule 20402040 c 4 0 p m ( 6 0 p m < 4 0 p m < 6 0 p m urethane tht 3 1 2 1 1 pht 2 5 2 8 chloralose tht 89+88 pht 8 7 2 9 0 sodium pentobarbital tht 68+13 pht 5 9 2 1 3 neurolept tht 136+114 pht 1322115 ether tht 9 0 2 8 6 pht 77+86 84+32 76f31 138290 101239 5 4 2 3 3 4 7 2 3 3 1 0 2 2 6 2 9 7 2 6 2 79 + 46 69 & 44 280+ 135 298+ 143 177+132 1702137 259+104 273+131 179+110 168+89 3 5 4 2 146 3 8 9 5 190 1 4 4 2 4 9 243+ 183 1872174 393+151 1322124 257+ 150 138+59 328+217 116+74 267+225 group had a significantly lower rebleeding frequency than venules in the other four groups (p<0.05). in fig. 2 the total frequency of rebleeding has been plotted against the total rebleeding time. it is apparent that, particularly in venules, sodium pentobarbital and ether differed from the other three anaesthetic agents used. the pentobarbital group showed a tendency to a long total re table iv. incidence of rebleeding through previously stable haemostatic plugs in different anaesthetic groups the low incidence of rebleeding after ether anaesthesia is apparent while the values for the other groups are comparable arteriole, 20< 40 pm 6 3 6 5 2 arteriole, 40< 60 p m 4 8 6 3 3 venule, 2 0 < 4 0 p m 18 12 19 14 3 venule,40-<60,um 20 16 18 18 7 total 48 39 49 40 15 arterioles 20 39p venules 2039p 0 1 * * i i 1 i 60 i20 180 240 total rebleedrng time (sec) fig. 2. total rebleeding time in transected mesenteric arterioles (open symbols) and venules (filled symbols) plotted a s a function of the frequency of rebleeding. the symbols representing each anaestetic group are as shown in fig. 1. bleeding time while the opposite is true of ether. there were, however, no statistically significant differences between the total rebleeding times in the different groups. discussion it has become increasingly accepted by workers studying, in particular, the cardiovascular system that anaesthesia has a profound effect on dif ferrent physiological functions. when interpreting data obtained from anaesthetised animals it is clearly important to estimate the extent of possible abnormality. in this study, ether was the only anaesthetic agent which significantly altered haemostatis; the haemostatic plug formation times from transected mesenteric venules were somewhat shorter and the incidence of rebleeding through haemostatic plugs in venules was significantly lower. ether also induced most hypotension of the various anaesthetics used. since, in addition to these dis advantages, assistance is required to maintain an even depth of ether anaesthesia, use of this agent appears unsatisfactory in these experiments. the technical difficulties associated with the use of neurolept or chloralose anaesthesia were not offset by any obvious advantage with either agent. the relatively stable acid-base status during urethane anaesthesia in rabbits has been reported by bito & eakins (5) and emphasis had also been placed on the wider safety margin of urethane vpsala j med s c i 79 influence of anaesthetic agents on haemostatic plugs 43 over barbiturate anaesthesia (21). from a circula tory point of view urethane seems to give more stable conditions (18). ph, pc0, and po, were stable during urethane anaesthesia, thus confirming the results of bito & eakins (5). the significant reduction in p 0 2 immediately after induction of pentobarbital anaesthesia confirms our earlier results (20). although both urethane and pentobarbital have on occasion been given intraperitoneally in haemo static and thrombotic studies (8, 12, 24), this route seems questionable when observations on the mesenteric microvasculature are being made. the present study shows that the haemostatic plug formed during pentobarbital anaesthesia tends to be unstable, this tendency being reflected as a longer total rebleeding time. born & philp (6) showed that the duration of platelet micro-emboli sation from sites of injury in rat pial vessels was significantly longer after urethane anaesthesia than after pentobarbital or ether. since control observations could not be made on unanaesthetised rats it is difficult to draw precise conclusions, particularly since an inhibitory effect of pento barbital on rabbit platelet reactivity in vitro and in vivo has recently been reported (20). the cytotoxic effect of urethane on vascular endothelium (10) and its haemolytic effect (7) are the theoretical objections to acceptance of it as the anaesthetic of choice for these studies. in the concentration used (20% w/v) urethane causes minimal haemolysis and any adenosine diphosphate released from red blood cells would be rapidly metabolised (13). since the ideal anaesthetic agent in these experiments should lie between the extremes of negligible rebleeding (if situations of decreased plug stability are under study) and a pronounced rebleeding tendency (if situations of enhanced plug stability are being examined), the compromise which fits best these circumstances would appear to be urethane. acknowledgments we are grateful t o anette mckenzie and inger eriksson for skilful technical assistance. 2. 3. 4 5 . 6 7. 8 . 9. 10. 11. 12. 13. 14. 15. 16. 11. 18. 19. references 1 . apitz, k.: diebedeutungder gerinnungund thrombosa fur die blutstillung. virchows arch path anat308: 540, 1942. 20 arfors, k.-e., arturson, g. & malmberg, p.: effect of prolonged chloralose anaesthesia on acid base balance and cardiovascular functions in dogs. acta physiol scand8i: 47,1965. arfors, k.-e. ,bergqvist, d., bygdeman, s . , mckenzie, f. n. & svensjo, e.: the effect of inhibition of the platelet release reaction on platelet behaviour in vitro and in vivo. scand j haemat 9: 323, 1972. bergqvist, d.: haemostatic plug formation in the rabbit mesentery. a methodological study. upsala j med sci, in press. bito, l. & eakins, k.: the effect of general anae sthesia on the chemical composition of blood plasma of normal rabbits. j pharm exptl ther 169: 277, 1969. born, g. v. r. & philp, r. b.: effect ofanaesthetics on the duration ofembolization ofplatelet thrombi formed in injured blood vessels. nature 205: 398, 1965. bree, m. & cohen, b.: effects of urethane anae sthesia on blood and blood vessels in rabbits. lab animal care 15: 254, 1965. didisheim, p.: inhibition by dipyridamole of arterial thrombosis in rats. thromb diath haemorrh 20: 257, 1968. dixon, w. j . & massey, f. j. jr: introduction to statistical analysis. mcgraw-hill book comp. inc., new york, 1957. doljanski, l. &rosin, a.: studies on the earlychanges in the livers of rats treated with various toxic agents, with special reference to the vascular lesions. i . the histology of the rat’s liver in urethane poisoning. amer j pathol. 20: 945, 1944. evans, g., packham, m., nishizawa, e., mustard, j. & murphy, e.: the effect of acetylsalicylic acid on platelet function. j exptl med 128: 877, 1968. herrman, r., frank, j.d. & marlett, d. l.: an in vivo technique for assessing the formation of a hemostatic plug. proc soc exptl biol med 128: %0, 1968. holmsen, i. & holmsen, h.: breakdown of adenine nucleotides in whole blood. 111. congr. int. soc. thromb. haemost, washington 1972, p. 2 0 6 . honour, a. j . & russell, r. w. r.: experimental platelet embolism. brit j exptl pathol 43: 350, 1962. hovig, t., rowsell, h. c., dodds, w. j . , jmgensen, l. & mustard, j. f.: experimental hemostasis in normal dogs and dogs with congenital disorders of blood coagulation. blood 30: 636, 1967. hugues, j.: contribution a i’etude des facteurs vasculaires et sanguins dans i’hemostase spontanee. arch int physiol41: 565, 1953. kjaerheim, a . & hovig, t.: the ultrastructure of haemostatic blood platelet plugs in rabbit mesenterium. thromb diath haemorrh 7: 1 , 1962. korner, p. i., uther, j. b. &white, s. w.: circulatory effects of chloralose-urethane and sodium pento barbitone anaesthesia in the rabbits. j physiol 199: 253, 1968. m a n , j., barboriak, j. j . & johnson, s.: relation ship of appearance of adenosine diphosphate, fibrin formation and platelet aggregation in the haemo static plug in vivo. nature205: 259, 1965. mckenzie, f. n . , svensjo, e. & arfors, k.-e.: effect upsala j med sci 79 44 21. 22. 23, 24, 25. d. bergqvist et al. of sodium pentobarbital anaesthesia on platelet behaviour in vitro and in v i v o . microvasc res 4 : 4 2 1972. murdock, h . : anaesthiesia in the rabbit. fed proc 28: 15.10, 1969. scheffe, h.: the analysis of variance. john wiley & sons, inc., new york, 1963. snedecor, g. w . & cocbran, w. g.: statistical methods. iowa state university press ames, 1967. webber, a. & johnson, s.: platelet participation in blood coagulation aspects of hemostasis. amer j pathol 60: 19, 1970. zucker, m . : platelet agglutination and vasoconstric tion as factors in spontaneous hemostasis in normal thrombocytopenic, heparinized and hypoprothtrombine mic rats. amer j physioll48: 275, 1947. r e c e i v e d m a y 16, 1973 address for reprints: david bergqvist department of experimental medicine pharmacia ab, box 604 s-751 25 uppsala 1 sweden u p s d a j med sri 79 upsala j med sci 84: 281-284, 1979 rupture of an abdominal aortic aneurysm into the vena cava s. bowald, b . almgren, i . eriksson, g. j a c o b s o n and l.-e. lorelius from the departments of general surgery and diagnostic radiology, university hospital, uppsala, sweden abstract aortic abdominal aneurysm combined with aorto-caval fistula is a rare condi tion with a high mortality. we discuss here the symptomatology and treatment of one such patient. introduction an aorto-caval fistula may occur either spontaneously from a ruptured athero sclerotic aortic aneurysm, or traumatically. spontaneous aorto-caval fistulae may be expected in 1-4 % of surgically treated aortic aneurysms ( 7 ) , leading to severe haemodynamic changes. the diagnosis is often overlooked preoperatively and the mortality rate is high even for patients who reach the operating table ( 5 ) . the first case was described by syme in edinburgh in 1 8 3 1 and a recent re view of the literature revealed 9 4 cases (5). we will relate our experience of one case. case report a previously healthy 68-year-old man was admitted to the department of s u r gery on april 29, 1978 because of sudden abdominal and low back pain, general weakness and frequent vomiting. he had been treated with analgesics for three days for his back. physical examination revealed a well-nourished, slightly pale man with tachycardia, but with no overt signs of cardiac failure. the blood pressure was 1 4 0 / 8 0 mm hg. a slightly tender abdominal mass was palpable and epigastric pulsations were seen. there were no signs of gastrointestinal bleeding but urinalysis disclosed numerous red blood cells. on the clinical suspicion of a dissecting aortic aneurysm, abdominal aortography was performed. this revealed a large aortic aneurysm just below the renal arteries and extend ing to the external iliac arteries. contrast medium could be seen outside tne aneurysm along the right border, indicating an aorto-caval fistula (fig.). 28 1 a f i g . 1 . e a r l y ( a ) and l a t e ( b ) a r t e r i a l p h a s e of a o r t o g r a p h y . arrows p o i n t t o l e a k a g e of c t r a s t medium from t h e aneurysm t o t h e i n f e r vena c a v a . ton , i o r b 282 an emergency 1aparotomy was immediately performed. early during the operation the patient went into severe heart failure with difficulty in oxygenation. the aorta was clamped just below the renal arteries. when the aneurysm was opened, heavy venous bleeding occurred from a 5-mm wide aorto-caval fistula. because of necrosis of the aneurysmal wall, the fistula was excised and the defect in the caval vein was repaired with a saphenous patch graft. the aneurysm was replaced with a dacron bifurcation graft. after closure of the fistula a sudden improvement in the central circulation was noted. postoperatively the patient improved rapidly. there was no haematuria and the chest x-ray showed no stasis. at follow-up three months later, the patient was in good condition. comments the symptoms of a large arterio-venous fistula are well known and the diag nosis of an aorto-caval fistula should in fact present no difficulty. in spite of this, most of these fistulas are not detected before operation (4). this may be due to a lack of awareness of this uncommon condition on the part of the physician, or to the absence of some characteristic symptoms ( 1 ) . in a patient who appears with an abdominal pulsatile mass, venous hypertension of both lower extremities, low back pain, an abdominal bruit, and gross or microscopic haema turia, an aorto-caval fistula should be suspected ( 1 , 6). a typical continuous murmur may be absent owing to vena cava obstruction produced by the aneurysm (2). in the early stage, when the fistula is small, there are no signs of peripheral oedema or venous distension ( 3 ) . the fistula enlarges with time, due to degenerative changes of the aneurysmal wall, and typical signs of high output cardiac failure will occur. the oedema of the lower abdomen and legs is of the pitting type and does not respond to digitalis or diuretics ( 3 ) . some patients show varying degrees of haematuria and renal failure. this is probably related both to decreased arterial perfusion and to renal vein congestion ( 3 ) . some patients have intensive vomiting, which may be due to congestion of the liver or gastric mucosa ( 3 ) . during the operation the haemodynamics must be carefully observed to main tain balance in fluid therapy. for this purpose the swan-ganz thermistor cathe ter is preferable, since it allows monitoring of pulmonary arterial pressure and pulmonary capillary wedge pressure, as well as cardiac output with the thermodilution technique. during surgery the risk of bleeding from con gested pelvic veins the cause of death in some cases ( 1 ) must be kept in mind. 283 references 1 . baker, w.h., sharzer, l.a. & ehrenhaft, j.l.: aortocaval fistula as a com 2. beall, a.c., cooley, d.a., morris, g.c. & debakey, m.e.: perforation of plication of abdominal aortic aneurysms. surgery 7 2 : 9 3 3 9 3 8 , 1 9 7 2 . arteriosclerotic aneurysms into inferior vend cava. arch surg 8 6 : 8 0 9 8 1 8 , 1 9 6 3 . 3 . cortis, b.s., jablokow, v.r., shah, a.n. & cortis, p.f.: spontaneous rup ture of an abdominal aortic aneurysm into the inferior vena cava: a case report and review of the literature. mt sinai j med (ny) 3 9 : 5 6 6 6 0 4 , 1 9 7 2 . fistula involving the abdominal aorta: report of four cases with successful repair. ann surg 1 4 7 : 6 4 6 6 5 8 , 1 9 5 8 . dominal aortic aneurysms into the vend cava nature’s aid to the surgeon? scand j thorac cardiovasc surg 1 2 : 6 5 6 9 , 1 9 7 8 . 6 . nennhaus, p.h. & javid, h.: the distinct syndrome of spontaneous abdominal aortocaval fistula. am j med 4 4 : 4 6 4 4 7 3 , 1968. 7 . olcott, c., holcroft, j.w., stoney, r.j. & wylie, e.j.: unusual problems of abdominal aortic aneurysms. am j surg 1 3 5 : 4 2 6 4 3 5 , 1 9 7 8 . 4 . debakey, m.e., cooley, d.a., morris, g.c. & collins, h.: arteriovenous 5 . neglh, p., book, k., eklund, a-e., eklof, b. & karp, w.: rupture of ab accepted february 2 0 , 1 9 7 9 address for reprints: staffan bowald, m.d. department of surgery university hospital s 7 5 0 1 4 uppsala sweden 284 upsala j med sci 84: 7 5 8 2 , 1979 the effect of short-term high-dose treatment with methenamine hippurate of urinary infection in geriatric patients with indwelling catheters 11. evaluation by m e a n s of a quantified urine sediment astrid norberg, bo norberg, ulf parkhede, hans gippert and rolf ekman the school of'nursing and drparfmenfs oj'education, i n f e r n a l medicine and clinical clirmistry, university of l u n d , s\r,eden. 3 m riker ltrborafories. shurholmen, sw'eden abstract the u r i n e s e d i m e n t of 12 g e r i a t r i c p a t i e n t s w i t h i n d w e l l i n g c a t h e t e r s was quan t i f i e d by t h e glutaraldehyde-cytocentrifuge method p r i o r t o , d u r i n g and a f t e r a c l i n i c a l t r i a l of methenamine h i p p u r a t e ( m h ) , 2 g x 3 d a i l y g i v e n f o r 34 days as t h e s o l e t h e r a p e u t i c a g e n t f o r u r i n a r y t r a c t i n f e c t i o n . c o n c e n t r a t i o n i n t h e u r i n e of t h e s e p a t i e n t s w a s 100 c e l l s / p l ( q , q 3 5 0 3 5 0 ) , i . e . t e n f o l d h i g h e r t h a n t h e u p p e r normal l i m i t s r e p o r t e d i n h e a l t h y p r o b a n d s . the median b a c t e r i u r i a i n t h e c o n t r o l p e r i o d was 1 2 x l o 5 b a c t e r i a / m l u r i n e , i n t e r q u a r t i l e r a n g e 10-60 x 10 500 x 10 b a c t e r i a / m l . n o t p r o m i n e n t and c o u l d n o t b e c o r r e l a t e d w i t h mh t r e a t m e n t , n o r w i t h c a t h e t e r changes. the r e p o r t e d o b s e r v a t i o n s s u g g e s t t h a t s h o r t t e r m h i g h d o s e t r e a t m e n t w i t h mh as s o l e t h e r a p e u t i c a g e n t r e d u c e d p y r u r i a and b a c t e r i u r i a i n t h e group of p a t i e n t s s t u d i e d . the median l e u k o c y t e 5 b a c t e r i a / m l and e x t r e m e i n d i v i d u a l v a l u e s 300 5 h e m a t u r i a , d e f i n e d a s '> 24 e r y t h r o c y t e s / p l u r i n e , w a s i n t r o d u c t i o n the c l i n i c a l r e l e v a n c e of b a c t e r i u r i a and p y u r i a i n p a t i e n t s w i t h i n d w e l l i n g c a t h e t e r s i s complex. these p a t i e n t s r e g u l a r l y a c q u i r e b a c t e r i u r i a due t o t h e a s c e n s i o n of g u t m i c r o b e s a l o n g t h e mucous l a y e r s u r r o u n d i n g t h e c a t h e t e r ( 2 , 6 , 7 , 8 ) . the b a c t e r i u r i a i s t h o u g h t t o r e f l e c t t h e sum of b a c t e r i a l i n f e c t i o n s of t h e u r i n a r y t r a c t and b a c t e r i a l growth i n t h e u r i n e ( c f . 1 0 ) . t h r e e f a c t o r s a r e thought t o c o n t r i b u t e t o t h e p y u r i a of p a t i e n t s w i t h indwel l i n g c a t h e t e r : 4 , 1 5 ) , t h e b a c t e r i a i n d u c e d i n f l a m m a t i o n of t h e u r i n a r y t r a c t ( 1 1 ) and o t h e r chemical o r p h y s i c a l i r r i t a n t s . the u r i n e becomes c l o u d y and f o u l s m e l l i n g due t o b a c t e r i u r i a , p y u r i a , mucous s e c r e t i o n s , b a c t e r i a l p r o d u c t s and s a l t p r e c i p i t a t i o n . t h e r e a c t i o n of t h e u r e t h r a l mucosa t o a l a r g e f o r e i g n body ( 3 , 75 it i s r e a s o n a b l e t o assume t h a t t h e u r i n e s e d i m e n t r e f l e c t s t h e t o t a l e f f e c t of f o r e i g n body, mucosal l e s i o n s , b a c t e r i a l i n v a s i o n and l e u k o c y t e e m i g r a t i o n . i n a p r e v i o u s s t u d y ( 1 3 ) , w e r e p o r t e d a s i m p l e and r a p i d method of s a m p l i n g , f i x i n g , s t a i n i n g and q u a n t i f y i n g t h e c e l l s and b a c t e r i a of t h e u r i n e from g e r i a t r i c p a t i e n t s w i t h an i n d w e l l i n g c a t h e t e r . the aim of t h e p r e s e n t s t u d y was t o e l u c i d a t e t h e problems of q u a n t i f y i n g t h e p r e v i o u s l y d e s c r i b e d g l u t a r a l d e h y d e c y t o c e n t r i f u g e s e d i m e n t and a p p l y t h i s u r i n e s e d i m e n t t o t h e e v a l u a t i o n o f s h o r t t e r m high-dose t h e r a p y w i t h methenamine h i p p u r a t e (mh) as t h e s o l e t r e a t ment of u r i n a r y i n f e c t i o n i n p a t i e n t s w i t h i n d w e l l i n g c a t h e t e r s . material and methods the s t u d y i n v o l v e d 14 i n p a t i e n t s a t t h e s o m a t o g e r i a t r i c wards of s a i n t lars h o s p i t a l , lund. t o s e v e r a l y e a r s b e f o r e t h e y e n t e r e d t h e s t u d y ( 1 4 ) . a week d u r i n g t h e p r e t r e a t m e n t c o n t r o l p e r i o d , days 10-17, d u r i n g t r e a t m e n t w i t h methenamine h i p p u r a t e (mh, h i p r e x r , r i k e r l a b o r a t o r i e s , loughborough, l e i c e s t e r . e u g l a n d ) , 2 g x 3 , d a i l y from day 18-52 and d u r i n g t h e p o s t t r e a t m e n t c o n t r o l p e r i o d , d a y s 66-73, as d e s c r i b e d i n t h e c l i n i c a l r e p o r t ( 1 4 ) . during days 1-9 d i f f e r e n t c y t o c e n t r i f u g e p r e p a r a t i o n s of t h e g l u t a r a l d e h y d e suspended l e u k o c y t e s and b a c t e r i a were e v a l u a t e d ( 1 3 ) . two p a t i e n t s were exc l u d e d due t o f r e q u e n t b l o c k a g e of t h e c a t h e t e r s a f t e r p r e s a m p l i n g p l u g g i n g , see below. they h a d a l l had i n d w e l l i n g c a t h e t e r s f o r a p e r i o d of 6 months u r i n e w a s sampled t w i c e the c a t h e t e r w a s p l u g g e d i m m e d i a t e l y a f t e r b r e a k f a s t , 15-30 m i n u t e s b e f o r e s a m p l i n g . i n p a t i e n t s no. 1, 3 , 4 u r i n e w a s o b t a i n e d a f t e r p l u g g i n g f o r 2 h r s b e f o r e b r e a k f a s t . the u r i n e , a p p r o x i m a t e l y 1 5 m l , w a s c o l l e c t e d i n a s t e r i l e t u b e and t h o r o u g h l y s h a k e n i n o r d e r t o d i s p e r s e t h e s o l i d s . the u r i n e ( 0 . 1 ml) w a s t h e n immediately t r a n s f e r r e d t o 0 . 9 m l 2% g l u t a r a l d e h y d e (taab l a b o r a t o r i e s , reading. england) i n p h o s p h a t e b u f f e r , 0 . 1 3 5 m , ph 7.4. the g l u t a r a l d e h y d e suspended c e l l s and b a c t e r i a , 0 . 2 m l , were spun down on a s l i d e 2-4 h r s a f t e r s a m p l i n g by means of a c y t o c e n t r i f u g e ( s h a n d o n e l l i o t c y t o s p i n ) a t 1 , 0 0 0 r . p . m . , 10 m i n . , u s i n g t w i n p r e p a r a t i o n s from each specimen. w a s always s t a i n e d w i t h t h e may-griinwald-giemsa s t a i n (mgg) and t h e o t h e r w i t h h a e m a t o x y l i n e o s i n , p e r i o d i c a c i d s c h i f f o r p a p a n i c o l a o u s t a i n , as p r e v i o u s l y d e s c r i b e d ( 1 3 ) . one of t h e p r e p a r a t i o n s it i s e v i d e n t from t h e above d e s c r i p t i o n of t h e p r e p a r a t i o n of t h e u r i n e s e d i m e n t t h a t t h e c e l l number on t h e s l i d e s h o u l d be l i n e a r l y c o r r e l a t e d t o t h e number of c e l l s i n t h e o r i g i n a l u r i n e sample: 1. g l u t a r a l d e h y d e ) . 2. 20 ul u r i n e w a s spun down on e a c h s l i d e (200 u l of a 10% u r i n e s u s p e n s i o n i n the r e l a t i o n between t h e a r e a of a high-power v i s u a l f i e l d of t h e z&ss 76 p h o t o m i c r o s c o p e u s e d ( r = 7 5 um) and t h e a r e a of t h e c e l l p r e p a r a t i o n on t h e s l i d e ( ~ = 3 , 0 0 0 um) w a s d e f i n e d by: 1 2 2 r 7 1 r it 1 , 6 0 0 the whole s l i d e p r e p a r a t i o n w a s scanned u n d e r t h e x10 l e n s . the a r e a w i t h t h e h i g h e s t c e l l d e n s i t y w a s f u r t h e r e v a l u a t e d u n d e r t h e xl00 l e n s , t o t a l mag n i f i c a t i o n ~ 1 , 0 0 0 . and t h e a v e r a g e c e l l number was t a k e n t o r e p r e s e n t t h e c e l l u l a r i t y of t h e p r e p a r a t i o n e x c e p t when t h e lower number w a s z e r o . then one t h i r d of t h e h i g h e s t c e l l number w a s a p p r o x i m a t e d t o r e p r e s e n t t h e c e l l d e n s i t y as a c o r r e c t i o n f o r t h e h e t e r o g e n o u s d i s p e r s i o n of c e l l s . when t w i n specimens d i f f e r e d i n c e l l den s i t y , t h e one w i t h t h e h i g h e r c e l l d e n s i t y was r e c o r d e d , s i n c e l o s s of c e l l s d u r i n g p r e p a r a t i o n a p p e a r e d t o b e more l i k e l y t h a n p r o d u c t i o n of excess c e l l s ( 1 3 ) . the extreme r a n g e s of c e l l s p e r high-power f i e l d were n o t e d s t a t i s t i c s . the r e s u l t s were s u b j e c t e d t o n o n p a r a m e t r i c a n a l y s e s a c c o r d i n g t o s i e g e 1 1956 ( 1 7 ) . results the t r a n s f o r m a t i o n c u r v e between c e l l s p e r high-power f i e l d and c e l l s p e r p 1 u r i n e i s shown i n f i g u r e 1 t o g e t h e r w i t h t h e normal l i m i t s f o r l e u k o c y t e concen t r a t i o n and e r y t h r o c y t e c o n c e n t r a t i o n r e p o r t e d by l i t t l e 1964 (11). the median l e u k o c y t e c o n c e n t r a t i o n of t h e p r e s e n t p a t i e n t s w a s 100 ( q , q3 50 350) l e u k o c y t e s / p l u r i n e d u r i n g t h e p r e t r e a t m e n t c o n t r o l p e r i o d ( t a b l e 1 1 , i . e . a t e n f o l d h i g h e r l e u k o c y t e c o n c e n t r a t i o n t h a n t h e u p p e r l i m i t s of l e u k o c y t e c o n c e n t r a t i o n i n h e a l t h y probands r e p o r t e d by l i t t l e 1964 (11). the r a n k sums s u g g e s t e d a r e l a t i v e r e d u c t i o n i n p y u r i a d u r i n g t h e l a t e p e r i o d of mh t r e a t m e n t (table l), b u t t h i s t e n d e n c y w a s n o t s i g n i f i c a n t ( 0 . 3 > p > 0 . 2 ) , as e v a l u a t e d by t h e friedman two-way a n a l y s i s of v a r i a n c e . the c o n c e n t r a t i o n of b a c t e r i a i n t h e u r i n e f o l l o w e d t h e same p a t t e r n a s t h e l e u k o c y t e c o n c e n t r a t i o n a l t h o u g h on a h i g h e r n u m e r i c a l l e v e l ( t a b l e 2 ) . median c o n c e n t r a t i o n d u r i n g t h e p r e t r e a t m e n t c o n t r o l p e r i o d w a s 1,200 b a c t e r i a / p1 u r i n e , i n t e r q u a r t i l e r a n g e 1,000 6,000 b a c t e r i a / p l u r i n e . s u g g e s t a r e l a t i v e r e d u c t i o n i n b a c t e r i a d u r i n g t h e l a t e p e r i o d of mh t r e a t m e n t ( t a b l e 2), b u t t h i s t r e n d was n o t s i g n i f i c a n t ( 0 . 3 > p > 0 . 2 ) , when e v a l u a t e d b y t h e friedman two-way a n a l y s i s of v a r i a n c e . the the r a n k sums i n c o n t r a s t t o p y u r i a and b a c t e r i u r i a , m i c r o s c o p i c h a e m a t u r i a d e f i n e d as 1 2 4 e r y t h r o c y t e s / p l u r i n e was n o t a p r o m i n e n t f e a t u r e i n t h e p a t i e n t s s t u d i e d ( f i g . 3 2 t a b l e 1. l e u k o c y t e s p e r high-power m i c r o s c o p i c f i e l d ( 1 8 x 10 ,um ) i n t h e $ 1 ~ t a r a l d e h y d e f i x e d c y t o c e n t r i f u g e p r e p a r e d q u a n t i f i e d u r i n e s e d i m e n t s o f g e r i a t r i c p a t i e n t s w i t h i n d w e l l i n g c a t h e t e r s b e f o r e , d u r i n g and a f t e r t r e a t m e n t w i t h methenamine h i p p u r a t e ( m h ) , 2 g x 3 d a i l y . median v a l u e s w i t h i n e a c h p e r i o d , b a s e d o n 3-5 m e a s u r e m e n t s o n d i f f e r e n t d a y s w i t h i n t h e p e r i o d ( c f . 1 3 ) . an i n d i v i d u a l measurement was b a s e d o n d o u b l e p r e p a r a t i o n s f r o m t h e same u r i n e s p e c i men. mm: m e d i a n o f m e d i a n s . q, q 3 : i n t e r q u a r t i l e r a n g e . r . : t h e r a n k sums o f t h e f r i e d m a n two-way a n a l y s i s o f v a r i a n c e ( 1 7 ) r e f l e c t t h e j e l a t i v e c h a n g e s i n p y u r i a d u r i n g t h e p e r i o d s t u d i e d , d a y s 1-73. p r e t r e a t m e n t i n i t i a l p e r i o d l a t e p e r i o d p o s t t r e a t m e n t c o n t r o l p e r i o d o f t r e a t m e n t o f t r e a t m e n t c o n t r o l p e r i o d p a t . sex days 10-17 days 18-35 days 36-52 days 66-73 1. f 2 . m 3 . f 4 . f 5 . f 6 . f 7. f 8 . m 9 . f 1 0 . f 11. f 1 2 . f 3 . 0 0 . 3 1 . 0 1 . 3 0 . 7 5 .o 8 . 0 0.7 1 . 3 7 .o 4 . 0 0 . 3 1 . 9 6 .9 75.0 0 . 9 0 . 3 2.5 5 3 . 0 2 . 2 0 . 3 5 . 0 4 . 2 3 . 9 1 . 0 2.2 8 . 0 0 . 7 0 . 0 4 . 0 30.0 1 .o 0 . 7 4 . 0 1 . 7 0 . 7 2 . 0 1 . 7 1 1 . 0 1 . 7 0 . 3 5 . 0 3 0 . 0 0 . 7 0 . 7 9 . 0 1 . 7 1 . 7 mm 1 . 3 3 . 2 1 . 4 1 . 7 3 0 . 0 3 2 . 5 2 3 . 5 3 4 . 0 r 0.7-4.5 1.4-6 .o 0 . 7 4 . 0 1 . 2 7 .o ql q3 j 2 ) . no e v i d e n c e was f o u n d t h a t e i t h e r s h o r t t e r m t r e a t m e n t w i t h h i g h d o s e m h , 2 g x 3 d a i l y , o r c a t h e t e r c h a n g e s i n c r e a s e d t h e i n c i d e n c e o f h a e m a t u r i a i n p a t i e n t s w i t h i n d w e l l i n g c a t h e t e r s . d u r i n g 20 c o n t r o l d a y s w i t h o u t mh, haemat u r i a was n o t e d i n 1 3 o u t o f 72 u r i n e s e d i m e n t s (18%). d u r i n g mh t r e a t m e n t f o r 34 d a y s , h a e m a t u r i a w a s n o t e d i n 27 o u t o f 1 0 6 u r i n e s e d i m e n t s ( 2 5 % ) . t h i s d i f f e r e n c e was n o t s i g n i f i c a n t ( 0 . 3 > p ; o . 2 ) , a s e v a l u a t e d by t h e x'-test f o r two i n d e p e n d e n t s a m p l e s . nor d i d t h e number o f c a t h e t e r c h a n g e s i n a n i n d i v i d u a l p a t i e n t c o r r e l a t e w i t h t h e number o f e p i s o d e s of h a e m a t u r i a ( n = l l , s p e a r m a n ' s r h o = +0.20). discussion the q u a n t i f i c a t i o n o f b a c t e r i a and c e l l s i n t h e u r i n e o f p a t i e n t s w i t h i n d w e l l i n g c a t h e t e r s i s d i f f i c u l t . one b a s i c c o n d i t i o n i s t h a t t h e c o n c e n t r a t i o n o f b a c t e r i a a n d l e u k o c y t e s a p p e a r s t o b e d e p e n d e n t o n u r i n e p r o d u c t i o n , i . e . e x c r e t i o n o v e r t i m e p r o v i d e s a b e t t e r e s t i m a t i o n t h a n p a r t i c l e c o n c e n t r a t i o n 78 3 2 t a b l e 2 . b a c t e r i a p e r high-power m i c r o s c o p i c f i e l d ( 1 8 x 10 vm ) i n t h e g l u t a r a l d e h y d e f i x e d c y t o c e n t r i f u g e p r e p a r e d q u a n t i f i e d u r i n e s e d i m e n t s of g e r i a t r i c p a t i e n t s w i t h i n d w e l l i n g c a t h e t e r s b e f o r e , d u r i n g and a f t e r t r e a t m e n t w i t h ,,,&henmine h i p p u r a t e ( m h ) , 2 g x 3 d a i l y . b a s e d on 3-5 measurements on d i f f e r e n t days w i t h i n t h e p e r i o d ( c f . 1 3 ) . an i n d i v i d u a l measurement w a s b a s e d on d o u b l e p r e p a r a t i o n s from t h e same u r i n e specimen. mm: median of medians. q q3: i n t e r q u a r t i l e r a n g e . r . : t h e rank sums of t h e friedman two-way a n a l y s i s of v a r i a n c e (17) r e f l e c t t d e r e l a t i v e changes i n p y u r i a d u r i n g t h e p e r i o d s t u d i e d , days 10-73. median v a l u e s w i t h i n e a c h p e r i o d , p r e t r e a t m e n t i n i t i a l p e r i o d l a t e p e r i o d p o s t t r e a t m e n t c o n t r o l p e r i o d of t r e a t m e n t of t r e a t m e n t c o n t r o l p e r i o d p a t . sex days 10-17 days 18-35 days 36-52 days 66-73 1. f 2 . m 3: f 4 . f 5. f 6 . f 7 . f 8. m 9. f 10. f 11. f 1 2 . f 15 150 1 5 125 15 8 20 10 15 7 150 1 5 9 105 23 30 60 12 19 3 5 1 7 7 1 8 9 40 1 2 100 2 15 8 15 8 15 8 6 a 1 3 105 30 1 5 33 55 10 1 3 40 35 35 55 mm 15 1 9 10 34 13-73 11-33 7-15 14-48 30.5 31.0 23.0 35.5 i1 9 3 j (11, 1 6 ) . under t h e e x p e r i m e n t a l c o n d i t i o n s of t h e p r e s e n t s t u d y , t h e c e l l con c e n t r a t i o n p e r p l u r i n e w a s e x p e c t e d t o p r o v i d e a c l o s e a p p r o x i m a t i o n o f e x c r e t i o n p e r u n i t t i m e ( c f . 1 1 ) . the r h e o l o g i c a l p r o p e r t i e s of c a t h e t e r u r i n e a r e a n o t h e r s t u m b l i n g b l o c k . t h i s u r i n e r e s e m b l e s t h i n g r u e l , t h a t i s n o t a homogenous s u s p e n s i o n . the cor p u s c l e s t e n d t o c o n c e n t r a t e i n t h e clumps, much l i k e c e l l c o n c e n t r a t i o n w i t h i n a f i b r i n coagulum d u r i n g b l o o d c l o t t i n g . it i s o b v i o u s t h a t t h e p r e s e n c e of a clump i n t h e sample w i l l produce an o v e r e s t i m a t i o n of t h e c o r p u s c u l a r i t y o f t h e u r i n e and c o n v e r s e l y t h e a b s e n c e of a clump w i l l p r o d u c e a n u n d e r e s t i m a t i o n . gram-stained smears of u r i n e c e n t r i f u g a t e s have b e e n r e p o r t e d t o c o r r e l a t e w i t h t h e numbers of v i a b l e o r g a n i s m s c u l t u r e d from t h e same u r i n e specimens ( 9 ) . it i s r e a s o n a b l e t o assume t h a t t h e p r e s e n t c y t o c e n t r i f u g e p r e p a r a t i o n f u r t h e r improved t h e c o r r e l a t i o n between u r i n e organisms and o r g a n i s m s o b s e r v e d on t h e s l i d e . u r i n a r y b a c t e r i a d e r i v e d from t h e s e p a t i e n t s w i t h i n d w e l l i n g c a t h e t e r s ( t a b l e 2) were i n agreement w i t h t h e b a c t e r i a l c o n c e n t r a t i o n s found by p r e v i o u s a u t h o r s by wans of q u a n t i t a t i v e u r i n e c u l t u r e s (i), i . e . a median p r e t r e a t m e n t b a c t e r i u r i a c o r r e s p o n d i n g t o 1 2 x l o 5 b a c t e r i a / m l u r i n e , i n t e r q u a r t i l e r a n g e 10 60 x 10 t h i s a s s u m p t i o n i s s u p p o r t e d by t h e f a c t t h a t t h e c o n c e n t r a t i o n s of 5 79 f i g . 1. the t r a n s f o r m a t i o n c u r v e bgtwefn c e l l s p e r high-power f i e l d (18 x 1 0 um ) and c e l l s p e r u l u r i n e . the shaded a r e a i n d i c a t e s t h e r e f e r e n c e r a n g e of e r y t h r o c y t e s and l e u k o c y t e s a c c o r d i n g t o l i t t l e 1964 (11). 0 number of urine specimens f i g . 2 . i n c i d e n c e and m a g n i t u d e of haema t u r i a i n 1 2 g e r i a t r i c p a t i e n t s w i t h indwel l i n g c a t h e t e r s p r i o r t o ( d a y s 10-17), d u r i n g (days 18-52) and a f t e r ( d a y s 66-73) t r e a t ment w i t h methenamine h i p p u r a t e ( m h ) , 2 g x 3 d a i l y . erythrocytes /pi urine 0 0.3 0.7 1-2 >2 i i j i , boctcriurio w p y u r i a '""1 m h t reotmcnt 5 4 f a t 1 17 35 52 1 3 d a y s f i g . 3. r e l a t i v e changes i n b a c t e r i u r i a and p y u r i a d u r i n g t r e a t m e n t w i t h methen amine h i p p u r a t e ( m h ) , 2 g x 3 d a i l y , i n p e r c e n t o f c o n t r o l v a l u e s . median v a l u e b a s e d o n t h e p e r i o d medians g i v e n i n t a b l e s 1 and 2 . ____ 80 b a c t e r i a / m l , extreme v a l u e s 300 500 x 105 b a c t e r i a l m l . the i n c i d e n c e and magnitude of h a e m a t u r i a was m o d e r a t e , c o n s i d e r i n g t h a t a l a r g e f o r e i g n body, t h e c a t h e t e r , was i n c l o s e c o n t a c t w i t h t h e mucosal membrane of t h e u r i n a r y t r a c t and t a k i n g i n t o a c c o u n t t h e p e r s i s t e n t p y u r i a b a c t e r i u r i a . mh h a s b e e n t h o u g h t t o b e of l i m i t e d v a l u e i n t h e t r e a t m e n t of e s t a b l i s h e d u r i n a r y t r a c t i n f e c t i o n ( 1 2 ) . if i t i s assumed t h a t mh r e d u c e s b a c t e r i a l growth i n t h e u r i n e w i t h o u t a f f e c t i n g i n f e c t i o n of t h e mucosa, mh t r e a t m e n t c o u l d n o t be e x p e c t e d t o r e d u c e b a c t e r i u r i a and p y u r i a p r i o r t o t h e h e a l i n g of t h e p r e e x i s t i n g mucosal i n f e c t i o n , i . e . a f t e r mh t r e a t m e n t f o r a t l e a s t 1 2 weeks. it i s e v i d e n t from t a b l e s 1, 2 and f i g u r e 3 t h a t a r e l a t i v e r e d u c t i o n of bac t e r i u r i a and p y u r i a o c c u r e d d u r i n g t h e l a t e p e r i o d of mh t r e a t m e n t , i n g l y , t h e v a l u e s from t h e p r e t r e a t m e n t c o n t r o l p e r i o d and t h e i n i t i a l t r e a t ment p e r i o d a r e grouped t o g e t h e r a s one e x t e n d e d c o n t r o l p e r i o d ( t a b l e s 1, 2 ) , t h e p y u r i a w a s a l m o s t s i g n i f i c a n t l y r e d u c e d d u r i n g t h e l a t e mh t r e a t m e n t p e r i o d (p=0.014), as e v a l u a t e d by t h e friedman two-way a n a l y s i s of v a r i a n c e . t i v e r e d u c t i o n of b a c t e r i u r i a was s t i l l n o t s i g n i f i c a n t (p=o.14). i f a c c o r d the rela i n view of t h e s i z e of t h e p r e s e n t m a t e r i a l (n=12) and t h e d i s p e r s i o n of i n d i v i d u a l v a l u e s , t h e r i s k of a c c e p t i n g a f a l s e n u l l h y p o t h e s i s ( i . e . no c u r a t i v e e f f e c t of m h ) seems g r e a t . it i s more r e a s o n a b l e t o assume from t h e cen t r a l t e n d e n c y d e s c r i b e d by t h e r a n k sums ( t a b l e s 1, 2) and t h e r e l a t i v i z e d medi ans ( f i g . 3 . 1 , t h a t s h o r t t e r m high-dose mh t r e a t m e n t r e d u c e d t h e b a c t e r i u r i a and t h e p y u r i a i n p a t i e n t s w i t h i n d w e l l i n g c a t h e t e r s . acknowledgements no. 1. 2. 3 . 4 . 5 . 6. 7 . t h i s work was s u p p o r t e d by t h e swedish medical r e s e a r c h c o u n c i l p r o j e c t 2294 and no. 5362 and g r a n t s from t h e medical f a c u l t y of lund. references a l l i n g , b . , brandberg, a . , s e e b e r g , s . & svanborg, a . : a e r o b i c and a n a e r o b i c m i c r o b i a l f l o r a i n t h e u r i n a r y t r a c t of g e r i a t r i c p a t i e n t s d u r i n g long t e r m care. j i n f e c t d i s 127:34-39, 1973. brehmer, b . & madsen, p . o . : route and p r o p h y l a x i s of a s c e n d i n g b l a d d e r i n f e c t i o n i n m a l e p a t i e n t s w i t h i n d w e l l i n g c a t h e t e r s . j u r o l 108:719-721, 1972. b r u c e , a.w., s i r a , s . s . , c l a r k , a.f. & awad, s . a . : the problem of c a t h e t e r e n c r u s t a t i o n . canad med a s s j 111:238-241, 1 9 7 4 . edwards, l . & t r o t t , p.a.: c a t h e t e r i n d u c e d u r e t h r a l i n f l a m m a t i o n . j u r o l 110:678-681, 1973. g r o s s , p . a . , harkavy, l . m . , barden, g.e. & f l o w e r , m.f.: the e p i d e m i o l o g y of n o s o c o m i a l e n t e r o c o c c a l u r i n a r y t r a c t i n f e c t i o n . am j med s c i 272: 75-81, 1976. k a s s , e.h.: b a c t e r i u r i a and t h e d i a g n o s i s of i n f e c t i o n s o f t h e u r i n a r y t r a c t . arch med 100:709-714, 1957. kass, e . h . & schneiderman, l . j . : e n t r y of b a c t e r i a i n t o t h e u r i n a r y t r a c t s of p a t i e n t s w i t h i n l y i n g c a t h e t e r s . n e w eng j med 256:556-557, 1957. 6-792854 81 8. 9 . 10. 11. 1 2 . 1 3 . 1 4 . 1 5 . 1 6 . 1 7 . k e r e s t e c i , a.g. & l e e r s , w-d.: i n d w e l l i n g c a t h e t e r i n f e c t i o n . canad med a s s j 109:711-713, 1973. l e w i s , j . f . & a l e x a n d e r , j . : microscopy of s t a i n e d u r i n e smears t o d e t e r mine t h e need f o r q u a n t i t a t i v e c u l t u r e . j c l i n microbial 4:372-374, 1976. lewis, j . f . & a l e x a n d e r , j . : the u r i n e c u l t u r e r e v i s i t e d . south med j 70:15-16, 1 9 7 7 . l i t t l e , p . j . : a comparison of t h e u r i n a r y w h i t e c e l l c o n c e n t r a t i o n w i t h t h e w h i t e c e l l e x c r e t i o n r a t e . b r j u r o l 36:360-363, 1964. n i l s s o n , s . : long-term t r e a t m e n t w i t h methenamine h i p p u r a t e i n r e c u r r e n t u r i n a r y t r a c t i n f e c t i o n . acta med scand 198:81-85, 1975. n o r b e r g , b. , n o r b e r g , a . , p a r k h e d e , u., g i p p e r t , h . & akerman, m . : the e f f e c t of s h o r t t e r m h i g h d o s e t r e a t m e n t w i t h methenamine h i p p u r a t e of u r i n e i n f e c t i o n i n g e r i a t r i c p a t i e n t s w i t h i n d w e l l i n g c a t h e t e r s . i . the p r e p a r a t i o n and morphology of a q u a n t i f i e d u r i n e s e d i m e n t . u p p s a l a j med s c i 84: 1979. n o r b e r g , b . , n o r b e r g , a . , p a r k h e d e , u . , g i p p e r t , h . & ekman, r . : the e f f e c t of s h o r t t e r m high-dose t r e a t m e n t w i t h methenamine h i p p u r a t e of u r i n e i n f e c t i o n i n g e r i a t r i c p a t i e n t s w i t h i n d w e l l i n g c a t h e t e r s . i n manus. p a i n t e r , m . r . , b o r s k i , a . a . , t r e v i n o , g.s. & c l a r k , w . e . j r . : u r e t h r a l r e a c t i o n t o f o r e i g n o b j e c t s . j u r o l 106:227-230, 1 9 7 1 . r o b e r t s , a . p . , robinson, r . e . & b e a r d , r.w.: some f a c t o r s a f f e c t i n g bac t e r i a l c o l o n y c o u n t s i n u r i n a r y i n f e c t i o n . b r med j 1:400-403, 1967. s i e g e l , s . : n o n p a r a m e t r i c s t a t i s t i c s f o r b e h a v i o r a l s c i e n c e s . mcgraw-hill, n e w york 1956. c l i n i c a l e v a l u a t i o n . r e c e i v e d f o r p u b l i c a t i o n 1 4 t h j u l y 1978 and i n r e v i s e d form 1 6 t h o c t o b e r , 1978. address f o r r e p r i n t s : bo n o r b e r g , m . d . department of i n t e r n a l medicine u n i v e r s i t y h o s p i t a l of lund s-221 85 lund, sweden 8 2 upsala j med sci 79: 94-96, 1974 roentgenologic determination of foetal maturity ove axelsson and anders hemmingsson f r o m the departments of gynecology a n d diagnosiic r a d i o l o g y , university hospital, u p p s a l u , s w e d e n abstract foetal length determination by roentgenologic measurement of the lumbar length is an unreliable method, with a large range of variation. it is important to be aware of this fact when the method is used for determining foetal maturity. it can be of some value, however, when no centres of ossifica tion can be seen in the knee joint. foetal maturity can also be determined by non-roentgeno logic methods. the biparietal diameter of the foetus can thus be measured with a relatively high degree of accuracy by the ultra sound technique which is simple and has no radiation effects on the mother or foetus. this method gives at a single examination about the same range of variation as the roentgenologic methods. it can, however, because of the lack of radiation hazards, be used in repeated examina tions, whereby the rate of growth of the foetus can be estimated. we have therefore used this method during the last year, with satisfactory results. introduction determination of the degree of foetal maturity can be of value t o the obstetrician, e.g. when the date of conception is uncertain or where induction of labour may come into question. the latter group includes pregnancies complicated by rhesus in compatibility, toxicosis o r diabetes mellitus. several roentgenologic methods are available for determining foetal maturity, one of which is the demonstration of centres of ossification (bishop, 1965; holmberg & liliequist, 1969). the presence of epiphyseal ossification centres in the knee joint is often used as a sign of foetal maturity, but their absence is of no great significance (bishop, 1965). calculations of the foetal length based on the length of the lumbar spine (zsebok, 1957; fagerberg & roonemaa, 1959; weishaar & port, 1964; margolis & woss, 1968), sitting height (zuppinger, 1952) or biparietal diameter (jacobs, 1953, among others) are other roentgenologic methods for determining the degree of maturity. further, ringertz (1971) has calculated the foetal weight from the lumbar length and abdominal diameter. in the last 12 years in our hospital the foetal length has been calculated by the method described by fagerberg & roonemaa i n 1959. the values obtained by this method have shown a relatively large range of variation, however, and its reliability in determining the maturity of the foetus has there fore not been quite clear. as non-roentgenologic methods for determining the foetal maturity are now available, e. g. measurement of the biparietal diameter by ultrasound (thompson et al., 1965) and analyses of the amniotic fluid (brosens & gordon, 1966; mandelbaum et al., 1967), we have investigat ed the reliability of the roentgenologic method of calculation of foetal length and foetal weight, on a one-year material. material and methods all cases examined during 1971 with a gestational time of over 32 weeks ( n = 164) and delivered within 7 days from the time of the roentgenologic examination were re viewed. the length of the lumbar spine was measured by the method of fagerberg & roonemaa (1959) and correlated to the length and weight of the infant post partum (referred to hereafter as “foetal length” and “foetal weight”). the length of the lumbar spine was measured on roentgenograms taken in the prone posi tion with a film-focus distance of 100 cm and a p . a . projection. the measurement was performed from the upper margin of l 1 to the lower margin of l 5 , follow ing the curve of the spine. the foetal length and foetal weight were measured immediately after delivery. from the regression line for lumbar length to foetal length, correction factors for calculating the foetal length were obtained. results the correlation of the lumbar length, measured on the roentgenogram, to the foetal length, is shown in fig. i . the standard deviation is 1.98 cm, which means that the foetal length can be given with an upsala j med sci 79 roentgenologic determination of foetal maturity 95 table. correction factors by which some lumbar lengths are multiplied to obtain the foetal lengths lumbar length multiplying foetal length ( m m ) factor (cm) f i in c n i 60 50 4 0 / / i ,' 1 a' -45 1 i i * 60 l i in mm 40 50 fig. 1 . regression lines for lumbar length ( l . i . ) to foetal length (f. i . ) . -, total material (164 cases). equation: y=23.029+0.483~. ---, 95% confidence level for the total material; ----, diabetes mellitus (9 cases); _ _ _ _ _ , rh incompatibility (13 cases); ----, toxicosis (20 cases). accuracy of 2 4 cm at the 95% confidence level. the regression line for the cases with diabetes mel litus and rh imcompatibility corresponds well with that for the whole material, while the line for the cases with toxicosis is steeper (fig. 1). the number 40 45 50 55 60 10.6 42.324.0 9.9 44.724.0 9.4 47.224.0 9.0 49.654.0 8.7 51.924.0 of cases in these sub-groups is small, however. the correlation coefficient for the whole material is 0.70. the correction factors by which the lumbar lengths are to be multiplied to obtain the foetal lengths can be seen in the table. these factors were calculated from the regression line for the whole material. the correlation between foetal weight and lum bar length is seen in fig. 2 . for the whole material the correlation coefficient is 0.72, with a standard deviation of 0.52 kg, corresponding t o k1.02 kg at the 95 95 level. the regression for the different diag nostic groups falls within this range. it may be noted, however, that the foetal weight for the dia betic cases is greater than that for the total mate rial when the lumbar length exceeds about 50 mm. l . i . in mm discussion / / i' "%, i i 1.8 3.0 4.0 f.w. in kg fig. 2 . regression lines for foetal weight (f. w.) to lumbar length (l. 1.). -, total material (159 cases). equation: y=39.19+4.9x , 95% confidence level for the total material; ---, diabetes mellitus (10 cases); , rh incompatibility (13 cases); ----, toxicosis (17 cases). calculation of the foetal length from the lumbar length gives an inexact result on account of the large standard deviation ( 4 4 cm at the 95% confi dence level). this deviation is of the same order of magnitude as that reported by fagerberg & roone maa (1959), k 3 . 4 cm, and by margolis & woss (1968), +5 cm. this means in gestational time about 5 4 weeks (lubchenco et al., 1966). the regression line for lumbar length to foetal length in this investigation is somewhat less steep than that obtained by fagerberg & roonemaa (1959), which means that the factors for determining the foetal length will be somewhat d'ifferent (see table). thus, for calculating the foetal length from the lumbar length the correction factors obtained here should be used. the correlation between lumbar length and foetal weight in this material is poor. the standard devi ation at the 95% confidence level is 21.02 kg, which means that the method is not practicable for upsala j med sci 79 96 0 . axelsson and a . hemmingsson determining the foetal weight. t h e method used by ringertz (1971), with measurement of the lumbar length and the abdominal diameter, gave a rather better result, with a standard deviation of k0.5 kg a t the 90% level. it is of interest, however, that the overnutrition of the foetus that takes place in diabe tes mellitus (gordon, 1962; margolis & woss, 1968, among others) is evident in the regression for lum bar length to foetal weight (see fig. 2 ) . as men tioned above, however, the number of cases in this group is small. references i . bishop, p. a.: radiologic studies of the gravid uterus, p. 85. hoeber medical division, harper & row, new york, 1965. 2. brosens, i. & gordon, h.: the estimation of maturity by cytological examination of the liquor amnii 7 3 : 88, 1966. 3. fagerberg, s . & roonemaa, j . : radiological de termination of foetal length by measurement of the lumbar spine. acta obstet gynec scand 38: 333, 1959. 4. gordon, h . : the infants of diabetic mothers. am j med sci 224: 35, 1962. 5 . holmberg, n. g . & liliequist, b.: mognadsbestam ning a v fostret in utero. obstetrik och gynekologi 5 : 57, 1969. 6. jacobs, j. b.: the value of the lateral pelvic roent genogram a s an index of fetal maturity and type of maternal pelvis. am j obstet gynec 65: 897, 1953. 7. lubchenco, l. o., hansman, ch. & boyd, e.: intrauterine growth in length and head circum ference as estimated from live births at gestational ages from 26 to 42 weeks. pediatrics 37: 403, 1966. 8. mandelbaum, b., l a croix, g . c. & robinson, a . r . : determination of fetal maturity by spectro-photo metric analysis of amniotic fluid. obstet gynec 29: 471, 1967. 9. margolis, a. j . & woss, r . g . : a method for radio logic detection of fetal maturity. am j obstet gynec 101: 383, 1968. 10. ringertz, h. g.: method for assessment of fetal weight. acta radio1 (diag.) (stockholm) 11: 545, 1971. 1 1 . thompson, h. e., holmes, j . h. gottesfeld, k. r. & taylor, e . s . , fetal development as determined by ultrasonic pulse echo techniques. am j obstet gynec 92: 44, 1965. 12. weishaar, j. & port, f . : beitrag zur rontgenolo gischen grossenbestimmung des feten. geburtsh frauenheilk 24: 1029, 1964. 13. zsebok, z.: neue rontgenmethode zur bestimmung von lange und entwicklungsgrad des intrauterinen fetus. zbl gynaek 79: 1295, 1957. 14. zuppinger, a.: rontgendiagnostik in d e r geburts hilfe. in schinz, h. r., baensch, e . , friedl, e. & uelinger, e.: lehrb.d. rontgendiag. band iv. thieme verlag, stuttgart 1952. received november 17. 1973 address for reprints: anders hemmingsson, m. d. department of diagnostic radiology university hospital s-750 14 uppsala sweden upsala j med sci 79 upsala j med sci 91: 67-76, 1986 fine needle biopsy and scintigram in the preoperative diagnosis of thyroid lesions sighild westman-naeser,' lars grimelius,' henry johansson3 and jan malmaeus3 'departments o pathology and 'surgery, university hospital, uppsala, malmo general hospital, malmo, sweden and f department of pathology, university of lund, abstract fine needle biopsies (fnb) of the t h y r o i d were examined from 860 p a t i e n t s . in 703 c a s e s f o l l i c u l a r c e l l s without atypia were found and i n t h i s group of p a t i e n t s the c l i n i c a l diagnosis was nodular goitre. operations were performed i n 138 p a t i e n t s and i n 97 cases the cytological f i n d i n g could be c o r r e l a t e d t o t h e histopathological diagnosis. in 33 of these patients thyroid carcinoma was h i s t o l o g i c a l l y v e r i f i e d . i n 26 of the carcinoma cases cytologic examination showed grave atypia or changes fndicating carcinoma. the cases i n which the cytological diagnoses were f a l s e l y negative are discussed. moderate c e l l u l a r atypia occurred i n one case w i t h papillary carcinoma. in two cases the cyto 1 ogical examination gave a f a l s e positive diagnosis of cancer, both represent i n g t h y r o i d i t i s of the lymphoid type. cold nodules were found i n 10/19 p a t i e n t s and a hot nodule i n ,1 p a t i e n t . in 3 p a t i e n t s the scintigrams were normal a n d i n another 5 inconclusive. the results indicate t h a t thyroid scintigrams can only be used as a supplement t o t h e physical examination and a guidance f o r fnb. t h e contribution of fnb i n t h e decision t o operate i s discussed and i t is concluded t h a t fnb i s a valuable adjunct i n preoperative diagnosis of thyroid lesions. the b e s t diagnostic r e s u l t s a r e obtained when there i s a c l o s e cooperation between c l i n i c i a n , r a d i o l o g i s t , c y t o l o g i s t and pathologist. the scintigrams i n patients w i t h thyroid carcinoma a r e a l s o presented. introduction palpable thyroid nodules may present d i f f i c u l t i e s i n the d i f f e r e n t i a l diag nosis between a malignant tumour and a benign thyroid lesion. the thyroid gland i s readily accessible f o r f i n e needle aspiration biopsy and thus a possi b i l i t y i s offered t o d i s t i n g u i s h morphologically between non-ma1 ignant and malignant lesions as well as between various types of malignant neoplasms (10,2,6). scintigraphy is often used t o l o c a l i z e areas suspected of malignancy, i . e . cold nodules. i t i s a valuable aid i n the guidance of the f i n e needle 67 b i o p s y . the frequency o f maiignancy on s c i n t i g r a p h i c a l l y l o c a l i z e d c o l d nod u l e s has been r e p o r t e d t o v a r y between 3 and 30 %, and i t i n c r e a s e s w i t h age (8,4,). the p r e s e n t study was undertaken t o analyze t o what e x t e n t these two methods have proven u s e f u l t o g u i d e t h e c l i n i c i a n i n t h e t r e a t m e n t o f p a t i e n t s w i t h t h y r o i d l e s i o n s . to e s t i m a t e t h e accuracy o f t h e c y t o l o g i c a l d i a g n o s i s , t h e r e s u l t s o b t a i n e d a f t e r f i n e needle a s p i r a t i o n b i o p s y were compared w i t h t h e h i s t o p a t h o l o g i c a l f i n d i n g s i n those cases t h a t were operated on. the p r e s e n t i n g s i g n s and symptoms i n p a t i e n t s w i t h a f i n a l d i a g n o s i s o f t h y r o i d malignancy were a l s o i n v e s t i g a t e d . material and methods the p r e s e n t study i n c l u d e s 896 p a t i e n t s w i t h suspected o r apparent t h y r o i d disease r e f e r r e d t o t h e department o f c l i n i c a l cytology f o r f i n e needle b i o p s y o r t o t h e department o f surgery f o r o p e r a t i o n d u r i n g t h e y e a r s 1979-80. a l t o g e t h e r 910 f i n e needle b i o p s i e s (fnb) o f t h e t h y r o i d glands were performed on 860 o f these p a t i e n t s a t t h e l a b o r a t o r y o f c l i n i c a l c y t o l o g y , u n i v e r s i t y , hospi t a l , uppsala. i n 36 p a t i e n t s t h e need f o r o p e r a t i o n was c l i n i c a l l y so & v i o u s t h a t n e i t h e r fnb n o r s c i n t i g r a m were regarded necessary. t h y r o i d s c i n t i g r a m s performed w i t h 99mtc were used i n more than h a l f o f t h e p a t i e n t s i n c l u d e d i n t h i s m a t e r i a l . i n t h i s study, however, o n l y t h e s c i n t i g r a p h i c f i n d i n g s i n t h e p a t i e n t s , who f i n a l l y g o t a d i a g n o s i s o f t h y r o i d carcinoma, w i l l be r e p o r t e d . the fnb and t h e c y t o l o g i c a l examinations were performed b y t h e same doctor. the smears were a i r d r i e d and s t a i n e d a c c o r d i n g t o may grunewald-giemsa. c y s t c o n t e n t s were c e n t r i f u g e d and two smears were prepared, one s t a i n e d as above, t h e o t h e r e t h a n o l f i x e d and s t a i n e d a c c o r d i n g t o papanicolau. f o r h i s t o l o g i c a l examination t h e s u r g i c a l l y removed t h y r o i d specimens ( f i x e d o r u n f i x e d ) were c a r e f u l l y s e c t i o n e d i n t o t h i n s l i c e s o f ji-1 cm. ten % n e u t r a l f o r m a l i n was used as f i x a t i v e . f i v e v m d e p a r a f f i n e d s e c t i o n s were s t a i n e d w i t h haematoxyl i n e o s i n o r a c c o r d i n g t o van gieson. a l l h i s t o p a t h o l o g i c a l examina t i o n s were performed b y t h e same p a t h o l o g i s t . the h i s t o l o g i c a l c l a s s i f i c a t i o n a c c o r d i n g t o t h e who was used. c l i n i c a l i n f o r m a t i o n r e l e v a n t t o t h e p r e s e n t study was o b t a i n e d from t h e m e d i c a l r e c o r d s o f t h e p a t i e n t s . results the h o s p i t a l s and d i f f e r e n t c l i n i c s from where t h e p a t i e n t s were r e f e r r e d f o r fnb a r e l i s t e d i n table 1. 68 table 1. the h o s p i t a l s and c l i n i c s r e f e r r i n g t h e p a t i e n t s (860) f o r f i n e needle biopsy hospital (hi __ c1 i nic nos. of b i o p s i e s u n i v e r s i t y h . uppsal a medic i ne oncology 257 20 1 surgery 116 otolaryngology 36 others 61 regional medicine and 159 community h* surgery general 80 p r a c t i t i o n e r s * total number of b i o p s i e s 910 in some patients t h e biopsy was r e p e a t e d 1-3 times a s the m a t e r i a l was presumed n o t t o be r e p r e s e n t a t i v e f o r the l e s i o n o r t o o s c a r c e t o permit a r e l i a b l e d i a g n o s i s * in t h e county of uppsala table 2. cytological f i n d i n g s i n 910 f i n e needle b i o p s i e s from 860 p a t i e n t s cytological f i n d i n g s nos. of b i o p s i e s a n d d i a g n o s i s fol 1 i c u l a r c e l l s without a t y p i a 703 fol 1 icul a r c e l l s w i t h a t y p i a s l i g h t t o moderate grave or c a n c e r grave i n a s s o c i a t i o n w i t h t h y r o i d i ti s lymphoid type granul omatous type t h y r o i d i t i s non-thyroid l e s i o n s 46 28 18 91 79 12 6 18 i n s u f f ic i en t ma t e r i a1 total 910 * lymphomas, m e t a s t a s e s of renal or s a l i v a r y gland carcinoma 69 i n t h e m a j o r i t y o f t h e fnb f o l l i c u l a r c e l l s w i t h o u t a t y p i a were observed ( t a b l e 2 ) . i n t h i s p a t i e n t group a c l i n i c a l o r h i s t o p a t h o l o g i c a l d i a g n o s i s o f n o d u l a r g o i t r e was u s u a l l y made. b i o p s i e s from 46 l e s i o n s showed c e l l s w i t h s l i g h t t o moderate a t y p i a . i n another 46 cases c o r r e s p o n d i n g t o 5 % o f t h e m a t e r i a l t h e c e l l u l a r a t y p i a was found t o b e grave o r suggesting malignancy. t h i s f i g u r e i n c l u d e s 18 cases o f lymphoid t h y r o i d i t i s w i t h grave c e l l u l a r a t y p i a s . i n a l l t h e s e 18 cases t h e c l i n i c a l d i a g n o s i s o f t h y r o i d i t i s was c l i n i c a l l y and i m m u n o l o g i c a l l y w e l l e s t a b l i s h e d . these p a t i e n t s were f o l l o w e d c l i n i c a l l y and have h i t h e r t o n o t been o p e r a t e d on. t h y r e o i d i t i s o f t h e lymphoid o r granulomatous t y p e was diagnosed i n 9 1 o f t h e b i o p s i e s (10 % ) . i n 1 8 p a t i e n t s t h e m a t e r i a l o b t a i n e d a t t h e b i o p s y d i d n o t p e r m i t a d i a g n o s i s and i t was n o t p o s s i b l e t o p e r f o r m another fnb. d u r i n g t h e i n v e s t i g a t i o n p e r i o d 138 p a t i e n t s underwent surgery o f t h e t h y r o i d gland, 102 o f which had been examined w i t h fnb. most o f t h e p a t i e n t s i n whom t h e c y t o l o g i c a l smears had shown a t y p i a , u s u a l l y o f a s l i g h t degree were n o t operated on. these p a t i e n t s were u s u a l l y e l d e r l y w i t h a l o n g h i s t o r y of m u l t i n o d u l a r g o i t r e . those where t h e a t y p i a was s u s p i c i o u s f o r malignancy and n o t a s s o c i a t e d w i t h lymphoid t h y r o i d i t i s were a l l operated. the main i n d i c a t i o n s f o r surgery a r e g i v e n i n table 3. table 3. i n d i c a t i o n s f o r o p e r a t i o n i n d i c a t i o n s nos. o f p a t i e n t s toxic ( h o t ) n o d u l e ( s 1 6 toxic g o i t r e 32 col d nodul e( s 1 11 nodule(s1 w i t h o u t c e l l u l a r a t y p i a * nodule(s) w i t h c e l l u l a r a t y p i a o r cancer 13 25 c1 i n i c a l l y s t r o n g l y suspected ma1 ignancy** 3 recurrence o f cancer or r e g i o n a l metastases 18 tracheal compression*** 30 t o t a l 138 * ** no fnb performed *** due t o a t o x i c g o i t r e based on f i n e needle b i o p s y (fnb) 70 t ab le 4 . c o rr el at io n b et w ee n cy to lo g ic al an d h is to p at h o lo g ic al di ag no si s in 9 7 p at ie n ts w it h d is ea se d th yr oi d gl an d c yt ol og ic al di ag no si s h is to pa th ol og ic al di ag no si s or f in d in g s g oi tr e a de n om a c ar ci no m a t hy ro i d i t i s c e ll u la r c e ll u la r fo ll ic h ur th le at y p ia at y p ia u la r -c el l f o ll ic u la r p ap il la ry m ed ul la ry a na pl as ti c (l ym ph oc yt ic t y p e) w it ho ut ty pe ty pe f ol l i cu l a r c e ll s w it h no a ty p ia 48 1 2 1 2* 3 1 sl ig h t at y p ia 2 2 m od er at e at yp i a 1 1 gr av e at y p ia 2 3 no s 1 1 c ar ci no m a no s po l 1 ic u l a r pa pi 1 1 a ry m ed ul l a ry a na pl a st ic 1 2 2 10 1 5 t hy ro i d i t i s 2 t ot al 51 2 5 1 7 19 1 6 5 * m ic ro in va si ve t yp e the c o r r e l a t i o n between t h e c y t o l o g i c a l f i n d i n g s and h i s t o p a t h o l o g i c a l d i a g n o s i s c o u l d be g i v e n f o r 97 p a t i e n t s ( t a b l e 4 ) . i n 33 o f these p a t i e n t s t h y r o i d carcinoma was h i s t o p a t h o l o g i c a l l y v e r i f i e d . i n 26 o f t h e carcinoma cases t h e c y t o l o g i c a l diagnoses were grave a t y p i a o r carcinoma. moderate c e l l u l a r a t y p i a o c c u r r e d i n one case which t u r n e d o u t t o be a p a p i l l a r y c a r c i noma. no c e l l u l a r a t y p i a was noted i n two cases w i t h f o l l i c u l a r carcinoma, i n 3 cases w i t h p a p i l l a r y and i n one case w i t h m e d u l l a r y carcinoma. the 6 cases w i t h f a l s e n e g a t i v e f i n d i n g s a t fnb w i l l be d e s c r i b e d more e x t e n s i v e l y below. i n 2 cases t h e r e was a f a l s e p o s i t i v e c a n c e r diagnosis, made on t h e c y t o l o g i c a l smears, b o t h r e p r e s e n t i n g cases w i t h t h y r o i d i t i s o f t h e lymphoid type. the h i s t o p a t h o l o g i c a l examination r e v e a l e d t h a t a mu1 ti f o c a l growth p a t t e r n o f t h e d i f f e r e n t types o f carcinomas was f r e q u e n t . o f t h e 7 cases w i t h f o l l i c u l a r carcinoma 5 showed a m u l t i f o c a l growth p a t t e r n and i n 2 t h e cancer had an a g g r e s s i v e growth e x t e n d i n g i n t o t h e a d j a c e n t c e r v i c a l t i s s u e . e i g h t o f t h e 19 cases w i t h p a p i l l a r y carcinoma appeared u n i l a t e r a l l y w i t h more o r l e s s s o l i t a r y l e s i o n s . i n 11 cases t h e p a p i l l a r y cancer was growing b i l a t e r a l l y , 3 % o f them had an i n f i l t r a t i v e growth. s i x p a t i e n t s w i t h p a p i l l a r y cancer had r e g i o n a l lymph node metastases a t t h e t i m e o f o p e r a t i o n . the case w i t h m e d u l l a r y cancer showed a m u l t i f o c a l growth. a l l a n a p l a s t i c cancers e x h i b i t e d an e x t e n s i v e growth p a t t e r n . the main symptoms presented a r e b r i e f l y summerized i n table 5 . table 5. c l i n i c a l symptoms presented i n cases o f t h y r o i d malignancy symptoms d i f f u s e growth o f t h e t h y r o i d g l a n d 15 nodular growth o f t h e t h y r o i d g l a n d a g o i t r e w i t h symptoms 10 c e r v i c a l nodules 5 l o c a l r e c u r r e n c e a f t e r p r e v i o u s 3 (compression, s tri d o r 1 s u r g e r y f o r t h y r o i d carcinoma t o t a l no. 41 n i n e t e e n o f t h e cancer p a t i e n t s i n t h i s study underwent p r e o p e r a t i v e s c i n t i grams w i t h t h e f o l l o w i n g r e s u l t s : i n one case t h e examination showed a t o x i c ( h o t ) nodule, i n 10 cases c o l d nodules were found, and 3 cases were normal. i n t h e r e m a i n i n g 5 cases t h e r e s u l t s were i n c o n c l u s i v e . 72 carcinoma cases with a f a l s e negative cytologic diagnosis: a 41-year-old woman w i t h a slowly ( s i n c e 10 y e a r s ) growing enlarge ment ( p a r t l y nodular) of b o t h thyroid lobes with s l i g h t toxic symptoms (medi c a l l y t r e a t e d ) . a scintiscan showed uptake o f tmg9 only in one palpable nodule of the r i g h t lobe. fnb showed f o l l i c u l a r c e l l s without atypia. the r i g h t lobe was removed a n d histopathological examination showed nodular g o i t r e i n addition t o a small focus of papillary carcinoma. a t reoperation the l e f t thyroid-lobe was removed a n d found t o contain small foci of carcinoma o f the same type. case 2. a 52-year-old woman with a b i l a t e r a l l y enlarged nodular thyroid gland located partly i n t r a t h o r a c i c a l l y . no toxic symptoms. scintigram showed lowered uptake in the r i g h t lobe. a t fnb no c e l l u l a r atypia was found. sub t o t a l b i l a t e r a l thyroidectomy was performed. the histopathological diagnosis was nodular g o i t r e with an encapsulated highly d i f f e r e n t i a t e d f o l l i c u l a r c a r c i noma of microinvasive type in the r i g h t lobe. a 36-year-old woman with an enlargement of t h e thyroid gland during t h e l a s t 3 years. no toxic symptoms b u t compression of the trachea. a s c i n t i gram indicated nodular c o l l o i d goitre. fnb showed f o l l i c u l a r c e l l s w i t h o u t atypia. bilateral subtotal thyroidectomy was undertaken. the histopatho logical examination showed nodular g o i t r e with multiple foci of papillary carcinoma in b o t h lobes. a 48-year-old woman w i t h a slowly ( s i n c e 2 y e a r s ) growing nodule i n t h e l e f t thyroid lobe. no toxic symptoms. scintigram showed no uptake in the l e f t and a normal r i g h t lobe. fnb indicated normal f o l l i c u l a r c e l l s . left -sided lobectomy was performed. the histopathology showed a highly d i f f e r e n t i a t e d papillary carcinoma. the r i g h t lobe was removed l a t e r and a l s o contained small foci of papillary carcinoma. a renewed examination of the cytological smears revealed a few c l u s t e r s of thyroid c e l l s with atypia and cytoplasmic nuclear inclusions indicating papillary carcinoma were found. case 5. a 63-year-old man with a mobile tumour above sternum since several years. the tumour a n d the l e f t thyroid lobe were removed a t a regional comun i t y hospital. the histopathological examination revealed a medullary c a r c i noma. the p a t i e n t was transferred t o uppsala university hospital f o r f u r t h e r examination. a scintigram showed focally decreased u p t a k e of tmg9 i n the r i g h t lobe. fnb d i d n o t reveal any c e l l u l a r atypia. bilateral thyroidectomy was performed. the following histopathological examination showed multiple foci of c e l l nests strongly suspected of representing medullary carcinoma i n t h e r i g h t lobe. the diagnosis of medullary cancer was v e r i f i e d by elevated l e v e l s of c a l c i t o n i n . case 6. a 52-year-old woman with a rapidly growing (since 2 months) nodule case 1. case 3. case 4 . 73 in the r i g h t thyroid lobe. fnb indi c a t e d f o l l i c u l a r c e l l s without atypia. after 3 months of medical treatment w i t h thyroid hormone the r i g h t lobe was resected. the histopathological examin a t i o n demonstrated a f o l l i c u l a r adenoma. however, behind t h i s adenoma a small f o l l i c u l a r carcinoma was l o c a t e d . later on a l e f t s i d e d lobectonly was per formed. a scintigram showed uptake in this lobe. histopathological examination did not show any signs of ma1 ignancy. discussion i n t h i s study 102 of 810 p a t i e n t s who underwent fnb were l a t e r operated and i n 97 a c o r r e l a t i o n between cytological and histopathological findings was possible. of t h e 33 cases w i t h histopathologically diagnosed cancer, 5 were n o t diagnosed by fnb a n d i n one the malignant c e l l s were n o t observed until the smears were reexamined. t h u s , false-negative diagnoses were noted i n 6 (18 % i of the cancer cases. the corresponding figures f o r false-negative diagnoses presented by other groups varies between 7.7 and 27.5 % ( s e e lowhagen e t a l . , 1981). the d i f f i c u l t y t o distinguish a f o l l i c u l a r adenoma from a highly differen t i a t e d f o l l i c u l a r carcinoma i s well-known (6). according t o t h i s research g r o u p i t i s n o t f e a s i b l e t o make t h i s d i s t i n c t i o n with fnb. they suggest t h a t fnb should only aim a t recognizing a f o l l i c u l a r neoplasm leaving t h e d e f i n i t e diagnosis t o t h e histopathological examination. this strategy seems rational a s the diagnosis of a f o l l i c u l a r neoplasm always warrants a surgical explora t i o n . by such management i t i s also possible t o minimize the risk of a f a l s e -negative or f a l s e p o s i t i v e diagnosis. the problems i n sampling the relevant i . e . malignant c e l l s i n small cancers are well i l l u s t r a t e d by cases 2 and 6 . i n case 2 a small microinvasive f o l l i c u l a r cancer was present in a nodular g o i t r e and i n case 6 a small f o l l i c u l a r cancer was found behind a "hot" f o l l i c u l a r adenoma. t h u s , fnb i s not a tool t o d e t e c t occult cancers which has been pointed o u t also by granberg e t a l . ( 3 ) . as i n a l l diagnostic procedures f a l s e p o s i t i v e findings should ideally n o t occur i n fnb. i t seems d i f f i c u l t t o avoid a low level of f a l s e positive find ings i f one wishes t o avoid missing t h e diagnosis of cancer (7). t h e r a t e s of f a l s e positive diagnoses presented in the l i t e r a t u r e amounts to 0-2 % (1,5). i n our material there were 2 cytological biopsies f a l s e l y interpreted t o show cancer c e l l s . both cases occurred i n p a t i e n t s w i t h lymphoid t h y r o i d i t i s , where a grave atypia of the f o l l i c u l a r c e l l s is common as is a lymphocytic i n f i l t r a t i o n i n the periphery of a cancer. the d i f f i c u l t y i n making a c o r r e c t diag nosis of lymphoid t h y r e o i d i t i s , especially i n a s o l i t a r y nodulus has been pointed out a l s o by lowhagen e t a1 (6). even histopathology may sometimes f a i l t o resolve d i f f e r e n t i a l d i a g n o s t i c problems i n t h e thyroid (9). 74 only half of t h e cases w i t h a histologically v e r i f i e d cancer had a radio nuclide scanning (99tc) indicating cold nodule(s1. in a b o u t 15 % the s c i n t i s c a n s were "normal" a n d i n a n o t h e r 25 % inconclusive. one p a t i e n t had a papillary cancer although a " h o t " nodule was seen, indicating t h a t malignancy c a n n o t be excluded w h e n technetium i s used as radioactive compound (11). our findings therefore p o i n t t o the limitation of thyroid scintiscans and underline t h a t scintigraphy alone cannot c l a s s i f y a nodule as malignant or benign. i t can only be considered an adjunct of physical examination and a guidance f o r fnb and cytological evaluation of a thyroid lesion. knowledge a b o u t t h e growth pattern of t h e cancers i s a l s o valuable when analyzing the r e s u l t s . this i s t r u e b o t h f o r the c y t o l o g i s t where the need f o r multiple biopsies i s evident a n d f o r the surgeon when deciding how radical the operation has t o be. in approximately half of the cases there were more than one area of cancer growth i n the thyroid gland. metastases a t the time of the primary operation was also a r e l a t i v e l y common finding. i t i s d i f f i c u l t t o evaluate t o w h a t extent the r e s u l t s of the fnb contributed t o the actual deci sion of surgical intervention or whether i t s main b e n e f i t was t o help the surgeon t o plan t h e operation. most of the patients already had c l i n i c a l symp toms suggesting malignancy. however, i n two groups of patients the cytological diagnoses probably made a s i g n i f i c a n t contribution t o the decision t o operate. these were the patients w i t h g o i t r e and/or adenomas a n d those w i t h enlarged cervical glands. t h e findings of the present study strongly indicate t h a t fnb i s a valuable a d j u n c t in thyroid d i a g n o s i s . i t i s a l s o obvious t h a t fnb should be considered complementary and optimal r e s u l t s can only be obtained when there i s a d i a g n o s t i c cooperation among the c l i n i c i a n , radiologist, c y t o l o g i s t and pathologist. references 1. 2 . 3. 4 . 5. 6. droese, m . : tumours. verh dtsch ges pathol 61:283-291, 1977. einhorn, j. & franzgn, s.: fine needle biopsy i n the diagnosis of thyroid disease. acta radio1 (stockh) 57:321-340, 1962. granberg, p . o . , hamberger, b . , lundell, g . , lowhagen, t. & willems, j.s.: preoperative evaluation o f the s o l i t a r y thyroid nodule. in: surgery of the thyroid a n d parathyroid glands (ed. e . l . kaplan) , churchill & livingstone, 1983. katz, a.d. & warren, j.z.: am j surg 132:459-462, 1976. l a n g , w . , atay, z . & georgii, a . : the cytological c l a s s i f i c a t i o n o f f o l l i c u l a r tumours i n the thyroid gland. virchows a r c h (pathol anat) lowhagen, t . , willems, j . s . , lundell, g . , sundblad, r . & granberg, p . o . : aspiration biopsy cytology i n t h e diagnosis of thyroid cancer. world j surg 5:61-73, 1981. a s p i r a t i o n biopsy cytology in the diagnosis of thyroid the malignant "cold" nodule of the thyroid. 378:199-211, 1978. 75 7. m i l l e r , j.m., hanberger, j.j. & k i n i , s.r.: the impact o f needle b i o p s y on t h e p r e o p e r a t i v e d i a g n o s i s o f t h y r o i d nodules. henry f o r d hosp med j 8. psarras, a., papadopoulos, s.n., livados, d., pharmakiotis, a.d. & kontras, d.a.: the s i n g l e t h y r o i d nodule. b r j surg 59:545, 1972. 9. sax&, e., f r a n s s i l a , k., bjarnasson, o., norrman, t. & r i n z e r t z , n.: observer v a r i a t i o n i n h i s t o l o g i c c l a s s i f i c a t i o n o f t h y r o i d cancer. acta p a t h o l m i c r o b i o l scand a 86:483-486, 1978. 10. soderstrom, n.: a s p i r a t i o n b i o p s y p u n c t u r e o f g o i t e r . acta med scand 144:237, 1952. 11. thompson, n.w.: the t h y r o i d nodule s u r g i c a l management. i n : endocrine surgery (ed. d.a. johnston & n.w. thompson), pp. 14-24. b u t t e r w o r t h s i n t e r n a t i o n a l medical reviews, 1983. 28:2-3, 145-148, 1980. address f o r r e p r i n t s : sighild westman-naeser, m.d. n a t i o n a l board o f h e a l t h and welfare department o f drugs box 607 76 upsala j med sci 87: 215-222, 1982 evaluation of parathyroid function in patients with hypergastrinaemia and pernicious anaemia 0. selking’, k. borch2, h. johansson’, s . ljunghal13 and l. wide4 from the departments of surgery’, medicine’ and clinical chemistry4, university hospital, u p p s a l a , and the department of surgery’, central hospital, eskilstuna, sweden abstract i n o r d e r t o e v a l u a t e t h e p o s s i b l e c a u s a l r e l a t i o n s h i p between r a i s e d serum g a s t r i n l e v e l s and t h e development o f p r i m a r y h y p e r p a r a t h y r o i d i s m (hpt) w h i c h i s suggested f r o m e x p e r i m e n t a l s t u d i e s we e v a l u a t e d p a r a t h y r o i d f u n c t i o n i n a group o f 3 2 p a t i e n t s w i t h h y p e r g a s t r i n a e m i a and p e r n i c i o u s anaemia. the v a l u e s f o r serum c a l c i u m and p a r a t h y r o i d hormone were d e t e r m i n e d as w e l l as t h e f a s t i n g u r i n a r y e x c r e t i o n s o f c y c l i c amp and c a l c i u m . there was no r e l a t i o n s h i p between t h e serum a a s t r i n l e v e l s and any o f t h e o t h e r s t u d i e d p a r a m e t e r s and t h e r e was no c o n s i s t e n t p a t t e r n s u g g e s t i n g p a r a t h y r o i d h y p e r f u n c t i o n . a r e t r o s p e c t i v e a n a l y s i s o f h o s p i t a l r e c o r d s f r o m 441 p a t i e n t s o p e r a t e d f o r p r i m a r y hpt showed a p r e v a l e n c e o f p e r n i c i o u s anaemia o f 1.8 %. t h i s f i g u r e i s h i g h e r t h a n t h a t f o u n d i n t h e u n s e l e c t e d age-matched p o p u l a t i o n (0.31 % i . how ever, t a k e n t o g e t h e r t h i s s t u d y does n o t s u p p o r t t h e h y p o t h e s i s t h a t h y p e r g a s t r i n a e m i a i s o f p a r t i c u l a r i m p o r t a n c e f o r t h e p a t h o g e n e s i s o f p r i m a r y hpt. introduction s e v e r a l p i e c e s o f e v i d e n c e s u g g e s t t h a t g a s t r i n m i g h t be i n v o l v e d i n t h e p a t h o g e n e s i s o f p r i m a r y h y p e r p a r a t h y r o i d i s m (hpt). i n p a t i e n t s w i t h p r i m a r y hpt r a i s e d g a s t r i n l e v e l s have been f o u n d i n an i n c r e a s e d f r e q u e n c y o f t e n p e r s i s t i n g even a f t e r p a r a t h y r o i d e c t o m y (5, 10, 16, 2 1 ) . when h y p e r g a s t r i n a e m i a was e x p e r i m e n t a l l y i n d u c e d i n r a t s h y p e r p l a s i a o f t h e parenchymal c e l l s o f t h e p a r a t h y r o i d s was seen ( 7 ) and i n v i t r o s t u d i e s s u g g e s t t h a t g a s t r i n m i g h t be a s t i m u l u s f o r s e c r e t i o n o f p a r a t h y r o i d hormone (pth) (22). i n o r d e r t o f u r t h e r e v a l u a t e t h e p o s s i b l e r e l a t i o n s h i p between h y p e r g a s t r i n a e m i a and p r i m a r y hpt we s t u d i e d p a r a t h y r o i d f u n c t i o n i n a group o f p a t i e n t s w i t h p e r n i c i o u s anaemia where a c h l o r h y d r i a and h y p e r g a s t r i n a e m i a i s an o b l i g a t o r y f i n d i n g . i n a d d i t i o n , t h e p r e v a l e n c e o f p e r n i c i o u s anaemia was d e t e r m i n e d among p a t i e n t s w i t h p r i m a r y hpt. 215 patients and methods altogether 32 p a t i e n t s (13 males, 19 females) w i t h a mean age of 67 2 10 ( s d ) (range 44-82) years were studied. they a l l h a d a verified history of perni cious anaemia based on typical haematological findings including marrow biopsy and a f u l l response t o treatment w i t h vitamin b12. their d u r a t i o n of disease was from 1 t o 2 1 years (mean 9.8 2 6.9 y e a r s ) . gastric acid secretion was investigated i n 27 p a t i e n t s and they were found t o have achlorhydria. all p a t i e n t s except one had normal values f o r serum c r e a t i n i n e , 4 h a d diabetes and 3 received regular treatment w i t h thiazides. the patients were studied as out-patients (central hospital, eskil s t u n a ) , where they reported a f t e r an overnight ( 1 2 hours) fasting. blood samples were taken i n evacuated tubes, whenever possible w i t h o u t tourniquet. urine was collected d u r i n g a 2-hour period as previously described (11). blood samples were centrifuged and serum was decanted. serum was frozen a t -8o'c and urine was frozen a t -2o'c until analyzed. calcium, c r e a t i n i n e and albumin concentrations were determined as p a r t of the c l i n i c a l routine a t t h e department of clinical chemistry. the serum calcium concentrations were adjusted for variations of the serum albumin content, the correction f a c t o r being n.019 mnol/l f o r each g/1 t h a t the individual albumin concentration deviated from the normal mean of 46 g/1. w i t h t h i s correction the normal range in our laboratory i s 2.20-2.60 mnol/l. ( 1 3 ) w i t h the g a s t r i n antibodies coupled t o bromcyanide-activated cellulose. the antiserum used was a generous g i f t from professor j rehfeld, copenhagen, denmark, and has been characterized previously (15). synthetic human gastrin i (shg ici chemicals, u . k . ) was used f o r preparation of standards and f o r 1251-labelling w i t h the chloramine-t method. the r e s u l t s are presented as pmol equivalents of shg/1. the normal value f o r serum gastrin i n our laboratory i s l e s s than 55 pmol/l . immunoreactive pth concentrations were measured by a radioimmunoassay method employing 1251-labelled bovine pth (inolex) and sheep antiserum (s478) against porcine and bovine pth. this assay measures i n t a c t human pth and the c-terminal 2/3 of the molecule. the antiserum r e a c t s predominantly w i t h a mid portion (44-68) of the hormone b u t not w i t h a n-terminal (1-34) fragment or a small c-terminal (53-84) fragment ( 9 ) . the reference range, as estimated from 50 healthy individuals i s 0.4-1.2 a r b i t r a r y units (arb. u/l) per l i t r e , whereas among 40 consecutive p a t i e n t s w i t h verified primary hpt t h e mean value was 2.3 1.6 arb. u/l ( 1 2 ) . descrihed (23). the normal range f o r the f a s t i n g urinary camp i s 0.2-0.7 serum gastrin concentrations were determined by a radioimnunosorbent method urinary cyclic amp was determined by a radioimmunoassay method as previously 216 lreol/mnol c r e a t i n i n e and f o r the f a s t i n g urinary calcium 0.05-0.50 mmol/mmol c r e a t i n i ne (11 1. a retrospective study of hospital records was also carried out of a l l p a t i e n t s operated a t our hospital d u r i n g the years 1959-1979 for primary hpt ( 1 ) . the prevalence of pernicious anaemia in t h i s material of 441 p a t i e n t s was compared t o figures from a recent survey of the apparently healthy population of approximately 20,700 individuals i n a nearby rural swedish d i s t r i c t (18). results all p a t i e n t s w i t h pernicious anaemia had c l e a r l y elevated l e v e l s of g a s t r i n , which ranged from 80 t o 5500 pmol/l w i t h a mean (+ sd) of 2000 1200 pmol/l. in 2 4 of t h e 32 cases values above 1000 pmol/l were detected. the mean values f o r serum calcium a n d pth a s well as f o r urinary camp and f a s t i n g calcium excretion a l l f e l l w i t h i n the normal range (table 1). table 1. in urine in p a t i e n t s w i t h pernicious anaemia and hypergastrinaemia. mean values ( + ) sd of parathyroid hormone ( p t h ) and i n serum and of f a s t i n g excretions of cyclic amp and calcium serum values urine values pth calcium camp calcium ( a r b . u/l) (nmol/l) ( pmol /mol (mnol /mol 0.84 + 0.61 2.45 + 0.09 0.61 + 0.41 0.25 + 0.17 c r e a t ) c r e a t ) pa ti en t s normal range 0.40 1-20 2.20 2.60 0.17 0.67 0.05 0.50 there was no relationship between gastrin and pth concentrations ( f i g . 1 ) nor between g a s t r i n and calcium values (fia. 2 ) . neither was there any relationship between gastrin a n d f a s t i n g urinary calcium or camp. two p a t i e n t s had serum calcium concentrations j u s t above the upper normal limit(2.62 mmol/l i n both cases) and one of them had also a s l i g h t l y elevated value f o r urinary camp. in 2 nomocalcaemic individuals raised l e v e l s f o r pth were found (1.31 and 3.55 a r b . u/l) and i n 2 others an elevated urinary camp (1.3 a n d 2.6 pmol/mmol c r e a t i n i n e ) b u t they had normal values for t h e f a s t i n g urinary calcium. among the 441 p a t i e n t s operated f o r primary hpt altogether 8 individuals (1.8 % i , mostly elderly females, had a verified diagnosis of pernicious anaemia. diagnosis of the blood disorder h a d preceded the detection of hpt w i t h periods from 2 t o 29 years. in the p o p u l a t i o n study 0.31 % o f a l l individuals had a diagnosis of pernicious anaemia (18) (table 2 ) . this difference is s t a t i s t i c a l l y s i g n i f i c a n t ( p <0.001. fisher's exact t e s t ) . 217 t 3-5 t 1.3 i 0 0 . i l j i 0 0 0 iq 5 0.7 q) 0 v) 0 0.5 o 0 0 0 0 a 8 ° p o 0 0 0 0 0 0 0 l 0 0 0 80 200 500 1000 2000 4000 6000 serum gastrin (pmolll) fig. 1 r e l a t i o n s h i p between t h e serum c o n c e n t r a t i o n s o f g a s t r i n and p a r a t h y r o i d hormone ( p t h ) i n p a t i e n t s with p e r n i c i o u s anaemia ( r = -0.0616, p> 0 . 7 ) . 0 0 0 0 0 0 0 0 0 0 0 .0 ([i 0 0 0 5 l 2.1 a, v) 80 200 500 1000 2000 40006000 serum gastrin (pmol/l) fig. 2 r e l a t i o n s h i p between t h e serum c o n c e n t r a t i o n s o f g a s t r i n and calcium in p a t i e n t s with p e r n i c i o u s anaemia ( r = 0 . 1 0 , p = 0 . 5 8 ) . 218 ii:5 t a b l e 2. h y p e r p a r a t h y r o i d i s m (hpt) i n u p p s a l a 1959-1979 compared t o d a t a f r o m a p o p u l a t i o n s u r v e y ( 1 8 ) . p r e v a l e n c e o f p e r n i c i o u s anaemia i n p a t i e n t s o p e r a t e d f o r p r i m a r y hpt p a t i e n t s ( n = 4 4 1 ) ma1 es females ma1 e s females n % n % n % n % p o p u l a t i o n s t u d y ( n = 20 7 0 0 ) 15-44 2 0.1 45-64 5 0.2 7 0.3 65-74 1 4.2 4 4 8 0.7 7 0.6 753 4.2 1 4 1.7 22 2.2 a l l ages 1 0.9 7 2.2 29 0.3 36 0.4 d i s c u s s i o n g a s t r i n bas been shown t o have t r o p h i c a c t i o n s b e s i d e s i t s s t i m u l a t o r y e f f e c t on t h e a c i d s e c r e t i n g p a r i e t a l c e l l s . it s t i m u l a t e s rna, dna and p r o t e i n s y n t h e s i s i n t h e mucosa a l o n g t h e i n t e s t i n a l t r a c t w i t h t h e e x c e p t i o n o f oesophagus and antrum. a l s o t h e p a n c r e a s i s a t a r g e t o r g a n f o r t h e t r o p h i c a c t i o n o f g a s t r i n ( 6 , 8 ) . p r e v i o u s e x p e r i m e n t a l work i n o u r group ( 7 1 s u g g e s t e d t h a t , i n t h e r a t , h y p e r g a s t r i n a e m i a c o u l d s t i m u l a t e t o p a r a t h y r o i d h y p e r p l a s i a . i n t h e s e e x p e r i m e n t s , t h e i n d u c t i o n o f h y p e r g a s t r i n a e r n i a t h r o u g h a n t r a l e x c l u s i o n was a s s o c i a t e d w i t h an i n c r e a s e d volume o f t h e p a r a t h y r o i d s owing t o h y p e r p l a s i a o f t h e parenchymal c e l l s . however, t h e serum c a l c i u m l e v e l s were n o t a f f e c t e d . i n t h e p r e s e n t s t u d y o f p a t i e n t s w i t h h y p e r g a s t r i n a e m i a and p e r n i c i o u s anaemia t h e r e was no p a t t e r n o f g e n e r a l p a r a t h y r o i d h y p e r f u n c t i o n . n a t u r a l l y we have no i n f o r m a t i o n as t o t h e h i s t o l o g i c a l appearance o f t h e p a r a t h y r o i d g l a n d s i n t h e s e cases. thus we do n o t know i f t h e r e m i g h t have been h y p e r p l a s i a o f t h e g l a n d s w i t h o u t h y p e r c a l c a e m i a as i n t h e work by g r i m e l i u s e t a l . ( 7 ) . i n t h a t s t u d y , however, t h e r a t s were exposed t o h y p e r g a s t r i n a e m i a f o r 1 4 weeks o n l y , w h i l e i n r h e p r e s e n t work t h e p a t i e n t s had a d u r a t i o n o f d i s e a s e f r o m 1 t o 2 1 y e a r s . i n a work by v a n t i n i e t a l . ( 1 9 ) an i n t r a v e n o u s i n f u s i o n o f p e n t a g a s t r i n i n d u c e d a s i g n i f i c a n t i n c r e a s e o f b o t h c a l c i t o n i n and pth l e v e l s , and a d e c r e a s e i n serum c a l c i u m l e v e l s was c o n c o m i t a n t l y seen. d e m o n s t r a t i o n o f h y p e r c a l c a e m i a i s a r e q u i s i t e f o r t h e d i a g n o s i s o f p r i m a r y hpt. i n a d d i t i o n , s e v e r a l methods can be used f o r e v a l u a t i o n o f p a r a t h y r o i d f u n c t i o n i n c l u d i n g d e t e r m i n a t i o n s o f t h e serum c o n c e n t r a t i o n s o f pth o r o f t h e u r i n a r y e x c r e t i o n s o f camp. f u r t h e r m o r e v a r i o u s i n d i r e c t e v i d e n c e o f p a r a t h y r o i d hormone a c t i v i t y based on t h e r e n a l h a n d l i n g o f c a l c i u m and phosphate has been r e p o r t e d t o be o f c l i n i c a l v a l u e ( 3 , 1 7 ) . the l a r g e number o f t e s t s t h a t have been d e v e l o p e d t o assess p a r a t h y r o i d f u n c t i o n i n d i c a t e s t h a t none o f them i s e n t i r e l y s a t i s f a c t o r y f o r c l i n i c a l purposes. 219 we have previously found t h a t determinations of b o t h t o t a l urinary cablp ( 2 3 ) and serum concentrations of pth (20) will separate most patients with hyper calcaemia and primary hpt from patients w i t h normal parathyroid function. a common picture i n primary hpt i s also a h i g h fasting urinary calcium (11, 141, due t o actions of pth o n bone. f o r a l l these parameters, which more o r l e s s closely r e f l e c t parathyroid function, the p a t i e n t s with pernicious anaemia and hyperaastrinaemia as a g r o u p presented values t h a t were closely compatible with those found i n apparently heal thy subjects. furthermore, there was no suggestion of any correlation between the individual serum l e v e l s of gastrin on one h a n d a n d serum pth o r calcium concentrations on the other. thus i t seems unlikely t h a t there i s any consistent relationship between the raised gastrin values i n pernicious anaemia, a n d parathyroid function. one patient displayed a pattern which was compatible w i t h mild primary hpt ( s l i g h t hypercalcaemia and elevated urinary c a m p ) . this was presumably a chance finding since the prevalence of hpt in the apparently heal thy elderly population seems t o be a t l e a s t one per cent ( 4 ) . in 3 other normocalcaemic cases e i t h e r pth o r urinary camp was elevated. these p a t i e n t s presented no other signs of hpt nor were they known t o s u f f e r from any other disease of possible importance. i t seems most l i k e l y t h a t the laboratory data i n these cases r e f l e c t the ineffec tiveness of any s i n g l e t e s t t o f u l l y discriminate normal i n d i v i d u a l s from those wi t h para thyroid hyperfunc ti on. operated f o r hpt w i t h regard t o the prevalence of pernicious anaemia, which was s i g n i f i c a n t l y higher i n those patients w i t h primary hpt than i n the unselected population. i t is, however, doubtful i f t h i s r e a l l y means an increased risk f o r a p a t i e n t w i t h pernicious anaemia t o develop hpt. pernicious anaemia, or any other disease one looks f o r , i s l i k e l y to be diagnosed i n an increased frequency i n any thoroughly investigated p a t i e n t material such as ours ( 2 ) . i t i s also reasonable t o assume t h a t pernicious anaemia was under-diagnosed i n the unselected population material i n the study by tyrberg 8 smedby ( 1 8 ) . some complex relationship between the two diseases cannot be excluded however. on the other h a n d , i f pernicious anaemia r e a l l y means an increased risk f o r developing hpt t h i s must be a weak factor as only 8 out of 441 patients developed b o t h diseases during an observation time of up t o 29 years. aemia, i n pernicious anaemia, i s n o t of p a r t i c u l a r importance f o r the develop m e n t of primary hyperparathyroidism. we a l s o carried out a retrospective analysis of our material of patients taken together the information i n t h i s study indicates t h a t hypergastrin financial assistance was given by the swedish medical research council ( p r o j e c t nos. 882-17x-04787-078, 102 and 4534). 220 references 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. adami, h-o., bergstrom, r. g r i m e l i u s , l., johansson, h., l j u n g h a l l , s., palmpr, m., rudberg, c . & i k e r s t r o m , g.: p a t h o l o g i c a l f i n d i n g s i n 4 4 1 p a t i e n t s o p e r a t e d f o r p r i m a r y h y p e r p a r a t h y r o i d i s m 1959-1979. to be p u b l i s h e d , 1982. berkson, j.: h o s p i t a l d a t a . b i o m e t r i c s b u l l 2: 47-53, 1946. broadus, a.e. & rasmussen, h.: c l i n i c a l e v a l u a t i o n o f p a r a t h y r o i d f u n c t i o n . am j pled 70: 475-478, 1981. c h r i s t e n s s o n , t., h e l l s t r i j m , k., wengle, b., a l v e r y d , a. & wikland, b.,: p r e v a l e n c e o f h y p e r c a l c a e m i a i n a h e a l t h s c r e e n i n g i n stockholm. a c t a med scand 200: 131, 1976. dale, j.k., farndon, j.r., hinshaw, w.m., d e l l e y , w.g., handwerger, s. & wells, s.a.: the i n c i d e n c e o f m u l t i p l e e n d o c r i n e n e o p l a s i a (men) t y p e i o r i1 i n p a t i e n t s w i t h p r i m a r y h y p e r p a r a t h y r o i d i s m . 1981. enochs, m.r. & johnson, l.r.: t r o p h i c e f f e c t s o f g a s t r o i n t e s t i n a l hormones: p h y s i o l o g i c a l imp1 i c a t i o n s . fed p r o c 36: 1942-1947, 1977. g r i m e l i u s , l . , johansson, h., l u n d q v i s t , g., o l a z a b a l , a . , p o l a k , j.h. & pearse, a.g.e.: the p a r a t h y r o i d g l a n d s i n e x p e r i m e n t a l l y i n d u c e d h y p e r g a s t r i n e m i a i n t h e r a t . scand j g a s t r o e n t e r 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q v i s t , g. & wide, l.: serum g a s t r i n d e t e r m i n a t i o n w i t h a r a d i o i m m u n o s o r b e n t t e c h n i q u e . c l i n chim a c t a 79: 357-362, 1977. flordin, b.e.c., peacock, m. & w i l k i n s o n , r.: h y p e r c a l c i u r i a and c a l c i u m s t o n e d i s e a s e . c l i n e n d o c r i n o l metab 1: 169, 1972. r e h f e l d , j.f., s t a d i l , f. & rubin, b.: p r o d u c t i o n and e v a l u a t i o n o f a n t i b o d i e s f o r t h e radioimmunoassay o f g a s t r i n . s c a n d j c l i n lab i n v e s t 30: s e l k i n g , u . , johansson, h. 8 l u n d q v i s t , g.: r e s p o n s e t o s e c r e t i n i n h y p e r p a r a t h y r o i d p a t i e n t s . a c t a c h i r scand 147: t r a n s b o l , i., jdrgensen, f.s., hornum, i. & k e i d i n g , n.: h y p e r c a l c a e m i a d i s c r i m i n a t i o n i n d e x : a m u l t i v a r i a t e a n a l y s i s o f p a r a t h y r o i d f u n c t i o n i n 107 h y p e r c a l c a e m i c p a t i e n t s . a c t a e n d o c r i n o l 86: 768-783, 1977. t y r b e r g , e. & smedby, b. : l a k a r t i d n i n g e n 79: 1116-1119, 1982. v a n t i n i , i., c o m i n a c i n i , l., p i u b e l l o , w., g h i d i n i , o., f a t t o v i c h , g., e d e r l e , a., b e n i n i , l., c o c c h e t t o , r., c a v a l l i n i , g., locassio, v. & scuro, l.a.: e f f e c t o f exogenous g a s t r o i n t e s t i n a l p e p t i d e s c o n t a i n i n g t h e t h e c t e r m i n a l t e t r a p e p t i d e of g a s t r i n on c a l c i u m , c a l c i t o n i n and p a r a t hormone serum l e v e l s i n man. h e p a t o g a s t r o e n t e r o l o g y 28: 43-48, 1982. wikstrom, b., l j u n g h a l l , s., wide, l. 8 f i k e r s t r o m , 6.: c l i n i c a l s t u d i e s o f p h o s p h a t e h a n d l i n g i n h y p e r c a l c a e m i a . s u b m i t t e d f o r p u b l . 1982. wilson, s.d., singh, r.b., k a l k h o f f , r.k. & go, v.l.w.: does h y p e r p a r a t h y r o i d i s m cause h y p e r g a s t r i n e m i a ? s u r g e r y 80: 231-237, 1976. c l i n i c a l , l a b o r a t o r y and h i s t o l i m i t a t i o n s o f t h e a p p l i c a t i o n o f f o u r f o l d t a b l e a n a l y s i s t o w o r l d j surg 5 : 463, 221-232, 1972. serum g a s t r i n and i t s 649-655, 1981. b l z b e h a n d l i n g och v t r d k v a l i t e t . 15-822858 22 1 22. windeck, r., brown. e.m., gardner, d.g. & aurbach, g.d.: e f f e c t o f 23. w i l i n d e r , o . , l j u n g h a l l , s. & w i b e l l , l.: u r i n a r y e x c r e t i o n o f c y c l i c amp g a s t r o i n t e s t i n a l hormones on i s o l a t e d b o v i n e p a r a t h y r o i d c e l l s . e n d o c r i n o l o g y 103: 2020-2026, 1978. i n h y p e r p a r a t h y r o i d i s m . scand j u r o l n e p h r o l 12: 67-71, 1978. address f o r r e p r i n t s : o r j a n s e l k i n g , m.d. department o f s u r a e r y u n i v e r s i t y h o s p i t a l s-750 1 4 uppsala 222 upsala j med sci 78: 169-180, 1973 the adequacy and compatibility of compartmental models of electrolyte exchange in the dog’s heart g. arturson,l t. groth,2 g. grotte,3 p. malmberg,4 r. samuelsson5 and u. sjostrand5 from rhe ‘burn center, rhe 3department of pediatric surgery, rhe 4deparrment of clinical physiology, university hospital; the uppsala university duta center and the 5department of physiology and medical biophysics, uppsula uniuersity, uppsala, sweden abstract the exchange of electrolytes (na, k, so,) and albumin in the dog’s heart was studied by using tracer technique in a modified heart-lung preparation. the data were analysed in order to investigate if the system could be characterized kinetically in terms of a few distinct compartments, and to what extent tracer measurements in systemic plasma and cardiac lymph provide information about the capillary and cellular exchange of electrolytes in the dog’s heart. the compartmental models used for sodium and potassium were easily conformed so a s to be compatible with the tracer data and it was also possible to make them consistent with independent relevant data from the literature. however, it was not possible to construct adequate models in the sense that the model parameters were well deter mined by the data. this was not due to inaccurate data but to the insufficiency of this type of data to identify adequate models of the required complexity. therefore, detailed quanti tative information on the electrolyte transport across cellular membranes cannot be obtained in experimental studies of the type presented in this investigation. the excitability of the heart muscle is heavily depend ent on the extraand intracellular concentrations of sodium and potassium ions. analysis of the rela tions between changes of these concentrations and the corresponding changes of excitability and con tractility would be considerably simplified if the heart could be characterized kinetically in terms of a few distinct exchanging compartments. this type of description necessarily involves simplifying assump tions concerning the number and interconnections of compartments, the relation of measurements in the body fluids to compartments etc., assumptions which limit the possibilities of drawing detailed conclusions. however, models of this kind provide simple concepts, which may be used for the descrip tion of the essential features of the system studied. for the complete identification of a multicompart ment system, it is necessary t o have data from each compartment (27). this condition is often not f u l filled, which means that some aspects of the model will be less well determined. it is therefore of primary importance t o test a proposed model not only for compatibility with the experimental data available, but also for the more strict condition of adequacy, which besides compatibility, requires that the para meters of the model are well determined by the data. compartmental analysis has been used in previous investigations of electrolyte transport in the dog’s heart. transcellular sodium and potassium exchange have been calculated and correlated to changes of cardiac frequency (8, 9, 16, 29). however, the models used in these studies were not tested for adequacy, a circumstance which limits the reliability of the results. in the present communication, the experimental data were gathered from a simultaneous study of 24na, 42k, 1311-albumin and 3 5 s 0 4 in systemic plasma and heart lymph. the compartmental analysis was performed, with due consideration to the requirement of model adequacy, in order t o investigate t o what extent this type of data provide information about capillary and cellular exchange of electrolytes in the dog’s heart. material a n d experimental procedure t h e experiments were performed o n modified heart-lung preparations (hlp), prepared according to areskog (1). in four young, anaesthetized vorsteh dogs, weighing about 20 kg. n e m b u t a p (30 mg/kg) was used as the anaesthetic agent. t h e fall in body temperature during the experiment was diminished by heating the operation table, irradiating the dog with a heat lamp and regulating the temperature of the reservoir and tubing system in the hlp. evaporation from the open thorax was diminished by means of a plastic foil cover and the hlp was continuously supplied with 5 . 5 % upsala i m e d sci 78 -170 g. arturson et al. h e a r t lung p r e p a r a r a t i o n ( d o g l ) m m hg 1 cm h20 1 i i c 5 i 9 12 i i i i i i i i 1 i u h v y -_ m . z n vi m n i 1 i 1 0 50 100 150 200 250 300 350 min. t radioactivity i n systemic blood ( c o n t i n u o s infusion) fig. i . experimental results for dog 1. glucose solution ( i ml/min), which was added to the reservoir. the heart rate (hr), the arterial systemic pressure (bp), the central venous pressure (cvp) and the temperature of the venous blood were measured. the cardiac output was meas ured continuously by means of an ultrasonic detector located on the tubing on the arterial side. the tracers 24na, 42k, 35s04 and 1311-albumin were added gimultaneously to the blood reservoir. the 1311-albumin had opsala j m e d sci 78 been separated one hour previously, according to the method of bill et al. ( 6 ) , in order to remove free 1311. 3 5 s 0 4 and lali albumin were given as a single injection. together with the initial addition of 24na and 42k, continuous infusions of these isotopes were started in order to keep the systemic plasma concentration as constant as possible. cardiac lymph was collected in heparinized plastic tubes as described previ ously (2). blood samples were taken from the reservoir and electrolyte exchange kinetics in the dog's heart 171 h e a r t lung preparation (dog21 \ , i k j 0 5 0 100 153 200 250 303 350 100 min , r a d i o a c t i v i t y in systemic b l o o d ( c o n t i n u o u s infuslon1 fig. 2. experimental results for dog 2. were centrifuged immediately in order to separate the plasma and the red blood cells. the experiments were discontinued when the systolic systemic prsssure fell below 40 mmhg. the concentrations of sodium and potassium in cardiac lymph and systemic blood plasma were determined with an eppendorf flame photometer. the activities of z4na, 42k and 1311-albumin in systemic plasma, red blood cells and cardiac ymph were measured by a gamma spectrometer (cf. 26). 3 6 s 0 , in the systemic plasma and the cardiac lymph was measured in a beckman cpm-2co scintillation counter (cf. 18) after the separation of "so, from the haemoglobin in a sephadexb c-50 column (cf. 19). the mean plasma concentration of the various isotopes was calculated as the mean of the values in all samples of systemic plasma and the separate concentrations (per cent) in the plasma, red blood cells and lymph samples were related upsnla j m e d sci 78 172 g . arturson et al. t o this mean concentration. in figs. 1 , 2 and 4 the radioactive data are calculated as successive means of three consecutive measurements. the magnitudes of the errors in the experi mental data were estimated from the errors of the radioactive measurements and the errors in the weighing procedurzs (cf. 26). during the first 15-20 min of the experimental period the errors were calculated to be about f 5 46 (s.d.) or even slightly larger, whereafter they decreased. experimental results the experimental results from two dogs are pre sented in figs. 1 and 2. the results from the other two dogs have been given in a previous communi cation (3). the cardiac output and the arterial blood pressure gradually decreased, whereas the central venous pressure was fairly constant in the range of 0-10 cm h,o during the experiments. the heart rate decreased somewhat, probably due to a slight decrease in body temperature during the experimental period. the lymph flow rate usually increased at the end of the experimental period. the na and k concentra tions in the systemic plasma and the lymph were practically constant throughout the experiments. these results indicate that the ionic fluxes in the heart muscle could be considered to be in steady state. for unknown reasons, in one dog the activity of 24na in the cardiac lymph increased t o values ex ceeding that of the systemic plasma (fig. 2). these data were therefore excluded from the subsequent computational analyses. as can be seen in figs. 1 and 2 , the 42k and 1311 albumin activities were fairly constant in the red blood cells throughout the experiments, whereas the 24na activity gradually increased. the initially high relative activity of the isotopes 24na, 42k and 1311 albumin in the packed red blood cells may be ex plained by an unavoidable amount of trapped plasma. the red blood cell compartment was con sidered to be in negligibly slow exchange with plasma and was therefore not taken into account during the subsequent compartmental analysis. is localized in the interface between the compart ments and the mixing of tracer is assumed to be very rapid within each compartment. the flux of tracer from one compartment to another is taken as being proportional to the concentration of tracer in the compartment which the tracer is leaving. thus the rate constants are lumped parameters, including the exchange of tracer by way of bulk flow, diffusion and other possible transport processes which can be described in this way. the various mechanisms of the transport of substances cannot therefore be resolved in this kind of analysis. the physical models generate kinetic equations, forming a system of linear differential equations of the first order. the equations are formulated in terms of the concentration of tracer c , (counts min-l g-l) in the various compartments ( i ) of volume vi (ml) and the rate constants ki, (mi-'), describing the transport of substance from compartment j t o compartment i. the system of equations was inte grated numerically by the use of a fourth-order runge-kutta method (see e.g. (5)). this approach has the apparent advantage over analytical methods (see 8) that cases with arbitrary plasma functions can be analysed, and not only cases in which the plasma concentration is constant with respect to time. the parameters (rate constants ki, and volume ratios yjv,) were estimated by conforming the models in a least-squares sense to the experimental tracer-concentration data from plasma and lymph samples, using a method due to powell ( 2 2 ) . the models were tested for compatibility with the tracer data, and the parameter estimates and the quantities derived from them were also tested for consistency with available independent information about the biological system studied. however, the aim of the analysis was not only to find models which were compatible with the experimental data but rather to define adequate models for the descrip tion of the transport of the various substances, i.e. to find compartmental models of minimal complexity, which, besides compatibility, also define parameters which are reliably determined by the data. the estimation situation was investigated by drawing likelihood contours. the region enclosed by the theory a n d computational methods sum-of-squares contour s=s(o) various types of compartmental models have been used (cf. fig. 3), all of which are based on the assump tion of a steady state, i.e. that the compartments are constant in time with respect to composition and volume. furthermore, the resistance to tracer flow i p s(e) q s(6) 1 + ~ where 6 is the best estimate of the parameters 8 , gives an approximate 100 (1 a ) yo confidence region, n p ) { n p upsala j m e d sci 78 electrolyte exchange kinetics in the dog’s heart 173 1 k21 k12 , i ki2 2 . k 2 1 _1 i compartmental m o d e l s l model l a 1 k i o r i l 1 m o d e l lb model 5 1 = f u n c t i o n a l p l a s m a compartment 2 f u n c t i o n a l i n t e r s t i t i a l c o m p a r t m e n t f u n c t i o n a l i n t e r s t i t i a l compartment il 3. f u n c t i o n a l l y m p h c o m p a r t m e n t 5: f u n c t i o n a l i n t r a c e l l u l a r c o m p a r t m e n t n 1 = f u n c t i o n a l i n t r a c e l l u l a r c o m p a r t m e n t i o r fig. 3. compartmental models used in the analysls. if the model is reasonably linear in 8 within the region of interest (4, 7). f,(p, n p ) is the c( significance level of the f distribution with p and n p degrees of freedom (p=number of parameters, n=number of data the kinetic equations for model l a are and the concentrating effect on the tracer due to evaporation during the experiment was simulated by an equivalent inflow ( k , , ) of tracer to this compart ment. points). dc; _ _ dt k,, c; + k , , vz vl c i + k , , c ; and model approaches and results 1311-albumin dci as 1311-albumin can be regarded as having a purely _ _ ~ (k12 + k,,) ci + k,, v; c ; dt v 2 extracellular distribution (14), the 1311-albumin tracer data were analysed in terms of an open two compartment model (model 1 a in fig. 3). the plasma compartment was not regarded as infinite the parameters kzl, klz, koz and vz/vl were estimated from the experimental plasma and lymph tracer data (representing the plasma and interstitial upsala j m e d sci 78 \ 174 g . arturson et al. 100 1311-a1bumln ........ __.._...... ....... 100 e 1311a l b u m i n ........ ...... ......... .. i p 50 tl 50 m s y s t e m i c p l a s m a cardiac l y m p h m o d e l l a 0 " 0 50 100 150 200 m i n % i i j c * s y s t e m i c p l a s m a x cardiac l y m p h m o d e l l a 0 50 100 150 200 m i n er s y s t e m i c p l a s m a f o r c i n g functmn a c a r d i a c l y m p h m o d e l l b and m o d e l 3 0 200 mm 50 100 150 s y s t e m i c p l a s m a , , 1 r d i a c lym; m o d e l 2a 0 0 0 50 100 150 200 m i n d i i " t s y s t e m i c p l a s m a i c a r d i a c l y m p h m o d e l 2 a 0 50 100 150 200 m i n u c a l c u l a t e d " c a p i l l a r y p l a s m a " forcing f""ctl0" cardiac lymph , 4 mode(: a n d model; 0 50 100 150 2 0 0 mi , fig. 4 . illustration of the results of conforming various models to the experimental data. figs. a-d and f refer to dog 2 and fig. e t o dog 1. upsala j m e d sci 78 l 3 l i a t b dog 2 m o d e l l a ko2[mm -11 a d 1 v2/v1=0055 2 v2/v1=o060 1 v 2 / v 1 = 0 0 7 0 5 v 2 / v 1 = 0 075 kl2=0 0009min-1 electrolyte exchange kinetics in the dog's heart 175 sol ; d o g 2 m o d e l l a 0 015 kl2[mnn-1] b 0 021 0 020 0 019 0 018 0 017 k21[min-1] 0 0009 0 0010 00011 0 0012 0 0 016 z l n a . d o g 1 model 3 0 0 1 3 1 1 l k 0 2 = 0 1 1 2 2k02=0115 3 k02=0117 0 0 3 9 i lk02'0119 1 5 k o 2 = 0 122 0035 0 0050 0 5 0 7 0 9 1 1 1 3 1 5 1 7 zlna ;dog 1 m o d e l l b o z o : 1 0 05 0 10 0 15 0 20 00055 00060 00065 0 0070 2 l n a , dog 1 m o d e l 3 w i t h c o n s t r a i n t s v c z l x v , . c , r l l * c c 0 21 0 0 5 s k ~ 2 s o 6 020 019 . 018 017 016 ' i 011 015 016 017 018 019 0 2 0 l 2 k , d o g 2 m o d e l 3 fig. 5 illustration of the multi-dimensional, 95 % confidence regions for the estimated model parameters by representative cross-sections. upsala j m e d sci 78 176 g , arturson et al. table i. parameter estimates f o r 131z-albumin using model l a and model 2a (dog 2 ) in the analysis of dogs i, 2 , 3 and 4 dog 2 dog 1 dog 3 dog 4 dog2a model model model model model model parameters l a l a 2a l a l a l a k,, (min-l) k,, (min-l) k,, (min-l) .k,,(min-') k,, (min-l) ko3 b i n ' ) tlag ( m i 4 v2l v, v2i v 3 derived .quantities cpici 0 0.0001 0 0.003 2 0.019 0.6 0.0010 0.0010 0 0.001 1 0.0010 0.0001 0.0009 0.0043 0 0.019 0.005 0.028 0.030 20 0 7 0.065 0.044 0.016 1.21 0 0.0008 0 0.018 26 0.050 1,2*0:2 1.0 0.9 1 . 1 0 0.0002 0 0.017 20 0.014 1.2 not corrected for evaporation, k,, = 0. a compartments). the influence of evaporation and other factors on the size of the plasma pool was estimated from data for stable sodium in plasma. in one dog (dog 2) there was a systematic decrease in the plasma-pool size, which was corrected for by a constant inflow of tracer (k,,=o.ool min-i); in the other dogs, the estimated pool size fluctuated irreg ularly but was regarded as approximately constant for the analysis (cf. k,, in table i and the comments below). in order to get a good fit in the interstitial compartment, it was also necessary t o introduce a time-lag parameter, t l a g . t l a g was chosen from the experimentai data by visual extrapolation. curvilinear interpolation of adjacent lymph values was used to calculate iymph values at times corre sponding to the plasma-sample times. the parameter estimates are given in table i and the corresponding simulated curves for dog 2 are shown in fig. 4 a , compared with the experimental data. despite the fact that the deviations in some regions are greater than the analytical errors (see page 172), model 1 a was accepted as a satisfactory description of the data, in view of the combined effects of biologically but mainly methodologicaiiy uncontrollable errors. the parameter confidence for model l a and dog 2 is illustrated in fig. 5 a . an alternative model (model 2a in fig. 3) was used in an attempt to describe the data by the introduction of a special lymph compartment con nected to the interstitial compartment, instead of upsala j m e d sci 78 the time-lag parameter of model l a . the kinetic equations for this model are: and d c j _ _ k,, c i i k,, 3 c,* dt v 3 (3) (4) only dog 2, which was judged to be the best ex periment, was analysed by this model and the para meters k,,, k,,, k,,, k,, and v , / v , were estimated from a least-squares fit of compartments 1 and 3 to the experimental plasmaand lymph-tracer data. by introducing the plausible but still questionable con straint c: ( t ) c,* ( t ) , t 20, it was also possible to estimate v,/ v, and v,/ v3 separately. the goodness of fit is illustrated in fig. 4 b (dog 2) and the least squares estimates of the parameters are given in table i. as can be seen from fig. 4 b , there is a need for still more complex models in order to describe the experimental data within the limits of the analytical errors, for example, by adding a series of small compartments t o explain the time lag. however, this work did not seem justified in view of the avail able data, as the time lag must be strongly influenced by the drainage of cardiac lymph. from the parameter estimates in table i, it fol lows, by the use of equations for mass transport and the steady-state assumption, that the concentration of unlabelled substance is the same in the plasma (c,) and the interstitial (c,) compartments. the amount of albumin brought back to the plasma per minute (influx rate) is of the order of 4 7 % of the amount transported from the plasma per minute (efflux rate), as calculated for dog 2. these conclu sions are not critically dependent on the choice of model l a or 2a for this case. the correction for evaporation in dog 2 is, however, rather critical for the estimation of the parameters k , , and vjv,, as can be seen in the last column of table i. this effect may be the explanation of the discrepancies between dog 2 and dogs 1, 3 and 4 concerning v,, and this is also the reason for the choice of dog 2 as the best experiment. 36soa in accordance with studies of the distribution of various ions and carbohydrate molecules in the electrolyte exchange kinetics in the dog's heart 177 table 11. parameter estimates for 3 5 s 0 4 using model l a and model 2a (dog 2 ) in the analysis of dogs 2, 3 and 4 dog 2 d o g 3 d o g 4 parameters model l a model 2a model l a model l a 0.0010 0.0010 0.0062 0.0085 0.041 0.047 0.012 0.01 1 0.066 0.12 0.15 6.25 12 0 0.0035 0.043 0.024 0.05 3 0 0.0010 0.006 0.052 0.02 1 3 1 .o 0.8 1 .o 1.2 mammalian heart by page & solomon (21) and page (20), and in the frog heart by danielson (lo), so, can be regarded as having a purely extracellular distribu tion. model l a was therefore used for the analysis of the 35s04 data. the least-squares estimates of the parameters are given in table i1 for dogs 2, 3 and 4 and the corresponding curves for dog 2 are shown in fig. 4c. the parameter estimates for dogs 3 and 4 are influenced by the same uncertainty concerning evaporation as in the case of lsii-albumin. the good ness of fit was acceptable for model l a but was not improved by using the more complex model 2a, as can be seen in fig. 4d. the 95% confidence region for the estimated parameters is shown in fig. 5b. from the values for dog 2 in table 11, it can be seen that k , , is larger than the corresponding value for i-albumin. independent lymph-flow data can be used to estimate the volumes of the functional plasma and interstitial pools. the functional plasma volume, v,, is of the same size, 42-54 ml, but the functional interstitial volume, v,, is larger (5 ml, compared with 3.5 ml for the corresponding value for 1311-albumin). it should be stressed that v , here signifies the part of the interstitial compartment drained by the cannula. the estimates of pool volumes are also subject t o large numerical errors. furthermore, it can also be concluded from the figures for dog 2 that the influx rate of a5s04 to plasma from the interstitial space approaches 100 % of the corresponding efflux rate from plasma. 12 732853 in this case, and also in the case of 42k, the electro lyte was infused continuously, in order to maintain a constant plasma level. the reason for this was primarily to simplify the mathematical analysis (cf. 8); however, it was not possible t o maintain the plasma level within + 5 % (cf. 8) and, instead of analytical methods, numerical methods had t o be used, with the advantage of accepting an arbitrary function for the plasma concentration of tracer. data for dog 2 were invalidated for some unknown measuring and/or technical reason (see experimental results). the data from dogs 1 , 3 and 4 were analysed. using model 3 (cf. 9), the kinetic equations of which are the dc,* vl dt vz v, __ = (k,, + k,, + k,,,) cz* + k,, c ; + k,, y4 c ; ( 6 ) d c ; _ _ k 2 , c ; + k , , 5 c , * dt v, (7) a quite satisfactory fit was obtained, as illustrated for dog 1 in fig. 4e. in this model the disappearance of the tracer from the interstitial fluids via the lymph system and other routes is described by a special rate constant ( k o z ) . this parameter cannot be resolved (see eqs. (6) and (7)), but only estimated in addition to k , , (intersti tium to plasma). since the measured plasma con centration is used as a governing function, c: ( t ) , the other parameters k , , and vl/vz can also only be estimated as the product k , , ( vl/ v,). the parameter estimates are given in table 111 for dogs 1 , 3 and 4 and the 95% confidence region is shown in fig. 5c for dog 1. as can be seen from this figure, the parameters k42 and k , , are unreliabfy determined by the tracer data, and, in view of the apparent discrepancy of the conclusions about the concentration of stable na in interstitial (c,) and intracellular space (c,) with common knowledge (c,14cc, cf. (28), (13)), model 3 is not an adequate model for the data. by introducing the con straints vc-4v,(cf. 8) and ci-14cc, i.e. k , , = 3.5k4,, it was also possible to fit the experimental tracer data, thus making model 3 compatible with these data. this model is, however, still not ade quate, as can be seen from fig. 5d (see also table 111). the exchange between the interstitial and intra cellular compartments (k,,, k 4 , ) is also in this case unreliably determined, while the transcapillary ex upsala j m e d sci 78 178 g . arturson et ai. table 111. parameter estimates for 24na using model i b and 3 f o r the analysis of dogs i , 3 and 4 table iv. parameter estimatesfor 4ak using model 3 in the analysis of dogs i , 2, 3 and 4 dog 1 model 3 with model model con parameters lb 3 straints dog 3 d o g 4 model model lb lb k,,x vl/vz(min-1)0.13 0.32 0.17 k l z + k,, (min-l) 0.14 0.34 0.18 k,, (min-l) 1.45 0.60 kza (min-l) 1.05 2.10 tlag b i n ) 6 6 6 v4/ v, 4 frozen 4 frozen 0.09 0.16 0.11 0.17 derived quantities 1 . 1 1 . 1 1 . 1 1.2 1 . 1 2.9 14 frozen c d c i ciicc change ( k , , , k , , ) and interstitial outflow (k,,) remain reliably determined. in fact, even the simplest model l b (see fig. 3) is compatible with the tracer data and, according t o fig. 5e, the parameters k,,+k,, and k z l ( vl/ v,) are reliably determined. 42k a three-compartment system in series (model 3 in fig. 3) was used in the analysis of the 42k data. this model differs from the open two-compart ment model introduced by conn & robertson (s), and used by others (11, 15, 17, 29) in various re spects. the plasma pool has not been considered as being infinite in size and has not been assumed to be in rapid exchange with the interstitial fluids, but has been described by an estimated “capillary plasma” forcing function. the governing function c: (the “capillary con centration”) was estimated as a mean value of the concentrations in arterial plasma and cardiac lymph (coronary lymph -coronary venous plasma; cf. (1 1)). this approximation seems justified for all the ions studied in view of their rapid transcapillary exchange (cf. 30), but was only considered for potassium be cause of the approximation for this ion has the greatest influence (see figs 4 d , e and f). the parameters (klz + k o j , ( k d v,/ v d ) , k24 and k p 2 were estimated by fitting c,* to the lymph-tracer data, with the assumption that these data were representative of the interstitial fluids. the ratio v4/ v, does not influence the c,* values, but only the values of the intra-cellular compartment. the least squares estimates are given in table iv for all the upsulu j m e d sci 78 parameters dog 1 d o g 2 d o g 3 d o g 4 k , , x v l / v 2 (min-l) 0.16 0.045 0.03 0.11 k,, + k,, (min-l) 0.19 0.052 0.03 0.14 k,, (min-l) 1.38 0.194 0.22 0.17 k,, (min-’) 1.26 0.192 0.34 0.81 tlag (min) 0 6 3 9 derived quantities c&i 1.2 1.2 1.0 1 . 3 dogs and the corresponding simulated curve c,* is shown in fig. 4 f for dog 2, compared with the experimental data. model 3 was accepted as being compatible with the tracer data. the estimated parameter values are greatly dependent on the approximation of the “capillary concentration”, as tested by repeating the calculations, using the pri mary plasma concentration values; the parameter values were then changed by factors ranging from 5 to 10. the steady-state equation for the intra-cellular compartment is thus, in order to fulfil the constraint cc n 30ci (cf. 13), the volume ratio v i / v c must be set equal t o -30 for dog 2. this is in conflict with common knowledge (v,-4vi, cf. 8). as was found in the analysis of the ,,na data, model 3 was not adequate, because of the large uncertainty in the estimated parameter values kz4 and k4,. the same conclusion applies t o model 3 for the description of the 42k data, as can be seen from fig. 5 f , where the 95% confidence limits for the parameters k , , and kq2 are shown for dog 2. an attempt t o fit model 3 t o the data of dog 2 with the constraints c, 3oci and vc-4vi did not produce an acceptable agreement with the experimental data. by adding a fourth compartment in series (model 5 in fig. 3), it is possible to fulfil the concentration and volume constraints; the steady-state equations for compartments 4 and 5 give the relations (9) electrolyte exchange kinetics in the dog’s heart 179 with the assumptions that c, = c, 30 c, and v,+ vs-4 v, it follows from the relations (9) and (10) that for example, for dog 2, where k , , k 4 2 , k,, 120k45, v , 3 0 v 4 and v5-120v4-4v2. a spectrum of possible solutions thus presents itself, the limits of which are determined by the un certainty of the parameter estimates of k,, and k , , (see fig. 7f). the introduction of a special lymph compartment (model 4 in fig. 3) does not present any new, or simpler solution of the present problems. the uncer tainty concerning the exchange on the cellular level remains. discussion the studies presented here show that several com partmental models for sodium and potassium ex change (model 3 and model 5 ) could easily be con formed so as to be compatible with the tracer data and it was also possible to make them consistent with independent relevant data from the literature. however, it was not possible to construct adequate models for the data, in the sense that the model parameters were reliably determined by the data. as the plots of the 95% confidence regions show (see figs. 5 c, d, f), the uncertainty in the rate constants between the interstitial and the intracellular compart ments is too large to permit any far-reaching conclu sions. the tracer data only allowed for the estimation of transcapillary exchange for albumin and sulphate ions. the results so far are consistent concerning (1) the slower exchange of albumin between plasma and interstitial fluids, compared with the correspond ing values for the sulphate ion (k=0.0011 +0.0002 (2 s.d.) min-l for i-albumin (dog 2) and k =0.0062 0.0002 min-’ for so, (dog 2)), and ( 2 ) the influx rate of albumin and so, to plasma (4-7 yo and ca. 100 %, respectively, of the efflux rate from plasma). the coefficient of transfer of albumin across the capillary walls was here estimated to be 0.0011 0.0002 min-i (dog 2), i.e. about 0.1 yo of the albumin in the plasma pool crossed the capillary walls each minute. this value is appreciably smaller than the values of 1 4 % previously reported from a similar study (2). the turnover time or the average time the albumin molecules spend in the interstitial space (ilk,,,) was here estimated as 53 minutes for dog 2, a value which is also significantly smaller than the mean value of 257 minutes given by the same authors. the differences are due to the circumstance that these authors regarded the plasma pool as infinite, which was not the case in the present study (cf. page 173). the barrier t o potassium exchange has been claimed to be localized in the cell membrane by some authors (8, 1 1 , 15, 29), while other authors (23, 30) have presented some evidence for its localization in the capillary wall. as pointed out above, our 4 2 k data do not allow any detailed conclusions about the exchange between interstitial and intracellular spaces, but the estimates of the rate constants, taking into account their large confidence limits (see table iv and fig. 5f), still suggest that the barrier is situated at the capillary wall. however, it should be noted that it was necessary to introduce one more com partment in series (see model 5 in fig. 3 and the comments on page 178), in order to achieve compati bility with both 4 2 k tracer data and data from the literature concerning the concentration and distribu tion relations in interstitial and intracellular potas sium spaces. whether this auxiliary compartment should be interpreted as a second intracellular com partment (cf. 24, 29) or as a reflection of slow mixing in an inhomogeneous interstitial compartment (cf. 20), cannot be answered by this study. it is thus also impossible to localize more precisely the potassium exchange barrier. the analysis of 24na data presented similar prob lems to those discussed above for potassium (cf. also sjostrand (25)). the data could not resoive such com plex models as discussed by conn & wood (9) for sodium exchange. in fact even the simplest two-com partment model (model 1 b in fig. 3) was compatible with our data. no adequate model could be found for the transfer kinetics of this substance. the uncertainty of the rate constants connecting interstitial and intra cellular compartments was too large (see fig. 5d) and could not be reduced to a meaningful level by introducing the further constraints presented by literature data on the concentrations and distribu tion volumes of sodium (see fig. 5 d). however, the 95 % confidence limits of these rate constants include the point estimates given for the same parameters by conn & wood (9) for a number of dogs. in summary, it can be concluded that 1 . experimental data of the type presented here upsala j m e d sci 78 180 g . arturson et al. a n d previously used by other authors in the study of electrolyte kinetics in the dog’s heart, cannot be described adequately in terms of a few distinct compartments; and 2. accordingly the same data, d o not provide suf ficient information for a reliable estimation of cellular exchange of sodium and potassium; 3. the transcapillary exchange of albumin and sulphate ions can be reliably estimated from the corresponding tracer data and compartmental mod els, which are adequate for these substances. acknowledgements t h e authors thank prof. lars garby for valuable criticism, a n d c. hallin, g. montin, a. persson, a. wallenstll, b. westerberg and b. d s t m a r k for skilful technical assistance. the investigation was supported financially by the swedish medical research council, grants b70-14x-2871-01a, b71 14x-4252-01, and 0. and e. ericsson’s research foundation. 14x-2871-02b, b72-14x-2871-03c, k73-17p-4141-01a, b74 references 1. areskog, n.-h.: some aspects of the hemodynamics of the heart-lung preparation of the dog. acta soc med upsal 67: 143-152, 1962. 2. areskog, n.-h., arturson, g. & grotte, g.: studies o n heart lymph. i . kinetics of 1311-albumin in dog heart-lung preparations. acta physiol scand 62: 209-217, 1964. 3. arturson, g., grotte, g., malmberg, p., samuelsson, r. & sjostrand, u.: simultaneous measurements of myo cardial lymph 42k, 24na, 35s0,-2 and 1311-albumin con centration during steady-state infusion. i n alfred benzon symposium i i : capillary permeability (copenhagen, 1969), pp. 319-323. munksgaard, copenhagen, 1970. 4. beale, e. m. l.: confidence regions in non-linear esti mation. s r stat soc 22: 41-76, 1960. 5. benyon, p. r.: a review of numerical methods for digitai simulation. simulation 11: 219-238, 1968. 6. bill, a , , marsden, n. & ulfendahl, h. r.: some applica tions of a new gel filtration method for molecular separa tion. scand j lab clin invest 12: 392-395, 1960. 7. box, g. e. p.: fitting empirical data. ann n y acad sci 86: 792-816, 1960. 8. conn, h. l. & robertson, j. s . : kinetics of potassium transfer in the left ventricle of the intact dog. amer j physiol 181: 319-324, 1955. 9. conn, h. l. & wood, j. c.: sodium exchange and distri bution in isolated heart of the normal dog. amer j physiol 197: 631-636, 1959. 10. danielson, b. g.: t h e distribution of some electrolytes in the heart. acta physiol scand 62: suppl. 236, 1964. 11. downey, h. f. & kirk, e. s.: coronary lymph: specific activities in interstitial fluid during uptake of 4*k. amer j physiol 215: 1177-1182, 1968. 12. downey, h. f. & kirk, e. s.: indications from coronary lymph and early extraction data that steady-state uptake of 42k is not primarily limited by the capillary wall. i n alfred benzon symposium 11: capillary permeability. munksgaard, copenhagen, 1970. upsala j m e d sci 78 13. folkow, b. & neil, e.: circulation. oxford university press, new york, 1971. 14. gitlin, d.: distribution dynamics of circulating and extra vascular 1311-plasma proteins. n y acad sci 70: 122-136, 1957. 15. grupp, g.: potassium exchange in the dog heart in situ. circul res 13, 219-289, 1963. 16. langer, g. a.: sodium exchange in dog ventricular muscle. relation to frequency of contraction and its possible role in the control of myocardial contractility. j gen physiol50: 1221-1239, 1967. 17. langer, g. a. & brady, a. j.: potassium in dog ventric ular muscle: kinetic studies of distribution and effects of varying frequency of contraction and potassium con centration of perfusate. circul res 18: 164177, 1966. 18. lyttkens, l. & sjostrand, u.: dual-isotope determination b y liquid scintillation spectrometry with special regard to samples with varying quenching. beckman instruments international s. a., geneva, 1971. 19. dstling, s. g.: permeability of human red cells during hypotonic fractional mass hemolysis in dextran. acta univ upsal 88: 1970. 20. page, e.: cat heart muscle in vitro. 111. the extracellular space. j gen physiol46: 201-213, 1962. 21. page, e. & solomon, a. k.: cat heart muscle in vitro. i. cell volumes and intracellular concentration in papillary muscle. j gen physiol 44: 327-344, 1960. 22. powell, m. j. d.: a method for minimizing a sum of squares of nonlinear functions without calculating deriv atives. computer journal 7: 303-307, 1965. 23. renkin, e. m . : transport of potassium-42 from blood to tissue in isolated mammalian skeletal muscles. amer j physiol 197: 1205-1210, 1959. 24. schreiber, s. s . , oratz, m. & rothschild, m . a.: ion diffusion delay in the beating mammalian heart. proc soc exper biol med 116: 164-167, 1964. 25. sjostrand, u.: analysis of ionic tracer movements during single heart cycles. acta physiol scand 61: suppl. 227, 1964. 26. sjostrand, u., lyttkens, l., dberg, p. a. & dstling, s. g.: spectrometry for determination of isotopic mix tures with special regard t o biological and clinical investi gations. acta soc med upsal 74: 219-246, 1969. 27. solomon, a. k.: compartmental methods of kinetic anal ysis. i n mineral metabolism. academic press, new york, 1960. 28. weidmann, s . : elektrophysiologie der herzmuskelfaser. h. huber, bern, 1956. 29. wood, j. c. & conn, h. l.: potassium transfer kinetics in the isolated dog heart. influence of contraction rate, ventricular fibrillation, high serum potassium and acetyl choline. amer j physiol 195: 451-458, 1958. 30. yudilevich, d. & martin de julian, p.: potassium, so dium and iodide transcapillary exchange in the dog heart. amer j physiol208: 959-967, 1965. received december 11, 1972 address for reprints: ulf sjostrand, m.d. department of anaesthesiology & intensive care regional hospital s-701 85 d r e b r o sweden upsala j med sci 81: 79-83, 1976 changes of reactive hyperaemia after clinical bed rest for seven days goran friman and elisabeth hamrin from the departments of infectious diseases and clinical physiology, university hospital, uppsala, sweden abstract as an indication of peripheral circulatory function reactive hyperaemh was studied in the forearm and calf muscle in 14 healthy young men before and after clinical bed rest for one week. blood flow was measured after different arterial occlu sion times with venous occlusion plethysmography. after bed rest peak flow values in the calf after arterial occlusion for 3 or 5 minutes decreased moderately (by about 20-23 %) and significantly. peak flow in the forearm decreased as well although not significantly. introduction immobilization and bed rest are known t o influence central circulatory function (5,20) and blood volume (13, but the effect on peripheral circulation seems to have been less well investigated. the aim of the present investigation was to establish the possible influence of the bed rest regimen used on a modern acute ward on peripheral circulation studied as reac tive hyperaemia in the forearm and calf muscles. the investigation is part of a more extensive study of the effect of clinical bed rest on a number of variables related to physical fitness (9). material and methods subjects fourteen healthy men aged 21-32 years, took part in the investigation. they were confined to bed for 7 days in a special room on a ward for infectious diseases. the aim was to achieve the same degree of physical activity and caloric intake as encountered by hospitalized patients. thus, the subjects were allowed to leave bed for personal hygiene. in addition, they sat in an armchair for a short period twice daily starting on the fifth day. apart from this no physical activity was permitted. eight of the subjects were on a standard hospital diet, while the other 6 had a starvation diet for the first 4 days, thereafter the standard diet. fluid intake was unrestricted. procedure measurements were made on three occasions: one week before the start of the bed rest period, a t the end of the bed rest period, and one month later during which time the subjects had maintained normal activity. measurements b l o o d f l o w . reactive hyperaemia in the forearm and calf muscles was measured by venous occlusion plethysmo graphy and expressed in terms of ml/min. 100 ml tissue. a more detailed description of the technique and procedure used for blood flow measurements with venous occlusion plethysmography has been given previously by graf t westersten (13) and graf (12). the forearm and calf plethysmograph, an air-filled rubber cuff, enclosed a 5 cm long segment of the muscular part of the extremity. a proximal occlusion cuff was applied to the upper arm or to the thigh, and a second occlusion cuff distal to the rubber cuff. the air-filled plethysmograph cuff was always in flated to 40-50 mm hzo. during ischaemia the proximal occlusion cuff was in flated to 100 mmhg above the systolic arterial blood pres sure in the brachial artery, while the distal occlusion cuff was inflated to 90 mmhg. reactive hyperaemia was rneas ured after different arterial occlusion times: 1 , 3 and 5 min. during blood flow measurements the proximal cuff was inflated to 60 mmhg (during reactive hyperaemia, this value was initially somewhat higher) and the distal cuff was simultaneously inflated to arterial occlusion pressure. the blood flow was recorded on a conventional amplifier and a direct-writing mingograph (siemens-elema, stockholm) for 2 min with measurements made every 10 seconds during the first minute. the recommendations given by graf (12) in order to avoid errors of measurements connected with the method were followed. two models of venous occlusion plethysmographs were used, the difference being the way the pressure was applied to the proximal and distal occlu sion cuffs on the extremities. mean values for the blood flow for subjects using the two different models were statistically tested, and no significant differences were found. blood volume. blood volume measurements were made by determination of the total amount of haemoglobin (thb) using the alveolar co method, as described by sjostrand upsala j med sci 81 80 g. friman and e . hamrin table i . mean values m . e . m . ofbloodflow at rest andpeakjlow during reactive hyperaemia in forearm and calf muscle after arterial occlusion f o r i , 3 and 5 rnin ( a . o . ) , and certain circulatory and anthropometric data, in 14 healthy men subjected t o clinical bed rest f o r one week a b c (before (end of (one month bed rest) bed rest) bed rest) forearm blood flow (ml/min . 100 ml tissue) rest 1 min a.o. 3 rnin a . o . 5 min a.o. calf blood flow (ml/min . 100 ml tissue) rest 1 min a.o. 3 rnin a . o . 5 rnin a.o. blood volume (1) total hemoglobin (g) red cell volume (1) plasma volume (1) body weight (kg) extremity circumference (cm) hb (g%) forearm calf handgrip knee extension plantar flexion isometric muscle strength (kp) 1 . 9 f 0 . 3 14.6f2.1 24.6f2.6 28.4f3.0 (n=13) 2.8k0.3 1 7 . l f 1.4 (n=13) 30.6f 1.4 36.52 1.7 (n= 13) 5.24f0.14 702k 20 14.65f0.28 2.08f 0.06 3.17f0.10 70.5k2.2 2 6 . 2 f 0 . 3 *35.8+0.5 *51.6k 2.5 69.6f 1.6 163.7f 6.3 2 . 0 f 0 . 4 11.72 1.3 22.4f2.6 (n = 13) 25.8k2.4 (n=13) 2.3-10.3 14.6f 1.0 ***24.5f1.1 **28.2k 1.5 (n=13) * * 4.96f0.12 * *664f 22 14.6950.23 * * 1.975 0.07 *2.99f 0.06 **69.4f2.0 26.1 f 0.3 **35.4f0.5 49.3 k 2.5 66.5f2.7 152.4f7.4 1.820.4 12.8k 1.1 24.7f 1.8 30.3k 1.8 2 . 8 2 0 . 4 **19.9f 1.3 ***32.1?1.3 ***37.712.1 **5.23f0.13 *688120 14.46f0.25 *2.04f 0.06 **3.26?0.08 69.842.0 26.220.3 35.5k0.5 48.8k 2.5 (n = 13) 69.84 1.9 (n=13) * 158.24 8.0 (n=13) *, **, and *** denote statistically significant differences (p<0.05,0.01 and 0.001, respectively), and refer to the values of the columns between which they are interposed; asterisks in column a refer to comparisons between values of column a and c. (21). the total blood volpume was then calculated from the thb and the haemoglobin concentration (hb) of blood from a cubital vein, punctured without venous occlusion. duplicate determinations of thb were always made with an interval of one day, the coefficient of variation for the single determination being about 4 % during the period of investigation. the determinations made at the end of the bed rest period were performed on the seventh day of bed rest and on the following day. in addition, haematocrit was determined and the plasma volume (pv) and red cell volume (rcv) were calculated. isometric muscle strength. the maximal isometric mus cle strength was tested under standardized conditions ac cording to bacmund & nordgren (2). in this paper only the values for handgrip, knee extension and plantar flexion are presented. statistical methods in order to reveal differences between the observations on the three occasions student’s t-test for paired observations was used. upsala j med sci 81 results all results are summarized in table i . bloodjlow in forearm and calf(fig. 1). in forearm the resting blood flow did not change during bed rest. the mean values for peak flow during reactive hyperaemia decreased, although not significantly, after arterial occlusion for 1, 3 as well as 5 min. in calf the resting blood flow decreased during bed rest, but the change was not statistically significant. the peak flow values recorded at the end of bed rest were significantly lower after arterial occlusion for 3 and 5 min ( p < o . o o l and 0.005, respectively), whilst the decrease after occlusion for 1 minute did not reach significance. at the one month control peak flows in calf with arterial occlusion for 1 , 3 and 5 rnin showed a signifi cant increase when compared with the values re peripheral circulation after bed rest 81 corded at the end of bed rest (p<0.005, 0.001 and 0.001, respectively). blood volume. the total blood volume was signifi cantly lower at the end of bed rest than before @<0.005) or one month later (p<o.ol). thb, pv and rcv showed the same pattern of response. body weight and extremity circumference. the body weight fell significantly during bed rest @<0.01) and had not returned to the initial level after one month. the calf circumference decreased sig nificantly during bed rest (p<o.ol), and the one month control value was still significantly lower than the initial value bc0.05). isometric muscle strength. the maximal strength of isometric plantar flexion was lower at the end of bed rest than when the subjects were ambulant; however, the difference was significant (pco.05) only when compared with the one month control value. the strength of knee extension did not change significantly. unexpectedly, the handgrip strength was signifi cantly lower at the one month control compared to the initial value (p<0.05), while the value at the end of bed rest attained an intermediate position. discussion in the present study we have found a significant change of reactive hyperaemia in the calf but not in the forearm of 14 healthy young men after bed rest for one week. in order to avoid errors of measure ment connected with venous occlusion plethysmo graphy the recommendations given by graf (12) have been closely followed, both in connection with the subjects and the evaluation of the inflow curves. the pressure system in the plethysmograph has been tested and some variation of the inflation pressures of the cuffs have been accepted, as in earlier works (3). delius et al. (6) found the same changes of reactive hyperaemia when measuring blood flow with venous occlusion plethysmography in the forearm and calf of 32 patients who had been operated upon for acquired or congenital heart disease. the authors drew the conclusion that the changes in blood flow after the operation could have been caused by a change in the sympathetic activity. several authors have reported alterations in reactive hyperaemia in skeletal muscle after passive changes of body posi tion from horizontal to different degrees of tilting and to the erect position. paterson (19) found that reac tive hyperaemia in the forearm decreased signifi cantly during tilting, a finding which he believed could be caused by increased sympathetic activity. mosley (17) found decreased reactive hyperaemia in the forearm of healthy subjects when changing from supine to erect position, a reaction which could be abolished by intra-arterial infusion of sympathetic adrenergic antagonists. using microneurography delius et al. (7) recorded increased sympathetic signals in human muscle nerves to forearm and calf after tilting from horizontal to 30-45 degrees. 6-162852 upsala j med sci 81 82 g. friman and e . hamrin it is not clear from the literature whether an in crease in sympathetic tone occurs in healthy sub jects as a result of bed rest for one week. however, hyatt (15) studied haemodynamic and body fluid alterations in healthy subjects during different periods of bed rest (10, 14 and 28 days), and in all the studies he found a decrease in plasma volume and orthostatic tolerance and an increase of peripheral vascular resistance. hyatt considered increased sympathetic tone to result from the decrease in plasma volume. since we have also found a signifi cant decrease in plasma volume, it seems reasonable t o us that this decrease may exert an effect on the low pressure baroreceptors and thus bring about a change in the sympathetic tone. the role of the low pressure baroreceptors during changes of blood vol ume have been clearly stated in different studies using lower body negative pressure (lbnp). thus, ardill et al. ( 1 ) found that lbnp significantly de creased the reactive hyperaemia in the forearm, and that sympathetic adrenergic antagonists prevented this effect. johnson et al. (16) found that moderate lbnp (to -20 mmhg) only reduced right atrial pressure and forearm blood flow, while further low ering of the lbnp also changed aortic mean pres sure, aortic pulse pressure and heart rate. according to this study the likely source of the stimulus for the reduction of the muscular blood flow is the low pressure baroreceptors. in recent experiments of similar design sundlof & wallin (22) have come to similar conclusions with recordings of sympathetic signals by microenurography . apart from a change in the neurogenic component regulating the vascular bed in skeletal muscle, changes in the local components have to be consid ered. according to folkow (8) myogenic activity is especially important in muscles which are responsi ble for posture. the soleus muscle is a tonic muscle, composed of mainly red (slow twitch) fibres (1 l ) , and according t o emg measurements (4) it plays a sig nificant role in the maintenance of balance and pos ture. during bed rest this muscle is not being used as a posturq muscle, because of changes gravity conditions. an alteration in rnyogenic activity as the main cause of the observed change in reactive hyperaemia might therefore explain the fact that the change was significant in the calf but not in the forearm. factors at the cellular and subcellular levels must also be taken into consideration. it has been shown in previous studies that immobilization may cause upsala j med sci 81 atrophy of skeletal muscle (14, 18). disuse atrophy (cf. denervation atrophy and atrophy after tenotomy, for review see goldspink (10)) is characterized by a reduction of the myofibril protein content, while the sarcoplasmic protein content seems to be uninfluenced (14). the observed de crease of calf muscle strength and circumference after bed rest in the present study is compatible withe the hypothesis that a reduction of the amount of contractile substance occurred despite the rather short immobilization time. however, other explana tions of the strength reduction such as a decrease of the atp content are also possible. the finding of a decreased calf circumference, on the other hand, could be explained just as well by a reduction in tissue water, a suggestion supported by the de creased plasma volume and body weight. a search of the literature revealed no definite evidence of whether the numbers or sizes of the blood vessels are subjected to a reduction proportional to that of the muscle cell in disuse atrophy. if such were the case a reduction of reactive hyperaemia would be expected with atrophy. acknowledgements the work was supported by the delegation for applied medical defence research (grant no. u65/1973) and by the county council of uppsala. miss margareta torgen and mrs ninni rosen gave technical assistance and miss ingrid lundh secretarial aid. references 1. 2. 3. 4. 5 . 6. ardill, b. l., bhatnager, v. m. & fentem, p. h.: reduction in the vasoconstriction produced by sympa thetic adrenergic nerves during reactive hyperaernia. cardiovasc res 1 : 327, 1967. backlund, l. & nordgren, l.: a new method for testing isometric muscle strength under standardized conditions. scand j clin lab invest 21: 33, 1968. bygdeman, s . & pernow, b.: venos ocklusions pletysmografi. in perifer cirkulation. kliniskt fysio logiska undersokningsmetoder (ed. pernow b.), pp. 55-80. almqvist & wiksell forlag ab, stockholm, 1970. carlsoo, s.: how man moves. heinemann ltd., london, 1972. deitrick, j . e., whedon, g. d. & shorr, e.: effects of immobilization upon various metabolic and physiologic functions of normal men. am j med 4: 3, 1948. delius, w., delius, k. & nystrom, s. 0.: periphere durchblutungsveranderbgen nach operativer kor rektur von herzvitien. z kreislaufforschg 58: 1260, 1%9. peripheral circulation after b e d rest 83 7. delius, w., hagbarth, k . e . , hongell, a. & wallin, b. g.: manoeuvres affecting sympathetic outflow in human nerves. acta physiol scand84: 82, 1972. 8. folkow, b. &neil, e.: circulation. oxford university press, london & toronto, 1971. 9. friman, g.: t o be published. 10. goldspink, g.: postembryonic growth and differentia tion of striated muscle. in the structure and function of muscle, vol. i. fed. g. m. bourne), pp. 179-236. academic press, new york & london, 1972. 11. gollnick, p. d . , sjodin, b., karlsson, j., jansson, e. & saltin, b.: human soleus muscle: a comparison of fiber composition and enzyme activities with other leg muscles. pflugers arch348: 247, 1974. 12. graf, k.: auswertung und messfehler okklusions pletysmografischer durchblutungsregistrierungen. acta physiol scand 60: 120, 1964. 13. graf, k. & westersten, a . : untersuchungen uber eigenschaften und verwendungsmoglichkeiten eines flexiblen extremitatenplethysmografen. acta physiol scand 46: 1, 1959. 14. helander, e.: on quantitative muscle protein determi nation. acta physiol scand41, suppl. 141, 1957. 15. hyatt, k . h.: hernodynamic and body fluid alterations induced by bed rest. in hypogravid and hypodynamic environments (ed. r. murray & m. mccally), pp. 187-209. national aeronautics and space administra tion washington dc 1971, nasa sp-269. 16. johnson, j. m., rowell, l. b., nierenberger, m. & eisman, m.: human splanchnic and forearm vaso constrictor responses to reductions of right atrial and aortic pressures. circ res34: 515, 1974. 17. mosley, j. g.: a reduction in some vasodilator re sponses in free-standing man. cardiovasc res 3: 14, 1969. 18. patel, a. n., razzak, 2. a. & dastur, d. k.: disuse atrophy in human skeletal muscles. arch neurol 20:413, 1969. 19. paterson, n. a. m.: the effects of increased vaso motor tone on reactive hyperaemia in the human forearm. aust j exp biol med sci45: 651, 1967. 20. saltin, b., blomqvist, g., mitchell, j. h., johnson, r. l., wildenthal, k. & chapman, c. b.: response to exercise after bed rest and after training. circula tion38, suppl. vii, 1968. 21. sjostrand, t.: a method for the determination of the total haemoglobin content of the body. acta physiol scand16:211, 1948. 22. sundlof, g. & wallin, g.: to be published. received december 15, 1975 address for reprints: elisabeth hamnn, m.b. department of clinical physiology university hospital s-750 14 uppsala sweden upsala j m e d sci 81 upsala j med sci 78: 150-152, 1973 stereospecific uptake of narcotic analgesics by a subcellular fraction of the guinea-pig ileum a preliminary communication lars terenius from the department o f pharmacology, university of uppsala, uppsala, sweden abstract longitudinal muscle of guinea-pig ileum was homogenized. precipitates from centrifugation were incubated with la belled dihydromorpbine and competitors. the uptake of label to a high-speed fraction was stereospecific (the analgesics levorphanol and levo-methadone were more active competbrs than their inactive dextro-counterparts). morphine, normorphine, dextromoramide, the antagonists nalorphine and naloxone inhibited uptake. codeine was inactive. introduction the mode of action of narcotic analgesics remains obscure in spite of the intense work which has been attributed to the problem. i t is even largely unknown a t which site(s) in the cns they exert their analgesic action. therefore, the longitudinal muscle of the guinea-pig ileum, which is anatomi cally well-defined and which is affected by mor phine-like analgesics in vitro (schaumann, 1957; weinstock, 1971) has considerable interest as a model tissue. the present communication analyses the inter action between narcotic analgesics and subcellular fractions of the longitudinal muscle from guinea pig ileum. evidence is provided that there is a specific interaction. part of this work has been presented a t the scandinavian pharmacological society meeting, uppsala, june 1972 (terenius, 1972). materials and methods guinea-pigs of either sex weighing at least 450 g were killed by a blow on the head. in a typical experiment (12-18 incubation flasks, see below) 5 animals were used. the ileum except for 10-15 cm of the terminal part was removed. longitudi nal muscle strips were prepared from 10-15 cm upsalu j med sci 78 segments by the method of rang (1964), carefully avoiding mesenteric fat. the muscle with adhering nerve plexi was finely cut with a pair of scissors. all subsequent operations were at 0” to 4oc. ten times the weight of ice-cold 0.32 m sucrose in bidistilled water was added. homogenization was done with an ultraturrax (kinematica, luzern) 2 times for 15 sec at setting 2 with cooling for 2 min inbetween. the homogenate was further dis integrated in a glass homogenizer with a loose fitting teflon pestle at low speed. it was then centrifuged at 2 x 1000 g for 10 min. the super natant was saved and centrifuged a t 120000 g for 30 min. in a few experiments the 2 x 1000 g supernatant was centrifugated at 10000 g for 30 min, see text. the respective pellets were re suspended in a small volume of 0.32 m sucrose. aliquots of the suspension containing about 1 mg protein were added to 4 ml of incubation buffer, nacl 124 mm, kc1 5 mm, kh2p0, 1.2 mm, cacl, 0.75 mm, mgso, 1.3 mm, n-2-hydroxy ethylpiperazine-n’-2-ethanesulfonic acid “hepes” 26 mm, ph 8.0 a t 25°c; this buffer is modified from bradford & thomas (1969) which in ap propriate cases also contained non-labelled test substances. after a pre-incubation period of 10 minutes a t 25°c with the test substances, d i h y d r ~ m o r p h i n e ~ h (specific activity 66.6 mci/ mg, radiochemical purity 95-98 % ) in 0.2 ml in cubation buffer was added, and the incubation was continued for 1 hour. the incubation was stopped by cooling the vessels on ice. the content was quantitatively transferred to centrifuge tubes and centrifuged for 30 min a t 120 000 g. aliquots (0.5 ml) of the supernatant were removed for radioactivity measurement. the remaining super natant was discarded, and the pellets were rapidly washed with ice-cold buffer. the tubes were im mediately inversed and allowed to stand in an in uptake of narcotic analgesics 151 table i. competition experiment showing the stereo specificity of the uptake of labelled dihydromorphine to the 120 000gparticulate fraction (expts. 1-2) or the 10 000 gparticulate fraction (expts. 3-4) of guinea-pig ileum the concentration of dihydromorphine was 0.55 x m, of levorphanol, dextrorphan and of the methadone antipodes 1 . 1 x lo-* m. each experiment represents one homogenate, each treatment was run in duplicate uptake (dpm/mg protein) competitor expt. 1 2 3 4 table 11. effect of various analgesics and analgesic antagonists on the uptake (dpmlmg protein) of labelled dihydromorphine (concentration 0.7 x m ) to a high-speedparticulate fraction of guinea-pig ileum a different homogenate was used for each substance. means + s . e . m . are given for 3 samples per group concentration (m) of competitor substance 0 10-9 10-8 10-7 morphine 9 8 1 f 5 4 794+50 585+3 508f55 dextromora mide 953+46 1 0 1 2 k 2 9 845+34 6 1 5 5 3 7 normorphine 1 0 6 9 5 1 6 9 0 5 i 6 7 9 8 5 2 0 6 5 8 i 4 8 nalorphine i 2 9 6 f 4 2 1 0 2 7 5 4 4 9 5 1 k 3 4 570+24 naloxone 1 1 2 1 1 3 1 805f32 5 7 1 k 3 2 3 9 5 5 1 3 codeine 1 2 5 6 + 1 2 1 2 2 5 5 1 8 1 2 1 9 5 6 2 n o n e 761 844 1 1 1 8 1 2 5 2 levorphanol 330 346 807 810 dextrorphan 653 674 1052 1 0 6 4 none 647 759 650 634 1019 1 0 5 6 999 1003 levo-methadone 432 424 483 436 665 736 619 626 dextro methadone 565 765 107 742 985 997 847 920 versed position for a t least one hour. the pellets were digested with soluenea (packard, downers grove, ill.) and the radioactivity content meas ured. protein was determined according to lowry et al. (1951) using bovine serum albumin as stand ard. results competition experiments were performed in order to establish the specificity of the interaction be tween dihydromorphine and the high-speed sub cellular fraction. it was found that the optical antipodes, levorphanol/dextrorphan and levo-/ dextro-methadone differentially inhibited the up take of dihydromorphine to the high-speed precipi tate, the analgetically more active levo-antipodes being the more active competitors (table i, expts. 1 and 2). in experiments 3 and 4 (table i) is shown that the stereospecific uptake is also present in the 10000 g precipitate. other narcotic analgesics (morphine, normor phine and dextromoramide) and the narcotic an tagonists (nalorphine and naloxone) were active competitors in the test system while codeine was without effect (table 11). discussion morphine-like compounds inhibit the contractions of the longitudinal muscle of the guinea-pig ileum (see schaumann, 1957; weinstock, 1971). this tissue should therefore contain morphine receptors and an interaction with such receptors might be observable in simple incubation experiments. i n view of the very strict steric requirements for activity on the ileum (schaumann e t al., 1953) (which seem identical to those for analgesic ac tivity) one might expect a similar selectivity in the binding properties of the receptor. the present work shows a stereoselective inter action with the opiates levorphanol and dextror phan as competitors and as well with the all synthetic, structurally completely different metha done antipodes. those isomers which are more potent on the ileum (schaumann et al., 1953) are the more active competitors (table i). also an other synthetic analgesic, dextromoramide, showed considerable competitive activity. on the other hand, codeine is practically inactive as a compe titor (table 11) as on the ileum preparation (kos terlitz & watt, 1968). all this evidence favors the assumption that the observed interaction is of fundamental importance for the action of mor phine-like drugs and that it could be ascribed to an interaction with a receptor. i t is also notable that the narcotic antagonists, nalorphine and naloxone, are active as competi tors (table 11). although most narcotic antago nists including nalorphine have agonistic proper ties, naloxone is held to be a pure antagonist (also on the ileum preparation, kosterlitz & watt, 1968) indicating that the antagonistic activity of nar cotic antagonists may depend on competition with the analgesics for the receptors. also normorphine is active as competitor (table 11), which is of interest since this compound has been implicated upsala j med sci 78 152 l. terenius in the action of morphine (beckett et al., 1956). preliminary experiments with further fractiona tion (combined with enzymological and morpho logical examination) indicate that particles rich in acetylcholinesterase and 5’-nucleotidase and not mitochondria are responsible for the uptake. acknowledgment technical assistance was given by mrs ulla staav. nor morphine was kindly donated by d r e. l. may at n.i.h., b-thesda, md, usa, naloxone by d r h. w. kosterlitz, university of aberdeen, scotland, and levorphanol and dextrorphan by hoffmann-la roche, basel, switzerland. the work was supported by the swedish medical re search council (proj. no. k72-14x-3756-01). references 1. beckett, a. h., casy, a . f. & harper, n. j.: anal gesics and their antagonists: some steric and chemical considerations. j pharm pharmacol 8: 874, 1956. 2. bradford, h. f. & thomas, a. j.: metabolism of glucose and glutamate by synaptosomes from mam malian cerebral cortex. j neurochem 16: 1495, 1969. 3. kosterlitz, h. w. & watt, a. j.: kinetic parameters of narcotic agonists and antagonists, with particular reference t o n-allylnoroxy-morphone (naloxone). brit j pharmacol 33:266, 1968. 4. lowry, 0. h., rosenbrough, n. j., farr, a. l. & ran dall, r. j.: protein measurement with the folin phenol reagent. j biol chem 193:265, 1951. 5. rang, h. p.: stimulant actions of volatile anaesthetics on smooth muscle. brit j pharmacol 22:356, 1964. 6. schaumann, 0.: morphin und morphinahnlich wirkende verbindungen. i n handbuch der experimentellen phar makologie, erganzungswerk (ed. 0. elchler & a. farah), vol. 12, pp. 170-189. springer, berlin, 1957. 7. schaumann, o., jcchum, k. & schaumann, w.: anal getika und darmmotorik. 11. wirkung auf den langs muskeltonus. arch pharmakol 217: 360, 1953. 8. terenius, l.: specific uptake of narcotic analgesics by subcellular fractions of the guinea-pig ileum. acta pharmacol toxicol 3i:suppl. i, 50, 1972. 9. weinstock, m.: sites of action of narcotic analgesic drugs: i n peripheral tissues. in narcotic drugs, bio chemical pharmacology (ed. d. h. clouet), pp. 394 407. plenum press, new york. received december 15, 1972 address for reprints: l. terenius department of pharmacology university of uppsala box 573, 751 23 uppsala sweden upsala j m e d sci 78 upsala j med sci 84: 247-254, 1979 the effects of endogenous hypergastrinemia and hypogastrinemia on the exocrine and endocrine rat pancreas h. johansson'.2, l. grirnelius3, ph.u. heitz', g. lundqvist4, p. peterson5, g. portela-gomes3 and e. wilande? from the depurtments of surgery', clinicul research 112, pathology3, and clinicul chemistry4, university hospital, uppsala, the department of histology5, uppsala university, uppsalu, sweden and the department of pathology6, university of basel, switzerland abstract the effects of endogenous hypergastrinemia and hypogastrinemia on the exo crine and endocrine pancreas were studied in the rat. hypergastrinemia was in duced by antral exclusion, and hypogastrinemia by antral resection. the studies were made 14 weeks after surgery. the total weight of the pancreas was increased both in hypergastrinemic and hypogastrinemic animals, due to hypertrophy of the exocrine cells. in contrast, the volume and total weight of the pancreatic islets were decreased. there was no numerical difference in the a-, d-, pp-cells between the hyper and hypogastrinemic animals, respectively, and the controls. the number of in sulin-producing (b-) cells was certainly reduced after the induction of hypo gastrinemia. there was, however, signs of increased b-cell activity, which might contribute to an underestimation of the number of b-cells with the tech nique used. these findings do not support the hypothesis that antral gastrin has trophic influence on either exocrine or endocrine pancreas. introduction gastrin is known to influence the gastrointestinal tract in many ways. its stimulatory action on the secretion of hydrochloric acid and pepsinogen and its trophic effect on the parietal cell area of the stomach are well defined ( 3 , 1 9 , 2 0 ) . pentagastrin, administered in pharmacological doses, has also been re ported to exert a trohpic action on the exocrine pancreas in rats ( 1 , 9 ) . hitherto, however, no such action has been found following various surgical procedures leading to an increased endogenous serum gastrin concentration ( 1 3 ) . in the experiments by the latter authors no increase in the total dry weight of the pancreas was observed. a link between gastrin and the endocrine pancreas has been discussed by larsson ( l o ) , who found islet cell hyperplasia in some patients with hypergastrinemia due to tumor production and in patients with atrophic gastritis. furthermore, indirect evidence of a gastro-insular axis 247 mediated by gastrin has been produced, as an altered glucose tolerance has been found in patients with hypergastrinemia compared to normogastrinemic patients (16,17). the present experiments were designed in order to further examine the effect of endogenous hypergastrinemia not only on the exocrine but also on the endo crine pancreas. material and methods adult male sprague-dawley rats (body weight 250-300 9) were used. they were fed on laboratory food. the animals were randomly assigned to the following g roups : (1) 5 rats with exclusion of the antral part of the stomach (ae) ( 2 ) 4 rats with resection of the antral part of the stomach (ar) ( 3 ) 4 control rats untreated ( c ) . antral exclusion and antral resection were performed by techniques described previously ( 5 ) . serum gastrin concentrations were determined by solid-phase radio-immuno assay ( 1 1 ) 12 weeks after operation, after a starvation period of 48 h. the animals were killed 14 weeks after the stomach operation. the pancreas was extirpated, dissected free from fat tissue and weighed. the specimens were then fixed in bouin‘s fluid for 18 h, dehydrated, cleared in xylene and em bedded in paraffin. deparaffinized 5 um thick sections were stained with hema toxylin-eosin and by the van gieson method. to calculate the percentage volume of the pancreatic islets, the point sam pling technique described by chalkley ( 2 ) was employed. at least 3000 inter sections (hits) were analysed on acinar or islet parenchyma of each pancreas. the weight of the endocrine parenchyma was calculated by multiplying its per centage volume by the weight of the entire pancreas. the densities of the exo crine and endocrine tissue were assumed to be equal. the density of the exocrine cells per unit volume exocrine tissue was deter mined in a microscope equipped with a square grid placed in the focal plane of the eyepieces at a magnification of x 400. in each case three randomly selected areas of exocrine tissue were examined and the nuclei or nuclear fragments within the square grid were counted. the nuclear sizes of 50 randomly selected exocrine cells were also determined with the aid of a mm scale placed in one of the eyepieces. as the observed nuclear frequency is influenced by the nu clear size, the formula of flodbrus was used to correct these values (7). the relative number of exocrine cells was calculated by multiplying the corrected number of exocrine cells per unit volume by the exocrine pancreatic weight. gomori’s aldehyde fuchsin technique (12) with ponceau fuchsin as a counter stain (8) was used to visualize b-(insu1in)cells. d-(somatostatin)cells were stained with the davenport alcoholic silver nitrate technique as modified by hellerstrom & hellman (6). a-(g1ucagon)cells and pp-(pancreatic polypeptide) cells were identified with the immunoperoxidase (pap) technique described by sternberger ( 1 8 ) . the sections were counterstained with hernechtrot. controls a s described by goldman (4) were used. differential counts were made in order to estimate the frequency of differ ent types of endocrine pancreatic cells (except pp-cells). at least 2000 endo crine cells were counted in each animal and with each of the various staining procedures. only cells with visible nuclei or nuclear fragments were included in the counts. the nuclear size of band non-b-cells was estimated in aldehyde-fuchsin stained sections (counterstained with ponceau fuchsin) as described above, at a magnification of 1 2 5 0 x. statistics: student’s t-test was used. results the mean body weight at sacrifice was slightly higher in the control group than in the two experimental groups (table 1 ) . the serum gastrin concentration was considerably increased in group ae and markedly decreased in group ar as compared with that in the controls (table 2). the mean pancreatic weight was much higher in both experimental groups than in the controls (table 3 ) . the observed number of exocrine cell nuclei per unit volume was highest in the control group and lowest in group ar. after correction concerning the ru clear size the differences were more pronounced (table 4). these findings indi cate a hypertrophy of the exocrine pancreas both after antral exclusion and after antral resection. the relative values of the total number of the exocrine cells were also highest in the control group and lowest in group ar (table 4 ) , indicating a numerical decrease of the exocrine cells after antral resection. the volume of the pancreatic islet tissue in relation to the total pancre atic volume was significantly lower in the experimental groups (table 5). the calculated weight of the islets was significantly decreased in both experimen tal groups in comparison with the control group (table 5 ) . the islet weights of the two experimental groups did not differ from each other. the amount of non parenchymal tissue (blood vessels, connective tissue, fat cells) was approxi mately equal in the three groups as judged by visual observation. in group ae and the control group the b-cells showed a strong and even staining reaction, while this reaction was highly variable in group ar. no dif ference in the staining of a , dor pp-cells was found between the different groups. the frequency of b-cells in group ar was significantly lower than in the controls (table 6), whereas the frequency of the other endocrine cell types 249 table 1. body weight at sacrifice group body weight (4) m c s.d. ae 4 1 7 f 3 4 ar 4 1 1 2 2 9 controls 4 6 3 f 3 5 table 2. serum gastrin group serum gastrin (mmol/l) ae 2 8 7 ? 2 6 ar 1 3 8 f 3 5 controls 1 7 0 f 1 9 table 3. weight of the pancreas group weight of pancreas (9) m f se ae 2 . 2 9 7 c 0 . 1 0 6 ar 2.319 2 0 . 1 0 7 controls 1 . 9 8 2 c 0 . 1 1 5 (aand d-cells) did not differ significantly between the three groups. pp cells were only seen in some islets, mainly located in the periphery. they varied in number, but were most often sparse. no differences in the distribu tion were observed between the different groups. the diameters of the band non-b-cell nuclei (table 7 ) did not differ sig nificantly between the three groups. however, in group ar the nuclear diameter of the weakly stained b-cells was significantly greater ( 6 . 0 5 c 0 . 1 3 ) than that of the heavily stained b-cells ( 5 . 5 f 0 . 1 0 ) . discussion in these experimental models endogenous hypergastrinemia was induced by antral exclusion and hypogastrinemia by antral resection. the total pancreatic weight was higher in both experimental groups than in the control animals. this enlargement of the pancreas was due to hypertrophy and not to hyperplasia of the exocrine cells. the cellular hypertrophy was especially prominent in group ar. the hypertrophied cells were also characterized by nuclear enlargement, 250 t a b l e 4 . d i a m e t e r s (p m) o f th e n u c l e i o f e x o c r i n e c e l l s a nd t h e o b s e r v e d an d c o r r e c t e d ( b y t h e f l o d e r u s f o r m u l a ) n u m b e r o f e x o c r i n e c e l l s p er u n i t v ol um e. t h e c o r r e c t e d v a l u e s r e p r e s e n t t ho se n u c l e i w h o s e c e n t e r s l a y w i t h i n th e se ct io n. t h e d i a m e t e r o f 5 0 r a n d o m l y n u c l e i w e r e e s t i m a t e d in e a c h c as e. g r o u p n u c l e a r d i a m e t e r s i g n i f i c a n c e o b s e r v e d n u m b e r o f c o r r e c t e d nu mb er o f s i g n i f i c a n c e * e x o c r i n e c e l l s p er e x o c r i n e c e l l s p er u n i t v o l u m e u n i t v o l u m e m f s e m f s e m f s e a e 5 .3 7 f 0 .1 0 < 0 .0 5 4 6 f 4 3 1 i 3 < 0 .0 5 ar 5 .6 2 f 0 .2 2 < 0 .0 5 3 6 f 1 2 3 t 1 < 0 .0 1 c o n t r o l s 5 .0 7 f 0. 05 5 9 f 4 4 1 f 3 * p v a l u e (t -t es t) f o r d i f f e r e n c e b e t w e e n th e e x p e r i m e n t a l g r o u p s an d t h e c o n t r o l g r o u p t a b l e 5 . r e l a t i v e i s l e t t i s s u e v o l u m e p er t o t a l p a n c r e a t i c v o l u m e (% ) a n d t o t a l c a l c u l a t e d w e i g h t o f i s l e t t is su e. t h e p o i n t s a m p l e t e c h n i q u e w a s us ed i n t he v o l u m e e s t i m a t i o n . t h e t o t a l i s l e t t i s s u e w e i g h t w a s c a l c u l a t e d f r o m th e t o t a l p a n c r e a t i c w e i g h t a n d i s l e t t i s s u e v ol um e. g r o u p i s l e t t i s s u e s ig n i f i c a n c e * t o t a l i s l e t t i s s u e s i g n i f i c a n c e * v o l u m e (% ) w e i g h t (m g) m f s e m f s e a e 0 .3 3 f 0 .0 6 < 0 .0 0 5 8 .8 0 f 1 .5 6 a r 0 .3 6 f 0 .1 0 0 .0 5 7 .5 1 f 2 .3 3 c o n t r o l s 0 .7 7 f 0 .0 7 1 8 .0 4 f 1 .0 8 < 0 .0 0 1 < 0 .0 0 1 * s e e t a b l e 4 t a b l e 6 . r e l a t i v e f r e q u e n c y o f i s l e t b , a a n d d c e l l s p er t o t a l n um be r o f i s l e t c e l l s s i g n i f i c a n c e * d c e l l s g r o u p b c e l l s s i g n i f i c a n c e " ace ll s s i g n i f i c a n c e * m ? s e m ? s e m ? s e a e 7 7 _ + 2 n s 2 1 _+ 1 ns 1 2 * 2 n s ar 5 5 c 3 ** < 0 .0 0 1 2 2 ? 2 n s 1 5 ? 3 n s c o n t r o l s 7 3 c 1 2 1 f 2 1 6 ? 3 * s e e t a b l e 4 ** t h i s v a l u e m a y be u n d e r e s t i m a t e d , f or r e a s o n s g i v e n i n t h e t e x t t a b l e 7. d i a m e t e r s (u m )* o f b an d no nbce ll n u c l e i o f p a n c r e a t i c is le ts . 10 0 r a n d o m l y s e l e c t e d n u c l e i w e r e e s t i m a t e d i n e a c h c e l l t y p e a n d g ro up . g ro u p b c e l l s s i g n i f i c a n c e * * n o n b c e l l s s i g n i f i c a n c e * * m c s e m ? s e ae 5 .1 2 f 0 .2 6 n s 4 .2 3 ? 0 .0 9 n s a r 5 .5 5 i 0 .1 2 n s 4 .6 4 ? 0 .1 4 n s c o n t r o l s 5 .5 4 f 0 .1 3 4 .4 1 c 0 .0 7 * i n n u c l e i w i t h a n o v o i d s h a p e t h e g e o m e t r i c a l m e a n v a l u e w a s e s t i m a t e d , ** s e e t a b l e 4 w h e r e a = t h e l o n g e s t a x i s , b = th e s h o r t e s t a x i s probably reflecting increased cellular activity. on the other hand the total volume of pancreatic islet tissue was decreased both after antral exclusion and after antral resection. these findings do not indicate a trophic effect of antral gastrin on the exocrine o r endocrine pancreas, since similar changes were observed in the two experimental groups. when adding the percentages of the different cell types in the same group, the content exceeded 100 % for group ae and the controls. this was due to the fact that with the staining procedures used a-, b-, and d-cells are visualized in different ways. the reason for this was that the conditions for differential counting varied with staining technique, as not only the cell type in question but also other endocrine cells could appear differently depending on the back ground counterstain. nevertheless, a comparison of the frequency of a particu lar type of cell between the individual groups is considered justifiable. the only difference noted between the two experimental groups was a lower frequency of b-cells in group ar, but on the other hand, the 3-cells in this group showed an increased rate of synthesis (nuclear enlargement) and release (decreased staining reaction to insulin). this lower frequency of b-cells might, however, be misleading, since some of the more or less degranulated cells might have been overlooked in the differential counting. the reason for this appar ently altered function of b-cells after antral resection is not known. it must be emphasized than in these experiments the duodenum was excluded from continuity with the gastrointestinal tract after both surgical procedures. it is therefore possible that some duodenal factor or factors might have been involved in the pathogenesis of the observed changes in both the endocrine and the exocrine pancreas. such factors might well include hormones, e.g. chole cystokinin, secretin, somatostatin and duodenal gastrin. it has been suggested that exogenous cholecystokinin may exert a trophic effect on the exocrine pan creas (14,15), while secretin and somatostatin are believed to counteract the trophic action of gastrin (13,21). in our study the secretion of these hormones was not analysed. however, it may be assumed that the secretion of cholecysto kinin and secretin was decreased, as stimulation by intraluminal contents of the duodenum is excluded. in order to test these speculations further, studies on the possible trophic action of duodenal hormones in the rat are planned. the findings of the present study with the experimental model used do not support the view that antral gastrin has a trophic effect on the rat pancreas. acknowledgements pure hpp and anti-hpp serum were a generous gift from dr r.e. chance, lilly the expert technical assistance of miss m. kasper is gratefully acknowledged. the study was supported by grants from the swedish medical research council research laboratories, indianapolis. (nos 1 0 2 , 4534 and 2 2 9 7 ) . 253 references 1. barrowman, j.a. & mayston, p.d.: the trophic effect of pentagastrin on the pancreas. in: gastrointestinal hormones (ed. j.c. thomson), pp. 231-247, univ of texas press, austin and london, 1975. 2. chalkley, h.w.: method for the quantitative morphological analysis of tissues. j nat care inst 4:347-353, 1943. 3. enochs, m.r. & johnson, l.r.: hormonal regulation of the growth of gastro intestinal tract; biochemical and physiological aspects. in: progress in gastroenterology (ed. g.b.j. glass), vol. 111, pp3-28, grune and stratton, new york, 1977. york, 1968. mental study in the rat with special reference to gastrin and some hyper gastrinemic states. acta univ upsal, no. 296. 1978.(dissertation.) 6. hellerstrom, c. & hellman, b.: some aspects of silver impregnation of the islets of the langerhans in the rat. acta endocrinol (kbh) 35:518-532, 1960. 7. hellman, b.: methodological aspects on the differential cell count of the islet tissue in the rat. acta pathol microbiol scand [a] 45:338-346, 1959. 8. hultquist, g.: nuclear size in the cells of langerhans’ islet during star vation in the rat. acta anat (basel) 49:281-287, 1962. 9. johnson, l.r.: the trophic action of gastrointestinal hormones. gastroenterology 70:278-288, 1976. 10. larsson, l-i.: two distinct types of islet abnormalities associated with endocrine pancreatic tumours. virshows arch [pathol anat] 376:209-219, 1977. 11. lundqvist, g. & wide, l.: serum gastrin determinations with a radioimmuno sorbent technique. clin chim acta 79:357-362, 1977. 12. maske, h.: eine neue metode zur darstellung der aund b-cellen in den langerhansschen inseln des pankreas. klin wochenschr 33:1058, 1955. 13. oscarson, j., hikanson, r., liedberg, g., lundqvist, g., sundler, f. & thorell, j.: variated serum gastrin concentration: trophic effects on the gastrointestinal tract. bull 16, dept of surgery, univ of lund, lund, sweden, 1978. 14. pehrsen, h., solomon, t. & grossman, m.i.: effect of chronic pentagastrin, cholecystokinin and secretin on pancreas of rats. am j physiol. in press 1978. exocrine pancreas in rats. j surg res 22:554-560, 1977. release. studies on insulin secretion and glucose tolerance in pernicious anemia. j clin invest 58:41-49, 1976. 17. rehfeld, j.f., holst, j.j. & kiihl, c.,: the effect of gastrin on basal and aminoacid-stimulated insulin and glucagon secretion in man. eur j clin invest 8:5-9, 1978. pp. 129-171, englewood cliffs, n.j. prentice-hall inc., 1974. 4. goldman, m.: fluorescent antibody methods, p. 159, academic press, new 5. gustavsson, s.: propulsion and mixing of small bowel contents. an experi 15. reber, h.a., johnson, f. & deveney, k.: trohpic effects of gastrin on the 16. rehfeld, j.f.: disturbed islet-cell function related to endogenous gastrin 18. sternberger, l.a.: in: immunocytochemistry (ed. a.g. osler & l. weiss), 19. walsh, j. & grossman, m.i.: gastrin. new engl j med 292:1324-1334, 1975. 20. walsh, j. & grossman, m.i.: gastrin. new engl j med 292:1377-1384, 1975. 21. wiseman, d.a. & johnson, l.r.: evidence that secretion does not have direct antitrophic effect on the rat stomach. proc soc exp biol med 153:277-279,1976. received august 10, 1979 address for reprints: h. johansson, m.d. department of surgery university hospital 5-750 14 uppsala, sweden 254 upsala j med sci 82: 21-26, 1977 acute effects of a new stimulatory luteinizing hormonere1 easi n g hormone an a i og u e d-s er (tbu ) 6ea lolr h on the gonadotrophin and gonadal steroid secretion in women with amenorrhoea sven johan nillius and l e i f wide from the departments of obstetrics and gynaecology and clinical chemistry, university hospital, uppsala, sweden abstract a new stimulatory luteinizing hormone-releasing hormone (lrh) analogue o-ser(tbu)6-ea10-lrh was administered subcutaneously in a dose of 10 pg into 10 women with amenorrhoea. the injection resulted in a 20-fold increase of the lh level in blood and a 5-fold increase of the fsh level with maxima at 4 and 6 h after the administration, respec tively. there was an evident biphasic pattern of lh release similar to that described after extended pituitary stimula tion by constant infusions of lrh with early and late peaks of lh combined with a gradual, progressive fsh release. the duration of the effect on lh and fsh was at least 12 and 24 h, respectively. all the women responded with evi dent oestradiol increases in blood during the last 18 h of the 24 h study period. a comparison between the effects of a single subcutaneous injection of 10 pg of the lrh analogue and 500 pg of lrh showed that the initial fsh and lh release was similar. however, the fsh and lh release dur ing the remainder of the study period was significantly greater after administration of the analogue and so was the oestradiol increase. thus, this study in women confirms previous studies in men that o-ser(tbu)6-ea10-lrh is a potent stimulatory lrh analogue with prolonged biological activity. introduction luteinizing hormone-releasing hormone (lrh) was isolated from porcine hypothalami, structurally determined and synthesized by schally and co workers in 1971 (14). during recent years numerous analogues of lrh have been synthesized in at tempts to ( a ) develop inhibitors of lrh useful for birth control and ( b ) to find superactive stimulatory analogues with prolonged biological activity (15, review). such stirnulatory analogues should be more useful for therapeutic use than lrh itself, which has effects on the gonadotrophin secretion of short duration. the plasma clearance of exogenous lrh is very rapid with initial half-life of 4 min (2, 10). present therapeutic regimens for treatment of hypogonadism in men and women therefore require 8-hourly injections of large doses of lrh over pro longed periods of time to be effective (6, 8, 9). there is need for a superactive lrh analogue with prolonged activity for simplifying treatment with lrh . in the present study, acute effects of a new stimulatory lrh analogue d-ser(tbu)6-ea10-lrh (hoechst 766) on the gonadotrophin and gonadal steroid secretion were investigated in ten women with amenorrhoea. in five of these women the ef fects of the lrh analogue were compared with those obtained after a large dose of synthetic lrh. patients and methods patients. ten 20-29-year-old women with amenorrhoea volunteered for this study. nine of them had secondary amenorrhoea of at least 2 years' duration, while one woman had primary amenorrhoea due to kallman's syndrome (hypogonadotrophic hypogonadism with anos mia). in seven of the women the amenorrhoea had oc curred in a setting of self-imposed weight loss. four of them had regained weight while three women were still underweight at the time of study. all the women had normal x-rays of the skull and pituitary fossa and their thyroid, adrenal, renal and hepatic function was normal. the patient with kallman's syndrome was found to have a moderate hyperprolactinaemia (26 p g per i) while all the other women had normal prolactin levels in serum. the gonadotrophin levels in serum were low or normal and the endogenous oestrogen production was low. lrh treatment. the lrh analogue o-ser(tbu)6-ea10 lrh (hoechst 766) was administered subcutaneously in a dose of 10 p g to all the women. venous blood sam ples were obtained before and during the first 24 h after l ' ~ \ < i / < i j m c d s 1 182 22 s . j . nillius and l . wide . l 3 2 1 -0 arnenorrhoelc w m n n:10 i& d-serltbu)6-eaa-lrh sc i m i s e m -3,n3/1 2 . 1 .o o j t . . . . . . . . . . . . . , . , . , . . . . . , 0 2 l 6 8 m fz u 16 b 20 22 2 l h fig. i . mean serum levels of lh and fsh before and after subcutaneous administration of 10 p g of d s e r ( n u ) ' ea'o-lrh into 10 women with amenorrhoea. the administration and assayed for fsh, l h , oestradiol and progesterone. five of the women were also studied by hormone assays before and after subcutaneous administration of 500 p g of lrh (hoechst) which was given either one month before or after the administration of the analogue. twenty-four hours before the administration of lrh or the analogue, the pituitary reserve capacity for gonadotrophin secretion was tested with 100 p g of lrh (7). hormone assay methods. immunoreactive fsh and lh in serum were assayed by a radioimmunosorbent technique with indirectly coupled antibodies (17). lh in serum was measured by utilizing human pituitary lh (12) labelled with lz5i and rabbit anti-human pituitary lh. the lh preparation had a biological activity of 9400 i u (2nd irp-hmg) per mg. fsh in serum was measured by utiliz ing human pituitary fsh ( 1 i ) labelled with 1251 and guinea pig anti-human pituitary fsh. the fsh preparation had a biological activity of 12000 iu (2nd irp-hmg) per mg. the results are expressed in pg per 1. immunoreactive oestradiol in serum was measured by a radioimmunological technique using an antiserum to an oestradiol-6-oxime-bsa conjugate ( 5 ) . progesterone was assayed by a similar radioimmunological technique. statistical methods. the hormone values were trans formed into logarithms in the statistical calculations. the mean values given in the text are geometric means. for calculation of differences between mean values, formulas based on the student's t-distribution were used. results mean lh and fsh levels in serum before and after subcutaneous administration of 10 p g of d-ser( t13u)6-ea10-lrh intor the ten women with amenorrhoea are shown in fig. 1. the lh and fsh results from the preceding intravenous lrh test are shown in fig. 2. upsulu i med sci 82 the mean pretreatment lh level (0.18 p g per i ) was below the normal range for women of ,fertile age. all the women responded to the lrh analogue with a rapid increase of the lh level in blood with a first peak (2.7 pg per 1) at 45-60 min after the administration. the lh level remained unchanged for the next hour and then increafed again to a second peak with the highest mean value (3.7 pg per 1) at 4 h after the administration. after that the lh level slowly declined. the mean lh level was still significantly elevated over the pretreatment level at 12 ( p < o . o o l ) but not at 24 h (p>0.05) after the administration of the analogue. the mean pretreatment fsh level (0.60 pg per i ) was within the lower normal range for women of fertile age. fsh progressively increased after the injection of the lrh analogue and reached a peak (3.1 p g per i) at 6 h after t h e administration. then the fsh level slowly declined. the mean fsh val ues at 12 (1.9 p g per 1) and 24 ( i . 1 p g per i ) were significantly higher (y<o.ool) than the mean pre treatment value. the mean oestradiol level in serum before and after administration of the lrh analogue is shown in fig. 3. the mean pretreatment level (162 pmol per 1) was similar to that found in the early follicular phase of the menstrual cycle. all the women re sponded with evident oestradiol increases in blood after 6 h with maximum values of between 230 and 1930 pmol per 1 (mean 610) at 12 h after the administration of the analogue. the mean oestradiol level at 24 h after the administration (405 pmol per 1) was 2 . 5 times higher than the pre treatment level. the mean progesterone levels be fore and at 12 and 24 h after the administration of the analogue were low (1.3, 1.4 and 1.2 nmol ? 0 i h f i g . 2. lh and fsh results from intravenous lrh tests in the 10 amenorrhoeic women before the administratiop of the lrh analogue. acute effects of a lrh analogue in amenorrhoea 23 amenorrtwcic m e n o j , , , , . . , , , , , . . . , , , , , . , , . , , l o 0 2 4 6 8 10 12 y 16 b 20 22 2 l h rag. 1.1 ti 1 2 nn-d/l f i g . 3 . mean oestradiol (e,) levels in blood before and after administration of 10 pg of d-ser(tbu)6-ea'o-lrh into 10 women with amenorrhoea. per 1, respectively) and did not differ significantly (p >0.05) * a comparison between effects on the gonado trophin secretion of subcutaneous administra tion of 10 p g of d-ser(t13u)6-ea10-lrh and 500 p g of lrh is i l h t r a t e d in fig. 4 , which shows mean l h and fsh results from the five women who were given both lrh and the analogue on separate occasions. it can be seen from the figure that the results from the preceeding intravenous lrh tests did not differ on the two occasions. after administration of 500 p g of lrh the lh level rapidly increased to a peak 45 to 60 min later. the lh level remained essentially unchanged for about four hours and then slowly declined. l h was still significantly higher than the pretreatment level at 8 h (p<0.02) but not at 12 h after the administra tion (p>0.05). the injection of the analogue re sulted in a similar initial l h increase in blood, later followed by a second increase to a peak at 4 h after the administration. the lh level then slowly de clined. increased l h levels were observed at 12 (p<o.001) but not at 24 h after the administration of the analogue (p>0.05). the fsh level progressively increased in similar fashion after injection of lrh and the analogue. peak levels were reached after 4 and 6 h, respective ly. the fsh levels at 6, 8, 12 and 24 h after the administration of the analogue were significantly higher @<0.001) than those obtained after 500 p g of lrh. there were no significant differences (p>0.05) between the mean fsh levels during the first 4 h after the administration. the mean oestradiol level in blood was signifi o j , , ~ , , . , . . . , , , , , . . . , . . . . . . t o x x x p. ,' ' \ -600 .ym f i g . 4. a comparison between effects of 10 fg of d-ser(tbu)6-ea'u-lrh and 500 pg of lrh on l h , fsh and oestradiol (e,) secretion in 5 women with amenorrhoea. results from the preceding intravenous lrh tests are shown to the left on the figure. x xp<0.01, x x xp<0.001. .3a) xx) , , . i 0 2 l 6 8 10 12 k 16 1 8 2 0 2 2 t h upsulu j med sci 82 24 s . j . nillius and l . wide l h lrh -w lrh s.c. lh m iv 0 0 1 0 p g o-*(tbu)-ea'-lrh s c m" l 4 p i 3 2 1 0 m prol/l -m .50 .uxl -33 ..m € 2 f i g . 5. lh, fsh and oestradiol (e2) levels before and after administration of 10 p g of d-ser(tbu)6-ea10-lrh and 500 k g of lrh in a 28-year-old woman with amen oj 1 0 orrhoea. cantly increased ( p < o . o o i ) over the pretreatment level at 6 h after the administration of both lrh and the analogue. peak levels were reached after 6 and 12 h respectively. the oestradiol levels at 12 and 24 h after administration of the analogue were significantly higher ( p < o . o o l ) than those obtained after 500 p g of lrh. the mean oestradiol level at 24 h after administration of 500 p g of lrh was not significantly different (p>0.05) from the pretreat ment level. there was no increase of the low pre treatment progesterone level at 12 and 24 h after the administration of either the analogue or lrh. blood levels of l h , fsh and oestradiol after administration of the analogue and lrh to an indi vidual patient with amenorrhoea are shown in fig. 5 . the gonadotrophin responses to intravenous lrh were similar on the two occasions. the sub cutaneous injection of 500 p g of lrh resulted in a rapid lh increase to a peak at 45 min after ad ministration followed by a plateau-like level for 2-3 hours and then a decline down to the pretreatment level which was reached at 12 h after the adminis tration. the initial lh peak reached at 30-60 min after subcutaneous injection of the analogue was lower than obtained after 500 p g of lrh. however, u p ~ a l o j med sci 82 the lh level after the analogue injection increased further after 2 h and reached a second peak at 4-6 h after the administration followed by a slower fall down to basal levels. the fsh increase after subcutaneous administra tion of 10 p g of the analogue was more pronounced than after 500 p g of lrh with a broad peak at 6-8 h and higher blood levels throughout the rest of the 24-hour-period. the oestradiol increase observed after injection of the analogue was also greater than that obtained after lrh (fig. 5). n o side-effects were observed after the injection of lrh or the analogue. one of the women fainted in connection with venipuncture 3 h after adminis tration of the analogue. discussion this study shows that the new lrh analogue d-ser(tbu)6-ea'o-lrh was capable of increasing the gonadotrophin secretion in amenorrhoeic wom en who had very low basal l h and low normal fsh levels. a single subcutaneous injection of 10 p g of the analogue evoked a 20-fold increase of the l h level in blood and a 5-fold increase of the fsh acute effects ofa lrh analogue in amenorrhoea 25 level with maxima at 4 and 6 h after the administra tion respectively. this analogue has previously proved to be high effective in raising gonadotro phin secretion in experimental animals and normal male volunteers (3, 13, 18). wiegelmann et al. (18) found significant l h increases after injection of 1 and 2.5 p g and dose-dependent lh increases after 5 and 10 p g of the analogue with peaks at 30 min after intravenous administration into normal men. fsh increases were only observed after the 5 and 10 pg doses with peaks after 120 min (18). kuhl et al. (3) found that a subcutaneous injection of 5 p g of the analogue was as effective as 100 p g of lrh. how ever, the analogue had a depot effect and the sub cutaneous dose of 5 p g was effective for 6-8 h in normal men (3). in the amenorrhoeic subjects of the present study the duration of the effect on fsh was at least 24 h and the lh level remained elevated over the basal level for at least 12 h after a single subcutaneous injection of 10 p g of the analogue. there was an evident biphasic pattern of lh release after the single injection of the lrh ana logue with early and late peaks of lh combined with a gradual, progressive fsh release. this pat tern of gonadotrophin release is similar to that de scribed by bremner & paulsen ( 1 ) after continous intravenous infusions of lrh into normal men. the results from their study suggested the existence of two functional pools of l h in the human pituitary, one that is acutely releasable and another that re quires longer stimulation to be released (1). in wom en, this biphasic pattern of lh response to ex tended stimulation by constant infusion of lrh is most evident during the early follicular phase of the cycle (16). the amenorrhoeic subjects had oestrogen levels similar to those found in the early follicular phase of the cycle. when they were given a single injection of the large dose of 500 p g of lrh, there was a pi onounced first initial peak of acutely releaseable lh followed by continued l h release for the next 3 4 hours but no evident second peak of lh release was observed in contrast to what was found after administration of the analogue. a single injection of 10 p g of the lrh analogue resulted in a comparable initial lh peak but then it also evoked a great re lease of l h from the second pool. this pool may be regarded as a pituitary reserve with a component of yet unmeasurable amounts of newly synthesized gonadotrophins (4). the great activation of the sec ond pool by the single dose of the lrh analogue shows that it exerts a more extended pituitary stimulation than a single large dose of lrh. this prolonged action of the lrh analogue may be due to a better binding to the pituitary receptors, a slower inactivation or a combination of both factors ( 1 5 ) . the gonadotrophins released into the blood by the lrh analogue stimulated the ovaries to an in creased steroid secretion and all the women had evident oestradiol elevations in blood during the last 18 hours of the 24 h study period with maximum at 12 h after the administration. the gonadal re sponse was more pronounced after administration of the analogue than after the large dose of lrh, another illustration of the more prolonged and po tent action of the lrh analogue. thus, the present study in women confirms pre vious studies in men and shows that d-ser(mu)' ea'o-lrh is a potent stimulatory lrh analogue with prolonged biological activity. the marked acute effects on the gonadotrophin and gonadal steroid secretion in amenorrhoeic women suggest that this lrh analogue will be useful therapeuti cally. it might be possible to simplify the present therapeutic regimens by using this potent and long-acting lrh analogue for chronic lrh treat ment. acknowledgements this study was supported by the swedish medical re search council (grant no. 13x-3145). we are indebted to dr mrs m. von der ohe, farbwerke hoechst ag, frankfurt (main) for generous supply of the lrh analogue and lrh, to professor paul roos, at the institute of biochemistry for supplying the highly puri fied fsh and lh preparations and to mrs anna-lena barmark, mr christer bengtsson, mrs birgitta bohman, mrs rose-marie lindquist and mrs ann sandberg for skilful technical assistance. references bremner, w. j . & paulsen, c. a,: two pools of luteinizing hormone in the human pituitary: evidence from constant administration of luteinizing hormone releasing hormone. j clin endocrinol metab 39: 81 1 , 1974. keye, w. r., kelch, r . p., niswender, g. d. & jaffe, r. b.: quantitation of endogenous and exogenous gonadotropin releasing hormone by radioimmunoas say. j clin endocrinol metab36: 1263, 1973. k u h l , h., kaplan, h.-g. & taubert, h.-d.: die wirkung eines neuen analogs des lh-rh, d-ser(tbu)6-ea1u-lh-rh, auf die gonadotropin freisetzung bei mannern. dtsch med wschr 101: 361, 1976. 26 s. j . nillius and l . wide 4. lasley, b. l., wang, c. f. & yen, s. s. c.: the effects of estrogen and progesterone on the functional capacity of the gonadotrophs. j clin endocrinol metab41: 820, 1975. 5 . lindberg, b. s., lindberg, p., martinsson, k . & johansson, e. d. b . : radioimmunological methods for the stimulation of oestrone, oestradiol-17 p and oestriol in pregnancy plasma. acta obstet gynecol scand, suppl. 32: 5, 1974. 6. mortimer, c. h., mcneilly, a . s., fisher, r. a , , murray, m. a. f. & besser, g. m.: gonadotrophin releasing hormone therapy in hypogonadal males with hypothalamic or pituitary dysfunction. br med j 4: 617, 1974. 7. nillius, s. j. &wide, l.: the lh-releasing hormone test in 31 women with secondary amenorrhoea. j ob stet gynaecol br commonw 79: 874, 1972. 8. nillius, s . j. & wide, l.: gonadotrophin-releasing hormone treatment for induction of follicular matura tion and ovulation in amenorrhoeic women with ano rexia nervosa. br med j 3: 405, 1975. 9. nillius, s. j., fries, h. &wide, l.: successful induc tion of follicular maturation and ovulation by pro longed treatment with lh-releasing hormone in women with anorexia nervosa. am j obstet gynecol 122: 921, 1975. 10. redding, t. w., kastin, a. j., gonzalez-barcena, d., coy, d. h., coy, e . j., schalch, d. s. & schally, a. v.: the half-life, metabolism and excretion of tritiated luteinizing hormone-releasing hormone (lh rh) in man. j clin endocrinol metab37: 626, 1973. 11. roos, p.: human follicle-stimulating hormone. acta endocrinol, suppl. 131: 1, 1968. 12. roos, p., nyberg, l., wide, l. & gemzell, c.: human pituitary luteinizing hormone. isolation and characterization of four glycoproteins with luteinizing activity. biochim et biophys acta405: 363, 1975. 13. sandow, j., koenig, w. & rechenberg, w. v.: en docrine activity of a highly active analogue of lh rh. v international congress of endocrinology, hamburg, july 18-24, 1976, abstract 51. 14. schally, a. v., arimura, a., kastin, a. j., matsuo, h., baba, y., redding, t. w., nair, r. m. g . , de beljuk, l . & white, w. f.: gonadotropin-releasing hormone: one polypeptide regulates secretion of luteinizing and follicle-stimulating hormones. science 173: 1036, 1971. is. schally, a. v . , kastin, a. j. & coy, d. h.: lh releasing hormone and its analogues: recent basic and clinical investigations. int j fertil21: 1 , 1976. 16. wang, c. f., lasley, b. l., lein, a. & yen, s. s. c.: the functional changes of the pituitary gonadotrophs during the menstrual cycle. j clin en docrinol metab42: 718, 1976. 17. wide, l., nillius, s. j., gemzell, c . & roos, p.: radioimmunosorbent assay of follicle-stimulating hormone and luteinizing hormone in serum and urine from men and women. acta endocrinol, suppl. 174: 1, 1973. 18. wiegelmann, w . , solbach, h . g., kley, h. k . , nieschlag, e., rudorff, k . h. & kriiskemper, h. l.: effect of new lh-rh analogue (d-ser(tbu)' ea"'-lh-rh) on gonadotrophin and gonadal i secretion i n men. hormone res 7: 1 , 1976. received februury 2, 1977 address for reprints: sven johan nillius, m.d. department of obstetrics and gynaecology university hospital s-750 14 uppsala 14 sweden ;teroid u m u l a j mrd s c i 82 upsala j med sci 79: 116-128, 1974 brain growth in children with marasmus a study using head circumference measurement, transillumination and ultrasonic echo ventriculography g u n n a r e n g s n e r , 2 s h o a d a g n e b e l e t e , ' i r e n e sjogren2 and bo vahlquist' from the ethiopian nutrition institute, addis a b a b a , ethiopia, i and the department of pediatrics, university hospitals2 u p p s a l a , sweden abstract brain growth was studied by making simultaneous meas urements of head circumference, transillumination and lateral ventricle indices in 102 children aged 2-24 months suffering from marasmus. the head circumference was significantly reduced, transillumination showed a slight to-moderate increase in the children 6-24 months of age, and echo encephalography showed a normal lateral ven tricle index. the results indicate a reduction of brain size which (particularly after the first 6 months of age) goes slightly beyond what may be inferred from the head circumference per se. the interpretation of the results, especially the relation between head circumference and brain size, is discused. in cases of severe protein-calorie malnutrition (pcm) of t h e marasmus type, there is not only a severe reduction of weight in relation to age but also a retardation of height in relation t o age, i.e. a true stunting of growth (4, 23). furthermore, studies in recent years have indicated a marked retardation of brain growth, demonstrated both in vivo by measuring head circumference (15, 24) and a t autopsy b y measuring brain weight ( 1 , 37). head circumference is claimed t o reflect brain size fairly closely (35, 40). this is not always true, however. in cases of severe p c m , alterations in the thickness of t h e scalp and t h e skull bone may cause changes in t h e head circumference/brain size ratio ( 1 1 , 28). this ratio may b e further changed b y abnormal accumulation of fluid on t h e surface of t h e brain (30) o r by enlargement of t h e brain ventricles (38). a s part of t h e work done a t the ethiopian nutrition institute (eni), addis ababa, various studies related t o brain growth in young children were carried o u t between 1969 and 1972. the aim of the study t o be presented in this article was twofold: ( i ) to measure the brain size in marasmic in fants and children by simultaneously recording the head circumference and performing transillumina tion and e c h o encephalography. (2) to demonstrate whether o r not, in infants with marasmus aged less than six months, a re cordable improvement in brain size takes place during nutrition rehabilitation. m a t e r i a l definition of marasmus the criteria used for including children in the study were as follows: ( a ) weight for age below 60% of the boston standard (50% percentile) and no apparent oedema, i.e. the de finition of marasmus adopted at the jamaica conference in 1969 (6). ( b ) no signs of any serious disease which could in itself be a major cause of severe malnutrition. chil dren with diarrhoea (arbitrarily defined as more than three loose stools a day) were also excluded. organization of the study and number of children examined the total number of children with marasmus examined was 102. the age and sex distribution and the place of examination are given in table i. a cross-sectional study was devoted to analysing the situation in the total material of 102 children, aged 2-24 months, before any medical or dietary rehabilita tion took place. each examination included in principle anthropometric and clinical observations, transillumina tion and ultrasonic echo ventriculography. a longitudinal study was carried out by performing follow-up examinations of the 53 children aged 2-6 months who were recruited fi-om the lidetta mother and child health (mch) centre in the bole area of addis ababa. each examination included in principle anthropo metric and clinical observations, transillumination and ultrasonic echo ventriculography . the interval between upsala j med sci 79 brain g r o w t h in chilurrn w i t h rnarasrnus i17 table i . a g e , s e x d i s t r i b u t i o n a n d p l a c e of e x a m i n a t i o n . c h i l d r e n w i t h rnarasmus boys girls age, months age, months place of examination n 2-3 4-5 6-8 9-11 12-14 15-17 18-24 n 2-3 4-5 6-8 9-11 12-14 14-17 18-24 total lidetta mother and child 53 healthcentre 24 10 12 2 2 9 1 7 8 4 nutrition re habilitation clinic,espc 2 9 5 4 8 6 6 2 0 1 6 4 3 6 49 total 5 3 1 0 1 2 7 4 8 6 6 4 9 1 7 8 5 6 4 3 6 102 two examinations was routinely i monthk7 days. on a few occasions (four in all) transillumination and echo ventriculography could not be performed and the next complete examination then usually took place 1 month later. the aim was originally to make at least three follow up studies of each of the 53 children. not unexpectedly, however, this proved impossible. ten of the children dropped out even after the first examination (fatal out come, home problems, unco-operative mothers). an other 14 were re-examined only one or two times. the majority of the children-29-were, however, re examined five times or more, in some cases up to nine times. ages of the children records containing accurate information concerning dates of birth were available for 28 out of 53 of the children aged less than 6 months (all attending the lidetta mch centre). for the 49 children aged 6-24 months (the majority attending the nutrition rehabilita tion clinic at the ethio-swedish pediatric clinic (espc) in addis ababa), birth records could be obtained in only 6 cases. for the children without birth records, we had to rely on information given by the mothers concerning the date of birth. thanks to the existence of a fairly de tailed religious calender in ethiopia, the birth dates of the majority of the children could be reconstructed with considerable accuracy. cross-checking, when pos sible, indicated that the information was correct to the week, often even to the day. it may be expected that a discrepancy between re corded age and true age will become greater with in creasing age. since, in this study, as in some others in this series, the main focus was on a limited age group ( c 2 4 months), the errors are, in most cases, probably small, although in individual cases larger aberrations cannot altogether be excluded. birth weights of the children. children with low birth weights ( ~ 2 5 0 0 g) represent a fairly large proportion of all the children born in de ’ veloping countries (39). they also represent a consider able proportion of all the children who have low weights for age during the first few months of life. in order to eliminate as far as possible this category of children, the following measures were taken: ( a ) no children under 2 months of age were in cluded in the series. ( b ) no children with available birth records indi cating a birth weight g2500 g or representing the out come of a twin pregnancy were included. feeding pattern n o attempt was made to record a detailed dietary history in every case. in most population groups in ethiopia, prolonged breast feeding-often up to ages of 18-24 months-is stiu the custom (19). this is true also of the area in addis ababa in which the non privileged families of our study lived. for the children below 12 months of age, the marasmic disease could primarily be attributed to “starvation at the breast”; for the older children lack of suitable weaning foods played a major role. in some of the children, earlier diseases (repeated diarrhoea etc.) contributed to their marasmic condition. socio-economic background the families to which the children with rnarasmus be longed, came throughout from the non-privileged stra tum which has been defined in another publication of this series (8). in brief, this means an income below often far below-us $13 per month, a period of educa tion for the parents which did not exceed 2 years (the vast majority were illiterate), poor lodgings, very primi tive living conditions and a high frequenty of unemploy ment and broken homes. nutrition rehabilitation program each of the 53 children aged 2 6 months included in the longitudinal study were also included in a nutrition rehabilitation program on an out-patient basis. the pro gram comprised regular check-up, nutrition education and free distribution of an infant formula based on full fat milk powder (“baby faffa”, produced at the ethiopian nutrition institute). each mother received 1.5 kg of baby faffa once a week. this amount was upsala j m e d sci 79 i18 g. engsner e t al. fig. i . an ordinary transillumination lamp (oculus) ( a ) with a black rubber adapter ( b ) fixed to the rub liberal also taking into account the fact that some of the severely malnourished children needed at least 150 calo ries and 3 g of protein per kilogram of body weight and 24 hours. the mothers were carefully instructed in how to feed the baby, using either a cup or a carefully cleaned bottle. methods all the children were examined by one and the same pediatrician (g. e . ) . interviews with the children’s guardians, mostly their mothers, were carried o u t with an ethiopian nurse or health officer acting as inter preter. the nurse or health officer also acted a s an assistant at the examinations. a n f h r o p o m e t r y . standard anthropometric data, in cluding body weight, length, arm circumference, head circumference and skinfold thickness (triceps), were re corded. for the methodological details, see w h o mono graph series no. 53 (17). scales and tapes were re gularly checked. a harpenden caliper was used. h e a d circumference was of particular importance in these studies. great care was taken to obtain accurate and reproducible results. the measurement was made her rim held against the baby’s head, to which rim is attached a circular scale plate ( c ) (34). with a steel tape to the nearest 0.1 cm. the tape was placed so as to measure the greatest occipito-frontal circumference. transillirminarion. the transillumination examinations were performed by using a transillumination lamp of the oculus type with a small 25-watt (7.5 v) lamp and a point scale fixed to the rim by a black rubber adapter (fig. 1 ) . the results of the examinations were ex pressed in scale points, according to the method de scribed by sjogren & engsner (34). the examinations were performed in a totally dark ened room after the examiner had adapted to darkness for 3-5 minutes. as a routine, all infants were examined over the fronto-temporal and parieto-occipital regions, on the right as well a s the left side of the head. in a minority of cases, a slight difference between the two sides was observed, but i t never exceeded 0.5 scale points. if a difference was noted, the mean value of the two sides was used. normally, newborn infants should transilluminate fronto-temporally up to scale point 2 or less, and parieto-occipitally up to scale point 1 or less. children aged more than 12 months should not illuminate the scale plate at all (34). ultrasonic e c h o ventriculography. the size of the lateral ventricles was measured by ultrasonic echo (31, wpsala j m e d sci 79 brain growth in children with marasrnus 119 i = t / d i,=m/d f i g . 2. schematic pneumogram compared wirh enlarged schematic echo ventriculogram, where m e is the mid line control, dx the echo encephalogram from the right, and s i n an inverted echo encephalogram from the left temporal region. the position of the lateral ventricle echoes correspond to the lateral surface of the lateral ventricles, and the echo-free zones to the widths of the lateral ventricles seen in the pneumogram (3 1). 32). the echo encephalograph used was a siemens apparatus (kraut-kramer system) with a polaroid land camera for instant recording of the oscillographic trac ings. the probe used had a frequency of 2 megacycles per second and a diameter of 24 mm. liquid paraffin was used a s a contact medium between the head and the probe. the head was not shaved prior to the ex amination. the summarized width of the right and left lateral ventricles (t) was expressed in relation to the dia meter of the child’s head ( d ) a s a lateral ventricle index ( 2 ) (fig. 2). two or more echo ventriculograms were regularly photographed; if there was a slight difference between them, the mean value of the lateral ventricle indices was recorded. normally this index should not exceed 0.33 or 33 7% ( m + 1 s.d.) of the diameter of the head in newborn babies and 0.29 in children aged 12 months (33). discussion of the methods some sources of error in the interpretation of the re corded data should be discussed. as regards the in fluence on head circumference measuremenis of the variation in thickness of scalp and skull bone, this question is dealt with at some length under “discus sion”. the same factors (thickness of scalp and skull bone), a s well a s the degree of mineralization of the bone and the thickness of the hair covering, may exert some influence on the outcome of transillumination. severe pcm may in itself cause, not only thinning, but also alteration of the mineralization of the bone (14). if rickets is also present, it may produce further de mineralization and thinning of the bone. in a separate study it could be demonstrated that florid rickets with marked craniotabes may increase transillumination by 0.5-1.0 scale points (10). however, pronounced rickets is said to be uncommon in marasmic children with stunted growth and this proved true also in our ma terial. dodge & porter (5) made the observation on autopsy material the transillumination of the skull bone, as such, will disappear at a thickness of 6 mm but that, when scalp and skull bone are examined together, it seems to disappear at a thickness of the skull bone of 2.5-3 mm. according to roche (29), the skull bone thickness in the naseon region will reach 6 mm around 18 months of age, whereas in other regions the thick ness of the bone at this age amounts to only 2.5 3.0 mm. these observations would indicate an age limit for transillumination in practical work at 18 months rather than at 12 months, which is the figure most frequently mentioned. as we shall see in the present material a s well as in a material of children with kwashiorkor (9), trans illumination, often of abnormal degree, could be ob served in children up to 24-36 months of age. in directly, this is a proof that the translucency of the integuments due to the thinning of tissues (in cases of kwashiorkor perhaps oedema) and the demineralization of bone must have been of common occurrence. as for the hair covering, this is, on an average, thicker in an ethiopian than in a swedish infant but shows the same temporary thinning a couple of weeks after birth. most ethiopian families, apart from the privileged ones, still practice shaving the heads of their childern from the time of baptism, i.e. at 4 weeks for boys and 6 weeks for girls. recordings in a limited number of infants of transillumination before and after hair shaving indicated an effect of the order of 0.3 scale points (10). the lateral ventricle index measured by ulrrasonic echo ventriculography is less influenced by the factors mentioned. pronounced thinning of scalp and bone was estimated to give a maximum index deviation of 0.01 0.02 only. results cross-sectional study anthropometry. the data are presented in table i i a . in addition to absolute figures, the percent age standard (17) is also given routinely for two parameters (weight/age and length/age). cor upsala j m e d s c i 79 120 g. engsner et al. table i1 a . anthropometn‘c data. children with marasmus included in the cross-sectional study age arm cirtriceps groups weight weight/age length length/age weight/length cumference skinfold (months) n (kg) % standard (cm) % standard 5% standard (cm) (mm) ~ ~~~ ~~ 23 27 3.02 56 [0.54? 1121 45 20 3.79 56 [0.72] [i21 68 4.20 52 co.641 1 91 9-1 i 10 5.04 54 [1.@1 [ i l l 12-14 12 5.34 53 11.261 1131 15-17 9 6.05 56 [1.12] [ i l l 18-24 12 6.68 55 11.341 1 81 54.0 14.41 55.5 16.21 60.0 15.81 61.2 16.21 64.2 [5.81 66.0 16.21 67.1 [6.61 92 87 89 85 85 83 78 “21 [ i 11 [ 91 [ 91 1101 81 1101 8.2 8.5 8.0 9.2 8.5 8.8 8.6 [ i .41 11 .21 11.81 12.41 12.61 1 3 ~ 1 12.81 4.6 4.4 4.0 3.8 4.0 4.4 4.8 12.41 p . 0 1 12.41 p.01 12.21 p . 1 1 11.41 a figures in brackets are 2 s.d. responding to the mode of selection (weight/age below 60% of standard; cf. above), the emaciation and the stunting of growth, as evidenced from the mean values for weight, length, head circum ference, arm circumference and skinfold thickness, are very pronounced. head circumference. mean values and standard deviations for head circumference i n relation to age are given in table i l b , fig. 3 a and 3b. these figures include, as background information, the mean values and distribution for head circum ference/age in healthy swedish children (18). the mean values for head circumference/age for the marasmic children lie at or slightly below m -2 s.d. the variation is considerable, however. even in the youngest age group, the deviation from normal is pronounced. in absolute values, the difference between means varies from 3.4 cm (age group 2-6 months, both sexes combined) to 2.8 cm (age group 18-24 months, both sexes com bined). transillumination. the results are presented in fig. 4. the controls were non-privileged ethiopian children with no or only mild pcm (8). the character of the control group means that the deviations observed in the children with marasmus are, if anything, slightly minimized. only in the age groups above 6 months there is a clearcut tendency to increased values. thus, as regards the values of fronto-temporal recordings, the differences between the marasmus group and the control group for the age groups 6-11, 12-17 and 18-24 months are highly significant (p<0.001) and, as regards the values of parieto-occipital re cordings, these differences in the age group 6-1 1 . 12-17 and 18-24 months are not significant (p>0.05). ultrasonic echo ventriculography. a positive identification of the lateral ventricle echoes was obtained in all of the 102 marasmic children ex amined. the results are presented i n fig. 5 . it is obvious that the marasmic state does not i n any way cause a deviation of the lateral ventricle in dex, thus implying that there is no change in lateral ventricle width in relation to head diameter. also, when a correlation for the abnormally low head circumference/age is made (using “head circumference age” (16) rather than chronological age), the means for the lateral ventricle index d o not show any significant deviation from normal. longitudinal study anthropometry. the anthropometric data, ex pressed as means of the percentage standard, are given in fig. 6 . in addition to group observations, four individ ual cases are also briefly presented with respect to changes in anthropometric data during nutrition rehabilitation (fig. 7). they are chosen so as to upsala j med sci 79 brain growth in children with marasmus 12 1 scale pm table i1 6. head circumference in relation to age. boys and girls separately age group (months) n boys n girls head circumference (cm) 1 wiem occifitauy 23 10 36.0 45 12 37.6 6 8 7 41.1 [2.4] 9-1 1 4 42.0 l2.8p 13.21 ~ 3 . 2 1 12-14 8 43.8 ~ 3 . 2 1 15-17 6 44.4 18-24 6 45.2 l2.81 l2.81 17 35.2 l2.21 8 38.0 12.21 5 40.6 6 41.6 4 43.1 l2.01 3 44.0 l2.41 6 44.6 l2.61 ~ 2 . 4 1 ~ 2 . 4 1 ~ figures in brackets are 2 s.d. represent various types with respect t o the suc cess of nutrition rehabilitation. case b . y . a girl, first child of a 17-year-old mother married to a labourer. delivery uncomplicated, birth weight 2950 g. breast fed, no other food given. the father had been without income for the last 2 months. the mother stated that the family actually earned a t most 10 us cents twice a week. she was favourable to the rehabili tation work and attended regularly. cuse s. w. a girl, third child of a 21-year-old mother. delivery uncomplicated, birth weight 3 150 g. breast fed, no other food given. the mother had earlier worked a s a bar head . crcum. 8 ference. . cm 50 /--/ swedish + 2 9 children m lo (eqys) 2 5 0 marasmus *2sd "a ~~ i " ' , ' 6 12 18 age. months f i g . 3. ( a ) head circumference related to age. children with marasmus compared with normal range (18). cross ( sectional study. boys only. t i l . l l , l l l l . ' i 6 18 ap,monihs 12 fig. 3. ( b ) head circumference related to age. children with marasmus compared with normal range (18). cross sectional study. girls only. girl but was now out of work, owing to the birth of the last child. she lived with a girl friend who worked at the same bar. the mother attended the nutrition rehabilitation program fairly regularly but probably gave some of the food to the other two children. case j . w . a boy, the second child of a 19-year-old unmarried woman. delivery uncomplicated, birth weight 2850 g. breast fed, no other food given. the mother's first child died at the age of 1 year when the mother was 18 years old. the mother lived with the child at a relative's house. the head of the extended family was a labourer, with a monthly income of about 5 us$. the mother was one of the most active members of the nutrition rehabilita tion group. cuse w . h . a boy, the first child of a 23-year-old, deserted mother. delivery uncomplicated, birth weight 3 100 g. breast fed, no other food given. the mother attended the nutrition rehabilitation program somewhat irregularly and was front0 temporplly scale p 3 s s i i i i i -0 marasmus m22sd fig. 4 . head transillumination related to age. children with marasmus compared with a control group of ethio pian children (8). cross-sectional study. boys and girls combined. upsala j med sci 79 122 g . engsner et a!. lateral ventricle index i a s /normal values :z 1 fig. 5 . echo encephalography. lateral ventricle index ( i ) related to age. children with marasmus compared to normal range (33). cross-sectional study. boys and girls combined. known to sell some of the food given to a neighbour. it was not possible to control or stop this practice. the mother had no other income. h e a d circumference. t h e results a r e presented in fig. 8 a and 8 6 . t h e s e figures include, as background information, t h e mean values and distribution for head circumference in relation t o age in healthy swedish children (18). the deviation of head circumference/age (fig. 8 a and 8 b ) from normal was less marked in t h e longitudinal group than in t h e larger cross-sectional o n e (excess mortality in children with t h e severest marasmus, who therefore were underrepresented in the longitudinal study). a slight tendency t o catch u p in head circumference during nutrition rehabilitation could be observed but was not very kdod]l 100 3 6 9 age, months 40 a weighuage w w w l e n g t h a length/@ 0 arm circumlerence/age fig. 6 . anthropometric data expressed as means of percentage of standard (17). children with marasmus. longitudinal study of 29 cases examined at monthly intervals during nutrition rehabilitation. boys and girls combined. impressive. this general rule was not without ex ceptions, however. in two of t h e individual cases (fig. 7 , b. y . and j. w . ) with better than average effects of nutrition rehabilitation, t h e catch up in head circumference w a s reasonably good. t ~ a n s j f l u ~ i n a t i o n . t h e results of t h e follow-up examinations a r e presented in fig. 9, which gives t h e means and standard deviations. t h e mean values d o not differ significantly from those ob served in non-privileged ethiopian children with n o o r only mild pcm (8). t h e r e is a slight tend ency f o r t h e values of t h e children with marasmus t o move towards zero faster t h a n in the group given f o r comparison. ultrasonic echo ventriculography. the results of the follow-up examinations are presented in fig. 10. as was t h e case with t h e larger, cross-sec tional material, t h e initial values come very close t o those observed in ethiopian children with n o or mild p c m (8) and in swedish children (33). dur ing t h e follow-up period of nutrition rehabilitation f o r 6 months o r more, t h e mean values for the lateral ventricle index manifest t h e same gradual slow decline a s is typical of healthy children of this age group. thus, n o significant deviation from normal could b e observed either initially or a t follow-up. d i s c u s s i o n brain weight a n autopsy material of brain weights of children with severe p c m was first published from uganda by brown (1). h e found in all t h e age groups examined (0-5 years) a numerical reduction in brain weight of the order of 15-20%. t h e dif ferences w e r e significant for t h e age groups above 1 year. similar results have been presented by winick & rosso in a small series (9 cases) of children from chile (40) a n d b y udani and co workers from india (37). h e a d circumference i n vivo, assessments of brain size have mostly been made b y measuring head circumference. the reduction of head circumference observed in cases of marasmus differs in degree and also with re spect t o catch-up growth in longitudinal studies o v e r longer time. stoch and smythe (36) followed their group of initially grossly undernourished children u p to the upsala j med sci 79 brain growth in children with marasmus 123 % stand. j. w. 6 3 9 age, months 40 mf -l fig. 7. anthropometric data, expressed as percentage of standard (17) during nutrition rehabilitation. four individual cases. a wnpn/age wmghlllongth b lengthiage 0 hwd circwntuence/ags age of 10-11 years. at that time, there was stifl a significant difference in the head circumference/ age relation (49.58 cm, as compared with 52.04 cm for american age mates). since the children in the test group were still markedly stunted i n growth, the head circumference/height relation was about normal. monckeberg (25) followed 14 children aged 8-9 months, who had been admitted to hospital "with severe marasmic malnutrition". renewed ex amination at the ages of 3-6 years showed values for head circumference definitely below normal. at the follow-up, the children were mainly normal, as regarded weight for age, but clearly subnormal, as regarded length for age; thus many of them were obese. the average head circumferencelage . .. ... pareto occipitally controls +hsd z marasmus m t 2 s d f i g . 8. head circumference related to age. children with marasmus compared with normal range (18). lon gitudinal study of 29 cases examined at monthly inter vals during nutrition rehabilitation. boys and girls se parately. fig. 9. head transillumination related to age. children with marasmus compared with a control group of ethio pian children (8). longitudinal study of 29 cases ex amined at monthly intervals during nutrition rehabilita tion. boys and girls combined. upsala j med sci 79 124 g. e n g s n e r et a [ . lateral ventricle azo 3 6 9age.months fig. 10. echo encephalography. lateral ventricle index ( i ) related to age. children with marasmus compared with normal range (33). longitudinal study of 29 cases examined at monthly intervals during nutrition re habilitation. boys and girls combined. relation was 2 . 4 k 1 . 4 cm below normal. the length deficit was 12.7&4%. in a series of papers, graham et al. (15, 16) have studied the growth and development of chil dren of families living in the “septic fringes” of lima, peru. they followed over a number of years more than a hundred children afflicted at an early age with severe malnutrition, almost all of the marasmus type. graham et al. do not give any absolute figures for head circumference etc. ; they introduced the term “development age” and “development ratio”, which make comparison with the results of other workers a bit compli cated. for our purpose, the most interesting con clusion of their work is expressed as follows (16, speculation): “catch-up growth, both i n height and in head size, can go on for many years after a period of severe malnutrition.” like many other workers in the field, they are of the opinion that “it may well be that the much slower rates of growth and the smaller statures achieved by chil dren in adverse situations are a convenient adapta tion for survival”. the head circumference measurements in o u r material show a reduction which for the cross sectional material in its entirety is significant @< 0.01>0.001). for four out of six different age groups the difference is probably significant @< 0.05>0.01). in absolute values, the differences observed are of the same magnitude as those ob served by stoch & smythe (36). for the longi tudinal material with children in the age group 2 4 months, the difference in head circumference, to begin with, is less pronounced and not signifi upsala 3 med sci 79 cant @>0.05). during nutrition rehabilitation, which was, on an average, slow and irregular, only a slight tendency to catch up was observed. throughout in our studies, only the ratio of head circumference to age has been given. we have thus refrained from giving the ratio of head cir cumference to length. the patterns of growth in crements for head circumference and for length differ markedly and there is only a weak correla tion between the two. winick & rosso (40) have given as equation for the relationship between head circumference and brain weight in normal children: total brain weight in g=(head circumference in cm -20.5)2+ 109.75. applying this equation to children with “neonatal malnutrition”, they concluded that “reduction in head circumference accurately reflects reduction in brain weight”. if so, it is hard to concieve that in cases of marasmus there could be a substantial increase of intracranial fluid with a corresponding further reduction of brain size. if a comparison is made between the observations by stoch & smythe on the head circumferences of south african children and by brown on the brain weights of ugandan children, it can be said that the two parameters tally reasonably well. interpretation of subnormal head circumference values 1. what is the normal head circumference in ethiopian children? there is good reason to postulate that in healthy ethio pian children the same norms can be adopted as those noted in swedish children. if ethnic differences were important, one would not expect privileged ethiopian children to follow the same standards a s those observed in swedish children. but in fact, they do; at least this is true for the age groups under consideration here (7, 8). 2 . what is the definition of a subnormal head circumference value in an individual subject? the range of head circumference values in healthy individuals of one and the same age is considerable. this range is mainly genetically determined. it bears only a moderate relation to body size in general. normal intelligence is rarely found if the brain weight is below 1000 g in malde adults and 900 g in female adults (12). the corresponding head circumference can be assumed to be around 50 and 49 cm respectively. in growing subjects, as a border-line in relation to microcephaly, a head circumference of m 3 s.d. has been given (2, 3). for a one-year-old boy, this cor responds to c . 43 cm, as compared with a normal brain g r o w t h in children w i t h m a r a s m u s i25 skull cavity (21), on the other. however, neither time nor equipment were available and a convincing reason for exposing children to x-rays was lacking. likewise, autopsy material was not accessible as a basis for com parison. earlier investigators, making use of radiographs, have made detailed studies of the thickness of the scalp (41) as well as of the bony cranium (29) in children of various ages. such data make it possible to calculate how much of the head circumference measured by a tape reflects the thickness of the integuments and how much remains for the “skull cavity circumference” as such. the former component may be calculated to be roughly of the order of 10 per cent of the total, i.e. a child with a head circumference of 45 cm should have a “skull cavity circumference” of the order of 40.5 cm. this is not only a theoretical speculation. at the autopsy of two adults, a plaster cast was made of the calvarium. the head circumference was, on an average, 56.3 cm, and the “skull cavity circum ference”, measured as the circumference of the plaster cast, was 50.2 cm, i.e. a difference of 10.8 per cent. on the basis of the data concerning thickness of scalp and bone (cf. above), a calculation has been made as to the effect on the head circumference of various degrees of reduction in thickness (table iv). a simul taneous reduction of 50 % of the scalp tissue and 25 % of the bone thickness means a decrease of the head circumference by 1.7 cm (3.5%). an even more pro nounced reduction of 75% of the soft tissue and 50% of the bone thickness means a decrease of the head circumference by 3 cm (6 %). the reduction of head circumference observed in our series of children with marasmus, i.e. an average of 3 . 4 cm in the age group 2-6 months, is almost certainly partially due to a thinning of the integuments. it is note worthy that a decrease of the thickness of the scalp by 50% and of the bone by 2 5 % may explain c . 50% of the reduction of head circumference observed. it is evident that part of the reduction of head cir cumference values in cases of marasmus may be ex plained by emaciation, but even if this is pronounced, hardly more than 50%. i t should be noted in this con text. that even after successful nutrition rehabilitation a head circumferencelage relation below normal may re main for many years (36). mean value of 46.5 cm. other authors (26, 27) are of the opinion that even head circumference values below m 2 s . d . imply suboptimal intelligence. it is obvious that also values “within the normal ,range” may be abnormal for a given child, e.g. if the genetic potential favours a m + 1 s.d. development, whereas the actual value (or values) observed is m 1 s . d . however, only under special conditions (longi tudinal observations, access to values in relatives, etc.) is it possible with some certainty to diagnose such a “relative subnormality”. 3. what do subnormal head circumference values observed in groups of children imply? the definition of subnormality in this case is simple; the mean value observed should be significantly below the standard (e.g. that of swedish children). however, the interpretation of the mechanisms behind such a subnormality in mean head circumference is not always quite simple. this is true the more there are conco mitant signs of severe malnutrition, perhaps of long standing (cf. below). 4 . how close is the correlation between head circumference and brain size? the interest in measuring head circumference stems mainly from the fact that in healthy individuals there is a fairly good correlation between head circumference and brain weight. however, this general rule has ex ceptions, particularly in sick and malnourished children. the four factors mentioned below (table 111) can all weaken the correlation. as sources of error, they act in different directions and it is not an easy task to find out in vivo to what extent the overall balance means an overestimation or an underestimation of the brain size. 5 . attempts to quantify the effect of abnormal thinness of scalp and bone thickness on head circumference our studies have only dealt with conditions in vivo. by making use of x-ray technique, it is possible to measure the thickness of the scalp (41) and the skull bone (29). on the one hand, and the volume of the table 111. f a c t o r s influencing t h e r e l a t i o n s h i p b e t w e e n head c i r c u m f e r e n c e a n d b r a i n s i z e ( a ) scalp tissue abnormally head circumference 6. h~~ can subnormaljo in head circumference be translated into subnormality in brain size? if the head circumference is assumed to reflect directly the cranial internal circumference and if the brain oc cupies the cavum cranii in the normal way, then it can ( b ) bone tissue abnormally head circumference easily be calculated that a reduction of head circum ference at the age of one year from 46 to 44 cm will mean a corresponding reduction of the brain weight ( c ) subdural/subabnormally headcircumference from c . 760 g to 660 g, i.e. a difference of 100 g (40). arachnoidal increased suggests however, a s we have seen, such a simple correlation may not exist, especially in cases of severe pcm. the fluid high brain size ( d ) cerebral venabnormally head circumference reduction may be less if the thinning of integuments is tricles large pronounced or greater if the subdural/subarachnoidal space is abnormally enlarged (cf. above). suggests erroneously low brain size suggestserroneously low brain size (and hair) thin thin suggests erroneously high brain size upsala j med s c i 79 126 g. engsner et a!. table i v . the eflect on head circumference b y alterations in thickness of scalp andlor s k u l l bone scalp-thickness reduction, % . . . 25 50 0 0 25 50 50 15 i skull-bone-thickness reduction, 92 . . . 0 0 25 50 25 25 50 50 age circumference (months) (cml calculated effect on head circumference, cm“ mean head 6 43.3 -0.5 -1.0 -0.5 -1.0 -1.0 -1.5 --2.0 -2.5 9 45.2 -0.6 -1.2 -0.6 -1.2 -1.2 -1.8 -2.4 -3.0 i2 46.5 -0.6 -1.2 -0.6 -1,2 -1.2 -1.8 -2.4 -3.0 24 48.0 -0.5 -1.0 -0.7 -1.4 -1.2 -1.7 -2.4 -2.9 the calculations have been made using the equation for a circle rather than that for an ellipse. the true shape of the skull may exhibit considerable individual variations. the approximation j u s t mentioned will influence the result of the calculations only to a very limited extent. transillumination the monckeberg group in santiago (30) examined 32 children in the age range 3-12 months with severe “third-degree marasmic malnutrition”, the growth deficit in all instances being more than 50% for their respective ages, according to iowa standards. since photographic recording was the aim, a strong light source had to be used (800 w). the opening through which the 1-second flash of light was concentrated had a diameter of 5 cm. around the opening a rubber ring was fitted. the trans illumination was considered positive (abnormal) if the light also was more than 8 cm i n diameter (five positions). by this criterion 28 out of 32 of the in fants with marasmus had a positive finding, as compared with one (a border-line value) out of 30 nutritionally normal age matees. in 26 out of 32 cases of marasmus, the existence of excess subarachnoidal fluid was verified by needle aspiration. a chemical analysis proved the fluid t o have the same composition as the cerebro spinal fluid. routinely, only c . 4 ml were as pirated, but in one case, in which aspiration was continued by mistake, 25 ml could be evacuated. the conclusion by the authors is “that malnutri tion during the first months of life is associated with a brain size smaller than cranial capacity, which would result in a secondary increase in cerebro-spinal fluid”. as far as we are aware, no other similar series with systematic transillumination (possibly fol lowed by aspiration) in cases of severe pcm has been published so far. our own studies indicated a slight-to-moderate increase of transillumination, in the age groups 6 24 months. it was demonstrable both from the fronto-temporal and parieto-occipital regions. the findings are by no means as dramatic as those just referred to by the monckeberg group. how should the difference between our ob servations in addis ababa and those of the monckeberg group in santiago be explained? is there a true geographical difference or is the dif ference explained by the criteria for selection of material and/or technique? the severity of the pcm in the santiago children may have been somewhat more pronounced than in ours. further more, it is not quite clear to what extent children with low birth weights may have formed parts of the material. the technique using a v e r y intense flash (800 w bulb) certainly exercised an influence. it is hard to see, however, that this could explain the results with very much more pronounced trans illumination in the children with marasmus, as compared with normal children. an important factor is the following. in the first year of life, the subarachnoidal space may be as wide as 0.5-1 cm also in children who are ap parently normal. there is very little in the litera ture on this matter, but it is well known to pe diatric neurologists and radiologists (gamstorp, personal communication). the same phenomenon is observed also i n foetal life (20). it prompts caution in interpreting x-ray pictures (pneumo graphy), in order to avoid an erroneous diagnosis of “cortical atrophy”. for the same reason, it seems justifiable to exercise caution also in the evaluation of positive needle aspiration in this age group. further studies from other regions are urgently needed to settle the question of the extent to upsala j med sci 79 brain growth in children with rnarasrnus 127 support t h e observations made by t h e chilean scientists, using transillumination. which severe marasmus is accompanied by a n ab normal accumulation of fluid in the subarachnoidal space. if such an abnormal accumulation of fluid is found, it would obviously mean that brain size is reduced t o an even greater extent than head circumference p e r se indicates. ultrasonic e c h o ventriculography we a r e not aware of any earlier studies with this technique, except for the preliminary data pub lished by our own group ( 3 8 ) . using chronological age, the mean values for the lateral ventricle index come very close to those of swedish children and also the range of observa tions falls within normal limits. using “head cir cumference age” (see above) instead, a consider able shift to the left would have occurred. h o w ever, as discussed under “results”, since the slope of t h e mean f o r the lateral ventricle index in healthy children which exists during the first year of life is rather modest, the change in posi tion of t h e mean values of the marasmic children in relation t o those of normal children is rather small and the difference in either case (“chrono logical age” and “head circumference age”) from the normal is not significant. it is of some interest in this context to recall that stoch and smythe (35) performed pneumo graphy in two cases of severe marasmus and found the results normal. furthermore, after the completion of o u r field work, we were informed of a multi-facetted study from brazil (22; in portu guese), involving also the taking of pneumoence phalogram in five children with marasmus, aged 4 14 months. t h e lateral ventricle size was normal in 4 of t h e 5 patients. this is in accordance with our own findings of normal lateral ventricle width measured by echo ventriculography. however, the findings relating t o the sub arachnoidal space are different. all 5 patients showed “cortical atrophy”, which was present also in 3 o u t of 4 patients on re-examination 4-5 months later. t h e r e are, however, definite diffi culties in making x-ray diagnoses of “cortical atrophy”, when it is not very pronounced. this is particularly true of children in the first year of life (cf. above). f o r these reasons, and because of the limited number of children involved, it is hard to evaluate t h e extent t o which the observation made b y the brazilian investigators can b e said t o a c k n o w l e d g e m e n t s the authors wish to express their sincere appreciation of the valuable support given by drs bo akerrtn, me hari gebre-medhin, uiia larsson, suzanne levine, goran sterky, teklesion woldemariam, mr erwin kopp and members of the staffs of the ethiopian nutrition institute, the ethio-swedish pediatric clinic and the lidetta mch centre. the study was financially supported by the swedish international development authority through the ethio pian nutrition institute and by grants from the swedish medical research council (b70-61 p-2924-01 and k72 19x-3788-01), uppsala university and the scandinavian institute of african studies. r e f e r e n c e s 1 . 2. 3. 4 . 5 . 6. 7. 8. 9. 10. 1 1 . 12. 13. brown, r . e . : organ weight in malnutrition with special reference to brain weight. dev med child neurol8: 512, 1966. b m k , j . a , , schut, j . w . & reed, s. c.: a clinical and genetical study of microcephaly. am j ment defic57: 637, 1953. davies, h . & kirman, b. h . : microcephaly. arch dis childh37: 623, 1962. dean, r . f. a , : the effects of malnutrition on the growth of young children. modern problems of pediatrics 5 : 10, 1960. karger, basel, switzerland and new york. dodge, p. r . & porter, p . : demonstration o f intra cranial pathology by transillumination. arch neurol 5: 30, 1961. editorial. classification of infantile malnutrition. lancet 11: 302, 1970. eksmyr, r.: anthropometry in privileged ethiopian pre-school children. acta paediatr scand 59: 157, 1970. engsner, g.: brain growth in privileged and non privileged ethiopian children. a study using head circumference measurement, transillumination and ultrasonic echo ventriculography. accepted for pub lication in j trop pediatr 19: 357, 1973. engsner, g . , habte, d., sjogren, i . & vahlquist, b.: brain growth in children with kwashiorkor. a study using head circumference measurement, trans illumination and ultrasonic echo ventriculography. accepted for publication in acta paediatr scand 1 974. engsner, g . : unpublished data. evans, d. e . , moodie, a. d. & hansen, j. d. l.: kwashiorkor and intellectual development. s afr med 545: 1413, 1971. ford, f. f.: diseases of the nervous system in in fancy, childhood and adolescence, 5th ed. charles c. thomas publishers, springfield, illinois, 1966. gamstorp, i.: personal communication. upsala j med sci 79 128 g. engsner e t a l . 14. garn, s. m.: malnutrition and skeletal development in the pre-school child. the national academy of science, national research council. pre-school child malnutrition: primary deterrent to human progress. washington, d.c., 1966. 15. graham, g. g.: the later growth of malnourished infants, effects of age, severity and subsequent diet. in calorie deficiencies and protein deficiencies (ed. r . a. mccance & e. m. widdowson), pp. 301 316. little, brown & co., boston, 1968. 16. graham, g. g. & adrianzen, b.: growth, inheri tance, and environment. pediatr res 5: 691. 1971. 17. jelliffe, d. b.: the assessment of the nutritional status of the community. who monograph series no. 53, 1966. 18. karlberg, p . , engstrom, i., lichtenstein, h . & svennberg, i . : the development of children in a swedish urban community. a prospective longi tudinal study. 111. physical growth during the. first three years of life. acta paediatr scand, suppl 187: 48, 1968. 19. knutsson, k . e. & mellbin, t.: breast feeding habits and cultural context. a study of three ethio pian communities. j trop pediatr 15: 40, 1969. 20. lanman, j . t., partanen, y., ullberg, s. & lind, j.: extracortical cerebrospinal fluid in normal hu man fetuses. pediatrics 21: 403, 1958. 21. mackinnon, i. l . , kennedy, j . a. & davies, t . v.: the estimation of skull capacity from roent genologic measurements. am j roentgenol radium ther nucl med 76: 303, 1956. 22. marcondes, e . et al.: desenvolvimento neuropsico motor da criangca desnutrida. 11. subnutrigcao. arquivos d e neuro-psiquiatria 28: 221, 1970. 23. mclaren, d. 8 . , pellet, p. l . & read, w. w. c.: a simple scoring system for classifying the severe forms of protein-calorie malnutrition of early child hood. l a n c e t l : 533, 1967. 24. monckeberg, f.: effect of early marasmic malnutri tion on subsequent physical and psychologica1 de velopment. in malnutrition, learning, and be havior (ed. n . s. scrimshaw & j. e. gordon), pp. 269-278. m i t press, boston, 1968. 25. discussion. i n calorie deficiencies and protein deficiencies (ed. r. a. mccance & e . m. widdow son), p. 315. little, brown & co., boston, 1968. 26. o’connell, e. j., feldt, r. h . & stickler, g. b.: head circumference, mental retardation, and growth failure. pediatrics 36: 62, 1965. 27. pryor, h. b. & thelander, h.: abnormally small head size and intellect in children. j pediatr 73: 593, 1968. 28. robinow, m.: field measurement of growth and development. in malnutrition, learning, and be havior (ed. n. s. scrimshaw & j . e. gordon), pp. 409-425. m i t press, boston, 1968. 29. roche, a. f.: increase in cranial thickness during growth. hum biol25: 81, 1953. 30. rozovski, j . , novoa, f., abarzua, j. & moncke berg, f.: cranial transillumination in early and severe malnutrition. br j nutr 25: 107, 1971. 31. sjogren, i.: echo encephalography in paediatrics practice with special regard to measurements of the ventricular size. acta paediatr scand, suppl. 178, 1967. 32. echo encephalographic measurement at ventri cular size in children. dev med child neurol 10: 145, 1968. 33. echo encephalography. evaluation based on records from 100 normal infants and children. am j dis child 119: 45, 1970. 34. sjogren, i . & engsner, g . : transillumination of the skull in infants and children. recording with a new point scale. acta paediatr s c a n d 6 i : 426, 1972. 35. stoch, m. b. & smythe, p. m.: does undernutri tion during infancy inhibit growth and subsequent intellectual development? arch dis childh 38: 546, 1963. 36. undernutrition during infancy, and subsequent brain growth and intellectual development. i n mal nutrition, learning, and behavior (ed. n . s . scrim shaw & j . e . gordon), pp. 278-289. mit press, boston, 1968. 37. udani, p. m., murkerjee, s . , parekh, u . c. & parwani, a , : relationship between body weights and brain weights in under-nourished children. pro ceedings of the nutrition society of india 10: 187, 1971. 38. vahlquist, b., engsner, g. & sjogren, i.: malnutri tion and size of the cerebral ventricles. acta paediatr scand 60: 533, 1971. 39. venkatachalam, p. s . & romanathan, k. s . : ef fect of protein deficiency during gestation and lacta tion o n body weight and composition of offspring. j nutr84: 38, 1964. 40. winick, m . & rosso, p.: head circumference and cellular growth of the brain in normal and marasmic children. j pediatr 74: 774, 1969. 41. young, r. w . : age changes in the thickness of the scalp in white males. hum biol31: 74, 1959. received january 14, 1974 address for reprints: gunnar engsner, m.d. department of pediatrics university hospital 750 14 uppsala sweden uvsala j med sci 79 upsala j med sci 92: 47-58, 1987 insulin receptor binding and metabolic effects of insulin in human subcutaneous adipose tissue in untreated non-insulin dependent diabetes mellitus peter arner, peter engfeldt, einar skarfors, hans lithell and jan bolinder the department of medicine, huddinge hospital, karolinska institute, stockholm, sweden, and the department of geriatrics, kungsgardet’s hospital, uppsala university, sweden a b s t r a c t insulin a c t i o n a t the t a r g e t tissue l e v e l i n non-insulin dependent diabetes m e l l i t u s was investigated using human adipose tissue. specific adipocyte r e c e p t o r binding o f insulin and t h e e f f e c t s o f the hormone on glucose o x i d a t i o n and lipolysis were d e t e r m i n e d in subcutaneous adipose tissue. the study included 25 p a t i e n t s w i t h u n t r e a t e d non insulin dependent diabetes m e l l i t u s and 38 h e a l t h y c o n t r o l subjects m a t c h e d f o r age, sex and body weight. insulin s t i m u l a t e d adipose tissue glucose o x i d a t i o n in a dose dependent way in t h e c o n t r o l subjects. on the other hand. a m a r k e d i n h i b i t i o n o f t h i s insulin e f f e c t was observed in t h e diabetics. a weak s t i m u l a t i o n was observed only a t high unphysiological hormone concentrations [> 0.7 n m o l / l l and the m a x i m a l insulin response was 6 t i m e s l o w e r t h a n t h a t in t h e c o n t r o l subjects. however, n e i t h e r s p e c i f i c insulin r e c e p t o r b i n d i n g nor t h e a n t i l i p o l y t i c e f f e c t o f insulin were i n h i b i t e d in diabetes. s i m i l a r r e s u l t s w i t h insulin binding and t h e m e t a b o l i c e f f e c t s o f insulin were obtained in non-obese normoinsulinemic d i a b e t i c s as compared t o m o d e r a t e l y obese hyperin sulinemic diabetics. it is concluded t h a t adipose tissue insulin resistance in non-insulin dependent diabetes m e l l i t u s only involves glucose m e t a b o l i s m and n o t antilipolysis. furthermore. it m a y solely be due t o postreceptor d e f e c t s in insulin a c t i o n and seems n o t t o be influenced b y obesity o r oversecretion o f insulin. i n t r o d u c t i o n in p a t i e n t s w i t h non-insulin dependent diabetes m e l l i t u s t h e hypoglycemic response t o insulin i s o f t e n attenuated. insulin resistance in t a r g e t tissues is an i m p o r t a n t cause o f h y p e r g l y c e m i a in t h i s f o r m o f diabetes. when t h e disease i s accompanied b y obesity the a c t i o n o f insulin m a y be f u r t h e r inhibited. insulin resistance in non-insulin dependent diabetes m e l l i t u s is usually a t t r i b u t e d t o a c o m b i n a t i o n o f i m p a i r e d hormone b i n d i n g t o s p e c i f i c c e l l s u r f a c e r e c e p t o r s and i n h i b i t i o n o f t h e signals f r o m t h e r e c e p t o r t o i n t r a c e l l u l a r m e t a b o l i c processes [14,15.18.22.261. it is suggested t h a t a l t e r a t i o n o f the r e c e p t o r is an e a r l y e v e n t t h a t leads t o a m i l d f o r m o f insulin resistance. w h i c h is subsequently aggravated b y i m p a i r e d i n t r a c e l l u l a r hormone signal c20.221. the investi g a t i o n o f t h e insulin a c t i o n in non-insulin dependent diabetes m e l l i t u s in m a n has been c e n t e r e d m a i n l y on hormone binding t o c i r c u l a t i n g blood cells, where insulin has l i t t l e . i f any, b i o l o g i c a l e f f e c t and on t h e over-all e f f e c t o f insulin on glucose m e t a b o l i s m 47 i n vivo. it is a t present unclear how insulin action i n this disease is altered i n the primary target tissues [fat, muscle and liver]. it is also not known i f insulin action i n target tissues d i f f e r s between obese and non-obese diabetics. the aim of the present study was accordingly t o establish whether insulin receptor binding and/or the metabolic e f f e c t s of the hormone were altered i n adipocytes i n non-insulin dependent diabetes mellitus. subcutaneous adipose tissue was obtained f r o m 25 untreated patients w i t h this f o r m of diabetes: 12 of the patients were non-obese and the others were moderately obese. comparison was made t o the finding i n 38 healthy control subjects w i t h normal glucose tolerance, who were matched w i t h the diabetics f o r age, sex and body weight. m a t e r i a l a n d methods patients: the series f o r the study consisted of 25 patients w i t h recently diagnosed and untreated non-insulin dependent diabetes mellitus. twelve were non-obese and 13 were moderately obese. moderate obesity was defined as a r e l a t i v e body weight of 115-150% of the average body weight. which was obtained f r o m tables i n documenta geigy [131. glucosuria was present i n a l l diabetics but ketoacidosis i n none. they were otherwise healthy and there were no signs o f physical i n a c t i v i t y secondary t o the illness. a f t e r the investigation the diabetics received diet therapy, either alone or i n combina t i o n w i t h sulphonylurea. thirty-eight healthy volunteers w i t h normal values f o r either intravenous glucose tolerance [25 g glucosel or oral glucose tolerance [ l o 0 g glucosel served as control subjects: 18 were classed as moderately obese and 20 as non-obese. some o f these subjects have been included i n previous reports as control subjects. the obese subjects had not followed any f o r m of slimming program during the six months preceeding the study. clinical data o f diabetic patients and control subjects are present in table 1 . they were matched w i t h respect t o age. sex and body-weight. the patients and control subjects consumed a diet consisting o f 40% carbohydrate, 40% fat, 20% protein and 7-8.5 mj, according a 24-h recall. a l l subjects and patients were informed i n detail about the study and their consent was obtained. the study was approved by the hospital's ethical committee. the investigation was performed a t 8 a.m. a f t e r an overnight fast a t the out-patient department. venous blood samples were taken f o r the determination of glucose [ 1 1 1 and serum immunoreactive insulin [301. gluteal specimens o f subcutaneous adipose tissue were obtained surgically. local anesthesia was induced w i t h prilocaine chloride. which was given i n such a way that it did not a f f e c t the metabolism o f adipose tissue [ll. adipocyte insulin receptor binding: isolated f a t cells were prepared by rodbell's method [291. they were incubated i n 0.5 m l o f krebs-henseleit bicarbonate b u f f e r [ p h 7.41 containing dialyzed bovine serum albumin [40 g/ll, glucose [5 m m o l / l l mono 1251-[tyr a ]-insulin l0.05 nmol/ll, and unlabelled insulin [o-50 nmol/ll. the f i n a l cell concentration was 8 % [vol/voll. a f t e r incubation f o r 60 m i n a t 24oc i n triplicate, 10 ml of ice-cold saline was added and the c e l l s were centrifuged through 1.2 ml of 14 48 a, 5 t . a, 5 e 0 h m + i (d h 0, n + i m c : 2 + i c j + i m c n + i n m c c \ j c m n rn u . a, n 0 . 1 d h j + i i0 0 c c c + i 0 c c d + i [9 j m + i c c n + i m j m \ 0 n m m rn e y c 0 n h j + i + i m m a j h ( d m + i m h. + i h. 0 c n m + i 0 h 0 c c m + i + i n h m n + i +i m c n h h n m +i + i o m m a n o n m + i m n m + i m m 1d \ h m w lo w 8 i\ (d + i m n + i c n c d + i m. a n + i i\ n c rn + i 0 m m \ 0 m c rn 0 c y 6 n 0 v 9 7 0 0 v 9 r 4-878571 49 silicone oil: they were then removed f r o m the o i l surface for r a d i o a c t i v i t y determination. non-specific binding was measured as the amount o f 1251-insulin remaining in the cell layer in the presence o f unlabelled insulin [20 umol/ll. a l l the values presented were corrected for non-specific binding. which amounted t o 2 4 % . the method has been described in detail c5.71. it was previously shown t h a t [a1 insulin binding reaches a maximum a f t e r 30 m i n and remains constant for a t least 90 min, [ b l insulin degradation i n the incubation medium is insignificant and [ c l the coefficient o f variation w i t h i n one subject for insulin binding is 8 % c5.71. / adipose tissue metabolism. explants o f adipose tissue [about 100 m g l were preincubat ed f o r 30 m i n and then incubated i n t r i p l i c a t e f o r 2 h a t 37oc in 1 m l o f krebs-henseleit bicarbonate buffer cph 7.41 t o which dialyzed bovine serum albumin [40 g/ll. glucose 1 4 9 [5 mmol/ll. [u cl-glucose [2 x 10 cpm/ll and insulin [o-7 nmol/ll were added. a mixture o f [co :o 1 [5:951 was used as gas phase. a f t e r incubation glycerol release and production were determined as described i n detail previously l5.71. these measures were used as indices o f lipolysis and glucose oxidation, respectively. the c o e f f i c i e n t o f variance within one subject f o r the metabolic studies was 8%. insulin degradation in the incubation medium. as determined by the tricholoroacetic acid precipitation method [51. was 5%. we have discussed i n detail previously why the metabolic e f f e c t s o f insulin are preferably detemined on segments o f f a t rather than on isolated f a t cells [71. 111 co2 2 2 adipocyte cellularity: the f a t cell volume and surface area. and the number o f f a t cells incubated were determined as described i n detail previously r5.71. chemicals: crystalline. glucagon-free porcine insulin was a generous g i f t f r o m v i t r u m ab [sweden]. mono 1251-[tyr a i-insulin [specific a c t i v i t y about 200 ci/gl. was f r o m novo [denmarkl. u c-glucose [specific a c t i v i t y 270 m c i / m m o l l was f r o m the radio chemical centre [england]. bovine serum albumin [ f r a c t i o n v1 was f r o m armour pharma ceutical company [england]. 14 1 4 statistical methods: the reported values are the mean se. student's unpaired t test was used f o r statistical comparison. results f a t cell size was enhanced i n the obese diabetic and obese non-diabetic groups: the values for diabetics and control subjects were however, similar [table 11. the fasting serum insulin level was t w i c e as high [p<0.021 for the obese diabetics than for the obese control subjects and 5 0 % higher [p<o.o51 in the whole diabetic group as compared t o the whole control group [table 11. basal metabolism o f adipose tissue is shown in fig. 1. lipolysis tended t o be slower and glucose oxidation was less pronounced i n the whole diabetic group as compared t o the whole group. 50 a c 01 fig. 1 basal glucose o x i d a t i o n and basal lipolysis in adipose tissue o f p a t i e n t s w i t h u n t r e a t e d non insulin-dependent diabetes m e l l i t u s [closed symbolsl and h e a l t h y c o n t r o l subjects w i t h n o r m a l glucose tole rance copen symbolsl. the incorpora t i o n o f r a d i o a c t i v e glucose i n t o c o 2 [glucose o x i d a t i o n ] is shown in the upper graphs and the r a t e o f g l y c e r o l release [lipolysisl is depicted i n the l o w e r graphs. a: the r e s u l t s w i t h a l l subjects. b: the results w i t h non-obese subjects. c: the r e s u l t s w i t h m o d e r a t e l y obese subjects. values are mean + se, student’s unpaired t t e s t was used f o r s t a t i s t i c a l cornpa r ison. when t h e m a t e r i a l was divided according t o body-weight it was observed t h a t the non-obese subjects accounted f o r the e n t i r e d i f f e r e n c e between diabetes and t h e c o n t r o l state. thus, in t h e non-obese diabetes group basal glucose o x i d a t i o n was reduced b y 2/3 and the basal r a t e o f lipolysis was doubled in comparison w i t h t h e non-obese c o n t r o l group. in obese subjects, on t h e o t h e r hand. basal lipolysis and basal glucose o x i d a t i o n were almost i d e n t i c a l in diabetes and the c o n t r o l state. o l i n 03 0 . 0 0 2 5 a 01 d 2 0 b i b c 1. 5 1 the e f f e c t o f insulin on glucose o x i d a t i o n is shown i n fig. 2. in t h e c o n t r o l subjects t h e r e was a m a r k e d dose-dependent insulin e f f e c t . the results w i t h non-obese and obese subjects were q u i t e similar. in t h e diabetics t h e r e was only a s m a l l insulin e f f e c t o c c u r r i n g solely a t h i g h unphysiological hormone concentrations [l 700 pmol/ll. since t h e dose-response curves in diabetics w e r e s c a t t e r e d it was n o t possible t o c a l c u l a t e ed50. in t h e whole diabetes group t h e a m p l i t u d e of the dose-response c u r v e was 6 t i m e s l o w e r t h a n i n t h e c o n t r o l group. the results w i t h non-obese and obese diabetics were similar. fig. 3 shows the a n t i l i p o l y t i c e f f e c t of insulin. i t is evident t h a t t h i s a c t i o n o f the hormone was n o t i n h i b i t e d in u n t r e a t e d non-insulin dependent diabetes m e l l i t u s . in t h e whole m a t e r i a l and in obese subjects the amplitudes o f the dose-response curves were i n t h e same order o f magnitude i n diabetes and the c o n t r o l state. furthermore, insulin s e n s i t i v i t y ii.e. the l e f t r i g h t position o f the dose-response curve1 was almost i d e n t i c a l in these groups: ed50 was 35 pmol/l. insulin s e n s i t i v i t y in non-obese diabetics was also the same as in non-obese c o n t r o l subjects = 35 pmol/ll. however, t h e a n t i l i p o l y t i c e f f e c t o f insulin was higher i n t h e non-obese diabetics as compared t o t h e non-obese c o n t r o l group a t a l l hormone concentrations tested. the l a t t e r m a y i n d i c a t e t h a t insulin responsiveness [i.e. the m a x i m u m hormone e f f e c t l was a c t u a l l y enhanced i n non-obese diabetics. this was f u r t h e r investigated i n table 2 w h i c h shows insulin responsiveness as c a l c u l a t e d f r o m each subject's dose-response curve. the absolute values f o r insulin responsiveness were almost t w i c e as h i g h in t h e non-obese diabetes group in comparison t o t h e non-obese c o n t r o l group. the r e l a t i v e values f o r t h e m a x i m u m a n t i l i p o l y t i c e f f e c t were, however, almost i d e n t i c a l i n b o t h groups, insulin m a x i m a l l y i n h i b i t e d basal lipolysis b y 35%. the l a t t e r i s probably due t o t h a t b o t h basal lipolysis [fig. 11 and insulin-induced a n t i l i p o l y s i s [fig. 31 were enhanced i n non obese diabetics. table 2. responsiveness o f the a n t i l i p o l y t i c e f f e c t o f insulin i n non-obese subjects w i t h u n t r e a t e d non-insulin dependent diabetes m e l l i t u s and in non-obese c o n t r o l subjects. p e r c e n t o f basal responsi yness ,umol/lo cells/2 h c o n t r o l diabetes p 2.00 + 0.41 3.49 + 0.47 < 0.01 34 + 5 36 + 7 in each subject glycerol release a t the m a x i m u m e f f e c t i v e insulin c o n c e n t r a t i o n was substracted f r o m t h e basal g l y c e r o l release. the obtained value represents responsiveness [ t h e m a x i m u m a n t i l i p o l y t i c e f f e c t l in absolute terms. this value was also d i v i d e d b y the basal value t o obtain t h e r e l a t i v e responsiveness. 52 e b c nmd,i, i@ 0 0017 007 0 3 5 7 1 7 1ns"lln n r n o l i l fig. 3: a n t i l i p o l y t i c e f f e c t o f insulin in human adipose tissue in u n t r e a t e d non-insulin dependent diabetes me11 i t u s and t h e c o n t r o l state. basal g l y c e r o l release minus insulin induced g l y c e r o l release was c a l c u l a t e d and p l o t t e d versus the insulin concentration. the corresponding insulin s e n s i t i v i t y p l o t is given i n the inset. in t h e l a t t e r graph the a n t i l i p o l y t i c e f f e c t o f insulin is expressed as percentage o f the response t o insulin a t the m a x i m u m e f f e c t i v e c o n c e n t r a t i o n 1%) and is p l o t t e d versus t h e insulin concentra t i o n (nmol/ll. see legends t o figs. 1 and 2 f o r f u r t h e r details. it is observed in t h e c o n t r o l subjects t h a t insulin i n h i b i t e d lipolysis a t lower hormone c o n c e n t r a t i o n t h a n a t w h i c h t h e r e was an e f f e c t o f insulin on glucose oxidation. s i m i l a r d i f f e r e n c e s in t h e s e n s i t i v i t i e s o f the e f f e c t s o f insulin on lipolysis and glucose o x i d a t i o n have previously been demonstrated in human adipose tissue (71. s p e c i f i c insulin b i n d i n g t o f a t c e l l s is shown in f i g u r e 4. there was n o evidence o f decreased binding i n u n t r e a t e d diabetes. the results w e r e s i m i l a r in non-obese and m o d e r a t e l y obese diabetes patients. the results o f t h e comparison between diabetes and c o n t r o l groups were t h e same whether expressed in t e r m s o f f a t c e l l number or in t e r m s o f f a t c e l l surface area. a "1 b 7 l"*"l,", " r n o l i i fig. 4: insulin binding t o isolated human adipocytes i n u n t r e a t e d non-insulin-dependent diabetes m e l l i t u s and t h e c o n t r o l state. s p e c i f i c % 1251-insulin b i n d i n g was d e t e r m i n e d and p l o t t e d versus the t o t a l insulin concentration. see legends t o figs. 1 and 2 for f u r t h e r details. 53 discuss1 on it is w e l l established t h a t non-insulin dependent diabetics are resistant t o t h e hypo g l y c e m i c e f f e c t o f insulin [15,22.261. in the present u n t r e a t e d p a t i e n t s w i t h t h i s disease t h e r e was a m a r k e d a l t e r a t i o n o f t h e a b i l i t y o f insulin t o s t i m u l a t e glucose o x i d a t i o n i n adipose tissue. a s m a l l insulin e f f e c t was observed solely a t h i g h unphysiological hormone concentrations. in contrast. adipose tissue o f c o n t r o l subjects responded t o insulin added a t physiological concentrations and t h e m a x i m a l insulin e f f e c t was 6 t i m e s g r e a t e r in these subjects than i n the diabetics. thus. the present data demonstrate a m a r k e d insulin resistance in adipose tisue in u n t r e a t e d non-insulin dependent diabetes mellitus. w h i c h suggests t h a t f a t tissue is involved i n t h e over-all i m p a i r m e n t o f insulin a c t i o n on glucose u t i l i z a t i o n i n t h i s disease. i n h i b i t e d a c t i o n o f insulin on glucose m e t a b o l i s m i n human adipose tissue in u n t r e a t e d non-insulin dependent diabetes has previously been observed i n hyperobese [average body w e i g h t > 150%1 p i m a indians 1191 and in a small group o f p a t i e n t s w i t h n o r m a l f a s t i n g insulin levels (71. however. the influence o f obesity and f a s t i n g hyperinsulinemia on insulin a c t i o n was n o t considered in these studies. non-insulin dependent diabetics are o f t e n obese and hyperinsulinemic. b o t h these f a c t o r s m a y induce insulin resistance independently o f diabetes 1223. in t h i s study a s i m i l a r degree o f adipose tissue insulin resistance was observed i n non-obese diabetics w i t h n o r m a l f a s t i n g insulin levels as in obese diabetics w i t h f a s t i n g hyperinsulinemia. these r e s u l t s i n d i c a t e t h a t n e i t h e r obesity nor enhanced insulin s e c r e t i o n are o f i m p o r t a n c e f o r adipose tissue insulin resistance in non-insulin dependent diabetes mellitus. the present results w i t h insulin a c t i o n on lipolysis in diabetics d i f f e r e d m a r k e d l y f r o m those w i t h insulin-induced glucose oxidation. the a n t i l i p o l y t i c e f f e c t o f insulin was n o t i n h i b i t e d i n adipose tissue o f u n t r e a t e d non-insulin dependent diabetics. in t h e whole m a t e r i a l insulin a c t i o n on lipolysis was almost i d e n t i c a l in diabetes and t h e c o n t r o l s t a t e b o t h regarding insulin s e n s i t i v i t y and insulin responsiveness [ m a x i m u m hormone e f f e c t ] . furthermore. n e i t h e r non-obese nor obese diabetics were r e s i s t a n t t o t h e a n t i l i p o l y t i c e f f e c t o f insulin. these d a t a s t r o n g l y support our o r i g i n a l hypothesis 12.71 t h a t a n t i l i p o l y s i s is n o t involved i n diabetes associated insulin resistance. f u r t h e r support f o r t h i s t h e o r y is t h e r e c e n t f i n d i n g o f n o r m a l a n t i l i p o l y t i c e f f e c t o f insulin in adipose tissue o f non-insulin dependent diabetics on long t e r m t r e a t m e n t w i t h d i e t plus sulphonylurea 1211. the f i r s t step i n the a c t i o n o f insulin is i t s binding t o s p e c i f i c c e l l s u r f a c e receptors. f r o m r e c e n t r e v i e w a r t i c l e s it would appear t o be a c o m m o n b e l i e f t h a t reduced r e c e p t o r number is an i m p o r t a n t f a c t o r underlying insulin resistance in insulin sensitive tissues in non-insulin-dependent diabetes m e l l i t u s [14.15.18.22,261. however. in t h e present study adipocyte insulin r e c e p t o r binding in a large group o f non-obese and obese p a t i e n t s w i t h u n t r e a t e d non-insulin dependent diabetes m e l l i t u s was n o t l o w e r t h a n in h e a l t h y subjects w i t h n o r m a l glucose tolerance, who were m a t c h e d f o r age. sex and body weight. these results are a t variance w i t h numerous studies w h i c h have demonstrated a de 54 creased number o f insulin receptors on circulating lymphocytes. monocytes and erythro cytes i n non-insulin dependent diabetes mellitus 13.12.16.23.24.27.281. however. evidence of decreased insulin binding t o circulating blood cells i s probably irrelevant since these cells are not natural target cells for insulin action. the results d i f f e r also f r o m those published by kolterman and co-workers who observed decreased adipocyte insulin binding i n untreated non-insulin dependent diabetics [zoi. in that investigation. however. the diabetic patients were significantly older than the control subjects. age matching o f the groups is probably important in any comparison i n respect o f adipocyte insulin binding. since several reports have demonstrated that insulin binding t o human f a t cells decrease w i t h age [8.21.251. it is also possible that regional variations in adipocyte insulin binding play a role. we investigated gluteal f a t cells and kolterman and co workers investigated abdominal f a t cells. we have recently observed that insulin binding varies between d i f f e r e n t adipose sites in man 14.51. o n the other hand, normal insulin binding t o adipocytes has recently been observed i n some rare groups of untreated non-insulin dependent diabetics. namely ones w i t h normal insulin secretion [71 and young hyper-obese pima indians [191. furthermore, normal insulin binding t o f a t cells was also observed i n non-insulin dependent diabetics receiving long-term diet and sulpho nylurea therapy [9.211. thus. when previous and present findings are considered together the adipocyte insulin receptor appears not t o be impaired i n any f o r m o f non-insulin dependent diabetes mellitus. this would seem t o call into question the role o f the insulin receptor i n the development o f insulin resistance i n this disease. it should be noted that i n the present binding experiments f a t cells were incubated a t 24oc. a t this temperature some of the insulin bound is internalized. in theory the l a t t e r f r a c t i o n may d i f f e r between diabetics and non-diabetics. however. i n human f a t cells we have recently observed [5.61 an excellent correlation [r>0.91 between insulin binding a t 24oc and 37oc [where a large portion of insulin is internalized] and between insulin binding a t 24oc and 16oc [where no insulin is internalized]. this indicates that differences in insulin internalization between diabetics and non-diabetics have l i t t l e bearing upon the present results w i t h insulin receptor binding. furthermore, the observa t i o n o f normal sensitivity o f the a n t i l i p o l y t i c e f f e c t of insulin argues further f o r t h a t the insulin receptor was normal i n our diabetics. since insulin sensitivity is thought t o r e f l e c t insulin action a t the receptor level [171. apparently, the presently observed inhibition of insulin-induced glucose metabolism is solely due t o a post-binding alteration o f insulin action. furthermore. this resistance t o insulin appears not t o involve the a n t i l i p o l y t i c e f f e c t of the hormone. the molecular mechanisms behind defects in insulin action on glucose metabolism a t the postreceptor level i n f a t cells of non-insulin diabetics are unclear. however, decreased a b i l i t y o f insulin t o stimulate adipocyte glucose transport has recently been observed i n this f o r m o f diabetes [10.191. such a defect may in p a r t explain the inhibition o f insulin-induced adipose tissue glucose metabolism found in this study. however, additional defects i n the intracellular action o f insulin may also cause the presently observed insulin resistance. particularly in the non-obese subjects where we also found a marked reduction o f basal glucose oxidation. in the l a t t e r respect it is o f interest t o compare adipose tissue metabolism i n our two diabetes groups. although insulin action did not d i f f e r between the two groups basal lipolysis and basal glucose oxidation were markedly altered in normoinsulinemic non-obese diabetics but unchanged i n mode r a t e l y obese hyperinsulinemic diabetics. it is thus possible t h a t adipose tissue metabolism may be regulated d i f f e r e n t l y in these two forms o f diabetes. acknowledgement this study was supported by grants f r o m the swedish medical research council [mfr 1034 and 54461. the swedish diabetes association. the nordic insulin foundation. the karolinska institute. and the osterman. wiberg. groschinsky. folksam and tore nilsson foundations. references 1. arner. p.. arner. 0. g ostrnan. j.: the e f f e c t o f local anaesthetic agents on lipolysis by human adipose tissue. l i f e sci 13: 161-169, 1973. 2. arner. p.. bolinder. j.. engfeldt. p. g ostman. j.: the antilipolytic e f f e c t of insulin i n human adipose tissue in obesity, diabetes mellitus, hyperinsulinemia. and starva tion. metabolism 30: 753-760. 1981. 3. beck-nielsen, h.: the pathogenetic role o f an insulin receptor defect i n diabetes mellitus o f the obese. diabetes 27: 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lipolysis. j b i o l c h e m 239: 375-380. 1964. 30. wide. l.. ax&, r. g porath. j.: radioimmunosorbent assay f o r proteins. c h e m i c a l couplings o f antibodies t o insoluble dextran. lmrnunochemistry 4: 381-386. 1967. address f o r reprints: jan bolinder d e p a r t m e n t o f medicine huddinge h o s p i t a l s-1 4 1 86 huddinge sweden 58 upsala j med sci 81: 193-200, 1976 the fluid double polar-nonpolar-polar leaflet model for biological membranes erik cerven from the institute of medical and physiological chemistry, biomedical center, university of uppsala, uppsala, sweden abstract a model for biological membranes is proposed accord ing to which the plasma membrane consists of two functionally different polar-nonpolar-polar leaflets separated by a polar space. the binding of water soluble proteins, integral lipoproteins and spanning proteins to a biological membrane as well as possible conformations of interphase peptides partitioned be tween polar and nonpolar layers are discussed. a model for the diffusion of water soluble proteins across adenylate kinase ( 5 ) , ndp kinase (2), atpase (3, 43), and protein kinase (6, 44). phenomena such as ghost resealing (c$: 26), secretion of proteins apparently without exocytosis (23, 32), and the transport of polynucleotides across intact plasma membranes (cf.: 36)are difficult to explain with the traditional lipid bilayer concept of a plasma membrane. this concept for biological mem branes in general has previously been questioned nonpolar layers of a membrane is described. three complete biological membranes containing two leaflets and an inter-leaflet space are defined. these are: 1: the inner nuclear membrane + the perinuclear space and the endoplasmatic cisternae + the outer nuclear membrane and the endoplasmatic reticulum, 2: the inner mitochondrial membrane + the mitochondrial intermembraneous space + the outer mitochondrial membrane and 3: the cytoplasmic leaflet of the plasma membrane + an intramembraneous space in the plasma membrane + the outer leaflet of the plasma membrane. on morphological ( 4 3 , chemical (28) and bio functional (28) grounds. perhaps the most con vincing objection is that lipids can be extracted from some biological membranes without altering the usual trilaminar appearance in electronmicro scopic pictures (18). also the nature of the ma terial in the sheet which does not st& with osmium remains undetermined because according to the traditional view strong hydro phobic bonds would be expected to be present thus preventing, for example, tangential splitting of this region on freeze-etching (6: 51). introduction the fluid mosaic model proposed by s.j. singer and g.l. nicholson in 1972 (46) represented con siderable progress in our understanding of several plasma membrane phenomena but it does not satisfactorily explain all of them, in particular several findings that have been made in this labo ratory during the past 10 years relating to the as sociation of cytoplasmic enzymes to the plasma membrane (4 l), albumin-inhibited leakage of such enzymes into the extracellular medium (54) and the apparent existence of an intramembraneous pool of solutes (4). all cells hitherto investigated in this laboratory display extracellular adenylate metabolism including atp synthesis (1, 5 , 42), the model as an approach to these and other questions as sociated with the traditional lipid bilayer concept, the fluid double polar-nonpolar-polar leaflet model for biological membranes illustrated in fig. 1 to 7 is proposed. according to this model, biological membranes consist of two functionally different polar-nonpolar-polar (pnp) leaflets each containing water soluble and integral (46) pro teins, lipids, and extended polypeptides partitioned between polar and nonpolar phases. one leaflet is in addition enriched in carbohydrate residues shared by the polar and nonpolar layers. the structure of the plasma membrane, for example, upsala j med sci 81 194 erik cervin p, '. i p5 r p? . i 4 - fig. 1. the principal structure of a biological mem brane. light areas indicate polar and dark areas indi cate nonpolar residues (p = polar layer, n = nonpolar layer, i = intermembraneous space). the indices and the arrow indicate the polarity of the two leaflets. the proportions between the different layers and the inter membraneous space are not necessarily the same as in this illustration, but may depend on the composition and the amount of the different chemical constituents. a b c d e fig. 2. association of watersoluble proteins to one leaflet of a biological membrane. the capitals a-e indicate increased complexity of the interactions be tween associated proteins and the membrane. from the cytoplasmic surface and outward is given by the following general scheme (see fig. 1). the layers of the n2 leaflet p1. polar bound (= associated and covalently bound to the nonpolar layer) residues. n2. fluid (9) nonpolar residues. p3. polar bound residues. the i4 space intermembraneous polar space containing solutes and enzymes but susceptible to conventional electron microscopic preparatory techniques. the layers of the n6 leaflet p5. polar bound residues. n6. fluid nonpolar residues. p7. polar bound residues. upsala j med sci 81 a. i ll jll i y p fig. 3. development of watersoluble membrane-as sociated proteins into integral (a-b) and spanning (b) proteins. the roman numbers i-iv indicate increased content of nonpolar amino acid residues involved in binding the integral protein to a membrane. a b c d e f g fig. 4. possible modes of binding of an integral protein spanning (46) across two leaflets of a biological mem brane e.g. the plasma membrane (a-d). stabilization of close spatial arrangement of two leaflets of a bio logical membrane by indirect (e) and direct (f) polar bonds and by fusion of the n2 and n6 leaflets (g). the binding between a membrane and globular proteins that do not become denatured when extracted (e.g. amino acid-binding proteins from bacteria, 10) is illustrated in fig. 2. the substitution of polar bonds involved in this type of binding for nonpolar bonds resulting in proteins integrated (46) in the nonpolar layer (e.g. a naiandk+ stimulated atpase, 1o)is illustrated in fig. 3 a and b. two types of spanning (46)proteins can be visualized in this model, mainly polar-nonpolar polar (pnp, see fig. 4a and b) and mainly polar nonpolar-polar-nonpolar-polar (pnpnp, see fig. 4 c and d) tentatively illustrated by glycophorin a (35) and the band 3 protein (25, 35) from ery throcytes respectively. the spanning proteins pre sumably hold the two leaflets of the plasma membrane together. this function could also be accomplished by indirect or direct polar bonds between the p3 and p5 layers as illustrated in fig. 4 e and f or by fusion (c$: 29) of the n2 and n6 layers (fig. 4 g). it is to be expected that a pnp leaflet heavily the fluid double proteolipid leaflet model f o r biological membranes 195 fig. 5. conformations of oligo-peptides partitioned between polar (p) and nonpolar (n) layers of a mem brane without reference to possible stabilizing interactions with adjacent molecules. oligopeptides with alternating polar and nonpolar amino acids represented by glu-leu-glu-leu (a-b) and leu-glu-leu-glu (c-d). part of a ring structure (e i) and possibilities for direct (e 11) or indirect (e 111) electrostatic stabilization illustrated by a conformation of the oligopeptide glu-glu-trp-leu-glu-glu. upsala j med sci 81 196 erik cewkn loaded with electronlucent and possibly displace able enzymes bound as illustrated in fig. 2 e would yield an electron microscopic picture reminiscent of a lipid bilayer. the reason for this is that polar and nonpolar residues of a mem brane are closer and therefore firstly more sus ceptible to cross-linking, secondly more electron dense and thirdly, since they are linked together by both polar and nonpolar bonds, also less extractable. however, on careful examination this type of trilaminar membrane (see fig. 2e) would be expected to reveal globular structures re sembling those found in mitochondrial and endo plasmatic reticulum membranes (47). the pro found effect on the interleaflet distance in bio logical membranes of conventional electron microscopic preparatory techniques (48) which are known to produce leakage of cytoplasmic proteins, shrinking and swelling of specimens, is documented in the case of mitochondria (49) chloroplasts (50) and plasma membranes (1 7). in all these cases, certain preparation techniques seem to preserve a broader interleaflet distance. this favours the view that inter-leaflet spaces are polar and excludes the possibility of strong non polar bonds in this region. tangentially extended polypeptides with polar residues directed outward and nonpolar residues directed inward in the proteolipid leaflets are imagined to constitute some kind of a lipid ske leton and in addition to mediate the attachment of watersoluble enzymes and carriers. these interphase polypeptides could be expected to require a secondary structure different from the a helix, the p-pleated sheat and the random coil, perhaps making them even more susceptible to cross-linking agents used in electron microscopy and to other frequently used denaturing proce dures. some possible configurations of such inter phase polypeptides are illustrated in fig. 5 . as shown in fig. 5 a-d, polypeptides with alternating polar and nonpolar residues are readily partitioned between the water and lipid phases of a biological membrane. exclusively nonpolar peptides may form a ring structure with the nitrogen atoms of the peptide bond directed towards the center. part of such a ring located in the interphase between polar and nonpolar layers upsala j med sci 8i is shown in fig. 5 e. numerous possibilities for stabilization of an interphase conformation by electrostatic interactions exist, some of which are illustrated in fig. 5 e. these interactions could be direct, between positively and negatively charged amino acid residues, or indirect, mediated by cations or anions that link similarly charged groups together. this model imposes functional similarities be tween the plasma membrane and the mitochond rial membranes because, in both cases, a pool of enzymes involved in adenylate metabolism (e.g. adenylate kinase, 5 , 16, 33, and ndp kinase, 2, 33) is located in the i4 space. this pool is acces sible from the outside through a n6 leaflet permeable to nucleotides (33) and containing carbohydrates (8, 37, 46). on the other hand the inner n2 leaflet is assumed to be impermeable to nucleotides necessitating the presence of various carriers in both inner mitochondrial (33) and plasma membranes. phenomena associated with intact cells such as crypticity (15, 19) of enzymes (15, 19) and receptors (14) could be explained by this similarity. weak interactions between the p3 and p5 layers are imagined to catalyze the fluid state dependent fusion of the n2 and n6 layers (see fig. 4 g) resulting in nonpolar intermembraneous bridges. the fusion points are possible sites for diffusion of cytoplasmic enzymes across the plasma membrane as illustrated in fig. 6 a. they could thus be responsible for the fact that albu min inhibits leakage of several cytoplasmic en zymes from some tumor cells without affecting or displacing other enzymes expressing their activity at the cell surface (54)indicating differential ac cessability of albumin to the plasma i4 and intra cellular spaces of macromolecules. on the other hand, preferential and specific entry of globular proteins through one pnp leaflet must be considered (see fig. 6 b) because some soluble enzymes are enriched in the mitochondrial i4 space (33) which because of continuous great changes of shape (21) can not have any fusion points between its two leaflets. if all transport of macromolecules across the plasma membrane not mediated by exoand endocytosis (38) occurred via the i4 space any function of enzymes em the fluid double proteoligid leaflet model f o r biological membranes 197 a fig. 6. diffusion of watersoluble proteins across non polar layers of a membrane. the diffusion of a protein across a double leaflet membrane is shown in a. the roman numbers 1-111 represent successive events while i v and v represent possible final events. succes sive events in the diffusion of a globular protein across a single leaflet membrane are shown in b. fig. 7 . the principal outline and polarity of three complete biological membranes, the inner nuclear membrane and the outer nuclear membrane combined with the endoplasmatic membranes, the mitochondrial membranes and the plasma membrane(s). the cor responding interleaflet spaces are the perinuclear space with the cisternae of the endoplasmatic reticulum, the intermembraneous space of the mitochondria and the intramembraneous space of the plasma membrane. n=nucleus, m=mitochondrion, p=plasma mem brane. the arrows indicate the polarity from the n2 to the n6 leaflet. bedded in or between the two leaflets would be disturbed. this simple mechanism for transport of macromolecules across biological membranes implies that only adhesion sites could be required since low entropy adhesion would favour dis placement of the underlying nonpolar fluid lipid residues. on the basis of ultrastructural evidence and biochemical characterization the polarity of at least three complete biological membranes can be determined (see fig. 7). by definition the associa tion of energy transducers such as coupling fac tors (33) and actin (30, 3 1) and covalently bound dna, characterizes a n2 leaflet as exemplified by the inner nuclear membrane (12, 13, cj: 34) the inner mitochondrial membrane (33, cj: 53) and the cytoplasmic leaflet of the plasma mem brane (24 cj: 22). also the presence of adenylyl cyclase (40) is a characteristic feature of some n2 leaflets. a comparatively high content of carbo hydrates characterizes a n6 leaflet as for example the outer nuclear membrane (7, 37), the endo plasmatic reticulum ( 4 3 , the outer mitochondrial membrane (8, 37)and the exterior leaflet of the plasma membrane (46). this characterization is supported by ultrastructural evidence indicating a globular appearance of the mitochondrial and endoplasmatic membranes (47) which resemble each other and are thinner than the plasma membrane (47). the facts that the nuclear mem branes are thin and rarely display a trilaminar structure (52) indicate that these membranes will be possible to characterize as stacked with glo bular enzymes in analogy with the structure of mitochondrial and endoplasmatic membranes (47). the associated i4 spaces in these cases are the perinuclear space combined (52) with the cisternae of the endoplastmatic reticulum, the intermembraneous space of mitochondria and a corresponding intramembraneous space in the plasma membrane (4). these three complete biological membranes containing n2 and n6 leaflets as well as an i4 space together with their polarity are illustrated in fig. 7. the nature and functions of the inter membraneous space with its associated enzymes and solutes is interesting in view of the fact that this space containing adenylate kinase and ndp upsala j med sci 81 198 erik cervkn kinase enlarges during oxidative phosphorylation in rat liver mitochondria (21). similar fluctuations of the i4 spaces of other biological membranes could be expected to be associated with their characteristic functions. the demonstration that the endoplasmatic cisternae also contain adeny late kinase and ndp kinase in vivo will support generalizations facilitating the interpretation of phenomena associated with any of these three complete biological membranes by their mutual analogy. discussion this model for biological membranes offers the possibility of a new approach to several plasma membrane phenomena. the involvement of gtp in adenylyl cyclase activity (39) is one example. the enhanced transport of amino acids across the nuclear n2 leaflet brought about by exogenous atp (27) may according to this hypothesis cor respond to similar phenomena in the i4 space and n2 leaflet of the plasma membrane. furthermore, various effects of treating intact cells with phospholipase c and other exogenous enzymes may be interpreted in terms of increased exposure of the i4 space and the n2 leaflet of the plasma membranes. the finding that certain amino acids apparent ly are deprotonated during transport across the plasma membrane (1 1) may indicate that under certain conditions the i4 space is capable of main taining a concentration of univalent cations, dif ferent from that on either side of the enclosing pnp-leaflets and may have important implica tions for the function of the mitochondria and the endoplasmatic reticulum. in the former case, swelling of the i4 spaceiscorrelated with oxidative phosphorylation and in the latter case inhibition of protein synthesis by puromycin is known to cause swelling of the endoplasmatic cisternae (20). the possibility that carriers generate a sodium gradient primarily across the n2 leaflet is interesting in view of the knowledge that inward transport of many metabolites is correlated with comigration of sodium (10). due to the fact that sodium participates in an electrochemical gradient upsala j med sci 81 (lo), such an arrangement would be possible to detect as a transient positive potential when in serting an electrode into a cell. if sodium-depend ent transport of solutes occurs primarily across the n2 leaflet, this transient positive potential would be detected primarily in cells that are characterized by a high rate of sodium-dependent transport and by an n6 leaflet that is semiperme able to sodium ions. the compartment idea for the subcellular loca tion of macromolecules and the idea of vesicles as the unique mediators of transmembraneous transport of macromolecules are concerned by this interpretation of the structure of biological membranes. several apparently unrelated obser vations dealing with, for example, the location of serum proteins in the nucleus (55) and the leakage of cytoplasmic proteins from intact cells (23, 32, 54)may be accounted for by assuming that the nuclear and plasma membranes and probably other intracellular membranes as well are perme able to certain macromolecules by diffusion as implied in this membrane model. this permeabi lity, is an intersting weakness of tumor cells be cause of the possibility of introducing macro molecular agents that are harmful for their proli ferative metabolism. acknowledgement this work was supported by grants from the swedish medical research council (project b7713x-228 -13b), the university of uppsala and a personal grant from uddeholms ab. i would like to thank drs. gunnar agren and gunnar ronquist for valuable criti cizm. references 1. agren, g., ponten, j., ronquist, g. & wester mark, b.: formation of 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720-731, 1972. 47. sjostrand, f.s.: a comparison of plasma mem brane, cytomembranes, and mitochondrial mem brane elements with respect to ultrastructural 529-552, 1975. 81-94, 1974. 468-473, 1972. 867-876, 1975. 7 4 : 594-605, 1968. features. j ultrastruct res 9: 561-580, 1963. 48. the problems of preserving molecular struc ture of cellular components in connection with electron microscopic analysis. j ultrastruct res 49. sjostrand, f.s. & barajas, l.: effect of modifica tions in conformation of protein molecules on structure of mitochondria1 membranes. j ultra struct res 25: 121-155, 1968. 50. sjostrand, f.s. & kretzer, f.: a new freeze drying technique applied to the analysis of the molecular structure of mitochondria1 and chloro plast membranes. j ultrastruct res 53: 1-28, 1975. 5 1. smith, v., ryan, j.w. & smith, d.s.: freeze-etch studies of the plasma membrane of pulmonary endothelial cells. j cell biol 56: 492-499, 1973. 52. stevens, b.j. & andrk, j.: the nuclear envelope. in handbock of molecuiar cytology (ed. a. lima-de-faria) pp. 837-87 1 north-holland publishing company, amsterdam & london 1972. 53. swift, h. & wolstenholme, d.r.: mitochondria and chloroplasts: nucleic acids and the problem of biogenesis (genetics and biology). in hand book of molecular cytology fed. a. lima-de faria) pp. 972-1046 north-holland publishing company, amsterdam & london 1972. 54. wernstedt, c., agren, g. & ronquist, g.: en zyme activities at the surface of intact ehrlich mouse-ascites tumor cells with albumin in the isotonic medium. cancer res: 35: 1536-1541, 1975. 5 5 . zardi, l., ziri, a. & santi, l.: a serum protein associated with chromatin of cultured fibro blasts. science 191: 869-870, 1976. 55: 271-280, 1976. received november 15, 1976 address for reprints: e. cerven institute of medical and physiological chemistry biomedical center university of uppsala box 575 s-751 23 uppsala sweden upsala j med sci 81 upsala j med sci 85: 5 9 4 6 , 1980 early changes in zinc and copper metabolism in rats with alloxan diabetes of short duration after local traumatization with heat goran hallmans and folke lithner departments qf pailwlogy und medicine, univer.uty qf umetr, umeri, s<<beden abstract local heat trauma was induced in rats with alloxan diabetes of 3 days dura tion. zinc and copper concentrations in serum, liver, heart and pancreas were estimated. after traumatization there was a decrease of the serum zinc concentration in all animals. the lowest concentration was found in the diabetic animals. serum copper concentrations were lower both in traumatized and nontraumatized diabetic animals compared with controls. the serum copper concentration was th nontraumatized animals in both slightly lower in traumatized compared w diabetic animals and controls. in the liver there was an increase of zinc and copper concentration in both groups of traumatized animals, but especially in the diabetic animals. i n pancreas there was a decrease in the zinc concentration in traumatized and nontraumatized diabetic animals compared with controls. introduction the effects of experimental local thermal cutaneous traumatization in non diabetic humans and animals have earlier been studied by others (see lithner, 1975). the skin of the lower legs in diabetics has an altered reaction compared with controls (10, 12, 13). this altered reaction is related to the occurrence of late diabetic lesions, such as microangiopathy and neuropathy, but not to the diabetic metabolic derangement per se. to determine the importance of the metabolic derangement, we have studied by different means the early inflammatory reaction to local heat trauma in alloxan diabetic rats and rabbits (5, 11, 13, 14) without finding any obvious differ ences between diabetic and nondiabetic animals. it is plausible, however, to suppose not only a late altered inflammatory reaction in diabetes (10, 11, 12, 13) but also an early one, and we have found a decrease of serum zinc concentrations after local thermal trauma to be sig nificantly lower in diabetic compared to nondiabetic rats (6). 59 in the present study, we have reported in more detail about the metabolism of zinc and copper in serum, liver, pancreas and heart of alloxan diabetic rats after local traumatization with heat. material and methods thirty male albino rats of the highly inbred r-strain were used ( 4 ) . diabe tes was induced in 16 animals at the age of 3 months with an intravenous injec tion of alloxan, 0.34 mmol/kg, as described earlier (9). no insulin was given after the injection of alloxan. all animals had blood glucose concentrations higher than 1 4 mmol/l, polyuria and glucosuria. no animal had ketonuria. methods of depilation and anesthesia were described previously (5, 1 4 ) . traumatization was performed 3 days after the alloxan injection, the tempera ture used was 6ooc for 5, 10 and 15 secs. traumatization was induced in the anesthetized animals by placing the end surface of an electrically heated cylindrical brass rod, 18 mm in diameter, against the skin ( 1 0 ) . inspection of the skin injury was performed as described previously (5, 6). immediately before the alloxan injection, 4 hrs after alloxan injection, immediately before the traumatization and 8 hrs after traumatization 0.5 ml blood was obtained from the tail of each rat. after clotting, samples of serum were taken. the animals were killed 24 hrs after the traumatization and samples were then taken from serum, liver, heart (myocardium, left ventricle) and pan creas. the samples were removed with stainless steel instruments, placed in weighed, acid-washed, borosilicate tubes and the wet weights were determined. all samples, except serum, were dried at llo°c for 3 days to constant weight. the dry weights were determined and the samples were ashed for 24 hrs at 55ooc. the ash was dissolved in 0.5 ml 3 mol/l hc1 overnight and then diluted with 2 ml of distilled water. if necessary, the samples were further diluted with 0.6 mol/l hc1 for zinc and copper determinations by atomic absorption spectro photometry. the analyses were performed on a varian aa-6db spectrophotometer at 213.9 nm and 324.7 nm for zinc and copper, respectively. reference samples of the minerals in 0.6 mol/l hc1 were used. samples of serum were diluted 10 times in 0.1 m hc1 before atomic absorption spectrophotometry with reference samples in 0.1 m hc1. differences between groups were tested using student's t-test for unpaired observations. the test was modified if the variances were significantly differ ent (p < 0.01; f-test). differences between groups were also tested with the nonparametric rank sum test of wilcoxon. p < 0.05 was chosen as the level for statistical significance. 60 results zinc and copper concentrations alloxan injection did not significantly affect the serum zinc concentrations (table 1). table 1. zinc concentration in serum before and after injection of alloxan or sodium chloride. the results are given as the mean +_ se. no significant dif ferences were observed between the four groups. before nacl injection 4 hrs after nacl injection before alloxan injection 4 hrs after alloxan injection concentrations number pg znlg serum n 1.33 _t 0.06 14 1 . 3 1 0.06 14 1.41 0 . 0 5 16 1.25 _t 0.09 16 eight hrs after traumatization a decrease in serum zinc concentration was found both in controls (p < 0.05) and in diabetic animals (p < 0.001). the de crease was most pronounced in the diabetic animals (fig. 1) and 24 hrs after traumatization the decrease persisted in them. 0 8 2 l hours after traumatization fig. 1. the effect of cutaneous traumatization (arrow) by heat on serum zinc concentration. the results are presented as the mean se. t-test results, control animals diabetic animals: 8 hrs p = 0.06. 24 hrs p < 0 . 0 2 . the copper concentrations in serum were only registered 24 hrs after trau matization and they were significantly lower both in traumatized and nontrau matized diabetic rats compared with controls (fig. 2). lower concentrations of serum copper were seen in traumatized diabetic and nondiabetic animals compared with nontraumatized animals, but only significantly lower in controls. 61 ~ l y cu/g serum 0.5 1.5 2.0 contra( animals n on-traumatized control animals traumatized diabetic animals non-traumatized diabetlc animals traumatized control animals non-traumatized control animals traumatized fig. 7 on -erurn copper concentration. the results are presented as the mean 2 se. 'i-test results are indicated in the figure. the effect of alloxan injection and cutaneous traumatization by heat 1 1 , ///////f/m& +,05 p.o.001 i an increase of the zinc concentrations in the liver was observed in diabetic animals compared with controls and in traumatized animals compared with non traumatized (fig. 3). the changes of the copper concentrations in the liver were similar to those of zinc, however, they were not significant except in traumatized diabetic animals compared with controls (fig. 4 ) . diabetic animals non-traumatize d' $401 phooi diabetic animals traumatized /////mu// fig. 3. the effect of cutaneous traumatization by heat on the zinc concentra tion in the liver. the results are presented as the mean 2 se. t-test results are indicated in the figure. 62 ~ l y cu/g liver dry weight 5 10 15 20 25 control animals contrd animals non-traumatized control animals traumatized diabetic animals non-traumatized diabetic animals diabetic animals traumatized ' 'ancreas 3eart fig. 4. the effect of cutaneous traumatization by heat on the copper concentra tion in liver. the results are presented as the mean : se. nontraumatized non traumatized traumatized 110.8 4.4 101.4 5.6 81.0 3.5*** 77.8 1.2 72.3 z 0.8 71.0 1.7 .ug culg dry weight in diabetic animals after traumatization there was a decrease in the pancreatic zinc concentrations compared with controls (table 2). only a slight decrease was seen in the copper concentrations of diabetic animals compared with controls. in the heart no differences were seen, neither in zinc nor in copper con centrations between groups (table 2). control animals table 2. zinc and copper concentrations in pancreas and heart bg/g dry weight). the results are given as the mean : se. the results of comparisons with non traumatized control animals (t-test) are indicated. diabetic animals i u g znlg dry weight 'ancreas leart nontraumatized nontraumatized traumatized traumatized 7.4 : 0.7 5.3 0.3* 6.6 0.4 6.1 : 0.5 21.8 0.6 20.1 : 0.9 20.3 1.7 19.4 1.0 traumatized 85.3 7.7* 70.2 1.2 * p < 0.05 *** p < 0.001 63 macroscopic and microscopic findings in traumatized areas a slightly higher, but not statistically significant, extent of purpura was seen in controls compared with diabetic animals 8 and 2 4 hrs after the trau; matization. similar findings were described previously (6). microscopically, no differences were found between controls and diabetic animals. there was extensive tissue damage to the skin after 10 and 15 secs of trau matization, often with necrosis through the entire thickness of the skin in cluding the panniculus carnosus. there was an accumulation of granulocytes and mononuclear cells surrounding this devitalized area. after 5 secs of traumati zation the epithelium showed signs of disruption and necrosis with accumulation of inflammatory cells, mostly granulocytes, in the upper dermis. signs of in flammation were sometimes seen to extend to the panniculus carnosus. discussion the serum zinc concentration decreased after heat trauma both in diabetic animals and in controls while the liver concentration of zinc increased. this relationship has been postulated to be due to the liberation of a leucocytic endogenous mediator (lem) from activated phagocytic cells (6) which, in the present study, were located in the traumatized areas. several functional dis turbances in diabetic granulocytes have been reported (see bagdade et al. 1978). the decrease of serum zinc concentrations was more pronounced in the dia betic animals, and this has been reported previously (6). this can explain the more pronounced increase of zinc concentrations in the liver of traumatized diabetic animals. however, an increase of the zinc concentrations in the liver was also seen in nontraumatized diabetic animals when compared with controls. this indicates that an increased concentration of zinc binding sites is found in the liver of the diabetic animal. this may be due to an increased synthesis or an incorporation of a zinc binding ligand in diabetic animals in response to the metabolic derangement or to the injection of alloxan. the increase of zinc concentrations in the liver in traumatized controls and in nontraumatized diabetic animals suggests that these animals need more zinc than what is available from the loosely bound serum zinc. partly, it comes from an increased absorption from the intestine ( 7 ) and partly probably from zinc mobilized from tissues of other organ systems. i n this study, however, no sig nificant decreases were observed in the zinc concentrations of pancreas or heart after traumatization, but the zinc concentrations of nontraumatized dia betic animals were decreased compared with the corresponding controls. most of the serum copper is strongly bound to ceruloplasmin, a n d -2-globu lin with oxidase activity ( 3 , 8 ) . a minor part of the serum copper is weakly 64 bound to albumin and amino acids (3). ceruloplasmin belongs to the group of acute phase proteins. elevation of ceruloplasmin and serum copper is usually induced by acute stress (1) but we should'nt expect it to be present as soon as 24 hrs after the thermal trauma, as in our study. the decrease of the serum copper concentration in the diabetic animals probably is due to a decrease of ceruloplasmin concentration. the liver plays an important role in copper homeo stasis and 3 of it's functions in this respect are: storage of copper, cerulo plasmin synthesis and excretion of copper through the bile (3). at least one of these functions has to be altered after traumatization in the diabetic animals which had increased copper concentrations in their livers. acknowledgements this study was supported by the swedish medical research council (no. 718). we wish to thank ann-marie nilsson for expert technical assistance. 1. 2. 3. 4. 5. 6. 7. 8 . 9. 1 0 . 11. 12. 13. 14. references beisel, w.r., pekarek, r.s. & wannemacher., r.w.: homeostatic mechanisms affecting plasma zinc levels in acute stress. in: trace elements in human health and disease, academic press 1:87-106, 1976. bagdade, j.d., stewart, m. & walters, e.: impaired granulocyte adherence. a reversible defect in host defense in patients with poorly controlled diabetes. diabetes 27:677-681, 1978. dowdy, r.p.: copper metabolism. am j clin nutr 22:887-892, 1969. hbgg, e . : renal lesions in rats with long-term alloxan diabetes. a semi quantitative light microscopic study with particular reference to the glomeruli. acta pathol microbiol scand 82 a:199-210, 1974. hallmans, g . , lithner, f. & hbgg, e.: early cutaneous reactions to local traumatization with heat in alloxan diabetic rats. upsala j med sci 83: 17-21, 1978. hallmans, g . , lithner, f. & hbgg, e.: local traumatization with heat cutaneous reactions and early effects on serum zinc concentration in rats with alloxan diabetes of very short duration. upsala j med sci 84:61-66, 1979. hallmans, g. & wing, k.: effects of early wound healing and wound treat ment with zinc tape on intestinal absorption and distribution of zinc in rats. acta chir scand 144:431-439, 1978. holmberg, c.g. & laurell, c.-b.: investigation in serum copper. acta chem scand 2:550-556, 1948. korec, r.: experimental diabetes mellitus in rat. puhlishing house of the slovak academy of sciences, bratislava, 1967. lithner, f.: cutaneous reactions of the extremities to local thermal trauma. acta med scand 198:319-325, 1975. lithner, f . & h;igg, e.: cutaneous reactions of alloxan diabetic rats to local thermal trauma. upsala j med sci 80:99-102, 1975. lithner, f.: skin lesions of the legs and feet and skeletal lesions of the feet in familial amyloidosis with polyneuropathy. acta med scand 199: 197-202, 1976. lithner, f.: lesions of the legs in diabetics and in patients with familial amyloidosis and polyneuropathy. acta med scand suppl 589, 1976. lithner, f . & hallmans, g.: cutaneous reactions to local traumatization with heat in alloxan diabetic rabbits with and without ketosis. upsala j med sci 83:23-28, 1978. received november 5, 1979. address for reprints: folke lithner, m.d. department of medicine university of umea s-901 85 umeh sweden 66 upsala j med sci 89: 171-177, 1984 sexual and contraceptive experience among teenagers in uppsala erik weiner, ines johansson, gisela helmius and viveca odlind department of obstetrics and gynaecology, university hospital, uppsala, sweden abstract a questionnaire was offered t o 181 sixteen-year-old boys (n=88) and girls (n=93) in different schools in the city of uppsala. the questionnaire was introduced to the students by a nurse midwife during a regular lesson and included over 100 questions dealing partly with sexual education, attitudes towards sex and own experience of sex and contraception. of the girls, 47% and of the boys, 31% answered that they had had intercourse. twenty-eight per cent of the girls and 21.5 % of the boys had had their first intercourse before the age of 15. contraception was used a t the f i r s t intercourse by 59 % of the girls and by 70 % of the boys. a t the very f i r s t intercourse, the condom was the most commonly used contraceptive method. increasing sexual experience changed the contraceptive pattern and a t their last intercourse 48 %j of the girls were on the pill and 33 % of the boys said that their girl friend was on the pill. introduction in order to provide adequate counselling and education in matters of sexual ity and contraception to teenagers it is important to know not only the habits but also the common attitudes towards these questions in the age group. previous studies have reported a gradual decrease in the age of the f i r s t intercourse among girls and boys (1, 2 , 3 ) . accordingly, society has increasingly provided clinics for teenagers, where not only contraception advice can be given but also a wider counselling can take place in matters that apply to adolescents. in collaboration with the institute of sociology a t the university of uppsala and the school gynaecologic clinic in uppsala, a study was performed in 171 1978 among high-school students. the aim of the s t u d y was to evaluate sexual experience and attitudes towards sexuality and contraception in a group of 16-year-old boys and girls. parts of the results have previously been published ( 4 ) and this report deals mainly with sexual and contra ceptive experience. the school gynaecologic clinic in uppsala is p a r t of the local school health care programme and s t a r t e d in 1973. the main interest of the clinic is to give contraceptive advice to both girls and boys b u t girls can also be exa mined and treated for various gynaecologic disorders by a trained gynae cologist who visits the clinic twice weekly. the clinic is mainly r u n by two nurse midwives, who always see the patients before they see the gynae cologist. usually, the midwives can give the patient all the advice and care she/he needs and supply contraceptives and very few of the patients have to see the gynaecologist. material a n d methods the students one hundred and eighty-one students participated in the present s t u d y , 88 boys and 93 girls. this sample was achieved by a cluster-sampling procedure in which all high schools in uppsala participated and their classes were arranged in clusters of around 30 students each. among the arranged 50 clusters, 7 from 5 different schools were sampled by use of a random number generator. the sample constituted 14% of the total number of first year high school students. the median age among the girls was 15.9 years (range 15 to 18) and among the boys 16.0 years (range 1 4 to 18). the questionnaire the questionnaire included more than 100 questions on topics such a s family background, attitudes and plans for the the f u t u r e , hobbies, knowledge of and previous educat.ion in s e x , attitudes towards s e x , own sexual experience and experience of contraceptive methods. the questionnaire was distributed among the students during a biology lesson without the students having previously been informed about the s t u d y . instead of their regular and expected teacher they were met by one of the nurse midwives from the school gynaecologic clinic, who carefully informed the students about the aim of the s t u d y , the questionnaire, the importance of serious answers and that they were to be guaranteed anonymity. the students had the oppor tunity to ask questions and they all agreed to participate in the s t u d y . i72 results most of the results a r e presented in the following tables. the head of the tables represents the question that the students were asked to answer. of the girls 44 (47 %) and among the boys 27 (31 %) said that they had a t least had intercourse once. table 1. how old were you a t the time of your first intercourse? 12 1 3 14 1 5 1 6 1 7 n g i r l s 1 3 22 13 4 1 44 boys 0 3 5 11 8 0 27 table 2 . how old was your partner a t your first intercourse? 12 13 14 1 5 1 6 1 7 18-22 n girls 0 1 2 6 12 8 1 5 44 boys 1 2 5 12 6 1 0 27 table 3. for how long had you known your first coital partner? 1 day 1 week 1 month <+! year >$ year n girls 3 4 11 11 1 5 44 boys 6 4 5 2 10 27 table 4. what kind of relationship did you have to your f i r s t coital partner? going steady good friend casual friend n girls 29 boys 11 10 7 4 43 9 27 12-848572 173 to the question 'where did your first intercourse take place?' almost 90 % of the girls replied t h a t it had been in their own house or a t the home of their boyfriend or of a mutual friend. among the boys, 45 % claimed that their f i r s t intercourse had taken place in a c a r , in a tent or otherwise outdoors. when asked about the degree of alcohol use a t the time of the first inter course, 59 % of the girls answered that they were not a t all d r u n k , 29 % said t h a t they were a little drunk and 11 % answered that they had been very d r u n k . among the boys, 37 % were not a t all d r u n k , 45 % were a little drunk and 18.5 % were very drunk a t the time of their f i r s t intercourse. the students were asked whether they now were going steady. among the girls 64 (70 %) said that they were and among the boys 75 (85 %). the majority of the girls said that they had been going steady for up to half a y e a r , several close to two years. none of the boys had been going steady for more than six months. table 5 . what type of contraceptive method did you use a t your first intercourse? none condom oral iud foam n girls 18 23 2 0 1 44 % 40.9 52.3 4.5 0 2 . 3 boys 8 13 4 2 0 27 % 26.9 48.1 1 4 . 8 7 . 4 0 table 6. which contraceptive method did you use during your last intercourse? none condom oral iud foam n girls 9 14 21 0 0 44 % 20.5 31.8 47.7 0 0 boys 7 11 9 0 0 27 % 25.9 40.7 33.3 0 0 174 the contraceptive method was provided to the girls either by the school gynaecologic clinic (42 %), a hospital clinic (23 %) or by a private doctor (11.5 7,). the boys had bought the condoms a t a shop (69 %) or got it from a friend ( 2 1 %). no boy had received contraceptives from the school clinic although it is open to both boys and girls. a s many a s 84 % of the girls and 70 % of the boys had used the condom on some occasions. no girl in the study had ever used the diaphragm, nor an iud. the students were also asked about their total number of partners with whom thay had had sexual intercourse. thirty-six per cent of the sexually active girls had only had intercourse with one boy and another 24 % had had two boys. a minority of the girls claimed to have had coitus ,with three boys or more. among the sexually active boys, 48 % had only been together with one girl and 17 % had had two girls. thirty-five per cent of the boys had had inter course with three or more girls. table 7. how many times did you have intercourse during the last month? 0 1-3 4-6 7-9 >10 n girls 23 4 7 1 9 44 boys 18 6 0 1 2 27 table 8. are you satisfied with your present frequency of intercourse? wants satiswants inexper. inexper. more fied less wants does not want n girls 10 31 2 25 2 90 boys 15 15 0 35 20 85 175 comment this study describes a random sample of 16-year-old students in a medium-sized swedish city. the answers were judged to be serious (4) and therefore the results can be assumed to give a true picture of the sexual and contraceptive habits and experiences in this group of adolescents. the proportion of girls and boys who claimed t o have had intercourse is well in agreement with other recent reports ( 3 , 6 ) . in a study from stockholm, 1978, the median age for the first intercourse was reported to be 15.8 years for girls and 17.4 years f o r boys (3). in the present study nearly 50 % of the 16-year-old girls and a third of the 16-year-old boys had had inter course. in a recent study f r o m uppsala, i t was reported that among 15-year-old students 71 % of the girls and 67 % of the boys had had inter course, suggesting that the age a t the first intercourse has decreased further (5). in a study f r o m i978 on finnish girls, the mean age a t the first intercourse was around 15 years (7). a majority of the girls said that they were going steady o r had known their first 'coital' partner for a t least one month, which is a fairly long time in the perspective of an adolescent (5). a less idyllic picture was given by the boys, who more often had had their f i r s t intercourse with a casual friend, more often were more or less drunk on the occasion and the intercourse had often taken place outside their homes. the girls often had had their f i r s t intercourse with a partner who was older whereas the boys chose partners of their own age. the use of contraceptives a t the f i r s t intercourse was relatively frequent as more than 60 % of the girls and 70 % of the boys claimed that contraception was used a t their very first sexual intercourse, the most common contra ceptive a t that time being the condom. the results suggest a shift in the type of contraceptive method to a more safe and efficient one after the first intercourse a s the proportion of users of oral contraceptives increased with increasing sexual experience. although almost 50 % of the girls were on the pill a t their last intercourse, a s large a proportion a s 20 % did not use any form of contraception, indicating the need of continued efforts to reach teenagers with education and information about contraceptive methods. when such advice is given i t has to be kept in mind that the sexual habits in this age group a r e very irregular (table 7 ) , and the motivation among many teenagers with sporadic sexual contacts to use a highly efficient method may be weak. therefore, good and repeated information also on barrier methods should be given to this age group. 176 acknowledgement this study was supported by the swedish national board of health and welfare, which is gratefully acknowledged. 1 . 2 . 3. 4. 5. 6 . 7. references crafoord, k . : symptom eller ilderstypiskt beteende. department of psychology, university of stockholm, 1972. (swe. ) karlsson, g . : sexuella vanor och attityder bland folkhogskoleelever . sociologisk forskning 1 , 1964. (swe. ) klackenberg-larsson, i . ti bjorkman, k . : tonsringars sexualdebut. socialmed tidskrift 55: 5-6, 1978. (swe.) lewin, b . : sexual attitudes and sexual experiences among teenagers in a swedish city. department of sociology, uppsala university, 1980. lewin, b . & helmius, g. : ungdom och sexualitet. department of socio logy, uppsala university, 1983. (swe . ) rahm, v . a . : flickorna t a r initiativet . journalen 2 : 230-232, 1982. (swe.) ruusuvaara, l . r . : teenage abortions. dissertation a t the university of helsinki, 1983. address for reprints : erik weiner, m.d. department of obstetrics & gynaecology university hospital s-751 85 uppsala sweden 177 upsala j med sci 97: 177-182 sternocostoclavicular hyperostosis presentation and long-term follow-up of three cases samwel nungu, claes olerud and lars rehnberg department of orthopaedics, uppsala university hospital, s751 85 uppsala, sweden abstract sternocostoclavicular hyperostosis is a rare disease characterized by recurrent pain and skeletal swelling in the upper part of the chest. the clinical manifestations are closely linked to pustulosis palmo-plantaris but the etiology is still obscure. we present three cases of sternocostoclavicular hyperostosis with a follow-up period of 9-22 years at our department. introduction sternocostoclavicular hyperostosis is a rare ailment which can be characterized by clinical and radiological manifestations in the sternum, clavicles, upper ribs and spine. the earliest literature about the ailment was published in 1970 by kapert and campbell (8). several cases have been monitored and reported, especially from japan by ishibashi et al. (7), sonozaki et al. (18), kojima ( l l ) , abe et al. (1) and from sweden by bjorksten et al. (3) and hradil et al. (6). the presenting and most striking clinical feature is recurrent throbbing pain accompanied by local tumescence, an increase in body temperature and a tenderness in the anterior upper part of the chest. weather changes and recurrent infections appear to aggravate the clinical picture. the radiological changes range from local to diffuse ossification and hyperostosis and sonozaki et al. (18) has proposed a radiological classification divided into three stages (table 1). table 1. stage classification of radiological findings according to sonozaki et al. (18). stage 1 localized. ossification is mild and localized to the costoclavicular ligaments. tomography or computed tomography may or may not show intraosseous new-bone foimation. stage 2. generalized. ossification is widely spread beyond the margins of the costoclavicular ligaments. the inferior margins of the first ribs appear irregular but no penosteal reaction is visible. hyperostotic. not only the inferior but also the superior margins of the clavicles show hyperostotic changes and the clavicles appear thickened and tumescent. stage 3. 177 in the early stages scintigraphy reveals an appreciable radionuclide uptake. the ailment is not characteristically accompanied by any specific laboratory findings, but later on, there can be slight changes in the erythrocyte sedimentation rate (esr), white blood count (wbc), c reactive protein (crp) and serum alkaline phosphatase (salp). tests for rheumatoid factor (rf) and both specific and unspecific immunological tests are usually negative. histopatho logy and cytology examinations may reveal non-specific, inflammatory cellular changes compatible with osteomyelitis or paget’s disease of the bone. case reports case 1 a 5 9 year old woman complained of periodically increasing pressure pain in the right sternoclavicular joint in 1969. initially her pain responded well to indomethacin. radiological examinations and laboratory tests, (esr, wbc, salp and rf) were normal. there was no increase in body temperature. she had been treated for recurrent pustulosis palmo-plantaris (ppp) several times from the mid 60‘s onwards at the department of dermatology. an infectious focus was suspected as well as a relationship between the ppp and the sternoclavicular clinical manifestations. radiological findings, such as a widening of the synchondrosis between the manubrium and the corpus sterni, ossification in front and behind the synchondrosis as well as periarticularly around the sternoclavicular joint and the first costosternal joint were recorded in 1974 four years after the earliest clinical manifestations. the manubrium also manifested stage 2 findings according to sonozaki et al. (18). in 1976 a histopathological analysis from the sternoclavicular joint was carried out and revealed an unspecific chronic osteomyelitic reaction with an esr of 43 m m h (the highest ever recorded). bone marrow cytology was normal and there was no evidence of malignancy. an oral examination in 1979 revealed severe paradontitis and a chronic sinusitis was diagnosed. haemophilus bacteria were cultured from the epipharynx. she was treated successfully with penicillins, wash-outs and a radical sinus operation coupled with dental extraction was performed in 1981. since then, she has had no complaints of either inflammatory symptoms in the anterior part of the chest or ppp. case 2 a 42 year-old woman presented with a pustulosis palmo-plantaris in 1965. she suffered from recurrent manifestations but all bacterial cultures proved negative. she was referred to our department in 1967 with a tender, warm and hard tumescence in the right sternoclavicular area which was accompanied by throbbing pain. she was afebrile and had an esr of 12 m d h . x-ray images demonstrated an expansive osteolytic lesion in the medial third of the right clavicle and a periosteal reaction localized mainly in the posterior aspect. malignancy (sarcoma or ewing’s tumor) was suspected and a total right clavicleectomy was performed. the histopathological examination showed a picture of unspecific chronic osteomyelitis. there was no evidence of malignancy. 178 since 1974 she has had episodes of back aches (cervical, thoracic and/or lumbar) but the first radiological manifestations were revealed in 1983 when unilateral sclerotic lesions in the vertebral bodies of l 111, l v and s i were observed. they also demonstrated overbridging sclerotic osteophytes between the left aspects of l i11 and l iv and the right of l v and s i. there was no evidence of lumbar disc collapse. her symptoms exacerbated during the night and morning hours but gradually improved towards the afternoon and evening hours. they also responded well to indomethacin therapy. in 1975, the same type of symptoms reappeared as in 1967 but this time in the left sternoclavicular area. x-ray images and bacterial cultures from the left sternoclavicular joint were negative. in 1987 a hard tumescence accompanied by a local increase in temperature was observed and x-ray images including tomography demonstrated ossification and hyperostosis, with irregular sclerotic lesions in the sternalmanubrium and osteolysis in the medial end of the left clavicle (stage 3 according to sonozaki et al.) (1 8). the symptoms could only be contained by continuous antibiotic (cephalexin) administration during a period of five months. however the pain worsened and a partial medial clavicle resection was performed. the histopathological examination revealed findings compatible with chronic osteomyelitis but no organisms cloud be cultured from the specimen. she has never been free from symptoms for more than 4 months each year. her esr has remained at a relatively high level (25-40 mm/h) since 1986. she has recently presented with the same inflammatory symptoms again in the proximal right side of the manubrium, incapacitating pains in the left sacroiliac and hip joints, pustulas on her palms as well as decrease in her cervicaland lumbarspine mobility. although multidisciplinary approaches have been implemented, no infectious focus has ever been revealed. case 3 in 1980, a 26-year-old woman presented with atypical dermatological manifestations on her right leg and left foot. several diagnoses such like psoriasis, neurodermatitis, recurrent chronic dermatitis, were postulated but the deimatologists could not arrive at one definitive diagnosis. in 1986 she was referred to our department because of pains and a tenderness in the left sternoclavicular joint. she had been out of work for more than one year. the pains worsened during the night and morning hours and varied with climatic changes. in 1987 a hard tumescence developed gradually in the sternoclavicular joint. scintigraphy revealed a high radionuclide uptake in the manubrium sterni and the sternoclavicular areas. radiological examination including computerized tomography of the left sternoclavicular joint demonstrated hyperostotic lesions in the clavicle with cortical destruction (stage 3 according to sonozaki et al.) (18). histopathological studies of the bone from the area have shown findings compatible with chronic osteomyelitis but a culture from the specimen proved negative. there was no evidence of malignancy. 179 table 2. clinical and laboratory characteristics of the three cases of sternocosto clavicular hyperostosis. case sex presenting follow-up side esr skin age (years) (years) manifestations 1 female 40 19 left low ppp 2 3 21 22 bilat varying ppp 26 9 left high atypical discussion our three patients (table 2) were all females and at the time of the debut of their symptoms aged 2 1 , 2 6 and 40 years respectively. these facts are not in agreement with the observations of resnik et al. (17), who stated that this disorder typically affects men or, less frequently, women in the fourth, fifth or sixth decades of life. our observations are in agreement with those of authors from japan, such as chigira et al. (4), kojima (1 1) and sonozaki et al. (18). our diagnosis of sternocostoclavicular hyperostosis is based on typical clinical symptoms, the histopathological picture of chronic osteomyelitis and the radiological and tomographic features described previously by bjorksten et al. (3), chigira et al. (4), kapert and campbell (8), kato et al. (9), kawai et al. (10) and kohler et al. (12). scintigraphy is an extremely useful tool especially in the early stages when radiological findings are not yet conclusive (16). laboratory tests have only been of value for the purpose of differential diagnosis. the esr which usually varies in inflammatory diseases is relatively unpredictable. it is known that psoriasis can also be accompanied by joint complications, which tend to follow the same irregular chronic pattern characterized by remissions and exacerbations, having an unpredictable onset and duration. in our three cases we found that pustulosis palmo-plantaris (in cases 1 and 2) proceeded the sternocostoclavicular manifestations by 3-5 years, as observed by enfors and molin (9, ishibashi et al. (7) and kato et al. (9). case 1 and 3 had atypical psoriasis-like lesions but the dermatologists could not arrive at a single diagnosis. the course and non-infectious genesis of the mentioned dermatological lesions are compatible with the well-established entity of “localized pustular psoriasis” (13). our three patients could not recall any close relative with psoriasis. case 1 did not have any dermatological lesions or pains in the upper part of the chest since 1981 (at the age of 51 years) when the radical removal of a chronic infectious focus was effected. scintigraphic (tc 99m) imaging 8 years postoperatively still revealed a striking radionuclide uptake in the sternum and the sternocostoclavicular joints as evidence of a low continuous level of activity. this supports ono (15) who postulated the possibility of a natural spontaneous remission of the clinical symptoms in elderly patients especially after the sixth decade. a distant infectious focus can function like a trigger mechanism or behave like an exacerbating factor of clinical manifestations in the sternocostoclavicular area (2). this 180 may also explain why random tonsillectomy as proposed by ono (14) can relieve clinical symptoms in certain cases. we have not yet localized any infectious foci in case 2 and 3 but the symptoms sometimes appear to come under control during long-term antibiotic therapy (cephalexin). treatment with cyclooxygenase inhibitors has proved to be very effective during the peak symptomatic period (4,19). in summary, sternocostoclavicular hyperostosis is a rare, recurrent disease which is easily confused with other chronic diseases, such as osteomyelitis, paget’s disease, ankylosing spondylitis etc. the diagnosis is based on typical clinical manifestations, radiological findings and negative bacterial cultures. in the acute stages, the patients respond to treatment with cyclooxygenase inhibitors. systematic broad spectrum antibiotic therapy is sometimes effective in relieving the pain. clavicle resection does not treat the symptoms radically but offers prolonged pain relief. our three cases seem to demonstrate a variety of joint and bone complications as sequels to dermatological disease. references 1 2. 3. 4. 5 . 6. 7. 8. 9. 10. 11. abe y., funayama, k., iwai, k. & irei, 0.: an analysis of sternocostoclavicular hyperostosis. report of 27 cases. orthop and traumat surg 27: 749-753,1984 andrews, g. c. & machacek, g. f.: pustular bacterids of the hands and feet. arch dermatol and syphilol32:837-847, 1935. bjorksten, b., gustavsson, k-h., eriksson, b., linderholm, a. & nordstrom, s . : chronic recurrent multifocal osteomyelitis and pustulosis palmo-plantaris. j pediatr 93:227-23 1, 1978. chigira, m., maehara, s., nagase, m., ogimi, t. & udagava, e.: sternocostoclavicular hyperostosis. a report of nineteen cases, with special reference to etiology and treatment. j bone joint surg (am) 68:103-112, 1986. enfors, w. & molin, l.: pustulosis palmaris et plantaris. a follow-up study of a ten-year material. acta derm venereol 151:289-294. 1971. hradil, e., gentz, c-f., matilainen. t., moller. h., sanzcn, l. & udcn, a,: skeletal involvement in pustulosis palmo-plantaris with special reference to the sternocostoclavicular joints. acta derm venereol68:65-73, 1988. ishibashi, a., nischiyama, m., endo, m., kawaji, w. & kato, t.: orthopaedic symptoms in pustular bacterid (pustulosis palmaris et plantaris): tietze’s syndrome and arthritis of manubriosternal joint due to focal infection. j derrnatol4:53-59, 1977. kapert, j. a. & campbell, p. e.: recurrent hyperostosis of the clavicles. an un-diagnosed syndrome. australian pediatric journal 697-104, 1970. kato, t., kambara, h. & hoshi, e.: case of bilateral clavicular osteomyelitis with palmar and plantar pustulosis. seikei geka 19590-593, 1968. kawai, k., doita, m., tateishi, h. & hirohata, k.: bone and joint lesions associated with pustulosis palmaris et plantaris. a clinical and histological study. j bone joint surg (br) 70:117-121. 1988. kojima, k.: clinical study of so-called steriiocostoclavicular hyperostosis. orthop and traumat surg 25:243-248, 1982. 181 12. 13. 14. 15. 16. 17. 18. 19. kohler, h., uehlinger, e., kutzner. j. & west, t. b.: sternocostoclavicular hyperostosis: painful swelling of the sternum, clavicles, and upper ribs. report of two new cases. ann intern med 87: 192-194, 1977. moschella, s . l. & hurley, h. j.: dermatology, vol. i . saunders co., philadelphia 1985. ono, t.: evaluation of tonsillectomy for pustulosis palmais et plantaris. nippon hifuka gakkai zasshi 86677-683, 1976. ono, t.: pustulosis palmaris et plantaris. rinsho derma. 24:1205-1213, 1982. otsuka, n., fukunaga, m., sone, t.. nagai, k., tomomitsu, t., yanagimoto, s . , muranaka, a., ono, s . & morita, r.: usefulness of bone imaging in diagnosis of sternocostoclavicular hyperostosis. clinical nuclear medicine. 11:651-652, 1986. resnick, d., vint, v. & poteshman, n. l.: sternocostoclavicular hyperostosis. a report of three new cases. j bone joint surg (am) 63:1329-1332,1981. sonozaki, h., azuma, a., okai, k., nakamura, k., fukuoka, s., taleishi, a., kurosawa, h., mannoji, t., kabata, k., mitsui, h., seki, h., abe, i., furusawa, s . , matsuura, m., kudo, a. & hoshino, t.: clinical features of 22 cases with "intersternocostoclavicular ossification". a new rheumatic syndrome. arch orthop traumat surg 95:13-22, 1979. vane, j. r.: inhibition of prostaglandins synthesis as a mechanism of action of aspirin-like drugs. nature (new biol) 231:232-235,1981. correspondence to: lars rehnberg department of orthopaedics, university hospital, s-751 85 uppsala, sweden. 182 upsala j med sci 92: 1-17, 1987 a psychophysical study of work-related stress using observer ratings m. wangenheim,' g. borg,' p. hoizrnann' 'bygghulsan research foundation, danderyd, department of rehabilitation medicine, university hospital, uppsala, and 'department of psychology, university of stockholm, stockholm, sweden abstract i n t w o e x p e r i m e n t a p e r c e i v e d e x e r t i o n s e r o f a e s a p a n e l of o b s e r v e r s r a t e d t h e w o r k e r p e r f o r m i n g d i f f e r e n t t a s k s . the f i r s t e x p e r i m e n t i n v o l v e d t h e l i f t i n g of b o x e s o f d i f f e r e n t w e i g h t s . the s e c o n d e x p e r i m e n t c o m p r i s e d combined s t a t i c and dynamic work of t h e a s s e m b l y l i n e t y p e d u r i n g a f u l l w o r k i n g day. the r e s u l t s show t h a t t h e o b s e r v e r s were a b l e t o d i s c r i m i n a t e between t h e d i f f e r e n t l o a d s i n a l l s t r e s s s i t u a t i o n s . s i n c e t h e l o a d s r a n g e d f r o m low t o maximal s t r e s s , a l l were a b l e t o make a s s e s s m e n t s o v e r t h e f u l l r a n g e . f u r t h e r m o r e , t h e r e l a t i o n s h i p between o b s e r v e r r a t i n g s a n d s e l f r a t i n g s a p p r o x i m a t e d a l o g a r i t h m i c f u n c t i o n w i t h t h e o b s e r v e r s t e n d i n g t o o v e r e s t i m a t e t h e low s t r e s s e s w h i l e t h e r e was c l o s e r a g r e e m e n t i n t h e r a t i n g of h i g h s t r e s s e s . 1 i n t r o d u c t i o n i n p r i n c i p l e , s e v e r a l d i f f e r e n t m e t h o d s a n d t y p e s o f m e a s u r e m e n t s c a n b e u s e d t o d e t e r m i n e p h y s i c a l s t r e s s . it i s p o s s i b l e t o p e r f o r m " a b s o l u t e " p h y s i c a l m e a s u r e m e n t s s h o w i n g w h a t p h y s i c a l f o r c e s a r e i n v o l v e d i n t h e w o r k , t h e a m o u n t o f t o r q u e , e n e r g y c o n s u m p t i o n e t c . s u c h p h y s i c a l m e a s u r e m e n t s a r e s o m e t i m e s f e a s i b l e , b u t t h e c a l c u l a t i o n s c a n p r o v e t o b e d i f f i c u l t t o p e r f o r m . a n o t h e r t y p e o f m e a s u r e c o n s i s t s i n p h y s i c a l m e a s u r e m e n t s o f t h e w o r k e r ' s p e r f o r m a n c e (13). i n t h i s c o n t e x t u s e i s o f t e n made o f m a x i m a l p e r f o r m a n c e s a n d f a t i g u e c u r v e s , o r o c c a s i o n a l l y o f t h e w o r k e r ' s p e r f o r m a n c e a t some i ' s u i t a b l e ' ' o r " p r e f e r r e d " i n t e n s i t y o f w o r k . p h y s i o l o g i c a l m e a s u r e m e n t s o f s t r e s s i n i n d u s t r y a r e p e r h a p s some o f t h e m o s t common o b j e c t i v e m e a s u r e m e n t s . m e t h o d s f o r d e t e r m i n i n g h e a r t r a t e , o x y g e n c o n s u m p t i o n , e m g ( w i t h t h e e x c e p t i o n o f s u r f a c e e m g ) a n d t h e l i k e a r e g e n e r a l l y v e r y r e l i a b l e ( 1 ) . two d i s a d v a n t a g e s o f t h e s e m e t h o d s a r e , h o w e v e r , t h a t t h e y r e q u i r e a p p a r a t u s a n d t i m e , a n d t h a t t h e m e a s u r e m e n t s o f t e n i n v o l v e some i n f r i n g e m e n t u p o n t h e o n g o i n g w o r k i n g p r o c e d u r e . t h u s a w o r k p e r f o r m a n c e c a n b e a n a l y s e d f r o m t h e p o i n t o f v i e w o f t h e a m o u n t o f e n e r g y u s e d o n t h e b a s i s o f o x y g e n c o n s u m p t i o n o r i n t e r m s o f p h y s i c a l s t r e s s o n j o i n t s , e.g. t h e e l b o w o r k n e e j o i n t , o r o n t h e b a c k , e.g. i n t r a s p i n a l p r e s s u r e e t c . i t may b e d i f f i c u l t t o s e e how t h e d i f f e r e n t p h y s i o l o g i c a l m e a s u r e s o f s t r e s s s h o u l d b e w e i g h e d i n r e l a t i o n t o e a c h o t h e r o r w h a t i m p o r t a n c e s h o u l d b e a t t a c h e d t o a s p e c i f i c m e a s u r e m e n t . an i m p o r t a n t q u e s t i o n i s j u s t how r e p r e s e n t a t i v e t h e m e a s u r e m e n t s a r e o f t h e u s u a l l y q u i t e c o m p l e x p r o c e d u r e b e i n g p e r f o r m e d . an e n t i r e l y d i f f e r e n t t y p e o f m e a s u r e m e n t o f p h y s i c a l s t r e s s c o n s i s t s i n p e r c e p t u a l e s t i m a t e s o f t h e m a g n i t u d e o f s t r a i n . i n a n u m b e r o f s t u d i e s made m o r e r e c e n t l y , t h i s t y p e o f m e a s u r e m e n t h a s p r o v e d t o b e a n e x c e l l e n t c o m p l e m e n t t o t h e o t h e r o n e s 1 2 , 4, 1 0 ) . 2 one m i g h t t h i n k t h a t t h e e f f e c t o f p h y s i c a l s t r e s s m u s t b e m e a s u r e d i n p h y s i o l o g i c a l v a r i a b l e s . t h i s i s n o t c o r r e c t , h o w e v e r , a l t h o u g h i t i s a r a t h e r common n o t i o n . b y a n a l o g y w i t h t h i s l i n e o f r e a s o n i n g , i t w o u l d o n l y b e p e r m i s s i b l e t o u s e p s y c h o l o g i c a l v a r i a b l e s f o r m e n t a l o r p s y c h o l o g i c a l s t r e s s . h o w e v e r , a s we k n o w , i t i s common, a n d o f t e n a l s o a p p r o p r i a t e , t o u s e p h y s i o l o g i c a l u n i t s o f m e a s u r e m e n t f o r m e n t a l s t r e s s . s i m i l a r l y , i t i s o f t e n e x p e d i e n t t o u s e p s y c h o l o g i c a l v a r i a b l e s , e . g . r a t i n g s o f p e r c e i v e d i n t e n s i t i e s , a s m e a s u r e s o f p h y s i c a l o r p h y s i o l o g i c a l s t r e s s . some o f t h e d i s a d v a n t a g e s o f u s i n g m e a s u r e m e n t s b a s e d o n m e n t a l p e r c e p t i o n a s i n d i c a t o r s o f w o r k r e l a t e d p h y s i c a l s t r e s s a r e t h e f a c t t h a t some i n d i v i d u a l s f i n d i t d i f f i c u l t t o m a k e r e l i a b l e e s t i m a t e s a n d t h a t c e r t a i n t y p e s o f s t r e s s d o n o t m a n i f e s t t h e m s e l v e s i n p e r c e p t u a l l y r e l e v a n t c h a n g e s . t h e r e a r e some a d v a n t a g e s , h o w e v e r , o n e b e i n g t h a t t h e i n d i v i d u a l s p o n t a n e o u s l y i n t e g r a t e s t h e d i f f e r e n t s t r e s s f a c t o r s t o w h i c h h e i s e x p o s e d . w h e r e t h e p h y s i o l o g i c a l a p p r o a c h g i v e s u s a n u m b e r o f d i s p a r a t e m e a s u r e m e n t s , h e r e we o b t a i n a n i n t e g r a t e d p e r c e p t i o n o r e x p e r i e n c e o f t h e s t r e s s , a " g e s t a l t " o f t h e c o m p l e t e s t r e s s p a t t e r n . i f a n y p a r t i c u l a r s t r e s s i s e s p e c i a l l y c r i t i c a l , i t w i l l b e e m p h a s i z e d t h e m o s t . a f u r t h e r a d v a n t a g e i n t h i s r e s p e c t i s t h a t t h e s u b j e c t i v e l y p e r c e i v e d s t r e s s w i l l d i r e c t l y r e f l e c t t h e c a p a c i t y o f t h e s u b j e c t a n d t h e r e b y c o n s t i t u t e a r e l a t i v e m e a s u r e . t h e m e t h o d s g e n e r a l l y u s e d i n p e r c e p t i o n p s y c h o l o g y o r p s y c h o p h y s i c s a r e t h e s o c a l l e d r a t i o e s t i m a t i n g m e t h o d s , i . e . m e t h o d s e m p l o y i n g s c a l e s w i t h a z e r o p o i n t a n d e q u a l d i s t a n c e s b e t w e e n p o i n t s o n t h e s c a l e ( 1 5 , 16). h o w e v e r , i n many f i e l d s t u d i e s u s e i s m a d e o f s i m p l e s c a l e s f o r e s t i m a t e s i n t h e f o r m o f c a t e g o r y s c a l e s , w h i c h o n l y a l l o w a r a n k i n g o f i n t e n s i t i e s a n d n o t ' m e a s u r e m e n t s " a p p r o a c h i n g r a t i o s c a l e d e t e r m i n a t i o n s . a r e v i e w o f d i f f e r e n t m e t h o d s u s e d i n p r a c t i c a l s i t u a t i o n s h a s b e e n p r e s e n t e d b y p e a r s o n ( 1 2 ) . 3 i n o r d e r t o r e d u c e t h e f u n d a m e n t a l d i s a d v a n t a g e s t h a t t h e c o n v e n t i o n a l r a t i n g s c a l e s a r e f r a u g h t w i t h , b o r g h a s e l a b o r a t e d a n e w m e t h o d o l o g y t h a t p r o d u c e s r a t i n g s o f a m e t r i c o r d e r a p p r o a c h i n g t h a t o f p h y s i o l o g i c a l m e a s u r e m e n t s . by i n s e r t i n g v e r b a l d e s c r i p t i o n s o f t h e t y p e " v e r y s l i g h t " a n d " s t r o n g " o n a r a t i o s c a l e f r o m 1 t o 1 0 a c c o r d i n g t o t h e p e r c e p t u a l i n t e n s i t y a s d e t e r m i n e d b y s p e c i a 1 p s y c h o p h y s i c a l a n d p h y s i o l o g i c a l m e a s u r e m e n t s , a m e a s u r i n g s c a l e h a s b e e n p r o d u c e d w h i c h a 1 l o w s q u a n t i t a t i v e d e t e r m i n a t i o n s a n d m a t h e m a t i c a l c o m p u t a t i o n s (5). t h e m e t h o d c a n b e u s e d f o r r a t i n g s b y t h e w o r k e r h i m s e l f w h e n h e p e r c e i v e s a p h y s i c a l s t r e s s ( 9 , 7 1 , a s w e l l a s b y o t h e r i n d i v i d u a l s o b s e r v i n g s u b j e c t s a t w o r k . t h e f a c t t h a t r a t i n g s b y o b s e r v e r s b a s e d o n v i s u a l i n f o r m a t i o n o b t a i n e d b y w a t c h i n g a s u b j e c t d o i n g w o r k c a n p r o d u c e r e l i a b l e m e a s u r e m e n t s o f p h y s i c a l s t r e s s i n t e n s i t i e s , h a s b e e n d e m o n s t r a t e d b y r u n e s s o n a n d f r y k h o l m (14). t h e a i m o f t h e e x p e r i m e n t s d e s c r i b e d b e l o w i s t o d e t e r m i n e t h e r e l a t i o n s h i p b e t w e e n o b s e r v e r r a t i n g a n d s e l f r a t i n g s ( b y t h e w o r k e r h i m s e l f ) o f w o r k r e l a t e d s t r e s s e s . t h e b a s i c i d e a o f t h e e x p e r i m e n t s w a s t o e x p o s e a w o r k e r t o a n u m b e r o f d i f f e r e n t s t r e s s p r o d u c i n g s i t u a t i o n s u n d e r w e 1 1 c o n t r o l l e d l a b o r a t o r y c o n d i t i o n s . t h e s e s i t u a t i o n s i n c l u d e d l i f t i n g o f b o x e s a s w e l l a s s i t u a t i o n s i n v o l v i n g a c o m b i n a t i o n o f s t a t i c a n d d y n a m i c w o r k o v e r a n 8 h o u r p e r i o d . i n e a c h s i t u a t i o n b o t h t h e w o r k e r a n d a p a n e l o f o b s e r v e r s r a t e d t h e s t r e s s o n b o r g ' s s c a l e . a 1 1 r a t i n g s w e r e e n t i r e l y i n d i v i d u a l . t h i s w a s f o l 1 o w e d b y a c o m p a r i s o n o f t h e w o r k e r ' s own r a t i n g s o f t h e p e r c e i v e d e x e r t i o n w i t h t h o s e o f t h e o b s e r v e r s . t h e e v a l u a t i o n s w e r e b a s e d o n t h e a s s u m p t i o n t h a t t h e s e l f r a t i n g s a r e t h e o b j e c t i v e l y c o r r e c t o n e s s i n c e t h e r e l e v a n c e o f r a t e d p e r c e i v e d e x e r t i o n ( r p e ) o n b o r g ' s s c a l e t o p h y s i o l o g i c a l s t r e s s p a r a m e t e r s i n c o n n e c t i o n w i t h t h e w o r k e r ' s o w n r a t i n g s h a s a 1 r e a d y b e e n d e m o n s t r a t e d (5). 4 method l i f t i n g o f b o x e s a p a n e l o f 6 8 i n d i v i d u a l s r a t e d t h e p e r c e i v e d e x e r t i o n o f a w o r k e r l i f t i n g a b o x w h o s e w e i g h t w a s u n k n o w n t o h i m a s w e l l a s t o t h e o b s e r v e r s . a l l p a r t i c i p a t i n g i n d i v i d u a l s h a d p r e v i o u s e x p e r i e n c e w i t h b o r g ' s s c a l e . t h e w e i g h t o f t h e b o x e s v a r i e d b e t w e e n 1 . 5 a n d 2 2 . 5 k g i n 6 8 p r o g r e s s i v e i n c r e m e n t s . a t f i r s t t h e b o x w a s l i f t e d , u s i n g a t w o h a n d g r i p , o n l y a f e w c e n t i m e t r e s f r o m t h e f l o o r , w i t h t h e k n e e s s t r a i g h t a n d t h e b a c k b e n t a t a n a n g l e o f 90'. n e x t t h e w o r k e r l i f t e d t h e b o x t o a p o s t u r e w i t h h i s b a c k b e n t f o r w a r d a t a 45' a n g l e . l a t e r o n t h e w o r k e r w a s a l l o w e d t o l i f t t h e b o x i n a n y m a n n e r d e s i r e d u n t i l a n u p r i g h t p o s t u r e w a s r e a c h e d w i t h h i s a r m s e x t e n d e d s t r a i g h t f o r w a r d w h i l e h o l d i n g t h e b o x . f i n a l l y , t h e b o x w a s a g a i n l i f t e d t o a 45' f o r w a r d i n c l i n a t i o n o f t h e b a c k , a t w h i c h t i m e t h e w e i g h t r a n g e a s w e l l a s t h e n u m b e r o f r e p e t i t i o n s o f e a c h l i f t w a s i n c r e a s e d t o t h e m a x i m u m ( 8 ) . a l l l i f t s l a s t e d 1 5 s e c o n d s f r o m t h e s t a r t i n g s i g n a l u n t i l t h e s i g n a l t o p u t t h e b o x d o w n w a s g i v e n . b e t w e e n t h e l i f t s t h e w o r k e r r e s t e d f o r 4 5 6 0 s e c o n d s . c o m b i n e d s t a t i c a n d d y n a m i c w o r k o v e r 8 h o u r s t h e s e c o n d e x p e r i m e n t i n v o l v e d a f o r m o f p h y s i c a l s t r e s s s i m i l a r t o a n a c t u a l i n d u s t r i a l s i t u a t i o n w o r k a t a c o n v e y o r b e l t . t h e e x p e r i m e n t c o m p r i s e d a f u l l w o r k i n g d a y d u r i n g t h e c o u r s e o f w h i c h t h e s a m e l o a d r e c u r r e d s e v e r a l t i m e s i n o r d e r t o e l u c i d a t e t h e e f f e c t o f c o n t i n u a l f a t i g u e . t h e d e v i c e u s e d c o n s i s t e d o f a c o n v e y o r b e l t t h a t f e e d s o u t b l o c k s a t o p t i o n a l i n t e r v a l s o f t i m e . t h e s e b l o c k s , w h o s e o u t e r d i m e n s i o n s a r e t h e s a m e f o r a l l w e i g h t s , w e r e m o v e d a b o u t 5 0 cm d o w n w a r d s , w h e r e u p o n t h e w o r k e r l i f t e d t h e m u p a g a i n . 5 t h e s e c o n d e x p e r i m e n t c o m p r i s e d 4 s e r i e s l a s t i n g 8 h o u r s . e a c h , s u c h s e r i e s c o m p r i s e d 9 d i f f e r e n t l o a d s i t u a t i o n s d e f i n e d b y 3 d i f f e r e n t w e i g h t s o f t h e b l o c k s : 0.1, 2 a n d 5 k g ; a n d 3 d i f f e r e n t r a t e s o f w o r k : 1 2 . 5 , 2 5 a n d 3 7 b l o c k s p e r m i n u t e . t h e s e 9 w o r k l o a d s w e r e v a r i e d w i t h i n e a c h s e r i e s . d u r i n g a1 1 e x p e r i m e n t s t h e w e i g h t s w e r e v a r i e d q u a s i r a n d o m l y . t h i s means t h a t t h e i n c r e m e n t s f r o m l o w e r t o h i g h e r w e i g h t s , a n d v i c e v e r s a , w e r e n o t a l l o w e d t o f o r m p r o g r e s s i v e s e r i e s . n o r w e r e t h e h e a v y o r l i g h t w e i g h t s a l l o w e d t o a c c u m u l a t e a t t h e b e g i n n i n g o r e n d o f t h e e x p e r i m e n t s . results l i f t i n g o f b o x e s t h e f i r s t e x p e r i m e n t a l s e r i e s p r o d u c e d r e s u l t s t h a t i n d i c a t e d a c l e a r c o r r e l a t i o n t h a t m a k e s i t p o s s i b l e t o u s e b o r g ' s s c a l e i n c o n n e c t i o n w i t h o b s e r v e r r a t i n g s , p r o v i d e d t h a t c e r t a i n p o i n t s a r e o b s e r v e d . t h e r e s u l t s show t h a t t h e o r d e r i n w h i c h t h e w e i g h t s a r e p r e s e n t e d s o m e t i m e s i n f l u e n c e s t h e r a t i n g o f t h e i n t e n s i t y o f t h e e f f o r t . t h e l a t e r i n t h e e x p e r i m e n t a w e i g h t i s p r e s e n t e d , t h e h e a v i e r i t i s r a t e d . t h i s may b e t h e r e s u l t o f i n c r e a s i n g f a t i g u e a s t h e e x p e r i m e n t s p r o g r e s s e d . c o n s e q u e n t l y , t h e r e c o v e r y p e r i o d o f 4 5 6 0 s e c p r o b a b l y was n o t l o n g e n o u g h . on t h e o t h e r h a n d , t h e p a n e l i s t s ' r a t i n g s s e e m e d t o r e f l e c t t h e s t r e s s r e s u l t i n g f r o m t h e w o r k a n d w e r e n o t m e r e l y a n a t t e m p t t o e s t i m a t e t h e w e i g h t o f t h e b o x . t h e p a n e l t e n d e d t o o v e r e s t i m a t e l o w l o a d s a n d u n d e r e s t i m a t e d h i g h o n e s ( f i g 1). t h i s c a n b e i n t e r p r e t e d a s a c e r t a i n d e g r e e o f o v e r e s t i m a t i o n o f t h e w o r k e r ' s m o v e m e n t s a n d u n d e r e s t i m a t i o n o f t h e w e i g h t o f t h e l o a d . 6 o m a e n v a n a u a i w o w o n a c n 6 i s t 2 . s 1 e . s 2 a . s f i g . 1. c o r r e l a t i o n b e t w e e r : r a t i n g s b y t h e o b s e r v e r s a n d b y t h e w o r k e r f o r t h e l i f t i n g o f b o x e s o f s i x d i f f e r e n t w e i g h t s i n t h e f i r s t t h r e e t e s t s i n t h e f i r s t e x p e r i m e n t . a l l o f t h e e x p e r i m e n t s p r o d u c e d r e l a t i v e l y g o o d c o r r e l a t i o n s b e t w e e n t h e r a t i n g s a r r i v e d a t b y t h e p a n e l a n d b y t h e w o r k e r h i m s e l f . t h e r e g r e s s i o n l i n e o f b e s t f i t f o l l o w e d a l o g a r i t h m i c f u n c t i o n ( f i g . 2 1 , w h i c h c a n b e c h a r a c t e r i z e d b y a f o r m u l a o f t h e t y p e : w o r k e r ' s r a t i n g = a x i n ( o b s e r v e r r a t i n g ) + b a, b = c o n s t a n t s y = 6 . 5 8 8 1 1 n x 3 . 5 1 1 r = 0.9765 7 w o r k e r r a t i n g s rnax 10 a 6 4 2 0 a o d ' .. 0 f 1 r i t t e i t ) s e c o n d 1 e . t t n i n o t e a l . .. * f i g . 2. c o r r e l a t i o n b e t w e e n r a t i n g s b y t h e o b s e r v e r s a n d t h e w o r k e r h i m s e l f , o n b o r g ' s s c a l e . s u m m a r y o f t h e f i r s t t h r e e t e s t s i n t h e f i r s t e x p e r i m e n t , l i f t i n g o f b o x e s o f d i f f e r e n t u n k n o w n w e i g h t s . t h e r e l a t i o n s h i p b e t w e e n t h e w o r k e r ' s r a t i n g a n d t h o s e o f t h e p a n e l i n t h e f o u r t h t e s t i s p r e s e n t e d i n f i g u r e 3 . a l i n e t h a t b e s t f i t s t h e s c a t t e r o f d o t s r u n s a c o u r s e t h a t i s p r a c t i c a l l y p a r a l l e l t o t h e o n e o b t a i n e d f o r t h e s t a t i c w o r k i n t h e f i r s t t h r e e t e s t s . 8 w o r k e r r a t i n g s y i 6.191nx + 5.06 8 6 . ..< . . .i.. 1 9 . . .,:h *.i.. , .. . 4 -. #.*.w ..& .. . .i .. 2 . .:.p 0 . . ... * ..r t" . i .., 2 / i'h.lt1 f. * , ..$.f w m -1 * ' # . + ,. ., . . i . . * 0 2 4 6 8 1 0 o b s e r v e r r a t i n g s f i g . 3 . c o r r e l a t i o n b e t w e e n r a t i n g s b y t h e o b s e r v e r s a n d b y t h e w o r k e r f o r t h e l i f t i n g o f b o x e s i n t h e f o u r t h t e s t i n t h e f i r s t e x p e r i m e n t . i f we t a k e a l o o k a t t h e m e a n r a t i n g s o f t h e i n d i v i d u a l m e m b e r s o f t h e p a n e l , we f i n d c o n s i d e r a b l e v a r i a t i o n ( t a b l e 1 ) . t h e m e a n f o r a l l m e m b e r s o f t h e p a n e l i s 4 . 3 4 , r a n g e d b e t w e e n 3 . 0 1 a n d 7 . 5 8 . h o w e v e r , t h e m a j o r i t y o f t h e p a n e l m e m b e r s u s e d 8 9 p o i n t s o f t h e 1 0 p o i n t s c a l e . i t c a n b e i n t e r p r e t e d t o m e a n t h a t t h e d i f f e r e n t o b s e r v e r s t e n d t o c o n c e n t r a t e t h e i r r a t i n g s w i t h i n a c e r t a i n p o r t i o n o f t h e s c a l e , t h e d i f f e r e n c e s b e i n g i n f l u e n c e d b y i n d i v i d u a l f a c t o r s . 9 t a b l e 1. means a n d d i s p e r s i o n s o f t h e r a t i n g s b y 8 o b s e r v e r s a n d b y t h e w o r k e r h i m s e l f f o r t h e 6 4 l o a d s i t u a t i o n s ( 8 w e i g h t s , 8 r e p e t i t i o n s ) i n t h e f o u r t h t e s t i n t h e f i r s t e x p e r i m e n t . worker 3 piean rating might (kg) 1.5 2.1 2.9 4.0 5.8 0.2 11.3 15.8 worker 's s.d observer panelists' m n ratings m s l -2 rrs3 m s 4 m s 5 m s 6 m s 7 0.92 0.92 0.76 1.33 0.71 1.24 1.24 1.05 2.63 2.63 3.38 5.13 1.75 2.69 3.25 2.63 2.25 3.38 4.00 5.25 2.50 3.03 2.88 2.63 3.38 4.00 5.50 5.63 4.25 3.88 4.00 3.88 3.25 4.13 5.75 6.00 3.13 3.63 3.13 1.75 3.43 4.71 5.14 5.00 3.43 3.57 3.29 4.00 4.38 5.50 7.50 6.25 4.50 4.13 4.50 3.38 4.00 5.00 7.75 7.63 5.25 5.56 5.75 6.88 5.50 7.50 8.25 9.00 7.88 6.38 8.13 8.00 3.01 3.24 4.32 3.85 4.07 5.02 5.98 7.58 rating 0.81 1.44 1.88 2.75 3.29 4.44 5.63 8.00 0.35 0.46 0.54 0.43 0.70 0.58 1.22 1.12 a s t u d y o f t h e m a n n e r i n w h i c h t h e w o r k e r a n d t h e o b s e r v e r s r a t e d t h e same l o a d w h e n i t r e c u r r e d d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t s h o w s t h a t t h e w o r k e r i s a b l e t o d i s c r i m i n a t e b e t w e e n t h e d i f f e r e n t 1 o a d s c o n s i d e r a b l y b e t t e r t h a n t h e p a n e l . a b o v e a l l , i t was d i f f i c u l t f o r t h e p a n e l t o d i f f e r e n t i a t e b e t w e e n l o w l o a d s . a t h i g h l o a d s t h e p a n e l i s t ' s r a t i n g s c o r r e l a t e d c o n s i d e r a b l y b e t t e r w i t h t h e a c t u a l w e i g h t o f t h e b o x a n d t h e w o r k e r ' s s e l f r a t i n g . 10 c o m b i n e d s t a t i c a n d d y n a m i c w o r k o v e r a p e r i o d o f 8 h o u r s t h e e x p e r i m e n t s s h o w e d t h a t a l l s u b j e c t s w e r e a b l e t o c l e a r l y d i s c r i m i n a t e b e t w e e n t h e d i f f e r e n t w e i g h t s . a p o s i t i v e l y i n c r e a s i n g f u n c t i o n f o r t h e d i f f e r e n t l o a d s was n o t e d , a s w e l l a s a f a t i g u i n g e f f e c t w i t h t h e p a s s a g e o f t i m e . on s t u d y i n g t h e t r e n d i n t h e a s s e s s m e n t o f w e i g h t r a t e v a r i a n t s o n b o r g ' s s c a l e i t was f o u n d t h a t , r e g a r d l e s s o f t h e r a t e , t h e a s s e s s m e n t i n c r e a s e s w i t h i n c r e a s i n g w e i g h t . t h i s i s w e l l i n l i n e w i t h s t u d i e s made o n b o r g ' s s c a l e i n c o n n e c t i o n w i t h i t s c o n s t r u c t i o n . a l l s u b j e c t s w e r e a b l e t o d i s c r i m i n a t e b e t w e e n t h e d i f f e r e n t w e i g h t s . on s t u d y i n g t h e r e l a t i o n s h i p b e t w e e n r a t i n g s o n b o r g ' s s c a l e a n d t h e a c t u a l a m o u n t o f w o r k d o n e i t was f o u n d t h a t b o t h t h e p a n e l a n d t h e w o r k e r s h o w e d a p o s i t i v e l y r i s i n g t r e n d i n r a t i n g s w i t h i n c r e a s i n g w o r k . a s t u d y o f t h e t r e n d w i t h r e g a r d t o how t h e same l o a d s w e r e r a t e d when t h e y o c c u r r e d a t d i f f e r e n t t i m e s o f t h e d a y , s h o w s t h a t t h e w o r k e r r a t e d t h e l o w l o a d s m o r e c o n s i s t e n t l y t h a n t h e p a n e l r e g a r d l e s s o f t h e t i m e o f d a y a t w h i c h t h e y o c c u r r e d ( f i g 4 ) . t h e h i g h l o a d s show a s l i g h t t e n d e n c y t o b e p e r c e i v e d t o b e h e a v i e r l a t e r d u r i n g t h e d a y . the mean r a t i n g s i n c r e a s e d f r o m 2 . 1 i n t h e f i r s t s e r i e s t o 2.5 i n t h e l a s t o n e . i n c o n t r a s t , t h e p a n e l s h o w s a p r o g r e s s i v e i n c r e a s e i n t h e e s t i m a t e d v a l u e s a t t h e r a t e s o f 1 2 . 5 a n d 3 7 b l o c k s / m i n . h e r e t h e a v e r a g e v a l u e p e r s e r i e s r o s e f r o m 1 . 4 f o r t h e f i r s t s e r i e s t o 2 . 9 f o r t h e l a s t o n e , i . e . a l m o s t d o u b l e t h e o r i g i n a l f i g u r e . t h i s i n d i c a t e s a c l e a r t e n d e n c y t o f a t i g u e d u r i n g t h e d a y . 11 perceived exertion borgs scale 5 4 3 2 1 a 7 w c en s? o n s e n v e n s 2 6 w o r k e r 2 5 o q s e r v e r s 1 2 . 5 o n s e r v e n s 12.5 w o r k e r tlme 8.45 9.45 10.45 12.00 13.00 14.00 15.00 16.19 f i g . 4. c o r r e l a t i o n b e t w e e n r a t i n g s b y t h e o b s e r v e r s a n d b y t h e w o r k e r f o r t h e d i f f e r e n t l o a d s o v e r t h e w h o l e p e r i o d o f w o r k i n t h e s e c o n d e x p e r i m e n t . t h e a s s e m b l y l i n e t y p e w o r k i n v o l v e d l i f t i n g o f b l o c k s o f d i f f e r e n t w e i g h t s f e d a t t h r e e r a t e s : 1 2 . 5 , 2 5 a n d 3 7 b l o c k s p e r m i n u t . t h e r a t i n g s s h o w t h a t t h e w o r k r e l a t e d s t r e s s d o e s n o t c o v e r a s w i d e a r a n g e o f v a r i a t i o n a s w a s a s s u m e d w h e n t h e e x p e r i m e n t s w e r e b e i n g p l a n n e d . t h i s i s p r o b a b l y d u e t o t h e f a c t t h a t t h e w e i g h t o f t h e a r m s t h e m s e l v e s c o n s t i t u t e s a l a r g e p o r t i o n o f t h e t o t a l l o a d . t h e w o r k e r ' s s e l f r a t i n g s r a n g e b e t w e e n 0 a n d 6 , w h i c h m e a n s t h a t h e a l s o u s e d t h e same p o r t i o n o f b o r g ' s s c a l e a s t h e o b s e r v e r s . a l o o k a t t h e i n d i v i d u a l v a r i a t i o n i n t h e o b s e r v e r s ' r a t i n g s s h o w s t h a t t h e a v e r a g e d i p e r s i o n f o r a l l 3 6 s i t u a t i o n s w a s 12 s.d. = 0 . 8 5 ( t a b l e 2 ) . f o r t h e i n d i v i d u a l s i t u a t i o n s , s.d. r a n g e s b e t w e e n 0 . 1 a n d 1 . 3 . f u r t h e r , t h e v a l u e o f t h e o b s e r v e r ' s a v e r a g e r a t i n g s r a n g e d b e t w e e n 0.7 a n d 4 . 1 o n b o r g ' s s c a l e . t h u s o n l y a b o u t 4 0 % o f t h e p o t e n t i a l o f t h e 1 0 p o i n t s c a l e h a s b e e n u t i l i z e d . i n d i v i d u a l l y , t h e p a n e l i s t s u s e d v a l u e s b e t w e e n 0 a n d 7 . t h e d e s i g n o f t h e e x p e r i m e n t w a s b a s e d o n a r e a l i n d u s t r i a l s i t u a t i o n , w o r k a t a c o n v e y o r b e l t . s u c h w o r k i s c h a r a c t e r i z e d b y m o d e r a t e p h y s i c a l s t r e s s . f u r t h e r m o r e , t h e w o r k e r w a s i n v e r y g o o d p h y s i c a l c o n d i t i o n . a s a r e s u l t , t h e w o r k l o a d w a s c o n c e n t r a t e d o n o n e p o r t i o n o f t h e 1 0 p o i n t s c a l e . t a b l e 2 . m e a n s a n d d i s p e r s i o n s o f t h e r a t i n g s b y 6 o b s e r v e r s a n d b y t h e w o r k e r h i m s e l f f o r t h e 3 6 l o a d s i t u a t i o n s ( 9 w e i g h t / r a t e c o r n b i n a t i o n s , 4 r e p e t i t i o n s ) i n w o r k i n v o l v i n g l i f t i n g b l o c k s a t a c o n v e y o r b e l t . s i n c e t h e c o m p l e t e r a n g e o f b o r g ' s s c a l e w a s n o t u s e d i n t h e e x e c u t i o n o f t h e s e c o n d e x p e r i m e n t o w i n g t o t h e f a c t t h a t v e r y h e a v y l o a d s d i d n o t o c c u r , t h e r e s u l t s s h o w a n a c c u m u l a t i o n o f v a l u e s o n t h e l o w e r p o r t i o n o f t h e s c a l e ( f i g . 5). t h i s , i n t u r n , m e a n s t h a t t h e c a l c u l a t e d r e g r e s s i o n l i n e m u s t b e i n t e r p r e t e d c a u t i o u s l y , a n d e x t r a p o l a t i n g t o h i g h e r v a l u e s o n t h e 1 0 p o i n t s c a l e i s i n a d m i s s i b l e . 13 w o r k e r r a t i n 0 0 m . 7 . 1 y1.246+1.923*lnx r 0 . 7 7 2 (n 36) t * & . , + a 1 8 8 i o i i e r v e r r a l l w o o 5. c o r r e l a t i o n b e t w e e n r a t i n g s b y t h e o b s e r v e r s a n d b y yt e w o r k e r h i m s e l f o n b o r g l s s c a l e . summary o f t h e e n t i r e s e c o n d e x p e r i m e n t w o r k o n a c o n v e y o r b e l t . d i s c u s s i o n i n a l l w o r k s i t u a t i o n s t h e o b s e r v e r s w e r e a b l e t o d i s c r i m i n a t e b e t w e e n t h e d i f f e r e n t l o a d s . i n c a s e s w h e r e t h e l o a d e n c o m p a s s e d t h e w h o l e s p e c t r u m o f w e i g h t s , i . e . w e i g h t s r a n g i n g f r o m v e r y l o w t o m a x i m a l , t h e o b s e r v e r s made u s e o f p e r c e p t u a l r a t i n g s o v e r t h e e n t i r e s c a l e . t h i s was a d v a n t a g e o u s t o e s t a b l i s h a r e l i a b l e r e l a t i o n s h i p b e t w e e n o b s e r v e r r a t i n g s a n d s e l f r a t i n g s . i n o t h e r c a s e s , s u c h a s w o r k a t t h e c o n v e y o r b e l t , t h e c u r v e e n c o m p a s s e d o n l y a p o r t i o n o f t h e s c a l e r a n g e . t h r o u g h o u t t h e e x p e r i m e n t s t h e c u r v e s f o l l o w e d a p o s i t i v e l y a c c e l e r a t i n g p o w e r f u n c t i o n w i t h i n c r e a s i n g w o r k l o a d s , a s h a d b e e n d e m o n s t r a t e d i n b o r g l s e a r l i e r s t u d i e s ( 4 ) . t h e c o r r e l a t i o n b e t w e e n t h e o b s e r v e r s ' r a t i n g a n d t h e w o r k e r ' s r a t i n g s was n o t l i n e a r , b u t f o l l o w e d a c u r v e d l i n e t h a t c o u l d b e b e s t d e s c r i b e d b y a l o g a r i t h m i c f u n c t i o n . a n o t h e r f i n d i n g 14 was t h a t t h e o b s e r v e r s t e n d e d t o o v e r r a t e t h e l o w l o a d s , w h e r e a s t h e r a t i n g s f o r t h e h i g h e r l o a d s s h o w e d b e t t e r a g r e e m e n t . i n p r i n c i p l e , w i t h t h e a i d t h e l o g a r i t h m i c f u n c t i o n , i t s h o u l d b e p o s s i b l e t o f i g u r e o u t how a w o r k e r w o u l d i n d i c a t e h i s own p e r c e p t i o n o f t h e s t r e s s i f t h e o b s e r v e r s ' r a t i n g o f t h e same w o r k i s k n o w n . t h e e x p e r i m e n t s a l s o s h o w e d t h a t t h e o b s e r v e r s t e n d e d t o r a t e a c c o r d i n g t o a c e r t a i n i n d i v i d u a l l y c o l o u r e d p a t t e r n . t h e m o s t n o t i c e a b l e f e a t u r e was t h a t c e r t a i n i n d i v i d u a l s t e n d e d t o d e v i a t e c o n s i s t e n t l y e i t h e r t o w a r d s l o w e r o r t o w a r d s h i g h e r r a t i n g s , s o c a l l e d " 1 o w r a t e r s " a n d " h i g h r a t e r s " . t h i s i s a f a i r l y we1 1 k n o w n e f f e c t m e n t i o n e d i n t h e 1 i t e r a t u r e a s t h e r a n g e e f f e c t . i t c a n b e c o r r e c t e d , i f s o d e s i r e d , b y c a l c u l a t i n g t h e i n d i v i d u a l d e v i a t i o n f r o m a mean f o r a n u m b e r o f o b s e r v e r s a n d t h e n c o r r e c t i n g a l l o f t h e i n d i v i d u a l s r a t i n g s b y a s i m p l e f a c t o r s o t h a t h i s mean a g r e e s w i t h t h e common mean. a n o t h e r t y p e o f p e r s o n a l d e v i a t i o n s e e m e d t o b e t h e r a n g e u s e d b y t h e o b s e r v e r , i . e . t h e d i f f e r e n c e b e t w e e n t h e h i g h e s t a n d t h e l o w e s t r a t i n g i n d i c a t e d . t h e r e w e r e i n d i v i d u a l s who u s e d t h e c o m p l e t e r a n g e o f t h e 1 0 p o i n t s c a l e a n d o t h e r s who t e n d e d t o r e s t r i c t t h e m s e l v e s t o a l i m i t e d a r e a o f t h e s c a l e . a l t h o u g h t h e o b s e r v e r s w e r e t r a n i n e d i n r p e , t h e s e d i f f e r e n c e s i n t h e u s e o f t h e s c a l e c o u l d b e c o n f i r m e d , i n c o n f o r m i t y w i t h t h e f i n d i n g s o f d a s ( 8 ) a n d s u r y ( 1 7 ) . i f a c o r r e c t i o n i s t o b e made, t h e b e s t s o l u t i o n w o u l d p r o b a b l y b e t o i n t r o d u c e a t e s t o f t h e r a t e r s ' a b i l i t y t o u s e d i f f e r e n t i a t e d s c a l e s o f t h e b o r g t y p e , l i k e t h e o n e s t h a t h a v e a l r e a d y b e e n u s e d b y h i m . a s p e c i a l c a s e p r e s e n t s i t s e l f when t h e " m a x i m u m l o a d " r a t i n g i s i n d i c a t e d o n b o r g ' s s c a l e . t h i s i n c r e m e n t o n t h e s c a l e i s t h e o n l y o n e w i t h o u t a n u m e r i c a l v a l u e f o r t h e m a g n i t u d e o f t h e l o a d . t h e p u r p o s e o f t h i s i s t o make i t p o s s i b l e f o r t h e o b s e r v e r t o i n d i c a t e e x c e p t i o n a l l o a d s w h o s e o c c u r r e n c e i s e x t r e m e l y r a r e . it i s a l s o c o n c e i v a b l e t o d e s i g n a t e l o a d s b e y o n d t h e c a p a b i l i t y o f t h e w o r k e r a s " m a x i m a l " . 15 i n t h e l i g h t o f t h e e x p e r i m e n t a l r e s u l t s , t h e s u i t a b i l i t y o f b o r g ' s c a t e g o r y s c a l e w i t h r a t i o p r o p e r t i e s f o r u s e i n w o r k a n a l y s e s b a s e d o n o b s e r v e r s ' r a t i n g s c a n b e a s s u m e d . h o w e v e r , a n i m p r o v e m e n t i n t h i s r e s p e c t c o u l d b e i n t r o d u c t i o n o f a c o r r e c t i o n f a c t o r f o r t h e o b s e r v e r s ' r a t i n g s . t h i s f a c t o r c o u l d b e s u i t a b l y f o r m u l a t e d a s t h e common r e g r e s s i o n 1 i n e w i t h l o g a r i t h m i c c h a r a c t e r i s t i c s . acknowledgements t h i s s t u d y i s b a s e d o n d a t a c o l l e c t e d i n 1 9 8 2 w i t h i n t h e f r a m e w o r k o f t h e arban p r o j e c t , s u p p o r t e d b y r e s e a r c h g r a n t s 7 9 / 8 2 f r o m t h e s w e d i s h w o r k e n v i r o n m e n t f u n d t o b y g g h a l s a n r e s e a r c h f o u n d a t i o n . 1. 2 . 3 . 4. 5. 6 . 7 . 8 . 9 . 1 0 . references b a s m a j i a n , i . v . : m u s c l e s a l i v e , c h a p t e r 2. w i l l i a m & w i l k i n s c o m p a n y , b a l t i m o r e , usa, 4 t h e d i t i o n , 1 9 7 9 . b o r g , g . a . v . : p h y s i c a l p e r f o r m a n c e a n d p e r c e i v e d e x e r t i o n . i n : s t u d i a p s y c h o l o g i a e t p a e d a g o g i c a , s e r i e s a l t e r a , i n v e s t i g a t i o n s x i , l u n d , 1 9 6 2 . b o r g , g . a . v & n o b l e , 6 . : p e r c e i v e d e x e r t i o n . i n : e x e r c i s e a n d s p o r t s c i e n c e s r e v i e w s ( v o l . 1 1 ) . ( e d . j.h. w i l m o r e ) . new y o r k . a c a d e m i c p r e s s i n c . , 1 9 7 4 . b o r g , g . a . w . : p h y s i c a l w o r k a n d e f f o r t . w e n n e r g r e n c e n t e r i n t e r n a t i o n a l s y m p o s i u m s e r i e s , v o l u m e 2 8 . p e r g a m o n p r e s s , 1 9 7 7 . b o r g , g . a . v . : a c a t e g o r y s c a l e w i t h r a t i o p r o p e r t i e s f o r i n t e r m o d a l a n d i n t e r i n d i v i d u a l c o m p a r i s o n s . i n : p s y c h o p h y s i c a l j u d g m e n t a n d t h e p r o c e s s o f p e r c e p t i o n . i e d . g . g . g e i s s l e r & p. p e t z o l d ) . b e r l i n : veb d e u t s c h e r v e r l a g d e r w i s s e n s c h a f t e n , 1 9 8 2 . b o r g , g . , h e r b e r t , a . & c e c i , r . : some c h a r a c t e r i s t i c s o f a s i m p l e r u n t e s t a n d i t s c o r r e l a t i o n w i t h a b i c y c l e e r g o m e t e r t e s t o f p h y s i c a l w o r k i n g c a p a c i t y . r e p o r t s f r o m t h e d e p a r t m e n t o f p s y c h o l o g y , t h e u n i v e r s i t y o f s t o c k h o l m , 1 9 8 4 . c o r l e t t , e . n . & b i s h o p , r . p . : a t e c h n i q u e f o r a s s e s s i n g p o s t u r a l d i s c o m f o r t . e r g o n o m i c s 1 9 , 2 : 1 7 5 1 8 2 , 1 9 7 6 . d a s , 6 . : a s t a t i s t i c a l i n v e s t i g a t i o n o f t h e e f f e c t o f p a c e a n d o p e r a t i o n o n p e r f o r m a n c e r a t i n g . i n t . j. p r o d . r e s . k i r k , n.s. & s a d o y a m a , t.: a r e l a t i o n s h i p b e t w e e n e n d u r a n c e a n d d i s c o m f o r t i n s t a t i c w o r k . u n p u b l i s h e d m . s c . r e p o r t , d e p a r t m e n t o f human s c i e n c e s , u n i v e r s i t y o f t e c h n o l o g y , l o u g h b o r o u g h , 1 9 7 3 . m a r k s , l . e . : i n d i v i d u a l d i f f e r e n c e s i n p e r c e i v e d e x e r t i o n a s s e s s e d b y t w o n e w m e t h o d s . p e r c e p t i o n & p s y c h o p h y s i c s y o 1 3 , n o 1: 6 5 7 2 , 1 9 6 5 . 3 4 , 3 : 2 8 0 2 8 8 , 1 9 8 3 . 16 11. m i h e v i c , p . m . : s e n s o r y c u e s f o r p e r c e i v e d e x e r t i o n : a 1 2 . p e a r s o n , r . g . : s u b j e c t i v e a s p e c t s o f p e r f o r m a n c e . i n : r e v i e w . m e d i c i n e a n d s c i e n c e i n s p o r t s a n d e x e r c i s e . 1 3 , h a n d b o o k o f i n d u s t r i a l e n g i n e e r i n g . ( e d . g . s a l v e n d y ) . w i l e y & s o n s , 1 9 8 2 . 1 3 . r o h m e r t , w . & l a u r i g , w . : e v a l u a t i o n o f w o r k r e q u i r i n g p h y s i c a l e f f o r t . i n s t i t u t e o f i n d u s t r i a l s c i e n c e , d a r m s t a d t p o l y t e c h n i c , 1 9 7 5 . 1 4 . r u n e s s o n , s . & f r y k h o l m , g.: y i s u a l p e r c e p t i o n o f l i f t e d w e i g h t . r e p o r t no. 2 6 8 , d e p t . o f p s y c h o l o g y , u n i v e r s i t y o f u p p s a l a , 1 9 7 9 . 1 5 . s t e v e n s , s . s . : on t h e p s y c h o p h y s i c a l l a w . p h y c h o l o g i c a l r e v i e w 5 4 , 1 5 3 1 8 1 , 1 9 5 7 . 1 6 . s t e v e n s , s . s . : i s s u e s i n p s y c h o p h y s i c a l m e a s u r e m e n t . p h y c h o l o g i c a l r e v i e w 7 8 : 4 2 6 4 5 0 , 1 9 7 1 . 1 7 . s u r y , r.j.: a c o m p a r a t i v e s t u d y o f p e r f o r m a n c e r a t i n g s y s t e m s . i n t . 3 . p r o d . r e s . v o l . 1 , no. 2 : 2 3 3 8 , 1 9 6 2 . 3 ~ 1 5 0 1 6 3 , 1 9 8 1 . a d d r e s s f o r r e p r i n t s : m i c h a e l w a n g e n h e i m r e s e a r c h f o u n d a t i o n f o r o c c u p a t i o n a l s a f e t y a n d h e a l t h i n t h e s w e d i s h c o n s t r u c t i o n i n d u s t r y b o x 9 4 s i 8 2 11 d a n d e r y d s w e d e n 287857 1 17 upsala j med sci 81: 167-173, 1976 acute myopericarditis a long-term follow-up study e. sanner, g. sigurdsson, d. gislasson, b. gudbrandsson and m. stefansson from the department of medicine, central hospital, eskilstuna, sweden abstract a follow-up study was made of 29 patients aged 21 to 45 years, some 15-158 months after acute myopericarditis. the mean follow-up period was 72.9 months. the follow-up investigation included recording of history, physical exami nation, laboratory tests, radiologic examination of the heart and lungs and electrocardiography. all hut one of the pa tients were fit for fulltime work. nine had residual cardiac symptoms, but the physical examination was negative in all but 2 of them. one patient had chronic cardiac insufficiency and hepatic enlargement. another had sinus tachycardia and cardiac enlargement of moderate degree and impaired working capacity in relation to heart size. cardiac murmurs without clinical significance were audible in three cases. the resting ecg was pathologic in only 6 cases. orthostatic ecg evoked ecg abnormalities in 6 more cases. exercise tolerance tests showed reduced working capacity in relation to heart volume in 5 of the 29 cases (17%). four of these 5 patients had cardiac enlargement. there was thus good correlation between increase in heart volume and reduction of physical capacity. the prognosis in regard to cardiac function was good, as was also found in other comparable series in which the observation time was somewhat shorter. introduction acute myopencarditis can occur in previously healthy persons in connexion with acute infections of varying aetiology. in many cases the precise aetiology cannot be established ( 1 , 16). in the diffe rential diagnosis cardiomyopathy , ischaemic heart disease, collagenosis, rheumatic fever, tuberculosis and sarcoidosis must be excluded. some reports have recently been published on the incidence and course of acute myopericarditis (11, 3, 7, 2, 6). the prognosis of acute myo pericarditis in regard to cardiac function is con sidered to be good, however, a few deaths and occasional cases of chronic cardiac insufficiency have been reported (11, 16, 13). because of the paucity of long-term observations, we have fol lowed up a selected series of patients who were treated at the medical clinic of eskilstuna for myopericarditis between 1958 and 1970 (one patient was hospitalized in stockholm). our primary inten tion was to study the long-term prognosis in regard to cardiac function. the observation periods ranged from 15 to 158 months (mean 72.9 months). material and methods patients: the records of 64 cases classified as acute myopericarditis were reviewed. this diagnosis was altered in 2 patients who died soon after admission (of carbon monoxide and thioridazine intoxication). other reasons for exclusion from the study were death from malignant disease in two cases and uncertain diagnosis in three. thirteen patients could not be traced (most had left the district). in order to exclude ischaemic heart disease, the 15 patients who were older than 45 years at the time of follow-up were not studied. the clinical material then consisted of 29 patients aged 21 to 45 years (mean 33.9 years), of whom 15 were men aged 21 to 43 (mean 31.7) years and 14 women aged 26 to 45 (mean 36.2) years. none of the patients had diabetes, hypertension, angina pectoris, collagenosis or rheumatic heart disease. no patient was under digitalis treatment at follow-up. observations in the acute stage the clinical manifestations of acute myopericarditis has been reviewed by klainer (10). in the present series the commonest clinical manifestations were raised erythro cyte sedimentation rate (24 cases), praecordial pain (22 cases), fever (21 cases), leukocytosis (16 cases), pharyn gitis (14 cases), anaemia (13 cases), raised serum trans ferase (glutamic oxaloacetate transferase) level (9 cases) and joint and muscle pains (8 cases). the antistreptolysin titre was elevated in only 5 of the 26 tested patients, the c-reactive protein test was positive in 3 of 8 cases and the antinuclear factor (anf) was found in 6 of 10 cases. no le cells were found in the 13 cases in which the tests were made. upsala j med sci 81 168 e . sanner et al. table i. physical signs and ecg changes during the acute illness case no. sex periecg changes cardial (minnesota rub murmur code) 1 d 2 p 3 0 4 p 5 d 6 8 7 d 8 0 9 0 10 6 11 0 12 d 13 0 14 d 15 d 16 0 17 d 18 6 19 p 20 p 21 0 22 d 23 d 24 9 25 6 26 d 27 d 28 0 29 0 9-2,5-2 + 84,5-2,4-2 8-7,5-3,4-2 + + 9-1, &7,5-2 + + 9-1,8-1,5-2 + 51 5-2,4-3 9-1,s-3,4-3 5-2,4-1 9-2 5-3,4-3 + 9-2.5-1 + 5-3' + 8-7.5-2.4-2 + + + 9-2' 5-2 9-2,5-2 3-3 5-2 + 8-7 7-3,5-2,4-1 9-2,s-3 9-2,8-7,5-2 + 8-7,7-2 9-2 9-2,s-1 9-2,s-2 9-2,5-2 + 9-2 physical findings auscultation of the chest revealed nothing abnormal in 16 of the 29 patients. in all of the remaining 13 patients various physical signs were found during the acute iu ness-pericardial rub in 8 cases, systolic murmur in 6 cases, diastolic murmur (aortic insufficiency) in one case (case 14) (table i). bacteriology beta-haemolytic streptococci were found in throat cultures from 7 of 19 tested patients (cases 2,6, 10, 13,24, 26 and 27). urine cultures were positive in 3 of 14 patients (cases 3, 13 and 24), two of them also had beta-haemolytic streptococci in throat cultures. blood cultures were done in 9 cases and faeces cultures in 3 cases. all were nega tive. virology attempts to isolate virus from faeces and throat were made in only a few cases and were negative. serology serologic studies (for neutralizing antibodies and com plement fixation) were done in 10 cases and were nega tive. mononucleosis tests were done in 3 cases and gave positive result in one case (case 1). radiologic examination chest radiographs (table 11) were taken during the acute illness in all patients but one (case 28), who was pregnant. the heart was somewhat enlarged in 5 cases, in one of them to 600 ml/m2 body surface. some pulmonary conces tion was seen in 4 cases and pleural effusion in 2 cases. in case 25 there were pleural adhesions from a healed tuberculous process. case 19 had acute broncho pneumonia. electrocardiography electrocardiograms were taken on admission and thereaf ter at least once weekly, using standard leads i, i1 and 111 and praecordial leads cr 1,2,4,5 and 7. in all cases there were some abnormality of the ecg during the acute ill ness. the minnesota code (4) was used for classification (table i). t-wave changes were the only abnormality in 6 cases. t-wave changes together with st-segment depres sion occurred in 8 cases, together with st-segment eleva tion in 8 cases. thus t-wave changes either alone or in combination with st-segment abnormalities occurred in 22 cases, one of them also having incomplete right bundle branch block (case 21). st-segment elevation occurred in 4 cases and disturbances of rhythm in 8 cases. one of table 11. radiographic cardiac and pulmonary findings during the acute illness heart volume case (ml) no. sex totallrelative" pulmonary changes 1 6 2 p 3 0 4 p 5 6 6 6 7 8 8 p 9 0 10 d 11 0 12 d 13 9 14 6 15 8 16 p 17 d 18 d 19 p 20 0 21 p 22 d 23 d 24 0 25 6 26 d 27 d 28 p 29 p 1 1201570 4701300 5701400 4801320 7801435 1 1901600 7501420 2801215 4541280 56013 10 6701360 7751440 4801290 1480 6551350 1470 5501315 8601460 8401480 4251275 5201300 4901295 1 0001465 6701325 6051310 9351390 1 0901520 no x-rays 600/300 bilateral pleural effusion pulmonary congestion pulmonary congestion pleural effusion pulmonary congestion pulmonary congestion and bronchopneumonia pleural adhesions pulmonary emphysema pleural adhesions no x-rays a mllm2 body surface. upsala j med sci 81 acute myupericarditis 169 table 111. heart volume, work load and working capacity in relation t o heart volume at fullow-up case age heart volume (ml) pulse 170lmin in relation to no. (yrs) sex totallrelative (kpmlmin)" heart volume work load at working capacity 1 21 d 9801490 990 2 26 0 3901270 390 3 28 0 5301360 740 4 31 p 4201270 750 5 35 8 10301530 930 6 28 d 8101430 1 250 7 42 8 7601415 1090 8 43 0 4001300 300 9 43 0 50513 15 625 10 24 d 6801370 1200 11 45 p 9001480 830 12 25 d 7701430 950 13 45 0 5801335 300 14 37 d 9501540 700 15 37 d 8901470 910 16 43 p 7501390 7 10 17 32 6 7201400 1210 18 25 d 9401480 930 19 33 0 4201250 600 20 27 p 5101320 600 21 44 p '6501370 600 22 24 d no x-rays 625 23 43 d 1070/515 1 320 24 31 0 7001330 625 25 40 d 6901340 775 26 30 d 12101520 1 020 low 27 33 8 12101580 450 low 28 38 0 5901360 665 29 30 p 6001380 435 low lowb low conversion factor from traditional units to si units 1 kmp/min=o. 163 watt. mean load in males=957 kpmlmink244 s.d. (n=15). mean load in females=584 kpmlmink166 s.d. (n= 14). neurocirculatory asthenia. these 8 cases had supraventricular tachycardia and 7 cases had sinus tachycardia, among which one having also com plete right bundle-branch block. only occasionally were sporadic monofocal ectopic ventricular extra systoles and ectopic supraventricular extra systoles detectable (not tabulated). atrio-ventricular conduction defects were not seen. methods in follow-up studies anamnesis the medical history was closely explored, particularly in regard to residual cardiopulmonary symptoms. a sociomedical investigation was made of the patient's cur rent work situation, with emphasis on the possibility of heart-disease sequelae. physical examination the general examination included auscultation of the heart and lungs and measurement of the blood pressure, using a cuff mercury manometer. laboratory tests these included analyses in venous blood of haemoglobin, haematocrit, leukocytes, fasting blood sugar, cholesterol, creatinine, bilirubin and serum enzymes (glutamic oxalo acetatetxansferase and lactate dehydrogenase). radiologic examination x-rays of the heart and lungs in erect position were com pared with films taken during the acute illness. all com parisons were made by the same roentgenologist. the total and the relative heart size (ml/m2 body surface) were calculated according to jonsell (9). the accepted upper limits of normal value were 500 d / m a for men and 450 ml/mz for women. electrocardiography a direct writing ink-recorder (elema-schonander, stock holm) was used with standard leads i, i1 and i11 and praecordial leads cr 1, 2, 4, 5 and 7 for resting and orthostatic ecg. in exercise tolerance tests ch leads were used with the indifferent electrode on the forehead. ecg tracings were made after ten minutes in supine posi upsala j med sci 81 170 e . sanner et at. table iv. heart volume, ecg and working capacity at follow-up (in cases with pathologicfindings) pulse ecg after working capacity case rate heart volume ecg 8 min standecg during w 170 in relation to no. sex atrest totallrelative atrest ing position work heart volume 2 0 110 3901270 normal pathologic normal normal 4 0 76 4201270 normal pathologic normal normal 5 d 85 10301530 pathologic pathologic pathologic low 6 6 75 8 101430 pathologic pathologic pathologic normal 8 0 86 4001300 normal pathologic normal normal 9 p 83 50513 15 pathologic pathologic pathologic normal 11 0 75 9001480 pathologic pathologic pathologic normal 13 0 115 5801335 normal pathologic pathologic low' 14 d 105 9501540 normal normal normal low 18 8 66 9401480 normal pathologic pathologic normal 21 0 108 6501370 normal pathologic normal normal 24 0 115 7001330 pathologicb pathologicb pathologicb normal 26 d 72 1 2101520 normal normal normal low 27 d 54 12lol580 pathologic pathologic pathologic low ' neurocirculatory asthenia. right bundle-branch block. tion, immediately after adopting the erect position and after eight minutes' standing. pulse rate and blood pres sure were registered simultaneously. exercise tolerance test in exercise tests the patient sat on an electrically braked bicycle as described by holmgren & mattsson (8). the work load was increased stepwise at 6-minute intervals. the pulse rate was measured every second minute of each increment. the work load at 170 beatslmin in steady state (w 170) was calculated according to sjostrand (12) and wahlund (15). the working capacity in relation to the heart volume was compared with findings in a series of healthy persons published by gerz6n et al. (6). deviation from the regres sion line exceeding 2 s.d. were classified as pathologic. physical examination abnormal physical findings occurred only in 5 pa tients. two of them (cases 12 and 16) had systolic cardiac murmurs without clinical significance. in case 14 moderate sinus tachycardia and slight hypertension were registered, but at repeated ex amination the pressure had normalized. in case 20 a grade 11-111 pansystolic murmur was heard in the third intercostal space, which was considered to be due to physiologic flow and with no haemodynamic significance. moderate hepatic enlargement was found in case 5. only these last 2 patients had both subjective and objective symptoms. laboratory tests results these showed moderate sideropenic anaemia in a subjective symptoms young woman (case 3). the serum protein level up which they associated with the myocarditis. sevase reading was elevated in the man with hepatic en had praecordial or pain, dysfunction. all but two of the patients underwent one (case 20) complained of sporadic pulse irreguthe laboratory tests and these two were asympto matic. larity and one (case 5) of dyspnoea and fatigue. nine patients (31 %) reported symptoms at followwas slightly subnormal and the alkaline phosphat sociomedical examination all but one of the patients were fully fit for work. in case 5 the working capacity was impaired as a result of hepatic dysfunction which presumably was of cardiac origin. this patient, however, worked part-time in his previous occupation. five patients had progressed to better positions, probably be cause of more advanced training. four had switched to less physically demanding work, as a result of the myocarditis. radiologic examination the relative heart size was increased in 5 male patients and one female. one of these patients (case 27) suffered from cardiac enlargement during the acute illness, and this had now increased (ta bles i1 and 111). electrocardiography pathologic resting ecg was found in only 6 patients (21%). in 5 of them previously observed t-wave upsala j med sci 81 acute rnyopericarditis 171 w1 70 kpm im i n 1600 1200 800 400 males kpnlrnin 1600 12 00 8 00 4 00 1 , . , . , . , . . . , . -1 w w m e 200 coo 600 800 lo00 m l females 1 , . : :’: , , , . , , .’ <* heart wlwe 200 400 600 800 1000 ml f i g . l a , b. the individual relation between working capacity and the heart volume as compared with normal regression lines ( f 2 s.d.). changes had diminished. complete right bundle branch block was still present in case 24 (table iv) . the orthostatic ecg was pathologic in 12 pa tients (41%), 6 of whom showed normal patterns during rest. the new changes in standing were in version or flattening of t-waves, except in the pa tient with right bundle-branch block, who showed this pattern also during rest. in case 13 the resting pulse rate of 115 beatslmin rose to 140 in standing position. the exercise ecg was pathologic in 8 patients (27%), all of whom also had pathologic orthostatic ecg. two patients with pathologic st-t changes during work had a normal resting ecg. complete right bundle-branch block was again seen in case 24 (table iv). working capacity the total work load a t pulse 170 beats/min (w 170) averaged 957 kpmlminf244 s.d. in the male pa tients and 584 kpmlminfl66 s.d. in the females (table 111). five patients (4 males and one female) showed low exercise tolerance in relation to cardiac volume (fig. l a , b). in one of them (case 27) the break point was well below a pulse rate of 170 beats/min (450 kpm/min at pulse 98 beatdmin). his working capacity was regarded as equivalent to the highest tolerated load for 6 minutes according to sjostrand (12). in cases 14 and 26 the low physical capacity was not accompanied by ecg changes (table iv). case 13 showed sinus tachycardia and low working capacity, probably because of neurocirculatory asthenia. her heart size was nor mal, in contrast to the other patients with impaired exercise tolerance (table iv). in only one of these 5 patients (case 5) did the follow-up examination also reveal subjective and objective physical sequelae and pathologic ecg. discussion during the acute infectious illness all 29 patients showed signs of cardiac disorder, in particular path ologic ecg a t rest, but also physical signs. a presumptive aetiologic diagnosis was made in 13 cases (45%): 7 had beta-haemolytic streptococci in throat swabs, 4 had elevated antistreptolysin titre without positive throat swabs, one had mo nonucleosis and one had urinary tract infection. in the remaining cases the symptoms and the physi cal findings during the acute illness indicated viral infection. a relevant point is that 15 of the 29 pa tients were treated before 1%6, when virologic studies were relatively rare at this hospital. our frequency of aetiologic diagnosis tallies with bergstrom e t al. (2) and gerzcn et al. (6). the physical examination at follow-up was nega tive in all but 5 of the patients, one of whom had hepatic dysfunction (case 5). repeated biopsies in this case yielded normal liver tissue, indicating a cardiac causation. the same patient also had dysp noea of effort and progressive cardiac enlargement with pleural effusion which necessitated diuretic medication. moderate sinus tachycardia was found in case 14. this patient also had moderate cardiac enlarge ment and impaired physical capacity in tolerance test, but had no subjective symptoms. systolic upsala j med sci 81 172 e . sanner et al. murmurs without any clinical significance was heard in 3 patients. one of them (case 20) com plained of sporadic pulse irregularity, but the ecg at follow-up showed sinus rhythm without extra systoles. of the 9 patients who reported residual symp toms, only cases 5 and 20 showed deviation from the normal at physical examination, and of these only case 5 had reduced capacity at function test. of the five patients with pathologic physical find ings, only cases 5 and 14 showed low exercise tolerance. similar observations were made by berg strom et al. (2). by contrast, bengtsson & lam berger (3) reported good correlation between the patients’ symptoms, ecg changes and reduction of working capacity. the cardiac volume in this series was increased at follow-up in 21% of cases. bengtsson & lamberger (3) reported 20% in a considerably larger case series. the follow-up resting ecg was pathologic in only 6 of the 29 cases. two of the six had low working capacity (cases 5 and 27). change to stand ing position gave pathologic ecg in 6 more cases, with altered s t and t tracings. this illustrates the value of the orthostatic test for providing sup plementary information. similar findings were published by others ( 1 1 , 3, 2, 6). the pulse rate in the orthostatic test rose by more than 20 beats/min in 1 1 of the 29 cases. one of them (case 13) had sinus tachycardia at rest and sympatheticotonic ecg changes appeared during standing, indicating neurocirculatory asthenia. the mean pulse increase in the orthostatic test was 15 beats/min, which tal lies with bergstrom et al. (2) and gerzcn e t al. (6). exercise tolerance tests produced no additional ecg changes are compared with the orthostatic tests. gerzkn et al. (6) found arrhythmia in 5 of 45 cases and in two others t-wave changes, which were not apparent a t rests or during the orthostatic test. levander-lindgren ( 1 1) reported that ecg changes arose during work in 22% of her cases. bergstrom e t al. (2) found abnormal ecg at rest in 33 %, in standing in 53 % and during work in 73 % of cases. corresponding figures reported by bengts son & lamberger (3) were 19, 10 and 30%. work tests thus can evoke otherwise unseen ecg changes, though this did not occur in our case series. in 4 of our patients with pathologic ortho static ecg the s t and t tracings were normal during work. the physical capacity was low in relation to the heart volume in 5 of the 29 cases (17%). the cor responding frequency reported by bergstrom e t al. (2) was 13%. two patients with subnormal physical capacity had normal ecg tracing (resting, standing and working). gerzcn et al. (6) reported residual cardiac symptoms in 35% of their cases, but work tests showed normal capacity in relation to total haemoglobin and to heart volume. physical working capacity in healthy persons is in linear relation to the heart volume. myocardial damage of fimctional significance may, however, impair the working capacity while the heart size tends to increase. calculation of working capacity in relation to heart size may therefore be helpful in detecting deviations from the normal. in our series 4 of the 5 patients whose working capacity was low in relation to heart size had cardiac enlargement. the fifth patient was classified as neurocirculatory asthenia. bengtsson & lamberger (3) similarly found good correlation between cardiac enlarge ment and impaired physical working capacity. the mean work loads a t a pulse rate of 170 beats/min (w 170) were similar to those found by frisk et al. (5). in 67 healthy males they reported 1050 kpm/min+ 125 s.d. in 58 healthy females they found 750 kpmlminf 100 s.d. in the general popu lation, however, the physical working capacity shows wide variations. it may have diminished in recent years. strom (14) reported a study from 1964-1965 in which healthy swedish students showed mean readings of 1 009 kpmlmin in 61 males and 555 kpmlmin in 22 females. our conclusion is that the prognosis in this series of patients with earlier myopericarditis is good. the results are in agreement with those in other com parable series in which the observation time was somewhat shorter. references 1 . bell, r . w. & murphy, w. m.: myocarditis in young military personel. amer heart j 74: 309, 1%7. 2. bergstrom, k., erikson, u . , nordbring, f., nordgren, b. & parrow, a.: acute non-rheumatic myopencarditis: a follow-up study. scand j infect dis 2: 7, 1970. 3. bengtsson, e. & lamberger, b.: five-year follow-up study of cases suggestive of acute myocarditis. amer heart j 72:751, 1966. 4. blackburn, h . , keys, a . , simonson, e., rautahaju, p. & punsar, s.: the electrocardiogram in population studies. a classification system. circulation 21: 1160, 1960. upsala j med s c i d l acute myopericarditis 173 5 . frisk, a. r., holmgren, a., strom, g. & viktorsson, k. e.: stockholms stads halsoundersokning 1954.111. viloekg, arbetsekg och fysisk arbetsformgga. nord med 58: 1437, 1957. 6. gerzcn, p., granath, a., holmgren, b. & zetterquist, s.: acute myocarditis. a follow-up study. brit heart j 34: 575, 1972. 7. helin, m., savola, j. & lapinleimu, k.: cardiac man ifestations during a coxsackie b5 epidemic. brit med j iii: 97, 1968. 8. holmgren, a. & mattsson, k. h.: a new ergometer with constant work load at varying pedalling rate. scand j clin lab invest6:137, 1954. 9. jonsell, s.: a method for the determination of the heart size by teleroentgenography (a heart volume index). acta radiol20: 325, 1939. 10. klainer, a. s.: clinical aspects of infectious heart disease. postgrad med 55: 124, 1974. 11. levander-lindgren, m.: studies in myocarditis. iv. late prognosis. cardiologia47: 209, 1965. 12. sjostrand, t.: exercise tests. in clinical physiology (ed. t. sjostrand), pp. 515-530. svenska bokforlaget bonniers, stockholm, 1967. 13. smith, w. g.: coxsackie b myopericarditis in adults. amer heart j 80: 34, 1970. 14. strom, g.: n b r a medicinska synpunkter pf~ fysisk trbing. medicinska foreningens tidskrift 45: 168, 1%7. 15. wahlund, h.: determination of the physical working capacity. acta med scand, suppl. 215: 1948. 16. wenger, n. k.: infectious myocarditis. postgrad med 44: 105,c1%8. received august 8, 1976 address for reprints: e. sanner, m.d. department of medicine central hospital s-63 1 88 eskilstuna sweden upsala f med sci8l upsala j med sci 97: 127-139 afferent activity in pulmonary stretch receptors before and after lung injury by peter radell and anders jonzon the department ofiphynology and medical biophysics, biomedical center, uppsala university and the department of pedtatrlcs, university hohpital, uppsala univeruty, uppsala, sweden abstract this study was undertaken to determine the effect of a lungin jury on the activity of slowly adapting pulmonary stretch receptors. comparisons of receptor activity were made at in hibition of inspiratory (phrenic nerve) activity. the inspira tory activity of these receptors was found to be decreased after lung-injury. introduction we have previously studied the effects of ventilatory variables in cats at inhibition of inspiratory activity (4,5,7). comparisons of the effects of different changes in ventilatory variables were made at inhibition of inspiratory activity, i.e. when the sum of afferent inputs (e.g. from chemo-receptors and mechano-receptors) inhibits inspiratory activity. we consider a ventilatory setting that barely inhibits inspiratory activity to be a useful biological reference point for comparisons ( "inhibition of inspiratory activity"). we found that inhibition of inspiratory activity occurred at a somewhat lower arterial pc02 after lung injury 127 (5), which would lead to correspondingly lower chemoreceptor activity. this implies that the activity of other afferents changes after lung injury if inhibition of inspiratory activity is maintained. the present investigation was under taken to determine whether the activity of the slowly adapting pulmonary stretch receptors (psr) was altered after a lung injury that included a reduction of pulmonary compliance. changes in pulmonary compliance alone are known to lead to changes in the activity of rapidly adapting pulmonary stretch receptors (3,9). the aim of the present study was to determine whether the psr changes its over-all activity or its phasic activity after lung injury. methods in five cats (2.2-3.5 kg), general anaesthesia was induced with chloroform and maintained with intermittent infusions of chloralose ( 7 . 2 g/l) as required. after endotracheal in tubation a ligature was tied around the endotracheal tube to prevent air leakage. a catheter was inserted through femoral artery until its tip lay in the thoracic aorta, and another catheter was introduced through the femoral vein until its tip lay in or near the right atrium. a mixture of glucose and sodium bicarbonate (two-thirds 5.5% glucose + one-third 0.6 m nahc03) was given intravenously throughout the experiment at a rate of 2 ml/kg b.w./h. body temperature was maintained in the normal range with use of a heating pad. 128 arterial samples were taken at intervals throughout the experiment via the femoral artery catheter. acid-base imbalances were corrected by administration of nahc03 or respirator adjustments. arterial blood pressure and in tratracheal pressure were measured with transducers (druck ag, gfr) placed at the level of the tips of the respective catheters. signals were amplified (hellige ag gfr) and recorded (recorder 330-p, hellige ag, gfr) and in addition intratracheal pressure and vagal impulses were recorded on an oscillograph (oscillograph 6608, se labs, uk). the cats were ventilated with a volume-controlled ventilator (siemens-elema 9 o o c ) . positive end-expiratory pressure (peep) of 4 5 cm h20 was used at a respirator rate of 60 breaths/minute. fi02 was increased to 0.5 after lung injury (see below). spontaneous breathing activity could be inhibited by increasing pre-set tidal volume until the phrenic nerve activity ceased (in hibition of inspiratory activity). a medial frontal incision was made in the pre-tracheal region, permitting exposure and dissection of vagal and phrenic nerves. these were immersed in mineral oil to prevent drying of the nerve and for electrical insulation. after dissection and removal of connective tissue, the left phrenic nerve was placed on two hook electrodes. phrenic nerve activity was amplified with the use of one channel of a neuro log system (nl 100; nl 103; nl 105; nl 115; nl 2 0 0 ) , and was fed through an integrator and recorded on the recorder (recor der 330-p, hellige ag, gfr). 129 from one of the cervical vagus nerves fine slips were dissected from the intact nerve after removal of the connec tive tissue. nerve activity was recorded between a single electrode and ground. filaments were split from the slip of the vagus nerve and impulse activity was analysed until the signal from a single slowly adapting psr was found. this receptor was recognized by its characteristic respiration synchronized activity and by the occurrence of enhanced ac tivity on airway occlusion of the expiratory tube, leading to an increased lung volume. impulses were processed by the other channel of the neurolog system, and a spike processor (digiti mer, uk) counted the impulses within the range of a window discriminator. this range could be adjusted to accept potentials of desired amplitude. the impulses were displayed on an oscilloscope and recorded on the oscillograph, together with the intratracheal pressure. thus psr impulses could be counted for inspiratory and expiratory phases or for shorter intervals of individual respiratory cycles. the remaining part of the vagus nerve was left intact, as well as the contralate ral vagus. protocol all observations were made at inhibition of inspiratory ac tivity and at a constant minute volume and ventilation frequency. when a psr was found, the acid-base status was checked and a baseline recording, at inhibition of inspiratory activity, was made after i.v. administration of 2 ml of pancuroniumbromide (pavulon) for muscle relaxation, so as to prevent disturbing muscle reflexes. recording was repeated 130 after lung injury induced by administration of xanthine oxidase, (50 units/ml, 0.2 ml/kg b.w. ) . this was given through a thin catheter into the distal part of the endotracheal tube. recordings were made at steady state and at inhibition of inspiratory activity, usually 10-15 minutes after admini stration of xanthine oxidase. in one animal, recordings were made after a single dose of xanthine oxidase and again after a second dose. in another animal the protocol was followed twice on two different, sepa rately isolated psr fibers. analysis of data afferent vagal nerve activity was computed as the mean impulse rate (impulses/s) recorded for inspiratory and expiratory phases over a period of ten respiratory cycles. mean values for the six fibers are presented in the figures. in some experiments the afferent activity was also computed for inter vals of 100 ms. inspiratory and expiratory phases were determined from simultaneous recording of intratracheal pressure. mean peak and end-expiratory pressures were cal culated analogously. results this study shows that the overall and phasic activity of psrs is altered after lung injury induced by xanthine oxidase. before lunginjury the psr activity was proportional to the 131 airway pressure, with lower activity during the expiratory phase and higher activity during the inspiratory phase (fig. 1). after lung injury the psr activity was less pronoun ced at the peak of inspiration than before the lung injury (fig.2). i i i > 0 fig.1. 05 10 1.5 s time intratracheal pressure (pit, lower panel) and activity (action potential, ap) from one slowly adapting pulmonary stretch receptor during mechani cal ventilation before lung injury. 132 i i i > 0 0.5 10 1.5 s time fig.2. intratracheal pressure (pit, lower panel) and activity (action potential, ap) from the same slowly adapting pulmonary stretch receptor as in fig.1 during mechanical ventilation after lung injury. the average impulse activity was calculated both for whole respiratory cycles and for inspiratory and expiratory phases. the total mean activity of six fibers before lung injury was 89 impulses per second and after lung injury 74 impulses per second. the inspiratory activity before lung injury was 56 impulses per second and after lung injury 45 impulses per second, (p<0.05), whereas the corresponding values for expira tory activity were 33 and 30 impulses per second respectively (difference non-significant) (fig.3). 133 i m p / s 100 80 60 4 0 20 0 insp. before xanthine oxidase 'otal nsp. after xanthine oxidase fig.3. mean impulse frequency of six different slowly adap ting pulmonary stretch receptors. the impulse frequ ency has been calculated for the whole respiratory cycle and for the inspiratory and expiratory phases before and after lung injury. when the psr activity was further subdivided into periods of 100 ms, this pattern became more pronounced. this was done for two fibers and representative curves are shown in fig. 4. thus, the results in figures 3 and 4 demonstrate that instillation of xanthine oxidase into the airways reduces the total number of impulses per respiratory cycle and that the reduction in impulse frequency is most marked during inspiration. 134 k p a : 10 ? 3 m m q 0 0.2 0.4 0 6 08 se c time k p a f 10 3 ln m 0 l q $ 5 c u m m l c l c 5 0 ',-x-x-.x imp i i i 0 0.2 0.4 0 6 0.8 sec time irnp i s 10 d ln w 0 irnp i s 10 0 fig.4. impulse frequency in one slowly adapting pulmonary stretch receptor before and after lung injury. the impulse frequency has been calculated for 100 ms intervals during one respiratory cycle. before lung injury the airway pressure at inhibition of in spiratory activity was 1.3 kpa at the peak of inspiration and the end-expiratory pressure 0.6 kpa. after lung injury the corresponding values were 1.5 and 0.6 kpa respectively (fig.5). thus the difference in peak pressure was significant 135 ly higher after lung injury (p<0.025). a 3 # m a q m a 1i: u m m t l l d l + l * kpa 1.5 1 .o 0.5 0 before itanthine ox i dase ns p. after ox i dase i ? s d pj xant hine fig.5. peak and end-expiratory intratracheal pressure before and after lung injury at inhibition of inspiratory activity. measurements were made simul taneously with the receptor activity presented in fig. 3 discussion the results of this study show that the activity of the slowly adapting pulmonary stretch receptors decreases after lung injury induced by instillation of xanthine oxidase into the airways. the inspiratory activitywas lower after lung injury, 136 although the airway pressure was higher. this led to a shift in the receptor activity from a phasic pattern to a more continuous one. saugstad el a1 (10) reported that instillation of xanthine oxidase into the airways produced a lung injury characterized byperivascular oedema, dilatation of lymph vessels, infiltra tion of neutrophils and filling of the alveoli with amorphous material, resulting in a reduction of lung compliance. we did not measure lung compliance in our experiments, butthe tidal volume and consequently the airway pressure had to be increased after the lung injury in order to maintain in hibition of inspiratory activity, which probably reflects a decrease in lung compliance. reductions in lung compliance have been shown to increase the activity of rapidly adapting pulmonary mechano-receptors ( 3 , 8 ) . these receptors are thought to stimulate inspiratory activity, possibly as sighs, in response to changes in lung compliance and/or functional residual capacity. it is difficult to determine their role in the present experiments. the slowly adapting pulmonary stretch receptors are thought to be involved in the control of the depth and rate of breathing (2); their major function may be to interrupt in spiration. thus, a change from phasic to continuous activity of these receptors could be of great importance in the control of breathing. a continuous discharge from psrs results in inhibition of inspiratory activity (1,6,9). 137 in newborn infants with severe respiratory distress requiring respirator treatment it is often seen that inhibition of spon taneous breathing requires that the arterial blood gases are within the normal range. during recovery, when the lung attains better compliance, while still mechanically ventila ted, spontaneous breathing usually reappears and may interfere with the mechanical ventilation. these observations might be explained by the findings in this study that the activity of the slowly adapting pulmonary stretch receptors was continuous ,after lung injury, when compliance was low, but phasic before lung injury, when compliance was high. conclusions it is concluded that the inspiratory activity of slowly adap ting pulmonary stretch receptors is reduced after lung injury. acknowledgements this study was supported financially by the foundation to the memory of sigurd and elsa golje, gillbergska stiftelsen and by the swedish medical research council (grant no.19m-7544). the skilful technical assistance of g.nilsson, e.ekstrom and b.kjallstrom is gratefully acknowledged. we are also indebted to susanne thorell and sabina albinsson for typing the manusc ript. 138 references 1. 2. 3 . 4 . 5. 6. 7. a . 9. 10. banzett r, lehr j & geffroy b: high-frequency venti lation lengthens expiration in the anesthetized dog. j appl physiol 1983;55: 329-334. euler c & trippenbach t: on the respiratory phase switching mechanisms. in: respiratory centres and afferent systems (ed. duron), collogue inserm, i976;59: 11-18. jonzon a, pisarri te, coleridge jcg & coleridge hm: rapidly adapting receptor activity in dogs is inver sely related to lung compliance. j appl physiol iga6;6i: igao-iga7. jonzon a, tantalean ja, norsted t & sedin g: airway pressures during positive pressure ventilation with superimposed oscillations. upsala j med sci 1990;95: 53-61. jonzon a, norsted t & sedin g: airway pressures du ring positive pressure ventilation with superimposed oscillations before and after lung injury. upsala j med sci 1992; this issue. man gcw, man sfp & kappagoda ct: effects of high frequency oscillatory ventilation on vagal and phrenic nerve activities. j appl physiol 1983;54: 502-507. norsted t, jonzon a & sedin g: pancuronium bromide does not lower airway pressures during intermittent positive pressure ventilation in young cats. acta anaesthesiol scand 1989;33: 21-25. pisarri te, jonzon a, schultz hd, coleridge hm & coleridge jcg: effect of high frequency oscillatory ventilation on pulmonary vagal afferents in dogs. fed proc i987;46: 662. pisarri te, jonzon a, coleridge jcg & coleridge hm: rapidly adapting receptors monitor lung compliance in spontaneously breathing dogs. j appl physiol 1990;6a: 1997-2005. saugstad od, becher gi grossman mi merker g, oddoy a & lachman b: acute and chronic effects of xanthine oxidase on lung thorax-compliance in guinea pigs. intensive care med 1987; 13: 30-32. 10-928572 139 upsala j med sci 98: 417-427, 1993 7. approaches towards a combined dataand knowledge-base for analytical quality specifications in clinical chemical laboratories carl henric de verdier' 'department of clinical chemistry, university of uppsala, uppsala, sweden introduction it is not realistic to believe that the management of every clinical chemical laboratory is willing to go through literature and reports and try to collect all clinical and technical background material for estimating numerical values of 'clinical goals' and for listing the 'quality specifications' for all kinds of measurements of their laboratory. as the clinical and especially the technical background material has a rapid turnover time it seems to be most suitable to collect the material in continuously updated computerized database systems. a complete database ought to cover several hundred different types of components and the measurement of each component may be applied in many different clinical situation. it is thus not possible in this report to build up an extensive database. i have, however, in this preliminary list tried to present a number of different and illustrative examples, which can be used as models for an extended list and finally a real database. a database for this purpose will always contain two types of data: 1. those describing the (analytical) measurement procedures and 2. those describing the clinical (and the normal) situations in which it is appropriate to apply the measurement procedure. quality assurance specifications and quality control procedures will come out as conse quences of these two types of data. we have found it most appropriate in this presentation to use system--measwand (analyte) as the primary key in our data base. in many clinical settings one kind of measurement (analysis) is not giving the diagnosis or the advice for treatement. i n such circumstances one or more clinical situtations must be defined. 41 7 furthermore, in one clinical situation, it may be necessary to use the measurand in combination with a set of other measurands from the clinic, the diagnostic radiology, department and the clinical laboratories. this may turn up to be a very complex assessment. examination of a few key-examples will, however, soon learn us to draw conclusions, valid for a number of analogous examples. i am confident that we in the future will see several groups working with combined data and knowledge-bases within this area and we feel sure that computerized such bases is an excellent medium for rapid exchange of information and knowledge. examples of combined dataand knowledge-bases for aqspecs the following list have been written using the dataprogram microsoft excel@ 3.0. this program does not allow writing characters as subscripts, instead they have been written within [ 1. the abbreviation c* has been used for the 'conventional true value'. the measurands are listed in alphabetic order. in each segment clinical goals calculated in different ways are listed first, followed by recommendations from proficiency testing bodies. a few examples have been given: s--creatinine, b--haemoglobin and s--urate, in which the clinical goals have been expressed as fractions of easily understandable physiological functions or by logical reasoning obtained pathological processes. references 1. 2. 3. 4. 418 bundeartzekammer. qualitetatssicherung der quantitativen bestimmungen im laboratorium. neue richtlinien der bundesiirtzekammer. dt h z t e b l 1988;85 (1 l):a-697-a-712. costongs gmpj, janson pcv, bas bm, et al. short-term and long-term intra-individual variations and critical differences of clinical chemical laboratory parameters. j clin chem clin biochem 1985;23:7-16. costongs gmpj, janson pcw, bas bm, hermans j, van wersch jwj, brombacher pj. short-term and long-term intraindividualvariation and critical differences of heamatological laboratory factors.. j clin chem clin biochem 1985;23:69-80. de verdier c-h. assessing analytical quality specifications. scand j clin lab invest 1994; (in press). 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. de verdier c-h, groth t, hyltoft petersen p. medical need for quality specifications a nordkem project for selecting the appropriate quality in clinical laboratories. scand j clin lab invest 1993;.53, suppl 215:29-37. fraser cg. the application of theoretical goals based on biological variation data in clinical chemistry. arch pathol lab med 1988;112:404-415. fraser cg. biological variation in clinical chemistry. an update: collated data, 1988-1991. arch pathol lab med 1992;116:916-923. fraser cg, hyltoft petersen p, lytken larsen m. setting analytical goals for random analytical error i n specific clinical monitoring situations. clin chem 1990;36: 1625-1628. fraser cg, hyltoft petersen p, ricos c, haeckel r. proposed quality specifications for imprecision and inaccuracy of analytical systems for clinical chemistry. eur j clin chem clin biochem 1992;30:311-317. geilenkeuser w-j, kruse r, rijhle g. ringversuche der deutschen gesellshaft fuer klinische chemie -interpretation der auswertung. d g klinische chemie 1992;23(2):57-81. groth t, ljunghall s, de verdier c-h. optimal screening for patients with hyperparathyroidism with use of serum calcium observations: a decision-theoretical analysis. scand j clin lab invest 1983;43:699-707. hyltoft petersen p, blaabjerg 0, irjala k, iccn a, bjoro k. upsala j med sci 1993;98 (3): in press. linnet k. analytical goal5 for p--bilirubins. upsala j med sciences 1993; 98(3): in press. linnet k. analytical goals for accuracy and precision of plasma creatinine determinations evaluated by reference method measurements.. upsala j med sciences 1993;98 (3): in press. lytken larsen m, hyltoft petersen p, fraser cg. quality specifications for haemo globin a l c assays i n the monitoring of diabetes. upsala j med sciences 19y3;98 (3): in press. national cholesterol educationprogram laboratory standard panel. current status of blood cholesterol measurement in clinical laboratories in the united states. clin chem 1988;34: 193-201. skendzel lp, barnett rn, platt r. medical useful criteria for analytical performance of laboratory tests. am j clin path01 1985;83:200-205. 419 18. 19. 20. 21. 22. thue g, sandberg s, fugelli p. clinical assessment of haemoglobin values by general practitioners related to analytical and biological variation. scand j ciin lab invest 1991;51:4.53-459. van waeg g, groth t. allopurinol kinetics in humans as a means to assess liver function: design of a loading test. a m j physiol 1989;257:r237-r24.5. westgard jo. charts of operational process specifications ("opspecs charts") for assessing the pecision, accuracy, and quality control needed to satisfy proficiency testing performance criteria. clin chem 1992;38:1226-1233. westgard jo, hyltoft petersen p, wiebe a. laboratory process specifications for assuring quality in the u.s. national cholesterol education program (ncep). clin chern 1991;37:656-661. wiggers p, dalhoj j, hyltoft petersen p, blaabjerg 0, h ~ r d e r m. screening for haemochromatosis: influence of analytical imprecision, diagnostic limit and preva lence on test validity. scand j clin lab invest 1991;51:143-148. correspondence: carl-henric de verdier, m.d., professor department of clinical chemistry university hospital s-7.51 85 uppsala, sweden 420 53 ianal. qual. sp. ieurop. work gr. 1 cv[a]<0.027 422 55 ianal. qual. sp. lgerman rinpers. icv[a]<0.06 i151 i i i . 18 19 20 21 22 -. numerical values (system. errors) note rejerences insensitive to changes in bias insensitive to changes in bias 19 19 26 27 28 29 30 31 32 33 i i 34 i 1s i i i numerical values (system. errors) note references deviation<o.lo c' 13 deviation< 0.098 c* 9 deviation<o.zo c* or <6.8j umovl imprecision included 20 deviation < 0 21 c* bias unchanged cv[a]<%cv[bw] 2 1 36 37 38 19 numerical values (?stein. errors) note references bias unchanged cv[a]<%cv[bw] 7 bias < 0.02 c* 11 -_ 40 41 42 d1 bias changed <0.007 (0.018) 9 deviation <0.25 m m o e imprecision included 20 deviation< 0.10 c* 1 i :; ibias < 0.03 14 controls/ser;2 pat samples i 21 i i 44 45 46 numerical values (system errors) r o t e rejerences 42 3 47 48 49 50 bias unchanged cv[a]<!acv@w] 7 deviation <0.38 c* cv[a]<1/4cvp] 7 bias=o 2 controlsfseries 21 bias < 0.03 2 controlsfseries 21 l a , i 53 deviation <o.oji c* s4 deviation <o. 10 c* 5.5 deviation < 0,18 c* 56 deviation< 0.03 c* 1 imprecision included 9 20 1 16 424 57 e i f g i 59 60 61 62 63 6 1 65 66 61 ivunterica1 values (systenr. errors) .vote references bias unchanged cv[a]<%cv[b\v] 7 deviation< 0.046 cv[a]<l/4cv[b] 7 bias unchanged icv[bw]=o.o-i; prob. 95% 5 bias unchanged j 2 pat samples 5 bias unchanged detect +o. 17; prob. 99% 8, 17 dev. <0,16c* when cv[a] 0.036 detect+o.l6:prob. 95% 14 i . _ 79 80 81 82 91 nuinericol values (system. errors) h b t e refirences bias unchanged bias unchanged the two analyses combined. prob.99%. predictive value 22 of a positive result 0.29. i 425 "i 8 1 85 86 87 88 89 90 91 nunterical volues (system errors) #ore references bias unchanged cv[aj<%cvpw] 8 bias unchanged cv[bw]=0.027; prob.95% 18 bias unchanged cv[bw]=o.o2j; prob.95% 8 bias unchanged cv[bw]=0.027; prob.95% 4 92 93 94 95 96 a7 deviation<o.olz c* 9 deviation <0.07 c* imprecision included 20 ,l 98 99 100 i ivunierica1 values ($,;sent. errors) /!vote references bias unchanged /detect+/-l%hbalc; prob.o.80 15 bias unchanged ldetect+/-2% hbalc; prob.0.99 15 i a b c d 1101 i 426 111 112 113 114 115 116 117 118 i s--potassium nuiiierical values (randoiir errors) clinical goals biol. approach within subj variatlon cv[a]<o 006 anal. qual. sp. europ. work. gr. cv[a]<o 021 anal. qual. sp. anal. qual. sp. german ringvers. us prof. test. 158 159 1 sn nuiiierical values (randoin errors) clinical goals ibiol. approach n i t h n subj variahon cv[a]<o 052 i 161 162 163 anal. qual. sp. europ. work. gr. cv[a]<0.063 anal. qual. sp. anal. qual. sp. german rinpers. us prof. test. 112 113 114 115 116 117 118 1 1 0 numerical values (vsteiir. errors) h'ote references bias unchanged , cv[a]<xcv[bw] 7 deviaiion<0.016 (0.048) c* 9 deviation <0.5 m m o l l imprecision included i 20 deviation<0.08 c* imprecision included 10 i 121 122 123 note references ivutirerica1 values (iysterir. errors) bias unchanged cv[a]<%cv[bw] 7 124 125 126 127 i bias changed <0.002 (0.006) 9 deviation <4 m o v l imprecision included 20 deviation <0.06 c* 1 i 152 deviation <0.04(0.084) 9 153 deviation <o.i7c* imprecision included 20 154 cv[a]<0.18 c* imprecision included 1 i"" 161 162 163 bias change < 0.053 9 deviation<0.09 c* or <0.71 mmovl imprecision included 20 deviation<o.24 c* imprecision included 10 16 935253 427 upsala j med sci 83: 153-162, 1978 health and disease at the age of sixty findings in a health survey of 60-year-old men in uppsala and a comparison with men 10 years younger urban waern from the department of internal medicine, university hospital, uppsala, sweden abstract a health investigation was performed among 331 men aged 60 years in uppsala. the investigation was performed in the same way as in 50-year-old men ( n = 2 3 2 2 ) in the same community, previously described. thus it was possible to make certain comparisons between these two populations of middle-aged men. a considerably higher morbidity, glven as point prevalence, was found for diseases of the cardiovascular system and of the endocrine organs, in the older population compared with the younger. parallel to this a higher consumption of pharmaceuticals was reported by the older men. only 39% among the older men versus 51 % of the younger men were smokers. nearly the same number (two-thirds) of men in both age groups were with out codable ecg abnormalities in their resting ecg. among the older men, however, there were more subjects having multiple pathological ecg flndings than in the youngeragegroup. it is concluded that it is possible to reach approximately 80% of the actual population in special health investiga tions, in middle-aged men. future studies, preferably in younger subjects, should aim at an early detection and primary prevention of cardiovascular and endocrine dis eases. introduction during recent decades numerous studies have been performed to elucidate factors that increase the risk of cardiovascular disease and death. cardiovascu lar disease has been accorded special importance due to its large and increasing contribution to morbidity and mortality in modern society. in sweden, studies have been carried out in gothenburg (34), stockholm and uppsala (is), among other places. this paper deals with general findings in a health survey of 60-year-old men in uppsala. they were investigated in much the same way as another group of men, at the age of 50 (15) in the same community. these men were born in the period 1920-24. they numbered 2 322 subjects and were investigated in 1970-1973. comparisons with this younger age group will be made, with respect to blood pressure (37) and glucose tolerance and serum lipid levels. some additional studies were made on the same population, including the occurrence of cardiac arrhythmias in daily life, alcohol intake and patterns of urinary electrolytes. these will be reported on elsewhere. material the population consisted of all men born in 1915 living in uppsala in 1975. they numbered 422. at the time of screening they were living within the same area as were the 50-year-old men of the previous study. the population of the corresponding area of uppsala on 1st january 1975 was 108 676. of the above-mentioned 422 men called for health examination, 331 arrived, which meant a participation rate of 78.4%. from the registers of the county council in uppsala, the names and addresses of all men born in 1915 were collected. in order to get a relevant list, this collection was made 3 months in advance of the forthcoming examina tion. methods a letter was sent to each man in the population one week in advance, inviting him to the health survey and explain ing its purpose. in the letter the subject was asked to fast and also refrain from smoking from midnight prior to the morning of the investigation. this started at 7.00 am. the examination was performed by the author, with the as sistance of the same registered nurse as in the study of 50-year-old men (15). it took place in the outpatient clinic of the department of internal medicine at the university hospital of uppsala. non-participants were all offered another appointment by direct telephone contact. the investigation started in the middle of august 1975 and was completed by the end of november the same year. questionnaire a self-administered questionnaire modified after collen (7) was used. it contained 145 questions, including family upsala j m e d sci 83 154 u . w a e r n table i. l a b o r a t o r y i n v e s t i g a t i o n s p e r f o r m e d in the h e a l t h i n v e s t i g a t i o n of 6 0 y e a r o l d m e n mean values, standard deviations and laboratory methods used by the laboratory system component n mean s.d. method serum serum urine serum serum urine urine blood serum urine serum urine urine urine serum serum blood albumin calcium calcium cholesterol creatinine creatinine ethanol glucose glut. trans magnesium phosphate phosphate potassium sodium triglycerides urate erythrocyte ferase 331 4 . 2 811 33 1 2.4 mmol/l 326 5.4 mmol/d 33 1 6.2 mmol/l 33 1 83.4 pmolll 327 1 1 980.8 pmol/d 36 251.7 mgll 33 1 5.4 mmol/l 3 24 327 33 1 326 326 326 329 33 1 330 0.33 pkat/l 3.8 mmol/d 0 . 9 mmolll 29.4 mmol/d 70.6 mmol/d 159.7 mmol/d 1.7 mmolll 269.9 pmol/l 8.1 mm/h sed. rate blood haematocrit 330 44.9 % 0.35 0.08 2.8 1.2 auto-analyzer n-70 brom cresol green binding technique atomic absorption method using internal atomic absorption method using internal standardization with strontium standardization with strontium 13.6 jaffk reaction after dialysis jaffk reaction after dialysis glucose oxidase method after zinc hydroxide 3 007.8 1.6 408.1 alcohol dehydrogenase method 0.27 1.7 0.3 8.9 24.4 61.5 1.0 64.9 7.2 preparation to szasz kinetic photometric method according atomic absorption method dialysis of sample and reduction of dialysis of sample and reduction of flame photometric method using internal flame photometric method using internal auto-analyzer n-24 a uricase method westergren’s method phosphomolybdate with elon phosphomolybdate with elon standardization with lithium standardization with lithium 3.2 micromethod in duplicate using international microcapillary centrifuge history, previous symptoms among diseases, smoking habits, and stress other things. personal interview this procedure included questions about marital and pro fessional status, consumption of medicines, dietary regi mens, etc. contacts with a physician and the reasons therefore were also noted. each subject was also asked if he took advantage of the general health survey in the county that is offered by the local health authorities every third year. blood pressure measurements blood pressure (j3p) was measured on the right arm after 10 min in the recumbent position and after another 2 min in the sitting position. the pulse rate was counted im mediately prior to the first bp measurement. a mercury wall manometer (kifa ercameter) was used. the cuff had a rubber bladder 12 cm wide and 35 cm long. systolic bp (sbp) and diastolic bp (dbp) were recorded to the nearest 5 mmhg. dbp was recorded at the disappearance of the korotkoff sounds (phase 5 ) . anthropometric measurements the height without shoes and the weight in undershorts were measured in whole centimetres and kilograms, re spectively. in order to define relative body weight, three weight indices were used: 1) an index described by lind berg et al. (24), 2) an index based on the findings of insurance holders in the usa ( 5 ) . 3) finally the heights and weights of the 50-year-old men in the same commun ity (15) were used as a reference to relative body weight index in the 60-year-old men. body fat was estimated with a harpender (9) caliper. three parts of the body were measured: to the right of the umbilicus, under the angle of the scapula and at the back of the mild-upper arm. all these measurements were made in the sitting position and were recorded in whole mil limeters. laboratory investigations all blood samples were taken in the fasting state. the laboratory investigations performed are shown in table i together with the methods used by our central laboratory. the mean values are also presented in this table. the morning urine was examined qualitatively for glu cose and albumin, using paper sticks. each participant was then asked to collect all urine for 24 h following screening, in a plastic bottle. an intravenous glucose tolerance test (ivgtt) was performed in a randomized subgroup of 67 (20.2%) of the subjects who denied diabetes mellitus and all other dis eases requiring chronic medication and dietary treatment. these findings will be described separately (38) and will include a comparison towards 50-year-old men concerning insulin secretion at ivgtt. wnsula j m e d sci 83 health and disease at sixty 155 table 11. percentage of positive replies t o some questions in the sev-administered questionnaire, in health surveys of 50and 60-year-old men in upp sala questions 50-year60-year old men old men ( n = 2 322) (n=331) does (did) your father have does (did) your mother have have you suffered from oppres sion of the chest when walking at normal speed on flat ground? 2.8 walking outdoors in cold weather? 3.5 chest when walking outdoors in cold weather? 3.3 have you been in hospital for myocardial infarction? 0.9 do you have the diagnosis angina pectoris established by a physician? 2.2 do you take nitroglycerin tablets? 1 . 1 are you a smoker? 51.0 have you gained more than 10 kg elevated blood pressure? 11.2 elevated blood pressure? 25.2 do you get chest pains when do you get oppression of the body weight since the age of 30? 6.6 do you feel absolutely well? 80.1 are you retired for medical reasons? 1 . 1 6.1 16.3 10.9 11.8 11.2 4.2 11.5 6.7 39.6 34.4 51.4 10.0 electrocardiogram a resting 12-lead electrocardiogram (ecg) was recorded in all men. this included standard leads i , 11, and 111, unipolar leads avf, avl and avr and finally leads v1-v6, the conventional amplification 1 mv=lo mm was used, with a paper speed of 50 mm/sec. the paper speed and amplification were frequently tested. the ecgs were in terpreted by two independent physicians at the depart ment of clinical physiology according to the goldmann criteria (13) and to the minnesota code (31). a long-term ecg recording covering 6 hours was made (8) but will be discussed elsewhere (23). results questionnaire and interview some results are summarized in table i1 concern ing questionnaire data obtained for the 60-year-old men, as compared with the 50-year-olds. for the questions concerning angina pectoris (ap) there was a consistently higher percentage of positive replies in the older age group. only 2 . 2 % of the younger men reported ap confirmed by a physi cian, compared with 1 1.5 % in the older group. this five-fold increase was also noted with respect to hospitalization for myocardial infarction. only 39.6% of the older men smoked as against 51.0% of the 50-year-olds. however, among the 60-year-old men 13.6% reported that they had stopped smoking after the age of 50. this would mean that 53.2% of those participating in the screening were smokers at the age of 50. nearly two-thirds (64.9%) of the 60-year-old men who were smokers at the time of screening were not interested in smoking withdrawal trials. concerning body weight changes after the age of 30, almost one third (34.2%) of the older men re ported an increase in body weight of more than 10 kg after the age of 30. in the younger group 6.6% reported a similar increase in a period of 20 years. half (50.8%) of the older men had access to a physician through their employer. slightly more than three-quarters of the participants (77.8 %) took advantage of the local health examination the last time they were called. table 111. prevalence of diagnoses, reported as s p e c 8 c diagnosis, in health surveys of 50and 60-year-old men in uppsala code number classification according to icd. only diagnoses established by a physician are included code number diagnosis 50-year-old men 60-year-old men ( n = 2 322) (n=331) subjects number subjects number per cent of men per cent of men 40 1 essential hypertension 412 ischaemic heart disease 250 diabetes mellitus 443 intermittent claudication 3 .l 81 12.4 41 0.6 15 4.2 14 0.9 21 4.8 16 0.1 3 0.9 3 156 u. waern table iv. prevalence of diagnoses, reported as groups of diagnoses in health surveys of 50and 60-year-old men in uppsala code number classification according to icd (international classification of diseases, 3rd rev. ed.). only diagnosis established by a physician are included 50-year-old men 60-year-old men (n=2 322) (n=331) code number groups of diagnoses subjects number subjects number per cent of men per cent of men 140-239 240-279 290-316 320-389 390458 460-5 19 520-577 710-738 tumours endocrine, metabolic mental disorders nervous diseases circulatory diseases respiratory diseases gastrointestinal diseases musculoskeletal disorders 0.2 1.2 1.5 0.8 5.9 0.3 0.1 0.7 5 1 .5 5 27 12.1 40 34 3.0 10 18 3.6 12 136 31.1 103 7 2.4 8 2 2.1 7 16 7.9 26 in tables 111 and iv the occurrence of various diagnostic groups and specific diagnoses are shown. an increase in general morbidity between the ages of 50 and 60 is apparent. of particular im portance, however, is the increase in diseases of the circulatory system and of endocrine and metabolic disorders. in table 111 the relative increase in specific diagnosis is seen. a five-fold increase in the preva lence of diabetes mellitus and a four-fold increase in the prevalence of hypertension was found. this tendency is also reflected by the increase in the use of medical preparations between the ages of 50 and 60. in the younger age group 224 men (9.6%) reported a daily drug intake, compared with 126 (38.1 %) in the older group. the consumption of various classes of drugs in the two age groups is shown in table v . about 10% of the men born in 1915 were taking diuretics and/or beta blocking agents. no increase was noted in the use of insulin, while the consump tion of oral antidiabetic agents was 10 times higher in the older age group. the higher prevalence of musculoskeletal dis table v. pharmaceutical classipcation, according to fass 1976, of drugs used by 50and 60-year-old men as revealed in health surveys in uppsala only daily drug consumptions are included pharma( n = 2 322) ( n =33 1) ceutical classification number of subjects number of subjects tion type of drug subjects per cent subjects per cent 50-year-old men 60-year-old men 02 a 02 b 02 b 02 c 02 e 02 f 02 h 06 c 10 d 10 j 05 10 j 10 11 a 1 1 b 1 1 c 12 b cardiac glycosides quinidine beta blocking agents vasodilators s ympathicol ytics diuretics lipid lowering agents anticoagulants steroids insulin oral antidiabetics sedatives neuroleptics antidepressives analgesics 8 33 8 33 53 23 1 7 10 9 6 10 10 16 0.3 1.4 0.3 1.4 2.3 1 .o 0.3 0.4 0.4 0.3 0.4 0.4 0.6 13 36 32 13 35 17 2 1 2 13 22 4 4 23 3.9 10.9 9.7 3.9 10.6 5.1 0.6 0.3 0.6 3.9 6.6 1.2 1.2 6.9 upsula j m e d sci 83 health and disease at sixty 157 j 100 s.d= 16.2 n.331 )o 180 >200 rnrnhg fig. 1 . distribution of supine systolic blood pressure in the total population. orders in the older group is reflected by the greater consumption of analgesics. the more extensive use of hypnotics and sedatives in the older age group should also be noted. blood pressure the distributions of sbp and dbp are shown in figs. 1 and 2. the mean sbp was 145 mmhg and the mean dbp 87 mmhg in the entire studied popu lation. in addition to 41 (12.4%) men who were known as hypertensives at the time of screening, 23 (6.9%) were found to have a dbp of 3105 mmhg. thus the prevalence of hypertension was 19.3 %, comprising the sum of these two population groups. when the who criterion (39) for hypertension was applied, the prevalence increased to 35.0% of the population. thirty-six persons (10.9%) could not say if they 70 i mean = 87.2 s.d= 11.7 n = 331 1 ilb 3 %p 110 mmhg 120 fig. 2 . distribution of supine diastolic blood pressure in the total population. had been told previously that their bp was elevated. this should be compared with the other questions in the self-administered questionnaire, where the possible answer “do not know” was given only 12 times altogether concerning previous diseases, stress symptoms etc. a derailed report of the group with hypertension will follow (37). anthropometric measurements the results obtained on application of three weight indices are given in table vi. using the index based on the 50-year-old men in the same community, 59.3 % of the 60-year-old men fell within _+ 10% of the “ideal” weight. when the u.s. insurance index ( 5 ) was used, this figure was 59.8%. using the standards applied by lindberg et al. (24), however, only 37.8% of the 60-year-old men fell within these limits. table v1. distribution of 50and 60-year-old men in uppsala according t o relative weight in p e r cent populations: so-year-old men, n=2322; 60-year-old men, n=331. weight index 1 according to lindberg et al. (24), weight index 2 according to build and blood pressure study, chicago (9, weight index 3 based on anthropometric studies of 50-year-old men in uppsala (15) index 1 index 2 index 3 weight index 50 year 60 year 50 year 60 year 50year 60year c0.90 4.4 5.7 17.2 13.0 28.0 22.4 0.90-0.99 14.7 9.4 32.3 32.0 34.2 35.1 1.00-1.09 28.8 28.4 28.3 27.8 23.9 24.2 1.1b1.19 27.3 26.0 15.0 18.7 9.2 13.0 21.20 24.9 30.5 7.3 8.4 4.7 5.4 upsulu j med sci 83 u . waern 158 15 10 c q m $ 5 ln mean =8 i n = 330 3 6 9 12 15 18 z20 8 e s r m m l h fig. 3. distribution of erythrocyte sedimentation rates in the total population. laboratory investigations the distributions of the values for erythrocyte sedimentation rate (esr) and of venous haematocrit are shown in figs. 3 and 4, respective ly. the mean esr was 8.1 mmlh, and 43 men (13.0%) had a value of i5 mm/h or more. the mean venous haematocrit was 44.9%. only 15 men (4.5 %) had a haematocrit below 40%. the values for fasting serum cholesterol are pre sented in fig. 5. the mean value was 6.20 mmol/l. subjects with values of 7.50 mmol/l or higher were re-tested ( n = 4 0 ) , and if they exceeded this limit again they were referred to the department of geriatrics for further evaluation. fig. 6 shows the distribution of serum tri glycerides. this shows skewness to the right. how ever, after logarithmic conversion the distribu tion was normal (fig. 7). the mean value was 1.68 mmol/l. subjects with a value of 2.4 mmol/l or higher ( n =46) were re-tested and followed the pro cedure mentioned above for serum cholesterol. < 36 lo h c t mean= ll 9 n = 330 sd. 3.2 h l0 per cent 3 0 fig. 4. distribution of venous haematocrit values in the total population. u p s a l u j med sci 83 20 15 c bl b ;lo c 4 5 l? 5 meanz6 2 n-331 sd=12 8 m m o l l l fs-chol fig. 5 . distribution of serum cholesterol values in the total population. finally, it should be mentioned that 36 ( 1 1 .o %) of the men had detectable amounts of ethyl alcohol in their urine. urinary electrolytes will be discussed in another article (37). electrocardiographic findings the ecg findings were considered pathological in 52 (15.7%) of the subjects. in a further 19 men (5.7%) the ecg was considered to be possibly pathological. in table vii the ecg results, coded according to the goldmann (13) criteria and to the minnesota code (31) are shown, together with ecg findings in 50-year-old men in the same community. no cod able abnormality was found in 67.1% of the men. the dominating findings were high qrs amplitude, which occurred in 1 1 . 8 % and qrs axis deviation in 8.5%. c al i < 0.7 1.5 mean=1.68 s.d=1.03 n.329 nj 2.5 z3.1 fs-tg mrnol/l fig. 6. distribution of serum triglyceride values in the total population. arithmetically plotted. health and disease at sixty 159 2o 1 i? rn 5 04 l 2 0 8 < o l 0 fs-tg log mmol/l fig. 7. distribution of serum triglyceride values in the total population. geometrically plotted. discussion the participation rate in this study was somewhat lower (78.4 %) than in the health examination of 50-year-old men in the same community (15), where it reached 81.7%. the somewhat lower figure could be explained by the larger number of older men who were already under the care of another physician. another reason for abstaining from the screening might have been access to health controls provided by employers. in the general health examination provided by the local health authorities, the male participation rate is about 70% for males aged 50 as well as 60 years (19). in a primary preventive study in gothenburg in 1970 (38) the participation rate reached 74% for men born in 1915. almost the same figure was re ported by isokoski (20) from a health survey in finland. in that study the highest participation was found for ages between 35 and 44 years, after which it levelled off. this is in accordance with the find ings reported by napier (10) in their tecumseh community study. the questionnaire seemed to be an adequate method of getting information which could in many ways be confirmed by the personal interview. con cerning hereditary questions it has been found (36) that middle-aged men in uppsala have a good knowledge of their parents’ age at and cause of death. as mentioned previously, very few ques tions were given the answer “do not know” in the self-administered questionnaire. one exception was that 36 (10.9%) of the subjects were uncertain about previous information on a high bp. this is surpris ing and suggests uncertainty on the part of the physicians handling hypertension rather than any thing else. the comparison between 50and 60-year-old men shows that the younger age group more often report a high bp in their parents than the older men. this might reflect the fewer opportunities of the older men’s parents to be examined by a physician. the prevalence of chest pain as revealed in the questionnaire seems somewhat higher than the cor responding figures in a study of men aged 55-59 in finland (30), but is comparable to that reported for 60to 64-year-old men in prague (1 1). the validity of the diagnosis ap given in a questionnaire has been discussed by lundman et al. (25). in this health examination additional information could be obtained at the personal interview in the cases with positive replies to the ap questions. between 10.9 table vii. frequency of some codable ecg abnormalities according to the minnesota code in health surveys of 50and 60-year-old men in uppsala a subject may be included more than once 50 years ( n = 2 322) code number subjects code number subjects item number of men per cent number of men per cent 60 years (n=331) no codable abnormality i , o 1 608 69.6 1, 0 q items i , 1-2 22 1 .o i , 1-2 qrs axis deviation 11, 1 70 3.0 11, 1 high qrs amplitude 111, 1 207 9.0 111, 1 s-t depression iv, 1 16 0.7 i v , 2-3 t-wave items v , 1-2 53 2.3 v, 1-2 a-v conduction defects vi, i 4 47 2.0 vi, 1-4 ventricular conduction defects v i i , 1-2 24 1.0 vii, 1-2 atrial fibrillation viii, 3 7 0.3 viii, 3 222 67. i 6 i .8 28 8.5 39 11.8 12 3.6 14 4.2 9 2.7 4 1.2 6 1.8 160 u . waern and 11.8% positive replies were noted to these questions, which corresponded well with the figure of 11.5% for subjects in whom ap was confirmed by a physician. the increased occurrence of ischaemic heart disease with age found in studies in uppsala is in accordance with other reports (2, 10, 21, 26, 32). the prevalence of diabetes was five times greater than in the 50-year-old men. this is in accordance with the findings of other authors (1, 4, 10, 28, 33) suggesting an age-related increase of prevalence, but contradicts the studies of ostrander et al. (29), who found the most marked increase in prevalence in the age groups 4 0 4 9 years. studies on smoking habits showed that only 39.6% of the 60-year-old men smoked, as against 51.0% of the 50-year-olds. this diminishing fre quency has been discussed by several authors (14, 16, 27, 35, 40). a more negative attitude amongst physicians towards tobacco smoking may have con tributed t o this age effect. this is evident, in fact, in the group of treated 60-year-old hypertensives, who smoked less than the population in general. measurements of haematocrit and esr have been performed in many screenings, due to the sim plicity of these tests and certainly for psychological reasons. however, the diagnostic value of these tests in this screening was not high. the mean esr values were 7.8 and 8.1 mm/h in the younger and older age groups, respectively. only 9.4% and 13.0% had values above 15 mm/h in the respective age groups. many authors (3, 6, 22) have discussed the com mon finding of higher esr values in aged popula tions, and regard a higher esr value as normal in aged persons. boyd (3), however, proposed that the probable upper normal limit in persons aged 65 years or more is 40 mm/h. gibson (12) denies this age-related increase of esr. the present study shows a negligible effect of age on the esr values. choosing an arbitrary limit of 20 mm/h, 5.2 % and 6.1 % of the younger and older age groups, respec tively, fell above this value. haematocrit was found to have the same propor tion of low values in both populations; thus 6.3 % in the younger and 4.5% in the older age groups had a haematocrit below 40 %. in the older group none of the men had a haematocrit below 40% without a known cause. the mean haematocrit was slightly higher in the older age group. the indices used in describing relative weight showed that the norwegian based index rqcom mended by lindberg et al. (24) classified relatively more men as overweight in both the 50and 60-year age groups than the other two indices used. this is probably explained by the relatively lean nor wegian population chosen as a reference for the former weight index. the two other indices used, one based on find ings in the build and blood pressure study in usa ( 5 ) and the other on findings in 50-year-old men in uppsala (15) yielded almost identical results in de scribing relative weight. when the latter was used there seemed to be more young men with an index of 0.9, and more older men with an index of 1.1. however, these differences were not significant and the proportions of subjects in the two populations with an index between 0.90 and 1.09 were nearly identical. a comparison of the ecg findings showed that almost the same proportion-approximately two thirds-in the two studied populations had no abnormality according to the minnesota code (3 1). no increase in the occurrence of q-wave items was found, but proportionately more men in the older group had qrs axis deviation and t-wave items than in the younger men. atrial fibrillation also showed a higher prevalence in the group of older men. among the groups with codable ab normalities, there were more men with multiple findings in the older than in the younger age group. most reports on ecg findings are based on hospitalized patients, which make epidemiological comparisons difficult. however, in a large material of 122,043 ecgs performed in the u.s. air force, hiss et al. (18) described various findings in differ ent age groups. proportionately more abnormali ties, especially t-wave changes, were registered in higher ages. the same has also been found by higgins et al. (17) in analysing results from the framingham study. the health examination of 60-year-old men gave results that, compared with those for 50-year-old men, may have been influenced by two factors. firstly the age factor, which manifested itself as increased morbidity and thus increased consump tion of medical preparations. this tendency is of general importance, as the proportion of aged peo ple is going to increase in sweden. two main diagnostic groups-diseases of the circulatory system and diabetes mellitus-merit special interest. primary preventive activities and upsalo j med s c i 83 health and disease at sixty 161 early detection of these diseases ought t o be given priority in future health screenings of younger groups. secondly, factors operating within a changing society may have influenced the results. thus the decrease in t h e number of smokers between t h e ages of 50 and 60, which is a common feature, might be explained in many ways. one contributory factor may b e the increased propaganda in the mass media against smoking. in the study of 50-year-old men in the same community (is), various primary preven tive activities were carried out. these included a smoking withdrawal programme, dietary informa tion and treatment of increased bp. some of these activities may have influenced t h e attitudes of o t h e r groups of middle-aged men. t h e relatively larger number of 60-year-old than 50-year-old men who are retired may t o a certain extent b e ascribed to age, but the more liberal rules as regards retirement, especially o n account of med ical disablement, probably accounts for s o m e of the increase. finally, t h e expansion of health screening exami nations and medical services offered b y employers, in particular, must b e considered. thus, it was noted paradoxically that many 60-year-old men who participated in this screening also reported frequent contacts with other medical authorities. neverthe less, the increased number of 60-year-old men already under t h e care of other physicians may have been t h e reason f o r part of t h e lack of participation in this health examination. a c k n o w l e d g e m e n t this work was supported by grants from the swedish national association against heart and chest disease and from the faculty of medicine at the university of uppsa la. r e f e r e n c e s 1 . andres, r., pozefsky, t., swerdloff, r. s. & tobin, d. j.: effect of aging on carbohydrate metabolism. in early diabetes. academic press, new york, 1970. 2. beaglehole, r., heiss, g., tyroler, h. a., cassel, j. c. & hames, c. g.: prevalence and incidence data in the assessment of the risk of coronary heart diseases. amer j epidemiol102,l: 55, 1975. 3. boyd, r. v. & hoffbrand, b. i.: erytrocyte sedimen tation rate in elderly hospital in-patients. brit med j i:901, 1966. 4. boyns, d. r., crossley, j. n., abrams, m. e., jar rett, r. j. & keen, h.: oral glucose tolerance and related factors in a normal population sample. i. blood 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community surveillance for coronary heart disease: the framingham cardio vascular disease survey. am 3 epidemiol 100, 6 : 425, 1974. 27. mckennel, a. c. & thomas, r. k.: adults’ and ado lescents’ smoking habits and attitudes. government social survey. h.m.s.o., london, 1967. 28. metz, r . , surmaczynska, b., berger, s. & sobel, g.: glucose tolerance, plasma insulin and free fatty acids in elderly subjects. ann intern med64: 1042, 1966. 29. ostrander, l. d., lamphiear, d. e. & block, w. d.: diabetes among men in a general population. preval ence and associated physiological findings. arch in tern med 136:415, 1976. 30. reunanen, a., pyorala, k., punsar, s . , heinonen, 0. p., puro, k . & aromaa, a,: the prevalence of angina pectoris and other chest pain in nine population groups in finland. diiodecim89: 668, 1973. 31. rose, g. a. & blackburn, h.: cardiovascular survey methods. who monographies, vol. 56. geneva, 1968. 32. rosenman, r. h., brand, r. j . , jenkins, d., friedman, m., straus, r. & wurm, m.: coronary heart disease in the western collaborative group study. jama233: 8 , 8 7 2 , 1975. 33. streeten, d. h. p., gerstein, m. m., marmor, b. m. & doisy, r. j.: reduced glucose tolerance in elderly subjects. diabetes 14: 579, 1965. 34. tibblin, g.: high blood pressure in men aged 50. a population study of men born in 1913. acta med scand, suppl. 470, 1%7. 35. todd, g. f.: (ed.) statistics of smoking in the united kingdom. tobacco research council, london, 1969. 36. waern, u., hedstrand, h. & aberg, h.: what middle-aged men know of their parents’ cause of death and age at death. a comparison between history and death certificate. scand j soc med4: 123, 1976. 37. waern, u. & aberg, h.: blood pressure in 60-year-old men. findings in a health survey and some compari sons with 50-year-old men in the same community. to be published. 38. waern, u. & boberg, j.: comparison of glucose toler ance, serum insulin, serum lipids, and skinfold thick ness between 50and 60-year-old men. to be published. 39. wilhelmsen, l., tibblin, g. & werko, l.: a primary preventive study in gothenburg, sweden. prev med 1 , 1 5 3 , 1972. 40. world health organization. series of technical re ports 168: 1 . who, geneva, 1959. 41. zeiner-henriksen, t.: rskevaner i den norske be folkning. tidsskr nor laegeforen 11:617, 1976. received february 15, 1978 address for reprints: urban waern, m.d. department of internal medicine university hospital s-750 14 uppsala sweden u[)sulu j m e d s c i 83 upsala j med sci 87: 127-134, 1982 release of insulin and pancreatic somatostatin in response to increased circulating growth hormone (gh) levels sven gustavsson' and gudmar lundqvist' departments of surgery' and clinical chemistry2, university hospital, uppsala, sweden abstract in an experimental study on 5 anaesthetized pigs the effect of increased circulating levels of growth hormone (gh) on the release of insulin and somato statin from the pancreas was investigated. blood was sampled simultaneously from the pancreatic venous effluent and from mixed venous blood. as measured by radioimmunoassay infusion of gh (20 pg x kg-i x h-') resulted in a significant (p < 0.01 ) increase both in insulin and somatostatin concentration in pancreatic venous blood. in mixed venous blood no significant changes in hormone levels were found. the results illustrate one possible mechanism for the close relation between circulating gh and endocrine pancreas. introduction considerable evidence is now available showing that growth hormone (gh) is capable of inhibiting its own secretion (12, 15, 8). involves a "short feed-back loop" operating on the hypothalamus /14/ is not finally proven. indirect evidences have, however, been obtained for such a hypothesis, where hypothalamic somatostatin seems to contribute to the gh release regulation (1 2). excess and hypothalamic somatostatin concentration in rats ( 1 3 ) , which might indicate an increase in somatostatin secretion during gh treatment. whether this mechanism a positive correlation is also demonstrated between gh so far no physiological significance has been attributed neither to the well-known extrapituitary actions of somatostatin, n o r to the localization of large amounts of somatostatin in the pancreatic islets and in the gastric mucosa. however, numerous reports on somatostatin as inhibitor of gastro intestinal (gi) hormone release points to a specific function for somatostatin as a modulator of gi hormone response, especially within the pancreatic islets but also in the stomach (7, 3 ) . the effect of gh o n pancreatic hormonal release is controversial. thus, gh does not appear to have any immediate effect on insulin secretion of the &cell 127 i n t h e i n v i t r o s t u d y of m a l a i s s e e t a l . ( l o ) , b u t t h e r e s u l t s of t a i & pek ( 1 6 ) i n d i c a t e t h a t g h may h a v e a t o n i c e f f e c t b o t h on i n s u l i n and g l u c a g o n r e l e a s e . c o n s t a n t l y e l e v a t e d l e v e l s of g h i n t u m o u r b e a r i n g rats r e s u l t e d i n an i n c r e a s e i n t h e f a s t i n g i n s u l i n l e v e l s a s w e l l a s i n t h e e x t r a c t a b l e p a n c r e a t i c i n s u l i n ( 1 1 ) . i n h i b i t o r y e f f e c t on i n s u l i n r e l e a s e ( 1 ) . m i g h t i n d u c e a change i n p a n c r e a t i c s o m a t o s t a t i n s e c r e t i o n o r n o t h a s n o t been s t u d i e d p r e v i o u s l y . however, j u d g i n g from c l i n i c a l s t u d i e s a d m i n i s t r a t i o n of g h h a s an whether c i r c u l a t i n g l e v e l s o f gh in t h e p r e s e n t p a p e r we a r e e v a l u a t i n g t h e h y p o t h e s i s t h a t p a n c r e a t i c somato s t a t i n r e l e a s e i s a f f e c t e d by t h e c i r c u l a t i n g l e v e l s o f gh i n t h e same way a s h y p o t h a l a m i c s o m a t o s t a t i n i s supposed t o be r e g u l a t e d . f o r t h e s e s t u d i e s we h a v e u s e d a n e x p e r i m e n t a l model which p e r m i t s s e l e c t i v e blood s a m p l i n g from t h e p a n c r e a t i c v e i n s d u r i n g and a f t e r gh i n f u s i o n . material and methods animals f i v e p i g s of swedish l a n d r a c e weighing 20-28 kg were used. a n a e s t h e s i a a n a e s t h e s i a was i n d u c e d by a n i n t r a m u s c u l a r i n j e c t i o n of k e t a m i n e ( k e t a l a g ) 250-500 mg and m a i n t a i n e d by r e p e a t e d i n t r a v e n o u s i n j e c t i o n s of p h e n o p e r i d i n ( l e a l g i n r , 4 mg e v e r y 30-45 min) and pancuron bromide ( p a v u l o n r , 4 mg e v e r y 30-45 min). a f t e r e n d o t r a c h e a l i n t u b a t i o n p o s i t i v e p r e s s u r e v e n t i l a t i o n was g i v e n w i t h a m i x t u r e of n20 and o2 ( 4 : l ) by means of a r e s p i r a t o r . a r t e r i a l blood p r e s s u r e was m o n i t o r e d t h r o u g h a c a t h e t e r ( i n f a n t f e e d i n g t u b e no 5 ) i n t r o d u c e d i n t o t h e common c a r o t i d a r t e r y of t h e l e f t s i d e . an i n f a n t f e e d i n g t u b e no 8 was i n s e r t e d i n t o t h e r i g h t atrium v i a t h e i n t e r n a l j u g u l a r v e i n o f t h e l e f t s i d e . t h i s c a t h e t e r was u s e d f o r measurement of t h e c e n t r a l venous p r e s s u r e and f o r blood s a m p l i n g (mixed venous b l o o d ) . s u r g i c a l p r o c e d u r e an u p p e r l a p a r o t o m y was performed u s i n g a m i d l i n e i n c i s i o n . a c a t h e t e r ( i n f a n t f e e d i n g t u b e no 5 ) was i n t r o d u e d i n t o t h e s u p e r i o r p a n c r e a t i c o d u o d e n a l v e i n i m m e d i a t e l y b e f o r e i t s e n t r a n c e i n t o t h e p o r t a l v e i n . t h i s c a t h e t e r was u s e d f o r blood s a m p l i n g from t h e p a n c r e a t i c venous e f f l u e n t . the c a t h e t e r was e x t e r i o r i z e d t h r o u g h a s t a b wound i n t h e abdominal w a l l and t h e laparotomy wound was c l o s e d . e x p e r i m e n t a l p r o t o c o l a f t e r a n i n i t i a l c o n t r o l p e r i o d of s a l i n e i n f u s i o n v i a an e a r v e i n and blood s a m p l i n g from b o t h c a t h e t e r s ( 4 5 , -30, -15 and 0 ) human growth hormone 128 (crescormone, kabi ab, sweden) was infused for 30 min via an ear vein in the dose 20 pgxkg-lxh-l. to the results of adamsson and efendic ( 1 ) to achieve an increase in circulating gh simulating the highest levels seen during a 24 h period in man and well within the range of circulating gh in acromegalic patients. blood was then sampled at 15, 30, 45, 60, 75 and 90 min after the beginning of the growth hormone administration. in 2 of the pigs a repeated infusion of growth hormone was given in the same dose starting 90 min after the beginning of the first infusion (fig. 1 ). protocol. the dose of administered gh was chosen according blood was sampled according to an identic experimental in different experiments the drip rate of blood from the catheter in the superior pancreatico-duodenal vein varied between 20-60/min. experiment the constancy of the drip rate (+ 10 $ ) was taken as evidence that the venous blood flow did not change. within each blood samples (5 ml each) were collected in chilled tubes with the addition after centrifugation at of heparin (143 usp-units) and trasylol (400 kie/ml). -4°c plasma was decanted and frozen at -20°c until radioimmunoassay. radioimmunoassae before radioimmunoassay of somatostatin plasma samples were extracted with acetone-petroleum-ether as suggested by arimura et al. (2). the radioimmuno assay was performed with a solid phase technique (9) with somatostatin anti bodies coupled to microcrystalline cellulose. the antiserum used, r 141, is well characterized with respect to reactivity against different parts of the somatostatin molecule as well as to the lack of reactivity against other gi peptides (2). tyr-1-somatostatin (beckman, geneva) was used for iodination with the lactoperoxidase method and synthetic somatostatin (beckman, geneva) was used for preparation of standards. insulin and gh were determined with solid phase radioimmunoassays (phadebas, pharmacia, sweden). statistical analysis the degree of significance for the difference between hormone levels before and after the beginning of the gh infusion was tested by analysis of variance. results the gh infusion in the selected dose resulted in a 10-fold increase in gh levels as measured in one of the pigs (fig. 1). the insulin concentration in pancreatic venous blood was significantly (p < 0.01 ) increased after the beginning of the gh infusion (table 1 ) . there was also noted a tendency to an increase in insulin in mixed venous blood although this increase did not reach 9-822x57 129 t a b l e 1 l e v e l s o f immunoreac b e f o r e and a f t e r t h e a n a l y s i s o f v a r i a n c e t i v e i n s u l i n (mu/l) i n p a n c r e a t i c venous blood b e g i n n i n g of t h e g h i n f u s i o n . mean + sem. r e v e a l e d f = 10.08, p = 0.004. animal no. b e f o r e g h i n f u s i o n a f t e r g h i n f u s i o n 10.7 + 2.7 23.6 + 6.0 121 + 28.0 7.8 + 1.8 9.3 + 1.8 20.5 + 8.6 114 + 13.9 741 + 188 21 + 15 18.0 + 2.9 t a b l e 2 l e v e l s of immunoreactive i n s u l i n (mu/l) i n mixed venous blood b e f o r e and a f t e r t h e b e g i n n i n g of t h e g h i n f u s i o n . mean + sem. a n a l y s i s of v a r i a n c e r e v e a l e d f = 3.86, p = 0.058. animal no. b e f o r e g h i n f u s i o n a f t e r g h i n f u s i o n 1 9.6 + 0.7 2 3.7 + 0.6 3 4.2 + 1.9 4 7.0 + 0.5 5 7.8 + 1.3 32.4 + 21 6.2 + 0.5 11.9 + 1.3 9.8 + 0.6 19.4 + 3.0 t a b l e 3 l e v e l s o f immunoreactive s o m a t o s t a t i n (pg/ml) i n p a n c r e a t i c venous blood b e f o r e and a f t e r t h e b e g i n n i n g of t h e g h i n f u s i o n . a n a l y s i s of v a r i a n c e r e v e a l e d f = 10.21, p = 0.007. mean + sem. animal no. b e f o r e gh i n f u s i o n a f t e r g h i n f u s i o n 799 + 47 458 + 44 391 + 56 331 + 106 429 + 56 1755 + 265 500 + 62 487 + 72 628 + 55 537 + 87 i30 t a b l e 4 insuun nw' 150 ko 50 0 l e v e l s o f immunoreactive s o m a t o s t a t i n (pg/ml) i n mixed venous blood b e f o r e and a f t e r t h e b e g i n n i n g of t h e gh i n f u s i o n . mean + sem. a n a l y s i s o f v a r i a n c e r e v e a l e d f = 0.73, p = 0.400. . . . . . . . . . . 't \ ', ** y' +-+-. *--+ c-+ animal no. b e f o r e g h i n f u s i o n a f t e r g h i n f u s i o n 567 + 103 284 + 64 275 + 31 244 + 18 232 l 29 611 + 161 240 + 37 295 + 48 250 + 1 1 329 l 23 f i g . 1 . a r e p r e s e n t a t i v e example of hormone l e v e l s i n one i g b e f o r e , d u r i n g and a f t e r gh i n f u s i o n i n t h e d o s e 20 pgxkg-'xh-l ( h a t c h e d a r e a ) 131 s t a t i s t i c a l s i g n i f i c a n c e ( t a b l e 2 ) . measured i n t h e p a n c r e a t i c venous e f f l u e n t i n c r e a s e d s i g n i f i c a n t l y ( p < 0.01). i n mixed venous blood no s i g n i f i c a n t change was observed i n t h e s o m a t o s t a t i n c o n c e n t r a t i o n ( t a b l e 4). venous blood o c c u r r e d s i m u l t a n e o u s l y . in 2 a n i m a l s a r e p e a t e d gh i n f u s i o n was a d m i n i s t e r e d , i n b o t h c a s e s f o l l o w e d by a r e p e a t e d i n c r e m e n t i n t h e i n s u l i n and s o m a t o s t a t i n l e v e l i n p a n c r e a t i c venous blood ( f i g . 1 ) a c c o r d i n g t o t a b l e 3 t h e s o m a t o s t a t i n l e v e l the i n c r e a s e i n i n s u l i n and s o m a t o s t a t i n i n p a n c r e a t i c discussion the i n c r e a s e i n i n s u l i n and s o m a t o s t a t i n found i n t h e p r e s e n t e x p e r i m e n t s as a r e s p o n s e t o gh i n f u s i o n was s t a t i s t i c a l l y s i g n i f i c a n t i n p a n c r e a t i c venous b l o o d b u t n o t i n mixed venous blood. thus t h e p r e s e n t r e s u l t s i l l u s t r a t e t h e b e n e f i c i a l v a l u e o f blood s a m p l i n g a l s o from p a n c r e a t i c v e i n s i n s t u d i e s of hormonal i n t e r p l a y , a s a l s o p o i n t e d o u t b e f o r e ( 5 , 6 ) . is e s p e c i a l l y n e c e s s a r y i n s t u d i e s of p o l y p e p t i d e s w i t h a r a p i d metabolism o r e l i m i n a t i o n , l i k e s o m a t o s t a t i n . t h i s e x p e r i m e n t a l d e s i g n the u s e o f human gh m i g h t be e r r o n e o u s due t o t h e well-known s p e c i e s d i f f e r e n c e s known f o r g h ( 4 ) . n o t e d between human and p o r c i n e gh (18) which j u s t i f i e s t h e p r e s e n t e x p e r i m e n t a l p e r f o r m a n c e . the p o s s i b i l i t i e s of d i r e c t measurement of t h e g h l e v e l s reached by g h i n f u s i o n is a n o t h e r r e a s o n f o r t h e u s e of human g h i n t h e s e e x p e r i m e n t s . the mechanism whereby g h a f f e c t s i s l e t f u n c t i o n i s n o t known and t h e e f f e c t of gh o n i n s u l i n s e c r e t i o n i s c o n t r o v e r s i a l . the p r e s e n t r e s u l t s w i t h an i n s u l i n r e s p o n s e d e t e c t e d i n p a n c r e a t i c venous b l o o d a f t e r gh i n f u s i o n a r e i n a c c o r d a n c e w i t h r e p o r t s of a s t i m u l a n t e f f e c t of g h o n i n s u l i n s e c r e t i o n ( 1 1 , 19). on t h e o t h e r hand a small b u t s i g n i f i c a n t d e c r e a s e of i n s u l i n r e l e a s e h a s a l s o been r e p o r t e d a f t e r gh i n f u s i o n i n man ( 1 ) . v a r i o u s d o s e s o f gh is u n c l e a r . however, m o l e c u l a r and immunological s i m i l a r i t i e s a r e whether t h i s d i s c r e p a n c y is due t o i n t h e p r e s e n t s t u d y t h e l e v e l of immunoreactive s o m a t o s t a t i n i n t h e pan c r e a t i c venous e f f l u e n t i n c r e a s e d s i g n i f i c a n t l y d u r i n g i n f u s i o n o f gh. t h i s i n c r e a s e was e v i d e n t i n a l l a n i m a l s b u t w i t h c o n s i d e r a b l e i n t e r i n d i v i d u a l v a r i a t i o n i n m a g n i t u d e , which j u s t i f i e s t h e p r e s e n t s t a t i s t i c a l e v a l u a t i o n . although l i t t l e i s known a b o u t t h e r o l e of s o m a t o s t a t i n p r o d u c i n g c e l l s w i t h i n t h e p a n c r e a t i c i s l e t s , it seems t o be r e a s o n a b l e t o s u g g e s t t h a t t h e p a n c r e a t i c d c e l l s r e a c t i n t h e same manner as h y p o t h a l a m i c s o m a t o s t a t i n c e l l s upon b l o o d gh a l t e r a t i o n s . the unchanged s o m a t o s t a t i n l e v e l i n mixed venous b l o o d , found by u s , do n o t s u p p o r t t h e h y p o t h e s i s t h a t p a n c r e a t i c s o m a t o s t a t i n d u r i n g p h y s i o l o g i c a l c o n d i t i o n s p a r t i c i p a t e s i n a feed-back c o n t r o l o f gh s e c r e t i o n from t h e p i t u i t a r y g l a n d . i t i s d i f f i c u l t t o f u r t h e r s p e c u l a t e on t h e complex i n t e r p l a y between gh o n one hand and i n s u l i n and p a n c r e a t i c s o m a t o s t a t i n on t h e o t h e r . an i n c r e a s e i n 132 somatostatin release would be followed by a diminished release of insulin in view of the well-known suppressive effect of this hormonal peptide on basal and stimulated insulin secretion. on the other hand, in this particular experimental model at least, stimulation of insulin by glucose is coupled to a decrease of somatostatin release into pancreatic veins ( 5 , 6). hypothetically the release of insulin may be the first event and the increase in somatostatin should then modulate the insulin response. however, such an hypothesis has to be tested by hormone determinations at closer intervals than was made in the present study. acknowledgement financial support was obtained from the swedish medical research council project no. 4574. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13 14. 15. references adamson, u. & efendic, s.: insulin-like and diabetogenic effects of growth hormone in healthy subjects, diabetics, and low insulin responders. j clin endocrinol metab 49:456-461, 1979. arimura,a., lundqvist, g., rothman, j., chang, r., fernandez-durango, r., elde, r., coy, d.h., meyers, c. & schally, a.v.: radioimmunoassay of somatostatin. metabolism 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assay for plasma somatostatin. clin chim acta 101:183-191, 1980. malaisse, w.j., malaisse-lagae, f., king, s. & wright, p.h.: effect of growth hormone o n insulin secretion. am j physiol 215:427-428, 1968. martin, j.m., akerblom, h.k. & garay, g.: insulin secretion in rats with elevated levels of circulating growth hormone due to mtt-w15 tumor. diabetes 17:661-667, 1968. molitch, m.e. & hlivyak, l.e.: growth hormone short-loop feedbook. horm metab res 12:559-560, 1980. patel, y.c.: growth hormone stimulates hypothalamic somatostatin. life sci 24:1589-1593 1979. reichlin, s.: regulation of somatotrophic hormone secretion. in: handbook of physiology iv, part 2, pp. 405-422, 1973. american physiological society, washington dc. siler, t.m., yen, s.s.c., vale, w. & guillemin, r.: inhibition by somatostatin on the release of tsh induced in man by thyrotropin releasing factor. j clin endocrinol metab 78:742-745, 1974. 1978. 40:612-618, 1975. 133 16. t a i , t.y. & p e k , s.: d i r e c t s t i m u l a t i o n by growth hormone o f g l u c a g o n and i n s u l i n r e l e a s e from i s o l a t e d r a t p a n c r e a s . e n d o c r i n o l o g y 99:669-677, 17. tannenbaum, g.s., epelbaum, j . , c o l l e , e . , b r a z e a u , p. & m a r t i n , j . b . : 1976. d i s s o c i a t i o n o f e f f e c t s o f s o m a t o s t a t i n a n t i s e r u m on growth hormone and i n s u l i n s e c r e t i o n . metabolism 27: suppl 1 : 1263-1 266, 1978. 18. t a s h j i a n j r , a . h . , l e v i n e , l. & wilhelmi, a . e . : immunochemical r e l a t e d n e s s o f p o r c i n e , b o v i n e , o v i n e and p r i m a t e p i t u i t a r y growth hormones. e n d o c r i n o l o g y 77:563-566, 1965. i n t e r n a t i o n a l congress s e r . 184:220-231 , 1968. received november 1 4 , 1981 address f o r r e p r i n t s : sven g u s t a v s s o n , m.d. department o f s u r g e r y u n i v e r s i t y h o s p i t a l s-750 14 uppsala sweden 19. t a y l o r , k.w.: r e g u l a t i o n of i n s u l i n s y n t h e s i s . e x c e r p t a medica 134 u p l a i med sci 79: 45-50, 1974 estimation of the maximal work rate sustainable for 6 minutes using a single-level load or stepwise increasing loads lars-olof nordesjo from the military medical examination centre ( m m u c ) and the department of clinical physiology (head: prof. t . sjostrand), karolinska sjukhuset, stockholm abstract twenty-seven young men participated in four different types of maximal work tests: the tornvall maximal ergometer test with a constant load and a work time of about 6 min, a maximal work test with two increases in load after 12 and 18 min work and a work time of about 21 min, a work test with increase in load every 6th minute until exhaustion, and a maximal work test on constant load during which maximal oxygen uptake was determined. a method based on the relationship between log load and log work time is proposed for estimation of physical work capacity expressed a s wmax ~ min from work tests with increasing loads. wm,, calculated in this manner differs very little from the results obtained from a work test with a constant load and a work time of about 6 min (the tornvall test) and the coefficient of correlation and standard error of residuals are of the same magnitude (r = 0 . 9 5 4 . 9 7 ; se = 6488 kpm x min-') as those obtained from a test-retest of thetorn vall test (r = 0.97; se = 59 kpm x min-i). the tornvall test also shows a very strong relationship with the maximal oxygen uptake (r = 0.88). on the basis of earlier studies (1, 4) and his own in vestigations tornvall (8) assumed that a linear rela tionship existed between the logarithms for maximal work time and load for work on a bicycle ergometer. this relationship may be expressed as follows: log t = u + p l o g n + e (1) where n = load, t = maximal work time at load n , tc and ,!? =constants, and e = a remainder term caused by disturbing factors. tornvall tested a number of individuals (including 52 conscripts and 28 other young men) at different loads and calculated the mean of the individual esti mates of ,!?. this was -4.959 for the conscript group and, with the aid of this value, tornvall stated that one could estimate the highest load an individual can sustain for 6 min ( w,,, by letting him work to exhaustion at an arbitrarily selected load and insert ing the values for t and n in the equation log t log 6 log wmax 6 rnin = 4.959 + log n in the case of work times of less than i min or more than 18 rnin a new test must be made at a more appropriate load, as it had not been confirmed whether the equation held good also for work of shorter or longer duration. tornvall also stated that the estimate became considerably more reliable if the range for permissible work times was reduced to 1-12 min. as a new system for registration of conscripts into the swedish armed forces was being investigated, it was suggested that the medical examination should include a work test ad modurn tornvall. the primary idea was that, to simplify the examination procedure in the new system, all draftees should be tested at the same load (1 400 kpm x min-')l. a pilot study, how ever, showed that about 25% could sustain this load for m x e than 12 rnin and about 9 % more than 18 min. in a discussion of the results of the pilot study the present author suggested that, by raising the load by 200 kpm x min-' after 12 min, the proportion of those exceeding the 18-min limit would be reduced and that, for those exceeding this limit, the test should not be interrupted but that the load should instead be raised by an additional 200 kpm x min-' every 6th minute. the first aim of the present study was t o test the tornvall procedure and to compare the results ob tained at a constant load and work time between 2 and 12 rnin with 1) a maximal work test in which the load is raised 1 kpmxmin-'=0.1634 watt; 1 watt=6.118 kpmxmin-l. upsala j m e d sci 79 46 lars-olof nordesjo table 1 . physical characteristics and selected data on physical working capacity for the 27 subjects and 18 year-olds at registration (n = 1718) ~~ the subjects boys of 18 variable 2 s.d. range x age, years 22.37 22 18.7-27.0 height, cm 180.6 6 . 9 169-192 177.2 weight, kg 67.8 7.8 54.5-85.0 66.4 heart volume, ml 734 98 520-910 673a wmsxamin. k p m x 1 6 1 6 241 1 180-2002 1 4 5 1 min-' maximal oxygen uptake 3.406 483 2.65-4.39 i x min-' ml x kg-l x min-' 50.4 6 . 4 37.5-63.4 calculated from micro x-rays taken in a standing position. after 12 min by 200 kpm x min-' and after 18 min by an additional 200 kpm x min-' and the total work time is at least 18 min, 2 ) a graded maximal work test in which the load is raised by 300 kpm xmin-' every 6th minute until exhaustion. another aim was to study the reliability of the original tornvall test, at a constant load and with a work time between 2 and 12 min, and the relation of this test t o maximal oxygen uptake. material the material consisted of 27 male university students. the subjects applied in response to an advertisement and re l o o d , kpm , min-' f i i 300 k p m x min-1 n t _ i c t i m e . m i n 1 i i i 0 g 12 18 2 4 30 fig. 1. sequence of change in load. nb=initial load work test type 6. n c = initial load work test type c. upsala j m e d sci 79 ttme. rill" t fig. 2. recalculation and addition of work times in work test type b. see text. ceived a remuneration which covered their travel expenses a n d offered a moderate stimulus. data of the 27 subjects will be seen in table i, from which it appears that the subjects were somewhat older and very slightly taller and heavier than 18-year-old draftees and that their working capacity measured as w,,, min was con siderably higher. methods height was measured to within 1 cm and weight to the nearest 0.1 kg. heart volume was calculated according to the formula proposed by kjellberg et al. (3) from anterio-posterior and lateral roentgenograms, taken with the tube at right angles t o the table. t h e subject was placed in a supine position with cranially extended arms. ecg was recorded at rest with 1 1 leads on an elema schonander mingograf 4 2 or 34. ch2 ch, were recorded during the first work test, ch, in the others. t h e tests were carried out on a n electrically braked ergo meter-elema-schonander model a m 368 (2). no changes were detected in the calibration when the ergometers were checked before, during and after the experiment. the length of the pedal lever was 17.5 cm. i n order that a work test should be judged as maximal, i t was required that the heart rate at end of work was at least 175 beats per rnin and that, on close supervision, the subject was deemed to be so exhausted as not t o be able t o continue t o work o r t o maintain the prescribed rate of revolutions (60 r.p.m.). in n o case was it necessary t o reject a test. during the test the subject was encouraged t o continue work until complete exhaustion. even if the expected work time, which was unknown t o the subject, had been attained, the encour agement continued with unchanged intensity. estimation of maximal work capacity 47 table 11. heart rate at termination of work, total work time, and physical work capacity ( wmax obtained from the various work tests final heart rate, work time, wmax e min' beats x min-' min kpm x min-' work test j . s.d. range x s.d. range 2 s.d. range preliminary test 193.2 7.74 175-210 11.24 6.10 2.5-20.3 1603 256 1 174-2085 (type b) type aia 190.5 7.84 175-200 6.54 1.64 4.4-10.7 i 6 1 6 241 1180-2002 type b 195.7 8.01 185-218 22.06 1.49 19.2-25.1 1649 261 1186-2 114 type 01; 190.7 7.66 1 7 6 2 0 4 5.84 1.00 4.17.7 1 6 1 0 256 1 122-2074 determination 191.3 7.66 182-208 16.26 1.19 14.5-19.7 of vozmax type 01: 190.4 7.56 176-200 5.79 1.05 4.17.7 1607 270 1 122-2074 type cb 192.2 6.81 185-208 24.72 3.67 19.8-31.3 1597 268 1073-2083 0 i = first series, 11 = second series. n-24. four variants of work tests were used: (a) a work test ad modum tornwall (at constant load), for which the result was calculated in accordance with equation (2). this test is called test a in the following. ( b ) a work test in which the load was raised by 200 kpm x min-' after 12 and again after 18 min (and, if necessary, additional increases by 200 kpm x min-' every 6th minute) as shown in fig. i . this test is called test b in the following. from the result of this test wm,, ~ min was estimated as follows. according t o equation (2) 12 rnin at load nisequiv alent (in respect of w,,,, to t i rnin at load n f 2 0 0 , where log ti = log 12 4.959 [log ( n + 200) -log n ] (3) t o t ; was added the time, t , (= 6 min), worked by the subject at load n + 200 and, analogously, the work time, t i , at load n + 4 0 0 was calculated which corresponded to ( t i + f,) rnin at load n + 200: log t i = log ( t i + t,) 4.959 [log ( n + 400) log ( n + ~ o o ) ] (4) to r i was added the time, t,, worked by the subject at load n+400. (if the load was raised three times, an additional recalculation and addition were made.) w,,, was ob tained from the equation log (ti + t h ) log 6 4.959 + l o g "+ ( h x 2oo)j (5) log wmax e min = where h = n u m b e r of increases of load. the manner of recalculation and addition of the work times is illustrated in fig. 2. (c) a work test in which the load was initially 300 o r 600 kpm x min-' but was raised by 300 kpm x min-' every 6th minute as shown in fig. 1. this test is called test c in the fol lowing. the result was calculated analogously to that for test b: log ti = log 6 -4.953 [log ( n + 300) -log n] (6) log ri=log(t;+6)-4.959 [ l o g ( n + 6 0 0 ) l o g ( n + 3 0 0 ) j (7) further recalculations were made analogously t o equations (6) and (7) until w,, could be obtained from the equa tion log (ti + t h ) log 6 4.959 + l o g ( n + h x 300) (8) log wmax e rnin = ( d ) a work test during which maximal oxygen uptake was determined. after a 10-min warm-up at 50% of the maximum load which the subject was calculated t o sustain for 6 minutes ( w m a x min) the load was raised to wmax min50 kpm x min-'. after 3 minutes at the higher load a mouthpiece was inserted and, after an additional minute, the collection of ex pired air (45-sec portions) in douglas bags started. duplicate analyses were made of the samples by the haldane technique (precision 1/(cd2),2n = 0.025 vol % 02) volm,x was indicated as the highest value of oxygen uptake obtained for each subject. experimental procedure at a preparatory visit the subject was given a general examina tion comprising, inter alia, heart-lung radiogram, ecg and physical examination, and a work test of type b in which the starting load for all subjects was 1 400 kpm x min-'. i n a first series a work test of type a and a work test of upsala j m e d sci 79 48 lars-olof nordesjo table 111. differences and relationships between the work tests work test x1 x2 n (kpm x min-') (kpm x min-') r (kpm x min-i) regression equation 4 2 . s.d.,. seb typea; type b 21 3 2 . g d 68.5 0.966 63.6 xi=o.892 x,+ 145 type ai type 011 21 6.3 62.0 0.971 59.0 x1=0.953 x,i145 type aii type c 24 10.0 86.0 0.948 87.7 x1 = 0.953 x2 + 85 type a11 vo2max 21 0.881 118.0 x1 = 445.9 x2 + 81 27 0.881 0.23e x2=0.00174 x1+0.62 ' se= standard deviation of residuals. i =first series, i1 = second series. p < 0.05. 1 x min-'. type b were performed. on the basis of the results from the test at the preparatory visit the load in test a was so chosen that the subject was expected to be exhausted after 6 min, and the starting load in test b so that the subject would be ex hausted after 21 min. (the loads were, however, rounded off t o the nearest 50 kpm x min-'.) t h e two tests were per formed at one week's interval and in random order. i n a second series 2-4) months after the first, a work test of type a was again carried out at the load (rounded off t o the nearest 50 kpm x min-') for which the expected maximal work time was 6 min, and the work test in which maximal oxygen uptake was determined was performed. the tests were executed in random order and with at least 3 days' interval (3-14 days). in a third series, 2-55 days after the second, test c was carried out with starting load 300 (or 600) kpmxmin-l. owing t o temporary illness, 3 subjects did not perform this test. means and standard deviations for the remaining 24 subjects have been reported earlier (5). results table i1 shows the mean, standard deviation and range for final pulse rate, actual work time and w,,, calculated from the various tests. in table 111 the various estimates of wma,6 are compared, and it will be seen that the differences in mean values are small. only in one case is the dif ference significant ( p < 0.05). the covariation between the different estimates of w,,, is very high. in all cases r is 0.95 or higher (see table 111). for test a the test-retest reliability was high (r = upsala j m e d sci 79 0.97) although 3 months had elapsed between test and retest. the relation between maximal oxygen uptake and w,,, , calculated from test a , was high (r = 0.91, p <0.001), and with maximal oxygen uptake as de pendent variable s, amounted to 0.234 1 x min-l (6.7 yo). discussion the result of test b was significantly higher ( 3 3 kpm x min-') than that of test a. correcting the result with regard to the mean difference, the stand ard error1 in estimation of the result of test a from the result of test b was 68.5 kpm x min-', which may be compared with the standard error (62.0 kpm x min-i) obtained when the result was estimated from the result of the second series with test a. the com parison is, however, not altogether fair as a con siderably longer time ( 2 4 ) months) had elapsed between the two tests of type a than between the first test of type a and the type b test (one week). probably the difference in standard error would have been greater if the time between the tests had been the same. the observation that the error tends t o be greater when the estimate is based on a test with duration about 22 min than on a test with duration about 64 min is in accordance with tornvall's ob i/""-"' 2(n 1) estimation of maximal work capacity 49 table iv. time in minutes to be added to the work time on the final b a d , when estimating w,,, from work test of type c *nation that the uncertainty is greater at work times exceeding 12 min. if the result is not corrected for the mean dif ference, the standard error1 will be 76.2 kpm x min-1. the results can, of course, also be corrected accord ing to the regression equation (table 111), this leads to a standard error of the same order of magnitude (63.6 kpm x min-'). it cannot be asserted that in all contexts test b can be used instead of test a with a work time close t o 6 min. but for the examination of draftees the advantage of, if necessary, raising the load after 12 and i8 min instead of testing on another day and at a more suitable load should more than outweigh the slight uncertainty of the result. the procedure has implied that the proportion of draftees with work times longer than 18 min has been reduced from 9 % to below 3%. in the present study the work times in test b varied between 19.2 and 25.1 min, so that no major con clusions can be drawn concerning the outcome of the test if carried out at a load which would have t o be raised more than twice. comparison of the results from tests c and a shows that w,,, (i can be estimated also from the for mer. this is an advantage, as one can then obtain four ordinary measures of physical working capacity from a single work test, i.e., in addition t o wmax6 also w170, hr,,, and the maximal oxygen uptake (koz,,,,j estimated from the pulse rate. a more important advantage is that, in the same way as in testing with w,,,, one can follow the change of blood pressure, ecg etc. on gradual raising of the load, but, in contradistinction t o the normal practice in the w,,, test, by continuing to exhaustion or until the test must be stopped for reasons of safety, one ob tains a more reliable estimate of the subject's maximal physical working capacity (or the highest load or pulse rate the subject can sustain without any un toward reactions). the calculation of w,,, may seem com plicated, but tables have been drawn up from which w,,, mfn can be directly read both for tests of type a and for tests of types b and c . it would require too much space t o reproduce these tables in this context, but table iv may be of some assistance in conjunc tion with test c. it shows the calculated time, t ; , t o be added to the time worked by the subject at the fi final load, kpm x min-' initial load (kpmxmin-')600 900 i 2 0 0 i500 1800 2 100 300 0.123 0.830 1.639 2.526 3.450 4.402 600 0.804 1.632 2.524 3.449 4.402 900 1.440 2.461 3.426 4.390 1 200 1.985 3.232 4.300 1 500 2.429 3.926 1800 2.795 nal load for different initial loads and for varying number of increases of load. it will be seen from the table that, when the load has been raised several times, the work a t the first loads has little signifi cance for the estimate of w,,, (i if the subject stops after 3 min at 1 500 kpm x min-l, t ; + t h will be 5.526, 5.524, 5.461 or 4.985 min depending on whether the initial load was 300, 600, 900 o r 1200 kpm x min-', which implies that w,,, is 1475.5, 1475.4, 1471.5 and 1444.6 kpm xmin-' respectively. the test-retest reliability has also been tested in a field study of screening character. as the initial load in all cases was 1 400 kpm x min-' the test for those who were exhausted within 12 min was of type a, while the test for the remainder was of type b, since the load was raised by 200 kprn xmin-' a t 12 min and if necessary, also after 18 and 24 min. even in this non-ideal situation the reliability was found t o be statisfactory ( r = 0.90, s, = 84.8 kpm x min-', n = 83). in another field study, likewise of screening character, a combination of tests a and b (initial load 1 400 kpm xmin-l in all cases in the same way as above) was compared with a work test of type c. with test c as independent variable the correlation was of the same order as in the laboratory study, whereas the standard error was rather higher ( r =0.90, s, = 105.5 kpm x min-l, n = 107). on this occasion the lactic acid level in the blood was determined a t the end of test a (or b) and the mean was found to be 12.2 mmol x 1-', s.d. 2.5 mmol x i-'. acknowledgements this work was supported by a grant from the delegation for applied medical defence research (project no. 18: 090/68). upsala j med sci 79 4 142854 50 lars-ofof nordesjo references i . grosse-lordemann, h. & miiller, e. a.: der einfluss der leistung und der arbeitsgeschwindigkeit auf das arbeitsmaximum und den wirkungsgrad beim rad fahren. arbeitsphysiol 9: 454-475, 1937. 2. holmgren, a. & mattsson, k . h.: a new ergometer with constant work load at varying pedalling rate. scand j clin lab invest 6: 137-140, 1954. 3 . kjellberg, s. r., lonroth, h. & rudhe, u.: the effect of various factors o n the roentgenological determination of the cardiac volume. acta radio1 35: 413-427, 1951. 4. miiller, e. a,: die abhangigkeit des arbeitsmaximums von der leistung bei verschiedenen personen. arbeits physiol 10: 67-73, 1938. 5. nordesjo, l.-0.: a comparison between the tornvall maximal ergometer test, submaximal ergometer tests and maximal oxygen uptake, forsvarsmedicin. in press, 1973. 6. nordesjo, l.-0. & schkle, r.: validity of an ergometer cycle test and measures of isometric muscle strength when predicting some aspects of military performance. forsvarsmedicin. in press, 1973. 7. sjostrand, t.: changes in respiratory organs of workmen a t ore smelting works. acta med scand, suppl. 196: 687, 1947. 8. tornvall, g.: assessment of physical capabilities. acta physiol scand 58: suppl. 201, 1963. 9. wahlund, h.: determination of physical working ca pacity. acta med scand 132: suppl. 215, 1948. received april 3 , 1973 address for reprints: l.-0. nordesjo military medical examination centre (mmuc) fack s-104 01 stockholm 60 sweden upsala j m e d sci 79 upsala j med sci 84: 255-258, 1979 estimation of parenchymal cell mass of parathyroid glands using a volumeter technique goran akerstrom', lars grimeliu?, conny fridh3 and henry johansson' from the departments of surgery' and pathology', university hospital, uppsala and the institute of technology3, stockholm, sweden abstract a volumeter technique was used to estimate the density of the two main tissue components of the parathyroid gland parenchymal and fat tissue. the difference in density between the two components was distinct ( 1 . 0 6 and 0.93 g/ml) and measurements of the weight and volume of parathyroid glands could therefore be utilized in calculating their parenchymal tissue content. the results of these measurements corresponded to those obtained at histopathological evaluation of parathyroid glands. the presented technique is simple and convenient and with slight improvement could be used for intraoperative characterization of para thyroid glands. introduction normal parathyroid glands contain parenchymal and fat cells and a minimal amount of stroma. the parenchymal cell mass probably best reflects the glandular endocrine activity. however, there are difficulties in estimating this cell mass, because of the irregular distribution of the parenchymal cells. histo pathological evaluation of the parenchymal cell content therefore has to be based on examination of a large number of sections. this is possible with an objective method using an image analysing computer technique (2) or semiquanti tatively by conventional ocular evaluation. both these methods have their limi tations, however, in that the image analysing computer is not available in many centres and the ocular evaluation requires great experience on the part of the examiner. a more suitable method for determining the parenchymal cell content of parathyroid glands is therefore greatly needed. as the two main glandular components, parenchymal and fat tissue, differ in density (according to the estimate of gilmour and martin ( 1 ) the respective densities are approximately 1.10 and 0.90 g/ml), it should be possible to use glandular density as an indirect measure of parenchymal cell content. this report presents a method for density determination of parathyroid glands, using values of weight and volume. the volumes of the glands were 255 measured in a sensitive volumeter. methods and material a volumeter was constructed according to the diagram in fig. 1. phosphate buffered saline (0.1 m, ph 7.2, 300 mosm/l) was used as the fluid in the volu meter measurements. to represent the density of pure parenchymal cell tissue, pieces of histologically verified, fat-free, solid parathyroid adenomas were weighed and measured in the volumeter. the density of intraglandular fat tissue was calculated from measurements of small pieces of fat taken from the sur roundings of the parathyroid glands. volumes were determined as the mean of five a [ 0-jgland in cylinder fig. 1. volumeter. in measuring the volume of the gland archimedes' principle is applied. part a of the apparatus can be disconnected when the gland to be measured is put in the cylin der. before disconnecting part a, the fluid is drawn below the cylin der (to a') with the piston in or der to keep the fluid in the system the connection b is a watertight, carefully ground glass-plate con nection. when the gland is in the apparatus and the surface of the fluid is adjusted (to a), the other surface of the fluid column is moved from bo to bl. the volume of the capillary from bo to bl is the volume of the gland. consecutive measurements on the same piece of tissue. to prevent dehydration due to evaporation the pieces of tissue were kept in a moist chamber between measurements. the reproducibility of the volumeter technique was evaluated and the maximal deviation from the mean value in series of repeated measurements was 1 . 2 3 % . the volumeter technique was tested on ten parathyroid glands taken from autopsy cases within 24 hours after death. the glands were cleared of surround ing fat and weighed, and their volume was estimated in the volumeter. after these measurements they were fixed in formalin and stained with hematocylin -eosin, and the parenchymal cell content was evaluated by conventional light microscopy on 10-12 sections of each gland. 256 f i g . 2 . diagram r e l a t i n g d e n s i t y o f p a r a t h y r o i d g l a n d s t o p a r enchymal c e l l c o n t e n t . parenchymal tissue the e f f e c t s upon t h e measurement r e s u l t s o f e v a p o r a t i o n due t o e x p o s u r e t o a i r between measurements were i n v e s t i g a t e d . results the d e n s i t i e s o f parenchymal and f a t t i s s u e were f o u n d t o be 1.059 * 0 . 0 0 5 g/ml ( n = 1 0 ) and 0 . 9 3 +_ 0 . 0 2 g/ml ( n = l o ) , r e s p e c t i v e l y . from t h e s e r e s u l t s t h e diagram i n f i g . 2 was c o n s t r u c t e d , t o r e l a t e t h e d e n s i t y of g l a n d s t o p a r enchymal c e l l c o n t e n t i n p e r c e n t . the r e s u l t s o f t h e s e c a l c u l a t i o n s of t h e parenchymal c e l l c o n t e n t were i n a g r e e m e n t w i t h t h e f i n d i n g s a t l i g h t m i c r o s c o p y on 10-12 s e c t i o n s of e a c h g l a n d . water loss d u e t o e v a p o r a t i o n of t i s s u e was found t o have a marked e f f e c t upon t h e measurements. the t o t a l w e i g h t of g l a n d s exposed t o t h e a i r f o r 15 min d e c r e a s e d by 10-15 % due t o w a t e r loss. t h i s w a t e r loss c o u l d b e minimized by k e e p i n g t h e tissues i n a moist chamber o r c o v e r e d w i t h a p i e c e of f a t t i s s u e be tween measurements . discussion i n t h i s s t u d y a v o l u m e t e r t e c h n i q u e was used t o estimate t h e d e n s i t y o f t h e t w o main t i s s u e components o f t h e p a r a t h y r o i d g l a n d parenchymal and f a t t i s s u e . the d i f f e r e n c e i n d e n s i t y between them was pronounced ( 1 . 0 6 and 0 . 9 3 g / m l ) . from t h e s e v a l u e s d e n s i t i e s r e p r e s e n t i n g 100 % and 0 % parenchymal c e l l c o n t e n t were c a l c u l a t e d and a d i a g r a m was c o n s t r u c t e d whereby g l a n d u l a r d e n s i t y c o u l d be u s e d f o r e s t i m a t i o n o f t h e c o n t e n t of parenchymal c e l l s . v a l u e s ob t a i n e d by t h e u s e o f t h i s diagram were i n agreement w i t h parenchymal c e l l con t e n t s o b t a i n e d by a c o n v e n t i o n a l method, u s i n g l i g h t m i c r o s c o p y on 1 0 1 2 sec t i o n s o f e a c h g l a n d . the d e s c r i b e d t e c h n i q u e i s a new way of e s t i m a t i n g t h e parenchymal c e l l mass o f t h e p a r a t h y r o i d s and is c o n s i d e r e d a u s e f u l advancement 257 in the histopathological characterization of these glands. the reproducibility of the volumeter measurements was good. however, effects of tissue evaporation necessitated procedures to prevent water loss. the volumeter technique is rapid compared to conventional histopathological methods for determination of the parathyroid parenchymal cell content. however, to get reproducible results measurements with the volumeter have to be made with great care, which makes them somewhat time-consuming. the method could be de veloped further, however, by electronic reading and automatic adjustments of the volumeter pistons. with such improvement it could well be of value for intra operative characterization of parathyroid glands. the density technique for histopathological evaluation of parathyroid glands has on basis of the preliminary results presented above now been further de veloped using a more convenient and reproducible density gradient column tech nique (3). acknowledgement this work was supported by a grant from the swedish medical research council, project no. b77-17x-04787-02. references 1. gilrnour. j.r. & martin, w . j . : the weight of the parathyroid glands. 2. grimelius, l., akerstrom, g., johansson, h. ei lundqvist, h.: estimation of j pathol bacteriol 44:431-462, 1937. parenchymal cell content of human parathyroid glands using the image ana lysing computer technique. am j pathol 93:793-799, 1978. 3. akerstrom, g., grimelius, l., johansson, h., lundqvist, h. & pertoft, h.: estimation of the parenchymal cell content of parathyroid glands by means of density gradients. (to be published). accepted january 15, 1979 address for reprints: goran akerstrom, m.d. department of surgery university hospital s-750 14 uppsala sweden 258 116 plenary lectures structure-function relationships for heparin-like polysaccharides m. h66k (uppsala, sweden) the d r a m a t i c e f f e c t s o f h e p a r i n 1 i k e p o l y s a c c h a r i d e s i n v a r i o u s b i o l o g i c a l systems a r e p r o b a b l y o f t e n due t o i n t e r a c t i o n s between t h e p o l y s a c c h a r i d e and a t a r g e t p r o t e i n . for example, t h e a c t i o n o f h e p a r i n as an a n t i c o a g u l a n t can be e x p l a i n e d by t h e s p e c i f i c i n t e r a c t i o n o f t h e p o l y s a c c h a r i d e w i t h a n t i t h r o m b i n i l l , a p r o t e a s e i n h i b i t o r a c t i n g on s e v e r a l o f t h e s e r i n e p r o t e a s e s i n t h e c o a g u l a t i o n cascade. b i n d i n g o f h e p a r i n t o t h e p r o t e i n g r e a t l y p o t e n t i a t e s t h e a c t i v i t y o f a n t i t h r o m b i n i l l , t h u s l e a d i n g t o an e f f e c t i v e b l o c k a g e o f t h e s e l e c t i v e p r o t e o l y t i c t r a n s f o r m a t i o n o f zymogens t o a c t i v e coagu 1 a t i o n f a c t o r s . i n j e c t i o n o f h e p a r i n i n t o t h e b l o o d o f an animal a f f e c t s n o t o n l y t h e co a g u l a t i o n system b u t a l s o t h e plasma c o n c e n t r a t i o n o f t r i g l y c e r i d e s . t h i s l i p o l y t i c e f f e c t o f h e p a r i n i s p r o b a b l y caused by t h e r e l e a s e o f l i p o p r o t e i n 1 ipase, a t r i g l y c e r i d e d e g r a d i n g enzyme, f r o m t i s s u e s i t e s t o t h e c i r c u l a t i n g b l o o d . l i p o p r o t e i n l i p a s e r e a d i l y b i n d s t o c e r t a i n p o l y a n i o n s and i s p r o b a b l y r e l e a s e d as a complex w i t h h e p a r i n . heparan s u l f a t e , a p o l y s a c c h a r i d e s t r u c t u r a l l y r e l a t e d t o h e p a r i n , o c c u r s a s s o c i a t e d w i t h t h e c e l 1 s u r f a c e o f s e v e r a l mammal i a n c e l l t y p e s . recent work i n o u r l a b o r a t o r y has shown t h a t p a r t o f t h e c e l l s u r f a c e heparan s u l f a t e i s bound t o s p e c i f i c b i n d i n g s i t e s i n t h e plasma membrane. i n t h e p r e s e n t communication t h e s t r u c t u r a l r e q u i r e m e n t s for b i n d i n g o f h e p a r i n 1 i k e p o l y s a c c h a r i d e s t o a n t i t h r o m b i n i i i , 1 i p o p r o t e i n l i p a s e and s p e c i f i c s i t e s o n t h e s u r f a c e o f r a t l i v e r c e l l s w i l l be d i s c u s s e d . b i n d i n g t o a n t i t h r o m b i n i l l . h e p a r i n i s t h e o n l y g l y c o s a m i n o g l y c a n t h a t b i n d s w i t h h i g h a f f i n i t y t o a n t i t h r m b i n i i i . furthermore, n o t a l l h e p a r i n m o l e c u l e s c o n i a i n t h e s t r u c t u r a l f e a t u r e s r e q u i r e d f o r h i g h a f f i n i t y b i n d i n g t o t h e p r o t e i n ; and h e p a r i n can i n f a c t be s e p a r a t e d i n t o two d i s t i n c t f r a c t i o n s h a v i n g h i g h and low a f f i n i t y f o r a n t i t h r o m b i n , r e s p e c t i v e l y . a n a l y s i s o f c h e m i c a l l y m o d i f i e d h e p a r i n s suggests t h a t t h e a n t j t h r o m b i n s t i m u l a t i n g a c t i v i t y i s l o s t when n s u l f a t e groups a r e removed from t h e polymer and r e s t o r e d on r e n s u l f a t i o n b u t n o t on n a c e t y l a t i o n o f t h e n d e s u l f a t e d p o l y s a c c h a r i d e . a h e p a r i n fragment w i t h a m o l e c u l a r w e i g h t o f about 5 0 0 0 con t a i n i n g t h e a n t i t h r o m b i n b i n d i n g s i t e and p o s s e s s i n g a n t i t h r o m b i n e n h a n c i n g a c t i v i t y c o u l d be i s o l a t e d a f t e r d i g e s t i o n o f t h e h e p a r i n a n t i t h r o m b i n complex w i t h a b a c t e r i a l h e p a r i n a s e . the s t r u c t u r e o f t h i s fragment w i l l be d i scussed. b i n d i n g t o 1 i p o p r o t e i n i ipase. beside h e p a r i n a l s o heparan s u l f a t e and dermatan s u l f a t e a r e c a p a b l e o f b i n d i n g t o 1 i p o p r o t e i n 1 ipase. the enzyme appears to b i n d much s t r o n g e r t o h e p a r i n t h a n t o t h e l o w s u l f a t e d g l y c o s aminoglycans; however, t h e a f f i n i t y o f t h e l i p a s e f o r t h e p o l y s a c c h a r i d e s does n o t depend s o l e l y on charge d e n s i t y o f t h e g l y c o s a m i n o g l y c a n as chon d r o i t i n s u l f a t e o n l y b i n d s s m a l l amount o f enzyme a l t h o u g h i t s c h a r g e den s i t y i s s i m i l a r t o t h a t o f dermatan [ s u l f a t e and heparan s u l f a t e . a n a l y s i s o f c h e m i c a l l y m o d i f i e d h e p a r i n s suggests t h a t n a c e t y l groups can be sub s t i t u t e d f o r n s u l f a t e groups w i t h o u t s i g n i f i c a n t l y a f f e c t i n g t h e a f f i n i t y f o r 1 i p o p r o t e i n 1 ipase. upshla j med sci 82 plenary lectures 117 a f t e r p a r t i a l d e g r a d a t i o n o f h e p a r i n , a fragment w i t h a m o l e c u l a r w e i g h t o f about 3 5 0 0 w i t h a p p r e c i a b l e a f f i n i t y f o r 1 i p o p r o t e i n 1 i p a s e c o u l d be i s o l a t e d . these r e s u l t s i n d i c a t e t h a t t h e b i n d i n g s i t e s f o r l i p o p r o t e i n 1 i p a s e and a n t i t h r o m b i n i i i i n t h e h e p a r i n m o l e c u l e a r e d i f f e r e n t . i n s u p p o r t o f t h i s c o n c l u s i o n t h e two h e p a r i n s p e c i e s separated by a f f i n i t y chromatography on a n t i t h r o m b i n s e p h a r o s e gave e s s e n t i a l l y i d e n t i c a l e l u t i o n p r o f i l e s when chromatographed on i m n o b i l ized 1 i p o p r o t e i n 1 i p a s e . b i n d i n g t o r a t l i v e r c e l l s . the heparan s u l f a t e s y n t h e t i s e d i n r a t l i v e r c e l l s u l t i m a t e l y appears a t t h e c e l l s u r f a c e where p a r t o f t h e m o l e c u l e s o c c u r s a s s o c i a t e d w i t h a " r e c e p t o r " . t h i s p o l y s a c c h a r i d e can be r e l e a s e d by h e p a r i n and by some heparan s u l f a t e s . p o l y s a c c h a r i d e s c a p a b l e o f r e l e a s i n g endogenous r a t l i v e r heparan s u l f a t e f r o m t h e c e l l s a l s o b i n d t o t h e c e l l s . r e s u l t s w i l l be p r e s e n t e d s u g g e s t i n g t h a t b i n d i n g o f heparan s u l f a t e t o c e l l s depends on t h e c o n t e n t o f e s t e r s u l f a t e b u t n o t on t h e presence o f n s u l f a t e groups i n t h e polymer. conclusions: the p r e s e n t e d r e s u l t s i n d i c a t e d i f f e r e n t s t r u c t u r a l r e q u i r ments f o r b i n d i n g of h e p a r i n r e l a t e d p o l y s a c c h a r i d e s t o a n t i t h r o m b i n i i i , 1 i p o p r o t e i n 1 i p a s e and r a t 1 i v e r c e l i s , r e s p e c t i v e l y . - upsala j med sci 82 upsala j med sci 92: 59-64, 1987 lkanssphenoidal therapeutic puncture of a cystic pituitary adenoma p. 0. lundberg, c. muhr, k . bergstrom, m. bergstrom, h. deuschl, p . enoksson, e. hagelquist, k.-a. thuomas and l. wide departments of neurology, diagnostic radiology, oto-rhino-laryngoiogy, ophthalmology, pathology and clinicul chemistry, university hospital, uppsala, sweden abstract transsphenoidal diagnostic puncture of skull base tumours has been a routine procedure in uppsala for a decade. bromocriptine is the treatment of choice for patients with prolactin secreting pitutiary adenomas. here we report on a patient with a pituitary adenoma who instead developed a n increase in tumour size after bromocriptine treatment. magnetic resonance imaging showed the tumor to be cystic. a transsphenoidal puncture was used therapeutically instead of surgery with excellent result. introduction bromocriptine treatment of prolactin-secreting pituitary adenomas usually results in a decrease of tumour size (27 out of 27 patients) (8). occasionally there is, however, an expansion of the tumour which may lead to progressive visual deterioration ( 3 ) and a demand for immediate surgery. the effect of the bromocriptine treatment is in most of the cases shrinkage of the tumour mass but sometimes a necrosis or cyst develops ( 2 , 5 ) . such cysts can be difficult to discover on computed tomography. magnetic resonance imaging (mri) on the other hand is an excellent method to diagnose necrotic and cystic changes in pituitary adenomas (5,7). transsphenoidal diagnostic puncture of skull base tumorus including pituitary adenomas has been a routine procedure at the university hospital in uppsala for more than 10 years (1, 8). in the present communication we describe that a transsphenoidal puncture also can be used as a therapeutic procedure. 59 case report a 38 year-old female patient, with two children born 1974 and 1977 had noticed galactorrhea since end of breast feeding 1978. her menstrual periods were regular up to 1982. endocrine evaluation 1985 revealed hyperprolactinemia 42-69 pg/l (normal < 20 pg/l) with no apparent diurnal variations. lh-rh and trh-tests showed normal increase in serum lh and tsh. serum oestradiol was below normal value for a woman of fertile age. no indication of thyroid or adrenal gland insufficiency was found. ct and mri disclosed an intraand suprasellar expansion reaching the chiasm. the visual fields were, however, normal. the patient was given 50 mg bromocriptine retard i.m. august 9, 1985, which led to a normalization of serum prolactin already 4 hours after the injection. six weeks later treatment with bromocriptine 2.5 mg twice a day orally was initiated. serum prolactin values remained normal. september 1985 the patients vision deteriorated with development of partial bitemporal hemianopsia and in october 1985 further progression of the visual field defects was observed. ct showed an increase in tumour size a few millimeters in the suprasellar region. mri with t1, t2 and proton density weighted images including a procedure now showed the tumour to be cystic with a thin cyst wall (fig. 1 a and b). october 29, 1985 a transnasal-transsphenoidal aspiration puncture of the cyst was performed. the position of the needle was during the puncture checked by fluoroscopy in the lateral projection. at 5 occasions with a few minutes interval a waterthin blood stained fluid, altogether 5 ml, was aspirated. at the 3 first occasions the patient experienced visual sensations in the form of differently coloured dots described to mimic pseudoisochromatic images. the patient also experienced a slight pain at both temples. an ophthalmologic examination performed after the procedure now demonstrated normal visual fields. a repeated mri the same afternoon revealed a considerable reduction in the tumour volume (fig. 2). there was no longer any suprasellar portion of the tumor. the upper tumour surface was concave and the sella was partially empty. after the therapeutic puncture the patient was kept on subtraction fig. 1 midsagittal mr image of the pituitary tumour with suprasellar extension a) t1 weighted image; b) subtraction image showing the cystic part of the tumour. 61 fig. 2 midsagittal mr image demonstrating the reduced size of the tumour immediately after the cyst puncture. subtraction image. fig. 3 at 10 months follow-up mri showed the same tumour size as in fig. 2. 62 bromocriptine treatment. galactorrhea and amenorrhea disappeared. at follow-up 3 and 10 months after the puncture mri showed a tumour appearance similar to that found immediately after the puncture (fig. 3). comments diagnostic puncture of skull base tumors, particularly pituitary adenomas, has been a routine procedure at our hospital. until now more than 80 patients have been investigated. the puncture is performed with a fine needle biopsy instrument. the needle with a mandrin is 15 cm long with an outer diameter of 0.9 mm. the needle is entered through the nose cavity below the anterior part of the middle turbinate. the final position of the needle is checked by fluoroscopy in the lateral projection. when the skull base is completely invaded by the tumour the needle passes without any pressure. otherwise the needle is rotated under gentle pressure until it is passed through the walls of sphenoid sinus into the tumour area. the needle is introduced about 1 cm into the tumour. at that time the position of the needle is checked by x-ray in the frontal and lateral projections. the fluid is aspirated after check of possible bleeding. care is taken not to loose the needle from the syringe during the procedure to avoid air embolism. so far few of the patients have experienced pain or other disagreeable sensations. there have been no serious complications. a slight bleeding after removal of the needle has on two occasions necessitated a tamponade for a few hours. a slight leakage of cerebrospinal fluid which stopped spontaneously occurred in two patients. signs of meningitis have never been observed. cysts within pituitary tumours may be recognized on ct. however, according to our experience of mri in more than 120 patients with tumours in the sellar region cysts or necrosis are better visualized with mri than with ct (5, 7). we also apply a technique of subtraction of two mr images with the same repetition time but different echo delays which gives further information about the tissue components of the tumour than the standard images (4). treatment of prolactin secreting pituitary adenomas with oral bromocriptine is recognized to be very effective with few side-effects. bromocriptine given in a slow release preparation i.m. results, however, in a more rapid decrease of serum prolactin. according to our experience in 20 patients it also gives fewer side-effects. 63 the effect of the injection lasts for 6 weeks and the treatment is thereafter continued with oral bromocriptine. we have observed in 5 further of our prolactinoma patients treated in such a way that magnetic resonance signal analysis revealed development of intratumor cysts (5). transsphenoidal therapeutic puncture of expanding cysts seems to be an alternative to open transsphenoidal surgery and very lenient to the patients. acknowledgements this study was supported by a grant from the swedish medical research council project no b86-39x-07004-02a. references 1. bergstrom, b., drettner, b. & stenkvist, b. : transnasal aspiration biopsy of central skull base destructions. acta cytol 1 7 425-430, 1973. 2. gen, m., uozumi, t., ohta, m., ito, a., kajiwara, h. & mori, s.: necrotic changes in prolactinomas after long term administration of bromocriptine. j clin endocrinol metab 59: 463-470,1984. 3. hall, k., prescott, r.w.g., kendall-taylor, p., patrick, d. & johnston, d.g.: long-term dopamine agonist therapy for prolactinomas: radiological aspects. in: trends in diagnosis and treatment of pituitary adenomas (eds. s.w.j. lamberts, f.j.h. tildens, e.a. van der veen & j. assies), pp. 139-146. free university press, amsterdam, 1983. 4. hemmingsson, a., bergstrom, k., ericsson, a., jung, b., sperber, g. & thuomas, k.-a.: structure enhancement in magnetic resonance imaging. acta radiol diagn 1986 (in press). 5. lundberg, p.o., bergstrom, k., thuomas, k.-a., muhr, c. & enoksson, p.: mri in tumours of the sellar region: evaluation of treatment with bromocriptine retard. acta radiol diagn 1986 (in press). 6. lundberg, p.0,. drettner, b., hemmingsson, a., stenkvist, b. & wide, l.: the invasive pituitary adenoma. arch neurol 34: 742-749, 1977. 64 upsala j med sci 81: 113-1 18, 1976 segmental arterial spasm in patients with total brain infarction lars-erik lorelius from the department of diagnostic radiology, university hospital, uppsala, sweden abstract an arteriographic investigation has shown that segmental spasm occurs in a relatively high frequency of patients with total brain infarction (12 of 30), and if spasm at the origin of arterial branches is included, the frequency is still higher (19 of 30). the phenomenon is possibly a sign of changed sympathetic tone, the pathophysiological significance of which is discussed. introduction during the last 20 years sporadic reports have been made of wave-like, regular, inconstant luminal variations in arteries at arteriography. this phe nomenon is usually limited to one segment of the artery and arteriographically it somewhat resembles fibromuscular hyperplasia. it differs from the latter condition, however, by the softness and strict re gularity of the waves. furthermore, these waves can be altered or made to disappear completely by another injection of contrast medium, with or without previous injection of a vasodilative agent. the length of the wave in the affected segment is correlated to the diameter of the artery. this ar teriographic phenomenon will be referred to in the following as a segmental spasm ( s s ) . the first t o report this observation was ratschow (13). h e believed it to be due to a dysfunction of the vessel, and claimed that occurrence of the phe nomenon on the arteriogram was related to pain. wickbom & bartley (18) found that arterial “spasm” were eliminated by priscol’ and claimed that a proneness to spasm was more marked in patients with raynaud’s disease. they also pointed out that the frequency of the phenomenon was inversely proportional to the age of the patient, which they a substance with a moderately effective adrenergic blocking function. 8-762852 attributed to increasing rigidity of the vessel wall with advancing age. lindbom (10) reported that spasms could be induced by raising the intra vascular pressure or by stretching the artery. the term “standing waves” was used by theander (16), who concluded from measurements on films that the length of the waves in the involved segment was directly related to the diameter of the artery, and also observed that in one case the vessel was wider when stationary waves were noted than when they were absent. from these findings and from physical arguments he considered that the phenomenon could not be due t o spasm but was caused by resonance in the vessel proximal to an occlusion. this theory was questioned by kohler (9) who made a physico-physiological analysis of the stand ing wave phenomenon and demonstrated that the arteriographic length of the assumed standing wave in human arteries did not correspond to the luminal variations. kohler considered that the changes were prob ably caused by a spastic circular constriction of the arterial wall, and were very likely an artificial phe nomenon produced by an irritation from the con trast medium in an especially sensitive vessel. mayall (11) postulated that the wavy contour was due to the formation of layers between the blood and the contrast medium, with rippling at the inter face between these layers. this theory was refuted, among other authors, by new (12), who pointed out that the phenomenon could also be perceived with a horizontal roentgen beam. ishikawa et al. (6, 7) reported a series of arteriographies of the lower ex tremity, in which accordion-like arterial shadows were present in about 10%. they claimed that this high frequency was related to the fact that the examination was performed with semiflexed legs and under spinal anesthesia, conditions which upsala j med sci 81 114 l . e . lorelius n 1 lalal “1 1 15 25 35 45 55 65 age n t spasm 15 25 35 45 age n m a l e 3 1, -15 25 35 45 55 6 5 age n female 4 1 2” 1 l 15 25 35 45 55 65 w e fig. 1 . age distribution in the total material and in the patients with spasm. the type of spasm with regard to pa tients age and sex. seemed to predispose to longitudinal contraction of the artery. they also pointed out that the phenom enon persisted after manual compression of the artery proximal to the site of injection. on electrical stimulation of the sympathetic nerves supplying the kidney in the rabbit, bergquist et al. (2) found lu minal variations in the renal artery reminiscent of those observed in clinical segmental spasm. seg mental spasm has been reported from practically all arterial areas except intracranial and coronary arteries-vital vascular areas in which an arterial spasm would endanger the patient’s life. segmental spasm has not been reported in patients examined under general anaesthesia. the causative mechanism of the phenomenon has been much discussed but little attention has been paid to its pathologic significance and its possible relation to the patient’s symptoms. its occurrence has been reported in raynaud’s disease ( l a ) , biir ger’s disease (15) and angiodyskinesia (1, 5, 8). n o uniform group of patients has been presented, how ever. in recent years some reports of nonocclusive ischemia in the small intestine in association with cardiovascular shock have been published (19,20). siegelmann et al. (14) demonstrated spasm of the superior mesenteric artery in the dog in, various types of cardiovascular shock in angiographic in vestigations; this spasm was located at the origin of arterial branches, and caliber variations were found in the main trunk or branches of the superior mesenteric artery. we have observed segmental spasm at some arteriographies in our clinic. a relatively large num ber of these cases have been patients with total brain infarction, for this reason we have re examined a series of arteriograms from patients with this conditions. material and methods (1) a retrospective investigation was performed on ab dominal arteriograms from 36 patients with clinical signs of total brain infarction (areflexia, hypothermia, per sistent respiratory arrest as tested by a 3-min apnoea test; isoelectric eeg on two occasions in each patient). on these patients cerebral 4 vessel-angiography was per formed to establish the diagnosis and renal aortography in order to survey the renal circulation with a view to pos fig. 2. patient 14, a woman aged 38 years. total cerebral infarction following intoxication. a semi-selective arterio gram clearly shows segmental spasm in the right renal artery. the patient was having an isoprenaline drip in fusion at the time of the examination. upsala j med sci81 arterial spasm in brain death 115 fig. 3. patient 21, a man aged 42 years. total cerebral infarction following an operation for subdural hematoma. selective angiography of the superior mesenteric artery. no drugs were given during the examination. note that in addition to segmental spasm there is a distinct spasm at the origin of arterial branches. sible kidney donation. six patients with atherosclerosis were excluded. of the remaining 30 patients, 14 were women and 16 men. the age and sex distributions are shown in fig. 1. (2) clinical data were obtained from the case journals. the pulse, blood pressure and temperature were recorded regularly. (3) clinico-chemical blood analysis of po,, ph and pco,, using a blood gas analyzer at 37°c (instrumentation lab. incorp., no. 313), were performed within 24 hours following the roentgen examination. hco, was measured at 37°c with a microcapillary technique on a radiometer p r meter 22 (radiometer, copenhagen). na+ and k+ were determined by flame photometry (il 343 flame photometer, instrumentation lab. inc., lexington, mass., usa). these data were also obtained from the case journals. cerebral 4 vessel-arteriography was performed by the seldinger technique, using a grey odman-ledin catheter with side-holes, introduced through the femoral artery. the tip of the catheter lay in the ascending aorta. at renal aortography 30 ml angiografin was injected at the level of the origins of the renal arteries, i.e. with the catheter tip located at l 1. all injections were given at a pressure of 5 kp per cmz, using a cisal i1 pressure syr inge. the exposure frequency during the renal aorto graphy was 3 frames per sec for 3 sec, followed by 1 frame every other sec for 8 sec. at selective arteriography of the superior mesenteric artery 25 ml angiografin was in jected at a pressure of 3 kp per cm2; the exposure fre quency was 2 frames per sec for 5 sec and 1 frame every other sec for 10 sec. this selective arteriography was per formed in patients 21 to 30, with the exception of patient 27. at all arteriographies the ecg, injection time and ex posure were registered with a mingograf e n 81 or mingo graf 800 direct recorder. the film-focus distance was 100 cm in all examinations and the same apparatus was used on all patients included in this investigation. the exposure data were 60-75 kv, 500-600 ma and 0, 10 sec. at the re-examination of the abdominal arteriograms, the renal and superior mesenteric arteries were examined with respect t o segmental spasm and the latter arteries were also examined for spasm at the origins of branches. spasm at the origins of branches was considered t o be present if at least two of the branches were slightly but clearly narrowed for a very short distance (1-2 mm). results (1) all examined patients showed clinical signs of total brain infarction, and this was also evident at cerebral 4 vessel-angiography. (2)-the systolic blood pressure was low in all cases (50-100 mmhg). the rectal temperature was also low and varied between 31 and 35.2"c, with the exception of one patient (no. 16), whose tempera ture was 39°c. this latter measurement was pos sible wrong. the heart rates varied from 50 t o 150 beats per min at the time of the angiographies. see tables i and 11. (3) the po,, pcoz, ph and serum electrolyte values are given in tables i and 11. at the time of the angiographies the patients were being treated with the drugs listed in table i. (4) of the 30 patients, one had segmental spasm in the right renal artery. eleven had segmental spasm in the superior mesenteric artery or its branches. sixteen patients had spasm at the origin of two or more branches of the superior mesenteric artery. nine patients had both segmental spasm and spasm at the origin of arterial branches. (see tables i and 11). only in 11 patients was no form of spasm observed. discussion in 6 patients atheromatosis was noted. these pa tients were relatively old and none of them showed signs of spasm, possibly because of rigidity of the vessel wall. they were therefore excluded from the subsequent analysis. the reason for the high fre quency of spasm in patients with total brain infarc tion can be assumed to be associated with the clini upsala j med s c i 81 + 1 f t ab le i. d at a fo r th e in di vi du al p at ie n ts : a g e, se x, d ia gn os is , d ru gs g iv en , b lo od p re ss u re , h ea rt r at e, te m pe ra tu re a n d c li n ic och em ic al a \ la bo ra to ry v al u es 2 2 s ys t. h r ? p u nf or tu na te ly t he c as e jo ur na l fo r pa ti en t 1 co ul d no t be f ou nd . s = se gm en ta l sp as m ; b s = sp as m a t or ig in o f br an ch es . d ru gs : a , a tr op in e; b , i ns ul in ; c , i so pr en al in ; d , a nt ib io ti cs ; e , p he ny to in ; f , f ur os em id e; g , c or ti co st er oi ds ; h , c hl or pr om az in e c n o. s ex (y rs ) s s b s d ia gn os is d ru gs (m m h g) m in ) (m m h g) ( m m h g) (m m ol /l ) ph (m m ol /l ) (m m ol /l ) "c f' 2 a ge b p (b ea ts / po , pc o , h c 0, n a+ k + t em p. 5 1 d 52 ic h em or r 2 0 20 + \ + su bd ur h em at d, g 70 50 22 .5 20 7. 5 13 4 3. 1 31 .0 3 8 40 + + fr ac t sk ul l g 90 70 17 2 33 24 7. 46 14 3 5. 7 34 .2 4 0 23 + + fr ac t sk ul l d, g 90 70 15 9 35 28 7. 45 14 4 4. 0 34 .5 16 + + fr ac t sk ul l d7 g 70 60 23 6 21 14 7. 34 13 7 3. 9 32 .8 su ba ra ch f 70 60 14 1 19 22 7. 55 13 8 3. 9 33 .0 5 p 6 0 34 7 0 15 + + su ba ra ch d 70 80 21 .5 21 .5 7. 52 15 1 4. 3 34 .8 30 + + su ba ra ch 90 80 25 6 34 16 7. 43 14 3 3. 7 35 .0 su ba ra ch h, c 10 0 15 0 10 8 42 20 7. 27 14 3 4. 0 34 .9 8 6 23 fr ac t sk ul l f 90 80 70 37 17 7. 38 13 9 2. 5 33 .7 9 d 10 d 52 11 d 46 op tc f, h 50 90 13 5 5. 1 35 .2 13 .5 7. 4 12 9 3. 7 34 .2 13 0 2. 2 33 .2 53 12 0 30 in to x ba rb e th yl c 10 0 50 98 19 12 7. 37 15 0 2. 9 32 .6 13 0 38 + fr ac t sk ul l f, g 70 12 0 16 5 57 23 7. 26 14 8 3. 9 35 .2 14 0 18 24 7. 47 13 2 3. 9 39 .0 e , f , g , h 10 0 10 0 11 9 30 op ic a ne w 15 6 16 0 29 17 6 41 + + su bd ur al h em or r e, f , g , h 80 90 99 41 28 7. 44 14 7 3. 6 33 .4 28 + fr ac t sk ul l g, h 70 70 10 3 18 21 .5 7. 7 14 8 3. 1 32 .5 op ic h em at 60 70 13 3 6. 1 33 .0 18 0 19 d 41 20 d 23 + fr ac t sk ul l e, g 90 80 16 4 49 27 .5 7. 21 14 3 3. 3 34 .0 42 + + op s ub d he m at 80 10 0 10 7 34 33 7. 58 14 6 3. 7 35 .0 ce r he m or r g 90 60 17 .5 14 6 4. 2 32 .0 21 6 22 d 45 + 35 + + su ba ra ch a, d 11 0 80 10 2 33 23 .5 7. 51 13 9 4. 0 31 .5 su ba ra ch 90 70 23 .5 15 8 4. 1 35 .0 23 0 24 0 40 25 0 22 + fr ac t s ku ll f 70 60 25 .5 15 8 4. 1 31 .0 19 + op a n ew c, g 10 0 80 12 6 45 22 7. 36 16 5 2. 4 34 .5 fr ac t sk ul l b 90 70 26 .0 15 3 1. 8 31 .5 26 6 27 d 32 + 49 + fr ac t sk ul l c 80 13 0 26 .0 14 5 4. 4 33 .0 90 70 67 34 24 .5 7. 6 15 4 4. 4 33 .2 26 .0 16 6 4. 0 34 .0 28 d 29 6 30 30 0 25 + en ce ph al it is c, d , g 80 80 60 70 78 30 su ba ra ch f, h su ba ra ch f, h 70 80 + fr ac t sk ul l d , g arterial spasm in brain death 117 table 11. shows the statistical datas f o r the different groups offindings with respect to arterial bloodpres sure, heart rate, arterial p o z , pcoz, h c 0 3 , p h , serum nu+, k + and body temperature hr bp (beats/ po, pco, hcobna+ kf temp. (mmhg) min) (mrnhg) (mmhg) (mmol/l) ph (mmol/l) (mmol/l) (“c) ss m n s.d. range s at origin m n s.d. range all spasm m n s.d. range n o spasm m n s.d. range 86 71 12 12 12 15 70-110 50-100 83 81 7 7 11 22 70-100 60-130 85 75 19 19 12 18 70-110 50-130 76 86 10 10 18 28 50-100 60-120 153 8 64 98-256 115 4 41 67-164 141 12 58 67-256 113 6 36 70-165 29.4 21.9 7.46 144 10 12 10 12 7.6 6.3 0.07 6 19.049.0 12.0-33.0 7.34-7.58 134-153 36.5 24.7 7.47 155 4 7 4 7 13.9 2.2 0.22 11 18.049.0 21.5-26.0 7.21-7.70 143 31.4 23.0 7.46 148 14 19 14 19 9.8 5.3 0.12 9 18.049.0 12.0-33.0 7.21-7.70 134 72 72 35.8 20.4 7.38 138 6 7 6 10 12.9 3.9 0.11 9 19.0-57.0 13.5-24.0 7.26-7.55 129-158 cal status. these patients comprise an extreme g r o u p t h e i r higher brain functions have been eliminated and their respiratory centre is out of function. their blood pressure and body tempera ture are low, and the heart rate is also usually low. the electrolyte balance is frequently disturbed and artificial ventilation is necessary, entailing a risk of changes in the acid-base status. this retrospective investigation revealed no difference between pa tients with and those without spasm with respect to blood pressure, temperature, blood gases, blood ph and serum sodium and potassium. four of five pa tients treated with a n isoprenaline drip infusion, l l of 13 patients treated with cortisone, and all 6 pa tients receiving antibiotics had segmental spasm. the material is too small, however to draw any con clusions from these observations. it is interesting that there appear to be no reports on segmental spasm in children, whereas adoles cents are well represented in various series. one possible explanation for this is that in children arteriographies are performed under general an aesthesia, which affects the vascular tone. preli minary results of direct nerve recordings in man show that the sympathetic activity in cutaneous 3.7 0.9 12 1.8-5.7 3.7 7 0.7 2 . 4 4 . 4 3.7 0.8 1.8-5.7 19 3.9 1.1 2.2-6.1 10 33.2 12 1.5 3 1.0-35.0 33.2 7 1.2 3 1 .o-34.5 33.2 19 1.3 31.0-35.0 34.6 10 1.8 3 1.5-39.0 nerves is inhibited on induction of anaesthesia (17) with fluothane. a similar anaesthesia might be the reason for the absence of segmental spasm in chil dren. the sympathetic activity is altered if the im pulses from higher centers to the vasomotor center are eliminated (3, 4). it is thus possible that the sympathico-adrenal activity in patients with total brain infarction has a completely different profile from that in other individuals; this is possibly to be regarded as dysfunction of the sympathetic nervous system. the simultaneous occurrence of spasm at the origins of branches of the superior mesenteric artery and segmental spasm in the same vascular area supports the theory that segmental spasm is caused by a change in tone of the arterial wall. the high frequency of spasm in a group of patients with total brain infarction may thus mean that segmental spasm is related t o changes in the sympathetic activity of the individual. applied to clinical routine, the occurrence of segmental spasm may thus, in certain circumstances, be a sign of altered vascular tone, the late effects of which are rela tively uninvestigated. thus the phenomenon may imply a change (possibly an increase) of the sym pathetic tone of the patient. it would be of interest upsala j m e d s c i 81 118 l . e . lorelius to investigate whether in healthy persons this phe nomenon is a manifestation of a sympathetic dys function, which could cause changes in the regional blood flow. segmental spasm should not be confused with fibromuscular hyperplasia. if any doubt should arise as to the nature of the arterial changes, it is re commended that a vasodilatant e.g. bradykinin be injected into the artery via the catheter inserted for the injection of contrast medium. a spasm might then be expected to change or disappear, while waves caused by an organic mechanism would be unaffected. references 1 . 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. amir-jahed, a. k.: angiodyskinesia. surg gynecol obstet 127: 609, 1968. bergquist, e., erikson, u. & ulfendahl, h.: sta tionary wave or segmental vasoconstriction. acta radiolll:497, 1971. cohen, m. i. & gootman, p. m.: periodicities in ef ferent discharge of splanchnic nerve of the cat. am j physiol218: 1092, 1970. folkow, b.: nervous control of the blood vessels. physiol rev 35: 629, 1955. haeger, k.: falsk claudicatio sive dysbasia inter mittens. lakartidningen 70: 1063, 1973. ishikawa, k . , mishima, y . , morioca, y . & hara, k.: accordionlike arterial shadows observed on the artenogram. angiology 24: 398, 1973. ishikawa, k., morioka, y . & mishima, y . : ac cordionlike arterial shadows on the femoral arterio grams. proceedings of the vii international congress of angiology, p. 832. barcelona, 1967. khobreh, m. & roy, p.: functional circulatory dis orders of lower extremities due to regional hemo cinetic imbalance. a new clinical and angiographic concept. surgery 61: 880, 1967. kohler, r.: regular alternating changes in arterial width in lower limb angiograms. acta radiol 3 : 529, 1965. lindbom, a . : arterial spasm caused by puncture and catheterization. acta radiol 47: 449, 1957. mayall, g. f.: arterial waves. clin radiol 15t355, 1964. new, p.: arterial stationary waves. am j roent genol radium ther nucl med 97: 488, 1966. ratschow, m.: die perlschnurarterie. zentralbl neurochir3: 154, 1955. siegelman, s . , sprayregel, s . & boley, s.: angio graphic diagnosis of mesenteric arterial vasoconstric tion. radiologv 112: 533. 1974. peripheral arteriography using the cathetel: method. acta radio147: 433, 1957. 19. williams, l. & jim-pok, k.: nonocclusive mesenteric ischemia. i n vascular disorders of the intestine (ed. by s. j. boley), p. 519. appleton-century-crofts, 1971. 20. wittenberg, j., athanasoulis, c., shapiro, j. & wil liams, l., jr: a radiological approach to the patient with acute, extensive bowel ischemia. radiology 106: 13, 1973. received march 20. 1976 address for reprints: lars-erik lorelius, m.d. department of diagnostic radiology university hospital s-750 14 uppsala sweden -_ 15. sutton, d.: arteriography. e & s livingstone ltd., edinburgh, 1962. 16. theander, g . : arteriographic demonstration of sta tionary arterial waves. acta radiol 53: 417, 1960. 17. wallin, g.: personal communication. 18. wickbom, i . & bartley, 0.: arterial “spasm” in upsala j med sci 81 upsala j med sci 79: 65-7 i , 1974 ultrastructure of human uterine epithelium at the time of imp i an ta t i on after post ovu i at ory administration of norethindrone ove nilsson and karl-gosta nygren from the department of anutomy, biomedical c e n t e r , s-75123 u p p s a l a , und department of obstetrics and g y n e c o l o g y , university h o s p i t a l , s-750 14 uppsulu, s w e d e n abstract the present investigation was performed to elucidate the mechanism of action of the previously demonstrated contraceptive effect in women of postovulatory ad ministration of a synthetic gestagen, norethindrone (net). seven women participated during 3 control cycles and during 8 treatment cycles, in which net was given orally after ovulation. daily peripheral plasma levels of progesterone, estradiol and net were assayed. an endometrial biopsy was taken in all cycles a t about the expected time of implantation. light microscopy revealed no consistent differences between non-treat ment and treatment cycles hut electron microscopy indicated that, after net treatment. the mitochondria treatment was found to have a certain contra ceptive effect although the efficacy was not suffi ciently high t o merit its use in clinical practice. one possible reason for the relatively low con traceptive efficacy of the net treatment, in spite of the drastic effect upon the progesterone levels, could be that the drug itself substitutes for the decreased progesterone levels (23). from previous investigations ( i , 2, 7, 8, 10, 17, 26) it is known that net influences the endometrium, but in these studies the administration of n e t did not follow the present schedule. therefore, the pres had grown larger and that nucleolar channel system ent investigation was designed t o study the impact had appeared. these changes suggest an i n c ~ a s e d of postovulatory n e t treatment upon the human progesterone-like influence upon the epithelium, despite uterine epithelium of normal at the time of the decreased progesterone plasma levels, caused by implantation, as visualized by lightand electronnet. it is assumed that these structural changes, caused by the postovulatory net treatment, might microscopy, in relation to the peripheral plasma change the functional properties of the endometrium levels of the ovarian steroids and of net. and thereby impair the possibilities for normal im plantation. material and methods introduction postovulatory administration of synthetic gestagens to women has been shown to markedly reduce peripheral plasma levels of progesterone ( 14). estradiol levels are also decreased but this de crease is not so pronounced (23). it was pro posed that the “luteolytic” effect of such treat ment might be utilized for postovulatory contra ception (14). the concept was that the hormonal effects of the treatment would induce changes in the endometrium significant enough to impair the possibility of normal implantation of the blastocyst when tested in clinical trials, in which a syn thetic gestagen, norethindrone (net), was given in various doses and dose schedules (23, 24) the (23). volunteers seven healthy regularly menstruating women, in the age range 25-30 years, participated in this investigation. five of the women had previously been pregnant and the remaining 2 women have both become pregnant since the present study. n o pathological changes were found upon pelvic examination (including a vaginal smear for cytology) before the start of the investigation. general design of the investigation a total of i i menstrual cycles were investigated. daily peripheral venous blood samples were collected, com mencing before the expected day of ovulation until after the day when implantation is expected to occur in a normal pregnancy. the concentrations of pro gesterone and estradiol were assayed in each blood sample. from the results of these assays the probable day of ovulation was calculated: after the mid-cyclic peak the first significant decrease in the estradiol level and the second day with a progesterone level above 5-742855 upsala j med sci 79 66 0. nilsson mnd k . g . n y w n i ng/ml plasma was called the day of ovulation (14). progesterone levels of 5 nglml plasma or more were re garded as postovulatory levels, i.e. a s an indication of ovulation (15). three of the eleven cycles served a s controls. in eight of the eleven cycles oral n e t treatment was given postovulatory and the n e t plasma levels were assayed. the treatment was aimed to start on the third postovulatory day, when the corpus luteum is functioning well and when, therefore, peripheral blood levels of progesterone have become high enough to permit the conclusion that ovulation has taken place (15). however, for practical reasons t h e results of both the progesterone and the estradiol assays were some times not available until a couple of days after the day when the blood samples were taken. therefore, preliminary calculation of the day of ovulation, which was i n some cycles based upon only one or two in creased progesterone plasma levels, sometimes re quired correction when all the results of the hormonal assays were available. in three of the cycles, treat ment had actually been started on the fourth and in one cycle on the fifth postovulatory day, as indicated in the table. in order to study the influence of varius schedules of treatment the doses of n e t ranged in total from 150 to 300 mg and the period of treatment from 2 to 4 days. this is also indicated in the table. the women were instructed to take their tablets in the evening and to give their blood sample in the morning. however, one woman (woman d, indicated in the table) took her n e t dose in the morning about one hour before the blood sample was taken. during each control and treatment cycle an endo metrial biopsy was obts,ned for lightand electron microscopy. the timing of biopsy removal was aimed to coincide with the probable time when implantation occurs in a normal pregnancy, i.e. about i week after the time of ovulation (9). for practical reasons the period of time elapsing from the calculated day of ovulation until the biopsy was taken varied between 6 and 8 days. all women were instructed to use mechanical and/or chemical contraceptives during the investigation. the biopsies were immediately fixed by immersion in a 2.5% solution of glutaraldehyde in soerensen phos phate buffer (ph 7.4) and were kept in the fixative for periods of one day up to several months. at embedding, small specimens were cut from the biopsies, rinsed in the buffer, and post-fixed for 3 hours in a solution of 1 % osmium tetroxide in soerensen phosphate buffer, ph 7.4. dehydration was performed in ethanol, and the specimens were embedded in epon 812. for light-microscopy, the sections were stained in a basic solution of toluidine blue. after microscopical examination appropriate areas of glandular or luminal epithelium were selected and trimmed for sectioning for electron-microscopy. for transmission electron microscopy, the sections were stained with uranyl acetate followed by lead citrate. for scanning electron microscopy (21), specimens with an endometrial sur face area of about i x 1 mm were cut, carefully rinsed in distilled water, and taken to 10% ethanol in water for freeze-drying. the specimens were frozen in iso pentane cooled by liquid nitrogen and dried in torr for 3 days at -79°c (solid carbon dioxide). the dry specimens were mounted for scanning electron microscopy and coated with carbon followed by gold. a jeol jsm-u3 scanning microscope was used. r e s u l t s hormonal assays all cycles in this investigation were found t o b e ovulatory, as judged by the levels of progesterone in plasma. the results of the assays of pro gesterone, estradiol and n e t a r e shown in the table, which summarizes the daily plasma levels of these hormones from the d a y after t h e cal culated day of ovulation until the day when the endometrial biopsy was taken. this gives a n estimation of the total amount of the hormones in plasma between the time of ovulation and t h e date of t h e biopsy. woman a had, initially, high progesterone levels and low estradiol levels compared with those found hormonal assays in most of the other treatment cycles, but respondprogesterone in p l a s m a was estimated by a com petitive protein binding assay, as described by johansed as expected to the n e t treatment. son (13). had comparatively low estradiol levels in both estradioi in p l a s m a was determined by a radiocvcles investisated. while in her second treatment y immunoassay as described by hotchkiss et ( 1 i ) , modified a s described by edqvist & johansson (4). norethindrone ( n e t ) in p l a s m a was assayed by a cycle progesterone levels were exceptionally low. t h e remaining 5 women, c through g, showed a radioimmunoassay as described by nygren et al. ( 2 5 ) . more consistent pattern. comparing control cycles and treatment cycles from t h e same women ( e , f and g) it was found endometrial morphology that the summarized progesterone levels were the endometrial biopsies were obtained with a genell lower i n the treatment cycles. estradiol levels curette without anesthesia and without dilation of the cervix. the biopsies were taken from the middle part were not changed but it be of the anterior wall of the corpus uteri. noted that in 2 of the women (f and g) the upsala j med sci 79 endometrium after norethinclrone treutment 67 table. peripherul plusmu concentrutions of progesterone, estradiol, und n e t , summurized jrom the day after ovulution until the d u y when the endometriul biopsy m ' u s tuken. the day oj'ovulution is cxilled duy 0 control cycles treatment cycles progesproges terone estradiol terone estradiol n e t biopsy (nglml (pglml n e t treatment biopsy (ng/ml (pg/ml (nglml woman on day plasma) plasma) mg on day on day plasma) plasma) plasma) a 100 4-6 7 b 100 5-7 8 l o o 3-4 6 c loo 3-4 7 d 100 4-5 7 e 7 87.1 1475 50 3-6 7 f 7 66.9 i226 50 4-6 8 g 6 73.2 i032 50 3-5 7 138 52.5 18.8 49.5 43.8 41.7 49.2 41.4 445 310 645 i285 i305 i445 i328 995 456 696 332 not assayed 1413" 349 219 i14 0 woman d took her n e t tablets about one hour before the blood sample was taken. hormonal levels were summarized from o n e more day in the treatment cycle as compared with the corresponding control cycle. o n t h e day of the biopsy the progesterone plasma level in woman e decreased from 14.5 ng/ml in the control cycle to 0.6 ng/ml in the treatment cycle, in woman f from 9. i ngfml t o 0.9 ng/ml and in women g from 15.5 ng/ml t o 5.5 ng/ml. corresponding estradiol plasma levels were decreased from 245 t o i10 pg/ ml in woman e , from 210 t o 60 pg/ml in woman f and from 250 t o 135 pglml i n woman c. to illustrate the hormonal patterns, the daily plasma levels of progesterone, estradiol and n e t in the control cycle and the treatment cycle of woman e a r e shown in fig. i . endometrial morphology light microscopy. t h e biopsies were examined with regard t o t h e amount of glycogen, stromal oedema, and decidual changes (9, 22, 27). micro scopy demonstrated slight differences among the biopsies, but n o difference was sufficiently con sistent to s e r v e a s a criterion of net-induced change. however, the glycogen content of t h e uterine epithelium appeared t o increase somewhat after the n e t treatment. electron microscopy. the ultrastructure of t h e luminal and glandular epithelium from biopsies obtained on the 6th-8th postovulatory d a y of t h e normal untreated women corresponds rather well to what has been previously reported (3, 5 , 6, 12, 18, 20, 21, 28, 29, 31): apical protrusions were ~ present although they contained only a few glyco 35. 30. 25. 20. ef ?, control cycle : *, days from ovulailon f i g . 1. woman e. daily peripheral plasma levels of progesterone, estradiol and n e t during one control cycle and one n e t treatment cycle. in both cycles an endometrial biopsy was taken 7 days after the calculated day of ovulation (=day 0). upsala j med sci 79 68 0. nilsson and k.-g. nygren fig. 2. luminal surface of normal endometrium, d a y 7 postovulatory. t h e microvilli of the secretory cells are noticed. several cells also possess apical pro trusions. x 10000. gen granules (fig. 2). t h e endoplasmic reticulum was well developed and the golgi apparatus was rather large. most mitochondria were in t h e normal size range and only a few giant mito chondria were observed. lysosome-like bodies occurred sparsely. the amount of glycogen gran ules varied but never reached the high levels present a few days earlier in the cycle when glycogen content reaches its peak (28). the ultrastructure of the cells of the luminal and glandular net-influenced uterine epithelium differed in some aspects from that of the control biopsies. apical protrusions were present but they generally contained more vesicles and glycogen granules than were observed in biopsies from untreated women (fig. 3). t h e mitochondria were generally larger (fig. 4), and giant mitochondria were more often present basally. t h e glycogen granules seemed t o be more numerous, often occurring in groups containing glycogen vacuoles. nucleolar channel systems were rather frequently observed (fig. 5 ) . no marked differences related t o the various dose schedules were found. obviously, this does not exclude t h e existence of functional differences although the technique used in the present in upsala j med sci 79 vestigation did not reveal a n y structural differences. t h e biopsies from woman a showed a glandular epithelium containing more dense granules than were usually observed. discuss ion t h e duration between the ingestion of n e t tablets and the blood sampling for the determination of plasma n e t levels was not entirely consistent, f o r practical reasons. after oral ingestion. n e t has been found t o have a plasma half-life, after its initial peak, of about 9 hours only (25). thus. the plasma levels of n e t recorded in the present investigation cannot be used for strictly quantitative comparison but merely as an indicator that the women took their tablets as intended. a decrease in progesterone and estradiol plasma levels was found during treatment cycles at the d a y of biopsy, as anticipated from previous ex perience with postovulatory n e t treatment (14, 23). o n e woman ( a ) had extremely high progesterone levels a s compared with the o t h e r women in this study. on examining the electronmicrographs, the uterine epithelium of this woman was found t o contain more dense granules than did the epithe lium from t h e other net-treated women. however, since this i s a n observation from o n e single wo man, no conclusion can a t present b e drawn from t h e finding. further, it must b e remembered that o u r knowledge of t h e inter-relationship between a hormonal plasma concentration and its effect upon the target tissues is meager. f o r instance, we d o not yet know the time lag between a change in t h e plasma level of a hormone and the structural response it may induce in the uterine epithelium. therefore, in the present communication only general trends in hormonal and structural changes have been considered. light microscopical findings indicating that the glycogen content of the gland cells increased after n e t treatment a r e not particularly conclusive, con sidering the inconsistency of t h e findings and t h e relatively few biopsies available. although electron microscopical observations imply ,various diffi culties (19), changes were found which seem to be sufficiently consistent t o warrant the con clusion that n e t did affect the uterine epithelium. t h e changes comprised t h e appearance of large mitochondria and nucleolar channel systems. both etidomcjtriurn ufter norethindrone treutmcnt 69 f i g . 3. luminal part of uterine epithelium, day 7 trusion is observed. i t contains vesicles a n d glycogen postovulatory after n e t treatment. a n apical progranules. x20000. these features a r e regarded, by most authors, as an effect of progesterone ( i . 7, 17, 30). since the serum levels of both progesterone and estradiol were lower in the treatment cycles at the time of biopsy, the progesterone-like action on the epithe lium is probably d u e to t h e synthetic gestagen. results from animal studies (16) suggest that the relation between the action of progesterone and n e t upon uterine hormone receptors might b e of great importance in this respect. several explanations for a contraceptive effect of the n e t treatment a r e possible. t h e tubal function might be affected. the proportion of tubal pregnancies, found in previous clinical trials with postovulatory n e t treatment was unexpected ly high (24), even though t h e actual number was too low t o provide conclusive statistical evidence of a n increased incidence of tubal pregnancies. furthermore, the luteotrophic effect of human chorionic gonadotropin ( h c g ) from the blastocyst upon the corpus luteum after successful implantation, might b e affected by the presence of n e t . however, both experiments with exogenous hcg administered to women treated with n e t after ovulation (14) and the normal hormonal and histological development of pregnancies occurring after such treatment (24) suggest that this is not a probable explanation. the most likely explana upsala j med sci 79 70 0. nilsson and k . g . n y g r e n f i g . 4 . basal part of glandular uterine epithelium, day 7 postovulatory after n e t treatment. the mito chondria generally are larger than at a similar stage of a control cycle. ~ 3 0 0 0 0 . tion is perhaps that the net-induced changes observed in the uterine epithelium might result in a secretion which is not optimal for t h e survival of the blastocyst or in a n extra-cellular coat on the luminal surface, counteracting the attachment of the blastocyst. it is possible that the use of a drug with a more potent progesterone-like action might in fluence t h e uterine epithelium more drastically, thereby giving a better contraceptive efficacy than postovulatory n e t treatment. on the other hand a drug having the same depressive effect a s n e t upon the function of the corpus luteum but which f i g . 5 . nucleolar channel system of glandular uterine epithelium, day 8 postovulatory after n e t treatment. x25 000. does not exhibit progesterone-like actions o n the uterine epithelium might also induce a more pronounced disturbance in the development of the endometrium. a c k n o w l e d g e m e n t this investigation was supported by grants from the international committee for contraception research of the population council (no. m71, 138), the ford foundation (grant to prof. gemzell, no. 66-405-a) and from the swedish medical research council (b74 17x-3495-03b). the authors are indebted to their technical staffs for skilled assistance. the assay of progesterone and estradiol by dr elof d. b. johansson is gratefully acknowledged. r e f e r e n c e s i . ancla, m., de brux, j . , belaisch, j . & musset, r.: influence de i’equilibre oestrogene-progesterone sur les ultrastructures de i’endometre humain. i . morphologie et evolution de corpuscules intra nucleaires presents dans les cellules glandulaires de i’endometre sous i’effet de la norethisterone et dans les sterilites essentielles. gynec obstet (paris) 63: 239, 1964. upsala j med sci 79 endometrium ufter norethindrone treatment 7 i 17. kohorn. e. i . . rice. s. i.. hemperly, s . & gordon, m.: the relation of the structure of progestational steroids to nucleolar differentiation in human endo metrium. j clin endocr34: 257. 1972. 18. morgenroth. k. & verhagen. a.: sublichtniikro skopische obertlachenstruktur des endometrium. arch gynaek 212: 30, 1972. 19. nilsson, 0.: electronmicroscopy of the glandular epithelium in the human uterus. i . follicular phase. j . ultrastruct res6: 413, 1962. 20. electron microscopy of the glandular epithelium in the human endometrium. 11. early and late luteal phase. j. ultrastruct res 6: 422. 1962. 2 1 . nilsson. 0. & nygren, k. g.: scanning electron microscopy of human endometrium. upsala j med sci 77: 3, 1972. 22. noyes, r. w . , hertig. a . t. & rock. j . : dating the endometrial biopsy. fertil steril 1: 3, 1950. 23. nygren, k. g . . johansson, e. d. b. & wide, l . : postovulatory contraception in women with large doses of norethindrone. contraception 5 : 445, 1972. 24. the contraceptive efficacy of 10 mg or 25 mg of norethindrone given orally on day 15-21 of the human menstrual cycle. contraception. in press. 2 5 . nygren, k . g . , lindberg. b . , martinsson, k . , b o w , w. t. k. & johansson. e. d. b.: radioimmuno assay of norethindrone: peripheral plasma levels after oral administration to humans and rhesus monkeys. contraception, i n press. 26. rice-wray, e., schulz contreras, m . & rosell, a . a , : endometrial changes with the administration of norethindrone. ginec obstet mex 17(98): 333, 1962. 27. rock, j . & barlet, m. k.: biopsy studies of human endometrium. j amer med ass 108: 2022, 1937. 28. sakuma, s.: glykogengenhalt undverteilung in der uterusschleimhaut der frau wahrend des nor malen zyklus im elektronenmikroskopischen bild. beitr path anat 140: 454, 1970. 29. sengel, a . & stoebner, p . : ultrastructure de i’endometre humain normal. 11. les glandes. z zellforsch 109: 260, 1970. 30. terzakis, j . a.: the nucleolar channel system of human endometrium. j cell biol27: 293, 1965. 31. wynn, r . m. & woolley, r. s.: ultrastructural cyclic changes in the human endometrium. 11. normal postovulatory phase. fertil steril 18: 721, 1967. 2. boschann, h. w. & kur, s.: the effect of 17 aethinyl-19-nortestosterone-enanthate. a new long acting gestagen on the endometrium and the atrophic vaginal epithelium. geburtsh frauenheilk 17: 928, 1957. 3. cavazos, f., green, j. a , , half, d. g . & lucas, f. v.: ultrastructure of the human endometrial glandular cell during the menstrual cycle. amer j obstet gynec 99: 833, 1967. 4. edqvist, l. e. & johansson. e. d. b.: radio immunoassay of estrone and estradiol in human and bovine peripheral plasma. acta endocr (kbh) 71: 716. 1972. 5 . ferenczy, a,, richart, r. m., agate, jr, f. j . , purkerson, mable l. & dempsey, e. w.: scanning electron microscopy of the human endometrial sur face epithelium. fertil steril23: 515, 1972. 6. ferenczy. a. & richart, r . m . : scanning and transmission electron microscopy of the human endometrial surface epithelium. j clin endocr metabol36: 999, 1973. 7. ferin, j. & delforge. j. p . : endometrial assessment of relative potency of contraceptive progestational compounds. mode of action of luteal supplementa tion. histochemical study. proc 3rd i n t congr hor monal steroids. hamburg sept. 1970. int congr ser 219. excerpta medica foundation (amsterdam) 878, 1971. 8. henzl. m.. jirasek, j., horsky, j . & presl, j.: proliferative effect of 17-alpha-ethinyl-19-nortesto sterone. arch gynaek 199: 335, 1964. 9 . hertig, a. t.: gestational hyperplasia of endo metrium. a morphologic correlation of ova, endo metrium and corpora lutea during early pregnancy. lab invest 13: 1153, 1964. 10. hertz, r., waite, j. h. & thomas, l. b . : pro gestational effectiveness of 19-nor-ethinyl-testo sterone by oral route in women. proc soc exp biol med 91: 418, 1956. 1 1 . hotchkiss, j., atkinson, l . e. & knobil, e.: time course of serum estrogen and luteinizing hormone (lh) concentrations during the menstrual cycle of the rhesus monkey. endocrinology 89: 177, 1971. 12. johannisson, e & nilsson, l.: scanning electron microscopic study of t h e human endometrium. fertil steril23: 613, 1972. 13. johansson, e. d. b.: a simplified procedure for the assay of progesterone. acta endocr (kbh), suppl. 147: 188, 1970. 14. -depression of the progesterone levels in women treated with synthetic gestagens after ovulation. acta endocr (kbh) 68: 779, 1971. 15. johansson, e. d. b., wide, l. & gemzell, c.: luteinizing hormone (lh) and progesterone in plasma and lh and oestrogens in urine during 42 normal menstrual cycles. acta endocr (kbh) 68: 502, 1971. 16. janne, o., kontula, k., rajakoski, e . , tanhuan paa, e. & vikho, r.: specificity of progesterone binding proteins. acta endocr (kbh), suppl. 177: 249. 1973. received december 23, 1973 a d d r e s s f o r reprints: k.-g. nygren, m. d. department of obstetrics & gynecology university hospital s-750 14 uppsala sweden upsala j med sci 79 upsala j med sci 84: 83-93, 1979 oxygen tension measurements in the intervertebral disc a methodological and experimental study arvid ejeskar and sten holm froin t h e depurtment qf' ortliopuedic sirrgery i , university of g t e h o r g , suhlgrensku sjukhuset. goteborg, su,eden abstract a polarographic membrane-covered electrode has been used for measurements of oxygen tension in the intervertebral disc. in vitro studies showed that the method measured partial pressures of oxygen with good reproducibility. however, in long continuous experiments, disc matrix adhering to the membrane influenced the readings negatively. the results of in vivo measurements in canine nucleus pulposus showed tensions of the order of 0.53 1.06 kpa ( 4 8 mm hg). no significant variations between different disc levels were found. introduction much of the work on the aetiology of low back pain has concentrated on the behaviour of healthy and degenerated intervertebral discs. the normal and pathological appearance, as well as the mechanical behaviour of the disc, have been the subject of numerous investigations (2, 4 , 9, 19, 23, 24). the bio mechanical changes with age and degeneration have also been described (21). a field of speculation has been the nutrition of the disc, particularly as it has been postulated that nutritional deficiency might lead to disc de generation (18, 20). the intervertebral disc is the largest avascular tissue in the body and one of the questions has been whether diffusion alone can ensure and adequate supply of nutrients to the cells in various parts of the disc (6, 16, 18). quantitative diffusion studies (27, 28) have elucidated the routes of solute transport and the metabolism of the sulphate ion. investigations on glucose-metabolizing enzymes (14) and sulphate incorporation (5, 13) in chondrocytes and articular cartilage have indicated that the cell metabolism is affected by variations in oxygen tension. metabolic studies on articular cartilage (8, 22) show that the chondrocytes follow a predominantly anaerobic pathway, but in a recent report (10) it was suggested that either chondrocytes can partially shift from anaerobic to aerobic metabolism, or that two different populations of chondrocytes are present: one anaerobic and a less common aerobic type. however, no experimental data are available in the literature on the oxygen exchange of the cells of the 83 intervertebral disc. in view of the above findings, and in order to obtain basic information on the oxygen situation, the present investigation was initiated to develop a method permitting in.vivo measurement of oxygen tension (po ) in the intervertebral disc. 2 materials and methods the oxygen electrode used in this study was a polarographic silver-platinum electrode (see fig. 1) similar to the one described by aust and drettner in 1972 (3). in our electrode the centrally located platinum thread had a thickness of 5 pm and the total length of the electrode was 15 centimetres. fig 1. the polarographic electrode 1. stainless steel cannula 2. silver cylinder (anode) the teflon membrane 1 2 3 5 3. nylon cuff employed when using 4 . glass capillary c( 5. platinum wire 1.0 mrn 6. membrane the electrode was connected to a ph-meter (phm 27b radiometer copenhagen) with a p02 monitor (pha 927b radiometer copenhagen) and operated at approxima tely 630 mv. the sensitivity was adjusted manually on the ph-meter (10 to -11 a/mm hg). two different membranes and application techniques were tested: a) a 12 um thick teflon membrane, keeping a layer of 0.9% saline between the membrane and the glass tip. the membrane was kept in place by a small nylon cuff. a special applicator was used for mounting the membrane (see fig. 2). fig. 2. the teflon membrane applicator. 1. electrode 2. teflon membrane 3 . saline 4 . nylon cuff b) a membrane made of rubber-modified polystyrene in toluene. the tip of the electrode was dipped in kc1 electrolyte and after drying the membrane was dip coated onto the electrode (26). the function of both membranes was tested during calibration. to avoid disturbances of membrane function during oxygen measurements,the electrode was placed in a protective cannula, which had a solid end and an opening at the tip of the electrode. 84 calibration: calibrations of the electrode were performed in test-tubes containing saline, one equilibrated with pure nitrogen and the others with different oxygen con centrations. the tubes were kept at constant temperature in a thermostated waterbath saturated saline and the zero of the p02 or when steady state was reached (calibration time), the electrode was trans ferred to the oxygen-saturated saline and the sensitivity wascorrected according to the barometric pressure. experimental tests of the electrode a . response time: with stirring. the electrode was first placed in the nitrogen scale was adjusted. after 5 minutes, the response time for the electrode was tested when transferring the elec trode from the nitrogen-saturated saline to saline equilibrated with oxygen and vice versa. b. linearity: although the electrode should be linear, a simple test of this characteristic was included. after calibration, measurements were made in test tubeseontaining saline saturated with a mixture of nitrogen and 0 . 9 % , 1.92, 2 . 5 % , 4 . 8 % and 1 0 . 3 % oxygen respectively. five experiments were performed at each tension level and the membranes were changed between each run. c. temperature dependence: the influence of temperature was tested as follows: after calibration at 37oc, measurements were made in tesetubescontaining saline equilibrated with either 1.9% or 5 . 0 % oxygen and placed in a water bath at 37oc. similar test tubes were kept at room temperature and results were obtained at the two tension levels. the electrode was kept for 5 minutes in each tube and afterwards recalibrated. this was repeated five times at both tension levels and the averages of the results with each membrane were taken as a measure of temperature dependence. d. stability: the stability of the electrode was tested by repeated calibrations during one hour, immersing the electrode each time for a period of 5 minutes in each test tube. deviations from the original values were registered after 5, 10, 2 0 , 3 0 and 60 minutes. ten such experiments were performed and the membranes were changed between each experiment. e. pressure dependence: the electrode was inserted into a rubber tube filled with saline equilibrated with 5 . 0 % oxygen, and then placed in a pressure chamber (see fig. 3 ) . the p02 of the saline solution was measured initially, after which increasing pressure was applied to the rubber tube. readings of p02 were done at 2 0 2 . 6 , 3 0 3 . 9 , 4 0 5 . 2 , 5 0 6 , 5 and 6 0 7 . 8 kpa. 85 to manometer fig. 3. apparatus for testing the pressure dependence. 1. electrode 2. rubber tube filled with saline o f p r e s s u r e 2omm f. effects of ph variations: measurements were made in saline following tension levels were used: ranging from 6.0 tc 8.0. g. elimination of adhering matrix: 3. cone of silastic solutions equilibrated with oxygen and the o x , 2 . 5 % , 4.1% and 10.3% with ph variations in a set of test tubes several nuclei pulposi were pooled and placed in a water bath at 37oc. the electrode was introduced with either of the two mem branes into the mixed nuclei gel. after 5, 10, 20, 30 and 6 0 minutes, the electrode was rinsed with one of the following solutions: saline, papain (merck, germany) or trypsin (merck, germany) (all at 37oc) and then rinsed with 0.9% saline and recalibrated. changes in calibration time and deviations in calibra tion were noted. h. introduction into an intervertebral disc: postmortem specimens of spines were used. the tough outer part of the disc was punctured ventrally with an outer cannula which had been designed to permit measurements at predetermined levels. the electrode was then introduced into the centre of the disc via the outer cannula and calibration before and after the procedure was compared and the differences were recorded. i n series 1 and 2 the teflon membrane was used, whereas i n series 3 and 4 the rubber-modified polystyrene membrane was used. series i: 36 introductions into canine discs. series 2: 30 introductions into human discs. series 3: 11 introductions into canine discs. series 4: 25 introductions into human discs. i. intradiscal oxygen tension in vitro: canine spines were taken, deep-frozen, thawed and refrozen for at least 24 hours and rethawed to ensure that the cells were dead. a) the discs were cut out leaving a tiny layer of the vertebral body on each side. these segments were immersed in a 0.9% saline with a small amount of heparin added in an open beaker. this was stored at 4oc, and after varying intervals specimens were removed and warmed in saline at 37oc in a water-bath. the electrode was introduced via the outer cannula into the disc and readings of oxygen tension were taken until steady state was reached. 0.05% solution of sodium azide in 86 b) another set of tests were performed with the disc in a 100% humidity chamber t o air, with one of the vertebral endplates trimmed away. the electrode in its protective cannula was placed in the central part of the nucleus at varying depths from 0.5 to 2.0 m from the air-exposed surface and continuous measurements were made until steady state was reached or for 3.5 hours. in vivo measurements c 20 adult labrador dogs were used. the animals were anaesthetized with pento thal (abbott, italy) (30 mg/kg body weight) and ventilated i n an engstroem respirator. blood gases and blood. pressure were controlled during the experi ment. laparotomy was performed and the ventral aspects o f the lumbar and lower thoracic discs were freed by minimal dissection and under careful haemostasis. the electrode was introduced into the central part of the nucleus pulposus and the oxygen tension was registered until steady state was reached. althogether, 63 intradiscal measurements were included in this study. results a. response time: with a well-fitting teflon membrane, 90% of full excursion was reached within 15-20 seconds and 100% after at most 3 minutes. the dip-coated membrane of the rubber-modified polystyrene material reached 95-100% of full excursion within 15-30 seconds, depending on the membrane thickness. b.+c. linearity and temperature dependence: the maximum deviation was i 2 . 1 % . the results are shown in fig. 4 . temperature dependence varied between different electrodes, ranging from 2.0% to 3.2% per degree centigrade. 90 fig. 4 . diagram showing the linearity of the electrode response. the bars represent s.d. i.-' ' o f , , , , -7-h 2 4 6 8 1 0 1 2 percent oxygen in the medium 87 d. stability: the results of the stability test are presented in table 1. the deviation increased with time and the average deviation in 5 . 0 % oxygen after one hour was 57.12 when the teflon membrane was used. with the polystyrene membrane the deviation after one h o u was t 3 . 3 2 . 100 q t m w u 3 e 5 : z w a= z ; 90 o l a 7 0 table 1. results of stability tests of the electrode using different membranes. h fig. 5 . deviation in cali bration after measurements .--.- ----_ in pooled samples of nuclei7 -a-----+ -0_ _ rinsing . o---o---.------.----- 0 pulposi and the effect of _ _ = polystyrene membrane o = teflon membrane ----rinsed in trypsin calibration deviation (kpa) time (min) tension (kpa) membrane 5 10 20 30 60 0 teflon .050 .053 .053 .053 .093 0 polystyrene .025 .030 .030 .035 .068 5.067 teflon .041 .053 . 0 9 2 .zoo 1 . 3 3 3 5.067 polystyrene .038 .050 .063 .120 .820 h. introduction into an intervertebral disc: series 1: in six cases membrane function was lost ( t :viation in cali ration of more than 1.333 kpa (10 mm hg) or instability). the remaining 30 specimens showed an average deviation of z 0 . 2 8 0 kpa o r 26.2x. 88 series 2: these introductions resulted in l o s s of function in two instances and a mean deviation of f0.253 kpa or 55.1% in the remaining 28 experiments. series 3: two introductions gave disturbed function. the average deviation in calibration was 20.140 kpa or 2 4 . 4 % . series 4 : there was no loss of membrane function in any of these experiments and the mean deviation was 20.093 kpa or ? 4 . 1 % . 1. intradiscal oxygen tension i n vitro: a) the oxygen tension in the disc increased slowly with storage time, reach ing tension levels corresponding to atmospheric air after 6 to 8 days. b) during the time of the experiment (3.5 hours) equilibrium was reached at depths down to 1.0 m, whereas in the deeper zones equilibrium was not quite achieved (see fig. 6). g 40 2, ._ -0 -.' fig. 6. results of continuous measure ments in discs, with one of the verte bral endplates removed. the electrode was placed in the nucleus pulposus at varying depths below the air-exposed surface. * = 0.5 mm 0 = 1.0 mm 0 = 2.0 mm i 1 2 3 t i m e (hrs) in-vivo measurements the oxygen tension values were consistently low, ranging from almost zero to 2.13 kpa (16 mm hg), with a mean value of 0.92 2 0.41 kpa (6.9 2 3.1 mm hd. no significant differences were found between the disc levels (see fig. 7). the mean value for equilibrium time, i.e. the time taken to reach steady state, was 19 minutes, in spite of the fact that in a majority of the experiments it was l e s s than 10 minutes. 14 e l2 e n i t fig. 7. results of in. vivo measure ments in canine nucleus pulposus at various disc levels. the bars represent s.d. i thl2 th13 li l 2 l 3 ll l 5 l 6 l7 intervertebral discs 7-792854 89 discussion the type of electrode used in this study has been used for p02 measurements in fluids, especially blood (7, 11) in maxillary sinus atmosphere (3) and in corpus vitreum (1) but not in other tissues. the reason for this has been that a probe of this size disturbs the circulation in the capillaries and therefore interferes with the normal supply of oxygen to the point of the measurements ( 2 5 ) . the adult intervertebral disc, however, is avascular and this argument therefore did not apply in this study. from a theoretical point of view, our electrode was working as a recessed electrode as it was dependent on the fact that tissue material completely filled the small cavity around the tip of the electrode. this also meant that the material around the measuring point was partly cut off from the surrounding tissue and the supply of oxygen might therefore be reduced. aust and drettner (3) stated that one of the difficulties in handling the electrode was to get a well-fitting membrane. this statement was confirmed in this study. a faulty membrane gave instability and rapid fluctuations or slowly decreasing or increasing recordings. the polystyrene membrane attached with the dip-coating technique was much easier to handle and its function was more stable compared to the teflon membrane. furthermore, it was easier to clean the electrode tip without distur bances. the stability of both types of memebranes seems to be sufficiently good for measurements during short periods, but might impose problems in long con tinuous experiments (3 hours or more). however, when the polystyrene membrane was used the stability was con siderably better than when the teflon membrane was employed. this effect might be due to the problems ofattaching the latter type of membrane. the response time was not as short as aust and drettner reported but in agreement with the findings of mapleson et a1 (12), who found that the response time varied from one membrane to another. however, the purpose of this investigation was to measure the oxygen tension which in fact cannot be expected to vary much when the gas tensions in the blood are kept constant. the response time of the order found was therefore not a major problem. the fact that the temperature dependence obtained was considerably higher (2-2.5 times) than was found by aust and drettner ( 3 ) might be due to the modification of the electrode. it is known that the normal human intradiscal pressure is higher than atmospheric pressure (17). in the canine discs, however, the situation is still unknown. according to our experience of disc punctures in vivo, the pressure in a normal nonchondro-dystrophoid canine intervertebral disc also seems to be higher than atmospheric pressure. these findings warranted the inclusion of a pressure test in the investigation. these changes in the external environment did not seem to influence the tension 90 measurements. it was quite obvious that the matrix of the disc interfered with oxygen tension measurements. the deviation in calibration increased markedly with time. in order toobtain acceptable calibrations after intradiscal introductions, a carefully performed rinsing procedure was necessary. the actual adherence mechanism of the matrix, however, was beyond the scope of this investigation. to be able to perform measurements in an intervertebral disc a robust elec trode that can withstand penetration of the very tough outer layers of the annulus fibrosus is required. in the experimental situation it must also resist the mechanical forces during introduction into the disc produced by the re sistant collagen fibre network and the irregularities in the endplates, which were neither flat nor parallel to each other. this mechanical stress on the electrode was probably an important reason for loss of membrane function. introduction into human discs caused less loss of membrane function than when canine discs were used. the anatomy of the canine spine was, however, such that penetration into the nucleus pulposus was more difficult compared to human discs, mainly because of less space between the vertebral bodies. oxygen tension measurements in discs stored to reach equilibrium showed a very slow increase of p02 with time. one reason may be blockage by coagulating blood in the capillaries underneath the vertebral endplate, preventing the oxygen from reaching the centre of the disc within the expected time. taking this into consideration, the measured times were long and some other blocking factor was probably present. in those cases where the electrode was introduced into the nucleus pulposus below the air-exposed surface (with one of the vertebral endplates removed), oxygen equilibrium was reached down to 1.0 m but in deeper layers the results obtained did not quite coincide with expected values. maroudas (15) found for articular cartilage an equilibrium time (90% of final equilibrium) of less than 3 hours for a tissue thickness of 2.0 mm (with one side exposed). in long continuous measurements matrix adherence might have been the main explanation for the discrepancy between the theoretical and the experimental values. another possible disturbing factor in this particular experimental set-up might be changes in the outermost layer of air-exposed tissue surface. our results indicate that a steep gradient in p02 must be present in the intervertebral disc. the periphery of the annulus is in contact with' the arterial blood pool, which has a tension of roughly 13.33 kpa (100mm hg). this means that even slight differences in depth of measurement will markedly influence the result. the variability of the measurements in vivo in canine discs, where diffusion distances to the centre of the nucleus are of the order of 8-10 xmn from the 91 annular edge, was therefore dependent upon the measuring site rather than being an expression o f an inherent error of the electrode system. the present in vivo po measurements were obtained in the central part of the nucleus pulposus, 1.5 mm to 2.0 mm from the vertebral endplate. theoretical calculations based upon fick’s law of diffusion (j. urban, personal communication) give values of almost zero in the most central part of the nucleus. accordingly, the oxygen tensions registered are in good agreement with those predicted when only taking into account diffusion from the blood pool below the hyaline cartilage endplate, neglecting the amount entering through the surrounding annulus periphery. 2 although the oxygen tension values in vivo (of the order of 0.53-1.06 kpa) were low they nevertheless seemed to be physiologically relevant, taking into account the relatively low cell density and long transport distances from the oxygen supply. the significance of the tension values cannot, however, be adequately interpreted without knowledge of the oxygen requirements of the disc tissue. acknowledgements these studies were supported by grants from the swedish work environment fund, carin trygger’s foundation, greta and einar asker’s foundation, riks farbundet mot rheumatism and goteborg’s liikaresallskap. references 1. alm, a. & bill, a.: the oxygen supply to the retina, i. effects of changes in intraocular and arterial blood pressures, and in arterial p 0 2 and pc0 on the oxygen tension in the vitreous body of the cat. acta physiol scan8 84:261-274, 1972. 2. armstrong, j. r.: lumbar disc lesions. e. & s. livingstones ltd, edinburgh, 1965. 3. aust, r. & drettner, b.: investigation of oxygen exchange in human paranasal sinuses with a small po -electrode. upsala j med sci 77:208-212, 1972. 4. benn, r. t. & wood, p. 6. n.: pain in the back. rheumatol and rehabil 14: 121-128, 1975. 5. brighton, c. t., lane j . m. & koh, j. k.: in vitro rabbit articular carti lage organ model 11. 35s incorporation in various oxygen tensions. arthritis rheum 17:245-252, 1974. disc. acta orthop scand 24:177-183, 1955. del p02 nei fluidi. boll soc ital biol sper 43:1247-1249, 1967. 268, 1937. gothenburg, sweden. acta orthop scand (suppl 118), 1969. metabolism in articular cartilage. j rheumatol 4:334-342, 1977. surfaces. in oxygen measurements of blood and tissues (ed. j. p. payne & d. w. hill), pp. 103-128, j & a churchill ltd, london, 1966. 12.mapleson, w. w., horton, j. n. & imrie, d. d.: the response pattern of polarographic oxygen electrodes and its influence on linearity and hysteresis. med biol eng 8:585-593, 1970. 6. brodin, h.: path of nutrition in articular cartilage and the intervertebral 7. brotzu, g. & meissl, a.: descrizion di microelettrode per la determinazione 8. bywaters, e. g. l.: metabolism of joint tissues. j pathol bacteriol 44:247 9. horal, j.: the clinical appearance of low back disorders in the city of lo.lane, j. m., brighton, c. t. & menkowitz, b. j.: anaerobic and aerobic ll.lubbers, d. w.: methods of measuring oxygen tensions of blood and organ 92 1 3 . 14. 15. 1 6 , 1 7 . 18. 19. 20. 21. 22. 2 3 . 24. 25. 26. 27. 28. marcus, r. f.: the effect of low oxygen concentration on growth, glycolysis and sulphate incorporation by articular chondrocytes in monolayer culture. arthritis rheum 16:646-656, 1973. marcus, r. e. & srivastava, v. m. l.: effect of low oxygen tensions on glucose-metabolizing enzymes in cultured articular chondrocytes. proc soc ~ x p biol med 143:488-491, 1973. maroudas, a.: glycosaminoglycan turn-over in articular cartilage. philos trans r soc lond biol 271:293-313, 1975. maroudas, a., stockwell, r. a., nachemson, a. & urban, j.: factors in volved in the nutrition of the human lumbar intervertebral disc: cellularity and diffusion of glucose in vitro. j anat 120:113-129, 1975. nachemson, a.: lumbar intradiscal pressure. experimental studies on post mortem material. acta orthop scand (suppl 43:1), 1960. nachemson, a., lewin, t., maroudas, a . & freeman, m. a. r.: in vitro diffusion of dye through the endplates and the annulus fibrosus of human lumbar intervertebral discs. acta ortop. scand 41:589-607, 1970. nachemson, a.: low back pain its etiology and treatment. clin med 78: 18-24, 1971. nachemson, a.: towards a better understanding of low-back pain: a review of the mechanics of the lumbar disc. rheumatol rehabil 14:129-143, 1975. naylor, a.: the biophysical and biomechanical aspects o f intervertebral disc herniation and degeneration. ann r c o l l surg eng 31:91-114, 1962. rosenthal, o . , bowie, m. a . & wagoner, g.: studies on the metabolism of articular cartilage. i. respiration and glycolysis in relation to its age. j cell physiol 17:221-233, 1940. rothman, r. h. & simone, f. a.: the spine. w. b. saunders co, philadelphia, 1975. schmorl, g. & junghanns, h.: the human spine in health and disease. grune & stratton, new york, 1971. silver, i.: the measurements of oxygen tension in tissues. in oxygen measurements of blood and tissues (ed. j. p. payne & d. w. hill), pp 133 154, j @ a churchill ltd, london, 1966. soutter, l. p., conway, m. j. & parker, d.: a system for monitoring arterial oxygen tension in sick new born babies. biomed eng 10 (7):257-260, 1975. urban, j . p. g.: fluid and solute transport in the intervertebral disc. ph. d. thesis, london university, 1977. urban, j., holm. s., maroudas, a. & nachemson, a.: nutrition of the inter vertebral disc: an in vivo study of solute transport. j clin orthop 129: 101-114, 1977. received july 1978. accepted october 1978 address for reprints: arvid ejeskar, m. d. department of orthopaedic surgery sahlgrenska sjukhuset s-413 45 giiteborg sweden 93 upsala j med sci 79: 97-102, 1974 choice of variables for predicting the heart volume lars backlund, kjell bergstrom, per ericsson, u n o erikson, bernhard huitfeldt and hans renck from the d e p a r t m e n t s of a n a e s i h e s i o l o g y , clinical physiology, internnl medicine a n d diagnostic r a d i o l o g y , university h o s p i t a l , u p p s a l u , s w e d e n abstract roentgenological heart volume determinations in the supine posture with calculation according to the formulae of both jonsell & kjellberg et al., and measurements of the height, weight, total haemoglobin, blood volume and physical work capacity, were made on 45 men of ages 64-86 years, previously prostatectomized and with no subjective symptoms of cardiopulmonary disease, and on 22 healthy men and 17 healthy women of ages 58-71 years from a health survey. statistical analysis of these data comprised simple correlation calculations and step wise regression. the body weight showed, as a rule, a stronger correlation than the body surface area with the heart volume. on testing a combination of variables, measures of body size in combination with blood vol ume appeared to have a stronger explanatory capacity than other combinations. measures of physical work capacity seemed to have a weaker predictive capacity in the age groups concerned. all correlations between heart volume and the other variables were weaker in a group with ecg anomalies possibly expressing heart disease than in the groups with a normal ecg. introduction roentgenological measurement of the heart volume is a very common procedure in clinical practice and is used in the diagnosis of heart diseases, in evaluating the course of a disease and in assess ing therapeutic effects. in certain cases, especially for diagnostic purposes, attempts are made to esti mate the size of the individual heart chambers, but in general a measurement of the total heart volume is considered adequate. in sports physiology and clinical work physiology heart volume determination is performed with the aim of correlating the size of the heart with the cardiac function. heart volume determination is also used in health surveys as a screening method for heart disease. with this ex tensive application of the procedure it is of great importance to have access to a method with good precision and t o use uniform and adequate models for prediction of normal values. a recent question naire study (3) showed, however, that within sweden alone several different methods are used, with variations both in the examination technique and in the formula for calculation of the volume. in normal persons the heart volume is related, among other things to body size and circulatory functional capacity. in calculating normal values, therefore, consideration must be taken of one or more such factors. the methods usually used in sweden for heart volume determination have been developed by jonsell (10) and kjellberg et al. ( 1 i , 12, 13). the volume according to the method of jonsell (vol,) is calculated from the formula: 71 ( f v r y (f-vl) 6 .p val,=a b c where a , b and c are the diameters illustrated in fig. 1, f is the film-focus distance (=125cm), v f r is the distance from the centre of the heart to the film in the frontal plane and v' is the corresponding distance to the film in the lateral plane. the formula corrects for degree of magnifi cation. the volume according to kjellberg et al. (vol,) is calculated from the formula: where c 1 and e l are c and e reduced for the degree of magnification and d , e and c are diameters as illustrated in fig. 1. the other notation is the same as in the previous formula. the body position and projection are also of importance. bergstrom et al. ( 1 , 2 ) found that the differences in mean values obtained with the differ ent projections were often considerable and larger in calculation according to jonsell's formula than 7 742855 upsala j med sci 79 98 l . backlund et al. in calculation by the formula of kjellberg. in their material of healthy young men, kjellberg’s formula gave, as a rule, somewhat higher values than jonsell’s formula. the range of variation was smaller with exposure in the recumbent than in t h e sitting position. angled projections did not result in smaller ranges of variation than straight projections. t h e heart volume is usually expressed in relation t o calculated body surface area as a measure of body size. reindell e t al. (14) have pointed o u t , however, that body weight has a stronger and more constant correlation than the calculated body sur face area with the heart volume. other methods of predicting the heart volume a r e to use rneas ures of t h e circulatory functional capacity, e.g. physical work capacity, o r measures of t h e volume of t h e circulatory system, e.g. total haemoglobin or blood volume (6, 7, 14). to increase the possi bilities of accurate prediction it is important to ascertain which variables have the strongest corre lation with t h e heart volume, but also to know how these correlations vary with age. reindell e t al. (14) have shown that both age and sex as well a s degree of physical fitness influence t h e correlations between physical work capacity and heart volume. at a given heart volume the oxygen pulse a t submaximal work is lower in women than in men a n d higher in well-trained athletes than in fig. 1 . schematic drawing showing the three diameters according to jonsell, (u, b and c ) and the three diameters according to kjellberg et al. (d, e and c ) . normal men. further, there is a weaker correla tion between oxygen pulse and heart volume in higher than in younger ages. t h e aim of the present study was t o find o u t which single variable and which combination of 2, 3 and 4 relevant variables, respectively, have the greatest explanatory value in prediction of t h e heart volume in elderly persons free from heart disease. m a t e r i a l the study comprised both a group of men with no subjective symptoms of cardiopulmonary disease, who 5-7 months previously had undergone prostatectomy with no postoperative complications, and a group of men and women from a health survey on a selection from the population of the urban district of uppsala. this latter group had undergone a comprehensive clin ical examination, including static and dynamic spiro metry and a submaximal work test, at which they had exhibited no signs indicative of respiratory or circula tory disease or other abnormality. none of them suf fered from any disease which could be assumed to be limiting for the physical work capacity or to affect the blood formation. in the group of prostatectomized patients all were clinicully free of cardiopulmonary dis ease. this group was divided into two sub-groups, one with a normal ecg both at rest and during exercise and the other with a pathological ecg at rest and/or during exercise. the sub-groups are referred to in the following as ecg-normal and ecg-abnormal. the ecg table i . anthropometric data f o r the material height, cm weight, kg age, yrs group n x range x range x range a. prostatectomized, b . prostatectomized, ecg-a bnormal 22 73.2 61-86 172.1 165-184 76.8 53-97 c. healthsurvey, men 22 64.6 59-71 176.0 164-184 75.3 59-95 d. health survey, women 17 63.0 5 9 4 9 162.9 150-178 63.5 47-79 ecg-normal 23 72.5 64-82 173.3 166-183 75.5 58-95 upsala j med sci 79 heart volume prediction 99 formula of both jonsell (val,) and kjellberg (vol,). in the calculations correction was made for the fact that the formula of kjeuberg et al. is intended for 30” angulation in the frontal projection, while in the present study the central beam was directed perpendicularly to the frontal plane ( i , 2). table 11. meon values, standard deviations and rangesfor m e a s i m s of’ heart volume, physical work cupucity und total hartnoglobin group a. prostatectomized, ecg-normal b . prostatectomized, ecg-a bnormal c. health survey, men d. health survey, women vari able ,f s.d. range n volj 985 226 6 6 6 1 3 7 7 23 volk 843 206 523-1280 23 wls,) 733 145 498-1 145 21 thb 695 127 509-1 140 21 v01j 967 219 663-1341 22 volk 810 132 623-1096 22 wise 713 154 34&l000 19 thb 672 142 442907 22 v01j 890 177 667-1336 22 volk 649 94 487827 22 wiso 798 170 497-1 108 20 thb 673 103 525998 22 volj 641 110 426904 17 voik 505 90 389712 17 wise 484 158 200810 16 thb 520 88 395734 17 changes consisted in bundle branch block, low-grade a-v block or unspecific st-t changes. table i gives the age distribution and certain anthropo metric data for the different groups. physiological methods the physical work capacity was determined by bicycle ergometry (graded loads of 6 min duration). w,,, and w,,o, defined as the work intensity at heart rates of 130 and 150 b e a t s h i n , respectively, were calculated by interor extrapolation (7). at the end of the work test capillary blood was taken for lactate determination, whereafter the variable w,,,,, defined a s the work load in kpmlmin performed at a lactate concentration of 5 meq/l, was calculated for the group of prostatecto mized patients (15). total haemoglobin (thb) was de termined by the alveolar co method, wherewith dupli cate determinations were performed on the “health survey” group (5) and single determinations on the group of prostatectomized patients (15). determinations of the haemoglobin concentration (hb) was performed on capillary blood and from the value of thb and hb the total blood volume (tbv) was calculated. roentgenological methods all patients were examined in the supine posture with antero-posterior and lateral projections and with the central beam directed at right angles to the frontal plane and lateral plane. the film-focus distance was 125 cm and frontal and lateral films were exposed simultaneously without ecg triggering. the subject breathed calmly and shallowly during the exposure. measurement on the films was performed by two of the authors in collaboration ( k . b. and u. e.) and in all cases the volume was calculated according to the computer analysis and statistical methods the body surface area (bsa), thb, tbv and heart volume were calculated from measured primary data by table 111. certain regression equations, giving re sidual variances and correlation coefficients ( r ) regr. residual coeff. const. variance r n a. prostatectomized, ecg-normal val,= 13.5 bw 36.7 171 volk= 9 . 5 b w + 128.0 183 volj= 1068.2 bsa1033.8 176 vol,= 748.9bsa572.6 185 val,= 0.6w1so585.1 233 v o ~ k = 0.2w,sn+ 742.4 219 volj= 54.7 tbv+ 627.4 201 volk= 80.9tbv+ 351.1 186 b. prostatectomized, ecg-abnormal val,= 8.7 bw + 301.7 208 volk= 4.4 bw + 472.1 129 volj= 563.3bsa101.1 215 volk= 313.3bsa+ 215.5 131 val,= 0.7w150+ 434.2 207 volk= 0.2w1,50+ 632.2 115 volj= -16.7 tbv + 1055.5 229 volk= 7.0tbv+ 772.3 138 c . health survey, men val,= 1.5 bw + vol,= 13.5 bw volj907.7bsa volk= 139.2bsa+ vol j = 0.5 wisnf val,= 115.4tbv+ volk= 66.9 tbv+ d. health survey, women vol j = 8 . 9 b w + volk= 6 . 9 b w + volk= 0. i wlso,of volj= 564.4bsa volk= 471.9bsa val,= 0.4w,,o+ volk= 0.3 w,sof val,= 44.5 tbv+ volk= 34.6 tbv+ 129.8 138 537.1 98 845.4 137 382.5 97 490.2 170 550.0 101 249.8 155 277.6 79 83.2 77 69.0 66 307.2 81 287.5 65 453.8 102 344.8 80 438.9 111 347.9 92 0.69 23 0.53 23 0.67 23 0.51 23 0.29 20 0.10 20 0.29 20 0.44 20 0.43 22 0.36 22 0.36 22 0.33 22 0.41 17 0.23 17 -0.08 22 0.06 22 0.67 22 0.14 22 0.68 22 0.20 22 0.48 20 0.21 20 0.56 22 0.61 22 0.76 17 0.73 17 0.72 17 0.74 17 0.53 16 0.56 16 0.31 17 0.29 17 upsala j med sci 79 100 l . backlund et al. table iv. some resrrlts f r o m s t e p w i s e regression, gi\>ing the mirltiple correlation coefficient ( r ) a s (in index of the explanatory value of the regression ~ dependregression coefficients ent vari able const. bw tbv w,30 w,,,, bsa r a. prostatectomized, ecg-normal, n =20 vol, 127.3 11.0 voij -263.0 11.4 62.8 v0lj -413.9 11.4 69.3 0.2 volj -322.6 12.3 62.3 0 . 4 -0.5 voij -958.6 44.5 -0.5 vol, 133.5 11.0 -0.01 b. prostatectomized, ecg-abnormal, n =22 vol, 301.7 8.7 vol, 1.9 7.7 0.7 voij 448.7 9.8 -43.9 voij 147.0 8 . 9 -52.1 0.7 voij 899.7 10.4 -134.9 1 . 1 -1.2 vol, 412.4 -88.0 -0.8 c . health survey, men, n=22 volj -129.8 13.5 volj -389.9 1 1 . 1 80.0 voij -399.1 11.5 87.6 -0.1 volj -148.4 12.8 0.1 voij -827.9 0.1 vol j 52.5 9 . 4 vol, 60.2 8.8 0.1 voij -313.1 0.1 voij -816.9 60.8 d. health survey, women, n = i6 volj -76.0 9 . 0 34.7 vol j 82.5 9.1 36.0 -0.02 voij -420.7 25.4 0.63 0.71 0.63 0.72 0.76 1003.7 0.73 0.43 0.47 0.51 0.56 0.67 737.0 0.46 0.67 0.77 0.68 0.77 716.3 0.73 866.9 0.69 0.78 0.81 0.79 0.81 566.8 0.76 546.9 0.75 means of a computer. the statistical analysis was also performed by a computer; in this analysis the regression line, residual variance and coefficient of correlation were calculated for each individual variable against each of the other variables. stepwise linear regression analysis (4) was performed in order to determine which variable and which combination of variables had the greatest value for predicting the heart volume. only persons for whom complete data had been obtained were included in these analyses. results the mean values and ranges for heart volume, w,,, and t h b in the different groups are pre sented in table 11. it can be seen in the table that voi, was higher than vol, in all groups (p,<0.05, p,<o.ol, p,<o.ool, p,<o.ool). it is also evident from the table that the heart volume was somewhat higher in the two groups of pro statectomized patients than in the healthy men from the health survey. this difference was sta tistically significant for vol,(p<o.ool). there was no significant differences in heart volume, how ever, between ecg-normal and ecg-abnormal prostatectomized patients. table i11 a-d presents certain regression equa tions, giving residual variance and coefficient of correlation as measures of the predictive value of the regression. it is evident from the table that the residual variance was generally somewhat lower and the coefficient of correlation somewhat higher when body weight (bw) was the independ ent variable than when this variable was body surface area (bsa). the explanatory value for bw and bsa was, in general, higher than for w,,, and tbv. the coefficient of correlation was usually higher in regressions with vol, than with vol, as the dependent variable. table iv a-d gives certain results from the stepwise regression analysis with the multiple correlation coefficient ( r ) as a measure of the prediction value of the regression. the starting point of the calculations was the finding that in the simple regression analysis volj and bw had shown a higher degree of correlation to different variables than volj and b s a , respectively. the table shows that all regressions had a considerably lower prediction value in the ecg-abnormal group of prostatectomized patients than in the other groups. it can also be seen in the table that bw alone has a relatively high explanatory value and that bw in combination with blood volume seems t o have a higher explanatory value than b s a in combination with blood volume. wis, as a further variable did not increase the predictive value of the regression noteworthily except in the group of ecg-abnormal prostatectomized patients. discussion groups c and d were taken from a statistically selected sample of the population in the age section concerned. the persons included comprise a relatively stringently selected group among those who fulfilled the criterion of having no manifest disease of importance for the ventilatory capacity, central circulatory capacity or blood formation. these groups (c and d) can thus be said t o re zlpsala j med sci 79 present healthy people in this age section. group a (prostatectomized patients with no manifest cardio pulmonary disease) reasonably can be considered to be representative of healthy men of the age range in question. groups a , c and d exhibited essentially the same pattern as regards the correla tion between heart volume and body dimensions and between circulatory functional capacity and blood volume. i n group b (ecg-abnormal pro statectomized patients) these correlations were weaker despite the absence of a history of or other clinical signs of heart disease. the im portance of ecg changes for correlations with the heart volume has been demonstrated previ ously by strandell (16). reindell et al. (14), among others, have found the heart volume to be more strongly correlated to body weight than to body surface area. this observation is also corroborated by the present investigation. thus there appear to be convincing arguments favouring the view that the total heart volume shall be related to body weight rather than to body surface area. situations with rapid changes in weight or other abnormal weight con ditions should probably comprise exceptions. in these extreme cases, however, it is doubtful whether any measure of body size at all can be of value for predicting the heart volume. a further improvement in prediction of the heart volume and establishment of normal values is obtained if the blood volume is added to the body weight. this is in agreement with previous results (16). the findings in the present study indicate, on the other hand, that the predictive value of physical work capacity is considerably smaller than has been reported earlier for younger age groups (8, 9, 16). in our material the correlation between physical work capacity, as a measure of the central circulatory capacity, and the heart volume, as an expression of the volume of the circulatory system, was relatively weak. thb and tbv can also be said to be measures of the volume of the circulatory system. in a larger sample of men and women of corresponding ages from the same health survey material, the corre lations between physical work capacity and thb and tbv, respectively, were studied and found to be weaker than those reported earlier for younger persons (5). both of these observations may in dicate that in a population such as this the physical work capacity is limited by other factors heart volume prediction i0 1 than the volume of the circulatory system and the oxygen transport capacity. in a comparison between the calculation proce dures of jonsell and kjellberg et al., the correlations between heart volume and other variables were found to be stronger throughout with the jonsell formula despite the fact that the variance was, as a rule, lower in calculations according to kjell berg et al. the implication of this observations cannot be stated with certainty, and further analy sis would seem to be motivated. a possible ex planation can be that determinations according to kjellberg for special body types and/or in the case of obesity have a poorer volume discrimina tory capacity than jonsell’s method. this might possibly explain the observation that the residual variances are lower for kjellberg’s method while the correlation coefficients are higher for measure ments according to jonsell. references i . 2 . 3. 4. 5 . 6 . 7. 8. 9. 10. bergstrom, k . , backlund, l., erikson, u . & gustafs son, b.: heart volume and its relation to measures of circulatory function in healthy young men. acta med scand 185: 471, 1969. bergstrom, k . , erikson, u. & gustafsson, b.: roentgenological determination of the heart volume. acta soc med upsal74: 81, 1969. bergstrom, k . & erikson, u.: rontgenologisk h j p t volymsbestamning. en enkat-undersokning. lakar tidningen 68; 2599, 1971, efroymson, m. a.: multiple regression analysis. in mathematical methods for digital computers, part v (17) (ed. a. ralston & s . wilf). john wiley and sons, 1960. ericsson, p.: the effect of iron supplementation on the physical work capacity in the elderly. acta med scand 188: 36 i , 1970. ericsson, p.: total haemoglobin and physical work capacity in elderly people. acta med scand 188: 15, 1970. ericsson, p. & irnell, l.: physical work capacity and static lung volumes in elderly people. acta med scand 185: 185, 1969. holmgren, a., jonson, b., levander, m., linder holm, h., sjostrand, t. & strom, g.: low physical working capacity in suspected heart cases due to inadequate adjustment of peripheral blood flow (vasoregulatory asthenia). acta med scand 158: 413, 1957. holmgren, a. & strandell, t.: the relationship between heart volume, total hemoglobin and physi cal working capacity in former athletes. acta med scand 163: 149, 1959. jonsell, s . : a method for the determination of the heart size by teleroentgenography ( a heart volume index). acta radio1 (stockh) 20: 325, 1939. upsala j med sci 79 102 l . backlund et al. 11. kjellberg, s. r., rudhe, u . & sjostrand, t.: the relations of the cardiac volume to the weight and surface area of the body, the blood volume and the physical capacity for work. acta radiol (stockh) 31: 113, 1949. 12. kjellberg, s . r., lonroth, h. & rudhe, u.: the effect of various factors on the roentgenological determination of the cardiac volume. acta radiol (stockh)35: 413, 1951. 13. larsson, h. & kjellberg, s . r.: roentgenological heart volume determination with special regard to pulse rate and the position of the body. acta radiol (stockh) 29: 159, 1948. 14. reindell, h., konig, k. & roskamm, h.: funktions diagnostik des gesunden und kranken herzens. george thieme verlag, stuttgart, 1967. 15. renck, h.: the elderly patient after anaesthesia and surgery. with special regard to certain respira tory, circulatory, metabolic and muscular functions. acta anaesthesiol scand, suppl. x x x i v . 1969. 16. strandell, t.: heart volume and its relation to some anthropometric data in old men compared with young men. acta med scand 176: 205, 1964. received november 15. 1973 address for reprints: lars backlund, m . d. dept. of clinical physiology university hospital s-750 14 uppsala sweden upsala j med sci 79 upsala j med sci 79: 1-6, 1974 ultrastructural changes of erythrocyte membranes isolated with colloidal silica solution (ludox) inger lindqvist, ove nilsson and gunnar ronquist from the institute of medical chemistry and the institute of human anatomy, uppsala, sweden abstract the surface morphology of membrane fractions of hu man erythrocytes isolated by gradient centrifugation with a colloidal silica solution (ludox) differed from the structure of control membranes isolated without the silica solution. the altered surface had a wrinkled ap pearance with a granular texture, compared with the smooth appearance of the controls. thus the colloidal silica solution changed the ultrastructure of erythrocyte membranes. possible mechanisms for the influence of the silica particles are suggested: secondary chemical bonds, e.g. hydrogen bonds, might well have been established between the silica particles and components of the eryth rocyte membrane like spectrin of the inner surface of the membrane. less probable is an attachment to posi tive sites of the outer side of the membranes as well as any association to the pitlike infoldings of the eryth rocyte surface. introduction gradient centrifugation in a silica solution of an iso lated membrane fraction of human erythrocytes resulted in a well defined single band comprising about 90% of the material (3). this membrane material contained some enzyme activities, among which that of glyceral dehyde-3-phosphate dehydrogenase could be detected, provided sulfhydryls were added to the medium. transmission electron microscopy demonstrated a par ticulate appearance of the membranes when silica was present. it was suggested that the particles observed were silica particles which had been attached to the mem braneous structure and, furthermore, that such an at tachment might have contributed to the inactivation of the enzyme observed when sulfhydryls were absent dur ing the preparation. an incorporation of silica particles into membranes might well influence their structural properties and the present investigation was undertaken to find out i f the surface configuration of the membrane is influenced by silica particles, as judged by scanning electron micro scopy. materials and methods special chemicals glutathione (reduced form) was purchased from sigma chemical company, st. louis, mo., usa, and dextran t40 from pharmacia fine chemicals, uppsala, sweden. the aqueous silica sols used were ludox hs, obtained from du pont de nemours, wilmington, delaware, usa, and nyacol, colloidal silica grade 2040 from nyanza inc., ashland, mass., usa. all chemicals were of ana lytical grade. preparation o f membrane fraction membrane fraction was prepared as described by ron quist (6). colloidal silica solutions 16% (wlv), with a ph of 7.4 and containing 65 mm naci, 12.5 mm kci, 1.5% (wlv), dextran t 40 and 1 mm gluthatione were prepared. the osmolarity of the solution was 290-300 mosm. batches of silica particles of defined diameters were obtained after density gradient centrifugation according to pertoft i% laurent (4). the following sizes were isolated: 100 a, 250 8, and 400-500 8,. the largest particles were sampled as a gel pellet from the bottom of tubes which had been emptied of the rest of the solution. the gel pellet was dissolved in the salt solu tion, and ph and osmolarity were adjusted. the silica preparation was carried out both with par ticles of the defined sizes and with uncentrifuged solu tions with a size range from 80 to 250 a . one volume of the membrane fraction was added to 4 vol of isotonic silica solution that contained dextran and sulfhydryls and was incubated for 25 min a t 20°c. the material was then washed three times, each time in 10 times its volume of an isotonic salt solution con taining 130 mm nac1, 25 mm kci, 1.5% dextran t40 and 1 mm glutathione before preparation for scanning electron microscopy. preparations for scanning electron microscopy were performed in different ways to test their influence on the ultrastructure. samples prepared both in the presence and absence of the colloidal silica solution were used. the following procedures were applied. 1. fixation and dehydration in a graded series of etha nol, air-drying. 2. as 1 , but after dehydration: ether, air-drying. 3. fixation in a solution of 2 . 5 % glutaraldehyde in sorensen’s phosphate buffer (ph 7.2), rinsing in distilled water, air-drying. 4. as 3, but after rinsing in distilled water: dehydra tion in a graded series of ethanol, air-drying. 1-742854 upsala j m e d sci 79 2 inger lindqvist et al. fig. 1 . membrane fraction, prepared without the colloidal solution of silica. x 2000. fig. 2. membrane fraction, prepared without the colloidal solution of silica. x 20000. upsula j med sci 79 erythrocyte membranes isolated with ludox 3 f i g . 3. membrane fraction, prepared by gradient centrifugation in a colloidal solution of silica. ~ 2 0 0 0 . , f i g . 4. membrane fraction, prepared by gradient centrifugation in a colloidal solution of silica. ~ 2 0 0 0 0 . upsala j med sci 79 4 inger lindqvist et al. f i g . 5 . membrane fraction, prepared by gradient centrihgation in a colloidal solution of silica particles with a size of about 250 p\. x 1ooooo. 5 . fixation in a solution of 1 % osmium tetroxide in sorensen’s phosphate buffer (ph 7 . 2 ) , rinsing in distilled water, air-drying. 6. a s 5 , but after rinsing in distilled water: dehydra tion in a graded series of ethanol, air-drying. 7. a s 6, but after dehydration: ether, air-drying. the samples were allowed to dry on small squares of clean glass, which then were fixed to the specimen stubs. the coating was performed with gold-palladium, and the specimens were examined in a jeol emu-3 scanning electron microscope. results membrane fractions prepared without the colloidal silica solution demonstrated a smooth surface (fig. i ) which in high magnification showed only minor irregularities (fig. 2 ) . even where stacks of folded membranes were observed, the smooth character of the membranes remained. membrane fractions prepared with the colloidal silica solution were irregularly wrinkled (fig. 3) and a granularity of the surface was evident at high magnification (fig. 4). application of two batches of silica particles, with diameters of 250 a and 500 a , respectively, 1 did not cause any difference in the appearance of the membrane of the two groups. at high magnification the 500 a particles were visible (fig. 6) but the 250 a ones were too small to be distin guished clearly (fig. 5). upsala j med sci 79 the various preparative methods did not influ ence the characteristic appearance of the mem branes. discussion the silica particles have a moderate negative charge under the present experimental conditions and can therefore react with positive groups of the membranes. an ionic bond is, however, not nec essarily the only possible one between the silica particles and the membrane since the silica par ticles can easily establish hydrogen bonds (h. per toft, 1973, personal communication). the inside of the erythrocyte membrane being favoured for the binding of silica particles is sug gested by the findings of steck et al. (7). they prepared a membrane fraction from human eryth rocytes in an alkaline buffer (ph 8.2) of low ionic strength and reported that the membranes appeared to bud spontaneously into the ghost interior which led to an accumulation of many small vesicles within each parent ghost. moreover, a high percent age of these vesicles were morphologically inside out when divalent cations were absent during the preparation. because of the very close similarity between this preparative procedure and the one we used, most of the membranes we observed in the scanning electron microscope ought to be inside o u t erythrocyte membrune isolated with ludox 5 fig. 6. membrane fraction, prepared by gradient centrifugation in a colloidal solution of silica particles with a size of about 500 a . x 100000. too and therefore having an exposed inside for the silica particles. one of the major proteins of the inside surface of the erythrocyte membrane has recently been characterised and named spectrin ( 2 ) . these authors also suggested that about 85% of the total sh-groups of the membrane is localized i n the spectrin molecules. because of the protective function exerted by sh-reagents like glutathione and dithiotreitol (cf. 3) in the membrane vis-ci-vis silica particles, it is reasonable to assume that the sh-groups of spectrin might have interacted direct ly or indirectly with the particles giving rise to the morphological change observed. the outer surface of the membrane on the other hand could have attracted the silica particles, since positive groups are probably present on all mam malian cells in spite of their net negative charge. although relatively little is known about the loca tion and distribution of positive groups on the cell surface, it would be theoretically possible for the silica particles to adhere to the cell surface between fixed anionic groups. if such were the case, one would expect that for steric reasons particles of a certain diameter would be favoured. but the quali tative result was the same whatever the particle size within the diameter range 80-500 a. another possible site for binding the silica par ticles is the small pitlike infoldings of the eryth rocyte surface (1). the pits have an outer diameter of about 300 a-compared with 80 8, to 500 p\ of the silica particles-and might well differ in charge properties from their surrondings. they seem to be sufficiently frequent to account for a change in the appearance of the erythrocyte surface-if the silica particles are bound in the pits. here too one would expect a favouring of a certain size of the silica particles fitting the size of the pits. the results were the same, however, with particles within the diameter range 80-500 a. these two possibilities for binding the silica par ticles seem therefore to be less probable. instead, hydrogen bonds could have been established bet ween the silica particles and components on the inner side of the membrane. these bonds might well have produced some modification of the mem brane structure giving rise to the observed change of the morphological appearance. acknowledgement this work was supported by the swedish medical re search council (projects no. 13x-228, and 12k-70). we are indebted to mr. bengt wieselblad, research engineer, for skilful technical assistance with the elec tron microscope. references 1 . harris, j. r . & agutter, p . : a negative staining study of human erythrocyte ghosts and rat liver nuclear membranes. j ultrastruct res 33:219. 1970. upsalo j med sci 79 6 znger lindqvist et al. 2. nicholson, g. l . , marchesi, v. t. & singer, s. j.: the localization of spectrin on the inner surface of human red blood cell membranes by ferritin-conjugated anti bodies. j cell bio151:265, 1971. 3. nilsson, 0. & ronquist, g.: enzyme activities and ultrastructure of a membrane fraction from human erythrocytes. biochim biophys acta 183: 1 , 1969. 4 . pertoft, h. & laurent, t. c.: modem separation meth ods of macromolecules and particles. in progress in separation and purification series (ed. t. gerrit sen), wiley, new york, 1969. 5 . pertoft, h.: personal communication, 1973. 6. ronquist, g.: formation of adenosine triphosphate by a membrane fraction from human erythrocytes. acta chem scand21: 1484, 1967. 7. steck, th. l., weinstein, r. s . , straus, j . h. & wal lach d. f. h.: inside-out red cell membrane vesicles: preparation and purification. science 168: 255, 1970. received september 15, 1973 address for reprints: inger lindqvist, b.sc. institute of medical chemistry box 575 s-75 1 23 uppsala sweden upsala j med sci 79 t new aspects on training bradycardia eva nylander and nils-holger areskog from the department of clinical physiology, university of linkoping, region hospital, linkoping, sweden dedicated to professor torsten teorell abstract r a t s were t r a i n e d by t r e a d m i l l running a f t e r chemical sympathectomy with 6-hydroxy-dopamine or d u r i n g c h r o n i c b e t a r e c e p t o r blockade. contrary t o un t r e a t e d t r a i n e d a n i m a l s , sympathectomizcd r a t s d i d n o t g e t a r e d u c t i o n of t h e i n t r i n s i c h e a r t r a t e a f t e r t r a i n i n g d e s p i t e an i n c r e a s e d h e a r t weight. i n c o n t r a s t , no c a r d i a c hypertrophy o c c u r r e d a f t e r t r a i n i n g d u r i n g b e t a a d r e n e r g i c blockade b u t t h e h e a r t r a t e d u r i n g e x e r c i s e was reduced i n t h e s e animals. i t ' i s concluded t h a t t h e t r a i n i n g i n d u c e d b r a d y c a r d i a c o n t a i n s a lowering o f t h e i n t r i n s i c h e a r t r a t e and t h a t t h i s i s n o t dependent on t h e s t i m u l a t i o n of c a r d i a c b e t a r e c e p t o r s or t h e magnitude of h e a r t r a t e i n c r e a s e d u r i n g e x e r c i s e . the r e s u l t s a l s o i n d i c a t e t h a t t h e r e i s not a c a u s a l r e l a t i o n s h i p between t h e t r a i n i n g i n d u c e d b r a d y c a r d i a and c a r d i a c h y p e r t r o p h y . the l a t t e r c o n c l u s i o n i s s u p p o r t e d by an e c h o c a r d i o g r a p h i c study i n humans where no c o r r e l a t i o n was found between i h r and c a r d i a c dimensions. introduction a low h e a r t r a t e ( h r ) a t r e s t and d u r i n g e x e r c i s e a t submaximal work l o a d s i s an e f f e c t of endurance t r a i n i n g t h a t h a s been r e c o g n i z e d s i n c e l o n g ago and s i n c e t h e beginning of t h i s c e n t u r y a t t r i b u t e d t o an a l t e r e d autonomic nervous a c t i v i t y , mainly an i n c r e a s e d v a g a l t o n e ( 1 7 ) . recent i n v e s t i g a t i o n s have shown t h a t a lowered r a t e of t h e c a r d i a c pacemaker ( i n t r i n s i c h e a r t r a t e , i h r ) i s p a r t of t h e e x p l a n a t i o n t o t h i s b r a d y c a r d i a seen i n p h y s i c a l l y t r a i n e d i n d i v i d u a l s (18,4,9). s t i l l , however, t h e mechanisms for t h e lowering o f ihr through t r a i n i n g a r e known, and t h e i n v e s t i g a t i o n s t o be r e p o r t e d were undertaken t o i d e n t i f y some f a c t o r s of importance f o r t h i s a d a p t a t i o n . we p r e f e r t o look upon p h y s i c a l t r a i n i n g as an a d a p t a t i o n t o an i n c r e a s e d p h y s i c a l a c t i v i t y r e s u l t i n g i n an i n c r e a s e d c a p a c i t y t o perform t h i s a c t i v i t y . hereby f o l l o w s t h a t t h e c i r c u l a t o r y and o t h e r a d j u s t m e n t s a t each e x e r c i s e o c c a s i o n , r e p e a t e d a t c e r t a i n 1-822855 1 i n t e r v a l s and f o r a s u f f i c i e n t l y l o n g d u r a t i o n induce t h e " t r a i n i n g e f f e c t s " . by manipulation of t h e c a r d i o v a s c u l a r a d a p t a t i o n a t each t r a i n i n g s e s s i o n ( e . g . t h e i n c r e a s e of oxygen consumption, s t r o k e volume or h r ) it would be p o s s i b l e t o draw c o n c l u s i o n s about t h e importance o f t h e s e d i f f e r e n t f a c t o r s f o r t h e development of t r a i n i n g e f f e c t s , i n t h i s c o n t e x t t h e t r a i n i n g brady c a r d i a and r e d u c t i o n of i h r . i n t h i s paper a r e summarized such l o n g i t u d i n a l t r a i n i n g s t u d i e s t h a t have been c a r r i e d o u t i n r a t s . i n a p r e v i o u s c r o s s s e c t i o n a l human s t u d y w e have found evidence f o r a t r a i n i n g i n d u c e d d e c r e a s e of ihr a l s o i n man ( 9 ) . a s i n many o t h e r s t u d i e s we a l s o found g r e a t e r r o e n t g e n o l o g i c a l h e a r t volumes i n t r a i n e d t h a n i n u n t r a i n e d p e r s o n s . we have now performed an e c h o c a r d i o g r a p h i c s t u d y of endurance a t h l e t e s t o i n v e s t i g a t e whether t h e r e i s any c a u s a l r e l a t i o n s h i p between i h r and c a r d i a c dimensions. material and methods r a t s were t r a i n e d by treadmill running. a method f o r r e c o r d i n g of ecg d u r i n g e x e r c i s e and an e x e r c i s e t e s t f o r r a t s w i t h s t e p w i s e i n c r e a s e d t r e a d m i l l speed t o t e s t t h e e f f i c i e n c y of t h e t r a i n i n g program were developed (5). one group of r a t s w a s t r a i n e d a f t e r chemical sympathectomy w i t h 6-hydroxy dopamine (6-oh-da)(10). l i t t e r m a t e u n t r e a t e d r a t s were t r a i n e d s i m u l t a n e o u s l y . both t r e a t e d and u n t r e a t e d r a t s , l i t t e r m a t e s t o t h e t r a i n i n g o n e s , were k e p t as s e d e n t a r y c o n t r o l s ( 1 6 ) . i n a s i m i l a r s t u d y one group of t r a i n i n g r a t s and one s e d e n t a r y group were i n s t e a d t r e a t e d w i t h m e t o p r o l o l , a c a r d i o s e l e c t i v e b e t a r e c e p t o r a n t a g o n i s t ( 1 ) o r a l l y d u r i n g t h e t r a i n i n g p e r i o d ( 1 1 ) . a f t e r a t r a i n i n g p e r i o d a l l r a t s were e x e r c i s e t e s t e d . i n o r d e r t o measure t h e i h r a d e n e r v a t i o n w a s performed i n c l u d i n g 6-oh-da t r e a t m e n t , c u t t i n g of t h e vagus n e r v e s and s p i n a l c o r d d e s t r u c t i o n ( p i t h i n g ) ( 1 5 ) . a t t h e end of t h e experiment t h e h e a r t s were weighed. i n a s e p a r a t e s t u d y h e a r t s from t r a i n e d and s e d e n t a r y r a t s were i n v e s t i g a t e d a l s o i n a modified langendorff p r e p a r a t i o n , t o e n s u r e t h a t t h e h e a r t r a t e s measured i n p i t h e d p r e p a r a t i o n s r e p r e s e n t t h e t r u e i n t r i n s i c hr ( 1 2 ) . i n t h e human study seven c o m p e t i t i v e c y c l i s t s of n a t i o n a l e l i t e c l a s s were i n v e s t i g a t e d . they had been t r a i n i n g r e g u l a r l y f o r 7-11 y e a r s and t r a i n e d 600-1000 km/week a t t h e t i m e of t h e s t u d y . p h y s i c a l c h a r a c t e r i s t i c s a r e given i n table 1. they performed a maximal e x e r c i s e t e s t with d e t e r m i n a t i o n of maxi 2 ma1 oxygen consumption with t h e douglas bag method and with r e c o r d i n g of h e a r t r a t e and b l o o d p r e s s u r e d u r i n g e x e r c i s e . on a s e p a r a t e day t h e t e s t w a s re p e a t e d a f t e r t h e a d m i n i s t r a t i o n of a t r o p i n e s u l p h a t e 0.04 mg/kg body weight and p r o p r a n o l o l h y d r o c h l o r i d e 0.25 mg/kg body weight as d e s c r i b e d p r e v i o u s l y (9). table 1 . p h y s i c a l c h a r a c t e r i s t i c s and maximal oxygen u p t a k e (vo2 max) i n seven c o m p e t i t i v e c y c l i s t s . go max 2 $0 max 2 age height weight y cm kg l / m i n ml/kg/min mean 24 179 68 4 . 9 72 + + + -+ -1 sd -3 -7 -5 t 0 . 6 +5 the e c h o c a r d i o g r a p h i c examination was performed by one i n v e s t i g a t o r ( e . n . ) b e f o r e t h e e x e r c i s e t e s t . a 3 mhz atl (advanced technology l a b o r a t o r i e s ) mechanical s e c t o r scanner was u s e d f o r both 2-dimensional imaging and m-mode. a l l r e c o r d i n g s were performed w i t h t h e s u b j e c t i n t h e l e f t l a t e r a l recumbent p o s i t i o n , e x c e p t f o r r i g h t v e n t r i c u l a r dimension which w a s s t u d i e d w i t h t h e s u b j e c t s u p i n e . the measurements on t h e m-mode r e c o r d i n g s were performed f o l l o w i n g t h e recommendations of t h e american s o c i e t y of echocardiography ( 1 4 ) . echocardiographic s t r o k e volume was e s t i m a t e d w i t h t h e cube method. l e f t v e n t r i c u l a r mass was c a l c u l a t e d a c c o r d i n g t o bennett & evans ( 2 ) . s t a n d a r d s t a t i s t i c a l methods were used. results e x e r c i s e h e a r t r a t e and i n t r i n s i c h e a r t r a t e i n r a t s . a t t h e e x e r c i s e t e s t a f t e r t h e t r a i n i n g p e r i o d t r a i n e d u n t r e a t e d animals had s i g n i f i c a n t l y lower hr t h a n t h e s e d e n t a r y ones. also a f t e r d e n e r v a t i o n and i n i s o l a t e d h e a r t s t h e r e remained a s i g n i f i c a n t hr d i f f e r e n c e between t r a i n e d and s e d e n t a r y r a t s ( f i g 1 ) . there was an e x e r c i s e hr d i f f e r e n c e a l s o between sympathectomized t r a i n e d and u n t r a i n e d r a t s . however, t h e t r e a t m e n t with 6-oh-da r e s u l t e d i n h i g h e r hr t h a n were found i n u n t r e a t e d animals so t h a t t h e hr of t r a i n e d t r e a t e d animals were s i m i l a r t o t h o s e of t h e u n t r e a t e d s e d e n t a r y ones ( f i g 2 ) . i n t h e s e a n i mals t h e r e w a s no s i g n i f i c a n t hr d i f f e r e n c e between t r a i n e d and u n t r a i n e d r a t s a f t e r c a r d i a c d e n e r v a t i o n o r i s o l a t i o n . 3 heart rate beatslmin 4oc 300 200 100. t ++ _ _ _ untraine trained i i i i i i i i i i i i i i i i i i i i i i i a ++ i i i i i 1 i 10 15 isoi!td 35 4 0 45 time after pithing,rnin t pithing 5 heart f i g 1 . heart r a t e s i n u n t r e a t e d t r a i n e d and u n t r a i n e d r a t s a f t e r denervftion (6-oh-da, vagotomy and p i t h i n g ) and i n t h e i s o l a t e d h e a r t , mean v a l u e s -sem. * p<0.05, ** ~ ( 0 . 0 1 . from ( 1 2 ) . the metoprolol t r e a t e d r a t s performed t h e e x e r c i s e t e s t t h r e e days a f t e r withdrawal of m e t o p r o l o l , when plasma c o n c e n t r a t i o n s of t h e drug were shown t o be n e g l i g i b l e . u n t r a i n e d r a t s had similar h e a r t r a t e s t o t h e u n t r e a t e d s e d e n t a r y ones. the t r a i n e d metoprolol t r e a t e d r a t s had s i g n i f i c a n t l y lower hr t h a n b o t h t h e s e d e n t a r y animals and t h e t r a i n e d u n t r e a t e d ones ( f i g 3 ) . i n t h i s s t u d y t h e r e was a s l i g h t b u t n o t s i g n i f i c a n t i h r d i f f e r e n c e between t r a i n e d and u n t r a i n e d r a t s , both i n t h e u n t r e a t e d and t h e m e t o p r o l o l t r e a t e d group. heart weights of r a t s a f t e r t r a i n i n g i n u n t r e a t e d a n i m a l s , t h e a v e r a g e h e a r t weight was s i g n i f i c a n t l y g r e a t e r i n t h e t r a i n e d t h a n i n t h e u n t r a i n e d group (1.079 g 0.020 (s.e.) vs. 0.935 o . o l l , p<o.ool). also t h e h e a r t weight / body weight r a t i o (hw/bw) w a s g r e a t e r i n t h e t r a i n e d a n i m a l s , 3.811 0100 t o . 0 6 2 vs. 3.293 0100 t 0 . 0 6 9 , p<o.ool). 4 heart rate b a t s p i n 6oc 550 i 500 450 l *** 0 normal 0 sympathectornired untrained trained ---- y 1 i 21 25.5 30 trsudrnilt speed rnlmin fig 2. heart r a t e s d u r i n g e x e r c i s e a t 3 d i f f e r e n t work l o a d s + i n t r a i n e d and u n t r a i n e d , u n t r e a t e d and sympathectomized r a t s , mean v a l u e s -sem. a s t e r i s k s denote s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s compared t o c o r r e s p o n d i n g un t r a i n e d group. * ~ ( 0 . 0 5 , ** p<o.ol, *** ~ ( 0 . 0 1 . from ( 1 2 ) . 6-oh-da t r e a t e d t r a i n e d r a t s a l s o had a s i g n i f i c a n t l y i n c r e a s e d hw/bw r a t i o i n one i n v e s t i g a t i o n ( 1 6 ) and both hw and hw/bw i n t h e o t h e r s t u d y ( 1 2 ) . r a t s t r a i n e d d u r i n g c h r o n i c b e t a b l o c k e r t r e a t m e n t d i d n o t e x h i b i t a c a r d i a c hypertrophy ( 1 1 ) . ihr and c a r d i a c dimensions i n endurance a t h l e t e s . heart r a t e s and e c h o c a r d i o g r a p h i c dimensions a r e l i s t e d i n table 2. the r e l a t i o n between i h r and l e f t v e n t r i c u l a r mass or l e f t v e n t r i c u l a r i n n e r dimension i s i l l u s t r a t e d i n f i g 4, showing t h a t t h e r e was n o s t r i k i n g c o r r e l a t i o n b e t ween t h e s e c a r d i a c dimensions and i h r . 5 heart f a t e b e a t d m i n 500 450 ** 400 t ** ? 3 ** -i trained untrained rn 0 untreated 0 0 metoprolol treated r +/ i i i rest before 15 exercise 20 25 treadmill speed m/min f i g 3. heart r a t e s a t r e s t and d u r i n g e x e r c i s e a t t h r e e d i f f e r e n t work l o a d s i n u n t r e a t e d rats an$ i n m e t o p r o l o l t r e a t e d r a t s a f t e r withdrawal of meto p r o l o l , mean v a l u e s -sem. * : s i g n i f i c a n t d i f f e r e n c e between t r a i n e d and corresponding u n t r a i n e d group . *: s i g n i f i c a n t d i f f e r e n c e between t r a i n e d metoprolol t r e a t e d and t r a i n e d u n t r e a k e d group. *,*: pco.05 *****: p(o.01 discussion the group of endurance a t h l e t e s i s homogenous as r e g a r d s p h y s i c a l c h a r a c t e r i s t i c s and work c a p a c i t y . thus it i s n o t r e p r e s e n t a t i v e of t h e g e n e r a l popula t i o n b u t w i t h i n t h i s group t h e r e w a s no c o r r e l a t i o n between i h r and lv mass o r lv i n n e r dimension. t h i s i s i n accordance with our animal experiments and f i n d i n g s by o t h e r s ( 2 1 ) of a poor c o r r e l a t i o n between h e a r t weight and h e a r t 6 table 2. heart r a t e s and e c h o c a r d i o g r a p h i c dimensions i n seven c o m p e t i t i v e c y c l i s t s . beatslmin beatslmin 1201 120. 100100, 80 * * * * 80, + * 60, 60 40, 40 20 20 hr r e s t ihr la r v i d ivs l v i d lvpw lv-mass sv b/min b/min mm mm mm mm mm g m l mean 52 8 1 37 1 8 1 2 58 1 2 380 130 -1 sd -5 + i 3 22 24 21 23 1 ?65 :15 + + + la = l e f t a t r i u m ; r v i d = r i g h t v e n t r i c u l a r i n n e r d i a m e t e r ; ivs = i n t e r v e n t r i c u l a r s e p t a 1 t h i c k n e s s , l v i d = l e f t v e n t r i c u l a r i n n e r d i a m e t e r ; lvpw = l e f t v e n t r i c u l a r p o s t e r i o r w a l l t h i c k n e s s , sv = e c h o c a r d i o g r a p h i c s t r o k e volume. r a t e . t h i s i n d i c a t e s t h a t ihr i s n o t determined by c a r d i a c dimensions only. the p r e s e n t animal experiments show t h a t an i n c r e a s e i n h e a r t weight may occur without a l o w e r i n g of ihr, as i n t h e sympathectomized t r a i n e d animals and t h a t a t r a i n i n g i n d u c e d b r a d y c a r d i a can develop without an i n c r e a s e of c a r d i a c mass, a s was seen i n t h e metoprolol group. all t h i s speaks a g a i n s t t h e b r a d y c a r d i a and r e d u c t i o n of i h r as d i r e c t e f f e c t s of an i n c r e a s e d lv dimension of myo c a r d i a l mass. ihr ihr lv mass g lvid mm f i g 4 . r e l a t i o n between ihr and l e f t v e n t r i c u l a r (lv) mass e s t i m a t e d by echocardiography ( l e f t ) and between ihr and l e f t v e n t r i c u l a r i n n e r dimension ( l v i d ) ( r i g h t ) i n seven c o m p e t i t i v e c y c l i s t s . 7 which c a r d i o v a s c u l a r a d j u s t m e n t s d u r i n g each e x e r c i s e s e s s i o n may t h e n a c t a s s t i m u l i f o r a r e d u c t i o n of e x e r c i s e and i n t r i n s i c hr? t h i s can n o t y e t be completely answered b u t t h e h y p o t h e s i s of t h e r e p e a t e d h e a r t r a t e i n c r e a s e above a c e r t a i n l e v e l can be r e j e c t e d s i n c e b e t a r e c e p t o r blockade d i d not p r e v e n t t h e development o f t r a i n i n g i n d u c e d b r a d y c a r d i a . likewise, 6-oh-da t r e a t e d animals t h a t c o n t r a r y t o t h e b e t a blocked ones have an i n c r e a s e d hr r e s p o n s e t o e x e r c i s e (16) d i d n o t g e t a r e d u c t i o n of e x e r c i s e hr o r i h r . chronic a t r o p i n e t r e a t m e n t , r e s u l t i n g i n r e p e a t e d p e r i o d s of t a c h y c a r d i a , i s a l s o r e p o r t e d not t o a l t e r t h e i n t r i n s i c r a t e of i s o l a t e d a t r i a i n r a t s ( 7 ) . a d i r e c t e f f e c t of catecholamines a t t h e c a r d i a c b e t a r e c e p t o r s i s a l s o un l i k e l y s i n c e t h e development of t r a i n i n g b r a d y c a r d i a was enhanced d e s p i t e a diminished b e t a s t i m u l a t i o n i n t r a i n e d r a t s r e c e i v i n g metoprolol. however, i n d i r e c t l y t h e a d r e n e r g i c n e r v e s a r e e s s e n t i a l f o r t h e r e d u c t i o n of i h r s i n c e i n t h e 6-oh-da t r e a t e d r a t s t r a i n i n g d i d n o t have t h i s e f f e c t . i n accordance with t h i s a r e t h e f i n d i n g s by p a y n t e r e t a 1 ( 1 3 ) t h a t a t r a i n i n g i n d u c e d r e s t i n g b r a d y c a r d i a d i d n o t occur i n immuno sympathectomized r a t s . it i s w e l l known from many animal s p e c i e s t h a t t h e hr i n c r e a s e s i n response t o a c u t e mechanical s t r e t c h of t h e a t r i a ( 8 ) . the h y p o t h e s i s h a s been s e t f o r t h by f r i c k e t a 1 ( 6 ) and blomqvist and lewis ( 3 ) t h a t t h i s hfi r e s p o n s e t o s t r e t c h i n g may b e reduced as an a d a p t a t i o n t o t h e r e p e a t e d a t r i a l d i l a t a t i o n r e s u l t i n g from t h e i n c r e a s e d venous r e t u r n d u r i n g e x e r c i s e and t h a t t h i s phenomenon e x p l a i n s t h e lowered s e t t i n g of i h r and a l s o hr a t r e s t and sub maximal e x e r c i s e a f t e r endurance t r a i n i n g . the r e s u l t s from t r a i n i n g a f t e r p r e t r e a t m e n t with 6-oh-da or t r a i n i n g d u r i n g b e t a r e c e p t o r blockade fit i n t o t h i s h y p o t h e s i s . i n 6-oh-da t r e a t e d animals t h e enhanced t a c h y c a r d i a d u r i n g e x e r c i s e s h o r t e n s d i a s t o l e , t h e r e b y p r e v e n t i n g an i n c r e a s e d d i a s t o l i c f i l l i n g and t h e r e d u c t i o n of i h r . the nega t i v e c h r o n o t r o p i c e f f e c t of t h e beta-blockade on t h e o t h e r hand would augment t h e a t r i a l d i l a t a t i o n because of i n c r e a s e d s t r o k e volumes d u r i n g e x e r c i s e . thanks t o t h e c o n t r i b u t i o n s of t e o r e l l ( 1 9 , 2 0 ) it i s known t h a t t h e r e e x i s t s i n t h e h e a r t n o t o n l y an e l e c t r o m e c h a n i c a l b u t a l s o a mechanoelectri c a l c o u p l i n g of e v e n t s . the r e p o r t e d r e s u l t s s u p p o r t t h e t h e o r y t h a t t h i s r e l a t i o n s h i p can undergo a d a p t a t i o n and imply t h a t a diminished hr response t o a t r i a l s t r e t c h c o u l d be t h e mechanism f o r t h e lower i h r s e t t i n g i n t r a i n e d i n d i v i d u a l s . 8 references ablad, b., borg, k . o . , c a r l s s o n , e . , ek, l . , j o h n s s o n , c . , malmfors, t. & regbrdh, c-g. a s u r v e y of t h e p h a r m a c o l o g i c a l p r o p e r t i e s o f m e t o p r o l o l i n a n i m a l s a n d man. a c t a pharmacol t o x i c o l 3 6 , s u p p l . 5:7-23, 1975. b e n n e t t , d.h. & evans, d.w. c o r r e l a t i o n of l e f t v e n t r i c u l a r mass d e t e r mined by e c h o c a r d i o g r a p h y w i t h v e c t o r c a r d i o g r a p h i c a n d e l e c t r o c a r d i o g r a p h i c v o l t a g e measurements. br h e a r t j 36:981-987, 1974. b l o m q v i s t , c . g . & lewis, s.f. p h y s i o l o g i c a l e f f e c t s o f t r a i n i n g : g e n e r a l c i r c u l a t o r y a d j u s t m e n t s . i n : p h y s i c a l c o n d i t i o n i n g a n d c a r d i o v a s c u l a r re h a b i l i t a t i o n . ed: cohen, l.s., mock, m.b. & r i n g q v i s t , i. j. wiley & sons i n c . , n e w york, 57-76, 1981. b o l t e r , c.p., hughson, r . l . & c r i t z , j . b . i n t r i n s i c r a t e and c h o l i n e r g i c s e n s i t i v i t y o f i s o l a t e d a t r i a from t r a i n e d and s e d e n t a r y r a t s . p r o c soc exp b i o l med 144:364-367, 1973. ekblom, b . , kilbom, 8 . , malmfors, t . , s i g v a r d s s o n , k . & s v a n f e l d t , e. sympathectomy and p h a r m a c o l o g i c a l b l o c k a d e i n t r a i n e d r a t s . acta p h y s i o l scand 89:283-285, 1973. f r i c k , m . h . , e l o v a i n t o , r.o. & somer, t . the mechanism o f b r a d y c a r d i a evokedby p h y s i c a l t r a i n i n g . c a r d i o l o g i a 5 1 : 46-54, 1967. hughson, r . l . , s u t t o n , j . r . , f i t z g e r a l d , j . d . & j o n e s , n.l. r e d u c t i o n o f i n t r i n s i c s i n o a t r i a l f r e q u e n c y a n d n o r e p i n e p h r i n e r e s p o n s e o f t h e e x e r c i s e d r a t . can j p h y s i o l pharmacol 55:813-820, 1977. j e n s e n , d. i n t r i n s i c c a r d i a c r a t e r e g u l a t i o n . appleton-century c r o f t s , n e w york. 151-162, 1971. l e w i s , s.f., n y l a n d e r , e . , gad, p. & areskog, n.-h.:non-autonomic compo n e n t i n b r a d y c a r d i a of e n d u r a n c e t r a i n e d men a t r e s t and d u r i n g e x e r c i s e . acta p h y s i o l scand 109:297-305, 1980. malmfors, t. & thoenen, h. ( e d ) . 6-hydroxydopamine a n d c a t e c h o l a m i n e n e u r o n s . north h o l l a n d p u b l i s h i n g company 15-74 , 1971. n y l a n d e r , e.! e f f e c t of b e t a a d r e n e r g i c r e c e p t o r b l o c k a d e on development of t r a i n i n g i n d u c e d b r a d y c a r d i a i n r a t s . acta p h y s i o l scand 112:449-454, n y l a n d e r , e . , s i g v a r d s s o n , k . & kilbom, 8 . t r a i n i n g i n d u c e d b r a d y c a r d i a and i n t r i n s i c h e a r t r a t e i n r a t s . eur j apql p h y s i o l , 1981. i n p r e s s . p a y n t e r , d . e . , t i p t o n , c.m. & tcheng, t.-k. response o f immunosympathec t o m i z e d r a t s t o t r a i n i n g . j appl p h y s i o l 42:935-940, 1977. sahn, d . j . , demaria, a . , k i s s l o , j . & weyman, a . the committee on m-mode s t a n d a r d i z a t i o n of t h e american s o c i e t y of e c h o c a r d i o g r a p h y . recommenda t i o n s r e g a r d i n g q u a n t i t a t i o n i n m-mode e c h o c a r d i o g r a p h y : r e s u l t s o f a s u r v e y o f e c h o c a r d i o g r a p h i c measurements. c i r c u l a t i o n 58:1072-2083, 1978. s h i p l e y , r . e . & t i l d e n , j . h . a p i t h e d r a t x e p a r a t i o n s u i t a b l e f o r a s s a y i n g p r e s s o r s u b s t a n c e s . p r o c soc exp u i o l med 64:453-455, 1947. s i g v a r d s s o n , k . , s v a n f e l d t , e . & kilbom, 8 . r o l e o f t h e a d r e n e r g i c n e r v o u s s y s t e m i n development o f t r a i n i n g i n d u c e d b r a d y c a r d i a . acta p h y s i o l scand 101:481-488, 1977. s t e i n h a u s , a.h. chronic e f f e c t s of e x e r c i s e . p h y s i o l rev 12:103-147, 1933. s u t t o n , j . r . , c o l e , a . , gunning, j . , h i c k i e , j . b . & s e l d o n , w.a. c o n t r o l o f h e a r t r r t e i n h e a l t h y young men. l a n c e t 2:1398-1400, 1967. t e o r e l l , t . e l e c t r o k i n e t i c c o n s i d e r a t i o n s of m e c h a n o e l e c t r i c a l t r a n s d u c t i o n . new york acad s c i 137:950-966, 1966. t e o r e l l , t . a f i x e d c h a r g e s y s t e m as a f o r m a l model f o r t h e b e h a v i o u r of smooth m u s c l e s a n d t h e h e a r t . u p s a l a j med s c i t i p t o n , c . m . , m a t t h e s , r . d . , tcheng, t.-k., dowell, r.t. & vailas, a . c . the u s e o f t h e langendorff p r e p a r a t i o n t o s t u d y t h e b r a d y c a r d i a o f t r a i n i n g . med s c i s p o r t s 9:220-230, 1977. 1981. 85:201-209, 1980. 7 . 2. 3. 4. 5. 6 . 7. 8. 9. 10. 1 1 . 12. 13. 14. 75. 16. 17. 18. 19. 20. 21. r e c e i v e d j a n u a r y 2 0 , 1 9 8 2 9 address f o r r e p r i n t s : d r eva nylander department of c l i n i c a l p h y s i o l o g y u n i v e r s i t y h o s p i t a l s-581 85 linkaping sweden 10 upsala j med sci 78: 160-165, 1973 a new factor in the etiology of chronic nonspecific tendovaginitis in the wrist sten oldberg from the department of medicine, county hospital, koping, sweden abstract the most frequent sites of tendovaginitis of the dorsal side of the hand are the first and third compartments. in the first compartment, the tendons of m. extensor pollicis brevis and abductor poll. longus are involved and the dis order is best known under the name de quervain’s disease. in the third compartment, the tendon to m. extensor poll. long. is involved and the damage to this tendon usually manifests itself as a rupture. local chronic irritation is considered to he one of the causes of the damage but it is not known clearly why these disorders primarily occur in middle-aged women. in the present material comprising women above the age of 50, an account is given of roentgenologically observed local bone and joint over growths which would appear to favour the development of the above-mentioned chronic, mechanical irritation of tendons and tendon sheaths. these new bone formations are regarded as part of a general growth disturbance which the author is referring to as involutional acromegaly. introduction among the long-lasting pain conditions of the wrist, stenosing tendovaginitis is very usual. the most common of them, best known under the name de quervain’s disease, was described by the author mentioned as early as 1885 and was lo calized to the tendon sheaths of m. abductor pol licis longus and m. extensor pollicis brevis, i.e. the first compartment of the dorsal side of the hand (16). usually, the diagnosis involves no prob lems: pain, local swelling and tenderness over the tendons mentioned are typical symptoms. further more, the patient usually reports indirect pain when extending these tendons which is most easily tested by means of ulnar flexion of the wrist with the fingers closed around a maximally flexed thumb-finkelstein’s test (4). a t operation, a thickening of lig. carpi dorsale and of the sheath is observed. i n some cases there is also a local thick ening of the tendon itself. histological examination has revealed fibrosis, hyaline degeneration and car tilage formation both in the ligament and in the sheath. according to most authors, these changes are the result of a chronic, mechanical irritation. upsala j m e d sci 78 as the tendons run in a shallow groove over the radial styloid it is clear that a bone fragment after a radial fracture can be one of the causes of the disease. as there is no history on direct traumati zation in most cases, however, som authors have considered the cause to be periosteal thickening or formation of exostosis on the radial epiphysis, so-called styloiditis (8, 17, 21). most publications indicate that women are pre-disposed to this dis order and that, usually, women above the age of 50 are involved. the relationship women/men in some series is 10-20/1 and the sex difference thus appears totally convincing (7, 17, 20). the suggestions made to explain this age and sex distribution appear less imposing: high age results in reduced tissue vitality, middle aged women are engaged in monotonous, manual domestic work “washerwoman’s sprain”, the tendons run a dif ferent course, “the degree of angulation is usually greater in women” (1, 2, 7). the question must be regarded as unanswered. changes of the kind, which in cases of de quervain’s disease affect the first compartment, occasionally occur in the third compartment which contains the tendon to m. extensor pollicis longus. usually, these manifest themselves as a rupture of the tendon in question. one of the causes mentioned has been vocational micro-traumata, e.g. “drummer’s palsy”, previous trauma o r ten don disease but in addition to these there is a small group in which the etiology is unknown. the course of the tendon is considered to be of importance in the pathogenesis of the latter cases. inclosed in the third dorsal tendon sheath, the tendon runs in a narrow oblique groove on the back of the radius. this groove is bordered radi ally by lister’s tubercle which forms a hypo mochlion for the tendon which then runs in a radial direction before its attachment at the end phalange of the thumb. in this shallow groove, the tendon and the tendon sheath would thus be exposed to a chronic irritation which would give tendovaginitis in the wrist 161 table i. review of 6 cases with de quervain’s fendovaginitis tendofrontal heberden arthros case vaginit hypernodes c.m.c. other no. age sin dxt ostosis sin dxt sin dxt diabetes symptoms therapy results peri hum-scap.dxt carpal 2 6 9 ( f i g . 1 ) + + + + + + + + + + + + tunnel syndrome hypo 1 76 + + + + + + + + ( + a ) arthritis 3 62 + + + + + + + + ? thyroid hypo thyroid 4 57(fig. 2) + (+) + + + + + + carpal tunnel syndrome 5 70 + + + + + + + + + hyper 6 69 + + + + + + + + + tension s a 1 icy 1 phenyl butazone + lev ax i n phenyl+ butazone thyroid cortison (+) op. s a 1 icy 1 + s a 1 icy 1 lasix + + a indicates a positive glucose tolerance test. rise to laceration. the fact that this takes place has been shown by a number of observations in connection with radial fracture with remaining bone fragments and rupture of the tendon at the site in question (10, 19). the mechanism remains to be explained, however, as no trauma has been shown and as roentgen does not reveal any frac ture. here, the situation is as remarkable as men tioned above for de quervain’s disease, i.e. rupture of the tendon to m. ext. poll. long. involves women in two thirds of the cases and usually wcmen above the age of 50 (7, 18). the problem when discussing the etiology of the two previously mentioned diseases, two factors appear to be essential: 1) the local irritation factor and 2) the predilection to women above the age of 50. the latter fact, of course, makes one consider endo crine disturbance. after the menopause there are often signs of a growth disturbance in the form of hyperostosis frontalis interna (h.f.i.), osteo arthrosis, especially in the first carpo-metacarpal (c.m.c.) joint and the distal interphalangeal joints in the form of heberden’s nodes (h.n.) and, fre quently, a decreased glucose tolerance, sometimes as a manifest diabetes. the material accounted for in this report has been analyzed with respect to 1 the above-mentioned factors. material as pre-requisites for the diagnosis de quervain’s disease, we required local swelling and tenderness over the tendons of the first dorsal compartment and a positive finkelstein test (17). i n the present material there were 6 such cases. often one will find cases where this triad is incomplete insofar as the local oedema seems to occur in the acute stage of the disease only. i n this connection, patients with local tenderness over the tendon sheaths and a positive finkelstein test have been regarded as potentia1 de quervain-cases in the borderline zone between being normal o r pathological. i n order to gain information on the general appearance of these symptoms, consecutive cases of women above the age of 50, who have visited the medical clinic, have been registered. one series com prising 100 cases was taken from the general clinic where the patients came for a variety of disorders, and is referred to in this connection as “normal cases”. another series comprising 50 cases consists of diabetic patients. the material also includes one case with rupture of the tendon to m. ext. poll. longus. results the findings in the 6 cases with complete de quervains’s disease are listed in table i. the symptoms in the potential de quervain cases have been registered in the consecutive series and for the “normal cases” the results were: tender ness in 11 % and positivie finkelstein tests in 16 % . the corresponding figures for the diabetic cases were 30 % and 22 % , respectively. upsala j m e d sci 78 11 732853 162 s . oldberg table 11. review of 13 cases with potential de quervain’s tendovaginitis local tenderness frontal arthrosis case + finkelstein pos. hyperheberden in c.m.c. joint dia no. sin. dxt. ostosis nodes sin. dxt. betes normal cases 1 2 + 3 4 5 6 + i f diabetic cases 8 + 9 + 10 + 11 + 12 13 + + + + + + + + + + + + (+) + i + ( + ) ? + + ( + ) + + + i+) + + + + + + (+) ( + i + + ( + i ( + ) + + + (+) + + + + + + + + + + + + i+) + + f + (+) + + + + ( + ) + + + + (+) i+) + + + + + + + fig. 1. case c.m.c. joint, multangulum hand. 2. osteoarl osteophytes majus and .hrosis i n the o n naviculare h.n. on the first > and right upsala j m e d sci 78 tendovaginitis in the wrist 163 fig. 2. case 4. osteoarthrosis in the first c.m.c. joint and osteophytes o n proc. styloid. radii dxt. i n the normal series, the number of patients with both symptoms was 11 % and in the diabetic cases 22 % . from these groups, 7 and 6 patients respectively have been examined in the manner previously described for the genuine de quervain cases. the results are shown in table 11. this material includes one case with rupture of the tendon to in. extensor poll. longus. a now 75-year-old woman who, since 1950, has been treated with tiotil for slight thyreotoxicosis has for some months been troubled by prickly pains and numbness i n the fingers. these symptoms were always worst at night and improved if she shook her hands. the patient was admitted to the medical department in august 1971. diag nosis: carpal tunnel syndrome. at examination, there was considerable tenderness over the tendons of the first dorsal compartment and clear atrophy of the thenar musculature, especially o n the right side. on her fingers, the patient had slight h. n. phalen’s test was positive bilaterally and finkelstein’s test positive on the right side. the glucose load was positive. i t was also found that the patient could not extend the end phalange of her left thumb. this had apparently not worried her too much but she reported that, 7 years ago, while she worked in her garden, she suddenly discovered that it was difficult for her to move the left thumb. simultaneously she ob served “a stretched string” at the location of the tendon to ext. poll. longus. t h e patient felt no pain and the “string” soon disappeared. since then, she has had diffi fig. 3. case 3. 75-year-old woman with rupture of the tendon of m. extensor poll. longus. osteophytes i n the groove for this tendon. culty in handling certain tools, “most of the time, the hand has been a t rest”. the patient has never felt that these difficulties have been so severe that she should consult a physician. roentgen: h.f.i., slight h.n., most pro nounced o n her right index finger, moderate arthrosis of the first c.m.c. joints, as well as a few osteophytes next t o the groove for ext. poll. long. sin. (fig. 3). the pa tient was treated with rest, diuretics and butazolidin and improved considerably subjectively. at the time being, the tendon rupture i n the patient’s left thumb did not call for any medical treatment. discussion frontal hyperostosis and osteoarthrosis of the first c.m.c. joints and of the distal inter-phalangeal joints in the form of h.n. thus appeared almost constantly in the present selected material com prising women above the age of 50. these women had symptoms which are characteristic of de upsala j m e d sci 78 164 s . oldberg quervain’s disease or of rupture of the tendon to ext. poll. long., respectively. although part of this material comprises known diabetic cases, there is a clear positive correlation with such a disturb ance of the carbohydrate metabolism. previous in formation with respect to the incidence of h.f.i. in a normal population varies considerably. most investigations show, however, that these hyperos toses occur almost exclusively in post-menopausal women where they have a n incidence of about 2 0 % (5, 9, 11). among diabetics of the same sex and age, the incidence has been about 30% (11). many authors are of the opinion that there are good reasons to consider h.f.i. as a n expression of an increased activity of the anterior lobe of the hypophysis in connection with involution (5, 11). in patients with frontal hyperostosis, some authors have also found signs of a general growth ten dency, among other things an enlargement of the atlas diameter (11) and osteoarthrosis of the first c.m.c. joints and of the distal inter-phalangeal joints (6, 11, 12). a similar bone growth and osteoarthrosis of the type mentioned above are common in cases of genuine acromegaly. the agreement includes a reduced glucose tolerance, often in the form of manifest but benign diabetes. the introduction of the term involutional acro megaly seems to be justified (13). the above mentioned bone and joint changes by themselves most often cause only slight and spontaneously disappearing troubles. t o the extent that adjacent tissues are subjected to pressure, however, the symptoms become more pronounced. i n this con nection, cranial nerves in shallow bone grooves are in the risk zone. i n patients with frontal hy perostosis visual field defects caused by osteo phytes in foramen opticum have been observed. after removal of the roentgenologically observed osteophytes there has been quick improvement (3). according to the present author, similar growth changes in the carpal region may contribute to the damage to n. medianus which characterizes the so-called carpal tunnel syndrome (1 3). the material presented here shows that bone and joint changes of the type referred to above as involutional acromegaly appear rather constantly in patients with symptoms of de quervain’s tendo vaginitis. considering that the first c.m.c. joint is one of the most frequently used joints in the body, it is likely that osteoarthrosis of this joint, perhaps with concomitant subluxation of meta carpale i and osteophyte formations in the vicinity can create the chronic irritation of the tendons of the first dorsal compartment. such an assump tion is supported by the corresponding localization of bone and joint changes and de quervain symp toms which is shown in the tables. the same mechanism with osteophytes on the tuberculum listeri and protuberances and constriction in the groove for the tendon to ext. poll. longus prob ably contribute to local irritation and degeneration of this tendon with rupture as a result. in con nection with acromegalic growth changes, hyper trophy of bone and periosteum as well as thicken ing of ligaments and tendon sheaths can be ob served. as mentioned before, this is a common finding also in cases of stenosing tendovaginitis. phalen (15) has stated that “the majority of cases of de quervain’s disease, trigger finger, trigger thumb, carpal tunnel syndrome and periarthritis of the shoulder all have a similar fundamental cause-a chronic, nonspecific tendosynovitis there must be some etiologic relationship”. several cases, above reported, illustrate this relationship. involutional acromegaly appears to be such an etiological factor. i n treating these diseases it is probably im portant to observe that there is considerable varia tion and a tendency towards spontaneous improve ment (7). just as in the case of genuine acro megaly the process seems capable of becoming “burnt out”. i n acute cases, rest has been used as well as antirheumatic medications and local steroid injections. in order to reduce the forma tion of oedema, diuretics and thyroid preparations have been used in some cases. if these and similar treatment methods prove to be insufficient, the patient must be subjected to surgery with relief of the pressure on the tendons. in many cases, this has led to immediate improvement of the symptoms but recurrences are not unusual. the uniformity of involutional acromegaly with re spect to etiology and the great number of benign symptoms involved would call for a certain de gree of conservatism. references 1. albert, s. m., rechtman, a. m. & wohl, m. a,: med 2. bunnel, s.: surgery of the hand, p. 450. j. b. lippin ciin n amer 45: 1625, 1961. cott co., philadelphia, 1948. upsulu j med sci 78 tendovaginitis in the wrist 165 3. falconer, m. a. & pierard, b. e.: brit j ophtal 34: 4. finkelstein, h.: j bone joint surg 12:509, 1930. 5. henschen, f.: morgagni's syndrome. oliver & boyd, 6. kellgren, j. h. & moore, r.: brit med j 1: 181, 7. lapidus, p. w.: surg clin n amer 33: 1317, 1953. 8. leger, l. & gauthier-villars, p.: presse med 74: 858, 9. moore, s.: hyperostosis cranii. thomas, springfield, 265, 1950. edinburgh and london, 1949. 1952. 1947. ill., 1955. 10. nalebuff, e. a.: surg clin n amer 49:811, 1969. 1 1 . oldberg, s.: acta soc med upsal 51: 1945. 12. acta med scand, suppl. 170:381, 1946. 13. acta soc med upsal 76: 179, 1971. 14. phalen, g. s.: cleveland clin quart 35: 1, 1968. 16. de quervain, f.: corr. blatt f . schweitz. arzte 25: 389, 17. ruelle, m. & navarre, m.: rev rheum 34: 714, 1967. 18. strandell, g.: acta chir scand 109: 81, 1955. 19. weinberg, e. d.: jama 142:979, 1950. 20. werner, h. h.: nord med 75:551, 1966. 21. winterstein, 0.: munch med wschr 74: 12, 1927. 15. jama 142:979, 1950. 1895. received december 12, 1972 address for reprints: s . oldberg, m.d. dzpartment of medicine county hospital s-731 01 koping sweden upsulu j m e d sci 78 upsala journal of medical sciences 2022, 127, e8829 http://dx.doi.org/10.48101/ujms.v127.8829 distribution of intraocular pressure in a swedish population maria häkkinen and curt ekström department of surgical sciences, ophthalmology, uppsala university, uppsala, sweden abstract background: increased intraocular pressure (iop) and pseudoexfoliation (pex) are major risk factors for open-angle glaucoma (oag), an age-related neurodegenerative disease of significant importance for public health. there are few studies on the distribution of iop in populations where pex is a common finding. methods: the distribution of iop was studied in 733 subjects 65–74 years of age, examined in a population survey in the rural district of tierp, sweden, 1984–86. the difference between the right and left eye and the effect of which eye was measured first were examined. odds ratios, adjusted for age and sex, according to mantel-haenszel (or mh ), were calculated to estimate predictors of increased iop, defined as a pressure ≥20 mm hg in either eye. the pressure was measured with goldmann applanation tonometry. automated perimetry was used to identify oag. results: the distribution of iop was close to that of other european-derived populations. the pressure in the first measured eye was higher than in the second measured eye. increased iop was related to oag and pex, or mh 8.97 (95% confidence interval [ci] 3.84–20.9) and 2.40 (95% ci 1.53–3.76), respectively. an iop ≥20 mm hg increased the risk of having been diagnosed with diabetes (or mh 1.83; 95% ci 1.08–3.09). conclusion: in this study of subjects 65–74-years-old in sweden, the distribution of iop was close to that of other european-derived populations. although the difference was small, the pressure in the first measured eye was higher than in the second eye. increased iop was strongly related to untreated oag and pex. article history received 13 june 2022 revised 28 july 2022 accepted 6 september 2022 published 3 october 2022 keywords diabetes; epidemiology; intraocular pressure; open-angle glaucoma; population survey; pseudoexfoliation; repeated applanation tonometry; risk factor introduction open-angle glaucoma (oag) is an age-related neurodegenerative disease of significant importance for public health, characterised by progressive loss of optic nerve fibres with typical appearance of the optic nerve head and consistent visual field defects. globally, glaucoma is the leading cause of irreversible blindness (1). in a swedish study, increased intraocular pressure (iop) and pseudoexfoliation (pex) were proved to be important risk factors for the development of oag (2). in pex, a fibrillar material is produced and accumulated in the anterior segment of the eye, thus increasing the pressure by impairing the outflow of aqueous humour (3). common sequence variants in the lysyl oxidase-like 1 gene, involved in elastic fibre formation, are closely related to pex (4). the distribution of intraocular pressure is well-known from numerous studies on different ethnicities. one of the earliest studies, conducted in ferndale in wales, reported a mean iop of 16.6and 15.9-mm hg for women and men, respectively (5). results from the studies in framingham and beaver dam on subjects 65–74 years old are presented in table 1 (6, 7). to the best of our knowledge, only two studies on the distribution of iop, using applanation tonometry, have been done in sweden, both from dalby in the south (8, 9). the first of these studies reported a mean iop in the right eye of 15.4 mm hg for the age group 60–69 years and 15.9 for the age group 70–79 years. pseudoexfoliation was an uncommon finding in the dalby population (9). a connection between pex and increased iop has been demonstrated in several population studies (10–12). however, there are few studies on the distribution of iop in populations where pex is a common finding, none of them from sweden (13– 15). the study in oulu, in the north of finland, reported a mean pressure of 16.2 mm hg in the right eye and 15.7 in the left eye (13). a follow-up study in skellefteå in northern sweden, where pex is common, found a mean iop of 16.3 mm hg in women and 15.3 in men at baseline, when the subjects were 66 years old (16). the objectives of the present research were to examine the distribution of iop in a swedish population with a high exposure to pex and to estimate predictors of increased pressure. the effect of which eye was measured first was also studied. the investigation took the form of a cross-sectional study on a defined population. contact curt ekström curt.ekstrom@neuro.uu.se © 2022 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article http://dx.doi.org/10.48101/ujms.v127.8829 mailto:curt.ekstrom@neuro.uu.se mailto:curt.ekstrom@neuro.uu.se http://creativecommons.org/licenses/by/4.0/ 2 m. häkkinen and c. ekström methods the tierp glaucoma survey in 1984–1986, a population survey was conducted in the rural district of tierp, south central sweden. its target population comprised 2,429 residents, aged 65–74-years-old. a sample of about one-third of the target population was randomly selected. of the eligible number of 838 individuals, 760 (91%) underwent a detailed eye examination, as described elsewhere (17). briefly, an interview was first held, covering medical and family history. the pressure was taken with a goldmann applanation tonometer mounted on a haag–streit slit lamp. in subjects 65–69-years old, whose date of birth was divided by the figure 2, the left eye was measured first, while in the rest of the sample, the right eye was measured first. as a rule, the pressure was taken with single tonometer readings. if the difference between the two eyes exceeded 2 mm hg, a control measurement was done, as described by bengtsson (8). in this case, the second reading was defined as the iop for that person. the visual fields were tested using the competer 350 automated perimeter (bara elektronik ab, lund, sweden). after perimetry, the pupils were dilated, and the slit lamp biomicroscopy, including a binocular assessment of the optic  discs and gonioscopy, was done. the presence of cataract was  ascertained based on retroillumination using indirect ophthalmoscopy with lens opacities evident on biomicroscopy. pseudoexfoliation was defined as the presence of characteristic white flakes on the lens capsule or on the pupillary border. the study population of the total number of 760 participants, 25 were treated for glaucoma. these subjects were excluded from the study, as was one subject with unreliable pressure readings of both eyes. one individual declined iop measurement (figure 1). the remaining 733 people, 381 women and 352 men, constituted the study population. the investigation was approved by the human subjects committee at the faculty of medicine, uppsala university, and adhered to the tenets of the declaration of helsinki. an informed consent was obtained from all participants. this report is in accordance with the original ethical approval. classification of oag consistent with the concept of foster et al. (18), glaucoma with pex was classified as oag. to qualify for a diagnosis of oag, a reproducible visual field defect was a prerequisite, consistent with glaucoma and not explicable on other grounds, as described elsewhere (17). twenty-four subjects fulfilled a diagnosis of definite oag. pseudoexfoliation in either eye was present in 117 subjects (16.0%), of whom five were diagnosed with oag. assessment of systemic predictors information on treated systemic hypertension, ischaemic heart disease, and diabetes mellitus was obtained at the interview or from medical records. in the case of a discrepancy between the figure 1. flow chart showing how the study population of 733 individuals was derived. iop: intraocular pressure. 838 population sample 25 under treatment 1 unreliable iop 733 study population 1 declined iop 78 non participation 760 examined table 1. percent distribution of intraocular pressure in the right eyes in individuals aged 65–74 years in the framingham eye study and the beaver dam eye study by sex. study iop (mm hg) sex <13 13–15 16–18 19–21 22–24 ≥25 mean framinghama females 10.5 25.4 35.8 19.7 5.0 3.6 17.0c males 13.4 25.3 34.3 17.4 4.7 4.9 16.7c beaver damb females 13.8 30.9 36.4 13.4 4.2 1.2 16.0 males 18.3 34.0 31.2 10.9 5.1 0.6 15.5 iop: intraocular pressure. aref. (6); bref. (7); the age groups 65–69 years and 70–74 years are combined; cthe mean relates to both eyes. distribution of intraocular pressure 3 self-reported history and the medical record, data from the latter source were used in this report. the participants were asked if they were current smokers or past smokers and when they stopped smoking. information on smoking was also acquired from medical records and family members. statistical methods a repeated measures anova was done to explore the covariation in iop between the right and left eye depending on which eye was measured first. predictors of increased iop, defined as a pressure ≥20 mm hg in either eye, were estimated using 2 × 2 tables, with odds ratios adjusted for age and sex strata, according to the mantel–hansel’s method (or mh ). to simultaneously assess several variables affecting the risk for increased iop, multiple logistic regression analyses were used, with an iop ≥20 mm hg as the dependent variable. results the distribution of the highest pressure in either eye was slightly drawn-out to the right, as shown in figure 2. most of the oag cases were found to the right. the median pressure was 17 mm hg (interquartile range 15–19), and the mean pressure was 16.9 (95% ci 16.7–17.2). the percent distribution in the right eyes is presented in table 2. there were small differences between individuals aged 65–70 years and 70–74 years, and between females and males. in subjects 65–69 years of age, the mean pressure was higher in the eye that was measured first (table 3). analysis of variance revealed a small but significant interaction between measuring the right eye first and the left eye second (p = 0.0025). the mean iop in right eyes with pex was 18.7 mm hg, compared with 16.0 in eyes without pex, with a clear overrepresentation of pressures above 21 mm hg in eyes with pex (table 4). the stratified analyses are presented in table 5. oag (or mh 8.97; 95% ci 3.84–20.9), pex (or mh 2.40; 95% 1.53–3.76), and diabetes (or mh 1.83; 95% ci 1.08–3.09) were related to an iop ≥20 mm hg, while age, sex, cataract, smoking, systemic hypertension, and ischemic heart disease were not. the factors in table 5 were tested in logistic regression models. the results figure 2. distribution of the highest pressure in either eye in the study population of 733 participants in the tierp glaucoma survey (3 mm hg pressure intervals). twenty-seven individuals were excluded. oag: open-angle glaucoma. table 2. percent distribution of intraocular pressure in right eyes in 731 participants in the tierp glaucoma survey by age and sex.a age (years) iop (mm hg) sex no. <13 13–15 16–18 19–21 22–24 ≥25 mean 65–69 f 202 9.4 29.2 34.7 21.8 3.0 2.0 16.6 65–69 m 187 12.3 32.6 33.2 12.8 6.4 2.7 16.4 65–69 total 389 10.8 30.8 33.9 17.5 4.6 2.3 16.5 70–74 f 178 17.4 26.4 37.6 9.0 5.6 3.9 16.3 70–74 m 164 16.5 37.8 27.4 12.8 4.3 1.2 15.7 70–74 total 342 17.0 31.9 32.7 10.8 5.0 2.6 16.0 65–74 f 380 13.2 27.9 36.1 15.8 4.2 2.9 16.5 65–74 m 351 14.2 35.0 30.5 12.8 5.4 2.0 16.1 65–74 total 731 13.7 31.3 33.4 14.4 4.8 2.5 16.3 iop: intraocular pressure; f: females; m: males. atwenty-nine subjects are excluded; the right eye was removed in two subjects. table 3. mean intraocular pressure in the right and left eye in 389 participants 65–69 years of age in the tierp glaucoma survey by the first measured eye.a eye right eye measured first left eye measured first no. iop (95% ci) no. iop (95% ci) right eye 200 16.7 (16.1–17.3) 189 16.3 (15.8–16.8) left eye 200 16.2 (15.7–16.7) 189 16.5 16.0–17.0 iop: intraocular pressure, mm hg; ci: confidence interval. afifteen subjects treated for glaucoma are excluded from the analyses. 4 m. häkkinen and c. ekström of a model including age, sex, oag, pex, smoking status, and diabetes were almost identical to that of the stratified analyses (data not shown). there was no indication of interaction in the models. discussion in this study, the distribution of iop was close to that of other european-derived populations of the same age (6, 7). likewise, in agreement with other population surveys (10–12), pex was associated with increased iop, defined as a pressure ≥20 mm hg in either eye. thus, the high prevalence of pex in the examined population (16%) had no significant impact on the distribution of iop. to the best of our knowledge, the current study, including a defined population, was the first to explore the effect of which eye is measured first. although the difference was small, in subjects 65–69 years of age, the mean pressure was higher in eyes randomly assigned to be measured first. furthermore, analysis of variance revealed a significant interaction between measuring the right eye first and the left eye second. it is well known that repeated applanation tonometry reduces the pressure (19–22). however, the reason for the pressure decreasing is not fully understood. one explanation presupposes a passing stage of initial tension in subjects being examined (23). interestingly, psychological stress has been proven to result in an increase of the iop in healthy individuals (24). the results of the present study support the idea of stress as the cause of this phenomenon. however, even if the findings present new knowledge, they do not have any apparent clinical implication other than a recommendation to repeat the measurements if there is a noteworthy difference in the iop between the two eyes. increased iop has frequently been related to oag in population surveys (15, 25–27). in fact, a strong association was demonstrated also in the current study, where an iop ≥20 mm hg increased the risk of having oag 9-fold (table 5). moreover, in accordance with the studies in framingham and beaver dam (6, 7), we did not find any relationship between age or sex and the distribution of iop. systemic hypertension has consistently been associated with an increased iop in many studies (7, 8, 28, 29). this was not the case in the present study, in which only individuals treated for hypertension based on the medical records were classified as exposed. in contrast, the blood pressure was measured in the other population studies referred to above. it is impossible to speculate on what effect the different methods might have had on the estimates. lack of statistical power may also have affected table 4. percent distribution of intraocular pressure in right eyes in 731 participants in the tierp glaucoma survey by the presence of pseudoexfoliation.a pex iop (mm hg) no. <13 13–15 16–18 19–21 22–24 ≥25 mean yes 79 11.4 25.3 25.3 13.9 10.1 13.9 18.7 no 652 14.0 32.1 34.4 14.4 4.1 1.1 16.0 iop: intraocular pressure; pex: pseudoexfoliation. atwenty-nine subjects are excluded; two subjects were missing their right eyes. table 5. odds ratios for intraocular pressure ≥20 mm hg in either eye in 733 participants in the tierp glaucoma survey, adjusted for age and sex.a characteristics no. of cases (n = 129) or m-h 95% ci age ≥70 yearsb no 76 1.00 yes 53 0.75 0.51–1.10 male sexc no 72 1.00 yes 57 0.83 0.57–1.22 open-angle glaucoma, either eyeb no 114 1.00 yes 15 8.97 3.84–20.93 pseudoexfoliation, either eye no 93 1.00 yes 36 2.40 1.53–3.76 cataract, either eye no 90 1.00 yes 39 1.04 0.68–1.59 smoking status never smoked 78 1.00 past smoker 28 1.61 0.93–2.78 current smoker 23 1.43 0.82–2.49 diabetes no 106 1.00 yes 23 1.83 1.08–3.09 hypertension, treated no 90 1.00 yes 39 1.16 0.76–1.77 ischaemic heart disease no 107 1.00 yes 22 1.32 0.79–2.22 ci: confidence interval; or m-h : mantel-haenszel adjusted odds ratio. atwenty-seven subjects are excluded from the analyses; badjusted for sex; cadjusted for age. distribution of intraocular pressure 5 the study in tierp. for this reason, the results should be interpreted with some caution. at present, a positive relationship between diabetes mellitus and iop is well established in the literature (29–32). in larger population surveys, diabetes was usually diagnosed either from a self-reported history of taking medication or the determination of plasma glucose levels in blood samples. in this study, an iop ≥20 mm hg increased the risk of having been diagnosed with diabetes by 83%. why subjects with diabetes have a higher iop is unclear. an explanation often mentioned implies that raised glucose levels induce an osmotic gradient, attracting fluid into the intraocular space, resulting in increased pressure (30). a genetic link between the two disorders has also been suggested (33). our study has several strengths, including its communitybased design, high participation rate, and the use of a detailed protocol. the eye pressures were taken by an experienced assistant, and all eye examinations conducted by the same glaucoma specialist, who was masked to the result of the pressure readings and the visual field testing. furthermore, a visual field defect was required for a diagnosis of oag. nevertheless, as with many epidemiologic studies, the research was limited in several respects. most importantly, compared with many other population studies, the tierp glaucoma survey was a small study, limiting its statistical power to provide reliable estimates on some of the predictors of increased iop. however, the iop measurements delivered sufficient data for an accurate description of the iop distribution in the examined population, which was the main issue of this study. furthermore, the study only involved individuals aged 65–74-years-old. nonetheless, there are no reports on significant age differences in the distribution of iop in other european-derived population. there is always a risk of misclassification of exposure in crosssectional studies when data are based on self-reports, which was the case regarding smoking habits. this type of information bias should be non-differential, thereby ‘diluting’ the relationship between increased iop and possible predictors in the analyses. the lack of association with systemic hypertension in this study was possibly an effect of non-differential misclassification. in conclusion, in this population-based study on individuals aged 65–74-years-old in sweden, the distribution of iop was close to that of the framingham and beaver dam studies. the pressure in the first measured eye was higher than the pressure in the second eye. increased iop was strongly related to untreated oag and pex. disclosure statement the authors report no conflict of interest. funding the swedish medical research council, crown princess margaretha’s foundation for the visually impaired, the glaucoma research fund at the department of ophthalmology, uppsala university hospital, and uppsala county council provided financial support for this study. notes on contributors maria häkkinen is a medical student at uppsala university, uppsala, sweden. curt ekström, md, phd, is a senior researcher at the department of surgical sciences, ophthalmology, uppsala university, uppsala, sweden. orcid curt ekström 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department of clinicul chemistry, u 1 7 i ~ ~ e r s i t y h o s p i t u l , uppsalu, sii,eden abstract nine a m e n o r r h o e i c women w i t h a n o r e x i a n e r v o s a (an) w e r e g i v e n l o n g t e r m t r e a t m e n t w i t h 500 ug of s y n t h e t i c l u t e i n i z i n g h o r m o n e r e l e a s i n g hormone (lrh) e v e r y 8 h . a l l t h e women h a d i m p a i r e d l u t e i n i z i n g hormone (lh) s e c r e t i o n a n d no e v i d e n c e of endogenous o e s t r o g e n p r o d u c t i o n . t h r e e o f them a l s o h a d d e f i c i e n t f o l l i c l e s t i m u l a t i n g hormone (fsh) s e c r e t i o n . the p i t u i t a r y r e s e r v e c a p a c i t y f o r g o n a d o t r o p h i n s e c r e t i o n w a s n o r m a l b u t t h e r e s p o n s e p a t t e r n t o lrh w a s s i m i l a r t o t h a t d e s c r i b e d i n p r e p u b e r t a l g i r l s . the c o n s t a n t a d m i n i s t r a t i o n o f lrh n o r m a l i z e d b a s a l lh and fsh s e c r e t i o n and i n d u c e d a c y c l i c a l g o n a d o t r o p h i n s e c r e t o r y p a t t e r n w i t h d i f f e r e n t i a l c h a n g e s o f t h e lh and fsh r e s p o n s e s t o lrh d u r i n g t h e t r e a t m e n t . lrh-induced g o n a d o t r o p h i n s e c r e t i o n p r o d u c e d f o l l i c u l a r growth and m a t u r a t i o n i n a l l t h e women. p r e s u m p t i v e o v u l a t i o n a l s o o c c u r r e d d u r i n g t h e 8 t r e a t m e n t c o u r s e s i n w h i c h o n l y lrh w a s a d m i n i s t e r e d . however, i n a d e q u a t e l u t e a l p h a s e s were o b s e r v e d d u r i n g 6 o f t h e s e 8 c y c l e s . combined t h e r a p y w i t h lrh and human c h o r i o n i c g o n a d o t r o p h i n (hcg) d u r i n g 5 t r e a t m e n t c o u r s e s r e s u l t e d i n normal o v u l a t o r y c y c l e s w i t h a d e q u a t e c o r p u s l u t e u m f u n c t i o n . i n t r o d u c t i o n a n o r e x i a n e r v o s a i s c h a r a c t e r i z e d e n d o c r i n o l o g i c a l l y by a n i m p a i r m e n t o f t h e g o n a d o t r o p h i n s e c r e t i o n from t h e a n t e r i o r p i t u i t a r y . the i m p a i r m e n t i s more m a r ked f o r lh t h a n f o r fsh s e c r e t i o n . the p i t u i t a r y r e s p o n s i v e n e s s t o lrh i s re duced i n a c u t e s t a g e s o f an and r e s t o r e d t o normal a f t e r c l i n i c a l improvement w i t h r e g a i n o f body w e i g h t (20, 28, 3 2 , 3, 1 8 ) . r e p e a t e d s t i m u l a t i o n w i t h lrh can a l s o r e s t o r e t h e p i t u i t a r y r e s e r v e c a p a c i t y f o r g o n a d o t r o p h i n s e c r e t i o n t o normal ( 1 6 , 1 9 , 3 6 ) . by l o n g t e r m t h e r a p y w i t h lrh it i s p o s s i b l e t o i n d u c e normal o v u l a t o r y m e n s t r u a l c y c l e s i n a m e n o r r h o e i c women w i t h an ( 1 6 , 1 9 ) . how e v e r , c y c l e s w i t h l u t e a l p h a s e d e f e c t s o f t e n o c c u r d u r i n g p r o l o n g e d t r e a t m e n t s w i t h only lrh ( 1 6 , 1 9 ) . here we a n a l y z e 13 m e n s t r u a l c y c l e s i n d u c e d by lrh i n 9 a m e n o r r h o e i c women w i t h an i n a n a t t e m p t t o d i s c l o s e t h e mechanism(s) b e h i n d t h e l u t e a l p h a s e d e f e c t s . 21 material and methods p a t i e n t s nine women, aged 20-33 (mean 2 5 . 4 ) y e a r s , w i t h symptoms of an v o l u n t e e r e d f o r t h e s t u d y . they a l l had s e c o n d a r y amenorrhoea of 8 monthst o 1 3 y e a r s (median 20 months) d u r a t i o n . s i x women were j u d g e d by an e x p e r i e n c e d p s y c h i a t r i s t t o f u l f i l l t h e d i a g n o s t i c c r i t e r i a o f t r u e an ( 4 ) w h i l e t h e o t h e r t h r e e were l a b e l l e d a s a n o r e c t i c b e h a v i o u r , c o n s i d e r e d a s a m i t i g a t e d form of an ( 7 ) . t h e i r mean body w e i g h t 4 2 . 8 kg ( r a n g e 3 6 4 7 ) which c o r r e s p o n d s t o 7 2 % ( r a n g e 63-88) o f mean i d e a l body w e i g h t ( 8 ) . no o r g a n i c c a u s e of t h e amenorrhoea and w e i g h t l o s s was found a t t h e c l i n i c a l i n v e s t i g a t i o n . buccal smear c h r o m a t i n complement showed a normal f e m a l e p a t t e r n . x-ray e x a m i n a t i o n s of t h e s k u l l and p i t u i t a r y f o s s a were normal. a l l t h e p a t i e n t s were e u t h y r o i d , a s j u d g e d by c l i n i c a l e x a m i n a t i o n and t h y r o i d f u n c t i o n t e s t s . the 24-hour u r i n a r y e x c r e t i o n of 17-hydroxycorticosteroids and 1 7 k e t o s t e r o i d s was normal i n a l l e x c e p t one p a t i e n t , who had low b a s a l l e v e l s which i n c r e a s e d n o r m a l l y a f t e r metyrapone. t e s t o s t e r o n e and p r o l a c t i n l e v e l s i n blood were w i t h i n t h e norm31 r a n g e s . a l l t h e p a t i e n t s had low o r a b s e n t endogenous o e s t r o g e n p r o d u c t i o n , a s j u d g e d b o t h by i n d i r e c t c l i n i c a l methods f o r e s t i m a t i n g o e s t r o g e n i c a c t i v i t y on t a r g e t o r g a n s (no w i t h d r a w a l b l e e d i n g a f t e r i n t r a m u s c u l a r p r o g e s t e r o n e e t c . ) and d i r e c t measurements o f o e s t r o g e n l e v e l s i n b l o o d . l r h t r e a t m e n t i n t r a v e n o u s lrh t e s t (100 ug, hoechst) were performed b e f o r e , d u r i n g and a f t e r t h e t r e a t m e n t s . the fsh and lh r e s p o n s e t o lrh was d e f i n e d a s t h e d i f f e r e n c e between t h e mean o f t h e two v a l u e s a t 30 and 45 m i n u t e s a f t e r t h e lrh i n j e c t i o n and t h e mean of t h e two c o n t r o l v a l u e s . f i v e hundred vg o f lrh ( h o e c h s t ) were a d m i n i s t e r e d s u b c u t a n e o u s l y o r i n t r a m u s c u l a r l y e v e r y 8 h d u r i n g t h e t r e a t m e n t . seven women were t r e a t e d w i t h o n l y lrh d u r i n g 8 t r e a t m e n t c y c l e s . during t r e a t m e n t f r e q u e n t d e t e r m i n a t i o n s of fsh, lh, o e s t r a d i o l (e ) and p r o g e s t e r o n e i n b l o o d were made. o e s t r o g e n m o n i t o r i n g w a s p e r f o r m e d by e s t i m a t i o n of t h e t o t a l u r i n a r y o e s t r o g e n (te) e x c r e t i o n i n a l l b u t two o f t h e women. the t r e a t m e n t w i t h lrh a l o n e was c o n t i n u e d u n t i l m e n s t r u a t i o n o c c u r r e d . four women were t r e a t e d w i t h lrh i n c o m b i n a t i o n w i t h hcg d u r i n g 5 t r e a t m e n t 2 c y c l e s m o n i t o r e d by d a i l y d e t e r m i n a t i o n s o f e 2 i n b l o o d o r te i n u r i n e . when o e s t r o g e n s l e v e l s c o n s i s t e n t w i t h f o l l i c u l a r m a t u r a t i o n were r e a c h e d , t h e lrh i n j e c t i o n s were i n t e r r u p t e d and a s i n g l e i n t r a m u s c u l a r i n j e c t i o n o f 6-9000 iu o f hcg ( p r e g n y l , organon) was a d m i n i s t e r e d . a f t e r t h a t , 1-4 i n j e c t i o n s of 1 5 0 0 6 0 0 0 i u of hcg were g i v e n a t i n t e r v a l s o f 3-7 d a y s . r 2 2 hormone a s s a y methods immunoreactive fsh and lh i n serum w e r e a s s a y e d by t h e radioimmunosorbent t e c h n i q u e w i t h i n d i r e c t l y c o u p l e d a n t i b o d i e s ( 3 3 ) . lh i n serum was measured by u t i l i z i n g human p i t u i t a r y lh ( 2 2 ) l a b e l l e d w i t h 1251 and r a b b i t antihuman p i t u i t a r y lh. the lh p r e p a r a t i o n had a b i o l o g i c a l a c t i v i t y of 9400 i u (2nd irp hmg) p e r mg. fsh i n serum was measured by u t i l i z i n g human p i t u i t a r y fsh ( 2 1 ) l a b e l l e d w i t h lz5i and g u i n e a p i g anti-human p i t u i t a r y fsh. the fsh p r e p a r a t i o n had a b i o l o g i c a l a c t i v i t y o f 12000 iu (2nd iw-hmg) p e r mg. the r e s u l t s w e r e e x p r e s s e d i n ng o f t h e p u r i f r e d lh and fsh p r e p a r a t i o n s p e r m l o f serum. com p a r i s o n s between fsh and lh l e v e l s were made i n r e l a t i o n t o t h e g e o m e t r i c mean fsh/lh r a t i o ( a p p r o x i m a t e l y = 1) o f t h e normal m e n s t r u a l c y c l e . immunoreactive e 2 w a s measured by a r a d i o i m m u n o l o g i c a l t e c h n i q u e u s i n g an a n t i s e r u m t o o e s t r a d i o l 6 o x i m e (11). p r o g e s t e r o n e was a s s a y e d by a s i m i l a r method ( 3 1 ) . one pg of e 2 / m l = 3.67 p m o l / l and l n g of p r o g e s t e r o n e / m l = 3.12 n m o l / l , results p r e t r e a t m e n t g o n a d o t r o p h i n l e v e l s i n serum b e f o r e and a f t e r i n t r a v e n o u s lrh a r e shown i n f i g . 1. the b a s a l lh l e v e l s were below t h e normal r a n g e i n a l l t h e p a t i e n t s w h i l e o n l y t h r e e of t h e p a t i e n t s had a b n o r m a l l y low b a s a l fsh l e v e l s . a l l b u t one o f t h e an p a t i e n t s r e s p o n d e d t o lrh w i t h e v i d e n t r e l e a s e o f lh and fsh. the mean lh r e s p o n s e w a s s i m i l a r t o t h a t of t h e c o n t r o l group o f women i n t h e e a r l y f o l l i c u l a r p h a s e of t h e m e n s t r u a l c y c l e w h i l e t h e fsh r e s p o n s e was 4 t i m e s l a r g e r ( f i g . 1). the p r e t r e a t m e n t fsh/lh r a t i o of t h e g o n a d o t r o p h i n r e s p o n s e s t o lrh w a s 1 . 0 4 i n comparison w i t h 0.22 f o r t h e c o n t r o l s u b j e c t s . t r e a t m e n t w i t h lrh a l o n e f i v e h u n d r e d pg of lrh was a d m i n i s t e r e d p a r e n t e r a l l y e v e r y 8 h o u r s t o 7 women u n t i l m e n s t r u a t i o n o c c u r r e d a f t e r 24-36 (mean 2 9 . 5 ) days of t r e a t m e n t . f o l l i c u l a r growth and m a t u r a t i o n , a s j u d g e d by i n c r e a s e d o e s t r o g e n l e v e l s i n u r i n e o r b l o o d , were i n d u c e d d u r i n g a l l t h e 8 t r e a t m e n t c y c l e s . o v u l a t i o n , as j u d g e d by p r o g e s t e r o n e v a l u e s o f more t h a n 3 ng/ml i n s i n g l e b l o o d samples t a k e n 3-10 days b e f o r e m e n s t r u a t i o n ( 9 ) , w a s a l s o i n d u c e d i n a l l t h e c y c l e s . b a s a l body t e m p e r a t u r e s were r e c o r d e d d u r i n g 5 t r e a t m e n t c y c l e s and a l l t h e c u r v e s were b i p h a s i c . the maximum p r o g e s t e r o n e c o n c e n t r a t i o n s o b s e r v e d w e r e l e s s t h a n . 1 0 ng/ml i n a l l b u t 2 c y c l e s (26.4 and 13.8 n g / m l ) . i n t h e f o u r c y c l e s w e r e b l o o d samples were o b t a i n e d a t l e a s t e v e r y 4 t h d a y , t h e maximum p r o g e s t e r o n e v a l u e was 7 . 7 ng/ml, s u g g e s t i n g i n s u f f i c i e n t c o r p u s l u t e u m func t i o n . the l e n g t h o f t h e l u t e a l p h a s e was 12-16 d a y s . hormone l e v e l s d u r i n g a p r e s u m p t i v e l y o v u l a t o r y m e n s t r u a l c y c l e i n d u c e d by lrh a l o n e are shcwn i n f i g . 2 . during t h e f i r s t t h r e e days of t r e a t m e n t t h e fsh r e s p o n s e s were l a r g e r t h a n t h e lh r e s p o n s e s . then t h e fsh r e s p o n s e s 23 lh 23 2 1 0, fm2s.e.m an-anorexia nervosa, n=9 c=control, healthy women, early follicular phase, n=x) c an [1 fiaf3 1 i i 0 c an t 0 304560 c an minutes f i g . 1. mean lh and fsh l e v e l s b e f o r e and a f t e r i n t r a v e n o u s lrh ( l e f t ) and mean lh and fsh r e s p o n s e s t o lrh ( r i g h t ) b e f o r e lrh t r e a t m e n t o f 9 women a n . d e c r e a s e d w h i l e t h e lh r e s p o n s e s p r o g r e s s i v e l y i n c r e a s e d . t h e r e w a s s l o w i n c r e a s e o f e 2 i n serum d u r i n g t h e f i r s t week o f t r e a t m e n t f o l l o w e d by a more r a p i d i n c r e a s e t o a peak l e v e l (350 pg/ml) c o n s i s t e n t w i t h f o l l i c u l a r matura t i o n on t r e a t m e n t day 13. a t t h a t time lh i n serum r e a c h e d a maximum w i t h a l e v e l s i m i l a r t o t h a t s e e n d u r i n g t h e m i d c y c l e peak i n t h e normal m e n s t r u a l c y c l e . the lh peak was n o t accompanied by an e v i d e n t fsh peak. a f t e r t h e lh peak t h e e 2 l e v e l d e c r e a s e d somewhat and a t t h e same t i m e i n c r e a s e d p r o g e s t e r o n e l e v e l s were o b s e r v e d , i n d i c a t i n g t h a t o v u l a t i o n presumably o c c u r r e d . pro g e s t e r o n e i n blood s l o w l y i n c r e a s e d to r e a c h a p l a t c a u of 1-14 ng/ml o n e w e e k l a t e r . i n c r e a s e d p r o g e s t e r o n e l e v e l s i n b l o o d were s e e n f o r 12-14 days b e f o r e m e n s t r u a t i o n . one week l a t e r , a new t r e a t m e n t c o u r s e w a s i n i t i a t e d ( f i g . 3 ) . the p r e t r e a t m e n t lrh t e s t r e s u l t e d i n s m a l l g o n a d o t r o p h i n i n c r e a s e s from l o w b a s a l v a l u e s w i t h a g r e a t e r r e s p o n s e o f lh t h a n o f fsh. the fsh r e s p o n s e i n c r e a s e d s l i g h t l y d u r i n g t h e f i r s t days of t r e a t m e n t b u t t h e fsh l e v e l s a f t e r lrh 24 1 5 10 15 20 25 dav of treatment fig. 2. basal lh and fsh levels i n serum as well as fsh and lh responses to lrh and serum levels of e 2 and progesterone before, during and after the first lrh treatment of a 22-year-old patient w i t h an. i p e fig. 3. lh, fsh, e and progesterone levels i n serum before, during and after the second lrh treatment of the an patient kl from fig. 2. 2 25 1 5 x) 15 20 25 day of treatment *_ j7 40 j 7 l p e q 0 f i g . 4 . b a s a l lh and fsh l e v e l s i n serum as w e l l as lh and fsh r e s p o n s e s t o lrh and serum l e v e l s of e 2 and p r o g e s t e r o n e b e f o r e and d u r i n g t h e f i r s t t r e a t m e n t w i t h lrh i n c o m b i n a t i o n w i t h hcg of a 29-year-old woman w i t h an. remained below t h e lh l e v e l s t h r o u g h o u t t h e t r e a t m e n t . t h e r e was a s m a l l and v e r y s l o w e 2 i n c r e a s e and t h e r e w a s no e v i d e n t m i d c y c l e e peak. on t r e a t m e n t day 1 9 , when t h e maximum lh r e s p o n s e t o lrh was o b s e r v e d , t h e e 2 l e v e l s was o n l y 1 2 0 pg/ml. a f t e r t h e lh peak, t h e r e w a s a s l o w i n c r e a s e of p r o g e s t e r o n e w i t h a maximum o f 8 . 2 ng/ml on day 29. 2 t r e a t m e n t w i t h lrh i n c o m b i n a t i o n w i t h h c g . f o l l i c u l a r m a t u r a t i o n and o v u l a t i o n were i n d u c e d by combined t h e r a p y w i t h lrh and hcg i n 5 t r e a t m e n t c y c l e s . the c o r p u s l u t e u m f u n c t i o n w a s a d e q u a t e , as j u d g e d by h i g h normal p r o g e s t e r o n e l e v e l s i n b l o o d . r e s u l t s from a combined lrh hcg t r e a t m e n t o f an i n f e r t i l e a m e n o r r h o e i c p a t i e n t w i t h a c o m p l e t e l a c k o f p i t u i t a r y r e s p o n s i v e n e s s t o lrh b e f o r e t h e t r e a t m e n t , a r e shown i n f i g . 4 . d u r i n g t h e p r o l o n g e d lrh t r e a t m e n t b o t h lh and fsh r e s p o n s e s t o lrh a p p e a r e d . the fsh r e s p o n s e was more marked t h a n t h e lh r e s p o n s e d u r i n g t h e f i r s t 3 days and a p r e p u b e r t a l l i k e fsh/lh r a t i o of t h e r e s p o n s e s were s e e n d u r i n g t h e f i r s t week o f t r e a t m e n t . then t h e lh r e s p o n s e p r o g r e s s i v e l y i n c r e a s e d w h i l e t h e fsh 26 lh nglrnl . . e . i -. . .' f i g . 5. l h , fsh, e2 and p r o g e s t e r o n e l e v e l s i n serum as w e l l a s lh and fsh r e s p o n s e s t o lrh b e f o r e and d u r i n g t h e second lrh t r e a t m e n t o f t h e an p a t i e n t bl from f i g . 4 . r e s p o n s e d e c r e a s e d . a t t h e same t i m e , t h e e s e c r e t i o n s t a r t e d t o r i s e . on t r e a t m e n t day 18, t h e lh l e v e l r e a c h e d a m i d c y c l e l i k e p e a k , which w a s f o l l o w e d by i n c r e a s e d p r o g e s t e r o n e l e v e l s i n b l o o d . the c h a r a c t e r i s t i c f a l l o f t h e e l e v e l c o n c o m i t a n t w i t h t h e i n c r e a s e o f t h e p r o g e s t e r o n e l e v e l , found a t o v u l a t i o n i n t h e normal m e n s t r u a l c y c l e , was s e e n d u r i n g t h e lrh i n d u c e d c y c l e o f t h i s p a t i e n t . the p r o g e s t e r o n e c o n c e n t r a t i o n was 1 0 . 7 ng/ml when hcg was admini s t e r e d , i n d i c a t i n g t h a t o v u l a t i o n had a l r e a d y o c c u r r e d a t t h a t t i m e . repeated i n j e c t i o n s o f h c g were t h e n g i v e n d u r i n g t h e l u t e a l p h a s e , which was s l i g h t l y p r o l o n g e d w i t h h i g h e2 and high-normal p r o g e s t e r o n e l e v e l s . 2 2 on m e n s t r u a l day 5 a new t r e a t m e n t w i t h o n l y lrh was i n s t i t u t e d ( f i g . 5 ) . b e f o r e t h i s second lrh t r e a t m e n t , t h e r e was o n l y a s m a l l lh r e s p o n s e b u t no e v i d e n t fsh r e s p o n s e t o i n t r a v e n o u s lrh. during t h e f i r s t day of t r e a t m e n t , t h e fsh r e s p o n s e r e a p p e a r e d b u t t h e fsh l e v e l s a f t e r lrh n e v e r became h i g h e r t h a n t h e lh l e v e l s . a f t e r a b o u t 1 0 days of t r e a t m e n t t h e r e was a v e r y s m a l l and slow i n c r e a s e o f e2 i n b l o o d . when t h e maximal lh r e s p o n s e t o lrh was o b s e r v e d on t r e a t m e n t day 1 9 , t h e e 2 l e v e l was o n l y 1 1 9 pg/ml. p r o g e s t e r o n e s t a r t e d t o i n c r e a s e s l o w l y and t h e r e was a f u r t h e r e 2 i n c r e a s e t o a p l a t e a u w i t h l e v e l s between 150 and 200 pg/ml. i n c r e a s e d p r o g e s t e r o n e l e v e l s i n b l o o d were o b s e r v e d f o r 13 days b e f o r e m e n s t r u a t i o n w i t h maximum of 8.5 ng/ml on t r e a t m e n t days 29 and 31. anorexia nerma long-term lrh treatment n = 6 mz5e.m a lh i responses to lrh at the beginning of treatment e2= 28 pghl i] -*ii , , i at follicular maturation e2= 345pg/ml f i g . 6 . mean fsh and lh r e s p o n s e s t o lrh i n 6 women w i t h an a t t h e b e g i n n i n g of t h e p r o l o n g e d lrh t r e a t m e n t and a t f o l l i c u l a r m a t u r a t i o n d u r i n g t h e t r e a t m e n t . changes i n t h e p i t u i t a r y r e s p o n s i v e n e s s t o lrh d u r i n g p r o l o n g e d lrh t r e a t m e n t . the a l t e r a t i o n s i n t h e p i t u i t a r y g o n a d o t r o p h i n r e s p o n s e s t o lrh d u r i n g c h r o n i c a d m i n i s t r a t i o n of lrh a r e summarized i n f i g . 6 , which i s a c o m p o s i t e i l l u s t r a t i o n of fsh and lh l e v e l s a f t e r lrh a d m i n i s t r a t i o n a t t h e b e g i n n i n g o f and a f t e r 10-19 days of t r e a t m e n t w i t h 500 pg o f lrh e v e r y 8 h o u r s i n 6 o f t h e women w i t h an. during t h e f i r s t days of t r e a t m e n t , a t a mean serum e 2 l e v e l of 28 pg/ml, t h e fsh l e v e l a f t e r lrh was t w i c e a s h i g h a s t h e lh l e v e l , an i n v e r t e d p r e p u b e r t a l l i k e r e s p o n s e p a t t e r n . a f t e r 14 days o f t r e a t m e n t , on a v e r a g e , a t a mean e 2 l e v e l ( 3 4 5 pg/ml) c o n s i s t e n t w i t h f o l l i c u l a r m a t u r a t i o n , t h e s i t u a t i o n had changed d r a m a t i c a l l y . the mean lh c o n c e n t r a t i o n i n b l o o d a f t e r lrh had r i s e n t o a l e v e l s i m i l a r t o t h a t o b s e r v e d d u r i n g t h e m i d c y c l e peak i n t h e normal men s t r u a l c y c l e . the lh l e v e l a f t e r lrh was 9 t i m e s h i g h e r t h a n t h e fsh l e v e l , which had d e c r e a s e d c o n s i d e r a b l y a t t h a t t i m e . the fsh and lh r e s p o n s e s t o lrh b e f o r e , d u r i n g and a f t e r two c o n s e c u t i v e lrh hcg t r e a t m e n t s o f one o f t h e an p a t i e n t s ( f i g . 7) i l l u s t r a t e t h e changes i n t h e p i t u i t a r y r e s p o n s i v e n e s s t o lrh which t a k e p l a c e d u r i n g p r o l o n g e d t r e a t m e n t s w i t h lrh. b e f o r e t h e f i r s t t r e a t m e n t c o u r s e , t h e b a s a l lh l e v e l s were v e r y low b u t t h e r e was a normal r e s p o n s e t o lrh. the b a s a l fsh l e v e l was normal and t h e fsh r e s p o n s e t o lrh w a s 10 t i m e s g r e a t e r t h a n t h e a v e r a g e fsh r e s p o n s e in h e a l t h y women i n t h e e a r l y f o l l i c u l a r p h a s e of t h e m e n s t r u a l c y c l e . during t h e t h e r a p y w i t h 500 ug o f lrh e v e r y 8 h o u r s , t h e r e w a s a p r o g r e s s i v e d e c r e a s e of t h e g r e a t fsh r e s p o n s e . a t f o l l i c u l a r m a t u r a t i o n on t r e a t m e n t day 10, t h e r e was a s l i g h t l y i n c r e a s e d lh r e s p o n s e b u t no l o n g e r any fsh r e s p o n s e t o lrh. hcg w a s 28 hcg 6000iu i m i 4 2 o 0 0 . i ..0 1 , , , l1 &&&,",: 1 3 5 7 9 11 13 bleeding 2 0 t o -fig. 7 . b a s a l lh and fsh l e v e l s i n serum a s w e l l a s lh and fsh r e s p o n s e s t o lrh b e f o r e , d u r i n g and a f t e r two c o n s e c u t i v e lrh t z e a t m e n t s of a 28-year-old woman w i t h a n . t h e n g i v e n t o i n d u c e o v u l a t i o n . a s e c o n d hcg i n j e c t i o n was g i v e n one week l a t e r t o s u p p o r t c o r p u s l u t e u m f u n c t i o n . m e n s t r u a t i o n o c c u r r e d 15 days a f t e r t h e f i r s t hcg i n j e c t i o n . a f t e r t h e f i r s t t r e a t m e n t , on m e n s t r u a l day 3, t h e b a s a l fsh l e v e l s were below t h e d e t e c t i o n l i m i t o f t h e a s s a y and t h e r e was no fsh r e s p o n s e t o lrh. four days l a t e r , t h e r e was a v e r y s m a l l b u t s i g n i f i c a n t fsh r e l e a s e a f t e r lrh from unmeasurable b a s a l l e v e l s . during t h e f i r s t days of lrh t h e r a p y , t h e fsh r e s p o n s e t o lrh i n c r e a s e d markedly and r e a c h e d a h i g h maximum on t r e a t m e n t day 4 , when t h e e 2 l e v e l was s t i l l low. the fsh r e s p o n s e t h e n p r o g r e s s i v e l y de c r e a s e d . on t r e a t m e n t day 10 t h e r e w a s no l o n g e r any fsh r e s p o n s e t o lrh. the lh l e v e l s 90 min a f t e r t h e 500 ug d o s e o f lrh i n c r e a s e d d u r i n g t h e t r e a t m e n t and were maximal a f t e r 1 2 1 4 d a y s . however, t h e s e lh l e v e l s were a p p a r e n t l y n o t h i g h enough t o i n d u c e o v u l a t i o n a s no p r o g e s t e r o n e i n c r e a s e was o b s e r v e d a t t h a t t i m e . hcg was t h e r e f o r e g i v e n t o i n d u c e o v u l a t i o n and pregnancy o c c u r r e d i n t h i s i n f e r t i l e p a t i e n t w i t h 13 y e a r s ' o f amenorrhoea. one h e a l t h y c h i l d was d e l i v e r e d a t t e r m . only two ( p a t i e n t kl and bl) o f t h e o t h e r 8 women were i n v o l u n t a r i l y s t e r i l e . they were t r e a t e d by lrh a l o n e o r i n c o m b i n a t i o n w i t h hcg f o u r t i m e s ( f i g s . 2-5) b u t d i d n o t c o n c e i v e d u r i n g t h e t r e a t m e n t s . 29 discussion this study shows that the impaired gonadotrophin secretion in women with 'an can be restored to normal by long-term treatment with lrii. constant administra tion of the single gonadotrophin-releasing hormone not only normalized basal fsh and lh secretion but also induced a cyclical gonadotrophin secretory pattern with differential changes of the lh and fsh responses to lrh during the treat ment. lrh-induced gonadotrophin secretion initiated follicular growth and a normal ovarian cycle with follicular maturation and ovulation could be produced in amenorrhoeic women who were devoid of ovarian activity before the treatment. the results suggest that the impaired gonadotrophin secretion in amenorrhoeic women with an is due to a supra-pituitary disturbance with deficient secretion of endogenous gonadotrophin-releasing hormone from the hypothalamus. the lh secretion was reduced in all the 9 patients with an but the pituitary reserve capacity for lh secretion was normal in all but one woman. the pitui tary fsh secretion was unimpaired in most patients and the pituitary capacity to release fsh in response to lrh was, on average, four times greater than in healthy women in the early follicular phase of the menstrual cycle. the pre treatment fsh/lh ratio, both in the basal state and after stimulation with lrh, was therefore much greater than in healthy women of fertile age and similar to that described in prepubertal children (10, 23, 5). one of the an patients had neither any lh nor fsh response to lrh before the treatment but her pituitary responsiveness was restored to normal after repeated stimulation with lrh. thus, unresponsiveness in a diagnostic lrh test does not necessarily indicate primary pituitary failure but may be due to dysfunction at the hypothalamic level with insufficient hypothalamic stimulation of the pituitary gonadotrophs by endo genous gonadotrophin-releasing hormone. nor does a lack of gonadotrophin re sponse to acute lrh stimulation preclude successful results of long-term lrh therapy as shown by the induction of both follicular maturation and ovulation by lrh in the patient with no pretreatment gonadotrophin responses to lrh (fig. 4 ) . striking changes in the pituitary responsiveness to lrh were seen during the lrh treatments. the maximal fsh responses were obtained during the first days of treatment and at that time fsh levels which were greater than or equal to the lh levels after lrh were observed in most patients. however, the fsh re sponses rapidly decreased during the treatment and this decrease became more marked when the oestrogen secretion from the ovaries started to rise. the lh responses to lrh, on the other hand, progressivley increased during the treat ment and, in most patients,reached maximal midcycle-peak levels at high oestro gen levels consistent with follicular maturation. after that increased progest erone levels began to appear in the blood, suggesting that ovulation occurred. during the luteal phase of the cycle, the lh responses decreased somewhat but . ... 30 remained much g r e a t e r t h a n t h e fsh r e s p o n s e s t h r o u g h o u t t h e r e m a i n d e r of t h e c y c l e . thus, t h e i n v e r t e d p r e t r e a t m e n t g o n a d o t r o p h i n r e s p o n s e s t o lrh changed d u r i n g t h e p r o l o n g e d lrh t r e a t m e n t and became s i m i l a r t o t h o s e s e e n i n h e a l t h y a d u l t women. the changes i n t h e p i t u i t a r y r e s p o n s i v e n e s s t o lrh may b e due t o t h e f a c t t h a t lrh s t i m u l a t e s s y n t h e s i s and r e l e a s e of p r e d o m i n a n t l y lh and t h a t modulatory f e e d b a c k e f f e c t s of t h e o v a r i a n hormones, p a r t i c u l a r l y e 2, t h e n a c t a t t h e p i t u i t a r y l e v e l t o g e t h e r w i t h s e l f p r i m i n g e f f e c t s of lrh t o c a u s e a f u r t h e r marked i n c r e a s e of t h e lh r e s p o n s i v e n e s s t o lrh ( 2 5 , 1, 35). these a l t e r a t i o n s are v e r y s i m i l a r t o t h o s e s e e n i n t h e normal f e m a l e p a s s i n g from t h e p r e p u b e r t a l t o t h e p o s t p u b e r t a l s t a g e ( 1 0 , 5 ) . s i m i l a r e n d o c r i n e and p h y s i c a l changes of p u b e r t y h a v e b e e n s e e n d u r i n g p r o l o n g e d lrh t h e r a p y i n hypogonado t r o p i c men ( 1 5 ) . it c o u l d b e c o n s i d e r e d t o b e a n a l o g o u s t o t a k i n g t h e s e p a t i e n t s through p u b e r t y by t h e h i g h d o s e lrh t r e a t m e n t . f o l l i c u l a r growth and m a t u r a t i o n were i n d u c e d d u r i n g t h e lrh t r e a t m e n t and p r e s u m p t i v e e v i d e n c e of o v u l a t i o n , i . e . i n c r e a s e d p r o g e s t e r o n e s e c r e t i o n , w a s a l s o o b t a i n e d . however, t h e p r o g e s t e r o n e v a l u e s d u r i n g t h e p r e m e n s t r u a l p e r i o d were r a t h e r low i n 6 of t h e 8 c y c l e s where o n l y lrh w a s a d m i n i s t e r e d , s u g g e s t i n g i n s u f f i c i e n t c o r p u s l u t e u m f u n c t i o n . the l u t e a l p h a s e s w e r e of normal l e n g t h and t h e l u t e a l p h a s e d e f e c t s were t h e r e f o r e more s i m i l a r t o t h a t d e s c r i b e d a s t h e i n a d e q u a t e l u t e a l p h a s e by sherman & korenman ( 2 7 ) t h a n t o t h e s h o r t l u t e a l p h a s e d e f e c t d e s c r i b e d by s t r o t t e t a l . ( 2 9 ) . subnormal p r e o v u l a t o r y fsh l e v e l s h a v e b e e n found d u r i n g m e n s t r u a l c y c l e s w i t h l u t e a l p h a s e d e f e c t s ( 2 9 , 2 6 , 2 7 ) . s t r o t t and co-workers p o s t u l a t e d t h a t a r e l a t i v e fsh d e f i c i e n c y d u r i n g t h e f o l l i c u l a r p h a s e r e s u l t s i n abnormal f o l l i c u l a r development and s u b s e q u e n t i n a d e q u a t e c o r p u s l u t e u m f o r m a t i o n o r f u n c t i o n ( 2 9 ) . p a t i e n t s of t h e p r e s e n t s t u d y , who had i n v e r t e d p r e t r e a t m e n t r e s p o n s e p a t t e r n s w i t h g r e a t fsh releases a f t e r lrh, responded promptly t o t h e lrh t r e a t m e n t w i t h marked e 2 i n c r e a s e s c o n s i s t e n t w i t h f u l l f o l l i c u l a r m a t u r a t i o n and t h e y h a d normal c o r p u s l u t e u m f u n c t i o n , as j u d g e d by t h e p r o g e s t e r o n e c o n c e n t r a t i o n i n b l o o d ( e . g . p a t i e n t kl, t r e a t m e n t i , f i g . 2 ) . p a t i e n t s w i t h low fsh r e s p o n s e s i n c o m b i n a t i o n w i t h h i g h e r lh r e s p o n s e s b e f o r e t h e t r e a t m e n t r e s p o n d e d s l o w l y w i t h much l o w e r p r e o v u l a t o r y o e s t r o g e n i n c r e a s e s f o l l o w e d by i n a d e q u a t e l u t e a l p h a s e s ( e . g . p a t i e n t b l , t r e a t ment 11, f i g . 5 ) . the r e s u l t s s u g g e s t t h a t a p o s s i b l e e x p l a n a t i o n f o r t h e l u t e a l p h a s e d e f e c t s might be an a b s o l u t e o r r e l a t i v e fsh d e f i c i e n c y which l e a d s t o d e f e c t i v e f o l l i c u l a r m a t u r a t i o n and s u b s e q u e n t i n s u f f i c i e n t c o r p u s l u t e u m func t i o n . an a l t e r n a t i v e e x p l a n a t i o n f o r abnormal f o l l i c u l a r development might b e t h e r e l a t i v e l y h i g h lh l e v e l s i n d u c e d by lrh d u r i n g t h e f o l l i c u l a r p h a s e of t h e t r e a t m e n t c y c l e s . the lh/fsh r a t i o w a s s i m i l a r t o t h a t d e s c r i b e d i n women w i t h t h e p o l y c y s t i c o v a r y syndrome ( 1 4 , 3 4 ) where f o l l i c u l a r m a t u r a t i o n i s i m p a r i e d . the r a i s e d lh l e v e l s may s t i m u l a t e t h e o v a r i e s t o an i n c r e a s e d androgen s e c r e t i o n . 31 which inhibits follicular development. ross and co-workers reported that small doses of hcg or lh to oestrogen-treated hypophysectomized immature female rats resulted in decreased granulosa cell proliferation and increased follicular atresia and showed that this inhibitory effect was mediated by local intra ovarian effects of androgens, secreted by the ovary in response to hcg and lh (12, 13). in regularly menstruating women, elevation of lh activity in blood by hcg administration during the early follicular phase has been shown to cause luteinization of the theca interna with degeneration of tertiary follicles and delay or suppression of ovulation (30, 6). it can not be excluded that the lrh induced lh elevations during lrh stimulation of follicular growth and maturation may have had deleterious effects on the follicular development and subsequent corpus luteum function. thirdly, it might be that during treatment with only lrh the lh peak levels at follicular maturation were not high enough for sufficiently long periods for normal ovulation and corpus luteum formation to occur. to secure an adequate preovulatory lh surge, hcg was therefore administered during five additional treatment cycles after induction of follicular maturation by lrh. a l l these lrh hcg treatment cycles were ovulatory with normal luteal phases, as judged by the progesterone values. in two of the cycles (e.g. fig. 4), the e2 and progesterone patterns in blood suggested that ovulation had already occurred when hcg was given. during the postovulatory phase additional hcg injections were given to support corpus luteum function. in the cycles where only lrh was given, the luteal phase lh levels after lrh were presumably high enough for further support of the corpus luteum. however, it seems necessary to continue the lrh treatment throughout the luteal phase as we observed short luteal phases during treatment cycles where the lrh injections were interrupted during the postovulatory phase of the cycle (17). one may question whether ovulation really occurred during the treatment cycles with signs of luteal phase insufficiency. the increased progesterone levels in blood are only indirect indices of ovulation and may be caused by luteinization of granulosa cells of the follicles without ovulation. in summing up results of treatment with lrh, schally and co-workers concluded that although ovulation can be induced with lrh in sterile women the percentage of ovulations and pregnan cies is relatively low (24). this might possibly be explained by a high percent age of cycles with luteal phase insufficiency. only three of the nine women in the present study were involuntarily sterile. one of them became pregnant during her second lrh-hcg treatment (fig. 7 ) and by that she proved that normal folli cular maturation can be induced by treatment with lrh alone in women with im paired gonadotrophin secretion and absent pretreatment ovarian activity. for treatment of anovulatory infertility, it may be necessary to combine lrh with hcg or lh to secure normal ovulation and adequate corpus luteum function. 32 acknowledgements t h i s work w a s s u p p o r t e d by t h e s w e d i s h m e d i c a l r e s e a r c h c o u n c i l ( g r a n t no. 13x-3145). we a r e i n d e b t e d t o d r s . f. enzmann a n d m . v a n d e r ohe, f a r b w e r k e h o e c h s t ag, f r a n k f u r t / m a i n , frg, f o r g e n e r o u s s u p p l y o f s y n t h e t i c lrh and t o mrs. anna-lena barmark, mrs. b i r g i t t a bohman, m r . c h r i s t e r b e n g t s s o n , miss m a r g a r e t a h o f s t e d t , m r s . ann s a n d b e r g , miss k e r s t i n wall f o r s k i l f u l 1 t e c h n i c a l a s s i s t a n c e and t o t h e n u r s e s a n d o t h e r p e r s o n e l a t w a r d 37 o f t h e d e p a r t m e n t o f o b s t e t r i c s and g y n a e c o l o g y , u n i v e r s i t y h o s p i t a l , u p p s a l a , f o r t h e i r k i n d h e l p . 1. 2. 3. 4. 5 . 6 . 7. 8. 9. 10. 11. 1 2 . 13. 1 4 . 15. references a i y e r , m.s., c h i a p p a , s.a. & f i n k , g . : a p r i m i n g e f f e c t o f l u t e i n i z i n g h o r mone r e l e a s i n g f a c t o r on t h e a n t e r i o r p i t u i t a r y g l a n d i n t h e f e m a l e r a t . j e n d o c r i n o l 62:573-588, 1 9 7 4 . aono, t . , k i n u g a s a , t . , yamamoto, t . , miyake, a . & k u r a c h i , k . : a s s e s s m e n t o f g o n a d o t r o p h i n s e c r e t i o n i n women w i t h a n o r e x i a n e r v o s a . a c t a e n d o c r i n o l 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e r s o l l , f.m. & w o r c e s t e r , j . : the u r i n a r y e x c r e t i o n o f i n t e r s t i t i a l c e l l and f o l l i c l e s t i m u l a t i n g hormone a c t i v i t y b y women w i t h d i s e a s e s o f t h e r e p r o d u c t i v e s y s t e m . j c l i n e n d o c r i n o l metab 18:1202-1215, 1 9 5 8 . m o r t i m e r , c . h . , m c n e i l l y , a.s., f i s h e r , r . a . , murray, m.a.f. & besser, g . m . : g o n a d o t r o p h i n r e l e a s i n g hormone t h e r a p y i n h y p o g o n a d a l males w i t h h y p o t h a l a m i c o r p i t u i t a r y d y s f u n c t i o n . b r med j 4:617-621, 1 9 7 4 . 3-792854 33 16. nillius, s . j . & wide, l.: gonadotrophin-releasing hormone treatment for in duction of follicular maturation and ovulation in amenorrhoeic women with anorexia nervosa. br med j 3:405-408, 1975. 17. nillius, s . j . & wide, l.: luteal function during menstrual cycles induced by gonadotrophin-releasing hormone in women with amenorrhoea. v interna tional congress of endocrinology, hamburg, july 18-24, 1976, abstract 784, p. 323. 18. nillius, s . j . & wide, l.: the pituitary responsiveness to acute and chronic administration of gonadotropin-releasing hormone in acute and recovery stages of anorexia nervosa, in: anorexia nervosa (ed. r.a. vigersky), pp. 225-241. raven press, new york, 1977. 19. nillius, s . j . , fries, h. & wide, l.: successful induction of follicular maturation and ovulation by prolonged treatment with lh-releasing hormone in women with anorexia nervosa. am j obstet gynecol 122:921-928, 1975. 20. palmer, r.l., crisp, a.h., mackinnon, p.c.b., franklin, m., bonnar,j. & wheeler, m.: pituitary sensitivity to 50 ijg lh/fsh-rh in subjects with anorexia nervosa in acute and recovery stages. br med j i:179-182, 1975. 131, 1-93, 1968. hormone. isolation and characterization of four glycoproteins with luteini zing activity. biochim biophys acta 405:363-379, 1975. 23. roth, j.c., kelch, r.p., kaplan, s.l. & grumbach, m.m.: fsh and lh response to luteinizing hormone-releasing factor in prepubertal and pubertal children, adult males and patients with hypogonadotropic and hypergonadotropic hypo gonadism. j clin endocrinol metab 35:926-930, 1972. 24. schally, a.v., kastin, a.j. & arimura, a.: the hypothalamus and reproduction. am j obstet gynecol 122:857-862, 1975. 25. schally, a.v., kastin, a.j. & coy, d.h.: lh-releasing hormone and its ana logues: recent basic and clinical investigations. int j fertil 21:l-30, 1976. 26. sherman, b.m. & korenman, s.g.: measurement of plasma lh, fsh, estradiol and progesterone in disorders of the human menstrual cycle: the short luteal phase. j clin endocrinol metab 38:89-93, 1974. 27. sherman, b.m. & korenman, s.g.: measurement of serum lh, fsh, estradiol and progesterone in disorders of the human menstrual cycle: the inadequate luteal phase. j clin endocrinol metab 39:145-149, 1974. tropin-releasing hormone in anorexia nervosa: effect of nutritional rehabi litation. j clin endocrinol metab 41:135-142, 1975. 29. strott, c.a., cargille, c.m., ross, g.t. & lipsett, m.b.: the short luteal phase. j clin endocrinol 30:246-251, 1970. 30. tamada, t. & matsumoto, s.: suppression of ovulation with human chorionic gonadotropin. fertil steril 20:840-848, 1969. 31. thorneycroft, i.h. & stone, s.c.: radioimunoassay of serum progesterone in women receiving oral contraceptive steroids. contraception 5:129-146, 1972. 32. warren, m.p., jewelewicz, r., dyrenfurth, i., ans, r., khalaf, s. & vande wiele, r.l.: the significance of weight loss in the evaluation of pituitary response to lh-rh in women with secondary amenorrhea. j clin endocrinol metab 40:601-611, 1975. follicle-stimulating horrnone and luteinizinghormone in serum and urine from men and women. acta endocrinol (suppl) 174, 1-58, 1973 stimulating hormone and luteinizing hormone in polycystic ovarian disease. j clin endocrinol 30:435-442, 197u. operating characteristics of the hypothalamic-pituitary system during the menstrual cycle and observations of biological action of somatostatin. recent prog horm res 31:321-363, 1975. 21. roos, p.: human follicle-stimulating hormone. acta endocrinol (suppl) (kbh) 22. roos, p., nyberg, l., wide, l. & gemzell, c.: human pituitary luteinizing 28. sherman, b.m., halmi, k.a. & zamudio, r.: lh and fsh response to gonado 33. wide, l., nillius, s . j . , gemzell, c. & ross, p.: radioimunosorbent assay of 34. yen, s.s.c., vela, p. & rankin, j.: inappropriate secretion of follicle 35. yen, s.s.c., lasley, b.l., wang, c.f., leblanc, h. & siler, t.m.: the 34 36. yoshimoto, y., moridera, k. imura, h.: restoration of normal pituitary gonadotropin reserve by administration of luteinizing-hormone-releasing hormone in patients with hypogonadotropic hypogonadism. new engl j med 2 9 2 : 2 4 2 2 4 5 , 1 9 7 5 . accepted january 1 9 , 1 9 7 9 . address for reprints: sven johan nillius, m.d. department of obstetrics and gynaecology university hospital s-750 14 uppsala sweden 35 upsala j med sci 82: 27-30, 1977 on the reproducibility of exercise tests in patients with atrial fibrillation hans &erg, gunnar strom and war werner from the departments of internal medicine and clinical physiology, university hospital, uppsala, sweden abstract fifteen patients with atrial fibrillation, mostly due to advanced valvular heart disease, were examined by a graded work test and an orthostatic test on two occasions, the reason being, to study the reproducibility of the test in such patients. in 6 of the 7 patients with the highest working capacity the agreement between the two tests was good regarding heart rates at maximum work load. in the 8 patients with a low working capacity the results of the two tests differed widely. in the orthostatic test the difference in increase of the heart rate after standing was less than 5 beatslmin in 3 patients in one of the tests, and in 2 patients in both tests. the exercise test in patients with atrial fibrilla tion must be evaluated with caution and has an acceptable reproducibility only in patients with fairly good functional capacity. introduction we have previously reported on exercise tests in patients with atrial fibrillation (2, 3). the average increase in ventricular rates at gradually increased exercise loads was often found to be close to a linear repationship. in particular, this was the case in patients with a relatively well maintained functional capacity of the heart. how ever, in patients with more severely impaired heart function there was a tendency of a slightly more pronounced increase in the heart rate from resting value to especially that of the first load. in the literature different results have been presented on this problem (4, 7, 12). in another study we have investigated the influ ence of different dose rates of digitalis on the heart rate and on the working capacity in patients with atrial fibrillation during exercise tests (3). by in creasing the digitalis dosage the heart rate at the same load decreased. the aim of the present study was to test the reproducibility of orthostatic and exercise tests in patients with atrial fibrillation. in such an investiga tion it is of critical importance that the cardiac state be stable and unchanged at, and in the interval between the two exercise tests. material and methods in 15 patients with atrial fibrillation exercise tests were done twice in each patient. the composition of the patient group is shown in table i. the group is dominated by patients with valvular heart disease, often referred to this hospital for consideration for cardiac surgery. the exer cise test was a part of this evaluation. most patients had fairly advanced valvular disease. the repeated test was only performed with the patient’s consent since this test was made mainly for scientific reasons. this second exercise test was performed only in those patients fulfilling certain criteria to ensure a clinically sta ble condition of the patient between the time of the two tests. these criteria were: (a) no change had occurred in clinical findings o r in the patient’s history of his capacity between the two tests. the patient was not in heart failure. (b) the time interval between the two tests had not exceeded 3 months and i t had frequently been much less. seven of the cases had the tests less than 2 weeks apart. (c) the resting ecgs were identical on both occasions. in 3 patients with more than 1 month between the tests chest x-rays were performed before the two exercise tests and were found to be identical. (d) the patient had the same drugs in identical dosages on the two occasions. serum potassium and other electro lytes were within normal limits at both tests. (e) n o other disease had occurred during the interval between the two tests. the orthostatic test preceding the exercise test was performed and described in more detail by sandberg (9). according to the recurrent nomenclature the test was considered ‘negative’ when the heart rate increased less than 20 beatslmin and ‘positive’ if between 20 and 29. the exercise test was performed according to the graded ‘steady state’ principle (10, 13). the exercise test was performed using a bicycle ergometer (5, 6) with the method and calculations having previously been described (2). the heart rate was determined a t rest, after 8 min uosciki j m i 4 sci 82 28 h . aberg et al. table i. composition of the case material diagnosis no. of patients age heart volume cc/m2 bsa male female total mean range mean range mitral stenosis 4 3 7 42 28-57 745 510-1 060 (650) disease 1 1 2 (49) valve disease 2 3 5 48 39-62 730 590-1 010 unknown 1 1 (36) (550) combined mitral mitral +aortic total 8 7 15 44.8 28-62 714 510-1 060 standing, and after 2, 4 and 6 min a t each load as the average of 25 consecutive heart intervals. the test was supervised by a physician at all times. the results of the exercise test was expressed in differ ent ways: as the work load at a heart rate of ll0/min (w,,,), as the highest work load which according to clini cal judgment of the patient’s symptoms and signs was permissible and possible to perform for 6 min (w,,,), and as the ability to reach ‘steady state’ as defined below. w,,, was calculated by slight interpolation or extrapolation, assuming a linear relationship between heart rate and work load within the small interval in question. in patients with atrial fibrillation and very low working capacity this is less precise (2). a ‘steady state’ was judged to be obtained when the difference between the heart rates a t 2, 4 and 6 min on the same load was 10 beats/min or less. results and comments the resting heart rates were fairly constant on the two occasions with few exceptions. in 13 of is pa tients the difference in resting heart rates were 10 beats/min or less and in 6 of 15 only 5 beatslmin or less. the resting values (the lowest of the 2) were below 70 beats/min in 9 patients and above 90 (the highest of the 2) in 3 patients. these rates were rather high, considering that all patients except one was on maintenance dose digitalis. with regard to the orthostatic test there was a difference between the two tests in 3 patients (from ‘positive’ t o ‘negative’ or vice versa). only in one was the difference greater. this patient had on the first occasion an increase of the heart rate of 26 beatslmin after 8 min standing (‘positive’ reaction) and on the second test an increase of 6 beatslmin (‘negative’ reaction). the other 2 had 18 and 16 on the first and 23 and 25 on the second test, respec tively. in s cases the difference was 5 beatslmin or less between the two tests of orthostatic reaction. the working capacity, measured as wllo, was generally low, with a few exceptions (table ii). the mean w,,, was 168 (range 18439) and 191 (range upsulu j med sci 82 23497) kpm/min at the two exercise tests. this was expected, as many patients had fairly advanced heart disease. in 8 patients with a w,,, less than 100 kpm/min in one or both of the exercise tests there were 4 patients whose difference between the two tests did not exceed 1/3 of the lowest value. in one the difference was about 40%. in the remaining 3 in this patient group with low working capacity the difference was as much as double or more the low w,,, in each pair. in the group with a working capacity w,,, above 110 kpmlmin, consisting of 7 patients, the agreement between the two exercise tests was slightly better. in 3 patients the results table 11. results of exercise tests a is the first exercise test and b the second one. defini tions are given in methods patients w,,, w,,, (kpm/min) (kpmlmin) a b a b 1 . a . m . 2. r . b . 3 . v . r . 4 . k . b . 5 . a . k . 6. v. p. 7. m. s. 8. n . e . 9. g . h . 10. e. 0. 1 1 . s. k. 12. e . w . 13. e. 0. 14. g. s . 15. s . h . mean s.d. 18 23 50 36 177 169 88 1 i6 180 82 23 30 308 391 167 86 206 248 75 260 3 i7 497 i92 139 37 47 239 376 439 367 168 191 123 155 150 1 50 200 200 200 250 250 300 400 400 400“ 450 500 500 700 337 158 1 50 1 50 200 200 200 250 250 300 400 400 600 500 500 500 700 3.53 175 a the patient showed aberrant ventricular ecg complexes. the first test was then interrupted. in spite of the same occurrence of aberration at the second test the exercise was allowed to continue. reproducibility of exercise in atrial fibrillation 29 table 111. heart rate resting, standing and during exercise a is the first exercise test. b-a is the difference between the second and the first test. w , is the first load and wz is the second load. patients 1-8 have the lowest w,,, and patients 9-15 have the highest w,,,. definitions in methods patients 1-8 patients 9-15 total mean s.d. mean s . d . mean s.d. resting a b -a a b-a a b-a a b-a w,,, a b-a standing w , w* 75.0 2.1 10.4 -1.6 104.8 4.9 125. i 5.8 129.4 10.3 15.3 4.0 9.9 6.7 19.0 10.9 25.4 12.0 22.1 11.6 77.4 -7.3 17.3 -0.2 107.6 -6.8 134.0 -5.4 154.2 -4.3 9.8 6.7 5.6 3.9 23.1 8.4 26.1 9.6 18.1 9.6 76.1 -2.2 13.6 -0.9 105.8 0.4 128.5 1.5 137.5 3.1 12.6 4.3 8.7 5.4 19.8 10.0 25 .o 11.0 22.4 11.3 were almost identical, 2 had a difference of about 1/3 and the remaining 2 had about 50% increase from the lowest value in the compared pair. w,,, values were identical in 13 of the 15 pa tients. in one patient, on the second occasion, the maximal load was chosen as 500, due to a mistake, instead of as planned 450 kpmlmin, while at lower loads that were the same there was a good agree ment between the two tests. in another patient, there were similar heart rates at 200 and 400 kpmlmin but on the first exercise test the in vestigator finished the test due to aberrant veptricu lar ecg-complexes. the second test was contiwed in spite of a similar aberration. in 3 patients with a w,,, of 500 kpmlmin and more the highest attained heart rates were almost identical at the two tests, 150 and 146 in one, 171 and 171 in another and 127 and 123 in the third, respectively. in 2 patients who had a w,,, of 400, one had heart rates of 141 and 134 but the other patient had 163 and 137. out of the 8 patients with a low w,,,, 2 had shown a good agreement between the heart rates at the two tests. in table i11 the dif ferences between the patients with a low and those with a higher w,,, are seen. it is apparent from the table that the s.d. of the differences between the two exercise tests is smaller in the group with a better w,,,. the only exception is at rest where the s.d. was higher in the group with a higher w,,,. a ‘steady state’ was achieved on both occasions in only 2 patients. in 9 patients a ‘steady state’ condition at the highest load was obtained during one exercise test but not the other. six of these patients obtained ‘steady state’ conditions on the second exercise test. discussion in many patients the case history is sufficient for a clinically appropriate judgment of the working capacity. however, i t is sometimes difficult to get a reliable description from the patient and also i t is often necessary to obtain a more objective and pre cise measurement on the functional capacity. this is for example necessary to evaluate results after medication, surgery, or in an attempt to find an optimal time for surgical intervention etc. the need of a reliable method in this respect is obvious. such a method is of course to a critical degree dependent upon a good reproducibility of the pro cedure. the ideal method should be well standardized, easy to repeat and the results should not be influenced by the training effect of repetition. the method used in the present study probably fulfils these requirements. a training effect of any significance is not probable considering the time lapse between the two tests and the fact that the test was repeated only once. a most important factor with regard to the purpose of this study, i.e. to evaluate the repro 30 h . aberg et al. ducibility of the exercise test is the stability of the patients with regard to their cardiac state. most patients had got their atrial fibrillation in the course of a progressive heart disease and it was difficult to ascertain that the patients were stable enough for the purpose of this investigation. i n spite of a careful selection of patients, the results of the exercise tests in the patients with a low working capacity indicate either that such stable conditions were not obtained or that the ar rhythmia was highly variable in itself. in the group of patients with a better heart function there was a relatively good reproducibility of the orthostatic test reaction as well as the exercise test. the ventricular heart rate in atrial fibrillation is dependent upon the conditions of the atrioventricu lar junction (7, 1 i ) . it has been shown that the rate of the atrial activity is not changed during exercise in patients with atrial fibrillation (1). in spite of the irregular atrial activity the atrial rate is so rapid that there is always an impulse on hand for propagation through the atrioventricular junction. therefore, the ventricular response in atrial fibrillation should be dependent upon the function of the atrioventricu lar node. thus, we must consider influences from pharmacologic agents and the autonomous nervous system as well as from the heart disease in itself. i n a previous paper we have shown that a rather high digitalis dosage is favourable for patients with atrial fibrillation (3). this has also been studied by red fors (8). in this study, however, there was no difference in drug therapy or electrolyte balance between the two tests. the alteration in atrioventricular function, particularly in those individuals with low working capacity, should therefore be caused either by vari ation in autonomic tone or by changes in the under lying heart disease. in conclusion, the reproducibility of exercise tests was acceptable in patients with a good work ing capacity. on the other hand, in the group with more severely impaired heart function, the repro ducibility was poor in spite of digitalization. references 1. aberg, h . & furberg, b.: atrial activity during exer cise in patients with atrial flutter or atrial fibrillation. ups j med sci80: 20, 1975. 2. aberg, h . , strom, g. & w e r n e r , i.: heart rate during exercise in patients with atrial fibrillation. acta med scand 191; 3 15, 1972. 3 . 4. 5. 6. 7. 8. the effect of digitalis on the heart rate during exercise in patients with atrial fibrillation. acta med scand 191:441, 1972. holmgren, a , , jonsson, b . , linderholm, h . , sjostrand, t. & strom, g . : physical working capacity in cases of mitral valvular disease in relation to heart volume, total amount of hemoglobin and stroke vol ume. acta med scand 167: 99, 1958. holmgren, a. & mattsson, k . h . : a new ergometer with constant load at varying pedalling rate. scand j clin lab invest6: 137, 1954. holmgren, a . & strandell, t.: on the use of chesthead leads for recording of electrocardiogram during exercise. acta med scand 169: 57, 1961. know, j. a . c.: the heart rate with exercise in pa tients with auricular fibrillation. br heart j ll: 119, i 949. redfors, a , : digoxinbehandling vid formaksflirnmer. relationen mellan dos, plasmakoncentration och ef fekt. akademisk avhandline., lund 1971. i 9. sandberg, l.: studies on electrocardiographic changes during exercise tests. acta med scand [suppl.] 36.5, 1961. 10. sjostrand, t.: changes in the respiratory organs of workmen at an ore smelting works. acta med scand [suppl.] 196: 687, 1947. 1 1 . soderstrom, n.: what is the reason for the ventricular arrhythmia in cases of auricular fibrillation? am heart j40:212, 1950. 12. varnauskas, e., cramkr, g . , malmcrona, r., dahl, l.-e., nystrom, b., wassen, a. & werko, l.: res toration of normal sinus rhythm in patients with mitral-valve disease and atrial fibrillation. nord med 62: 1109, 1959. 13. wahlund, h.: determination of the physical working capacity. acta med scand [suppl.]215, 1948. received september 2, 1976 address for reprints: h a n s aberg, m.d. department of internal medicine university hospital s-750 14 uppsala 14 sweden upsula .i m e d sci 82 upsala j med sci 8 1 : 85-92, 1976 dietary and drug treatment of hyperlipidaemia a feasibility study in asymptomatic middle-aged men hans hedstrand from the departments of internal medicine and geriatrics, university hospital, uppsala, sweden abstract 83 middle-aged men with different types of hyperlipopro kinaemia were recruited from a health examination survey. they were treated with diet for 3 months and with diet and drugs in combination over a 2-year period (63 men used drugs). the serum lipid reductions after the dietary period were 14 % and 27 % for serum cholesterol and triglycerides, respectively. after 2 years the corresponding reductions were 21 % and 42 %, indicating an additional effect of diet and drugs. there was an average body weight reduction of 4.3% during the first 3 months which was maintained over the 2-year period. special considerations in treating asymptomatic individuals are discussed. prospective epidemiological studies have shown an association between elevated levels of cholesterol (8, 20) and triglycerides (tg) in serum ( 2 ) and de velopment of coronary heart disease (chd). significant positive linear correlations between serum cholesterol and/or tg concentrations and risk of chd have been demonstrated. during the last 20 years several feasibility studies (5, 11, 13) as well as primary preventive trials have been performed aiming at lowering serum choles terol by dietary regimen (4, 12, 17, 19) or drug ther apy (15, 16). the results as t o the lipid lowering ef fects have vaned in different populations (14). the aim of the present study was to investigate the effect of dietary and drug treatment on serum lipid levels in a group of asymptomatic middle-aged men with hyperlipidaemia. material all men, born 1920-1924, who lived in the city of uppsala were invited to a health examination survey with the main purpose of identifying and correcting risk factors for car diovascular disease (6). the investigation was conducted at the department of medicine, university hospital. the participation rate was 83.9 %. a total number of 174 asymptomatic men with “pri mary” hyperlipidaemia, defined as mean value of two samples of serum cholesterol and/or tg above the 80th percentile of the examined population, were invited to a special lipid clinic. one subject did not come t o the lipid clinic. five men came to the first visit but did not come for the complete serum lipoprotein (lp) analysis. the result of the classification of the l p pattern in the remaining 168 subjects is shown in table i. all 168 subjects were offered treatment after the l p analysis. those with a normal l p pattern according to the limits used were given dietary prescriptions only and their serum lipids were reexamined after one year. this group comprised 30 men. all subjects with hyperlipoproteinae mia (hlp) were initially treated with a dietary regimen for 3 months, after which they were offered additional therapy with lipid-lowering drugs. the results presented here concern the effect of the therapy on serum lipid concentrations in the group of 83 men with hlp repre senting all men who a t the follow-up had participated for a t least 24 months after initiation of therapy. methods at the first visit to the lipid clinic the subjects were intro duced to the programme and its long-term aspects were explained. the wives were invited together with the men to the second visit a t the lipid clinic. results of the l p determinations were then presented and a dietary informa tion was given by a dietitian, often to a group of three or four couples together. during the following three months of dietary treatment serum lipid analyses were performed at monthly intervals. the subjects were seen 2 weeks after each determination when they were informed of the result and had the possi bility to see the dietitian if they desired. diet prescriptions. the subjects were advised to avoid food high in ( a ) saturated fatty acids, e.g. fat meat and pork, dairy products such as butter, cream, cheese and whole milk and hard margarine. ( b ) cholesterol, e.g. egg yolks and liver. (c) simple sugars (monoand disaccharides) e.g. refined sugar, sweets, sweet desserts, sweet beverages and sweet cakes (hlp type i1 b and iv). the subjects were advised to use foods containing ( a ) little or no saturated fatty acids, e.g. skimmed milk, lean meat, low-fat cheese. upsala j m e d s c i 8 1 86 h . hedstrand table i . lipoprotein patterns of 168 asymptomatic middle-aged m e n with serum lipids above the 80th percentile of the same population lipoprotein no. of subjects pattern subjects (%i normal i1 a i1 b 111 iv v total 37 22.0 38 22.6 25 14.9 15 8.9 51 30.4 2 1.2 168 100.0 ( b ) polyunsaturated fatty acids, e.g. vegetable oils (with more than 5 0 % linoleic acid), liquid margarine (containing more than 6 0 % of linoleic acid), soft margarine (contain ing 2 5 4 5 % linoleic acid). the composition of the recommended diet should result in a p/s ratio of 2, total fat 30-35 % of the total calories and less than 300 mg cholesterol daily. the importance of using at least 50 ml of oil or liquid margarine daily was stressed. it was recommended to be used for frying, in dressings and in sauces. the subjects were advised that alcohol consumption should not exceed one bottle of beer a day. the daily caloric allowance was specified only when the subject had an actual over ideal weight > 1.20. drug prescriprions. the subjects were offered drugs after the diet period, i.e. after 3 months. the drugs used were clofibrate (atromidin@, ici-pharma, goteborg, sweden) and nicotinic acid (nicangin@, draco, lund, sweden). the men were informed about the most com mon side effects of the drugs. clofibrate was given in a dose of i g twice daily, as a rule in the morning and in the evening and not necessarily after meals. treatment with nicotinic acid was started with 0.25 g three times daily after meals. the dosages were increased by further 0.25 g three times daily every fourth day until a total dose of 3 g daily was reached. the intention was to use clofibrate as the first drug in h l p type iv and nicotinic acid in h l p type i1 a and i1 b. laborafory methods. at the screening examination the blood samples were drawn after an overnight fast. at the lipid clinic the blood samples were taken at the same time but the subjects were recommended to fast from 8 p.m. the evening before. cholesterol and tg analyses were performed by semiautomatic techniques in a technicon auto-analyzer type i1 (18). the separation and quantita tive determination of l p density fractions by preparative ultracentrifugation as well as the analyses on agarose elec trophoresis have been described in detail elsewhere (7). the l p patterns were classified according t o fredrick son et al. using the recommendations by beaumont e t al. (1). the upper “normal” limits used, based on the 85th percentile of a local material of healthy men of different ages, were 200 mg/loo ml for low density lipoprotein ratio polyunsaturated fatty acids to saturated fatty acids. (ldl) cholesterol and 1.40 mmol/l for very low density lipoprotein (vldl) tg (3). these limits were used by the lipid clinic and were close to those obtained in a sample of healthy men from the present population study (7). sfatistical calculafions. conventional methods were used for calculation of mean value and standard deviation (s.d.). significances of differences between mean values were estimated with student’s two-tailed t-test. when test ing the means of serum tg concentrations the logarithm transformed values were used because the distribution of tg was skewed to the right (6). the differences between two values in the same subjects were estimated with the paired observation test using the hewlett-packard 9100 calculator programme. the accepted level of significance was p<0.05. results subjects with normal lp pattern the screening values (mean of two samples) of serum cholesterol and tg for 30 subjects with nor mal lp pattern at the lipid clinic were 299k33 mg1100 ml and 2.50k0.79 mmol/l, respectively. the average reduction between screening and the first analysis at the lipid clinic was 47 mg/100 ml(l4.9%) for serum cholesterol and 0.57 mmol/l (18.8%) for serum tg although no lipid lowering regimen had been prescribed. table 11. serum cholesterol concentrations ( m e a n m . e . m . ) in 83 asymptomatic middle-aged men with different types of hyperlipoproteinaemia ( h l p ) at initial examination and after 3 months of diet therapy and after 24 months of diet and drug therapy (drugs were used in 63 subjects) months 0 3 24 h l p type i1 a ( n =20) mean value, mg/loo ml 3372 10 308f 11 2 6 0 5 9 mean change, mg/100 ml 2 9 f 2 . 3 7 5 f 2 . 5 mean change, % 8 . 6 f 3 . 0 2 1 . 3 f 3 . 0 h l p t y p e i i b (n=17) mean value, mgll00 ml 3 2 1 f 8 2 7 7 2 7 2 4 4 2 8 mean change, mg/100 ml 4 4 f 2 . 1 7 7 f 2 . 4 mean change, % 1 3 . 2 f 2 . 4 23.5f2.8 h l p type iii ( n = l o ) mean value, mgll00 ml 3032 19 242k 17 2 2 9 f 9 mean change, mg/100 ml 6 2 k 5 . 9 7 5 f 4 . 1 mean change, % 1 9 . i f 5 . 6 2 3 . 4 f 2 . 8 h l p f y p e iv (n =36) mean value, mg/loo ml 2 7 8 f 6 2 3 5 f 7 2 2 1 f 6 mean change, mg/loo ml 4 3 f 0 . 9 56k1.1 mean change, % 1 5 . 4 f 1 . 8 1 9 . 4 f 2 . 2 upsala j m e d sci 81 dietary and drug treatment of hyperlipidaemia 87 table 111. serum triglyceride concentrations ( m e a n 5 s . e . m . ) in 83 asymptomatic middle-aged m e n with different types of hyperlipoproteinaemia ( h l p ) at initial examination and after 3 months of diet therapy and after 24 months of diet and drug therapy (drugs were used in 63 subjects) months 0 3 24 h l p type i1 a (n =20) mean value, mmol/l mean value, (log) mean change, mmol/l mean change, % h l p t y p e i i b ( n = 1 7 ) mean value, mmol/l mean value, (log) mean change, mrnol/l mean change, % hlp type i l l ( n = i o ) mean value, mmol/l mean value, (log) mean change, mmol/l mean change, % h l p type iv ( n =36) mean value, mmol/l mean value, (log) mean change, mmol/l mean change, % 2.37 0.368k0.018 3 .oo 0.471f0.019 4.04 0.593k0.036 3.36 0.513f0.018 2.12 0.316f0.021 0.26k 0.03 9.7f4.7 2.18 0.326f0.025 0.82f 0.03 26.9f3.4 2.40 0.347f0.058 1.64k0.09 40.1f6.0 2. i9 0.316f0.024 1.18f0.02 33.7f 3.4 1.47 0.160f0.019 0.90f0.02 36.5k3.1 1.63 0.195f0.030 1.37f0.04 44.4k4.6 1.65 0.21 lf0.025 2.3950.12 56.4f4.5 1.85 0.246f 0.023 1.5 1 f 0 . 0 3 42.4f3.2 at the lipid clinic the subjects and their wives were given simple dietary recommendations by the physi cian only. when these men were reexamined one year later they still showed significantly lower serum cholesterol and t g concentrations than at the initial screening. the average reductions were 31 mg/loo ml(9.9 %) for cholesterol @<0.001) and 0.50 mmol/l (17.2%) for tg @<0.01) compared to the screening levels. after one year 90 % of the subjects had lower serum cholesterol and 77 % had lower tg than ini tially. the mean body weight of the group was unchanged between the two visits at the lipid clinic one year apart. subjects with hyperlipoproteinaemia effect of dietary treatment. after 3 months of dietary treatment the serum cholesterol concentration was 262 mg/100 ml and the serum tg concentration was 2.19 mmol/l in the whole group of 83 men included in the 2-year study. these values corresponded t o a reduction of 13.7 % a n d 27.2 %for serum cholesterol and tg, respectively. the degree of serum lipid changes was depending on the type of serum lp pattern the patient had before the treatment started (tables 11-111, figs. 1-2). the greatest reductions, both in serum cholesterol and tg, were obtained in type 111 and the lowest in type i1 a . the changes of individual serum lipid values are presented in figs. 4-7. a few subjects with type i1 a had a higher serum cholesterol value after three months of dietary therapy compared with the initial values. ten men with type i1 a had unchanged or higher serum tg values. among patients with type i1 b pattern only one had an increased serum choles terol value and none had higher serum tg after three months. two men with type 111 had slightly higher serum cholesterol values while all serum tg values were considerably reduced during the dietary period. all but 2 individuals with type iv had lower serum cholesterol and tg values after the three months of dietary treatment. the average body weight reduction was 4.3 % in the whole group during the first 3 months. the weight was reduced in all types of hlp (table iv, fig. 3). the reduction was greatest in subjects with type i1 b, 4.6 kg, and lowest in those with type i1 a, i .7 kg. e f f e c t of combined lipid lowering therapy. sixty upsala j med sci 81 88 0 -20 -lo -60 h . hedstrand 3 12 2lmonths ‘10 change fig. 1 . effect of lipid lowering therapy on serum cholesterol over a 2-year period in 83 asymptomatic middle-aged men with type i1 a hyperlipoproteinaemia (n=20, 0-0), type i1 b (n=17, 0-o), type i11 (n=lo, a-a), and type iv (n=36, a-a). (mean fs.e.m.) three of the 83 men (75.9%) who were followed over the 2-year period were treated with lipid-lowering agents. in 44 men (69.8%) drug therapy was started immediately after the dietary period, i.e. after 3 months. in 8 subjects drugs were introduced at 6 months and in another 8 men between 6 and 12 months. only 3 men were given medication later than one year after the hypolipidaemia treatment was started. table iv. body weight (mean 3 . e . m . ) in 83 asymptomatic middle-aged men with different types of hyperlipoproteinaemia (hlp) a t initial examina tion and after 3 months of diet therapy and after 24 months of diet and drug therapy (drugs were used in 63 subjects) months 0 3 24 hlp t y p e i1 a ( n =20) mean value, kg mean change, kg mean change, % hlp type i1 b (n=17) mean value, kg mean change, kg mean change, % hlp t y p e i l l ( n = i o ) mean value, kg mean change, kg mean change, % hlp type iv (n=36) mean value, kg mean change, kg mean change, % ~ 73.25 2.1 79.9f 3 . 3 82.6k3.5 81.3k 1.5 ~~ 71.5k2.0 1.7$0.1 2 . 2 f 0 . 5 75.3k 3.0 4 . 6 f 0 . 2 5 . 6 f 0 . 7 78.7f 3 . 3 3.9k0.2 4 . 7 f 0 . 7 77.7k 1.4 3.8f0.1 4 . 7 f 0 . 5 ~~ 70.8f 1.9 2.4k0.2 3.1k0.9 74.6k2.8 5.4k 0.3 6.4k 1.3 78.5k2.8 4.1k0.4 4.7k 1.4 76.2k 1.4 5 . l k 0 . 1 6.2k0.7 3 12 24 months 0 -10 -20 i i -30 %change f i g . 2. effect of lipid lowering therapy on serum triglyce rides over a 2-year period in 83 asymptomatic middle-aged men with hyperlipoproteinaemia. symbols as in fig. 1 . nicotinic acid was given to 36 subjects. their initial hlp types were type i1 25 cases, type 111 2 cases and type iv 9 cases. clofibrate was given to 27 men, 7 with type i i , 4 with type i11 and 16 with type iv. in type i1 a and i1 b 32 men (86.5 %) used drugs. the percentage was lower, 69.4 %, in type iv where 25 subjects were given drug therapy. there were 9 men who declined drug therapy and 11 men were not offered drug therapy because the serum lipid concentrations on dietary treatment were already down to levels considered satisfactory. the mean serum lipid values after 3 months of dietary treatment in these 11 subjects were 197 mg/loo ml and 1.66 mmol/l for cholesterol and tg, respectively. the lp pattern in 3 of these men was classified as type i11 and as type iv in 5. after 2 years the average serum cholesterol and tg concentrations were 236 mg/100 ml and 1.68 mmolll respectively in the whole group of 83 men. these reductions corresponded to 2 1.2 % and 43.1 %. the serum lipid reductions in the 63 men 3 12 24 months i 1 -10 %change fig. 3. reduction of body weight in 83 asymptomatic middle-aged men with hyperlipoproteinaemia during 2 years of lipid lowering therapy. symbols as in fig. 1 . upsala j med sci 81 dietary and drug treatment of hyperlipidaemia 89 who were on diet and drug therapy in combination were 22.9 % and 45.2 % for serum cholesterol and tg, respectively. the serum lipid changes in the four groups with different initial lp patterns are shown in tables 11-111 and figs. 1-2. the serum cholesterol reduc tion was similar, around 75 mg/loo ml, in all h l p types except for type iv. in all types the reduction was around 20% after 2 years. the greatest reduc tion of serum tg was obtained in type 111, 2.39 mmol/l, which corresponded to 56.4 %. the individual changes of serum lipid concentra tions after 2 years are shown in figs. 4-7. in type i1 a (fig. 4) 5 patients, all treated with drugs, had serum cholesterol values above the limit used as cut-off point (291 mg/100 ml) at the screening. the serum tg values were below the cut-off point (2.64 rnmol/l) in all patients. in type i1 b two individuals, both on drug therapy, had serum cholesterol values above the cut-off limit while the serum tg values were below the screening limit in all patients (fig. 5). in a few subjects the serum lipids were higher than after the dietary period. all the subjects with type 111 had serum lipid values below the cut-off limits (fig. 6). the serum cholesterol values of all men with type iv were below the cut-off values (fig. 7). however, there were 14 men with higher values after 2 years than they had after the dietary period. most of them had initially low serum cholesterol concentrations. nine of them were without drug therapy. in 11 subjects the serum tg had increased after the first 3 months. three individuals had values above the screening limit. the serum lipid values of the 11 men without drug therapy were 210+31 mg/loo ml and 1.60k0.44 mmol/l for serum cholesterol and serum tg, respec tively, corresponding to a reduction of 20.8% and 46.8 %. the 9 men who declined advice of drug therapy had significantly higher serum tg values after 2 years than the remaining subjects. the values were 2.20 mmol/l and 1.62 mmol/l, respectively ( p < o . o o i ) . the serum cholesterol values were not significantly different. the average body weight reduction in the total group after 2 years was 4.4 kg corresponding to 5.3 %. the reductions in the different types of h l p are shown in table iv and fig. 3 . the initial reduc tions after 3 months were maintained throughout the chol tg 3 24 months rnmo 1 1 i 3 24 i i months f i g . 4 . serum cholesterol and triglyceride concentrations in 20 men with type i1 a hyperlipoproteinaemia after 3 months of dietary therapy and after 2 years of diet and drugs in combination. o=diet, o=diet+clofibrate, a =diet+nicotinic acid, = diet +dofibrate +nicotinic acid c h o l rng / 1 ooml 1 t g rnrnc i : ',4 months fig. 5 . serum cholesterol and triglyceride concentrations in 17 men with type i i b hyperlipoproteinaemia after 3 months of dietary therapy and after 2 years of diet and drugs in combination. symbols as in fig. 4. upsulu j m e d sci 81 90 h . hedstrand chol r n g l l o o rnl 1 r 300 200 months tg rnrnol 3 24 r i months f i g . 6. serum cholesterol and triglyceride concentrations in 10 men with type 111 hyperlipoproteinaemia after 3 months of dietary therapy and after 2 years of diet and drugs in combination. symbols as in fig. 4. 2 years. the greatest reduction, 5.4 kg, was obtained in subjects with type i i b and the lowest, 2 . 4 kg, in those with type i1 a . out of the 168 men in whom the lp pattern was classified (table i), 9 men (5.4%) dropped out during the following 18 months. if the six men who did not come for the l p analysis, are included the total drop-out rate over a 18-month period was 8.6% of those invited to the lipid clinic. side effects of drug therapy. nicotinic acid was discontinued in 19 men (52.8%) because of side effects. the reasons were: flushing in 13 cases (com bined with pruritus in 3 cases) and gastrointestinal irritation in 5 cases. one withdrawal was caused by psychosomatic complaints, this patient complained of blurred vision. in 9 men nicotinic acid treatment was discon tinued within 2 months, in 6 of them because of flushing. in the remaining 10 subjects the drug was withdrawn on average after 1 1 months (range 6-22). liver function tests were checked regularly. in 3 cases there was a slight elevation of alkaline phos phatase. no case of hyperuricaemia was seen. clofibrate was well tolerated by all patients. when nicotinic acid had to be withdrawn, clofibrate was given instead. discussion the hyperlipidaemia found in this population was moderate. the l p pattern was classified as normal in 22 % of the subjects with lipid values over the 80th percentile at the initial examination. in 30 of them a lipid reduction of 15 % in serum cholesterol and 19 % in serum t g had occurred between the screening and the first visit to the lipid clinic, i.e. usually within 3 months. this reduction was obviously caused by dietary changes induced by the information of ele vated serum lipids. a moderate but significant serum lipid reduction was maintained over a one-year period. this indicates that among subjects with hyperlipidaemia there is a number of individuals, perhaps 20 %, with moderate hyperlipidaemia in whom a substantial lipid reduction can be achieved by simple dietary recommendations. the actual serum cholesterol reduction of 10% in these 30 men can be compared to that of the national diet heart study where the cholesterol level was reduced by 1 1 % over a one-year period (13). how. ever, more impressive reductions have been re ported in patients with chd. in male survivors of myocardial infarction a serum cholesterol reduction of 17.6 % over a 5-year period was obtained with diet therapy alone (9). chol mg/100ml i 1 $ 24 0 i months tg mmol/l 1 3 i 24 i months f i g . 7 . serum cholesterol and triglyceride concentrations in 36 men with type iv hyperlipoproteinaemia after 3 months of dietary therapy and after 2 years of diet and drugs in combination. symbols as in fig. 4. upsala j med sci 81 there are several advantages, reviewed by levy et al. (lo), in defining the type of h l p that is causing the hyperlipidaemia. thus, it is well known that the different types of hlpdiffer in responsiveness t o the therapy. this was illustrated during the diet therapy by a serum cholesterol reduction of 19% in subjects with h l p type i11 compared to 9 9% in men with h l p type i i a . the serum tg reductions ranged from 10% in type i i a to 40% in type 111. the average serum lipid reductions obtained by diet therapy were maintained or increased over the 2-year period when drugs were added. sixty-three men were given drug therapy. the effect of drugs may to some extent have been counteracted by decreased adherence to diet. on the other hand, the unchanged mean weight in all types of h l p may indicate that the instituted dietary regimen was fol lowed satisfactorily. the reductions obtained in the present study by diet and drugs in combination (23 9% for serum cholesterol and 45% for tg) could be compared to those of 18 % for serum cholesterol and 28 '3% for serum tg reported from a primary preven tive trial using clofibrate without advising any change in the usual diet ( 1 5 ) . concerning drug therapy, most subjects accepted this therapy after the diet period. however, there were individuals who wanted to continue with diet alone for a longer period. no attempts were made to dissuade them. later it was generally easier t o moti vate them to accept drug therapy. there were also 9 men who for different reasons were not willing to use drugs. nicotinic acid was discontinued due to side effects in over half of the subjects who had received this medication. flushing was the most common side effect. some subjects could not overcome the initial flushing. others discontinued the drug because of flushing accentuated by irregularities in work and meals. this is an important aspect when a drug causes discomfort when not taken regularly, espe cially when introducing medications in asympto matic individuals. clofibrate, on the other hand, was well tolerated. this drug has also the ad vantage of being administered twice daily why the subjects do not have to take their medication during work. a high participation rate at the screening examina tion and few drop-outs during the therapeutical trial indicates that middle-aged men are concerned about their health. a low frequency of side effects to the therapy is of particular importance when dealing dietary and drug treatment ofhyperlipidaemia 91 with asymptomatic individuals, who may already have a tendency to blame the regimen, diet or drug, to cause every possible complaint. this study indicates that an acceptable degree of dietary adherence and patient cooperation can be accomplished in the treatment of asymptomatic middle-aged men with hyperlipidaemia. the serum lipid reductions can be maintained at least over a 2-year period. acknowledgements this study was supported by uppsala lans landsting, pharmacia ab and grants from the swedish medical re search council (19x-3116). references 1 . beaumont, j . l., carlson, l. a,, cooper, g. r., fejfar, z., fredrickson, d. s . & strasser, t.: classifi cations of hyperlipidaemias and hyperlipoproteinae mias. bull wld hlth org43.891, 1970. 2. carlson, l . a. & bottiger, l . e.: ischaemic heart 3. 4. 5 . 6 7. 8. 9. disease in relation to fasting values of plasma triglyc erides and cholesterol. lancet i : 865, 1972. carlson, l. a. & ericsson, m.: quantitative and qual itative serum lipoprotein analysis. i . studies in healthy men and women. atherosclerosis. in press, 1975. dayton, s . , pearce, m. l., hashimoti, s . , dixon, w. j . & tomiyasu, u.: a controlled clinical trial of a diet high in unsaturated fat in preventing complications of atherosclerosis. circulation 3 9 4 0 : suppl. 2, 1969. evans, d. w., turner, s . m. & ghosh, p.: feasibility of long-term plasma-cholesterol reduction by diet. lancet i : 172, 1972. hedstrand, h.: a study of middle-aged men with particular reference to risk factors for cardiovascular disease. upsala j med sci, suppl. 19, 1975. hedstrand, h . & vessby, b.: serum lipoprotein con centration and composition in healthy 50-year old men. upsala j med sci, in press. kannel, w. b., garcia, m. j., mcnamara, p. m. & pearson, g.: serum lipid precursors of coronary heart disease. human pathol2: 129, 1971. leren, p.: the effect of plasma cholesterol lowering diet in male survivors of myocardial infarction. acta med scand. suddi. 466. 1966. , _ _ 10. levv. r. i . . fredrickson. d. s . . shulman. r.. bil 1 1 . 12. heimer, d. w.,breslow, j. l . , stone, n. j., lux, s. e., sloan, h. r., krauss, r . m. & herbert, p. n.: dietary and drug treatment of primary hyperlipoproteinaemia. ann int med 77: 267, 1972. mcgandy, r. b., hall, b., ford, c. & stare, f. j.: dietary regulation of blood cholesterol in adolescent males: a pilot study. amer j clin nutr 25: 61, 1972. miettinen, m., turpeinen, o., karvonen, m. j., elosuo, r. & paavilainen, e.: effect of cholesteroi lowering diet on mortality from coronary heart disease and other causes. a twelve-year clinical trial in men and women. lancetll: 835, 1972. upsala j m e d sci 81 92 h . hedstrand 13. national diet heart study. final report. circulation 37: suppl. i, 1968. 14. national heart foundation of australia. dietary fat and coronary heart disease: a review. med j australia 58: 1155, 1971. 15. oliver, m. f.: a primary prevention trial using clofibrate t o lower hyperlipidaemia. i n atheroscle rosis: proceedings of the second international symposium (ed. r. j. jones). springer-verlag, berlin, heidelberg and new york, 1970. 16. rifkind, b.: drugs affecting lipid metabolism. milano, sept. 1974. 17. rinzler, s. h.: primary prevention of coronary heart disease by diet. bull ny acad med44: 936, 1%8. 18. rush, r. l., leon, l. & turrell, j.: automated simultaneous cholesterol and triglyceride determina tion on the auto-analyzer i1 instrument. advances in automated analysis. thurman associates i : 503, 1971. 19. stamler, j.: acute myocardial infarction-progress in primary prevention. brit heart j33, suppl. 145, 1971. 20. westlund, k . & nicolaysen, r.: ten-year mortality and morbidity related to serum cholesterol. a follow up of 3 751 men aged 4 0 4 9 . scand j clin lab invest, suppl. 127, 1972. recieved j a n u a r y 5, 1976 address for reprints: hans hedstrand, m.d. department of international medicine university hospital s-750 14 uppsala sweden upsala j m e d sci 81 upsala j med sci 84: 181-187, 1979 reaction to cold of patients with coronary insufficiency christer backman, sonja holm and h%kan linderholm from the department of clinical physiology, university of u m e d , u m e d , sweden abstract twenty s i x p a t i e n t s w i t h a n g i n a p e c t o r i s and coronary i n s u f f i c i e n c y a s judged by an e x e r c i s e e c g t e s t were examined. about h a l f of t h e p a t i e n t s had more pronounced ecg changes i n a c o l d room a t 1 5 o c t h a n a t room t e m p e r a t u r e . they worked l e s s , t h e i r s u b j e c t i v e r a t i n g of e x e r t i o n d u r i n g e x e r c i s e was h i g h e r and t h e h e a r t performed l e s s work, e x p r e s s e d as t h e h e a r t r a t e b l o o d p r e s s u r e p r o d u c t . the o t h e r h a l f of t h e p a t i e n t s was n o t much i n f l u e n c e d by c o l d . during an e x e r c i s e t e s t i n t h e s u p i n e p o s i t i o n almost a l l p a t i e n t s g o t more pronounced ecg changes, worked l e s s and t h e h e a r t performed l e s s work t h a n i n t h e s i t t i n g p o s i t i o n . it i s s u g g e s t e d t h a t c o l d exposure as w e l l a s a supine body p o s i t i o n may t o a c o n s i d e r a b l e p a r t e x e r t t h e i r e f f e c t , i . e . lower t h e a n g i n a l t h r e s h o l d and i n c r e a s e ecg changes, by i n c r e a s i n g t h e c e n t r a l blood volume and t h e d i a s t o l i c volume of t h e l e f t h e a r t and t h u s c e t e r e s p a f i b u s sumption. t h e myocardial oxygen con i n t r o d u c t i o n p a t i e n t s w i t h coronary h e a r t d i s e a s e o f t e n e x p e r i e n c e a d e t e r i o r a t i o n i n t h e i r c o n d i t i o n i n c o l d weather ( 4 , 5 ) . many p a t i e n t s s t a t e t h a t t h e i r symptoms a r e provoked by a c o l d wind. some p a t i e n t s observe t h a t an e x e r t i o n which can be maintained w i t h o u t d i f f i c u l t y i n warm weather may provoke an a t t a c k of an g i n a p e c t o r i s i n t h e c o l d . other p a t i e n t s may n o t be much i n f l u e n c e d by c o l d ( 5 , 1 3 ) . an examination of t h e way i n which p a t i e n t s w i t h coronary h e a r t d i s e a s e r e a c t t o c o l d under s t a n d a r d i z e d c o n d i t i o n s i s t h e r e f o r e o f i n t e r e s t . i n t h i s way experiments may be performed t o e l u c i d a t e t h e mechanism of a c t i o n of c o l d i n t h e s e p a t i e n t s . 181 material and methods twenty s i x p a t i e n t s from 41 t o 6 1 y e a r s of age (mean 50 y e a r s ) were exam i n e d because of angina p e c t o r i s . they were chosen o u t of a l a r g e r number be c a u s e t h e y showed ecg changes t y p i c a l of c o r o n a r y i n s u f f i c i e n c y d u r i n g and a f t e r an e x e r c i s e t e s t . they were n o t chosen because o f p a r t i c u l a r d i f f i c u l t i e s i n c o l d weather. most of them were under t r e a t m e n t w i t h beta-blocking drugs. the put,iecc: performed two o r t h r e e e x e r c i s e t e s t s on a b i c y c l e ergometer under d i f f e r e n t c o n d i t i o n s . 1). cycling i n t h e s i t t i n g p o s i t i o n i n a room a t a t e m p e r a t u r e of about +22oc. a t e m p e r a t u r e of about 1 5 o c . i n t h e c o l d room t h e p a t i e n t s were d r e s s e d i n w a r m c l o t h e s , g l o v e s and a cap c o v e r i n g t h e head except f o r t h e f a c e . before t h e c y c l i n g s t a r t e d t h e s u b j e c t s had s t a y e d for about two minutes i n t h e c o l d room. s u p i n e p o s i t i o n . 2 ) , cycling i n t h e s i t t i n g p o s i t i o n i n a room a t 3 ) . most of t h e p a t i e n t s a l s o worked on t h e b i c y c l e ergometer i n t h e during t h e e x e r c i s e t e s t ( 1 6 ~ 2 ) t h e work w a s i n c r e a s e d s t e p w i s e w i t h i n d i v i d u a l l y chosen increments e v e r y s i x minutes u n t i l t h e p a t i e n t w a s unable t o c o n t i n u e o r t h e ecg change o r o t h e r signs l e d t o an i n t e r r u p t i o n of t h e t e s t . heart r a t e , r e s p i r a t o r y frequency, blood p r e s s u r e , and t h e s u b j e c t i v e r a t i n g of e x e r t i o n ( 2 ) were r e c o r d e d a t t h e end of each work l o a d . the maximum work l o a d performed was t a k e n t o be t h e h e a v i e s t l o a d a t which t h e s u b j e c t worked f o r s i x minutes w i t h an increment p r o p o r t i o n a l t o t h e com p l e t e d p e r i o d a t t h e n e x t h i g h e r l o a d (17). the t o t a l work performed w a s c a l c u l a t e d a s t h e sum of work l o a d s m u l t i p l i e d by t i m e . the ( h e a r t r a t e ) ( s y s t o l i c blood p r e s s u r e ) product was c a l c u l a t e d f o r t h e maximum work l o a d and used a s an e x p r e s s i o n of h e a r t work l o a d (14). f o r e a c h p a t i e n t e q u a l work l o a d s were used under t h e d i f f e r e n t c o n d i t i o n s of e x e r c i s e . a group o f 17 p a t i e n t s o f mean age 54 ( r a n g e 36-70) y e a r s w i t h a n g i n a pec t o r i s and f a i r l y comparable w i t h t h e 26 p a t i e n t s d e s c r i b e d were used as con t r o l s i n t h e room t e m p e r a t u r e c o l d room s t u d y . they performed two e x e r c i s e t e s t s i n t h e s i t t i n g p o s i t i o n a t a room t e m p e r a t u r e o f about +22oc w i t h an i n t e r v a l of l e s s t h a n one month. a s c o n t r o l s i n t h e s i t t i n g s u p i n e s t u d y 26 h e a l t h y s u b j e c t s of mean age 70 ( r a n g e 61-83) y e a r s (17) were used. results fourteen of t h e 26 p a t i e n t s (group 1) g o t more pronounced st d e p r e s s i o n s (0.5 1 mm) d u r i n g t h e e x e r c i s e t e s t i n t h e c o l d room compared w i t h t h e t e s t a t room t e m p e r a t u r e ( " c o l d r e s p o n d e r s " ) . the remaining 12 p a t i e n t s (group 2 ) d i d n o t g e t more st d e p r e s s i o n s i n t h e c o l d room t h a n a t room t e m p e r a t u r e 182 on e q u a l work l o a d . f i g . 1 ( l e f t s i d e ) and table 1 show r e s u l t s o f group 1 and group 2 as w e l l a s t h e whole group ( 1 + 2 ) a t room t e m p e r a t u r e and i n t h e c o l d room. the average of t h e maximum work l o a d performed, t h e maximum h e a r t r a t e r e a c h e d , t h e h e a r t r a t e s y s t o l i c b l o o d p r e s s u r e product as an e x p r e s s i o n of t h e h e a r t work a t maximum work l o a d , t h e t o t a l work performed d e c r e a s e d , and t h e r a t i n g of sub j e c t i v e e x e r t i o n i n c r e a s e d s i g n i f i c a n t l y i n group 1 b u t n o t i n group 2 . the p e r c e n t a g e d i f f e r e n c e between t h e e x e r c i s e performance as w e l l as t h e s u b j e c t i v e r a t i n g of e x e r t i o n o f t h e group 1 p a t i e n t s a t room t e m p e r a t u r e and i n t h e c o l d room w a s s t a t i s t i c a l l y s i g n i f i c a n t l y g r e r 5 e r t h a n t h e d i f f e r e n c e between t h e two work t e s t s , b o t h a t room t e m p e r a t u r e , i f t h e 17 p a t i e n t s of t h e con t r o l group w i t h a n g i n a p e c t o r i s ( t a b l e 1). max. work load 120 1 '00 c l--j performed, max. heart rate, 140 1 100 hrxsbp beats. kpa 3 2 10-3, min *it i body position sitt sitt temp. *22'c -15'c t sitt sup *22t f i g . 1 . r e s u l t s of t h e e x e r c i s e t e s t s a t room t e m p e r a t ~ r e , + 2 2 ~ c , and i n t h e c o l d room, -15oc, s i t t i n g on t h e b i c y c l e ergometer f o r angina p e c t o r i s pa t i e n t s who responded w i t h i n c r e a s e d ecg changes i n t h e c o l d , group l(.) and f o r non-responders, group 2 ( = ) , l e f t s i d e of t h e f i g u r g . the r i g h t hand s i d e of t h e f i g u r e shows r e s u l t s from t e s t s performed a t +22 c i n t h e s i t t i n g and supine p o s i t i o n f o r group 1 + 2 ( a ) . the symbols i n d i c a t e means and v e r t i c a l b a r s s t a n d a r d e r r o r of t h e mean. 183 table 1. maximumwork l o a d performed, t o t a l work performed, maximum h e a r t r a t e , and r a t i n g of s u b j e c t i v e e x e r t i o n d u r i n g work on e q u a l l o a d i n t h e s i t t i n g po s i t i o n a t room t e m p e r a t u r e , rt i n a c o l d room, and 1+2. the r e s u l t s of work t e s t i1 a r e g i v e n a s d i f f e r e n c e s between work t e s t i and i1 i n p e r c e n t of work t e s t i , i . e . 1 0 0 * ( i i i ) / i . about +22oc i n t h e two work t e s t s . mean and sem a r e g i v e n . +22oc, (work t e s t i ) and d u r i n g a second t e s t cr 1 5 o c , (work t e s t 11) f o r t h e angina p e c t o r i s group 1 , 2 the c o n t r o l group of a n g i n a p e c t o r i s p a t i e n t s e x e r c i s e d a t room t e m p e r a t u r e v a r i a b l e group n work t e s t i work t e s t i1 ,% change from work t e s t i rt +22'c c r 1 5 o c rt +22oc maximum work l o a d , bj group 1 14 96 10 group 2 1 2 96 ; 11 c o n t r o l group 17 78 8 group 1 + 2 26 9 6 ; 7 2 1 7.6xx'0 1 ; 3 . 2 11 8.ox 0 +_ 3 . 9 t o t a l work performed wxmin group 1 1 4 1026 3 171 26 8.4xxy0 group 2 1 2 9 4 1 ; 193 -4 ; 9 . 5 + group 1+2 26 987 7 126 13 6 . 9 c o n t r o l group 1 7 800 130 -1 6 . 4 max h e a r t r a t e s t r o k e /min group 1 1 4 125 4 group 1+2 26 125 7 4 c o n t r o l group 17 123 7 group 2 1 2 126 8 r a t i n g o f e x e r t i o n group 1 group 2 group 1+2 c o n t r o l group 14 15 a.7 1 2 16 ; 0 . 5 26 1 5 ; 0 . 4 17 ' 14 0.4 -16 ? 3.4xx300 5 ; 3 . 1 xx,oo -10 2 . 5 + 0 4 2.2 n = number of s u b j e c t s , x and o i n d i c a t e 0.05 >f 7 0 . 0 1 , xx and 0 0 i n d i c a t e 0.01>pr0.001, x and xx i n d i c a t e t h e p r o b a b i l i t y t h a t t h e d i f f e r e n c e between work t e s t i and work t e s t i1 i s caused by random f a c t o r s . o and 00 i n d i c a t e t h e p r o b a b i l i t y t h a t t h e d i f f e r e n c e between t h e p e r c e n t a g e change between work t e s t i and work t e s t i1 of t h e p a t i e n t group and t h e c o r r e s p o n d i n g p e r c e n t a g e change between t h e two work t e s t s of t h e c o n t r o l group i s caused by random f a c t o r s . the s y s t o l i c blood p r e s s u r e d u r i n g m a x i m u m work l o a d was n o t s i g n d i f f e r e n t between t h e groups o r t h e d i f f e r e n t c o n d i t i o n s of e x e r c i s e 184 f i c a n t l y the mean s y s t o l i c p r e s s u r e s were s l i g h t l y h i g h e r d u r i n g t h e e x e r c i s e i n t h e c o l d room. angina p e c t o r i s was r e c o r d e d e a r l i e r i n t h e c o l d room t h a n a t room tempera t u r e i n 11 of 1 4 s u b j e c t s of group 1 and i n 6 of 1 2 s u b j e c t s of group 2 . a l though s u g g e s t i v e t h i s d i f f e r e n c e i s n o t s t a t i s t i c a l l y s i g n i f i c a n t . s e v e r a l of t h e p a t i e n t s i n b o t h groups performed a n e x e r c i s e t e s t i n t h e s u p i n e p o s i t i o n . there was no s i g n i f i c a n t d i f f e r e n c e between t h e s u b j e c t s from group l and group 2 and t h e r e f o r e t h e r e s u l t s a r e g i v e n f o r a l l t h e s e p a t i e n t s t o g e t h e r on t h e r i g h t hand s i d e o f f i g . 1 , cf a l s o table 2 . table 2 . r e s u l t s from e x e r c i s e t e s t s performed a t room t e m p e r a t u r e , +22oc, s i t t i n g (work t e s t i ) and s u p i n e (work t e s t 11) f o r t h e a n g i n a p e c t o r i s pa t i e n t s of groups 1 , 2 , 1 + 2 and a c o n t r o l group of h e a l t h y males ( 1 7 ) v a r i a b l e group n work t e s t i work t e s t i1 % change from work t e s t i s i t t i n g supine maximum work l o a d , y group 1 9 88 1 1 . 5 group 1 + 2 1 9 93 ; 8.4 c o n t r o l group 26 136 5 . 4 group 2 1 0 97 1 2 . 6 37 1 0 .2xx 33 ; 5 . 5 30 7 6.ixxx 18 2.0n xx t o t a l work performed wxmin group 1 7 964 263 45 i 1 0 . g x x group 2 7 864 ; 185 52 7 1 0 . g n group 1+2 1 4 914 y 158 48 < 7.3= c o n t r o l group 26 1 5 9 1 107 28 l.oxx max h e a r t r a t e s t r o k e / m i n group 1 11 123 1 1 . 7 17 i 2 . 9 r group 2 1 0 126 ; 9 . 1 8 7 1.9 group 1+2 21 125 ; 5 . 0 1 2 7 2.0 c o n t r o l group 26 153 3 . 4 9 3.9= symbols a s i n table 1 the maximum work l o a d performed, t h e t o t a l work performed, t h e maximum h e a r t r a t e and t h e h e a r t work l o a d , c a l c u l a t e d as t h e h e a r t r a t e s y s t o l i c blood p r e s s u r e p r o d u c t , were a l l lower i n t h e supine p o s i t i o n . the impairment of e x e r c i s e performance i n t h e s u p i n e p o s i t i o n , e x p r e s s e d a s t h e p e r c e n t a g e of t h e r e s u l t i n t h e s i t t i n g p o s i t i o n , f o r t h e a n g i n a p e e t o r i s 185 p a t i e n t s was n o t s t a t i s t i c a l l y s i g n i f i c a n t l y g r e a t e r t h a n t h a t found i n t h e c o n t r o l group of 26 h e a l t h y o l d s u b j e c t s , s e e table 2. there was no s i g n i f i c a n t d i f f e r e n c e between s y s t o l i c blood p r e s s u r e d u r i n g maximum e x e r c i s e l o a d i n t h e s i t t i n g and s u p i n e p o s i t i o n s . st d e p r e s s i o n s were i n a l l c a s e s b u t t h r e e more pronounced i n t h e s u p i n e t h a n i n t h e s i t t i n g po s i t i o n . i n a few c a s e s t h e e x e r c i s e t e s t i n t h e c o l d room was of s i g n i f i c a n c e f o r t h e d i a g n o s i s of coronary i n s u f f i c i e n c y , as t h e r e were no s i g n i f i c a n t e c g changes a t room t e m p e r a t u r e , while ecg changes t y p i c a l of coronary i n s u f f i c i e n cy appeared d u r i n g and a f t e r t h e e x e r c i s e t e s t i n t h e cold room. e x e r c i s e i n t h e s u p i n e p o s i t i o n w a s even more e f f e c t i v e i n t h i s r e s p e c t . discussion s e v e r a l p a t i e n t s i n group 1 were v e r y i n c a p a c i t a t e d d u r i n g t h e w i n t e r and i n t h e s e p a t i e n t s t h e examination of t h e r e a c t i o n t o an e x e r c i s e t e s t i n t h e c o l d room was of i n t e r e s t f o r t h e e v a l u a t i o n of t h e i r degree of i n c a p a c i t y . p a t i e n t s o f group 2 demonstrated a low s e n s i t i v i t y t o c o l d . it i s p o s s i b l e t h a t t h e i r r e a c t i o n t o c o l d i s t h e r e s u l t of an a d a p t a t i o n t o c o l d (1). about h a l f of t h e a n g i n a p e c t o r i s p a t i e n t s r e a c t e d t o c o l d exposure or approximately t h e same p r o p o r t i o n as t h a t r e p o r t e d by o t h e r a u t h o r s ( 5 , 1 3 ) . s e v e r a l mechanisms have been d i s c u s s e d a s an e x p l a n a t i o n f o r t h e e f f e c t of c o l d i n p a t i e n t s w i t h coronary i n s u f f i c i e n c y , such as r e f l e x c o n t r a c t i o n of coronary a r t e r i e s a s r e s u l t of t h e cold s t i m u l u s t o t h e s k i n (5,ll,l2) i n c r e a s e d v a s c u l a r r e s i s t a n c e and i n c r e a s e d blood p r e s s u r e ( 4 , 7 ) and t h e r e f o r e i n c r e a s e d work f o r t h e h e a r t ( 9 ) . another e x p l a n a t i o n ( 1 0 ) might be t h a t c o l d r e s u l t s i n a g e n e r a l vasocon s t r i c t i o n of cutaneous v e s s e l s and consequent r e d i s t r i b u t i o n o f t h e blood v o l ume , w i t h an i n c r e a s e i n t h e c e n t r a l blood volume ( 6 ) and probably i n c r e a s e i n c e n t r a l venous p r e s s u r e s , a t l e a s t i n i t i a l l y . i n t h i s way an e f f e c t i s o b t a i n e d which may be similar t o t h a t o b t a i n e d by changing from an u p r i g h t t o a su p i n e body p o s i t i o n ( 3 ) o r by t h e i n t r a v e n o u s a d m i n i s t r a t i o n of d e x t r a n ( 8 ) . the s i m i l a r i t i e s between t h e changes o b t a i n e d i n c o l d and i n t h e s u p i n e p o s i t i o n axe s u g g e s t i v e . it i s l i k e l y t h a t c o l d as w e l l a s t h e supine p o s i t i o n r e s u l t s i n some i n c r e a s e i n t h e d i a s t o l i c volume of t h e l e f t h e a r t . changes i n v e n t r i c u l a r volume a r e of c r i t i c a l importance i n d e t e r m i n a t i n g myocardial oxy gen consumption, s i n c e an i n c r e a s e means t h a t an augmented w a l l t e n s i o n w i l l be r e q u i r e d by t h e h e a r t f o r t h e same e x t e r n a l work (15 ) . 186 references 1. le b l a n c , j . , dulac,s., cot6,j., and girard,b.: autonomic nervous system and a d a p t a t i o n t o c o l d i n man. j appl p h y s i o l 39:181-186, 1975. 2. borg,g. and linderholm,h.: e x e r c i s e performance and p e r c e i v e d e x e r t i o n i n p a t i e n t s w i t h coronary i n s u f f i c i e n c y , a r t e r i a l h y p e r t e n s i o n and vasoregu l a t o r y a s t h e n i a . a c t a med scand 187: 17-26,1970. h o l d d u r i n g l e g e x e r c i s e . europ j c l i n i n v e s t 4:201-206,1974. e f f e c t s o f a r e d u c t i o n i n e n v i r o n m e n t a l t e m p e r a t u r e on t h e c i r c u l a t o r y r e s p o n s e t o e x e r c i s e i n man. new eng j med 280: 7-11, 1969. 5 . freedberg,a.s. , spieg1,e.d. , & riseman,j.e.f.: e f f e c t of e x t e r n a l h e a t and c o l d on p a t i e n t s w i t h a n g i n a p e c t o r i s : evidence f o r t h e e x i s t e n c e o f a re f l e x f a c t o r . h e r heart j 27: 611-622, 1944. t h e d i s t r i b u t i o n of blood w i t h i n t h e human body. 1950. s t r e a m of c o l d a i r . cardiovasc res 10:691-696,1976. t h e a n g i n a l t h r e s h o l d . c i r c u l a t i o n 43: 824-835, 1971. sponses t o c o l d exposure i n man. h e r j c a r d i o l 26: 38-45, 1970. c i l a r c t med res rep 8 : 48-56, 1977. i n c r e a s e i n c o r o n a r y v a s c u l a r r e s i s t a n c e i n p a t i e n t s w i t h ischemic h e a r t d i s e a s e . n eng j med 295: 1333-1337, 1976. 12. neill,w.a., duncan,d.a., k l o s t e r , f . , e t a l . : response of coronary c i r c u l a t i o n t o cutaneous c o l d . am j med 56: 471-476, 1974. 13. riseman,j.e.f., s t e r n , b . : a s t a n d a r d e x e r c i s e t o l e r a n c e t e s t f o r p a t i e n t s w i t h a n g i n a p e c t o r i s on e x e r t i o n . h e r j med s c i 188: 646-659, 1934. 1 4 . robinson,b.: r e l a t i o n of h e a r t r a t e and s y s t o l i c blood p r e s s u r e t o t h e on s e t of p a i n i n a n g i n a p e c t o r i s . c i r c u l a t i o n 35: 1073-1083, 1967. 15. r o l e t t , e . l . , yurchag,p.m., hood,w.b.jr., g o r l i n , r . : p r e s s u r e volume c o r r e l a t e s of l e f t v e n t r i c u l a r oxygen consumption i n t h e hypervolemic dog. c i r c u l a t res 17: 499-518, 1965. 3. bygdeman,s., wahren,j.: i n f l u e n c e o f body p o s i t i o n on t h e a n g i n a l t h r e s 4 . e p s t e i n , s . e . , stampfer,m., beiser,g.d. , g o l d s t e i n , r . & braunwald,e.: 6 . glaser,e.m., b e r r i d g e , f.r., and prior,k.m.: e f f e c t s of h e a t and c o l d on c l i n s c i 9 : 181-187, 7 . hayward,j.m., holmes,w.f., gooden,b.a.: c a r d i o v a s c u l a r r e s p o n s e s i n man t o 8. k h a j a , f . , sanghi,v., mark,a., p a r k e r , j . o . : e f f e c t of volume expansion on 9 . leon,d.f., amidi,m. & l e o n a r d , j . j . : l e f t h e a r t work and t e m p e r a t u r e r e 10. linderholm,h.: the c o l d environment and coronary i n s u f f i c i e n c y . nord com 11. mudge,g.h.jr., grossman,w., m i l l s , r . m . j r . , lesch,m., braunwald,e.: r e f l e x 1 6 . s j i j s t r a n d , t . , ed. : c l i n i c a l p h y s i o l o g y . svenska b o k f o r l a g e t , stockholm, 17. s t r a n d e l 1 , t . : heart r a t e and work l o a d a t maximal working i n t e n s i t y i n old 1967. men. a c t a med scand. suppl. 414,1964. received a p r i l 2 , 1 9 7 9 address f o r r e p r i n t s : p r o f e s s o r hzkan linderholm department of c l i n i c a l physiology u n i v e r s i t y of umeh s-901 85 umeh,sweden 187 upsala j med sci 91: 89-98, 1986 studies of drug-treated diabetes in the county of jamtland, sweden, based on prescription of insulin and oral antidiabetic drugs olov w;linder, ingmar bergstrom, goran boethius and lars ugander department of internal medicine and pulmonary medicine, ostersund hospital, ostersund, sweden absract epidemiological data on drug-treated diabetes mellitus was obtained by ana 1.yzing prescriptions of insulin and oral antidiabetics in the county of jamtland sweden. during a 12-year period, from 1971 to 1982, the preval.ence of diabetes increased from 1.5% to 2.276, while there was no significant change in the an nual incidence of around 2.7/1000 inhabitants. women displayed a considerable higher prevalence and incidence than men in the over 70 age-group, while in the50 69 age-group there was a male domi nance. a significant change in the prescription pattern with an increased use of insulin and decreased use of oral antidiabetics was observed during the periad. more patients were started on oral antidiabetics during the first part o f the year than during the latter. introduction by recording drug prescription data valuable information can be obtained concerning the morbidity of a certain disease, provided the drugs are prescri bed on one well defined indication (2). this is the case for antidiabetic drugs and therefore prescription of insulin and oral antidiabetics should reflect the morbidity of drug-treated diabetes. in the county of jamtland, sweden, an individual-hased drug prescription project has seen in operation since 1970. at the start of this project about 2 % of the county inhahitants were prescribed antidiabetic drugs ( 3 , 4 ) . h e r e we report data on prescription of antidiabetic drugs and diabetes morbidity during the following 12-year period from 1971 to 1982. methods jamtland has a relatively stable population of around 135000 inhabitants. the age distribution differs from that of sweden as a whole, in so far that the 89 county has relatively more people in the older age groups. in 1 9 8 2 , 1 3 . 3 5 of the population was over 70 years of age compared to 11.52 in the whole of sweden. since 1970 all drug prescriptions to inhabitants of the county born on four certain days of the month have been recorded. thirteen per cent of the in habitants of the county, i.e. about 17000 inhabitants, have thus been conti nuously monitored. drugs dispensed to occasional visitors to the county have not been registered. the recording concerns, inter alia, the patient's identity number, the year and week of dispensation, the prescribing physician, total amount of the drug and the dosage. a detailed presentation of the method was given by boethius and wiman (5). prescriptions of insulin and oral antidiabetics were analyzed retrospecti vely for the 12-year period, from 1971 to 1982. the prevalence of drug-treated diabetes was obtained by counting the number of individuals diabetic drugs during one particular year. the incidence was calculated from the number of individuals who obtained their first prescription of antidiabetic drugs during that year. patients who changed to insulin within six months after the start of oral treatment have been regarded as inuslin-treated. a few pat ients, who started on insulin but changed to tablets within six months, were considered tablet-treated. prescribed anti the population at risk was the average population of the county at the end of each year according to vital statistics. the figures thus obtained somewhat underestimate the real prevalence and incidence of drug-treated diabetes since the drugs used by diabetics at geriatric clinics are not recorded. defined daily dose (ddd) is the average dose for a particular drug (2). recommended doses for insulin are 40 ie and oral antidiabetics, e.g. glibencla mide 5 mg. data concerning sale figures were obtained from the national cor poration o f swedish pharmacies. results prevalence and incidence the prevalence of drug-treated diabetes increased gradually from 1.55 in 1971 to 2.2% in 1982 (table 1 ) . women displayed a higher prevalence than men throughout the whole period. after correcting the figures according to sex and age of the total swedish population, the prevalence for 1982 was 1 . 9 % for men and 2 . 2 % for women. 90 table 1. prevalence of drug-treated diabetes in the county of jimtland. men % women l total % 1971 1.3 1.6 1 . 5 -73 1.6 1 . 7 1.6 -75 1.7 1.9 1.8 -77 1.8 2.0 1.9 -79 1.9 2 . 2 2 . 1 -81 1.9 2 . 4 2.1 -82 2.1 2 . 3 2 . 2 during the observation period, the prevalence was fairly constant in the age groups below 60 years, but over this age there was a gradual increase (fig 1). in the youngest age-group (0-19 years), the average prevalence was 0.11%. in the age group between 50 and 69 years the prevalence was higher for men than for women, whi1.e the reverse was seen in the over 70 age group. in women a rapid in crease in diabetes prevalence occurred after the age o f 70. at the end of the observation period the prevalence was about 3 % in women aged 60 to 69 years, whereas in women over 70 years the corresponding figure was about 9%. no significant change in the annual incidence was observed during the {i 10 period (fig 2 ) . the incidence o f diabetes treated with oral drugs was somewhat higher in women than in men ( 2 . 4 and 2.1/1000 respectively). this tendency was also noted for insulin-treated diabetes (0.39 and 0.35/1000). z w v z w j 5 a 3 w a -71 -73 -75 -77 -79 -81-82 year the annual incidence was low in all age groups given insulin (fig 3). fig 1. in the age group 0 19 years it was prevalence of diabetes rnellitus, treated 0.16/1000. for those treated with oral antidiabetics, there was a conwith insulin o r oral antidiabetics in various age groups during the period 1971 1982 (0-0 men; women). siderable increase with age. the highest incidence (12.5/1000) was found in females over 80 years o f age. during the period no significant change in incidence was observed in any of the age groups. 91 f -71 -73 -75 -77 -79 -81 0 y e a r b i 4 b il -71 73 -75 -77 year -79 -81 fig.2. annual incidence o f diabetes mellitus treated with insulin ( a ) o r oral anti diabetics ( b ) during the period 1971 1982. 1 0 0 5 m w t . i z 0 0 . ln c z 10 w t a4 u. 0 i 5 5 1 a u d .r i rn b i 0 20 30 lo 50 60 70 00 , , , , , , i , , 9 19 29 39 l9 59 69 79 09 a g e groups ( y e a r s ) b u d .o 4 14 219 i 9 1 9 49 $9 719 s'9 age groups ( y e a r s ) fig. 3. average annual incidence o f diabetes mellitus treated with insulin ( a ) or oral antidiabetics ( 6 ) in various age groups during the period 1971 1982. sales o f insulin and oral antidiabetics (excluding hospitals) sales o f insulin (excluding hospitals) were higher in the county o f jamt land than in sweden as a whole and increased slowly during the period (fig.4). sales o f oral antidiabetic drugs declined in the county in the middle o f the 7 0 ' s and have since then been somewhat. lower than in the rest o f sweden (fig.5). i l -75 -76 -77 -78 -79 -80 -81 -82 year fig.4. -74 -75 -76 -77 -78 -79 -80 -81 8 2 year fig.5. sales o f insulin (excluding hospitals) in the county o f jwmtland ( 0-0 ) and cluding hospitals) in the county o f i n sweden as a whole ( 0-0 ) during the jsmtland ( ) and in sweden as sales o f oral antidiabetics ( e x 92 period 1974 1982. number of defined daily doses (ddd) per 1000 inhabitants 1974 1982. number and day. inhabitants and day as a whole ( c-0 during the period o f ddd per 1000 change in prescription pattern the prescribing of various types of insulin from 1970 to 1982 is shown in fig.6. a steady increase was observed during the period for medium-acting in sulin, while there was a decline in the use o f long-term and regular insulin. amongst the oral antidiabetic drugs chlorpropamide was largely replaced by glibenclamide (fig.7). the prescribing o f phenformin diminished after 1973 and it was withdrawn from the swedish market in 1978. 100 % 50 '10 medium -. -,.. ong-term \. -.-..-____._ 1 1 ~ 1 1 1 ~ 1 1 1 ~ -70 -72 -74 -76 -78 -80 -82 y e a r fig.6. prescription of various types of in sulin in the county of jsmtland 1970 1982. percentage distribution based on the number of ddd. l o o *i, 50 % -. metformin -.-. .//.b.-.-.-./ / phenformin 'l-., ,.' \. ' glibenctamide chlor propamide \. -70 -72 -74 -76 -78 -80 -82 year fig.7. prescription of various types of oral antidiabetics in the county of jamt land 1970 1982. percentage distri bution based on the number of ddd. the relation between prescription of insulin and oral antidiabetics varied with age and with time (fig.8). at the beginning of the 70's combined anti diabetic therapy (insulin + oral drugs, sulfonyl urea + biguanid drugs) was frequently adopted. at the end of the period hardly any patients were prescri bed insul.in in combination with oral drugs and very few used combinations of oral drugs. in 1982 40% of drug-treated diabetics were prescribed insulin and 605 oral drugs. 93 19 29 39 1 9 59 6 9 7 9 1975 50% 1 1 10 i p (0 5 0 6? 7;o 8; 19 29 39 19 59 69 7 9 1982 50 *i. fig. 8. prescription in various age groups; insulin (i), oral antidiabetics (m), insulin,+ oral drugs (m), and sulfonyl urea + biguanid (b). per centage distribution o f pat ients. the change in diabetes treatment in individual pat ients during the period is illustrated in table 2 . several patients changed from oral drugs to insulin while the reverse was l e s s common. a change from one to two o r a l drugs was instituted fairly frequently at the earliest part o f the observation period but then became l e s s common. i i 2 0 3p l p 5,o 60 7,o 8,o i"g 29 3 9 1 9 5; 6'9 ;9 age groups ( y e a r s ) table 2 . changes o f treatment in individual patients. change over -72 -73 -74 -75 -76 -77 -78 -79 -80 -81 -82 oral drug-> insulin 7 11 8 13 11 12 16 4 15 11 1 3 i nsu 1 i n-> one oral drug--) newly diagnosed diabetics 52 51 45 60 41 47 4 3 38 5 5 42 49 diabetics oral drug 3 2 3 1 0 1 2 2 0 1 1 two 4 8 5 i i 5 4 5 5 1 4 3 registered 287 31 8 335 365 378 94 seasonal variation there was no significant seasonal variation in the total sales of insulin o r oral antidiabetics during the period studied (fig.9). however, when con sidering each patient's first purchase of antidiabetic drugs, more patients were found to start therapy with oral antidiabetics during the first half of the year than during the latter (fig.10). this difference was statistically significant (p<o.oi, chi-square test). patients starting treatment with insulin were too few to permit any valid conclusions. 1000 1 w m 6 i v cc 2 :: 500 t i a: i 3 z 1 9 17 25 3 3 41 49 4 12 20 28 36 4 4 5 2 period of weeks fig.9. seasonal distribution in the total pur chases of insulin (0-0) and oral anti diabetics (0-0) during the period 1971 1982. m w m 6 i v a: 3 a u 0 a: w m 5 z 5 0 40 30 20 10 1 9 17 2 5 33 41 4 9 4 12 20 28 36 4 4 52 period of weeks fig.10. seasonal distribution for the start of medication with insulin (o-e) and oral antidiabetics (0-0) during the period 1971 1982. discussion in this study a continual increase in the prevalence of drug-treated diabetes was observed from 1 . 5 % in 1972 to 2.2% in 1982. this was due to an increase in diabetes prevalence in the over 60 age groups, whilst no change was observed below 60 years of age. few other reports on the change i n d i a b e tes prevalence over such a long period of time are available. in a study on drug utilization in the county of gotland, sweden, 1.7% of the population used such drugs in 1972 compared to 2.5% in 1981 (9). in czechoslovakia annual reports on the total number of diabetics have been available for several years. an increase in diabetes preval.ence from 1.9% in 1972 to 3.1% in 1980 was observed (14). the most recent diabetes epidemiological study performed in sweden is that from the community of ystad, based on both inand out-patient registers and 95 thus i n c l u d e s a l l t y p e s o f d i a b e t e s ( 1 0 ) . when t h e f i g u r e s were s t a n d a r d i z e d a g a i n s t t h e t o t a l swedish p o p u l a t i o n , t h e d i a b e t e s p r e v a l e n c e was 2.01% f o r men and 1.95% f o r women. i n a s t u d y f r o m t i e r p , sweden, a l s o i n c l u d i n g a l l t y p e s o f d i a b e t e s , t h e p r e v a l e n c e was 2.3% a f t e r age-adjustment as above ( 8 ) : i n t h e p r e s e n t s t u d y c o r r e s p o n d i n g v a l u e s were 1.9% f o r men and 2.2% f o r women a t t h e end o f t h e o b s e r v a t i o n p e r i o d . however, t h e s e f i g u r e s o n l y com p r i s e d r u g t r e a t e d d i a b e t e s , no e s t i m a t i o n i s a v a i l a b l e on t h e p r e v a l e n c e o f d i e t t r e a t e d d i a b e t e s i n jamtland. p r o b a b l y t h i s f i g u r e i s s i m i l a r t o t h a t o b t a i n e d f o r g o t l a n d (20%) and t i e r p ( 2 5 % ) b u t r e g i o n a l d i f f e r e n c e s may e x i s t d e p e n d i n g on t h e a v a i l a b i l i t y o f d i e t i t i a n s . as e x p e c t e d t h e use o f a n t i d i a b e t i c d r u g s i n c r e a s e d r a p i d l y w i t h age. e i g h t p e r c e n t o f t h e i n h a b i t a n t s i n j a m t l a n d o v e r 70 y e a r s o f age were p r e s c r i b e d s u c h d r u g s a t t h e end o f t h e o b s e r v a t i o n p e r i o d . f o r t h e age g r o u p above 75 y e a r s t h e c o r r e s p o n d i n g f i g u r e was 7 . 3 % i n t i e r p ( 8 ) and 11% i n g o t l a n d ( 9 ) . women d i s p l a y e d a h i g h e r p r e v a l e n c e f o r d i a b e t e s t h a n men t h r o u g h o u t t h e whole p e r i o d . t h i s i s i n a c c o r d w i t h o t h e r swedish s t u d i e s ( 8 , 9 ) and was due t o a h i g h p r e s c r i p t i o n o f o r a l a n t i d i a b e t i c s t o women o v e r 70 y e a r s o f age. thus, a t t h e end o f t h e p e r i o d , t h e use o f a n t i d i a b e t i c s was a l m o s t t h r e e t i m e s h i g h e r i n women o v e r 70 y e a r s compared t o t h o s e i n t h e l o w e r age g r o u p ( 6 0 69 y e a r s ) . the p r e v a l e n c e and i n c i d e n c e o f d i a b e t e s i n t h e youngest age g r o u p ( 0 19 y e a r s ) was 0.11% and 0.16/1000 i n h a b i t a n t s and y e a r . these v a l u e s a r e l o w e r t h a n t h o s e r e p o r t e d by d a h l q u i s t e t a 1 ( 7 ) f o r t h e age g r o u p 0 15 y e a r s i n j s m t l a n d ( p r e v a l e n c e 0.175%, i n c i d e n c e 0.21/1ooo). our v a l u e s s h o u l d however b e c o n s i d e r e d a s a p p r o x i m a t e , s i n c e t h e number o f p a t i e n t s i s few. we have n o t been a b l e t o t r a c e d a t a on t h e i n c i d e n c e o f a d u l t d i a b e t e s i n sweden. i n a s t u d y f r o m f i n l a n d ( 1 3 ) t h e a n n u a l i n c i d e n c e o f t y p e i 1 d i a b e t e s was e s t i m a t e d a t 4/1ooo i n h a b i t a n t s . the f i g u r e f o r d i a b e t e s t r e a t e d w i t h o r a l d r u g s was a p p r o x i m a t e l y 2/100o, w h i c h i s i n good agreement w i t h t h a t r e p o r t e d h e r e (2.3/1000). by comparison, t h e r o c h e s t e r s t u d y (12), i n c l u d i n g a l l t y p e s o f d i a b e t e s , r e p o r t e d an a n n u a l i n c i d e n c e o f 1.7/1000. very few (0.4/1000 i n h a b i t a n t s and y e a r ) o f t h o s e w i t h n e w l y d i a g n o s e d d i a b e t e s were t r e a t e d w i t h i n s u l i n f r o m t h e s t a r t . however, a h i g h p r o p o r t i o n o f t h o s e (2.3/1000) t r e a t e d w i t h o r a l a n t i d i a b e t i c s changed o v e r t o i n s u l i n t h e r a p y . the r e a s o n f o r t h i s was p r o b a b l y secondary f a i l u r e a f t e r some y e a r s o f t r e a t m e n t w i t h o r a l d r u g s . a t t h e end o f t h e p e r i o d , as many a s 40% o f t h e d r u g t r e a t e d p a t i e n t s had i n s u l i n p r e s c r i b e d w h i l e 60% had o r a l d r u g s . i n t h e s t u d i e s f r o m t i e r p and g o t l a n d , 2 2 % and 37% r e s p e c t i v e l y o f t h e d r u g t r e a t e d p a t i e n t s used i n s u l i n ( 8 . 9 ) . thus t h e p r o p o r t i o n o f i n s u l i n t r e a t e d p a t i e n t s seemed t o be f a i r l y h i g h i n j a m t l a n d . 96 during the first part of the period combination therapy was frequently used. similarly in the gotland survey 1982, 28% of the patients were receiving a combination therapy. at the end of the period very few patients in jiimtland used this therapy. however, in gotland combination therapy was as common in 1982 as in 1972 (9). considerable changes also occurred during the period with respect to the type of antidiabetic drugs prescribed. thus glihenclamide, introduced in 1971, was soon the dominating oral antidiabetic drug in jbrntland. the rapid fall in prescriptions of phenformin in the middle of the 70's and its withdrawal from the swedish market in 1978 was due to the potential risk o f lactic acidosjs. the prescription of long-term insulin steadily decreased during the period in favour o f medium-acting insulin. these changes in prescription pattern are in accordance with revised principles for diabetes treatment in general. and with recommendations issued by the local drug committee. during the observation period, there was a steady increase in sales of insulin and an increased prescription of insulin compared to oral drugs, which was especially evident in older patients. there was a corresponding decrease in sales of oral drugs. these changes are also in accord with modern principles for diabetes therapy, which favour insulin above oral drugs in adult diabetes ( 1 1 ) . the decline in the sale of oral drugs probably also reflects the ap pointment of dietitians in the county in the middle o f the 70's. many diabetics could then be treated by diet alone, o r a reduced dose of antidiabetic drugs. information from northern ireland, czechoslovakia and sweden suggests that there may be a reciprocal relationship between dietery and oral treatment ( 1 ) . the total number of prescriptions of antidiabetic drugs dispensed to county inhabitants during the period studied did not show any seasonal. variation. this is in accord with the previous study in jamtland by boethius ( 6 ) . however, more patients started oral antidiabetic therapy during the first part of the year than during the latter. this fact indicates a seasonal variation in the start of oral therapy for diabetes and possibly also for the detection o f type i1 diabetes. a similar seasonal variation, with high incidence during the winter months, is wel.1-known for childhood diabetes in sweden and several other countries (7). it shows the importance of environmental factors, such as virus infections, for the pathogenesis of type i diabetes. such factors have not been considered of importance for the onset of type i1 diabetes. thus, our finding was unexpected and will be the subject of further investigations. acknowledgements support for this project was received from the county council of jamtland and the national corporation of swedish pharmacies. the valuable assistance o f arne solberger is gratefully acknowledged. 6 t 86857 1 97 references 1 . 2. 3. 4. 5. 6. 7 . 8 . 9. 10. 11. 1 2 . 1 3 . 14. bergman, u., elmes, p., halse, m., halvorsen, t., hood, h., lunde, p.k.m., sjoqvist, f., wade, o.l. & westerholm, 8.: the measurement of drug consumption. drugs for diabetes in northern ireland, norway and sweden. eur j clin pharmacol 8:83-89, 1975. bergman, u. & sjoqvist, f.: measurement of drug utilization in sweden: methodological and clinical implications. in: drug utilization studies: implications for medical care (ed. f. sjoqvist k i. agenas). acta med scand suppl 683, 15-22, 1984. bergstrom, i., boethius, g. & wiman, f.: sjukdomspanorama utlast ur en l a k e r n e d e l s r e g i s t r e r i n g . lakartidningen suppl iv 69: 34-37, 1972. bergstrorn, i.: diabetes in jamtlands ian, sverige. in: nordiskt symposium, diabetes och dess behandling (ed. l. hallberg & g. blohrne) pp 193-200. goteborg 1974. boethius, g. & wiman, f.: recording of drug prescription in t,he county o f jimtland, sweden. i. methodological aspects. eur .1 clin pharmacol 12:31 35, 1977. boethius, g . : recording o f drug prescription in the county o f jamtland, sweden: pattern of drug usage in 16600 individuals 1970-75. acta med scand 202:241-251, 1977. dahlquist, g . , gustavsson, k . h . , holmgren, g., hagglof, b . , larsson, y . , nilsson, k.u., samuelsson, g., s t e r k y , g., thalme, b. & wall, 5.: the incidence o f diabetes in swedish children 0-14 years o f age. a prospective study 1977-1980. acta paediat scand 71:7-14, 1982. hallqvist, j. k smedby, 6 . : forekomst och medicinskt omhandertagande av diabetes i en definierad population. lakartidningen 78:3706-3708, 1981. idman. l., bergman, u., dahlen, m . , martinsson, l. & wessling, a.: got landsstudie: hog forskrivning av diabetesmedel men sven hog morbiditet. lakartidningen 82:1051-1054, 1985. lithman, t., schersten, b. k tallroth, g.: dataregistrering av forekomst av diabetes i en kornmun i sodra sverige. 3. nordiska kongressen i all manmedicin, tammerfors, fin]-and, 1983. luft, r.: diabetes. underlag til.1 vbrdprogram for primarvgrden. social styrelsen v.%xiprogramn%nden 1982. melton. l.j., palumbo, p.j. & chu, c-p.: incidence of diabetes mellitiis by clinical type. diabetes care 6:75-86, 7983. reunanen, a.: prevalence and incidence of type 2 diabetes in finland. acta endocrinol suppl 262, 31-35, 1984. stika, l.: patterns in drug utilization national and international as pects: antidiabetic drugs. in: drug utilization studies: 1mpli.cations for medical care (ed. f. sjoqvist & j. agenas). acta med scand suppl 683, 53-57. 1984. correspondence address: olov wglinder department o f internal medicine osteisund hospital s-831 83 ostersund sweden 98 upsala journal of medical sciences 2021, 126, e7609 http://dx.doi.org/10.48101/ujms.v126.7609 poorly controlled ambulatory blood pressure in outpatients with peripheral arterial disease nina dahlea,b, emma skauc,d, jerzy leppertc, johan ärnlöve,f and pär hedbergc,g acentre for clinical research, uppsala university, falun, sweden; bprimary health care center britsarvet-grycksbo, county of dalarna, falun, sweden; ccentre for clinical research, uppsala university, västmanland county hospital, västerås, sweden; ddepartment of cardiology, danderyd university hospital, stockholm, sweden; edivision of family medicine and primary care, department of neurobiology, care sciences and society, karolinska institutet, huddinge, sweden; fschool of health and social studies, dalarna university, falun, sweden; gdepartment of clinical physiology, västmanland county hospital, västerås, sweden abstract background: patients with peripheral arterial disease (pad) are generally less intensively managed than patients with coronary heart disease (chd), despite that their risk of complications is believed to be equivalent. identification of pad patients at risk of poorly controlled blood pressure (bp) could lead to improved treatment, thus lowering the risk of cardiovascular (cv) complications. we aimed to describe the prevalence of poorly controlled cardiovascular (cv) risk factors, focusing on bp, in outpatients with pad diagnosed in a vascular ultrasound laboratory. methods: consecutive outpatients with carotid and/or lower extremity pad were included (n = 402) and examined with blood sampling, clinical bp, and 24-h ambulatory bp measurements. a poorly controlled clinical bp was defined as ≥140/90 mmhg, ambulatory bp ≥130/80 mmhg, low-density lipoprotein ( ldl)-cholesterol level ≥2.5 mmol/l, and glycated hemoglobin (hba1c) level >53 mmol/mol in those with diabetes. results: most of the patients had poorly controlled clinical (76.6%) and ambulatory bp (51.7%) profiles. antihypertensive medications were prescribed in 84% of the patients. however, >40% of them used only 0–1 medication, and <25% of them used three or more agents. clinical bp, a low number of medications, body mass index, and the presence of diabetes independently predicted a poorly controlled ambulatory bp. nearly one-third of the patients were smokers, and most of the cohort had an ldl-cholesterol level of ≥2.5 mmol/l. an hba1c level of >53 mmol/mol was present in 55% of diabetic patients. conclusion: poorly controlled clinical and ambulatory systolic bp profiles were common. in addition, suboptimal control of other important cv risk factors was detected. the findings of this study highlight the need for better preventive efforts against cv risk factors in outpatients with pad. article history received: 13 february 2021 revised: 29 march 2021 accepted: 29 march 2021 published: 29 april 2021 keywords carotid artery disease; cardiovascular risk factors; hypertension; smoking; hyperlipidemia; preventive efforts contact nina dahle nina.dahle@regiondalarna.se © 2021 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article introduction peripheral arterial disease (pad) is a common clinical manifestation of systemic arterial atherosclerosis, affecting arteries other than the coronary arteries, intracranial arteries, and the aorta (1, 2). atherosclerosis is often generalized, and many affected patients also have coronary artery disease (cad) (2). patients can present with a spectrum of symptoms, such as claudication, but most are asymptomatic, and all face a substantially increased risk of major cardiovascular (cv) events and deaths (3, 4). pad is the third leading cause of cv morbidity, after cad and stroke (5). it is considered to be equivalent to the coronary heart disease risk, warranting aggressive secondary prevention against cv risk factors. however, patients with pad are generally less intensively managed compared with those with cad (1, 6–9). the overall prevalence of hypertension in adults is about 30–45%, and it becomes more common with advancing age (10). among registered diagnoses in hospital care and primary health care in sweden, hypertension is the diagnosis with the highest prevalence (11, 12). antihypertensive treatments are well established, safe, and highly effective (13). however, many patients with hypertension have inadequate control of their bp or undergo no treatment at all (10). some of the factors found to be associated with poorly controlled bp in the general population include diabetes, older age, obesity, multi-drug regimens, lack of information on hypertension, and living alone (14–18). there is growing evidence that ambulatory bp measurement (abpm) is a strong predictor of organ damage and cv outcomes. it can provide important clinical information beyond clinical bp measurements, such as revealing nocturnal http://dx.doi.org/10.48101/ujms.v126.7609 mailto:nina.dahle@regiondalarna.se http://creativecommons.org/licenses/by/4.0/ 2 n. dahle et al. dipping patterns (19–25). however, how ambulatory bp is controlled and how abpm can be useful in patients with pad is not well studied. to further identify patients with pad at risk of poor bp control could lead to individualized and improved bp treatments, and thereby a lower risk for cv complications (15). in  this study, we aimed to describe the prevalence of poorly controlled cv risk factors, focusing on ambulatory bp in consecutive outpatients with pad diagnosed in a vascular ultrasound laboratory. methods study population analyses were based on patients included in the peripheral arterial disease in västmanland (padva) study (26). all patients visiting the ultrasound laboratory of the department of vascular surgery at the västmanland county hospital in västerås, sweden, from april 2006 to february 2011, were considered for inclusion. reasons for referral include claudication (45%), transient ischemic attack or stroke (26%), aortic aneurysm (8%), heart murmur (5%), suspected renal artery stenosis or renovascular hypertension (4%), and others (12%). every patient was examined with ultrasonography to identify any stenosis in the internal carotid artery (ica). patients with symptoms of claudication also underwent ankle bp measurement to calculate the ankle–brachial index (abi) and ultrasonography of the arteries in the symptomatic leg. the patients were invited to participate in the padva study if they met at least one of the following inclusion criteria: 1) mild to severe stenosis or occlusion of the ica, 2) symptoms of claudication combined with abi ≤0.90 in the symptomatic lower extremity, or 3) symptoms of claudication combined with ultrasonographic evidence of arterial occlusive disease in the same extremity. in total, 452 patients (73.6%) accepted the invitation to join the study. everyone in the study was offered abpm, of whom 35 individuals refused. we excluded patients with <10 daytime or <5 night-time abpm readings (n = 15) (27), leaving 402 patients for analysis. the study was approved by the ethics committee of uppsala university, sweden (dnr 2005:382). all participants gave their written informed consent to participate. the study is registered with clinicaltrials.gov number nct01452165. examination protocol all patients were invited to the department of clinical physiology and were examined according to a standard examination protocol, comprising a questionnaire, including the number of persons in household (living alone vs. cohabitating), educational level (low level was defined as primary school or less), smoking status (smoking defined as regular smoking within the past year), medical history, medication, and physical activity (physically inactive was defined as mostly sedentary with more demanding activities, such as walking, biking, gardening < 2 h per week). selfreported diagnoses of cv disease and diabetes mellitus were confirmed from the medical records. participants fasted overnight, and venous blood samples were taken by trained staff and immediately sent to the accredited laboratory of clinical chemistry, västmanland county hospital, västerås. the estimated glomerular filtration rate (egfr) was calculated from creatinine levels standardized by isotope dilution mass spectrometry (synchron lx or unicel dxc instruments; beckman coulter, inc., brea, ca, usa) using the chronic kidney disease epidemiology collaboration (ckd-epi) formula (28). glycated hemoglobin (hba1c) mono was determined using a tosoh glycohemoglobin analyzer g7 (tosoh, japan) and calibrated against the swedish mono-s method. the hba1c was calculated from hba1c mono using the formula: 10.45 × (hba1c mono level) – 10.62 (international federation of clinical chemistry and laboratory medicine, ifcc standard). the treatment target level for diabetics (≤53 mmol/mol) was based on the european society of cardiology (esc) guidelines from 2003 (29). the serum total cholesterol (tc) concentration was determined using a unicel dxc 800 or synchron lx20 analyzer (beckman coulter, inc.). levels of low-density lipoprotein cholesterol (ldl-cholesterol) were calculated from those of tc, high-density lipoprotein cholesterol (hdl-cholesterol), and triglycerides using the friedewald equation: (ldl-cholesterol = tc – hdl-cholesterol – triglyceride levels × 0·45). the treatment target levels of ldl-cholesterol are based on the esc guidelines from 2003 (<2.5 mmol/l) and 2019 (<1.4 mmol/l) (29, 30). based on a standard 12-lead surface electrocardiography (ecg), left ventricular (lv) hypertrophy was defined as the sokolow–lyon voltage >35 mv or cornell voltage >28 mv in men and >20 mv in women. ankle blood pressure and carotid ultrasound blood pressure in both arms and ankles was measured in all included participants in a supine position after at least 5 min rest. the ankle bp was measured in the bilateral dorsalis pedis and posterior tibial arteries using an inflatable leg-cuff, an aneroid sphygmomanometer, and a handheld doppler instrument with a 5-mhz probe. the abi was calculated by dividing the highest ankle pressure by the highest bp of both arms. an abnormal abi was defined as ≤0.90 or ≥1.40 in either leg. carotid artery ultrasonography has been described in detail (26). briefly, grading of ica lesions into normal artery, plaque without flow disturbance, mild/moderate/severe stenosis, or occlusion was based on gray-scale images, color flow doppler scans, and spectral doppler blood flow velocities. clinical and ambulatory bps clinical bp was measured manually by trained technicians and was obtained from the non-dominant arm or from the other arm if the systolic bp was >10 mmhg higher. the bp was measured from participants in the supine position after at least 5 min rest http://clinicaltrials.gov ambulatory blood pressure in outpatients 3 and was rounded up to the nearest 2 mmhg. using the arm from which clinical bp was obtained, the abpm 04 instrument (meditech ltd., budapest, hungary) was applied for 24-h abpm with readings taken every 20 min (31). three different cuff-sizes were available and selected depending on the size of the patient’s upper arm. dayand night-time periods were assessed from the time of awakening and sleeping entered by the patient in a diary card. the clinical bp was defined as poorly controlled if systolic bp was ≥140 mmhg or diastolic bp was ≥90 mmhg. the  corresponding definition for ambulatory bp was a 24-h ambulatory systolic bp of ≥130 mmhg or diastolic bp ≥80 mmhg (10, 32). white coat hypertension was defined as a poorly controlled clinical bp combined with a well-controlled ambulatory bp, whereas masked hypertension was defined as the reverse condition, that is, a well-controlled clinical bp in combination with a poorly controlled ambulatory bp (20). statistics data are presented as mean ± standard deviation or frequency and (percentage). to investigate potential predictors of a poorly controlled ambulatory bp, we used logistic regression analysis. in a multivariable model, we included available variables that have been proposed or established in previous studies: age, sex, body mass index (bmi), smoking habit, educational level, living alone, physical activity, diabetes, egfr, previous myocardial infarction (mi), stroke, heart failure, abnormal abi, ica stenosis, number of antihypertensive medications, lv hypertrophy, and clinical systolic and diastolic bp (14–18). statistical analyses were performed using r 3.5.3 (r foundation for statistical computing, 2019, vienna, austria; http://www.rproject.org). two-sided p  values <0.05 were considered to be statistically significant results baseline characteristics nearly a third of the patients (28%) were smokers (table 1). among patients with a poorly controlled ambulatory bp, diabetes mellitus was more frequent (29%) compared with the those with well-controlled ambulatory bp (19%). ica stenosis table 1. characteristics of patients with peripheral arterial disease, overall and stratified by well-controlled (<130/80 mmhg) and poorly controlled (≥130/80 mmhg) ambulatory 24-h blood pressure (bp). all patients; n = 402 well-controlled ambulatory (amb) bp; n = 194 poorly controlled amb bp; n = 208 age (years) 69.9 ± 7.1 69.6 ± 7.2 70.1 ± 7.1 male (sex) 240 (59.7%) 118 (60.8%) 122 (58.7%) body mass index (kg/m2) 27.0 ± 4.2 26.3 ± 3.9 27.7 ± 4.3 smoking 112 (28.1%) 55 (28.9%) 57 (27.4%) low education level 229 (57.0%) 116 (59.8%) 113 (54.3%) living alone 105 (26.2%) 58 (29.9%) 47 (22.8%) physically inactive 108 (26.9%) 51 (26.3%) 57 (27.4%) diabetes mellitus 97 (24.1%) 37 (19.1%) 60 (28.8%) abnormal ankle-brachial index 225 (56.0%) 109 (56.2%) 116 (55.8%) internal carotid artery stenosis 303 (75.4%) 139 (71.6%) 164 (78.8%) previous myocardial infarction 78 (19.4%) 39 (20.1%) 39 (18.8%) previous stroke 42 (10.4%) 22 (11.3%) 20 (9.6%) heart failure 29 (7.2%) 19 (9.8%) 10 (4.8%) total cholesterol (mmol/l) 4.56 ± 1.16 4.47 ± 1.11 4.65 ± 1.20 low-density lipoprotein (ldl) cholesterol (mmol/l) 2.68 ± 0.94 2.63 ± 0.90 2.73 ± 0.98 ldl cholesterol ≥2.5 mmol/l 210 (53.3%) 100 (52.4%) 110 (54.2%) ldl cholesterol ≥1.4 mmol/l 385 (97.7%) 185 (96.9%) 200 (98.5%) estimated glomerular filtration rate (ml/min/1.73 m2) 73.9 ± 17.5 73.8 ± 16.4 73.9 ± 18.4 glycated hemoglobin (mmol/mol) 42.3 ± 10.3 41.0 ± 8.3 43.4 ± 11.8 left ventricular hypertrophy 66 (16.7%) 23 (12.0%) 43 (21.0%) angiotensin-converting enzyme inhibitor/angiotensin receptor blocker 230 (57.2%) 108 (55.7%) 122 (58.7%) betablockers 200 (49.8%) 103 (53.1%) 97 (46.6%) diuretics 96 (23.9%) 50 (25.8%) 46 (22.1%) calcium inhibitor 145 (36.1%) 64 (33.0%) 81 (38.9%) statins 329 (81.8%) 161 (83.0%) 168 (80.8%) blood pressure clinical systolic blood pressure (sbp) (mmhg) 153 ± 21 143 ± 17.7 162 ± 19.7 clinical diastolic blood pressure (dbp) (mmhg) 76.7 ± 9.69 74.5 ± 8.8 78.8 ± 10 amb 24-h sbp (mmhg) 131 ± 13.9 120 ± 7.09 141 ± 10.3 amb 24-h dbp (mmhg) 68 ± 8.49 64.5 ± 6.93 71.2 ± 8.55 values are presented as mean ± sd or frequency (percentage). there were missing values in the following categories: smoking (n = 4), living alone (n = 2), heart failure (n = 1), total cholesterol (n = 2), ldl cholesterol (n = 8), egfr (n = 1), and lv hypertrophy (n = 6). http://www.r-project.org http://www.r-project.org 4 n. dahle et al. and lv hypertrophy were more prevalent among patients with poorly controlled ambulatory bp. most of the patients (53%) had an ldl-cholesterol level of ≥2.5 mmol/l, and almost all reported a level of ≥1.4 mmol/l. of the 97 patients with diabetes mellitus, 53 patients (55%) had an hba1c level of >53 mmol/mol, whereas this was seen only in six (2%) of the 305 patients without documented diabetes. clinical and ambulatory bps most of our patients with pad had poorly controlled bps, especially clinical bp (76.6%), but also ambulatory bp (51.7%) (figure 1). a clinical systolic bp of ≥140 mmhg was found in 307  (76.4%) patients, and a clinical diastolic bp of ≥90 mmhg was observed in 58 (14.4%) patients. if a poorly controlled clinical bp was re-defined as systolic bp >140 mmhg or diastolic bp >90 mmhg, the prevalence rate was found to be 70.4% (n  =  283). results of abpm revealed that 120 (29.9%) patients exhibited white coat hypertension and 20 (5.0%) patients showed masked hypertension. an ambulatory 24-h systolic bp of ≥130 mmhg was detected in 207 (51.5%) patients and a 24-h diastolic bp of ≥80 mmhg observed in 40 (10.0%). antihypertensive medications were prescribed in 84% of patients. the number of drugs was remarkably similar in patients with well-controlled and poorly controlled ambulatory bp (figure 2). more than 40% of the patients were taking only 0  – 1 medication, while less than 25% of patients were using three or more agents. predictors of poorly controlled ambulatory bp in a logistic regression analysis, higher clinical systolic bp, higher bmi, fewer antihypertensive medications, and diabetes mellitus were independent predictors of a poorly controlled ambulatory bp independently (table 2). clinical diastolic bp was not associated with poorly controlled ambulatory bp. in figure 3, the probability of poorly controlled ambulatory bp is illustrated depending on the diabetic status and levels of clinical systolic bp and of bmi after adjustment for the other variables listed in table 2. patients with diabetes mellitus had similar clinical figure 1. flowchart showing the prevalence of well and poorly controlled levels of clinical and ambulatory 24-h blood pressure (bp) in 402 outpatients with peripheral arterial disease. figure 2. the distribution of the number of prescribed antihypertensive medications according to ambulatory blood pressure levels. ambulatory blood pressure in outpatients 5 systolic bp profiles compared with those without diabetes (154 ± 20 vs. 153 ± 21 mmhg; p = 0.604) but a higher ambulatory 24-h systolic bp (135 ± 15 vs. 129 ± 13 mmhg; p = 0.002). discussion we found that both clinical and ambulatory systolic bps were poorly controlled in our outpatients with pad, and that many of these patients appeared to have undergone suboptimal treatment with few antihypertensive medications. patients with diabetes had an increased risk of poorly controlled ambulatory bp. moreover, a high proportion of the patients were still smokers and with hyperlipidemia, further emphasizing that preventive care against cv risk factors could be improved in such patients. among the patients with poorly controlled clinical bp, one-third had white coat hypertension, whereas only 5% of the patients exhibited masked hypertension. bmi, diabetes mellitus, number of medications, and clinical systolic bp were independent predictors of a poorly controlled ambulatory bp. the results of this study suggest that a substantial proportion  of outpatients with pad have undertreated systolic hypertension. in contrast, diastolic bps were better controlled, which is in accordance with previous findings in patients with pad and likely derives from the stiffness of arteries caused by atherosclerosis (32). elevated bp is the most important risk factor for death and disability worldwide, accounting for almost 10 million deaths in 2015 (13). only a few previous studies have investigated ambulatory bp levels in patients with pad. skoglund et al. evaluated clinical and ambulatory bp profiles in 98 male patients with lower extremity arterial disease (33). compared with their findings, our patients had similar mean clinical systolic and diastolic bps (153/77 vs. 151/79 mmhg), whereas the mean ambulatory 24-h bps were lower in our population table 2. clinical characteristics independently associated with a poorly controlled 24-h ambulatory blood pressure (i.e. ≥130/80 mmhg) in patients with peripheral arterial disease. odds ratio (95% ci) p age (for every 10-year increase) 0.79 (0.52–1.21) 0.281 male (sex) 1.00 (0.60–1.66) 0.998 body mass index 1.09 (1.02–1.17) 0.009 smoking 1.23 (0.68–2.23) 0.486 low education 0.79 (0.48–1.31) 0.370 living alone 0.81 (0.47–1.42) 0.464 physically inactive 1.04 (0.60–1.80) 0.900 diabetes 2.03 (1.10–3.75) 0.024 estimated glomerular filtration rate (for every 10-unit increase) 0.94 (0.80–1.10) 0.412 previous myocardial infarction 1.18 (0.60–2.33) 0.632 stroke 0.79 (0.35–1.78) 0.563 heart failure 0.35 (0.11–1.11) 0.074 abnormal ankle–brachial index 1.11 (0.64–1.93) 0.708 internal carotid artery stenosis 1.44 (0.77–2.69) 0.251 number of medications 0.67 (0.52–0.86) 0.001 left ventricular hypertrophy 1.87 (0.95–3.68) 0.071 clinical systolic blood pressure (for every 10-unit increase) 1.81 (1.52–2.15) <0.001 clinical diastolic blood pressure (for every 10-unit increase) 1.00 (0.74–1.35) 0.998 values are odds ratios and 95% confidence intervals (ci) for every one unit increase in continuous independent variables, unless stated otherwise. analysis is based on 388 patients in a multivariable logistic regression model. figure 3. probability of poorly controlled ambulatory 24-h blood pressure (i.e. ≥130/80 mmhg) according to the clinical systolic blood pressure, body mass index (bmi) in kg/m2, and occurrence of diabetes mellitus. the probabilities are adjusted for the variables shown in table 2. 6 n. dahle et al. (131/68 vs. 142/78 mmhg). this might have been because of a higher rate of antihypertensive medications (84% vs. 70%) in our population. data on optimal bp treatment target levels in patients with pad are conflicting. possibly, lowering bp too far in these patients might reduce perfusion in lower limbs and increase the risk of pad-related events. in a reanalysis of data from the antihypertensive and lipid-lowering treatment to prevent heart attack trial (allhat ), the association of clinical bps with incident pad events (hospitalization, procedures, medication, or death related to pad) was evaluated. the authors found a u-shaped association of systolic bp with pad events. higher rates of lower extremity pad events were observed with high (>160 mmhg) as well as with low (<120 mmhg) systolic bp and with low diastolic bp (<70 mmhg) (34). in contrast, a metaanalysis of small prospective studies suggested that antihypertensive treatment was not associated with worsening symptoms or outcomes in pad, and there was a trend toward improvement in leg ischemia (35). although optimal bp target levels specific for pad are unclear, the evidence so far has not been strong enough to modify guidelines for patients with pad (34). the suboptimal treatment of hypertension remains worrying, because patients with pad are at high risk of cvrelated mortality (9, 36) but are less intensively treated and have a higher cv mortality than those with mi (37). a combination of drugs is recommended as first-line therapy against hypertension for most patients according to  the latest guidelines (10). in a recent swedish nationwide registry study of long-term prophylactic treatment patterns in pad, 60% of the patients had any antihypertensive treatment (8), which is considerably lower than the 84% in our study population. nonetheless, there was a high prevalence of poorly controlled hypertension in our patient population, and more than 40% of patients were using only one or no bplowering agents. in the general population, white coat hypertension can be found in up to 30–40% of subjects with an elevated in-clinic bp level, and masked hypertension can be observed in around 15% of patients with a normal in-clinic bp level (10). ambulatory bp in our patients revealed fewer cases with masked hypertension (5%) and a similar proportion with white coat hypertension (30%). these findings suggest that abpm is of minor importance in detecting masked hypertension in patients with pad. instead, abpm may be more useful in detecting white coat hypertension when attempts to reduce clinical bps fail. the clinical features that predicted a high ambulatory bp were not surprising. the prevalence of hypertension is known to be increased in individuals with other cv risk factors, such as obesity and diabetes (30, 31). that high clinical bp increases the risk of poorly controlled ambulatory bp and that the use of more hypertensive medications reduces bp, was also expected. in parallel with poorly controlled bp profiles, it is obvious that the control of other important cv risk factors was weak in our population. nearly 30% of our patients were smokers, which is markedly more common than in the general population, where the prevalence of smoking in sweden has decreased from 14% to 7% between 2006 and 2018 according to the public health agency of sweden. smoking is a particularly strong risk factor for pad (1), and smoking duration seems to be a risk factor for women after only 10 years of smoking (36). smoking cessation is highly beneficial and should be prioritized to reduce cv events and mortality rates (2). the latest european guidelines recommend lipid-lowering treatments in all patients with pad, including a maximum tolerated dose of statins, plus ezetimibe or in combination with a proprotein convertase subtilisin/kexin type 9 (pcsk9) inhibitor if needed. the use of statins and addition of a pcsk9 inhibitor to further lower the ldl-cholesterol level seem to provide a favorable effect on lower limb prognosis, in addition to further reduction of cv events (38, 39). a nationwide swedish study found that 74% of patients with mi used statins, but only 53% of patients with pad did so (37). among our patients, more than 80% were using statins; however, the majority of these individuals still had excessive levels of ldl-cholesterol (table 2). diabetes mellitus is strongly associated with an elevated risk of pad and with worse outcomes (1, 10). here, outpatients with pad and concomitant diabetes had an increased risk of poorly controlled ambulatory bp. that many of our outpatients with pad were still smokers and with ldl-cholesterol levels of ≥2.5 mmol/l, and that many with diabetes had hba1c levels >53 mmol/mol, in addition to poorly controlled hypertension, suggests the need for improved preventive care in such individuals. this research study was limited to consecutive outpatients of european origin who were found to have lower extremity and/or carotid artery disease in a visit to a vascular ultrasound laboratory. the invited patients who declined to join the study (n = 162) did not differ in age (p = 0.68) or sex (p = 0.93) compared with the participants. however, if more burdened with disease, these dropouts may have been a source of bias. the participants who declined abpm (n = 35) or were excluded due to few abpm readings (n = 15) did not differ significantly from the included participants regarding age, sex, bmi, smoking, diabetes, previous myocardial infarction, or stroke (all p-values >0.095). data on medication were based on selfreported information, which might be a cause of informational bias. however, good agreement between patient interview and computerized pharmacy records has been found in the elderly population (40). the clinical bps were measured on one occasion only. furthermore, we did not have any information regarding previous efforts to intensify antihypertensive treatments in these patients. in our outpatients with pad, diagnosed in a vascular ultrasound laboratory, poorly controlled clinical and ambulatory systolic bp profiles were common. those receiving a higher number of antihypertensive agents had a better bp control, thus suggesting room for improvement. in addition, we found a suboptimal control of other important cv risk factors in these patients, including smoking, hba1c levels in those with diabetes mellitus, and ldl-cholesterol levels. this study will help in motivating intensified preventive efforts against cv complications in outpatients with pad. ambulatory blood pressure in outpatients 7 acknowledgements the authors thank the participants and staff of the padva study. disclosure statement the authors declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding the research study was supported by sparbanksstiftelsen nya, the county of västmanland, and the swedish medical association. notes on contributors nina dahle, md, phd student in medical sciences at uppsala university, sweden. primary health care center britsarvetgrycksbo, falun, sweden. emma skau, md, phd student in medical sciences at uppsala university, sweden. department of cardiology, danderyd university hospital, stockholm, sweden. jerzy leppert, md, professor, centre for clinical research, uppsala university, västerås, sweden. johan ärnlöv, md, professor at the division of family medicine and primary care, department of neurobiology, care sciences and society, karolinska institutet, huddinge, and school of health and social studies, dalarna university, falun, sweden. pär hedberg, md, senior consultant at the department of clinical physiology, västerås, sweden. associate professor at the centre for clinical research, uppsala university, västerås, sweden. orcid nina dahle https://orcid.org/0000-0003-1444-2515 emma skau https://orcid.org/0000-0002-5625-1146 jerzy leppert https://orcid.org/0000-0003-1433-0329 johan ärnlöv https://orcid.org/0000-0002-6933-4637 pär hedberg https://orcid.org/0000-0001-5731-966x references 1. criqui mh, aboyans v. epidemiology of peripheral artery disease. circ res 2015; 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35: 2864–72. doi: 10.1093/eurheartj/ehu080 39. stock jk. the challenge of peripheral arterial disease: how do we improve outcome? atherosclerosis 2018; 270: 196–8. doi: 10.1016/j. atherosclerosis.2017.12.031 40. sjahid si, van der linden pd, stricker bh. agreement between the pharmacy medication history and patient interview for cardiovascular drugs: the rotterdam elderly study. br j clin pharmacol 1998; 45: 591–5. doi: 10.1046/j.1365-2125.1998.00716.x http://dx.doi.org/10.1056/nejmra060433 http://dx.doi.org/10.1161/hypertensionaha.114.03883 http://dx.doi.org/10.3109/02813439308994897 http://dx.doi.org/10.1093/eurheartj/ehu016 http://dx.doi.org/10.1093/eurheartj/ehu016 http://dx.doi.org/10.1161/hypertensionaha.114.03882 http://dx.doi.org/10.1161/hypertensionaha.114.03882 http://dx.doi.org/10.1056/nejmoa1712231 http://dx.doi.org/10.1161/hypertensionaha.114.03292 http://dx.doi.org/10.1002/ejhf.95 http://dx.doi.org/10.1097/mbp.0000000000000309 http://dx.doi.org/10.1097/mbp.0000000000000309 http://dx.doi.org/10.7326/0003-4819-150-9-200905050-00006 http://dx.doi.org/10.1016/s0195-668x(03)00347-6 http://dx.doi.org/10.1093/eurheartj/ehz455 http://dx.doi.org/10.1097/hjh.0b013e328363e964 http://dx.doi.org/10.1097/hjh.0b013e328363e964 http://dx.doi.org/10.3109/00365599.2012.676755 http://dx.doi.org/10.1161/circulationaha.118.033348 http://dx.doi.org/10.1161/circulationaha.118.033348 http://dx.doi.org/10.1371/journal.pone.0178713 http://dx.doi.org/10.1371/journal.pone.0178713 http://dx.doi.org/10.1097/hjr.0b013e32831c1383 http://dx.doi.org/10.1177/2047487319893046 http://dx.doi.org/10.1093/eurheartj/ehu080 http://dx.doi.org/10.1016/j.atherosclerosis.2017.12.031 http://dx.doi.org/10.1016/j.atherosclerosis.2017.12.031 http://dx.doi.org/10.1046/j.1365-2125.1998.00716.x upsala j med sci 98: 445-486, 1993 10. vocabulary for describing the metrological quality of a measurement procedure with an alphabetic index rene dybkzr department of clinical chemistry frederiksberg hospital, frederiksberg, danmark laboratory medicine is characteristic in having practitioners with various educational backgrounds. current terms have therefore not been chosen according to a systematic plan and seldomly with adequate knowledge of modern metrological and statistical terminology work. the following vocabulary comprises sections of salient concepts related to quality, metrology, statistics, metrological performance characteristics, and reference measure ment systems. when a definition is given with a reference in brackets, the sign "=" indicates that the definition is taken verbatim, the sign "m" shows that editorial differences have been introduced for consistency, and no sign means that the reference is a source in general. if no reference is given, the definition is materially different from the set of references given att the end of the vocabulary. the notes are not necessarily taken directly from any source of the definition unless followed by a reference in [ 1, which then also applies to the definition unless it has a reference already. it should be emphasized that definitions of concepts and their terms are continuously being developed and changed so that it is always useful to consult the latest editions of relevant documents from standards bodies. the terminological evolution also means that disagreement may exist between the standards or recommendations from different organizations and even from different committees within a given body. 445 i the present selection has taken the international vocabulary of basic and general terms in metrology (see bipm et az.) as the highest authority, followed by iso, cen, and i professional international unions. 1 quality who health for all by the year 2000 ensuring the quality of services target 31 by 1990, all member states should have built effective mechanisms for ensuring quality of patient care within their health care systems. 1.1 quality totality of features and characteristics of a product or service that bear on its ability to satisfy stated or implied needs [ = i s 0 8402:1986] 1.2 quality policy overall quality intentions and directions of an organization as regards quality, as formally expressed by top management [ = i s 0 8402:1986] 1.3 quality management that aspect of the overall management function that determines and implements the quality policy notes 1. the attainment of desired quality requires the commitment and participation of all members of the organization whereas the responsibility for quality management belongs to top management. 2. quality management includes strategic planning, allocation of resources and other systematic activities for quality such as quality planning, operations and evaluations. [ = i s 0 8402:1986] 1.4 quality system organizational structure, responsibilities, processes, and resources for implementing quality management [ = i s 0 8402:1986] 446 1.5 quality assurance all those planned and systematic actions necessary to provide adequate confidence that a product or service will satisfy given requirements for quality [ = i s 0 8402:1986] 1.6 quality control operational techniques and activities that are used to fulfil requirements for quality [ = i s 0 8402:1986] notes 1. in order to avoid confusion, care should be taken to include a modifying term when referring to a subset of quality control. 2. quality control involves operational techniques and activities aimed both at monitoring a process and at eliminating causes of unsatisfactory performance. 1.7 quality audit systematic and independent examination to determine whether quality activities and related results comply with planned arrangements and whether these arrangements are implemented effectively and are suitable to achieve objectives [ = i s 0 840219861 notes 1. quality audits are carried out by independent staff. 2. one purpose of a quality audit is to evaluate the need for improvement or corrective action. the concept should not be confused with "surveillance" or "inspection" performed for the sole purpose of process control or product acceptance. 2 measurable quantity 2.1 system demarcated part of the perceivable or conceivable universe, material or immaterial, that may be regarded as a set of elements and a set of relationships between these elements, and that may be observed at a given calendar time note this concept is allied to "object", i.e. any part of the perceivable or conceivable world [ i s 0 1087:1990]. 447 2.2 set well-defined finite or infinite collection of elements note the elements may be physical objects, numbers, concepts, or sets. 2.3 measurable quantity quantity attribute of a phenomenon, body or substance that may be distinguished qualitatively and determined quantitatively [ = vim, 19931 2.4 component definable part of a system notes 1. components can be mechanical, physical, chemical, mathematical, or functional. 2. components are sometimes divided in analyte, concornitant(s), and solvent. 2.5 elementary entity relevant structure of a chemical component such as an atom, molecule, ion, electron, other particle, or specified group of such particles note the elementary entity may be chosen as convenient, not necessarily as physically real, individual particles, for example 0,5 ca2+. 2.6 kindsf-quantity quantity in a general sense definition of how to obtain a value of a quantity by measuring a quantity of its system or of its components or both examptec. length; volume; amount-of-substance; mass concentration; volume fraction nore this concept is called "quantity in a general sense" by vim. 2.7 generic quantity description of an attribute of a generic system 448 note a generic system is a system lacking specification of space or calendar time or both. 2.8 value of a measurable quantity value 4 y magnitude of a measurable quantity generally expressed as a unit of measurement multiplied by a number note a measurable quantity that cannot be expressed as a unit of measurement multiplied by a number may be expressed by reference to a conventional reference scale or to a measurement procedure or to both. [= vim, 19931 2.9 unit of measurement unit measurable quantity, defined and adopted by convention, with which other measurable quantities of the same kind are compared in order to express their magnitudes relative to that quantity [= vim, 19931 2.10 numerical value of a measurable quantity numerical value number by which the unit of measurement is multiplied in the expression of the value of a measurable quantity [= vim, 19931 2.11 truth <metrology> quality of being in accordance with the definition of a measurable quantity that may be represented by a distribution of true values 2.12 true value of a measurable quantity true value cl value consistent with the definition of a given measurable quantity [= vim, 19931 449 2.13 conventional true value of a measurable quantity conventional true value p value attributed to a measurable quantity and accepted, sometimes by convention, as having an uncertainty of measurement appropriate for a given purpose [= vim, 19931 note quasi-synonyms are "assigned value", "best estimate of the value", "accepted reference value". 3 metrology 3.1 metrology science of measurement note metrology includes all aspects both theoretical and practical with reference to measurements, whatever their uncertainty, and in whatever fields of science or technology they occur. [ = vim, 19931 3.2 measurement set of operations having the object of determining a value of a quantity note the operations may be performed automatically. [ = vim, 19931 3.3 principle of measurement scientific basis of a measurement [ = vim, 19931 3.4 method of measurement logical sequence of operations, described generically, used in the performance of measurements [ = vim, 19931 3.5 measurement procedure set of operations, described specifically, used in the performance of particular measurements according to a given method [ = vim, 19931 450 3.6 measurand measurable quantity subject to measurement note a specification of a measurand may require statements about quantities such as time, temperature and pressure. [:: vim, 19931 3.7 measuring instrument device intended to be used to make measurements, alone or in conjunction with supplementary device(s) [ = vim, 19931 3.8 measuring system complete set of measuring instruments and other equipment assembled to carry out specified measurements [ = vim, 19931 note a measuring system may include materials, such as chemical or biological substances and material measures. 3.9 indication of a measuring system indication value of a measurable quantity provided by a measuring system notes 1. the value read from the displaying device may be called the "direct indication"; it is multiplied by the instrument constant to give the indication. 2. the measurable quantity may be the measurand, a measurement signal, or another quantity to be used in calculating the value of the measurand. [= vim, 19931 3.10 input measurable quantity input quantity xs measurable quantity whose value is used in calculating a value for the output measurable quantity according to a function examples indication of the measuring system; quantity represented by a calibrator; any influence quantity 45 1 notes 1. all input quantities may be regarded as random variables. 2. the measurand can be considered an output quantity. 3.11 observed value of a measurable quantity observed value value of a measurable quantity obtained as the outcome of a single observation note one or more observed values of each input measurable quantity may be needed and may have to be corrected before applying a specified function to obtain a result of a measurement. 3.12 result of a measurement result y value attributed to a measurand, obtained by measurement [ = vim, 19931 notes 1. when a result is given, it should be made clear whether it refers to the indication the uncorrected result the corrected result and whether several values are averaged. 2. a complete statement of the result of a measurement includes information about the uncertainty of measurement. [ = vim, 19931 3.13 sampling process of taking samples, usually qualified by a description of the sampling procedure [rso/drs 3534-2:19861 3.14 sample one or more parts taken from a system and intended to provide information on the system or to serve as a basis for a decision on the system 452 notes 1. unless otherwise specified, the sample is assumed to be representative of a "static system". 2. the system from which a sample is taken may not be of the same type as that of the measurand. 3. for a "dynamic system", the calendar time of sampling has to be specified. 3.15 primary sample collection of one or more parts initially taken from a system [= iupac, 19901 3.16 laboratory sample primary sample or a subsample of it as prepared for sending to or as received by the laboratory and intended for measurement note the laboratory sample is the final sample of sample collection and the initial sample in the laboratory. 3.17 analytical sample sample prepared from the laboratory sample and from which analytical portions may be taken 3.18 analytical portion portion of material taken from the analytical sample and on which the measurement of an appropriate measurable quantity is actually carried out 3.19 analytical run analytical series set of measurements performed under repeatability conditions 4 statistics 4.1 probability real number in the scale of 0 to i attached to a random event f = isofdis 3534-1:1990] 453 note probability can be related to a long-run number fraction of occurrence or to a degree of belief that an event will occur. for a high degree of belief, the probability is near 1. 4.2 random variable variate x , y variable that may take any of the values of a specified set of values and with which is associated a probability distribution [ = iso/dis 3534-1:1990] 4.3 probability distribution of a random variable probability distribution function that gives the probability that a random variable takes any given value or belongs to a given set of values note the probability on the whole set of values of the random variable equals 1. [ = iso/dis 3534-1:1990] 4.4 location <statistics > typical magnitude of a distribution of values examples arithmetic mean; median; mode 4.5 dispersion <statistics> degree of scatter shown by a set of values examples standard deviation; central 0,95-interfractile interval 4.6 population totality of items under consideration [ = iso/dis 3534-1:1990] note in the case of a random variable, the probability distribution is considered as defining the population of that variable. 454 4.7 population parameter parameter quantity used in describing the distribution of a random variable in a population [ = iso/dis 3534-1:1990] 4.8 expectation of a random variable expectation e k ) , e(y) expected value mean sum of products of values xi of a discrete random variable x and their respective probabilities pi’ the sum being extended over all values xi that can be taken by x e ( x ) = ~ p r y i 4.9 variance of a random variable variance of a probability distribution variance a2,, wc), v(y) expectation of the square of the centred random variable u2x = e{(x e ( x ) i 2 ) [ = iso/dis 3534-1:1990] 4.10 standard deviation of a random variable standard deviation of a probability distribution standard deviation positive square root of the variance of a random variable 0 [z iso/dis 3534-1:1990] 4.11 coefficient of variation of a random variable coefficient of variation of a probability distribution coefficient of variation o/e(x), u / p ratio of the standard deviation to the expectation of a non-negative random variable [ = iso/dis 3534-1:1990] do not use: relative standard deviation (deprecated) 455 4.12 statistic function of the sample random variables [ = iso/dis 3534-1:1990] notes 1. the value of the statistic obtained by using the observed values or results of measurements in this function may be used in a statistical test or as an estimate of a population parameter such as an expectation of a random variable or a standard deviation of a random variable. 2. a statistic is itself a random variable. 4.13 estimator statistic used to estimate a population parameter [ = iso/dis 3534-1: 19901 4.14 estimate, noun value of an estimator obtained as a result of an estimation [ = iso/dis 3534-1:1990] 4.15 number fraction distribution relative frequency distribution empirical relationship between the values of a random variable and their number fractions note the term "frequency distribution" is often used for the different concepts "number distribution" and "number fraction distribution". 4.16 arithmetic mean average 2, y sum of values divided by the number of values [ = iso/dis 3534-1:2990] note the term "mean" or "expectation" should be used only for the population parameter. 456 4.17 median with n values arranged in non-decreasing order of magnitude 1 to n, the ( ( n + 1)/2)th value of n odd values or a value between (n/2)th and (n/2 + 1)th even values with the arithmetic mean of these values chosen if not otherwise specified [ x iso/dis 3534-1:1990] note an alternative definition is: 0,s-fractile. 4.18 sample variance sum of the squared deviations of results of measurements from their arithmetic mean divided by one less than the number of results note the sample variance is an unbiased estimator of the population variance [e iso/dis 3534-1:1990] 4.19 fractile of a random variable fractile x p variate value equal to or below which lies a stated number fraction of the cumulative number fraction distribution 4.20 confidence interval closed interval within which the value of a population parameter may be expected to lie with a stated probability note this approximate definition is valid for a two-sided confidence interval. 4.21 statistical coverage interval interval for which it can be stated with a given level of confidence that it contains at least a specified proportion of the population [ = iso/dis 3534-1:1990] 451 4.22 tolerance interval variate values between and including the tolerance limits giving upper and lower limits to permissible values 4.23 statistical outlier outlier observed value in a sample, so far separated in value from the remainder as to suggest that it may be from a different population [m iso/dis 3534-1:1990] 5 error 5.1 error of a result of a measurement error of measurement error result of a measurement minus a true value of the measurand [ = vim, 19931 notes 1. since a true value cannot be determined, in practice, a conventional true value is used [ = vim, 19931. error is the sum of random error and systematic error [iso/dis 3534-1:1990]. if several components of error are listed, “error” may be named “total error”. 2. 3. 5.2 deviation value minus its reference value [ = vim, draft 2. ed.] 5.3 random error of a result of a measurement random error e result of a measurement minus the mean that would result from an infinite number of measurements of the same measurand carried out under repeatability conditious e = y -‘(yi, r,) [ = vim, 19931 notes 1. since the mean is not known, in practice, the arithmetic mean is usually substituted. 458 2. it is not possible to correct for random error as it varies unpredictably in successive results. 5.4 systematic error of a result of a measurement systematic error mean that would result from an infinite number of measurements of the same measurand carried out under repeatability conditions minus a true value of the measurand [ = vim, 19931 notes 1. since the mean (or expectation) is not known, in practice, the arithmetic mean is usually substituted. 2. since a true value cannot be determined, in practice, a conventional true value is used. 3. the systematic error of successive results remains constant or varies in a predictable way. 5.5 correction value added algebraically to the uncorrected result of a measurement to compensate for systematic error [ = vim, 19931 notes 1. the correction is equal to the negative of the estimated systematic error. 2. some systematic errors may be estimated, and compensated by applying appropriate corrections. however, since the systematic error cannot be known perfectly, the compensation cannot be complete. 5.6 correction factor numerical factor by which the uncorrected result of a measurement is multiplied to compensate for systematic error [ = vim, 19931 note since the systematic error cannot be known perfectly, the compensation cannot be complete. [ = vim, 19931 18-935253 459 5.7 mistake blunder unauthorized departure from the prescribed measurement procedure note the mistake can take the form of an omission or an incorrect action. this is to be distinguished from "error of measurement". 6 accuracy 6.1 accuracy of measurement accuracy closeness of the agreement between the result of a measurement and a true value of the measurand [ = vim, 19931 do not use: precision (deprecated) notes 1. accuracy of measurement can be measured on an ordinal scale such as (poor, fair, good). 2. accuracy is usually expressed numerically by statistical measures of the inverse concept "inaccuracy of measurement". 3. the term "accuracy", when applied to a set of results, describes a combination of "random error of measurement" and "systematic error of measurement". 6.2 trueness of measurement trueness closeness of the agreement between the average value obtained from a large run of results of measurements and a true value notes 1. trueness of measurement can be measured on an ordinal scale such as (low, medium, high). 2. trueness is usually expressed numerically by the statistical measure "bias" that is inversely related to trueness. 460 6.3 bias of measurements bias difference between the expectation of the results of measurements and a true value do not use: inaccuracy notes 1. bias is a "systematic error of results of measurements" and it may have several components. since a true value cannot be determined, in practice, a "conventional true value" is used. 2. 6.4 precision of measurements precision closeness of the agreement between independent results of measurements obtained under prescribed conditions [z iso/dis 3534-1:1990] notes 1. precision of measurements can be measured on an ordinal scale such as (low, medium, high). 2. precision is usually expressed numerically by statistical measures of the inverse concept "imprecision of measurements". 3. precision depends only on the distribution of "random errors of measurement". 6.5 precision conditions statement of conditions of measurement under which independent results of measure ments of the same measurand are obtained, specifying which sources of variation operate note the term "independent results" means that a given result must not be influenced by any previous result. 7 components of a result 7.1 overall expectation of results of measurements overall expectation ev), m expectation of results of measurements of a given measurand as obtained from all 46 i laboratories using the same measurement procedure in a collaborative assessment experiment note the overall expectation is the sum of a true value of the measurand and the bias of the measurement procedure, e(y) = p + 6, that is the overall expectation depends solely on a true value and the measurement procedure. in some cases, however, an in dependent true value does not exist and the overall expectation is exclusively defined by the measurement procedure. 7.2 bias of results of a measurement procedure bias of a measurement procedure difference between the expectation of all results of measurements made by a stated measurement procedure and a true value of a given measurand to be expected to apply to all measurements made by that procedure 6 = e f l ) p = m p notes 1. the bias of a measurement procedure may depend on the value of the measurand. 2. a n estimator of this bias is the difference between the general average and a conventional true value, d = y f i . 7.3 laboratory deviation b difference between the expectation of the results of measurements of a laboratory for a given measurand obtained under stated precision conditions and the overall expectation of results of measurements notes 1. the laboratory deviation may depend on the value of the measurand. 2. an estimator of this deviation is the difference between the average in the laboratory and the general average of results of measurements, b =ji y’ 3. the laboratory deviation is given specific names for typical precision conditions. a 7.4 laboratory bias of results of a measurement procedure laboratory bias a difference between the expectation of all results of measurements made in a given 462 laboratory using a specified measurement procedure and a true value of the measurand a = e(yj /.l notes 1. the laboratory bias may depend on the value of the measurand. 2. an estimator of this bias is the difference between the average of all results obtained by a given laboratory and a conventional true value, a a = j i . f i . 7.5 aberrant-sample bias of the result of measurement aberrant-sample bias s difference between the expectation of the results of measurements on a given sample of unusual composition and the expectation for a measurand of the same magnitude pertaining to a sample having a usual composition 7.6 bias caused by an undetected mistake a4 difference between the result of a measurement on a given sample in a given run and the expectation of repeated results of the same measurand, in excess of any random error 8 repeatability (see also 7) 8.1 repeatability conditions conditions of measurement where independent results of measurements of the same measurand are obtained with the same measurement procedure in the same laboratory by the same observer using the same equipment, used under the same conditions within a short period of time note the term "independent results" means that a given result must not be influenced by any previous result. 46.3 8.2 laboratory component of bias of results of a measurement procedure laboratory component of bias bi, r difference between the laboratory bias of results of a measurement procedure and the bias of results of a measurement procedure bi, notes 1. the laboratory component of bias is related to the expectation of results of measurements, e(y) = rn, not to a true value. the laboratory component of bias is considered constant under repeatability conditions, r, in one laboratory for one measurand, but its value may depend on the value of the measurand. an estimator of this bias is the difference between the average of all results of measurements obtained by a given laboratory and the general average, b i , = a i , 6 = e(yiir) e ( y ) = e(yiir) m 2. 3. = a i , d = yi, a t = yi, -3;. 8.3 repeatability of results of measurements repeatability closeness of the agreement between the results of successive measurements of the same measurand carried out under the same conditions of measurement notes 1. repeatability conditions (see 8.1) include: the same measurement procedure, the same observer, the same location, repeatability depends solely on the dispersion of random error and does not relate to a true value. repeatability can be measured on an ordinal scale of measurement such as (poor, good, excellent). the same measuring instrument, used under the same conditions, repetition over a short period of time 2. 3. [ = vim, 19931 464 8.4 within-laboratory repeatability variance of results of measurements within-laboratory repeatability variance 2 2 i,rj w variance of the distribution of random errors within a single laboratory under repeatability conditions notes 1. an alternative definition, which includes the dispersion of true values is: variance of the distribution of results of measurements within a single laboratory under repeatability conditions. 2. the value may depend on the value of the measurand. 3. an estimator of u2, is the sample within-laboratory repeatability variance, s2,. 8.5 repeatability variance of results of measurements repeatability variance average of the within-laboratory repeatability variances of results of measurements taken over all t h e laboratories using a given measurement procedure in a precision experiment azr = uzw notes 1. it is assumed that for a given measurement procedure any variation in the variances between the laboratories is small. 2. the value of the repeatability variance may depend on the value of the measurand. 3. an estimator of 02r is the sample repeatability variance, s2,. 9 intermediate precision (see also 7) 9.1 intermediate precision conditions conditions of measurement where independent results of measurement of the same measurand are obtained with the same measurement procedure in the same laboratory subject to stated changes in one or more sources of variation such as time, observer, and measuring system note the term "independent results" means that a given result must not be influenced by any previous result. 46 5 9.2 intermediate precision of results of measurements intermediate precision closeness of agreement between the results of measurements of the same measurand, where the measurements are carried out in the same laboratory by the same measure ment procedure, but with stated changes in one or more sources of variation such as time, observer, and measuring system notes 1. results are here usually understood to be corrected results. 2. the notes 1, 2, and 3 to precision of measurements apply analogously. 10 reproducibility (see also 7) 10.1 reproducibility conditions conditions of measurement where independent results of measurement of the same measurand are obtained with the same measurement procedure in different laboratories with different observers using different equipment notes 1. the term "independent results" means that a given result must not be influenced by any previous result. 2. the definition in vim, 1993, allows principle of measurement, methods of measurement, and conditions of use as added possible sources of variation. 3. the conditions changed must always be specified. 10.2 reproducibility of results of measurements reproducibility closeness of the agreement between the results of measurements of the same measurand, where the measurements are carried out by the same measurement procedure, but in different laboratories with different observers using different equipment notes 1. a valid statement of reproducibility requires specification of the conditions changed. 2. results are here usually understood to be corrected results. 3. the notes 1, 2, and 3 to precision of results of measurements apply analogously. 4. the definition in vim, 1993, allows principle of measurement, method of measurement, and conditions of use as added possible sources of variation. 466 10.3 between-laboratory variation b r distribution of the individual laboratory-component-of-bias of results of measurements obtained under defined precision conditions. 10.4 between-laboratory variance of results of measurements between-laboratory variance (jzbr = cjzl variance of the distribution of the laboratory components of bias of results of a measurement procedure obtained under reproducibility conditions notes 1. the distribution is assumed to be at least approximately gaussian. 2 . the between-laboratory variance may depend on the value of the measurand. 3. an estimator of a2l is the sample between-laboratory variance, s2l. 10.5 reproducibility variance of results of measurements reproducibility variance ( j r sum of the repeatability variance of results of measurements and the between-laboratory variance of results of measurements c j ~ ~ = a2r + azl notes 1. the reproducibility variance may depend on the value of the measurand. 2. an estimator of uzr is the sample reproducibility variance, s2r = s2r + szl. 2 10.6 between-procedure variation 6p distribution of individual bias of results of measurement procedures under defined precision conditions 467 11 analytical reliability metrological reliability 11.1 analytical reliability metrological reliability ability of a measurement procedure to perform a required function under stated conditions for a stated period of time [=: i s 0 8402:1986] 11.2 analytical performance characteristic metrological performance characteristic performance characteristic property in the set of properties that is necessary for assessing the suitability of a measurement procedure for any given purpose and where each property can be given an experimentally determined value examples analytical sensitivity; repeatability standard deviation; limit of detection 11.3 analytical calibration function metrological calibration function calibration function signal of the measuring system as a function of the stated value of the rneasurand note the stated value may be the assigned value of a reference material. 11.4 analytical sensitivity si slope of the analytical calibration function notes 1. the analytical sensitivity may vary with the magnitude of the measurand involving the component i or with influence quantities. 2. the term "analytical sensitivity" must not be used as a synonym for "limit of detection". 468 11.5 analytical measuring function measuring function value of the measurand as a function of the signal of the measuring system related to that measurand and derived from the analytical calibration function note the analytical measuring function, in principle, is the inverse of the analytical calibration function, but this relationship may not hold if the measurement procedure is not specific for one analyte or where analytical interference occurs. 11.6 influence quantity measurable quantity that is not the measurand but that affects the result of measurement [= vim, 19931 example bilirubin concentration in the measurement of hemoglobin concentration in human blood plasma 11.7 analytical specificity ability of a measurement procedure to determine solely the measurable quantity it purports to measure note the analytical specificity is usually expressed in terms of the effect of any component of the sample other than the analyte causing an indication of the measuring instrument and thereby introducing an error of measurement. 11.8 analytical interference systematic error of measurement caused by an analytical interferent 11.9 analytical interferent influence quantity which does not by itself produce a signal in the measuring system, but which causes an enhancement or depression of its indication note sone analytical chemists prefer that an analytical interferent can itself produce a signal. 11.10 reagent blank measurement measurement on a material lacking natural sample 469 note the result of the measurement is used as a correction for that part of the indication caused by the reagent(s). 11.11 matrix blank measurement measurement on a material containing matrix material, but lacking analyte notes 1. if matrix material is unavailable, the measurement may be made on sample material, by a modified procedure omitting a reagent or inactivating the analyte. the result of the measurement is used as a correction for that part of the indication caused by the sample matrix. 2. 11.12 recovery measurement subtracting of the indication of a measuring instrument obtained by measurement on an analytical sample from the indication of another analytical sample of the same laboratory sample containing an added amount of the analyte and comparing with the added amount. 11.13 repeatability standard deviation ur standard deviation of results of measurements obtained under repeatability conditions [z iso/dis 3534-1:1990] notes 1. synonyms of "repeatability standard deviation" are "within-run standard deviation", "within-series standard deviation", "intra-run standard deviation", and "intra-serial standard deviation". 2. the estimator of "repeatability standard deviation" is the "sample repeatability standard deviation", s,. 11.14 repeatability limit r; '0,95 value less than or equal to which the absolute difference between two single results of measurements, obtained under repeatability conditions, is expected to be with a probability of 0,95 [z iso/dis 3534-1:1990] 470 note in practice, the estimate of the repeatability is taken as 2 4 sr = 2,8 s,, see repeatability standard deviation. 11.15 intermediate precision standard deviation standard deviation of results of measurements obtained under defined intermediate precision conditions note the estimator of "intermediate precision standard deviation" is the "sample intermediate precision standard deviation". 11.16 reproducibility standard deviation or standard deviation of results of measurements obtained under reproducibility conditions [iso/dis 3534-1:1990] note the estimator of "reproducibility standard deviation" is the "sample reproducibility standard deviation", sr. 11.17 reproducibility limit r; r0,95 value less than or equal to which the absolute difference between two single results of measurements, obtained under reproducibility conditions, is expected to be with a probability of 0,95 [= iso/dis 3534-1:1990] note in practice, the estimate of the reproducibility limit is taken as 2 a s r = 2,8 sr, see reproducibility standard deviation. 11.18 reproducibility standard deviation divided by analytical sensitivity a r / s j 11.19 limit of detection detection limit lowest result of a measurement by a given measurement procedure that can be accepted 47 i with a stated confidence level as being different from the value obtained on matrix blank material note an experimental procedure for determining the limit of detection consists in using the average value of, say, 20 results of measurements on matrix blank material and adding three times their sample standard deviation. at these low values with probably non gaussian distribution, the corresponding confidence level should be taken to be about 0,90. 11.20 lower limit of determination lowest result of a measurement, that can be obtained by a stated measurement procedure, and that can be given with a statement of uncertainty of measurement. 11.21 higher limit of determination highest result of a measurement, that can be obtained by a stated measurement procedure, and that can be given with a statement of uncertainty of measurement 11.22 uncertainty of measurement parameter, associated with the result of a measurement, that characterizes the dispersion of the values that could reasonably be attributed to the measurand [ = vim, 19931 notes 1. the parameter may be, for example, a standard deviation (or a given multiple of it), or the half-width of an interval having a stated level of confidence. uncertainty of measurement comprises, in general, many components. some of these components may be evaluated from the statistical distribution of the results of series of measurements and can be characterized by experimental standard deviations. the other components, which can also be characterized by standard deviations, are evaluated from assumed probability distributions based on experience or other information. 3. it is understood that the result of the measurement is the best estimate of the value of the measurand, and that all components of uncertainty, including those arising from systematic effects, such as components associated with corrections and reference standards, contribute to the dispersion. 2. 472 11.23 robustness of a measurement procedure robustness ability to yield acceptable results of measurements in spite of deviation from details of the measurement procedure 11.24 transferability of a measurement procedure transferability ability of a measurement procedure to allow observers in different laboratories to perform measurements giving results in accordance with the stated analytical performan ce characteristics 11.25 interchangeability of a measurement procedure interchangeability ability of one measurement procedure to be used in place of another to fulfil the same requirements [iso/iec guide 2:1986] 11.26 commutability of a measurement procedure commutability ability of a measurement procedure to produce results of measurement that, within the uncertainty of measurement, are in a constant relationship with the results of another stated measurement procedure when both are applied to natural samples and reference materials 11.27 compatibility suitability of products, processes or services for use together under specific conditions to fulfil relevant requirements without causing unacceptable interactions [ = iso/iec guide 2:1986] 12 practicability 12.1 practicability of a measurement procedure practicability properties of a measurement procedure concerning number rate of results of measure ments, production time of one result, cost, requirements of technical skill, requirements of services and environment, reliability of measuring system, and safety 473 13 reference measurement system 13.1 reference measurement procedure thoroughly investigated and described measurement procedure having analytical performance characteristics, especially bias and expressions of precision of measure ments, permitting its use for assessing the accuracy of other procedures and charac terizing reference materials [is0 guide 30:1981] 13.2 measurement standard standard etalon material measure, measuring instrument, reference material or measuring system intended to define, realize, conserve or reproduce a unit of measurement or one or more values of a quantity to serve as a reference [:: vim, 19931 13.3 primary measurement standard primary standard measurement standard that is designated or widely acknowledged as having the highest metrological qualities and whose value is accepted without reference to other standards of the same [generic] quantity, within a specified context [:: vim, 19931 13.4 secondary measurement standard secondary standard measurement standard whose value is assigned by comparison with a primary standard of the same [generic] quantity [:: vim, 19931 13.5 reference measurement standard reference standard measurement standard generally having the highest metrological quality available at a given location or in a given organization, from which measurements made there are derived [:: vim, 19931 13.6 working measurement standard working standard measurement standard that is used routinely to calibrate or check material measures, 474 measuring instruments or reference materials notes 1. a working standard is usually calibrated against a reference standard. 2. a working standard used routinely to ensure that measurements are being carried out correctly is called a check standard. [vim, 19931 13.7 reference material r m material or substance one or more of whose property values are sufficiently homogene ous and well established to be used for the calibration of an apparatus, the assessment of a measurement procedure, or for assigning values to materials [ = i s 0 guide 30:1992] 13.8 certified reference material crm reference material, accompanied by a certificate, one or more of whose property values are certified by a measurement procedure which establishes traceability to an accurate realization of the measurement unit in which the property values are expressed, and for which each certified value is accompanied by an uncertainty at a stated level of confidence note some reference materials and certified reference materials have properties which, because they cannot be correlated with an established chemical structure or for other reasons, cannot be determined by exactly defined physical and chemical measurement procedures. such materials include certain biological materials such as vaccines to which an international unit has been assigned by the world health organization. [a i s 0 guide 30:1992] 13.9 calibration material calibrator reference material used for calibration 13.10 control material material used for the purposes of internal quality control or external quality assessment and subjected to measurement according to the same or part of the same measurement procedure as that used for unknown samples in order to monitor analytical performance 19 935253 475 notes 1. analyte and matrix should closely resemble those of unknown samples. 2. control material may or may not meet the requirements of reference materials. 13.11 blank material material lacking the analyte or another component necessary to produce an indication of a measuring system that is specific to the analyte note reagent blank material contains no natural sample. sample blank material contains natural sample, but a modified measurement procedure yields no signal for the analyte. 13.12 calibration set of operations that establish, under specified conditions, the relationship between values of measurable quantities indicated by a measuring instrument or measuring system, or values represented by a material measure or a reference material, and the corresponding values realized by measurement standards [= vim, 19931 13.13 traceability property of the result of a measurement or the value of a measurement standard whereby it can be related to stated references, usually national or international measurement standards, through an unbroken chain of comparisons having stated uncertainties [= vim, 19931 14 analytical quality assurance metrological quality assurance 14.1 see 1.5 14.2 internal quality control set of procedures undertaken by the laboratory staff for continuous monitoring of operations and results of measurements in order to decide whether the results are reliable enough to be released and to eliminate causes of unsatisfactory performance notes 1. internal quality control primarily monitors the batchwise trueness of results of 476 measurements on control materials and precision on replicate measurements of natural samples. internal quality control in the wider sense applies to all stages of activity in the production of results of measurements from assessing clinical needs, via preparation of the patient, collection of sample, and measurement to reporting of results. 2. 14.3 interlaboratory measurement comparisons interlaboratory test comparisons organization, performance and evaluation of measurements on the same or similar items or materials by two or more laboratories in accordance with predetermined conditions [= iso/iec guide 2:1986] 14.4 laboratory proficiency testing proficiency testing external quality assessment system for objectively checking laboratory results of measurements by an external agency, and including comparison of a laboratory’s results at intervals with those of other laboratories, the main object being the establishment of trueness [= iso/remco n231:1991] note the main purposes of laboratory proficiency testing are to establish between-laboratory comparability of results and long term stability of analytical performance. 14.5 technical specification document that prescribes technical requirements to be fulfilled by a product, process or service notes 1. a technical specification should indicate, whenever appropriate, the procedure(s) by means of which it may be determined whether the requirements given are fulfilled. 2. a technical specification may be a standard, a part of a standard or independent of a standard. [ = iso/iec guide 2:1986] 14.6 requirement provision that conveys criteria to be fulfilled [ = iso/iec guide 2:1986] 477 note in a standard, requirements will be indicated by the auxiliary "shall". 14.7 regulation document providing binding legislative rules, that is adopted by an authority [ = iso/iec guide 2:1986] 15 schemes of recognition 15.1 good laboratory practice glp organizational process and the conditions under which laboratory studies are planned, performed, monitored, recorded, and reported [ = oecd:1981] 15.2 certification of conformity action by a third party, demonstrating that adequate confidence is provided that a duly identified product, process or service is in conformity with a specific standard or other normative document [ = iso/iec guide 2: 19861 15.3 laboratory accreditation formal recognition that a testing laboratory is competent to carry out specific tests or specific types of tests note the term "laboratory accreditation" may cover the recognition of both the technical competence and the impartiality of a testing laboratory or only its technical competence. accreditation is normally awarded following successful laboratory assessment and is followed by appropriate surveillance. [ = iso/iec guide 2:1986] who health for all by the year 2000 health technology assessment target 38 before 1990, all member states should have established a formal mechanism for the systematic assessment of the appropriate use of health technologies and their effectiveness, efficiency, safety and acceptability, while reflecting national health policies and economic restraints. 478 references abbreviations of international organizations bipm iec ifcc i s 0 iupac iupap oecd oiml international bureau of weights and measures international electrotechnical commission international federation of clinical chemistry international organization for standardization international union of pure and applied chemistry international union of pure and applied physics organization for economic co-operation and development international organization of legal metrology bipm, iec, ifcc, iso, iupac, iupap, oiml. international vocabulary of basic and general terms in metrology (vim). 2nd ed. geneva: iso, 1993 (in press). iso. terms and definitions used in connection with reference materials. i s 0 guide 30, 1st ed. geneva: iso, 19815 pp. iso. quality vocabulary. i s 0 8402, 1st ed. geneva: iso, 1986:12 pp. iso. statistics vocabulary and symbols. part 2. statistical quality control. iso/dis 3534-2. geneva: iso, 1986:iii i30 pp. iso. statistics vocabulary and symbols. part 1. probability and general statistical terms. iso/dis 3534-1. geneva: iso, 1990230 pp. iso. harmonized proficiency testing protocol. iso/remco n 231, 1991:46 pp. iso, iec. general terms and their definitions concerning standardization and related activities. iso/iec guide 2. geneva: iso, 1986:iii + 45 pp. iupac. nomenclature for sampling in analytical chemistry. recommendations 1990. pure appl chem 1990;62: 1193-1208. 479 oecd. oecd principles of good laboratory practice. env/chem/mc/81.14.1981: 15 44. vim. see. bipm et al. 480 alphabetical index aberrant-sample bias of the result of measurement 7.5 accuracy of measurement 6.1 analytical calibration function 11.3 analytical interference 11.8 analytical interferent 11.9 analytical measuring function 11.5 analytical performance characteristic 11.2 analytical portion 3.18 analytical reliability 11.1 analytical run 3.19 analytical sample 3.17 analytical sensitivity 11.4 analytical series 3.19 analytical specificity 11.7 arithmetic mean 4.16 average 4.16 between-laboratory variance of results of measurements 10.4 between-laboratory variation 10.3 between-procedure variation 10.6 bias caused by an undetected mistake 7.6 bias of a measurement procedure 7.2 bias of measurements 6.3 bias of results of a measurement procedure 7.2 blank material 13.11 blunder 5.7 calibration 13.12 calibration function 11.3 calibration material 13.9 calibrator 13.9 certification of conformity 15.2 certified reference material 13.8 48 1 coefficient of variation of a probability distribution 4.11 coefficient of variation of a random variable 4.11 commutability of a measurement procedure 11.26 compatibility 11.27 component 2.4 confidence interval 4.20 control material 13.10 conventional true value of a measurable quantity 2.13 correction 5.5 correction factor 5.6 detection iimit 11.19 deviation 5.2 dispersion 4.5 elementary entity 2.5 error of a result of a measurement 5.1 error of measurement 5.1 estimate, noun 4.14 estimator 4.13 etalon 13.2 expectation of a random variable 4.8 fractile of a random variable 4.19 generic quantity 2.7 good laboratory practice 15.1 higher limit of determination 11.21 indication of a measuring system 3.9 influence quantity 11.6 input measurable quantity 3.10 input quantity 3.10 482 interchangeability of a measurement procedure 11.25 interlaboratory measurement comparisons 14.3 interlaboratory test comparisons 14.3 intermediate precision conditions 9.1 intermediate precision of results of measurements 9.2 intermediate precision standard deviation 11.15 internal quality control 14.2 kind-of-quantity 2.6 laboratory accreditation 15.3 laboratory bias of results of a measurement procedure 7.4 laboratory component of bias of results of a measurement procedure 8.2 laboratory deviation 7.3 laboratory proficiency testing 14.4 laboratory sample 3.16 limit of detection 11.19 location 4.4 lower limit of determination 11.20 matrix blank measurement 11.11 measurable quantity 2.3 measurand 3.6 measurement 3.2 measurement procedure 3.5 measurement standard 13.2 measuring function 11.5 measuring instrument 3.7 measuring system 3.8 median 4.17 method of measurement 3.4 metrological calibration function 11.3 metrological performance characteristic metrological reliability 11.1 metrology 3.1 11.2 20935253 483 mistake 5.7 number fraction distribution 4.15 numerical value of a measurable quantity 2.10 observed value of a measurable quantity 3.1 1 outlier 4.23 overall expectation of results of measurements 7.1 parameter 4.7 performance characteristic 11.2 population 4.6 population parameter 4.7 practicability of a measurement procedure 12.1 precision conditions 6.5 precision of measurements 6.4 primary measurement standard 13.3 primary sample 3.15 primary standard 13.3 principle of measurement 3.3 probability 4.1 probability distribution of a random variable 4.3 proficiency testing 14.4 quality 1.1 quality assurance 1.5 quality audit 1.7 quality control 1.6 quality management 1.3 quality policy 1.2 quality system 1.4 quantity 2.3 quantity in a general sense 2.6 random error of a result of a measurement 5.3 484 random variable 4.2 reagent blank measurement 11.10 recovery measurement 11.12 reference material 13.7 reference measurement procedure 13.1 reference measurement standard 13.5 reference standard 13.5 regulation 14.7 relative frequency distribution 4.15 repeatability conditions 8.1 repeatability limit 11.14 repeatability of results of measurements 8.3 repeatability standard deviation 11.13 repeatability variance of results of measurements 8.5 reproducibility conditions 10. i reproducibility limit 11.17 reproducibility of results of measurements 10.2 reproducibility standard deviation 11.16 reproducibility standard deviation divided by analytical sensitivity 11.18 reproducibility variance of results of measurements 10.5 requirement 14.6 result of a measurement 3.12 robustness of a measurement procedure 11.23 sample 3.14 sample variance 4.18 sampling 3.13 secondary measurement standard 13.4 secondary standard 13.4 set 2.2 standard 13.2 standard deviation of a probability distribution 4.10 standard deviation of a random variable 4.10 statistic 4.12 statistical coverage interval 4.21 48 5 statistical outlier 4.23 system 2.1 systematic error of a result of a measurement 5.4 technical specification 14.5 tolerance interval 4.22 traceability 13.13 transferability of a measurement procedure 11.24 true value of a measurable quantity 2.12 trueness of measurement 6.2 truth 2.11 uncertainty of measurement 11.22 unit of measurement 2.9 value of a measurable quantity 2.8 variance of a probability distribution 4.9 variance of a random variable 4.9 variate 4.2 within-laboratory repeatability variance of results of measurements 8.4 working measurement standard 13.6 working standard 13.6 correspondence: renc dybkzr department of clinical chemistry frederiksberg hospital dk-2000 frederiksberg, danmark 486 upsala j med sci 81: 175-178, 1976 dimensions of the rabbit tenuissimus muscle c. michael childs' and karl-e. arfors from the department of experimental medicine, pharmacia ab, uppsala, sweden abstract a study of the physical dimensions of the rabbit tenuissimus muscle is described and compared with previous work on the same muscle from the cat. the muscle in the rabbit was found to be larger than in the cat, but was consistent in its dimensions and suitable for use as a model for microvascu lar research. introduction the cat tenuissimus muscle has been analysed by eriksson & myrhage (2). a similar study is de scribed of the rabbit tenuissimus muscle. data are presented which show the rabbit muscle to be more substantial than its counterpart in the cat, and reasons are suggested for differences found in muscle fibre diameter. material and methods new zealand white rabbits fed on a standard diet (tekno san pellets, al3 ferrosan, malmo, sweden) were used in this study. tenuissimus muscle from the legs of 40 rabbits (mean weight 1.0 kgk0.3 kg s.d.) (table i) were ex amined, after the rabbits were anaesthetised with urethane and the tenuissimus muscle exposed. examination of the muscle was camed out in several ways. the muscle length from origin to insertion and its width were measured in vivo with vernier calipers and its thickness with a micrometer eyepiece x 16 on a stereo microscope (leitz). before removal from the rabbit, some muscles were fixed either by bathing with 3 % glutaraldehyde (buffered with 0.075 % sodium cacodylate at ph 7.2) or by cannulation of the aorta and intra-arterial perfusion of the muscle with this solution. other muscles were perfused with indian ink (pelikan) after cannulation of the main artery supplying the muscle 1 cm before it entered the muscle. in this way all sections of the vascular bed were filled with carbon particles, making possible the measurement of vascular lengths and diameters by microscopy. attempts were made to ensure maximal vas ' present address: department of surgery, university of aberdeen, aberdeen, scotland. odilatation in some of these muscles by electrical stimula tion or by adding papaverine to the ink. after removal from the rabbit, each muscle was pre pared for measurement of vascular dimensions by fixation for a further 15 hours in 3 % glutaraldehyde and dehydra tion with ethanol. some of the muscles which had been perfused with ink were then placed in a mixture of 85% benzylbenzoate and 15% wintergreen oil (6, 8). by this procedure the muscle becomes quite transparent and ink filled vessels can be visualised. the diameters of ink-filled vessels, including capillaries, were measured under a microscope using a measuring eyepiece (x 12.5, leitz) and x6.5 x12.5 or x 2 3 objective lenses. the eyepiece was calibrated by viewing a micrometer slide. the lengths of vessels were measured by moving the microscope table under a cross-wire eyepiece, the table being connected to a potentiometer calibrated over a micrometer slide so that the distance the table moved was indicated as vascular length. the rest of the ink-perfused muscles, and those which had not been perfused, were embedded in epoxy resin (shell epon 812) for sectioning (4). 3 pm thick sections were cut with a glass microtome knife and stained with toluidine blue. wax embedding was found to give inade quate support to the muscle which disintegrated when sec tioned. for estimation of the distribution of capillaries relative to muscle fibres, photomicrographs (leitz ortho mat) were taken of these sections through x2.5 or x10 (leitz) microscope objectives. areas of muscle sections were measured from enlargements of these photographs with a digitizer (hd9107 a, hewlett packard). results the muscle was found to be 6.4 cm long in the left leg (k0.9 cm s.d.) with a variation in length per kg body weight of from 4.0 cm to 9.3 cm. its greatest thickness was found to be 1.1 mm (k0.2 mm s.d.) and the muscle was shaped like an aerofoil with its narrower angle anterior (fig. 1). the central vessels were located in the thickest part of the muscle run ning parallel to its edges (fig. 2). the width of the muscle was found to vary, being greater at the distal end of the muscle (mean 4.4 mmk0.8 mm s.d.) than a t its proximal end (mean 4.2 mmk0.7 mm s.d.) or mid-way along it (mean 4.2 vpsala j med sci 81 176 c . m . childs and k . e . arfors table i. summary of experimentalfindings measurements of length, width and thickness of the mus cle were made before removal from the rabbit no. of muscles mean value rabbit body weight, kg tenuissimus length left leg, mm 34 left leg, mm/kg b.wt. right leg, mm 26 right leg, mm/kg b.wt. 26 left leg, proximal, mm 26 left leg, mid-level 46 left leg, distal 42 right leg, proximal 25 tenuissimus length muscle width right leg, mid-level 37 right leg, distal 34 muscle width, mm/kg b.wt. left leg, proximal 25 left leg, mid-level 46 left leg, distal 42 right leg, proximal 25 right leg, mid-level 37 right leg, distal 35 1.050.3 s.d. 64.4f8.8 s.d. 65.1f 8.9 s.d. 65.1f11.9 s.d. 4.2k0.8 s.d. 4.250.8 s.d. 4.4k1.1 s.d. 4.3k0.8 s.d. 4 4k0.7 s.d. 4.5k0.8 s.d. 4.32 1.0 s.d. 4.35 1 .o s.d. 4.5k1.1 s.d. 4.4f1.1 s.d. 4.5f 1.1 s.d. 4.521.1 s.d. mmf0.8 mm s.d.) the width of the muscle was not significantly related to the weight of the rabbit, varying from 2.1-8.0 mmlkg body weight. there was no significant difference between the dimen sions of muscles from left and right legs. the ratio of the part of the muscle lying anterior to the central vessels to the part lying posterior to them was calculated and found to be 0.58 at its upper end, 0.59 at mid-level and 0.60 at the distal end of the muscle. the density of 10 muscles was calculated after weighing each and determining its volume by dis placement of saline in a 2 ml pipette. the mean density was found t o be 1.20 g/cc ( f o . l s.d.). areas of muscles from 2 rabbits were measured from photographs, as described above, to determine the cross-sectional area of muscle fibres (table 11). the cross-sectional area of each fibre was calcu lated to be 1022 pm2 (k.50 s.d.) corresponding to 978 muscle fibres per mm2 and a fibre diameter of about 36 pm. comment distance between arterior muscle in all its physical dimensions, the rabbit tenuis left leg, proximal 25 2.5f0.7 s.d. simus muscle was found to be more substantial than left leg, mid-level 46 its counterpart in the cat. at its thickest part it was nearly twice as thick (0.8-1.4 mm compared with left leg, distal 42 0.3-0.6 mm). like the cat muscle, however, it had right leg, proximal 25 right leg, mid-level 37 right leg, distal 35 2.6f0.7 s.d. an antenor part, accounting for just over half its edge and central vessels, mm 2.4ko.6 s.d. 2.750.7 s.d. 2.5f0.7 s.d. 2.6f0.6 s.d. ratio of anterior section to whole muscle width left leg, proximal left leg, mid-level left leg, distal right leg, proximal right leg, mid-level right leg, distal tenuissimus thickness, mm cross-sectional area of each calculated muscle fibre muscle fibre, pm2 diameter=36pm 25 46 42 25 37 33 14 4 0.58f0.1 s.d. 0.59k0.1 s.d. 0.60f0.08 s.d. 0.60f0.07 s.d. 0.60f0.08 s.d. 0.58f0.08 s.d. 1.1 f0.2s.d. 1 022f50 s.d. width, which tapered to become less than 0.1 mm thick at the anterior edge. this was clearly the most suitable area for blood flow studies a s transillumina tion of the muscle would be unlikely t o be satisfactory towards the thickest part. an illustra tion of the shape of the muscle cross section is given in fig. 1. the width of the muscle was found t o vary along its length between 4.2 and 4.4 mm (mean values) compared with 3-5 mm for the cat. measurements of length, thickness and width central outline fig. 1. diagram of a cross-section of the tenuissimus muscle. in calculations the area of this cross-section was approximated to that of the two triangles (shown as dotted vessels lines). upsala j med sci 81 muscle dimensions 177 table 11. data f r o m 2 rabbits f o r calculation of cross-sectional area of musclejibres and calculation of the area of muscle cross-section served by each capillary calculated cross sectional area of muscle muscle area measured number of number of per fibre per capillary cum3 muscle fibres capillaries (pm') (i*.m2) 53 187 54 29 985 1 8 3 4 37 225 38 17 980 2 190 23 893 22 14 1 0 8 6 1 707 13 436 13 9 1035 1 493 mean 1 022 (f50 s.d.) 1 806 ( f 2 9 2 s.d.) were made in unfixed muscles in situ so that no distortion occurred. some difficulty was experienced in obtaining satisfactory measurements for muscle fibre diame ter. it is reported by brhnemark & eriksson (1) that shrinkage or swelling of muscle fixed in 3% glutaraldehyde and embedded in epoxy resin is less than 5 % , but this was not the experience of this study. the change in muscle width during prepara tion vaned from a decrease of 1.7 mm t o an increase of 1.2 mm, representing an average change of width of 22% (table 111). a further problem was that there was sometimes shrinkage of muscle fibres leading to their separation along connective tissue planes. these changes did not occur with every muscle, however, and it was possible to calculate fibre central vessels running parallel to muscle fibres muscle insertion fig. 2. diagram showing the main artery and vein supply ing the muscle at about the mid-point in its length. diameter from muscles that showed no change in overall dimensions during fixation and whose fibres showed no separation because of shrinkage. calcu lation of average fibre diameter showed this to be 36 pm. eriksson & myrhage (2) found the diameter of type a fibres in the cat tenuissimus to be 55 p m compared with 41 p m for type b fibres and 26 for type c fibres. the mean diameter, taking the pro portion of each fibre type into account was found to be 44 pm. this value is higher than that found for the rabbit in this study and probably represents a species difference in fibre size as well as in the proportion of each fibre type present. no attempt was made in this study to differentiate between muscle fibres types. it has been suggested (2, 3 , 5) that connective tissue makes up between 20 and 30% of the bulk of skeletal muscle. photographs of sections of muscle in this study suggested, however, that connective tissue in the tenuissimus formed a much smaller proportion of the total bulk than this. if muscle fibre diameter is calculated, from the data obtained from the rabbit, on the assumption of an 8 : 2 ratio be tween muscle tissue and connective tissue plus vas cular tissue, the diameter of each fibre decreases to 32 pm. in their study of skeletal muscle fibre density in the gastrocnemius muscle (table iv), schmidt nielsen & pennycuik ( 7 ) found that this muscle contained only 14% of type b fibres, with a total fibre density of 518 fibres mm-2. eriksson & myr hage (2) found that the cat tenuissimus contained 48% of the smaller type b fibres but did not calcu late the density per square millimeter. in this study muscle fibre density was found to be 978 mm-2 and suggests that the relatively high percentage of type b fibres found by eriksson & myrhage ( 2 ) in the cat 12 -762853 upsala j med sci 81 178 c. m . childs and k . e . arfors table ill. mid-/eve/ muscle width changes with preparation each pair of values is from a separate rabbit ~ muscle width before after change in preparation preparation width (m) (pm) (w) 4.1 2.4 2.8 3.7 4.1 4.9 5.7 3.2 4.2 3.6 4.5 4.4 3.6 4.8 4.4 3.9 4.1 4.2 4.0 3.8 5.0 3.3 5.1 3.1 3.7 4.0 4.2 2.8 mean, 4.3 y m -1.7 +0.9 +0.8 -1.5 -0.6 -0.1 +l.2 -0.5 +o. 1 -0.2 -1.7 -2.0 +0.3 -1.4 mean, 0.93 y m tenuissimus is also present in the rabbit and ac counts for the greater number of muscle fibres per unit area. it is also likely that a species difference also accounts for the lower value for fibre diameter of 36 p m found in this study. it is not likely that fibre shrinkage during preparation is responsible because great care was taken to ensure that the overall dimensions of the muscles actually used was unchanged by preparation and that there was no microscopic evidence of muscle fibre separation in the areas measured. in the aerofoil shape of the rabbit tenuissimus muscle, cross section is treated as two triangles joined at their bases, its area can be estimated. calculated from the mean values found for width and thickness (table i) the cross-sectional area is 2.31 mm2. with a muscle fibre density of 978 mm-2 a cross section would contain 2 259 fibres reflecting the rabbit muscle’s greater size than the cat tenuis simus where eriksson & myrhage found 1 375 fibres in a cross section, conclusion this study of the rabbit tenuissimus muscle pro vides information for comparison with already published data for the cat. no attempt has been made at fibre-typing the muscle, although the re sults suggest it to be of a similar mixed type to that table iv. distribution o f f i b r e types in the tenuis simus muscle of the cat (eriksson & myrhage, 1972) ( 2 ) mean type of muscle fibre a b c value distribution of fibres, muscle fibre diameter, number of capillaries % 38 48 14 p m 55 41 26 44 surrounding one muscle fibre 3.5 3.6 3.8 3.6 in the cat but with a different distribution of fibre types. although physically more substantial than its counterpart in the cat, the rabbit tenuissimus is confirmed as a muscle of sufficiently small dimen sions to be of value in microvascular research. acknowledgement we thank mr ove forsberg for skilful technical as sistance. references 1. brlnemark, p. i. & eriksson, e.: method for studying qualitative and quantitative changes of blood flow in skeletal muscle. acta physiol scand84: 284-288, 1972. 2. eriksson, e. & myrhage, r.: microvascular dimen sions and blood flow in skeletal muscle. acta physiol 3. hammersen, f.: the pattern of the terminal vascular bed and the ultrastructure of capillaries in skeletal muscle. in oxygen transport in blood and tissue (ed. d. w. lubbers eta].), pp. 184-195, 1968. 4. luft, j. h.: improvements in epoxy resin embedding methods. biophys biochem cytol j 9:409414, 1961. 5. pfaff, g. h.: a quantitative study of the capillary sup ply in certain mammalian skeletal muscles. anat rec 46: 401406, 1930. 6. romeis, b.: mikroskopische technik, p. 200, old enbourg, miinchen, 1948. 7. schmidt-nielsen, k. & pennycuik, p.: capillary den sity in mammals in relation to body size and oxygen consumption. am j physiol2w: 746-750, 1961. 8. spalteholz, w.: die verteilung der blutgefasse im muskel. k. s. ges d wiss 24: 507-532, 1888. scmd86: 211-222, 1972. received august 5, 1976 address for reprints: karl-e. arfors, ph.d. pharmacia ab department of experimental medicine box 181 s-75104 uppsala sweden upsala j med sci 81 upsala j med sci 78: 181-188, 1973 effects of lndomethacin on the transcapillary leakage of macromolecules and the efflux of prostaglandins in the paw lymph following experimental scalding injury gosta arturson and carl-evert jonsson from the burn center, department of plastic surgery, university hospital, (lppsala, sweden abstract transport of macromolecules (dextrans and proteins) from blood to lymph and efflux of prostaglandins into lymph were studied in dogs following scalding injury of the paw and treatment with indomethacin. indomethacin inhibited the efflux of prostaglandins following scalding injury, in dicating an inhibition of the biosynthesis of prostaglandins. a pronounced suppression of the increased lymph flow and transcapillary transoort of macromolecules following scalding was found after treatment with indomethacin. the increased microvascular permeability in scalded tissue was not significantly altered by indomethacin. these results indicate that the major effect of indomethacin on the microcirculation in the scalded tissue is a reduction of the capjllary surfaee area available for exchange due t o a re duced number of capillaries perfused with blood. the re sults d s o support the hypothesis that some of the vascular reactions following thermal injury may be mediated by prostaglandins. introduction prostaglandins, a group of biologically active lipids (for reviews see 9, 20, 22, 41) have been demon strated in tissue fluids following chemical in flammation (44, 4 3 , scalding injury (2, 24) and anaphylaxis (16). recently it was shown that scald ing injury in guinea pig is followed by an increased biosynthesis of prostaglandins (1 8). prostaglandin e, (pge,) is the major prostaglandin found in inflammatory fluids. the vascular effects of this compound fulfil many of the criteria as demanded by a chemical mediator of the inflammatory re sponse (37). evidence has accumulated for structural changes of the blood-lymph-barrier, i.e. increased micro vascular permeability (4, 29), increased capillary surface area due to dilatation of resistance vessels as well as increased tissue osm2tic forces in the burn wound (8). recently it was shown that drugs like aspirin and indomethacin inhibit the biosynthesis of pros taglandins (33, 39), suggesting that these drugs owe their anti-inflammatory effects to this mech anism. in the present communication we report effects of indomethacin (47) on prostaglandin efflux in paw lymph and on microvascular reactions follow ing scalding injury. material and methods six healthy dogs of the vorsteh-type, weighing between 17 and 28 kg and of the same breed, were used. all experi ments were done using sodium pentobarbital anaesthesia (nembutalo, abbott). f o r induction of anaesthesia a dose of 30 mg per kg of body weight was given. small additional doses were given when required. t h z dog was placed on an operating table equipped with electric heat ing to maintain a normal body temperature; this was con trolled throughout the experiment. free airway was ascertained by endothracheal intubation. ringer solution was given as a slow intravenous infusion during the ex periments in a dose of 5 ml per kg of body weight per hour. peripheral lymph was drained from the dog's hind paw via a short pe 50 intramedic polyethylene cannula (clay adams, inc., new york). th: operative procedures and methodological details have been published elsewhere (4). lymph flow was facilitated by regular, passive move ments of the paw. lymph was collected in ice-chilled poly ethylene tubes for assay of dextran, protein and prosta glandins. the thermal trauma was inflicted by immersing the animal's paw for 10 sec in water at 100°c oj for 20 sec in water at 70°c. this was done about 180 min affer clamping of the renal pedicles. four dogs were treated with indomethicin (10 o r 2 0 m g / kg body weight) intravenously immediately after scalding and onhour later except one dog which only received a single dose of indomethacin (20 mg/kg body weight). lndomethacin (merck, scharp and dohme, rahway, n.j., usa) was dissolved in 0.15 m p3tassium phosphate buf fer, p h 7.4, immediately prior to administration. two dogs served as controls and received n o treatment af:er scalding other than ringer solution. u p ~ ! u j m e d sci 78 182 g . arturson and c.-e. jonsson permeability studies the local microvascular permeability in the scalded tissue was studied before and at different times after the trauma. d:xtran was used as test substance. rheomacrodexo,, 10% in 0.9 saline, average molecular weight, m , = 40 000 (range 5 000-90 000) was given slowly in a dose of 0.5 g / kg bady weight. this low dose produces only very small changes in the plasma volum-, (15). before t h s dextran in fusion, th: renal pedicles were clamped to prevent th? rapid elimination of dextran molecules of low molecular weight via the kidneys. after an equilibration period of 120 min the molecular weights of dextran molecules in lymph and plasma were determined and the lymph/plasma concentration ratio ( c , / c p ) of different molecular sizes was calculated. the total concentration of dextran in plasma and lymph was determined by the anthrone method (23, 40). this methjd has an error of 1.8% s.d. (40). the total concentration of protein in lymph was deter mined according to a modified folin method (3). the molecular weight distribution of dextran was estimated by gel chromatography adapted t o automated routine and computer analysis ( 6 ) . the sensitivity, repro ducibility and resolving power of the gel chromatography method has been thoroughly investigated (13, 27). the accuracy of the determination of dextran molecular weight distribution is in the range of 3% s.d. (granath, personal communication). in all experiments the regional transport of dextran across thz blood-lymph barrier was calculated according to: c , j c , . v j t = t where c,, and c , (in mg/ml) is the concentration of dextran i n lymph and plasma respectively, and v the volume (in ml) of the lymph collected during the sampling period t (in min). t will then be equal to the net transport of th: substance from blood to the collected lymph can nula in mg/min at unit plasma concentration ( = 1 mg/ml). the physical dimension of t is actually ml/min. the cp value used in this ca!culation corresponded t o the plasma concentration at the middle of the lymph sampling period. it was not possible to correct for the time delay bstween thactual transport through the capillary wall and the collection from the lymph cannula. determination of prostaglandins lymph was precipitated in absolute ethanol containing 0.1 % d,l a-tocopherol as antioxidant and stored at 20°c before processing. lipid extracts were prepared as de scribed by unger et al. (38) and subjected to silicic acid chromatography (30) prior to bioassay. the silicic acid columns (unisil, 0.5 g, 100-200 mesh, clarkson chemical corp., williamsport, penns.) were made up in ethyl acetate-benzene (1 : 9, v/v) and eluted with 20 ml of ethyl acetate-benzene (1 : 9) followed by 20 ml of ethyl acetate. thes-, fractions were evaporated under reduced pressure and assayed for smooth muscle stimulating activity on colon of the gerbil (42). the recovery was determintd in each sample by addition of *h-pge, (0.1 pc, specific activity 87.3 c/mmole, new england nuclear corp., boston, mass.), to the lymph-ethand mixture and deter upsala j m e d sci 78 mination of radioactivity in aliquots of the ethyl acetate fractions and was about 50% ( x = 49.9, s.d. = 9.1,. n = 74). when 50 ng pge, was added to 5 ml human plasma and processed as described the recovery of smooth muscle stimulating activity was similar (n = 4 5 % , s.d. = & 1 8 , n = 17). indomethacin (100 p g / m l plasma) did not affect the recovery of smooth muscle stimulating ac tivity of pge, (35, 39). in all assays t h t colon of the gerbil responded to 0.5 ng pge, added to the organ bath. levels below 2 ng per sample could not be detected and were thus regarded as zero values. smooth muscle stimu lating activity was expressed in terms of pge, after cor rection for recovery of radioactivity. radioactivity was determined in a packard liquid spectrometer (model 3320). 10 ml of instagel (packard instr.) was used as scintillator. corrections for counting efficiency were made by use of external standardization. results transcapillary leakage of macromolecules no significant differences were found in lymph flow, in protein o r in dextran concentration of the lymph drained from the different paws before in fliction of the thermal trauma. the changes in sieving ratio ( c l / c p ) in the scalded areas were found to be related to the severity of the burn trauma, being somewhat less pronounced after scalding for 20 sec at 70°c than for 10 sec a t 100°c (figs. 1 and 2). the micro vascular permeability and the lymph flow were maximal about 1-4 hours after scalding. after immersion of a dog’s paw for 20 sec in water of 70°c the lymph flow increased about 10 times, the protein concentration of the lymph in creased from 0.7 to about 3.0 g/100 ml and the relative amount of dextran transported transcapil lary (t-values) increased 20 times (fig. 1). the lymph plasma dextran concentration ratio ( c l / c p ) for molecular weights of 10 000-80 000 increased immediately after the trauma and reached max imal values 1-3 hours post burn (fig. 1). after scalding of another dog’s paw for 20 sec at 70°c and treatment with indomethacin (10 mg/ kg body weight), repeated twice, immediately and 60 min after the burn, a pronounced decrease of the lymph flow and a slight reduction of the total protein concentration in the lymph compared with non-treated dog was observed (fig. 1). the relative amount of dextran transported transcapillary from the blood to the extravascular space after scald ing was reduced to between 25 and 35% of the amount transported transcapillary in the non treated dog (fig. 1). the c , / c p ratios for dif ferent molecular weights of dextran were about effects of indomethacin on the transcapillary leakage o f macromolecules 183 [controd [treatment with indomcthacinl min ( e a t r a n ) 1.2 1.0 0.8 0.4 0.2 0 i h o u r p o s t burn m o i weight x l o 3 i 300 min o 2 1 o j & ' ' " t " r m ~ 1 0 200 300 min p r p h u r n 0 20 60 60 80 m o i w e i g h t x 10.' min o 5 1 1 ° j bl h o v r p o s t burn 0 5 moi weigh1 x l o ' [friatment with indomethacin 1 mllrnin o b1 paw burn u ( n . 2 0 ~ ) k m p ii mplkp b.w. 3 hours posl bvrn 0 5 "1 \\i hour posl burn o j , l , p r e b u r n 0 20 40 60 80 moi w e i g h t i 16.' fig. 1. lymph flow, total protein concentration of lymph, t-values, efflux of prostaglandins and c , / c , ratios for dextran in lymph from paws scalded for 20 sec at 70°c in two dogs, one without treatment and one treated with indomethacin 10 mg/kg body weight immediately and one hour after scalding. _ _ _ _ _ , lymph flow 0-0, protein conc. fig. 2. lymph flow, total protein concentration of lymph, t-values, efflux of prostaglandins and c , / c , ratios for dextran in lymph from paws scalded for 10 sec at 100°c in two dogs, one without treatment and ons treated with indomethacin 20 mg/kg body weight immediately and one hour after scalding. _ _ , lymph flow; 0-0, protein conc. upsala j m e d sci 78 184 g . arturson and c.-e. jonsson \treatment with indomethacin i 0 100 200 300 10mg 10mg/kg b.w. 0 0 min cone. j l j f , , , . , , , , . , , , , , , , , , , , . . , , , . . . . i b h mg/min at unit cp 0.2 (dextran) 01 u 0 100 2 00 300 mi n n g pge2 o 2 ' k h j 200 300 0 100 mi n n g pge2 o 2 ' k h j 200 300 0 100 rnin 3 hours post burn 1 hour post burn 0 0 20 40 60 80 mol weight x fig. 3. lymph flow, total protein concentration of lymph, t-values, efflux of prostaglandins and c,/c, ratios for dextran in lymph from a paw scalded for 10 sec a t 100°c in one dog, treated with indomethacin 10 mg/kg body weight immediately and one hour after scalding. _ _ , lymph flow; 0----0, protein conc. equally increased in both the scalded non-treated and treated paws (fig. 1). figs. 2 and 3 show results from a comparative study in three dogs of the microcirculation in scalded tissue and with treatment with indo methacin a t two dose levels. the scaldings were made in exactly the same way in all three dogs, i.e. immersion of the paws for 10 sec in water of 100°c. the lymph flow, the total protein con centration of lymph and the 7'-values increased immediately after scalding and reached the same level in the non-treated dog and the dog treated with indomethacin in the low dose (10 mg/kg body weight). following scalding and treatment with indomethacin in a dose of 20 mg/kg body weight the increase in lymph flow and the t values were reduced to 30-40% of those found in the scalded non-treated dog (figs. 2 and 3). the sieving ratios for dextran molecules of upsala j m e d sci 78 various sizes in lymph/plasma (c,/c,) increased following the thermal trauma in a similar,way in all three dogs irrespective of treatment or not (figs. 2 and 3). a study of the microcirculation in scalded tissue with and without treatment with indomethacin made on one and the same dog is shown in fig. 4. a burn trauma on the right hind paw resulted in increases in lymph flow, transcapillary transport of dextran (t-values) and in the c l / c p ratio for all dextran molecules in the preparation. two hours after the first trauma a second one was made in exactly the same way on the left hind paw and the dog was treated with indomethacin (20 mg/kg). this resulted in less pronounced in creases in lymph flow and t-values compared t o the scalded non-treated paw (fig. 4). no signifi cant differences were found in the c,/c, ratios obtained from the two paws (fig. 4). efflux of prostaglandins in lymph after scalding of the dog's paw for 20 sec a t 70°c and for 10 sec at 100°c and without treat ment there was a rapid increase in the concen tration of prostaglandins in the lymph drained from scalded tissue with a decline after about 150 min. this was followed by a second increase of the efflux of prostaglandins in the lymph 3-5 hours after the trauma (figs. 1 and 2). after treatment with indomethacin in a dose of 10 o r 20 mg/kg body weight, administered immediately after scalding and one hour later, prostaglandins were not recovered in the paw lymph (figs. 1 , 2 and 3). in the study with and without treatment with indomethacin made on one and the same dog total inhibition of the efflux of prostaglandins was found following treatment (fig. 4). also on the previously scalded non-treated paw the amount of prostaglandins in the lymph decreased to zero values concomitant with the intravenous adminis tration of indomethacin two hours after the first thermal trauma. discussion i n the acute phase of a burn injury there is a rapid loss of intravascular fluid into the burned area. the fluid loss has been ascribed to func tional disturbances in the blood lymph barrier, increased effective filtration area and increased tissue osmotic forces (8). by selecting appropriate effects of indomethacin on the transcapillary leakage of macromolecules 185 (dextran) 0 50 100 150 min m i n [ treat m e n t with indomet hact n 1 mllmin paw burn 0.2\;] 0 0 0'23 0 0 1.01 rnin 120 rnglkg b.w. 0 50 100 150 rnin 0 50 100 1% min fig. 4. lymph flow, total protein concen tration of lymph, t-values, efflux of prosta glandins and c l / c , ratios for dextran in lymph following scalding for 20 sec at 70°c of two paws of a dog, one paw without 2 hours post burn ted pre burn and one and two hours after 0 20 4 0 60 80 0 20 4 0 60 80 the trauma, respectively. -, lymph flow: 0-0, protein conc. i m o l . weight x l o j mol. weight x l o 3 treatment and one paw treated with 20 mg indomethacin per kg body weight immedi ately after scalding. t h e c , / c , ratios for dextran molecules of various sizes are plot2 hours post burn thermal stimuli to experimental animals two phases of vascular reactions have been discerned, an immediate and a delayed phase (32). vaso active-compounds have been suggested t o mediate some of these vascular reactions following injury (37, 46). attempts have been made to modify the vas cular reactions following thermal injury by drugs. spector & willoughby (36) demonstrated that by treating rats with mepyramine maleate, a power ful antihistamine drug, prior t o moderate thermal injury, the immediate vascular reactions, expressed as leakage of trypan blue and oedema formation, were suppressed. administration of salicylate sup pressed both phases. these observations indicate that histamine is involved only in the immediate response to thermal injury suggesting that his tamine may initiate vascular reactions which are maintained by other mechanisms. the occurrence of pge-compounds in inflam matory fluids (2, 16, 24, 44, 45) and the vascular effects of these compounds suggest that they participate in the inflammatory response. paren terally administered pge-compounds induce hypo tension due to vasodilatation and reduced periph eral resistance (11, 14, 28). intradermally ad ministered pge-compounds elicit erythema and oedema (10, 25, 34) due to vasodilatation. in rats and guinea pigs, preinjected with protein bound dyes, pge-compounds induce extravasation of color on intradermal administration (10, 21, 2 6 ) . this effect has been claimed to be due to in creased capillary permeability. in canine adipose tissue pge, is a powerful vasodilatating agent with comparatively small effects on capillary per meability (1 1). recently it was demonstrated that anti-inflam matory drugs like aspirin, sodium salicylate and indomethacin inhibit the synthesis of prostaglan dins (12, 17, 33, 39). this was found both in cell free homogenates and in the whole animal. in the present investigation the effect of indo methacin on the efflux of prostaglandins in lymph drained from scalded tissue and the effect on the transcapillary leakage of macromolecules into the burn oedema was studied simultaneously. the efflux of prostaglandins in the lymph was assumed to reflect the biosynthesis of prostaglandins in the tissue. the sieving ratio for dextran molecules of various sizes in lymph/plasma (c,/c,) was meas upsala j m e d sci 78 186 g . arturson and c.-e. jonsson ured and used as a n index of the microvascular permeability. recently arturson ( 5 ) demonstrated a moderate suppression of the increased micro vascular permeability after scalding by 0-(p-hy droxyethy1)-rutosides (hr) a compound with anti oedematous effects (48). in the present investigation the scalding injury resulted in efflux of prostaglandins (cf. 2 , 24) con comitant with a n increased lymph flow and an increased concentration of both protein and dex tran in the lymph drained from scalded tissue (cf. 4). the sieving ratio ( c l / c p ) and the regional transport of dextran across the blood-lymph bar rier also increased. this indicates an increase of the microvascular permeability after scalding (cf. 4, 7 ) . in all dogs except one treatment with indo methacin resulted in a suppression of the increased lymph flow, a slight reduction of the total con centration of protein in lymph and a pronounced reduction of dextran transported from the blood to the extravascular space as compared to scalded non-trcated dogs. the sieving ratios for dextran molecules of various sizes in lymph/plasma were unaffected by the treatment with indomethacin. no smooth muscle stimulating activity was found in lymph fluid after scalding and subsequent treat ment with indomethacin in any dogs. the inhibition of prostaglandin efflux indicate an inhibition of prostaglandin biosynthesis by indomethacin. i t seems improbable that in the tissue with less rapid lymph drainage there was an increased metabolism of prostaglandin e-com pounds into metabolites without biological activity ( 1 ) . this is supported by the finding that no smooth muscle stimulating activity was recovered after scalding and subsequent treatment with indo methacin in a low dose (10 mg/kg twice) which did not affect the lymph flow (cf. fig. 3). the mechanism underlying the effects of indo methacin on the microcirculation in scalded tissue is not clear. i t could be either a decreased capil lary surface area available for exchange due to a reduced number of capillaries perfused with blood, or t o a suppression of the increased micro vascular permeability caused by the thermal trauma, or both. the very small changes of the sieving ratios for dextran molecules of various sizes in lymph/plasma ( c l / c p ) and the pro nounced suppression of the lymph flow as well as the amount of dextran transported transcapillary upsala j m e d scr 78 following treatment with indomethacin indicate that a reduction of the effective capillary surface area is the most important mechanism for the ef fect of indomethacin. lately a modulatory role of prostaglandins on the sympathetic nervous transmission has been implied (19). evidence exists to suggest that pros taglandins inhibit the release of norepinephrine (43). blocking of the local formation of prosta glandins leads to a considerably increased release of norepinephrine on sympathetic stimulation (31). in the same way it may be suggested that after scalding injury and treatment with indomethacin prostaglandins are not formed in sufficient amounts to oppose the increased secretion of catecholamines. however, removal of the vaso dilatory effect of prostaglandin e-compounds may also be one explanation. as it has not been demonstrated that indomethacin exclusively in hibits prostaglandin biosynthesis, other effects of the drug itself cannot be excluded. no suppressive effect on the lymph flow or the transcapillary macromolecular transport was ob served in the dog with a paw immersed for 10 sec in water of 100°c and treated with indomethacin in the low dose (fig. 3) although no pge, was found in the lymph. this might indicate either that the efflux of prostaglandins in lymph is not a satisfactory estimate of the prostaglandin bio synthesis in tissues or that the changes of the microvasculature are not mediated via pge,. the fact that clear suppressive effects on both lymph flow and transcapillary transport of dextrans were found following scalding for 10 sec at 100°c and treatment with indomethacin in high dose (fig. 2) favours the hypothesis that som: of the vascular reactions after scalding injury are mediated via the prostaglandin system. acknowledgement this work was supported by swedish medical research council (projects no. b73-40y-2370-06 and b73-40x-676 08) and pharmacia ab, uppsala, sweden. references anggbrd, e.: the biological activities of three meta bolites of 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pharm pharmac 21: 126, 1969. upsala j m e d sci 78 188 g . arturson and c.-e. jonsson 45. identification of prostaglandin e, in rat inflam matory exudate. pharmacol res com 2:297, 1970. 46. winkelmann, r. k.: molecular inflammation of the skin. j investig dermatol 57: 197, 1971. 47. winter, c. a., risky, e. a. & nuss, g . w.: anti inflammatory and antipyretic activities of indometha cin, l-(gchlorobemoyl)-5-methoxy-2-methylindole-3 acetic acid. j pharmac exp thiherap 141: 369, 1963. 48. wismer, r.: the action of tri-hydroxyethyl-rutoside on the permsability of the capillaries in man. praxis 52: 1412, 1963. received december 19, 1972 address for reprints: carl-evert jonsson departmmt of plastic surgery university hospital s-750 14 uppsala 14 sweden upsala j m e d sci 7 8 upsala journal of medical sciences 2022, 127, e7823 http://dx.doi.org/10.48101/ujms.v127.7823 violence and sexual risk taking reported by young people at swedish youth clinics sofia hammarströma,b, siw alehagenc and helena kilanderc,d,e,f aregion västra götaland, knowledge center for sexual health, gothenburg, sweden; bdivision of society and health, department of health, medicine and caring sciences, linköping university, linköping, sweden; cdivision of nursing sciences and reproductive health, department of health, medicine and caring sciences, linköping university, linköping, sweden; ddepartment of obstetrics and gynaecology, eksjö hospital, jönköping, sweden; ejönköping academy for improvement of health and welfare, jönköping university, jönköping, sweden; fdepartment of women’s and children’s health, karolinska institutet, sweden abstract background: early identification of sexual risk taking and exposure to violence is fundamental when seeking to strengthen young people’s health. the purpose of this study was to study factors associated with sexual risk taking and ill health, as well as to study gender differences, and the associations amongst exposure to multiple forms of violence, sexual risk taking and ill health. methods: this was a cross-sectional study based on data from 3,205 young people answering a questionnaire belonging to the sexual health identification tool (sexit 2.0), during consultations at 12 youth clinics in sweden. the analyses are based on descriptive statistics and nominal multiple regression analysis. results: male, transgender and non-binary youths reported significantly more events of sexual risk taking and ill health compared to women. those who reported sexual initiation before the age of 15 (or 2.87, ci 1.81–4.56), three or more sexual partners in the past 12 months (or 2.68, ci 1.70–4.22) and to have ever experienced an unintended pregnancy (or 2.29, ci 1.32–3.97) were more than twice as likely to report exposure to physical, emotional and sexual violence. transgender, non-binary youths and women were more exposed to multiple violence (or 3.68, 13.50) compared to men. conclusions: transgender and non-binary youths are exposed to significantly more violence compared to women and men. experiences of sexual risk taking and ill health demonstrated strong associations with exposure to multiple violence. article history received 20 april 2021 revised 26 november 2021 accepted 1 december 2021 published 21 january 2022 keywords gender identity; transgender youth; violence; risk taking; sexual health; re-victimisation; poly-victimisation; cumulative violence introduction globally, young people defined as the 10–24 years age group are disproportionally burdened by sexual and reproductive health problems, such as sexually transmitted infections (stis), unintended pregnancies (1) and exposure to sexual and genderbased violence (2). these experiences not only affect health and well-being directly, but, as experiences during a critical development phase in life, may also lead to long-term negative effects (3). early detection of young people in need of care or at risk of sexual ill health or violence victimisation is important in order to provide adequate care and preventive measures (4). sexual risk taking is commonly defined as risking an unintended pregnancy and/or sti by not using a condom or other contraception. previous experience of chlamydia infection and unintended pregnancy can also be considered risk factors, as they are associated with repeated chlamydia infections (5, 6) or unintended pregnancies (7). other factors found to be associated with sexual risk taking are substance use (8–10), early sexual initiation (<15 years) (11) and multiple sexual partners (5, 12). additionally, some experiences may increase the risk of unprotected sex through limited or absent ability to negotiate safer sex, such as transactional sex and violence victimisation (13). concerning violence, the consequences on health include depression (14, 15), anxiety (15), post-traumatic stress disorder (14, 15), substance abuse (14, 16), self-injury (14), suicidal behaviour (17) and poorer self-rated health (16), amongst others. in a swedish study, the prevalence of violence victimisation was shown to be 10 times more common amongst youths than amongst adult samples (18). most studies have focused on a single type of violence (physical/emotional/sexual) or a specific situation, i.e. dating violence, bullying or child sexual abuse. however, violence exposure is rarely an isolated event (8), and physical, emotional and sexual violence often co-occur (18, 19). young people (20, 21) as well as adults (22) with multiple violence exposure report more ill health than those suffering from repeated exposure to only one type. there are gender contact helena kilander helena.kilander@rjl.se © 2022 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article http://dx.doi.org/10.48101/ujms.v127.7823 mailto:helena.kilander@rjl.se http://creativecommons.org/licenses/by/4.0/ 2 s. hammarström et al. patterns where men report more physical abuse, whilst women report more exposure to emotional and sexual abuse (18). however, knowledge of the impact of multiple forms of violence on the lives of young people is limited, and the connection to sexual risk taking and ill health has been little explored. blom et al. (23) found an increased risk for experience of pregnancy, chlamydia, early sexual initiation and ≥3 sexual partners in both men and women exposed to multiple forms of violence. as their measure of multiple violence included sexual violence, a question warranting exploration is whether the elevated risks are predominantly related to being exposed to multiple forms of violence, or if they are primarily attributed to sexual violence victimisation. the associations between exposure to multiple forms of violence and sexual risk taking have, to our knowledge, not been studied amongst transgender and non-binary youths. in this study, we use ‘transgender and non-binary’ to refer to young people whose sex assigned at birth does not match their current gender identity. studies suggest that transgender and nonbinary youths are more exposed to discrimination and violence victimisation than their cis-gendered peers (24–26), resulting in both poor mental health (25–27) and negative effects on sexual health, such as hiv (28), avoidance of sexual situations out of fear, abandoning safer sex practices at times of mental ill health and lack of access to sexual health services (29). swedish studies suggest that female youth clinic visitors are more exposed to violence (31%) (19) than young women in general (21%) (23). this makes youth clinics a suitable arena not only to study relationships amongst violence victimisation, sexual risk taking and sexual ill health but also to clinically address the associated health needs. sweden has a nationwide system of youth clinics, providing a wide range of services related to sexual and reproductive health and rights (srhr) and psychosocial health concerns, and accessible to young people between the ages of 12 and 25 (30). most visitors are women (89%), often explained by a focus on contraceptive services (30). data on youth clinic utilisation are lacking. one study found that 16% of youths had visited a youth clinic in the past 3 months (31). in recent years, many youth clinics have introduced a tool, sexit (sexual health identification tool), aiming to systematically address sexual risk taking, ill health and violence exposure with all visitors, through a questionnaire (32). this provides a unique opportunity to study a large sample of youth clinic visitors from different parts of sweden, regarding multiple forms of violence and sexual risk taking. the aim of this study was to study factors associated with sexual risk taking and ill health, as well as to study gender differences, and the associations amongst exposure to multiple forms of violence, sexual risk taking and ill health. materials and methods design/setting this study is based on quantitative results from a questionnaire belonging to the sexit 2.0 (32), within a framework of quality  improvement (qi) and collaboration between youth clinics and the knowledge centre for sexual health in the southwest of sweden. the qi involved implementation of sexit in clinical practice. procedure and participants to reach a range of youth clinics covering different socio demographics in sweden, youth clinics were invited from participating counties with traditionally different statistics in abortion and stis. information about the study was sent to the heads of 12 youth clinics participating in the qi, and all agreed to participate. youth clinic visitors were offered the opportunity to answer the questionnaire in connection to their index visit. after the  visitor had answered the questionnaire, a healthcare professional (hcp) had a dialogue with the visitor about the responses to make a risk assessment regarding the visitor’s needs for support or treatment. in addition, the hcps were trained to have a dialogue about sexuality and to use the handbook, which apart from suggesting follow-up questions also clarifies which answers indicate risk exposure and recommends relevant measures. data from the questionnaires were evenly collected over 6 months by all youth clinics in the period 2017–2019. sexit 2.0 sexit was developed to facilitate the identification of young people exposed to, or at risk of, sexual ill health and includes three components: 1) a training of hcps, 2) a questionnaire for visitors and 3) a handbook for hcps to support the dialogue and risk assessment. the questionnaire includes 20 items concerning age, gender identity, sexual orientation, living situation, alcohol and drug uses, experiences of violence, age at time of first sexual initiation and number of sexual partners during the past 12 months. furthermore, the questionnaire includes items about the experience of unintended pregnancy and stis, use of contraception and protection against stis, sex for reimbursement and experiences of coercing/forcing someone sexually. the development of sexit 2.0 was based on a previous pilot study (32) where the instrument was developed, validated and pilot-tested at three swedish youth-clinics. based on the results of the pilot implementation, the authors further developed sexit by including items regarding physical and emotional violence, witnessing violence, protection against sti and pregnancy and experience of stis other than chlamydia, resulting in version 2.0. the content validity of sexit 2.0 was established by an expert panel. most of the variables in the questionnaire have been previously validated (32). the questionnaires did not gather social security numbers, names or contact details. each questionnaire had a unique code to enable digital processing, but the code was not connected to an individual. as no personal data were collected in the study, the swedish ethical review authority found no need for ethical approval (#: 2019/03628). localized igg4-related disease manifested on the tongue 3 variable definitions the gender identity alternatives ‘transgender’, ‘other gender’ and ‘do not wish to categorise myself ’ were summarised into the category ‘transgender and non-binary’. variables associated with sexual risk taking and ill health according to previous research were dichotomised as follows: alcohol use two or more times a month, ever drug use (8–10), sexual initiation before 15 years of age (11), three or more sexual partners during the past 12 months (12, 23), ever experience of own or partner’s unintended pregnancy (1), ever experience of chlamydia (5, 6), ever experience of sex for reimbursement and ever experience of coercing/forcing someone sexually (9). the response option ‘don’t know and yes’ for the items regarding unintended pregnancy, chlamydia and coercing/ forcing someone sexually was considered as sexual risk taking or ill health. experiences of multiple violence were defined as experiences of more than one category of violence, including physical, emotional and sexual violence. physical violence was defined as being beaten or hurt. emotional violence was defined as being threatened, harassed or bullied. sexual violence was defined as being victim of coerced/forced sex, i.e. unwanted sexual experiences including vaginal/oral/anal sex or touching someone’s genitals. data analysis of the 3,576 questionnaires, 16 were excluded due to missing values of gender and/or age. visitors who answered that they had had not initiated sex were asked to finish the questionnaire before the questions on sexual history started. these participants (n = 355) were excluded from the analysis, resulting in a total of 3,205 questionnaires. the internal attrition rate in individual items was below 3%. data grouped by gender were analysed by using descriptive statistics. categorical data were analysed with chi-square test. if more than 20% of the cells have an expected value <5, we used fischer’s exact test when calculating p-values. the mann– whitney u test is used in ordinal scales with three or more alternatives when calculating p-values. we analysed gender differences in dichotomised variables associated with sexual risk taking as well as violence victimisation. multinomial regression analysis was used to yield a model predicting the most important explanatory variables associated with the seven different groups of violence victimisation. the variables alcohol and drug use, sexual initiation before 15 years of age, three or more sexual partners during the past 12 months, to have ever experienced their own or partner’s unintended pregnancy and to have ever had chlamydia were found significant in the model fitting of the multinomial regression analysis and therefore included. seven different groups of violence were entered as dependent variables, and the independent variable no violence was entered as a reference. in the next step, gender was included for analysing gender differences with men entered as reference. age as a continuous variable was included as a covariate in the analysis. the multinomial regression analysis indicated an overall classification of 67%, and the results are presented as odds ratio (or) with 95% confidence interval (ci). the variables regarding sex for reimbursement as well as experiences of coercing/forcing someone sexually were not included in the analysis due to limited data. statistical analyses were performed using ibm spss statistics version 22.0. results demographics, sexual risk taking and ill health around 80% of the participants were heterosexual women aged 18–25 years old. some gender differences could be observed: being bisexual was more common amongst participants in the transgender group and amongst women compared to men. across genders, most participants reported living with their parents. however, more women reported living with a partner, compared to the other gender groups (table 1). half of all participants reported using alcohol twice a month or more. experience of ever having used drugs differed from 19 to 41% between gender groups (table 2). men reported significantly more use of alcohol and drugs compared to both women and the transgender group (tables 1 and 2). the reported age of first sexual initiation ranged from 10 to 24 years of age (mean 16, sd 1.7). furthermore, 18% of all participants reported sexual initiation before the age of 15 years, and this was more common amongst the participants in the transgender group. the number of sexual partners in the past 12 months varied between 0 and 38 (mean 2.7, sd 3.1). men reported significantly more sexual partners during the past 12 months (table 1), and more often three or more partners compared to women and the transgender group (55% vs 35%, p = 0.002) (table  2). men and transgendered participants reported experience of  unintended pregnancy to a greater extent compared to women (16% vs 9%, p = 0.020) (table 2). furthermore, men and transgendered participants reported never having used protection against unintended pregnancy more often, compared to women (10–13% vs 5%, p = 0.002) (table 1). amongst reported variables associated with sexual risk and ill health, the three most commonly reported variables across all gender groups were alcohol use twice a month or more, three or more sexual partners during the past 12 months, and ever having used drugs (table 2). experience of ever having been reimbursed for sex was reported to the same extent by men and women (1%) but was more common amongst participants in the transgender group (6%) (table 2). furthermore, experience of ever having reimbursed someone else for sex was only reported by men (3%), and coercing/forcing someone sexually was more common amongst men (6% vs 3%) (table 2). experience of emotional, physical, sexual violence and gender differences in total, experience of emotional violence was the most frequently reported type of violence victimisation (21%), 4 s. hammarström et al. table 1. demographics, sexual history and analysis of gender differences. women n (%) men n (%) transgender n (%) women vs. men/women vs. trans/men vs. trans p-value total number of participants, n = 3,205 age, years (n = 3,168) 13–17 18–25 mean age (sd) sexual orientation (n = 3,178) heterosexual homosexual bisexual none of the categories living arrangement (n = 3,168) alone with parents in foster family/institution with friends with partner other arrangement use of alcohol (n = 3,159) ≥4 times/week 2–3 times/week 2–4 times/month ≤1 time/month never median (iqr) use of drugs (n = 3,179) yes, during the past 30 days yes, during the past 12 months yes, more than 12 months ago never median (iqr) sexual debut <15 (n = 3,113) no yes no. of sexual partners during the past 12 months (n = 3,156) 0 1–2 3–4 5–10 11–20 ≥21 median (iqr) ever had chlamydia infection (n = 3,162) yes, during the past 12 months yes, more than a year ago never don’t know median (iqr) 2,682 (84) 659 (25) 1,994 (75) 19 (sd 2.49) 2,334 (88) 13 (0.5) 270 (10) 40 (1.5) 488 (18.4) 1,568 (59) 10 (0.4) 141 (5.3) 377 (14.2) 72 (2.7) 18 (0.7) 271 (10.3) 1,090 (41.3) 1,003 (38) 257 (9.7) 3 (1) 75 (2.8) 179 (6.7) 260 (9.8) 2,149 (80.7) 4 (0) 2,149 (82) 464 (18) 55 (2) 1,760 (66) 443 (17) 336 (13) 48 (2) 6 (0) 2 (1) 131 (4.9) 199 (7.5) 2,176 (82) 146 (5.5) 3 (0) 453 (14) 80 (18) 366 (82) 20 (sd 2.33) 412 (91.5) 22 (5) 15 (3) 2 (0.5) 101 (22.9) 274 (62) 2 (0.5) 24 (5.4) 33 (7.5) 8 (1.8) 10 (2.2) 68 (15.1) 195 (43.2) 139 (31.8) 39 (8.6) 3 (1) 40 (9) 69 (15.5) 75 (16.8) 262 (58.7) 4 (1) 342 (79) 93 (21) 19 (4) 179 (41) 110 (25) 102 (23) 24 (6) 5 (1) 2 (2) 18 (4.1) 29 (6.6) 320 (72.6) 74 (16.8) 3 (0) 70 (2) 29 (42) 40 (58) 18 (sd 2.6) 45 (64) 6 (9) 16 (23) 3 (4) 9 (12.9) 43 (61.4) 3 (4.3) 3 (4.3) 6 (8.6) 6 (8.6) 1 6 (8.7) 24 (34.8) 26 (37.7) 12 (17.4) 4 (1) 2 (2.9) 11 (15.7) 7 (10) 50 (71.4) 4 (1) 47 (72) 18 (28) 6 (9) 39 (57) 12 (17) 10 (14) 2 (3) 0 (0) 2 (1) 5 (7) 4 (6) 52 (75) 8 (12) 3 (0) <0.001/<0.001/<0.001b <0.001/0.001/<0.001b <0.001/ns/0.006c <0.001/0.040/0.004c ns/0.003/nsb <0.001/ns/0.002c <0.001/ns/nsc use of condom against sti (n = 3,122) always often seldom never median (iqr) ever experienced unintended pregnancy, own or partner (n = 3,168) yes no don’t know median (iqr) use of protection against unintended pregnancy (n = 3,117) always often seldom never median (iqr) 518 (20) 693 (26) 715 (27) 696 (27) 3 (2) 181 (7) 2,429 (91) 49 (2) 2 (0) 1,924 (73) 418 (16) 153 (6) 124 (5) 1 (1) 56 (13) 157 (36) 138 (32) 83 (19) 3 (1) 39 (9) 371 (84) 32 (7) 2 (0) 230 (53) 128 (30) 30 (7) 42 (10) 1 (1) 17 (26) 14 (21) 24 (36) 11 (17) 3 (2) 8 (12) 56 (84) 3 (4) 2 (0) 40 (59) 10 (15) 9 (13) 9 (13) 1 (2) ns/ns/nsc <0.001/ns/nsc <0.001/0.002/nsc atransgender (trans) includes identification as transgender, non-binary and those not willing to categorise themselves by gender. buse of pearson’s x2. cuse of mann–whitney test. in items with ≤5, use of fischer’s exact test. missing values 26–92 per item. sd = standard deviation, iqr = interquartile range. violence and sexual risk taking reported by young people 5 compared to sexual violence (15%) and physical violence (12%) (table 3). some gender differences were observed; physical violence was considerably more reported by men and participants in the transgender group compared with women (23–24% vs 10%, p = 0.001). both emotional violence and sexual violence were more common amongst participants in the transgender group and women compared to men (5–6% vs 1%, p = 0.008) (table 3) (or 9.80, 12.70) (table 4). experiences of both physical, emotional and sexual violence were more reported by participants in the transgender group (9%) in comparison to both men and women (9% vs 2–4%, p = 0.020) (table 3) (or 13.50) (table 4). multiple forms of violence and the relationship to variables associated with sexual risk taking and ill health participants reporting both physical, emotional and sexual violence, or the combination of both physical and sexual violence reported higher proportions of variables associated with sexual risk taking, compared to participants reporting other combinations of multiple violence or no violence. ever having used drugs, alcohol use twice a month or more, and three or more sexual partners during the past 12 months were the most commonly reported variables associated with sexual risk taking amongst visitors reporting experiences of multiple violence (figure 1). regarding violence victimisation, higher age increased the odds of participants reporting sexual or emotional violence (or 1.07). most combinations of violence resulted in raised odds ratios for drug use (or 1.77–5.59) and sexual initiation before the age of 15 (or 2.07–2.87) (table 4). participants reporting both physical, emotional and sexual violence were more than twice as likely to report three or more sexual partners in the past 12 months, experience of unintended pregnancy and sexual initiation before the age of 15 years. participants reporting physical violence or multiple violence were two to four times as table 2. frequency and gender differences in reported variables associated with sexual risk taking. variables women men transgendera women vs. men/women vs. transa/ men vs. transan = 2,682 n = 453 n = 70 % % % p-value alcohol use ≥2 times a month 53 60 45 0.001/ns/0.010 ever used drugs 19 41 29 <0.001/0.054/0.040 ≥3 sexual partners during past 12 months 35 55 35 <0.001/ns/0.002 sexual initiation <15 years of age 18 21 28 ns/0.030/ns ever had chlamydia 18 27 25 <0.001/ns/ns ever had unintended pregnancyb 9 16 16 <0.001/0.020/ns received imbursement for sex 1 1 6 ns/0.003/0.001 given imbursement for sex 0 3 0 <0.001/ns/ns coercing/forcing someone sexuallyc 3 6 3 0.001/ns/ns use of pearson’s x2 test. use of fischer’s exact test in items ≤5. atransgender (trans) includes identification as transgender, non-binary and those not willing to categorise themselves by gender. bincludes own or partner’s experience of unintended pregnancy. cincludes feeling that the sex was against the partner’s will, through pressure. figure 1. reported variables associated with sexual risk taking amongst visitors reporting experiences of multiple violence versus no violence. 6 s. hammarström et al. likely to report ever having used drugs (or 2.29, 4.74). in addition, participants reporting experiences of both physical, emotional and sexual violence or physical violence and sexual violence had considerably higher ors of three or more sexual partners in the past 12 months (or 2.68–2.85) and experience of unintended pregnancy (or 2.29–3.30) (table 4). discussion our results show that young people reported multiple forms of violence and sexual risk taking during consultations at youth clinics, showing them to be a vulnerable group of young people and reflecting gender inequalities in health. those reporting experiences associated with sexual risk taking and ill health were more likely to report violence victimisation. participants reporting experiences of taking drugs were more than four times as likely to report both physical, emotional and sexual violence. those who reported ≥3 sexual partners during past 12 months, early sexual initiation and unintended pregnancy were twice as likely to experience physical, emotional and sexual violence. taken together, experience of both physical, emotional and sexual violence was overrepresented amongst transgender, non-binary youth and women. their reported experiences of violence demonstrated strong associations with sexual risk taking. this demonstrates the urgent need to identify the vulnerable groups when visiting youth clinics, especially since previous research has identified lower self-rated health amongst young people who report both physical, emotional and sexual violence (33). through our research design, including different combinations of violence exposure, we can confirm that previously demonstrated associations between sexual risk taking and multiple forms of violence (23) were consistent also in the combination of only physical and emotional violence. this indicates that sexual risk taking is an important factor for experiencing multiple forms of violence amongst young people. however, our study was cross-sectional and cannot establish causality regarding when violence and sexual risk taking occurred. a likely hypothesis is that vulnerable adolescents have the double disadvantage of being at risk for both violence and sexual ill health. it has been suggested that polyvictimisation in adolescents to a large degree explains trauma symptoms, including sexual concerns (3). there is also evidence, suggesting that childhood abuse is associated with later high-risk sexual behaviours (34, 35) and re-victimisation (36), causing a dangerous circle of risk taking and victimisation. regarding gender inequalities in health, this study shows that the male and transgendered visitors were markedly more at risk than their female counterparts in almost every variable measured. men demonstrated higher alcohol consumption, more drug use and more sexual partners than other genders. men also reported more experience of unintended pregnancy, previous chlamydia infection, of giving reimbursement for sex and of coercing/forcing someone sexually, compared to women. table 3. reported experiences of violence and analysis of gender differences. violence victimisation (responding participants) women n (%) men n (%) transgendera n (%) women vs. men/women vs. transa/ men vs. transa p-value 2,682 (84) 453 (14) 70 (2) physical violence (n = 3,205) <0.001/<0.001/ns no 2,421 (90) 351 (77) 53 (76) yes 261 (10) 102 (23) 17 (24) emotional violence (n = 3,205) <0.001/0.008/<0.001 no 2,086 (78) 392 (86) 45 (64) yes 596 (22) 61 (14) 25 (36) sexual violence (n = 3,137) <0.001/0.005/<0.001 no 2,176 (83) 411 (93) 51 (74) yes 450 (17) 31 (7) 18 (26) physical and emotional violence (n = 3,205) 0.010/0.007/ns no 2,600 (97) 428 (94) 63 (90) yes 82 (3) 25 (6) 7 (10) physical violence and sexual violence (n = 3,205) ns/ns/ns no 2,661 (99) 447 (99) 69 (99) yes 21 (1) 6 (1) 1 (1) emotional violence and sexual violence (n = 3,205) <0.001/ns/0.008 no 2,560 (95) 450 (99) 66 (94) yes 122 (5) 3 (1) 4 (6) physical, emotional and sexual violence (n = 3,205) 0.030/0.020/0.008 no 2,585 (96) 444 (98) 64 (91) yes 97 (4) 9 (2) 6 (9) use of pearson’s x2. in items with ≤5, use of fischer’s exact test. atransgender (trans) includes identification as transgender, non-binary and those not willing to categorise themselves by gender. violence and sexual risk taking reported by young people 7 table 4. odds ratio (or) for associations between violence victimisation, gender and variables associated with sexual risk taking and ill health. method = enter no violence as reference in gender, enter men as reference b s.e. wald p-value or ci (95%) physical violence (n = 126) age 0.03 0.04 0.63 0.426 0.96 0.88–1.05 alcohol use ≥2 times a month 0.06 0.22 0.09 0.761 1.06 0.69–1.64 ever used drugs 0.82 0.22 13.66 <0.001* 2.29 1.47–3.55 ≥3 sexual partners during past 12 months 0.31 0.21 2.08 0.148 1.36 0.89–2.08 sexual initiation <15 years of age 0.75 0.23 10.48 <0.001* 2.12 1.34–3.35 ever had chlamydia 0.32 0.22 2.06 0.151 1.38 0.88–2.16 ever had unintended pregnancya 0.33 0.29 1.29 0.255 1.40 0.78–2.51 women −1.58 0.21 54.02 <0.001* 0.20 0.13–0.31 transgender −0.56 0.63 0.77 0.378 0.57 0.16–0.19 emotional violence (n = 327) age 0.07 0.02 7.24 0.007* 1.07 1.02–1.13 alcohol use ≥2 times a month −0.02 0.13 0.04 0.838 0.97 0.74–1.27 ever used drugs 0.57 0.15 13.19 <0.001* 1.77 1.30–2.41 ≥3 sexual partners during past 12 months 0.04 0.14 0.11 0.735 1.05 0.79–1.38 sexual initiation <15 years of age 0.79 0.15 26.44 <0.001* 2.20 1.63–2.98 ever had chlamydia −0.34 0.17 3.77 0.052 0.73 0.52–1.03 ever had unintended pregnancya 0.22 0.22 1.04 0.306 1.25 0.81–1.93 women 0.82 0.23 12.88 <0.001* 2.28 1.45–3.58 transgender 1.20 0.48 6.30 0.012* 3.34 1.30–8.57 sexual violence (n = 230) age 0.07 0.03 4.90 0.027* 1.07 1.01–1.15 alcohol use ≥2 times a month 0.21 0.17 1.55 0.213 1.23 0.88–1.72 ever used drugs 0.74 0.17 17.49 <0.001* 2.10 1.48–2.98 ≥3 sexual partners during past 12 months 0.36 0.16 5.02 0.025* 1.44 1.04–1.99 sexual initiation <15 years of age 0.72 0.18 15.25 <0.001* 2.07 1.43–2.98 ever had chlamydia −0.17 0.19 0.73 0.390 0.84 0.57–1.24 ever had unintended pregnancya 0.18 0.26 0.51 0.471 1.20 0.72–2.02 women 1.46 0.33 18.65 <0.001* 4.30 2.21–8.34 transgender 2.07 0.56 13.64 <0.001* 7.99 2.65–24.06 physical and emotional violence (n = 114) age −0.01 0.04 0.08 0.774 0.98 0.90–1.07 alcohol use ≥2 times a month −0.52 0.22 5.26 0.022* 0.59 0.37–0.92 ever used drugs 1.28 0.23 29.61 <0.001* 3.61 2.27–5.74 ≥3 sexual partners during past 12 months 0.23 0.23 1.01 0.315 1.26 0.80–1.98 sexual initiation <15 years of age 0.43 0.25 2.94 0.086 1.54 0.94–2.55 ever had chlamydia 0.01 0.25 0.002 0.965 1.01 0.61–1.66 ever had unintended pregnancya 1.23 0.26 22.26 <0.001* 3.44 2.06–5.76 women 0.00 0.28 0.00 1.000 1.00 0.57–1.74 transgender 1.36 0.52 6.69 0.010* 3.91 1.39–11.01 physical violence and sexual violence (n = 28) age 0.08 0.09 0.80 0.369 1.08 0.90–1.29 alcohol use ≥2 times a month −0.19 0.45 0.17 0.672 0.82 0.34–2.00 ever used drugs 1.72 0.44 15.25 <0.001* 5.59 2.35–13.25 ≥3 sexual partners during past 12 months 1.04 0.44 5.54 0.019* 2.85 1.19–6.84 sexual initiation <15 years of age 0.77 0.45 2.93 0.087 2.17 0.89–5.29 ever had chlamydia 0.17 0.44 0.15 0.691 1.17 0.50–2.82 ever had unintended pregnancya 1.19 0.47 6.47 0.011* 3.30 1.31–8.30 women 0.44 0.52 0.70 0.400 1.560 0.55–4.38 transgender 1.40 1.14 1.50 0.220 4.05 0.43–37.93 emotional violence and sexual violence (n = 129) age 0.02 0.04 0.26 0.610 1.02 0.94–1.11 alcohol use ≥2 times a month 0.08 0.21 0.14 0.708 0.92 0.60–1.41 ever used drugs 1.15 0.21 28.52 <0.001* 3.17 2.07–4.86 ≥3 sexual partners during past 12 months 0.32 0.21 2.32 0.127 1.38 0.91–2.09 sexual initiation <15 years of age 0.79 0.22 12.25 <0.001* 2.21 1.41–3.44 ever had chlamydia 0.57 0.21 6.82 0.009* 1.76 1.15–2.71 8 s. hammarström et al. except for drug use, these gender differences are not in accordance with studies of youth in the general population in sweden (23, 37), where gender differences are small and sometimes reversed. for example, previous chlamydia infection and unintended pregnancy are usually reported by a majority of women (23, 37). in summary, our results could illustrate a gender aspect that men visiting youth clinics are a selection of more sexually risk-taking men. previous studies have shown that men visiting youth clinics receive less counselling on protection in connection to sti tests and abortion, compared to women (37). this is particularly troublesome with our results in mind and a major impediment to gender-equal sexual health. it is worth noting that the associations between variables related to sexual risk taking and violence victimisations demonstrated in table 4, to a great extent reflect the female population that was in majority. in comparison to all genders, the transgender and non-binary group particularly stand out regarding early sexual initiation and experience of receiving reimbursement for sex. our results reveal that the transgendered and non-binary participants suffered from high rates of exposure to multiple forms of violence, including physical, emotional and sexual violence, compared to other genders. this confirms and extends previous studies (24– 26) by adding their vulnerability to multiple forms of violence. it is a complex process to compare results from different studies as definitions, age groups and data collection procedures often differ. in comparison to studies of young people in the general population (23), the youth clinic population was found to be exposed to violence to a similar degree (33). although our sample is not random, it is intended to be representative for swedish youth clinics in general, and our results are comparable with other studies conducted in the swedish youth clinic setting concerning demographic aspects such as gender (30, 33), age (8) and sexual orientation (38). however, the prevalence of physical, emotional and sexual violence observed in our study was lower than in another swedish study using anonymous questionnaires in the youth clinic setting. the difference could be explained by the difference in response rates between the studies but could also be because our study used responses provided during consultation and not anonymously. responses delivered during consultation and not anonymously are likely to be underestimated due to the sensitive nature of the questions and the risk of social desirability (39). on the other hand, a strength is that the design provides valuable information on what young people are prepared to discuss with hcps. a strength of our study is the relatively large sample size compared to the sparse research on young people (13–25 years of age). furthermore, most studies on multiple violence have been conducted either on children (20, 21) or in adult populations (22). sexit must be used systematically with every visitor, but as we lack information on visitors who were never offered the questionnaire or who chose not to respond, we cannot calculate the external attrition rate and there is a risk of selection bias. on the other hand, a strength of our study is the low internal attrition rate in individual items. concerning the variables, despite the item of alcohol consumption being designed primarily for clinical purposes and perhaps too rough, it still yielded similar results as studies using audit-c in the youth clinic setting (10). a strength of our study is the collection and reporting of data on non-binary and transgendered people, often excluded in research explained by small numbers, but sometimes also on ignorance when collecting and reporting data (40). it is worth noting that the gender inequalities in our results may be underestimated as the item of gender identity may not capture individuals with a transgender history who are currently identifying as a man or woman. furthermore, our analysis did not take sexuality into consideration, despite it being confirmed in many studies that homo and bisexual individuals are more exposed to violence than heterosexuals (19, 41). lastly, sexit does not account for number of victimisation events within each form of violence, which can be of importance when measuring victimisation (3). table 4. (continued) odds ratio (or) for associations between violence victimisation, gender and variables associated with sexual risk taking and ill health. method = enter no violence as reference in gender, enter men as reference b s.e. wald p-value or ci (95%) ever had unintended pregnancya 0.12 0.32 0.15 0.697 1.13 0.60–2.14 women 2.28 0.59 14.72 <0.001* 9.80 3.05–31.47 transgender 2.54 0.85 8.90 0.003* 12.70 2.39–67.44 physical, emotional violence and sexual violence (n = 112) age 0.07 0.04 2.31 0.128 1.07 0.98–1.17 alcohol use ≥2 times a month −0.83 0.23 12.52 <0.001* 0.43 0.27–0.68 ever used drugs 1.55 0.23 44.61 <0.001* 4.74 3.00–7.49 ≥3 sexual partners during past 12 months 0.98 0.23 18.04 <0.001* 2.68 1.70–4.22 sexual initiation <15 years of age 1.05 0.23 20.10 <0.001* 2.87 1.81–4.56 ever had chlamydia 0.25 0.24 1.11 0.292 1.29 0.80–2.08 ever had unintended pregnancya 0.83 0.28 8.28 0.003* 2.29 1.32–3.97 women 1.30 0.39 11.04 0.001* 3.68 1.70–7.96 transgender 2.60 0.60 18.52 <0.001* 13.50 4.12–44.19 b = b-coefficient. s.e. = std error. wald = wald-test. or = odds ratio. ci = confidence interval. a = includes own or partner’s experience of unintended pregnancy. *p=< 0.05. violence and sexual risk taking reported by young people 9 implications and future studies the associations found between sexual risk taking and experience of multiple forms of violence, and the urgent health needs found in the youth clinic population have implications for both clinical work and research. if only a single form of violence is considered in research, a misinterpretation of the association between sexual risk taking and violence is likely. in the clinical context, the association between sexual risk taking and violence victimisation suggests that screening for multiple forms of violence and sexual risk taking should be offered to all visitors in order to identify young people in need of care and support. conclusions young people with experiences of sexual risk taking and ill health report significantly more multiple violence victimisation. transgender and non-binary youths are exposed to considerably more violence compared to women and men. acknowledgements the authors are grateful to the statistician bo rolander at futurum, region jönköping county, for statistical advice and support. funding this study was funded by grants from jönköping county region. disclosure statement the authors report no conflict of interest. notes on contributors helena kilander, rnm and phd, is a midwife and adjunct senior lecturer at the department of obstetrics and gynaecology, region jönköping county and department of health, medicine and caring sciences, linköping university and postdoc at the department of women’s and children’s health, karolinska institute. siw alehagen, rnm and phd, is a midwife and associate professor at division of nursing sciences and reproductive health, department of health, medicine and caring sciences, linköping university. sofia hammarström, mph and phd, 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http://www.fsum.nu/wp-content/uploads/2018/03/statistik.pdf http://dx.doi.org/10.1177/1403494818774965 http://dx.doi.org/10.1080/13625187.2018.1564815 http://dx.doi.org/10.1177/1403494820921690 http://dx.doi.org/10.1177/1524838010378299 http://dx.doi.org/10.1177/1524838010378299 http://dx.doi.org/10.1016/j.chiabu.2004.10.015 http://dx.doi.org/10.1016/j.chiabu.2014.10.004 http://dx.doi.org/10.1016/j.chiabu.2014.10.004 https://www.folkhalsomyndigheten.se/contentassets/ebe239903fd346cba9cb12bfb83e0bfd/sexuality-health-young-people-sweden.pdf https://www.folkhalsomyndigheten.se/contentassets/ebe239903fd346cba9cb12bfb83e0bfd/sexuality-health-young-people-sweden.pdf https://www.folkhalsomyndigheten.se/contentassets/ebe239903fd346cba9cb12bfb83e0bfd/sexuality-health-young-people-sweden.pdf http://dx.doi.org/10.1177/0886260516681878 http://dx.doi.org/10.1007/s11135-011-9640-9 http://dx.doi.org/10.1007/s11135-011-9640-9 http://dx.doi.org/10.1080/19317611.2019.1667938 https://www.folkhalsomyndigheten.se/publicerat-material/publikationsarkiv/s/sexuell-och-reproduktiv-halsa-och-rattigheter-i-sverige-2017/?pub=60999 https://www.folkhalsomyndigheten.se/publicerat-material/publikationsarkiv/s/sexuell-och-reproduktiv-halsa-och-rattigheter-i-sverige-2017/?pub=60999 https://www.folkhalsomyndigheten.se/publicerat-material/publikationsarkiv/s/sexuell-och-reproduktiv-halsa-och-rattigheter-i-sverige-2017/?pub=60999 upsala j med sci 97: 141-148 airway obstruction, obesity and c 0 2 ventilatory responsiveness in the sleep apnea syndrome thorarinn gislason' and ritva tammivaara* from the departments of 'pulmonary medicine and 2clinical physiology, akademiska sjukhuset, university of uppsala, uppsala, sweden abstran in 32 patients with sleep apnea syndrome (sas), pulmonary function, blood gases and the ventilatory response to co, (co, vr) were studied before and 6 months after uvulopalato pharyngoplasty. nine of the sas patients had airway obstruction (ao-sas), defined as fev1.o i 72 % of the predicted value. they had a significantly higher paco,, lower pao, and a lower co, vr than the remaining sas patients. preoperatively 4 sas patients were hypercapnic (paco, >5.8 wa) and compared with the normocapnic ones they were more obese; in 3 of them fev,.o was i72%. the hypercapnic sas patients had a significantly lower co, vr. the co, vr was significantly correlated to a 0 and the degree of oxygen desaturation during sleep, but not to the number of episodes of apnea and hypopnea nor their length. the vr to co, did not predict the postoperative outcome. postoperatively 2 hypercapnic obese ao-sas patients showed a large decrease in episodes of apnea and hypopnea and an increase in co, vr, and became normocapnic. other patients showed no consistent changes in co, vr postoperatively. introduction the sleep apnea syndrome (sas) is a frequently diagnosed disorder which is characterized by loud snoring and repeated upper airway obstructions during sleep (1,2). many aspects of the inter relationship between sas, pulmonary function, obesity, hypercapnea and the ventilatory response to co, (co, vr) are still unclear. aubert-tulken and coworkers found a shift to the left in the curve of the co, vr after tracheostomy in a 41-year-old male patient who was hypercapnic and had pulmonary airway obstruction (3). guilleminault and cumminskey reported that among 5 eucapnic, nonobese men with sas the co, vr was doubled after tracheostomy (4). among 8 hypercapnic sas patients 4 became eucapnic after treatment (5). in 19 closely followed sas patients there was no change in the slope of the co, vr during successful treatment with continuous positive airway pressure (cpap) (6). however, in 10 of the patients, who were initially hypercapnic, there was an increase in the co, vr measured as the position on the co, vr 141 curve (6). we have so far found no reports on the possible results of uvulopalatopharyngoplasty (uppp) on hypercapnea and the co, vr. the aim of this prospective study was to evaluate the co, vr among sas patients in relation to airway obstruction, obesity, the severity of the sas, and arterial blood gases, before and after uppp. methods patients; this study comprised all sas patients who underwent uppp for sas between september 1984 and april 1986 at akademiska sjukhuset, uppsala, sweden. in all there were 31 men and 3 women, with a mean age of 49 years (range 30-68). fourteen were current smokers and one had stopped smoking less than one year previously. the study protocol had been approved by the ethics committee of the medical faculty of uppsala university. the clinical characteristics of the sas patients and the operative procedure have been described in detail elsewhere (7). five had a history of chronic obstructive pulmonary disease and 1 had bronchial asthma. two were treated with beta-2 agonists, and 2 with theophylline preparations. all but 1 were habitual snorers, and all-night polysomnographic studies had confirmed the sas diagnosis. an apnea was defined as a 10 second or longer complete cessation of both nasal and oral air flow and a hypopnea was defined as a marked decrease in oro-nasal air flow for at least 10 seconds, followed by a fall in baseline oxygen saturation by at least 4% or arousal. an index of apneas (a) and hypopneas (h), was calculated as the total number of such events per hour of sleep (1). preoperatively these 34 sas patients had a mean (a+h) index of 44 (median 30, range 9 101). six months postoperatively all 34 patients came for follow-up studies and 22 patients (65%) showed a decrease in the (a+h) index by more than 50% and were therefore classed as responders to uppp (7). sixteen patients had an (a+h) index below 10 postoperatively. all but 4 sas patients were overweight, with a body mass index (bmi) of 2 29 kg/m2 and the mean bmi for all patients was 32.7 (sd 5.7) kg/m2 (table 1). lunp function tests and blood gases: lung volumes and maximal flow volume curves were determined by standard spirometry and body plethysmography. arterial ph, po,, and pco, were measured with a coming 168 ph, po, and pco, blood gas analyzer (coming medical, halstead, england). oxygen saturation was measured continuously during sleep with a biox i11 pulse oximeter. the number of apneas and hypopneas per hour of sleep that caused a fall in sao, below 85% were especially recorded. testing of ventilatory regulation: all tests of the vr to hyperoxic hypercapnea were performed at the same time of day by a modification of the rebreathing method described by read (8), while the patients were awake and at rest. we have previously described the co, rebreathing test in detail (9). the vr slope was calculated as the change in ventilation (av l/min) caused by a rise in end tidal pco, by 1 kpa (=7.5 mmhg). the position of the vr line (l/min) was expressed as the 142 minute ventilation at a pco, of 8 kpa (6). table 1. sas patients with airway obstruction (ao-sas) compared with the other sas patients. bmi: body mass index; fevl.o: forced expiratory volume in one second; v c vital capacity; rv: residual volume; tlc: total lung capacity; frc functional residual capacity. a: apneas; h: hypopneas; e v f erythrocyte volume fraction. ao-sas other sas patients (n=9) (n=23) mean (s.d.) mean (s.d.) p-value age 54 bmi (kg/m2) 37.0 evf (%) 46 pulmonary function tests fev1,o (% pred.) 65 rv (% pred) 129 tlc (% pred) 84 fev,.pc 71 blood gases pao, ( h a ) 9.1 paco, (kpa) 5.8 av/apc02 (i/min/kpa) 13.1 sleep data (a+h) index (no. per hour) mean length of a+h ( s e c ) a+h with sao, < 85% postoperative reduction 58 21 (no. per hour) 32 in (a+h) index (%) 58 47 (10) 31.4 (4.9) 44 (3) 97 (12) 102 (30) 100 (10) 77 (6) 10.7 (1.2) 5.0 (0.5) 24.8 (10.1) 39 (26) 24 (7) 9 (15) 58 (38) =o. 1 <0.01 =0.2 <0.05 <0.001 <0.05 <0.002 <0.01 <0.01 =0.09 =0.4 <0.005 =0.97 statistics: statistical probability was tested by a student’s t test on unpaired values, except in comparisons of changes in vr, when paired values were used. the correlations between different parameters were assessed by least square linear regression. results among the 34 patients treated by uppp, all but 2 underwent representative lung function tests. flow parameters revealed airway obstruction (ao), defined as an fev,, of 272% of the 143 predicted value, among 9 sas. these patients will be referred to as ao-sas. compared with the remainder they were more overweight ( ~ ~ 0 . 0 1 ) and had lower pao, and higher paco, at rest (p<o.ol) (table 1). the ao-sas patients had a somewhat, but not significantly higher (a+h) index, but their mean length of a+h was the same. the ao-sas patients desaturated more often during sleep than the other sas patients. there was no difference regarding the postoperative results between the ao-sas patients and the others, as both groups showed a 58% decrease in the (a+h) index postoperatively. f%v,,o was not predictive of the uppp results and 5 of the ao-sas patients were later classed as responders to uppp, while four were non-responders (7). the pulmonary function tests 6 months postoperativly revealed only minor individual changes in fev,,, and all those classed preoperatively as ao-sas patients still had an fev,.o of 572% of the predicted value. after uppp they continued to be more hypoxemic (pao,: 8.6 (1.3 ) h a ) than the remainder (pa0,: 11.0 (1.6) kpa, p<o.ol) and their medication was similar. this was true both for responders to uppp and non-responders. pac02: preoperatively 4 sas patients were hypercapnic, with paco, >5.8 kpa (cases no 1,2,17 and 31 in ref. 7). compared with the normocapnic patients they were more obese and showed a tendency to a higher (a+h) index, but the length of a+h was the same in these 2 groups (table 2). three of the hypercapnic patients also had an fev,.o value i 7 2 % , but the fourth had a high fev, o, so the difference was not significant for this small sample (p=o. 1). table 2. hypercapnic (n=4) and normocapnic (n=28) sas patients. (abbreviations see table 1). hypercapnic normocapnic p-value bmi (kg/mz) evf (%) pao, &pa) fevl.0 (% p w fev,.flc(%) rv (% pred) av/ae'co, (l/min/kf'a) (a+h) index (no. per hour) a+h with sao, < 85% (no. per hour) 41.2 (7.6) 48 (4) 9.1 (1.6) 76 (13) 77(11) 156 (43) 11 (7) 65 (28) 43 (15) 31.9 (4.8) 44 (3) 10.4 (1.2) 91 (21) 7x71 100 (27) 23 (10) 40 (26) <0.003 =0.06 =0.06 =0.2 =0.6 <0.001 <0.02 =0.09 <0.002 ventilatory response to co2; altogether 32 sas patients underwent a test of the co, vr before uppp and all but two were tested again 6 months postoperatively (table 3). the mean slope of the co, vr curve was 21 (10) l/min/kf'a and the mean ventilation at 8 kpa was 47 (23) 144 l/min (table 3). there was a significant negative correlation between the number of a+h causing an sa02 desaturation < 85% and the co, vr (vr = 35.3 +(-0.95 x no. of desaturations), p<0.02). the co, vr was also significantly correlated to obesity (vr = 37.6 +(0.21 x bmi) p<0.05) and also to the degree of a 0 (vr = 71.6 + 0.81 x fev1, p<0.05). the 9 ao-sas patients displayed a significantly lower co, vr than the others (p<0.005), estimated both as the slope and as minute ventilation at 8 kpa (table 3). the 4 hypercapnic sas patients also exhibited a very low co, vr (1.8, 18.0, 13.0 and 9.6 v m i m a ) , and this was significantly lower than in the rest of the patients with sas (table 2). the co, vr was not significantly correlated to (a+h) index or the mean length of apneas and hypopneas. table 3. the ventilatory response to co, in vmin, preand postoperatively. no statistical difference was observed. preoperat ivel y postoperatively mean (sd) mean(sd) mean (sd) mean (sd) slope (av/kpa) vent. at 8 kpa slope (avikpa) vent. at 8 kpa all patients (n=30) 21 (10) 47 (23) 24 (14) 45 (22) ao-sas patients (n=8) 13 ( 6 ) 31 (18) 13 (7) 33 (20) other sas patients (n=22) 24 (10) 53 (22) 28 (15) 49 (22) responders to uppp (n=20) 23 (1 1) 53 (22) 26 (16) 49 (23) nonresponders to uppp (n=10)18 (8) 37 (22) 19 (8) 36 (16) postoderative findings: there was no consistent change postoperatively in the co, vr, either among responders or nonresponders to uppp, or among ao-sas patients (table 3). postoperatively one hypercapnic patient had no apneas or hypopneas and the slope of his co, vr increased from 1.8 to 7.3 vminlkpa, and paco, decreased from 7.5 to 5.8 kpa. another patient showed a 35% reduction in (a+h) index postoperatively and his paco, fell from 5.9 to 5.4 kpa; the slope of the co, vr curve increased from 18 to 25 vminma and ventilation at 8 kpa from 36 to 45 vmin. the remaining 2 patients showed only a minor decrease in the (a+h) index postoperatively and were still hypercapnic. smokers vs. nonsmokers: among the sas patients the smokers were more hypoxic (pao, = 9.7 (1.4) kpa vs.10.6 (1.1) kpa, p< 0.05) than the nonsmokers and they also had higher paco, values (5.5 (0.9) kpa vs. 5.0 (0.4 ) kpa, p< 0.05). their fev,.o was also somewhat lower (fevl.o = 83 (18) vs. 95 (19) % pred, p=0.08). there was no difference between the smokers and nonsmokers as to sleep data, the co, vr or the erythrocyte volume fraction. 145 discussion the findings in our study group of sas patients clearly illustrate the great subject variability in the severity of this disorder and their heterogeneous ventilatory and pulmonary status. the c 0 2 vr varied considerably among sas patients, but was significantly related to a 0 and obesity. we used ventilation as a parameter of chemosensitivity, but in patients with obstructive lung disease ventil-ation may be limited by expiratory airflow and not actual drive to breathe (10). however, as the airway obstruction in our patients was not so great (7), we consider that the low co, vr among the ao-sas patients, can only partly be explained by mechanical limitation imposed by lower airway dysfunction. this connection between airway obstruction, a low co, vr and hypercapnea has been pointed out in a previous case report (4) and also by bradley and coworkers (1 1). they found that among 50 consecutive sas patients the presence of diffuse airway obstruction was an important predisposing factor to the development of co, retention and all of their seven hypercapnic patients had a 0 (i 1). one of our hypercapnic patients had no signs of a 0 (fevl.o=95 % (of pred), but he suffered from extreme obesity (bmi=40.9 kg/m2> (7). in a group of 28 sas patients with airway obstruction hypercapnea was much more common among sas patients with high lifetime alcohol comsumption (12). our data on alcohol consumption are only based on information from our standard interview and we know that among our 4 hypercapnic sas patients, 2 reported heavy alcohol intake, 1 never drinker, and no information was available from the fourth. patients with low co, vr more frequently desaturated below 85%, but other data from sleep studies were not related to the co, vr. these results are similar to the results from kunitomo et al, but they reported that the awake hypercapnic ventilatory drive was significantly inversely correlated with maximal desaturation during rem sleep but showed no correlation with any other sleep desaturation parameters (13). in our study this is at least partly because these patients had greater pulmonary obstruction and their baseline awake sao, position is closer to the steep portion of the oxyhemoglobin dissociation curve. there was no consistent change in the co, vr after uppp, either in the group of sas patients as a whole or in the different subgroups (table 3). this is contrary to a previous finding of a shift in the vr curve to the left and an increase in the slope of the vr curve after tracheostomy (3) and also to a report on 5 initially normocapnic men, for whom an increase in the slope was observed 3 months after tracheostomy (4). in the largest study in this field hitterto it was found that among 19 sas patients treated with cpap, only those 10 patients with elevated daytime c 0 2 showed a progressive increase in ventilation at 8 kpa, but there was no change in the slope of the co, vr (6). one possible explanation for the lack of increase in the mean co, vr in our study might be that so few of our patients were hypercapnic. another reason may be that compared to cpap and tracheostomy, uppp does not eliminate all apneas and hypopneas as effectively. the patients in references 3 and 4 were all tracheostomized and this might possibly have influenced the vr 146 independently of the relief of apneas and hypopneas. after tracheostomy there is a decrease in dead space ventilation, which increases the alveolar ventilation and might lead to lower paco,, at least during sleep. although 16 of our sas patients had an (a+h) index below 10 postoperatively (7) they might still be suffering from partial upper airway obstruction (14). the mechanisms that cause a decrease in the co, vr might therefore still be operative. a small subgroup among our sas patients with airways obstruction and extreme obesity also had hypercapnea and a low co, vr. two of them who showed postoperatively the greatest improvement in the (a+h) index, became normocapnic and in their cases the low co, vr was partly reversible. a possible explanation for this is that increased endogenous opioid activity might occur as an adaptive reaction (15). acknowledgements supported by grants from the swedish national association against heart and chest diseases, stockholm, the king oscar i1 jubilee foundation, stockholm and the thuring foundation, stockholm. 1. 2. 3. 4 . 5 . 6. 7 . 8 . 9. 10. references guilleminault, c., cumminsky, j., & dement, w.c.: sleep apnea syndromes: recent advances. adv intern med 26:347-74, 1980. krieger, j.: sleep apnea syndromes in adults. bull eur physiopath respir 22:147-89, 1986. aubert-tulkens, g., willems, b., veriter, cl., coche, e. & stanescu, d.c.: increase in ventilatory response to c 0 2 following tracheostomy in obstructive sleep apnea. bull eur physiopath respir 16587-93, 1980. guilleminault, c., & cumminsky, j.: progressive improvement of apnea index and ventilatory response to c 0 2 after tracheostomy in obstructive sleep apnea syndrome. am rev respir dis 126:14-20, 1982. rapoport, d.m., stuart, m.g., epstein, h., & goldring, r.m.: hypercapnea in obstructive sleep apnea syndrome. chest 89:627-35, 1986. berthon-jones, m. & sullivan, c.e.: time course of change in the ventilatory response to c 0 2 with long-term cpap therapy for obstructive sleep apnea. am rev respir dis 135:144-7, 1987. gislason, t., lindholm, c-e., almqvist, m., bimng, e., boman, g., eriksson, g . , larsson, s.g., lidell, c., & svanholm, h.: uvulopalatopharyngoplasty in the sleep apnea syndrome predictors of results. arch otolaryngol 144:45-51, 1988. read, d.j.c.: a clinical method for assessing the ventilatory response to carbon dioxide. aust ann med 16:20-32, 1967. gislason, t., sandhagen, b., & boman, g . : transcutaneous c 0 2 monitoring in adults with sleep-related breathing disorders. upsala j med sci 94:171-81, 1989. plotkowski, l.m., hannhart, b, elfassi, r., sautegeau, a., peslin, r., & sadoul, p.: role 147 of the mechanical impairment on the ventilatory response to c02 in chronic airway obstruction. clin respir physiol 2351-6, 1987. 1 1 . bradley, t.d., rutherford, r., lue, f., moldofsky, h., grossman, r.f., zamel, n., & phillipson, e.a.: role of diffuse airway obstruction in the hypercapnia of obstructive sleep apnea. am rev respir dis 134:920-4, 1986. 12. chan, s.c., gurnstein, r.r., bye, p.t.p., woolcock, a.j., & sullivan, c.e.: obstructive sleep apnea with severe chronic airflow limitation. am rev respir dis 140:1274-8, 1989. 13. kuniotomo, f., kimura, h., tatsumi, k., kuriyama, t., watanabe, s . , & honda, y.: abnormal breathing during sleep and chemical control of breathing during wakefulness in patients with sleep apnea syndrome. am rev respir dis 139:164-9, 1989. 14. polo, o., brissaud, l., fraga, j., dejeaa, y., & billiard, m.: partial upper airway obstruction in sleep after uvulopalatopharyngoplasty. arch otolaryngol head neck surg 115~1350-54, 1989. 15. gislason, t., almqvist, m., boman, g., lindholm, c-e., & terenius, l.: increased csf odioid activity in sleep apnea syndrome-regress after successful treatment. chest 96:250-54, 1989. correspondence and request for reprints: thorarinn gislason, m.d dept. of pulmonary medicine vifilsstadir, 210 gardabaer, iceland 148 upsala j med sci 79: 5 1-54, 1974 the lntramedullary pressure during the bone marrow trauma of total hip replacement surgery g. hallin, j . modig, l. nordgren and s . olerud from the departments of orthopaedic surgery, anaesthesiology and clinical physiology, university hospital, uppsala, sweden abstract it is known that complications occur frequently in con nection with extensive intramedullary operative proce dures. the destruction of the medullary circulation is followed by a partial cortical necrosis, which certainly will diminish if the intramedullary pressure during the operation is reduced. there are also general complica tions with cardio-pulmonary dyshnction possibly caused by pulmonary fat embolism. the same types of complica tion are likely to occur in connection with total hip re placement surgery, especially during the femoral phase of the operation. in this study it was found that very high intramedullary pressures were always produced when the prosthetic stem was forced down into the femoral cavity fdled with acrylic cement, while the duration of the pressure elevations after this procedure showed greater variation. the importance of the high intramedullary pressures for the extent of devasculari zation and cortical necrosis is discussed, as well as its possible role as an aetiologic factor in the development of general complications such as fat embolism and sud den circulatory crises. introduction intramedullary operative procedures are known to involve certain risks of complication, local as well as general. danckwardt-lilliestrom has shown ex perimentally that cortical necrosis occurs after intramedullary reaming of the rabbit tibia and dog femur, and also that high intramedullary pressure (imp) can easily be produced ( 2 ) . the extent of necrosis is significantly reduced if the rise of imp is eliminated by a vent (3). the cortical necrosis is followed by a periosteal reaction with new bone formation (4), which is also the case in humans after intramedullary reaming and nailing. on the basis of clinical experience, it is advised that ex , tensive intramedullary procedures should be avoided during the immediate post-traumatic phase, when the risk of general complications such as fat embolism is increased (13). this may result from release into the general circulation of medul lary components such as fat and tissue thrombo plastic products. this release has been shown to occur by olerud et al. (7). there is reason to assume that the same type of effects will follow total hip replacement operations where acrylic bone cement is used. several authors have reported general complications such as blood pressure falls following the cementing (8, 1 i , 12), and also very serious reactions with car diac arrest (9, 1 i ) . on continuous recording of the intra-arterial pressure, phillips et al. (8) found that this pressure fell during the cementing of the fem oral prosthesis, while no such effect seemed to be related to the acetabular phase of the operation. sloof et al. (10) recently reported experiments on the dog femur, where the total filling with acrylic cement into the intramedullary cavity gave clear cortical necrosis with an abundant periosteal reac tion. in addition to a considerable local thermal effect on the bone, it may be postulated that very high imp's were obtained, contributing to the ex tent of necrosis. t h e purpose of this study it seems important to extend our knowledge con cerning the variation of imp during intramedullary operations. the purpose of this study was to record the imp during the femoral phase of total hip replacement operations, and to correlate the variations of this pressure with the different rou tine procedures used in preparing the femoral shaft and cementing of the prosthetic stem. upsala j med sci 79 52 g. hallin et al. fig. 1 . the position of the cannula in the distal femur, which is externally rotated and adducted during the broaching and cementing procedure. simultaneously with this study the following parameters were also recorded: fibrin trapping and platelet aggregation in the lungs using '251-labelled fibrinogen and 5'cr-labelled platelets monitored with an external detector, cardiac output, pul monary vascular resistance, venous admixture in the lungs and arterial blood pressure. fat particles, thromboplastic products, residual monomeres of the acrylic cement were also measured in pul monary arterial blood. some of these results are published elsewhere (5, 6j. methods measurements were made in eight patients during total hip replacement by the charnley technique. a specially constructed cannula furnished with a screw-thread at one end was inserted mediaily into the distal femoral meta physis through an anteromedial approach in the intra muscular septum. by screwing the cannula into the intra medullary cavity a steady, tight position was achieved (fig. 1). t h e cannula was connected t o a pressure trans ducer ( e m t 34, elema-schonander, stockholm, swe den) by a manometer connecting tube (portex 205, 4 feet). the pressure was registered with a direct-writing ink-jet recorder (mingograf 34, elema-schonander, stockholm, sweden). the system was filled with physio logical saline, which was allowed t o stream through the cannula into the intramedullary cavity for some minutes. the base line was adjusted t o the tip of the cannula with the thigh in position for reaming. the pressure record ing was begun when a stable pressure with pulse-syn chronous oscillations was obtained. the initial pressure value was taken as that prevailing in the cavity after the femoral neck had been transsected but prior t o any further preparation of the shaft. recording was then con tinued until the initial value was regained after the cementing of the prosthetic stem. the base line was checked every second minute. the highest pressure values greatly overloaded the transducer, whosednominal maximal value was 300 mmhg. the amplifier and writer were adjusted t o record 100 or 800 mmhg at full signal. however, the transducers used here could not register more than 630 and 680 mmhg, respectively, and this was checked by calibration before and after every re cording. results fairly good agreement was found between the re sults from the eight patients. fig. 2 shows the b fig. 2. (a). initial pressure level with pulse-synchronous sscillations before preparation of the femoral shaft. [ b ) opening and widening of the marrow cavity with tape reamers and broaches. ( c ) filling the cavity with cement. a catheter with suction reduces the pressure during this phase. the catheter is then withdrawn. ( d ) high pressure peak when the prosthetic stem is forced down into the cement filled marrow cavity. the small vacuum effect occurs when the prosthesis-holder is removed. (e) further pressure on the prosthesis gives additional peaks. to the right are seen the sharp peaks produced by hammer-blows. v) the pressure tends to remain at a moderately elevated level for a while after application of the prosthesis. (g) the initial pressure level is regained after a delay of 1-10 min. (for further details see table i.) upsala j med sci 79 bone marrow trauma and pressure in hip replacement surgery 53 table 1 marrow cavity marrow pressure cavity return to mmhg pressure initial pat. (initial >100mmhg value no. value) max (time in sec) (after min.) >640 >680 >630 >650 >630 >650 >650 >650 6 40 32 30 4 7 8 7 3 5 10 9 1 7 10 5 essential parts of one patient’s record, depicting the different phases of the operation. fig. 2a shows the basal imp with pulse-synchronous oscillations. the moderate pressure elevations of about 100 mmhg, seen in fig. 2b, occurred when the medullary cavity was opened and widened with tape reamers and broaches. when the acrylic bone cement was introduced into the femoral shaft, the cavity was always drained by a catheter to get the cavity thoroughly filled. no elevation of the pressure could be expected (fig. 2 c ) until the catheter was plugged with the semi-fluid plastic material. the catheter was then removed. very high pressure peaks, always over 600 mmhg, were seen in all cases. when the prosthetic stem was forced down into the cement-filled femoral shaft (fig. 2 d ) . the true peak values could not be re corded due to overloading of the instrument. the vacuum effect seen after the high peak occurred when the prosthesis-holder was removed. ham mer-blows gave short, sharp peaks as long as the prosthesis was moving downwards and these did not cease until the stem had stuck to the calcar region (fig. 2 e l . table i shows the variation in height and dura tion of the pressure elevations found in all cases. the initial values varied between 30 and 50 mmhg, and the maximal values always exceeded 600 mmhg. the duration of the pressure rise varied to a greater extent. the very high pressure recorded on insertion of the prosthetic stem had a duration of 6 t o 40 sec. the pressure was then stabilized for a while at about 70 mmhg. within 1-10 min it slowly fell t o the initial level again. , the lowest pressure elevations were recorded in a patient with a very wide medullary cavity and osteoporotic bone. discussion this study showed that high imp can be produced during the femoral phase of total hip replacement surgery. the pressure elevations can be related to definite phases of the operation, and with the re commended technique they seem to be a con stantly occurring phenomenon. the pressure increase would seem to be due to the piston-like effect that is induced during certain parts of the operative procedure, especially when the acrylic cement and later the prosthetic stem are pressed down into the intramedullary cavity. the force and rate of these insertions may be of im portance-the greater the force applied the higher the imp produced. the pressure-relieving catheter is probably of no value at this stage as it will be partly filled with semi-fluid plastic material. a high pressure in the upper part of the intra medullary cavity and thereby against the cement has the advantage, that the cement is well distrib uted over uneven surfaces and into crevices on the inner side of the femur, which increases the possibility of stable fixation (1). theoretically at least a high imp can, however, have certain deleterious effects. crushed tissue from the intramedullary cavity and its wall on reaming can be forced out first into haversian and volkmanns canals and then further into the general circulation, depending on the number of open vessels available. in the former case areas of cortical tissue may be shut off from the circula tion. being necrotic the bone may later undergo resorption, thereby contributing to loosening of the prosthesis. a periosteal reaction occurring around the upper femur under certain circum stances may be an expression of such cortical necrosis ( 3 , 10). furthermore, it is possible that crushed medul lary components which are forced out into the general circulation by a high imp (6, 7) may cause a general reaction ( 5 ) . the magnitude of such an “injection” of medullary fat and other thrombo plastic material is difficult to estimate but can probably vary within wide limits. evacuation of the deepest part of the intramedullary cavity with a suction catheter will obviously reduce the upsala j med sci 79 54 g. hallin e t a / . amount of crushed material and thereby the risk of large quantities being forced into the blood stream. the fall in blood pressure that often occurs after insertion of the femoral prosthesis may pos sibly be explained by this phenomenon. it has been suggested that monomeres released from the cement may enter the blood stream and result in a fall in blood pressure. with the aim of studying this question more closely, an investiga tion is being made to determine the blood con centrations of fat and of monomeres of the acrylic material both during the acetabular and the fem oral procedures. furthermore, comprehensive in vestigations of the effects on the respiration and circulation are in progress in an attempt to eluci date the pathophysiological course of events in this type of operation (modig et al., unpublished data). if the necessary precautions are observed, i.e. if a pressure relieving hole or catheter is used and the femoral prosthesis is not inserted too rapidly or with too great force, complications due to ex cessively high imp should no longer need to be feared. references 1. charnley, j.: acrylic cement in orthopaedic surgery, pp. 95-103. e. & s. livingstone, edinburgh and london, 1970. 2. danckwardt-lilliestrom, g . : reaming of the medul lary cavity and its effect on diaphyseal bone. acta orthop scand, suppl. 128, 1969. 3. danckwardt-lilliestrom, g . , lorenzi, l. & olerud, s . : intracortical circulation after intramedullary reaming with reduction of pressure in the medullary cavity. j bone joint surg (am)52: 1390, 1970. 4. danckwardt-lilliestrom, g . , lorenzi, l. & olerud, s.: intramedullary nailing after reaming. an in vestigation on the nealing process in osteotomized rabbit tibias. acta orthop scand, suppl. 134, 1970. 5 . modig, j . , olerud, s. & malmberg, p.: sudden pul monary dysfunction in prosthetic hip replacement surgery. a case report. acta anaesthesiol scand (in press). 6. modig, j . , olerud, s . , malmberg, p. & busch, c.: medullary fat embolization during total hip replace ment surgery. (a preliminary report.) injury 5: 161, 1973. 7. olerud, s., danckwardt-lilliestrom, g . & lorenzi, l.: do medullary components appear in the femoral vein during the reaming of the tibia? eur surg res 1: 243, 1969. 8. phillips, h., cole, p. v. & lettin, a. w. f.: cardio vascular effect of implanted acrylic bone cement. br med j3:460, 1971. 9. powell, j. n . , mc grath, p. j., lahiri, s. k . & 10. 11. 12. 13. hill, p . : cardiac arrest associated with bone cement. br med j 3: 326. 1970. slooff, t . j . j . h . : the influence of acrylic ,cement. acta orthop scand 42: 465, 1971. thomas, t . a , , sutherland, i. c . & waterhouse, t. d.: cold curing acrylic bone cement. anaesthesia 26:298, 1971. wagner, j . , burny, f . , greens, j., de marneffe, r. & blaimont, p . : etudes clinique et experimentale sur i’action hypotensive du plastique acrylique. 11. con grts international de chirurgie orthopkdique, mexico 1969, bruxelles, 1970. wehner, w.: die fettembolie. veb verlag volk und gesundheit, berlin, 1968. received october 18, 1973 address for reprints: jan modig department of anaesthesiology university hospital s-750 14 uppsala sweden upsala j med sci 79 upsala j med sci 79: 103-105, 1974 improved polymeric contrast agents for roentgenologic examination of the gastrointestinal tract 1. preliminary report on the chemistry of the polymers lars bjork, u n o e r i k s o n . bjorn i n g e l m a n and birgltta zaar from t h e d e p a r t m e n t s of diagnostic radiology a n d clinical c h e m i s t r y , university hospital, u p p s a l a , a n d pharmucia ab, u p p s a l a , s w e d e n abstract improved water-soluble iodine-containing polymers in tended as contrast substances for use in the roent genologic examination of the gastrointestinal tract have been synthesized. these polymers have a high solubility in water even at relatively low ph values, and, therefore, do not precipitate in the stomach. i n t r o d u c t i o n in an earlier publication in znvestigative radio logy we reported on polymeric water-soluble iodine-containing contrast agents intended for use in roentgenologic examination of the gastrointesti nal tract ( i ) . in contrast to the iodine-containing contrast agents used a t present, these polymeric contrast substances retain their solubility in water even a t relatively low ph values and therefore d o not precipitate in the stomach. t h e molecular weight and molecular dimensions a r e considerably larger than for the monomeric contrast substances used a t present f o r this purpose. aqueous contrast solutions of t h e polymers a r e less hypertonic than solutions of the currently available contrast substances at comparable iodine concentrations. t h e diffusion coefficients of the polymers a r e lower than the diffusion coefficients of the mono meric contrast agents. the advantages of water soluble polymeric contrast agents of this type in certain instances in the roentgenologic examination of the gastrointestinal tract were briefly presented in o u r earlier paper ( i ) . in this report, improved polymeric contrast substances f o r this purpose are described. t h e synthesis method has been modified. compared with o u r earlier polymers the improved polymers have an even higher solubility in water at low ph values. t h e average molecular weights m , and the molecular weight distributions have been changed slightly. preparation qf wuter-soluble polymeric contrast sub stances f o r examination qf the gastrointestinal tract t h e structure of the polymers reported in our previous paper ( i ) is presented in the schematic fig. l a . in this figure -adenotes iodine substituted benzene derivatives (preferably 2, 4, 6 triiodobenzoic acid derivatives) a n d -bdenotes intermediate hydroxyl-bearing aliphatic bridges. t h e group -ahad for example t h e structure given in fig. 1 b o r in l c . f o r contrast agents for oral use, the bridge -bbetween t h e iodine containing aromatic groups contained several a ) a 8 a b a b a b a b a c o o h cooh oh oh oh i oh d ) c h , a h c h 2 o c h 2 h h c h , o ( c h 2 ) 4 0 c h 2 c h c h 2 0 c h 2 c h c h 2 i fig. 1 . ( a ) basic structure o f the polymeric contrast substances. -aindicates iodine-substituted benzene derivatives, mainly 2,4,6-triiodobenzoic acid derivatives. -bindicates hydroxyl-bearing aliphatic bridges. ( 6 ) example of group -a( c ) example of group -a ( d ) example of the hydroxyl-bearing aliphatic bridge -b-. in the substance 730e, described in this report, group -awas represented by the example given in ( c ) . the bridge -bi n this substance was of the type shown in ( d ) , but the hydroxyl groups were partly replaced by glycerol ether groups. wpsala j med sci 79 104 l . bjijrk et al. -1. 10,000 30.000 hydroxyl groups so that the contrast polymer would be readily soluble in water, even a t low p h values. thus, the bridge -bwas preferably of t h e type shown in fig. id. in order to further increase t h e solubility at low p h values, additional hydroxyl groups have now been introduced into the molecules by treat ing t h e polymers with glycidol in an alkaline aqueous solution. in this reaction the hydroxyl groups of the bridge -ba r e partly replaced by glycerol ether groups. substitution a t the ends of t h e molecules can also occur. t h u s , a s a result of the reaction with glycidol the number of hydroxyl groups in the polymeric contrast mole cules is increased. as an example, t h e synthesis of substance 730 e is described below. t h e group -ain sub stance 7 3 0 e has t h e formula given in fig. i l'. t h e bridge -bi n this substance is principally of the type shown in id, but the hydroxyl groups have been partly replaced by glycerol ether groups. synthesis of substance 730 e 245.6 grams of 5-acetylamino-2,4,6-triiodo-n methyl-isophtalic acid monoamide were suspended in 140 ml of 4 n aqueous n a o h . at 3 0 t , 51.2 ml of glycidol were slowly added dropwise during 5 hours with continuous.stirring. t h e reac tion mixture was allowed t o stand for an addi tional i hour a t 30°c and then f o r 16 hours a t about 20"c, after which 10 ml of 5 n aqueous n a o h was added. then 70 ml of 1,4-butane dioldiglycide ether were added dropwise, with continuous stirring, during 5 hours, a t 30°c, t h e reaction mixture was allowed t o stand for another i hour a t 30°c and then for 18 hours a t about 20°c. thereafter, 100 ml of water w a s added and the mixture was stirred during 2 hours a t 30°c. t h e n 20 ml of glycidol were added dropwise, with continuous stirring, during 2 hours, a t 30°c. t h e reaction mixture was allowed t o stand for 2 hours a t 30°c and then for 23 hours a t about 20°c. with stirring, 6 n aqueous hci was added drop wise t o adjust the p h t o 1.6. t h e solution ivas kept a t this p h for 2 hours. n o precipitate was obtained. t h e solution was neutralized with 4 n aqueous n a o h to p h 7.0. 400 ml of water w a s added and then 2500 ml of acetone in order t o precipitate the pofymer. after o n e d a y , the super natant w a s separated from t h e syrup-like lower phase containing the sodium salt of the polymeric polyacid formed in the reaction. 200 ml of water was added to the lower phase and then i000 ml of acetone. after o n e d a y , t h e supernatant was separated from t h e lower phase whereafter 3 more reprecipitations with acetone were carried o u t in t h e same manner. t h e precipithte w a s dried a t 50°c in a vacuum. t h e substance ob tained was designated 730e and was used for the animal experiments described in part i 1 of this report. t h e yield was 292 grams. t h e product contained 0.8% naci. t h e iodine content of t h e sodium salt of the polymeric contrast substance obtained w a s 35.7%. t h e weight average mo lecular weight (au), determined by light scatter ing, was about 5000. fig. 2 shows the smoothed o u t integral molelcular weight distribution curve of substance 7 3 0 e determined by dr granath by gel chromatography o n a mixture of sephadex upsala j med sci 79 improvedpolymeric contrust agents. i. 105 fig. 3. differential molecular weight distri bution curve. substance 730 e. 10,000 20,000 30,000 m w g50 fine and sephadex g7s fine. the con centration of the contrast substance in the eluted fractions was estimated from t h e absorbance at 242 nm. fig. 3 shows t h e differential molecular weight distribution curve obtained by graphical derivation of the integral distribution curve shown in fig. 2 . ( t h e method has earlier been used for t h e determination of molecular-weight distribution curves for other polymers and has been described by granath & kvist ( 2 ) and arturson & granath (3 .) discussion t h e new polymeric contrast substances, for example s,ubstance 730 e , have improved solubility properties in water at low p h values. a relatively low average molecular weight was chosen for these polymers in these introductory tests. t h u s , the average degree of polymerization is low but can b e increased. t h e molecular weight distribu tion was such that the upper limit lay well below the limit of permeability of the renal glomeruli. thus, should t h e contrast agent escape from the gastrointestinal tract, e . g . , into t h e abdominal cavity through a perforation in the intestinal wall, it would b e easily excreted in the urine. if a higher average degree of polymerization is chosen molecules with a size above t h e renal threshold , c a n b e removed by fractionation methods. as these improved polymeric contrast sub stances have the desired chemical and physical chemical properties, tests in animals have been started. in part 11 of this report, preliminary studies of substance 730 e in animals are described. r e f e r e n c e s 1. bjork, l., erikson, u . & ingelman, b . : poly meric contrast media for roentgenologic examina tion of gastrointestinal tract. investigative radio logy5: 142, 1970. 2. granath, k . & kvist. b.: molecular weight distribution analysis by gel chromatography on sephadex. j chromatogr 28: 69, 1967. 3 . arturson, g . and granath, k . : dextrans as test molecules in studies of the functional ultrastructure of biological membranes. clin chim acta 37: 309, 1972. received december 2 2 , 1973 address for reprints: u . eriksson, m. 0. department of diagnostic radiology university hospital s-750 14 uppsala sweden upsala j m e d sci 79 upsala j med sci 98: 429-441, 1993 8. discussion per hyltoft petersen,' torgny groth2 and carl henric de verdier' ' departmtyt of clinical chemistry, university hospital, odense denmark, and -unit for biomedical systems analysis, and 3department of clinical chemistry, university of cippsala, sweden one main purpose of this nordkem project has been to combine the efforts and results from the two previous nordkem-projects on "assessing quality requirements in clinical chemistry" (13), and "quality control in clinical chemistry efforts to find an efficient strategy" (3), in order to establish a more relevant basis for analytical quality management in laboratory medicine. in order to obtain a broad and reliable basis for the evaluation, a number of mostly scandinavian experts were invited for critical comments and ideas in a two day meeting (the friibergh herrghrd seminar), from which a report has been published (2). one conclusion was that the project group should describe and summarize the most significant knowledge of analytical goals/analytical quality specifications and analytical quality control, and invite a number of scientists, who were involved in clinical and analytical projects. from these projects aspects on analytical goals/quality specifications and control could be extracted in order to obtain contributions to approaches presented in the final report. these ussociutedprojects were chosen according to the participants knowledge about current projects within the field. representatives from the associated projects were invited two years later to another two-day meeting for information and coordination, whereafter reports have been prepared within the individual projects. a uniform terminology is essential in order to communicate in an effective way and great efforts have been made to collect and suggest suitable terms and abbreviations. two major projects, dealing with transferability of laboratory data and reference intervals, have all the time been an integral part of the total project. 429 8.1. terminology there is a pronounced lack of internationally accepted terminology within this area. to avoid undefined concepts and expressions renc dybkzr has participated in the project group and prepared two documents: a more general one about 'truth, accuracy, error, and uncertainty' (8), and a 'vocabulary for describing the metrological quality of a measurement procedure' (7). the concepts and expressions have been defined according to internationally accepted authorities or organizations such as bipm, iso, iupac, ifcc, etc. some terms, which w e find necessary to use in this report, lack generally accepted definitions. we have collected these separately under the headline 'explanation of some other concepts and terms used in this report' (5). it is our expectation that these terms usually collected from authorities and organization on a high hierarchical level should be accepted by the clinical laboratory community. it is a risk that less well authorized concepts and terms may intimidate the laboratory people from their use. in such cases it is more important to start the quantitative characterization of goals and procedures of measurement and quality assurance than to wait for more fancy concepts and terms. 8.2. transferability of laboratory data it is easy to recognize a prolonged trend from the tendency of the practitioners and the physicians to use their own, in practical work acquired, "database" for reference values towards the use of scientifically acquired "databases" which use methodologies including reference method technology, statistical techniques and multicentre studies. this new tendency makes it important to try to reduce between-laboratory analytical variation (sbl) or, expressed in other words, to urge the laboratories to reduce their long term bias. although our experiences are limited to a regional study including creatinine and urate in 17 laboratories for three 14-week periods (18) and a nordic study including creatinine, calcium and cholesterol in 7 laboratories for a 4-week period and a later 2-week period (chapter 5.2) (11, 12), it is justified to conclude that application of a carefully selected bias assessment and correction procedure is motivated for several analytical methods. the alternatives may be to try to reduce the r u n d m error to an unrealistic extent and to instal new measurement procedures which may be more expensive and less practical. 430 8.3. external quality assurance for proteins the nordic protein project includes all the three elements of analytical quality in a structured combination: i) specification of analytical quality ii) creation of analytical quality iii) control of analytical quality. thereby it comes close to a model for total analytical quality management, with planning, implementation, registration, control, and evaluation. the basis for analytical quality specifications, however, is not clinical but rather biological. this choice relates to several considerations: i) i t is very difficult to find clinical situations for all the proteins where the result is determining ii) the use of specific proteins varies from country to country iii) the need for common reference intervals seems to be considerable. moreover, the projects on reference intervals in denmark and finland made the concept of quality specifications for sharing common reference intervals most relevant. creation of analytical quality relates to the analytical procedures and the basis for standardization. as evaluation of analytical procedures, equipments, and reagents are performed mainly by industry, i t is difficult to influence on this directly. guidelines are, however, given i n the project for improving performance or for choosing better methods. the need for common calibration was eminent in the project and solved by use of the nordic calibrator. this is further improved by transferring values from the ifcc reference preparation, whereby the traceability is secured. finally, the project designs a control system which challenges trueness of measurements by controlling the calibration with use of optimal (clear) control samples, and identical samples, except for turbidity. furthermore, the presentation of data relates directly to differences, both for estimation of bias related to calibration (measured minus conven tional true value) and unspecific signals from turbidity (turbid minus clear sample). 8.4. ethical aspects according to our opinion it is unethical to provide an analytical service and sell it without stating its 'analytical quality' and 'service quality'. see chapter 1, fig. 1.1 (4). this question was considered similarly i n the general scandinavian recommendations on quality control and quality assurance in clinical chemistry issued by the board of the scandinavian society for clinical chemistry in 1990 (21). how the analytical quality should 43 1 be presented to the customers in practice is another problem. as stated by prof. doumas, president of aacc, in a recent discussion (6) it seems to be be most practical to provide, this information in the 'laboratory guide for physicians' from the laboratory. certification and accreditation are procedures through which the management of the laboratory tries to guarantee that the aqspecs are followed. it involves stated activities to ascertain the traceability of the methods and to implement internal and external quality control and quality assurance systems. clinical goals and aqspecs are essential elements in these activities. 8 5 economical aspects in the attempts by i s 0 and its national standardization organizations to advertise their i s 0 guides for quality it is stressed that the characterization of the quality of a product or a service is important for its marketing and selling. this is a truth that has been known for decades in industry and it seems now to spread slowly to clinical laboratories along with the introduction of market economy within the health care sector. it is natural that for analyses with lower systematic and random errors, higher analytical specificity, lower detection limits and shorter turnaround time (19) a higher price could be charged. this requires, however, massive information and motivation from the laboratories directed towards the clinicians, the practitoners, their staff, and the adminitrators within health care. nordkem has recently carried out a project about 'cost management in clinical chemistry laboratories' (16). unfortunately, in this phase of that project, the relation between cost and quality was not discussed. 8.6. the associated projects in general the participants in the associated projects found it easy to grasp the concept of influence of analytical quality on the outcome of a clinical strategy. the process of assessing this influence was, however, a critical point, and especially the concrete assessment, where analytical disturbances were assumed or simulated, seemed to be rather difficult. in addition to the calculations, the approach of splitting up the process in clinical problems, biological variation, and analytical variation (considering both trueness and precision), was a hard nut to crack. some of the associated projects did not succeed in the total process of combining all the elements, partly because the basic problem was too 432 complicated for a clear and straight forward evaluation, and partly because the process was too cumbersome. the associated projects provided, however, valuable approaches to the problems and the variety of approaches has widened the whole concept of the project. in order to demonstrate that there are many aspects on clinical goals and aqspecs all the associated projects have been included in this report. fig. 8.1 illustrates what has been achieved by the associated projects. this scheme shows that the involved projects cover the whole spectrum of aspects related to quality specification. some of the projects are more detailed and therefore difficult to give a place between the boxes. the scheme provides, however, some indications of the expansion of the concept as it has been extended considerably compared to the original scheme in chapter 1. (fig. 1.1) the associated projects can be incorporated in different kinds of schemes or structures. in the list of contents of this book they have been order according to table 8.1. table 8.1. the associated projects, structure 6.1 approaclies ,from clinical situations 6.1.1 new approaches 6.1.2 known approaches 6.1.3 other approaches 6.2 6.3 otlier projects approaches from biological and methodological data in order to provide unotlier qppe of overview the associated projects have been listed in fig. 8.2 in a scheme comparable to fig. 4.3.1. contributions covering more than one approach of goal setting are listed accordingly. it is further seen that the fraction of bimodal approaches is comparatively smaller and that the ’time series’ are represented only by one single project. in contrast, the approach ’other situations’ covers a broad spectrum from ’decision models’, ’selection of measurement’, and ’definition of measurand’ over ’contamination’ and ’detection limit’ to ’technical’ approaches as well as ’quality improvement’. all are not listed in fig. 8.2 but presented under different headlines in table 8.2. 433 models for evaluation of type of quantity magid definition of type of quantity stennian i 1 i clinical model von eyben etal., lyfken et a/., brandslund & sigsgaard, arends et a/., groduni et a/., thue & sandberg, nsrgaard et a/. selection and validation of type of quantity nilsson ehle mdller & hamfelt, penffila e t a l . brandslund & sigsgaard detection limit antonsen e t a / . approach from clinical situation i \ approach from biological quality improvemeny arends e t a / . , nsrgaard et a/ linnet, linnet, groth 8, de verdier, penffi/s, sorfo & kaihola databases de verdier et a/., fraser ef a/. reference intervals djurhus et a/., hyltoff et a/. internal control arends et a/.. linnet. ' p reanaly tical contamination von eyben et a/. i i t characteristics of measurement procedure i i i linnet, hyltoff ef a/.,' kofstad i quality assurance programs internal and external control olafsdoffir & gudmundsson groth & d e verdier penttila et a/. sotto 8, kaihola t h e & sandberg hyltoft et a/. i i laboratory analytical specification fig. 8.1. a scheme describing the various associated projects in relation to the structure presented in fig. 1.1. the boxes taken over from fig. 1.1 are shadowed. the scheme illustrates that the associated projects cover the whole spectrum of aspects related to clinical goals and aqspecs. 434 s i n g l e p o i n t t w o p o i n t m e a s u r i n g m on itoring several point otiiers monitoring brandslund et a1 a , a n t i t r y p s i n sorto & tiaihola g l u c o s e + creatinine hyltoft et al plasma proteins d j u r h u u s et al k ' arends et al. a p p brandslund et al a , a n t i ttypsin nnrgaard et ai g o t h & d c verdier ca, creatinine, cholesterol thue & sandberg iiaemoglobin lytken et al h b a , , hbai, penttila et a l i i i j a , ~ grodum et nl cortisol von eyben et al contamination, ld-1 antonsen et al detcction limit kofstad pil,po,, pco, blaabjerg et al reference s e r u m u n i m o d a l bimodal c h a n g e w i t h i n s i i bj c c t time s e r i e s others fig. 8.2. this figure shows the associated projects in a structure comparable to fig. 4.3.1 table 8.2. the table demonstrates different associated projects and how they can be distributed under six specified headlines. decision models magid: general rnodels selection and vali dation of measure ments nilsson ehle: cholesterol moller & harnfelt: myoglobin penttila et al. afp +kcg brandslund et al.: ai -a definition of components stemnun: specificity, h cg aqspecs for external quality assessment olafsdottir rnundsson: cholesterol grotli & de verdier: ca, cii olesterol, creutinine sort0 & kui/iola: glucose, creatinine & gud nomencla ture & ter minology dybker: temiinology de verdier et al.: explana tion of some concepts databases de verdier et al.:bio logy + control frmer: biology 435 in the majority of involved projects the link has been obtained between goals/quality specifications and quality control, whether established or only indicated. thereby, the purpose of the project has been fulfilled and it has also been confirmed that it is possible within a broad area. most projects are related to external control where six of these are established in external quality assessment schemes. it may be mentioned that the hba,, projects ( n ~ r g a a r d et a/.; penttila et a/.) are restricted by lack of ideal control materials and that the project on contamination (von eyben et az.) does not recommend traditional control but rather a correction procedure. hba,, is an analyte of great importance for the guidance of treatment of diabetes. it also provides an excellent example of how bias and imprecision can be combined in a two-dimensional plot illustrating their contributions to the total error. fig. 8.3 presents results achieved from the three associated projects dealing with this problem. additional comments on the associated projects * applicability the well known approaches and models are applicable in a series of clinical situations where a single quantity is determining for a medical decision. the clearest applications are the bimodal model in pure screening situations, eg. tshand pku-screenings. the same model may also be relevant in diagnostic situations where there is a clear distinction between the two classification groups. in screenings and decisions where the basic concept is purely unimodal, eg. cholesterol screening (nilsson-ehle), the unimodal model is relevant. which concept should be applied must be thoroughly investigated prior to the assessment. for a predetermined change in a quantity the assessment is quite clear, whereas, there are actions of clinicians on change i n test results that are more doubtful. do they indicate how the clinicians think that they are expected to answer or how they really use t h e data? * new approaches to goal setting the associated projects have revealed that various approaches are possible and relevant in order to derive aqspecs. here, it becomes clear that decision models for assessment of clinical strategies, definition of the quantity to be measured, and the selection and validation of the right quantity to measure (fig. 8.1) are prerequisites for the outcome. furthermore, the preanalytical problems must be assessed and compensated for when 436 b” l 0.3 or b,, or b,, 0.7 -/ _-____---- 5 % a t 0 o 0.84 for ase= 3s, 0.0 0.1 0.2 0.3 0.4 0 5 imprecision ( s a ) percent hba1, fig. 8.3. comparison between different qitality specifications for the measurement of t i b a , , combining imprecision i s , ) along the abscissa u i t h bias along the ordinate. the part of the figure trhoi)e tlie upper abscissa describes (he clunical situation. where an increase of per cent hba,, of 1 or 7 between t a o samplings is considered here the measurements are considered to be inade i n the same laboratory ( b i a s b,,, ) 01between two insti-wrients (b,,,) or between laboratories (b,, ). the part of the figtire h t & ~ the tippetabscissa i s related directlh to the bias ( r , ) i n each individual laboraton. here tlie meastrremeiits are considered 10 be made in different laboratories (bias =: b,, ) . the f’oiir upwards coti\ex curies illustrate i ) the clinical goal suggested b? itarrts i 15). i i ) l>.tken larsen el d. with ,i per cent h b a i l 1 . bidirectional change and 0 80 cotifideiice l e \ d (9,15j. i i i ) lytken larsen l‘/ d. with 1 per cent hba,, -2, bidirectional change and 0.99 confidence level ( 1 5 ) and i \ ) 1f)ltoft petersen t / t r l . with s per cent tiba,, 2 . imidirectional change and 0.90 confidence level ( 14 ). 1 he two downwards c o n l e x curses i1lustrate.i) quality specifications calciilated from data obtained from the reference interval ( i o i and from a clinical strategy assuming that the clinician reacts for increase above per cent ifba,< 7 . 5 . rlie two horizontal hatched lines demonstrate the maximal allowable limits for u,,, or b,,,, ( 17). the tmo dots (a) along the upper abscissa indicate published values for actual ineastired laltie of s,,. the lower value is published bq penttila c/ l i / . ( 2 0 ) and the higher by lytken larsen o / t i / . ( 1 5 ) . ‘t‘he trio similar upper dots marked p arid l correspond. respectilely. to a situation where the same s,-values are combined with a systematic errors ( . z s e ) = 3 s , with the control ntle1 ,, this g i \ e s a probability for error detectioii (pc,,) ~~. 0.84 ( 2 2 ) . the filled square in at the bottom of the of the figure indicates the recommendation for s,? by a 1js expert coininittee ( i ). 437 possible, and the aspects of limit of detection must be considered. validation of the use of laboratory data is necessary (hamfelt, nilsson-ehle). the problems related to selections of the right classification (uniand bimodal) is demonstrated for al-antitrypsin, where choice of the bimodai concept may lead to the recommendation of typing of z-types instead of measuring s--al-antitrypsin (brandslund). * new models for assesstilent of quuli& specifications two models are principally new (grodum, nclrgaard) but one of them is a transformation of time-series data to a simple bimodal concept leaving only one new model (nclrgaard). here the problem of transferability is very concrete, as the patients are judged according to the same decision limits but may be classified differently due to divergencies between two (or more) measurement procedures. the assessment model is very simple, as the clinical outcome is the degree of disagreement between the results. * proposals for creation of better unalytical quality arends et al. have proposed internal reference material, nclrgaard et al. materials for calibration and control and linnet finally reference methods. 8.7. strategic approaches towards laboratory aqspecs our experiences during these four project years have tought us that there are no simple solutions to how the laboratory aqspecs should be settled. clinical goals must be the origin, either they come from a description of the clinical situation which is preferred or are estimated from data on 'biological variation'. the laboratory has to investigate what can be achieved with the present analytical equipment or with other equipments possible to procure. the internal quality control system and the external quality assurance system have to be selected and they should eliminate most erroneous analytical results (give high error detection and low false rejection). it is thus obvious that the three elements: clinical goals, characteristics of the measurement procedure, and quality control/assurance must be involved in the calculations. in chapter 7 we have tried to collect data from a few illustrative laboratory investigations. the important thing is not to try to find the most correct way of doing it but to start to provide documented laboratory aqspecs in order to gain experiences. 438 at the recent aacc forum on "accuracy and precision goals in clinical chemistry testing: can they be defined by medical relevance?" (6) prof. b. t. doumas, president of aacc, asked if it is feasible to set such goals and if it is the right approach. the joint answer from aacc, cap, and nccls was yes. this is also the answer from the nordkem project group 5/89. there are still many difficulties and much work has to be done but the advantages with acquiring and using aqspecs are considerable. 8.8. references 4. 5 . 6. 7. 8. 9. baynes jw, bumn hf, goldstein d, et al. report of the expert committee on glycosylated hemoglobin. diabetes care 1984;7:602-606. de verdier c-h. quality specifications in laboratory medicine. the nordkem project and its aims. upsala j med sci 1990;95:173-183. de verdier c-h, aronsson t, nyberg a.eds. quality control in clinical chemistry efforts to find an efficient strategy. scand j clin lab invest 1984;44, suppl 172: 1-241. de verdier c-h, groth t, hyltoft petersen p. 1. background. in the medical need for quality specifications i n clinical laboratories. upsala j med sciences 1993; 98(3): in press. de verdier c-h, groth t, hyltoft petersen p. explanation of some other concepts and terms used in this report. in the medical need for quality specifications i n clinical laboratories. upsala j med sciences 1993:98(3). chapter 11. in press. dournas bt. accuracy and precision goals in clinical chemistry testing: can they be defined by medical relevance? special assignments and closing comments. clin cem 1993;39(7):1544 dybkzr r. vocabulary for describing the metrological quality of a measurement procedure. in the medical need for quality specifications in clinical laboratories. upsala j med sciences 1993;98(3). chapter 10. in press. dybkzr r. truth, accuracy. error, and uncertainty. in the medical need for quality specifications in clinical laboratories. upsala j med sciences 1993;98(3). chapter 3. in press. fraser cg, hyltoft petersen p, lytken larsen m. setting analytical goals for random analytical error in specific clinical monitoring situations. clin chem 1990;36: 1625-1628. 439 10. gowans ems, hyltoft petersen p, blaabjerg 0, hoerder m. analytical goals for the acceptance of common reference intervals for laboratories throughout a geographical area. scand j clin lab invest 1988;48:757-764. groth t, de verdier c-h. analytical quality goals and assessment to ensure transfer ability of laboratory results. clin chim acta 1993;222: 129-139. groth t, de verdier c-h. transferability of clinical laboratory data. in the medical need for quality specifications in clinical laboratories. upsala j med sciences 1993;98(3). chapter 5.2. in press. hoerder m, ed. assessing quality requirements in clinical chemistry. scand j clin 11. 12. 13. lab invest 1980;40 suppi. 155:1-144. 14. hyltoft petersen p, lytken larsen m, fraser cg. the quality needed for measuring glycated haemoglobin. an application. upsala j med sci 1990;95 (3):185-190. lytken larsen m, fraser cg, hyltoft petersen p. a comparison of analytical goals for haemoglobin a l c assays derived using different strategies. ann clin biochem 15. 199 1 ;28:272-278. 16. magid e, heikkinen r, et al. cost management in clinical chemistry laboratories. report and guidelines prepared by a working group for the nordic clinical chemistry project, nordkem., helsinki: nordkem; isbn 951-47-8193; issn 0783-2907, 1993: 1-90. 17. noergaard jensen 0, de fine olivarius n, hyltoft petersen p, klitgaard na, h0rder m. discrepancy in glycated hba,, analysed at different local laboratories and a selected central reference laboratory. upsala j med sci 1993(3). chapter 6.1.1.1. in press. olafsdottir e, aronsson t, groth t, de verdier c-h. transferability of clinical lab oratory data within a health care region. scand j clin lab invest 1992;52:679-687. 18. 19. pellar tg, ward pj, tuckerman jf, henderson ar. the freckle plot daily turnaround time chart): a technique for timely and effective quality improve ment of test turnaround times. clin chem 1993;39(6): 1054-1059. penttila im, garert j, jalkunen a, rantanen t. quality specifications for glycated hemoglobin ale. in the medical need for quality specifications in clinical labora tories. upsala j med sci 1993(3). chapter 6.2.2. in press. scandinavian society for clinical chemistry. general scandinavian recommenda tions on quality control and quality assurance in clinical chemistry. scand j clin lab invest 1990;50:225-227. 20. 21. 440 22. westgard jo, barry pl. cost-effective quality control: managing the quality and productivity of analytical processes, washington, d.c.: aacc press, 1986: 1-230. correspondense: per hyltoft petersen department of clinical chemistry odense university hospital dk-5000 odense c denmark 44 1 upsala journal of medical sciences 2023, 128, e9212 http://dx.doi.org/10.48101/ujms.v128.9212 original article ventilation with the esophageal-tracheal combitube during general anaesthesia: assessing complications in 540 patients nicole harrisona, sahra pajendab, lukasz szarpakc, anna-maria buschsieweked, mostafa somrie, michael frassd, bernhard panningf and oliver robakd adepartment of medicine i, division of infectious diseases and tropical medicine, medical university of vienna, vienna, austria; bdepartment of medicine iii, division of nephrology, medical university of vienna, vienna, austria; cdepartment of emergency medicine, medical university of warsaw, poland; ddepartment of medicine i, intensive care unit, medical university of vienna, vienna, austria; edepartment of anaesthesiology, bnai zion medical centre, haifa, israel; fdepartment of anaesthesiology, medical university of hannover, hannover, germany abstract background: the esophageal-tracheal combitube (etc) was developed for the management of difficult airways but can also be used for general anaesthesia. methods: this clinical study collected data from patients undergoing anaesthesia with the etc in order to assess the rate of complications. results: five hundred forty patients were ventilated with the etc. in 94.8% (512/540), insertion was performed for the first time by the respective physician. the following minor complications were observed: 38.7% sore throat, 30.9% blood on tube as sign of mucosal lesions and 17.0% cyanotic tongue. experience decreased the risk of mucosal lesions (odds ratio [or]: 2.3, 95% confidence interval [ci]: 1.5–3.5). a higher than recommended volume of the oropharyngeal cuff was associated with blood on the etc (or: 1.5, 95% ci: 1.0–2.3) and tongue cyanosis (or: 2.3, 95% ci: 1.4–3.7). ventilation for more than 2 h was associated with tongue cyanosis (or: 2.2, 95% ci: 1.6–3.1) and tongue protrusion (or: 1.4, 95% ci: 1.1–1.9). conclusion: we conclude that the combitube may be used for short procedures requiring general anaesthesia, but the high rate of minor complications limits its value when other alternatives such as a laryngeal mask airway are available. the tested method appears safe regarding major complications, but minor complications are common. adherence to recommended cuff volumes, experience with the etc and limiting its use to surgeries lasting less than 2 h might reduce the rate of complications. article history received 14 december 2022 revised 16 march 2023 accepted 16 march 2023 published 21 april 2023 keywords combitube; etc; general anaesthesia; complications background the combitube (esophageal tracheal combitube, etc; medtronic, dublin, ireland) was developed for the management of difficult airway situations (1, 2). these are clinical situations in which a trained anaesthesiologist experiences difficulties with bag-mask ventilation, difficulties with endotracheal intubation (eti) or both (3). however, its ease of usage makes the etc applicable for utilisation in elective surgery, similar to the laryngeal mask airway (lma) (4). in contrast to the lma (5–8), there are only few studies for the use of the etc during general anaesthesia (9, 10). moreover, several authors recommended the use of the etc for elective surgery (9, 11–14). the advantages of the etc include inserting and securing an airway without the need for neck or head positioning, minimised risk of aspiration, firm anchorage of the device in the oropharynx after inflation of the respective balloon, application of high respiratory pressures and the fact that ventilation works equally well both in the tracheal and the oesophageal position (2, 15, 16). several smaller studies already evaluated the use of the etc during routine surgery with an overall of 524 patients (9, 10, 12, 17–20). primary concerns are uncertainties about the complication rate of the device, the difficulty of insertion and the feasibility of utilisation during general anaesthesia. while only few major complications are described in the literature, vezina et al. found that utilisation of the etc during blind insertion into the airway of critically ill emergency patients resulted in a 4.3% incidence rate of major complications (emphysema, tracheal injury, oesophageal perforation and upper airway bleeding) (21). similar complication rates are also found after conventional eti (22). however, several confounding factors have to be considered when assessing the etc in an emergency setting. the traumatic impact of chest compressions as well as trauma or illness leading to intubation can result in contact oliver robak oliver.robak@meduniwien.ac.at © 2023 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. http://dx.doi.org/10.48101/ujms.v128.9212 mailto:oliver.robak@meduniwien.ac.at http://creativecommons.org/licenses/by/4.0/ n. harrison et al. complications. therefore, assessing the etc as an airway device in the standardised situation of an elective surgical procedure allows for an elimination of these confounding factors and for an evaluation of complications directly associated with etc. we therefore collected data from patients undergoing routine anaesthesia with the etc in order to assess the rate of complications and whether the etc can be used for mechanical ventilation in daily practice. insertion of an etc is not a routine strategy for airway management in elective surgery in our institute. the reason for the application of this device for elective surgery was that operators should be prepared for emergency situations. methods this clinical study was approved by the local ethics committee of the medical university of hannover, germany (no. 3846/2005). data were collected at the department of anaesthesiology at the medical university of hannover. this was a clinical study including patients with etc used for airway management. patients were given oral and written information and signed informed consent at least 2 days prior to intubation. patients’ age, gender, body mass index (bmi) and type of planned surgeries were recorded. the patients’ airways were classified according to mallampati (23). patients were monitored by routine non-invasive measures including electrocardiogram, pulse oxymetry, temperature, non-invasive blood pressure measurement and capnography. the patient’s mouth was opened with one hand, and the etc was inserted parallel to the patient’s chest, so as to facilitate insertion until the black rings on the etc were positioned at the level of the patient’s maxillary teeth. depending on the preference of the respective physician, the etc was either positioned blindly and thereby usually entering the oesophagus or by using a laryngoscope intentionally inserting it into the esophagus. the etc is available in two different sizes: 37 f sa (small adult) for patients up to 180 cm and 41 f for patients above 180 cm (24). however, in literature, the 37 f version was used more often because of its ease of insertion (9, 12, 17, 24–26), and the fact that it was successfully used up to a patient’s height of 198 cm (25, 27). the volumes used to inflate the oropharyngeal and oesophago-tracheal cuffs of the 37 f and the 41 f were 85 and 100 ml, respectively, for the proximal balloon (28) and 10 ml for the distal balloon as recommended by the manufacturer. the oropharyngeal and esophago-tracheal cuffs were inflated successively one after another. correct placement of the respective device was confirmed by auscultation, oxymetry and end-tidal co2-concentration (etco2) measurements. in case of failure to ventilate sufficiently or place the etc correctly, a conventional endotracheal airway was used as rescue device. block volumes of both cuffs were recorded. with the etc in place, tongue cyanosis and protrusion were recorded if observed. tongue protrusion measurement by help of a measuring tape was part of the routine etc use. after removal, the etc was inspected for traces of blood. patients were questioned about pain in the throat or oesophagus during postoperative rounds. 24 h later, patients were asked again with regard to potential discomfort. minor complications were defined as lasting for less than 24 h, while major complications lasted for more than 24 h. besides anesthesiologists, emergency physicians and specialists in internal medicine inserted the etc. statistical analysis data were tested for normal distribution, and analysis was performed by univariate logistic regression; a p < 0.05 was considered statistically significant. results were presented as odds ratios (ors) with 95% confidence intervals. descriptive data were presented as mean with standard deviation (sd). analysis was done with spss version 23 (ibm, armonk, ny, usa). results general characteristics of study population five hundred forty patients underwent general anaesthesia for an elective surgery and were ventilated with the etc during the entire procedure without switching to another airway device. to ensure that the observed complications were associated with etc, only these patients were included in the final analysis. the database contained a further 88 patients where the etc was initially used but then switched to another device. the reason for switching was that surgery lasted for more than 2 h. as observations as well as complications in these patients could have been caused by both airways, those patients were excluded from analysis. in 94.8% (512/540), insertions of the etc were performed for the first time by the respective physician. the characteristics of the study population (n = 540), the medical procedures and the characteristics of the etc are summarised in table 1. observations and complications with etc during general anaesthesia observations and minor complications were documented in 86% (464/540) of patients ventilated exclusively with etc during general anaesthesia. the most frequent observation was protrusion of the tongue because of etc in 50.4% of patients (272/540). a significant protrusion of more than one centimetre was observed in 10.64% (57/540). on average, the tongue protruded 0.57 (sd ±0.63) centimetres during the procedure. the most often observed complication was sore throat occurring in 38.7% (209/540) of patients after use of etc with 5.2% (28/540) of them complaining about severe pain. after removal of etc, in 30.9% (167/540), blood was detected on the tube. in 24.1% of patients (130/540), only traces of blood were noted; in 5.2% (28/540), a moderate amount and in 1.7% (9/540), a significant amount of blood was found on the tube. a cyanotic tongue was observed in 17.0% (92/540) of patients. only one case was ventilation with combitube during general anaesthesia 3 reported where severe tongue cyanosis prompted a change to a different airway device. all complications disappeared within 24  h. no major complications or severe injuries (e.g. arterial bleeding, stridor, fractures, etc.) were noted in any of the patients ventilated with etc. risk factors for complications with etc using univariate logistic regression, risk factors were calculated for the occurrence of the following complications: tongue cyanosis, protrusion of tongue, blood on the tube and sore throat (see table 2). size and weight of the patient were significantly associated with the risk for tongue cyanosis (or: 0.96 and 0.98, respectively) or protrusion (or: 0.95 and 0.98, respectively), but not the bmi. female patients showed a higher risk for protrusion of tongue (or: 2.4) and for suffering from sore throat (or: 1.9). an american society of anesthesiologists physical status classification system (asa) of iii or higher, meaning that the patient was severely ill was significantly associated with an increased risk for blood on the tube (or: 2.0). experience with the use of etc (more than three prior intubations using this device) was associated with a decreased risk for mucosal lesions (or: 1.6). the use of etc by a physician other than an anaesthesiologist was associated with an increased risk for blood on the tube (or: 2.3). a longer duration of the etc in situ showed an increase for tongue cyanosis (or: 2.2) and for tongue protrusion (or: 1.4). a block volume of the oropharyngeal cuff that exceeded the recommended volume was associated with an increase of tongue cyanosis (or: 2.3) and of blood on the tube (or: 1.5). on the other side, a higher block volume for the esophago-tracheal cuff (more than 10 ml) was associated with a decreased risk for tongue cyanosis (or: 0.5) and tongue protrusion (or: 0.5). reasons for switching etc to other airway device the database contained 88 cases where the etc was initially used for airway management but then changed to another airway device. etc was changed for logistic reasons (e.g. long duration of surgery, n = 24) or was just intended for bridging before a tracheotomy or placement of a permanent airway device (n = 8). in 46 cases out of the 88 mentioned above, etc had to be changed to another airway device because of problems with the etc (reason unknown, n = 10). the following problems were noted: leakage of the etc despite increase of the cuff volume (n = 16), insufficient ventilation (n = 16), high inspiratory ventilatory pressure (n = 10), hiatus hernia (n = 1), minor injury of the soft palate (n = 1), severe gastric reflux (n = 1) and severe tongue cyanosis (n = 1). considering the characteristics of these 46 patients, there were no major differences concerning age, sex, weight or size compared to patients with successful use of etc (data not shown). however, the majority of patients was classified as asa physical status iii (95.7%, n = 44). most elective procedures in this group were pacemaker implantations (65.1%, n = 28) or percutaneous ethanol injections of malignancies (27.9%, n = 12). the experience of the physician, the experience with table 1. general characteristics of study population, medical procedures, and etc. characteristics of study population (n = 540) mean ± sd or total number and percentage age (n = 532), years 56.9 ± 17 sex (n = 540) male female 430 (79.6%) 110 (20.4%) weight (n = 502), kg 77.8 ± 17.2 size (n = 502), cm 173.1 ± 9.4 bmi (n = 502) 25.8 ± 4.9 asa physical status (n = 517) asa i (healthy) asa ii (minor illness) asa iii (severe illness) asa iv (life-threatening illness) asa v (moribund) 5 (1%) 89 (17.2%) 400 (77.4%) 12 (2.3%) 11 (2.1%) type of surgery or medical procedure (n = 540) pacemaker implantation percutaneous ethanol injection electroconvulsive therapy magnetic resonance imaging percutaneous transhepatic choledochal drainage endoscopic retrograde cholangiopancreatography other surgeries 200 (37.0%) 165 (30.6%) 50 (9.3%) 17 (3.1%) 15 (2.8%) 4 (0.7%) 80 (14.8%) 9 (1.7) indication for etc (n = 530) training special indication* anticipated difficult airway difficult intubation rapid-sequence induction 344 (64.9%) 63 (11.9%) 57 (10.8%) 50 (9.4%) 16 (3%) qualification of physician (n = 533) anaesthesiologist other physicians 396 (74.3%) 137 (25.7%) experience with etc (n = 538) beginner (1–3 times etc use) experienced (>3 times etc use) 210 (39%) 328 (61%) etc size (n = 508) 37 f (sa) 41 f 459 (90.4%) 49 (9.6%) insertion method (n = 517) without laryngoscope with laryngoscope 142 (27.5%) 375 (72.5%) number of number of attempts (n = 456) successful at first trial** more than one trial necessary 418 (91.7%) 38 (8.3%) duration of etc in situ (n = 471) <1 h 1 to 2 h >2 h 175 (37.2%) 191 (40.6%) 105 (22.3%) block volume oropharyngeal cuff (n = 452), ml 37 f (85 ml recommended) 41 f (100 ml recommended) 92.5 ± 19 103.8 ± 13 block volume esophago-tracheal cuff (n = 441), ml 37 f (10 ml recommended) 41 f (10 ml recommended) 8.8 ± 2.1 11.9 ± 3.8 asa: american society of anesthesiologists physical status classification system; bmi: body mass index; etc: esophageal-tracheal combitube; sd: standard deviation. *special indication: teeth assessment, vocal cord protection, electroconvulsive therapy, mri, avoiding movement of cervical spine; **successful ventilation assessed by lung auscultation. n. harrison et al. etc or the insertion method did not have a significant impact on having to change etc to another airway device. discussion overall, this study proved that etc is a safe device for ventilation during general anaesthesia in minor elective procedures. although minor complications like protrusion of the tongue or sore throat occurred frequently, major complications like symptoms lasting for more than 24 h or even rupture of the oesophagus, as reported previously (22, 29), had not been observed. the success rate for ventilation with etc was about 92% taking into account that a change to another airway device was necessary in 46 cases (46/540). furthermore, one should keep in mind that in 94.8% (512/540), insertion of the etc was performed for the first time by the respective physician. this success rate was slightly lower than reported by other studies, where ventilation was achieved in 97% (9) or even 99% (17) of patients with etc. major reasons for not maintaining ventilation with the etc were leakage despite increase of the cuff volume (n = 16), insufficient ventilation (n = 10) or high inspiratory ventilation pressure (n = 10). protrusion or cyanosis of the tongue was observed in 50.4 and 17.0% of patients, respectively. the pressure exerted by the oropharyngeal cuff might impair venous blood flow from the tongue leading to cyanosis. in line with this, in the present study, we found that a higher than recommended cuff volume was associated with a higher risk for tongue cyanosis. interestingly, a higher than recommended volume (>10 ml) of the oesophagotracheal cuff reduced the risk for tongue cyanosis significantly. we do not have a verisimilar explanation for this observation. another important factor influencing tongue cyanosis and protrusion was the duration the etc was left in situ. the etc is recommended for procedures lasting up to 8 h (30). a longer duration necessitates a change of the etc to another airway device, especially because tracheal secretion cannot be removed by suction if placed in the oesophageal position. theoretically, compression and cyanosis of the tongue could theoretically lead to postoperative oedema and subsequent airway compromise. however, out of 540 patients, only one case of severe tongue cyanosis was observed that led to removal of the etc. the most common minor complication described by other studies is superficial mucosal lesions (17, 19, 31). in previous studies, the incidence of traces of blood on the etc after removal ranged from 10% (12) to 47% (9, 10, 32). compared to the lma or eti, the etc causes slightly more lesions (19). most plausible reasons for this comprise laceration during insertion of the etc, over-inflation of the pharyngeal cuff causing mucosal lesions and rigidity and anterior flexion of the etc during insertion (lipp manoeuvre) (21, 33–35). most comparisons to lma were done in the prehospital setting. in a series of 470 patients reported by rumball and colleagues (36), the etc was rated best when compared with the pharyngeal tracheal lumen airway (ptla), the lma and bagvalve ventilation with mask and oral airway. the study was performed by emergency medical technicians (emts) in patients with cardiorespiratory arrest. successful insertion rate was significantly higher (p < 0.01) with the etc (86% vs. 73% lma) table 2. risk factors for complications with etc. risk factor for complication tongue cyanosis protrusion of tongue blood on tube sore throat total n or 95% ci total n or 95% ci total n or 95% ci total n or 95% ci age 428 1.0 0.99–1.0 421 1.0 0.99–1.0 433 1.0 1.0–1.03 384 1.0 0.99–1.0 sex (female) 431 1.5 0.9–2.6 425 2.4 1.4–4.2 434 1.1 0.7–1.7 385 1.9 1.1–3.1 size 425 0.96 0.94–0.99 418 0.95 0.93–0.98 425 1.0 0.98–1.0 377 1.0 0.98–1.0 weight 425 0.98 0.96–0.99 418 0.98 0.97–0.99 425 1.0 0.99–1.0 377 1.0 0.98–1.0 bmi 425 0.95 0.9–1.0 418 0.97 0.93–1.0 425 1.0 0.96–1.1 377 0.97 0.93–1.0 asa 417 1.5 0.8–2.9 411 0.7 0.4–1.2 418 2.0 1.1–3.5 372 1.2 0.7–2.1 etc size 403 1.6 0.6–4.0 397 0.7 0.3–1.5 407 0.6 0.2–1.5 361 0.6 0.3–1.5 insertion method 423 1.1 0.7–1.9 417 1.1 0.7–1.7 426 1.0 0.6–1.5 377 1.0 0.6–1.6 number of insertion attempts 384 0.9 0.4–2.4 378 1.1 0.5–2.5 382 1.1 0.5–2.5 340 1.5 0.7–3.4 qualification of physician 430 0.7 0.4–1.2 424 0.8 0.5–1.2 433 2.3 1.5–3.5 383 1.2 0.8–1.9 experience with etc 431 0.9 0.5–1.4 425 0.9 0.6–1.3 434 1.6 1.1–2.3 385 1.0 0.7–1.5 duration of etc in situ 412 2.2 1.6–3.1 405 1.4 1.1–1.9 412 0.9 0.7–1.2 361 1.2 0.9–1.6 block volume oropharyngeal cuff 419 2.3 1.4–3.7 410 1.1 0.7–1.7 420 1.5 1.0–2.3 364 1.3 0.9–2.1 block volume esoph.-trach. cuff 414 0.5 0.2–0.9 406 0.5 0.3–0.8 416 0.9 0.6–1.4 356 0.6 0.3–1.0 asa: american society of anesthesiologists physical status classification system; bmi: body mass index; ci: confidence interval; etc: esophageal-tracheal combitube; or: odds ratio; sd: standard deviation. asa: i-ii (healthy or minor illness) versus iii-v (very ill to moribund); etc size: 37f versus 41f; insertion method: with versus without laryngoscope; number of number of attempts: one attempt versus more than one attempt; qualification: anaesthesiologist versus other physicians; experience: beginner (1–3) versus experienced (>3); duration of etc in situ: <1 h, 1–2 h, >2 h; block volume oropharyngeal cuff: recommended volume versus more than recommended (>85 ml for 37f, >100 ml for 41f); block volume esophago-tracheal cuff: recommended volume versus more than recommended (>10 ml). bold values = statistically significant data. ventilation with combitube during general anaesthesia 5 despite the fact that some of the emts had been previously trained to use the lma in the operating room. mean pao 2 and mean exhaled volume were highest with etc confirming previous studies. no aspiration with the etc was noted in autopsies (36). tanigawa et al. have studied emergency intubation of 12,020 cases of prehospital cardiac arrest (31). successful insertion rates on the first attempt were 82.7% with the oesophageal gastric tube airway (egta), 82.4% with the etc and 72.5% with the lma (p < 0.0001). the rate of failed insertions was 8.2% with the egta; 6.9% with the etc and 10.5% with the lma (p < 0.0001). successful ventilation could be achieved in 71% with the egta; in 78.9% with the etc and 71.5% with the lma (p < 0.0004). six cases of aspiration were reported in the lma group, whereas nine cases of soft-tissue injuries, including oesophageal perforation, were reported in the etc group (31). the authors conclude that the etc appears to be the most appropriate choice among the airway devices examined. in this study, we noted blood on the tube as a sign for mucosal trauma in 30.9% of patients. this is a higher rate than reported by other studies (9, 12) although a laryngoscope was used for insertion in the majority of cases (72.5%). the qualification or the experience of the physician conducting the intubation decreased the risk of mucosal lesions. experience with the etc of at least three prior intubations was associated with a decreased risk for blood on the etc. an increased volume of the oropharyngeal cuff was associated with a higher risk for blood on the etc as a higher volume can cause more injury to the mucosa. patients with an asa physical status of iii or higher had also an increased risk for bleeding as a result of mucosal lesions. only one case was observed with an injury of the soft palate prompting removal of the etc. interestingly, women were at higher risk for tongue protrusion and for sore throat after use of etc. this might be related to the female anatomy of the pharynx; however, we do not have sufficient data to support this claim. complications might often occur because of the individual anatomy of the patient, which was not evaluated in this study. overall, sore throat occurred in 54.3% of patients and was therefore the second most common complication associated with the etc. a major strength of this study is the large cohort of patients. during the primary analysis, only patients who were ventilated with the etc during the entire procedure were evaluated in order to ensure that all observed complications were directly associated with the etc. a total of 540 patients were included, which is a considerably higher sample size than in other studies (9, 10, 12, 17, 20). another advantage is the controlled setting of an elective surgery with standardised procedures compared to an emergency setting with several confounding factors. many complications described in such studies (21) can be attributed to the associated risks of cardio-pulmonary resuscitation including chest compressions or trauma. a further outstanding difference of this study as compared to other studies about etc in elective surgery is the heterogeneity of the study population as well as the variety of elective procedures where etc was used for ventilation. in contrast to other studies, the majority of patients (82%) suffered from severe illnesses and co-morbidities (asa physical status iii or higher). this might partly explain the slightly lower success rate as well as the higher rate of minor complications. as a possible limitation, it can be argued that a true prospective study with a more strict protocol would have been ideal. however, this study deals with complications primarily. in this way, it appears even more objective since there was no danger of bias at the time of documentation in regard to correctly reporting complications, as the investigators had not been involved in the process of airway management. furthermore, there are several prospective studies published so far with the use of the etc in elective cases. overall, the evaluation of etc in general anaesthesia is limited by the fact, that etc was developed primarily for use in emergency situations. of course, emergency situations are associated with different complications than elective surgeries; therefore, this study can only evaluate the etc in this specific context. considering this large cohort of patients including severely ill persons, this study showed that the etc is a safe airway device for a broad range of minor elective surgeries reporting no major complications and a 92% success rate for maintaining ventilation during the entire procedure. we conclude that the combitube may be used for short procedures requiring general anaesthesia but the high rate of minor complications limits its value when other alternatives such as a lma are available. the tested method appears safe regarding major complications but minor complications are common. adherence to recommended cuff volumes, experience with the etc and limiting its use to surgeries lasting less than 2 h might reduce the rate of complications. disclosure statement michael frass has received royalties for the invention of the combitube. notes on contributors or, bp, nh, sp, ls and mf designed the study and drafted the manuscript. or, mf and bp developed the theory and performed the statistical analyses. ms, sp, nh and or verified the analytical methods. mf and ls supervised the findings of this work. all authors discussed the results and contributed to the final manuscript. orcid nicole harrison http://orcid.org/0000-0001-5971-0662 lukasz szarpak http://orcid.org/0000-0002-0973-5455 mostafa somri http://orcid.org/0000-0002-3814-1402 michael frass http://orcid.org/0000-0003-4140-4258 oliver robak http://orcid.org/0000-0002-3238-194x references 1. frass m, frenzer r, zdrahal f, hoflehner g, porges p, lackner f. the esophageal tracheal combitube: preliminary results with a new airway for cpr. ann emerg med. 1987;16:768–72. doi: 10.1016/s0196-0644(87)80571-1 http://orcid.org/0000-0001-5971-0662 http://orcid.org/0000-0001-5971-0662 http://orcid.org/0000-0002-0973-5455 http://orcid.org/0000-0002-0973-5455 http://orcid.org/0000-0002-3814-1402 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https://doi.org/10.1016/j.annemergmed.2003.12.034 https://doi.org/10.1016/j.annemergmed.2003.12.034 https://doi.org/10.1016/j.joms.2007.08.009 https://doi.org/10.1136/emj.4.4.219 https://doi.org/10.1080/10903129808958850 https://doi.org/10.1046/j.1365-2044.1998.00302.x https://doi.org/10.1097/00000542-200205000-00008 https://doi.org/10.1046/j.1365-2044.2001.01918-5.x https://doi.org/10.1007/bf03013236 https://doi.org/10.1080/10903129708958776 upsala journal of medical sciences 2021, 128, e9392 http://dx.doi.org/10.48101/ujms.v128.9392 increased plasma endostatin and gdf15 in indolent non-hodgkin lymphoma josefin hidmana,b,c, anders larssona,d, måns thuline and torbjörn karlssona,f adepartment of medical science, uppsala university, uppsala, sweden; bcentre for clinical research västmanland, västmanland county hospital, uppsala university, uppsala, sweden; cdepartment of medicine, västmanland county hospital, västerås, sweden; ddepartment of clinical chemistry, uppsala university hospital, uppsala, sweden; edepartment of mathematics, uppsala university, uppsala, sweden; fdepartment of haematology, uppsala university hospital, uppsala, sweden abstract background: increased microvascular density correlates with more advanced disease and unfavorable overall survival in non-hodgkin lymphoma (nhl), suggesting that angiogenesis is important for disease progression. however, studies of anti-angiogenic agents in nhl patients, have generally not shown favorable outcomes. the aim of this study was to investigate whether plasma levels of a subset of angiogenesis-associated proteins are increased in indolent b-cell derived nhl (b-nhl) and to investigate whether the levels differ between patients with asymptomatic versus symptomatic disease. methods: plasma levels of growth differentiation factor 15 (gdf15), endostatin, matrix metalloproteinase 9 (mmp9), neutrophil gelatinase-associated lipocalin (ngal), long pentraxin 3 (ptx3), and galectin 3 (gal-3) were measured by elisa in 35 patients with symptomatic indolent b-nhl, 41 patients with asymptomatic disease, and 62 healthy controls. bootstrap t-tests were used to assess the relative differences in biomarker levels between groups. group differences were visualized using a principal component plot. results: mean plasma endostatin and gdf15 levels were significantly higher in symptomatic and asymptomatic lymphoma patients than in controls. symptomatic patients had higher mean mmp9 and ngal than controls. conclusions: the finding of increased plasma endostatin and gdf15 in patients with asymptomatic indolent b-nhl suggests that increased angiogenic activity is an early event in indolent b-nhl disease progression. article history received 6 february 2023 revised 10 april 2023 accepted 11 april 2023 published 9 may 2023 keywords growth differentiation factor 15; endostatin; matrix metalloproteinase 9; neutrophil gelatinaseassociated lipocalin; non-hodgkin lymphoma; angiogenesis introduction around 90% of all lymphomas are non-hodgkin lymphoma (nhl), and 85–90% of these are derived from b-cells (b-nhl) (1). non-hodgkin lymphoma is subdivided into aggressive and indolent types. indolent nhl comprises a heterogeneous group of lymphomas that are considered incurable, except for patients with localized disease who can be cured by radiotherapy. there is individual variation in the course of the disease; some lymphomas progress rapidly or transform to a more aggressive disease, but most patients with indolent nhl have a good prognosis in which the disease requires no treatment or intermittent active treatment only. treatment of asymptomatic nhl patients does not prolong overall survival (os) (2). the most common types of indolent nhl are follicular lymphoma, small lymphocytic lymphoma, and marginal zone lymphoma (3). in follicular lymphoma symptomatic or highburden disease is defined as any tumor mass >7 cm, three or more nodal sites >3 cm, splenomegaly below the umbilical line, pleural or peritoneal effusions, cytopenias, b-symptoms or >5000 circulating lymphoma cells/mm³; these findings and symptoms are indications for treatment. these criteria for symptomatic disease can be extrapolated to the management of patients with other types of indolent nhl (2, 4). for patients with more advanced disease meeting criteria for treatment, regimens are based on the backbone of an anti-cd20 agent, which in follicular lymphoma is combined with chemotherapy (2). angiogenesis is of importance for solid tumor progression and the process has been well characterized at the molecular level (5). it is also important in nhl, but there are conflicting data on the correlation between increased angiogenesis and prognosis (6). a recent meta-analysis suggests that increased angiogenesis, as evaluated by microvascular density (mvd), is associated with decreased os in patients with indolent nhl (7). in cancer, disturbances in the extracellular matrix (ecm) metabolism play an important role in the creation of a microenvironment favoring tumor growth and angiogenesis (8,  9). the ecm provides structural tissue support as well as exerting a variety of biochemical functions (9). tumor cells promote degradation of the ecm, which stimulates angiogenesis via the release of cytokines such as vascular endothelial growth original article contact josefin hidman josefin.hidman@medsci.uu.se supplemental data for this article can be accessed here. © 2023 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. http://dx.doi.org/10.48101/ujms.v128.9392 mailto:josefin.hidman@medsci.uu.se http://dx.doi.org/10.48101/ujms.v128.9392 http://creativecommons.org/licenses/by/4.0/ 2 j. hidman et al. factor (vegf) sequestered in the ecm and via matrix remodeling. local tumor expansion is also dependent on proteolytic degradation of its surrounding ecm by matrix metalloproteinases (mmps) (9). to grow beyond a certain volume, a malignant tumor is dependent on blood supply via newly formed vessels. the initiation of blood vessel expansion into the tumor is referred to as the angiogenic switch (10). therapies using anti-angiogenic agents in nhl patients have not shown the same favorable outcomes as seen in treatment of patients with solid tumors (6). the aim of this study was to investigate plasma levels of six angiogenesis-associated proteins in an attempt to reveal their  possible roles in indolent b-nhl progression. we have recently shown that the plasma level of growth differentiation factor  (gdf15) is increased in multiple myeloma (mm) and  in  chronic  lymphatic leukemia (cll) (11, 12). plasma matrix  metalloproteinase 9 (mmp9) and the pro-angiogenic glycoprotein galectin 3 (gal-3) are also increased in cll (12). increased levels of the angiogenesis-associated proteins endostatin, neutrophil gelatinase-associated lipocalin (ngal), and long pentraxin 3 (ptx3) have been observed in different types of nhl, in cll and in nk/t-cell lymphoma (13–15). plasma levels of gdf15, endostatin, mmp9, ngal, ptx3, and gal-3 were determined by elisa and compared between patients with symptomatic indolent b-nhl, patients with asymptomatic disease, and healthy controls. this is to the best of our knowledge the first study investigating gdf15 in patients with indolent b-nhl. material and methods patients blood plasma drawn from 76 newly diagnosed indolent b-nhl patients and stored in the uppsala-umeå cancer consortium (u-can) biobank was used for elisa analyses of gdf15, endostatin, mmp9, ngal, ptx3, and gal-3. blood was sampled at the time of diagnosis. the patients were divided into two groups according to current guidelines (2, 4) – those with symptomatic lymphoma (n = 35) and those with asymptomatic disease (n = 41). whether a patient was considered symptomatic or not was decided at the discretion of the treating physician. laboratory and clinical data and pathology reports from the time of the lymphoma diagnosis were obtained from each patient’s individual chart. of the 76 patients, 23 (30%) were diagnosed with follicular lymphoma, 23 (30%) with waldenstrom’s macroglobulinemia, 6 (8%) with small lymphocytic lymphoma, 3 (4%) with marginal zone lymphoma, one with lymphoplasmacytic lymphoma and one with hairy cell leukemia, and 19 (25%) with indolent b-cell lymphoma not otherwise specified (nos). of the patients with b-cell lymphoma nos one was diagnosed by splenectomy, five by lymph node biopsy or fine needle aspiration, eight by bone marrow aspiration or biopsy, one from peripheral blood, and four from biopsy of extra nodal tissue. ten patients (13%) had stage i lymphoma according to the ann arbor staging system, 6 patients (8%) had stage ii lymphoma, 10 (13%) had stage iii lymphoma, and 50 (66%) had stage iv lymphoma. blood plasma from 62 ageand sex-matched individuals was used as control. written informed consent was obtained from all study participants. this study was approved by the research ethics committee of uppsala university and the swedish research ethics committee, respectively (epn 2010/98, 2014/233, ups01367, and 2020-00164). laboratory analyses elisa analyses were performed using commercial sandwich kits (r&d systems, minneapolis, mn, usa) for gdf15 (dy957), endostatin (dy1098), mmp9 (dy911), ngal (dy1757), gal-3 (dy1154), and ptx3 (dy1826). a monoclonal antibody specific for the different peptides was coated onto microtiter plates. samples and standards were pipetted into the wells, after which the peptide was bound to the immobilized antibodies. a biotinylated peptide-specific antibody was added after washing. a streptavidin-hrp conjugate was added to the wells after incubation and washing and a substrate solution was added after another incubation and washing cycle. the absorbance was measured in a spectramax 250 (molecular devices, sunnyvale, ca, usa). concentrations were determined by comparing the optical density of the samples with the standard curve. all assays were calibrated against highly purified recombinant human peptides. measurements were performed blinded, without knowledge of the clinical diagnoses. statistics statistical analyses were calculated using version 4.2.1 of r (r foundation for statistical computing, vienna, austria). to assess the relative differences in biomarker levels, age, c-reactive protein (crp), albumin, creatinine, hemoglobin, and lactate dehydrogenase between groups, bootstrap t-tests were used. group differences were visualized using a principal component plot (16). all p-values were adjusted for multiplicity using the benjamini–hochberg procedure (17). results among the 76 lymphoma patients included in this study the majority had follicular lymphoma (30%) or waldenstrom’s macroglobulinemia (30%), while 25% were diagnosed with lowgrade b-nhl not otherwise specified. the median age for patients with symptomatic and asymptomatic lymphoma was 69 and 71 years, respectively. the control group consisted of 62 individuals with a median age of 60 years. the two groups of lymphoma patients did not differ significantly in terms of age, crp, albumin, creatinine, hemoglobin, or lactate dehydrogenase (table 1). a principal component analysis revealed that the expression patterns of the six plasma proteins studied were more homogenous in controls and in patients with asymptomatic lymphoma than in symptomatic patients, whose expression pattern was more heterogeneous (figure 1). endostatin and gdf15 in indolent nhl 3 mean endostatin level was significantly higher in patients with symptomatic lymphoma than in controls (p < 0.01), and significantly higher in patients with asymptomatic disease than in controls (p < 0.01). there was no significant difference in mean endostatin between the two lymphoma patient groups (figure 2a). mean gdf15 level was significantly elevated in patients with symptomatic and asymptomatic lymphoma in comparison with controls (p < 0.05 in both cases), but did not differ significantly between the two patient groups (figure 2b). patients with symptomatic lymphoma had significantly higher mean levels of mmp9 and ngal than controls (p < 0.05 in both cases; figures 2c–2d). there were no significant differences in the mean levels of ptx3 and gal-3 comparing the two lymphoma cohorts and controls (supplemental material, figures 1–2) discussion increased mvd in lymph nodes and bone marrow correlates with a more advanced disease stage and unfavorable os in nhl, suggesting that angiogenesis is important for lymphoma progression (7). in an attempt to elucidate a possible role for increased angiogenesis in the transition of indolent b-nhl from an asymptomatic to a symptomatic state, we compared plasma levels of six angiogenesis-associated proteins between a group of patients with asymptomatic indolent b-nhl and a group of patients with symptomatic disease. principal component analysis revealed that patients with  symptomatic b-nhl expressed these proteins more heterogeneously than both those with asymptomatic disease and controls (figure 1). we have recently observed similar heterogeneous protein expression patterns of angiogenesisrelated proteins in patients with cll (12) and mm (hidman, unpublished results) compared with controls. this similarity probably reflects the close biological relationship between indolent b-nhl, cll, and mm. table 1. clinical and laboratory characteristics of patients with symptomatic and asymptomatic b-cell derived nhl. age, hemoglobin, lactate dehydrogenase, albumin, creatinine, and c-reactive protein are expressed as median, range. n/a = not applicable. variable symptomatic b-nhl (n = 35) asymptomatic b-nhl (n = 41) p age (years) 69 (40–89) 71 (41–92) 0.2 male gender (%) 49 51 n/a hemoglobin (g/l) 124 (51–158) 127 (110–157) 0.31 lactate dehydrogenase (µkat/l) 2.9 (1.8–7.1) 2.6 (1.5–6.9) 0.74 albumin (g/l) 37 (16–49) 38 (30–45) 0.2 creatinine (µmol/l) 66 (35–207) 64 (43–104) 0.31 crp (mg/l) 2.1 (1.3–61) 1.8 (1.1–82) 0.64 ann arbor stage i (n) 8 2 n/a ann arbor stage ii (n) 4 2 n/a ann arbor stage iii (n) 4 6 n/a ann arbor stage iv (n) 21 29 n/a follicular lymphoma (n) 16 7 n/a waldenstrom’s macroglobulinemia (n) 5 18 n/a small lymphocytic lymphoma (n) 0 6 n/a marginal zone lymphoma (n) 2 1 n/a lymphoplasmacytic lymphoma (n) 1 0 n/a hairy cell leukemia (n) 0 1 n/a b-cell lymphoma not otherwise specified (n) 13 6 n/a figure 1. a pca plot showing expression of the six plasma proteins studied. controls (yellow) and patients with asymptomatic lymphoma (purple) have a more homogeneous expression pattern than patients with symptomatic lymphoma (blue), who express the proteins more heterogeneously. figure 2a. plasma endostatin is significantly higher in patients with symp tomatic and asymptomatic lymphoma than in controls (p < 0.01 in both cases). figures 2a-d. boxplots visualizing levels of gdf15, endostatin, mmp9, and ngal in ng per ml, in controls and in patients with asymptomatic and symptomatic lymphoma. medians are shown as thick lines, the bottoms and tops of the boxes represent the first and third quartiles, and the whiskers show the smallest and largest non-outliers. outliers are shown as circles. https://dx.doi.org/10.48101/ujms.v128.9392 4 j. hidman et al. growth differentiation factor 15 is a pro-angiogenic protein that belongs to the transforming growth factor β superfamily. it is expressed in the placenta during physiological conditions as well as being secreted by activated macrophages in response to cellular stress signals such as inflammation, tissue injury, and hypoxia (18), and stimulates proliferation and migration of endothelial colony-forming cells (19). there are several other studies published showing that gdf15 stimulates angiogenesis in vitro (20, 21). increased levels of circulating gdf15 are seen in patients with various types of cancer, for example, prostate and colorectal cancer (18). growth differentiation factor 15 levels are also increased in mm and are associated with poor prognosis (11, 22, 23). our finding of higher plasma gdf15 in patients with asymptomatic indolent b-nhl, compared with controls, points to an increased angiogenesis even in pre-symptomatic disease. matrix metalloproteinases are a family of proteolytic enzymes, which degrade components of the ecm. in cancer, mmps are known to support tumorigenic processes such as angiogenesis, metastasis, proliferation, and invasion. degradation of the ecm by mmps promotes angiogenesis by several mechanisms including release of pro-angiogenic cytokines (such as vegf) sequestered in the matrix. matrix metalloproteinase activity is essential for angiogenesis and mmp inhibition reduces capillary growth (24). a study in rat skeletal muscle showed that decreased mmp activity was associated with a reduction in the number of microscopically observable capillary basement membrane breaks (25). thus, it is conceivable that mmp activity facilitates vascular sprouting and capillary growth by making the vascular basement membrane permeable to migrating and proliferating endothelial cells. matrix metalloproteinases are expressed in several types of solid tumors (24), and increased expression of mmp9 has also been seen in patients with cll and high-grade nhl (12, 26). endostatin is formed by mmp proteolytic cleavage of type xviii collagen. this type of collagen is abundantly expressed in basement membranes, including those in blood vessels (27). increased circulating levels of endostatin are seen in patients with several different types of cancer (28). endostatin exerts its effect on angiogenesis by binding vegf receptors and inhibiting the angiogenic effect of vegf, but the net effect on tumor angiogenesis is determined by the balance between antiand pro-angiogenic factors (28). since endostatin is formed by proteolytic degradation of collagen type xviii by mmp9 (27), our observation of higher plasma endostatin in patients with asymptomatic lymphoma suggests that increased angiogenic activity is an early event in indolent b-nhl progression. interestingly, we observed that endostatin was increased in plasma from asymptomatic b-nhl patients compared with controls, but mmp9 was not. there are several possible explanations for this. firstly, we cannot exclude an increased mmp9 activity in asymptomatic patients, since we measured plasma mmp9 concentration and not protein enzymatic activity. secondly, it is possible that the increased endostatin is a result of collagen xviii degradation by proteases other than mmp9. neutrophil gelatinase-associated lipocalin, also known as lipocalin-2, is a protein expressed by neutrophils and multiple tissues in humans, including adipose, lymphatic, and respiratory tissue. its expression is high in cancer tissue such as that from breast, pancreatic, and ovarian cancer (29). moreover, treatment-naïve cll patients have elevated levels of ngal in serum (14). ngal has the ability to form heterodimers with mmp9, thus preventing mmp9 degradation, and increased ngal-mmp9 complex formation has been figure 2b. plasma gdf15 is significantly higher in patients with symptom atic and asymptomatic lymphoma than in controls (p < 0.05 in both cases). figure 2c. patients with symptomatic lymphoma have significantly higher plasma mmp9 than controls have (p < 0.05). figure 2d. patients with symptomatic lymphoma have significantly higher plasma ngal than controls do (p < 0.05). endostatin and gdf15 in indolent nhl 5 detected in certain cancers (29). plasma ngal and tumor ngal mrna have been shown to be increased in a murine hypoxic tumor model, and ngal expression is increased in human and murine cancer cell lines cultured under hypoxic conditions (30). neutrophil gelatinase-associated lipocalin stimulates angiogenesis in vitro by upregulation of vegf expression, mediated by hif-1α signaling (31). it has also been shown to promote angiogenesis in rodent brain endothelial cells, by iron and reactive oxygen species-related pathways (32). we found increased levels of plasma ngal in patients with symptomatic, indolent b-nhl in comparison with controls, indicating that ngal is involved in the increased angiogenesis observed in these patients. long pentraxin 3, an acute phase reactant, is involved in several aspects of cancer progression, such as angiogenesis and immune modulation (33). the pro-angiogenic glycoprotein gal-3 is upregulated in anaplastic large cell lymphoma cells (34) and it has recently been shown that plasma gal-3 is increased in cll (12). in this study, we found no significant differences in mean plasma levels of ptx3 and gal-3 when comparing the two lymphoma cohorts and the controls, indicating that these two proteins are not involved in indolent b-nhl angiogenesis and disease progression. despite previous findings of increased mvd correlating with a more advanced disease and decreased os in patients with b-nhl, treatment with vegf inhibitors and other anti-angiogenic agents has generally not shown the positive outcome seen in treatment of solid tumors (6). lenalidomide is a potent inhibitor of angiogenesis in vitro (35), which could be an explanation for the positive effects on response rate and progression-free survival observed in relapsed indolent b-nhl when adding lenalidomide to rituximab (36). further indications that angiogenesis is of importance for lymphoma progression include findings that addition of the vegf-a antibody bevacizumab to rituximab treatment prolonged pfs in patients with relapsed follicular lymphoma (37), and that the anti-angiogenic multikinase inhibitor sorafenib given in monotherapy to patients with relapsed lymphoma has shown disease stabilization in patients with indolent b-nhl (38). in summary, we found that four proteins related to angiogenesis, that is gdf15, endostatin, mmp9, and ngal, are increased in plasma from patients with symptomatic b-nhl. this finding of increased gdf15 in patients with b-nhl has to our knowledge not previously been shown. since we did not have access to bone marrow and lymph node biopsies from the lymphoma patients, we could not perform immunohistochemical analyses for vascular markers. this is an obvious limitation of our study. we suggest that increased plasma gdf15 reflects an increased angiogenic activity and not an inflammatory response primarily, since there was no difference in mean crp between the two lymphoma patient cohorts (table 1). it is possible that gdf15 could be a marker for more advanced disease since it is secreted in response to cellular stress, but the fact that we did not find a significant difference in levels of albumin, hemoglobin or lactate dehydrogenase between the two lymphoma patient groups makes this less probable (table 1). the rationale behind our decision to compare protein expression between the two lymphoma cohorts was to identify angiogenesis-associated proteins that might be involved in the transition from asymptomatic to symptomatic disease. a comparison between the different lymphoma subtypes was not performed because of the limited number of patients. blood samples were taken at diagnosis only, so no longitudinal follow up was done. these are also limitations of our study. our discovery of increased plasma endostatin and gdf15 in asymptomatic indolent b-nhl indicates that increased angiogenic activity plays a role in the transition from asymptomatic to symptomatic lymphoma disease. it is possible that the increased endostatin reflects a transformation of the intratumoral capillary basement membranes to a more cell-permeable state, and that gdf15 stimulates endothelial cell migration through the breaks in capillary basement membranes and cell proliferation, creating new vessels. hence, increased plasma endostatin and gdf15 probably reflect high intratumoral concentrations of these two molecules. previous studies on anti-angiogenic agents in patients with indolent b-nhl have only included patients with relapsed or refractory disease. the results of our study indicate increased angiogenic activity in an early disease stage, emphasizing that  studies of anti-angiogenic treatment in patients with asymptomatic indolent b-nhl could be of interest. disclosure statement the authors report no conflict of interest. funding this study was supported by grants from the swedish government (national agreement on medical education and research), västmanland county cancer research fund (västmanlands cancerfond), and lions cancer research fund uppsala-örebro. notes on contributors josefin hidman, md, is a consultant physician at the department of medicine, västmanland county hospital, västerås and a phd student at the department of medical sciences and centre for clinical research västmanland, uppsala university, sweden. anders larsson, md, phd, is a professor at the department of clinical chemistry, uppsala university hospital and at the department of medical sciences, uppsala university, sweden. måns thulin, phd, is an associate professor at the department of mathematics, uppsala university, sweden. torbjörn karlsson, md, phd, is an associate professor at the department of haematology, uppsala university hospital and at the department of medical sciences, uppsala university, sweden. 6 j. hidman et al. orcid josefin hidman https://orcid.org/0000-0001-6660-8199 anders larsson https://orcid.org/0000-0003-3161-0402 torbjörn karlsson https://orcid.org/0000-0003-4707-8633 references 1. armitage jo, gascoyne rd, lunning ma, cavalli f. non-hodgkin lymphoma. lancet 2017; 390(10091): 298–310. doi: 10.1016/ s0140-6736(16)32407-2 2. lumish m, falchi l, imber bs, scordo m, von keudell g, joffe e. how we treat mature b-cell neoplasms (indolent b-cell lymphomas). j hematol 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https://doi.org/10.1016/j.clml.2014.02.010 https://doi.org/10.1016/j.clml.2014.02.010 https://doi.org/10.1111/j.1365-2141.2012.09139.x upsala j med sci 84: 95-104, 1979 on the relation between peripheral atherosclerosis and serum lipoproteins uno erikson', martin ericsson* and r u n e persson' from the departments of diagnostic radiology1 and geriatrics', university hospital, uppsala, s a w l e n abstract the occurrence and degree of peripheral atherosclerosis in 30 male patients with symptoms of intermittent claudication were studied by arteriography. the changes observed in the angiograms were codified. in all patients the concen trations of triglycerides and cholesterol were determined in whole serum and in the three major lipoprotein classes very low density, low density and high density lipoproteins. material and were also correlated to the codified angiographic findings in each individual patient. these data were compared with those of a control positive significant correlation was not found between the arteriographic changes and the serum concentrations of lipids and lipoproteins, which might be explained by an advanced stage of the disease where such relationships might not appear. introduction an increased incidence of hyperlipidaemia and hyperlipoproteinaemia in patients with peripheral arterial disease (pvd) has been noted by several authors (1,4,5,15,16,17,22,24). most of these reports, however, give quanti tative information only about total plasma lipids and since most plasma lipids are transported in the form of lipoproteins, it is of major importance that these will be determined. there appear to be no reports in the literature on the relation between angiographic changes in peripheral arteries and serum lipoprotein levels. this investigation was therefore undertaken to study the correlation between atherosclerotic manifestations and the concentrations of triglycerides and cholesterol in whole serum and in the three major lipopro tein classes (very low density /vldl/, low density /ldl/ and high density lipoproteins / h d l / ) . in this connection the recent report ( 2 ) that hyper lipoproteinaemic patients with pvd showed regression o f the atherosclerotic changes after treatment with lipid-lowering agents is of interest. it is always important to measure the atherosclerotic changes objectively. 95 various densitometric and computerized methods have been suggested for this purpose. these are not very commonly used, however, and are still a matter of evaluation ( 1 1 , 1 2 ) . it seems justified at present to assess the arteriograms by a more simple technique which we have used at our hospital for a long time, and the same type of gradation has been applied both for peripheral arteries and for the coronary arteries. in the study reported here w e have tried to make an objective evaluation of the arteriographic changes i n the distal aorta, the pelvic arteries and the arteries in the legs. these changes have been correlated to the concentrations of triglycerides and cholesterol in whole serum, vldl, ldl and hdl. material and methods mate r i a 1 thirty men of ages 39-76 years, with a mean age of 60 years, were investi gated. all patients had symptoms of intermittent claudication. they were hospitalized at the department of geriatrics in uppsala and referred to the department of diagnostic radiology of the university hospital in uppsala. five patients were overweight, defined as a weight/height index above 1.10 (weight in kg divided by height in cm -100). from thyroid dysfunction, diabetes, obstructive liver disease or nephrotic syndrome and none was being treated with any f3-blocking or lipid-lowering agent none of the patients suffered methods laboratory chemical methods blood was sampled in the morning after an overnight fast on the day of admission to hospital, in order to eliminate any effect of hospital diet. lipid-lipoprotein analysis the ultracentrifugal lipoprotein separation was performed as described by carlson (10). whole serum as well as isolated lipoprotein fractions were extracted manually with isopropanol. cholesterol and triglyceride concentra tions were then determined by automated techniques, using an autoanalyser model 2 (technicon corporation) (21). the cholesterol values obtained with this method agreed well with those obtained by the sperry-webb method. the trigly ceride values for whole serum were about 0.2 mmol/l higher than those reported by carlson (7). reference group control subjects for lipoprotein comparisons (n = 61) have been described previously (8). 96 radiological methods all patients underwent peripheral arteriography with use of the seldinger technique. a catheter was introduced into the femoral artery under local anaesthesia, its tip being placed in the distal aorta just above the bifurca tion. the investigation comprised the distal aorta and the arteries on the right and left side, including the distal part of the lower leg. in most patients special oblique projections were also used with regard to the deep femoral artery. an automatic film changer (aot, siemens elema, solna, sweden) was used. the film frequency was 1 exposurels. the contrast medium was injected with a sisal 2 pressure syringe (siemens elema, solna, sweden). i n all investiga tions angiografin (schering ag, w.germany) was used, in an amount of 30 ml which was injected at 15 ml/s. the film-focus distance was 100 cm. the angiograms were analysed by a standardized method. the diameter of the superficial femoral artery was measured at the level of the lesser trochanter and 20, 10 and 0 cm above the knee joint. for measurement of the diameters a magnifying glass with an inbuilt scale and with an accuracy of 0.1 mm wasused. the greatest and the smallest diameters were also noted in the different sec tions of the arteries. the changes in the different regions of the arteries from the aorta to the arteries of the distal lower leg were codified according to the following system. no changes = 0; diameter reduced by not more than 50% = 1; diameter reduced by more than 50% = 2; reduction of the diameter by more than 50% in several places in the arterial region = 3; and total occlu sion = 4 (13). the development of collaterals was also graded, using a 3-grade scale: 1 = a very small number of collaterals, 2 = a moderately large number and 3 = a large number. the length of any total arterial occlusion was measured. the following arteries in addition to the aorta were studied: the common, internal and external iliac arteries, deep and superficial femoral arteries, popliteal artery, anterior and posterior tibia1 arteries and the fibular artery, i.e. nine different arterial regions. thus the maximum sum of codes was 36. statistical analysis was performed as described by snedecor (23). results the results of the radiographic analysis by means of the code system are presented in the form of histograms in fig. 1. the lengths of the occlusions are shown in fig. 2. surprisingly long occlusions, up to 56 cm, were observed. occlusions as short as 3 cm were also found. i n most cases a large number of 97 l f o x .c o r n d (3 n 7 4. 0 0 0 0 < m e 4 7 c n 7 0 0 0 0 0 a p ) n l 98 d m n v 0 c v) u o o o a m c u l m n l 0 c t o o 0 < m e 4 7 m " a rl m o .d c a p w a m n n 0 n 30 1 2 3 4 :"$ 2 0 1 0 + code 0 1 2 3 4 n 30 a f i 2 0 -i 2 0 1 0 10 code code 0 1 2 3 4 fig. 1 (continuation) length of the occlusion cm 11 20 0 0 1 2 3 4 fig. 2 . the arterial occlusions found in 24 of the 30 patients are shown; mean value and range. dx sin collaterals had developed (fig. 3), indicating that the occlusions were of long duration. in fig. 4 the diameters at different levels of the superficial femoral artery and the popliteal artery are given. it is seen that the dia meters were clearly reduced at all levels. total occlusions were most common i n the superficial femoral artery and were relatively few in the arteries of the lower leg. the lipid analysis showed that 70% of the patients with pvd had a normal lipoprotein pattern according to the system modified by fredrickson (3). seven per cent had type iv, 20% type i1 a and 3% type i11 a . the cholesterol 99 n 15 10 5 8 6 4 2 1 2 3 i i i i --+ i t t 1 i _ i i i 1 1 1 1 1 1 --i 1 i i t i 1 i i i 7 i i i i i i i i i i i b i i i i i i 1 i i i i 1 i i i i i i i i i i i i i i i i i 1 i i i 1 i i i i i i i i i i i i i i i -i. i i i 0 mm a 1 fig. 3. the three classes of collaterals: 1 = a small number, 2 = a moderate number and 3 = abundent collaterals. fig. 4. the arterial dia meters at four different levels, and che maximal and minimal diameters. the dia meters are markedly reduced in many places. lesser 20 1 0 o m a x min troch. cm above t h e minor knee joint 0 q concentration in the h d l fraction was significantly lower in patients with pvd than in the controls (table 1). table 1. the concentrations of triglycerides (tg mmo1/1) and cholesterol (chol mmol/l) in total serum and in the three lipoprotein classes v l d l , l d l and h d l (mean 2 sem) in control subjects and in patients with peripheral atherosclero sis. xxx indicates a statistically significant difference (p < 0.001). total v l d l l d l h d l tg c h o l tg chol tg chol tg chol con tro 1 s n = 6 1 1.76 6.45 0.95 0.49 0.52 4.28 0.24 1.39 +.02 + . 1 3 2 . 0 1 t.05 +.08 + . 1 5 +.07 +.05 pvd n = 30 2.35 6.68 1.55 0.80 0.58 4.46 0.23 1.08xxx +.38 2 . 2 6 2.41 +.23 +.02 2 . 2 1 5 . 0 1 2.05 100 the results of correlation analysis of the angiographic code for the right + left limb versus the mean triglyceride and cholesterol concentrations in total serum and in the three lipoprotein classes (vldl, ldl, hdl) in the 30 patients with pvd are presented in table 2. table 2. correl-ation coefficients (r-values) of the scoring code (the sum of the codes from the right and left legs) versus the mean triglyceride (tg) and cholesterol (chol) concentrations in total serum and in the three lipoprotein classes vldl, ldl and hdl in the patients with peripheral atherosclerosis. x indicates a statistically significant correlation (p < 0.05). total vldl ldl hdl tg chol tg chol tg chol tg chol -. 23 -.43x -.22 . 2 7 -.05 -.18 -.22 -.07 there was a slight significant negative correlation between total choleste rol and the code symbolizing the atherosclerotic changes. ever, to the patient with a type 111 pattern of lipoprotein abnormality. this was due, how discusson arteriographic changes in the lower extremity have been described earlier by lohr and collaborators (18). they found that both early atherosclerosis in younger patients and atherosclerosis in elderly people were most common in the superficial femoral artery but were also present in the anterior and posterior tibular arteries and in the fibular artery. they did not, however, examine the relation between blood lipoproteins and atherosclerotic changes as on arteriograms. but they did report that among the patients with early athero sclerosis there was a high frequency of smokers, and that about 25% of the patients had diabetes mellitus. their angiographic results are in accordance with ours. when evaluating our results it must be kept in mind that arteriographic findings are always difficult to assess objectively. we have tried to avoid subjective judgement as far as possible. but codifying in itself involves an element of subjective evaluation. nevertheless we have found this method use fut. the arterial diameters at the different standardized points are more reliable and the error of the method is 0.1 mm (14). however, cannot always be identical with the maximal and minimal diameters, so we have also included these diameters to better characterize the arterial changes. we have also included the length of the occlusion and have tried to assess the number of collaterals. a l l this together should increase the value these measurement points, 101 of the classification of the arterial changes, that is the degree of periphe ral vascular disease in the legs. the angiographic technique is also important. a sufficiently large dose of contrast medium and a sufficiently long series of exposures, with a large num ber of films, must be used to be sure that all parts of an artery are visua lized. this is important as otherwise the number of collaterals may be under estimated and the length of a total arterial occlusion overestimated (19). this is especially true when an arterial region is filled in the retrograde direction via the collaterals. if the investigation time is too short an occlusion may be judged longer than it is in reality. we have tried in our investigations to avoid this and for this reason have avoided a so called stepwise technique, which means that the patient is automatically moved to gether with the table in relation to the x-ray tube, so that the same amount of contrast medium can be used almost throughout the whole series of investi gation. this technique is very unreliable and is unsuitable for a study such as the present one. it should not be used in any preoperative investigation. values are not available for normal arterial diameters at different levels. but a comparison may be made with a material mostly of men reported by erikson (14). stump and the healthy, intact leg were compared. from these arteriograrns it is obvious that in our 30 patients with atherosclerotic disease the arterial dia meters were greatly reduced. the degree of magnification was the same in the two materials. it seems that our arteriographic method is fairly objective and will describe the atherosclerotic changes fairly well. it thus seems justified to use this method for examining the relation between atherosclero sis and blood lipoprotein concentrations, even though a densitometric techni que is probably superior. this comprised a study of traumatic amputees in which the amputation it was a surprising finding that there was a very slight significant nega tive correlation between total cholesterol and the atherosclerotic changes. however, the arteriographies in our material obviously revealed all the typi cal signs of atherosclerosis, namely diametric reduction, occlusion and col laterals. the cholesterol concentration in the hdl fraction was significantly lower in our material than in the controls. this phenomenon has also been observed in survivors of myocardial infarction (8, 9). the role of a low con centration of hdl cholesterol in the development of atherosclerotic manifesta tions is not known. it is of interest, however, since hdl may transport cholesterol out of tissues (20), even from the arterial wall (6). our correlation data do not exclude the possibility of a causal relation ship between abnormalities in lipoprotein metabolism and atherosclerotic changes, as the stage of the disease in the individual patient was not known. 102 in view of the regression of early femoral atherosclerosis observed in hyper lipoproteinemic patients after treatment with lipid-lowering agents (2), our patients might have been in a fairly advanced stage of the disease, where correlations between lipoproteins and atherosclerotic manifestations do not necessarily appear. references n. 2. 3. 4. 5. 6. 7. 8. 9. ballantyne, e . & lawrie, t . d . v . : cyperlipoproteicaemia and peripheral vascular disease. clin chim acta 47:269-277, 1973. barndt, r., blankenharn,, d.h., crawford, d.w. & brooks, s.h.: regression and progression of early femoral atherosclerosis in treated hyperlipopro teinemic patients. ann intern med 86: 139-146, 1977. beaumont, j.-l., carlson, l.a., cooper, g.r., fejfar, z., fredrickson,d.s. & strasser, t.: classification of hyperlipidaemias and hyperlipoproteinae mias.who bull 43: 891, 1970. beyrer, k., may, r. & sailer, s.: blutlipide und glukosetoleranz bei 100 patienten mit arteriosklerotischem verschluss an dem unterem extremitgten. wien klin wochenschr 80:392-394, 1968. bliss, b.p., kirk, c.j.c. & newall, r.g.: abnoralities in glucose toler ance, lipid and lipoprotein levels in patients with atherosclerotic peri pheral arterial disease. angiology 23:$9-75, 1972. bondjers, g. & bjijrkerud, s.: cholesterol elimination in vitro from normal and atherosclerotic arterial tissue. eur j clin invest 4:368, 1974. carlson, l.a.. determination of serum triglycerides. j atlieroscler res 3: 334-336, 1963. carlsson, l..a. & ericsson, h.: quantitative and qualitative serum lipo protein analysis. part 1. studies in heblthy men and women. atherosclero sis. 21:417-433, 1975. carlson, l.a. & ericsson, m.: quatitative and qualitative serum lipopro tein analysis. p x t 2. studies in male survivors of myocai-dial infarction. atherosclerosis 21:435-450, 1975. 10. carlson, k,: lipoprotein fractionation. j clin pathol, suppl 5: 26: 32-37, 1973. 11. crawford, d . w . , brooks, s.h., selzer, r.h., barndt, r., beckenbach, e.s. & blankenhorn, d.h.: computer densitemetry for angiographic assessment of arterial cholesterol content and gross pathology in human atherosclerosis. j lab c l i n med 89:378-392, 1977. 12. crawford, d.w., brooks, s.h.,barndt, r.& blankenhorn, d . h . : measurement of atilefosclerotic luminal irre ularit and obstruction by radiographic densitometry. invest radiol f2:307-313,1977. findings in occlutions of arteries of the extremities. vasc surz 3:201-210, 1969. 14. erikson, u.: cirtulation in traumatic amputation stumps. an angiographical and physiological investigation. acta radiol suppl 238, 1965. 15. greenhalgh, r.m., rosengarten, d.s., mervart, i., lewis, s., galnan, j . s . & martin, p.: serumlipids and lipoproteins in peripheral vascular disease. lancet (11) 947-950,1971. tein%nien bei peripheren arterrellen verschlueskzankheiten. mgnch med wochenschr 115:66?, 1973. 13. delius, w. & erikson, u.: correlation between angiographlc and hemodynamic 16. kremer,g.j., niemczyk, h., poeplau, w. & nicolescu, r.f.: hyperlipopro 17. leren,p. & haabrekke, o.:blood lipids in patients with atherosclerosis 18. tiihr, e. & schulte-herbruggen, g.: angiographische befunde der arterio ' obliterans of the lower limbs. acta med scand 189:5:1, 1971 b. sklerose unter rerucksichtigung der frtihsklerose. fortschr geb roentgenstr nuklearmed 112:39-48,1971 103 19. 2c. 21. 22. 23. 24. madejski, t. & tobik,s.: arteriographic errors in occlusive diseases of lower limbs. surgery 55:210-213, 1964. norum, k.r., glomset, j.a. & gjone, e.: familial lecithin-cholesterol acyl transferase deficiqnc . in the metabolic basis of inh rited disease (ed. j.b.strandbury, j.b. h&gaa$den & d.s.fredricson) p . 537, mcgraw hill, new pork, 1972.. rush, r.l., leon, l. & turrel, j.: automated simultaneous cholesterol and t:prg%yceride deteminations on the auto analyserrii instrument. advances i 1 automated analysis, thurman a s s . 1:503, 1971. skrede, s. & kvarnstein, b.: hyperlipidemia in peripheral atherosclerotic arterial disease. acta chir scand 141:333-340, 1973. snedecor, g.s.: statistical methods. the iowa state univ presg. iowa,u.s.a. 1961. wollenweber, j., doenecke, p., greten, h., hild, r., nobbe, f., schmidt, f.h. & wagner, e.: zur hhfigkeit von hyperlipidki, hyperurikki, diabetes mellitus, hypertoni und ubergewicht bei arterieller verschlusskrankheit. dtsth med wochenschr 96:103-107, 1971. accepted january 20, 1979. address for reprints: uno erikson, m . d . department oe -diagnostic radiolo?; universoty hospital s-750 14 uppsala sweden 104 upsala j med sci 81: 123-128, 1976 relation between erythrocyte and plasma lithium concentrations as an index in psychiatric disease l e i f lyttkens, u l f soderberg and lennart wetterberg from the psychiatric research center, ullerzker hospital, university of uppsala, and department of psychiatry, karolinska institute, s t . goran's hospital, stockholm, sweden abstract in longitudinal and transverse studies, lithium was measured in plasma, serum and red blood cells (eryth rocytes) of healthy male and female subjects as well as in patients of both sexes suffering from manic-depressive disease or schizophrenia. the results confirm that lith ium in erythrocytes is lower than in plasma in all groups. the lithium concentration gradient between plasma and erythrocytes is not caused by a slow rate of diffu sion through the erythrocyte membrane. the new result of the present study is the importance of sex, disease and age on the erythrocyte/plasma lithium ratio, which is significantly higher in female subjects with manic-depressive disease. this difference persists even during long-term lithium therapy. older female schizophrenics also have a higher ratio of erythrocyte to plasma lithium than males of the same age. the findings emphasize the importance of endocrine in vestigation in mental disease and support the view that plasma lithium in humans does not always reflect the intracellular levels. the erythrocyte plasma ratio may also be of value in revealing diagnostic subgroups within the classical psychiatric framework. introduction even if lithium is assumed to be distributed mainly within intraand extracellular water compartments of the body, its distribution is not uniform for several reasons. it can be transported actively through cell membranes and it influences and com petes with several other ions such as sodium, po tassium, calcium and magnesium. (for review, see johnson, 1975 (1 l).) the distribution is strongly dependent on the lithium level in the plasma and possibly also on the duration of the lithium treat ment. the therapeutic level of lithium is low and less than 1 % of plasma sodium is exchanged for lithium. the lithium tissue/serum distribution ratios also unexpectedly exceed 1 . o in several tissues, such as muscle, bone, brain and kidney (21), al though intracellular sodium is known to be low. in addition, since blood lithium varies rapidly be tween doses, and its transport into cells vanes from tissue to tissue and is sometimes slow, part of the uneven distribution depends on the absence of a true distribution equilibrium. several reports have claimed that manic patients retain more lithium than healthy controls even after a single dose (1). this is the basis for attempts to link the clinical picture of patients receiving lithium and the erythrocyte/plasma concentra tion ratio. it has been preliminary reported that female manic-depressive patients had higher erythrocyte/plasma lithium ratio than healthy volunteers (14, is), and that manic patients with acute attacks have such an elevated ratio (7). correlations with response to treatment (4, 18) and with disease have been proposed (25), but also criticized (20). part of the disagreement may de rive from the observed dependence of the erythro cytelplasma lithium ratio on the plasma lithium level (12). fully aware of these difficulties, we further extended our previous material (14, 15) to include healthy male subjects and patients from both sexes with schizophrenia, i n order to investi gate whether or not some disagreement in the litera ture could be traced to distribution variations aris ing from differences in sex, age and disease; especially so, since preliminary findings revealed the possibility that differences in lithium distribu tion between erythrocytes and plasma are not ex clusive to manic-depressive disease. material the studies included longitudinal and transverse assays of lithium in plasma, serum and red blood cells. lithium upsala j med sci81 124 l . lyttkens et ai. mcqll 0.6 0.4 .e, .5 -t 0.2 0 t plasma -10rbc lithium 8 meq x 3 doily fig. 1 . concentration of lithium in plasma and erythrocytes in 8 healthy females in response to lith ium administration for 2 weeks. was analysed with a perkin-elmer 306 atomic absorption spectrophotometer. plasma and haemolysed blood were diluted 50 to 100 times for the analyses. the venous blood samples were drawn in the morning with the sub jects fasting before the first dose of lithium was given. lithium was administered as lithium carbonate. in the healthy individuals and in the patients comprising the longitudinal studies, a dose of 8 meq three times daily was given. the patients comprising the transverse studies were given dosages that gave plasma levels between 0.6 and 1.2 meq/l. material healthy persons two groups of apparently healthy subjects were exam ined, one comprising 8 female nurses and the other 8 male ambulance drivers from a hospital a t ludvika, dalecarlia. their ages varied between 20 and 40 years. no other medication than lithium was taken during the trial period. blood samples were taken in the morning, twice before lithium was given and 13 times during the following 18 days. in addition the sedimentation rate, leukocyte count, platelet count, plasma creatinine and glucose in whole blood were assayed before, during and after the lithium test. there was no restriction in diet or special control of salt intake. upsala j med sci 81 pafients the patients were all treated at ullergker hospital, uppsala, sweden. thirty-seven patients with manic depressive disease (23 women, 14 men) were examined; most of them had symptoms (in-patients), but some were in remission and were having lithium as prophylaxis (out patients). their ages ranged from 20 to 68 years. these patients had received lithium therapy for periods varying from 2 months to several years. a further 8 males and 8 females diagnosed as having chronic schizophrenia (in patients) were treated in therapeutic trial at the hospital with dosages comparable to those given to the above mentioned healthy subjects. blood samples were taken in the same sequence as in the healthy subjects. the schizophrenic patients, however, also received their usual antipsychotic medication during the trial period. the diet was not restricted and there was no special control of salt intake. results healthy subjects during the test period lithium concentrations in plasma and erythrocytes followed the same time course, though the level was lower in erythrocytes than in plasma (see fig. 1). a steady state between lithium in erythrocytes and plasma 125 table 1 . half-lives (t+) of lithium in plasma during the elimination phase subjects number (hours) half-life healthy females 8 19f 1 healthy males 8 23+1 schizophrenic females 8 19f2 schizophrenic males 8 21f3 plasma and erythrocytes was rapidly attained, and no significant delay in the erythrocyte level was noted with the present sampling routine. the plasma levels reached 95% of the steady state within 4 days of treatment with lithium. after dis continuation of lithium therapy the half-life was computed from the slope of the concentration curve obtained during the elimination period the first point being the measurement 24 hours after the last dose. the half-lives thus obtained were not significantly different in the two sexes (men 23k2 and women 19f2 hours; table i). likewise the groups of healthy persons showed no sex difference (table 11). the sedimentation rate, leukocyte and platelet counts, creatinine in plasma and glu cose in blood were not significantly affected by the lithium intake. pa tien ts the relation between the erythrocyte and plasma levels of lithium in the manic-depressive patients of both sexes can be seen in table 11. females with manic-depressive disease showed a probably significant higher erythrocyte/plasma ratio of lith ium than healthy females (p<o.os) and manic depressive males @<o.os). the results of the longitudinal study have been taken from the last day of sampling in the steady state phase (fig. 1 ) . in the case of the manic-depressive patients, the day of sampling varied in relation to the first day of treatment, but in all cases the sampling was performed during the steady state. in this group, no classification was made according to the activity of the disease, the duration of lithium therapy or the age of the patients, since the numbers in each such subgroup would have been too small t o permit reliable statistical analysis. on the other hand, in the group of schizophrenic patients it was possible to select the patients so as to permit a further classification according t o age. in the sample of schizophrenic patients there was no sex difference in the ratio of lithium in erythrocyte to lithium in plasma in the younger age groups (30-37 years), but in the higher age group this was considerably higher in the women (table 111). the half-lives of plasma lithium in the schizo phrenic groups did not differ from those of the healthy volunteers, although the standard devia tions were greater in the diseased than healthy subjects (table i). in addition, we found in indi vidual patients with schizophrenia and schizo affective disease high levels of lithium in erythro cytes, and in 3 patients with the latter disease, the erythrocytes levels were repeatedly almost 100% of the plasma levels (16). comment the results of this study confirm previous investi gations (7, 8 , 17, 19) that the concentrations of lithium in erythrocytes are lower than in plasma. the new result of the present study is the finding of the importance of sex, disease and age in affect table 11. distribution of lithium between plasma and erythrocytes at steady state lithium m e q / l f s e m rbcxloo subjects number (range) plasma rbc plasma healthy females 8 33 (23-47) 0.57f0.05 0.23k0.03 39+3 healthy males 8 35 (23-49) 0.48f0.02 0.17f0.02 3 4 f 3 manic-depressive females 23 38 (22-53) 0.66f0.04 0.34f0.03 5054 manic-depressive males 14 40 (23-66) 0.63k0.03 0.24 f0.03 3 8 f 4 age upsala i med sci 81 126 l . lyttkens et al. table 111. distribution of lithium (steady state levels) between plasma and rbc lithium steady state meql1ls.e.m. r b c x l o o age subjects number (range) plasma rbc plasma healthy females 8 33 (2347) 0.53f0.12 0.20i0.08 38+3 schizophrenic schizophrenic schizophrenic schizophrenic healthy males 8 35 (23-49) 0.47k0.06 0.17+0.05 34+3 females 4 36 (35-37) 0.37f0.01 0.15fo.01 4ol-5 females 4 60 (56-64) 0.66+0.10 0.37+0.10 52l-7 males 4 34 (30-37) 0.42+0.01 0.16i0.01 38+4 males 4 61 (59-63) 0.49i0.01 0.16f0.03 3 2 i 3 ing the erythrocyte/plasma ratio. the difference in lithium concentration between plasma and erythro cytes cannot be attributed to a slow rate of diffu sion through the erythrocyte membrane, at least not in our longitudinal studies in which the healthy subjects and schizophrenics were given lithium for 2 weeks. although not fully conclusive, the re sults in the manic-depressive sample seem t o indi cate that the difference persists even during long term therapy. one possible explanation for the low levels of lithium in erythrocytes is that erythrocytes contain less water and that the lithium concentrations are only seemingly lower when measured in relation to the total blood cell volume. since the water content of red blood cells is about 70% of that in plasma the difference found in this study between the lithium levels in plasma and erythrocytes can only partly be explained by a difference in distri bution volume. other explanations for the mech anism underlying the lower intracellular concentra tion must thus be sought, and possible candidates are the factors determining the transport and the difference between intraand extra-cellular electro lyte concentration. in the frog skin, there is a n active sodium trans port process by which sodium is transported from the outside to the inside of the skin. here the lithium ion can be actively transported by this “sodium pump” and lithium thereby competes with sodium in the transport process (29). the lower content of lithium in erythrocytes indi cates the presence of an active outward transport mechanism. the differences in erythrocytelplasma upsala j med sci 81 ratio observed might then be explained by changes in the capacity of the transport mechanism, a situation that has also been postulated in patients with unipolar depression (19) and has been shown in bipolar manic-depressive psychosis (10). in this context it is worthy of note that the dog, which has a different sodium pump in the erythrocyte membrane than man, shows erythrocyte levels of lithium comparable to plasma levels (13). this postulated close relation between lithium and sodium transport mechanisms indicates that dif ferences in sodium intake might explain our find ings. as shown by mendels and frazer (19), a change in sodium intake might cause a change in erythrocytes and plasma levels of lithium, but the erythrocyte/plasma ratio will, however, remain unchanged. thus the free salt diet cannot have been a source of error in our study. preliminary evidence for genetic control of lithium ion distribu tion across erythrocyte cell membrane has been presented (6), indicating that our differences in erythrocyte/plasma ratio in manic-depressive women might be correlated to a great predisposition to manic-depressive illness. lithium has also been shown to alter both calcium (27) and magnesium (3, 20) plasma levels. the significance of these changes is still unclear, but it is known that these two latter ions affect active transport of other ions, and it is conceivable that lithium may alter intracellular electrolyte concentrations in this way. in the apparently healthy subjects, there was no sex difference in the erythrocytelplasma lithium concentration ratio and in the group of schizo phrenics, a sex difference was only found in the lithium in erythrocytes and plasma 127 older age group. the significance of this finding is not clear, but would seem t o warrant a study of the possible effects of hormonal conditions on this ratio. in the present investigation we found two significant sex differences, one among the manic depressives, where the women had a higher erythrocyte lithium level than the men, and the other among the schizophrenics previously men tioned. in addition to the differences in endocrine state, these findings could be caused by differences in disease pattern, patterns of physical activity, reaction of the patients to the symptoms, effects of hospitalization, diet and administration of other drugs. from all these possibilities, we favour the differences in hormonal factors between males and females as a primary cause, since there are clear indications that the endocrine status influ ences lithium distribution in animals ( 2 5 ) , and the distribution of sodium both in humans and animals (2, 5, 28). the differences found in our study, however, may conceivably be related to specific differences in the erythrocytes, e.g. their age, and if this is the case they would not reflect the con ditions in other organs in the body. within the range of our assay, there is no significant deviation from a rectilinear relationship between the erythrocyte and plasma lithium concentration as in the work of lee et al. (12), where the erythrocyte lithium was compared with plasma lithium over a wide range. in our study, it is not possible to explain the relative levels of lithium in erythrocytes as caused by dif ferences in lithium concentration in the plasma. previously reported leukocytosis and changes in blood glucose found in patient samples (9, 17, 24) were not verified in the apparently healthy subjects in this study. thus, these reported changes are un likely to have been caused by the lithium intake alone, unless long-term treatment differs from a two-week test. acknowledgements this work was supported by grants from the swedish medical research council 3371 and the drug companies aco and hassle, sweden. references 1 . almy, g . l . & taylor, m. a.: lithium retention in mania. arch gen psychiatry 29: 232, 1973. 2. aikawa, j . k.: the nature of myxoedema: alterna tions in the serum electrolyte concentrations and radiosodium space and in the exchangeable sodium and potassium contents. ann int 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mania: before and after lithium carbo nate treatment. arch gen psychiat 23: 310, 1970. 10. hokin-neaverson, m., spiegel, d. a. & lewis, w . c.: deficiency of erythrocyte sodium pump activity in bipolar manic-depressive psychosis. life sciences is: 1739, 1974. 1 1 . johnson, f. n.: lithium research and therapy. academic press, 569 pp. 1975. 12. lee, g., lingsch, c., lyle, p. t. & martin, k.: lithium treatment strongly inhibits choline transport in human erythrocytes. br j clin pharmac 1:365, 1974. 13. ljungberg, s. & paalzow, l.: some pharmacological properties of lithium. acta psychiat scand, suppl. 207: 68, 1969. 14. lyttkens, l . , soderberg, u . & wetterberg, l.: in creased lithium erythrocyte/plasma ratio in manic depressive psychosis. lancet i, 40, 1973. 15. relationen mellan intraoch extracellulart litium som index vid psykisk sjukdom. nordisk psykiatrisk tidskrift 27: 392, 1973. 16. -unpublished observation. 17. maggs, r.: electrolyte changes in lithium therapy mania. proc. iv world congress of psychiatry. excerpt. medica foundation i.c.s. no. 150, part 3 , 150:2211, 1968. 18. mayfield, d. & brown, r . g.: the clinical labora tory and electroencephalographic effects of lithium. j psychiat res 4 : 207, 1966. 19. mendels, j . & frazer, a,: intracellular lithium con centration and clinical response: towards a mem brane theory of depression. j psychiat res 10:9, 1973. 20. nielsen, j.: magnesium-lithium studies. 1. serum and erythrocyte magnesium in patients with manic states during lithium treatment. acta psychiat scand 40: 190, 1964. 21. rybakowski, j . , chlopocka, m., kapelski, z., her upsala j med sci 81 128 l . lyttkens et al. nacka,b., szajnerman, z. & kasprzak, k.: red blood cell lithium index in patients with affective disorders in the course of lithium prophylaxis. int pharma copsychiat 9: 166, 1974. 22. schou, m . : lithium studies. 111. distribution be tween serum and tissues. acta pharmacol (kbh) 15: 115, 1958. 23. shopsin, b., friedman, r. & gershon, s.: lithium and leukocytosis. clin pharmacol therap 12: 923, 1971. 24. shopsin, b., stem, s. & gershon, s.: altered carbo hydrate metabolism during treatment with lithium carbonate. arch gen psychiat 26: 566, 1972. 25. soderberg, u.: konshormonberoende effekter och omsattningen av iitium pb experimentdjur. medicinsk riksstamma xxx, 391, 1973. 26. soucek, k., zvolsky, p., krulik, r., silip, v., vina rova, e. & dostal, t.: the levels of lithium in serum and in red blood cells and its ratios in manic-depres sive patients. act nerv super (praha) 16: 193, 1974. 27. tupin, j. p., schagenhauf, g. f. & creson, d. l.: lithium effects on electrolyte excretion. am j psychiat 125: 536, 1968. 28. zarrow, m . & money, w. l.: involution of the adre nal cortex of rats treated with thiouracil. endocrin ology 44: 345, 1949. 29. zerahn, k.: studies of the active transport of lithium in the isolated frog skin. acta physiol scand 33: 347, 1954. received december 10, 1975 address for reprints: professor lennart wetterberg, m.d. department of psychiatry s:t gorans sjukhus box 12500 s-11281 stockholm sweden upsala j med sci 81 upsala j med sci 81: 93-95, 1976 preoperative recurrent laryngeal nerve paralysis in patients subjected to thyroid surgery karl erik arosenius and sven grevsten from the department of surgery, central hospital, falun, sweden abstract results (table i and 11) all cases of preoperative recurrent nerve paralysis have been analysed in an unselected material of patients sub jected to thyroid surgery at the surgical clinic in falun during the period of 1969-74. the preoperative finding of recurrent nerve paralysis as a malignant sign is discussed. introduction hoarseness is a sign of malignancy, when found during the preoperative clinical examination of patients who present with a goitre (1). this is a classical statement. hoarseness is due to loss of function in the re current laryngeal nerve and/or the superior la ryngeal nerve and is usually caused either by cns damage or mechanically by section, pressure, or traction somewhere along the course of the nerve. this damage need not always be due to malignancy ( 2 ) . to illustrate this, we have studied the occurrence of pre-operative recurrent laryngeal nerve paralysis in a series of patients subjected to thyroid surgery during the 5-year period, 1969 to 1974. among 445 patients without carcinoma 5 cases of recurrent laryngeal nerve paralysis-one was partial-were found preoperatively. in 4 of these the paralysis has persisted in one case the nerve function recovered completely. at operation, 3 patients had large adenomas, one patient a nodular goitre and in one patient a bulging carotid artery was found. in 3 cases the nerve was seen to be compressed by an adenoma; in one of these the adenoma was calcified. the pathological diagnosis in 4 cases was nodular colloid goitre. in one case no pathological findings were present. in 2 cases x-ray examination showed extreme tracheal compression. in 3 patients the history of hoarseness began less than 2 weeks before admission; in one of these the nerve function returned later. in another 2 patients hoarseness had been present for longer. the scinti gram performed on the patient with a bulging carotid artery showed a small impression on the thyroid lobe on the side of the paralysis. of the 31 patients with thyroid cancer, there were 5 cases of recurrent laryngeal nerve paralysis. material and method this studv is based on the records of patients admitted discussion for thyroid surgery to the surgical clinic at falun between the recurrent nerve innervates the intrinsic 1969 and 1974. during this period a total of 476 thyroid muscles of larynx and passes the inferior thyroid operations were performed. thyroid cancer was present in 31 cases and benign goitre in 445. the vocal cord func tion of all patients was checked preoperatively. those patients with recurrent nerve paralysis have been artery in a variable fashion (3). when the nerve is injured, ordinary hoarseness occurs and the ipsilatera1 vocal cord is primarily followed up for 1 to 5 years and their vocal cord function immobilized in the paramedian or intermediate has been checked at intervals after the operation. nnsitinn 1 3 . 4. 5) was found, were recorded. muscle. it accompanies the superior thyroid artery upsala j med sci 81 94 k . e . arosenius and s . grevsten table i . preoperative recurrent laryngeal nerve paralysis in benign goitre preoperative tracheal patient’s onset of paralysis paralysis (+). pad from the compres age, less than 14 days postoperative operative sion on operative findings, sex before admission recovery (r) spicement x-ray gross pathology 42 p + sin.+r hoarseness and acute growth 54 0 79 0 67 0 60 0 dx+ (partial) tumor to the right in jugulum. scintogram: small impression on the right thyroid lobe. + bilat. + dx t onset paralysis 30 years earlier at the end of a preg nancy-“goitre already then” + sin. + cystic thyroid not per tissue+ formed nodular colloid goitre with haemorrhage not per formed nodular colloid extensive goitre compres sion nodular colloid not per big adenoma on left side bulging carotic artery on right side in jugulum. otherwise nothing. nodular goitre and “thin recurrent nerve” right-sided thyroid goitre formed adenoma, “big as a fist”. “the recurrent nerve firmly pressed by the adenoma where it is calcified.” nodular colloid extensive the goitre bigger goitre compreson left side sion “the recurrent nerve pressed flat behind the biggest adenoma”. medially. it is usually covered by the fascia of the inferior constrictors, but in 15% it is found in the thyroid sheath and in another 6% the nerve courses among the branches of the superior thyroid artery (3, 6). the first effect of laryngeal nerve injury is a re duction in the vocal cord range, particularly in the higher register. the affected cord moves but lies at a lower level than normal (7). the vocal cord is also irregular or wavy in outline (8). this is easy t o foresee clinically. simple hoarseness due to recur rent laryngeal nerve damage is the commonest sign and can be explained anatomically. in about 40% (3, 9, 10) (some variation of the figures for different authors) the nerve divides into two or more branches, 5 cm or more below the level of the cricothyroid joint. on entering the larynx the branches may be as much as 6 cm apart (11). furthermore, in 10% the nerve penetrates the thyroid gland before entering larynx (3, 12). the branches of the recurrent nerve thus form a delta before entering the larynx and this branch table 11. preoperative recurrent laryngeal nerve paralysis in patients subjected t o thyroid surgery in the y e a r s 1969-74 at the surgical clinic in faiun thyroid cancer benign goitre total n % n % n 9% n o recurrent nerve paralysis 26 83 440 99 466 98 31 100 445 100 476 100 recurrent nerve paralysis 5 17 5 1 10 2 upsala j m e d scirl preoperative recurrent laryngeal nerve paralysis 95 delta can b e partly or totally fixed in t h e thyroid tissue. local swelling of the gland in the region of the delta can exert considerable traction on t h e diverging branches of t h e nerve and produce distor tion particularly at the branching point, i.e. affect ing the whole nerve. an unbranched nerve would of course move more easily and adapt itself to the change of form i n the surrounding tissue. general or specific local growth of the thyroid gland can thus damage the recurrent nerve. t h e lesion can b e caused by tense, diffusely growing processes like cancer or riedel’s goitre but local changes such as local cancer, cystic adenoma, nodular goitre or acute subcapsular haemorrage can also cause similar damage. in our material of patients subjected t o thyroid surgery from 1969 to 1974 (table 11) (cancer pa tients included), we have found 10 cases with pre operative paralysis of the recurrent nerve. 5 of those 10 had no cancer. although 50% of the cases of paralysis belong to the cancer group, we think it is an exaggeration to describe the preoperative find ing of recurrent nerve paralysis as a sign of malig nancy. a statistical reservation must be made con cerning the figures in this study. the figures are collected from a patient material in a goitre region. the local goitre frequency clearly affects the prob able cause of preoperative recurrent nerve paraly sis. consequently in a region with less goitre a preoperative nerve paralysis may b e more relevant as a malignant sign. it is worth noting therefore that “locality” may influence the clinical significance of the presence of preoperative recurrent laryngeal nerve paralysis. r e f e r e n c e s i . cole, w. h., slaughter, d. p. & majazakis, j. d.: carcinoma of the thyroid gland. surg gynecol obstet 89: 349, 1949. 2. widstrorn, a , : vocal cord palsies following surgery for benign non-toxic goitre. acta otolaryngol75: 370, 1975. 3. kratz, r. c.: the identification and protection of the laryngeal motor nerves during thyroid surgery. a new microsurgical technique. laryngoscope 83: 59, 1973. 4. wagner, r.: die median stellung der stimrnband der bei der recurrenslahmung. arch path01 anat physiol 124: 127, 1890. 5 . dedo, h. h.: the paralyzed larynx: an electromyo 6 . 7. 8. 9. 10. 11. 12. mossman, d. a. & de wesse, m. s . : the external laryngeal nerve as related to thyroidectorny. surg gynecol obstet 127: 101 1, 1968. nordland, m.: the larynx as related to surgery of the thyroid. based on an anatomical study. surg. gyne col obstet 91: 449, 1950. lawson, l. j . : the superior and recurrent laryngeal nerves. quart bull northwestern univ med sch 22: 356, 1948. lawson, l. j . : recurrent laryngeal nerve paralysis. a revised concept based on the dissections of 100 cadavers. ann otol rhinol laryngol61: 567, 1952. rustad, w. h. revised anatomy of recurrent laryngeal nerves. surgical importance based on the dissection of 100 cadavers. j clin endocrinol metab 14: 87, 1954. king, b . t. & gregg, r . l.: an anatomical reason for the various behaviours of paralyzed vocal cords. ann otol rhinol laryngeol57: 925, 1948. berlin, d. d.: the recurrent laryngeal nerves in total ablation of the normal thyroid gland. an ana tomical and surgical study. surg gynecol obstet 60: 19, 1935. received january 19, 1976 address for reprints: sven grevsten, m.d. department of surgery university hospital s-750 14 uppsala sweden ~. graphic study in dog and humans. laryngoscope 80: 1455, 1970. upsala j med sci 81 upsala j med sci 98: 487-491, 1993 11. explanation of some other concepts and terms used in this report carl-henric de verdier,' torgny groth2 and per hyltoft petersen3 'department of clinical chemistry, and 2unit for biomedical systems analysis, uniuersity of uppsala, uppsala, sweden and 3departrnent of clinical chemistry, odense uniuersity hospital, 5000 odense, denmark analytical procedure an analytical procedure is made up of 1) an analytical measurement procedure consisting of the measurement procedure proper, calibration, calculation and correction procedures; 2) an analytical control procedure. this terminology was in principle introduced already in (1) and is preferred here in order to clearly distinguish between the overall procedure and its component parts. analytical control procedures can be defined by specification of control rule(s), control limits, control material, and the number of control observations. different types of variations abbreviations: st sb sa 'bw 'bb spea 'aw sab stbw total standard deviation (total sd) total biological sd total analytical sd biological within-subject sd biological between-subject sd pre-analytical sd analytical within-series sd analytical between-series sd total within-subject sd (including biological and analytical variation) cv, total coefficient of variation cvb total biological coefficient of variation. etc. 21 -935253 487 sawl sabl s~~ 'a0 analytical within-laboratory sd, can be calculated in different ways, e.g. as i) average for an individual laboratory or ii) average for several laboratories. analytical between-laboratory sd total analytical sd (including analytical within-lab and between-lab variation) inherent stable analytical random error (estimated from saw or sawl dependent on problem) definitions according to analysis of variation (anova): st2 = sb2 + sa 2 -k sprea 2 2 2 2 2 2 sb 'bw + 'bb sa2 = saw + sab 2 2 2 'tb; = 'a + 'bw ' 'prea sal2 = sabl + 'awl 2 2 anova is carried out according to references (2, 3). similar system for abbreviations of variations have been described by dot, mir6, and fuentes-arderiu ( 5 ) . different types of bias of a measuring instrument according to chapter 1 and vim (4), bias (of a measuring instrument) is defined as a "systematic error of t h e indication of a measuring instrument". in accordance with ref. (8) we have used bias to describe long term stable systematic errors and ase for short term systematic errors not included in "averaging the error of indication over an appropriate number of repeated measurements". abbreviations: b a analytical bias bcorr correctable bias; that part of bias (systematic error), which is well defined and estimated. noncorrectable bias; that part of bias which can not be well estimated. bnoncorr 488 b w l difference in bias (within a laboratory or for a given instrument) between two time points = [ba(tl) ba(t2)]. difference in bias between two laboratories. difference in bias between two instruments. bhl 'h1 the concepts of "analytical stability" and "analytical instability" are introduced in references (8, 9) in order to describe variation in analytical performance over a longer time period expressed as distances between (k; 1) points in a two-dimensional co-ordinate system. reference interval reference range a (biological) reference interval is, if other characteristics are not indicated, defined as a 0.95-interfractile interval (6, 7). the statistical definition of range is "the difference between the highest and the lowest value in a distribution". reference range is according to this definition the difference between the highest and lowest value in a distribution of reference values. errors of a measuring instrument the total error, te, is composed of the following terms: te = biascorr + biasnoncorr + ase * sao t z * are * sao where ase is a temporary increase of the systematic error, not detected/eliminated by the quality control system of the laboratory. this increase in error is expressed as multiples of sao . can have a positive or negative value and be of the order (0; asedetect). are is a temporary increase of the random error, not detected/eliminated by the quality control system of the laboratory. the change in random error is expressed as multiples of sao and is in the order (0; aredetect). z is a multiplier related to the portion of the distribution exceeding the quality requirement, often set as 2.65 to fix the maximum defect rate to 5 %. compare ref. (8) (chapter 5.2). 489 quality concepts different quality concepts are explained in chapter 11. we are using both the defined concept "quality assurance" and the concept "quality assessment" with regard to analytical quality e.g. in the context of "internal quality assurance" and "external quality assessment and assurance". "external quality assessment" refers to a system of retrospectively and objectively comparing results from different laboratories by means of an external agency. the main object of eqa is to establish the degree of agreement of results, both amongst laboratories and with an absolute standard where one exists" (11). we have used the abbreviation eqaa to represent the type of active "external quality assessment and assurance" program described in ref. 8. according to a document produced by eccls, references 1. 2. 3. 4. 5. 6. 7. 8. aronsson t, de verdier, c-h, groth,t. factors influencing the quality of analytical methods a systems analysis with use of computer simulation. clin chem 1974;20:738-748. aronsson t, bjpirnstad p, johansson sg, leskinen e, raabo e, de verdier c-h. interlaboratory quality control with investigation of different methodological characteristics. scand j clin lab invest 1978; 3853. aronsson t, bjpirnstad p, leskinen e, uldall a, de verdier c-h. assessment of analytical quality in nordic clinical chemistry laboratories using data from contemporary national programs. scand j clin lab invest 1984; 44 (suppl 172):lls 124. bipm, iec, ifcc, iso, iupac, iupap, oiml. international vocabulary of basic and general terms in metrology, 2nd ed. iso, 1993:l-59. dot d, mird j, fuentes-arderiu x. biological variation of the leucocyte differential count quantities. scand j clin lab invest 1992; 52:607-611. ifcc, icsh. approved recommendation (1986) on theory of reference values. part 1. the concept of reference values. j clin chem clin biochem 1987;25:337-342. ifcc, icsh. approved recommendation (1987) on theory of reference values. part 6. presentation of observed values related to reference values. j clin chem clin biochem 1987;25:657-662. groth t, de verdier c-h. transferability of clinical laboratory data. upsala j med sciences 1993;98 (3): in press. 490 9. 10. 11. olafsdottir e. aronsson t, groth t, de verdier c.-h. transferability of clinical laboratory data within a health care region. scand j clin lab invest 1992;52:679 687. westgard jo. charts of operational process specifications ("opspecs charts") for assessing the pecision, accuracy, and quality control needed to satisfy proficiency testing performance criteria. clin chem 1992;38: 1226-1233. european committee for clinical laboratory standards (eccls) 3rd draft, standard for quality assurance programmes. part 1: general principles and terminology. eccls documents 3 (9, 1983. correspondence: carl-henric de verdier, md., professor department of clinical chemistry university hospital s-751 85 uppsala, sweden fax: t 4 6 18 552562 49 1 computed tomography of the brain, hepatotoxic drugs and high alcohol consumption in male alcoholic p full terms & conditions of access and use can be found at https://www.tandfonline.com/action/journalinformation?journalcode=iups20 upsala journal of medical sciences issn: 0300-9734 (print) 2000-1967 (online) journal homepage: https://www.tandfonline.com/loi/iups20 computed tomography of the brain, hepatotoxic drugs and high alcohol consumption in male alcoholic patients and a random sample from the general male population sture mützell to cite this article: sture mützell (1992) computed tomography of the brain, hepatotoxic drugs and high alcohol consumption in male alcoholic patients and a random sample from the general male population, upsala journal of medical sciences, 97:2, 183-194, doi: 10.3109/03009739209179295 to link to this article: https://doi.org/10.3109/03009739209179295 published online: 18 jan 2010. submit your article to this journal article views: 159 view related articles citing articles: 4 view citing articles https://www.tandfonline.com/action/journalinformation?journalcode=iups20 https://www.tandfonline.com/loi/iups20 https://www.tandfonline.com/action/showcitformats?doi=10.3109/03009739209179295 https://doi.org/10.3109/03009739209179295 https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/action/authorsubmission?journalcode=iups20&show=instructions https://www.tandfonline.com/doi/mlt/10.3109/03009739209179295 https://www.tandfonline.com/doi/mlt/10.3109/03009739209179295 https://www.tandfonline.com/doi/citedby/10.3109/03009739209179295#tabmodule https://www.tandfonline.com/doi/citedby/10.3109/03009739209179295#tabmodule upsala j med sci 97: 183-194 computed tomography of the brain, hepatotoxic drugs and high alcohol consumption in male alcoholic patients and a random sample from the general male population sture miitzell department of family medicine, university hospital of uppsala, uppsala, sweden abstract computed tomography (ct) of the brain was performed in a random sample of a total of 195 men and 21 1 male alcoholic patients admitted for the first time during a period of two years from the same geographically limited area of greater stockholm as the sample. the same medical, social and neuroradiological methods were used for examination of the alcoholic inpatients as for the random controls. laboratory tests were performed, including liver and pancreatic tests. toxicological screening was performed and the consumption of hepatotoxic drugs was also investigated and the following were the types of drugs used: antiarrhythmics, antiepileptics, antiphlogistics, mixed analgesics, barbiturates, sulphonamides, benzodiazepines, clomethiazole and phenothiazine derivatives, all of which are metabolised by the liver. the group of male alcoholic inpatients and the random sample were then subdivided with respect to alcohol consumption and use of hepatotoxic drugs: group ia, men from the random sample with low or moderate alcohol consumption and no use of hepatotoxic drugs; ib, men from the random sample with low or moderate alcohol consumption with use of hepatotoxic drugs; iia, alcoholic inpatients with use of alcohol and no drugs; and iib, alcoholic inpatients with use of alcohol and drugs. group iib was found to have a higher incidence of cortical and subcortical changes than group ia. group ib had a higher incidence of subcortical changes than group ia, and they differed only in drug use. groups iib and iia only differed in drug use, and iib had a higher incidence of brain damage except for anterior horn index and wide cerebellar sulci indicating vennian atrophy. significantly higher serum (s) levels of bilirubin, gamma-glutamyl transpeptidase (ggt), aspartate aminotransferase (asat), alanine amino-transferase (alat), creatine kinase (ck), lactate dehydrogenase (ld) and amylase were found in iib. the results indicate that drug use influences the incidence of cortical and subcortical aberrations, except anterior horn index. it is concluded that the groups with alcohol abuse who used hepatotoxic drugs showed a picture of cortical changes (wide transport sulci and clear-cut or high-grade cortical changes) and also of subcortical aberrations, expressed as an increased widening of the thud ventricle. 183 introduction in recent years, there have been a large number of computed tomography (ct) studies (2-4,7 9,12,13,15,17,19,23,25-27,29,31) of the morphological changes in the alcoholic brain. in these reports, the criteria for diagnosing alcoholism have varied widely. some researchers have focused on heavy social drinkers (3,7-9) and there are reports based on zimberg’ s scale (23), the criteria of the national council on alcoholism (17), or special diagnostic criteria which were quantitatively determined, one, for example, being an intake of more than 50 g of ethanol per day (19). others have excluded cases of delirium tremens, withdrawal seizure or korsakoff‘ s syndrome from their studies (7,8). background factors, including sex , age, duration of alcohol drinking or complications, also vary from report to report; only male patients were studied in some reports (2 4,19,29) while a few females were included in others (7-9,12,13,23,27). the criteria for exclusion of complications such as epilepsy, drug dependence other than alcoholism, head injury, intracranial hematoma, severe liver dysfunction and hepatocerebral syndrome differ among the reports. some authors have tried to assess the cerebral morphological status among persons without alcohol problems by comparison with various control groups. however, it is clear from the selection criteria of the groups that they cannot be considered representative of a general population. controls but smaller than those of alcoholics (18). in a 6-year follow-up of 50 patients with primary dependence on sedative and hypnotic drugs, 26 patients (52%) were abusing drugs andfor alcohol at the time of follow-up. ct of the brain was performed in 33 of these 50 patients, and in 17 patients, 10 of whom were currently abusing drugs, there were indications of cortical atrophy (1). rumbaugh et al. (30) reported on 6 drug abusers with dilatation of the ventricles and subarachnoid spaces, suggesting that cerebral atrophy can be related to drug abuse. caballeria et al. (6) found that systemic effects of alcohol may be exacerbated in patients receiving cimetidine and leo et al. (20) found that hepatic cytosolic retinal dehydrogenase activity was increased 8-fold after phenobarbitone and ethanol administration, but no extrahepatic induction was observed. the purpose of the present interdisciplinary study was to compare a random sample of men from the general population with alcoholic inpatients with regard not only to the incidence and location of morphological cerebral changes but also to the laboratory findings in relation to drinking habits and to the use of hepatotoxic drugs in combination with alcohol intake. populations. benzodiazepine users, as a group, have been found to have larger ventriclebrain ratios than a special feature of this study is that the same instrument has been applied in different 184 material and methods the study comprised a sample from the general population and alcoholic inpatients consisting of men living in the catchment area of the karolinska hospital, a geographically limited area in the northern part of stockholm (solna and sundbyberg) with about 80,000 inhabitants. the sample was drawn randomly from the national register covering all swedish inhabitants. the sample was stratified with regard to age and it was intended that a sample of 200 men should be drawn, with 40 in each of the age-groups 20-29,30-39,40-49,50-59 and 60-65 years, in order to achieve the same degree of precision for all age-groups in the estimation of different variables. the initial random sample drawn consisted of 209 men aged 20-65 years. of this sample, 2 persons had died, 5 had moved more than 20 miles away from stockholm, and 10 were living permanently abroad at the time of investigation and were thus excluded from the study. nine persons refused to be examined and 2 refused to take part in the ct investigations. thus, of the initial sample of 209 men, 181 were available for investigation. to increase the sample to 200, a supplementary sample of 19 men was drawn in exactly the same way as before, and all these men were available for investigation. the non-participant group was small and the drop-outs (1 1 %) did not differ from the examined persons with respect to social status, age, education, civil status, employment status or data from official registers (ps.05). five men refused to undergo ct examination of the brain, and ct scans for a total of 195 men were therefore available. the patient group comprised 21 1 men consecutively admitted to the magnus huss clinic for alcohol diseases at the karolinska hospital. all were born on an uneven date and were staying at the clinic for at least one week for voluntary treatment. all alcoholic patients admitted for the first time during a period of 2 years from the same geographically limited area as the sample were investigated. the same medical, social and neuroradiological methods were used for examination of the 21 1 alcoholic inpatients as for the 195 random controls. laboratory tests were performed, including liver and pancreatic tests. blood samples were drawn in the morning after an overnight fast in the random sample and in the alcoholic inpatient group. toxicological screening with the following assays was performed: serum concentrations of barbiturates, other sedatives and alcohol and urinary concentrations of meprobamate, benzodiazepines, alkaloids, phenothiazines, tricyclic antidepressants, amines stimulating the nervous system and salicylic acid. the amount of alcohol consumed, indications of alcohol dependence and alcohol-related complications were thoroughly assessed by means of a standardised procedure combining a self-administered questionnaire and a check-up interview. two measures of alcohol consumption were used the amount of alcohol consumed in the previous week in grams of absolute alcohol per day and the typical peak consumption in the last six months in grams of pure alcohol per day. the consumption in the last week was recorded, as it was considered that the subjects’ recall would be poorer for the period further back in time. the drug consumption case history is composed of two parts. the first part focuses on drugs consumed during the last 24 months before admission and all prescription for the patient, data on type of drug and prescribing doctor. drugs are coded in accordance with the swedish 185 pharmacological register (fass). the second part consists of a pedicine chart/questionnaire with special emphasis on psychotropic and addictive drugs, and the consumption of hepatotoxic drugs was also investigated. the following were the types of drugs used: antiarrhythmic agents, antiepileptic agents, antiphlogistics, mixed analgesics, barbiturates, benzodiazepines, phenothiazine derivatives, sulphamethizole and sulphamethoxypyridazine derivatives and clomethiazole, all of which are metabolised by the liver (table 1). this latter part of the drug consumption history is designed for the patient’s own completion. patients are asked to record the drugs which they are either using at present o r have used during the last 24 months. altogether 180 different drugs are itemised. table 1. use of hepatototoxic drugs combined with alcohol consumption in the two groups ib and iib. pharmacological groups and the doses of each drug in grams per day (g/d) after the generic name. minimum time of drug consumption in months for each drug group. pharmacological group ib: random sample group iib: alcoholic groups and h e doses in g/d duration of drug duration of drug consumption in consumption in low alcohol + druos inuatients + drugs (n=31) months (n-) months antiarhytmic drugs quinidine bisulph. 0.4; verapamil hydmchlor. 0.32 antiepileptics phenytoin sod. 0.4 antiphlogistics ibuprofen 1.2 mixed analgesics dolerone 2 tabletdday barbiturates amylobarbitone 0.2; hexapropymate 0.4 nitrazepam 0.01; diazepam 0.02; chlordiazepxide 0.03; oxazepam 0.03 phenothiazine derivatives promethazine 0.05; chlorprothixene 0.05; alimemazine 0.08 sulphamethizole 0.4 g, sulphamethoxypyridazine 0.1 g clomethiazole benzodiazepines sulfapral4 tablets/day hemineurin 0.9 24 24 24 24 24 24 24 1 0 1 24 0 0 0 0 4 24 10 24 10 24 8 24 0 0 7 6 n 31 40 186 the validity of the drug consumption case history may be open to question. on the other hand, with regard to psychotropic drugs, many of the patients have shown an impressive knowledge of the names, doses and effects of the various drugs. the group of male alcoholic inpatients and the random sample were then subdivided with respect (ia) men from the random sample with low or moderate alcohol consumption and no use of drugs (n=164); (ib) men from the random sample with low or moderate alcohol consumption with use of drugs (n=31); (iia) alcoholic inpatients with use of alcohol but no drugs (n=171); ( i d ) alcoholic inpatients with use of alcohol and drugs (n=40); to alcohol consumption and use of hepatotoxic drugs: computed tomography the tomographic images were evaluated with regard to ventricular, cortical and cerebellar changes. an anterior horn index, i.e. evan’ s ratio, was obtained by dividing the width of the anterior horns by the largest inner skull diameter. values exceeding 0.3 i were considered pathological. a transverse diameter of the third ventricle exceeding 6 mm was also considered pathological. a four-step scale of degenerative cortical changes was used, based on a general assessment of the tomographs by the radiologist with regard to observations of widened sulci. in this scale, l=normal, i.e. no sulci visible or sulci less than 3 mm in natural size, 2=suspected degenerative changes, i.e. up to 5 sulci exceeding 3 mm in diameter, 3= clear-cut changes, i.e. more than 5 sulci exceeding 3 mm in diameter and appearing in at least 2 cuts, and 4=high-grade changes, i.e. marked widening of a large number of sulci in all lobes. the inter-rater reliability of the scale has been found to be 0.81 (1 1). results groups ia-iib characteristics of the 2 groups of alcoholic inpatients and the 2 groups from the random sample who used and did not use drugs are presented in table 2. the 40 alcoholic inpatients and drug users in group iib were significantly heavier. ethanol was present in the blood in 2% of the random sample using no drugs, and in 10% of those using drugs, who had consumed 32 g and 49 g of alcohol per day respectively in the last week before examination in the hospital. ethanol was present in the blood in 42% and 40% respectively of the inpatients not using and using drugs, who had consumed 250 g and 251 g per day respectively in the last week before admission to hospital. there were significantly more smokers in the 3 groups ib, iia and id. 13-928572 187 twelve per cent of the sample who did not use drugs (group ia) and 19% of those who used drugs (group ib) were registered by the temperance board. among the alcoholic inpatients who did not use drugs (group iia) and those who used drugs (group id), 66% were registered by the ' temperance board. table 2. characteristics of the 4 groups of men with different drinking habits with and without the use of hepatotoxic drugs. group i a group ib group iia group iib random sample random sample alcoholic alcoholic low alcohol low alcohol inpatients inpatients (n=164) (n=31) (n=171) ( n 4 0 ) no drugs + drugs no drugs + drugs age (ye@ 44+14 4 7 ~ 1 3 44+14 45+13 alcohol intake previous week in g absolute alcohovday 32k5 1 4 9 ~ 8 8 250+_115**** 251+126**** registered by the temperance board (%) 12 19 66**** 66**** smokers (%) 44 58**** 74**** go**** alcohol in blood on arrival at hospital (%) 2 degrees of significance in comparison with low alcohol-no drugs group by student's t-test and chi-square test. *p<0.05; **p<o.ol; ***p<o.ool; ****p<o.oool. liver and pancreatic tests the results of alcohol-related liver and pancreatic tests in the subgroups are presented in table 3. significantly higher serum levels of bilirubin, ggt, asat, alat, ck, ld and amylase were found in the alcoholic group using drugs (group izb) than in the other groups. in group iia, with a high alcohol consumption and no drug use, only serum alkaline phosphatase (alp), ggt, asat, alat and amylase were elevated. in group ib, the men from the random sample using drugs, significantly higher values of serum alp, ggt and ld were found, but these values lay within the given reference ranges, except ggt. in the men from the random sample who used drugs combined with alcohol (group ib), the drugs taken included antiarrhythmic agents (quinidine, verapamil), antiepileptics (epanutin), antiphlogistics (ibuprofen) dextropropoxyphene (doleron) and benzodiazepine derivatives, all of which can cause increases in liver enzymes such as serum ggt, asat, alat and ld ( see table 1,3). seventy per cent of the random sample using drugs (group ib) had pathological ggt values and 60% pathological values of alat. the most imprtant of these drugs are anticoagulants, antiepileptic agents and barbiturates (10,16). 188 the drug profile of the 40 patients in group iib and the men in group ib is shown in table 1. none of the subjects used cardiovascular drugs such as digitalis, antihyperlipidemics, antidiabetics, or uricosuric drugs. the most frequently used non-psychotropic group of drugs was antacids. with regard to psychotropic drugs in group iib, 25% of the subjects had experience of amylobarbitone and hexapropymate, and 26% of benzodiazepines like diazepam (table 1). as regards non addictive psychotropic drugs, 19% of the men reported using phenothiazine derivatives and 17% used clomethiazole, which is a drug usually prescribed for alcoholic abstinence. table 3. alcohol-related liver and pancreatic disturbances in the 4 groups as reflected by mean values of serum bilirubin, alp, ggt, asat, alat, ck, ld and amylase. reference values in parenthesis. group ia group ib group iia group iib random sample random sample alcoholic alcoholic low alcohol low alcohol inpatients inpatients no drugs + drugs no drugs i drugs (n=164) (n=31) (n= 17 1) (n=40) s-bilirubin 12+7 16+12 *** (4-21 u m o l / l ) llk7 9k4 s-alp (0.8-4.0 ukat/l) 2.94.9 3.3k1.0 * 4.9k7.8 *** 4.3k2.0 **** s-ggt (<loo ukat/l) 0.524.42 1.21k1.14 **** 4.40+8.21**** 6.94k9.76 **** s-asat (c0.70 ukat/l) 0.42k0.33 0.44k0.22 1.26+1.01**** 2.26k1.91 **** (<0.70 ukat/l) 0.42k0.42 0.46k0.26 1.159.9 1 **** 1.6651.38 **** (0.6-2.7 ukat/l) 2.4~1.7 3.0+4.3 2.3k2.1 3.4k5.0 * (2.7-6.4 ukat/l) 5.6k0.9 6.2k2.1 ** 7.3k1.8 8.2+2.1**** s-alat s-ck s-ld s-amylase (1.1-5.0 ukat/l) 3.3k1.6 2.89.8 3.8k1.9 ** 6.4k8.8 **** significance levels tested in comparison with group ia by student’s t-test and chi-square test. *p<0.05; **p<o.ol; ***p<o.ool; ****p<o.oool. ct findings in groups ia-iib cortical changes were found in 80% of group iib(p<o.oool compared with group ia) and 62% of group iia(p<0.0001 ), and wide transport sulci in 28%(p<0.0001) of group iib and 22% (p<o.oool) of group iia. users of hepatotoxic drugs had a higher incidence of cortical changes than non-users in groups ia and iia vs ib and iib respectively (table 4). 189 in group iia, 40% (p<o.oool) had a pathological anterior horn index, but in the groups that used drugs only 32-33% (p<0.05) had a pathological anterior horn index. the incidence of an enlarged third ventricle varied from 32 to 53% in the drug-using groups and was 35% in group iia. wide cerebellar sulci indicating vermian atrophy were observed in 35% of the men in groups iib and iia @<o.oool) but were found in only 6% of group ib and 1% of group ia (table 4). table 4. ct variables used in the 4 groups with different drinking habits with and without the use of hepatotoxic drugs. ct measures cortical wide transport sulci cortical changes (subjective rating: clear-cut or high-grade) subcortical anterior horn index > 0.3 1 width 3rd ventricle > 6 mm verrnian atrophy group ia group ib group iia group iib random sample random sample alcoholic alcoholic low alcohol low alcohol inpatients inpatients no drugs + drugs no drugs -+ drugs (n=164) (n=31) (n=17 1) ( n 4 0 ) % % % % 9 32**** 35**** 53**** 1 6 35**** 35**** degrees of significance tested in comparison with low alcohol-no drugs group by chi-square test. *p<0.05; **p<o.ol; ***p<o.ool; ****p<o.oool. discussion regarding potential bias, the ct images were assessed by 2 radiologists independently and the assessments were made blindly, i.e. without the radiologists' having any knowledge of the alcohol consumption of the patient in question. the study was performed on a representative sample of men from greater stockholm, and the sample was completely unselected, randomly chosen and age stratified, and drawn by statistics sweden, a government agency. the subjects knew that they were to undergo a health examination, and even though they may have consumed some alcohol the day before, they had not taken any on the day they came to the hospital. blood samples were drawn in the morning after an overnight fast in the random. sample and in the alcoholic inpatient group. none of the subjects was drunk at the time of the tests or had cerebral oedema according to the ct findings. they were thoroughly assessed concerning the amount of alcohol consumed, indications of alcohol dependence and alcohol-related complications, 190 by means of a standardised procedure combining a self-administered questionnaire and a structured interview. in exactly the same way as with alcoholic inpatients (24), a sample of 40 was selected randomly from the 200 men who answered the questions concerning alcohol habits. these men were asked to answer the same questions 6-24 months after the first occasion, and a test-retest analysis was performed using spearman’s rank-order correlation and kendall’s tau-b. kappa does not utilise the ordinal information of the variable, and we therefore used kendall’s tau-b with values between 0.56 and 1.00. the methods used in the assessment of the ct images of the brain are well known and have k e n well documented for several years (3,4,25,26). at the magnus huss clinic of the karolinska hospital, the same methods have been used for these purposes since 1976, both in alcoholics and in healthy control persons, with each subject assessed blindly by two examiners. the reliability has been 2 0.81 (1 1). in consideration of the above, there should be no important sources of bias regarding the population sample and the alcoholic inpatients or the ct examinations of the brain. the drugs used are known to be metabolised by the liver and the findings in the subjects with combined drug abuse and alcoholism show the clinical importance of alcohol and drug interactions. many of the central nervous effects of various drugs are potentiated by simultaneous intake of alcohol, and the elimination rates of many drugs are influenced both by chronic alcohol consumption and by possible liver damage (21). lader et al. (18) performed ct scanning in 20 patients who had taken benzodiazepines for a long period. the mean ventricular/brain area as measured by planimetry was larger than the mean values in an age and sex-matched group of control subjects, but was smaller than that in a group of alcoholics. there was no significant correlation between the ct findings and the duration of benzodiazepine therapy. the present findings are in accordance with reports in the literature concerning cerebral changes on ct and the effects of combination of alcohol and drugs, namely that simultaneous alcohol and drug intake has an additive action on the central nervous system. bergman (5) found that alcoholic patients had p r e r results in neuropsychological tests and that these were related to ct changes. in bergman’ s study, the subjects in the alcoholic group were severely alcoholic, whereas our heavy drinkers from the random sample of the male population of greater stockholm were not social outcasts but had a high alcohol consumption. from the purely ct aspect, they resembled the social outcasts in bergman’s study, the difference being that they drank smaller quantities of alcohol but used drugs at the same time. their affinity for ethanol, sensitivity to 4-methylpyrazole, and electrophoretic migration) in the human stomach. at the high ethanol concentrations prevailing in the gastric lumen during alcohol consumption, the sum of their activities could account for significant oxidation of ethanol. in vivo, therapeutic doses of cimetidine (but not famotidine) increased blood ethanol levels when ethanol was given orally but not when it was given intravenously, indicating a significant contribution of the gastric adh to the bioavailability and thereby the potential toxicity of ethanol. hernandez-munoz et al. (14) found two types of alcohol dehydrogenase isoenzymes (differing in 191 roine et al. (28) found that aspirin may increase the bioavailability of ingested ethanol in humans, possibly by reducing ethanol oxidation by gastric alcohol dehydrogenase. lieber (22) says that until recently it was commonly believed that the primary pathway for hepatic ethanol metabolism is due almost exclusively to the activity of cytosolic alcohol dehydrogenase, with a minor contribution from peroxisomal catalase. it is now recognised, however, that liver microsomes (through meos) participate in ethanol metabolism. caballeria et al.(6) found that the areas under the curve of blood ethanol concentrations after oral alcohol intake were twice as large with cimetidine as without. the results indicate that gastric alcohol dehydrogenase activity partly governs the systemic bioavailability of ethanol. consequently, systemic effects of alcohol may be exacerbated in patients receiving cimetidine, which is metabolised by the liver. the same instrument has been applied in different populations in this study and the most interesting and new finding seems to be the differences in levels of markers between drug-users and non drug-users. the pathological values often found in alcoholics could to a large extent be ascribed to concomitant drug use. that alcohol and drugs can interact and that the combination may be dangerous is well known. the importance of this in relation to the use of biomedical markers as screening instruments has probably not received sufficient attention. in the present study, the greatest cortical and subcortical changes were found in the men of the small group iib, who both had a high alcohol consumption and used drugs daily, but an exception was the anterior horn index, where group iia, with high alcohol intake and no drug use, had a pathological index in 40%. in the random sample, the greatest cortical and subcortical changes were found in the men of the small group ib, who both had a relatively high alcohol consumption and used drugs daily. what we have found is that high alcohol consumption and drug use does not influence the anterior horn index so much as the other measures of brain damage. from a purely scientific viewpoint, this means that even though there may be major ct changes in some patients, the use of alcohol in combination with drugs is the most important factor which gives pathological cortical and subcortical changes in the population and influences the changes in the alcoholic inpatient groups. combination with drugs are at greater risk and have more pathological ct scan items except the anterior horn index and also have more pathological liver and pancreatic tests, which has not previously been shown in the literature. it is concluded that the alcoholic inpatients and the men in the random sample who use alcohol in references 1. 2. 3 . allgulander, c., borg, s. & vikander, b.: a 4-6 year follow-up of 50 patients with primary dependence on sedative and hypnotic drugs. am j psych 141: 1580-1582,1984. begleiter, h., porjesz, b. & tenner, m.: neuroradiological and neurophysiological evidence of brain deficits in chronic alcoholics. acta psychiatr scand. suppl286:3-13,1980. bergman, h., borg, s., hindmarsh, t., idestrom, c.-m. & miitzell, s.: computed tomography of the brain, clinical examination and neuropsychological assessment of a random sample of men from the general population. acta psychiatr scand. suppl286:47-56, 1980. 192 4. 5 . 6. 7. 8. 9. bergman, h., borg, s . , hindmarsh, t., idestrom, c.-m. & miitzell, s.: computed tomography of the brain and neuropsychological assessment of male alcoholic patients and a random sample from the general male population. acta psychiatr scand. suppl286:77-88, 1980. bergman, h.: cognitive deficits and morphological cerebral changes in a random sample of social drinkers. recent developments in alcoholism, vol3, 1985. caballeria, j., baraona, e., rodamilans, m. & lieber, c.s.: effects of cimetidine on gastric alcohol dehydrogenase activity and blood ethanol levels. gastroenterology 96:388-392,1989. cala, l.a., jones, b., mastaglia, f.l. & wiley, b.: brain atrophy and intellectual impairment in heavy drinkers: a clinical, psychometric and computerized tomography study. aust n z j med 8:147-153,1978. cala, l.a., jones, b., wiley, b. & mastaglia, f.l.: a computerized axial tomography (cat) study of alcohol induced cerebral atrophy: in conjunction with other correlates. acta psychiatr cala, l.a. & mastaglia, f.l.: computerized axial tomography in the detection of brain damage. med j aust 2: 193-198,1980. s c a d . suppl286:31-40, 1980. 10. drug hterferences and drug effects in clinical chemistry. the national corporation of swedish pharmacies. 4th edition. editors: tryding, n. & roos, k.-a., 1986. 11. elofsson, s.a., gamberale, f., hindmarsh, t., iregren, a., isaksson, i., knave, b., lydahl, e., mindus, p., person, h.e., philipson, b., steby, m., struwe, g., soderman, e., wennberg, a. & widen, l.: exposure to organic solvents. a cross-sectional epidemiologic investigation on occupationally exposed car and industrial spray painters with special reference to the nervous system. scand j work environ health 6:239-273, 1980. 12. von gall, m., becker, h., artmann, h., lerch, g. & nemeth, n.: results of computer tomography on chronic alcoholics. neuroradiology 16:329-33 1, 1978. 13. gotze, p., kiihne, d. hansen, j. & knipp, h.p.: hirnatrophische veranderungen bei chronischem alkoholismus: eine klinische und computertomographische studie. arch psychiatr nervenkr 226:137-156, 1978. 14. hernandez-munoz, r., caballeria. j.. baraona. e.. uddal, r.. greenstein, r. & lieber. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27, c.s.: human gastric alcohol dehydrogenase: its'inhibzon by &-receptor antagonists, &d its effect on the bioavailability of ethanol. alcohol clin exp res 24: 946-950, 1990. huckman, m.s., fox, j.h. & ramsey, r.g.: computed tomography in the diagnosis of degenerative diseases of the brain. semin roentogenol 12:63-75, 1977. keso, l. & salaspuro, m.: laboratory tests in the establishment and treatment of alcohol problems. nord med 101:306-312, 1986. kroll, p., seigel, r., 0' neill, b. & edwards, r.p.: cerebral cortical atrophy in alcoholic men. j clin psychiatry 41:417-421, 1980. lader, m. & peterson, h.: long-term effects of benzodiazepines. neuropsychopharmacol20, lee, k., moller, l., hardt, f., haubek, a. & jensen, e.: alcohol-induced brain damage and liver damage in young males. lancet : ii(8164):759-761, 1979. leo, m.a., kim, c.i., lowe, n. & lieber, c.: increased hepatic retinal dehydrogenase activity after phenobarbital and ethanol administration. biochem pharmacol38:97103, 1989. lieber, c.s.: medical disorders of alcoholism: pathogenesis and treatment. (ed.) lieber c.s. philadelphia: w.b.saunders, 1982. lieber, c.s.: interaction of ethanol with drugs, hepatotoxic agents, carcinogens and vitamins. lusins, j., zimberg, s., smokler, h. & gurley, m.a.: afcoholism and cerebral atrophy: a study of 50 patients with ct scan and psychologic testing. alcohol clin exp res 4:406-411, 1980. miitzell, s.: the reliability of anamnestic questionnaires for alcoholic inpatients. hygiea acta societatis medicorum suecarae 87: 310,1978. miitzell, s.: alcohol consumption in a population sample of urban men with neuropsychological assessment and computed tomography of the brain. acta univ ups 139, 1988. thesis. mutzell, s. & tibblin, g.: high alcohol consumption, liver toxic drugs and brain damage a population study. ups j med sci 94: 305-315, 1989. newman, s.e.: the eeg manifestations of chronic ethanol abuse: relation to cerebral cortical atrophy. ann neurol3: 299-304,1978. suppl4: 527-533, 1983. alcohol alcohol 25~157-176, 1990 193 28. roine, r., gentry, r.t., hernandez-munoz, r., baraona, e. & lieber, c.s.: aspirin increases blood alcohol concentrations in humans after ingestion of ethanol. jama 264: 2406 2408, 1990. 29. ron, m.a., acker, w. & lishman, w.a.: morphological abnormalities in the brains of chronic alcoholics: a clinical, psychological and computerized axial tomographic study. acta psychiatr scand. 286: 41-46, 1980. 30. rumbaugh, c.l., fang, h.c.h., wilson, g.h., higgins, r.e. & mestek, m.f.: cerebral cx findings in drug abuse: clinical and experimental observations. j computer assisted tomography 4: 330-334, 1980. 3 1 . wilkinson, d.a. & carlen, p.l.: neuropsychological and neurological assessment of alcoholism: discrimination between groups of alcoholics. j stud alcohol 41: 129-139, 1980. correspondence: sture miitzell, md phd department of family medicine akademiska hospital s-751 85 uppsala, sweden 194 upsala j med sci 84: 188-194, 1979 method for measurement of maximal isometric muscle strength with special reference to the fingers bengt nordgren, alf elmeskog and agne nilsson from the departments of medical rehabilitation and clinical physiology, university hospital, and the ala foundation, uppsala, sweden introduction in daily life, both in industrial and household work, as well as i n certain leisure activities, good function of the hand and fingers is of great importance. this function depends upon several factors, of which a main one is muscle strength. in order to study the finger function, a method has been developed for measurement of the maximal isometric muscle strength in the fingers. it has been considered of importance that the measurement procedure (including calibration and reading of the instrument) shall be easily handled by the examiner. methods for measurement of muscle strength have been described previously by assmussen et al. (1959, 1961), tornvall (1963), backlund & nordgren (1968) and hijgk & tornvall (1970), among others. method the apparatus used is a combination of standard and laboratory-constructed a mechano-elastic transducer (pressduktor@, asea; dahle 1954) i s used as equipment. the pressure transducer. the pressduktor has been used previ.ously in muscle strength measurements by backlund & nordgren (1968). measurement of finger strength: the muscle strength in the fingers varies between about 0.05 and 15 kp (i.e.wo.5 and 150 newton), thus including comparatively very low strengths and percentually large range of variation. the pressduktor used for measuring the finger strength has been adjusted to a pressure of 20 kp, which gives a measurement range between -20 kp (output signal -1.3 v) and +20 kp (output signal +1.3 v). the finger strength press duktor is mounted in a holder (fig. 1). the force applied influences the press duktor via a lever system, where the lever for the finger force applied is 8.5 times longer than the lever for the transducer. when these low finger strengths were measured hysteresis in the system gave rise to problems. 188 fig. 1. to the left the pressduktor with the lever and the tightening screw nuts; to the right the adjustable finger grip. several reasons for this became evident. one of them was the summation effect of several very minor backlashes, i.e. the system must have absolutely tight fittings. the use of a hinge joint on fixation of the lever gave hysteresis. instead, therefore,grooves were made on both the lever and the holder. into these grooves two thin steel plates were welded (one in the horizontal and one in the vertical plane). for transferring the force of the lever and for suspension, a constant and defined lever length was also obtained (fig. 2 a and b). the application of tension to the pressduktor and the tightening of the screw-nuts (fig. 1) at the connection between the pressduktor and the lever mean that the lever's own weight has no effect on the finger force applied. here also a thin steel plate was used for transference of force between the pressduktor and the lever. by the use of these steel plates (0.5 ran thick) fig. 1 shows measurement of the strength on opposition between the thumb and index finger. the grip surfaces are grooved to fit the finger and thumb. the middle bar is attached to the lever connected to the pressduktor, while the upper and lower bars are firmly attached to one another. their position can be varied by the screw under the lowest finger-grip bar. for testing the finger abduction strength (fig. 3) the tested fingers are inserted between the middle bar and one of the two fixed bars, which is adjusted to give a space of a few mm between the fingers. abduction and adduction are tested with an angle of 0' in the metacarpophalangeal joint, i.e. in an extended posit ion. 189 1 1 200 l 214 190 16 y pig. 2 a. diagram of the pressduktor with the lever for measurement of the finger strength. the figures are in mm. ,fig. 2 b. section of the lever illustrating transference of power in measurement of finger strength. fig. 3 . grip for measurement of the strength on opposition between digits i and 11. the width o f the grip is adjusted by means o f the screw, 190 fig.s 4 a and b show the values recorded on the potentiometer recorder (servogor re 520 z) with different suspended weights (both pull ( 0 ) and push ( 0 ) sympols). the linear relationships correspond for both pull and push, with no tendency to hysteresis around the origin. mm 100mm 60 60 20 20i i i i i i i 0.2 0.4 0.6 kp 2 6 lo kp fig. 4 b. the linearity on application of force (pull 0 , push 0 ) to the fig. 4 a. the linearity on application pressduktor. of force (pull 0 , push 0 ) to the press duktor. with forces of more than 10 kp (e.g. in several persons on opposition between the thumb and index finger) the apparatus for measurement of the hand grip can be suitably used at such forces the finger strength pressduktor gets a slight curvilinearity. occasional overloading of the pressduktor measuring system has not altered its properties. apparatus set-up the set-up of the apparatus also allows measurements of the strength of the hand grip, proand supination and volar flexion and dorsiextension of the hand as described by bbcklund and nordgren (1968). the measurement procedure thus gives a more complete picture of the function of both the fingers and the hand and forearm. fig. 5 shows the apparatus set up with the recorder and selector panel (to the right on the table) for the different tests. the holder of each press duktor is suspended on a steel wire. in this vay it can be adjusted vertically and rotated around the metal tube. the electronics are constructed such that the signals from the different pressduktors can be selected on the panel. for the finger strength three different sensitivity ranges can be chosen (the greatest degree of amplifica tion for the adduction test). in consideration of the variations in muscle strength between the examined persons it is a l s o possible by means of a 191 fig. 5. the setup of the apparatus. special selector to dupulicate o r halve the pen recording. there is a possi bility of compensation (rotation potentiometer) in each of the five selectors. calibration by changing the magnitude of the output signal from the respective pressduktors can be performed by means of special rotation potentiometers. the muscle strength applied is read directly on the recorder (kp; for rotatory tests kpcm) by means of graded rules. experience of the stability of the measurement equipment has shown that the output signal only needs to be altered twice a year, at the most. results reproducibility in repeated measurements of the finger strength. table i presents the variation in muscle strength in the fingers at repeated ___. measurements on the same female subject at intervals of a few days. table i. repeated tests of the maximal isometric muscle strength of the fingers in the same person on four different days ( x = mean). right side left side x (kp) range ( z of x) 2 (kp) range (% of ?) opposition digit. 1-11 digit. 1-111 digit. i-iv digit. i v adduction digit. 1-11 digit. 11-111 digit . 111-iv digit. iv-v abduct ion digit. 1-11 digit. 11-111 digit. 111-iv digit. iv-v 192 9.3 8.4 6.1 4.2 8.9 4.8 2.8 1.7 1.3 1.3 0.7 0.9 95-106 89-107 82-111 77-132 97-101 80-119 76-113 72-114 91-106 89-114 89-119 65-131 8.1 8.1 6 . 1 4.3 7.9 4.4 2.9 1.6 1.3 1.3 0.8 0.9 93-111 98-105 82-115 81-127 86-114 76-115 70-130 70-114 85-108 89-104 80-107 74-116 finger muscle strength in normal persons table i1 gives the mean values (x) and standard deviations (s) in 14 men with a mean age of 34 years (range 22-55 hears) and 13 women with a mean age of 27 years (range 19-46 years), with no signs of disease. table 11. isometric maximal muscle strength in the fingers in a control series of healthy persons, kp, (x = mean, s = standard deviation). gight left x s x s women eight &eft x s x s opposition digit. 1-11 16.5 7.4 14.9 4.9 digit. 1-111 14.2 5.1 15.1 6.1 digit. i-iv 12.1 6.4 12.9 6.0 digit. i-v 8.9 2.6 9.8 4.0 10.4 3.6 9.9 3.6 9.9 3.5 8.9 3.6 6.7 3.6 6.8 2.5 5.5 2.0 5.2 2.5 adduc t ion digit. 1-11 7.1 2.5 6.7 2.3 5.5 1.5 4.7 1.7 digit. 111-iv 1.4 0.6 1.6 1.0 1.3 0.6 1.9 1.7 digit. iv-v 1.2 0.8 1.3 1.0 1.2 0.5 1.3 0.8 digit. 11-111 2.2 1.4 2.6 1.2 2.7 1.5 2.9 2.5 abduction digit. 1-11 1.11 0.55 1.05 0.61 0.92 0.43 0.81 0.50 digit. 111-iv 0.63 0.42 0.65 0.45 0.65 0.40 0.67 0.40 digit. iv-v 0.63 0.48 0.60 0.20 0.64 0.33 0.64 0.34 digit. 11-111 1.02 0.61 0.97 0.57 0.90 0.37 0.79 0.37 discussion the variation in repeated finger strength measurements on the same person (table i) is greater on the ulnar than on the radial side, which may be an expression of the better muscular coordination on the thumb side. asmussen et al. (1961) measured the thumb pressure, which corresponds to adduction of digits 1-11 in the present study, and also pinching of digits 1-11, corresponding to opposition of digits 1-11 in this study (table 11). they found higher values for thumb pressure than for pinching, while opposition gave higher values than adduction in the present study. the values for adduction are lower than the values of asmussen et al. for thumb pressure, while those for opposition are higher than the values of asmussen et al. for pinching. the reasons for these discrepancies in the results cannot be explained with certainty, but differences in the measurement technique may be a possible reason. the movement on adduction and opposition between digits i and i1 merge into one another and variations in the perfor mance of the test may therefore play a role. the function of the finger musculature, e.g. in association with nerve 193 injuries, can be tested with this method. opposition and abduction of digit i, and the lumbrical muscles 1-111, depend upon the median nerve. abduction and adduction between digits i1 and v are dependent upon the interosseous muscle and the abductor and opponens muscles of digit v. these muscles are innervated exclusively by the ulnar nerve, and thus constitute a test of the function of this nerve. summary a method for measurement of the maximal isometric muslce strength in the fingers is presented. details of the apparatus designed for avoidance of hysteresis in mechanical components on measurement of low strengths are given. the measurement of low strengths is described. the measurement procedure is simple both for examiner and for the subject. values for finger muscle strength in normal persons are presented. references 1. asmussen, e., heebdll-nielsen, k. & mollbeck, sv.: methods for evaluation of muscle strength. communications from the testing and observation institute of the danish national association for infantile paralysis, helferup, denmark, no 5, 1959. men and women. communications from the testing and observation institute of the danish national association for infantile paralysis, hellerup, denmark, no 11, 1961. strength under standardised conditions. scand j clin lab invest 21: 33-41, 1968 industrial work performance in mentally retarded persons. acta pediatrica scand, suppl 217, 1971. of new type. iva (periodical of the royal swedish academy of engineering sciences) 25: 221, 1954. isometric muscle strength in man. scand j rehab med 1: 139-142, 1969 seand j rehab med 2: 125-132, 1970. retarded persons, europa medica physica 8: 1-4, 1972. 58: suppl 201, 1963. 2. asmussen, e. & heebdll-nielsen, k.: isometric muscle strength of adult 3 . backlund, l. & nordgren, l.: a new method for testing isometric muscle 4. bbckstriim, l. & nordgren, b.: correlations between muscular strength and 5. dahle, 0.: the torductor and the pressduktor, two magnetic straingages 6. hciik, 0. & tornvall, g.: apparatus and methods for determination of 7. nordgren, b.: physical capabilities in a group of mentally retarded adults. 8. nordgren, b.: physiological aspects on the habilitation of young mentally 9. tornvall, g . : assessment of physical capabilities. acta physiol scand adress for reprints: bengt nordgren, m.d. department of medical rehabilitation & physical medicine university hospital s-750 14 uppsala 14 sweden upsala j med sci 78: 167-168, 1973 growth of nervous tissue in the regenerated rabbit ear chamber j. falk, k.-e. arfors and f. n. mckenzie from the departments of toxicology and experimental medicine, pharmacia ab, uppsala, sweden, and department of surgery, university of aberdeen, aberdeen, scotland abstract highly specific nerve staining using thiocholine techniques has been applied to a study of nerve regeneration in tita nium rabbit ear chambers. early and extensive ingrowth of nerves rich in true cholinesterase activity was demon strated. introduction since its description (lo), the regenerated rabbit ear chamber preparation has been extensively used in studies of vascular and cellular behaviour in the microcirculatory area of conscious animals. de spite widespread use of the method, the question of innervation of the regenerated tissue has re ceived scant attention. we considered it of in terest, therefore, to briefly report our experience in applying modern histochemical techniques to a study of nerve ingrowth in chamber tissue. materials and methods titanium ear chambers were aseptically inserted into sandy-lop rabbits according t o the method previously described by arfors et al. (1). cholinesterase activity was displayed histochemically by gomori's ( 6 ) modification of koelle & friedenwald's (8) method using ear chamber tissues of three weeks, two and four months of age. normal rabbit ear perichondrium from the same rabbits treated in identical fashion t o the chamber tissue served as control. the tissues stained by the cholinesterase method were used fresh and were sufficiently thin (about 100 p m ) to obviate the need for sectioning. after excision of the ear chamber the top cover glass was removed and the regenerated tissue stained in situ o n the bottom plate. incubation with acetyl thiocholine iodide as substrate was continued at 37°c for 18-24 hours. t o inhibit pseudo cholinesterase and thus demonstrate true cholinesterase activity, a portion of e a r chamber tissue was pretreated with 1 x m tetraisopropylpyrophosphorarnide (iso ompa, 3.42 mg/l of 40% sodium sulphate) for 30 min. the same concentration of 1.50-ompa was included in ihz incubation medium. during dehydration and clear ing procsdures, the ear chamber and perichondrial tissues were kept under a cover glass to minimise tissue distor tion. nerve staining by silver impregnation was also done using a modification ( 5 ) of bielschowsky's (2) method. in this instance prior fixation in 10% buffered formalin (ph 7.0) was required. the mounted preparations were viewed using transmit ted or phase contrast light. photographs were taken with a reichzrt automatic camera using kodak ektachrome film. results since the tissues used in this study were not sectioned, ingrowing nerves could be followed as uninterrupted dark brown lines. occasional blood vessels could be faintly distinguished due to the presence of small amounts of pseudocholines terase in the smooth muscle of the vascular wall. i n the three week old chamber, a single nerve trunk could be seen entering the chamber tissue. after giving off a small branch it extended some 2 mm towards the centre of the chamber. this regenerated nerve was shown to contain true cholinesterase; enzymatic activity persisted after inhibition of pseudocholinesterase by iso-ompa. nerve regeneration followed a similar pattern and was developed to a similar extent in the two and four-month-old ear chambxs. at both ages, the regenerated tissue was completely interwoven by a meshwork of nerve fibres with a maximal diameter of around 5 p m (fig. 1). the larger nerves were seen to be made up of several neuro fibrils and in this respect they resembled the nerves seen in perichondrial tissue. not surpris ingly, however, no thick nerve bundle entered the narrow chamber space, most being single fibrils which seemed to pursue a random course to cnd blindly in the chamber tissue or more often in relation to a blood vessel. the nerve fibres were upsula j med sci 78 168 j . falk et al. apparently unmyelinated but had an investing neurolemmal sheath. schwann cell nuclei could be readily identified as local deviations of the neurofibrils but nodes of ranvier were not ob served. silver impregnation of ear chamber tissue showed the striking development of the peri vascular nerve plexus particularly well (fig. 2 ) . excessive and uneven staining with silver were, however, throublesome. discussion using intravital staining with methylene blue dye, clark et al. (3) demonstrated the ingrowth of an unmyelinated nerve accompanying an arteriole three weeks after insertion of a rabbit ear cham ber. the present study using specific histochemical techniques confirms and extends these findings. the thiocholine method gives highly selective nerve staining, the only other deeply staining elements being fat cells, sebaceous glands and hair roots, none of which are found in regenerated chamber tissue. arterial smooth muscle also shows some activity due to the presence of pseudocholin esterase (4), but counterstaining is required to dis play the vascular wall clearly. our results show that differentiation of the capillary loops into definitive arterioles and venules is quickly fol lowed by innervation of these structures. by two months a meshwork of ramifying nerve fibres has developed. true cholinesterase activity appears to be present from the outset and enzyme activity was evenly distributed along the nerve fibre. the staining features of the nerves suggest that they are postganglionic fibres of the autonomic ner vous system. most fibres terminate in relation to blood vessels but some nerve strands appear to end blindly, a n observation which was also made by grant & thompson (7) using normal rabbit ear preparations. silver impregnation techniques suffer the dis advantage when applied to ear chamber tissue that the abundance of reticular fibres may lead t o staining of non-nervous tissue with consequent difficulty in precise interpretation. however, as shown by richardson (9) using rabbit intestinal tissue, silver staining was particularly useful in demonstrating the richness of the perivascular nervous network which quickly develops in ear chamber tissue (fig. 2). as a final point it may be mentioned that the histological appearances of the derivatives of the different germ layers found in regenerated cham ber tissue do not differ from that described in unspecialised connective tissue found elsewhere in the body. there seems, therefore, no a priori reason to presumthat the physiological responses observed in fully vascularised and stable ear chamber tissues should be atypical. references 1. arfors, k.-e., jonsson, j. a. & mckenzie, f . n.: a titanium rabbit ear chamber: assembly, insertion and results. microvasc res 2: 516, 1970. 2. bielschowsky, m.: die silber impragnation der axen cylinder. neurologisches centralblatt 21: 579, 1902. 3. clark, e. r., clark, e. l. & williams, r. g.: micro 4. 5 . 6. 7. 8. 9. 10. scopic observations in the living rabbit of the new growth of nerves and the establishment of nerve controlled contractions of newly formed arterioles. amer j anat 55:47, 1934. coupland, r. e. & holmes, r. l.: the use of cholin esterase techniques for the demonstration of periph eral nervous structures. quart j micr sci 98: 327, 1957. davenport, h. a., windle, w. f. & buch, r. h.: block staining of nervous tissue. iv. embryos. stain technology 9: 5, 1934. gomori, g.: microscopic histochemistry; principles and practice. the university of chicago press, chicago, 1952. grant, r. t. & thompson, r. h. s . : cholinesterase and the nerve supply to blood vessels in the rabbit’s external ear. j anat (london) 97:7, 1963. koelle, g. b. & friedenwald, j. s . : a histochemical method for localising cholinesterase activity. proc soc exp biol med 70:617, 1949. richardson, k. c.: studies on the structure of auto nomic nerves in the small intestine correlating the silver impregnated image in light microscopy with the permanganate-fixed ultrastructure in electronmicro scopy. j anat (london) 94:457, 1960. sandison, j. c.: a new method for the microscopic study of living growing tissues by the introduction of a transparent chamber in the rabbit’s ear. anat rec 28: 281, 1924. received january 23, 1973 address for reprints: dr karl-e. arfors department of experimental medicine pharmacia ab box 604 s-751 25 uppsala 1 sweden upsala j med sci 78 fig. 1. thiocholine stain of four month old ear chamber fig. 2. silver impregnation of four month old ear chamber tissue showing the rich meshwork of regenerated nerve tissue showing the complex perivascular network of nerve fibres which develop, phase contrast light linear magnififibrils surrounding a 40 ,urn diameter venule. transmitted cation x 275. light, linear magnification x 700. 1 i t 732853 upsala j med sci 78 upsala j med sci 78: 189-190, 1973 effect of norhydroxyprogesterone caproate on cervical sperm penetration and secretion of ovarian steroids in the human female lars carlborg from the department of obstetrics and gynecology (head: prof. carl gernzell), university hospital, uppsala, sweden abstract compound sh 8.0582 (19-norhydroxyprogesterone 1701 caproate) is a dep8t gestagen which in experiments o n laboratory animals seemed to have a slightly higher inhibi tory affinity for peripheral reproductive organs than for gonadotropin. in this study it was injected on day 5 of the menstrual cycle in healthy young subjects. the action was recorded by daily quantitative determination of sperm penetration in the cervical mncus. the influence on ovarian function was estimated by determination of uri nary total oestrogens and urinary pregnanediol or plasma progesterone. doses of 5 mg or more tended to prolong the cycle length, prevented cervical sperm penelration and decreased ovarian steroid secretion. lower doses did not consistently influence any of these parameters. there was no certain specificity for the cervix. introduction compound sh 8.0582 (19-norhydroxyprogesterone 17a caproate) was synthesized by schering ag, berlin. in routine bioassays on laboratory animals it was found to possess a strong gestagenic action on perip!ieral reproductive organs and a mild sup pressive action on pituitary gonadotropins. i t was also found that the compound, when given intra muscularly in oil, had a more prolonged action than had progesterone or most synthetic deri vatives of progesterone. the effect was estimated to last for about 14 days (unpublished data, scher ing ag, berlin). i n the human, a single dose of 200 to 500 mg caused no changes in liver function or carbohydrate metabolism. to elicit a signifi cant biological response in the endometrium, doses below 50 mg were sufficient ( 5 ) . in post-meno pausal women 40 to 200 mg reduced the gonado tropin output. therefore it was of interest to determine whether the compound would have a high af finity for the peripheral reproductive organs (inter ference with cervical sperm penetration) without disturbing the ovarian function (estimated by de termination of oestrogens and progesterone in the human female). if this were the case the drug could possibly be used as a novel contraceptive injected early in the menstrual cycle. materials and methods regularly menstruating women between 20 and 30 years of age were chosen as test subjects. they had not used any hormonal contraceptives for at least 3 cycles before the experiments. the compound was given in oil in a single intramuscular injection on day 5 of the cycle at doses ranging from 2.5 to 20 mg. daily samples of cervical mucus were taken and tested for quantitative sperm penetration in vitro (2). t h e daily basal body temperature was recorded in all cycles. the total urinary oestrogen excretion was measured on days 12 to 18 (1). the occurrence of ovulation was esti mated either by determination of urinary pregnanediol ex cretion ( 3 ) o n days 10 and 22, o r by the determination of plasma progesterone concentration on days 9, 13, 17 and 23 (4). results 20 rng ( 2 subjects): bleeding was delayed by 17 days and an immediate thermogenic effect lasted for about 2 weeks. all hormone determinations were low. sperm penetration tests were negative or became positive after 19 days. 10 mg ( 2 subjects): bleeding was postponed by 5 days. with regard to temperature, sperm pene tration, and hormone determinations, the data were similar to those obtained after the 20 mg dose. 5 rng (5 subjects): in one cycle, bleeding was delayed by 20 days but otherwise the cycle length was unaffected. ovulation probably occurred in the remaining 4 cycles. no sperm penetration was found in 3 subjects and the last subject with unim paired cervical function conceived and delivered a healty infant in due course. upsala j m e d sci 78 190 l. carlborg 3 mg ( 4 subjects): in this group all subjects had an untreated control cycle before the test period, and the determination of sperm penetration, uri nary oestrogens, and plasma progesterone were within the normal range. administration of the compound did not affect the length of the cycle and no major hormonal changes were detected. only in 2 subjects was there a marked inter ference with sperm penetration. one subject with a positive sperm penetration conceived and deliv ered a healthy infant in due course, 2.5 mg ( 4 subjects): there was no interferance with cycle length and all cycles appeared to be ovulatory. however, in 3 cycles there was no sperm penetration. discussion gestagens injected early in the menstrual cycle have been tried as a means of contraception (6, 7). the treatment generally resulted in amenor roea for 3 to 5 months. i t is possible that the postponed bleeding could have been preceded by an ovulation-a situation which would reduce the reliability of the contraceptive method. the pres ent study was undertaken to determine whether a long-acting gestagen (sh s.oss2) given early in the cycle would act as a contraceptive without impairing menstruation. the limited data show that doses as low as 5 mg did postpone the ex pected bleeding. after an interval of up to 2 weeks when a thermogenic action of the compound was manifest a positive cervical sperm penetration could bz demonstrated. ovulation might possibly have occurred after the schedded investigational intervals. the inter est was thus focused on doses less than 5 mg. the data revealed that there was no consistent influ ence on cycle length, ovarian estrogen and pro gesterone production or inhibition of sperm pcne t r a t i x iil the cervical mucus. the inability to act as a contraceptive a t these doses was evidenced by two conceptions. to prevent sperm penetration, higher doses were needed but with thc increased risk of disturbance of a menstrual rhythm. it was shown that the lowest doses needed to interfere with cervical sperm penetration were also liable to postpone uterine bleeding and to interfere with ovarian steroid production. the mechanism for the latter phenomenon was beyond the scope of the study. upsala j m e d sci 78 acknowledgements the author is indebted to schering ag, berlin, germany from which company generous financial support was received, to professor carl gemzell for reviewing the manuscript, to docent e. d. b. johansson for determina tion of plasma progesterone and to miss ulla geifalk for skilled technical assistance. references 1. brown, j. b., macleod, s. s . , macnaughtan, c., smith, m. a. & smyth, b.: a rapid method for estimating oestrogens in urine using a semiautomatic extractor. j endocr 42: 5, 1968. 2. carlborg, l.: determination of sperm migration rate in small samples of cervical mucus. acta endocr (kbh) 62: 132, 1969. 3. cox, r. i.: gas chromatography in the analysis of urinary pregnanediol. j chromatogr 12: 242, 1963. 4. johansson, e. d. b.: progesterone lev& in peripheral plasma during the luteal phase of the normal human menstrual cycle mzasured by a rapid competitive protein binding technique. acta endocr (kbh) 61: 592, 1969. 5. nevinny-stickel, j.: die unterschiedliche morphologische wirkung verschiedener synthetischer gestagenz auf das endometrium. neuntes symp. d. dtsch. ges. f . endo krin. wiesbaden u. mainz 3.-5.5. 1962, 177. 6. tyler, t. e., levin, m., elliot, j . & dolman, h.: present status of injectable contraceptives: result of seven years study, fertil steril 21: 469, 1970. 7. zanartu, j., pupkin, m., rosenberg, d., davansens, a , , guerrero, r., rodriquez-bravo, r . & garcia huidobro, m.: long term effects of medroxy pro gesterone acetate in human ovarian morphophysiology and sperm transport. fertil steril 21: 525, 1970. received march 2, 1973 address for reprints: lars carlborg, m.d. kliniken for gynekologisk radioterapi akademiska sjukhuset s-750 14 uppsala 14 sweden upsala j med sci 87: 135-142, 1982 a clincal study of 30 gastric carcinoids henry johansson and erik wilander d e p a r t m e n t s of surgery a n d p a t h o l o g y , university hospital, u p p s a l a , sbt'eden abstract the c l i n i c a l p i c t u r e i n 30 p a t i e n t s operated on f o r g a s t r i c c a r c i n o i d s was s t u d i e d r e t r o s p e c t i v e l y . there were 1 2 men and 18 women, w i t h an age range of 32-79 y e a r s (mean 57 y e a r s ) . the tumours were l o c a t e d i n the corpus a r e a of t h e stomach i n more than h a l f of t h e p a t i e n t s and i n o n e t h i r d were r e l a t i v e l y small ( < 1 cm). four p a t i e n t s had m u l t i p l e tumours. metastases were found i n e i g h t p a t i e n t s , mostly t h o s e w i t h l a r g e r primary tumours. in no case was the c a r c i n o i d syndrome present. of the stomach, such a s polyps, u l c e r and carcinoma. barium c o n t r a s t s t u d y and gastroscopy d i d n o t r e v e a l t h e t r u e n a t u r e of the g a s t r i c d i s e a s e and even biopsy of t h e stomach f a i l e d t o g i v e a c o r r e c t p r e o p e r a t i v e d i a g n o s i s i n f o u r of f i v e p a t i e n t s . since six p a t i e n t s had a c h y l i a p r e o p e r a t i v e l y , i t i s empha s i z e d t h a t the p o s s i b i l i t y of a g a s t r i c c a r c i n o i d , e s p e c i a l l y i n the corpus a r e a , i s more l i k e l y i n a s s o c i a t i o n with this c o n d i t i o n . the p a t i e n t s presented symptoms s i m u l a t i n g t h o s e of more common a f f e c t i o n s introduction a g a s t r i c c a r c i n o i d i s a r a r e type of malignant b u t slowly growing e p i t h e l i a l tumour of the stomach. i t has been d e s c r i b e d i n s e v e r a l c a s e r e p o r t s ( 2 ) , b u t few comprehensive i n v e s t i g a t i o n s have been made on the c l i n i c a l p i c t u r e of p a t i e n t s w i t h this tumour (2,5). the p r e s e n t s t u d y was undertaken w i t h t h e aim of g a i n i n g f u r t h e r knowledge about t h e age and sex d i s t r i b u t i o n of t h e p a t i e n t s , t h e symptomalogy, and t h e s i z e , l o c a t i o n and frequency of m e t a s t a s e s i n an u n s e l e c t e d m a t e r i a l of g a s t r i c c a r c i n o i d s . material a n d methods all a v a i l a b l e g a s t r i c c a r c i n o i d s recorded i n the cancer r e g i s t r y of t h e national board of health and welfare i n sweden between 1958 and 1974 and i n t h e l o c a l r e g i s t e r a t t h e department of pathology i n uppsala between 1975 and 135 1980 were collected. the tumours were a l l obtained a t surgical resection and no autopsy material was included i n the investigation. the paraffin blocks were c u t i n t o about 4 um thick sections and stained w i t h haematoxylin-eosin a n d with the grimelius argyrophil s t a i n ( 3 ) . the findings in routinely stained sections a t l i g h t microscopy and the frequency of argyrophil tumour c e l l s were decisive in the characterization of the tumours. only tumours w i t h a morphology of fore-gut carcinoids ( 1 3 ) and with an argyrophil reaction i n the majority of the tumour c e l l s were accepted f o r the study. with these c r i t e r i a , the material comprised a t o t a l of 30 patients with carcinoids. the location of the tumours w i t h i n the stomach was established from the morphology of the g a s t r i c mucosa bordering the tumours , the surgical records a n d the g a s t r i c x-ray report. tumours in the pyloric area were a l l closely adjacent t o brunner's gland. these tumours were included i n the study provided t h a t a t l e a s t some p a r t of the tumour was growing in the area o f the antral mucosa. the s i z e of the tumours was given in the histopathological reports in a few cases, b u t otherwise was estimated by measuring the l a r g e s t diameter of the tumour in the paraffin block. in f o u r patients with y l t i p l e tumours the s i z e of the l a r g e s t tumour was noted. hospital in which the p a t i e n t was operated u p o n . clinical information was obtained from the medical case records a t the results sex and age d i s t r i b u t i o n o f the 30 p a t i e n t s , 12 were men and 18 were women. the age range of the p a t i e n t s was 32-79 years and the mean age 57 years (m:61; f:55). tumour location the d i f f e r e n t locations of the tumours among men a n d women a r e given in table i. i t i s seen t h a t most tumours were found in the corpus area o f the stomach a n d t h a t there was a female preponderance in a l l locations (except the c a r d i a ) . there were no noteworthy variations in age d i s t r i b u t i o n i n r e l a t i o n t o tumour location, except t h a t the three women with multiple carcinoids in the corpus were considerably younger (range 32-41 years, mean 37 y e a r s ) than the r e s t of the patients. 136 table i . locations of the g a s t r i c carcinoids i n 30 p a t i e n t s , d i s t r i b u t e d by sex. area of stomach men women total cardi a 1 1 corpus 6 10 16') antrum 3 4 7 pylorus 2 4 6 total 12 18 30 x ) one o f the men and three of the women with carcinoids located i n the corpus had multiple tumours. tumor s i z e ten of the 30 tumours were 1 cm i diameter o r smaller. the l a r g e s t tumours were found i n the a n t r a l area. f o u r patients (one male a n d three females) presented multiple tumours ( 2 , 3, 5 and more than 5, respectively). they were a l l located in the corpus of the stomach, and three of them were 1 cm or l e s s i n diameter. metastatic spread metastatic turnour g r o w t h was observed in eight of the 30 p a t i e n t s ; a l l h a d lymph node i n f i l t r a t i o n a n d three of the e i g h t a l s o had metastases of the l i v e r . there was a r e l a t i o n between the s i z e of the primary tumour and the frequency o f metastases (table 1 1 ) , in t h a t tumour spread was more common in association with l a r g e r tumours. table i 1 . the occurrence of lymph node a n d / o r l i v e r metastases in associa tion with g a s t r i c carcinoids of d i f f e r e n t locations i n r e l a t i o n t o tumour s i z e . area of stomach 0-1 cm 1-2 cm 2-3 cm 3-5 cm 5cm total 1/10 3/13 o/ 1 3/3 1/3 8/30 137 symptomatology a r e g i v e n i n t a b l e 111. b e s i d e s g a s t r i c p a i n , t h e most pronounced c l i n i c a l s i g n s were haematemesis and melaena. these l a t t e r symptoms c o u l d be e x p l a i n e d b y t h e presence o f u l c e r a t i o n o f t h e mucosa c o v e r i n g t h e tumour, s i n c e u l c e r a t i o n was observed i n 18 o f t h e 30 cases a t l i g h t m i c r o s c o p y . two p a t i e n t s underwent x-ray e x a m i n a t i o n o f t h e stomach f o r i n v e s t i g a t i o n o f anaemia o f unknown cause, and had no s u b j e c t i v e g a s t r o i n t e s t i n a l symptoms. seven p a t i e n t s had c o n c o m i t a n t a c h y l i a . t h i s was diagnosed p r e o p e r a t i v e l y i n s i x p a t i e n t s , and h a l f o f t h e s e had an a s s o c i a t e d p e r n i c i o u s anaemia. the g a s t r i c c a r c i n o i d s o c c u r r i n g i n an a c h y l i c stomach were a l m o s t e x l u s i v e l y l o c a t e d i n t h e corpus a r e a and tended t o be m u l t i p l e ( 1 0 ) . there was no c l i n i c a l s i g n s o f c a r c i n o i d syndrome o r o t h e r e n d o c r i n e a b n o r m a l i t y r e c o r d e d i n any o f t h e p a t i e n t s . the main symptoms w h i c h o c c u r r e d b e f o r e t h e o p e r a t i o n f o r g a s t r i c c a r c i n o i d t a b l e 111. m a i n symptoms l e a d i n g t o a d m i s s i o n t o h o s p i t a l and f u r t h e r i n v e s t i g a t i o n o f 30 p a t i e n t s w i t h g a s t r i c c a r c i n o i d s . tumours l o c a t e d i n : symptoms c a r d i a corpus antrum p y l o r u s t o t a l g a s t r i c p a i n melaena haematemesis anaemia vomi ti ng w e i g h t 1 oss ti redness d y s p e p s i a d i a r r h o e a d y s p h a g i a 1 6 8 8 5 4 2 1 1 1 1 5 12 1 11 9 1 9 2 8 3 7 2 6 3 4 1 3 2 p r e o p e r a t i v e i n v e s t i g a t i o n and d i a g n o s i s i n v e s t i g a t i o n s c o n s i s t e d m a i n l y o f x-ray e x a m i n a t i o n o f t h e stomach and g a s t r o s c o p y , sometimes w i t h a s i m u l t a n e o u s g a s t r i c b i o p s y . g a s t r i c x-ray was n o t p e r f o r m e d i n f i v e p a t i e n t s . i n t h r e e o f them t h e reason was an emergency e x p l o r a t i v e l a p a r o t o m y f o r a m a j o r haematemesis. i n one p a t i e n t g a s t r o s c o p y r e v e a l e d a tumour of t h e c a r d i a and i n one p a t i e n t t h e g a s t r i c c a r c i n o i d was an a c c i d e n t a l f i n d i n g d u r i n g o p e r a t i o n f o r c h r o n i c g a l l b l a d d e r d i s e a s e . four p a t i e n t s had normal g a s t r i c r a d i o g r a p h s . they were o p e r a t e d on e i t h e r because o f p a t h o l o g i c a l f i n d i n g s a t g a s t r o s c o p y ( 3 p a t i e n t s ) o r because o f s e v e r e a c c o r d i n g t o i n f o r m a t i o n i n t h e c l i n i c a l case r e c o r d s , t h e p r e o p e r a t i v e haematemesis of u n k n o w n origin (one p a t i e n t ) . gastric biopsies were performed in f i v e p a t i e n t s . the histopathological diagnoses of the g a s t r i c t i s s u e material were ulcera tion (one c a s e ) , g a s t r i t i s (one c a s e ) , undifferentiated carcinoma ( 2 cases) and carcinoid (one c a s e ) . after re-examination of the cases diagnosed a s undiffer i t became entiated carcinoma, including grimelius staining of the tumour obvious t h a t the specimens originated from g a s t r i c carcinoids. a survey of the putative preoperative diagnoses arrived a t n a t i o n and/or gastroscopy, sometimes with g a s t r i c biopsy, i s g the most common preoperative diagnosis was g a s t r i c polyp(s) ( 9 rom x-ray exami ven i n table iv. p a t i e n t s ) . in one of these patients multiple g a s t r i c carcinoids were subsequently disclosed a n d in another p a t i e n t multiple adenomatous mucosal polyps were found i n addi tion t o a g a s t r i c carcinoid. table iv. putative preoperative diagnosis leading t o surgery i n p a t i e n t s with g a s t r i c carcinoids. preoperative no. of comments diagnoses pa t i ents polyp(s) ulcer carcinoma not known tumour, n o t specified 3 pyloric s t e n o s i s 1 neuri noma ( ? ) 1 g a l l stone 1 carci noid 1 2 cases with multiple polyps explorative laparotomy because of haematemesis the g a s t r i c tumour was an accidental finding a t opera ti on total 30 type of operation tumours were extirpated by a b i l l r o t h i or i1 g a s t r i c resection, especially those located i n the more d i s t a l p a r t of the stomach. many smaller tumours i n the corpus area were removed by local resection. a subtotal o r t o t a l gastrec tomy was performed i n some p a t i e n t s w i t h large o r multiple tumours. the g a s t r i c carcinoids were removed by d i f f e r e n t surgical methods. most 139 survival and follow-up mortality of 7 per cent. three patients died from metastatic disease and one developed a local recur rence. two of the 30 p a t i e n t s died a f t e r surgery, corresponding t o a post-operative twenty-two p a t i e n t s were observed f o r one year o r more. during t h i s period of 15 patients followed f o r a period of f i v e years o r longer, 10 a r e s t i l l a l i v e and symptom-free. discussion by d e f i n i t i o n , carcinoids a r e endocrine tumours in which the tumour c e l l s contain endocrine secretory granules a t the electron microscopic l e v e l . since the grimelius s i l v e r s t a i n causes a reaction i n almost a l l g a s t r o i n t e s t i n a l endocrine c e l l s ( 4 ) , though without discriminating between them, t h i s staining procedure was used t o i d e n t i f y the tumours (11,lz). although the chemical back ground of the argyrophil reaction i s unclear, i t i s k n o w n from u l t r a s t r u c t u r a l studies t h a t the s i l v e r s t a i n i s s p e c i f i c f o r endocrine secretory granules (4), a n d tumours which lack an argyrophil reaction in the majority of t h e i r c e l l s cannot be regarded unquestionably as endocrine; f o r t h i s reason a few tumours which were primarily c l a s s i f i e d as g a s t r i c carcinoids in the swedish cancer registry were n o t accepted f o r the study. i t i s emphasized t h a t s t r i c t c r i t e r i a f o r the d e f i n i t i o n of endocrine tumours improves the p o s s i b i l i t y of identifying t h e i r c h a r a c t e r i s t i c s . although based on a retrospective study, o u r findings demonstrate the d i f f i c u l t i e s i n the diagnosis of g a s t r i c carcinoids. most p a t i e n t s presented symptoms such as pain, haematemesis a n d / o r melaena, anaemia, vomiting a n d loss of weight, which a r e common signs i n much more frequent pathological conditions of the stomach such as ulcer disease and carcinoma. thus, the infrequent g a s t r i c carcinoids can hardly be suspected from the c l i n i c a l symptoms. the presence of g a s t r i c carcinoids, since with these methods the tumours resemble polyps, ulceration or g a s t r i c carcinoma. the most accurate diagnostic procedure appears t o be g a s t r i c biopsy, b u t even w i t h t h i s the diagnosis seems unreliable, since discrimination from undifferentiated carcinoma of the stomach, in p a r t i c u l a r , can be d i f f i c u l t . additional staining of the biopsy material with the grimelius argyrophil s t a i n i s apparently useful, b u t a p r e r e q u i s i t e for the use of this procedure i s , of course, t h a t a possible diagnosis of g a s t r i c carcinoid i s considered. possible, be treated primarily with surgery just l i k e carcinoids of the g a s t r o i n t e s t i n a l in general (1,7,8). in g a s t r i c carcinoids l e s s than 1 cm in b o t h x-ray of the stomach and gastroscopy seem i n s u f f i c i e n t f o r establishing when the diagnosis i s confirmed, carcinoids of the stomach should, i f 140 d i a m e t e r complete l o c a l e x c i s i o n may be adequate i f t h e r e a r e no s i g n s o f i n v a s i o n o f t h e m u s c u l a r i s p r o p r i a o r lymph node i n v o l v e m e n t . tumours l a r g e r t h a n 1 cm s h o u l d b e t r e a t e d b y g a s t r i c r e s e c t i o n o r g a s t r e c t o m y . i f lymph node metas t a s e s a r e found, t h e g a s t r i c s u r g e r y s h o u l d be combined w i t h e x c i s i o n o f a l l a c c e s s i b l e nodes and such tumour cases s h o u l d t h u s be t r e a t e d as f r a n k carcinumas. it has been c l a i m e d t h a t g a s t r i c c a r c i n o i d s a r e m o s t l y l o c a t e d i n t h e a n t r a l a r e a ( 6 ) . the f i n d i n g s i n t h e p r e s e n t s t u d y a r e n o t i n agreement w i t h t h i s o b s e r v a t i o n , as more t h a n h a l f of t h e tumours were f o u n d i n t h e corpus of t h e stomach. the c a r c i n o i d syndrome i s seldom seen i n a s s o c i a t i o n w i t h c a r c i n o i d tumours o f t h e stomach (14). i n o u r s e r i e s none o f t h e p a t i e n t s p r e s e n t e d symptoms i n d i c a t i n g such a syndrome. when i t does appear, t h e g a s t r i c c a r c i n o i d syndrome d i f f e r s f r o m t h e c l a s s i c a l type. p a t i e n t s w i t h t h e g a s t r i c c a r c i n o i d syndrome have a t y p i c a l f l u s h , and d i a r r h o e a i s u s u a l l y n o t a p r o m i n e n t symptom. a l t h o u g h r e l a t i v e l y s l o w l y growing, c a r c i n o i d s a r e m a l i g n a n t tumours w i t h an a b i l i t y t o m e t a s t a s i z e . i n t h e p r e s e n t s e r i e s m e t a s t a s e s were p r e s e n t a t t h e t i m e o f o p e r a t i o n i n 26% o f t h e cases. a r e l a t i o n was f o u n d between tumour s p r e a d and s i z e o f tumour, i n t h a t metastases were more f r e q u e n t i n t h e l a r g e r tumours. the s u r v i v a l r a t e s were a l s o i n f l u e n c e d b y t h e tumour s i z e and s p r e a d o f t h e d i s e a s e . the 5-year s u r v i v a l r a t e , as f a r c o u l d b e e s t i m a t e d , was 67 p e r c e n t . recent i n v e s t i g a t i o n s have shown t h a t g a s t r i c c a r c i n o i d s o c c u r r e l a t i v e l y f r e q u e n t l y i n a s s o c i a t i o n w i t h a c h y l i a , e s p e c i a l l y t h o s e w h i c h a r e l o c a t e d i n t h e c o r p u s a r e a o f t h e stomach and t e n d t o be m u l t i p l e ( 9 , l o ) . it has a l s o been suggested t h a t g a s t r i c c a r c i n o i d s w h i c h d e v e l o p i n an a c h y l i c stomach may have a p a t h o g e n e s i s s i m i l a r t o t h a t proposed f o r carcinoma o f t h e stomach i n a s s o c i a t i o n w i t h a c h y l i a and p e r n i c i o u s anaemi ( 1 0 ) . these o b s e r v a t i o n s s t r e s s t h e i m p o r t a n c e o f i n c r e a s e d a l e r t n e s s t o t h e p o s s i b i l i t y o f a g a s t r i c c a r c i n o i d i n p a t i e n t s w i t h p a t h o l o g i c a l f i n d i n g s i n t h e stomach and an a s s o c i a t e d a c h y l i a (and p e r n i c i o u s anaemia). acknowledgement t h i s work was s u p p o r t e d by g r a n t s f r o m t h e m e d i c a l research c o u n c i l ( p r o j . no. 102). references 1. aranha, g . v . & greenlee, h.b.: s u r g i c a l management o f c a r c i n o i d turnours of t h e g a s t r o i n t e s t i n a l t r a c t . am s u r g aug. 429-435, 1980. 2. c h r i s t o d o u l o p o u l o s , j.b. & k l o t z , a.p.: c a r c i n o i d syndrome w i t h p r i m a r y c a r c i n o i d tumour o f t h e stomach. g a s t r o e n t e r o l o g y 40, 429-440, 1961. 3. g r i m e l i u s , l.: a s i l v e r n i t r a t e s t r a i n f o r a 2 c e l l s i n human p a n c r e a t i c a c t a s o c med u p s a l i e n s i s 73, 243-276, 1968. 4. g r i m e l i u s , l. & w i l a n d e r , e.: s i l v e r s t a i n s i n t h e s t u d y of e n d o c r i n e c e l l s o f t h e g u t and pancreas. i n v e s t c e l l p a t h o l 3, 3-12, 1980. 141 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. marks, c . : c a r c i n o i d turnours. a c l i n i c o p a t h o l o g i c s t u d y . g . k . hall & co., c e l l s of t h e g u t and pancreas. i n v e s t c e l l pathol 3 , 3-12, 1980. oota, k. & s o b i n , l . h . : h i s t o l o g i c a l t y p i n g of g a s t r i c and oesophageal turnours. who. no. 1 8 geneva, 1977. t i l s o n , m . d . : c a r c i n o i d syndrome. surg c l i n north am 4 , 409-429, 1979. welch, j . p . & malt, r . a . : management of c a r c i n o i d tumours of g a s t r o i n t e s t i n a l t r a c t . surg gynecol o b s t e t 145, 223-227, 1977. wi l a n d e r , e . : a c h y l i a , p e r n i c i o u s anaemia, e c l c e l l s and g a s t r i c c a r c i n o i d s . virchows arch ( p a t h o l anat) 387, 371-373, 1980. wilander, e . : achylia and the development o f g a s t r i c c a r c i n o i d s . virchows arch ( p a t h o l anat) 394, 151-160, 1981. wilander, e . , g r i m e l i u s , l . , lundqvist, g . & skoog, v . : p o l y p e p t i d e horrno nes i n a r g e n t a f f i n and a r g y r o p h i l g a s t r o d u o d e n a l e n d o c r i n e tumours. am j p a t h o l 96, 519-530, 1979. wilander, e . , portela-gomes, g . , g r i m e l i u s , l. & westermark, p . : argent a f f i n and a r g y r o p h i l r e a c t i o n s of human g a s t r o i n t e s t i n a l c a r c i n o i d s . g a s t r o e n t e r o l o g y 73, 733-736, 1977. williams, e . d . & s a n d l e r , m . : the c l a s s i f i c a t i o n of c a r c i n o i d turnours. lancet 1:238-239, 1963. wilson, h . , cheek, r . c . , sherman, r . t . & s t o r e z , e . h . : c a r c i n o i d tumours. i n : c u r r e n t problems i n surgery. chicago: year medical p u b l i s h e r s 1970. address f o r r e p r i n t s : e r i k wilander, m . d . department of pathology u n i v e r s i t y o f uppsala p . o . box 553 s-751 22 uppsala sweden 142 upsala j med sci 81: 201-212, 1976 the migrating thermodynamic quantum hypothesis for cytoplasmic streaming, sodium pumping and other cell biological phenomena, deduced from biofunctional considerations of the ultrastructure of brush border microvilli erik cerven from the institute of medical and physiological chemistry, biomedical center, university of uppsala, uppsala, sweden. abstract an attempt is made to reconcile experimental data dealing with, inter alia, cytoplasmic streaming in characean algae, contraction in actomyosin systems, na+and -k+ stimulated atpase activity and the ultrastructure of brush border microvilli. i t is postu lated that myosin molecules transfer energy from atp to a n actin-containing filament and that a high energy conformation is subsequently propagated along the filament. a t regularly spaced intervals corresponding to the length of an actin-tropomyosin subunit, the propagation of high energy involves re jection of a pressure pulse in the direction of cyto plasmic streaming. proteins in solution capable of storing the thermodynamic energy represented by the pressure pulse will either migrate in the opposite direc tion or conserve the quantized cytoplasmic flow generated by the actin-containing filaments. at sites where actin filaments are attached to the plasma membrane the high energy is propagated in another direction leading to expulsion of sodium ions and neutralization of the vectorial pressure pulse. introduction in 1956-57 (9, 12) it was discovered by darkfield microscopy that protoplasma droplets from characeae contain fibrils, rapidly moving for ward or in circle, longitudinally dissociating and associating. the multiply associated state was also observed to display undulating movement. cytoplasmic particles that came close to these fibrils were captured and without brownian motion translocated in the opposite direction and it was therefore concluded that the observed fibrils are responsible for cytoplasmic streaming in this and other species (9, 10, 12.) the moving fibrils generating cytoplasmic streaming have recently been shown by decoration (22) with myosin to contain f-actin oriented as if able to pull myosin filaments in the direction of the cyto plasmic flow (14). during recent years, actin and actin-like pro teins have been found in a number of nonmuscle cells (4, 15, 17, 19, 24,28, 32), including bacteria (21), and their interaction with myosin from distant unrelated species suggests that actin has maintained its original molecular properties during the course of evolution pointing to a general function not restricted to cytoplasmic streaming or muscular contraction (15). in cases where a transmembraneous gradient of sodium is suggested to play a main role in a physiological function, such as propagation of action potentials in nerve cells and sodium-dependent transport of certain metabolites in intestinal epithelial cells (25), actin-containing filaments are found in close association with the plasma membrane (1 9, 24). electron microscopic investigations of nerve cell axones (19) and brush border microvilli (24) in dicate that the actin-containing filaments may be involved in excitation (19) and in functional co operation with proteins in the plasma membrane of brush border microvilli. in the latter case this is because the attachment sites are regularly spaced at a distance that corresponds to the length of an actin-tropomyosin subunit (16, 22, 24). in volvement of actin-containing filaments in plasma membrane phenomena is also suggested by the findings that release from “contact inhibition” of cultured cells may be accompanied both by a decreased transmembraneous potential (8) and a diminished association of actin-containing fila upsala j med sci 81 202 erik cervkn ments to the plasma membrane (17). furhter, a mutant strain of e. coli lacking the ability to accumulate potassium from a potassium-depleted medium is characterized by a mutant actin that fails to polymerize at that particular potassium concentration (2 1). when taken together, these and other findings suggest that sub-plasma membraneous actin-containing filaments may be part of phenomena involved in generating a transmembraneous electrochemical gradient of sodium and potassium without reference to the mechanism of this involvement. the polarity of the actin-containing filaments in brush border microvilli (see fig. 1) has recently been determined by decoration (22) with myosin subfragment 1, by means of immunologic loca lization of a-actinin predominantly in the distal tips of the microvilli and by ultrastructural evid ence for myosin filaments in the proximal terminal web (24). the diverging observations on micro villar contraction, which has been described as either shortening or undulating (cf 29) move ment, could be unified by assuming that actin containing filaments behave in brush border microvilli as they do in protoplasma droplets from churuceue (c$ 9, 10, 12) carrying with them a cytoplasmic flow. the direction of this cyto plasmic flow can be deduced from the polarity of the actin-containing filaments in brush border microvilli by analogy with the corresponding filaments in churuceue (14). this deduction is complicated by the fact that the mechanism generating cytoplasmic streaming must be inhi bited, or directed towards other functions, along the entire length of at least some actin-containing filaments, in order to ascertain restreaming into the cell. evidence has been obtained that transport of certain amino acids and sugars against a gradient into brush border microvilli is dependent on co migration of sodium (25), a phenomenon that is a fundamental principle for the transport of many metabolites into different types of cells (3). in order to ascertain restreaming in brush border microvilli it will therefore be postulated in the present hypothesis that the mechanism generating cytoplasmic streaming is used in the plasma upsala j med sci 81 i 1 ,minal web actin core fig. i. schematic illustration of the anatomic structure of a cut microvillus (a, cj: 24) and the direction of cytoplasmic streaming deduced by analogy with characean algae (b, c j : 14). membrane for generating a transmembraneous gradient of sodium. the direction of the cyto plasmic flow that will result from this assumption, is illustrated in fig. 1 b. actin-containing fila ments are attached to the plasma membrane in regularly spaced intervals at a distance that cor responds to the length of one actin-tropomyosin subunit (24) indicating functional cooperation between the filaments and membraneous proteins. since no other binding sites have been observed it will be assumed in this hypothesis that both cyto plasmic streaming and sodium expulsion are governed from these regularly spaced intervals. an additional function of the observed attache ment-sites (24) is probably that their presence counteract the propulsion of the actin core into the cell interior that would occur in their absence as a result of cytoplasmic streaming in the distal direction. when brush border microvilli are demembra nated by treatment with triton-x 100 (23) the subsequent addition of c a + + and atp results in propulsion of the actin core into the cell interior (23). this finding does not support that the slid ing filament model for muscular contraction (7, the migrating thermodynamic quantum hypothesis for celt biological phenomena 203 relaxed state contraction fig. 2. schematic illustration of the sliding filament model according to which contraction of skeletal muscle is mediated by active translocation of myosin filaments along the actin-containing filaments by a mechanism that implies atpase activity for every translocation step past an actin dimer. (cj: 7). see fig. 2)is applicable to contraction of brush border microvilli, since no myosin was reported to be located along the axis of propulsion (23) or is even found in the vicinity of the villous part of the actin core in contracted intact brush border microvilli preparations (29), necessitating the postulation of invisible myosin (29). filaments reminiscent of striated muscle myosin are instead located perpendicular to the actin core in the underlying terminal web (24) initiating the shortening and undulating movement of microvilli at distance. they apparently do not migrate distally towards the a-actinin-containing tips of microvilli (corresponding to the z-line of muscle (24)) as they should do according to the sliding filament model for muscular contraction. an alternative interpretation of myosin action is also required in view of failures of the sliding filament model to explain contraction in actomyosin pre parations lacking myosin filaments (6) and in re generated actomyosin containing conversely oriented actin filaments (6). further, the excess of actin in comparison with myosin in many non muscle cells (15) and the mechanochemical coup ling in actomyosin systems catalyzed by globular myosin subfragments (27), evoke the suspicion that the sliding filament model is not generally applicable to contractile phemomena ( 1 5 ) . this model has also been criticized on the basis of ul trastructural observations on contracted striated muscle (30). it is very difficult to devise a mechanism by which the sliding filament model for muscular contraction can explain the cytoplasmic stream ing in brush border microvilli that is deduced from their shortening or undulating (cf.: 29) movement by analogy with the movement of the corresponding fibrils in protoplasma droplets from characeae (qf: 9, 12). since this model implies that myosin filaments sometimes return to a relaxed position it also implies that cyto plasmic streaming should be reversed at times contrary to what is known about this pheno menon in other systems (9, 10, 12, 14). if the sliding filament model is modified by assuming that myosin molecules of the myosin filaments upon reaching the a-actinin-containing distal tips of microvilli are detached and subsequently pas sively transported into the cell, other questions still remain unanswered. these questions relate to energetic considerations dealing with the con siderable amounts of atp that must be trans ported to and consumed in microvilli in order to mediate on one hand the myosin atpase cata lyzed translocation of myosin (7) and on the other hand the na+-andkt -stimulated atpase catalyzed (3, 20) generation of a transmem braneous electrochemical gradient of sodium and potassium. the sliding filament (7), the screwing filament ( 1 1) and the undulating filament (3 1) hypotheses are all unable to provide any physio logical function to the observed contraction of brush border microvilli. the reason for this is that if any of these hypotheses is adapted, too complex and multiple interactions would be necessary in the binding of the filaments to the plasma membrane. in addition, any active me chanism for the assumed cytoplasmic straming in brush border microvilli would yield energy-re quiring frictional flow. one experimental fundament for the hypothesis that will be presented here is the original observa tion that cytoplasmic particles move closely along the protoplasmic filaments of characean algae without brownian motion (9, 12). only a mecha nism capable to non-randomize and vectorize (structurize) this random motion can thus be as sumed to be responsible for cytoplasmic stream ing. any active mechanism such as the myosin atpase catalyzed sliding of filaments (7), pro pelling of filaments (1 l), or undulation of fila ments ( 3 1) must accordingly be rejected because it would on the contrary enhance brownian motion in the vicinity of these filaments. upsala j med sci 81 204 erik cervkn thermal motion of solvent molecules can be absorbed.by proteins in small amounts resulting in e.g. strained bond angles (cj.3). it is postulated in the present hypothesis that increased utilization of similar or other possibilities of storing thermo dynamic energy results in a defined conformation of a protein. by further postulating that this energy-storing conformation of a protein in one single step reverts to a low energy conformation a possibility is created to structurize the brownian motion of the surrounding solvent molecules. since the thermodynamic energy is rejected as a pressure pulse when used for generating cyto plamic streaming it must be assumed that a “con densed” form of a protein equivalents its energy storing, low entropy conformation. this idea is compatible with calculations indicating that all components of a protein do not oscillate simul taneousely in all degrees of freedom of motion of the protein (26). a protein oscillating between two main conformations may be considered to have one main degree of freedom of motion, at least represented by the difference of entropy between the “condensed” and the “loose” form. according to the present hypothesis, the one step release of thermodynamic energy from the low entropy conformation of a protein (or pro teins) in the actin-containing filaments into the surrounding medium is brought about by the transfer to this conformation of the energy re siding in a high energetic phosphate bond in an adjacent protein of the actin-containing filament. by this assumption a concept of propagating high energy equivalent to a phosphate bond in the actin-containing filament is introduced, which may explain why myosin apparently effects contraction of brush border microvilli at dis tance and why atp enhances cytoplasmic streaming in several other cells (12). this idea is not contradicted by recent reports that in actin, high energy may be stored in two interchangeable forms, bound atp and a labile conformation of protein (1 6). furthermore the idea is compatible with considerations indicating that the nucleotide binding region of actin is located in a p-pleated sheet (16), because this conformation of a poly peptide would be the most efficient in generating a pressure pulse. since a low entropy conformation of a protein imposed by absorption of thermo dynamic energy from the surrounding medium is labile and tends to reject this energy the postula tion of such a conformation may be a require ment for the concept of a unidirectional propa gation of high energy pulses which will be “pulled” by the thermodynamically labile con formation. it is further assumed in this hypothesis that a thermodynamically labile conformation of a protein that in addition binds phosphate in a high energetic bond tends to “push” the phosphate bond to adjacent proteins. it is inherent in the hypothesis that is outlined here that absorption of thermodynamic energy resulting in a defined low entropy conformation would confer on the propagating high energy pulse the possibility of an equivalent active physiological function at equiva lent sites along the actin-containing filaments. the hypothesis it is postulated that components of the actin containing filaments formed in vivo can exist in two low energy conformations, l,, l, and two l a ! i i fig. 3. energy content of proteins that are of the same magnitude of size as the proteins constituting the actin-con taining filaments. (l,-lj is equivalent to (hi-h,) and defines the energy content of a protein that is exchangeable with the surrounding medium as one thermodynamic quan tum (q), that is, a single pressure pulse or a defined region of increased thermal motion of solvent molecules. the capital h denotes a high energy content derived from and ex changeable with a terminal phosphate bond of atp. this high energy may reside in a protein in the form of a high energetic phosphate bond or an unstable conformation of a protein ( c j : 16). the.capital l denotes that a protein is lacking this particular form of high energy content. the lines in this figure denote possible but not obligate energy transi tions of a protein submitted to the postulates of this hypo thesis. upsala j med sci 81 the migrating thermodynamic quantum hypothesis f o r cell biological phenomena 205 high energy conformations, h,, h, (see fig. 3 and page 209 for explanation of the notations used). the energetic difference q (=one thermo dynamic quantum) between l,, h, and l,, h,, respectively, is equal and exchangeable with the surrounding medium as described by the follow ing reactions: l2p + qs-+p (1 ) h2p + qs -h2p the thermodynamic quantum is generated by the actin-containing filaments as a mainly uni directional cytoplasmic pressure pulse which is equivalent to a defined region of increased thermal motion of cytoplasmic solvent molecules. the energetic difference between l,, l, and h,, hi, re spectively is comparable to the energy content of the terminal phosphate bond of atp. the main function of myosin is to transfer energy from atp to the actin-containing filaments which as reflected by their polarity have the capability of propagating this energy in one direction. the un idirectional propagation of high energy is a re quirement for the generation of thermodynamic quanta. mainly two combinations of energy con tent in adjacent proteins of the actin-containing filament are unstable and responsible for the un idirectional propagation of high energy as described by the following reactions: h2 (xfl (y) -+ l2 (x)h2 (y) + qs hi (xf2 (y) -+ l1 (x)h2 (y) (3) (4) these reactions have a physiological signi ficance at the junction between two adjacent actin-tropomyosin subunits where they are in pi 5-c a. fig. 4 . a: schematic illustration of an actin-tropomyosin subunit. the equilibrium arrows indicate that the propagation of high energy in the intermediate part of the subunit not necessarily is unidirectional. b to c illustrate reaction (5). the notations used are explained on page 209. a b -+c d e e--f fig. 5 . schematic illustration of sodium pumping from a sodium-depleted (int) medium across a plasma membrane (m) to a sodium-enriched medium without reference to the pos sible occurrence of proteins linking the carrier and the actin containing filaments together. filled circles represent sodium ions and empty circles represent carrier sites. the transloca tion of the carrier sites as illustrated in this figure only indicate differential accessability to either side of the mem brane without any preference of molecular mechanism for this phenomenon. a illustrate reaction ( 6 ) . b to c illustrate reaction (7). c to d illustrate reaction (8). d and e illustrate reaction (9). e to f illustrate reaction (10). the notations used are explained on page 209. volved in either the generation of cytoplasmic streaming or sodium carrier function. cyto plasmic streaming is generated as illustrated in fig. 4 and can be described by reaction (3) in the following form (see fig 4 b-c): h2 (di)l1 (pii) -+ l2 (di)h2 (pii) + 6s (5) in order to ascertain that the propagating high energy pulse does not give rise to any pressure pulse when transferred from a sodium carrier molecule to pi1 it must be assumed that reaction (2) is involved in this transfer. it is further postu lated that association and dissociation of sodium to the carrier sites are equilibrium reactions and that each energy content according to fig. 3 of the carrier is preferentially represented by a sodium-associated or -dissociated carrier mole cule exposing its carrier sites to either a sodium depleted or sodium-enriched medium. by applying reactions (1) to (4), the consequences of these postulates for the sequential mode of action of a sodium carrier molecule will be described by the following reactions (see fig. 5 , and appendix for deduction). nalt + l1 (c) 2 id2 (c, naifnl) + qs ( 6 ) (7) + l1 (c) + naint l2 (c, nalt) + qs (d1)l2 ('9 nalt) l1 (di)h2 (c, na+ ext ) (8) (9) t "2 (c, nalxt) + qs "t h1 (c) + naext hi (cf2 (pii) -+ l1 (c)h2 (pii) (10) upsala j med sci 81 206 erik cervbn by the reactions (7) to (10) one cycle of the carrier is completed leading to expulsion of sodium ions and the propagation of one high energy pulse in the distal direction. in order to ascertain a sodium carrier function it must be assumed that the high energy pulse is preferenti ally transferred to a carrier which is associated with sodium as described in reaction (8). this property is inherent in the sequence of reactions that have been described in that the high energy pulse leaves the carrier in a sodium-dissociated l, conformation (reaction (10)) which according to reaction (7) (see appendix for interpretation) has a high affinity for sodium. since ll0, is unstable, efficient pumping of potassium would be impossible if this ion were to associate with an l, conformation of the carrier in a potassium depleted external medium. by making the same postulates as were made for sodium, relating to different conformations of the carrier, the following transitions will operate in potassium pumping, in the sequence correspond ing to the reactions ( 6 ) to (9): (see appendix for additional evidence). since ouabain-binding is involved in reaction (9b) (see appendix) electrogenic action of the carrier is ascertained in the present hypothesis by competition of endogenous ouabain (1, 18, 35) with the potassium-binding sites when these are exposed to the external side of the plasma mem brane. this view is further supported by the find ing that high external potassium ‘concentrations result in a diminished transmembraneous poten tial (32). the “field” of migrating thermodynamic quanta generated by the actin-containing fila ments will induce unusual properties of the sur rounding medium as exemplified by the behaviour of proteins in solution that are capable of oscil lating in harmony with the quantized flow. ac cording to boltzmanns law, s=k in a($: 5), highly ordered states are less probable, and there fore accumulation of either high entropy forms or upsah j med sci 81 low entropy forms in adjacent coordinates (x-1), (x), (x+ 1) of the field is not favoured. some con sequences of this statement for proteins in solu tion, capable of storing the energy represented by q are described by the following reactions where the sequence of notations indicate adjacent loca tion in the direction of quantized cytoplasmic flow. -? -+ (q, + lzp) qs + -q, (1 1) these reactions are all unidirectional because they occur in a field of migrating thermodynamic quanta. fusion of the energy components in parenthesis in reaction (1 1 ) to (14) is either ac companied by migration of the protein in the backward direction or by extension of the quan tized field. discussion according to this hypothesis atp enhances cytoplasmic streaming (12) by increasing the number of high energy pulses per filament length while filamentous myosin probably influences cytoplasmic streaming by increasing synchrony in the quantized field surrounding the actin-con taining filaments. a tendency of the protoplasmic fibrils of characeae to join in circles (9, 12) is explained partly from considerations of fuel economy since only thermodynamic energy would be required to maintain the rotation of such circles provided the propagation of high energy pulses is unrestricted. there are profound energetic differences be tween on one hand the present hypothesis and on the other hand the sliding filament hypothesis for myosin-actin interaction (7) and the na’ and k + -stimulated atpase hypothesis for generation of a transmembraneous sodium gradient (cf.: 1, 3). these differences are illustrated by the follow ing calculations which are based on an estimated number of 50 actin-tropomyosin subunits, (cf.: 24) the migrating thermodynamic quantum hypothesis f o r cell biological phenomena 201 each containing 13 actin monomers ( 2 2 ) in their turn constituting spheres with a diameter of 5 5 8, (22), in one actin-containing filament along the entire length of one microvillus. according to the present hypothesis the hydrolysis of 2 atp mole cules optimally results in 50 cycles of the sodium carrier, and depending on the degree of quantiza tion and on the magnitude of the oscillations of the proteins involved in generating a pressure pulse, results in a cytoplasmic translocation (flow) perhaps in the range of l o a to 50xloa. the latter estimation is based on the difference of width of the interchain cleft between tetrameric oxyand deoxyhemoglobin (mw: 64 500) which is 7 8, (29) and which may be representative of the magnitude of oscillations of a protein of approximately that molecular weight (mwactin: 42000). on the contrary, in order to bring about a comparable physiological effect according to the sliding filament and the na+-andkt stimulated atpase hypotheses, loai55a (0.2) to 50 x 10/55 (9.1) atp molecules (see text to fig. 3 and ref. 7) and 50 atp molecules, respectively, are hydrolyzed (&: 7). the present hypothesis does not contradict the idea that a na' -and-k' -stimulated atpase molecule is responsible for generation of an elec trochemical gradient, but extends this idea in that it includes a mechanism for the reutilization of high energetic phosphate bonds. this consider ably increases the efficiency for generation of an electrochemical gradient of sodium and potas sium. it is notable that when the sequential mode of action of the carrier is deduced from the postulates of this hypothesis one "solution" (see appendix) is compatible with several experimental data. in addition this solution is also compatible with fundamental thermodynamic principles in that a low entropy form of the carrier is as sociated with potassium and dissociated from sodium on the potassium-depleted and sodium enriched side of the membrane. this is a less probable state and should represent lower en tropy according to boltzmanns law s = k inn. a high affinity for internal sodium is also accounted for in the present hypothesis since the low entropy form, l,, is more unstable when adjacent to a high energetic phosphate bond in accordance with the same fundamental thermodynamic prin ciples. the finding that mitochondria may be located in horizontal planes at the level of the z-band of striated muscle (2) is compatible with the postu late of the present hypothesis that high energetic phosphate bonds propagate towards regions en riched in a-actinin when combined with the generally held view that mitochondria are found where they are best needed. since the propagating high energy would leave the filament at this site, low energetic phosphate metabolites would ac cumulate resulting in positive chemotaxis of mito chondria. in skeletal muscle, the length of an actin tropomyosin subunit determined by x-ray dif fraction is approximately 365 a (7) and the inter crosslink distance of equivalent spatial orientation between actin and myosin filaments is 429 8, (7). this diff'erence of length is interpreted in the pre sent hypothesis to indicate that myosin is ar ranged in the myofilaments so to, as far as pos sible, avoid contact with equivalent sites on the actin-containing filaments. this arrangement would guarantee, firstly minimal interference with the quantum flow generating mechanism located between adjacent actin-tropomyosin subunits, and secondly, transfer of atp from myosin to the actin-containing filaments irrespective of their spatial orientation and the degree of shortening. this application of the present hypothesis is, in contrast with the sliding filament model for mus cular contraction, compatible with the repetedly demonstrated fact that atp weakens the inter action between myosin and actin (&: 2 2 ) . the contraction of regenerated actomyosin containing conversely oriented actin filaments (6) is explained by reactions (1 1) to (14) according to which proteins capable of storing the energy represented by q are able to migrate opposite to the quantized flow generated by the actin-con taining filaments. these reactions imply that con versely oriented actin-containing filaments slide past each other inside the regenerated actomyosin threads (cf.: 6) where the degree of quantization is high. the reassembly of myosin filaments simul upsala j med sci 81 208 erik cervpn taneously with contraction in regenerated acto myosin ( 6 ) and the tendency of myosin molecules to join in the a-band of striated muscle proximal (see notations, page 209) to the actin-containing filaments is explained by postulating that myosin molecules are capable of storing the energy repre sented by q . by this assumption the occurrence of myosin-like filaments in the i-band proximal to the z-band (30) and the apparent migration of mate rial from the i-band to the a-band during contrac tion (30) would also be explained. the present hypothesis gives an alternative ex planation to the phenomenon that the atp level remains unchanged after contraction in skeletal muscle (cf.: 18). it is further suggested that creatine and phosphocreatine interconversion constitute a mechanism for recycling of the high energy pulse when it leaves the distal ends of the actin-containing filaments. the main effect of atp hydrolysis in muscular contraction would consequently be to supply energy in the form of thermal motion of cytoplasmic solvent molecules. since no possibilities for restreaming close to the z-band of the sarcomere of skeletal muscle can be discerned the propagation of high energy pulses along the actin-containing filaments in this system only results in the establishment of a quantized field without accompanying quantized cyto plasmic flow. contraction of skeletal muscle will therefore be described by the following reaction: h2(d1)l1(p11)(x)~ l2(di)h2(pii)(x-1) (15) the ubiquity of actin and actin-like proteins in animal and plant kingdoms (cf. : 15) suggests that the evolution of many proteins has occurred under the influence of the quantum-generating mecha nism described here. these conditions would counteract the establishment of protein mutants that do not oscillate in harmony with the quan tum flow generated by actin-containing filaments, because the presence of such “distorsion” mole cules would render impossible the optimal func tion of cellular enzymes, adapted to a quantized environment millions of years ago. experimental support for this assumption is again found in electron-microscopic studies on brush border upsala j med sci 81 microvilli. in some of these studies the actin-core supposed to carry cytoplasmic flow in the distal direction apparently originates in the cell body (29). since cytoplasmic enzymes are of the same size as the proteins constituting the actin core they would tend to obstruct the flow if they were to follow with it. however, the reaction (1 1) im plies that proteins oscillating in harmony with the quantized flow are either stationary or translo cated in the opposite direction. entrance into microvilli of cytoplasmic enzymes adapted to a quantized flow would then be counteracted by both the central actin core and the terminal web, a network of actin-containing (29) quantum-gene rating filaments situated in the distal part of the cell but proximal to microvilli (see fig. 1). these considerations thus seem to be compa tible with the view that actin-containing filaments in generating thermodynamic quanta constitute a pace-maker for cellular life and evolution and pro foundly influence the behaviour of most cellular proteins. summary statements a hypothesis for cell biological phenomena is presented according to which: 1. “condensed”, low entropy forms of a protein are capable of generating a pressure pulse when reverting in one single step to high entropy forms. 2. the actin-containing filaments generate vectorial pressure pulses each representing a de fined thermodynamic energy content, thus explain ing cytoplasmic streaming. 3. proteins of actin-containing filaments gene rate vectorial pressure pulses in connection with the vectorial propagation of protein-bound high energy pulses along the filament. these pheno mena are mutually dependent. 4. the evolution of many proteins has occurred under the influence of the quantum flow generated by the actin-containing filaments, making them capable of oscillating in a quantized field, storing and rejecting the thermodynamic energy repre sented by the pressure pulse. 5 . the protein(s) responsible for generation of a transmembraneous gradient of sodium and potas sium is (are) in this sence adapted to a quantized environment. 6 . a sequential mode of action of a sodium and potassium carrier molecule can be deduced from the postulates of the hypothesis. one of four solu tions in this deduction is compatible with experi mental data and fundamental thermodynamic principles. 8. a physiological significance is ascribed to the contraction of brush border microvilli. con cominantly with contraction, sodium ions are pumped out of the cell thus creating an electro chemical gradient which is necessary for sodium dependent transport of certain metabolites across the microvillous plasma membrane. when reach ing the intracellular space the transported meta bolites are carried further into the cell by a sub membraneous cytoplasmic flow in the microvilli. 9. the high energetic phosphate bond of atp is reutilized several times for sodium pumping when propagated along an actin-containing filament bound to carriers in the plasma membrane, thus considerably increasing the efficiency of the sodiumand potassium-pumping mechanism. 10. the main effect of atp hydrolysis in muscu lar contraction is to supply energy in the form of thermal motion of cytoplasmic solvent molecules. notations l,, l,, hi, and h,: energy content of proteins as defined in fig. 3. the sequence of these denotions indicate the vectorial propagation of a high energy pulse. q: the thermodynamic quantum, that is, the energy differnce (l, l2) rejected as a pres sure pulse from actin-containing filaments in connection with the unidirectional propaga tion of high energy residing in a labile phos phate bond of atp. -q: the energy difference (l,-l,) 0: a sign denoting energy comparable to and exchangeable with the energy residing in a terminal phosphate bond of atp. p: index denoting the energy that resides in pro tein s: index denoting the energy that resides in solu tion (x-1), (x) and (x+ 1): indices occurring after p and s and denoting coordinates in a field om mig rating thermodynamic quanta in the direction of quantized cytoplasmic streaming. +: migration of energy in the direction of quanti zed cytoplasmic streaming (= forward, distal) +: migration of energy in the direction opposite to quantized cytoplasmic streaming (=back ward, proximal) p: proximal part (in the direction of quantized cytoplasmic streaming) of an actin-tropmyo sin subunit. the part of an actin-containing filament located immediately distal to a bind ing site of the filament to the plasma mem brane in brush border microvilli. d : distal part (in the direction of quantized cyto plasmic streaming) of an actin-tropomyosin subunit. the part of an actin-containing fila ment located immediately proximal to a bind ing site of the filament to the plasma mem brane in brush border microvilli. i: the intermediate part of an actin-tropomyosin subunit. c: sodium and potassium carrier molecule lo cated in a biological membrane. (x), (y): indices denoting location of energy or ions. i, 11: sequence of actin-tropomyosin subunits in the migrating thermodynamic quantum hypothesis f o r cell biological phenomena 209 upsala j med sci 81 210 erik cervth the direction of quantized cytoplasmic streaming. (c,na:,,), (c,na:,,): indices denoting asso ciation of ions to the carrier sites when ex posed to the sodium-depleted (int) or the sodium-enriched (ext) side of a membrane. appendix the reactions (6) to (10) are deduced as follows. since h,~c~l2~plr~+ll~c~hz~p~l~ is the final re action, the sequence of transitions of the carrier must be l2+h2+hl-+ll. since adsorption of so dium is an equilibrium reaction h,(c)+hl(c) is either accompanied by adsorption of nati,, or dislocation of na:,,. in the former case ll& associated with na:,,and li(c)+lz(c)or hzli(c)-*l2hz(c) is necessary for its dislocation. l1(c,i.la+e,t) is thus on average thermodynamically unstable. on the contrary, when hz(c)+hi(c) is accompanied by dislocation of na:,t(c), l,(,, is an interior sodium-dissociated form. the thermo dynamically spontaneous transition h2l,(,)-+ l2h2(,-) is then impossible because h2(c) is as sociated with na:,,. on average l1(,, will therefore be unstable increasing the affinity of the carrier for sodium when the carrier sites are exposed to the cell interior. this sequential mode of action of the sodium carrier is illustrated in reactions (6) to (10) because it is compatible with several expe rimental findings and interpretations made by re searchers in this field as given by the following statements (1): 1. a single cation carrier has an absolute requirement for na’ to go to its ‘‘acti vated” form. 2. external atp is not hydrolyzed. 3. the temperature dependent step is neither the phosphorylation nor the dephosphorylation step (reaction (8) and (10)). 4. carriers have inwardly oriented cation sites of high affinity for na’. when a corresponding deduction is made for potassium pumping, one “solution” is rejected because in that case the l, conformation is labile, making potassium accumulation from a potas sium depleted medium improbable. this sequen also improbable according to boltzmanns law, ce, l1(c)+l2(c,natint kiext)+hz(c,natext kqnt)sh1(c)is upsala j med sci 81 s=k i&, which implies that highly ordered states are not favoured. the other solution which is illustrated in the text as the sequence (6b) to (9b) is compatible with inter alia the following experi mental findings and interpretations (1): 1. the cation carrier can return to its inactivated state either as a free carrier or in combination with k+. 2. k + but not na’ decreases labeling with 32p from at32p at low temperatures (re action (6), (6b), (8) and (8b)). in this case k + but not na’ stabilizes l,(,,, and the terminal phos phate bond is looked upon as corresponding to oubain competitively inhibits potassium-bind ing (18, 35) when the carrier sites for this ion are exposed to the external side of the plasma mem brane (1 8) indicating that oubain-binding is in volved in reaction (9b). this is compatible with experimental data indicating that oubain binds to a phosphorylated form of the enzyme (34). hl(dx).)* addendum possible application of the present hypothesis to anabolic phosphorylation of a d p each actin-tropomyosin subunit in an actin-con taining filament is considered to represent a func tional unit attached to a sodium carrier molecule in the plasma membrane. when the concentration of na’,, is high and na:,, is low, the l2(e) and hl(,, conformations are favoured. under these conditions, a high atp/adp ratio will favour the generation of a transmembraneous gradient be cause of imposed unidirectional components for transitions of the carrier as illustrated by the on the contrary, if na’,,, is high and na’,, is low, the h1(,, and l,(,, conformations will be stabilized. under these conditions, a low atp/adp ratio will favour the reverse action of the carrier as illustrated by another sequence of transitions, l~,,, liil(c, nt h , ( ~ , -+ l,(,, c l ~ ( ~ ) . the actin-tropomyosin subunit may thus oe re garded as a coupling factor (cj: is), the main function of which could be to bind adp and ortophosphate in an optimal spatial relationship atp sequence l,(,, + k ( c ) z h l ( c ) + l x , l 2 ( c ) . a d p + p , ’ the migrating thermodynamic quantum hypothesis f o r cell biological phenomena 2 1 1 to the carrier. when the carrier associated with a coupling factor is operating in reverse due to a high transmembraneous gradient of the trans ported ions, atp will be formed from adp and ortophosphate. these functional considerations support the idea derived from structural similari ties (1 6 ) that an actin-tropomyosin subunit is equivalent to the coupling factors of mitochondria (and chloroplasts). in both cases atp is formed simultaneousely with the reflux of ions participat ing in a transmembraneous gradient. these ions are evidently sodium (and potassium) in the plasma membrane and protons in the mito chondrial membrane (cj: chemiosmotic hypo thesis for oxidative phosphorylation, 18). in addition to the already established photo phosphorylation and the oxidative phosphoryla tion of a d p the present hypothesis is theoreti cally compatible with a third mechanism for generating atp. this mechanism which may be denoted thermodynamic phosphorylation relates to the well-known tendency of actin filaments to depolymerize at high potassium concentrations (cj: 6 ) . thermodynamic phosphorylation implies that potassium is accumulated and sodium pumped out of a cell by means of a high energy pulse propagating along an actin filament. when a critical potassium concentration is reached the actin filaments are enzymatically depolymerized to single actin-tropomyosin subunits, still at tached to the plasma membrane. the sodium potassium gradient is then used for generation of atp by the reverse action of the carrier mole cules with their associated actin-tropomyosin coupling factors. at the critical potassium con centration both polymerized and depolymerized actin-tropomyosin filaments may coexist and these mechanisms may be operating simultane ously. this type of phosphorylation may be phylogenetically older than photophosphorylation and oxidative phosphorylation. the above considerations may be applicable to the phenomena that tumor cells can survive in a low oxygen environment practically devoid of fuel for conventional generation of atp (36) and that fibroblasts exposed to a low oxygen tension begin to move about (d: 12, 36). acknowledgement this work was supported by grants from the swedish medical research council (project b77-13x-228 13b), the university of uppsala and a personal grant from uddeholms ab. i would like to thank drs. gunnar agren and gunnar ronquist for generous support and valuable criticizm. references 1. 2. 3. 4. 5 . 6. 7. 8. 9. 10. 11. 12. albers, r.w.: biochemical aspects of active transport. i n ann rev biochem. vol 36 part i1 (ed. p.d. boyer), pp 727-756 ann rev inc, palo alto, california, 1967. bennett, h.s.: the structure of striated muscle as seen by the electron microscope. i n the struc ture and function of muscle, volume i (ed. g.h. bourne)p 150, academic press new york and london, 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science 123: 309-314, 1956. received november 15, i976 address for reprints: e. cerven institute of medical and physiological chemistry biomedical center university of uppsala box 575 s-751 23 uppsala sweden upsala journal of medical sciences 2022, 127, e8913 http://dx.doi.org/10.48101/ujms.v127.8913 tissue-based diagnosis of systemic amyloidosis: experience of the informal diagnostic center at uppsala university hospital justina damjanovic vesterlunda,b, elisabet ihsea,b, ulrika thelandera,b, alice zancanarob, gunilla t. westermarkc and per westermarka,b aclinical pathology, uppsala university hospital; bdepartment of genetics, immunology and pathology; cdepartment of medical cell biology, uppsala university, uppsala, sweden abstract diagnosis of systemic amyloidosis is a clinical challenge and usually relies on a tissue biopsy. we have developed diagnostic methods based on the presence of amyloid deposits in abdominal subcutaneous fat tissue. this tissue is also used to determine the biochemical type of amyloidosis, performed by western blot and immunohistochemical analyses with the aid of in-house developed rabbit antisera and mouse monoclonal antibodies. mass spectrometric methods are under development for selected cases. the diagnostic outcome for 2018-2020 was studied. during this period, we obtained 1,562 biopsies, of which 1,397 were unfixed subcutaneous fat tissue with varying degrees of suspicion of systemic amyloidosis. of these, 440 contained amyloid deposits. the biochemical nature of the amyloid was determined by western blot analysis in 319 specimens and by immunohistochemistry in further 51 cases. article history received 21 july 2022 revised 17 august 2022 accepted 18 august 2022 published 27 september 2022 keywords amyloidosis; systemic; monoclonal antibodies; subcutaneous fat tissue; biopsy introduction systemic amyloidoses are generally life-threatening diseases, earlier believed to be very rare but are now increasingly often diagnosed. several therapeutic drugs have been developed during the last years, and the interest in the disorders is steadily growing. amyloid is characterized by misfolding of proteins that aggregate into cross β-sheet fibrils (1). in humans, such fibrillar deposits are mainly extracellular, exerting detrimental effects on cells, which may undergo apoptosis or necrosis. close to 40 different human proteins have been identified as amyloid forming in vivo, and more are expected to be discovered (2). the proteins are usually small (below 200-300 amino acid residues long) and vary substantially in their native folding. despite this, irrespective of biochemical origin, the fibrils are very similar in molecular arrangement, appearance, and properties. amyloid can be localized or systemic. the amyloidogenic protein in the systemic forms, where deposits appear in many different organs, is synthesized by one or a few cell types and transported in a native or near-native form via the bloodstream to a target place, where the typical fibrils form. there is an increasing understanding of how a single protein develops into a fibril. a missense mutation is a common cause of misfolding, characteristic of the many hereditary types of amyloidosis. however, a mutation is not a prerequisite, and wild-type proteins can also be amyloidogenic. the spreading of amyloid likely depends on seeding, by which a misfolded protein or protein aggregate recruits molecules of the same kind, catalyzes their misfolding, and propagates the formation of fibrils (3). presently, 17 different proteins are known to give rise to systemic amyloidoses (2). three biochemically different systemic amyloidoses are predominating. al amyloidosis, where the fibril protein is derived from a monoclonal immunoglobulin light chain, depends on a plasma cell clone, often small and usually in the bone marrow. the fibril protein in aa amyloidosis is a large n-terminal fragment of the acute phase protein serum amyloid a (saa), expressed by the liver at acute or chronic inflammations. the third of the most prevalent systemic amyloidoses is of a transthyretin (ttr) nature. plasma ttr, mainly synthesized by the liver, forms the fibrils in most hereditary forms of amyloidosis, but wild-type ttr is also amyloidogenic and is the fibril protein in the age-associated wild-type (attrwt) amyloidosis, earlier called senile systemic amyloidosis (4). symptoms from systemic amyloidoses vary considerably in all biochemical forms. in many types of the disease, polyneuropathy, cardiomyopathy, cardiac arrhythmia, or renal problems are common, but virtually all organs may be affected in various combinations. this variability, in combination with the relative rarity of systemic amyloidosis, makes the diagnosis difficult (5), and many patients meet several doctors before a diagnosis can be established (6). coming to a definite diagnosis contact per westermark per.westermark@igp.uu.se © 2022 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article http://dx.doi.org/10.48101/ujms.v127.8913 mailto:per.westermark@igp.uu.se mailto:per.westermark@igp.uu.se http://creativecommons.org/licenses/by/4.0/ 2 j.d. vesterlund et al. can take considerable time, which is unfortunate since late diagnosis is associated with less successful treatment results. the problem is particularly severe in al amyloidosis. for more than 20 years, we have built up a diagnostic facility center at uppsala university hospital. the present paper describes the procedures and their background used at our laboratory in uppsala. we report on some trends seen by our biopsy material over the years and analyze the results more in detail for the latest 3-year period for which all data are available. the study was conducted in agreement with the declaration of helsinki and does not need an ethical approval according to the swedish ethics review authority (dnr. 2021-06773-01). tissue-based diagnosis of amyloidosis in uppsala development of subcutaneous abdominal fat biopsy for diagnosis of amyloidosis when comprehensive studies on systemic amyloidosis started, the aa variant dominated in sweden and other european countries, and this disease most often followed rheumatic diseases. the interest in amyloidosis was low, and aa amyloidosis was often missed clinically, even at autopsy (7). a finding that aa amyloid deposits usually occur around fat cells in the subcutaneous tissue (8), initially described in 1909 (9) but later denied (10), prompted us to study whether fine-needle aspiration biopsy could be a method to obtain a correct diagnosis of amyloidosis. since the result was successful in the single studied patient (11), we extended the study to more patients and showed that the method is a risk-free, reliable, and straightforward alternative to rectal biopsy, usually used at that time (12, 13). fine-needle biopsy of abdominal fat tissue later became the most common initial method to diagnose systemic amyloidosis worldwide (14-16). while fine needle aspiration biopsy of subcutaneous fat tissue is an excellent method to diagnose aa and al amyloidosis, it is less successful for the attr forms, particularly wild-type (wt). one reason is that the amount of attr amyloid in fat tissue is often sparse. however, an even more important explanation for the results with fine needle aspiration is that the attr deposits are associated with collagen that is not easily included in the aspirated material. for this reason, we have largely discontinued the use of fine needle aspiration biopsy from subcutaneous tissue for amyloidosis diagnosis. we now recommend a slightly more invasive method for this tissue. we first introduced a small surgical biopsy (about 1 cm3) (17). further development has been the introduction of punch biopsies, including subcutaneous tissue (18). demonstration of small amyloid deposits in sections of fat tissue is often difficult. we, therefore, introduced squeeze preparations of unfixed tissue, which has made the identification of deposits much more successful (19, 20). one further advantage of this method is that some types of amyloid, e.g. attrwt, stain better with congo red when not going through formalin fixation and paraffin embedding steps. minimal amyloid deposits can be identified in squeeze preparations (figure 1). determination of amyloid type the specific treatments for amyloidosis require the determination of the exact type of disease. consequently, there is a big need for safe methods to diagnose amyloidosis and determine its type. these demands have resulted in the more or less independent development of different methods at several specialized laboratories in the world (21). variants of immunohistochemistry (ihc) and, later, mass spectrometry (ms) are most commonly used. fine needle biopsy of abdominal fat tissue can be used for the determination of the biochemical type of amyloidosis. the first example of this was probably performed in uppsala in 1979, when double immunodiffusion was utilized to identify protein aa in micro-extracts of subcutaneous adipose tissue (22). some laboratories still use subcutaneous fat tissue obtained by fine needle aspiration for typing, but now with a modern, mass spectrometric technique (23, 24). immunological methods used in uppsala development of specific antibodies it is a general experience that most commercial antibodies are unsuitable for clinical diagnostic work of amyloidosis (21, 25, 26). one reason for this failure is that such antibodies usually are raised against normally folded proteins. therefore, it is necessary to develop antibodies that react not only with natively folded proteins but also with their amyloid fibril form. for polyclonal antibodies, this can be done by using partly degraded and thereby solubilized fibrils as immunogens (27, 28) or by raising antibodies against short peptides corresponding to a part of the protein incorporated in the amyloid (29). recombinant protein may also be used. the antibodies we developed for amyloid typing have been raised against various antigens (table 1). initially, we developed rabbit antisera, of which we are still using some but we are trying to replace these gradually with mouse monoclonal antibodies (mabs), the first being anti-protein aa mab sne5 from 1995 (unpublished) and the latest anti-al kappa figure 1. abdominal fat tissue fragment in a squeeze preparation containing a very small attr particle (arrow), stained with congo red. bar = 50 μm. tissue-based diagnosis of systemic amyloidosis 3 mab pwkap (to be published). the most important reason to leave polyclonal antibodies is the indefinite availability and the uniform properties of mabs. western blot analysis as described above, we obtain unfixed fat tissue specimens, and this has a historical explanation. our earliest trials to establish the type of systemic amyloidosis were by double immunodiffusion or by amino acid sequence analysis (30). later, after attempts with enzyme-linked immunosorbent assay (elisa) (17), we adopted sodium dodecylsulfate-polyacrylamide gel electrophoresis (sds-page) with western blot analyses of extracts of subcutaneous fat tissue (19, 20), which we found to be a more sensitive method. this is still the method that we use for all adipose samples, except for those with very small amyloid deposits. for routine clinical analyses, we use the antisera 1898, a126, a147, and the mabs pwlam and pwkap (table 1), always with known control materials that often give characteristic band patterns. the antisera 1898 and a126 react with all attr and aa materials, respectively, if there is enough amyloid material in the fat tissue (≥score 1-1.5 on a scale of 0-4). a147, directed against a linear epitope at the beginning of the constant region of al kappa, labels at least most cases of this type. the mab pwlam reacts with a not fully identified epitope at the junction between the variable and constant region in al lambda proteins. this antibody has been validated by ihc, showing 50% sensitivity and 100% specificity for al lambda amyloid (31). the sensitivity by western blot analysis is higher in our experience, although no exact figure has been determined. in addition to these antibodies, we have generated other rabbit antisera, not reported here, which we use for unreactive materials. immunohistochemistry (ihc) ihc has been and still is the most common method to determine the type of amyloidosis in most laboratories. ihc is used both on sections of formalin-fixed and paraffin-embedded materials and of frozen sections, the latter particularly in renal pathology. we also use ihc for adipose tissue samples, particularly for attrwt materials. the subcutaneous amount of amyloid is often very low in this amyloid type, and it is common to find only one or very few but usually distinctive deposits in the squeeze preparations. western blot or mass spectrometry are not helpful for such biopsies. removing the cover glass followed by ihc with our monoclonal attr antibody 7x often solves the problem. like other clinical pathology departments working with amyloidosis, we receive paraffin blocks of tissue with identified amyloid deposits to determine the biochemical type. usually, there is a substantial amount of amyloid in such specimens. we use traditional ihc but with our antibodies. aa and attr cases offer no problems. approximately 75% of al cases can be diagnosed, and with the recent development of an al kappa mab pwkap, we expect the number of undiagnosed cases to decrease. mass spectrometry mass spectrometry for the determination of amyloid type was pioneered by researchers at the mayo clinic and is now an established method in many laboratories (32, 33). the technique is most commonly based on formalin-fixed and paraffinembedded materials, from which amyloid is dissected with laser dissection microscopy (ldm). after solubilization, the crude material is enzymatically fragmented into peptides, usually with trypsin which cleaves after the basic amino acid residues arginine and lysine. the obtained ms data are after that analyzed by proteomics. ms can also be used for non-fixed amyloid samples. particularly, fat tissue obtained by fine needle aspiration is used for amyloid type determination in some laboratories, as mentioned above. most laboratories using ihc for type determination turn to ms as a next option when ihc results are uncertain. since we receive surgically or punch adipose tissue biopsies, we have the opportunity to directly extract these for ms. we are presently developing a method with initial sds-page followed by the analysis of low molecular protein bands in order to obtain a concentration of pertinent proteins. trends in uppsala biopsy materials 2006-2021 since our organized amyloid clinical work started, the number of obtained biopsies has increased steadily (figure 2). fresh fat tissue biopsies constitute the majority of them. for the three table 1. rabbit antisera and mouse monoclonal antibodies developed for clinical typing of human systemic amyloidoses antibody fibril protein immunogene antibody type antibody used in reference sne5 aa purified protein aa m-mab ihc unpublished a126 aa peptide pc, rabbit wb (17) a147 al, κ peptide pc, rabbit wb (17) pwkap al, κ dam m-mab ihc, wb unpublished pwlam al, λ dam m-mab ihc, wb (55) 1898 attr recombinant ttr50-127 pc, rabbit ihc, wb (56) 7x attr dam m-mab ihc, wb (57) a82/01 aapoaiv peptide pc, rabbit ihc (56) a159 aapoai peptide pc, rabbit ihc (58) dam: alkali-degraded amyloid; pc: polyclonal; m-mab: mouse monoclonal antibody; ihc: immunohistochemistry; wb: western blot. 4 j.d. vesterlund et al. years 2018-2020, 1,397 samples (89.4%) were from subcutaneous fat tissue, obtained either surgically or with a punch. with a few exceptions, the biopsies were taken without a previous proven diagnosis of amyloidosis. the remaining 165 biopsies contained formalin-fixed and paraffin-embedded materials, diagnosed with amyloidosis elsewhere and sent to us to determine the type. these materials varied in nature and included endomyocardial, renal, liver, skin, and many other kinds of specimens. subcutaneous fat tissue biopsies we have developed a unique principle way to diagnose systemic amyloidosis based on fat tissue biopsies, and the results for the three-year period (2018-2020) are shown in table 2. about one-third of them (440) contained amyloid. the amount of amyloid varied, ranging from a situation with almost no normal tissue structures remaining to very tiny deposits. western blot analysis revealed the fibril protein in 72.5% of the cases. nineteen further biopsies were rich in amyloid but not possible to type with our antibodies. the remaining 102 biopsies contained small amounts of amyloid, often only as one or a few but distinct deposits. for further characterization of these materials, we applied ihc and used the original squeezeprepared slides. since the deposits often were minimal and could be difficult to find again (figure 1), their coordinates in the microscope were noted before removing the cover glass. the slide was then taken back to water before ihc was performed in the usual way. in this way, it was possible to identify attr as the amyloid fibril protein in 51 cases (figure 3). in 51 other cases which were highly suspected to be of attr nature, this method did not give a convincing result due to the minimal amount of amyloid, often only one dot-like deposit, a common finding in attr amyloidosis. type a and type b attr amyloidosis. the attr antiserum 1898 was raised against recombinant ttr50-127. in contrast to a commercially available product, this antiserum reacts with both figure 2. the number of biopsies obtained for amyloid diagnosis at the uppsala laboratory for 2002-2020. there was a relatively even increase until 2015, with a more substantial increase during the last years. table 2. tissue biopsies for amyloid diagnosis 2018-2020 total number of biopsies 1,562 abdominal fat biopsies (unfixed) 1,397 abdominal fat biopsies with amyloid 440 (31.5%) type determined (western) 319 (72.5%) type determined (ihc) 51 (11.6%) total number typed 370 (84.1%) type not determined 70 (15.9%) too little material 51* not classified amyloid type 19 *almost all had microscopic appearance of attr amyloid and a disease history supporting attrwt. figure 3. a sharply delined amyloid particle in a subcutaneous fat tissue biopsy. the amount of amyloid (arrow) was too sparse to analyze by western blot. therefore, the congo red stained squeeze preparation was subjected to ihc with our anti-attr antibody 7x, resulting in evident labeling of the deposit. insert: the same particle at higher magnification, visualized in polarized light. green-red-yellow birefringence due to the remaining congo red staining is obvious. immunolabeling was shown with 3,3’-diaminobenzidine. bar = 50μm. tissue-based diagnosis of systemic amyloidosis 5 full-length and fragmented attr species (34, 35). by western blot analysis, we are able method, we are able to distinguish between attrv type a and type b fibrils (figure 4). patients with these variants behave like having two different disorders (36). most importantly, patients with type a disease incorporate more wt ttr in their amyloid fibrils, often leading to progressive amyloid cardiomyopathy (37, 38), which is associated with shorter survival than patients with type b fibrils (36). the appearance of the two patterns is particularly seen in patients with the ttrv30m mutations and is very rare in other variants. however, pattern a is by far the most common in other mutations than ttrv30m (39) and is the only one seen in attrwt amyloidosis (unpublished result). we have tried to determine the attr fibril type in all fat pad biopsies with at least a moderate (1-1.5 + on a 0-4+ scale) amount of amyloid and were able to do so in 146 biopsies. while there were no patients with type a fibrils below the age of 50, the age spectrum among the type b patients was remarkably wide (mean age 53.7, range 28-88), with nine patients over 70 years and one as old as 88 years. proportions of the three major systemic amyloidoses. al amyloidosis was the most prevalent type in our material, as in reports of most other amyloid laboratories. notably, the amount of amyloid in al materials was always moderate (2+) to very rich (4+). the rarity of aa amyloidosis with only 4 biopsies from patients with this type is in accordance with the findings in other western countries (40). additional biopsy materials in addition to the biopsies from subcutaneous adipose tissue, we received 165 materials, most formalin-fixed and paraffinembedded, where amyloid had been found at other laboratories and sent to us for typing by ihc. these are not reported in detail here, but one notable aspect has come up repeatedly: a question regarding localized vs. systemic amyloidosis. in almost all cases, fat tissue biopsies were taken due to suspicion of systemic amyloidosis. however, the question of a localized form arose in some situations. most biopsies from localized amyloidoses were received as formalin-fixed and paraffin-embedded tissue blocks. insulin amyloid. five cases of iatrogenic localized, insulinderived (ains) amyloid were identified in our 3-year material. there was no suspicion of this amyloid type in any of the patients, and four materials were obtained paraffin-embedded after that amyloid unexpectedly had been discovered in other pathology departments, while the fifth subcutaneous material was received unfixed and contained large amounts of amyloid. amyloid formation is not a rare effect of repeated subcutaneous injections of insulin at one site. for unknown reasons, insulin is then misfolded and deposited as fibrillary aggregates instead of being released into the circulation (41, 42). the deposits create one or several nodules containing heavy amyloid deposits and are strictly local except for one published case, in whom ains amyloid was identified in draining lymph nodes (43). if the phenomenon is not known to the clinician or pathologist, the deposits may be misdiagnosed as a sign of systemic amyloidosis. analysis of the amyloid with immunological or mass spectrometric methods elucidates the nature of the material. the formation of insulin-derived amyloid is likely an underappreciated event and may cause blood sugar control problems (44). al amyloidosis occurs both as localized and systemic disorders, and the distinction is essential since while the systemic form is life-threatening, the localized is usually not. localized al amyloidosis develops at a site of clonal expansion of plasma cells secreting an amyloidogenic immunoglobulin light chain that forms fibrils close to the production site. localized al amyloidosis rarely develops into a systemic disease (45). localized al amyloid does often, but not always, have a characteristic microscopic appearance with bright birefringent deposits, the presence of groups of plasma cells, and giant cells that may cover a surface of single amyloid particles (figure 5) (46). even at light microscopy, the deposits can look fibrillary and organized. a number of localized al amyloidosis specimens are sent to us each year from other pathology laboratories for type-determination. discussion systemic amyloidoses are all comparably rare and often diagnosed several years after the onset of the disease. a correct diagnosis, including the amyloid type, is required for proper treatment. treatment options for the different variants have figure 4. typical findings at western blot analysis of attr amyloid containing fat tissue biopsies demonstrating ttr monomers (m) and dimers (d) as well as ttr fragments (fr). type a, with a mixture of full-length and fragments of ttr, seen to the right, is found in most mutated forms as well as in wild-type attr amyloidosis. type b to the left is found in many patients with attrv30m amyloidosis. 6 j.d. vesterlund et al. emerged rapidly during the last years (47, 48), and the demands for an accurate diagnosis have significantly increased. the clinical diagnosis of amyloidosis is still based on tissue biopsy. amyloid is often easy to recognize microscopically, but there are many pitfalls. the usual way is to stain tissue material with the dye congo red, introduced in amyloid diagnostics one hundred years ago, nowadays in combination with polarization microscopy. although principally simple, the staining procedure has to be strictly controlled since other tissue components can look like amyloid in overstained materials. experience is necessary, and the concentration of amyloid diagnostics to a limited number of examiners is, therefore, important. this is even more apparent regarding the type-determination of amyloid deposits. to overcome the difficulties in diagnosing systemic amyloidosis and develop the most efficient treatment procedures for the different variants, referral centers for systemic amyloidosis have been established in many countries, particularly in europe, usa, japan, and australia. in sweden, a center for caring for patients with attrv amyloidosis was set up in umeå decades ago. patients with all other types of systemic amyloidosis have been investigated and treated at local hospitals, usually by hematologists and cardiologists. our laboratory has acted as an informal swedish referral center for biopsy diagnosis of amyloidosis. the swedish national board of health and welfare has recently decided to create up to four national highly specialized health care units for systemic amyloidosis, of which two should be able to perform advanced tissue-based diagnostic work (49). a variety of diagnostic principles have been developed, each with advantages and drawbacks. attrwt amyloidosis offers particular difficulties. the deposits in peripheral tissue, such as in a subcutaneous fat pad, can be minute and very difficult to identify, and determining the amyloid type of such small particles is a challenge, well illustrated in our material. ihc solved the nature in many cases, but for the remaining patients in whom endomyocardial biopsy was unsuitable, the attr diagnosis had to rely on the typically sparse deposits in subcutaneous fat tissue combined with clinical methods, including imaging. this should also be true for patients with negative fat tissue biopsy but with a strong suspicion of attrwt amyloidosis. the difficulties with attrwt amyloidosis have made a cardiac scintigraphy method attractive. this method is based on the affinity for skeletal markers to attr amyloid-containing hearts.  in europe, [99mtc]tc-labeled 3,3-disphos-phono-1,2propanodicarboxylic acid (dpd) is most commonly used. the method is fairly specific for attr amyloidosis when al disease has been excluded. however, the affinity of dpd is not due to the amyloid itself, as was originally believed but depends on irregularly distributed dense clouds of microcalcifications (50). therefore, dpd scintigraphy is a surrogate method rather than a way to truly demonstrate amyloid. this fact is particularly important to know when trying to show therapeutic effects on the degree of heart involvement. the field of systemic amyloidosis is developing rapidly, and treatment has become more individualized. it is probable that not only the biochemical type will have to be determined in the future but also subtypes, as is already the case with attrv30m amyloidosis. a reasonable subject might be al amyloidosis, in which the amyloid protein originates from one out of around 50 possible immunoglobulin light chain genes. there are indications that certain gene products are associated with depositions in specific organs (51). since al proteins are cleaved and mainly consist of the variable region and a usually short part of the constant segment, fragmentation may also be important in amyloid fibril protein properties (52, 53). there are other, even more, rare forms of systemic amyloidosis, some hereditary due to mis-sense mutations and some of wildtype nature. so far, we have not seen such examples in our recent material. however, the first aapoaiv case described was found in our material (54), and more unexpected cases will probably turn up. in summary, immunological methods to determine the type of amyloidosis are safe and cheap but demand experience and specific antibodies that are not commercially available. for some materials, mass spectrometry is necessary. there are also cases with very little amyloid in the subcutaneous fat tissue for which other methods are of value, particularly for attrwt amyloidosis. disclosure statement the authors have no conflicts of interest to declare that are relevant to the content of this article. funding the study was supported by the patient organizations famy västerbotten, famy norrbotten and amyl. figure 5. localized al amyloidosis of immunoglobulin light chain lambda origin. a sharply delimited immunolabeled amyloid particle (am) is surrounded by multinuclear giant cells (black arrow) that are often difficult to recognize. groups of immunolabeled lambda-light chain plasma cells (red arrow) are evident. these characteristics make it possible to suggest that the process is localized and not part of systemic disease. anti-al antibody pwlam binding visualized with 3,3’-diaminobenzidine. bar = 20 μm. tissue-based diagnosis of systemic amyloidosis 7 notes on contributors justina damjanovic vesterlund, msc, research engineer, unit of clinical pathology, department of laboratory medicine, uppsala university hospital. elisabet ihse, phd, researcher, department of immunology, genetics and pathology, uppsala university, and molecular biologist, unit of clinical pathology, department of laboratory medicine, uppsala university hospital. ulrika thelander, md, specialist physician of pathology, unit of clinical pathology, department of laboratory medicine, uppsala university hospital and phd student, uppsala university. alice zancanaro, msc, phd student, uppsala university. gunilla t. westermark, phd, professor of medical cell biology, department of medical cell biology, uppsala university. per westermark, md, phd, emeritus professor of pathology, uppsala university and consultant pathologist, unit of clinical pathology, department of laboratory medicine, uppsala university hospital. orcid elisabet ihse https://orcid.org/0000-0001-7648-3246 ulrika thelander https://orcid.org/0000-0002-6647-0967 alice zancanaro https://orcid.org/0000-0002-8362-6572 gunilla t. westermark https://orcid.org/0000-0003-1151-9986 per westermark 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amyloidosis. thesis, uppsala university. 2004. 57. westermark gt, ihse e, westermark p. development of mouse monoclonal antibodies against human amyloid fibril proteins for diagnostic and research purposes. methods mol biol. 2018;1779:401–14. 58. mucchiano gi, häggqvist b, sletten k, westermark p. apolipoprotein a-1-derived amyloid in atherosclerotic plaques of the human aorta. jpathol. 2001;193(2):270–5. http://dx.doi.org/10.1002/ajh.25915 http://dx.doi.org/10.1080/13506129.2022.2052838 http://dx.doi.org/10.1080/13506129.2022.2052838 http://dx.doi.org/10.1080/13506129.2022.2093635 https://www.socialstyrelsen.se/kunskapsstod-och-regler/regler-och-riktlinjer/nationell-hogspecialiserad-vard/oversikt/systemisk-amyloidos/ https://www.socialstyrelsen.se/kunskapsstod-och-regler/regler-och-riktlinjer/nationell-hogspecialiserad-vard/oversikt/systemisk-amyloidos/ https://www.socialstyrelsen.se/kunskapsstod-och-regler/regler-och-riktlinjer/nationell-hogspecialiserad-vard/oversikt/systemisk-amyloidos/ http://dx.doi.org/10.1080/13506129.2022.2095618 http://dx.doi.org/10.1080/13506129.2022.2095618 upsala j med sci 98: 443-444, 1993 9. conclusions carl-henric de verdier,' torgny groth2 and per hyltoft petersen3 'department of clinical chemistry, and 2unitfor biomedical systems analysis, university of uppsala, uppsala, sweden and 'department of clinical chemistry, odense university hospital, so00 odense, denmark in all clinical quality work the first steps ought to be to define the clinical goals and the quality specifications. in order to be meaningful they ought to be expressed as total allowable errors (tea ) in quantitative terms. this project deals mainly with anazytical goals and specifications. three types of approaches have been used: 1) the state of the art approach 2) the biological approach 3) the clinical usefulness approach. the state of the art approach aimes primarily at comparison with other laboratories. the biological approach gives easily available and more general goals/specifications. clinical investigations are used in clinical work and it should thus be most meaningful to try to use the third alternative. guidelines for this approach are, however, not generally available and we have in this report tried to indicate different ways to obtain goals/specifications following this approach. statistical, graphical and computer methodologies have been discussed and applied. terminology is important for understanding and for transfer of information. this nordkem project report provides suggestions for terms and abbreviations and stresses that a universally accepted terminology within this area is highly needed. twentytwo associated nordic projects are accounted for in the project report. they have brought up important questions for discussion. two chapters deal with special topics. one, originating from the nordic protein project, describes the quality necessary for sharing common reference intervals for 9 specific proteins in the nordic countries. the other, originating from a "transferability project" 17-935253 443 , describes how 'long term bias' in the different laboratories can be assessed and serve as the prerequisite for making corrections. analytical quality specifications (aqspecs) should be provided by the clinical laboratories and the report gives many examples of how they can be estimated. correspondence: carl-henric de verdier, m.d., professor department of clinical chemistry university hospital s-751 85 uppsala, sweden 444 upsala j med sci 84: 3 7 4 6 , 1979 evaluation of a computer programme for interpretation of 12-lead electrocardiograms johan landelius and lars nordgren f r o m the department o j clinical physialogy, university hospital, uppsula, s w e d e n abstract a 12-lead electrocardiogram (ecg) interpretation programme (cardionics, brussels) was tested for computer diagnosis of ecg. the computer performance was evaluated on an ecg population from hospital patients by a method giving results which will allow the clinician to judge the usefulness of the computer diagnosis in clinical practice. diagnoses made by experienced ecg readers were in essential agreement with the computer diagnoses in 83.5 % of 4 9 3 ecgs. cli nically significant disagreements due to differences in criteria occurred in 6.0 % of the tracings,whereas such disagreements due to programme errors were found in 10.5 %. introduction several computer programmes for interpretation of electrocardiograms (ecg) are available today. evaluation of such programmes is time-consuming and in some aspects difficult, especially concerning their performance in the clini cal situation. firstly it is essential to define the composition of the tested population of ecgs in terms of the prevalence of certain abnormalities. the ideal would be to have access to an archive of ecgs accepted generally by in vestigators and including ecgs with abnormalities due to diseases of a known type and at a known stage, as well as ecgs from normal subjects. the perfor mance of a computer programme could then be determined in relation to a par ticular ecg abnormality, to a certain disease, and to a normal as well as a mixed ecg population. secondly, it is important to analyse disagreements between the computer diagnosis and the correct diagnosis, that is the diagnosis made by manual in terpretation, and to classify them into those due to differences in criteria and those due to programme errors. the latter will consist either of mismea surements or of deficient programme logic. a s pointed out by bailey et al. (11, criteria differences do not indicate any technical deficiency in the programme. 37 thirdly, it is also necessary to consider the possibility of human reader errors and failures in the recording quality in studies of this kind. material and methods a material of 510 unselected ecgs was obtained at random from patients sent to our department from different wards and outpatient units of the university hospital in uppsala. the tracings were collected from consecutive cases exami ned in one of four routine ecg stations between june and september, 1974. the tracings represent a hospital population and many of the patients had cardio pulmonary diseases. each recording consisted of the standard 12-lead system (i, 11, 111, avr, a m , avf, v -v ) and the orthogonal vectorcardiographic leads x, y and z as described by frank (2). 1 6 a cardionics cart was used for the recordings, which were made both on a strip chart and on high fidelity fm analogue tape. the tape recordings were sent to cardionics, brussels,where they were digitized and computerized by the mount sinai hospital programme (1973), whereafter the computer printout of each ecg diagnosis as well as a d-a-converted printout of the same ecg tracing we re returned to our department. six ecgs had to be excluded two of them were lost for unknown reasons and four were lost in the technical process. of the 504 ecgs remaining for further analysis, 10 were excluded because of faulty lead connections, as discussed later, and one was discarded because it could not be classified. this left 493 ecgs, obtained from 4 9 3 patients (281 male and 212 female). the age range of the men was 19 to 85 years (mean 58; s.d. 14) and of the women 17 to 89 years (mean 54; s.d. 17). each ecg was interpreted in detail by the authors, neither of whom was aware of the computer interpretation or of the interpretation of the other reader at the time of the first reading. the two manual interpretations of each ecg were then combined by the two readers to fit a set of ecg criteria common to the department and in agreement with international rules (3). this combined "depart mental" interpretation is referred to in the following as the "reader diagno sis''. the individual reader error appeared to be small but was not further ana lysed. the vectorcardiographic leads were not considered by the readers in their manual interpretation but were used by the computer programme, mainly to deter mine loop areas and vector angles. the reader diagnosis was then compared with the diagnosis produced by the computer. the comparison was made essentially along the lines proposed by bai ley et al. (1). their definitions for agreement and disagreement were modified as follows. 1. reader-computer agreement. "agreement" was defined as an identical interpretation by the reader and the computer (group a). no further analysis was performed. 38 2. reader-computer disagreement. trdisagreement" was divided into (a) minor disagreement importance (group b); (b) major disagreement probably of clinical importance (group c); and (c) major disagreement definitely of clinical importance (group probably of no clinical d) * in each of groups b, c and d, the disagreements were classified into those due to criteria differences and those due to programme error. the complete set of criteria used by the computer programme was available. most of the abnormal ecgs in this study contained multiple abnormalities. in groups c and d, only those diagnostic statements which carried clinical significance were noted and submitted to statistical analysis. as mentioned above, 10 tracings had faulty lead connections. this was done intentionally by the clinical technologists in order to test the computer. in seven of these, two precordial leads had been exchanged and as a result the computer gave a false statement of anterior myo cardial infarction. in the remaining three, extremity leads had been exchanged. two of these were correctly identified as "lead wire inversion" but in the third case the computer report was: "unusual electrical axis, compatible with left ventricular hypertrophy". thus the programme seemed to have difficulty in cases of faulty lead connections. this emphasizes the need for correct recor ding of the ecg, and also calls for supplementation of the programme logic. statistics the following formulas were used, as proposed by rautaharju et al. ( 4 ) . a=true positives, b=false positives, c=false negatives, d=true negatives sensitivity (se) = 100 a/(a+c), specificity (sp) = 100 d/(b+d), accuracy of positive tests (ap) = a/(a+b) , accuracy of negative tests (an) = d/(c+d), n error ratio = (b+c)/a, over-all diagnostic accuracy (da) = p(1) se(1) 1=1 n = number of diagnostic test categories (i) p(1) = fraction of statements in category (i) se(1) = fraction of correctly diagnosed statements in category (i) none of these indices gives a fully representative and relevant picture of dis agreement and failure. the basic limitation is that all events are classified as of equal importance, the more advanced concepts of statistical decision theory being disregarded. however, a more thorough discussion of statistical principles is beyond the aim of this study. results out of 4 9 3 ecg tracings, the reader diagnosis in 2 3 4 ecgs comprised one or 39 more clinically significant abnormalities ( 4 7 . 5 % abnormal and 5 2 . 5 z normal). the exact number of ecgs in each kind of reader statement is given in the fi' gures. the results are expressed in bar graphs. the first bar in each figure shows the sensitivity of the computer diagnosis in relation to the reader diag nosis, and the third bar shows the specificity. the total number of diagnoses for each graph was 4 9 3 , except for the graphs of myocardial infarction and arrhythmia, where the totals were 5 0 6 and 5 2 9 respectively, due to the occur rence of more than one statement for a given ecg. in the final statistical analysis, groups a and b were both regarded as agreements, and groups c and d as disagreements. left ventricular neg hypertrophy (lvh) for lvh by readers by program by readers 7 8 92.2 :.t ?5.9 0.5 99 5 myocardial inlorction neg (mi) tolol tor mi by readers by program by readers 5 3 i6 0 w.7 19 8 6 3 h program ermrs criteria differences program ermrs criteria differences 0 agreements 0 agreements fig. 1. left ventricular hypertrophy (lvh). in each bar graph the number (n=) written above the first bar represents the total number of times the readers made the diagnostic statement listed. the number (n=) over the middle bar re presents the total number of times the programme made the diagnostic statement in question. the number (n=) over the third bar represents the total number of tracings in which the readers did not make the diagnostic statement under con sideration. each of the three bars may be subdivided into three segments. the bottom segment (no lines) indicates the per cent of cases in which the readers were in agreement with the computer programme. the middle segment (oblique li nes) indicates the per cent of cases in which the readers and the programme disagreed due to criteria differences. the top segment (horizontal lines) indi cates the percentage of cases in which disagreements between readers and com puter programme resulted from programme errors (in this figure, none). fig. 2. myocardial infarction. explanation, see fig. 1. left ventricular hypertrophy (lvh). there were 5 1 reader diagnoses of lvh. the criteria used by the computer programme were very similar, although not i dentical, to those of the readers. there were no disagreements dueto programe 40 errors. the sensitivity was 92 % (47/51), and the specificity 99.5 % (4401442). right ventricular hypertrophy (rvh). a reader diagnosis of rvh was made in only five cases. only two of these were correctly identified by the computer but the disagreements were due to criteria differences, the programme prefer ring statements such as "right intraventricular conduction delay". the speci ficity of the computer diagnoses was 99.5 % (4861488). myocardial infarction. the computer programme utilized several degrees of severity in its diagnostic language, based on the amplitude and width of q waves, the location of q, combinations of q, r-wave progression and combina tions of q waves or q equivalents and st-t changes in different leads. i n the reader diagnosis a two-graded scale of severity was used. this was based on much the same criteria but certain differences did exist. programme errors, though rather infrequent, occurred more often for myocardial infarction than for hypertrophy. in one case the computer erroneously reported diaphragmatic myocardial infarction, based on a t wave obscured in both shape and amplitude by atrial flutter waves. in another case the computer did not measure s-t segment elevation correctly because s-t displacements were measured by a j point with a fixed time relation to the preceding qrs complex. the sensitivity of the computer diagnosis was 83 % (59/71) and its specificity 96.3 % (4191435). programme errors were more common than criteria differences regarding diaphrag matic infarctions, while the reverse was true for anterior or antero-lateral infarctions. arrhythmia. there were no difficulties in diagnosing arrhythmia in the ma nual interpretations in any of the e c g s . the computer reported "undetermined rhythm" whenever a tracing failed to satisfy the computer's logic or criteria for aspecific rhythm diagnosis. this happened in several cases, mostly cases of atrial fibrillation, but these disagreements were usually classified as group b, as we felt the additional diagnoses in these cases to be clinically more significant and as the computer made correct statements of the latter diagnoses. however, in five cases of a false computer diagnosis of "undeter mined rhythm", the report was classified as group c or d on the ground of mis measurement. to summarize the over-all performance of the computer programme regarding arrhythmia, there were no disagreements due to criteria differences. the sensitivity and specificity of the computer diagnosis, determined by a two-group classification procedure, were 82 % (71187) and 97.5 % (431/442), respectively. the over-all diagnostic accuracy, determined by a multi-group classification procedure, was 95.1 %. atrial fibrillation. out of 41 tracings of atrial fibrillation, the compu ter programme correctly identified 31 (76 %). the specificity was 99.8 % (4511 1 4 5 2 ) . in eight cases the computer stated "sinus rhythm", mostly in combina tion with "sinus arrhythmia", "sinus arrest" or "supraventricular ectopic 41 table 1. frequency of different rhythm diagnoses made by reader sinus rhythm 4 4 2 ectopic atrial rhythm 4 av nodal rhythm 2 atrial flutter 4 atrial fibrillation 4 1 supraventricular ectopic beats 1 2 ventricular ectopic beats 2 4 5 2 9 beats" and in two cases it stated "undetermined rhythm". the computer falsely reported atrial fibrillation in one case where the tracing showed a normal s i nus rhythm suddenly changing into ectopic atrial rhythm. other supraventricular arrhythmias. the computer programme falsely reported junctional rhythm or junctional tachycardia in eight cases of normal sinus rhythm, where the p waves were not detected. this gave a specificity of 9 8 . 4 % ( 4 8 3 / 4 9 1 ) . two actual cases of av nodal rhythm were correctly stated as such. concerning atrial flutter, the computer mismeasured two out of four cases but did not give any false positive answers. ventricular arrhythmia. there were 2 4 reader diagnoses of ventricular arrhythmia, i.e. premature ventricular contractions (pvc). the sensitivity of the computer was 8 8 % ( 2 1 / 2 4 ) , the disagreements all being due to programme errors. the specificity was 9 9 . 8 % ( 4 6 8 / 4 6 9 ) . in one case the computer measu red an artifact and stated pvc. there were no ecgs with series of pvcs or more complex ventricular arrhythmia. first degree av block. the reader and the computer used the same criteria for the diagnosis of first degree av block regarding the time interval. never theless, three computer diagnoses were judged as false positives on the basis of criteria differences, because the computer stated "first degree av block" in combination with "undetermined rhythm". the logical procedure would have been to supress all av block statements in the presence of the statement "un determined rhythm". i n one case the computer did not detect normal p waves. the sensitivity of the computer diagnosis was 97 % ( 3 7 / 3 8 ) , and the specifi city 9 8 . 9 % ( 4 5 0 1 4 5 5 ) . second degree av block. the computer did not recognize the two cases of atrial flutter. there were no other tracings containing second or third degree av block. left bundle branch block (lbbb). there were 11 reader diagnoses of lbbb. the computer made one false positive and three false negative statements in this respect, giving a sensitivity of 7 3 % ( 8 / 1 l ) and a specificity of 9 9 . 8 % ( 4 8 1 / 4 8 2 ) . two false negative reports were due to programme errors. 42 a r r h y t h m i a (total) by readers by progr. 18 82 1 ~ 8 1 13 87 neg. for a r r h y t h m i a by readers n 4 4 2 2 . 5 97.5 primary st and neg. lor t-wave changes (st-t) st-t by readers by program by readers i1 4 5 2 93.4 3.0 0.6 96.4 ' ~ 3 0 program errors @!j criteria dillerences 4 0 agreements fig. 3. arrhythmia. explanation, see fig. 1. fig. 4 . primary s-t segment and t wave changes. explanation, see fig. 1. overall performance (tracings) 521493 program errors 301493 criteria differences l11ll93 agreements program errors criteria differences 5 0 agreements overoll performance (diagnostic statements; n 6 8 1 ) clinically relevant obnormolity by readers by program no clinically relevant obnarmolit y by readers n259 progrom ermrs criteria dillerences 6 0 agreements fig. 5. over-all performance (tracings). explanation, see fig. 1. fig. 6. over-all performance (diagnostic statements). explanation, see fig. 1. 43 right bundle branch block (rbbb). out of ten cases of rbbb the computer m i s interpreted one as rvh, due to criteria differences, giving a sensitivity of 90 % ( 9 / 1 0 ) . no false positive reports were made. intraventricular conduction delay. due to mismeasurement, the computer re ported preexcitation in one tracing where the correct diagnosis should have been intraventricular conduction delay. in 11 tracings out of 12 the reader and computer diagnoses agreed, the sensitivity of the computer diagnosis being 92 z and its specificity 9 9 . 6 % ( 4 7 9 / 4 8 1 ) . primary s-t segment and t wave changes. the sensitivity of the computer diagnosis was 83.4 z ( 1 6 1 / 1 9 3 ) and its specificity 98.0 % ( 2 9 4 / 3 0 0 ) . most dis agreements were due to programme errors, where the computer seemed to be in accurate in detecting the shape of the s-t segment and a l s o abnormal s-t de pressions or elevations. the reason for this might have been that judgements concerning the s-t segment are based on deviations from a j point fixed in temporal relation to the preceding qrs complex. the computer also had diffi culties regarding the shape of the t wave, especially concerning coupling bet ween this shape and slight changes in amplitude. miscellaneous. there were 11 cases with left anterior hemiblock, for all of which complete agreement was found, and there were no false positive re ports. this was also the result for 24 cases of marked left axis deviation, two cases of right axis deviation, two cases of counter-clockwise rotation and three cases of clockwise rotation. there were five pacemaker ecgs. in two of these there was considered to be disagreement, on criterional grounds, as the computer did not report the presence of spontaneous beats, which in one case revealed extreme t-wave inversion in precordial leads. over-all performance. in 411 cases ( 8 3 . 5 x ) the readers and the computer were in agreement, while disagreements were noted in the remaining 82 cases. detailed analysis revealed that in 6 . 0 z ( 3 0 / 4 9 3 ) these disagreements were based upon the use of different diagnostic criteria. in the remaining 52 cases ( 1 0 . 5 w ) they resulted from programme errors such as mismeasurement, pattern recognition failures or deficient programme logic. regarding the over-all efficiency of the computer diagnosis, as expressed by a two-group classifica tion procedure, the sensitivity was 83.5 % ( 3 5 5 / 4 2 5 ) and the specificity 82.6% ( 2 1 4 / 2 5 9 ) . the error ratio was 0 . 3 , the accuracy of positive tests (ap) 0 . 9 and the accuracy of negative tests ( a n ) 0.8. when expressed by a multi-group classification procedure, the over-all diagnostic accuracy (da) was 83.5 % . discussion there are limitations to the use of the present ecg population for evalua ting a computer programme. some diagnoses were very uncommon and some were 44 not represented at all. however, one aim was to test the computer against a re presentative sample of our own day-by-day routine clinical population of ecgs. the method of statistical analysis of the result also has its limitations, mainly in that no differential weight is given to different ecg diagnoses. some differentiation in this respect was made in our investigation, however, since the ecgs were initially classified according to their clinical signifi cance in a broad sense. with the present method of analysis, unnecessary dis cussion about diagnostic criteria is avoided. discrepancies regarding criteria can of course influence the decision whether to use the services of a computer programme or not. on the other hand, such difficulties can be overcome in co operation with the computer programme constructor. more serious are disagree ments due to programme errors, where the user has to find out firstly, whether the programme is adequate for the kind of subject population in question and secondly, whether the rate of programme errors can be accepted. some authors have not only used sensitivity and specificity, as defined above, as measures of diagnostic efficiency, but have also used what is often called mean performance (mp) and association index (ai), where mf' = a(se+sp) and a 1 = se+sp-100. rautaharju et al. ( 4 ) have claimed that all of these con cepts are often misunderstood in the sense that they have not been validated against the composition of the test ecg population used. we feel that m p and a1 are not of much value in determining the efficiency of the present compu ter programme a s used on the present population. the sensitivity of the computer diagnosis was the same whether two-group 2 or multi-group classification procedures were used, and the values were fairly satisfactory. likewise, the specificity was acceptable and the values were within the range commonly reported in other similar studies of computer pro gramme performance. the error ratio (0.3) was not satisfactory, however, as it implied that one-third of the diagnostic statements made by the computer were false. the accuracy of positive tests was acceptable but the accuracy of negative tests was disappointing and reflected an inability to detect arrhyth mias and st-t aberrations, in particular. we consider that the results of this study adequately describe the performance of the tested computer program me as applied to the type of population of ecgs used here. acknowledgement the authors are indebted to mr. wauquez, cardionics, brussels, for his kind help in providing us with the equipment and computer services necessary for this study. 45 references 1. bailey, j.j., itscoitz, s.b., hirschfield jr, j.w., grauer, l.e. & horton, m.r.: a method for evaluating computer programs for electrocardiographic interpretation. i. application to the experimental ibm program of 1971. circulation 50:73-79, 1974. 2. frank, e.: an accurate, clinically practical system for spatial vector cardiography. circulation 13: 737-749, 1956. 3. goldman, m.j.: principles of electrocardiography, lange medical publica tions, los altos, california, 1973. 4 . rautaharju, p.m., blackburn, h.w. and warren, j.w.: the concepts of sensi tivity, specificity and accuracy in evaluation of electrocardiographic, vectorcardiographic and polarocardiographic criteria. j electrocardiology 9:275-281, 1976. received in final form november 23, 1978 address for reprints: dr johan landelius dept of clinical physiology university hospital 5-750 14 uppsala sweden 46 upsala journal of medical sciences 2023, 128, e8832 http://dx.doi.org/10.48101/ujms.v128.8832 study protocol: the cross-sectional uppsala weight gain in pregnancy study (viga study) theodora kunovac kallak, alice zancanaro, katja junus, anna-karin wikström, inger sundström poromaa and susanne lager department of women’s and children’s health, uppsala university, uppsala, sweden abstract background: more than two in five swedish women are overweight or obese when becoming pregnant. maternal overweight or obesity and excessive pregnancy weight gain are associated with several adverse pregnancy outcomes. the underlying mechanisms that link maternal adiposity, diet, exercise, pregnancy weight gain with pregnancy outcome are incompletely understood. methods: we describe the design for a cross-sectional study of pregnant women at uppsala university hospital, sweden. all participants delivered by elective cesarean section before the onset of labor. at inclusion, participants answered two questionnaires concerning their dietary and exercise habits. fasting maternal blood samples (buffy coat, plasma, serum) were collected. during the cesarean section, biopsies of maternal subcutaneous and visceral adipose tissues were obtained. placental tissue was collected after delivery. all biological samples were processed as soon as possible, frozen on dry ice, and stored at −70 °c. pregnancy outcomes and supplementary maternal characteristics were collected from medical records. results: in total, 143 women were included in the study. of these women, 33.6% were primiparous, 46.2% had a pre-pregnancy body mass index (bmi) over 25 kg/m2, and 11.2% of the offspring were born large for gestational age (lga). complete collection, that is both questionnaires and all types of biological samples, was obtained from 81.1% of the participants. conclusions: this study is expected to provide a resource for exploration of the associations between maternal weight, diet, exercise, pregnancy weight gain, and pregnancy outcome. results from this study will be published in peer-reviewed, international scientific journals. this study was approved by the regional ethics review board in uppsala (approval no 2014/353) and with an amendment by the swedish ethical review authority (approval no 2020-05844). article history received 21 october 2022 revised 14 december 2022 accepted 12 january 2023 published 10 february 2023 keywords placenta; adipose tissue; biomarkers; pregnancy outcome; maternal obesity; fetal growth; birth weight; diet; exercise; excessive pregnancy weight gain introduction worldwide, the proportion of overweight women is increasing, with many women being overweight or obese when becoming pregnant. in 2020, over 40% of swedish women were considered overweight or obese at registration for antenatal care, as measured by body mass index (bmi) (1). obesity or being overweight before and during pregnancy increases the risk of a variety of complications in both mother and child. obese women, on average, take longer to get pregnant (2) and are more likely to suffer from miscarriages or stillbirths. in addition, these women are more likely to develop gestational diabetes, preeclampsia, depression, and require delivery by emergency cesarean section (3, 4). infants of obese mothers are at higher risk of developing several complications throughout their life. as such, they risk being born large for gestational age (lga), having congenital anomalies (5), cardiovascular disease, stroke, asthma, type 2 diabetes (6), as well as developing certain types of cancers (7, 8). furthermore, maternal obesity has an intergenerational effect: children of obese mothers are at a higher risk of becoming obese themselves (3, 6). like maternal obesity, excessive pregnancy weight gain is also associated with pregnancy complications. excessive pregnancy weight gain has been linked with an increased risk of maternal development of gestational hypertension or preeclampsia. women gaining excessive weight during pregnancy are also more likely to give birth to a lga or macrosomic baby (9). it has also been shown that women with excessive pregnancy weight gain tend to retain more weight postpartum (10). there are guidelines for recommended rate and total weight gain during pregnancy from the united states (us) institute of medicine (11). these guidelines propose that underweight women (bmi < 18.5 kg/m2) should gain a total of 12.5–18 kg during pregnancy. the recommended weight gain is then progressively lower for women of normal weight contact susanne lager susanne.lager@kbh.uu.se supplemental data for this article can be accessed here. © 2023 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original research article http://dx.doi.org/10.48101/ujms.v128.8832 mailto:susanne.lager@kbh.uu.se http://dx.doi.org/10.48101/ujms.v128.8832 http://creativecommons.org/licenses/by/4.0/ 2 t.k. kallak et al. (bmi  18.5–24.9 kg/m2; recommended weight gain 11.5–16 kg), overweight (bmi 25.0–29.9 kg/m2; recommended weight gain 7–11.5 kg), and obesity (bmi ≥ 30 kg/m2; recommended weight gain 5–9 kg). however, among women in sweden (regardless of bmi category), the actual range of weight gain is broader than the recommended (12). in addition to bmi and weight gain during pregnancy, maternal exercise habits may also influence pregnancy outcome. the swedish healthcare system recommends 2.5 h of exercise a week for expectant mothers. the benefits of exercise for pregnant mothers are not clear. some evidence suggests that exercise has a positive effect on the physical and the psychological well-being of expecting mothers. mothers who exercise pre-pregnancy have a lower risk of developing preeclampsia and gestational diabetes (13). women exercising during their pregnancy have a slightly lower risk of lga or small for gestational age baby (sga) (14), and a lower risk of developing prenatal depression (15). dietary and exercise interventions have been shown to lower the risk of excessive pregnancy weight gain, but do not reduce the risk of preeclampsia or macrosomia (16). although adverse shortand long-term consequences of maternal obesity and excessive pregnancy weight gain for children are well-established, much remains to be understood regarding the underlying mechanisms. the overall hypothesis for this study was that there is an interplay between maternal adipose tissue and the placenta affecting pregnancy outcome. to test this hypothesis, the viga study (viktuppgång under graviditet [in swedish, ‘weight gain in pregnancy’]) was established to provide careful characterization of a cohort of pregnant women with extensive biological sample collection. the viga study has the potential to substantially contribute to an understanding of the mechanisms concerning diet, exercise, maternal weight, pregnancy weight gain, and how these affect pregnancy outcomes. methods aim, design and setting of the study the aim of the viga study was to establish a pregnancy database and biobank at uppsala university. our study focuses on pregnancy weight gain and further facilitates research relating to maternal obesity in pregnancy. by combining biological samples, clinical data, and questionnaires, the viga study intends to explore physiological and endocrinal consequences of maternal weight gain and obesity during pregnancy. the study design was cross-sectional, with two questionnaires and biological samples collected at time of delivery (figure 1). data on maternal and pregnancy characteristics were collected from medical charts. the study was conducted at uppsala university hospital in uppsala, sweden during 2014–2016. before initiation of data collection, the study was approved by the regional ethics review board in uppsala (approval no 2014/353) and later amended by the swedish ethical review authority (approval no 2020-05844). characteristics of participants women undergoing elective cesarean section at uppsala university hospital were eligible for participation in the viga study. additional participation requirements were: having a singleton pregnancy, being 18 years of age or older, no known blood-borne infections, as well as understanding swedish (spoken and written). before signing the consent form, the study was carefully described by the recruiting midwife/staff and the potential participant had an opportunity to ask questions. data collection upon recruitment, participants answered two questionnaires. the first questionnaire consisted of 12 questions regarding eating habits. the second questionnaire focused on physical activity and consisted of 17 questions. validated questionnaires were provided by the swedish food agency (17). minor changes to the questions were implemented to reflect diet and physical activity during pregnancy. specifically, the first 10 questions of the dietary questionnaire focused on eating habits during the last 9 months instead of the last 12 months. question 11 asked whether the respondent attempted changing her eating habits during pregnancy, rather than during the last 12 months. in the physical activity questionnaire questions 21 and 27 were changed to reflect activity during pregnancy, rather than the last year or present level of activity. the complete questionnaires (in swedish) are available in supplementary table 1. medical records were reviewed, along with information collected throughout pregnancy in the maternal health care system and at the delivery ward. sociodemographic information, obstetrical medical history, current medication, pregnancy complications, biochemical analyses, and neonatal information were also recorded. biological sample collection after inclusion, fasting maternal blood samples for buffy coat, plasma, and serum were collected. serum samples were allowed to clot before being centrifuged and the serum was collected. plasma samples were obtained in ethylenediamine tetraacetic acid -tubes. after centrifugation, buffy coat and plasma were separated and stored. during cesarean section, biopsies of maternal subcutaneous and visceral adipose tissue were taken. after delivery, a full thickness section from the central part of the placenta was obtained. collected tissues were washed in sterile phosphate-buffered saline and then frozen immediately on dry ice. all samples were stored at −70 °c until further processing and analysis. results current findings in total, 143 women were included in the viga study ( table 1). a majority of participants were born in sweden and were cohabiting with a partner. eighteen women (12.6%) conceived with the help of in vitro fertilization. a total of 46.2% of the http://dx.doi.org/10.48101/ujms.v128.8832 study protocol 3 women were overweight or obese when registered for maternal health care during early pregnancy. fifty-eight women (40.6%) had an adequate weight gain according to the us institute of medicine recommendations based on their bmi category. most of the remaining women (n = 62; 43.4%) gained more weight than recommended and had an excessive weight gain, while 19 women (13.3%) gained less weight and had an inadequate weight gain. most of the women had a healthy pregnancy. among recorded complications were five women with preeclampsia or hypertension and three women with diabetes. a large majority gave birth at term and approximately 11.2% of the children were born lga, while only one was born sga. a total of 139 women (97.2%) reported eating habits and 136 (95.1%) reported physical activity during pregnancy. serum was collected from 133 (93.0%) women, but plasma and buffy coat from only 131 (91.6%) women. placental biopsies were collected from 139 (97.2%) women, with visceral and subcutaneous adipose tissue biopsies of 133 (93.0%) and 136 (95.1%) during cesarean section. the complete collection, those answering both questionnaires and donating all six types of biological samples, was obtained from 81.8% of the participants. using data from the swedish national board of health and welfare (18), a comparison of participant characteristics in the viga study was done for all women in uppsala county and all women in sweden giving birth during the same time period (presented in table 2). this comparison suggests that, in general, participants of the viga study were on average 3.4 years older than the average age of women giving birth in uppsala or sweden. the viga study participants were more often multiparous and gave birth to more high birth weight infants. ongoing studies several projects are ongoing or planned utilizing the database and biological samples collected in the viga study. these projects include determination of placental rna levels using sequencing, exploring potential associations between rna levels and maternal pre-pregnancy bmi, pregnancy weight gain or fetal growth. in another project, a mass spectrometry-based metabolomics analysis of placenta and maternal plasma is currently being performed. this analysis is expected to generate data on amino acids, anthocyanins, fatty acids, flavonoids, lipids, oligolignols, organic acids, phenols, sterols, and sugars. maternal adipose tissue will be analyzed using the same approach. in addition, maternal plasma proteins related to metabolism have been measured by proteomics using proximity extension assays (19). this assay simultaneously measures 92 different proteins, focusing on cell adhesion, cell surface receptor signaling pathways, cellular metabolic processes, and regulation of phosphorylation. in an upcoming study, maternal plasma proteins related to inflammation will be assessed using the same proximity extension assays technology. further studies figure 1. an overview of viga study. in the cross-sectional viga study, 143 women were included. for most of the women data from medical records, two questionnaires, as well as an extensive biological sample collection have been obtained. 4 t.k. kallak et al. will be conducted to more completely describe the associations between maternal adiposity, pregnancy weight gain, diet, exercise, and fetal growth. discussion there is a need to better understand the impact of pregnancy weight gain and overweight/obesity as it relates to pregnancy health and outcome, with shortand long-term consequences for mother and child. this is in order to provide adequate care for overweight or obese pregnant women. our project aim was establishment of a database and biobank for excessive maternal weight gain and obesity during pregnancy. excessive weight gain and obesity are associated with pregnancy complications, such as accelerated fetal growth (3, 9, 20, 21). accelerated fetal growth is associated with complications during delivery (22, 23). in addition, accelerated fetal growth has longstanding implications for the future health of an individual (6–8). therefore, understanding the underlying mechanisms of accelerated fetal growth, along with how excessive weight gain and obesity impacts pregnancy, may further lead to strategies for managing these conditions. strengths and limitations our study has several strengths. the database contains an extensive characterization of participants. maternal health information, pregnancy, and child outcomes have been obtained from medical records. complementing the information from medical records, the participants filled out two validated questionnaires regarding their dietary and exercise habits. this information allows for additional observations concerning laboratory data associated with the biological samples. such observations are rarely included in studies of biological samples focusing upon accelerated fetal growth or maternal obesity/weight gain. another strength of the study is the extensive biological sample collection, including maternal adipose tissue. finally, a notable study strength is that most of the data and biological samples have been collected from a majority of study participants. a study limitation is the low number of participants due to selecting those delivering by planned cesarean section, possibly resulting in a recruitment bias. when compared with the general population giving birth in sweden during the same time period, participants in viga were older, less primiparous, and also gave birth to a higher proportion of large infants. however, only including women delivering through planned cesarean section is also an important study strength. this is because previous reports found that exposure to labor may affect placental rna levels and activity of cellular signaling pathways (24–27). therefore, the biological samples collected may better reflect the in utero environment rather than labor effects. although the table 1. characteristics of viga study participants. variable viga study (n = 143) maternal age, years 33.7 (5.2) primiparous 48 (33.6%) born in sweden 122 (85.3%) university education 94 (65.7%) missing 5 (3.5%) cohabiting with a partner 131 (91.6%) pre-pregnancy bmi, kg/m2 24.7 (17.3 – 44.1) missing 4 bmi ≥ 25 kg/m2 66 (46.2%) in vitro fertilization 18 (12.6%) weight gain during pregnancy, kg 13.7 (4.3) inadequate weight gain* 19 (13.3%) adequate weight gain* 58 (40.6%) excessive weight gain* 62 (43.4%) missing 4 (2.8%) preeclampsia 2 (1.4%) hypertension 3 (2.1%) diabetes 3 (2.1%) gestational age, weeks 38.9 (36.9 – 41.6) infant sex, female 70 (49.0%) infant birth weight, grams 3606 (527) small for gestational age 1 (0.70%) large for gestational age 16 (11.2%) missing 4 (2.8%) parametric distributed data is provided as mean (standard deviation) while non-parametric data is given as median (min–max). frequencies are given as total number (percentages). bmi, body mass index. *inadequate (less than the recommended), adequate (within the recommended range), and excessive (more than the recommended) weight gain calculated based on pre-pregnancy bmi category and us institute of medicine recommendations (11). table 2. comparison of viga study participants with pregnant women in the general population of uppsala county and sweden during the same time period. variable viga study 2014–2016 (n = 143) uppsala county ‡ 2014–2016 (n = 11,383) sweden ‡ 2014–2016 (n = 328,688) maternal age (mean) 33.7 years 30.3 years 30.3 years primiparous (%) 33.6% 42.9% 43.0% pre-pregnancy bmi (mean) * 25.3 kg/m2 24.9 kg/m2 24.9 kg/m2 underweight (bmi <18.5) 2.8% 2.4% 2.7% normal weight (bmi 18.5–25) 49.0% 57.4% 58.3% overweight (bmi 25–30) 33.6% 26.0% 25.5% obese (bmi ≥ 30) 11.9% 14.3% 13.6% infant birth weight ** < 2,500 g 0.7% 5.7% 4.8% > 4,500 g 5.6% 3.7% 3.6% small for gestational age 0.7% 2.4% 2.6% large for gestational age 11.2% 4.4% 3.5% *missing bmi data from four viga study participants (2.8%). **missing birth weight data from four viga study participants (2.8%). ‡information on uppsala county and sweden retrieved from the swedish national board of health and welfare (18). bmi, body mass index. study protocol 5 study is limited by no of participants and the recruitment bias, we anticipate the study has a capacity to contribute to an increased understanding of maternal excessive weight gain and obesity effects upon pregnancy outcome. this is achieved through careful characterization, the extensive data, and biological samples available from the participants. summary in conclusion, the viga study biobank and database allows for valuable laboratory discoveries regarding maternal excessive weight gain and overweight/obesity, as well as accelerated fetal growth. it is hoped that such discoveries may ultimately be used for the improvement of care for pregnant women. acknowledgements the authors are grateful to all the participating women and staff at the delivery unit of uppsala university hospital, uppsala, sweden. figure 1 was created by a.z. in biorender. competing interests and funding the authors have no conflicts of interest to report. this study was funded by the swedish state under an agreement between the swedish government and county councils (the alf agreement). analyses of biological samples were funded by grants from åke wiberg foundation, gillbergska foundation, kronprinssessan lovisa foundation, lisa och johan grönberg foundation, and magnus bergvall foundation. ethics approval and consent to participate the regional ethics review board in uppsala approved the study protocol (approval no 2014/353) and the swedish ethical review authority approved the amendment to extend the analysis of collected biological samples (approval no 202005844). all participating women gave written consent after careful explanation of the study. notes on contributors theodora kunovac kallak, msc, phd, associate senior lecture, uppsala university alice zancanaro, msc, phd-student, uppsala university katja junus, md, phd, uppsala university anna-karin wikström, md, phd, professor, uppsala university inger sundström poromaa, md, phd, professor, uppsala university susanne lager, msc, phd, senior lecture, uppsala university orcid theodora kunovac kallak https://orcid.org/0000-0002-21128674 alice zancanaro https://orcid.org/0000-0002-8362-6572 katja junus https://orcid.org/0000-0003-4088-400x anna-karin wikström https://orcid.org/0000-0001-6431-3303 inger sundström poromaa https://orcid.org/0000-00022491-2042 susanne lager https://orcid.org/0000-0003-3556-065x references 1. socialstyrelsen. statistics on pregnancies, deliveries and newborn infants 2020. health med care. 2021. contract no. 2021-12-7653. 2. gesink law dc, maclehose rf, longnecker mp. obesity and time to pregnancy. hum reprod. 2007;22(2):414–20. doi: 10.1093/humrep/ del400 3. poston l, caleyachetty r, cnattingius s, corvalan c, uauy r, herring s, et al. preconceptional and maternal obesity: epidemiology and health consequences. lancet diabetes endocrinol. 2016;4(12):1025–36. doi: 10.1016/s2213-8587(16)30217-0 4. ma rcw, schmidt mi, tam wh, mcintyre hd, catalano pm. 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http://dx.doi.org/10.1016/j.placenta.2014.10.006 http://dx.doi.org/10.1016/j.placenta.2014.10.006 http://dx.doi.org/10.2353/ajpath.2007.070528 http://dx.doi.org/10.1016/j.placenta.2014.11.005 http://dx.doi.org/10.1093/molehr/gam083 upsala j med sci 97: 149-155 intravenous theophylline after beta2-agonist treatment in severe acute asthma. effect on patients who are not pre-treated with theophylline christer janson and gunnar boman department of lung medicine, uppsala university, akademiska sjukhuset, uppsala, sweden abstract the effect of i.v. theophylline after high-dose 02-agonist treatment in severe acute asthma was studied in 30 patients from a multicentre study who reported not having taken theophylline during the last 24 hours. one hour after the start of inhaled or i.v. salbutamol treatment, all patients received 6 mg/kg of i.v. theophylline. the plasma concentration 30 minutes after the start of the theophylline infusion was 78 _+ 13 ymolll (mean f sd). the mean change (a) in peak expiratory flow (pef) was 8 f 6% of the predicted 30 minutes after the theophylline infusion and 7 f 5% 60 minutes after it. the increase in pef was greater in this patient group than in a group of 101 patients from the same multicentre study who were on theophylline medication and were therefore given a reduced dose (3 mg/kg) (7 f 5 vs. 4 f 6% of the predicted value, pc0.01). the proportion of patients with an increase in pef of 2 10% of the predicted at discharge was 27% (8/30) in the patient group in this investigation and 14% (14/101) in the group who was on theophylline treatment. i ntr 0 d u ctf on inhaled 02-agonists and systemically administered corticosteroids are recognized as the cornerstones of the modern treatment of acute asthma (2,16). theophylline has subsequently been degraded to the position of a third-line drug in the current guidelines on the management of acute asthma (2,16). in fact, there are several studies in which no additional bronchodilatory effect has been found when theo phylline has been administered with inhaled 0-agonists (1,6,15). a further disadvantage of theophylline is the risk of potential serious side-effects owing to the narrow therapeutic interval of the drug (4). in a swedish multicentre study, i.v. theophylline was given 60 minutes after inhaled and i.v. 02-agonist treatment (1 7). most of the patients in the study were taking theo phylline as maintenance treatment and were therefore given a reduced theophylline dose. in a subanalysis we found that only a small proportion of these patients had a clinically significant additional improvement after theophylline (9). since the time of the multicentre study there has, however, been a change in therapy practice. as a result, most patients admitted because of acute asthma today are not on theophylline. the aim of this investigation was therefore to study the effect of i.v. 149 theophylline after 02-agonist treatment in acute asthma on the subgroup of patients who had not taken theophylline during the last 24 hours before admission to the emergency room. material and methods between september 1985 and january 1987 the swedish society of chest medicine conducted a multicentre study on the effect of i.v. versus inhaled salbuta mol treatment in severe acute asthma (17). one hour after the start of salbutamol treatment (ventoline, glaxo), i.v. theophylline was given to both patient groups. an infusion of aminophylline (teofyllamin, aco) in a dose corresponding to 6 mg/kg of theophylline was given to patients who had not taken theophylline during the last 24 hours. the dose was reduced to 3 mglkg in those patients who had taken oral theophylline. in this analysis we have only included those patients who were not taking oral theo phylline before treatment and were thus given a dose corresponding to 6 mg/kg of theophylline. we have excluded the patients who reported having taken broncho dilatory drugs for which no method of determining the plasma level was available; they included fenoterol, orciprenaline, isoprenaline and ipratropium bromide. this analysis thus comprised 30 adult patients (13 men and 17 women, mean age 54 years, range 18-73) who attended the emergency room with a severe acute attack of asthma (peak expiratory flow rate (pef) of 2 5 0 % of the patient's predicted normal value ( 7 ) , pulse rate of 2 100 beatslmin.). a s initial treatment 15 patients received inhaled salbutarnol (0.15 mglkg x 2) and 15 received i.v. salbutamol (5 pglkg). the infusion time for the i.v. aminophylline was 20-30 minutes. blood samples for the determination of plasma theophylline levels were taken before the start of treatment and 30 minutes after the start of the aminophylline infusion. plasma theophylline levels were determined at the department of clinical pharmacology at sahlgren's university hospital, gothenburg, using a high-pressure liquid chromatographic method (5). using this method, the lowest detectable level is 1 ymolll. the coefficient of variation is 4% for a plasma level of 3 ymolll and 3% at 55 pmolll. plasma levels of salbutamol and terbutaline were determined 55 minutes after the start of the salbutamol treatment. from the result of the salbutamol and terbutaline assay an estimation of the total l32-agonist plasma concentration was made. the methods for this drug concentration assay and for the estimation of the total 02-agonist plasma level have been described in detail elsewhere (8). peak expiratory flow (pef), pulse rate and blood pressure were followed from arrival at the emergency room until 60 minutes after the start of the theophylline treatment. the informed consent of all patients was obtained and the study was approved by all the relevant local ethics committees. 150 statistics differences between subgroups of the population were analysed using the mann whitney u test. spearman's rank correlation test was used to analyse the correlation of the change (a) in pef after the theophylline treatment with the other variables. a p-value of < 0.05 was regarded as statistically significant.the results are expressed as mean f sd. r e s u l t s measurable plasma levels of theophylline before treatment were found in 20 of the 30 patients (3f2 pmolll, range 1-9). the mean p-theophylline concentration 30 minutes after the start of the theophylline infusion was 78 f 13 pmol/l (range 52-102) (fig. 1). n i e 5. y q) c * c a .i i i 0 q) c i i n 2v 0 i figure 1 .p-theophylline concentration 30 minutes after the start of i.v theophylline treatment (6 mg/kg) in 30 patients with severe acute asthma (individual values, median, inter-quartile range (2575%) and range). following the theophylline infusion, a pef was 8 f 6% of the predicted value (40 f 33 vmin) after 30 minutes and 7 f 5% of the predicted (37 f 30 i/min) after 60 minutes (table 1). 151 table 1.peak expiratory flow, pulse rate and blood pressure before and 30 and 60 minutes after treatment with 6 mg/kg of i.v. theophylline (n=30). (mean + sd) (* p<0.05, ** p<o.ol, *** p<o.ool, compared with before treatment.) before 30 min 60 min peak expiratory flow (% of predicted) 45+16 53+15*** 53+15*** pulse rate (beatshin) 99+21 104+21** 1 0 3 f l 9 * systolic blood pressure (mm hg) 137+25 133+21 131f19 diastolic blood pressure (mm hg) 85+15 82+14 84+14 (vmin) 229f82 269+86*** 266f82*** patients treated with inhaled salbutamol displayed a significantly greater increase in pef before the theophylline infusion than those treated with i.v. salbutamol (pc0.05). there was, however, no significant difference in a pef after the theophylline treat ment between the inhalation and i.v. salbutamol group (fig. 2). at discharge (60 minutes), 8 patients had an increase of 2 10% of the predicted value. four of these patients had received inhaled salbutamol and 4 i.v. salbutamol. apef (“a of pred.) t 20 15 10 5 -i i inhalation intravenous (n=15) (n=15) figure 2.change in pef (mean and se) 55 minutes after inhaled or intravenous salbutamol (open bars) and 60 minutes after the addition of intravenous theophylline (cross-hatched bars). 152 no significant correlations were found between the post-theophylline increase in pef and age, sex, pef before treatment, a pef before theophylline, p-02-agonist concentration or a p-theophylline after treatment. the increase in pef after theophyline treatment was significantly greater in the study group in this investigation than in a group of 101 patients from the same multicentre study who were on theophylline medication and were therefore given a reduced theophylline dose (3 mg/kg) (8 f 6 vs. 4 f 6% after 30 minutes and 7 f 5 vs. 4 * 6% of the predicted after 60 minutes, pc0.01). the number of patients with an increase in pef of 2 10% of the predicted at discharge was 8 (27%) in this investigation and 14 (14%) in the group who were on theophylline treatment. the average increase in pef before the theophylline infusion was 13% of the predicted in both groups. d i s c u s s i o n when designing the swedish society of chest medicine's multicentre study, the main purpose was to compare the effect of inhaled and intravenous 02-agonist treatment in severe acute asthma. a further aim was to see whether theophylline was of any clinical use after high-dose 02-agonist treatment. in our first report, our conclusion was that it was doubtful whether theophylline had any clinical effect after high-dose inhaled 82-agonists (1 7). this conclusion was, however, criticized as being based on data which was not totally convincing (3). this caused us to make a subanalysis of the effect of i.v. theophylline on patients who were taking oral theo phylline as maintenance treatment. in this investigation, there was a small group of patients who did improve rather well after i.v. theophylline. this group was characterized by having subclinical p4heophylline;concentrations before treatment (9). the swedish multicentre study was conducted from 1985 to 1987. since then therapy practice has changed. maintenance treatment with theophylline has markedly declined at least in sweden (10). a large proportion of patients who currently seek emergency treatment are therefore not on theophylline. these patients can be given a larger theophylline dose without increasing the risk of toxic side-effects. the negative correlation between plasma concentration before treatment and the treatment effect reported earlier (9) indicates that patients without theophylline pre-treatment might be most suitable for emergency treatment with i.v. theophylline. our results partly support this hypothesis. consequently the proportion of patients whose pef increased by 10% or more compared with the patients predicted value after i.v. theophylline was 27% (8/30) in this patient group compared with 14% (14/101) of the patients who had taken theophylline before emergency treatment. on the basis of previous dose-effect studies, a dose of 5-6 mg/kg theophylline is usually recommended in the treatment of acute asthma in patients who are not taking theophylline (12,16). in this study nearly all the patients reached a p theophylline concentration of above 55 pmol/l, while none reached a concentration 153 above the upper limit of the therapeutic interval (1 10 pmol/l). we can thus confirm that a theophylline dose of 6 mg/kg is a suitable loading dose in the emergency treatment of patients who are not on theophylline medication. as would be the case in a clinical setting, the question of whether the patients had taken theophylline before arrival or not was based on their own reports. in several of the patients we found small but measurable levels of theophylline. these might have been caused by theophylline taken earlier than 24 hours before arrival. in some patients the low theophylline levels might also have been caused by drinking tea as tea contains small levels of theophylline (1 3). the theophylline concentrations measured were below the level at which they could have influenced the clinical out come. as in our previous report (9), the effect of theophylline appears to be relatively un related to the 02-agonist treatment. as a result, we found no significant difference between the effect in patients treated with inhaled or i.v. salbutamol. there was no significant correlation between the estimated p-02-agonist concentration before treatment and the effect of the theophylline infusion. in both our investigations the re sponse to theophylline was also unrelated to the patient's age, sex, pre-treatment pef or the level of change in pef after 02-agonist treatment. there was also no difference in pef response to salbutamol between the patient group with and the group without t heophylli ne pre-treatment. lately there has been a discussion as to whether theophylline, apart from being a bronchodilator, also has other effects of possible therapeutic value. in one american study, theophylline treatment was reported to decrease the need for hospital ad mission despite the fact that there was no difference in the effect on lung function between the patients who were given theophylline and the control group (18). the author suggests that this might be caused by an anti-inflammatory effect. an editorial commenting on the study speculates on a possible future rise for theophylline in asthma treatment (11). our results are a further indication that it is perhaps not possible to completely count-out theophylline in the treatment of severe acute asthma. we conclude that after high-dose 02-agonist treatment there is a subgroup of patients in whom i.v. theophylline produces a further bronchodilation of probable clinical value. the factors causing this interindividual difference in theophylline response are not known at present. acknowledgements this investigation was part of a multicentre study conducted by the swedish society of chest medicine. the investigation was supported financially by bror hjerpstedt foundation, uppsala, the swedish heart-lung foundation, stockholm, and ab glaxo lakemedel, molndal, sweden. 154 1. 2. 3. 4. 5. 6. 7 8. 9. references bowler, s.d., mitchell, c.a., armstrong, j.g. & scicchitano, r. nebulised feno terol and i.v. aminophylline in acute severe asthma. eur j respir dis 70: 280 283, 1987. british thoracic society, research unit of the royal college of physicians of london, king's fund centre & national asthma campaign. guidelines for mana gement of asthma in adults: i i acute severe asthma. br med j 301: 797-800, 1990. crompton, g.k. nebulized or intravenous beta2 adrenoceptor agonist therapy in acute asthma? eur respir j 3: 125-1 26, 1990. eason, j. & markowowe, h.l.j. aminophylline toxicity how many hospital asthma deaths does it cause? respir med 83: 219-226, 1989. fagerstrom, p.o., mellstrand, t. & svedmyr, n. absorption of theophylline from conventional and sustained release tablets. int j clin pharmacol ther toxicol fanta, c.h., rossing, t.h. & mcfadden jr, e.r. treatment of acute asthma. is combination therapy with sympathornimetics and methylxanthines indicated? am j med 80: 5-10, 1986. gregg, i. & nunn, a.j. peak expiratory flow in normal subjects. br med j 3: 282 284, 1973. janson, c., boe, j., boman, g., mossberg, b. & svedmyr, n. bronchodilator intake and plasma levels at admission for severe acute asthma. eur respir j 5: janson, c., boman, g. & boe, j. which patients benefit from adding theophylline to 02-agonist treatment in severe acute asthma? ann allergy 69: 107-1 10, 1992 19: 131-138, 1981. 80-85, 1992. 10. johansson, h. & nordenstam, i. svensk iakemedels statistik 1989 (swedish drug sale statistics). apoteksbolaget, stockholm, 1990. 11. mcfadden jr, e.r. methylxanthines in the treatment of asthma: the rise, the fall, and the possible rise again. ann intern med 11 5: 323-324, 1991. 12. mitenko, p.a. & ogilvie, r.i. rational intravenous doses of theophylline. new engl j med 289: 600-603, 1973. 13. rall, t.w. central nervous system stimulants: the methylxanthines. in: goodman and gilman's the pharmacological basis of therapeutics (ed. a. goodman gilman, l.s. goodman, t.w. rall & f. murad), pp. 589-603. macmillan, new york, 1985. 14. self, t.h., abou-shala, n., burns, r., stewart, c.f., ellis, r.f., tsiu s.j. & kellermann, a.l. inhaled albuterol and oral prednisone therapy in hospitalized adult asthmatics. does aminophylline add any benefit? chest 98: 131 7-1321, 1990. 15. siegel, d., sheppard, d., gelb, a. & weinberg, p.f. aminophylline increases the toxicity but not the efficacy of an inhaled beta-adrenerg agonist in the treatment of acute exacerbation of asthma. am rev respir dis 132: 283-286, 1985. 16. soler, m., imhof, e. & perruchoud, a.p. severe acute asthma. respiration 57: 17. swedish society of chest medicine. high-dose inhaled versus intravenous salbutamol combined with theophylline in severe acute asthma. eur respir j 3: 18. wrenn, k., slovis, c.m., murphy, f. & greenberg, r.s. aminophylline therapy for acute bronchospastic disease in the emergency room. ann intern med 115: 241 247, 1991. 11 4-1 21, 1990. 163-1 70, 1990. adress for reprints: dr christer janson, department of lung medicine akademiska sjukhuset, s-751 85 uppsala, sweden i 1 -928572 155 upsala j med sci 79: 72-83, 1974 quantitative determination of pulmonary microembolism ' in the dog comparison of in vivo and in vitro methods christer busch from the department of forensic medicine, university of uppsala, uppsala, sweden abstract external monitoring and in vitro measurement of pulmo nary microembolism using 5'cr-labelled platelets and 1251-labelled fibrinogen were compared. the micro enbolism was induced in dogs by intravenous infusion of thrombin and the radioactivities were recorded by an external detector as well as at intervals in biopsy material. external detection proved to give lower esti mates of the amount of trapped radioactivity than in vitro measurements. this was particularly so for 51cr. thus only about 2/13 of the in vitro amount of trapped "cr and about 1/3 of the i z 5 i radioactivity were revealed by external detection. external detection, however, records the changes continuously and there fore offers advantages over the biopsy technique, which requires open chest surgery and artificial respiration with consequent alterations in intrathoracic pressure and circulatory conditions. introduction in previous communications from this laboratory, methods for the quantitation of intravascular plate let and fibrin deposition in various organs of rats, rabbits, dogs and humans have been presented ( 3 , 4 , 5, 6 , 7 , 12, 13). isotope-labelled platelets and fibrinogen were injected in advance and the organ deposition following intravenous infusion of throm bin was measured in vivo by external detec tors o r i n vitru by radioactivity measurements in tissue specimens. the in vivo technique was used for continuous registration of deposition of fibrin and platelets in, and their elimination from, the lung after intra venous (i.v.) infusion of thrombin in normal dogs with inhibited fibrinolysis. the method proved to be very useful in elucidating the dynamics of pul monary microembolism, thought it can be postulat ed that in viho measurement o f lung tissue ra dioactivity should give more accurate quantitative information. the aim of the present study was to determine the quantitative differences between the in vivo and the in vitro techniques and hence to define the applicability of each of them. thus, the pulmo nary distribution of radioactivity before and after an i.v. infusion of thrombin into dogs given 99mtc-albumin, t r p l a t e l e t s and t-fibrinogen was studied by the two techniques. material and methods animals. twenty-four healthy german pointer dogs of both sexes, weighing 15-24 kg, were used. labelling of platelets. the day before the experi ment autologous platelets werelabelledwith 5 c r by a modi fication (4) of the method described by aas & gardner ( i ) . each dog was given a suspension of labelled plate lets containing 1 5 4 0 pci labelledfibrinogen. human fibrinogen prepared accord ing to blomback & blomback (2) was used. the label ling was performed according to rosa et al. ( i i ) . each dog was given an i.v. injection of about 60 pci of 9 labelled fibrinogen (5 pci/mg fibrinogen) 2 0 4 0 min before commencement of the thrombin (or saline) infusion. in previous experiments no differences in the kinetics of human and dog fibrinogen were observed (4). labelled albumin. human serum albumin labelled with 9 4 m t ~ (ab atomenergi, studsvik) was used to determine the plasma volume per gram of lung tissue. immediately after passage through an ion exchange column (dowex i-x8, 50-100 'mesh) about 100 pci was injected i.v. ( 1 mci/mg). this was allowed to mix with the plasma vol ume for 5 rnin before the animals were killed (9). thrombin. bovine thrombin (topostasin@, roche) dissolved in saline was used. blood samples were collected through a catheter inserted in the femoral artery (see below). one-ml portions were taken into small ellermann plastic tubes for determination of radioactivity. by i.v. injection. upsola j med sci 79 quantitutive determination o j p u l m o n a v y microembolism in dog 7 3 fin sections were stained with haematoxylin-eosin and the mallory ptah method for demonstration of fibrin. radioautogruphs were prepared from sections adja cent to those used for light microscopy; they were dipped in ilford g5 emulsion and the film was exposed for 3 weeks a t 4°c. the sections were stained with haema toxylin-eosin. % c m 0 1 2 3 4 5 6 c m fig. 1 . absorption of 51cr-radioactivity (left) and of 1251 (right) in water at various distances from the ori fice of the collimator. isocount lines are expressed as percentage of the counting rate at t h e orifice of the collimator. haemutocrit (hct) was determined in triplicate in microhaematocrit tubes after centrifugation at 10 000 g for 5 min. no correction for trapped plasma was made. calculations. in both in vivo and in vitro methods the amount of radioactivity of each isotope in the lung (il(r)) was calculated as il(t)=llt) -clfi where l ( t ) is the counting rate over the lung (or the radio activity per gram lung tissue) at time t and c ( t ) the amount of circulating radioactivity recorded by the detector (or in the lung tissue sample) at time t , estimated as where p ( o ) and p ( t ) are the plasma radioactivities at times the aid of the haematocrits. ments the following standardization was introduced: o and t , calculated from whole blood activities with to allow comparison of results from different experi thus, the amount of radioactivity estimated with either of the described methods was expressed in per cent of the preinfusion radioactivity over (or in) the lung. this calculation requires two assumptions, first that the pulmonary blood volume does not change during the experiment, and second that the extravascular radio activity can be neglected. these assumptions are dis cussed later. statisticul m e t h o d s mean values, standard deviations, linear regressions and correlation coefficients were calculated by con ventional statistical methods. differences between mean values were tested by means of student’s /-test. general experimental procedure the dogs were anaesthetized by intravenous adminis tration of thiopental sodium (pentohalsodiumm, abbott), 10 mg per kg body weight (b.w.). a cuffed endotracheal tube was inserted and the dogs were placed supine. a polyethylene catheter was advanced into the right atrium via the right external jugular vein and another was inserted in the femoral artery. in previous experiments the dogs were anaesthetized with a chloralose dissolved in 10 ml of saline per kg b.w. (4) and to obtain comparative conditions in the present experiments each dog was given this amount of saline before the thrombin infusion (see below). in addition the dogs were pretreated i.v. with 100 mg per kg b.w. of tranexamic acid (amca, kabi. stockholm) to prevent fibrinolysis. an infusion of thrombin, 310 nih units per kg b.w., was given in 30 min through the jugular vein catheter. the dogs submitted to a left sided thoracotomy were given only 150 nih units per kg b.w. because with the higher dose the mortality rate in open chest conditions is high. fig. 2. schematic diagram of the position of the dog’s chest a n d o f t h e detector. lb=lead bricks, sb=sand bags,ll= left lung, r l =right lung, h=heart, lc=lead collimator, morphological m e t h o d s light microscopy. specimens were taken from differ , ent parts of both lungs. formalin fixed 5 thich parafc = n a i (ti)crystal,a=aorta. upsala j med sci 79 74 christer busch 1 0 0 0 1 r = o 803 v w c w fig. 3 . relationship between the estimates of the amount of 51cr-radioactivity trapped in the lungs a s measured in vivo and in x 1 0 0 1, . 2 0 0 l o o 600 * * 1000 n 2 300 w 2 0 0 2 0 0 0 i l ( ' i r e , vitro. 2 -l n c w biopsy arterial blood samples were drawn at regular inter vals for determination of radioactivity, plasma fibrinogen and platelet count. radioactivity measurements recording by exfernal detector. the detector consisted of a 1"xl" sodium iodine crystal with a photomulti plier (harshaw) and a 24 mm lead cylindrical collimator with an inner diameter of 50 mm. the distance between the crystal and the collimator orifice was 63 mm. the sensitivity of 51crand 1251-radioactivity in water a t various distances from the front of the collimator was measured using a point source of radioactivity, giving the isoresponse curve a s shown in fig. 1 . with this detector the radioactivity was monitored continuously over the right lung a s described earlier (4, 5). the dogs were kept immobile by lead bricks. the detector was placed immediately cranial to the edge of the intercostal angel and immediately t o the right of the midline and was directed 10-20" laterally to avoid disturbance from the heart and major vessels. the chest was also rotated about 20" to the left to shift the mediastinal structures from the area used for detection (fig. 2). the signals from the detector were passed t o a linear amplifier, analysed by a discriminator and recorded by a scaler (canberra industries, connecticut, usa). i u w i2 o 0 1 w n 1 a > z ll w + x w 1 2 5 1 r = 0.815 o 0 8 two or three channels were used simultaneously and set for i z 5 i , 9 9 m t ~ and 51cr respectively. normally more than 400 counts per 100 seconds were recorded. the background was less than 1/6 of this value. the counting rates in each channel was corrected for over lapping between the channels and for background. for 99mtc correction was also made for decay; this was not done for rzsi and w r , with their long halflives (60 and 28 days respectively). at autopsy the position of the organs in relation to the detector was checked. radioactivity in tissue and blood samples was meas ured in a gamma counting system (gammatrix c , stock holm) with a well crystal and three windows set for 99mtc, 51cr and iz5i. normally more than 2000 counts were registered in each channel. the background was usually less than 1/10 of the total counting rate. cor rections for overlapping between the channels, for decay of 99mtc and for background were made as described above. recording by gamma camera.' one dog was placed as described above (supine with the thorax rotated ' this part of the investigation was performed with the kind and skilful assistance of k.-j. vikterlof and k.-w. beckman, department of radiophysics, regional hospital, orebro, sweden. fig. 4 . relationship between the estimates of the amount of 1251-radioactivity trapped in the lungs as measured in vivo and in i l ( ' i r e l vitro. 1 0 0 200 3 0 0 b i o p s y llpsala j med sci 79 quantitative determination of pulmonary microembolism in dog 75 5 30 60 m i n thrombin f i g s . 5 and 6 . pulmonary deposition of t r (fig. 5 ) and 1z5i (fig. 6) (il(r)re, as measured in vitro (-) and in v i v o (---) as well as the percentual changes recorded by the about 20" to the left) and was given about 150 pci of 13'i-labelled human fibrinogen i.v. a 1 000-hole, diver gent collimator was placed over the thorax in the same direction as t h e detector (described above). the radioactivity distribution was recorded by a gamma camera (selektronik, denmark). the information was stored, treated and presented by an on-line com puter system (nucab 2530) incorporating a pdp 8/e computer with 12 k core memory and a cartridge memory for storing programme and pictorial informa tion. the activity distribution was registered as con secutive 64 x64 matrices during 100-second intervals. the entire right lung, the base of the same lung and the heart were selected as "regions of interest" and the radioactivity of these regions detected during the 100-second intervals was listed digitally and analogously plotted by the computer. e x p e r i m e n t s a n d r e s u l t s i . comparison of in vivo and in vitro determi nation of pulmonary microembolism a . six dogs were given 51cr-labelled platelets and '251-labelled fibrinogen as described. t h e y were ventilated by a n engstrom respirator and a left sided thoracotomy was performed so as t o expose the base of t h e left lung f o r biopsies. t h e radio % 300 200 1 0 0 1 2 5 6 30 thrombin 6 0 ~ min detector without correction for plasma decrease of the radioactivity (-). activity over the right lung was recorded con tinuously and after administration af a m c a t h e thrombin w a s infused in 30 min. small biopsies (0.3-1.0 g) were taken repeatedly a t intervals of 5-10 min during t h e infusion and in o n e dog for a further 50 min. t h e biopsies were taken from the peripheral parts of t h e lower lobe with the aid of vessel clamps and ligatures. t h e samples were weighed a n d t h e radioactivities determined. as c a n b e seen in figs. 3 and 4, a n almost linear relationship between t h e estimates of trapped radioactivity of both slcr and lz5i was obtained. t h e correlation coefficient was 0.803 for t r (n=19) and 0.815 (n=27) for ln51. t h e uptake reg istered b y external detection was considerably lower than that measured in vivo. t h u s , f o r t r , the in vitro values were about 6.5 times higher than the in vivo estimates for lz5i about times higher. in figs. 5 and 6 t h e estimates of pulmonary de position of w r and lz5i (il(t)re,) in o n e of the dogs as measured in vitro and in vivo, as well a s t h e percentual changes recorded b y the external detector without correction f o r decrease of plasma radioactivity, a r e plotted against time. t h e quanti upsala j med sci 79 76 christer busch table i . the rcrtios bet,t,een the skr-rudioactivity per gram tissue at the end of the thrombin infusion and that per gram plasma before the infirsion (mean ?s.d.) no. of dog no. tissue 1 2 3 4 5 samples skin 0.064k0.005 0.038+0.010 0.007k0.003 0.052r+0.014 5 skeletal muscle 0.014t0.003 0.040?0.016 0.001 20.007 0.008k0.007 5 bone-cartilage 0.153~0.021 0.092t0.022 0.065?0.012 0.162t0.053 5 lung, ventral part 6.559-el.086 3.885to.615 5.759?0.335 2.163t0.195 3.291 50.955 15 lung, dorsal part 5.138k1.347 3.692k0.495 5,61820.394 2.135-eo.283 3.576?1.117 is tative differences between the techniquesare shown, a s well as the unexplained deviation of the amount of label in the tissue specimens. b. five dogs were treated in the s a m e way as those under “ a ” except that they were allowed to breathe spontaneously and a higher dosage of thrombin (310 nih units per kg b.w.) was used. after completion of t h e thrombin infusion the dogs were killed with an overdose of thiopental sodium. small pieces (0.3-1.0 g) were taken from the ventral and t h e dorsal parts of the right lung (15 samples from each region) as well a s from the skin, skeletal muscle (intercostal) and bone and cartilage from the ribs ( 5 samples from each tissue). samples were also taken from different parts of the lung for morphological studies. t h e radioactivities of t h e tissue and corresponding plasma samples were determined. t h e ratio be tween tissue radioactivity per gram and the pre infusion plasma activity per gram was calculated for each sample. t h e radioactivity of both isotopes was seen to b e evenly distributed in different parts of the lung (tables i and 11) and t h e microemboli were found t o b e localized in small pulmonary arterioles and capillaries (fig. 7 and 8). no emboli were found in the large vessels o r in the heart at autopsy. t h e amount of radioactivity in the thoracic wall tissues did not increase during the thrombin infusion (tables i and 11). 11. distribution of 99mtc-albumin, 51cr-platelets and iz5z-fibrinogen before the thrombin infusion t h r e e dogs given ”cr-platelets and l z 5 1 f i brino gen were injected i.v. with 100 p c i gymtc-albumin. ionogenic 99mtc was removed by a n ion exchange column (dowex 1-x8, 50-100 mesh) immediately before the infusion. after s-min mixing, which was followed by t h e external detector, the dogs were killed with a n overdose of thiopental sodium. at this time the 99mtc-albumin should have been distributed homogeneously in the vascular com partment (9). t h e lungs were removed and i5 small pieces from the ventral and 15 from the dorsal parts of t h e right lung were transferred into plastic tubes after gentle drying o n filter paper. furhermore, tissue samples from the thoracic wall (0.5-1.0 g), liver, kidney and spleen were collected analogously with those mentioned a b o v e , and the radioactivity was measured. t h e content of each label per gram was determined in the tissue specimens and in plasma. table 11. the ratio between the ~z51-radioactivity per gram tissue at the end of the thrombin infusion and that per gram plasma immediately before the thrombin infusion ( m e a n 5 s . d . ) dog no. no. of tissue i 2 3 4 5 samples 0.043?0.004 5 skin 0.018~0.001 0.01450.002 0.01 1 k0.002 skeletal muscle 0.014?0.001 0.01520.002 0.011 ?0.001 0.02520.002 5 bone-cartilage 0.059?0.013 0.030k0.009 0.044?0.004 0.019?0.010 5 lung, ventral part 2.08920.284 1.115ko.176 1.499?0.170 2.65121.402 1.32650.220 15 lung, dorsal part 1.78320.326 1.14150.151 1.34620.362 2.926t0.965 1.57450.242 is upsala j med sci 79 quantitative &termination of pulmonary microembolism in dog 77 f o r none of t h e labels did the tissue/plasma ra tios show a significant difference between different parts of the lung (tables 111-v). t h e ratio for lz5i were lower than those of 99mtc in the lungs, liver a n d kidneys. t h e 51cr ratios were higher than those for 1451 and 9 y m t ~ in t h e spleen, liver and b o n e c a r t i l a g e a s well as in the lung (table 111). 111. statistical evaluation of in vivo external de tection of pulmonary microembolism a . three dogs' were injected with 5lcr-labelled platelets and lz51-labelled fibrinogen as in previous experiments. after a n injection of 20 ml saline thrombin was infused as before into the jugular vein in 30 min (310 nih units per kg b.w.). ex ternal detection was carried out o v e r right lung for a further 5 1/2 hours. during this time t h e animals were given another 50 ml of saline. b. five dogs1 were injected with amca 100 mg per kg b.w. about 15 min before an infusion of , fig. 7. fibrin microemboli in pulmoi arterioles and capillaries (ptah, x 1 iary 150). thrombin identical t o that above. after t h e 30-min infusion another 100 mg of amca per kg b.w. dissolved in 50 ml of saline was given o v e r 5 1/2 hours. t h e radioactivity was monitored a s in group a. the amount of 51cr and lz5i radioactivity trapped in t h e lung a s measured by t h e in vivo method (il(t)re,) was plotted against time (figs. 9 a n d 10). c. t w o dogsz were given 20 ml saline and another t w o dogs 50 mg amca per kg b.w. a s a single, rapid i.v. injection. 1251-fibrinogen was also given in advance and external detection was then performed as described (fig. 1 i ) . one of t h e main purposes of the in vivo method was t o measure differences in the rate of elimi nation of the labelled microemboli from the i the results of these experiments have been pre sented in detail elsewhere (4). * the results of these experiments have been pre sented in detail elsewhere ( 5 ) . upsala j med s c i 79 7 8 christer busch lung. in figs. 9 and 10 it is visually evident that amca treatment delayed the elimination of both 1251-fibrin and 5*cr-platelets. this effect was most pronounced from one to 3 hours after completion of the thrombin infusion. the tails of the curves show high coefficients of variation and/ or low numbers of observation. fig. 8 . radioautograph of microemboli (within arrows) in small pulmonary vessels (haematoxylin-eosin, ~ 3 0 0 ) . fig. i i shows that even a lower dose of amca than was used in previous experiments and given as a single rapid injection caused retardation of the elimination as compared with dogs with nor mal fibrinolysis. in dogs given 200 mg amca per kg b.w. the pre-infusion counting rate was usually not regained during the 6-hour observation period. table 111. the ratios between 51cr-rudioa~tivity per grum tissue and per grum piusma before the thrombin infusion (mean fs.d.) dog no. n o . of tissue i 2 3 samples skin skeletal muscle bone-cartilage liver kidney spleen lung, ventral part lung, dorsal part 0.03 1 k0.021 0.03120.014 0.19520. 160 3.92450.643 0.262 20.0 19 25.074 0.491 k0. 193 0.660 20.405 0.014?0.007 0.015?0.003 0.04620.018 0.69720.006 0.174-to.013 12.364 0.191 20.038 0.163 50.050 0.017k0.003 0.01320.002 0.064 k0.023 0.56320.018 0.134k0.019 17.823 0.26220.046 0.22520.023 5 5 5 2 2 1 15 15 upsala j med sci 79 quantitative determination of pulmonary microembolism in dog 19 table i v . the ratios between ‘251-radioactivity per gram tissue and p e r gram plasma before the thrombin infusion ( m e a n + s . d . ) ~~ ~~ dog no. n o . of tissue i 2 3 samples skin skeletal muscle bone-cartilage liver kidney spleen lung, ventral part lung, dorsal part 0.02520.002 0.01 520.003 0.02720.00 i 0.13720.001 0.17620.013 0.097 0.160?0.024 0.156?0.022 0.020 20. 004 0.01620.003 0.03 1 20.002 0.108~0.001 0.22620.004 0.170 0. i1520.01 i 0.11520.019 differences in the elimination rate can also be expressed as differences in the percentual amount of the trapped radioactivity at various time points after the maximum value. such figures (il(t)re,/ 1l(0.5)re,) for groups a and b are shown in ta ble vi. thus, significant differences in the ratios were found at 60, 90 and 120 min after commencement of the thrombin infusion for both isotopes, while the tail values did not differ significantly. iv. recording by gamma camera one dog was given 150 pci 1311-fibrinogen and a 30-min thrombin infusion. after about 1 hour’s registration of the radioactivity changes about 1 mci of 99mtc-labelled macroaggregated human serum albumin (ae3 atomenergi, studsvik) was injected into the jugular vein catheter s o that the contours of the lungs and heart were visualized. the entire right lung, the base of the same lung and the heart were taken as “regions of interest” and the radioactivity recorded i n 0.023+0.002 5 0.018 20.002 5 0.05120.012 5 0.191 20.014 2 0 . 1 6 2 ~ 0 . 0 1 1 2 0.157 i 0.1 1320.013 15 0.115t0.013 15 these regions during the 100-second intervals was listed digitally and plotted analogously by the computer. the results are given in fig. 12. it is evident that the gamma camera technique was less effici ent in revealing the true increase of pulmonary radioactivity than the lead collimated detector placed over a smaller field of the right lung. the pictures also show that the shifting of the chest to the left also shifts the mediastinal struc tures, thus giving a large free field for detection at the base of the right lung. discussion the present study has shown that quantitative determination of labelled pulmonary microemboli by external detection in dogs gives lower estimates of the trapped radioactivity than in vitro measure ment. the differences are probably due to the fact that the radioactivities measured emanate from table v. the ratios between 99mtc-radioactivity per gram tissue and per gram plasma before the thrombin infusion (mean l s . d . ) dog no. n o . of tissue i 2 3 samples skin 0.0 18 20.003 skeletal muscle 0.01 5 20.004 bone-cartilage 0.032t0.014 liver 0.127?0.002 kidney 0.292 to.0 i0 spleen 0.070 lung, ventral part 0.18020.025 lung, dorsal part 0.161 20.018 0.041 20.018 0.021 20.005 0.040 20.004 0. 12720.001 0.335 c0.010 0.149 0.15520.010 0.15020.009 0.02220.002 5 0.01 9?o.o01 5 0.056 20.0 i 2 5 0.217?0.013 2 0.314t0.014 2 1 0.14520.016 15 0.14020.013 15 upsala j med sci 79 80 christer busch 9 =saline + thrmbb 0 = amca + thrombin 10 zoo =saline +thrombin 0 =amca + thrombin $ l 2 150 . l u ? * k? 9 2 figs. 9 and 10. pulmonary deposition (il(t)re,) of 5'cr-radioactivity (above) and lz51-radioactivity (beiow) in dogs pretreated with amca, 200 mg per kg b . w . ( 0 ) and dogs with normal fibrinolysis (0). 100 , 5 0 hours thrombin different sources in the two techniques. thus, the external detector records activity originating in all thoracic tissues, the limits and extent of the tissue field depending on the collimating properties of the detector and on the energy of the radio active isotope employed (fig. i ) . the counting rate is an expression of the sum of radioactivity from the chest wall, peripheral lung tissue (where the deposition actually occurs), large vessels and perhaps also from the right heart (in spite of the preventive measures). the biopsy specimens consist of peripheral lung tissue only, thus with no disturbance form the blood of large vessels. thur the decrease of radio activity in the blood of large vessels gives a smaller net accretion when measured with the external technique than when measured in vitro on tissue samples. this difference was more pronounced for 51cr than for only about 2/13 of the in vitro estimate was recorded externally, as compared with about 1/3 for the it is conceivable that the 51cr radioactivity with its higher gamma energy (0.320 mev), emanates to a greater extent from the large central vessels than the radio activity of iz5i (0.035 mev) (fig. i ) . the pulmonary microemboli were shown to be homogeneously distributed in the arterioles and capillaries, while at autopsy no emboli were found in the heart or large pulmonary arteries. further more, no significant increments of the 51cr or lz5i radioactivities were observed in the various parts of the chest wall. thus an uneven distribution of deposits in the tissues mentioned did not influence the measurements. the related differences are also illustrated in upsala j med sci 79 quantitative determination of pulmonary microembolism in dog 8 1 w d > r > t 0 z? 4 z & t o be about 30% (6). in the present experiments this amount should be considerably smaller, since only 2 0 4 0 min elapsed between the administratiop of t h e labelled fibrinogen and the thrombin infusion. a large amount of 51cr radioactivity was recover i-, , , , , , , , ed in the spleen, liver and bone-cartilage. further 2 3 4 more, the lung/plasma ratio of 51cr was higher hours thrombin 1 fig. / i . pulmonary uptake and elimination of lz5i in dogs pretreated with a m c a , 50 mg per kg b.w. (-) and in dogs pretreated with saline (-). figs. 7 and 8, where the estimates of pulmonary trapping of t h e radioactivities are presented as well as the changes recorded by the detector without correction f o r plasma decrease. in spite of the occasional variations in t h e tissue sample radio activities the in vitro and in v i v o methods a r e seen to describe t h e same time course, which further indicates that they reflect the same patho physiological events. t h e distribution of the different isotopes be fore t h e thrombin infusion in the present study revealed some interesting facts. firstly, the tissue/ plasma ratios for all isotopes were homogeneously distributed in the different parts of the lung. second ly, the ratios of 99mtc were higher than those of iz5i in t h e lungs. kidneys and liver. this than that of the two other isotopes indicating that this isotope and/or labelled platelets o r fragments of platelets a r e retained in these organs on re-infusion of t h e suspension of y r platelets. t h e accumulation of 51cr in the liver, spleen and bone might b e d u e t o the action of the re ticuloendothelial system, while the accretion in the lung might indicate trapping of aggregates formed during the labelling procedure. these high lung/ plasma ratios introduce a n error in the estimation of the amount of circulating 51cr activity be fore the thrombin infusion ( p ( t j 0 ) . since, how ever, the tissue/plasma ratio after the thrombin infusion ( l ( t ) / p ( t ) , ) was about 14 times that be fore the infusion ( l ( t ) o / p ( t ) o ) , and the correction made f o r circulating radioactivity ( c ( t ) w a s rather small because of t h e decreased plasma radioactivity ( p ( t ) ) , this error was neglected. the increase in counting rate over the lung during the thrombin infusion might theoretically be d u e t o a n increase in t h e pulmonary plasma volume. in a n earlier study using 12si-labelled table vi. the ratio between the cimount of 5lcr and iz5i tripped in the lungs a t various time points after the thrombin infusion a t the amount at the end o f t h e infusion (il(t)re,/ll(0.5)re, ( m e a n m . d . ) 1251 51cr saline+ a m c a + saline + a m c a + minutes after thrombin thrombin thrombin thrombin thrombin infusion &ks.d. x,?s.d. p x 3 2 s . d . x,ks.d. p start of the 60 0.327k0. 122 0.869k0.046 <0.001 0.427k0.052 0.698k0.069 <0.01 90 0.274k0.082 0.781 k0.066 <0.001 0.314k0.045 0.54220.097 <0.02 120 0.312k0.146 0.672k0.099 <0.02 0.15220.092 0.36420.062 c0.05 180 0.397k0.200 0.59720.123 non-sign. 0.154k0.058 0.244k0.037 non-sign. 240 0.379k0.245 0.574+0.133 non-sign. 0.13220.070 0.212?0.055 non-sign. 6 142855 upsala j m e d sci 79 82 christer busch w 3 a > ll 0 z 75l 75 i 0 i i 500 1000 1500 2000 2500 3000 3500 seconds 751 t h rom 81 n albumin and external detection, no such increment was observed, however (4). the gamma camera system did not prove to be more sensitive than recording by the conventional detector. the former requires high doses of high energy radioisotopes for adequate discrimination and again the influence of decreasing plasma radio activity, especially in the large vessels, must affect the recordings. this is supported by the fact that some improvement was obtained by se lecting only the base of the right lung as the “region of interest”. 1311 is not an ideal radioisotope for studies with gamma camera. it is possible that the use of more suitable isotopes such as iz31 which has not been available to us could increase the sensi tivity and hence make monitoring by gamma camera suitable and possible for study also of human microembolism. the results of the external detection in this study are i n accordance with those of saldeen (13), who used a similar technique on rabbits given 1311-labelled fibrinogen and thrombin i.v. only a small, though significant increase was detected over the lung. coombey & tyler (8), using 1251-fibrinogen, infused 50 units of throm bin via a sublingual vein i n 24 min into rats. the fig. 12. gamma camera pic tures of the anatomical rela tionship in the dog’s chest and the “regions of inter est” ( , left). the radio activity changes in each re gion (% of preinfusion values) are shown to the right. radioactivity was recorded externally over the lung. the increase was about 30% above the baseline level. according to o u r experience from rats (6), however, such a thrombin infusion will give a true in vitro. increase in lung radioactivity of several hundred percent, which is another example of the differences between in vitro and in vivo tech niques. in conclusion, external determination is a re producible method particularly suited for con tinuous study of the dynamics of pulmonary micro embolism. the method can also be used for simul taneous registration of the radioactivity in other organs such as the liver and kidneys (5). it pro vides definite advantages over the biopsy tech nique, which limits the number of observations and occasionally shows considerable variations between different samples. these variations might be due to altered intrathoracic pressure and cir culatory conditions following the thoracotomy and artificial respiration. the sensitivity of the external method is limited, however, and in states with an expected low de gree of intravascular coagulation the biopsy tech nique should be used. in investigations of humans with intravascular coagulation and microembolism the biopsy method llpsala j med sci 79 quantitative determination of pulmonary microembolism in dog 8 3 is not practicable. h e n c e t h e external m e t h o d h a s r e c e i v e d n o v e m h e r 5 , 1973 been e m p l o y e d in t h e s t u d y of p u l m o n a r y fibrin and platelet deposition a s s o c i a t e d with t r a u m a . in t h e s e cases e v i d e n c e of transient (10) or progressive (3) a c c u m u l a t i o n o f isotope h a s b e e n o b s e r v e d . address for reprints: department of forensic medicine 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quantitation of fibrin deposition and elimination in organs of rats injected with labelled fibrinogen and albumin. thromb diathes haemorrh (stuttg.) 29: 94, 1973. busch, c. & rammer, l.: quantitation of fibrin deposition and elimination in organs of rats in jected with labelled fibrinogen by isolation of the fibrin from water soluble tracer. thromb diathes haemorrh (stuttg.) 29: 108, 1973. coombey, d. & tyler, h . m.: quantitative moni toring of intra-vascular coagulation and fibrinolysis in the lungs of rats. thromb diathes haemorrh (stuttg.) 27: 241, 1972. huggins, r. a,, smith, e. l. & deavers, s . volume distribution of evans blue dye and iodinated albumin in the dog. am j physiol2m: 35 i , 1963. modig. j., busch, c., olerud, s. & saldeen, t.: evidence of platelet and fibrin deposition in the lungs during intra-medullary orthopaedic trauma. to be published. rosa. u., scasselatti, g . a. & pennisi, f.: label ling of human fibrinogen by electrolytic iodination. biochem biophys acta (amst.) 86: 519, 1964. saldeen, t.: experimental investigation on fat em bolism and intravascular coagulation. acta path microbiol scand 66: 271, 1966. saldeen, t. quantitative determination of intra vascular coagulation in the lungs of experimental animals. scand j haemat6: 205. 1969. s-75 1 23 uppsalh’i sweden upsala j med sci 79 upsala journal of medical sciences 2021, 126, e7848 http://dx.doi.org/10.48101/ujms.v126.7848 accuracy in detecting major depressive episodes in older adults using the swedish versions of the gds-15 and phq-9 johnny pellasa,b* and mattias damberga,b adepartment of public health and caring sciences, uppsala university, uppsala, sweden; bcentre for clinical research, uppsala university, västmanland county hospital, västerås, sweden abstract objectives: the purpose of this study was to evaluate the diagnostic accuracy at different cut-off values for the swedish versions of the 15-item geriatric depression scale (gds-15) and patient health questionnaire (phq-9) compared with a structured clinical psychiatric interview in older adults. methods: community-dwelling participants (n = 113) aged 65 years or older completed the swedish versions of the gds-15 and phq-9 and were then interviewed using the mini international neuropsychiatric interview (mini) to establish the presence or absence of current major depressive episodes (mdes). areas under the curve (auc) were calculated for each scale, as well as the sensitivity, specificity, and youden’s index for different cut-off values. results: seventeen participants met the criteria for mdes. the auc was 0.97 for the gds-15 and 0.95 for the phq-9. a cut-off of ≥6 on the gds-15 yielded a sensitivity of 94%, a specificity of 88%, and a youden’s index of 0.82. a cut-off of ≥5 on the phq-9 yielded a sensitivity of 100%, a specificity of 81%, and a youden’s index of 0.81. the proposed cut-off of ≥10 on the phq-9 produced excellent specificity of 95% but a lower sensitivity of 71%. conclusions: this study indicates that the swedish versions of the gds-15 and phq-9 have comparable accuracy as screening instruments for older adults with mdes. however, the proposed cut-off of 10 on the phq-9 might be too high when applied to older individuals in sweden, and further investigations in larger samples in different healthcare settings are warranted. article history received 27 april 2021 revised 18 august 2021 accepted 14 september 2021 published 20 october 2021 keywords depression; geriatric; validation; screening; rating scales introduction in sweden, the prevalence of depression in adults aged 60 years or above is about 6% in community settings (1) and 15% in primary care (2). depression in older adults increases the risk of mortality and morbidity (3), leads to functional impairments (4), and reduces quality-of-life (5) and is therefore important to identify and treat. using depression questionnaires may increase the depression detection rate and thereby increase the number of people with depression receiving treatment (6). the swedish agency for health technology assessment and assessment of social services recommends the use of the 15-item geriatric depression scale (gds-15) for depression screening in older adults (7, 8) and the patient health questionnaire (phq-9) for adults in general (9, 10). the gds was originally a 30-item questionnaire for depression screening in older adults but was modified into a shorter questionnaire consisting of 15 items (11). a meta-analysis revealed a sensitivity of 89% and a specificity of 77% for the gds-15 with a cut-off of >5 (12). the gds-15 has been translated into swedish, and a 20-item version, the gds-20, has also been developed, which includes additional items of insomnia, anxiety, panic, pain, and somatization (13). the swedish gds-15 has acceptable reliability and validity at different levels of cognitive functioning (14). a study by sacuiu and colleagues (15) reported a sensitivity of 71% and a specificity of 93% when using a cut-off of 9, but the diagnosis was made using another rating scale, and the sample consisted of older individuals who had made a suicide attempt 1 year before and thus constituted a psychiatric sample that may not apply to individuals living in community settings. the phq-9 is a nine-item screening questionnaire used to identify depression and to measure its severity (16). a metaanalysis revealed a sensitivity of 88% and a specificity of 78% at a cut-off of 10 or above (9). the swedish phq-9 has acceptable reliability and validity (17). there are, to our knowledge, no published studies that have compared the swedish versions of the phq-9 and gds-15 in older individuals and no reports of the diagnostic accuracy that have used a structured clinical interview as a reference test for contact johnny pellas johnny.pellas@regionvastmanland.se © 2021 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article http://dx.doi.org/10.48101/ujms.v126.7848 mailto:johnny.pellas@regionvastmanland.se http://creativecommons.org/licenses/by/4.0/ j. pellas and m. damberg the swedish versions of phq-9 or gds-15 in older adults. therefore, the aim of the present study was to evaluate the diagnostic accuracy at different cut-off values for the swedish gds-15 and phq-9 compared with a structured clinical interview in older adults. materials and methods design the present study was a retrospective, cross-sectional diagnostic accuracy study. the sample was a convenience sample pooled from two separate trials: 1) n = 77 participants were included from the psychiatric syndromes in late life – assessment and treatment study, pllat, a trial aimed at validating swedish versions of psychiatric measures, using data collected in 2019–2020, and 2) n = 36 participants were included from the covidep-study, a trial of telephone-based psychological treatment for depressive symptoms in older individuals in isolation during covid-19, using data collected in 2020 (clinicaltrials id nct04508868). both studies received ethical approval from the swedish ethical review authority (registration numbers 2019-00944 and 2020-02079). all participants were recruited from the county of västmanland in sweden and were residing in the community. all participants provided written informed consent. participants the participants were approached through organizations for senior citizens in the county of västmanland as well as via advertisements in local newspapers. the inclusion criteria were 1) 65 years old or above, 2) fluent in spoken and written swedish, and 3) willing to participate in the trial. for the covidep-study, the participants also experienced low mood and/or diminished interest in activities. the exclusion criteria for both the trials were a current substance use disorder, current diagnosis of dementia/major neurocognitive disorder, and current diagnosis of a neurological condition or severe visual impairment (not able to read the questionnaires). in the pllat-study, there was also a lower limit of 25 points on the cognitive screening test mini-mental state examination, which could not be applied in the covidep-trial because all contact was by telephone. materials mini international neuropsychiatric interview the mini international neuropsychiatric interview 7.0 (mini) (18), a structured clinical interview, was used as a reference test to assess the presence or absence of major depressive episodes (mdes) and/ or other common psychiatric disorders according to the diagnostic and statistical manual of mental disorders 5th edition (dsm-5). the mini has high reliability and validity (18) and a sensitivity of 95% and a specificity of 84% compared with the structured clinical interview for dsm-iv-axis-i disorders (scid-i) (9). geriatric depression rating scale 15 the gds-15 (11) is a questionnaire used to identify depression in older individuals with scores ranging from 0 to 15. patient health questionnaire-9 the phq-9-item is a questionnaire used to identify depression and its severity, with scores ranging from 0 to 27, with higher scores indicating higher depression severity (16). procedure the procedure differed between the two samples because of the covid-19 pandemic. the participants from the pllat-trial came to the study research clinic and filled out the rating scales. they were then interviewed by a clinical psychologist (demographic data and mini) on the same day. participants from the covidep-trial performed rating scales at home and were interviewed by a clinical psychologist by telephone (demographic data and mini). only the participants who were interviewed within 2 weeks of performing the rating scales were included from the covidep-trial. a research nurse scored the rating scales to ensure that the psychologists were blinded to the results. all interviews in the pllat-trial were performed by the same psychologist (the corresponding author), whereas additionally four psychologists performed the interviews in the covidep-trial. all psychologists were trained in administering the mini. the diagnosis of depression was made using the mini algorithm for current mdes, according to the dsm-5. analyses plan diagnostic accuracy was calculated with sensitivity and specificity for different cut-off values, as well as the area under the curve (auc). we chose 70% as the minimum level of sensitivity and specificity. optimal cut-off values were determined using youden’s index (sensitivity + specificity – 1). results a total of 113 participants were included in the study. participant flow is described in figure 1. based on the diagnostic procedure, 17 participants were classified as having a current mde. baseline demographic and clinical characteristics are documented in table 1. none of the participants were receiving specialized psychiatric care. the auc was 0.97 for the gds-15 and 0.95 for the phq-9. sensitivity and specificity values for different cut-off points are shown in table 2, illustrating the cut-off values with at least 70% sensitivity and 70% specificity, as well as the proposed cut-off values. according to youden’s index, the optimal cut-off was 6 points and above for the gds-15 and 5 points and above for the phq-9 prioritising sensitivity. cross tabulation for the proposed cut-off value of ≥6 for the gds-15 appears in table 3, for the proposed cut-off value of ≥10 for the phq-9 in table 4, and for the optimal cut-off value of ≥5 for the phq-9 in table 5. accuracy of the swedish gds-15 and phq-9 table 1. demographic and clinical characteristics. total sample (n = 113) age, mean (sd) years 75.65 (6.1) women, n (%) 83 (73.5) major depressive episode, n (%) 17 (15) gds-15, mean (sd) 3.24 (3.8) phq-9, mean (sd) 4.39 (5.5) sd: standard deviation; gds-15: geriatric depression rating scale 15-item short form; phq-9: patient health questionnaire 9. table 2. sensitivity, specificity and youden’s index of gds-15 and phq-9 at different cut-off points. instrument and cut-off point sensitivity (%) (ci) specificity (%) (ci) youden’s index gds-15 ≥4 100 (80–100) 76 (66–84) 0.76 ≥5 100 (80–100) 81 (72–88) 0.81 ≥6 94 (71–100) 88 (79–93) 0.82 ≥7 88 (64–99) 91 (83–96) 0.79 ≥8 82 (57–96) 93 (86–97) 0.75 ≥9 71 (44–90) 97 (91–99) 0.68 phq-9 ≥4 100 (80–100) 72 (62–81) 0.72 ≥5 100 (80–100) 81 (72–88) 0.81 ≥6 88 (64–99) 83 (74–90) 0.71 ≥7 88 (64–99) 86 (78–93) 0.74 ≥8 88 (64–99) 93 (86–97) 0.81 ≥9 82 (57–96) 93 (86–97) 0.75 ≥10 71 (44–90) 95 (88–98) 0.66 gds-15: geriatric depression rating scale 15-item short form; phq-9: patient health questionnaire 9; ci: 95% confidence interval. note. bold cut-off values indicate the optimal balance of sensitivity and specificity based on youden’s index prioritising sensitivity, whereas italicized cut-off values represent the proposed cut-off values. table 3. cross tabulation of major depressive episode and the geriatric depression scale 15 at ≥6 points. gds-15 major depressive episode total yes no ≥6 16 12 28 <6 1 84 85 total 17 96 113 gds-15: geriatric depression rating scale 15-item short form. table 5. cross tabulation of major depressive episode and the patient health questionnaire 9 at ≥5 points. phq-9 major depressive episode total yes no ≥6 17 18 35 <6 0 78 78 total 17 96 113 phq-9: patient health questionnaire 9. table 4. cross tabulation of major depressive episode and the patient health questionnaire 9 at ≥10 points. phq-9 major depressive episode total yes no ≥10 12 5 17 <10 5 91 96 total 17 96 113 phq-9: patient health questionnaire 9. discussion this is, to our knowledge, the first study to compare the diagnostic accuracy of the swedish gds-15 and phq-9 in older adults and the first study to use a structured clinical interview as a reference standard for these tests in swedish. the results indicate that the gds-15 and phq-9 have comparable diagnostic accuracy in classifying older adults with mdes. however, the proposed cut-off of 10 on the phq-9 might be too high for the application to older adults in sweden, a conclusion in line with studies of older adults in other countries that have reported an optimal cut-off of 6 (19, 20), and despite other studies that have found the proposed cut-off value to be optimal (21). our findings highlight the importance of further studies of the appropriate cut-off on the phq-9 because it is widely used and recommended for use in primary health care in sweden (10). the difference between the cut-off values in different countries may reflect cultural differences but may also reflect the use of different settings, populations, and age groups. in this study, none of the participants received psychiatric care and were all recruited from the community. there are several limitations to this study. firstly, the total sample was pooled from two trials with differences in the procedure; 77 participants performed the mini face-to-face directly after filling in the rating scales, whereas 36 participants filled in the rating scales at home and performed the mini over assessed for eligibility (n = 145) -covidep (n = 65) -pllat (80) included (113) -covidep (n = 36) -pllat (n = 77) current major depressive episode (n = 17) no current major depressive episode (n = 96) excluded (n = 32) -declined to participate/ did not consent (n = 17) -did not meet inclusion criteria or met exclusion criteria (n = 3) -missing data (n = 9) -> 2 weeks between index test and reference test (n = 3) figure 1. participant flow. j. pellas and m. damberg the telephone within 2 weeks. however, the mini has been found to produce equivalent results when administered via the telephone compared with in-person interview (22), and a maximum of 2 weeks between the index test and reference test has been allowed in a recent meta-analysis of the gds (12). secondly, in the pllat-trial, all interviews were administered by the same psychologist, whereas additionally four psychologists administered the interviews in the covidep-trial. although all psychologists were experienced in administering the mini and the interview is highly structured, we did not investigate the inter-rater reliability. thirdly, the samples differed in that the covidep-participants were recruited for a depression treatment trial and thereby subjectively depressed, whereas the pllatsubjects were not recruited based on subjective feelings of depression. this might contribute to a spectrum effect and was accounted for by including participants with subclinical depressive symptoms in the control group and not excluding participants with other psychiatric conditions. none of the participants were receiving specialized psychiatric care, suggesting that no cases of more severe depression were included. finally, although using a structured clinical interview as a reference test is considered a strength in diagnostic accuracy studies, it might be considered potentially problematic when used with older individuals because the symptoms in depression might differ from those in younger and middle-aged adults, with older adults more often fulfilling the criteria for minor depression than major depression (7). there is, however, no consensus about the differences in symptoms of depression between younger and middle-aged adults and older adults (23), nor is there any consensus on specific diagnostic criteria for depression in older individuals. nonetheless, future studies could use, for example, a diagnostic procedure based on the longitudinal, expert, all data-procedure as a reference standard, as suggested by others (7). in summary, our study indicates that the swedish versions of the gds-15 and phq-9 are viable options for case finding of mdes in older adults. however, while the cut-off of ≥6 on the gds-15 seems optimal, the cut-off on the phq-9 may need to be lowered to ≥5 instead of ≥10. further studies are needed to evaluate the accuracy of the gds-15 and phq-9 for older adults in different care settings. acknowledgements we wish to thank research nurses, marie stenius-svensson and angelica norling, for helping with the data collection and providing practical assistance throughout this study. data access data are not available because of legal issues. disclosure statement the authors declare no conflicts of interest. funding this study was funded by a research grant from the county of västmanland. notes on contributors johnny pellas, msc. clinical psychologist at västmanlands sjukhus västerås, sweden, and phd student at uppsala university, sweden. mattias damberg, md, phd. senior consultant physician at västmanlands sjukhus västerås, sweden, and associate professor at uppsala university, sweden. orcid johnny pellas https://orcid.org/0000-0002-0707-0832 references 1. karlsson b, 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http://dx.doi.org/10.1017/s1041610219001650 http://dx.doi.org/10.1046/j.1525-1497.2001.016009606.x http://dx.doi.org/10.3399/bjgp09x454070 http://dx.doi.org/10.3389/fpsyt.2020.555011 http://dx.doi.org/10.1016/j.ijge.2016.05.002 http://dx.doi.org/10.1136/bmj.l1476 http://dx.doi.org/10.1017/s1041610217001752 http://dx.doi.org/10.1016/j.jagp.2017.06.011 upsala j med sci 92: 65-73, 1987 chronic renal failure in khartoum, sudan e. m. osman,' 0. i. abboud' and b . g. danielson2 'university of khartoum, khartoum, sudan and 2department of internal medicine, university hospital, uppsala, sweden abstract a study o f t h e c l i n i c a l p r e s e n t a t i o n and c o n c i e v a b l e causes o f c h r o n i c r e n a l f a i l u r e ( c r f ) i n 6 1 sudanese p a t i e n t s i n khartoum i s presented. the c l i n i c a l f e a t u r e s i n v o l v e d almost a l l t h e systems, however, g a s t r o i n t e s t i n a l and c a r d i o v a s c u l a r s i g n s and symptoms predominated. the causes o f c h r o n i c r e n a l f a i 1 u r e i n sudan and sweden a r e a l s o s t u d i e d f o r comparison. the causes o f c r f i n sudan a r e c h r o n i c g l o m e r u l o n e p h r i t i s , o b s t r u c t i v e nephropathy ( s t o n e disease), h y p e r t e n s i o n and d i a b e t e s m e l l i t u s i n t h a t o r d e r (1). the main causes o f crf i n sweden a r e c h r o n i c g l o m e r u l o n e p h r i t i s , diabe t e s m e l l i t u s and c h r o n i c p y e l o n e p h r i t i s . o f t h e 6 1 sudanese p a t i e n t s 16 have kidney t r a n s p l a n t s , o n l y one i n sudan, t h r e e p a t i e n t s a r e on r e g u l a r h e m o d i a l y s i s , n i n e p a t i e n t s a r e on i n t e r m i t t e n t p e r i t o n e a l d i a l y s i s , 1 6 a r e on c o n s e r v a t i v e t r e a t m e n t and 17 d i e d d u r i n g t h e course o f t r e a t m e n t . introduction renal f a i l u r e i n i t s m i l d and c h r o n i c forms has v a r y i n g f a c e t s i n i t s c l i n i c a l p r e s e n t a t i o n . w i t h a b l o o d urea o f 100 mg% one may be i n reasonably good h e a l t h and can u s u a l l y l e a d a normal l i f e (8). b u t as a r e s u l t o f a supervening i l l n e s s , r e n a l f u n c t i o n can r a p i d l y d e t e r i o r a t e ; and t h i s has t o be recognised e a r l y on. p a t i e n t s w i t h r e n a l f a i l u r e can be asymptomatic o r may have symptoms from any organ o r organ system. the p r e s e n t paper i s a r e v i e w o f t h e c l i n i c a l p r e s e n t a t i o n o f 6 1 sudanese p a t i e n t s who were t r e a t e d i n t h e medical department o f soba u n i v e r s i t y hos p i t a l khartoum. 5 -878571 65 a comparison o f t h e causes o f crf i n sudan and sweden i s a l s o presented. materials and methods over a p e r i o d o f two y e a r s , jan 1984 feb 1986, 6 1 sudanese p a t i e n t s o f afro-arab o r i g i n , aged between 15 and 75 y e a r s (mean 4 0 . 1 y e a r s ) were s t u died. there were 38 men and 23 women. the m a j o r i t y o f t h e p a t i e n t s were i n p a t i e n t s . most o f these p a t i e n t s were r e f e r r e d from o t h e r h o s p i t a l s i n t h e c o u n t r y as o u r h o s p i t a l i s one o f t h e main r e f e r r e n c e c e n t r e s i n t h e c o u n t r y , which has one m i l l i o n square m i l e s s u r f a c e area and a p o p u l a t i o n o f 22 m i l l i o n people. the h o s p i t a l has no c a s u a l t y ward so most p a t i e n t s came t o t h e o u t p a t i e n t c l i n i c s o r r e c e i v e d i n t h e main c a s u a l t y ward i n khartoum h o s p i t a l i n t h e c i t y c e n t r e . the c r i t e r i a f o r i n c l u s i o n i n t h i s study was t h e f i n d i n g o f a p e r s i s t e n t l y r a i s e d b l o o d u r e a and c r e a t i n i n e , supported by t h e f i n d i n g o f small kidneys on x-rays o r ultrasonography. the c r e a t i n i n e c l e a r a n c e was i n f e r r e d from t h e serum c r e a t i n i n e concen t r a t i o n (4). h y p e r t e n s i o n i s d e f i n e d as a b l o o d p r e s s u r e (bp) o f >160/90 mm hg. m a l i g n a n t h y p e r t e n s i o n i s diagnosed when t h e d i a s t o l i c bp i s >130 mm hg o r t h e p a t i e n t had papilloedema. the d a t a concerning causes o f crf i n sweden was o b t a i n e d from y e a r l y s t a t i s t i c s , from t h e swedish n e p h r o l o g i c a l s o c i e t y , 1984. results f i g u r e 1 shows t h e sex d i s t r i b u t i o n and age i n c i d e n c e . males outnumbered females and t h e peak o f i n c i d e n c e o f age o c c u r r e d between 30 and 40 years. tables 1 and 2 g i v e a summary o f t h e o b s e r v a t i o n s . the commonest modes o f p r e s e n t a t i o n (see table 1) were: f a t i g u e , a n o r e x i a , nausea and v o m i t t i n g ; a n k l e s w e l l i n g , b r e a t h l e s s n e s s , p u f f i n e s s o f t h e face, cough and insomnia i n t h a t o r d e r . 66 i n t h i s s e r i e s 17 d i e d o u t o f t h e 61 p a t i e n t s d u r i n g t h e course o f t r e a t ment. no p o s t mortem was done f o r reasons o u t o f hand. the m o r t a l i t y was 28%, f a r l e s s t h a n i n s e r i e s s t u d i e d b e f o r e (3). . d u r i n g f o l l o w u p (some p a t i e n t s were n o t o f f e r e d immediate d i a l y s i s be cause o f l a c k o f vacancies) g a s t r o i n t e s t i n a l symptoms predominate (table 2). number of patients m f a17 age (years) f i g . 1. table 1. sex d i s t r i b u t i o n and age i n c i d e n c e i n 6 1 p a t i e n t s w i t h crf. main c o m p l a i n t s a t t h e t i m e o f p r e s e n t a t i o n i n o r d e r o f frequency. camp1 a i n t s ~ ~~ no o f p a t i e n t s percentage 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 f a t i g u e anorexia, nausea, v o m i t t i n g ankle s w e l l i n g breath1 essness p u f f i n e s s o f t h e f a c e cough insomnia i t c h i n g chest p a i n d i a r r h o e a p a r a s t h e s i a , h o t sensations i n f e e t and hands e p i s t a x i s bad v i s i o n p a i n i n t h e j o i n t s headache 51 46 33 30 23 20 19 18 13 9 9 5 5 3 3 82 75 52 49 36 32 31 29 21 15 15 8 8 5 5 67 e p i s t a x i s was t h e p r e s e n t i n g f e a t u r e i n 1 4 p a t i e n t s , and some o f t h e s e pa t i e n t s were r e f e r r e d from e a r , nose and t h r o a t (ent) departments: one p a t i e n t d i s c o v e r e d l a t e r t o be s u f f e r i n g from a l p o r t ' s syndrome, a f t e r s t u d y i n g h i s r e n a l b i o p s y and audiometry, was under t h e c a r e o f an ent s p e c i a l i s t f o r deafness. p e r i c a r d i t i s o c c u r r e d i n 11 p a t i e n t s , and a l l were d i a l y z e d . table 2. observed c l i n i c a l f e a t u r e s d u r i n g t h e course o f i l l n e s s c l i n i c a l f e a t u r e s i n v o l v i n g no o f p a t i e n t s percentage i g a s t r o i n t e s t i n a l t r a c t 1 anorexia, nausea, v o m i t t i n g 55 2 d i a r r h o e a 10 3 hiccough 5 4 haematamesi s 1 t e r m i n a l 1 f a t i g u e 55 2 insomnia 25 3 convulsions 2 4 myoclonus 2 5 tremors f l a p p i n g 6 6 p e r i p h e r a l neuropathy 11 1 hypertension m a l i g n a n t 7 non-malignant 29 2 p e r i c a r d i t i s 11 3 e p i s t a x i s 1 4 i 1 neuromuscular system i11 c a r d i o r e s p i r a t o r y system i1 i v i n v o l v i n g s k i n 1 purpura 2 3 i t c h i n g 22 4 pigmentation 9 2 uraemic f r o s t 25 v miscellaneous c l i n i c a l f e a t u r e s anaemia; normochronic normocytic 42 90 16 8 1 90 4 1 3 3 10 19 19 8 1 19 22 3 4 1 36 15 69 p e r i p h e r a l neuropathy o c c u r r e d i n 11 p a t i e n t s and was so troublesome t h a t a l l o u r p a t i e n t s a p p l i e d hena (a l o c a l dye from p l a n t s w i t h a soothening e f f e c t ) t o t h e i r hands and f e e t . it g i v e s a r e d b l a c k c o l o u r and i s v e r y c h a r a c t e r i s t i c o b s e r v a t i o n i n t h e p a t i e n t s . one p a t i e n t had severe neuropathy w i t h p a r a l y s i s o f t h e l o w e r l i m b s , ame l i o r a t e d by p e r i t o n e a l d i a l y s i s . i t c h i n g was observed i n 22 p a t i e n t s and i t d i d n o t disappear a f t e r d i a l y s i s . however, 3 p a t i e n t s were symptom f r e e a f t e r t r a n s p l a n t a t i o n . nine o f o u r p a t i e n t s became more d a r k , t h i s i s n o t a w e l l recognized f e a t u r e i n 68 b l a c k s ; o u r p a t i e n t s a r e o f afro-arab o r i g i n and some have v e r y f a i r c o l o u r . normochromic n o r m o l y t i c anaemia o c c u r r e d i n 42 p a t i e n t s (table 2). other causes had been excluded. f i g u r e 2 d e p i c i t s t h e b l o o d p r e s s u r e l e v e l a t admission, most o f t h e hy p e r t e n s i v e p a t i e n t s responded t o drugs and d i a l y s i s . 0 'y i ....* n o o c ll..... f i g . 2. blood p r e s s u r e i n 6 1 p a t i e n t s a t p r e s e n t a t i o n . f i g u r e s 3 and 4 show t h e b l o o d u r e a and c r e a t i n i n e a t t h e t i m e o f s e e i n g t h e p a t i e n t s . four p a t i e n t s showed v e r y h i g h l e v e l s and one male 28-years-old w i t h a b l o o d u r e a o f 600 mg% and c r e a t i n i n e o f 40 mg% d i e d d u r i n g d i a l y s i s due t o h y p e r t e n s i v e encephal opathy. f i f t y n i n e p e r c e n t o f t h e p a t i e n t s had h y p e r t e n s i o n and o f these 19% had ma1 i g n a n t hypertension. the i n c i d e n c e o f h y p e r t e n s i o n i n o u r p a t i e n t s i s l o w e r t h a n o t h e r s ( 3 ) . three o f o u r p a t i e n t s had a d u l t p o l y c y t i c disease as t h e p r i m a r y cause o f crf and two p a t i e n t s had s a l t l o o s i n g nephropathy; 69 250 200 150 4 0 . 35. 30 25 ~ ........ 4 : . ...... . . 0 . . 50 0 1 f i g . 3. b l o o d urea l e v e l i n 6 1 p a t i e n t s a t p r e s e n t a t i o n . serum creatinine (mg%) i:: ..... 1 0 1 ':fff ..................... ..... 5 t : : : . 0 1 f i g . 4. serum c r e a t i n i n e l e v e l i n 6 1 p a t i e n t s a t p r e s e n t a t i o n . table 3 compares t h e causes o f crf i n sudan and sweden. chronic glomerulo n e p h r i t i s i s t h e commonest cause o f c r f b o t h i n sudan and sweden, however, o b s t r u c t i v e nephropathy i s t h e second common cause i n sudan which i s n o t t h e case i n sweden. diabetes m e l l i t u s i s t h e second common cause o f c r f i n sweden. 70 table 3. causes o f c h r o n i c r e n a l f a i l u r e (crf) i n sudan and sweden i n percentage (%) cause sudan ( n = 61) sweden* ( n = 524) (%i (%i chronic g l o m e r u l o n e p h r i t i s o b s t r u c t i v e nephropathy h y p e r t e n s i v e n e p h r o s c l e r o s i s diabetes m e l l i t u s chronic p y e l o n e p h r i t i s p o l y c y s t i c disease o f t h e kidney systemic diseases other causes and unknown 33 18 13 12 3 5 16 27 11 22 13 7 9 11 *according t o s t a t i s t i c s from t h e swedish n e p h r o l o g i c a l s o c i e t y , 1984. d i s c u s s i o n f a t i g u e was t h e p r e s e n t i n g symptom i n 82% o f o u r p a t i e n t s w i t h end stage r e n a l f a i l u r e . t h i s was v o l u n t e e r e d by t h e p a t i e n t s and many o f them had been under medical c a r e and were s u p p l i e d w i t h v i t a m i n e s u n t i l t h e b l o o d u r e a was done and t h e y were r e f e r r e d t o us. although many people complain o f f a t i g u e i n t h e h o t weather o f sudan due t o excessive sweating e t c , c l i n i c i a n s s h o u l d be a l e r t and l o o k f o r o t h e r f e a t u r e s o f crf, e s p e c i a l l y now where t e s t s l i k e b l o o d u r e a a r e a v a i l a b l e i n b i g c i t i e s . f a t i g u e i n crf i s a t t r i b u t e d t o anaemia, d e h y d r a t i o n , p r o t e i n c a l o r i e mal n u t r i t i o n and accumulation o f uraemic t o x i n s (2). g a s t r o i n t e s t i n a l symptoms o c c u r r e d i n 75% as t h e p r e s e n t i n g f e a t u r e , how e v e r , t h e y predominate d u r i n g f o l l o w u p . these g a s t r o i n t e s t i n a l f e a t u r e s have d e l e t e r i o u s e f f e c t on r e n a l f u n c t i o n and s h o u l d be l o o k e d f o r and t r e a t e d p r o m p t l y because i n a p l a c e l i k e ours i t i s always cheaper t o r e v e r s e t r e a t a b l e causes and keep p a t i e n t s w i t h esrd s t a b l e on c o n s e r v a t i v e t r e a t m e n t (7). nine p a t i e n t s had p e r i p h e r a l neuropathy, s i x w i t h o b j e c t i v e s i g n s m a i n l y i n t h e l o w e r limbs, one p a t i e n t was c r i p p l e d , however, he improved a f t e r p e r i t o n e a l d i a l y s i s and i s now w a l k i n g w i t h a s t i c k . the p e r i p h e r a l neuro p a t h y seems t o be r e l a t e d t o t h e gfr and d u r a t i o n o f crf (9, 10). 71 a l l t h e 6 1 p a t i e n t s w e r e anaemic, however, 69% had t h e t y p i c a l normo chromic normocytic anaemia o f crf ( 6 ) . g a s t r o i n t e s t i n a l b l e e d i n g was seen i n o n l y one p a t i e n t who was v e r y ill and d i e d b e f o r e i n s t i t u t i n g therapy. the i n c i d e n c e o f h y p e r t e n s i o n i n o u r p a t i e n t s i s lower t h a n o t h e r obser v e r s ' (3). i n a s t u d y done by makene i n oar-es-salaam where a l l h i s 42 p a t i e n t s were p u r e a f r i c a n s 91% had h y p e r t e n s i o n and 24% o f those p a t i e n t s g o t m a l i g n a n t h y p e r t e n s i o n . i n o u r s e r i e s as mentioned e a r l i e r t h e p a t i e n t s a r e o f afro-arab o r i g i n and t h r e e had p o l y c y s t i c kidneys and t w o had s a l t l o o s i n g nephropathy. the l a s t two decades had seen improvement i n d i a l y s i s and t r a n s p l a n t (2, 5). however, i n a p l a c e l i k e sudan, where p r i o r i t y goes t o m a l n u t r i t i o n and endemic diseases l i k e m a l a r i a , t u b e r c u l o s i s and s c h i s t o m a t o s i s , o u r d u t y as n e p h r o l o g i s t s i s t o p r e v e n t and f i n d remediable causes b e f o r e o u r p a t i e n t s succumb t o esro. the comparison o f t h e causes o f c r f i n sudan and sweden shows t h a t c h r o n i c g l o m e r u l o n e p h r i t i s i s t h e commonest cause o f crf i n b o t h c o u n t r i e s . obstruc t i v e nephropathy ( s t o n e disease) i s a common cause o f crf i n sudan. t h i s i s a t t r i b u t e d t o : 1. l a c k o f f a c i l i t i e s , e.g. x-rays and u l t r a s o u n d , 2. l a t e p r e s e n t a t i o n o f p a t i e n t s . three o f o u r p a t i e n t s p r e s e n t e d w i t h c r f w i t h o u t been seen b e f o r e and had no x-rays examination i n t h e i r l i f e . a v a i l a b i l i t y o f such f a c i l i t i e s and e a r l y r e f e r r a l o f p a t i e n t s w i l l d e t e c t p a t i e n t s b e f o r e t h e y develop crf. t h i s p r e v e n t s c r f , postpones d i a l y s i s and i t s c o s t s (7) and makes l i f e e a s i e r f o r t h e p a t i e n t and t h e people i n charge o f h e a l t h care. diabetes m e l l i t u s i s n o t a common cause o f c r f i n sudan as i n sweden. probably o u r d i a b e t i c p a t i e n t s i n sudan succumb t o o t h e r c o m p l i c a t i o n s and do n o t l i v e l o n g enough t o develop crf. acknowledgment i would l i k e t o t h a n k o r b j o r n wikstrom f o r r e v i s i n g t h e m a n u s c r i p t and p r o v i d i n g d a t a from sweden. my g r a t i t u d e a l s o t o m r s i v a kulhanek who typed t h e manuscript. 72 references abboud, 0.1.: chronic r e n a l f a i l u r e i n t h e sudan. md t h e s i s , u n i v e r s i t y o f khartoum, sudan. h a r i s o n ’ s p r i n c i p l e o f i n t e r n a l medicine (ed. r.g. p e t e r s d r o f , r.d. adams, e. braunwald, k.j. i s s e l b a c h e r , j.b. m a r t i n & j.d. wilson). mcgraw-hill book company, new york, 1983. makene, w.j.: the c l i n i c a l p r e s e n t a t i o n o f crf as seen i n dar-es-salaam. east a f r i c a n medical j o u r n a l 49:844-853, 1972. morgan, d . b . & w i l l , e . j . : s e l e c t i o n , p r e s e n t a t i o n and i n t e r p r e t a t i o n o f b i o c h e m i c a l d a t a i n r e n a l f a i l u r e . kindey i n t 24(4):438-445, 1983. p a r f f e r y , p.s. & g a u l t , m.h.: c l i n i m e t r i c s i n nephrology. nephron 42:l-5, 1986. replacement of renal f u n c t i o n by d i a l y s i s (ed. drukker, parsons, maher). m a r t i n s n i j h o f f p u b l i s h e r s , boston, 1983. roberts, s.d., maxwell, d.r. & gross, t.l.: c o s t e f f e c t i v e c a r e o f end stage r e n a l disease. ann i n t med 92(2):243-248, 1980. rosenheim, m.l.: problems o f c h r o n i c p y e l o n e p h r i t i s . b r i t med j 1:1433, 1963. siddique, j. & k e r r , d.n.s.: complications o f r e n a l f a i l u r e and t h e i r response t o d i a l y s i s . b r i t med b u l l 27:153, 1971. the p e r i p h e r a l nervous system i n d i a l y z e d uremic p a t i e n t s : r e g r e s s i v e motor u n i t changes. i n : c o n t r i b u t i o n s t o nephrology (ed. g.m. b e r l y n e , s. g i o v a n n e t t i ) v o l 45, pp. 42-59. karger, basel, 1985. 1. 2 . 3. 4. 5. 6. 7. 8. 9. 10. address f o r r e p r i n t s : d r e l f a d i l m. osman u n i v e r s i t y h o s p i t a l department o f i n t e r n a l medicine 5-751 85 uppsala sweden 73 upsala journal of medical sciences 2023, 128, e9234 http://dx.doi.org/10.48101/ujms.v128.9234 the aura-gain laryngeal mask for airway management in neonatal inguinal hernia surgery. a feasibility study ali-reza modiri, robert frithiof, tomas luther and peter frykholm department of surgical sciences, section of anaesthesiology and intensive care medicine, uppsala university, uppsala, sweden background the benefits of the laryngeal mask airway (lma) for elective procedures requiring general anesthesia in children are well documented. besides easy insertion, the incidence of perioperative respiratory adverse events has been found to be lower when the lma is compared to endotracheal intubation (1). however, there is a paucity of studies in small infants and neonates. in our tertiary pediatric anesthesia center, lma is the most commonly used airway  adjunct. when a new second generation lma became available, we decided to perform a feasibility study of using the aura-gain lma in infants undergoing inguinal hernia surgery. aim to investigate the feasibility and safety of using the aura-gain lma in infants undergoing inguinal hernia surgery. methods the design was prospective observational. the regional ethics committee granted permission (dnr 2019-04418); informed consent was obtained from both parents. neonates of post menstrual age ≤ 60 weeks scheduled for inguinal surgery were eligible for inclusion. exclusion criteria were moderate or severe respiratory disease or congenital heart disease requiring treatment. primary outcome was the successful completion of surgery. secondary outcomes were time to correct placement, fiberoptic view of the larynx through the lma, leak pressure, and adverse events associated with airway management. for the secondary outcomes, we started the clock when a decision to insert the lma was made by the attending anesthesiologist and stopped it when an endtidal co 2 trace of a complete breath was visualized on the anesthesia monitor. we recorded visualization of the larynx using a fiberoptic endoscope inserted past the major bend of the auragain, using the cormacklehane scale adapted for flexible laryngoscopy through the lma, and we measured the leak pressure by increasing the peak pressure in steps of two up to a maximum of 30 cmh 2 0. the leak pressure was defined as the pressure when the difference between the inspired and expired tidal volumes was more than 10%. the anesthesia technique included i.v. or mask induction according to the availability of easy i.v. access, maintenance with sevoflurane in oxygen targeting fio 2 0.3, flow 0.2–1 l min-1. an 8 french feeding tube was inserted through the side-port of the lma, initially used for suctioning the stomach and then left open during the procedure. near the end of the procedure, sevoflurane was turned off, flows increased and bolus doses of propofol 0.5 mg kg-1 were given until the end-tidal sevoflurane concentration was ≤0.3, at which point the lma was removed. results we included 20 neonates, with mean gestational age 50.2 ± 4.9 weeks, body weight 5.6 ± 1.0 kg (range 4.3–7.7) (table 1). all operations were performed according to plan. all lmas were inserted at the first attempt. mean time to correct position from decision to intubate was 14.1 ± 6.3 s. we recorded a modified cormack lehane grade 1 in 17 infants and grade 2 in three infants respectively. there was no case of airway obstruction due to malpositioning or folding of the epiglottis. there were neither any adverse events at induction nor during lma insertion. pressure support ventilation was used initially in all infants, but in three cases a small dose of atracurium was given on request from the surgeon due to difficult operating conditions during pneumoperitoneum. there was one case of transient stridor after extubation, resolving spontaneously after a brief period of observation in the operating room. discussion this small observational study corroborates previous reports of low rates of adverse events when lmas are used in children. drake-brockman et al. reported a rate of perioperative respiratory adverse events (prae) of 18% vs 55% in infants with lma vs endotracheal tube (ett), respectively, in a randomized controlled trial (rct) in older children (1). in an observational trial comparing spinal anesthesia to general anesthesia with lma, the authors reported one case of bradycardia and one case of laryngospasm in the ga cohort (2). in the present study, the age and size of the infants were lower or similar compared to the contact peter frykholm peter.frykholm@surgsci.uu.se © 2023 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. prospective observation study http://dx.doi.org/10.48101/ujms.v128.9234 peter.frykholm@surgsci.uu.se mailto:peter.frykholm@surgsci.uu.se http://creativecommons.org/licenses/by/4.0/ 2 a.-r. modiri et al. former studies (1, 2). despite this we found an even lower rate of adverse events and observed no serious praes such as laryngospasm or hypoxemia. furthermore, we had no cases of mucosal damage or bleeding and the majority of children breastor bottle-fed within an hour of extubation. all lmas were inserted on the first attempt in contrast to two randomized controlled trials comparing the auragain to the lma supreme in slightly older children, in which first attempt success-rate for the auragain was 96 and 86% respectively (3, 4). in a randomized controlled study comparing the lma supreme with the proseal lma in infants, the rate of mucosal hyperemia, mucosal damage or blood on the lma was 5% vs 8.3% in the two respective groups (5). furthermore, the mean oropharyngeal leak pressures (olp) were 17.2 and 24.1 cmh 2 0 in the two abovementioned rcts comparing the auragain to the lma supreme (3, 4). lopezgil et al. found that four different methods for detecting olp were well correlated (6). therefore we could speculate that the slightly lower mean olp of 16.8 cmh 2 0 found in the present study may be attributed to the lower age of the infants rather than the measurement method. the main limitation of the present study is that the sample size is small. furthermore, we did not compare the aura-gain to other lmas or ett, and the above discussion on adverse events must therefore be interpreted with caution. the strength of the study is that it was performed in a homogenous cohort of small infants <60 weeks post menstrual age, which is younger than other published studies. conclusion we conclude that the auragain may be used for neonatal hernia surgery, since we found it easy to insert to correct position and there were no serious adverse events. acknowledgements the authors declare no conflicts of interest. the study was financed by departmental sources only. notes on contributors ali-reza modiri, ph.d. research assistant, dept of surgical sciences, uppsala university. robert frithiof, m.d., ph.d., associate professor, dept of surgical sciences, uppsala university. tomas luther, m.d., dept of surgical sciences, uppsala university. peter frykholm, m.d., ph.d., associate professor, dept of surgical sciences, uppsala university. orcid robert frithiof https://orcid.org/0000-0003-2278-7951 tomas luther https://orcid.org/0000-0003-4449-3831 peter frykholm https://orcid.org/0000-0001-6402-136x references 1. drake-brockman tfe, ramgolam a, zhang g, hall gl, von ungern-sternberg bs. the effect of endotracheal tubes versus laryngeal mask airways on perioperative respiratory adverse events in infants: a randomised controlled trial. lancet. 2017;389:701–8. doi: 10.1016/s0140-6736(16)31719-6 2. ceccanti s, cervellone a, pesce mv, cozzi da. feasibility, safety and outcome of inguinal hernia repair under spinal versus general anesthesia in preterm and term infants. j pediatr surg. 2021;56:1057–61. doi: 10.1016/j.jpedsurg.2020.09.064 3. jagannathan n, hajduk j, sohn l, huang a, sawardekar a, gebhardt er, et  al. a randomised comparison of the ambu auragain and the lma supreme in infants and children. anaesthesia. 2016;71:205–12. doi: 10.1111/anae.13330 4. shariffuddin ii, teoh wh, tang e, hashim n, loh ps. ambu® auragain™ versus lma supreme™ second seal™: a randomised controlled trial comparing oropharyngeal leak pressures and gastric drain functionality in spontaneously breathing patients. anaesth intensive care. 2017;45:244–50. doi: 10.1177/0310057x1704500215 5. oba s, turk hs, isil ct, erdogan h, sayin p, dokucu ai. comparison of the supremetm and prosealtm laryngeal mask airways in infants: a prospective randomised clinical study. bmc anesthesiol. 2017;17:1–6. doi: 10.1186/s12871-017-0418-z 6. lopez-gil m, brimacombe mb, keller c. a comparison of four methods for assessing oropharyngeal leak pressure with the laryngeal mask airway (lmatm) in paediatric patients. pediatr anesth. 2001;11:319–21. doi: 10.1046/j.1460-9592.2001.00649.x table 1. patient/procedure characteristics and observations using the aura-gain laryngeal mask airway (mean ± sd or absolute number). gestational age (weeks) 50.3 ± 4.9 weight (kg) 5.6 ± 1.0 duration of anesthesia (min) 96 ± 18 duration of surgery (min) 34 ± 14 laparoscopic/open hernioraphy insertion time (seconds) 14.1 ± 6.3 modified cormack-lehane grade 1 vs 2 17 vs 3 oropharyngeal leak pressure (cmh2o) 16.8 ± 4.8 time to first feeding in pacu 63 ± 49 pacu; post anesthesia care unit. https://orcid.org/0000-0003-2278-7951 https://orcid.org/0000-0003-2278-7951 https://orcid.org/0000-0003-4449-3831 https://orcid.org/0000-0003-4449-3831 https://orcid.org/0000-0001-6402-136x https://orcid.org/0000-0001-6402-136x http://dx.doi.org/10.1016/s0140-6736(16)31719-6 http://dx.doi.org/10.1016/j.jpedsurg.2020.09.064 http://dx.doi.org/10.1111/anae.13330 http://dx.doi.org/10.1177/0310057x1704500215 http://dx.doi.org/10.1186/s12871-017-0418-z http://dx.doi.org/10.1046/j.1460-9592.2001.00649.x upsala j med sci 79: 7-17, 1974 a convection-diff usion model of capillary permeability with reference to single-injection experiments €30 aberg and jarl v. hagglund from the institute of physiology and medical biophysics, biomedical centre, university of uppsala, sweden abstract a theoretical model of the transport of neutral molecules across the capillary wall has been developed. the model is applicable to single-injection experiments. the mutual coupling between the diffusion and convection of solute molecules in extracellular pores under the influence of restriction bas been considered as responsible for the capillary permeability. the extraction, which is experimentally measurable, and solute flows across the capillary membrane, have been calculated with the continuity equation in the steady state a s well a s in the case of relaxation phenomena in the membrane-concentra tion profile due to non-steady-state properties. explicit equa tions of the steady-state extraction, suitable for experimental application, are presented, showing the effect of the convection in increasing the capillary permeability and thus in opposing the restriction caused by the solute-membrane interaction. non-steady-state computations were performed on an analog computer and showed that back transport from the membrane compartment to the blood stream may be partly responsible for the regain of solute molecules which have earlier passed into the capillary wall. introduction it has been suggested that the transport of lipid insoluble molecules across the capillary wall occurs through discrete pores with dimensions comparable to those of small molecules (16, 19,20). the existence of both small and large pores has been discussed (12). the small pores, which are considered to pre dominate, have in skeletal muscle, for instance, an estimated radius in the range of 3 to 4 nm. other organs show slightly different values. on the basis of electron-microscopic evidence, it has been pro posed that the intercellular junctions are the ana tomical counterparts of the small pores (15). the transport mechanism in the capillary mem brane for small molecules has been assumed t o be diffusion, modified by a restriction factor due t o the finite dimensions of the pores (19, 22). the contribu i tion t o the solute flux due to convection (i.e. solvent drag), in accordance with starling’s hypothesis of transcapillary solvent circulation, has usually been treated as a small correction (12, 16). in experimental investigations of the capillary permeability, what is called the single-injection technique has been widely used (6, 7, 8). in this method, a bolus, containing an impermeable refer ence substance and permeable test substances, is injected into a n afferent artery, and the blood is sampled and analysed at the corresponding efferent vein. by comparing the venous concentrations of the test substances and the reference solute, the amounts of the test substances absorbed can be calculated as an indirect measure of the capillary permeability. the aim of the present investigation is to study a capillary-membrane model which takes into account the combined effect of convection and diffusion in restricted pores as transcapillary transport mechan isms. the interaction of convection on the diffusion equation has been studied in several other theoretical and experimental situations (11, 13, 14, 17, 21, 23, 24). the present membrane model has been adapt ed to single-injection experiments by adding a single channel model of the blood capillary system. calcula tions have been made for the steady-state case of the convection-diffusion equation. however, since the time-varying bolus concentration may induce a diffusional non-steady state, this situation is also treated and discussed. nomenclature for convenience, the various notations used through out the paper are listed as follows. these symbols will also be defined in the text when they appear. ad true pore area (cm2) a, apparent pore area for a solute (cm2) a molecular radius (cm) upsala j m e d sci 79 8 b. aberg and j . v. hagglund c = c ( x , t ) cfl k n n p p q 4 r r t t t l l z v x a x 17 concentration of solute in the blood tissue barrier as a function of x and t (mole/cm3) concentration at intersegmental mem brane barrier n (mole/cm3) concentration gradient at intersegmental membrane barrier n (mole/cm4) plasma concentration of solute (mole/ cm3) maximum plasma solute concentration of the bolus (mole/cm3) tissue solute concentration (mole/cm3) diffusion coefficient (cm*/sec) thickness of blood-tissue barrier (cm) extraction total extraction solute flow as a function of x and t (mole/(cm2 sec)) solute flow at intersegmental membrane barrier n (mole/(cm2 sec)) restriction coefficient avogadro's number (number/mole) index referring t o the number of the intersegmental membrane barriers permeability of the capillary wall (cm/ sec) steady-state capillary permeability (cm/ sec) scale factor = k d / a x 2 plasma-volume flow (cm3/sec) scale factor = k v / 2 a x gas constant (dyne cm/(mole degree)) pore radius (cm) absolute temperature ( o k ) time (sec) half-width of the bolus concentration linear transendothelial flow velocity (cm/ sec) space dimension (cm) membrane-segment thickness (cm) viscosity (poise) (set) the normalized quantities used in this paper have not been included in the above list but are summa rized in eq. (9). model of the plasma-tissue barrier for the transport of neutral lipid-insoluble molecules through the endothelial wall, a pore model has been upsala j m e d sci 79 blood blood-tissue barrier tissue i ' 0 d fig. 1. the convection-diffusion model of the blood-tissue barrier. the convection velocity u disturbs the concentration profile in the membrane of thickness d. the bolus concentra tion caused by the bolus injection is co(f). the tissue concentra tion cd is assumed to be zero. adopted. inside the pores, the transport is one dimensional, and the barrier extends from x = o on the plasma side to x = d on the interstitial-tissue side of the pores (fig. 1). a test solute is considered, which for simplicity has a transport rate across the blood-cell membranes which is much slower than both the transport rate across the capillary wall and the transit time of the solute through the capillary system (see the next section). the time-(t)-dependent plasma concentration of this solute is c o ( t ) , and the membrane concentration is designated c ( x , t ) . the corresponding tissue concentration cd is assumed to be zero, which means assuming a perfectly stirred extracellular space of infinite extension. the free diffusion coefficient of the solute is d and a constant convection (flow) velocity v is considered to be present within the pores. the solute flux j,(x, t ) in the pores is then: where k is the restriction coefficient attributed t o the narrowness of the pores. renkin (cf. 5 , 22) assumed that the restriction was caused by a steric hindrance at the entrance of a pore and by frictional forces between the solute and the pore walls. he calcu lated the restriction coefficient and interpreted it as the apparent pore area a, for the solute over the true pore area a,: convection-diffusion model of capitlary permeability 9 ( 2 ( 1 a / r ) z (1 a / r ) 4 ) { 1 2.104alr a ks-' = a, + 2.09(a/r)3 0 . 9 5 ( a / ~ ) ~ } ( 2 ) where a/r is the ratio of molecular radius t o pore radius. when convection is considered, the frictional forces between the solute and the water must also taken into account. this was done by forster (lo), who showed that the restriction influences the diffusional and convectional contributions (first and second terms in eq. (1)) in the same proportion. he found that k =d,,,/(d + ds,,) where d,., is the solute-diffusion coefficient against the membrane and d , as above, the value in free solution. when the transport flux is not in the steady state, the following differential equation, obtained from the continuity equation, describes the concentration within the pores: (3) thus, in the non-steady state the concentration is calculated from eq. (3) and the solute flux is then obtained from eq. (1). finally, it has been assumed that the solute flux does not influence the plasma concentration c o ( t ) , which means assuming that the amount of substance lost through the capillary walls is small, as com pared with the amount of substance in the plasma, and also that the plasma compartment is well stirred. model o f the blood capillary system in order to adapt the membrane model t o single injection experiments, a model of the blood capillary system is needed to describe the blood flow through the organ under investigation. the simplest model is to approximate the blood capillaries t o a single channel (fig. 2). at the arterial end of the channel, the solute flow from the plasma towards the tissue compartment is j,(o, t ) . the water flow with velocity v is thought t o be filtered out from the vascular bed at the arterial end and re-absorbed at the venous end, according t o starling's principle of paracapillary circulation. all events in the reabsorptive part, as solute reabsorption from the tissue to the blood or a possible loss of solute from the blood t o the tissue are neglected. all the solute flow entering the tissue blood ecs arterial end (filtration channel) venous end (solute reabsorption neglected) elimination -js(d,t) a fig. 2. the four-compartment model of the solute transport from the plasma across the capillary wall to the extracellular space (ecs) and its elimination. the capillary system is modelled by a single channel with a circulating solvent flow u across the endothelial membrane (cf. fig. 1 ) . see text for further explanation. compartment, jjd, r ) , can thus be thought of as being eliminated. the volume flow of lymph is usually very small, as compared with the total volume flow of plasma q in the capillary system, which means that putting the plasma inflow at the arterial end equal to that at the venous end is a good approx imation. if the arterial and venous solute concentra tions are called c o ( t ) and c,(t) respectively, then a material balance gives or calculation of j,(o, t ) from eq. (l), using eq. (3) as described above, thus makes it possible t o obtain the experimentally measurable quantity c,(t) from eq. ( 5 ) . extraction in single-injection techniques, the experimental result is often expressed as the extraction e ( t ) , which is a measure of the loss of solute into the tissue, as com pared with the amount of solute in the bolus (8). upsala j meci sci 79 10 b. ldberg and j . v. hagglund using the result from eq. ( s ) , we obtain: (7) where p ( t ) is the membrane permeability. this means that in our model the extraction is propor tional t o the permeability of the capillary wall. i n the experimental situation, the extraction is time-dependent, due to non-steady state phenomena, and different methods of dealing with this situation have been discussed, for instance, the method of calculating the “early extraction” e(0) (18). we have used a different approach by introducing the fotal extraction, defined as roc j o the total extraction et means the total loss of solute from the plasma stream, divided by the amount of solute in the injected bolus. in the present model, et will vary with the half-width tlrz of the arterial bolus concentration c,(t), as long as the transport equation (3) is in the non-steady state. in the steady state, the extraction is independent of time, and eq. (8) is re duced to eq. (7). normalizations in order to give a comprehensive theoretical descrip tion, the following normalizations are introduced: normalization table x = x / d t = t k . did2 tliz = t l l z . k . d / d 2 (see below) (see below) (9) and where 6 , is the maximum of c,(t). v denotes the convection velocity v divided by the diffusional velocity obtained with zero convection. correspond upsala j m e d sci 79 ingly, js(x, t) is the ratio of the solute flux and the maximum diffusional flux (without convection). e and e,, can be interpreted as the extraction’or total extraction divided by the extraction obtained from pure diffusion. the normalized extraction e is equal t o the permeability p ( t ) normalized t o the permea bility with only diffusion. these relations are easily established if we consider that j,(o, t ) / c o ( t ) = kd/d in the case of pure diffusion and use eq. (7). steady-state approximation the calculations are substantially simplified if the approximation to the steady-state transport equation is made, which means neglecting the time derivative in eq. (3) ( a / a t =o). since c , ( t ) varies with time, c(x, t ) will vary with time also in the steady-state approximation, but the variation of c o ( t ) in this approximation is much slower than the relaxation time of eq. (3). the validity of this steady-state approximation will be further discussed below. using the normalizations introduced in the pre ceding section and assuming the steady-state con dition, eqs. (i), (3) and (7) (or (8)) are transformed into a2c(x, t) ac(x, t) ax2 ax ___ =”- and eqs. (10) and (12) are valid also in the non-steady state, but eq. (1 1) includes the steady-state approxima tion. the boundary conditions of eq. (11) can be ex pressed in normalized quantities, such as (cf. fig. 1) c(0, t) =c,(t) and c(1, t) = o the differential equation (1 1) is easily solved, together with the boundary conditions (eq. (13)), and the expression c(x, t) is then used in eq. (lo), which, in combination with eq. (12), gives an expression of the extraction e. the details of this procedure are given in the appendix and the result is (eq. (a7)): v e = l e x p ( v ) convection-diffusion model of capillary permeability 1 1 a graphical representation of eq. (14) (continuous curve) is given in fig. 3. the inset diagram in fig. 3 shows the relation between v and the concentration profile within the plasma-tissue membrane, as cal culated from eq. (a5) in the appendix. the figure illustrates that, as expected, the convection increases the extraction. at high filtration rates, the convection term predominates over the diffusion and the extrac tion approaches proportionality with v. in experimental work, using the single-injection technique, the ratio of the capillary-wall permea bility and the free diffusion coefficient of the solute has been used to express the transport properties of the blood tissue barrier ( 2 ) . in the present model, the corresponding quantity can be given as the actual permeability p divided by the permeability of free diffusion (did), a ratio which is proportional to the ratio of the extraction e ( t ) and the free diffusion coefficient d . then from eqs. (9) and (14): (15) v p . did = e ( t ) * d . q / ( d * ad) = k 1 -exp ( v) this expression is illustrated in fig. 4 (continuous curves) with k calculated from eq. ( 2 ) . different solute molecules correspond to different values of the molecule-to-pore radius a / r , but also to differ norm. total extraction t 5 /i / d 0 1 2 3 4 5 6 norm. filtration velocity fig. 3 . the steady-state extraction e as a function of the covection velocity v (continuous curves). the inserted dia gram shows the concentration profiles in the capillary mem brane corresponding t o different v-values. note that v = 0 correspond to pure diffusion and that for high values of v the extraction is proportional to v, indicating a negligible contribution by diffusion to the transcapillary transport. w / d i. fig. 4. the ratio of steady-state capillary permeability p and the free diffusion permeability d / d as a function of the solute to molecular-radius ratio for various values of the convection velocity v (-) and v (---). the ordinate is proportional t o the extraction divided by the free diffusion coefficient. on the constant v curves, the convection velocity u is con stant, while the molecular radius varies according to the abscissa. i t will be seen that for a certain value of u different molecules correspond t o different values of v and subsequently to different shapes of the capillary membrane-concentration profile (cf. the inset diagram in fig. 3). ent v, since the free diffusion coefficient which varies with the molecular size, is included in the normaliza tion (v = vd/d). if we introduce the stoke-einstein relation between the free diffusion coefficient and the molecular ra dius (a), an experimentally more convenient expres sion of the permeability is obtained: d = r t / ( 6 n . q n . a ) (16) where r = t h e gas constant, t = the absolute tempera ture, 7 = the viscosity and n = avogadro’s number. then, from eqs. (9), (15) and (16) we have v ( a / r ) 1 exp ( vajr) p d i d = k eq. (17) is illustrated in fig. 4 by dashed curves. in an experiment with constant convection velocity v but different probe molecules, the results will fall on one of these constant v curves. such a n experi ment may thus give rise to (a) increasing, (b) approxi mately constant or (c) decreasing values of p d / d as upsala j m e d sci 79 12 b. jberg and j. v. hagglund 1 0 5 0 alasrno conc [ll integrated solute flow (31 10 5 0 i t i m e 0 1 2 3 4 5 fig. 5 . the plasma solute concentration co(t) caused by a single injection, the solute flow jjo, t) across the capillary wall and the integrated solute flow j'&j,(o, t) d t as functions of time t. the integrated solute flow represents the instantaneous absorption from the plasma. the curves were calculated by analog computation and are given for v = 1 and fortl/,=0.9, which means an almost steady-state condition. note that the solute flux (curve (3)) at each time is almost proportional to the plasma concentration (cruve (1)) in this case. a function of ajr. in other words, the consequence of convection is to oppose the restriction, but finally with increasing molecular size the restriction will predominate and reduce the permeability towards zer 0. non-steady-state calculations when the time relaxation of the concentration profile within the capillary wall is appreciable, as compared with the time course of the bolus concentration c o ( t ) , the complete diffusion equation must be solved (eqs. (3) and (9)). this equation, together with the appropriate boundary conditions according to eq. (13), and eq. (lo), have been solved by means of analog computa tions. the bolus concentration c,(t) was taken from a single-injection experiment as the venous con upsala j m e d sci 79 1 05 0 'loma conc. [ 1 1 ill . . n integrated solute flow [ 2 1 12) i-', i \\ \ i \ non steady-state 1 12) i-', i \\ \ i \ -----------05 0 steady state 1 0 solute flow (31 12 6 1 ' -time 0 01 02 03 fig. 6. the same as fig. 5 but for t1/,=0.02, which means that the transport equation is in the non-steady srafe. in this case, there is a back transport from the capillary mem brane t o the plasma (curve ( 3 ) ) during the descending part of the plasma solute concentration (curve ( i ) ) . this is also reflected in the fact that the integrated solute flow is descend ing at the same time. centration of an essentially non-permeable solute, namely, evans blue bound to albumin (1). this curve corresponds to the so-called lagged normal density curve in indicator-dilution experiments (3). the bolus concentration was generated on a function generator, and the degree of non-steady state was simulated by varying the time width t t l z at half the maximum value of the bolus concentration, i.e. at the method of the analog solution was to use a finite-difference approximation of the equations, as described in more detail in the appendix. the analog computer used was a donner 3000 and the results were recorded on a servo recorder three-channel watanabe multicorder, model h.s. figs. 5 and 6 show the results for v = 1 and f o r two values of the half-width, t,,, =0.9 and t,,, =0.02 respectively, corresponding t o an almost steady-state condition (fig. 5) and to the non-steady state (fig. 6). the solid curves are the bolus concentration co(t) and the solute flow j,(o, t) across the capillary wall, and the dashed curves are the integrated solute c,(t) = e,/2 (4). convection-diffusion model of capillary permeability 1 3 region of 4 back transport \ + + + i t v = 4 + \ ++ + + . ++\\ + + 4. +/' + \ + \ \\ continuous lines steady-state approx \ \ \ + v-2 \ + f + t + + i : t + + + : t i + \ + b * t '/2 10 0.01 01 1 flow at each instant, j;j,(o, t ) d t . for the steady state case (fig. 5), the solute flux is proportional to the bolus concentration and the extraction can easily be calculated from eq. (12). on the other hand, in the non-steady state, there is non-proportionality corresponding t o a non-constant extraction during the passage of the bolus. furthermore, at the latter part of the bolus the solute is transported back to the plasma from the accumulated solute in the plasma-tissue membrane. this phenomenon, which is associated with non-steady-state behaviour, will in the following text be called back transport, in contrast to the term re-absorption, which we shall connect with the transport of solute from the extra cellular space back to the plasma compartment. as already mentioned, the re-absorption was neglected in the present model by assuming zero tissue con centration. finally, the total extraction was calculated for different values of t , , , and v, according to the equa tion (see eqs. (8) and (9)) using the analog procedure already mentioned. the results are summarized in fig. 7, and illustrate that high values of t , , , correspond to the steady-state approximation, eq. (14) and fig. 3, and that smali t , , , values imply the non-steady state. the transition fig. 7. the total extraction et as a function of tl;, for different values of the convection velocity v. t h e dots and crosses were obtained from analog computations, and the continuous lines from the steady-state approxima tion (eq. (14) and fig. 3). back trans port (cf. fig. 6 ) occurs approximately to the left of the oblique dashed line. region between the validity of the steady-state ap proximation and the non-steady-state condition is roughly given by the oblique dashed line, t o the left of which back transport is obtained. it will be seen that this transition occurs where t,12 is of the order of 1, which is also to be expected from the normalized diffusion equation (eq. (18)). discussion the validity of the model the models of the capillary permeability and the blood-capillary spatial distribution are, of course, somewhat oversimplified, as compared with the actual biological situation. at the membrane level, for instance, the possibility of unstirred layers in the plasma as well as in the extracellular space (9) have not been included. the assumption of zero tissue concentration, c, = 0 , means neglecting the effect of gradients in the tissue space and disregarding the re absorption back t o the blood capillary from the tissue and the exchange between capillaries. the validity of the approximation of zero extracellular concentration is thought t o be better at the beginning of the venous outflow curves, and also better for larger molecules, which pass through the capillary wall at a relatively slow rate. furthermore, the distribution of the pore geo metry, as concerns radius, length and tortuousness, has not been treated, nor has the distribution of the convection velocity. the pore radius and convec upsala j m e d sci 79 14 b. aberg and j. v. hagglund tion velocity should therefore be regarded as average values of a more complicated situation. the shape of c o ( t ) at the pores is supposed to be fairly well described by the lagged normal density curve (3), but in a biological capillary net there is certainly a distribution of transit times (4) which complicates the absorption from the blood and the exchange properties between the capillaries. experimental applicability of the model the main features of the present model are the assumption of a significant convectional flow across the endothelial wall and the introduction of the concept of the non-steady state, with relaxation of the membrane-concentration profile during the pas sage of the bolus in single-injection experiments. these features may be subject t o experimental tests, using the model. in the diffusional non-steady state, it has been shown that during the latter part of the venous out flow curves there is a back transport from the membrane compartment to the plasma compartment (fig. 6). this back transport may in many experi mental situations be difficult to distinguish from the re-absorption from the extracellular space back to the plasma, a phenomenon which was neglected in the present model. as mentioned above, the non steady state is roughly obtained when tl,, < 1 and the steady state when t,,, > 1 (fig. 7). considering that tli2 = t l l z k d / d 2 (eq. (9)), it will be seen that the steady state is likely t o occur for small molecules with a high restriction factor k (eq. (2)) and a high value of the free-diffusion coefficient d (eq. (16)). another important consequence is that the non steady state seemingly occur for large molecules with sufficiently small d and k values. thus, the experimental situation may roughly be covered by the following three categories: (a) small molecules probably means a fast steady-state transport with relatively early re-absorption, (b) medium molecules mean the steady state with re-absorption appearing later, and (c) large molecules imply the non-steady state and negligible re-absorption. one method of investigating these possibilities would be to use different test molecules. it has been shown that for glucose and raffinose, the extraction is constant, with respect t o time, over the main part of the venous outflow curves, which means that these molecules fit well in the second category above (1). the possibility of the non-steady state may be further illustrated by the following numerical ex upsala j m e d sci 79 ample. assuming tllz = 1 sec, k = 0.1 ( a / r = 0.5) and d = cm2/sec and using the relation t l l z k d / d 2 < 1, we find that d > 3 pm is a condition for the non steady state. it may be mentioned that pappenheimer considered the microscopic thickness of the capillary wall to be between 0.1 and 1 pm (19). as mentioned above, however, the membrane thickness d may be increased by the presence of unstirred layers at the blood capillary wall and in the extracellular space. the constancy of the extraction over a major part of the venous concentration curve may be considered as a pre-requisite for the use of the steady-state equations. for this case, the convection velocity v can be estimated from fig. 4, using several probe molecules with constant or maybe also different values of u. appendix steady-state solution the steady-state approximation is described by eqs. (lo)-( 1 3 ) : boundary conditions c(0, t) =co(t) and e(1, t) = o (al) = ( 1 1) (a2) = (1 3) js(x, t) = &(x, t)/ax + v ' c (a3) = (10) the solute concentration inside the plasma-tissue membrane is obtained by solving the differential equation (al), together with the boundary condi tions (eq. (a2)), which gives ( 4 5 ) now combining eqs. (a5) and (a3) gives the solute flux which is independent of x. convection-diffusion model of capillary permeability 15 i 2 c t .\ cg= 0 0 a x z a x 3 a x l a x s a x d n ; o $ l 2 3 l 5 6 fig. 8 . the convection-diffusion model of the solute transport across the capillary wall, as approximated for analog computa tion (cf. fig. 1 ) . the plasma-tissue barrier is divided into 6 segments, each ax wide. the finite-difference method means straight-line approximation of the concentrations within the segments. finally, the extraction is obtained from eqs. (a6) and (a4): v e= 1 -exp(-v) (a7) = (14) non-steady-state computations the non-steady-state solutions were obtained by means of analog computations. the non-normalized equations were used for this case. normlized equa tions can be solved with equal ease but need the introduction of time-scaling, which is implicit in the non-normalized solution. the solute concentration c = c(x, t ) and solute flux j, = j,(x, t ) within the i i 1 plasma-tissue membrane are given by the following equations: boundary conditions c(0, t ) = c,(t) and c(d, t ) = 0 (a9) =(13) ac j s ( o , t ) = k * d ax + k * u * c (a10)=(1) to solve these equations on an analog computer, a conventional finite-difference approximation meth od was adopted. the plasma-tissue barrier was di vided into six segments of thickness ax, as shown in fig. 8. the firstand second-order derivatives with respect to x of the concentration c, at intersegmental barriers of number n can be expressed as (&/ax), = ( c , + ~ cn-,)/2ax for n = 1 , 2 , ... 5 (a1 1) (ac/ax),-l/z =(c, -cjax for n = 1, 2, ... 6 (a12) (azc/ax2), = (c,+~ 2c, + c,-1)/ax2 for n = 1 , 2, ... 5 (a1 3) the insertion of eqs. (all)-(a13) in eqs. (a8) (a10) and approximating js(o, t ) by the solute flow ( j s ) 1 / 2 at n = 1/2 yields fig. 9 . analog-computer diagram for the model in fig. 8, derived from eqs. (a14) (a18). the function generator was used to c5 simulate the plasma solute concentration c&). the parametersp and q (eq. (a 16)) were adjusted on precision potentiometers. upsala j med sci 79 16 b. aberg and j . v. hagglund boundary conditions co =c,(r> and cs = o (‘415) where the following parameters were introduced: p = k . d l a x 2 and the relations between the normalized values of the convection velocity and of the half-bolus time width, on the one hand, and of the parameters p and q, on the other hand, are given by v = 12q/p (‘419) and eqs. (a14)-(a18) are suitable for analog program ming, and the analog-computer diagram of these equations which was used is shown in fig. 9. for the calculations, the following initial conditions were applied to the computer: c,=o at t < o for n=o, i , ... 5 (‘421) the degree of the non-steady state was varied by changing the parameterp (eq. (a20)), and the convec tion velocity was then chosen by the parameter q , according to eq. (a19). acknowledgements the authors are greatly indebted t o professor t. teorell for initiating this work and for much valuable advice and many stimulating discussions. we also wish t o thank miss c. ferner for valuable technical assistance. this work was supported by grants from the medical faculty of the university of uppsala, the swedish medical research council (project no. 14x-629) and the national institutes of health (grant no. 5 r01 h e 12960-08). references i . aberg, b.: manuscript in preparation. 1974. 2. alvarez, 0. a. & yudilevich, d. l.: heart capillary permeability to lipid-insoluble molecules. j physiol (london) 202: 45, 1969. upsula j m e d sci 79 3. bassingthwaighte, j. b . , ackerman, f. h. & wood, e. h.: applications of the lagged normal density curve as a model for arterial dilution curves. circ res, 18: 398, 1966. 4. bassingthwaighte, j. b.: plasma indicator dispersion in arteries of the human leg. circ res 19: 332, 1966. 5. beck, p. e. & schultz, j. s.: hindered diffusion in micro porous membranes with known pore geometry. science 170: 1302, 1970. 6 . chinard, f. p., vosburgh, g. j. & enns, t.: transcapil lary exchange of water and other substances in certain organs of the dog. am j physiol 183: 221, 1955. 7. crone, c.: om diffusionen av nogle organiske non elektrolyter fra blod ti1 hjernevzv. (thesis). munksgaard, kabenhavn, 1961. 8. the permeability of capillaries in various organs a s determined by use of the “indicator diffusion” method. acta physiol scand. 58: 292, 1963. 9 . crone, c. & garlick, d.: the penetration of inulin, sucrose, manitol and tritiated water from the interstitial space in muscle into the vascular system. j physiol (london) 210: 387, 1970. 10. forster, r . e.: the transport of water in erythrocytes. in current topics in membranes and transport (ed. f. bronner & a. kleinzeller), vol. 2, pp. 41-98. academic press, new york, 1971. 1 1 . garby, l.: studies on transfer of matter across mem branes with special reference t o the isolated human amniotic membrane and the exchange of amniotic fluid. acta physiol scand, suppl. 137, 1957. 12. grotte, g.: passage of dextran molecules across the blood-lymph barrier. acta chir scand, suppl. 211, 1956. 13. hertz, g.: uber trennung von gasgemischen durch diffusion in einem stromenden gase. z. physik 19: 35, 1923. 14. jacobs, m. h.: diffusion processes. ergebn biol. 12: 1 , 1935. 15. karnovsky, m. j . : the ultrastructural bases of trans capillary exchange, in biological interfaces: flows and exchanges. little, brown & co., boston, 1968. 16. landis, e. m. & pappenheimer, j. r.: exchange of sub stances through the capillary walls. in handbook of physiology, sect. 2: circulation (ed. w. f. hamilton), vol. 11, pp. 961-1034. amer. physiol. soc., washington, d.c., 1963. 17. manegold, e. & solf, k.: uber kapillarensysteme, xiv. 1 . die dynamik osmotischer zellen. kolloid z 54: 179, 1932. 18. martin, p. & yudilevich, d.: a theory for the quantifica tion of transcapillary exchange by tracer-dilution curves. am j physiol 207: 162, 1964. 19. pappenheimer, j. r., renkin, e. m. & borrero, l. m.: filtration, diffusion and molecular sieving through peri pheral capillary membranes. a contribution t o the pore theory of capillary permeability. am j physiol 167: 13, 1951. 20. pappenheimer, j. r.: passage of molecules through capillary walls. physiol rev 33: 387, 1953. 21. rapoport, s. i.: ionic accumulation by water flow through a membrane. acta physiol scand 64: 361, 1965. 22. renkin, e. m.: filtration, diffusion, and molecular sieving through prorous cellulose membranes. j gen physiol38: 225, 1954. convection-diffusion model of capillary permeability 17 23. teorell, t.: zur quantitativen behandlung der membran permeabilitat. z elektrochem 55: 460, 1951. 24. general physico-chemical aspects of drug distribution. in advances in the biosciences 5 (ed. g . rasp&), pp. 21 37. pergamon press, oxford, 1969. receiaed september 14, 1973 adress for reprints: go b e r g inst. of physiology and medical biophysics bio-medical center box 572 s-751 23 uppsala sweden 2 742854 upsala j m e d sci 79 upsala j med sci 98: 2 15-220, 1993 3. truth, accuracy, error and uncertainty renc dybkzr department of clinical chemistry frederiksberg hospital, frederiksberg, danmark the year 1993 should witness the final version of some very important documents on metrology and statistics that are revising our understanding and vocabulary concerning the components of a result of measurement and its uncertainty (see bibliography). the quite voluminous draft texts are neither easy reading nor fully harmonized, and they represent different philosophical approaches. the following may serve as an introduction to further study. a vocabulary, given in a separate paper, should ease the assimilation of any unfamiliar concepts and terms. this discussion is limited to those properties of systems that can be expressed on a difference scale or a ratio scale, comprising the measurable quantities, hereinafter called quantities, where a value is a numerical value multiplied by a unit. due to residual uncertainty of any definition of a quantity in the real world, especially prominent in biology, the true magnitude of a quantity can only be represented by a distribution of true values, but definitions of theoretical metrological concepts usually select a true value, p , as a representation of truth; in practice, a conventional true value, 9, is substituted. a quantity being subjected to measurement is called a measurand, y, and it can be considered as an output quantity, that is a function of a set of input quantities, xg such as calibrator quantities and influence quantities so that y = h(x, , x, , ..., x,), where y and xg are all considered to be random variables. observations on any xg yield an estimate, and all such estimates allow calculating an observed value of y according to the function h. one or more observed values yield a result of a measurement, y , as described by the measurement procedure. it is assumed that any result has been cor rected for known error. 215 the traditional analysis of error defines error of a result of a measurement as the difference y p . repeated measurements of the same measurand yield different results due to the variable error that forms a distribution having a location corresponding to the systematic error and a dispersion caused by the random error. the distribution of errors may be said to be convoluted with the true distribution to give the distribution of results for the measurand. ' the systematic error is the expectation of all results minus a true value, e(y) 1.1, whereas the random error is y e(y) . in practice j 0 and y 7 respectively are used as estimates of the theoretical values. the accuracy of a result is now defined as the closeness of agreement between the result and a true value; it therefore depends on a combination of random and systematic errors, not only on the latter. for the closeness of agreement between the expectation of a large series of results and a true value, the term trueness of results has been coined, whereas precision as before is the closeness of agreement between independent results under prescribed conditions. the three concepts can only be expressed on a few-value ordinal scale of measurement such as (poor average good). the inverse measures of trueness and precision in the form of population parameters or their statistical estimates are bias of results, e(y) p or jj 8, and variance of the distribution of results, u2 or 2, respectively. the variance is often replaced by the standard deviation, u or s, or the coefficient of variation, u/e(y) or s h . the theoretical statistical linear model of a result, y , may be stated as y = e(y) + b + s + m + e where e(y) = m is the overall expectation of all results from all laboratories obtained by a given measurement procedure for a given measurand; it is equal to 1.1 + 6 where 6 is the inherent (unknown) bias of the measurement procedure under any set of precision conditions; b is the laboratory (i) deviation under given precision conditions; it is equal to ewj) '0') ; another parameter may be defined on the above variables, namely 216 a which is the laboratory bias of results; it is equal to e(yi) p or 6 + b ; s is an aberrant-sample bias of result caused by an unusual analyte form or an unexpected influence quantity; its detection may require a reference measurement procedure, but this bias is not considered further here; m is the bias caused by an undetected mistake; it may be detected by an outlying result, but is not considered further here; e is a random error of a result occurring in every single result under repeatability conditions (r), te. intra-run; it is equal to y e ( y j i r); the distribution of e is assumed to be approximately gaussian with an expectation of zero. each of the parameters mentioned above has a variance. up2 is often neglected and is often taken to be zero. within a laboratory, i, the variance of the random error e is called the within-laboratory repeatability variance, u 2, and if its magnitude does not vary much between laboratories, a mean is calculated, uw2, and called the repeatability variance, a:. the laboratory deviation, b, varies with the precision conditions as follows. w under repeatability conditions, r, (one laboratory, operator, measuring system, and run) b is called the laboratory component of bias of results, bi,r, and considered to be 2 constant, l e . with a variance of zero. consequently, the total variance of y is uw . under intermediate precision conditions, i, where one or more sources of random variation are added in either or all of the groups time, operator, and calibration of equipment and reagents, the term b may be divided into elements that remain constant and other that become variables with expectations and variances; for, e.g., element 2 we have e(b2iii) and e2i,r respectively. the former changes the constant b , and thus ek i r), the latter adds to the total random variance so that u2 = ow2 + u2,*, . with each new source of variation being introduced, the residual b is being further transformed, acquiring another value, b j , i , where i has to be specified, and the total variance is increased. y i , ~ 4 i under reproducibility conditions, r, several laboratories are involved in applying the same measurement procedure, increasing the number of sources of variation. the 217 individual laboratory component of bias, b,,, or bi,*, is substituted by the behveen laboratory variation, b,. with a sufficient number of laboratories, its distribution is assumed to be approximately gaussian with an expectation, e(b ir), equal to zero and a between-laboratory variance, ub ’, also symbolized ul2. the overall expectation of the population of results from all laboratories may be taken to be e(y) = p + 6 and the total variance of the random error, called the reproducibility variance, is ar2 = ul2 + “r r 2 the effects of additional sources of variation can be seen and treated analogously when changes in measurement procedure, method of measurement, and even principle of measurement are introduced during laboratory proficiency testing or certification exercises for reference materials. the description above concerns populations and their parameters, 8. in practice, sample estimators, b, are used. 4 the classical description of uncertainty of measurement of a result was based on an analysis of error with systematic errors being summed linearly and random errors quadratically; the first sum was added to the positive square root of the latter. the newer procedure establishes an uncertainty budget giving an estimate of the uncertainty a socalled standard uncertainty, u for each input quantity. some are estimated as standard deviations, u = s, calculated from the observed number fraction distribution of values or stated on the certificate of a calibrator (type a evaluation). others are estimated non-statistically, but are also characterized by standard deviations, assuming some specified type of distribution, such as for influences of environment, algorithms, inhomogeneous sample, imperfect measurement procedures (type b evaluation). the former group corresponds to random errors, the latter to both random and systematic ones. the combined standard uncertainty, u,, is calculated from the sum of the estimated variances, ux with due consideration of any correlation between input quantities, xg. and the type of function, h , providing the value of the output quantity. multiplying the combined standard uncertainty, u,, by a coverage factor, k, yields an expanded uncertainty, u. this allows an interval to be calculated, [(y k uj; (y + k u 3 ] , such that a stated number fraction of all intervals obtained by calculation from repeated measurement results will contain a true value of the measurand; the g’ 218 number fraction (or probability) depends on k . in any case, the conditions under which a result and its uncertainty are obtained must be specified; terms such as "inter-run" and "reproducibility conditions" need explanation in view of different definitions being offered by various standards. bibliography abbreviations of international organizations bipm iec international electrotechnical commission ifcc international federation of clinical chemistry i s 0 international organization for standardization iupac iupap oiml international bureau of weights and measures international union of pure and applied chemistry international union of pure and applied physics international organization of legal metrology bipm, iec, ifcc, iso, iupac, iupap, oiml. international vocabulary of basic and general terms in metrology (vim). 2 ed. geneva: iso, 1993 (in press). iso. statistics vocabulary and symbols. part 2. statistical quality control. iso/dis 3534-2. geneva: iso, 1986:iii + 30 pp. iso. statistics vocabulary and symbols. part 1. probability and general statistical terms. iso/dis 3534-1. geneva: iso, 199030 pp. iso. accuracy (trueness and precision) of measurement methods and results. part 1. general principles and definitions. iso/dis 5725-1. geneva: iso, 1991:24 pp. idem. part 2. a basic method for the determination of repeatability and reproducibility of a standard measurement method. iso/dis 5725-2. geneva: iso, 1991:67 pp. idem. part 4. basic methods for estimating the trueness of a test method. iso/dis 5725 4. geneva: iso, 1991:35 pp. 3 -935253 219 idem. part 3. intermediate measures of the precision of a measurement method. iso/dis 5725-3.2. geneva: iso, 1992:49 pp. bipm, iec, ifcc, iso, iupac, iupap, oiml. guide to the expression of uncertainty in measurement. geneva: iso, 1993 (in press). correspondence: renc dybkar department of clinical chemistry frederiksberg hospital dk-2000 frederiksberg, danmark 220 upsala j med sci 97: 195-200 pulse pressure, mean blood pressure and impaired glucose tolerance-a study in middle-aged subjects jan cederholm1,2 and lars wibell' i departments of internal medicine and 2farnily medicine, university hoapital, uppsala, sweden a b s t r a c l in a study of 695 middle-aged subjects, without antihypertensive agents, and without more pronounced obesity, both pulse pressure (pp) and mean blood pressure (mbp) were strongly related to 2-h blood glucose in 75 g ostts (p < 0 . 0 0 1 ) . all hypertensives (dbp 290 mm hg) were separated into 39 with higher pp (260 m hg) and 137 with lower pp (<60 mm hg). the high pp hypertensives, compared with the low pp hypertensives and all 519 normtensives, had higher frequency of impaired glucose tolerance (igt; who-criteria), 3 3 % , 606, and 4%, respectively (p <0.001), and also higher mean 2-h blood glucose, 5.9, 4.5, and 4,2 mmo1.1-1, respectively (p <0.001). these differences were independent of mbp levels. similarly, all 54 hypertensives with higher mbp (2110 mm hg) had more igt and higher 2-h glucose than the 122 hypertensives with lower mbp (<110 m hg) or the normtensives, 30%, 5% and 4%, respectively (p < 0 . 0 0 1 ) , and 5.8, 4.4, 4.2 m~l.l-~, respectively (p <0.001), independently of pp. thus, both high pp and high mbp were related to igt, independently of each other. i n t r o d u c t i o n blood pressure can be divided into two components: a steady component, represented by mean blood pressure, and a pulsatile component, represented by pulse pressure (3,4,9). it was the aim of this study to analyse the independent relationships between blood glucose and these two components, in middle-aged untreated subjects. 195 m a t e r i a l a n d m e t h o d s a sample of 695 subjects, 47-54-years-old, was obtained from a health survey in uppsala, previously described, with a participation rate of 71% (1). all subjects in the health survey with mre pronounced obesity or antihypertensive agents were excluded from the present study. the hypertensives, 176 subjects (45.5% males, 54.5% females), were the subjects with diastolic blood pressure (dbp) 290 mm hg and no antihypertensive agents. the normotensives were 519 subjects (50.3% males, 49.7% females). body mass index (bmi) was computed as weight height-2 (kg. m 2 ) and expressed as relative bmi (rbmi, % ) , based on ideal bmi values. blood pressure (bpi was measured sitting after >15 min of rest and no previous smoking, using korotkoff fifth phase sounds, with a mercury sphygmomanometer (cuff size 12.5 x 35 cm), by the same observer. pulse pressure (pp) was the difference of systolic bp and diastolic bp. mean blood pressure (mbp) was diastolic bp + one-third of pp. all subjects with rbmi >130% at the health survey were excluded from the present study, to avoid the problem of greater arm circumference in obese subjects. oral glucose tolerance tests (cgtt) were performed in the morning after 10 hours of fasting (11). venous whole blood glucose was measured at 0-h and 2-h levels by a glucose oxidase method (ysi model 23 a m ) . impaired glucose tolerance (igt) was diagnosed according to strict who-criteria, based on two subsequent ogtts (11). subjects with manifest diabetes mellitus were excluded from the present study. a questionnaire was used to obtain information on smoking and physical activity during leisure time and at work, the latter evaluated with a 4-point scale as used in the gothenburg studies. statistical analysis. analyses were performed with the sas program. a p value of p <0.05 was considered statistically significant. student's t-test, chi-square statistics and pearson's correlations were used. multiple regression analysis (prcc glm) was used (table 1) with t-values of the predictors and the coefficient of determination (r ) given. analysis of covariance (pro= glm) yielded mean values (table 3 ) , after adjustment for confounding covariates. 2 n-way analysis of frequency distribution (prcc freq) yielded the odds ratio (table 3) , after adjustment for n confounding covariates, with cochran mantel haenszel correlation and general association statistics. 196 table 1. multiple regressions, pp and mbp as dependent variables (n=695). ........................................................................... pulse pressure mean blood pressure t-va lue t-va lue predictors: 2-h glucose body mass index age 4.8 *** 3.1 ** 5.2 *** 6.9 *** 4.2 *** 1.9 r e s u l t s in all 695 participants, the correlation coefficients (r) were: pp-sbp 0.84, pp-dbp 0.11, and mbp-sbp 0.89, mbp-dbp 0.91. other correlations were: pp-mbp 0.52, sbp-dbp 0.62. all correlations were mainly similar in females and mles. table 1 shows that 2-h glucose was independenly related to both pp and mbp (p <0.001). hypertensives with low or high pp levels. all hypertensives were divided in two groups, with the mean + 1 sd value of pp (60 mm hg) as the dividing level (table 2). group 2 hypertensives (high pp level) had higher 2-h glucose than group 1 hypertensives (low pp level) or normotensives, also after adjustment for mbp levels (table 3, left part). the frequency of igt (table 4, left part) was clearly increased in group 2 hypertensives, 33%, compared with group 1 hypertensives, 7 % , and normtensives, 4%, also differing as odds ratios for igt, adjusted for mbp levels (p <0.001). hypertensives with low or hiqh mbp levels. all hypertensives were also divided in two groups, wih roughly the mean + 1 sd value of mbp (110 mm hg) as the dividing level (table 2). group 4 hypertensives (high mbp) had higher mean 2-h glucose than group 3 hypertensives (low mbp) and normotensives, even when adjusted for pp levels (table 3, right part), and the frequency of igt was higher in group 4 hypertensives, 30 %, than in group 3 hypertensives, 5%, or normtensives, 4%, independently of pp according to adjusted odds ratios (p <o. 001) . all differences of 2-h glucose and odds ratios above remained (p <0.01), when adjustment also was made for bmi, simultaneously with the other covariates above (not in the table). group 4 hypertensives included 16 igt subjects, and 13 of them were also found among the group 2. 197 hypertensives (ht) versus normtensives (nt) : sp <0.001, &p <0.01. se = st err group2-ht vs groupl-ht or group3-ht vs group4-ht: $p <0.001. d i s c u s s i o n the main findings in this study was, that hypertensives with high pp levels or high mbp levels had increased prevalence of igt (one-third), and that pp and mbp levels were, independent of each other, related to igt and postload 2-h blood glucose. blood pressure has been divided in two components (3,9). the steady component, mbp, can be estimated as the product of cardiac output and vascular resistance (mainly in small arteries). the pulsatile component, pp, is determined by other mechanisms, and related to the ratio of ventricular ejection time (stroke volume) and vascular compliance (visco-elastic properties of mainly large arteries). the dominant factor causing high pp seems to vary greatly with age. younger subjects with high pp have mainly increased velocity of ventricular ejection and stroke volume, with normal arterial compliance (9). middle-aged may have both increased stroke volume and decreased arterial compliance (lo), while old subjects have mainly decreased compliance ( = increased arterial stiffness) (9). pp was strongly correlated to sbp (r=0.85) and an obvious marker for systolic hypertension. on the contrary, pp is the variable least related to dbp, 'classical hypertension'. pp was clearly related to postload glucose in chicago (5), weakly in paris (3). small studies of hypertensives with high pp had increased rates of manifest diabetes (2,5). pp was also indepedently related to age in the present study, as in paris and chicago above the ages of 45-55 (3,4), since arteries stiffen with increasing age. 198 we found mbp independently related to 2-h blood glucose, as in the paris study (3). notably, our hypertensives with high mbp seemed to include many hypertensives with high pp. the corr coeff pp-mbp was 0.52 in the present study. 2-h glucose (ml/l) pp (mm hg) mbp (mm hg) numbers freq. igt ( 8 ; ) odds ratio igt (95% bounds) 5.8/ 4.5as 5.2/ 4.3as 5.7/ 4.4bs 5.5/ 4.2bs ;t0.3/+0.1 k0.3/+0.1 ;t0.3/+0.2 +o. 2/+0.1 58kl.1/47*0 .f%olto.3/43&1.@ 113r0.6/106~0.p 112+0.6/96+0.$s 39/137 39/519 54/ 122 54/519 33/6.6 33/4.1 30/4.9 30/4.1 5.2 4.6 5.2 6.4 (2.2-12.5)& (2.0-10.8p (1.7-16. of (3.3-12.6p %djusted for mbp, age and sex (concerning group 2). 'adjusted for pp, age and sex (concerning group 4). p <0.001. se = st err ' p <0.01. a possible bias was the single blood pressure measurement here, as in paris (3) and partly chicago ( 4 ) , which might result in loss of power in the analysis. it has been shown, however, that a single blood pressure measurement can predict which individuals are more likely to develop cardiovascular diseases (4). moreover in chicago, relationships were similar with a single, the mean of 3, or the lowest of 3 measurements used (4). furthermore, as our associations were strongly significant (p <0.001), we believe this bias was mitigated in the present study. hyperinsulinaemia in igts is related to hypertension, possibly due to increased sympathetic nervous activity (71, which might either increase the contractibility of the heart or increase smooth muscle tone and arterial stiffness (6,8). high pp has been related to left ventricular hypertrophy in hypertensives and to coronary heart disease above the age of 45 (4). do hypertensives with igt and high pp have changes in ventricular ejection, or do they have increased arterial stiffness due to atherosclerosis, microangiopathy or sympathetic activity? this could have therapeutical implications. ace-inhibitors and calcium entry blockers, for example, seem useful in igt, and might also improve pp and arterial compliance (9). 199 r e f e r e n c e s 1. 2. 3. 4. 5. 6. 7. 8. 9. cederholm, j. & wibell, l.: glucose intolerance in middle-aged subjects a cause of hypertension? acta med scand 217: 363-372, 1985. colandrea, m.a., friedman, g.d. & nichaman, m.z.: systolic hypertension in the elderly. circulation 41:239-242, 1970. darne, b., girerd, x., safar, m., cambien, f. & guize, l.: pulsatile versus steady component of blood pressure: a cross-sectional analysis and a prospective analysis on cardiovascular mortality. hypertension 13: 392-400, 1989. dyer, a.r., stamler, j., shekelle, r.b., schoenberger, j.a. & stamler, r: pulse pressure i. level and associated factors in four chicago studies. j chon dis 35: 259-273, 1982. ferguson, j.j. & randall, o.s. systolic, diastolic and combined hypertension. arch intern med 146: 1090-1093, 1986. moore, r.d. effects of insulin upon ion transport. biochirn biophys acta 737: 1, 1983. o'hare, j.a.: the enigma of insulin resistance and hypertension. am j med 84: 505-510, 1988. petrin, j., egan, b.m. & julius, s.: increased beta-adrenergic tone enhances arterial compliance in hyperkinetic borderline hypertension. j hypertension 7 (suppl 6): s78-s79, 1990. ricandri, c.l., agabiti-rosei, e., fariello, r., beschi, m & boni, e.: aortic rigidity and plasma catecholamines in essential hypertensive patients. clin exp hypertens a4: 1073-1078, 1982. 10. safar, m.e.: editorial review. pulse pressure in essential hypertension: clinical and therapeutical implications. j hypertension 7 : 769-77 i 1989. 11. who study group on diabetes. diabetes mellitus. who tech rep ser 727. who, geneva, 1985. address to: dr j cederholm department of internal medicine university hospital 5-75185 uppsala sweden 200 upsala j med sci 79: 5 5 6 2 , 1974 nocturnal secretory patterns of fsh, gh, lh and tsh p. 0. osterman, g. wallin and l. wide departments of neurology, neurophysiology and clinical chemistry, university hospital. uppsala, sweden abstract tsh (17) have been reported, whereas in other the nocturnal secretory patterns of foiiicie-stimulating investigations (8, 18) such a rhythm has not been hormone (fsh), growth hormone (gh), luteinizing observed. an episodic secretion of fsh and lh hormone (lh) and thyroid-stimulating hormone (tsh) has been found in some studies (15, 2 5 ) , whereas were studied in 1 female and 4 male volunteers. immunoin other investigations the plasma have been reactive fsh, gh, lh and tsh in serum were assayed 11-hydroxycorticosteroids in plasma were also measured of only one or two hypophyseai hormones have in the male volunteers by a fluorimetric method. been determined at the same time. the dresent by a radioimmunosorbent technique. non-conjugated rather constant (20). the piasma levels were taken half from lo pemto 8 a.m. polygraphic monitoring of sleep stages was per formed. in all the subjects a marked elevation of serum gh oecurred within 60 minutes affer the onset of investigation was undertaken to ascertain whether there is any correlation between the nocturnal secretory patterns of fsh, gh, lh and tsh or sleep and additional, isolated gh peaks were observed in 3 subjects. the serum concentrations of fsh, lh and tsh also fluctuated, but the variations were small between the secretory patterns of these hormones and different stages of sleep. compared with the fluctuations of gh. the serum levels of fsh, lh and tsh respectively were not cor related with the depth or stages of sleep. the observed fluctuations, in the serum levels of all hormones assayed, are larger than could be expected from intra-assay errors and probably represent changes in the secretion rate of the hormones. a lack of concordance between the secretory patterns of fsh, gh, lh and tsh in dicates that different mechanisms may be involved in their release. introduction during recent years several studies have been made on the secretory patterns of hypophyseal hormones. the occurrence of a circadian rhythm, and an episodic secretion of acth and cortisol are well established (7, 12). the secretory pattern of the growth hormone (gh), which is characterized by sporadic peaks and a marked elevation of plasma g h at the onset of deep sleep, is also well known (9, 27). however, the results from studies on the plasma levels of follicle-stimulating hormone (fsh), luteinizing hormone (lh) and thyroid-stimulating hormone (tsh) are more divergent. a circadian rhythm of the secretion of fsh (9, l h (26) and materials and methods four healthy male volunteers, in the age range 19-24, and one 24-year-old, healthy female volunteer were studied. none of them took any medicine, tobacco or alcohol during the study. the female (mo) had a normal menstrual cycle and did not take oral contraceptives. blood samples were obtained from her 9 and 10 days respectively after the onset of the preceding menstrual period. all the subjects reported a normal sleepwaking cycle. all had normal eeg activity during sleep and wakefulness. three of the subjects (cb, jb and mo) spent 3 nights, and two (hh and bs) spent 4 con secutive nights in a quiet room in the neurophysio logical department. only a weak light was used in the room from 11 p.m. to 7 a.m. the subjects were fasting after 7 p.m. electroencephalographic, electrooculographic and electromyographic recordings were made con tinuously from 10 p m . to 7 a.m. on the last 2 nights. because of a technical defect, sleep-stage scoring for the female volunteer was possible only for the last night of the study. for the female subject blood sampling took place on the last 2 nights of the study and for the males on the last night. a flexible venous cannula was inserted into an antecubital vein and connected to a 2-foot long plastic tube, so as to permit blood drawing without touching the subject’s arm. in 3 cases the system was filled with heparinized isotonic saline between samples, and in 2 cases the cannula was kept patent with a slow drip of isotonic saline. in each case upsala j med sci 79 56 p . 0. osterman et al. blood samples were taken half hourly from 10 p.m. to 8 a.m. from the female subject a total volume of approximately 200 ml of blood was removed on each of the 2 nights of the study. plasma ll-hydroxycorti costeroids were not determined in the female volunteer in order to avoid removal of more blood. a total volume of approximately 300 ml of blood was removed from each male subject. blood samples for assay of plasma 1 1-hydroxycorticosteroids were run into heparinized tubes, whereas fsh, gh, l h and tsh were determined in serum. after centrifugation plasma and serum were stored at -20°c until assayed. plasma 1 1-hydroxycorticosteroids were assayed by a fluorimetric method measuring non-conjugated 1 l-hydro xycorticosteroids (14). during the period of investigation the intra-assay error estimated from duplicate determina tions was 4 4 % (coefficient of variation). immunoreactive fsh, gh, l h and tsh in serum were assayed by a radioimmunosorbent technique (3 1, 32, 33). fsh in serum was measured by utilizing human pituitary fsh (23) labelled with lz5i and guinea pig anti-human pituitary fsh. the fsh preparation had a biological activity of 14 ooo iu (2nd irp-hmg) per mg. the results were expressed in ng per ml. one ng of fsh was equivalent to 369 ng of ler-907 in the immunoassay. gh in serum was measured by utilizing human pituitary g h (24) labelled with lz5i and rabbit anti-human pituitary gh. the results were expressed in ng per' ml using the who international reference preparations of growth hormone, 66/217, as a reference. lh in serum was measured by utilizing human pituitary lh (23) labelled with 1251 and rabbit anti human pituitary lh. the lh preparation had a biolo gical activity of 14 000 iu (2nd irp-hmg) per mg. the results were expressed in ng per ml. one ng of l h was equivalent to 83 ng of ler-907 in the immunoassay. tsh in serum was assayed by utilizing human pituitary tsh (national pituitary agency) labelled with lz5i and rabbit anti-human pituitary tsh. the activity was expressed in p u per ml using medical research council htsh research standard a as a reference. in all radioimmunoassays the standards were dis solved in sera with undetectable or low levels of the hormones to be assayed. the samples were assayed i n duplicate. the results of the immunoassays were cal culated by the use of a logit transformation as pro posed by rodbard e t al. (22). the percentage standard error of the mean of the two determinations was between 4 and 6%. for recordings of eeg, neck muscle emg and eye movements ag-agc1 surface electrodes were glued to the skin. two symmetrical eeg channels were re corded with electrodes in position p3-01 and p4-oz. for recordings of eye movements one electrode was placed a t the lateral corner of one eye and another electrode above the same eye. the emg electrodes were placed symmetrically over the muscles on the back of the neck. the 4 signals were amplified in a standard eeg apparatus (elema, sweden) and from there fed to a 4 channel fm tape recorder (p1-6200, usa), upsala j med sci 79 where they were stored for later analysis. eeg ana lysis was made visually and 4 sleep stages were separated. stage 1 : light sleep with low voltage background, sleep spindles and k-complexes. no rapid eye movements. stage 2: intermediary stage with a fair amount of high voltage slow waves and superimposed spindles. stage 3: deep sleep dominated by high voltage, slow delta waves. rapid eye movernenf ( r e m ) stage: low voltage, fast activity without spindles and with simulta neously occurring rapid eye movements. absence of emg activity in the neck muscles. results the results are shown in figs. 1-3. onset of sleep occurred between 10 and i 1 p.m. in 2 sub jects and between midnight and 1 a.m. in 3 sub jects. each person slept well for several hours during the night, but the blood sampling procedure occasionally awakened the subjects. in 2 men (cb and bs) rem sleep constituted about 10% and in 2 men and one woman (jb, h h and mo) rem sleep constituted about 15% of total sleep. the serum or plasma concentrations of the 5 hormones assayed were, throughout, within the normal range as determined in this laboratory in fertile men and women. in the 4 male volunteers, in whom plasma i l hydroxycorticosteroids were determined, maximum levels occurred during the early morning hours. the pattern of plasma 1 1-hydroxycorticosteroid levels was in all the subjects characterized by sharp increases followed by falls in the plasma level. the lowest levels occurred between 2 and 4 a.m. it can be seen on the graphs that in all the subjects the serum concentrations of fsh, g h , l h and tsh fluctuated. each point on the graphs represents the mean of duplicate determinations. the variation between the points in each curve is caused by the error of the mean of the duplicates (intra-sample variation) and a possible true varia tion between the samples (inter-sample variation). the variance of the results obtained during the period of investigation was calculated for each curve. the difference between this value and the variance of the mean of the duplicate determinations (intra-sample variation) gave an estimate of the inter-sample variance. the median and range of the inter-sample variation, expressed as percentage standard deviation was 12 (4.2-17.3) for fsh, 22.7 (16.4-30.7) for l h , 15.3 (10.0-22.3) for tsh and 95.5 (71.8-130) for gh. thus, an nocturnal secretory patterns of f s h , g h , lh and tsh 57 rem 1 . 2 3 awake rem . 1 . 2 . 3 c.a. d j. 8. d” 2 0 . 10. 5 . a . 3 . 3 . 2 2 , . l h nglml 3 1 1.0 0.5 o.s1 lh n g h l 1stipulml-t gh nglml 30 ’ 20 . 10 5 l 3 2 1.0 tsh uulml jwi gh nglml 20 10 5 a 3 2 58 p . 0. osterman et al. 2.0 1.0 0.5 0.3 h.h. d 1.0 0.5 0.3 b.s. d 2 1.0 0.5 awake 1 2 3 e h pu/ml yln gh nglml -lo 0.5 j awoke rem * 1 2 3 tsh pu/ml y g h nglml 10 5 i 3 w i 1 hydroxy cwticosterolds 22 24 02 04 06 08 time of day 22 2.4 02 04 06 08 time of day fig. 2. patterns of serum fsh, lh, tsh, gh and lationship to sleep stages in male subjects hh and bs. plasma 11-hydroxycorticosteroid levels and their reblood samples taken half hourly from 10 p.m. to 8 a.m. vpsala j med sci 79 nocturnal secretory patterns of f s h , g h , l h and fsh 59 u.o. 0 r e m 1 2 3 u.o. q 1.0. 0.5 j 22 2l 02 ol 06 08 tsh uulml )-* gh ng'ml -lo 1 2.0. j 1.0 1 1/22 0.5 2 2 2 4 02 04 06 08 time of day fig. 3. patterns of serum fsh, lh, tsh and gh levels in the female subject. blood samples taken half hourly two consecutive nights, 9 and 10 days respectively evident inter-sample variation was found for all four pituitary hormones. in all the subjects a significant elevation of serum g h concentration occurred within 60 minutes after the onset of sleep. a peak in serum g h concentration preceded the g h peaks associ ated with the onset of sleep in 2 subjects (hh and bs). in the female subject a second gh peak at 3 a.m. followed the peak which appeared at the onset of sleep. the variation in the level of l h was greater than those of the fsh and tsh levels. serum levels of fsh, l h and tsh were not correlated (p>0.05) with the depth or stages of sleep. the secretory patterns of fsh, l h and tsh were not constant ,between nights, in the female subject fsh ng/ml n-x lh ng/ml 2.0 1 0.5 22 2 4 02 ol 06 08 0.5 i 22 21 02 04 06 08 time of day after the onset of the preceding menstrual period. sleep stages recorded the last night. studied. furthermore no relationship was observed between the secretory patterns of fsh, g h , lh and tsh in any of the 5 subjects. discussion several studies have shown that cortisol is secreted in a series of brief episodes separated by intervals during which there is either no, or only low, secretion. in men and women with a >>normal>> sleep-waking cycle most of the secretory bursts occur in the early morning hours, with maximum plasma cortisol levels between 4 and 8 a.m. (7, 12, 19, 30). in the present investigation the pattern of plasma 1 1-hydroxycorticosteroid levels was, in all 4 men in accordance with that de upsala j med sci 79 60 p . 0. osterman el al. scribed previously for plasma cortisol. thus, all the men had a .normal. pattern of plasma 11 hydroxycorticosteroid levels in spite of a certain amount of stress caused by frequent blood sampling. krieger et al. (12) found a very good correlation between the times of peaks of plasma corticosteroid levels and those of plasma acth levels. the time at which the acth peak occurred coincided in most cases with that of the plasma 11-hydroxy corticosteroidpeak. however, there was no apparent proportionality between plasma corticosteroid and plasma acth levels. in all the subjects a marked elevation of serum g h was observed at the onset of sleep. in 3 sub jects, 2 male and one female a second g h peak was observed. the peak at 10.30 p.m. in subject h h may be due to the stress of the initial vene punction, that at midnight in subject bs is pro bably part of the nocturnal peak. the observed secretory pattern of g h is similar to that previously reported (9, 10, 21, 27). evidently, the peak of serum g h at the onset of sleep is very constant, whereas additional nocturnal peaks are often missing. dierschke et al. (4) found, in ovariectomized rhesus monkeys, wide rhythmic oscillations in the plasma l h levels. in intact female monkeys no such fluctuations were observed. similarly, in castrated male and female rats fairly regularly occurring peaks of lh were found, whereas the serum fsh levels were more stable (6). yen et al. (34) reported, in women of fertile age, fluc tuations in the serum level of l h which were rather regular in frequency and magnitude. however, these parameters varied according to the stage of the menstrual cycle. in the present study the fluctuations of fsh and l h occurred irregularly, and were of varying magnitude. this finding is in accordance with observations reported in recent investigations in adult men (2, 13, 16, 25) and in one investigation on women of fertile age (15). the fluctuations found for fsh were smaller than for l h which agrees with previous studies (15, 32, 34). there was no correlation between the levels of fsh and l h , and the depth or stages of sleep. rubin et al. (25) observed that in adult men the l h values were 14% higher during rem sleep than during other stages of sleep; but release of fsh was not clearly related to the stages of sleep. on the other hand, boyar et al. (1) did not find, in adult men and prepubertal children, any signifi cant difference between mean lh concentration, regardless of whether the subjects were asleep or awake; and no relation between lh secretory activity and the stages of sleep was observed. in pubertal children, however, the mean, sleep l h concentration was significantly higher than the mean, awake lh concentration ( i ) . vanhaelst et al. (28) observed, in 7 of 8 euthy roid subjects, a peak of tsh between 4 and 6 a.m. moreover, all the subjects exhibited a rhythm of higher frequency, lasting from 1 to 2 hours. the findings of these authors are at variance with those of webster et al. (29), who demonstrated, in normal subjects and in patients with myxedema, occasional significant variations in serum tsh concentration, but no convincing pattern of cir cadian rhythm or other mode of variation of tsh secretion. the present study supports the results of webster et al. (29). fluctuations of the serum concentrations of tsh were observed, but there was no evidence of a regular periodicity. the observed fluctuations, in the serum levels of all hormones assayed, are larger than could be expected from intra-assay errors. the fluctuations of the radioimmunoassayed hormones, fsh, gh, l h , tsh, are not synchronized. therefore it is unlikely that the fluctuations are due to variations of substances in serum, causing nonspecific inter ference with the assay technique. kohler et al. ( l l ) , and coble et al. (3) have shown that the metabolic clearance rates of l h and fsh are rather constant, and are not influenced by gonadal function. it is likely that the observed fluctuations in the serum levels of fsh, g h , l h and tsh probably represent changes in the secretion rate of the hormones. takahashi et al. (27) did not find any correlation between the secretory patterns of cortisol and gh. rubin et al. (25) observed no relation between the peaks of fsh and l h in adult men; nor did krieger et al. (13) detect any relation between the patterns of fsh, g h and l h in adult men. yen et al. (34) failed t o observe any relation between the secretory patterns of fsh and l h in women of fertile age, but in postmenopausal women the fluctuations of fsh and l h usually were coincidental with minor asynchrony. in the study by midgley & jaffe (15), in women of fertile age, the changes in the fsh level often paralleled those of l h . in this study, no correlation was found between upsala j med sci 79 nocturnal secretory p a t t e r n s of f s h , g h , l h and f s h 61 the nocturnal secretory patterns of fsh, g h , l h a d tsh. the mechanisms, regulating the secretion of the different hypophyseal hormones, are still un known. the lack of concordance between the secretory patterns of fsh, g h , l h and tsh indicates that different mechanisms may be in volved in their release. it is not known whether the observed fluctuations in the trophic hormone levels are necessary for the maintenance of a normal function of the target organs. further similar studies on patients with hypothalamo-hypo physeal diseases may prove valuable for under standing the importance of the observed secretory patterns. acknowledgements we are indebted to dr paul roos at the institute of biochemistry, uppsala for supplying the highly purified lh, fsh and g h preparations, and to mrs anna-lena barmark, mr christer bengtsson and miss vera star felt for skilful technical assistance. this study was supported by the swedish medical research council (grant no. 13x-3145) and by a grant from the medical faculty of the university of uppsala. references 1. boyar, r., finkelstein, j., roffwarg, h., kapen, s., weitzman, e . & hellman, l.: synchronization of augmented luteinizing hormone secretion with sleep during puberty. new eng j med 287: 582, 1972. 2. boyar, r., perlow, m., hellman, l., kapen, s. & weitzman, e.: twenty-four hour pattern of luteinizing hormone secretion in normal men with sleep stage recording. j clin endocr35: 73, 1972. 3. coble, y . d., kohler, p. o., cargille, c. m. & ross, g. t.: production rates and metabolic clearance rates of human follicle-stimulating hormone in premenopausal and postmenopausal women. j clin invest 48: 359, 1969. 4. dierschke, d. j., bhattacharya, a. n., atkinson, l. e. & knobil, e.: circhoral oscillations of plasma lh levels in the ovariectomized rhesus monkey. endocrinology 87: 850, 1970. 5. faiman, c. & winter, j. s. d.: diurnal cycles in plasma fsh, testosterone and cortisol in men. j clin endocr 33: 186, 197 1. 6 . gay, v . l. & sheth, n. a.: evidence for a periodic release of l h in castrated male and female rats. endocrinology 90: 158, 1972. 7. hellman, l., nakada, f., curti, j., weitzman, e. d., kream, j., roffwarg, h., ellman, s., fukushima, d. k. & gallagher, t. f.: cortisol is secreted episodically by normal man. j clin endocr 30: 411, 1970. 8. hershman, j . m. & pittman, j . a.: utility of the radioimmunoassay of serum thyrotrophin in man. ann intern med 74: 481, 1971. 9. honda, y., takahashi, k., takahashi, s., azumi, k., h i e , m., sakuma, m., tsushima, t. & shizume, k.: growth hormone secretion during nocturnal sleep in normal subjects. j clin endocr 29: 20, 1969. 0. hunter, w . m., rigal, w. m. & sukkar, m. y.: plasma growth hormone during fasting. in: proc. int. symp. growth hormone, milan, italy, p. 408. excerpta medica, amsterdam, 1968. 1. kohler, p. o., ross, g. t. & odell, w. d.: meta bolic clearance and production rates of human luteinizing hormone in preand postmenopausal women. j clin invest 47: 38, 1968. 12. krieger, d. t., allen, w., rizzo, f. & krieger, h. p.: characterization of the normal temporal pattern of plasma corticosteroid levels. j clin endocr 32: 266, 1971. 13. krieger, d. t . , ossowski, r., fogel, m. & allen, w.: lack of circadian periodicity of human serum fsh and l h levels. j clin endocr 35: 619, 1972. 14. mattingly, d.: a simple fluorimetric method for the estimation of free 1 i-hydroxycorticoids in human plasma. j clin path 15: 374, 1962. 15. midgley, a. r. & jaffe, r. b.: regulation of human gonadotropins: x. episodic fluctuation of l h during the menstrual cycle. j clin endocr 33: 962, 1971. 16. nankin, h. r. & troen, p.: repetitive luteinizing hormone elevations in serum of normal men. j clin endocr33: 558, 1971. 17. nicoloff, j . t., fisher, d. a. & appleman, m. d.: the role of glucocorticoids in the regulation of thyroid function in man. j clin invest 49: 1922, 1970. 18. nillius, s. j. & wide, l.: effects of oestrogen on serum levels of lh and fsh. acta endocr (koben havn)65: 583, 1970. 19. otth, d. n., island, d. p. & liddle, g. w.: experi mental alteration of the circadian rhythm in plasma cortisol (17-ohcs) concentration in man. j clin endocr 27: 549, 1967. 20. peterson, n. t . , midgley, a. r. & jaffe, r. b.: regulation of human gonadotropins. 111. luteinizing hormone and follicle stimulating hormone in sera from adult males. j clin endocr 28: 1473, 1%8. 21. quabbe, h-j., schilling, e. & helge, h.: pattern of growth hormone secretion during a 24-hour fast in normal adults. j clin endocr 26: 1173, 1966. 22. rodbard, d., rayford, p. l., cooper, j. a. & ross, g. t.: statistical quality control of radioimmuno assays. j clin endocr 28: 1412, 1968. 23. roos, p.: human follicle-stimulating hormone. acta endocr (kobenhavn) suppl. 131: 1, 1968. 24. roos, p., fevold, h. r. & gemzell, c. a.: pre paration of human growth hormone by gel filtration. biochim biophys acta 74: 525, 1963. 25. rubin, r. t., kales, a., adler, r., fagan, t. & odell, w.: gonadotropin secretion during sleep in normal adult men. science 175: 196, 1972. 26. saxena, b. b., leyendecker, g., chen, w., gandy, vpsulu j med sci 79 62 p. 0. osterman et al. h. m. & peterson, r. e.: radioimmunoassay of follicle-stimulating (fsh) and luteinizing (lh) hor mones by chromatoelectrophoresis. acta endocr (kobenhavn) suppl. 142: 185, 1969. 27. takahashi, y., kipnis, d. m. & daughaday, w. h.: growth hormone secretion during sleep. j clin invest 47: 2079, 1968. 28. vanhaelst, l., van cauter, e., degaute, j. p. & golstein, j.: circadian variations of serum thyro tropin levels in man. j clin endocr 35: 479, 1972. 29. webster, b . r . , guansing, a. r. & paice, j . c.: absence of diurnal variation of serum tsh. j clin endocr 34: 899, 1972. 30. weitzman, e. d . , fukushima, d., nogeire, c., roffwarg, h., gallagher, t. f. & hellman, l.: twenty-four hour pattern of the episodic sercretion of cortisol in normal subjects. j clin endocr 33: 14, 1971. 31. wide, l.: radioimmunoassays employing immuno sorbents. acta endocr (kobenhavn) suppl. 142: 207, 1%9. 32. wide, l., nillius, s. j . , gemzell, c. & roos, p.: radioimmunosorbent assay of follicle-stimulating hormone and luteinizing hormone in serum and urine from men and women. acta endocr (koben havn) suppl. 174: 1 , 1973. 33. wide, l. & porath, j.: radioimmunoassay of pro teins with the use of sephadex-coupled antibodies. biochim biophys acta 130: 257, 1966. 34. yen, s. s. c., tsai, c. c., naftolin, f., vanden berg, g. & ajabor, l.: pulsatile patterns of gonado tropin release in subjects with and without ovarian function. j clin endocr34: 671, 1972. received june 8,1973 address for reprints: per olof osterman, m.d. department of neurology university hospital s-750 14 uppsala 14 sweden upsala j med sci 79 upsala journal of medical sciences 2021, 126, e7649 http://dx.doi.org/10.48101/ujms.v126.7649 the influence of nutritional state on the fatty acid composition of circulating lipid fractions: implications for their use as biomarkers of dietary fat intake sion a. parrya#, fredrik rosqvista,b#, sarah petersa, rebecca k. younga, thomas cornfielda, pamela dysona,c and leanne hodsona,c* aoxford centre for diabetes, endocrinology and metabolism, university of oxford, churchill hospital, oxford, united kingdom; bdepartment of public health and caring sciences, clinical nutrition and metabolism, uppsala university, uppsala, sweden; cnational institute for health research oxford, biomedical research centre, oxford university, hospital trusts, oxford, united kingdom abstract background: the fatty acid (fa) composition of blood can be used as an objective biomarker of dietary fa intake. it remains unclear how the nutritional state influences the fa composition of plasma lipid fractions, and thus their usefulness as biomarkers in a non-fasted state. objectives: to investigate the associations between palmitate, oleate and linoleate in plasma lipid fractions and self-reported dietary fa intake, and assess the influence of meal consumption on the relative abundance of these fa in plasma lipid fractions (i.e. triglyceride [tg], phospholipids [pls] and cholesterol esters [ces]). design: analysis was performed in plasma samples collected from 49 (34 males and 15 females) participants aged 26–57 years with a body mass index (bmi) between 21.6 and 34.2 kg/m2, all of whom had participated in multiple study visits, thus a pooled cohort of 98 data sets was available for analysis. a subset (n = 25) had undergone nutritional interventions and was therefore used to investigate the relationship between the fa composition of plasma lipid fractions and dietary fat intake. results: significant (p < 0.05) positive associations were observed between dietary polyunsaturated fat and linoleate abundance in plasma ce. when investigating the influence of meal consumption on postprandial fa composition, we found plasma tg palmitate significantly (p < 0.05) decreased across the postprandial period, whereas oleate and linoleate increased. a similar pattern was observed in plasma pl, whereas linoleate abundance decreased in the plasma ce. conclusion: our data demonstrate that the fa composition of plasma ce may be the lipid fraction to utilise as an objective biomarker when investigating recent (i.e. previous weeks-months) dietary fa intakes. in addition, we show that the consumption of a high-fat meal influences the fa composition of plasma tg, pl and ce over the course of the postprandial period, and therefore, suggest that fasting blood samples should be utilised when using fa composition as a biomarker of dietary fa intake. article history received: 17 february 2021 revised: 16 april 2021 accepted: 11 may 2021 published: 13 july 2021 keywords postprandial; fatty acids; biomarker; lipid fractions; fatty acid composition introduction the high prevalence of metabolic diseases such as cardiovascular disease (cvd) and type 2 diabetes (t2d) are recognised as a global health issue (1). the relationship between dietary fat quantity and quality and metabolic health is highly debated with some suggesting that increased intake of saturated fat is associated with an increased risk for the development of cardiometabolic diseases (e.g. cvd and t2d), whereas others suggest that no relationship exists (2–5). the conflicting findings may be partly attributable to the methods of dietary assessment used in epidemiological studies, which typically involve the use of food-frequency questionnaires or food diaries, which have known limitations (6–8). using the fatty acid (fa) composition of blood and tissues as a biomarker of dietary fa intake is an additional and objective method of dietary assessment (9, 10). typically, the fa composition of lipid fractions in blood is measured in the fasting state to avoid the potentially confounding influence of recent dietary fa intake (11, 12). however, although it is often assumed that non-fasting samples cannot be used to investigate biomarkers of dietary fa intake, this assumption has not been fully investigated; it remains unclear as to whether nutritional state influences the fa composition of circulating lipid fractions. for large-scale contact leanne hodson leanne.hodson@ocdem.ox.ac.uk supplemental data for this article can be accessed here. © 2021 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article #sap and fr contributed equally to this work. http://dx.doi.org/10.48101/ujms.v126.7649 mailto:leanne.hodson@ocdem.ox.ac.uk https://ujms.net/index.php/ujms/article/view/7649/13684 http://creativecommons.org/licenses/by/4.0/ 2 s.a. parry et al. observational studies, where it can be logistically challenging to obtain fasting samples, it would be useful to determine whether nutritional state influences circulating fa composition as it may potentially reduce the burden on researchers and participants. although a number of observational studies have previously obtained non-fasted samples from participants (13–17), or have obtained samples after only a relatively short fasting period (minimum of 4 h fasting) (18, 19), it remains unclear whether the presence of recently ingested fat influenced their findings. therefore, the aim of this study was to investigate: (1) the association between the dietary saturated (sfa), monounsaturated (mufa) and polyunsaturated (pufa) fas in plasma lipid fractions and self-reported dietary fa intakes, and (2) the influence of meal consumption on the relative abundance of specific sfa, mufa and pufa in plasma lipid fractions across a 6-h postprandial period. materials and methods participants participants were recruited from the oxford biobank (www. oxfordbiobank.org.uk) (20) or from the wider oxfordshire area through advertisement. based on data provided at screening, all volunteers were non-diabetic and free from any known disease, were not taking medication known to affect lipid or glucose metabolism, and did not consume alcohol above recommended limits. some, but not all, of the data reported in this work constitute a reanalysis of previously published studies (21, 22) and ongoing dietary intervention trials (clinicaltrials.gov identifiers: nct03090347 and nct03587753). data in this manuscript were from a total of 49 (34 males and 15 females) participants aged 26–57 years with a body mass index (bmi) between 21.6 and 34.2 kg/m2 (table 1). twenty-five participants were enrolled in one of two dietary intervention studies, both of which involved changing their relative intakes of fat and carbohydrate. the 25 participants were representative of the study population, as they were aged 38–54 years, with a bmi between 22.0 and 34.2 kg/m2. data from this subset were used to investigate the relationship between dietary fa intake and the fasting fa composition of circulating plasma lipid fractions. however, within this subset, four food diaries were missing or incomplete, leaving 46 complete sets of data for analysis. of the total 49 participants, the remaining 24 participants were enrolled in a randomised crossover study, involving two postprandial study days separated by a 2-week washout period, during which they were asked to maintain their habitual diet and physical activity patterns (21). as all participants (n = 49) included in this study took part in two postprandial study visits, this gave a total of 98 data sets to investigate the temporal changes in plasma fa composition in response to meal consumption. a flow chart of participant recruitment and experimental procedures is presented in supplementary fig. 1. all studies were approved by the respective research ethics committee, and all subjects provided written informed consent. prior to study days, subjects were asked to refrain from strenuous physical activity and to not consume alcohol for a minimum of 24 h. after an overnight fast, subjects came to the clinical research unit at the oxford centre for diabetes, endocrinology and metabolism, and a fasting venous blood sample was taken. subjects were then given a standardised test meal, and venous blood samples were taken at regular intervals for 6 h after meal consumption. dietary interventions and assessments a subset (n = 25) of participants included in this study underwent dietary interventions. of these, 16 participated in a randomised crossover trial in which they underwent two dietary interventions: 1) a 4-week low-fat, high-carbohydrate diet enriched in free-sugars and 2) a 4-week high-fat, lowcarbohydrate diet enriched in sfa (22). the remaining nine participants were included in an ongoing study (nct03090347) and underwent either a 2-week low-fat, high-carbohydrate diet enriched in free-sugars (n = 8) or a 2-week high-fat, lowcarbohydrate diet enriched in sfa (n = 1) (supplementary fig. 1). dietary intakes were assessed by food diaries collected on 3 days, including a weekend day. participants taking part in dietary interventions were instructed to maintain their usual body weight, physical activity levels and alcohol intakes throughout the intervention periods, and were contacted weekly by a member of the research team to aid adherence. dietary intakes were analysed using the nutritics dietary analysis online software (dublin, uk), by a registered dietitian to determine energy and nutrient intakes. standardised test meal on the study day, participants consumed a standardised test meal consisting of 40 g cereal (kellogg’s rice krispies), 200 g skimmed milk and a chocolate drink containing 40 g oil, providing 591 kcal, with ~64% energy as fat, ~30% energy as carbohydrate and ~6% energy as protein. the oil used was either olive oil (meal a) or 15 g sunflower oil plus 25 g palm oil (meal b). the fa composition of meal a was ~13% palmitate, ~64% oleate, ~11% linoleate, ~3% stearate and ~9% minor fa, whilst the fa composition of meal b was ~32% palmitate, ~35% oleate, ~27% linoeate, ~5% stearate and ~1% minor fa. all subjects consumed the same meal twice, separated by a period of 2–11 weeks. table 1. baseline characteristics of participants. age (years) 46±7 sex (m/f) 34/15 bmi (kg/m2) 26.6±3.1 glucose (mmol/l) 5.0±0.6 insulin (mu/l) 8.3±4.5 homa-ir 1.9±1.1 total cholesterol (mmol/l) 4.3±0.7 hdl cholesterol (mmol/l) 1.2±0.4 triglycerides (tgs) (mmol/l) 0.9±0.4 non-esterified fatty acid (µmol/l) 519±275 data are mean ± sd. n = 49. m, male; f, female; hdl, high-density lipoprotein; homa-ir, homeostatic model assessment for insulin resistance. http://www.oxfordbiobank.org.uk http://www.oxfordbiobank.org.uk http://clinicaltrials.gov https://ujms.net/index.php/ujms/article/view/7649/13684 https://ujms.net/index.php/ujms/article/view/7649/13684 postprandial plasma fatty acid composition 3 analytical procedures whole-blood was collected into heparinised tubes, and plasma was immediately separated for analysis by centrifugation at 4°c. plasma glucose, triglycerides (tgs), total cholesterol and highdensity lipoprotein (hdl) cholesterol were analysed enzymatically (ilab 600/650 clinical chemistry, werfen). fatty acid composition total lipids were extracted from plasma by using chloroform– methanol (2:1, v/v) (23), and plasma lipid fractions (tg, phospholipid [pl] and cholesterol esters [ces]) were separated by solid-phase extraction (24), followed by methylation using acidified methanol. the fa profile of extracted samples was then determined via gas chromatography with flame ionisation detection (25). fas were identified by comparing sample retention times to a known standard, and results are expressed as mol%. statistical analysis data were analysed using spss (version 25.0). the specific fas investigated were restricted to the major dietary sfa, mufa and pufa (i.e. palmitate, oleate and linoleate, respectively). normality of variables was assessed by shapiro–wilk test and visually by histograms. the spearmans rank correlation coefficient was used to assess the associations between the specific fas in lipid fractions and the relative percentages of energy intake from sfa, mufa and pufa calculated from 3-day diet diaries. differences in fa abundance in response to meal consumption were analysed using one-way repeated measures (time) anova. where a significant main effect of time was noted, bonferroni post-hoc comparisons were made for postprandial vs. fasting time points (0 min). data are presented as mean and standard deviation (sd). results relationship between dietary fa intake and the fa composition of circulating plasma lipid fractions the fasting fa composition of plasma tg, pl and ce fractions is presented in figure 1. as data were obtained from participants undertaking various dietary interventions, some of which involved increasing dietary carbohydrates/free sugars, and some increasing dietary fat/saturated fat, there were wide variations in self-reported intakes of total fat, sfa, mufa and pufa, which is reflective of the specific dietary interventions which were undertaken (table 2). we assessed the association between dietary fa intake and the abundance of specific fa that represent the major dietary sfa, mufa and pufa sources, in the respective plasma lipid fractions. we found no significant associations between dietary fa and the abundance of palmitate, oleate and linoleate in plasma tg or pl (table 3). however, significant (p < 0.05) positive associations were observed between dietary pufa and linoleate in plasma ce, whilst there were no associations between dietary sfa and the abundance of plasma ce palmitate, or dietary mufa and plasma ce oleate (table 3). m yr is ta te pa lm ita te pa lm ito le at e st ea ra te o le at e li no le at e aa ep a dh a m yr is ta te pa lm ita te pa lm ito le at e st ea ra te o le at e li no le at e aa ep a dh a 0 10 20 30 40 50 a b u n d a n c e ( m o l % ) a b u n d a n c e ( m o l % ) 1.9% 1.6% 0.5% 0.5% a b m yr is ta te pa lm ita te pa lm ito le at e st ea ra te o le at e li no le at e aa ep a dh a a b u n d a n c e ( m o l % ) c 0 10 20 30 40 0.4% 0.6% 1.1% 0 10 20 30 40 50 60 0.9% 0.8% 1.0% 0.3% figure 1. fasting plasma fa composition for (a) tg (n = 98), (b) pl (n = 76) and (c) ce (n = 96). aa, arachidonic acid; epa, eicosapentaenoic acid; dha, docosahexaenoic acid. data are mean ± sd. 4 s.a. parry et al. temporal changes in fa composition following meal consumption as fa composition is typically measured in the fasting state, we investigated whether the consumption of a meal influences the relative abundance of palmitate, oleate and linoleate in circulating lipid fractions. we achieved this by feeding participants a high-fat test meal, of known fa composition, and assessing changes in plasma palmitate, oleate and linoleate over the course of the postprandial period in the respective lipid fractions. the consumption of the test meal significantly (p < 0.05) decreased the abundance of palmitate in plasma tg, with time points 240–360 min being significantly (p < 0.05) lower than time 0 (fasting), and the greatest differences (i.e. ~1.3 mol%) being apparent between 240 min and 0 min (figure 2a). conversely, meal consumption significantly (p < 0.05) increased the abundance of oleate, and linoleate in plasma tg, with the oleate peaking at 240 min, which was ~3.8 mol% greater than 0 min (figure 2b), and linoleate peaking at time point 360 min, which was ~1.1 mol% greater than 0 min (figure 2c). for plasma pl, the general trend was similar to the changes observed in plasma tg, although not as striking. the relative abundance of palmitate was significantly (p < 0.05) decreased by the consumption of the test meal, with significant differences apparent between time points 240–300 min and 0 min, with a nadir at 300 min, which was ~0.7 mol% lower than 0 min (figure 3a). the relative abundance of oleate was not influenced by meal consumption (figure 3b). the abundance of linoleate was significantly increased (p < 0.05) following meal consumption, with time points 120 min onwards significantly greater than 0 min, and peaking at 300 min (i.e. ~1.2 mol% greater than 0 min) (figure 3c). in contrast to the fa composition of plasma tg and pl, the consumption of the test meal did not influence the relative abundance of palmitate in plasma ce (figure 4a). there was, however, a significant (p < 0.05) main effect of time for oleate in plasma ce, although bonferroni post hoc comparisons revealed no statistically significant differences between any postprandial time table 3. spearmans rank correlation coefficients between the abundance of palmitate, oleate, and linoleate in circulating lipid fractions and the relative percentages of energy intake from dietary saturated fatty acids (sfa), polyunsaturated fatty acids (pufa) and monounsaturated fatty acids (mufa). mol (%) dietary sfa (%te) dietary mufa (%te) dietary pufa (%te) triglyceride (tg) palmitate -0.029 phospholipid (pl) palmitate -0.003 cholesterol ester (ce) palmitate 0.031 tg oleate -0.057 pl oleate -0.364 ce oleate -0.210 tg linoleate 0.211 pl linoleate 0.198 ce linoleate 0.372* *p < 0.05. n = 46 for tg, n = 27 for pl and n = 44 for ce. table 2. dietary fat intake as a proportion of total energy (te) intake. total fat (%te) 31.5±13.7 saturated fat (%te) 12.3±7.0 polyunsaturated fat (%te) 4.7±2.5 monounsaturated fat (%te) 10.5±5 data are mean ± sd. n = 46. 0 60 120 180 240 300 360 20 21 22 23 24 25 26 27 28 29 time (minutes) 0 60 120 180 240 300 360 time (minutes) 0 60 120 180 240 300 360 time (minutes) p a lm it a te ( m o l % ) time, p < 0.001 * * * a 34 37 40 43 46 49 52 o le a te ( m o l % ) time, p < 0.001 * * * * * b 12 14 16 18 20 22 24 26 l in o le a te ( m o l % ) time, p = 0.001 * c figure 2. temporal changes in the relative abundance of (a) palmitate, (b) oleate and (c) linoleate in plasma tg in response to the consumption of a high-fat test meal. data are presented as mean ± sd. n = 98. *p < 0.05 compared to fasting (time 0). the dotted line at time 0 denotes the consumption of the experimental test meal. postprandial plasma fatty acid composition 5 points and 0 min (figure 4b). the abundance of linoleate in plasma ce significantly (p < 0.05) decreased following meal consumption, reaching a nadir at 300 min, which was ~2.6 mol% lower than 0 min (figure 4). discussion the fa composition of blood and tissues has frequently been used as a biomarker of dietary fa intake (9). typically, the fa composition of blood lipids is measured in blood samples taken figure 4. temporal changes in the relative abundance of (a) palmitate, (b) oleate and (c) linoleate in plasma ce in response to the consumption of a high-fat test meal. data are presented as mean ± sd. n = 96. *p < 0.05 when compared to fasting (time 0). the dotted line at time 0 denotes the consumption of the experimental test meal. 10 12 14 16 p a lm it a te ( m o l % ) time, p = 0.092a 16 18 20 22 24 26 28 o le a te ( m o l % ) time, p < 0.001b 40 43 46 49 52 55 l in o le a te ( m o l % ) time, p < 0.001 * * * * * * c 0 60 120 180 240 300 360 time (minutes) 0 60 120 180 240 300 360 time (minutes) 0 60 120 180 240 300 360 time (minutes) 28 30 32 34 36 p a lm it a te ( m o l % ) time, p = 0.001 * * a 9 10 11 12 13 o le a te ( m o l % ) time, p = 0.066b 18 20 22 24 26 l in o le a te ( m o l % ) time, p < 0.001 * * * * *c 0 60 120 180 240 300 360 time (minutes) 0 60 120 180 240 300 360 time (minutes) 0 60 120 180 240 300 360 time (minutes) figure 3. temporal changes in the relative abundance of (a) palmitate, (b) oleate and (c) linoleate in plasma pl in response to the consumption of a high-fat test meal. data are presented as mean ± sd. n = 76. *p < 0.05 when compared to fasting (time 0). the dotted line at time 0 denotes the consumption of the experimental test meal. 6 s.a. parry et al. from individuals after an overnight fast. however, some largescale observational studies have utilised non-fasting samples (13–17), and it remains unclear if feeding influences the postprandial fa composition of plasma lipid fractions. we therefore aimed to assess the associations between fa composition in plasma lipid fractions and dietary fa intake in participants who had undergone dietary interventions to investigate biomarkers of dietary fa intake. in addition, we aimed to determine the influence of meal consumption on the postprandial fa composition of plasma tg, pl and ce. overall, our findings suggest that the fa composition of plasma tg or pl does not reflect dietary fat intakes over the short term (2–4 weeks), whereas the relative abundance of plasma ce linoleate was positively associated with self-reported intakes of pufa. thus, plasma ce linoleate may represent a valid biomarker of dietary pufa even in situations where individuals have recently altered their dietary fa intakes. moreover, our data indicate that the consumption of a high-fat test meal acutely influences the fa composition of plasma tg, pl and ce. therefore, it would seem prudent to suggest that fasting blood samples should be utilised when using fa composition as a biomarker of dietary fa intake, as the consumption of a high-fat meal may confound measurements of fa composition taken in a non-fasting state. measurement of fa composition in various tissue and plasma lipid pools provides an objective assessment of dietary fa intake, which may strengthen data obtained from self-reported dietary records which have limitations (e.g. underreporting) (7, 8, 26). it has been suggested that due to the slow turnover of adipose tissue (i.e. half-life of ~600 days), adipose tissue fa composition may be most reflective of long-term (i.e. 2–3 years) fat intake (27–29). in contrast, the fa composition of red blood cells, plasma ce and pl has been shown to reflect dietary fat intakes within weeks (29–31). epidemiological investigations have generally used the fa composition of various plasma/ serum lipid fractions as biomarkers of dietary fa intake (32, 33), which may be because blood sampling is relatively simpler than adipose tissue biopsies. however, there is heterogeneity between the metabolism and turnover of circulating lipid fractions, which is reflected in their fa composition, and debate continues as to which plasma lipid fraction represents the most accurate biomarker of dietary fat intake. we therefore investigated the relationship between self-reported dietary fat intake and the relative abundance of palmitate, oleate and linoleate (representative of the major sfa, mufa and pufa) in plasma tg, pl and ce. in line with previous observations (9), we found a positive association between the abundance of linoleate in plasma ce and dietary pufa intake, but observed no significant associations relative abundance of palmitate or oleate in plasma ce and dietary sfa and mufa, respectively. we also observed no associations between specific fas in the plasma tg or pl fraction and self-reported intake of dietary sfa, mufa and pufa. it is plausible that positive associations are observed more often for pufa than other fas as linoleate represents an essential fa, whereas the in vivo synthesis of palmitate and oleate may influence their circulating abundance independently of dietary intake. equally, palmitate and oleate are relatively ubiquitous in foods, which, when combined with the inability of fa composition measures to establish quantitative intakes, may make it challenging to separate individuals with low and high intakes of these fa. when comparing the utility of fas in various lipid fractions as biomarkers of dietary fa intake, furtado et al. (12) reported that combining plasma tg and non-esterified fa (nefa) fractions was more reflective of dietary fa intake than total plasma, ce or pl fa composition. the difference between our findings and those of furtado et al. (12) is likely due to differences in study design. participants in our study completed 2–4 weeks dietary interventions at the time of assessment, whereas those studied by furtado et al. (12) had not undertaken a dietary intervention and were consuming their habitual diet, which was assessed using a food frequency questionnaire examining intakes over the previous year. it is therefore plausible that the strength of the correlation for the combined tg and nefa fraction was driven by nefa fa composition, which has been suggested to be reflective of adipose tissue fa composition (34). we found that the abundance of palmitate, oleate and linoleate in plasma tg, pl and ce was influenced by a high-fat meal in a manner which reflected, to a degree, the fa composition of the test meal. changes in fa abundance were specific to lipid fractions. the plasma tg and pl fractions were influenced to a greater extent than plasma ce. it is unsurprising that the composition of circulating tg was altered during the postprandial period as ingested fas are initially incorporated into chylomicron-tg prior to entering the circulation (35), and the incorporation of dietary fa into hepatic very low-density lipoprotein (vldl) tg also occurs relatively soon after ingestion (25). thus, our data are in-line with others who have previously demonstrated that changes in the non-fasting fa composition of plasma tg are reflective of the recently ingested meal fa composition (36–38). we also show that the fa composition of plasma pl and ce is influenced by the intake of a high-fat mixed meal with significant differences apparent from 60-min onwards, dependent on the fraction and fa. recently, shokry et al. demonstrated that the abundance of some fa in plasma tg and nefa, including linoleate, changed during a 7-h postprandial period following a high-calorie mixed macronutrient test meal (45 g of fat and 97 g of carbohydrate); the plasma pl fa composition was unaffected (38). this finding is in contrast to our observations, but may be explained in part by the difference in the fa composition of the meals, with shokry et al. feeding a test meal containing 26.4 g of linoleate (i.e. over 50% of the fat component). meal consumption has also been shown to influence the fa composition of plasma pl when assessed using lipidomic methodologies (39, 40). our observations are in line with karupaiah et al. who reported changes in plasma ce within 7-h, which reflected the fat composition of the consumed meal (41). thus, regardless of methodology (gc or lipidomics), it would seem that changes in both pl and ce species have been observed in the non-fasting compared with the fasting state, but the degree and manner of change may be dependent on the composition of the test meal. postprandial plasma fatty acid composition 7 in the present work, the abundance of linoleate increased in the tg and pl fractions but decreased in the ce. these differences may be explained by differences in fa incorporation time between the fractions, as the synthesis of ce involves the enzymatic transfer of an fa from pl and cholesterol precursors, typically from the sn-2 position of pl, which is commonly occupied by a pufa (9). using stable isotope tracer methodology, we have previously shown the incorporation of linoleate in plasma pl is greater than palmitate following a high-fat mixed meal (21), demonstrating the metabolic heterogeneity of specific fa in lipid fractions. it is therefore plausible that the incorporation time of linoleate in plasma ce from pl is longer than we have investigated, and that the abundance of linoleate in plasma ce may have increased at a time point later than the 6-h period examined here. our study has a number of limitations, including: all subjects were free from known metabolic disease, and results may therefore not be reflective of other metabolic phenotypes. for instance, individuals with metabolic-associated fatty liver disease (mafld) demonstrate increased de novo lipogenesis (dnl) relative to their non-mafld counterparts (42), which may lead to an increased palmitate abundance in plasma lipid fractions. participants consumed a single test meal, which was high in fat; therefore, we cannot exclude the possibility that non-fasting fa composition would change further if we had given a subsequent/second meal (i.e. more reflective of habitual dietary pattern in most individuals). moreover, it remains unclear if the responses observed were mediated by the quantity, along with the quality of fa in the meal; it could be speculated that a lower fat meal may result in less notable/obvious responses/ changes. our test meals were devoid of marine n-3 fa; thus, we are unable to comment on the stability of these fa across the postprandial period, but it would be of interest to investigate this given their usefulness as biomarkers (43–45). similarly, we only assessed the most abundant sfa, mufa and pufa; therefore, our findings cannot be extrapolated to fa of lower abundance, for example, myristate, pentadecanoic acid, stearate and arachidonic acid. we did not assess the fa composition of plasma nefa, which whilst being a potentially good marker of long-term dietary fa intake (as it reflects at fa composition) (9), likely does not reflect shortto medium-term dietary intake. in addition, we and others have previously shown that during the postprandial period chylomicron-derived dietary fa spillover contributes 10–50% of fa within the systemic nefa pool (46–48). thus, similar to plasma tg fa composition with meal consumption, the fa composition of plasma nefa would be highly influenced over the postprandial period by the fat content and fa composition of the recently consumed meal. in conclusion, our data demonstrate that the fa composition of plasma tg and ce does not reflect short-term (i.e. previous weeks) dietary fa intakes, and that the fa composition of plasma ce may be the lipid fraction to utilise as an objective biomarker when investigating recent dietary fa intakes over this period. in addition, we show that the consumption of a high-fat meal influences the fa composition of plasma tg, pl and ce over the course of the postprandial period, with responses appearing to be specific to fas in the different lipid fractions. thus, the fa composition of plasma lipid fractions during the postprandial period (i.e. 1–6 h post meal) may not reflect fasting values. based on these observations, it would be prudent to suggest that fasting blood samples should be utilised when using fa composition as a biomarker of dietary fa intake. acknowledgements we thank louise dennis, rachel craven-todd and all cru staff for excellent nursing provision, and niall dempster and lia anguelova (oxford centre for diabetes, endocrinology and metabolism, university of oxford, oxford) for providing medical support during study days. this work was supported by the nihr biomedical research centre, oxford, and we also thank the volunteers from the oxford biobank, nihr oxford biomedical research centre, for their participation. the oxford biobank (www.oxfordbiobank.org.uk) is also a part of the nihr national bioresource, which supported the recalling process of the volunteers. conflict of interest all authors declare no conflict of interest. disclosure statement the authors have no conflicts of interest to declare. funding lh is a british heart foundation senior research fellow in basic science (fs/15/56/31645). work contributing to this manuscript was funded by the world sugar research organization, and the biotechnology and biological sciences research council uk (bb/ n005600/1 and bb/n015665/1). during this work, fr was supported by the henning and johan throne-holsts foundation, swedish society for medical research, swedish society of medicine and the foundation blanceflor. notes on contributors sion a. parry, sion a. parry, phd is a researcher at the oxford centre for diabetes, endocrinology and metabolism (ocdem), university of oxford, united kingdom. fredrik rosqvist, fredrik rosqvist, phd was a postdoctoral researcher at the oxford centre for diabetes, endocrinology and metabolism (ocdem), university of oxford, united kingdom at the time of the work, now a researcher at the department of public health and caring sciences, clinical nutrition and metabolism, uppsala university, sweden. sarah peters, sarah peters was an undergraduate medical student undertaking a final honours scheme research project at the oxford centre for diabetes, endocrinology and metabolism http://www.oxfordbiobank.org.uk 8 s.a. parry et al. (ocdem), university of oxford, united kingdom at the time of the work. rebecca young, rebecca young was an undergraduate medical student was an undergraduate medical student undertaking a final honours scheme research project at the oxford centre for diabetes, endocrinology and metabolism (ocdem), university of oxford, united kingdom at the time of the work. thomas cornfield, thomas cornfield, bsc was a research technician at the oxford centre for diabetes, endocrinology and metabolism (ocdem), university of oxford, united kingdom at the time of the work. pamela dyson, pamela dyson, phd is a research dietitian at the oxford centre for diabetes, endocrinology and metabolism (ocdem), university of oxford, united kingdom. leanne hodson, leanne hodson, phd is a professor of metabolic physiology and british heart foundation senior research fellow at the oxford centre for diabetes, endocrinology and metabolism (ocdem), university of oxford, united kingdom. orcid sion a parry 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upsala j med sci 82: 167-181, 1977 the plasma membrane consists of two po la r-no n po la r-po lar leaf lets interpretation of some data, mainly from the erythrocyte erik cerven from the institute of medical and physiological chemistry, biomedical center, university of uppsala, uppsala, sweden abstract the implications of a double polar-nonpolar-polar leaflet construction of the plasma membrane are investigated. experimental data from transmission electron microscopic and enzymologic characterization of plasma membranes are advantageously interpreted in these terms compared to interpretation in terms of lipid bilayer. x-ray diffraction and electron spin resonance studies do not differentiate between the present and previous models for the structure of plasma membranes but electron spin resonance data that fail to indicate a statistical distribution of spin labels also fail to support the fluid mosaic model for cell mem branes. results from experiments involving vectorial di gestion and labelling of plasma membranes as well as freeze fracture electron microscopic data are compatible with the present model. the molecular composition of the human erythrocyte membrane is investigated whereby the band 111 protein and glycophorin are suggested to be the structural proteins of the outer leaflet and the spectrins those of the inner leaflet. introduction the erythrocyte membrane is the most thoroughly investigated of biological membranes. several approaches have been used in elucidating its structure, ranging from early estimations of lipid content (47) t o recent x-ray photo-electron spectroscopy (69). the vast amount of information available on particularly the human erythrocyte membrane provides a basis for testing the recently developed concept that the plasma membrane con sists of two polar-nonpolar-polar leaflets (15). this concept was originally developed from considera tions of structural and functional similarities and dissimilarities between various biological mem branes. in particular it required knowledge about the nucleotide metabolism associated with intact cells (3, 4, 5, 6 , 29, 90) and elaboration (16) of the idea (58) that actin-containing filaments attached to the plasma membrane (52, 74) are analogous to the coupling factors (cf. 58) of mitochondria and chloroplasts. it is the aim of this work t o examine data that are difficult to reconcile with the fluid mosaic model for the structure of cell membranes (95) and that tend to support the concept that such membranes consist of two parallel polar-nonpo lar-polar leaflets. primarily, data dealing with the human erythrocyte membrane will be discussed. some previous models f o r the structure of the human erythrocyte membrane the lipid bilayer concept for the structure of biolog ical membranes (see ref. 104 for a short review on membrane models) (fig. 1) was originally de veloped from considerations of the surface area of intact erythrocytes compared with that occupied by the lipids extracted from erythrocytes when spread as a monolayer on an aqueous surface (47). accord protein mspholiro fig. 1 . illustration of the fluid mosaic model for the structure of cell membranes according to which intra membranous particles revealed by freeze-fracturing rep resent globular proteins surrounded by unperturbed lipid bilayer (ref. 95). 12-772856 upsala j m e d s c i 8 2 168 e . cerve'n electron beam electron optical system 4 image c r / r l trilaminar strkture fig. 2. formation of a trilaminar image seen in the trans mission electron microscope. ing to this model (47, 88, 94, 9 9 , a trilaminar ap pearance of the erythrocyte membrane under the transmission electron microscope is attributed to lack of affinity of stains to the nonpolar residues be tween polar residues of the bilayer (88). fracture planes generated by the freeze fracture procedure for electron microscopic preparation are attributed to lack of cooperativity of van der waals forces between methyl groups of contrapositioned lipids in the bilayer (25). proteins spanning across two leaflets of the erythrocyte membrane (cf. 95) are claimed to be detected by the occurrence of intra membranous particles after freeze fracturing. the stability of pure lipid bilayers ( i 04) and the apparent lack of interdependence of protein and lipid conformation in biological membranes as in terpreted from measurements of circular dichroism (94) have contributed significantly to the develop ment of this concept (94, 95). however, lipid in bilayers is presumably equilibrated between mem branes of various cellular systems by way of a small aqueous pool (104). it is therefore difficult to ex plain the fact that membranes from different organ elles are characterized by different lipid composi tion (23, 70) and by characteristic enzymatic activi ties and proteins. this is because the fluid mosaic model for cell membranes implies that both lipid and protein move about, lacking close and specific contact in the planes of membranes (39, 95) that may in addition be capable of fusion (cf. 8). with this model it is also difficult to imagine any genetic control mechanism responsible for species-specific lipid composition of biological membranes (cf. 70, 120). very complex mechanisms including re strained mobility of integral proteins imposed by specifically associated fibrous proteins have been upsala j m e d sci 82 developed (74) in order to overcome some of these difficulties. the unit membrane hypothesis (88), according to which all biological membranes are represented by lipid bilayers with various amounts of loosely and peripherally associated protein, was partly based on electron microscopic observations of kmn0, or os0,-stained and dehydrated membranes. how ever, the thickness of the erythrocyte membrane, as determined by this methodology, is uncertain be cause of extensive losses of both protein and lipid when osmium and dehydration are used for electron microscopic preparation (67). in dehydrated prep arations, cross-linked with glutaraldehyde and stained with low concentrations of osmium, thereby avoiding extensive losses of protein, no trilaminar structure can be seen and the membrane thickness is about 160 a (67). great variations of membrane thickness as measured by independent techniques (see table i) indicate that the degree of hydration and the amounts of associated water-soluble pro teins are important factors. table 1. the thickness of the (human) erythrocyte membrane determined by various methods for comparison, the thickness of a lipid bilayer may be 45-50 a determined by x-ray diffraction (27, 104) and 45f5 8, determined by electron microscopy (72, 88). see ref. 71 for a more exhaustive constellation of data method thickness ref. birefringence 5 0 0 0 8 , 71 leptoscope (rabbit, rat steer) 160-220 a 114 argon ion etching combined with iron signals determined by electron spectroscopy 1 000 a 69 energy transfer 65-100a 82 electron microscopy glutaraldehyde and high con excluded volume 600 a 44 centrations of osmium tetroxide 70 a 67 centrations of osmiurn tetroxide 160 a 67 embedding in water-soluble polymers and staining with uranylacetate and lead citrate 70-110a 52, 81 the nonpolar core estimated from lipid content assuming even distribution in one glutaraldehyde and low con surface layer 27 a 33 double polar-nonpolar-polar leaflet construction of the plasma membrane 169 0 polar residues nonpolar residues 0 aqueous phase protein @ oil p h a y protein 0 k h b l x \ ( glycopfloteln f i g . 3 . the structure of a complete biological membrane according to ref. 15. the indexed capitals p (polar), n (nonpolar) and z (intramembranous) serve to denote the location of different components. for example, actin is a p, component of the plasma membrane, ndp-kinase and adenylate-kinase are presumably i, components of the mitochondrial and the plasma membranes (ref. 15). definition of a complete biological membrane the nucleotide metabolism associated with the surface of many intact cells is similar to that as sociated with intact mitochondria (4, 6 , 15, 29). furthermore, actin-containing filaments, the sub unit structure of which is reminiscent of that of the coupling factors of the inner mitochondrial mem brane ( 5 8 ) , are closely associated with the inner leaflet of the plasma membrane of many cells (52, 74). the similarity of the two mitochondrial mem branes and the inner and outer leaflets of the plasma membrane is further emphasized by the asymmetric distribution of carbohydrates in both instances (10, 74, 75, 102). in addition, covalently bound dna is included as a marker for the inner leaflets in both these in stances (cf. 15, 45, 50). by these criteria, the two mitochondrial membranes and the two leaflets of the plasma membrane may be designated as com plete biological membranes (15, see fig. 3). since the outer leaflet of the plasma membrane contains identity markers and receptors of importance for cellular interactions in multicellular systems, there is reason to regard one complete biological mem brane as a functional unit composed of one leaflet carrying endogenous information (dna) and/or energy transducing activity and one leaflet mediat ing informational exchange with the surrounding medium (exogenous information). these functions may be more or less developed in particular in stances. for example, endogenous information may be more important than the informational exchange function (exogenous information) in the complete biological membrane represented by the inner nuclear membrane + the outer nuclear membrane combined with the endoplasmatic membranes of animal cells. structure of a single leaflet nonpolar residues of amino acids and lipids tend to be shielded from hydrogen binding water-soluble compounds (104). according t o this fundamental thermodynamic principle (the hydrophobic effect), a monolayer of phospholipids in contraposition to nonpolar amino acid residues of a peptide in a p-pleated sheet-configuration is stable, as il lustrated in fig. 4 a . this structure is maximally stabilized in an aqueous environment if every sec ond amino acid residue of the peptide is polar. however, clusters of 15-17 nonpolar amino acids are stabilized in the form of an a-helix embedded in the nonpolar core (see fig. 4b) because of the hydrophobic effect (104). the peptide may bind another layer of phospholipids after it has traversed the membrane, as illustrated in fig. 4 c . similar arrangements of phospholipid, protein and other biological compounds may be imagined to exist throughout the three-dimensional membrane. this structure would be extremely stable owing to the contribution of cooperative nonpolar bonds, hydrogen bonds and salt bridges, as illustrated in f+6riocipid a 0 c f i g . 4 . transsection of a monolayer of phospholipid and a peptide in ,&pleated sheet configuration (a and c) and a peptide segment of 15-17 exclusively nonpolar amino acid residues stabilized in a-helix form in the nonpolar core ( b ) . upsala j mrd sci 82 170 e . cerve‘n phospholipid 00 corbohydrate glycolipid a,a protein cholesterol q o charges f i g . 5 . illustration of the possible contribution of lipid, protein and sugar to the structure of a single leaflet. broad lines denote compounds located close to and thin lines far from the reader. this structure is composed of a nonpolar core ( i ) , two planes of hydrogen bonding ( 2 ) and two planes of salt bridges (3). the binding of aqueous phase and oil phase proteins to this type of structure is illustrated in fig. 3. fig. 5. the possible contribution of different com pounds t o these three types of binding is listed in table 11. the association of water-soluble and in tegral proteins as well as pore formation in one leaflet has been described previously (15). sugar residues are assumed (cf. 15) to profoundly influence a membrane on account of their nonpolar surfaces and their property to structunze water (cf. 35) thus presumably forming part of the planes of hydrogen bonding. the presence of sugar in bio logical membranes presumably make them more permeable to polar solutes by influencing the hydrophobic effect. this is illustrated tentatively by the decreased surface tension in lecithin monolayers brought about by sialic acid in combi nation with metal ions (26). antigens and receptors residing in glycosyl residues would thus be shielded from detection until the membrane structure is dis turbed by proteolytic or lipolytic cleavage. thus treatment of fat cells with phospholipase c in creases the insulin-binding capacity (24). some experimental data in support of this type of structure (see fig. 5) may be accrued from the lit erature. for example the low extractability of camel erythrocyte phospholipid with ether/methanol ( 1 10) in contrast to that of human erythrocyte phos pholipid of similar composition and of similar amount/surface area (30, 61) may suggest that pro tein is important in binding the phospholipids to the camel erythrocyte membrane which is charac terized by a high protein to lipid ratio. if the thermo dynamic principles denoted the hydrophobic effect are strictly applied to glycophorin (the sequenced main sialoglycoprotein of the human erythrocyte (62)) then the polar thr-74, ser-92 and under some conditions weakly acidic tyr-93 amino acid residues are preferably excluded from the nonpolar segment thought t o extend through the lipid bilayer (cf. 104). the remaining segment, composed of 17 nonpolar amino acid residues and stabilized in an a-helix form due to the nonpolar environment (104) would table 11. examples of atoms and groups in different biological compounds possibly contributing to the nonpolar core and the planes of hydrogen bonding and of salt-bridges in a biological membrane compound nonpolar core bonding bridges plane of hydrogen plane of salt cholesterol phospholipid sphingolipid glycolipid and carbohydrate peptides and protein water metal ions -ch,, -chb, -c=c-oh -chz-, -ch,, -c=c=o, -c=c-, -nhz -inositol -chz-, -ch,, -c=c=o, -oh, -nh, -c=c -ch,, -ch,, -c=c-, =0, -oh, -nh, -c=c nonpolar surfaces of carbohydrate nonpolar residues and atoms, a-helix p-pleated sheet =0, -nh, polar residues, interphase peptide of unknown configuration polar “pockets” -oh p-0-, -choline+ , -serine -nh: -p-0-, -choline+ -fucosate-, -sialate polar and ionic residues -oh +, ++ upsala j med sci82 double polar-nonpolar-polar leaflet construction of the plasma membrane 17 1 be 25.5 a long (1.5 &residue, cf. 9) in good agree ment with the postulated width of the nonpolar core according to the present model. the abun dance of basic proteins in some biological mem branes (e.g. myelin, ref. 34) is interesting because the positively charged residues of arginine and lysine are separated by 5 and 4 atoms respectively of similar size from a plane of hydrogen bonding (see fig. 5) while a negatively charged phosphorous oxygen of phospholipid is separated from such a plane by 4-5 atoms, indicating the possibility of favourable conditions for electrostatic interactions between these residues. however, ionic solutes are probably important contributors to the planes of salt bridges (see fig. 5). ca++ ions have been reported to bind to the carboxyl groups of protein in the human erythrocyte membrane and these binding sites are shielded by amino groups (42, 43). in accordance with these findings, the carboxyl groups of glutamic and aspartic acid residues would be separated from a plane of hydrogen bonding by only 2 and 1 atom(s) respectively and shielded from the surrounding aqueous medium by the amino groups of phospholipid and protein (see fig. 5 and table 11). analytical freeze-cleaving has re vealed that cholesterol may be enriched three-fold in the outer leaflet compared with the inner leaflet of the erythrocyte membrane (40). this finding is not compatible with the existence of a transmem branous nonpolar lipid core which would facilitate the rapid equilibrium of the nonpolar cholesterol molecule between the two leaflets in the absence of specific binding to protein and/or lipid. structural similarities between the present model and the bilayer model, of importance for experi mental determination of physical parameters can be pointed out. these include the orientation of the axis of lipid perpendicular to the plane of the mem brane and increased mobility of methyl groups in comparison with methylene groups. the latter are probably being more rigidly held by the planes of hydrogen bonding and fixed by adjacent nonpolar amino acid residues. if ions are excluded from the planes of hydrogen bonding and if cholesterol is present in the membrane (conferring on the lipids a preferred extended configuration, 18) then the re gion of comparatively low electron density would be 28-33 a (see fig. 5, the nonpolar amino acid resi dues contribute 2-5 a). the presence of adsorbed ions in the planes of salt bridges would confer on these regions a higher electron density than that of the surrounding buffer. the planes of salt bridges containing electron-dense phosphorous and metal ions are probable reflection planes, which when detected by wide-angle x-ray analysis give rise to diffraction patterns. for example the bragg spacing related to lipid in the bovine erythrocyte membrane as determined by wide-angle x-ray diffraction was 43 8, while that of extracted phospholipid was 51.5 8, (27). this discrepancy has led t o the postulation of “tilted” phospholipid in the assumed lipid bilayer phase of that particular biological membrane (27). however, according to the present model for the plasma membrane the centres of the reflecting planes are between 28 and 53 8, (most probably 4 0 4 3 8,) apart (see fig. 5) in better agreement with the 43 8, spacing reported from the bovine erythro cyte membrane. thus, physical data which tend to support the lipid bilayer model, are also applicable t o the present model for biological membranes. however, biological membranes are regarded’ as delicate structures with optimized functions which have been acquired during the course of evolution. they are characterized by cooperative effects (98) and vectorial non-random motion in the plane of the membrane (7, 17), phenomena which are difficult to reconcile with the fluid mosaic model for cell mem branes. the present model is compatible with: 1. translational rearrangements in the plane of a membrane coupled or not coupled to translational rearrangements in an adjacent membrane. 2. vectorial movement of protein and lipid in “channels” (in the plane of a membrane) formed by the asymmetric apposition of protein, carbohydrate and lipid. 3. rearrangements along an axis perpendicular to the plane of a membrane permitting transmem branous diffusion of macromolecules in their water-soluble form across one or two membranes (see figure 6 in ref. 15). 4. adsorption of water-soluble enzymes, e.g. pro teases and phospholipases with or without rear rangements and digestion of protein and lipid. 5. shrinking of the membrane surface by folding, lipid bilayer formation and/or by exclusion of aqueous phase proteins. 6. expansion of the membrane surface by integra tion of aqueous phase proteins in the plane of the membrane, /3-pleated sheet formation or by unfold ing of the entire membrane. 7. cooperative effects mediated by conforma upsalu j m e d sci 82 172 e . cerven tional changes of lipid and structural protein and/or membrane-associated water (cf. 37) or by modified permeability properties of either of the two mem branes. 8. receptor-effector transducing mechanisms (e.g. the adenylcyclase system) across two mem branes by ( a ) cooperative effects (see above), ( b ) proteins spatially linked in the intramembranous space (see fig. 3), (c) penetration of the first mes senger through one leaflet combined with allosteric regulation of enzymes in the other leaflet. 9. dissociability of regulatory and catalytic sub units of hormone-sensitive enzyme complexes lo cated in different membranes. 10. specific o r preferred interactions between protein and lipid, conferring on biological mem branes individuality in each organelle and each cell line derived from different species. thus, biological membranes may contain a significant amount of information and may be of semiconservative nature since lipid and proteins are partly complementary constituents of the membranes. fusion the demonstration that fusion may occur (8), for instance between cells and extracellular vesicles, does not contradict the present hypothesis for the structure of plasma membranes. on the contrary, some experimental data from fusion experiments may be interpreted advantageously by assuming that the plasma membrane is two membranes. this is because the lipid vesicles may fuse either with the outer leaflet only, or with regions of the plasma membrane where the two leaflets are already fused. for example fusion with the outer leaflet would re sult in loading of the intramembranous space with compounds enclosed in the bilayer vesicles and leakage as has been reported (8) of these com pounds through a “semiporous” outer leaflet. fu sion of human lymphocytes with mono-layered liposomes containing a fluorescent probe detectable only when diluted, resulted in fluorescence that was inhibited by 0.1 % triton x-100, thus suggesting either that the vesicles were adsorbed to the cell surface (116) o r that the fluorescent probe was di luted in the entire cell (116) o r in the intramem branous space after fusion of the vesicles with the outer leaflet. liposome-cell interactions involve adsorption of positively charged vesicles to the negatively charged cell surface (8, 65), endocytosis (8), and possible exchange of lipid between the cell surface and the liposomes (8). complex interactions are presumably also involved in the fusion of human erythrocytes with lipid lp-x vesicles accumulating in serum during some hepato-biliary diseases since the fusion process (detected by enlarged surface area) does not occur in the absence of serum (1 11). the dilution of the intramembranous particles con taining glycoprotein (see below) in these fusion ex periments (111) may indicate that the intramem branous particles contain reserve material for form ing membrane area. in agreement with this inter pretation, antigens residing in the band 3 protein (see below) were detectable close to the intramem branous particles but not elsewhere (84), probably reflecting cryptic location at these other sites. fu sion of cells mediated by liposomes and virus mem branes has sometimes been used as an argument for the occurrence of unperturbed lipid bilayer in cell membranes. however, if such were the case, the same argument would, contrary to what is generally observed, favour extensive fusion of cells in the absence of liposomes. fusion of intracellular vesicles with the plasma membrane (exocytosis) may in addition involve either displacement of the inner membrane or re lease of trapped compounds in the intramembran ous space, followed by diffusion through the outer leaflet. present model applied to the human erythrocyte membrane according t o the present model, the two leaflets of the erythrocyte membrane which stain with osmium-tetroxide represent two nonpolar layers containing cholesterol and other lipids with carbon double bonds that induce precipitation of electron dense osmium compounds (117). in addition, amino groups of protein (12) and polar heads of phos pholipid (88) may contribute to osmium stain ing. glutaraldehyde is believed to preserve a trilaminar appearance of the erythrocyte membrane of intact cells by cross-linking the proteins of the inner leaflet (cf. 67, 100). the inner leaflet of human erythrocytes is prefer entially visualized by glutaraldehyde fixation fol lowed by embedding in water-soluble polymers and staining with uranyl-acetate and lead-citrate (5 1 ) . the interleaflet space of the plasma membrane in such preparations where extensive extraction of lipid is avoided may be more than 70 a (51, 80) upsala j med sci 82 double polar-nonpolar-polar leaflet construction of the plasma membrane 173 which is indicative of a polar character. this in terpretation is supported by the facts that the inter leaflet space in such preparations is usually more than 100 8, in tight junctions where the outer membrane leaflets of two adjacent cells adhere (80) and these preparations when stained with acidified silicotungstic acid reveal substantial gaps between tissue cells (81) probably corresponding to the outer leaflet containing carbohydrate and the intramem branous spaces. the profound difference between the appearance of the plasma membrane and the intracellular mem branes under the electron microscope is well documented by several preparation techniques (5 1, 80, 96). it is also amply demonstrated by conven tional preparation of vesicles derived from yeast cell plasma membranes and mitochondria (cf. 45). a thin electron-lucent rim in one leaflet of the plas ma membrane can be seen in some electron microscopic images prepared by embedding in water-soluble polymers (51, 80, compare fig. 5). divalent cations may contribute to the electron density of both leaflets of the human erythrocyte membrane by association with negatively charged residues (42, 43). the main contribution to a trilaminar image of the erythrocyte membrane would, however, be the ad ditive electron density of components located at different depths of the transverse section (see fig. 2). this superposition effect probably diminishes the importance of the specific affinity of stains to different chemical compounds and increases the importance of close spatial location of the compo nents (cf. 15). the fracture plane of the erythrocyte membrane is presumably created by shear forces, generated during freezing, between contracting (18, 104) nonpolar layers and the expanding aqueous phase. the outer leaflet is probably anchored in the ex tracellular ice by glycoprotein, while the inner leaflet is anchored in the intracellular ice by water soluble protein (see fig. 3) leading to predominant intramembranous splitting of the plasma mem brane . the lipid bilayer model may be included in this system as a special structure adoptable by a single leaflet or by fusion of two leaflets (see figure 4 in ref. 15). in addition one leaflet may be formed by proteins or by protein and lipid in close association. perturbation of a delicate structure such as that illustrated in fig. 5 could be expected to induce a lipid bilayer structure in one leaflet and consequent multiple fracture planes of the plasma membrane. this interpretation is particularly attractive in freeze-etch preparations where the cell surface may be identified (figure 10a and c in ref. 32). the erythrocyte membrane is u delicate structure the lipids of intact human erythrocytes are digested neither with pancreatic phospholipase a, (89), nor with phospholipase c from bucillus cereus (89). however, lipids of the corresponding permeable ghosts are extensively digested by these and other lipolytic enzymes (46, 89, 112, 120). hydrolysis of 2 0 4 8 % of the total phospholipid of intact human erythrocytes by phospholipase a, from nuju nuja orland sphingomyelinase from staphylococcus au reus results in ultrastructural alterations of both the inner and outer leaflets of the membrane (112). similarly, digestion of intact rat erythrocytes with sphingomyelinase from staphylococcus uureus and phospholipase c from bacillus cereus under non lytic conditions profoundly affects the appearance of both leaflets, leading to a significantly lower den sity of the intramembranous particles (46). lipo lytic enzymes from different sources digest phos pholipids of intact erythrocytes from different species in a specific manner reflecting the protec tion afforded by adjacent molecules in the mem brane (110, 112, 120). the degree of hydrolysis and the morphological effects of lipolytic enzymes upon erythrocytes influenced by the sequence of diges tion ( i 12), the intracellular atp concentration (46), the presence or absence of extracellular albumin (49), and protecting effects of non-digestible lipid and protein ( 1 10, 112, 120). the complexity of these interactions is difficult to reconcile with the fluid mosaic model for cell membranes, according to which most of the erythrocyte surface is formed by unperturbed lipid bilayer. molecular composition of the two leaflets of the human erythrocyte membrane the major proteins of the human erythrocyte mem brane have been characterized by their electro phoretic migration in sds-polyacrylamide gels (102) whereby mainly 7 protein-staining (bands i vii) and 3 carbohydrate-staining (the sialoglycopro tein, glycophorin) bands have been distinguished. by various techniques, including protease digestion and the labelling of intact erythrocytes and resealed ghosts with radioactive markers, only band i11 and upsain .i med sci82 174 e . cerve'n glycophorin of the main proteins have been shown to be exposed at the outer surface (102). these proteins, both of which are hydrophobically as sociated with lipid, constitute about 30% (102) of the total protein mass and the remaining 70% are not to a similar extent detectable from the outside (102). some of these remaining 70 % detectable in leaky ghosts (102) o r inside-out vesicles are susceptible to crosslinking agents (100) and may form a continuum in the intact membrane (12, 102). two of them, the band i and i1 proteins, constitute about 30% of the total protein mass (102) and are jointly designated spectrin. it has been estimated that the amount of spectrin is sufficient to cover the inside of the erythrocyte (12). immunological methods have established that spectrin is located on the cyto plasmic side of the membrane (76). the solubility of these proteins in 80 % ethanol (20) and their capabil ity for both polar and nonpolar interactions with phospholipid (53) suggest that they may be as sociated with lipid in siru. they can be extracted from human ghosts by incubation with cholate (22), edta (87) or neutral and alkaline buffers of low ionic strength (87). these procedures lead to ve siculation of ghosts, solubilization of up to 90 % of the membrane proteins (87) and/or alteration of the trilaminar structure obtained by osmium-staining the pelleted material obtained from high-speed centrifugation (22,87). extraction with water under gentle stirring seems to specifically elute spectrin leaving a residue of atypical trilaminar membranes loosely associated with osmiophilic droplets which may represent phospholipid (87). in this connection it is noteworthy that phospholipid-glycoprotein aggregates ( 1 15) as well as proteins (36) and lipid bilayers (72) may give rise to trilaminar images in the electron microscope. the fact that osmium staining and dehydration leads to significant losses of protein and lipid (67) is also pertinent. spectrin is an elongated (fibrous) protein in aqueous solution (20) but fibrous networks have never been observed by freeze-etching to cover the inside of erythro cytes or ghosts (108) although other fibrous proteins are detectable with this technique (106). part of the spectrin is firmly associated with camel erythrocyte ghosts and is experimentally defined as an integral (not extractable by edta o r slight modifications of ionic strength) protein in that system (30). camel erythrocyte ghosts contain five times more integral protein per unit surface area than d o human ghosts and a higher density of intramembranous particles which are also larger (30). extraction of spectrin from camel erythrocyte ghosts leads to conforma tional changes in the membrane as evidenced by decreased surface area and increased density observed in phase contrast microscope (85). ve siculation of human ghosts by incubation with pro tamine is accompanied by the appearance of multi ple fracture planes some of which form in the inner leaflet (the author's interpretation of figure iou and c in ref. 32) and the shedding of protein-free lipid vesicles (32). the conclusion that lipid bilayer is formed from structures such as that illustrated in fig. 5 during vesiculation is (contrary to the fluid mosaic model) compatible with the increased fluid ity of lipids (and decreased mobility of protein) in the ghost membrane as measured by electron spin resonance (1) under conditions which lead to ve siculation (cf. 32). other evidence (cf. 101, 102) indicates that the band v (actin-like polypeptide) and the band vi (glyceraldehyde 3-phosphate dehydrogenase, gapdh) proteins are components of the inner leaflet. the band iv and vii proteins are however more difficult to locate. the band i, 11, v, vi and vii proteins can be extracted from ghosts by incu bation with cholate (22) indicating that this proce dure preferentially solubilizes the inner leaflet. af ter extraction, the remaining thin (22) trilaminar structure is predominantly composed of the band 111 and iv proteins, the sialoglycoproteins and some lipid (22). this cholate-extracted cholesterol and lipid-depleted structure should be expected to exhibit staining properties markedly different from those of the native membrane. incubation in 8 m urea also seems to elute preferably the proteins of the inner leaflet leaving mainly the band i11 protein associated with about 50 % of the lipid (54). on the other hand, triton x-100 and tween 20 preferably solubilize the proteins of the outer leaflet (22, 59). the contours of the ghosts can be seen with the aid of the phase contrast microscope during elution with cholate or triton x-100 (22). ghost-like structures can also be seen after these procedures by electron-microscopic examination (22). these structures are globular after extraction with triton x-100 and trilaminar after extraction with cholate (22). these data justify the suggestion that spectrin is the structural (48) protein of the inner leaflet while the band i11 protein and glycophorin are upsala j med sci 82 double polar-nonpolar-polar leaflet construction of the plasma membrane 175 the structural proteins of the outer leaflet of the human erythrocyte membrane. (the hydrophobic segment of glycophorin is only about 25.5 a (see above) which is not sufficient for spanning two leaflets. see also page 176 for an evaluation of ex perimental data concerning the localization of pro teins in native membranes.) if spectrin is not as sociated with lipid in situ, then a highly ordered network of hydrogen bonds and salt bridges must be assumed to exist in the inner leaflet in order to account for the low permeability of the erythrocyte membrane towards various polar solutes. since spectrin may be partly extracted from human ghosts (full extraction requiring sonication) with appar ently maintained structure (31), it is reasonable to assume that other proteins as well contribute to the formation of the inner leaflet. the extracellular activity of gapdh and other glycolytic enzymes which is firmly associated with intact cells (3, 6, 90) may reflect the “semiporous” properties of the outer leaflet. it is notable in this connection that phosphate added t o hela cells and erythrocytes may first enter the 1,3 diphosphoglycerate and atp pools (77) indicating that phosphate transport is actually mediated by gapdh, which presumably holds a transmem branous position in the inner leaflet. the outer leaflet of the human erythrocyte membrane may be regarded as the diffusion barrier inferred from studies of glucose and galactose transport (86), while the carrier mechanism for these solutes is situated in the inner leaflet and is destroyed con comitantly with the proteolytic cleavage of spectrin by proteolytic enzymes included in resealed ghosts (66). this is also supported by the finding that gapdh (and aldolase) most of which is bound to the inner (101) surface of the ghost membrane is more available to its substrate than are a number of cytoplasmic enzymes (e.g. lactate dehydrogenase and pyruvate kinase) under various conditions (28) indicating that this enzyme (and aldolase) constitute or is located exterior to a diffusion barrier (presum ably the inner leaflet). these interpretations are corroborated by the demonstration that the en zymatic properties of gapdh are not significantly different in the membrane-associated and s o h bilized states (91). the na+and k+-stimulated atpase is presuma bly located in the inner leaflet (cf. 63). ouabain and potassium are regarded as being capable of influenc ing the activity of this enzyme by binding to the outer surface of the inner leaflet after passage through the outer leaflet. the intramembranous particles conditions which labilize the spectrin (the band i and i1 proteins) tend to facilitate aggregation of the intramembranous particles (3 1). cross-linking of spectrin with glutaraldehyde inhibits particle ag gregation (31) (as well as solubilization of these proteins by sonication in the absence of divalent metal ions (31)). these and other findings (84) sug gest that spectrin is involved in creating o r forming the intramembranous particles seen by freeze fracture (31, 84). there is also much evidence correlating the intramembranous particles with glycophorin, the main sialoglycoprotein of the hu man erythrocyte membrane (107). however, re cently it was shown that mutant erythrocytes which lack this protein still contain intramembranous particles (13). ferritin-conjugated antibodies di rected against antigens residing in glycophorin as well as in the band 111 protein bind to the surface of human erythrocytes in a pattern that is reminiscent of the pattern formed by the intramembranous particles (84, 107). thus spectrin, glycophorin, the band i11 protein and other proteins and lipid (60) as well, may be involved in forming the intra membranous particles which are tetrameric or heterogeneous when visualized by rotatory replica tion (64). several difficulties attend the correlation of morphological observations on freeze-fractured specimens with other parameters. these include variations due to the choice of freeze fracture equipment and methodology (60), the proportion of cross fractures to tangential fractures (60) and altered fracture planes (99). the findings that fixa tion (79) cooling before freezing ( i 18) and the pres ence of dimethylsulphoxide or glycerol (57) affect the particle frequency of various biological mem branes indicate that particles are impermanent structures. they d o not necessarily represent pro teins of high molecular weight since they are found in protease-digested membranes lacking high mo lecular weight proteins (1 19) and in artificial mem branes containing hydrophobic peptides (92). the fluid mosaic model for cell membranes pre dicts that the density of the intramembranous parti cles representing intramembranous protein (94, 95) should increase when lipid but not protein is re moved (21, 60) from the membrane by digestion upsulu j med s c i 82 176 e . cerven with phospholipase c. this is because the smooth fracture planes are considered to represent the no,npolar region of the membrane built up of lipid bilayer (95) in which the integral proteins are freely (39) diffusible (95) and appear after freeze-etching as intramembranous particles. contrary to this pre diction, a decreased density of the particles follow ing digestion with phospholipase c was reported (60). ghosts digested maximally with phospholipase c very rarely split tangentially (60). when they do, smooth fracture faces are occasionally (60) but not always (1 12) produced. if the membrane-associated particles of ghosts are permanent intramembranous structures, it is unlikely that their higher density would counteract the tangential splitting of the plasma membrane, since aggreagated particles have been visualized repeatedly (84, 107). exogenously applied proteins of high molecular weight (e.g. actin, m w : nx43000; ferritin, m w : 450 000; the tetramer of phytohemagglutinin, mw: 126000; the tetramer of concanavalin a, m w : 110000) bound to the cell surface may be visualized by the freeze fracture procedure (83, 84, 106, 107). if it is assumed that endogenous globular and fi brous peripheral proteins of high molecular weight (e.g. actin, m w : nx43000; spectrin, m w : 200 000-240 000, and the tetramer of gapdh, m w : 140000) are also visualized by this procedure, then they must be confined to the intramembranous particles. this is because both the intracellular and extracellular surfaces are comparatively smooth (see ref. 83). these considerations are compatible with the view that proteins experimentally defined as peripheral proteins (94, 95) partly form the inner leaflet of intact erythrocytes and minimally perturbed ghosts. the significance of water-soluble proteins in forming membrane area is also adduced from the observation that extensively washed and resealed ghosts are smaller than slightly washed and resealed ghosts (55). it follows from these considerations that the outer leaflet cannot be a lipid bilayer, since such structures are cleaved smoothly by freeze-fracture, and no fracture planes are seen within the outer leaflet under ordinary conditions. current status of previous reports proteins spanning two leaflets of the plasma mem brane (95) were included in the original version of the present model for biological membranes with some reservations (15). however, other features of the model such as the postulated complex interac tions between various residues forming the planes of hydrogen bonding and salt bridges and also the transmembranous diffusion of water-soluble protein must be taken into account. such phenomena may partly compensate for the apparent vectorial label ling and digestion of intact cells, resealed ghosts, resealed inside-out and right side-out vesicles and leaky ghosts (cf. 102). the interdependency of pro tein and lipid conformations in erythrocyte mem branes is documented by electron-spin resonance studies (93, 103, 113). the complex pattern of diges tion of intact erythrocytes with lipolytic enzymes (110, 112, 120) is tentatively explained by polar interactions between protein and lipid. in this con nection is may be added that iodination of intact mitochondria with the lactoperoxidase method re sults in labelling of one component apparently re lated to the inner membrane (1 1) and the same pro cedure applied to intact erythrocytes may cause labelling of hemoglobin (109). thus, the existence of proteins spanning two polar-nonpolar-polar leaflets with those properties required by the pres ent model for the structure of plasma membranes and other biological membranes is not corroborated by the available evidence. the globular structures preserved by some electronmicroscopic preparatory techniques (67, 68, 96, 97) may be interpreted as foldings of one leaflet around globular water-soluble structures (15). other arrangements are equally possible, the characterization of which depends on further de finement of the preparatory techniques (97). general considerations by analogy with the inhibitory effect of proteolytic enzymes on glucose and galactose carrier function in erythrocytes (66), the function of giant axons is similarly disturbed by proteolytic action on the in ner leaflet of the axolemma (105). a thermodynamic analysis of nerve cell depolarization across, two membranous leaflets separated by a polar space has been presented and found to describe satisfactorily the experimental data ( 5 6 ) . the interpretation of data from nerve cell axons is complicated by the occurrence of serial membranes in addition to the two leaflets of the plasma membrane. however, according to the double leaflet model for biological membranes only the inner leaflet is involved in energy transducing activity (15). therefore, the periaxonal space deduced from experimental devia upsulu j med sci 82 double polar-nonpolar-polar leaflet construction of the plasma membrane 177 tions from theoretically expected depolarization currents ( 2 ) may partly represent the intramem branous space. the possibility of a sodium gradient primarily across the inner leaflet (cf. 15) is an attrac tive explanation why the generalizing of the concept of sodium-dependent transport of certain metabo lites across cellular membranes (cf. 19) fails ex perimentally (73). the main criticism that can be directed against the lipid bilayer concept for biological membranes involves the fact that no other structure which ex plains the experimental data has seriously been con sidered. the controlled references in the experi ments have of necessity been lipid bilayers or pro tein in aqeous media. for example the fact that fracture planes are generated in the nonpolar core of lipid bilayer ( 2 5 ) seems t o suggest that non cooperative hydrophobic interactions are always responsible for the fracture planes in biological membranes. estimation of the amount of a a-helix, /3-structure and random coil in biological mem branes rests on the use of water-soluble reference peptides without considerations of additional in teractions possibly occurring in biological mem branes. interpretation of x-ray diffraction data in volves a comparison with electronmicroscopic pictures that are considered to represent bilayers. interpretation of electron spin resonance data sometimes rests on the assumption that the lipid is statistically distributed in the native membrane which is assumed to be a lipid bilayer. however, electron spin resonance studies should be in terpreted with care, since a statistical distribution of spin labels and lipid is not to be expected a priori (93, 113). acknowledgement i thank dr gunnar agren and dr gunnar ronquist for generous support and for reading the manuscript. this work was supported by grants from the swedish research council (project b77-13x-228-13b), the university of uppsala and a personal grant from uddeholms ab. references 1 . adams, d., markes, m. e., leivo, w. j. & carra way, k . l.: electron spin resonance analysis of ir reversible changes induced by calcium perturbation of erythrocyte 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hundred and fourteen children (60% girls) adopted from india through five major adoption organizations, were recruited consecutively. this paper describes the environment of the children in india and in sweden, discusses the certainty of the ages and reports their condition at arrival in sweden. the median age at arrival was 9.3 months, 62% being below one year of age (range 3-72 months). infectious diseases similar in kind and frequency to those noted in child populations in developing countries, were found. heighdage and weightlage mean values were approximately -2 standard deviation scores (sds) of the nchs/who standard, which is similar to the anthropometric status of indian average children. there were no significant sex differences. thirty-seven birth weights were known, the majority below 2 500 g. psychomotor retardation was found in 29% of the children. in the children with stunting and in those with weightlage < 3 sds at arrival there were high percentages of psychomotor retardation, anaemia and combined wasting and stunting. therefore these children should be regarded as a risk group and be followed up with special care. introduction during the period 1969-87 more than 27 ooo children were adopted from developing countries to sweden, 4 800 of them from india. from 1975 onwards 1 300-1 700 children arrived yearly, an average of 300 from india. retrospective studies of the somatic development and social adaptation after arrival have been carried out (9, 11, 14). these studies have shown rapid initial catch-up growth and favourable development in general. however, it has been found that indian adopted girls undergo earlier sexual maturation and in some cases attain shorter final height than indian and swedish girls (1, 16, 17). menarcheal age was positively correlated to the age at arrival and to the velocity of catch-up growth (16, 17). 79 final height was positively related to menarcheal age and to height at arrival (17). the pubertal growth component was found to be normal, lower final height being associated with a shortened childhood growth phase due to early pubertal onset, as well as to lower height before onset of puberty (18). these studies indicate that the later growth and development of adopted children is influenced by their conditions before adoption as well as during the initial period after arrival in sweden. a longitudinal prospective study can add information not obtainable by retrospective investigations. limitation of the study population to children adopted from one country enables comparison with national data. the aim of the present study was to follow a group of adopted children from their arrival in sweden and during the following 2 years, in order to record their growth, development and clinical condition, and to relate these parameters to sex, conditions before adoption, and to age, health and nutritional status at arrival. this paper describes the environment of the adopted children in india and sweden, discusses the certainty of the age data and describes the anthropometric and clinical condition at arrival in sweden. material and methods the parents of 132 children were invited to take part in the study, upon the arrival of the children in sweden. they were consecutively recruited through 5 adoption organizations: the adoption centre (ac), the children above all-adoptions (bfa), the indo-swedish association for intercountry adoptions (isia), the family association for international adoptions (ffia), and the swedish association for adopted children's welfare (saw). the parents of 114 children (86%) were willing to take part. the rest answered that they were unable to participate. due to the sensitivity of the issue of adoption experienced by many parents, the authors were advised by the adoption organizations neither to encourage enlistment in the study too insistently, nor to demand specification of the reason for non-participation. the adoptive parents answered a questionnaire regarding their child's social and health conditions in india, and also about their own age and education and the number of their previous biological and/or adopted children. the doctor carrying out the first health examination was supplied with a standardized examination form. the subsequent examinations during the 2 years of follow up were also standardized, and will be described in detail in a subsequent paper. the data collection started in 1985. 80 telephone contact with the parents was taken when necessary. the parents were offered medical advice regarding their children when needed. the parents' informed consent for data processing was obtained. anthropometric measurements were carried out according to routines used in swedish child health practice. the anthropometric data are expressed in standard deviation scores (sds) according to the method recommended by who, using the reference data from the national center for health statistics (nchs) (15, 26, 27). the nchs reference data are also applicable to indian children (2, 25). when analyzing anthropometric data in relation to age at arrival, the age groups recommended by who were applied whenever possible. the calculation of standard deviation scores (sds) for the anthropometric indices heighvage, weighdage, weighuheight and head circumference/age was done according to the procedure recommended by who (15, 26): individual's value median value of reference population standard deviation value of reference population sds =-------------------------------------------------------------------------------------------- the cdc anthropometric software package (13) based on the nchs reference population was used for the sds calculations. according to who recommendations malnutrition was classified as "stunting" for height/age <-2 sds, and as "wasting" for weight/height <-2 sds (15, 26, 27). psychomotor development was related to accepted standards (12). statistical analysis using student's t-test, bivariate linear regression, calculation of pearson's correlation coefficient and non-parametric analysis of variance (kruskal-wallis test) were carried out using the sas system (19, 20 21). when not otherwise stated, percentages are calculated on the total material of 114 children. results environment in india and sweden. the age at arrival and the sex distribution is seen in table 1. the median age at amval was 9.3 months, 62% being below 1 year and 81 % below 2 years. the age ranged from 3-72 months. the girls dominated (60%). 6 928571 81 table 1. a g e at arrival age at arrival boys girls total (months) n n n % 11 31 40 71 62.2 12 23 8 13 21 18.4 24 35 2 9 11 9.7 36 5 6 11 9.7 , total n 46 68 114 100.0 ' % 40.4 59.6 100 mean age (months) 14.3 15.8 15.2 median age (months) 8.6 9.5 9.3 range (months) 3-62 3-72 3-72 most of the children were born in or near big cities (calcutta 33%, bombay 15%, nagpur 15% and madras 9%). the remaining came from all over india. information regarding one or both biological parents was available for 52 children. for 45 of these only the mother was known. four of these 45 mothers had died and all the remaining lacked social support in various ways, e.g. they were unmarried, abandoned or widows. six children had living married parents. in one case, only the father was known. for the majority (62%) the first institutional contact eventually leading to adoption was with a children's home, to which the children were brought by parents or relatives. eighteen percent were born or left in a hospital and later transferred to a foster home or children's home. fourteen percent of the children were foundlings, e.g. abandoned in the street. most of the children (77%) were adopted from children's homes. twenty-two percent came from foster homes, and 1% from a hospital. for 36 children (32 %) information regarding the previous medical history was available. this could refer to the period just before departure or earlier. the adoptive parents reported a summary of this information. ten of the 36 children were reported to have had protein energy malnutrition, 11 acute respiratory infections, 9 diarrhoeal disease and 5 anaemia. a range of other diseases was also reported for a few or single cases: intestinal parasites, urinary, ear, eye and skin infections, hepatitis b, tuberculosis, vitamin a deficiency and rectal prolapse. most children had more than one disease. the adoptive families were located all over sweden, in both rural and urban areas. there were 112 families in all, consisting of 108 couples and four single mothers. all the 108 adoptive couples were married. two couples adopted 2 children each, the rest of the families adopted 1 child. 82 the mean age of the fathers (n=108) was 36 years (range 25-48 years) and that of the mothers (n= 112) was 35 years (range 25-50 years). eighteen mothers were 40 years or older. for 7 of these mothers the adopted child was the first child. forty-seven percent (51/108) of the fathers and 49% (55/112) of the mothers were first time parents. the 4 single mothers were among these. the mean age of the first time fathers was 34 years (range 25-48), and of the first time mothers 33 years (range 25-44). nine families had previous biological children (7 families had 1 child and 2 families had 2 children). three of these families also had previously adopted children. fifty-one families had previously adopted children (48 families had one child and 3 families had 2 children). eighty percent (43/54) of the previously adopted children came from india, the others from sri lanka, indonesia, thailand, bolivia, chile and sweden. socio-economic classification of the adoptive parents has been carried out according to the method based on occupational categories developed by statistics sweden (23). compared with swedish figures for 1986 of classification of parents, the distribution of socio-economic classes was similar among the adoptive parents, except that no unemployed or unclassifiable parents were found among the adoptive parents (table 2) (6). table 2 . classification of adoptive families ( n = 112) study population swedish families with children 1986 (6) n % % homogeneous families~ a. b. c. workers or assistant intermediate non-manual employees, professionals or executives 35 31 self-employed (other non-manual employees 39 35 than professionals) 1 1 non-homogeneous families one parent group a, the other group b one parent group c, the other any other occupation other^ 24 21 13 12 33 29 4 19 5 10 ~ total 112 100 100 i.e. both parents belong to the same occupational group unemployed or unclassifiable 83 certainty of the cbildren's age. the childrens' age was based on official documents issued by medical and civil authorities in india. additional information was sometimes available from medical or other personnel at institutions where the children were cared for. the degree of certainty of the age was based on the information given to the adoptive parents as well as on the judgement of the doctor carrying out the first health examination after arrival. based on this the age data of 54% were classified as "certain", 33% as "fairly certain" and 9% as "uncertain". anthropometric evaluation. the majority (75 %) of the first examinations after arrival were carried out within 1 week, and 86% within 2 weeks. sixty-four percent of the examinations were carried out at pediatric clinics, 26% at infectious diseases clinics, and 10% at other clinics. figure 1 and table 3 show the distribution and the means of the weightlage, height/age and the head circumferencehge. -2 s.d. 1 b 3 6 9 1 2 18 2 3 l 5 6 7 8 months years figure 1 . head circumference (triangles), height (squares) and weight (dots) at arrival for the adopted boys and girls, in relation to the nchs reference (median and +/2 sd indicated). the mean values for weightjage, heightlage and head circumference/age were about -2 sds, but with great variation. the mean weightlheight was approximately -1 sds. many children showed extremely low values, particularly among the young infants and the older children. 84 table 4 shows the rate of stunting and wasting according to the definitions used; 47% were stunted, 1.8% wasted and 6.4% showed a combination of both conditions. there were no significant sex differences. the age distribution of the anthropometric variables and the nutritional status are seen in tables 5 and 6. the children in the age groups 0-5 months and 218 months had the lowest weight/age and heighuage. analysis of variance (kruskal-wallis test) showed significant differences between the means of the age groups for heighuage and for weighuage, while for weight/height and head circumference/age there were no significant variations. six of the 7 children with both stunting and wasting were found in the oldest age group. table 3. anthropometry at arrival boys (n=46) girls (n=68) total (n=114) weight/age mean sds range sds n % <-2 sds % <-3 sds height/age mean sds range sds n % <-2 sds % <-3 sds weightlheight mean sds range sds n % < -2 sds % < 3 sds -2.06 45 49 27 (-4.02)(-0.26) -2.08 45 51 22 (-5.26)-(+o. 14) -0.84 45 9 0 (-2.82)-(+0.90) head circumference/age mean sds -1.83 range sds (-3.91)(-0.17) n 34 % <-2 sds 44 % < 3 sds 9 -2.38 (-5.21)(-0.14) 66 67 24 -2.33 (-6.20)( + 0.90) 66 56 27 -1.02 64 8 0 (-2.27)-(+0.91) -2.07 59 54 15 (-4.62)-(+0.84) -2.25 111 60 25 (-5.21)(-0.14) -2.23 111 54 25 (-6.20)-( +0.90) -0.94 109 8 0 (-2.82)-( +0.91) -1.98 93 51 13 (-4.62)-( + 0.84) 85 table 4. distribution of stunting and wasting (n=109) normal stunted wasted stunted & total n % n % n % n % n % wasted boys 21 46.7 20 44.4 1 2.2 3 6.7 45 100.0 girls 28 43.7 31 48.4 1 1.6 4 6.3 64 100.0 all children 49 45.0 51 46.8 2 1.8 7 6.4 109 100.0 table 5. height/age, weight/heighf and head circurnference/age. mean sds values on arrival for all children for the various age groups, f o r weight/age, weightiage heightiage weightlheight head circ/age n sds n sds n sds n sds 0-5 22 -2.39 22 -2.61 20 -0.62 18 -1.88 6-11 48 -2.13 48 -1.83 48 -0.91 41 -1.85 12-17 13 -1.74 13 -1.75 13 -0.86 11 -1.88 1828 -2.58 28 -2.84 28 -1.26 23 -2.35 kruskalwallis test' p < 0.05 p < 0.01 ns ' non-parametric analysis of variance (20), 5 % significance level ns table 6. age distribution o f nutritional status ( n = 1 0 9 ) normal stunted wasted stunted & total age wasted (months) n % n % n % n % n % 0-5 7 35.0 12 60.0 1 5.0 0 0 20 100.0 6-11 25 52.0 21 43.8 1 2.1 1 2.1 48 100.0 12-17 8 61.5 5 38.5 0 0 0 0 13 100.0 2 18 9 32.1 13 46.5 0 0 6 21.4 28 100.0 86 there were birth weight data for 37 children (19 boys and 18 girls). eighty-one percent (30/37) had a birth weight below 2 500 g. at arrival in sweden 17 were < 6 months of age, 19 were 6-17 months of age and 1 218 months of age. sixty-five percent (24137) were stunted and 8.6% (3/35) were wasted. there was no sex difference regarding weight/age, height/age and weight/height at arrival, and therefore the sexes were combined into one group. linear regression analysis showed that birth weight correlated significantly with weighdage at arrival (p < 0.001, r=0.38) and height/age at arrival (p<o.001, r=o.36), but not significantly with weight/height at arrival). clinical examination. the clinical assessment after arrival showed that 54% had pathological findings, most commonly malnutrition (as judged clinically) (45 %), acute respiratory infections (8.8%), diarrhoea (4.4%), molluscum contagiosum (8.3%), scabies (9.7%), otitis media or otosalpingitis (15%), external otitis (3.7%) and throat infections (4,4%). frequently the same child had several pathological findings. anaemia (b-haemoglobin c 100 g/l) (5) was found in 15 % , elevated esr ( ~ 2 0 mm/h) in 26%, leucocytosis (215 x 109/1) in 40%, abnormal differential count (mainly lymphocytosis and eosinophilia) in 60%. hbsag was positive in 8.3%. the stools were abnormal in 44%, with bacterial, parasitic or combined infections (table 7). table 7. results of the stool examination of the children in "the 2-year study" ( n = 1 1 2 1 1 3 ) , at arrival in sweden. diagnosis' % salmonella 15.9 giardia lamblia 13.3 trichuris trichiura 9.7 campylobacter 7.1 ascaris 6.2 strongyloides stercoralis 4.4 ancylostoma duodenale 3.5 hymenolepis nana 3.5 s higella 1.8 e. histolytica 1.8 one child may have several infections 87 of 108 children assessed regarding their psychomotor development, 29% were found to be retarded according to accepted development standards (12). psychomotor retardation occurred mainly among the stunted children (47%), compared with 7.8% in the non-stunted group. corresponding figures for anaemia were 23% and 4.0%, respectively. twenty five percent (28/111) of the children (27% of the boys and 24% of the girls) had weight/age < -3 sds. this indicator was selected since it reflects both height for age and weight for height (27). the majority of those with height for age <-3 sds and 5 of the 9 with weight for height < -2 sds, were found in this group. retarded psychomotor development was more common (54% compared to 21 % among the rest). anaemia was also more frequent. otherwise there were no clear differences regarding disease frequencies (table 8). table 8. characteristics of children with w e i g h t / a g e (-3 s d s compared with those 2-3 s d s weight/age < -3 sds weight/age 2 3 sds (n=24-28) (n =72-83) n % n % height/age <-3 sds weightlheight < -2 sds anaemia (b-hb < 100 g/l) hbsag positives salmonella s higella campylobacter e. histolytica giardia lamblia ascaris trichuris trichiura acute respiratory infection diarrhoeal disease urinary tract infection scabies conjunctivitis throat infections otitis tuberculosis retarded psychomotor development 21 75 5 19 6 21 3 12 5 18 2 7 2 7 0 0 4 14 2 7 4 14 0 0 2 7 1 4 3 11 0 0 0 0 4 14 1 3 14 54 7 8 4 5 10 12 5 6 13 16 0 0 6 7 2 2 10 12 5 6 7 9 11 13 3 4 1 1 8 10 2 2 4 5 13 16 0 0 17 21 88 discussion the study population was recruited consecutively during a certain period. the age and sex distributions in the studied population were very similar to those of all indian children adopted in sweden during the recruitment period. the 5 major swedish adoption organizations undertaking adoptions of children from india were included. therefore the study population could be considered as representative for indian children adopted in sweden during the period mentioned. there is no reason to believe that the defaulting 18 children were different from those in the study population. as many as 87% of the children were estimated as having a "certain" or "fairly certain" age. this seems reasonable since the majority of the children were very young at arrival, and it is easier to obtain reliable birth data for younger children. furthermore, the margin of error in age estimation is naturally less the younger the child. the adoptive families were selected to provide a good emotional and social environment to the children. the majority of the children with known background came initially from socially deprived situations. the children's homes and foster homes, from which most of the children were adopted, were as a rule well-ordered but certainly not affluent. it is therefore not surprising to find that the diseases in india, as well as at arrival, corresponded to those usually found in developing countries. moreover the anthropometric indices were similar to those of an average indian population, characterized by chronic malnutrition among a considerable proportion of the children (3, 8, 22). similar findings are reported also from earlier studies of children adopted from developing countries. the relatively high prevalence of psychomotor retardation has likewise been noted earlier. (9, 14). the mean weight and height for age at arrival were approximately 2 sds lower than for affluent indian children (2, 2 5 ) but were similar to indian national average data which included also less privileged children (8). the distribution of wasting and stunting was similar to that found in india except for a higher percentage of combined wasting and stunting (table 4) ( 2 2 ) . no significant differences regarding anthropometry were found between the sexes, which is in accordance with the findings of sastry et al. ( 2 2 ) . in our study population there were data on birth weight for only 37 children. the sub-sample is naturally limited to children delivered in an institution where birth weight is recorded. as many as 30 (81 %) had low birth weight ( < 2 500 8). the indian low birth weight average is 30% ( 2 4 ) , with a wide range depending on socio-economic conditions (7). our sub-sample is probably biased, including many socially deprived mothers. malnutrition and pregnancy complications may 6 t-928571 .s9 have caused these mothers to deliver in a hospital or similar health service, thereby enabling the birth weight to be recorded. birth weight has been found to predict weight and height in early childhood (4, 10). the high proportion of stunting seen already in the children who were youngest at arrival (0-5 months) fits with the high percentage of low birth weight in this group (table 6). our study sample of 37 children showed significant positive correlations between birth weight, and height as well as weight at arrival, confirming the studies mentioned above. the low height/age in the youngest age group (table 5 ) indicates that stunting may have been present already before birth. the lower rate of stunting in the age group 6-17 months may be due to mortality selection of the weakest and to nutritional catch-up in the survivors. the high rate of stunting seen in the oldest children conforms to rates found in india (22). the wide ranges of the anthropometric indices at arrival (table 3) probably reflect the differences in the children's pre-adoptive environment, of which we only have limited knowledge. the stunted children showed a high rate of anaemia and psychomotor retardation. the children with very low weight for age (a group which contained many of the most severely stunted and/or wasted) also included a high proportion of children with psychomotor retardation and anaemia. stunted and very low weight children should be regarded as risk groups and routinely be given a more prolonged and intensive follow up after arrival. acknowledgements the authors wish to thank the adoptive parents and their children for their participation and interest in the study. we also thank the adoption organizations mentioned above (ac, bfa, isia, ffia and saw) for their co-operation. moreover we are grateful to kerstin goransson of the uppsala university data centre for her help with the data processing and to professor gunnar eklund, the karolinska institute, stockholm and tore lundstrom of the regional oncologic centre, the uppsaldorebro health care region, for their statistical advice. the study was supported by grants from the swedish council for social research (sfr), stockholm, and from uppsala university. references 1. 2. 3. 4. 90 adolfsson, s . , & westphal, 0.: early pubertal development in girls adopted from far eastern countries. pediatr res 15:82, 1981. aganval, k.n., et al.: growth performance of affluent indian children (under-fives). growth standard for indian children. nutrition foundation of india. sci report 11, 1991. banik, n.d.: quoted in: eveleth, p.b. & tanner, j.m.: worldwide variation in human growth. cambridge univ press, 1976. binkin, n.j., yip, r., fleshood, l. & trowbridge, f.l.: birth weight and childhood growth. pediatrics 6:828-834, 1988. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. fernlund, p., fex, g., hanson, a. stenflo, j. & lundh, b.: laurells klinisk kemi i praktisk medicin. sjatte upplagan. studentlitteratur, lund, 1991. gellerstedt, l.: kvinnor, man och barn i klasstrukturen. valfardsbulletinen 2: 18-21, 1989. gopalan, c.: women and nutrition in india. some practical considerations. bull nutr found of india, 4: 1-4, 1989. growth and physical development of indian infants and children. indian council of medical research, icmr, tech rep ser no. 18, new dehli, 1972. gunnarby, a., hofvander, y., sundelin, c. & sjolin, s.: utlandska adoptivbarns halsotillstand och anpassning till svenska forhallanden. lakartidningen 79; 1679-1705, 1982. hofvander, y.: international comparisons of postnatal growth of low birth weight infants with special reference to differences between developing and affluent countries. acta paediatr scand suppl 296, 1982. hofvander, y. & sundelin, c.: transnational adoptions. in: advances in international maternal and child health. vol 2. (eds. d.b. jelliffe & e.f.t. jelliffe), pp. 111-116, oxford university press, oxford, 1982. illingworth, r.s. : the normal child. churchill livingstone, edinburgh, 1979. jordan, m.d.: the cdc anthropometric software package. version 3.0. tutorial guide and handbook. anthropometric analysis software based on the cdc standard deviation-derived growth reference curves derived from nchskdc reference population. the centers for disease control, center for health promotion and eduvation, division of nutrition, statistics branch, 1600 clifton road, atlanta, georgia 30333, 1986. magnusson, b. & proos, l.: viktoch langdutveckling hos adoptivbam frin u-lander. lakartidningen 76:3192-3 193, 1979. measuring change in nutritional status. guidelines for assessing the nutritional impact of supplementary feeding programmes for vulnerable groups. world health organization, geneva, 1983. proos, l.a., hofvander, y. & tuvemo, t.: menarcheal age and growth pattern of indian girls adopted in sweden. i. menarcheal age. acta paediatr scand 80:852-858, 1991. proos, l.a., hofvander, y. & tuvemo, t.: menarcheal age and growth pattern of indian girls adopted in sweden. 11. catch-up growth and final height. indian j pediatr 58: 105-1 14, 1991. proos, l.a., karlberg, j., hofvander, y. & tuvemo, t.: pubertal growth of indian girls adopted in sweden. submitted to acta paediatr scand. sas institute inc. sas language guide for personal computers, version 6.03 edition. sas institute inc., cary, nc, 1988, sas institute inc. sawstat guide for personal computers. version 6 edition. sas institute inc., cary, nc, 1987. sas institute inc. sas procedures guide for personal computers, version 6.03. sas institute inc., cary, nc, 1988. sastry, j.g., vijayaraghavan, k. & rao, n.p.: indian preschool children a profile on stunting and wasting. j trop pediatr, 5:237-240, 1989. swedish socio-economic classification. statistics sweden. report on statistical co ordination, 4, 1982. the state of the world's children 1992. unicef. oxford university press, oxford, 1992. vijayaraghavan, k., singh, d. & swaminathan, m.c.: heights and weights of well nourished indian school children. indian j med res 59548-654, 1971. waterlow, j.c., buzina, r., keller, w., lane, j.m., nichaman, m.z. & tanner, j.m.: the presentation and use of height and weight data for comparing the nutritional status of groups of children under the age of 10 years. bull worlds health organ 55:489-498, 1977. who working group. use and interpretation of anthropometric indicators of nutritional status. bull world health organ 64:929-941, 1986. 7 928571 91 address for reprints: dr lemm a.proos international child health unit department of pediatrics uppsala university children's hospital sweden s-751 85 uppsala 92 upsala journal of medical sciences 2023, 128, e9473 http://dx.doi.org/10.48101/ujms.v128.9473 maternal, pregnancy and neonatal outcomes in triplet pregnancies in sweden – a nationwide cohort study mia-maria ekströma, eleonor tiblada,b, mikael normanc, olof stephanssona,b and michaela granforsa,b aclinical epidemiology division, department of medicine solna, karolinska institutet, stockholm, sweden; bdivision of obstetrics, department of women’s health, karolinska university hospital, stockholm, sweden; cdepartment of clinical science, intervention and technology, karolinska institutet, stockholm, sweden abstract background: triplet pregnancies carry a high risk of pregnancy-related complications. the primary aim of this study was to describe maternal, pregnancy, and neonatal outcomes in expectantly managed triplet pregnancies in sweden. the secondary aim was to compare outcomes in expectantly managed triplet pregnancies with triplet pregnancies where fetal reduction had been performed with the only indication to reduce the number of fetuses. methods: nationwide cohort study based on linkage of data from three national swedish registers. triplet pregnancies with delivery at gestational age ≥ 22+0 weeks between 2014 and 2019 were included. results: in the main cohort of expectantly managed triplet pregnancies (n = 106), 98% (312/318) of infants were liveborn with a mean gestational age at birth of 32+3 weeks and a mean birthweight of 1,726 g. nine percent (n = 29) suffered from severe neonatal morbidity, and 4% (n = 12) died during the neonatal period. in the reduced cohort (n = 13 pregnancies), all infants were liveborn (n = 22). mean gestational age at birth (36+0 weeks) and mean birthweight (2,444 g) were higher than in the expectantly managed cohort (p < 0.01 for both comparisons). there were no cases of severe neonatal morbidity (p = 0.24) or mortality (p = 1.00). conclusion: overall neonatal survival from 22+0 weeks of gestation in expectantly managed triplet pregnancies in sweden was high. nine out of 10 infants did not suffer from severe neonatal morbidity. fetal reduction was performed in only a very small number of cases and was associated with higher gestational age at birth and higher birth weight. article history received 5 march 2023 revised 25 may 2023 accepted 30 may 2023 published 17 july 2023 keywords triplet pregnancy; multifetal gestation; maternal outcome; perinatal outcome; fetal reduction introduction during the last decades, multifetal birth rates have increased significantly in high-income countries, mainly due to assisted reproductive technologies (art) (12–3). the swedish regulation on in vitro fertilization (ivf) was revised in 2003, and single embryo transfer was set as normal routine to reduce the risk of multifetal pregnancies (4). the proportion of triplet deliveries in sweden has been stable at about 0.2‰ for the past two decades (5). the risk of miscarriage as well as of fetal and neonatal death and neonatal and infant morbidity, mainly related to chorionicity and preterm birth, is increased in multifetal pregnancies (6). maternal complications associated with multifetal pregnancy are, for example, gestational diabetes, hypertensive disorders of pregnancy, intrahepatic cholestasis, anemia, major obstetric hemorrhage, and cesarean section (789–10). fetal reduction is a procedure where the number of fetuses in a multifetal pregnancy is reduced by one or more (11). fetal reduction has been shown to reduce the risk of preterm birth but carries an increased risk of miscarriage. a combined observational study and systemic review showed an increased risk of miscarriage after fetal reduction, but a decrease in very preterm birth (defined as gestational age [ga] <32+0 weeks) from 28 to 10% (12). other retrospective studies have shown similar results, with significantly prolonged pregnancies after fetal reduction compared to expectantly managed triplet pregnancies (13, 14). it is still uncertain whether multifetal pregnancy reduction carries a significant benefit in the context of contemporary maternity and neonatal care (15). information regarding expected outcomes nationwide or for countries with similar maternal and infant care is important when counseling women pregnant with triplets regarding the possibility of reduction or expectant management. the primary aim of this study was to describe maternal, pregnancy, and neonatal outcomes in expectantly managed triplet pregnancies using national data from sweden. the secondary aim was to compare these outcomes in expectantly managed triplets with triplet pregnancies where fetal reduction had been performed. contact michaela granfors michaela.granfors@ki.se supplemental data for this article can be accessed here. © 2023 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article http://dx.doi.org/10.48101/ujms.v128.9473 mailto:michaela.granfors@ki.se http://dx.doi.org/10.48101/ujms.v128.9473 http://creativecommons.org/licenses/by/4.0/ 2 m.-m. ekström et al. materials and methods in this nationwide cohort study of triplet pregnancies, triplet deliveries at ga 22+0 weeks or later from 1 january 2014 to 31 december 2019, registered in the swedish pregnancy register, were included. the expectantly managed cohort included triplet pregnancies with a delivery of three infants, still-, or liveborn. the reduced cohort consisted of triplet pregnancies where fetal reduction had been performed exclusively in order to reduce the number of fetuses and not due to fetal complications. the reduced cohort was identified from the fetal therapy register and linked to the swedish pregnancy register. data from both cohorts were linked to the swedish neonatal quality register (snq) to retrieve detailed information on neonatal outcomes (1617–18). antenatal care in sweden, including fetal therapy, is standardized and free of charge. high-risk pregnancies and deliveries are centralized to university hospitals. according to swedish law, civil registration of all deliveries or pregnancy losses at ga 22+0 weeks or later is mandatory, while a pregnancy loss until ga 21+6 is defined as miscarriage (19). swedish national guidelines from 2016 recommend active perinatal care for preterm births ≥ 23+0 weeks, whereas at 22+0–6 weeks, active care may be considered (20). all pregnant women are invited to an early second‐trimester ultrasound screening at 18–20 weeks of gestation, and approximately 97% of the pregnant population participate (21). in addition, during the years 2014–2019, an estimated up to one-half of the pregnant population underwent a first trimester screening ultrasound (16). the swedish pregnancy register is a national quality register, established in 2013 (16). it receives data from different sources. most variables are obtained by direct transfer from the electronic medical records, which included approximately 91% of all births in sweden (17 of 21 regions) during the study period. the fetal therapy register is a register at the center for fetal medicine at karolinska university hospital, stockholm. manual registration in the fetal therapy register is done for all women undergoing invasive fetal therapy in sweden. since 2013, highly specialized invasive fetal therapy has been centralized to one center in sweden, karolinska university hospital. reduction of multiple pregnancies in non-monochorionic pregnancies is formally not centralized to karolinska university hospital, but according to correspondence to all university hospitals in sweden in 2020, multifetal reduction has probably not been performed outside karolinska university hospital in recent years. snq is a national quality register, established in 2001 (17). it includes all infants born alive in sweden who are admitted for neonatal care within 27 days after birth (around 11,500 annual admissions corresponding to 10% of all births in sweden) and delivery room deaths. completeness in snq for preterm infants in sweden has been found to be excellent (17). study population there were 112 triplet pregnancies with the icd-10 (international classification of diseases) code o30.1 (triplet pregnancy) at any time during pregnancy and/or the term ‘birth of three’ in the swedish pregnancy register (figure 1). six of those pregnancies were confirmed triplets by ultrasound early in pregnancy, but already at later ultrasound examinations before ga 22+0 weeks, less than three fetuses were registered. as we did not know the underlying cause for this, and with the intention to study only ongoing triplet pregnancies confirmed at ga ≥22+0 weeks, these six pregnancies were excluded. thus, 106 triplet pregnancies formed the expectantly managed cohort. figure 1. flow diagram of the study protocol. outcomes in triplet pregnancies in sweden 3 for the reduced cohort, data on all triplet pregnancies where fetal reduction had been performed before ga 22+0 weeks during the study period were identified from the fetal therapy register (n = 21). six pregnancies, where fetal reduction had been performed due to fetal indications such as twin-to-twin transfusion syndrome or fetal malformation, and two pregnancies with miscarriage before ga 22+0 weeks after fetal reduction were excluded from the cohort (figure 1). thus, only ongoing pregnancies at ga ≥22+0 weeks after fetal reduction were included. the final reduced cohort consisted of 13 pregnancies. the sample selection is illustrated as a flowchart in figure 1. maternal outcomes obtained from standardized delivery records were the mode of delivery and estimated blood loss.  further, we analyzed blood transfusion, preeclampsia, and significant pregnancy complications presented as a composite variable including maternal blood loss in need of transfusion, placental abruption, preeclampsia, gestational hypertension, gestational diabetes, and liver disease during  pregnancy. icd-10 diagnostic codes and procedure codes  used to identify these outcomes are presented in supplementary table 1. pregnancy outcomes were stillbirth, ga at birth, birth weight, and small for gestational age (sga; defined as a birthweight more than two sds below the mean in a swedish reference for normal fetal growth) [20]. neonatal outcomes included neonatal mortality and the incidence of severe neonatal morbidity, analyzed as a composite variable which included intraventricular hemorrhage (ivh) grade 3 and 4, sepsis, necrotizing enterocolitis (nec), bronchopulmonary dysplasia (bpd), and/or treated retinopathy of prematurity (rop). other outcomes were: apgar at 5 min, neonatal resuscitation at birth, umbilical artery ph, and admission to the neonatal care unit. in snq, the definition of neonatal resuscitation included the need of mask ventilation, administration of continuous positive airway pressure (cpap), intubation, cardiac compressions, correction of acidosis, or administration of epinephrine. as a core outcome set for multiple pregnancies according to the core outcomes in women’s health (crown) initiative has not been established yet, we chose important outcomes that have been reported in previous studies on multifetal pregnancies (22). statistical analyses descriptive statistics were applied to present outcome variables in the cohorts. outcomes were presented as numbers and percentages, mean ± standard deviation (sd), or median and interquartile range (iqr). chi-square and fisher’s exact test were applied to analyze differences in categorical data. mannwhitney u-test was carried out for the comparison of continuous, table 1. maternal characteristics in the expectantly managed and the reduced cohort, respectively. maternal characteristics expectantly managed cohort n = 106 pregnancies reduced cohort n = 13 pregnancies p age (years), mean ± sd 33 ± 6 34 ± 4 0.41a data missing, n (%) 0 1 (7.7) bmi, kg/m2, mean ± sd 26 ± 5 23 ± 4 0.046a data missing, n (%) 0 3 (23.1) educational level, y, n (%) ≤ 9 7 (6.6) 0 (0) 0.19b 10–12 32 (30.2) 2 (15.4) ≥12 41 (38.7) 9 (69.2) data missing, n (%) 26 (24.5) 2 (15.4) country of birth, n (%) sweden 53 (50.0) 7 (53.8) 0.97b non-sweden 36 (34.0) 4 (30.8) data missing, n (%) 17 (16.0) 2 (15.4) nulliparous, n (%) 47 (44.3) 6 (46.1) 0.98b data missing, n (%) 5 (4.7) 0 art, n (%) 33 (31.1)d 8 (61.5)e < 0.01b data missing, n (%) 16 (15.1) 3 (23.1) smoking, early pregnancy, n (%) 4 (3.8) 0 1.0c data missing, n (%) 19 (17.9) 2 (15.4) any significant pre-existing maternal disease, n (%)f 6 (5.7) 2 (15.4) 0.23c data missing, n (%) 13 (12.3) 1 (7.7) sd: standard deviation; bmi: body mass index; art: assisted reproductive technology; ivf: in vitro fertilization. acalculated with mann whitney u-test. bcalculated with chi-square test. ccalculated with fisher’s exact test. dovulation stimulation n = 13; ivf n = 20. eovulation stimulation n = 4; ivf n = 4. fcardiovascular disease, pre-gestational diabetes, history of thrombosis, sle, kidney disease or chronic hypertension; according to check-boxes filled out at first antenatal visit. 4 m.-m. ekström et al. non-parametric data, between the two cohorts. the statistical software ibm spss statistics 25 was used for all statistical analyses. a p-value <0.05 was considered to be statistically significant. ethical approval ethical permission for this study was given by the swedish ethical review authority with registration number: 2019-05160. results in the expectantly managed cohort of 318 fetuses, six infants were stillborn (figure 2). the reduced cohort consisted of 13 pregnancies with initially 39 fetuses before fetal reduction. of those, the number of fetuses was reduced from three to two in nine pregnancies, and from three to one in four pregnancies, respectively. all 22 infants were liveborn (figure 2). maternal age, parity, smoking in early pregnancy, level of education, country of birth, and pre-pregnancy comorbidity were not significantly different between women with expectantly managed and reduced triplet pregnancies (table 1). in contrast, the utilization of art was significantly less common in the expectantly managed (31% [33/106]) than in the reduced cohort (62% [8/13], p < 0.01). moreover, maternal bmi in early pregnancy was slightly higher in expectantly managed (26 ± 5 kg/m2) compared to reduced triplet pregnancies (23 ± 4 kg/m2; p = 0.046). pregnancy and maternal outcomes are presented in table 2. in the expectantly managed cohort, stillbirth of at least one infant occurred in three pregnancies. there was one pregnancy each with three, two and one stillborn fetus(es), respectively. almost all women delivered by cesarean section (97% [103/106]). the median estimated blood loss was 700 ml (iqr 500–1090 ml), and 15% (16/106) of the women received a blood transfusion. nine percent (9/106) of the women suffered from preeclampsia, and 33% (35/106) suffered from significant pregnancy complications. in contrast, in the reduced cohort, no infant was stillborn (p = 1.00), a lower proportion of women was delivered by cesarean section (31% [4/13], p < 0.01), and the median estimated blood loss was lower (440 ml [iqr 333–813 ml], p = 0.02). the proportion of pregnancy complications was lower than in the expectantly managed cohort, but without reaching statistical significance (7.7%, p = 0.11).there were no cases of maternal venous or pulmonary embolism and no cases of maternal death during pregnancy or the postnatal period in table 2. maternal and pregnancy outcomes in the expectantly managed and the reduced cohort, per pregnancy. outcome per pregnancy expectantly managed cohort n = 106 pregnancies reduced cohort n = 13 pregnancies p pregnancies with stillbirth of at least one infant, n (%) 3 (2.8) 0 1.00a cesarean section, n (%) 103 (97.2) 4 (31) < 0.01a total postpartum hemorrhage (ml), median (iqr) 700 (500–1,090) 440 (333–813) 0.02b data missing, n 3 (2.8) 5 (38.5) blood transfusion to mother, n (%) 16 (15.1) 0 0.21a preeclampsia, n (%) 10 (9.4) 1 (7.7) 1.00a any significant pregnancy complicationc, n (%) 35 (33.0) 1 (7.7) 0.11a iqr: interquartile range. acalculated with fisher’s exact test. bcalculated with mann-whitney u test. cmaternal blood loss in need of blood transfusion, placental abruption, preeclampsia, gestational hypertension, gestational diabetes, liver disorder in pregnancy. figure 2. number of pregnancies, fetuses and liveand stillbirths in expectantly managed and reduced triplet pregnancies. expectantly managed cohort n = 106 pregnancies live birth n = 312 infants stillbirth n = 6 infants reduced from 3 to 2 n = 9 pregnancies live birth n = 18 infants live birth n = 4 infants reduced from 3 to 1 n = 4 pregnancies reduced cohort n = 13 pregnancies outcomes in triplet pregnancies in sweden 5 either group. overall, consideration should be given to the very small number of pregnancies in the reduced cohort. pregnancy and neonatal outcomes for liveborn infants (n = 312) are presented in table 3. in the expectantly managed cohort, the mean ga at birth was 32+3 weeks (min – max: 22+0 – 36+1 weeks). six percent (17/312) of the infants were born before 28+0 weeks of gestation. the mean birthweight was 1,726 ± 474 g, and 34% (106/312) were born sga. nine percent (29/312) had an apgar score <7 at 5 min, and 92% (287/312) of the infants were admitted to neonatal care, where the median length of stay was 21 days (iqr 14–37 days). sixty-three percent (196/312) of the infants needed resuscitation at birth, and 9% (29/312) suffered from severe neonatal morbidity. four percent (12/312) of the infants died during the first 28 days of life, whereof 10 during the early neonatal period (0–7 days). seven of the 12 infants who died had been born extremely preterm (22+0–26+1 weeks), two other infants had lethal malformations, and the remaining three had been born between 28 and 32 weeks of gestation (whereof two with extreme fetal growth restriction). in the reduced cohort – compared to the expectantly managed cohort – mean ga at birth was significantly higher (36+0 weeks [min – max: 32+2 – 40+0 weeks], p < 0.01) as was the mean birthweight (2,444 ± 631 g, p < 0.01), but there was no significant difference in the proportion of sga births (18%; p = 0.32). fewer infants were admitted to neonatal care (32%; n < 0.01), with a shorter median stay (14 days [iqr 8–16]; p < 0.01). fewer infants needed resuscitation at birth (18%; p < 0.01), and none suffered from severe neonatal morbidity (p = 0.24). all infants were alive at 28 days of life (p = 1.0). discussion in a country with excellent access to high-quality maternity, delivery, and neonatal care, all infants in a nationwide cohort of expectantly managed triplet pregnancies were born preterm, whereof 6% extremely and 23% very preterm (22+0 – 27+6 weeks and 28+0 – 31+6 weeks of gestation, respectively). perinatal survival rates were high, with 98% being liveborn, and 96% of liveborn infants being alive at 28 days of life. nine out of 10 infants did not suffer from severe neonatal morbidity. fetal reduction was performed in only a very small number of cases. in the reduced cohort, no infants were born extremely or very table 3. pregnancy and neonatal outcomes in the expectantly managed and the reduced cohort, per liveborn infant. outcome per liveborn expectantly managed cohort n = 312 liveborn infants reduced cohort n = 22 liveborn infants p gestational age at birth mean (± sd), days 227 ± 17.4 252 ± 17.8 <0.01a median (min – max), weeks + days 33 + 1 (22+0 – 36 +1) 36 +6 (32+2 – 40+0) weeks, n (%) 22+0 to 27+6 17 (5.4) 0 28+0 to 31+6 73 (23.4) 0 32+0 to 36+6 222 (71.2) 12 (54.5) ≥37+0 0 10 (45.5) data missing, n (%) 0 0 birth weight (grams) mean ± sd 1,726 ± 474 2,444 ± 631 <0.01a data missing, n (%) 2 (0.6) 1 (4.5) small for gestational age, n (%) 106 (34.0) 4 (18.2) 0.32b data missing, n (%) 2 (0.6) 3 (13.6) boys, n (%) 168 (53.8) 14 (63.6) 0.37c data missing, n (%) 0 2 (9.1) apgar score at 5 min, n (%) <4 9 (2.9) 0 0.06c <7 20 (6.4) 0 data missing, n (%) 11 (3.5) 4 (18.2) resuscitation at birth (mask or cpap ventilation, intubation, heart massage, correction of acidosis, epinephrine), n (%) 196 (62.8) 4 (18.2) <0.01b admission for neonatal care, n (%) 287 (92.0) 7 (31.8) <0.01c length of stay at neonatal care unit in days (of those admitted), median (iqr) 21 (14–37) 14 (8–16) <0.01a any severe neonatal morbidity (bpd, nec, ivh 3 and 4, sepsis and/or treated rop), n (%) 29 (9.3) 0 0.24c alive at 28 days of life, n (%) 300 (96) 22 (100) 1.00b small for gestational age: a birth-weight z score more than 2 sds below the mean in a swedish reference for normal fetal growth. na: not applicable; cpap: continuous positive airway pressure; iqr: interquartile range; bpd: bronchopulmonary dysplasia; nec: necrotizing enterocolitis; ivh: intraventricular hemorrhage; rop: retinopathy of prematurity. a calculated with mann-whitney u test. b calculated with fisher’s exact test. c calculated with chi-square test. 6 m.-m. ekström et al. preterm, and mean birthweight and gestational age at birth were significantly higher compared to the expectantly managed cohort. there were no cases of severe neonatal morbidity or mortality and only one case of significant maternal pregnancy complication. however, consideration should be given to the very small number of pregnancies in the reduced cohort. the proportion of pregnancies conceived by art was high in the expectantly managed cohort (31%). this is in sharp contrast to the overall proportion of pregnancies conceived by art in deliveries in sweden, which was 3.8% during the years 2014–2019 (23). despite the routine of single embryo transfer in the case of ivf in sweden, multiple pregnancies still occur (24). further, women can travel abroad for art procedures with multiple embryo transfer. the proportion of pregnancies conceived by art was significantly higher in the reduced cohort (62%), which is in line with previous studies, also showing that women who had a fetal reduction were substantially more likely to have used art than were those who did not undergo fetal reduction (25, 26). in the expectantly managed cohort, the proportion of cesarean sections was high (97%), which is in line with international guidelines, suggesting a planned cesarean section at 35 weeks of gestation in uncomplicated triplet pregnancies (27). a high proportion of women suffered from significant pregnancy complications (33%), which is comparable to other studies (7). in the reduced cohort, there was a significantly lower proportion of cesarean sections, and a non-significant lower proportion of maternal pregnancy complications probably due to the small sample size, which is expected and also in line with earlier studies (28). it might be considered controversial to test for a group difference concerning the proportion of cesarean sections, as it can be confounding by indication. however, to give birth vaginally or by cesarean is important from a clinical perspective, and thus, data are shown. in contrast to our study, most other studies report deliveries from 24+0 rather than 22+0 weeks of gestation. nevertheless, the rate of stillbirth and neonatal death in expectantly managed triplets in our study (2 and 4%, respectively) is comparable to or even slightly lower than in other studies. curado et al. found an incidence of stillbirth of 2% in trichorionic triamniotic (tcta) and 5% in dichorionic triamniotic (dcta) triplets (due to mainly twin to twin transfusion syndrome, ttts) (29). the rate of neonatal death has earlier been described to be twice that of stillbirth in both dcta and tcta triplets, which was also in line with our findings (29). while the rate of stillbirth is higher in triplets compared to singletons, several studies have found that neonatal morbidity and mortality in triplets were comparable to matched twins and singletons, highlighting the significance of prematurity and birthweight for these outcomes (15, 30, 31). there were no significant differences in the proportion of severe neonatal morbidity or neonatal mortality between the expectantly managed and the reduced cohort. however, the non-significance is most probably due to the small number of neonates in the reduced cohort. a main strength of the study is the nationwide design. the 106 triplet pregnancies in the expectantly managed cohort cover 84% of all triplet pregnancies ≥ 22+0 weeks of gestation when compared to data from statistics sweden during the years 2014–2019 (5). the coverage rate for transfer of data from the electronic medical records to the swedish pregnancy register during the study period was 91%, which may explain the main part of the difference. to be mentioned as a second strength, also data on fetal reduction from the fetal therapy register can be considered as nationwide. thus, this study provides combined data from three registers with prospectively registered information. the coverage rates of the registers are exceptionally high, with high quality of data. firstly, the significant limitation in our study is the lack of information on chorionicity. secondly, it was not possible to follow triplet pregnancies including miscarriages from early gestation through registers in sweden, as a first trimester ultrasound is not offered to all pregnant women. in contrast, it was possible to identify basically all triplet pregnancies with a delivery ≥ 22+0 weeks of gestation in the country. thirdly, the number of pregnancies in the reduced cohort was small. triplet pregnancies are rare events, and fetal reductions with the only indication to reduce the number of fetuses are even more rare, especially in a country where single embryo transfer is routine in the case of ivf. however, the study population represents almost the whole swedish population of expectantly managed and reduced triplets, delivered ≥ 22+0 weeks of gestation. thus, that fetal reduction with the only indication to reduce the number of fetuses was a rare event in our study (and thus in sweden) is an important finding itself. fourthly, data on some outcomes were missing to a higher extent in the reduced compared to the expectantly managed cohort, as for example apgar score at 5 min of age (missing in 4/22 [18.2%] and 11/312 [3.5%] of infants, respectively), making comparisons between the cohorts more difficult. according to statistics sweden, the proportion of triplet deliveries ≥ 22+0 weeks of gestation was 0.018% during the years 2014–2019 in sweden (5). the proportion of triplet pregnancies in our study was 0.017%. in contrast, although declining from the 1998 peak with a proportion of 0.19% in the us, the proportion of triplet deliveries in the us was still almost fivefold that in sweden in 2019 (0.084%) (32). preventing triplet pregnancies by a routine of single embryo transfer in case of ivf or intracytoplasmic sperm injection (icsi) and by careful monitoring in case of ovarian stimulation is the method of choice to reduce maternal and infant morbidity and mortality. however, triplet pregnancies will always occur to some proportion, and knowledge about management and outcomes is important. with the limitation that we do not have any data about miscarriage rates before ga 22+0 weeks in expectantly managed triplets in our cohort, our data regarding triplets born ga ≥ 22+0 weeks are in line with previous studies (15, 26, 28, 33). when the top priority is liveborn infants, expectant management seems to be the best choice. when the priority is to minimize prematurity – and probably also neonatal and long-term morbidity – the most advisable option seems to be fetal reduction. women (and partners) with a triplet pregnancy ultimately must make decisions with potentially lifelong consequences, and careful counseling is of utmost importance (28). outcomes in triplet pregnancies in sweden 7 conclusion while overall neonatal survival from 22+0 weeks of gestation in expectantly managed triplet pregnancies in sweden was high, all infants were born preterm. every tenth infant and every third mother suffered from severe neonatal morbidity or pregnancy complications, respectively. fetal reduction was performed in only a very small number of cases and was associated with higher gestational age at birth and higher birth weight. in an international context, rates of multifetal births are very low in sweden. funding mn was supported by grants from a regional agreement on clinical research (alf) between region stockholm and karolinska institutet (2020-0443) and the childhood foundation of the swedish order of freemasons. os was supported by the swedish research council (2019-02175). acknowledgments we are grateful for the support from the swedish pregnancy register and snq. notes on contributors statement mg and et conceived and designed the study together with mn and os. mme, et, and mg performed the analyses. mme and mg wrote the paper. all authors 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according to chorionicity: systematic review and meta-analysis. ultrasound obstet gynecol 2019;54(5):589–95. doi: 10.1002/uog.20209 30. shah ps, kusuda s, håkansson s, reichman b, lui k, lehtonen l, et al. neonatal outcomes of very preterm or very low birth weight triplets. pediatrics 2018;142(6):e20181938. doi: 10.1542/peds.2018-1938 31. mitsiakos g, gialamprinou d, chatziioannidis i, pouliakis a, kontovazainitis cg, chatzigrigoriou f, et al. are neonatal outcomes of triplet pregnancies different from those of singletons according to gestational age? j perinat med 2021;49(9):1145–53. doi: 10.1515/ jpm-2020-0558 32. martin ja, hamilton be, osterman mjk, driscoll ak. births: final data for 2019. natl vital stat rep 2021;70(2):1–51. 33. shiva m, mohammadi yeganeh l, mirzaagha e, chehrazi m, bagheri lankarani n. comparison of the outcomes between reduced and nonreduced triplet pregnancies achieved by assisted reproductive technology. aust n z j obstet gynaecol 2014;54(5):424–7. doi: 10.1111/ ajo.12225 https://doi.org/10.1093/hropen/hoaa032 https://doi.org/10.1093/hropen/hoaa032 https://doi.org/10.1503/cmaj.160722 https://doi.org/10.1111/1471-0528.13348 https://doi.org/10.1002/pd.5852 https://doi.org/10.1002/uog.20209 https://doi.org/10.1542/peds.2018-1938 https://doi.org/10.1515/jpm-2020-0558 https://doi.org/10.1515/jpm-2020-0558 https://doi.org/10.1111/ajo.12225 https://doi.org/10.1111/ajo.12225 upsala j med sci 98: 323-330, 1993 6.1 .1.7 quality specifications in the clinical laboratory: how good are our methods, and what are they good for? peter nilsson-ehle department of clinical chemistry university hospital, lund the technical performance of laboratory techniques can be clearly defined and docu mented in terms of precision, accuracy, detection limits etc. at present, much interest focuses on how to define clinical requirements for assay performance in order to obtain an optimal combination of analytical quality, accessibility, simplicity, speed of operation and economy in our laboratories (e g 1, 2). from such considerations it is possible to describe how good our methods are. however, quality specifications in a broader sense also require that we define and explain to our customers what our analyses are good for, and how they may, or may not, be helpful in various clinical settings (fig 1). clinical b-\ needs clinical perf o r m an ce predictive values etc. technical performance precision accuracy etc. figure 1. interactions between clinical specialities and the laboratory in defining analytical quality specifications and assessing the clinical usefulness of laboratory measurements. 323 lack of such information does not necessarily mean that the analyses in question loose their market or popularity. rather, the risk is that the clinical use of a method is influenced or even governed by information or concepts which are not relevant for or directly applicable in medical practice. the "cholesterol debate" provides us with a recent example. different perspectives of medical problems for health authorities, health economists and colleagues engaged in occupational medicine and pharmaceutical industries, epidemiological concepts such as (biochemical) risk factors, risk ratios and relative risk are crucial to describe the health situation in a population and analyze needs for activities or intervention on the national, community or local level. for those engaged in medical practice, however, these concepts offer little guidance in the assessment of the individual patient's situation and treatment. in this situation, the ability of laboratory tests to differentiate between normal and abnormal, to predict prognosis, to monitor the development of disease and to document the effects of treatment are of prime importance. thus, information related to biological variation, diagnostic sensitivity and specificity and, especially, the predictive values of laboratory data is in focus. if these different perspectives are not recognized, laboratory services may be used ineffi ciently. a n important didactic task for clinical laboratories is to provide easily accessible, clear background information to illustrate the usefulness of various analyses in different clinical situations, i e what different measurements are good for. the cholesterol debate during the last decade, intervention programmes of various designs (including general screening programmes for plasma cholesterol) have been advocated to lower plasma cholesterol levels in the population in order to remedy the high incidence of cardiovascular disease in western countries. in general terms, the arguments for such programmes have been that the plasma cholesterol level is a major risk factor for cardiovascular disease, that cholesterol concentrations can be lowered by non pharmaceutical or pharmaceutical means, and that such lipid lowering does indeed prevent manifestations of atherosclerosis. let us scrutinize the underlying information. 324 the relationship between plasma cholesterol and cardiovascular disease there is ample documentation that the plasma cholesterol level is one of the three major risk factors for premature cardiovascular disease, as is frequently demonstrated in diagrams of relative risk vs. cholesterol concentrations (fig 2 a). nevertheless, less than 50% of myocardial infarctions in sweden occur in subjects with plasma cholesterol levels > 6,s mmol/l (fig 2 b), the limit recommended for active intervention by authorities in most countries (4,5). thus, screening for and successful intervention against s-cholesterol concentrations above 6,5 mmol/l may at best prevent a minority of cases of e g myocardial infarction. when a variable expressing absolute rather than relative risk is introduced, the impact of isolated hypercholesterolemia on cardiovascular disease is considerably less impressive. fig 2 c illustrates "cardiovascular survival" vs. plasma cholesterol concentrations. its message is that most middle-aged men, even those with moderately elevated cholesterol levels, wih not suffer from cardiovascular disease during the next 5 10 years. expressed in other terms, the incidence of cardiovascular manifestations is only about 5 % over the observation period. consequently, the predictive value of elevated s-cholesterol concentrations for cardiovascular disease is extremely low. ' 490 630 830 cholesterol conc (mmol/i) figure 2a. 325 cholesterol conc (mmolll) figure 2b. "cardiovascular health" 490 690 830 cholesterol conc (mmolll) figure 2c. figure 2. schematic representations of the relationships between plasma cholesterol levels and cardiovascular disease expressed as (a) relative risk vs cholesterol concentrations, (b) incidence of myocardial infarctions in cholesterol concentration intervals (bars) as compared to the frequency distribution of cholesterol concentrations in the general population, and (c) percentage of middle-aged men with moderate hypercholesterolemia who did not develop serious cardiovascular symptoms during a 5 year follow up. figs 3 a and b are modified from ref 3 and 5, and fig 3 c is based on data from refs 3 and 6 . effects of lipid-lowering therapy a number of large-scale intervention studies have demonstrated that efficient lipid lowering therapy is indeed associated with a lowered incidence of atherosclerotic manifes tations. the results are generally expressed as a 20 40% reduction of cardiovascular 326 events. fig 3 shows schematically two popular, and strongly suggestive, illustrations of the effects of intervention. cumulative incidence incidence per 1000 person-years 10 v) c a > a > 0 0 p 3 z c. 5 c & e time (years) drug placebo figure 3. schematic representations of the effects of intervention on cardiovascular morbidity and mortality in middle-aged men with hypercholesterolemia. based on data from ref 6. again, however, presentation of the overall results may convey a different message. fig 4 a shows 100 middle-aged men with elevated cholesterol levels. given the incidence of cardiovascular manifestations in this particular group, five of them will suffer from a cardiovascular event over an observation period of 5 years (fig 4 b). if all 100 men are treated with an efficient lipid-lowering drug (fig 4 c), cardiovascular events will be registered in tree subjects. thus, the program has resulted in the prevention of cardiovascular events in two subjects (40% therapeutic efficacy). the therapeutic gain, consequently, is around one case per 250 treatment years. 327 6 . in a couple of these studies there has been an excess mortality from apparently unrelated causes such as suicide, accidents etc. although it has been suggested that this may represent a side effect of cholesterol lowering, the general pattern (relation to dose, time frame etc) is not typical of a traditional drug side effect. the clinical situation the individual patient's attitude and inclination to adopt a specific therapy is largely governed by the information and advice given by his physician. the message of elevated s-cholesterol to a middle-aged man with isolated, moderate hypercholesterolemia can, depending upon which perspective is in focus, be phrased in two ways: 0 "your cholesterol is elevated, and you r u n a 3 4 fold increased risk to develop a cardiac condition. with diet and drugs, we can reduce that risk by almost 50%". "your cholesterol is elevated, but your chances to escape cardiac trouble over the next 5 10 years is still about 95%. with diet and drugs, those chances will increase to 97%. hopefully, there will be no side effects of the treatment". 0 although neither is incorrect, it is quite conceivable that these messages will result in different schemes of management. conclusions technical specifications of the performance of chemical analyses and documentation of the different sources of variation are basic components of quality assurance programmes. information on the usefulness of laboratory data in different clinical settings is another important aspect of providing laboratory services of high quality. predictive values of positive and negative tests are informative, but may be difficult to use efficiently in our relation with clinicians. new efforts and initiatives in this area would promote the rational use of laboratory resources. these considerations are especially important as the technical development now allows a large number of analyses to be performed outside the major hospital laboratories. some possibilities to simplify and clarify available information in order to make it relevant 329 for the typical clinical situation have been illustrated using plasma cholesterol measurements as an example. the analysis demonstrates that screening programmes in the general population would be costly and ineffective mainly because the incidence of cardiovascular events in subjects with isolated, moderate hypercholesterolemia is low. it should be pointed out that determination of cholesterol (and hdl and triglycerides) is recommended in subjects presenting additional risk factors such as genetic disposition, smoking, hypertension, obesity, diabetes etc. in these cases, the overall relative risk may be increased 30-fold, and the incidence of cardiovascular disease is high enough to warrant an integrated assessment of the risk profile as a base for intervention. references 1. 2. 3. 4. 5. 6. svensk forening for klinisk kemi. att mata kolesterol en handbok for mottagningar och laboratoriepersonal. almqvist och wikseli, uppsala 1990. nilsson-ehle, p., nordin, g., nilsson, j.e. & tryding, n. kolesterol svirare att mata an att sanka? ukartidningen 1989;86:1263 1269. martin, m.j., hulley, s.b., browner, w.s., kuller, l.h. & wentworth, d. serum cholesterol, blood pressure and mortality: implications from a cohort of 361 662 men. lancet 1981;11:933-936. socialstyrelsens kommittk for lakemedelsinformation. behandling av hyperlipidemi. information frdn socialstyrelsens lakemedelsavdelning 1988;5: 152-157. hjart-lungfonden: riktlinjer for behandling av hyperkolesterolemi. svenskt tryck, stockholm, 1988. frick, m.h., elo, o., haapa, k. et al. helsinki heart study: primary prevention trial with gemfibrozil in middle-aged men with dyslipoproteinemia. new engl j med 1987;3 17: 1237-1245. correspondence: peter nilsson-ehle department of clinical chemistry university hospital s-221 85 lund, sweden 330 upsala j med sci 79: 18-20, 1974 effect of an enzyme-resistant phosphopeptide on calcification of embryo chicken bone in vitro conny edeno department of pathology i and department of medical chemistry, university of goteborg, sweden abstract cultivation of embryonic chicken bone in vitro enables substances to be added to the culture medium in order to ascertain how they affect the histological development of the bone. this method has been adopted for studying an enzyme-resistant phospbopeptide extracted by mel lander from casein. by cultivating paired bones that from one side can be used as a control for the contra lateral bone. the test group was given calcium complexly bound to the phosphopeptide and the control group calcium as caci,. studies of bones from embryos of different incubation ages after cultivation for various periods in media containing different concentration of calcium revealed that similar degress of ossification and rates of osteoid tissue formation were achieved when the phospbopeptide was the source of calcium as when it was caci,. these experiments have demonstrated that calcium bound to a phosphopeptide can be utilized in the ossifcation process just as well as readily soluble inorganic calcium. it has long been known that phosphoproteins are present in such products a s milk-there in the form of casein-and that considerable amounts of phosphorylated peptides are formed when these proteins a r e hydrolyzed in the human intestinal tract (1). using experimental animals with rickets fed special diets, mellander discovered an anti rachitic effect of such phosphorylated peptides ( 2 ) . this effect has been interpreted a s the result of increased intestinal absorption of calcium com plexly bound to phosphorylated peptides ( 3 ) . we have in the present investigation studied their direct action on the ossification of embryonic chicken bone in an in v i t r o system. most previous trials with bone cultivation have been done with chicken bone. t h e y have revealed that bone from 6-day chick embryos incubated a t 36°c c a n be explanted, that histologically they continue t o differentiate normally, and that they upsala j med sci 79 exhibit considerable longitudinal growth (4). bone tissue from 6-day chick embryos have also been used for studying the effects in v i t r o of various hormones on longitudinal growth and ossification (5, 6 , 7, 8, 9, 10, 11, 12, 13). such experiments, all using a mixture of plasma and chicken embryo extract a s culture medium, have disclosed that both longitudinal growth and ossification are significantly affected by insulin, thyroxin, cortisone, vitamin a and parathyroid hormone. embryo chicken bone, incubated at 36", de velop in v i v o from commencing differentiation a t 6 days t o a bone with well developed marrow cavity at 12 to 16 days of age (14). pilot studies in this laboratory o n embryonic chick tibiae aged 6 t o 17 d a y s and cultivated for 1 to 15 days demonstraeted that 10 to 14 days was a suitable age for studying ossification and the formation of osteoid tissue. t e c h n i q u e s dissection was carried out with a cataract knife and tweezers under a dissection microscope. all bones were measured before and after cultivation by means of trowell's grid technique (15). according to franks, the ambient gas phase may well be air for cultivation of embryonic organs, so we chose air (16). the medium was .eagle's minimum essential medium for spinner cul tures>> with 10% calf serum (17). to avoid cells attaching themselves to the grid, we placed the bones on milli pore paper which accompanied them to fixation. the specimens were fixed i n 10% formalin neutralized with calcium, dehydrated, embedded in paraffine, sectioned longitudinally in 5 to 10 slices, and stained with haema toxylin-eosine according to van gieson and von kossa. first the tibia was dissected out from 6 embryos in each age group of 10, 12 and 14 days. thus each embryo yielded 2 bones, one of which was cultivated in medium plus calcium chloride and the other in medium plus phosphopeptide. caseinphosphopeptide-effect on chicken bone in vitro i9 the amounts of cacl, and phosphopeptide were such as to yield concentrations of calcium of 100 mg/ litre. two of the 6 embryos in each age group were for 5 , 10 and 15 days respectively. results m e specimens were examined after staining and judged on the basis of the degree of ossification and the amount of osteoid tissue. both variables were classified according to a scale from 0 to 3 , where 0 implies no ossification or osteoid tissue and 3 denotes marked ossification and plenty of osteoid tissue respectively. with respect to both controls and experimental bones it appeared that: (i) both ossification and osteoid tissue were equal to 3 in the group incubated for 14 days regardless of how long they were cultivated; (ii) both ossification and osteoid tissue were rated 0.5 in the group incubated for 10 days, regardless of how long they were cultivated; and (iii) osteoid tissue rated 2.5 in the experimental group and 2.7 in the controls while ossification was 3 in the experimental group and 2 in the controls among embryos incubated for 12 days. the differences observed between experimental and control groups were confined to those embryos incubated for 10 days and cultivated for 10 days and to those incubated for 12 and cultivated for 5 days. embryos 10 to 12 days old are evidently in a critical state with respect to ossification and formation of osteoid tissue. hence we examined 20 additional embryos incubated for 10 days and cultivated for 6 and 8 days, as described above. the results were as follows: it will be seen that no differences in amount of osteoid tissue and degree of ossification could be demonstrated between embryonic chicken bones cultivated in a medium containing 100 mg calcium per litre in the form of cacl, and similar cul tures in a medium containing calcium complexly bound to a phosphopeptide. incub. cultiv no. of time, time ca source bones days days cacl, 10 10 6 phospho-peptide 10 10 6 cacl, 10 10 8 i phospho-peptide 10 10 8 to exclude the possibility that a concentration of 100 mg of calcium per litre was too high to allow any differences to appear, we carried out another experiment in which the calcium con centration was reduced to 25 mg per litre. for this latter experiment we chose embryos incubated for 9 days and cultivated them for 8 days in the same way as before. it turned out that longitudinal growth in the experimental group was 1 . 1 t o . 5 mm and 1.35 0.6 mm in the control group. the amount of osteoid tissue in the two groups was 2.2 and 2.3 respec tively. no bone exhibited any calcification. consequently not even a calcium concentration which is extremely low for tissue cultivation pur poses could expose any differences between the phosphopeptide and calcium chloride in either ossification or the rate of osteoid tissue formation in embryonic chicken bone. references 1. 2 . 3. 4 . 5 . 6 . 7. mellander, 0. & folsch, g.: enzyme resistance and metal binding of phosphorylated peptides. i. e. f. n., vol. 1 1 , p. 569 (ed. e. j . bigwood). pergamon press, oxford and new york, 1972. mellander, i. & olsson, n.: the influence of pep tide bound calcium and phosphorous on bone cal cification in rickets. numero extraordinario del bolitin medico del hospital infantil, vol. xiii, mellander, 0.: nutritional factors (other than vita min d) influencing the intestinal absorption of calcium and strontium. transfer of calcium and strontium across biological membranes, section v. academic press, new york, 1963. fell, h . b. & robisson, r.: the growth, development and phosphatase activity of embryonic avian femora and limb-buds cultivated in vitro. biochem j 23: 765, 1929. chen, j . m.: the effect of insulin on embryonic limb bones cultivated in vitro. j physioll25: 148, 1954. hay, m. f.: the effect of growth hormone and insulin on limb-bone rudiments of the embryonic chick cultivated in vitro, j physiol 144: 490, 1958. zwilling, e.: micrornelia as a direct effect of insulin. evidence from in vitro and in vivo experiments. j morph 104: 159, 1959. 243-246, 1956. growth, osteoid ossifi mm tissue cation 1.0k0.4 2.9 2.9 1.420.4 2.9 2.9 0.9k0.4 2.9 2.9 1.0k0.5 2.9 2.9 upsala j med sci 79 20 conny edeno 8. buno, w. & goyena, h.: effect of cortisone on growth in vitro of femur of chick embryo. proc soc exp biol med89: 622, 1955. 9. sobel. h . & freund, 0.: the action ofcortisone on the embryonic cartilage and muscle in vorro. experi entia14:421, 1958. 10. fell, h. b. & mellanby, r.: the biological action of thyroxine on embryonic bones grown in tissue culture. j physiol127: 427, 1955. 11. fell. h. b. & thomas, l.: the influence of hydro cortisone on the action of excess vitamin a on limb bone rudiments in culture. j e x p med: 114: 343 1961. 12. lawson, k.: 11. growth rate. j embryo1 morph 9: 42, 1961. 13. gaillard, p. j . : parathyroid gland tissue and bone in v i f r o . 11. koninkl nederl akad wetenschap proc c 58: 579, 1955. 14. fell, h. b. & robison, r.: the development of the calcifying mechanism in avian cartilage and osteoid tissue. biochem j 28: no. 6,2243 15. trowell, c . a , : a modified technique for organ culture in v i f r o . exp cell res6: 246, 1954. 16. franks, l. m.: a factor in normal human serum that inhibits epithelial growth in organ cultures. e x p cell res 17: 579, 1959. 17. eagel, h.: science 130: 432, 1959. received june 36. 1 573 address for reprints: conny edeno department of pathology i university of goteborg s-413 45 goteborg sweden upsala j med sci 79 upsala j med sci 97: 157-167 effects of omeprazole and ranitidine on plasma gastrin concentration and stomach gastrin content in rats rein seensalu,' kinfe girma,2 bengt romel12 and goran nilsson2 'division of gastroenterology & hepatology, department of medicine and department of surgery and research center, karolinska institute, huddinge university hospital, huddinge and 2department of physiology, faculty of veterinary medicine, university of agricultural sciences, uppsala, sweden abstract prolonged fasting and longer time between dosing and sampling reduced the plasma gastrin concentrations after omeprazole (80 pmol/kg x 2 for 14 days) treatment in male rats whereas the amounts of tissue gastrin were essentially unchanged during these initial experiments. after 28 days omeprazole (80 pmol/kg x 2) or ranitidine (375 pmol/kg x 4) that produced corresponding inhibition of acid secretion, increased the tissue gastrin content by 114 and 59 %. a low dose of omeprazole (20 pmol/kg x 2) also raised the gastric gastrin content (41 %), whereas no change was noted on treatment with a low dose of ranitidine (125 pmol x 4). following recovery for 28 days no significant increases in gastrin were observed. 1, 3, 7, 14 or 28 days of treatment with omeprazole (80 pmol/kg x 2) gradually increased the gastric gastrin content beeing significantly raised already after 3 days. we conclude that a) measuring the tissue gastrin content may be the preferable method when changes in gastrin following long-term treatment with acid inhibiting drugs are to be determined, b) the amount of gastrin in the stomach increases rapidly following treatment with omeprazole and is approximately doubled following 28 days of treatment and c) after treatment for 28 days omeprazole was found to cause greater elevations in the tissue gastrin content than ranitidine despite similar degrees of basal acid inhibition. introduction it is well known from animal and human studies that a reduction of the acid output will increase the plasma gastrin concentration (3, 6, 12, 14,23). the elevated secretion of gasmn will exert a trophic effect on the acid-producing portion of the stomach (8, 14) and may also increase the number of fundic ecl (enterochromaffin-like) cells (10-1 1, 20-22, 27). in addition, long-term treatment with some acid-suppressing drugs has produced carcinoid (4, 5, 7, 19) or other (26) gastric tumours in rats. the occurrence of carcinoid tumours in rats following treatment with the selective h+/k+-atpase inhibitor omeprazole has been ascribed to the elevated intragasmc ph and the subsequent increase in gastrin release (4). however, the concept that an increased antral 157 ph during treatment with acid suppressing drugs is the only factor responsible for the gastrin increase is questioned by others (3, 16-18,25). thus, further studies are motivated on such drugs, on gastrin production and release. in the present investigation we have therefore studied the influence of the h2-receptor antagonist ranitidine and omeprazole on tissue and plasma concentrations of gastrin, using doses of ranitidine and omeprazole that evoke similar degrees of inhibition of the basal acid output. acid secretory experiments were performed in this study to confirm the inhibitory effects on acid secretion found in a previous investigation by us (24). in the present study we also investigated how low and high doses of omeprazole and ranitidine affect the tissue gastrin concentration following four weeks of treatment and four weeks of recovery from such treatment. we also determined how the gastric tissue concentration gradually changes during the treatment period with a high dose of omeprazole. the effects of h2-receptor antagonists (5, 10,20-23,27, ) and omeprazole (2, 3, 8,9-10,20-23, 27) on gastrin concentrations in rats have been studied previously. in many of those studies gastrin concentrations in plasma and tissues were determined under different conditions, which complicates comparisons between the studies. to evaluate factors that are of importance in this context we have studied the influence on plasma and tissue gastrin concentrations during omeprazole treatment when the animals have free access to food and during different periods of food deprivation, as well as the effect of changing the interval between the last dose and sample collection. materials and methods animals male sprague-dawley rats, weighing 200-325 g were used in the acid secretory experiments. the rats were provided with a stainless steel cannula in the proximal part of the stomach under pentobarbitone anaesthesia. after surgery the rats were allowed to recover for three weeks before acid secretory experiments were started. before each experiment the rats had free access to water but were fasting for 18-20 hours. male sprague-dawley rats weighing 170-200 g at the start of the experiments were used in the gastrin studies. the animals were weighed twice a week and the average weight gain was calculated. the rats, all of which had free access to water, were fed with standard rat food (r3, ewos ab, sijdertalje, sweden) containing 22.0 % protein, 51.5 % carbonhydrate and 5.0 % fat, 13 mj/kg. when fasting, rats had free access to water and were placed in mesh wire bottom cages 2 24 hours before they were killed. 158 drugs and drug administration the drugs omeprazole ( generously supplied by ab hassle, sweden) and ranitidine (glaxo operations uk ltd., england) were dispersed or dissolved in hydroxypropylmethylcellulose (dow coming corp., midland, usa). when hydroxypropylmethylcellulose (methocel) was used as vehicel or in control experiments 2 m d m l of nahco3 was dissolved in 0.5% of methocel and naoh added in order to adjust ph to 9.0. the omeprazole suspensions were kept in a refrigerator (+4oc) and renewed every 6th day. fresh ranitidine solutions were prepared twice weekly to assure stability. on the basis of body weight determinations, the amounts of drugs to be administered were regularly adjusted. drugs were given orally by gavage with a flexible plastic tube in volumes varying between 2.7 and 3.3 ml. in all experiments the drugs or the vehicle were always administered twice (omeprazole at 7 am and 7 pm) or four (ranitidine at 1 am, 7 am, 1 pm and 7 pm) times daily in order to accomplish a persistent 24 hour inhibition during the treatment period. samples from 9-10 rats were collected for each observation on gastrin concentrations. treatment of plasma and gastric tissue after conclusion of the drug administration, rats were killed by decapitation. blood was collected from the jugular and carotid vessels in heparinized glass tubes, cooled and centrifuged (5000 rpm) for 10 minutes at +4oc. the plasma was then frozen and stored at -20% after bleeding, the abdominal wall was opened by a midline incision and the stomach was taken out. blood vessels and mesenteric tissue along the major and minor curvatures were removed. the stomach was opened along the major curvature and gently rinsed with saline. following determination of the weight the stomach was frozen on dry ice and then stored at -2ooc until assayed for gastrin content by radioimmunoassay. the gasmn content of the whole stomach was determined. the stomach was homogenized and then boiled in water for 10 minutes (w/v=l/lo). after cooling, the mixture was filtered through gauze and the filtrate was adjusted to 25 ml and then stored at -2ooc. radioimmunological procedures gastrin was determined by radioimmunoassay according to a method described previously (13, using antibodies (4562) generously supplied by professor jens rehfeld, copenhagen, denmark. synthetic human gastrin 17 i (generously supplied by dr j s morley, ici ltd., england) was used as standard. human gastrin i labelled with 1125, purchased from milab, malmo, sweden, was used as tracer. gasmn determinations were performed in duplicate and in serial dilutions. plasma gastrin concentrations are expressed in p d m l and the gastrin amounts in tissues in pgstomach. 159 acid secretion studies before acid secretory experiments the gastric cannula was opened and carefully rinsed to allow a free passage of gastric juice. basal and pentagastrin (650 nmol/kg/h in s.c. infusion) stimulated gastric acid secretions were followed for 24 hours in rats given ranitidine (375 pmol/kg every sixth hour), omeprazole (80 pmol/kg every twelfth hour) or vehicle (3.0 ml, 2 or 4 times daily). on the seventh day, during ongoing drug administration, the acid secretion was determined in ranitidine treated rats hours 3,6,9,12,15,18,21 and 24 or during hours 4,8,12,16,20 and 24 in rats given omeprazole. pentagastrin was given continously during the hours of sample collection. at collection of gastric juice the volume was measured and the acidity was determined by titration with 0.1 m naoh to ph 7.0 using an automatic endpoint titrator system (radiometer, copenhagen, denmark). gastrin studies before starting other studies, we determined how gastrin in plasma and stomach tissue were influenced by the conditions under which samples were collected. thus we examined how feeding and food deprivation for various periods of time influenced the concentrations (series a). we also studied how variations in the interval between the last drug administration and sampling affected the gastrin concentrations (series b). the results from these initial studies were used in planning for more extensive studies in which the stomach gastrin content was determined after 4 weeks of treatment with omeprazole and ranitidine and following a subsequent recovery period of 4 weeks (series c). in series d we investigated how a high dose of omeprazole influences gastric gastrin content following 1,3,7, 14 and 28 days of administration. statistical evaluations all values are expressed as mean k sem. statistical evaluation of the results in series a and b was carried out by regression analysis. in series c analysis of variance and bonferroni's test was carried out for multipel comparisons and in series d student's t-test was used for comparisons between groups. results ranitidine (375 pmolkg x 4) inhibited the 24 hour basal and pentagastrin stimulated acid secretions with 82k2% (mean+sem) and 7 9 f l % (fig. la). the corresponding inhibition for omeprazole (80 pmolkg x 2) was 8 m 5 % and 78k2% (fig. 1 b). 160 fig. 1. basal (a a ) and pentagastrin stimulated ( 0 0) acid out-put during 24 hours in gastric fistula rats (n 6-10) during the seventh day of administration of a) 375 pmol/kg (a@) ranitidine or 3.0 ml of methowl( a 0 ) at hours 0,6, 12 and 24 or during administration of b) 80 pmol/kg of omeprazole (a 0 ) or 3.0 ml of methocel (ao) at hours 0 and 12. gastric juice were collected at hours 3,6,9,12,15,18,2 1,24 (ranitidine) or 4,8,12,16,20, 24 (omeprazole). 400 1 2oo l-lakf& 4 8 1 2 16 20 2 4 hours no significant differences in body or stomach weights were seen between the different treatment groups in series a c (results not illustrated). in series a rats were given omeprazole (80 pmovkg x 2) for 14 days and were killed 12 hours after the last dose of omeprazole. the animals had access to food until killed (0) or they had been fasting for 2, 6, 12 or 24 hours. as can be seen in fig. 2a, plasma gastrin concentrations gradually and significantly decreased as the period of fasting was prolonged (t=-7.29, p10.001), whereas the amounts in gastric tissue (fig. 2b) at the corresponding points of time were essentially unchanged (t=1.65, ns). the rats in series b had free access to food and the concentration of gastrin in plasma was significantly (t=-5.29, p10.001) lowered as time between dosing and sampling was prolonged (fig. 3a). the amounts of gasmn in the stomach were not greatly influenced (t=1.75, ns), although some increase of gastrin content was seen as the period between sampling and last administration of the drug was increased (fig. 3b). the time point 0 hour after drug administration was omitted in the statistical analysis. the rationale for that is that the time between administration and sampling of plasma and stomach tissue was too short to allow omeprazole to interfere with acid and gastrin secretion. effects of treatment with omeprazole (20 or 80 pmol/kg x 2) or ranitidine (125 or 375 pmolkg x 4) for 28 days and following 28 days of recovery were studied in series c . after 18-20 hours of fasting the treated animals were killed 6 (ranitidine) or 12 (omeprazole) hours after the last dose. the high dose of omeprazole (fig. 4) and ranitidine (fig. 5 ) significantly increased the tissue content of gastrin. the largest amounts were found when animals were given the high omeprazole dose for four weeks. however, the high dose of ranitidine caused a gastrin increase that was closer to that produced by the low dose of omeprazole. 161 no change in tissue gastrin content was noted in rats treated with the low dose of ranitidine (fig. 5). after 4 weeks of recovery, the amounts of tissue gastrin in omeprazole treated rats were, somewhat, but not significantly, raised whereas the amounts in ranitidine-treated rats were unchanged when compared to the recovery controls. t 0 2 6 12 24 hours 0 2 6 1 2 2 4 h o u e 0 2 6 1 2 2 4 hours 0 2 6 1 2 2 4 hours fig 2. (a) plasma gastrin concentration and (b) gasmc gastrin content, expressed as mean k sem, in rats treated with omeprazole 80 pmolkg twice daily for 14 days. the rats (n=9-10) were killed after having free access to food (0 hour) or after 2, 6, 12 or 24 hours of food deprivation. fig 3. (a) plasma gastrin concentration and (b) gastric gasmn content in rats having free access to food (n=9-10) treated with omeprazole 80 prnolkg twice daily for 14 days and killed 0,2,6,12 or 24 hours after the administration of the last dose. results are expressed as mean if: sem. 162 treatment lor 28 days recovery for 28 days ... ,1 t treatment for 28 days recovery for 28 days 3 1 ... p ... methocel i omeorazole i 1 3 7 days of treatment 1 4 fig 4. stomach gastrin content in rats (n=10) treated with 20 or 80 pmol/kg of omeprazole or with 3 ml of methocel every twelfth hour for 28 days and following recovery for 28 d a y s . t h e r e s u l t s a r e expressed as mean k sem. *** indicates significant ( ~ 1 0 . 0 0 1 ) difference between methocel and omeprazole treated rats. bonferroni’s test. fig 5. stomach gastrin content in rats (n=10) treated with 125 or 375 p m o l k g of ranitidine or with 3 ml of methocel every sixth hour for 28 days and following recovery for 28 days. *** indicates significant (p<o.001) difference between methocel and ranitidine-treated rats. bonferroni’s test. ... t fig.6. increase in stomach gastrin content in rats (ng7-10) treated with 80 p m o l k g of omeprazole or with 3 ml of methocel every twelfth hour for 1, 3, 7, 14 or 28 days. * (p‘p.05) and *** (p10.001) inhcate significant dlfferences between m e t h o c e l and omeprazole-treated rats after 3 and 7 28 days of treatment respectively. student’s t-test. 2 8 163 the results from series d are illustrated in fig. 6. the amounts of gastrin gradually increased and were 31 per cent larger after 3 days (p10.05), which can be compared to the increase of 114 per i cent (~10.001) following 28 days of treatment. it should be noted that the gastrin values following 28 days of treatment are the same as in series c. discussion in the present study attempts were made to evaluate how duration of fasting or variation of the time period between last drug administration and sampling influence gasmn in plasma and tissue. it is previously known that deprivation of food in rats decreases the plasma and antral gastrin concentrations (13). recently wu et a1 (28) demonstrated a time dependent decrease in tissue gastrin concentration and antral messenger rna(mrna) level in starving untreated rats. a significant decrease of both antral gastrin and gastrin mrna concentration occurred already after 12 hours’ starvation. in the present study we have demonstrated that fasting for 2 24 hours following 14 days of treatment with omeprazole gradually and considerably (92% after 24 hours) reduces the plasma gastrin concentrations, whereas the amounts of antral gastrin measured at corresponding times are essentially unchanged. it seems reasonable to assume that a prolonged administration of omeprazole to rats causes an increase of tissue gasmn concentration that may be relatively less sensitive to alterations in the feeding state. when the dosing intervals were varied and the animals had free access to food plasma gasmn levels remained more stable. some increase in plasma gastrin concentration was seen when omeprazole exerted its optimal effect (after 2-6 hours), whereas concentrations gradually and significantly decreased as the period between sampling and the last administration of the drug was increased. the above results underline the importance of well established sampling routines for the collection of plasma and gasmc tissues for gasmn determination. the lack of such routines will contribute to extensive variations in results in individual studies and great difficulties in comparing results from different laboratories. taken together, the results of this methodological part of the study suggest that, when long-term effects on gastrin changes are studied results from determinations of tissue gastrin concentrations seem to be relatively independent of temporary variations in degrees of food stimulation, intragastric ph changes and other factors that may influence gastrin release. in a previous study of ours (24) we gave rats various doses of omeprazole and ranitidine and determined the effects on acid secretion. in the present study we have confirmed some of the results on acid secretion from that study and we have selected doses of omeprazole and ranitidme that caused comparable inhibitions of the basal acid output. when these doses were administered twice (omeprazole) or four (ranitidine) times per day during 28 days the stomach gasmn content of the omeprazole-treated rats reached higher values than that in the ranitidine treated animals. larsson (1 1) and ryberg (23) and their coworkers concluded that when acid secretion is inhibited to a similar degree, ranitidine is capable of inducing hypergastrinemia of the same magnitude as omeprazole. therefore in their view a direct action on the gastrin cells by either of the two 164 compounds seems less probable. however, in another study of the same group (22) previously used doses of omeprazole and ranitidine (23) failed to cause a corresponding degree of acid inhibition. our studies of gastrin in the stomach and plasma (24) following treatment with omeprazole and ranitidine indicate larger increases in gastrin levels following omeprazole treatment. therefore we cannot exclude the possibility that omeprazole or ranitidine may influence the ability of the gastrin cells to secrete gastrin by other mechanisms than by affecting the intraluminar ph of the stomach. such a concept is supported by results from ohe et a1 (16-18) who studied gastrin release in perfused rat stomachs and by results from decktor et a1 (3) and simoens et a1 (25) in studies on gastric fistula rats and dogs. when omeprazole (80 pmolkg) was given twice daily for various periods of time the amounts of gastric gastrin gradually increased and following 4 weeks of treatment they were 114 per cent above the control level. a slight but insignificant increase in tissue gastrin content was seen already after one day of omeprazole administration whereas administration for 3 days caused a marked and significant increase. these findings are in agreement with the results of brand and stone (1) who studied the time course of stimulation of gastrin secretion and synthesis in rats after intraperitoneal administration of omeprazole. they found that antral gastrin mrna levels were significantly increased after 2 days (but not after one day) of omeprazole administration. following four weeks of recovery from omeprazole treatment, the amounts of gastric gastrin were slightly and insignificantly increased whether the rats had been treated with the high or the low dose of omeprazole. a similar tendency was found in studies presented by creutzfeld et al(2) and larsson et a1 (9) following 42 (2) and 35, but not 70 (9), days of recovery after omeprazole treatment. however, since the doses of omeprazole, length of omeprazole treatment, number of administrations per day and routines for collection of blood samples differed between these studies, no direct comparisons can be made. no sustained elevations of the gastrin concentrations were found after 4 weeks in rats treated with ranitidine when compared with the vehicle treated recovery controls. it should be noted that the gastrin contents in control rats treated with the vehicle four times per day were somewhat higher than in recovery controls treated twice per day. no certain explantion can be given to this difference. in summary, the present results indicate the importance of controlled experimental conditions in studies of gastrin in plasma and gastric tissue. in studies of long-term effects on the gastrin mechanism determinations of tissue concentration may offer more stable and less time dependent results. during omeprazole treatment increased amounts of gastrin were found in gastric tissue already following 3 days of treatment and they gradually increased thereafter. following 28 days of treatment, omeprazole produced higher tissue amounts of gastrin than ranitidine at doses selected to cause similar inhibition of gastric secretion. 165 acknowledgments the authors wish to express their gratitude to mrs annika wagman and mrs elisabeth dugner for their technical assistance and to mrs margareta tope1 and ms catrine olsson for typing the manuscript. this investigation was supported by grants from the swedish medical research council (04x-3521), the swedish society of medicine and "forenade liv" mutual group life insurance company, stockholm, sweden. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. references brand, s. & stone, d: reciprocal regulation of antral gastrin and somatostatin gene expression by omeprazole-induced achlorhydria. j clin invest 82: 1059-1066, 1988. creutzfeldt, w., stiickman f., conlon j.m., folsch u.r., bonatz g &wiilfrath m: effect of shortand long-term feeding of omeprazole on rat gastric endocrine cells. digestion 35 (suppl 1): 84-97 1986. decktor, d.l., pendelton r.g., kellner a.t & 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gastrin-17 in intact and antrectomized rats. gastroenterology 99: 935-42, 1990. ryberg, b, tielemans y, axelson j, carlsson e, hkanson r & mattsson h: thropic effects of continous infusion of leois-gastrin-17 in the rat. gastroenterology 98: 33-38, 1990. ryberg, b., bishop a.e., bloom s.r., carlsson e., hilkansson r., larsson h., mattsson h., polak j.m & sundler f: omeprazole and ranitidine, antisecretagogues with different modes of action, are equally effective in causing hyperplasia of enterochromaffin-like cells ryberg, b., mattsson h., larsson h & carlsson e: correlation between inhibition of gastric acid secretion, plasma gastrin, and oxyntic mucosal histidine decarboxylase activity in the rat. scand. j. gastroenterol. 24: 287-292, 1989. seensalu, r., girma k., rome11 b & nilsson g: time-course of inhibition of gastric acid secretion in gastric fistula rats by omeprazole and ranitidine, eur j pharmacol 180, 145 152, 1990. simoens, c., woussen-colle m.c & de graef j: effects of acute suppression of acid secretion by omeprazole on postprandial gastrin release in conscious dogs. gastroenterology 97: 837-845, 1989 streett, c.s., cimprich r.e & robertson j.l: pathologic findings in the stomachs of rats treated with the h2-receptor antagonist tiotidine. scand. j. gastroenterol 19 (suppl 101): tielemans, y., h&kansson r., sundler f & willems g: proliferation of enterochromaffinelike cells in omeprazole-treated h ypergastrinemic rats. gastroenterology wu, v, sumii k, taxi a, sumii &, walsh j: regulation of rat antral gastrin and somatostatin gene expression during stravation and after refeeding. gastroenterology in rat stomach. regul pept 25: 235-246, 1989. 109-1 17, 1984. 96: 723-729, 1989. 101: 1552-1558, 1991. address for reprints: dr rein seensalu division of gastroenterology & hepatology department of medicine, k63 huddinge university hospital s-141 86 huddinge sweden 167 upsala journal of medical sciences 2021, 127, e8941 http://dx.doi.org/10.48101/ujms.v127.8941 admission of patients with chest pain and/or breathlessness from the emergency department in relation to risk assessment and copeptin levels – an observational study lee ti davidsona, emilia gauffinb, preben henangerb, maciej wajdaa, daniel wilhelmsa, bertil ekmanb, hans j. arnqvistb, martin schillingc and simona i. chisalitab adepartment of emergency medicine in linköping, local health care services in central östergötland, region östergötland & department of biomedical and clinical sciences, linköping university, sweden; bdepartment of endocrinology in linköping, and department of health, medicine and caring sciences, linköping university, linköping, sweden; cclinicum and innovations centrum, department of emergency medicine and department of clinical and experimental medicine, linköping university, linköping, sweden article history received 1 august 2022 revised 8 november 2022 accepted 10 november 2022 published 26 december 2022 keywords emergency department, chest pain, breathlessness, copeptin, mr-proadm, mr-proanp background chest pain and/or breathlessness are common symptoms in patients attending the emergency department (ed), and both have a broad spectrum of underlying diseases (1, 2). a critical issue for the emergency physician at the ed is to decide which patients should be admitted to a ward and which patients can be discharged home (1, 3–5). the rapid emergency triage treatment scale (retts©) (6) is used to identify and prioritize patients who need acute treatment and to reduce waiting time at the ed 6, 7). based on their presenting symptoms and vital signs, patients are allocated to five triage priority categories (blue, green, yellow, orange, and red), based on cut-off levels for vital signs and chief complaint algorithms, resulting in different recommended times for physician assessment (6). the national early warning score (news) is a track and trigger system developed on hospital wards by assessing patients’ vital signs in order to detect patients at high risk of serious adverse events, such as unplanned intensive care unit (icu) admission, cardiac arrest, or in-hospital death within 24 h (8–14). news has been validated in eds and in hospital settings (11, 12, 15). triage scores characterize a patient’s clinical state at a single time point. when vital signs are still unremarkable, patients at risk of deterioration might be missed. copeptin, mr-proadm, and mr-proanp are surrogate markers for arginine vasopressin (avp), adrenomedullin (adm), contact emilia gauffin emilia.gauffin@liu.se supplemental data for this article can be accessed here. © 2022 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article abstract background: one of the most critical decisions that emergency department (ed) physicians make is the discharge versus admission of patients. we aimed to study the association of the decision in the ed to admit patients with chest pain and/or breathlessness to a ward with risk assessment using the rapid emergency triage and treatment system (retts), the national early warning score (news), and plasma levels of the biomarkers copeptin, midregional proadrenomedulin (mr-proadm), and midregional proatrial natriuretic peptide (mr-proanp). methods: patients presenting at the ed with chest pain and/or breathlessness with less than one week onset were enrolled. patients were triaged according to retts. news was calculated from the vital signs retrospectively. results: three hundred and thirty-four patients (167 males), mean age 63.8 ± 16.8 years, were included. of which, 210 (62.8%) patients complained of chest pain, 65 (19.5%) of breathlessness, and 59 (17.7%) of both. of these, 176 (52.7%) patients were admitted to a ward, and 158 (47.3%) patients were discharged from the ed. in binary logistic models, age, gender, vital signs (o 2 saturation and heart rate), news class, and copeptin were associated with admission to a ward from the ed. in receiver-operating-characteristics (roc) analysis, copeptin had an incremental predictive value compared to news alone (p = 0.002). conclusions: emergency physicians’ decisions to admit patients with chest pain and/or breathlessness from the ed to a ward are related to age, o 2 saturation, heart rate, news category, and copeptin. as an independent predictive marker for admission, early analysis of copeptin might be beneficial when improving patient pathways at the ed. http://dx.doi.org/10.48101/ujms.v127.8941 mailto:emilia.gauffin%40liu.se?subject= mailto:emilia.gauffin@liu.se http://dx.doi.org/10.48101/ujms.v127.8941 http://creativecommons.org/licenses/by/4.0/ 2 l.t. davidson et al. and atrial natriuretic peptide (anp), respectively (16, 17). these biomarkers have shown to improve diagnostic performance and risk stratification of patients presenting with chest pain and/or breathlessness at the ed (18–20). avp is secreted from the posterior pituitary gland and mediates vasoconstriction and water retention. in an ed, adding copeptin analysis to troponin improves the diagnostic reliability of acute coronary syndrome compared to troponin alone (21). stretching of myocytes leads to the secretion of anp from the heart’s atria, which promotes natriuresis, diuresis, vasodilation, and inhibition of the renin angiotensin aldosterone system (22). mr-proanp has similar diagnostic and prognostic performance to other natriuretic peptides in heart failure (23, 24). adm is secreted from the endothelial cells in the vessel wall as a result of ischemia and is, hence, associated with endothelial dysfunction (18). higher levels of mr-proadm are associated with adverse outcomes in patients with cardiovascular diseases (23–25). it is vital that the physician can identify the patients who need to be admitted to a ward, so these patients can achieve the best possible care. moreover, fast assessment of patients without need of in-hospital care shortens the waiting time at the ed, which has been shown to correlate with adverse outcomes (26). risk assessment tools such as retts and news are used at ed to prioritize and assess the patients. our hypothesis is that biomarkers such as copeptin, mr-proadm, and mr-proanp could aid emergency physicians by identifying patients in need of admission. this study aimed to investigate the association of vital signs and risk assessment using retts, news, and biomarkers with the decision on admission to a ward from the ed in patients with chest pain and/or breathlessness. methods study design we conducted a single-center observational study in the ed at a tertiary care teaching hospital with a primary catchment area of 460,000 inhabitants and 48,000 ed visits annually during the period 2013–2017. non-critically ill patients 18 years or older presenting at the ed with chest pain, breathlessness, or both, with the onset within 7 days, were enrolled in this study after providing a written informed consent. exclusion criteria included advanced renal failure, advanced malignancy, liver failure, st-segment elevation myocardial infarction or new left bundle branch block, and severely ill patients (pronounced shortness of breath, massive chest pain, or clinically unstable patients for whom there was no doubt about requiring urgent medical attention). blood pressure, respiratory rate, heart rate, body temperature, and oxygen saturation evaluated by pulse oximetry were recorded. patients were triaged by a nurse according to the rapid emergency triage and treatment system (retts) (figure 1 in the supplementary material). the triage priorities (in order of acuity) are red, orange, yellow, green, and blue, with red being the highest priority and blue being the lowest. the patients were dichotomized as either low (blue, green, or yellow) or high (red or orange) priority in the statistical analyses. the news was retrospectively calculated using the recorded vital signs, i.e. respiratory rate, oxygen saturation, temperature, blood pressure, pulse rate, and level of consciousness, as described previously by the royal college of physicians (13). using the resulting combined score, patients were classified into three news categories representing low (0–4 points), moderate (5–6 points), and high (≥7 points) risk (figure 2a and 2b in the supplementary materials). because data on supplemental oxygen were not available, the results presented here correspond to news-potentially minus 2, i.e. adjusted news. all patient management was performed by clinical routine. biochemical analyses blood samples were collected on presentation at the ed. plasma was separated by the core laboratory and kept frozen at −70°c for later analysis. copeptin, mr-proadm, and mr-proanp were analyzed using a highly sensitive time-resolved amplified cryptate emission technology assay (b·r·a·h·m·s, kryptor, ag, hennigsdorf, germany). the assay has a lower detection limit of 1.7 pmol/l and an inter-assay precision of 5.2% cv for copeptin. for mr-proadm, the assay has an analytical detection limit of 0.04 nmol/l and an inter-assay variability of 3.3%. for mr-proanp, the assay has a detection limit of 2.1 pmol/l and an inter-assay variability of 3.0%. blood samples for c-reactive protein (crp), creatinine, and high sensitivity troponin t (hstnt) were analyzed at the central laboratory by clinical standard. all clinical information including the final diagnosis was collected from cambio cosmic® digital medical records by two figure 1. roc curve for prediction of admission to a ward. blue = news. green = news + copeptin. admission of patients with chest pain 3 physicians (s.i.c. and l.t.d.), with a follow-up of 90 days after the initial presentation. main outcome the outcome of this study was admission to a ward from the ed, coded as a binary variable: admitted to a ward or discharged from the ed. ethics all participants gave their written informed consent. this study was approved by the regional ethical review board in linköping, sweden (diary number 2011/501-31). the study protocol followed the principles expressed in the declaration of helsinki. statistical analysis the statistical analyses were performed using ibm spss statistics v.28.0.1.0. means and standard deviation, median and interquartile ranges, counts, and percentages were reported as appropriate. the mean and median between groups were compared using either t-test or median test for continuous numerical data and chi-square test for categorical variables. p-values < 0.05 were considered statistically significant. the biomarkers mr-proanp, mr-proadm, and copeptin were dichotomized by cut-off values used in previous studies (23, 27, 28). data were analyzed with binary logistic regression to estimate adjusted odds ratio (ors) for the single outcome variable identified above. first, univariable analysis was performed for age, gender, vital signs, news, retts, copeptin, mr-proadm, or mr-proanp. thereafter, three different models of multivariable analysis were performed: (1) age, gender, vital signs, copeptin, mr-proadm, and mr-proanp; (2) age, gender, news moderate/high versus low class, copeptin, mr-proadm, and mr-proanp; and (3) age, gender, retts high versus low priority, copeptin, mr-proadm, and mr-proanp. finally, a supplemental analysis was performed adjusting the previous models for creatinine and hstnt. receiver-operating-characteristics (roc) curves were constructed for news separately and for news combined with continuous copeptin to determine the prognostic performance with area-under-the-curves (aucs) for prediction of admission to a ward. we used stata (mp v17.1, college station, usa) to test for differences between the aucs. results in total, 334 patients with a mean age of 63.8 ± 16.8 years were included in the study. baseline data for the whole population and the admitted and discharged patients are presented in table 1. of all patients, 210 complained of chest pain (62.8%), 65 (19.5%) of breathlessness, and 59 (17.7%) of chest pain and breathlessness. after examination at the ed, 176 (52.7%) patients were admitted, and 158 (47.3%) patients were discharged. the admitted patients had a hospitalization time of 1 (0; 3) day (median; interquartile range (iqr)). no patients were admitted to the icu. patients admitted to a ward tended to be older and predominately male compared with those who were discharged from the ed. there was a significant difference in oxygen saturation (o 2 saturation), respiratory rate, and heart rate between admitted and discharged patients (table 1). in all, 89 (26.6%) had at least one prior episode of ischemic heart disease (e.g. unstable angina, myocardial infarction, coronary artery bypass grafting, or percutaneous coronary intervention), whereas most of the patients (240, 72.1%) had at least one comorbidity (e.g. hypertension, ischemic heart disease, atrial fibrillation, heart failure, stroke, transient ischemic attack, thromboembolism, diabetes mellitus, renal dysfunction, asthma, chronic obstructive pulmonary disease, systemic inflammatory diseases, or malignancy). the final diagnoses are presented in table 2. in total, 68 patients were admitted to a ward for observation and thereafter discharged with a non-specific diagnosis. of these, 51 (75%) had a copeptin value below 10 nmol/l. using the retts triage model, 97 (34.9%) of the patients were classified as low priority (blue, green, or yellow) and 181 (65.1%) as high priority (red or orange) (table 1). in the group assigned retts high priority, the number of patients admitted to a ward was higher than those discharged home (111 [71.2%] vs. 70 [28.8%], p = 0.017). in the news track and trigger system model, most patients assigned to the moderate (13 [7.8%] vs. 3 [2.0%], p = 0.02) and high (14 [8.4%] vs. 1 [0.7%], p = 0.001) risk groups were admitted. levels of copeptin, mr-proadm, and mr-proanp were significantly higher in the patients admitted to a ward (table 1). in table 3, the levels of the biomarkers are presented according to group of retts and news. none of the discharged patients died within 90 days of follow-up, while there were five deaths in the group admitted to a ward. the association of vital signs, news, and biomarkers with admission to a ward or discharge from the ed was tested in multivariate binary regression models (tables 4a-c). in model 4a, age (or 1.031 [1.006–1.057]), o 2 saturation (or 0.860 [0.775–0.954]), heart rate (or 1.020 [1.003–1.037]), and copeptin >10 nmol/l (or 2.254 [1.116–4.549]) were associated with admission to a ward. when the compound scale news was used instead of single vital signs, an association with admission to a ward was found (or 3.592 [1.082–11.991]) (table 4b), while no significant association between retts and the need for admission could be found (p = 0.204) (table 4c). even in these models, copeptin remained significantly associated with the outcome of admission/discharge (or 2.308 [1.163–4.582] and or 2.644 [1.272–5.497]) (table 4b-c). after adjusting model 4a–c for creatinine and hstnt (supplementary materials table 1), only copeptin maintained the significance in all models (or 2.662 [1.064–6.657], or 2.795 [1.150–6.792], and or 3.792 [1.444–9.954], respectively), whereas age, saturation, heart rate, and news lost their predictive value for admission to a ward. 4 l.t. davidson et al. in roc analysis, news showed an auc of 0.649 (0.591–0.707) and the combination of news and copeptin showed an auc of 0.711 (0.652–0.769), see figure 1. adding copeptin had a significant incremental predictive value when compared to analysis of news separately (p = 0.002). discussion in this study, we found that age, gender, vital signs, news, and copeptin were associated with the decision of admission to a ward for patients presenting at the ed with chest pain and/or breathlessness. vital signs such as heart rate, respiratory rate, o 2 saturation, temperature, blood pressure, and consciousness objectively indicate the immediate well-being of patients and are an imperative component of patient assessment and management (29). in our study, o 2 saturation, heart rate, and age were independently associated with the decision on admission to a ward. in geriatric patients, knowledge of pulse oximetry values has been shown to affect the decision on admission (30). heart rate on admission is associated with prognosis in patients with heart failure (31). in a large, unselected ed population, age and vital signs were significantly related to 1-day mortality, 30-day mortality, and icu admission (32). in the same study, respiratory rate and oxygen saturation were associated with higher odds of mortality than changes in systolic blood pressure and pulse rate. the patients in our study were risk-stratified according to the retts priority triage model, which is used to identify and prioritize the order in which patients need to be dealt with by the ed physician (6, 7). in our study, the proportion of patients classified in the red retts priority class was 4.0%, which is similar to results presented by ljunggren et al. (32). by contrast, the proportion of patients allocated to the orange retts priority class, 61.2%, was considerably higher than the 6.7% reported by the same authors (32). it is conceivable that in our study, there might have been overtriage due to the broad triage criteria in retts. it has to be noted that in retts, a higher triage level can be applied by triage nurses’ discretion. ljunggren et al. suggested that in retts, the most commonly used triage system in sweden, future triage systems should also include age (32). with news, a lower number of patients were included in the high and moderate risk classes: 4.8% and 5.1% vs. 90.1% in the low news risk class. it should be pointed out that the emergency table 2. diagnosis at discharge from the hospital in patients who were admitted to a ward. diagnosis copeptin ≤ 10 nmol/l copeptin >10 nmol/l total (n) non-specific diagnosis 51 (75.0%) 17 (25.0%) 68 acute coronary syndrome 13 (43.3%) 17 (56.7%) 30 other cardiac diagnosis 16 (35.6%) 29 (64.4%) 45 acute lung infection 2 (15.4%) 11 (84.6%) 13 table 1. baseline characteristics of the study population. variables all population discharged from ed admitted to a ward p-value number of patients 334 158 176 age, years (mean ± sd) 63.79 ± 16.87 57.26 ± 18.30 69.66 ± 12.97 < 0.001 male/female, n (%) 167 (50.0)/167 (50.0) 68 (43.0)/90 (57.0) 99 (56.3)/77 (43.8) 0.016 chest pain, n (%) 210 (62.8) 105 (66.4) 105 (59.6) 0.119 breathlessness, n (%) 65 (19.5) 32 (20.3) 33 (18.8) 0.131 chest pain and breathlessness, n (%) 59 (17.7) 21 (13.3) 38 (21.6) 0.047 news low, n (%) 283 (90.1) 144 (97.3) 139 (83.7) < 0.001 news moderate, n (%) 16 (5.1) 3 (2.0) 13 (7.8) 0.020 news high, n (%) 15 (4.8) 1 (0.7) 14 (8.4) 0.001 retts low (yellow, green, and blue), n (%) 97 (34.9) 52 (42.6) 45 (28.8) 0.017 retts orange, n (%) 170 (61.2) 69 (56.6) 101 (64.7) 0.165 retts red, n (%) 11 (4.0) 1 (0.8) 10 (6.4) 0.018 retts high (red + orange), n (%) 181 (65.1) 70 (57.4) 111 (71.2) 0.017 blood pressure systolic, mmhg (mean ± sd) 147.36 ± 23.79 146.43 ± 21.98 148.20 ± 25.33 0.49 blood pressure diastolic, mmhg (mean ± sd) 83.32 ± 14.35 84.05 ± 13.62 82.68 ± 14.98 0.39 temperature, °c (mean ± sd) 36.93 ± 0.68 37.00 ± 0.60 36.86 ± 0.74 0.79 saturation, % (mean ± sd) 96.46 ± 3.88 97.75 ± 2.36 95.30 ± 4.57 < 0.001 respiratory rate/minute (mean ± sd) 18.95 ± 5.21 18.32 ± 4.79 19.51 ± 5.52 0.04 heart rate/minute (mean ± sd) 80.34 ± 20.14 75.76 ± 15.40 84.45 ± 22.88 < 0.001 length of hospital stay, days (median (iqr)) 1 (0–3) 0 (0.0) 2 (1–5) < 0.001 previous ischemic heart disease 89 (26.6) 26 (16.5) 63 (35.8) < 0.001 comorbidities 240 (72.1) 94 (59.9) 146 (83.0) < 0.001 copeptin, nmol/l (median (iqr)) 6.20 (3.69–13.49) 4.94 (3.34–8.11) 9.58 (4.05–26.58) < 0.001 mr-proadm, nmol/l (median (iqr)) 0.68 (0.53–0.90) 0.61 (0.33–0.47) 0.73 (0.58–1.06) < 0.001 mr-proanp, pmol/l (median (iqr)) 91.05 (57.29–179.43) 72.75 (47.04–115.10) 126.75 (73.33–244.08) < 0.001 creatinine, µmol/l (median (iqr)) 79 (65–97) 75 (65–94) 81 (66.0–102) 0.004 high sensitivity troponin t, µmol/l (median (iqr)) 9 (6–19) 7 (5–10) 13 (7–27) 0.246 missing values: news 20, retts 56, systolic bp 1, diastolic bp 9, temperature 11, saturation 1, respiratory rate 10, copeptin 20, mr-proadm 25, and mr-proanp 25. admission of patients with chest pain 5 physicians were aware of vital signs and the retts priority classes, but not specifically of the news score and were blinded towards the biomarkers studied. in the multivariate regression model including age and sex, there was no significant difference between high (red and orange) and low (green, blue, and yellow) retts levels regarding admission to a ward, whereas a significant difference was found for news risk levels between patients admitted to a ward and those discharged home. this suggests that the track and trigger system news is a more sensitive tool for risk stratification than triage priority using the retts model. few studies have previously considered the predictive value of news for admission to a ward (9, 33). mitsunaga and colleagues found that news values in the ed effectively predicted admission to a ward and in-hospital mortality in elderly patients (9). other studies have investigated the discriminative power of news in the ed for admission to an icu ward and for all-cause mortality (10, 15, 34, 35). recent studies have shown that the swedish version of news had excellent inter-rater reliability, and the median scores for patients admitted to the icu were higher than for those not admitted. patients classified as medium or high risk by news experienced a twofold or threefold increase, respectively, in odds of in-hospital death or 30-day mortality compared to low-risk patients (11, 12). in our study, copeptin was independently correlated to admission to a ward in addition to age, gender, vital signs, and news, whereas mr-proadm and mr-proanp were not. this suggests that copeptin could be used at the ed to aid emergency physician evaluating the need of admission. in a study of patients presenting with non-specific complaints, elevated copeptin was associated with increased 30-day mortality (36). in a large, multicenter, unselected ed cohort of patients, schuetz and colleagues showed that combining clinical information and measured copeptin, mr-proadm and table 3. median (iqr) levels of copeptin, mr-proadm, and mr-proanp across the categories of news and retts. biomarkers retts low retts high p-value news low news moderate + high p-value copeptin (nmol/l) 4.94 (3.59–11.67) 8.56 (4.03–22.10) 0.030 5.71 (3.48–11.54) 27.92 (10.48–56.26) <0.001 mr-proadm (pmol/l) 0.63 (0.50–0.83) 0.70 (0.57–1.07) 0.360 0.65 (0.52–0.88) 1.07 (0.78–1.56) 0.003 mr-proanp (nmol/l) 89.49 (47.61–174.70) 106.80 (65.33–236.50) 0.035 88.40 (55.54–163.10) 204.80 (70.93–354.60) 0.006 table 4. binary regression analyses for associations of age, gender, vital signs (a), news (b), retts (c), and biomarkers with admission to a ward. variables in the predicting model univariable multivariable or (95%ci) p-value or (95%ci) p-value age 1.051 (1.035–1.067) < 0.001 1.031 (1.006–1.057) 0.014 gender 1.702 (1.103–2.625) 0.016 1.365 (0.763–2.444) 0.295 systolic blood pressure 1.003 (0.994–1.012) 0.497 0.998 (0.986–1.010) 0.734 temperature 0.746 (0.537–1.037) 0.081 0.647 (0.403–1.040) 0.072 saturation 0.781 (0.712–0.856) < 0.001 0.860 (0.775–0.954) 0.004 respiratory rate 1.049 (1.001–1.100) 0.045 0.985 (0.918–1.056) 0.667 heart rate 1.023 (1.011–1.035) < 0.001 1.020 (1.003–1.037) 0.021 copeptin > 10 nmol/l 4.569 (2.668–7.826) < 0.001 2.254 (1.116–4.549) 0.023 mr-proadm > 0.75 pmol/l 2.500 (1.553–4.023) < 0.001 0.797 (0.382–1.664) 0.546 mr-proanp > 120 nmol/l 3.513 (2.149–5.741) < 0.001 1.369 (0.642–2.919) 0.416 b. univariable or (95%ci) p-value multivariable or (95%ci) p-value age 1.051 (1.035–1.067) < 0.001 1.034 (1.011–1.058) 0.004 gender 1.702 (1.103–2.625) 0.016 1.665 (0.953–2.909) 0.073 news high + moderate vs. low 6.993 (2.385–20.502) < 0.001 3.592 (1.082–11.991) 0.037 copeptin > 10 nmol/l 4.569 (2.698–11.416) < 0.001 2.308 (1.163–4.582) 0.017 mr-proadm > 0.75 pmol/l 2.500 (1.553–4.023) 0.005 0.774 (0.386–1.551) 0.470 mr-proanp > 120 nmol/l 3.513 (2.149–5.741) < 0.001 1.689 (0.814–3.507) 0.160 c. univariable or (95%ci) p-value multivariable or (95%ci) p-value age 1.051 (1.035–1.067) < 0.001 1.038 (1.013–1.063) 0.003 gender 1.702 (1.103–2.625) 0.016 1.906 (1.055–3.442) 0.033 retts high vs. low 1.832 (1.113–3.017) 0.017 1.464 (0.813–2.637) 0.204 copeptin > 10 nmol/l 4.569 (2.668–7.826) < 0.001 2.644 (1.272–5.497) 0.009 mr-proadm > 0.75 pmol/l 2.500 (1.553–4.023) 0.005 0.467 (0.212–1.030) 0.059 mr-proanp > 120 nmol/l 3.513 (2.149–5.741) < 0.001 1.886 (0.858–4.145) 0.114 notes: ed: emergency department; bp: blood pressure; mr-proadm: midregional proadrenomedulin; mr-proanp: midregional proatrial natriuretic peptide; crp: c-reactive protein; retts: rapid emergency triage and treatment system; news: national early warning score; or: odds ratio; ci: confidence interval. 6 l.t. davidson et al. procalcitonin strongly predicted high initial triage priority, icu admission, and 30-day mortality (37). the clinical examination is fundamental, and the patients will still need to be prioritized, for example, by vital parameters or news, but levels of copeptin could give additional aid in the decision-making. in our study, the roc-curve for the model including copeptin showed a significant incremental predictive value for admission to the use of news solely. in our study, 68 patients were discharged from a ward with a  non-specific diagnosis. many patients have probably unnecessarily been admitted to a ward due to lack of reliable risk stratification methods. analysis of copeptin might have helped the physician to discharge some of these patients directly from the ed. previous studies have shown that ed physicians receive little training on how to make disposition decisions, and that the applied research in this area has focused on the implementation of decision rules or algorithms for narrow, predefined patient groups, such as those presenting with chest pain (38–40). cardel et al. have studied in ‘real time’ how experienced ed physicians make discharge decisions and reported that they most often relied on clinical judgment, rather than evidence-based guidelines (5). this aspect could also have implications for the results of our study. a strength of our study is the broad inclusion criteria consistent with daily practice. we believe that the unselected feature of the cohort makes it representative of patients typically seen in clinical practice in the ed with a well-distributed gender balance, i.e. the same number of male and female participants. however, the sample size is small compared to the number of patients presenting with chest pain and/or breathlessness each year at the ed. further study limitations include the fact that the decision on admission to a ward was taken by several ed physicians with different levels of experience and may, therefore, be subject to  variation. also, physicians could not be blinded to the triage  score, which might affect initial clinical management due  to priority recommendations connected to triage. finally, information on the use of oxygen was not available for the retrospective calculation of news, reducing the maximum score to 18 out of 20. therefore, in accordance with the royal college of physicians, a weighting score of two was added. this could result in the misclassification of news categories. furthermore, the use of hstnt as diagnostic biomarker in the clinical pathway for chest pain patients directly related to the decision to admit might have outweighed the potential benefit of news in our study with the majority of patients (90.1%) presenting with low news scores. conclusions in conclusion, emergency physicians’ decisions to admit patients with chest pain and/or breathlessness from the ed to a ward are related to age, o 2 saturation, heart rate, news category, and copeptin. as an independent predictive marker for admission, early analysis of copeptin might be beneficial when improving patient pathways at the ed. further prospective studies evaluating the value of copeptin on top of clinical judgement and news seem motivated. acknowledgments we acknowledge helene kimme for excellent support in recruiting the patients at the ed. ethics approval this study was conducted according to the guidelines of the  declaration of helsinki and approved by the ethics  committee  of linkoping university (protocol code 2011/501-31). an informed consent was obtained from all subjects  involved in the study. a written informed consent has been obtained from the patient(s) to publish this paper. disclosure statement authors declare no conflict of interest. funding research was funded by region östergötland, rö-285131, rö351681, rö-340001, rö-430481, rö-533731, rö-534451, rö599931, rö-623391, and rö-936926. author contributions conceptualization, s.c., m.s., and h.a.; methodology, s.c., m.s., and h.a.; formal analysis, s.c. and e.g.; investigation, s.c., m.s., and l.t.d.; resources, s.c.; data curation, s.c., p.a., and l.t.d.; writing – original draft preparation, s.c., l.t.d., e.g., and h.a.; writing – review and editing, s.c., e.g., h.a., m.s., l.t.d., b.e., d.w., and p.a.; visualization, s.c. and e.g.; supervision, s.c.; project administration, s.c. and m.s.; funding acquisition, s.c. all authors have read and agreed to the published version of the manuscript. notes on contributors lee ti davidson, md, is an emergency and internal medicine specialist at linköping university hospital and is doing her research study at the institute of biomedical and clinical sciences, linköping university, linköping, sweden. emilia gauffin, md, is a resident physician at the department of clinical genetics at linköping university hospital and a phd student at linköping’s university. preben henanger, intern physician at the hospital of arvika, sweden. maciej wajda, md at department of emergency medicine and department of biomedical and clinical sciences, linköping university, linköping, sweden. admission of patients with chest pain 7 daniel wilhelms, md, phd at department of emergency medicine and department of biomedical and clinical sciences, linköping university, linköping, sweden. bertil ekman, md, phd at departments of endocrinology in norrköping and linköping, and associate professor at the department of health, medicine and caring sciences, linköping university, linköping, sweden. hans arnqvist, md, phd is a specialist in internal medicine and endocrinology at department of endocrinology in linköping and senior professor at department of biomedicine and clinical sciences, linköping university, linköping, sweden. ulf martin schilling, md, phd, is a consultant in emergency medicine and internal medicine. simona i. chisalita, md, phd, is senior consultant endocrinology, diabetology and internal medicine at the department of health, medicine and caring sciences and associate professor at the faculty of medicine and health sciences, linköping university, linköping, sweden orcid lee ti davidson https://orcid.org/0000-0001-6961-7517 emilia gauffin https://orcid.org/0000-0002-4084-1010 preben henanger https://orcid.org/0000-0002-0402-4099 daniel wilhelms https://orcid.org/0000-0001-6347-3970 bertil ekman https://orcid.org/0000-0001-8732-7361 hans arnqvist https://orcid.org/0000-0002-0368-3308 martin schilling https://orcid.org/0000-0002-0957-4604 simona chisalita https://orcid.org/0000-0002-9982-3554 references 1. goodacre s, cross e, arnold j, angelini k, capewell s, nicholl j. the health care burden of acute chest pain. heart 2005;91:229–30. doi: 10.1136/hrt.2003.027599 2. mockel m, searle j, muller r, slagman a, storchmann h, oestereich p, et al. chief complaints in medical emergencies: do they relate to underlying disease and outcome? 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discrimination of mild hyperparathyroidism with total serum calcium, ionized calcium and parathyroid hormone measurements lars benson', sverker ljunghall', torgny groth3, hans falk3, andreas hvarfnef, jonas rastad4, leif wide' and goran akerstrom4 departmenls of 'clinical chemistry, 2medicine, 4surgery, university hospital, and 'unit of biomedical systems analysis, uppsala university, uppsala, sweden a b s t r a c t t h e s e r u m c o n c e n t r a t i o n s o f calcium, a l b u m i n a n d p a r a t h y r o i d hormone ( p t h ) a n d t h e plasma levels o f i o n i z e d calcium w e r e d e t e r m i n e d in 1 2 4 h e a l t h y s u b j e c t s , 8 9 p a t i e n t s w i t h p r i m a r y h y p e r p a r a t h y r o i d i s m ( h p t ) , 2 3 o f whom h a d t h e s y n d r o m e o f m u l t i p l e e n d o c r i n e neoplasia t y p e 1 (men-1) a n d 4 3 p a t i e n t s who h a d hypercalcaemia o f o t h e r causes t h a n h p t ( n o n h p t ) , in most cases d u e t o w i d e s p r e a d malignancies. t h e t o t a l s e r u m calcium was c o r r e c t e d f o r t h e s e r u m a l b u m i n c o n c e n t r a t i o n (cam). h e a l t h y females o v e r t h e age o f 5 0 h a d h i g h e r cam, t h a n y o u n g e r females a n d t h e women o f a l l ages also h a d , h i g h e r s e r u m p t h l e v e l s t h a n males. f o r a l l s t u d y g r o u p s b o t h t h e i n t r a a n d i n t e r d i u r n a l v a r i a t i o n s w e r e small f o r a l l t h e s t u d i e d v a r i a b l e s . d i s c r i m i n a n t f u n c t i o n a n d optimal d i s c r i m i n a t o r y l i m i t s w e r e c a l c u l a t e d w i t h t h e h e l p o f c o m p u t e r p r o g r a m s . a c o n s i d e r a t i o n o f a l l t h e i n d i v i d u a l s in t h e d i s c r i m i n a n t a n a l y s i s , r e v e a l e d t h a t measurements o f cam alone s e p a r a t e d most h p t p a t i e n t s b o t h f r o m t h e h e a l t h y s u b j e c t s a n d f r o m t h e non-hpt p a t i e n t s . however, w h e n o n l y those who h a d b o r d e r l i n e values ( d e f i n e d as cam between 2 . 4 5 a n d 2 . 7 5 m m o l / l ) w e r e i n c l u d e d it t u r n e d o u t t h a t measurements o f i o n i z e d calcium m a r k e d l y i m p r o v e d t h e d e l i n e a t i o n o f m i l d h p t f r o m t h e h e a l t h y s u b j e c t s a n d t h a t , in a d d i t i o n , pth measurements h e l p e d t o e x c l u d e those w i t h n o n h p t hypercalcaemia. t h e optimal d i s c r i m i n a t o r y l e v e l s o f s e r u m calcium w e r e c a l c u l a t e d as t h e l e v e l s w h i c h c a u s e d t h e minimum loss in t e r m s o f m i s c l a s s i f i c a t i o n w h e n a t t e n t i o n was p a i d t o t h e r e l a t i v e i m p o r t a n c e o f false p o s i t i v e t o false n e g a t i v e c l a s s i f i c a t i o n s a n d t o t h e p r e v a l e n c e o f hpt. t h e optimal d i s c r i m i n a t o r y l e v e l f o r serum calcium f o r a w e i g h t i n g r a t i o between false p o s i t i v e t o false n e g a t i v e of 1:1, a n d a p r e v a l e n c e of hpt o f 1 &, was c a l c u l a t e d t o b e 2 . 6 8 mmol/i a n d f o r a p r e v a l e n c e o f 5 0 % 2 . 5 6 mmol/i. in t h e l a t t e r s i t u a t i o n a w e i g h t i n g r a t i o o f 1o:l f o r false p o s i t i v e t o false n e g a t i v e g a v e a level o f 2 . 6 3 m m o l l l w h i l e a w e i g h t i n g r a t i o o f 1 : 1 0 c o r r e s p o n d e d t o a n optimal d i s c r i m i n a t o r y l e v e l o f 2 . 4 7 mmol/i. 10-878572 147 i n t r o d u c t i o n p r i m a r y h y p e r p a r a t h y r o i d i s m ( h p t ) i s a common d i s o r d e r , k n o w n t o a f f e c t o n e p e r c e n t o f t h e p o p u l a t i o n a b o v e 60 y e a r s o f age a n d o c c u r r i n g a t e v e n h i g h e r f r e q u e n c i e s in o l d e r i n d i v i d u a l s (11,36,49). t h e high p r e valence o f t h e disease c a l l s for a c c u r a t e s c r e e n i n g a n d d i a g n o s t i c methods w h i c h c a n e a s i l y p r o v i d e a n i d e n t i f i c a t i o n o f t h e diseased p a t i e n t s w i t h a minimal n u m b e r o f misclassifications. t h e i d e n t i f i c a t i o n o f p a t i e n t s w i t h h p t in t h e c l i n i c a l r o u t i n e r e l i e s p r i m a r i l y o n t h e d e m o n s t r a t i o n o f hypercalcemia. in a p r e v i o u s s t u d y s i n g l e measurements o f t o t a l s e r u m calcium w e r e u s e d t o c a l c u l a t e optimal d i s c r i m i n a t i n g l i m i t s f o r t h e d i a g n o s i s o f p r i m a r y hpt ( 1 8 ) . in t h e p r e s e n t r e p o r t we h a v e e x t e n d e d t h i s a n a l y s i s t o i n c l u d e measurements o f t h e s e r u m con c e n t r a t i o n s o f p a r a t h y r o i d hormone ( p t h ) a n d of ionized calcium, d e t e r m i n a t i o n s o f w h i c h h a v e become more w i d e l y a v a i l a b l e during r e c e n t y e a r s . p r a c t i c a l l y a l l s t u d i e s d e a l i n g w i t h t h e d i a g n o s i s o f h p t a r e based o n m a t e r i a l s w h e r e t h e m a j o r i t y o f p a t i e n t s h a v e m a r k e d hypercalcaemia. in c l i n i c a l p r a c t i c e , h o w e v e r , most p a t i e n t s w i t h s u s p e c t e d h p t nowadays h a v e o n l y m i l d l y e l e v a t e d s e r u m calcium v a l u e s ( 2 0 . 5 0 ) . t h e p r e s e n t s t u d y t h e r e f o r e d e v o t e s p a r t i c u l a r a t t e n t i o n t o t h e p o t e n t i a l v a l u e o f i o n i z e d calcium a n d pth measurements in t h e d e l i n e a t i o n o f m i l d hpt. in many i n s t a n c e s t h e r e may n o t b e a g r e a t need t o e s t a b l i s h t h e d i a g n o s i s o f hpt in s u c h b o r d e r l i n e cases. sometimes, h o w e v e r , a p r e c i s e c l a s s i f i c a t i o n c o u l d b e i m p o r t a n t . for example we r e c e n t l y r e p o r t e d ( 2 ) t h a t in t h e d o m i n a n t l y i n h e r i t e d s y n d r o m e o f m u l t i p l e endocrine, neoplasia t y p e 1 (men-1) h p t i s a p p a r e n t l y t h e f i r s t m a n i f e s t a t i o n . t h e d e m o n s t r a t i o n o f m i l d h p t t h e r e f o r e i s t h e e a r l i e s t o p p o r t u n i t y t o disclose t h e c a r r i e r o f t h e men-1 t r a i t . p a t i e n t s w i t h r e c u r r e n t r e n a l stones c o n s t i t u t e a n o t h e r g r o u p w h e r e it i s highly d e s i r a b l e t o o b t a i n d e f i n i t e e v i d e n c e f o r o r a g a i n s t hpt d e s p i t e u n d e c i d e d hypercalcemia . a n o t h e r c l i n i c a l p r o b l e m in t h e d i a g n o s i s o f hpt c o n s i s t s o f t h e e x c l u s i o n o f o t h e r causes o f hypercalcemia, p r i m a r i l y m a l i g n a n t d i s o r d e r s . a l t h o u g h these a r e g e n e r a l l y e v i d e n t c l i n i c a l l y , a d d i t i o n a l i n v e s t i g a t i o n s a r e sometimes r e q u i r e d . i48 reference sample groups h e a l t h y s u b j e c t s from a h e a l t h s u r v e y in uppsala c o u n t y 98 a p p a r e n t l y h e a l t h y indivi d u a l s w e r e r e c r u i t e d t o r e p r e s e n t a " h e a l t h y r e f e r e n c e sample g r o u p " ; 5 2 men a n d 46 women, a g e d between 16 t o 9 2 , w i t h a mean age of 46 f. 20 y e a r s (mean f s d ) , a n d equal d i s t r i b u t i o n o f t h e sexes in a l l age g r o u p s . t w e n t y s i x a p p a r e n t l y h e a l t h y employees from t h e h o s p i t a l s t a f f , 10 men a n d 1 6 women, a g e d between 20-60 y e a r s , p a r t i c i p a t e d in s t u d i e s o f t h e i n t r a a n d , i n t e r d i u r n a l v a r i a t i o n s . patients w i t h h p t : 89 c o n s e c u t i v e p a t i e n t s o p e r a t e d f o r hpt, 2 7 men a n d 62 women, a g e d between 19-83 w i t h a mean o f 60 f 17 y e a r s w e r e s t u d i e d b e f o r e o p e r a t i o n : 66 o f them h a d s p o r a d i c hpt a n d s e r u m calcium was normalized p o s t o p e r a t i v e l y in a l l cases. t h e o t h e r 2 3 , 1 6 women a n d seven men, h a d h p t as a p a r t o f men-1, n i n e o f them h a d p e r s i s t e n t o r r e c u r r e n t hypercalcaemia a f t e r p r e v i o u s o p e r a t i o n s . f i v e o f them also h a d a n e n d o c r i n e t u m o r o f t h e pancreas, in t h r e e cases w i t h l i v e r metastases. neck e x p l o r a t i o n c o n f i r m e d p a r a t h y r o i d h y p e r p l a s i a . s u b t o t a l o r t o t a l p a r a t h y roidectomy w i t h a u t o t r a n s p l a n t a t i o n was p e r f o r m e d . serum calcium r e t u r n e d t o t h e normal r a n g e p o s t o p e r a t i v e l y in a l l cases. p a t i e n t s w i t h hypercalcemia o f o t h e r o r i g i n t h a n hpt ( n o n h p t ] : f o r t y t h r e e p a t i e n t s , 21 men a n d 2 2 women, aged 38-78 w i t h a mean age o f 60 f. 15 y e a r s h a d hypercalcemia o f c l i n i c a l l y o b v i o u s causes o t h e r t h a n hpt. m a l i g n a n c y was t h e most common cause, b e i n g e n c o u n t e r e d in 3 0 pa t i e n t s , 13 men a n d 17 women. renal c a n c e r was seen in s e v e n cases, f i v e h a d cancer mammae, f i v e h a d lung c a n c e r , f i v e h a d myeloma, t h r e e cancer o f t h e pancreas, t h r e e lymphomas, one h a d t h y r o i d c a n c e r a n d one p a t i e n t h a d a leiomyosarcoma. in most cases b o n e metastases w e r e e v i d e n t . o t h e r causes of hypercalcemia w e r e e n c o u n t e r e d in 13 p a t i e n t s , s i x men a n d s e v e n women. sarcoidosis was seen in s e v e n p a t i e n t s , whose s e r u m calcium v a l u e s n o r m a l i z e d u p o n t r e a t m e n t w i t h s t e r o i d s . t w o p a t i e n t s h a d t h y r o t o x i cosis, a n d became normocalcemic in response t o medical t r e a t m e n t . immobili zation d u e t o t e t r a p l e g i a was t h e cause o f hypercalcaemia in f o u r p a t i e n t s . 149 l a b 0 r a t 0 ry met h od s blood specimens w e r e c o l l e c t e d b e t w e e n 07.00-09.00 o n t h e m o r n i n g f o l l o w i n g a n o v e r n i g h t f a s t . f o r each p a t i e n t t h e mean v a l u e o f a l l s u c h measurements was c a l c u l a t e d a n d u s e d as t h e basal value. f o r t h e s t u d y o f v a r i a t i o n during t h e d a y , specimens w e r e also c o l l e c t e d b e f o r e meals a t 1 2 . 0 0 a n d 16.00 h o u r s . no d i e t r e s t r i c t i o n s a p a r t f r o m a b a n o n m i l k a n d cheese w e r e imposed. i o n i z e d calcium ( c a l l : whole b l o o d was c o l l e c t e d a n a e r o b i c a l l y in 5 ml h e p a r i n i z e d t u b e s a n d a n a l y z e d w i t h i n a few h o u r s f o r i o n i z e d calcium w i t h a n ion-selective e l e c t r o d e ( m i c r o l y t e , kone i n s t r u m e n t s , f i n l a n d ) . all samples w e r e measured in d u p l i c a t e . t h e a n a l y z e r has a n automatic t h r e e p o i n t c a l i b r a t i o n p r o c e d u r e u s i n g w a t e r s t a n d a r d s a d j u s t e d f o r i o n i c s t r e n g t h a n d p h . a f t e r each sample a m i d d l e s t a n d a r d i s measured f o r assessment o f drift. t h e t e m p e r a t u r e o f t h e e l e c t r o d e b l o c k i s m a i n t a i n e d a t 3ooc. in p a t i e n t samples t h e a v e r a g e a n a l y t i c a l w i t h i n r u n s t a n d a r d d e v i a t i o n was 0 . 0 1 2 mmol/i a t t h e level o f 1 . 1 5 mmol/i a n d 0.019 mmol/l a t t h e l e v e l o f 1.45 mmol/l. t h e l i f e s p a n o f a n e l e c t r o d e i s 3 6 m o n t h s . a f t e r c h a n g e o f e l e c t r o d e , t h e v a l u e s (n=178) f r o m a r e f e r e n c e p o p u l a t i o n of h e a l t h y i n d i v i d u a l s w e r e compared w i t h p r e v i o u s v a l u e s (n=100) in t h e same i n d i v i d u a l s . a d i f f e r e n c e o f 0.01 mmol/l was o b s e r v e d f o r t h e mean v a l u e s , w i t h o u t c h a n g e in i m p r e c i s i o n . t h e r e a r e n o e x t e r n a l c o n t r o l s t a n d a r d s f o r i o n i z e d calcium, a n d e x t e r n a l q u a l i t y assessment schemes h a v e s t r e s s e d t h e u s e o f p r o t e i n e n r i c h e d s o l u t i o n s ( 4 4 ) . in t h e absence o f a n e x t e r n a l c o n t r o l sample, r e c o n s t i t u t e d l y o p h i l i z e d c o n t r o l sera ( v a l i d a t e , general d i a g n o s t i c s ) f r o m t h e same b a t c h a c t u a l l y n o t m a n u f a c t u r e d f o r t h i s p u r p o s e a n d n o t always w i t h i n t h e r e f e r e n c e r a n g e f o r h e a l t h y s u b j e c t s , h a v e b e e n u s e d as a n e x t e r n a l c o n t r o l o v e r time w i t h a c v o f 1 . 4 % a t 1 . 2 0 mmol/i a n d 2 . 3 % a t 1 . 6 0 mmol/l. no e f f o r t was t a k e n t o i m p r o v e s t a n d a r d i z a t i o n o f w a t e r , t e m p e r a t u r e , p h o r g a s c o n t e n t ( 9 ) . serum was o b t a i n e d a f t e r c l o t t i n g a n d c e n t r i f u g a t i o n , a n d was e i t h e r a n a l y z e d in t h e o r d i n a r y l a b o r a t o r y r o u t i n e t h e same d a y , o r s t o r e d a t +4o o v e r n i g h t a n d a n a l y z e d t h e n e x t d a y . t o t a l s e r u m calcium ( c a t ) c o n c e n t r a t i o n s w e r e d e t e r m i n e d by a n atomic a b s o r p t i o n t e c h n i q u e ( perkin-elmer 3 0 3 0 ) . t h e a n a l y t i c a l p r o c e d u r e f o r d e t e r m i n a t i o n o f s e r u m calcium h a d a n a v e r a g e w i t h i n r u n s t a n d a r d d e v i a t i o n o f 0 . 0 3 8 mmol/l, a n d a n a v e r a g e b e t w e e n r u n s t a n d a r d d e v i a t i o n o f 150 0.022 mmol/l, giving a t o t a l a n a l y t i c a l s t a n d a r d d e v i a t i o n o f 0.044 mmol/l. a l l v a l u e s r e f e r t o a c o n c e n t r a t i o n l e v e l o f 2.46 mmol/i, a n d t h e a n a l y t i c a l e r r o r was assumed t o b e t h e same f o r a l l c o n c e n t r a t i o n l e v e l s e x p r e s s e d as a c o e f f i c i e n t o f v a r i a t i o n (cv=0.018). t h e a n a l y t i c a l b i a s o f t h e p r o c e d u r e was a b o u t -1% compared w i t h t h e r e f e r e n d u m v a l u e o f t h e r e g i o n a l e x t e r n a l q u a l i t y assessment p r o g r a m . serum a l b u m i n was d e t e r m i n e d by a b r o m c r e s o l b i n d i n g t e c h n i q u e a n d c a l i b r a t e d w i t h p u r i f i e d human a l b u m i n s o l u t i o n . t h e a n a l y t i c a l w i t h i n r u n s t a n d a r d d e v i a t i o n was 0.42 g / l a n d t h e b e t w e e n r u n s t a n d a r d d e v i a t i o n 0.71 g / l , giving a t o t a l a n a l y t i c a l s t a n d a r d d e v i a t i o n o f 0.83 g / l . c o r r e c t i o n o f cat f o r s e r u m a l b u m i n c o n c e n t r a t i o n ( c a m ) : a s s e r u m calcium i s b o u n d t o a l b u m i n , a c o r r e c t i o n ( m o d i f i c a t i o n ) o f s e r u m calcium v a l u e s was made f o r d e v i a t i o n s o f t h e a c t u a l a l b u m i n f r o m t h e r e f e r e n c e mean v a l u e o f 42 g / l by t h e f o l l o w i n g formula u s e d in o u r l a b o r a t o r y : cam = cat 0.019 (s-albumin 421 mmol/i radioimmunoassay o f p a r a t h y r o i d hormone ( p t h ) in s e r u m serum specimens w e r e k e p t a t 2ooc until analyzed. t h e p t h concen t r a t i o n was d e t e r m i n e d by a radioimmunoassay s y s t e m e m p l o y i n g 121 i labelled b o v i n e p t h ( i n o l e x ) a n d sheep a n t i s e r u m ( s 478) a g a i n s t p o r c i n e a n d b o v i n e pth. t h i s a n t i s e r u m r e a c t s w i t h a m i d p o r t i o n (44-68) o f human p t h but has also a high a f f i n i t y ( 0 . 6 x 1013 i/mmol) f o r i n t a c t human pth (21 1 . t h e assay p r o c e d u r e u s e d s o l i d phase-coupled a n t i s h e e p l g g t o s e p a r a t e b o u n d a n d f r e e labelled pth. t h e s e r u m specimens a n d t h e a n t i s e r u m w e r e f i r s t i n c u b a t e d f o r 2 4 h a t 4oc followed by a 48 h i n c u b a t i o n w i t h l a b e l l e d pth . microsepharose-coupled h o r s e a n t i s h e e p l g g ( d e c a n t i n g suspension 2, pharmacia ab, sweden) was t h e n a d d e d a n d t h e i n c u b a t i o n was p r o l o n g e d f o r 3 h. t h e p a r t i c l e s w e r e c e n t r i f u g e d d o w n during 5 m i n a t 2000 g a n d t h e p e l l e t was washed once w i t h saline c o n t a i n i n g 0.5 % tween-20. b o v i n e pth d i l u t e d in human sera w i t h low pth l e v e l s was u s e d as labora t o r y s t a n d a r d a n d t h e c o n c e n t r a t i o n o f p t h in human s e r u m was e x p r e s s e d in a r b i t r a r y u n i t s ( a r b u l l ) . a b o u t 2.5 a r b u w e r e e q u i v a l e n t t o 1 0 u n i b s c r e s e a r c h s t a n d a r d f o r human p t h 75/479. a l l specimens w e r e assay e d in d u p l i c a t e . t h e t o t a l assay c o e f f i c i e n t o f v a r i a t i o n in 42 assays was 9.2%. w i t h a n a v e r a g e w i t h i n a s s a y c o e f f i c i e n t of v a r i a t i o n o f 3.6% a t a l e v e l o f 0.7-0.8 a r b u/i. 151 t h e disappearance o f s e r u m p t h f o l l o w i n g p a r a t h y r o i d e c t o r n y was i n v e s t i g a t e d in 1 0 p a t i e n t s w i t h adenomatous h p t , basal values o f 0 . 8 1 1.24 a r b u / l a n d a normal k i d n e y f u n c t i o n . a mean r e d u c t i o n o f 17% was f o u n d w i t h i n t h e f i r s t 1 5 m i n u t e s , w h i c h d e m o n s t r a t e d a c a p a b i l i t y o f t h e assay system r a p i d l y t o d e t e c t changes in s e r u m c o n c e n t r a t i o n s ( f i g . 1 ) . 0 -5 -10 -15 2 0 -25 4 fig. 1 . r e d u c t i o n o f s e r u m p a r a t h y r o i d hormone ( p t h ) c o n c e n t r a t i o n s a f t e r p a r a t h y r o i d e c t o m y in 10 p a t i e n t s w i t h adenornatous h y p e r p a r a t h y r o i d i s m . ( b a r s i n d i c a t e sem). 152 c o m p u t a t i o n a l methods a l l s t a t i s t i c a l c a l c u l a t i o n s w e r e p e r f o r m e d o n a b a s f 7 / 7 3 i b m / m v s computer system a t uppsala u n i v e r s i t y data c e n t e r . t h e s t a t i s t i c a l a n a l y s i s system p a c k a g e (sas i n s t i t u t e i n c . , n o r t h c a r o l i n a u s a ) was u s e d f o r d e s c r i p t i v e s t a t i s t i c s (means a n d s t a n d a r d d e v i a t i o n s o f v a r i o u s r e f e r e n c e sample g r o u p s ; biological i n t r a a n d i n t e r d i u r n a l v a r i a t i o n ; c r o s s p l o t t i n g ; s t a t i s t i c a l g o o d n e s s o f f i t t e s t s ) , l i n e a r r e g r e s s i o n analysis; a n d n o n l i n e a r parameter fitting. stepwise d i s c r i m i n a n t a n a l y s i s was p e r f o r m e d w i t h t h e bmdp p r o g r a m package (biomedical computer programs, p-series, u n i v e r s i t y o f c a l i f o r n i a , 1 9 7 7 ) . a p r o g r a m f o r a n a l y s i s o f v a r i a n c e o n a luxor abc-80 c o m p u t e r was u s e d t o c a l c u l a t e t h e a n a l y t i c a l w i t h i n , a n d b e t w e e n r u n v a r i a t i o n . optimal d i s c r i m i n a t o r y l i m i t s w e r e c a l c u l a t e d w i t h t h e h e l p o f a p r o g r a m developed a t t h e u n i t o f biomedical systems a n a l y s i s ( 1 8 ) . t h i s p r o g r a m ca icu lates : ( a ) t h e e x p e c t e d f r e q u e n c y o f false n e g a t i v e a n d false p o s i t i v e outcomes in c o n n e c t i o n w i t h c l a s s i f i c a t i o n , u s i n g a s p e c i f i e d d i s c r i m i n a t o r y level; ( b ) a measure o f loss r e l a t e d t o misclassification, ( c ) t h e optimal d i s c r i m i n a t o r y l i m i t , ( e ) , a n d ( d ) t h e d i a g n o s t i c s e n s i t i v i t y , s p e c i f i c i t y a n d t h e p r e d i c t e d v a l u e o f a p o s i t i v e a n d n e g a t i v e t e s t r e s u l t . input data t o t h e p r o g r a m a r e : ( a ) f r e q u e n c y d i s t r i b u t i o n s o f t h e r e f e r e n c e p o p u l a t i o n s ; in t h i s case d i s t r i b u t i o n s r e p r e s e n t i n g h e a l t h y i n d i v i d u a l s , p a t i e n t s w i t h s p o r a d i c hpt o r h p t as p a r t o f men-1, as well as p a t i e n t s w i t h non-hpt hypercalcemia ; ( b ) p r e v a l e n c e o f t h e diseases, e x p r e s s e d as n u m b e r r a t i o s ; ( c ) n u m e r i c a l w e i g h t s , w1 a n d w2, r e p r e s e n t i n g t h e r e l a t i v e c o s t s o f m a k i n g misclassifications; [ d ) p r e a n a l y t i c a l a n d a n a l y t i c a l v a r i a t i o n e x p r e s s e d as c o e f f i c i e n t s o f v a r i a t i o n : “pre-a a n d c v a , r e s p e c t i v e l y ; t h e v a r i a n c e s o f f r e q u e n c y d i s t r i b u t i o n s r e p r e s e n t i n g t h e d i f f e r e n t r e f e r ence p o p u l a t i o n s a r e calculated f r o m : 153 2 2 2 + ' a t o t a l b i o l + p r e a 2 a n d t h e r e f o r e 2 2 2 b i o l t o t a l (' p r e a w h e r e s i s t h e t o t a l b i o l o g i c a l s t a n d a r d d e v i a t i o n ( i n c l u d i n g i n t r a , a n d i n t e r i n d i v i d u a l v a r i a t i o n ) ; spre-a i s t h e p r e a n a l y t i c a l s t a n d a r d d e v i a t i o n , i.e. t h e v a r i a t i o n r e l a t e d t o specimen h a n d l i n g ; a n d sa i s t h e a n a l y t i c a l s t a n d a r d d e v i a t i o n . in t h e p r o g r a m t h e c a l c u l a t i o n s a r e p e r f o r m e d t o estimate t h e "tail-areas" c u t o f f by a s p e c i f i e d d i s c r i m i n a t o r y l i m i t f r o m t h e d i f f e r e n t d i s t r i b u t i o n s , giving v a l u e s f o r t h e n u m b e r o f false p o s i t i v e s ( f p ) a n d false n e g a t i v e s ( f n ) . f n ) / $ o w h e r e 4 i s t h e loss u n d e r ideal c o n d i t i o n s , b i o l a s p r e v i o u s l y d e s c r i b e d ( 1 8 ) t h e loss i s c a l c u l a t e d as a = ( w l x f p + w 2 x cva=o a n d cvpre-a =o . since t h e w e i g h t i n g f a c t o r s a r e g i v e n in r e l a t i v e n u m b e r s , t h e loss s h o u l d b e r e g a r d e d as a r e l a t i v e loss. t h e c a l c u l a t i o n s a r e automatically r e p e a t e d f o r a n u m b e r o f d i f f e r e n t values f o r t h e d i s c r i m i n a t o r y level, in o r d e r t o allow d e t e r m i n a t i o n o f t h e optimal value. results d e s c r i p t i v e s t a t i s t i c s : t a b l e 1 summarizes t h e mean v a l u e s f o r t h e s t u d i e d v a r i a b l e s in a l l t h e f i v e g r o u p s o f s u b j e c t s . t h e mean calcium v a l u e s w e r e h i g h e r in t h e p a t i e n t s w i t h n o n h p t hypercalcaemia t h a n in t h e h p t p a t i e n t s . t h e hpt p a t i e n t s h a d a h i g h e r mean v a l u e f o r p t h t h a n a l l o t h e r g r o u p s . t h e r e w e r e small, but s t a t i s t i c a l l y s i g n i f i c a n t , agea n d s e x r e l a t e d d i f f e r e n c e s w i t h i n t h e g r o u p o f normal s u b j e c t s ( t a b l e 2 ) . women o v e r t h e age o f 5 0 h a d h i g h e r cam values t h a n y o u n g e r females. o n t h e o t h e r h a n d males o v e r t h e age o f 5 0 showed lower cat ( b u t n o t cam) v a l u e s t h a n did t h e y o u n g e r men. no d i f f e r e n c e s w e r e n o t e d f o r c a l b e t w e e n t h e s u b g r o u p s . females o f a l l ages h a d c l e a r l y h i g h e r v a l u e s f o r p t h t h a n males. t h e a v e r a g e t o t a l i n t r a d i u r n a l v a r i a t i o n s ( i n c l u d i n g b i o l o g i c a l , p r e a n a l y t i c a l a n d a n a l y t i c a l ) f o r t h e d i f f e r e n t g r o u p s a r e g i v e n in t a b l e 3. t h e r e w e r e n o s i g n i f i c a n t v a r i a t i o n s o v e r t h e d a y in a n y o f these g r o u p s f o r a n y o f t h e v a r i a b l e s s t u d i e d . t a b l e 4 g i v e s t h e i n t r a i n d i v i d u a l v a r i a t i o n s b o t h w i t h i n a n d b e t w e e n d a y s . a s c a n b e seen in t h e t a b l e t h e v a r i a t i o n s f o r t h e calcium measure 154 ments w e r e somewhat g r e a t e r in t h e hypercalcaemic i n d i v i d u a l s t h a n in t h e h e a l t h y s u b j e c t s . n a t u r a l l y , f o r a l l s t u d i e d v a r i a b l e s t h e i n t e r d i u r n a l v a r i a t i o n s w e r e g r e a t e r t h a n t h e i n t r a d i u r n a l but g e n e r a l l y t h e d i f f e r e n c e s were small. o n t h e b a s i s o f these measurements it c o u l d b e c a l c u l a t e d t h a t t h e biological sd f o r t h e c a l v a l u e s was 0.045 mmol/i f o r b o t h h e a l t h y s u b j e c t s a n d p a t i e n t s w i t h h p t w h i l e f o r cam it was 0.055 m m o l / l in b o t h g r o u p s . s i m i l a r l y t h e b i o l o g i c a l v a r i a t i o n f o r p t h was c a l c u l a t e d t o b e 0.14 a r b u / i in t h e h e a l t h y s u b j e c t s a n d 0.09 a r b u / i in t h e p a t i e n t s w i t h h p t . t a b l e 1. mean v a l u e , s t a n d a r d d e v i a t i o n ( s d ) , a n d s t a n d a r d e r r o r o f t h e mean (sem) f o r t h e measured v a r i a b l e s in t h e d i f f e r e n t s u b j e c t g r o u p s . n mean sd s em plasma i o n i z e d calcium ( m m o l / l ) ( c a t ) h e a l t h y 93 1.203 0.047 0.005 hpt, s p o r a d i c 52 1.400 0.17 0.024 h p t /men 12 1.416 0.133 0.038 non-hpt 43 1.513 0.242 0.037 t o t a l s e r u m calcium ( m m o l / l ] ( c a t ) h e a l t h y 98 2.425 0.082 0.009 h p t , s p o r a d i c 65 2.836 0.287 0.035 h p t / men 23 2.745 0.192 0.040 non-hpt 43 3.103 0.538 0.081 serum a l b u m i n l a / l l h e a l t h y 98 43.06 2.86 0.30 hpt, s p o r a d i c 65 38.50 3.83 0.28 h p t / m e n 23 39.78 4.55 0.62 non-hpt 43 35.32 5.49 0.89 a l b u m i n m o d i f i e d s e r u m calcium ( m m o l / l ) (cam) heal thy 98 2.415 0.079 0.008 hpt, s p o r a d i c 65 2.908 0.301 0.037 h p t / m e n 23 2.767 0.205 0.043 non-hpt 43 3.245 0.526 0.079 serum p t h ( a r b u l l ) heal thy 98 0.75 0.17 0.02 hpt, s p o r a d i c 65 1.27 0.68 0.05 h p t /men 23 1.20 0.50 0.07 non-hpt 43 0.80 0.25 0.04 155 t a b l e 2. values in h e a l t h y s u b j e c t s s e p a r a t e d w i t h r e g a r d t o age a n d sex (mean f sd) . age < 50 y e a r s > 50 y e a r s a l i (n = 50) ( n = 48) c a l ( m m o l i i ) men 1.21 ? 0.05 1.20 f 0.06 1.21 f 0.05 women 1.20 ? 0.04 1.21 f 0.04 1.20 f 0.04 c a t men 2.45 f 0.07 women 2.41 f 0.10 a l b u m i n ( g l l ) men 44.2 f 2.9 women 44.0 f 3.5 cam men 2.40 f 0.07 women 2.38 ? 0.08 p t h ( a r b u / l ) men 0.67 2 0.17 women 0.82 f 0.18 2.40 f 0.08*) 2.42 f 0.09 42.2 f 3.3*) 41.9 f 1.8**) 2.40 f 0.09 2.43 f 0.08**) 0.71 i 0.14 0.85 f 0.14 2.43 f 0.07 2.40 f 0.09 43.5 f 3.2 43.0 f 3.0 2.40 k 0.07 2.40 f 0.08 0.68 t 0.16 0.83 f 0.16***) * ) p < 0.05 compared w i t h men < 5 0 y e a r s . * * ) p < 0.05 compared w i t h women < 50 y e a r s . * * * ) p < 0.001 compared w i t h males. r e l a t i o n s h i p s b e t w e e n c a l a n d cam b o t h t h e h e a l t h y s u b j e c t s ( r = 0.33; p = 0.00 ) a n d t h e h p t p a t i e n t s ( r = 0.90; p < o . o o l ) d i s p l a y e d highly s i g n i f i c a n t c o r r e l a t i o n s b e t ween t h e v a l u e s f o r cam a n d c a l . however, t h e slope f o r t h e r e g r e s sion e q u a t i o n was s t e e p e r f o r t h e h p t p a t i e n t s t h a n f o r t h e h e a l t h y s u b j e c t s ( f i g . 2). t h e r e w e r e n o s i g n i f i c a n t d i f f e r e n c e s as r e g a r d s t h e r e l a t i o n s h i p s b e t w e e n cam a n d c a l ( d a t a n o t s h o w n ) b e t w e e n h p t p a t i e n t s a n d those w i t h o t h e r causes o f hypercalcaemia . within t h e r a n g e o f cam v a l u e s b e t w e e n 2.45 a n d 2.75 m m o l l l t h e r e was n o s i g n i f i c a n t c o r r e l a t i o n b e t w e e n t h e cam a n d c a l v a l u e s f o r a n y o f t h e s t u d y g r o u p s (fig. 3). 156 t a b l e 3. a v e r a g e t o t a l i n t r a d i u r n a l v a r i a t i o n s in h e a l t h y s u b j e c t s ( n = 52), a n d p a t i e n t s w i t h h y p e r p a r a t h y r o i d i s m ( n = 50) a n d o t h e r hypercalcaernia ( n = 2)). h e a l t h y s u b j e c t s hpt non-hpt h o u r mean sd mean sd mean sd 08.00 12.00 16.00 08.00 12.00 16.00 08.00 12.00 16.00 08.00 12.00 16.00 08.00 12.00 16.00 c a l ( m m o l / l ) 1.15 0.045 1.15 0.042 1.14 0.051 cat i m m o l / l ) 2.45 0.102 2.45 0.082 2.47 0.095 a l b u m i n (911) 42.1 3.1 42.9 2.6 42.9 3.3 cam ( m m o l l l l 2.46 0.07 2.45 0.06 2.46 0.06 pth ( a r b u / i 1 0.80 0.14 0.80 0.14 0.81 0.13 1.40 1.40 1.38 2.90 2.94 2.90 40.0 40.5 39.5 2.91 2.98 2.91 1.16 1.17 1.18 0.179 0.183 0.198 0.375 0.388 0.362 3.7 4.2 3.4 0.39 0.39 0.36 0.53 0.53 0.55 1.37 0.145 1.36 0.142 1.32 0.149 2.80 0.357 2.79 0.340 2.77 0.304 33.7 2.4 33.1 3.3 33.2 3.7 2.97 0.38 2.93 0.35 2.91 0.34 0.78 0.27 0.78 0.26 0.80 0.27 r e l a t i o n s h i p s between cam a n d p t h t h e r e w e r e n o s i g n i f i c a n t c o r r e l a t i o n s between t h e cam a n d p t h values e i t h e r in t h e g r o u p o f h e a l t h y s u b j e c t s ( r = 0.04) o r among t h o s e w i t h non-hpt hypercalcaemia ( f i g . 4). 157 t a b l e 4. a v e r a g e t o t a l i n t r a i n d i v i d u a l v a r i a t i o n w i t h i n a n d between d a y s . w i t h i n d a y between-day (sd) (sd) c a l ( m m o l / l ) h e a l t h y 0.019 0.027 h p t 0.026 0.031 non-hpt 0.041 0.060 cat (rnmol/l) h ea i thy 0.043 0.032 h p t 0.054 0.071 non-hpt 0.050 0.111 a l b u m i n ( g l l ) h e a l t h y 0.221 1.187 h p t 1.502 1.750 non-hpt 0.992 1.110 cam ( m m o l / l ) heal thy 0.049 0.07 h p t 0.051 0.07 non-hpt 0.063 0.12 p t h ( a r b u / i ) h e a l t h y 0.044 0.16 h p t 0.050 0.12 non-hpt 0.045 0.05 when a l l t h e cam v a l u e s f o r t h e hpt p a t i e n t s w e r e c o n s i d e r e d a highly s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between cam a n d p t h was e v i d e n t ( r = 0.62; p<o.ool) as seen in f i g . 4. t h e r e was n o s i g n i f i c a n t d i f f e r e n c e in t h i s r e s p e c t b e t w e e n h p t associated w i t h men-1 a n d t h e s p o r a d i c f o r m . when o n l y cam v a l u e s below 2.75 m m o l / l w e r e c o n s i d e r e d t h e r e was, h o w e v e r , n o s i g n i f i c a n t c o r r e l a t i o n ( r = 0.20; p = 0.30) b e t w e e n t h e v a l u e s in t h e h p t p a t i e n t s ( f i g . 5 ) . 158 ca i (mmol/l) 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 r e a // a 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.8 1.4 1.2. 1.0 0.8 0.8 0.4 0 0 0 . ow 0 0 ow 0 0 0000 0 0 0 0 . . . .. a . . * 0 . . . 0:. n 2 . 0 0 d 0 0 " o m n . 0 " 0 0 = healthy subjects = hpt 1 .oj i 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 gi cam (mmol/l) f i g . 2 . r e l a t i o n s h i p between a l b u m i n c o r r e c t e d s e r u m calcium (cam) a n d plasma i o n i z e d calcium ( c a l l c o n c e n t r a t i o n s in h e a l t h y s u b j e c t s a n d p a t i e n t s w i t h h y p e r p a r a t h y r o i d i s m ( h p t ) . t h e i n t e r r u p t e d l i n e denotes t h e r e g r e s s i o n l i n e f o r t h e h e a l t h y s u b j e c t s a n d t h e s o l i d l i n e t h a t ' f o r t h e h p t p a t i e n t s . . 0 . . 0 . 0 = healthy s = hpt 3 = non-hpt n 2.20 2.40 2.80 2.80 3.00 3.20 3.40 3.80 3.80 4.00 4.20 cam (mmolll) f i g . 3 . r e l a t i o n s h i p b e t w e e n cam a n d c a l f o r v a l u e s o f cam b e t w e e n 2 . 4 5 a n d 2.75 mmol/l. 159 0 healthy 0 hpt 11 non-hpt 0 0 0 0 0 0 0 0 0 0 . 0 0 0 0 0 0 . 0 0 0 0 n 0 1 2.45 2.50 2.55 2.60 2.65 2.70 2.75 cam (mmolll) fig. 4. r e l a t i o n s h i p b e t w e e n p t h a n d a l b u m i n c o r r e c t e d s e r u m calcium (cam) in h e a l t h y s u b j e c t s , p a t i e n t s w i t h h p t a n d p a t i e n t s w i t h o t h e r causes o f hypercalcemia ( n o n h p t ) . 0 = healthy = sporadic hpt a = hptlmen . . . 0 0 . . 0 b " i * * * * .. 0 0 0 0 0 0 * * 0 0 0 0 0 0 0 0 0 0 . " 2 0 0 1 1.30 1.35 1.40 1.45 1.15 1.20 1.25 ca i (mnov0 f i g . 5 . r e l a t i o n s h i p b e t w e e n a l b u m i n c o r r e c t e d s e r u m calcium (cam) a n d p t h in h e a l t h y s u b j e c t s a n d h p t p a t i e n t s . o n l y i n d i v i d u a l s w i t h cam v a l u e s b e t w e e n 2.45 a n d 2 . 7 5 mmol/l a r e i n c l u d e d . 160 t a k e n as a g r o u p , p a t i e n t s w i t h hypercalcaemia d u e t o o t h e r causes t h a n h p t h a d lower v a l u e s for p t h f o r c o r r e s p o n d i n g cam v a l u e s t h a n t h e h p t p a t i e n t s but t h e r e was a c o n s i d e r a b l e o v e r l a p , p a r t i c u l a r l y f o r cam v a l u e s below 3.0 m m o l l l ( f i g . 4 ) . r e l a t i o n s h i p b e t w e e n c a l a n d pth f o r t h e h e a l t h y s u b j e c t s t h e r e was n o r e l a t i o n s h i p b e t w e e n t h e c a l a n d p t h v a l u e s ( r = 0.15; p = 0.15). f o r t h e h p t p a t i e n t s , h o w e v e r , t h e r e was a p o s i t i v e c o r r e l a t i o n ( r = 0.63; p < o . o o l ) w h e n a l l values w e r e c o n s i d e r e d , but n o t in o n l y t h o s e w i t h cam v a l u e s below 2.75 m m o l l l ( f i g . 6 ) . d i s c r i m i n a n t a n a l y s i s a s c a n b e seen f r o m f i g s . 2-6 t h e r e w e r e o v e r l a p s f o r cam, c a l a n d p t h between b o t h h e a l t h y s u b j e c t s a n d h p t p a t i e n t s as w e l l as between t h e l a t t e r g r o u p a n d t h o s e w i t h o t h e r causes o f hypercalcaemia. a n a t t e m p t was t h e r e f o r e made by means of d i s c r i m i n a n t a n a l y s i s t o find t h e most e f f i c i e n t c o m b i n a t i o n o f t h e l a b o r a t o r y t e s t s f o r t h e s e p a r a t i o n o f h p t f r o m h e a l t h y s u b j e c t s , a n d t o d i f f e r e n t i a t e b e t w e e n h p t a n d o t h e r causes o f e l e v a t e d s e r u m calcium levels. t h i s a n a l y s i s was c a r r i e d o u t in t w o steps. in t h e f i r s t , a l l indivi d u a l s w e r e i n c l u d e d . t h e r e a f t e r , o n l y those w i t h cam v a l u e s b e t w e e n 2.45 a n d 2.75 m m o l l l w e r e e v a l u a t e d . in b o t h t h e s e a n a l y s e s c o n s i d e r a t i o n was g i v e n t o t h e agea n d sex v a r i a t i o n s f o r cam a n d p t h in t h e h e a l t h y s u b j e c t s . t h e a n a l y s i s was p e r f o r m e d f o r a p r e v a l e n c e o f 50 % a n d w i t h e q u a l c o s t w e i g h t s g i v e n t o t h e false p o s i t i v e s a n d false n e g a t i v e s . t h e r e s u l t o f t h e d i s c r i m i n a n t f u n c t i o n analyses a r e g i v e n in t a b l e 5. in t h e f i r s t p a r t o f t h i s a n a l y s i s , w h e n t h e whole r a n g e o f v a l u e s w e r e i n c l u d e d , it a p p e a r e d t h a t measurements o f cam s u f f i c e d t o p r o v i d e a n optimal s e p a r a t i o n o f t h e g r o u p o f p a t i e n t s w i t h h p t f r o m t h e h e a l t h y s u b j e c t s . all t h e h e a l t h y s u b j e c t s h a d a cam below 2.657 but 1 4 p a t i e n t s w i t h h p t w e r e e r r o n e o u s l y c l a s s i f i e d h e a l t h y w i t h t h i s l i m i t . in t h i s model n e i t h e r measurements o f c a l n o r p t h i m p r o v e d t h e d i s c r i m i n a t o r y p o w e r . 161 pth (arb u/i) 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 . . . . 0 0 0 0. 0 0 0 0 0 . 6 0 0 , 0 0 0 0 = healthy = sporadic hpt a = hptlmen . . 0 0 0 .* 0 .. 0 * * .. * a . 0 0 0 0 0 0 0. . . 0 . 4 8 0 2.45 2.50 2.55 2.60 2.65 2.70 2.75 cam (mmol/l) f i g . 6. r e l a t i o n s h i p b e t w e e n plasma i o n i z e d calcium ( c a l ) a n d p t h f o r t h e same i n d i v i d u a l s as in fig. 5. when, h o w e v e r , t h e model was r e s t r i c t e d t o i n c l u d e o n l y s u b j e c t s w i t h v a l u e s f o r cam b e t w e e n 2.45 a n d 2.75 m m o l l l i t a p p e a r e d t h a t c a l allowed c o n s i d e r a b l e a d d i t i o n a l s e p a r a t i o n o f t h e t w o g r o u p s . t h u s , t h e u s e o f t h e combined measurements r e s u l t e d in o n l y t w o misclassifications ( t a b l e 5 a l l ) . in b o t h these s i t u a t i o n s t h e d i v i s i o n o f t h e m a t e r i a l w i t h r e g a r d t o age a n d sex o n l y caused m i n o r a l t e r a t i o n s o f t h e c l a s s i f i c a t i o n s . f o r t h e d i f f e r e n t i a l d i a g n o s i s between h p t a n d o t h e r causes o f h y p e r calcemia w i t h i n t h e whole r a n g e o f cam v a l u e s it t u r n e d o u t t h a t c a l , pth, a n d cam a l l c o n t r i b u t e d t o t h e s e p a r a t i o n o f t h e t w o p a t i e n t g r o u p s ( t a b l e 5b). t h e r e l a t i v e i m p o r t a n c e was g r e a t e s t f o r cam a n d smallest f o r pth. o u t of 1 3 4 p a t i e n t s 26 were misclassified, most o f them h a v i n g cam v a l u e s below 3.0 m m o l / l ( f i g . 5 ) . when o n l y p a t i e n t s 162 t a b l e 5. r e s u l t s o f d i s c r i m i n a n t a n a l y s i s . symbols w i t h i n p a r e n t h e s e s d e n o t e t h e measurements chosen by t h e c o m p u t e r p r o g r a m . t h e c l a s s i f i c a t i o n i n t o g r o u p s a , a n d b i s p e r f o r m e d w i t h u s e o f a c l a s s i f i c a t i o n f u n c t i o n z = f (cam, cal, p t h ) as d e t e r mined in t h e d i s c r i m i n a n t a n a l y s i s f o r t h e a c t u a l g r o u p s . a n i n d i v i d u a l i s allocated ( c l a s s i f i e d ) t o g r o u p a f o r z<o a n d g r o u p b f o r z>o. a. d i s c r i m i n a t i o n w i t h hpt (6) , 'a b b e t w e e n h e a l t h y s u b j e c t s ( a ) a n d p a t i e n t s c l a s s i f i e d g r o u p a b i. all subjects a ) a l l s u b j e c t s ( c a m ) ' ) c o r r e c t g r o u p a 91 0 c o r r e c t g r o u p b 14 7 4 c o r r e c t g r o u p a 50 0 b ) men ( & m i 2 ) c o r r e c t g r o u p b 1 15 c ) women < 50 y e a r s (cam, p t h ) 3 ) c o r r e c t q r o u p a 2 2 1 i . ' c o r r e c t g r o u p b 1 7 4 ) d ) women > 5 0 y e a r s (cam) c o r r e c t c l r o u p a 18 0 . c o r r e c t g r o u p b 1 2 51 i i . s u b i e c t s w i t h cam 2.45 2.75 mmol/l 5) a ) a l l s u b j e c t s [cam, c a l ) c o r r e c t g r o u p b 1 31 b ) men ( c a m j 6 ) c o r r e c t g r o u p a 2 9 1 c o r r e c t g r o u p a 1 6 0 c o r r e c t g r o u p 8 0 8 c ) women < 50 y e a r s ( c a l , p t h ) 7 ) c o r r e c t g r o u p a 5 0 c o r r e c t g r o u p b 0 8 8 ) d ) women > 50 y e a r s (cam, c a l ) c o r r e c t g r o u p a 8 1 c o r r e c t g r o u p b 1 19 1 1 -878572 t a b l e 5. ( c o n t i n u e d ) . b. d i s c r i m i n a t i o n between p a t i e n t s w i t h hpt ( b ) a n d o t h e r causes o f hypercalcaemia ( c ) . a n i n d i v i d u a l i s allocated t o g r o u p b f o r z<o a n d g r o u p c f o r z>o. classified g r o u p i. a l l subiects a) a l l s u b j e c t s (cam, cal, p t h ) 1) c o r r e c t g r o u p b 74 14 b ) men (cam)21 c o r r e c t g r o u p c 7 14 c o r r e c t g r o u p c 12 34 c o r r e c t g r o u p b 14 3 c ) women < 50 y e a r s ( p t h ) 3) c o r r e c t qroui) b 7 1 c o r r e c t g r o u p c 0 5 d) women > 50 y e a r s (cam, pth)‘) c o r r e c t g r o u p b 57 6 c o r r e c t g r o u p c 6 14 ii. s u b i e c t s w i t h cam 2.45 2.75 m m o l / l all s u b j e c t s (car)’) c o r r e c t g r o u p b 22 10 c o r r e c t g r o u p c 2 5 1 ) z = 6.76xcam-7.13xcal-3.34xpth 2) 2 = 2.967 ( > = g r o u p c ) 3) 2 = 0.806 ( > = g r o u p b) 4) z = 4.oxcam-3.72xpth-8.64 5) = 1.293 ( > = g r o u p c ) 164 who h a d cam v a l u e s below 2.75 mmol/i w e r e a n a l y z e d t h e c o m p u t e r p r o g ramme o n l y selected c a l measurements t o separate t h e t w o g r o u p s most e f f i c i e n t l y ( t a b l e 5b 1 1 ) . optimal d i s c r i m i n a t o r y levels o f cam f o r s c r e e n i n g o f hpt in t h e r e f e r e n c e sample g r o u p o f h e a l t h y s u b j e c t s t h e d i s t r i b u t i o n o f cam v a l u e s was close t o gaussian as j u d g e d by s t a t i s t i c a l goodness-of f i t t e s t s ( t h e shapiro-wilks t e s t f o r n < 50) a n d t h e kolmogorov-smirnow t e s t f o r n 2 50). in t h e h p t p a t i e n t g r o u p s , h o w e v e r , t h e fit was less close. similar o b s e r v a t i o n s w e r e made f o r t h e c a l values. since i t a p p e a r e d from t h e s e c a l c u l a t i o n s a n d p l o t s t h a t t h e f r e q u e n c y d i s t r i b u t i o n s f o r t h e h p t p a t i e n t s w e r e f a i r l y gaussian o v e r t h e r i g h t h a n d s i d e o f t h e c u r v e but n o t o n i t s e x t r e m e l e f t t h e f o l l o w i n g p r o c e d u r e was c a r r i e d o u t : t h e assembled cam values w e r e r e o r g a n i z e d o n t h e a s s u m p t i o n t h a t t h e y c o n s t i t u t e d a p a r t o f a gaussian d i s t r i b u t i o n , w h e r e t h e lowest v a l u e s h a d b e e n omitted. a t h e o r e t i c a l gaussian d i s t r i b u t i o n f u n c t i o n was t h e n f i t t e d t o t h e t r u n c a t e d f r e q u e n c y d i s t r i b u t i o n giving estimates o f t h e location ( c ) a n d s t a n d a r d d e v i a t i o n (sd). f i g u r e 7 shows a gaussian f u n c t i o n so f i t t e d u s i n g a n o n l i n e a r p a r a meter estimation p r o c e d u r e f o r t h e f r e q u e n c y d i s t r i b u t i o n s f o r cam in t h e hpt p a t i e n t s . from t h i s f i g u r e it c a n b e seen t h a t t h e mean v a l u e a n d s t a n d a r d d e v i a t i o n (c = 2.81 f 0.23 m m o l / l ) f o r t h e t h e o r e t i c a l d i s t r i b u t i o n a r e somewhat l o w e r t h a n t h e c o r r e s p o n d i n g v a l u e o b t a i n e d in a s t r a i g h t f o r w a r d c a l c u l a t i o n o f t h e h p t p o p u l a t i o n (c = 2.91 * 0.30 m m o l / l ) . t h e optimal d i s c r i m a t o r y levels o f cam w e r e c a l c u l a t e d f r o m t h e idealized c u r v e s as f u n c t i o n s o f w e i g h t i n g r a t i o s ( f a l s e p o s i t i v e o r false n e g a t i v e ) a n d f o r d i f f e r e n t p r e v a l e n c e s ( f i g . 8 ) . as c a n b e seen t h e optimal d i s c r i m i n a t o r y l e v e l f o r a w e i g h t i n g r a t i o 1:l v a r i e d f r o m 2.73 mmol/l a t a p r e valence o f 0.1 % t o 2.56 mmol/l w h e n t h e p r e v a l e n c e was assumed t o b e 50%. f u r t h e r m o r e , f o r a p r e v a l e n c e o f 5 0 % t h e optimal d i s c r i m i n a t o r y l i m i t was 2.63 mmol/l f o r a w e i g h t i n g r a t i o o f 1o:l f o r false p o s i t i v e t o false n e g a t i v e , but 2.47 mmol/l f o r t h e r e v e r s e w e i g h t i n g r a t i o . 165 numbers ** *** **** 2.30 2.40 2.50 2.60 2.70 2.80 2.90 3.00 3.10 3.20 3.30 cam (mmol/d f i g . 7. h i s t o g r a m f o r t h e a c t u a l v a l u e s o f cam in p a t i e n t s w i t h h y p e r p a r a t h y r o i d i s m . t h e f i t t e d t h e o r e t i c a l d i s t r i b u t i o n i s i n d i c a t e d by d o t t e d lines. 2.80 = 1 0 2.70 u a, > a, 2.60 f .e 2.50 5 5 3 2.40 e 0 a c .# . v) . .# 0" h prevalence 1 0.00 1 0.0 1 0.05 0.10 0.50 1o:l 5: 1 2: 1 1:l 1:2 1:5 1:lo weighting ratio false positive/false negative fig. 8 . t h e optimal d i s c r i m a t o r y level f o r s e r u m calcium (cam) as a f u n c t i o n o f w e i g h t i n g r a t i o false p o s i t i v e : false n e g a t i v e f o r d i f f e r e n t p r e v a l e n c e s o f h y p e r p a r a t h y r o i d i s m . 166 discuss i o n t h e p r e s e n t s t u d y was c o n d u c t e d w i t h two major aims. t h e f i r s t was t o e s t a b l i s h t h e e x t e n t t o w h i c h measurements o f plasma i o n i z e d calcium a n d s e r u m pth c o n t r i b u t e d t o t h e s e p a r a t i o n o f p a t i e n t s w i t h m i l d h p t f r o m h e a l t h y i n d i v i d u a l s w i t h cam v a l u e s in t h e u p p e r p a r t o f t h e r e f e r e n c e r a n g e as well as in t h e d i f f e r e n t i a l d i a g n o s i s o f m a n i f e s t hypercalcemia. t h e o t h e r was t o fix t h e optimal d i s c r i m i n a t o r y l i m i t f o r s e r u m calcium in t h e d e l i n e a t i o n o f m i l d p r i m a r y h p t f r o m h e a l t h y s u b j e c t s in t h e u p p e r p a r t o f t h e r e f e r e n c e r a n g e . as a b a s i s f o r these c a l c u l a t i o n s i n f o r m a t i o n was c o l l e c t e d f o r assessment o f b o t h i n t r a i n d i v i d u a l a n d i n t e r d i u r n a l v a r i a t i o n s f o r t h e s t u d i e d v a r i ables. d i u r n a l v a r i a t i o n s we f o u n d o n l y small, s t a t i s t i c a l l y i n s i g n i f i c a n t v a r i a t i o n s o f t h e s e r u m calcium a n d p t h c o n c e n t r a t i o n s t h r o u g h t h e d a y . a p e a k v a l u e f o r s e r u m calcium in t h e m o r n i n g h a s b e e n d e s c r i b e d (13.25) w h i l e a n ear,ly m o r n i n g n a d i r o f t o t a l calcium a n d i o n i z e d calcium has also b e e n r e c o r d e d (25,29). a d i u r n a l p a t t e r n o f s e r u m immunoreactive pth has b e e n r e p o r t e d in b o t h normal s u b j e c t s a n d p a t i e n t s w i t h hpt (14,25,29,41) a n d also a r e l a t i o n s h i p b e t w e e n p t h a n d sleep stages (27,39), f o u n d n o e v i d e n c e f o r r h y t h m i c e p i s o d i c v a r i a t i o n o f calcium o r pth during t h e d a y . in t h e p r e s e n t s t u d y l i t t l e a t t e n t i o n was p a i d t o t h e i n f l u e n c e o f meals. a l t h o u g h it w o u l d b e o f some b e n e f i t t o h a v e a l l specimens f o r calcium a n d pth t a k e n in t h e m o r n i n g w i t h t h e p a t i e n t f a s t i n g it seems t h a t f o r most p r a c t i c a l c l i n i c a l p u r p o s e s a random sample during t h e d a y i s s u f f i c i e n t . t h e i n t e r d i u r n a l v a r i a t i o n s w e r e o n l y moderately g r e a t e r t h a n t h e i n t r a d i u r n a l v a r i a t i o n s w h i c h w e r e c o m p a r a t i v e l y modest. t h e m a j o r i t y o f these v a r i a t i o n s c o u l d b e e x p l a i n e d by p r e a n a l y t i c a l a n d a n a l y t i c a l v a r i a t i o n s , i.e, t h e b i o l o g i c a l v a r i a t i o n s w e r e small. t h e 95 % c o n f i d e n c e i n t e r v a l f o r a s i n g l e s e r u m calcium v a l u e o f 2.50 mmol c o u l d b e c a l c u l a t e d t o b e 2.38 2.62 mmol/l. t h u s t h e c l i n i c a l r o u t i n e w h i c h r e q u i r e s s e v e r a l samples o n a l t e r n a t e d a y s m i g h t b e u n n e c e s s a r y in s u b j e c t s w h e r e t h e f i r s t v a l u e is lower t h a n 2.50 mmol /i. 167 a a e a n d sex v a r i a t i o n s t h e o b s e r v a t i o n o f h i g h e r cam v a l u e s f o r e l d e r l y h e a l t h y women agrees w i t h r e c e n t f i n d i n g s in a l a r g e h e a l t h s u r v e y ( 3 6 ) . but i s in c o n t r a s t t o some e a r l i e r r e p o r t s w h e r e lower v a l u e s f o r t o t a l calcium in e l d e r l y p e r s o n s w e r e r e c o r d e d (15,26) o r n o c h a n g e w i t h age was o b s e r v e d . h o w e v e r , n o sex o r age d i f f e r e n c e was f o u n d f o r c a l in accordance w i t h p r e v i o u s r e p o r t s ( 4 2 ) . we did n o t find a n y s i g n i f i c a n t age-dependent d i f f e r e n c e s f o r t h e s e r u m p t h c o n c e n t r a t i o n s . h i g h e r p t h l e v e l s in o l d e r p e r s o n s h a v e p r e v i o u s l y b e e n r e p o r t e d ( 1 0,16,22,34,47). however, m a r c u s e t al . ( 3 4 ) r e p o r t e d t h a t w h e n t h e v a l u e s w e r e c o r r e c t e d f o r g l o m e r u l a r f i l t r a t i o n r a t e a d i f f e r e n c e r e l a t e d t o age was n o t s i g n i f i c a n t in h e a l t h y s u b j e c t s . c l e a r l y s i g n i f i c a n t s e x d i f f e r e n c e s w e r e n o t e d in t h e p r e s e n t s t u d y , f o r s e r u m pth w i t h a 20 % h i g h e r values in females. similar f i n d i n g s h a v e n o t p r e v i o u s l y b e e n g e n e r a l l y o b s e r v e d ( 3 0 ) . t h e reasons f o r t h e s e x d i f f e r ences a r e n o t o b v i o u s s i n c e t h e y r e l a t e t o b o t h p r e a n d postmenopausal females. a n a p p a r e n t c l i n i c a l consequence o f t h e o b s e r v a t i o n s t h a t b o t h cam a n d p t h a r e h i g h e r in h e a l t h y post-menopausal females i s t h a t t h e r e w i l l b e a n i n c r e a s e d n u m b e r o f diagnosed h p t in t h i s g r o u p i f i d e n t i c a l c u t o f f p o i n t s a r e u s e d as in males a n d y o u n g e r females, g i v e n t h e e x i s t e n c e o f a n u m b e r o f p a t i e n t s w i t h m i l d h p t who o t h e r w i s e remain u n r e c o g n i z e d . t h i s m i g h t t o some e x t e n t e x p l a i n t h e a p p a r e n t l y h i g h e r p r e v a l e n c e o f h p t in postmenopausal women ( 1 1 1. d e l i n e a t i o n o f hpt t h e a v a i l a b i l i t y o f measurements o f plasma i o n i z e d c a l c i u m a n d s e r u m pth in c l i n i c a l p r a c t i c e has a r o u s e d g r e a t e x p e c t a t i o n s f o r i m p r o v e d d i a g n o s i s o f hpt, r e g a r d i n g b o t h t h e d i f f e r e n t i a l d i a g n o s i s o f hypercalcemia a n d t h e s e p a r a t i o n f r o m t h e u p p e r r e f e r e n c e r a n g e o f h e a l t h y s u b j e c t s . s e v e r a l s t u d i e s h a v e d e s c r i b e d cases w h e r e a n e l e v a t e d plasma ionized calcium was f o u n d t o g e t h e r w i t h a v a l u e w i t h i n t h e r e f e r e n c e r a n g e f o r t o t a l s e r u m calcium, a n d w h e r e s u b s e q u e n t n e c k e x p l o r a t i o n r e v e a l e d hpt (7,23,32). i t seems logical t h a t in some p a t i e n t s p r o t e i n b i n d i n g o f f r e e calcium v a r i e s so t h a t t h e t o t a l s e r u m calcium v a l u e m i g h t b e misleading ( 3 7 ) . however, if ionized calcium i s s u b s t i t u t e d f o r t o t a l s e r u m calcium mea s u r e m e n t s in t h e c l i n i c a l r o u t i n e it seems l i k e l y t h a t a n o v e r l a p w i l l o c c u r b e t w e e n t h e u p p e r p a r t o f t h e d i s t r i b u t i o n o f t h e r e f e r e n c e v a l u e s o f 168 h e a l t h y i n d i v i d u a l s a n d t h e l o w e r p a r t o f t h e c o r r e s p o n d i n g d i s t r i b u t i o n c u r v e f o r p a t i e n t s w i t h hpt. a t least in t h e p r e s e n t s t u d y t h e o v e r l a p b e t w e e n t h e t w o p o p u l a t i o n s was o f a similar m a g n i t u d e as r e g a r d s i o n i z e d calcium v a l u e s a n d t o t a l , a l b u m i n c o r r e c t e d values. t h e t e c h n i q u e s for measurement o f t h e free, i o n i z e d , f r a c t i o n o f s e r u m calcium h a v e o n l y r e c e n t l y become a v a i l a b l e f o r g e n e r a l c l i n i c a l r o u t i n e use. many e a r l i e r s t u d i e s w e r e t h e r e f o r e p e r f o r m e d f o r t h e p u r p o s e o f d e v e l o p i n g f o r m u l a s w h e r e t h i s f r a c t i o n c o u l d b e estimated f r o m measurements o f t h e t o t a l s e r u m calcium a n d c a l c i u m b i n d i n g p r o t e i n s , p r i m a r i l y a l b u m i n (12,31,35,38). in c l i n i c a l p r a c t i c e it has g e n e r a l l y b e e n c o n s i d e r e d con v e n i e n t t o a d j u s t t h e s e r u m calcium a c c o r d i n g t o t h e v a l u e o f t h e s e r u m a l b u m i n from t h e r e f e r e n c e mean v a l u e . s u c h a c o r r e c t i o n has been demon s t r a t e d t o i m p r o v e t h e d i a g n o s i s o f hpt ( 4 3 ) . we f o u n d , as b r a u m a n e t al. ( 6 ) t h a t t h e slope f o r i o n i z e d calcium o n t o t a l ( m o d i f i e d ) s e r u m calcium was steeper f o r t h e hypercalcaemic p a t i e n t s t h a n f o r t h e h e a l t h y c o n t r o l s , i.e. t h e p a t h o l o g i c a l c o n d i t i o n leading t o a r i s e in t h e plasma i o n i z e d calcium, did n o t p r o p o r t i o n a l l y i n c r e a s e t h e f r a c t i o n b o u n d t o albumin. p a t i e n t s w i t h hpt c o n s e q u e n t l y h a d h i g h e r values f o r c a l , f o r c o r r e s p o n d i n g v a l u e s o f cam. measurements o f pth did n o t completely s e p a r a t e p a t i e n t s w i t h m i l d h p t f r o m h e a l t h y s u b j e c t s . t h e r e i s a c o r r e l a t i o n b e t w e e n t h e g l a n d u l a r mass, s e r u m p t h a n d s e r u m calcium in p a t i e n t s w i t h p r i m a r y hpt. t h u s p a t i e n t s w i t h t h e m i l d e s t hypercalcemia h a v e t h e smallest g l a n d s , o f t e n w i t h modest h y p e r p l a s i a , a n d t h e y also h a v e s e r u m pth values close t o or e v e n w i t h i n t h e r e f e r e n c e r a n g e of h e a l t h y i n d i v i d u a l s ( 5 0 ) . f u r t h e r m o r e , a t t h e c e l l u l a r l e v e l p a t i e n t s w i t h t h e m i l d e s t h y p e r c a l c e m i a h a v e a close t o normal r e s p o n s e t o a l t e r a t i o n s o f t h e a m b i e n t calcium c o n c e n t r a t i o n s as r e g a r d s t h e release o f p t h (40.45). e v a l u a t i o n s o f s e r u m pth c o n c e n t r a t i o n s m u s t t a k e i n t o c o n s i d e r a t i o n t h e f a c t s t h a t s e v e r a l f r a g m e n t s o f p t h a r e c i r c u l a t i n g , a n d t h a t t h e r e l a t i v e amount o f d i f f e r e n t f r a g m e n t s may b e a l t e r e d by v a r i o u s d i s o r d e r s (1,4,19). t h e c l i n i c a l u s e f u l n e s s o f each assay m u s t t h e r e f o r e b e e s t a b l i s h e d e m p i r i c a l l y ( 7 ) . i t h e a n t i s e r u m u s e d in t h e p r e s e n t s t u d y has a high a f f i n i t y f o r i n t a c t pth. a s t h e r e was a f a i r l y r a p i d e l i m i n a t i o n o f immunoreactive hormone from t h e c i r c u l a t i o n in p a t i e n t s o p e r a t e d f o r hpt ( f i g . 1 ) it seems u n l i k e l y t h a t f r a g m e n t s w i t h a slow e l i m i n a t i n g r a t e s t r o n g l y i n f l u e n c e d t h e assay. 169 p r e v i o u s i n v e s t i g a t i o n s w i t h t h e same assay h a v e in p a t i e n t s w i t h i m p a i r e d r e n a l f u n c t i o n d e m o n s t r a t e d a close c o r r e l a t i o n between s e r u m levels o f p t h , b o n e r e s o r p t i o n s u r f a c e s a n d bone f o r m a t i o n r a t e s ( 3 4 ) . a r a p i d increase in t h e pth response t o l o w e r i n g o f s e r u m calcium w i t h e d t a has also b e e n o b s e r v e d ( 3 ) . a l l t h e s e o b s e r v a t i o n s i n d i c a t e t h a t t h e assay d e t e c t s m a i n l y i n t a c t pth a n d o n l y t o a lesser e x t e n t c a r b o x y l t e r m i n a l f r a g m e n t s o f p t h w h i c h a r e k n o w n t o b e s l o w l y eliminated f r o m t h e c i r c u l a t i o n . i t has been r e p o r t e d t h a t assay system d e t e c t i n g t h e c t e r m i n a l r e g i o n o f p t h h a v e a g r e a t e r c a p a c i t y t o d i s c l o s e p r i m a r y h p t o n basal mea surements o f pth t h a n d o those system w h i c h a r e n o t capable o f d e t e c t i n g r a p i d changes in s e c r e t i o n ( 8 ) . from d e t e r m i n a t i o n s o f p t h alone less t h a n h a l f o f a l l p a t i e n t s w i t h h p t were c l e a r l y s e p a r a t e d f r o m t h e h e a l t h y s u b j e c t s a n d t h e s e p a r a t i o n a g a i n s t o t h e r causes o f hypercalcaemia was less t h a n 75 %. when t h e simultaneously measured calcium c o n c e n t r a t i o n s w e r e also c o n s i d e r e d b e t t e r s e p a r a t i o n s w e r e o b t a i n e d , but t h e d e l i n e a t i o n s w e r e still n o t complete, p a r t i c u l a r l y in t h o s e w i t h m i l d hypercalcaemia. p t h i s p r i m a r i l y eliminated t h r o u g h g l o m e r u l a r f i l t r a t i o n , a n d i m p a i r e d r e n a l f u n c t i o n w i l l t h e r e f o r e i n a p p r o p r i a t e l y r a i s e t h e pth levels. s u c h a r i s e in p t h l e v e l s i s more p r o m i n e n t w i t h assays d i r e c t e d t o w a r d s t h e c a r b o x y t e r m i n a l p a r t o f t h e hormone. in g e n e r a l , w i t h o u r assay system moderately i m p a i r e d r e n a l f u n c t i o n , w i t h o u t s e c o n d a r y h y p e r p a r a t h y r o i d i s m , does n o t r a i s e t h e pth levels above t h e normal r a n g e ( 3 4 ) . in p a t i e n t s w i t h hypercalcaemia o f o t h e r o r i g i n t h a n h p t r e n a l f u n c t i o n i s o f t e n i m p a i r e d ( 3 3 ) a n d t h e s e r u m c o n c e n t r a t i o n s o f p t h may t h e r e f o r e b e e l e v a t e d e v e n above t h e r e f e r e n c e r a n g e a l t h o u g h t h e s e c r e t i o n o f p t h i s in f a c t s u p p r e s s e d by t h e hypercalcaemia. a moderate r e d u c t i o n o f g l o m e r u l a r f i l t r a t i o n , as e v i d e n c e d by a n i n c r e a s e o f t h e s e r u m c r e a t i n i n e v a l u e s , was p r e s e n t in s e v e r a l o f o u r p a t i e n t s w i t h n o n h p t hypercalcaemia, a f a c t w h i c h m i g h t e x p l a i n some o f t h e o v e r l a p between t h e p t h v a l u e s b e t w e e n t h e h y p e r calcaemic p a t i e n t g r o u p s . one p a t i e n t , w i t h sarcoidosis, w i t h a serum c r e a t i n i n e v a l u e a r o u n d 300 umol/i e v e n h a d a m a r k e d l y e l e v a t e d p t h v a l u e ( 2 . 2 a r b u / l ) . w i t h o u t p e r f o r m i n g n e c k e x p l o r a t i o n in all hypercalcaemic p a t i e n t s it c o u l d n o t b e d e f i n i t e l y e x c l u d e d t h a t some o f t h e m did n o t also h a v e hpt. p a t i e n t s w i t h o u t m a l i g n a n t d i s o r d e r s , h o w e v e r , d i s p l a y e d n o r m a l i z a t i o n o f t h e i r hypercalcaemia w h e n t h e i r u n d e r l y i n g disease r e c e d e d ( s a r c o i d o s i s , 170 t h y r o t o x i c o s i s , immobilisation) a n d also in most o f t h o s e w i t h malignancies t h e c l i n i c a l p i c t u r e was c h a r a c t e r i s t i c o f hypercalcaemia o f o t h e r o r i g i n t h a n hpt. when t h e c l i n i c a l e x p e r i e n c e s o f v a r i o u s r e s e a r c h c e n t e r s a r e compared t h e i n v e s t i g a t e d p a t i e n t p o p u l a t i o n s m u s t also b e c o n s i d e r e d . a t o u r h o s p i t a l , we h a v e f o r s e v e r a l y e a r s b e e n i n t e r e s t e d in p a t i e n t s w i t h m i l d h y p e r calcaemia a n d also h a d a l i b e r a l a t t i t u d e t o w a r d s s u r g i c a l t r e a t m e n t o f p a t i e n t s w i t h s u s p e c t e d m i l d hpt. in o u r c o n s e c u t i v e s e r i e s o f p a t i e n t s t h e r e a r e t h e r e f o r e many b o r d e r l i n e s e r u m calcium v a l u e s . t h e d i s c o v e r y o f a g r e a t o v e r l a p o f pth values b e t w e e n t h e p a t i e n t s w i t h m i l d h p t a n d t h e h e a l t h y r e f e r e n c e p o p u l a t i o n s h o u l d b e r e g a r d e d in t h i s light. if o n l y p a t i e n t s w i t h high s e r u m calcium v a l u e s , above 2 . 9 0 m m o l / l , h a d been con s i d e r e d as h a v i n g hpt, t h e d i a g n o s t i c d i f f i c u l t i e s w i t h r e g a r d t o b o t h s e p a r a t i o n t o w a r d s t h e h e a l t h y s u b j e c t s a s well as a g a i n s t o t h e r causes o f hypercalcaemia w o u l d h a v e been m u c h less t h a n we now e x p e r i e n c e d . d i s c r i m i n a t o r v a n a l v s i s t h e d i s c r i m i n a t o r y a n a l y s i s was c a r r i e d o u t in t w o s t e p s , i n c l u d i n g f i r s t a l l i n d i v i d u a l s in t h e t w o g r o u p s t o b e compared. in a second s t e p o n l y those w i t h b o r d e r l i n e cam v a l u e s ( i . e . 2 . 4 5 2 . 7 5 m m o l / l ) w e r e c o n s i d e r e d . t h e r a t i o n a l e f o r t h e l a t t e r r e s t r i c t i o n was t h a t o t h e r w i s e p a t i e n t s w i t h t h e most abnormal biochemical d e v i a t i o n s w o u l d a f f e c t t h e d i s c r i m i n a t o r y f u n c t i o n o u t o f p r o p o r t i o n t o t h e d e s i r e d goal. t h e c l i n i c a l p r o b l e m , o b v i o u s l y , does n o t c o n s i s t in s e p a r a t i n g a h p t p a t i e n t w i t h a s e r u m calcium v a l u e o f 3 . 5 4 m m o l / l f r o m t h e h e a l t h y p o p u l a t i o n but r a t h e r t o d i s c l o s e t h e m i l d e s t f o r m o f hpt. in t h e s t a t i s t i c a l analyses it was a p p a r e n t t h a t measurements o f cam alone c o u l d s e p a r a t e t h e m a j o r i t y o f h p t p a t i e n t s f r o m t h e h e a l t h y s u b j e c t s . t h i s i s almost s e l f e x p l a n a t o r y as t h e m a j o r i t y o f h p t p a t i e n t s h a v e c l e a r c u t hypercalcaemia. in s u c h i n s t a n c e s a d d i t i o n a l measurements ( o f c a l o r p t h ) c a n n o t allow o f f u r t h e r s e p a r a t i o n . however, w h e n o n l y t h e b o r d e r l i n e i n d i v i d u a l s w e r e a n a l y z e d cal p r o v e d t o b e a u s e f u l a d d i t i o n a l determina t i o n . a l t h o u g h t h e r e was a g e n e r a l c o r r e l a t i o n b e t w e e n cam [ a n d c a l l a n d pth f o r a l l t h e h p t p a t i e n t s t h e m i l d p a r a t h y r o i d h y p e r f u n c t i o n in those w i t h t h e m i l d e s t hypercalcaemia did n o t r e s u l t in s i g n i f i c a n t e l e v a t i o n s o f t h e p t h l e v e l s a n d in t h i s area p t h measurements did n o t p r o v i d e a n y f u r t h e r d i s c r i m i n a t i o n . 171 a l t h o u g h t h e r e w e r e some age a n d sex d i f f e r e n c e s f o r b o t h cam a n d p t h these w e r e a p p a r e n t l y n o t s u c h as t o i m p r o v e t h e d i s c r i m i n a t i o n in t h e p r e s e n t material. f o r t h e d i f f e r e n t i a l d i a g n o s i s o f hypercalcaemia it t u r n e d o u t t h a t measurements o f p t h w e r e v a l u a b l e , p a r t i c u l a r l y in i n d i v i d u a l s w i t h t h e h i g h e s t s e r u m calcium values. in a d d i t i o n c a l v a l u e s w e r e h i g h e r f o r t h e h p t p a t i e n t s t h a n f o r t h e non-hpt hypercalcaemics, p a r t i c u l a r l y w i t h i n t h e l o w e r r a n g e o f r a i s e d s e r u m calcium values. t h i s i s most l i k e l y e x p l a i n e d by a n " o v e r c o r r e c t i o n " o f t h e cam values f o r t h e decreased s e r u m a l b u m i n , o f t e n f o u n d in p a t i e n t s w i t h hypercalcaemia ( 5 ) . optimal d i s c r i m i n a t o r y l i m i t b e f o r e c a l c u l a t i o n o f t h e optimal d i s c r i m i n a t o r y l i m i t s b e t w e e n t h e h e a l t h y s u b j e c t s a n d p a t i e n t s w i t h m i l d h p t a n a d j u s t m e n t o f t h e f r e q u e n c y d i s t r i b u t i o n c u r v e for cam in t h e h p t p a t i e n t s was p e r f o r m e d . t h e reason f o r t h i s m a n o e u v r e was t h e o b s e r v a t i o n t h a t t h e calcium v a l u e s a p p r o a c h e d a gaussian d i s t r i b u t i o n e x c e p t in t h e lower p a r t w h e r e t h e r e was a l a c k o f v a l u e s below 2.60 m m o l l l t o achieve a b e l l s h a p e d c u r v e . t h e c o n c e p t o f llnormocalcemic p r i m a r y h p t " has b e e n d i s c u s s e d f o r s e v e r a l y e a r s (23,46,48). many p a t i e n t s r e p o r t e d t o h a v e normocalcemic p r i m a r y h p t a p p e a r t o h a v e h p t w i t h a m i l d , sometimes f l u c t u a t i n g , h y p e r calcemia (24,28). h o w e v e r , s e v e r a l o b s e r v a t i o n s i n d i c a t e t h a t h p t c o u l d e x i s t also in p a t i e n t s who n e v e r d i s p l a y r a i s e d s e r u m calcium values. f o r i n s t a n c e s e r i e s o f p a t i e n t s h a v e b e e n p r e s e n t e d w h e r e n e c k e x p l o r a t i o n h a s b e e n c a r r i e d o u t d e s p i t e c o n s t a n t normocalcemia (17.23) a n d w h e r e d e f i n i t e h p t h a s b e e n c o n f i r m e d h i s t o p a t h o l o g i c a l l y . in a r e c e n t s e r i e s a p p r o x i m a t e l y 40 p a t i e n t s o p e r a t e d a t o u r h o s p i t a l h a d s e r u m calcium v a l u e s b e t w e e n 2.60 a n d 2.80 mmol/l. hpt c o u l d b e o p e r a t i v e l y v e r i f i e d in a l l p a t i e n t s ( 5 0 ) . t h i s s u g g e s t s t h a t some i n d i v i d u a l s w i t h s e r u m v a l u e s below 2.60 m m o l / l also h a v e hpt. t h i s d i s c u s s i o n o f "normocalcemic h p t i' o n l y s e r v e s as a n e x p l a n a t i o n o f t h e mathematical a n a l y s i s a n d does n o t c a r r y a n y i m p l i c a t i o n s as t o w h e t h e r s u c h p a t i e n t s s h o u l d b e s o u g h t o r e v e n less w h i c h t r e a t m e n t i s optimal. o n t h e b a s i s o f t h e s e r e s u l t s we c o u l d c a l c u l a t e a n optimal d i s c r i m i n a t o r y l i m i t f o r h p t o f 2.68 m m o l l l , assuming a p r e v a l e n c e of 1% a n d a w e i g h t i n g r a t i o o f 1:l b e t w e e n f a l s e p o s i t i v e s a n d false n e g a t i v e s . a s m e n t i o n e d 172 above, h o w e v e r , o n l y p o s i t i v e e x p l o r a t i o n s w e r e p e r f o r m e d in c o n s e c u t i v e p a t i e n t s w i t h s u c h calcium values. if o t h e r p r e r e q u i s i t e s a r e c o r r e c t , t h i s finding i n d i c a t e s t h a t t h e p r e v a l e n c e o f h p t in a g e n e r a l p o p u l a t i o n m u s t b e c o n s i d e r a b l y h i g h e r t h a n 1%. in s u p p o r t of t h i s v i e w , a u t o p s y s t u d i e s h a v e f o u n d adenomatous h p t in as many as 2.4 % o f i n d i v i d u a l s above t h e age o f 70, a n d e v i d e n c e of h y p e r p l a s t i c p r i m a r y h p t in f u r t h e r 7 % ( 5 0 ) . from fig. 8 it c a n b e seen t h a t t h e w e i g h t i n g r a t i o o f 1 : l (i.e. e q u a l i m p o r t a n c e i s p a i d t o false p o s i t i v e a n d false n e g a t i v e c l a s s i f i c a t i o n s ) f o r a s e r u m calcium v a l u e of 2.60 m m o l / l c o r r e s p o n d s t o a p r e v a l e n c e in t h e p o p u l a t i o n o f a b o v e 10 %. f i r s t d e g r e e r e l a t i v e s o f p a t i e n t s w i t h t h e men-1 s y n d r o m e h a v e a 50 % r i s k o f b e i n g c a r r i e r s o f t h e men-1 t r a i t . in a p o p u l a t i o n w i t h a 50 % p r e v a l e n c e o f h p t t h e optimal d i s c r i m i n a t o r y l i m i t f o r s e r u m calcium in t h e p r e s e n t s t u d y was 2.56 mmol/l i f it was c o n s i d e r e d e q u a l l y i m p o r t a n t t o a v o i d f a l s e l y p o s i t i v e a n d falsely n e g a t i v e c l a s s i f i c a t i o n s . it i s a moot p o i n t w h e t h e r t h e w e i g h t i n g r a t i o in t h i s p a r t i c u l a r s e t t i n g i s d i f f e r e n t from o t h e r s i t u a t i o n s w h e r e h p t i s suspected. whatever s t a n d p o i n t i s t a k e n in t h e i n d i v i d u a l c l i n i c a l s i t u a t i o n it i s a p p a r e n t f r o m these c o n s i d e r a t i o n s t h a t in men-1 r e l a t i v e s s e r u m calcium v a l u e s e v e n w i t h i n t h e r e f e r e n c e r a n g e m i g h t b e compatible w i t h h p t a n d t h e r e f o r e necessitate f u r t h e r i n v e s t i g a t i o n s . acknowledgement t h i s s t u d y was s u p p o r t e d by t h e s w e d i s h medical research c o u n c i l . references 1. 2. 3 . 4. 5. a r n a u d , c.d., goldsmith, r.s., b o r d i e r , p.j., sizemore, g.w., l a r s e n , j.a. & g i l k i n s o n , j.: i n f l u e n c e o f immunoheterogeneity o f c i r c u l a t i n g p a r a t h y r o i d hormone o n t h e r e s u l t s of radioimmunoassays o f s e r u m in man. am j med 56:785-793, 1974. benson, l., l j u n g h a l l , s . , a k e r s t r o m , g. & o b e r g , k.: h y p e r p a r a t h y r o i d i s m p r e s e n t i n g as t h e f i r s t lesion in m u l t i p l e e n d o c r i n e neoplasia t y p e 1 (men-1). am j med, in p r e s s . benson, l., rastad, j., wide, l., a k e r s t r o m , g. e l j u n g h a l l , s.: s t i m u l a t i o n f o r p a r a t h y r o i d hormone s e c r e t i n o by e d t a i n f u s i o n a t e s t o f t h e d i f f e r e n t i a l d i a g n o s i s o f h y p e r p a r a t h y r o i d i s m . a c t a endo c r i n o l o g i c a . 11 1 : 498-506, 1986. b e r s o n , s.a. & yalow, r.s.: lmmunochemical h e t 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e s o f p r i m a r y hpt. world j s u r g 1986; a c c e p t e d f o r p u b l i c a t i o n . 624:69-72, 1979. 176 upsala journal of medical sciences 2022, 127, e8833 http://dx.doi.org/10.48101/ujms.v127.8833 ulcerative colitis progression: a retrospective analysis of disease burden using electronic medical records david dahlgrena, lars agréusb, jan stålhammarc and per m. hellströmd adepartment of pharmaceutical biosciences, translational drug discovery and development, uppsala university, uppsala, sweden; bdepartment of neurobiology, care sciences and society, karolinska institutet, stockholm, sweden; cdepartment of public health and caring sciences, family medicine and preventive medicine, uppsala university, uppsala, sweden; ddepartment of medical sciences, uppsala university, uppsala, sweden abstract background: ulcerative colitis (uc) is a debilitating inflammatory bowel disease. present knowledge regarding uc disease progression over time is limited. objective: to assess uc progression to severe disease along with disease burden and associated factors. methods: electronic medical records linked with swedish national health registries (2005–2015) were used to identify disease progression of uc. odds of all-cause and disease-related hospitalization within 1 year were compared between patients with disease progression and those without. annual indirect costs were calculated based on sick leave, and factors related to uc progression were examined. results: of the 1,361 patients with moderate uc, 24% progressed to severe disease during a median of 5.2 years. severe uc had significantly higher odds for all-cause (or [odds ratio] 1.47, 95% ci [confidence interval]: 1.12–1.94, p < 0.01) and uc-related hospitalization (or 2.47, 95% ci: 1.76–3.47, p < 0.0001) compared to moderate disease. average sick leave was higher in patients who progressed compared to those who did not (64.4 vs 38.6 days, p < 0.001), with higher indirect costs of 151,800 sek (16,415 €) compared with 92,839 sek (10,039 €) (p < 0.001), respectively. uc progression was related to young age (or 1.62, 95% ci: 1.17–2.25, p < 0.01), long disease duration (or 1.09, 95% ci: 1.03–1.15, p < 0.001), and use of corticosteroids (or 2.49, 95% ci: 1.67–3.72, p < 0.001). conclusion: disease progression from moderate to severe uc is associated with more frequent and longer hospitalizations and sick leave. patients at young age with long disease duration and more frequent glucocorticosteroid medication are associated with progression to severe uc. article history received 16 june 2022 revised 15 august 2022 accepted 18 september 2022 published 18 october 2022 keywords ulcerative colitis; health economics; inflammation; outcomes research; inflammatory bowel disease introduction ulcerative colitis (uc) is a chronic inflammatory bowel disease (ibd) with a relapsing-remitting course of disease activity of the colon (1). the incidence of uc in the uppsala region, sweden, is reported to 20 cases per 100,000 inhabitants (2). uc is a debilitating disease with unpredictable course, which is severely impacting on patients’ health-related quality of life (3–6). uc is limited to the colon. the treatment is based on which part of the colon that is affected as well as the severity of the disease activity (1, 7). the pharmacological treatment options are based on 5-aminosalicylates, glucocorticosteroids, various immunosuppressants, and targeted immunomodulators (8), whereas surgical colectomy today is seen as a rescue in medically treatment resistant cases. the primary goal of therapy is to attain clinical remission, defined as symptomatic remission with no remaining glucocorticosteroid therapy. despite a plethora of treatment options, there are unmet medical needs in uc, particularly regarding the impact of uc and its treatment in activities of daily living (4, 9). uc has an onset often in adolescence or early adulthood resulting in sizeable long-term medical healthcare, reduced quality of life, and ability to work (5,  10–12). now, confirmative data delineating the progressive nature of uc are limited as well as outcomes associated with uc disease progression. the primary objective of this study was to assess progression rate from moderate to severe uc, and the secondary objective was to investigate the medical healthcare resource burden associated with disease progression and to identify factors connected to disease progression in a cohort of swedish uc patients. materials and methods study design this study data were retrieved from electronic medical records (emrs) in uppsala county council, sweden. emrs were cross contact dr david dahlgren david.dahlgren@farmbio.uu.se © 2022 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article http://dx.doi.org/10.48101/ujms.v127.8833 mailto:david.dahlgren@farmbio.uu.se http://creativecommons.org/licenses/by/4.0/ 2 d. dahlgren et al. analyzed with data from the swedish national patient registry, cause of death registry, prescribed drug registry, and national socioeconomic registry. data were pseudonymized as regards age, sex, prescriptions, diagnoses, laboratory test results, referrals, and clinical variables as retrieved from emrs in primary care and secondary care settings. this study complies with applicable laws, regulations, and guidance regarding patient protection, including privacy. this study was approved by the uppsala ethics review board and filed under pyg-998351, november 28, 2014. data collection the primary data source was emrs from primary and secondary care centers/hospitals in uppsala county council. data from approximately 30 primary care centers in the uppsala county council including two hospitals (enköping, uppsala) were collected. in a few cases, the same variables were collected from emr and one of the health registries. if data then deviated, the emr database and prescription registry were considered gold standards for outcomes and prescribed medications, respectively. the primary source emr data were linked to the different health registries through the individual swedish personal identity number (pin), which is unique for each swedish citizen. extracted emr data were stored in a key code file retaining the possibility for data verification, and an analysis file for statistical operations. thus, personal data in the analysis file were pseudonymized by replacing the pin with a unique internal patient identification number. for posterity, the key code file is safely preserved, accessible only for the principal investigator. patients with a diagnosis of uc (k51) were identified from the emr using the pygargus customized extraction program (cxp, version 4.0) data extraction tool. the cxp has been used since 2005 in swedish and norwegian healthcare systems and in several previously published research studies with high accuracy (13–15). the integrated emr data of uppsala county council allow for retrospective data capture over at least 7–8 years from primary care and secondary hospital care. patient data from the swedish national patient registry were collected from inpatient and outpatient specialist medical care across all of sweden. key variables collected include diagnosis, surgery, sex, age, region, hospital visits, gastroenterology surgery specialist visits, as well as hospital admissions and discharges. detailed information is available on all medical procedures and surgeries performed in both inpatient and outpatient settings. the cause of death registry contains information on the death cause of all swedish residents and non-swedish citizens living in sweden, as well as swedish citizen whose deaths did not occur in sweden. the main variables in the registry include personal identification numbers; home districts; sex; date of death; underlying cause of death; nature of injury; multiple causes of death; and if the death was alcohol-, narcotic-, or diabetes-related. in the present study, the cause of death registry was used to censor patients who died during follow-up. the censoring date was set to january 1, 2015. data on prescribed drugs were obtained from emr supplemented by linking individual patient identifiers to the national prescribed drug registry and were used to identify the total ibd population. the national prescribed drug registry contains data on all prescription drugs dispensed at swedish pharmacies (16). the registry covers prescriptions from both primary and specialist care level and includes data on prescription date, dosage, pack size, healthcare drug prescriber, and costs associated with the drug prescription. in our study, prescription data were retrieved from emr as well as the national prescribed drug registry. the difference between the two sources is that prescription data from emr include prescribed medications, whereas the national prescribed drug registry records dispensed medications. drugs administered during hospitalization are recorded in emr. the national socioeconomic registry includes information on all individuals over 16 years of age registered in sweden. this longitudinal database integrates information on educational level, marital status and family situation, occupational status, retirement, economic compensation, and social benefits. our study obtained information from this registry on unemployment, sick leave, early retirement, and disability. study population and follow-up patients of 18 years or older with a uc diagnosis from july 2005 until january 2015 were identified using international statistical classification of diseases and related health problems, 10th revision codes (k51 uc including all subdiagnoses: k51.0 ulcerative [chronic] pancolitis, k51.2 ulcerative proctitis, k51.3 ulcerative proctosigmoiditis, k51.4 pseudopolyps of colon, k51.5 left-sided colitis, k51.8 other specified uc, k51.9 uc, unspecified). moderate disease activity was defined as having a partial mayo score of 2–4 being treated with prednisolone <40  mg/day or its equivalent. severe disease was defined as having a partial mayo score of 5–9 and being treated with prednisone ≥40 mg/day or its equivalent. patients were followed from the date of eligibility with moderate uc until the date of progression to severe uc. outcomes the rate of disease progression from moderate to severe uc was determined over a 5.2-year median follow-up period. factors associated with disease progression were measured 1 year prior to index date, including age, sex, disease duration, medication use (e.g., corticosteroid and 5-aminosalicylic acid), and serum c-reactive protein (s-crp) levels. index date for patients was defined as the date of progression from partial mayo score 2–4 to score 5–9. for patients who did not progress, the index date was the earliest date qualifying as moderate uc. over the median 5.2-year follow-up, the burden of healthcare resource utilization was assessed by determining the presence disease progression in ulcerative colitis 3 of all-cause hospitalization, uc-related hospitalization, and ucrelated surgery within 1 year before and after the index date among patients who progressed to severe uc and those who did not progress. the average annual number of sick leave days and the annual indirect costs associated with sick leaves were calculated for both cohorts in patients aged 18–65 years. statistical data analyses statistical analysis software, version 9.3 (sas institute, cary, nc, usa), was used for data management and statistical analyses. for the primary endpoint, kaplan–meier analysis was used to depict the progression rate from moderate to severe uc. the secondary endpoints including demographic and clinical factors associated with disease progression were analyzed with a logistic regression model. frequency and proportion of elevated serum crp (>30 mg/l) and fecal calprotectin (>50 mg/g) were compared between the two groups. for patients whose disease progressed versus those patients who did not progress, risks of all-cause hospitalization, uc-related hospitalization, and ucrelated surgery were assessed as dependent variables using multivariate logistic regression models controlling for age, gender, and duration of disease; baseline corticosteroid and 5-aminosalicylic acid use; and baseline all-cause hospitalization. odds ratios (or) with 95% confidence intervals (ci) were estimated from logistic regression models. indirect costs were calculated within 2 years after patients entered a moderate disease condition based on annual number of sick leave days. indirect costs were calculated by multiplying the average annual number of sick leave days × average annual swedish salary, adjusting for gender (assuming 250 work days per year). annual sick leave days were obtained from the national socioeconomic registry. the average annual salary in 2014 was 350,400 sek for women and 403,200 sek for men (excluding social fees 47%) (17). indirect costs were converted to 2020 value of sek and reported in euro (€) for comparison. chisquare or kruskal–wallis tests were used for comparisons between patients with or without progressing disease. results demographics and clinical characteristics in total, 2,450 patients diagnosed with uc (k51) were retrieved from the emr over the period 2005–2015. the vast majority of the diagnoses were made at gastroenterology, internal medicine, and surgery secondary care units and only few at other care units. largely, 60% of patients were classified as unspecified uc according to the icd-10, the majority of which with moderate disease activity. of all uc patients registered in the emr, 9% were not retrievable through extraction with the cxp 4.0 (table 1). there were also numerous comorbidities with arthritis, asthma bronchiale, and cancer being the most prevalent; however, none of which exceeding 8% in the study cohort (table 2). rate of disease progression and associated factors out of 1,361 patients identified with moderate uc in the emr, 321 (24%) developed severe uc over a median follow-up period of 5.2  years (interquartile range: 4.8 years) (figure 1). during the follow-up, 121 of the 1,361 (9%) patients used biologic therapies, table 1. demographics, diagnosis setting, and clinical characteristics of study cohort with uc extracted from electronic medical records by the customized extraction program, version 4.0. variable all uc patients, n = 2,450 (%) gender female 1,185 (48.4) male 1,265 (51.6) diagnosed with uc, n (%) missing 231 gastroenterology care unit 633 (28.5) internal medicine care unit 415 (18.7) other specialist unit 105 (4.7) pediatric care unit 140 (6.3) surgery care unit 926 (41.7) disease extension ulcerative proctitis 354 (14.6) left-sided colitis 205 (8.5) extensive colitis 400 (16.5) unspecified uc 1,491 (60.2) disease severity mild 668 (27.2) moderate 1,361 (55.6) severe 421 (17.2) medical treatment mesalazine 2,352 (96.0) glucocorticosteroids 1,788 (72.9) immunomodulators 613 (25.0) antitumor necrosis factor-α 318 (12.9) uc, ulcerative colitis. table 2. number of comorbidities to uc in the study cohort. comorbidities number (% of cohort) anxiety 140 (6.0) arthritis 175 (7.7) asthma bronchiale 181 (7.7) bronchitis 6 (0.3) cancer 139 (6.2) chronic obstructive pulmonary disorder 38 (1.6) depression 41 (1.7) diabetes mellitus 133 (5.6) hyperthyroidism 22 (0.9) hypothyroidism 53 (2.2) malnutrition 1 (0.04) renal insufficiency 4 (0.16) multiple sclerosis 10 (0.4) myasthenia gravis 1 (0.04) osteoporosis 25 (1) primary sclerosing cholangitis 73 (3.1) psoriasis 61 (2.5) pyoderma gangrenosum 7 (0.3) rheumatoid arthritis 15 (0.6) thromboembolism 69 (2.9) thyroiditis 7 (0.3) uveitis 49 (2) 4 d. dahlgren et al. more specifically 48 out of the 321 (15%) patients in the progression group and 73 of 1,040 (7%) among those in the non-progression group. the odds for progression from moderate to severe uc were 1.62 (95% ci: 1.17 to 2.25, p < 0.01) times greater in patients below 30 years of age and 1.38 (95% ci: 1.01 to 1.90, p = 0.04) times greater in patients between 30 and 50 years of age compared with those who were aged over 50 years (table 3). in addition, glucocorticosteroid use (or 2.49, 95% ci: 1.67 to 3.72, p < 0.0001), elevated s-crp (or 1.59, 95% ci: 1.21 to 2.08, p  <  0.01), and long disease duration (or 1.09, 95% ci: 1.03 to 1.15, p < 0.01) were associated with significantly greater odds for progression from moderate to severe disease. healthcare resource utilization and work productivity associated with disease progression over the 5 years of follow-up, higher rate of all-cause hospitalization (41% vs 33%, p < 0.01) and uc-related hospitalization (25% vs 11%, p < 0.01) were found in the patient group who progressed to severe uc as compared to those who did not progress (figure 2). among patients with disease progression to severe uc, the odds were 1.47 times greater for all-cause hospitalization (95% ci: 1.12 to 1.94, p < 0.01) and 2.47 times greater for uc-related hospitalization (95% ci: 1.76 to 3.47, p < 0.001). there was no difference in uc-related surgery between the two groups. unemployment among those progressing to severe uc was only numerically greater than for those who did not (42% vs 37%, p = 0.18; figure 3), while the average sick leave days were significantly higher among patients with progressive disease (64% vs 39%, p < 0.001; figure 4). the higher frequency of sick leave days in the progression group translated to higher indirect costs with 151,800 sek (16,415 €) vs 92,839 sek (10,039 €) compared with the non-progression group (p < 0.001; figure 5). discussion our present study investigated the rate of progression from moderate to severe uc in a cohort of swedish patients 2005– 2015. emr data linked to multiple national registries were analyzed in order to reflect real-world practice in patients with uc. using the icd classification of diagnoses, our study shows that approximately one-fourth of patients with moderate uc progress to severe uc over a period of 5 years. the odds of a patient experiencing a uc-related hospitalization were two-fold greater in those with progressive disease resulting in a sizeable medical and societal burden; specifically, the loss of work productivity results from absenteeism. logistic regression analysis of factors associated with the disease indicates that younger age, long disease duration, and glucocorticosteroid use were associated with progression to severe uc. the disease progression rate in our present study is in agreement with the disease extension rate reported in a recent meta-analysis (18). results of the meta-analysis showed that the rate of disease extension from proctitis or left-sided colitis to extensive uc was 18% at 5 years and 31% at 10 years (18). furthermore, in three different population-based cohort studies, 21–24% of uc patients showed progressive disease from limited to extensive colitis, and 5–10% underwent a colectomy during a figure 1. probability of progressing from moderate to severe ulcerative colitis over the median 5.2-year follow-up. the numbers at risk are given for each time point of interest. uc, ulcerative colitis. table 3. demographic and baseline clinical factors associated with progression from moderate to severe uc over a median 5.2-year follow-up. variable no progression (n = 1,040) progression (n = 321) odds ratio (95% ci) p value aged <30 years, n (%) 276 (26.5) 104 (32.4) 1.62 (1.17–2.25) <0.01 aged 30–50 years, n (%) 355 (34.1) 115 (35.8) 1.38 (1.01–1.90) 0.04 aged >50 years, n (%) 409 (39.3) 102 (31.8) 1.00 (reference) – female, n (%) 488 (46.9) 155 (48.3) 1.16 (0.90–1.51) 0.26 male, n (%) 552 (53.1) 166 (51.7) 1.00 (reference) – uc disease duration, y (mean ± sd) 4.9 ± 2.7 5.8 ± 2.6 1.09 (1.03–1.15) <0.01 glucocorticosteroids, n (%) 867 (83.4) 316 (98.4) 2.49 (1.67–3.72) <0.0001 5-asa, n (%) 489 (47.0) 155 (48.3) 1.25 (0.95–1.65) 0.11 crp: elevateda, n (%) 362 (34.8) 161 (50.2) 1.59 (1.21–2.08) <0.01 crp: not elevated, n (%) 492 (47.3) 141 (43.9) 1.00 (reference) – 5-asa, 5-aminosalicylic acid; ci, confidence interval; crp, c-reactive protein; sd, standard deviation; uc, ulcerative colitis; y, years. aelevated crp was defined as >30 mg/l. disease progression in ulcerative colitis 5 figure 2. disease burden in patients progressing from moderate to severe ulcerative colitis. uc, ulcerative colitis. figure 3. proportion of unemployed patients. figure 4. sick leave within 1 year. 6 d. dahlgren et al. forthcoming 5to 10-year period (19–21). several meta-analyses (22–25) report an increased risk of colitis-associated colorectal cancer in patients with uc compared to the general population. this increased risk has been found to be related to young age at disease onset, prolonged disease duration, extent of disease, and male sex. in our current study, young age, prolonged disease duration, and extended corticosteroid use were all found to be significant factors associated with disease progression. these results are in line with those reported for patients with chronic disease activity, frequent relapses, need for systemic glucocorticosteroid use, and young age at diagnosis (26–28). as shown in our study, these same factors are associated with considerable work disability (29). uc is often diagnosed in adolescence or early adulthood in the working-age population, and substantial work productivity losses among patients with moderate to severe disease have been reported (5, 11, 29). our study confirms that a greater proportion of patients with progressive disease were more frequently unemployed compared with those who had a nonprogressive disease. hence, the average annual number of sick leave days was significantly greater in those with progressive disease resulting in 1.6 times higher indirect costs in patients with progressive disease as compared to those maintaining moderate disease activity. to this end, the use of medical healthcare resources was found to be higher in patients with progressive uc as the prevalence of all-cause and uc-related hospitalization was significantly greater in this patient group. though the overall disease progression rate may not be deemed to be aggressive, the cost of disease progression in uc is significant in terms of medical costs as well as societal burden. despite the availability of antibody-based biologics to treat uc during the study period, biologic use was only 9% overall in this uc population during a median of 5-year follow-up. even among patients with aggressive disease activity progressing to severe uc, the use of biologics was 15%. the use of biologics reported in other population-based cohort studies with assessment of disease extension has also been relatively low ranging from 5 to 11% (20, 21, 30), consistent with the overall use of biologics. in agreement with this, the use of biologics, in general, was reported to be 7.4% in a recent study, estimating the annual societal cost of uc in sweden (31). these findings suggest that patients with uc are being managed predominantly with 5-aminosalicylates and corticosteroids to induce remission and control symptoms against a suggested background treatment with azathioprine treatment in 23% of this disease population (20). guidelines for uc treatment support a treat-to-target approach, in which the therapeutic goal includes not only control of symptoms and improved quality of life but also the prevention of disease progression, bowel damage, surgery, and disability (32–36). the ultimate targets of treatment in uc remain to be defined but may include clinical and patient-related outcomes (pros) such as resolution of rectal bleeding and normalization of bowel habits; endoscopic and histological mucosal healing; and normalization of biomarker targets such as serum crp and fecal calprotectin levels. to accomplish treat-to-target goals, therapy needs to begin early, be aggressive and optimized, and then patients need to be monitored regularly until predefined response goals are achieved. given the burden associated with uc progression, broader use of biologics earlier in the disease course is demanded to accomplish these treat-to-target goals. one of the strengths of the current study is the linkage of several databases including the most proximal emr to provide a comprehensive and robust evaluation of data in a large cohort of patients with moderate to severe uc (37). however, there are some limitations that need to be considered when interpreting the results of the present study. although clinical outcomes reported as partial mayo scores and the icd classification are available, some clinical factors are not fully captured, such as endoscopic findings with disease location. also, since the proportion of patients on biologics treatment was low and the proportion of patients with fecal calprotectin analysis was low (38% in progression group and 22% in non-progression group), these two factors were not included in the regression model. figure 5. costs associated with sick leave. sek, swedish krona; €, euro. disease progression in ulcerative colitis 7 however, despite the low frequency of reported fecal calprotectin values, numerically more patients with uc progression compared with no progression had elevated fecal calprotectin, being 87% versus 79%. in this swedish uc population, progression of moderate to severe uc occurred in approximately one-fourth of the patients with moderate disease. the disease burden associated with hospitalizations and sick leave and their associated costs were significantly higher in patients who progressed to severe uc compared with those who did not. longer disease duration, young age, glucocorticosteroid use, and elevated serum crp levels were markedly associated with disease progression. early identification of disease progression and intervention with appropriate effective treatment should be implemented early to prevent the considerable disease burden associated with uc progression. acknowledgments the basic study was funded with further writing support by abbvie. we thank wan-ju lee, phd, ryan clark, pharmd, and yuri sanchez gonzalez, phd, for essential basic work with the manuscript and fruitful collaboration. source data verification and database monitoring was performed by clinical reports ab, stockholm, sweden. disclosure statement david dahlgren has no disclosures. per m. hellström, lars agréus, and jan stålhammar received consultant fees from abbvie. funding this study was funded by abbvie inc. abbvie participated in the study design, research, data collection, analysis and interpretation of data, writing, reviewing, and approving the publication. all authors had access to the data results and participated in the development, review, and approval of this article. notes on contributors david dahlgren, phd, is an expert in gastrointestinal injury, biopharmaceutics and translational in vivo models at the department of pharmaceutical biosciences, faculty of medicine and pharmacy, uppsala university. lars agréus, md, phd, senior professor of general medical gastroenterology, specialist in general medicine, department of neurobiology, care sciences and society, karolinska institutet, stockholm. jan stålhammar, md, phd, associate professor, specialist in general medicine, department of public health and caring sciences, family medicine and preventive medicine, uppsala university, uppsala, faculty of medicine and pharmacy, uppsala university, uppsala, sweden. per m. hellström, md, phd, study lead, professor, senior consultant of gastroenterology, gastroenterology and hepatology unit, department of medical sciences, faculty 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http://dx.doi.org/10.14309/ajg.0000000000000183 http://dx.doi.org/10.14309/ajg.0000000000000152 http://dx.doi.org/10.1038/ajg.2015.233 http://dx.doi.org/10.1053/j.gastro.2020.01.006 http://dx.doi.org/10.48101/ujms.v127.8260 http://dx.doi.org/10.48101/ujms.v127.8260 upsala journal of medical sciences 2022, 127, e7860 http://dx.doi.org/10.48101/ujms.v127.7860 blood pressure screening in midlife aids in prediction of dementia later in life linn moberga, jerzy lepperta, simon liljeströma, mattias rehna, lena kilanderb and abbas chaboka acentre for clinical research, uppsala university, västmanland county hospital, västerås, sweden; bdepartment of public health and caring sciences/geriatrics, uppsala university, uppsala, sweden abstract background: there is substantial evidence that midlife hypertension is a risk factor for late life dementia. our aim was to investigate if even high blood pressure at a single timepoint in midlife can predict an increased risk for all-cause dementia, alzheimer’s disease (ad), or vascular dementia ( vad) later in life. methods: the community-based study population comprised 30,102 dementia-free individuals from the westmannia cardiovascular risk factors study. the participants were aged 40 or 50 years when the health examination took place in 1990–2000. diagnose registers from both hospitals and primary healthcare centers were used to identify individuals who after inclusion to the study developed dementia. the association between midlife high blood pressure (defined as systolic blood pressure >140 and/or diastolic blood pressure >90 mmhg) at a single timepoint and dementia was adjusted for age, gender, body mass index (bmi), fasting blood glucose, education, smoking, and physical activity level. multivariate binary cox regression analyses were used. results: after a mean follow-up time of 24 years resulting in 662,244 person/years, 761 (2.5%) individuals had been diagnosed with dementia. midlife high blood pressure at a single timepoint predicted allcause dementia (hazard ratio [hr]: 1.22, 95% confidence interval [ci]: 1.02–1.45) and vad (hr: 2.10, 95% ci: 1.47–3.00) but not ad (hr: 1.06, 95% ci: 0.81–1.38). conclusion: this study suggests that even midlife high blood pressure at a single timepoint predicts allcause dementia and more than doubles the risk for vad later in life independently of established confounders. even though there was no such association with ad, this strengthens the importance of midlife health examinations in order to identify individuals with hypertension and initiate treatment. article history received 30 april 2021 revised 06 october 2021 accepted 07 november 2021 published 03 january 2022 keywords midlife hypertension; screening; dementia; prediction; long-term follow-up; prevalence introduction the prevalence of dementia is increasing worldwide, and as of today, there is no disease-modifiable treatment (1–5). this fact has increased the interest in prevention, and potentially modifiable midlife vascular risk factors have been identified (2, 3, 5, 6). there are several subtypes of dementia where alzheimer’s disease (ad) is the most common followed by vascular dementia ( vad), and altogether, they account for 80% of all dementia cases (5). coexisting cerebrovascular disease lowers the threshold for ad to become clinically manifest. thus, both subtypes are associated with vascular risk factors (7). midlife hypertension has been highlighted since research indicates that antihypertensive treatment lowers the incidence of dementia later in life (8–10). also, a number of observational cohort studies have confirmed that midlife hypertension (age 40–64) increases the risk for dementia or cognitive decline later in life (11–17). most of these studies measured blood pressure repeatedly throughout life and did not differentiate between various dementia subtypes. screening for cardiovascular risk factors was highlighted during the 1980–1990s in the western countries. the county of  västmanland, situated near stockholm, sweden, started, in  1990, a screening program in a large population: the westmannia cardiovascular risk factors study (wictory) with the overall aim of lowering the cardiovascular mortality rate in the county. with data from this cohort, we aimed to investigate whether the presence of high blood pressure at a single timepoint at a health examination in midlife is a predictive factor for the development of all-cause dementia, ad, or vad later in life. contact linn moberg linn.moberg@regionvastmanland.se © 2022 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article http://dx.doi.org/10.48101/ujms.v127.7860 mailto:linn.moberg@regionvastmanland.se http://creativecommons.org/licenses/by/4.0/ 2 l. moberg et al. materials and methods study population the intention of wictory was to offer a free screening for cardiovascular risk factors to all inhabitants aged 40 or 50 during 1990–2000 (born 1940–1959) and residing in the county of västmanland. of the 56,977 eligible individuals, 34,269 (60%) completed the health examination. all participants gave an informed consent. a detailed overview of the selection procedure for the present study is shown in figure 1. reasons for exclusion were as follows: missing records of systolic or diastolic blood pressure (dbp), previouly recorded dementia diagnosis, or incorrect social security numbers (pins). finally, individuals who had emigrated or moved to another swedish county (source: swedish tax agency population registration) were excluded if they did not already have a record of dementia in our registers, since we were unable to know if they got a dementia diagnosis elsewhere than in västmanland in 2008 or later (our national registers reach 2007, see ‘diagnoses of dementia’ below). in total, 4,167 individuals were excluded, presented as lost to follow-up in the following text. the final study population comprised 30,102 dementia-free individuals from the wictory cohort with age range 60–80 years and mean follow-up time of 24 years. the uppsala regional ethical review board approved the study by waiving informed consent (dnr. 2007/165). baseline examinations the screening procedure has been presented in detail elsewhere (18). briefly, data for the following measurements from the wictory cohort health examination in 1990–2000 were collected for this study: office blood pressure, body mass index (bmi), fasting blood glucose (fb-glucose), educational level, and smoking and physical activity habits. blood pressure was measured after 15 min of rest in a sitting position in the upper arm. a manual sphygmomanometer at a suitable size was used, and the average of two measurements was documented. bmi was calculated as the body mass divided by figure 1. flow chart of study population. moved from county of västmanland -abroad, n = 213 -another county, n = 3,781 no diagnosed dementia n = 7,003 individuals with non-high blood pressure** n = 22,847 participants in health survey n = 34,269 eligible study population n = 34,096 included in analysis n = 30,102 excluded: -missing blood pressure data at baseline, n = 142 -diagnosed dementia at baseline, n = 16 -incorrect social security number, n = 15 individuals with high blood pressure* n = 7,255 diagnosed dementia n = 509 no diagnosed dementia n = 22,338 diagnosed dementia n = 252 *>140 and/or >90, **<140/90 high blood pressure in midlife predicts dementia 3 the square of the body height (kg/m2). blood glucose was measured after 2 h refraining from eating and was analyzed using a factory-calibrated photometer. education was selfreported as number of years post-elementary school. smoking habits were self-reported in a questionnaire with five alternatives (never, previous, 1–14 cig/day, 15–25 cig/day, and >25 cig/day). physical activity level was self-reported and defined as low impact exercise for at least 30 min, and a fivegrade scale was used (exercising daily, 3–4 times/week, 1–2 times/week, 1–2 times/month, and exercising rarely or never). diagnoses of dementia the duva (datalager för uppföljning och verksamhetsanalys), 2008–2020, was our main diagnoses register since it comprises data from both primary healthcare centers and hospitals in västmanland county. in addition, hospital diagnoses registers from the national board of health and welfare (nbhw ) were used for the years 1963–2007. individuals from the cohort were considered to have a diagnosis of dementia if they had a record of any of the icd-10-se diagnoses of dementia shown in table 1. since icd-10-se was implemented in 1997, a conversion of the diagnoses to icd-9-se was made to be used for the earlier registers. for this, conversion tables supplied by the nbhw were applied, and included icd-9-se diagnoses are also shown in table 1. finally, the national register for death causes was used. both primary and secondary diagnoses were searched for in all registers. every visit to a doctor where a dementia diagnosis was recorded had been documented. thus, many individuals had repetitions of diagnoses and sometimes subtypes of dementia. individuals were included in analyses for ad or vad if they ever had a record of any of the diagnoses mentioned. statistical analysis high blood pressure was defined as systolic blood pressure (sbp) >140 mmhg and/or dbp >90 mmhg. individuals were dichotomized into a high blood pressure group and a non-high blood pressure group (reference category). regarding confounders, continuous variables were used for age, bmi, and fb-glucose. others were dichotomized into previous/current smoker or non-smoker (reference), physical activity less than one time per week/never or at least one time per week (reference), and educational level ≤1 year or >1 year of education after elementary school (reference). baseline characteristics were analyzed using independent sample t-tests (continuous variables) and chi-square tests (categorical variables). the association between midlife high blood pressure and dementia was first analyzed using univariate cox regression models, with all-cause dementia, vad, and ad as dependent variables and blood pressure as independent variable. after univariate analysis, multivariate cox regression models were used to further investigate the effect of putative confounding variables. dementia was used as dependent variable with blood pressure, bmi, fb-glucose, smoking, physical activity, educational level, age at inclusion, and gender as independent variables. a series of sensitivity analyses were conducted to test the robustness of the results. altered cut-offs were used (sbp > 130 mmhg and/or dbp > 80 mmhg, and sbp > 150 mmhg and/or dbp 100 mmhg) and analyzed with multivariate cox regression models. all statistical analyses were performed using ibm spss version 26, with p-values ≤ 0.05 considered statistically significant. results out of the 30,102 individuals included in the analysis, 7,255 (24.1%) had values defined as high blood pressure at the index investigation. after a mean follow-up time of 24 years (which resulted in 662,244 person/years), a total of 761 (2.5%) individuals were diagnosed with dementia: 252 in the high blood pressure group and 509 in the non-high blood pressure group. mean age for first diagnosis of dementia was 71 years. participants who later were diagnosed with dementia had a higher mean age at inclusion and overall, a more accentuated risk profile including significantly higher sbp, dbp, bmi, fb-glucose, and lower education ( table 2). they also had a higher mortality compared to the group with no dementia, 32% versus 13%, respectively. data regarding mortality among those lost to follow-up were missing. when comparing by blood pressure status, 18% had died in the high blood pressure group versus 13% in the non-high blood pressure group. mortality in the total cohort was 14%. the group lost to follow-up had a significantly lower mean age, sbp, dbp, and bmi. out of the 761 individuals diagnosed with dementia, 166 (22%) had received a diagnosis of vad and 343 (45%) a diagnosis of ad. since some individuals had more than one dementia diagnosis, there was an overlap between the two groups. thirtyseven individuals had a diagnosis of both vad and ad, resulting in 472 (62%) individuals ever diagnosed with ad or vad. unadjusted values of midlife high blood pressure at a single timepoint were associated with a significantly increased risk for all-cause dementia (hr: 1.69, 95% confidence interval [ci]: 1.45– 1.96, p-value < 0.001), vad (hr: 2.91, 95% ci: 2.15–3.96, p-value < 0.001), and ad (hr: 1.51, 95% ci: 1.20–1.89, p-value < 0.001). in the multivariate analysis, midlife high blood pressure at a single timepoint did significantly increase the risk for both all-cause dementia (hr: 1.22, 95% ci: 1.02–1.45, p-value 0.026) and vad  (hr: 2.10, 95% ci: 1.47–3.00, p-value < 0.001). for ad, there  was no statistically significant association (hr: 1.06, 95% ci: 0.81–1.38, p-value 0.940). all results are shown in table 3. table 1. dementia diagnoses. vascular dementia f01, f010, f011, f012, f013, f018, f019 alzheimer’s disease f000, f001, f002, f009, g30, g300, g301, g308, g309 other dementia types f020, f021, f022, f023, f024, f028, f03-p, f039, f051, f107a, g310, g318a icd-9-se 290.0, 290.1, 290.2, 290.3, 290.4, 290.8, 290.9, 291.2, 331.0, 331.1 4 l. moberg et al. sensitivity analyses with altered cut-offs revealed no major differences from the primary results. concerning all-cause dementia, adjusted hazard ratios for a lower cut-off (sbp > 130 mmhg and/or dbp > 80 mmhg) was 1.22 (95% ci: 1.03–1.45) and a higher cut-off (sbp > 150 mmhg and/or dbp > 100 mmhg) was 1.34 (95% ci: 1.08–1.67). sensitivity analyses for vad revealed adjusted hazard ratios for a lower cut-off was 1.73 (95% ci: 1.16–2.57) and for a higher cut-off = 1.70 (95% ci: 1.11–2.60). sensitivity analyses for ad were statistically non-significant. discussion in this community-based prospective cohort study of more than 30,000 individuals with a mean follow-up of 24 years, midlife high blood pressure at a single timepoint predicted all-cause dementia and more than doubled the risk for vad later in life when adjusted for age, gender, education, and other established midlife vascular risk factors. however, midlife high blood pressure at a single timepoint was not associated with the risk of ad when adjusted for the same confounders. hypertension has been suggested to be the largest potentially preventable risk factor for cerebrovascular damage (5). hypertension changes the structure of the cerebral vessels and consequently in the neurovascular unit in several ways. first, it promotes atherosclerosis in both extraand intracranial arteries, which, in turn, increases the risk for thromboembolic stroke, intracranial hypoperfusion, and vessel rupture causing hemorrhagic stroke (5, 19). after a stroke, there is a 30% risk to develop dementia (20, 21). also, silent (asymptomatic) infarcts increase the risk for dementia (20). midlife high blood pressure has been associated with increased white matter hyperintensity volumes (12), and autoregulation of cerebral blood flow is also disturbed in cases of long-lasting hypertension (5). in addition, hypertension is the main risk factor for microbleeds, which also contribute to the pathogeneses leading to cognitive impairment (5, 22). together, all these mechanisms contribute to cerebral ischemia, which increases the risk for primarily vad but also allcause dementia. there is emerging evidence of a neuropathological overlap between cerebrovascular disease and ad (7, 19, 23). a neuropathological study has shown that in 40% of clinically diagnosed ad cases, cerebrovascular lesions are present suggested to have lowered the threshold to dementia (7). considering this, it is surprising that we could not show an association between single value of midlife high blood pressure and ad later in life. previous cohort studies measuring repeated blood pressures have, however, shown this association (24). on the other hand, some previous cohort studies with repeated blood pressures presented results in line with ours regarding ad (15, 25). table 2. baseline characteristics at health examination by later dementia status, overall, and lost to follow up. characteristics dementia n = 761 no dementia n = 29,341 p-values dementia/no dementia total n = 30,102 lost to follow-up n = 4,167 p-values total/lost to follow-up age, mean (sd), years 48.9 (3.3) 45.6 (5.0) p < 0.001 45.7 (5.0) 44.9 (5.0) p < 0.001 female, n (%) 401 (52.7) 15,249 (52.0) n.s. 15,650 (52.0) 2,133 (51.2) n.s. sbp, mean (sd), mmhg 134 (18) 130 (17) p < 0.001 130 (17) 128 (16) p < 0.001 dbp, mean (sd), mmhg 85 (11) 83 (10) p < 0.001 83 (10) 81 (10) p < 0.001 bmi, mean (sd), kg/m2 25.9 (3.9) 25.6 (3.9) p < 0.05 25.6 (3.9) 25.4 (3.7) p < 0.01 fb-glucose, mean (sd), mmol/l 5.8 (1.3) 5.6 (1.3) p < 0.001 5.6 (1.3) 5.6 (1.1) n.s. smokers, n (%) 248 (32.8) 8,576 (29.7) n.s. 8,824 (29.7) 1,215 (29.6) n.s. physically active, n (%) 623 (84.0) 24,343 (84.8) n.s. 24,966 (84.8) 3,331 (84.0) n.s. elementary school only (%) 178 (27.6) 4,984 (19.1) p < 0.001 5,162 (19.3) 711 (19.1) n.s. dead, n (%) 241 (31.7) 3,924 (13.4) p < 0.001 4,165 (13.8) – n.a. sbp: systolic blood pressure; dbp: diastolic blood pressure; bmi: body mass index; fb-glucose: blood glucose after 2 h fasting; smokers: previous/current smoker; physically active: 30 minutes at least one time/week; n.s.: not significant; n.a.: not available; sd: standard deviation. table 3. cox proportional hazard models of midlife high blood pressure1 at a single timepoint and risk for all-cause dementia, vascular dementia, and alzheimer’s disease later in life. dementia type unadjusted hr (95% ci) adjusted for age at inclusion, gender, education, bmi, fb-glucose, smoking, and physical activity level, hr (95% ci) all-cause dementia (n = 761) 1.69 (1.45–1.96)*** 1.22 (1.02–1.45)* vad (n = 166) 2.91 (2.15–3.96)*** 2.10 (1.47–3.00)*** ad (n = 343) 1.51 (1.20–1.89)*** 1.06 (0.81–1.38)† ci: confidence interval; ad: alzheimer’s disease; vad: vascular dementia; bmi: body mass index; fb-glucose: fasting blood glucose; hr, hazard ratio. 1sbp >140 mmhg and/or dbp > 90 mmhg. *p < 0.05; ***p < 0.001; †n.s. high blood pressure in midlife predicts dementia 5 the validity of using registers of diagnoses might be debatable. in this particular study design, we have not examined the patients ourselves as many previous cohort studies have done (13, 15, 16), which may question the validity in the recorded diagnoses. for reference, it has been suggested that generally, 85–95% of all inpatient registered diagnoses are correct (26). however, regarding dementia, this number is suggested to be only 26% (27). in the present study, diagnoses were collected both from hospitals and primary healthcare centers, meaning that the correct number should reasonably be higher since dementia diagnoses most often are identified by primary care physicians. the fact that hospitals in general were included might have resulted in lower numbers of specific dementia diagnoses compared to unspecified ones. thus, the relatively low proportion of ad and vad (62% of all-cause dementia) in our study might be false due to unrecorded or misclassified cases. this might have contributed to an underestimation of the effect of midlife high blood pressure at a single timepoint on the development of ad and vad later in life, but not all-cause dementia. however, this fact alone does probably not explain the unsignificant results regarding ad since its association to hypertension is not as strong as for vad. the dementia prevalence of 2.5% in this cohort with individuals aged 60–80 years, mean age 71 for first diagnosis, is considered reliable. according to a swedish report from 2006, the prevalence of demented individuals aged 65 is 1%, and the figure is expected to be doubled for every 5 years (28). moreover, mortality rates in the high blood pressure group were significantly higher than in the total cohort (18% vs. 14%). considering hypertension being the largest risk factor for cardiovascular disease and all-cause mortality (24), death caused by hypertension might be a competing risk for the possibility of diagnosing dementia. strengths of this study include the large community-based population and a long follow-up time. another strength is our differentiating into dementia subtypes, thus contributing to clarify the associated risk for each subtype. at this period of time, brain computed tomography (ct) scan was recommended in the guidelines for dementia investigation, indicating that the  differentiation between vascular and neurodegenerative disorders should be reasonably correct. furthermore, we were able to adjust for several established midlife confounders in the multivariate analyses, and primary results were confirmed by sensitivity analyses. continuous variables were used when possible to minimize residual confounding. there are several limitations to this study. first, we did not take into account whether individuals were on antihypertensive medication at baseline or during follow-up. this information is available from the nbhw in the prescribed drug register starting 2006 but was not retrieved for the present study. it would be beneficial for future cohort studies to separate individuals with high blood pressure at baseline into those who are on antihypertensive treatment and those who are not. also, it would be of great value to investigate compliance to prescribed medication by using data from the prescribed drugs register. another source of limitation was that it was clearly obvious in the registers that round offs had been made when measuring blood pressure. this is the reason why we chose >140/90 mmhg and not ≥140/90 mmhg as our definition of high blood pressure. this can also be considered a strength since we, with this definition, probably have minimized the risk of false high blood pressure values. we did not use the term hypertension since this diagnosis cannot be confirmed by measurements from a single timepoint (24). regarding analysis for vad and ad, icd-9 codes were not taken into account since before 1997, differentiated dementia diagnoses were not available. finally, our results cannot be generalized to other populations because an overwhelming majority of study participants were scandinavians. in conclusion, our study suggests that midlife high blood pressure at a single timepoint predicts all-cause dementia and more than doubles the risk for vad later in life when adjusted for established confounders. regarding ad, only unadjusted values showed association between midlife high blood pressure at a single timepoint and ad later in life. we were unable to investigate the validity in using diagnosis registers from primary healthcare centers regarding dementia. this would be desirable in future studies. also, our study design using midlife high blood pressure values from a single time-point has to be replicated in future studies. learning from these, we could investigate when midlife health examinations should be initiated in order to start treatment at an optimal age. disclosure statement the authors report no conflicts of interest. funding this work was supported by the regional research council uppsala-örebro, sweden, under grant [rfr-73931]. notes on contributors linn moberg, md, is a medical intern at västmanland county hospital, västerås, sweden. jerzy leppert, md, phd, is a professor of family medicine at the centre for clinical research, uppsala university, västmanland county hospital, västerås, sweden. simon liljeström, phd, is a statistician at the centre for clinical research, uppsala university, västmanland county hospital, västerås, sweden. mattias rehn, bsc, med, is a systems administrator at the centre for clinical research, uppsala university, västmanland county hospital, västerås, sweden. lena kilander, md, phd, is a professor of geriatrics at the department of public health and caring sciences, uppsala university, uppsala, sweden. abbas chabok, md, phd, is an associate professor of surgery at  the centre for clinical research, uppsala university, västmanland county hospital, västerås, sweden. 6 l. moberg et al. orcid linn moberg https://orcid.org/0000-0003-1038-1196 jerzy leppert https://orcid.org/0000-0003-1433-0329 simon liljeström https://orcid.org/0000-0002-1463-2346 mattias rehn https://orcid.org/0000-0001-9927-2660 lena kilander https://orcid.org/0000-0001-6600-9110 abbas chabok https://orcid.org/0000-0001-9662-5045 references 1. world alzheimer report 2018 – 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http://dx.doi.org/10.1161/circulationaha.114.011147 http://dx.doi.org/10.1056/nejmoa022066 http://dx.doi.org/10.1056/nejmoa022066 http://dx.doi.org/10.1161/01.str.29.1.75 http://dx.doi.org/10.1371/journal.pone.0091637 http://dx.doi.org/10.1371/journal.pone.0091637 http://dx.doi.org/10.1212/01.wnl.0000271090.28148.24 http://dx.doi.org/10.1212/01.wnl.0000271090.28148.24 http://dx.doi.org/10.1093/eurheartj/ehy339 http://dx.doi.org/10.1159/000330020 http://dx.doi.org/10.1186/1471-2458-11-450 http://dx.doi.org/10.1007/bf03324718 http://www.sbu.se/upload/publikationer/content0/1/demens_sammanfattning.pdf http://www.sbu.se/upload/publikationer/content0/1/demens_sammanfattning.pdf upsala journal of medical sciences 2023, 128, e9290 http://dx.doi.org/10.48101/ujms.v128.9290 validation of myocarditis diagnoses in the swedish patient register for analyses of potential adverse reactions to covid-19 vaccines rolf gedeborga,b, lennart holmc, nils felteliusc, anders sundströmd, kai m eggerse, marja-leena nurminend, maria grünewaldf, nicklas pihlströmf, björn zetheliusc and rickard ljungc,g adepartment of efficacy and safety 1, division of licensing, medical products agency, uppsala, sweden; bdepartment of surgical sciences, uppsala university, sweden; coffice of use and information, division of use and information, medical products agency, uppsala, sweden; ddepartment of drug safety, division of use and information, medical products agency, uppsala, sweden; edepartment of medical sciences, uppsala university, uppsala, sweden; fstatistics group, department of efficacy and safety 2, division of licensing, medical products agency, uppsala, sweden; ginstitute of environmental medicine, karolinska institutet, stockholm, sweden abstract background: coronavirus disease 2019 (covid-19) mrna vaccines are associated with an increased risk of myocarditis using hospital discharge diagnoses as an outcome. the validity of these register-based diagnoses is uncertain. methods: patient records for subjects < 40 years of age and a diagnosis of myocarditis in the swedish national patient register were manually reviewed. brighton collaboration diagnosis criteria for myocarditis were applied based on patient history, clinical examination, laboratory data, electrocardiograms, echocardiography, magnetic resonance imaging and myocardial biopsy. poisson regression was used to estimate incidence rate ratios, comparing the register-based outcome variable to validated outcomes. interrater reliability was assessed by a blinded re-evaluation. results: overall, 95.6% (327/342) of cases registered as myocarditis were confirmed (definite, probable or possible myocarditis according to brighton collaboration diagnosis criteria, positive predictive value 0.96 [95% ci 0.93–0.98]). of the 4.4% (15/342) cases reclassified as no myocarditis or as insufficient information, two cases had been exposed to the covid-19 vaccine no more than 28 days before the myocarditis diagnosis, two cases were exposed >28 days before admission and 11 cases were unexposed to the vaccine. the reclassification had only minor impact on incidence rate ratios for myocarditis following covid-19 vaccination. in total, 51 cases were sampled for a blinded re-evaluation. of the 30 randomly sampled cases initially classified as either definite or probably myocarditis, none were re-classified after re-evaluation. of the in all 15 cases initially classified as no myocarditis or insufficient information, 7 were after re-evaluation re-classified as probable or possible myocarditis. this re-classification was mostly due to substantial variability in electrocardiogram interpretation. conclusion: this validation of register-based diagnoses of myocarditis by manual patient record review confirmed the register diagnosis in 96% of cases and had high interrater reliability. reclassification had only a minor impact on the incidence rate ratios for myocarditis following covid-19 vaccination. article history received 16 january 2023 revised 17 march 2023 accepted 9 april 2023 published 11 may 2023 keywords covid-19 vaccines; myocarditis; diagnosis; validation study introduction a large study based on nationwide health registers in denmark, finland, norway and sweden has demonstrated that both first and second doses of mrna coronavirus disease 2019 (covid-19) vaccines are associated with an increased risk of myocarditis and pericarditis (1). the primary outcome was a hospital discharge diagnosis indicating myocarditis in the respective country’s patient register. it is important to determine the accuracy of these diagnoses to support the validity of the estimated associations between mrna covid-19 vaccine exposure and myocarditis. clinically, the diagnosis of acute myocarditis is based on symptoms (mainly acute chest pain), electrocardiogram (ecg), echocardiography, serum biomarkers for myocardial injury, magnetic resonance imaging (mri) and/or endomyocardial biopsy (2). there are no previous international consensus case definitions for myocarditis as adverse events following immunisation. such criteria have, however, recently been proposed by the brighton collaboration (3). the aim of this study was to validate the accuracy of a myocarditis diagnosis when based on icd-10 hospital discharge diagnoses in the swedish national patient register, applying the contact rolf gedeborg rolf.gedeborg@lakemedelsverket.se supplemental data for this article can be accessed here. © 2023 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article http://dx.doi.org/10.48101/ujms.v128.9290 mailto:rolf.gedeborg@lakemedelsverket.se http://dx.doi.org/10.48101/ujms.v128.9290 http://creativecommons.org/licenses/by/4.0/ 2 r. gedeborg et al. proposed brighton collaboration criteria in a structured manual review of patient records. methods study population to enable pharmacoepidemiological studies of the covid-19 vaccines, the swedish medical products agency has set up a regularly updated dynamic nationwide register-based study cohort (covacsafe-se). this research database has been generated from individual-level linkage of covid-19 vaccination exposure data to other national health data registers (4). it primarily included all individuals permanently residing in sweden on 31 december 2020 and has been continuously updated with, e.g. information on exposure to covid-19 vaccines and diagnoses from the swedish national patient register. for validation of icd-10 diagnoses of myocarditis, registered in the swedish national patient register and linked to covacsafese, we selected individuals 12–39 years old having an incident hospital discharge diagnosis of myocarditis identified during the study period from 27 december 2020 to 13 november 2021. these selection criteria are identical to those in the nordic study on sars-cov-2 vaccination and myocarditis (1) but cover a longer time period. all cases of myocarditis with a prior exposure to the covid-19 vaccine and a random selection of cases unexposed to the covid-19 vaccine were eligible for the validation procedure. our primary validation was restricted to individuals 12–39 years old because young men and adolescent boys appeared to be at the highest risk in the nordic study (1). individuals with any record of myocarditis from inpatient or specialised outpatient hospital care from 1 january 2017 to 26 december 2020 were considered as prevalent cases and therefore excluded from the study population. the study was approved by the swedish ethical review authority. register-based diagnosis of myocarditis from covacsafe-se cases of myocarditis were identified from primary or secondary hospital discharge codes (icd-10-se) i400, i401, i408, i409, i411, i418 or i514 after an in-hospital stay. diagnoses in covacsafe-se originate from exact person-based linkage to the swedish national patient register using the unique personal identification number (5). this register is maintained by the national board of health and welfare and reporting to the register of all in-hospital care and out-patient specialist care is mandated by law. during the study period, discharge diagnoses were coded according to the swedish clinical modification of the 10th revision of the international statistical classification of diseases and related health problems (icd-10-se) (6). patient record review patient records covering the hospital separation where a myocarditis diagnosis occurred first were requested from that specific hospital department. brighton collaboration criteria for the level of evidence were applied based on patient history, clinical examination, laboratory data, electrocardiograms, echocardiography, magnetic resonance imaging and myocardial biopsy to verify the diagnosis of myocarditis. a structured electronic questionnaire was developed to capture the basic information required for the application of the brighton collaboration criteria for the level of evidence (see variable list in table s1) (3). according to these criteria, each case reviewed was classified as (1) ‘definitive case’, (2) ‘probable case’, (3) ‘possible case’, (4) having ‘insufficient information’ or (5) ‘not a case’. the brighton collaboration levels of evidence is an ordinal scale, and the consequences of reclassifications are different depending on where on the scale the reclassification occurs. a pragmatic case definition based on the brighton collaboration levels would be either level 1–2 (definite or probable) or level 1–3 (definite, probable, or possible). each patient record was reviewed by one of four raters. all four had several years of experience from working with pharmacovigilance at the swedish medical products agency. three were medical doctors specialised in rheumatology/internal medicine, anaesthesiology/ critical care and pharmacology, respectively. one had a clinical background as anaesthesia nurse and research nurse. effective blinding to vaccination status was deemed unfeasible. if the rater was uncertain how to interpret clinical information or classify a case, a contracted senior cardiologist at the uppsala university hospital was consulted. if there remained uncertainty, the case was discussed in the group of raters to reach consensus. interrater reliability to evaluate interrater reliability, we randomly selected 15 records (or all if fewer were available) from each brighton collaboration level for re-evaluation in a new round of patient record review. the same group of raters was used, but each rater could only reassess patient records reviewed by another rater in the first round of manual record review. also in this second round of review, the contracted senior cardiologist could be consulted at the discretion of the individual rater. statistics based on the results of the patient record review we described the extent and pattern of reclassification and calculated the positive predictive value of a register-based diagnosis with 95% confidence interval (ci). to determine the sensitivity of risk estimates in the previously published nordic myocarditis study (1) to the reclassification of the outcome, we compared the results of using the registerbased outcome definition to an outcome definition requiring brighton collaboration level of evidence of at least possible (level 1–3) or probable (level 1–2). as only a random selection of myocarditis cases unexposed to the covid-19 vaccine was  validated, a random selection of unexposed myocarditis cases not validated was reclassified proportionate to the reclassification in the validated subset. starting follow-up on validation of myocarditis 3 27  december 2020, we used poisson regression to estimate incidence rate ratios (irrs) with 95% cis, comparing rates of myocarditis in 28-day risk periods after the administration date of the first and second dose of a covid-19 vaccine to rates in unvaccinated periods, using time-varying exposures. identical modelling of covariates as in the nordic study was used (1). results out of 404 cases of myocarditis 12–39 years old identified during the study period, patient records were requested for all 157 exposed cases and for a random sample of 191, out of the 247 cases unexposed to the covid-19 vaccine. patient records could be retrieved for 154 (98.1%) of the exposed cases and 188 (98.4%) of the randomly selected cases unexposed to the covid-19 vaccine (figure s1). in this population, 273 were males and 69 were females. overall, unexposed cases tended to be more non-specific with a more complex clinical presentation. a lower proportion had elevated markers for myocardial damage and in more than half of these cases, no results from echocardiography were available (table 1). the characteristics of the randomly selected unexposed cases were comparable to the source population of unexposed cases (table s2). reclassification after patient record review after the patient record review, 95.6% (327/342) of the cases registered as myocarditis in the swedish national patient register were confirmed as having myocarditis (definite, probable or possible), while 4.4% (15/342) were reclassified as not having myocarditis (table 2). of these, two cases had been exposed to the covid-19 vaccine no more than 28 days before the myocarditis diagnosis, two cases were exposed >28 days before admission and 11 cases were unexposed to vaccine. the overall positive predictive value was 0.96 (95% ci 0.93–0.98), identical when restricted to males and 0.94 (95% ci 0.86–0.98) when restricted to females. an even stricter case definition of myocarditis (definite or probable) yielded a positive predictive value of 0.94. impact of reclassification on risk estimates when the regression analysis to estimate the risk for myocarditis associated with covid-19 was rerun in the age group 16–24 years old, with a case definition requiring a brighton collaboration level of evidence of at least possible, the results were only marginally different compared to using the original register diagnosis (figure 1). an even stricter case definition, requiring a brighton collaboration level of evidence of at least probable, produced essentially identical results. a similar pattern was seen in the analysis of the age group 25–39 years old (figure 2). interrater reliability in total, 51 cases were sampled for a blinded re-evaluation ( table 3). of the 30 randomly sampled cases initially classified as either definite or probable myocarditis none were table 1. case severity by exposure status. clinical severity measures exposed to covid-19 vaccine ≤ 28 days before admission for myocarditis n = 106 exposed to covid-19 vaccine > 28 days before admission for myocarditis n = 48 not exposed to covid-19 vaccine before admission for myocarditis n = 188 deaths 0 0 0 length of hospital stay (days), median (iqr) 3 (2–4) 2 (1–4) 3 (1.75–4) troponin i/t, n (%) elevateda 100 (94.3) 45 (93.8) 166 (88.3) missing 0 0 6 (3.2) ejection fraction, n (%) < 40 0 3 (6.3) 7 (3.7) 40–54 18 (17.0) 6 (12.5) 19 (10.1) ≥ 55 46 (43.4) 20 (41.7) 61 (32.4) missing 42 (39.6) 19 (39.6) 101 (53.7) athe cut-off for elevated troponin was analysisand hospital-specific. table 2. classification in brighton collaboration levels of diagnostic certainty for myocarditis from the primary patient record review, by exposure status. brighton collaboration diagnostic certainty level exposed to covid-19 vaccine ≤ 28 days before admission for myocarditis (n = 106) exposed to covid-19 vaccine > 28 days before admission for myocarditis (n = 48) not exposed to covid-19 vaccine before admission for myocarditis (n = 188) definitive case, n (%) 44 (41.5) 20 (41.7) 74 (39.4) probable case, n (%) 57 (53.8) 26 (54.2) 100 (53.2) possible case, n (%) 3 (2.8) 0 (0) 3 (1.6) insufficient information, n (%) 0 (0) 1 (2.1) 3 (1.6) not a case, n (%) 2 (1.9) 1 (2.1) 8 (4.3) 4 r. gedeborg et al. re-classified outside the brighton collaboration levels of evidence of at least probable (level 1–2). in all, 21 cases were initially classified as brighton collaboration level 3–5, of which all were sampled for re-evaluation. among these 21 cases, eight out of the 12 reclassifications by a new rater had a direct impact on the binary outcome variable. after reevaluation 12 of these 21 cases were classified within the brighton collaboration levels of evidence of at least possible (level 1–3). discussion approximately 4% of the cases registered as myocarditis in the swedish national patient register during the study period were reclassified as not having myocarditis. the proportion reclassified was slightly higher in the group unexposed to the covid-19 vaccine compared to those exposed to the vaccine. when the risk for myocarditis related to covid-19 vaccines was estimated, differences in the strength of association were minor when figure. 1. estimated association between covid-19 vaccines and myocarditis events within 28 days of exposure in the age group 16–24 years, comparing different definitions of the outcome variable. squares represent incidence rate ratios with lines representing 95% confidence intervals, and arrows truncation of these intervals. a single vaccine name indicates first dose of that vaccine (eg, bnt162b2) and the risk of the outcome after the first dose. vaccine names in combination indicate a vaccine schedule of first dose of the first vaccine and a second dose of the second vaccine (eg, bnt162b2, bnt162b2) and the risk of the outcome after the second dose. the poisson regression model adjusted for age group and sex, previous sars-cov-2 infection, health care worker status, nursing home resident, and comorbidity variables. figure. 2. estimated association between covid-19 vaccines and myocarditis within 28 days of exposure in the age group 25–39 years, comparing different definitions of the outcome variable. squares represent incidence rate ratios with 95% cis. a single vaccine name indicates first dose of that vaccine (eg, bnt162b2) and the risk of the outcome after the first dose. vaccine names in combination indicate a vaccine schedule of first dose of the first vaccine and a second dose of the second vaccine (eg, bnt162b2, bnt162b2) and the risk of the outcome after the second dose. the poisson regression model adjusted for age group and sex, previous sars-cov-2 infection, health care worker status, nursing home resident, and comorbidity variables. validation of myocarditis 5 using an outcome measure based on the patient record review, compared to using the original register diagnosis. before the covid-19 pandemic, swedish national register data have indicated a slightly increasing trend in the background incidence of myocarditis in subjects aged ≥16 years during the period 2000–2014 (7). during a 1-year follow-up, 6.4% were newly diagnosed with either heart failure or dilated cardiomyopathy. the frequency of severe outcomes was higher in older patients and occurred in the immediate post-discharge period. during the pandemic, myocarditis has been described as a rare cardiovascular complication to both the covid-19 infection and covid-19 vaccines (1, 8, 9). using a register-based diagnosis of myocarditis may, however, raise concerns regarding the validity of the diagnosis. validation is therefore important to support interpretation of findings in register-based studies. there are currently no internationally accepted consensus criteria for myocarditis. we used the recently proposed brighton collaboration criteria as the basis for the manual patient record review (3). other criteria used, such as those issued by the us centers for disease control and prevention (cdc), are similar but not identical (10). it may be difficult to apply myocarditis criteria to information extracted from patient records. endomyocardial biopsy results are considered as a key component but are rarely justified in cases of uncomplicated myocarditis and has limited sensitivity (11). in our validation, only few patients were subjected to biopsy. this diagnostic criterion is therefore in reality of low value for studies using data from routine care. the reporting in routine patient records of results from cardiac magnetic resonance imaging (cmr) is not always easily matched to published cmr criteria for myocarditis (12). echocardiographic results may be borderline and subjective. findings on the ecg may be unspecific and difficult to interpret. clinical symptoms are not systematically reported. it is therefore important to look for interrater variability in a validation based on patient record review such as in the present study. the patient record review was not straightforward. interrater reliability was very high in the brighton collaboration level 1 and level 2. however, interrater reliability was much lower in the 21 cases initially classified as level 3 to level 5, most likely related to the evaluation of electrocardiograms. this is not unexpected as substantial variability in ecg interpretation has been observed for physicians at all training levels, even after educational interventions (13–15). several ratings changed after re-evaluation by a new rater, which in a proportion of cases also changed the value of the binary myocarditis variable. in general, the re-evaluation mostly changed the value from a non-case to a more definite case (definite, probable or possible case). the application of the brighton collaboration level of evidence criteria for myocarditis should be done after careful training of raters and with good support during the review process, but it may still be expected to generate some interrater variability. the assessment of patient cases with limited or borderline support for the myocarditis diagnosis is a challenge both in the clinical context and in a patient record review such as in our study. importantly, our study did not identify any major concern with using the myocarditis diagnosis registered in the swedish national patient register as outcome variable in epidemiological studies. if interrater variability has affected the estimated positive predictive value, it is likely to have resulted in an underestimation of the quality of the registrer diagnosis. in a previous single-centre validation 507 electronic case records with a discharge diagnosis of myocarditis were systematically reviewed, and 421 (83.0%) could be verified as acute myocarditis (7). the evaluation also of false negatives requires wider sampling criteria. in a us validation of cdc’s vaccine safety datalink case criteria, they were found suboptimal by not including the icd-10 diagnosis i51.4 (myocarditis unspecified) (16). the reduced sensitivity noted with the 15-day risk period in the cdc criteria compared to a 30-day risk period is, however, no concern for our study, since we applied a 28-day risk window. our study has some notable limitations. the patient record review was not blinded to vaccine exposure status. it was considered unfeasible to reliably blind the reviewers. redaction of the extracts from patient records would signal vaccination status or if too extensive threaten the overall clinical assessment of the case. furthermore, our study design does not allow evaluation of potentially false negative cases as this would require a large sample of health-care contacts with plausible symptoms but without a diagnosis of myocarditis in the patient register. this limits the ability to generate estimates of sensitivity and specificity, which would have been helpful for quantitative bias analysis. some caution must also be exerted before generalisation to other countries since coding practices for hospital discharge diagnoses may differ, and to age groups not represented in our study population. table 3. interrater reliability analysis of myocarditis diagnosis when classified according to brighton collaboration level. reclassification table for 51 patient records randomly selected (15 for each brighton collaboration level, or all available if fewer) from the primary validation. re-evaluation of brighton collaboration level was done in a second round of patient record review by another rater blinded to the first assessment. brighton collaboration level from first round of patient record review brighton collaboration level from second round of patient record review 1. definitive case 2. probable case 3. possible case 4. insufficient information 5. not a case 1. definitive case 14 1 0 0 0 2. probable case 1 14 0 0 0 3. possible case 0 3 2 0 1 4. insufficient information 0 1 1 1 1 5. not a case 0 3 2 0 6 6 r. gedeborg et al. conclusion this validation of register-based diagnoses of myocarditis by manual patient record review confirmed the register diagnosis in 96% of cases and had high interrater reliability. among the  few cases not classified as definite or probable interrater reliability was much lower, mostly due to substantial variability in electrocardiogram interpretation. reclassification had only minor impact on the incidence rate ratios for myocarditis following covid-19 vaccination. disclosure statement dr sundström reported participating in research funded by governmental agencies, universities, astellas pharma, janssen biotech, astrazeneca, pfizer, roche, (then) abbott laboratories, (then) schering-plough, ucb nordic and sobi, with all funds paid to karolinska institutet, outside the submitted work. dr grünewald reported being involved in the european medicines agency regulatory assessment of comirnaty; being previously employed at a drug development consultancy firm with cross-product responsibilities and being involved on a project for pertussis vaccines funded by sanofi pasteur, merck sharp & dohme corp, and glaxosmithkline at the swedish agency of infectious disease control. dr ljung reported receiving grants from sanofi aventis paid to his institution outside the submitted work and receiving personal fees from pfizer outside the submitted work. funding this research was conducted as a pharmacovigilance activity by the medical products agency, which is a swedish government agency. the study did not receive any external funding. orcid rolf gedeborg https://orcid.org/0000-0002-8850-7863 rickard ljung https://orcid.org/0000-0002-0654-4530 björn zethelius https://orcid.org/0000-0002-1738-0834 anders sundström https://orcid.org/0000-0003-2337-3371 kai eggers https://orcid.org/0000-0002-8806-5778 nils feltelius https://orcid.org/0000-0003-1460-4078 maria grünewald https://orcid.org/0000-0002-9280-8140 references 1. karlstad ø, hovi p, husby a, härkänen t, selmer rm, pihlström n, et al. sars-cov-2 vaccination and myocarditis in a nordic cohort study of 23 million residents. jama cardiol 2022; 7(6): 600–12. doi: 10.1001/ jamacardio.2022.0583 2. caforio al, pankuweit s, arbustini e, basso c, gimeno-blanes j, felix sb, et al. current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the european society of cardiology working group on myocardial and pericardial diseases. eur heart j 2013; 34(33): 2636–48, doi: 10.1093/eurheartj/eht210 3. sexson tejtel sk, munoz fm, al-ammouri i, savorgnan f, guggilla rk, khuri-bulos n, et al. myocarditis and pericarditis: case definition and guidelines for data collection, analysis, and presentation of immunization safety data. vaccine 2022; 40(10): 1499–511. doi: 10.1016/j. vaccine.2021.11.074 4. ljung r, sundström a, grünewald m, backman c, feltelius n, gedeborg r, et al. the profile of the covid-19 vaccination register safety study in sweden (covacsafe-se). ups j med sci 2021; 126, e8136. doi: 10.48101/ ujms.v126.8136 5. ludvigsson jf, andersson e, ekbom a, feychting m, kim jl, reuterwall c, et al. external review and validation of the swedish national inpatient register. bmc public health 2011; 11: 450. doi: 10.1186/1471-2458-11-450 6. the national board of health and welfare (socialstyrelsen). classification icd-10 stockholm, sweden. 2022. available from: https://www. socialstyrelsen.se/statistik-och-data/klassifikationer-och-koder/icd-10/ [cited 13 september 2022]. 7. fu m, kontogeorgos s, thunström e, zverkova sandström t, kroon c, bollano e, et al. trends in myocarditis incidence, complications and mortality in sweden from 2000 to 2014. sci rep 2022; 12(1): 1810. doi: 10.1038/s41598-022-05951-z 8. ammirati e, lupi l, palazzini m, hendren ns, grodin jl, cannistraci cv, et al. prevalence, characteristics, and outcomes of covid-19-associated acute myocarditis. circulation 2022; 145(15): 1123–39. doi: 10.1161/ circulationaha.121.056817 9. husby a, hansen jv, fosbøl e, thiesson em, madsen m, thomsen rw, et al. sars-cov-2 vaccination and myocarditis or myopericarditis: population based cohort study. bmj (clin res ed) 2021; 375: e068665. doi: 10.1136/bmj-2021-068665 10. gargano jw, wallace m, hadler sc, langley g, su jr, oster me, et al. use of mrna covid-19 vaccine after reports of myocarditis among vaccine recipients: update from the advisory committee on immunization practices united states, june 2021. morb mortal wkly report 2021; 70(27): 977–82. doi: 10.15585/mmwr.mm7027e2 11. cooper lt, baughman kl, feldman am, frustaci a, jessup m, kuhl u, et al. the role of endomyocardial biopsy in the management of cardiovascular disease: a scientific statement from the american heart association, the american college of cardiology, and the european society of cardiology endorsed by the heart failure society of america and the heart failure association of the european society of cardiology. eur heart j 2007; 28(24): 3076–93. doi: 10.1093/eurheartj/ ehm456 12. ferreira vm, schulz-menger j, holmvang g, kramer cm, carbone i, sechtem u, et al. cardiovascular magnetic resonance in nonischemic myocardial inflammation: expert recommendations. j am coll cardiol 2018; 72(24): 3158–76. doi: 10.1016/j.jacc.2018.09.072 13. pawlukiewicz aj, geringer mr, davis wt, nassery dr, april md, streitz mj, et al. interrater agreement of the heart score history component: a chart review study. j am coll emerg physicians open 2022; 3(3): e12732. doi: 10.1002/emp2.12732 14. mehta s, granton j, lapinsky se, newton g, bandayrel k, little a, et al. agreement in electrocardiogram interpretation in patients with septic shock. crit care med 2011; 39(9): 2080–6. doi: 10.1097/ ccm.0b013e318222720e 15. cook da, oh s-y, pusic mv. accuracy of physicians’ electrocardiogram interpretations: a systematic review and meta-analysis. jama intern med 2020; 180(11): 1461–71. doi: 10.1001/jamainternmed.2020.3989 16. sharff ka, dancoes dm, longueil jl, johnson es, lewis pf. risk of myopericarditis following covid-19 mrna vaccination in a large integrated health system: a comparison of completeness and timeliness of two methods. pharmacoepidemiol drug saf 2022; 31(2): 921–925. doi: 10.1002/pds.5439 https://orcid.org/0000-0002-8850-7863 https://orcid.org/0000-0002-8850-7863 https://orcid.org/0000-0002-0654-4530 https://orcid.org/0000-0002-0654-4530 https://orcid.org/0000-0002-1738-0834 https://orcid.org/0000-0002-1738-0834 https://orcid.org/0000-0003-2337-3371 https://orcid.org/0000-0003-2337-3371 https://orcid.org/0000-0002-8806-5778 https://orcid.org/0000-0002-8806-5778 https://orcid.org/0000-0003-1460-4078 https://orcid.org/0000-0003-1460-4078 https://orcid.org/0000-0002-9280-8140 https://orcid.org/0000-0002-9280-8140 https://doi.org/10.1001/jamacardio.2022.0583 https://doi.org/10.1001/jamacardio.2022.0583 https://doi.org/10.1093/eurheartj/eht210 https://doi.org/10.1016/j.vaccine.2021.11.074 https://doi.org/10.1016/j.vaccine.2021.11.074 https://doi.org/10.48101/ujms.v126.8136 https://doi.org/10.48101/ujms.v126.8136 https://doi.org/10.1186/1471-2458-11-450 https://www.socialstyrelsen.se/statistik-och-data/klassifikationer-och-koder/icd-10/ https://www.socialstyrelsen.se/statistik-och-data/klassifikationer-och-koder/icd-10/ https://doi.org/10.1038/s41598-022-05951-z https://doi.org/10.1161/circulationaha.121.056817 https://doi.org/10.1161/circulationaha.121.056817 https://doi.org/10.1136/bmj-2021-068665 https://doi.org/10.15585/mmwr.mm7027e2 https://doi.org/10.1093/eurheartj/ehm456 https://doi.org/10.1093/eurheartj/ehm456 https://doi.org/10.1016/j.jacc.2018.09.072 https://doi.org/10.1002/emp2.12732 https://doi.org/10.1097/ccm.0b013e318222720e https://doi.org/10.1097/ccm.0b013e318222720e https://doi.org/10.1001/jamainternmed.2020.3989 https://doi.org/10.1002/pds.5439 upsala j med sci 97: 107-1 14 tubuglomerular feedback control in long-looped nephrons topical minireview hans r ulfendahl, ann goransson-nyberg, peter hansel1 and mats sjoquist department of physiology & medical biophysics, university of uppsala, biomedical center, uppsala, sweden abstract the tubuloglomerular feedback mechanism is highly activated in juxtamedullary nephrons and considered to play a major role in intrarenal regulation of glomerular filtration rate. the vasculature of juxtamedullary nephrons is highly vasoreactive with a high ability for vasodilation. this vasoreactivity is a prerequisite for an important influence of the tubuloglomerular feedback mechanism on the medullary blood flow and its regulation. the renal medulla is of particular interest since the processes which regulate the osmolar concentration of the extracellular fluid by forming a dilute or concentrated urine primarily occur in this region. the nephrons, which constitute the functional units of the kidney, are not a homogenous group of structures. instead, those nephrons which originate closer to the cortico medullary border (juxtamedullary nephrons), as opposed to those which originate closer to the renal surface (superficial nephrons), are the nephrons which primarily are involved in the processes giving rise to a dilute or concentrated urine. the underlying reason for this is that only juxtamedullary nephrons have long loops of henle that protrude all the way down into the inner medulla and, furthermore, give rise to vessels (vasa recta) which perfuse the medulla. these structures produce and preserve the progressive axial osmotic concentration gradient from the cortico-medullary border to the tip of the papilla. the present paper evaluates some special features of the juxtamedullary nephrons and points out some differences in functional aspects between the two nephron sub-populations. we (8) have found that the interlobular arteries in rat kidneys behave as resistance vessels. according to calculations made by other authors (9), this is also true for dog kidneys. the blood pressure drop along an interlobular artery amounts in normotensive, normohydrated adult rats to 40-45 mm hg. this pressure drop is a prerequisite for different haemodynamic 8-928572 107 conditions for the outermost (superficial) and innermost (juxtamedullary) nephrons within the cortex. there are indications that the glomerular capillary hydrostatic pressure is about the same in both superficial and juxtamedullary glomeruli (l), which means that the pressure-drop in afferent arterioles of superficial glomeruli is smaller than in those of juxtamedullary glomeruli. in fact, we propose (3) that the superficial afferent arterioles are nearly maximally dilated, while the juxtamedullary afferent arterioles are significantly constricted in normotensive, normohydrated rats. we have also discovered that the tubuloglomerular feedback mechanism (tgf) is normally strongly activated in juxtamedullary nephrons (3). in young sprague-dawley rats the papilla was exposed by excising the ureter and prepared for micropuncture procedures. the single nephron glomerular filtration rate (sngfr) was measured by quantitative sampling of urine in a loop of henle with an oil blockade distal to the sampling site. in this situation the urine flow to the macula densa is blocked, resulting in a stop-flow condition. in rats prepared in the same manner the sngfr was also measured with a modified hanssen technique (12) where a free-flow condition exists at the macula densa. during stop-flow conditions the sngfr of juxtamedullary glomeruli amounted to 84.1 +_ 8.5 nlmin-' (n=15, mean +_ 1sem) and during free-flow conditions it amounted to 27.7 & 2.9 nlmin-' (n=7). in other experiments (3) on the same animal model free-flow and stop-flow pressures were measured in proximal and distal tubules on the renal surface and in the loop of henle in the papilla. from the values obtained, it was possible to estimate the net driving force for glomerular filtration at the beginning of glomerular capillaries of juxtamedullary nephrons. the results indicate a much higher net driving force during stop-flow (47 mm hg) than during free flow conditions (19 mm hg). in a third series of experiments (1 1) it was possible to calculate the urine flow rate during free-flow conditions by measuring the linear velocity of small dye boli injected into the loops of henle of juxtamedullary nephrons. the passage of the boli and the diameter of the loop lumen were recorded with a video-technique. according to the calculations the urine flow rate during free-flow conditions, 3.9 k 0.37 nlmin" (n=8), differs markedly from that during stop-flow conditions, 8.0 f 0.54 nlmin-' (n=8). all the previously given results firmly support our proposal of a strongly activated tgf in intact juxtamedullary nephrons under normal physiological conditions. our present view on the relationship between the sngfr and the urine flow rate at the macula densa is summarized in fig. 1. in this figure, points a and d represent the sngfr values obtained with the micropuncture techniques and points b and e are yielded from the modified hanssen technique. point c is derived from values given by schnennann et al. (10). the value 108 of f is uncertain, as we have no experimental values available. the points b and e thus represents the operation points for the flow characteristics of normohydrated, normotensive rats. we have thus found a great influence of tgf on the glomerular filtration rate in juxtamedullary nephrons. the question then arises as to whether the blood flow to these nephrons is simultaneously influenced by the tgf. up to date we have not been able to find a reliable method to determine the blood flow in single juxtamedullary nephrons so it has not been possible to directly study the relation between the tgf and blood flow in these nephrons. we have therefore chosen to examine the medullary blood flow which mirrors the blood flow in the juxtamedullary nephrons since only these nephrons give rise to vessels (vasa recta) which perfuse the medulla. nl . m i d . g-' k w superficial nephron nl 0 arb.units flow rate at rnacula densa 0 ~i, arhunits flow rate at rnacula densa fig. 1. schematic influence of urine flow rate at the macula densa on the single nephron glomerular filtration rate (sngfr). points a and d represent the values obtained with a blocked urine flow at the macula densa and b and e those with a normal flow. thus b and e constitute the operation points for the two nephron populations during normal conditions. points c and f are expected values for high urine flow rates. kw = kidney weight (from acta physiol scand 14: 203-209, 1982). 109 in one series of experiments in adult sprague-dawley rats we estimated the plasma flow in the inner and outer medulla by the extraction of radioactively labelled rubidium (%rbcl). the method has previously been described by karlberg et al. (7). simultaneously, we also estimated the sngfr with a modified hanssen technique (described by sjijquist et al. (12)) in three layers of the cortex, namely the outer, the middle and the inner cortex. in order to study the vasoreactivity of the vessels in the juxtamedullary vasculature, we continuously, intravenously infused the calcium entry blocker verapamil (isoptin, knoll ag, germany) or the angiotensin converting enzyme inhibitor captopril (sq14225, squibb & sons, nj, usa). the results from these experiments are depicted in the left panel of fig. 2. neither verapamil (0.6 mg-h-'-kg-' body weight) nor captopril (3 mg.h"-kg" body weight) caused any change in the sngfr of the different nephron populations. thus no change in the distribution of glomerular filtration rate between different nephron populations were indicated. a redistributing effect of the drugs might, however, have been camouflaged by the drop in arterial blood pressure which for verapamil was from 127 & 3 mm hg to 113 f 4 mm hg and for captopril from 120 f 3 mm hg to 102 f 5 mm hg (4). the right panel of fig. 2 gives the plasma flows in the outer and inner medulla. in both medullary regions verapamil and captopril markedly increased the plasma flow. captopril treatment caused an increase of about 90 % in the inner medullary plasma flow, which is in agreement with earlier findings by eloy et al. (2). an estimation of the cortical plasma flow showed that captopril increased the flow by only 10-20 %. the results thus strongly indicate that the vasculature of the juxtamedullary nephrons is highly vasoreactive and has a high potential to dilate, which must be a prerequisite for the tgf to potently influence the medullary plasma flow. in another series of experiments we studied the influence of verapamil and captopril on the medullary red cell flux with a video-technique (5, 6) . in young munich-wistar rats the papilla was exposed and with a highly sensitive tv-camera we followed the number of fluorescently labeled (fitc) red cells passing vas rectum per minute. the same vessels were investigated before and during infusion of drugs. figure 3 shows the results thereby obtained. two different doses of verapamil were used and in both cases the red cell flux increased in the vasa recta; with the lower dose (0.6 mg-h-'.kg-' body weight) the increase was 30 f 6 % (p<o.ol) and with the higher dose (2.4 mg-h-l-kg-' body weight) the corresponding value was 39 k 7 % (p<o.ol). in the animals treated with captopril (3 mg.h-'-kg-' body weight) the red cell flux increased by 40 k 4 % (pm.01). these results confirm our theory of a high vasoreactivity in the juxtamedullary vasculature. 110 o =c o n tr o l @ =v er ap am il 10 b m gl kg b w h ) nl lm 1n .g k w m =c af 't o pr il i3 0m gl kq b w h l 3c q c 0 y u 20 '" ll l l w z ln 1c o ut er c or te x in ne r c or te x t l 0 =c o nt ro l m = v e r a p a m ll ( 0 6 m g/ kg b w h ) .= ca pt o pr il (3 0 m q/ kq b w h l pu llm im m g tis su e o ut er m ed ul la in ne r m ed ul la p' 00 4 t 14 i e p 1 2 3 3c 10 0 l c a 06 d a 02 c on t. v er . c ap t. lo nt . v er . c ap t. n = 6 5 8 6 5 8 c on t. v er . c ap t. n: 6 5 8 c on t. ve r. c ap t. 6 5 8 f ig . 2 . t he s n g fr in th e in ne r an d ou te r c or te x (l ef t p an el ) a nd p la sm a fl ow r at es in o ut er a nd in ne r m ed ul la (r ig ht p an el ) d ur in g co nt ro l c on di ti on s an d du ri ng c on ti nu ou s i nf us io ns o f ve ra pa m il an d ca pt op ri l. k w = k id ne y w ei gh t. 40 30 20 10 0 v0.6 v2.4 cap fig. 3. percentage increase in red cell flux (~qrbc) in the vasa recta after continuous intravenous infusions of verapamil at two different doses (v0.6, 0.6 mg-h-'-kg-'; v2.4, 2.4 mgh-'.kg-') and captopril (cap, 3.0 mg.h-'.kg-') (values from am j physiol 2 5 4 f492-f499, 1988 and acta physiol scand 134: 9-15, 1988). finally, table 1 shows the urinary excretion of sodium and potassium and of osmotically active particles during control conditions and during infusions of verapamil and captopril. both drugs increased the excretion of sodium, potassium and osmotically active particles. a slight increase in urine flow and a distinct rise in the urine osmolality was evident. according to a theoretical concept by thurau et al (13) the increase in medullary plasma flow induced by the two drugs might have been expected to give a so-called "washing-out" effect of the concentration gradient in the renal medulla and thus decrease the concentrating ability of the kidney. the expected relationship between medullary blood flow and concentrating ability was thus not evident in this study, instead the increase in plasma flow was accompanied by an increase in ion-excretion and in concentrating ability which may be due to a direct effect of the drugs on ion transport in tubular structures. the inhibition of transport mechanisms may counteract and overcome the washing-out effect. 112 table 1. renal excretion data during control conditions and during continuous intravenous infusion of verapamil and captopril. ~ excretion parameter v (plvmin) control 2.12 f 0.30 u, (m) unav (pmovmin) u,v (pmovmin) uosm (mosm) i 1316k 120 134 f 30 0.14 f 0.03 0.31 k 0.09 uosmv (posdmin) i 2.72 f 0.45 7 4 f 7 i 88 f 12 11 272 f 29' 0.21 f 0.04' 231 k 42" 0.24 f 0.04" 0.66 f 0.15" i/ 0% f 0.14" i 1743 f 160' 4.53 f 0.65' 1603 f 157' 4.50 f 0.56" urine flow rate (v), urinary concentration of sodium (u,j and potassium (u,), sodium and potassium excretion (u,,v, u,v), urine osmolality (uosm) and excretion of osmotically active particles (ljosmv). * p<0.05 vs control. acknowledgements this study was supported by the swedish medical research council (project nos 140 and 9283). references 1. aukland, k., tonder-heyeraas, k. & naess, g.: capillary pressure in the deep and superficial glomeruli of the rat kidney. acta physiol scand 101: 418-427, 1977. 2. eloy, l., griinfeld, j., bayle, f., bankir, l., ramos-frendo, b. & tnnh-trang-tan, m.: papillary plasma flow in rats. 11. hormonal control. pflugers arch 398: 253-258, 1983. 3. ericson, a.-c., sjiiquist, m. & ulfendahl, h.r.: heterogeneity in regulation of glomerular function. acta physiol scand 114: 203-209, 1982. 4. goransson, a. & sjiiquist, m.: the effect of a converting enzyme inhibitor on autoregulation and intrarenal distribution of glomerular filtration in the rat. acta physiol stand 124: 515-523, 1985. 113 5 . 6. 7. 8. 9. 10. 11. 12. 13. hansell, p., sjwuist, m. & ulfendahl, h.r.: the calcium entry blocker verapamil increases red cell flux in the vasa recta of the exposed renal papilla. acta physiol scand 134: 9-15, 1988. hansell, p., sjijquist, m. & ulfendahl, h.r.: effect of a converting enzyme inhibitor on vasa recta blood flow in rat kidney. am j physiol 254: f492-f499, 1988. karlberg, l., kqlskog, o., ojteg, g. & wolgast, m.: renal medullary blood flow studied with the 86-rb extraction method. acta physiol scand 115: 11-18, 1982. kallskog, o., lindbom, l.o., ulfendahl, h.r. & wolgast, m.: hydrostatic pressures within the vascular structures of the rat kidney. pfliigers arch 363: 205-210, 1976. ofstad, j., morkrid, l. & willassen, y.: diameter of the afferent arteriole in the dog kidney estimated by the microsphere method. scand j clin lab invest 35: 767-774, 1975. schnerman, j., persson, a.e.g. & agerup, b.: tubuloglomerular feedback. non-linear relation between glomerular hydrostatic pressure and loop of henle perfusion rate. j clin invest 52: 862-869, 1973. sjijquist, m. & ulfendahl, h.r.: the tubuloglomerular feedback mechanism in juxtamedullary nephrons. in: the juxtaglomerular apparatus (ed. a.e.g persson & u. boberg), pp. 201-210. elsevier science publishers (biomedical division), amsterdam , 1988. sjwuist, m., goransson, a., kallskog, 0. & ulfendahl, h.r. 1984. the influence of tubuloglomerular feedback on the autoregulation of filtration rate in superficial and deep glomeruli. acta physiol scand 122: 235-242, 1984. thurau, k., deetjen, p. & kramer, k.: hamodynamik des nierenmarks. 11. wechselbezieung zwischen vasculiiren und tubuliiren gegenstromsystem bei artenllen druchsteigerungen, wasserdiurese und osmotischer diurese. pfliigers arch 270: 270-275, 1960. offprint requests to: hans r ulfendahl, department of physiology & medical biophysics, university of uppsala, biomedical center, po box 572, s-751 23 uppsala, sweden. 114 upsala journal of medical sciences 2021, 126, e7590 http://dx.doi.org/10.48101/ujms.v126.7590 the association between bmi and 90-day mortality in patients with and without diabetes seeking care at the emergency department per wändella, axel c. carlssona,b, anders larssonc, olle melanderd,e, torgny wessmand,e, johan ärnlöva,f and toralph ruged,e adepartment of neurobiology, care sciences and society, karolinska institutet, huddinge, sweden; bacademic primary health care centre, stockholm region, stockholm, sweden; cdepartment of medical sciences, uppsala university, uppsala, sweden; ddepartment of emergency and internal medicine, skånes university hospital, malmö, sweden; edepartment of clinical sciences malmö, lund university & department of internal medicine, skåne university hospital, malmö, sweden; fschool of health and social studies, dalarna university, falun, sweden abstract background: the impact of body mass index (bmi) on mortality varies with age and disease states. the aim of this research study was to analyse the associations between bmi categories and shortand long-term mortality in patients with or without diabetes seeking care at the emergency department (ed) with acute dyspnoea. population and methods: patients aged ≥18 years at ed during daytime on weekdays from march 2013 to july 2018 were included. participants were triaged according to the medical emergency triage and treatment system-adult score (metts-a), and blood samples were collected. totally, 1,710 patients were enrolled, with missing values in 113, leaving 1,597 patients, 291 with diabetes and 1,306 without diabetes. the association between bmi and short-term (90-day) and long-term (mean follow-up time 2.1 years) mortality was estimated by cox regression with normal bmi (18.5–24.9) as referent category, with adjustment for age, sex, metts-a scoring, glomerular filtration rate, smoking habits and cardiovascular comorbidity in a fully adjusted model. the bonferroni correction was also used. results: regarding long-term mortality, patients with diabetes and bmi category ≥30 kg/m2 had a fully adjusted hazard ratio (hr) of 0.40 (95% confidence interval [ci]: 0.23–0.69), significant after the bonferroni correction. amongst patients without diabetes, those with underweight had an increased risk but only of borderline significance, whilst risks in those with overweight or obesity did not differ from reference. regarding short-term mortality, risks did not differ from reference amongst patients with or without diabetes. conclusions: we found divergent long-term mortality risks in patients with and without diabetes, with lower risk in obese patients (bmi ≥ 30 kg/m2) with diabetes, but no increased risk for patients without diabetes and overweight (bmi: 25–29.9 kg/m2) and obesity. article history received 3 february 2021 revised 24 august 2021 accepted 24 august 2021 published 16 september 2021 keywords diabetes; bmi; mortality; triage level; emergency department introduction globally, body mass index (bmi) has increased during the last few decades (1), with an increasing rate of overweight and obesity, although a plateau phase seems to be reached in many high-income countries (2). obesity has been proposed as the main upstream driver of cardiometabolic risk factors and associated outcomes (3), such as hypertension, diabetes and myocardial infarction (mi) (4). moreover, obesity is regarded as one of the leading risk factors for mortality in the world (5). despite that overweight and obesity are associated with an increased risk of mortality (6), cardiovascular mortality, especially coronary heart disease (chd), has decreased in western countries (7), including in sweden (8), during the last few decades. the association between bmi and mortality has been explored in many articles. a jor u-shaped association between bmi and both all-cause and cardiovascular mortality has been reported (9, 10). in a study of the risk of being among the lowest 5% in different anthropometrical measures, both bmi and percent body fat showed an increased mortality risk, whilst all measures showed an excess mortality risk in the highest quartile (11). a paradoxical risk association has been observed between bmi and mortality, particularly for patients with diabetes, with a higher mortality risk in the bmi range 18.5–24.9 kg/m2 (12). in a contact per wändell per.wandell@ki.se © 2021 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article http://dx.doi.org/10.48101/ujms.v126.7590 mailto:per.wandell@ki.se http://creativecommons.org/licenses/by/4.0/ 2 w. per et al. recent review, the lowest mortality risk was found for men in the bmi range 31–35 kg/m2, and for women in the bmi range 28–31 kg/m2 (13). however, the bmi-mortality paradox in diabetes is still controversial, with some results in agreement (14), and others questioning it (15, 16), and the impact of obesity on health can be very different according to age, gender, patient characteristics and the presence of comorbidities, as well as the state of acute or non-acute illness (17). thus, the role of bmi in patient outcome, whether in long term or short term, is still not clear. the aim of this study was to determine the effect of bmi on shortand long-term mortality rates in elderly acute ill patients, with or without diabetes, admitted to the emergency department (ed). as a model, we chose a cohort of patients admitted to the ed because of acute dyspnoea, the ‘acute dyspnea study’ (adys) (18), known to include severely ill patients with a high level of comorbidity. methods population and methods patients with acute dyspnoea admitted to the ed of sus malmö were included in the ‘acute dyspnea study’ (adys) between march 2013 and january 2019. patients ≥18 years who presented to the ed during daytime, 6:45 am to 4:30 pm working days with acute dyspnoea as their main complaint, were asked for consent to take part in the study by a research nurse. critically ill patients who directly were transferred from the resuscitation room to an intensive care unit (icu) were excluded, as were patients with lower degrees of consciousness as these patients were not able to give consent nor able to partake in answering the questionnaire. vital parameters were registered as were medical triage priority level according to the validated medical emergency triage and treatment system (metts). a total of 1,710 patients were enrolled, in which 113 of these were excluded because of missing values, leaving 1,597 patients for further analyses. body mass index data were present amongst 1,553 patients, and the 44 missing values were imputed for further analyses (see below). of the included 1,597 patients, 291 patients had diabetes and 1,306 were without diabetes. patients with diabetes in this study included both patients with type-1 and type-2 diabetes, and patients were only defined as diabetic if they had been previously diagnosed with diabetes in the electronic patient record. after inclusion, all patients were interviewed by a research nurse about their health, medication, symptoms and social situation according to a standardised and approved questionnaire. patients were questioned regarding smoking habits and categorised as non-smokers, former smokers (cessation 1 month ago or longer) or active smokers (regularly smoking the past month or longer), disease history associated with dyspnoea (i.e. congestive heart failure, chronic obstructive pulmonary disease (copd), asthma, coronary artery disease, atrial fibrillation, restrictive lung disease, cancer, thromboembolic disease or rheumatic disease) and current medications. information on weight and height was taken from the medical records if this was recently documented; otherwise, this information was collected at the ed by the research nurse. the research nurses reviewed the patient’s medical records in order to confirm the details with the support of senior physicians whenever uncertainties occurred. originally metts-a uses five clinical priority levels with increasing clinical priority: blue (lowest clinical priority – not life-threatening), green, yellow, orange and red (highest clinical priority – life-threatening). the lowest clinical priority level, blue, was not used in the clinical triage of the patients included here because of the local triage routines, with the lowest clinical triage priority here being green. body mass indexwas categorised into <18.5 kg/m2 (underweight), 18.5–24.9 kg/m2 (normal weight), 25–29.9 kg/m2 (overweight) or ≥30 kg/m2 (obesity) (19). high sensitivity c-reactive protein (crp) was analysed using a particle-enhanced turbidimetric assay, and the creatinine level was analysed using an integrated database management system (idms) calibrated enzymatic assay (20). lactate and glucose were analysed with routine methods. these methods are accredited by swedac and are included in the swedish external quality assurance programme for crp and creatinine, respectively, and are routinely used. within an hour of presentation at ed, blood was sampled, serum and plasma were separated and subsequently stored at −80°c for future analysis. the assays were performed blinded without knowledge of clinical data. the chronic kidney disease epidemiology collaboration (ckd-epi) equation was used to estimate the glomerular filtrate rate (gfr). ethical considerations this study obtained ethical approval from ‘regionala etikprövningsnämnden epn’, lund, sweden. dnr 2014/82. all included patients provided their written informed consent to take part in the study. statistics baseline values expressed as numbers, with percentages, or mean values with standard deviation (sd), and chi-square tests or analysis of variance (anova) were used for comparisons. the association between the short-term mortality (90-day mortality) and long-term mortality (after total follow-up time of the study, 2.1 ± 1.5 years), respectively, and bmi categories was analysed by cox proportional hazard model in univariate analyses and in models adjusted for age, sex (model a), and the clinical triage scoring system (model b), and cardiovascular co-morbidity (established coronary disease, heart failure and hypertension), smoking habits and gfr (model c). we categorised the patients into those with or without diabetes, and performed interaction analyses. individuals with normal bmi (18.5–24.9) was used as the referent category. we  imputed values for bmi (missing in 44 patients), for the completeness of data for further analyses. we also performed a sensitivity analysis for patients with diabetes merging bmi levels 25–29.9 and ≥30 kg/m2 using models a and c, as the hazard ratio (hr) showed similar values for both these categories. the association between bmi and 90-day mortality in patients 3 the follow-up time stretched from the time of presentation at the ed to death within or the end of follow-up (90 days postpresentation and total mortality 2.1 ± 1.5 years). a p-value of <0.05 was considered to be statistically significant, and we also performed a bonferroni correction (with 0.05 divided by 9 for statistical significance of outcomes using each group of models per patient group as a unit). the dataset was handled, and cox proportional hazard models were all computed with ibm spss statistics 25.0 software (spss inc., chicago, il). results general data baseline data show that patients with diabetes were older, with higher mean bmi, and with higher lactate, glucose and creatinine levels (table 1). patients with diabetes also were more often admitted to hospital care, and more often were diagnosed with heart failure, chd, stroke and hypertension than those without diabetes. mortality, according to the bmi interval for 90-day mortality, shows the highest risk for diabetes patients in the normal bmi interval, that is, 18.5–24.9 kg/m2, with 21%, and for patients without diabetes in the underweight interval, that is, <18.5 kg/m2 with 22% (table 2). there were no deaths reported amongst the two patients with diabetes in the bmi interval <18.5 kg/m2. 90-day mortality there was no significant interaction for the mortality between patients with and without diabetes (p < 0.07). patients with diabetes and in the bmi intervals 25–29.9 and ≥30 kg/m2 showed no statistically decreased mortality risk (table 3). patients without diabetes had the highest risk in the bmi interval <18.5 kg/m2; however, it is not statistically significant after the bonferroni correction (bonferroni-adjusted p-level < 0.006). long-term mortality there was a significant interaction between patients with and without diabetes as regards long-term mortality (p < 0.0001). in patients with diabetes, a significantly lower mortality risk was found in the bmi interval ≥30 kg/m2 in all three models (table 4), also statistically significant after the bonferroni correction (bonferroni adjusted p-level < 0.001). in patients without diabetes and bmi < 18.5 kg/m2, the results showed a statistically borderline higher risk after the bonferroni correction (bonferroni-adjusted p-level = 0.006). discussion we found different patterns between individuals with and without diabetes as regards mortality in different bmi intervals. regarding long-term mortality, patients with diabetes and bmi ≥ 30 kg/m2 showed a significantly lower risk than the reference, whilst patients without diabetes and underweight showed higher risk but only of borderline significance. as regards diabetes patients with obesity, that is, bmi ≥ 30 kg/m2, the statistically significant lower risk are in line with the bmi–mortality paradox, that is, with lower mortality risks at higher bmi intervals, in contrast with results from large population studies showing an excess risk in the overweight and obesity interval (9, 21). in contrast, we could not find any statistically significant association between underweight and an increased mortality, but there were only two patients within this bmi interval. table 1. baseline characteristics of patients with acute dyspnoea, with and without diabetes seeking care at a hospital emergency department. variable diabetes (n = 291) no diabetes (n = 1,306) female 44%*** 58% age at survey (years) 74 (14)*** 69 (19) body mass index (kg/m2) 30 (7)*** 26 (6) systolic blood pressure (mmhg) 148 (27) 146 (29) diastolic blood pressure (mmhg) 80 (18) 82 (16) respiratory rate (frequency) 25 (7) 24 (7) c-reactive protein (crp, mg/l) 30 (53) 35 (64) lactate (mmol/l) 2.1 (1.2)*** 1.7 (1.04) glucose level (mmol/l) 11.0 (5.9)*** 6.9 (2.4) creatinine (µmol/l) 125.3 (100.6)*** 93.0 (69.4) metts-a triage red – most acute (%) 16 12 orange (%) 32 31 yellow (%) 49 50 green – least acute (%) 4 7 admitted to hospital care (%) 69*** 46 cancer (%) 24* 18 chronic obstructive pulmonary disease (%) 29 30 chronic heart failure (%) 53*** 29 coronary artery disease (%) 50*** 24 stroke (%) 17*** 9 hypertension (%) 63*** 37 90-day mortality (%) 14 12 missing data points were less than 4% for all included characteristics, except  for diastolic blood pressure and lactate where around 8% of data  points were missing. means and standard deviations, or percentages, *p < 0.05, ***p < 0.001; for differences between individuals with or without diabetes. table 2. data on a 90-day mortality by bmi categories in patients seeking care for dyspnoea at the emergency department with (n = 291) or without (n = 1,306) diabetes. bmi categories patients with diabetes number of mortality events or numbers at risk (%) patients without diabetes number of mortality events or numbers at risk (%) 90-day mortality: bmi < 18.5 kg/m2 0/2 (0) 18/83 (22) bmi 18.5–24.9 kg/m2 16/76 (21) 59/580 (10) bmi 25–29.9 kg/m2 10/89 (11) 41/328 (13) bmi ≥ 30 kg/m2 11/114 (10) 27/281(10) total mortality: bmi < 18.5 kg/m2 1/2 (50) 32/83 (39) bmi 18.5–24.9 kg/m2 37/76 (49) 136/580 (23) bmi 25–29.9 kg/m2 31/89 (35) 66/328 (20) bmi ≥ 30 kg/m2 25/114 (22) 52/281(18) bmi: body mass index. 4 w. per et al. one of the german studies of patients with type-2 diabetes revealed a u-shaped curve, with the lowest mortality rates for individuals with bmi at around 31 kg/m2 (22). another study on patients with type 2 diabetes revealed a lower mortality rate among individuals with overweight and obesity class i (bmi 30–34.9 kg/m2) compared to individuals with normal bmi (23). a systematic review and meta-analysis confirmed these findings, with a higher mortality in underweight, that is, <18.5 kg/m2, and a lower mortality in overweight and mild obesity compared with normal bmi (24). for patients without diabetes, we found no statistically significant results, but a borderline significance for underweight after the bonferroni correction. overweight and obesity were not associated with an increased mortality rate. large studies have shown a uor j-shaped curve, with the lowest bmi risk in individuals in the normal bmi interval (21). the research findings with a similar mortality in patients with overweight and obesity as in patients with normal weight thus in some respects could be seen as a marker of the obesity paradox. underweight has been associated with an increased mortality in earlier, large population studies (9–11, 25). furthermore, the association between underweight and increased mortality seems to be more pronounced in individuals with respiratory diseases (21). actually, patients in this study actually had dyspnoea. the obesity paradox has certainly been questioned. one of the reviews on the obesity paradox concluded on studies supporting this hypothesis: ‘these studies have numerous limitations due to their mainly retrospective nature and to numerous confounding factors, such as associated pathologies, antidiabetic treatments, smoking habit, lack of data about distribution of body fat or weight history’ (16), and another review on the same topic shows ‘conflicting evidence for the role of overweight and obesity in all-cause mortality may largely be a result of differences in study populations, epidemiological methods, and statistical analysis’ (15). the reason for findings related to the obesity paradox has been discussed. amongst patients with congestive heart failure, one of the studies found that increasing bmi was associated with lower mortality (26), especially for in-hospital mortality (27). another study found the obesity paradox to be present, table 3. the associated risk between non-normal bmi and 90-day mortality in patients with or without diabetes seeking care at a hospital emergency department. bmi categories patients with diabetes (hr, 95% ci) patients without diabetes (hr, 95% ci) model a model b model c model a model b model c bmi < 18.5 2.11 (1.24–3.60) 2.11 (1.24–3.60) 2.33 (1.75–3.11) bmi 18.5–24.9 referent referent referent referent referent referent bmi 25–29.9 0.54 (0.24–1.20) 0.68 (0.29–1.60) 0.54 (0.21–1.38) 1.23 (0.83–1.84) 1.23 (0.82–1.84) 1.22 (0.87–1.72) bmi ≥ 30 0.46 (0.21–1.02) 0.50 (0.22–1.11) 0.50 (0.22–1.13) 1.20 (0.75–1.88) 1.23 (0.77–1.95) 1.24 (0.77–1.98) bmi: body mass index; ci: confidence interval; hazard ratio (hr): . analyses include imputed data for missing values for bmi. model a includes age and sex; model b includes model a and metts-a triage; model c includes model b and cardiovascular comorbidity (established coronary disease, heart failure and hypertension), smoking and gfr. with the bonferroni correction, the results of patients without diabetes were not statistically significant (corrected p-level < 0.006 was not satisfied). table 4. the associated risk between non-normal bmi and mortality after the total follow-up time in the study (after 2.1 years, ± 1.5 years) in patients with or without diabetes seeking care at a hospital emergency department. bmi categories patients with diabetes (hr, 95% ci) patients without diabetes (hr, 95% ci) model a model b model c model a model b model c bmi < 18.5 0.67 (0.09–4.97) 0.95 (0.12–7.25) 1.32 (0.17–10.38) 1.67 (1.13–2.47) 1.64 (1.34–2.00) 1.76 (1.43–2.17) bmi 18.5–24.9 referent referent referent referent referent referent bmi 25–29.9 0.66 (0.41–1.08) 0.83 (0.49–1.39) 0.82 (0.47–1.42) 0.86 (0.64–1.15) 0.88 (0.65–1.18) 0.82 (0.60–1.10) bmi ≥ 30 0.41 (0.24–0.69)* 0.44 (0.26–0.75)* 0.40 (0.23–0.69)* 0.95 (0.68–1.31) 0.99 (0.72–1.37) 0.94 (0.67–1.31) bmi: body mass index; ci: confidence interval; hazard ratio (hr): . analyses include imputed data for missing values for bmi. model a includes age and sex; model b includes model a and metts-a triage; model c includes model b and cardiovascular comorbidity (established coronary disease, heart failure and hypertension), smoking and gfr. with the bonferroni correction, the results in the diabetes group were statistically significant (corrected p-level < 0.006 was satisfied); however, in the non-diabetes group the values were borderline significant (corrected p-level = 0.006). *p < 0.05 with the bonferroni correction. the association between bmi and 90-day mortality in patients 5 however, not in patients with diabetes (28), contradictory to the study findings. yet, another study confirmed not only the association between higher bmi and lower mortality in patients with heart failure but also an association between being underweight and higher mortality (29). however, this study also concluded that the influence is complex, and that the effect of bmi was depending on the ejection fraction and the presence or absence of copd (29). in contrast, for patients with copd, obesity was associated only with mortality from respiratory causes and only in the bmi interval > 40 kg/m2 (30). excess mortality in low bmi could be explained by cancer mortality or smoking habits, but adjusting for this or by categorising into causes of mortality has shown that the bmi– mortality paradox still exists (12). smoking has been shown to increase the mortality risk in the lower bmi interval (9). for patients with copd, underweight is shown to be associated with increased mortality (30). the patients in this study represent a special group, that is, patients seeking care at a hospital ed with symptoms of dyspnoea, and within this specific group it might be more dangerous being to lean. an earlier study in patients with acute mi found that excess weight at the time of mi was associated with a lower mortality, whilst in the long term it was associated with recurrent re-infarction and cardiac death (31). a review also confirmed the obesity paradox in mi, but also finding that this was true for long-term mortality (32), in contrast to other studies. thus, in general, there are contradictory findings related to the obesity paradox. an increased risk with underweight is regularly shown (9, 21); however, the relation between higher bmi intervals, that is, overweight and obesity, and mortality differs between studies, thus seemingly supporting the critical views in some reviews (15, 16). more studies on the possible effects of the obesity paradox in specific groups are warranted. there are some limitations with this study. this is an observational study, and we had data only at baseline and not at follow-up. the sample was rather small with a small number of deaths, with a low statistical power. however, the sample represents a specific group in a specific setting, that is, patients with dyspnoea seeking care at the ed. we have no data on individual bmi trends. furthermore, we imputed bmi values, which could have distorted the results, especially in the diabetes group, and this is why the results should be interpreted with some caution. furthermore, the number of underweight patients was rather small, especially in the diabetes group with only two patients, and the association with mortality was not statistically significant after the bonferroni correction, even if the mortality rate was highest within this group. the main aim was originally to study the association between bmi and shortterm mortality; however, we also added the total registered mortality. furthermore, the research study was not designed to analyse the work or effect of emergency care on patient outcomes, as there are too many unknown variables for analysis. we cannot generalise to other patientageand ethnic groups. strengths of the study include the longitudinal study design and the use of real-world clinical data from a well-characterised cohort consisting of acute ill patients. conclusions we found for diabetes patients with overweight or obesity a lower overall mortality, in line with the obesity paradox, whilst those without diabetes no increased mortality was found for obesity, and the higher risk for underweight showed borderline significance after the bonferroni correction. taken together, our data suggest that obesity in this specific patient group seems to have a protective effect on patients with diabetes, and with no increased mortality risk for patients without diabetes compared with those with normal weight. disclosure statement the authors report no conflicts of interest. funding no funding was received for this research work. notes on contributors per wändell, md and phd, is a specialist in family medicine and senior professor in family medicine at karolinska institutet, department of neurobiology, care sciences and society, division of family medicine and primary care. axel c. carlsson, phd, is a pharmacist and associate professor in family medicine at karolinska institutet, department of neurobiology, care sciences and society, division of family medicine and primary care. anders larsson, md´and phd, is a senior consultant in clinical chemistry and professor in clinical chemistry at uppsala university, department of medical sciences, clinical chemistry. olle melander, md and phd, is a professor of internal medicine at lund university and a consultant at the department of internal medicine, skåne university hospital. torgny wessman, torgny wessman, md and phd student, is a specialist in general medicine and emergency medicine at lund university, faculty of medicine, department of clinical sciences malmö. he is also a senior consultant at the emergency department, skåne university hospital, malmö. johan ärnlöv, md and phd, is a specialist in family medicine and a professor in family medicine at karolinska institutet, department of neurobiology, care sciences and society, division of family medicine and primary care. toralph ruge, toralph ruge, md and phd, is a specialist in family medicine and emergency medicine and an associate professor in emergency medicine at lunds university. orcid per wändell http://orcid.org/0000-0001-5169-2965 axel c. carlsson http://orcid.org/0000-0001-6113-0472 http://orcid.org/0000-0001-5169-2965 http://orcid.org/0000-0001-5169-2965 http://orcid.org/0000-0001-6113-0472 http://orcid.org/0000-0001-6113-0472 6 w. per et al. anders larsson http://orcid.org/0000-0003-3161-0402 olle melander https://orcid.org/0000-0002-2581-484x torgny wessman http://orcid.org/0000-0002-7314-2240 johan ärnlöv http://orcid.org/0000-0002-6933-4637 toralph ruge http://orcid.org/0000-0002-1170-5183 references 1. finucane mm, stevens ga, cowan mj, danaei g, lin jk, paciorek cj, et al. national, regional, and global trends in body-mass index since 1980: systematic analysis of health examination surveys and epidemiological studies with 960 country-years and 9.1 million participants. lancet. 2011;377:557–67. doi: 10.1016/s0140-6736(10)62037-5 2. collaboration ncdrf. worldwide trends in body-mass index, underweight, overweight, and obesity from 1975 to 2016: a pooled analysis of 2416 population-based measurement studies in 128.9 million children, adolescents, and adults. 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faculty of health and medicine, school of medicine and public health, university of newcastle, callaghan, nsw, australia; cgastrointestinal unit, massachusetts general hospital, harvard medical school, boston, ma, usa; ddepartment of medical epidemiology and biostatistics, karolinska institutet, stockholm, sweden; edivision of epidemiology and public health, school of medicine, university of nottingham, city hospital, nottingham, uk; fceliac disease center, department of medicine, columbia university college of physicians and surgeons, new york, usa abstract background: eosinophilic esophagitis (eoe) is a relatively new diagnosis, where until recently a specific international classification of disease code was missing. one way to identify patients with eoe is to use histopathology codes. we validated the clinicopathological eoe diagnosis based on histopathology reports and patient charts to establish these data sources as the basis for a nationwide eoe patient cohort. methods: through the epidemiology strengthened by histopathology reports in sweden (espresso) study, we randomly selected 165 patients from five swedish health care regions with a histopathologic diagnosis of eoe. patients were assigned a histopathology diagnosis of eoe if they had ≥15 eosinophils per high-power field or, in the absence of eosinophil quantification, the pathologist interpreted the biopsy as consistent with eoe. patient charts were scrutinized to see if the other diagnostic criteria were fulfilled. of the 131 received patient charts, 111 (85%) had sufficient information to be included in the study. results: of the 111 validated patients, 99 had eoe, corresponding to a positive predictive value of 89% (95% confidence interval = 82–94%). dysphagia was the most common symptom (n = 78, 70%), followed by food impaction (n = 64, 58%) and feeding difficulties (n = 37, 33%). twelve patients had coexisting asthma (11%) and 16 allergic rhinitis (14%). seventeen patients underwent esophageal dilatation (15%), of which seven had more than one dilatation. ninety-seven (87%) patients had a proton-pump inhibitor treatment ≤2 years before or after the diagnosis. forty-two patients (38%) had been prescribed inhalation steroids and 64 (58%) had undergone esophageal radiology. conclusion: histopathology reports from the espresso cohort with esophageal eosinophilic inflammation are suggestive of eoe. introduction eosinophilic esophagitis (eoe) has been recognized relatively recently and was first proposed as a distinct clinicopathologic entity in 1993–1994 (1, 2). time trends in eoe incidence and prevalence have shown an exponential rise in the past 25 years (3). eoe is a chronic, local immune-mediated esophageal disease of the squamous esophagus. it is clinically characterized by symptoms related to esophageal dysfunction, endoscopic findings of rings, linear furrows, exudates, edema, strictures, narrowing and crepe-paper mucosa on biopsy ≥15 eosinophils per high-power field (hpf, 60 eosinophils/mm2), and eosinophilia isolated to the esophagus (4). endoscopy can also be macroscopically normal (5). the diagnosis of eoe requires that clinical manifestations and pathologic data be interpreted in tandem (6). the diagnosis of eoe has traditionally been limited to proton-pump inhibitor (ppi) non-responders, but guidelines from an international consensus meeting in 2017 acknowledged that ppi therapy is an appropriate and effective treatment for a significant proportion of eoe patients. thus, ppi non-response as a diagnostic criterion for eoe has since been removed (6). eoe and gastroesophageal reflux disease (gerd) may coexist with significant overlap in symptoms, and therefore, it can sometimes be difficult to distinguish between eoe and gerd on clinical grounds alone (7, 8). in register-based research, patients of interest are usually identified through a relevant international classification of contact jonas f. ludvigsson jonasludvigsson@yahoo.com supplemental data for this article can be accessed here. © 2021 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article article history received: 1 march 2021 revised: 11 july 2021 accepted: 12 july 2021 published: 13 august 2021 keywords eosinophilic esophagitis; inflammation; validation; histopathology http://dx.doi.org/10.48101/ujms.v126.7687 jonasludvigsson@yahoo.com mailto:jonasludvigsson@yahoo.com http://dx.doi.org/10.48101/ujms.v126.7687 http://creativecommons.org/licenses/by/4.0/ l. röjler et al. disease (icd) code. however, in the case of eoe, no specific icdcode was available until 2012 in sweden, and this prohibits studies of long-term prognosis in eoe. we envisioned using an alternative means to identify eoe patients: histopathology reports with a systematized nomenclature of medicine-clinical terms (snomed-ct) code t62 (esophagus) in combination with m47150 (eosinophilic inflammation) from the epidemiology strengthened by histopathology reports in sweden (espresso) cohort study (9). in the current paper, we aimed not only to validate esophageal eosinophilia against eoe diagnosis in a randomly selected group of patients but also to characterize these patients with regards to symptoms, investigations, histopathology, laboratory data, differential diagnoses, smoking and alcohol consumption, and treatment. materials and methods study population swedish biopsy data are categorized according to the snomedct system, a system of comprehensive health and clinical terminology used in many countries. in a nationwide project, we collected gastrointestinal histopathology report data from all pathology departments in sweden (n = 28) from the time period 1965 to 2017; this cohort forms the espresso study (9) and contains more than 6 million biopsy reports. through searching espresso for individuals with a biopsy from the esophagus (t62) that showed inflammation with eosinophil infiltration (m47150) (n = 1,663), we aimed to establish a cohort of patients with eoe. from this cohort, an external biostatistician randomly selected 165 patients from 17 hospitals in five health care regions in sweden that included both local and university hospitals: örebro (departments of medicine, surgery, and ear-nose-throat (ent) and private clinic läkargruppen), karlskoga (medicine and surgery), lindesberg (surgery), norrtälje (surgery and endoscopy), karlstad (surgery), torsby (surgery), skövde (ent and surgery), stockholm karolinska (ent, gastroenterology, and endoscopy), stockholm södersjukhuset (medicine), eksjö (endocrinology and gastroenterology), arvika (surgery), danderyd (emergency department), lidköping (medicine), falun (medicine), and jönköping (surgery). the departments responsible for each patient with a t62 topography code and an m47150 morphology code (mainly internal medicine, surgery, or ent) were contacted, and patient charts were requested, which included discharge notes, histopathology reports, laboratory data, endoscopy notes, radiology reports, and surgery notes. between october 2017 and august 2018, we received clinical data from 131 individuals (79%). the patient charts of these individuals were then reviewed using a standardized form based on similar validation studies of inflammatory bowel disease (ibd) (10) and microscopic colitis (11), but adjusted to the unique conditions of eoe. additional symptoms, laboratory data, radiology, endoscopies, concomitant diseases (such as allergy), gastrointestinal infections, and ongoing treatment (medical or diet) were examined (see supplementary material for list of variables). the patients had sought health care for their medical complaints between december 2000 and january 2017, and the charts originated from patients’ hospital visit appointments between 1989 and 2017.  biopsies from the esophagus were categorized as ‘upper esophagus’ or ‘lower esophagus’. biopsies taken from the midesophagus as well as those taken 0–35 cm from the teeth were considered as ‘upper esophagus’, and biopsies taken from >35  cm were considered as ‘lower esophagus’. when data on location were inexact, biopsies were classified as from the ‘upper esophagus’. relevant data (a symptom, an examination, or other information) were considered absent if not explicitly reported in the patient charts. for instance, a patient without a record of dysphagia in the patient chart was interpreted as having ‘no dysphagia’. case definition lp and ig classified cases as definite, likely eoe (these two categories were merged for the positive predictive value [ppv] calculations) or negative for eoe. likely was defined as borderline number of eosinophils, but where other supporting features such as typical endoscopic appearance or highly suspicious clinical symptoms were present. in case of uncertainty, jjg was consulted. patients with insufficient patient chart data were excluded from the study (figure 1). ethical approval this study was approved by ethical review board in stockholm (july 19, 2017; reference number: 2017/1497-32). in accordance with the ethics approval, no patient was contacted in person since this study was part of a larger register-based project and aimed to verify already collected histopathology data (12). statistics this is a cross-sectional descriptive study. the random selection of patients was performed using r (version 3.6.1, r foundation for statistical computing, vienna, austria). ppvs and 95% confidence intervals (cis) were calculated using epitools (https:// epitools.ausvet.com.au/cipropor tion?page=cipropor tion, accessed october 2020) and the wilson score interval. when p-values were calculated for comparison between symptoms in true and false-positive eoe individuals, pearson chi-square and fishers exact test were used. our alpha level (significance level) was 0.05. sample size determination to detect a ppv of 90% with a 95% ci of 85–95%, we needed 139 patients (epitools.ausvet.com.au). from experience, we know that not all charts can be found or are delivered, and we, hence, requested 165 patient charts from the clinics. https://ujms.net/index.php/ujms/article/view/7687/13775 https://epitools.ausvet.com.au/ciproportion?page=ciproportion https://epitools.ausvet.com.au/ciproportion?page=ciproportion http://epitools.ausvet.com.au validation of eosinophilic esophagitis 3 results we received patient charts from 131 individuals with a histopathology code for eosinophilia in the esophagus (m47150). in 111 (85%), there were sufficient data to confirm or reject the diagnosis of eoe. of the 111 patients, 91 (82%) had definite eoe and eight (7%) likely eoe. twelve patients (11%) were defined as ‘not eoe’ based on the lack of an adequate number of eosinophils (<15/ hpf) (n = 9), another primary esophageal disorder such as barrett’s esophagus (n = 2), and dysphagia primarily attributable to poor dentition (n = 1). lack of clinical notes was the only reason for excluding 20 patients from the study. the outcome of the chart review is illustrated in figure 1. hence, of the 111 patients with available data, 99 had eoe, corresponding to a ppv of 89% (95% ci = 82–94%). two out of 111 patients had a family history of eoe. clinical characteristics and presenting symptoms the median age at first diagnostic biopsy was 63 years (range 12–87 years; one child). of the 111 patients included in the study, the majority were males (n = 86 (78%)). duration of symptoms was listed in 79 patient charts, and 23% (n = 25) of the patients had had symptoms for >10 years. smoking (current or past) was noted in 15 patients (14%) and alcohol use in 24 (22%) (table 2). body mass index (bmi) was recorded in nine patients (range 20–34.2 kg/m2). twelve patients had coexisting asthma (11%) and 16 allergic rhinitis (14%). atopic dermatitis was noted in five patients (5%). thirteen patients (12%) had a record of food allergies. dysphagia was the most common symptom (n = 78, 70%), followed by food impaction (n = 64, 58%) and feeding difficulties (n = 37, 33%) (table 1). comparing patients with true eoe versus those with falsepositive eoe (not confirmed through patient chart review) revealed that food impaction (p = 0.015) was more common in true-positive eoe (supplementary table s1). radiology and endoscopic examinations of the 111 eoe patients, 64 (58%) underwent not only esophageal radiology (true eoe vs. not eoe: n = 56 vs. 8), mainly barium esophagram (n = 41; 36 vs. 5) but also computed tomography (n  =  5; 4 vs. 1) and conventional x-ray (n = 3; 3 vs. 0). fifteen patients (14%; 13 vs. 2) underwent unspecified diagnostic radiology. many of the examinations were performed in the setting of acute food impaction (n = 17), and six examinations demonstrated strictures of the esophagus. one patient had a perforation after dilatation, and one had esophageal achalasia. twenty-five patients (23%) underwent esophageal manometry and ph registration. in the manometry examinations, 13 were normal, two showed suspected achalasia (these two patients fulfilled our eoe criteria), five hypomotility, two hypertensive peristalsis, one insufficient lower esophageal sphincter, and two were inconclusive. ph registration indicated normal ph in eight patients, reflux in three (ph < 4 for a significant part of the day), mild reflux in three, and 11 were without result. all patients underwent endoscopy with biopsy at least once. in 54 patients (49%), the location and number of biopsies could be determined from the endoscopy reports. among these, the majority (54%) had biopsies taken from both the upper and lower esophagus, although some patients had biopsies taken figure 1. study flowchart of patients with a histopathology diagnosis of eosinophilic esophagitis (eoe). table 1. patient symptoms reported in charts among 111 patients with a histopathology code consistent with eosinophilic esophagitis (eoe). symptom all patients (n = 111) patients with true eoe (n = 99) patients with no eoe (n = 12) dysphagia, n (%) 78 (70) 71 (72) 7 (58) food impaction, n (%) 64 (58) 61 (62) 3 (25) feeding difficulties, n (%) 37 (33) 33 (33) 4 (33) vomiting, n (%) 19 (17) 19 (19) 0 (0) abdominal pain, n (%) 17 (15) 15 (15) 2 (17) weight loss, n (%) 12 (11) 10 (10) 2 (17) other pain, (%) throat 2 (2) 2 (2) 0 (0) sternal 12 (11) 12 (12) 0 (0) epigastric 2 (2) 1 (1) 1 (8) abdominal 4 (4) 4 (4) 0 (0) headache/backpain 2 (2) 1 (1) 1 (8) myoclonus unspecified location 1 (1) 0 (0) 0 (0) eating slowly 6 (5) 6 (6) 0 (0) https://ujms.net/index.php/ujms/article/view/7687/13775 l. röjler et al. from only the upper (20%) or lower (26%) esophagus. when this could be determined from the endoscopy reports, the mean number of biopsies taken was 2 (range 1–9) from the upper esophagus and 2 (range 1–8) from the lower esophagus. five patients (5%) had a record of helicobacter pylori positivity. no patient was positive for giardia. a detailed laboratory data can be found in the supplementary material. treatment treatment data are presented in table 2. thirteen patients (12%) were on ppi therapy at the time of diagnostic biopsy, and 97  patients (87%) had a ppi trial ≤2 years before or after the diagnosis. dietary advice had been recorded in five patients, but none of these were recommended an empiric elimination diet (e.g. six-food elimination diet, sfed) or elemental diet. dietary advice instead included avoidance of confirmed allergens or foods with specific textures (e.g. large pieces of meat). one patient was already avoiding meat products due to dysphagia. the most common eoe treatments were swallowed steroids and esophageal dilation. seven patients (6%) had received systemic steroids (betamethasone or prednisolone). swallowed inhalation steroids were prescribed to 42 patients (38%). the most common swallowed steroids were mometasone furoate (nasonex, n = 27), fluticasone propionate (n = 11), and budesonide (n = 4). three patients (3%) had budesonide/formoterol prescribed but for asthma indication. one patient had intraesophageal steroid injection at the time of dilatation. in addition, six patients (5%) were prescribed montelukast. no patient had a record of biological treatment. seventeen patients (15%) underwent therapeutic esophageal dilatation; seven (6%) of these had undergone ≥2 dilatations. in the year before biopsy, 13 (12%) patients had been prescribed antibiotics for indications of respiratory tract infection, skin wound, prophylaxis for bladder tumor resection, otitis media, erysipelas, perforated esophagus, and unknown reasons (n = 7). twenty patients (18%) had a record of non-steroidal anti-inflammatory drug use ≤1 year before or after the diagnosis of eoe. discussion a major criterion for the diagnosis of eoe is the presence of at least 15 eosinophils/hpf in the esophageal mucosa, regardless of the results of ppi treatment outcome (4, 13). eosinophils are found throughout the gastrointestinal mucosa but are typically not present in the normal esophagus (14). based on this information, we hypothesized that the presence of eosinophils in adequate numbers to trigger a histopathologic diagnosis of eoe would, in the absence of other plausible explanations for esophageal eosinophilia, be highly predictive of a valid clinical diagnosis of eoe. in the current study, we examined a random subset of 111 patients with a histopathology report with eosinophilia in the esophagus and found that 99 (89%) had a clinicopathological diagnosis of eoe – meeting all three criteria for eoe. this validity is similar to having a physician-assigned diagnosis in the swedish patient register (15). main findings and comparison with earlier literature in accordance with earlier research (3, 4), most patients (78%) in our nationwide cohort study were male. the majority had typical symptoms and presentation of eoe, including chronic dysphagia (70%) and a history of prior food impaction (58%), with very few patients reporting prominent heartburn. taken together, these results suggest a patient population quite distinct from that afflicted with chronic gerd although these two medical conditions often overlap. with these symptoms, it is likely that patients suffer from a reduced health-related quality of life (16). moreover, it is probable that the prevalence and severity of esophageal symptoms reported in our study are underestimated because lack of any record for a specific symptom in the patient chart was interpreted as missing. slow eating and excessive chewing may represent coping strategies that eoe patients consciously or subconsciously employ to avoid food impaction and ease dysphagia (17). extended mealtime (slow eating) was only reported in six patients (5%) with eoe in our study. this symptom may be more common but overshadowed by dysphagia and food impaction, which patients may perceive as more serious concerns. we also cannot rule out table 2. drugs prescribed to eosinophilic esophagitis (eoe) patients within 2 years before or after the first biopsy of eoe. medication n = 111 (%) ppi treatment, n (%) 97 (87) ppi at the time of first biopsy, n (%) 13 (12) response to ppi treatment, n (%) 50 (45) other antacids, n (%) ranitidine 6 (5) aluminum-hydroxide antacid 12 (11) oral steroids, n (%) betamethasone 4 (4) prednisolone 4 (4) swallowed steroids, n (%) mometasone 27 (24) fluticasone/flixotide 11 (10) budesonide 4 (4) antihistamines, n (%) desloratadine 6 (5) loratadine 2 (2) others: montelukast, n (%) 6 (5) omalizumab, n (%) 1 (1) azathioprine, n (%) 0 (0) other biologics, n (%) 0 (0) other drugs, n (%) (list restricted to drugs prescribed to at least three patients) paracetamol, simvastatin, amlodipine, cocillanaetylmorphine, acetylsalicylic acid, betamethasone, enalapril, zopiclone, phenoxymethylpenicillin, metformin, metoprolol, propiomazine, atorvastatin, ipratropium, ciprofloxacin, paracetamol/codeine, levothyroxine, insulin, oxycodone, ramipril, pivamdinocillin ppi: proton-pump inhibitor. https://ujms.net/index.php/ujms/article/view/7687/13775 validation of eosinophilic esophagitis 5 that swedish physicians do not ask questions specifically about the amount of time taken to eat a meal, especially when they have confirmed the presence of dysphagia. the average age at first biopsy in our study was 63 years, which is higher than expected. eoe has been described in all ages, but most studies examining eoe in adults have reported an average age of diagnosis of 30–50 years (18–23). there are several possible explanations for the more advanced age observed in our study. first, we documented only the date of the index esophageal biopsy in which increased eosinophils were noted leading to the histopathological diagnosis, and not the date from which symptoms first became apparent, although we found that 53% of patients had experienced symptoms for >2 years and 23% for >10 years. for cases with adequate follow-up assessment, just under half (45%) were documented to achieve a good clinical response. this finding agrees with published data describing rates of ppi-induced remission in 30–50% (24). also in line with previous studies (25), 38% of eoe patients in our study were treated with swallowed steroids (mometasone, fluticasone, and budesonide). the high rate of mometasone use in our study population may reflect that mometasone is listed, along with fluticasone, as a topical steroid option with more local effect and less systemic exposure compared with budesonide in swedish eoe clinical practice guidelines (26). there may be several reasons for the low rate of dietary therapies. first, it may reflect that the majority of patients in our study were primarily treated by a gastroenterologist or internist (rather than an allergist), specialties that may have a lower comfort level with recommending dietary therapy. second, patients opting for dietary therapy will undergo numerous repeat endoscopies during the process of empiric elimination and step-wise food reintroduction. for example, it is estimated that patients choosing an empiric sfed will undergo a mean of seven upper endoscopies with a predicted success rate of 55– 60% in ultimately defining their dietary trigger(s) (27). the frequency of therapeutic elimination diets in our study may also be underestimated given that dieticians have not traditionally recorded interventions in physician patient charts. only a minority of patients had a record of allergic comorbidity. twelve patients (11%) had asthma, which was lower than expected considering the strong allergic component in eoe (28). in another study conducted in the us (29), 24% of adult eoe patients and 52% of pediatric eoe patients were diagnosed with asthma, figures considerably higher than in our study. the most likely reason for this discrepancy is that physicians in routine health care (as opposed to those in a research setting) may not register the presence of comorbidity, or that such diseases are cared for outside the clinic that managed the eoe diagnosis. the major strengths of our study include its populationbased design and real-world setting. however, this paper also has a number of limitations. first, we used routine care data, which means that some information for comorbidities, medication, bmi, smoking, and alcohol consumption may not have been documented by the treating physician (as opposed to a research setting). a second limitation is our attrition rate. of the 131 patient charts, only 111 had sufficient data to be included in the analyses. none of the hospitals included in this study was situated in the north of sweden, and, hence, our study has limited information on geographical differences of eoe within sweden. third, we acknowledge that an eoe icd code was introduced in the swedish patient register in 2012. much of our data, however, originate from before 2012; taking advantage of biopsy reports to identify eoe in the early 2000s allows researcher to carry out cohort studies of long-term prognosis of eoe, which is not feasible if limited to patients diagnosed in 2012 or later. fourth, in a subset of patients without exact data on the number of eosinophils/hpf, an eoe diagnosis was sometimes accepted when the pathologist had interpreted that the biopsy was consistent with eoe. this is not according to current practice (30), but was done since a large proportion of esophageal biopsies were carried out in the early 2000s when current diagnostic criteria were not yet established. fifth, we lacked data on the location of a subset of esophageal biopsies, and we urge caution when interpreting findings related to upper as opposed to lower esophagus. sixth, because of restrictions imposed by the ethics review board, we were unable to rereview the slides of the patients examined for suspected eoe. still, other studies have shown a 90% accuracy when reanalyzing biopsies for number of eosinophils per hpf (31). seventh, ideally, data on histopathological features other than number of eosinophils per hpf should have been reported in this review (such as basal zone hyperplasia, eosinophil abscesses, eosinophil surface layering, dilated intracellular spaces, surface epithelial alteration, dyskeratotic epithelial cells, and lamina propria fibrosis) (32). unfortunately, such information was rarely available in the biopsy reports (which tended to focus on eosinophil numbers) and, hence, not recorded in this study. finally, in many eoe patients, there was a lack of detailed data on endoscopic appearance. conclusion histopathology reports from espresso cohort indicating eosinophilic inflammation in the esophagus are suggestive of eoe. disclosure statement jonas f. ludvigsson coordinates a study on behalf of the swedish ibd quality register (swibreg). this study has received funding from the janssen corporation. the other authors declare that they have no conflicts of interest. funding örebro university hospital and karolinska institutet. notes on contributors lovisa röjler, md. resident physician at the department of pediatrics at örebro university hospital, sweden. ida glimberg, medical student at örebro university, sweden. l. röjler et al. marjorie m. walker, bmbs frcpa. professor of anatomical pathology at the university of newcastle, new south wales, australia. john j. garber, md. gastroenterologist at the massachusetts general hospital, and harvard medical school, boston ma, usa. jonas f. ludvigsson, md, phd. senior consultant at the department of pediatrics at örebro university hospital, and a professor of clinical epidemiology at karolinska institutet, stockholm, sweden. orcid ida glimberg https://orcid.org/0000-0002-8738-5673 marjorie m. walker https://orcid.org/0000-0002-7788-0056 f. ludvigssona 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10.2500/aap.2014.35.3768 30. dellon es, liacouras ca, molina-infante j, furuta gt, spergel jm, zevit n, et al. updated international consensus diagnostic criteria for eosinophilic esophagitis: proceedings of the agree conference. gastroenterology. 2018;155:1022–33.e10. doi: 10.1053/j.gastro.2018.07.009 31. dellon es, erichsen r, pedersen l, shaheen nj, baron ja, sorensen ht, et al. development and validation of a registry-based definition of eosinophilic esophagitis in denmark. world j gastroenterol. 2013;19:503–10. doi: 10.3748/wjg.v19.i4.503 32. collins mh, martin lj, alexander es, boyd jt, sheridan r, he h, et al. newly developed and validated eosinophilic esophagitis histology scoring system and evidence that it outperforms peak eosinophil count for disease diagnosis and monitoring. dis esophagus. 2017;30:1–8. doi: 10.1111/dote.12470 https://orcid.org/0000-0002-8738-5673 https://orcid.org/0000-0002-8738-5673 https://orcid.org/0000-0002-7788-0056 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illinois abstract twenty-seven non-infected delayed union or non-union fractures of the shaft of the tibia in 26 patients were operated on with a full cortical thickness inlay graft taken from the injured leg. the initial trauma was moderate in 9 fractures and severe in 18. fifteen fractures were closed injuries and 12 open. the fracture pattern was longitudinal in 5 cases, transverse in 11 and communited in 11. surgery was performed 12 to 72 weeks (mean: 30 weeks) after the fractures had been sustained. all fractures healed after the bone grafting procedure. the time until union was established after surgery ranged from 6 to 37 weeks (mean: 17 weeks). the healing time was neither influenced by the initial type of fracture pattern (p > 0.05) nor by the fracture age at surgery (r = 4 . 2 1 ) . of 13 patients re-examined 8 to 16 years after surgery, the clinical results were classified as excellent in 8, as fair in 2, and as poor in 3 patients. introduction delayed union and non-union are well known complications in fractures of the shaft of the tibia. albert (2) reviewed 395 tibial fractures and found an incidence of delayed union or non union of 29 per cent. brumsback (4) reported that 22 per cent of 120 tibial fractures had to be operated on to obtain union. urist et al. (14) estimated that approximately one-third of all tibial fractures will present problems with union. factors such as initial fracture displacement, comminution, associated soft-tissue injuries and wound infections are known to influence fracture healing (7,12). in displaced, compound, comminuted tibial fractures, carpenter et al. (5) reported an incidence of non-union of 75 per cent. over the years, many surgical procedures have been described for the management of non union of diaphyseal fractures. they include different bone grafting procedures using onlay, barrelstave and cancellous bone graft, intramedullary nailing and compression plate osteosynthesis. in recent years electrical stimulation has also been applied. in 1930, albee (1) described a sliding inlay graft technique for treatment of non-union of diaphyseal fracture taking a full cortical thickness graft from the fractured bone. the advantage of this procedure is that the graft provides both internal fixation and osteogenetic stimulus (9). 169 we have employed a modification of albee's procedure in the treatment of delayed union and non-union of the shaft of the tibia and the purpose of this study was to review our experience with this surgical procedure. i patients and methods during the 8 year period from 1969 through 1977, 27 fractures of the shaft of the tibia in 26 patients were operated on for delayed union or non-union with a modification of albee's sliding full cortical thickness inlay bone graft technique at the departments of orthopedics, sahlgren hospital, goteborg, and borfis hospital, sweden. the medical records and avail able radiographs of these 26 patients were reviewed. from the medical records and radiographs, information was collected about the patient's age and sex, the type of initial trauma, the fracture pattern, the soft-tissue injury, the primary treatment, the surgical treatment and the healing time. the initial trauma causing the fracture was classified as severe or moderate (7). severe trauma was defined as fractures sustained in traffic accidents or from falls from a height. for the remaining fractures, the trauma was classified as moderate. type of fracture pattern and soft-tissue injury were classified according to a modification of the classification used by edwards (7). the fracture patterns were thus classified as trans verse, oblique or comminuted when there was one or more intermediary fragment. fractures with only minor puncture wounds were included in the group of open injuries. healing time was defined as the time when the fracture was determined, clinically and radiologically, to be united sufficiently well not to require further immobilization. in none of the patients did deformation or refracture occur after this time. statistics: student's t-test for small samples was used and a p-value less than 0.05 was con sidered significant. surgical technique: the anesthetized patient is placed in the supine position on the operating table and a tourniquet is applied around the thigh. a slightly medially curved incision, about figure 1 . modification of albee's bone grafting procedure. from the anteromedial slide of the tibia a full cortical thickness bone graft of unequal length on either side of the fracture is taken out, turned 180 degrees and put back into the trough. 170 15 cm long, is made just medial to the anterior aspect of the tibia at the fracture site. the subcutaneous tissue and the periosteum are then incised along the skin incision and the periosteum is separated from the anteromedial side of the tibia "en-bloc'' with the sub cutaneous tissue and the skin. reflecting the periosteum together with the soft-tissue, the tibia and the fractures are then exposed and, using an oscillating saw, a full cortical thickness graft, about 15 cm long and 1.5 cm wide, is taken out from the anteromedial side of the tibia (figure 1). the graft, which must be of unequal length on either side of the fracture, is then turned 180 degrees and put back into the trough. the asymmetrical length of the graft will guarantee that the fracture space is covered by intact graft bone. when the graft fits f m l y into the trough, internal fixation is not necessary. if this is not the case, however, internal fixation of the graft using screws may be necessary. at this stage, especially if there is still a considerable fracture gap, one may also have to consider cancellous bone grafting, taking bone grafts from the proximal end of the tibia, or preferably, from the iliac crest. as suggested by phemister (13), however, it is recommended that the fibrous pseudoaxthrosis be left intact. the periosteum is then sutured back and a hemovac is routinely placed under the subcutaneous tissue and the operation wound is closed with loosely interrupted skin sutures. after wound dressing and after having removed the tourniquet, a long, non-weight-bearing, cast is applied. the long leg cast is kept until it is obvious, both clinically and radiologically, that fracture union has started. at that time, a patellar tendon-bearing full weight-bearing cast is applied which is kept on until union is solid. results initial descriptive data. of the closed injuries, 4 fractures were longitudinal, 9 transverse and 2 comminuted, and of the open injuries, 1 fracture was longitudinal, 2 transverse and 9 comminuted (table 1). sixteen fractures were initially treated with closed reduction and application of a long leg cast and 11 with open reduction, internal fixation and a long leg cast. all 12 open injuries were initially treated with debridement and 11 wounds were closed primarily and 1 was treated with delayed primary closure. table 1. initial descriptive data on 27 fractures of the shaft of the tibia in 26 patients (20 male and 6 female) operated on for delayed union or non-union age (years) fracture pattern range 17-74 longitudinal mean 38 transverse comminuted 5 11 11 initial trauma primary treatment moderate 9 closed reduction 16 severe 18 open reduction and internal fixation 11 soft-tissue injuries initial treatment of soft-tissue injuries closed injuries 15 debridement and primary closure 11 open injuries 12 delayed primary closure 1 12-928572 171 bone grafting procedure: the fracture age when the bone grafting procedure was performed varied from 12 to 72 weeks (mean: 30 weeks) and was 20 weeks or more in 74 per cent of the fractures and 32 weeks or more in 33 per cent of the fractures (table 2). the sedimentation rate at operation was available for 21 patients (81 per cent) and was 15 or less in 19 patients and 20 or more in 2 patients. at the time of surgery, 6 patients (23 per cent) received pro phylactic antibiotic treatment and in all but 3 fractures additional cancellous bone grafting performed. table 2. data on bone grafting procedure fracture age at surgery (weeks) internal fixation of the bone graft range 12-72 none 13 mean 30 screws 12 cerclage 2 sedimentation rate at surgery additional cancellous bone grafting range 2-42 yes 24 mean 11 no 3 healing time: data on the healing time after surgery are summarized in figure 2 and table 3. all fractures healed after the bone grafting procedure. the time until union was established varied from 6 to 37 weeks (mean: 17 weeks) after surgery (table 3). the initial fracture pattern did not influence the healing time after surgery significantly ( p s . 0 5 ) although there was a tendency toward shorter healing time in longitudinal fractures. further, there was no correlation between healing time after surgery and fracture age at surgery (correlation coefficient = -0.21). table 3. healing time after surgery in relation to initial soft-tissue injury and type of fracture soft-tissue injury number healing time and (weeks) types of fracture range mean all fractures closed injuries all fractures longitudinal transverse comminuted open injuries all fractures longitudinal transverse comminuted 27 15 4 9 2 12 1 2 9 6.4-37.0 7.6-28.7 7.6-20.0 12.1-28.7 15.3-23.6 6.4-37.0 12.9 12.1-19.3 6.4-37.0 17.7 17.8 13.0 19.7 19.5 17.5 12.9 15.7 18.4 172 per cent 100 50 lo 2o 30 40 figure 2. percentage of united fractures in relation weeks to weeks after surgery. clinical results: in all, it was possible to re-examine 16 patients (62 per cent) 8 to 16 years (mean: 13 years) after they had sustained the fracture. of the patients who were not re examined, 7 were dead and 3 could not be traced. at the time of re-examination, 2 patients had been operated on with a total hip joint replacement and 1 for a hip fracture. in 12 patients, total ankle joint motion (summary of plantar flexion and extension) was restricted less than 20 degrees and in 4, 20 degrees or more. of 13 patients who had not had hip surgery, all except one could walk unlimited distances and 10 were walking without limping while 3 had a slight limp. the clinical results in patients who had not had hip surgery were also classified by summary of the clinical findings and the patient's own assessment of their walking ability. in 8 patients who could walk unlimited distances without limping, and in whom total ankle joint motion was restricted less than 20 degrees, the results were classified as excellent; in 2 patients who could walk unlimited distances without limping but in whom total ankle joint motion was restricted 20 degrees or more as fair, and in 3 patients who could not walk unlimited distances and/or limped, they were classified as poor. of the patients in whom the clinical results were classified as excellent, 4 had sustained the fracture by moderate trauma and 4 by severe trauma, whereas all 5 patients in whom the results were classified as fair or poor had sustained the fracture by severe trauma. discussion when evaluating fractures of the shaft of the tibia in which healing is not progressing at normal speed, it is essential to asses whether one is dealing with a delayed union or a non union. delayed union, by definition, means that a fracture has not united in the time period when that type of fracture is usually united. non-union, on the other hand, means that the healing process has ceased (3). in clinical practice, it is often difficult, however, to apply these definitions correctly. as a guideline for the clinical management of fractures of the shaft 173 of the tibia, it has been suggested that delayed union be defined as absence of fracture healing, assessed both clinically and radiologically, at the fracture age of 20 weeks, and non union as when a fracture is not united after 32 weeks (10). according to these definitions, 74 per cent of the fractures reported on in this study would be classified as having delayed union or non-union. in non-infected non-union of tibia1 fractures several surgical procedures are possible alternatives, including internal fixation using a compression plate, an intramedullary nail, and a full cortical thickness inlay graft. it is usually recommended that internal fixation procedures, especially in "atrophic pseudoarthrosis", can be combined with cancellous bone grafting (1 1). to our knowledge, there are not yet enough published reports on these different techniques to allow conclusions as to whether any of these methods of treatment is superior. the compression plate offers rigid immobilization, which is often desirable, especially in "hypertrophic pseudoarthrosis", but has the disadvantage of causing bone resorption with an increased risk of re-fracture later. the plate also constitutes a foreign body, thereby increasing the risk of infection. further, after union is established, the plate has to be removed and thus a second operation is required. the intramedullary nail does not have these disadvantages to the same extent and seems to be a suitable method for hypertrophic pseudoarthrosis in transverse undisplaced fractures. if the fracture is comminuted it can, however, be difficult to obtain adequate fixation. the full cortical thickness inlay graft has the advantage of offering internal fixation and stimulation of osteogenesis without osteosynthesis. to obtain internal fixation the graft must fit firmly into the trough, and this can be more easily achieved by using a twin saw adjusted to the right width, as suggested by albee (1930), than when a single-bladed saw is used. it is also essential to aim for optimal fit of the layers of the graft to the layers of the host and that the graft contain both cortex and marrow in sufficient amounts. the cortex of the graft will mainly provide internal fixation of the fracture fragments and the marrow will stimulate osteogenesis. further, resuturing of the periosteum over the graft will reduce the risk for dislocation of the graft. in 52 per cent (14/27) of the fractures in this study it was considered necessary to use internal fixation of the graft, probably due to the fact that a single-bladed saw had been used when taking out the graft and an accurate fit of the graft into the trough had probably not been obtained. in all except 3 fractures additional cancellous bone grafting was performed to obtain sufficient amount of meduilary bone. by combining these two procedures, which is probably not routinely necessary, all the fractures healed. as the healing time after surgery was not influenced by the fracture pattern this procedure also seems to work well i n non infected transverse and comminuted fractures, usually caused by severe trauma, which are known commonly to present healing problems. the clinical results were probably influenced more by the severity of the initial trauma than by the bone grafting procedure. of 9 patients who had sustained the fracture by severe trauma the clinical results were classified as excellent in 4 and as fair or poor in 5, whereas in 4 patients who had sustained the fracture by moderate trauma the clinical results were classified as excellent in all cases. 174 conclusions in summary, we believe that the results of this study of treatment of 27 non-infected delayed union or non-union of fractures of the shaft of the tibia with a full cortical thickness inlay graft warrant the conclusion that this is a reliable surgical procedure which merits consider ation in cases of healing problems in tibial fractures. our present treatment policy for this condition is to use this method for non-infected atrophic pseudoarthrosis and in displaced fractures which cannot be treated with closed intramedullary nailing. in hypertrophic pseudo arthrosis of the middle third of the tibia in undisplaced transverse fractures, however, we now prefer rigid closed intramedullary nailing. acknowledgement the authors wish to thank monica lindh for typing the manuscript. references 1. albee, f.h. principles of the treatment of non-union of fracture. surg gynecol obstet 2. albert, m. delayed union in fractures of the tibia and fibula. j bone joint surg 3. brashear, h.r.: diagnosis and prevention of non-union. j bone joint surg. 47 a:174-8, 1965. 4. brumsback, j.e. mean disposition of tibial fractures. am j surg 71532-3, 1946. 5. carpenter, e.b., dobbie, j.j. & sewers, c.f. fractures of the shaft of the tibia and fibula. a comparative end-results from various types of treatment in a teaching hospital. a.m.a. arch surg 64:433-456, 1952. 6. dietrichson, g.j.f. and storen, g. posterolateral approach. a back-door to infected tibial shaft fractures. acta chir scand 129:4714, 1965. 7. edwards, p. fractures of the shaft of the tibia: 492 consecutive cases in adults. acta orthop scand, suppl76,1965. 8. freeland, a.e. & mutz, s.b. posterior bone-grafting for infected ununited fracture of the tibia. j bone joint surg 58-a:653457, 1976. 9. hohl, m. surgical treatment and technique. j bone joint surg 47-a:179-90, 1965. 10. johner, r. & wruhs, 0. classification of tibial shaft fractures and correlation with results after rigid internal fixation. clin orthop 178:7-25, 1983. 11. miiller, m.e., allgower, m., schneider, r. & willenegger, h. manual of internal fixation. springer-verlag, 1979. 12. nicoll, e.a. fractures of the tibial shaft: a survey of 705 cases. j bone joint surg 46 13. phemister, d.b. treatment of ununited fractures by onlay bone grafts without screw or tie fixation and without breaking down the fibrous union. j bone joint surg 29:94640, 1947. 14. urist, m.r., mazet, r. and mclean, f.c. pathogenesis and treatment of delayed union and non-union; survey of 85 ununited fractures of shaft of the tibia and 100 control cases with similar injuries. j bone joint surg 36-a:93148; 980, 1954. 3~289-320, 1930. 26~566-78, 1944. b:373-87, 1964. 175 correspondence to: goran hansson, md department of orthopedics uppsala university hospital s-751 85 uppsala, sweden 176 upsala j med sci 97: 1 15-126 ariway pressures during positive pressure ventilation with superimposed oscillations before and after lung injury in the cat by a. jonzon, t. norsted, and g. sedin the department of pediatrics, university hospital and the department of physiology and medical biophysics, university of uppsala, uppsala, sweden abstract this study was made to determine how oscillations superimposed on intermittent positive pressure ventilation (ippv) influence the arterial blood gases, ph and the airway pressures during adequate alveolar ventilation i.e. at inhibition of inspiratory activity, before and after experimentally induced lung injury in the anaesthetized cat. two ippv frequencies were studied. the lung was injured by instillation of xanthine oxidase into the upper airways during ippv. the peak, mean and end-expiratory intrapleuraland airway (intratracheal) pressu res at two levels were measured and the arterial blood gases and ph were determined at inhibition of inspiratory activity with and without superimposition of oscillations on the ventilatory pattern. before lung injury, superimposed oscilla tions lowered the airway pressures only at an ippv rate of 15 breaths per minute (b.p.m.). after lung injury, such oscilla tions increased the airway pressures only at 15 b.p.m. the airway pressures were always lower at 60 than at 15 b.p.m. 115 introduction we have previously shown that superimposition of oscillations on intermittent positive pressure ventilation (ippv) lowers the mean intratracheal airway pressure at ippv frequencies of 15 breaths per minute (b.p.m.) but not at 100 b.p.m. (3). the pressure reduction with superimposed oscillations may be of value during ventilation of newborn infants, since high airway pressures during ippv together with oxygen administration are considered to play an important pathogenetic role in broncho pulmonary dysplasia (5). in infants with interstitial emphyse ma, superimposed oscillations are of advantage in most cases ( 4 , 8 ) . experimental studies of the effects of ventilatory patterns on the airway pressures and arterial blood gases in the presence of a lung disease require a good and stable experi mental model which allows repeated measurements under stan dardized conditions. recently saugstad et a1 (7) demonstrated that injection of xanthine oxidase into the trachea results in lung damage with formation of perivascular oedema, dilatation of lymph vessels and infiltration of neutrophils with reduced lung compliance. the lung damage in this experi mental model has some histopathological characteristics in common with that which may be seen in prolonged neonatal ventilation ( > 5 days). the model could therefore be appli cable in studies of the way in which ippv with and without superimposed oscillations influences the airway pressures and arterial blood gases after lung injury. the present study was undertaken to determine whether pressure 116 oscillations superimposed on ippv influence the arterial blood gases and ph and the airway pressures differently at inhibi tion of inspiratory activity in cats with normal and injured lungs. methods subjects and preparation seven cats, weighing between 2.5 and 4.7 kg, were studied. anaesthesia was induced with chloroform and maintained with intermittent injections of chloralose (merck ag, g f r ) . catheters were introduced through the femoral vein and artery into the inferior vena cava and aorta. the left phrenic nerve was exposed through a frontal, medial incision in the neck. an endotracheal tube was inserted just below the larynx and a ligature was placed so that no air could leak between the endotracheal tube and the tracheal wall. two equally long catheters had previously been attached to the endotracheal tube so that one had its tip 1 cm above the carina and the other had its tip 5 cm further down. to measure the pleural pressure, another catheter was inserted through the rib cage, without letting any air into the pleural space. measurements airway pressures were measured at the tip of the endotracheal tube ("tip pressuret1) and also about 1 cm above the carina and 5 cm below the tip of the endotracheal tube ("distal pres surev1). pleural pressure was measured through a catheter 117 inserted into the pleural space. arterial blood pressure was ' measured through a catheter placed in the abdominal aorta. all catheters were connected to identical transducers (druck ag, gfr) and amplifiers (hellige ag, gfr) . all signals were ampli fied with an 8-channelled medical amplifier system (hellige ag, gfr) and fed to a recorder (recorder 330-p, hellige ag, gfr). measurements of arterial blood gases and ph were made with an automatic acid-base analyser (radiometer, denmark). the phrenic nerve activity was recorded by placing the left phrenic nerve on bipolar hook electrodes. the nerve and electrodes were immersed in mineral oil. for amplification a neurolog system (digitimer, u.k.) was used. ventilators a siemens elema servo ventilator (sv 9ooc) was used in the experiments. a positive end-expiratory pressure (peep) of 0.5 kpa was applied. the set peep was not altered when oscillatory ventilation was superimposed. oscillations of the ventilation gas were accomplished by attaching metal bellows to the tubing between the ventilator and the endotracheal tube. the bellows were controlled by a motor on which the stroke volume and the number of strokes per minute could be set independently. the stroke volume of the bellows in these experiments was 19 ml. the number of strokes was 570-600/minute. 118 experimental procedure before any measurements were made a check was made to see that all airway pressures were at zero during expiratory rest with the cat breathing spontaneously and also that the acid-base status was normal and that the base excess (be) was above -5 meg/l. the experiments were performed during ventilation with 15 or 60 breaths per minute with the ventilator in volume controlled mode. the inspiratory time was always 3 3 % of the cycle and a plateau of 10 % was used. first the cat was ventilated at a frequency of 15 or 60 per minute, and the minute ventilation was slowly increased until the phrenic nerve activity was inhibited. about 20 seconds after inhibition of inspiratory activity, measurements of the peak, mean and end-expiratory airway and pleural pressures were made. a blood sample was drawn for determination of arterial blood gases and ph. oscillations were then superim posed and the tidal volume of the ventilator was reduced until the phrenic nerve activity reappeared; it was then again slowly increased until the phrenic nerve activity was inhib ited and the same measurements were repeated. subsequently 10 u/kg b.w. of xanthine oxidase was injected into the airways and the fraction of inspired oxygen ( f i 0 2 ) was increased to 0 . 4 . after 30 minutes the same procedure was repeated, with ventilation to inhibition without and with superimposed oscillation. 119 histopathology of the lungs after the experiment the lungs were perfused with a mixture of formaldehyde (10%) and glutaraldehyde ( 4 % ) through a catheter inserted into the pulmonary artery. the lungs were then removed and placed in a 4 % solution of formaldehyde. histological examination revealed a non-homogeneous distribu tion of atelectatic areas with capillary dilatation, oedema, and fluid accumulation in the alveolar spaces. in some animals there was also local infiltration of leucocytes in the atelectatic areas. results before lung injury, without oscillations, at inhibition at inhibition of inspiratory activity, without oscillations the arterial pozl p c 0 2 , ph and be were the same at 15 and 60 b.p.m. the peak and mean airway pressures were lower at 60 than at 15 b.p.m. (p<o.ol). before lung injury, with oscillations, at inhibition when oscillations were superimposed the arterial blood gases and ph were the same as without superimposed oscillations at inhibition of inspiratory activity. with oscillations, as without the peak and mean airway pressures were lower at 60 than at 15 b.p.m. (p<o.ol). at 15 b.p.m. the peak and mean airway pressures were lower with than without superimposed oscillations (p<o.o1). 120 n o r m a l l u n g s a o " i 18 j o f 15 60 i n j u r e d lungs 15 60 15 60 v e n t i l a t o r y frequency fiq. 1 normal lungs i n j u r e d lungs n 0 0 a m a, . l c l q 8 6 l----- l 15 60 15 60 ventilatory frequency arterial p o 2 and p c 0 2 , ph and base excess (be) during ventila tion at 15 and 60 b.p.m. at inhibition of inspiratory activity without superimposed oscillations (whole lines) and with superimposed oscillations (dotted lines). triangles represent experiments made after lung injury. e n d o f e t -t u b e n o r m a l lu n g s in ju r e d lu n g s k p a * 0 z i 5 0 2 -0 l k7 ;l 1 5 a "-# y f iv e c m b e lo w ti p o f e t -t u b e n o r m a l lu n g s ln ju r e d lu n g s u 15 6 0 k p a * a 0 6 i = 1 a 0 4 a 0 2 w w ip p v ip p v + o sc ip p v ip p v + 0 5 c u 0 u 60 6 0 15 6 0 v e n ti la to ry f re q u en cy v e n ti la to ry fr e q u e n c y p le u r a l n o r m a l lu n g s in ju r e d l u n g s i -0 l ip p v 'o s c i lpp v 60 15 6 0 v e n ti la to ry f re q u en cy fi g. 2 p e a k , m e a n a nd e n d e x p i r a t o r y a i r w a y a nd p l e u r a l p r e s s u r e s d u r i n g v e n t i l a t i o n a t 1 5 a nd 6 0 b. p. m. , a t i n h i b i t i o n of i n s p i r a t o r y a c t i v i t y w i t h o u t s u p e r i m p o s e d o s c i l l a t i o n s ( w h o l e l in es ) an d w i t h s u p e r i m p o s e d o s c i l l a t i o n s ( d o t t e d l in es ). t h e a i r w a y p re ss ur es a r e m e a s u r e d a t t h e d i s t a l e nd of t h e e n d o t r a c h e a l (e t) t u b e a nd 5 c m b el ow t h e e nd of t h e e t tu be . t r i a n g l e s r e p r e s e n t e x p e r i m e n t s m a d e a f t e r lu ng in ju ry . after lung injury, without oscillations, at inhibition the arterial pc02 was lower after than before lung injury (p<o.ol 0.05). p o 2 was always higher after than before lung injury due to the higher f i 0 2 . arterial ph and be were the same as before lung injury. the peak and mean airway pressures were lower at 60 than at 15 b.p.m. (p<o.ol). pleural pressure was lower at 60 than at 15 b.p.m. (~~0.05). after lung injury, with oscillations, at inhibition the arterial pc02 was lower after than before lung injury (p<o.ol 0.05). po2 was always higher after than before lung injury, because of the higher f i 0 2 . arterial ph and be were the same as before lung injury. the peak and mean airway pressures were lower at 60 than at 15 b.p.m. (p<o.ol). pleural pressure was lower at 60 than at 15 b.p.m. (pco.05). the peak and mean airway pressures were higher with than without superimposed oscillations at 15 (p<o.ol) but not at 60 b.p.m.. discussion this study shows that airway pressures at inhibition of inspiratory activity differ before and after lung injury. thus, the airway pressures were higher after than before lung injury at a ventilatory frequency of 60 b.p.m., but the same under both conditions at a ventilatory frequency of 15 b.p.m. superimposition of oscillation lowered the airway pressures before but not after lung injury. 9-928572 123 attempts have been made to standardize animal models of bronchopulmonary dysplasia to be used in experimental studies ( 6 ) . of all species studied, only the newborn baboon has been kept on a ventilator long enough to mimic stages 2 and 4 of this condition ( 6 ) . even if damage caused by long-term ventilation may differ from that induced by chemical agents, the latter is the only alternative in most experimental situations. we therefore considered several alternatives such as oxygen injury, oleic acid injury and injury caused by xanthine oxidase. instillation of oleic acid into the lungs results in a very unstable condition, usually with progressive deterioration of lung function, and does not permit valid comparisons of different respirator settings. administration of 100% oxygen needs 4 to 5 days to cause injury with pulmonary oedema and alveolar damage. we have therefore chosen the technique used by saugstad et a1 (7) with instillation of xanthine oxidase into the airways. the adverse effect of xanthine oxidase may at least partly be explained by formation of superoxide radicals (2), and the amount of oxygen radicals formed is dependent on the hypoxanthine concentration, oxygen concentra tion and the presence of xanthine oxidase (1). we have made no attempt to characterize in detail the lung injury caused by instillation of xanthine oxidase into the airways, but have verified that the histopathological lung changes in our cats resemble those described by saugstad et a1 (7). we have also considered it important to document the fact that during the period of the study there were no undue changes in acid-base status which could have influenced the inspiratory activity 124 and its inhibition. we chose the model described by saugstad et a1 (7) in our attempt to determine whether superimposed oscillations could be of advantage after a lung injury that has some features in common with a neonatal lung disease seen after some days of treatment. during this phase the lung disease may necessitate the use of high ventilatory pressures to achieve adequate alveolar ventilation. x-ray of the lung may then show variable aeration and sometimes localized densities. our results do not support the hypothesis that superimposed oscillations could be beneficial in reducing airway pressures after lung injury induced with xanthine oxidase. this injury is non homogenous with capillary dilatation, oedema and fluid accumulation in alveolar spaces and would probably cause localized densities on chest x-ray examination. clinical results indicate, however, that superimposed oscillations are beneficial during ventilation of lungs (8) with interstitial emphysema without localized densities. conclusions 2. in cats with injured lungs: 1. oscillations superimposed on ippv do not lower the airway pressure at inhibition of inspiratory activ ity. a low arterial pc02 is needed to inhibit inspiratory activity. 125 acknowledgements this study was supported by grants from the swedish medical research council (19m-7544), stiftelsen allmanna bb:s minnes fond, the samaritan foundation and the sigurd and elsa golje memorial fund. the authors thank siemens ab, stockholm for kindly providing the servoventilator 9ooc. they are also indebted to barbro kjallstrom, harry johansson, gunno nilsson and erik ekstrom for technical assistance. we thank susanne thorell and sabina albinsson for typing assistance. references 1. 2. 3. 4. 5. 6. 7. 8. fridovich i. quantitative aspects of the production of superoxide anion radical by mild xanthine oxidase. j biol chem, 245:4053, 1970. johnson kj, fanton jc, kaplan j, ward p. in vivo damage of rat lungs by oxygen metabolites. j clin invest, 67:983, 1981. jonzon a, tantalean j, norsted t, sedin g. airway pressures during positive pressure ventilation with superimposed oscillations. upsala j med sci, 95:53 61, 1990. ng kpk, easa d management of interstitial emphysema by high frequency low positive pressure hand venti lation in the neonate. j pediatr, 95:117-118, 1979. phillip ags, oxygen plus pressure plus time: the etiology of bronchopulmonary dysplasia. pediatrics, 55 : 44-50, 1975. recommendations of the workshop on bronchopulmonary dysplasia. j pediatr, 95:920, 1979. saugstad od, hallman m, becher g, oddoy a, lium b, lachmann b. respiratory failure caused by intratrac heal saline. additive effect of xanthine oxidase. biol neonate, 54: 61-67, 1988. sedin,g., jonzon,a. kaniklides,c. superimposed high frequency oscillatory ventilation in the treatment of neonatal interstitial pulmonary emphysema. in manuscript. 126 upsala journal of medical sciences 2022, 127, e8841 http://dx.doi.org/10.48101/ujms.v127.8841 are off-target effects of imatinib the key to improving beta-cell function in diabetes? nils welsh department of medical cell biology, uppsala university, uppsala, sweden abstract the small tyrosine kinase (tk) inhibitor imatinib mesylate (gleevec, sti571) protects against both type 1 and type 2 diabetes, but as it inhibits many tks and other proteins, it is not clear by which mechanisms it acts. this present review will focus on the possibility that imatinib acts, at least in part, by improving beta-cell function and survival via off-target effects on beta-cell signaling/metabolic flow events. particular attention will be given to the possibility that imatinib and other tk inhibitors function as inhibitors of mitochondrial respiration. a better understanding of how imatinib counteracts diabetes will possibly help to clarify the pathogenic role of beta-cell signaling events and mitochondrial function, and hopefully leading to improved treatment of the disease. article history received 20 june 2022 revised 18 july 2022 accepted 27 july 2022 published 14 september 2022 keywords pancreatic beta-cells; type 1 diabetes; type 2 diabetes; imatinib; tyrosine kinase inhibitor; mitochondria introduction for some 20 years now, the small tyrosine kinase inhibitor (tki) imatinib mesylate (gleevec, sti571) has been successfully used for the treatment of chronic myeloid leukemia and other malignancies. imatinib targets certain oncogenes, such as bcrabl, leading to apoptosis and decreased proliferation of malignant cells. in this context, it exerts its effects by binding to the abl tyrosine kinase (tk) when it is in its closed and inactive conformation, which results in occlusion of the substrate site, a distorted adenosine triphosphate (atp)-binding site, and therefore, a dramatically reduced catalytic activity (1). somewhat surprisingly, imatinib also protects against diabetes in animal models (2–10), and it has now been reported that imatinib mitigates both type 1 and type 2 diabetes in humans (11–13). as imatinib inhibits many cellular tk that have not been mutated into oncogenes, for example, c-abl, arg, pdgfr, c-kit, ddr1, flt-3, c-src, and lck, a multitude of imatinib-induced effects in nontransformed cells has been observed (14–18). in addition, imatinib is known to bind and inhibit also non-tk proteins, for example, the quinone oxidoreductase-2 enzyme, the atpsensitive k+ channel, and the v-atpase (14, 19, 20). this occurs most probably by binding to atp-pockets, in which the purine/ pyrimidine moiety of imatinib fits. as such pockets are present in  many proteins, the complexity of imatinib-induced effects increases considerably. consequently, specific pivotal mechanisms by which imatinib ameliorates diabetes are not easily identified, and it is possible that many modest effects in concert result in the observed antidiabetic action. indeed, a multitude of processes, such as peripheral insulin sensitivity, autoimmunity, inflammation, autophagy, fibrosis/amyloidosis, and arteriosclerosis (21–27), have been suggested to be modulated by imatinib, thereby providing a number of possible causes that either individually or synergistically promote improved metabolic control in diabetes. however, among the many pleiotropic actions of imatinib may of particular interest be imatinib’s role as a beta-cell protective drug, promoted by a direct effect on beta-cell signaling, and not via peripheral or indirect effects, a notion supported by findings both in vitro (2, 3, 28–33) and in vivo (5, 6, 13, 32). as beta-cells have a decisive role in glucose homeostasis, and loss of beta-cell function and survival is a crucial step in the pathogenesis of both type 1 and type 2 diabetes, it is tempting to speculate that this beta-cell protection effect could be an explanation of particular importance to the antidiabetes actions of imatinib. therefore, this short review will focus on possible mechanisms, both tk-dependent and tkindependent, by which imatinib and other tkis modulate signaling events and metabolic flow in insulin producing cells, and how this improves beta-cell function and survival. imatinib-targeted tks and their down-stream effectors in beta-cell survival at first glance, it appears counterintuitive that a drug designed to promote apoptosis (of cancer cells) also promotes cell survival and improved function (of beta-cells), but it is important to note that non-malignant cells do not express imatinib-sensitive oncogene proteins and are therefore not highly prone to apoptosis when exposed to the drug. instead, imatinib inhibits contact nils welsh nils.welsh@mcb.uu.se © 2022 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. review article http://dx.doi.org/10.48101/ujms.v127.8841 mailto:nils.welsh@mcb.uu.se http://creativecommons.org/licenses/by/4.0/ 2 n. welsh in primary cells a multitude of non-oncogenic tks, among which c-abl has received particular attention. this non-receptor and multifunctional tk (34) is inhibited by imatinib with an inhibitor concentration 50 (ic 50 ) of 100–300 nanomolar, and in other cells than beta-cells, it is well known that c-abl activation in response to different types of stress promotes apoptosis, an event which  can be inhibited by treatment with imatinib (35–37). c-abl-induced apoptosis seems to be achieved via interactions with, for example, atm (38), tip1 (39), parkin (40), stress-activated protein kinases (41), p53 (42), p73 (43), and nf-kappab (44). these proteins play important roles in tumor suppression (atm, p53, p73), microtubule cytoskeletal organization (tip1), protein ubiquitination (parkin), and inflammation (nf-kappab). it was also observed that c-abl is partially localized to the endoplasmic reticulum (er), and that er stress promotes c-abl activation and translocation to mitochondria where apoptotic signals leading to caspase 3 activation are initiated (45). much less is known about the role of c-abl in insulin-producing beta-cells, but the c-abl-activation seems to promote apoptosis/ dysfunction in this cell type as sirna-mediated c-abl knockdown has been reported to reduce cell death rates of mouse islet cells (3) and the mouse insulin producing cell line βtc-6 (46) or improve beta-cell function of the mouse insulin producing cell line nit-1 (29) and the rat insulin producing cell line ins-1 as well as the human insulin producing cell line endoc-βh1 (30). some beta-cell c-abl down-steam targets, direct or indirect, have been inferred via sirna and co-immunoprecipitation experiments, namely, the phosphatidylinositol-3, 4, 5-triphosphate (pip 3 ) phosphatase inositol polyphosphate phosphatase like 1 (inppl1) and the phosphatidylinositol-3-kinase (pi3k) pathway, using endoc-βh1 cells and the mouse insulin producing cell line min6 (28), the transcription factor nkx2.2 (29), pdgfr/lrp1-induced erk phosphorylation (30), the estrogen-related nuclear receptor gamma (errgamma), using ins-1 cells (47), and ire-1alpha, one of the three arms of the unfolded protein response (upr), using ins-1 cells and mouse and human islet cells (32). in the first case, we observed that c-abl co-immunoprecipitated with inppl1, and that the c-abl/inppl1 interaction was associated with decreased pi3k/pip 3 signaling (28). pip3-signaling is known to reduce cellular apoptosis. consequently, imatinib inhibited the c-abl-induced suppression of the pi3k pathway, and the resulting imatinib-induced increase in akt phosphorylation fits well with improved beta-cell survival, for example, via increased phosphobad or beta-catenin signaling (28). second, c-abl downregulation resulted also in increased expression of nkx2.2, a positive regulator of insulin gene expression, which was associated with higher levels of the glucose transporter glut-2 (29). these findings support a role of imatinib as a stimulator of beta-cell function, in addition to merely maintaining survival. the mechanisms by which c-abl downregulation resulted in increased nkx2.2 were, however, not further characterized. third, c-abl appears to reduce pdgfr/lrp1-induced erk phosphorylation, which could explain the pronounced activation of erk that occurs in response to imatinib at basal conditions (30). erk is known to positively regulate both beta-cell mass and function (48), which concurs with the finding that an erk inhibitor partially blocked imatinib-induced protection against beta-cell death (30). fourth, using the c-abl inhibitor gnf2 on ins1 cells, it was demonstrated that basal c-abl activity may suppress the expression of the orphan nuclear receptor errgamma, and that this leads to reduced glutaminase 1 activity and a weaker glutathione-dependent defense against oxidative stress (48). fifth, activated c-abl seems to directly bind to and activate ire1alpha in beta-cells, leading to xbp1 splicing, er stress, and beta-cell death in nod mice (32). therefore, imatinib, acting as a c-abl inhibitor, protected against beta-cell er stress and subsequent cell death by blocking the c-abl/xbp1 interaction (32). on the other hand, it is noteworthy that imatinib also causes er stress in cardiomyocytes via a c-abl-independent pathway leading to cardiomyopathy, a known complication to imatinib therapy (49). furthermore, it has recently been demonstrated that c-abl promotes a type of ire1alpha activation that mediates a dna-damage response, rather than the canonical upr that occurs in response to er stress (50), indicating that the complex role of imatinib and c-abl in er stress may involve opposite actions. nevertheless, it appears that c-abl inhibition may positively modulate different beta-cell pro-survival and profunction events. this is in line with the notion that c-abl is a modulating, rather than a master switch type, tk that fine tunes the cellular outcome in response to stress (34). on the other hand, it cannot be excluded that hitherto unknown counterregulatory events become activated by imatinib-induced tki inhibition, and that such putative events may blunt or even nullify the pro-survival signals. other classical imatinib tk targets, such as c-kit and the pdgf receptor, have received less attention than c-abl when it comes to protection against beta-cell death and diabetes, but one report states that c-kit is not necessary for imatinib-induced protection against diabetes in nod mice (8). imatinib-targeted non-tks and their role in beta-cell survival as micromolar concentrations of imatinib are required for protection against beta-cell death and dysfunction in vitro, and as only high nanomolar concentrations are required for inhibition of the classical imatinib targets in vitro, it is possible that non-tk targets with ic 50 s in the micromolar range, i.e. offtargets, mediate a substantial part of the protective actions. indeed, it has been observed that imatinib, in min6 cells, binds to and inhibits the atp-sensitive k+-channel with an ic 50 of 9.4 micromolar (19), probably via binding to the atp-pocket. in the same study, it was also observed that the non-c-abl inhibitor sunitinib promoted the same effect, suggesting that the effects were not mediated by c-abl. along the same line, we recently observed that both imatinib and sunitinib moderately inhibited complex i and ii of the respiratory chain at micromolar concentrations when supplemented to isolated rat kidney mitochondria (33). the molecular details of imatinib binding to the respiratory complexes have not been characterized, but both complexes have nad-binding pockets, and it is therefore possible that the purine moiety of many tkis will fit as well. are off-target effects 3 indeed, using isolated rat liver mitochondria, it was observed that 18 out of 31 tkis promoted toxic effects to the mitochondria, suggesting that the inhibition of respiration is a common trait of tkis (51). in myocardial cells, it was observed in vitro that sunitinib induced mitochondrial inhibition at 5–10 micromolar concentrations, and that this resulted in oxidative stress and apoptosis (52), and in vivo, it has been observed that respiration was reduced, oxidative stress increased, and mitochondrial proliferation decreased (53). in hek293 cells, sorafenib also has been demonstrated to inhibit respiration (54). thus, it appears that multiple tkis promote mitochondrial inhibition, and that this could be more or less a class effect (figure 1). imatinib-induced inhibition of respiration in human beta-cells (endoc-βh1 and human islets) resulted in lowered respiration rates, a decreased atp/amp ratio and amp-activated protein kinase (ampk) activation (33). interestingly, the ampk activator aicar mimicked, whereas the ampk inhibitor compound c counteracted the imatinib effect on survival (33), suggesting that imatinib acts on beta-cell survival, to some extent, via ampk activation. ampk is activated in response to lowered atp/amp ratios and rescues cells by increasing autophagy, catabolism, and energy conservation (55), and we have previously observed that ampk activation in endoc-βh1 cells promotes human beta-cell survival in response to pro-inflammatory cytokines, which are proapoptotic factors able to reduce beta-cell atp-levels (56). it may be that ampk mediates its protective actions, at least in part, via the downregulation of thioredoxin interacting protein (txnip) (33), a protein that is known to promote beta-cell dysfunction and death (57) (figure 1). imatinib has recently been found to preserve beta-cell function in adults with recent-onset type 1 diabetes resulting in an initial lowering of the exogenous insulin dose as well as hba 1c levels, an effect not associated with any clear alterations in immunity (13). the beneficial effects of imatinib were lost upon discontinuation of the treatment, suggesting that imatinib needs to be administered in the long term in order to protect beta-cells from dysfunction (13). in type 2 diabetes, imatinib also promotes a glucose-lowering effect (11, 12). interestingly, not only imatinib but also other tkis have been reported to promote antidiabetes effects. in different case reports, erlotinib, dasatinib, sunitinib, and sorafenib lowered the blood glucose in patients suffering from both cancer and diabetes (58–61). as these tkis have different tk target profiles, it can be hypothesized that the antidiabetic actions result from mitochondrial inhibition, or some other ‘off-target’ effect, rather than from inhibition of specific tks with low ic 50 values. further support for this hypothesis may be provided by a recent report stating that dasatinib, a bcr-abl tki, which is also considered to be a potent senolytic agent when combined with quercetin (62), promotes more pronounced antidiabetic actions than imatinib in humans with type 2 diabetes (63). interestingly, it has recently been shown that dasatinib in myotubes inhibits mitochondrial function more efficiently than imatinib at equimolar concentrations (64). conclusions and future directions increasing evidence suggests that imatinib and other tkis improve beta-cell function and survival, and although events via c-abl signaling cannot be ruled out, it may be that this occurs mainly via ‘off-target’ effects that are tk-independent. therefore, it is warranted to further characterize mitochondrial ‘off-target’ effects of tkis, and to compare them with those of other mitochondria-targeting drugs, for example, metformin (65) or the mitochondrial k(atp)-channel opener nnc 55-0321 (66). finally, it is noteworthy that the moderately efficient antidiabetes agent imatinib protected against recent-onset type 1 diabetes equally well or even better than many immune-targeting therapies (13), which raises the interesting question whether the more potent mitochondrial inhibitor dasatinib would perform even better as a treatment for type 1 diabetes. declaration of interest the author has no conflicts of interest that are relevant to the content of this article. funding this work was supported in part by the swedish diabetes association, the family ernfors fund, barndiabetesfonden, and exodiab. notes on contributor nils welsh, md/phd, uppsala university, department of medical cell biology, uppsala, sweden. orcid nils welsh https://orcid.org/0000-0001-5926-5407 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investigates the predictive value of the systemic immune-inflammation index (sii), which was calculated as platelet × neutrophil/lymphocyte ratio, for all-cause mortality in patients with hypertrophic cardiomyopathy (hcm). methods: a total of 360 hcm patients were enrolled. they were divided into three groups based on the tertiles of baseline sii. the association between sii and all-cause mortality was analyzed. results: there were 53 hcm patients who died during a mean follow-up time of 4.8 years (min: 6 days and max: 10.8 years), and the mortality rate was 3.0 per 100 person years. the cumulative mortality rate was significantly different among the three tertiles of sii (p = 0.004), and the mortality rate in tertile 3 was much higher than that in the first two tertiles. in reference to tertile 1, the fully adjusted hazard ratios of all-cause mortality were 1.02 for the tertile 2 (95% confidence interval [ci]: 0.45–2.31, p = 0.966) and 2.31 for tertile 3 (95% ci: 1.10–4.87, p = 0.027). no significant interactions between sii and other variables were observed during subgroup analysis. the discriminative power was better for mid-term outcome than that for shortterm or long-term outcomes. sensitivity analyses including patients with normal platelet and white blood cell count have revealed similar results. conclusion: sii was a significant risk factor for all-cause mortality in hcm patients. however, the discriminative power was poor to moderate. it could be used in combination with other risk factors in mortality risk stratification in hcm. article history received 4 august 2021 revised 29 september 2021 accepted 20 october 2021 published 3 december 2021 keywords systemic immuneinflammation index; all-cause mortality; hypertrophic cardiomyopathy; inflammation; risk stratification introduction hypertrophic cardiomyopathy (hcm) is the most frequent genetically transmitted heart disease with an estimated prevalence of 1:500 to 1:200 (1). hcm is characterized with a wide range of clinical features, which ranged from completely asymptomatic and normal lifespan to deleterious arrythmia, sudden cardiac death (scd), severe thromboembolism, and end-stage heart failure (hf), resulting in hcm-related premature death (2–3). although the overall prognosis is relatively good when managed in line with current clinical practice guidelines (4–5), excess mortality for hcm patients was still observed in different studies (6–8). therefore, the desire to better risk stratify patients who were at high risk of an adverse outcome is an essential component in disease management. previous studies have revealed the existence of both local  and systemic inflammation in hcm patients (7–9). also, several studies indicated that markers of inflammation predicted adverse outcomes in hcm, such as high-sensitivity c reactive protein (crp) (7), monocyte to high density lipoprotein cholesterol ratio (m/hdl-c) (8), and neutrophil to lymphocyte ratio (nlr) (10). a novel immune and inflammation index, namely, systemic immune-inflammation index (sii), which is calculated from platelet, neutrophil, and lymphocyte counts, has been examined as a prognostic factor for clinical outcomes in cancer patients (11–12) and in patients with cardiovascular diseases, such as coronary artery disease (13–14), hypertension (15), pulmonary embolism (16), and acute ischemic stroke (17). however, to date, the predictive ability of sii for mortality has not been reported in hcm. the present study investigated the prognostic value of sii for mortality in hcm patients from a tertiary referral center. methods study patients from december 2008 to november 2018, 537 patients with a diagnosis of hcm were consecutively enrolled at west china hospital of sichuan university, chengdu, china. all patients original article contact sen he hesensubmit@163.com supplemental data for this article can be accessed here. © 2021 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. http://dx.doi.org/10.48101/ujms.v126.8124 mailto:hesensubmit@163.com http://dx.doi.org/10.48101/ujms.v126.8124 http://creativecommons.org/licenses/by/4.0/ 2 z. wang et al. underwent 2d transthoracic echocardiography examinations by standard techniques, and the diagnosis of hcm was based on the presence of increased left ventricular (lv ) wall thickness (≥15 mm) that was not solely explained by abnormal loading conditions (18). based on the inclusion and exclusion criteria (figure 1), a total of 360 adult hcm patients were finally included. this study was conducted according to the principles of the declaration of helsinki and was approved by the biomedical research ethics committee, west china hospital of sichuan university (approval number: 2019-1147). this study has been registered on the website of chinese clinical trial registry (http://www.chictr.org.cn/showproj.aspx?proj=48695). an informed consent was waived due to the retrospective nature of the study. other detailed information has been reported in three recently published studies (19–21). clinical evaluations baseline characteristics were collected from medical records by  experienced physicians, and these characteristics mainly included medical history, therapy, 12-lead echocardiogram, doppler echocardiography, and peripheral blood parameters. a twice-entry method was used for data entry. when values of the two entries were consistent, they would enter the database; otherwise, the raw data would be checked. platelet, neutrophil, and lymphocyte were tested using a sysmex xn-9000 analyzer (sysmex corporation, kobe, japan), and the values were collected at the time of hospital admission. in this system, normal ranges of platelet count, white blood cell count (wbcc), neutrophil count, and lymphocyte count are 100–300*109/l, 4–10*109/l, 1.8–6.3*109/l, and 1.1–3.2*109/l, respectively. sii was calculated as total peripheral platelets count (p) × neutrophil-to-lymphocyte ratio (n/l) (sii = p × n/l ratio) (22). study endpoints the endpoint was all-cause mortality, which included hcmrelated death, other cardiac death (e.g. myocardial infarction), non-cardiac death, and unexplained death. specifically, hcmrelated death was defined as a composite of hf-related death, stroke-related death (scd), and other specific hcm-related death. follow-up was carried out via medical records or telephone contact with the patients themselves and/or referring relatives. all patients were followed from the first evaluation up to the endpoint or the most recent evaluation. statistical analysis to quantify, in a simple form, the relationship between sii and all-cause mortality, the patients were divided into three groups according to baseline sii, which were categorized separately as follows: tertile 1 (<246.04×109/l), tertile 2 (246.04–424.90×109/l), and tertile 3 (≥424.91×109/l). for continuous variables, data were presented as median with interquartile range (iqr) since all variables were skewed distribution based on shapiro–wilk test. for categorical variables, data were presented as number (percentage). kruskal–wallis test, chi-square test, or fisher exact  test were used, as appropriate, to compare baseline characteristics among the three groups. the kaplan–meier method was used to evaluate the association between sii and all-cause mortality, and log-rank tests were used for comparisons. several cox proportional hazard regression models were constructed to assess the prognostic value. variables for inclusion were carefully chosen to ensure parsimony of multivariate models. two multivariate models were constructed. model 1, the basic model, adjusted for demographic data, including age and gender. model 2, the final model, baseline variables that were considered to be related to the study endpoint based on previous meta-analysis were pre-specifed, which included nyha function class, syncope/presyncope, maximal lv wall thickness (mwt), and resting lv outflow track obstruction (lvoto) (23), and were foreced into the final model. besides, variables that showed a significant relationship with mortality in univariate analysis (p < 0.05) were entered into the multivariate model as well and  then were sought using a backward stepwise modeling approach (p = 0.05 for inclusion and p = 0.10 for exclusion) to include some of them in the final model. additionally, stratified analysis was condcuted to assess the consistency of association between sii and all-cause mortality in various subgroups, and their interaction effect was tested. meanwhile, the robustness of  the main results was assessed by calculation of e-values. e-values could assess the potential for unmeasured confounding figure 1. study flow diagram. http://www.chictr.org.cn/showproj.aspx?proj=48695 sii and mortality in hcm 3 between sii and all-cause mortality, and it quantifies the required magnitude of an unmeasured confounder that could negate the observed association between sii and all-cause mortality (24). furthermore, a time-dependent receiver operating characteristic curve was generated to evaluate the discriminative power of sii in predicting all-cause mortality over time. finally, we assessed the relation between sii and all-cause mortality in patients with normal platelet and wbcc as a sensitivity analysis in case some other unknown reasons affecting the components of sii had not been ruled out. the statistical analyses were performed with the use of r  software, version 4.1.0 (r project for statistical computing). for  all statistical analyses, a two-sided p-value of 0.050 was considered statistically significant. results baseline characteristics the present study comprised 360 patients (male, 53.89%) with a median age of 56.00 (iqr, 44.00–66.00) years, and sii ranged from 6.55 to 2,856.84×109/l (median, 336.95×109/l; iqr, 216.57– 502.28×109/l). according to the tertiles of baseline sii, there were 119 patients in the lowest tertile, 118 in the second tertile, and 123 in the highest tertile. the levels of platelet count, wbcc, neutrophil count, and monocyte count significantly increased across the sii tertiles. the prevalence of hypertension and the use of aspirin significantly also increased across the sii tertiles. these patients with higher sii had significantly lower levels of hemoglobin and lymphocyte count, as well as lower prevalence of palpitation. other detailed data about baseline characteristics are presented in table 1. correlation sii negatively correlated with hemoglobin (r = −0.23, p < 0.001) and lymphocyte count (r = −0.29, p < 0.001) and positively correlated with platelet count (r = 0.44, p < 0.001), wbcc (r = 0.57, p < 0.001), neutrophil count (r = 0.73, p < 0.001), and monocyte count (r = 0.26, p < 0.001). these correlations were somewhat weak to moderate. besides, sii showed no correlation with creatinine, uric acid, blood lipid parameters, and echo data (supplementary table 1). study endpoints during a follow-up period of 1,744.0 person-years (pys) (median, 4.8 years; iqr, 2.8–6.8 years), there were 53 (14.7%) all-cause mortalities with a mortality rate of 3.0 (95% confidence interval [ci]: 2.2–3.8) per 100 pys. the specific causes of deaths were as follows: 14 hf-related deaths, 11 stroke-related deaths, 8 scds, 2 hcm-related postoperative deaths, 1 other cardiac death, and 12 non-cardiac deaths. the cause of death could not be determined in five patients. association between sii and mortality the all-cause mortality rates were 2.0 (95% ci: 0.9–3.1), 2.2 (95% ci: 1.0–3.3), and 5.1 (95% ci: 3.3–7.0) per 100 pys in the tertile 1, tertile 2, and tertile 3, respectively (table 2). the cumulative mortality rate was significantly different among the three tertiles of sii (log-rank p = 0.004, figure 2), and the mortality rate in tertile 3 was much higher than that in the first two tertiles. univariate cox regression analysis indicated that sii was a significant risk factor for future all-cause mortality (table 2). other variables that could predict all-cause mortality have been shown in supplementary table 2. in reference to tertile 1, fully adjusted hrs were 1.02 for tertile 2 (95% ci: 0.45–2.31, p = 0.966) and 2.31 for tertile 3 (95% ci: 1.10–4.87, p = 0.027) (table 2). due to the similar mortality rates in the first two tertiles, we combined them into one to perform the stratified analysis (tertiles 1–2 vs. tertile 3). it was found that the mortality risk was consistently higher in tertile 3 than in tertiles 1–2 in all  subgroups. no interaction effect was observed between sii  and  other variables for mortality prediction (figure 3), which suggested that sii was an independent predictor for allcause mortality in hcm patients, and other variables were not confounders or effect modifier. in addition, the e-value estimates for the effects of sii on all-cause mortality were 4.05 (lower limit of ci, 1.43) for tertile 3. this suggested that the main findings should be robust, unless an unmeasured confounder existed with a higher relative risk than the above-mentioned e-values. discriminative power of sii for all-cause mortality we assessed the discriminative power of sii for all-cause mortality at different timepoints. the time-dependent area under curve (auc) at 1-year follow-up was 0.580. with time prolongation, auc increased to a maximum of 0.722 at 5.4-year follow-up and then gradually decreased to 0.517 at 10-year follow-up, indicating a dynamic change of the discriminative ability (figure 4). sensitivity analysis sensitivity analysis including only patients with normal platelet and wbcc (n = 269, all-cause mortality = 39) revealed similar results with the main analyses. kaplan–meier analysis demonstrated significantly higher incidence of all-cause mortality across the three tertiles (log-rank p = 0.018, supplementary figure 1). with tertile 1 as reference, unadjusted hrs were 1.38 for tertile 2 (95% ci: 0.54–3.51, p = 0.495) and 2.81 for tertile 3 (95% ci: 1.24–6.36, p = 0.013). although the association between sii and all-cause mortality was reduced after adjustment for confounders, it did not change materially. fully adjusted hrs were 1.27 for tertile 2 (95% ci: 0.45–3.55, p = 0.650) and 2.22 for tertile 3 (95% ci: 0.92–5.35, p = 0.074) when compared to tertile 1. time-dependent aucs showed similar findings with the main analyses (at 1-year: 0.525, at 5.4-year: 0.709, and at 10-year: 0.500) (supplementary figure 2). 4 z. wang et al. discussion in this retrospective cohort study of patients with hcm, we evaluated the ability of sii, a novel inflammatory biomarker, to predict the all-cause mortality. higher sii was significantly associated with increased risk of all-cause mortality. the discriminative power in predicting mid-term outcome was better than that for short-term or long-term outcomes. table 1. baseline characteristics. variables all (n = 360) sii, tertile 1 (n = 119) sii, tertile 2 (n = 118) sii, tertile 3 (n = 123) p gender: male 194 (53.89%) 71 (59.66%) 58 (49.15%) 65 (52.85%) 0.257 age (years) 56.00 [44.00, 66.00] 56.00 [44.00, 64.50] 55.00 [44.25, 69.00] 56.00 [45.00, 66.00] 0.844 family history of hcm 32 (8.89%) 12 (10.08%) 12 (10.17%) 8 (6.50%) 0.519 family history of scd 14 (3.89%) 2 (1.68%) 4 (3.39%) 8 (6.50%) 0.147 myha iii-iv 59 (16.39%) 18 (15.13%) 15 (12.71%) 26 (21.14%) 0.189 symptoms dyspnea 204 (56.67%) 71 (59.66%) 60 (50.85%) 73 (59.35%) 0.298 chest pain 209 (58.06%) 72 (60.50%) 72 (61.02%) 65 (52.85%) 0.352 syncope/presyncope 120 (33.33%) 42 (35.29%) 48 (40.68%) 30 (24.39%) 0.024 palpitation 143 (39.72%) 62 (52.10%) 44 (37.29%) 37 (30.08%) 0.002 medical history prior te 15 (4.17%) 2 (1.68%) 5 (4.24%) 8 (6.50%) 0.159 vascular disease 29 (8.06%) 9 (7.56%) 10 (8.47%) 10 (8.13%) 0.967 hypertension 116 (32.22%) 26 (21.85%) 43 (36.44%) 47 (38.21%) 0.012 diabetes mellitus 29 (8.06%) 6 (5.04%) 9 (7.63%) 14 (11.38%) 0.190 atrial fibrillation 62 (17.22%) 22 (18.49%) 21 (17.80%) 19 (15.45%) 0.805 therapy aspirin 72 (20.00%) 11 (9.24%) 27 (22.88%) 34 (27.64%) 0.001 clopidogrel 21 (5.83%) 8 (6.72%) 4 (3.39%) 9 (7.32%) 0.378 warfarin 38 (10.56%) 15 (12.61%) 10 (8.47%) 13 (10.57%) 0.585 statin 110 (30.56%) 30 (25.21%) 41 (34.75%) 39 (31.71%) 0.265 beta blocker 274 (76.11%) 92 (77.31%) 99 (83.90%) 83 (67.48%) 0.011 acei/arb 72 (020.00%) 24 (20.17%) 23 (19.49%) 25 (20.33%) 0.985 intervention of obstruction 0.519 none 313 (86.94%) 100 (84.03%) 101 (85.59%) 112 (91.06%) alcohol septal ablation 41 (11.39%) 16 (13.45%) 15 (12.71%) 10 (8.13%) septal myectomy 6 (1.67%) 3 (2.52%) 2 (1.69%) 1 (0.81%) devices 0.460 none 320 (88.89%) 101 (84.87%) 108 (91.53%) 111 (90.24%) pacemaker 14 (3.89%) 5 (4.20%) 4 (3.39%) 5 (4.07%) icd 26 (7.22%) 13 (10.92%) 6 (5.08%) 7 (5.69%) blood index hgb (g/l) 139.00 [128.00, 151.00] 141.00 [129.00, 152.00] 140.50 [132.00, 151.75] 134.00 [120.00, 148.00] 0.005 platelet count (109/l) 146.00 [112.00, 185.00] 112.00 [88.50, 135.00] 153.50 [124.00, 185.50] 177.00 [148.50, 224.50] <0.001 wbcc (109/l) 6.26 [5.15, 7.44] 5.44 [4.64, 6.37] 6.31 [5.22, 7.24] 7.37 [5.98, 8.66] <0.001 lymphocyte count (109/l) 1.69 [1.36, 2.08] 1.85 [1.52, 2.26] 1.81 [1.47, 2.17] 1.42 [1.07, 1.72] <0.001 neutrophil count (109/l) 3.78 [3.05, 4.90] 2.93 [2.44, 3.51] 3.77 [3.21, 4.60] 5.22 [4.03, 6.58] <0.001 monocyte count (109/l) 0.35 [0.28, 0.46] 0.32 [0.26, 0.39] 0.35 [0.27, 0.46] 0.41 [0.30, 0.53] <0.001 creatinine (μmol/l) 80.00 [67.00, 92.65] 80.00 [66.20, 87.85] 80.00 [67.32, 90.92] 80.40 [68.00, 95.00] 0.454 uric acid (μmol/l) 362.50 [299.08, 433.48] 357.00 [301.75, 421.50] 366.50 [308.25, 433.50] 367.10 [291.20, 446.15] 0.723 tg (mmol/l) 1.28 [0.97, 1.96] 1.20 [0.96, 1.87] 1.46 [1.01, 2.16] 1.24 [0.90, 1.72] 0.087 hdl-c (mmol/l) 1.27 [1.04, 1.54] 1.30 [1.11, 1.54] 1.23 [1.01, 1.54] 1.28 [1.02, 1.52] 0.339 ldl-c (mmol/l) 2.44 [1.87, 2.94] 2.56 [1.81, 3.07] 2.37 [1.95, 2.85] 2.37 [1.83, 2.92] 0.395 echocardiographic parameters lvedd (mm) 43.00 [40.00, 47.00] 43.00 [39.50, 46.00] 43.00 [40.00, 47.75] 43.00 [39.00, 47.50] 0.566 la diameter (mm) 40.00 [36.00, 45.00] 40.00 [36.00, 45.00] 40.00 [36.00, 45.00] 40.00 [35.00, 45.00] 0.800 mwt (mm) 19.00 [17.00, 22.00] 19.00 [17.00, 22.00] 19.00 [16.00, 22.00] 19.00 [16.50, 22.00] 0.540 lvef (%) 69.00 [64.00, 73.00] 69.00 [64.50, 73.50] 68.00 [64.00, 71.00] 69.00 [63.00, 73.00] 0.117 resting lvotg ≥30 mm hg 156 (43.33%) 54 (45.38%) 55 (46.61%) 47 (38.21%) 0.362 values are median (iqr) or n (%). sii: systemic inflammatory-immune index; hcm: hypertrophic cardiomyopathy; scd: sudden cardiac death; nyha: new york heart association; te: thrombo-embolic event; ace: angiotensin-converting enzyme; arb: angiotensin receptor blocker; icd: implantable cardioverter defibrillator; hgb: hemoglobin; wbcc: white blood cell count; tg: triglyceride; hdl-c: high-density lipoprotein cholesterol; ldl-c: low-density lipoprotein cholesterol; lvedd: left ventricular end-diastolic dimension; la: left atrial; mwt: maximal left ventricular wall thickness; lvef: left ventricular ejection fraction; lvotg: left ventricular outflow tract gradient. sii and mortality in hcm 5 sensitivity analysis of patients with normal platelet and normal wbcc demonstrated similar results with the main analysis. our study suggested that sii might be a potentially useful noninvasive predictor for mortality in hcm. sii is determined by three components, namely, neutrophils, lymphocytes, and platelets, which reflect host inflammatory and immune status. it was initially proposed by hu et al. as a powerful prognostic indicator for poor outcome in patients with hepatocellular carcinoma (22). after that, series of studies regarding the association between sii and clinical outcome in a variety of neoplastic diseases have been conducted, and all studies consistently confirmed the previous finding (11–12). due to high levels of platelets and neutrophils while low level of lymphocytes, a higher sii indicates an elevated inflammatory and a suppressed immune response, both of which have played decisive roles in the pathogenesis, progression, and metastasis of neoplastic diseases (25, 26). from this point of view, increased sii is biologically plausible to associate with adverse prognosis for patients with neoplastic diseases. it is widely acknowledged that the inflammation has played a central role in cardiovascular diseases, especially in coronary artery disease. previous studies have shown that sii is a potential indicator for clinical endpoints for patients with coronary artery disease (13, 14). the same was found in patients with hypertension (15), acute ischemic stroke (17), and chronic hf (27). however, no previous study has discussed the predictive value of sii in hcm. in the present cohort study of hcm, we firstly proved its predictive value for all-cause mortality, which consisted of scd, hf-related death, stroke-related death, cancer-related death, etc. the precise mechanisms of poor prognosis for some hcm patients have not been fully elucidated, but the chronic inflammation is possibly involved. myocardial tissue changes, such as inflammatory cell infiltration and neutrophil extracellular traps formation due to myocyte disarray in the early stage, platelet activation, microvascular thrombosis and fibrosis pathway activation in the intermediate stage, myocardial fibrosis, and remodeling in the late phase, might be related to the phenotypic variability of hcm (28). also, some systemic inflammatory biomarkers were also found to be significantly associated with adverse outcomes in hcm. in a retrospective study of 490 hcm patients, zhu et al. demonstrated that patients with higher levels of plasma highsensitivity c-reactive protein were at higher risk of all-cause mortality and cardiovascular death (7). in another study, ekizler et al. showed that hcm patients with higher m/hdl-c had higher risk of malignant arrhythmic events and cardiovascular death (8). in a prospective observational study, ozyilmaz et al. revealed that a higher nlr was associated with a higher 5-year risk of scd in patents with hcm (10). in corroboration with that, our previous studies also found a relationship between nlr (29), red blood cell distribution width (30), and all-cause mortality, and the present study further extended the inflammatory biomarkers into sii. in addition, we observed that the discriminative power of sii for all-cause mortality was not stable at different timepoints, and it was better for mid-term outcome than that for short-term or long-term outcomes. whether this dynamic change is a chance finding or not, it will require further studies. sii is a biomarker comprehensively reflecting the inflammatory and immune mechanism. however, the discriminative power was moderate table 2. associations of sii with all-cause mortality. sii tertile 1 sii tertile 2 sii tertile 3 no. of patients (n) 119 118 123 endpoints (n) 12 13 28 follow-up (pys) 598.7 597.7 547.6 mortality rates (95% ci)* 2 [0.9, 3.1] 2.2 [1, 3.3] 5.1 [3.3, 7] unadjusted hrs (95% ci), p 1 1.09 [0.50, 2.40], 0.822 2.53 [1.29, 4.98], 0.007 adjusted hrs (95% ci), p model 1 1 1.00 [0.45, 2.20], 0.998 2.52 [1.28, 4.96], 0.008 model 2 1 1.02 [0.45, 2.31], 0.966 2.31 [1.10, 4.87], 0.027 model 1 with adjustment for age and gender. model 2 with adjustment for age, gender, syncope/presyncope, dyspnea, nyha iii-iv, uric acid, tg, monocyte count, la diameter, lvedd, lvef, mwt, and resting lvoto. pys: person-years; ci: confidence interval; hrs: hazard ratios; other abbreviations as in table 1. per 100 pys. figure 2. kaplan–meier analysis showing cumulative all-cause mortality by baseline sii tertiles. 6 z. wang et al. at  best, highlighting the difficulty of risk stratification in hcm patients if based on only a single risk factor due to the heterogeneity of hcm per se. in the present study, the all-cause mortality rate was 14.7%, and the cardiovascular mortality rate was about 10.0%. in another hcm cohort from fuwai hospital, bejing, china, the all-cause mortality and cardiovascular mortality rates were 7.8 and 6.1%, respectively (7). in a multiple european centers-based study, the authors focused on a composite study endpoint, which included scd or equivalent end point, hf-related death or equivalent end point (heart transplant), other cardiovascular causes, and other unknown causes. the composite study endpoint occurred in 721 hcm patients with a prevalence of 14.7%, and the cardiovascular mortality rate was approximately 10.0% (6). in another study, based on two american centers, marron et al. showed that the event rate for all-cause mortality figure 3. stratified analyses of all-cause mortality. note: each stratification adjusted for age, gender, dyspnea, uric acid, tg, monocyte count, lvedd, la diameter, and lvef, except the stratification factor itself. the p-value for interaction represents the likelihood of interaction between variable and sii tertiles. abbreviations as in tables 1 and 2. †adjusted hr = 0. sii and mortality in hcm 7 and hcm-related mortality was 8.0 and 4.0%, respectively (5). for a turkey hcm cohort, the all-cause mortality rate was 5.8%,  and the cardiovascular death rate was 3.6%. however, there was a high prevalence (10.3%) of malignant arrhythmic events, including an occurrence of scd, sustained ventricular tachycardia/ventricular fibrillation, or implantable cardioverter– defibrillator discharge in their study (8). evidently, there are still discrepancies with regard to the poor outcomes across different studies. although there are different baseline characteristics and ethnicities in these studies that might partially be the cause, the generally poor prognosis is still present. notably, most of the hcm patients in our study had wellpreserved systolic function with the median value of lv ejection fraction (lvef) at 69%. however, hcm is characterized by normal to hyperdynamic lvef. only a small portion of patients (4–9%) would develop systolic dysfunction with lvef < 50% and progressed into end-stage hcm (31). in our study, the prevalence of end-stage hcm was 4.4%. there were some slight differences with regard to the values of echocardiographic parameters, including lv end-diastolic diameter, left atrial diameter, and mwt and lvef between our study and previous studies (7, 8, 10), which might be caused by different ethnicities, different gender composition, and the variety of comorbidities. but, all supported the characteristics of non-dilated ventricles in hcm. additionally, we did not find correlations between sii  and those aforementioned echocardiographic diameters, and  those data showed homogeneity across the three sii tertiles. sii represents a systemic inflammatory index. there is limited data about how sii would directly influence cardiac structure and cardiac function. a previous study, investigating the relationships between several inflammatory biomarkers and myocardial fibrosis, systolic and diastolic functions, and the degree of cardiac hypertrophy in hcm patients, also found no  correlations between systemic inflammation and systolic function. however, only some circulating inflammatory markers were associated with myocardial fibrosis (e.g. interleukin-6, interleukin-4, and monocyte attractant protein-1), the degree of hypertrophy (e.g. fractalkine), or diastolic dysfunction (e.g.  interferon-γ-inducible protein 10, interleukin-10, and transforming growth factor-β1) (32). those inflammatory markers belong to cytokines/chemokines, which may mediate the pathological process of cardiac hypertrophy and fibrosis in hcm directly. therefore, the correlation between the systemic inflammatory index derived from peripheral blood cells and the cardiac structure and cardiac function needs further studies. the present study revealed the predictive value of sii for allcause mortality in hcm patients. sii is a readily available inflammatory biomarker that could be easily obtained from complete blood cell test. patients with higher sii might be given a closer clinical monitoring and more aggressive therapy. our study, however, has certain limitations. first, the hcm patients enrolled in our study come from a single tertiary referral center, and the study was carried out retrospectively. besides, the relatively small sample size might reduce the statistical power.  second, we focused on the all-cause mortality but not  the hcm-related mortality due to the limited number of specific causes. the latter one might better reflect the clinical importance of sii in hcm, but we can still get some useful information. third, although we have adjusted for certain potential confounding factors, some other well-established risk factors, such as non-sustained ventricular tachycardia and b-type natriuretic peptide, were not included due to incomplete data, which might reduce the strength of our findings. but, we have collected most of the variables as much as possible, which had showed significant predictive value for all-cause mortality and cardiovascular mortality in hcm patients in previous studies and included them in a multivariate model for adjustment. fourth, our study only demonstrated a possible association of an inflammatory state and the clinical severity of hcm rather than a causal relationship between sii and the prognosis of hcm. further large-scale studies are warranted to validate the present findings and clarify the prognostic utility of sii in hcm. conclusion our study indicates that a higher sii is associated with increased risk for all-cause mortality in hcm patients, but the discriminative power is only poor to moderate. further studies are needed to ascertain the predictive value of sii for adverse outcomes, especially for the specific hcm-related cardiovascular events. the index might be used in combination with other risk factors to improve the risk prediction of adverse outcomes for hcm patients. funding this study was supported by the national key r&d program of china (grant number: 2017yfc0910004), the national clinical research center for geriatrics, west china hospital, sichuan university (grant number: z20192010), and the national natural science foundation of china (grant number: 81600299). figure 4. time-dependent aucs. note: the curve was based on the aucs, which was calculated every 0.2 years (from 1 to 10 years). in the figure, the solid line depicts the aucs, and the ribbon represents 95% ci. abbreviations as in tables 1 and 2. 8 z. wang et al. disclosure statement the authors declare that they have no conflicts of interest. notes on contributors ziqiong wang, md. attending physician at department of cardiology, west china hospital of sichuan university, chengdu, china. haiyan ruan, md. attending physician at department of cardiology, traditional chinese medicine hospital of shuangliu district, chengdu, china/department of cardiology, west china hospital of sichuan university, chengdu, china. liying li, md. med candidiate at department of cardiology, west china hospital of sichuan university, chengdu, china. xin wei, md. associated professor. cardiologist at department of cardiology, west china hospital of sichuan university, chengdu, china/department of cardiology and national clinical research center for geriatrics, west china hospital of sichuan university, chengdu, china. ye zhu, md. professor. cardiologist at department of cardiology, west china hospital of sichuan university, chengdu, china. jiafu wei, md. associated professor. cardiologist at department of cardiology, west china hospital of sichuan university, chengdu, china. xiaoping chen, md, professor. cardiologist at department of cardiology, west china hospital of sichuan university, chengdu, china. sen he, md, phd. associated professor. cardiologist at department of cardiology, west china hospital of sichuan university, chengdu, china. orcid ziqiong wang https://orcid.org/0000-0002-9777-2650 sen he https://orcid.org/0000-0002-0446-2865 references 1. semsarian c, ingles j, maron ms, maron bj. new perspectives on the prevalence of hypertrophic cardiomyopathy. j am coll cardiol. 2015;65:1249–54. doi: 10.1016/j.jacc.2015.01.019 2. maron bj. clinical course and management of hypertrophic cardiomyopathy. n engl j med. 2018;379:655–68. doi: 10.1056/nejmra1710575 3. cirino al, ho c. hypertrophic cardiomyopathy overview. genereviews®, 1993; pp. 1–16. available from: http://www.ncbi.nlm.nih.gov/pubmed/ 20301725 [cited 1 august 2021]. 4. elliott pm, gimeno jr, thaman r, shah j, ward d, dickie s, et al. historical trends in reported survival rates in patients with hypertrophic cardiomyopathy. heart. 2006;92:785–91. doi: 10.1136/hrt.2005.068577 5. maron bj, rowin 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sweden; cdepartment of endocrinology in linköping, and department of biomedical and clinical sciences, linköping university, linköping, sweden; ddepartment of endocrinology in linköping, and department of health, medicine and caring sciences, linköping university, linköping, sweden abstract background: predicting the risk of readmission or death in patients at the emergency department (ed) is essential in identifying patients who would benefit the most from interventions. we aimed to explore the prognostic value of mid-regional proadrenomedullin (mr-proadm), mid-regional pro-atrial natriuretic peptide (mr-proanp), copeptin, and high-sensitivity troponin t (hs-tnt) to identify patients with a higher risk of readmission and death among patients presenting with chest pain (cp) and/or shortness of breath (sob) in the ed. methods: this single-center prospective observational study included non-critically ill adult patients with a chief complaint of cp and/or sob who visited the ed at linköping university hospital. baseline data and blood samples were collected, and patients were followed up for 90 days after inclusion. the primary outcome was a composite of readmission and/or death from non-traumatic causes within 90 days of inclusion. binary logistic regression was used and receiver operating characteristics (roc) curves were constructed to determine the prognostic performance for predicting readmission and/or death within 90 days. results: a total of 313 patients were included and 64 (20.4%) met the primary endpoint. mr-proadm > 0.75 pmol/l (odds ratio [or]: 2.361 [95% confidence interval [ci]: 1.031 – 5.407], p = 0.042) and multimorbidity (or: 2.647 [95% ci: 1.282 – 5.469], p = 0.009) were significantly associated with readmission and/or death within 90 days. mr-proadm increased predictive value in the roc analysis to age, sex, and multimorbidity (p = 0.006). conclusions: in non-critically ill patients with cp and/or sob in the ed, mr-proadm and multimorbidity may be helpful for the prediction of the risk of readmission and/or death within 90 days. article history received 18 january 2023 revised 1 march 2023 accepted 20 march 2023 published 3 may 2023 keywords emergency department; chest pain; shortness of breath; copeptin; mr-proadm; mrproanp; readmission; multimorbidity introduction in sweden, about 15–20% of all hospitalizations are hospital readmissions occurring within 30 days after discharge (1). readmission is associated with increased morbidity and mortality and related annual costs were estimated to be sek 2.3 billion (usd 114 million) (2). preventing avoidable readmissions can profoundly improve the quality of life for patients as well as healthcare’s financial systems. therefore, hospital readmission and death are considered quality healthcare measures (3). identifying high-risk patients at the emergency department (ed) is essential to prevent shortand long-term deterioration. several scoring systems and triage scales were developed to identify patients at a high risk of catastrophic deterioration in which vital parameters play an important role (4). identifying patients at high risk but without apparent derangement in vital parameters is tricky. there is no ideal decision-making tool for seemingly non-critical ed patients, and physicians’ assessments of disease severity are inconsistent (5). furthermore, the prediction of readmission is complex and not all relevant information may be available at the ed, which results in many published tools/scoring systems with often limited predictive value (6). blood biomarkers can be measured quickly and accurately and are more objective than personal judgment. biomarkers of physiological stress might help to determine patients at generally increased risk of poor outcomes (7–9) and potentially help identify patients with increased risk of readmission or death after the immediate illness requiring the visit to the ed. mid-regional proadrenomedullin (mr-proadm) is the stable portion of the pro-hormone adrenomedullin (adm), a potent contact lee ti davidson lee.ti.chong@liu.se supplemental data for this article can be accessed here. © 2023 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article http://dx.doi.org/10.48101/ujms.v128.9300 mailto:lee.ti.chong@liu.se http://dx.doi.org/10.48101/ujms.v128.9300 http://creativecommons.org/licenses/by/4.0/ 2 l. t. davidson et al. vasodilator peptide expressed in various tissues. it involves fluid-electrolyte homeostasis by acting on the renin-angiotensinaldosterone system (raas) and hypothalamic-pituitaryadrenal axis (10). mid-regional pro-atrial natriuretic peptide (mr-proanp) is a pro-hormone of the atrial natriuretic peptide (anp) produced in the cardiac atrium. anp causes vasodilatation, promotes natriuresis and diuresis, and suppresses the raas and sympathetic nervous system (11). copeptin, a more stable peptide of arginine vasopressin (avp), is co-secreted from the  pituitary gland in equimolar amounts to avp upon hemodynamic, osmotic, and various other stress-related stimuli (12). mr-proadm, mr-proanp, and copeptin are surrogate markers reflecting the mature peptides released into the blood circulation. high plasma levels of these prohormones were described in various diseases and found to correlate with disease severity, for example, myocardial infarction, heart failure, respiratory problems, and sepsis (7, 10–15). elevated plasma levels of mr-proadm, mr-proanp, and copeptin are physiological stress markers, indicating severe underlying disease, which is highly valuable for risk stratification and prognostic information for ed patients (15–18). in this study, we aimed to evaluate the prognostic value of mr-proadm, mr-proanp, copeptin, high-sensitivity troponin t (hs-tnt), and baseline information available at ed presentation to identify seemingly non-critically ill patients with chest pain (cp) and/or shortness of breath (sob) at risk of readmission or death within 90 days. material and methods study design in this single-center prospective observational study, between the years 2013 and 2017, we included a sample of noncritically ill adult patients (18 years old and above) presenting to the ed with cp, sob, or both, with onset within 7 days (19).  the study was performed at linkoping university hospital, sweden, a tertiary care teaching hospital with almost 50,000 annual ed patients. the following were the exclusions criteria: 1. patients presenting with st elevated myocardial infarction or a newly discovered left bundle branch block on electrocardiogram (ecg). 2. critically ill patients (airway compromise, saturation < 90%, respiratory rate > 30 or < 8, heart rate > 120 or < 40 beats per min, systolic blood pressure < 90 mmhg, massive cp, clinically unstable patients for whom urgent medical attention was needed). 3. patients diagnosed with renal failure with an estimated glomerular filtration rate of less than 20 ml/min/1.73 m². 4. patients with an advanced focal or spread malignancy. 5. patients with liver disease or liver failure. 6. trauma patients. 7. patients with reduced decision-making ability. all patients were triaged according to the rapid emergency triage and treatment system (retts©) (supplementary 1) as described in a previous study (19). patients’ symptom characteristics and vital parameters, that is, blood pressure, respiratory rate, heart rate, body temperature, peripheral oxygen saturation, and ecg, were collected at triage. the medical history, body weight, and height were recorded. emergency physicians conducted routine assessments and physical examinations of patients. information about baseline comorbidities was collected from the medical records (please refer to supplementary 2 for a sample of patients’ comorbid conditions in the study). laboratory analysis blood samples were collected at ed presentation and frozen at  −70°c until analysis. blood samples for c-reactive protein (crp), lactate, and hs-tnt were analyzed by clinical practice at linköping university hospital’s central laboratory. in cases where the results were unavailable on the first presentation, hs-tnt data were obtained using the analyzed frozen blood sample. hs-tnt levels analyzed from frozen blood samples correlated well with hs-tnt analyzed at a presentation by the  central laboratory (spearman’s, r s = 0.92, p < 0.001 [supplementary 3]). mr-proadm, mr-proanp, and copeptin were analyzed using a highly sensitive time-resolved amplified cryptate emission technology assay (b.r.a.h.m.s, kryptor, ag, hennigsdorf, germany). the assay had an analytical detection limit of 0.04 nmol/l, 2.1 and 1.7 pmol/l, while the inter-assay variability was 3.3, 3.0, and 5.2% for mr-proadm, mr-proanp, and copeptin, respectively. the results of the study markers were unavailable to the attending physician and no intervention was conducted based on these data. the patients’ assessments were left to the discretion of the attending physicians. patient follow-up information was collected from the electronic health records (cambio cosmic®, linköping, sweden) used by all regional hospitals, which were integrated with other medical health records and death reports in the state. follow-up was performed until 90 days after the first ed presentation. main outcome the primary outcome was a composite of the first unplanned readmission or death within 90 days after study recruitment. death was defined as death from any non-traumatic cause. endpoint data were collected from the electronic health records, as described here. association of physiological stress markers at the emergency department 3 ethics all participants gave their written informed consent. the study was approved by the regional ethical committee of linköping (diary number 2011/501-31) and conformed to the principles outlined in the declaration of helsinki. statistical analysis data analyses were conducted using spss software (ibm spss statistics, version 26). a p-value (2-tailed) of <0.05 was accepted as statistical significance. given the exploratory nature of this study, no sample size calculation was performed. the primary outcome was binarily coded; 0 = survivor and no readmission, and 1 = readmission and/or death. comorbidities were defined as a known medical illness, coded as a binary variable, and dichotomized according to the number of comorbidities: up to two comorbidities (non-multimorbidity group) and more than two comorbidities (multimorbidity group), according to the swedish national board of health and welfare’s definition (20). the biomarkers mr-proadm, mr-proanp, copeptin, and hs-tnt were dichotomized at a pre-specified cut-off level as in previous studies (19, 21, 22). baseline population characteristics were presented for the total cohort and stratified based on the primary outcome. descriptive statistics were expressed as means and standard deviations or medians with interquartile ranges for continuous variables and as frequencies and percentages for nominal variables. categorical variables were compared using the chi-square test, while the student’s t-test and mann–whitney u-test were used to compare continuous variables. univariable logistic regression analysis was first performed to identify potential explanatory variables (p < 0.05) that could be considered for inclusion in the multivariable binary logistic regression analysis in which a backward elimination was performed. a base model was created, including age and sex as compulsory variables and multimorbidity. mr-proadm, mrproanp, copeptin, and hs-tnt were added separately to the base model, followed by a combination of studied markers. the adjusted odds ratios (or) and 95% confidence intervals (ci) were reported as a measure of association. receiver operating characteristics (roc) curves were constructed separately and then compared for the base model; age, sex, and multimorbidity, and for the base model combined with binary mr-proadm > 0.75 nmol/l to determine the prognostic performance with area-under-the-curves (aucs) for prediction of readmission and/or death within 90 days. results in total, 313 patients were included in the analysis, of which 64 (20.4%) met the outcome, that is, a composite of readmission and/or death within 90 days. the population characteristics at inclusion are presented in table 1. the mean age of the participants was 64 ± 17 years, and 158 (50.5%) were men. of all included patients, 195 (62.3%) presented with cp only, 61 (19.5%) presented with sob only, and 57 (18.2%) presented with both complaints. a total of 165  (52.7%) of all patients were admitted for in-hospital care, of whom 58 (35.2%) were admitted for more than 3 days. none of the patients needed intensive care unit (icu) admission. within the 90-day follow-up, 61 (19.5%) patients were readmitted, and five (1.6%) deceased, of which two happened at readmission, resulting in the final composite endpoint outcome consisting of 64 (20.4%) patients. a total of 33 (51.6%) of the patients who were readmitted and/or died had multimorbidity. of the 249  (79.6%) survivors with no readmission events during the 90-day follow-up period, 202 (81.0%) had no more than two comorbidities. the most frequent pre-existing comorbidity was hypertension (47.6%), followed by ischemic heart disease (26.5%) (supplementary 2). there was no significant difference regarding baseline vital parameters except for lower peripheral oxygen saturation in patients who were readmitted or died within 90 days. at baseline, patients who were either readmitted and died had higher plasma concentrations of crp,  creatinine, lactate, mrproadm, mr-proanp, copeptin, and hs-tnt (table 1). peripheral oxygen saturation, multimorbidity, use of angiotensin-converting enzyme inhibitors/angiotensin receptors blockers (ace-i/arb), use of diuretics, hemoglobin level, creatinine, lactate, were identified as potential explanatory variables by the univariable binary logistic regression analysis. after adjustment, multimorbidity remained a possible explanatory variable. the result of the multivariable binary logistic regression analyses: a base model with age and sex as  compulsory variables, and multimorbidity, added with mr-proadm, mr-proanp, copeptin, and hs-tnt alone or in combination, are presented in table 2a–f. hs-tnt was significantly associated with outcome added to age, sex, and multimorbidity (or: 2.622, 95% ci: 1.255 – 5.471, p = 0.010) (table 2c). however, this significant association was lost when mr-proadm was added to the model (table 2e). multimorbidity was the only baseline variable that remained significantly associated with readmission and/or death within 90 days; p  =  0.009. patients with multimorbidity had an or of 2.6 (95% ci: 1.282 – 5.469) of being readmitted or dying within 90 days compared with those  with fewer than two or no comorbidity (table 2a–f ). mr-proadm > 0.75 pmol/l was the only stress marker that remained significantly associated with  readmission and/or death within 90 days (or: 2.4, 95% ci:  1.031 – 5.407, p = 0.042) (table 2e–f ). in roc analysis, the base predictive model consisting of age, sex, and multimorbidity showed an auc of 0.686 (95% ci: 0.607 – 0.764), and the addition of mr-proadm to the base model showed an auc of 0.764 (95% ci: 0.695 – 0.833), see figure 1. adding mr-proadm had a significantly increased predictive value compared with  the base predictive model, that is, age, sex, and multimorbidity, separately (auc difference 0.078, 95% ci: 0.022 – 0.134, p = 0.006). 4 l. t. davidson et al. discussion this study found that mr-proadm and multimorbidity were related to readmission and/or death in non-critically ill patients with cp and/or sob 90 days after ed presentation. there was no  significant difference regarding baseline vital parameters except for lower peripheral oxygen saturation in patients who were readmitted or died within 90 days. the baseline plasma concentrations of hemoglobin, creatinine, crp, mr-proanp, hs-tnt, and copeptin were higher but not independently associated with the risk of readmission and/or death within the 90-day follow-up period. our findings for mr-proadm were consistent with those of earlier studies that evaluated the utility of prognostic markers in patients at the ed. in selected, potentially critically ill populations, mr-proadm was identified as a stress marker with the ability to  predict adverse coronary events, clinical deterioration after ed admission, readmission, and all-cause mortality, and as a triage biomarker for hospitalization reduction (8, 18, 23–25). mr-proadm was considered a better predictor of poor outcomes independent of other biomarkers combined (mr-proanp, copeptin, hs-tnt, n-terminal pro-b-type natriuretic peptide, c-terminal pro-endothelin-i, or crp) (7, 9, 21, 23, 25, 26). additionally, mr-proadm assessment could accurately identify disease progression in patients with infection at the ed and safely increase outpatient management without increasing the number of readmissions or death (25). however, most of these studies were conducted in selected ed patients with specific diseases or age groups. our study supports earlier results and shows that mr-proadm may be able to predict poor outcomes even in unselected, non-critically ill ed patients at the earliest point of care. in our study, having more than two medical diagnoses defining multimorbidity was the only available baseline information in the ed significantly associated with readmission and/or death within 90 days. the number and disease conditions included in multimorbidity studies vary (27). at the same time, it is accepted that the cumulative effects of illnesses are compounded by the addition of further comorbidities with increasing levels of disability and utilization of healthcare resources (28). multimorbidity increases the risk of hospital admission, more extended hospital stays, readmission, and mortality (29). the mere addition of a disease condition is a table 1. baseline characteristic of the study population. variables overall readmission and/or death within 90 days p yes no number of patients, n (%) 313 64 (20.4) 249 (79.6) male/female, n (%) 158 (50.5)/155 (49.5)  37 (57.8)/27 (42.2)   121 (48.6)/128 (51.4)  0.188 age, years (mean ± sd) 64 ± 17 70 ± 16 63 ± 17 0.003 bmi, kg/m² (mean ± sd) 28.0 ± 5.5 28.6 ± 5.3 27.6 ± 5.6 0.252 multimorbidity, n (%) 80 (25.6) 33 (51.6) 47 (19.0) <0.001 ace-i/arb, n (%) 124 (39.9) 37 (58.7) 87 (35.1) 0.001 diuretic, n (%) 76 (24.4) 30 (47.6) 46 (18.5) <0.001 chest pain only, n (%) 195 (62.3) 32 (50.0) 163 (65.5) 0.023 shortness of breath only, n (%) 61 (19.5) 17 (26.6) 44 (17.6) 0.109 chest pain and shortness of breath, n (%) 57 (18.2) 15 (23.4) 42 (16.9) 0.224 retts blue & green (no emergency), n (%) 6 (2.3) 0 6 (3.0) 0.184 retts yellow (within 120 min), n (%) 87 (33.5) 18 (31.0) 69 (34.1) 0.657 retts orange & red (20 min to urgent), n (%) 167 (64.2) 40 (69.0) 127 (62.9) 0.393 systolic blood pressure (mean ± sd) 147 ± 24 144 ± 23 149 ± 24 0.155 temperature, °c (mean ± sd) 36.9 ± 0.7 37.0 ± 0.7 36.9 ± 0.6 0.519 peripheral oxygen saturation, % (mean ± sd) 97 ± 4 95 ± 5 97 ± 3 0.008 respiratory rate/min (mean ± sd) 19 ± 5 20 ± 6 17 ± 5 0.056 heart rate/min (mean ± sd) 80 ± 20 82 ± 21 79 ± 19 0.092 hemoglobin, g/l;(mean ± sd) 139 ± 16 133 ± 19 140 ± 14 0.002 c-reactive protein, crp, mg/l (median [iqr]) 10 (5–10) 10 (5 – 14) 7 (5 – 10) 0.001 creatinine, μmol/l (mean ± sd) 84 ± 27 98 ± 38 80 ± 23 <0.001 lactate, mmol/l (mean ± sd) 1.2 ± 0.5 1.3 ± 0.7 1.2 ± 0.5 0.024 copeptin, pmol/l (median [iqr]) 6.2 (3.7 – 13.4) 10.3 (4.2 – 26.6) 5.4 (3.5 – 9.7) 0.044 hs-tnt, g/l (median [iqr]) 9 (6 – 19) 19 (8–33) 7 (4 – 12) <0.001 mr-proadm, nmol/l (mean ± sd) 0.8 ± 0.5 1.1 ± 0.7 0.7 ± 0.4 <0.001 mr-proanp, pmol/l (median [iqr]) 91 (58 – 180) 132 (76 – 283) 79 (53 – 130) <0.001 admission, n (%) 165 (52.7) 44 (68.8) 121 (48.6) 0.004 length of hospital stay, > 3 days, n (%) 58 (35.2) 22 (34.4) 36 (14.5) <0.001 bmi: body mass index; iqr: inter quartile range; ace-i/arb: angiotensin converting enzyme inhibitors/angiotensin receptors blockers; retts: rapid emergency triage and treatment system, color-coded triage with increasing acuity from green to red; hs-tnt: high-sensitivity troponin t; mr-proadm: midregional pro-adrenomedullin; mr-proanp: midregional pro-a-type natriuretic. association of physiological stress markers at the emergency department 5 simplified method to provide a proxy for identifying frailty, chronic disease burden, and functional status. in contrast to the immediate predictive value of copeptin in critically ill patients (30), we found that copeptin had no predictive value for readmission and/or death within 90 days in this non-critically ill patient population. this was in line with previous studies showing that copeptin had no added value for prognostic information over that provided by troponin or mrproadm (26, 31). a study conducted in a population of critically ill patients admitted to the icu found that the primary determinant of elevated copeptin was the severity of critical illness, as reflected by organ failure and hemodynamic alterations, not the underlying etiologic (17). our study focused on a population of non-critically ill patients that may have contributed to copeptin’s negative result. hs-tnt is known as an organ-specific marker used to diagnose myocardial infarction. studies showed that a hs-tnt level above the 99th percentile was common in ed patients with cp but with no myocardial infarction (32) and chronic myocardial injury defined as stable hs-tnt levels > 14 ng/l was a strong marker for death and cardiovascular events (33). crp and lactate were important in early triage, and previous studies showed their predictive effect on readmission and adverse events (34–37). however, in our population, neither vital signs nor established markers crp, lactate, nor hs-tnt were significantly associated with readmission and/or death within 90 days. by our findings, the prognostic value of vital parameters, crp, lactate, and hs-tnt in non-critically ill patients seems beneficial mainly in the short term. age and gender are important predictors of various prognostic models for diseases, readmission, and death (38–40). a study population with heart failure showed that women had a 2.5 times greater risk for rehospitalization within 90 days than men (39). at the same time, another study from the population-based longitudinal study swedish adoption/twin study of ageing (satsa) showed that higher age and male sex increase the risk of readmission (38). in our study, including adults 18 years and above, the mean age was higher in patients who were readmitted and/or died within 90 days. however, in our non-critically ill ed population with undifferentiated cp and/or sob, neither age nor gender had a significant predictive value for  readmission and/or death within 90 days after adjustment for multimorbidity, mr-proadm, mr-proanp, hstnt, and copeptin. most readmission risk prediction models have a poor predictive ability (6) and focus on specific conditions or ages (41). many are based on data available only after discharge, table 2. multivariable binary logistic analysis for association of age, sex, and multimorbidity with mr-proadm, mr-proanp, hs-tnt, and copeptin, respectively, and in combination, to readmission and/or death within 90 days. variables in predicting models or (95% ci) p 2a age 0.996 (0.974 – 1.019) 0.752 sex 1.478 (0.807 – 2.707) 0.206 multimorbidity 3.184 (1.600 – 6.337) <0.001** mr-proadm > 0.75 pmol/l 2.514 (1.235 – 6.118) 0.011* 2b age 0.995 (0.969 – 1.021) 0.690 sex 1.443 (0.791 – 2.633) 0.232 multimorbidity 3.653 (1.857 – 7.186) <0.001** mr-proanp > 120 nmol/l 2.009 (0.897 – 4.500) 0.090 2c age 0.996 (0.973 – 1.020) 0.743 sex 1.262 (0.687 – 2.321) 0.453 multimorbidity 3.156 (1.593 – 6.252) <0.001** hs-tnt > 14 g/l 2.622 (1.255 – 5.471) 0.010* 2d age 1.013 (0.990 – 1.036) 0.283 sex 1.463 (0.790 – 2.709) 0.226 multimorbidity 3.361 (1.699 – 6.646) <0.001** copeptin > 10 nmol/l 0.979 (0.492 – 1.951) 0.952 2e age 0.989 (0.966 – 1.014) 0.390 sex 1.380 (0.738 – 2.579) 0.313 multimorbidity 2.878 (1.427 – 5.802) 0.003* mr-proadm > 0.75 pmol/l 2.153 (1.009 – 4.592) 0.047* hs-tnt > 14 g/l 1.859 (0.846 – 4.088) 0.123 2f age 0.991 (0.964 – 1.019) 0.527 sex 1.657 (0.855 – 3.209) 0.134 multimorbidity 2.647 (1.282 – 5.469) 0.009* mr-proadm > 0.75 pmol/l 2.361 (1.031 – 5.407) 0.042* mr-proanp >120 nmol/l 1.224 (0.490 – 3.061) 0.665 hs-tnt > 14 g/l 2.244 (0.920 – 5.476) 0.076 copeptin > 10 nmol/l 0.476 (0.211 – 1.075) 0.074 or: odds ratio; ci: confidence interval; mr-proadm: midregional proadrenomedullin; mr-proanp: midregional pro-a-type natriuretic; hs-tnt: high-sensitivity troponin t. figure 1. roc curve for prediction of readmission and/or death within 90 days. blue = age + sex + multimorbidity, vs. red = age + sex + multimorbidity + mr-proadm > 0.75 pmol/l (p = 0.006) 6 l. t. davidson et al. requiring burdensome data collection beyond that readily available from routine clinical care records (42), rendering their application difficult, if possible, in the immediate emergency visit. cumulative comorbidities, that is, multimorbidity, and mr-proadm, are potential variables that can be used early in the patient’s pathway to identify patients at risk of readmission and/or death within 90 days. the strengths of our study include its prospective nature and real-life ed patients with various underlying diseases. we used the cumulative number of comorbidities as a proxy for  morbidity burden to simplify ed decisions, making it as non-cumbersome as possible for patients’ early care. to our knowledge, this is the first study of the association between stress biomarkers and unplanned readmission and/or death within 90 days among non-critically ill ed patients. we excluded  easily identifiable critically ill patients, those with pronounced sob, massive cp, or st-segment elevation confirming myocardial infarction, and clinically unstable patients for whom urgent medical attention was needed. this  was deliberate considering the difficulties in the identification and risk stratification of critically ill patients without apparent derangement in the vital parameters. this is a significant difference in patient selection compared with previous blood marker studies conducted in the ed (7–9, 23). our study had limitations. we used specific cut-offs, which  might decrease the analytical power compared with continuous analysis. however, we used known cut-off values as in the previous study, which makes it easier for application in daily clinical practices. given the exploratory nature of this  study, no  sample size estimation was performed,  and no  causal relationship could be determined. as this was a single-center study, the results may not apply to other settings. conclusions in non-critical patients presenting with undifferentiated cp and/ or sob in the ed, multimorbidity is the only baseline information of importance for predicting readmission and/or death within 90 days. mr-proadm may be a helpful biomarker for identifying ed patients with cp and/or sob at higher risk  of  readmission and/or death within 90 days. mr-proadm might facilitate ed discharge decisions and increase safe outpatient management. considering our findings, further studies to determine the prognostic value of mr-proadm in the unselected non-critically ill ed population seem warranted. acknowledgments the authors would like to acknowledge helene kimme for her help with patient recruitment and preben henanger for help with the data collection. disclosure statement authors declare no conflict of interest. funding grants from the region östergötland; rö-610991, rö-351681, rö-340001, rö-430481, rö-533731, rö-534451, rö-599931, rö-421461, rö-966396. notes on contributors conceptualization, s.c., l.t.d., m.s., ha; methodology, l.t.d, s.c., m.s., ha; formal analysis, l.t.d, s.c.; investigation, l.t.d, s.c., m.s.; resources, l.t.d, s.c., m.s; data curation, l.t.d, s.c.; writing – original draft preparation, l.t.d, f.n, writing – review and editing, l.t.d, f.n, h.a, m.s, s.c.; visualization, l.t.d, f.n; supervision, f.n; project administration, l.t.d, m.s, s.c.; funding acquisition, l.t.d, m.s, s.c. all authors have read and agreed to the published version of the manuscript. lee ti davidson, md, is a ph.d. student and a consultant in emergency and internal medicine at the department of emergency, linköping university hospital, and is doing her research study at the institute of biomedical and clinical sciences, linköping 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http://dx.doi.org/10.1136/bmj.l5358 http://dx.doi.org/10.1136/bmjopen-2018-028302 http://dx.doi.org/10.1161/circoutcomes.117.003885 upsala journal of medical sciences 2021, 126, e7603 http://dx.doi.org/10.48101/ujms.v126.7603 the relationship between nlrp3 rs10159239 and vaspin rs2236242 gene variants and obstructive sleep apnea buğra kergeta*, ferhan kergetb, çiğdem yüce kahramanc, alperen aksakald and ömer araza adepartment of pulmonary diseases, ataturk university school of medicine, erzurum, turkey; bdepartment of infection diseases and clinical microbiology, health sciences university erzurum regional education and research hospital, erzurum, turkey; cdepartment of medical genetics, ataturk university school of medicine, erzurum, turkey; ddepartment of pulmonary diseases, health sciences university erzurum regional education and research hospital, erzurum, turkey abstract background: in obstructive sleep apnea (osa), recurrent upper airway obstruction and apnea/hypopnea episodes result in endothelial dysfunction, which leads to the release of many proinflammatory cytokines and reactive oxygen species (ros). ros induces nlrp3, a protein involved in the synthesis of interleukin (il)-1 and il-18; vaspin is a serine protease inhibitor that has an important role in suppressing the activation of nlrp3 inflammasome. in this study, we aimed to investigate the effect of nlrp3 rs10159239 (rs9239) and vaspin rs2236242 (rs6242) single nucleotide polymorphisms (snps) on osa development. methods: this study included 220 individuals who underwent polysomnography (118 patients with osa and 102 healthy controls). nlrp3 rs9239 and vaspin rs6242 mutation frequencies were analyzed. results: the nlrp3 rs9239 snp genotype analysis revealed no statistically significant differences between the osa and control groups. in the vaspin gene analysis, the rs6242 aa genotype was significantly more frequent in the osa group compared with the control group, while the at genotype was more frequent in controls (p = 0.004, p = 0.02). comparison of rs6242 allele levels showed that the a allele was significantly more frequent in osa patients than in controls (p = 0.03). the aa genotype was significantly more frequent in patients with severe osa than in patients with mild or moderate osa and the control group (p = 0.001 for all). serum vaspin levels were significantly lower in carriers of the aa genotype than those with at and tt genotypes (p = 0.001). conclusion: the vaspin rs6242 snp aa genotype increased susceptibility to osa, while the at genotype appeared to be protective. the lower plasma vaspin levels in osa compared with the control group and in patients with the aa genotype suggest that vaspin may be a protective biomarker for osa. article history received 6 february 2021 revised 22 april 2021 accepted 4 may 2021 published 2 june 2021 keywords: obstructive sleep apnea; polymorphism; vaspin; nlrp3; elisa introduction obstructive sleep apnea (osa) is characterized by recurrent episodes of apnea/hypopnea and frequent reductions in blood oxygen saturation resulting from upper airway obstruction (1). in osa patients, repeated oxygen desaturations and subsequent endothelial dysfunction induce the release of strong proinflammatory biomarkers, particularly tumor necrosis factor alpha (tnf-α), interleukin-6 (il-6), c-reactive protein (crp), leptin, and reactive oxygen species (ros) (2–4). ros play an important role in the exacerbation of inflammation in osa and increase in correlation with disease severity (5). ros are also important in the synthesis of nod-like receptor protein 3 (nlrp-3) (6). nlrp3, adapter protein asc-1, and caspase-3 precede the synthesis of il-1 beta and il-18, which are key mediators of inflammation. a study investigating nlrp3, il-1 beta, and il-18 levels in osa patients showed that the levels of proinflammatory cytokines generated due to oxidative stress increased independently of nlrp3 synthesis (7). on the other hand, in studies on diabetes mellitus and coronary artery disease, which are frequent complications in osa, nlrp3 levels were found to increase in correlation with disease severity (8,9). in patients with the nlrp3 rs10159239 (rs9239) a allele, the increased nlrp3 expression was associated with a significant increase in the severity of coronary artery disease (8). the serine protease inhibitor vaspin, a novel adipocytokine synthesized by white adipose tissue, plays an important role  primarily in the anti-inflammatory balance, as well as overcoming insulin resistance and preventing obesity (10,11). a comparative analysis of serum vaspin levels in osa patients and a control group showed that patients with severe osa had significantly lower serum vaspin levels compared with controls (12). studies of vaspin rs2236242 (rs6242) single nucleotide contact buğra kerget bjkerget1903@gmail.com © 2021 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article http://dx.doi.org/10.48101/ujms.v126.7603 mailto:bjkerget1903@gmail.com http://creativecommons.org/licenses/by/4.0/ 2 b. kerget et al. polymorphism (snp) demonstrated that individuals carrying the a allele had higher risk of developing type 2 diabetes mellitus (13). in another recent study, patients with the vaspin a allele were also found to be at higher risk of developing coronary artery disease (14). however, the serum vaspin level was not associated with genotype frequency in either of these studies. the oxidative stress, endothelial dysfunction, and insulin resistance that occur as a result of recurrent apnea/hypopnea episodes in osa predispose to many diseases, especially coronary artery disease and diabetes. as this study aimed to elucidate the role of nlrp3 rs9239 and vaspin rs6242 snps in the development of osa, patients with comorbidities were not included in order to eliminate the confounding effect of comorbidities. materials and methods study design this single-center case–control study included individuals aged 20 years and over who presented to the chest diseases department of our hospital with complaints of snoring, witnessed apnea, and/or excessive daytime sleepiness and underwent polysomnographic evaluation between august 2020 and april 2021. a written informed consent was obtained  from all patients. this study was designed and conducted in accordance with the ethical guidelines set forth  in the declaration of helsinki, and the study protocol  was  approved by the local ethics committee (b.30.2.ata.0.01.00/63). this research was supported by the  ataturk university  scientific  research project office (project number 6607). study population a total of 243 adults who underwent polysomnography were screened for inclusion in the study. exclusion criteria were defined as the presence of chronic or clinically significant infectious or inflammatory conditions in the past month, asthma, chronic obstructive pulmonary disease, malignancy, current or past smoking history, invasive surgical intervention in the past month, uncontrolled hypertension, high fasting blood glucose, diabetes, cerebrovascular disease, kidney disease, and coronary artery disease. according to these criteria, 1 patient with a history of upper respiratory tract disease in the last month, 12 patients in whom coronary artery disease was detected during follow-up, and 10 patients with high fasting blood glucose were excluded. as a result, a total of 220 patients were included in the study. controls were matched with osa patients by age and gender. based on the results of polysomnography, 118 patients with a apnea/hypopnea index (ahi) > 5 were regarded as having osa and were classified by severity as mild (ahi: 5–15), moderate (ahi: 16–30), or severe (ahi: >30). the remaining 102 individuals who had ahi values < 5 were included in the control group. polysomnography all participants underwent full polysomnography monitoring using a compumedics e-series sleep system (compumedics sleep, melbourne, australia). surface electrodes were used  to  record electroencephalography, right and left electrooculographies, and submental electromyography. ventilatory airflow through the nose and/or mouth was  measured, and inductive plethysmography bands were  used to monitor body position and respiratory movements  of the chest and abdomen. arterial oxygen saturation was measured transcutaneously via finger-tip oximeter. apnea was defined as continuous cessation of airflow for  ≥ 10 sec. hypopnea was defined as at least 50% reduction  in airflow for ≥ 10 sec together with oxygen desaturation of ≥3% or an arousal from sleep on eeg. apneas were classified as obstructive, central, or mixed according to  the standard american academy of sleep medicine criteria (1). molecular analysis dna isolation protocol blood samples were collected into ethylenediaminetetraacetic acid (edta) tubes. dna of the patients was extracted using qiaamp dna mini kit (qiagen, hilden, germany) according to the manufacturer’s protocol. dna quality was measured using nanodrop (nd-1000, thermo fischer scientific, wilmington, de, usa). analysis of rs10159239 and rs2236242 single-nucleotide olymorphisms allele-specific snptype assays were run using the fluidigm  flex six™ genotyping ifc (fluidigm corp., south san francisco, ca, usa). specific target amplification (sta) was performed to increase the number of molecular targets at the beginning. the determined thermal cycle program was run using the bioer gene pro thermal cycler (95°c for 15 min followed by 14 cycles of 95°c for 15 sec and 60°c for 4 min). snptype assay mixes and sample mixes were prepared according to the manufacturer’s protocol. after all the dynamic array was loaded with a 4 μl of each 10× assay mix and 5 μl of each sample mix. dynamic array was placed on the ifc controller hx (fluidigm), and the loading process was completed. then, dynamic array was placed on the biomark system (fluidigm), which performs the thermal cycling and fluorescent image acquisition. a data collection software was used on biomark system. genotyping application, rox passive reference, and snptype-fam and snptype-hex probe types were selected. the snptype e flexsix v1 protocol was used for thermal cycling and image  capture. at the end, we assessed the genotypes of the samples. nlrp3 rs10159239, vaspin rs2236242, and osas 3 measurement of biochemical markers peripheral venous blood samples were collected after 15 min of rest into tubes containing edta. vaspin levels were measured using the enzyme-linked immunosorbent assay (elabscience human elisa kit, uk). statistical analysis the data were analyzed using ibm spss statistics for windows version 24.0 (ibm corp., armonk, ny). comparisons of characteristics between cases and controls were analyzed by χ2 test for categorical variables and independent sample t‐test or mann–whitney u test for continuous variables, as appropriate. the difference in allele and genotype frequencies was compared  between control and osa using pearson’s χ2 test. statistical significance of the observed genotype frequencies was evaluated according to the hardy–weinberg rule and compared to the expected genotype frequencies. independent-samples t  test was used to compare demographic data and laboratory parameters between groups. a p-value less than 0.05 was considered statistically significant. hardy–weinberg equilibrium the hardy–weinberg equilibrium (hwe) is a principle that states that genetic variation in a population will remain constant from one generation to the next in the absence of disruptive factors, such as non-random reproduction, mutations, and natural selection. therefore, the hwe defines an idealized state, and genetic variations in nature  can  be defined as deviations from this equilibrium state (15). for example, in hwe, there is (a) and (a)allele p = allele frequency of (a) and q = allele frequency of (a), the expected genotype frequency under hwe is p2 for the aa genotype, 2pq for the aa genotype, and q2 for the aa genotype. p2+ 2pq+ q2= 1 i = (total number of a allele)/2 × n q = (total number of a allele)/2 × n after these calculations, our expected and observed genotype and allele frequencies are compared by χ2 test. if there is no significant difference in expected and observed parameters, we could say there is hwe for our study group. results the mean age in the osa and control groups was 48.2 ± 23.6 years and 47.4 ± 22.1 years, respectively. of the 118 patients in the osa group, 62 (52.5%) were men and 52 (47.5%) were women, while the 102 controls included 55 men (53.9%) and 47  women (46.1%). there was no statistically significant difference in age or sex between the osa and control groups. the osa group showed significantly higher body mass index (bmi), ahi, triglycerides, and oxygen desaturation index (odi) compared with the control group (p = 0.04, 0.001, 0.001, 0.001, respectively) (table 1). the nlrp3 rs9239 snp analysis revealed that there was no statistically significant difference between the osa and control groups (χ2: 0.98, df: 2, p = 0.66). (table 2). the frequency of the at genotype was higher in the control group than in the osa group, while the aa genotype was higher in the osa group (odds ratio [or]: 0.445, 95% ci: 0.219–0.91, p = 0.02 and or: 2.21; 95% ci: 1.46–2.68, p = 0.004, respectively) (table 3). obesity-adjusted analysis showed that the difference in aa genotype frequency persisted (or: 1.96, 95% ci: 0.98–2.57, p = 0.03), but there was no statistically significant difference in the at genotype (or: 0.63, 95% ci: 0.315–1.22, p = 0.06). in the evaluation of allele frequency, it was also observed that the frequency of the a allele was higher in the osa group than in the control group. the vaspin rs6242 allele and genotype frequencies in the study groups and comparison with the hw equilibrium are shown in table 4. both the control and osa groups showed statistically significant differences in hw equilibrium analysis (p = 0.016, p = 0.006). table 1. comparison of laboratory test results in patients with obstructive sleep apnea syndrome (osa) and controls control (n = 102) mean ± sd all osa patients (n = 118) mean ± sd p age (years) 47.4 ± 22.1 48.2 ± 23.6 0.42 bmi (kg/m2) 24.9 ± 7.4 28.6 ± 7.6 0.04 ahi 3.1 ± 1.2 52.6 ± 31.8 0.001 rem ahi 3.1 ± 1.7 28.5 ± 24.2 0.001 triglycerides (mg/dl) 116.1 ± 47.2 143.2 ± 41.1 0.001 hdl cholesterol (mg/dl) 41.1 ± 8.2 42.3 ± 11.1 0.12 ldl cholesterol (mg/dl) 120.2 ± 42.4 124.5 ± 47.9 0.16 cholesterol (mg/dl) 181.4 ± 30.4 187.2 ± 35.1 0.23 odi 1.6 ± 0.8 34.1 ± 10,4 0.001 fasting blood glucose (mg/dl) 79.2 ± 15.6 82.5 ± 18.4 0.18 hgba1c (%) 5.7 ± 1.4 6 ± 1.2 0.06 vaspin (ng/ml) 1,89 ± 1,76 1,02 ± 0,74 0.01 bmi: body mass index, ahi: apnea/hypopnea index, hdl: high-density lipoprotein, ldl: low-density lipoprotein, odi: oxygen desaturation index, rem: rapid eye movement sleep. bold indicates statistically significant parameters. 4 b. kerget et al. table 2. comparison of nlrp3 rs9239 genotype numbers between osa and control groups ag, n (%) aa, n (%) gg, n (%) p control (n = 102) 68 (66.6) 11 (10.9) 23 (22.5) 0.66 osa (n = 118) 82 (69.5) 12 (10.1) 24 (20.4) χ2: 0.98, df: 2, osa:obstructive sleep apnea, a: adenine, g: guanine. table 5. comparison of genotype distributions in osa patients according to disease severity control (n = 102) n (%) mild osa (n = 31) n (%) moderate osa (n = 31) n (%) severe osa (n = 56) n (%) p at 61 (59.8) 13 (41.9) 7 (22.6) 5 (8.9) 0.001* aa 34 (33.3) 11 (35.5) 19 (61.3) 47 (83.9) 0.001** tt 7 (6.9) 7 (22.6) 5 (16.1) 4 (7.2) > 0.05 p*: comparison of at genotype in control vs. other groups, p**: comparison of aa genotype in severe osa vs. other groups and in control vs. light osa group; osa: obstructive sleep apnea, a: adenine, t: thymine. bold indicates statistically significant parameters. table 4. vaspin rs6242 allele/genotype frequencies and test of hardy–weinberg equilibrium control osa f(a) 0.63 0.82 f(t) 0.37 0.18 – o e o e tt 7 13.9 9 3.8 at 61 47.5 25 34.8 aa 34 40.4 84 79.39 χ2: 8.259, df: 2, p = 0.016 χ2: 10.142, df: 2, p = 0.006 osa: obstructive sleep apnea, a: adenine, t: thymine; f: observed frequency of each allele (t or a), o: observed genotype numbers, e: expected genotype numbers under a hardy–weinberg equilibrium assumption; χ2: chi-square values. table 3. comparison of vaspin rs6242 genotype / allele numbers between osa and control groups control osa or (95% ci) p or (95% ci)* p* tt (n) 7 (6.9%) 9 (7.6%) – – – – at (n) 61 (59.8%) 25 (21.2%) 0.445 (0.219–0.91) 0.02 0.63 (0.315–1.22) 0.06 aa (n) 34 (33.3%) 84 (71.2%) 2.21 (1.46–2.68) 0.004 1.96 (0.98–2.57) 0.03 allele t (n) 75 43 – – a (n) 129 193 0.581 (0.381–0.912) 0.03 osa: obstructive sleep apnea, a: adenine, t: thymine, or: odds ratio, ci: confidence interval. *: adjusted for obesity. bold indicates statistically significant parameters. figure 1. evaluation of rs6242 genotype frequency and serum vaspin level. p*: comparison of serum vaspin level in aa genotype with at and tt genotype (p = 0.001). the aa genotype was significantly more frequent in patients with severe osa than in patients with mild or moderate osa and the control group (p = 0.001 for all) (table 5). the at genotype did not differ in frequency based on osa severity but was significantly more frequent in the control group (p = 0.001 for all). in the comparison of serum vaspin level in aa genotype with at and tt genotype, a statistically significant higher was observed in aa genotype compared to at and tt (p = 0.001 for all) (figure 1). discussion no significant differences were observed between the osa and control groups in the nlrp3 rs9239 genotype analysis. however, in the hwe analysis of vaspin rs6242 allele/genotype frequency, the at genotype was more frequent than expected in the control group, and the aa genotype was more frequent in the osa group. nlrp3 rs10159239, vaspin rs2236242, and osas 5 in our study excluding individuals with comorbidity, the aa genotype was observed to increase susceptibility to osa compared to the control group, independently of bmi. furthermore, comparisons based on osa severity showed that the aa genotype was more frequent in patients with severe osa as well. individuals with the at genotype had a lower risk of osa. in the comparison of serum vaspin levels in the aa genotype group with the at and tt genotypes, higher levels were observed in the aa genotype. the frequency of the a allele was higher in the osa group than in the control group, suggesting that it may be an important risk factor for osa susceptibility. osa is characterized by recurrent upper airway obstruction during sleep, increased respiratory effort in response to this obstruction, and frequent sleep interruptions (16). successive episodes of apnea lead to increased sympathetic nervous system activity, oxidative stress, intrathoracic pressure fluctuations, sudden increases in systemic blood pressure, and hypoxia, all of which contribute to endothelial dysfunction (17–19). many cytokines such as tnf-alpha, il-1, il-2, il-4, il-6, il-18, monocyte chemoattractant protein-1 (mcp-1), vimentin, and platelet-derived growth factor (pdgf) are produced by macrophages and t lymphocytes activated due to endothelial dysfunction and oxidative stress (20–22). inflammatory cytokines contribute to a further progression of endothelial dysfunction and the development of comorbidities such as atherosclerosis and insulin resistance, which are commonly seen in osa (21,23). it is worthy of note that continuous positive airway pressure (cpap) therapy reduces levels of inflammatory cytokines in osa patients (24). oxidative stress is known to be the main activator of nlrp3 synthesis, which, in turn, plays an important role in the synthesis of powerful proinflammatory cytokines, primarily il-1 and il-18 (25). in a study on patients with copd, another condition in which oxidative stress is important, it was found that nlrp3 levels increased during acute exacerbations and decreased in correlation with clinical stability (26). moreover, studies on patients with idiopathic pulmonary fibrosis with long-term hypoxia and accompanying comorbidities demonstrated that pirfenidone suppressed lung inflammation and fibrosis development by blocking the nlrp3 inflammasome (27). in studies investigating plasma nlrp3 levels in osa patients, it was observed that the proinflammatory cytokine il-1 and il-18 levels increased independently of nlrp3 levels (7). our literature search yielded few studies on the nlrp3 rs9239 snp. in one study, a higher frequency of the a allele was associated with higher incidence of coronary artery disease and was also correlated with disease severity (8). coronary artery disease was among the exclusion criteria for the patient and healthy control groups of our study. detection of the ag allele in all 120 subjects in our study suggests that nlrp3 rs9239 snp is not a risk factor associated with osa development. moreover, the absence of coronary artery disease in carriers of the a allele raises the need for more comprehensive studies evaluating the correlation between this snp and coronary artery disease. research into the suppression of the strong proinflammatory effect of nlrp3 highlighted the activity of vaspin, an adipose tissue-derived serine protease inhibitor. vaspin suppresses the activation of the nlrp3 inflammasome and also reduces il-1 and tnf-alpha synthesis, in which nlrp3 plays an important role. in vitro studies demonstrated that vaspin also reduces the generation of mitochondrial free radicals (28). analysis of plasma vaspin levels in osa patients showed that after excluding those with diabetes mellitus and coronary artery disease, the vaspin level was lower among osa patients than in controls (12). in another study, vaspin levels increased in correlation with bmi, waist circumference, waist/hip ratio, triglyceride level, and fasting blood glucose (28). in that study, which had no group homogenization in terms of parameters that may affect vaspin level, it was observed that vaspin increased in correlation with osa severity and regressed with cpap therapy. studies on the vaspin rs2236242 snp have shown that the frequency of type 2 diabetes mellitus was higher in individuals with the a allele than the control group (13). in a study of women in upper egypt, the a allele was found to be protective against obesity and diabetes mellitus, and carriers of this allele had lower plasma vaspin concentrations (29). in another study on its association with coronary artery disease, there was a significant correlation between the at genotype and coronary artery disease, while the tt genotype was more frequent in controls. when compared in terms of plasma vaspin levels, the tt genotype had relatively higher vaspin levels than the at genotype, but as there were only three patients with the aa genotype, the analysis was weak (14). the osa group in our study, which did not include patients with diseases associated with rs6242 snp, such as coronary artery disease and diabetes mellitus, showed a higher frequency of the aa genotype compared with the control group. the at genotype was detected at a higher rate in the control group compared with the osa group, suggesting that this genotype may be protective against osa. the statistically significant difference caused by increased at genotype frequency in the control group and increased aa frequency in the osa group observed in the hw equilibrium analysis also supported this. however, in the analysis adjusted for obesity and gender, this difference disappeared. when this is evaluated together with studies, suggesting that the a allele may be protective against obesity, it can be expected that bmi may be higher in those with the at genotype compared to the aa genotype. analysis of patients according to osa severity showed that the aa genotype was more frequent in the severe group. in addition, serum vaspin levels were lower in individuals with the aa genotype compared with those with tt and at genotype. this result can also be interpreted as evidence that vaspin, which has been reported as anti-inflammatory in studies conducted with osa, is synthesized at lower levels in individuals with the a allele. this, in turn, may increase the susceptibility to osa, in which impaired inflammatory/antiinflammatory balance plays an important role. 6 b. kerget et al. although patients with comorbidities were excluded in order to avoid their effect on nlrp2 rs9239 and vaspin rs6242 snp genotype analysis, the most important limitation of our study was the inability to ensure bmi homogeneity between the osa and control groups. this obscured the susceptibility and protective effect of the vaspin rs6242 snp between the  control and osa groups. large-scale studies with bmi-homogeneous samples are needed to further elucidate this relationship. in summary, there was no difference in the osa development according to nlrp3 level, whereas the osa was significantly more prevalent among individuals with the rs6242 aa genotype for vaspin. osa was more severe in individuals with the aa genotype. oxidative stress and endothelial dysfunction caused by this condition may lead to more critical conditions in osa such as coronary artery disease, diabetes mellitus, and cerebrovascular diseases. therefore, vaspin rs6242 genotype analysis in osa patients may help to closely monitor these patients in the early period and prevent possible comorbidities. disclosure statement the authors declare that they have no conflicts of interest. notes on contributors buğra kerget, md. pulmonology specialist at the department of  pulmonary diseases, assistant professor at the ataturk university, erzurum, turkey. ferhan kerget,  md. infectious disease specialist at the department of infectious diseases and clinical microbiology, health sciences university erzurum regional education and research hospital , erzurum, turkey. çiğdem yüce kahraman, md. medical genetic specialist at the department of medical genetic, assistant professor at the ataturk university, erzurum, turkey. alperen aksakal,  md. pulmonology specialist at the department of pulmonary diseases, health sciences university erzurum regional education and research hospital , erzurum, turkey. ömer araz,  md. pulmonology specialist at the department of  pulmonary diseases, professor at the ataturk university, erzurum, turkey. orcid buğra kerget https://orcid.org/0000-0002-6048-1462 ferhan kerget https://orcid.org/0000-0002-5160-4854 çiğdem yüce kahraman https://orcid.org/0000-0003-1957-9596 alperen aksakal https://orcid.org/0000-0001-6883-3314 ömer araz https://orcid.org/0000-0002-3476-4506 references 1. kapur vk, auckley dh, chowdhuri s, kuhlmann dc, mehra r, ramar k, et al. clinical practice guideline for diagnostic testing for adult obstructive sleep apnea: an american academy of sleep medicine clinical practice guideline. j clin sleep med. 2017;13:479–504. doi: 10.5664/ jcsm.6506 2. bhushan b, guleria r, misra a, 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children’s health, uppsala university, akademiska sjukhuset, uppsala, sweden abstract background: the purpose of this study was to investigate whether treatment of a depressive episode with intermittent theta burst stimulation (itbs) over the dorsomedial prefrontal cortex (dmpfc) had any effects on heart rate variability (hrv). we also investigated if changes in hrv covaried with symptom change after itbs and if hrv could predict symptom change. methods: we included 49 patients with a current depressive episode. all were randomized to receive a double-blind treatment course with active or sham itbs over the dmpfc. hrv data were obtained from 1 h of night data before and after the itbs. the standard deviation of the rr interval (sdnn) was chosen as primary outcome measure. depressive, negative, and anxiety symptoms as well as self-rated health were assessed by clinicians or by self-report. results: the group×time linear mixed model revealed no effect of itbs on sdnn (estimate = −1.8, 95% confidence interval [ci]: −19.9 to 16.2). there were neither correlations between hrv and depressive, negative, or anxiety symptom change after itbs nor with self-assessed health. no predictive value of hrv was found. conclusions: treatment for depression with dorsomedial itbs had neither negative nor positive effects on the cardiac autonomic nervous system. article history received 3 august 2022 revised 9 november 2022 accepted 31 january 2023 published 27 march 2023 keywords depressive disorder; noninvasive brain stimulation; autonomic nervous system; sympathetic; parasympathetic; heart– brain connection introduction lower levels of heart rate variability (hrv) are generally associated with a wide range of diminished health aspects (1, 2). the interest for hrv in depression stems to a large extent from the association between depression and the risk of cardiovascular disease (3, 4). unmedicated patients with depression exhibit significantly lower levels of hrv when compared to healthy controls, with effect sizes ranging from –0.096 to –0.462 (5). medicated patients with depression have even lower hrv, probably partly due to the medications’ anticholinergic properties (6–10). it seems that hereditary factors do not contribute to the low hrv in depression, but psychosocial or life-style factors might explain part of the differences (5, 11, 12). whether lower hrv mainly constitutes a state or a trait marker in depression is thus still an open question, and another way of approaching this issue has been to investigate if changes in depressive symptoms are accompanied by changes in hrv. for pharmacological or psychotherapeutic interventions in depression, it appears that symptom change is unrelated to changes in hrv, even if there are inconsistencies in the literature (8, 13). these findings favor the role of hrv as a trait marker in  depression. initiation or cessation of antidepressant pharmacological treatment has, however, been shown to decrease or increase hrv, respectively – further pointing out the important role of medication effects (10). repetitive transcranial magnetic stimulation (rtms) (14, 15) is a non-pharmacological treatment option for depression and might therefore provide an opportunity to study depressive symptom change and hrv without confounding medication effects. various rtms treatment protocols are being used, of which intermittent theta burst stimulation (itbs) (16) has been of special interest since it can reach the same efficacy with a shorter duration of the daily treatment protocol (17). the most commonly used treatment target for rtms in depression is the dorsolateral prefrontal cortex (dlpfc), which is part of the central autonomic modulation network, also including the dorsomedial prefrontal cortex (dmpfc), insula, amygdala, and other structures (18). the anterior cingulate cortex (acc) is another area implicated in central autonomic modulation, and it is located just beneath the dmpfc (19). using angled magnetic stimulation coils allows for modulating the acc when applied over the dmpfc (20). it could therefore be expected that stimulation of both the dlpfc and the dmpfc would affect cardiac autonomic regulation. indeed, while rtms seems to increase hrv during the actual stimulation in healthy individuals (21–24), there is a scarcity of studies on the cardiac autonomic effects of rtms in depression following a full treatment course. an open study reported increased hrv after rtms (25) but the only sham-controlled study measuring the concomitant effect of itbs found no enduring effects on contact johan bengtsson johan.bengtsson@neuro.uu.se © 2023 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article http://dx.doi.org/10.48101/ujms.v128.8949 mailto:johan.bengtsson@neuro.uu.se http://creativecommons.org/licenses/by/4.0/ 2 j. bengtsson et al. hrv, albeit acute changes during the itbs session, thus converging with the findings in healthy individuals (26). broadening the diagnostic perspective to schizophrenia, a recent large, multi-center and sham-controlled study found no effects of an rtms treatment course on heart rate (27). regarding hrv and concomitant symptom change in depression after rtms, there are only few reports of such associations. the open study mentioned above reported a correlation between posttreatment depression scores and post-treatment hrv, but there were no comparisons with baseline values (25). another study reported a non-significant correlation between heart rate decelerations during the first treatment session and greater symptom reduction (26). this finding has been argued to open up for heart rate decelerations as a predictor for treatment response in depression (28), but to the best of our knowledge, there are no studies on hrv as a predictor for treatment response after rtms. the results of hrv as a predictor of treatment response for other treatment options in depression have hitherto been mixed (29–31). in summary, there is only one open study reporting an increase in hrv after a full treatment session of rtms. this finding would be strengthened if replicated with a shamcontrolled design. also, since other treatment options for depression, such as tricyclic antidepressant agents (tcas), clearly diminish hrv, a lack of such negative treatment side effects of rtms would be beneficial. the present study aimed at investigating the effect on hrv of a treatment course with itbs, targeting the dmpfc in patients with depression. further aims were to investigate the correlation between symptom change and change in hrv and also to investigate whether baseline hrv would predict symptom change. materials and methods participants the 49 participants were a subsample of patients from a randomized controlled trial (clinicaltrials.gov identification number nct02905604) of dorsomedial prefrontal itbs for negative symptoms in schizophrenia or depression, and from an add-on brain-imaging study (32, 33). the transdiagnostic approach was based on the observed overlap between negative and depressive symptoms (34). inclusion criteria were originally designed for the above-mentioned trial, but for the current study, they were as follows: written informed consent, being 18–59 years old, a diagnosis of unior bipolar depression established by a psychiatrist in clinical routine care and verified through a mini international neuropsychiatric interview (m.i.n.i., swedish translation of version 6.0.0) (35), less or equal than 40 points on the motivation and pleasure scale-self-report (map-sr) (36), and no changes in medication during the past month. exclusion criteria were standard rtms such as metals implanted in the head, epilepsy, pacemakers, vagus nerve stimulators, medication pumps, etc. (37). other exclusion criteria were any condition implicating a substantial risk of non-compliance or loss of follow-up, active substance use disorder (except nicotine and caffeine), and pregnancy. the patients with schizophrenia recruited to the main trial were excluded from the current study (n = 16). see figure 1 for a flow chart. the study was conducted in accordance with the helsinki declaration and approved by the research ethical review board in uppsala. the trial was conducted between 2016 and 2020 at uppsala university hospital in sweden. excluded (n= 46) ♦ not meeting inclusion criteria (n = 20, including 16 with schizophrenia) ♦ declined to participate (n = 24) ♦ other reasons (n = 2) assessed for eligibility (n = 98) randomized (n = 52) enrollment allocation follow-up analysis allocated to active itbs (n = 26) ♦ received allocated intervention (n = 26) allocated to sham itbs (n = 26) ♦ received allocated intervention (n = 26) discontinued intervention (adverse event) (n = 1) analysed (n = 23 + 1 last observation carried forward) ♦ excluded from analysis (incomplete hrv data) (n= 2) discontinued intervention (withdrew consent) (n = 1) analysed (n = 25) figure 1. consort flow chart of the subsample constituting the current study. http://clinicaltrials.gov no effects of itbs on hrv in depression 3 rtms procedures the dmpfc was localized with neuronavigation (33) or defined as 25% of the distance between the nasion and the inion (n = 11). the itbs was delivered with a magnetic stimulator magpro x100. the coil was an angled combined active/placebo coil (cool-db80 a/p) with two identical sides, of which one side was shielded, so that the majority of the magnetic field did not reach the participant. upon entering a randomization code into the stimulator, the operator was instructed by the software which side to position toward the participant’s head (tangentially to the scalp, handle toward the right side of the participant). resting motor threshold was defined as the lowest magnetic stimulator output needed for a visually observable muscle contraction in the musculus extensor hallucis longus in the foot in 50% of the trials, determined by an automated maximum likelihood strategy (38, 39). the active itbs was a modified version of earlier protocols (39, 40), with 20 trains of stimulation with right-left stimulation and 20 trains with left-right stimulation. a second identical treatment session was delivered after a 15 min break (41, 42). stimulation was applied at 90% of resting motor threshold with a total of 2,400 pulses per day. the sham itbs comprised an identical protocol but with the shielded side of the coil toward the participant, whereby only a weak magnetic field was applied. in addition, two transcutaneous electrical nerve stimulation (tens) electrodes were placed under the coil of both the active and sham participants’ foreheads. in order to also mimic the sensation of the magnetic stimulation in the sham group, a current of maximum 4 ma (scaled to the magnetic stimulator output intensity) was delivered synchronous with the magnetic pulses. the treatment was delivered by a research nurse, and the symptom raters were not present during the treatment sessions. stimulation intensity was ramped up to minimize discomfort, aiming at 10 consecutive weekdays of treatment at target intensity (32). heart rate variability the two-electrode heart rate recorder firstbeat bodyguard 2 (firstbeat technologies ltd., jyväskylä, finland) was used to monitor heart rate. the device was attached across the chest by a research nurse on the morning the day before the first treatment day. after this visit, the participants wore the device until the next day, also during the night, and returned it when they came back for their first treatment session. the same procedure was repeated the day after the last treatment, 2 weeks after baseline. sampling rate was 1,000 hz with a resolution of 1  ms. since the aim of this study was to assess the potential lasting effects on hrv, an hour of data were extracted during the night (midnight to 5 am). this hour was chosen from a period where heart rate seemed stable, when inspected visually. the chosen hour varied between subjects depending on visual inspection of where there were fewest artifacts. this hour could also vary between each participant’s baseline and follow-up data, but was aimed to be the same. within the chosen time period, the following hrv metrics were calculated: the standard deviation of the rr interval (sdnn), the root mean square of successive rr interval differences (rmssd), high frequency hrv (hf), low frequency hrv (lf), lf/hf ratio, and the rr triangular index (2). lf and hf were logaritmized. as an open study has previously reported changes in sdnn after rtms, this metric was chosen as the primary outcome measure to enable comparisons (25). if artifact correction was necessary after visual inspection, the inbuilt algorithm in the software kubios hrv standard (version 3.0.2) was used. the algorithm compares the length of each interbeat interval (ibi) to an average of the surrounding ibis and classifies ibis that differ from a selected threshold as potential artifacts. in a subset of patients (n = 37), we also extracted hrv data from a 5-min daytime period when the participants were resting, sitting in a supine position just after the device was attached. this data collection was performed both the day before the first treatment session (baseline) and 4 weeks after baseline. from this period, we extracted hf-hrv data. these data were used for a separate analysis of the lasting effects of itbs on hrv. the data were only available in a subset of patients due to slightly different designs of the scheduled time frames of the main trial (32) and the add-on brain imaging study (33). this was done in order to evaluate the effect of itbs on two different standard hrv metrics – sdnn and hf-hrv. clinical assessments since the original trial aimed at treating negative symptoms with a transdiagnostic approach, patients were assessed at the baseline visit (the day before the first treatment day) by a trained physician with the clinical assessment interview for negative symptoms  (cains) (43, 44) and the brief psychiatric rating scale  – extended (bprs-e) (45). participants also filled in selfreports: sociodemographic data, nicotine consumption, the alcohol use disorders identification test (audit) (46), the drug use disorders identification test (dudit) (47), montgomeryåsberg depression rating scale – self-report (48), and selfassessed health with the eq-vas (49). the same interviews and symptom ratings were repeated the day after the last treatment session (i.e. 2 weeks from baseline), and also 4 weeks after baseline. after that, the concealed treatment allocation was unblinded, and participants who had been receiving sham itbs were offered an open treatment course. statistics all data were assessed for normality by visual inspection of histograms, q-q plots, and with the shapiro–wilk test. means and standard deviations were reported if variables were found to be normally distributed and medians and inter-quartile ranges if the data distributions were assessed to be skewed. to investigate differences in demographic variables between the groups, we used the independent-samples t-test for continuous normally distributed variables, mann–whitney u-test for continuous non-normally distributed variables, and chi square or fisher’s exact test for dichotomous and rank variables. 4 j. bengtsson et al. in the primary analysis, we used a linear mixed model using maximum likelihood estimation with random intercept per subject to assess group and time effects of itbs on sdnn (or hfhrv in the subsample of 37 patients). for the correlation between hrv metrics and symptom change, pearson correlation coefficients were computed to assess correlations between delta values of symptoms and delta values of sdnn. the symptom ratings from the assessment 2 weeks after baseline were used, except for eight participants, where the ratings 4 weeks after baseline were used (due to slightly different time frames for the participants also participating in the brain imaging study mentioned above). to assess the predictive value of baseline sdnn on symptom change, we used a linear regression model with delta values of symptoms as the dependent variable and baseline sdnn as the independent variable. here, we only analyzed the active itbs data from both the 25 participants receiving active itbs at first and the data from the participants from the sham group who decided to enter an open phase with active itbs (n = 17), resulting in a sample of 42 participants. the procedures for the itbs sessions, hrv assessments, and symptom ratings were identical for both the active and sham groups. the symptom ratings from the assessment 2 weeks after baseline were used, except for six participants, where the ratings 4 weeks after baseline were used (due to the same reason as mentioned above). we assessed linearity between the variables through visual inspection. the presence of influential outliers was also inspected visually as well as with the cook’s distance. multicollinearity was assessed with the variance inflation factor. the durbin–watson statistic was used to test the independency of the residuals’ values, and pp plots were used to inspect the distribution of the residuals. the residuals’ variance indicated no severe heteroscedasticity. spss version 26 was used for the statistical calculations. results demographics all demographic data are presented in table 1. overall, the two randomized groups were similar, but there were more patients in the sham group with a significant difference regarding body mass index (bmi) and prescription of positive chronotropic drugs. however, there were no significant differences in baseline sdnn between the groups (independent t-test, t = 0.29, p = 0.774). for bmi, a linear regression model revealed no significant impact of bmi on baseline sdnn (β = 0.016, p = 0.914). there was one participant in the active group on antidiabetic treatment. hrv and symptom data see table 2 for hrv and symptom burden data. the sham group had lower heart rate both at baseline and at follow-up. the difference was borderline significant at baseline (independent sample t-test, p = 0.052) and significant at follow-up (p = 0.028). there were no significant differences in  sdnn between the groups, neither at baseline nor at follow-up. regarding the effect of itbs on sdnn, there was no interaction effect of group×time (estimate = −1.8, 95% confidence interval [ci]: −19.9 to 16.2). no effects on the secondary hrv variables were noted. there were no significant symptom reduction and no correlations between change in sdnn and symptom change on table 1. demographic and clinical characteristics (n [%] if not stated otherwise). variable active (n = 25) sham (n = 24) pa age (years), median (iqr) 27 (14) 27 (12) 0.674 sex, n (female/male) 14/11 14/10 0.869 bmi (kg/m2), median (iqr) 25 (12) 23 (7) 0.040 bipolar depression 1 (4) 3 (13) 0.349 comorbidity anxiety disorders 9 (36) 11 (46) 0.484 comorbidity neuropsychiatric disorders 6 (24) 8 (33) 0.470 supported housing 2 (8) 5 (21) 0.247 level of education primary school 3 (12) 5 (21) 0.463 high school 12 (48) 15 (63) 0.308 higher education 10 (40) 3 (13) 0.051 in current employment or studying 15 (60) 15 (63) 0.858 sheehan disability scale, median (iqr) 19 (10) 18 (11) 0.865 nicotine useb 7 (28) 9 (38) 0.478 audit total score, median (iqr) 3 (3) 5 (6) 0.643 dudit total score, median (iqr) 0 (8) 0 (3) 0.940 psqi sleeping time (h), mean (sd)c 8 (2) 7 (2) 0.096 baseline hrv data starting time, mean time a.m. (sd in min) 2:28 (68) 2:39 (58) 0.529 follow-up hrv data starting time, mean time a.m. (sd in min) 2:38 (71) 2:42 (67) 0.701 difference between starting times (baseline – follow-up hrv data), min, mean (sd) 22 (29) 27 (41) 0.611 prescriptions ssri 6 (24) 5 (21) 0.791 snri 7 (28) 9 (38) 0.478 tca 5 (20) 2 (8) 0.417 antidepressant combination 10 (40) 7 (29) 0.426 lithium 6 (24) 4 (17) 0.725 antipsychotic 4 (16) 5 (21) 0.725 positive chronotropic drugd 2 (8) 9 (38) 0.018 negative chronotropic drugd 1 (4) 4 (17) 0.189 iqr: interquartile range; bmi: body mass index; sd: standard deviation; audit: alcohol use disorders identification test; dudit: drug use disorders identification test; psqi: pittsburgh sleep quality index; ssri: selective serotonin reuptake inhibitor; snri: serotonin and norepinephrine reuptake inhibitors; tca: tricyclic antidepressant agent; hrv: heart rate variability. aindependent-samples t-test/mann–whitney u-test for continuous variables, chi square for dichotomic variables, and fisher’s exact test if n < 5 in any cell with dichotomic variables. btobacco or swedish snuff. ctwo missing. dpositive chronotropic drugs included dexamphetamine, levothyroxine, and methylphenidate. negative chronotropic drugs included betablockers, guanfacine, and thiamazole. no effects of itbs on hrv in depression 5 cains, madrs-s, bprs anxiety subscale, or self-assessed health. in the correlation analyses, we analyzed the active and sham groups together, but subgroup analyses of the active and sham group separately revealed no differences (data not shown). see figure 2 for a graphical presentation of the correlations. regarding the predictive value of sdnn, there was no effect of baseline values of sdnn on any of the symptom categories, see table 3. the main analysis of the effect of itbs on hrv was also run in the subsample of patients (n = 37) with 5 min daytime resting hrv data available (before the treatment course and 4  weeks after baseline). in this model, we used logarithmic hf-hrv as the dependent variable. the results did not differ substantially from the main analysis (estimate = −0.24, 95% ci: −1.60 to 1.12). discussion to the best of our knowledge, this is so far the largest study investigating the effect on hrv after a treatment course of itbs targeting the dmpfc in depression. we did not detect any significant effects on the hrv metrics. neither there were any correlations between change in hrv and symptom change detected, nor there was any predictive value of baseline hrv on symptom change. first, it has to be pointed out that we specifically assessed hrv before and after the itbs treatment course and therefore cannot draw any conclusions about whether itbs has an acute and transient effect on hrv. comparisons with studies on healthy individuals are hindered due to the fact that no healthy  individuals receive longer treatment courses. of the figure 2. scatter plots for delta values of symptoms and delta values of hrv. note that a positive value of eq-vas indicates health improvement, whereas positive values on the other scales represent increased symptom load. r = pearson correlation coefficient for the regression line (similar results using spearman’s correlation coefficients). hrv: heart rate variability; sdnn: standard deviation of the rr interval; cains: clinical assessment interview for negative symptoms; madrs-s: montgomery asberg depression rating scale self-rating; bprs: brief psychiatric rating scale; eq-vas: euroqol group visual analogue scale. asum of items anxiety and tension. table 2. effect of itbs on hrv and symptoms. variable baseline follow-up group×time estimate ci lower ci upper active (n = 25) sham (n = 24) active (n = 25) sham (n = 24) ibi (ms) 868.6 (147.5) 941.0 (132.5) 857.6 (132.5) 933.3 (131.0) −3.3 −110.3 103.6 hr (bpm) 71.0 (11.8) 65.0 (9.0) 71.4 (9.9) 65.4 (8.5) 0.0 −7.8 7.8 sdnn (ms) (chosen outcome) 37.7 (26.5) 39.9 (27.1) 35.6 (18.2) 39.6 (18.7) −1.8 −19.9 16.2 rmssd (ms) 37.1 (35.2) 40.8 (39.5) 32.9 (23.2) 37.6 (27.1) −1.0 −26.0 24.1 loglf 6.1 (1.3) 6.2 (0.9) 6.1 (1.4) 6.4 (0.8) −0.2 −1.1 0.7 loghf 5.7 (1.6) 5.8 (1.6) 5.7 (1.4) 5.8 (1.4) 0.0 −1.2 1.2 lf/hf 2.1 (1.9) 2.3 (1.7) 2.1 (2.1) 2.8 (2.2) −0.5 −2.0 1.1 rr triangular index 10.2 (7.8) 10.9 (7.5) 9.7 (4.6) 10.5 (5.2) −0.3 −5.3 4.8 artifacts corrected (% of beats removed) 1.3 (5.7) 0.1 (0.2) 0.4 (1.5) 0.3 (0.4) −1.1 −3.4 1.3 symptom ratings cains total score 27.4 (7.8) 31.1 (7.4) 21.2 (9.8) 28.6 (8.2) −3.7 −10.2 2.8 madrs-s total score 29.2 (8.3) 30.1 (7.3) 24.8 (9.6) 28.0 (9.4) −2.3 −9.1 4.6 bprs anxiety subscalea 6.7 (2.9) 7.7 (2.6) 6.0 (2.6) 6.4 (2.6) 0.7 −1.5 2.8 eq-vas 33.8 (16.8) 34.8 (15.0) 43.8 (19.3) 40.4 (16.4) 4.4 −8.9 17.8 means and standard deviations for descriptive values. estimates and 95% confidence intervals for linear mixed model results. itbs: intermittent theta burst stimulation; hrv: heart rate variability; ci: confidence interval; ibi: interbeat interval; ms: milliseconds; hr: heart rate; bpm: beats per minute; sdnn: standard deviation of the rr interval; rmssd: square root of the mean squared differences of successive rr intervals; rr: ; loglf: logarithmic low frequency; loghf: logarithmic high frequency; cains: clinical assessment interview for negative symptoms; madrs-s: montgomery asberg depression rating scale self-rating; bprs: brief psychiatric rating scale; eq-vas: euroqol group visual analogue scale. asum of items anxiety and tension. 6 j. bengtsson et al. sham-controlled studies on healthy individuals, there have been no effects that lasted over time (22, 50–54). regarding depression, the only earlier sham-controlled study investigated the acute effects of itbs in 15 patients with depression and mainly focused on heart rate decelerations, which complicates comparisons with our investigation of the enduring effects of a treatment series with itbs (26). concerning hrv in that study, it was found that hrv was significantly more affected by active itbs than sham. the sham condition though (shielded coil placed over the vertex) did not produce any comparable pain stimuli as the active itbs, which leaves the possibility to open that these observations are connected to the pain effects of the stimuli, since arousal effects have been shown to be important (55). the treatment target in the same study was the dlpfc and not the dmpfc as in our study. even if both of these structures are connected to the acc, which is an important component of the central autonomic network (18, 19), it cannot be ruled out that differential locations implicate separate effects on hrv. indeed, a study from the same research group indicates this (56). nevertheless, taken together, these differences hamper further comparisons between the two studies. as for non-sham-controlled studies of the effect of rtms on hrv, an open study found increased sdnn between baseline and follow-up 2 weeks later in 30 patients with depression treated with rtms (targeting dlpfc using 5-cm-rule, 15 hz, 100% of resting motor threshold (rmt), 1,500 pulses per session). they assessed hrv before and after the treatment, which possibly eliminates the potential confounding effects of the simultaneous pain or sensational stimuli of rtms. however, since the study was not sham-controlled, there can be no speculations of the specific effects of rtms on hrv (25). in our study, we did not observe any changes at all, regardless of active or sham treatment. however, our studies differ in many aspects. the sample in the study by udupa et al. consisted of drug-naïve patients without psychiatric or somatic comorbidities and with a milder depression score rating at baseline, whereas our sample consisted of patients on medication, with comorbidities and higher baseline depression rating scores. the hrv assessments also differ in that we used an hour of night data, whereas their study used 5 min of resting data as well as different assessment conditions. comparisons between the studies must therefore be cautious. regarding the correlation between hrv change and symptom change, we did not find any such correlations. in the literature, this question has been of interest as a way of approaching the causal interactions between hrv and symptom change. in an open study of 27 patients receiving rtms, there was no correlation between symptom reduction and hrv or other autonomic parameters (57). adding three patients to the same sample yielded a correlation between follow-up values of lf/hf and depressive symptoms at follow-up, but there were no comparisons with baseline values (25). we could not find any predictive value of hrv regarding treatment response. here, we analyzed all open treatment sessions in our study (n = 42). earlier efforts of using hrv as a predictor of treatment response in depression have not led to any conclusive results, even if there are positive findings of other treatment modalities scattered in the literature (29, 30). results from the only sham-controlled rtms study investigating this issue in 15 patients with depression could not find any correlations between decelerations of heart rate early in the treatment course and clinical response after the treatment, even if there was a trend toward such an effect (26). more research is needed before hrv can achieve acceptable prediction properties in the field of depression treatment. there are some limitations of our study. even though our sample is, to our knowledge, the so far largest sample investigating the effects of a sham controlled treatment course of itbs on hrv, it is still a small sample size. it has been argued that interpreting cis is better than post-hoc analyses to judge the robustness of ‘null’ results (58). our results yielded quite wide cis for all the outcome measures (see table 2). the cis for sdnn spanned from a decrease by approximately 20 ms to an increase by 16 ms. it can therefore be stated that itbs at least does not affect sdnn more or less than that. regarding the hrv data extraction, we could not ascertain that the participants actually slept. however, the time frame of midnight to 5 am is a time when most people do sleep, and the participants in both groups reported a sleeping time of around 7 h. furthermore, the mean starting time for the hrv data did not differ between the groups. the baseline and the follow-up hrv data were in some cases extracted from different times during the night, but the mean difference was only 25 min in the whole sample (see table 1). however, hrv might also fluctuate according to sleep phase, which we were unable to assess (59). this might have introduced uncontrolled variability and thus be a source for our negative findings. our converging results from the subsample analysis of a standard 5 min hrv recording however strengthen our results somewhat. future studies assessing hrv during night would merit from a more detailed sleep phase assessment. we chose sdnn as our primary outcome. our main interest  has not been the parasympathetic nervous system in isolation, and sdnn originates from both sympathetic and parasympathetic signaling (60). sdnn is encouraged for use in longer recordings, even if a 24-h recording would have been preferable. furthermore, it has the advantage of being more easily interpreted and is also used in the important prediction table 3. hrv as a predictor for symptom change after itbs. variable sdnn b beta p cains 0.061 0.146 0.355 madrs-s −0.022 −0.055 0.732 bprs anxietya 0.004 0.031 0.843 eq-vas 0.063 0.078 0.621 linear regression results. dependent variable: delta values of symptoms (n = 42). hrv: heart rate variability; itbs: intermittent theta burst stimulation; sdnn: standard deviation of the rr interval; cains: clinical assessment interview for negative symptoms; madrs-s: montgomery asberg depression rating scale self-rating; bprs: brief psychiatric rating scale; eq-vas: euroqol group visual analogue scale. asum of items anxiety and tension. no effects of itbs on hrv in depression 7 study of hrv and cardiovascular risk (61). in the main analysis, we also analyzed a subsample (n = 37) using the vagal measure of hf-hrv, and the results did not differ to that of sdnn. some argue that baseline statistical comparisons between the groups in randomized data are not necessary (62). however, if the study sample is restricted, there is still a risk of imbalance between the groups. in the current study, there were no significant differences between the treatment groups regarding known baseline factors that might affect the outcome. we therefore refrained from controlling for factors such as age and sex. the participants’ medications were quite diverse, possibly reflecting the heterogeneity of the treatment-resistant depression concept and the aim of recruiting a clinically representative sample. importantly, all drug regimens were kept stable during this study, and even though these medications could have an impact on hrv, it is therefore unlikely that they would contribute to a change in hrv during the study period. some drugs prescribed (most notably tcas) could cause a high occupancy of the muscarinic receptors in the heart and might thereby diminish the vagal possibilities to modulate heart rate, thus rendering these participants unable to change their hrv. there were however no substantial differences in tca prescription between the active and the sham groups. eight participants’ symptom ratings originated from 4 weeks after baseline rather than 2 weeks after baseline. however, at least some of the symptoms assessed are not subject to rapid fluctuations but rather quite stable. furthermore, excluding these participants did not materially change the results (data not shown). the use of delta values in correlational analyses may introduce measurement error from both baseline and follow-up assessments with subsequent unreliability. our results from these analyses are therefore to be interpreted with caution, and the reader is referred to figure 1 for a visual interpretation. our sample consisted of patients recruited to a transdiagnostic study of the effects of itbs on negative symptoms. the results are therefore not necessarily generalizable to a wider group of patients with depression. the patients who come in contact with rtms are however often labeled as ‘treatment resistant’, and even if these patients can be defined in many ways (63, 64), our sample is probably comparable to this group. some of these patients are already exposed to treatments with well documented effects on their cardiac autonomic nervous system functioning, for example, tcas (6). our results indicate that rtms lacks such effects. we conclude that treatment with dorsomedial itbs did not increase hrv, but it did not have any negative effects on the cardiac autonomic nervous system. acknowledgment we wish to acknowledge statistician hans arinell for help with the statistical analyses. disclosure statement the authors report no conflict of interest. funding this study was funded by unrestricted research grants from the  märta and nicke nasvell foundation, professor bror  gadelius  foundation, selander foundation, söderströmkönigska foundation, and linné foundation. rb was supported by the swedish research council under grant 2016-02362, and the gullstrand research fellow grant from uppsala county council. jp was supported by the swedish brain foundation. jb  was also supported by the lennander foundation and granted conference attendance from anna maria lundin’s scholarship committee. notes on contributors johan bengtsson, conceptualization, methodology, formal analysis, investigation, writing – original draft, visualization. erik olsson, conceptualization, methodology, writing – review & editing. jonas persson, conceptualization, methodology, writing – review & editing. robert bodén, conceptualization, methodology, investigation, writing – review & editing, supervision. orcid johan bengtsson https://orcid.org/0000-0002-6043-0761 erik olsson https://orcid.org/0000-0002-1591-7407 jonas persson 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islets frozen in the presence of either a simple salt solution (hanks' balanced salt solution) or a complete tissue culture medium (rpmi 1640). moreover, the addition of 1 0 % human serum to the freezing solutions was evaluated. collagenase isolated islets were kept in culture for three days, before being cooled at a rate of 5oc/min or 25oc/min t o -7ooc, at which temperature the islets were transferred t o liquid nitrogen. all freezing media were supplemented with 2 m dimethylsulphoxide as cryoprotectant. the islets were rapidly thawed a t 37oc and subsequently cultured for another three days. the recovery of islets was higher when the more rapid cooling rate was used and the addition of serum further improved t h e recovery. compared to non-frozen cultured islets there was a loss of cells in all groups of cryopreserved islets, as measured by their d n a content, and this was accompanied by a lowered insulin content. all groups of frozen-thawed islets responded to a high glucose stimulus in vitro w i t h a 5-9 fold increase in insdin secretion. there was no obvious advantage of using a complete tissue culture medium for islet cryopreservation, but the addition of serum had some beneficial effects. data obtained from non-frozen control islets suggest that human serum slightly impairs the function of mouse pancreatic b-cells. introduction we have recently demonstrated that cryopreserved mouse pancreatic islets cooled at a rapid rate (25oc/min) are able to synthesize (pro)insulin and release insulin, in response t o glucose i n vitro, at rates not different from those of non-frozen cultured control islets (1 5). such cryopreserved islets significantly reduced alloxan-induced hyper glycemia when transplanted into syngeneic mice. i t was also found that the function of more rapidly cooled islets was better preserved than that of islets cooled at a rate of 5oc/min. in two previous studies we observed that a prerequisite for a successful 177 cryopreservation of isolated i s l e t s is t h a t t h e i s l e t s a r e maintained f o r a period in c u l t u r e b e f o r e f r e e z i n g (12,161. the aim of t h e present s t u d y was to i n v e s t i g a t e w h e t h e r t h e viability of cryopreserved p a n c r e a t i c i s l e t s could be improved by using a c o m p l e t e tissue c u l t u r e medium (rpmi 1640)(10) instead of a simpler salt solution (hanks' balanced salt solution)(z) during t h e f r e e z i n g procedure. w e have r e c e n t l y found t h a t supplementation of t h e c u l t u r e medium with human serum promotes t h e development of islet-like s t r u c t u r e s in v i t r o (14) when using a r e c e n t l y described method f o r t h e c u l t u r e of human f e t a l p a n c r e a s (13). f u t u r e a t t e m p t s t o cryopreserve human i s l e t s intended f o r t r a n s p l a n t a t i o n to human insulin -dependent diabetics may involve t h e addition of human serum to media. therefore, we examined t h e e f f e c t s of adding human s e r u m n o t only t o t h e f r e e z i n g medium but also t o t h e medium used during c u l t u r e of t h e i s l e t s before and a f t e r freezing. materials and methods islet isolation, c u l t u r e and cryopreservation. p a n c r e a t i c islets w e r e isolated by a collage nase digestion method (5) from male, adult nmri mice (anticimex ab, sollentuna, swe den). prior to f r e e z i n g t h e i s l e t s were kept f o r t h r e e days free-floating in t i s s u e c u l t u r e medium rpmi 1640 (flow laboratories ltd., irvine, scotland) supplemented with 100 u/ml benzylpenicillin ( a s t r a lakemedel, sodertalje, sweden), 0.1 mg/ml s t r e p t o m y c i n (glaxo laboratories, greenford, england) and 1 0 % pooled h e a t i n a c t i v a t e d human s e r u m (v/v) (blood c e n t e r , university hospital, uppsala, sweden). the c u l t u r e s w e r e maintained a t 37oc in a n a t m o s p h e r e of humidified a i r + 5% c 0 2 . t h e c u l t u r e medium was changed a f t e r t w o days. cooling was performed using a programmable t e m p e r a t u r e controller (planer mini f r e e z e r , model r 202/200r, planer products ltd., sunbury-on-thames, england). groups of about 75 i s l e t s w e r e t r a n s f e r r e d at room t e m p e r a t u r e t o s t e r i l i z e d glass ampoules containing 0.4 ml f r e e z i n g medium. the medium was e i t h e r hanks' solution or medium rpmi 1640 with or without 1 0 % (v/v) human serum. in all f r e e z i n g media 2 m dimethyl sulphoxide (me2so; sigma chemicals, st louis, mo, usa) was added as c r y o p r o t e c t a n t . first, t h e i s l e t s w e r e cool'ed at a r a t e of 8oc/min from room t e m p e r a t u r e t o o°c where t h e t e m p e r a t u r e was maintained for 20 min t o allow p e r m e a t i o n of t h e c r y o p r o t e c t a n t . cooling was t h e n resumed at e i t h e r 5oc/rnin or 25oc/min down t o -7ooc, a f t e r which t h e ampoules w e r e immersed in liquid nitrogen. a f t e r s t o r a g e f o r 60 min at -196oc t h e i s l e t s w e r e rapidly t h a w e d by s t i r r i n g t h e glass ampoules in a 37'c w a t e r bath. the resulting warming rate was about 240°c/min. when t h e ice of t h e f r e e z i n g medium had just m e l t e d t h e islets, with t h e f r e e z i n g medium w e r e poured i n t o c u l t u r e dishes containing 1 0 ml of t h e s a m e c u l t u r e medium as t h a t used before t h e freeze-thawing experiments. this l e a d to a rapid dilution of t h e me2so t o less t h a n 0.08m. finally, 5 m l of c u l t u r e medium w a s removed f r o m t h e dishes and 178 t h e i s l e t s w e r e c u l t u r e d f o r a n o t h e r 3 days before testing. in a previous study w e have shown t h a t t h i s me2so concentration does not impair t h e b-cell function of i s l e t s main t a i n e d in c u l t u r e in t h e presence of me2so (11). the non-frozen c o n t r o l islets w e r e obtained from t h e s a m e islet isolations as t h e frozen-thawed islets, but maintained in t i s s u e c u l t u r e in medium rpmi 1640 + 10% human serum f o r 6 days, with medium exchanges every second day. islet recovery. the islet recovery a f t e r cryopreservation was c a l c u l a t e d by counting t h e number of islets in a stereomicroscope just before t r a n s f e r to t h e f r e e z i n g ampoules and subsequently on day t h r e e a f t e r thawing. the recovery of t h e non-frozen c o n t r o l i s l e t s was c a l c u l a t e d by counting t h e i s l e t s present in t h e c u l t u r e dishes on day 3 and day 6 of culture. islet dna c o n t e n t . on day 3 a f t e r thawing i.e. on e x p e r i m e n t a l day 6, frozen-thawed and c o n t r o l i s l e t s in grou'ps of 30 w e r e ultrasonically disrupted in 0.2 ml redistilled water. the dna c o n t e n t of t h e resulting aqueous homogenates w a s measured by fluorophoto m e t r y (4,7) islet insulin c o n t e n t . a 50 p1 f r a c t i o n of t h e islet homogenates was mixed with 125 p1 of acid e t h a n o l (0.18 m hc1 in 96% (v/v) ethanol), e x t r a c t e d overnight at +4oc and t h e n s t o r e d at -2ooc. t h e insulin c o n c e n t r a t i o n in t h e s a m p l e s was d e t e r m i n e d by radio immunoassay (31, using mouse crystalline insulin as s t a n d a r d (novo, copenhagen, denmark) and 1251-labelled insulin as tracer (novo). islet insulin release. t r i p l i c a t e groups of 10 i s l e t s w e r e incubated f o r 2 consecutive hours at 37oc (02:coz; 95:5) in a slowly shaking w a t e r b a t h in s e a l e d glass vials ( 6 ) containing 0.25 m l of a krebs-ringer bicarbonate buffer (8), supplemented with 2 mg/ml bovine albumin (miles laboratories, slough, england) and 10 mm n-2-hydroxyethylpipera zine-n'-2ethanesulphonic acid (hepes; sigma), h e r e a f t e r r e f e r r e d t o as krbh. during t h e first hour t h e incubation medium contained 1.67 mm glucose. a f t e r t h i s incubation t h e medium was c a r e f u l l y removed and r e p l a c e d by 0.25 ml of krbh supplemented with 16.7 mm glucose. t h e insulin c o n c e n t r a t i o n in t h e incubation media was d e t e r m i n e d by radioimmunoassay as described above. s t a t i s t i c a l analysis. r e s u l t s a r e expressed as means t sem. groups of d a t a w e r e compared using student's unpaired t-test. results in g e n e r a l t h e recovery of cryopreserved i s l e t s was higher a f t e r cooling at a r a t e of 25oc/min as compared t o t h a t a f t e r cooling at 5oc/min (table 1). the highest values 12-878572 179 c i m 0 t a b l e 1. e ff ec ts o f hu m an s er u m ( h s) a dd it io n an d c oo li ng r at e on i sl et r ec o v er y , is le t d n a c o n te n t an d i sl et i ns ul in c o n te n t of m ou se p an cr ea ti c is le ts c ry op re se rv ed i n th e p re se n ce o f 2 m m e2 so . f re ez in g c on di ti on is le t re co ve ry (c oo li ng r at e; m ed iu m ) (% ) is le t d n a c o n te n t (u g d n a /1 0 is le ts ) is le t in su li n co n te n t (n g in su li n/ lo i sl et s) 5o c /m in ; h an ks ' 62 .5 f 8 .8 (7 )* * 0. 33 f 0 .0 41 (7 )* * 71 .5 t 11 .7 (7 )* ** 5o c /m in ; h an ks ' + 1 0% h s 71 .5 ? 9 .0 (7 ) 0. 30 ? 0 .0 28 (7 )* ** 72 .8 t 1 1. 7 (7 )* ** 5o c /m in ; r p m i 16 40 41 .2 f 1 0. 7 (7 )* ** 0. 41 ? 0 .0 37 (6 ) 50 .7 ? 9 .0 (7 )* ** 5o c /m in ; r pm j 16 40 + 1 0% h s 68 .6 4 9 .5 (7 ) 0. 33 ? 0 .0 29 (7 )* * 67 .4 ? 1 3 (7 )* ** 80 .2 ? 1 5 (7 )* ** 25 o c /m in ; h an ks ' 78 .5 f 4 .2 (7 )' 0. 30 f 0 .0 22 (7 )* ** 25 o c /m in ; h an ks + 1 0% h s 89 .0 f 2 .0 (8 ) 0. 35 ? 0 .0 22 (8 )* * 1 0 8 4 7 .8 (8 )* ** 25 o c /m in ; r p m i 16 40 74 .7 f 2 .0 (8 )* *+ + 0. 37 t 0 .0 23 (8 )* 85 .8 ? 5 .6 (8 )* ** 25 o c /m in ; r p m i 16 40 + 1 0% h s 85 .6 4 2 .0 (8 ) 0. 38 f 0 .0 25 (8 )* 93 .0 ? 1 0 (8 )* ** n on -f ro ze n cu lt u re d i sl et s 87 .2 ? 2 .6 (9 ) 0. 48 f 0 .0 33 (9 ) 22 3 f 1 5 (9 ) p an cr ea ti c is le ts w er e is ol at ed f ro m n m r i m ic e an d cu lt u re d f o r th re e da ys i n m ed iu m r p m i 16 40 + 1 0 % h s pr io r to f re ez in g. t he is le ts w er e fr o ze n a t th e co ol in g ra te a nd i n th e m ed iu m g iv en i n th e f ir st c ol um n. a ft er s to ra g e fo r 12 ho ur s at 19 6o c t h e f ro ze n sa m p le s w er e ra pi dl y th aw ed a t 37 o c a nd t h e is le ts c u lt u re d f o r an o th er t h re e da ys . n on -f ro ze n is le ts f ro m t h e s am e is ol at io ns se rv ed a s co nt ro ls . t h e nu m be r of i sl et s w er e co u n te d i n a st er eo m ic ro sc o p e ju st b ef o re t ra n sf er t o th e f re ez in g a m po ul es , an d su b se qu en tl y on d ay t h re e af te r th aw in g, a nd t h e is le t re co v er y w as e xp re ss ed a s th e p er ce n ta g e of i sl et s re m ai ni ng . t h e re co v er y o f th e c o n tr o l is le ts w as c al cu la te d b y co m pa ri ng t h e n um be r of i sl et s p re se n t on d ay 3 a nd d ay 6 o f cu lt ur e. i sl et d n a c o n te n t w as m ea su re d fl uo ro -p ho to m et ri ca ll y in w at er h om og en at es o f th e is le ts , an d th e in su li n co n te n ts a ss es se d by r ad io im m un oa ss ay o f ac id et h an o l ex tr ac ts o f th e i sl et s. d at a ar e gi ve n as m ea ns ? s e m , w it h nu m be r of o bs er va ti on s w it hi n pa re nt he si s. * , * * an d ** * d en o te p< o .o 5, p <o .o l an d p< o .o o l re sp ec ti ve ly , co m p ar ed t o n on -f ro ze n is le ts u si ng u np ai re d tte st . + an d + ' d en o te p (0 .0 5 an d p< o .o l w he n co m pa ri ng i sl et s fr o ze n a t th e sa m e co ol in g ra te a nd i n th e s am e sa lt s ol ut io n bu t w it h or w it ho ut h s, u si ng u np ai re d tte st . t a b l e 2 . e ff ec ts o f h u m an s er u m ( h s ) ad d it io n a n d c oo li ng r at e on g lu co se -s ti m u la te d i ns ul in r el ea se o f m ou se p an cr ea ti c is le ts c ry o p re se rv ed i n t h e p re se n ce o f 2 m m e2 s o . f re ez in g c on di ti on (c o o li n c ra te : m ed iu m ) i n s u l i n r e l e a s e 1. 67 m m d u c o se 16 .7 m m e tlu co se s t i m u la t i on f ac to r " (n g in su li n/ lo i sl et s x 60 m in ) (n g in su li n/ lo "i sl et s x 60 m in ) (1 6. 7 vs 1 .6 7 m m g lu co se ) 5o c /m in ; h an ks ' 5o c /m in ; h an ks ' + 1 0 % h s 5o c /m in ; r p m i 16 40 s °c /m in ; r p m i 16 40 + 1 0 % h s 25 o c /m in ; h an ks ' 25 °c /m in ; h an ks ' + 1 0 % h s 25 o c /m in ; r p m i 1 6 4 0 25 o c /m in ; r p m i 1 6 4 0 + 1 0 % h s n on -f ro ze n cu lt u re d i sl et s 1. 9 f 0 .2 2. 0 f 0 .5 1. 6 f 0 .4 2. 9 f 0 .6 3. 4 f 0 .8 3. 3 f 0 .4 2. 9 f 0 .5 3. 7 f 0 .7 12 .2 f 3 .0 12 .5 f 4 .1 9. 8 f 2 .1 11 .2 f 4 .5 12 .5 f 3 .1 20 .5 f 5 .5 19 .9 t 3 .3 22 .0 f 4 .0 27 .8 f 7 .8 41 .7 ? 4 .9 6. 3 f 2 .1 5. 0 f 0 .8 5. 9 f 1 .3 4. 3 f 0 .7 6. 3 f 1 .2 7. 5 f 1 .1 8. 4 f 1 .2 7. 4 & 1 .8 5. 0 f 1 .2 t h e gr ou ps o f is le ts w er e tr ea te d a s d es cr ib ed i n t ab le 1 . i sl et i ns ul in r el ea se e x p er im en ts w er e p er fo rm ed b y in cu b at in g th e i sl et s in g ro up s of t e n i n k r b h b u ff er c o n ta in in g 1 .6 7 m m g lu co se a n d a lb um in ( 2 m g/ m l) a t 37 o c ( 0 2 :c 0 2 , 95 :5 ). a ft er 6 0 m in t h e m ed iu m w as r em o v ed a n d t h e i sl et s in cu b at ed f o r an o th er 6 0 m in i n m ed iu m c o n ta in in g 1 6. 7 m m g lu co se . t h e st im u la ti o n f ac to r w as d et er m in ed i n e ac h i nd iv id ua l ex p er im en t by c al cu la ti n g t h e r at io o f in su li n re le as e b et w ee n th e s ec o n d a n d f ir st i n cu b at io n h ou r. d at a ar e g iv en a s m ea n s f s e m , w it h n u m b er o f o b se rv at io n s w it hi n p ar en th es is . *, ** a n d * ** d en o te p < 0. 05 , p< o .o l an d p <o .o o l re sp ec ti v el y , co m p ar ed t o n on -f ro ze n is le ts u si ng u n p ai re d t -t es t. + , + + an d + + + d en o te p < 0. 05 , p< o .o l an d p <o .o o l w he n co m p ar in g t h e s am e g ro u p o f is le ts i n cu b at ed a t 1. 67 m m a n d 1 6. 7 m m gl uc os e, u si ng u np ai re d tte st . w e r e obtained when human serum was added t o e i t h e r hanks' solution or rpmi 1 6 4 0 used f o r f r e e z i n g islets at a r a t e of 25oc/min. in t h e s e t w o cases t h e r e c o v e r y figures w e r e very close t o t h a t of t h e non-frozen c o n t r o l islets. t h e number of i s l e t s d e c r e a s e d by approximately 13% during c u l t u r e between day 3 and day 6. in all test situations t h e addition of human serum yielded a higher r e c o v e r i e s when c o m p a r e d t o t h e s a m e group of i s l e t s f r o z e n without s e r u m , t h e dna c o n t e n t in all groups of cryopreserved i s l e t s was reduced by 20-30% (table 1). t h e addition of human serum to t h e f r e e z i n g medium did not a f f e c t t h e islet dna content. the insulin c o n t e n t of t h e cryopreserved islets was lowered to 50% or less of t h e value found in t h e non-frozen c u l t u r e d i s l e t s (table 1). t h e addition of human s e r u m t o t h e f r e e z i n g media slightly increased t h e insulin c o n t e n t of t h e i s l e t s in t h e various groups of f r o z e n islets, however t h e d i f f e r e n c e s did not a t t a i n s t a t i s t i c a l signifi cance. t h e basal insulin release of t h e non-frozen c o n t r o l i s l e t s at 1.67 mm glucose, was e l e v a t e d compared t o all groups of frozen-thawed i s l e t s (table 2). when t h e i s l e t s w e r e challenged with high glucose (16.7 mm) t h e insulin s e c r e t i o n was increased in all groups of i s l e t s by 5-9 times. however, in t h e t w o groups of i s l e t s cooled at a rate of 5'c/min, in t h e absence of human s e r u m , t h e high-glucose s t i m u l a t e d insulin release was not significantly d i f f e r e n t from t h e basal secretion. t h e r a t e of insulin release at t h e high glucose c o n c e n t r a t i o n was lower in t h e cryopreserved i s l e t s t h a n in t h e con trols, e x c e p t f o r t h e i s l e t s cooled at a r a t e of 25oc/min in rpmi 1 6 4 0 plus human serum. i t should be noted t h a t t h e stimulation f a c t o r of insulin r e l e a s e in response to t h e high glucose challenge in t h e c o n t r o l i s l e t s was fairly low d u e t o t h e high basal insulin release. discussion cryopreservation a p p e a r s to be t h e only s u i t a b l e method f o r prolonged s t o r a g e of isolated p a n c r e a t i c i s l e t s in v i t r o intended f o r t r a n s p l a n t a t i o n t o insulin-dependent diabetics. during t h e last 5-1 0 y e a r s a number of d i f f e r e n t cryopreservation protocols have been presented employing various cooling r a t e s , modes of addition and r e m o v a l of t h e c r y o p r o t e c t a n t and warming rates (for a review see bank, (1)). although t h e o r e t i c a l calculations c a n be made on how a particular cell preparation should b e cryopreserved (91, when designing t h e o p t i m a l freeze-thawing conditions f o r a given cell population t h e d i f f e r e n t s t e p s in t h e cryopreservation procedure must o f t e n be t e s t e d separately. this communication i s s u c h a s t e p in t h e e f f o r t s to find t h e best cryopreservation proce d u r e for isolated mouse p a n c r e a t i c islets. in t h i s c o n t e x t i t was also of i n t e r e s t t o s t u d y t h e e f f e c t of human s e r u m addition to t h e f r e e z i n g medium, s i n c e t h i s biological supple ment will most probably b e used in conjunction with human islet cryopreservation. overall, t h e use of rpmi 1640 compared to hanks' solution as t h e f r e e z i n g medium a f f o r d e d of no c l e a r a d v a n t a g e f o r t h e viability of t h e frozen-thawed islets. the addition 182 of human s e r u m increased t h e islet r e c o v e r y a f t e r cryopreservation, which i n d i c a t e s t h a t t h e p r e s e n c e of s e r u m h a s a beneficial e f f e c t for t h e survival of t h e islet cells during t h e freeze-thawing manoeuvres. t h e reason for t h i s is obscure. i t could be specula t e d t h a t serum h a s a supportive role f o r islet viability a s during tissue c u l t u r e , but it cannot be excluded t h a t f a c t o r s in t h e s e r u m have c r y o p r o t e c t i v e properties. t h e islet insulin c o n t e n t was markedly reduced a f t e r cryopreservation, which confirms our previous findings (12,15). however , when comparing t h e insulin c o n t e n t of t h e c o n t r o l i s l e t s cultured with 1 0 % human serum with t h a t of i s l e t s c u l t u r e d in t h e presence of 10% calf serum (12,151, i t is obvious t h a t human serum r e d u c e s t h e insulin c o n t e n t s of mouse p a n c r e a t i c islets. i t may b e t h a t human serum is slightly t o x i c to mouse islets, leading t o a passive insulin s e c r e t i o n or a l t e r n a t i v e l y a n impaired insulin biosynthesis. the finding of a n increased insulin s e c r e t i o n by t h e c o n t r o l i s l e t s at t h e low glucose c o n c e n t r a t i o n speaks in favour of a n e f f e c t on t h e s e c r e t o r y apparatus. if t h e addition of 10% human serum t o i s l e t s in c u l t u r e slightly impairs t h e i r viability, i t c a n be e x p e c t e d t h a t t h e i r functional c a p a c i t y will b e f u r t h e r reduced by cryopreservation, s i n c e t h e prefreezing s t a t u s of t h e islets is c r u c i a l f o r t h e i r ability t o r e s i s t t h e stresses of t h e freeze-thawing process. in conclusion t h e d a t a of t h e present s t u d y suggest t h a t t h e use of a c o m p l e t e t i s s u e c u l t u r e medium (rpmi 1640) as f r e e z i n g medium does not i n c r e a s e t h e viability of cryo preserved p a n c r e a t i c islets. on t h e o t h e r hand, t h e addition of 10% human serum t o t h e f r e e z i n g medium supported t h e survival of t h e cryopreserved islets, but human s e r u m may, to s o m e degree, impair t h e viability of mouse p a n c r e a t i c i s l e t s in culture. w e have, however, found t h a t t h i s supplement may s t i m u l a t e t h e g e n e r a t i o n of p a n c r e a t i c b-cells from human f e t a l p a n c r e a t i c glands (14). acknowledgements w e t h a n k eva forsbeck, astrid nordin and margareta engkvist f o r e x c e l l e n t t e c h n i c a l assistance and agneta snellman for c a r e f u l preparation of t h e manuscript. financial support from t h e swedish medical r e s e a r c h council (12x-109; 17x-7501; 12p-7680), t h e swedish diabetes association, t h e nordic insulin fund, t h e juvenile diabetes founda t i o n international, t h e novo company, t h e o.e. and edla johansson fund, t h e ake wiberg fund, ernfors diabetes foundation, t h e c l a s groschinsky memorial fund, t h e swedish society of medicine and t h e syskonen svenssons fond, is g r a t e f u l l y acknowledged. references 1. bank, h.l.: cryobiology of isolated i s l e t s of langerhans c i r c a 1982. cryobiology 2. hanks, j.m. & wallace, r.e.: relation of oxygen and t e m p e r a t u r e in t h e preservation of t i s s u e by refrigeration. proc.soc.exp.bio1.med. 71:196-200, 1949. 3. heding, l.g.: determination of t o t a l s e r u m insulin (ili) in insulin-treated patients. diabet ologia 8: 26 0 -266, 1 9 7 2. 4. hinegardner, r.t.: an improved f l u o r o m e t r i c assay for dna. anal.biochem. 39:197 5 . howell, s.l. & taylor, k.w.: potassium ions and t h e s e c r e t i o n of insulin by i s l e t s of langerhans incubated in vitro. bi0chem.j. 108:17-24, 1968. 20:119-128, 1983 -201, 1971. i83 6 . 7 . 8. 9. 1 0 . 1 1 . 1 2 . 1 3 . 1 4 . 1 5 . 1 6 . keen, h., field, j.b. & p a s t a n , i.h.: a s i m p l e m e t h o d f o r in v i t r o m e t a b o l i c s t u d i e s using s m a l l volumes of t i s s u e a n d medium. m e t a b o l i s m 12:143-174, 1 9 6 3 . kissane, j.m. & robins, e.: t h e f l u o r o m e t r i c m e a s u r e m e n t of deoxyribonuqleic a c i d in a n i m a l t i s s u e s w i t h s p e c i a l r e f e r e n c e to t h e c e n t r a l n e r o v u s s y s t e m . j.bio1. c h e m . 233:184-188, 1 9 5 8 . krebs, h.a. & h e n s e l e i t , k.: u n t e r s u c h u n g e n u b e r d i e h a r n s t o f f b i l d u n g im tierkor per. hoppe-seylers z. physiol.chem. 210:33-66, 1 9 3 2 . mazur, p.: f r e e z i n g o f living cells: m e c h a n i s m s a n d implications. am.j. physiol. moore, g.e., g e r n e r , r.e. & franklin, h.a.: c u l t u r e of n o r m a l h u m a n l e u k o c y t e s . j.am.med.assoc. 199:87-92, 1 9 6 7 . sandler, s. & a n d e r s o n , a.: short a n d l o n g t e r m e f f e c t s of d i m e t h y l s u l f o x i d e on m o u s e p a n c r e a t i c islet b-cell f u n c t i o n in vitro. cryobiology 19:299-305, 1 9 8 2 . sandler, s. & a n d e r s o n , a.: t h e s i g n i f i c a n c e o f c u l t u r e f o r s u c c e s s f u l c r y o p r e s e r v a t i o n of i s o l a t e d p a n c r e a t i c i s l e t s of langerhans. cryobiology 2 1 5 0 3 5 1 0 , 1 9 8 4 . sandler, s., a n d e r s o n , a., schnell, a., mellgren, a., t o l l e m a r , j., borg, h., p e t e r s son, b., g r o t h , c-g. & h e l l e r s t r o m , c.: t i s s u e c u l t u r e of h u m a n f e t a l pancreas: d e v e l o p m e n t a n d f u n c t i o n of b-cells in v i t r o a n d t r a n s p l a n t a t i o n of e x p l a n t t o n u d e mice. d i a b e t e s 34:1113-1119, 1 9 8 5 . s a n d l e r , s., a n d e r s o n , a., schnell l a n d s t r o m , a., t o l l e m a r , j., p e t e r s s o n , b., g r o t h , c-g. & h e l l e r s t r o m , c.: tissue c u l t u r e of h u m a n f e t a l p a n c r e a s : e f f e c t s of h u m a n s e r u m on t h e d e v e l o p m e n t a n d e n d o c r i n e f u n c t i o n of islet-like cell c l u s t e r s . manu s c r i p t , 1 9 8 6 . s a n d l e r , s., kojima, y. & a n d e r s o n , a.: c r y o p r e s e r v a t i o n of m o u s e p a n c r e a t i c islets: e f f e c t s of f a s t cooling o n islet b-cell f u n c t i o n in v i t r o a n d a f t e r islet t r a n s p l a n t a t i o n . t r a n s p l a n t a t i o n , i n press, 1 9 8 6 . sandler, s., nilsson, b., borg, l.a.h., s w e n n e , i., p e t e r s s o n , b., h e l l e r s t r o m , c. & a n d e r s o n , a.: s t r u c t u r e a n d f u n c t i o n of c r y o p r e s e r v e d m o u s e p a n c r e a t i c islets. in: islet isolation, c u l t u r e a n d c r y o p r e s e r v a t i o n . f e d e r l i n , k a n d b r e t z e l , r.g. (eds.) t h i e m e s t u t t g a r t , p p 1 3 8 1 5 1 , 1 9 8 1 . 247:c125-c142, 1 9 8 4 . a d d r e s s f o r c o r r e s p o n d e n c e : d r s t e l l a n s a n d l e r d e p a r t m e n t of medical c e l l biology biomedicum, p.o.box 571 s-751 2 3 uppsala, sweden 184 upsala journal of medical sciences 2021, 126, e7688 http://dx.doi.org/10.48101/ujms.v126.7688 mortality in eosinophilic esophagitis – a nationwide, population-based matched cohort study from 2005 to 2017 lovisa röjlera, john j. garberb, bjorn roelstraetec, marjorie m. walkerd and jonas f. ludvigssona,c,e,f adepartment of pediatrics, örebro university hospital, sweden; bgastrointestinal unit, massachusetts general hospital, harvard medical school, boston ma, usa; cdepartment of medical epidemiology and biostatistics, karolinska institutet, stockholm, sweden; ddepartment anatomical pathology university of newcastle faculty of health and medicine school of medicine and public health callaghan, nsw, australia; edivision of epidemiology and public health, school of medicine, university of nottingham, city hospital, nottingham, uk; fceliac disease center, department of medicine, columbia university college of physicians and surgeons, new york, new york, usa abstract background: there is a lack of knowledge about mortality in eosinophilic esophagitis (eoe). therefore, this study aimed to examine the mortality in eoe. methods: a nationwide, population-based matched cohort study was conducted of all eoe patients in sweden diagnosed between july 2005 and december 2017. individuals with eoe (n = 1,625) were identified through prospectively recorded histopathology codes from all gastrointestinal pathology reports in sweden, representing 28 pathology departments (the espresso study). each individual with eoe was then matched with up to five reference individuals from the general population (n = 8,003) for age, sex, year of birth, and place of residence. we used the cox proportional hazard modeling to estimate the adjusted hazard ratio (ahr) and 95% confidence interval (95% ci) while adjusting for other potential confounders. in sensitivity analyses, mortality in eoe patients was compared with mortality in their siblings. results: through december 2017, 34 deaths were confirmed in eoe patients (4.60 per 1,000 person-years) compared with 165 in reference individuals (4.57 per 1,000 person-years). this rate corresponds to an ahr of 0.97 (95% ci = 0.67–1.40). hrs were similar in males (ahr = 1.00 [0.66–1.51]) and females (ahr = 0.92 [0.38–2.18]). we observed no increased risk in mortality due to esophageal or other gastrointestinal cancers in patients with eoe (ahr = 1.02 [0.51–2.02]). mortality was similar in eoe patients and their siblings (ahr = 0.91 [0.44–1.85]). conclusion: in this nationwide, population-based matched cohort study in sweden, there was no increased risk of death in patients with eoe compared with their siblings and the general population. article history received 01 march 2021 revised 19 july 2021 accepted 20 july 2021 published 31 august 2021 keywords death; cancer; eosinophilic esophagitis; mortality; population-based introduction eosinophilic esophagitis (eoe) is a chronic inflammatory condition of the esophagus that is associated with recurrent food impaction and the development of esophageal strictures. the incidence of eoe appears to be increasing for reasons that are obscure (1). a meta-analysis of 18 studies describing the incidence of eoe in adults and children reported an overall incidence rate of 4.4 new cases of eoe per 100,000 individuals/ year (2), and the disease is now estimated to affect 1 in 2,000 persons in north america and europe (3, 4). despite the growing recognition of eoe as an important clinical entity associated with significant disease burden and health care costs (5), the natural history of eoe is not fully known. the most well-recognized complication of eoe is a progression from a predominantly inflammatory to a fibrostenotic phenotype (6), which is associated with the development of esophageal strictures or diffuse esophageal narrowing (7). straumann and colleagues described the clinical course of 30  adult eoe patients who were followed for an average of 7.2 years. the authors found no cases of esophageal malignancy or death from any cause (8). another study followed 13 eoe patients for an average of 13.6 years. similarly, this study did not observe any cases of malignancy or death during the 13.6-year follow-up (9). importantly, neither the us (10) nor international (11) guidelines and consensus documents specifically address mortality in eoe. mortality associated with eoe is presumed to be relatively low, but this has not been specifically examined in a large, geographically restricted cohort of eoe patients. inflammation has been hypothesized to play a role in the development of several chronic conditions associated with contact jonas f ludvigsson jonasludvigsson@yahoo.com supplemental data for this article can be accessed here. © 2021 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article http://dx.doi.org/10.48101/ujms.v126.7688 mailto:jonasludvigsson@yahoo.com http://dx.doi.org/10.48101/ujms.v126.7688 http://creativecommons.org/licenses/by/4.0/ 2 lovisa röjler et al. increased mortality, including cardiovascular disease (cvd) (12) and cancer (13). because eoe is a life-long disease that typically afflicts young, otherwise healthy individuals, patients may experience many years of persistent and chronic immune activation and inflammation. while the direct effects of longstanding esophageal inflammation are recognized to contribute to esophageal fibrosis (6), the long-term risk of mortality associated with eoe remains elusive. we, therefore, sought to examine the mortality in 1,625 patients with biopsy-verified eoe and to compare them with 8,003 reference individuals from the general population, as well as with 2,142 siblings of the 1,625 eoe patients. methods study population eosinophilic esophagitis swedish biopsy data are categorized according to the snomed-ct classification system (systematized nomenclature of medicine clinical terms), a standardized health care terminology used in many countries. through the previously described espresso cohort (14), we requested all electronic gastrointestinal (gi) histopathology reports of esophageal biopsies (t62) obtained at 28 centers across sweden from october 12, 2015 through april 10, 2017. we limited eoe to incident disease from july 1, 2005 onwards since data on medications were available only since that date. cases of eoe (n  = 1,625; figure 1) were identified based on the presence of inflammation that included eosinophils (m47150). data from our group show that the presence of eosinophilic inflammation on histopathology has a positive predictive value of 89% for the eoe diagnosis (15). for a more detailed review of our data collection, we refer to our previous publication on the espresso study (14). reference individuals each individual with eoe was matched with up to five age, sex, county of residence, and birth year reference individuals (n = 8,003) from the swedish total population register ( tpr) (16). reference individuals had to be free from eoe at the time  of matching but could still be included in the study if they developed eoe in the future. however, if they developed eoe later, their follow-up was excluded from the control group. sibling comparators we identified siblings of the eoe patients (n = 2,142) through the swedish multigeneration register, a sub-section of the tpr. sibling data were available on all individuals born after 1932 and who were registered as residents of sweden in 1961 or later. to minimize intrafamilial confounding (mainly genetic and early environmental factors) that could potentially influence both the risk of eoe and mortality, sibling comparators were examined. outcome measure date of death was retrieved from the tpr. this register is maintained by the swedish government and contains data on  life events, including birth, death, family relationships, and migration within as well as migration to and from sweden. it covers essentially 100% of deaths (16). the swedish cause of death register (17), a comprehensive and virtually complete record of all deaths in sweden since 1952, contains  data on the cause of death. our primary outcome was overall mortality, but we also examined death from cvd,  cancer, and other diseases (including gi and infectionrelated deaths). eoe, eosinophilic esophagitis. figure 1. flowchart of study participants. mortality in eosinophilic esophagitis 3 other covariates level of education was divided into the following categories: compulsory (≤9 years), upper secondary (10–12 years), and college or university (≥13 years). data were retrieved from the longitudinal integrated database for health insurance and labour market studies (lisa) (18). to assess the potential importance of drug treatment in eoe and mortality, we estimated separate hrs for death in eoe  patients with and without steroids and proton-pump inhibitors (ppis). medication data were available through the swedish prescribed drug register (19), and the analysis was, therefore, restricted to incident eoe patients from july 1, 2005 or later, when the register started. steroid use was defined as having an atc code of h02ab (systemic), r03ba01, r03ba02, r03ba05, r03ba08, and r01ad09 (all r-codes represented swallowed/topical steroids) from first eoe diagnosis or january 1, 2006 (we allowed half a year since the start of the register to make sure this was the first ever steroid prescription) up until death. we defined ppis similarly but through atc code a02bc. statistical analysis the study design was a population-based, nationwide matched cohort study. in our primary analysis, eoe patients and general population reference individuals were matched (1:5) at the time of diagnosis for age, sex, county of residence, and calendar year. study follow-up was done from the date of diagnosis of eoe (or index date for matched reference individuals) to the date of death, emigration, or end of follow-up on december 31, 2017, whichever came first. for the reference individuals, there was also the possibility that they might develop eoe during followup; if this occurred, they were excluded as reference individuals and moved to the eoe group. cox proportional hazard modeling was used to calculate the hazard ratio (hr) and 95% confidence interval (95% ci) for overall and cause-specific mortality while accounting for the matching variables. absolute risks (deaths per 1,000 person-years of follow-up) were calculated for the complete follow-up period. in exploratory analyses, the association was examined according to strata defined by years of follow-up (divided into three groups), age at first eoe diagnosis (<18, 18 to <50, and ≥50 years), sex, and education level (table 1). table 1. demographics for patients with eosinophilic esophagitis and reference controls (general population, normal biopsy, and siblings). eoe population reference individuals siblings n (%) n (%) n (%) total 1,625 (100.00) 8,003 (100.00) 2,142 (100.00) male 1,221 (75.14) 6,004 (75.02) 1,097 (51.21) female 404 (24.86) 1,999 (24.98) 1,045 (48.79) age at start of follow-up (years) mean (sd) 37.71 (20.34) 37.35 (20.16) 37.81 (19.81) median (iqr) 39.00 (19.00–53.00) 38.00 (19.00–52.50) 39.00 (21.00–53.00) <18 247 (15.20) 1,226 (15.32) 257 (12.00) 18 to <50 824 (50.71) 4,100 (51.23) 1,131 (52.80) ≥50 554 (34.09) 2,677 (33.45) 754 (35.20) years of follow-up (years) mean (sd) 4.55 (2.43) 4.51 (2.44) 4.59 (2.35) median (iqr) 4.09 (2.71–6.07) 4.07 (2.64–5.93) 4.21 (2.84–5.99) <1 32 (1.97) 210 (2.62) 49 (2.29) 1 to <5 996 (61.29) 4,886 (61.05) 1,283 (59.90) ≥5 597 (36.74) 2,907 (36.32) 810 (37.82) start of follow-up 2005–2011 421 (25.91) 2,075 (25.93) 547 (25.54) 2012–2013 436 (26.83) 2,155 (26.93) 630 (29.41) 2014–2015 566 (34.83) 2,782 (34.76) 717 (33.47) 2016–2017 202 (12.43) 991 (12.38) 248 (11.58) reason for end of follow-up death 34 (2.09) 165 (2.06) 35 (1.63) emigration 13 (0.80) 126 (1.57) 9 (0.42) december 31, 2017 1,578 (97.11) 7,709 (96.33) 2,092 (97.67) diagnosed with eoe 0 (0.00) 3 (0.04) 6 (0.28) education compulsory school (≤9 years) 254 (15.63) 1,511 (18.88) 315 (14.71) upper secondary school (10–12 years) 574 (35.32) 2,802 (35.01) 782 (36.51) college or university (≥13 years) 488 (30.03) 2,044 (25.54) 638 (29.79) no data 309 (19.02) 1,646 (20.57) 407 (19.00) eoe, eosinophilic esophagitis; sd, standard deviation; iqr, interquartile range. 4 lovisa röjler et al. in the secondary analysis, the rate of mortality in eoe patients was compared with their siblings and to patients with normal esophageal biopsies. sibling analyses were stratified as per family (one stratum per family). the power of this approach is that it automatically controls for covariates that are shared in the family (family situation, genetics, etc.). finally, we also explored mortality in eoe according to steroid use (see appendix for a list of relevant atc codes). in a posthoc analysis, we also compared mortality in eoe patients with systemic steroids vs those with swallowed/topical steroids. all analyses were adjusted for age, sex, county of residence, and year of biopsy. statistics were carried out using r statistical software (version 3.5.2; r foundation for statistical computing, vienna, austria) and the survival package (version 2.43, therneau, t (2015), https://cran.r-project.org/package=survival). statistical significance was set to p < 0.05. cis were computed by inversion of the likelihood ratio test statistic. ethics this study was approved by the stockholm ethics review board. an informed consent was waived by the board, given that the study was strictly register based (20). patient and public involvements patients or the public were not involved in the design, or conduct, or reporting, or dissemination plans of our research. results background data of eoe patients and reference individuals in total, 1,625 patients were diagnosed with eoe over 12 years (2005–2017) (table 1). the median age at diagnosis was 39 years (interquartile range [iqr] 19–53). half of the patients were between 18 and 50 years old, with 15.2% of eoe patients diagnosed before the age 18 and 34% being ≥50 years at the time of diagnosis. consistent with previous descriptions, an observed male predominance (75.1%) was noted. the median duration of follow-up was 4.09 years (iqr 2.71–6.07), with 42 (2.6%) eoe patients being followed for ≥10 years. some 370 (22.87%) of eoe patients had a record of steroid use after eoe diagnosis. a patient chart review from the espresso cohort has revealed that in a random subset of 54 eoe patients with data on the location of esophageal biopsy, 54% had biopsies taken both in the upper/mid part and the lower part of the esophagus (15). mortality among eoe patients and general population reference individuals in the eoe group, 34 deaths were confirmed (4.6 per 1,000 person-years) compared with 165 in population comparators (4.57 per 1,000 person-years) (table 2). these risk estimates correspond to an hr of 0.97 (95% ci = 0.67–1.40) (figures 2 and 3; etable 1 shows crude hrs and etable 2 shows adjusted hrs). individuals with eoe died from a wide range of causes, including cancer, cvd, pulmonary disease, and infections. in the group of older patients (≥50 years at diagnosis), there were 31 deaths, corresponding to an adjusted hr (ahr) of 0.98; 95% ci = 0.66–1.44. in the 18–49-year age group, there were three deaths (ahr = 0.98; 95% ci = 0.28–3.38), and in the younger age group (≤17 years), no deaths occurred (figure 3). mortality rates in patients with eoe did not differ by sex. of the males with eoe, there were 4.99 deaths (95% ci = 3.46–7.00) per 1,000 person-years versus 4.75 (95% ci = 4.00–5.60) per 1,000 person-years in male reference individuals (table 2), which corresponds to an ahr death of 1.00 (95% ci = 0.66–1.51) (figure 3). in females with eoe, the mortality rate was 3.36 (95% ci = 1.58–6.54) per 1,000 person-years compared with 3.99 (95% ci = 2.88–5.42) per 1,000 person-years in female reference individuals (table 2), which corresponds to an ahr death of 0.92 (95% ci = 0.38–2.18) in eoe patients versus population reference individuals (figure 3). education level did not influence risk estimates (strata according to attained education: ≤9 years: ahr = 1.39 (95% ci  =  0.73–2.63); 10–12 years: ahr = 1.08 (95% ci = 0.48–2.40); and ≥13 years: ahr = 1.10 (95% ci = 0.32–3.74)). length of follow-up was divided into three categories: <1, 1 to <5 years, and ≥5 years. there were no differences in mortality in any of these groups (<1 year ahr = 0.41 [95% ci = 0.13–1.33]; 1 to <5 years ahr = 1.09 [95% ci = 0.71–1.69]; and ≥5 years ahr = 1.59 [95% ci = 0.59–4.28]) (figure 3). in the most recent subset of patients (the 202 eoe patients diagnosed since january 2015), the ahr was 0.92 (95% ci = 0.27–3.20) (figure 3). similar ahrs were found when the followup was restricted to the first 2 years (to make the different calendar periods more comparable as an earlier follow-up would otherwise influence mortality much more in the later calendar period with the shortest follow-up, namely, 2015–17) (etable 2). cause-specific mortality rates of death were specifically analyzed for cvd and cancer (table 3). for the eoe patients, there were six deaths (per 7,400 person-years) attributable to cardiovascular causes, which corresponds to an ahr of 0.75 (95% ci = 0.32–1.79) as compared with reference individuals. there were 10 deaths due to cancer (per 7,400 person-years) in the eoe cohort, which corresponds to an ahr of 1.06 (95% ci = 0.53–2.11) compared with reference individuals. the number of deaths attributable to infections and gi causes was too few to allow meaningful analyses, and these were, therefore, grouped with all other causes, of which there were 18 cause-specific deaths (per 7,400 person-years) with an ahr of 1.01 (95% ci = 0.60–1.68). additional analyses finally, we compared 1,247 patients with eoe who had ≥1 sibling (n = 2,142) (figure 1). the mortality in the eoe patients did not https://cran.r-project.org/package=survival mortality in eosinophilic esophagitis 5 differ from that in their siblings (ahr = 0.91 [95% ci = 0.44–1.85]) (etable 2). adjusting our sibling analyses for education yielded a similar ahr (1.16; 95% ci = 0.54–2.46). restricting our cohort to eoe patients with a record of steroids, we found no association with death (hr = 0.98; 95% ci  = 0.43–2.22). an interaction test found a higher mortality in eoe patients with systemic steroids (hr = 2.01; 0.94–4.29) than in those with topical/swallowed steroids (hr = 0.14; 95% ci  =  0.02–1.05) (p = 0.016), but none of the risk estimates was statistically significant compared to the general population. the hr for death was 1.00 (95% ci = 0.66–1.51) in eoe patients without a record of steroid medication. discussion in this nationwide population-based matched cohort study of more than 1,600 patients with eoe, we found neither increased risk of death nor any increase in death from cancer or cvd. these results are reassuring for patients who often suffer from food impaction and feeding difficulties, in which ongoing table 2. mortality incidence rates with 95% ci per 1,000 person-years for patients with eosinophilic esophagitis and reference controls (general population, normal biopsy, and siblings). eoe population reference individuals siblings n total 1,625 8,003 2,142 n events 34 165 35 incidence proportion (%) 2.09 2.06 1.63 person-years 7,399 36,112 9,823 incidence rate/1,000 person-years (95% ci) 4.60 (3.30–6.26) 4.57 (3.92–5.29) 3.56 (2.57–4.84) years of follow-up <1 1.85 (0.67–4.46) 4.15 (2.96–5.68) 3.75 (1.93–6.76) 1 to <5 5.60 (3.80–7.99) 5.04 (4.19–6.03) 3.32 (2.16–4.93) ≥5 4.58 (2.15–8.91) 3.46 (2.30–5.05) 4.19 (2.07–7.81) sex males 4.99 (3.46–7.00) 4.75 (4.00–5.60) 3.61 (2.30–5.46) females 3.36 (1.58–6.54) 3.99 (2.88–5.42) 3.51 (2.20–5.37) age at start of follow-up (years) <18 0.00 (0.00–0.00) 0.21 (0.07–0.59) 0.46 (0.11–1.70) 18 to <50 0.88 (0.32–2.13) 0.90 (0.55–1.41) 0.83 (0.33–1.81) ≥50 14.74 (10.41–20.36) 14.73 (12.55–17.20) 10.71 (7.52–14.87) education compulsory school (≤9 years) 16.30 (10.36–24.65) 9.40 (7.38–11.81) 8.21 (4.74–13.47) upper secondary school (10–12 years) 3.73 (2.05–6.37) 4.69 (3.64–5.96) 4.19 (2.55–6.56) college or university (≥13 years) 2.40 (1.06–4.92) 2.38 (1.56–3.50) 1.41 (0.57–3.09) start of follow-up 2005–2010 3.60 (1.92–6.31) 4.12 (3.13–5.34) 2.18 (1.08–4.07) 2011–2014 5.59 (3.70–8.15) 5.19 (4.27–6.26) 4.43 (2.99–6.37) 2015–2017 3.11 (1.13–7.49) 3.18 (1.94–4.98) 3.34 (1.36–7.32) start of follow-up (with max 2 years of fu) 2005–2010 5.11 (1.86–12.30) 4.51 (2.65–7.27) 2.61 (0.81–7.28) 2011–2014 2.92 (1.29–5.99) 5.86 (4.44–7.61) 4.29 (2.36–7.33) 2015–2017 3.61 (1.31–8.69) 2.95 (1.70–4.83) 2.94 (1.07–7.07) 95% ci, 95% confidence interval; p-years, person-years; fu, follow-up. table 3. causes of death, number of events, number of person-years, and crude and adjusted hazard ratios. cause of death model reference population reference individuals siblings all hr 34/7.40 165/36.11, 1.01 (0.70–1.46) 35/9.82, 0.76 (0.38–1.51) ahr 34/7.40 165/36.11, 0.97 (0.67–1.40) 35/9.82, 0.91 (0.44–1.85) cardiovascular hr 6/7.40 40/36.11, 0.73 (0.31–1.73) 9/9.82, 0.00 (0.00–inf ) ahr 6/7.40 40/36.11, 0.75 (0.32–1.79) 9/9.82, 0.00 (0.00–inf ) cancer hr 10/7.40 45/36.11, 1.09 (0.55–2.15) 12/9.82, 0.81 (0.25–2.59) ahr 10/7.40 45/36.11, 1.06 (0.53–2.11) 12/9.82, 1.05 (0.31–3.61) other hr 18/7.40 80/36.11, 1.10 (0.66–1.83) 14/9.82, 1.28 (0.51–3.22) ahr 18/7.40 80/36.11, 1.01 (0.60–1.68) 14/9.82, 1.81 (0.62–5.27) hr, crude hazard ratio; ahr, adjusted hazard ratio. 6 lovisa röjler et al. inflammation, albeit local, often leads to strictures that cause mechanical obstruction. in the present study, cases of eoe were identified based on the presence of inflammation that included eosinophils. an accurate understanding of the long-term prognosis of eoe is critical to helping providers and patients decide among current medical and dietary treatments (21), as well as to guide priorities of wider research and therapeutic development. over the past decade, important progress has been made toward understanding the genetic and environmental basis of the disease (22). moreover, the definition and diagnostic criteria of eoe have evolved from this work (11). the natural history of eoe has been studied (23–25), and a range of possible outcomes described, with fibrostenotic progression being the primary complication of undiagnosed or untreated disease (6). however, one important area in the natural history of eoe that remains relatively unexamined is whether this disease carries an increased risk of mortality. mortality in eoe has been presumed to be low, but up to this point, long-term outcomes in eoe specific to mortality have only been described in a small number of eoe patients followed longitudinally (8, 9). through the espresso study (14), we identified more than 1,600 eoe patients and had an 80% power to detect a 25% increased risk of death at a significance level of 0.05. in the present study, we found no difference in overall mortality (hr = 0.97) in eoe patients when compared with reference individuals. in addition, the mortality rate in eoe patients did not appear to be significantly different in comparison with the general population based on the age at diagnosis, duration of followup or socioeconomic status. a high rate of concurrent atopic disease (60–70%) (26) occurs in eoe and eoe patients often have atopic dermatitis, asthma, or both (26). patients with severe atopic dermatitis may be at increased risk of death (27). of note, however, danish researchers have shown that (cardiovascular) mortality in atopic dermatitis is neutral in mild disease. meanwhile, the excess mortality seen for severe disease completely disappears with adjustment for background factors (28). multiple epidemiologic studies have confirmed increased mortality with asthma (29). although eoe  and asthma sometimes coexist and are mechanistically figure 3. incidence and adjusted hazard ratios for death in patients with eosinophilic esophagitis compared with general population reference individuals. shaded areas denote 95% confidence intervals. 95% cis are only given for eoe until 8 years after diagnosis since there were no deaths in this group after that. figure 2. kaplan–meier curves for death in eosinophilic esophagitis compared with general population reference individuals. mortality in eosinophilic esophagitis 7 overlapping atopic diseases, it has not yet been established where eoe fits in the spectrum of possible mortality. we have shown here that despite often severe symptoms, patients with eoe are at no increased risk of death or, at most, have a marginally increased risk (in this study, the upper 95% ci was 1.4). consistent with previous research (30), there appears to be no increased mortality due to cancers, including gi cancers (e.g. esophageal carcinoma). this finding is notable because one of the strongest risk factors for esophageal adenocarcinoma is barrett’s esophagus (31), which is thought to arise primarily from molecular genetic changes induced by chronic inflammation (acid reflux, alcohol, and smoking) in the esophageal epithelium (32). a close relationship and overlap between acid reflux and eoe have been reported, including clinical presentation and response to proton pump inhibitor (ppi) therapy (33, 34). one might expect that chronic inflammation, which is due to eoe, might also predispose to metaplastic changes giving rise over time to esophageal cancers. our data should provide clarity and reassurance to patients given that we observed no increased risk in mortality due to esophageal or other gi cancers in patients with eoe (hr = 1.02 [95% ci = 0.51–2.02]). the major strength of our study is that it represents the first effort to examine specifically mortality rates in eoe. second, it uses a large cohort of eoe patients identified through a validated histopathologic method from a nationwide population-based register. the cohort of 1,625 eoe patients that we identified through histopathologic criteria was highly representative of descriptions of eoe in the literature, including a 3:1 male-tofemale ratio, that the disease occurs most frequently in the early and middle decades of life, and with typical rates of exposure to conventional therapies, such as swallowed steroids (38%) and ppi antacids (87%) (15). a set of reference individuals from the general population was generated and matched for age, sex, country of residence, and year of birth. in addition to comparing eoe patients with the general population, unique access to the swedish multigeneration register (a part of the larger tpr) enabled the identification of 2,142 siblings of eoe patients to compare mortality. eoe is strongly driven by both genetic and environmental factors (35), which can be difficult to isolate because genetically related family members very often live in the same house and have shared dietary and environmental exposures. the use of sibling comparators in these analyses minimizes these overlapping and potentially confounding intrafamilial factors and allows better control over isolating the direct effect of having eoe on the risk of death, which was overall similar in individuals with eoe and their siblings. eoe was identified through a computerized search of all 28 pathology departments in sweden. thus, we were able to minimize selection bias that is often typical of tertiary referral centers. moreover, this study was preceded by a careful validation of the diagnosis in 111 individuals with a history of eoe. the positive predictive value (ppv) for eoe was 89% (15), and gastric reflux as a cause of eosinophilic inflammation was ruled out. an 89% ppv is similar to that found for physicianassigned diagnoses (85–95%) in the swedish national patient register (36), and because biopsy is a prerequisite for the diagnosis, the sensitivity for diagnosed eoe in sweden should be close to 100%. additional strengths include the completeness of the tpr (16) and the cause of death register (17) to ascertain the primary outcome of death. data on both eoe and death were prospectively recorded in swedish registers. some limitations of the study should be addressed. first, awareness of eoe in hospitals outside the university system has increased only recently, and because most of our cases were diagnosed after 2010, we had a limited follow-up (mean duration was 4.6 years). this follow-up is less than the only other existing studies that have examined mortality in eoe. in one study, 30 patients were followed for an average of 7.2 years (8) and in another 13 patients were followed for an average of 13.6 years (9). however, these studies only had an estimated total followup of roughly 216 and 177 person-years, respectively, which can be compared with a follow-up of 7,400 person-years in our study. importantly, 42 (2.6%) individuals in our study were actually followed for over 10 years. still, we acknowledge that our study has not only little information on long-term risk of death beyond 10 years with eoe but also limited power to examine follow-up specific hrs. other limitations are our lack of data on disease severity and symptoms. however, we found similar mortality hrs in those with and without steroid medications and, therefore, cannot draw any firm conclusions based on risk estimates; medications may also mirror disease activity itself. in conclusion, in the first nationwide study of more than 1,600 individuals with biopsy-verified eoe, we found no association between eoe and death. the overall low mortality associated with eoe was confirmed after adjusting for factors known to be associated with mortality risk, as well as by minimizing intrafamilial factors through analysis of a large cohort of eoe siblings. disclosure statement jonas f. ludvigsson coordinates a study on behalf of the swedish ibd quality register (swibreg). that study has also received funding from the janssen corporation. the other authors declare that they have no conflicts of interest. availability of data and materials other researchers can apply for our data through the swedish national board of health and welfare. funding this study has received funding from örebro university hospital and karolinska institutet. notes on contributors lovisa röjler, md. resident physician at the 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http://dx.doi.org/10.1136/gutjnl-2015-310991 http://dx.doi.org/10.1136/gutjnl-2015-310991 http://dx.doi.org/10.1016/j.jaci.2017.07.010 http://dx.doi.org/10.1016/j.jaci.2017.07.010 http://dx.doi.org/10.1186/1471-2458-11-450 http://dx.doi.org/10.1186/1471-2458-11-450 upsala journal of medical sciences 2022, 127, e8803 http://dx.doi.org/10.48101/ujms.v127.8803 increased accuracy in diagnosing diverticulitis using predictive clinical factors johanna sigurdardottira,b, abbas chaboka,b, philippe wagnerb and maziar nikberga,b adepartment of colorectal surgery, västmanlands hospital, västerås, sweden; bcentre for clinical research region, västmanland uppsala university, västerås, sweden article history received 29 may 2022 revised 4 october 2022 accepted 15 december 2022 keywords colonic diverticulitis; c-reactive protein; diagnosis; diverticular disease; scoring system introduction diverticular disease is a common disorder in the western world with over 50% prevalence in the 60 years and older age group (1). while it is among the most common causes for referrals to emergency departments (eds) (2), only about 25% of patients have the classic triad of symptoms and laboratory results: abdominal pain, fever, and leukocytosis (3, 4). according to previous reports, the clinical diagnosis has an accuracy of 34– 72% (5–9). the gold standard for diagnosing acute diverticulitis in the acute setting is laboratory work-up (white blood cell [wbc] count, neutrophilic count, and c-reactive protein [crp]), and a computed tomography (ct) scan (10). several differential diagnoses, such as irritable bowel syndrome, inflammatory bowel disease, and colonic malignancy, are important to differentiate from acute diverticulitis. ct is currently the most commonly used radiological method for diagnosing acute diverticulitis with high sensitivity (95%) and specificity (96%) (11). ct reveals possible complications and can exclude other possible diagnoses (12). however, it exposes the patient to radiation, and the intravenous contrast used might be allergenic and nephrotoxic. with the incidence and prevalence of acute diverticulitis increasing in younger patients (13), the risk of numerous ct scans increases along with radiation exposure. as many of these patients are diagnosed and treated in an outpatient setting, it is important to differentiate acute diverticulitis from other diagnoses. andeweg et al. (14) have proposed a scoring system for diagnosing acute colonic diverticulitis, and lameris et al. (8) have developed decision rule for aiding a clinical diagnosis of acute diverticulitis. however, to our knowledge, no scoring system or decision rule is in frequent use (8, 14). the purpose of this study was to identify predictors that increase clinical accuracy in diagnosing acute diverticulitis and formulate a risk score (rs) to predict the probability of acute diverticulitis. material and methods a database was used from a previous study by thorisson et al. (15), which focused on the radiological aspects of diagnosing acute colonic diverticulitis. the aim of that study was to evaluate whether contact j. sigurdardottir johanna.sigurdardottir@regionvastmanland.se © 2022 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article abstract background: the aim of this study was to identify clinical factors leading to increased diagnostic accuracy for acute colonic diverticulitis. methods: patients with clinical suspicion of acute colonic diverticulitis verified with computed tomography (ct) from two hospitals in sweden between 9 january 2017 and 31 october 2017 were prospectively included. symptoms, comorbidities, and laboratory results were documented. candidate variables were analyzed using logistic regression, and the final variable set that yielded the most accurate predictions was identified using least absolute shrinkage and selection operator regression and evaluated using the area under the receiver operating characteristic (roc) curve. results: in total, 146 patients were included (73% women; median age 68 years; age range, 50–94 years). the clinical diagnostic accuracy was 70.5%. in the multiple logistic regression analysis, gender (female vs male odds ratio [or]: 4.82; confidence interval [ci], 1.56–14.91), age (or, 0.92; 95% ci, 0.87–0.98), pain on the lower left side of the abdomen (or, 15.14; 95% ci, 2.65–86.58), and absence of vomiting (or, 14.02; 95% ci, 2.90–67.88) were statistically significant and associated with the diagnosis of ct-verified diverticulitis. with seven predictors (age, gender, urinary symptoms, nausea, temperature, c-reactive protein, and pain left lower side), the area under the roc curve was 0.82, and a formula was developed for calculating a risk score. conclusion: we present a scoring system using common clinical variables that can be applied to patients with clinical suspicion of colonic diverticulitis to increase the diagnostic accuracy. the developed scoring system is available for free of charge at https://phille-wagner.shinyapps.io/diverticulitis_risk_model/. http://dx.doi.org/10.48101/ujms.v127.8803 http://johanna.sigurdardottir@regionvastmanland.se mailto:johanna.sigurdardottir@regionvastmanland.se http://creativecommons.org/licenses/by/4.0/ https://phille-wagner.shinyapps.io/diverticulitis_risk_model/ 2 j. sigurdardottir et al. non-enhanced low-dose ct was as sensitive as standard-dose ct in detecting acute colonic diverticulitis. that was a prospective observational study, conducted between 9 january 2017 and 31 october 2017 in two hospitals in sweden (västmanlands hospital västerås and dalarnas hospital mora, a rural district hospital). together, these hospitals have a catchment area of 340,000 people. included patients underwent two ct scans (with and without contrast) and therefore included only patients 50 years and older. patients with a clinical suspicion of acute diverticulitis were eligible for inclusion. the inclusion criteria were as follows: patients with clinical suspicion of acute diverticulitis, wbc count above 10 × 10⁹/l or crp >25 mg/l (if wbc is not elevated), and age group 50 years and older. the exclusion criteria were as follows: pregnancy, contraindications to receive intravenous contrast medium such as renal failure or allergy, and patients who were unable for whatever reason to give informed consent (such as a language barrier or dementia). patients who met the inclusion criteria were asked to participate. inclusion was done in the ed as well as laboratory work-up and ct scans. clinical evaluation of patients on admission in the ed, symptoms were documented regarding abdominal pain; location of pain; anorexia; vomiting; changes in bowel movement; history of fever during the symptomatic period; duration of symptoms in hours or days; previous history of acute diverticulitis; comorbidities such as heart and vascular disease; lung, kidney or liver diseases; diabetes mellitus; or immunosuppression. the following results of the physical examination were documented by the on-call surgical resident on a data collection form: weight, height, vital parameters, signs of direct, indirect tenderness, and peritoneal irritation. the findings of the abdominal examination were documented on a four-grade intensity scale as none, slight, moderate, or strong tenderness. the patient was asked to mark his/her pain experience on a visual analogue scale (vas) where 0 is none and 10 is the worst pain imaginable. laboratory and radiological evaluation wbc count, neutrophilic count, and the concentration of crp were recorded. after examination by a surgeon or a resident in surgery, all patients in the study underwent a ct scan with iodine-based intravenous contrast material with an individualized dosage based on the patient’s age, height, and weight. all ct scans were performed using a 64-slice general electric optima ct 1,600 machine (ge healthcare, marlborough, ma, usa) (15). the contrast medium used was omnipaque™ (ge healthcare) with a concentration of 350 mg iodine/ml (15). findings on ct scans were assessed for signs of acute diverticulitis regarding inflamed diverticula of the colon, colonic wall thickness over  5 mm, pericolic fat stranding as well as complications due to acute diverticulitis and other diagnoses. complications of acute diverticulitis were defined as abscesses (intramural, pericolic, or pelvic collections) or perforations (extra luminal  gas located pericolic, retroperitoneal, or in the peritoneal cavity). statistics means and standard deviations (sds) were calculated for continuous variables. descriptive odds ratios (ors) generated by using standard logistic regression were used to quantify associations between independent variables and outcomes, both separate for each variable and combining all in a multivariable analysis. the area under the receiver operating characteristic (auc-roc) curve was calculated for each variable individually, including 95% confidence intervals (cis). the risk factors were analyzed through univariable and multiple regression to evaluate the separate and combined contribution of these risk factors in predicting the risk of acute diverticulitis and in developing an rs. l 1 penalized logistic regression, least absolute shrinkage and selection operator (lasso) was used to combine variables to create the rs, considering the relation between the number of figure 2. area under the receiver operating characteristic curve for the combined set of the seven best predictors of acute diverticulitis (age, gender, urinary symptoms, nausea, temperature in degrees celsius, crp >5, and pain on the lower left side). 146 patients with suspected acute left sided diverticulitis 103 patients with acute left sided diverticulitis 43 patients with other diagnoses figure 1. flowchart of the cohort.[aq6] contained perforations in 12, perforation with free air in three and abscess in six. the most common symptoms for patients with clinical suspicion for acute diverticulitis were left-sided abdominal pain (96%), nausea (33%), and change in bowel habits (obstipation 26% and loose bowels 25%), as shown in table 2. a history of previous diverticulitis was found in 41 patients (40%). most of the patients had a good eastern cooperative oncology group performance status (0–1 in 66% of patients). the mean age in the acute diverticulitis group was 66 years (sd 9.1), compared with 70 years (sd 10.4) in the group with other diagnoses. history of pain duration for patients with acute diverticulitis was 3.6 days (sd 2.8) compared with 4.2 days (sd 5.2) for those with another diagnosis. a statistically significant difference between groups was seen in gender (female vs male or, 4.82; 95% ci, 1.56–14.91), age (or, 0.92; 95% ci, 0.87–0.98), pain vs no pain on the lower left side of the abdomen (or, 15.14; 95% ci, 2.65–86.58), and absence vs presence of vomiting (or, 14.02: 95% ci, 2.90–67.88). table 3 shows the differences between groups. predictor selection and evaluation variables with statistically significant auc values were gender, absence of vomiting, moderate rebound tenderness, and crp >5 (table 3). the inclusion criteria for the laboratory results were wbc count > 10 × 10⁹/l and/or crp >25 mg/l. to identify patients with only increased wbc count, we used the crp value <5. to simplify presentation, and present risk increases, as opposed to risk decreases, we defined the variable as crp >5 mg/l. crp was also included in different sensitivity analyses with different cut-offs, as continuous and using regression increased accuracy in diagnosing diverticulitis 3 table 1. final diagnosis of patients suspected of having acute colonic diverticulitis (findings based on clinical and radiological results). final diagnosis number % left-sided diverticulitis 103 70.5 uncomplicated 82 complicated 21 no cause found 17 11.6 colitis 8 5.5 appendicitis 7 4.8 small bowel obstruction 3 2.1 pyelonephritis/kidney stone 2 1.4 basal pneumonia 1 0.7 cholecystitis 1 0.7 malignancy 1 0.7 gynecologic cause 1 0.7 spleen infarction 1 0.7 perforation due to fishbone 1 0.7 total 146 100 figure 3. least absolute shrinkage and selection operator (lasso) regression with up to 14 variables. the area under the receiver operating characteristic curve decreases as the logarithm of the penalty λ increases and the variables become fewer. events and number of variables, to prevent overfitting and reduce model complexity. all 14 variables were included in the initial analysis to select the variable set that yielded the most accurate predictions. a range of different penalties was used, and results were evaluated using 10-fold cross-validation of the auc statistic and presented in a graph. the final variable set was also analyzed using an ordinary logistic regression model, and ors, cis, and p-values corresponding to a two-sided test of a null association with outcome were presented in table 4. the sensitivity and specificity of different cut-offs for the rs resulting from the lasso regression were also presented using a roc curve and a corresponding standard auc. the resulting formula for generating the rs for a patient is given together with a table for users to gauge the risk of diverticulitis for a patient, based on the score. the analyses were done using statistical program r (16) and ibm spss statistics (ibm corp. released 2016. ibm spss statistics for windows, version 24.0. armonk, ny, usa). p-values from two-tailed tests below the level of 0.05 were considered statistically significant. results study population one hundred and forty-six patients were included in the study, all with clinical suspicion of acute diverticulitis. figure 1 shows a flow-chart of the study population. the median age was 68 years (73% female: age range, 50–94 years). the mean body mass index was 28.6 kg/m2 (sd 4.6), and the mean symptom duration was 3.8 days (sd 3.7). the final diagnosis was based on clinical and radiological findings, as shown in table 1. the three most common final diagnoses apart from acute diverticulitis were no cause found (12%), colitis (6%), and acute appendicitis (5%). clinical evaluation for patients with acute diverticulitis diverticulitis was found in 103 of 146 patients with a diagnostic accuracy in terms of positive predictive value of 70.5%. acute diverticulitis was complicated in 21 patients (20%), with 4 j. sigurdardottir et al. table 2. patient characteristics and symptoms. variables total acute diverticulitis other diagnosis 146 103 (70.5) 43 (29.5) gender male 40 22 18 female 106 81 25 age group 8 50–59 40 32 8 60–69 41 30 11 70–79 50 34 16 80+ 15 7 8 previous acute diverticulitis* 53 41 12 uncomplicated 45 35 10 complicated 9 7 2 comorbidity* cardiovascular 46 33 13 lung 12 8 4 liver 2 2 0 dm 2 1 1 kidney 1 0 1 other 18 13 6 ecog performance status 0 119 86 33 1 14 10 4 2 8 4 4 3 2 1 1 4 0 0 0 missing 3 2 1 symptoms* pain left side 141 101 40 pain right side 5 2 3 obstipation 38 27 11 diarrhea 36 24 12 nausea 48 32 16 vomiting 16 4 12 urinary symptoms 12 10 2 vas scale 0–4 29 19 10 5–7 65 51 14 8–10 32 21 11 missing 20 12 8 bmi (kg/m2, n = 131)# 28.6 (4.6) 29.0 (4.6) 27.8 (4.3) clinical signs body temperature, °c# 37.4 (0.7) 37.5 (0.7) 37.1 (0.7) localization of abdominal tenderness* left lower quadrant 129 93 36 suprapubic 68 53 15 left right quadrant 40 25 15 rebound tenderness none 37 22 15 minimal 62 44 18 medium 36 29 7 severe 11 8 3 laboratory results wbc (×10⁹/l)# 11.8 (4.4) 11.6 (3.2) 12.3 (6.5) neutrophiles (×109/l) (n = 134)# 8.7 (3.2) 8.67(3.0) 8.9 (3.7) crp (mg/l)# 82 (61) 90 (60) 63 (62) *patients can have a history of both uncomplicated and complicated ad, more than one comorbidity, more than one symptom, and pain can be in more than one location on the physical examination. #continuous values are presented as mean (standard deviation). other values in number of patients and parentheses are percentages. ecog: eastern cooperative oncology group; vas: visual analogue scale; bmi: body mass index; wbc: white blood cell count; crp: c-reactive protein. splines, to determine which form yielded the most effective predictions. the discriminatory accuracy (da) of each variable separately was low (auc ranging between 0.60 and 0.65). combining them using lasso regression to select the best set of predictors yielded a considerable increase in da with respect to diagnosing ad. the auc observed was 0.82 using crossvalidation and 0.88 (95% ci, 0.82–0.94) without using crossvalidation, for 12 of 14 variables (age, gender, fever, nausea, obstipation, diarrhea, temperature, rebound tenderness, crp  >5, wbc count, previous acute colonic diverticulitis, and pain on the left lower side of the abdomen). all combinations of the 14 variables using different penalties in the lasso regression are shown in figure 3. to get a more manageable and useful set of predictors, we settled for a set of seven predictors for the rs and a slight decrease in da, to a cross-validated auc of 0.82, corresponding to a standard auc of 0.85 (0.78-0.92) as seen in figure 2. the variables selected in the lasso regression for this set were age, gender, urinary symptoms, nausea, temperature, crp >5, and pain on the left lower side of the abdomen. formula for generating the rs the following formula was developed for calculating the rs for acute diverticulitis: − ∗ − ∗ ∗ ∗ ∗ ∗ ∗ rs gender age urinary vomit temp crp tender = 14.6 + 0.65 0.029 + 0.18 + 1.48 + 0.34 + 1.86 5 + 1.36 where gender takes the value of 1 for women and 0 for men, age is the patient age in years, urinary takes the value of 1 if the patient has urinary symptoms and 0 if they do not, vomit takes the value of 1 if the patient has not vomited and 0 otherwise, temp is the patient’s temperature in degrees celsius, crp5 takes on the value of 1 if the patient’s crp is >5, and tender takes on the value of 1 if there is a pain on the left lower side. in table 4, the rs gives the likelihood of acute diverticulitis based on the variables. discussion the current study proposes that usage of common symptoms such as left-sided abdominal pain, gender, age, change in bowel habits, and absence of vomiting may improve diagnosis in patients with clinical suspicion of acute diverticulitis. the high diagnostic accuracy in terms of the auc at 0.82 found in this study indicates that even in a selected population of patients with clinical suspicion of diverticulitis, factors remain that facilitate improved diagnostic accuracy. andeweg et al. (14) proposed a scoring system with several predictive clinical and laboratory factors. we used their nomogram on our cohort and found an auc of 0.70, i.e. a fair ability to differentiate between those with verified acute diverticulitis and those identified as having other diagnoses. by using our scoring system, the accuracy of the diagnosis for our cohort was increased further to reach 0.79. however, andeweg’s nomogram was developed for use in a different clinical setting and patient cohort, a major difference increased accuracy in diagnosing diverticulitis 5 being that our cohort consists of patients with clinical suspicion of left-sided acute diverticulitis. therefore, we hypothesized and subsequently verified that accuracy could be increased if we created an rs tailored to this setting. in sweden, patients with abdominal pain that require workup are either referred to the ed by their general practitioner or attending ed directly. all patients are evaluated by an on-call physician (often surgeon or surgical resident), and most undergo a diagnostic ct scan with contrast as well as laboratory work-up; the present study is consistent with this approach. a strength in this study is that each patient had a ct scan, which was re-evaluated by an experienced gastrointestinal radiologist, verifying that the included patients were correctly diagnosed. however, some patients with acute diverticulitis may not have been included in the study. there are limitations with this study. one of which is a limited and selected cohort and the need for the scoring system to be validated before being used. we have tried to address these concerns in the text below. we used the lasso regression to select relevant variables for our rs and to estimate their association with outcome (17, 18). table 3. symptoms and physical examination. the diagnostic value of acute diverticulitis using odds ratios (ors) with 95% confidence intervals (cis) and logistic regression. patient characteristics simple regression analysis multiple regression analysis total n = 146 or (95% ci) auc (%) (95% ci) or (95% ci) p gender female 106 2.65 (1.23–5.71) 0.60 (0.50–0.71) 4.82 (1.56–1,491) 0.01 male 40 1.00 (reference) 1.00 (reference) bmi (n = 90)# 29 (4.6) 1.06 (0.97–1.16) 0.57 (0.46–0.67) age# 68 (9.7) 0.95 (0.92–0.99) 0.63 (0.53–0.73) 0.92 (0.87–0.98) 0.01 previous diverticulitis no 93 1.00 (reference) 0.56 (0.46–0.66) yes 53 1.71 (0.79–3.71) symptoms pain lower left side no 9 1.00 (reference) 0.56 (0.45–0.66) 1.00 (reference) <0.001 yes 137 5.41 (1.29–22.73) 15.14 (2.65–86.58) nausea no 110 1.00 (reference) 0.56 (0.46–0.66) 1.00 (reference) 0.58 yes 36 2.02 (0.81–5.04) 1.44 (0.39–5.28) obstipation no 108 1.00 (reference) 0.50 (0.40–0.61) 1.00 (reference) 0.09 yes 38 1.03 (0.46–2.33) 3.05 (0.83–11.25) diarrhea no 110 1.00 (reference) 0.52 (0.42–0.63) 1.00 (reference) 0.44 yes 36 0.79 (0.35–1.76) 0.64 (0.21–1.98) urinary symptoms no 134 1.00 (reference) 0.53 (0.42–0.63) 1.00 (reference) 0.22 yes 12 2.20 (0.46–10.50) 4.28 (0.43–42.63) vomiting no 130 9.58 (2.88–31.85) 0.62 (0.51–0.73) 14.02 (2.90–67.88) <0.001 yes 16 1.00 (reference) 1.00 (reference) vas (n = 91)# 6 (2.2) 1.01 (0.84–1.20) 0.49 (0.37–0.61) clinical signs body temperature, °c# 37.4 (0.7) 2.07 (1.21–3.52) 0.65 (0.55–0.75) rebound tenderness vas none 37 1.00 (reference) 0.60 (0.50–0.70) 1.00 (reference) 0.84 minimal 62 1.67 (0.71–3.92) medium 36 2.82 (0.98–8.11) 1.06 (0.60–1.88) severe 11 1.81 (0.41–7.99) laboratory results# wbc ×109/l 11.4 (4.4) 0.97 (0.89–1.04) 0.50 (0.39–0.61) 0.91 (0.82–1.01) 0.09 neutrophiles ×109/l 8.6 (3.2) 0.97 (0.86–1.09) 0.49 (0.36–0.61) crp (>5) mg/l 71 (61) 1.01 (1.00–1.02) 0.65 (0.56–0.76) #continuous values are presented as mean (standard deviation) in the total column. other values are presented in number of patients. bmi: body mass index; vas: visual analogue scale; wbc: white blood cell count; crp: c-reactive protein. 6 j. sigurdardottir et al. table 4. table for translating risk scores into risk of acute diverticulitis in a clinical population of patients with suspected diverticulitis. risk score risk (%) –2.94 5 –2.20 10 –1.73 15 –1.39 20 –1.10 25 –0.85 30 –0.62 35 –0.41 40 –0.20 45 0 50 0.20 55 0.41 60 0.62 65 0.85 70 1.10 75 1.39 80 1.73 85 2.20 90 2.94 95 this was done because of our somewhat limited sample size, proportion of non-events, and the number of potential variables to consider when generating the rs. since we attempted this  analysis using a regular regression approach, we would likely have to run the risk of overfitting our rs to our study population and limited the ability to generalize these findings and the use of the score in other patient settings. an indication of overfitting was the difference between the cross-validated and the regular auc, where the former is more robust to these  issues. however, we cannot rule out, even with our more robust approach, that results were affected to some extent by  overfitting bias, even if it should have mitigated most of these issues. another limitation of the study is the selected cohort. when interpreting results from the present study, it is important to keep in mind that the study population is one where acute diverticulitis is already suspected, as is seen in the clinical diagnostic accuracy of 70%, which is higher than that in other studies (8, 19). because of this bias, variables selected for predicting a verified acute diverticulitis diagnosis may not be the same as in other studies conducted in a more general clinical population or without a suspected clinical acute diverticulitis diagnosis. the difference here may be that variables on which the clinical suspicion of acute diverticulitis is based may have reduced variability in our cohort compared with more general clinical populations, which makes these variables less useful for prediction in our cohort. one such example may be pain in the left lower quadrant, which is likely to be present in most clinically suspected cases of acute diverticulitis. the fact that it is one of the symptoms also indicates that it is a useful predictor of acute diverticulitis in a general clinical population, as those with pain in the left lower quadrant are much more likely to have a verified acute diverticulitis than are those who do not. the fact that lower left quadrant pain was prevalent in our cohort, as well as associated with a large difference in risk, is evident from table 3, where we can see that pain in the left lower side substantially increases the probability of the patient having acute diverticulitis. however, as this variable is present in most cases, it also is a poor predictor of acute diverticulitis in our cohort, as is clear from the rather low auc value. a somewhat surprising result of the present study was that the limit of crp to produce the best prediction of verified acute diverticulitis was as low as 5. one would possibly expect the risk of acute diverticulitis to increase with increasing crp. however, in the present study, where many with suspected acute diverticulitis have elevated crp, average crp levels may not substantially differ between those with and without a confirmed acute diverticulitis diagnosis. instead, a more useful difference may be found in those patients with crp <5, where all but one did not have acute diverticulitis. this is similar to the reasoning above, where studying only suspected cases changes the distribution and association of known predictors to outcome. acute diverticulitis is a common diagnosis, and the gold standard for confirming the diagnosis in these patients is a ct scan. the scoring system, when validated in other cohorts, is not intended to be used instead of a radiological diagnosis, but rather, to be used as a complementary tool in the diagnostic arsenal. not all centers have access to a ct scan all hours of the day and may need to refer their patients to get a ct scan. in some cases, the patients need to travel to do a ct scan. in those cases, if the clinician has a high suspicion of ad and the patient is clinically stable, this scoring system may aid in the decision to do a ct scan and when. however, we impress upon the fact that a scoring system does not differentiate between diagnoses as a ct scan does, and the patient’s clinical status is the dominant factor in deciding when and how the diagnosis is done. the authors emphasize that the scoring system is not a guarantee for the diagnosis ‘acute diverticulitis’ but a calculation of the risk of having acute diverticulitis. this is important to remember when using the system. another instance where this system may be in aid of diagnosing diverticulitis is for patients with recurring acute diverticulitis presenting numerous times with similar symptoms. if clinically stable, perhaps these patients need not do a ct scan each time, and with aid from the scoring system, a more selective approach can be undertaken. this was a selected group of patients with clinical suspicion of acute diverticulitis. we sought to design a scoring system for ad to increase the clinical diagnostic accuracy. the developed scoring system is available for free at https://phille-wagner.shinyapps. io/diverticulitis_risk_model/. conclusion common symptoms and clinical findings can predict and improve diagnosis in patients with suspicion of acute diverticulitis. we present a scoring system that can increase the https://phille-wagner.shinyapps.io/diverticulitis_risk_model/ https://phille-wagner.shinyapps.io/diverticulitis_risk_model/ increased accuracy in diagnosing diverticulitis 7 diagnostic accuracy of acute diverticulitis and may lead to a reduction of repeated ct scans. ethics approval this study was approved by the regional ethics committee and followed the 2013 declaration of helsinki guidelines (registration number dnr 2016/411 and clinical trials registration number ntc03443011). informed consent patients received oral and written information about the study and radiation risks. a written informed consent was required for participation. conflict of interest the authors declare they have no conflict of interest. acknowledgments the authors would like to thank dr. arnar thorisson, md, phd, radiologist, radiology department, region, västmanland, sweden, for ct evaluations, dr. helan laurell, md, phd, colorectal unit, department of surgery, landstinget dalarna, mora, sweden, for assistance with inclusion of patients in mora, and,  finally, malin engdahl, surgical department, region, västmanland, for practical assistance. funding this research was supported by research grants from the county of västmanland and the uppsala-örebro regional research council, sweden. notes on contributors johanna sigurdardottir, md is a surgeon at the department of colorectal surgery, västmanlands hospital västerås, sweden and a phd student at the centre for clinical research of uppsala university, västmanland’s hospital västerås, sweden. abbas chabok, md, phd is a surgeon at the department of colorectal surgery, västmanlands hospital västerås, sweden and an associate professor at the centre for clinical research of uppsala univsersity, västmanland’s hospital västerås, sweden. philippe wagner, phd is a biostatistician at the centre for clinical research of uppsala univsersity, sweden. maziar nikberg, md, phd is a surgeon at the department of colorectal surgery, västmanlands hospital västerås, sweden and an associate professor at the centre for clinical research of uppsala univsersity, västmanland’s hospital västerås, sweden. orcid johanna sigurdardottir https://orcid.org/0000-0002-9671-6518 abbas chabok https://orcid.org/0000-0001-9662-5045 philippe wagner https://orcid.org/0000-0002-9293-3371 maziar nikberg https://orcid.org/0000-0002-5949-3810 references 1. peery af, keku to, martin cf, eluri s, runge t, galanko ja, et al. distribution and characteristics of colonic diverticula in a united states screening population. clin 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http://dx.doi.org/10.1002/(sici)1097-0258(19970228)16:4%3c385::aid-sim380%3e3.0.co;2-3 http://dx.doi.org/10.1002/(sici)1097-0258(19970228)16:4%3c385::aid-sim380%3e3.0.co;2-3 http://dx.doi.org/10.1155/2015/590346 upsala journal of medical sciences 2021, 126, e7819 http://dx.doi.org/10.48101/ujms.v126.7819 original article contact curt ekström curt.ekstrom@neuro.uu.se © 2021 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. association between open-angle glaucoma and alzheimer’s disease in sweden: a long-term population-based follow-up study curt ekströma , ida puhtoa and lena kilanderb aophthalmology, department of neuroscience, uppsala university, uppsala, sweden; bgeriatrics, department of public health and caring sciences, uppsala university, uppsala, sweden abstract background: open-angle glaucoma (oag) and alzheimer’s disease (ad) are two age-related neurodegenerative diseases of significant public health importance. epidemiological studies have indicated that there might be an association between the disorders. methods: predictors of ad, including mixed and unspecified dementia, were analysed in a cohort of 712 residents aged 65–74 years, examined in a population survey in the rural district of tierp, sweden, from 1984 to 1986. to expand the sample size, 821 people were recruited by means of glaucoma case records established at the eye department in tierp from 1978 to 2007. in this way, the cohort comprised 1,533 people, representing more than 21,000 person-years at risk. medical records were reviewed to identify subjects diagnosed with dementia. those with a follow-up duration shorter than 2 years were excluded. results: by the conclusion of the study, in august 2020, 307 subjects had received a diagnosis of ad, including mixed and unspecified dementia. of these cases, 55 were affected with definite oag at baseline. higher age and ischemic heart disease were the only predictors of ad identified. in multivariate analysis, adjusting for age, participation in the population survey and competing events, no association was found between oag and ad (hazard ratio 1.08; 95% confidence interval: 0.80–1.47). conclusion: in this long-term follow-up study of subjects aged 65–74 years old in sweden, oag was not associated with ad. article history received: 19 april 2021 revised: 18 may 2021 accepted: 24 may 2021 published: 05 july 2021 keywords alzheimer’s disease; open-angle glaucoma; normal-tension glaucoma; dementia; epidemiology; risk factor introduction open-angle glaucoma (oag) and alzheimer’s disease (ad) are two progressive, age-related neurodegenerative diseases of significant public health importance. glaucoma is characterised by loss of optic nerve fibres with typical appearance of the optic nerve head and consistent visual field defects, whilst ad is known for its ongoing cognitive decline and behavioural impairment. neuropathologically, hallmarks of ad include the accumulation of large extracellular b-amyloid plaques and intracellular fibrillar tangles of abnormally phosphorylated tau protein within the central nervous system (1). polymorphism of the apolipoprotein e (apoe) gene, involved in the transport of lipids, is an important risk factor for late onset ad (2). at the same time, increased intraocular pressure (iop) is closely related to oag (3). similarities between oag and ad have raised the question of whether they share common risk factors or if one condition has an influence on the other. two main hypotheses have been proposed to explain a possible connection between the disorders. the first hypothesis includes a common neurodegenerative process involving the activation of enzymatic caspases and the production of neurotoxic amyloid b (4). the findings of optic nerve degeneration and the loss of retinal ganglion cells in ad (5,  6) agree with this assumption. the second hypothesis implicates optic nerve damage as a consequence of an abnormally high-pressure difference across the lamina cribrosa in ad, with decreased cerebrospinal fluid pressure (7). berdahl et al.’s study supports this concept (8). they found lower cerebrospinal fluid pressure in patients with oag than in controls. numerous researchers have examined a possible association between oag and ad, albeit with conflicting results. in a case– control study in bavaria, bayer et al. found an increased rate of glaucoma in ad patients (9). likewise, tamura et al. reported a high frequency of oag in japanese patients with ad (10). interestingly, they also found a connection between apoe genotypes and oag. in a 3-year follow-up study, helmer et al. revealed a relationship between oag and incident dementia (11). moreover, two register-based studies in taiwan found a 40 and 47% increased risk, respectively of ad, in subjects with a diagnosis of oag (12, 13). another taiwanese study demonstrated a 52% increased risk in normal-tension glaucoma (ntg) compared with subjects without glaucoma (14). finally, a case–control study recently reported that cognitive impairment was more prevalent in oag subjects with an iop ≤21 mmhg than in those with an iop ≥25 mmhg (15). http://dx.doi.org/10.48101/ujms.v126.7819 mailto:curt.ekstrom@neuro.uu.se http://creativecommons.org/licenses/by/4.0/ https://orcid.org/0000-0002-8265-6518 https://orcid.org/0000-0001-6606-9110 2 c. ekström et al. in contrast, several large-scaled cohort studies have failed to confirm the results (16–19). furthermore, in a cross-sectional study from singapore, glaucoma was not associated with cognitive dysfunction (20). clearly, additional research is essential for better understanding of the relationship between these common diseases. the purpose of the present research was to explore the relationship of oag with ad, including mixed and unspecified dementia, in a swedish cohort. the investigation took the form of a long-term follow-up study on a defined population. study results have previously been reported briefly (21). methods the study population residents in two rural districts in uppsala county, south-central sweden, registered with a glaucoma case record at the eye department in tierp, or who had participated in the tierp glaucoma survey, were eligible for the study. the inclusion criteria embraced all subjects 65–74 years of age. the tierp glaucoma survey in 1984–1986, a population survey was conducted in the rural district of tierp. its target population comprised 2,429 residents, aged 65–74 years old. a representative sample of about onethird of the target population was randomly selected. of the eligible number of 838 residents, 760 (91%) underwent a detailed eye examination, as described elsewhere (22). the study was primarily designed to address the distribution and determinants of oag. however, a vast amount of information was collected, including data on health problems. briefly, an interview was first held, covering medical and family history. information was also obtained from medical records. visual fields were tested using the competer 350 automated perimeter (bara elektronik ab, lund, sweden). after perimetry, the pupils were dilated, and the slit lamp biomicroscopy, including a binocular assessment of the optic discs and gonioscopy, was undertaken. pseudoexfoliation was defined as the presence of characteristic white flakes on the lens capsule or on the pupillary border. the cohort a total of 78 residents did not participate in the population survey. of these, one joined the cohort after being examined in 1993. thus, this part of the cohort was comprised of 761 subjects. to expand the sample size, 923 patients were recruited by means of glaucoma case records established in 1978–2007. those enrolled had a diagnosis of ocular hypertension, glaucoma, suspicious optic discs or a positive family history. in addition, more than 200 subjects had participated as controls in a case–control study on risk factors for oag (unpublished data). apart from visual field testing, they underwent an eye examination equal to that of the population survey. information about the visual fields was obtained from medical records. the examination day was defined as the index date for the participant in the population survey. for the rest of the cohort, the first visit at the eye department at the age of 65–74 years was chosen as the index date. of the total number of 1,684 individuals, 46 were diagnosed with either angle-closure glaucoma or secondary glaucoma. these individuals were excluded from the study, as they were 16 subjects with dementia or mental retardation, six with incomplete data and five for other reasons. only subjects who completed a follow-up time of at least 2 years (‘immortal person–time’) were accepted. consequently, 68 subjects were removed from the study. ten people did not want to participate (figure 1). the remaining 1,533 constituted the study cohort, whose characteristics are presented in table 1. the regional ethical review board of uppsala university approved the study. the tenets of the declaration of helsinki were observed. assessment of dementia follow-up started after the baseline examination and ended on 31 august 2020. medical records were reviewed to identify subjects diagnosed with dementia. if the word dementia was found anywhere in the text, pertinent parts of the records were copied and de-identified. permanent impairment in cognitive and social function, persisting for at least 6 months, had to be figure 1. flow chart showing how the study cohort of 1,533 individuals was derived. acg, angle-closure glaucoma; sg, secondary glaucoma. the miscellaneous group included five subjects not examined in tierp. open-angle glaucoma and alzheimer’s disease 3 present for a diagnosis of dementia. a geriatrician (lk), who was not cognisant of the baseline data, approved the diagnoses using all available information. diagnoses of ad fulfilled the national institute on aging and the alzheimer’s association criteria (23). thus, a history free from abrupt onset and computed tomography without sign of any major cerebrovascular disease was mandatory for a diagnosis of ‘pure ad’. cases with insidious onset and having a slowly progressive course, showing evidence of cerebrovascular lesions according to tomography, were identified as having mixed ad and vascular dementia. most patients with an onset after 80 years of age were designated as having unspecified dementia. for this study, ‘pure ad’ was combined with mixed and unspecified dementia into a single category. classification of open-angle glaucoma consistent with the concept of foster et al. (24), glaucoma with pseudoexfoliation was classified as oag. ntg was defined as a variant of oag in cases where not more than one iop reading exceeding 21 mmhg had ever been recorded and none of the readings were above 24 mmhg. to qualify for a diagnosis of oag, a reproducible visual field defect was a prerequisite, consistent with glaucoma and not explicable on other grounds. likewise, subjects with end-stage disease in either eye were included among the oag cases. in all, 264 subjects fulfilled a diagnosis of definite oag, 42 in the population sample and 222 in the rest of the cohort. pseudoexfoliation in either eye was present in 152 (57.6%) of the subjects with oag. statistical methods ageand sex-standardised morbidity ratios (smrs) were estimated. follow-up time was calculated from the index date to the date of the dementia diagnosis (n = 357), death (n = 1,049), migration out of uppsala county (n = 46) or the end of the study (n = 81), whichever occurred first. ‘immortal person–time’ was removed from the follow-up time for all calculations (25). following standardised analyses, cox proportional hazards models were developed to assess the effect of more than one predictor on the risk of ad, censoring those who died, migrated from the county, received other types of dementia diagnosis or remained in the cohort at the end of the follow-up period. adjustments were made for the influence of censuring due to death (competing events). only predictors with a substantial number of exposed cases were used in the multivariate analyses. the proportional hazard assumptions were tested using timedependant covariates. the effect of the covariates on survival was independent on time, apart from participation in the population survey. consequently, a time-dependant variable was included in the cox models. results the median follow-up time for survivors was 18.9 years (range 13.6–34.8 years). by the end of the study, 357 cases of dementia had been identified, 307 of whom had ‘pure ad’, mixed dementia or unspecified dementia. of the 307 cases, 55 had definite oag in either eye at baseline. ‘pure ad’ or mixed dementia was found in 50 subjects, whilst 257 were diagnosed with unspecified dementia. higher age was the only variable found to be associated with ad. subjects aged ≥70 years experienced a 1.69-fold (95% confidence interval [ci]: 1.35–2.13) increased risk, compared with those <70 years (table 2). adjusting for age, the risk of developing ‘pure ad’ or mixed dementia in oag patients was lower than the risk for any type of dementia, smr 0.61 (95% ci: 0.24–1.54) and 1.12 (95% ci: 0.85–1.48), respectively. there was no association between participation in the population survey and ad (smr table 1. characteristics of the cohort, by age and gender. age group no. of people (n = 1,533) person–years (n = 21,676) female (%) male (%) female (%) male (%) 65-69 years 416 (48) 312 (47) 6,978 (53) 4,588 (54) 70-74 years 455 (52) 350 (53) 6,194 (47) 3,916 (46) 65-74 years 871 (100) 662 (100) 13,172 (100) 8,504 (100) mean follow-up time: 14.1 years (standard deviation: 7.0 years). table 2. associations of potential risk factors and alzheimer’s disease including mixed and unspecified dementia in a cohort of 1,533 individuals, adjusted for age and sex. baseline characteristics no. of cases smr (95% ci) (n = 307) age ≥ 70 yearsa no 126 1.00 yes 181 1.69 (1.35–2.13) female sexb no 103 1.00 yes 204 1.23 (0.97–1.55) participation in the population survey no 180 1.00 yes 127 0.83 (0.66–1.05) open-angle glaucoma, either eye no 252 1.00 yes 55 1.16 (0.86–1.56) pseudoexfoliation, either eye no 215 1.00 yes 92 1.05 (0.82-1.34) smoking status never smoked 224 1.00 past smoker 57 0.97 (0.70–1.35) current smoker 26 0.84 (0.55–1.30) diabetes mellitus no 276 1.00 yes 31 1.02 (0.70–1.48) hypertension, treated no 196 1.00 yes 111 1.15 (0.91–1.45) ischaemic heart disease no 256 1.00 yes 51 1.27 (0.94–1.72) ci: confidence interval; smr: standardised morbidity ratio. aadjusted for sex. badjusted for age. 4 c. ekström et al. 0.83; 95% ci: 0.66–1.05). ntg was present in 11 subjects, one of whom was diagnosed with unspecified dementia. cox proportional hazards models included oag, age, sex, participation in the population survey, smoking habits, systemic hypertension and ischaemic heart disease (table 3). no association was found between oag and ad (hazard ratio [hr] 1.08; 95% ci: 0.80–1.47). every year of higher age increased the risk by 16% (hr 1.16; 95% ci: 1.12–1.21). in a separate model, ischemic heart disease was found to be associated with ad, including mixed and unspecified dementia (hr 1.43; 95% ci: 1.05–1.94). inclusion of birth year in the models had no effect on the estimates (data not shown). discussion to our knowledge, this study on a defined population is the longest follow-up study on oag and the risk of developing ad yet to be reported. definite oag was found to be unrelated to ad. however, although we had access to a sizable cohort, only 55 ad cases had been exposed to definite oag, limiting the ability to reveal a small increase in risk and to analyse sub-groups. in fact, applying a confidence level of 95% and a power of 80%, this study had the capacity to detect a 53% increased risk. contrary to the present study, a meta-analysis of six studies on oag and ad by xu et al. recently showed a 17% increased risk of ad (26). there are several explanations for the discrepancy between our study and other studies. clearly, insufficient statistical power downgrades the chance of detecting an enlarged risk. moreover, the study in tierp was a long-term follow-up study, whilst most of the studies demonstrating a connection with ad referred to previously were either case– control studies or register-based studies (9–11, 13–15). it is well known that case–control studies are more susceptible to bias than cohort studies. register-based studies, on the other hand, are reliant on the quality of the registers used. of interest, in the review by xu et al., there was no association between oag and ad when the analysis was restricted to cohort studies (26). furthermore, the studies showing a relationship between oag and ad, where information on the iop was provided, were generally characterised by an iop within the normal range (10–12). in contrast, increased iop was a typical finding of oag diagnosed in the tierp population (27). in the present study, 11 out of 264 patients with oag were found to have ntg. of note, two studies recently have reported a relationship of ntg with ad (14, 15), though a previous danish study did not (28). there are evidence suggesting that ntg and ad share common risk factors. a cross-sectional study from south korea found a higher risk for vascular and metabolic comorbidities in subjects with oag and a baseline iop ≤15 mmhg (29). moreover, a swedish cohort study confirmed a relationship between vascular risk factors and dementia (30). in fact, if ntg is related to ad, it is possible that some of the divergence between the result in tierp and other studies might be explained by variances in the characteristics of oag cases. results from the study in tierp have previously been reported briefly (21). although the follow-up time for surviving individuals increased by 4 years in the present study and an additional 31 cases of dementia were identified, the risk associated with oag was almost identical, demonstrating stability in the data over time. also, the increased number of cases facilitated a sensitivity analysis, showing inconsistency with risk estimates. thus, the risk of ‘pure ad’ or mixed dementia in oag was substantially lower than the risk of any type of dementia (smr 0.61 and 1.12, respectively). however, considering the low number of ‘pure ad’ or mixed cases, the finding should be interpreted with caution. the study in tierp involved people aged 65–74 years old, whilst the other studies referred to in this report covered a broader age span. nonetheless, there are no reports of age differences in ad risk associated with oag. our study has several strengths, including its communitybased design, long-term follow-up with risk factors measured at baseline before disease diagnosis and sizeable cohort, nearly half of which was randomly selected. furthermore, the same glaucoma specialist conducted all the eye examinations. a geriatrician (lk), ‘masked’ to baseline information, approved the dementia diagnoses. moreover, a reproducible visual field defect or end-stage disease was required for a diagnosis of oag. nevertheless, as with many epidemiologic investigations, the research is limited in several respects. first, baseline examination dates extended over nearly 30 years, which might give rise to bias. however, adjustment for year of birth in the regression models did not change the estimates. second, some misclassification of oag diagnoses cannot be ruled out. if oag increases the risk of ad, this type of information bias should be non-differential, thereby ‘diluting’ the effect of oag in the analyses. on the other hand, if oag has no effect, non-differential misclassification would not bias the result (31). third, dementia diagnoses were usually based on clinical judgement by general practitioners, after which an experienced geriatrician (lk) classified the cases by reviewing the medical records. the extent to which doctors make a diagnosis of dementia in cognitive impaired patients varies. consequently, an unknown number of people with dementia were likely to have remained undiagnosed or were not diagnosed until they had reached the stage of advanced disease. there is no reason to believe that decisions made by general practitioners were in any way connected with the exposure being studied, and therefore had any effect on risk estimates. finally, ‘pure ad’ or mixed dementia was diagnosed in 14% of the cases, which is much lower than expected (32). a probable table 3. association of open-angle glaucoma with alzheimer’s disease including mixed and unspecified dementia in a cohort of 1,533 individuals. oag no. of subjects no. of cases hazard ratio (95% confidence interval) age-adjusteda multivariateb no 1,269 252 1.00 1.00 yes 264 55 1.14 (0.85–1.53) 1.08 (0.80–1.47) oag: open-angle glaucoma. aadjusted for age (continuous variable) and competing events (deaths). badjusted for age (continuous variable), participation in the population survey, including a time-dependent variable, and competing events (deaths). open-angle glaucoma and alzheimer’s disease 5 explanation for the paucity of ad cases is that specialists in  geriatrics were involved in only a small proportion of cases. moreover, strict diagnostic criteria were applied to establish a diagnosis of ad for this study. we believe that the distribution of  dementia diagnoses justifies our decision to combine ad, mixed dementia and unspecified dementia into a single category, as many patients classified with unspecified dementia most likely  had ad. the proportion of ad, mixed ad and vascular  dementia, as well as unspecified dementia, stands at  approximately 70% in the national swedish dementia registry (33). in conclusion, in this long-term follow-up study of subjects aged 65–74 years old in sweden, definite oag was not related to ad. thus, we were unable to confirm the association found in some previous studies. disclosure statement the authors report no conflict of interest. funding the swedish medical research council, crown princess margaretha’s foundation for the visually impaired, the glaucoma research fund at the department of ophthalmology, uppsala university hospital and uppsala county council provided financial support for this study. notes on contributors curt ekström, md, phd, is a senior researcher at the department of neuroscience, ophthalmology, uppsala university, uppsala, sweden. ida puhto is a medical student at uppsala university, uppsala, sweden. lena kilander, md, phd, is a 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[updated 20 february 2019]. available from: https://www.ucr. uu.se/svedem/in-english [cited 10 february 2021]. http://dx.doi.org/10.1136/bjo.86.2.238 http://dx.doi.org/10.1111/aos.14114 http://dx.doi.org/10.1111/j.1755–3768.1991.tb04842.x http://dx.doi.org/10.1111/j.1755–3768.2011.02125.x http://dx.doi.org/10.1111/j.1755–3768.2011.02125.x http://dx.doi.org/10.1111/aos.13487 http://dx.doi.org/10.1159/000330020 https://www.ucr.uu.se/svedem/in-english https://www.ucr.uu.se/svedem/in-english upsala journal of medical sciences 2023, 128, e9340 http://dx.doi.org/10.48101/ujms.v128.9340 assessing stress using repeated saliva concentration of steroid hormones in dementia care dyads: results from a controlled pilot care music intervention azita emamia,b, tö res theorellc,d, hyejin kime, lars berglundf, helena hallinderb and gabriella engströ mf athe university of washington, school of nursing, seattle, wa, usa; bdepartment of neurobiology, care sciences and society, division of occupational therapy, karolinska institutet, stockholm, sweden; cdivision of international public health, karolinska institutet, stockholm, sweden; dstress research institute, department of psychology, stockholm university, stockholm; edepartment of adult health and gerontological nursing, rush university college of nursing, chicago, il, usa; fdalarna university school of health and welfare, falun, sweden abstract background: stress-related biomarkers have the potential to provide objective measures of whether interventions directed at people with dementia (pwd) and their family caregivers (fcg) are successful. the use of such biomarkers has been limited by logistical barriers to sample collection. objective: explore saliva concentration of steroid hormones in dementia care dyads during a music intervention. methods: consecutive pwd attending a memory evaluation center and their fcg were allocated to either an intervention-with-music or a non-intervention control group. all were living at home. stress biomarkers, salivary cortisol and dehydroepiandrosterone sulfate (dhea-s) samples were collected by the pwd and their fcg, in the morning and evening, 5 days a week, for 8 consecutive weeks. biomarker concentrations of the intervention and the control groups were compared at week 8, in an intention-to-treat approach with adjustment for baseline value. results: twenty-four pwd in the intervention group and 10 in the control group, and their fcg were included in the analyses. the mean number of morning saliva collections was similar in the intervention and the control groups, ranging from 4.3 to 4.9 per participant weekly during the first 7 weeks, declining to 3.3 during week 8. median log morning cortisol (pg/ml) among caregivers was lower in the intervention group than in the control group (8.09 vs. 8.57, p = 0.0133). conclusion: this study demonstrates that music intervention was associated with lower morning saliva cortisol concentrations for fcgs. article history received 19 january 2023 revised 23 february 2023 accepted 7 march 2023 published 5 may 2023 keywords caregivers; dementia; music; saliva; stress; cortisol; dhea-s introduction as the global population ages, the number of people living with dementia (pwd) is increasing, making the disease a major public health issue. in the early to middle stages of the disease there is a prevailing preference among pwd and their family caregivers (fcgs) for in-home care, which results in increased stress for these dyads (1–3). unmet physical and emotional needs (e.g. activities of daily living, pain or discomfort, negative emotions) are causes of high stress among pwd, which can lead to a deterioration in the behavioral and psychological symptoms of dementia (bpsd). for caregivers, studies have consistently shown high levels of biological stress levels (4, 5). music has been extensively reported as an effective, low-cost intervention that benefits both pwd and their caregivers by helping diminish bpsd at high stress times such as personal care and feeding encounters. however, most studies relied on fcg’s self-report rather than an objective assessment. recently, attention has focused on developing objective measures that use stress biomarkers. research shows that active individualized music listening as part of stroke rehabilitation accelerated the healing process of the brain (6). methodologically, the invasive nature of the blood collection traditionally required for stress biomarker analysis has been a barrier. in addition to being invasive and uncomfortable, blood collection is expensive, requires a trained phlebotomist, presents infection risk, and is impractical for high-volume, daily, serial collections at home. this led to an interest in saliva collection techniques for assessing cortisol and dehydroepiandrosterone sulfate (dhea-s) (7, 8). at-home saliva collection has the potential to be an objective, easily implemented, non-invasive way of measuring changes in stress levels that reflect the impact of music or other intervention. contact azita emami emamia@uw.edu & sondean@uw.edu © 2023 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article http://dx.doi.org/10.48101/ujms.v128.9340 mailto:emamia@uw.edu mailto:sondean@uw.edu http://creativecommons.org/licenses/by/4.0/ 2 a. emami et al. stress biomarker daily cycles stress triggers the adrenal glands to release cortisol, together with other hormones, in order to maintain homeostasis as summarized by kristenson et al. (9) cortisol follows a diurnal rhythm. the levels of cortisol surge immediately after awakening and continue rising for 30 to 45 min. cortisol then declines rapidly and steadily decreases throughout the day until reaching its nadir around bedtime. recent research shows that dhea-s is correlated with stress levels and the body’s ability to counter adverse effects of stress, which is defined as long-lasting energy-mobilization without effective recuperation (10). a greater ratio of cortisol-to-dhea-s during acute stress responses among persons with higher longlasting stress levels than among those with lower levels (11) makes salivary dhea-s a relevant stress biomarker in this study. such a ratio indicates an adverse balance between energy mobilization and regeneration that may be particularly harmful in caregivers for persons with dementia since many acute stress situations can be expected to arise in their daily routine (10). ouanes et al. (12) have shown that high cortisol levels and a high cortisol/dhea-s ratio in cerebrospinal fluid are associated with cognitive decline. during interventions aimed at reducing stress levels and increasing capacity to handle stress, a diminished cortisol-todhea-s ratio is a favorable development. most studies to date using salivary biomarkers of stress have made only single-point measures before and after an extended period of intervention, rather than the longitudinal daily evaluation of salivary biomarkers that would more accurately capture fine-grained changes in response to music or other intervention programs (7, 13–15). in this study, we aim to explore saliva concentration of steroid hormones in dementia care dyads during a music intervention. a prior preliminary proof-of-concept study examined only an intervention group (16). methods sample and setting we used a two-group, non-randomized open trial to explore the effects of music on physiological markers of stress, cortisol and dhea-s in saliva, among pwd and their fcg. pwd and fcg, who visited a memory care center, were approached by a study coordinator via phone. we collected the data between november 2018 and march 2020. eligible pwd and their fcg were allocated as dyads in a 1:1 ratio to a music-based intervention group or comparison group (figure 1). a total of 72 dyads (pwd and fcg) were approached. of those 72, 18 dyads declined to participate and the remaining 58 dyads (32 for intervention group and 26 for comparison group) consented to participate in this study. a total of 34 dyads (24 in the intervention group and 10 in the comparison group) were included in the analysis. we included pwds who 1) were diagnosed with dementia; 2) were in early to severe stage of dementia as defined by a global deterioration scale (gds) score of 4 or greater (17); 3) lived with a fcg; and 4) provided written informed consent (or proxy consent from the caregiver) to participate. fcg participants were included if they 1) were at least 18 years of age and 2) provided written informed consent to participate. we excluded pwd and fcg who have active mental disorders (e.g. severe depression and anxiety disorder) or those who previously participated in other music therapies or music interventions. saliva collection and intervention education after obtaining an informed consent form, both the intervention and the control group dyads received an education session on saliva collection from the study coordinator. the saliva collection has been described elsewhere (16, 18, 19). briefly, the education session provided written instructions on each step of saliva figure 1. participant flow diagram. drop out n = 6 couples • pwd admitted to facility (n = 2 couples) • caregiver personal and medical problem (n = 4 couples) approached couples n = 72 intervention group couples consented n = 32 intervention and saliva collection couples started n = 26 pwd admitted to facility (n = 2 couples) unable to start due to covid-19 (n = 15 couples) couples included in the analyses n = 24 couples included in the analyses n = 10 saliva collection couples started n = 11 control group couples consented n = 26 drop out (n = 1 couple) repeated measures of saliva hormones 3 collection and hands-on training. participants were instructed to collect saliva over an 8-week period. participants collected their saliva in the morning and in the evening for 5 days, from sunday evening to friday morning. participants were allowed to collect the samples based on their own diurnal cycle rather than at specific times. collecting saliva is a simple and non-invasive procedure that does not require much equipment or technical knowhow. this allows for multiple samples to be taken from the same individual over time. the cortisol levels in a person with normal cortisol regulation capacity upon waking provide an indication of the expected stress level during the day. therefore, the difference between cortisol levels in the morning and evening can serve as a proxy measure of the awakening response. a trained research assistant who completed the dementiaspecific online education session (e.g. how to communicate with pwd) picked up the saliva every morning at participants’ homes and delivered the samples to the biobank at the karolinska institutet for handling until they were shipped for analyses. with covid-19 lockdowns and social distancing measures, it became increasingly difficult to pick up the saliva samples at the participants’ homes, leading to cancellation of the study in march 2020. at that time, not all participants had completed the full 8-week saliva collection. the samples were assayed using radioimmunoassay at the truly labs, lund, sweden. in addition to the saliva collection session, the intervention group also received an education session about the music-based intervention. the music-intervention has been described elsewhere (16, 18, 19). briefly, the study coordinator provided information about the music-based intervention (e.g. benefits of music and how to access the intervention). pwd and fcg participated in the online, in-home music intervention whenever they wanted or could attend, together or alone at their convenience, throughout the study. they were encouraged to listen together and to create a daily routine for the music listening. measures mean values (picograms per milliliter; pg/ml) of four indices were used for each week of the study period to measure the levels of stress in the body among pwd and fcg. firstly, cortisol concentrations (i.e. primary indicator of stress) were measured using the morning samples and evening samples. secondly, dhea-s concentrations were measured using participants’ morning samples. thirdly, diurnal cortisol variation was assessed by subtracting evening cortisol concentrations from morning cortisol concentrations. fourthly, the ratio of cortisol to dhea-s (i.e. indicator of adverse chronic stress) was measured using participants’ morning saliva samples. statistical analysis analyses were performed using sas® version 9.4 (sas institute inc., cary, nc, usa). the level of statistical significance for twotailed hypothesis testing was set at 0.05. no adjustments for multiple tests were made. firstly, data were screened for anomalies to check for outliers and missing data. participants who did not complete the questions regarding a given sample were considered to have missing saliva data for that sample. saliva samples with insufficient saliva for the assay were treated as missing. due to highly skewed distributions of saliva markers, all values were transformed using natural logarithms. the appropriate descriptive statistics were used based on the levels of measurement and data distribution (means with standard deviations [sd], median with interquartile ranges [iqr], frequency [n], percentage [%], and geometric means). all analyses were made separately for pwd and for their fcgs. the analysis was based on primary endpoints (pes), which are the mean values of the last available week (i.e. last observation carried forward [locf] analysis). carrying forward an intermediate value is a conservative estimate of outcome trajectory when treated patients are assumed to improve gradually. since true baseline values (i.e. measurements before intervention) did not exist, mean values from the first week of the study period were used as ‘pseudo’ baseline values. the analysis was based on intention-to-treat with intervention/control group comparisons of pes at 8 weeks with adjustments for baseline values. the distribution of residuals in the regression models of pes on intervention/control group and baseline values, was examined for normality with shapiro–wilk’s test, where w > 0.95 indicates normal distribution. for normally distributed pes, linear regression models of pes on intervention/control group and baseline values were estimated. means adjusted for baseline values, by intervention/control group, p-values for test of the null hypotheses and 95% confidence intervals (cis) for comparisons of adjusted intervention/control group means were presented. for non-normally distributed pes, intervention/ control group comparisons were performed using willetts’s residual method (20). the residuals in the linear regression of pes on baseline values were calculated and compared between intervention and control groups using wilcoxon two-sample test. the medians were adjusted for baseline values, by intervention/control groups; p-values for tests of null hypotheses and 95% moses cis for median differences between the intervention and control groups were calculated. to examine the robustness of the locf method we did a sensitivity analysis using multiple imputation with the fully conditional specification method based on the intervention/ control group variable and previous values of each pe (50 imputations), for calculation of estimated effects and p-values. number of weeks elapsed and number of saliva collections per week were compared between the groups with wilcoxon two-group test. due to shorter data collection time for the control group, a sensitivity analysis was performed with week 6 as the last available observation week (i.e. observations from weeks 7 and 8 were disregarded). results table 1 reports baseline characteristics of pwd and the fcg. among the participating dyads who started the saliva collection, 4 a. emami et al. 92% (n = 24) in the intervention group and 90% (n = 10) in the control group were included in the analyses (figure 1). median participation time was 8.0 weeks for the intervention group and 6.5 weeks for the control group (p = 0.06). there were no statistically significant differences between the intervention and control groups with regard to timing of saliva collection. the collection of the morning sample occurred between 07.20 and 07.45 in the intervention group fcgs and between 07.32 and 07.42 in the control group fcgs. corresponding timing of saliva collection for pwd in the intervention group occurred between 07.41 and 08.07 and in the control group between 07.46 and 08.18. of five potential morning samples for each person per week, the mean number of saliva collections per fcg was similar between the intervention and control groups, ranging from 4.3 to 4.9 per week during the first 7 weeks and declined to 3.3 during week 8 (see figure 2). results for pwd were similar. stress biomarkers in dementia care dyads table 2 shows medians with iqrs for log morning cortisol in the pwd and fcg intervention and control groups. the baseline values are shown at first and then the corresponding values at follow-up, with adjustment for baseline (making intervention and control group comparable) and locf. then the adjusted group difference at follow-up is presented as 95% confidence limits and p-value for the group difference. in the pwd group, the log morning cortisol minus evening cortisol results as well as the log dhea-s and the log cortisol/dhea-s results are presented with medians as well. in the remaining analyses means with sds are presented. the reason for choice of median versus mean is that median was used when a variable’s distribution differed from normality and vice versa. table 1. baseline sample characteristics for persons with dementia and the family caregivers in the intervention and control group. variable persons with dementia family caregivers all (n = 34) intervention (n = 24) control (n = 10) all (n = 34) intervention (n = 24) control (n = 10) age m ± sd (range) ― 79 ± 8.5 (60–92) 79 ± 5.0 (72–96) ― 73 ± 11.5 (37–90) 77 ± 6.0 (69–86) sex n (%) female ― 7 (29) 4 (40) ― 16 (67) 6 (60) male ― 17 (71) 6 (60) ― 8 (33) 4 (40) activities of daily livinga m ± sd (range) 2.4 ± 1.7 (0–6) 2.4 ± 1.7 (0–6) 2.4 ± 1.7 (0–6) ― ― ― months since dementia diagnosis (range)b 20.2 ± 14.6 (2–60) 20.1 ± 14.0 (5–57) 20.3 ± 16.7 (2–60) ― ― ― global deterioration scalec m ± sd (range) 4.8 ± 1.0 (4–7) 4.9 ± 1.1 (4–7) 4.4 ± 1.0 (4–6) ― ― ― gdsc, n (%) moderate cognitive decline 17 (50) 12 (50) 5 (50) ― ― ― moderately severe cognitive decline 8 (24) 4 (17) 4 (40) ― ― ― severe cognitive decline 7 (21) 6 (25) 1 (10) ― ― ― very severe cognitive decline 2 (6) 2 (8) 0 (0) ― ― ― perceived general healthb, n (%) excellent ― ― ― 1 (3) 0 (0) 1 (10) very good ― ― ― 11 (34) 8 (35) 3 (30) good ― ― ― 13 (38) 11 (48) 2 (20) fair ― ― ― 8 (24) 4 (17) 4 (40) poor ― ― ― 0 (0) 0 (0) 0 (0) note: m ± sd = mean ± standard deviation. gds: global deterioration scale. aadl scores of pwd were reported by their fcg. bone participant in the intervention group was excluded due to the incompleteness of the item. cgds scores of pwd were reported by the study coordinator. figure 2. number of saliva tests per family caregiver by treatment week for intervention and control groups. mean values with error bars (std) and number of remaining caregivers by treatment week. repeated measures of saliva hormones 5 although we did not find significant differences for pwd ( table 2), fcg in the intervention group showed lower adjusted log morning cortisol locf than the control group (p  = 0.013, figure 3). adjusted medians for intervention and control groups were 8.09 and 8.57 log pg/ml, respectively. a total of 95% cis for difference between adjusted medians was −0.91 to −0.12. no other adjusted locf differences between groups were statistically significant: the corresponding nonsignificant results for log morning cortisol/dhea-s are shown in figure 4. in the sensitivity analysis with week 6 as the last available observation week, results were similar. fcg in the intervention group showed lower adjusted log morning cortisol locf than the control group (p = 0.047) in this sensitivity analysis and adjusted medians for intervention and control groups were 7.54 and 8.02 (log pg/ml), respectively. when we used multiple imputation with 50 imputations for estimated effects and p-values the results were similar to the locf analysis. for example, for morning cortisol among fcg the p-value was 0.0205 and the difference between groups were comparable to locf results. table 2. saliva concentrations of steroid hormones at baseline and locf1 for persons with dementia and family caregivers in intervention and control groups. variable group baseline mean (std)/ median (interquartile range) locf1 mean (std)/ median (interquartile range) adjusted locf1 mean/ median adjusted group difference at follow-up p lower 95% cl upper 95% cl persons with dementia log morning cortisol (pg/ml) intervention n = 24 8.05 (5.96–8.34) 7.85 (6.03–8.35) 8.04 control n = 10 8.62 (8.03–9.29) 8.58 (8.19–8.88) 8.10* 0.38 −0.644 0.169 log morning dhea-s (pg/ml) intervention n = 24 7.95 (0.91) 7.89 (1.01) 8.18 control n = 10 8.90 (0.68) 8.79 (0.97) 8.16** 0.94 −0.506 0.545 log morning cortisol/ morning dhea-s intervention n = 24 −0.60 (1.43) −0.62 (1.54) −0.51 control n = 10 −0.33 (1.13) −0.13 (1.05) −0.48** 0.90 −0.603 0.535 log morning cortisol (pg/ml) minus log evening cortisol (pg/ml) intervention n = 24 1.42 (0.89–1.90) 1.36 (0.66–1.84) 1.42 control n = 10 1.18 (0.42–1.62) 1.01 (0.37–1.66) 1.73* 0.45 −0.555 0.635 log evening cortisol (pg/ml) intervention n = 24 6.10 (1.48) 6.28 (1.71) 6.76 control n = 10 7.84 (1.64) 8.14 (1.56) 6.98** 0.55 −0.977 0.529 family caregivers log morning cortisol (pg/ml) intervention n = 24 8.07 (6.25–8.55) 8.06 (6.05–8.35) 8.09 control n = 10 8.22 (7.91–8.50) 8.60 (8.37–8.76) 8.57* 0.013 −0.910 −0.124 log morning dhea-s (pg/ml) intervention n = 24 8.30 (7.38–8.66) 7.78 (7.14–8.54) 8.30 control n = 10 7.78 (6.84–8.55) 7.72 (7.50–8.51) 8.51* 0.44 −0.947 0.506 log morning cortisol/ morning dhea-s intervention n = 24 −0.12 (−1.62 to 0.34) −0.34 (−0.97 to 0.43) −0.42 control n = 10 0.51 (−0.20 to 1.25) 0.93 (0.20–1.10) 0.28* 0.36 −1.135 0.454 log morning cortisol (pg/ml) minus log evening cortisol (pg/ml) intervention n = 24 1.62 (0.80) 1.59 (0.86) 1.51 control n = 10 1.19 (1.00) 1.47 (0.75) 1.64** 0.64 −0.683 0.425 log evening cortisol (pg/ml) intervention n = 24 6.28 (4.52–6.95) 6.18 (4.63–7.34) 6.45 control n = 10 6.73 (6.44–7.09) 6.72 (6.50–7.06) 6.74* 0.86 −0.657 0.476 1last observation carried forward, *non-normal distribution, wrm = willett’s residual method/median, **normal distribution, lr = linear regression/mean. locf: last observation carried forward; cl: confidence limit ; dhea-s: dehydroepiandrosterone sulfate. 6 a. emami et al. discussion findings for the family caregivers fcgs participating in the music intervention had lower saliva morning cortisol concentrations than the control group in the final week. the geometric mean at the final week was 2,039 pg/ml in the intervention group, and 3,463 pg/ml in the control group, adjusted for baseline value level. kristenson et al. (9) emphasize that chronic stress is associated with disturbed regulation of cortisol excretion, often resulting in a ‘flattened’ circadian cortisol rhythm with relatively low morning and high evening cortisol. previous work (16), based on only a music intervention group, showed a statistically significant difference between morning and evening cortisol levels among the caregivers. there was little evidence of disturbed regulation of saliva cortisol due to chronic stress despite the well-known burden of providing at-home care for a person with dementia. thus, the participants, in particular the caregivers, should be regarded as subjects with retained ability to regulate cortisol levels. what we could hypothesize in this study is a lowered morning cortisol as well as a lowered evening cortisol and a lowered cortisol/dhea-s level with a lowered stress level. prior work (16) based on the intervention group in this study showed pronounced day-to-day variations. this is an important factor that should be considered in all such studies, because these variations complicate the analysis of whether there are significant change patterns. in the present study this was handled by averaging all data collected during a week. an extra sample was collected in the morning, half an hour after awakening. in three cases when the first morning sample failed, this second sample was used for analysis. the most common pattern in healthy individuals is that there is a rise in cortisol concentration between awakening and half an hour later. we decided not to analyze this ‘morning rise’ (much smaller than the normal morning-evening difference), which is a recommended stress measure, since it was impossible to follow the strict rules surrounding this kind of assessment (21). the most likely explanation for the cortisol level findings in the caregiver group is that there was a lower morning stress level in the caregivers participating during the 8 weeks of intervention with music at times of personal care than in the corresponding control group. the morning routines may be particularly stressful for the caregivers, which could be the reason why the findings are more pronounced in the morning than in the evening. in the previous report (16) there is anecdotal evidence describing decreased within-dyad conflicts as a consequence of the music intervention. as reported in a separate article (19) caregiver ratings of stress and depression improved more in the intervention than in the control group. there were pronounced differences between individual couples in the intervention group, as reported previously (16). our individual analyses indicated that approximately one-fourth of the dyads may have markedly benefited from the music intervention (16). brown et al. (5) published the results of a randomized evaluation of the effects for fcg of a mindfulness training program that was compared with a program with improved social support. the programs lasted for 2 months and saliva cortisol was assessed six time points during one day, at each of the 3 study phases (pre and post-intervention, and 3-month follow-up. no differences were found between the intervention groups. whether our positive findings for the caregiver group with regard to morning cortisol are due to the interventions or differences in the schedules for saliva cortisol collection is not known. we hypothesized that the ratio between cortisol and dhea-s would improve due to the intervention with music during periods of personal care. the comparison with the control group did not confirm this. however, as reported (16), one-fourth of the caregivers in the intervention with music group showed such improvement, although the difference was not statistically significant (p = 0.36). this needs to be examined in more depth in the future. figure 3. log morning cortisol by treatment week for family caregivers in intervention and control groups. locf median values with error bars (95% confidence interval). figure 4. log morning cortisol/dhea-s by treatment week for family caregivers in intervention and control groups. locf median values with error bars (95% confidence interval). repeated measures of saliva hormones 7 findings in the persons with dementia there were no statistically significant differences in any of the study variables in the pwd group. for morning cortisol there was greater decrease (improvement) in the intervention group than in the control group, but the difference in improvement was not statistically significant (p = 0.38). one reason for the lack of statistically significant group differences for the pwd group may be that there was rather rapid disease progression in some of the people. these pwds are also vulnerable to other kinds of illnesses, for example massive infections. such episodes represent stressful events that cause hormonal levels (16) to fluctuate. study intention and limitations the present study had a number of limitations that should be considered when assessing the outcomes. • the covid-19 pandemic resulted in a reduced size and collection duration for the control group, complicating and compromising the attempt to establish statistical significance of the intervention effects. • cohort sizes were small. • the study populations were non-randomized. however, the pwd groups in the intervention and control conditions were comparable in age, activities of daily living (adl) functional level, and months since diagnosis. • the median period of data collection for the control group was shorter than for intervention group (8.0 and 6.5 weeks, respectively). however, the significance of the intervention/ control group difference in the caregivers was retained when the comparison period was limited to the initial 6 weeks. • data from the intervention group were not collected during the same year as those from the control group. the saliva concentration of cortisol, particularly in the morning, may show seasonal variations. persson et al. (22) studied saliva cortisol measures in 24 working men and women over the course of a year. the differences in morning saliva cortisol were pronounced and statistically significant between seasons. since all the saliva sampling in the present study took place from january to april, when the differences are not so pronounced, this is unlikely to have caused significant bias.  • we did not gather data on the timing or frequency with which the fcgs chose to utilize a music intervention, so it is impossible to determine if personal care encounters increased with rising levels of dementia and if music was deployed more or less frequently based on an increase in such encounters. conclusion we have previously shown that serial, in-home saliva collection on a daily basis by study participants over a multi-week period is feasible (18). in this study we demonstrate that music intervention was associated with lower morning saliva cortisol concentrations for fcgs. a strength of this study is that cortisol and dhea-s were measured 5 days per week throughout an 8-week period, offering insight into participants’ diurnal cortisol and dhea-s levels in general. this process enabled collection of a large number of observations for each participant, and established the potential for using this methodology to objectively measure responses in stress levels to music and other interventions directed at modifying the adverse bpsd. having such an objective yardstick is important, because progressively worse bpsds are a frequent reason for pwds being institutionalized. developing successful interventions requires a reliable, repeatable means of measuring the outcomes of such interventions for both pwds and their caregivers. evidence suggests that music, by stimulating the neural pathways in the brain, affects physiological responses and mediates and moderates behavior in pwd (23, 24). we have demonstrated a viable alternative to the use of self-report questionnaires for capturing the impact of a music intervention on caregivers and particularly on pwd. as the latter become progressively less able to self-report, data become skewed to increasingly reflect the perceptions of the caregiver rather than the physiological response of the pwd. this adds urgency and importance to the need for developing objective measures of stress. in a forthcoming paper we will examine day-to-day variability of salivary cortisol and dhea-s levels both among pwd and fcgs and evaluate how a music intervention for stress affects variability in pwd and fcgs. furthermore, to facilitate design of future pwd and fcg dyad studies we will present how many sample days one needs for  establishing salivary cortisol and dhea-s differences between intervention and control groups for a given number of dyads. a more definitive result will require a larger randomized control trial (rct ) that controls for confounding variables such as seasonality, as well as acute infections and other temporary stressors such as  changes in care personnel, family conflicts, and medical encounters. acknowledgments this work was supported by amf insurance company in sweden, section of elderly research (emami) and the robert g. and jean a. reid endowed fund (emami) from the university of washington school of nursing. the authors would like to thank the participants who volunteered for this study and brian weiss for professional manuscript editing. disclosure statement the authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. 8 a. emami et al. notes on contributors azita emami, project organization and planning, data collection, manuscript writing. töres theorell, project organization and planning, statistical analysis, manuscript writing. hyejin kim, data collection, manuscript writing. lars berglund, statistical analysis, manuscript writing. helena hallinder, manuscript writing. gabriella engström, project organization and planning, data collection, manuscript writing. ethical approval ethical approval was obtained from the karolinska institute’s institutional review board, dnr: 2018/1596-31/2. verbal consent was obtained from all participants. orcid azita emami https://orcid.org/0000-0003-2608-9691 gabriella engström https://orcid.org/0000-0002-2729-995x töres pg theorell https://orcid.org/0000-0002-3845-3545 hyejin kim https://orcid.org/0000-0003-2211-209x references 1. boscart vm, mcneill s, grinspun d. dementia care in canada: nursing recommendations. can j aging. 2019;38:407–18. doi: 10.1017/ s071498081800065x 2. førsund lh, grov ek, helvik as, juvet lk, skovdahl k, eriksen s. the experience of lived space in persons with dementia: a systematic meta-synthesis. bmc geriatr. 2018;18:33. doi: 10.1186/ s12877-018-0728-0 3. low lf, fletcher j. models of home care services for persons with dementia: a narrative review. int psychogeriatr. 2015;27:1593–600. doi: 10.1017/s1041610215000137 4. gouin jp, hantsoo l, 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https://doi.org/10.3390/healthcare9060698 https://doi.org/10.3233/nre-208011 upsala journal of medical sciences 2023, 128, e8869 http://dx.doi.org/10.48101/ujms.v128.8869 accuracy of a faecal immunochemical test in patients under colonoscopy surveillance of colorectal adenoma and cancer louise olssona,b and daniel sjöbergc aschool of medical sciences, örebro university, örebro, sweden; bcamtö, örebro university hospital, örebro, sweden; ccentre for clinical research in dalarna, falun, sweden abstract background: surveillance of colorectal neoplasia place great strain on colonoscopy resources, and faecal immunochemical tests (fit) are under-investigated for this purpose. the aim of this study was to report the outcome of fit among patients scheduled for post-polypectomy and post-resection colorectal cancer (crc) surveillance. methods: patients scheduled for colonoscopy surveillance at five endoscopy units in mid-sweden in 2016– 2020 were eligible. they provided a faecal sample from 2 separate days, which were analysed by ifobt quikread go® (aidian oy). both the colonoscopies, and the fit analyses were conducted by staff blinded to the other. results: out of 216 included patients, 157 (73%) underwent both a complete colonoscopy and had at least one fit analysed prior to the examination. the indication for surveillance was previous adenoma in 69 (44%) and post-resection crc in 88 (56%) patients. two (1%) in the crc surveillance group were diagnosed with a metachronous crc, whereas 49 (56%) patients in the crc surveillance, and 17 (25%) in the adenoma group had no pathology identified at colonscopy (p < 0.001). the proportion of patients diagnosed with adenomas requiring surveillance according to european society of gastrointestinal society (esge) guidelines 2020 was 6 (7%) in the post-crc resection versus 7 (10%) in the adenoma surveillance group (p = 0.4). based on one fit and at cut-off 10 µg hb/g, sensitivity for crc was 100%, specificity 83% (95% confidence interval [ci]: 77–89), positive predictive value (ppv) 7% (−2 to 16) and negative predictive value (npv) 100%. all patients with an adenoma requiring surveillance had a fit below this cut-off. adding a second fit decreased the specificity. conclusion: larger studies to evaluate the accuracy and consequences of using fit for surveillance of colorectal neoplasia are needed. fit may be more interesting for post-resection crc surveillance than follow-up of adenoma. article history received: 5 july 2022; revised: 27 february 2023; accepted: 5 march 2023 published: 23 june 2023 keywords colorectal cancer; adenoma; faecal immunochemical test; surveillance; colonoscopy introduction post-polypectomy and post-resection colorectal cancer (crc) surveillance claim a large proportion of available colonoscopy resources, estimated to 18% of colonoscopies in the uk (1). lately, as new guidelines state more strict criteria for adenoma surveillance (2), the number of patients included in surveillance programme due to adenomas will probably decrease (3), but on the other hand, as more individuals are included in screening programmes, the influx for surveillance will continue. there is, therefore, a growing interest in using quantitative faecal immunochemical tests (fit) as a triage test for colonoscopy in surveillance of adenoma patients (4). such a surveillance regime would expose fewer patients to the discomfort of bowel cleansing and colonoscopy itself, and come at a lower cost. however, data on the consequences of using fit, at various cutoffs for faecal haemoglobin, in patients subjected to colonoscopy surveillance specifically is still very scarce. the aim of this study was to compare the outcome of fit in patients scheduled for colonoscopy, for post-polypectomy and post-resection crc surveillance. methods participants, inclusion and sample collection this is a prospective study on patients ≥18 years referred for colonoscopy surveillance after polypectomy of an adenoma, or radical resection of crc at five endoscopy units (örebro university hospital, and falun hospital in mid-sweden, and södersjukhuset, aleris sabbatsberg hospital and ersta hospital in stockholm). patients were included from june 2016 to january 2020 but recruitment periods differed between the sites and the inclusion rate varied within each site. exclusion criteria were surveillance due to ibd, chronic radiation proctitis and hereditary syndromes hereditary nonpolyposis colorectal cancer, adenomatous polyposis coli (hnpcc, apc). the dates and the quality of the previous colonoscopies were not available. contact l. olsson louise.olsson@oru.se © 2023 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article http://dx.doi.org/10.48101/ujms.v128.8869 mailto:louise.olsson@oru.se http://creativecommons.org/licenses/by/4.0/ 2 l. olsson and d. sjöberg eligible patients were contacted by phone well before their scheduled colonoscopy, and those interested in participating were mailed written study information, a consent form, sampling instructions and two fit sampling devices (quikread go® fob sampling set, aidian oy, espoo, finland), and envelopes with pre-paid stamps. they were asked about current symptoms and medication (anticoagulants), but received no instructions on diet or the use of drugs prior to sample collection. patients were to collect the faecal samples from 2 different days and fill in the dates of collection. they were to return the samples on the same day, or keep them in the refrigerator for at most 3 days before sending them off. index test the faecal samples were sent to unilabs laboratory, accredited according to iso 15189, at eskilstuna hospital, sweden. no clinical information accompanied the samples. the samples were analysed on the day of arrival using the quikread go® instrument (aidian oy, finland). this is a point-of-care quantitative immunochemical test device (5). all technicians involved received specific training for the study. a positive calibration control was done for every 50 analyses, and all were performed according to a specific standard operating protocol (sop) from the manufacturer. during the study period, the instrument provided numerical results of haemoglobin concentration in the range 15 to >200 µg hb/g faeces. in order to get numerical results also for the interval 10– 15 µg hb/g faeces, the manufacturer carried out supplementary analyses of the haemoglobin absorbance obtained from the instrument. concentrations below the latter interval were reported as <10 µg hb/g faeces. a checklist for reporting on fit is provided in appendix 1 (6). reference standard the overwhelming majority of colonoscopies were performed by gastroenterologists and according to routine standards. the study relied on clinical judgement insofar as any examination would be interrupted if the bowel cleansing was considered too poor. information on all documented intraluminal findings, bowel cleansing, and completeness of the examination (intubation of caecum or ileum) was extracted from the colonoscopy reports by one or two experienced endoscopy nurses at each site. neither the endoscopists, nor the nurses had any information on the fit outcome. biopsy specimens were sent for pathology assessment according to routines in the regular clinical setting and the findings were retrieved from the pathology reports. categorisation of findings the colonoscopy findings were categorized as crc, inflammation, diverticulosis and ‘no pathology’, the first two were confirmed by pathology reports. all available data on polyps/adenomas, i.e. number of lesions, macroscopic appearance, size and pathologists´ assessment of grade of dysplasia and cellular architecture were categorized independently by both researchers (lo, ds) according to guidelines of the european society of gastrointestinal endoscopy from 2020 and 2013, respectively (2, 7). the classification from 2013 involved categorisation into a low risk group (1–2 tubular adenomas < 10 mm and with low grade dysplasia) and a high risk group (adenomas with villous architecture or high grade dysplasia, ≥10 mm or ≥2 adenomas). the classification from 2020 involved adenomas requiring surveillance (at least 1 adenoma ≥10 mm or with high grade dysplasia, or ≥5 adenomas, or any serrated polyp ≥ 10 mm or with dysplasia) and adenoma in need of no surveillance (complete removal of 1–4 < 10 mm adenomas with low grade dysplasia, irrespective of villous components or any serrated polyp <10 mm without dysplasia). data were not complete on all aspects needed for classification of all lesions and some had to be classified as ‘undetermined polyps’. any disagreement between the researchers were resolved in consensus. if patients had several neoplastic findings, they were categorized according to the most advanced lesion. statistical analysis this is a feasibility study and no specific sample size calculation was done in advance. outcome of first faecal sample provided by each patient was denoted as ‘one fit’, and the highest numerical outcome of any of two analyses was denoted as ‘highest value/2 fits’. all faecal haemoglobin concentrations are reported as µg hb/g faeces. cut-off was set at 10 µg hb/g faeces, but the outcome is also reported for commonly used categories <10, 10–14.9, 15–19.9 and ≥20 µg hb/g. these cut-offs were specified in advance. sensitivity, specificity, positive and negative predictive values were reported with 95% confidence intervals. the chi-square test was used to compare proportions and the mann–whitney and t-tests to compare continuous variables. all analyses were executed in spss 22 (ibm, chicago, il, usa). a two-sided p-value <0.05 was considered statistically significant. a checklist for reporting according to the stard guidelines is in appendix 2 (8). the study was approved by the ethical review board in stockholm, april 2016 (d-nr 2016/711-32). results in all, 216 patients were included, but 37 patients provided no faecal sample, and 12 only after the colonoscopy (figure 1). ten patients provided faecal samples but nine did not undergo colonoscopy and one examination was incomplete. in total, 157 (73%) patients provided at least one stool sample and had a complete colonoscopy, and they were included in the analyses. the reason for colonoscopy was crc surveillance for 88 (56%) and adenoma surveillance for 69 (44%) patients (table 1). median age at colonoscopy for patients undergoing crc surveillance was 70 (range 40–85) and for adenoma surveillance colonoscopy surveillance of colorectal adenoma and cancer 3 69 (range 50–88) years (p = 0.37). for crc surveillance, there were 44 men and 44 women, and for adenoma surveillance, there were 46 (67%) men and 23 (33%) women (χ2 = 4.4; p = 0.04). in all, 32 (20%) patients reported symptoms at the time of their scheduled colonoscopy (table 1). there was no difference in the proportion reporting symptoms by indication (adenoma surveillance 16 [23%] vs. cancer surveillance 16 [18%]; p = 0.44), by age (above median age 20 [25%] patients vs. below median age 12 [15%; p = 0.12), or by sex (men 19 [21%] vs. women 13 [19%]; p = 0.8). the frequency of reported symptoms was as follows: diarrhoea 9 (28%), constipation 6 (19%), fresh blood 5 (16%), change of bowel habits 4 (12%), abdominal pain 3 (9%), bloating 3 (9%), and difficulties in emptying 2 (6%). findings the findings of colonoscopy are summarized in table 2. two patients had crc and 66 (42%) had a clean colon. in all, 51 (32%) patients had adenomas (13 requiring surveillance and 38 requiring no surveillance) whereas for 31 (20%) patients, data extracted from the colonoscopy reports was insufficient to admit any further classification than ‘undetermined polyps’. in 24 of these 31 cases, the polyps had not been sent for microscopic evaluation, and most of these polyps were depicted as diminutive, or minimal. the size of 13 adenoma classifying for surveillance according to esge 2020 was median 10 (range 2–20) mm, size available for 31/38 adenoma categorized as ‘no surveillance’ was median 5 (range 1–15) and for 16/31 in the undetermined group, size was median 4 (range 1–14) mm. a larger proportion of patients in the post-resection crc group had no pathology at colonoscopy compared with the adenoma surveillance group (56% vs. 25%) (χ2 = 16.4; p = 0.002) (table 2). the adenoma surveillance group was characterized figure 1. study flow chart. patients scheduled for colonoscopy surveillance and included in fit study n = 216 no index test n = 49 (23%) provided no stool sample 37 fit after colonoscopy 12 no reference test n = 10 (5%) no colonoscopy 9 incomplete colonoscopy 1 analysed n = 157 (73%) colonoscopy surveillance due to colorectal cancer 88 colonoscopy surveillance due to colorectal adenomas 69 colonoscopy �ndings cancer 2 (1%) adenoma 51 (32%) undetermined polyps 31 (20%) inflammation 2 (1%) diverticulosis 5 (3%) no pathology 66 (42%) fit outcome: median (range) one fit (n = 157): 3 (0-200) highest/2 fits (n = 148): 4 (0-200) table 1. basic characteristics of included patients (n = 157). variable n % age groups (years) 65 50 32 66–72 53 34 73 54 34 sex men 90 57 women 67 43 endoscopy unit sabbatsberg aleris 13 8 falun 33 21 örebro 53 34 södersjukhuset 47 30 ersta 11 7 reason for surveillance cancer 88 56 adenoma 69 44 reported symptoms yes 34 22 no 123 78 bowel cleansing complete 148 94 some remarks 9 6 anticoagulants yes 17 11 no 135 86 missing data 5 3 4 l. olsson and d. sjöberg both by a larger proportion of adenoma not qualifying for surveillance (32%) and undetermined polyps (28%), but there was no difference in the proportion of adenoma qualifying for surveillance between the post-resection crc (6/88 = 7%) and the adenoma surveillance group (7/69 = 10%), (χ2 = 0.6; p = 0.4). among patients with adenoma and undetermined polyps, 20/82 (24%) reported any symptom versus 12/66 (18%) among patients with no pathology detected on colonoscopy (χ2 = 0.8; p  = 0.4). neither of the two patients with crc reported any symptom. the findings on adenoma were compared using the esge 2013 and esge 2020 classifications and out of 24 patients categorized as high risk according to esge 2013, 11/24 (46%) did not fulfill the criteria for the surveillance group according to esge 2020 (table 3). outcome of fit the number of days from collection of the first faecal sample to colonoscopy was median 13 (range 1–133). the number of days from faecal collection until analysis was median 2 (range 0–7) for both the first and second fit. in all, 148 (94%) provided two fits (figure 1). outcome of the first fit was median 3 (range 0–200) µg hb/g faeces, of the second fit it was median 4 (range 0–200), and the highest value/2 fits was median 5 (range 0–200). among 32 patients who reported symptoms, first fit was median 2 (range 0–35), versus median 3 (range 0–200) in 125 patients who reported no symptoms (χ2 = 0.1; p = 0.8). the first fit from the two patients with crc showed 66 and 85 µg hb/g, respectively, whereas first fit from patients with adenomas qualifying for surveillance according to esge 2020 all showed <10 µg hb/g (table 4). twelve patients in this adenoma group provided two faecal samples but only 1/12 had an outcome above this cut-off. for patients with no pathology at colonoscopy, first fit was <10 µg hb/g in 58 (88%). based on one fit and cut-off 10 µg hb/g for positivity, 29/157 (18%) colonoscopies would have been carried out, both crc identified, and all 13 adenoma for surveillance missed. based on the highest value/2 fits and cut-off at 10 µg hb/g for positivity, 41 (28%) colonoscopies would have been carried out, both crc identified and 11/12 (92%) adenoma for surveillance would have been missed. a summary of the calculated accuracy is in table 5. higher cut-off values increased specificity, but adding a second fit decreased specificity. budget impact the estimated cost for one colonoscopy in sweden, 2022 is approximately 790 € (9). the cost for all 157 scheduled surveillance colonoscopies equals (157 × 660) 124,030 €. for one fit and cutoff at 10 µg/g, 128/157 (82%) were negative, theoretically lowering the costs for colonoscopy by (128 × 790) 101,120 €. the costs for the colonoscopies would have been 22,910 € to detect 2/2 crc and 0/13 adenomas qualifying for surveillance. an estimated total cost for one fit of 10–20 € would add another 1,570–3,140 € to the total costs. this would entail a reduction of costs close to 80% for surveillance in this particular group. discussion this study on post-polypectomy and post-resection crc surveillance, the first fit of both of the two patients diagnosed with crc had high concentrations of faecal haemoglobin well above cut-off at 10 µg/g faeces, whereas more than 80% of patients had a negative (<10 µg/g faeces) test. this included 13 patients diagnosed with adenomas qualifying for surveillance. there is only a limited number of studies on fit for surveillance of colorectal neoplasia available. for adenoma surveillance specifically, cross et al. recruited 6,000 patients after a positive gfobt and follow-up colonoscopy, as part of a screening programme in southern england in 2012–2013 (10). table 2. distribution of colonoscopy findings by indication for surveillance (n = 157). colonoscopy findings cancer surveillance n = 88 adenoma surveillance n = 69 total n = 157 n % n % n % cancer 2 (2) 0 (0) 2 (1) adenoma* and polyps1 34 (39) 48 (70) 82 (52) surveillance * 6 (7) 7 (10) 13 (8) no surveillance* 16 (18) 22 (32) 38 (24) undetermined1 12 (14) 19 (28) 31 (20) inflammation 1 (1) 1 (1) 2 (1) diverticulosis 2 (2) 3 (4) 5 (3) no pathology 49 (56) 17 (25) 66 (42) total 88 (100) 69 (100) 157 (100) *classification according to esge 2020. 1insufficient data for further classification of these polyps. number in italics indicate subgroups of adenomas and polyps. table 3. classification of adenoma according to guidelines from esge 2020 (surveillance, no surveillance) versus esge 2013 (high risk, low risk) (n = 51). adenoma classification esge 2020 surveillance no surveillance total esge 2013 high risk 13 (54) 11 (46) 24 (100) low risk 0 27 27 (100) total 13 (25) 38 (75) 51 (100) values in parentheses are row percentages. colonoscopy surveillance of colorectal adenoma and cancer 5 after polypectomy, patients with an intermediate risk of crc (3–4 small adenomas or one adenoma ≥10 mm) had annual fit (oc-sensor diana) and a colonoscopy after 3 years. overall programme sensitivity of fit at cut-off 10 μg/g was 72% for crc and 57% for advanced adenoma (aa); 3-year positivity at this cut-off was 29%. in other words, replacing a colonoscopy surveillance after 3 years with annual fit could reduce colonoscopies by 71%, but would miss 30–40% of crcs and a large proportion of aa. but, importantly, sensitivity for crc of first fit in this study was 52% (95% confidence interval [ci]: 32–71), and 33% (95% ci: 29–38) for aa. a prospective, double-blind study from israel published in 2010 included 1,071 consecutive, asymptomatic patients scheduled for colonoscopy surveillance after resection of crc, or adenoma polypectomy, or at increased risk of crc due to family history (11). participants provided three stool samples for oc-micro i-fobt analysis. first test was positive in 8%, cumulative positivity of the first two tests rose to 12%, and was 15% of all three tests. at the lowest threshold of 50 ng hb ⁄ml of buffer, and using only the first i-fobt, sensitivity for crc was 100% and 65% for all significant neoplasms (crc or advanced adenomatous polyps). finally, a study from the netherlands conducted in 2006–2009 including 1,041 participants scheduled for colonoscopy surveillance due to personal history of adenoma/crc, or family history of crc, reported an overall positivity rate of 11% at cut-off 50 ng/ml using oc-sensor (12). sensitivity for crc was 80% (95% ci: 28–99) and 28% (95% ci: 19–38) for advanced adenoma. this study is the first to report a sensitivity of 100 and 0% for crc and aa, respectively, of one fit. the intensity of colonoscopy surveillance of colorectal adenoma has been reduced lately, and one specific example is the previously recommended colonoscopy surveillance at intervals of 3 years for individuals with an estimated intermediate risk of crc. for this group, atkin et al. found that, if baseline colonoscopy was of high quality, and if there were no proximal polyps, high-grade or large adenoma ≥20 mm, the risk of crc was even lower than that of the general population (13). since 2020, esge has adopted more strict criteria for colonoscopy surveillance after 3 years and it is now recommended when at least one adenoma ≥10 mm, or with high‐grade dysplasia, or ≥5 adenomas, or any serrated polyp ≥10 mm or with dysplasia is detected, i.e. villous architecture and merely three adenoma are no longer indications for surveillance (2). a study from austria contrasted these new guidelines with the 2013 recommendations (7) and, as part of a quality assurance programme, found the proportion of individuals assigned to 3-year colonoscopy fell from table 4. outcome of one fit and max value/ two fits (µg hb/g) by colonoscopy findings. colonoscopy findings 9.9 10–14.9 15–19.9 ≥20 total one fit cancer 0 0 0 2 (100) 2 adenoma* and polyps1 65 (79) 5 (6) 3 (4) 9 (11) 82 surveillance* 13 (100) 0 0 0 13 no surveillance* 31 (82) 3 (8) 2 (5) 2 (5) 38 undetermined1 21 (68) 2 (6) 1 (3) 7 (23) 31 inflammation 1 (50) 0 1 (50) 0 2 diverticulosis 4 (80) 0 1 (20) 0 5 no pathology 58 (88) 1 (2) 1 (2) 6 (9) 66 total 128 (82) 6 (4) 6 (4) 17 (11) 157 (100) max value/two fits cancer 0 0 0 2 (100) 2 adenoma* and polyps1 58 (73) 6 (8) 4 (5.0) 12 (15) 80 surveillance* 11 (92) 1 (8) 0 0 12 no surveillance* 26 (70) 3 (8) 3 (8) 5 (14) 37 undetermined1 21 (68) 2 (6) 1 (3) 7 (23) 31 inflammation 1 (50) 0 0 1 (50) 2 diverticulosis 4 (80) 0 1 (20) 0 5 no pathology 44 (75) 5 (8) 3 (5) 7 (12) 59 total 107 (72) 11 (7) 8 (5) 22 (15) 148 (100) fit: faecal immunochemical tests. *adenoma classification according to esge 2020. 1insufficient data for further classification of these polyps. values in parenthesis are row percentages. table 5. accuracy and predictive values for colorectal cancer of one fit and max value/2 fits at cut-off 10, 15 and 20 µg hb/g faeces. one fit cut-off sensitivity specificity (95% ci) ppv (95% ci) npv ≥10 100 83 (77–89) 7 (−2 to 16) 100 ≥15 100 86 (81–92) 9 (−3 to 20) 100 ≥20 100 90 (86–95) 12 (−4 to 27) 100 highest/2 fits ≥10 100 73 (66–80) 5 (−2 to 11) 100 ≥15 100 80 (74–87) 7 (−2 to 16) 100 ≥20 100 86 (81–92) 9 (−2 to 21) 100 fit: faecal immunochemical tests; ci: confidence interval; ppv: positive predictive value; npv: negative predictive value. 6 l. olsson and d. sjöberg 10.4 to 4.9%, a relative reduction of 47% (3). this is very close to our findings in this study (11/24; 46%). the austrian researchers also reported maintained, or even improved, risk stratification because the point estimate of crc mortality was higher in the surveillance group according to 2020 guidelines compared with the high-risk group adenoma of guidelines from 2013 (hazard ratio (hr): 2.6; 95% ci: 1.6–4.0 vs. hr: 1.7; 95% ci: 1.1–2.6). in addition, there was no difference in all-cause mortality between the no surveillance group of 2020 guidelines as compared with low-risk group of 2013 (hr: 1.06, 95% ci: 1.01–1.11 vs. hr: 1.05, 95% ci: 0.99–1.10). this clearly indicates that there has been an overuse of colonoscopy surveillance. concerning surveillance after resection of crc, a systematic review and meta-analysis reported a cumulative incidence of metachronous crc over 16 years of 2.2% (95% ci: 1.8–2.9), the overwhelming majority being detected within the first 36  months (14). in addition, the cumulative incidence of anastomotic crc was 2.7% (95% ci: 1.9–3.9). the literature search included studies from inception and up to 2018, and only a smaller part provided data from the era of colonoscopies of high quality. however, even in a recent study of dutch patients who had undergone preoperative colonoscopies in 2013–2016, five metachronous crc, and five anastomotic recurrences (10/572 ~1.7% lesions) were detected at colonoscopy after mean 13 months (15). in all, there is limited evidence underpinning current guidelines on colonoscopy surveillance after crc, but colonoscopy is in general recommended 1 year after resection, and then after 3 and 5 years (16). adherence to guidelines on colonoscopy surveillance intervals is a well-known problem and estimated to merely 49% (17, 18) and shorter intervals are often recommended by physicians (19). interestingly, the study outlined here on surveillance of adenoma with an estimated intermediate risk (13) was made possible only due to the fact that 5,019/11,944 (42%) eligible patients did not attend surveillance, compared with 6925 (58%) individuals who complied with the recommendations. common reasons among patients are fear of pain and discomfort, and ‘concerns about bowel preparation’ (20). in hypothetical scenarios presented to the english public, fit was preferred over colonoscopy for both adenoma surveillance, and work-up of symptoms (21, 22). incremental costs per additional advanced adenoma detected by colonoscopy versus by fit at cut-off 10 μg/g was estimated to £8,863 (95% ci: 7,018–10,939) and per additional crc to £243,094 (95% ci: −1,242,531 to 1,990,865) in the largest study on fit for surveillance so far by cross et al. (10). a simulation study based on the dutch screening programme among asymptomatic individuals of 55 to 75 years found that adding colonoscopy surveillance to fit screening was not cost-effective based on the dutch icer threshold, and increased the colonoscopy demand substantially (23). both examples illustrate the resource-consuming aspect of primarily colonoscopy-based surveillance. limitations of this study are the low number of participants of a convenience series. merits are the blinded collection of data, a  meticulous classification of adenomas/polyps, and the comparison of fit surveillance of adenoma and crc in a setting representing routine care. overall, our findings are consistent with previous studies, i.e. there is a significant difference in the ability of fit to detect crc and adenoma (24). however, a few circumstances underline fit surveillance as an important topic for further studies. firstly, there is little hard evidence on the effect of colonoscopy surveillance on crc incidence and crc mortality, and the reduction of crc risk compared with the general population was quite recently shown to be limited to high-risk groups only (25). it has also been put forward that surveillance may be limited to adenoma sized ≥20 mm or high-grade dysplasia, in particular for healthcare systems with limited capacity, as this would reduce the number of colonoscopies substantially with little effect on crc mortality (26). the findings were also reproduced by the austrian researchers (3). this is interesting, as fit level is associated with adenoma size (27). secondly, adherence to surveillance is crucial, and fit may make up a first option for patients who hesitate to undergo colonoscopy or bowel cleansing for psychological reasons, for patients with severe comorbidity, or for those living far away from nearest endoscopy unit (28). thirdly, health economic aspects favour fit surveillance, and fourthly, free up the colonoscopy resource for symptomatic patients or screening. another possibility is to integrate fit into existing colonoscopy surveillance programmes to prolong or personalize the colonoscopy intervals (29). in all, we conclude further studies on fit surveillance of colorectal neoplasia are warranted. disclosure statement aidian oy provided a quikread go® instrument for analysis. the authors declare no conflict of interest. funding the study was supported by afa försäkring. acknowledgements the authors would like to thank the staff at the participating centres for granting the recruitment of patients. notes on contributors louise olsson, md, phd, associate professor of surgery, consultant, head of hta unit (camtö), örebro. daniel sjöberg, md, phd, post-doc, consultant gastroenterologist, falun. orcid louise olsson https://orcid.org/0000-0002-8708-7502 daniel sjöberg https://orcid.org/0000-0002-9082-8017 https://orcid.org/0000-0002-8708-7502 https://orcid.org/0000-0002-8708-7502 https://orcid.org/0000-0002-9082-8017 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2019;17:2285–93. doi: 10.1016/j.cgh.2019.02.026 16. kahi cj, boland cr, dominitz ja, giardiello fm, johnson da, kaltenbach t, et al. colonoscopy surveillance after colorectal cancer resection: recommendations of the us multi-society task force on colorectal cancer. gastroenterology. 2016;150:758–68 e11. doi: 10.1053/j. gastro.2016.01.001 17. djinbachian r, dubé a-j, durand m, camara lr, panzini b, bouchard s, et al. adherence to post-polypectomy surveillance guidelines: a systematic review and meta-analysis. endoscopy. 2019;51:673–83. 18. dong j, wang lf, ardolino e, feuerstein jd. real-world compliance with the 2020 u.s. multi-society task force on colorectal cancer polypectomy surveillance guidelines: an observational study. gastrointest endosc. 2023; 97: 350–6 e3. doi: 10.1016/j.gie.2022.08.020 19. johnson mr, grubber j, grambow sc, maciejewski ml, dunn-thomas t, provenzale d, et al. physician non-adherence to colonoscopy interval guidelines in the veterans affairs healthcare system. gastroenterology. 2015;149:938–51. doi: 10.1053/j.gastro.2015.06.026 20. kerrison rs, sheik-mohamud d, mcbride e, whitaker kl, rees c, duffy s, et al. patient barriers and facilitators of colonoscopy use: a rapid systematic review and thematic synthesis of the qualitative literature. prev med. 2021;145:106413. doi: 10.1016/j.ypmed.2020.106413 21. bonello b, ghanouni a, bowyer hl, macrae e, atkin w, halloran sp, et al. using a hypothetical scenario to assess public preferences for colorectal surveillance following screening-detected, intermediate-risk adenomas: annual home-based stool test vs. triennial colonoscopy. bmc gastroenterol. 2016;16:113. doi: 10.1186/s12876-016-0517-1 22. von wagner c, verstraete w, hirst y, nicholson bd, stoffel st, laszlo h. public preferences for using quantitative faecal immunochemical test versus colonoscopy as diagnostic test for colorectal cancer: evidence from an online survey. bjgp open. 2020;4. doi: 10.3399/ bjgpopen20x101007 23. greuter mje, de klerk cm, meijer ga, dekker e, coupe vmh. screening for colorectal cancer with fecal immunochemical testing with and without postpolypectomy surveillance colonoscopy: a cost-effectiveness analysis. ann intern med. 2017;167:544–54. doi: 10.7326/m16-2891 24. imperiale tf, gruber rn, stump te, emmett tw, monahan po. performance characteristics of fecal immunochemical tests for colorectal cancer and advanced adenomatous polyps: a systematic review and meta-analysis. ann intern med. 2019;170:319–29. doi: 10.7326/m18-2390 25. cross aj, robbins ec, pack k, stenson i, kirby pl, patel b, et al. long-term colorectal cancer incidence after adenoma removal and the effects of surveillance on incidence: a multicentre, retrospective, cohort study. gut. 2020;69:1645–58. doi: 10.1136/gutjnl-2019-320036 26. wieszczy p, kaminski mf, franczyk r, loberg m, kobiela j, rupinska m, et al. colorectal cancer incidence and mortality after removal of adenomas during screening colonoscopies. gastroenterology. 2020;158:875– 83 e5. doi: 10.1053/j.gastro.2019.09.011 27. o’reilly sm, macnally s, o’donoghue d, mooney t, fitzpatrick p, mulcahy he, et al. correlation of fecal immunochemical testing levels with pathology results in a national colorectal cancer screening program. clin transl gastroenterol. 2021;12:e00277. doi: 10.14309/ctg.0000000000000277 28. pilonis nd, bugajski m, wieszczy p, rupinski m, pisera m, pawlak e, et al. participation in competing strategies for colorectal cancer screening: a randomized health services study (piccolino study). gastroenterology. 2021;160:1097–105. doi: 10.1053/j.gastro.2020.11.049 29. wassie mm, young gp, winter jm, cock c, bampton p, rahman m, et al. multiple negative fecal immunochemical tests reduce risk of advanced neoplasia in a colonoscopy surveillance program. clin gastroenterol hepatol 2023 jan 5;s1542-3565(23)00002-2. doi: 10.1016/j. cgh.2022.12.024 https://doi.org/10.1136/gutjnl-2011-301848 https://doi.org/10.1055/a-1185-3109 https://doi.org/10.1002/ueg2.12119 https://doi.org/10.3310/hta23010 https://doi.org/10.1515/cclm-2021-0655 https://doi.org/10.1097/cej.0000000000000016 https://doi.org/10.1055/s-0033-1344548 https://doi.org/10.1136/bmj.h5527 https://vardgivare.skane.se/patientadministration/avgifter-och-prislistor/prislistor-sodra-sjukvardsregionen/ https://vardgivare.skane.se/patientadministration/avgifter-och-prislistor/prislistor-sodra-sjukvardsregionen/ https://doi.org/10.1136/gutjnl-2018-317297 https://doi.org/10.1136/gutjnl-2018-317297 https://doi.org/10.1111/j.1365-2036.2009.04202.x https://doi.org/10.1186/1471-230x-12-94 https://doi.org/10.1016/s1470-2045(17)30187-0 https://doi.org/10.1016/s1470-2045(17)30187-0 https://doi.org/10.1053/j.gastro.2018.12.006 https://doi.org/10.1016/j.cgh.2019.02.026 https://doi.org/10.1053/j.gastro.2016.01.001 https://doi.org/10.1053/j.gastro.2016.01.001 https://doi.org/10.1016/j.gie.2022.08.020 https://doi.org/10.1053/j.gastro.2015.06.026 https://doi.org/10.1016/j.ypmed.2020.106413 https://doi.org/10.1186/s12876-016-0517-1 https://doi.org/10.3399/bjgpopen20x101007 https://doi.org/10.3399/bjgpopen20x101007 https://doi.org/10.7326/m16-2891 https://doi.org/10.7326/m18-2390 https://doi.org/10.1136/gutjnl-2019-320036 https://doi.org/10.1053/j.gastro.2019.09.011 https://doi.org/10.14309/ctg.0000000000000277 https://doi.org/10.1053/j.gastro.2020.11.049 https://doi.org/10.1016/j.cgh.2022.12.024 https://doi.org/10.1016/j.cgh.2022.12.024 8 l. olsson and d. sjöberg appendix 1. checklist for reporting on faecal immunochemical tests. specimen collection and handling quikread go® aidian oy, råsta strand väg 13 c 169 79 solna name of specimen collection device and supplier (address). essential quik read fob probe description of specimen collection device (vial with probe/stick, card, other). essential single faecal samples description of specimens used if an in vivo study (single or pooled faeces, artificial matrix with added blood, etc.). essential for laboratory evaluations probe details of faecal collection method (sampling technique and number of samples). essential patients who collected the specimens from the samples (patient, technician, etc.). essential two faecal specimens from two separate days number of faecal specimens used in the study (single, pooled, individual patient faeces). essential for laboratory evaluations 10 mg mean mass of faeces collected.* essential buffer into which specimen is taken by probe volume of buffer into which specimen is taken by probe, applicator stick or card.* essential kept at most 3 days in fridge temperature at home before sending to the laboratory by ordinary post (1 day) time and storage conditions of faecal specimen from ‘passing’ to sampling, including time and temperature (median and range). essential for laboratory evaluations specimen were analysed on the day of arrival to the laboratory time and storage of collection devices from specimen collection to analysis, including time and temperature (median and range). a concise description of process from collection to analysis is recommended. essential analysis quikread go® aidian oy, råsta strand väg 13 c 169 79 solna tel +468-623 04 00 one instrument was used during the study name of analyser, model, supplier (address), number of systems if more than one used. essential one number of times each sample was analysed. essential 75–1000 ng hb/ml buffer, 15–200 µg hb/g faeces no reassays or dilutions analytical working range* and whether samples outside this range were diluted (factor) and reassayed. essential for laboratory evaluations quikread go ifobt. the reference material has been verified with a method comparable to the international council of standardisation in haematology (isch) reference method. source of calibrator(s) (supplier with address), number of calibrator(s), how concentrations were assigned* and details of calibration process including frequency. essential for laboratory evaluations ~ 340 samples analysed. mean (sd) was 11,2 (30,4) µg hb/g faeces. analytical imprecision*, ideally with number of samples analysed, concentrations, and mean, sd and cv. essential for all studies quality management quik read fob positive control cat.no 06027 hb in buffert >1000 ng/ml or > 200 μg hb/g faeces. one control performed per kit (50 tests) source (address) or description of internal quality control materials, number of controls, assigned target concentrations and ranges, how target concentrations were assigned, rules used for acceptance and rejection of analytical runs. desirable for laboratory evaluations no participation in external quality assessment schemes: (name and address of scheme), frequency of challenges, performance attained. desirable for laboratory evaluations unilabs inc, dep of clinical chemistry, mälarsjukhuset s-631 88 eskilstuna sweden accreditation held by the analytical facility (address). desirable for laboratory evaluations 5 biomedical scientists the number, training and expertise of the persons performing the analyses and recording the results. essential colonoscopy surveillance of colorectal adenoma and cancer 9 result handling electronic and manual recording, single reading mode of collection of data – manual recording or via automatic download to it system, single or double reading. desirable μg hb/g faeces units used, with conversion to μg hb/g faeces if ng hb/ml used. essential assigned by instrument 15 μg hb/g faeces and then complementary analyses by the manufacturer cut-off concentration(s) if used and explanation of how assigned locally or by manufacturer.* essential yes were the analysts blinded (masked) to the results of the reference investigation and other clinical information. essential 10 l. olsson and d. sjöberg appendix 2. stard guidelines for reporting diagnostic accuracy studies. section & topic no item reported on page # title or abstract 1 identification as a study of diagnostic accuracy using at least one measure of accuracy (such as sensitivity, specificity, predictive values, or auc) 2 abstract 2 structured summary of study design, methods, results, and conclusions (for specific guidance, see stard for abstracts) 2 introduction 3 scientific and clinical background, including the intended use and clinical role of the index test 3 4 study objectives and hypotheses 3 methods study design 5 whether data collection was planned before the index test and reference standard were performed (prospective study) or after (retrospective study) 4 participants 6 eligibility criteria 4 7 on what basis potentially eligible participants were identified (such as symptoms, results from previous tests, inclusion in registry) 4 8 where and when potentially eligible participants were identified (setting, location and dates) 4 9 whether participants formed a consecutive, random or convenience series 4, 14 test methods 10a index test, in sufficient detail to allow replication 4 10b reference standard, in sufficient detail to allow replication 5 11 rationale for choosing the reference standard (if alternatives exist) 12a definition of and rationale for test positivity cut-offs or result categories of the index test, distinguishing pre-specified from exploratory 6 12b definition of and rationale for test positivity cut-offs or result categories of the reference standard, distinguishing pre-specified from exploratory 6 13a whether clinical information and reference standard results were available to the performers/ readers of the index test 4 13b whether clinical information and index test results were available to the assessors of the reference standard 5 analysis 14 methods for estimating or comparing measures of diagnostic accuracy 7 15 how indeterminate index test or reference standard results were handled 8, figure 1 16 how missing data on the index test and reference standard were handled 8, figure 1 17 any analyses of variability in diagnostic accuracy, distinguishing pre-specified from exploratory 7 18 intended sample size and how it was determined 6 results participants 19 flow of participants, using a diagram fig 1 20 baseline demographic and clinical characteristics of participants table 1 21a distribution of severity of disease in those with the target condition table 1 21b distribution of alternative diagnoses in those without the target condition table 1 22 time interval and any clinical interventions between index test and reference standard 9 test results 23 cross tabulation of the index test results (or their distribution) by the results of the reference standard table 4 24 estimates of diagnostic accuracy and their precision (such as 95% confidence intervals) table 5 25 any adverse events from performing the index test or the reference standard not reported discussion 26 study limitations, including sources of potential bias, statistical uncertainty, and generalisability 12–16 27 implications for practice, including the intended use and clinical role of the index test 12–16 other information 28 registration number and name of registry 7 29 where the full study protocol can be accessed 30 sources of funding and other support; role of funders 16 upsala journal of medical sciences 2023, 128, e9014 http://dx.doi.org/10.48101/ujms.v128.9014 a long-term follow-up study of labor market marginalization in psychiatric patients with and without personality disorder hanna spangenberg, mia ramklint and adriana ramirez department of medical sciences, uppsala university, uppsala, sweden abstract background: personality disorders (pds) in adulthood are considered stable over time and are likely to have lasting psychosocial impact on the affected individual, including in areas like vocational functioning. the aim of this study was to study labor market marginalization (lmm) and receipt of social welfare benefits during 13 years from age 18 to 25 years in a sample of former psychiatric patients with and without pd. methods: this study followed-up 186 former psychiatric patients who were thoroughly assessed in 2002– 2004, including for pd, and compared them with controls. participants were divided into three groups: former patients with pd, without pd, and a matched control group from the general population. register data on employment, sick leave absence, disability pensioning, education, days of psychiatric care, income, and receipt of social welfare benefits in 2003–2016 were collected. results: former patients had more days of unemployment, sick leave absence, and disability pensioning and received more social welfare benefits than controls during the study period. differences between patients with and without pd were smaller than expected, but significant as regards receipt of social welfare benefits. pd also had an effect on income at age 30 years. conclusions: early onset of psychiatric disorders impairs vocational functioning up to 13 years after diagnosis, and most in those with pd. article history received: 15 september 2022 revised: 9 june 2023 accepted: 29 june 2023 published: 31 july 2023 keywords personality disorder; young adulthood; social functioning; labor market marginalization; vocational functioning introduction personality disorders (pds) are associated with a wide range of functional impairments affecting psychosocial functioning, including in the occupational setting (1, 2). the impact of pd on work life has been studied as regards any association between pd and labor market marginalization (lmm), and there is evidence that pd increases the risk of lmm (3, 4). there is also evidence, suggesting that especially borderline pd entails worse psychosocial function, though improvement in psychosocial function is often seen over time across all pds (5). there is no scientific consensus on the definition of lmm. from a social insurance perspective, lmm can be conceptualized as based on either medical assessments (resulting in sick leave or disability pension) or non-medical assessments (resulting in unemployment) (6). studying lmm is important, as occupation, education, and income are traditional indicators of socioeconomic position (7), and there is substantial evidence showing that socioeconomic position has a major impact on health outcomes and mortality (7–9). furthermore, there is evidence suggesting a reciprocal association between personality functioning and aspects of functional impairment, such as psychosocial functioning, indicating that interventions aimed at either domain will positively affect the other (10). this underscores the importance of studying the psychosocial effects of functional impairments in pd, such as lmm. some research focuses on specific areas of lmm; a few studies have been performed of the association between pd and longterm sick leave. a norwegian study found a significant association among schizotypal, paranoid, and borderline pd and risk of long-term sick leave (11). studies have shown ambiguous results regarding the association between pd and disability pensioning. in a swedish register study, pd and schizophrenia/non-affective psychoses were found to bring larger increases in risk of disability pensioning and long-term sick leave than other mental disorders (3). a norwegian study found a strong association between pd and disability pensioning in young adults. the association was stronger than for mood disorders (12). another british study found a strong association between probable pd with comorbid mental disorders and disability pensioning, but a weak association between probable pd without comorbid mental disorders and disability pensioning (13), whereas a finnish study found that pd increased the risk of early retirement on health grounds more than twice as much as anxiety disorders, and to an equal or slightly larger degree as contact hanna spangenberg hanna.spangenberg@neuro.uu.se supplemental data for this article can be accessed here. © 2023 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original research article http://dx.doi.org/10.48101/ujms.v128.9014 mailto:hanna.spangenberg@neuro.uu.se http://dx.doi.org/10.48101/ujms.v128.9014 http://creativecommons.org/licenses/by/4.0/ 2 h. spangenberg et al. depression (14). there is evidence suggesting that pd increases the risk of unemployment (4, 15). a swedish study showed that males with pd in young adulthood had approximately 10 additional days of unemployment per year compared with peers without any mental disorder, which was comparable to the effects of other common mental disorders (4). another indicator of socioeconomic position is education. there is evidence suggesting an inverse relation between pd and level of education (1). an australian study found severity of pd in young adults to be associated with not having a degree or vocational qualification and with receiving welfare benefits (16). although there are a number of studies of lmm and socioeconomic position in pd, there is a scarcity of studies of these outcomes where pd has been assessed reliably and using gold standard diagnostics. in 1983, spitzer proposed a new procedure for psychiatric diagnostics: ‘longitudinal evaluation (l), done by experts (e), all (a) data (d) available’ (lead) (17). the lead procedure has been used as an index of validity in studies of psychiatric diagnostics (18, 19) and has been shown to produce stable and valid predictors of clinical status in pd over time (20). in sweden, the lead procedure is considered the gold standard for psychiatric diagnostics, as the swedish health technology institute uses it for this purpose, based on a systematic literature review (21). few studies have used a lead procedure in analyses of psychosocial outcomes, such as lmm, in pd. the aim of this study was to evaluate lmm after 13 years in a clinical sample of psychiatric patients with pd reliably diagnosed in young adulthood through the lead procedure. methods participants this study was a follow-up of a clinical cohort from 2002 to 2004 comprising all patients aged 18–25 years who sought care at a specific psychiatric out-patient clinic in uppsala, sweden. in the original study (22), 217 patients came for an appointment and were invited to participate, and 200 (92%) agreed. each participant underwent a psychiatric diagnostic assessment in accordance with the lead procedure over three patient visits. a clinical interview was conducted by a psychiatrist during the first visit, and the structured clinical interview for dsm-iv axis i disorders clinical version, scid-i-cv (23), was conducted by the same psychiatrist during the second visit. psychosocial problems were evaluated by a social worker during the third visit, in accordance with dsm-iv axis iv, within nine categories (problems with the primary support group, problems related to the social environment, educational problems, occupational problems, housing problems, economic problems, problems with access to healthcare services, problems related to interaction with the legal system/crime, and other psychosocial and environmental problems). the total burden of stress was rated on a scale from 1 (none) to 6 (catastrophic) within each domain. a team conference was held after three visits, at which all the collected information was presented, and diagnoses were established in accordance with the diagnostic and statistical manual of mental disorders, 4th edition (dsm-iv) (24). the psychiatrists performed assessment of pds, including the structured interview for dsm-iv axis ii disorders (scid-ii) (25), for 188 participants (94%), after treatment of axis i disorder had been finalized, in order to minimize the state effect of current axis i disorders. interrater reliability between the two psychiatrists who conducted all clinical and diagnostic interviews was measured for six randomly selected scid-i interviews and six randomly selected scid-ii interviews (kappa coefficients of 1.0 and 0.89, respectively). the psychiatrists were both trained in accordance with the scid manual. pd diagnosis in 188 subjects was based on observations starting at the preliminary diagnostic process (anamnesis and selected questionnaires, scid-i, an interview with a social worker), continuing during the first treatment process, and ending during assessment of pd (scid-ii) performed by the same psychiatrist who made the initial assessments. scid-ii assesses a total of 101 symptom criteria on a scale from 1 to 3 (1 = absent, 2 = subthreshold, and  3 = threshold), resulting in a score of 101–303. aside from symptom criteria, general pd criteria were evaluated during the interviews. two individuals later withdrew consent and were excluded from the study. in 2016, participants who had undergone pd diagnostics in the original study (n = 186) were divided into two groups based on the presence or absence of pd at baseline, yielding one group of 52 individuals who had one or more pds at baseline (pd group) and one group of 134 individuals who had no pd at baseline (non-pd group). within the pd group, the number of pd diagnoses per individual was 1–4: 25 individuals (48%) had one pd, 19 (10.2%) had two pds, six (3.2%) had three pds, and two (1%) had four pds. the mean scid-ii score in the pd group was 164 (standard deviation [sd] 19), and that in the non-pd group was 132 (sd 15) (t = 10.82, p < 0.01). there were no significant differences in employment status or study enrollment between the two former patient groups at baseline. in the pd group, 11 individuals (21%) were employed at baseline, and 29 individuals (56%) were studying. in the non-pd group, 27 individuals (20%) were employed at baseline and 89 individuals (66%) were studying. further characteristics of the two former patient groups are shown in table 1. a control group was established for the study by statistics sweden, by matching each participant in the patient groups for sex, age, and place of residency in 2002–2003 with five control subjects (n = 930). data on participants and controls were obtained from national registers from statistics sweden and the swedish national board of health and welfare. registers statistics sweden is the agency responsible for holding national register data in sweden. statistics sweden compiles data from various sources, such as the swedish national tax agency and the swedish social insurance agency, in its registers (26, 27). this study used data from the longitudinal a long-term follow-up study of labor market marginalization 3 integrated database for health insurance and labor market studies (lisa), withheld by statistics sweden. the swedish national board of health and welfare maintains national registers on swedish healthcare and social services, such as the swedish national inpatient register (ipr). the quality of  parts of these registers, including the ipr, has been validated (28). for this study, data on participants and controls for the years 2003–2016 were collected from lisa, and the swedish national board of health and welfare’s ipr register. names and personal identity numbers of participants and controls were omitted from the retrieved register data to protect their privacy. the variables used in the study are described below and are illustrated in supplementary table 1. the study was not pre-registered, and the data are not available for external use. variables unemployment data on unemployment were collected from lisa. data were analyzed as days of unemployment during the study period. the  data collected covered days where individuals were registered as part-time unemployed, full-time unemployed, or registered in labor market measures for the unemployed. sick leave data on sick leave were collected from lisa. sick leave was defined as sick leave absence >14 days, as statistics sweden only records information about sick leave from day 15 in lisa, due to how the swedish social security system is devised. sick leave absence is paid for by the employer until day 15; sick leave episodes <15 days are therefore not reported to lisa. exceptions to this rule can be made if new sick leave episodes occur within 5 days of a previous episode. this can be registered in lisa, as it can be paid for by the social insurance agency, instead of by the employer (26). sick leave can be granted for full-time, 100%, or part-time, 75, 50, or 25%. to receive sick leave payments, an individual must have been at least partially employed during the preceding year. if there are no grounds for sick leave payment, for instance, due to unemployment, a medical certificate can mean the individual is eligible for social welfare benefits instead. in the current study, the net number table 1. description of study group at baseline 2002–2003. descriptive personality disorder, pd n = 52, n (%) no personality disorder, non-pd n = 134, n (%) χ2 p females, n (%) 38 (73) 110 (82) 0.64 0.42 axis i disorder χ2 p affective disorder 42 (81) 100 (75) 0.78 0.38 anxiety disorder 42 (81) 87 (65) 4.42 0.04 psychotic disorder 0 (0) 2 (1) 0.78 0.38 eating disorder 17 (33) 35 (26) 0.80 0.37 substance abuse 8 (15) 6 (4) 6.40 0.01 other psychiatric disorder 5 (10) 8 (6) 0.77 0.38 number of axis i disorders χ2 p 0 0 (0) 6 (4) 26.50 0.001 1 7 (13) 43 (32) 2 13 (25) 53 (40) 3 15 (29) 19 (14) 4 10 (19) 9 (7) 5 7 (13) 4 (3) personality disorder paranoid 9 (17) schizoid 2 (4) schizotypal 1 (2) borderline 15 (29) histrionic 1 (2) narcissistic 1 (2) antisocial 4 (8) avoidant 29 (56) dependent 1 (2) obsessive-compulsive 5 (10) t p scid-ii score, mean (sd) 164 (19) 132 (15) 10.82 0.01 t p psychosocial problems score, mean (sd) 16 (6) 13 (4) 3.29 <0.01 sd: standard deviation; pd: personality disorders. https://ujms.net/index.php/ujms/article/view/9014/15789 4 h. spangenberg et al. of sick leave days during the study period was used in all analyses. disability pensioning data on disability pensioning were obtained from lisa. to receive a disability pension in sweden, an individual’s work ability must be expected to be at least 25% reduced for at least 1 year. the work disability can be complete, 100%, or partial, 75, 50, or 25%, and can be permanent or time-limited, for example, lasting 3 years. since 2009, it is permitted to work up to 12.5% of full-time and still retain full compensation (26). in the current study, the net number of days of disability pensioning was calculated by multiplying the degree of disability compensation with the number of days on disability pension. social welfare benefits data on social welfare benefits were collected from lisa. in sweden, social welfare benefits are granted to people who are unable to financially support themselves or their children. social welfare benefits aim to cover the regular expenses in a household. recipients of social welfare benefits are obliged to actively search for work opportunities or take part in educational programs aiming to increase their chances of employment in the future. if an individual is not in a position to apply for work due to medical conditions but does not fulfill the criteria for sick leave payments, a medical certificate can grant the individual social welfare benefits without the requirement of applying for work (29). for this study, information was collected on the amount of money that individuals had received as social welfare benefits during the study period. income data on total income were collected from lisa. the variable on income which was used for analyses covered the individuals’ declared income. social welfare benefits, sick leave payments, and study grants are not included in this register variable. those who are self-employed can choose to declare income under a different variable. education data on the highest attained level of education at age 30 years were collected from lisa. data were dichotomized into two groups: up to secondary education (≤12 years) and postsecondary education (>12 years). disease burden due to psychiatric disorders days admitted to hospital for psychiatric care were calculated using data from the ipr. the ipr records day of admittance and day of discharge for all admittances to hospitals in sweden. admittances recorded in the ipr are assigned a code unique to the admitting department (e.g. psychiatry, geriatrics, and pediatrics). the ipr also links each admittance to diagnoses in the international classification of diseases (icd) (28). for this study, days registered as being in care at departments with an ipr code referring to psychiatric care were used. missing data data were missing for participants who had resided abroad at any time during the study period. therefore, these participants were excluded, as well as their matched controls. participants who had died during the study period were also excluded from analyses, as well as their matched controls. other controls who had resided abroad or had died during the study period were also excluded. statistics chi-squared tests were performed for proportional differences between the study groups. when comparing the two study groups with the control group, the wald chi-square test was used. ibm spss statistics for macintosh, version 28.0, was used for all computations. the level of significance was set at 5%. ethics approval this study was approved by the regional ethics committee in uppsala (dnr 2017/251). results the characteristics of the two former patient groups are shown in table 1. there were significant differences between these groups regarding prevalence of anxiety disorders and substance abuse. the pd group had significantly more comorbid axis i disorders and a higher mean psychosocial problem score at baseline than the non-pd group. the pd group also had a significantly higher mean scid-ii score. five individuals (9.6%) were excluded from the pd group because of death or residency abroad during the study period. the corresponding figure in the non-pd group was 13 (9.7%). their matching controls (n = 90) were excluded. furthermore, there were 86 controls who had resided abroad or died during the study period, who were also excluded. labor market outcomes and receipt of psychiatric care days of unemployment, sick leave, and disability pensioning were added up and merged into the new variable, lmm. the mean number of days in lmm for the pd group was 838 (95% confidence interval [ci] 627; 1,121), that for the non-pd group was 665 (95% ci 502; 881), and that for the control group was 440 (95% ci 380; 508). at age 30 years, 76.1% of the pd group had attained an education beyond high school (>12 years). the corresponding figure in the non-pd group was 85.1%, and that in the control group was 68.2%. during the study period, 44.7% of the pd group a long-term follow-up study of labor market marginalization 5 had ever been admitted to psychiatric care. the  corresponding figure was 22.3% for the non-pd group and 3.7% for the control group. the pd group had a mean yearly income at age 30 years of 173 (× 100 us dollars) (95% ci 131; 227). the non-pd group had a mean yearly income at age 30 years of 219 (× 100 us dollars) (95% ci 191; 251), and controls 236 (× 100 us dollars) (95% ci 225; 248). in the pd group, 42.6% of individuals had received social welfare benefits at some point during the study period. the corresponding figure for the non-pd group was 23.1%, and that for controls was 13%. for the pd group, the mean amount of social welfare benefits received during the study period was 92 (× 100 us dollars) (95% ci 47; 182), that for the non-pd group was 17 (× 100 us dollars) (95% ci 8; 34), and that for controls was 14 (× 100 us dollars) (95% ci 10; 21). when comparing the two study groups (pd and non-pd) with controls, significant differences were found in regards days in lmm, ever receipt of social welfare benefits, and ever admittance to psychiatric care. when comparing the two study groups with each other, the pd group was found to have received significantly more social welfare benefits during the study period. the pd group also had significantly more participants who had ever been admitted to psychiatric care during the study period compared with the non-pd group. the pd group had a lower income at age 30 years compared with the non-pd group and controls, but the finding was only significant compared with controls. both study groups had a higher level of education at age 30 years compared with controls, but this finding was only significant for the non-pd group. the results are shown in figure 1 and table 2. discussion the results indicated that mental disorder in young adulthood increased lmm up to 13 years after diagnosis, with the two table 2. labor market outcomes, educational level, and receipt of psychiatric care 2003–2016 for the groups pd, non-pd, and controls. outcome pd group n = 47 mean (sd) non-pd group n = 121 mean (sd) controls n = 754 mean (sd) wald χ2 post hoc test days in lmm in 2003–2016 (95% ci) 838 (627; 1,121) 665 (502; 881) 440 (380; 508) 18.3 pd > controls** non-pd > controls* amount of social welfare benefits received in 2003–2016 (95% ci) (× 100 us dollars)ç 92 (47; 182) 17 (8; 34) 14 (10; 21) 26.9 pd > non-pd* pd > controls* income at age 30 years (95% ci) (× 100 us dollars/year) ç 173 (131; 227) 219 (191; 251) 236 (225; 248) 6.3 pd < controls* n (%) n (%) n (%) wald χ2 post hoc test ever receipt of social welfare benefits in 2003–2016 (%) 20 (42.6) 28 (23.1) 98 (13.0) 29.4 pd > non-pd* pd > controls** non-pd > controls** education > 12 years at age 30 years, n (%) 35 (76.1)a 103 (85.1) 514 (68.2) 14.0 non-pd > controls*** ever admitted to psychiatric care, n (%) 21 (44.7) 27 (22.3) 28 (3.7) 98.6 pd > non-pd*** non-pd > controls** pd > controls*** sd: standard deviation; pd: personality disorder; ci: confidence interval; lmm: labor market marginalization. *p < 0.05, **p < 0.01, ***p < 0.001. çconverted from swedish kronor (sek) to us dollars (usd) using an exchange rate of 1 sek = 0.1005 usd, based on data from may 2022. an = 46 due to incomplete register data. figure 1. days of labor market marginalization (unemployment, sick leave, and disability pensioning) in 2003–2016 in the groups pd, non-pd, and controls. pd: personality disorder. 6 h. spangenberg et al. study groups having more days of lmm and more often having received social welfare benefits compared with controls. the significant differences in lmm in the study were mainly between former patients (pd group and non-pd group) and controls, though the former patient pd group showed more distinct differences regarding the receipt of social welfare benefits. the pd group also had a lower level of yearly income at age 30 years than the other groups. there could be several reasons for these findings. first, there was a certain degree of overlap in pd symptoms between the two former patient groups when the data were approached with a dimensional view of pd. also, both former patient groups were heavily burdened with axis i comorbidity at baseline. although the pd group had significantly more axis i disorders, the long-term outcomes in the non-pd group were most likely negatively affected by this burden. it is striking that the former patient groups had worse outcomes than controls, although both groups had more individuals with education >12 years at age 30 years than the controls. age 30 years was chosen as point of reference in a similar study by jonsson et al. (30). it would be of interest to compare participants with controls later on in life. it would have been of interest to account for social covariates at baseline, for example, parental socioeconomic situation. upon selecting data to be retrieved from statistics sweden and the national board of health and welfare, there was a concern that more individual data would risk breaches to anonymity of participants. we therefore refrained from gaining information on some factors from baseline which would have been of interest to account for, such as country of birth. sweden is a country with relatively small economic gaps, where 45% of the population has higher education (31), and it could therefore be assumed that no great differences in parental socioeconomic situation existed between groups at baseline. another issue to address when interpreting results is the effect that outliers had on mean values. as figure 1 shows, the mean value in the non-pd group might be increased because of the outliers with extremely high lmm values. these extreme values were also present in the control group. however, as the control group was much larger (n = 930), the effect of extreme values would be lower there. we do not know the reasons for these extreme lmm values, but they may have been caused by somatic disease, as they were also present in the control group. avoidant pd was the most common pd in the study. anxiety disorders, such as social phobia, were significantly more common in the pd group than in the non-pd group. the diagnostic and statistical manual of mental disorders, 5th edition (dsm-5) (32), describes an overlap between avoidant pd and social phobia, and that the two diagnoses may stem from the same or similar personality trait. in the current study, pd diagnoses were assessed after finalization of treatment of axis i disorders. this decreased the risk of misdiagnosis between axis i and ii, but there was a risk that individuals actually suffering from avoidant pd were diagnosed with social phobia, which would have affected results. also, comorbidity between pd and axis i disorder is common (33). it is possible that the higher degree of axis i comorbidity in the pd group would account for the findings. in the current study, it was not possible to use axis i disorders as a covariate in the analyses, due to the characteristics of the register data used. our findings are in line with those of previous studies of lmm in pd (1, 3, 4, 11, 12, 14, 15). this study adds further validity to previous findings, thanks to the use of gold standard diagnostics based on the lead procedure. the findings are important for various reasons. first, there is a scientific discourse on the validity of pd diagnoses (34). the findings in the current study indicate validity of pd diagnostics in young adulthood, as worse outcomes in psychosocial functioning were found in the pd group over time. second, lmm as an aspect of socioeconomic position is important for many health outcomes and may in itself lead to worsened mental health (35–37). individuals in the pd group received a mean amount of 9,208 us dollars in social welfare benefits during the study period (corresponding to on average 658 us dollars per year), which was significantly more than the other groups. the pd group had a lower yearly income at age 30 years than other groups. the variable on income which was used for analyses covered the individuals’ declared income; however, social welfare benefits, sick leave payments, and study grants were not included in this register variable. furthermore, individuals who are self-employed can choose to declare income in a manner which will become registered under a different variable in lisa. we therefore ran analysis of the other variable (kuink) at age 30 years for those who had been registered as self-employed in lisa. the difference in results for these individuals when comparing the declared income variable with income declared as the variable kuink was minimal. in sweden, future pension is largely based on income during working years, meaning lower income results in lower future pension payments. the worse labor market outcomes in the pd group might therefore have life-long impacts. the pattern of increased lmm found in the current study among those with pd calls for urgency in addressing and, when possible, treating pd. this study did not look into why pd affects lmm. previous studies have found that pd is associated with dysfunctional behaviors at work, leading to adverse work outcomes such as being fired, laid off, or experiencing problems in interactions with coworkers and bosses (15). further studies of this are suggested. there are a number of limitations to the study. first, it is problematic that the pd group was assessed as a single entity, as pd diagnoses differ in areas of functional impairment; something which is easy for an individual with a certain pd can be difficult for an individual with another pd. comorbidity between pds as defined in the dsm-iv (24) is common (34). occurrence of more than one pd in the same individual is often interpreted by clinicians as an indication of severe pd. in the dsm-5 (32), section iii, a dimensional approach to pds is suggested as an alternative model of pd (38). this model has been suggested to be useful for the study of psychosocial functioning in pd, as it places a general transdiagnostic a long-term follow-up study of labor market marginalization 7 personality dysfunction severity factor at the core of the pd entity (2). in the current study, the dichotomic pd construct was used primarily, but an attempt at a more dimensional approach to the pd construct was adopted by analyzing the scid-ii scores and comparing these between the two former patient groups. the two groups differed significantly in total scid-ii scores, but there was a certain degree of overlapping scores. it would have been of interest to analyze outcomes based on a dimensional pd approach through the scid-ii score instead of the dichotomized pd categories. however, this was not possible due to the characteristics of the retrieved register data. another limitation to the current study was the small sample size, especially in the pd group (n = 52). small effect sizes in such small groups lead to non-significant results, making it hard to draw conclusions. however, using the lead procedure for pd produces valid and reliable diagnoses. the two former patient groups differed significantly in mean scid-ii scores, increasing the reliability of findings. it was within the scope of the study to use this strength to make up for the low power created by the small sample size. there are many strengths to using swedish national registers for data collection. some of the registers have been validated (28), and they are generally considered to have high quality. however, the registers are not created for scientific purposes and reflect changes in politics and society in sweden over time. this has resulted in many changes to the variables over time, which sometimes complicates the study of register data over longer periods of time. one could argue that merging sick leave, disability pensioning, and unemployment into a compound lmm variable, instead of studying each variable on its own, limits the interpretability of findings. however, the compound lmm variable reflects lowered vocational functioning and ability of financial self-support, irrespective of the underlying reason, which was a main aim of the study. a compound lmm variable has been used in previous swedish long-term studies of psychosocial outcomes in psychiatric disorders (39). another weakness of the study concerns the quality of the specific data collected from the registers. days of sick leave, disability pensioning, and unemployment were merged into the new variable lmm and analyzed together. however, social welfare benefits were analyzed separately, as there were no data on the number of days that social welfare benefits had been received, only on the amount of money received. it would have been useful to have access to data on the number of days with social welfare benefits, to enable comparison with collected data on lmm, but such data were not available. there was also a limitation in the data collection of variables regarding disability pension. the registers include information on the net number of days on disability pension. however, this was overlooked when collecting the data, with the gross number of days being collected instead. the net number of days was calculated by multiplying the gross number of days with the fraction of received disability pension. this yielded a margin of error, as data on gross number of days and fraction of received sickness pension are compiled by statistics sweden from different data sources. the error was small and is not likely to have had any significant impact on the findings. the number of days admitted to hospital for psychiatric care was used to reflect the disease burden of psychiatric disorders other than pd in this study. this could also have been studied using the icd code that admittances were assigned in the ipr. however, there would have been several limitations to that strategy. first, the risk of invalid diagnoses being made by clinicians during admittance would have had to be considered. second, it is more common in psychiatric care than in somatic care to have missing primary diagnoses in the ipr after the year 2000 (28). another limitation of the study is that the control group was matched for sex, age, and living location at study baseline. however, the occurrence of pd in the control group is not known. the prevalence of pd in the general population is disputed, with studies suggesting it to be 9–12% (40, 41). the control group for the current study was chosen to match participants and, hence, is not representative of the general population. it is therefore difficult to estimate the proportion of pd in the control group. when interpreting the results, it should be considered that a certain proportion of individuals in the control group are likely to have fulfilled diagnostic criteria for pd, which could have given this subgroup similar results to those of the pd group. this would have had an attenuating effect on the results. in conclusion, in this study, former psychiatric patients with and without pd diagnosed in young adulthood, followed up after 13 years, had more days of lmm and received more social welfare benefits than controls. findings were mostly significant between former patient groups and controls, but the pd group stood out as regards receipt of social welfare benefits. results suggest that pd diagnosed in young adulthood impairs social functioning in the long term. acknowledgments the authors wish to thank mr. hans arinell for statistical support. disclosure statement the authors declare no conflicts of interest. the views expressed in the article are the authors’ own and not an official position of the institution or funders. funding this study was funded by the märta and nicke nasvell foundation, the anna-britta gustafsson foundation, ratiopharm ab, the fredrik and ingrid thuring foundation, and swedish medical association and medical training and research agreement (alf) funds from uppsala university hospital. 8 h. spangenberg et al. data availability the analyze code can be made available upon request. data will not be made publicly available but will be made available upon reasonable request. notes on contributors hanna spangenberg, m.d., phd. department of medical sciences, uppsala university. mia ramklint, m.d., professor. department of medical sciences, uppsala university. adriana ramirez, m.d., phd. department of medical sciences, uppsala university. orcid hanna spangenberg https://orcid.org/0000-0002-6129-0879 mia ramklint https://orcid.org/0000-0001-8203-8755 adriana ramirez https://orcid.org/0000-0002-1599-2925 references 1. huang y, 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https://doi.org/10.1146/annurev-clinpsy-032511-143131 https://doi.org/10.1177/002214650704800403 https://doi.org/10.1177/1403494819852847 https://doi.org/10.2147/prbm.s207971 https://doi.org/10.2147/prbm.s207971 https://doi.org/10.1521/pedi.2011.25.2.136 https://doi.org/10.1007/s00787-021-01825-3 https://doi.org/10.1007/s00787-021-01825-3 https://doi.org/10.1192/bjp.2018.202 https://doi.org/10.1192/bjp.2018.202 https://doi.org/10.1177/0033294117692807 upsala journal of medical sciences 2023, 128, e9194 http://dx.doi.org/10.48101/ujms.v128.9194 increased levels of a subset of angiogenesis-related plasma proteins in essential thrombocythemia sofia vikmana,b, anders larssona,c, måns thulind and torbjörn karlssona,b adepartment of medical sciences, uppsala university, uppsala, sweden; bdepartment of haematology, uppsala university hospital, uppsala, sweden; cdepartment of clinical chemistry, uppsala university hospital, uppsala, sweden; ddepartment of mathematics, uppsala university, uppsala, sweden abstract background: increased local angiogenesis is important for the growth and dissemination of cancer. the myeloproliferative neoplasm essential thrombocythemia (et) is known to involve increased bone marrow angiogenesis. blood levels of several angiogenesis-related proteins are increased in different types of cancer. the aim of this study was to investigate whether a subset of such proteins was elevated in treatment-naïve et patients. methods: blood plasma from 41 et patients and 43 healthy aged-matched controls was analyzed for eight different angiogenesis-related proteins. results: the et cohort displayed a more homogenous expression pattern of these proteins compared with controls. five of the eight proteins were significantly increased in et patients. conclusion: increased plasma levels of matrix metallopeptidase 9 (mmp9) and endostatin have not previously been reported in et. in our patients, mmp9 levels correlated positively with janus kinase 2 (jak2) v617f allele burden and leukocyte count. article history received 12 december 2022 revised 21 february 2023 accepted 22 february 2023 published 27 march 2023 keywords essential thrombocythemia; angiogenesis; extracellular matrix; matrix metallopeptidase 9; endostatin introduction essential thrombocythemia (et) is a chronic myeloproliferative neoplasm characterized by clonal megakaryocyte proliferation and thrombocytosis and is associated with hemorrhagic and thrombotic complications (1). other features of the disease are leukocytosis, splenomegaly, and risk of fibrotic or leukemic transformation. approximately 60% of et patients are positive for the janus kinase 2 (jak2) v617f mutation (1), which leads to a gain-of-function of this non-receptor tyrosine kinase. hematopoietic cells that harbor the jak2 v617f mutation have a proliferative advantage over non-mutated cells. a smaller fraction of et patients has mutations in the calreticulin (calr) or mpl proto-oncogene, thrombopoietin receptor (mpl) genes. only 10–20% of them harbor none of these three driver mutations (1) this group is referred to as having triple negative et. angiogenesis is increased in several types of cancer, including et, where increased bone marrow angiogenesis is observed (2). angiogenesis is of importance for tumor progression and the process has been well characterized at the molecular level. vascular endothelial growth factor (vegf) is a family of proteins, which regulates angiogenesis and lymphatic angiogenesis. vegf exerts its effect on angiogenesis by binding and activating specific transmembrane receptor tyrosine kinases (vegfr) predominantly expressed on endothelial cells (3). the vegfvegfr interaction stimulates proliferation and migration of endothelial cells. three different vegf receptors (r1, r2 and r3) have been identified; activation of vegfr2 by vegf-a is the most  important ligand–receptor interaction for angiogenesis. proliferation and migration of lymphatic endothelial cells depend on activation of vegf receptors 2 and 3 by the growth factors vegf-c and -d (4). alternative splicing of vegf and vegfr premrna gives rise to different vegfand vegfr protein isoforms, which can have opposing functions in vessel formation (5). extracellular matrix (ecm) is the non-cellular tissue component and is mainly composed of proteins such as collagen, polysaccharides and water. the ecm provides structural tissue support as well as exerting a variety of biochemical functions. in cancer there is a disturbance in ecm metabolism, which plays an important role in the creation of a tumor microenvironment (6). tumor cells promote degradation of ecm, which stimulates angiogenesis via the release of cytokines such as vegf sequestered in the ecm. local tumor expansion is also dependent on proteolytic degradation of the surrounding ecm by matrix metallopeptidases (also known as metalloproteinases) (7). besides the different vegf proteins and their receptors and matrix metallopeptidases (mmps), other proteins such as endostatin, growth differentiation factor 15 (gdf15), pentraxin 3 (ptx3) and selectins are involved in angiogenesis, both in health and disease. contact torbjörn karlsson torbjorn.a.karlsson@medsci.uu.se © 2023 the author(s). published by upsala medical society. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. original article http://dx.doi.org/10.48101/ujms.v128.9194 mailto:torbjorn.a.karlsson@medsci.uu.se http://creativecommons.org/licenses/by/4.0/ 2 s. vikman et al. in this study, we investigated plasma levels of eight angiogenesis-related proteins in an attempt to reveal their possible roles in angiogenesis in et. plasma levels of endostatin, gdf15, matrix metallopeptidase 9 (mmp9), ptx3, endothelial selectin (e-selectin), platelet selectin (p-selectin) and soluble vegf receptors (svegfr) 1 and 2 were determined by elisa and compared between et patients and healthy controls. patients and methods written informed consent was obtained from all study participants. the study was approved by the research ethics committee of uppsala (refs: 2010/98, 2014/233, ups-01367, and 2021-03316). blood plasma from 41 newly diagnosed treatment-naïve et patients (32% male) was used for elisa-analyses of endostatin, e-selectin, p-selectin, gdf15, mmp9, ptx3, svegf receptors and c-reactive protein (crp). plasma was generated from whole blood left for 30 min before centrifugation at 2,400 g for 7 min at room temperature. edta was used as anticoagulant. time from blood sampling to storage was less than 4 h. samples were stored in the uppsala-umeå cancer consortium (u-can) biobank at −80°c before analysis. laboratory and clinical data and pathology reports from the time of et diagnosis were obtained from each patient’s individual chart. the sysmex xn-9000 cell counter (kobe, japan) was used to obtain complete blood count. the control group consisted of 43 ageand sex-matched healthy individuals (37% male). zzstatistical analyses were calculated using version r 3.6.3 of the r software package (r foundation for statistical computing, vienna, austria). bootstrap t-tests were used to assess the relative differences in biomarker levels between groups (8). correlations were computed and tested using the spearman rank correlation. throughout, p-values were adjusted for multiplicity using the benjamini–hochberg procedure (9). results we analyzed blood plasma from 41 newly diagnosed treatment-naïve et patients and 43 controls, for levels of crp, endostatin, gdf15, mmp9, ptx3, e-selectin, p-selectin, svegfr1 and svegfr2. median (range) age was 60.5 (25–93) for et patients and 60 (43–79) for controls. prior to the et diagnosis, 33% (13/41) of the patients had been diagnosed with arterial or venous thromboembolic disease. the jak2 v617f mutation was found in 73% (30/41) of the et patients, with a median (range) allele burden of 22% (0.13–49.7%) a calr mutation was found in 10% (4/41) of the et patients, and none of the group was mpl positive. clinical and laboratory characteristics of the et patients and controls are summarized in table 1. leukocyte and platelet counts were significantly higher in the et group (p < 0.001 in both cases), whereas mean crp did not differ between et patients and controls. mean endostatin (p < 0.001), gdf15 (p  <  0.001), mmp9 (p < 0.01), e-selectin (p < 0.05) and p-selectin (p < 0.001) plasma levels were higher in the et group (table 1, figure 1). no differences in pxt3, svegfr1, or svegfr2 were observed between the two groups (table 1). a principal component analysis revealed that the et cohort expressed the proteins more homogenously than did the controls (figure 2). plasma mmp9 correlated positively and significantly with jak2 v617f allele burden (p < 0.05) and leukocyte count (p < 0.001) but not with platelet count (table 2). discussion in an attempt to reveal whether a subset of angiogenesis-related proteins might play a role in the increased bone marrow angiogenesis observed in et, we compared plasma levels of eight angiogenesis-related proteins between et patients and controls. as expected, the platelet count was higher among et patients. the et group also showed a significantly higher leukocyte count in comparison to controls. leukocytosis is a common feature in et (1). five of the proteins were significantly increased in the et patients: endostatin, gdf15, mmp9, and e-selectin and p-selectin. increased levels of circulating mmps are observed in several types of cancer (10) and their activity enhances angiogenesis via ecm degradation. mmp9 is secreted by a variety of cells, including neutrophils and macrophages (10). it degrades several components of the ecm, including collagen xviii, which is expressed in the basal lamina of vessel walls (11). endostatin is formed by mmp9 degradation of collagen xviii (11), and so an elevated level of endostatin reflects increased mmp9 activity (12). mmp9 is essential for formation of new blood vessels in vivo (13), and we observed significantly higher plasma levels of both mmp9 and endostatin in et patients compared with controls. haas and co-workers have previously shown decreased mmp table 1. clinical characteristics and laboratory data for the control and et populations. variable controls et p age (years)a 60 (43–79) 60.5 (25–93) ns jak2 v617f allele burden+ (%)a n.a 22 (0.13–49.7) na hb (g/l)b 140 (11) 141 (15) ns leukocytes(×109/l)b 6.4 (1.6) 7.9 (3.5) <0.001 platelets (×109/l)b 251 (80) 587 (284) <0.001 crp (mg/l)b 1.0 (1.7) 1.5 (2.8) ns gdf15 (ng/ml)b 521 (204) 720 (737) <0.001 ptx3 (ng/ml)b 1,432 (971) 1,850 (1,385) ns e-selectin (ng/ml)b 9,618 (4,823) 11,450 (6,936) <0.05 p-selectin (ng/ml)b 44,058 (15,388) 135,959 (62,207) <0.001 svegfr1 (ng/ml)b 632 (437) 564 (596) ns svegfr2 (ng/ml)b 3,002 (1,105) 2,891 (1,049) ns variables are expressed as amedian (range) or bmedian (interquartile range). et: essential thrombocythemia; na: not applicable; ns: not significant; jakv617f allele burden+: proportion of positive for janus kinase 2 allele showing the v617f mutation; crp: c-reactive protein; gdf15: growth differentiation factor 15; svegfr1: soluble vascular endothelial growth factor receptor 1; svegfr2: soluble vascular endothelial growth factor receptor 2. activity to be associated with a reduction in the number of microscopically observable capillary basement membrane breaks (14). thus, it is conceivable that increased mmp9 activity facilitates vascular sprouting and capillary growth by making the sub-endothelial basement membrane permeable to migrating and proliferating endothelial cells. endostatin is primarily an anti-angiogenic agent (15), but the net effect on tumor angiogenesis is determined by the balance between anti and pro-angiogenic factors. the anti-angiogenic effect of endostatin might be counteracted by mmp9-induced release of vegf sequestered in the ecm (10). a certain mmp9 gene polymorphism (gln279arg) has been associated with et (16), but to the best of our knowledge, this study is the first that reports increased plasma mmp9 levels in a population consisting exclusively of et patients. polymorphisms in the promoter region of the mmp9 gene have been reported to increase gene expression (17, 18), but the gln279arg polymorphism observed in et is not caused by a mutation in the mmp9 gene promoter. this polymorphism affects mmp9 substrate binding (16), and it is unlikely to increase mmp9 gene expression. since neutrophils are the main source of secreted mmp9 in cancer (19), we believe that the increased plasma mmp9 in et is related to the apparent leukocytosis observed in our patients. absolute neutrophil count was analyzed in only a few of the patients in our et cohort, but it is known that leukocytosis in myeloproliferative neoplasms is attributed to neutrophilia (20). this notion is supported by the finding of a positive correlation between mmp9 and leukocyte count. leukocytes and megakaryocytes stem from a common myeloid progenitor cell (21). thus, it is likely that the leukocytosis is a result of  clonal myelopoiesis rather than cancer-associated inflammation since there was no difference in mean crp between et patients and controls. interestingly, petzold and co-workers have recently published data that show a role for neutrophils in mobilization of platelets from megakaryocytes (22). neutrophil-dependent thrombopoiesis requires a direct physical contact between neutrophils and paravascular bone marrow megakaryocytes. this cell-to-cell contact is dependent on binding of the chemokine-receptor cxcr4 to its ligand cxcl12, and so it is conceivable that neutrophilia can exacerbate thrombocytosis in et. gdf15, a member of the transforming growth factor β superfamily, is a pro-angiogenic protein. it is abundantly figure 1. boxplots visualizing plasma levels of (a) mmp9 and (b) endostatin in ng/ml in controls and et patients. medians are shown as thick lines, bottom and top boxes represent the first and third quartiles, whiskers show the smallest and highest non-outliers, and circles represent outliers. in (a) mmp9 in et > controls (p < 0.01) and in (b) endostatin in et > controls (p < 0.001). abbreviations: et, essential thrombocythemia; mmp9, matrix metallopeptidase 9. figure 2. principal component analysis plot showing homogenous expression of the eight angiogenesis-related proteins analyzed in essential thrombocythemia (et) patients compared with controls. controls, yellow dots. et, blue dots. table 2. correlation coefficients (spearman rank order correlation) of mmp9 with leukocyte and platelet counts and jak v617f allele burden. variable rs (variable vs. mmp9) p leukocyte count 0.68 <0.001 jak2 v617f 0.42 <0.05 platelet count 0.11 ns mmp9: matrix metallopeptidase 9; jak2 v617f: ratio (%) between v617f mutant and wild type cells (allele burden); ns: not significant. angiogenesis-related plasma proteins in essential thrombocythemia 3 4 s. vikman et al. expressed in the placenta during physiological conditions and secreted by activated macrophages as a response to cellular stress signals, such as inflammation, tissue injury and hypoxia (23); its effect is to stimulate proliferation and migration of endothelial colony-forming cells (24). gdf15 exerts its pro-angiogenic effects at least partly by stimulation of vegf-a synthesis (25). increased levels of circulating gdf15 are seen in patients with various types of cancer including myeloproliferative neoplasms and in multiple myeloma increased gdf15 is associated with poor prognosis (26, 27). our current finding of increased plasma gdf15 in et suggests a possible role for this protein in et-related increased bone marrow angiogenesis. selectins are glycoproteins that are important in immune and inflammatory responses (28). these glycoproteins mediate cell–cell contacts by interaction with their receptors, for example, binding of leukocytes to endothelial cells. whereas p-selectin is expressed on the surface of platelets and endothelial cells, e-selectin is mainly expressed in endothelium. pand e-selectin are both involved in angiogenesis (29, 30). p-selectin is increased in et (31, 32), but there is conflicting evidence regarding e-selectin and et. our data showing increased plasma e-selectin in et are in contrast to the results of bilgir and co-workers but in line with the finding of others (31– 33). soluble vegf receptor 1 is commonly regarded as a decoy receptor for vegf-a, hence acting as an anti-angiogeneic agent. for example, svegfr1 is known to maintain corneal avascularity (34), but there are published data indicating that it is required for normal vascular development (35). lymphangiogenesis, which is an important factor in tumor progression, is induced by vegf-c binding its transmembrane receptor vegfr3. the receptor vegfr2 exists in a soluble form, which is a result of alterative splicing. soluble vegfr2 binds vegf-c, thus inhibiting vegf-c/ vegfr3 mediated signaling, which leads to decreased lymphatic endothelial cell proliferation (4). we did not observe any difference in plasma levels of svegfr1 when comparing et and controls. this is in contrast to other reports on svegfr1 in myeloproliferative neoplasms (36). the reason for these conflicting data is currently unknown. serum vegf-a is increased in et (37), which could be relevant given its potential involvement in an alternative way of vessel endothelialization, that is not dependent on local endothelial cell migration and proliferation. this process involves vegf-dependent recruitment of circulating cd34+/ vegfr2+ endothelial precursor cells into growing capillaries, where they differentiate into mature endothelium (38). vegf-a binds to and activates vegfr2 expressed on endothelial cells, and so decreased svegfr2 could possibly stimulate migration and differentiation of cd34+/vegfr+ endothelial precursor cells via an increased vegf-a effect on these cells. the mechanism behind this effect would be less circulating svegfr2 able to bind and neutralize vegf-a. in one study svegfr2 was decreased in et (37), but in our et cohort mean plasma svegfr2 did not differ significantly between et patients and controls. ptx3 and crp, both belong to the pentraxin protein superfamily. however, in contrast to the hepatic protein crp, ptx3 is produced by different cell types including endothelial cells. ptx3 has dual roles in angiogenesis, since it has both antiand pro-angiogenic properties (39, 40). in this study, mean plasma ptx3 did not differ significantly between the et and control cohorts. principal component analysis of plasma proteins in leukemia and hodgkin lymphoma has revealed distinct and homogenous expression patterns compared with controls (41, 42). in line with these findings, we here report that the expression pattern of eight angiogenesis-related proteins is more homogenous in et than in controls. this can at least partly be explained by the clonality of the disease. an obvious limitation of this explorative study is that it is retrospective and performed at a single centre. in summary, it reveals that a subset of angiogenesis-related proteins is increased in plasma from treatment-naïve et patients compared with healthy controls and hence that these proteins may be involved in the increased bone marrow angiogenesis observed in this neoplasm. increased plasma endostatin and mmp9 in et have not been reported previously, and further studies are necessary to define their possible roles in angiogenesis in myeloproliferative disorders. disclosure statement the authors declare no conflict of interest. funding this study was supported by grants from the swedish government (national agreement on medical education and research) and from lions cancer research fund uppsala-örebro. notes on contributors anders larsson, md, phd, is a professor at the department of  clinical chemistry, uppsala university hospital and at the department of medical sciences, uppsala university, sweden. måns thulin, phd, is an associate professor at the department of mathematics, uppsala university, sweden. torbjörn karlsson, md, phd, is an associate professor at the department of haematology, uppsala university hospital and at the department of medical sciences, uppsala university, sweden. sofia vikman, md, phd, is a consultant physician at the department of haematology, uppsala university hospital and at the department of medical sciences, uppsala university, sweden. orcid anders larsson https://orcid.org/0000-0003-3161-0402 måns thulin https//orcid.org/0000-0002-2756-3933 torbjörn karlsson https://orcid.org/0000-0003-4707-8633 sofia vikman https://orcid.org/009-007-9095-288x https://orcid.org/0000-0003-3161-0402 https://orcid.org/0000-0003-3161-0402 https//orcid.org/0000-0002-2756-3933 http://orcid.org/0000-0002-2756-3933 https://orcid.org/0000-0003-4707-8633 https://orcid.org/0000-0003-4707-8633 https://orcid.org/009-007-9095-288x https://orcid.org/009-007-9095-288x angiogenesis-related plasma proteins in essential thrombocythemia 5 references 1. tefferi a, pardanani a. essential thrombocythemia. n engl j med. 2019;381:2135–44. doi: 10.1056/nejmcp1816082 2. panteli k, zagorianakou n, agnantis nj, bourantas kl, bai m. clinical correlation of bone marrow microvessel density in essential thrombocythemia. acta haematol. 2005;114:99–103. doi: 10.1159/000086583 3. apte rs, chen ds, ferrara n. vegf in signaling and disease: beyond discovery and development. cell. 2019;176:1248–64. doi: 10.1016/j. cell.2019.01.021 4. stacker sa, williams sp, karnezis t, shayan r, fox sb, achen mg. lymphangiogenesis and lymphatic vessel remodelling in cancer. nat rev cancer. 2014;14:159–72. doi: 10.1038/nrc3677 5. bowler e, oltean s. alternative splicing 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